Creator Good stuff continue




Camelgate. Introduction to Camels. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Présentation des chameaux. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. About Camel Milk. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Lait de chamelle. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Camel Milk Products. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Les produits de lait de chamelle. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Camel Meat. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Viande de chameau. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Our Team. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Notre équipe. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Our Services. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Nos services. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Milk Commercialisation. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Commercialisation du lait de chamelle. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Links. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Liens. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. Links. The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. Links. The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Contact. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Contact. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. Contact. The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. Contact. The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Picture Gallery. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Galerie d'images. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. Galery. The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. Galery. The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Publications. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Publications. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. PUb. The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. PU. The platform for consulting and knowlege in camel dairy production
lang 3


Camelgate. Bookshelf. The platform for consulting and knowlege in camel dairy production
lang 0


Camelgate. Librairie. The platform for consulting and knowlege in camel dairy production
lang 1


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 2


Camelgate. . The platform for consulting and knowlege in camel dairy production
lang 3Weltwoche.ch - Artikel drucken

http://www.weltwoche.ch/artikel/print.asp?AssetID=9348&Category...

Diese Woche Camburger & Co. Mathias Plأ¼ss Forschung auf Weltniveau: Die ETH widmet sich der Kamelmilch. Dass die ETH Zأ¼rich in Quantenchemie und Turbinentechnik brilliert, hatten wir gewusst. Doch nun ereilt uns die أ¼berraschende Nachricht, dass das Polytechnikum auch in der nicht gerade landesأ¼blichen Disziplin der Kamelkunde eine Vorreiterrolle spiele. Das Kamel, sei es in seiner ein- oder zweihأ¶ckrigen Form, ist perfekt ans Leben in der Wأ¼ste angepasst. Bei Trockenheit kann es bis zu dreissig Tage ohne Wasser auskommen. Im Sandsturm verschliesst es wohlweislich seine etwas hoch getragene Nase. Fأ¼r den Menschen besonders wichtig ist, dass der Passgأ¤nger selbst in Notzeiten, wenn Schafs- und Ziegenzitzen unbarmherzig versiegen, zuverlأ¤ssig Milch produziert, bis zu acht Liter am Tag. Darum ist das Kamel vor allem in den Trockengebieten Ostafrikas ein zentraler Ernأ¤hrungs- und Wirtschaftsfaktor. Mit dem Handel ist das allerdings so eine Sache: Oft fehlt es an Verarbeitungstechniken, um Kamelprodukte verkaufstauglich zu machen. Gerade hier leistet die ETH Entwicklungshilfe: Sie liefert – Weltpremiere! – eine Anleitung zur Produktion von Kamelkأ¤se. Bislang fehlte es nأ¤mlich an einem geeigneten Stoff, der Kamelmilch schadlos zur Gerinnung bringt. Verwendet man gewأ¶hnliches Kuhlab, wird der Kamelkأ¤se bitter. Ein Forscherteam vom Institut fأ¼r Lebensmittelwissenschaft hat es nun geschafft, einem Schimmelpilz die Produktion eines geeigneten Gerinnungsenzyms beizubringen – Gentechnik sei Dank. Mit diesem Stoff, so heisst es, gelinge ein perfekter Kamelkأ¤se. Der Kopf des Teams ist Zakaria Farah, ein gebأ¼rtiger Somali und promovierter Chemiker, der sich an der ETH seit 15 Jahren mit den Feinheiten der Kamelmilch auseinander setzt. Zusammen mit Dromedarexperten aus der ganzen Welt hat er seine Erkenntnisse nun in einem Buch verأ¶ffentlicht (Milk and Meat from the Camel, vdf-Hochschulverlag, 230 S., Fr. 47.–) – ein Meilenstein der Kamelforschung. Den Hأ¶hepunkt des Werks bilden zweifellos die gut vierzig Rezepte, die auszuprobieren uns leider in Ermangelung geeigneter Zutaten nicht vergأ¶nnt war: Kamelbierwurst, Camel Irish Stew, Kamelkأ¤se, Corned Camel, Kamelcamembert und – unser Liebling: Camburger. Ein kleiner Dأ¤mpfer zum Schluss: Die ETH hأ¤lt keine eigenen Dromedare, wie man uns auf Anfrage versicherte. Die fأ¼r die Forschung benأ¶tigte Kamelmilch werde in gekأ¼hltem Zustand aus Kenia eingeflogen. (c) 2004 by Die Weltwoche, Zأ¼rich - E-mail: webmaster@weltwoche.ch

1 of 1

11.12.2004 16:14

pdf

322

Wangoh, Camel rennet

Extraction of camel rennet and its comparison with calf rennet extract
By J. WANGOH1, Z. FARAH and Z. PUHAN Laboratorium fأ¼r Milchwissenschaft, Eidgenأ¶ssische Technische Hochschule Zأ¼rich, ETH Zentrum, CH-8092 Zأ¼rich, Switzerland 1. Introduction Camel milk requires more calf rennet than cow milk to coagulate and the relative amount of rennet needed varies widely (2, 8, 14, 16). Extracts of adult camel abomasa have been used to coagulate cow milk with success (5, 6, 7). However, these enzymes have not been tried on camel milk. Rennet extracts from lamb and cow calves were found to be more effective with the milk of the respective species (12), while pig chymosin and pepsin respectively, were found to have a higher milk clotting activity in pig milk than in cow milk (9). Accordingly, it would not be surprising if camel rennet is more effective on camel milk than calf rennet. This work was therefore aimed at extracting camel rennet and testing its ability to coagulate camel and cow milk compared to calf rennet extract, chymosin and pepsin.

Preparation of milk substrates. Camel and cow milk powders were reconstituted according to IDF Standard (13). Determination of milk clotting activity. Thrombelastograph D (Hellige GmbH, FRG) at 32آ°C using 10ml milk and 200 آµI enzyme preparation was used during extraction of enzymes from the abomasa and the subsequent activation. IDF Standard method, Appendix A (13) was used for all the other enzyme activity determinations. To compare the activity of a particular enzyme in different milks, the stock solution of an enzyme was diluted to give a clotting time of 5.5 min in milk, and the activity of the enzyme was calculated for that milk. Separation of extract into fractions. The IDF Standard method (13) was used to separate the rennet extracts into fractions with the modification that the flow rate was set at 1 ml/min and the optical density (OD) of the eluate was continuously monitored at 226 nm and recorded by LKB 2238 Uvicord SII UV Monitor and LKB 2210 Potentiometric Recorder (LKB-Produkter AB, Sweden), respectively. 1-ml Samples were taken at intervals during elution and their clotting activity was determined in both cow and camel milk. 3. Results and discussion Enzymes extraction and activation The clotting activity of the extracts from both cow and camel calf abomasa during extraction and before activation is shown in Fig.1.

2. Materials and methods Abomasa. Camel calf abomasa were obtained from Ol Maisor Ranch in Kenya. Milk fed calves were slaughtered at the age of 3-4 weeks and their abomasa removed, dry salted and sun dried. Commercial cow calf abomasa were obtained from Winkler AG (Switzerland). Camel and cow milk powders. Camel milk was obtained from Ol Maisor Ranch in Kenya, held at 4 آ°C and transported to the laboratory within 24 h where it was defatted, freeze dried and kept until use. The cow milk used was Extra Low Heat spray dried skim milk powder obtained from Milchpulverfabrik Sulgen (Switzerland). Enzymes. Microbial chymosin was obtained from Gist Brocades (France) and porcine pepsin from Siegfried, Zofingen (Switzerland). Preparation of rennet extracts. Extraction was done after cutting the dry cow or camel calf abomasa into 1cm2 slices, soaking them in 6 % NaCI solution (1:10, w/v) containing 2 % boric acid and stirring continuously over 4 days at 5آ°C. The mixture was then filtered and centrifuged at 1,500 rpm for 15 min. The pH of the supernatant was then lowered from 5.5 to 4.7 with 1 N HCI and the extracts held at 25 آ°C for 24 h to activate 'the zymogens. The pH was thereafter raised to 5.5 with 1 N NaOH and the mixture centrifuged to obtain the final rennet extract.

Fig. 1: Clotting activity during extraction of camel and calf rennet
1

Permanent address: Department of Food Science, Technology and Nutrition, University of Nairobi, Box 29053, Nairobi, Kenya

Milchwissenschaft

48 (6) 1993

Wangoh, Camel rennet The main increase in clotting activity occurred during the first 24 h. Under similar experimental conditions, for rennet extracts from lamb and kid abomasa (1) and from cow abomasa (15). maximum clotting activities were obtained between 24-48 h of extraction. Fig. 2 shows the change in clotting activity during activation of the zymogen in the solutions after extraction.

323

Fig. 3: Clotting activity of calf rennet fractions with cow and camel milk

Fig. 2: Clotting activity of the extracts during activation at pH 4.7 The shape of the activation curves was similar for both extracts. The main increase in clotting activity was observed in the first 8 h of activation, 60 % for calf and 73 % for camel rennet extract. In commercial rennet production, activation requires 14-36 h at pH 4.6 and 5آ°C (15). Under laboratory conditions, however, maximum activation has been achieved already in 4-10 h at pH 4.7 and 25 آ°C (3,17).

Activity of the rennet extracts and their fractions Typical absorbance curves together with clotting activity on both cow and camel milk samples as determined during elution of rennet extracts are shown in Figs 3 and 4. The absorbance patterns for both camel and calf rennet extract were similar. However, the maximum clotting activity of the first fraction from calf rennet extract did not coincide with maximum absorbance at 226 nm, e.g. with the highest protein concentration. Similar results have been reported in the literature (4). In contrary, in camel rennet extract the maximum absorbance coincided well with that of the clotting activity. Additionally, the first fraction of camel rennet extract showed two, and the second fraction one active peak, whereas in calf rennet extract only one active peak was detected in each fraction. The first fraction of calf rennet extract coagulated cow milk readily but caused no coagulation after more than 1 h in camel milk, while the second fraction coagulated camel milk much faster than cow milk (Fig. 3). Both milks responded equally well to the first fraction of camel rennet, but
Milchwissenschaft

Fig. 4: Clotting activity of camel rennet fractions with cow and camel milk camel milk responded better to the second fraction though the activity of the fraction was low (Fig. 4). Since it is known that in calf rennet the first fraction contains chymosin and the second pepsin, pure commercial chymosin and porcine pepsin were tested on their ability to coagulate both cow and camel milk. Porcine pepsin was selected because it is being used for cheese manufacture (11). Furthermore bovine and porcine pepsin were shown to have a similar milk clotting activity per mg protein and to differ only in their general proteolytic activity (10, 11). Camel milk was coagulated 5 times faster by porcine pepsin and 7 times slower by chymosin than cow milk (Table 1). The ability of the extracts to coagulate both cow and camel milk were then tested. The analysis of variance and multiple range analysis for clotting time of cow and camel milk by calf and camel rennet extracts are shown in Table 2.

48 (6) 1993

324

Wangoh,

Camel rennet

Table 1: Estimation of activity of pure chymosin porcine pepsin in cow and camel milk Enzyme Chymosin Pepsin Milk Cow Camel Cow Camel Activity of stock enzyme, RU/ml 72.5a 10.9b 4.5a 22.1b Stock enzyme 1/250 1/40 1/78 1/15 Activity ratio (a:b) 6.7:1 1 :4.9 Clotting time, see 345.0 365.7 352.8 330.2 Standard deviation* 4.1 3.7 5.3 4.9 *Three independant determinations Camel milk was clotted slightly, but not significantly better by camel rennet than cow milk. Camel milk was, however, clotted slower by cow rennet extract. Interactions between milks and rennet extracts were significant. These results can be explained by the fact that the coagulation of camel milk by cow rennet extract was primarily due to the pepsin content of the cow rennet as shown in Fig. 3. The activity of the pepsin fraction compensated in this case the low activity of chymosin fraction in clotting of camel milk as also confirmed in Table 1. The large variations in the ability of cow rennet to coagulate camel milk reported in literature (2, 8, 14, 16) may be explained by differences in the pepsin content of the rennet used. The better coagulation of camel milk by camel rennet (Table 2) cannot be explained at present time. However, it could be the result of better suitability of camel rennet for coagulating camel milk. Similar observations were made with rennet and milk of other species (9, 12).

It was concluded that the coagulation of camel milk by calf rennet is primarily due to the pepsin content of the calf rennet. Therefore, camel milk should be coagulated with camel rennet or pepsin as it is not coagulated readily by calf chymosin. Acknowledgements We would like to sincerely thank Mr. Jasper Evans, proprietor, Ol Maisor Ranch, Kenya, who removed and processed the abomasa and supplied the camel milk . 5. References (1) ANIFANTAKIS, E., GREEN, M.L.: J. Dairy Res. 47221-230 (1980) (2) BAYOUMI, S.: Kieler Milchwirtschaftliche Forschungsberichte 42 3-8 (1990) (3) BISCHOFSBERGER, T., PUHAN, Z.: Milchwissenschaft halt 34 (10) 614-617 (1979) (4) BERANKOVA, E., SAJDOK, J., RAUCH,P., KAS, J.: Neth. Milk Dairy J. 42 337-340 (1988) (5) EL-ABBASSY, F.: Egyptian J. Dairy Sci. 15 87- 92 (1987) (6) EL-ABBASSY, F., WAHBA, A.: Egyptian J. Dairy Sci. 14 181-186 (1986) (7) EL-BATAWY, M.A., AMER, S.N., IBRAHIM, S.A: Egyptian J. Dairy Sci. 15 93-100 (1987) (8) FARAH, Z., BACHMANN, M.R.: Milchwissenschaft 42 689-692 (1987) (9) FOLTMANN, B., JENSEN, A.L., LONBLAD, P., SMIDT, E., AXELSEN, N.H.: Comp. Biochem. Physiol. 68B 9-13 (1981) (10) FOX, P.F.: J. Dairy Sci. 36 427-433 (1969) (11) GREEN, M.L.: J. Dairy Res. 39 261-273 (1972) (12) HERIAN, K., KRCAL, Z.: Prumysi-potravin 22 (5) 137-139 (1971) (13) International Dairy Federation: FIL/IDF Standard 110A (1987) (14) MOHAMED, M.A., MURSAL, A.I., LARSSONRAZNIKIEWICZ, M.: Milchwissenschaft 44 278280 (1989) (15) PLACEK, C.: Industrial Engineering and Chemistry 52 (1) 2-8 (1960) (16) RAMET, S.P.: World Animal Review 61 11-26 (1987) (17) RAND, A.G., ERNSTROM, C.A.: J. Dairy Sci. 47 181-187 (1964)

4. Conclusions The camel rennet extract coagulated camel milk slightly better than cow milk, while calf rennet extract coagulated camel milk less readily. The chymosin fraction of calf rennet extract had very little activity on camel milk while the pepsin fraction coagulated it much more readily than cow milk. Further tests showed that camel milk was coagulated 5 times faster than cow milk by pepsin, but 7 times slower by chymosin.

Table 2:

Analysis of variance and multiple range for clotting time of cow and camel milk with calf and rennet extracts Anova Multiple range analysis F-ratio Significance level 21.453 1.989 40.917 9.282 0.0000 0.1776 0.0000 0.0077 Milk Camel Cow Cow Camel Rennet Camel Camel Cow Cow CT(min) 4.17a 4.36ab 4.67b 5.21c SS 2.9492 0.1367 2.8125 0.6380 1.0998 4.6870 df 2 1 1 1 16 19 Mean square 1.4746 0.1367 2.8125 0.6380 0.6874

Source Main effect Milk Rennet Interactions Residual Total

CT = Clotting time abc = Means joined by the same letters are not significantly different P (95 %)
Milchwissenschaft

48 (6) 1993

325

6. Summary WANGOH, J., FARAH, Z., PUHAN, Z.: Extraction of camel rennet and its comparison with calf rennet extract. Milchwissenschaft 48 (6) 322-325 (1993). 86 Camel rennet (extraction) Camel rennet was extracted from camel calf abomasa by the method used for bovine rennet. The clotting activity was determined during extraction and activation. Both camel and cow abomasa extracts were fractioned and the clotting activity of the fractions compared. Camel rennet coagulated camel milk slightly faster than cow milk, while calf rennet extract coagulated camel milk less readily than cow milk. The chymosin fraction of calf rennet showed weak activity on camel milk while the pepsin fraction coagulated the same much more readily than cow milk. The first fraction of camel rennet coagulated cow and camel milk equally well, whereas the second fraction showed higher clotting activity with camel milk. It is concluded that the coagulation of camel milk by calf rennet is primarily due to the pepsin content of the calf rennet. The reported large variations in the ability of bovine rennet in coagulating camel milk can be explained by the differing pepsin content of the rennet used. Camel milk should therefore be coagulated with camel rennet or pepsin.

WANGOH, J., FARAH, Z., PUHAN, Z.: Gewinnung von Kamellab und der Vergleich mit Kأ¤lberlab. Milchwissenschaft 48 (6) 322-325 (1993). 86 Kamellab (Extraktion) Aus Labmagen von jungen Kamelen wurde Lab extrahiert nach der gleichen Methode, wie sie bei der Herstellung von bovinem Kأ¤lberlab angewendet wird. Die Gerinnungsaktivitأ¤t wurde wأ¤hrend der Extraktion und Aktivierung bestimmt. Kamellab zeigte mit Kamelmilch eine kأ¼rzere Gerinnungszeit als mit Kuhmilch, wahrend Kأ¤lberlab mit Kamelmilch lأ¤ngere Gerinnungszeiten als mit Kuhmilch aufwies. Die Fraktionierung von Kأ¤lberlab ergab, dass Chymosin nur eine geringe und das Pepsin eine gute Gerinnungsaktivitأ¤t mit Kamelmilch aufweist. Die erste Fraktion aus dem Kamellab koagulierte gleich gut mit Kuh- und Kamelmilch, und die zweite Fraktion ergab eine hأ¶here Gerinnungsaktivitأ¤t mit Kamelmilch. Daraus wurde gefolgert, dass die Gerinnung von Kamelmilch mit Kأ¤lberlab weitgehend von dessen Pepsingehalt abhأ¤ngig ist. Die in der Literatur erschienenen Angaben Ober grosse Unterschiede in der Gerinnung von Kamelmilch mit Kأ¤lberlab sind hأ¶chstwahrscheinlich auf den unterschiedlichen Pepsingehalt im Kأ¤lberlab zurأ¼ckzufأ¼hren. Die Ergebnisse der Untersuchungen lassen den Schluss zu, dass fأ¼r die Gerinnung von Kamelmilch am besten Kamellab oder Pepsin eingesetzt werden soll.

pdf

3.

Farah, Camel milk

763

Effect of heat treatment on whey proteins of camel milk
By Z. FARAH
Labor fأ¼r Milchwissenschaft, Eidg. Technische Hochschule, Zأ¼rich, Switzerland

1. Introduction Camel milk is an important component of the human diet in many parts of the world. It contains all essential nutrients and the composition is similar to that of cow milk (l). The present knowledge about the milk production potential of camels (Camelus dromedarius) is very limited. Data available show, however, that a healthy camel on good feed can produce 2,000 J of milk per lactation period (2). Even higher milk yields have been recorded (3). Most of the camel milk is drunk fresh. It is also consumed when slightly sour. Heat processing as a means of preserving camel milk is not known. The heat treatments commonly used such as pasteurization and sterilization cause denaturation of the whey proteins. This phenomenon has been extensively studied because of its importance in understanding the changes in the properties of milk that occur with heat treatment (4, 5). However, most of these studies are limited to cow milk due to its wide-spread industrial processing and commercialization. This investigation was undertaken to determine the degree of denaturation of the whey proteins, when camel milk is subjected to various heat treatments. 2. Materials and methods 2. 1 Milk samples Camel milk samples were taken at Ngare Ndare Camel Farm', which is situated just north of the Equator in Kenya's Laikiba District and at an altitude of between 1,730 to 1,890 m above sea level. The animals of the indigenous breed (Camelus dromedarius) were fed all year round exclusively by grazing. The milk samples were collected from 10 individual camels on 3 different occasions. For each occasion, the 10 milk samples were mixed to one batch and skimmed. Each batch was divided into 250 ml portions. One portion was kept as a control (raw milk) and the rest was individually heated at 63, 80 and 90 آ°c for 30 min in a water bath in 500 ml round bottom flasks equipped with a condenser and thermometer. After heating, the flasks were cooled to room temperature. For comparison, bulk cow milk was used.

2.2 Nitrogen distribution
The nitrogen distribution in the milk was determined by the procedure of ASCHAFFENBURG and DREWRY (6). The following N-fractions were determined: total protein nitrogen (TN), non casein nitrogen (NCN) and non protein nitrogen (NPN) soluble in 12 % trichloracetic acid. Denaturation of whey proteins was calculated by the difference of the whey protein nitrogen (NCN minus NPN) before and after heating of the milk.

2.3 Electrophoresis
The non casein nitrogen filtrate containing the whey proteins was dialysed against water, lyophilized and then examined by polyacrylamide gel electrophoresis (PAGE). The electrophoresis was performed in a vertical slab gel apparatus of DESAGA (Heidelberg) according to the procedure of SMITH (7). The concentration of acrylamide in the resolving gel was 10%. Electrophoresis was run at 20 mA for 20 min and then at 1 00 mA until the marker dye (bromphenolblue) was 0.5 cm from the anodic end of the slab (about 3 h). Cooling water of 12 آ°c was circulated in the electrophoretic chamber. The slabs were stained for 1 h with Coomassie brilliant blue G 250 in 3.5 % perchloric acid, according to REISNER et al. (8) and destained with 7.5 % (v/v) acetic acid. Polyacrylamide gel electrophoresis containing sodium dodecyl sulphate (50S-PAGE) was performed as described by LAEMMLI (9). The resolving gel contained 12.5 % acrylamide. 3. Results and discussion The distribution of N-fractions in raw milk as well as in milk samples heated at 63, 80 and 90 آ°c for 30 min are presented in Table 1. The value of NCN, which consists of whey proteins and NPN expressed as percentage of the total milk nitrogen, varied in raw milk from 23 to 24 %, showing no pronounced difference between camel and cow milk. The amount of NPN in both milks ranging from 5.6 to 6.6 % was not affected by the heat treatment of milk. Denaturation susceptibility of the whey proteins was expressed as percentage denaturation relative to the control raw milk.

Tab 1: Effect on heat treatment on the distribution of N-fractions in camel and cow milk TN Batch No. 1 Temperature for 30 min raw 63QC 80آ°C 90آ°C raw 63QC 80QC 90QC raw 63آ°C 80آ°C 90QC mg/100g Cow Camel 522 433 NCN mg/100g Camel 106 93 79 70 124 113 95 81 130 115 97 79 % of TN Camel 24 22 18 16 23 21 17 15 24 21 18 14 mg/100g Camel 29 28 29 29 31 32 32 32 30 30 30 30 NPN % of TN Camel 6.6 6.5 6.6 6.6 S.7 5.8 S.8 5.8 S.6 5.6 5.6 5.6 mg/100g Cow Camel 88 77 82 65 22 SO 17 41 97 93 90 81 26 63 18 49 91 100 81 85 26 67 23 49 Percentage denatured WPN mg/100g Cow Camel 7 75 81 7 73 81 7 70 74 16 3S 47 13 32 53 15 33 51

2

530

543

3

550

548

Cow 122 115 55 51 129 122 57 SO 124 113 58 55

Cow 23 22 11 10 24 23 11 9 23 21 11 10

Cow 34 33 33 34 32 32 31 32 33 32 32 32

Cow 6.5 6.3 6.3 6.5 6.0 6.0 5.8 6.0 6.0 5.8 5.8 5.8

Milchwissenschaft 41 (12) 1986

764

Farah, Camel milk

The amount of denaturated whey proteins in the cow milk is in agreement with the values reported in the literature (10, 11). The lowest time-temperature combination (63 آ°C/30 min), which represents the conditions of conventional pasteurization caused little whey protein denaturation, while higher heat treatment at 80 and 90آ°C for 30 min which is more excessive than pasteurization, resulted in a 70 to 81 % denaturation of the whey proteins . The camel milk whey protein showed generally a higher heat stability than cow milk. After an initial higher denaturation of whey proteins in camel milk at 63آ°C, the heal stability of the camel milk whey proteins increased in comparison to cow whey proteins markedly with temperature during heat treatment. The degree of denaturation of the whey proteins varied in camel milk from 32 to 35%at80آ°C and 47 to 51 % at 90آ°C heating temperature. This means that the susceptibility of camel whey protein to heat denaturation under the applied conditions is nearly twofold lower than that of the cow whey proteins, as shown in Table 1. By means of polyacrylamide electrophoresis it has been possible to give visual evidence of heat effect on the whey proteins, thus confirming the results of the distribution of the N-fractions. Fig. 1 gives the whey protein gel patterns of the raw and heated cow and camel milk. The obtained electrophoretic patterns of the individual whey proteins in cow milk agree with the results of previous investigations (10, 11, 12). Pasteurization temperature (63آ°C) caused no visible change in the whey protein gel pattern. At 80آ°C (slot C) immune globulins and serum albumin disappeared from the electrophoresis pattern. Portions of خ²-lactoglobulins (A and B) and خ±-lactalbumin remain undenaturated at 80آ°C, but disappear after heat treatment of 90آ°C (slot D). Following earlier investigations of electrophoretic separation of camel milk (13), camel milk whey proteins showed lower mobility than cow milk whey protein (Fig. 1). The main whey protein bands of camel milk are designated by the numbers 1, 2, 3, and 4. The electrophoretic patterns in slots E F and G show 1 broader band in the upper part of the gel (component 1) followed by 2 sharp bands and I faint band in the lower part of the gel (components 2, 3 and 4). The gel patterns indicate that a pronounced heat effect can be observed in the sample of 90آ°C (slot H) where band intensities decrease and component 2 disappears. In order to estimate the molecular weight of the individual whey proteins of camel milk, the proteins were further examined by PAGE in the presence of sodium dodecyl sulphate

Fig. 2: 50S-PAGE patterns of whey protein filtrates prepared from camel milk heated at various temperatures for 30 min. E, raw; F, 63آ°C; G, 80آ°C, H, 90آ°C. 5, standard marker proteins from top to bottom: bovine albumin (mol. wt. 66,000); egg albumin (mol. wt. 45,000); glyceraldehyde phosphate dehydrogenase (mol. wt. 36,000); carbonic anhydrase (mol. wt. 29,000); trypsinogen (mol. wt. 24,000); trypsin inhibitor (mol. wt. 20,000); خ±-lactalbumin (mol. wt. 14,000).

(SDS, Fig. 2). Comparing with the standard mixture of proteins in slot 5, band I and band IV are identical with standard bovine serum albumin (molecular weight 66,000) and خ±-lactalbumin (molecular weight 14,000). The estimated molecular weights of bands II and III were 43,000 and 23,000 respectively. These 2 protein bands are presumably خ²-lactoglobulins. It must be noted, however, that the sequence of the bands in Fig. 2 (I-IV) is not identical with the sequence of bands in Fig. 1 (1-4). The electrophoresis indicates that the degree of susceptibility to heat denaturation of the individual camel whey proteins is not as pronounced as it is in cow whey proteins. It seems that the differences in heat stability among camel milk whey proteins are smaller than among the cow milk whey proteins. Thus, a more intensive heat treatment of camel milk is necessary to obtain the same degree of denaturation as in cow milk.

Origin

-

Serum albumin خ±-Lactalbumin Fig. 1: Polyacrylamide gel patterns of whey protein filtrates prepared from camel and cow milk heated for 30 min at various temperatures. Cow milk: A, raw; B, 63 آ°C; C, 80 "'C; D, 90 آ°C Camel milk: E, raw; F, 63آ°C; G, 80آ°C; H, 90آ°C.

-

خ²-Lactoglobulin

B– A–

Milchwissenschaft 41 (12) 1986

Farah, Camel milk

765 des Nicht-Caseinآ· und des Nicht-Protein-Stickstoffes in der rohen Kamelmilch am Gesamtstickstoff variierte von 23 bis 24 %, bzw. von 5,6 bis 6,6 %. Die Anwendung der Elektrophorese in Natriumdodecylsulfat-Polyacrylamidgel (SDS-PAGE) zur Identifizierung von Kamelmolkenproteinen zeigte neben خ±-Laktalbumin und Serumalbumin die Anwesenheit von zwei Molkenproteinen unterschiedlichen Molekulargewichtes. Diese kأ¶nnen als Homologe der خ²-Laktoglobuline der Kuhmilch betrachtet werden. Die Molkenproteine der Kamelmilch sind bedeutend weniger empfindlich fأ¼r Hitzedenaturierung als diejenigen der Kuhmilch. Unter den gewأ¤hlten experimentellen Bedingungen war der Grad der Hitzedenaturierung der Molkenproteine bei der Kamelmilch annأ¤hernd zweimal geringer als bei Kuhmilch.

From the results of this investigation it can be concluded that the heat sensitivity of whey proteins in camel milk is considerably lower than in cow milk. At the time being no adequate explanation can be offered for this relative heat stability. Further studies are needed to elucidate the mechanism involved in the heat denaturation of camel milk whey proteins.

Acknowledgement This work was supported in part by the Swiss Technical Cooperation and Humanitarian Aid in Berne. The author expresses his thanks to Dr. W. Schulthess, Dept. of Food Technology and Nutrition, University of Nairobi, Kenya, for providing the camel milk.

FARAH, Z.: Influence du traitement thermique sur les protأ©ines du sأ©rum du lait de chamelle. Milchwissenschaft 41 (12) 763-765 (1986). 24 Lait de chamelle (denaturation thermique)

4. References
(1) YAGIL, R.: FAD Animal Production and Health Paper, Rome 2614-19 (1982) (2) KNOESS, K.H. in: Camels, International Foundation for Science (IFS) Symposium, Sudan 201-214 (1979) (3) KNOESS, K.H.: World Animal Rev. 22 3-8 (1977) (4) JENNESS, R., PATTON, S.: Principles of Dairy Chemistry. John Wiley and Sons, New York City 323-357 (1959) (5) FOX, P.F.: Developments in Dairy Chemistry - 1. Applied Science Publishers, London, New York 189-228 (1982) (6) ASCHAFFENBURG, R., DREWRY, I.: XV. Intern. Dairy Congr.3 1631-1637 (1959) (7) SMITH, I.: In: Chromatographic and Electrophoretic Techniques, Vol. 2, 2nd ed., Medical Books, London 399-418 (1 968) (8) REISNER, AX et 01.: Anal. Biochem. 64 509 (1975) (9) LAEMMLI,I.U.K.: Nature 227 680-685 (1970) (10) LARSON, B.L., ROLLERI, G.D.: J. Dairy Sci. 38 351 (1955) (11) MELACHOURIS, N.T., TURCKY, S.L.: J. Dairy Sci.91105411057 (1966) (12) AKROYD, P.: In: Chromatographic and Electrophoretic Techniques, Vol. 2, 2nd ed., Medical Books, London, p. 410 (1968) (13) FARAH, Z., FARAH-RIESSEN, M.: Milchwissenschaft 40 669-671 (1985)

FARAH, Z.: Influjo del tratamiento tأ©rmico en las proteأ­nas de suero de leche de camella. Milchwissenschaft 41 (12) 763-765 (1986). 24 Leche de camella (denaturaci6n tأ©rmica)

5. Summary
FARAH, Z.: Effect of heat treatment on whey proteins of camel milk. Milchwissenschaft 41 (12) 763-765 (1986). 24 Camel milk (heat denaturation) Heat denaturation of the whey proteins of camel and cow milk was compared. The milk was heated to 63, 80 and 90 "c for 30 min and nitrogen distribution was determined in raw and heated milk. The whey proteins were also examined by polyacrylamide gel electrophoresis. Non casein nitrogen and non protein nitrogen in raw camel milk expressed as percentage of the total milk nitrogen varied from 23 to 24 % and 5.6 to 6.6 %, respectively. Application of electrophoresis in sodium dodecyl sulphate polyacrylamide gel(SDS-PAGE) to identify camel whey proteins revealed beside خ±-lactalbumin and bovine serum albumin, the presence of two whey proteins of different molecular weights, which can be regarded as possibly homologous to bovine خ²-lactoglobulins. Compared with cow milk, camel whey proteins exhibited markedly lower sensitivity to heat denaturation. Under the selected experimental conditions the rate of heat denaturation of camel milk whey proteins was approximately twofold lower than cow milk whey proteins.

FARAH, Z.: Einfluss der Hitzebehandlung auf die Molkenproteine der Kamelmilch. Milchwissenschaft 41 (12) 763-765 (1986). 24 Kamelmilch (Hitzedenaturierung) Die Hitzedenaturierung der Molkenproteine von Kamel- und Kuhmilch wurde verglichen. Die Milch wurde bei; 63,80 und 90 "c wأ¤hrend 30 min erhitzt und die Stickstoffverteilung in der rohen und in der erhitzten Milch bestimmt. Die Molkenproteine wurden auch mit Hilfe der Polyacrylamidgel-Elektrophorese untersucht. Der Anteil

Milchwissenschaft 41 (12) 1986

pdf

J. Dairy Sci. 86:498–508  American Dairy Science Association, 2003.

5′-Flanking Regions of Camel Milk Genes Are Highly Similar to Homologue Regions of Other Species and Can be Divided into Two Distinct Groups
S. R. Kappeler, Z. Farah, and Z. Puhan
Laboratory of Dairy Science, Institute of Food Science, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland

ABSTRACT The concentrations of individual casein and whey proteins in camel milk differ markedly to respective protein concentrations in bovine milk. The ratio of خ²casein to خ؛-casein is considerably higher in camel milk. خ²-Lactoglobulin is absent, but whey acidic protein and peptidoglycan recognition protein have been detected. Genomic sequences upstream to milk-protein genes, which are known to regulate the expression of milk proteins to a great extent, were determined for 10 camel milk-protein genes and compared to respective sequences in other mammals. Multiple sequence alignment showed closest relationships to homologous sequences from other mammals. Comparison of milk protein regulative regions revealed two distantly related groups with pronouncedly different transcription factor site probabilities. The GC-content in sequences of the ï¬پrst group was considerably higher than in sequences of the second group and combined occurrence of CAAT and TATAA boxes was rare, suggesting that the ï¬پrst group represented mostly the housekeeping gene type, probably regulated by cellular signal transduction pathways, whereas the second group helped to regulate genes speciï¬پcally expressed in terminally differentiated cells of the lactating alveolar epithelium. A core region of the composite response element, which primarily controls milk protein gene activity, was found by a search for elements conserved within all 5′-flanking sequences analyzed, and it is assumed, that the presence of this element determines gene expression in the lactating mammary gland, and binding sites for general activator and repressor factors, surrounding the milk protein gene speciï¬پc element, are important for regulation of gene activity. (Key words: camel, gene expression, milk protein, transcription factor binding site)

Abbreviation key: 5′-flanking region = gene sequence 5′-flanking to the transcriptional start site of the milk-protein genes examined, TF = transcription factor. Abbreviations for transcription factors follow the TRANSFAC entries (Wingender et al., 2000). INTRODUCTION Milk proteins may basically be divided into watersoluble proteins, which often have a function in protecting the lactating udder and the newborn against environmental stress, and into amphipatic proteins, which help to preserve the suspension- and emulsionlike character of milk. The quantitative distribution of individual proteins greatly differs between species, and the distribution in camel milk is unique in particular. Some important proteins of bovine milk, such as خ²-lactoglobulin and lysozyme C, are not found in camel milk, whereas other proteins are present, such as the whey acidic protein and the peptidoglycan recognition protein, which were not detected in bovine milk. These proteins most probably function as protective or immunomodulating factors, and some of them are also found as a part of the body immune system. The different distribution of these factors in bovine and camel milk is probably a result of the harsh conditions in the natural habitat of the camels. The immunological situation, which faces the lactating camel and its offspring, requires modiï¬پcations in the response to environmental stimuli. Our interest in this study was to understand the factors that regulate the expression of the genes that correspond to milk proteins and lead to the observed variation in the distribution of these proteins between species. In particular, we intended to ï¬پnd out whether the mechanisms described to regulate the milk protein gene expression in other species also apply to the homologous camel genes. During the past decade, a new level in understanding of the processes that regulate the tissue-speciï¬پc expression of milk-protein genes has arisen, with molecular and cellular investigations in the regulation of the mammary gland during pregnancy, lactation,

Received January 31, 2002. Accepted March 26, 2002. Corresponding author: S. R. Kappeler; e-mail: stefan.kappeler@ alumni.ethz.ch.

498

ANALYSIS OF CAMEL MILK-PROTEIN GENES

499

and involution. Expression of milk-speciï¬پc genes in the lobuloalveolar epithelium of the lactating mammary gland was reported to be regulated by hormonal and environmental stimuli, which are transmitted through a set of transcription factors that bind to enhancer elements located on the proximal or distal 5′flanking region to the transcriptional start site (Rosen et al., 1998). The minimal requirements to elicit a sufï¬پcient lactogenic response include prolactin, glucocorticoids, and insulin (Nagaiah et al., 1981), which may be substituted by the insulin-like growth factor. Progesterone, which is involved in mammogenesis, was shown to repress casein gene expression during pregnancy through a DNA-binding factor of 65 kDa (Lee and Oka, 1992). Epidermal growth factor (Teng, 1999), which is by itself secreted into milk and stimulates the gastrointestinal development of the newborn (Brown et al., 1989), also contributes to mammogenesis (Gallego et al., 2000), counteracting transforming growth factor خ². On the morphological level, it was found that signals from the basement membrane cooperate with hormonal factors mentioned to maintain the lobuloalveolar structure and the basal-apical polarization of the epithelial cells, which is required for expression and secretion of milk proteins (Aggeler et al., 1991; Close et al., 1997). In particular, the basement membrane protein laminin was shown to be required for activation of the prolactin-receptor by prolactin, acting via membrane anchored خ²1-integrins (Edwards et al., 1998), as well as factors from the extracellular membrane suppressing histone deacetylases (Rosen et al., 1999). In studies, where stage- or tissue-speciï¬پc levels of milk proteins and corresponding mRNA were compared, good correlations were found, indicating that quantitative control of expression occurs on the transcriptional level (McClenaghan et al., 1995). Our ï¬پnding that the protein composition of camel milk is markedly different from the composition in bovine milk over the course of the lactational cycle, prompted us to investigate whether this differential expression pattern can be followed back to differences between the 5′-flanking regions of camel and bovine milk-protein genes. The data gained allowed us to start a comparative statistical analysis of putative transacting factor binding sites in 5′-flanking regions of homologous milk genes from different species, and to examine, which of the proposed regulative elements is likely involved in the regulation of the examined camel milk-protein genes.

MATERIALS AND METHODS Camel Milk Collection and Quantitative Analysis of Milk Proteins Milk from different breeds and individuals was frozen at −20آ°C for transport and stored at −70آ°C until analysis. Caseins and whey proteins were separated, identiï¬پed and characterized as described (Kappeler et al., 1999b, 1999a). DNA Sequence Analysis Coding sequences for camel milk proteins were determined by using a cDNA library constructed from a Somali breed camel, as described in Kappeler et al (1998). Genomic DNA was extracted from white blood cells of an Arabian camel by conventional phenol-ethanol puriï¬پcation. An average length of about 40 kDa was found after 0.5%-agarose gel separation. Five Genome Walker libraries were created as recommended by the manufacturer (K1807-1; Clontech, Palo Alto, CA). Sequence speciï¬پc primers were designed, based on information from exon I and intron I regions sequenced previously. PCR-ampliï¬پcation products were sequenced on an ABI Prism 310 Genetic Analyzer using BigDye chemistry. The 5′ flanking sequences were entered in the EMBL/GenBank database under the accession numbers AJ409277 (خ±S1-CN), AJ409278 (خ±S2CN), AJ409279 (خ²-CN), AJ409280 (خ؛-CN), AJ409281 (خ±-LA), AJ409282 (lactoferrin), AJ409283 (lactophorin), AJ409284 (lactoperoxidase), AJ409286 (peptidoglycan recognition protein), and AJ409285 (whey acidic protein). Computational Sequence Analysis The GCG version 10 software package (Genetics Computer Group, Madison, WI) was used to create a tree illustrating the relatedness of the 5′-upstream sequences examined. First, 50 proximal, 5′-flanking regions (≤1120 bp) of milk-protein genes were aligned with the pileup function, using gap weight 1 and gap length weight 0. The penalties for gap creation and extension were set to a low value, since sequences 5′upstream to vertebrate genes usually exhibit a high variability in regard to insertion and deletion of DNA fragments. A Tamura distance-corrected matrix of the aligned sequences was created with the distances function. Third, a phylogenetic tree was created out of the matrix according to the unweighted pair group method using arithmetic averages. Additional sequences used for the analyses (see Figure 1) included DNA regions from the following EMBL/GenBank acJournal of Dairy Science Vol. 86, No. 2, 2003

500

KAPPELER ET AL.

Figure 1. Phylogenetic tree of 5′-flanking regions adjacent to the transcriptional start site of milk-protein genes. Length of the branches corresponds to the number of substitutions. The distance between human mucin 1 and rat gamma-casein was 54.70 substitutions per 100 base pairs.

Journal of Dairy Science Vol. 86, No. 2, 2003

ANALYSIS OF CAMEL MILK-PROTEIN GENES

501

cession numbers. The information in brackets indicates the corresponding protein and the number of base pairs in front of the transcriptional start site included into analysis. AC005962 (human lactoperoxidase, 2000), AC007785 (human peptidoglycan recognition protein, 2000), AC063956 (human alpha s2-casein, 2000), AF027807 (human beta-casein, 1194), AF044256 (porcine lactoferrin, 1366), AF072711 (human carboxyester lipase, 2000), AF107201 (equine beta-lactoglobulin, 2000), AL121936 (human butyrophilin 1A, 2000), D16108 (murine lactophorin (GlyCAM-1, PP3, 2000), D85424 (human alpha s1-casein, 1180), E12614 (porcine beta-casein, 2000), L11749 (murine mammary tumor virus, LTR, 1361), M10936 (rat beta-casein, 783), M55158 (bovine beta-casein, 1722), M61170 (human mucin 1, gene product of the human polymorphic epithelial mucin gene (PEM), 2000), M74778 (murine lactoferrin, 2000), M75887 (bovine kappa-casein, 2000), M90645 (bovine alphalactalbumin, 1951), M94327 (bovine alpha s2 casein type A (CASAS2), 1509), U02884 (murine fatty acid binding protein, 1515), U16175 (murine mucin 1, 2000), U25810 (bovine lysozyme C, 2000), U28757 (porcine lysozyme C, 2000), U38816 (murine whey acidic protein, 2000), U57623 (human fatty acid binding protein, 1247), U67065 (murine butyrophilin 1A, 2000), U69534 (murine epidermal growth factor, 2000), X01153 (rat whey acidic protein, 1190), X03584 (rat alpha s1-casein, 682), X03589 (rat gamma-casein, 679), X12817 (ovine beta-lactoglobulin, 801), X13484 (murine beta-casein, 2000), X15735 (rabbit beta-casein, 2000), X59856 (bovine alpha s1-casein, 2000), X83391 (bovine lactophorin (GlyCAM-1, PP3), 1014), X98558 (porcine fatty acid binding protein, 1607), Y00726 (guinea pig alpha-lactalbumin, 1195), Y12088 (murine peptidoglycan recognition protein, 1532), Z33882 (caprine kappa-casein, 2000), Z48305 (bovine beta-lactoglobulin, 2000). Repetitive elements were detected in the examined 5′-sequences using RepeatMasker version 2, based on the Repbase database (Smit and Green, 2000). Potential binding sites for transcription factors, socalled TF-sites, were detected in the 50 5′-flanking regions examined, according to the method of (Schug and Overton, 1997). The TRANSFAC (transcription factor) database (Wingender et al., 2000) version 3.3 was used and the following parameters applied: no allowable mismatch, a minimum element length of six bases and a minimum lg-likelihood of 6. Where possible, the 2000-bp upstream to the transcriptional start site were examined, to be able also to detect more distantly located binding sites. The reliability of TFsites found was 31%, calculated as follows: (خ£nP − خ£nR)/خ£nP with خ£nP as the number of sites found per

base pair of 5′-flanking sequence (the total number of base pairs analyzed was 78,598), and خ£nR as the number of sites found per base pair in 200 kbp of randomly generated DNA sequence. Because factors with a large number of TRANSFAC-compiled binding sites were overrepresented by these string-based searches, redundancies were ï¬پltered out. The frequencies of occurrence within 1000 bp of the 5′-flanking regions were calculated for all TF sites. The same was done with randomly generated DNA sequences. The values obtained from the randomly generated sequences, considered as a background resulting from weakly deï¬پned binding sites, were subtracted from the former. The resulting binding site proï¬پles contained information about the grinding potential of known transcription factors to the analyzed promoter regions, under relinquishment, of positional information. A correlation factor for every pair of promoter regions was calculated as the sum of products of the number of background-subtracted, nonredundant binding sites of known transcription factors, according to the formula خ£(nxA-nxR)(nxB-nxR) for x = 1 to z, where A represents a 5′-sequence, B another 5′-sequence, R a random sequence, n the nonredundant number of binding sites for a certain transcription factor and z the total number of transcription factors compiled in the TRANSFAC library (Wingender et al., 2000). A search for conserved motifs within the 5′-upstream sequences was done using MEME (multiple expectation maximization for motif elicitation) Version 3.0 with analysis of both strands and a limiting range for width variability between 6 and 50 bp, expecting at least one occurrence of every motif in each sequence (Bailey and Gribskov, 1998). A set of the following 26 5′-upstream sequences was chosen, of which the gene products are abundant in milk: Bos taurus: خ±S1-CN, خ±S2-CN, خ²-CN, خ؛-CN, خ±-LA, خ²-LG, lactoferrin, PP3 component (lactophorin). Camelus dromedaries: خ±S1-CN, خ±S2-CN, خ²-CN, خ؛-CN, خ±-LA, lactoferrin, PP3 component (lactophorin), peptidoglycan recognition protein, whey acidic protein. Capra hircus: خ؛-CN. Equs caballus: خ²-LG. Homo sapiens: خ±S1-CN, خ±S2-CN, خ²-CN. Ovies aries خ²-LG. Sus scrofa: خ²-CN, fatty acid binding protein, lactoferrin. Where possible, the 2000 bp upstream to the transcriptional start site were examined, to be able also to detect more distantly located binding sites. Rodent sequences were not included, due to the more distant evolutionary relationship, and presumably modiï¬پed TF binding site preferences. In the following, each motif resulting from MEME analysis was searched for TF binding sites, mainly by TESS analysis (Schug and Overton, 1997).
Journal of Dairy Science Vol. 86, No. 2, 2003

502

KAPPELER ET AL.

Table 1. Average amount [mg 1−1] of some casein and whey proteins in mature milk from different species. nd = not detected. ↓ indicates a downregulation of gene expression between colostral and mid-lactational milk. ↑ indicates an upregulation in case of mastitis. Data from (Aguirre et al., 1998; Cals et al., 1994; Cuilliere et al., 1997; Hennighausen et al., 1994; Kappeler et al., 1999b; Ragona et al., 2000). Milk protein خ±S1-Casein خ±S2-Casein خ²-Casein خ؛-Casein خ±-Lactalbumin خ²-Lactoglobulin Whey acidic protein Lactophorin (PP3 component) Lactoferrin Lactoperoxidase Peptidoglycan recognition protein Lysozyme C nd nd Camel 5000 2200 15,000 800 3500 nd 157 950 95 ↓↑ 107 ↑ nd ∼100 ↓↑ Cow 12,000 3000 10,000 3500 1260 3500 nd 300 140 ↓↑ 30 nd 274 ↓ Human minute minute 4670 minute 3400 nd nd nd 565 ↓↑ 6↓ Rodents 1600 nd 4500 nd nd nd 1500 nd nd 465 nd nd Function Formation of casein micelle Formation of casein micelle Formation of casein micelle Formation and rennet coagulation of casein micelles Regulatory subunit of lactose synthetase Binding of fatty acids and retinol Probably an epithelial growth regulator, similar to WDNM1 Lipolysis inhibition Anti-inflammational, nutritive, iron uptake, regulative Anti-inflammational, bacteriolytic activity Anti-inflammational Bacteriolytic activity, N-acetylmuramidase

RESULTS The protein composition of camel and cow milk differed in regard to both casein and whey proteins. Camel milk contained signiï¬پcantly higher concentrations of خ²-CN and lower amounts of خ؛-CN (Table 1). خ²-LG, lysozyme C and lactoperoxidase were not detected in mid- to late-lactational camel milk, and the corresponding cDNA of the former two proteins was not found by screening of a lactating mammary gland cDNA library. On the other hand, whey acidic protein, generally described as a major constituent of rodent milk, and peptidoglycan recognition protein, an intracellular protein binding to gram-positive bacteria, and presently not known to be a milk constituent, were detected in major amounts in camel whey, both on the cDNA and protein level. Lactophorin, the proteose peptone 3 component of whey, was detected in higher concentrations than in bovine milk (Kappeler et al., 1999b). A comparative analysis was started for elements regulating genes, which express camel milk proteins. Fewer than or equal to 2 kb of the regions upstream to the transcriptional start sites of 10 camel genes, for which we found the corresponding mRNA in the lactating udder, were sequenced, and compared to homologous regions from other species. Percent identity to homologous bovine sequences were between 56 and 74%, to rodent sequences between 45 and 61%, and to human sequences between 50 and 76%. The overall GC-content of the camel sequences analyzed was about 44%, similar to homologous sequences from other species. Multiple alignment between the 5′-flanking regions of milk-protein genes from several species revealed
Journal of Dairy Science Vol. 86, No. 2, 2003

two groups of distantly related sequences (Figure 1). The nucleic acid composition of the two groups, thereafter designated as group I and group II, differed markedly, with group I having an average GC content of 54%, and group II having an average GC content of 38%. Interspersed elements and long terminal repeats covered about 16.5% of group I sequences, and 20.5% of group II sequences. Correlation analysis of transcription-factor binding site proï¬پles of the different 5′-upstream sequences produced a relational matrix with similar relationships between binding site preferences as the relationships found with multiple sequence alignment (Figure 2). The most abundant potential binding sites found in the 5′-flanking regions examined were those for the glucocorticoid receptor, transcription factors of the ets proto-oncogene family, especially MAF (mammary activating factor, predominantly in group I sequences) and PEA3, furthermore those for NF-1 (nuclear factor 1), YY1 (yin yang 1), and the progesterone receptor (Table 2). Sites for TBP/TFIID, proteins of the octamer binding family, among them Pit-1a (a pituitary gland tissue-speciï¬پc activator), GATA-binding proteins, F2F (repressor of the prolactin promoter), C/EBP خ² (CCAAT enhancer binding protein خ²) and, to a minor extent, C/EBP خ´ were predominantly found in group II promoter sequences, whereas binding sites for the ubiquitous activators Sp1, AP-2 and PU.1, as well as MAF and PPAR (peroxisome proliferator-activated receptor) were mostly found in the GC-rich promoter sequences of group I. MEME analysis of the examined promoter regions revealed six motifs, which occurred in all promoter sequences at least once (Figure 3). A positional over-

ANALYSIS OF CAMEL MILK-PROTEIN GENES

503

Figure 2. Simpliï¬پed correlation matrix of 5′-flanking regions ≤2.0 kbp adjacent to the transcriptional start site of milk-protein genes. The average number of binding sites between the examined homologous promoter regions from different species was used for the calculation of the correlation strength between non-homologous promoter regions, as described in Materials and Methods. Strong correlation (Sum of products > 200) is indicated as “++â€‌, values > 100 with “+â€‌, negative values with “−â€‌.

view of these motifs on the sequences is presented in Figure 4. They were in the following compared to the consensus sequences of known TF binding sites, especially to those reported to be involved in the regulation of milk protein genes. The ï¬پrst and the ï¬پfth motif contained elements exclusively reported for the regulation of milk protein genes. The mammary activating factor was reported as a regulative factor in the mouse mammary tumor virus long terminal repeat (Reuss and Cofï¬پn, 2000), and the milk protein binding factor was described in the regulation of the sheep خ²-LG gene (Watson et al., 1991). The second motif found by MEME analysis corresponded to the core of a composite response element, which was described to be a main regulator of milk protein gene activity (Rosen et al., 1999). The motif contained the STAT5 consensus sequence and putative binding sites for C/EBP خ² and the yin yang 1 repressor, as well as a glucocorticoid receptor half site. The third motif corresponded to an RNA polymerase II promoter sequence. This motif was not clearly noticeable in front of the transcriptional start sites of group I sequences (Figure 1). The fourth motif contained a binding site for the estrogen receptor

and the sixth motif was detected in repeated arrangements in group I sequences. The detection of glucocorticoid receptor half sites (Lechner et al., 1997) was difï¬پcult by the method chosen, probably due to the variability is glucocorticoid response elements. NF-1 sites were not found within the detected motifs, most likely because of differences in the local arrangements of this ubiquitous factor in composite response elements of group 1 and group II 5′-upstream sequences. DISCUSSION The protein composition of camel and cow milk differs in some fundamental aspects. We assume that the immunological challenges, which face these two ruminating species in their respective natural environment, has led to adaptations in the milk composition. We were not able to isolate خ²-LG, the primary constituent of bovine whey, from camel milk, nor lysozyme C, and did not detect the respective sequences in a lactating mammary gland cDNA library by plaque screening or the PCR method. We also were not able to isolate lactoperoxidase from midlactational camel
Journal of Dairy Science Vol. 86, No. 2, 2003

504

KAPPELER ET AL.

Figure 3. Conserved motifs, i.e., multilevel consensus sequences, detected by MEME analysis of 26 5′-flanking regions to genes abundantly expressed in the lactating mammary gland and sorted according to the output ï¬پle. Transcription factor binding sites were attributed to the motifs, which have previously been reported to be involved in the regulation of gene expression in the course of lactation.

milk, although this enzyme can be isolated from bovine milk throughout lactation. Nevertheless, we were able to isolate and sequence a corresponding clone from a cDNA library produced from udder tissue four
Table 2. Most prominent TF sites with possible involvement in the regulation of milk protein genes after subtraction of background and sorting out of redundancies. The numbers represent the percentage of a particular site, as compared to the total number of sites detected. Factor Group I Group II All sequences

ETS (MAF; PEA3) MAF GR GR half sites TFIID TBP C/EBPbeta C/EBPdelta NF-1 YY1 PU.1 AP-2 Oct Sp1 PPAR Pit-1a PR F2F GATA C/EBPalpha MPBF NF-kappaB Pit-1 COUP STAT5

5.49 1.00 6.89 0.27 0.94 1.38 1.39 0.37 2.38 0.91 1.97 12.44 0.00 12.54 2.07 0.00 0.88 0.26 0.00 0.30 0.61 0.61 0.00 0.26 0.02

(% of sites detected) 5.55 5.97 0.11 0.43 5.47 6.40 1.66 1.31 7.87 6.10 6.41 5.18 5.15 4.26 3.72 2.86 2.04 2.32 2.58 2.21 0.85 1.31 0.10 4.39 8.24 5.68 0.00 1.83 0.30 0.94 5.55 3.99 1.84 1.65 3.24 2.46 2.88 1.33 1.49 1.19 0.00 0.21 0.00 0.20 0.48 0.30 0.07 0.14 0.06 0.03

weeks after parturition. This enzyme is probably rapidly downregulated in the camel mammary gland, as it was also observed in human tissue. On the other hand, high concentrations of lactophorin (the PP3 component), whey acidic protein and the peptidoglycan recognition protein were found in camel whey. Furthermore, we found a considerably higher ratio of خ²-CN to خ؛-CN in camel milk from different breeds that reported for cow milk (Table 1). In the course of this study, we sequenced the proximal (≤2 kbp) regions 5′-upstream to the transcriptional start of 10 camel milk protein genes, to ï¬پnd a rationale to the different expression pattern of milk protein genes in the lactating mammary gland of camels and cows. We supposed these regions to be sufï¬پcient to direct the stage- and tissue-speciï¬پc expression of the respective genes, as it was shown for corresponding sequences in other mammals (Faerman et al., 1995; Lee et al., 1998). Alignment of the 50 5′-upstream sequences analyzed (Figure 1) showed in a ï¬پrst step, that all 5′flanking regions of camel milk-protein genes were most closely related to their homologous counterparts from other species, independently from the gene expression level in the mammary gland of the other species (Figure 1). Interestingly, the camel 5′-upstream sequences also showed the observed relatedness in those cases, where the respective genes were reported to be expressed to a very different level in the lactating mammary glands of the different species, e.g., in the case of camel and bovine خ؛-CN (Kappeler et al., 1998). This sequence relatedness gave indication that the

Journal of Dairy Science Vol. 86, No. 2, 2003

ANALYSIS OF CAMEL MILK-PROTEIN GENES

505

Figure 4. A summary of the motifs from Figure 3 showing an optimized (nonoverlapping) tiling of all of the motif occurrences with a positional P-value (the probability of a single random subsequence of the length of the motif scoring at least as well as the observed match) ≤0.0001 for each of the sequences in the training set. The product of motif probabilities on a sequence is given as “Expectâ€‌ value. Motif occurrences are indicated by numbers, motif lengths on a sequence as empty spaces, and the motif direction with plus or minus.

regulation of gene expression has to be restricted to small conserved areas within the 5′-upstream sequences, and that mutations in these areas will initiate, abolish, or modulate the tissue- and stage-speciï¬پc expression of milk protein genes. Additionally, two very distantly related groups of 5′flanking regions were discerned, designated as group I and group II (Figure 1). Group I sequences enclosed some of the whey and the milk-fat globule membrane protein gene sequences, group II sequences encompassed casein and some whey and butyrophilin gene sequences. The higher GC-content of group I sequences, and the scarcity in combined CAAT and TATAA boxes towards the transcriptional start sites let us assume that group I 5′-flanking regions predominantly were regulating housekeeping genes, which became highly expressed in the lactating mammary gland of some species, whereas group II genes were speciï¬پcally expressed in the end-differentiated cells of the lobuloalveolar epithelium. Our interest at this point was to see if binding site probabilities for the different transcription factors were similar for the 50 sequences examined. A position

independent pattern search strategy was developed, as described in Materials and Methods, because similar regulative elements, especially with regard to hormone responsive elements, have been localized on different positions relative to the transcriptional start site (Rosen et al., 1998). A correlation matrix was generated out of the background corrected binding site probabilities. A simpliï¬پed view of this matrix is shown in Figure 2 combining the results of homologous sequences. The resulting matrix showed the same division into two weakly related groups as the multiple sequence alignment before, with the exception of binding site probabilities for wap 5′-flanking sequences, which also exhibited good TF-site correlation results with some group II sequences. Figure 2 shows, nonetheless, that the majority of the different 5′-flanking regions weakly correlate with each other, indicating that most sequences contain some common regulative elements. Some TF-sites were detected on nearly all sequences with a background-corrected frequency of more than one per 1000 bp. Most abundantly, binding sites for the glucocorticoid receptor and for transcription factors of the Ets
Journal of Dairy Science Vol. 86, No. 2, 2003

506

KAPPELER ET AL.

family were found on sequences of both groups (Table 2). Ets factors, especially MAF and PEA3, were localized on regulative sequences of milk-protein genes and probably mediate signals of the epidermal growth factor and of insulin to the prolactin gene (Jacob et al., 1999). Glucocorticoid receptor sites in the ï¬پrst group were often mere half sites, as described to exist particularly on milk-protein gene promoter regions (Lechner et al., 1997), whereas sites in the second group preferably consisted of near-palindromic sequences. Binding sites for the peroxisome proliferator activated receptor (PPAR) were detected on most group I sequences. This fatty-acid activated nuclear receptor controls genes involved in the lipid metabolism and is enhanced by prolactin during adipogenic conversion of cells (Nanbu et al., 2000). The خ³2-isoform was reported to be downregulated (Gimble et al., 1998) or upregulated (Jain et al., 1998) during pregnancy and lactation, and a role in response to physiologic and pathologic stimuli, which alter lipid metabolism, was suggested. This indicates that the factor may help to regulate group I gene expression in differentiating and involuting mammary tissue. The ubiquitous transcription factors of the octamer binding family were reported to bind to elements in the proximal 5′ sequence of casein genes (Groenen et al., 1992) and in the long terminal repeat of the mouse mammary tumor virus (Brueggemeier et al., 1991), enhancing the activity of the mediators of hormonal and local signals. Octamer binding sites were abundantly detected in all group II sequences, but only weakly in group I sequences. There are a number of observations on the histological level that genes belonging to either of the two groups show strikingly different stage- and cell-speciï¬پc expression patterns. A mutually exclusive histological localization of lactoferrin mRNA on one hand, خ±-LA and خ±S1-CN mRNA on the other hand was reported in the alveolar epithelium (Molenaar et al., 1992), depending on the lactational status of the cells. Whereas خ±-LA and خ±S1-CN mRNA have been detected in terminally differentiated, lactating alveoli, lactoferrin mRNA was almost exclusively found in emerging and regressing alveoli. Lactoferrin expression also did not require basement membrane signals and its expression was even repressed in epithelial cells with basal-apical orientation, but stimulated in nonpolarized cells (Close et al., 1997). This ï¬پnding was in contrast to the expression of genes that belong to typical milk proteins, such as خ±-LA, caseins and also the whey acidic protein, although we found the 5′-flanking sequences of wap genes to belong to group I (Figure 1). Fatty acid binding protein, also known as mammaryderived growth inhibitor, was localized in the vacuolar, nonlactating cell type (Erdmann and Breter,
Journal of Dairy Science Vol. 86, No. 2, 2003

1993), whereas butyrophilin mRNA was detected in lactating cells (Molenaar et al., 1995). Most genes of the second group are solely expressed in alveolar epithelial cells of the late-pregnant and lactating mammary gland, whereas the ï¬پrst group, with the exception of the whey acidic protein, was made up of genes, which are known to be expressed in a broader range of tissues. Fatty acid binding protein, for example, participates in the intracellular transport of fatty acids in different tissues, and peptidoglycan recognition protein is a protein of the innate immune system of vertebrates and invertebrates. Additionally, many proteins of the ï¬پrst group are likely involved in feedback regulation of protein and fat secretion into the alveoli and thus need to be expressed in a manner different to caseins and related proteins, to execute these functions. Altogether, a majority of the proteins, which help to preserve the structural properties of milk, seem to be expressed in terminally differentiated cells under control of a TATA-like promoter. Proteins, which protect against environmental stress, on the other hand, rather seem to be constitutively expressed and merely upregulated in the lactating mammary gland. Established models describing the regulation of milk-protein gene transcriptional control are mainly based on studies with genes for rodent whey acidic protein and rodent and bovine خ²-CN. Additionally, the long terminal repeat of the murine mammary tumor virus, which directs expression to lactating mammary epithelial cells, is well investigated. Information about the regulation of other genes expressing milk proteins is rare. DNase I hypersensitivity and footprinting experiments, as well as electrophoresis mobility shift assays and transgenic studies, helped to identify binding sites for transcription factors and hormone responsive elements (Rosen et al., 1999). A crucial role in the regulation of milk genes plays a pathway, by which the prolactin signal is effected via phosphorylation of the membrane-bound prolactin-receptor, inducing tyrosine phosphorylation of STAT5 A and B isoforms by Janus kinase, dimerization, nuclear translocation, and DNA binding (Rosen et al., 1999). Short forms of the prolactin-receptor and of STAT5 have been reported, which were found in nonlactating mammary tissue and act as dominant negative regulators, repressing expression of target genes (Edwards et al., 1998). STAT5 binding sites were shown to reside within a composite response element, which also included sites for NF-1 and GR in the rodent whey acidic 5′-flanking region, and for GR, C/EBP خ² and خ´ isoforms, and YY1 in the bovine خ²-CN 5′-flanking region (Doppler et al., 1995). The core region of this element was

ANALYSIS OF CAMEL MILK-PROTEIN GENES

507

retrieved as the second motif from a MEME analysis of 26 regions 5′-upstream to genes highly expressed in the lactating mammary gland of different species (Figure 3). The motif was lost when sequences were included in the MEME training set, which are not highly expressed in the lactating mammary gland, supporting the idea of a crucial role for this response element in the control of milk protein gene expression. We conclude by comparing our results from TF-site analysis and from MEME detection of conserved elements, that genes lacking the composite response element, which combines the STAT5 binding site with TF-sites for other activating and repressing factors, will not be expressed in the lactating mammary gland of a particular species. However we did not succeed to associate the variances in milk protein levels between species to a structural disparity in the corresponding 5′-flanking regions. We presume that the ï¬پne-tuning of milk protein gene expression will probably reside in the arrangement of binding sites for ubiquitous factors, such as NF-1 or the Octamer family, nearby the conserved elements. Additionally, superior regulative regions and mRNA stability are likely involved in the control of milk protein gene expression. ACKNOWLEDGMENTS The authors thank the laboratory group of the central veterinary laboratory of Dubai for providing genomic camel DNA. REFERENCES
Aggeler, J., J. Ward, L. M. Blackie, M. H. Barcellos-Hoff, C. H. Steuli, and M. J. Bissell. 1991. Cytodifferentiation of mouse mammary epithelial cells cultured on a reconstituted basement membrane reveals striking similarities to development in vivo. J. Cell Sci. 99:407–418. Aguirre, A., P. N. Castro, F. J. De La, and F. O. Castro. 1998. Expression of human erythropoietin transgenes and of the endogenous WAP gene in the mammary gland of transgenic rabbits during gestation and lactation. Transgenic Res. 7:311–317. Bailey, T. L., and M. Gribskov. 1998. Methods and statistics for combining motif match scores. J. Comput. Biol. 5:211–221. Brown, C. F., C. T. Teng, B. T. Pentecost, and R. P. DiAugustine. 1989. Epidermal growth factor precursor in mouse lactating mammary gland alveolar cells. Mol. Endocrinol. 3:1077–1083. Brueggemeier, U., M. Kalff, S. Franke, C. Scheidereit, and M. Beato. 1991. Ubiquitous transcription factor OTF-1 mediates induction of the MMTV promoter through synergistic interaction with hormone receptors. Cell 64:565–572. Cals, M. M., M. Guillomot, and P. Martin. 1994. The gene encoding lactoperoxidase is expressed in epithelial cells of the goat lactating mammary gland. Cell. Mol. Biol. 40:1143–1150. Close, M. J., A. R. Howlett, C. D. Roskelley, P. Y. Desprez, N. Bailey, B. Rowning, C. T. Teng, M. R. Stampfer, and P. Yaswen. 1997. Lactoferrin expression in mammary epithelial cells is mediated by changes in cell shape and actin cytoskeleton. J. Cell Sci. 110:2861–2871. Cuilliere, M. L., M. Abbadi, C. Mole, P. Montagne, M. C. Bene, and G. Faure. 1997. Microparticle-enhanced nephelometric immu-

noassay of alpha-lactalbumin in human milk. J. Immunoassay 18:97–109. Doppler, W., T. Welte, and S. Philipp. 1995. CCAAT/enhancer-binding protein isoforms beta and delta are expressed in mammary epithelial cells and bind to multiple sites in the beta-casein gene promoter. J. Biol. Chem. 270:17962–17969. Edwards, G. M., F. H. Wilford, X. Liu, L. Henninghausen, J. Dijiane, and C. H. Streuli. 1998. Regulation of mammary differentiation by extracellular matrix involves protein-tyrosine phosphatases. J. Biol. Chem. 273:9495–9500. Erdmann, B., and H. Breter. 1993. Irregular distribution of mammary-derived growth inhibitor in the bovine mammary epithelium. Cell. Tissue Res. 272:383–389. Faerman, A., I. Barash, R. Puzis, M. Nathan, D. R. Hurwitz, and M. Shani. 1995. Dramatic heterogeneity of transgene expression in the mammary gland of lactating mice: A model system to study the synthetic activity of mammary epithelial cells. J. Histochem. Cytochem. 43:461–470. Gallego, M. I., N. Binart, G. W. Robinson, R. Okagaki, K. T. Coschigano, J. Perry, J. J. Kopchick, T. Oka, P. A. Kelly, and L. Hennighausen. 2000. Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects. Dev. Biol. 229:163–175. Gimble, J. M., G. M. Pighetti, M. R. Lerner, X. Wu, S. A. Lightfoot, D. J. Brackett, K. Darcy, and A. B. Hollingsworth. 1998. Expression of peroxisome proliferator activated receptor mRNA in normal and tumorigenic rodent mammary glands. Biochem. Biophys. Res. Commun. 253:813–817. Groenen, M. A., R. J. Dijkhof, J. J. van der Poel, R. van Diggelen, and E. Verstege. 1992. Multiple octamer binding sites in the promoter region of the bovine alpha s2-casein gene. Nucleic Acids Res. 20:4311–4318. Hennighausen, L., R. McKnight, T. Burdon, M. Baik, R. J. Wall, and G. H. Smith. 1994. Whey acidic protein extrinsically expressed from the mouse mammary tumor virus long terminal repeat results in hyperplasia of the coagulation gland epithelium and impaired mammary development. Cell Growth Differ. 5:607–613. Jacob, K. K., E. Wininger, K. DiMinni, and F. M. Stanley. 1999. The EGF response element in the prolactin promoter. Mol. Cell. Endocrinol. 152:137–145. Jain, S., S. Pulikuri, Y. Zhu, C. Qi, Y. S. Kanwar, A. Yeldandi, V. M. S. Rao, and J. K. Reddy. 1998. Differential expression of the peroxisome proliferator-activated receptor gamma (PPARgamma) and its coactivators steroid receptor coactivator-1 and PPAR-binding protein RBP in the brown fat, urinary bladder, colon, and breast of the mouse. Am. J. Pathol. 153:349–354. Kappeler, S., M. Ackermann, Z. Farah, and Z. Puhan. 1999a. Sequence analysis of camel (Camelus dromedarius) lactoferrin. Int. Dairy J. 9:481–486. Kappeler, S., Z. Farah, and Z. Puhan. 1999b. Alternative splicing of lactophorin mRNA from lactating mammary gland of the camel (Camelus dromedarius). J. Dairy Sci. 82:2084–2093. Kappeler, S., Z. Farah, and Z. Puhan. 1998. Sequence analysis of Camelus dromedarius milk caseins. J. Dairy Res. 65:209–222. Lechner, J., T. Welte, J. K. Tomasi, P. Bruno, C. Cairns, J. A. Gustafsson, and W. Doppler. 1997. Promoter-dependent synergy between glucocorticoid receptor and Stat5 in the activation of beta-casein gene transcription. J. Biol. Chem. 272:20954–20960. Lee, C. S., and T. Oka. 1992. Progesterone regulation of a pregnancyspeciï¬پc transcription repressor to beta-casein gene promoter in mouse mammary gland. Endocrinology 131:2257–2262. Lee, W. K., S. J. Kim, S. Hong, T. H. Lee, Y. M. Han, O. J. Yoo, K. S. Im, and K. K. Lee. 1998. Expression of a bovine beta-casein/ human lysozyme fusion gene in the mammary gland of transgenic mice. J. Biochem. Mol. Biol. 31:413–417. McClenaghan, M., A. Springbett, R. M. Wallace, C. J. Wilde, and A. J. Clark. 1995. Secretory proteins compete for production in the mammary gland of transgenic mice. Biochem. J. 310:637– 641. Journal of Dairy Science Vol. 86, No. 2, 2003

508

KAPPELER ET AL. mammary gland cells share functional elements. J. Virol. 74:8183–8187. Rosen, J. M., C. Zahnow, A. Kazansky, and B. Raught. 1998. Composite response elements mediate hormonal and developmental regulation of milk protein gene expression. Biochem. Soc. Symp. 63:101–113. Rosen, J. M., S. L. Wyszomierski, and D. Hadsell. 1999. Regulation of milk protein gene expression. Annu. Rev. Nutr. 19:407–436. Schug, J., and G. C. Overton. 1997. Subject: TESS (Transcription Element Search Software on the WWW). http://www.cbil. upenn.edu/tess. Accessed Dec. 28, 2001. Smit, A. F. A., and P. Green. 2000. Subject: RepeatMasker. http:// ftp.genome.washington.edu/RM/RepeatMasker.html. Accessed Dec. 28, 2001. Teng, C. T. 1999. Regulation of lactoferrin gene expression by estrogen and epidermal growth factor: molecular mechanism. Cell Biochem. Biophys. 31:49–64. Watson, C. J., K. E. Gordon, M. Robertson, and A. J. Clark. 1991. Interaction of DNA-binding proteins with a milk protein gene promoter in vitro: identiï¬پcation of a mammary gland-speciï¬پc factor. Nucleic Acids Res. 19:6603–6610. Wingender, E., X. Chen, R. Hehl, H. Karas, I. Liebich, V. Matys, T. Meinhardt, M. Pruess, I. Reuter, and F. Schacherer. 2000. TRANSFAC: An integrated system for gene expression regulation. Nucleic Acids Res. 28:316–319.

Molenaar, A. J., S. R. Davis, L.-J. W. Jack, and R. J. Wilkins. 1995. Expression of the butyrophilin gene, a milk fat globule membrane protein, is associated with the expression of the alpha-S1 casein gene. Histochem. J. 27:388–394. Molenaar, A. J., S. R. Davis, and R. J. Wilkins. 1992. Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue. J. Histochem. Cytochem. 40:611–618. Nagaiah, K., F. F. Bolander, Jr., K. R. Nicholas. T. Takemoto, and Y. J. Topper. 1981. Prolactin-induced accumulation of casein messenger RNA in mouse mammary explants: A selective role of glucocorticoid. Biochem. Biophys. Res. Commun. 98:380–387. Nanbu, W. R., Y. Fujitani, Y. Mashuho, M. Muramatu, and H. Wakao. 2000. Prolactin enhances CCAAT enhancer-binding protein-beta (C/EBP beta) and peroxisome proliferator-activated receptor gamma (PPAR gamma) messenger RNA expression and stimulates adipogenic conversion of NIH-3T3 cells. Mol. Endocrinol. 14:307–316. Ragona, L., F. Fogolari, L. Zetta, D. M. Perez, P. Puyol, K. De Kruif, F. Loehr, H. Rueterjans, and H. Molinari. 2000. Bovine betalactoglobulin: Interaction studies with palmitic acid. Prot. Sci. 9:1347–1356. Reuss, F. U., and J. M. Cofï¬پn. 2000. The mouse mammary tumor virus transcription enhancers for hematopoietic progenitor and

Journal of Dairy Science Vol. 86, No. 2, 2003

pdf

Farah, Camel milk casein

669

Separation and characterization of major components of camel milk casein
By Z. FARAH and M. FARAH-RIESEN Labor fأ¼r Milchwissenschaft, Eidg. Technische Hochschule Zأ¼rich, Switzerland 1. Introduction
Development and research activities on domestic animals are mostly concentrated on species and breeds of animals available in the temperate zones of Europe and North America. This results in a relative neglect of several species of animals native to the tropics and subtropics. The camel (Camelus dromedarius) is certainly one of the most neglected species of the domestic animals. The majority of the studies conducted on camels concentrate mainly on its anatomical features and physiological adaptations to desert conditions. Information about camels as milk animals is very limited. Camel milk is an important component of the human diet in many parts of the world. People unfamiliar with camels are surprised to learn that a normal camel on good feed can produce 2'000 litres of milk per lactation period (1), even higher milk yields have been recorded (2). Available information concerning camel milk is mainly related to the general composition of the mil k without detailed characterization of the individual compounds. This paper presents the results of the characterization of camel milk and the separation of its main casein compounds by polyacrylamid gel electrophoresis and the urea fractionation method.

chloric acid according to REISENER (6) and destained with 7.5 % (v/v) acetic acid. The sodium dodecyl sulphate polyacrylamid gel (SOSPAGE) system was essentially that was used by LAEMMLI (7). The resolving gel contained 12.5 % acrylamid. By the latter method the molecular weights of the casein fractions were estimated by comparing their mobility in SOS-gel with those of marker proteins with known molecular weights. 3. Results and discussion 3.1 Electrophoretic patterns of camel milk Samples of milk from six individual camels as well as their pooled milk were examined to determine whether they have similar composition. As can be seen in Fig. 1 no difference can be observed and the electrophoretic patterns show the same main bands of equal intensity and mobility for both pooled and individual milks. Compared with cow milk, camel milk has considerably lower electrophoretic mobility. The electrophoretic pattern shows three sharp and distinguishable main bands in camel milk. As firmly established data on structure of camel milk proteins are not available, we avoid to use the commonly accepted system of milk protein nomenclature at this stage of the investigation. The camel mil k protein bands are therefore given the interim designation B, A, C according to their increasing electophoretic mobility. In comparison with cow milk, the first 2 bands (B and A) can be regarded as a possible homologue to bovine-(j and a-caseins. The last band (C) seems to be whey protein as it does not appear in the whole casein fraction (Fig. 2).

2. Materials and methods 2.1 Milk samples Camel milk samples were taken at Ndare Ngare Camel Farm which is situated just North of the equator in Kenya's Laikiba district and at an altitude of between 1'730 and 1'890 m above sea level. The animals of indigenous breed (Camelus dromedarius) were fed all year around exclusively by grazing. The milk samples comprised herd bulk milk and milks from individual camels. The milk was used both fresh and lyophilized for the analysis.

2.2 Preparation of casein and its fractions The whole casein was precipitated from skimmed fresh milk with 1 N HCI at pH 4.5-4.6 and 30uC. The precipitate was washed twice with water (pH maintained at 4.5-4.6), dissolved at pH 7.0-7.2 with 1 N NaOH re-precipitated and washed. The casein was then lyophilized and stored at - 20آ°C. Following the procedure of ASCHAFFENBURG (3), the total casein obtained by acid precipitation was dispersed in 3.3 M urea and the pH reduced to 4.6. Under these conditions (j-casein remains in solution whilst all other casein components are precipitated. (j-casein was isolated from the solution as described by ASCHAFFENBURG (3) and acasein was obtained from the precipitate by urea fractionation according to HIPPEL et al. (4).

2.3 Electrophoresis Polyacrylamid gel electrophoresis (PAGE) was performed in a vertical slab gel apparatus of Desaga (Heidelberg). The acrylamid gel (7.5 % in 5 M urea), the buffer solution and samples were prepared according to the procedure of I. SM ITH (5). The electrophoresis was run at 20 milliampere for 20 minutes and then 100 milliampere until the marker dye (bromphenol blue) was 0.5 cm from the anodic end of the slab (about 3 hours). Cooling water of 12آ°C was circulated in the electrophoretic chamber. The slabs were stained for 1 hour with Coomassie brilliant blue G 250 in 3.5 % per-

Fig. 1: Polyacrylamid gel patterns of camel milk and cow milk: 1, 2, pooled camel milk; 3, 4, 5, 6, 7, 8, milk from six individual camels; 9, cow milk.

No protein bands homologous to bovine K-casein could clearly be detected in the electrophoretic pattern. Attempts to improve the separation by incorporating mercaptoethanol

Milchwissenschaft 40 (11) 1985

670

Farah, Camel milk casein

Fig. 2: Polyacrylamid gel patterns of camel milk and camel milk casein fractions: 1, fraction A; 2, fraction B; 3, whole camel casein; 4, camel milk.

Fig. 3: 50S-PAGE patterns of: 1, fraction B; 2, camel milk casein; 3, camel milk; 5, standard marker proteins from top to bottom: bovine albumin, egg albumin, glyceraldehyde phosphate dehydrogenase, carbonic anhydrase, trypsinogen, trypsin inhibitor, خ±-lactalbumin.

in the electrophoretic procedure, were unsuccessful. On the other hand, addition of rennet to camel milk caused a clotting reaction with strong coagulum. This suggests the possible occurrence of خ؛casein or a homologous protein in camel milk and makes further studies necessary. 3.2 Fractionation of casein by means of aqueous urea solutions In order to separate the main camel casein compounds, the method of the differential solubility in urea of the casein compounds normally applied to cow milk, was utilized. According to HIPP et al. (4), fractionation of the casein is achieved by stepwise dilution of a solution of all the casein components in strong urea. Our first attempt to separate the whole camel milk casein following this method gave no clear separations and the fractions obtained were no homogeneous when examined by electrophoresis. Nevertheless, a combination of this procedure with that of ASCHAFFENBURG (3) gave a fairly good separation as shown in Fig. 2. In this figure camel milk, whole camel casein, fraction B and fraction A are compared. Fraction B, which was isolated by the ASCHAFFENBU RG (3) t1-casein preparation method, is free from impurities and shows no traces of fraction A. Fraction A obtained as a-casein by the urea method of HIPP et al. (4), shows some slow moving material. This material could not be removed with the employed fractionation method.

Fig. 4. Calibration curve obtained when the logarithm of the marker protein molecular weights were plotted against their relative mobilities in 50S-PAGE of 12.5 % acrylamid concentration. The molecular weights of the fraction A and B are interpolated from their relative mobilities. Marker proteins: 1, bovine albumin (mol. wt. 66'000); 2, egg albumin (mol. wt. 45'000); 3, glyceraldehyde phosphate dehydrogenase (mol. wt. 36'000); 4, carbonic anhydrase (mol. wt. 29'000); 5, trypsinogen (mol. wt. 24'000); 6. trypsin inhibitor (mol. wt. 20'000); 7, خ±-lactalbumin (mol. wt. 14'000).

3.3 SDS-Electrophoresis 50S dissociates proteins into their constituant polypeptide chains and has been used for the separation of the proteins according to their molecular weights (8). 50S-PAGE patterns of camel milk and camel milk caseins are presented in Fig. 3. As can be seen, the 50S electrophoresis exhibits the same main protein bands in camel milk and camel milk casein as in Fig. 2. The marker proteins with molecular weights between 14'800 and 66'000 were excellently separated in the selected acrylamid gel concentration (12, 5 %). When the electrophoretic mobilities were plotted against the logarithm of the marker protein molecular weights, a linear relation was obtained (Fig. 4). The molecular weights of the two camel casein fractions B and A estimated from this calibration curve, are 32'000 and

35'500 respectively. This is considerably higher than the possible homologous bovine caseins which are normally reported in the literature as 24'000 for t1-casein and 22'000 to 27'000 for a-casein (9). These first results on camel milk and camel milk casein fractions do not allow firm conclusions. I t is however interesting to note that camel milk exhibits less protein bands in electrophoresis. The bands seem to present relatively homogeneous components. Acknowledgement This work was supported in part by the Swiss Technical Coآ· operation and Humanitarian Aid. The authors express their thanks to Dr. W. Schulthess, Dept. of Food Science and Technology, University of Nairobi" Kenya,for providing the camel milk.
Milchwissenschaft 40 (11) 1985

Farah, Camel milk casein

671 cow milk the overall electrophoretic pattern of camel milk exhibits less protein bands which seem to present more homogeneous proteins. FARAH, Z., FARAH-RIESEN, M.: Trennung und Charakterisierung der Casein-Hauptbestandteile in Kamelmilch. Milchwissenschaft 40 (11) 669-671 (1985). 12 Kamelmilch (Casein) Kamelmilchen (Camelus dromedarius) wurden mit Hilfe der Harnstoff-Fraktionsmethode und der Polyacrylamidgel-Elektrophorese auf ihre Caseinzusammensetzung untersucht. Das elektrophoretische Bild von sechs einzelnen Kamelmilchproben sowie der Mischungen zeigte drei Hauptbanden gleicher Intensitأ¤t. Die Wanderungsgeschwindigkeiten der Proteinbanden sind langsamer als bei Kuhmilch. Die Proteinbanden wurden vorlأ¤ufig mit B, A und C bezeichnet und kأ¶nnen in dieser Reihenfolge als Homologe von (1
4. References
(1) KNOESS, K.H. in: Camels, International Foundation for Science (IFS) Symposium, Sudan, 201-214 (1979) (2) KNOESS, K.H.: World Anim. Rev. 22 3-8 (1977) (3) ASCHAFFENBURG, R.: J. Dairy Res. 30259-260 (1963) (4) HIPP, N.J. et al.: J. Dairy Sci. 35 272-281 (1952) (5) SMITH, I.: Chromatographic and electrophoretic techniques, Vol. 2, 2nd ed., Medical Books, London, 399-418 (1968) (6) REISNER, A.H. et al.: Anal. Biochem. 64 509 (1975) (7) LAEMMLI, U.K.: Nature 227680-685 (1970) (8) HAMES, B.D.: Gel electrophoresis of proteins, I.R.L. Press, London, 14-18 (1981) (9) McKENZIE, H.A.: Milk proteins, Vol. 2, Academic Press, New York, 151-163 (1971)

6. Summary
FARAH, Z., FARAH-RIESEN, M.: Separation and characterization of the major components of camel milk casein. Milchwissenschaft 40 (11) 669-671 (1985). 12 Camel milk (casein) Milks from camels (Camelus dromedarius) were examined for their casein composition by the urea fractionation method and polyacrylamid gel electrophoresis. The electrophoretic patterns of milk from 6 individual camels as well as their pooled milk showed three main protein bands of equal intensity. Compared with cow milk all the bands of camel milk have considerably lower electrophoretic mobilities. The bands were given the interim designation B, A and C and can be regarded as possibly homologous to bovine l3-casein, (I<-casein and whey protein, respectively. The molecular weights of the two separated casein fractions B and A obtained by means of sodium dodecyl sulfate polyacrylamid gel electrophoresis (50S-PAGE) are 32'000 and 35'500, respectively. Compared with

pdf

Journal of Dairy Research (2001) 68 1–7 Printed in the United Kingdom

1

Studies on the supply of immunoglobulin G to newborn camel calves (Camelus dromedarius)
Bï‌¹ ROLF KAMBER", ZAKARIA FARAH#*, PETER RUSCH" ï‌،ï‌®ï‌¤ MICHAEL HASSIG" " Clinic for Obstetrics, Veterinary Faculty, University of Zurich, CH-8057 Zurich, Switzerland # Laboratory of Dairy Science, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zurich, Switzerland (Received 6 December 1999 and accepted for publication 9 October 2000)

Sï‌µï‌­ï‌­ï‌،ï‌²ï‌¹. A major problem in camel productivity is the high mortality rate of camel calves in the ï¬پrst 3 months. The causes for mortality are mainly poor management practice and infectious diseases. The purpose of this research, carried out on a ranch in Kenya, was to determine the immunoglobulin G (IgG) concentration in camel colostrum as well as the extent of the calves’ passive immunization by maternal antibodies. IgG concentration in colostrum and in the serum of the calf were measured during the ï¬پrst 3 d of life. Evaluation was carried out by comparing the respective values with those for horses and cattle. The average IgG concentration in the camel colostrum was higher than that found in literature for horses and cattle. IgG concentration in the serum of the camel calves reached its maximum 24 h after birth. In 39 % of the examined calves, this maximum concentration was below 4 g\l, which is considered to be the critical value in horses and cattle. 61 % of the calves achieved an IgG concentration of over 4 g\l. Since there is no correlation between IgG level in colostrum and early mortality, the results indicate that low colostrum intake during the ï¬پrst 24 h of life and not low IgG concentration in colostrum is presumably one of the main causes of early calf mortality. Therefore, it was recommended that the care of the newborn calves by herdsmen should be improved. Kï‌¥ï‌¹ï‌·ï‌¯ï‌²ï‌¤ï‌³ : Immunoglobulin G, camel, Camelus dromedarius, colostrum, passive immunization.

Camels are slow reproducers. A female camel is sexually mature at the age of 4–5 years. Pregnancy is just over 12 months and the calving interval in pastoral production systems is normally 24 months or more. Female camels can remain fertile up to the age of 25 years and it is often reported that they produce 8–10 calves during a lifetime. In pastoral production systems, however, only a small proportion of the breeding female can reach this production performance (Schwartz & Dioli, 1992 ; Farah, 1996). Beside this natural productivity limitation, the main factor affecting herd growth is calf mortality, which is high during the postnatal and pre-weaning stages. In a survey carried out in eastern Sudan, Agab & Abbas (1998) reported a 48 % mortality rate among calves under 6 months of age and 14n6 % after that time.
* For correspondence : zakaria.farah!ilw.agrl.ethz.ch

2

R. Kï‌،ï‌­ï‌¢ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

Calf mortality between 30 and 50 % has also been reported in Kenya (MukasaMugerwa, 1981), Tunisia (Burgemeister, 1974) and Somalia (Hussein, 1987). All these studies showed the main reasons for the high postnatal mortality to be poor management practice and diseases. The newborn calf has no natural protection against diseases, as there is no antibody transfer from the mother during fetal development. The calf can obtain immediate immunization soon after birth only through the colostrum, which has a very high concentration of antibodies. Therefore, it is vital for the calf to suckle as soon and as much as possible. Unfortunately there is a common belief among many pastoralists that colostrum causes diarrhoea and, consequently, is unsuitable for the newborn calf. This wide spread practice of withholding the colostrum from the newborn calves, depriving them of essential antibodies, is certainly a crucial factor in the frequently reported high calf mortality in pastoral production systems. The present investigation was undertaken to examine the immunoglobulin G (IgG) concentration of camel colostrum as well as the extent of the calf’s passive immunization by maternal antibodies. IgG concentration in colostrum and in the serum of the calf during the ï¬پrst 3 d of life were compared with the respective values in horses and cattle. ï‌­ï‌،ï‌´ï‌¥ï‌²ï‌©ï‌،ï‌¬ï‌³ ï‌،ï‌®ï‌¤ ï‌­ï‌¥ï‌´ï‌¨ï‌¯ï‌¤ï‌³ Sample collection The study was carried out in Ol Maisor Ranch in Kenya’s Laikipia district. The camels (Camelus dromedarius) were fed all year around exclusively by grazing. The supply of water was ad libitum and, in addition to pasture, the camels were provided with a mineral lick containing P, Ca, NaCl and trace elements. On return from pasture at about 18.30, lactating camels are separated from their calves until 06.30 the following morning, when they are milked. The investigations were carried out during a 6-month period between October 1993 and April 1994. At the time of the survey, the camel herd amounted to 350 animals of three different breeds (Turkana, Somali and Pakistani) and their crossbreeds. During the study, 39 camel births took place on the ranch, 31 of which were included in the investigation. In 26 cases colostrum samples were taken before ï¬پrst suckling of the calf. From 29 calves it was possible to take blood samples before the ï¬پrst suckling. The age of the dam was between 5 and 18 years at the time of delivery. The following samples were taken : $ milk from the dam immediately after delivery and 6, 12, 24, 48 and 72 h later, $ blood from the dam at the time of delivery, $ blood from the newborn calf immediately after birth but before the ï¬پrst colostrum intake, and additionally after 24, 48 and 72 h. The blood samples were taken from the jugular vein using a Becton-Dickinson Vacutainer, centrifuged within 1 h of sampling and the serum was immediately frozen at k18 mC. The colostrum samples were taken as composite milk (four teats) and stored in sterile milk tubes at k18 mC until analysis. Sample analysis The IgG concentration was determined in the laboratory of the Clinic of Obstetrics, University of Zurich, by means of radial immune diffusion (RID), as described by Mancini et al. (1965) and the anti-camel IgG antibodies were produced according to Kamber (1996). Ten blood samples of camels were pooled and IgG was

Immunoglobulin G in newborn camel calves
70 60 50 IgG (g/l) 40 30 20 10 0 0 24 48 Hours after delivery 72

3

Fig. 1. Average immunoglobulin G (IgG) concentration in the colostrum of the camels during the ï¬پrst 72 h after giving birth.

isolated by FPLC using a protein A-sepharose column. The purity of IgG was determined by SDS–PAGE. Two rabbits were immunized three times along with complete and incomplete Freud’s adjuvant. Blood was collected 4 weeks after the last injection. The content of anti-camel IgG was determined by an Ouchterlony test using 1 % agarose H and barbital buffer. The pre-immune serum gave negative results and one rabbit did not produce antibodies. The data were collected using EpiInfo (WHO) and evaluated with Statview 2 (SAS Institute). For continuous variables the paired t-test was used and for nominal values the د‡# test. The signiï¬پcance threshold was set at an خ± error value of 5 %, P l 0n05. The standard deviation is indicated in the corresponding text. ï‌²ï‌¥ï‌³ï‌µï‌¬ï‌´ï‌³ The IgG concentration in the colostrum of the 26 dams at the time of birth was 58n6p15n4 g\l and 24 h post-partum, it decreased to 38n8p17n9 g\l. This decrease in the IgG concentration of the colostrum within the ï¬پrst 24 h was signiï¬پcant (P 0n0001). After 24 h the concentration continued to decrease markedly and was only 16n5p11n5 g\l after 72 h. The changes in the IgG concentration in the colostrum of the dams during the ï¬پrst 72 h are shown in Fig. 1. The IgG concentration in the serum of the dams at the time of birth was 17n6p2n7 g\l, the value ranging between 13n3 and 22n3 g\l. There was no relationship between the IgG concentration in the serum of the dams and the IgG concentration in the colostrum. From 29 of the 31 calves it was possible to take pre-colostral blood samples. The average IgG content of the pre-colostral calf serum was 0n2p0n3 g\l. Twenty-four of the 29 calves examined had a pre-colostral serum IgG concentration below 0n2 g\l. The concentrations for the remaining ï¬پve calves ranged between 0n5 and 1n3 g\l (Fig. 2). The IgG concentration in the blood serum of calves increased signiï¬پcantly within the ï¬پrst 24 h and the mean increase was 7n8 g\l. After the ï¬پrst 24 h there was no further increase and the mean concentration then decreased slowly. The changes in the IgG concentration in the serum of the calves over 72 h are shown in Fig. 3. As shown in Fig. 4, the distribution of the IgG concentration 24 h after the birth allows us to distinguish between two groups : group A with an IgG concentration above and group B with an IgG concentration below 4n0 g\l, which is considered to be the threshold value in horses and cattle (McGuire et al. 1977 ; Rumbaugh et al.

4
24 20 Calf numbers 16 12 8 4 0

R. Kï‌،ï‌­ï‌¢ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³
24

1 0آ·1 0آ·5

2 0آ·6 IgG (g/l)

1 0آ·8

1 1آ·3

Fig. 2. Frequency of different immunoglobulin G (IgG) concentrations in the serum of camel calves before colostrum intake (n l 29). 10 8 IgG (g/l) 6 4 2 0

0

24 48 Hours after birth

72

Fig. 3. Immunoglobulin G (IgG) concentration in calf’s serum during the ï¬پrst 72 h after birth. 9 8 7 6 5 4 3 2 1 0

Calf numbers

1

2

5

6آ·5 8آ·5

9آ·5 10آ·5 11آ·5 13آ·5 15آ·5 16آ·5 17آ·5 20آ·5 25آ·5

IgG in serum (g/l) Fig. 4. Frequency of different immunoglobulin G (IgG) concentrations in the individual calf’s serum 24 h after birth (n l 31) ;
below 4n0 g\l, above 4n0 g\l.

1979 ; Dudan & Hirni, 1989 ; Bostedt & Thein, 1990 ; LeBlanc, 1990). For group A the value for the IgG concentration at 24 h after birth was 12n5p5n9 g\l (n l 19), while for group B it was only 1n4p0n6 g\l (n l 12). The concentrations before the ï¬پrst intake of milk (time 0) were not signiï¬پcantly different for the two groups. In group A, the serum IgG concentration rose in the ï¬پrst 24 h from 0n2p0n2 to 12n5p5n9 g\l. In group B, this increase was signiï¬پcantly lower, from 0n2p0n4 to 1n4p0n6 g\l.

Immunoglobulin G in newborn camel calves
14 12 10 IgG (g/l) 8 6 4 2 0 0 24 48 Hours after birth 72

5

Fig. 5. Course of immunoglobulin G (IgG) concentration in the serum of two calf groups during the ï¬پrst 72 h after birth ; $ above 4 g\l, # below 4 g\l.

In the subsequent variations, the concentration of IgG in the serum of group B remained well below the limit of 4 g\l and showed no further signiï¬پcant change. The IgG values for group A, on the other hand, although decreasing continuously after 24 h, still amounted to 11n1p4n1 g\l 72 h after the birth (Fig. 5). There was no signiï¬پcant difference between the birth weights of the two groups of calves and the weight was 38n9p7n3 kg. ï‌¤ï‌©ï‌³ï‌£ï‌µï‌³ï‌³ï‌©ï‌¯ï‌® The main objective of the present investigation was to ï¬پnd out whether inadequate IgG concentration in the colostrum, or inadequate passive immunization of the calves, could be a reason for high calf mortality (Roy, 1990). According to some researchers, low IgG concentrations in the colostrum lead to a poorer passive immunization of calves (McGuire et al. 1977 ; Schmidt et al. 1982 ; Hancock, 1985 ; Morris et al. 1985). The IgG concentrations in the camel colostrum were higher on average than the values found in the literature for other domestic animals. In colostrum from horses IgG concentration was 15–50 g\l and in cattle it was between 34 and 39 g\l, whereas in sheep it ranges between 17 and 20 g\l (Perryman & Crawford, 1979 ; Eder, 1987 ; Buschmann, 1990). This means that the concentration of IgG in camel colostrum is similar to, or even higher than, that of other domestic animals. In the present study the lowest IgG concentration measured in the camel colostrum was 20n9 g\l. A relationship between the IgG concentration in the camel colostrum and that in the serum of the calves could not be determined, due to the unknown amount of colostrum intake. It can, however, be assumed that the limiting factor in the passive immunization of camel calves is not the level of IgG concentration in colostrum but rather the quantity of colostrum received by the newborn calves. An elevated precolostral IgG titre may be evidence of prior intra-uterine infection. The pre-colostral titres had no influence on the subsequent development of the IgG concentrations in the calf sera. Also Schenker’s (1987) ï¬پnding that calves with elevated pre-colostral IgG titres show a lower birth weight could not be conï¬پrmed. If a 24 h limit value of 4 g\l is applied to the camel calves studied, the IgG concentrations in serum of 12 calves (39 %) with only 1n4p0n6 g\l must be considered as insufficient. For 19 calves (61 %), on the other hand, adequate passive immunization occurred, as their average IgG concentration 24 h post-natum was 12n5p5n9 g\l and ranged from 4n7 to 26n0 g\l. The ranch on which the investigations took place had in recent years a calf

6

R. Kï‌،ï‌­ï‌¢ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

mortality rate of 20–30 %. Of the 31 calves examined up to the age of 3 weeks, two (approximately 6 %) died during the period of this investigation. In neither case was the cause of death an infectious disease (fracture\ileus). Thus, no link could be established between mortality and insufficient IgG supply. The same applies to perinatal health problems without a fatal outcome. The fact that an intervention study on the subject of supplying the calves with colostrum was taking place affected the herdsmen’s attitude to this particular aspect. It must be assumed, therefore, that the calves were given much less attention before this study, thus receiving less colostrum. Through the continuous inspection visits to the herds, the calves’ problems could be quickly recognized and remedied during the investigation, which otherwise was not necessarily the case. The care of the calves by the herdsmen has been found to be one of the main problems. The husbandry conditions of the calves must be optimized. This can be achieved by bringing the females into a big enclosed area before calving. It is then not necessary to tie up the newborn calves. They will be able to develop initiative themselves in deciding when to suck, and would not be exclusively dependent on the caring behaviour of the mothers. If the calves are not able to suck by themselves within 12 h after birth, assistance from the herdsmen is needed. The absorption of IgG after this time decreases signiï¬پcantly, as shown in this study. Calves which after birth cannot stand up by themselves must be put on their feet at an early stage and led to the udder. If, despite all efforts, a calf has not yet suckled in the ï¬پrst 6 h after the birth, the dam must be milked and the calf given milk from a bottle. In addition, if possible, a reserve of frozen colostrum should be set up at the ranch in case a dam does not produce enough milk or does not allow the calf to suck. The authors are grateful to Mr J. O. Evans and Mrs D. Atkins for providing us with all the facilities needed to carry out the ï¬پeldwork at Ol Maisor Ranch. ï‌²ï‌¥ï‌¦ï‌¥ï‌²ï‌¥ï‌®ï‌£ï‌¥ï‌³
Agab, H. & Abbas, B. 1998 Epidemiological studies on camel diseases in eastern Sudan. Camel Newsletter 14 53–57 Bostedt, H. & Thein, P. 1990 Foal diseases, general part. In Neonatology of Animals, pp. 140–158 (Eds K. Walser and B. Hartwig). Stuttgart : Ferdinand Enke Verlag Burgemeister, R. 1974 Husbandry of Dromedary in South Tunisia. PhD thesis, Institute of Tropical Veterinary Medicine, Giessen Buschmann, H. 1990 Immunology. In Neonatology of Animals, pp. 30–35 (Eds K. Walser and B. Hartwig). Stuttgart : Ferdinand Enke Verlag Dudan, F. & Hirni, H. 1989 Intensive care of new-born foals. Tierarztliche Praxis (Suppl) 4 63–84 W Eder, H. 1987 Blood and Lymph, pp. 160–208 (Eds A Scheunert and A Trautmann). Berlin : Paul Parey Verlag Farah, Z. 1996 Camel Milk, Properties and Products, pp. 14–19. St. Gallen, Switzerland : SKAT, Swiss Centre for Development Co-operation in Technology and Management Hancock, D. D. 1985 Assessing efficiency of passive immune transfer in dairy herds. Journal of Dairy Science 68 163 Hussein, M. A. 1987 Traditional practices of camel husbandry and management in Somalia. Camel Forum 9 11–12 Kamber, R. 1996 Studies on the Supply of Immunoglobulin G to Newborn Camel Calves (Camelus dromedarius). PhD thesis, University of Zurich, Switzerland LeBlanc, M. M. 1990 Immunologic considerations. In Equine Clinical Neonatology, pp. 275–294 (Eds A. M. Koterba, W. H. Drummond and P. C. Kosch). Philadelphia : Lea & Febiger Mancini, G., Carbonnara, A. O. & Heremans, I. F. 1965 Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2 235–254 McGuire, T. C., Crawford, T. B., Hallowell, A. L. & Macomber, L. E. 1977 Failure of colostral immunoglobulin transfer as an explanation for most infections and deaths of neonatal foals. Journal of the American Veterinary Medical Association 170 1302–1304 Morris, D., Meirs, A. & Merryman, G. 1985 Passive transfer failure in horses : incidence and causative factors on a breeding farm. American Journal of Veterinary Research 46 2294–2299

Immunoglobulin G in newborn camel calves

7

Mukasa-Mugerwa, E. 1981 The Camel (Camelus dromedarius) : A Bibliographical Review. Addis Ababa, Ethiopia : International Livestock Centre for Africa Perryman, L. E. & Crawford, T. B. 1979 Diagnosis and management of immune system failures in foals. Proceedings of the American Association of Equine Practitioners 25 235–245 Roy, H. B. 1990 The Calf : Management of Health, volume 1, 5th edn. London : Butterworths Rumbaugh, G. E., Ardans, A., Ginno, D. & Sommerhausen-Smith, A. 1979 Identiï¬پcation and treatment of colostrum-deï¬پcient foals. Journal of the American Veterinary Medical Association 174 273–276 Schenker, C. 1987 Frequency and Effect of Prenatal Infections in New-born Calves. PhD thesis, University of Zurich, Switzerland Schmidt, F. W., Kim, J. W., Derenbach, J. & Langholz, H. J. 1982 Colostral immunity and performance in suckling calves. Tierarztlich Umschau 37 485–489 W Schwartz, H. J. & Dioli, M. 1992 The One-humped Camel in Eastern Africa : A Pictorial Guide to Diseases, Health Care and Management. Berlin : Josef Margraf Scientiï¬پc Books

pdf

412

Farah, Camel milk butter

Manufacture and characterization of camel milk butter
By Z. FARAH, T. STREIFF and M.R. BACHMANN Laboratory of Dairy Science, SWISS Federal Institute of Technology, Zurich. Switzerland 1. Introduction Camels (Camelus dromedarius) are Important as daily animals in certain regions of the world. Most of the camel milk is drunk fresh or when it has just turned sour. Among many camel rearing societies, there is a common belief that butter cannot be made from camel milk. This belief has been supported by some authors (1) while others (2, 3) claim that butter can be made from camel milk. The present study was undertaken in order to gain a clear picture of this rather contradictory situation. Experiments on butter from camel milk were made in rural areas in North Eastern Kenya, where a majority of the population subsists almost entirely on camel milk. The butter obtained, along with camel milk samples, was analyzed at the Dairy Laboratory of the Swiss Federal Institute of Technology (ETH) Zurich. 2.5 Chromatography of fatty acids Milk from 2 different camel herds was used for the analysis. For each herd 10 samples were collected from 10 Individual camels Samples were kept refrigerated and transported to our laboratory within 36 h. Upon arrival, fat of each sample was extracted by a mixture of chloroform and methanol (2:1 v/v). The solvent was removed in a stream of nitrogen and the fat stored in sealed tubes at -20آ°C until analyzed. Fatty acids analysis of milk was carried out as described by PRABUCKI (6). The methyl esters were prepared by the procedure of CHRISTOPHERSON (7). The analysis was performed on a Hewlett Packard 5830 gas chromatograph with a flame ionization detector. The columns (2 m x 1/8") were packed with Chromosorb W-AW, 100/120 mesh containing 3 % SP-231 0 and 2 % SP-2300. The samples were injected at an oven temperature of 75آ°C; then the oven temperature was raised to 220آ°C at a rate of 5 آ°C/min. The detector temperature was 250آ°C. 3. Results Centrifugation and churning Due to the low fat content of cream after the first pass of centrifugation, the cream had to be separated a second time. As a result the separation temperature decreased from 65آ°C at the first to 55آ°C at the second centrifugation pass. In 5 milk samples with a fat content between 2.5 and 3.8 %, the butter fat content of the skim milk obtained after the second centrifugation varied between 0.2-0.9 %. Cow milk separated in the same centrifuge Yielded skim milk with a butter fat content of 0.1 -0.2 %. After centrifugation the cream samples of varying fat content were churned immediately at different
Camel milk cream samples churned under varying cream fat content and churning temperature Cream

2. Materials and methods 2.1 Milk samples Camel milk was obtained from the following sources: (a) Camel herd of the University of Nairobi, (b) Ngare Ndare ranch, Laikipla District, Kenya, (c) individual herds owned by nomads around the town of Garissa, North Eastern Province, Kenya. 2.3 Starter culture As starter culture for sour cream freeze-dried mesophilic lactic culture O-CH-143 obtained from Chr. Hansen's Laboratory, Denmark, was used 2.3 Cream separation and churning Milk was heated to 65آ°C and separated with an AlfaLaval hand centrifuge, type 24 S. After the first pass, the cream was immediately separated a second time. The cream was divided into small portions of 1 I on average, and the fat content was adjusted to 20.5, 25 and 30 %. Some cream samples were inoculated with 2 % starter culture and incubated at room temperature (27آ°C). Churning took place in a domestic glass hand churn of a 2 I capacity. The filling level was approximately 1 I each time. The churning temperatures were varied between 15 and 36آ°C. The butter grains were washed twice with 500 ml drinking water at ambient temperature (27آ°C). The butter samples were frozen for further analysis. 2.4 Butter analysis Cream and butter fat contents were determined by the Gerber method (4). The following physical and chemical constants of the butter were determined according to the Swiss Manual for Food Analysis (5): Melting point (capillary method), refractive index (Zeiss Butyro-Refractometer), iodine value, saponification value, Reichert-Meissl value and Polenske value.

Table 1:
Sample No

fat
%

Churning temperature آ°C

Churning time

Butler fat yield
%

mln 13 18 16 11 10 35 38 6 50 10 5 5

Sweet cream:

1 2 3 4 5 6 7 8
9 Sour cream:

20.0 22.5 22.5 22.5 25.0 25.0 25.0 30.0 30.0 20.0 25.0 30.0

25 15 20 25 25 32 36 25 30 25 25 25

81.1 80.8 84.0 85.3 78.2 60.7 65.6 64.8 58.7 59.6 55.0 55.2

10 11 12

Milchwissenschaft 44 (7) 1989

Farah, Camel milk butter

413

churning temperatures. Results of the churning trials are shown in Table 1 and Fig. 1. Increasing of the cream fat content decreased the churning time in both sour and sweet cream (Fig. 1). At the same cream fat content sour cream yielded a lower butter fat content than sweet cream (Fig. 1). The highest fat yields In butter (8085.3%) with cream of 20-25% fat were obtained at churning temperatures between 15 and 20آ°C. The corresponding values for cow milk cream vary between 8-12 آ°C, being on average 10آ°C lower (8).

higher than the corresponding values in cow milk butter. Due to the high melting point, reading with the butyrorefractometer had to be made at 46آ°C and corrected to 40 آ°C. The mean value of the reading was 49.1. Compared with cow milk butter, camel milk butter was found to have low Reichert-Meissl, Polenske and saponification values, but a higher iodine value. Fatty acid analysis The molar percentages of fatty acid composition of milk fat from 2 different camel herds along with those of cow milk (10) are presented in Table 3. Each herd consisted of 10 camels. Herd A was fed exclusively by free grazing, whereas the camels of herd B grazed during the day and upon return from pastures in the evening, they were fed a supplement consisting of a mixture of maize and barley. In both groups milk samples were taken in September during the dry season. Apart from the dietary supplement to herd B, management and pasture quality was similar for both herds. Compared to cow milk fat (Table 3), camel milk fat contains less short chain fatty acids, but relatively high concentrations of C14:0 and C16:0 acids. Long chain unsaturated fatty acids occur to about the same extent as in cow milk fat. Compared to herd A camel milk fat from herd B which received supplement showed an Increased concentration in C18:0 and C18:1 and a decreased concentration in the yield of fatty acids C16:0 and C14:0, This seems to follow the effect of dietary supplements on the yields of fatty acids generally observed In bovine milk fat (11).

11 min

Churning time (min)
_ Sweet cream ~ Sour cream 6min

I

22.5

25

30

Cream fat content (%)
Fig 1 Effect 01 cream fat content on fat yield (churning temperature 25آ°C)

Butter constants Some chemical and physical constants of the butter obtained were determined. Mean values of 10 butter samples are presented In Table 2. Values for cow milk fat are also included for comparison (9). The mean melting point is at 41.4 آ°C and is on average 8آ°C
Table 2: Physical and chemical constants of 10 camel butter samples Sample No 1 2 3 4 5 6 7 8 9 10 Mean Cow milk butter Melting point 41.5 40.6 40.7 40.9 41.3 40.6 41.5 41.5 42.5 42.5 41.4 28-38 Refractometer readings 49.5 48.5 49.0 49.5 49.5 49.0 49.5 49.5 48.5 48.5 49.1 39-46

4. Discussion and conclusions Although the experiments were carried out in rural areas under difficult working conditions, a reasonable

Saponification value 201 201 202 200 200 200 200 200 200 200 200.4 220-233

Iodine value 51.3 51.0 50.5 47.8 47.3 47.8 48.0 50.1 47.8 48.0 48.96 25-38

Reichert-Meissl value 2.27 2.25 214 2.16 2.09 2.12 2.00 2.22 2.00 2.00 2.12 24-34

Polenske value 0.68 0.60 0.60 0.62 0.60 0.58 0.60 0.67 0.62 0.62 0.62 1.5-5

Table 3: Milk fat From Camel herd A Camel herd B Cow

Fatly acid composition (mole %) of milk fats from 2 camel herds along with cow milk fat for comparison Molar percentage mean values 01 tatty acids from 10 individual animals for each camel herd C120 C140 C141 C15:0 C15:1 C160 C161 C170 C171 C180 C181 0.26 1.03 0.80 3.50 14.20 9.70 10.00 1.29 0.80 1.29 1.98 0.50 0.26 0.20 31.50 28.50 24.60 1030 720 220 0.78 1.10 052 083 11.40 17.60 14.90 24.60 12.20 25.10

C40 1.81 1.10 8.85

C60 0.78 0.20 3.30

C8:0 0.52 1.10 2.60

C100 C10:1 1.29 0.20 4.07

C182 1.10 4.40 2.20

C183 1.29 1.38 0.70

C200 0.78 1.38 -

Milchwissenschaft 44 (7) 1989

414

Farah. Camel milk butter

efficiency of separation has been achieved. In order to find out optimum churning conditions, cream was churned at different temperatures. At low cream temperature (10 to 12آ°C) no butter grams could be recovered above 36 آ°C butter yield began to decrease. Hence, the temperature trials were made between 15 and 36آ°C. The highest butterfat yield was obtained at a churning temperature of 25آ°C from cream with 22.5 % fat content. The time taken to churn at this temperature was 11 min. The reason for this different churning behaviour of camel milk fat in comparison with cow milk fat can partly be attributed to the high melting point of camel milk fat. This seems to shift the ideal ratio of solid to liquid fat in globules at a given temperature towards a point higher than that of cow milk fat. The different churnability can also be attributed to the reported small size of camel milk fat globules (12). Small globules have a larger surface in relation to their mass which tends to increase their resistance. Compared to cow milk butter, camel milk butter is white in colour and is more sticky and greasy in consistency. The fatty acid analysis by gas chromatography confirmed the results of the butterfat constants. As indicated the analysis represents mean values of the fatty acids from 10 individual animals for each camel herd. Fatty acid composition is influenced to some degree by environmental and physiological factors such as diet, stage of lactation and genetic differences within the species. Within this limitation the general pattern of the camel milk fatty acids found in the present investigation are comparable to those of GLASS et al. (13) obtained by gas chromatography technique, but differ from the data reported by OHINGRA (14) who analyzed milk fat of not specified Indian camels using a fat fractionation method. From the present investigation it can be concluded, that butter can be prepared from camel milk. Owing to the nature of its milk fat, camel cream has different churning properties compared to cream from cow milk.

(10) FOX, PF.: Development in Dairy Chemistry - 2. Appl. Sci. Publishers, London, New York, p. 15 (1983) (11) FOX, P.F.: Development In Dairy Chemistry - 2. Appl. SCI Publishers, London, New York. p. 60 (1983) (12) Y AGIL, R.: FAO Animal Production and Health Paper, Rome 26 17 (1982) (13) GLASS, RL et al.: Camp. biochem. physiol. 22 415-425 (1967) (14) OHINGRA, DR Biochem. J. 28 73-78 (1934)

6. Summary
FARAH, Z., STREIFF, T., BACHMANN, M.R: Manufacture and characterization of camel milk butter. Milchwissenschaft 44 (7) 412-414 (1989). 44 Butter composition (camel milk) Experiments on butter manufacture from camel milk were carried out in rural areas In North Eastern Kenya. The milk was heated to 65آ°C and separated with an Alfa-Laval hand centrifuge. In order to find optimum churning conditions. cream of varying fat content was churned at different temperatures. Under the selected experimental conditions the highest butter fat yield (85.3 %) was found at 25آ°C from cream with a fat content of 22.5 %. The churning time at this temperature was 11 min. Chemical and physical constants of the butter obtained as well as the fatty acid composition of milk fat tram 2 different camel herds were also studied. Compared with cow milk butter, camel milk butter was found to have low Reichert-Meissl, Polenske, and saponification values, but a higher melting point, refractive index and Iodine value. The fatty acids of camel milk differ markedly from those at cow milk fat in the lower content of short chain fatty acids. FARAH, Z., STREIFF. T., BACHMANN. M.R.: Herstellung und Charakterisierung van Kamelmilch-Butter. Milchwissenschaft 44 (7) 412-414 (1989) 44 Butterzusammensetzung (Kamelmilch) Es wurden Versuche zur Herstellung von Butter aus Kamelmilch in einer lأ¤ndlichen Region im Nordosten Kenias durchgefأ¼hrt. Die Milch wurde auf 65آ°C erhitzt und mit einer Handzentrifuge der Marke Alfa-Laval separiert. Um die idealen Butterungsbedingungen zu ermitteln, wurde der Rahm bei verschiedenen Temperaturen und unterschiedlichem Rahmfettgehalt verbuttert. Unter den gewأ¤hlten experimentellen Bedingungen wurde die hأ¶chste Ausbeute an Butterfett (85,3%) bei einer Butterungstemperatur von 25آ°C und einem Rahmfettgehalt von 22.5% erreicht. Bei dieser Temperatur betrug die Butterungszeit 11 min. Einige chemische und physikalische Konstanten der gewonnenen Butter sowie die Zusammensetzung der Fettsأ¤uren in Milch von zwei verschiedenen Kamelherden wurden ebenfalls untersucht. Verglichen mit Kuhmilchbutter weist Kamelmilchbutter niedrigere Reichert-Meissl-, Polenskeund Verseifungszahlen, aber hأ¶heren Schmelzpunkt, Brechungsindex sowie eine hأ¶here Jodzahl auf. Kamelmilchfett unterscheidet sich van Kuhmilchfett im niedrigeren Gehalt an kurzkettigen Fettsأ¤uren. FARAH. Z., STREIFF, T, BACHMANN'. M.R.: Fabrication
et caracterisation de beurre du lait de chamelle. Milchwissenschaft

Acknowledgements The authors express their thanks to Prot. A. Prabucki for his valuable technical assistance and Prof. Z. Puhan for stimulating discussion.

5. References
(1) DICKSON, H.R.P, in The Arab at the Desert. London, George Allan and Unwin Ltd., p. 409 (1951) (2) KNOSS, K.H.: World Animal Rev. 57 11-21 (1986) (3) YAGIL. R.: FAO Animal Production and Health Paper, Rome, 2622-24 (1982) (4) SCHNEIDER, K., ROEDER, H.: Ole praktische Milchprأ¼fung und die Kontrolle van Molkereiprodukten, 12. Aufl. Verlag Wyss, Bern (1979) (5) Schweiz. Lebensmittelbuch, 2. Band-spezieller Teil, Kapitel Rahm und Butter, Eidg. Drucksachen- und Materialzentrale, Bern (1967) (6) PRABUCKI, A.: Anleitung zur gaschromatographlschen Bestimmung van Fettsأ¤uren-Methylestern. ETH Zأ¼rich (unverأ¶ffentlicht) (7) CHRISTOPHERSON, FW, GLASS. RL J. Dairy Science 52 1289-1290 (1969) (8) HUNZIKER. O.R., In: The Butter Industry. La Grange, Illinois, p. 286 (1927) (9) TOPEL, A.: Chemie und Physik der Milch, 2. Aufl. VEBVerlag, Leipzig, p. 90 (1981)

44 (7)412-414 (1989).

44 Composition du beurre (Iait de chamelle) FARAH, Z., STREIFF, T., BACHMANN, M.R.: Producciأ³n y caracterizaciأ³n de manteca de leche de camella. Milchwissenschaft 44 (7) 412-414 (1989). 44 Composiciأ³n de manteca (Ieche de camella)

Milchwissenschaft 44 (7) 1989

pdf

The Creaming Properties and Size Distribution of Fat Globules in Camel Milk Z. FARAH Institute of Food Science Swiss Federal Institute of Technology CH-8092 Zأ¼rich, Switzerland M. RأœEGG Federal Dairy Research Institute CH-3097 Liebefeld-Bem, Switzerland

ABSTRACT The natural creaming of raw and heated camel milk was studied. Compared with cow (Bos taurus) milk, camel (Camelus dromedarius) milk showed very slow creaming rate in both raw and heated milk at refrigerator and room temperatures. Fat globule size distribution was similar in cow and camel milks. The creaming ability was also studied in various combinations of skim milks and creams of raw camel and cow milks. All systems that contained skim camel milk creamed poorly. It was concluded that insufficient quantity of agglutinin in camel milk was mainly responsible for the slow rate of creaming. (Key words: camel milk. fat globules, creaming properties) INTRODUCTION Camels (Camelus dromedarius) are important dairy animals in certain regions of the world Most of the camel milk is drunk fresh or when it has just soured. Traditional camel rearing societies apparently have problems in converting camel milk into butter, and it is often stated that conversion is not possible (3, 13). A recent study on butter manufacture from camel milk run in rural areas in Kenya showed, however, that butter can be made from camel milk (4). Cream was separated with a hand centrifuge and churned under varying conditions. The highest milk fat yield (8S.3%) was found at 2Sآ·C from cream with a fat content of 22.5%.

The main reason for the reported difficulties in obtaining camel butter was the different churning behaviour of camel fat in comparison with cow (Bos taurus) milk fat. An earlier study (4) reported that, upon standing, camel milk creams by gravity less rapidly and completely than cow milk. For proper understanding of this behaviour, the creaming ability and the size distribution of fat globules in camel milk were investigated MATERIALS AND METHODS Milk Samples Bulked fresh camel milk and cow milk were supplied by the university herd in Nairobi, Kenya. The experiments on creaming rate were carried out immediately after milk collection. Milk used for fat globule size determination was pooled, kept refrigerated and transported to our laboratory within 36 h. Measurement of Creaming Rate Six pooled camel milk samples were heated in water baths for 30 min at SS, 60, 62, 68, 70, and 77آ·C. Duplicate samples of 100 ml for each temperature were poured into measuring cylinders (i.d 30 mm, height 250 mm) and left to cool. On reaching ambient temperature, two drops of nigrosin solution were added to all samples to allow clear optical distinction between the aqueous (blue) and fat (white) phases. The milk samples were left to cream at 4آ·C, and the cream layer was measured from the scale of the measuring cylinder after 5 and 24 h. Untreated raw milk was creamed under the same conditions in parallel to the heated samples. For comparison, the same studies were carried out with cow milk.

Received December 28, 1989. Accepted June 29, 1990. Journal of Dairy Science Vol. 74, No. 9, 1991 2901

2902

FARAH AND RأœEGG

Agglutinin Test in Camel Milk Fresh camel milk: and fresh cow milk of 3.7 and 3.5 milk: fat percentage, respectively, were used. To increase the concentration of the agglutinating substance in skim milk:, the milk: was heated at 45آ°C for 60 min and separated at 5000 x g with an Alfa-Laval hand centrifuge type 24S (Alfa-Laval, Inc., Kansas City, MO). Various combinations of skim milks and creams were prepared by mixing skim milk and cream to produce a mixture containing 4% fat. Cream layer volume measurement was made after 24 h at 4 آ°C, and the fat percentage in 50-ml portions of skim milk withdrawn from the bottom of the measuring cylinder was estimated by the Gerber method (10). Size Distribution of Fat Globules in Camel Milk

Distribution of fat globule size was deterFigure 1. Creaming rate of raw and heated camel mined in whole milk and cream supplies of 30% milk and cow milk. Camel milk (3.3% fat content) fat content. Photomicrographs were made using after 5 h (â—ڈ) and 24 h (*). Cow milk (3.4% fat a Wild-Leitz Light Microscope M20, equipped content) after 5 h (+) and 24 h (â–،). with a camera MKA4 (Wild-Leitz, Heerbrugg, Switzerland). The diameter of the fat globules was measured on prints with a final rate was rapidly reduced, mainly because of magnification of 1000 times. An eyepiece protein denaturation. Compared with cow milk:, micrometer was used for calibration. On eight camel milk shows a very slow creaming rate at photographs, 1803 particles were counted. The all temperatures. Creaming layers varied from .5 diameters were classified into size classes of I- to 2 ml at 4 آ·C. Creaming of camel milk' samples J.UD class widths. The number and volume at room temperatures was not significantly frequencies and various statistical parameters of different from that creamed at 4آ·C. Extending the size distribution were calculated using a the creaming time up to 48 h did not produce program written in GW-BASIC for MS-DOS any significant increase in creaming layer. computers. (The listing of the program is availThe creaming phenomenon in milk, particuable on request from M. Rأ¼egg.) larly in cow milk, has been extensively investigated. The main factor responsible for rapid RESULTS AND DISCUSSION formation of a cream layer on cow milk has been Creaming Capacity of Camel Milk shown to be a heat denaturable protein adsorbed Preliminary experiments were made in which on cold fat globules, which has the different creaming conditions of camel milk characteristics of a euglobulin. This protein, were studied in order to ascertain which heating know as fat agglutinin, promotes clustering of temperature and creaming conditions merited globules (6). To test whether the low creaming capacity of investigation. The results of the~ selected experimental conditions are presented in Figure camel milk could be due to the deficiency in 1. The creaming rate of cow milk: was agglutinin, the creaming ability of raw cow milk, consistent with findings reported in the literature raw camel milk, and various combinations of (11). Heating up to 62آ·C for 30 min increased skim milks and creams was studied. Results of the cream volume over that of raw milk. At cream volume and fat percentage are presented in Table 1. It is presumed that higher temperatures, the creaming

Heating temperature آ°C

Journal of Dairy Science Vol. 74, No. 9, 1991

CAMEL MILK CREAMING PROPERTIES

2903

TABLE 1. Creaming of camel and cow milks and of various combinations of their cream and skim milks in 100-ml measuring cylinders at 4آ°C Sample Cow milk Camel milk Cow skim plus cow cream Camel skim plus camel cream Cow skim plus camel cream Camel skim plus cow cream the warm separation at 4SoC yielded agglutininrich skim milk and relatively agglutinin poor cream (6). Mixing a cream with its own skim milk yielded cream layer approximately equal to the original unseparated milk. The milk from camel cream and cow skim milk creamed much better than camel milk and the mixture of cow cream and camel skim milk. This could be an indication that camel milk lacks the agglutinating substance required to cluster fat globules. The creaming behaviour of camel milk appears to be similar to that of buffalo and goat milks (1, 2, 7), which show poor creaming ability due to an insufficient quantity of agglutinin. To examine whether the observed difference in creaming power between camel and cow milk could be related to differences in the size of the fat globules, the size distribution of camel milk fat globules was determined by light microscopy. Figure 2 shows the volume frequency distribution of fat globules in camel milk compared with the distribution found in cow milk (12). The results show similar globule size distribution in cow and camel milks. From the light microscopy data, a mean diameter of 2.61 J.1m, a volume to surface mean diameter of 4.40 J.1m, a weight mean diameter of S.l1 J.1m, and a distribution width of 40.1 % can be calculated. These values are comparable with those reported for cow milk (8, 9, 12). A chisquare heterogeneity test (2 X k contingency table) applied to the data in Figure 2 revealed a chi-square value of only 1.7 [chisquare (P = .9S, n = 4) = 9.S]. This indicates that the size distributions were not significantly different. Therefore, the difference observed in the creaming behaviour between cow and camel milk cannot be explained by differences in the size of fat globules. Cream layer after 24h [ml] 12 1 11 1 7 3 Fat in lower 50ml after 24h [%] 0.8 3.2 0.7 3.4 2.0 2.9

Comprehensive information on camel fat globules is not available. The only reference cited in the literature is the work of Knoess et al. (S), who analyzed camel milk of five different animals and found an average fat globule size diameter ranging from 2.31 to 3.93 J.1m. However, no details on the total particles counted were given, nor was the method used in the study disclosed.

Figure 2. Size distribution of fat globules in camel milk (â–،) and cow milk (â—ڈ). Values for cow milk are from Walstra et al. (12).

Journal of Dairy Science Vol. 74, No. 9, 1991

2904

FARAH AND RأœEGG

From the results of the present investigation, it can be concluded that natural creaming of camel milk differs markedly from that of cow milk. The rate of creaming of camel milk is very slow, both in raw and heated milk at refrigerator and room temperatures. The present study reveals no relationship between the average size distribution of fat globules and the observed poor creaming properties of camel milk. The results indicate that an insufficient quantity of agglutinin in camel milk may be responsible for the slow creaming rate. However, it is also possible that camel milk contains the protein required to cluster fat globules but that structural features of the protein are such as to preclude adsorption to the fat globules. Other factors, such as electrical charges on the globules, ionic distribution, and interfacial tension between milk sera and fat globules may also contribute to the poor creaming ability of camel milk.

REFERENCES 1 Abo-E1naga, I. G. 1966. Factors affecting creaming of cow's and buffalo's milk. Milchwissenschaft 21:429. 2 Abo-Elnaga, I. G., G. M. El-Sadek, and A. M. El-Sobry. 1966. Clustering of fat globules in cow's and buffalo's milk, creaming mechanism and physical arrangement of globules in gravity cream. Milchwissenschaft 21:210. 3 Dickson, H.R.P. 19S1. The Arab of the desert. George Allan and Unwin Ltd. London, Engl. 4 Farah, Z., T. Streiff, and M. R. Bachmann 1989. Manufacture and characterization of camel milk butter. Milchwissenschaft 44:412. S Knoess, K. H., M. R. Makhutum, and M. Hafeez. 1986. Milk production potential of the dromedary, with special reference to the Province of Punjab, Pakistan. World Anim. Rev. 56:11. 6 Mulder, H., and P. Walstra. 1974. Page 147 in The milk fat globule. Commonw. Agric. Bur., Farmham, Royal Buckinghamshire, Engl. 7 Parkash, S., and R. Jenness. 1968. The composition and characteristics of goat milk. Dairy Sci. Abstr. 30: 67. 8 Precht, D., and K. H. Peters. 1984. Pfropfenbildung in Schlagrahm bei verschiedenen Filtrierungsbedinugen. Milchwissenschaft 39:652. 9 Precht, D., K. H. Peters, and J. Petersen. 1987. Qualitأ¤tsverbesserung von Schlagsahne durch Zusatz von Carrageen und Milchinhaltsstoffen. n. Einfluss von Zusأ¤tzen auf die physikalischen Eigenschaften geschlagener Sahne. Milchwissenschaft 42:776. 10 Schneider, K., and H. Roder, 1979. Die praktische Milchprأ¼fung, die Kontrolle von Molkereiprodukten. 12. Auflage, Verlag Wyss, Bern. 11 Walstra, P., and R. Jenness. 1984. Dairy chemistry and physics. John Wiley & Sons, New York, NY. 12 Walstra, P., H. Oortwijn. and J. J. de Graaf. 1969. Studies on milk fat dispersions. I. Methods of determining globule-size distribution. Neth. Milk Dairy J. 23:12. 13 Wilson, R. T. 1984. The camel. Longman. London, Engl.

ACKNOWLEDGMENTS The authors express their thanks to C. N. Karue of the University of Nairobi for providing the camel milk samples and to M. Farah for her valuable technical assistance.

Journal of Dairy Science Vol. 74, No. 9, 1991

pdf

K. Ulmer and A. Fischer

85

6
Traditional slaughter, carcass dressing and processing of camels
K. Ulmer and A. Fischer

The following chapter uses Kenya as an example to describe the traditional way camels are slaughtered, deboned and processed. There is every likelihood that the slaughter, deboning and processing methods described can be adapted to other countries in Africa and Asia.

6.1

Locations for slaughter of camels
In rural areas facilities for slaughter are often non-existent or very simple. In most cases camels are slaughtered in the open air, on bare ground, without any roof to give protection from dust and sun. Simple scaffolds or trees are used to hang the carcasses up for processing. The place where camels are slaughtered in Isiolo, Kenya, can be cited as an example. Here camels are slaughtered in the open air, at a location which is quite separate from the slaughterhouse used for cattle. The smooth concrete floor is surrounded by a six-foot-high fence. The slaughter takes place on the ground, so the meat is soon contaminated with dust and dirt. Hanging racks are available for hanging up the cuts. There is also running water for cleaning, with a hole in the ground for drainage. Even in public slaughterhouses there are usually no technical facilities for carrying out the hygiene measures that are urgently needed. Inadequate energy and water supplies often make it difficult to clean and disinfect the slaughterhouse and equipment and dispose of offal and effluent. This means there is a very high risk of contamination of the meat. Adequate cold storage rooms are rarely available. This is why fresh meat is many times of poor quality and has a short shelf life in most developing countries.

86

Traditional slaughter, carcass dressing and processing of camels

Fig. 6.1: Place where camels are slaughtered in Isiolo, Kenya

6.2

The traditional method of slaughter for camels
The traditional slaughter of camels requires the meat to be â€‌halalâ€‌, in accordance with Islamic custom. In this type of slaughter the animals are not stunned. The camel is first put into a sitting position, the head is secured in a caudal position (i.e. turned towards the tail), and the main blood vessels between the neck and the thorax are severed with a single cut. Death occurs immediately. Although the Koran forbids consumption of blood, in some parts of Kenya the blood is collected and consumed. If the cut in the neck is not expertly executed, however, this traditional method of slaughter can be cruel and causes great suffering to the animal, since death then does not occur immediately – which is a strong argument for prior stunning of the animal. The camel’s skin is removed, starting from the backbone and going down both sides of the carcass to the belly. The skin is laid on the ground with the flesh side uppermost. The hump is then split lengthwise and removed. The shoulders are separated and the ribs are cut away from the vertebrae. Next, the gastrointestinal tract is removed. The backbone is then cut out, so that the carcass collapses in on itself. The hind legs are split in the pelvis and divided up into smaller cuts in the joints. Camels are usually slaughtered early in the morning, when the outside temperatures are relatively low, about 10آ؛C. In public slaughterhouses there is usually a post-mortem inspection for camels as well as cattle. After the post-mortem inspection the meat is taken away very quickly by dealers in special meat transport cases on handcarts or donkey-drawn carts.

K. Ulmer and A. Fischer

87

Fig. 6.2: Traditional slaughter of the onehumped camel (Field, 1995)

6.3

Traditional dressing of the carcass
In the traditional method of slaughter the carcass is already divided into cuts at the end of the slaughter procedure. As already mentioned in the previous chapter, after skinning the hump is first removed, then the shoulders are detached and the ribs are cut away from the spinal column. The spinal column is then removed, so that the carcass collapses in on itself. The hind legs are split in the pelvis and divided up into smaller cuts in the joints. There is no further dressing of the carcass. The meat is cut directly off the bone. There is no grading into high-quality and lower-quality cuts. This means that standardization is impossible and there is no guarantee of consistent quality by uniform grading of cuts of meat. The only sorting that takes place is into meat with or without bones, fat and offal.

Fig. 6.3: Traditional marketing of camel meat in rural areas of Kenya

88

Traditional slaughter, carcass dressing and processing of camels

6.4

Traditional meat products in Africa and Asia
The range of traditional product groups is fairly narrow. The traditional products in Africa and Asia are mainly dried products, which are made by a lowtechnology approach. Because of the climatic conditions and lack of cold storage facilities it is virtually impossible to keep meat or meat products fresh for any length of time. The drying of meat, taking advantage of existing natural factors such as temperature, humidity and air movement, is the oldest method of food preservation. Examples of typical dried meat products are biltong, odka, qwanta, kilishi and pastirma. For the first three of these the meat is cut into strips, then dry-salted or rubbed with a paste of spices and dried in the sun on straw mats. For odka and qwanta the meat is then heated in oil and dried in the sun again. So as to be able to keep the products for up to 12 months, they are covered with oil and stored in a closed container. To make kilishi, muscle meat is cut into slices which are dried for a short time in the sun. The slices are then dipped into a mixture of water, flour and various spices and dried in the sun again with this coating. Dried products are frequently smoked over a fireplace, to improve their flavor and microbiological stability. Another dried product is pastirma. Here the dry-salting process takes several days, since large pieces of meat are used. After the dry-salting, the meat is pressed for several days to remove water and give it an attractive shape. During the subsequent drying process, which usually takes place in the shade and involves air-drying, the pieces of meat are pressed again. The dried muscle meat is then coated with a paste made from water, salt, garlic, fenugreek seeds, paprika and mustard, and dried again. In spite of the simple procedures used to make traditional dried products, the seasonal weather conditions often make it difficult to achieve consistent quality. Thus, the products often vary widely in degree of drying and processing time, and this results in poorer microbiological stability and reduced keeping quality.

For more on: Method for hygienic slaughter of camels, Dressing of the camel carcass, Meat products from camel meat, read : “Milk and meat from camel: Hand book on products and processingâ€‌ Z. Farah and A. Fischer (Editors)




pdf

Journal of Dairy Research (1992) 59 229-231 Printed in Great Britain

229

Heat coagulation of camel milk
By ZAKARIA FARAH AND DEBORAH ATKINS*
Laboratorium fأ¼r Milchwissenschaft, Institut fأ¼r Lebensmittelwissenschaft, ETH-Zentrum, CH-8092 Zأ¼rich, Schweiz

* Ol Maisor Farm, PO Box 9, Rumuruti, Kenya
(Received: 30 July 1991 and accepted for publication 28 October 1991)

Camel milk is an important component of the human diet in many parts of the world. It contains all essential nutrients and the composition is similar to that of cows' milk (Yagil, 1982). Present knowledge about the milk production potential of camels (Oamelu8 dromedariu8) is very limited. Data available show, however, that a camel on good feed can produce 2000 I milk per lactation (Yagil, 1982), and even higher milk yields have been recorded (Knoess, 1980). Camel milk is drunk fresh or in the form of fermented milk. Heat processing, such as pasteurization and sterilization, as a means of preserving camel milk is unknown. Information on the heat stability of camel milk is therefore scarce. In an earlier study (Farah, 1986), camel milk was heated to 63, 80 and 90آ°C for 3o min and the distribution of N between the total protein, non-casein N and non-protein N fractions was determined. The whey proteins were also examined by PAGE. The camel milk whey protein showed generally higher heat stability than that from cows' mille In order to study the ability of camel milk to withstand higher processing temperatures, the heat coagulation time (HCT) was determined in the range 100-130آ°C and pH 6-:~-7'1, and compared with measurements on cows' milk.

EXPERIMENTAL Milk samples Camel milk samples were taken at Ol Maisor Camel Farm, which is situated just north of the equator in Kenya's Laikipia District at an altitude of between 1767 and 1889 m above sea level. The animals were of indigenous breed and were fed throughout the year exclusively by grazing. The milk samples were collected from ten individual camels. The pH of the ten samples and that of a pooled sample were determined. The milks were then kept refrigerated at 4آ°C and transported to our laboratory within 24 h. Upon arrival, the milk samples were skimmed and analysed. For comparison, bulk cows' milk from our Zurich laboratory was used.

Determination of heat stability Milk was adjusted to various pH in the range 6.3 - 7.1 by adding 0.1 M NaOH or 0آ·1 MHCI. HCT was determined in a thermostatically controlled oil bath at 100, 120 and 130آ°C according to the method of Davies & White (1966).

230

Z. FARAH AND D. ATKINS

Fig. I. Heat coagulation time-pH curves for (camel milk at â–،. 100آ°C; ∆. 120آ°C and O. 130 آ°C, and for cows' milk at 130آ°C (X).

RESULTS AND DISCUSSION The heat stability of milk can be defined in terms of the time required to induce coagulation at a given temperature. For bovine milk, the most widely used temperature for heat coagulation is 130 or 140آ°C. Preliminary experiments showed that in camel milk the HCT at 140آ°C was too short (< 1 min) for the present assay. Coagulation times were therefore determined at. 100, 120 and 130 آ°C. Fig. I shows the HCT-pH curves for pooled camel and cows' milks. All ten individual camel milk samples gave similar HCT-pH curves. The HCT-pH curve of cows' milk is in agreement with findings reported previously (Rose, 1963; Fox, 1982). It. showed a marked maximum around pH 6.7 and a minimum near pH 6.8. The heat stability increased above pH 6.9. The shape of the HCT-pH curve for camel milk at low temperature was different from those at high temperatures. The milks heated at. 130 and 120آ°C were very unstable at all pH and coagulated in 2-3 min. At 100 آ°C the HCT initially increased with pH, remained constant between pH 6.4 and 6.7 and then increased progressively with increasing pH. Milks from different species differ in their heat stability. Compositional differences and heat-induced interaction between the caseins and whey proteins, particularly خ؛-casein and خ²lactoglobulin, are reported to be responsible for these differences (Haynes & Fox, 1975; Fox & Hoynes, 1976; Ganguli, 1979). Casein fractions homologous with bovine a- and ft-casein were isolated and identified by PAGE and ion-exchange chromatography (Farah & Farah-Riesen, 1985; Larsson- Raإ؛nikiewicz & Mohamed, 1986). In these studies no protein fraction corresponding to K-casein could be clearly detected. It is possible that camel casein contained so little خ؛-casein that it escaped detection or was obscured by other casein fractions. On the other hand, foul' whey proteins have been isolated from camel milk: two proteins similar to serum albumin and خ±-lactalbumin, and two novel milk proteins of no structural similarity to other milk proteins. The evidence for the presence of خ²-lactoglobulin in camel milk is conflicting (Farah, 1986; Beg et al. 1984, 1987).

Heat coagulation of camel milk

231

The present study found that the heat stability of camel milk differs markedly from that of cows' milk خ؛-Casein and خ²-lactoglobulin play an important role in the stability of bovine milk. Therefore, the absence or deficiency of these two proteins in camel milk might be a cause of its poor stability at high temperatures. However, this remains to be confirmed.

REFERENCES
BEG, O. U. VON BAHR-LINDSTRأ–M, H. ZAIDI, Z.H. & Jأ–RNVALL, H. 1987 Characterisation of a heterogeneous camel milk whey non-casein protein. FEBS Letters 216 270-274 DAVIES. T. & WIIITE, J. C. D. 1 966 The stability of milk protein to heat. I. Subjective measurement of heat stability of milk. Journal of Dairy Research 33 6i-81 FARAH, Z. 1986 Effect of heat treatment on whey proteins of camel milk. Milchwissenschaft 41 763-765 FARAH, Z. & FARAH-RIESEN, M. 1985 Separation and characterization of major components of camel milk casein. Milchwissen8chaft 40 66B-6i 1 Fox, P. F. 1982 Heat-induced coagulation of milk. In Developments in Dairy Chemistry-l Proteins pp. 189-228 (Ed. P. F. Fox). London: Applied Science Publishers Fox, P. F. & Haynes, M. C. T. 1976 Heat stability characteristics of ovine, caprine and equine milks. Journal of Dairy Research 43 433-442 GANGULI, N. C. 1979 Stability of buffalo casein micelles. Journal of Dairy Research 46 401-405 Hoynes,M. C. T. & Fox. P. F. 1975 Some physico-chemical properties of porcine milk. Journal of Dairy Research 42 4:3-.56 KNOESS, K. H. 1H80 Milk production of the dromedary. In Camels pp. 201-214. International Foundation for Science Provisional Report No. (j LARSSON-RAZNIKIEWICZ, M. & MOHAMED, M. A. 1986 (Analysis of the casein content, in camel (Camelus dromedarius) milk. Swedish Journal of Agricultural Research 16 13-18 ROSE, D. 1963 Heat stability of bovine milk: a review. Dairy Science Ab8tracts 25 45-52 Y AGIL, R. 1982 Camels and camel milk. Rome: Food and Agriculture Organization (FA 0 Animal Production and Health Paper No. 26 pp. 14-19) BEG, O. D., Von BAHR-LINDSTROM, H., ZAIDI, Z. H. & Jأ–RNVALL, H. 1984 A small camel-milk protein rich in cysteine/half-cystine. Bioscience Reports 4 1065-1070

pdf

J. Dairy Sci. 87:2660–2668  American Dairy Science Association, 2004.

Expression of the Peptidoglycan Recognition Protein, PGRP, in the Lactating Mammary Gland
S. R. Kappeler, C. Heuberger, Z. Farah, and Z. Puhan
Laboratory of Dairy Science, Institute of Food Science, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland

ABSTRACT The peptidoglycan recognition protein, PGRP, known as an intracellular component of neutrophils, has been isolated from camel (Camelus dromedarius) milk by acid precipitation followed by heparin-sepharose afï¬پnity chromatography of the supernatant. The mean concentration in milk was about 120 mg/L. It decreased during lactation by 19% and increased in the event of severe mastitis by 45%. The protein bound to lactic acid bacteria and other gram-positive bacteria with an afï¬پnity similar to that reported for the human and murine orthologs, although the isoelectric point of the molecule was distinctly higher at pH 9.02. The N-terminus of mature camel PGRP was determined as NH2ArgGluAspProPro-CO2H. Calculated and measured molecular masses were both 19.1 kDa, excluding the possibility of posttranslational modifcation or binding of cation ligands. The peptide probably builds a homotrimer at high concentration. The corresponding mRNA was isolated from lactating mammary gland tissue, and 5.3 kbp of the corresponding gene was sequenced. Similarities were found to the camel lactoferrin gene with regard to sites of expression and to the region 5′ upstream to the gene. (Key words: camel milk, lactating mammary gland, peptidoglycan recognition protein) Abbreviation key: MALDI-MS = matrix-assisted laser desorption/ionization mass spectrometry, pglyrp = gene for the peptidoglycan recognition protein (short pglyrp is synonymous to murine tumor-associated gene 7 and human tnfsf3l), PGRP = peptidoglycan recognition protein (the short variant is synonymous to murine tumor-associated gene 7 and bovine oligosaccharidebinding protein). INTRODUCTION The ability of milk to protect the offspring against various diseases is based on factors that belong primar-

Received October 6, 2003. Accepted March 27, 2004. Corresponding author: S. R. Kappeler; e-mail: stefan.kappeler@ alumni.ethz.ch.

ily to the innate and adaptive immune systems. The cellular and humoral components of the latter system, such as leukocytes, immunoglobulins, and the complement system, are selectively imported from serum by intracellular routing (Praetor et al., 1999). On the other hand, the components of the ï¬پrst line host defense are mainly synthesized in the lactating mammary gland, conferring antimicrobial protection to the newborn. Among these factors, pattern-recognition proteins, such as lactoferrin, which binds to lipopolysaccharides of gram-negative bacteria, and lysozyme C, which binds and hydrolyzes peptidoglycans, preferably of gram-positive bacteria, exhibit antimicrobial, immunomodulating, and antiinflammatory activities, and are synthesized in the lobuloalveolar tissue of the lactating mammary gland. Lysozyme C, a 1,4-خ²-N-acetylmuramidase closely related to خ±-lactalbumin, is expressed in the functionally differentiated, lactating epithelium, in contrast to lactoferrin, which is predominantly expressed in developing, resting, and regressing tissue (Molenaar et al., 1992). Short peptidoglycan recognition protein (PGRP) is a soluble, conserved pattern-recognition protein of vertebrates and invertebrates that binds to bacterial peptidoglycan with similar speciï¬پcity, but with a higher afï¬پnity than lysozyme C (Liu et al., 2000). It has been previously detected in different mammalian tissues, such as spleen and lung (Kustikova et al., 1996), but it has not yet been isolated from milk. Expression of the gene for short peptidoglycan recognition protein (pglyrp) has been conversely discussed; the expression of the human, bovine, and murine variants exclusively in polymorphonuclear leukocytes and bone marrow stem cells, accompanied by intracellular storage in cytoplasmic granules, was demonstrated (Liu et al., 2000; Tydell et al., 2002). Low levels of pglyrp expression have been detected in different human and murine tissues (Kang et al., 1998; Liu et al., 2001). The corresponding cDNA has ï¬پrst been cloned from 2 mammary adenocarcinoma cell lines (Kustikova et al., 1996), and a majority of murine expressed sequence tag entries related to pglyrp in GenBank are derived from mammary gland tumors. The 19 kDa peptide is basic, monomeric, and probably aggregates to homo-trimers at higher concen-

2660

PGRP IN CAMEL MILK

2661

trations (Fornhem et al., 1996; Kiselev et al., 1998; Liu et al., 2000). Speciï¬پc binding with nanomolar afï¬پnity to polymeric, noncrosslinked peptidoglycans was shown for murine PGRP (Liu et al., 2000). The molecule showed weak or no afï¬پnity to other microbial polysaccharides or to peptidoglycans of low molecular weight (Yoshida et al., 1996; Liu et al., 2000). A bacteriostatic activity toward gram-positive bacteria was reported, without any bacteriolytic or otherwise bactericidal effect, and an antiinflammatory modulation of the immune response was observed. However, in a more recent report (Tydell et al., 2002), a microbicidal effect of bovine PGRP was observed, and the protein was also effective against gram-negative bacteria and a fungal pathogen. Our interest was to characterize camel PGRP and its binding to lactic acid bacteria and pathogenic bacteria. The regulation of pglyrp expression in the lactating mammary gland of camels is furthermore correlated with data from 68 individual milk samples, with regard to health status, incidence of bacterial infection, and lactational stage of the animals. MATERIALS AND METHODS Milk Sample Collection A total of 68 samples of camel milk were collected at 2 breeding stations in Isiolo and Rumuruti in Kenya. Milk from the four quarters of the camel udders were collected separately and examined for total SCC (California mastitis test) and for bacterial growth on blood agar and Edwards agar, and in Todd-Hewitt broth. The lactational stage of the camels was between 2 and 13 mo. Additionally, 5 porcine mastitis-negative milk samples were collected on a local farmyard on the day of parturition and 3 wk thereafter. Pooled human and bovine mastitis-free raw milk samples from midlactation were obtained from a women’s hospital in Zurich and from a Swiss brown cattle farm near Zurich. The milk was centrifuged at 10,000 أ— g and at 4آ°C for 30 min, and ï¬پltered through nylon mesh. The skimmed milk was acidiï¬پed by addition of 1% (vol/vol) acetic acid and incubated at 37آ°C for 10 min. Following, it was neutralized with 1/10 vol. of 1 M sodium acetate, and centrifuged at 10,000 أ— g and at 4آ°C for 5 min. The supernatant, containing soluble whey proteins, was ï¬پltered through a 0.45-آµm screen. Heparin-Sepharose Afï¬پnity Chromatography Soluble whey proteins (10 mL) were loaded on a 1mL Heparin-Sepharose HiTrap column (GE Healthcare Bio-Sciences, Little Chalfont, UK). The column was washed with 20 mL of 0.01 M sodium phosphate and

0.1 M sodium chloride at pH 7.4. Elution was performed at ambient temperature applying a linear gradient from 0.1 to 0.7 M sodium chloride over 37 min. The column effluent was monitored at 280 nm. The collected fractions were separated by 4 to 12% gradient SDS-PAGE, and proteins were detected by Coomassie staining. An additional puriï¬پcation step was carried out, prior to micro-sequencing and molecular mass determination, by reversed-phase HPLC on an analytical silica-coated C18-column (5 آµm, 250 أ— 4.6 mm). Elution was performed at ambient temperature by applying a linear gradient from 0.1% trifluoroacetic acid in double-distilled, nanoï¬پltered water to 0.1% trifluoroacetic acid in acetonitrile, at a flow rate of 1 mL/min over 60 min. Amino Acid N-Terminal Sequence Analysis Protein (0.5 nmol) collected from the effluent of the C18-column was used directly for N-terminal sequencing. Eluted proteins were checked for purity by SDSPAGE, applied on a trifluoroacetic acid-treated polyvinylidene-fluoride cartridge ï¬پlter and dried under continuous nitrogen flow. Automated Edman degradation over 35 cycles was performed using an ABI 471A sequencer (Applied Biosystems, Foster City, CA) equipped with a 120A HPLC for online reversed-phase C18-HPLC analysis of phenylthiohydantoinyl AA derivatives. Mass Determination of PGRP The molecular mass of camel PGRP was determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Protein from the effluent of the C18-column was co-crystallized with an equal volume amount of خ±-cyano-4-hydroxy-cinnamic acid (5 g/L) in 0.2% trifluoroacetic acid. Co-crystallized PGRP (3 pmol) was applied to the target, and air dried at ambient temperature. A time-of-flight mass spectrometer in linear mode was used (Voyager Elite, Applied Biosystems). Spectra were recorded with a nitrogen ultraviolet laser at 337.1 nm and an acceleration voltage of 25 kV. The instrument was calibrated with porcine myoglobin (monomeric, 16.953 kDa). Quantiï¬پcation The volumes V of the ï¬پrst and the third fraction collected from heparin-sepharose chromatographic runs of camel soluble whey proteins were determined (mL). Absorptions at 280 nm were measured, and the PGRP and lactoferrin concentrations C (mg/L) in the respective milk samples were calculated as A280 أ— Mr أ— خµâˆ’1 أ— 10 أ— V−1 using extinction coefï¬پcients خµ of 39,050
Journal of Dairy Science Vol. 87, No. 8, 2004

2662

KAPPELER ET AL.

M−1 ط’cm−1 for mature PGRP, and of 84,540 M−1ط’cm−1 for mature lactoferrin, as determined from the primary sequence of the mature protein. The milk samples were classiï¬پed with regard to lactational stage, udder health, and bacterial infections. Average concentrations and standard errors thereof were calculated for the classes built. Correlation analyses were done according to Pearson for the comparison of quantitative values and according to Spearman for the comparison of quantitative vs. qualitative values. cDNA Sequence Analysis Udder tissue of a lactating camel, Somali breed, was used for polyA-mRNA isolation (Oligotex Direct mRNA Kit, Qiagen GmbH, Hilden, Germany). The tissue was prepared immediately after biopsy. A sample of mRNA (2 آµg) was used for cDNA synthesis with the Universal RiboClone cDNA Synthesis System (Promega Corp., Madison, WI) with an oligo(dT)15 primer and EcoR I adapters. One-ï¬پfth of the resulting cDNA was ligated to 1 آµg of dephosphorylated خ»-gt11 arms. The ligated DNA was packaged in vitro with an Escherichia coli C Packagene خ» DNA extract (Promega). All work was done according to the manufacturer’s instructions. Phages were plated on E. coli LE 392 (Promega). The library titer was estimated at 2.6 أ— 105 pfu/mL. A volume of 100 آµL was ampliï¬پed and produced a lysate with a titer of 1 أ— 108 pfu/mL. Speciï¬پc DIG-11-dUTP-labeled probes (Roche) were PCR-ampliï¬پed and used for library screening by nucleic acid hybridization of plaque lifts. A degenerated oligonucleotide primer (IUB standard) 5′-CCCGCCTGCGGTTCNA THGTNCC-3′ was synthesized using the information from N-terminal sequencing. The Swiss-Prot database (www.expasy.org) was searched for entries similar to the sequenced N-terminus. The greatest similarity was found with entry Q62185, which encodes the murine tumor-associated protein TAG7, which is the murine ortholog of short PGRP. The corresponding GenBank/ EBI Data Bank entry X86374 was used for synthesis of the reverse oligonucleotide primer 5′-TGATGTTCC AGCCTCGGCCTTCAT-3′. The short PCR product obtained was used for identiï¬پcation of a plaque. The excised insert was ligated into a pBR322-derived plasmid and sequenced. Genomic Sequence Analysis A 3.3-kbp genomic sequence encompassing the region from exon I to exon III of the camel pglyrp gene was PCR ampliï¬پed twice using the exon I speciï¬پc oligonucleotide primer 5′-GTGCGCTACGTGGTGGTGTCGCACAC-3′ and the exon III speciï¬پc oligonucleotide primer 5′-GC
Journal of Dairy Science Vol. 87, No. 8, 2004

CACACCACAAGCCAGCAGATTCTG-3′, low-sheared genomic DNA from white blood cells of an Arabian camel and PfuTurbo DNA polymerase (Stratagene). The products were ligated into a pBR322-derived plasmid and sequenced. For sequence determination of the region 5′ upstream to the transcription start site, the same genomic DNA was completely digested with DraI. A GenomeWalker library (K1807-1; BD Biosciences, San Jose, CA) was constructed and the sequence speciï¬پc primers 5′-GAG GGCGAGGAGAGCCCAGACGAGAAGCAC-3′ and 5′AGCACGCAGTGCCGGGTCATGGCAGGCAGA-3′ were designed based on information from exon I sequenced previously. A 1.7-kbp band was obtained by nested PCR, using PfuTurbo DNA polymerase and Perfect Match PCR enhancer (Stratagene, La Jolla, CA). A TESS (Transcription Element Search System) (Schug and Overton, 1997) search for potential transcription factor binding sites was done with the TRANSFAC database (version 3.3; Wingender et al., 2000), and the following parameters were used: no allowable mismatch, a minimum element length of 6 bases and a minimum log likelihood of 6. The reliability of transcription factor sites found was 31%, calculated as follows: (خ£nP − خ£nR)/خ£nP, with خ£nP as the number of sites found per base pair of 5′-flanking sequence, and خ£nR as the number of sites found per base pair in 400,000 bp of randomly generated DNA-sequence.

Bacterial Binding Assay The lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii, spp. bulgaricus, the yeast Kluyveromyces marxianus, the lysozyme-sensitive strain Micrococcus luteus DSM 20030, the gramnegative enterobacteriaceae, Enterobacter agglomerans, Escherichia coli, and Staphylococcus aureus were used to study the binding of camel PGRP to bacteria. Inoculated cultures were harvested in the exponential phase at OD600 = 0.4, and washed twice with 10 mM phosphate, 20 mM NaCl (pH = 7.4). Between 20 and 120 آµL of culture was centrifuged and resuspended in 40 آµL of the PBS containing 0.8 آµg of PGRP and then incubated for 30 min at room temperature. The bacteria were centrifuged at 4,000 أ— g, 4آ°C, for 10 min, and the absorption of the supernatant was determined at 280 nm. The bacterial pellet was resuspended in 40 آµL of 10 mM phosphate, 1 M NaCl (pH = 7.4) to dissociate bound PGRP from the bacteria. The solution was centrifuged again and the absorption of the supernatant measured as before.

PGRP IN CAMEL MILK

2663

Figure 1. Isolation of the peptidoglycan recognition protein (PGRP) by heparin-sepharose chromatography. A) Elution proï¬پles of camel (black line) and bovine (gray line) whey heparin-binding proteins; B) porcine (black line) and human (gray line) whey. Fractions were collected and separated by SDS-PAGE. a) camel fractions I–III, b) bovine fractions I–V, c) porcine fractions I–III ( Ib corresponds to fraction I, but from porcine colostral milk), and d) human fractions I–III.

RESULTS Isolation of PGRP from Camel Milk The protein was eluted from heparin-sepharose at 290 mM NaCl (Figure 1a, fraction I). The second large peak obtained was at 640 mM NaCl and contained lactoferrin (Figure 1a, fraction III). A protein with characteristics similar to those of PGRP was not isolated from bovine or human milk, and is not described in the literature as a milk component. A band with an apparent molecular weight of 19 kDa was eluted at the same ionic strength when the column was loaded with porcine whey, and presumably corresponded to porcine PGRP (Fornhem et al., 1996). The band was more intense when colostral porcine milk was used for chromatography (Figure 1c, fraction Ib). Camel whey fraction II, which was eluted at about 400 mM NaCl, contained a

protein of about 59 kDa with an unknown N-terminal sequence NH2-NRLVG-CO2H. A band of the same size was also present in fraction IV of bovine whey. cDNA and Peptide Analysis The N-terminal sequence of the reverse-phase puriï¬پed camel protein was determined as NH2-REDPP AXGSI VPRRE WRALX SEERE RLTTP VRYPV-CO2H and was similar to the predicted N-terminal sequence of human short PGRP (Kang et al., 1998). A cDNA clone of approximately 700 bp that corresponded to the Nterminal sequence was obtained from a cDNA library prepared from C. dromedarius lactating mammary gland tissue (Panel B of Figure 2; GenBank/EBI Acc. No. AJ131676). The 5′-untranslated region contained a partial Kozak box (Kozak, 1989) in front of the start
Journal of Dairy Science Vol. 87, No. 8, 2004

2664

KAPPELER ET AL.

Figure 2. A) 5′-flanking region of the camel gene for the peptidoglycan recognition protein (pglyrp). Possible binding sites of transcription factors known to control the expression of milk protein genes are underlined (not presenting glucocorticoid half sites): STAT5 = signal transducer and activator of transcription 5; NF-1 = nuclear factor 1; C/EBP = CAAT/enhancer-binding protein; TBP = TATA-binding protein; TFIID = Trans-acting regulatory factor IID. B) pglyrp cDNA sequence. Deduced peptide in bold, with periplasmic leader sequence in italics. The open reading frame of the cDNA sequence extended from A43 to A621, preceded by a partial Kozak-box (underlined). Polyadenylation signal in italics from A696 to A701. Numbering of the amino acid sequence begins with the ï¬پrst residue of the mature protein. Residues underlined are conserved in camel, murine, silkworm, and human short PGRP, human long PGRP, and T3 lysozyme.

site of protein translation. The open-reading frame coded for a peptide of 193 AA residues with a calculated molecular mass of 21.377 kDa. The combination of protein and cDNA sequencing data allowed for the prediction of the primary sequence of mature camel PGRP as a peptide 172 AA residues in length. The calculated molecular mass of 19.143 kDa was conï¬پrmed by a molecular mass of 19.117 آ± 0.05 kDa, as measured by MALDI-MS. Both masses were identical within measuring error, excluding the possibility of posttranslational translation (e.g., by glycosylation or by phosphorylation, or ligand binding, such as Zn2+, which is required for amidase activity of the structurally related T3 and T7 lysozymes). The Ami_2 domain, shared with the T3/T7 lysozymes and several other proteins (Letunic et al., 2002), extended from residues 30 to 156. Sequence identity of the mature peptide was 79.4% to bovine short PGRP, 75.4% to human short PGRP, 75.2% to murine short PGRP, 37.8% to silkworm PGRP, 29.7%
Journal of Dairy Science Vol. 87, No. 8, 2004

to T3 lysozyme, and 38.6% to the C-terminal domain of the long variant of human PGRP. In all of these proteins, 19 residues were conserved, most of them small and hydrophobic. The calculated isoelectric point of camel PGRP was at pH 9.02, and thus distinctly higher than the calculated isoelectric points of the human and murine homologues, which were at pH 8.24 and 7.22, respectively, but lower than porcine and bovine PGRP, which had isoelectric points higher than pH 10.5 (Fornhem et al., 1996) and 9.38 (Tydell et al., 2002). The basic nature of the protein was mostly due to a high content of arginine, but not of lysine. The arginine residues were weakly conserved between mammalian PGRP homologues. Conserved basic residues were only histidine residues His61, His94, and His147. The afï¬پnity to bacteria and heparin was completely abrogated when we partially protected the free amino groups. We partially sequenced the cDNA to porcine pglyrp (GenBank/EBI Data Bank Acc. No.

PGRP IN CAMEL MILK

2665

Table 1. Background-corrected frequencies of the most prominent TF sites per 1000 bp of 5′-flanking sequence, (calculated as described in Kappeler et al., 2003). Short PGRP Camel 7.8 5.3 5.1 3.4 1.7 1.7 1.1 1.0 0.6 0.6 0.3 0.1 Human Murine 5.9 2.8 7.0 1.5 6.0 1.7 0.5 3.5 2.2 1.4 1.8 0.1 1.5 2.3 1.2 0.6 2.2 0.1 1.2 0.8 0.3 Long PGRP Human 5.3 5.9 5.7 0.2 Camel 3.6 8.6 4.4 0.8 0.1 Lactoferrin Murine 2.8 0.9 1.3 0.8 0.4 1.7 1.1 0.4 1.6 0.1 0.6 Porcine 4.9 3.7 6.6 1.2 0.8 0.9 0.2 Lysozyme C Bovine 10.3 2.7 2.3 2.3 4.8 1.9 4.7 0.7 4.9 Porcine 5.9 0.3 0.2 4.8 1.7 1.1 4.8 2.2 0.7 2.4

Species Gene product Factor Glucocorticoid receptor Activator protein 2 Sp1 c-Ets-2 TBP C/EBP beta Mammary activating factor TFIID Nuclear factor 1 Nuclear factor خ؛B Pituitary gland speciï¬پc factor 1a Yin and Yang 1 repressor Octamer DNA-binding protein
1

1 Sp1 = Simian virus 40n protein 1; TBP = TATA-binding protein; C/EBP = CAAT/enhancer-binding protein; TFIID = trans-acting regulatory factor IID.

AJ310355) using PCR primers deduced from the camel cDNA sequence. This sequence was 84% identical to the corresponding camel sequence, and the translated peptide sequence was 82% identical. All residues, which were highly conserved amongst mammalian PGRP proteins (Tydell et al., 2002), were also found in the porcine sequence. Genetic Organization and 5′-Flanking Sequence The genetic organization of the camel pglyrp gene was similar to the human short pglyrp gene, with 2 intron sequences separating 3 exon sequences (Panel A of Figure 2). The ï¬پrst intron in both genes was considerably longer than the corresponding second intron of the silkworm ortholog (2606 vs. 188 bp), due to a high frequency of interspersed elements, mainly of the short mammalian interspersed repetitive element and the long L1 and L2 types. Analysis of the 1598-bp 5′ flanking to camel pglyrp revealed a housekeeping gene type structure, with a weak TATA-box promoter sequence and a site potential for transcription factors similar to that of the corresponding region of camel lactoferrin (Table 1). A high frequency of sites for the glucocorticoid receptor, which is essential for gene expression in the lactating mammary gland (Rosen et al., 1999), was found in the 5′-flanking regions to the camel and murine gene, but not in the corresponding region to the human gene. A STAT5 site, which is indispensable for induction of gene transcription in the mammary gland by prolactin was only found in the 5′ region adjacent to camel pglyrp (Panel A of Figure 2). All sequences, especially the one 5′ adjacent to camel pglyrp, had a high probability to bind members of the erythroblast trans-

formation speciï¬پc family of transcription factors, which probably effect the insulin signal. The transcription factors presented underneath the intercepting line in Table 1 had a lower frequency of occurrence in the 5′flanking sequence to camel pglyrp, but have been discussed to be involved in the regulation of gene expression in the lactating udder or in the regulation of pglyrp expression in other species. Camel pglyrp Expression and Regulation Our interest was to ï¬پnd whether the expression of pglyrp in the lactating mammary gland was influenced by the health status of the udder or by the lactational stage of the mammary gland. We therefore collected and analyzed the milk of the 4 mammary quarters of each udder separately. The pathological status of 68 quarters from 17 lactating camels were recorded, the milk was tested on growth of different pathogenic strains and the total number of leukocytes and alveolar
Table 2. Number of microbial infections and elevated cell counts. Item Individual udders examined Quarter samples tested Streptococcus infection Staphylococcus infection Enterobacter infection Micrococcus infection Subclinical mastitis Elevated number of cells Inflammation of udder quarter N 17 68 6 6 1 1 14 9a 16a

a In 4 cases of quarter inflammation and elevated cell number, a bacterial infection was not found; another source of infection is therefore likely.

Journal of Dairy Science Vol. 87, No. 8, 2004

2666

KAPPELER ET AL.

Table 3. Expression levels of the peptidoglycan recognition protein gene (pglyrp) and the lactoferrin gene compared at different lactational stages. Lactational period Early lactation (1–4 mo) Mid lactation (5–8 mo) Late lactation (9–13 mo) N 4 19 3 PGRP [mg/L] 130 آ± 24 111 آ± 7 105 آ± 7 Lactoferrin [mg/L] 68 آ± 14 91 آ± 9 114 آ± 17

ferrin concentration increased more pronouncedly in infected milk than the PGRP concentration. This was also observed in cases of inflammation without evidence of bacterial origin. Four samples of irritated tissue without elevated cell counts showed a strong increase in the lactoferrin concentration, but only a slight increase in the PGRP concentration. Binding to Lactic Acid Bacteria

epithelial cells was counted (Table 2). In many camels, some quarters of the udder were seriously infected, whereas neighboring quarters remained healthy. The cell number was elevated in milk contaminated with nonbacterial pathogens and Streptococcus, but not in milk contaminated with other types of bacteria. Altogether, severe mastitis was diagnosed in 13.2% of cases, subclinical mastitis in 20.6%, and a quarter inflammation, as judged by a veterinarian, was diagnosed in 23.5%. Furthermore, PGRP and lactoferrin from each quarter were separated by heparin-sepharose afï¬پnity chromatography and quantiï¬پed. Mean values of milk from pathologically healthy and mastitis-free quarters of different lactational stages are presented in Table 3. The concentration of PGRP insigniï¬پcantly decreased over the course of lactation. On the other hand, the concentration of camel lactoferrin increased by about 70% toward the end of lactation (Pearson r = 0.433; P < 0.001). A positive correlation with the severity of mastitis was observed for the concentrations of both proteins in milk (Table 4; Spearman r = 0.500; P < 0.0001 for PGRP, r = 0.547; P < 0.0001 for lactoferrin). The assessed quarter inflammation also correlated positively with both protein concentrations (Spearman r = 0.367; P < 0.005 for PGRP, r = 0.496; P < 0.0001 for lactoferrin). An increase of both concentrations was also found in the case of Streptococcus infections (Spearman r = 0.535; P < 0.0001 for PGRP, r = 0.532; P < 0.0001 for lactoferrin), in contrast to infections from other bacteria. The lacto-

A bacterial binding assay was carried out to determine the afï¬پnity of PGRP to lactic acid bacteria and to pathogenic bacteria often associated with clinical mastitis. Strong binding of PGRP was found to M. luteus (Figure 3, panel A). The afï¬پnity to lactic acid bacteria and to Staph. aureus was weaker. At OD600 = 0.8, Staph. aureus bound 22% of the PGRP supplied, corresponding to about one-third of the binding capacity of M. luteus. No binding afï¬پnity was found to gram-negative bacteria and the yeast K. marxianus. The relationship found between bound and free PGRP in the assay was linear. DISCUSSION We used heparin-sepharose chromatography to separate antimicrobial components of camel milk. We were able to isolate PGRP, a protein not yet described as a milk constituent, but did not detect lysozyme C, either on the protein or at the mRNA level, although this protein is present in human and bovine milk. Porcine milk probably contained both PGRP and lysozyme C, as concluded from SDS-PAGE results. Antimicrobial proteins in milk most likely have a primary function in the protection of the lactating mammary gland, but may also be required to build up a functioning immune system in the newborn (Goldman, 2002). The extreme habitat and body metabolism of camels may have brought about a different immunological answer to pathogenic challenges compared with other mammals. A peculiarity, for example, is the expression of singlechain Ig. This hypothesis may also explain the absence of lysozyme in camel milk. The regulation of antimicrobial gene expression in the lactating mammary gland depends on the primary function of the protein in milk. Lactoperoxidase, which serves as a bactericidal enzyme in milk, is rapidly downregulated in human and camel milk after parturition, but continuously expressed in bovine milk (unpublished data). The concentration of lactoferrin, on the other hand, is upregulated during lactation, changing its function as an iron-depleted, iron-scavenging agent in colostral milk toward an iron-saturated protein, which is bactericidal through an N-terminal proteolytic cleavage product (Kappeler et al., 1999). In camel milk,

Table 4. Expression levels of the peptidoglycan recognition protein gene (pglyrp) and the lactoferrin gene compared for different pathological ï¬پndings. Pathological status of the udder N PGRP [mg/L] 107 118 155 156 110 135 87 121 147 آ± آ± آ± آ± آ± آ± آ± آ± آ± 5 6 17 16 15 135 87 11 13 Lactoferrin [mg/L] 95 109 234 237 105 99 73 156 211 آ± آ± آ± آ± آ± آ± آ± آ± آ± 7 5 49 47 8 99 73 47 38

No detectable inflammation 29 Mild inflammation 4 Severe inflammation 6 Streptococcus infection 6 Staphylococcus infection 6 Enterobacter infection 1 Micrococcus infection 1 Inflammation of nonbacterial origin 4 Elevated cell number 9

Journal of Dairy Science Vol. 87, No. 8, 2004

PGRP IN CAMEL MILK

2667

Figure 3. Camel milk peptidoglycan recognition protein (PGRP) binds to gram-positive lactic acid bacteria and pathogenic bacteria. A) Different bacterial strains and the yeast Kluyveromyces marxianus were tested on their ability to bind PGRP in low-salt (20 mM) buffer. Numbers below the illustrations indicate the OD600 values of the assays. The protein was released by increasing the salt concentration to 1 M. Strong binding was found to the lysozyme-sensitive Micrococcus luteus. Other gram-positive bacteria exhibited about half of the binding capacity of M. luteus. Gram-negative bacteria and K. marxianus did not bind PGRP. B) Binding of 20 آµg/mL PGRP to varying amounts of M. luteus. The graph shows a linear decrease of unbound protein (∆) in the low-salt supernatant and a proportional increase of bound protein (C) in the high-salt supernatant, when the bacterial concentration was elevated. The total of both values (G) was about 80% of the protein added (x).

PGRP was found to be upregulated in cases of mastitis concomitantly with lactoferrin, pointing to a function in udder protection. Furthermore, the higher protein level in early lactation indicates a role in the passive immunity of the newborn. Camel PGRP avidly bound to lactic acid bacteria, and the protein was markedly upregulated in the case of streptococcal infection, but not in other instances of mastitis. It is therefore suggested that the main function of the protein in milk is to control lactic acid bacteria. No afï¬پnity was observed toward gram-negative bacteria and the yeast K. marxianus, and a binding speciï¬پcity similar to that for murine PGRP was found (Liu et al., 2000). Altogether, our ï¬پndings indicate a host defense function only in relation with gram-positive bacteria. In contrast, a microbicidal activity against gram-negative bacteria and even yeast has been reported for the bovine ortholog (Tydell et al., 2002). The porcine PGRP was originally isolated from nematodeinfected pigs (Fornhem et al., 1996), and a ï¬پve- to sevenfold increase was reported for murine PGRP after lipopolysaccharide stimulation (Kiselev et al., 1998), indicating stimulation by and activity against several invaders. We therefore suggest that mammalian PGRP alters its target speciï¬پcity under appropriate conditions, maybe by formation of heteromeric complexes with other components, as suggested for D. melanogaster PGRP long variant C (Choe et al., 2002). It is also possible that mammalian PGRP is able to transmit a pathogenic signal to the immune system similar to the closely related D. melanogaster PGRP short variant A (Michel et al., 2001). An intriguing characteristic of the bovine ortholog is the formation of a basic bactericidal oligopeptide, Cterminal to a highly conserved leucine residue (Tydell et al., 2002). Similar basic peptides are expressed by the invertebrate immune system after transduction of the pathogenic signal by different PGRP variants (Michel et al., 2001; Choe et al., 2002; Gottar et al., 2002; Ramet et al., 2002). In milk, a direct response to a pathogenic challenge is required, and it is therefore likely that a corresponding cleavage product is also formed from camel PGRP. The PGRP in camel ripe and porcine colostral milk is a 19-kDa monomeric protein. The protein probably aggregates to multimeric complexes, as proposed for murine short PGRP, because we always found a faint band with an apparent molecular weight of 48 to 50 kDa after SDS-PAGE analysis, even when we puriï¬پed fraction I from afï¬پnity chromatography by reversedphase HPLC. The intensity of the band changed proportionally to the intensity of the 19-kDa band (Figure 1a, fraction I). Furthermore, we did not detect the binding of a divalent ligand, as found in the related phage lysoJournal of Dairy Science Vol. 87, No. 8, 2004

2668

KAPPELER ET AL. Gottar, M., V. Gobert, T. Michel, M. Belvin, G. Duyk, J. A. Hoffmann, D. Ferrandon, and J. Royet. 2002. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416:640–644. Kang, D., G. Liu, A. Lundstrom, E. Gelius, and H. Steiner. 1998. A آ¨ peptidoglycan recognition protein in innate immunity conserved from insects to humans. Proc. Natl. Acad. Sci. USA 95:10078– 10082. Kappeler, S. R., M. Ackermann, Z. Farah, and Z. Puhan. 1999. Sequence analysis of camel (Camelus dromedarius) lactoferrin. Int. Dairy J. 9:481–486. Kappeler, S. R., Z. Farah, and Z. Puhan. 2003. 5′-flanking regions of camel milk genes are highly similar to homologue regions of other species and can be divided into two distinct groups. J. Dairy Sci. 86:498–508. Kiselev, S. L., O. S. Kustikova, E. V. Korobko, E. B. Prokhortchouk, A. A. Kabishev, E. M. Lukanidin, and G. P. Georgiev. 1998. Molecular cloning and characterization of the mouse tag7 gene encoding a novel cytokine. J. Biol. Chem. 273:18633–18639. Kozak, M. 1989. The scanning model for translation: An update. J. Cell Biol. 108:229–241. Kustikova, O. S., S. L. Kiselev, O. R. Borodulina, V. M. Senin, A. V. Afanas’eva, and A. A. Kabishev. 1996. Cloning of the tag7 gene expressed in metastatic mouse tumors. Russ. J. Gen. 32:540–546. Letunic, I., L. Goodstadt, N. J. Dickens, T. Doerks, J. Schultz, R. Mott, F. Ciccarelli, R. R. Copley, C. P. Ponting, and P. Bork. 2002. Recent improvements to the SMART domain-based sequence annotation resource. Nucleic Acids Res. 30:242–244. Liu, C., E. Gelius, G. Liu, H. Steiner, and R. Dziarski. 2000. Mammalian peptidoglycan recognition protein binds peptidoglycan with high afï¬پnity, is expressed in neutrophils, and inhibits bacterial growth. J. Biol. Chem. 275:24490–24499. Liu, C., Z. Xu, D. Gupta, and R. Dziarski. 2001. Peptidoglycan recognition proteins: A novel family of four human innate immunity pattern recognition molecules. J. Biol. Chem. 276:34686–34694. Michel, T., J. M. Reichhart, J. A. Hoffmann, and J. Royet. 2001. Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 414:756–759. Molenaar, A. J., S. R. Davis, and R. J. Wilkins. 1992. Expression of alpha-lactalbumin, alpha-S1-casein, and lactoferrin genes is heterogeneous in sheep and cattle mammary tissue. J. Histochem. Cytochem. 40:611–618. Praetor, A., I. Ellinger, and W. Hunziker. 1999. Intracellular trafï¬پc of the MHC class I-like IgG Fc receptor, FcRn, expressed in epithelial MDCK cells. J. Cell Sci. 112:2291–2299. Ramet, M., P. Manfruelli, A. Pearson, B. Mathey-Prevot, and R. A. Ezekowitz. 2002. Functional genomic analysis of phagocytosis and identiï¬پcation of a Drosophila receptor for E. coli. Nature 416:644–648. Rosen, J. M., S. L. Wyszomierski, and D. Hadsell. 1999. Regulation of milk protein gene expression. Annu. Rev. Nutr. 19:407–436. Schug, J., and G. C. Overton. 1997. TESS: Transcription Element Search Software on the WWW. University of Pennsylvania, PA. Tydell, C. C., N. Y. Yount, D. Tran, J. Yuan, and M. E. Selsted. 2002. Isolation, characterization, and antimicrobial properties of bovine oligosaccharide binding protein: A microbicidal granule protein of eosinophils and neutrophils. J. Biol. Chem. 277:19658–19664. Wingender, E., X. Chen, R. Hehl, H. Karas, I. Liebich, V. Matys, T. Meinhardt, M. Pruess, I. Reuter, and F. Schacherer. 2000. TRANSFAC: An integrated system for gene expression regulation. Nucleic Acids Res. 28:316–319. Yoshida, H., K. Kinoshita, and M. Ashida. 1996. Puriï¬پcation of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J. Biol. Chem. 271:13854–13860.

zymes, although the porcine protein was described as a zinc-binding protein (Fornhem et al., 1996). Camel PGRP was eluted at 290 mM NaCl from the afï¬پnity column, and we suppose that a combination of afï¬پnitybinding and the high isoelectric point at 9.02 has led to the retention on the column, since binding of murine PGRP, with an isoelectric point at 7.22, to soluble peptidoglycan was not inhibited by heparin, indicating a low afï¬پnity to heparin (Liu et al., 2000). We thus cannot rule out the presence of short PGRP in human or bovine milk, since the isoelectric point of both proteins is one or two degrees of magnitude lower than the isoelectric point of the camel and porcine variants. Camel or porcine PGRP may serve as a helpful substrate for further characterization of mammalian PGRP structure and function, since it is abundantly available and can easily be puriï¬پed from milk by heparin-sepharose afï¬پnity chromatography. CONCLUSIONS Major differences have been found in the composition of protective factors in camel milk compared with milk from true ruminants and humans. Intriguing dissimilarities are the presence of PGRP, a protein not yet detected in milk, and the absence of lysozyme C. The different composition probably comes from the evolutionary distance between the species and the extreme ecological habitat of camels. The newly detected protein probably has a main function in the protection of the lactating mammary gland but may also contribute to the passive immunization of the newborn camel. ACKNOWLEDGMENTS We thank P. Vogeli and M. Younan for providing آ¨ swine and camel milk and the staff from CVRL, UAE for providing genomic camel DNA. REFERENCES
Choe, K. M., T. Werner, S. Stoven, D. Hultmark, and K. V. Anderson. 2002. Requirement for a peptidoglycan recognition protein (PGRP) in relish activation and antibacterial immune responses in Drosophila. Science 296:359–362. Fornhem, C., C. G. Peterson, and K. Alving. 1996. Isolation and characterization of porcine cationic eosinophil granule proteins. Int. Arch. Allergy Immunol. 110:132–142. Goldman, A. S. 2002. Evolution of the mammary gland defense system and the ontogeny of the immune system. J. Mammary Gland Biol. Neoplasia 7:277–289.

Journal of Dairy Science Vol. 87, No. 8, 2004

pdf

15

An introduction to the camel
Z. Farah

Present distribution and economic potential
According to FAO statistics there are about 19 million camels in the world, of which 15 million are found in Africa and 4 million in Asia. Of this estimated world population, 17 million are believed to be one-humped dromedary camels (Camelus dromedarius) and 2 millions two-humped (Camelus bactrianus). Approximately 11 million dromedaries, representing two thirds of the world’s camel population, are in the arid areas of Africa, particularly in North East Africa, i.e. Somalia, Sudan, Ethiopia and Kenya.
Table 1: Estimated camel populations of Africa and the world (FAOSTAT Database 2001)
Country Africa: Algeria Chad Djibouti Egypt Ethiopia Kenya Libya Mali Mauritania Other regions: China India Iraq 326 1030 76 Mongolia Pakistan Saudi Arabia 360 800 400 240 725 70 120 1070 830 72 467 1230 Morocco Niger Nigeria Senegal Somalia Sudan Tunisia West Sahara 36 415 18 4 6200 3200 231 105 Camel population (in 1000) Country Camel population (in 1000)

16

An introduction to the camel

Most of these animals are kept by pastoralists in subsistence production systems. They are very reliable milk producers during dry seasons and drought years when milk from cattle sheep and goats is scarce. At such times camel can contribute up to 50% of the nutrient intake of the pastoralists. In recent years the picture of â€‌movingâ€‌ nomads has changed to some extent. With growing urbanisation the demand for milk among the urban population has been increasing. On the other hand the demand for a number of goods such as grain, oil, sugar and clothes increased among the pastoralists and the milk sales became the most important part of cash income for many camel owning pastoralists. Camel meat is also an important by-product mainly as a source of income. Sale of live camels, usually males and unproductive females for slaughter, is very common in East Africa and there are now increasing numbers of camel butcheries in many urban centres. There is also a growing export trade of slaughter camels to the Arabian Peninsula. The camel is also a means for transportation and for domestic use as drawing water from wells, rivers and dams. From a global perspective, the economic significance of camel production is minimal in comparison with that of other domestic animals. Nevertheless, in Africa, especially in East Africa and Sahel countries, the camel population makes a significant contribution to national economies. However, it is difficult to evaluate this economic contribution as most of the camel products are traded in the informal sector. Owing to the increasing human population and declining per capita production of food in Africa, there is an urgent need to develop marginal resources, such as arid land, and optimise their utilisation through appropriate livestock production systems of which camel production is the most suitable without doubt.

Traditional husbandry and management
Camels are held by nomads in arid regions. The pastoral land is mainly covered with annual grass, acacias, euphorbias and dwarf bushes. The annual rain fall varies between 100 and 400 mm, the amount of rain varying from year to year and the rains being restricted to widely separated areas. This type of pasture permits only extensive types of animal production. Because of its high mobility, its modest fodder requirement and its water regulation perfectly adapted to the environment, the camel is better suited than any other domestic animal to use this type of pasture. According to the nomads, camels can survive in times of extreme need for up to 30 days without water. This depends, however, on the grazing and prevailing temperatures.

Z. Farah

17

The salt requirement of camels is very high and is six to eight times higher than that of other domestic animals. The salt requirement is only partly satisfied by grazing. When the herdsmen observe that the camels are restless, with reduced appetite and milking performance, they take this as a sure sign of salt deficiency. The camels are then driven to salty water sources and watered repeatedly. Alternatively, salt-containing earth collected from other areas is given to the animals. Studies of many nomadic people in several countries show that female animals constitute 70% to 80% of a camel herd. The high number of female animals is needed to satisfy the large milk requirement of the nomad economy. The number of male animals in the herd is reduced in two ways and at two points in the camel’s life cycle. A percentage of male calves are slaughtered at birth or within few weeks of it. This allows more milk for female calves and for family consumption or sales. Males not slaughtered at birth are allowed to grow until they are about 4 years old. At this age the majority are castrated, fattened, sold and slaughtered for meat. Reproduction of the herds is achieved by selection of suitable male camels. According to the traditional husbandry, these should have the following characteristics:
• The bull or its father should have had predominately female progeny with good milk performance; • It should be fully grown and strong; • It should be a good fighter able to overcome other males.

It would be difficult to evaluate to what extent these selection criteria influence the quality of the progeny. One restriction arises from the fact that only the characteristics of the father, and not the characteristics inherited from mother, are taken into account in the selection. In general, breeds of camels are not as differentiated and classified as breeds of other domestic species. In most camel rearing societies, breed classifications are based on names of the ethnic group, clan as well as on the geographical localities where these camels are raised, rather than upon phenotypical characteristics. In Kenya for example there are three main types of camel classified as Somali breed, Rendille/Gabbra breed and Turkana breed. The Somali breed camels are primarily owned by Somali people of North-Eastern province of Kenya and are generally much larger than the other breeds found in the country. Adult females average 500–600 kg and males 600–800 kg. Average milk daily yield is 5 kg to 8 kg during a lactation of 10 to 12 months. The Rendille/Gabbra breed is found mainly in Marsabit district amongst the

18

An introduction to the camel

Rendille and Gabbra tribes. It is generally smaller than Somali breed camels. Live weights average 350–450 kg and 400–500 kg for females and males respectively. Milk yields average 3 to 4 kg, over a lactation of 12 months. The Turkana breed is the smallest camel found in Kenya averaging 350 kg for females and 400–450 kg for males. Milk yields are much lower than from other breeds and are in average 2 kg to 3 kg per day over a lactation of 9 to 10 months. The small body size and small feet make Turkana camels very swift and able to climb steep lava hills.

Fig. 1: Somali breed bull

In recent years dromedaries from Pakistan have been introduced in some camel farms in Kenya in order to improve milk production through cross breeding. Camels are slow reproducers. A female camel is sexually mature at the age of 4–5 years. Pregnancy is just over 12 months and the calving interval in pastoral production systems is normally 24 months or more. Female camels can remain fertile up to the age of 25 years and it is often reported that they produce 8–10 calves during a lifetime. In pastoral production systems, however, only a small proportion of the breeding females can reach this production performance. A major problem in camel productivity is the high mortality rate of camel calves in the first 3 months. The causes for mortality are mainly poor management practice and infectious diseases. The new-born calf has no natural protection against diseases, as there is no antibody transfer from the mother during foetal development. The calf can

Z. Farah

19

obtain immediate immunisation soon after birth only through the colostrum, which has a very high concentration of antibodies. Therefore, it is vital for the calf to suckle as soon and as much as possible. Unfortunately there is a common belief among many pastoralists that colostrum causes diarrhoea and, consequently, is unsuitable for the new-born calf. This wide spread practice of withholding the colostrum from the new-born calves, depriving them of essential antibodies, is certainly a crucial factor in the frequently reported high calf mortality in pastoral production systems. The milking of camels is a process that varies according to the different pastoral groups. Camels may be milked once or several times a day. In general, it is normal practice among most nomadic tribes to milk their camels in the early morning before animals are taken to grazing and at night when they return from grazing. Before milking, the calf is allowed to suckle until the milk is flowing and then the camel can be milked. Without this stimulation, the dam cannot be milked. The milker stands on one leg, puts the milk pot on the upper part of the other leg, and milks with one or two hands. Sometimes, milking may be done by two persons, each milking two teats. To prevent calves from suckling while at pasture, it is a usual practice to tie up one or more teats with special strings.
Fig. 2: Somali breed female with calf

20

An introduction to the camel

Fig. 3: Milking of camels

Fig. 4: Adult female Somali breed. (Piers Simpkin)

Z. Farah

21

Fig. 5: Adult female Turkana breed. (Piers Simpkin)

Fig. 6: Adult female Somali/Turkana crossbreed. (Piers Simpkin)

22

An introduction to the camel

Fig. 7: Adult female Pakistan breed. (Piers Simpkin)

Fig. 8: Pregnant Somali heifer (L) and pregnant mature Rendille/Gabbra female (R). (Chris Field)

pdf

Journal of Dairy Research (1998) 65 209–222

Printed in Great Britain

209

Sequence analysis of Camelus dromedarius milk caseins
Bï‌¹ STEFAN KAPPELER*, ZAKARIA FARAH ï‌،ï‌®ï‌¤ ZDENKO PUHAN Laboratorium fur Milchwissenschaft, Institut fur Lebensmittelwissenschaft, W W Eidgenossische Technische Hochschule, CH-8092 Zurich, Schweiz W W (Received 6 May 1997 and accepted for publication 4 September 1997) Sï‌µï‌­ï‌­ï‌،ï‌²ï‌¹. خ±s -, خ±s -, خ²- and خ؛-caseins from Somali camels (Camelus dromedarius) were " # puriï¬پed by acid precipitation at pH 4n4, crudely separated into an خ±-CN and a خ²-CN fraction and further puriï¬پed by reversed-phase HPLC. Fragments of tryptic digests were sequenced. Amino acid patterns obtained were used to screen a cDNA library constructed from mRNA from lactating udder tissue. Full length clones corresponding to the four caseins were sequenced. The numbers of residues in the sequences deduced were خ±s -CN 207, خ±s -CN 178, خ²-CN 217, خ؛-CN 162. Percentage " # similarity to bovine proteins was خ±s -CN A 39, خ±s -CN 56, خ²-CN 64, خ؛-CN 56. Acid" # precipitated casein of pooled milk was separated by reversed-phase HPLC and monitored at 220 nm, and its composition, estimated from peak integration, was (g\kg total casein) خ±s -CN 220, خ±s -CN 95, خ²-CN 650, خ؛-CN 35. Degrees of " # phosphorylation and glycosylation were determined by laser ionization mass spectrometry and sequence pattern analysis. Molecular masses determined were (kDa) خ±s -CN A, 24n755 and 24n668 ; خ±s -CN B, 25n293 ; خ±s -CN 21n993 ; خ²-CN, 24n900 ; " " # خ؛-CN 22n294–22n987. The pH values of the most probable isoelectric points were : خ±s " CN A 6P 4n41, خ±s -CN B 6P 4n40, خ±s -CN 9P 4n58, خ²-CN 4P 4n66, خ؛-CN 1P, with ten " # sialic acid residues bound, 4n10. The worldwide camel population is estimated to be " 18 million animals (FAO, 1989) : " 16 million dromedaries (Camelus dromedarius), the rest Bactrian (Camelus bactrianus). Two-thirds of the dromedaries live in the arid area of Africa, particularly in northeastern Africa. Camel milk is one of the main components of the human diet in these regions. Its consumption is not limited to the pastoral nomads ; it is also sold in urban centres (Schwarz & Dioli, 1992). Camel milk is consumed fresh or as fermented products. The manufacture of butter, cheese and heat sterilized milk is not yet well developed. Since the early 1980s, interest has grown in both the physicochemical and technological characteristics of camel milk. The available information indicates that there are signiï¬پcant differences between cows’ and camel milk proteins in properties such as electrophoretic mobility (Farah & Farah-Riesen, 1985), size of casein micelles (Farah & Ruegg, 1989), and rennet coagulation (Farah & Bachmann, 1987). $ Separation of the acid-precipitated protein fraction of camel milk was ï¬پrst reported by Larsson-Raz! nikiewicz & Mohamed (1986). In the present investigation, خ±s -, خ±s -, خ²- and خ؛-caseins from Somali camels were " # isolated for mass determination, quantitative analysis, and partial sequencing of
* For correspondence.

210

S. Kï‌،ï‌°ï‌°ï‌¥ï‌¬ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

tryptic digests. Using amino acid sequence data, a cDNA library was screened for full-length clones corresponding to the four different caseins, and the respective clones were sequenced. ï‌­ï‌،ï‌´ï‌¥ï‌²ï‌©ï‌،ï‌¬ï‌³ ï‌،ï‌®ï‌¤ ï‌­ï‌¥ï‌´ï‌¨ï‌¯ï‌¤ï‌³ Isolation of individual caseins Milk of individual Somali camels and pooled milk of a herd with Somali, Turkana and Pakistani camels was collected during milking at Ol Maisor Ranch, Rumuruti, Kenya, immediately frozen at k20 mC for transport and stored at k28 mC until analysis. After thawing, the milk was skimmed at 1000 g and 4 mC for 15 min. The casein fraction was isolated by acid precipitation at 37 mC for 20 min with acetic acid (1 : 10, v\v, 100 ml\l) followed by addition of 1 ï‌­-sodium acetate pH 8n0 (1 : 10, v\v) and centrifugation at 4000 g for 5 min. Supernatant and pellet were frozen and stored at k28 mC. For crude preparation of an خ±- and a خ²-CN fraction, the casein pellet from 1 l fresh milk was dissolved in 200 ml 10 ï‌­-urea, diluted with 460 ml double distilled water and the pH adjusted to 7n5 with 1 ï‌­-NaOH. The solution was diluted with 200 ml double distilled water and adjusted to pH 5n0 with 1 ï‌­-HCl. The ï¬پrm precipitate consisted mainly of خ±- and خ؛-CN (Hipp et al. 1952). After centrifugation at 600 g for 5 min, the supernatant was saturated with ammonium sulphate for precipitation of خ²-CN. Both precipitates were lyophilized. Individual caseins were separated by HPLC (LaChrom ; Merck, D-64293 Darmstadt, Germany) on an analytical reversed-phase C column (GromSil 200 ") ODS-4 HE, 5 آµ, 250i4n6 mm ; Grom, D-71083 Herrenberg, Germany) (Fig. 1). Before analysis, 1 g acid precipitated casein was dissolved in 5 ml 10 ï‌­-urea–140 mï‌­sodium citrate–35 mï‌­-1,3-bis[tris(hydroxymethyl)-methylamino]propane–780 mï‌­خ²-mercaptoethanol–200 mï‌­-Tris-HCl buffer, pH 8n0, stirred for 1 h and passed through a hydrophilic 0n45 آµï‌­ ï¬پlter (Millipore, Bedford, MA 01730, USA) (Groen et al. 1994). Solvent A was trifluoroacetic acid (TFA, 1 ml\l) in water, solvent B was TFA (1 ml\l) in acetonitrile. After injection of 10–50 آµl ï¬پltrate, elution was performed by a linear gradient from 0 to 350 ml solvent B\l over 15 min, followed by a linear gradient from 350 to 450 ml B\l over 35 min, a linear gradient from 450 to 1000 ml B\l over 5 min, a 3 min hold at pure B, then from 1000 to 0 ml B\l over 2 min. The flow rate was 1 ml\min and runs were performed at room temperature (Fig. 1). For large scale isolation of individual caseins, a semi-preparative column (GromSil 300 ODS-5 ST, 5 آµ, 250i20 mm ; Grom) was used to separate the proteins of the crude خ±- and خ²-CN fractions. After injection of 1 ml ï¬پltrate, elution was performed by a 9 min hold with pure solvent A, followed by a linear gradient from 0 to 400 ml solvent B\l over 3 min and a linear gradient from 400 to 430 ml solvent B\l over 28 min. The flow rate was 9n5 ml\min and runs were performed at 30 mC. The column effluent was monitored with a diode array detector (L-7450 ; Merck) from 200 to 300 nm. Proteins eluted were collected manually and lyophilized. Amino acid sequencing Proteins collected from the effluent of the semi-preparative column were used directly for N-terminal sequencing. Analysed sequences were compared with the Swissprot database (http:\\www.ebi.ac.uk\searches\blitz.html) using default values. From each of the peaks corresponding to خ±s -, خ±s -, خ²- and خ؛-CN, 1 mg lyophilizate " # was dissolved in 1 ml acetonitrile–double distilled water (2 : 3, v\v) containing 400 mï‌­-ammonium carbonate buffer, pH 9 and digested overnight at 37 mC with

Sequence analysis of camel milk caseins

211

25 آµg trypsin (Sequencing grade, Boehringer, D-68305 Mannheim, Germany). Peptides were separated using the same analytical column and a linear gradient from pure solvent A to pure solvent B over 180 min. The flow rate was 1 ml\min and runs were performed at room temperature. Peptides eluted from the column were collected manually and dried by vacuum centrifugation with a Speed-Vac SVC100 (Savant Instruments, Hicksville, NY 11741-4306, USA). Samples were dissolved in 100 آµl acetonitrile–water (1 : 1, v\v), and 20–100 آµl was applied on to a TFA-treated cartridge ï¬پlter and dried under a continuous nitrogen flow. Automated Edman degradation (Matsudaira, 1989) was performed using an ABI 471A sequencer (PE Applied Biosystems, Foster City, CA 94404, USA) with on-line phenylthiohydantoinyl reversed-phase C HPLC analysis. ") Mass determination of HPLC separated proteins Molecular masses of proteins were measured by matrix-assisted laser desorption ionization–mass spectrometry. Vacuum-dried samples were dissolved in acetonitrile– double distilled water (2 : 3, v\v) containing 10 ml TFA\l and co-crystallized (1 : 1, v\v) with خ±-cyano-4-hydroxycinnamic acid (5 g\l) in TFA (2 ml\l water). For analysis, a time of flight mass spectrometer in linear mode was used (Voyager Elite, PerSeptive Biosystems, Framingham, MA 01701, USA). Quantiï¬پcation Protein peaks from the HPLC runs described above were integrated at 220 nm. Relative amounts of peaks corresponding to the different caseins were calculated and the results were compared with weights of the lyophilized fractions. PolyA-mRNA isolation and construction of a cDNA library Udder tissue of a Somali camel (1 g) was taken in the morning after milking and immediately homogenized with a rotor–stator homogenizer (Kinematica, CH-6014 Littau, Switzerland), PolyA-mRNA was isolated with the OligotexTM Direct mRNA Kit (Quiagen, D-40724 Hilden, Germany) according to the manufacturer’s instruction for large scale preparation of mRNA. Total yield was 21n6 آµg mRNA and the A : A ratio was 2n4. #'! #)! A sample of mRNA (2 آµg) was used for synthesis of cDNA using the Universal RiboClone2 cDNA Synthesis System (Promega, Madison, WI 53711-5399, USA) with an oligo(dT) primer and EcoR I adaptors. One-ï¬پfth of the resulting cDNA was "& ligated to 1 آµg dephosphorylated خ» gt11 arms (Promega). The ligated DNA was in vitro packaged using an Escherichia coli C Packagene2 خ» DNA extract (Promega). All these procedures were carried out according to the manufacturer’s instructions. Phages were plated on Esch. coli LE 392 (Promega) and the titre of the library estimated at 2n6i10& pfu\ml. Then 100 آµl of the library was ampliï¬پed and gave a lysate with a titre of 1i10) pfu\ml. Sequencing of cDNA clones corresponding to the individual caseins The cDNA library was screened for cDNA corresponding to خ±s -, خ±s -, خ²- and خ؛" # CN by nucleic acid hybridization (Maniatis et al. 1989). Plaque lifts, hybridization and signal detection were performed with the digoxigenin (DIG) system of Boehringer, using uncharged nylon membranes, DIG EasyHyb solution, anti-DIGAP Fab fragments and CSPD2, according to the manufacturer’s instructions. Speciï¬پc probes were synthesized by the polymerase chain reaction (PCR) with DIG-

212

S. Kï‌،ï‌°ï‌°ï‌¥ï‌¬ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

11-dUTP to screen for cDNA corresponding to خ±s -, خ±s -, خ²- and خ؛-CN. Degenerate " # PCR primers were designed with the help of amino acid sequences obtained from sequencing the N-terminus and tryptic digests of the caseins (see above). The following primer pairs were designed (IUB code for mixed base sites). خ±s -CN forward : 5h-TAYCCNGARGTNTTYCARAAY-3h, derived from the " sequence Tyr–Pro–Glu–Val–Phe–Gln–Asn at the N-terminus of خ±s -CN. " خ±s -CN reverse : 5h-NGGRTGNGCDATRTAYTGCAT-3h, derived from the " sequence Met–Gln–Tyr–Ile–Ala–His–Pro, part of a prominent fragment of the خ±s -CN tryptic digest eluted at 123 min. " خ±s -CN forward : 5h-AARCAYGARATGGAYCA-3h, derived from the sequence # Lys–His–Glu–Met–Asp–Gln, at the N-terminus of خ±s -CN. # خ±s -CN reverse : 5h-TGRTCCCANGGRTTCAT-3h, derived from the sequence # Met–Asn–Pro–Trp–Asp–Gln, part of a major fragment of the خ±s -CN tryptic # digest eluted at 101n5 min. خ²-CN forward : 5h-GARAARGARGARTTYAARACN-3h, derived from the sequence Glu–Lys–Glu–Glu–Phe–Lys–Trp at the N-terminus of خ²-CN. خ²-CN reverse : 5h-RTCNGGNACNGGYTCYTGRAA-3h, derived from the sequence Phe–Gln–Glu–Pro–Val–Pro–Asp, part of a major fragment of the خ²-CN tryptic digest eluted at 53 min. خ؛-CN forward : 5h-GARGTNCARAAYCARGARCAR-3h, derived from the sequence Glu–Val–Gln–Asn–Gln–Glu–Gln at the N-terminus of خ؛-CN. خ؛-CN reverse : 5h-GATCTCAGTCGAAGTAATTTG-3h, derived from a sequenced PCR fragment of 320 bp, which was synthesized using genomic DNA of a Bactrian and bovine primers JK 501 and JK 302 (Schlee & Rottmann, 1992). The base lengths of the probes against خ±s -, خ±s -, خ²- and خ؛-CN cDNA were 528, 271, " # 597 and 486 respectively. Positive plaques were picked and veriï¬پed by PCR, using the appropriate primer pairs from before. For each protein, the cDNA insert of one positive plaque was excised with partial EcoR I digest, ligated into a pGEM-7Z vector (Promega) and transformed into Esch. coli XL1-Blue (Stratagene, La Jolla, CA 92037, USA). Plasmids were puriï¬پed for fluorescent sequencing with the Wizard Plus kit from Promega. Fluorescent sequencing of the cDNA was carried out using an ALF automated device (Pharmacia, S-751 82 Uppsala, Sweden), internal Cy5TM-dATP labelling (Pharmacia) and primer walking starting from commercial SP6 and T7 primers (Promega). Sequences were analysed using the gcg\egcg programme package (Genetics Computer Group, Madison, WI 53711, USA). ï‌²ï‌¥ï‌³ï‌µï‌¬ï‌´ï‌³ ï‌،ï‌®ï‌¤ ï‌¤ï‌©ï‌³ï‌£ï‌µï‌³ï‌³ï‌©ï‌¯ï‌® Caseins and cDNA were isolated from camels of the Somali breed, the most common (" 6 million animals). The sequences described in Fig. 2 a and Figs 3–5 were considered to represent the most frequent of presumed genetic variants of camel milk caseins. Proteins of fractions I, II, III, V and VII (Fig. 1) were partly sequenced. Fraction I consisted of خ؛-CN, II and III of خ±s -CN, V of خ±s -CN and VII of خ²-CN. Amino acid " # sequencing of the fragment eluted at 123 min of the tryptic digest of fraction III revealed a major insert (Glu–Gln–Ala–Tyr–Phe–His–Leu–Glu ; in bold in Fig. 2 b) between Gln"&% and Pro"&& of the mature protein (Fig. 2 a). Measured protein mass

Sequence analysis of camel milk caseins
3آ·5 3آ·0 1000

213

2آ·0 A220 500 1آ·5 1آ·0 0آ·5 I 0 VIII 0

II

III

IV V

VI

0

10

20

30 Retention time, min

40

50

60

Fig. 1. Reversed-phase HPLC chromatogram of acid-precipitated camel milk casein. Peaks I, II, III, V, VII and VIII were collected for further analysis. Peaks IV and VI were not identiï¬پed. – – –, Solvent B gradient.

was " 25n7 kDa. From these results, we propose an خ±s -CN B 6P with 215 amino " acids, a molecular mass of 25n773 kDa and an isoelectric point at pH 4n40 (Fig. 2 b). Long and short variants of خ±s -CN also occur in ovine milk (Ferranti et al. 1995). The " authors suggest that those variants are a result of differential splicing of the heterogeneous nuclear RNA transcribed from the خ±s -Cn gene rather than gene " products of two different alleles. The minor peak VI to the left of peak VII (Fig. 1) is likely to represent a variant of خ²-CN. Comparing the خ³-CN sequence of camel milk obtained by Beg et al. (1986 a) with the خ²-CN sequence shown here reveals a single exchange : Glu"*& for Gln"*&. The fragment sequenced by Beg may therefore belong to a خ²-CN B. Molecular masses of fraction VIII were 13n9, 15n7, 15n75 and 15n9 kDa and presumably belonged to hydrophobic خ³-CN. cDNA sequences were translated into casein precursor proteins containing signal peptides (Fig. 2 a, Figs 3–5), which lead proteins into the rough endoplasmic reticulum and are subsequently cleaved off (Burgess & Kelly, 1987). Signal peptide sequences of all examined proteins were highly conserved between camel and cow (Fig. 6). The signal peptides of the calcium-sensitive caseins خ±s -, خ±s - and خ²-CN were " # all 15 amino acids in length and similar in amino acid sequence. We assume that this similarity is a result of the common evolutionary origin of the respective genes (Rosen, 1987) and of the similar protein destinies. The most likely motifs for post-translational phosphorylation, Ser–X–SerP\Glu, occur six times in خ±s -CN (serines at 18, 68, 70, 71, 72 and 73), nine times in خ±s -CN " # (serines at 8, 9, 10, 32, 53, 108, 110, 113 and 121), four times in خ²-CN (serines at 15, 17, 18 and 19) and twice in خ؛-CN (serines at 141 and 159) (Figs 2–5). Ser"&* in خ؛-CN is towards the end of the protein, a position that is less frequently modiï¬پed. Phosphorylation of the sites proposed is in agreement with the molecular masses measured by matrix-assisted laser desorption ionization–mass spectrometry (Table

Concn of solvent B, ml/l

2آ·5

VII

214
(a) cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA cDNA cDNA cDNA cDNA (b)

S. Kï‌،ï‌°ï‌°ï‌¥ï‌¬ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

Fig. 2. (a) cDNA sequence of camel milk خ±s -CN A and corresponding protein, with mature protein in " bold. The open reading frame of the cDNA sequence is from A%' to G("" and the polyadenylation signal in bold from A"!(( to A"!)#. Numbering of the amino acid chain starts from the ï¬پrst residue of the mature protein. P, phosphorylated serines. (b) Proposed amino acid sequence of camel milk خ±s -CN B " 6P. Insert shaded and amino acid residues in bold. P, phosphorylated serines.

1). The measured molecular masses of خ±s -, خ±s - and خ²-CN are a multiple of one " # phosphate group (80 Da) higher than the molecular masses calculated from the unmodiï¬پed amino acid chain. The most frequent form of خ²-CN had only three phosphate groups bound instead of the four groups predicted. The degree of

Sequence analysis of camel milk caseins
cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA cDNA cDNA cDNA

215

Fig. 3. cDNA sequence of camel milk خ±s -CN and corresponding protein, with mature protein in bold. # The open reading frame of the cDNA sequence is from A'" to T'$* and the polyadenylation signal in bold from A*%% to A*%*. Numbering of the amino acid chain starts from the ï¬پrst residue of the mature protein. P, phosphorylated serines.

phosphorylation of خ؛-CN could not be determined by this method because this protein is also glycosylated. Respective pH values of isoelectric points for camel milk proteins compared with the most frequent variants of respective cows’ milk proteins are given in Table 1. Isoelectric focusing of camel milk caseins revealed a narrower pH range than for cows’ milk caseins, within which the major bands appeared. Focusing of the different bands near pH 4n6 was in good agreement with the calculated values. Although camel milk caseins were less phosphorylated than cows’ milk caseins, the pH values of their isoelectric points were similar. However, the amount of micellar calcium phosphate may be lower than in cows’ milk. Another post-translational modiï¬پcation found in caseins is glycosylation of Thr residues in خ؛-CN. This occurs for Thr near Arg\Lys, Thr or Pro, and is likely to be inhibited by Ile (Pisano et al. 1994). Based on this study and in comparison with the glycosylation of bovine خ؛-CN, we propose glycosylation of ï¬پve Thr residues (threonines at 105, 109, 149, 152 and 153 ; Fig. 5). If all sites have two sialic acid residues bound, the isoelectric point will be lowered to 4n1 (with one SerP). SDS-

216
cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA cDNA cDNA cDNA cDNA

S. Kï‌،ï‌°ï‌°ï‌¥ï‌¬ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

Fig. 4. cDNA sequence of camel milk خ²-CN and corresponding protein, with mature protein in bold. The open reading frame of the cDNA sequence is from A%$ to C($) and the polyadenylation signal in bold from A"!($ and A"!(). Numbering of the amino acid chain starts from the ï¬پrst residue of the mature protein. P, phosphorylated serines.

PAGE and mass spectroscopic studies revealed that most of the خ؛-CN analysed was of relatively low molecular mass, making the low glycosylated form predominant and the protein very basic. This ï¬پnding disagrees with the high sialic acid content reported by Mehaia (1987). Sequence comparisons of cows’ and camel milk caseins are shown in Fig. 7. There are few pronounced structural differences when camel and cows’ milk caseins are compared. Although خ±s -CN of camel and cows’ milks have a low percentage " similarity in primary structure, similarities in the secondary structure (a series of خ±helical regions followed by a C-terminus with little deï¬پned secondary structure) predominate. In camel milk خ±s -CN, hydrophilicity of the N-terminal end is slightly " more pronounced. The deletions that shorten camel milk خ±s -CN compared with #

Sequence analysis of camel milk caseins
cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA Protein cDNA cDNA cDNA

217

Fig. 5. cDNA sequence of camel خ؛-CN and corresponding protein, with mature protein in bold. The open reading frame of the cDNA sequence is from A&& to C'!! and the polyadenylation signal in bold from A()) to A(*$. Numbering of amino acid chain starts from the ï¬پrst residue of the mature protein. Chymosin cleavage site, Phe*(–Ile*) ; P, phosphorylated serine ; G, glycosylated threonines. Species Camel Cow Camel Cow Camel Cow Camel Cow Casein as1-CN as1-CN as2-CN as1-CN b-CN b-CN -CN -CN Sequence of signal peptide

Fig. 6. Sequence comparison of signal peptides from camel and cows’ milk caseins. Conserved residues are shaded.

cows’ milk خ±s -CN A occur in an خ±-helical region between bovine Glu%* and Asn)$ (Fig. # 7). They go together with the loss of the phosphorylated serine cluster Ser&', Ser&( and Ser&). This loss may have implications in micelle assemblage and stability as well as in the nutritional behaviour of the caseins (Ferranti et al. 1995). As with cows’ milk

218

Table 1. Physicochemical characteristics of dromedary milk caseins compared with cows’ milk caseins†
Molecular mass, kDA

Species Camel خ±s -CN A

Casein†

Residues 207 21n993 24n900 22n294– 22n987 24n275 24n755 24.668 4n78

Calculated from amino acid chain Measured

Isoelectric point of amino acid chainâ€، 6 Ser-P

Charged amino acid residue modiï¬پcations

Isoelectric point of modiï¬پed protein 4n41

Percentage similarity to bovine proteinsآ§ 39 56 64 56 4n26 4n58 4n78 4n76 4n49 4n11

"
199 178 207 217 209 162 169 18n974 5n97 22n975 21n266 24n348 24n651 23n583 18n254 4n76 5n81 8n68 5n17 5n01 8n27

Cow Camel Cow Camel Cow Camel Cow خ؛-CN A

خ±s -CN B " خ±s -CN # خ±s -CN A # خ²-CN خ²-CN A2 خ؛-CN

S. Kï‌،ï‌°ï‌°ï‌¥ï‌¬ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

8 Ser-P 9 Ser-P 11 Ser-P 3 Ser-P 5 Ser-P 1 Ser-P, 10 ThrNANA 1 Ser-P, 12-Thr NANA

3n97

NANA, N-acetylneuraminic (sialic) acid. † Data on cow caseins after Eigel et al. (1984). â€، According to the gcg programme (Genetics Computer Group, Madison, WI 53711, USA). آ§ Result from blitz, applying gap penalty 1.

a-helical

a-helical

a-helical a-helical, only camelphosphorylation cluster a-helical

Camel as1-CN A Bovine as1-CN Bآ آ آ  a-helical a-helical deletion in camel as1-CN A

Camel as1-CN A Bovine as1-CN Bآ آ آ  a-helical phosphorylation cluster

Camel as2-CN Bovine as2-CN Aآ آ آ  a-helical phosphorylation cluster a-helical a-helical

a-helical a-helical a-helical cysteines phosphorylation cluster, lost in camel as2-CN a-helical

Camel as2-CN Bovine as2-CN Aآ آ آ  a-helical a-helical phosphorylation cluster a-helical b-pleated hydrophobic C-terminal domain

Camel b-CN Bovine b-CN A2 b-pleated b-pleated

Camel b-CN Bovine b-CN A2 b-pleated b-pleated b-pleated cysteine a-helical cysteine

Sequence analysis of camel milk caseins

Camel -CN Bovine -CN A b-pleated cleavage⇓site glycosylated threonine residues b-pleated

Camel -CN Bovine -CN A

Fig. 7. Sequence comparison of cows’ and camel milk caseins. At top, probable secondary structures are shown. On the following line, protein characteristics are described. Sequence deletions are indicated as dashed lines. Data of cows’ caseins are from the Swissprot database (http:\\expasy.hcuge,ch\sprot\sprot-top.html). S, phosphorylated serine, T, glycosylated threonine ; FI, FM, cleavage sites in خ؛-caseins.

219

220
Camel Cow

S. Kï‌،ï‌°ï‌°ï‌¥ï‌¬ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

Fig. 8. Sequence comparison of the chymosin-sensitive region of خ؛-CN from camel and cows’ milk. Conserved residues are shaded.

خ±s -CN, camel milk خ±s -CN is the most hydrophilic of the four caseins and shows the # # most distinct secondary structures, mainly خ±-helices (Fig. 7). The two Cys residues also occur at about position 40. The site of cleavage of camel milk خ؛-CN by chymosin is Phe*(–Ile*) (Fig. 5), leaving a macropeptide of 6n774 kDa, 65 amino acids in length with an isoelectric point of the unmodiï¬پed peptide at pH 4n13. In bovine خ؛-CN, the site of cleavage is Phe"!&–Met"!', leaving a macropeptide of 6n707 kDA, 64 amino acids in length with an isoelectric point of the unmodiï¬پed peptide at pH 3n87. The amino acid sequence from His*) to Lys""# is involved in binding and cleavage of bovine خ؛-CN by chymosin (Visser et al. 1987). The proline residues in this sequence are thought to stabilize the correct conformation of خ؛-CN in the active site cleft of chymosin and the basic residues are thought to bind to acidic residues at either end of the active side cleft (Plowman & Creamer, 1995). All proline residues are conserved in the camel milk خ؛-CN as shown in Fig. 8 and the bovine residue Leu"!$ is replaced by Pro*&. This additional proline residue may help to stabilize a conformation of خ؛-CN in the active site cleft of camel chymosin different from the conformation of cows’ milk خ؛-CN in the cleft of bovine chymosin. Histidine residues in the sequence His*)–Pro–His– Pro–His"!# of cows’ milk خ؛-CN are replaced by more basic arginine residues in camel milk خ؛-CN (Fig. 8). Since arginine remains protonized at higher pH values and has a longer and more flexible side chain, it can be speculated that these residues will still bind to the acidic centres of chymosin at a higher local pH and that the camel milk خ؛-CN backbone does not need to be bound as tightly to chymosin as was shown for cows’ milk خ؛-CN (Plowman & Creamer, 1995). Models for secondary structure patterns created with BCM Protein (Baylor College of Medicine, Houston, TX 77030, USA ; http:\\dot.imgen.bcm.tmc.edu: 9331\pssprediction\pssp.html) are similar in cows’ and camel milk خ؛-CN, with an Nterminal خ±-helix containing one Cys followed by خ²-pleated sheets and a second Cys (Fig. 7). Both Cys residues are at positions similar to those in cows’ milk protein, and are likely to be involved in intermolecular crosslinks (Richardson et al. 1992). Of خ؛-CN forms already sequenced porcine خ؛-CN is most similar in overall structure, revealing a cleavage site highly similar in primary and secondary structure. Porcine chymosin acting on porcine milk was shown to have 6–8 times higher proteolytic speciï¬پcity compared with bovine chymosin (Houen et al. 1996). A similar ability to cleave camel milk خ؛-CN with very high speciï¬پcity can be assumed for camel chymosin acting on camel milk. The amounts of the individual caseins in g\kg total casein were calculated from Fig. 1 : خ±s -CN 220, خ±s -CN 95, خ²-CN 650, خ؛-CN 35. Only scanty amounts of خ؛-CN were " # found in camel milk. خ؛-CN is considered to have a terminating function in micelle growth (Horne et al. 1989), as well as a stabilizing effect on the micelle by its net charge and by steric hindrance of aggregation by its hydrophilic C-terminal end (Holt & Horne, 1996). The glycosylated forms may have a stronger impact on both these functions than the non-glycosylated form, which seemed to be predominant in camel milk, owing to steric repulsion of charged sialic acid groups and to increased hydrophilicity. After cleavage of the macropeptide, hydrophobic forces and electrostatic interactions of bivalent cations with negatively charged groups promote

Sequence analysis of camel milk caseins

221

coagulation and stabilize the curd (Dalgleish, 1983 ; Mora-Gutierrez et al. 1993). Since خ²-CN predominated in the camel milk studied and phosphorylation of خ±s - and خ±s " # CN was lower than in cows’ milk, we assume that hydrophobicity is the driving force in coagulation of camel milk. It has been shown that camel milk casein is less stable at elevated temperatures than cows’ milk (Farah & Atkins, 1992). This may be an effect of the high خ²-CN content. On the other hand, in milks with amounts of خ؛-CN higher than in cows’ milk, e.g. in buffalo milk with 130–200 g خ؛-CN\kg total casein (El-Din & Aoki, 1993), curd ï¬پrmness was found to be higher than in cows’ milk (Bayoumi, 1990). Moreover, milks of transgenic mice producing bovine خ؛-CN were shown to have a linear correlation between the amount of خ؛-CN and curd ï¬پrmness ! (Gutie! rrez-Adan et al. 1996), and an inverse correlation with micelle size. The mean diameter of camel milk casein micelles is larger than that of casein micelles in bovine milk (Buchheim et al. 1989). Milks of animals traditionally used for technological processing always have higher amounts of خ؛-CN and lower amounts of خ²-CN than camel milk. It may be assumed that lack of selective breeding of camels for milk of good technological quality is responsible for the high خ²-CN and the low خ؛-CN content. Technological difficulties in processing camel milk are probably due more to different proportions of the individual caseins compared with cows’ milk than to structural differences of the proteins. It has been shown that a high content of خ²-CN and a low content of خ؛-CN adversely affect some of the processing characteristics of casein micelles (Schmidt & Koops, 1977), such as stability towards ethanol, homogenization and heat. Moreover, the proportion of whey proteins seemed to differ from that in bovine milk. Whey separated and N-terminal sequenced during the investigations contained a high proportion of a heterogenous whey protein (Beg et al. 1987) and خ±-lactalbumin (Beg et al. 1985), minor amounts of whey acidic protein (Beg et al. 1986 b), but no خ²-lactoglobulin. The functions of these proteins are not precisely known. Therefore, the impact of the whey protein distribution on technological and nutritional properties cannot be determined, nor can the reason for these differences be considered. Nevertheless, it must be assumed that the many differences in protein composition between the ruminant milks and camel milk will have a major impact on technological properties, and that a lower ratio of خ²-CN to خ؛-CN would be favourable for curd coagulation and heat sterilization. The authors thank Professor Hans Bruckner and Martin Leitenberger of the $ University of Hohenheim, D-70593 Stuttgart, Germany and Dr Michael Hassig of $ the Kantonales Tierspital, University of Zurich, CH-8057 Zurich, Switzerland for $ $ advice and for protein sequencing, Dr Mario Younan and Ernesto Beretta for biopsy of udder tissue, Christian Hulsebusch for leaving his ï¬پeld laboratory to us, the Evans $ family and Mrs Deborah Atkins for providing facilities at Ol Maisor Ranch. ï‌²ï‌¥ï‌¦ï‌¥ï‌²ï‌¥ï‌®ï‌£ï‌¥ï‌³
Bï‌،ï‌¹ï‌¯ï‌µï‌­ï‌©, S. 1990 Studies on composition and rennet coagulation of camel milk. Kieler Milchwirtschaftliche Forschungsberichte 42 3–8 $ $ Bï‌¥ï‌§, O. U., ï‌¶ï‌¯ï‌® Bï‌،ï‌¨ï‌²-Lï‌©ï‌®ï‌¤ï‌³ï‌´ï‌²ï‌¯ï‌­, H., Zï‌،ï‌©ï‌¤ï‌©, Z. H. & Jï‌¯ï‌²ï‌®ï‌¶ï‌،ï‌¬ï‌¬, H. 1985 The primary structure of خ±lactalbumin from camel milk. European Journal of Biochemistry 147 233–239 $ $ Bï‌¥ï‌§, O. U., ï‌¶ï‌¯ï‌® Bï‌،ï‌¨ï‌²-Lï‌©ï‌®ï‌¤ï‌³ï‌´ï‌²ï‌¯ï‌­, H., Zï‌،ï‌©ï‌¤ï‌©, Z. H. & Jï‌¯ï‌²ï‌®ï‌¶ï‌،ï‌¬ï‌¬, H. 1986 a Characterization of a camel milk protein rich in proline identiï¬پes a new خ²-casein fragment. Regulatory Peptides 15 55–62 $ $ Bï‌¥ï‌§, O. U., ï‌¶ï‌¯ï‌® Bï‌،ï‌¨ï‌²-Lï‌©ï‌®ï‌¤ï‌³ï‌´ï‌²ï‌¯ï‌­, H., Zï‌،ï‌©ï‌¤ï‌©, Z. H. & Jï‌¯ï‌²ï‌®ï‌¶ï‌،ï‌¬ï‌¬, H. 1986 b A camel milk whey protein rich in half-cysteine. Primary structure, assessment of variations, internal repeat patterns, and relationships with neurophysin and other active polypeptides. European Journal of Biochemistry 159 195–201 $ $ Bï‌¥ï‌§, O. U., ï‌¶ï‌¯ï‌® Bï‌،ï‌¨ï‌²-Lï‌©ï‌®ï‌¤ï‌³ï‌´ï‌²ï‌¯ï‌­, H., Zï‌،ï‌©ï‌¤ï‌©, Z. H. & Jï‌¯ï‌²ï‌®ï‌¶ï‌،ï‌¬ï‌¬, H. 1987 Characterization of a heterogeneous camel milk whey non-casein protein. FEBS Letters 216 270–274

222

S. Kï‌،ï‌°ï‌°ï‌¥ï‌¬ï‌¥ï‌² ï‌،ï‌®ï‌¤ ï‌¯ï‌´ï‌¨ï‌¥ï‌²ï‌³

Bï‌µï‌£ï‌¨ï‌¨ï‌¥ï‌©ï‌­, W., Lï‌µï‌®ï‌¤, S. & Sï‌£ï‌¨ï‌¯ï‌¬ï‌´ï‌©ï‌³ï‌³ï‌¥ï‌«, J. 1989 [Comparative studies on the structure and size of casein micelles in the milk of different species.] Kieler Milchwirtschaftliche Forschungsberichte 41 253–265 Bï‌µï‌²ï‌§ï‌¥ï‌³ï‌³, T. L. & Kï‌¥ï‌¬ï‌¬ï‌¹, R. B. 1987 Constitutive and regulated secretion of proteins. Annual Review of Cell Biology 3 243–293 Dï‌،ï‌¬ï‌§ï‌¬ï‌¥ï‌©ï‌³ï‌¨, D. G. 1983 Coagulation of renneted bovine casein micelles : dependence on temperature, calcium ion concentration and ionic strength. Journal of Dairy Research 50 331–340 Eï‌©ï‌§ï‌¥ï‌¬, W. N., Bï‌µï‌´ï‌¬ï‌¥ï‌², J. E., Eï‌²ï‌®ï‌³ï‌´ï‌²ï‌¯ï‌­, C. A., Fï‌،ï‌²ï‌²ï‌¥ï‌¬ï‌¬, H. M., Hï‌،ï‌·ï‌،ï‌¬ï‌«ï‌،ï‌², V. R., Jï‌¥ï‌®ï‌®ï‌¥ï‌³ï‌³, R. & Wï‌¨ï‌©ï‌´ï‌®ï‌¥ï‌¹, R. Mï‌£L. 1984 Nomenclature of proteins of cow’s milk : ï¬پfth revision. Journal of Dairy Science 67 1599–1631 Eï‌¬-Dï‌©ï‌®, M. Z. & Aï‌¯ï‌«ï‌©, T. 1993 High performance of gel and ion-exchange chromatography of buffalo casein. International Dairy Journal 3 141–147 FAO 1989 Statistics Yearbook. Rome : Food and Agriculture Organization Fï‌،ï‌²ï‌،ï‌¨, Z. & Aï‌´ï‌«ï‌©ï‌®ï‌³, D. 1992 Heat coagulation of camel milk. Journal of Dairy Research 59 229–231 Fï‌،ï‌²ï‌،ï‌¨, Z. & Bï‌،ï‌£ï‌¨ï‌­ï‌،ï‌®ï‌®, M. R. 1987 Rennet coagulation properties of camel milk. Milchwissenschaft 42 689–692 Fï‌،ï‌²ï‌،ï‌¨, Z. & Fï‌،ï‌²ï‌،ï‌¨-Rï‌©ï‌¥ï‌³ï‌¥ï‌®, M. 1985 Separation and characterization of major components of camel milk casein. Milchwissenschaft 40 669–671 $ Fï‌،ï‌²ï‌،ï‌¨, Z. & Rï‌µï‌¥ï‌§ï‌§, M. W. 1989 The size distribution of casein micelles in camel milk. Food Microstructure 8 211–216 Fï‌¥ï‌²ï‌²ï‌،ï‌®ï‌´ï‌©, P., Mï‌،ï‌¬ï‌¯ï‌²ï‌®ï‌©, A., Nï‌©ï‌´ï‌´ï‌©, G., Lï‌،ï‌¥ï‌؛ï‌؛ï‌،, P., Pï‌©ï‌؛ï‌؛ï‌،ï‌®ï‌¯, R., Cï‌¨ï‌©ï‌،ï‌®ï‌¥ï‌³ï‌¥, L. & Aï‌¤ï‌¤ï‌¥ï‌¯, F. 1995 Primary structure of ovine خ±s -caseins : localization of phosphorylation sites and characterization of genetic variants " A, C and D. Journal of Dairy Research 62 281–296 Gï‌²ï‌¯ï‌¥ï‌®, A. F., ï‌¶ï‌،ï‌® ï‌¤ï‌¥ï‌² Vï‌¥ï‌§ï‌´, R., ï‌¶ï‌،ï‌® Bï‌¯ï‌¥ï‌«ï‌¥ï‌¬, M. A. J. S., ï‌¤ï‌¥ Rï‌¯ï‌µï‌·, O. L. A. M. & Vï‌¯ï‌³, H. 1994 Case study on individual animal variation in milk protein composition as estimated by high-pressure liquid chromatography. Netherlands Milk and Dairy Journal 48 201–212 ! ! Gï‌µï‌´ï‌©ï‌¥ï‌²ï‌²ï‌¥ï‌؛-Aï‌¤ï‌،ï‌®, A., Mï‌،ï‌§ï‌،, E. A., Mï‌¥ï‌،ï‌¤ï‌¥, H., Sï‌¨ï‌¯ï‌¥ï‌­ï‌،ï‌«ï‌¥ï‌², C. F., Mï‌¥ï‌¤ï‌²ï‌،ï‌®ï‌¯, J. F., Aï‌®ï‌¤ï‌¥ï‌²ï‌³ï‌¯ï‌®, G. B. & Mï‌µï‌²ï‌²ï‌،ï‌¹, J. D. 1996 Alterations of the physical characteristics of milk from transgenic mice producing bovine خ؛-casein. Journal of Dairy Science 79 791–799 Hï‌©ï‌°ï‌°, N. J., Gï‌²ï‌¯ï‌¶ï‌¥ï‌³, M. L., Cï‌µï‌³ï‌´ï‌¥ï‌², J. H. & Mï‌£Mï‌¥ï‌¥ï‌«ï‌©ï‌®, T. L. 1952 Separation of خ±-, خ²-, and خ³-casein. Journal of Dairy Science 35 272–281 Hï‌¯ï‌¬ï‌´, C. & Hï‌¯ï‌²ï‌®ï‌¥, D. S. 1996 The hairy casein micelle. Evolution of the concept and its implications for dairy technology. Netherlands Milk and Dairy Journal 50 85–111 Hï‌¯ï‌²ï‌®ï‌¥, D. S., Pï‌،ï‌²ï‌«ï‌¥ï‌², T. G. & Dï‌،ï‌¬ï‌§ï‌¬ï‌¥ï‌©ï‌³ï‌¨, D. G. 1989 Casein micelles, polycondensation, and fractals. In Food Colloids, pp. 400–406 (Royal Society of Chemistry Special Publication no. 75) $ Hï‌¯ï‌µï‌¥ï‌®, G., Mï‌،ï‌¤ï‌³ï‌¥ï‌®, M. T., Hï‌،ï‌²ï‌¬ï‌¯ï‌·, K. W., Lï‌¯ï‌®ï‌¢ï‌¬ï‌،ï‌¤, P. & Fï‌¯ï‌¬ï‌´ï‌­ï‌،ï‌®ï‌®, B. 1996 The primary structure and enzymic properties of porcine prochymosin and chymosin. International Journal of Biochemistry and Cell Biology 28 667–675 Lï‌،ï‌²ï‌³ï‌³ï‌¯ï‌®-Rï‌،ï‌؛! ï‌®ï‌©ï‌«ï‌©ï‌¥ï‌·ï‌©ï‌£ï‌؛, M. & Mï‌¯ï‌¨ï‌،ï‌­ï‌¥ï‌¤, M. A. 1986 Analysis of the casein content in camel (Camelus dromedarius) milk. Swedish Journal of Agricultural Research 16 13–18 Mï‌،ï‌®ï‌©ï‌،ï‌´ï‌©ï‌³, T., Sï‌،ï‌­ï‌¢ï‌²ï‌¯ï‌¯ï‌«, J. & Fï‌²ï‌©ï‌´ï‌³ï‌£ï‌¨, E. F. 1989 Molecular Cloning, 2nd edn. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory Press Mï‌،ï‌´ï‌³ï‌µï‌¤ï‌،ï‌©ï‌²ï‌،, P. T. 1989 A Practical Guide to Protein and Peptide Puriï¬پcation for Microsequencing. San Diego, CA : Academic Press Mï‌¥ï‌¨ï‌،ï‌©ï‌،, M. A. 1987 Studies of camel casein micelles : treatment with soluble and immobilized neuraminidase. Carbohydrate Polymers 7 361–369 Mï‌¯ï‌²ï‌،-Gï‌µï‌´ï‌©ï‌¥ï‌²ï‌²ï‌¥ï‌؛, A., Fï‌،ï‌²ï‌²ï‌¥ï‌¬ï‌¬, H. M. & Kï‌µï‌­ï‌¯ï‌³ï‌©ï‌®ï‌³ï‌«ï‌©, T. F. 1993 Comparison of calcium-induced associations of bovine and caprine caseins and the relationship of خ±s -casein content to colloidal stabilization : a " thermodynamic linkage analysis. Journal of Dairy Science 76 3690–3697 Pï‌©ï‌³ï‌،ï‌®ï‌¯, A., Pï‌،ï‌£ï‌«ï‌¥ï‌², N. H., Rï‌¥ï‌¤ï‌­ï‌¯ï‌®ï‌¤, J. W., Wï‌©ï‌¬ï‌¬ï‌©ï‌،ï‌­ï‌³, K. L. & Gï‌¯ï‌¯ï‌¬ï‌¥ï‌¹, A. A. 1994 Characterisation of Olinked glycosylation motifs in the glycopeptide domain of bovine خ؛-casein. Glycobiology 4 837–844 Pï‌¬ï‌¯ï‌·ï‌­ï‌،ï‌®, J. E. & Cï‌²ï‌¥ï‌،ï‌­ï‌¥ï‌², L. K. 1995 Restrained molecular dynamics study of the interaction between bovine خ²-casein peptide 98-111 and bovine chymosin and porcine pepsin. Journal of Dairy Research 62 451–467 ! Rï‌©ï‌£ï‌¨ï‌،ï‌²ï‌¤ï‌³ï‌¯ï‌®, T., Oï‌¨, S. & Jï‌©ï‌­ï‌¥ï‌®ï‌¥ï‌؛-Fï‌¬ï‌¯ï‌²ï‌¥ï‌³, R. 1992 Molecular modelling and genetic engineering of milk proteins. In Advanced Dairy Chemistry—1. Proteins, pp. 545–577 (Ed. P. F. Fox). London : Elsevier Applied Science Rï‌¯ï‌³ï‌¥ï‌®, J. M. 1987 Milk protein gene structure and expression. In The Mammary Gland, pp. 301–322 (Eds M. C. Neville and C. W. Daniel). London : Plenum Sï‌£ï‌¨ï‌¬ï‌¥ï‌¥, P. & Rï‌¯ï‌´ï‌´ï‌­ï‌،ï‌®ï‌®, O. 1992 Identiï¬پcation of bovine خ؛-casein C using the polymerase chain reaction. Journal of Animal Breeding and Genetics 109 153–155 Sï‌£ï‌¨ï‌­ï‌©ï‌¤ï‌´, D. G. & Kï‌¯ï‌¯ï‌°ï‌³, J. 1977 Properties of artiï¬پcial casein micelles. 2. Stability towards ethanol, dialysis, pressure and heat in relation to casein composition. Netherlands Milk and Dairy Journal 31 342–351 Sï‌£ï‌¨ï‌·ï‌،ï‌²ï‌´ï‌؛, H. J. & Dï‌©ï‌¯ï‌¬ï‌©, M. 1992 The One-Humped Camel (C. dromedarius) in Eastern Africa : a Pictorial Guide to Diseases, Health Care, and Management. Weikersheim : Josef Margraf Vï‌©ï‌³ï‌³ï‌¥ï‌², S., Sï‌¬ï‌،ï‌®ï‌§ï‌¥ï‌®, J. C. & ï‌¶ï‌،ï‌® Rï‌¯ï‌¯ï‌©ï‌ھï‌¥ï‌®, P. J. 1987 Peptide substrates for chymosin (rennin). Interaction sites in خ؛-casein related sequences located outside the (103–108)-hexapeptide region that ï¬پts into the enzyme’s active-site cleft. Biochemical Journal 244 553–558

pdf

FOOD MICROSTRUCTURE, Vol. 8 (1989), pp. 211-216 Scanning Microscopy International, Chicago (AMF 0 'Hare), IL 60666 USA THE SIZE DISTRIBUTION OF CASEIN MICELLES IN CAMEL MILK Z. Farah1 and M.W. Rأ¼egg2
1

Department of Food Science, Swiss Federal Institute of Technology, CH-8092 Zأ¼rich, Switzerland, and 2 Federal Dairy Research Institute, CH -3097 Liebefeld, Switzerland

Abstract The size distribution of casein micelles in camel milk has been determined by electron microscopy. Individual and pooled samples were cryo-fixed by rapid freezing and freeze-fractured. Electron micrographs of the freeze-fracture replica revealed a relatively broad size distribution, with an average micelle diameter around 280 nm in the volume distribution curve. The distribution was significantly broader than that of the particles of cow's or human milk and showed a greater number of large particles. The submicelles were also somewhat larger than those observed in cow's and human milk (approx. 15, 10 and 7 nm, respectively). The average values for the gross composition of camel milk were similar to those of cow's milk. Partition of mineral salts between the serum and micellar phase of camel milk was studied by means of ultrafiltration. The proportion of soluble forms of the minerals expressed as percentage of their total concentrations were 33% for calcium, 69% for magnesium, 52% for phosphorus and 60% for citrate.

Introduction Ac cording to F AO statistics, there are 17 million camels in the world, of which 12.2 million are in Africa and 4.8 million in Asia (22). The camel is a potentially important source of milk. Indeed, in some countries hosting large camel populations, camel milk is one of the main components of the human diet. Milk production varying between 1,800 and 12,700 kg during a lactation period between 9 and 18 months has been reported (13). Information on the characteristics of camel milk is limited. Data available show, however, significant differences between cow and camel milk proteins in properties such as electrophoretic mobility, molecular size (8) and rennet coagulation (7). While a considerable amount of data is available on micellar casein of bovine milk, very little is known about casein micelles of camel milk. Ali and Robinson (2) have analyzed the size distribution of casein micelles in six samples of camel milk. They determined a number average diameter of 160 nm on electron micrographs of ultra-thin sections. This value, however, overestimates the true mean, because particles with diameters smaller than 14 nm could not be measured. It was therefore considered useful to determine the complete size distribution of casein micelles in camel milk by using freeze-fracture replica of cryo-fixed samples and to compare it to that observed in milk of other species. The freeze-fracture technique allows counting and sizing of the smallest casein micelles including submicelles. Other basic data on the chemical composition of camel milk are also given.

Initial paper received February 22, 1989 Manuscript received June 22, 1989 Direct inquiries to Z. Farah Telephone number: 41-1-256 5378

Materials and Methods Milk samples Camel milk samples were taken at Ngare Ndare Camel Farm which is situated just north of the equator in Kenya's Laikipia District, at an altitude of 1,730 to 1,890 m above sea level. The animals of indigenous breed (Camelus dromedarius) were all fed exclusively by grazing. The milk samples A and B were collected from 10 individual camels, on two different occasions. On each occasion, the 10 milk samples were pooled, kept refrigerated, and transported to our laboratory within 36 hours. Upon arrival, the milk samples were skimmed, freeze-dried and stored in sealed plastic bags until analysis. Two individual fresh milk samples (numbers 52 and 56) were also used for the analysis. For these samples the time

Key words: camel milk, casein micelles, electron microscopy, size distribution, amino acids, mineral salts

211

Z. Farah and M. Rأ¼egg Table 1. Average chemical composition of camel and cow's milk camel milk Component Dry matter proteinc Total N Casein N Non-casein N Non -protein N Lactose Fat Ash Calcium total Calcium dissolved Magnesium total Magnesium dissolved Phosphorus total Phosphorus dissolved Citrate totald Citrate dissolved
a

cowa sx 0.7 0.29 21 x 13 3.5 431b 76b 24b 5.5b 0.37 0.32 0.04 9 0.8 4 3
c

unit g I 100 g g 1100 g 6.7 mg/100 g % of TN % of TN % of TN g 1100 g g 1100 g g/100 g mg/100 ml % of total mg/100 ml % of total mg/100 ml % of total mg/100 ml % of total
b

x 12.2 3.11 418 76 24 6.7 5.24 3.15 0.80 157 33 8.3 69 104 52 177 60

4.6 3.8 0.72 117 32 11 66 66 53 175 92
d

Walstra and Jenness (20);

Jenness and Patton (12);

N x 6.38;

as citric acid.

elapsed between collection and examination was 36 hours. Chemical analysis Total solids, fat, protein, lactose and ash were determined according to AOAC standard methods (4). The nitrogen distribution in the milk was determined by the procedure of Aschaffenburg and Drewry (5). The following N fractions were determined: total protein nitrogen (TN), non-casein nitrogen (NCN) and non-protein nitrogen (NPN), soluble in 12% trichloracetic acid. The amount of casein nitrogen (CN) was calculated by difference. In order to study the distribution of salts between the dissolved and colloidal phases in milk, it was filtered through a diaflo ultrafiltration membrane (Amicon PM10). The ultrafiltration was carried out under nitrogen at a pressure of 0.35 MPa. In both the original milk and the collected ultrafiltrate the following minerals were determined: calcium and magnesium by atomic absorption spectrophotometry (19), phosphorus by the phosphomolybdate method described in the International Dairy Federation Standard (11) and citrate enzymatically by using a commercially available test kit (Boehringer, Mannheim, West Germany, catalog number 139076). For amino acid analysis, casein was precipitated from skimmed milk with 0.01 mol/l acetic acid at pH 4.5 - 4.6. The precipitate was washed three times with water and freeze-dried. 20-30 mg of this acid casein were hydrolyzed with 6 mol/l HCI for 24 hours at 110آ°C under vacuum. The hydrolysate was analyzed on a model Liquimat III amino acid analyzer (Kontron Instruments AG, Zurich) according to the procedure of Amado et al. (3).

Electron microscopy The reconstituted and fresh skimmed milk samples were cryo-fixed using the propane jet-freezing technique. This technique basically involves the rapid freezing (approximately 10,000 K.s-1) of a very low mass specimen in a jet of liquid propane at 88 K (14, 15). Freeze-fracture replicas were then obtained as described earlier (16). Fourteen to sixteen electron micrographs of each sample were taken at a magnification of approximately 20,000x and the negatives were enlarged 2.6 times for counting and classifying the particles. The total surface area of milk observed for the four samples was 742 micrometers2. 6,618 particles were counted on this surface. A diameter class width of 20 nm was chosen for the classification of the particles on the prints. A transparent sheet with bars corresponding to the different size classes was placed over the prints. The size class of each particle was found by fitting it into the appropriate diameter range. Particles smaller than about 5 nm in diameter were not considered. Statistical analysis Conversion of the observed size distribution of plane sections into real distribution of spherical particles was made using a method proposed by Goldsmith (10). The original FORTRAN program was modified and translated into GWBASIC for use on MS- DOS microcomputers. Copies of the program are available on request from one of the authors (M.R.). A slice thickness of 5 nm was assumed. Preliminary calculations revealed rather broad size distributions with relatively low frequencies in the larger size classes. The class width was therefore increased from 20 to 40 nm.

212

Casein micelles in camel milk Amino acid composition of whole casein from camel and cow's milk % amino acid Constituent camel cowa Aspartic acid 7.28 6.52 Threonine 4.87 4.42 Serine 5.39 5.75 Glutamic acid 21.26 20.35 Proline 11.62 10.33 Glycine 0.90 2.27 Alanine 1.98 2.80 Valine 5.43 6.48 Cysteine 0.02 0.65 Methionine 2.70 2.51 Isoleucine 6.23 5.54 Leucine 10.89 8.41 Tyrosine 3.84 5.59 Phenylalanine 4.01 4.73 Lysine 6.53 7.33 Histidine 2.44 2.70 Arginine 4.63 3.62 a Alais and Blanc (1); recalculated on a %-basis. Tryptophane was not determined. ------------------------------------------------The equations used to calculate the various mean diameters (dn, dv, dvs, dvm)' the distribution width (cs) and volume fraction (v) are explained in detail elsewhere (16). It should be remembered, that the number average and volume average diameters dn and dv are sensitive to shape, errors at both ends of the distribution function, and total number of particles. These values are meaningful only for corresponding symmetrical distribution curves. The weighted mean diameters dvm and dvs are more useful averages for the characterization of the casein micelle distributions. The distribution width Cs corresponds to the coefficient of variation of the surface-weighted distribution (21). Distribution curves from different samples were compared using a standard chi-square test for multiway frequency tables.

Fig. 1. Freeze-fractured casein micelles in camel milk (cm: casein micelles; sm: submicelles).

Fig. 2. Number of particles observed in freeze-fractured camel, cow's and human milk. The ordinate is logarithmic and gives the number of particles per mm2 fractured area and per nm class width. The amino acid compositions of pooled camel and cow's milk casein are presented in Table 2. A similar pattern can be observed for both species. The most pronounced differences were found for glycine and cysteine, both being significantly lower in camel milk casein. Size distribution of casein micelles Fig. 1 shows a typical electron micrograph of casein particles in freeze-fracture replica of camel milk. The mean diameter of the submicelles was on the average 15 nm. This is a rough estimate, because of uncertainties in the technique (plastic deformation of proteins etc.). The average number of particles observed on such freeze fractured surfaces is shown graphically in Fig. 2. The ordinate gives the normalized frequency of particles per unit area, i.e., the average number of particles per mm2 fractured area and per nm class width. The distribution is significantly broader than that of cow's or human milk and shows a greater number of large particles.

Results and Discussion Chemical composition of camel milk Table 1 shows data on the chemical composition of the camel milk used for this study. Values for cow's milk from the literature (12, 20) are presented for comparison. In general, the gross composition of camel and cow's milk is similar. The values of CN, NCN and NPN expressed as percentage of the total N appear also to lie in the same ranges. Concentrations of calcium, magnesium, phosphorus and citrate, along with their partition between the dissolved and colloidal phases are also given in Table 1. As generally reported in the literature (9, 12), about one third of the calcium and phosphate, 75% of magnesium, and 90% of the citrate of fresh cow's milk are present in the serum phase. In camel, milk the distribution of calcium, magnesium and phosphorus is similar. However, the amount of citrate in the serum phase was found to be lower in camel milk.

213

Z. Farah and M. Rأ¼egg Table 3. Size distribution of casein micelles in camel milk com pared to cow's milk herd milk Parameter Average micelle diameter dn ' number average dv ' volume average dvs' volume/surface av. dvm' weight average Distribution width Cs % of dvs Volume fractionc, v Submicelles nm b b b b dn , number average * A and B: pooled samples, freeze-dried and reconstituted; 52 and 56: fresh samples. b 14-16 nm; a From Rأ¼egg et al. (16) and Schmidt et al. (17,18); 15 14- 16 10- 11 % % 86 3.2 83 2.6 93 2.4 104 2.9 93 2.8 80-100 2.4-3.2 60-100 2.0-4.0 nm nm nm nm 28 63 165 288 28 57 131 222 27 51 113 212 26 50 114 237 27 55 129 243 26- 28 50- 63 113-165 212-288 21- 24 44- 50 90-100 104-140 unit A* B* individual 52* 56* pooled data ranges camel Cowa

c calculated from size distribution

Fig. 3 (at left). Size distribution of casein particles in camel milk compared to cow's and human milk (volume frequency histogram). Fig. 4 (to the right). Cumulative particle volume distribution of casein micelles in camel milk (pooled data from two individual and two herd milks). The differences between the distribution curves of the two individual camel milks and the herd milk samples were most pronounced in the diameter range of about 200 to 500 nm. However, the differences were statistically not significant. The particles in the lowest size class with diameters smaller than 40 nm comprise about 80% of the observed total number of particles but represent only 4-8% of the mass or volume of the casein in camel milk. It is therefore meaningful to consider the weight or volume frequency distribution. Fig. 3 shows the volume frequency of the pooled data of the four milk samples, compared again with the distributions found in cow's and mature human milk (16). The volume distribution curve of casein micelles in camel milk is broad and shows a maximum around 280 nm. As can be deduced from the cumulative distribution curve in Fig. 4, micelles with diameters between 125 and 310 nm comprise about 50% of the volume or mass of the casein. Some statistical data derived from the distribution curves, such as mean diameters, width of the distribution, and volume fraction are summarized in Table 3. For comparison, the ranges of the corresponding values for cow's milk are also included. In earlier investigations, camel milk, after rennet addition, was found to coagulate 2 - 3 times slower than cow's milk. The coagulum obtained was a precipitate in the form of flocks and no homogeneous clot formed (7). The present investigation revealed a relatively broad size distribution of casein micelles in camel milk with a greater number of large micelles

214

Casein micelles in camel milk than in cow's milk. The poor rennetability could be related to these differences in the size of casein particles. Coagulation time varies with the micelle size and reaches an optimum in the medium and small size micelles. This appears to be related to the availability of k -casein. The content of k -casein decreases with increasing micelle size (6, 20). From the results obtained it can be concluded that camel milk casein differs from cow's milk casein in terms of micellar size distribution. However, it would be premature to discuss the impact of this difference in relation to the preparation of products from camel milk. Various biochemical aspects must also be considered and additional studies are necessary to correlate any special feature of product structure with the findings in this investigation. Acknowledgements The authors express special thanks to Dr. E. Wehrli, ETH Zurich, for the preparation of the freeze-fracture replicas and to Mrs. M. Farah and U. Moor for their help in the analysis of the micrographs. References 1. Alais C, Blanc B. (1975). Milk proteins, biochemical and biological aspects. World Rev. Nutr. Diet. 20, 66-166. 2:" Ali MZ, Robinson RK. (1985). Size distribution of casein micelles in camel's milk. J. Dairy Res. 52, 303-307. - 3. Amado R, Rothenbأ¼lhler E, Arrigoni E, Solms J. (1983). Abbau von Purinderivaten zu Glycin durch saure Hydrolyse. Mitt. Gebiete Lebensm. Hyg. 74, 2330. 4. AOAC. (1970). Official Methods of Analysis. Ass. Offic. Anal. Chern. Washington D. C., 11th ed. Methods nr. 16.032, 16.034, 16.035, 16.051 and 16.052. 5. Aschaffenburg R, Drewry I. (1959). New procedure for routine determination of various non -casein proteins in milk. XV. Int. Dairy Congr. 3, 16311637. 6. Eksterend B, Larsson M, Perlmann C. (1980). Casein micelle size and composition related to the enzymatic coagulation process. Biochim. Biophys. Acta 660, 361-366. - 7. Farah Z, Bachmann M. (1987). Rennet coagulation properties of camel milk. Milchwissenschaft 42, 688-692. - 8. Farah Z, Farah M. (1985). Separation and characterization of major components of camel milk casein. Milchwissenschaft 40, 669-671. 9. Fox PF. (1982) Developments in Dairy Chemistry. I. Applied Science Publishers, London, p. 157. 10. Goldsmith PH. (1967). The calculation of true particle size distributions from the sizes observed in a thin slice. Brit. J. Appl. Phys. 18, 813830. 11. International Dairy Federation (1967). IDF Standard No. 42. IDF, Square Vergote 41, B-1040 Brussels, Belgium. 12. Jenness R, Patton S. (1959). Principles of Dairy Chemistry. John Wiley & Sons, London, pp. 2, 115, 170. 13. Knoess KH. (1979). Milk production of the dromedary. In: Camels. IF Symposium Sudan, pp. 20114. Mأ¼ller M. (1979). Elektronenmikroskopische Techniken auf der Basis von der Gefrierfixation. Jahrbuch der Schweiz. Naturforsch. Ges., Wiss. Teil, 34-39. 15. Mأ¼ller M, Meister N, Moor H. (1980). Freezing in a propane jet and its application in freeze fracturing. 1980. Mikroskopie (Wien) 36, 129-140. 16. Rأ¼egg M, Blanc B. (1982). Structure and properties of the particulate constituents of human milk. A review. Food Microstructure 1, 25 -4 7. 17. Schmidt DG, Koops J, Westerbeek D. (1977). Properties of artificial casein micelles. I. Preparation, size distribution and composition. Neth. Milk Dairy J. 31, 328-341. - 18. Schmidt DG, Walstra P, Buchheim W. (1973). The size distribution of casein micelles in cow's milk. Neth. Milk Dairy J. 27, 128-142. 19. Schweizerisches Lebensmittelbuch. Kapitel Milch (1987). Methode Nr. 5.1.1. Eidgenأ¶ssische Drucksachen - und Materialzentrale, CH -3000 Bern, Switzerland. 20. Walstra P, Jenness R. (1984). Dairy Chemistry and Physics. John Wiley & Sons, New York. P. 7, 43, 233. 21. Walstra P, Oortwijn H, de Graf JJ. (1969). Studies on the milk fat dispersions. I. Methods for determining globule-size distributions. Neth. Milk Dairy J. 23, 12-36. 22. Yagil R. (1982). Camels and camel milk animal production and health. Paper FAO 26, 14.

Discussion with Reviewers W. Buchheim: Apparently reconstituted (freeze-dried) skim milk was used for electron microscopy work. Is there any danger that freeze-drying might affect size, shape, and distribution of micelles? P. Resmini: It is written that both fresh and freezedried milk samples have been analyzed, but no data are reported concerning these two different products. Freeze-fracturing techniques suggest that the usual freeze-drying of liquid milk may modify the structure of casein micelles, due to the low freezing rate that promotes ice crystal formation inside the micelles, therefore freeze-drying of milk does not seem to be a suitable technique for ultrastructure studies of casein. Please comment. Authors: The freeze-dried samples were reconstituted to 12.2 % dry matter at 30 - 35آ°C. There is a certain risk that freezing and thawing or reconstitution of the freeze-drying affects the structure of casein particles. To our knowledge, no statistically significant differences between size distribution in fresh and reconstituted preparations has been reported in the literature and no significant difference was observed in the present investigation.

W. Buchheim: In my opinion, the number and sizes of micelles and non-micellar casein, visible in Fig. 1 contradict the frequency values given in Fig. 2 because the micrograph shows approximately equal number of small particles and cross-sections of large micelles, instead of 100- or 1000-fold. Authors: Fig. 1 is not a "random picture". A sector has been chosen which shows both large and small micelles. Therefore, the size distribution on this Fig. cannot be used to estimate the real distribution. The area of Fig. 1 represents about 7.9 micrometers2.

215

Z. Farah and M. Rأ¼egg This is only about 1/100th of the total area that has been measured. W. Buchheim: In case that the amount of non-micellar casein ("submicelles") has been overestimated, some average values (e.g., dn, dv' and even dvs) would be too small. According to reviewer's own experience (see e.g., Food Microstructure 5(1), 181192, (1986آ» direct determination of dvs from micrographs (via circumferences and areas of particles) is the best way for testing such possible discrepancies. Authors: The unweighted mean diameter dn and to some extent the other measures of the mean which are based on the lower moments of the distribution function are sensitive to both ends of the distribution as well as to the total number of the particles counted. The higher the power of the moments, the less is the sensitivity to the uncertainty in the estimation of the smallest particles. dvm is therefore the most robust estimate of the mean diameter. Considering the very broad size distribution of the casein particles in camel milk, the meaning of an "average diameter" should not be overestimated. W. Buchheim: I have some doubts as to how meaningful size values for so-called submicelles are. Protein molecules are plastically deformed when freeze-fractured, so that we identify primarily only their existence in the plane of cleavage. Slightly modified fracturing and shadowing conditions may influence their apparent size so that measurements of "diameters" and comparisons in different experiments are questionable. P. Walstra: Conclusions about the size of submicelles are, in my opinion, rather questionable because of the uncertainties in the technique. Authors: We agree with the reviewers' comment. The diameter of the submicelles is a rough estimate. It has mainly been added for comparison and because of the pronounced difference to that of cow's milk.

216

pdf

Z. Farah

25

1
Milk
Z. Farah

1.1

Milk production
It is difficult to estimate the daily milk yield of a camel under pastoralist conditions owing to the inconsistency of milking frequency. Milk yield also varies with species, breed, stage of lactation, feeding and management conditions. The length of lactation can vary from 9 to18 months. This depends mainly on the husbandry practices, which are largely determined by the need for milk, more being required in the dry months than in the wet months when other sources of food are available. Estimates of milk yields from various countries are given in Table 1.1. The data are highly speculative and should be considered as guidelines for milk yields under pastoral conditions. It must also be noted that throughout lactation calves are still suckling and therefore the actual volumes of milk secreted are higher than the figures presented in the table.
Table 1.1: Milk yields of camels reported from various sources
Country Algeria Ethiopia India Kenya Pakistan Somalia Tunisia Average daily yield in kg 4 5 6.8 4.5 8 5 4 Lactation length in months 9–16 12–18 18 11–16 16–18 9–18 9–16 Calculated yield in kg per 365 days 1460 1825 2482 1643 2920 1825 1460

26

Milk

Camel’s milk is generally opaque white. It has a sweet and sharp taste, but sometimes it can be salty. The taste generally depends on the type of fodder and availability of drinking water. The pH of camel’s milk ranges from 6.2 to 6.5 and the density from 1.026 to 1.035. Both density and pH are lower than those of cow’s milk. Compared to cow’s milk, camel’s milk sours very slowly and can be kept longer without refrigeration.

1.2

Milk composition
The composition of camel’s milk quoted from various sources and the corresponding values from other animal species are presented in Table 1.2 below. There are greater variations in constituents of camel’s milk than in cow’s milk. Camels are known to produce diluted milk in hot weather when water is scarce. The main difference between cow’s and camel’s milk lies in the different physicochemical characteristics of the individual components (protein, lipids, ash, etc.).
Table 1.2 : Gross composition of milk from various animal species
Species Moisture Camel Cow Goat Sheep Human 86–88 86–88 87–88 79–82 88.0–88.4 Percentage composition Fat 2.9–5.4 3.7–4.4 4.0–4.5 6.9–8.6 3.3–4.7 Lactose 3.3–5.8 4.8–4.9 3.6–4.2 4.3–4.7 6.8–6.9 Protein 3.0–3.9 3.2–3.8 2.9–3.7 5.6–6.7 1.1–1.3 Ash 0.6–1.0 0.7–0.8 0.8–0.9 0.9–1.0 0.2–0.3

Milk proteins
Proteins represent one of the greatest contributions of milk to human nutrition. They perform a variety of functions in living organisms ranging from providing structure to reproduction. The main components of milk proteins are casein and whey. Casein is found in no products other than milk. Casein is precipitated when milk sours or when acid or rennin added. In cheese-making, most of the casein is recovered with the milk fat. In camel’s milk, the value of casein is the lower limit of casein content of cow’s milk and varies between 72% and 76% of total protein. Casein is present in milk in the form of finely divided particles similar to clay in muddy water. The particles contain, beside the protein, considerable amounts of calcium phosphate. The most observed particles in cow’s milk casein have a

Z. Farah

27

diameter from 40 to 160 nanometre (1 nanometre = 10-7cm). In camel’s milk, casein particles range in diameter from 20 to more than 300 nanometre. The whey protein content in camel’s milk varies between 22% and 28% of total protein, which is slightly more than in cow’s milk.

Milk fat
Milk fat serves nutritionally as an energy source, acts as a solvent for the fatsoluble vitamins and supplies essential fatty acids. About 99% of milk fat is a mixture of fatty acids (triglycerides) of varying chain length from 4 to 20 carbon atoms. The fatty acids are divided according to the linkage of the carbon atoms into saturated and unsaturated fatty acids. In saturated fatty acids the carbon atoms are linked in chain by single bonds, in unsaturated fatty acids by one or more double bonds. The bulk of the fat in milk exists in the form of small spherical globules of varying sizes. The surface of these fat globules is coated with a thin layer known as a fat globule membrane, which acts as an emulsifying agent for the fat suspended in milk. The membrane protects the fat and prevents the globules coalescing into butter grains and can be broken by mechanical action. The fat content of camel’s milk varies between 2.9% and 5.4% and the average size of the fat globules is about the same as cow’s milk fat globules. According to present knowledge, the main differences between the fat in cow’s milk and camel’s milk are as follows:
1. Natural creaming of camel’s milk differs markedly from that of cow’s milk. On standing, camel’s milk creams less rapidly and completely than cow’s milk and no skimmable cream can be obtained even after standing for several days. 2. Compared to cow’s milk fat, camel’s milk fat contains less short-chain fatty acids. Long chain unsaturated fatty acids occur to about the same extents in both. 3. Butter can be obtained from camel’s milk only at high churning temperature of 20آ°C to 25آ°C. These values are considerably higher than that of cow’s milk, which normally vary between 8آ°C and 12آ°C. 4. The mean melting point of camel’s butter is around 41.5آ°C and is on average 8آ°C higher than that of corresponding values in cow’s milk butter.

Lactose
Lactose is the major carbohydrate fraction in milk and is a source of energy for the young calf. It is made up of two sugars, glucose and galactose, which are fermented to lactic acid when milk goes sour. The lactose content in camel’s milk ranges from 4.8% to 5.8% and is slightly higher than the lactose in cow’s milk. It seems that the lactose content in camel’s milk is relatively constant throughout lactation.

28

Milk

Mineral salts and vitamins
Milk mineral salts are mainly chlorides, phosphates and citrates of sodium, calcium and magnesium. Although salts comprise less than 1% of the milk, they influence its rates of coagulation and other functional properties. The mineral content of camel’s milk expressed in ash ranges from 0.6% to 0.8%. There is still little information about the mineral content of camel’s milk. Data available however, indicate that camel’s milk is rich in chloride and phosphorous, and low in calcium. Camel’s milk contains less vitamin A, B1, B2, E, folic acid and pantothenic acid than cow’s milk while the content of vitamin B6 and B12 is about the same level. The content of niacin and vitamin C is substantially higher than that of cow’s milk. In particular the high level of vitamin C in camel’s milk has been confirmed by several studies. The availability of a relatively fair amount of vitamin C (range reported in the literature 25–60 mg/l) in camel’s milk is of significant relevance from the nutritional standpoint in the arid areas where fruits and vegetables containing vitamin C are scarce.

Further reading Milchwissenschaft. (1985) 40 (11), 669-671 Journal of Dairy Research (1998) 65, 209-222 Food Microstructure (1989) 8, 211-216 Journal of dairy research.(1992) 59, 229-231 Journal of Dairy Science (1991) 74 , 2901-2904 International Journal of vitamin and Nutrition Research (1992) 62, 30-33

pdf

International Dairy Journal 9 (1999) 481}486

Sequence analysis of camel (Camelus dromedarius) lactoferrin
Stefan R. Kappeler*, Manfred Ackermann, Zakaria Farah, Zdenko Puhan
Laboratory of Dairy Science, Institute of Food Science, Swiss Federal Institute of Technology, CH-8092 Zurich, Switzerland Received 25 January 1999; accepted 5 May 1999

Abstract The aim of this study was to characterise camel lactoferrin in terms of primary structure and molecular weight. The protein was eluted from a heparin-sepharose column at a sodium chloride concentration of 0.5 M, and corresponded to bovine lactoferrin in terms of N-terminal sequence and the molecular weight of 80.16}80.73 kDa. Lactoferrin cDNA was PCR ampli"ed, using a cDNA library from lactating mammary gland of a Somali camel. The sequenced clone had a length of 2337 bp and an open reading frame of 2124 bp, which coded for a protein of 708 amino acid residues. The mature protein had a length of 689 amino acid residues, a calculated molecular weight of 75.250 kDa and a calculated isoelectric point at pH 8.14. Primary structure identity to bovine lactoferrin was 74.9%. Concentration of lactoferrin in whole, late-lactational milk was 220 mg l\, which was higher than the lactoferrin concentration in comparable bovine milk, which was 140 mg l\. 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Camelus dromedarius; Lactoferrin

1. Introduction Lactoferrin is a protein of the innate immune system, which is also expressed in the lactating mammary gland. It is found in milk and di!erent other body secretions, and in neutrophil leukocytes (Masson, 1970). The concentration in milk strongly depends on the species and the stage of lactation. Industrial scale puri"cation from whey is carried out by cation exchange, and use as a preserving agent in food, drugs and cosmetics has been proposed (Saito, Takase, Tamura, Shimamura & Tomita, 1994). Lactoferrin belongs to the family of transferrins, together with blood serotransferrin (siderophilin), egg white ovotransferrin (conalbumin), melanotransferrin of malignant melanoms, the porcine inhibitor of carbonic anhydrase, and other proteins. The common property of this protein family is the binding of two metal cations, preferably Fe>, at structurally closely related binding sites. Most proteins of the transferrin type are needed for storage or transport of iron. It can be assumed, that lactoferrin in colostral milk acts as an iron scavenger, which depletes the milk from free iron and thereby slows down microbial growth.

* Corresponding author.

Brock (1997) proposed, that the in vivo function of apolactoferrin is the prevention of iron-mediated lipid peroxidation, a property, which was already demonstrated with monocytes. This function is based on the ability of lactoferrin, to bind to cell membranes. The higher a$nity for iron, as compared to other transferrins, would enable it to function at the reduced pH found in the stomach and upper intestine. The high resistance of apolactoferrin to proteolysis, compared with other apotransferrins, would enable it to maintain its iron-binding potential in the face of proteolytic activity in the gut. Since diferric lactoferrin was reported to be even more resistant to proteolysis, it was supposed, that the iron}lactoferrin complex would resist degradation, and was sequestered by hepatocytes, or was excreted from the gut. A higher lactoferrin concentration also could help to prevent lipid peroxidation by free radicals in an infected udder, which has an elevated iron content. Iron-saturated lactoferrin, which is found in milk from the second week to the end of the lactational period, may primarily prevent microbial growth in the gut. This would help the new-born, which is easily infected, to survive the "rst weeks, until its own immune system becomes developed, and the gut becomes adapted to food digestion. Iron-saturated lactoferrin could also be a source of iron for the suckling, once the protein is degraded in the gut.

0958-6946/99/$ - see front matter 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 1 7 - X

482

S.R. Kappeler et al. / International Dairy Journal 9 (1999) 481}486

Camel milk was frequently reported to have a high antimicrobial activity, and was shown to slow down the growth of pathogenic bacteria more than bovine milk (Elagamy, Ruppanner, Ismail, Champagne & Assaf, 1992). Antimicrobial properties were partially attributed to well characterised proteins, such as lactoferrin, lactoperoxidase, lysozyme and immunoglobulin A. These proteins were shown to have higher concentrations or higher activity in camel milk, as compared to bovine milk. In this study we determined the basic physico-chemical and structural parameters of lactoferrin, of which the relationship between structure and function is well characterised in human and bovine counterparts.

Molecular masses of proteins were measured by matrix assisted laser desorption/ionization mass spectrometry (Kappeler et al., 1998). 2.3. Quantixcation Lactoferrin eluted from the Heparin-Sepharose column was collected, tenfold diluted in distilled water and the absorbency measured at 280 nm. For calculation of the protein concentration, an extinction coe$cient of 84540 M\ cm\ was used for camel lactoferrin and of 102890 M\ cm\ for bovine lactoferrin (Gill & Von Hippel, 1989). One litre whole milk was estimated to yield 0.8 l whey. 2.4. cDNA Sequence analysis PolyA-mRNA isolation and construction of a cDNA library was done as described in Kappeler et al. (1998). Overlapping fragments of the lactoferrin cDNA, which together covered the complete sequence, were produced by the polymerase chain reaction (PCR). The following protocol was applied to most of the reactions: 2 l of the -cDNA library were taken as templates in 50 l PCR assays with 2.5 units Taq Polymerase (Amersham Pharmacia), which was blended with 0.05 units Pfu Polymerase (Stratagene, La Jolla, CA), and 5 l 10; TaqPlus Precision incubation bu!er (Stratagene), 20 nmol of each dNTP (Amersham Pharmacia) and 50 pmol of speci"c primers. A series of 30 cycles was run with initial 2 min denaturation at 943C, followed by 10 s denaturation at 943C, 30 s annealing at 553C and 2 min 30 s elongation at 683C. Elongation prolongation was 20 s per cycle. A "nal 10 min incubation step at 723C was added to increase the concentration of full-length products. Each PCR product was generated twice and ligated into a pGEMà¯،-T Easy vector (Promega, Madison, WI) according to the manufacturer's instructions. In case of base reading ambiguities, a third PCR product was generated. Two -gt11 vector speci"c general primers were constructed to cover the 5- and 3-ends of lactoferrin cDNA: -gt11 forward: 5-GACGACTCCTGGAGCCCGTCAGTA-3, -gt11 reverse: 5-CACCAGACCAACTGGTAATGGTAG-3. The following PCR products were generated, mostly with the help of highly conserved regions in the cDNA sequences of other species (mixed base sites according to IUB code): A 0.4 kbp PCR product was generated with 5-CTGTCCCATAGACCTCTGCCGCTA-3, and gt11 reverse.

2. Materials and methods 2.1. Isolation of lactoferrin from whey Pooled milk of Arabian camels was stored at !703C until analysis. After thawing, the milk, which had a pH of about 6.6, was skimmed at 1000 g, 43C for 15 min. Whey was obtained by acid precipitation of casein at pH 4.6 and 373C for 20 min, using 0.1% acetic acid, followed by addition of 10 mM sodium acetate for neutralisation, and centrifugation at 4000 g for 5 min. The supernatant was dialysed twice against double distilled water for 5 h at 43C, and once against 10 mM sodium phosphate bu!er at pH 7.4 for 14 h at 43C, using an autoclaved SPECTRA/POR membrane tubing with a molecular cuto! of 6}8 kDa (Spectrum Medical Industries, Inc., Los Angeles, CA). Prior to chromatography, samples were "ltered through a hydrophilic 0.45 m membrane (ME25; Schleicher and Schuell, Dassel, Germany). A Heparin-Sepharose HiTrap column (1 ml; Amersham Pharmacia, Uppsala, Sweden) was loaded with 40 ml whey. The column was washed with 10 ml PBS (10 mM sodium phosphate, 20 mM sodium chloride, pH 7.4). Elution was performed at ambient temperature by a linear gradient from 0.02 to 1 M sodium chloride over 40 min. The column e%uent was monitored with an UV detector (L-7300; Merck, Darmstadt, Germany) at 280 nm. Proteins eluted were collected manually and lyophilised. Fractions were further puri"ed, prior to micro-sequencing and molecular mass determination, by reversed-phase C HPLC (Kappeler, Farah & Puhan,  1998). Elution was performed by a linear gradient from 0.1% TFA in double distilled, nano"ltered water, to 0.1% TFA in acetonitrile, over 60 min. 2.2. Physico-chemical characterisation Proteins collected from the e%uent of the C -column  were used directly for N-terminal sequencing (Kappeler et al., 1998).

S.R. Kappeler et al. / International Dairy Journal 9 (1999) 481}486

483

A 0.8 kbp PCR product was generated with 5-GTTCRRTGGTGTRCCRTMTCCMMA-3, and 5-GTCTTTGAACAGCAGGTCCTTCTG-3. A 1 kbp PCR product was generated with 5-TTCCAGCTCTTTGGCTCYCC-3, and 5-TTGAACAGAAGGTTTTTGGT-3. A 0.4 kbp PCR product was generated with 5-CCAGGCAAGTTTTGCTTGTTCCAG-3, and gt11 reverse. The ligation products were dialysed and transformed into E. coli XL1-Blue (Stratagene) by electroporation with a Gene-Pulserà¯، (BioRad, Hercules, CA) at 2.5 kV, 25 FD, and 200 in 0.2 cm cuvettes. The transformed bacteria were plated overnight at 373C on IPTG/XGal/Ampicillin-selective agar. White colonies were picked and grown overnight in 20 ml LB-Ampicillin 100 (Maniatis, Sambrook & Fritsch, 1989). Plasmid DNA was puri"ed for #uorescent sequencing with the Wizard Plus SV Minipreps DNA Puri"cation System (Promega). Fluorescent sequencing was carried out using an ALF automated device (Amersham Pharmacia) with standard operating procedures. Sequencing samples were prepared, using the Cy52+-dATP labelled, vector speci"c primers: Cy5-SP6: 5-TACTCAAGCTATGCATCCAACGCG-3, and Cy5-T7: 5-ACTCACTATAGGGCGAATTGGGCC-3 and the Thermo Sequenase cycle sequencing kit RPN 2438 (Amersham Pharmacia) according to the manufacturer's instructions. The following 25 cycles were applied: 953C, 30 s, 503C, 30 s, 723C, 50 s. Where sequencing results di!ered, a third PCR product was sequenced. Overlapping sequences were detected using the FASTA module of the gcg/egcg programme package (Genetics Computer Group, Madison, WI). Consecutive sequences were joined and vector speci"c sequences removed. In this way, complete cDNA sequences were obtained. 2.5. Computational sequence analysis Alignments of DNA and protein sequences and DNA similarity searches were performed using the gcg/egcg programme package (Genetics Computer Group). Protein sequence similarity searches against the Swissprot database (Swiss Institute of Bioinformatics, Geneva, Switzerland) were made using a Smith and Waterman algorithm with default values (Barton, 1997). A low-resolution model of the tertiary structure of camel milk lactoferrin was obtained by comparative modelling (Guex & Peitsch, 1997). The primary structure,

which was revealed by amino acid and cDNA sequencing, was threaded over resolved tertiary structures of di!eric lactoferrins from other species. Multiple sequence alignments were made for improvement of modelling reliability. Energy minimisation of the model was done with force "eld computation by GROMOS96. 3. Results 3.1. Primary structure PCR ampli"cation products of a full-length cDNA clone of camel lactoferrin were sequenced (EMBL/GenBank2+ accession number AJ131674). The clone was 2337 bp long, and contained a 5-untranslated region of 22 bp and a 3-untranslated region of 191 bp. The 5untranslated region contained a partial Kozak-box (Kozak, 1989) in front of the translational start site ATG, with a purine at !3 bp, and cytosines at !1 bp, !5 bp and !8 bp. The 3-untranslated region contained a polyadenylation signal AATAAA. The open reading frame ranged from A to G, and coded for a polypeptide of 707 aa residues. The start site of the mature protein was determined by similarity as Ala. The 19 aa signal peptide was to 94.7, 84.2 and 78.9% identical to the respective signal sequences of bovine, porcine and human lactoferrin. The mature protein was 689 aa residues long, and had a molecular weight of 75.250 kDa (Table 1), without postranslational modi"cations. The isoelectric point of the unmodi"ed peptide was at pH 8.14. The protein shared 74.9% sequence identity with bovine, 74.5% with porcine, and 74.0% with human lactoferrin. The pronounced homology gave indication for a nearly identical biological function of the proteins. 3.2. Glycosylation N-linked glycans contribute about 4}11% (3}9 kDa) to the total mass of bovine lactoferrin, which is about
Table 1 Physicochemical characteristics of camel and bovine lactoferrin Camel Amino acid residues Molecular mass (kDa) based on mass spectrometry Molecular mass (kDa) based on amino acid sequence Isoelectric point Concentration in milk (mg l\) Sequence identity 689 80.16}80.73 75.250 8.14 220 74.9% Bovine 689 84.0 76.143 8.18 140

Data on bovine lactoferrin after Schanbacher, Goodman and Talhouk (1993), Yip and Hutchens (1997). Calculated with the gcg programme (Genetics Computer Group, Madison, WI 53711, USA).

484

S.R. Kappeler et al. / International Dairy Journal 9 (1999) 481}486

84.0 kDa (Spik, Coddeville, Mazurier, Bourne, Cambillaut & Montreuil, 1994); Yip & Hutchens, 1997). Glycosylation enhances the solubility of the secreted protein and may help to bind at speci"c cell types, such as liver parenchymal cells (Ziere, Bijsterbosch & Van Berkel, 1993). Camel milk lactoferrin was found to contain 6.2% carbohydrates in colostral milk and 5.6% in milk collected 15}30 days after parturition (Mahfouz, ElSayed, Abd El-Gawad, El-Etriby & Abd El-Salam, 1997). The content of N-acetyl-glucosamine in camel milk lactoferrin was markedly higher than in ruminants' milk lactoferrins (3.35% in colostral camel milk compared to about 1.75% in colostral ruminants' milk, Mahfouz et al., 1997). In our study, the carbohydrate content of lactoferrin from end-lactational milk was 6.2}6.8% of total protein mass, calculated as a di!erence between the protein mass measured by MALDI-MS and the protein mass of the amino acid sequence (Table 1). Possible glycosylation sites, based on pattern analysis (Gavel & Von Heijne, 1990), are Asn, Asn, Asn and Asn. In bovine lactoferrin, four of "ve sites with N-glycosylation potential, Asn, Asn, Asn, and Asn, are glycosylated (Spik et al., 1994), and contribute to an overall carbohydrate content of 11.2%. Degree of glycosylation in human lactoferrin is about 6.40% (Spik et al., 1994), and thus similar to camel lactoferrin. Human lactoferrin contains 2 glycosylated sites, Asn and Asn, with glycans of the N-acetyllactosaminic type, which were also found in camel lactoferrin. By comparison with bovine and human lactoferrin, glycosylation of two of the four possible sites of camel lactoferrin is proposed (Fig. 1). 3.3. Concentration in camel milk Colostral camel milk was reported to have an extremely high lactoferrin content of 5.10 g l\ on the second day after parturition, compared to about 0.50 g l\ in bovine colostral milk. After 30 days of milking, the lactoferrin level in camel milk went down to 0.34 g l\, whereas in bovine milk, only about 0.06 g l\ were found (Abd El-Gawad, El-Sayed, Mahfouz & Abd El-Salam, 1996). In our studies, we used an extinction coe$cient of 84540 M\ cm\ at 280 nm to calculate a lactoferrin concentration of 0.22 g l\ in a milk sample, which was taken at the end of the lactation period, 360 days after parturition. In a sample of pooled cow milk, lactoferrin concentration was 0.14 g l\. If it is assumed, that the main function of lactoferrin in milk is the inhibition of bacterial growth, a di!erently composed micro#ora in the gut of the new-born could be a reason for the apparently higher lactoferrin concentration in camel milk. Nevertheless, it has to be taken into account, that the concentration of protective proteins in milk also depends on the milk yield, which was about 5 l d\ for the camels, and about 15 l d\ for the cattle studied. It also has to be considered that the

immunological situation, with regard to the placenta type, the colostrum, the development and stimulation of the immune system in the calf, and the nutritional properties in general, will strongly di!er between both species, as a result of the di!erent habitats, in which the animals live, adaptation of camels to a sub-optimal food supply and quality, di!erences in the way, the o!spring is raised, and the more distant paleontological relationship. 3.4. Tertiary structure and ligand binding The polypeptide chain of transferrins consists of about 700 amino acids and is folded into two, tandemly arranged, asymmetrical metal binding sites, designated as N- and C-lobes, which probably evolved by gene duplication. The sequence of the camel lactoferrin N-lobe, which extended from Val to Arg, shared 39.8% sequence identity with the sequence of the C-lobe, which ranged from Val to Arg. Under physiological conditions, transferrins bind one Fe> cation in each lobe with a low dissociation constant of about 10\ (Brock, 1997). Cation binding requires synergistic binding of a bicarbonate anion, probably for charge compensation. In bovine and probably also in camel lactoferrin, the side chains of Asp, Tyr, Tyr and His are involved in binding of the cation in the N-lobe (Baker et al., 1998; Fig. 1). Two oxygens from the bidentate CO\ anion are suggested to complete a dis torted octahedral geometry (Anderson, Baker, Norris, Rice & Baker, 1989). In the N-lobe, the side chains of Thr, Arg, and Tyr, and two backbone hydrogens of Ala and Gly, are involved in binding of the bicarbonate anion. Lactoferrin retains its iron binding potential at pH values below pH 5.5, and even in the presence of citrate, in contrast to the other known transferrins (Brock, 1997). The primary structure and a modelled tertiary structure of the binding sites of bovine and camel lactoferrin were found to be nearly identical. We therefore assume that cations are bound by both lobes of the camel protein with similar a$nities as in bovine lactoferrin. 3.5. Bacteriostatic activity of the N-terminal end A high amount of Arg and Lys are clustered at the N-terminal end of lactoferrin, near and between a loop, which is formed by disulphide bonding of Cys and Cys (Fig. 1). The N-terminus of human and bovine lactoferrin was found to have strong bacteriostatic activity on gram-negative bacteria, as a result of non-speci"c binding to the negatively charged outer bacterial membrane, and subsequent release of lipopolysaccharides, thereby altering the permeability properties (Ellison, Giehl & LaForce, 1988). The N-terminal part of camel lactoferrin contained 13 basic residues (Fig. 1), at sites more similar to bovine lactoferrin than to human

S.R. Kappeler et al. / International Dairy Journal 9 (1999) 481}486

485

Fig. 1. Schematic drawing of the relationship between structure and function in camel and bovine lactoferrin. Signal sequences in italics. Functional residues in bold. -Helical regions designated as & ', -pleated regions as & '. E indicate probably glycosylated asparagine residues (in bold).

lactoferrin. The isoelectric point of the N-terminal fragment from Ala to Ala was at pH 10.98, compared to pH 11.57 for the bovine fragment at the same position, which had one more basic residue. As in bovine lactoferrin, most residues were found in or near the loop. Based

on the high homology between camel and bovine lactoferrin in general, and particularly on the similar grouping of the N-terminal basic residues, we suggest a similar e!ect of camel lactoferrin as observed of bovine lactoferrin. The bacteriostatic activity of camel lactoferrin on

486

S.R. Kappeler et al. / International Dairy Journal 9 (1999) 481}486 Barton, G. J. (1997). SCANPS Version 2.3.1, User Guide. UK: University of Oxford. Brock, J. H. (1997). Lactoferrin Structure*Function Relationships. In T. W. Hutchens, & B. Lonnerdal, Lactoferrin: Interactions and K Biological Functions (pp. 3}23). Totowa, NJ: Humana Press. Elagamy, E. I., Ruppanner, R., Ismail, A., Champagne, C. P., & Assaf, R. (1992). Antibacterial and antiviral activity of camel milk protective proteins. Journal of Dairy Research, 59, 169}175. Ellison, R. T., Giehl, T. J., & LaForce, F. M. (1988). Damage of the outer membrane of enteric gram-negative bacteria by lactoferrin and transferrin. Infection and Immunity, 56, 2774}2781. Gavel, Y., & von Heijne, G. (1990). Sequence di!erences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering. Protein Engineering, 3, 433}442. Gill, S. C., & Von Hippel, P. H. (1989). Calculation of protein extinction coe$cients from amino acid sequence data. Analytical Biochemistry, 182, 319}326. Guex, N., & Peitsch, M. C. (1997). SWISS-MODEL and the SwissPdbViewer: an environment for comparative protein modelling. Electrophoresis, 18, 2714}2723. Kappeler, S., Farah, Z., & Puhan, Z. (1998). Sequence analysis of Camelus dromedarius milk caseins. Journal of Dairy Research, 65, 209}222. Kozak, M. (1989). The scanning model for translation: an update. Journal of Cell Biology, 108, 229}241. Mahfouz, M. B., El-Sayed, E. M., Abd El-Gawad, I. A., El-Etriby, H., & Abd El-Salam, A. M. (1997). Structural studies on colostrum and milk lactoferrins from di!erent species. Egyptian Journal of Dairy Science, 25, 41}53. Maniatis, T., Sambrook, J., & Fritsch, E. F. (1989). Molecular Cloning (2nd ed.). New York: Cold Spring Harbor Laboratory Press. Masson, P. L. (1970). La lactoferrine, proteine des secretions externes et des leucocytes neutrophiles. Brussels: Editions Arsca S.A. Saito, H., Takase, M., Tamura, Y., Shimamura, S., & Tomita, M. (1994). Physicochemical and antibacterial properties of lactoferrin and its hydrolysate produced by heat treatment at acidic pH. In T. W. Hutchens, S. V. Rumball, & B. Lonnerdal, Lactoferrin Structure and K Function (pp. 21}31). New York: Plenum Press. Schanbacher, F. L., Goodman, R. E., & Talhouk, R. S. (1993). Bovine mammary lactoferrin: implications from messenger ribonucleic acid (mRNA) sequence and regulation contrary to other milk proteins. Journal of Dairy Science, 76, 3812}3831. Spik, G., Coddeville, B., Mazurier, J., Bourne, Y., Cambillaut, C., & Montreuil, J. (1994). Primary and three-dimensional structure of lactotransferrin (lactoferrin) glycans. In T. W. Hutchens, S. V. Rumball, & B. Lonnerdal, Lactoferrin Structure and Function (pp. 21}31). K New York: Plenum Press. Yip, T. T., & Hutchens, T. W. (1997). A$nity Mass Spectrometry. In T. W. Hutchens, & B. Lonnerdal, Lactoferrin: Interactions and BioloK gical Functions (pp. 39}58). Totowa, NJ: Humana Press. Ziere, G. J., Bijsterbosch, M. K., & Van Berkel, T. J. (1993). Removal of 14 N-terminal amino acids of lactoferrin enhances its a$nity for parenchymal liver cells and potentiates the inhibition of beta-very low density lipoprotein binding. Journal of Biological Chemistry, 268, 27069}27075.

di!erent bacterial strains was found to have equal strength as the activity of bovine lactoferrin (Elagamy et al., 1992). 3.6. Food preservation Lactoferrin was discussed to be a promising choice for preservation in food and cosmetics, since it is highly stable towards heat treatment and at low pH conditions (Saito et al., 1994). It helps to establish a favourable micro#ora, promotes growth of bi"dobacteria, and may therefore "nd attraction for use in functional food products. The antimicrobial peptides formed upon gastric digestion of lactoferrin are also promising candidates as additives for food preservation. Primary structures of peptides formed from camel lactoferrin should be studied and activity of such peptides on inhibition of bacterial growth tested, to get better understanding of the action of lactoferrin in camel milk. The higher amounts of lactoferrin in camel milk are of advantage for natural preservation of the milk in arid regions, where technology for milk preservation is often not available.

Acknowledgements The authors thank Mr. and Mrs. Breitling of the Kamelfarm Fatamorgana in Rotfelden, Germany, for providing milk samples, and the laboratory group of Prof. M. Teuber for giving the opportunity to do some molecular-biological work in their laboratory.

References
Abd El-Gawad, I. A., El-Sayed, E. M., Mahfouz, M. B., & Abd ElSalam, A. M. (1996). Changes of lactoferrin concentration in colostrum and milk from di!erent species. Egyptian Journal of Dairy Science, 24, 297}308. Anderson, B. F., Baker, H. M., Norris, G. E., Rice, D. W., & Baker, E. N. (1989). Structure of human lactoferrin: crystallographic structure analysis and re"nement at 2.8 A resolution. Journal of Molecular s Biology, 209, 711}734. Baker, E. N., Anderson, B. F., Baker, H. M., MacGillivray, R. T., Moore, S. A., Peterson, N. A., Shewry, S. C., & Tweedie, J. W. (1998). Three-dimensional structure of lactoferrin. Implications for function, including comparisons with transferrin. In G. Spik, D. Legrand, J. Mazurier, A. Pierce, & J.-P. Perraudin, Advances in Lactoferrin Research (pp. 1}14). New York: Plenum Press.

pdf

Farah, Camel milk

Rennet coagulation properties of camel milk
By Z. FARAH and M. R. BACHMANN Labor fأ¼r Milchwissenschaft, Eidg. Technische Hochschule, Zأ¼rich, Switzerland

1.Introduction According to FAO statistics there are 17 million camels in the world, of which 12.2 million are in Africa and 4.8 million in Asia (1). The camel is a potentially important source of milk. Indeed, in some countries hosting large camel populations, camel milk is one of the main components of the human diet. The present knowledge about milk production potential is very limited. However, milk production varying between 1,800 and 12,775 kg during a lactation period between 9 and 18 months has been reported (2). Most camel milk is consumed fresh or when it has just turned sour. Reports on the possibility of obtaining cheese from camel milk are scarce and often contradictory. Some authors report the existence of rennet coagulated cheese made from camel milk while others categorically state that cheese cannot be made from camel milk (3, 4, 5). The present investigation was therefore undertaken to obtain a better insight into the problem of rennet coagulation of camel milk. The study describes the action of rennet on camel milk as well as the effect of pH, temperature, and added calcium chloride on the coagulum development. The release of non protein nitrogen (NPN) by the action of rennet on camel milk has also been studied. 2. Materials and methods 2.1 Milk samples Camel milk samples were taken at Ngare Ndare Camel Farm) which is situated in Kenya's Laikipia District at an altitude of 1,730 to 1,890 m above sea level. The animals of indigenous breed (Camelus dromedarius) were fed year round exclusively by grazing. The milk samples were collected from 10 individual camels. After measuring the pH of the individual samples the milks were mixed to form one batch, skimmed and prepared for coagulation studies in the laboratory. For comparison bulk cow milk was used. 2.2 Milk coagulation activity determination The rennet was commercial powder from Chr. Hansen with an activity of 1:100,000. Rennet solution of 0.4% was prepared and an appropriate amount of this solution was taken to give a visually observed coagulation time of approximately 5 min in cow milk. For measurement of the milk coagulation time the following two methods were used. Visual method 25 ml samples of milk were measured into a 125 ml beaker placed in a water bath at 35آ°C for 30 min. After the addition of appropriate volume of rennet solution, the coagulation time was measured with the visual method described by HOSTETTLER (6). The coagulation time was defined as the time required for the first appearance of graininess in the moving film of milk on the surface of the glass walls.

Formagraph method The coagulation time for pooled camel and cow's milk samples were measured on Formagraph Type 11700 (Foss Electric, Denmark) according to the procedure of McMAHON and BROWN (7). The milks were adjusted to pH 6.65 and equilibrated at 35 آ°C. 2.3 Electron microscopic examination For electron microscopic examination freeze dried reconstituted camel and cow milks were used. After adjusting the pH to 6.65 the milk were equilibrated at 35 آ°C and coagulation monitored by visual observation. Samples were taken before and after adding the rennet at time intervals up to the visually observed coagulation time. Samples were prepared for electron microscopy with the freeze-fracturing technique and without adding any cryoprotectives. The freeze-fracturing was carried out in a modified Batzers BAF 300 unit, at object temperature of -150 آ°C. Details of the procedure have been described by MأœLLER et al. (8). Electron micrographs were made at 25.000 X magnification. The negatives were enlarged 3-5 times, but magnification at 75,000 was used for the interpretation of the results. 2.4 Effect of temperature The milk samples were adjusted to pH 6.65 by slow addition of 1 M HCI, placed in a water bath, and the coagulation time was measured at temperatures over the range at 25 to 40 آ°C. 2.5 Effect of pH All samples were equilibrated at 35 آ°C and the coagulation time determined at pH between 6.25 and 7.00. 2.6 Effect of added calcium All the samples were adjusted to pH 6.65 and placed in a water bath at 35آ°C. Calcium sensitivity was evaluated by measuring the coagulation time after addition of an appropriate amount of calcium chloride. 2.7 Measurement of NPN released by the action of rennin on milk This method is patterned after that described by NITSCHMANN (9). An aliquote rennet solution low enough to give a coagulation time of around 20 min in cow milk was added to 25 ml portions of camel and cow milk samples incubated in a water bath at 35آ°C. At pre-determined intervals trichloroacetic acid was added to each portion to obtain a final concentration of 12 % (w/v) in order to stop the reaction and precipitate the milk proteins. The trichloroacetic acid soluble NPN, split-off during the action of rennet as well as the total nitrogen (TN) were determined by the Kjeldahl method (10).

Milchwissenschaft 42 (11) 1987

Farah, Camel milk

3. Results and discussion pH values and coagulation times of camel and cow milk are presented in Table 1. The lowest pH measured in the 10 individual camel milks was 6.48 and the highest 6.70. The most frequently observed pH values were 6.55 and 6.65. which agreed with camel milk pH values reported in the literature (11). Coagulation times for each individual camel milk sample as well as those of mixtures were measured and compared with bulk cow milk adjusted to pH 6.65. The coagulum obtained was a precipitate in the form of flocks Table 1: The pH values and coagulation times of 10 Individual camel milk samples at 35 آ°c pH 6.48 6.55 6.55 6.56 6.60 6.63 6.65 6.65 6.65 6.70 6.65 (adjusted) 6.65 (adjusted) Coagulation time (sec) 540 600 660 600 720 720 780 720 840 1,020 840 300 Fig. 1: Formagramm of duplicate camel (A), and cow (B milk samples. (pH adjusted 6.65 Rcow = 330 sec, Rcamel = 930 sec)

Camel milk sample No. 1 2 3 4 5 6 7 8 9 10 Mixture 1-10 Cow milk

and no clot was formed. In all the samples the visually observed coagulation time of camel milk was 2 to 3 times longer than that of cow milk. Typical tracing by Formagraph of duplicate cow and camel milk samples are shown in Fig. 1. The coagulation time (R) was determined by measuring the distance from the origin to the point where the baseline began to increase in width. Comparison of the coagulation time measured by the 2 methods showed that the coagulation point for the visual method occurred prior to that of the Formagraph. This is due to the requirement of minimal gel formation to induce pendulum movement by the Formagraph before coagulation can be detected (7). However, the coagulation time recorded by the Formagraph is consistent with that of the visual method and shows that under the applied experimental conditions camel milk coagulated approximately 3 times slower than cow milk.

By the Formagraph method the progress of the curd formation can be detected. This is expressed as the time from the start of gel development until a width of 20 mm is reached (7). Following this definition no curd firmness could be measured in camel milk as this width of Formagraph was not reached, due to the failure of curd formation. The primary aim of the electron microscopic examination was to give a visual illustration of the coagulation process and to compare the structural changes of cow and camel casein micelles during milk coagulation. Electron micrographs of the casein micelles of camel and cow milk are presented in Fig. 2. Both camel and cow casein micelles appeared as almost spherically shaped particles, composed of numbers of submicelles. Cow casein micelles are dispersed over the field (Fig. 2, A) whereas camel casein micelles are more aggregated and grouped together (Fig. 2, B). In all the examined fields the size of the camel casein particles appears bigger. In the present investigation the size distribution of the casein micelles has not been studied. Due to this limitation, no comparison can be made here between the size of cow and camel casein micelles. In a study on particle size distribution of casein micelles in camel milk by transmission electron microscopy ALI and ROBINSON (12) reported a range of size of casein micelles between 14 to 560 nm with most of the micelles having diameters ranging between 28 to 240 nm.

Milchwissenschaft 42 (11) 1987

Farah, Camel milk

The effects of temperature, pH and calcium chloride on coagulation time are shown in Figs. 3-5. In both camel and cow milk coagulation time was reduced with decreasing pH,

Fig. 2: Electron micrographs (x 75,000) of freeze-fractured casein micelles in camel and cow milk. Scale bar = 100 nm. A - cow milk before rennet addition B - camel milk before rennet addition C - cow milk after rennet addition (100% coagulation time) D - camel milk after rennet addition (120 % coagulation time) The visually obtained coagulation times of cow and camel milk were 5 and 15 min respectively. In the cow milk the onset of aggregation as observed in electron micrographs began approximately at 60% of the coagulation time. At 80% of the coagulation time many casein micelles were already linked together forming the beginning of a network. At the coagulation time the aggregation process was more advanced and a continuous cross-linked network of casein micelle chains could be detected (Fig. 2,C). In camel milk the electron microscopic observation did not show any micellar aggregation until about 120 % of the coagulation time (Fig. 2, D). At that time the formation of flocks was already well advanced. In contrast to cow milk, where at the coagulation point a network of mostly fused micelles is formed, camel casein micelles appear to form a less compact and looser network linked merely by contact with very little change in the original micellar structure (Fig. 2, D).

Fig. 3: Influence of temperatures on the coagulation time of camel and cow milk at pH 6.65

Fig.5: Effect of added calcium on the coagulation time of camel and cow milk at 35آ°C and pH 6.65

Milchwissenschaft 42 (11) 1987

Farah, Camel milk

with increasing temperature and added calcium. This means that the response to changes in pH, temperature and calcium concentration is the same for camel and cow milk, but the difference in the coagulation time still remains. The rate of liberation of NPN from casein by the action of rennet was measured by monitoring the increase in 12 % trichloroacetic acid (TCA) soluble in N-compounds. As Fig. 6 shows, in both camel and cow milk the amount of NPN released by the action of rennet increased at first, reaching a maximum at the coagulation point, and declined then at a slow constant rate. This is consistent with the findings of MEHAIA (13), who reported the release of NPN soluble in 12% trichloroacetic acid in camel milk. It seems reasonable to assume that a primary reaction of the bovine type occurred between rennet and camel milk caseins. The nature of the released fragments have not been examined. However, MEHAIA (13) reported the existence of glycoآ· and non-glyco-خ؛-cascin in camel casein. Our earlier attempt to isolate خ؛-casein-like protein from camel milk was however unsuccessful (14). Other investigations also reported difficulties in detecting خ؛-casein in camel milk (15). The main objective of this work was to examine the rennet coagulation and some of its properties in camel milk. From the obtained results it can be concluded that camel milk casein is accessible to rennin and the effects of variables like pH, temperature and calcium chloride on the coagulation time are similar to that of cow milk but not as pronounced. The action of rennet on camel milk leads to coagulation in the form of flocks with no evidence of gel formation.

(5) CHAPMAN, M.).: World Animal Rev. 5514-19 (1985) (6) HOSTETTLER, H., STEIN, J.: Schweiz. Milchztg. 81 Wiss. Beil. 20 24 (1955) (7) McMAHON, D.)., BROWN, R.).: J. Dairy ScL 651639 (1982) (8) MأœLLER, M. et 01.: Microskopie (Wien) 36 129140 (1980) (9) NITSCHMANN, H., BOHREN, H.U.: Helv. Chem. Acta 38 1953-1963 (1955) (10) International Dairy Federation: International Standard No. 20 (1982) (11) SHALASH, M.R.: in: Camels. Intern. Foundation for Science (IFS) Symposium Khartum, Sudan 285-306 (1979) (12) All, M.Z., ROBINSON, R.K.: J. Dairy Res. 52 303--307 (1985) (13) MEHAIA, M.A.: J. Dairy ScL 69 (suppl. 1) 89 (0119) (1986) (14) FARAZ, Z., FARAH-RIESEN, M.: MiJchwissenschaft 40 669671 (1985) (15) LARSSONآ·RAZNIKIEWICZ, M., MOHAMED, A.M.: Swedish ). Agric. Res. 1613-18 (1986) 5. Summary FARAH, Z., BACHMANN, M.R.: Rennet coagulation properties of camel milk. Milchwissenschaft 42 (11) 689-692 (1987). 51 Camel milk (cheesemaking) The action of rennet on camel milk has been compared with cow milk. The coagulation time was determined and the structural changes of the casein micelles during coagulation were examined by electron microscopy. The effect of pH, temperature, and added calcium chloride on the coagulation time as well as the release of NPN by the action of rennet was also studied. The coagulum obtained from camel milk was a precipitate in the form of flocks, and no clot was formed. With the same amount of rennet the coagulation time of camel milk was two to three fold longer than that of cow milk. In both, camel and cow milk coagulation time was reduced with decreasing pH, with increasing temperature and added calcium. This means, that the response to changes in pH, temperature and calcium concentration is the same for camel and cow milk, but the difference in the coagulation time still remains. Camel and cow milk showed similar NPN liberation patterns. This suggests that a primary phase reaction of bovine type occurs between rennet and camel casein.

Acknowledgement This work was supported in part by the Swiss Directorate for Technical Cooperation and Humanitarian Aid, Berne. The authors express their thanks to Dr. W. Schulthess of the University of Nairobi (Kenya) for providing the camel milk samples and to Dr. E. Wehrli of the Electron Microscopy Unit EHT Zurich for his valuable technical assistance.

4. References (1) FAO: Production Year Book, FAO Rome (1978) (2) YAGIL, R.: FAO Production and Health Paper, Rom 269 (1982) (3) GAST, M.L. et 01.: in: FAO Animal Production and Health Paper, Rom 26 19-24 (1982) (2) (4) RAO, M.B., GUPTA, R.C., DASTUR, N.N.: Ind. ). Dairy ScL 2372-78 (1970)

Milchwissenschaft 42 (11) 1987

Farah, Camel milk

FARAH, Z., BACHMANN, M.R.: Labgerinnungseigenschaften der Kamelmilch. Milchwissenschaft 42 (11) 689-692 {19B]}. 51 Kamelmilch (Kأ¤seherstellung) Es wurde die Wirkung des Labenzyms auf Kamelmilch mit Kuhmilch verglichen. Die Gerinnungszeit wurde ermittelt und die Strukturverأ¤nderung der Caseinmicellen wahrend der Gerinnung untersucht. Der Einfluss des pH-Wertes, der Temperatur und des zugesetzten Calciumchlorids auf die Gerinnungszeit sowie die Spaltung des NichtProteinstickstoffs durch die Einwirkung des Labenzyms wurde ebenfalls verfolgt. Das erhaltene Koagulum der Kamelmilch bestand in einer Ausfallung in Form von Flocken und es bildete sich keine Gallerte. Bei gleichbleibender Labkonzentration war die Gerinnungszeit van Kamelmilch zwei- bis dreimal langer als bei Kuhmilch. In beiden Milchen verminderte sich die Gerinnungszeit mit der Abnahme des pH-Wertes, der Zunahme der Temperatur und der zugesetzten Menge Calciumchlorid. Dies bedeutet, dass die Reaktion auf Verأ¤nderung des pH, der Temperatur und der Calciumkonzentration gleich ist, dass jedoch der Unterschied in der Gerinnungszeit der beiden Milchen bestehen bleibt. Kamel- und Kuhmilch zeigten ein أ¤hnliches NichtProtein-Stickstoff-Abspaltungsmuster. Dies lأ¤sst vermuten, dass zwischen Labenzym und Kamel-Casein eine dem KuhCasein أ¤hnliche Primأ¤rphasenreaktion stattfindet. FARAH, Z., BACHMANN, M.R.: Proprithes de coagulation presure du lait de chamelle. Milchwissenschaft 42 (11) 689-692 (1987). 51 Lait de chamelle (fromagerie) FARAH, Z., BACHMANN, M.R.: Propriedades de coagulacion por el cuajo de leche de camella. Milchwissenschaft 42 (11) 689-692 (1987). 51 Leche de camella (queseda)

Milchwissenschaft 42 (11) 1987

pdf

'LVV (7+ 1R 

&RPSRVLWLRQDO DQG 6WUXFWXUDO $QDO\VLV RI &DPHO 0LON 3URWHLQV ZLWK (PSKDVLV RQ 3URWHFWLYH 3URWHLQV
$ ',66(57$7,21 )RU WKH 'HJUHH RI '2&725 2) 7(&+1,&$/ 6&,(1&(6 6XEPLWWHG WR WKH (,'*(1g66,6&+( 7(&+1,6&+( +2&+6&+8/( 6:,66 )('(5$/ ,167,787( 2) 7(&+12/2*< =XULFK 3UHVHQWHG E\ 67()$1 .$33(/(5 'LSO 1DWZ (7+ %RUQ RQ  -XO\  6ZLVV &LWL]HQ

$FFHSWHG RQ WKH 5HFRPPHQGDWLRQ RI 3URI 'U =GHQNR 3XKDQ ([DPLQHU 'U =DNDULD )DUDK &RH[DPLQHU 3URI 'U *HUDOG 6WUDQ]LQJHU &RH[DPLQHU =XULFK 

7R P\ IDPLO\ HVSHFLDOO\ P\ SDUHQWV PXFK DSSUHFLDWLRQ

$&.12:/('*(0(176 , ZRXOG OLNH WR WKDQN 3URI = 3XKDQ DQG 'U = )DUDK IRU JLYLQJ PH WKH FKDQFH WR FDUU\ RXW WKLV WKHVLV DQG IRU WKHLU JXLGDQFH DQG DGYLVH LQ WKH FRXUVH RI WKH ZRUN , WKDQN 3URI 6WUDQ]LQJHU IRU WDNLQJ RYHU WKH H[WHUQDO H[DPLQDWLRQ RI WKLV ZRUN , DP LQ GHEW WR 3URI 7HXEHU DQG WKH /DERUDWRU\ RI )RRG 0LFURELRORJ\ ZKR DOORZHG PH WR FDUU\ RXW DOO PROHFXODU ELRORJLFDO H[SHULPHQWV KDYLQJ XQUHVWULFWHG DFFHVV WR WKHLU ODERUDWRULHV DQG HTXLSPHQW , ZLVK WR WKDQN 3URI %UآپFNQHU RI WKH 8QLYHUVLW\ *LHVVHQ DQG 'U *UHLOLQJ DQG 0U 0 /HLWHQEHUJHU RI WKH 8QLYHUVLW\ +RKHQKHLP 6WXWWJDUW ZKR KHOSHG PH LQ PDQ\ ZD\V LQ WKH FRXUVH RI WKH WKHVLV 'RLQJ UHVHDUFK DERXW DQ DQLPDO ZKLFK OLYHV LQ DULG UHJLRQV EXW QRW LQ 6ZLW]HUODQG , ZDV KDSS\ DQG WKDQNIXO WKDW , FRXOG GR SDUW RI P\ UHVHDUFK DW WKH &HQWUDO 9HWHULQDU\ 5HVHDUFK /DERUDWRU\ 'XEDL XQGHU DVVLVWDQFH RI 'U 'U KDELO 8 :HUQHU\ DQG KLV JURXS (VSHFLDOO\ , ZRXOG OLNH WR WKDQN 'U : +|O]HU DQG 'U - .LQQH ZKR KHOSHG PH WR FDUU\ RXW P\ H[SHULPHQWV , DOVR ZRXOG OLNH WR WKDQN WKH (YDQV IDPLO\ DQG 0V ' $WNLQV IRU JLYLQJ PH WKH SRVVLELOLW\ WR GR UHVHDUFK DW 2O 0DLVRU )DUP 5XPXUXWL .HQ\D DV ZHOO DV WR 'U 0
,

$FNQRZOHGJHPHQWV
, ,,, ,,, ,9 9,,                                       

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خ؛&DVHLQ 3ULPDU\ 6WUXFWXUH 6HFRQGDU\ 6WUXFWXUH &K\PRVLQ 6HQVLWLYH 6LWH RI خ؛&DVHLQ 5HQQHW &RDJXODWLRQ &RQVLGHUDWLRQV IRU &DPHO 0LON 3URFHVVLQJ  5HQQHWLQJ HQ]\PHV /LWHUDWXUH 3ULPDU\ 6WUXFWXUHV )XQFWLRQDO $VSHFWV

,,
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

 





  

,,, $%%5(9,$7,216 DD $E $%76 $6 خ±V&1 خ±V&1 خ²&1 خ؛&1 N'D +3/& /,1( /3V 0$/',06 0)* 0)*0 25) 3$*( 3&5 SIX 3*53 33 6'6 6,1( 7)$ :$3

$EEUHYLDWLRQV

$PLQR $FLG $QWLERG\ آ¶D]LQRELVHWK\OEHQ]RWKLD]ROLQHVXOSKRQLF DFLG $PLQRVlXUHQ خ±&DVHLQ FDOFLXP VHQVLWLYH W\SH  خ±&DVHLQ FDOFLXP VHQVLWLYH W\SH  خ²&DVHLQ خ؛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

,9 6800$5< 7KH SUHVHQW VWXG\ DLPV WR FRQWULEXWH LQ FKDUDFWHULVDWLRQ RI WKH FDPHO PLON SURWHLQ IUDFWLRQ 3URWHLQV IURP PLON RI 6RPDOL DQG $UDELDQ FDPHO EUHHGV &DPHOXV GURPHGDULXV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خ±V خ±V خ² DQG خ؛&1 DQG F'1$ FORQHV FRUUHVSRQGLQJ WR WKH IRXU FDVHLQV ZHUH VHTXHQFHG 7KH QXPEHU RI UHVLGXHV ZHUH خ±V&1  خ±V&1  خ²&1  خ؛&1  6LPLODULW\ WR ERYLQH SURWHLQV ZDV خ±V&1 $   خ±V&1   خ²&1   خ؛&1   $FLG SUHFLSLWDWHG FDVHLQ RI SRROHG PLON ZDV VHSDUDWHG E\ UHYHUVHGSKDVH +3/& DQG PRQLWRUHG DW  QP DQG LWV FRPSRVLWLRQ HVWLPDWHG IURP SHDN LQWHJUDWLRQ ZDV خ±V&1   خ±V&1   خ²&1   خ؛&1   'HJUHHV RI SKRVSKRU\ODWLRQ DQG JO\FRV\ODWLRQ ZHUH GHWHUPLQHG E\ PDVV VSHFWURPHWU\ DQG VHTXHQFH SDWWHUQ DQDO\VLV 0ROHFXODU PDVVHV GHWHUPLQHG ZHUH خ±V&1 $  N'D DQG  N'D خ±V&1 %  N'D خ±V&1  N'D خ²&1  N'D خ؛&1   N'D 7KH S+ YDOXHV RI WKH PRVW SUREDEOH LVRHOHFWULF SRLQWV ZHUH خ±V &1 $ 3  خ±V&1 % 3  خ±V&1 3  خ²&1 3  خ؛&1 3 ZLWK  VLDOLF DFLG UHVLGXHV ERXQG  ,W ZDV FRQFOXGHG WKDW WKH ORZ JHO ILUPQHVV RI UHQQHWHG FDPHO PLON DV FRPSDUHG WR UHQQHWHG PLON RI WUXH UXPLQDQWV LV D UHVXOW RI WKH GLIIHUHQW FRPSRVLWLRQ RI WKH FDVHLQ IUDFWLRQ DQG PDUNHG YDULDWLRQV LQ WKH خ؛&1 SULPDU\ VWUXFWXUH 7KH VWUXFWXUH RI FK\PRVLQ DQG SHSVLQ P51$ IURP JDVWULF PXFRVD RI FDPHOV ZDV GHWHUPLQHG WR XQGHUVWDQG VSHFLILFLW\ RI LQWHUDFWLRQ ZLWK خ؛&1 &K\PRVLQ ZDV  DD UHVLGXHV ORQJ ZLWK D PROHFXODU ZHLJKW RI  N'D DQG DQ LVRHOHFWULF SRLQW DW S+  ,W VKDUHG  VHTXHQFH VLPLODULW\ ZLWK

9

6XPPDU\

ERYLQH FK\PRVLQ 6SHFLILFLW\ SRFNHWV IRU LQWHUDFWLRQ ZLWK WKH FK\PRVLQ VHQVLWLYH UHJLRQ LQ خ؛&1 ZHUH IRXQG WR KDYH PRUH SURQRXQFHG K\GURSKRELF RU K\GURSKLOLF FKDUDFWHULVWLFV WKDQ WKRVH LQ ERYLQH FK\PRVLQ DQG ZHUH WKXV SUREDEO\ EHWWHU VXLWHG IRU K\GURO\VLV RI WKH VFLVVLOH ERQG LQ FDPHO خ؛&1 3HSVLQ ZDV  DD UHVLGXHV ORQJ ZLWK D PROHFXODU ZHLJKW RI  N'D DQG DQ LVRHOHFWULF SRLQW DW S+  ,W VKDUHG  VHTXHQFH VLPLODULW\ ZLWK SRUFLQH SHSVLQ DQG ZDV VXSSRVHG WR KDYH D VLPLODU DFWLYLW\ ,W ZDV FRQFOXGHG WKDW WKH KLJKHU DFWLYLW\ RI FDPHO FK\PRVLQ LQ PLON UHQQHWLQJ FRPSDUHG WR ERYLQH FK\PRVLQ ZDV EDVHG RQ KLJKHU VSHFLILFLW\ WRZDUGV LWV QDWXUDO VXEVWUDWH ZKLFK LV FDPHO PLON خ؛&1 DQG WKDW ODUJHVFDOH SURGXFWLRQ VKRXOG EH HQYLVDJHG :KH\ SURWHLQV RI FDPHO PLON ZHUH H[DPLQHG ZLWK IRFXV RQ WKHLU UHODWLYH GLVWULEXWLRQ DV FRPSDUHG WR ZKH\ SURWHLQV RI FRZ PLON DQG ZLWK VSHFLDO LQWHUHVW LQ SURWHLQV ZLWK SRVVLEOH DQWLPLFURELDO DFWLYLW\ $ QRYHO PLON SURWHLQ ZDV LVRODWHG E\ KHSDULQVHSKDURVH DIILQLW\ FKURPDWRJUDSK\ IURP FDPHO ZKH\ ZKLFK ZDV IRXQG DW D KLJK FRQFHQWUDWLRQ RI  PJ O DQG KDG  VLPLODULW\ ZLWK KXPDQ SHSWLGRJO\FDQ UHFRJQLWLRQ SURWHLQ 3*53 7KH SURWHLQ ZDV  DD UHVLGXHV ORQJ ZLWK D PROHFXODU PDVV RI  N'D DQG DQ LVRHOHFWULF SRLQW DW S+  ,Q DQDORJ\ WR KRPRORJRXV SURWHLQV IURP WKH LPPXQH V\VWHP RI YHUWHEUDWHV DQG LQYHUWHEUDWHV DFWLYLW\ DJDLQVW JUDPSRVLWLYH DQG SUREDEO\ DJDLQVW JUDPQHJDWLYH EDFWHULD DQG D SRWHQWLDO DQWLWXPRXU DFWLYLW\ ZDV SURSRVHG $ FDPHO ZKH\ SURWHLQ ZLWK  DPLQR DFLG VHTXHQFH VLPLODULW\ WR ODFWRSKRULQ IURP ERYLQH ZKH\ DQG  VLPLODULW\ WR WKH PRXVH 0XV PXVFXOXV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

9,, =86$00(1)$6681*

=XVDPPHQIDVVXQJ

0LW GHU YRUOLHJHQGHQ $UEHLW P|FKWHQ ZLU ]XP EHVVHUHQ 9HUVWlQGQLV GHU 3URWHLQIUDNWLRQ YRQ .DPHOPLOFK EHLWUDJHQ 0LOFK YRQ 6RPDOLVFKHQ XQG $UDELVFKHQ .DPHOHQ ZXUGH GXUFK 6lXUHIlOOXQJ LQ HLQH .DVHLQ XQG HLQH 0RONHQIUDNWLRQ DXIJHWUHQQW %HLGH )UDNWLRQHQ ZXUGHQ PLW +LOIH GHU 3KDVHQXPNHKU&KURPDWRJUDSKLH ZHLWHU DXIJHWUHQQW $XVVHUGHP ZXUGHQ 0RONHQSURWHLQH PLWWHOV +HSDULQ $IILQLWlWVFKURPDWRJUDSKLH LVROLHUW 'LH 5HLQKHLW GHU )UDNWLRQHQ ZXUGH PLW 6'63$*( آپEHUSUآپIW 3URWHLQNRQ]HQWUDWLRQHQ ZXUGHQ PLWWHOV $EVRUSWLRQVVSHNWURPHWULH EHL  QP JHPHVVHQ RGHU GXUFK ,QWHJUDWLRQ GHU 6LJQDOH GLH ZlKUHQG GHU 3KDVHQXPNHKU&KURPDWRJUDSKLH EHL  QP DXIJH]HLFKQHW ZXUGHQ 'LH 6WUXNWXU GHU LVROLHUWHQ 0LOFKSURWHLQH XQG GHU /DEHQ]\PH ZXUGH GXUFK 0DVVHQVSHNWURPHWULH (GPDQ$EEDX XQG F'1$ 6HTXHQ]LHUXQJ EHVWLPPW 6RZRKO GLH =XVDPPHQVHW]XQJ GHU .DVHLQ XQG 0RONHQIUDNWLRQHQ DOV DXFK VWUXNWXUHOOH (LJHQVFKDIWHQ GHU HLQ]HOQHQ 3URWHLQH XQWHUVFKLHGHQ VLFK VWDUN YRQ 5HVXOWDWHQ GLH DXV GHU 8QWHUVXFKXQJ YRQ .XKPLOFK EHNDQQW ZDUHQ 'LH 3ULPlUVWUXNWXU YRQ خ±V خ±V خ² XQG خ؛&1 ZXUGH GXUFK (GPDQ$EEDX YRQ WU\SWLVFKHQ )UDJPHQWHQ XQG GXUFK F'1$6HTXHQ]LHUXQJ EHVWLPPW خ±V&1 KDWWH  خ±V&1  خ²&1  XQG خ؛&1  $6 'LH bKQOLFKNHLW ]X .XKPLOFKSURWHLQHQ ZDU  IآپU خ±V&1  IآپU خ±V&1  IآپU خ²&1 XQG  IآپU خ؛&1 6lXUHJHIlOOWHV .DVHLQ YRQ 0LVFKPLOFK ZXUGH DXVVHUGHP PLWWHOV 3KDVHQXPNHKU&KURPDWRJUDSKLH JHWUHQQW XQG EHL  QP DXIJH]HLFKQHW $XIJUXQG GHU 6LJQDOLQWHQVLWlW ZXUGH GHU UHODWLYH *HKDOW YRQ خ±V&1 DXI   YRQ خ±V&1 DXI   YRQ خ²&1 DXI  XQG YRQ خ؛&1 DXI  GHV *HVDPWNDVHLQV JHVFKlW]W 6HLWHQNHWWHQ GHU 3URWHLQH GLH P|JOLFKHUZHLVH SRVWWUDQVODWLRQDO SKRVSKRU\OLHUW RGHU JO\NRV\OLHUW ZDUHQ ZXUGHQ GXUFK 0DVVHQVSHNWURPHWULH XQG GXUFK GLH $QDO\VH YRQ (UNHQQXQJVPXVWHUQ LQ GHU 3ULPlUVWUXNWXU EHVWLPPW )آپU خ±V&1 $ ZXUGHQ 0DVVHQ YRQ  XQG  N'D JHPHVVHQ خ±V&1 % خ±V&1 خ²&1 XQG خ؛&1 KDWWH 0DVVHQ YRQ  N'D  N'D  N'D XQG  N'D 'HU LVRHOHNWULVFKH 3XQNW YRQ خ±V&1 $ 3 ZDU EHL S+  YRQ خ±V&1 % 3 EHL  YRQ خ±V&1 3 EHL  YRQ خ²&1 3 EHL  XQG YRQ خ؛&1 3 PLW  JHEXQGHQHQ 6DOLFLOVlXUHUHVWHQ EHL  'LH JHULQJH *DOOHUWIHVWLJNHLW YRQ ODEEHKDQGHOWHU .DPHOPLOFK YHUJOLFKHQ PLW ODEEHKDQGHOWHU 0LOFK YRQ HFKWHQ :LHGHUNlXHUQ ZXUGH DXI GLH YHUVFKLHGHQDUWLJH =XVDPPHQVHW]XQJ

9,,, GHU .DVHLQIUDNWLRQ XQG DXI GHXWOLFKH 8QWHUVFKLHGH LQ GHU خ؛&16WUXNWXU ]XUآپFNJHIآپKUW 'LH P51$ YRQ &K\PRVLQ XQG 3HSVLQ DXV GHU 6FKOHLPKDXW GHV /DEPDJHQV HLQHV .DPHOV ZXUGH VHTXHQ]LHUW XP GLH VSH]LILVFKH ,QWHUDNWLRQ GHU (Q]\PH PLW خ؛&1 ]X DQDO\VLHUHQ .DPHO&K\PRVLQ KDWWH HLQH /lQJH YRQ  $6 HLQ 0ROHNXODUJHZLFKW YRQ  N'D XQG HLQHQ LVRHOHNWULVFKHQ 3XQNW EHL S+  'LH bKQOLFKNHLW ]X ERYLQHP &K\PRVLQ ZDU   6RJHQDQQWH 6SH]LIL]LWlWVWDVFKHQ IآپU GLH ,QWHUDNWLRQ PLW GHU FK\PRVLQVHQVLWLYHQ 5HJLRQ YRQ خ؛&1 ZXUGHQ JHIXQGHQ PLW K\GURSKLOHQ RGHU K\GURSKREHQ (LJHQVFKDIWHQ GLH DXVJHSUlJWHU ZDUHQ DOV GLHMHQLJHQ LQ ERYLQHP &K\PRVLQ 'DUDXV ZXUGH JHVFKORVVHQ GDVV .DPHO&K\PRVLQ ]XU +\GURO\VH YRQ .DPHOخ؛&1 ZDKUVFKHLQOLFK EHVVHU JHHLJQHW LVW DOV ERYLQHV &K\PRVLQ .DPHO3HSVLQ KDWWH HLQH /lQJH YRQ  $6 HLQ 0ROHNXODUJHZLFKW YRQ  N'D XQG HLQHQ LVRHOHNWULVFKHQ 3XQNW EHL S+  'LH bKQOLFKNHLW ]X 6FKZHLQH3HSVLQ ZDU   (V ZXUGH DQJHQRPPHQ GDVV GLH $NWLYLWlW EHLGHU (Q]\PH lKQOLFK ZDU 'LH K|KHUH $NWLYLWlW YRQ .DPHO&K\PRVLQ GLH IآپU GLH *HULQQXQJ YRQ .DPHOPLOFK LP 9HUJOHLFK ]XU $NWLYLWlW YRQ ERYLQHP &K\PRVLQ QDFKJHZLHVHQ ZRUGHQ ZDU :DQJRK  ZXUGH DXI GLH KRKH (Q]\P6XEVWUDW6SH]LILWlW YRQ &K\PRVLQ XQG خ؛&1 ]XUآپFNJHIآپKUW XQG HV ZXUGH DQJHUHJW ]X SUآپIHQ RE VLFK GDV (Q]\P IآپU GLH .lVHSURGXNWLRQ HLJQHQ ZآپUGH 'LH 0RONHQSURWHLQH GHU .DPHOPLOFK ZXUGHQ LP +LQEOLFN DXI LKUH .RQ]HQWUDWLRQ LP 9HUJOHLFK ]X 0RONHQSURWHLQHQ GHU .XKPLOFK XQWHUVXFKW PLW EHVRQGHUHP ,QWHUHVVH DQ 3URWHLQHQ PLW P|JOLFKHU DQWLPLNURELHOOHU $NWLYLWlW (LQ 3URWHLQ ZXUGH PLWWHOV +HSDULQ6HSKDURVH$IILQLWlWVFKURPDWRJUDSKLH DXV .DPHOPRONH LVROLHUW GDV NHLQH bKQOLFKNHLW ]X LQ GHU /LWHUDWXU EHVFKULHEHQHQ 0RONHQSURWHLQHQ KDWWH 'LH .RQ]HQWUDWLRQ LQ 0RONH ZXUGH DXI  PJ O JHVFKlW]W 'DV 3URWHLQ KDWWH  VWUXNWXUHOOH bKQOLFKNHLW ]XP PHQVFKOLFKHQ 3HSWLGRJO\FDQ (UNHQQXQJVSURWHLQ 3*53 HLQH /lQJH YRQ  $6 HLQH 0DVVH YRQ  N'D XQG HLQHQ LVRHOHNWULVFKHQ 3XQNW EHL S+  $XIJUXQG GHU :LUNXQJVZHLVH YRQ VWUXNWXUHOO YHUZDQGWHQ 3URWHLQHQ YRQ :LUEHOWLHUHQ XQG ,QYHUWHEUDWHQ ZXUGH YRUJHVFKODJHQ GDVV GDV 3URWHLQ JHJHQ JUDPSRVLWLYH XQG ZDKUVFKHLQOLFK DXFK JUDPQHJDWLYH %DNWHULHQ ZLUNVDP LVW .DPHO/DFWRSKRULQ HLQ 0RONHQSURWHLQ PLW  bKQOLFKNHLW ]X ERYLQHP /DFWRSKRULQ XQG  ]XP PXULQHQ =HOODGKlVLRQV0ROHNآپO *O\&$0 ZXUGH ]X FD  LQ HLQHU ODQJHQ 9DULDQWH $ PLW  $6 XQG HLQHU 0DVVH YRQ  N'D XQG ]X FD  LQ HLQHU NXU]HQ 9DULDQWH % PLW  $6 XQG

,;

=XVDPPHQIDVVXQJ

HLQHU 0DVVH YRQ  N'D JHIXQGHQ 'LH +HWHURJHQLWlW ZDU GLH )ROJH YRQ GLIIHUHQ]LHOOHP 6SOLFLQJ XQG 9HUOXVW GHV ]ZHLWHQ ([RQV LQ 9DULDQWH % %HLGH 3URWHLQH ZDUHQ YHUPXWOLFK GUHLIDFK SKRVSKRU\OLHUW ,P *HJHQVDW] ]X ERYLQHP /DFWRSKRULQ XQG ]X GHQ 0DXV XQG 5DWWHQYDULDQWHQ YRQ *O\&$0 ZDU GDV .DPHOSURWHLQ QLFKW JO\FRV\OLHUW 6SH]LILVFKH ,QWHUDNWLRQ PLW ]XFNHUELQGHQGHQ 3URWHLQHQ ZLH VLH EHL *O\&$0 EHULFKWHW ZXUGHQ NRQQWHQ GHVKDOE DXVJHVFKORVVHQ ZHUGHQ XQG HLQH )XQNWLRQ GHV 3URWHLQV LQ GHU VSH]LILVFKHQ (UNHQQXQJ YRQ =HOOW\SHQ RGHU LQ GHU 6XSSUHVVLRQ YRQ 5RWDYLUHQ LVW QLFKW ZDKUVFKHLQOLFK 'LH .RQ]HQWUDWLRQ LQ .DPHOPLOFK ZDU HWZD  PJ O ZlKUHQG GLH .RQ]HQWUDWLRQ YRQ ERYLQHP /DFWRSKRULQ LQ .XKPLOFK OHGLJOLFK HWZD  PJ O EHWUXJ (V ZXUGH YHUPXWHW GDVV GDV 3URWHLQ HLQH lKQOLFKH )XQNWLRQ LQ 0LOFK ZLH VHLQ ERYLQHV 3HQGDQW DXVآپEWH GDV GLH =XVDPPHQEDOOXQJ GHU )HWWNآپJHOFKHQ XQWHUGUآپFNW ]% ZlKUHQG GHU $XVVFKHLGXQJ LQ GLH 0LOFKGUآپVHQ XQG GDV GLH VSRQWDQH /LSRO\VH GHV 0LOFKIHWWV GXUFK /LSRSURWHLQ /LSDVH KHPPW /DFWRIHUULQ ZXUGH DXV .DPHOPRONH PLW +LOIH GHU +HSDULQ $IILQLWlWVFKURPDWRJUDSKLH LVROLHUW 'LH 3URWHLQVHTXHQ] GLH YRQ GHU F'1$ 6HTXHQ] DEJHOHLWHW ZXUGH KDWWH  bKQOLFKNHLW PLW ERYLQHP /DFWRIHUULQ KDWWH HEHQVR ZLH GLHVHV 3URWHLQ HLQH /lQJH YRQ  $6 HLQH 0DVVH YRQ  N'D XQG HLQHQ LVRHOHNWULVFKHQ 3XQNW EHL S+  'DV 3URWHLQ ZDU ZDKUVFKHLQOLFK ]ZHLIDFK JO\FRV\OLHUW XQG KDWWH ]ZHL P|JOLFKH =HQWUHQ IآپU GLH %LQGXQJ YRQ (LVHQ XQG .DUERQDW $QDORJ ]X ERYLQHP /DFWRIHUULFLQ ZXUGH HLQ 1WHUPLQDOHV SURWHRO\WLVFKHV $EEDXSURGXNW PLW DQWLPLNURELHOOHP 3RWHQWLDO YRUJHVFKODJHQ 'LH .RQ]HQWUDWLRQ GHV 3URWHLQV EHWUXJ  PJ O XQG ZDU K|KHU DOV LQ .XKPLOFK PLW  PJ O 'LH 3ULPlUVWUXNWXU YRQ /DFWRSHUR[LGDVH ZXUGH DXV LKUHU F'1$6HTXHQ] DEJHOHLWHW XPIDVVWH  $6 KDWWH HLQH 0DVVH YRQ  N'D XQG HLQHQ LVRHOHNWULVFKHQ 3XQNW EHL S+  'LH bKQOLFKNHLW ]XP ERYLQHQ +RPRORJ ZDU   bKQOLFK ZLH GLHVHV 3URWHLQ ZDU /DFWRSHUR[LGDVH DXV .DPHOPLOFK YHUPXWOLFK YLHUIDFK JO\FRV\OLHUW KDWWH HLQH +lP*UXSSH NRYDOHQW XQG HLQ .DO]LXPLRQ HOHNWURVWDWLVFK JHEXQGHQ (LQH YHUJOHLFKEDUH $NWLYLWlW LP /DFWRSHUR[LGDVH6\VWHP ZXUGH YHUPXWHW 'LH XQWHUVFKLHGOLFKH =XVDPPHQVHW]XQJ YRQ 0RONH XQG .DVHLQ LP 9HUJOHLFK ]X 0LOFK YRQ :LHGHUNlXHUQ LVW YHUPXWOLFK HLQH )ROJH YRQ HUEOLFKHQ )DNWRUHQ XQG 8PZHOWHLQIOآپVVHQ

;

  ,1752'8&7,21

,QWURGXFWLRQ

7KH $UDELDQ FDPHO &DPHOXV GURPHGDULXV LV WKH PRVW LPSRUWDQW OLYHVWRFN DQLPDO LQ WKH VHPLDULG DUHDV RI 1RUWKHUQ DQG (DVWHUQ $IULFDQ FRXQWULHV 2I  PLOOLRQ FDPHOV ZRUOGZLGH GURPHGDULHV DQG EDFWULDQV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

 DULG FRXQWULHV LW LV ZLWKRXW GRXEW WKDW WKHUH VKRXOG EH HQYLVDJHG D VHULRXV VWHS XS LQ FDPHO PLON UHVHDUFK
                   

)LJ  5HVHDUFK VWXGLHV SXEOLVKHG DERXW FDPHO PLON LQ WKH \HDUV  WR  'XH WR WKH LPSRUWDQFH RI FDPHOV DV PLON VXSSOLHUV UHVHDUFK DERXW FDPHO PLON VKRXOG WDFNOH WHFKQRORJLFDO SUREOHPV ILUVW LQ RUGHU WR XQGHUVWDQG WKH WHFKQRORJLFDO SURSHUWLHV RI WKH PLON 0DLQ TXHVWLRQV ZKLFK DULVH ZKHQ WU\LQJ WR PDNH SURGXFWV IURP FDPHO PLON DQG ZKLFK FDQQRW EH DQVZHUHG VDWLVIDFWRULO\ XQWLO QRZ DUH • :K\ LV WKH FXUG IRUPHG E\ IHUPHQWDWLRQ RU UHQQHW FRDJXODWLRQ RI FDPHO PLON PXFK ZHDNHU WKDQ WKH FXUG IRUPHG E\ FRDJXODWLRQ RI PLON RI UXPLQDQWV" • :K\ DUH SURWHLQV RI FDPHO ZKH\ PRUH UHVLVWDQW WR SURORQJHG KHDW WUHDWPHQW WKDQ SURWHLQV RI ERYLQH ZKH\" 'XH WR WKLV SURRI RI SDVWHXULVDWLRQ E\ WKH PRVW FRPPRQ PHWKRGV HJ GHQDWXUDWLRQ RI DONDOLQH SKRVSKDWDVH LV QRW IHDVLEOH ZLWK FDPHO PLON • :K\ LV FDPHO PLON DW KLJK KHDW WHPSHUDWXUH OHVV KHDW VWDEOH WKDQ FRZ PLON )DUDK $WNLQV " • :K\ LV JURZWK RI PDQ\ VWUDLQV RI ODFWLF DFLG EDFWHULD UHWDUGHG $EX 7DUERXVK  DQG  .DPRXQ " 7KH UHDVRQV IRU WKH GLIIHUHQW SURSHUWLHV RI FDPHO PLON DQG FRZ PLON PD\ EH PDQLIROG 7KH VXUYLYDO RI WKH RIIVSULQJ LQ D JLYHQ HQYLURQPHQW GHSHQGV RQ WKH LPPXQRORJLFDO VLWXDWLRQ ZLWK UHJDUG WR WKH SODFHQWD W\SH WKH FRORVWUXP WKH GHYHORSPHQW DQG VWLPXODWLRQ RI WKH LPPXQH V\VWHP LQ WKH FDOI DQG WKH QXWULWLRQDO SURSHUWLHV LQ JHQHUDO &HUWDLQO\ WKH ORQJ SHULRG RI WKH



,QWURGXFWLRQ

SDOHRQWRORJLFDO GHYHORSPHQW PD\ KDYH FDXVHG QRWLFHDEOH GLIIHUHQWLDWLRQ 7KH IDPLO\ RI WKH $UDELDQ DQG WKH %DFWULDQ FDPHOV EHORQJ WR WKH VXERUGHU RI 7\ORSRGD WRJHWKHU ZLWK WKH OODPD DQG YLFXQD IDPLOLHV %HFDXVH DOO PHPEHUV RI WKH &DPHOLGDH VXERUGHU UXPLQDWH WKH\ ZHUH SXW LQWR D FORVH SDOHRQWRORJLFDO UHODWLRQVKLS ZLWK WUXH UXPLQDQWV 0RVW DQLPDOV XVHG IRU PLON SURGXFWLRQ DUH PHPEHUV RI WKH %RYLGDH IDPLO\ RI 5XPLQDQWDH HJ FDWWOH VKHHS EXIIDORHV DQG JRDWV 5HFHQW UHVHDUFK XVLQJ PRUSKRORJLFDO DQG PROHFXODU ELRORJLFDO PHWKRGV VKRZHG WKDW WUXH UXPLQDQWV DUH FORVHU UHODWHG WR &HWDFHD EDOHHQ DQG WRRWK ZKDOHV WKDQ WR 7\ORSRGD DQG 6XLIRUPDH ZKLFK DUH PRUH GLVWDQWO\ UHODWHG )RU WKLV UHDVRQ WKH RUGHU RI &HWDFHD DQG WKH RUGHU RI $UWLRGDFW\OD HYHQ WRHG XQJXODWHV ZHUH SXW LQWR RQH RUGHU &HWDUWLRGDFW\OD *UDXU DQG +LJJLQV  7\ORSRGD DQG 6XLIRUPDH DUH SUREDEO\ FORVHU UHODWHG WR HDFK RWKHU WKDQ WR 5XPLQDQWDH 0DUWLQDW HW DO 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• 4XDQWLILFDWLRQ DQG VWUXFWXUDO DQDO\VLV RI FDPHO PLON FDVHLQV • 4XDQWLILFDWLRQ RI WKH PDLQ ZKH\ SURWHLQV خ±ODFWDOEXPLQ DQG ODFWRSKRULQ • 6WUXFWXUDO DQDO\VLV RI GLIIHUHQW IXQFWLRQDO ZKH\ SURWHLQV ODFWRIHUULQ ODFWRSHUR[LGDVH DQG SHSWLGRJO\FDQ UHFRJQLWLRQ SURWHLQ

 • 6WUXFWXUDO DQDO\VLV RI UHQQHWLQJ HQ]\PHV FK\PRVLQ DQG SHSVLQ DQG FRPSDULVRQ WR WKH KRPRORJRXV SURWHLQV IURP FDWWOH $V WKHUH LV OLWWOH UHVHDUFK DOUHDG\ GRQH LQ WKLV DUHD DQG WKH PLON SURWHLQ IUDFWLRQ LV D FRPSOH[ VXEMHFW WR LQYHVWLJDWH WKH UHVXOWV RI WKH SUHVHQW ZRUN FDQ RQO\ JLYH OLPLWHG LQVLJKW LQWR WKH QDWXUH RI FDPHO PLON SURWHLQV EXW ZLOO JLYH KLQWV ZKLFK TXHVWLRQV KDYH WR EH DGGUHVVHG LQ IXUWKHU UHVHDUFK RQ WKLV VXEMHFW ,Q WKH SUHVHQW WKHVLV RQO\ &DPHOXV GURPHGDULXV SURWHLQV ZHUH VWXGLHG &DPHO SURWHLQV GHVFULEHG DUH QDPHG ZLWKRXW DQQRWDWLRQ RI VSHFLHV LQ WKH IROORZLQJ WH[W WR IDFLOLWDWH HDVH RI UHDGLQJ

  /,7(5$785( 5(9,(:

/LWHUDWXUH 5HYLHZ

7KLV UHYLHZ ZDV ZULWWHQ ZLWK WKH LQWHQWLRQ WR FRYHU VRPH JHQHUDO DVSHFWV ZLWK UHJDUG WR FDPHO PLON DQG QRW WR JLYH EDFNJURXQG LQIRUPDWLRQ DERXW WKH SURWHLQV VWXGLHG /LWHUDWXUH FRQFHUQLQJ LQGLYLGXDO SURWHLQV ZLOO EH GLVFXVVHG LQ WKH UHVSHFWLYH FKDSWHUV RI آ³5HVXOWV DQG 'LVFXVVLRQآ´ 7D[RQRP\ 7KH &DPHOLGDH EHORQJ WR WKH RUGHU RI $UWLRGDFW\OD HYHQ WRHG XQJXODWHV DQG WKH VXERUGHU 7\ORSRGD SDG IRRWHG DQLPDOV 7KH\ DUH GLYLGHG LQWR WKH JHQXV &DPHOXV ZLWK WKH WZR ROGZRUOG VSHFLHV &DPHOXV GURPHGDULXV $UDELDQ FDPHO DQG &DPHOXV EDFWULDQXV %DFWULDQ FDPHO WKH JHQXV /DPD ZLWK WKH QHZZRUOG VSHFLHV /DPD JODPD OODPD /DPD JXDQLFRH JXDQDFR DQG /DPD SDFRV DOSDFD DQG WKH JHQXV 9LFXJQD ZLWK WKH QHZ ZRUOG VSHFLHV 9LFXJQD YLFXJQD YLFXQD &DPHOLGV VSUHDG IURP 1RUWK $PHULFD ZKHUH WKHLU DQFHVWRUV RULJLQDWHG WR 6RXWK $PHULFD $VLD DQG $IULFD LQ WKH ODWH WHUWLDU\ DJH ,Q 1RUWK $PHULFD WKH\ EHFDPH H[WLQFW 7KH ROGZRUOG VSHFLHV &DPHOXV GURPHGDULXV DQG &DPHOXV EDFWULDQXV FDQ EH FURVVHG DQG WKH IHPDOH RIIVSULQJ LV PRVW OLNHO\ IHUWLOH LQ WKH ILUVW JHQHUDWLRQ EXW WKH PDOHV ) VHHP WR EH VWHULOH 7KH $UDELDQ FDPHO ZDV SUREDEO\ GRPHVWLFDWHG LQ WKH UHJLRQ RI WRGD\آ¶V
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آƒ& WR  آƒ& WKXV VDYLQJ D FRQVLGHUDEOH DPRXQW RI ZDWHU GXULQJ WKH GD\WLPH PDLQO\ LQ WKH DIWHUQRRQ LQ FRQWUDVW WR RWKHU DQLPDOV ZKLFK UHJXODWH WKHLU ERG\ WHPSHUDWXUH E\ UHLQIRUFHG VZHDWLQJ 7KH ERG\ WHPSHUDWXUH LV ORZHUHG LQ WKH QLJKW WLPH EHQHDWK D OHYHO ZKLFK LV SK\VLRORJLFDOO\ WROHUDEOH IRU RWKHU PDPPDOV 6FKZDUW] 'LROL  &DPHOV KDYH GHYHORSHG D V\VWHP RI LQFUHDVHG DQG GLOXWHG PLON GHOLYHU\ LQ WLPHV RI ZDWHU GHSULYDWLRQ WR SUHYHQW WKH QHZERUQ IURP GHK\GUDWLRQ 


/LWHUDWXUH 5HYLHZ

7DEOH  *URVV FRPSRVLWLRQ RI FDPHO PLON DQG FDPHO FRORVWUXP FRPSDUHG WR FRZ DQG KXPDQ PLONV $EX/HKLD  *RUEDQ ,]]HOGLQ  )DUDK 

&DPHO PLON

&RZ PLON

3URWHLQ J O )DW J O /DFWRVH J O 0LQHUDOV PJ O &DOFLXP &RSSHU ,QRUJDQLF 3KRVSKDWH ,URQ .DOLXP 0DJQHVLXP 0DQJDQH 6RGLXP =LQF 9LWDPLQV PJ NJ $VFRUELF DFLG & &REDODPLQ % )ROLF $FLG 1LDFLQ % 3DQWRWKHQLF $FLG 3\ULGR[LQ % 5HWLQRO $ 5LERIODYLQ % 7KLDPLQ % 7RFRSKHURO ( 7RWDO 6ROLGV J O

  

 DYHUDJH  DYHUDJH 

        

        

          

          

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 :KHUHDV RQO\  QPRO O ZHUH PHDVXUHG LQ IUHVK FRZ PLON  QPRO O ZHUH GHWHFWHG LQ FDPHO PLON $OKRPLGD 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 7KLV JLYHV LQGLFDWLRQ WKDW WKH FRPSRVLWLRQ RI FRQVWLWXHQWV



/LWHUDWXUH 5HYLHZ

ZLWK EXIIHULQJ FDSDFLW\ LV GLIIHUHQW EHWZHHQ FDPHO DQG FRZ PLON 7LWUDWDEOH DFLGLW\ LV EHWZHHQ DQ HTXLYDOHQW RI  ODFWLF DFLG LQ IUHVK PLON ZKLFK LV VOLJKWO\ ORZHU WKDQ WKH PHDQ YDOXH RI  IRU FRZ PLON DQG VHHPV WR GHSHQG RQ WKH EUHHG :DQJRK  :KHUHDV FRZ PLON SRVVHVVHV D SURQRXQFHG KHDW VWDELOLW\ PD[LPXP DW S+  DQG D PLQLPXP DW S+  ZKHQ GHWHUPLQHG DW  آƒ& ZLWK VWDELOLW\ GHFUHDVLQJ DW S+ ORZHU WKDQ  DQG LQFUHDVLQJ DW S+ KLJKHU WKDQ  FDPHO PLON GRHV QRW VKRZ DQ LQFUHDVHG VWDELOLW\ DW S+  +HDW VWDELOLW\ RI FDPHO PLON LV PXFK ORZHU WKDQ RI FRZ PLON +HDW FRDJXODWLRQ WLPH IRU FRZ PLON DW  آƒ& LV DERXW  PLQ DW S+  ZKHUHDV FDPHO PLON FRDJXODWHV DW WKLV WHPSHUDWXUH DQG S+ LQ  WR  PLQ )DUDK $WNLQV  7KHUH LV QR LQIRUPDWLRQ DYDLODEOH FRQFHUQLQJ WKH HWKDQRO VWDELOLW\ RI FDPHO PLON 7KH IUHH]LQJ SRLQW RI FDPHO PLON ZDV IRXQG WR EH EHWZHHQ  آƒ& DQG  آƒ& :DQJRK  ,W LV ORZHU WKDQ WKH IUHH]LQJ SRLQW RI FRZ PLON ZKLFK LV EHWZHHQ  آƒ& DQG  آƒ& $ KLJKHU VDOW RU ODFWRVH FRQFHQWUDWLRQ LQ WKH FDPHO PLON ZKLFK ZDV VWXGLHG DV FRPSDUHG WR FRZ PLON PD\ KDYH FRQWULEXWHG WR WKLV UHVXOW 9DOXHV IRU VSHFLILF JUDYLW\ PHDVXUHG E\ .DPRXQ  DQG :DQJRK  GHSHQGHG RQ WKH EUHHG FKRVHQ DQG YDULHG EHWZHHQ  NJ O DQG  NJ O 7KHVH YDOXHV DUH VLPLODU WR YDOXHV IRU FRZ PLON ZKLFK DUH EHWZHHQ  NJ O DQG  NJ O 2WKHU DXWKRUV UHSRUWHG ORZHU YLVFRVLW\ DQG GHQVLW\ IRU FDPHO PLON WKDQ IRU FRZ PLON .DPRXQ  $ PHDQ YDOXH IRU YLVFRVLW\ RI (J\SWLDQ FDPHO PLON ZDV  P3D V ZKLFK LV KLJKHU WKDQ WKH PHDQ YDOXH RI  P3D V IRU FRZ PLON +DVVDQ HW DO  7KHVH FRQWUDGLFWRU\ UHVXOWV PD\ EH H[SODLQHG E\ GLIIHUHQFHV LQ KXVEDQGU\ PDLQO\ LQ ZDWHU VXSSO\ +DVVDQ HW DO  DOVR REVHUYHG VWURQJ VHDVRQDO YDULDWLRQV LQ PLON YLVFRVLW\ VSHFLILF JUDYLW\ DQG WLWUDWDEOH DFLGLW\ 0LON RI KHLIHUV GHSULYHG IURP ZDWHU IRU VHYHUDO GD\V ZDV UHSRUWHG WR EH PRUH GLOXWH SUREDEO\ WR SURWHFW WKH FDOI IURP GHK\GUDWLRQ GXULQJ GU\ SHULRGV 
 7KH VL]H GLVWULEXWLRQ RI FDVHLQ PLFHOOHV LV LQ WKH UDQJH RI  QP WR  QP ZLWK D PD[LPXP LQ YROXPH IUHTXHQF\ EHWZHHQ  QP DQG  QP )DUDK 5آپHJJ  7KH GLVWULEXWLRQ LV VLJQLILFDQWO\ EURDGHU WKDQ WKDW RI FRZ PLON ZKHUH LW LV LQ WKH UDQJH RI  QP WR  QP ZLWK D PD[LPXP EHWZHHQ  QP DQG  QP 7KH QXPEHU RI ODUJH PLFHOOHV LV VLJQLILFDQWO\ KLJKHU ZKLFK LV XQIDYRXUDEOH IRU IRUPDWLRQ RI D ILUP FRDJXOXP LQ PLON SURFHVVLQJ $ QHJDWLYH FRUUHODWLRQ EHWZHHQ PHDQ PLFHOOH VL]H DQG خ؛&1 FRQWHQW RI FRZ PLON ZDV UHSRUWHG 6FKOLPPH  6PDOO PLFHOOHV RI DERXW  QP FRQWDLQHG  خ؛&1 ODUJH PLFHOOHV RI DERXW  QP FRQWDLQHG PHUH  خ؛ &1 6LQFH WKHVH GDWD DOVR FRUUHODWH LQ KXPDQ PLON ZKLFK KDV D PHDQ PLFHOOH GLDPHWHU RI  QP WR  QP DQG D UDWKHU KLJK خ؛&1 FRQWHQW LW ZDV DVVXPHG WKDW FDPHO PLON ZDV ORZ LQ خ؛&1 RU GHYRLG RI WKLV SURWHLQ )DUDK  $ PDUNHGO\ KLJKHU UHVLVWDQFH RI ZKH\ SURWHLQV WRZDUGV KHDW WUHDWPHQW ZDV UHSRUWHG )DUDK  'HJUHH RI GHQDWXUDWLRQ YDULHG LQ FDPHO PLON IURP  WR  DW  آƒ& ,Q FRZ PLON  WR  RI ZKH\ SURWHLQV ZHUH GHQDWXUHG DW WKLV WHPSHUDWXUH $FWLYLW\ RI SODVPLQ DQG SODVPLQRJHQ LQ FDPHO PLON RI HDUO\ ODFWDWLRQ ZDV VKRZQ WR EH EHORZ WKH OHYHO LQ FRZ PLON %DHU HW DO 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 0LON RI QRQUXPLQDQWV VXFK DV KXPDQ KRUVH RU SLJ PLONV UHYHDOHG KLJK SURSRUWLRQV RI JO\FRSURWHLQV ZKLFK ZHUH FRQQHFWHG WR WKH PLON IDW JOREXOH PHPEUDQH 7KHVH JO\FRSURWHLQV ZHUH VLPLODU LQ VWUXFWXUH DQG IXQFWLRQ WR LQWHVWLQDO PXFLQV ZLWK PROHFXODU PDVVHV XS WR  N'D DQG ZLWK XS WR  FDUERK\GUDWH FRQWHQW 6FKOLPPH  7KH PDLQ IXQFWLRQ RI WKHVH SURWHLQV PD\ EH LQ SUHYHQWLRQ RI IDW JOREXOH DJJUHJDWLRQ DQG RI OLSRO\VLV 7KH\ PD\ DOVR KDYH DQ DQWLYLUDO DQG DQWLPLFURELDO HIIHFW $ KLJK SURSRUWLRQ RI WKHVH SURWHLQV LV DOVR OLNHO\ WR RFFXU LQ FDPHO PLON VLQFH D IDU WKLFNHU 0)*0 ZDV REVHUYHG WKDQ LQ PLON RI RWKHU DQLPDOV .QRHVV HW DO 



/LWHUDWXUH 5HYLHZ

7HFKQRORJ\ DSSOLHG IRU 3URFHVVLQJ RI &DPHO 0LON 7KH IDU JUHDWHVW DPRXQW RI FDPHO PLON LV FRQVXPHG DV D IUHVK RU DV D QDWXUDOO\ IHUPHQWHG SURGXFW آ³6XVDآ´ D SURGXFW FRQVXPHG LQ 1RUWK(DVWHUQ $IULFD LV PDGH E\ LQFXEDWLRQ RI PLON LQ VPRNH VDQLWLVHG ZRRGHQ EXFNHWV IRU DERXW RQH WR WKUHH GD\V 7KH FRQVLVWHQF\ RI IHUPHQWHG FDPHO PLON LV WKLQ $ IORFFXOHQW SUHFLSLWDWH LV IRUPHG UDWKHU WKDQ D ILUP FRDJXOXP 6WXGLHV FDUULHG RXW LQ .HQ\D VKRZHG WKDW WKH TXDOLW\ RI آ³6XVDآ´ FDQ EH LPSURYHG XVLQJ VHOHFWHG PHVRSKLOLF VWDUWHU FXOWXUHV UDWKHU WKDQ VSRQWDQHRXV EDFWHULDO FRQWDPLQDWLRQ IRU IHUPHQWDWLRQ 7KH 6RPDOL FRQVXPHUV SUHIHUUHG WKLV QRYHO SURGXFW WR WKH WUDGLWLRQDO SURGXFW )DUDK HW DO  *URZWK RI EDFWHULDO VWUDLQV XVHG IRU FRZ PLON IHUPHQWDWLRQ PD\ EH LQKLELWHG E\ WKH QDWXUDO DQWLPLFURELDO DFWLYLW\ RI FDPHO PLON (ODJDP\ HW DO  6WURQJHU LQLWLDO JURZWK ZDV UHSRUWHG IRU /DFWREDFLOOXV DFLGRSKLOXV $EX7DUERXVK  7KLV FRXOG EH GXH WR D KLJKHU FRQWHQW RI QRQ SURWHLQ QLWURJHQ LQ FDPHO PLON %D\RXPL  %XWWHU LV WUDGLWLRQDOO\ SURGXFHG E\ VNLPPLQJ RI FUHDPHG XS IDW DQG VXEVHTXHQW FKXUQLQJ 7KLV WHFKQLTXH FDQQRW EH DSSOLHG WR FDPHO PLON IDW VLQFH WKH PLON VKRZV OLWWOH WHQGHQF\ WR FUHDP XS %XWWHU ZDV SURGXFHG E\ KHDWLQJ WKH PLON DW  آƒ& IRU  PLQ DQG VHSDUDWLQJ WKH FUHDP E\ FHQWULIXJDWLRQ 7R REWDLQ D UHDVRQDEOH EXWWHU \LHOG FDPHO FUHDP ZDV FKXUQHG DW WHPSHUDWXUH EHWZHHQ  آƒ& DQG  آƒ& 7KH FRUUHVSRQGLQJ WHPSHUDWXUH IRU FRZ PLON FUHDP DUH EHWZHHQ  آƒ& WR  آƒ& 7KH UHDVRQ IRU WKLV GLIIHUHQFH LV WKH KLJK PHOWLQJ SRLQW RI FDPHO PLON IDW ZKLFK LV DW  آƒ& WR  آƒ& 7KLV VHHPV WR VKLIW WKH LGHDO UDWLR RI VROLG WR OLTXLG IDW DW JLYHQ WHPSHUDWXUH WRZDUGV D SRLQW KLJKHU WKDQ WKDW RI FRZ PLON IDW )DUDK 5آپHJJ  ,W ZDV VKRZQ WKDW FUHDPLQJ RI FDPHO PLON IDW ZDV PDUNHGO\ LPSURYHG E\ GLVVROXWLRQ LQ VNLP FRZ PLON &RZ PLON IDW GLVVROYHG LQ VNLP FDPHO PLON RQ WKH RWKHU KDQG VKRZHG D VKDUS GHFUHDVH LQ WKH DELOLW\ WR FUHDP XS ,W FDQ WKHUHIRUH EH FRQFOXGHG WKDW DJJOXWLQLQ LPPXQRJOREXOLQ 0 WKH IDFWRU ZKLFK SURPRWHV FUHDPLQJ RI FRZ PLON IDW LV ORZ RU GHYRLG LQ FDPHO PLON )DUDK 5آپHJJ  &KXUQLQJ RI FDPHO PLON IDW PD\ EH DJJUDYDWHG E\ WKH PXFK ORZHU UDWLR RI OLSLG GURSOHW WR 0)*0 LQ FDPHO PLON IDW JOREXOHV 7KH DYHUDJH PRLVWXUH FRQWHQW RI FDPHO EXWWHU LV   DQG WKXV PXFK ORZHU WKDQ WKH FRQWHQW LQ FRZ PLON EXWWHU ZKLFK LV  +DJUDVV HW DO  7KLV PD\ H[SODLQ WKH VWLFN\ WH[WXUH RI FDPHO PLON EXWWHU &DPHO PLON EXWWHU PD\ EH PRUH VXVFHSWLEOH WR OLJKW R[LGDWLRQ GXH WR WKH KLJKHU DPRXQW RI QRQ VDWXUDWHG WULJO\FHULGHV ,W ZRXOG EH ZHOO ZRUWK\ WR VWXG\ WKH VHQVLWLYLW\ RI FDPHO PLON IDW WRZDUGV OLSRO\VLV DQG R[LGDWLRQ +DYLQJ LQ PLQG WKDW LQVRODWLRQ LQ FDPHO NHHSLQJ FRXQWULHV LV KLJK DQG WKH WRWDO VXUIDFH RI PLON IDW LV ODUJHU VLQFH WKH YROXPH WR VXUIDFH UDWLR RI FDPHO

 PLON IDW JOREXOHV LV RQO\  آµP )DUDK 5آپHJJ  0HKDLD  FRPSDUHG WR D YDOXH RI  آµP IRU FRZ PLON IDW JOREXOHV OLJKW R[LGDWLRQ RI IUHVK FDPHO PLON PD\ EH D FRQFHUQ VLQFH PLON LV RIWHQ VWRUHG LQ WUDQVSDUHQW FRQWDLQHUV 6LPLODUO\ WR KRUVH PLON WKH UHQQHWLQJ FDSDELOLW\ RI FDPHO PLON LV SRRU %D\RXPL 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 &KHHVH \LHOG LV LQ WKH UDQJH RI  RI PLON GU\ PDVV FRPSDUHG WR DERXW  IRU FRZ PLON 7KLV UHVXOW PD\ EH H[SODLQHG E\ WKH ORZHU DPRXQW RI WRWDO VROLGV WKH SRRU UHQQHWDELOLW\ WKH VPDOOHU IDW JOREXOHV WKH VRGLXP FRQFHQWUDWLRQ ZKLFK LV RIWHQ KLJKHU WKDQ LQ FRZ PLON DQG WKH KLJKHU SURSRUWLRQ RI ZKH\ SURWHLQV +LJKHU FKHHVH \LHOG ZDV REWDLQHG ZLWK VRSKLVWLFDWHG WHFKQRORJ\ DGGLWLRQ RI &D&O DQG IRXUIROG KLJKHU FK\PRVLQ FRQFHQWUDWLRQV WKDQ XVHG LQ FRZ PLON 5DPHW  +LJKHU FKHHVH \LHOGV ZHUH DOVR REWDLQHG ZKHQ WKH PLON ZDV EOHQGHG ZLWK PLON IURP UXPLQDQWV 5DPHW  0RVW VWXGLHV RQ FKHHVH SURGXFWLRQ IURP FDPHO PLON UHSRUW WKH SURGXFWLRQ RI D ORZ IDW FKHHVH ZLWK VOLJKWO\ ELWWHU WDVWH )DUDK  ,W FDQ EH DVVXPHG WKDW WKLV W\SH RI FKHHVH ILQGV OLWWOH FRQVXPHU DFFHSWDELOLW\ LQ FDPHO NHHSLQJ FRXQWULHV ZKHUH FKHHVH KDV WR EH LQWURGXFHG DV D QRYHO SURGXFW 'LIIHUHQW VWXGLHV VKRZHG WKDW VHDVRQDO YDULDWLRQV LQ FDPHO PLON SURGXFWLRQ DUH JUHDW DQG PXFK RI VXUSOXV PLON LV FROOHFWHG GXULQJ WKH UDLQ\ VHDVRQ 3URFHVVLQJ FDPHO PLON LQWR SDVWHXULVHG DQG IHUPHQWHG SURGXFWV ZLOO WKHUHIRUH EH RI JUHDW DGYDQWDJH DOORZLQJ WKH FDPHO VPDOOKROGHU WR FRPPHUFLDOLVH KLV PLON )DUDK  &DPHO PLON LV FRPPHUFLDOO\ SDVWHXULVHG LQ 6DXGL$UDELD DQG 0DXULWDQLD 3UREOHPV PD\ DULVH IURP WKH ORZ KHDW FRDJXODWLRQ WLPH RI FDPHO PLON DQG D WHQGHQF\ WR IORFFXODWH 7KHUH DUH DOVR HQYLURQPHQWDO DQG VRFLRHFRQRPLFDO IDFWRUV ZKLFK PDNH PLON SURFHVVLQJ E\ SDVWHXULVDWLRQ D GLIILFXOW WDVN LQ DULG FRXQWULHV $EHLGHUUDKPDQH 5HHG  $ SUREOHP ZKLFK DULVHV IURP WKH KLJKHU JHQHUDO KHDW VWDELOLW\ RI FDPHO ZKH\ SURWHLQV LV WKDW WKH PRVW FRPPRQO\ XVHG PHWKRGV IRU GHWHUPLQDWLRQ



/LWHUDWXUH 5HYLHZ

RI SDVWHXULVDWLRQ DOO IDLO 7KH LQDFWLYDWLRQ RI SKRVSKDWDVH DQG ODFWRSHUR[LGDVH GR QRW RFFXU WR WKH VDPH H[WHQW 3RVLWLYH UHDFWLRQ RI WKH IRUPHU E\ VKRUW WLPH KHDW GHQDWXUDWLRQ LV DEOH WR GHWHFW  UDZ PLON LQ SDVWHXULVHG FRZ PLON 7KH ODWWHU LV DEOH WR GHWHFW  UDZ PLON LQ KLJK WHPSHUDWXUH WUHDWHG FRZ PLON %RWK UHDFWLRQV GR QRW ZRUN LQ FDPHO PLON 0RQWHW 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 FRQWHQW RI FDPHO PLON 9LWDPLQ ' LV LPSRUWDQW IRU ERQH IRUPDWLRQ RI FKLOGUHQ $ KLJK DPRXQW LQ FDPHO PLON ZRXOG WKHUHIRUH EH GHVLUDEOH $ KLJKHU OHYHO RI YLWDPLQ $ DQG ' FRXOG EH DFKLHYHG E\ DSSURSULDWH IHHGLQJ ZKHUHDV WKH YLWDPLQV RI WKH %JURXS DUH PDLQO\ SURYLGHG E\ WKH PLFURIORUD RI WKH UXPHQ 7KH KLJK FRQWHQW RI YLWDPLQ & QLDFLQ DQG FDUQLWLQ LV QXWULWLRQDOO\ LPSRUWDQW IRU FDPHO PLON FRQVXPHUV VLQFH IUXLWV DQG YHJHWDEOHV DUH VFDUFH LQ DULG DUHDV )DUDK  7KH OLSLG IUDFWLRQ LQ FDPHO PLON LV FKDUDFWHULVHG E\ D KLJK SURSRUWLRQ RI ORQJ FKDLQ IDWW\ DFLGV ZKLFK DFFRXQWV IRU   FRPSDUHG WR  LQ ERYLQH PLON $EX/HKLD 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 &DPHO PLON LV XVHG LQ WKH WUDGLWLRQDO PHGLFLQH RI 1RUWKHUQ DQG (DVWHUQ $IULFDQ FRXQWULHV IRU WUHDWPHQW RI LQIODPPDWLRQ DQG ZRXQGV 
 5DZ PLON LV DOVR XVHG LQ WKH WKHUDS\ RI GLDUUKHD PDLQO\ RI QHZERUQ FKLOGUHQ DQG RI SHSWLF XOFHUV &RPSOHWH KHDOLQJ RI  RI KXPDQ SDWLHQWV VXIIHULQJ IURP JDVWURLQWHVWLQDO XOFHUV DQG WUHDWHG ZLWK FDPHO PLON ZDV UHSRUWHG FRPSDUHG WR  DIWHU WUHDWPHQW ZLWK FRZ PLON /R]RYLFK  $ KLJKHU DQWLPLFURELDO SRWHQWLDO RI UDZ FDPHO PLON FRPSDUHG WR UDZ FRZ PLON KDV EHHQ UHSRUWHG )DUDK  7KHUH DUH DOVR UHSRUWV RI FDPHO PLON XVHG LQ QRQFRQYHQWLRQDO FDQFHU WKHUDS\ DQG WUHDWPHQW RI QHXURGHUPLWLV DQG GLDEHWHV )HUPHQWHG FDPHO PLON VKXEDW LV VXFHVVIXOO\ XVHG LQ WKH WUHDWPHQW RI SHSWLF XOFHUV LQ 5XVVLD 6XNKRY HW DO 

   0$7(5,$/6 $1' 0(7+2'6 3URWHLQ $QDO\VLV

0DWHULDOV DQG 0HWKRGV

6DPSOH 3UHSDUDWLRQ 0LON RI LQGLYLGXDO 6RPDOL DQG $UDELDQ FDPHOV ZDV FROOHFWHG GXULQJ PLONLQJ DW 2O 0DLVRU 5DQFK 5XPXUXWL .HQ\D DQG DW .DPHOIDUP )DWDPRUJDQD 5RWIHOGHQ *HUPDQ\ LPPHGLDWHO\ IUR]HQ DW  آƒ& IRU WUDQVSRUW DQG VWRUHG DW  آƒ& XQWLO DQDO\VLV $IWHU WKDZLQJ WKH PLON ZKLFK KDG D S+ RI DERXW  ZDV VNLPPHG DW  J  آƒ& IRU  PLQ 7KH FDVHLQ IUDFWLRQ ZDV LVRODWHG E\ DFLG SUHFLSLWDWLRQ RI  O PLON DW S+  DQG  آƒ& IRU  PLQ XVLQJ  DFHWLF DFLG IROORZHG E\ DGGLWLRQ RI  P0 VRGLXP DFHWDWH IRU QHXWUDOLVDWLRQ DQG FHQWULIXJDWLRQ DW  J IRU  PLQ &DVHLQ SHOOHW DQG ZKH\ ZHUH IUR]HQ DQG VWRUHG DW  آƒ& )RU FUXGH SUHSDUDWLRQ RI DQ خ± DQG D خ²&1 IUDFWLRQ WKH FDVHLQ SHOOHW ZDV GLVVROYHG LQ  PO  0 XUHD GLOXWHG ZLWK  PO GRXEOH GLVWLOOHG ZDWHU DQG WKH S+ DGMXVWHG WR  ZLWK  0 VRGLXP K\GUR[LGH 7KH VROXWLRQ ZDV GLOXWHG ZLWK  PO GRXEOH GLVWLOOHG ZDWHU DQG DGMXVWHG WR S+  ZLWK  0+&O +LSS HW DO  7KH ILUP SUHFLSLWDWH FRQVLVWHG PDLQO\ RI خ± DQG خ؛ &1 $IWHU FHQWULIXJDWLRQ DW  J IRU  PLQ WKH VXSHUQDWDQW ZDV VDWXUDWHG ZLWK DPPRQLXP VXOSKDWH IRU SUHFLSLWDWLRQ RI خ²&1 %RWK SUHFLSLWDWHV ZHUH O\RSKLOLVHG  J DFLG SUHFLSLWDWHG FDVHLQ RU O\RSKLOLVDWH ZHUH GLVVROYHG LQ  PO VDPSOH EXIIHU ZLWK  0 XUHD  P0 VRGLXP FLWUDWH  P0 %LV>WULVK\GUR[\PHWK\O PHWK\ODPLQR@SURSDQH  P0 خ²PHUFDSWRHWKDQRO DQG  P0 7ULV K\GUR[\PHWK\ODPLQRPHWKDQ+&O DW S+  DQG VWLUUHG IRU  K :KH\ ZDV GLDO\VHG WZLFH DJDLQVW GRXEOH GLVWLOOHG ZDWHU IRU  K DW  آƒ& DQG RQFH DJDLQVW  P0 VRGLXP SKRVSKDWH EXIIHU DW S+  IRU  K XVLQJ DQ DXWRFODYHG 63(&75$325 PHPEUDQH WXELQJ ZLWK D PROHFXODU FXWRII RI  WR  N'D 6SHFWUXP 0HGLFDO ,QGXVWULHV ,QF /RV $QJHOHV &$  86$ 3ULRU WR FKURPDWRJUDSK\ VDPSOHV ZHUH ILOWHUHG WKURXJK D K\GURSKLOLF  آµP PHPEUDQH 0( 6FKOHLFKHU 6FKXHOO  'DVVHO *HUPDQ\ 53& +3/& &KURPDWRJUDSK\ ,QGLYLGXDO FDVHLQV ZHUH VHSDUDWHG E\ +3/& /D&KURP 0HUFN ' 'DUPVWDGW *HUPDQ\ RQ D VLOLFDFRDWHG DQDO\WLFDO UHYHUVHGSKDVH & FROXPQ *URP6LO  2'6 +( آµ  [  PP *URP ' +HUUHQEHUJ *HUPDQ\ 6ROYHQW $ ZDV  YY 7)$ LQ GRXEOH GLVWLOOHG QDQRILOWHUHG ZDWHU VROYHQW % ZDV  YY 7)$ LQ DFHWRQLWULOH $IWHU LQMHFWLRQ RI  آµO WR  آµO FDVHLQ ILOWUDWH HOXWLRQ ZDV SHUIRUPHG E\ D OLQHDU

 JUDGLHQW IURP  WR  VROYHQW % RYHU  PLQ IROORZHG E\ D OLQHDU JUDGLHQW IURP  WR  % RYHU  PLQ 7KH IORZ UDWH ZDV  PO PLQ DQG UXQV ZHUH SHUIRUPHG DW DPELHQW WHPSHUDWXUH :KH\ SURWHLQV ZHUH VHSDUDWHG E\ LQMHFWLRQ RI  آµO ILOWUDWH (OXWLRQ ZDV SHUIRUPHG E\ D  PLQ KROG ZLWK  VROYHQW % D OLQHDU JUDGLHQW IURP  WR  VROYHQW % RYHU  PLQ IROORZHG E\ D OLQHDU JUDGLHQW IURP  WR  % RYHU  PLQ 7KH IORZ UDWH ZDV  PO PLQ  DQG UXQV ZHUH SHUIRUPHG DW DPELHQW WHPSHUDWXUH )RU ODUJH VFDOH LVRODWLRQ RI LQGLYLGXDO FDVHLQV D VLOLFDFRDWHG VHPLSUHSDUDWLYH UHYHUVHGSKDVH & FROXPQ *URP6LO  2'6 67  آµ  [  PP *URP ZDV XVHG WR VHSDUDWH WKH SURWHLQV RI WKH FUXGH خ± DQG خ²&1 IUDFWLRQV $IWHU LQMHFWLRQ RI  PO ILOWUDWH HOXWLRQ ZDV SHUIRUPHG E\ D  PLQ KROG DW  VROYHQW % IROORZHG E\ D OLQHDU JUDGLHQW IURP  WR  % RYHU  PLQ DQG D OLQHDU JUDGLHQW IURP  WR  % RYHU  PLQ 7KH IORZ UDWH ZDV  PO PLQ DQG UXQV ZHUH SHUIRUPHG DW  آƒ& 7KH FROXPQ HIIOXHQW ZDV PRQLWRUHG ZLWK D GLRGH DUUD\ GHWHFWRU / 0HUFN IURP  WR  QP 3URWHLQV HOXWHG ZHUH FROOHFWHG PDQXDOO\ DQG O\RSKLOLVHG +HSDULQ6HSKDURVH $IILQLW\ &KURPDWRJUDSK\  PO ZKH\ ZHUH ORDGHG RQ D  PO +HSDULQ 6HSKDURVH +L7UDS FROXPQ $PHUVKDP 3KDUPDFLD   8SSVDOD 6ZHGHQ 7KH FROXPQ ZDV ZDVKHG ZLWK  PO RI  P0 VRGLXP SKRVSKDWH  P0 VRGLXP FKORULGH EXIIHU DW S+  (OXWLRQ ZDV SHUIRUPHG DW DPELHQW WHPSHUDWXUH E\ D OLQHDU JUDGLHQW IURP  WR  0 VRGLXP FKORULGH RYHU  PLQ 7KH FROXPQ HIIOXHQW ZDV PRQLWRUHG ZLWK DQ 89 GHWHFWRU / 0HUFN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 ZDV SHUIRUPHG XVLQJ DQ $%, $ VHTXHQFHU 3( $SSOLHG %LRV\VWHPV )RVWHU &LW\ &$  86$ HTXLSSHG ZLWK D $ +3/& IRU RQOLQH UHYHUVHGSKDVH & +3/& DQDO\VLV RI SKHQ\OWKLRK\GDQWRLQ\O DD GHULYDWLYHV )URP HDFK RI WKH SHDNV FRUUHVSRQGLQJ WR خ±V خ±V خ² DQG خ؛&1  PJ O\RSKLOLVDWH ZDV GLVVROYHG LQ  PO RI D EXIIHU ZLWK  YY DFHWRQLWULOH



0DWHULDOV DQG 0HWKRGV

 YY GRXEOH GLVWLOOHG ZDWHU FRQWDLQLQJ  P0 DPPRQLXP FDUERQDWH S+  DQG GLJHVWHG RYHUQLJKW DW  آƒ& ZLWK  آµJ WU\SVLQ 6HTXHQFLQJ JUDGH %RHKULQJHU ' 0DQQKHLP *HUPDQ\ 3HSWLGHV ZHUH VHSDUDWHG XVLQJ WKH VDPH DQDO\WLFDO FROXPQ DQG D OLQHDU JUDGLHQW IURP  WR  VROYHQW % RYHU  PLQ 7KH IORZ UDWH ZDV  PO PLQ DQG UXQV ZHUH SHUIRUPHG DW DPELHQW WHPSHUDWXUH 3HSWLGHV HOXWHG IURP WKH FROXPQ ZHUH FROOHFWHG PDQXDOO\ DQG GULHG E\ YDFXXPFHQWULIXJDWLRQ ZLWK D VSHHG YDF 69& 6DYDQW ,QVWUXPHQWV 1HZ
 DQG WKH UHVXOWV ZHUH FRPSDUHG ZLWK ZHLJKWV RI WKH O\RSKLOL]HG IUDFWLRQV DQG OLWHUDWXUH YDOXHV )DUDK   '1$ $QDO\VLV

3RO\$P51$ ,VRODWLRQ 8GGHU WLVVXH RI D ODFWDWLQJ 6RPDOL FDPHO  J ZDV WDNHQ LQ WKH PRUQLQJ DIWHU PLONLQJ DQG LPPHGLDWHO\ KRPRJHQLVHG ZLWK D URWRUVWDWRU KRPRJHQLVHU .LQHPDWLFD &+ /LWWDX 6ZLW]HUODQG 3RO\$P51$ ZDV LVRODWHG ZLWK WKH 2OLJRWH[â„¢ 'LUHFW P51$ .LW 4XLDJHQ ' +LOGHQ *HUPDQ\ DFFRUGLQJ WR WKH PDQXIDFWXUHUآ¶V LQVWUXFWLRQ IRU ODUJH VFDOH SUHSDUDWLRQ RI P51$ 7RWDO \LHOG ZDV  آµJ P51$ DQG WKH $$ UDWLR ZDV  ,Q WKH VDPH ZD\ SRO\$P51$ ZDV SUHSDUHG IURP  آµJ RI PXFRVD WLVVXH IURP WKH URXJK DQG WKH VPRRWK SDUW RI WKH UHQQHW VWRPDFK RI D  \HDUV ROG $UDELDQ FDPHO V LQVWUXFWLRQV 3KDJHV ZHUH SODWHG RQ ( FROL /(  3URPHJD 7KH WLWUH RI WKH OLEUDU\ ZDV HVWLPDWHG DW [ SIX PO  آµO RI WKH OLEUDU\ ZHUH DPSOLILHG DQG SURGXFHG D O\VDWH ZLWK D WLWUH RI [ SIX PO 6HTXHQFH $QDO\VLV 7KH F'1$ OLEUDU\ ZDV VFUHHQHG IRU F'1$ FRUUHVSRQGLQJ WR خ±V خ±V خ² DQG خ؛&1 E\ QXFOHLF DFLG K\EULGLVDWLRQ 0DQLDWLV HW DO  3ODTXH OLIWV K\EULGLVDWLRQ DQG VLJQDO GHWHFWLRQ ZHUH GRQH ZLWK WKH GLJR[LJHQLQ ',* V\VWHP RI %RHKULQJHU XVLQJ XQFKDUJHG Q\ORQ PHPEUDQHV ',* (DV\+\E VROXWLRQ DQWL',*$3 )DE IUDJPHQWV DQG &63'آ® DFFRUGLQJ WR WKH PDQXIDFWXUHU
V LQVWUXFWLRQV 6SHFLILF SUREHV ZHUH V\QWKHVLVHG E\ WKH



0DWHULDOV DQG 0HWKRGV

SRO\PHUDVH FKDLQ UHDFWLRQ 3&5 ZLWK ',*G873 WR VFUHHQ IRU F'1$ FRUUHVSRQGLQJ WR خ±V خ±V خ² DQG خ؛&1 'HJHQHUDWH 3&5 SULPHUV ZHUH GHVLJQHG ZLWK WKH KHOS RI DPLQR DFLG VHTXHQFHV REWDLQHG IURP VHTXHQFLQJ WKH 1WHUPLQXV DQG WU\SWLF GLJHVWV RI WKH FDVHLQV VHH DERYH 7KH IROORZLQJ SULPHU SDLUV ZHUH XVHG ,8% FRGH IRU PL[HG EDVH VLWHV

خ±V&1 
7$<&&1*$5*7177<&$5$$<
 GHULYHG IURP WKH VHTXHQFH 7\U3UR *OX9DO3KH*OQ$VQ DW WKH 1WHUPLQXV RI خ±V&1 DQG 
1**57*1*&'$757$<7*&$7
 GHULYHG IURP WKH VHTXHQFH 0HW*OQ 7\U,OH$OD+LV3UR SDUW RI D SURPLQHQW IUDJPHQW RI WKH خ±V&1 WU\SWLF GLJHVW HOXWHG DW  PLQ خ±V&1 
$$5&$<*$5$7**$<&$
 GHULYHG IURP WKH VHTXHQFH /\V+LV*OX 0HW$VS*OQ DW WKH 1WHUPLQXV RI خ±V&1 DQG 
7*57&&&$1**577&$7
 GHULYHG IURP WKH VHTXHQFH 0HW$VQ3UR 7US$VS*OQ SDUW RI D PDMRU IUDJPHQW RI WKH خ±V&1 WU\SWLF GLJHVW HOXWHG DW  PLQ خ²&1 
*$5$$5*$5*$577<$$5$&1
 GHULYHG IURP WKH VHTXHQFH *OX/\V *OX*OX3KH/\V7US DW WKH 1WHUPLQXV RI خ²&1 DQG 
57&1**1$&1**<7&<7*5$$
 GHULYHG IURP WKH VHTXHQFH 3KH*OQ *OX3UR9DO3UR$VS SDUW RI D PDMRU IUDJPHQW RI WKH خ²&1 WU\SWLF GLJHVW HOXWHG DW  PLQ خ؛&1 
*$5*71&$5$$<&$5*$5&$5
 GHULYHG IURP WKH VHTXHQFH *OX9DO *OQ$VQ*OQ*OX*OQ DW WKH 1WHUPLQXV RI خ؛&1 DQG 
*$7&7&$*7&*$$*7$$777*
 GHULYHG IURP D VHTXHQFHG 3&5 IUDJPHQW RI  ES ZKLFK ZDV V\QWKHVLVHG XVLQJ JHQRPLF '1$ RI D %DFWULDQ FDPHO DQG ERYLQH SULPHUV -.  DQG -.  6FKOHH 5RWWPDQQ 
7KH EDVH OHQJWKV RI WKH SUREHV DJDLQVW خ±V خ±V خ² DQG خ؛&1 F'1$V ZHUH    DQG  UHVSHFWLYHO\ 3RVLWLYH SODTXHV ZHUH SLFNHG DQG YHULILHG E\ 3&5 XVLQJ WKH DSSURSULDWH SULPHU SDLUV IURP EHIRUH )RU HDFK SURWHLQ WKH F'1$ LQVHUW RI RQH SRVLWLYH SODTXH ZDV H[FLVHG ZLWK SDUWLDO

 (FR5 ,GLJHVW OLJDWHG LQWR D S*(0= YHFWRU 3URPHJD GLDO\VHG DQG WUDQVIRUPHG LQWR ( FROL ;/%OXH 6WUDWDJHQH /D -ROOD &$  86$ E\ HOHFWURSRUDWLRQ ZLWK D *HQH3XOVHUآٹ %LR5DG DW  N9  آµ)' DQG  آں LQ  FP FXYHWWHV 7KH WUDQVIRUPHG EDFWHULD ZHUH SODWHG RYHUQLJKW DW  آƒ& RQ ,37*;*DO$PSLFLOOLQVHOHFWLYH DJDU :KLWH FRORQLHV ZHUH SLFNHG DQG JURZQ RYHUQLJKW LQ  PO /%$PSLFLOOLQ  0DQLDWLV  3ODVPLG '1$ ZDV SXULILHG IRU IOXRUHVFHQW VHTXHQFLQJ ZLWK WKH :L]DUG 3OXV 69 0LQLSUHSV '1$ 3XULILFDWLRQ 6\VWHP 3URPHJD )OXRUHVFHQW VHTXHQFLQJ RI WKH F'1$ ZDV FDUULHG RXW XVLQJ DQ $/) DXWRPDWHG GHYLFH $PHUVKDP 3KDUPDFLD LQWHUQDO &\آŒG$73 ODEHOOLQJ $PHUVKDP 3KDUPDFLD DQG SULPHU ZDONLQJ VWDUWLQJ IURP FRPPHUFLDO 63 DQG 7 SULPHUV 3URPHJD 2YHUODSSLQJ IUDJPHQWV ZKLFK ZHUH SURGXFHG E\ SRO\PHUDVH FKDLQ UHDFWLRQ 3&5 ZHUH XVHG IRU VHTXHQFH DQDO\VLV RI UHQQHWLQJ HQ]\PHV DQG ZKH\ SURWHLQV 7KH IROORZLQJ SURWRFRO ZDV DSSOLHG WR PRVW RI WKH UHDFWLRQV  آµO RI WKH خ»F'1$ OLEUDU\ RU  آµO RI VLQJOH VWUDQGHG F'1$ ZHUH WDNHQ DV WHPSODWHV LQ  آµO 3&5 DVVD\V ZLWK  XQLWV 7DT 3RO\PHUDVH $PHUVKDP 3KDUPDFLD ZKLFK ZDV EOHQGHG ZLWK  XQLWV 3IX 3RO\PHUDVH 6WUDWDJHQH DQG  آµO [ 7DT3OXV 3UHFLVLRQ LQFXEDWLRQ EXIIHU 6WUDWDJHQH  QPRO RI HDFK G173 $PHUVKDP 3KDUPDFLD DQG  SPRO RI VSHFLILF SULPHUV  F\FOHV ZHUH UXQ ZLWK LQLWLDO  PLQ GHQDWXUDWLRQ DW  آƒ& IROORZHG E\  VHF GHQDWXUDWLRQ DW  آƒ&  VHF DQQHDOLQJ DW  آƒ& DQG  PLQ  VHF HORQJDWLRQ DW  آƒ& (ORQJDWLRQ SURORQJDWLRQ ZDV  VHF SHU F\FOH $ ILQDO  PLQ LQFXEDWLRQ VWHS DW  آƒ& ZDV DGGHG WR LQFUHDVH WKH FRQFHQWUDWLRQ RI IXOO OHQJWK SURGXFWV (DFK 3&5 SURGXFW ZDV JHQHUDWHG WZLFH DQG OLJDWHG LQWR D S*(0آٹ7 (DV\ YHFWRU 3URPHJD DFFRUGLQJ WR WKH PDQXIDFWXUHUV LQVWUXFWLRQV ,Q FDVH RI EDVH UHDGLQJ DPELJXLWLHV D WKLUG 3&5 SURGXFW ZDV JHQHUDWHG 7ZR خ»JW YHFWRU VSHFLILF JHQHUDO SULPHUV ZHUH FRQVWUXFWHG IRU VHTXHQFH DQDO\VLV RI IXOOOHQJWK F'1$ SURGXFWV RI WKH UHVSHFWLYH FORQHV خ»JW IRUZDUG آ¶*$&*$&7&&7**$*&&&*7&$*7$آ¶ خ»JW UHYHUVH آ¶&$&&$*$&&$$&7**7$$7**7$*آ¶ 7KH IROORZLQJ 3&5 SURGXFWV ZHUH JHQHUDWHG PRVWO\ ZLWK WKH KHOS RI KLJKO\ FRQVHUYHG UHJLRQV LQ WKH F'1$ VHTXHQFHV RI RWKHU VSHFLHV PL[HG EDVH VLWHV DFFRUGLQJ WR ,8% FRGH



0DWHULDOV DQG 0HWKRGV

&K\PRVLQ $  NES 3&5 SURGXFW RI FK\PRVLQ H[RQ  ZDV JHQHUDWHG ZLWK آ¶*7***&&&7**&7$&$*&$*آ¶ DQG آ¶7**7*$7&<&$6<*&&<7***$*$*آ¶ DQG JHQRPLF FDPHO '1$ 7KLV VHTXHQFH ZDV XVHG WR JHQHUDWH D  NES 3&5 SURGXFW RI WKH FK\PRVLQ 25) ZLWK آ¶7*$&&$**7&&$**7&&$**$7*&آ¶ DQG آ¶**6*$&$*<*$**77<.75*7&$*6*آ¶ DQG F'1$ IURP VWRPDFK PXFRVD 3HSVLQ $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶.5*$677***$6&&5**$$*$$&&آ¶ DQG آ¶5*$7&77&&7***$**7**&7**$آ¶ DQG PXFRVD

F'1$

IURP

VWRPDFK

3HSWLGRJO\FDQ 5HFRJQLWLRQ 3URWHLQ $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶&&&*&&7*&**77&1$7+*71&&آ¶ DQG آ¶7*$7*77&&$*&&7&**&&77&$7آ¶ DQG F'1$ IURP PDPPDU\ JODQG WLVVXH 7KLV VHTXHQFH ZDV XVHG WR JHQHUDWH D  NES 3&5 SURGXFW ZLWK آ¶&&*$*7*&$*$*$$$**&7$$&$&آ¶ DQG خ»JW UHYHUVH DQG D  NES 3&5 SURGXFW ZLWK آ¶&&$7&77&7&&*$7&$**$$*77*آ¶ DQG خ»JW IRUZDUG DQG F'1$ IURP PDPPDU\ JODQG WLVVXH /DFWRSKRULQ $  NES DQG D  NES 3&5 SURGXFW ZHUH JHQHUDWHG ZLWK آ¶*&&$*&77**&&*&&$&&7&7&7&آ¶ DQG آ¶**&$7*$***$$7$**&7777&$*آ¶ DQG F'1$ IURP PDPPDU\ JODQG WLVVXH 7KLV VHTXHQFH ZDV XVHG WR JHQHUDWH D  NES 3&5 SURGXFW ZLWK آ¶&&$&&7&7&7&*&&$*&&77$$7*آ¶ DQG خ»JW IRUZDUG DQG D  NES 3&5 SURGXFW ZLWK آ¶$$$*7&&$7**777&7&7&$7**7آ¶ DQG خ»JW IRUZDUG DQG F'1$ IURP PDPPDU\ JODQG WLVVXH 7R REWDLQ WKH LQWURQ VHTXHQFHV RI ODFWRSKRULQ WKH 3&5 UHDFWLRQ DV GHVFULEHG EHIRUH ZDV DSSOLHG DQG  آµO JHQRPLF '1$ ZDV XVHG DV D WHPSODWH ZKLFK ZDV LVRODWHG IURP $UDELDQ FDPHOV XVLQJ D 4,$DPS %ORRG .LW 4LDJHQ *PE+  +LOGHQ *HUPDQ\ DFFRUGLQJ WR WKH PDQXIDFWXUHUVآ¶ LQVWUXFWLRQV

 $ 3&5 SURGXFW ZKLFK FRQWDLQHG LQWURQ  ZDV JHQHUDWHG ZLWK آ¶$7*$$$77&77&*&7*7&&7*&7*آ¶ DQG آ¶&7*$*$&7&&$7*7$*$777&$7&آ¶ $ 3&5 SURGXFW ZKLFK FRQWDLQHG LQWURQ  ZDV JHQHUDWHG ZLWK آ¶*$7*$$$7&7$&$7**$*7&7&$*آ¶ آ¶*$&&7*$7**77*&7&$7*$7*$&آ¶ $ 3&5 SURGXFW ZKLFK FRQWDLQHG LQWURQ  ZDV JHQHUDWHG ZLWK آ¶&$$7&$*$$*$*$&&$$$*$$&7&آ¶ آ¶7$7*$7777$7*$*7*$*&7&&$&آ¶ /DFWRIHUULQ $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶&7*7&&&$7$*$&&7&7*&&*&7$آ¶ خ»JW UHYHUVH DQG F'1$ IURP PDPPDU\ JODQG WLVVXH $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶*77&557**7*75&&5707&&00$آ¶ DQG آ¶*7&777*$$&$*&$**7&&77&7*آ¶ DQG F'1$ IURP PDPPDU\ JODQG WLVVXH $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶77&&$*&7&777**&7&<&&آ¶ DQG آ¶77*$$&$*$$**77777**7آ¶ DQG F'1$ IURP PDPPDU\ JODQG WLVVXH $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶&&$**&$$*7777*&77*77&&$*آ¶ خ»JW UHYHUVH DQG F'1$ IURP PDPPDU\ JODQG WLVVXH /DFWRSHUR[LGDVH $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶&77&7*&$7&7&$7&$&&7$*&$&آ¶ خ»JW UHYHUVH DQG F'1$ IURP PDPPDU\ JODQG WLVVXH $  NES 3&5 SURGXFW ZDV JHQHUDWHG ZLWK آ¶**$*&$<$$&&**&7**&&$*$*$$آ¶ DQG آ¶*7**&&$$$*&**$$**&5$$**7*آ¶ DQG F'1$ IURP PDPPDU\ JODQG WLVVXH $  NES 3&5 SURGXFW ZDV FRQVWUXFWHG ZLWK آ¶*7*&7$**7*$7*$*$7*&$*$$*آ¶ خ»JW UHYHUVH DQG F'1$ IURP PDPPDU\ JODQG WLVVXH 7UDQVIRUPDWLRQ EOXHZKLWH VFUHHQLQJ EDFWHULDO FXOWXUH DQG SODVPLG SXULILFDWLRQ ZDV GRQH LQ WKH VDPH ZD\ DV ZLWK WKH S*(0= YHFWRU )OXRUHVFHQW VHTXHQFLQJ ZDV FDUULHG RXW XVLQJ DQ $/) DXWRPDWHG GHYLFH $PHUVKDP 3KDUPDFLD ZLWK VWDQGDUG RSHUDWLQJ SURFHGXUHV 6HTXHQFLQJ VDPSOHV ZHUH SUHSDUHG XVLQJ WKH &\آŒG$73 ODEHOOHG YHFWRU VSHFLILF SULPHUV

 &\63 &\7

0DWHULDOV DQG 0HWKRGV

آ¶7$&7&$$*&7$7*&$7&&$$&*&*آ¶  DQG آ¶$&7&$&7$7$***&*$$77***&&آ¶

DQG WKH 7KHUPR 6HTXHQDVH F\FOH VHTXHQFLQJ NLW 531  $PHUVKDP 3KDUPDFLD DFFRUGLQJ WR WKH PDQXIDFWXUHUآ¶V LQVWUXFWLRQV 7KH IROORZLQJ  F\FOHV ZHUH DSSOLHG  آƒ&  VHF  آƒ&  VHF  آƒ&  VHF :KHUH VHTXHQFLQJ UHVXOWV GLIIHUHG D WKLUG 3&5 SURGXFW ZDV VHTXHQFHG 2YHUODSSLQJ VHTXHQFHV ZHUH GHWHFWHG XVLQJ WKH )$67$ PRGXOH RI WKH JFJHJFJ SURJUDPPH SDFNDJH *HQHWLFV &RPSXWHU *URXS 0DGLVRQ :,  86$ &RQVHFXWLYH VHTXHQFHV ZHUH MRLQHG DQG YHFWRU VSHFLILF VHTXHQFHV UHPRYHG ,Q WKLV ZD\ FRPSOHWH F'1$ VHTXHQFHV ZHUH REWDLQHG  &RPSXWDWLRQDO 6HTXHQFH $QDO\VLV

$OLJQPHQWV RI '1$ DQG SURWHLQ VHTXHQFHV DQG '1$ VLPLODULW\ VHDUFKHV ZHUH SHUIRUPHG XVLQJ WKH JFJHJFJ SURJUDPPH SDFNDJH *HQHWLFV &RPSXWHU *URXS *HQRPLF '1$ ZDV VFUHHQHG IRU LQWHUVSHUVHG HOHPHQWV XVLQJ 5HSHDW0DVNHU 6PLW  3UREDELOLW\ FDOFXODWLRQV IRU LQWURQH[RQ MXQFWLRQV ZHUH PDGH E\ D FRPELQHG OLQHDU GLVFULPLQDQW UHFRJQLWLRQ IXQFWLRQ XVLQJ LQIRUPDWLRQ DERXW VLJQLILFDQW WULSOHW IUHTXHQFLHV LQ YDULRXV IXQFWLRQDO SDUWV RI VSOLFLQJ VLWH UHJLRQV DQG SUHIHUHQFHV RI RFWDQXFOHRWLGHV LQ SURWHLQ FRGLQJ DQG LQWURQ UHJLRQV 6RORY\HY HW DO  3URWHLQ VHTXHQFH VLPLODULW\ VHDUFKHV DJDLQVW WKH 6ZLVVSURW GDWDEDVH 6ZLVV ,QVWLWXWH RI %LRLQIRUPDWLFV *HQHYD 6ZLW]HUODQG ZHUH PDGH XVLQJ D 6PLWK :DWHUPDQ DOJRULWKP ZLWK GHIDXOW YDOXHV %DUWRQ  6HFRQGDU\ VWUXFWXUH SUHGLFWLRQV ZHUH PDGH XVLQJ QHDUHVW QHLJKERXU DQDO\VLV ZLWK ORFDO DOLJQPHQWV 6DODPRY 6RORY\HY  3RWHQWLDO IRU 2JO\FRV\ODWLRQ ZDV DQDO\VHG XVLQJ WKH 1HW2*O\F  3UHGLFWLRQ 6HUYHU WUDLQHG RQ PXFLQ W\SH 2JO\FRV\ODWLRQ VLWHV LQ PDPPDOLDQ SURWHLQV ZKLFK DUH JO\FRV\ODWHG E\ WKH 8'3*DO1$F SRO\SHSWLGH 1DFHW\OJDODFWRVDPLQ\OWUDQVIHUDVH IDPLO\ +DQVHQ HW DO 

 /RZ UHVROXWLRQ PRGHOV RI WKH WHUWLDU\ VWUXFWXUHV RI FDPHO PLON SURWHLQV ZHUH REWDLQHG E\ FRPSDUDWLYH PRGHOOLQJ *XH[ 3HLWVFK  7KH SULPDU\ VWUXFWXUHV ZKLFK ZHUH UHYHDOHG E\ DPLQR DFLG DQG F'1$ VHTXHQFLQJ ZHUH WKUHDGHG RYHU WHUWLDU\ VWUXFWXUHV RI SURWHLQV ZLWK KLJK VLPLODULW\ LQ SULPDU\ VWUXFWXUH 0XOWLSOH VHTXHQFH DOLJQPHQWV ZHUH PDGH IRU LPSURYHPHQW RI PRGHOOLQJ UHOLDELOLW\ (QHUJ\ PLQLPLVDWLRQ RI WKH PRGHO ZDV GRQH ZLWK IRUFH ILHOG FRPSXWDWLRQ E\ *52026

  5(68/76 $1' ',6&866,21

5HVXOWV DQG 'LVFXVVLRQ



&DVHLQV

/LWHUDWXUH 7KH SURWHLQ IUDFWLRQ RI FRZ PLON FRQVLVWV WR DERXW  RI FDVHLQV )RXU GLIIHUHQW JHQH SURGXFWV DUH GHVLJQDWHG DV خ±V خ±V خ² DQG خ؛FDVHLQV ZKLFK WRJHWKHU IRUP PLFHOODU VWUXFWXUHV RI  QP WR  QP E\ QRQFRYDOHQW DJJUHJDWLRQ خ±V&1 خ±V&1 DQG خ²&1 DUH VWUXFWXUDOO\ UHODWHG DQG FRQWULEXWH WR    DQG  RI WRWDO FDVHLQ خ³&1 D WHUP XVHG IRU EUHDNGRZQ SURGXFWV RI خ²&1 FRQWULEXWHV WR DERXW  RI WKH FDVHLQ IUDFWLRQ DQG خ؛&1 D SURWHLQ ZKLFK LV VWUXFWXUDOO\ QRW UHODWHG WR WKH RWKHU FDVHLQV FRQWULEXWHV WR DERXW  RI WRWDO FDVHLQ $OO FDVHLQV VKDUH D GLVWLQFW DPSKLSKLOLF QDWXUH 7KH 1WHUPLQDO UHJLRQ RI خ؛&1 DQG WKH &WHUPLQDO UHJLRQV RI خ±V خ±V DQG خ²&1 DUH SURQRXQFHG K\GURSKRELF 7KH 1WHUPLQDO UHJLRQV RI خ±V خ±V DQG خ²&1 DUH K\GURSKLOLF DQG FRQWDLQ FOXVWHUHG SKRVSKRVHULQHV ZKLFK ELQG LQRUJDQLF FDOFLXP SKRVSKDWH ZLWK H[WUHPHO\ ORZ GLVVRFLDWLRQ UDWHV 7KH &WHUPLQDO UHJLRQ RI خ؛&1 LV K\GURSKLOLF DQG SRO\2JO\FRV\ODWHG $OO FDVHLQV KDYH D KLJK SUROLQH FRQWHQW ZKLFK SUHYHQWV IRUPDWLRQ RI VHFRQGDU\ VWUXFWXUHV PDLQO\ RI خ±KHOLFHV LQ WKH LQWDFW SURWHLQ &\VWHLQH ZDV QRW IRXQG LQ خ±V خ±V DQG خ²&1 RI DQ\ VSHFLHV DQDO\VHG 7KH WZR F\VWHLQHV IRXQG LQ ERYLQH خ؛&1 ZHUH VKRZQ WR IRUP LQWHUPROHFXODU FURVVOLQNV LQ PLFHOOHV ZKLFK PD\ VXSSRUW WKH ULJLGLW\ RI WKH VWUXFWXUH DQG KHOS LQ JURZWK WHUPLQDWLRQ RI PLFHOOHV 6ZDLVJRRG  ,Q FDPHO PLON ODUJH PLFHOOHV ZKLFK XVXDOO\ FRQWDLQ ORZ DPRXQWV RI خ؛&1 ZHUH IRXQG LQ KLJKHU DPRXQWV WKDQ LQ FRZ PLON )DUDK 5آپHJJ 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خ؛&1 3URWHRO\WLF FOHDYDJH RI WKH JO\FRPDFURSHSWLGH ZKLFK LV WKH &WHUPLQDO SDUW RI خ؛&1 E\ WKH UHQQHWLQJ HQ]\PHV FK\PRVLQ DQG

 SHSVLQ OHDGV WR DJJUHJDWLRQ RI FDVHLQ PLFHOOHV ZKLFK VXEVHTXHQWO\ IRUP D PLFHOODU QHWZRUN REVHUYHG DV D ILUP FRDJXOXP 7KH FRDJXODWLRQ SURFHVV LV SURPRWHG E\ DFLGLILFDWLRQ RI WKH PLON WR S+  ZKLFK OHDGV WR FKDUJH QHXWUDOLVDWLRQ DQG E\ DGGLWLRQ RI FDOFLXP VDOWV &DPHO PLON VKRZV OLWWOH WHQGHQF\ WR FRDJXODWH XSRQ UHQQHW WUHDWPHQW DQG DFLGLILFDWLRQ RQO\ D ZHDN FXUG LV REWDLQHG )DUDK  7KH PLON WHQGV WR IORFFXODWH ZKLFK LV RI GLVDGYDQWDJH IRU PDQXIDFWXULQJ RI FKHHVH DQG IHUPHQWHG PLON SURGXFWV $GGLWLRQ RI XS WR  &D&O LQFUHDVHV WKH VWUHQJWK RI WKH FRDJXOXP VOLJKWO\ %D\RXPL 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آ±  ZHUH WKHUHIRUH FRQVLGHUHG WR UHSUHVHQW WKH PRVW IUHTXHQW RI SUHVXPHG JHQHWLF YDULDQWV RI FDPHO PLON FDVHLQV 7KHVH VHTXHQFHV KDYH EHHQ GHSRVLWHG WR WKH *HQ%DQNآŒ(%, 'DWD %DQN ZLWK DFFHVVLRQ QXPEHUV $- خ±V&1 $ $- خ±V&1 $- خ²&1 < خ؛&1 :H GHFLGHG WR SUHVHQW DQG GLVFXVV WKH UHVXOWV RI FDPHO PLON FDVHLQ DQDO\VLV ZLWKLQ RQH FKDSWHU WR WDNH UHVSHFW WR WKH PDQLIROG LQWHUDFWLRQV EHWZHHQ WKH IRXU FDVHLQV

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

6SHFLHV

&DVHLQ

$PLQR DFLG UHVLGXHV

&DPHO

خ±V&1 $ 

&RZ

خ±V&1 % 

&DPHO

خ±V&1



&RZ

خ±V&1 $ 

&DPHO

خ²&1



&RZ

خ²&1 $ 

&DPHO

خ؛&1



&RZ

خ؛&1 $



1$1$ 1DFHW\OQHXUDPLQLF VLDOLF DFLG D 'DWD RQ FRZ FDVHLQV DIWHU (LJHO HW DO  E &DOFXODWHG ZLWK WKH JFJ SURJUDPPH *HQHWLFV &RPSXWHU *URXS 0DGLVRQ :,  86$ F 6LPLODU DQG LGHQWLFDO UHVLGXHV DV DOLJQHG LQ )LJ 



    F'1$ $*777*&7*&77&77&&&$*7&77***77&$$**7&77*$&&$&&$7*$$*&77&7&$7&&77$&&7*&&77*7* 3URWHLQ 0HW/\V/HX/HX,OH/HX7KU&\V/HX9DO       F'1$ *&7*77*&*&77*&&$**&&7$$$7$7&&7&7&$**7$&&&$*$$*7&777&$$$$7*$$&&$*$&$*&$7$*$* 3URWHLQ $OD9DO$OD/HX$OD$UJ3UR/\V7\U3UR/HX$UJ7\U3UR*OX9DO3KH*OQ$VQ*OX3UR$VS6HU,OH*OX 3    F'1$ *$$*7&&7&$$&$$$$*$$$*$77&77*$*77$*&$*7**777&$&&&$77&$*777$*$&$**$*$$&$7&*$7 3URWHLQ *OX9DO/HX$VQ/\V$UJ/\V,OH/HX*OX/HX$OD9DO9DO6HU3UR,OH*OQ3KH$UJ*OQ*OX$VQ,OH$VS





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

)LJ  F'1$ VHTXHQFH RI FDPHO PLON خ±V&1 $ DQG FRUUHVSRQGLQJ SURWHLQ PDWXUH SURWHLQ LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR * DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 1XPEHULQJ RI WKH DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ 3 SRWHQWLDOO\ SKRVSKRU\ODWHG VHULQH UHVLGXHV



5HVXOWV DQG 'LVFXVVLRQ

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









    F'1$ **&$$*$&$$***&&7$&&&&777$77&&&$&7*7*$$&$&$*$*&$*&7&7&&$7&$*7*$**$$7&$$&7*$$ 3URWHLQ *O\/\V7KU$UJ$OD7\U3UR3KH,OH3UR7KU9DO$VQ7KU*OX*OQ/HX6HU,OH6HU*OX*OX6HU7KU*OX  3 3 3    F'1$ *77&&&$&$*$**$$7&$$&$*$$*7$77&$&7$$*$$$$&7*$$77*$&7*$$*$$*$$$$**$7&$&&$$$$$ 3URWHLQ 9DO3UR7KU*OX*OX6HU7KU*OX9DO3KH7KU/\V/\V7KU*OX/HX7KU*OX*OX*OX/\V$VS+LV*OQ/\V  3     F'1$ 777&7*$$&$$$$7&7$&&$$7$77$7&$*$&$77&&7&7**&&$*$*7$7&7&$$*$&7*777$7&$$7$7&$* 3URWHLQ 3KH/HX$VQ/\V,OH7\U*OQ7\U7\U*OQ7KU3KH/HX7US3UR*OX7\U/HX/\V7KU9DO7\U*OQ7\U*OQ      F'1$ $$$$&7$7*$&7&&$7**$$7&$&$7&$$*$*$7$&7777$$*$77&77*$$77$$&7*&77&7$&77*$77$7* 3URWHLQ /\V7KU0HW7KU3UR7US$VQ+LV,OH/\V$UJ7\U3KH(QG F'1$ F'1$ F'1$ F'1$     *&7&$$&7**$$$$7&*$7&77&7*&$*777&77$7&7$&&$&777$&77&$7&&7$&&**&$7*777$*$*$*$    &&&$77$$7$$*$7$*$$$7$77*$**$$$$**$$*$&7*7*&$*$$7$777&&7*$$*7$777$7$&&$7&&&*     77$*77&$7*77*$*7$7$&7***7&7*7$77*7**77$7$7$&*$$&77$*&7*$7*$77$77*$$$$7*7777    &$&7$&7&777*$*77$7$*$$&7$&$777&77777&&$7*$$$$7$$$$777&$&&*77*&7*7&*

)LJ  F'1$ VHTXHQFH RI FDPHO PLON خ±V&1 DQG FRUUHVSRQGLQJ SURWHLQ PDWXUH SURWHLQ LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR 7 DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 1XPEHULQJ RI WKH DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ 3 SRWHQWLDOO\ SKRVSKRU\ODWHG VHULQH UHVLGXHV



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









    F'1$ &&&$$$$&7$$**$*$&&$7&$77&&7$$*&*&$$$*$$$7*&&&77*&77&$*7&7&&$*7$*7*&&&777$&7 3URWHLQ 3UR/\V7KU/\V*OX7KU,OH,OH3UR/\V$UJ/\V*OX0HW3UR/HX/HX*OQ6HU3UR9DO9DO3UR3KH7KU     F'1$ *$$$*&&$*$*&&7*$&7&7&$&7*$7&7&*$$$$7&7*&$&&77&&7&7*&&7&7*&7&&$*7&777*$7*7$& 3URWHLQ *OX6HU*OQ6HU/HX7KU/HX7KU$VS/HX*OX$VQ/HX+LV/HX3UR/HX3UR/HX/HX*OQ6HU/HX0HW7\U      F'1$ &$*$77&&&&$*&&7*77&&7&$*$&&&&&$7*$77&&7&&7&$*7&&&7*&7*7&&&777&7&$*77&$$$*7& 3URWHLQ *OQ,OH3UR*OQ3UR9DO3UR*OQ7KU3UR0HW,OH3UR3UR*OQ6HU/HX/HX6HU/HX6HU*OQ3KH/\V9DO      F'1$ &7*&&7*77&&&&$*&$$$7**7*&&&7$&&&&&$*$*$*&&$7*&&7*7*&$$*&&*77&7*&&&77&&$**$* 3URWHLQ /HX3UR9DO3UR*OQ*OQ0HW9DO3UR7\U3UR*OQ$UJ$OD0HW3UR9DO*OQ$OD9DO/HX3UR3KH*OQ*OX      F'1$ &&7*7$&&7*$&&&&*7&&*****&7&&$&&&7*7*&&7&$$&&$&77*7&&&7*7*$77*&&7$$*$$*$777& 3URWHLQ 3UR9DO3UR$VS3UR9DO$UJ*O\/HX+LV3UR9DO3UR*OQ3UR/HX9DO3UR9DO,OH$OD(QG F'1$ F'1$ F'1$ F'1$ F'1$    $$$*77$$7$&&7&&7&&7&$&7777*$$77*$&7*&*$&7**$$$7*7**&$$&7777&$$7&777*7$7&$7*     77$&&$$$$&*$77777$$$7$$&7&7&&$7**$$$$$$$7*$$$&7*7$*7&7777$777$7777$7*&777$&     $7**&$&7&$7&77$$777*$$777*$&7&$$$$7777$$77&$$&7$$7*&&$7$$$*77&$$7*77$$*77**     $$$7$&&$7$$*&77$7&$$$$$7*777$7$$$$$7&$777*7*7$$7777*&77$77$77$777&777$$*$$7   &7$777&&$$$&&$*7&$&77&$$7$$$&7$$7&&777$**&$7

)LJ  F'1$ VHTXHQFH RI FDPHO PLON خ²&1 DQG FRUUHVSRQGLQJ SURWHLQ PDWXUH SURWHLQ LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR & DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 1XPEHULQJ RI WKH DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ 3 SRWHQWLDOO\ SKRVSKRU\ODWHG VHULQH UHVLGXHV



5HVXOWV DQG 'LVFXVVLRQ

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

)LJ  F'1$ VHTXHQFH RI FDPHO خ؛&1 DQG FRUUHVSRQGLQJ SURWHLQ PDWXUH SURWHLQ LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR & DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 1XPEHULQJ RI DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ &K\PRVLQ FOHDYDJH VLWH 3KH ,OH 3 SRWHQWLDOO\ SKRVSKRU\ODWHG VHULQH *O\ JO\FRV\ODWHG WKUHRQLQHV $UURZ LQGLFDWHV FK\PRVLQ FOHDYDJH VLWH

 (OXWLRQ 3DWWHUQ RI &DVHLQV LQ 5HYHUVHG3KDVH &KURPDWRJUDSK\ 3URWHLQV RI IUDFWLRQV , ,, ,,, 9 DQG 9,, )LJ  ZHUH SDUWLDOO\ VHTXHQFHG )UDFWLRQ , FRQVLVWHG RI خ؛&1 ,, DQG ,,, RI خ±V&1 9 RI خ±V&1 DQG 9,, RI خ² &1 $PLQR DFLG VHTXHQFLQJ RI WKH IUDJPHQW HOXWHG DW  PLQ IURP WKH WU\SWLF GLJHVW RI IUDFWLRQ ,,, UHYHDOHG D PDMRU LQVHUW *OX*OQ$OD7\U3KH +LV/HX*OX )LJ  EHWZHHQ *OQ DQG 3UR RI WKH PDWXUH SURWHLQ )LJ  0HDVXUHG SURWHLQ PDVV ZDV DERXW  N'D )URP WKHVH UHVXOWV ZH SURSRVH DQ خ±V&1 % 3 ZLWK  DPLQR DFLGV D PROHFXODU PDVV RI  N'D DQG DQ LVRHOHFWULF SRLQW DW S+  )LJ  /RQJ DQG VKRUW YDULDQWV RI خ±V&1 DOVR RFFXU LQ RYLQH PLON )HUUDQWL HW DO 
 

  





  





,

,,

,,, ,9 9

9,

9,,,

  



   



5HWHQWLRQ 7LPH >PLQ@









)LJ  5HYHUVHGSKDVH & +3/& FKURPDWRJUDP RI DFLG SUHFLSLWDWHG FDPHO PLON FDVHLQ 3HDNV , ,, ,,, 9 9,, DQG 9,,, ZHUH FROOHFWHG IRU IXUWKHU DQDO\VLV 3HDNV ,9 DQG 9, ZHUH QRW LGHQWLILHG *UDGLHQW RI VROYHQW % DV GDVKHG OLQH 7KH DXWKRUV VXJJHVWHG WKDW WKRVH YDULDQWV ZHUH D UHVXOW RI DOWHUQDWLYH VSOLFLQJ RI WKH KHWHURJHQHRXV QXFOHDU 51$ WUDQVFULEHG IURP WKH خ±V&1 JHQH UDWKHU WKDQ JHQH SURGXFWV RI WZR GLIIHUHQW DOOHOHV

&RQFHQWUDWLRQ RI 6ROYHQW % PO O



9,,



$ >$8@



5HVXOWV DQG 'LVFXVVLRQ

53.<3/5<3(9)41(3'6,((9/1.5.,/'/$9963,4)54(1,'(/.'7 3 51(37('+,0('7(5.(6*6666((996677(4.',/.('03645
    

)LJ  3URSRVHG DPLQR DFLG VHTXHQFH RI FDPHO PLON خ±V&1 % 3 ,QVHUW VKDGHG DQG DPLQR DFLG UHVLGXHV LQ EROG 3 SRWHQWLDOO\ SKRVSKRU\ODWHG VHULQHV 7KH PLQRU SHDN 9, WR WKH OHIW RI SHDN 9,, )LJ  LV VXJJHVWHG WR UHSUHVHQW D YDULDQW RI خ²&1 &RPSDULQJ WKH خ³&1 VHTXHQFH RI FDPHO PLON REWDLQHG E\ %HJ HW DO  D ZLWK WKH خ²&1 VHTXHQFH VKRZQ KHUH UHYHDOHG D VLQJOH H[FKDQJH *OX IRU *OQ 7KH IUDJPHQW VHTXHQFHG E\ %HJ PD\ WKHUHIRUH EHORQJ WR D QRYHO خ²&1 YDULDQW % 0ROHFXODU PDVVHV RI IUDFWLRQ 9,,, ZHUH    DQG  N'D DQG SUHVXPDEO\ EHORQJHG WR K\GURSKRELF خ³ &1 6SHFLHV
&DPHO &RZ &DPHO &RZ &DPHO &RZ &DPHO &RZ

&DVHLQ
خ±V&1 خ±V&1 خ±V&1 خ±V&1 خ²&1 خ²&1 خ؛&1 خ؛&1

6HTXHQFH RI VLJQDO SHSWLGH
0.//,/7&/9$9$/$ 0.//,/7&/9$9$/$ 0.)),)7&//$99/$ 0.)),)7&//$9$/$ 0.9/,/$&59$/$/$ 0.9/,/$&/9$/$/$ 0.6))/997,/$/7/3)/*$ 00.6))/997,/$/7/3)/*$

)LJ  6HTXHQFH FRPSDULVRQ RI VLJQDO SHSWLGHV IURP FDPHO DQG FRZV
PLON FDVHLQV &RQVHUYHG UHVLGXHV DUH VKDGHG 6LJQDO 6HTXHQFHV F'1$ VHTXHQFHV ZHUH WUDQVODWHG LQWR FDVHLQ SUHFXUVRU SURWHLQV FRQWDLQLQJ VLJQDO SHSWLGHV )LJV  آ±  ZKLFK OHDG SURWHLQV LQWR WKH URXJK HQGRSODVPLF UHWLFXOXP DQG DUH VXEVHTXHQWO\ FOHDYHG RII %XUJHVV .HOO\  6LJQDO SHSWLGH VHTXHQFHV RI DOO H[DPLQHG SURWHLQV ZHUH KLJKO\

 FRQVHUYHG EHWZHHQ FDPHO DQG FRZ )LJ  7KH VLJQDO SHSWLGHV RI WKH FDOFLXPVHQVLWLYH FDVHLQV خ±V خ±V DQG خ²&1 ZHUH DOO  DPLQR DFLGV LQ OHQJWK DQG VLPLODU LQ DPLQR DFLG VHTXHQFH :H DVVXPH WKDW WKLV VLPLODULW\ LV D UHVXOW RI WKH FRPPRQ HYROXWLRQDU\ RULJLQ RI WKH UHVSHFWLYH JHQHV 5RVHQ  DQG RI WKH VLPLODU SURWHLQ WDUJHW DQG IXQFWLRQ 3KRVSKRU\ODWLRQ &DVHLQV DUH SUHIHUHQWLDOO\ SKRVSKRU\ODWHG E\ PDPPDU\ JODQG FDVHLQ NLQDVH ZKLFK UHFRJQLVHV WKH SDWWHUQ >6HU 7KU@آ±;DD>6HU3 *OX $VS@آ±;DD ZLWK *OX RU $VS DW SRVLWLRQ  DQG  HQKDQFLQJ SKRVSKRU\ODWLRQ 6ZDLVJRRG  7KH PRWLI ZDV IRXQG VL[ WLPHV LQ خ±V&1 6HU 6HU 6HU 6HU 6HU DQG 6HU QLQH WLPHV LQ خ±V&1 6HU 6HU 6HU 6HU 6HU 6HU 6HU 6HU DQG 6HU IRXU WLPHV LQ خ²&1 6HU 6HU 6HU DQG 6HU DQG WZLFH LQ خ؛&1 6HU DQG 6HU )LJV  آ±  6HU LQ خ؛&1 ZDV IRXQG WRZDUGV WKH HQG RI WKH SURWHLQ D SRVLWLRQ WKDW LV OHVV IUHTXHQWO\ SKRVSKRU\ODWHG 0RGLILFDWLRQ RI WKH SURSRVHG VLWHV LV LQ DJUHHPHQW ZLWK WKH PROHFXODU PDVVHV PHDVXUHG E\ PDWUL[ DVVLVWHG ODVHU GHVRUSWLRQLRQLVDWLRQ PDVV VSHFWURPHWU\ 7KH PHDVXUHG PROHFXODU PDVVHV RI خ±V خ±V DQG خ²&1 ZHUH D PXOWLSOH RI RQH SKRVSKDWH JURXS ZKLFK DFFRXQWV IRU  'D KLJKHU WKDQ WKH PROHFXODU PDVVHV FDOFXODWHG IURP WKH XQPRGLILHG DPLQR DFLG FKDLQ 7DEOH  7KH PRVW IUHTXHQW IRUP RI خ²&1 KDG RQO\ WKUHH SKRVSKDWH JURXSV ERXQG LQVWHDG RI WKH IRXU JURXSV SUHGLFWHG 7KH GHJUHH RI SKRVSKRU\ODWLRQ RI خ؛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خ؛&DVHLQ $QRWKHU SRVWWUDQVODWLRQDO PRGLILFDWLRQ IRXQG LQ FDVHLQV LV JO\FRV\ODWLRQ RI 7KU UHVLGXHV LQ خ؛&1 7KLV RFFXUV IRU 7KU QHDU $UJ/\V 7KU RU 3UR DQG LV OLNHO\ WR EH LQKLELWHG E\ ,OH 3LVDQR HW DO  3UHGLFWLRQ RI 2 JO\FRV\ODWLRQ ZDV GRQH E\ WKH PHWKRG RI +DQVHQ HW DO  :KHUHDV ERYLQH خ؛&1 KDG KLJK JO\FRV\ODWLRQ SUREDELOLW\ DW 7KU 7KU 7KU



5HVXOWV DQG 'LVFXVVLRQ

2JO\FRV\ODWLRQ SRWHQWLDO 2JO\FRV\ODWLRQ SRWHQWLDO



D





E











  6HTXHQFH 3RVLWLRQ







)LJ  2JO\FRV\ODWLRQ SRWHQWLDO RI WKUHRQLQHV LQ خ؛FDVHLQ 7KH SRWHQWLDO RI WKH UHVLGXHV LV VKRZQ DV D VROLG EDU ZLWK D YDOXH IURP  QR SRWHQWLDO WR  KLJK SRWHQWLDO 7KH WKUHVKROG ZKLFK GHSHQGV RQ WKH SULPDU\ VWUXFWXUH RI WKH SURWHLQ LV VKRZQ DV D GDVKHG OLQH 7KH SUREDELOLW\ RI JO\FRV\ODWLRQ LV WKH GLIIHUHQFH EHWZHHQ WKH SRWHQWLDO DQG WKH WKUHVKROG D &DPHO خ؛FDVHLQ E %RYLQH خ؛FDVHLQ

 7KU 7KU 7KU 7KU DQG 7KU JO\FRV\ODWLRQ SRWHQWLDO RI FDPHO خ؛ &1 ZDV KLJK DW 7KU 7KU 7KU 7KU 7KU 7KU 7KU DQG 7KU )LJ  6HU LQ FDPHO خ؛&1 DQG 6HU 6HU DQG 6HU RI ERYLQH خ؛&1 DOVR KDG D KLJK JO\FRV\ODWLRQ SRWHQWLDO DQG WKH ERYLQH خ؛&1 VHULQH UHVLGXHV ZHUH UHSRUWHG WR EH JO\FRV\ODWHG VRPHWLPHV 6ZDLVJRRG  7KH SRVLWLRQV LQ FDPHO خ؛&1 ZHUH SUHGRPLQDQWO\ WRZDUGV WKH &WHUPLQDO HQG RI WKH JO\FRPDFURSHSWLGH ZKHUHDV JO\FRV\ODWLRQ SRWHQWLDO RI ERYLQH خ؛&1 ZDV KLJK WRZDUGV WKH 1WHUPLQDO HQG 7KLV GLIIHUHQFH LV VXJJHVWHG WR EH D VWUXFWXUDO FRQVHTXHQFH RI D GHOHWLRQ ZKLFK PDNHV FDPHO خ؛&1 VHYHQ DPLQR DFLGV VKRUWHU WKDQ WKH ERYLQH FRXQWHUSDUW )LJ  ,W LV DVVXPHG WKDW ERYLQH خ؛&1 DQG FDPHO خ؛&1 KDYH GLIIHUHQW DIILQLWLHV WRZDUGV FDPHO DQG ERYLQH FK\PRVLQ 7KHVH GLIIHUHQFHV EHWZHHQ FDPHO DQG ERYLQH خ؛&1 PD\ DOVR KDYH LPSOLFDWLRQV RQ خ؛&1 WHUWLDU\ VWUXFWXUH PLFHOOH DVVHPEO\ DQG VWUXFWXUH K\GURSKLOLF VKLHOGLQJ RI WKH PLFHOOH FRUH E\ WKH JO\FRV\ODWHG SDUW RI خ؛&1 DQG FKDUJH QHXWUDOLVDWLRQ E\ FDOFLXP VDOWV 7RWDO JO\FRV\ODWLRQ SRWHQWLDO ZDV VLPLODU LQ ERYLQH DQG FDPHO خ؛&1 0LFURKHWHURJHQHLW\ IRXQG LQ خ؛&1 RI RWKHU VSHFLHV GXH WR GLIIHUHQFHV LQ SDWWHUQ DQG FRPSRVLWLRQ RI FDUERK\GUDWH PRLHWLHV LV DOVR H[SHFWHG LQ FDPHO خ؛&1 7KH FDUERK\GUDWHV DWWDFKHG LQ ERYLQH خ؛&1 DUH WULVDFFKDULGHV RI WKH W\SH 1$1$خ±→*DO خ²→1$*$خ² RU WHWUDVDFFKDULGHV ZKLFK KDYH DQ DGGLWLRQDO خ±→1$*$ OLQNHG 1$1$ UHVLGXH ,I ILYH RI WKH WKUHRQLQHV ZLWK JO\FRV\ODWLRQ SRWHQWLDO KDYH WZR VLDOLF DFLG UHVLGXHV ERXQG DQG RQH VHULQH LV SKRVSKRU\ODWHG WKH LVRHOHFWULF SRLQW ZLOO EH ORZHUHG WR  6'63$*( DQG PDVV VSHFWURVFRSLF VWXGLHV UHYHDOHG WKDW PRVW RI WKH خ؛&1 DQDO\VHG ZDV RI UHODWLYHO\ ORZ PROHFXODU PDVV PDNLQJ WKH ORZ JO\FRV\ODWHG IRUP SUHGRPLQDQW DQG WKH SURWHLQ YHU\ EDVLF 7KLV ILQGLQJ GLVDJUHHV ZLWK WKH KLJK VLDOLF DFLG FRQWHQW UHSRUWHG E\ 0HKDLD  ,W LV DVVXPHG WKDW SHDNV LQ WKH 0$/', VSHFWUXP ZLWK KLJKHU PROHFXODU PDVVHV ZHUH QRW GHWHFWHG GXH WR SHDN EURDGHQLQJ E\ خ؛&1 PLFURKHWHURJHQHLW\ 3ULPDU\ 6WUXFWXUH 6LPLODUO\ WR ERYLQH FDVHLQV FDPHO خ±V&1 DQG خ²&1 ZHUH GHYRLG RI F\VWHLQH UHVLGXHV DQG خ±V&1 DQG خ؛&1 ERWK FRQWDLQHG RQO\ WZR F\VWHLQHV 7KH SUROLQH FRQWHQW LQ FDPHO FDVHLQV ZDV VOLJKWO\ KLJKHU WKDQ LQ ERYLQH FDVHLQV ZLWK  LQ خ±V&1  LQ خ±V&1  LQ خ²&1 DQG  LQ خ؛&1 FRPSDUHG WR  LQ ERYLQH خ±V&1 %  LQ ERYLQH خ±V&1 $  LQ ERYLQH خ²&1 $ DQG  LQ ERYLQH خ؛&1 $ 7KLV KLJKHU SUROLQH FRQWHQW LQ FDPHO FDVHLQV PD\ OHDG WR GHVWDELOLVDWLRQ RI VHFRQGDU\ VWUXFWXUHV LQ D PRUH SURQRXQFHG ZD\ WKDQ LW RFFXUV LQ ERYLQH FDVHLQV



5HVXOWV DQG 'LVFXVVLRQ

7KH JHQH VWUXFWXUH RI خ±V خ±V DQG خ²&1 LV FKDUDFWHULVHG E\ PDQ\ VKRUW H[RQ VHTXHQFHV ZKLFK DUH XVXDOO\ LQWHUUXSWHG E\ ORQJ LQWURQV 7KH P51$ RI ERYLQH خ±V&1 LV RQO\  ES ORQJ WKH UHODWHG KQ51$ FRPSULVHV  ES 7KH LQVHUWHG VHTXHQFH $VQ WR *OX LQ ERWK YDULDQWV RI FDPHO خ±V&1 ZDV SUREDEO\ WKH UHVXOW RI DQ DGGLWLRQDOO\ H[SUHVVHG H[RQ EHWZHHQ ERYLQH H[RQ  DQG H[RQ  DQG WKH GHOHWLRQ RI ERYLQH *OX WR *OX LQ FDPHO خ±V &1 $ UHIHUUHG WR ERYLQH H[RQ  7KH LQVHUW RI *OQ WR +LV LQ FDPHO خ±V &1 $ DQG % ZDV QRW DW DQ LQWURQH[RQ MXQFWLRQ DQG WKHUHIRUH OLNHO\ WKH UHVXOW RI DQ LQVHUWLRQ LQ WKH FDPHO خ±V&1 JHQH 7KH LQWURQH[RQ VWUXFWXUH RI WKH ERYLQH خ±V&1 JHQH LV QRW \HW UHVROYHG 7KH P51$ RI ERYLQH خ²&1 LV  ES ORQJ WKH KHWHURJHQHRXV QXFOHDU 51$ SUHFXUVRU  ES &DPHO DQG ERYLQH خ²&1 KDG D VLPLODU OHQJWK ZLWK IHZ LQVHUWLRQV LQ FDPHO خ²&1 )LJ  7KH VKRUW LQVHUWLRQV ZHUH D UHVXOW RI PXWDWLRQV LQ WKH FDPHO خ²&1 JHQH UDWKHU WKDQ D FRQVHTXHQFH RI DOWHUQDWLYH VSOLFLQJ &DPHO خ؛&1 ZDV VHYHQ UHVLGXHV VKRUWHU WKDQ WKH ERYLQH FRXQWHUSDUW 'HOHWLRQ RI 9DO WR $OD LV VXJJHVWHG WR EH WKH UHVXOW RI D PXWDWLRQ LQ WKH WKLUG H[RQ RI WKH خ؛ &1 JHQH ZKLFK FRGHV IRU WKH ODUJHVW SDUW RI WKH PDWXUH SURWHLQ LQ ERWK VSHFLHV 7KH ERYLQH خ؛&1 P51$ LV  ES ORQJ WKH FRUUHVSRQGLQJ KQ51$ PRUH WKDQ  ES ,Q FRQWUDVW WR WKH RWKHU FDVHLQ JHQHV WKH JHQH GRHV QRW FRQVLVW RI VKRUW H[RQ VHTXHQFHV ZKLFK SDUWLDOO\ PD\ KDYH HYROYHG E\ H[RQ GXSOLFDWLRQ LQ WDQGHP GLUHFWLRQ 7KH GHOHWLRQ RI 9DO WR $OD LV DOVR IRXQG LQ SRUFLQH خ؛&1 DQG WKH FOHDYDJH VLWH 3KH,OH DOVR FRUUHVSRQGV WR WKH SRUFLQH FOHDYDJH VLWH 1RQHWKHOHVV SRUFLQH خ؛&1 FRQWDLQV DQ LQVHUW LQ WKH JO\FRPDFURSHSWLGH ZKLFK ZDV QRW IRXQG LQ WKH FDPHO SURWHLQ 7KH JO\FRV\ODWLRQ SRWHQWLDO RI SRUFLQH خ؛&1 ZDV PRUH HYHQO\ GLVWULEXWHG DORQJ WKH ZKROH JO\FRPDFURSHSWLGH VHTXHQFH WKDQ LQ FDPHO DQG ERYLQH خ؛&1 6HFRQGDU\ 6WUXFWXUH 6HTXHQFH FRPSDULVRQ RI FRZ DQG FDPHO PLON FDVHLQV DUH VKRZQ LQ )LJ  )HZ SURQRXQFHG VWUXFWXUDO GLIIHUHQFHV ZHUH IRXQG ZKHQ FDPHO DQG FRZ PLON FDVHLQV ZHUH FRPSDUHG $OWKRXJK خ±V&1 RI FDPHO DQG FRZ PLON KDG D ORZ SHUFHQWDJH VLPLODULW\ LQ SULPDU\ VWUXFWXUH VLPLODULWLHV LQ WKH VHFRQGDU\ VWUXFWXUH D VHULHV RI خ±KHOLFDO UHJLRQV IROORZHG E\ D &WHUPLQXV ZLWK OLWWOH GHILQHG VHFRQGDU\ VWUXFWXUH SUHGRPLQDWHG ,Q FDPHO PLON خ±V&1 K\GURSKLOLFLW\ RI WKH 1WHUPLQDO HQG ZDV VOLJKWO\ PRUH SURQRXQFHG 7KH GHOHWLRQV WKDW VKRUWHQHG FDPHO PLON خ±V&1 FRPSDUHG ZLWK FRZ PLON خ±V &1 $ RFFXUUHG LQ DQ خ±KHOLFDO UHJLRQ EHWZHHQ ERYLQH *OX DQG $VQ )LJ  7KH\ ZHQW DORQJ ZLWK WKH ORVV RI WKH SKRVSKRU\ODWHG VHULQH FOXVWHU 6HU 6HU DQG 6HU  7KLV ORVV PD\ KDYH LPSOLFDWLRQV LQ PLFHOOH

&DPHO aV&1 $ %RYLQH aV&1 %  

3KRVSKRU\ODWLRQ &OXVWHUV aaaaaa bb bb aaaaaaaaa aaaaa 53.<3/5<3(9)41(3'6,((9/1.5.,/(/$9963,4)54(1,'(/ .'751(37('+,0('7(5.(6*6 666  53.+3,.+4*/34 (9/1(1//5))9$3)3(9)*.(.91(/6.',*6(67('4$0(',.40($(6,666 

&DPHO aV&1 $ %RYLQH aV&1 %

aaaaaaaaaaaa aaaaaaaaa aaaaaaa bb aaaaa ((996677(4.',/.('03645
&DPHO aV&1 $ %RYLQH aV&1 %

'HOHWLRQ LQ &DPHO aV&1 $ bbbb bbbb aaaaa *(39.9974 3)34))4/*$63<9$:<<334904<,$+366<'73(*,$6('**.7'9034:: .(30,*914(/$<)<3(/)54)<4/'$<36*$:<<93/*74<7'$36)6',313,*6(16(.7 703/:

 



&DPHO aV&1 %RYLQH aV&1 $

3KRVSKRU\ODWLRQ &OXVWHU &\VWHLQHV 3KRVSKRU\ODWLRQ &OXVWHU ORVW LQ aaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaa aaaaaa .+(0'4*666((6,19644.).49..9$,+36.(',&67)&(($951,.( 9(6$(937( .170(+9666((6, ,64(7<.4(.10$,136.(1/&67)&.(9951$1(((<6,*666((6$(9$7((9.,79'

 

&DPHO aV&1 %RYLQH aV&1 $

&DPHO aV&1 3KRVSKRU\ODWLRQ &OXVWHU aaaaaaaaaaaaaaaaaaaaaaaaaa bbb bbbb aaaaa 1.,64)<4.:.)/4
   

&DPHO aV&1 %RYLQH aV&1 $

aa aaaaaaaaaaaaaaaa aaaaaaaaa aaaaaaaaaaaaa 7..7(/7(((.'+4.)/1.,<4<<47)/:3(
&DPHO b&1 %RYLQH b&1 $

3KRVSKRU\ODWLRQ &OXVWHU aaaaaaaaaaaa aa aaaaa bb bbb 5(.(().7$*($/(6,666((6,7+,1.4.,(.).,((4447('(44'.,<7)34346/9<6+7(3,3<3,/341 5(/((/193*(,9(6/666((6,75,1. .,(.)46((4447('(/4'.,+3)$4746/9<3)3*3,316 /341  

&DPHO b&1 %RYLQH b&1 $

+\GURSKRELF &WHUPLQDO 'RPDLQ bbbb aaaaa )/33/43$909 3)/43.90'93.7.(7,,3.5.(03//463993)7(646/7/7'/(1/+/3/3//46/0<4,3 ,33/747399933)/43(90*96.9.($0$3.+.(03)3.<39(3)7(646/7/7'9(1/+/3/3//46:0+43+

   

&DPHO b&1 %RYLQH b&1 $

bb 43934730,3346//6/64).9/39344093<345$0394$9/3)4(393'395*/+39343/939,$ 43/33790)33469/6/646.9/3934.$93<345'03,4$)//<4(39/*395* 3)3,, 9



&DPHO k&1 %RYLQH k&1 $

&\VWHLQH aaaaaaaa bb bbbbb bbbbbb bbbb (9414(437&)(.9(5//1(.79.<)3,4)9465<36<*,1<<4+5/$93,114),3<31<$.39$,5/+$4,34 4(414(43,5&(.'(5))6'.,$.<,3,4<9/65<36<*/1<<44.39$/,114)/3<3<<$.3$$9563$4,/4

 

&\VWHLQH

'HOHWLRQ

&DPHO k&1 %RYLQH k&1 $

آ¸ 7KU6HU *O\FRV\ODWLRQ 3RWHQWLDO bbbbb bbbb &4$/31, '3379(55353536),$,33..74'.7913$,179$79(339,37$(3$91799,$($66 :49/61793$.6&4$43770$5+3+3+/6)0$,33..14'.7(,37,17,$6*(3767377($9(679$7/('63

   

5HVXOWV DQG 'LVFXVVLRQ

&DPHO k&1 %RYLQH k&1 $

bbbb bbbbbb (),77673(77794,767(, (9, (633(,17949767$9

)LJ  6HTXHQFH FRPSDULVRQ RI FRZ DQG FDPHO PLON FDVHLQV %RYLQH FDVHLQV DFFRUGLQJ WR WKH 6ZLVVSURW GDWDEDVH 0RGLILHG UHVLGXHV DQG F\VWHLQHV LQ EROG آ¸ 6LWH RI SURWHRO\WLF FOHDYDJH E\ DVSDUWLF SURWHDVHV LQ خ؛FDVHLQ

 DVVHPEODJH DQG VWDELOLW\ DV ZHOO DV LQ WKH QXWULWLRQDO EHKDYLRXU RI WKH FDVHLQV )HUUDQWL HW DO  6LPLODUO\ WR FRZ PLON FDPHO PLON خ±V&1 ZDV WKH PRVW K\GURSKLOLF DPRQJ WKH IRXU FDVHLQV DQG KDG D KLJK SRWHQWLDO IRU VHFRQGDU\ VWUXFWXUHV PDLQO\ خ±KHOLFHV )LJ  7KH WZR F\VWHLQH UHVLGXHV DOVR RFFXUUHG DW DERXW SRVLWLRQ  &K\PRVLQ 6HQVLWLYH 6LWH RI خ؛&DVHLQ 7KH VLWH RI FOHDYDJH RI FDPHO PLON خ؛&1 E\ FK\PRVLQ LV 3KH,OH )LJ  OHDYLQJ D PDFURSHSWLGH RI  N'D  DPLQR DFLGV LQ OHQJWK ZLWK DQ LVRHOHFWULF SRLQW RI WKH XQPRGLILHG SHSWLGH DW S+  ,Q ERYLQH خ؛&1 WKH VLWH RI FOHDYDJH LV 3KH0HW OHDYLQJ D PDFURSHSWLGH RI  N'D  DPLQR DFLGV LQ OHQJWK ZLWK DQ LVRHOHFWULF SRLQW RI WKH XQPRGLILHG SHSWLGH DW S+  7KH DPLQR DFLG VHTXHQFH IURP +LV WR /\V LV LQYROYHG LQ ELQGLQJ DQG FOHDYDJH RI ERYLQH خ؛&1 E\ FK\PRVLQ 9LVVHU  ,W LV DVVXPHG WKDW WKH SUROLQH UHVLGXHV LQ WKLV VHTXHQFH VWDELOLVH WKH FRUUHFW FRQIRUPDWLRQ RI خ؛&1 LQ WKH DFWLYH VLWH FOHIW RI FK\PRVLQ DQG WKH EDVLF UHVLGXHV DUH WKRXJKW WR ELQG WR DFLGLF UHVLGXHV DW HLWKHU HQG RI WKH DFWLYH VLGH FOHIW 3ORZPDQ  $OO SUROLQH UHVLGXHV ZHUH FRQVHUYHG LQ FDPHO PLON خ؛&1 DV VKRZQ LQ )LJ  DQG WKH ERYLQH UHVLGXH /HX ZDV UHSODFHG E\ 3UR. 7KLV DGGLWLRQDO SUROLQH UHVLGXH PD\ KHOS WR VWDELOLVH D FRQIRUPDWLRQ RI خ؛&1 LQ WKH DFWLYH VLWH FOHIW RI FDPHO FK\PRVLQ GLIIHUHQW WR WKH FRQIRUPDWLRQ RI FRZ PLON خ؛&1 LQ WKH FOHIW RI ERYLQH FK\PRVLQ +LVWLGLQH UHVLGXHV LQ WKH VHTXHQFH +LV3UR+LV3UR+LV RI FRZ PLON خ؛ &1 ZHUH UHSODFHG E\ PRUH EDVLF DUJLQLQH UHVLGXHV LQ FDPHO PLON خ؛&1 )LJ  6LQFH DUJLQLQH UHPDLQV SURWRQLVHG DW KLJKHU S+ YDOXHV DQG KDV D ORQJHU DQG PRUH IOH[LEOH VLGH FKDLQ LW FDQ EH VSHFXODWHG WKDW WKHVH UHVLGXHV ZLOO VWLOO ELQG WR WKH DFLGLF FHQWUHV RI FK\PRVLQ DW D KLJKHU ORFDO S+ DQG WKDW WKH FDPHO PLON خ؛&1 EDFNERQH GRHV QRW QHHG WR EH ERXQG DV WLJKWO\ WR FK\PRVLQ DV LW ZDV VKRZQ IRU FRZ PLON خ؛&1 3ORZPDQ  0RGHOV IRU VHFRQGDU\ VWUXFWXUH SDWWHUQV ZHUH VLPLODU LQ ERYLQH DQG FDPHO PLON خ؛&1 ZLWK DQ 1WHUPLQDO خ±KHOL[ FRQWDLQLQJ RQH &\V IROORZHG E\ خ² SOHDWHG VKHHWV DQG D VHFRQG &\V )LJ  %RWK &\V UHVLGXHV ZHUH DW SRVLWLRQV VLPLODU WR WKRVH LQ ERYLQH PLON خ؛&1 DQG DUH VXJJHVWHG WR EH LQYROYHG LQ LQWHUPROHFXODU FURVVOLQNV 5LFKDUGVRQ HW DO  2I خ؛FDVHLQV DOUHDG\ VHTXHQFHG SRUFLQH خ؛&1 ZDV PRVW VLPLODU LQ RYHUDOO VWUXFWXUH UHYHDOLQJ D FOHDYDJH VLWH KLJKO\ VLPLODU LQ SULPDU\ DQG VHFRQGDU\ VWUXFWXUH 3RUFLQH FK\PRVLQ DFWLQJ RQ SRUFLQH PLON ZDV VKRZQ WR KDYH  WLPHV KLJKHU SURWHRO\WLF VSHFLILFLW\ FRPSDUHG ZLWK ERYLQH FK\PRVLQ +RXHQ HW DO



5HVXOWV DQG 'LVFXVVLRQ

 $ VLPLODU DELOLW\ WR FOHDYH FDPHO PLON خ؛&1 ZLWK YHU\ KLJK VSHFLILFLW\ LV DVVXPHG UHQQHW FRDJXODWLRQ RI FDPHO PLON ZLWK FDPHO FK\PRVLQ

&DPHO $UJ3UR$UJ3UR$UJ3UR6HU3KH,OH$OD,OH3UR3UR/\V/\V &RZ +LV3UR+LV3UR+LV/HX6HU3KH0HW$OD,OH3UR3UR/\V/\V

)LJ  6HTXHQFH FRPSDULVRQ RI WKH FK\PRVLQVHQVLWLYH UHJLRQ RI خ؛&1 IURP FDPHO DQG FRZ PLON &RQVHUYHG UHVLGXHV DUH VKDGHG 5HQQHW &RDJXODWLRQ 7KH TXDQWLWDWLYH GLVWULEXWLRQ RI LQGLYLGXDO FDVHLQV LQ WRWDO FDVHLQ DV FDOFXODWHG IURP )LJ  ZHUH خ±V&1   خ±V&1   خ²&1   خ؛&1   خ؛&1 ZDV FRQVLGHUHG WR KDYH D WHUPLQDWLQJ IXQFWLRQ LQ PLFHOOH JURZWK +RUQH HW DO  DV ZHOO DV D VWDELOL]LQJ HIIHFW RQ WKH PLFHOOH E\ LWV QHW FKDUJH DQG E\ VWHULF KLQGUDQFH RI DJJUHJDWLRQ E\ LWV K\GURSKLOLF &WHUPLQDO HQG +ROW HW DO  7KH JO\FRV\ODWHG IRUPV PD\ KDYH D VWURQJHU LPSDFW RQ ERWK WKHVH IXQFWLRQV WKDQ WKH QRQJO\FRV\ODWHG IRUP ZKLFK VHHPHG WR EH SUHGRPLQDQW LQ FDPHO PLON RZLQJ WR VWHULF UHSXOVLRQ RI FKDUJHG VLDOLF DFLG JURXSV DQG WR LQFUHDVHG K\GURSKLOLFLW\ $IWHU FOHDYDJH RI WKH PDFURSHSWLGH K\GURSKRELF IRUFHV DQG HOHFWURVWDWLF LQWHUDFWLRQV RI ELYDOHQW FDWLRQV ZLWK QHJDWLYHO\ FKDUJHG JURXSV SURPRWH FRDJXODWLRQ DQG VWDELOLVH WKH FXUG 'DOJOHLVK  0RUD*XWLHUUH] HW DO  6LQFH خ²&1 SUHGRPLQDWHG LQ WKH FDPHO PLON VWXGLHG DQG SKRVSKRU\ODWLRQ RI خ±V DQG خ±V&1 ZDV ORZHU WKDQ LQ FRZ PLON ZH DVVXPH WKDW K\GURSKRELFLW\ LV WKH GULYLQJ IRUFH LQ FRDJXODWLRQ RI FDPHO PLON ,W KDV EHHQ VKRZQ WKDW FDPHO PLON FDVHLQ LV OHVV VWDEOH DW HOHYDWHG WHPSHUDWXUH WKDQ FRZ PLON )DUDK $WNLQV  7KLV PD\ EH DQ HIIHFW RI WKH KLJK خ²&1 FRQWHQW 2Q WKH RWKHU KDQG LQ PLONV ZLWK DPRXQWV RI خ؛&1 KLJKHU WKDQ LQ FRZ PLON HJ LQ EXIIDOR PLON ZLWK  J خ؛&1NJ WRWDO FDVHLQ (O'LQ $RNL  FXUG ILUPQHVV ZDV IRXQG WR EH KLJKHU WKDQ LQ FRZ PLON %D\RXPL  0RUHRYHU PLONV RI WUDQVJHQLF PLFH SURGXFLQJ ERYLQH خ؛&1 ZHUH VKRZQ WR KDYH D OLQHDU FRUUHODWLRQ EHWZHHQ WKH DPRXQW RI خ؛&1 DQG FXUG ILUPQHVV *XWLpUUH]$GiQ HW DO  DQG DQ LQYHUVH FRUUHODWLRQ ZLWK PLFHOOH VL]H 7KH PHDQ GLDPHWHU RI FDPHO PLON FDVHLQ PLFHOOHV LV ODUJHU WKDQ WKDW RI FDVHLQ PLFHOOHV LQ ERYLQH

 PLON %XFKKHLP HW DO  0LON RI DQLPDOV WUDGLWLRQDOO\ XVHG IRU SURFHVVLQJ WR FKHHVH KDV KLJKHU DPRXQWV RI خ؛&1 DQG ORZHU DPRXQWV RI خ² &1 WKDQ FDPHO PLON ,W PD\ EH DVVXPHG WKDW ODFN RI VHOHFWLYH EUHHGLQJ RI FDPHOV IRU PLON ZLWK IDYRXUDEOH FKHHVHPDNLQJ SURSHUWLHV LV UHVSRQVLEOH IRU WKH KLJK خ²&1 DQG WKH ORZ خ؛&1 FRQWHQW &RQVLGHUDWLRQV IRU &DPHO 0LON 3URFHVVLQJ 7HFKQRORJLFDO GLIILFXOWLHV LQ SURFHVVLQJ FDPHO PLON DUH SUREDEO\ GXH WR GLIIHUHQW SURSRUWLRQV RI WKH LQGLYLGXDO FDVHLQV FRPSDUHG ZLWK FRZ PLON UDWKHU WKDQ WR VWUXFWXUDO YDULDWLRQV ZLWKLQ WKH SURWHLQV ,W KDV EHHQ VKRZQ WKDW D KLJK GHJUHH RI خ²&1 DQG D ORZ GHJUHH RI خ؛&1 DGYHUVHO\ DIIHFWV VRPH RI WKH SURFHVVLQJ FKDUDFWHULVWLFV RI FDVHLQ PLFHOOHV 6FKPLGW .RRSV  VXFK DV VWDELOLW\ WRZDUGV HWKDQRO KRPRJHQLVDWLRQ DQG KHDW WUHDWPHQW ,W PXVW EH DVVXPHG WKDW WKH GLIIHUHQFHV LQ SURWHLQ FRPSRVLWLRQ EHWZHHQ WKH UXPLQDQW PLONV DQG FDPHO PLON ZLOO KDYH D PDMRU LPSDFW RQ WHFKQRORJLFDO SURSHUWLHV DQG WKDW D ORZHU UDWLR RI خ²&1 WR خ؛&1 ZRXOG EH IDYRXUDEOH IRU FXUG FRDJXODWLRQ DQG KHDW VWHULOLVDWLRQ  5HQQHWLQJ HQ]\PHV

/LWHUDWXUH &K\PRVLQ (&  DQG SHSVLQ (&  WKH UHQQHWLQJ HQ]\PHV RI WKH PDLQ VWRPDFK DUH DVSDUWLF SURWHDVHV DQG WKXV EHORQJ WR WKH EURDG $$ FODQ RI SHSWLGDVHV 5DZOLQJV %DUUHWW  $VSDUWLF SURWHDVHV DUH IRXQG LQ HXNDU\RWHV UHWURYLUXVHV DQG VRPH SODQW YLUXVHV (XNDU\RWLF DVSDUWLF SURWHDVHV DUH PRQRPHUV RI DERXW  N'D ZKLFK DUH IROGHG LQWR D SDLU RI WDQGHPO\ DUUDQJHG GRPDLQV ZLWK HJ LQ FDPHO FK\PRVLQ DERXW  VLPLODULW\ ,W LV DVVXPHG WKDW WKH WZR GRPDLQV HYROYHG IURP GXSOLFDWLRQ RI DQ DQFHVWUDO JHQH HQFRGLQJ D SULPRUGLDO GRPDLQ 7KH RYHUDOO VHFRQGDU\ VWUXFWXUH FRQVLVWV DOPRVW HQWLUHO\ RI SOHDWHG VKHHW DQG LV ORZ LQ خ±KHOLFHV (DFK GRPDLQ FRQWDLQV DQ DFWLYH VLWH FHQWUHG RQ D FDWDO\WLF DVSDUW\O UHVLGXH ZLWK D FRQVHQVXV VHTXHQFH >K\GURSKRELF@$VS7KU*O\>6HU7KU@ ZKLFK KHOSV WR PDLQWDLQ WKH FRUUHFW د•ORRS FRQIRUPDWLRQ RI WKH VLWH DQG ZLWK PXOWLSOH K\GURSKRELF UHVLGXHV QHDU WKH DVSDUWLF UHVLGXH 7KH WZR FDWDO\WLF FHQWUHV DUH DUUDQJHG IDFHWRIDFH LQ WKH WHUWLDU\ VWUXFWXUH RI FRUUHFWO\ IROGHG SURWHLQV 7KH GLVWDQFH EHWZHHQ WKH DVSDUWLF VLGHFKDLQV LV DERXW  c LQ ERYLQH FK\PRVLQ 7KH UHVLGXHV ZHUH UHSRUWHG WR EH H[WHQVLYHO\ K\GURJHQ ERQGHG FRQFRPLWDQWO\ ZLWK WKH DGMDFHQW WKUHRQLQH UHVLGXHV WR WKH FRUUHVSRQGLQJ UHVLGXHV RI WKH RWKHU GRPDLQ RU WR QHLJKERXULQJ DWRPV RI WKH RZQ GRPDLQ WR IL[ WKH FRUUHFW SRVLWLRQ 2SWLPDO DFWLYLW\ RI DQ DVSDUWLF



5HVXOWV DQG 'LVFXVVLRQ

SURWHDVH LV DFKLHYHG ZKHQ RQH RI WKH DVSDUWLF UHVLGXHV LV SURWRQDWHG DQG WKH RWKHU QHJDWLYHO\ FKDUJHG 7KXV WKH S+ RSWLPXP GHSHQGV RQ WKH HOHFWURVWDWLF PLFURHQYLURQPHQW FUHDWHG E\ WKH UHVLGXHV VXUURXQGLQJ WKH DVSDUWLF UHVLGXHV 7KH DFWLYH VLWHV DUH HPEHGGHG ZLWK ORZ DFFHVVLELOLW\ LQ WKH PLGGOH RI D FOHIW DERXW  c LQ OHQJWK ZKLFK VHSDUDWHV WKH WZR GRPDLQV DQG ZKLFK LV FRYHUHG E\ D IODS WKDW H[WHQGV DERXW IURP /HX WR ,OH LQ WKH 1WHUPLQDO GRPDLQ RI ERYLQH DQG FDPHO FK\PRVLQ 7KH %IDFWRU RI WKLV IODS LV ORZ ZKLFK LQGLFDWHV KLJK IOH[LELOLW\ ,Q FU\VWDOORJUDSKLF VWXGLHV ROLJRSHSWLGH SURWHDVH LQKLELWRUV ZHUH VKRZQ WR ELQG ZLWKLQ WKH FOHIW 7KH IODS ZDV IRXQG WR FORVH GRZQ RYHU WKH LQKLELWRUV H[FOXGLQJ VROYHQW ZKLOH EHFRPLQJ FRQVLGHUDEO\ OHVV IOH[LEOH 'DYLHV  $ VKRUW 1WHUPLQDO SHSWLGH RI DERXW  DD LV FOHDYHG IURP WKH SURHQ]\PH RI HXNDU\RWLF DVSDUWDWH SURWHDVHV E\ DQ DXWRFDWDO\WLF SURFHVV DW DFLGLF S+ )LJ  7KLV SHSWLGH LV RI GLVWLQFWO\ EDVLF QDWXUH ZLWK LVRHOHFWULF SRLQWV DW S+  IRU FDPHO FK\PRVLQ DQG DW S+  IRU FDPHO SHSVLQ ,Q SRUFLQH SHSVLQ WKH SHSWLGH ZDV VKRZQ WR EH EXULHG DORQJ WKH VXEVWUDWH ELQGLQJ FOHIW LQ WKH SURHQ]\PH WKHUHE\ LQKLELWLQJ WKH HQ]\PH E\ EORFNLQJ WKH DFWLYH VLWH ZLWK 6HU DQG /HX DQG E\ GLVSODFLQJ D ZDWHU PROHFXOH ZKLFK LV ERXQG LQ WKH DFWLYH HQ]\PH ZLWK WKH KLJKO\ FRQVHUYHG /\V 6LHOHFNL HW DO  6L[ RI WKH EDVLF DD UHVLGXHV RI WKH SRUFLQH SHSWLGH ZHUH VKRZQ WR IRUP LRQ SDLUV ZLWK DFLGLF UHVLGXHV ZKLFK DUH LQYROYHG LQ VXEVWUDWH ELQGLQJ LQ PDWXUH SRUFLQH SHSVLQ /RZHULQJ WKH S+ SRVVLEO\ SURWRQDWHV WKHVH DFLGLF UHVLGXHV FRQIRUPDWLRQDO FKDQJHV DQG ORVV RI خ±KHOLFDO VWUXFWXUH DUH IROORZHG E\ FOHDYDJH RI WKH DPLGH ERQG EHWZHHQ /HX DQG ,OH ZLWK IXUWKHU VWUXFWXUDO UHDUUDQJHPHQW DQG SURWHRO\WLF VWHSV OHDGLQJ WR WKH PDWXUH HQ]\PH 'DYLHV  7KH SURSHSWLGH ZDV IRXQG WR EH D SUHUHTXLVLWH IRU FRUUHFW IROGLQJ RI WKH HQ]\PH /L HW DO  ,W VHUYHV IRU SURWHFWLRQ RI FHOOXODU SURWHLQV IURP GLJHVWLRQ EHIRUH WKH HQ]\PH LV WUDQVIHUUHG IRU H[DPSOH LQWR D O\VRVRPDO FRPSDUWPHQW RU LV VHFUHWHG ZKLFK LV LQ WKH FDVH RI WKH UHQQHWLQJ HQ]\PHV FK\PRVLQ DQG SHSVLQ IURP WKH PXFRVD OD\HU RI WKH VWRPDFK LQWR WKH SHSWLF MXLFHV $VSDUWLF SURWHDVHV DUH HQGRSHSWLGDVHV ZLWK KLJK VXEVWUDWH VSHFLILFLW\ 7KH SHSWLGH VXEVWUDWH LV QXPEHUHG DZD\ IURP WKH VFLVVLOH ERQG 3 WR 3Q RQ WKH 1WHUPLQDO VLGH RI WKH DPLQR DFLG FKDLQ DQG 3آ¶ WR 3آ¶Q RQ WKH &WHUPLQDO VLGH 6SHFLILFLW\ SRFNHWV RI WKH FRUUHVSRQGLQJ HQ]\PH VXEVLWHV DUH ODEHOOHG 6 WR 6Q DQG 6آ¶ WR 6آ¶Q UHVSHFWLYHO\ 6XEVWUDWHV VXFK DV خ؛&1 RU DQJLRWHQVLQRJHQ D SURWHLQ FOHDYHG E\ UHQLQ DUH XVXDOO\ ERXQG LQ D خ² SOHDWHG VWUXFWXUH ZLWK D WXUQ DW 3آ¶ WR 3آ¶ 7KH DFWLYH VLWH FOHIW DFFRPPRGDWHV D KHSWD WR QRQDSHSWLGH VXEVWUDWH DQG SUREDEO\ DOVR WKH VDPH WDUJHW VLWH RI

 D SURWHLQ VXFK DV خ؛&1 LQ FK\PRVLQ 3 DQG 3آ¶ ZHUH IRXQG WR EH FULWLFDO LQ VXEVWUDWH ELQGLQJ 'DYLHV  ,W LV DVVXPHG WKDW ELQGLQJ RI DQ DSSURSULDWH SHSWLGH OHDGV WR D FRQIRUPDWLRQDO VWUDLQ LQ WKH VFLVVLOH SHSWLGH ERQG ZKLFK LV WZLVWHG RXW RI SODQDULW\ 7KLV WZLVWLQJ UHGXFHV WKH GRXEOH ERQG FKDUDFWHU RI WKH DPLGH ERQG LQ D ZD\ WKDW WKH QLWURJHQ DWRP RI 3آ¶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 (Q]\PHV RI WKH DVSDUWLF SURWHDVH IDPLO\ DUH RI FRPPHUFLDO LQWHUHVW LQ SURFHVVLQJ RI PLON VR\D DQG FRFRD $OEHUW HW DO  &K\PRVLQ LV WKH SUHIHUUHG HQ]\PH LQ WKH FKHHVHPDNLQJ SURFHVV VLQFH VSHFLILFLW\ IRU خ؛&1 LV KLJK JHQHUDO SURWHRO\WLF DFWLYLW\ LV ORZ DQG RSWLPDO DFWLYLW\ LV DFKLHYHG DW PLOGO\ DFLGLF FRQGLWLRQV :LOOLDPV HW DO  =\PRJHQV RI WKH QDWXUDO UHQQHWLQJ HQ]\PHV FK\PRVLQ DQG SHSVLQ DUH LVRODWHG IURP WKH PXFRVD OD\HU RI WKH IRUWK VWRPDFK RI WKH XQZHDQHG FDOI DQG LQ WKH FDVH RI SHSVLQ DOVR RI WKH DGXOW DQLPDO 3HSVLQ H[KLELWV EURDGHU SURWHRO\WLF DFWLYLW\ WKDQ FK\PRVLQ ZLWK D ORZHU S+ RSWLPXP DQG LV OHVV VXLWDEOH IRU FKHHVH SURGXFWLRQ VLQFH ELWWHU K\GURSKRELF SHSWLGHV ZKLFK DUH IRUPHG E\ SURWHRO\WLF DFWLRQ RQ خ±V DQG خ²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خ؛&1 ZRXOG EH DEOH WR VKLIW WKH HTXLOLEULXP WRZDUGV WKH DFWLYH VWDWH RI WKH HQ]\PH %LQGLQJ RI WKH FOXVWHU +LV3UR+LV3UR+LV ZKLFK FRUUHVSRQGV WR ERYLQH FK\PRVLQ +LV WR +LV )LJ  ZDV VKRZQ WR LQGXFH FRQYHUVLRQ IURP WKH VHOILQKLELWHG WR WKH DFWLYH VWDWH SUREDEO\ E\ VWUXFWXUDO WUDQVIRUPDWLRQ RI WKH HQ]\PH *XVWFKLQD HW DO  6HOILQKLELWLRQ LV VXSSUHVVHG LQ SHSVLQ 6WHULF KLQGUDQFH E\ 3KH ZKLFK FRUUHVSRQGV WR 9DO LQ ERYLQH FK\PRVLQ LQKLELWV LQWHUDFWLRQ RI 7\U ZLWK WKH DFWLYH VLWH 3HSVLQ KDV WKHUHIRUH JUHDWHU



5HVXOWV DQG 'LVFXVVLRQ

JHQHUDO SURWHRO\WLF DFWLYLW\ WKDQ RWKHU PDPPDOLDQ DFLGLF SURWHDVHV $ VLWH GLUHFWHG PXWDWLRQ RI 9DO WR 3KH LQ UHFRPELQDQW FDOI FK\PRVLQ OHG WR D WZRIROG LQFUHDVH LQ .0 YDOXHV DV FRPSDUHG WR ZLOGW\SH FK\PRVLQ ZLWKRXW DOWHULQJ NFDW YDOXHV DQG LQFUHDVHG WKH QXPEHU RI UHVLGXHV SDUWLFLSDWLQJ LQ VSHFLILFLW\ VXEVLWHV 6 DQG 6 JLYLQJ IXUWKHU VXSSRUW IRU WKH FHQWUDO UROH RI WKH VLGHFKDLQ DW SRVLWLRQ 9DO LQ FK\PRVLQ UHVSHFWLYHO\ 3KH LQ SHSVLQ LQ SURWHRO\WLF DFWLYLW\ 6WURS HW DO  (Q]\PDWLF FRDJXODWLRQ RI PLON E\ SURWHRO\WLF HQ]\PHV LV DQ DSSDUHQW WZR VWHS SURFHVV 7KH ILUVW VWHS LV FKDUDFWHULVHG E\ K\GURO\VLV RI خ؛&1 DW WKH VXUIDFH RI FDVHLQ PLFHOOHV خ؛&1 FRYHUV WKH SUHGRPLQDQWO\ K\GURSKRELF FRUH RI WKH PLFHOOHV E\ D &WHUPLQDO JO\FRPDFURSHSWLGH ZKLFK SUHYHQWV PLFHOODU DJJUHJDWLRQ E\ VWHULF KLQGUDQFH DQG FKDUJH UHSXOVLRQ 7KLV &WHUPLQDO SDUW LV VSHFLILFDOO\ FOHDYHG GXULQJ WKH HQ]\PDWLF UHDFWLRQ DW D K\GURSKRELF FOHDYDJH VLWH ZKLFK LV 3KH0HW LQ ERYLQH خ؛&1 ,Q WKH VHFRQG VWHS RI WKH UHQQHWLQJ SURFHVV DQ H[SRQHQWLDO LQFUHDVH LQ FRDJXODWLRQ RI FDVHLQ PLFHOOHV LV REVHUYHG $JJUHJDWLRQ VWDUWV IURP WKH PRPHQW ZKHQ DERXW  WR  RI خ؛&1 LV FOHDYHG $W WKLV WLPH D VXIILFLHQW SDUW RI WKH K\GURSKRELF PLFHOOH VXUIDFH LV VXSSRVHG WR KDYH WKH SRWHQWLDO WR SDUWLFLSDWH LQ WKH DJJUHJDWLRQ SURFHVV $ VXEVWDQWLDO UHGXFWLRQ LQ FRDJXODWLRQ WLPH DQG DQ LQFUHDVH LQ FXUG VWUHQJWK LV DFKLHYHG E\ S+ UHGXFWLRQ DQG E\ DGGLWLRQ RI IUHH &D 'DOJOHLVK  5HQQHW FRDJXODWLRQ RI FDPHO PLON ZDV IRXQG WR IROORZ D VLPLODU PHFKDQLVP DV UHSRUWHG IRU FRZ PLON 0HKDLD  &DPHO خ؛&1 ZDV IRXQG WR FRQWDLQ D GLVWLQFWO\ GLIIHUHQW FOHDYDJH VLWH IRU DVSDUWLF SURWHDVHV FRPSDUHG WR ERYLQH خ؛&1 )LJ  7KH H[FKDQJH RI WKH DPLQR DFLG /HX WR WKH LPLQR DFLG 3UR ZKLFK SUREDEO\ KDV LPSOLFDWLRQV RQ SHSWLGH IROGLQJ LV PRVW VLJQLILFDQW ,W ZDV RI LQWHUHVW WR VHH LI WKH DPLQR DFLG VXEVWLWXWLRQV LQ خ؛&1 FRXOG EH FRUUHODWHG ZLWK FKDQJHV LQ WKH VSHFLILFLW\ SRFNHWV RI DVSDUWLF SURWHDVHV IURP WKH FDPHO VWRPDFK 3ULPDU\ 6WUXFWXUHV 3&5 DPSOLILFDWLRQ SURGXFWV RI SDUWLDO F'1$ FORQHV RI FDPHO FK\PRVLQ DQG SHSVLQ ZHUH VHTXHQFHG )LJV  DQG  7KH FORQH IRU FDPHO FK\PRVLQ (0%/*HQ%DQNâ„¢ DFFHVVLRQ QXPEHU $- ZDV  ES ORQJ DQG FRQWDLQHG D آ¶XQWUDQVODWHG UHJLRQ RI  ES DQG D آ¶XQWUDQVODWHG UHJLRQ RI  ES 2QO\ D SDUWLDO FRQVHQVXV UHJLRQ DFFRUGLQJ WR .R]DN  ZDV IRXQG LQ IURQW RI WKH WUDQVODWLRQDO VWDUW ZLWK D SXULQH DW آ± ES DQG F\WRVLQHV DW  ES DQG آ± ES 7KH RSHQ UHDGLQJ IUDPH UDQJHG IURP $  WR & 3URSUDH


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



)LJ  F'1$ VHTXHQFH RI FDPHO FK\PRVLQ DQG FRUUHVSRQGLQJ SURWHLQ ZLWK PDWXUH SURWHLQ VWDUWLQJ IURP *O\ LQ EROG 25) IURP $ WR &



5HVXOWV DQG 'LVFXVVLRQ

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



)LJ  F'1$ VHTXHQFH RI FDPHO SHSVLQ DQG FRUUHVSRQGLQJ SURWHLQ ZLWK PDWXUH SURWHLQ IURP /HX LQ EROG 2SHQ UHDGLQJ IUDPH IURP $ WR &

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â„¢ DFFHVVLRQ QXPEHU $- ZDV  ES ORQJ DQG FRQWDLQHG D آ¶XQWUDQVODWHG UHJLRQ RI  ES DQG D آ¶XQWUDQVODWHG UHJLRQ RI  ES $V ZLWK FK\PRVLQ F'1$ RQO\ D SDUWLDO FRQVHQVXV UHJLRQ DFFRUGLQJ WR .R]DN  ZDV IRXQG LQ IURQW RI WKH WUDQVODWLRQDO VWDUW ZLWK D SXULQH DW آ± ES DQG F\WRVLQHV DW آ± ES DQG آ±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خ؛&1 LV QRW VXLWDEOH IRU UHOHDVH RI FDOI FK\PRVLQ IURP LWV VHOILQKLELWHG VWDWH $V PHQWLRQHG EHIRUH D FRQVLGHUDEOH YDULDWLRQ LQ



5HVXOWV DQG 'LVFXVVLRQ

SULPDU\ VWUXFWXUH ZDV IRXQG ZKHQ FDPHO DQG ERYLQH خ؛&1V ZHUH FRPSDUHG 1RWLFHDEOH PRGLILFDWLRQV LQ FDPHO خ؛&1 ZHUH WKH GHOHWLRQ RQ WKH 1 WHUPLQDO VLGH RI WKH FOHDYDJH VLWH )LJ  WKH GLIIHUHQW ORFDOLVDWLRQ RI VLWHV ZLWK KLJK SUREDELOLW\ RI JO\FRV\ODWLRQ )LJ  WKH UHSODFHPHQW RI D OHXFLQH ZKLFK ZDV UHSRUWHG WR EH KLJKO\ IOH[LEOH 3ORZPDQ &UHDPHU  ZLWK D ULJLG SUROLQH DW 3 DQG UHSODFHPHQW RI ZHDNO\ EDVLF KLVWLGLQH UHVLGXHV ZLWK KLJKO\ EDVLF DUJLQLQH UHVLGXHV DW 3 3 DQG 3 7KH UHJLRQ DURXQG WKH FOHDYDJH VLWH ZKLFK LV SURSRVHG WR KDYH D خ²VKHHW VWUXFWXUH LQ H[WHQGHG FRQIRUPDWLRQ :LOOLDPV HW DO  LV SUREDEO\ OHVV IOH[LEOH LQ FDPHO خ؛&1 GXH WR WKH H[FKDQJH RI OHXFLQH WR SUROLQH :H DVVXPH WKDW LQ JHQHUDO VXEVWUDWH VSHFLILFLW\ RI FK\PRVLQ LV HVSHFLDOO\ KLJK IRU خ؛&1 RI WKH VDPH VSHFLHV 7KH JHQHUDO SURWHRO\WLF DFWLYLW\ RI SRUFLQH FK\PRVLQ IRU H[DPSOH ZDV UHSRUWHG WR EH DERXW WHQ WLPHV ORZHU WKDQ WKH DFWLYLW\ RI ERYLQH FK\PRVLQ ZKHUHDV LWV FRZ PLON FORWWLQJ DFWLYLW\ ZDV RQO\ IRXU WLPHV ORZHU :KHQ WHVWHG DJDLQVW VRZ PLON WKH FORWWLQJ DFWLYLW\ ZDV DERXW VL[ WR HLJKW WLPHV JUHDWHU WKDQ WKDW RI FDOI FK\PRVLQ +RXHQ HW DO  1HYHUWKHOHVV FDPHO FK\PRVLQ ZDV IRXQG WR FORW FDPHO DQG FRZ PLON HTXDOO\ ZHOO :DQJRK  6SHFLILFLW\ SRFNHWV RI FDPHO FK\PRVLQ ZKLFK ELQG PRUH IOH[LEOH DUJLQLQH VLGHFKDLQV PD\ DOVR EH VXLWHG WR KRVW WKH PRUH ULJLG KLVWLGLQH VLGHFKDLQV RI ERYLQH خ؛&1 ,W FDQ EH DVVXPHG WKDW WKH SURWRQDWLRQ VWDWXV RI WKH KLVWLGLQHV ZLOO EH LPSRUWDQW IRU ELQGLQJ WR FDPHO FK\PRVLQ ,Q WKLV FRQWH[W LW ZRXOG EH RI LQWHUHVW WR NQRZ WKH S+ RSWLPXP RI FDPHO FK\PRVLQ IRU VSHFLILF K\GURO\VLV RI WKH FOHDYDJH VLWHV LQ FDPHO DQG ERYLQH خ؛&1 +LJK DFWLYLW\ RI ERYLQH SHSVLQ LQ FDPHO PLON UHQQHWLQJ KRZHYHU FDQ EH H[SODLQHG E\ WKH KLJK JHQHUDO SURWHRO\WLF DFWLYLW\ RI SHSVLQ 7KH SULPDU\ VWUXFWXUH RI ERYLQH SHSVLQ LV QRW \HW NQRZQ EXW LW FDQ EH DVVXPHG WKDW WKH SURWHLQ GRHV FRQWDLQ D EXON\ UHVLGXH ZKLFK VXSSUHVVHV VHOI LQKLELWLRQ DV LW ZDV IRXQG LQ FDPHO DQG SRUFLQH SHSVLQ ZLWK 3KH )LJ  &DPHO FK\PRVLQ FRQWDLQHG D 9DO DW WKH FRUUHVSRQGLQJ VLWH LQ WKH VDPH ZD\ DV ERYLQH FK\PRVLQ ,W FDQ EH DVVXPHG WKDW FDPHO FK\PRVLQ LV LQ D FKHPLFDO HTXLOLEULXP RI DQ DFWLYH DQ D UHVWLQJ VWDWH LQ D VLPLODU ZD\ DV ERYLQH FK\PRVLQ 6HYHUDO VWXGLHV WULHG WR XQYHLO WKH PHFKDQLVP IRU VSHFLILF ELQGLQJ RI ERYLQH خ؛&1 DQG ERYLQH FK\PRVLQ &RPSDUDWLYH PRGHOOLQJ *LOOLODQG HW DO  3ORZPDQ  NLQHWLF VWXGLHV 9LVVHU HW DO  6WURS HW DO  *XVWFKLQD HW DO  DQG VWUXFWXUDO DQDO\VLV RI LQKLELWRU FRPSOH[HV *URYHV HW DO WR EH SXEOLVKHG VKRZHG WKDW VSHFLILFLW\ RI ELQGLQJ GHSHQGV RQ DOWHUQDWLQJ ELQGLQJ RI K\GURSKRELF DQG SRVLWLYHO\ FKDUJHG UHVLGXHV RI

bVKHHW
آ«آ«آ«آ«آ«آ«آ«آ«

6LJQDO 6HTXHQFH آ¸     

bbbbbbbbbbb bbb &DPHO &K\PRVLQ 05&/99//$$/$/64$6*,75,3/+.*.7/5.$/.(5*//(')/4544<$966.<66/*.9$5 (3/76
3URSHSWLGH

آ¸

6

آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«



$VS 6آ¶ SRVLWLYHO\ FKDUJHG SDWFK IODS UHJLRQ ZLWK 7\U  bbbbb bbbbbbbb bbbbb aaaa b aaa bbbbbbbbbbb bbbbbbbbbbbb bb &DPHO &K\PRVLQ *.,<,*7334()799)'7*66'/:936,<&.619&.1++5)'35.667)51/*.3/6,+<*7*60(*)/*<'797961,9 &RZ &K\PRVLQ *.,
    

آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«

9DO3KH 6آ¶ 6آ¶ bbbbbbbbbbb aaaaa bbbb aaa aaaaaa bbbb bbbb aab &DPHO &K\PRVLQ '31479*/67(43*(9)7<6()'*,/*/$<36/$6(<6939)'100'5+/9$5'/)69<0'51*4 *60/7/*$,'36<< &RZ &K\PRVLQ ',4479*/674(3*'9)7<$()'*,/*0$<36/$6(<6,39)'10015+/9$4'/)69<0'51*4 (60/7/*$,'36<< 3LJ &K\PRVLQ '$+479*/674(36',)7<6()'*,/*/*<3(/$6(<7939)'100+5+/9$4'/)$9<0651'( *60/7/*$,'36<< &DPHO 3HSVLQ '914,)*/6(7(3*6)/<<$3)'*,/*/$<36,666**739)'1,:'(*/,6('/)69
    

bVKHHW

آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ« &WHUPLQDO

GRPDLQ آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«

$VS 66 6 bbbbbbbb b bbbbbbbbbb bbbb bbbbb bbb aaaaaaaaa bb bbb &DPHO &K\PRVLQ 7*6/+:9397/44<:4)79'697,1*9$9$&9**&4$,/'7*769/)*366',/.,40$,*$7(15<*()'91&*1/560 &RZ &K\PRVLQ 7*6/+:9397944<:4)79'697,6*999$&(**&4$,/'7*76./9*366',/1,44$,*$7414<*()','&'1/6<0 3LJ &K\PRVLQ 7*6/+:939704/<:4)79'697,1*999$&1**&4$,/'7*760/$*366',/1,40$,*$7(64<*()','&*6/660 &DPHO 3HSVLQ 7*6/1:93969(*<:4,79'6,70(*(6,$&66*&4$,9'7*76//$*37'$,61,46<,*$6('6<*'0996&66,66/ 3LJ 3HSVLQ 7*6/1:93969(*<:4,7/'6,70'*(7,$&6**&4$,9'7*76//7*376$,$1,46',*$6(16'*(09,6&66,'6/

    

آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«آ«



6 6 bbbbb bbbbb aaaabbbb bbbb bbb bbb aaaa bbbbbb bbbbbb &DPHO &K\PRVLQ 3799)(,1*5'<3/636$<76.'4*)&76*)4*'116 (/:,/*'9),5(<<69)'5$1159*/$.$, &RZ &K\PRVLQ 3799)(,1*.0<3/736$<764'4*)&76*)46(1+6 4.:,/*'9),5(<<69)'5$11/9*/$.$, 3LJ &K\PRVLQ 3799)(,6*50<3/336$<714'4*)&76*)4*'6.6 4+:,/*99),4(<<69)'5$1159*/$.$, &DPHO 3HSVLQ 31,9)7,1*94<3/636$<,/(6''6&76*)(*0'/6 66((/:,/*'9),54<)79)'5$1149*/$$9$4$ 3LJ 3HSVLQ 3',9)7,1*94<3/636$<,/4'''6&76*)(*0'93766*(/:,/*'9),54<<79)'5$11.9*/$39$

    

5HVXOWV DQG 'LVFXVVLRQ

)LJ  6FKHPDWLF GUDZLQJ RI WKH UHODWLRQVKLS EHWZHHQ VWUXFWXUH DQG IXQFWLRQ LQ FDPHO ERYLQH DQG SRUFLQH FK\PRVLQ DQG FDPHO SHSVLQ 1XPEHULQJ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQV )XQFWLRQDO UHVLGXHV LQ EROG خ±KHOLFDO UHJLRQV GHVLJQDWHG DV آ³خ±آ´ خ²SOHDWHG UHJLRQV DV آ³خ²آ´ آ¸ &OHDYDJH VLWHV RI WKH VLJQDO SHSWLGH DQG SURSHSWLGH 1XPEHULQJ RI VSHFLILFLW\ SRFNHWV DV IRXQG LQ FDPHO FK\PRVLQ

 WKH FK\PRVLQ VHQVLWLYH UHJLRQ LQ خ؛&1 E\ K\GURSKRELF DQG QHJDWLYHO\ FKDUJHG VSHFLILFLW\ SRFNHWV LQ FK\PRVLQ 7KLV SDWWHUQ LV DOVR WUXH DQG HYHQ SURQRXQFHG IRU WKH ELQGLQJ UHJLRQ RI FDPHO خ؛&1 ZKLFK LV RQ WKH 1 DQG &WHUPLQDO VLGH RI WKH FOHDYDJH VLWH 3KH,OH 7KH ULJLG VWUXFWXUH RI WKH UHJLRQ ZKLFK LV VXSSRVHG WR EH D SUHUHTXLVLWH IRU FDWDO\WLF DFWLRQ RI FK\PRVLQ E\ GLVWRUWLRQ RI DQ DPLGH ERQG DQG VXEVHTXHQW K\GURO\VLV LV HYHQ PRUH SURQRXQFHG LQ WKH H[WHQGHG FK\PRVLQ VHQVLWLYH UHJLRQ RI FDPHO خ؛&1 E\ UHSODFHPHQW RI 7KU /HX DQG 7KU ZLWK 3UR 3UR DQG 3UR ,Q DQ DWWHPSW WR ILQG SRVVLEOH VSHFLILFLW\ SRFNHWV IRU FDPHO خ؛&1 ZH PRGHOOHG WKH WHUWLDU\ VWUXFWXUH RI FDPHO FK\PRVLQ ZLWK WKH KHOS RI WKH UHVROYHG &06 VWUXFWXUH RI ERYLQH FK\PRVLQ *LOOLODQG HW DO  E\ WKH PHWKRG RI *XH[ 3HLWVFK  DQG VXSHUSRVHG WKH VHTXHQFH RI WKH FK\PRVLQ VHQVLWLYH UHJLRQ RI FDPHO خ؛&1 RYHU WKH HQHUJ\ PLQLPLVHG VWUXFWXUH RI WKH ERYLQH UHJLRQ DV GHVFULEHG E\ 3ORZPDQ &UHDPHU  7KH د• DQG دˆDQJOHV RI 3UR ZHUH WXUQHG WR WKH PRVW SUREDEOH DQJOH RI آ±آƒ DQG آƒ )LJ  7KH SUROLQH UHVLGXHV ZHUH IRXQG WR LQWURGXFH D NLQN LQ WKH FK\PRVLQ VHQVLWLYH UHJLRQ RI خ؛&1 ZKLFK PD\ KHOS WR SUHVHQW WKH K\GURSKRELF FOHDYDJH VLWH WR WKH FDWDO\WLF FHQWUH RI FK\PRVLQ .XPRVLQVNL HW DO  7KH FK\PRVLQ VHQVLWLYH UHJLRQV RI FDPHO DQG ERYLQH خ؛&1 ZHUH ODLG LQWR WKH FOHIW RI WKH UHVSHFWLYH FK\PRVLQ YDULDQWV ZLWK WKH KHOS RI WKH UHFHQWO\ UHVROYHG FK\PRVLQ&3 LQKLELWRU FRPSOH[ *URYHV HW DO WR EH SXEOLVKHG DV VKRZQ LQ )LJ  :H IRXQG WKDW WKH ELQGLQJ SRFNHWV RI WKH DD UHVLGXHV LQ WKH YLFLQLW\ RI WKH FOHDYDJH VLWH ZHUH PXFK VLPLODU LQ FDPHO DQG ERYLQH FK\PRVLQ ZLWK WKH H[FHSWLRQ RI 6آ¶ ZKLFK ZDV GHSULYHG IURP WZR W\URVLQH UHVLGXHV LQ FDPHO FK\PRVLQ 7DEOH  7KLV UHGXFWLRQ LQ ELQGLQJ VLWHV IRU 3آ¶ PD\ EH H[SODLQHG E\ WKH H[FKDQJH RI 0HW WR ,OH DW 3آ¶ DQ DPLQR DFLG ZKLFK LV QRW DEOH WR LQWHUDFW ZLWK WKH DURPDWLF VLGHFKDLQV RI W\URVLQH UHVLGXHV LQ FRQWUDVW WR PHWKLRQLQH %LQGLQJ RI ,OH LV VXSSRVHG WR GHSHQG ODUJHO\ RQ K\GURSKRELF LQWHUDFWLRQ ,W ZLOO EH QHFHVVDU\ IRU FDWDO\WLF DFWLYLW\ RI WKH DVSDUWDWH UHVLGXHV LQ FK\PRVLQ WKDW ,OH LV EURXJKW LQWR FORVH FRQWDFW ZLWK WKH 6آ¶ VSHFLILFLW\ SRFNHW E\ HOHFWURVWDWLF LQWHUDFWLRQV RI RWKHU خ؛&1 VLGHFKDLQV ZLWK WKH UHVSHFWLYH VSHFLILFLW\ SRFNHWV :KHQ ZH H[DPLQHG SRVVLEOH LQWHUDFWLRQV RI 3آ¶ 3 3 DQG 3 LQ FDPHO خ؛&1 ZH IRXQG PRUH DVSDUWLF DFLGV ZLWK SUREDELOLW\ IRU LRQ SDLU ELQGLQJ LQ FDPHO FK\PRVLQ WKDQ WKRVH IRXQG LQ ERYLQH FK\PRVLQ IRU ELQGLQJ RI WKH UHVSHFWLYH UHVLGXHV RI ERYLQH خ؛&1 ,W LV QRW SRVVLEOH WR PDNH DQ DFFXUDWH SUHGLFWLRQ RI WKH UHVLGXHV LQYROYHG LQ HOHFWURVWDWLF ELQGLQJ RI WKH FK\PRVLQ VHQVLWLYH UHJLRQ RI FDPHO DQG ERYLQH خ؛&1 DV ORQJ DV DQ H[SHULPHQWDOO\ UHVROYHG VWUXFWXUH RI خ؛&1 LV QRW DYDLODEOH 1HYHUWKHOHVV GLVWLQFW GLIIHUHQ



5HVXOWV DQG 'LVFXVVLRQ

D

/\V

E /\V

$UJ

+LV

)LJ  6SDFHILOOLQJ PRGHO RI WKH FK\PRVLQ VHQVLWLYH UHJLRQV $UJ/\V RI D FDPHO خ؛&1 DQG +LV/\V RI E ERYLQH خ؛&1 3RODU DQG ZHDNO\ EDVLF DD UHVLGXHV DUH OLJKW VKDGHG VWURQJO\ EDVLF DD UHVLGXHV DUH GDUN VKDGHG FHV LQ ELQGLQJ RI FDPHO DQG ERYLQH خ؛&1 FDQ EH SUHGLFWHG GXH WR WKH KLJKHU SUROLQH FRQWHQW RI WKH H[WHQGHG FK\PRVLQ VHQVLWLYH UHJLRQ LQ FDPHO خ؛&1 DQG WKH SURWRQDWLRQ RI WKH DUJLQLQH UHVLGXHV DW QHXWUDO S+ LQ FRQWUDVW WR WKH KLVWLGLQH UHVLGXHV RI ERYLQH خ؛&1 ,Q WKLV FRQWH[W LW ZRXOG EH LQWHUHVWLQJ WR XQGHUVWDQG ZK\ KLVWLGLQH DQG DUJLQLQH DUH IRXQG RQO\ LQ WKH DFWLYDWRU UHJLRQ 33 RI خ؛FDVHLQV IURP GLIIHUHQW VSHFLHV DQG O\VLQH RQO\ LQ 3آ¶ 3آ¶ DQG 3آ¶ )LJ  7KH FRPSLODWLRQ GHPRQVWUDWHV WKH KLJK FRQVHUYDWLRQ RI WKH FK\PRVLQ VHQVLWLYH UHJLRQ EH\RQG YDULRXV PDPPDOLDQ RUGHUV DQG WKH LPSRUWDQFH RI DUJLQLQH DQG KLVWLGLQH UHVLGXHV RQ 3آ¶ SRVLWLRQV DQG O\VLQH RQ 3 SRVLWLRQV 2QO\ KLSSRSRWDPXV DQG URGHQW VHTXHQFHV UHYHDOHG GLVWLQFW YDULDWLRQV LQ WKLV UHJLRQ *XWLpUUH]$GiQ HW DO  UHSRUWHG ORQJ UHQQHWLQJ WLPHV RI  WR  PLQ DQG D ORZ FXUG VWUHQJWK IRU PXULQH PLON WUHDWHG ZLWK FDOI FK\PRVLQ )LJ  VKRZV WKDW WKH GHOHWLRQ RQ WKH 1WHUPLQDO VLGH RI WKH FOHDYDJH VLWH LV IRXQG LQ DOO RUGHUV VWXGLHG ZLWK WKH H[FHSWLRQ RI UXPLQDQWV &DPHO KRUVH KXPDQ DQG PRXVH PLON ZHUH IRXQG WR KDYH ORQJHU UHQQHWLQJ WLPHV DQG ORZ FXUG VWUHQJWK ZKHQ WUHDWHG ZLWK FDOI FK\PRVLQ %D\RXPL 

 D
IODS *OX $VS 3
 $VS *OX 3 3 3 *OX $VS $VS

$VS $VS $VS $VS

E
IODS 3 3

3

*OX

$VS 3
 $VS $VS $VS

$VS $VS

)LJ  $ SLFWRULDO YLHZ RQ WKH LQWHUDFWLRQ RI FK\PRVLQ DQG WKH FK\PRVLQ VHQVLWLYH VLWH LQ خ؛FDVHLQ &K\PRVLQ LQ ULEERQOLNH UHSUHVHQWDWLRQ &K\PRVLQ VHQVLWLYH UHJLRQ RI خ؛FDVHLQ DQG DFLGLF DPLQR DFLGV ZKLFK SRVVLEO\ LQWHUDFW ZLWK خ؛&1 DUH VKRZQ DV ZLUHIUDPH D ,QWHUDFWLRQ RI FDPHO FK\PRVLQ DQG $UJآ±/\V RI FDPHO خ؛FDVHLQ E ,QWHUDFWLRQ RI ERYLQH FK\PRVLQ DQG +LVآ±/\V RI ERYLQH خ؛FDVHLQ



5HVXOWV DQG 'LVFXVVLRQ

7DEOH  &RPSDULVRQ RI FK\PRVLQ VHQVLWLYH UHJLRQ LQ FDPHO DQG ERYLQH خ؛ FDVHLQ DQG UHVLGXHV RI UHVSHFWLYH VSHFLILFLW\ SRFNHWV LQ FDPHO DQG ERYLQH FK\PRVLQ

&DPHO خ؛ 6SHFLILFLW\ VXEVLWHV LQ FDPHO FK\PRVLQ &1 UHVLGXH        آ¶ آ¶ آ¶ آ¶ آ¶ $UJ $UJ $UJ $UJ 3UR 6HU 3KH ,OH $OD ,OH 3UR /\V

%RYLQH 6SHFLILFLW\ VXEVLWHV LQ ERYLQH FK\PRVLQ خ؛&1 UHVLGXH $UJ +LV +LV +LV /HX 6HU 3KH 0HW $OD ,OH 3UR /\V $VS *OX $VS *OQ 7KU *O\ 7KU 6HU 7\U *O\ 7KU *O\ 7KU *OQ *O\ 6HU 7\U ,OH *O\ *O\ +LV 7\U 7\U ,OH *O\ 6HU ,OH /HX 7\U 6HU $OD *OQ 6HU 7\U 3UR 6HU /HX $OD *OQ 7\U $VS

*OX $VS $VS *OX *OX $VS $VS 7\U 7KU 7\U *O\ 7KU 3KH *O\ 6HU 7\U ,OH 7\U *O\ 7KU *O\ +LV ,OH *O\ 6HU /HX 7\U 6HU $OD 6HU 7\U 3UR 6HU /HX $OD *OQ 7\U $VS $VS *OX

 5HVLGXHV PRUH GLVWDQW WR WKH FOHDYDJH VLWH LQ خ؛&1 ZHUH GLVFXVVHG WR EH LQYROYHG LQ ELQGLQJ WR SHULSKHUDO VLWHV LQ FK\PRVLQ ,W ZDV REVHUYHG WKDW WKH % YDULDQW RI ERYLQH خ؛&1 ZDV DVVRFLDWHG ZLWK ILUPHU FKHHVH FXUGV VKRUWHU UHQQHWLQJ WLPHV D VPDOOHU DYHUDJH PLFHOOH VL]H KLJKHU PLON FDVHLQ DQG خ؛&1 FRQFHQWUDWLRQV DQG ORZHU ZKH\ SURWHLQ FRQFHQWUDWLRQV ZKHQ FRPSDUHG WR WKH $ YDULDQW -DNRE  DQG WKH & YDULDQW H[KLELWHG ORQJHU UHQQHW FRDJXODWLRQ WLPHV WKDQ YDULDQWV $ DQG % -DNRE  6HTXHQFH FRPSDULVRQ UHYHDOHG K\GURSKRELF UHVLGXHV LQ YDULDQW % DW SRVLWLRQV ZKHUH YDULDQW $ FRQWDLQHG D JO\FRV\ODWHG 7KU DQG DQ DFLGLF $VS DQG D KLVWLGLQH LQ YDULDQW & DW WKH SRVLWLRQ RI $UJ 7KH HIIHFWV UHSRUWHG IRU YDULDQW % DUH OLNHO\ WR UHVXOW IURP DOWHUDWLRQV RQ WKH OHYHO RI JHQH H[SUHVVLRQ DV ZHOO DV RQ HQ]\PDWLF VXLWDELOLW\ DV D VXEVWUDWH IRU ERYLQH FK\PRVLQ &DPHO خ؛&1 FRQWDLQHG K\GURSKRELF UHVLGXHV DW WKH FRUUHVSRQGLQJ VLWHV LQ WKH VDPH ZD\ DV ERYLQH خ؛&1 % $UJ LQ ERYLQH خ؛&1 $ DQG % ZDV SURSRVHG WR LQWHUDFW ZLWK $VS LQ D VWUXFWXUDO DUUDQJHPHQW ZKHUH QR LQWHUDFWLRQ RI +LV ZLWK DQ DFLGLF FK\PRVLQ UHVLGXH ZDV SRVVLEOH 3ORZPDQ HW DO  ,QWHUDFWLRQ RI +LV LQ ERYLQH خ؛&1 & ZLWK $VS ZDV QRW SRVVLEOH ZKLFK JDYH H[SODQDWLRQ IRU WKH H[WHQGHG UHQQHWLQJ WLPH ,W ZDV DOVR VKRZQ WKDW LQWHUDFWLRQ RI $UJ ZDV QRW SRVVLEOH ZKHQ $VS ZDV UHSODFHG E\ $VQ DV LW ZDV WKH FDVH LQ FDPHO FK\PRVLQ ,W LV SRVVLEOH WKDW LQWHUDFWLRQ RI DOO DUJLQLQHV RI WKH FDPHO خ؛&1 FK\PRVLQ VHQVLWLYH UHJLRQ LV EURXJKW DERXW E\ UHSODFHPHQW RI /HX ZLWK 3UR DW SRVLWLRQ 3 :LOOLDPV HW DO  SURSRVHG DQ LQWHUDFWLRQ RI $VS LQ WKH OHVV WKHUPRVWDEOH ERYLQH FK\PRVLQ YDULDQW $ ZLWK +LV 9DULDQW $ H[KLELWV D  KLJKHU FORWWLQJ DELOLW\ WKDQ FK\PRVLQ % ZKLFK PD\ EH H[SODLQHG E\ VWURQJHU LQWHUDFWLRQ RI WKH 1 WHUPLQDO FK\PRVLQ VHQVLWLYH UHJLRQ RI خ؛&1 ZLWK WKLV YDULDQW ,Q FDPHO FK\PRVLQ D JO\FLQH ZDV IRXQG DW WKLV SRVLWLRQ DV LQ ERYLQH FK\PRVLQ % $UJ DW SRVLWLRQ 3 LV OLNHO\ WR LQWHUDFW HLWKHU ZLWK *OX RU ZLWK $VS LQ FDPHO FK\PRVLQ VR WKHUH LV QR QHHG IRU UHSODFHPHQW RI *O\ +LJK FRQVHUYDWLRQ RI WKH O\VLQH DW SRVLWLRQ 3آ¶ WKURXJKRXW GLIIHUHQW PDPPDOLDQ RUGHUV SRLQW WR WKH LPSRUWDQFH RI WKLV UHVLGXH IRU RSWLPDO ELQGLQJ WR FK\PRVLQ )LJ  DOWKRXJK LW ZDV VKRZQ WKDW WKH SHSWLGH +LV WR /\V ZDV VXIILFLHQW IRU D K\GURO\VLV UDWH VLPLODU WR IXOOOHQJWK PDWXUH ERYLQH خ؛&1 $ SURQRXQFHG GLSROH PRPHQW ZDV IRXQG EHWZHHQ WKH 1 DQG &WHUPLQDO GRPDLQV RI PDPPDOLDQ DVSDUWLF SURWHDVHV 7KLV GLSROH ZDV IRXQG WR EH PRUH SURQRXQFHG DQG KDYH D GLIIHUHQW RULHQWDWLRQ LQ ERYLQH FK\PRVLQ WKDQ LQ RWKHU DVSDUWLF SURWHDVHV LQFOXGLQJ SHSVLQ $ SDWFK RI WKH SRVLWLYHO\ FKDUJHG UHVLGXHV /\V /\V +LV $UJ $UJ DQG /\V RQ WKH VXUIDFH RI



5HVXOWV DQG 'LVFXVVLRQ

3RVLWLRQ

333333333 333333333  








31,'3379(55353536) 31,'3379(55353536) 319<337*$55353+$6) 319<3379$55353+$6) 316+337995531/+36) 31,+3379$++3+3536) 31,+337/$++3+3536) 3',133793&5553+36) 31$<33799553+/+36) 317<737995+3+/3$6) 31,<367995+353+36) 31,<367995+3<5536) 7',+46704<+4$.+36) 31,+43.9*5+6+3)) 31)346$*93<$,3136) 31)3439*93+3,3136) 317$3$.6&4'43770$5+3+3+/6) 317$3$.6&4'43770$5+3+3+/6) 317$3$.6&4'43770$++3+3+/6) 31793$.6&4'4377/$5+3+3+/6) 31$93$.6&4'437$0$5+3+3+/6) 31793$.6&4'43770$++3+3+/6) 31793$.6&4'43770$5+3+3+/6) 61793$.6&4$43770$5+3+3+/6) 61793$.6&4$43770$5+3+3+/6) 31793$.6&4$4377075+3+3+/6) 31793$.6&4$4377/$5+3+35/6) 31793$.6&4$4377/$5+3+35/6) 31793$.)&4$43770$5+3+35/6) 31793$.)&4343770$5+3+35/6) 31796$56&4$43770$5+3+3+/6) 31793$56&4343770$5+3+3+/6) 31793$.6&4$43770$553+35/6) 31793$76&4$43$79$5+3+35/6) /1$96$.3&4$33770$55353+/6) ,$,33..74'.7913$,179$79( ,$,33..74'.79,3$,179$79( ,$,33..14'77$,3$,16,$79( ,$,33..14'.7$,3$,16,$79( ,$,33..,4'.,,,37,17,$79( ,$,33..74'.79,3,,17,$7$( 7$,33..74'.7$,3,,17,$79( /$,33;.'41.79,3,,17,$7;( ,$,33..,4'.76,67,17,96$( ,9,33..,4'.7*137,17,$7$( ,$,33.;/4(.79,3.,17,$79( ,$,33../4'.79531,17,$79( 0$,/6..,/*.$7,/67'$,$$3( 0$,/31.04'.$9737717,$$9( /$0371(14'17$,37,'3,73,9 /$,371(.+'17$,3$617,$3,9 0$,33..'4'.7(,37,17,$6$( 0$,33..'4'.7(,37,17,$6$( 0$,33..'4'.7(,37,17,$6$( 0$,33..'4'.7(93$,17,$6$( 0$,33..'4'.7(,3$,17,$6$( 0$,33..'4'.7(,3$,17,$69( 0$,33..'4'.7(,37,179$6$( 0$,33..14'.7(,37,17,$6*( 0$,33..14'.7(,37,17,$6*( 0$,33..14'.7(,37,17,969( 0$,33..14'.7',37,17,$79( 0$,33..14'.7',37,17,$79( 0$,33..14'.7',36,17,$7$( 0$,33..14'.7',36,17,$7$( 0$,33..14'.7',36,17,$7$( 0$,33..14'.7',36,17,$7$( 0$,33..14'.7'637,17,$79( 0$,33..64'.7'+37,176$79( 0$,33..'4'.7'737,17,979(

$UDELDQ &DPHO *XDQDFR &ROODUHG SHFFDU\ 3LJ +XPDQ )LQEDFN ZKDOH 6SHUP ZKDOH +LSSRSRWDPXV 3DQGD 6QRZ OHRSDUG *UHY\ ]HEUD 7DSLU *XLQHD SLJ 5DELW 0RXVH 5DW 0RXQWDLQ JRDW 0XVNR[ 5XSLFDSUD *RDW 6KHHS 'DOO VKHHS 6DLJD WDWDULFD &DWWOH (XURSHDQ ELVRQ :DWHU EXIIDOR 0XOH GHHU 5HLQGHHU 5HG GHHU (ODSKXUXV &HUYXV GXYDXFHOLL 6DPEDU *LUDIIH &KLQHVH PXQWMDN &KHYURWDLQ

)LJ  6HTXHQFH FRPSDULVRQ RI WKH FK\PRVLQ VHQVLWLYH UHJLRQ RI خ؛&1 IURP GLIIHUHQW VSHFLHV %DVLF DPLQR DFLG UHVLGXHV LQ EROG

 WKH 1WHUPLQDO OREH RI ERYLQH FK\PRVLQ ZDV GLVFXVVHG WR KDYH IDYRXUDEOH HOHFWURVWDWLF LQWHUDFWLRQV ZLWK WKH QHJDWLYHO\ FKDUJHG UHVLGXHV RQ WKH VXUIDFH RI FDVHLQ PLFHOOHV *LOOLODQG HW DO 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 %DVHG RQ VWUXFWXUDO FRPSDULVRQ ZH VXSSRVH WKDW FDPHO FK\PRVLQ LV EHWWHU VXLWHG IRU UHQQHW FRDJXODWLRQ RI FDPHO PLON WKDQ FDOI FK\PRVLQ ZKLFK ZDV VWXGLHG HOVHZKHUH 5DPHW  0HKDLD  $OWKRXJK ILUP FRDJXODWLRQ RI WKH PLON ZDV UHSRUWHG LQ VRPH VWXGLHV D FRQVLVWHQW TXDOLW\ ZDV QRW REWDLQHG LQ WKH FKHHVH PDNLQJ SURFHVV DQG DGGLWLRQ RI HOHYDWHG DPRXQWV RI &D&O ZDV D SUHUHTXLVLWH 8VH RI FDPHO FK\PRVLQ PD\ KHOS WR SUHYHQW WKH IRUPDWLRQ RI ELWWHU SHSWLGHV GXULQJ FKHHVH ULSHQLQJ ZKLFK LPSDLU WKH WDVWH DQG WKHUHIRUH FRXOG KHOS WR SURPRWH FRQVXPHU DFFHSWDELOLW\ RI FKHHVH



5HVXOWV DQG 'LVFXVVLRQ

SURGXFWV LQ FDPHO NHHSLQJ FRXQWULHV /DUJHVFDOH SURGXFWLRQ RI WKLV HQ]\PH HJ E\ UHFRPELQDQW PHWKRGV VKRXOG EH HQYLVDJHG WKHUHIRUH  0DMRU :KH\ 3URWHLQV

/LWHUDWXUH %RYLQH ZKH\ LV GHILQHG DV WKH VXSHUQDWDQW RI FDVHLQ SUHFLSLWDWLRQ DW S+  0DMRU SURWHLQ FRPSRQHQWV RI ERYLQH ZKH\ DUH خ²ODFWRJOREXOLQ ZLWK  RI WRWDO ZKH\ SURWHLQ خ±ODFWDOEXPLQ ZLWK  DQG EORRG VHUXP DOEXPLQ ZLWK  6FKOLPPH  $OWKRXJK WKH SUHFLSLWDWLRQ SRLQW RI FDPHO PLON FDVHLQ ZDV VKRZQ WR EH ORZHU WKDQ WKDW RI FRZ PLON FDVHLQ :DQJRK  S+  ZDV FKRVHQ IRU VWXGLHV RQ FDPHO ZKH\ IRU EHWWHU FRPSDULVRQ WR ERYLQH ZKH\ 5HYHUVHG3KDVH &KURPDWRJUDSK\ RI :KH\ 3URWHLQV :KH\ RI DFLG SUHFLSLWDWHG FDPHO PLON ZDV VHSDUDWHG E\ UHYHUVHGSKDVH& FKURPDWRJUDSK\ )LJ  3HDNV ZHUH LGHQWLILHG E\ 1WHUPLQDO VHTXHQFLQJ :KH\ DFLGLF SURWHLQ :$3 ZDV HOXWHG DW  PLQ خ± ODFWDOEXPLQ DW  PLQ WR  PLQ DQG ODFWRSKRULQ DW  PLQ WR  PLQ $EVRUSWLRQ RI ODFWRSKRULQ DW  QP ZDV ZHDN 7KH SUHVXPHG ORZ OHYHO LQ W\URVLQH WU\SWRSKDQ DQG F\VWHLQH ZDV FRQILUPHG E\ VHTXHQFH DQDO\VLV ,QWHJUDWLRQ RI SHDN DUHDV ZDV GRQH DW  QP خ±ODFWDOEXPLQ DFFRXQWHG IRU  RI WRWDO SHDN DUHD ODFWRSKRULQ IRU   DQG ZKH\ DFLGLF SURWHLQ :$3 IRU   6'63$*( UHYHDOHG WKDW EORRG VHUXP DOEXPLQ DQG RWKHU SURWHLQV FRHOXWHG ZLWK خ±ODFWDOEXPLQ DV PLQRU IUDFWLRQV 0LQRU ZKH\ SURWHLQV ZHUH QRW GHWHFWHG E\ 53& FKURPDWRJUDSK\ RI WRWDO ZKH\ ,I LW LV DVVXPHG WKDW FDPHO PLON FRQWDLQV DQ DYHUDJH RI  PJ O ZKH\ SURWHLQV WKH DPRXQW RI خ±ODFWDOEXPLQ WRJHWKHU ZLWK FRHOXWHG SURWHLQV LV DERXW  PJ O WKH DPRXQW RI ODFWRSKRULQ DERXW  PJ O DQG RI :$3 DERXW  PJ O 7KH PDMRU FDPHO ZKH\ SURWHLQV خ±ODFWDOEXPLQ ODFWRSKRULQ DQG :$3 ZHUH DOUHDG\ VHTXHQFHG E\ %HJ   E  ,Q WKLV VWXG\ ZH FRUUHFWHG WKH VHTXHQFH RI ODFWRSKRULQ DV SUHVHQWHG E\ %HJ  VLQFH WKLV SURWHLQ PD\ KDYH LQWHUHVWLQJ IHDWXUHV WR H[SODLQ VRPH FKDUDFWHULVWLFV RI FDPHO PLON ,Q D IXUWKHU VWXG\ LW ZRXOG DOVR EH RI LQWHUHVW WR FKDUDFWHULVH WKH SRWHQWLDO



   

D

,,



    

$ >$8@

                              

,,, ,

    

5HWHQWLRQ 7LPH >PLQ@ E





,,

     

,

,,,

  

5HWHQWLRQ 7LPH >PLQ@

)LJ  5HYHUVHGSKDVH & +3/& FKURPDWRJUDP RI FDPHO PLON ZKH\ SURWHLQV 3HDNV , ,, DQG ,,, ZHUH FROOHFWHG IRU IXUWKHU DQDO\VLV *UDGLHQW RI VROYHQW % DV GDVKHG OLQH D $EVRUSWLRQ DW  QP E $EVRUSWLRQ DW  QP

&RQFHQWUDWLRQ RI VROYHQW % PO O



$ >$8@

&RQFHQWUDWLRQ RI VROYHQW % PO O



7DEOH  3K\VLFRFKHPLFDO FKDUDFWHULVWLFV RI FDPHO DQG FRZ PLON ZKH\ SURWHLQVD
0ROHFXODU PDVV >N'D@ EDVHG RQ $PLQR DFLG VHTXHQFH $PLQR DFLG VHTXHQFH 0DVV VSHFWUR PHWU\ ,VRHOHFWULF SRLQWE EDVG RQ &KDUJHG PRGLIL FDWLRQV $PLQR DFLG RI VHTXHQFH ZLWK DPLQR PRGLILFDWLRQV DFLG UHVLGXHV &RQFHQ WUDWLRQ LQ PLON >PJ O@ 6LPL ODULW\ WR FRUUHV SRQGLQJ ERYLQH SURWHLQVF

6SHFLHV

3URWHLQ

$PLQR DFLG UHVLGXHV

&DPHO           QG آ§        QG QG QG QG QG QG      

خ±ODFWDOEXPLQ  QG     QG QG QRQH  6HU3  6HU3 QG QG QG QG QG QG QG QG

 QG 

!       QG   



&RZ

خ±ODFWDOEXPLQ



&DPHO

3*53



&DPHO

ODFWRSKRULQ $



  



&RZ

ODFWRSKRULQ



&DPHO

ODFWRIHUULQ

 QG  QG

&RZ

ODFWRIHUULQ



&DPHO

ODFWRSHUR[LGDVH



&RZ

ODFWRSHUR[LGDVH



&DPHO

:$3



5HVXOWV DQG 'LVFXVVLRQ

&RZ

خ²ODFWRJOREXOLQ % 

D 'DWD RQ ERYLQH ZKH\ SURWHLQV DIWHU (LJHO HW DO  +HUQiQGH] HW DO  'XOO HW DO  'H :LW +RR\GRQN  E &DOFXODWHG ZLWK WKH JFJ SURJUDPPH *HQHWLFV &RPSXWHU *URXS 0DGLVRQ :,  86$ F 6LPLODU DQG LGHQWLFDO UHVLGXHV

 SURWHDVHLQKLELWRU :$3 LQ WHUPV RI HQ]\PDWLF DFWLYLW\ DQG WHUWLDU\ VWUXFWXUH  /DFWRSKRULQ /LWHUDWXUH /DFWRSKRULQ LV D PDMRU SURWHLQ FRPSRQHQW RI FDPHO ZKH\ ZKLFK ZDV ILUVW GHVFULEHG E\ %HJ HW DO  DQG FKDUDFWHULVHG DV F\VWHLQH IUHH DQG ZLWK 1 WHUPLQDO KHWHURJHQHLW\ LQ WKH DPLQR DFLG VHTXHQFH ,W LV VWUXFWXUDOO\ FORVHO\ UHODWHG WR ERYLQH ODFWRSKRULQ ZKLFK LV D PLQRU SURWHLQ FRPSRQHQW RI ERYLQH ZKH\ %RYLQH ODFWRSKRULQ ZDV IRXQG WR EH D K\GURSKRELF SKRVSKR JO\FRSURWHLQ ZLWK DQ DSSDUHQW PDVV RI  N'D DQG D FRQFHQWUDWLRQ LQ PLON RI DERXW  PJ O -RKQVHQ HW DO  7KH SURWHLQ ZDV VKRZQ WR KDYH JRRG HPXOVLI\LQJ TXDOLWLHV &RXUWKDXGRQ HW DO  DQG WR LQKLELW VSRQWDQHRXV OLSRO\VLV E\ OLSRSURWHLQ OLSDVH *LUDUGHW HW DO 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 7KLV WHUP ZDV QRW XVHG IRU GHVLJQDWLRQ RI WKH FDPHO SURWHLQ EHFDXVH WKH SURWHLQ ZDV QRW LVRODWHG IURP SURWHRVH SHSWRQH DQG EHFDXVH ERYLQH 33 FRQVLVWHG RI VHYHUDO SURWHLQV RI ZKLFK ODFWRSKRULQ ZDV MXVW WKH PDLQ IUDFWLRQ )XUWKHUPRUH WKH WHUP 33 LV DOVR XVHG IRU GHVLJQDWLRQ RI RWKHU SURWHLQV WKDQ WKH PLON SURWHLQ 7KH WHUP آ³ODFWRSKRULQآ´ ZDV LQWURGXFHG E\ .DQQR  D WR GHVFULEH D ZKH\ FRPSRQHQW ZLWK DIILQLW\ WR VROXEOH JO\FRSURWHLQ DQWLVHUXP 7KLV WHUP ZDV FKRVHQ IRU GHVLJQDWLRQ RI WKH SURWHLQ VWXGLHG VLQFH LW ZDV XVHG SDUWLFXODUO\ IRU GHVFULSWLRQ RI WKLV SURWHLQ IDPLO\ 7KH WHUP آ³ODFWRJO\FRSKRULQآ´ DV SURSRVHG E\ *LUDUGHW /LQGHQ  FRXOG QRW EH XVHG IRU FDPHO ODFWRSKRULQ EHFDXVH WKH SURWHLQ ZDV QRW JO\FRV\ODWHG



5HVXOWV DQG 'LVFXVVLRQ

/DFWRSKRULQ *HQH 6WUXFWXUH 7KH JHQRPLF VWUXFWXUH RI WKH ODFWRSKRULQ JHQH ZDV DQDO\VHG E\ VHTXHQFLQJ RI 3&5 SURGXFWV ZKLFK VSDQQHG IURP H[RQ  WR H[RQ  (0%/*HQ%DQNâ„¢ DFFHVVLRQ QXPEHU $- 6LPLODUO\ WR WKH JHQH VWUXFWXUHV RI ERYLQH ODFWRSKRULQ DQG PXULQH *O\&$0 WKH FRGLQJ VHTXHQFH RI WKH FDPHO ODFWRSKRULQ JHQH ZDV LQWHUUXSWHG E\ WKUHH LQWURQ VHTXHQFHV DW * 7 DQG * )LJ   7KHVH SRVLWLRQV FRUUHVSRQGHG WR WKH SRVLWLRQV LQ WKH PXULQH *O\&$0 JHQH DQG WKH ERYLQH ODFWRSKRULQ JHQH ,QWURQ , ZDV  ES ORQJ LQWURQ ,,  ES DQG LQWURQ ,,,  ES 7KH 6,1( %RY$ VHTXHQFH ZKLFK LV SUHVHQW LQ ERYLQH LQWURQ , WKH PLFURVDWHOOLWH VHTXHQFH $& ZKLFK LV SUHVHQW LQ ERYLQH LQWURQ ,, DQG WKH /,1(/ ZKLFK LV SUHVHQW LQ ERYLQH LQWURQ ,,, ZHUH QRW IRXQG LQ WKH FDPHO JHQH )LJ 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 ZDV FRQILUPHG 1WHUPLQDO KHWHURJHQHLW\ ZDV IRXQG WR EH WKH UHVXOW RI DOWHUQDWLYH VSOLFLQJ 7KH VHTXHQFH ZKLFK FRUUHVSRQGHG WR H[RQ ZDV GHOHWHG LQ D PLQRU IUDFWLRQ RI FDPHO ODFWRSKRULQ 3&5 DPSOLILFDWLRQ SURGXFWV RI WZR IXOO OHQJWK F'1$ FORQHV RI  ES DQG  ES ZHUH VHTXHQFHG )LJ  %RWK F'1$ FORQHV FRQWDLQHG D آ¶XQWUDQVODWHG UHJLRQ RI  ES DQG D آ¶XQWUDQVODWHG UHJLRQ RI  ES 7KH آ¶XQWUDQVODWHG UHJLRQ FRQWDLQHG D SDUWLDO .R]DNER[ &&&&$&& ZLWK F\WRVLQHV DW   DQG  ES DQG DQ DGHQLQH DW  ES LQ IURQW RI WKH WUDQVODWLRQDO VWDUW $7* 7KH آ¶



D      



/,1(/

E      





6,1($OX



F   





 



/,1(/ 6,1( %RY$

0LFURVDWHOOLWH

/,1(/

)LJ  6FKHPDWLF FRPSDULVRQ RI WKH JHQRPLF VWUXFWXUHV RI D &DPHOXV GURPHGDULXV ODFWRSKRULQ JHQH (0%/ $& $- E 0XV PXVFXOXV *O\&$0 JHQH (0%/ $& ' F %RV WDXUXV ODFWRSKRULQ JHQH (0%/ $& ; %DUV LQGLFDWH WUDQVFULEHG H[RQ VHTXHQFHV ,QWHUVSHUVHG HOHPHQWV DUH LQGLFDWHG E\ GDVKHG OLQHV 1XPEHULQJ RI EDVHV DFFRUGLQJ WR IXOO OHQJWK VHTXHQFHV DV GHVFULEHG LQ WKH (0%/*HQ%DQN''%- 1XFOHRWLGH 6HTXHQFH 'DWDEDVH



5HVXOWV DQG 'LVFXVVLRQ

F'1$ /SK F'1$ /SK $ /SK % F'1$ /SK $ /SK % F'1$ /SK $ /SK % F'1$ /SK $ /SK % F'1$ /SK $ /SK % F'1$ /SK $ /SK % F'1$ F'1$

    &*77*&7*7&*&&$**$$$$&$*$7&&7*&7&&$*&&&&$&&$7*$$$77&77&*&7*7&&7*&7*&7**&&$*& 0HW/\V3KH3KH$OD9DO/HX/HX/HX$OD6HU       77*$&&7&&*&&7&7&77*&&$*&&77$$7*$*&&$$$$*$7*$$$7&7$&$7**$*7&7&$*&&&$&$*$7$&& /HX7KU6HU$OD6HU/HX$OD6HU/HX$VQ*OX3UR/\V$VS*OX,OH7\U0HW*OX6HU*OQ3UR7KU$VS7KU /HX7KU6HU$OD6HU/HX$OD6HU/HX$VQ$    7&7*&&&$**7&$7&$7*$*&$$&&$7&$**7&7&&$*7*$**$&&777&7$7**$*&&77&&$7&7&&$*$*$$ 6HU$OD*OQ9DO,OH0HW6HU$VQ+LV*OQ9DO6HU6HU*OX$VS/HX6HU0HW*OX3UR6HU,OH6HU$UJ*OX OD$OD*OQ9DO,OH0HW6HU$VQ+LV*OQ9DO6HU6HU*OX$VS/HX6HU0HW*OX3UR6HU,OH6HU$UJ*OX 3 3 3 3     *$7&7**777&&$$$*$&*$7*77*7*$7&$$$7&7*&&$**$*$&$&&$*$$7&$*$$7&&&$$*&7*&77&$& $VS/HX9DO6HU/\V$VS$VS9DO9DO,OH/\V6HU$OD$UJ$UJ+LV*OQ$VQ*OQ$VQ3UR/\V/HX/HX+LV $VS/HX9DO6HU/\V$VS$VS9DO9DO,OH/\V6HU$OD$UJ$UJ+LV*OQ$VQ*OQ$VQ3UR/\V/HX/HX+LV     &&&*7*&&$&$**$*$*&$*777&$*$$$7$&7*&&$&7&$$7&$*$$*$*$&&$$$*$$&7&$&7&&7****&7 3UR9DO3UR*OQ*OX6HU6HU3KH$UJ$VQ7KU$OD7KU*OQ6HU*OX*OX7KU/\V*OX/HX7KU3UR*O\$OD 3UR9DO3UR*OQ*OX6HU6HU3KH$UJ$VQ7KU$OD7KU*OQ6HU*OX*OX7KU/\V*OX/HX7KU3UR*O\$OD



 

 

 

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

)LJ  F'1$ VHTXHQFH RI FDPHO PLON ODFWRSKRULQ $ DQG FRUUHVSRQGLQJ SURWHLQ YDULDQWV $ /SK $ DQG % /SK % ZLWK PDWXUH SURWHLQV LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR $ DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 7KH VHTXHQFH IURP $ WR 7 FRUUHVSRQGV WR H[RQ  DQG LV GHOHWHG LQ FDPHO PLON ODFWRSKRULQ % 1XPEHULQJ RI WKH DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ 3 SRWHQWLDOO\ SKRVSKRU\ODWHG VHULQH UHVLGXHV

 XQWUDQVODWHG UHJLRQ FRQWDLQHG D SRO\DGHQ\ODWLRQ VLJQDO $77$$$ LQ WKH ORQJHU FORQH ZKLFK FRUUHVSRQGHG WR $77$$$ LQ WKH VKRUWHU FORQH 7KH ORQJHU FORQH FRQWDLQHG DQ RSHQ UHDGLQJ IUDPH IRU D SHSWLGH RI  DD UHVLGXHV DQG WKH VKRUWHU IRU D SHSWLGH RI  DD UHVLGXHV 7KH VWDUW VLWH RI ERWK PDWXUH SURWHLQV ZDV FRQILUPHG E\ 1WHUPLQDO SURWHLQ VHTXHQFLQJ WR EH 6HU 7KH  DD VLJQDO SHSWLGHV FRQIRUPHG WR WKH XVXDO SDWWHUQ IRU VLJQDO SHSWLGHV 1LHOVHQ HW DO  DQG KDG  VHTXHQFH VLPLODULW\ WR WKH VLJQDO SHSWLGHV RI ERYLQH ODFWRSKRULQ DQG *O\&$0 SURWHLQV 7ZR PDWXUH ODFWRSKRULQ YDULDQWV ZHUH IRXQG DQG GHVLJQDWHG DV YDULDQWV $ DQG % 9DULDQW $ FRQVLVWHG RI  DD UHVLGXHV DQG YDULDQW % RI  DD UHVLGXHV &RPSXWDWLRQDO DQDO\VLV %DUWRQ  RI FDPHO ODFWRSKRULQ $ UHYHDOHG VHTXHQFH VLPLODULWLHV RI  WR ERYLQH ODFWRSKRULQ DQG  WR PRXVH *O\&$0 7KH VHTXHQFH RI %HJ HW DO  ZDV FRUUHFWHG E\ LQVHUWLRQ RI 0HW WR 6HU IRU YDULDQW $ DQG 6HU WR 6HU IRU YDULDQW % )LJ  7KH H[FKDQJH RI /HX LQ WKH VHTXHQFH RI %HJ HW DO WR ,OH LQ YDULDQW $ DQG RI /HX WR ,OH LQ YDULDQW % UHVSHFWLYHO\ FRXOG EH GXH WR DQ DOOHOLF YDULDQW RU WR DQ HUURU LQ SURWHLQ VHTXHQFLQJ 7KH LQVHUWHG VHTXHQFH ZDV ULFK LQ 6HU7KU SDWWHUQV ZKLFK JDYH LQGLFDWLRQ IRU SURWHLQ SKRVSKRU\ODWLRQ DQG ODFNHG F\VWHLQH UHVLGXHV
&DPHO :KH\ SURWHLQ %HJ  6/1(3.',0< 0(36,65(' &DPHO :KH\ SURWHLQ %HJ  6/1 $$49(, 0(36,65(' &DPHO ODFWRSKRULQ $ 6/1(3.'(,<0(6437'76$49,061+4966('/60(36,65(' &DPHO ODFWRSKRULQ % 6/1 $$49,061+4966('/60(36,65('

)LJ  &RUUHFWHG 1WHUPLQDO VHTXHQFHV RI ODFWRSKRULQ $ DQG % FRPSDUHG WR WKH VHTXHQFHV SURSRVHG E\ %HJ HW DO  7KH SULPDU\ VWUXFWXUHV RI FDPHO DQG ERYLQH ODFWRSKRULQ ZHUH KLJKO\ VLPLODU 3HUFHQW VHTXHQFH VLPLODULW\ RI FDPHO ODFWRSKRULQ WR ERYLQH DQG FDSULQH ODFWRSKRULQ ZDV PXFK KLJKHU WKDQ WR UDW DQG PXULQH *O\&$0 7DEOH  7KLV UHVXOW FRXOG SDUWO\ EH GXH WR D FORVHU HYROXWLRQDU\ UHODWLRQVKLS EHWZHHQ FDPHOV DQG FDWWOH EXW LW DOVR FRXOG JLYH LQGLFDWLRQ IRU D FORVHU IXQFWLRQDO UHODWLRQVKLS RI WKH FDPHO DQG ERYLQH ODFWRSKRULQV LQ PLON &DPHO



5HVXOWV DQG 'LVFXVVLRQ

ODFWRSKRULQ % H[KLELWHG OHVV VHTXHQFH VLPLODULW\ GXH WR D JDS LQ WKH VHTXHQFH SLOHXS SURGXFHG E\ WKH GHOHWLRQ DOUHDG\ PHQWLRQHG *O\FRV\ODWLRQ DFFRXQWV IRU DERXW  WR  RI SURWHLQ PDVV RI ERYLQH ODFWRSKRULQ 1J HW DO  .DQQR  E *LUDUGHW HW DO  SKRVSKRU\ODWLRQ IRU DERXW  1J HW DO  WR  3kTXHW HW DO 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 (QGRSODVPDWLF VLJQDO SHSWLGDVH FXWV OODPD SUH ODFWRSKRULQ WKUHH DD UHVLGXHV SULRU WR WKH FOHDYDJH VLWH RI WKH FDPHO DQG ERYLQH FRXQWHUSDUWV DQG ERWK RI WKH VHTXHQFHG *O\&$0 SURWHLQV DUH FOHDYHG RQH DPLQR DFLG EH\RQG WKLV VLWH 7KH GLIIHUHQW FOHDYDJH VLWHV DGGLWLRQDOO\ GHPRQVWUDWH D KLJK YDULDELOLW\ RI WKH 1WHUPLQDO SDUW RI WKH ODFWRSKRULQ*O\&$0 IDPLO\ ,I WKLV YDULDELOLW\ LV WKH UHVXOW RI D YDULDWLRQ LQ IXQFWLRQDOLW\ EHWZHHQ WKH 1WHUPLQL RU LI LW UHVXOWV IURP D SUHYDOHQW ORZ 1 WHUPLQDO IXQFWLRQDOLW\ RI WKH SURWHLQV FRXOG QRW EH GHFLGHG EHFDXVH WKH WHUWLDU\ VWUXFWXUHV DUH QRW \HW UHVROYHG DQG WKH IXQFWLRQDO GRPDLQV QRW \HW GHWHUPLQHG 1HYHUWKHOHVV PDQ\ UHVLGXHV RI WKH 1WHUPLQDO SDUW ZHUH KLJKO\ FRQVHUYHG )LJ  7KLV JDYH LQGLFDWLRQ WKDW WKLV SDUW RI WKH SURWHLQ ZDV RI IXQFWLRQDO LPSRUWDQFH $ IXQFWLRQ ZKLFK GHSHQGHG RQ WKH 1WHUPLQDO VHTXHQFH DV IRXQG LQ ERYLQH ODFWRSKRULQ DQG FDPHO ODFWRSKRULQ $ ZDV H[SHFWHG WR EH ORVW RU VLJQLILFDQWO\ DOWHUHG LQ FDPHO ODFWRSKRULQ %

7DEOH  3K\VLFRFKHPLFDO DQG VHTXHQFH FKDUDFWHULVWLFV RI PDWXUH VHFUHWHG *O\&$0 DQG ODFWRSKRULQ

6SHFLHV

3URWHLQ

&DOFXODWHG 0HDVXUHG 0: 0: >N'D@ >N'D@

$PLQR ,VRHOHFWULF ,VRHOHFWULF 6LPLODULW\ WR &RQFHQWUDWLRQ DFLG SRLQW SRLQW FDPHO PLONVHUXP UHVLGXHV FDOFXODWHG PHDVXUHG ODFWRSKRULQ $ >PJ O@

&DPHOXV GURPHGDULXV /DFWRSKRULQ $  &DPHOXV GURPHGDULXV /DFWRSKRULQ %            QG   QG QG QG D   E

 

 

 

QG

     

 F QG QG G

%RV WDXUXV

/DFWRSKRULQ



&DSUD KLUFXV

/DFWRSKRULQ

5DWWXV QRUYHJLFXV

*O\&$0

0XV PXVFXOXV

*O\&$0

D 6آ،UHQVHQ HW DO  E *LUDUGHW /LQGHQ  F -RKQVHQ HW DO  G 6LQJHU 5RVHQ 

&DPHO $ &DPHO % /ODPD &RZ *RDW 0RXVH 5DW                                

 6/1(3.'(,<0(6437'7 6$49, 061+4966('/60(36,65('  6/1 $$49, 061+4966('/60(36,65('  6/96/1(3.'(,<0(643  ,/1.3('(7+/($437'$ 6$4), 51/4,61('/6.(36,65(' ,61/4,67('/6.(36,65('  ,/1(3('(7+/($437'$ 6$4),  /3*6.'(/40.7437'$,3$$467376<76((6766.'/6.(36,)5((  93*6.'(/+/57437'$,3$64)7366+,6.(6766.'/6.(6),)1((

 

 

&DPHO $ &DPHO % &RZ *RDW 0RXVH 5DW

     

/96.''99,.6$55+41413.//+3934(66)517$746((7.(/73*$$77/ /96.''99,.6$55+41413.//+3934(66)517$746((7.(/73*$$77/ /,6.(4,9,56654346413./3/6,/.(.+/51$7/*6((77(+736'$677 /,6.(319,56354341413./3/6,/.(.4/51$7/*6((77(+$36'$677 /,6.'199,(67. 3(14($ 4'*/56*664/((7753776$$776 /96('19*7(67. 3464($ 4'*/56*6644(( 776$$ 76



&DPHO $ &DPHO % &RZ *RDW 0RXVH 5DW

     

(*./9(/7+.,,.1/(1705(70')/.6/)3+$6(99.34 (*./9(/7+.,,.1/(1705(70')/.6/)3+$6(99.34 (*./0(/*+.,051/(179.(7,.
5HVXOWV DQG 'LVFXVVLRQ

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

 6HFRQGDU\ 6WUXFWXUH 7KH DPLQR DFLG FRPSRVLWLRQ RI ODFWRSKRULQ ZDV VLPLODU WR خ± DQG خ²FDVHLQV DOWKRXJK WKH SUROLQH FRQWHQW ZDV ORZHU  LQ FDPHO ODFWRSKRULQ FRPSDUHG WR  LQ FDPHO خ²FDVHLQ 6LPLODUO\ WR FDVHLQV FDPHO DQG ERYLQH ODFWRSKRULQ ZHUH FKDUDFWHULVHG E\ DQ DFLGLF 1WHUPLQDO SDUW RI WKH SURWHLQ ZKLFK ZDV ULFK LQ *OX 6HU 7KU DQG FRQWDLQHG FOXVWHUHG SKRVSKRVHULQHV ZKHUHDV WKH &WHUPLQDO SDUW ZDV ULFK LQ K\GURSKRELF UHVLGXHV ZLWK WKH GLIIHUHQFH WKDW ODFWRSKRULQ ZDV FRQVLGHUHG WR IRUP D & WHUPLQDO DPSKLSKLOLF KHOL[ ZLWK PL[HG EDVLF DQG DFLGLF UHVLGXHV RQ WKH SRODU VLGH *LUDUGHW /LQGHQ  )LJ  7KLV VWUXFWXUDO SURSHUW\ ZDV PRUH SURQRXQFHG LQ ERYLQH ODFWRSKRULQ DQG URGHQWVآ¶ *O\&$0 SURWHLQV WKDQ LQ WKH FDPHO KRPRORJXH VLQFH WKH ODWWHU SURWHLQ FRQWDLQHG D KHOL[ EUHDNLQJ 3UR LQ YDULDQW $ DQG 3UR LQ YDULDQW % QHDU WKH &WHUPLQXV ZKLFK LQGXFHG D NLQN WRZDUGV WKH &WHUPLQDO HQG RI WKH KHOL[ 7KH UHFHQWO\ UHVROYHG SULPDU\ VWUXFWXUH RI FDSULQH ODFWRSKRULQ /LVWHU HW DO  FRQWDLQHG 3UR DQG 3UR LQ WKH FRUUHVSRQGLQJ UHJLRQ ZKLFK LQGLFDWHG WKDW PRGLILFDWLRQ RI WKH SULPDU\ VHTXHQFH DW WKLV VLWH ZDV QRW DUELWUDU\ 7KH VLGH FKDLQ RI 7KU LQ YDULDQW $ DQG 7KU LQ YDULDQW % IXUWKHUPRUH SURWUXGHG LQWR WKH K\GURSKRELF SDUW RI WKH KHOL[ 7KH &WHUPLQDO SDUW RI ERYLQH ODFWRSKRULQ ZDV GLVFXVVHG WR EH LQYROYHG LQ ELQGLQJ RI SKRVSKROLSLGV RI WKH PLON IDW JOREXOH PHPEUDQH 0)*0 'LIIHUHQW SURWHLQV VXFK DV 3$6  FHFURSLQV DQG PDJDLQLQV ZHUH VKRZQ WR LQWHUDFW ZLWK SKRVSKROLSLG PHPEUDQHV E\ DPSKLSKLOLF KHOLFHV 6آ،UHQVHQ HW DO  $OWKRXJK FDPHO ODFWRSKRULQ ZDV LVRODWHG IURP ZKH\ WKH SUHGLFWHG VHFRQGDU\ VWUXFWXUH JDYH LQGLFDWLRQ IRU ELQGLQJ WR 0)*0 SKRVSKROLSLGV %RYLQH ODFWRSKRULQ ZDV GHWHFWHG LQ ZKH\ DQG LQ 0)*0 EXW QRW LQ FDVHLQ .HVWHU %UXQQHU  .DQQR  D 6آ،UHQVHQ HW DO  $QRWKHU 0)*0 ELQGLQJ SURWHLQ ERYLQH 3$6 ZDV VKRZQ WR LQWHUDFW ZLWK SKRVSKROLSLGV WKURXJK WKH SRODU VLGH RI LWV &WHUPLQDO DPSKLSKLOLF KHOL[ $QGHUVHQ HW DO 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آµP FRPSDUHG WR ERYLQH PLON IDW JOREXOHV DQG D GLIIHUHQW FRPSRVLWLRQ RI SKRVSKROLSLGV )DUDK  0DMRU FRPSRQHQWV RI



5HVXOWV DQG 'LVFXVVLRQ

D

$OD

*OQ

3UR E *OX

7KU

)LJ  3LFWRULDO YLHZ RQ WKH خ±KHOLFDO &WHUPLQDO SDUW RI D FDPHO DQG E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 ZKHUHDV ERYLQH 0)*0 PDLQO\ FRQVLVWV RI  SKRVSKDWLG\OHWKDQRODPLQH  SKRVSKDWLG\OFKROLQH DQG  VSKLQJRP\HOLQ 7KH SURSRUWLRQ RI SKRVSKDWLG\OFKROLQH ZKLFK GRHV QRW ELQG WR 3$6 LV PXFK VPDOOHU LQ FDPHO PLON WKDQ LQ FRZ PLON 7KH SURSRUWLRQ RI QRQ VDWXUDWHG  IDWW\ DFLG LQ PLON IDW LV DERXW WKUHH WR IRXU WLPHV KLJKHU WKDQ LQ FRZ PLON $EX/HKLD  )DUDK  7KHUH FRXOG EH D IXQFWLRQ RI WKH KHOL[EUHDNLQJ SUROLQH WR LQGXFH D YDULDWLRQ LQ WKH WHUWLDU\ VWUXFWXUHV RI FDPHO DQG ERYLQH ODFWRSKRULQV ZKLFK PD\ EH LPSRUWDQW IRU VSHFLILF ELQGLQJ WR WKH PLONVآ¶ SKRVSKROLSLG PL[WXUH

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@آ±;DD>6HU3 *OX $VS@آ±;DD ZLWK *OX RU $VS DW SRVLWLRQ  DQG  HQKDQFLQJ SKRVSKRU\ODWLRQ 6ZDLVJRRG  6HU 6HU 6HU 6HU DQG 6HU LQ YDULDQW $ DQG 6HU 6HU 6HU 6HU DQG 6HU LQ YDULDQW % UHVSHFWLYHO\ ILW WR WKH FRQVHQVXV SDWWHUQ 6HU 6HU 6HU DQG 6HU LQ YDULDQW $ DQG 6HU 6HU 6HU DQG 6HU LQ YDULDQW % ZHUH ORFDWHG LQ D SUHGLFWHG ORRS VXUURXQGHG E\ خ±KHOLFDO UHJLRQV DQG KDG KLJK SKRVSKRU\ODWLRQ SRWHQWLDO 5HVSHFWLYH 6HU 6HU 6HU DQG 6HU RI ERYLQH ODFWRSKRULQ DUH SDUWLDOO\ SKRVSKRU\ODWHG 6آ،UHQVHQ 3HWHUVHQ  LQ FDPHO YDULDQW $ DQG 6HU LQ YDULDQW % UHVSHFWLYHO\ ZHUH  6HU SUREDEO\ QRW SKRVSKRU\ODWHG EHFDXVH WKH VHTXHQFH 6HU/\V$VS GLG &DPHO /DFWRSKRULQ $

       خ²خ²خ²خ² خ±خ±خ±خ±خ± خ±خ±خ±خ±خ± خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ± 6/1(3.'(,<0(6437'76$49,061+4966('/60(36,65('/96.''99,.6$55+41413.//+39      خ±خ±خ±خ± خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ± 34(66)517$746((7.(/73*$$77/(*./9(/7+.,,.1/(1705(70')/.6/)3+$6(99.34 

%RYLQH /DFWRSKRULQ

       خ±خ± خ±خ±خ± خ±خ±خ±خ±خ±خ±خ± خ±خ±خ±خ±خ± خ²خ²خ²خ²خ² خ±خ±خ±خ± ,/1.3('(7+/($437'$6$4),51/4,61('/6.(36,65('/,6.(4,9,56654346413./3/6,/       خ±خ± خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ±خ± .(.+/51$7/*6((77(+736'$677(*./0(/*+.,051/(179.(7,.
)LJ  6HFRQGDU\ VWUXFWXUH SUHGLFWLRQV IRU FDPHO ODFWRSKRULQ $ DQG ERYLQH ODFWRSKRULQ خ±KHOLFDO UHJLRQV GHVLJQDWHG DV آ³خ±آ´ خ²SOHDWHG UHJLRQV DV آ³خ²آ´ 6HULQH UHVLGXHV ZLWK SKRVSKRU\ODWLRQ SRWHQWLDO LQ EROG



5HVXOWV DQG 'LVFXVVLRQ

QRW ILW WR WKH FRQVHQVXV SDWWHUQ IRU PDPPDU\ JODQG FDVHLQ NLQDVH DOWKRXJK IXOO SKRVSKRU\ODWLRQ RI WKH KRPRORJRXV 6HU LQ ERYLQH ODFWRSKRULQ ZDV UHSRUWHG 7KLV VHULQH ZDV IRXQG LQ DQRWKHU ORRS UHJLRQ WKDQ WKH RWKHU VHULQH UHVLGXHV ZLWK SKRVSKRU\ODWLRQ SRWHQWLDO )LJ  LQGLFDWLQJ D VSHFLDO IXQFWLRQ RI WKLV VHULQH HJ LQ ELQGLQJ RI FDOFLXP &DPHO ODFWRSKRULQ $ ZDV RI D GLVWLQFW DFLGLF QDWXUH ZLWK DQ LVRHOHFWULF SRLQW DW S+  WKH LVRHOHFWULF SRLQW RI YDULDQW % DW S+  ZDV VLPLODU WR ERYLQH ODFWRSKRULQ 7DEOH  7KUHHIROG SKRVSKRU\ODWLRQ ZRXOG GHFUHDVH WKH LVRHOHFWULF SRLQW RI YDULDQW $ WR S+  DQG RI YDULDQW % WR S+  3RWHQWLDO IRU %LQGLQJ RI IUHH &DOFLXP 7KH VHTXHQFH SUHVHQWHG E\ %HJ HW DO  ZDV FRPSOHPHQWHG ZLWK  DPLQR DFLGV LQ YDULDQW $ DQG ZLWK  LQ YDULDQW % )LJ  7KH FRUUHFWHG SURWHLQ VHTXHQFHV ZHUH ULFK LQ JOXWDPLF DFLG O\VLQH VHULQH DQG OHXFLQH DQG KDG RQO\ PLQRU DPRXQWV RI DURPDWLF DQG VXOSKDWHG UHVLGXHV 7KH LQVHUWHG VHTXHQFH WXUQHG RXW WR KDYH D KLJK SRWHQWLDO IRU SKRVSKRVHULQH FOXVWHUV 7KLV VWUXFWXUH DOVR RFFXUV LQ FDVHLQV DQG H[KLELWV KLJK ELQGLQJ DIILQLWLHV IRU &D %HUQRV HW DO  ,W ZDV VXJJHVWHG WKDW ODFWRSKRULQV PD\ ELQG FDOFLXP LQ WKH PLON 6آ،UHQVHQ 3HWHUVHQ  WKHUHE\ FRQWUROOLQJ WKH VROXELOLW\ RI QRQ FDVHLQ FDOFLXP SKRVSKDWH $GGLWLRQDO VXSSRUW IRU WKLV LGHD LV JLYHQ E\ WKH ILQGLQJ WKDW ELQGLQJ RI /VHOHFWLQ WR SODVPD *O\&$0 LV FDOFLXPGHSHQGHQW 6XJXUL HW DO 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 7KH VHTXHQFHV ZHUH IRXQG WR KDYH D VLPLODUO\ ORZ SRWHQWLDO IRU 2JO\FRV\ODWLRQ E\ 8'3*DO1$F SRO\SHSWLGH 1DFHW\OJDODFWRVDPLQ\O 7UDQVIHUDVH DV KDV ERYLQH ODFWRSKRULQ LQ FRQWUDVW WR PRXVH DQG UDW *O\&$0 VHTXHQFHV )LJ  :KHUHDV PRXVH *O\&$0 UHYHDOHG D VWURQJ 2JO\FRV\ODWLRQ SRWHQWLDO RI 6HU 7KU 7KU 6HU 7KU 6HU 7KU 7KU 7KU 7KU DQG 7KU WKHUH ZDV RQO\ D ORZ 2JO\FRV\ODWLRQ SRWHQWLDO RI 7KU 7KU DQG 7KU IRXQG LQ FDPHO



2JO\FRV\ODWLRQ SRWHQWLDO 2JO\FRV\ODWLRQ SRWHQWLDO 2JO\FRV\ODWLRQ SRWHQWLDO



D

  E

  F









  6HTXHQFH SRVLWLRQ





)LJ  2JO\FRV\ODWLRQ SRWHQWLDO RI WKUHRQLQHV DQG VHULQHV LQ SURWHLQV RI WKH *O\&$0ODFWRSKRULQ IDPLO\ D &DPHO /DFWRSKRULQ $ E %RYLQH /DFWRSKRULQ F 0XULQH *O\&$0 7KH SRWHQWLDO RI WKH UHVLGXHV LV VKRZQ DV D VROLG EDU ZLWK D YDOXH IURP  QR SRWHQWLDO WR  KLJK SRWHQWLDO 7KH WKUHVKROG ZKLFK GHSHQGV RQ WKH SULPDU\ VWUXFWXUH RI WKH SURWHLQ LV VKRZQ DV D GDVKHG OLQH 7KH SUREDELOLW\ RI JO\FRV\ODWLRQ LV WKH GLIIHUHQFH EHWZHHQ WKH SRWHQWLDO DQG WKH WKUHVKROG



5HVXOWV DQG 'LVFXVVLRQ

ODFWRSKRULQ $ DQG RI 6HU 7KU DQG 7KU LQ ERYLQH ODFWRSKRULQ 7KH SDWWHUQ $VQ;[[6HU7KU ZKLFK LV D SUHUHTXLVLWH IRU 1JO\FRV\ODWLRQ DQG ZKLFK RFFXUV DQG SURYRNHV JO\FRV\ODWLRQ LQ WKH ERYLQH YDULDQW DW $VQ *LUDUGHW HW DO  ZDV QRW IRXQG LQ FDPHO ODFWRSKRULQ 5HODWLRQ WR WKH 0XFLQW\SH 3URWHLQV *O\&$0 D JO\FRV\ODWLRQ GHSHQGHQW FHOO DGKHVLRQ PROHFXOH IRXQG LQ EORRG VHUXP LV D OLJDQG IRU O\PSKRF\WH /VHOHFWLQ ZKLFK LV H[SUHVVHG RQ KLJK HQGRWKHOLDO YHQXOHV LQ SHULSKHUDO O\PSK QRGHV DQG DW VLWHV RI FKURQLF LQIODPPDWLRQ %HUJ HW DO  ,W PHGLDWHV WKH UHFUXLWPHQW RI O\PSKRF\WHV IURP EORRG LQWR WKHVH WLVVXHV DV D VROXEOH SUREDEO\ PXOWLPHULF SURWHLQ ZLWK KLJKO\ G\QDPLF DGKHVLRQ RI WKH FHOOV GXH WR ORZ DIILQLW\ DQG UDSLG NLQHWLFV RI VHOHFWLQ LQWHUDFWLRQV 1LFKROVRQ HW DO  /VHOHFWLQ D SURWHLQ ZLWK D OHFWLQ GRPDLQ KDV KLJK DIILQLW\ IRU PXFLQV ZLWK PXOWLSOH 2OLQNHG FDUERK\GUDWHV RI WKH ODFWRVDPLQ W\SH %LQGLQJ GHSHQGV RQ VLDO\ODWLRQ VXOIDWLRQ DQG IXFRV\ODWLRQ *O\&$0 ZDV IRXQG WR FRQWDLQ VXOIDWHG 2 OLQNHG FDUERK\GUDWHV RI WKH W\SH 6LDخ±→*DOخ²→)XF خ±→*DO1DF 7KH VDPH SURWHLQ LV DOVR VHFUHWHG LQ PRXVH PLON 'RZEHQNR HW DO  1LVKLPXUD HW DO  EXW ZLWK D GLIIHUHQW JO\FRV\ODWLRQ VWUXFWXUH WKDQ LQ EORRG VHUXP *O\&$0 KDV PXFK KLJKHU SUREDELOLW\ RI EHLQJ JO\FRV\ODWHG WKDQ ODFWRSKRULQV )LJ  2JO\FRV\ODWLRQ RI WKH PRXVH KRPRORJXH VKRXOG EH H[SHFWHG DW  UHVLGXHV *O\FRV\ODWLRQ RI FDPHO ODFWRSKRULQ $ ZDV RQO\ H[SHFWHG DW 7KU ZKHUHDV WKH ERYLQH SURWHLQ KDG PHGLXP KLJK SUREDELOLW\ RI JO\FRV\ODWLRQ DW 7KU ,PSOLFDWLRQV RI VHFRQGDU\ DQG WHUWLDU\ VWUXFWXUHV PD\ IDYRXU RU SUHYHQW JO\FRV\ODWLRQ RI WKHVH VLWHV )LJ  %RYLQH ODFWRSKRULQ ZDV UHSRUWHG WR EH SDUWLDOO\ 2 JO\FRV\ODWHG DW 7KU FRPSOHWHO\ 1JO\FRV\ODWHG DW $VQ DQG FRPSOHWHO\ 2 3HWHUVHQ  &DPHO ODFWRSKRULQ GLG JO\FRV\ODWHG DW 7KU 6آ،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 DV FRPSDUHG WR ERYLQH ODFWRSKRULQ WR IXOILO WKLV

 SURSRVHG IXQFWLRQ RI ODFWRSKRULQ *O\FRV\ODWLRQ RI WKH ERYLQH SURWHLQ PD\ DOVR EH SDUW RI LQIDQW SURWHFWLRQ DJDLQVW YLUDO RU EDFWHULDO LQIHFWLRQ ,Q D VLPLODU ZD\ DV WKH 7DPP+RUVIDOO SURWHLQ LQ XULQH ERYLQH ODFWRSKRULQ PD\ KHOS WR LQKLELW EDFWHULDO DGKHVLRQ WR KRVW WLVVXH 6آ،UHQVHQ 3HWHUVHQ  6WLPXODWLRQ RI WKH JURZWK RI ELILGREDFWHULD E\ ERYLQH ODFWRSKRULQ ZDV DOVR GHPRQVWUDWHG *LUDUGHW /LQGHQ  1H[W WR EDFWHULDO LQIHFWLRQV URWDYLUDO LQIHFWLRQ LV DQ LPSRUWDQW FDXVH RI LQIDQW GLDUUKRHD ZKLFK FDQ OHDG WR GHDWK RI WKH DQLPDO 7KH PLON PXFLQ FRPSOH[ ZDV VKRZQ WR SURWHFW WKH LQIDQW E\ VSHFLILF LQWHUDFWLRQ ZLWK WKH YLUXV LQ D VLPLODU ZD\ DV LQWHVWLQDO PXFLQV 


5HVXOWV DQG 'LVFXVVLRQ

2OLJRPHULVDWLRQ %RYLQH ODFWRSKRULQ ZDV UHSRUWHG WR EH D FRPSOH[ RI DERXW  N'D 6آ،UHQVHQ HW DO  ZKLFK VHJUHJDWHG WR D FRPSOH[ RI DERXW  N'D XSRQ GLVVROXWLRQ LQ  0 JXDQLGLQH K\GURFKORULGH 1J HW DO  7KH KLJK PROHFXODU FRPSOH[ ZRXOG DFFRXQW IRU D GHFDPHU WKH ORZ PROHFXODU FRPSOH[ IRU D GLPHU &DPHO ODFWRSKRULQ ZDV IRXQG WR KDYH DQ DSSDUHQW PDVV RI  N'D E\ 6'63$*( DQG RI DERXW  N'D E\ 6HSKDGH[ * FKURPDWRJUDSK\ %HJ HW DO  $ FRPSOH[ ZLWK D KLJKHU PROHFXODU PDVV ZDV QRW IRXQG 7KH SURWHLQ ZDV WKHUHIRUH SUREDEO\ GLPHULVHG LQ ZKH\ $ PRGHO RI *O\&$0 IRUPLQJ D GLPHU E\ DVVRFLDWLRQ RI WKH &WHUPLQDO KHOLFDO SDUWV ZDV SURSRVHG E\ /DVN\ HW DO  'LPHULVDWLRQ RI ERYLQH ODFWRSKRULQ ZDV SURSRVHG WR RFFXU LQ D VLPLODU ZD\ *LUDUGHW /LQGHQ  3URWHRO\WLF &OHDYDJH %RYLQH ODFWRSKRULQ ZDV IRXQG WR EH VXVFHSWLEOH IRU SURWHRO\WLF FOHDYDJH E\ SODVPLQ DW $UJ6HU SURGXFLQJ DQ  N'D DQG DQ  N'D IUDJPHQW DV MXGJHG E\ 6'63$*( .DQQR 2JDZD  6آ،UHQVHQ 3HWHUVHQ  3URWHRO\WLF IUDJPHQWV RI FDPHO ODFWRSKRULQ ZHUH IRXQG LQ WLQ\ DPRXQWV LQ FDPHO ZKH\ $ PLQXWH SHDN DW  PLQ )LJ  SUREDEO\ FRQWDLQHG WKH K\GURSKLOLF 1WHUPLQDO SDUWV RI ERWK ODFWRSKRULQ YDULDQWV DV MXGJHG E\ 1 WHUPLQDO VHTXHQFLQJ &DPHO PLON ZDV VKRZQ WR EH ORZ LQ SODVPLQ DFWLYLW\ %DHU HW DO  (LWKHU SODVPLQ FRQFHQWUDWLRQ LQ FDPHO PLON LV ORZ RU SODVPLQ LV UHSUHVVHG E\ VHULQH SURWHDVH LQKLELWRUV VXFK DV WKH SXWDWLYH WU\SVLQW\SH SURWHDVH LQKLELWRU ZKH\ DFLGLF SURWHLQ :$3 &DPHO SODVPLQ PD\ DOVR KDYH D GLIIHUHQW WXUQRYHU UDWH WKDQ ERYLQH SODVPLQ 7KH DPLQR DFLG VHTXHQFH DW WKH VLWH ZKHUH WKH ERYLQH SURWHLQ LV FOHDYHG /\V6HU LQ YDULDQW $ DQG /\V6HU LQ YDULDQW % UHVSHFWLYHO\ FRXOG EH D WDUJHW IRU SODVPLQ FOHDYDJH EXW ZDV IRXQG LQ D خ±KHOLFDO UHJLRQ ZKHUHDV WKH ERYLQH VLWH ZDV DW WKH &WHUPLQDO HQG RI D خ²IROGHG VWUXFWXUH )LJ  7KLV GLIIHUHQFH PD\ UHQGHU WKH VLWH RI FDPHO ODFWRSKRULQ OHVV VXVFHSWLEOH IRU SURWHRO\WLF FOHDYDJH ,QKLELWLRQ RI /LSRO\VLV $Q LPSRUWDQW IXQFWLRQ RI ODFWRSKRULQ LQ PLON VHHPV WR EH WKH FRQWLQXHG PDLQWHQDQFH RI WKH IDW GLVSHUVLRQ (DUO\ LQGLFDWLRQ WKDW SURWHRVH SHSWRQH ZDV WKH FRPSRQHQW UHVSRQVLEOH IRU PLON IRDPLQJ ZDV JLYHQ E\ -HOHQ  6WURQJ IRDPLQJ RI ZKH\ GHSOHWHG IURP PDLQ SURWHLQV DQG ODFWRVH ZDV REVHUYHG WKH IRDP TXDOLW\ ZDV QHJDWLYHO\ DIIHFWHG E\ DGGLWLRQ RI FDOFLXP K\GUR[LGH 6KLPL]X HW DO  VKRZHG WKDW WKH KLJK HPXOVLI\LQJ DFWLYLW\ RI SURWHRVH SHSWRQH FRPSRQHQW  ZKLFK PDLQO\ FRQVLVWV RI ODFWRSKRULQ

 ZDV QRW QHJDWLYHO\ DIIHFWHG E\ H[WHQGHG KHDW WUHDWPHQW DW  آƒ& (PXOVLRQV UHPDLQHG VWDEOH IURP S+  WR S+  $GGLWLRQ RI  P0 &D&O RU  P0 1D&O LQFUHDVHG WKH HPXOVLI\LQJ DFWLYLW\ 7KH DPRXQW RI DGVRUEHG ERYLQH ODFWRSKRULQ ZDV DERXW  PJ J RLO LQ DQ H[SHULPHQWDO OLSLGZDWHU HPXOVLRQ 6XUIDFH WHQVLRQ ZDV UHGXFHG WR D OHYHO EHORZ  P1 P 7KH & WHUPLQDO SURWHRO\WLF SURGXFW IRUPHG E\ SODVPLQ DQG SUREDEO\ FRQWDLQLQJ DQ DPSKLSKLOLF KHOL[ GLG QRW DGVRUE VWURQJO\ WR WKH K\GURSKRELF LQWHUIDFH خ²ODFWRJOREXOLQ ZDV QRW DEOH WR UHGXFH WKH VXUIDFH WHQVLRQ WR D VLPLODU OHYHO /DFWRSKRULQ VKRZHG D PXFK VWURQJHU DGVRUSWLRQ WR WKH OLSLGZDWHU LQWHUIDFH WKDQ خ²ODFWRJOREXOLQ &RXUWKDXGRQ HW DO 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آƒ& *LUDUGHW HW DO  %RYLQH ODFWRSKRULQ ZDV IRXQG WR SUHYHQW WKH OLSRO\WLF DFWLRQ RI SRUFLQH SDQFUHDWLF OLSDVH 33/ D VHULQH OLSDVH VLPLODU WR OLSRSURWHLQ OLSDVH E\ FRPSHWLWLYH DGVRUSWLRQ WR WKH K\GURSKRELF SKDVH LQ D OLSLGZDWHU HPXOVLRQ DQG GLVSODFHPHQW RI OLSDVH LQWR WKH DTXHRXV SKDVH ,Q WKLV PRGHO V\VWHP WKH SURWHLQ GLG QRW LQWHUDFW GLUHFWO\ ZLWK WKH OLSDVH WR H[HUW WKLV IXQFWLRQ *LUDUGHW HW DO  33/ FDQQRW DGVRUE RQ D OLSLGZDWHU LQWHUIDFH DW D VXUIDFH WHQVLRQ EHWZHHQ  P1 P LQ WKH DEVHQFH RI FROLSDVH  LQKLELWLRQ RI OLSRO\WLF DFWLYLW\ ZDV DFKLHYHG ZLWK D IROG KLJKHU ZY 33 FRQFHQWUDWLRQ FRPSDUHG WR WKH LQKLELWRU\ HIIHFW RI خ²ODFWRJOREXOLQ 7KLV ZRXOG FRUUHVSRQG WR DERXW D  IROG KLJKHU ODFWRSKRULQ FRQFHQWUDWLRQ LI LW LV DVVXPHG WKDW DERXW KDOI RI 33 FRQVLVWV RI WKLV DFWLYH FRPSRQHQW %RWK DGVRUSWLYH SURWHLQV KDYH VLPLODU PROHFXODU PDVVHV ,Q WKH خ²ODFWRJOREXOLQ33/V\VWHP OLSRO\WLF DFWLYLW\ ZDV IXOO\ UHVWRUHG XSRQ DGGLWLRQ RI FROLSDVH ZKLFK ELQGV 33/ WR WKH K\GURSKRELF SKDVH DQG ORZ FRQFHQWUDWLRQV RI GLIIHUHQW ELOH VDOWV HJ  P0 WDXURGHR[\FKRODWH /LSRO\WLF DFWLYLW\ RI 33/ ZDV RQO\ UHVWRUHG LQ WKH ODFWRSKRULQ33/V\VWHP XSRQ DGGLWLRQ RI FROLSDVH DQG  P0 WDXURGHR[\FKRODWH UHDFWLYDWLRQ ZLWK RWKHU ELOH VDOWV ZDV QRW SRVVLEOH ,W ZDV FRQFOXGHG WKDW ERYLQH ODFWRSKRULQ ZDV PRUH GLIILFXOW WR GHVRUE IURP WKH RLOZDWHU LQWHUIDFH WKDQ خ²ODFWRJOREXOLQ



5HVXOWV DQG 'LVFXVVLRQ

/DFWRSKRULQ ZDV VKRZQ WR SUHYHQW OLSRO\VLV LQ FRQWUDVW WR خ²&Q3 I  DOVR QDPHG 33 D SURWHRO\WLF EUHDNGRZQ SURGXFW RI خ²&1 ZKLFK VHHPHG WR DFWLYDWH OLSRO\WLF DFWLYLW\ *LUDUGHW /LQGHQ 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 $ KLJKHU OHYHO RI QDWXUDO SUHVHUYLQJ DJHQWV PD\ EULQJ DERXW WKH ORQJHU VWRUDJH OLIH RI UDZ FDPHO PLON FRPSDUHG WR UDZ FRZ PLON )DUDK  $SSOLFDWLRQV 7KH FDPHO SURWHLQ LV D JO\FRV\ODWLRQ IUHH DQG PRQR RU GLPHULF PHPEHU RI WKH ODFWRSKRULQ*O\&$0 IDPLO\ ZKLFK LV HDVLO\ SXULILHG LQ ODUJH TXDQWLWLHV IURP FDPHO ZKH\ 7KLV SURSHUWLHV PDNH WKH SURWHLQ D JRRG VXEVWUDWH IRU IXQFWLRQDO FKDUDFWHULVDWLRQ FU\VWDOOLVDWLRQ DQG ;UD\ VWUXFWXUH DQDO\VLV )XUWKHUPRUH WKH SURWHLQ FRXOG EH VWXGLHG IRU LWV SUHVXPHG TXDOLWLHV DV DQ HPXOVLI\LQJ DJHQW DQG LQKLELWRU RI OLSRO\VLV LQ IRRG SURGXFWV VXFK DV EXWWHU RU LFH FUHDP  0LQRU :KH\ 3URWHLQV

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 %UXFHOORVLV WU\SDQRVRPLDVLV DQG RWKHU GLVHDVHV DUH ODWHQW GDQJHUV WR WKH KHDOWK RI WKH KHUGV WKH SDVWRUDOLVWV DQG WKH FRQVXPHUV +HQFH QDWXUDO LQKLELWLRQ RI SDWKRJHQ JURZWK LQ PLON LV RI LPSRUWDQFH &DPHO PLON ZDV IUHTXHQWO\ UHSRUWHG WR KDYH KLJK DQWLPLFURELDO DFWLYLW\ DQG ZDV VKRZQ WR LQKLELW SDWKRJHQ JURZWK PRUH WKDQ ERYLQH PLON (ODJDP\ HW DO 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 )XUWKHUPRUH D F'1$ FRUUHVSRQGLQJ WR O\VR]\PH ZDV QRW IRXQG E\ VFUHHQLQJ ZLWK ROLJRQXFOHRWLGHV GHULYHG IURP FRQVHUYHG UHJLRQV RI WKH DOUHDG\ NQRZQ &W\SH O\VR]\PHV :H VXSSRVH WKDW WKH O\VR]\PH DFWLYLW\ PHDVXUHG E\ RWKHU DXWKRUV (ODJDP\ HW DO  KDV



5HVXOWV DQG 'LVFXVVLRQ

WR EH DVVLJQHG WR RWKHU HQ]\PHV HJ WR WKH 3HSWLGRJO\FDQ 5HFRJQLWLRQ 3URWHLQ ZKLFK LV GLVFXVVHG LQ WKLV WKHVLV  3HSWLGRJO\FDQ 5HFRJQLWLRQ 3URWHLQ /LWHUDWXUH $ QRYHO SURWHLQ IDPLO\ ZDV GHVFULEHG UHFHQWO\ ZKLFK LV LQYROYHG LQ SULPDU\ LPPXQH UHVSRQVH RI YHUWHEUDWHV DQG LQYHUWHEUDWHV RQ JUDPSRVLWLYH EDFWHULD DQG RWKHU LQYDGLQJ RUJDQLVPV VXFK DV QHPDWRGHV DQG ZRUNV E\ QRQFORQDO SDWWHUQ UHFRJQLWLRQ 


    F'1$ *77*&7*7&**&*$7&7*&&7*&&$7*$&&&**&$&7*&*7*&77&7&*7&7***&7&7&&7&*&&&7&&7&$*& 3URWHLQ 0HW7KU$UJ+LV&\V9DO/HX/HX9DO7US$OD/HX/HX$OD/HX/HX6HU       F'1$ &7&**$*&**&7&*$*$$*$&&&*&&**&&7*&**&7&&$7&*7*&&&&*&&*$*$*7**$***&&&7**&*7&& 3URWHLQ /HX*O\$OD$OD$UJ*OX$VS3UR3UR$OD&\V*O\6HU,OH9DO3UR$UJ$UJ*OX7US$UJ$OD/HX$OD6HU    F'1$ *$*7*&$*$*$$$**&7$$&$&**&&**7*&*&7$&*7**7**7*7&*&$&$&7*&***&$*&&$&7*&*$&$&& 3URWHLQ *OX&\V$UJ*OX$UJ/HX7KU$UJ3UR9DO$UJ7\U9DO9DO9DO6HU+LV7KU$OD*O\6HU+LV&\V$VS7KU     F'1$ &&**&77&*7*&*&*&$*&$**&&&$*$$&*7*&$$$*&7$&&$7*7*&**$$&&7***&7**7*&*$&*7***& 3URWHLQ 3UR$OD6HU&\V$OD*OQ*OQ$OD*OQ$VQ9DO*OQ6HU7\U+LV9DO$UJ$VQ/HX*O\7US&\V$VS9DO*O\     F'1$ 7$&$$&77&&7*$7&**$*$$*$7***&7&*7*7$&*$$**&&*$**&7**$$&$7&$$***&*&&&$&*&$**7 3URWHLQ 7\U$VQ3KH/HX,OH*O\*OX$VS*O\/HX9DO7\U*OX*O\$UJ*O\7US$VQ,OH/\V*O\$OD+LV$OD*O\









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

)LJ  F'1$ VHTXHQFH RI FDPHO PLON SHSWLGRJO\FDQ UHFRJQLWLRQ SURWHLQ 3*53 DQG FRUUHVSRQGLQJ SHSWLGH ZLWK PDWXUH SURWHLQ LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR $ DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 1XPEHULQJ RI WKH DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ



5HVXOWV DQG 'LVFXVVLRQ

D

&DPHO 3LJ +XPDQ 0RXVH 0RWK 6LONZRUP &DPHO +XPDQ 0RXVH 0RWK &DPHO +XPDQ 0RXVH 0RWK &DPHO +XPDQ 0RXVH 0RWK

075+&9//9:$//$//6/*$$5

('33$&*6,9355(:5$/$6(&5(5/ *3$1,3,9655(:*$/$6( 6$1/ 065560//$:$/36//5/*$$4(7('3$&&63,9351(:.$/$6(&$4+/ 0/)$&$// $//*/$7 6&6),9356(:5$/36(&665/ 0(,/)9/))9)9796*' &* 997.'(:'*/73,+9(
   

75395<9996+7$*6+&'73$6&$44$41946<+951/*:&'9*<1)/,*  6/3/5<9996+7$*66&173$6&444$5194+<+0.7/*:&'9*<1)/,*  *+395<99,6+7$*6)&163'6&(44$5194+<+.1(/*:&'9$<1)/,*  $539(/9,,4+79767&17'$$&$4,951,46<+0'1/1<:',*66),,*  ('*/9<(*5*:1,.*$+$*37:13,6,*,6)0*1<01593335$/5$$41 ('*/9<(*5*:1)7*$+6*+/:1306,*,6)0*1<0'593734$,5$$4* ('*+9<(*5*:1,.*'+7*3,:1306,*,7)0*1)0'593$.5$/5$$/1 *1*.9<(*$*:/+9*$+7<* <15.6,*,7),*1<11'.374.6/'$/5$ //$&*9$/*$/561<(9.*+5'9437/63*'5/<(,,47:6+<5$ //$&*9$4*$/561<9/.*+5'9457/63*14/<+/,41:3+<563 //(&*965*)/561<(9.*+5'9467/63*'4/<49,46:(+<5( //5&*9(5*+/7$1<+,9*+54/,67(63*5./<1(,55:'+)/'1 075+&9//9:$//$//6/*$$5('33$&*6,9355(:5$/$6(&5(5/75 $.94).35$7 7( $594).45(6 7' *:&'9*<1)/ 395<9996+7$*6+&'73$6&$44$41946<+951/ +& 6$7.36419*95(,54:+.(4*:/'9*<+), $, )9 +& 6$7.36419*95(,54:+.(4*:/'9*<+), $, )9              

E

&DPHO 7 7 &DPHO 7 7 &DPHO 7 7 +LQIOXHQ]DH &DPHO 7 7 +LQIOXHQ]DH &DPHO 7 7 +LQIOXHQ]DH

,*('*/9<(*5*:1,.*$+$*37:13,6,*,6)0*1<01593335$/5$$  ,.5'*79($*5'(/$9*6+$ .*<1+16,*9&/9**,''.*.)'$1)73$  ,.5'*79($*5'(0$9*6+$ .*<1+16,*9&/9**,''.*.)'$1)73$  '*69*7*549*(,*$+9 .*+14169*,&/9**,7$6*.1+*(<7($  4 1//$&*9$/*$/561<( 9.*+5'9437/  4046/56// 97//$ .<(*69/5$++'9$3 .  4046/56// 97//$ .<(*$9/5$++(9$3 .  4:46/<.// 4(/($ (+3.$/,&*+5'/63'/1*'*9,73.(:/.  63* ' 5/<( ,,47:6+<5$ $&36)'/.5::(.1(/976'5* $&36)'/.5::(.1(/976'5* '&3&)'9:6:/'6(4991/'+/<.(    

)LJ  6HTXHQFH DOLJQPHQW RI SHSWLGRJO\FDQ UHFRJQLWLRQ SURWHLQV DQG UHODWHG SURWHLQV D 3*53 SURWHLQV IURP &DPHO &DPHOXV GURPHGDULXV 3LJ 6XV VFRUID 0DQ +RPR VDSLHQV 0RXVH 0XV PXVFXOXV 0RWK 7ULFKRSOXVLD QL DQG 6LONZRUP %RPE\[ PRUL 3RVLWLRQV ZLWK FRQVHUYHG DPLQR DFLGV DUH GDUN VKDGHG 3RVLWLRQV ZLWK DPLQR DFLGV RQO\ FRQVHUYHG LQ YHUWHEUDWHV DUH OLJKW VKDGHG E 6LPLODULW\ RI FDPHO 3*53 WR UHODWHG 1 DFHW\OPXUDPR\O/DODQLQH DPLGDVHV IURP 7 DQG 7 YLUXVHV DQG IURP +DHPRSKLOXV LQIOXHQFDH

 WUDQVODWLRQDO VWDUW VLWH $7* FRQVLVWHG RI D SXULQH DW آ± ES DQG F\WRVLQHV DW  ES  ES  ES DQG ES ([SUHVVLRQ RI WKH PXULQH KRPRORJXH RI FDPHO 3*53 ZDV UHSRUWHG WR EH UHJXODWHG DW WKH SRVWWUDQVFULSWLRQDO OHYHO .LVHOHY HW DO  7KH آ¶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 7KH  DD VLJQDO VHTXHQFH KDG  VLJQDO VHTXHQFH VLPLODULW\ ZLWK WKH VLJQDO VHTXHQFH RI KXPDQ 3*53 EXW GLG QRW UHYHDO VLJQLILFDQW VLPLODULWLHV WR PRXVH DQG PRWK 3*53 VLJQDO VHTXHQFHV 7KH PDWXUH SURWHLQ KDG  VLPLODULW\ ZLWK KXPDQ 3*53  ZLWK PXULQH 3*53 DQG  ZLWK PRWK 3*53 :HDNHU VLPLODULWLHV ZHUH FDOFXODWHG IRU 7 DQG 7 1DFHW\OPXUDPR\O/DODQLQH DPLGDVHV ZKLFK ZHUH   DQG   UHVSHFWLYHO\  UHVLGXHV ZHUH FRQVHUYHG LQ DOO SURWHLQV VWXGLHG )LJ  &DPHO 3*53 SURWHLQ KDG D KLJK FRQWHQW RI VPDOO DD UHVLGXHV RI  DQG ZDV ULFK LQ DUJLQLQH EXW SRRU LQ O\VLQH DOWKRXJK WKH LVRHOHFWULF SRLQW ZDV KLJKO\ EDVLF 7KH DUJLQLQH UHVLGXHV ZHUH ZHDNO\ FRQVHUYHG DPRQJ 3*53 IURP GLIIHUHQW VSHFLHV 0XULQH 3*53 ZDV UHSRUWHG WR H[LVW LQ WZR PDMRU IRUPV DV D PRQRPHU DQG DV WULPHU .LVHOHY HW DO  $ WULPHULF SURWHLQ PD\ KDYH KLJKHU DIILQLW\ WR WDUJHW VLWHV RQ EDFWHULDO RU HXFDU\RWLF FHOO ZDOOV DQG HYHQ EH DEOH WR DJJOXWLQDWH FHOOV 7KH XQHYHQ QXPEHU RI F\VWHLQHV LQ PXULQH DQG KXPDQ 3*53 LQGLFDWHV SRVVLELOLW\ RI FRYDOHQW LQWHUPROHFXODU FURVVOLQNLQJ 0DVV GHWHUPLQDWLRQ RI FDPHO 3*53 ZDV GRQH E\ 0$/',06 DQG 6'63$*( %RWK PHWKRGV RQO\ JDYH LQGLFDWLRQ IRU D PRQRPHULF SURWHLQ $IILQLW\ &KURPDWRJUDSK\ DQG 4XDQWLILFDWLRQ 7KH SHSWLGRJO\FDQ UHFRJQLWLRQ SURWHLQ 3*53 ZDV LVRODWHG IURP FDPHO ZKH\ E\ KHSDULQVHSKDURVH FKURPDWRJUDSK\ RI D PLON VDPSOH WDNHQ DW WKH HQG RI WKH ODFWDWLRQ SHULRG DERXW  GD\V DIWHU SDUWXULWLRQ $ VLQJOH EDQG DV MXGJHG E\ 6'63$*( ZDV REWDLQHG E\ HOXWLRQ IURP  0 1D&O WR  0 1D&O )LJ  SHDN , +HSDULQ ELQGLQJ SURWHLQV IURP ERYLQH ZKH\ ZHUH HOXWHG XQGHU WKH VDPH FRQGLWLRQV +HSDULQ LV D KLJKO\ VXOIDWHG JO\FRVDPLQRJO\FDQ DWWDFKHG WR D FRUH SHSWLGH ZLWK 6HU*O\ DQG SURGX



5HVXOWV DQG 'LVFXVVLRQ



D

,













,,















    



5HWHQWLRQ 7LPH >PLQ@









 

E

























        



5HWHQWLRQ 7LPH >PLQ@

)LJ  +HSDULQ DIILQLW\ FKURPDWRJUDSK\ RI D FDPHO DQG E FRZ PLON ZKH\ 3HDNV , DQG ,, ZHUH FROOHFWHG IRU IXUWKHU DQDO\VLV 3HDN , FRQVLVWHG RI 3*53 DQG SHDN ,, RI ODFWRIHUULQ *UDGLHQW RI VROYHQW % DV GDVKHG OLQH

&RQFHQWUDWLRQ RI VROYHQW % PO O

$ >$8@

&RQFHQWUDWLRQ RI VROYHQW % PO O

$ >$8@

 FHG E\ PDVW FHOOV ZKHUH LW LV VWRUHG LQ VHFUHWRU\ JUDQXOHV DQG UHOHDVHG XSRQ LPPXQRORJLFDO VWLPXODWLRQ ,W LV RQO\ LQ D IHZ FDVHV WKH QDWXUDO VXEVWUDWH RI SURWHLQV ERXQG E\ KHSDULQVHSKDURVH DIILQLW\ FKURPDWRJUDSK\ 0RVW SURWHLQV ELQG WR UHODWHG JO\FRVDPLQRJO\FDQV HJ WR H[WUDFHOOXODU PDWUL[ DVVRFLDWHG RU PHPEUDQH ERXQG KHSDUDQVXOIDWH SHSWLGRJO\FDQV 9ORGDYVN\ HW DO  (OXWLRQ IURP WKH KHSDULQVHSKDURVH FROXPQ ZDV DW D LRQLF VWUHQJWK ZKLFK ZDV KLJKHU WKDQ LQ D SK\VLRORJLFDO VRGLXP FKORULGH VROXWLRQ  0 1D&O ,W ZDV WKHUHIRUH FRQFOXGHG WKDW 3*53 ERXQG VSHFLILFDOO\ WR KHSDULQ $ SURWHLQ ZLWK VLPLODU FKDUDFWHULVWLFV DV 3*53 ZDV QRW LVRODWHG IURP ERYLQH PLON E\ KHSDULQ FKURPDWRJUDSK\ LQ WKH SUHVHQW VWXG\ DQW QRW GHVFULEHG LQ WKH OLWHUDWXUH 7KH H[WLQFWLRQ RI 3*53 ZDV UHFRUGHG DW  QP XVLQJ DQ H[WLQFWLRQ FRHIILFLHQW RI  0 FP &DOFXODWHG FRQFHQWUDWLRQ RI WKH SURWHLQ LQ WKH PLON DQDO\VHG ZDV  PJ O )XQFWLRQDO $VSHFWV RI 3*53 7KH DQWLPLFURELDO V\VWHP RI PLON FRQWDLQV FRPSRQHQWV ZKLFK DUH LPSRUWHG IURP EORRG VHUXP VXFK DV GLIIHUHQW W\SHV RI LPPXQRJOREXOLQV DQG FRPSRQHQWV SURGXFHG LQ WKH ODFWDWLQJ XGGHU VXFK DV 0)*0 ERXQG PXFLQV ODFWRIHUULQ ODFWRSHUR[LGDVH DQG O\VR]\PH :KHUHDV WKH FRQFHQWUDWLRQ RI EORRG VHUXP GHULYHG DQWLPLFURELDO SURWHLQV LV HOHYDWHG LQ PLON IURP LQIHFWHG XGGHU FRQFRPLWDQWO\ ZLWK DQ HOHYDWHG OHXNRF\WH OHYHO WKH FRQFHQWUDWLRQ RI DQWLPLFURELDO SURWHLQV ZKLFK RULJLQDWH IURP DOYHRODU FHOOV LQ WKH PDPPDU\ JODQG LV KRUPRQDOO\ UHJXODWHG DQG XVXDOO\ GHSHQGV RQ WKH VWDJH RI ODFWDWLRQ 7KH FRQFHQWUDWLRQV RI O\VR]\PH DQG ODFWRIHUULQ LQ FDPHO PLON ZHUH IRXQG WR GHFUHDVH UDSLGO\ ZLWKLQ WKH ILUVW PRQWKV RI ODFWDWLRQ %DUERXU HW DO  $EG (O*DZDG HW DO 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 3URWHLQV ZKLFK SDUWLFLSDWH LQ WKH LQQDWH KXPRUDO LPPXQH V\VWHP DUH VXSSRVHG WR UHFRJQLVH FRUH VWUXFWXUHV RI WKH PROHFXOHV ZKLFK DUH KLJKO\ FRQVHUYHG EHWZHHQ VSHFLHV 7KH LGHQWLILFDWLRQ RI QRQVHOI PROHFXOHV E\ FRPPRQ VWUXFWXUHV W\SLFDO IRU WKH PROHFXOH FODVV ZDV WHUPHG SDWWHUQUHFRJQLWLRQ .DQJ HW DO  3*53 RI VLONZRUP ZDV VKRZQ WR UHFRJQLVH PXOWLSOH UHSHDWLQJ XQLWV RI WKH JO\FDQ SRUWLRQ RI SHSWLGRJO\FDQV EXW QRW خ²JOXFDQ



5HVXOWV DQG 'LVFXVVLRQ

FKLWLQ DQG WKH GLJO\FRVLGLF FRUH VWUXFWXUH RI SHSWLGRJO\FDQV 
 WKH XQGHUO\LQJ EDVDO ODPLQD %DVHG RQ WKH ILQGLQJ WKDW FDPHO 3*53 VSHFLILFDOO\ ERXQG WR KHSDULQ ZH VXJJHVW WKDW 3*53 PD\ DGKHUH WR WKH H[WUDFHOOXODU PDWUL[ RI HQGRWKHOLDO FHOOV 7KH PDLQ FRPSRQHQWV RI WKLV PDWUL[ DUH FROODJHQV ODPLQLQ ILEURQHFWLQ HODVWLQ KHSDUDQ VXOIDWH SURWHRJO\FDQV GHUPDWDQ VXOIDWH SURWHRJO\FDQV DQG VPDOOHU DPRXQWV RI FKRQGURLWLQ VXOIDWH SURWHRJO\FDQV 9ORGDYVN\ HW DO 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 7KH SURWHLQ SUREDEO\ ZDV UHFRJQLVHG E\ D VSHFLILF UHFHSWRU ,QGXFWLRQ RI FHOO GHDWK ZDV LQGHSHQGHQW RI 71)LQGXFHG DSRSWRVLV DQG UHVXOWHG LQ FRQVLGHUDEOH '1$ IUDJPHQWDWLRQ 7KH IXQFWLRQV RI 3*53 DV D F\WRNLQH DQG DV DQ DSRSWRVLV LQGXFLQJ IDFWRU PD\ EH UHODWHG DQG DQ LPPLQHQW UROH RI WKH SURWHLQ LQ WXPRXU JURZWK ZDV UHSRUWHG .LVHOHY HW DO  3*53 F'1$ ZDV DOVR IRXQG LQ LUUDGLDWHG FRORQ WLVVXH 0DUUD HW DO (0%/ ,' $$ XQSXEOLVKHG 3*53 LV VHFUHWHG IURP FHOOV RI WKH O\PSK V\VWHP DQG IURP H[RFULQH JODQGV LQ D VLPLODU IDVKLRQ DV O\VR]\PH & ZKLFK LV DQ 1DFHW\OJOXFRVDPLQHخ²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 6HTXHQFH VLPLODULW\ EHWZHHQ 3*53 DQG D JRXS RI WKUHH 1 DFHW\OPXUDPR\O/DODQLQH DPLGDVHV GHVLJQDWHG DV 7 7 DQG +DHPRSKLOXV LQIOXHQ]DH O\VR]\PH GHPRQVWUDWHG D FRPPRQ RULJLQ RI WKHVH SURWHLQV ,Q FRQWUDVW WR WKH 3*53 IDPLO\ WKLV YLUDO DQG EDFWHULDO O\VR]\PH IDPLO\ SURYHG WR K\GURO\VH PXUDPR\OSHSWLGH ERQGV ,QRX\H HW DO  7KH UHDFWLRQ FHQWUH FRQWDLQHG D ]LQF LRQ DQG ZDV KLJKO\ FRQVHUYHG =LQF



5HVXOWV DQG 'LVFXVVLRQ

ELQGLQJ UHVLGXHV +LV 7\U +LV DQG &\V ZHUH RQO\ SDUWLDOO\ FRQVHUYHG LQ 3*53 )LJ 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 2Q WKH RWKHU KDQG %LILGREDFWHULXP ORQJXP  ZDV UHSRUWHG WR JURZ IDVWHU LQ FDPHO PLON WKDQ LQ FRZ PLON ZKHUHDV JURZWK RI RWKHU %LILGREDFWHULXP VWUDLQV ZDV UHWDUGHG $EX 7DUERXVK 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


    F'1$ $*7&*&&7&$**$&&&&$*$&$7*$$*&7&77&77&&&&*&&&7*&7*7&&&7&****&&&77**$&7*7*7&7* 3URWHLQ 0HW/\V/HX3KH3KH3UR$OD/HX/HX6HU/HX*O\$OD/HX*O\/HX&\V/HX       F'1$ *&7*&&7&7$$*$$$$*7*77&*$7**7*&$&&$&$7&$&&$*&$*$*7&*7&$$$$7*7*&&&$$7**&$$&** 3URWHLQ $OD$OD6HU/\V/\V6HU9DO$UJ7US&\V7KU7KU6HU3UR$OD*OX6HU6HU/\V&\V$OD*OQ7US*OQ$UJ    F'1$ $**$7*$$$$$$*7*&*7**7&&&7&7*7&$&&7*&*7$$$*$$*$&$7&7&*&777*$$7*&$7&&$**&&$7& 3URWHLQ $UJ0HW/\V/\V9DO$UJ*O\3UR6HU9DO7KU&\V9DO/\V/\V7KU6HU$UJ3KH*OX&\V,OH*OQ$OD,OH     F'1$ 7&*$&$*$$$$**&$*$7*&7*7*$&&&77*$&**7**777**7*7$7*$&*&$**&&7**$&&&&7$&$$*&7* 3URWHLQ 6HU7KU*OX/\V$OD$VS$OD9DO7KU/HX$VS*O\*O\/HX9DO7\U$VS$OD*O\/HX$VS3UR7\U/\V/HX     F'1$ &**&&*$7$*&**&$*$**7&7$7***$&$*$$$$&$$7&&&&$$$&&&$&7$77$7*&&*77*&&$77*&&$$$ 3URWHLQ $UJ3UR,OH$OD$OD*OX9DO7\U*O\7KU*OX$VQ$VQ3UR*OQ7KU+LV7\U7\U$OD9DO$OD,OH$OD/\V









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



5HVXOWV DQG 'LVFXVVLRQ

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

)LJ  F'1$ VHTXHQFH RI FDPHO PLON ODFWRIHUULQ DQG FRUUHVSRQGLQJ SURWHLQ ZLWK PDWXUH SURWHLQ LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR * DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 1XPEHULQJ RI WKH DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ *O\ UHVLGXH ZLWK JO\FRV\ODWLRQ SRWHQWLDO

 /DFWRIHUULQ EHORQJV WR WKH IDPLO\ RI WUDQVIHUULQV WRJHWKHU ZLWK EORRG VHURWUDQVIHUULQ VLGHURSKLOLQ HJJ ZKLWH RYRWUDQVIHUULQ FRQDOEXPLQ PHODQRWUDQVIHUULQ RI PDOLJQDQW PHODQRPV WKH SRUFLQH LQKLELWRU RI FDUERQLF DQK\GUDVH DQG RWKHU SURWHLQV 7KH FRPPRQ SURSHUW\ RI WKLV SURWHLQ IDPLO\ LV WKH ELQGLQJ RI WZR PHWDO FDWLRQV SUHIHUDEO\ )H DW VWUXFWXUDOO\ FORVHO\ UHODWHG ELQGLQJ VLWHV 0RVW SURWHLQV RI WKH WUDQVIHUULQ W\SH DUH QHHGHG IRU VWRUDJH RU WUDQVSRUW RI LURQ /DFWRIHUULQ ZDV GLVFXVVHG WR VHUYH IRU LURQ VFDYHQJLQJ LQ ERG\ VHFUHWLRQV %URFN  ,W LV IRXQG LQ PLON DQG GLIIHUHQW RWKHU ERG\ VHFUHWLRQV DQG LQ QHXWURSKLO OHXNRF\WHV 0DVVRQ  3ULPDU\ 6WUXFWXUH 3&5 DPSOLILFDWLRQ SURGXFWV RI D IXOO OHQJWK F'1$ FORQH RI FDPHO ODFWRIHUULQ ZHUH VHTXHQFHG )LJ  (0%/*HQ%DQNâ„¢ DFFHVVLRQ QXPEHU $- 7KH FORQH ZDV  ES ORQJ DQG FRQWDLQHG D آ¶XQWUDQVODWHG UHJLRQ RI  ES DQG D آ¶XQWUDQVODWHG UHJLRQ RI  ES 7KH آ¶XQWUDQVODWHG UHJLRQ FRQWDLQHG D SDUWLDO .R]DNER[ .R]DN  LQ IURQW RI WKH WUDQVODWLRQDO VWDUW VLWH $7* ZLWK D SXULQH DW آ± ES DQG F\WRVLQHV DW آ± ES  ES DQG آ± ES 7KH آ¶XQWUDQVODWHG UHJLRQ FRQWDLQHG D SRO\DGHQ\ODWLRQ VLJQDO $$7$$$ 7KH RSHQ UHDGLQJ IUDPH UDQJHG IURP $ WR * DQG FRGHG IRU D SRO\SHSWLGH RI  DD UHVLGXHV 7KH VWDUW VLWH RI WKH PDWXUH SURWHLQ ZDV GHWHUPLQHG E\ VLPLODULW\ DV $OD 7KH  DD VLJQDO SHSWLGH KDG  VHTXHQFH VLPLODULW\ WR WKH VLJQDO VHTXHQFH RI ERYLQH ODFWRIHUULQ DQG  WR WKH VLJQDO VHTXHQFH RI KXPDQ ODFWRIHUULQ 0DWXUH ODFWRIHUULQ ZDV  DD UHVLGXHV ORQJ DQG WKH XQPRGLILHG SHSWLGH KDG D PROHFXODU ZHLJKW RI  N'D 7DEOH  7KH LVRHOHFWULF SRLQW RI WKH XQPRGLILHG SHSWLGH ZDV DW S+  7KH SURWHLQ VKDUHG  VHTXHQFH VLPLODULW\ ZLWK ERYLQH DQG ZLWK KXPDQ ODFWRIHUULQ DQG  ZLWK SRUFLQH ODFWRIHUULQ +LJK VLPLODULW\ LQ SULPDU\ VWUXFWXUHV LQGLFDWHG WKDW WKHUH ZHUH RQO\ VPDOO YDULDWLRQV LQ IXQFWLRQDO DVSHFWV *O\FRV\ODWLRQ 1OLQNHG JO\FDQV FRQWULEXWH DERXW  WR   WR  N'D WR WKH WRWDO PDVV RI ERYLQH ODFWRIHUULQ ZKLFK LV DERXW  N'D 6SLN HW DO  *O\FRV\ODWLRQ HQKDQFHV WKH VROXELOLW\ RI WKH VHFUHWHG SURWHLQ DQG PD\ KHOS WR ELQG DW VSHFLILF FHOO W\SHV VXFK DV OLYHU SDUHQFK\PDO FHOOV =LHUH HW DO  &DPHO PLON ODFWRIHUULQ ZDV IRXQG WR FRQWDLQ  FDUERK\GUDWHV LQ FRORVWUDO PLON DQG  LQ PLON FROOHFWHG  WR  GD\V DIWHU SDUWXULWLRQ 0DKIRX] HW DO  7KH FRQWHQW RI 1DFHW\OJOXFRVDPLQH LQ FDPHO PLON ODFWRIHUULQ ZDV PDUNHGO\ KLJKHU WKDQ LQ UXPLQDQWVآ¶ PLON ODFWRIHUULQV  LQ FRORVWUDO FDPHO PLON FRPSDUHG WR DERXW  LQ FRORVWUDO UXPLQDQWVآ¶ PLON 7KH



5HVXOWV DQG 'LVFXVVLRQ

FDUERK\GUDWH FRQWHQW RI ODFWRIHUULQ IURP HQGODFWDWLRQDO PLON ZDV    RI WRWDO SURWHLQ PDVV FDOFXODWHG DV D GLIIHUHQFH EHWZHHQ WKH SURWHLQ PDVV PHDVXUHG E\ 0$/',06 DQG WKH SURWHLQ PDVV RI WKH DPLQR DFLG VHTXHQFH 7DEOH  3RVVLEOH JO\FRV\ODWLRQ VLWHV EDVHG RQ SDWWHUQ DQDO\VLV *DYHO 9RQ +HLMQH  DUH $VQ $VQ $VQ DQG $VQ ,Q ERYLQH ODFWRIHUULQ WKUHH RI ILYH VLWHV ZLWK 1JO\FRV\ODWLRQ SRWHQWLDO $VQ $VQ DQG $VQ DUH JO\FRV\ODWHG DQG FRQWULEXWH WR DQ RYHUDOO FDUERK\GUDWH FRQWHQW RI   *O\FRV\ODWLRQ RI ERYLQH ODFWRIHUULQ ZDV RQO\ IRXQG DW WKH &OREH 'HJUHH RI JO\FRV\ODWLRQ LQ KXPDQ ODFWRIHUULQ LV DERXW  6SLN HW DO  DQG WKXV VLPLODU WR FDPHO ODFWRIHUULQ +XPDQ ODFWRIHUULQ FRQWDLQV  JO\FRV\ODWHG VLWHV $VQ DQG $VQ ZLWK JO\FDQV RI WKH 1 DFHW\OODFWRVDPLQLF W\SH ZKLFK ZHUH DOVR IRXQG LQ FDPHO ODFWRIHUULQ %\ FRPSDULVRQ ZLWK ERYLQH DQG KXPDQ ODFWRIHUULQ JO\FRV\ODWLRQ RI $VQ DQG $VQ RU $VQ LQ FDPHO ODFWRIHUULQ LV SURSRVHG )LJ  &RQFHQWUDWLRQ LQ &DPHO 0LON &RORVWUDO FDPHO PLON ZDV UHSRUWHG WR KDYH DQ H[WUHPHO\ KLJK ODFWRIHUULQ FRQWHQW RI  J O RQ WKH VHFRQG GD\ DIWHU SDUWXULWLRQ FRPSDUHG WR DERXW  J O LQ ERYLQH FRORVWUDO PLON $IWHU  GD\V RI PLONLQJ WKH ODFWRIHUULQ OHYHO LQ FDPHO PLON ZHQW GRZQ WR  J O ZKHUHDV LQ ERYLQH PLON RQO\ DERXW  J O ZHUH IRXQG $EG (O*DZDG HW DO 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 7HUWLDU\ 6WUXFWXUH DQG /LJDQG %LQGLQJ 7UDQVIHUULQV DUH PRQRPHULF VWUXFWXUDOO\ KLJKO\ FRQVHUYHG SURWHLQV QHDUO\ LGHQWLFDO LQ VHFRQGDU\ DQG WHUWLDU\ VWUXFWXUH DQG ZLWK KLJK KRPRORJ\ RI GLVXOSKLGH ERQGLQJ 6FKDQEDFKHU HW DO  &DPHO DQG ERYLQH ODFWRIHUULQ

&DPHO %RYLQH

&OXVWHUHG EDVLF UHVLGXHV LURQ bbbbb aaaaaaaaaaaaaa bbbbbbbb aaaaaaaaaa bbbaaaaaaaa b $6..695:&7763$(66.&$4:4550..95*3697&9..765)(&,4$,67(.$'$97/'**/9<'$*/'3<./5 $35.195:&7,643(:).&55:4:50../*$36,7&955$)$/(&,5$,$(..$'$97/'**09)($*5'3<./5 

&DPHO %RYLQH

LURQ FDUERQDWH FDUERQDWH bbbbbbbbb bbb bbbbbbbb aaaaaaaaaaa aaaaa 3,$$(9<*7(11347+<<$9$,$..*71)4/14/4*/.6&+7*/*56$*:1,30*//53)/':7*33(3/4.$9$ 39$$(,<*7.(6347+<<$9$99..*61)4/'4/4*5.6&+7*/*56$*:,,30*,/53


&DPHO %RYLQH

LURQ aa bbb bbb aaaaaaaaaa bbbbaaaaaaa aaaa .))6$6&93&9'*.(<31/&4/&$*7*(1.&$&664(3<)*<6*$).&/4'*$*'9$)9.'679)(6/3$.$'5' .))6$6&93&,'54$<31/&4/&.*(*(14&$&665(3<)*<6*$).&/4'*$*'9$)9.(779)(1/3(.$'5'






&DPHO %RYLQH

LURQ abbbb bbb bbbbb aaaaaaaaaaaaaaa 4<(//&31175.39'$)4(&+/$5936+$99$5691*.('/,:.//9.$4(.)*5*.36*)4/)*63$*4.'//) 4<(//&/1165$39'$).(&+/$4936+$99$569'*.('/,:.//6.$4(.)*.1.656)4/)*633*45'//)

&DPHO %RYLQH

+LQJH 5HJLRQ bbbbb aaaaaaaaaaaaaaaaaaaaaaaaaaaabbbbbbbaaaaaaaaaaaaaa bbbbbb .'6$/*//5,66.,'6*/


&DPHO %RYLQH

LURQ LURQ aaaaaaaaaaa bbbaaaaaaaaa bbbbbbb bbbbbbbb 77('&,$/9/.*($'$/6/'**<,<,$*.&*/939/$(64463(66*/'&9+539.*
&DPHO %RYLQH

FDUERQDWH bbbb aaaaaaaaaaaa bbb aaaaa a /5*..6&+7$9'57$*:1,30*//6.17'6&5)'()/646&$3*6'356./&$/&$*1((*41.&93166(5<<* /.'..6&+7$9'57$*:1,30*/,9147*6&$)'())646&$3*$'3.65/&$/&$*''4*/'.&9316.(.<<*



&DPHO %RYLQH

LURQ LURQ aaaaaaaaa bbbbbaaaaaaa aaaaa bbbbbb bbb bbb bbbbb <7*$)5&/$(19*'9$)9.'979/'17'*.17(4:$.'/./*')(//&/1*75.397($(6&+/$9$31+$9965 <7*$)5&/$('9*'9$)9.1'79:(171*(67$':$.1/15(')5//&/'*75.397($46&+/$9$31+$9965






&DPHO %RYLQH

aaaaaaaaaaaaaaaa bbb aaaaaaaaaaaaaaaaa ,'.9$+/(49//544$+)*51*5'&3*.)&/)46.7.1//)1'17(&/$./4*.77<((
&DPHO %RYLQH

aaaaaaaaaa 6763//($&$)/05 6763//($&$)/75



5HVXOWV DQG 'LVFXVVLRQ

)XQFWLRQDO UHVLGXHV LQ EROG خ±KHOLFDO UHJLRQV GHVLJQDWHG DV آ³خ±آ´ خ²SOHDWHG UHJLRQV DV آ³خ²آ´

)LJ  6FKHPDWLF GUDZLQJ RI WKH UHODWLRQVKLS EHWZHHQ VWUXFWXUH DQG IXQFWLRQ LQ FDPHO DQG ERYLQH ODFWRIHUULQ



7KU

*O\

$UJ 6HU $OD 7\U &2 )H $VS

+LV

7\U

)LJ  /LJDQGELQGLQJ FHQWUH RI ERYLQH ODFWRIHUULQ )H LQ VSDFH ILOOLQJ YLHZ &2 DQG FRRUGLQDWHG DPLRQ DFLG UHVLGXHV DV ZLUH IUDPH +\GURJHQ ERQGV DV GDVKHG OLQHV 'DWD IURP 0RRUH HW DO  VKDUHG  DQG  VHTXHQFH VLPLODULW\ UHVSHFWLYHO\ ZLWK ERYLQH VHURWUDQVIHUULQ $OO WUDQVIHUULQV KDYH D SRO\SHSWLGH FKDLQ RI DERXW  DPLQR DFLGV ZKLFK LV IROGHG LQWR WZR WDQGHPO\ DUUDQJHG DV\PPHWULFDO PHWDO ELQGLQJ VLWHV GHVLJQDWHG DV 1 DQG &OREHV 7KHVH OREHV DUH FRQQHFWHG E\ D KHOLFDO KLQJH ZKLFK LQ FDPHO ODFWRIHUULQ H[WHQGV IURP



5HVXOWV DQG 'LVFXVVLRQ

*OX WR *OQ )LJ  7KH VHTXHQFH RI WKH 1OREH ZKLFK H[WHQGV IURP 9DO WR $UJ VKDUHV  VHTXHQFH VLPLODULW\ ZLWK WKH VHTXHQFH RI WKH & OREH ZKLFK UDQJHV IURP 9DO WR $UJ SUREDEO\ DV D UHVXOW RI JHQH GXSOLFDWLRQ 7KH WZR OREHV ZHUH UHSRUWHG WR ELQG LURQ V\QHUJLVWLFDOO\ EXW WKH &OREH ZDV IRXQG WR KDYH D ORZHU GLVVRFLDWLRQ FRQVWDQW WKDQ WKH 1OREH %DNHU HW DO  %RWK OREHV DUH IROGHG LQWR WZR GRPDLQV ZKLFK IRUP D FOHIW ZKHUH WKH PHWDO FDWLRQ LV ERXQG 8QGHU SK\VLRORJLFDO FRQGLWLRQV WUDQVIHUULQV ELQG WZR )H FDWLRQV ZLWK ORZ GLVVRFLDWLRQ FRQVWDQWV RI DERXW  %URFN  &DWLRQ ELQGLQJ UHTXLUHV V\QHUJLVWLF ELQGLQJ RI D ELFDUERQDWH DQLRQ SUREDEO\ IRU FKDUJH FRPSHQVDWLRQ ,Q ERYLQH DQG FDPHO ODFWRIHUULQ WKH VLGH FKDLQV RI $VS 7\U 7\U DQG +LV ZHUH IRXQG WR EH LQYROYHG LQ ELQGLQJ RI WKH FDWLRQ LQ WKH 1OREH )LJ  7ZR R[\JHQV IURP WKH ELGHQWDWH &2 DQLRQ DUH VXJJHVWHG WR FRPSOHWH D GLVWRUWHG RFWDKHGUDO JHRPHWU\ $QGHUVRQ HW DO 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 7KH ELQGLQJ VLWHV RI ERYLQH DQG FDPHO ODFWRIHUULQ ZHUH IRXQG WR EH KLJKO\ VLPLODU &RQVLGHULQJ WKH QHDUO\ LGHQWLFDO VWUXFWXUDO JHRPHWU\ ZH DVVXPH WKDW FDWLRQV DUH ERXQG E\ ERWK 1 DQG &OREH RI WKH FDPHO SURWHLQ ZLWK VLPLODU DIILQLWLHV DV LQ ERYLQH ODFWRIHUULQ %DFWHULRVWDWLF $FWLYLW\ RI WKH 1WHUPLQDO (QG $ KLJK DPRXQW RI $UJ DQG /\V DUH FOXVWHUHG DW WKH 1WHUPLQDO HQG RI ODFWRIHUULQ QHDU DQG EHWZHHQ D ORRS ZKLFK LV IRUPHG E\ GLVXOSKLGH ERQGLQJ RI &\V DQG &\V )LJ 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 7KHVH ELQGLQJ DIILQLWLHV ZHUH HTXDOO\ KLJK IRU GLIHUULF ODFWRIHUULQ DQG LURQ GHSOHWHG DSRODFWRIHUULQ 7KH 1WHUPLQDO SDUW RI FDPHO ODFWRIHUULQ FRQWDLQHG  EDVLF UHVLGXHV )LJ  DW VLWHV PRUH VLPLODU WR ERYLQH ODFWRIHUULQ WKDQ WR KXPDQ ODFWRIHUULQ 7KH LVRHOHFWULF SRLQW RI WKH 1WHUPLQDO IUDJPHQW IURP

 $OD WR $OD ZDV DW S+  FRPSDUHG WR S+  IRU WKH ERYLQH IUDJPHQW DW WKH VDPH SRVLWLRQ ZKLFK KDG RQH PRUH EDVLF UHVLGXH $V LQ ERYLQH ODFWRIHUULQ PRVW UHVLGXHV ZHUH IRXQG LQ RU QHDU WKH ORRS %DVHG RQ WKH KLJK KRPRORJ\ EHWZHHQ FDPHO DQG ERYLQH ODFWRIHUULQ LQ JHQHUDO DQG SDUWLFXODUO\ RQ WKH VLPLODU JURXSLQJ RI WKH 1WHUPLQDO EDVLF UHVLGXHV ZH VXJJHVW D VLPLODU HIIHFW RI FDPHO ODFWRIHUULQ DV REVHUYHG RI ERYLQH ODFWRIHUULQ 7KH EDFWHULRVWDWLF DFWLYLW\ RI FDPHO ODFWRIHUULQ RQ GLIIHUHQW EDFWHULDO VWUDLQV ZDV IRXQG WR KDYH HTXDO VWUHQJWK DV WKH DFWLYLW\ RI ERYLQH ODFWRIHUULQ (ODJDP\ HW DO 

D

E

&\V ,OH $OD

&\V

3KH

$OD

)LJ  6SDFH ILOOLQJ YLHZ RI WKH ORRS IRUPHG E\ F\VWLQ QHDU WKH 1WHUPLQDO HQG RI ODFWRIHUULQ %DVLF UHVLGXHV GDUN VKDGHG D 0RGHO RI WKH ORRS UHJLRQ IURP FDPHO ODFWRIHUULQ E /RRS UHJLRQ RI ERYLQH ODFWRIHUULQ UHFRQVWUXFWHG IURP ;UD\ FU\VWDOORJUDSKLF GDWD 0RRUH HW DO  %DFWHULRVWDWLF %UHDNGRZQ 3URGXFWV (YHQ VWURQJHU DQWLEDFWHULDO DFWLYLW\ ZDV VKRZQ IRU SHSWLGHV RI ODFWRIHUULQ ZKLFK ZHUH SURGXFHG E\ DVSDUWLF SURWHDVH GLJHVWLRQ RU KHDW WUHDWPHQW DW ORZ S+ LPLWDWLQJ WKH FRQGLWLRQV LQ WKH VWRPDFK 7KH DQWLPLFURELDO SHSWLGHV ZHUH IRXQG WR FRUUHVSRQG WR WKH 1WHUPLQDO HQG RI ODFWRIHUULQ 7RPLWD HW DO  6DLWR HW DO  'LIIHUHQW 1WHUPLQDO DQWLPLFURELDO SHSWLGHV ZHUH

$35.195:&7,643(: ).&55:4:50../*$36,7&955$)$ /(&,5$ ,$(..$'$97/ ERYLQH ODFWRIHUULQ آƒآƒ آƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒ ODFWRIHUULFLQ % آƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒآƒآƒ آƒآƒ آƒآƒآƒ QG SHSWLGH

*5555694:&$ 9643($7.&)4:45105.95*3396&,.5'63,4&, 4$,$(15$'$97/ KXPDQ ODFWRIHUULQ آƒآƒآƒآƒآƒآƒآƒآƒآƒ آƒ آƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒآƒآƒآƒ آƒ ODFWRIHUULFLQ +



$3..*95:&9 ,67$(<6.&54:46.,55713,)&,55$63 7'&,5$ ,$$.5$'$97/'**/ SLJ ODFWRIHUULQ آƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒآƒ SURSRVHG ODFWRIHUULFLQ آƒآƒآƒآƒآƒآƒآƒآƒ آƒ آƒآƒ آƒآƒآƒ QG SHSWLGH

$6..695:&7763 $(66.&$4:4550..95*3697&9..765)(&, 4$,67(.$'$97/ FDPHO ODFWRIHUULQ آƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒ آƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒآƒ آƒآƒآƒآƒآƒآƒآƒآƒ آƒ SURSRVHG ODFWRIHUULFLQ 5HVXOWV DQG 'LVFXVVLRQ

)LJ  3ULPDU\ VWUXFWXUHV RI ODFWRIHUULQ IURP GLIIHUHQW VSHFLHV %HQHDWK WKH DPLQR DFLG VHTXHQFH RI WKH 1 WHUPLQDO HQG WKHUH DUH VFKHPDWLF GUDZLQJV RI ERYLQH DQG KXPDQ ODFWRIHUULFLQ DQG SURSRVHG VWUXFWXUHV RI SRUFLQH DQG FDPHO ODFWRIHUULFLQ $QWLPLFURELDO SHSWLGHV IRUPHG XSRQ JDVWULF GLJHVWLRQ GHVLJQDWHG E\ آƒ %DVLF UHVLGXHV LQ EROG 'LVXOSKLGH ERQGV LQGLFDWHG E\ FRQQHFWLQJ OLQHV QG SHSWLGHV ZKLFK DUH IRUPHG XSRQ GLJHVWLRQ DUH DOVR VKRZQ DOWKRXJK WKHVH SHSWLGHV H[KLELW ZHDNHU DQWLPLFURELDO DFWLYLWLHV WKDQ ODFWRIHUULFLQV

 REWDLQHG E\ GLJHVWLRQ RI KXPDQ DQG ERYLQH ODFWRIHUULQ VLQFH KXPDQ ODFWRIHUULQ GRHV QRW FRQWDLQ D K\GURSKRELF GLSHSWLGH VXLWHG IRU GLJHVWLRQ E\ DVSDUWLF SURWHDVHV EHWZHHQ &\V DQG &\V $QWLPLFURELDO SHSWLGHV ZHUH VWUXFWXUDOO\ VLPLODU WR ZHOO FKDUDFWHULVHG DQWLPLFURELDO SHSWLGHV VXFK DV PDJDLQLQV IURP IURJ VNLQ RU FHFURSLQV IURP WKH KHPRO\PSK RI LQVHFWV 7KH ERYLQH SHSWLGH GHVLJQDWHG DV ODFWRIHUULFLQ % )LJ  ZDV WZHOYH WLPHV PRUH HIIHFWLYH LQ WKH LQKLELWLRQ RI JUDPSRVLWLYH DQG JUDPQHJDWLYH EDFWHULDO JURZWK WKDQ XQGLJHVWHG ERYLQH DSRODFWRIHUULQ )XUWKHUPRUH ELILGREDFWHULD ZKLFK GRPLQDWH WKH QHRQDWDO LQWHVWLQDO PLFURIORUD ZHUH OHVV LQKLELWHG WKDQ PDQ\ RSSRUWXQLVWLF SDWKRJHQV /DFWRIHUULQ PD\ WKHUHIRUH KHOS WR SURPRWH WKH HVWDEOLVKPHQW RI ELILGREDFWHULD LQ WKH QHRQDWDO PLFURIORUD $ FDPHO ODFWRIHUULFLQ LV SURSRVHG LQ DQDORJ\ WR ERYLQH DQG KXPDQ ODFWRIHUULFLQ )LJ  EDVHG RQ SUREDEOH SHSVLQ FOHDYDJH VLWHV 7KLV ODFWRIHUULFLQ ZRXOG KDYH PRUH VLPLODULW\ WR KXPDQ ODFWRIHUULFLQ ZKHUH WKH 1WHUPLQXV LV QRW FOHDYHG RII VLQFH SUREDELOLW\ RI FOHDYDJH EHWZHHQ &\V DQG &\V RI FDPHO ODFWRIHUULQ LV ORZ *URZWK LQKLELWLRQ RI WKLV SHSWLGH ZDV DVVXPHG WR EH LQ WKH UDQJH RI KXPDQ ODFWRIHUULFLQ ZKHUH  PJ O RI SHSWLGH LV QHHGHG WR LQKLELW JURZWK RI ( FROL FRPSOHWHO\ UDWKHU WKDQ WR EH LQ WKH UDQJH RI ERYLQH ODFWRIHUULFLQ ZKHUH PHUH  PJ O DUH VXIILFLHQW IRU FRPSOHWH LQKLELWLRQ RI EDFWHULDO JURZWK 7RPLWD HW DO 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



5HVXOWV DQG 'LVFXVVLRQ

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 LV IRXQG LQ PLON WHDUV DQG VDOLYD ,W FRQWULEXWHV WR WKH QRQLPPXQH KRVW GHIHQFH V\VWHP H[HUWLQJ EDFWHULFLGDO

 DFWLYLW\ PDLQO\ RQ JUDPQHJDWLYH EDFWHULD ,W LV VXSSRVHG WKDW WKH PDLQ IXQFWLRQ LQ PLON LV WKH SURWHFWLRQ RI WKH XGGHU IURP PLFURELDO LQIHFWLRQV 8HGD HW DO  7KH ILQGLQJ WKDW ODFWRSHUR[LGDVH UHPDLQHG DFWLYH LQ WKH JDVWULF MXLFH IURP D QHZERUQ EHLQJ IDLUO\ UHVLVWDQW WRZDUGV SURWHRO\WLF GLJHVWLRQ DQG DFLGLF S+ OHG WR WKH DVVXPSWLRQ WKDW WKH HQ]\PH PD\ DOVR EH D NH\ SOD\HU LQ WKH GHIHQFH RI WKH VXFNOLQJ DQLPDOآ¶V LQWHVWLQDO V\VWHP IURP EDFWHULDO LQIHFWLRQ LQ WKH LQLWLDO VWDJHV RI OLIH 3DXO 2KOVVRQ  /DFWRSHUR[LGDVH ZDV DOVR UHSRUWHG WR KDYH D JHQHUDO JURZWKSURPRWLRQ DQG DQWLWXPRXU DFWLYLW\ 8HGD HW DO 


5HVXOWV DQG 'LVFXVVLRQ

LQ DOYHRODU FDPHO WLVVXH DQG H[KLELWV KLJK VHTXHQFH VLPLODULW\ WR ERYLQH ODFWRSHUR[LGDVH 8SRQ FRPSDULVRQ RI VWUXFWXUDO GDWD ZH FDQ H[SHFW WKDW WKH ZD\ RI DFWLRQ LQ DFWLYDWLRQ RI WKH ODFWRSHUR[LGDVH V\VWHP DQG LQ LQKLELWLRQ RI VHYHUDO PDLQO\ SDWKRJHQLF EDFWHULD ZLOO EH QHDUO\ LGHQWLFDO WR WKH ERYLQH HQ]\PH 3ULPDU\ 6WUXFWXUH 3&5 DPSOLILFDWLRQ SURGXFWV RI D FDPHO ODFWRSHUR[LGDVH F'1$ FORQH ZHUH VHTXHQFHG )LJ  (0%/*HQ%DQNâ„¢ DFFHVVLRQ QXPEHU $- 7KH FORQH ZDV  ES ORQJ DQG FRQWDLQHG D آ¶XQWUDQVODWHG UHJLRQ RI  ES DQG D آ¶XQWUDQVODWHG UHJLRQ RI  UHDGDEOH ES 7KH WUXQFDWLRQ DW WKH آ¶HQG ZDV SUREDEO\ D UHVXOW RI LQFRPSOHWH UHYHUVH WUDQVFULSWDVH DFWLRQ PD\EH GXH WR VHFRQGDU\ VWUXFWXUHV LQ WKH FRUUHVSRQGLQJ P51$ 7KH آ¶XQWUDQVODWHG UHJLRQ FRQWDLQHG D SRO\DGHQ\ODWLRQ VLJQDO $$7$$$ 7KH RSHQ UHDGLQJ IUDPH VWDUWHG DW $ DQG H[WHQGHG WR 7 ,Q DQDORJ\ WR WKH ERYLQH SURWHLQ DQG FDOFXODWHG XSRQ WKH PHWKRG RI 1LHOVHQ HW DO  FOHDYDJH RI WKH VLJQDO SHSWLGH SUREDEO\ RFFXUV DW WKH 1WHUPLQDO VLGH RI *OQ 7KH  DD VLJQDO SHSWLGH KDG  VHTXHQFH VLPLODULW\ WR WKH VLJQDO VHTXHQFH RI ERYLQH ODFWRSHUR[LGDVH DQG  WR WKH VLJQDO VHTXHQFH RI KXPDQ ODFWRSHUR[LGDVH :H VXSSRVH E\ FRPSDULVRQ WR ERYLQH ODFWRSHUR[LGDVH WKDW WKH VHFUHWHG SURWHLQ LV VXEVHTXHQWO\ FOHDYHG SUREDEO\ E\ DXWRFDWDO\WLF K\GURO\VLV HLWKHU EHWZHHQ *OQ DQG /\V RU EHWZHHQ 6HU DQG 9DO )LJ  %RYLQH ODFWRSHUR[LGDVH ZDV UHSRUWHG WR EH KHWHURJHQHRXV LQ PROHFXODU ZHLJKW 7ZR PDMRU IRUPV ZHUH GHVFULEHG FRQVLVWLQJ RI  N'D DQG  N'D UHVSHFWLYHO\ 'XOO HW DO 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c 'DYH\


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



5HVXOWV DQG 'LVFXVVLRQ

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

)LJ  F'1$ VHTXHQFH RI FDPHO PLON ODFWRSHUR[LGDVH DQG FRUUHVSRQGLQJ SURWHLQ ZLWK PDWXUH SURWHLQ LQ EROG 7KH RSHQ UHDGLQJ IUDPH RI WKH F'1$ VHTXHQFH LV IURP $ WR 7 DQG WKH SRO\DGHQ\ODWLRQ VLJQDO LQ EROG IURP $ WR $ 1XPEHULQJ RI WKH DPLQR DFLG FKDLQ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH PDWXUH SURWHLQ *O\ SRWHQWLDOO\ JO\FRV\ODWHG DUJLQLQHV

 )HQQD  7KLV SURWHLQ LV SRVWWUDQVODWLRQDOO\ SURFHVVHG LQWR D KHDY\ DQG D OLJKW FKDLQ LQ FRQWUDVW WR ODFWRSHUR[LGDVH ZKLFK UHPDLQV DV D VLQJOH FKDLQ SHSWLGH $ P\HORSHUR[LGDVH PROHFXOH FRQVLVWV RI WZR KHDY\ DQG WZR OLJKW FKDLQV ZLWK QHDUH[DFW WZRIROG V\PPHWU\ FRYDOHQWO\ OLQNHG E\ D VLQJOH GLVXOILGH DW &\V 5HVLGXHV $OD WR *O\ DUH H[FLVHG IURP WKH PDWXUH FKDLQ E\ SRVWWUDQVODWLRQDO SURFHVVLQJ )LJ 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c DQG FRYDOHQW OLQNDJH ZRXOG UHVXOW LQ D PDMRU DOWHUDWLRQ LQ WHUWLDU\ VWUXFWXUH FRPSDUHG WR P\HORSHUR[LGDVH )XUWKHUPRUH &\V ZKLFK LV IUHH LQ P\HORSHUR[LGDVH DQG H[FKDQJHG WR WKH FRUUHVSRQGLQJ *O\ LQ ODFWRSHUR[LGDVH LV ZLWKLQ  c GLVWDQFH WR 6HU DQG PD\ KDYH D VLPLODU IXQFWLRQ LQ P\HORSHUR[LGDVH DV D IUHH &\V LQ ODFWRSHUR[LGDVH %RWK SURWHLQV FRQWDLQ D KHPH E SURWRSRUSK\ULQ ,; LQ WKH FDWDO\WLF FHQWUH ,Q FRQWUDVW WR RWKHU NQRZQ SHUR[LGDVHV IURP EDFWHULDO IXQJDO RU SODQW RULJLQV PDPPDOLDQ SHUR[LGDVHV ELQG WKH KHPH SURVWKHWLF JURXS FRYDOHQWO\ ZKLFK KHOSV WR PDLQWDLQ D PRUH ULJLG WHUWLDU\ VWUXFWXUH $QGHUVVRQ HW DO  7KH PDPPDOLDQ HQ]\PHV ZRUN RSWLPDOO\ DW D EURDG UDQJH LQ S+ DQG LRQLF VWUHQJWK 7KH\ DUH DOVR DEOH WR R[LGLVH KLJK SRWHQWLDO VXEVWUDWHV VXFK DV SVHXGRKDOLGHV HJ WKLRF\DQDWH LQ FRQWUDVW WR RWKHU SHUR[LGDVHV 'H3LOOLV HW DO  ,Q P\HORSHUR[LGDVH WKH KHPH S\UUROH ULQJ & PHWK\O JURXS LV FRYDOHQWO\ DWWDFKHG WR $VS E\ HVWHU OLQNDJH WKH PHWK\O JURXS RI WKH $ ULQJ WR *OX E\ HVWHU OLQNDJH DQG WKH YLQ\O JURXS RI WKH $ ULQJ WR 0HW E\ VXOIRQLXP LRQ OLQNDJH )LJ  :KHUHDV DVSDUWLF DQG JOXWDPLF DFLGV DUH IRXQG DW WKH FRUUHVSRQGLQJ SRVLWLRQV LQ ODFWRSHUR[LGDVH PHWKLRQLQH LV UHSODFHG E\ JOXWDPLQH 'H3LOOLV HW DO 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 D K\SRWKHVLV VXSSRUWHG E\ WKH VWXG\ RI $QGHUVVRQ HW DO



5HVXOWV DQG 'LVFXVVLRQ

 $VS LV DGMDFHQW WR +LV DQG *OX LV QHDU $UJ 7KHVH WZR UHVLGXHV DUH LQYROYHG LQ WKH FDWDO\WLF SURFHVV RQ WKH GLVWDO SRFNHW RI WKH DFWLYH VLWH )HQQD HW DO  ,Q WKH UHVWLQJ HQ]\PH WKH IHUULF KHPH KDV KLJKVSLQ SHQWDFRRUGLQDWH VSHFWUDO FKDUDFWHULVWLFV 7KH ILIWK OLJDQG LV +LV  LQ P\HORSHUR[LGDVH ZKLFK FRUUHVSRQGV WR +LV LQ ODFWRSHUR[LGDVH $ VL[WK OLJDQG ZDV H[FOXGHG E\ ;UD\ FU\VWDO VWUXFWXUH DQDO\VLV 'DYH\ )HQQD 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 &DPHO ODFWRSHUR[LGDVH UHYHDOHG IRXU SRVVLEOH 1JO\FRV\ODWLRQ VLWHV DW $VQ $VQ $VQ DQG $VQ 3RVVLEOH 1JO\FRV\ODWLRQ VLWHV RI ERYLQH ODFWRSHUR[LGDVH DUH DW $VQ $VQ $VQ $VQ DQG $VQ 7KH FDUERK\GUDWH FRQWHQW RI ERYLQH ODFWRSHUR[LGDVH ZDV UHSRUWHG WR EH DERXW  'H :LW 9DQ +RR\GRQN  ZKLFK FRUUHVSRQGV WR DERXW  N'D 7DEOH  7KLV FRXOG DFFRXQW IRU IRXU WR ILYH JO\FRV\ODWHG VLWHV 7KH VWUXFWXUDOO\ UHODWHG KXPDQ P\HORSHUR[LGDVH LV JO\FRV\ODWHG DW $VQ DQG DW $VQ E\ VLQJOH 1DFHW\OJOXFRVDPLQHV DQG DW $VQ E\ D IXFRV\ODWHG 1 DFHW\OODFWRVDPLQLF W\SH JO\FDQ )HQQD HW DO  %DVHG RQ WKHVH GDWD JO\FRV\ODWLRQ RI $VQ DQG $VQ LQ FDPHO ODFWRSHUR[LGDVH LV SURSRVHG 1HYHUWKHOHVV JO\FRV\ODWLRQ RI $VQ DQG $VQ PD\ DOVR RFFXU 7HUWLDU\ 6WUXFWXUH DQG &DWDO\WLF $FWLYLW\ $QWLPLFURELDO DFWLYLW\ RI ODFWRSHUR[LGDVH LV SHUIRUPHG E\ D VRFDOOHG ODFWRSHUR[LGDVH V\VWHP /3V\VWHP LQ ZKLFK K\GURJHQ SHUR[LGH +2 LV UHGXFHG DQG D KDOLGH HJ LRGLGH ,أک RU EURPLGH %Uأک RU D SVHXGRKDOLGH HJ WKLRF\DQDWH 6&1أک LV VXEVHTXHQWO\ R[LGLVHG )HUUDUL HW DO  &RPSOH[ IRUPDWLRQ RI ODFWRSHUR[LGDVH ZLWK 6&1أک D PROHFXOH ZLWK ORZ FKDUJH GHQVLW\ ZDV UHSRUWHG WR EH IDYRXUHG ERWK WKHUPRG\QDPLFDOO\ DQG NLQHWLFDOO\ &KDQJH LQ IUHH HQWKDOS\ ZDV  N- PRO IRU WKH UHDFWLRQ ZLWK &Oأک ZKLFK LV D JRRG VXEVWUDWH IRU P\HORSHUR[LGDVH 7KH YDOXH IRU WKH UHDFWLRQ ZLWK 6&1أک ZDV  N- PRO )HUUDUL HW DO  7KH NLQHWLF

&DPHO %RYLQH

3URVHTXHQFH آ¸ aaaaaaaaaaaa aaaaaaaa aaaaaaaaa aaaaaaaaaaa 4 77$$$06($9549.9+91.$)/'6575/.$$066(937754/6(
&DPHO %RYLQH +XPDQ

آ¸ aa bbb aaaaaaaaaaaaa 7471,7'36/'/7$/6:(9*&'939699.&'.163<57,7*'&11*5+3$/*$$14$/$5:/3$(<('*/6/3)* 7/7197'36/'/7$/6:(9*&*$393/9.&'(163<57,7*'&115563$/*$$15$/$5:/3$(<('*/$/3)* 97&3(4'.<57,7*0&1155637/*$615$)95:/3$(<('*)6/3<*

  



&DPHO %RYLQH +XPDQ

+HP OLQNHG $VS GLVWDO +LV &D ELQGLQJ $VS aaaaaaaaaaaa aaaaaaaaaaaaa :75*..51*)3/3/$5(961.,9*
  

&DPHO %RYLQH +XPDQ

&D ELQGLQJ UHJLRQ bb bb aaaa aaaaaa *'1&)3,0)351'5.9074*.&03))5$*)9&3133<46/$5(4,1$/76)/'$6/9<*6(36/$66/5'/663/ *'1&)3,0)3.1'3./.74*.&03))5$*)9&3733<46/$5(4,1$976)/'$6/9<*6(36/$65/51/663/ 433&)3/.,331'35,.14$'&,3))56&3$&3*6 1,7,514,1$/76)9'$609<*6((3/$51/510614/

  

&DPHO %RYLQH +XPDQ

'LVWDO $UJ +HP ELQGLQJ *OX aaaa aaaaaaaaaaaaaaaaaaaaa */0$914():'+*/$<33)91..363&(9,177$493&)/$*'65$6(4,//$76+7///5(+15/$5(/../13 */0$914($:'+*/$
  

&DPHO %RYLQH +XPDQ

3UR[LPDO +LV aaaaaaaaaaaaaaaaaaaaa aaaaa aaaaa aaaaa bbb +:'*(./<4($5.,/*$)04,,7)5'
&DPHO %RYLQH +XPDQ

bbb aaaaaa aaaaaaaa aaaa aa /'(1<43:*3(3(/3/+7/))17:5,9.'**,'3/95*//$..6.)064.5007*(/51./)433<7,+*)'/$$ /'(1<43:*3($(/3/+7/))17:5,,.'**,'3/95*//$..6./014'.0976(/51./)437+.,+*)'/$$ /'15<430(313593/659))$6:599/(**,'3,/5*/0$73$./15414,$9'(,5(5/)(4905, */'/3$

&DPHO %RYLQH +XPDQ

aaaaaaa aaaaaa aaaaaaa aaaaaaaaaa aaaaaa a ,+,45&5'+*03*<16:5*)&'/64347/.(/+$9/.1../$..//'/<573'1,',:/**,$(349.5*59*3/ ,1/45&5'+*03*<16:5*)&*/643.7/.*/479/.1.,/$../0'/<.73'1,',:,**1$(309(5*59*3/ /104565'+*/3*<1$:55)&*/343(79*4/*79/51/./$5./0(4<*7311,',:0**96(3/.5.*59*3/



&DPHO %RYLQH +XPDQ

aaaaaaaaaaaaa aaaaaaa aaaaaaa bbb bbb /$&//*54)54,5'*'5)::(13*9)7..44.6/4./6)65/9&'17+,7.93/+3)4$16<3+*)9'&6$,'./ /$&//*54)44,5'*'5)::(13*9)7(.45'6/4.96)65/,&'17+,7.93/+$)4$11<3+')9'&679'./ /$&,,*74)5./5'*'5)::(1(*9)604454$/$4,6/35,,&'17*,7796.11,)0616<35')91&67/3$/

5HVXOWV DQG 'LVFXVVLRQ

&DPHO %RYLQH +XPDQ

'/63:$69(1 '/63:$65(1 1/$6:5($6

  

)LJ  6FKHPDWLF GUDZLQJ RI WKH UHODWLRQVKLS EHWZHHQ VWUXFWXUH DQG IXQFWLRQ LQ FDPHO DQG ERYLQH ODFWRSHUR[LGDVH 1XPEHULQJ VWDUWV IURP WKH ILUVW UHVLGXH RI WKH SUHVXPHG ORQJ YDULDQW RI ODFWRSHUR[LGDVH +HDY\ DQG OLJKW FKDLQ RI KXPDQ P\HORSHUR[LGDVH XQGHUQHDWK IRU EHWWHU FRPSDULVRQ RI IXQFWLRQDO UHVLGXHV /LJKW FKDLQ RI P\HORSHUR[LGDVH IURP 9DO WR $OD KHDY\ FKDLQ IURP 9DO WR 6HU )XQFWLRQDO UHVLGXHV LQ EROG خ±KHOLFDO UHJLRQV GHVLJQDWHG DV آ³خ±آ´ خ²SOHDWHG UHJLRQV DV آ³خ²آ´ آ¸ 3RVVLEOH FOHDYDJH VLWHV RI WKH SURSHSWLGH

 SUHIHUHQFH IRU 6&1أک ZDV GLVFXVVHG WR EH D FRQVHTXHQFH RI VKDSH DQG DPLQR DFLG FRPSRVLWLRQ DW WKH GLVWDO VLGH RI ODFWRSHUR[LGDVH KHPH 'H :LW 9DQ +RR\GRQN  $ YHU\ ORZ .0 YDOXH RI  آµ0 ZDV UHSRUWHG IRU 6&1أک 5HDFWLRQ NLQHWLFV DUH QRW OLPLWHG E\ +2 VLQFH WKH .0 IRU WKLV FRVXEVWUDWH ZDV ORZHU WKDQ IRU 6&1أک DQG IRU KDOLGHV )HUUDUL HW DO  7KH DUFKLWHFWXUH RI WKH KHPH SRFNHW RI SHUR[LGDVHV LV KLJKO\ FRQVHUYHG ,Q PDPPDOLDQ SHUR[LGDVHV WKH KHPH LV GLVWRUWHG DV D UHVXOW IURP FRYDOHQW ELQGLQJ $W WKH SUR[LPDO VLGH RI WKH SRFNHW WKH IHUULF LRQ LV FRRUGLQDWHG E\ +LV LQ ODFWRSHUR[LGDVH DQG +LV LQ P\HORSHUR[LGDVH ZKLFK IXQFWLRQ DV DQ D[LDO ILIWK OLJDQG 7KH GLVWDO VLGH RI P\HORSHUR[LGDVH ZKLFK SURYLGHV D FDWDO\WLF VXUIDFH IRU WKH UHDFWLRQ FDVFDGH LV VKRZQ LQ )LJ  7KH FKDQQHO RI P\HORSHUR[LGDVH LV DERXW  c LQ GLDPHWHU OHDGLQJ WR D PXFK QDUURZHU HQWUDQFH WR WKH GLVWDO SRFNHW QHDU WKH KHPH S\UUROH ULQJ ' 7KH XSSHU VXUIDFH RI WKH GLVWDO SRFNHW LV FRPSRVHG RI $UJ DW WKH HQWUDQFH IROORZHG E\ +LV DQG *OQ DW WKH EDFN +LJK RFFXSDQF\ ELQGLQJ ZDV IRXQG DW WKH HQWUDQFH WR WKH GLVWDO SRFNHW EHWZHHQ S\UUROH ULQJ ' DQG $UJ +RUL HW DO  7KLV LQWHUDFWLRQ ZDV SUHVXPHG WR EH HOHFWURVWDWLF LQ QDWXUH VLQFH WKHUH LV DQ DFFXPXODWLRQ RI IRXU SRVLWLYH FKDUJHV IURP WKH IHUULF LURQ DWRP RI WKH KHPH DQG IURP $UJ ,W LV VXSSRVHG WKDW $UJ LV LQYROYHG LQ LQLWLDO ELQGLQJ RI QHJDWLYHO\ FKDUJHG VXEVWUDWHV 3HUR[LGDVHV XQGHUJR D WZR HOHFWURQ R[LGDWLRQ E\ +2 WR IRUP DQ HQ]\PH LQWHUPHGLDWH QDPHG FRPSRXQG , ZKLFK FRQWDLQV DQ R[RIHUU\O SRUSK\ULQ د€FDWLRQ UDGLFDO ZKLFK LV UHDGLO\ FRQYHUWHG LQWR DQ R[RIHUU\O SURWHLQ UDGLFDO +LV LV WKRXJKW WR GRQDWH D SURWRQ LQ LWV IXQFWLRQ DV DQ DFLGEDVH FDWDO\VW ZKLFK SURPRWHV KHWHURO\WLF FOHDYDJH RI SHUR[LGH ZKLOH $UJ SOD\V D VHFRQGDU\ UROH LQ KHOSLQJ WR VWDELOLVH WKH UHVXOWDQW R[RIHUU\O FHQWHU RI FRPSRXQG , 'DYH\ )HQQD  $ QXFOHRSKLOLF VXEVWUDWH VXFK DV 6&1أک GRQDWHV DQ HOHFWURQ WR FRPSRXQG , ZKLFK \LHOGV WKH FRUUHVSRQGLQJ VXEVWUDWH IUHH UDGLFDO DQG DQ R[RIHUU\O KHPH LQWHUPHGLDWH )H2 RU )H5 ZKLFK LV QDPHG FRPSRXQG ,, $ VXEVHTXHQW UHGXFWLRQ E\ D VHFRQG QXFOHRSKLOLF VXEVWUDWH PROHFXOH OHDGV WR IHUULF SHUR[LGDVH 3HUR[LGDVHV DOVR UHDFW ZLWK WKH VXSHUR[LGH UDGLFDO DQLRQ WR IRUP FRPSXQG ,,, D UHVRQDQFH K\EULG EHWZHHQ )H2أک DQG )H2 1DWXUDO VXEVWUDWHV RI ODFWRSHUR[LGDVH LQ PLON DUH WKLRF\DQDWH DQG LRGLGH RI ZKLFK PLON FRQWDLQV WUDFH DPRXQWV 6&1أک LV SURYLGHG PDLQO\ E\ FRQVXPSWLRQ RI SODQWV RI WKH IDPLO\ &UXFLIHUDH .DOH D FDEEDJH FRQWDLQV XS WR  J NJ WKLRJOXFRVLGHV ZKLFK DUH UHDGLO\ FRQYHUWHG LQWR 6&1أک E\ HQ]\PDWLF K\GURO\VLV %LEL  &RZ PLON FRQWHQW RI 6&1أک ZDV UHSRUWHG



5HVXOWV DQG 'LVFXVVLRQ

+LV $UJ

+HPH

)LJ  0\HORSHUR[LGDVH D FORVH KRPRORJXH RI ODFWRSHUR[LGDVH VKRZQ DV D PRQRPHU ZLWK RQH KHDY\ DQG RQH OLJKW FKDLQ 'LVWDO VLGH RI WKH KHPH SRFNHW ZKLFK FRQWDLQV WKH FDWDO\WLF FHQWUH LQ GLUHFWLRQ RI YLVLRQ 'LVWDO +LV DQG $UJ GDUN VKDGHG +HPH PHGLXP JUH\ VKDGHG 5HVLGXHV ZLWKLQ  c GLVWDQFH RI WKH KHPH DUH GUDZQ DV 9DQGHU:DDOV VXUIDFH RWKHU UHVLGXHV DUH GUDIWHG LQ ULEERQ YLHZ ;UD\ FU\VWDOORJUDSKLF GDWD IURP )HQQD HW DO 

 WR YDU\ EHWZHHQ  PJ O DQG  PJ O ZLWK PXFK ORZHU FRQFHQWUDWLRQV LQ ZLQWHU WLPH GHSHQGLQJ RQ IHHGLQJ 'H :LW 9DQ +RR\GRQN  &RQFHQWUDWLRQ RI K\GURJHQ SHUR[LGH LV ORZ LQ PDVWLWLV IUHH PLON ,W FDQ EH JHQHUDWHG E\ R[LGDWLRQ RI [DQWKLQH E\ ;DQWKLQH R[LGDVH RU VXSSOLHG E\ FDWDODVHQHJDWLYH EDFWHULD VXFK DV VWUDLQV RI ODFWREDFLOOL ODFWRFRFFL RU VWUHSWRFRFFL ZKLFK QDWXUDOO\ RFFXU LQ PLON %LEL  7KH UHDFWLRQ SURGXFWV RI WKLRF\DQDWH R[LGDWLRQ 26&1أ° DQG +26&1 DUH LQ FKHPLFDO HTXLOLEULXP DW S+  DQG DUH DEOH WR R[LGLVH IUHH VXOSKK\GULGH JURXSV RI WKH F\WRSODVPLF PHPEUDQH RI JUDPQHJDWLYH EDFWHULD VLPLODUO\ WR K\SRLRGLGH 2,أک 7KH VWUXFWXUDO GDPDJH RQ EDFWHULDO FHOO PHPEUDQHV UHVXOWV LQ GLIIXVLRQ RI SRWDVVLXP LRQV DPLQR DFLGV DQG SRO\SHSWLGHV RXW RI WKH FHOO ZKLOVW XSWDNH RI JOXFRVH DQG RWKHU PHWDEROLF VXEVWUDWHV LV LQKLELWHG *UDPSRVLWLYH EDFWHULD VXFK DV VWUHSWRFRFFL DUH SUREDEO\ EHWWHU SURWHFWHG IURP WKH DFWLRQ RI ODFWRSHUR[LGDVH E\ WKHLU ULJLG FHOO ZDOO 1HYHUWKHOHVV D EDFWHULRVWDWLF HIIHFW RQ PDQ\ JUDPSRVLWLYH EDFWHULD ZDV DOVR UHSRUWHG ZKLFK ZDV REVHUYHG LQ UHWDUGHG DFLG SURGXFWLRQ 'H :LW 9DQ +RR\GRQN  7KH DGYDQWDJH RI 26&1أ° DQG +26&1 FRPSDUHG WR RWKHU K\SHUR[LGLVHG VXEVWUDWHV LV WKDW WKH\ DUH LQHUW WRZDUGV PDPPDOLDQ FHOOV DQG PLON FRPSRQHQWV GXH WR ORZ FKDUJH GHQVLW\ /DFWRSHUR[LGDVH LV QRW DEOH WR R[LGLVH WKH FKORULGH DQLRQ &Oأک ZKLFK LV SUHVHQW DW KLJK FRQFHQWUDWLRQV LQ PLON DQG GLJHVWLYH VHFUHWLRQV LQ FRQWUDVW WR WKH RWKHU PDPPDOLDQ SHUR[LGDVHV ,W ZDV UHSRUWHG WKDW WKH ODFWRSHUR[LGDVHFKORULGH FRPSOH[ KDV D KLJK GLVVRFLDWLRQ FRQVWDQW RI  P0 ZKHUHDV FRPSOH[HV ZLWK WKH SUHIHUUHG VXEVWUDWHV WKLRF\DQDWH DQG LRGLGH KDYH ORZ .'YDOXHV RI  P0 DQG  P0 UHVSHFWLYHO\ )HUUDUL HW DO  7KH GLIIHUHQW VXEVWUDWH VSHFLILFLW\ RI ODFWRSHUR[LGDVH FRPSDUHG ZLWK P\HORSHUR[LGDVH DV ZHOO DV DQ DQRPDORXV HOHFWURQLF DEVRUSWLRQ VSHFWUXP UHODWLYH WR WKRVH RI RWKHU KHPH EFRQWDLQLQJ SURWHLQV LV WKRXJKW WR EH WKH UHVXOW RI DQ XQXVXDOO\ FRQVWUDLQW KHPH SRFNHW LQ ODFWRSHUR[LGDVH +X HW DO  7KH UHDVRQ IRU WKH ORZ DIILQLW\ RI ODFWRSHUR[LGDVH WRZDUGV &Oأک LV VXSSRVHG WR EH LQ WKH SURWHFWLRQ RI PDPPDOLDQ RUJDQV DQG PLON FRPSRQHQWV 1HYHUWKHOHVV LW ZDV VKRZQ WKDW DURPDWLF PROHFXOHV DUH DEOH WR ELQG DW WKH GLVWDO KHPH SRFNHW RI P\HORSHUR[LGDVH +RUL HW DO  ,W ZDV VXJJHVWHG WKDW WKH KHPH S\UUROH ULQJ ' DQG WKH VLGH FKDLQV RI WKH GLVWDO 3KH $UJ 3KH DQG 3KH IRUP D K\GURSKRELF VXUIDFH DW WKH HQWUDQFH WR WKH GLVWDO FDYLW\ ZKLFK FDQ ELQG DURPDWLF VXEVWUDWH PROHFXOHV ,Q WKLV ZD\ PRQR RU GLFKORULQDWHG ELSKHQ\OV 3&%V ZKLFK DUH DFFXPXODWHG LQ WKH ERG\ E\ GLJHVWLRQ RI DQLPDO IDWV DUH PHWDEROLVHG WR GLK\GUR[\ FRPSRXQGV DQG IXUWKHU R[LGLVHG WR



5HVXOWV DQG 'LVFXVVLRQ

UHDFWLYH PHWDEROLWHV ZKLFK FDQ UHDFW ZLWK FHOO '1$ 2DNOH\ HW DO  /DFWRSHUR[LGDVH DQG P\HORSHUR[LGDVH PD\ WKHUHIRUH SOD\ D FUXFLDO UROH LQ WKH FKHPLFDO LQGXFWLRQ RI EUHDVW FDQFHU -RVHSK\  /DFWRSHUR[LGDVH LV HDVLO\ LQKLELWHG E\ LUUHYHUVLEOH ELQGLQJ RI QLWULWH D]LGH F\DQLGH DQG FDUERQ PRQR[LGH +X HW DO  )HUUDUL HW DO  5LERIODYLQ YLWDPLQ % IRXQG LQ FDPHO PLON DW ORZ FRQFHQWUDWLRQV RI  PJ O WR  PJ O ZDV VKRZQ WR SURPRWH OLJKW LQGXFHG LQDFWLYDWLRQ RI ODFWRSHUR[LGDVH +HUiQGH] HW DO  3KRWRFKHPLFDO LQDFWLYDWLRQ RI ERYLQH ODFWRSHUR[LGDVH LQ PLON ZDV  DIWHU  K LUUDGLDWLRQ ZLWK  OX[ DQG ZDV LQKLELWHG E\ F\VWHLQH LQGLFDWLQJ WKDW ULERIODYLQ GDPDJHG ODFWRSHUR[LGDVH E\ R[LGDWLYH DFWLRQ 3RWHQWLDO IRU 0LON 3UHVHUYDWLRQ 7KH /3V\VWHP LV DFWLYH DJDLQVW PDLQO\ JUDPQHJDWLYH SV\FKURWURSKLF DQG PHVRSKLOLF EDFWHULD EXW DOVR DJDLQVW D EURDG UDQJH RI SDWKRJHQV LQFOXGLQJ YLUXVHV DQG PRXOGV RUJDQLVPV IRXQG LQ PLON RI SRRU K\JLHQLF TXDOLW\ 'H :LW 9DQ +RR\GRQN  ,W ZDV REVHUYHG WKDW WKH NHHSLQJ TXDOLW\ RI UDZ FRZ PLON VWRUHG DW  آƒ& DQG SDVWHXULVHG DIWHU WKUHH WR IRXU GD\V ZDV PXFK EHWWHU WKDQ WKDW RI WKH VDPH PLON SDVWHXULVHG RQ WKH ILUVW RU RQ WKH VHYHQWK GD\ ZKHQ DOVR VWRUHG DW  آƒ& EHIRUH DQG DIWHU SDVWHXULVDWLRQ 5DYDQLV /HZLV  )URP WKHVH UHVXOWV LW ZDV FRQFOXGHG WKDW WKH QDWXUDO /3V\VWHP LQ UDZ FRZ PLON LV PRVW HIIHFWLYH XS WR IRXU GD\V 7KHUHDIWHU +2 LV VXSSRVHG WR EH WKH OLPLWLQJ IDFWRU 0D[LPDO SHUIRUPDQFH RI WKH /3V\VWHP LV DFKLHYHG E\ DGGLWLRQ RI HTXLPRODU FRQFHQWUDWLRQV RI K\GURJHQ SHUR[LGH DQG D KDOLGH XVXDOO\  P0 RI HDFK %LEL  7KH RSWLPDO S+ UDQJH RI WKH /3V\VWHP LV DW S+  WR  *UDPQHJDWLYH FDWDODVHSRVLWLYH EDFWHULD VXFK DV SVHXGRPRQDGV FROLIRUPV VDOPRQHOODH DQG VKLJHOODH DUH NLOOHG E\ WKH /3V\VWHP SURYLGHG +2 LV SURYLGHG LQ VXIILFLHQW FRQFHQWUDWLRQV $Q DFWLYDWHG /3V\VWHP ZDV DOVR VKRZQ WR EH HIIHFWLYH DJDLQVW GLIIHUHQW /LVWHULD VWUDLQV %LEL  $FWLYDWLRQ RI WKH /3V\VWHP H[WHQGV WKH VWRUDJH WLPH RI UDZ FRZ PLON DW  آƒ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

 GLVWDQFH WUDQVSRUWV DQG LQDGHTXDWH FOHDQLQJ RI PLON KDQGOLQJ HTXLSPHQW HJ GXH WR ZDWHU VKRUWDJH %LEL  0DLQO\ WKH FROOHFWLRQ RI HYHQLQJ PLON ZDV UHSRUWHG WR EH SUREOHPDWLF VLQFH FRROLQJ LV RIWHQ QRW IHDVLEOH 7R HVWLPDWH WKH SRWHQWLDO RI WKH /3V\VWHP IRU FDPHO PLON LW ZRXOG EH QHFHVVDU\ WR PHDVXUH WKH DFWLYLW\ RI ODFWRSHUR[LGDVH LQ SRROHG FDPHO PLON 7KH 6&1أک FRQFHQWUDWLRQ LQ GLIIHUHQW IHHGLQJ DUHDV VKRXOG DOVR EH VWXGLHG )XUWKHU RQ EDFWHULDO VWUDLQV VKRXOG EH IRXQG ZKLFK SURPRWH WKH IRUPDWLRQ RI +2 LQ FDPHO PLON (ODJDP\ HW DO  UHSRUWHG WKDW WKH /3V\VWHP RI FDPHO PLON ZDV EDFWHULFLGDO DJDLQVW (VFKHULFKLD FROL DQG 6DOPRQHOOD W\SKLPXULXP EXW RQO\ EDFWHULRVWDWLF WRZDUGV /DFWRFRFFXV ODFWLV DQG 6WDSK\ORFRFFXV DXUHXV 7KH OLPLWHG LQWHUHVW LQ WKH /3V\VWHP IRU WKH SUHVHUYDWLRQ RI PLON PD\ EH H[SODLQHG E\ SUREOHPV ZLWK LQKLELWLRQ RI VWDUWHU FXOWXUHV DQG E\ GDQJHU RI PLON SRLVRQLQJ LI WKH FRQFHQWUDWLRQV RI WKH DGGHG FKHPLFDOV DUH VXUSDVVHG

  &21&/86,216 $1' 287/22.

&RQFOXVLRQV $QG 2XWORRN

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خ؛&1 FRQWHQW RI FDVHLQ PLFHOOHV FRPELQHG ZLWK D KLJK خ²&1 FRQWHQW DQG ZLWK VWUXFWXUDO GLYHUJHQFHV EHWZHHQ FDPHO DQG ERYLQH خ؛&1 8VH RI UHFRPELQDQW FDPHO FK\PRVLQ ZDV VXJJHVWHG WR EH D SURPLVLQJ DOWHUQDWLYH LQ FDPHO PLON UHQQHW FRDJXODWLRQ ZLWK IRFXV RQ LPSURYHPHQW RI FXUG ILUPQHVV 7KH KLJK SURSRUWLRQ RI خ²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خ²ODFWRJOREXOLQ ZKLFK PD\ UHVXOW LQ LQWROHUDQFH RI DQ LQIDQW WRZDUGV FRZ PLON +DPEUآ XV  D ORZ DPRXQW RI ODFWRSHUR[LGDVH ZKLFK VHHPV WR EH GRZQUHJXODWHG LQ KXPDQ PLON HDUO\ LQ ODFWDWLRQ D KLJK DPRXQW RI خ±ODFWDOEXPLQ ZKLFK KDV D KLJK QXWULWLRQDO YDOXH RI ODFWRIHUULQ ZKLFK LV DOVR IRXQG DW D KLJK OHYHO LQ KXPDQ PLON DQG RI QRQSURWHLQ QLWURJHQ VLPLODUO\ WR KXPDQ PLON FRXOG EH RI DGYDQWDJH LQ XWLOLVDWLRQ RI FDPHO PLON LQ GDLU\ SURGXFWV IRU LQIDQWV DQG SHRSOH ZLWK DOOHUJ\ DJDLQVW FRZ PLON SURGXFWV $V D ORQJ WHUP JRDO LQ FDPHO UHVHDUFK EUHHG FODVVLILFDWLRQ DQG GHWHUPLQDWLRQ RI JHQHWLF DQG QXWULWLRQDO IDFWRUV ZKLFK LQFUHDVH PLON \LHOG DQG LPSURYH FRQVLVWHQF\ RI PLON TXDOLW\ VKRXOG EH HQYLVDJHG *HQHWLF



&RQFOXVLRQV $QG 2XWORRN

FKDUDFWHULVDWLRQ LQ WHUPV RI ODUJHVFDOH F'1$ VHTXHQFH DQDO\VLV FKURPRVRPDO PDSSLQJ RI JHQHV DQG PLFURVDWHOOLWH VHTXHQFHV VWXG\ RI UHJXODWLRQ RI JHQH H[SUHVVLRQ DQG GHWHFWLRQ RI PDUNHUV IRU EUHHG FODVVLILFDWLRQ FRXOG KHOS WR ILQG VXLWDEOH DQLPDOV IRU PLON DQG PHDW SURGXFWLRQ DV ZHOO DV IRU WUDQVSRUW DJULFXOWXUDO DQG UDFLQJ SXUSRVHV DQG FRXOG EH D ILUVW VWHS LQ XQGHUVWDQGLQJ WKH JHQHWLF DQG SK\VLRORJLFDO UHVHUYRLU RI WKH FDPHO DV WKH OLYHVWRFN DQLPDO ZKLFK LV EHVW DGDSWHG WR DULG UHJLRQV



  5()(5(1&(6

5HIHUHQFHV

$EG (O*DZDG ,$ (O6D\HG (0 0DKIRX] 0% $EG (O6DODP $0  &KDQJHV RI ODFWRIHUULQ FRQFHQWUDWLRQ LQ FRORVWUXP DQG PLON IURP GLIIHUHQW VSHFLHV (J\SWLDQ -RXUQDO RI 'DLU\ 6FLHQFH   $EHLGHUUDKPDQH 10 5HHG ':  3DVWHXUL]LQJ FDPHOآ¶V PLON DQG PDNLQJ FDPHOآ¶V FKHHVH LQ 0DXULWDQLD 6SLULW RI HQWHUSULVH 7KH  5ROH[ $ZDUGV  $EX/HKLD ,+  &RPSRVLWLRQ RI FDPHO PLON 0LOFKZLVVHQVFKDIW   $EX/HKLD ,+  3K\VLFDO DQG FKHPLFDO FKDUDFWHULVWLFV RI FDPHO PLONIDW DQG LWV IUDFWLRQV )RRG &KHPLVWU\   $EX7DUERXVK +0  *URZWK EHKDYLRU RI /DFWREDFLOOXV DFLGRSKLOXV DQG ELRFKHPLFDO FKDUDFWHULVWLFV DQG DFFHSWDELOLW\ RI DFLGRSKLOXV PLON PDGH IURP FDPHO PLON 0LOFKZLVVHQVFKDIW   $EX7DUERXVK +0  &RPSDULVRQ RI DVVRFLDWLYH JURZWK DQG SURWHRO\WLF DFWLYLW\ RI \RJXUW VWDUWHUV LQ ZKROH PLON IURP FDPHOV DQG FRZV -RXUQDO RI 'DLU\ 6FLHQFH   $EX7DUERXVK +0  *URZWK YLDELOLW\ DQG SURWHRO\WLF DFWLYLW\ RI ELILGREDFWHULD LQ ZKROH FDPHO PLON -RXUQDO RI 'DLU\ 6FLHQFH   $OEHUW $ %OXQGHOO 7/ 'KDQDUDM 9 'RQDWH /( *URYHV 0 *XUXSUDVDG . 1XJHQW 3* 2USUD\RRQ 3 3LWWV -( 5XILQR 6 6ULQLYDVDQ 1 :LOOLDPV 0 :LOVKHU -  3URWHLQ HQJLQHHULQJ DVSDUWLF SURWHLQDVHV 6LWHGLUHFWHG PXWDJHQHVLV ELRFKHPLFDO FKDUDFWHULVDWLRQ DQG ;UD\ DQDO\VLV RI FK\PRVLQV ZLWK VXEVWLWXWHG VLQJOH DPLQR DFLG VXEVWLWXWLRQV DQG ORRS UHSODFHPHQWV $GYDQFHV LQ ([SHULPHQWDO 0HGLFLQH DQG %LRORJ\   $OKRPLGD $6  7RWDO IUHH VKRUWFKDLQ DQG ORQJFKDLQ DF\O FDUQLWLQH OHYHOV LQ $UDELDQ FDPHO PLON &DPHOXV GURPHGDULXV $QQDOV RI 1XWULWLRQ DQG 0HWDEROLVP  

 $O6DOHK $$ +DPPDG <$  %XIIHULQJ FDSDFLW\ RI FDPHO PLON (J\SWLDQ -RXUQDO RI )RRG 6FLHQFH   $QGHUVHQ 0+ %HUJOXQG / 5DVPXVVHQ -7 3HWHUVHQ 7(  %RYLQH 3$6 ELQGV خ±Yخ² LQWHJULQ DQG DQLRQLF SKRVSKROLSLGV WKURXJK WZR GRPDLQV %LRFKHPLVWU\   $QGHUVRQ %) %DNHU +0 1RUULV *( 5LFH ': %DNHU (1  6WUXFWXUH RI KXPDQ ODFWRIHUULQ FU\VWDOORJUDSKLF VWUXFWXUH DQDO\VLV DQG UHILQHPHQW DW c UHVROXWLRQ -RXUQDO RI 0ROHFXODU %LRORJ\   $QGHUVVRQ /$ %\ONDV 6$ :LOVRQ $(  6SHFWUDO DQDO\VLV RI ODFWRSHUR[LGDVH (YLGHQFH IRU D FRPPRQ KHPH LQ PDPPDOLDQ SHUR[LGDVHV -RXUQDO RI %LRORJLFDO &KHPLVWU\   $QGUHHYD 16 -DPHV 1*  :K\ GRHV SHSVLQ KDYH D QHJDWLYH FKDUJH DW YHU\ ORZ S+" $Q DQDO\VLV RI FRQVHUYHG FKDUJHG UHVLGXHV LQ DVSDUWLF SURWHLQDVHV ,Q 6WUXFWXUH DQG )XQFWLRQ RI WKH $VSDUWLF 3URWHLQDVHV *HQHWLFV 6WUXFWXUHV DQG 0HFKDQLVPV HG %0 'XQQ SS  3OHQXP 3UHVV 1HZ V PLON UHODWLRQ WR ZKH\ O\VR]\PH DQG VWDJH RI ODFWDWLRQ -RXUQDO RI )RRG 3URWHFWLRQ   %DUWRQ *-  6&$136 9HUVLRQ  8VHU *XLGH 8QLYHUVLW\ RI 2[IRUG 8. %D\RXPL 6  6WXGLHV RQ FRPSRVLWLRQ DQG UHQQHW FRDJXODWLRQ RI FDPHO PLON .LHOHU PLOFKZLUWVFKDIWOLFKH )RUVFKXQJVEHULFKWH  



5HIHUHQFHV

%HJ 28 9RQ %DKU/LQGVWU|P + =DLGL =+ -|UQYDOO +  7KH SULPDU\ VWUXFWXUH RI خ±ODFWDOEXPLQ IURP FDPHO PLON (XURSHDQ -RXUQDO RI %LRFKHPLVWU\   %HJ 28 9RQ %DKU/LQGVWU|P + =DLGL =+ -|UQYDOO +  D &KDUDFWHULVDWLRQ RI D FDPHO PLON SURWHLQ ULFK LQ SUROLQH LGHQWLILHV D QHZ خ² FDVHLQ IUDJPHQW 5HJXODWRU\ 3HSWLGH   %HJ 28 9RQ %DKU/LQGVWU|P + =DLGL =+ -|UQYDOO +  E $ FDPHO PLON ZKH\ SURWHLQ ULFK LQ KDOIF\VWLQH 3ULPDU\ VWUXFWXUH DVVHVVPHQW RI YDULDWLRQV LQWHUQDO UHSHDW SDWWHUQV DQG UHODWLRQVKLSV ZLWK QHXURSK\VLQ DQG RWKHU DFWLYH SRO\SHSWLGHV (XURSHDQ -RXUQDO RI %LRFKHPLVWU\   %HJ 28 %DKU/LQGVWU|P + =DLGL =+ -|UQYDOO +  &KDUDFWHUL]DWLRQ RI D KHWHURJHQHRXV FDPHO PLON ZKH\ QRQFDVHLQ SURWHLQ )(%6 /HWWHUV   %HUJ (/ 0XOORZQH\ $7 $QGUHZ '3 *ROGEHUJ -( %XWFKHU (&  &RPSOH[LW\ DQG GLIIHUHQWLDO H[SUHVVLRQ RI FDUERK\GUDWH HSLWRSHV DVVRFLDWHG ZLWK /VHOHFWLQ UHFRJQLWLRQ RI KLJK HQGRWKHOLDO YHQXOHV $PHULFDQ -RXUQDO RI 3DWKRORJ\   %HUQRV ( *LUDUGHW -0 +XPEHUW * /LQGHQ *  5ROH RI WKH 2 SKRVSKRVHULQH FOXVWHUV LQ WKH LQWHUDFWLRQ RI WKH ERYLQH PLON DOSKDV EHWD NDSSDFDVHLQV DQG WKH 33 FRPSRQHQW ZLWK LPPRELOL]HG LURQ ,,, LRQV %LRFKLPLFD HW %LRSK\VLFD $FWD   %LEL :  1DWXUDO DFWLYDWLRQ RI WKH ODFWRSHUR[GLDVHWKLRF\DQDWH K\GURJHQ SHUR[LGH /3 V\VWHP IRU SUHVHUYDWLRQ RI PLON GXULQJ FROOHFWLRQ LQ GHYHORSLQJ FRXQWULHV 'LVV (7+ 1U  6ZLVV )HGHUDO ,QVWLWXWH RI 7HFKQRORJ\ =XULFK 6ZLW]HUODQG %RRWK .6 .LPXUD 6 /HH +& ,NHGD6DLWR 0 &DXJKH\ :6  %RYLQH P\HORSHUR[LGDVH DQG ODFWRSHUR[LGDVH HDFK FRQWDLQ D KLJK DIILQLW\ VLWH IRU FDOFLXP %LRFKHPLFDO DQG %LRSK\VLFDO 5HVHDUFK &RPPXQLFDWLRQV   %URFN -+  /DFWRIHUULQ 6WUXFWXUH  )XQFWLRQ 5HODWLRQVKLSV ,Q /DFWRIHUULQ ,QWHUDFWLRQV DQG %LRORJLFDO )XQFWLRQV HGV 7: +XWFKHQV % /|QQHUGDO SS  +XPDQD 3UHVV 7RWRZD 1- 86$

 %XFKKHLP : /XQG 6 6FKROWLVVHN -  9HUJOHLFKHQGH 8QWHUVXFKXQJHQ ]XU 6WUXNWXU XQG *U|آ‰H YRQ &DVHLQPLFHOOHQ LQ GHU 0LOFK YHUVFKLHGHQHU 6SHFLHV .LHOHU 0LOFKZLUWVFKDIWOLFKH )RUVFKXQJVEHULFKWH   %XUJHVV 7/ .HOO\ 5%  &RQVWLWXWLYH DQG UHJXODWHG VHFUHWLRQ RI SURWHLQV $QQXDO 5HYLHZ RI &HOO %LRORJ\   &RXUWKDXGRQ -/ *LUDUGHW -0 &KDSDO & /RULHQW ' /LQGHQ *  6XUIDFH DFWLYLW\ DQG FRPSHWLWLYH DGVRUSWLRQ RI PLON FRPSRQHQW  DQG SRUFLQH SDQFUHDWLF OLSDVH DW WKH GRGHFDQHZDWHU LQWHUIDFH ,Q )RRG 0DFURPROHFXOHV DQG &ROORLGV HG ( 'LFNLQVRQ SS  5R\DO 6RFLHW\ RI &KHPLVWU\ /RQGRQ 8. 'DOJOHLVK '*  &RDJXODWLRQ RI UHQQHWHG ERYLQH FDVHLQ PLFHOOHV GHSHQGHQFH RQ WHPSHUDWXUH FDOFLXP LRQ FRQFHQWUDWLRQ DQG LRQLF VWUHQJWK -RXUQDO RI 'DLU\ 5HVHDUFK   'DOJOHLVK '*  7KH HQ]\PDWLF FRDJXODWLRQ RI PLON ,Q $GYDQFHG 'DLU\ &KHPLVWU\ 3URWHLQV HG 3) )R[ SS  (OVHYLHU $SSOLHG 6FLHQFH /RQGRQ 8. 'DYH\ &$ )HQQD 5(   c UHVROXWLRQ ;UD\ FU\VWDO VWUXFWXUH RI WKH ELVXEVWUDWH DQDORJXH LQKLELWRU VDOLF\OK\GUR[DPLF DFLG ERXQG WR KXPDQ P\HORSHUR[LGDVH D PRGHO IRU D SUHUHDFWLRQ FRPSOH[ ZLWK K\GURJHQ SHUR[LGH %LRFKHPLVWU\   'DYLHV '5  7KH VWUXFWXUH DQG IXQFWLRQ RI WKH DVSDUWLF SURWHLQDVHV $QQXDO 5HYLHZ RI %LRSK\VLFV DQG %LRSK\VLFDO &KHPLVWU\   'H3LOOLV *' 2]DNL 6 .XR -0 0DOWE\ '$ 2UWL] GH 0RQWHOODQR 35  $XWRFDWDO\WLF SURFHVVLQJ RI KHPH E\ ODFWRSHUR[LGDVH SURGXFHV WKH QDWLYH SURWHLQERXQG SURVWKHWLF JURXS -RXUQDO RI %LRORJLFDO &KHPLVWU\   'H :LW -1 9DQ +RR\GRQN $&0  6WUXFWXUH IXQFWLRQV DQG DSSOLFDWLRQV RI ODFWRSHUR[LGDVH LQ QDWXUDO DQWLPLFURELDO V\VWHPV 1HWKHUODQGV 0LON 'DLU\ -RXUQDO   'RZEHQNR ' .LNXWD $ )HQQLH & *LOOHWW 1 /DVN\ /$  *O\FRV\ODWLRQGHSHQGHQW FHOO DGKHVLRQ PROHFXOH  *O\&$0  PXFLQ LV



5HIHUHQFHV

H[SUHVVHG E\ ODFWDWLQJ PDPPDU\ JODQG HSLWKHOLDO FHOOV DQG LV SUHVHQW LQ PLON -RXUQDO RI &OLQLFDO ,QYHVWLJDWLRQ   'XOO 7- 8\HGD & 6WURVEHUJ $' 1HGZLQ * 6HLOKDPHU --  0ROHFXODU FORQLQJ RI F'1$V HQFRGLQJ ERYLQH DQG KXPDQ ODFWRSHUR[LGDVH '1$ DQG &HOO %LRORJ\   ']LDUVNL 5 7DSSLQJ 5, 7RELDV 36  %LQGLQJ RI EDFWHULDO SHSWLGRJO\FDQ WR &' -RXUQDO RI %LRORJLFDO &KHPLVWU\   (LJHO :1 %XWOHU -( (UQVWURP &$ )DUUHOO +0 +DZDONDU 95 -HQQHVV 5 :KLWQH\ 5 0F/  1RPHQFODWXUH RI SURWHLQV RI FRZ PLON ILIWK UHYLVLRQ -RXUQDO RI 'DLU\ 6FLHQFH   (ODJDP\ (, 5XSSDQQHU 5 ,VPDLO $ &KDPSDJQH &3 $VVDI 5  $QWLEDFWHULDO DQG DQWLYLUDO DFWLYLW\ RI FDPHO PLON SURWHFWLYH SURWHLQV -RXUQDO RI 'DLU\ 5HVHDUFK   (ODJDP\ (, 5XSSDQQHU 5 ,VPDLO $ &KDPSDJQH &3 $VVDI 5  3XULILFDWLRQ DQG FKDUDFWHUL]DWLRQ RI ODFWRIHUULQ ODFWRSHUR[LGDVH O\VR]\PH DQG LPPXQRJOREXOLQV IURP FDPHOآ¶V PLON ,QWHUQDWLRQDO 'DLU\ -RXUQDO   (O'LQ 0= $RNL 7  +LJK SHUIRUPDQFH RI JHO DQG LRQH[FKDQJH FKURPDWRJUDSK\ RI EXIIDOR FDVHLQ ,QWHUQDWLRQDO 'DLU\ -RXUQDO   )$2 )RRG DQG $JULFXOWXUH 2UJDQL]DWLRQ RI WKH 8QLWHG 1DWLRQV  6WDWLVWLFDO 'DWDEDVHV RI  )$2 5RPH ,WDO\ )DUDK =  (IIHFW RI KHDW WUHDWPHQW RQ ZKH\ SURWHLQV RI FDPHO PLON 0LOFKZLVVHQVFKDIW   )DUDK = %DFKPDQQ 05  5HQQHW FRDJXODWLRQ SURSHUWLHV RI FDPHO PLON 0LOFKZLVVHQVFKDIW   )DUDK = 5آپHJJ 0:  7KH VL]H GLVWULEXWLRQ RI FDVHLQ PLFHOOHV LQ FDPHO PLON )RRG PLFURVWUXFWXUH   )DUDK = 6WUHLII 7 %DFKPDQQ 05  3UHSDUDWLRQ DQG FRQVXPHU DFFHSWDELOLW\ WHVWV RI IHUPHQWHG FDPHO PLON LQ .HQ\D -RXUQDO RI 'DLU\ 5HVHDUFK  

 )DUDK = $WNLQV '  +HDW FRDJXODWLRQ RI FDPHO PLON -RXUQDO RI 'DLU\ 5HVHDUFK   )DUDK =  &DPHO PLON 3URSHUWLHV DQG 3URGXFWV 6.$7 6W *DOOHQ 6ZLW]HUODQG )HQQD 5 =HQJ - 'DYH\ &  6WUXFWXUH RI WKH JUHHQ KHPH LQ P\HORSHUR[LGDVH $UFKLYHV RI ELRFKHPLVWU\ DQG ELRSK\VLFV   )HUUDQWL 3 0DORUQL $ 1LWWL * /DH]]D 3 3L]]DQR 5 &KLDQHVH / $GGHR )  3ULPDU\ VWUXFWXUH RI RYLQH خ±VFDVHLQV ORFDOLVDWLRQ RI SKRVSKRU\ODWLRQ VLWHV DQG FKDUDFWHULVDWLRQ RI JHQHWLF YDULDQWV $ & DQG ' -RXUQDO RI 'DLU\ 5HVHDUFK   )HUUDUL 53 *KLEDXGL (0 7UDYHUVD 6 /DXUHQWL ( 'H *LRLD / 6DOPRQD 0  6SHFWURVFRSLF DQG ELQGLQJ VWXGLHV RQ WKH LQWHUDFWLRQ RI LQRUJDQLF DQLRQV ZLWK ODFWRSHUR[LGDVH -RXUQDO RI LQRUJDQLF ELRFKHPLVWU\   )RONPDQ - 6KLQJ <  &RQWURO RI DQJLRJHQHVLV E\ KHSDULQ DQG RWKHU VXOIDWHG SRO\VDFFKDULGHV ,Q +HSDULQ DQG 5HODWHG 3RO\VDFFKDULGHV HGV '$ /DQH , %M|UN 8 /LQGDKO SS  3OHQXP 3UHVV 1HZ


5HIHUHQFHV

*LUDUGHW -0 /LQGHQ * /R\H 6 &RXUWKDXGRQ -/ /RULHQW '  6WXG\ RI PHFKDQLVP RI OLSRO\VLV LQKLELWLRQ E\ ERYLQH PLON SURWHRVHSHSWRQH FRPSRQHQW  -RXUQDO RI 'DLU\ 6FLHQFH   *LUDUGHW -0 6DXOQLHU ) 'ULRX $ /LQGHQ * &RGGHYLOOH % 6SLN *  +LJK SHUIRUPDQFH HOHFWURSKRUHVLV FKURPDWRJUDSK\ RI ERYLQH PLON FRPSRQHQW  JO\FRSURWHLQV -RXUQDO RI 'DLU\ 6FLHQFH   *LUDUGHW -0 &RGGHYLOOH % 3ODQFNH < 6WUHFNHU * &DPSDJQD 6 6SLN * /LQGHQ *  6WUXFWXUH RI JO\FRSHSWLGHV LVRODWHG IURP ERYLQH PLON FRPSRQHQW 33 (XURSHDQ -RXUQDO RI %LRFKHPLVWU\   *LUDUGHW -0 /LQGHQ *  33 FRPSRQHQW RI ERYLQH PLON D SKRVSKRU\ODWHG ZKH\ JO\FRSURWHLQ -RXUQDO RI 'DLU\ 5HVHDUFK    *RUEDQ $06 ,]]HOGLQ 20  0LQHUDO FRQWHQW RI FDPHO PLON DQG FRORVWUXP -RXUQDO RI 'DLU\ 5HVHDUFK   *UDXU ' +LJJLQV '*  0ROHFXODU HYLGHQFH IRU WKH LQFOXVLRQ RI FHWDFHDQV ZLWKLQ WKH RUGHU DUWLRGDFW\OD 0ROHFXODU %LRORJ\ DQG (YROXWLRQ   *URHQHQ 0$0 'LMNKRI 5-0 9DQ GHU 3RHO --  &KDUDFWHUL]DWLRQ RI D *O\&$0OLNH JHQH JO\FRV\ODWLRQGHSHQGHQW FHOO DGKHVLRQ PROHFXOH  ZKLFK LV KLJKO\ DQG VSHFLILFDOO\ H[SUHVVHG LQ WKH ODFWDWLQJ ERYLQH PDPPDU\ JODQG *HQH   *URYHV 05 'KDQDUDM 9 3LWWV -( %DGDVVR 0 1XJHQW 3 +RRYHU ' %OXQGHOO 7/ 7KH  c FU\VWDO VWUXFWXUH RI FK\PRVLQ LQKLELWRU FRPSOH[HG ZLWK D UHGXFHG LQKLELWRU VKRZV WKDW WKH DFWLYH VLWH EHWDKDLUSLQ IODS LV UHDUUDQJHG ZKHQ FRPSDUHG WR WKH QDWLYH VWUXFWXUH 7R EH SXEOLVKHG *XH[ 1 3HLWVFK 0&  6:,6602'(/ DQG WKH 6ZLVV3GE9LHZHU $Q HQYLURQPHQW IRU FRPSDUDWLYH SURWHLQ PRGHOOLQJ (OHFWURSKRUHVLV   *XVWFKLQD ($ 0DMHU 3 5XPVK /' *LQRGPDQ /0 $QGUHHYD 16  3RVW ;UD\ FU\VWDOORJUDSKLF VWXGLHV RI FK\PRVLQ VSHFLILFLW\ $GYDQFHV LQ ([SHULPHQWDO 0HGLFLQH DQG %LRORJ\  

 *XWLpUUH]$GiQ $ 0DJD ($ 0HDGH + 6KRHPDNHU &) 0HGUDQR -) $QGHUVRQ *% 0XUUD\ -'  $OWHUDWLRQV RI WKH SK\VLFDO FKDUDFWHULVWLFV RI PLON IURP WUDQVJHQLF PLFH SURGXFLQJ ERYLQH خ؛FDVHLQ -RXUQDO RI 'DLU\ 6FLHQFH   +DJUDVV $( +DVVDQ $$ 6RU\DO .$ 0HUYDW $6 (O6KDEUDZ\ 6$  &KHPLFDO FRPSRVLWLRQ RI IDW DQG EXWWHU RI FDPHOآ¶V PLON (J\SWLDQ -RXUQDO RI )RRG 6FLHQFH   +DPEUآ XV /  1XWULWLRQDO $VSHFWV RI 0LON 3URWHLQV ,Q $GYDQFHG 'DLU\ &KHPLVWU\ 3URWHLQV HG 3) )R[ SS  (OVHYLHU $SSOLHG 6FLHQFH /RQGRQ 8. +DQVHQ -( /XQG 2 (QJHOEUHFKW - %RKU + 1LHOVHQ -2 +DQVHQ - (6 %UXQDN 6  3UHGLFWLRQ RI 2JO\FRV\ODWLRQ RI PDPPDOLDQ SURWHLQV 6SHFLILFLW\ SDWWHUQV RI 8'3*DO1$FSRO\SHSWLGH 1 DFHW\OJDODFWRVDPLQ\OWUDQVIHUDVH %LRFKHPLFDO -RXUQDO   +DVVDQ $$ +DJUDVV $( 6RU\DO  .$ (O 6KDEUDZ\ 6$  3K\VLFRFKHPLFDO SURSHUWLHV RI FDPHO PLON GXULQJ ODFWDWLRQ SHULRG LQ (J\SW (J\SWLDQ -RXUQDO RI )RRG 6FLHQFH   +HUiQGH] 0&0 9DQ 0DUNZLMN %: 9UHHPDQ +-  ,VRODWLRQ DQG SURSHUWLHV RI ODFWRSHUR[LGDVH IRUP ERYLQH PLON 1HWKHUODQGV 0LON DQG 'DLU\ -RXUQDO   +LSS 1- *URYHV 0/ &XVWHU -+ 0F0HHNLQ 7/  6HSDUDWLRQ RI خ± خ² DQG خ³&DVHLQ -RXUQDO RI 'DLU\ 6FLHQFH   +RUL + )HQQD 5( .LPXUD 6 ,NHGD6DLWR 0  $URPDWLF VXEVWUDWH PROHFXOHV ELQG DW WKH GLVWDO KHPH SRFNHW RI P\HORSHUR[LGDVH -RXUQDO RI %LRORJLFDO &KHPLVWU\   +ROW & +RUQH '6  7KH KDLU\ FDVHLQ PLFHOOH (YROXWLRQ RI WKH FRQFHSW DQG LWV LPSOLFDWLRQV IRU GDLU\ WHFKQRORJ\ 1HWKHUODQGV 0LON DQG 'DLU\ -RXUQDO   +RUQH '6 3DUNHU 7* 'DOJOHLVK '*  &DVHLQ PLFHOOHV SRO\FRQGHQVDWLRQ DQG IUDFWDOV )RRG &ROORLGV  



5HIHUHQFHV

+RXHQ * 0DGVHQ 07 +DUORZ .: /RHQEODG 3 )ROWPDQQ %  7KH SULPDU\ VWUXFWXUH DQG HQ]\PLF SURSHUWLHV RI SRUFLQH SURFK\PRVLQ DQG FK\PRVLQ ,QWHUQDWLRQDO -RXUQDO RI %LRFKHPLVWU\ DQG &HOO %LRORJ\   +X 6 7UHDW 5: .LQFDLG -5  'LVWLQFW KHPH DFWLYHVLWH VWUXFWXUH LQ ODFWRSHUR[LGDVH UHYHDOHG E\ UHVRQDQFH 5DPDQ VSHFWURVFRS\ %LRFKHPLVWU\   ,QRX\H 0 $UQKHLP 1 6WHUQJODQ] 5  %DFWHULRSKDJH 7 O\VR]\PH LV DQ 1DFHW\OPXUDP\O/DODQLQH DPLGDVH -RXUQDO RI %LRORJLFDO &KHPLVWU\   -DNRE (  %H]LHKXQJHQ ]ZLVFKHQ GHP JHQHWLVFKHQ 3RO\PRUSKLVPXV GHU 0LOFKSURWHLQH XQG GHU /DEIlKLJNHLW YRQ 0LOFK 'LVV (7+ 1U  6ZLVV )HGHUDO ,QVWLWXWH RI 7HFKQRORJ\ =XULFK 6ZLW]HUODQG -DNRE (  *HQHWLF SRO\PRUSKLVP RI PLON SURWHLQV ,QWHUQDWLRQDO 'DLU\ )HGHUDWLRQ %XOOHWLQ   -HOHQ 3  :KLSSLQJ VWXGLHV ZLWK SDUWLDOO\ GHODFWRVHG FKHHVH ZKH\ -RXUQDO RI 'DLU\ 6FLHQFH   -RKQVHQ /% 6آ،UHQVHQ (6 3HWHUVHQ 7( %HUJOXQG /  &KDUDFWHUL]DWLRQ RI D ERYLQH PDPPDU\ JODQG 33 F'1$ UHYHDOV KRPRORJ\ ZLWK PRXVH DQG UDW DGKHVLRQ PROHFXOH *O\&$0 %LRFKLPLFD HW %LRSK\VLFD $FWD   -RKQVHQ /% 3HWHUVHQ 7( %HUJOXQG /  7KH ERYLQH 33 JHQH LV KRPRORJRXV WR WKH PXULQH *O\&$0 JHQH *HQH   -RVHSK\ 3'  7KH UROH RI SHUR[LGDVHFDWDO\]HG DFWLYDWLRQ RI DURPDWLF DPLQHV LQ EUHDVW FDQFHU 0XWDJHQHVLV   .DPRXQ 0  &KHHVH SURGXFWLRQ IURP FDPHOآ¶V PLON 2SWLRQV 0HGLWHUUDQHHQQHV $   .DPRXQ 0  'URPHGDU\ PLON SURGXFWLRQ TXDOLW\ DQG VXLWDELOLW\ IRU WUDQVIRUPDWLRQ 2SWLRQV 0HGLWHUUDQHHQQHV %  

 .DQJ ' /LX * /XQGVWU|P $ *HOLXV ( 6WHLQHU +  $ SHSWLGRJO\FDQ UHFRJQLWLRQ SURWHLQ LQ LQQDWH LPPXQLW\ FRQVHUYHG IURP LQVHFWV WR KXPDQV 3URFHHGLQJV RI WKH 1DWLRQDO $FDGHP\ RI 6FLHQFHV RI WKH 86$   .DQQR &  D &KDUDFWHUL]DWLRQ RI PXOWLSOH IRUPV RI ODFWRSKRULQ LVRODWHG IURP ERYLQH PLON ZKH\ -RXUQDO RI 'DLU\ 6FLHQFH   .DQQR &  E 3XULILFDWLRQ DQG VHSDUDWLRQ RI PXOWLSOH IRUPV RI ODFWRSKRULQ IURP ERYLQH PLON ZKH\ DQG WKHLU LPPXQRORJLFDO DQG HOHFWURSKRUHWLF SURSHUWLHV -RXUQDO RI 'DLU\ 6FLHQFH   .DQQR & 2JDZD +  3URWHRO\WLF GHJUDGDWLRQ RI ODFWRSKRULQ IURP ERYLQH PLON E\ SODVPLQ DQG WU\SVLQ -DSDQHVH -RXUQDO RI =RRWHFKQLFDO 6FLHQFH   .DSSHOHU 6 )DUDK = 3XKDQ =  6HTXHQFH DQDO\VLV RI &DPHOXV GURPHGDULXV PLON FDVHLQV -RXUQDO RI 'DLU\ 5HVHDUFK   .DZDPXUD . 6DWRZ + 'R ,/ 6DNDL 6 7DNDGD 6 2ELQDWD 0  0RGXODWLRQ RI WKH WUDQVIHUUHG PRXVH . FDVHLQ JHQH LQ PRXVH / FHOOV E\ JOXFRFRUWLFRLG KRUPRQH -RXUQDO RI %LRFKHPLVWU\   .HVWHU -- %UXQQHU -5  0LON IDWJOREXOH PHPEUDQH DV SRVVLEOH RULJLQ RI SURWHRVHSHSWRQH JO\FRSURWHLQV -RXUQDO RI 'DLU\ 6FLHQFH   .LVHOHY 6/ .XVWLNRYD 26 .RURENR (9 3URNKRUWFKRXN (% .DELVKHY $$ /XNDQLGLQ (0 *HRUJLHY *3  0ROHFXODU FORQLQJ DQG FKDUDFWHUL]DWLRQ RI WKH PRXVH WDJ JHQH HQFRGLQJ D QRYHO F\WRNLQH -RXUQDO RI %LRORJLFDO &KHPLVWU\   .QRHVV .+ 0DNKXGXP $- 5DILT 0 +DIHH] 0  0LON SURGXFWLRQ SRWHQWLDO RI WKH GURPHGDU\ ZLWK VSHFLDO UHIHUHQFH WR WKH SURYLQFH RI 3XQMDE 3DNLVWDQ :RUOG $QLPDO 5HYLHZ   .R]DN 0  7KH VFDQQLQJ PRGHO IRU WUDQVODWLRQ DQ XSGDWH -RXUQDO RI &HOO %LRORJ\  



5HIHUHQFHV

.XPRVLQVNL 7) %URZQ (0 )DUUHOO +0  7KUHHGLPHQVLRQDO PROHFXODU PRGHOLQJ RI ERYLQH FDVHLQV D UHILQHG HQHUJ\PLQLPL]HG خ؛FDVHLQ VWUXFWXUH -RXUQDO RI 'DLU\ 6FLHQFH   .XVWLNRYD 26 .LVHOHY 6/ %RURGXOLQD 25 6HQLQ 90 $IDQDVآ¶HYD $9 .DELVKHY $$  &ORQLQJ RI WKH WDJ JHQH H[SUHVVHG LQ PHWDVWDWLF PRXVH WXPRUV 5XVVLDQ -RXUQDO RI *HQHWLFV   /DVN\ /$ 6LQJHU 06 'RZEHQNR ' ,PDL < +HQ]HO :- *ULPOH\ & )HQQLH & *LOOHWW 1 :DWVRQ 65 5RVHQ 6'  $Q HQGRWKHOLDO OLJDQG IRU /VHOHFWLQ LV D QRYHO PXFLQOLNH PROHFXOH &HOO   /L + =KDQJ < 'RQJ <
 0DVVRQ 3/  /D ODFWRIHUULQH SURWHLQH GHV VHFUHWLRQV H[WHUQHV HW GHV OHXFRF\WHV QHXWURSKLOHV (GLWLRQV $UVFD 6$ %UXVVHOV %HOJLXP 0DWVXGDLUD 37  $ SUDFWLFDO JXLGH WR SURWHLQ DQG SHSWLGH SXULILFDWLRQ IRU PLFURVHTXHQFLQJ $FDGHPLF 3UHVV ,QF 6DQ 'LHJR 86$ 0HKDLD 0$  6WXGLHV RI FDPHO FDVHLQ PLFHOOHV WUHDWPHQW ZLWK VROXEOH DQG LPPRELOLVHG QHXUDPLQLGDVH &DUERK\GUDWH 3RO\PHUV    0HKDLD 0$  (Q]\PDWLF FRDJXODWLRQ RI FDPHO PLON $ VWXG\ XVLQJ VROXEOH DQG LPPRELOL]HG FK\PRVLQ 0LOFKZLVVHQVFKDIW   0HKDLD 0$  7KH IDW JOREXOH VL]H GLVWULEXWLRQ LQ FDPHO JRDW HZH DQG FRZ PLON 0LOFKZLVVHQVFKDIW   0RQWHW '  3URRI RI SDVWHXUL]DWLRQ RI FDPHO PLON  /HFWXUH KHOG GXULQJ D VHPLQDU RQ D SURMHFW SURSRVDO IRU D MRLQHG (8,*$' SURJUDPPH RQ FDPHO KXVEDQGU\ LQ (DVWHUQ $IULFD 0RRUH 6$ $QGHUVRQ %) *URRP &5 +DULGDV 0 %DNHU (1  7KUHHGLPHQVLRQDO VWUXFWXUH RI GLIHUULF ERYLQH ODFWRIHUULQ DW  $ UHVROXWLRQ -RXUQDO RI 0ROHFXODU %LRORJ\   0RUD*XWLHUUH] $ )DUUHOO +0 .XPRVLQVNL 7)  &RPSDULVRQ RI FDOFLXPLQGXFHG DVVRFLDWLRQV RI ERYLQH DQG FDSULQH FDVHLQV DQG WKH UHODWLRQVKLS RI DVFDVHLQ FRQWHQW WR FROORLGDO VWDELOLVDWLRQ D WKHUPRG\QDPLF OLQNDJH DQDO\VLV -RXUQDO RI 'DLU\ 6FLHQFH   0RUULVRQ :5  7KH GLVWULEXWLRQ RI SKRVSKROLSLGV LQ VRPH PDPPDOLDQ PLONV /LSLGV   1J :& %UXQQHU -5 5KHH .&  3URWHRVHSHSWRQH IUDFWLRQ RI ERYLQH PLON ODFWHXP VHUXP FRPSRQHQW   D ZKH\ JO\FRSURWHLQ -RXUQDO RI 'DLU\ 6FLHQFH   1LFKROVRQ 0: %DUFOD\ $1 6LQJHU 06 5RVHQ 6' 9DQGHU 0HUZH 3$  $IILQLW\ DQG NLQHWLF DQDO\VLV RI /VHOHFWLQ &'/ ELQGLQJ WR JO\FRV\ODWLRQGHSHQGHQW FHOODGKHVLRQ PROHFXOH -RXUQDO RI %LRORJLFDO &KHPLVWU\  



5HIHUHQFHV

1LHOVHQ + (QJHOEUHFKW - %UXQDN 6 YRQ +HLMQH *  ,GHQWLILFDWLRQ RI SURNDU\RWLF DQG HXNDU\RWLF VLJQDO SHSWLGHV DQG SUHGLFWLRQ RI WKHLU FOHDYDJH VLWHV 3URWHLQ (QJLQHHULQJ   1LVKLPXUD 7 7DNHVKLWD 1 6DWRZ + .RKRPRWR .  ([SUHVVLRQ RI WKH P& JHQH HQFRGLQJ *O\&$0 LQ WKH ODFWDWLQJ PRXVH PDPPDU\ JODQG -RXUQDO RI %LRFKHPLVWU\   1RSSH : +DQVVHQV , 'H &X\SHU 0  6LPSOH WZRVWHS SURFHGXUH IRU WKH SUHSDUDWLRQ RI KLJKO\ DFWLYH SXUH HTXLQH PLON O\VR]\PH -RXUQDO RI &KURPDWRJUDSK\   2DNOH\ ** 'HYDQDER\LQD 8 5REHUWVRQ /: *XSWD 5&  2[LGDWLYH '1$ GDPDJH LQGXFHG E\ DFWLYDWLRQ RI SRO\FKORULQDWHG ELSKHQ\OV 3&%V LPSOLFDWLRQV IRU 3&%LQGXFHG R[LGDWLYH VWUHVV LQ EUHDVW FDQFHU &KHPLFDO UHVHDUFK LQ WR[LFRORJ\   3kTXHW ' 1HMMDU < /LQGHQ *  6WXG\ RI D K\GURSKRELF SURWHLQ IUDFWLRQ LVRODWHG IURP PLON SURWHRVHSHSWRQH -RXUQDO RI 'DLU\ 6FLHQFH   3DXO .* 2KOVVRQ 3,  7KH FKHPLFDO VWUXFWXUH RI ODFWRSHUR[LGDVH ,Q 7KH /DFWRSHUR[LGDVH 6\VWHP &KHPLVWU\ DQG %LRORJLFDO 6LJQLILFDQFH HGV .0 3UXLWW -2 7HQRYXR SS  0DUFHO 'HNNHU ,QF 1HZ
 5DPHW -3  3URFHVVLQJ FDPHO PLON LQWR FKHHVH 5HYXH 0RQGLDOH GH =RRWHFKQLTXH   5DYDQLV 6 /HZLV 0-  2EVHUYDWLRQV RQ WKH HIIHFW RI UDZ PLON TXDOLW\ RQ WKH NHHSLQJ TXDOLW\ RI SDVWHXULVHG PLON /HWWHUV LQ $SSOLHG 0LFURELRORJ\   5DZOLQJV 1' %DUUHWW $-  )DPLOLHV RI DVSDUWLF SHSWLGDVHV DQG WKRVH RI XQNQRZQ FDWDO\WLF PHFKDQLVP 0HWKRGV LQ (Q]\PRORJ\    5LFKDUGVRQ 7 2K 6 -LPpQH])ORUHV 5  0ROHFXODU PRGHOOLQJ DQG JHQHWLF HQJLQHHULQJ RI PLON SURWHLQV ,Q $GYDQFHG 'DLU\ &KHPLVWU\ 3URWHLQV HG 3) )R[ SS  (OVHYLHU $SSOLHG 6FLHQFH /RQGRQ 8. 5RVHQ -0  0LON SURWHLQ JHQH VWUXFWXUH DQG H[SUHVVLRQ ,Q 7KH 0DPPDU\ *ODQG SS  (G 0& 1HYLOOH &: 'DQLHO 3OHQXP 3XEOLVKLQJ &RUSRUDWLRQ /RQGRQ 8. 6DLWR + 7DNDVH 0 7DPXUD < 6KLPDPXUD 6 7RPLWD 0  3K\VLFRFKHPLFDO DQG DQWLEDFWHULDO SURSHUWLHV RI ODFWRIHUULQ DQG LWV K\GURO\VDWH SURGXFHG E\ KHDW WUHDWPHQW DW DFLGLF S+ ,Q /DFWRIHUULQ 6WUXFWXUH DQG )XQFWLRQ HGV 7: +XWFKHQV 69 5XPEDOO % /|QQHUGDO SS  3OHQXP 3UHVV 1HZ


5HIHUHQFHV

6FKPLGW '* .RRSV -  3URSHUWLHV RI FDVHLQ PLFHOOHV  6WDELOLW\ WRZDUGV HWKDQRO GLDO\VLV SUHVVXUH DQG KHDW LQ UHODWLRQ WR FDVHLQ FRPSRVLWLRQ 1HWKHUODQGV 0LON DQG 'DLU\ -RXUQDO   6FKZDUW] +- 'LROL 0  7KH RQHKXPSHG FDPHO & GURPHGDULXV LQ (DVWHUQ $IULFD D SLFWRULDO JXLGH WR GLVHDVHV KHDOWK FDUH DQG PDQDJHPHQW 9HUODJ -RVHI 0DUJUDI :HLNHUVKHLP *HUPDQ\ 6KLPL]X 0
 6WURS 3 6HGODFHN - 6W\V - .DGHUDENRYD = %ODKD , 3DYOLFNRYD / 3RKO - )DEU\ 0 .RVWND 9 1HZPDQ 0 )UD]DR & 6KHDUHU $ 7LFNOH ,- %OXQGHOO 7/  (QJLQHHULQJ HQ]\PH VXEVLWH VSHFLILFLW\ SUHSDUDWLRQ NLQHWLF FKDUDFWHUL]DWLRQ DQG ;UD\ DQDO\VLV DW c UHVROXWLRQ RI 9DO3KH VLWHPXWDWHG FDOI FK\PRVLQ %LRFKHPLVWU\   6XJXUL 7 .LNXWD $ ,ZDJDNL +
 HGV '$ /DQH , %M|UN 86$

5HIHUHQFHV

8 /LQGDKO SS  3OHQXP 3UHVV 1HZ
:DQJRK - )DUDK = 3XKDQ =  ([WUDFWLRQ RI FDPHO UHQQHW DQG LWV FRPSDULVRQ ZLWK FDOI UHQQHW H[WUDFW 0LOFKZLVVHQVFKDIW   :DQJRK -  &KHPLFDO DQG 7HFKQRORJLFDO 3URSHUWLHV RI &DPHO &DPHOXV GURPHGDULXV 0LON 'LVV (7+ 1U  6ZLVV )HGHUDO ,QVWLWXWH RI 7HFKQRORJ\ =XULFK 6ZLW]HUODQG :LOOLDPV 0* :LOVKHU - 1XJHQW 3 0LOOV $ 'KDQDUDM 9 )DEU\ 0 6HGODFHN - 8XVLWDOR -0 3HQWWLOD 0( 3LWWV -( %OXQGHOO 7/  0XWDJHQHVLV ELRFKHPLFDO FKDUDFWHUL]DWLRQ DQG ;UD\ VWUXFWXUDO DQDO\VLV RI SRLQW PXWDQWV RI ERYLQH FK\PRVLQ 3URWHLQ (QJLQHHULQJ   :LOVRQ 7 $UD\D $ 0HODNX $  7KH 2QH+XPSHG &DPHO $Q $QDO\WLFDO DQG $QQRWDWHG %LEOLRJUDSK\ 8162 7HFKQLFDO 3XEOLFDWLRQ 8PEHUOHLJK 8.
 OLYHU FHOOV DQG SRWHQWLDWHV WKH LQKLELWLRQ RI EHWD YHU\ ORZ GHQVLW\ OLSRSURWHLQ ELQGLQJ -RXUQDO RI %LRORJLFDO &KHPLVWU\  

  &855,&8/80 9,7$(

&XUULFXOXP 9LWDH

  آ±   آ±   آ±  $XWXPQ  6SULQJ 

 آ± 

 آ± 

%RUQ DV VHFRQG FKLOG RI 5REHUW DQG 6XVDQQH .DSSHOHU 0RVEHUJHU LQ =XULFK 6ZLW]HUODQG 3ULPDU\ 6FKRRO %DGHQ 6ZLW]HUODQG 6HFRQGDU\ 6FKRRO %DGHQ 6ZLW]HUODQG +LJK 6FKRRO %DGHQ 6ZLW]HUODQG &RPSOHWHG ZLWK D 0DWXUD $ 0LOLWDU\ (GXFDWLRQ DW WKH 'LYLVLRQ IRU $UWLOOHU\ )UDXHQIHOG 6ZLW]HUODQG 6WXG\ RI (QJOLVK /DQJXDJH DW ,QWHUQDWLRQDO +RXVH +DVWLQJV 8. &RPSOHWHG ZLWK &DPEULGJH )LUVW &HUWLILFDWH LQ (QJOLVK $UHOV +LJKHU &HUWLILFDWH LQ 6SRNHQ (QJOLVK DQG &RPSUHKHQVLRQ DQG 2[IRUG ([DPLQDWLRQ +LJKHU /HYHO 6WXG\ RI *HQHUDO %LRORJ\ DW WKH 6ZLVV )HGHUDO ,QVWLWXWH RI 7HFKQRORJ\ =XULFK 6ZLW]HUODQG &RPSOHWHG ZLWK D 'LSORPD 7KHVLV LQ 3ODQW *HQHWLFV WLWOHG آ³&LUFDGLDQ 2VFLOODWLRQV RI $WJUS DQG $WJOS WUDQVFULSWV LQ $UDELGRSVLV WKDOLDQD DQG ,PPXQRF\WRFKHPLFDO /RFDOLVDWLRQ RI WKH &RUUHVSRQGLQJ 3URWHLQV LQ 6LQDSLV DOEDآ´ DQG ZLWK D 'LSORPD LQ 1DWXUDO 6FLHQFHV &HOO %LRORJ\ 0ROHFXODU %LRORJ\ 0LFURELRORJ\ 3ODQW 3K\VLRORJ\ %LRFKHPLVWU\ DQG %LRWHFKQRORJ\ $VVLVWDQW 5HVHDUFK :RUN DW WKH ,QVWLWXWH RI )RRG 7HFKQRORJ\ 6ZLVV )HGHUDO ,QVWLWXWH RI 7HFKQRORJ\ =XULFK 6ZLW]HUODQG

pdf

Journal of Dairy Research (1990), 57, 281-283 Printed in Great Britain

281

SHORT COMMUNICATION Preparation and consumer acceptability tests of fermented camel milk in Kenya
By ZAKARIA FARAH, THOMAS STREIFF AND MARC R. BACHMANN Laboratory of Dairy
Science, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zأ¼rich, Switzerland

(Received 3 April 1989 and accepted for publication 16 October 1989)

There are estimated to be 600000 camels (Camelus dromedarius) in Kenya (Wandera, 1985). Almost 80% of these are kept by pastoral tribes living in arid areas in eastern and north-eastern parts of the country. In these regions, camels are important dairy animals. A camel in north-east Kenya can be expected to yield about 4 kg milk daily as compared with 0.5-1.5 kg for a cow in the same area. Most of the camel milk is consumed in the form of fermented milk. The milk is allowed to ferment naturally at ambient temperature and without prior heat treatment until it turns sour. The resulting fermented camel milk is known as Susa. Due to the spontaneous nature of the fermentation, this traditional method results in a product with varying taste and flavour and is often of poor hygienic quality. In addition, because of the limited scale of production, the product can be sold only in the immediate vicinity of the herd. For production of fermented milk under controlled conditions, thermophilic or mesophilic lactic acid cultures are normally used. In warm countries, mesophilic lactic cultured milk offers some advantages, as it can be incubated at ambient temperature (20-30 آ°C) and the fermentation stops at 1-1.2 % lactic acid, eliminating the need for cooling to stop further souring as occurs in the case of yoghurt (Kurwijila, 1980). Considering these advantages, Kurwijila (1980) developed, in Kenya, fermented cows' milk using mesophilic lactic cultures. Tests conducted with adult Kenyans showed high consumer acceptability. The present investigation was undertaken in Garissa, a north-eastern province town of Kenya, where a majority of the population subsists almost entirely on camel milk. The objective of this study was to develop fermented camel milk using mesophilic lactic culture and to test the acceptability of the product in comparison with the traditional fermented camel milk.

EXPERIMENTAL Milk samples Fresh camel milk was obtained from herds owned by nomads around the town of Garissa.

282

Z. FARAH AND OTHERS

Cultures Multiple strain, mixed type, lyophilized, mesophilic lactic cultures, O-CH:143 (homofermentative) and B-CH:40 (heterofermentative) were obtained from Chr. Hansen's Laboratorium, Denmark. Preparation of fermented milk Two 10-litre churns of fresh whole camel milk were placed in a container filled with water and heated until the milk attained 85آ°C. This temperature was maintained for 30 min. After cooling in a water bath to ambient temperature, each milk churn was inoculated with 2 % of a 24 h culture and incubated at a room temperature of 27آ°C for 24 h. Chemical analysis For each milk the following parameters were determined; acidity by titration expressed in Soxhlet-Henkel degrees (آ° SH), fat by the Gerber method, total solids by calculation after Fleischmann (1896) from the values of fat content, and specific gravity using a lactometer. The same determinations were also made with the traditionally fermented camel milk, Susa, from the local market. Sensory evaluation In the sensory tests, the two fermented camel milk samples were compared with a traditionally fermented camel milk, Susa. Two groups of people were selected for the sensory evaluation. Group A consisted of 13 Somali nomads with no formal education. All claimed to consume Susa regularly. Group B consisted of nine Somalis and three Canadians. They all worked as senior officers in the Provincial and District Administrations. Seven of the group reported consuming Susa regularly and five only occasionally. As the panellists had no previous experience of testing products, the rating test was simplified and limited in respect of consumer preference. Each person was asked to taste the three coded samples and score each product for preference on a threepoint scale ranging from' most preferred' (preference score = 1) to 'least preferred' (preference score = 3). The instructions were given orally in both the Somali and English languages. The panellists were told that they were testing camel milk, but the identities of the individual samples were revealed only after the tests were completed.

RESULTS AND DISCUSSION Compared with cows' milk, the consistency of fermented camel milk is thin. After fermentation a precipitate in the form of flocs was formed rather than a coagulum. In a preliminary experiment an attempt was made to improve the consistency by the addition of cows' milk powder. However, this had to be omitted in the final experiments, as sensory evaluation and conversation with consumers of camel milk revealed that mixing camel with cows' milk affected the typical camel milk taste and was undesired. Results of chemical and sensory analysis arc presented in Table 1. The values of total solids and fat content were the same in all the samples. Homofermentative culture O-CH:143 showed less titratable acidity. Fermented milk made with mesophilic lactic cultures was clearly preferred by both groups. Within the two

Fermented camel milk

283

Table 1. Test panel preference scores of three fermented samples of camel milk
Fermented samples of camel milk Mesophilic lactic cultures Sensory evaluation Group A, n = 13 Mean score s.d. Group B, n = 12 Mean score s.d. Chemical analysis Total solids, % Fat content, % Acidity آ°SH O-CH:143 B-CH:40 Susa

2.08 0.76 1.58 0.67 12.7 4.1 36.2

1.39 0.77 1.50 0.52 12.7 4.0 40.0

2.31 0.86 2آ·92 0.29 12.5 4.0 40.0

starter cultures used, the homofermentative culture O-CH:143 was least preferred. Both groups described the two prepared cultured milks as particularly good Susa with uniform fresh taste. The study shows that the traditional Susa can be improved by using selected mesophilic lactic acid culture. Seasonal variations in camel milk production are great in north-eastern Kenya, and' much surplus milk is wasted during the rainy season (R. Muriuki, pers. comm.). The method described here for fermented milk production can be introduced in rural areas. It allows small-holder producers to process surplus milk on-farm or in centralized small-scale units. Obtaining starter culture could be a limiting factor for large-scale production of fermented milk. However, simple commercial systems for producing frozen starter cultures which maintain their activity for years are in operation in Kenya (Kurwijila, 1983; Schulthess, 1988). We thank the staff of Garissa Community Service Centre in Kenya for providing us with all the facilities during our field work. REFERENCES
FLEISCHMANN, W. 1896 The Book of the Dairy, pp. 71-72. KURWIJILA, R. L. N. 1980 Low cost optimization of the flavour, consistency and keeping quality of fermented milk with particular reference to consumer acceptability in Kenya. M.Sc. Thesis, University of Nairobi KURWIJILA, R. L. N. 1983 Maintenance of reactivated, lyophilised mixed type lactic cultures by subculturing and by cold storage. Indian Journal of Dairy Science 36 338-343 SCHULTHESS, W. 1988 [Appropriate milk product development-an example in Kenya.] LebensmittelTechnologie 21 226-228 W ANDERA, J. G. 1985 Camel pastoralism in Kenya. In Significance and Prospects of Camel Pastoralism in Kenya pp. 61-62 (Ed. S. E. Migot-Adholla), Nairobi, Kenya: Institute for Development Studies

pdf

30
Internat. J. Vit. Nutr. Res. 62 (1992) 30-33 Received for publication September 5, 1991

Z. FARAH et al: Vitamin Content of Camel Milk
Camel milk Vitamin A Vitamin B2 Vitamin E Vitamin C

Vitamin Content of Camel Milk
Z. FARAH1, R. RETTENMAIER2 and D. ATKINS3
1

Laboratory of Dairy Science, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zأ¼rich (Switzerland) 2 R + D Department of Vitamins and Fine Chemicals, F. Hoffmann-La Roche Ltd., CH-4002 Basle (Switzerland) 3 Ol Maisor Farm, P.O. Box 9, Rumuruti (Kenya)

Summary: The content of vitamin C, vitamin B2 and fat-soluble vitamins E and A in camel milk was studied. The milk samples were collected from 20 individual camels (Camelus dromedarius) in two different occasions. The study showed that camel milk contains considerably less vitamin A and B2 than cow milk while the content of vitamin E was about the same level. The level of vitamin C was in average three times higher than that of cow milk.

Introduction

According to FAO Statistics there are 17 million camels (Camel us dromedarius) in the world, of which 12.2 million are in Africa and 4.8 million in Asia [1]. The camel is an important source of milk. Indeed, in some countries hosting large camel populations, camel milk is one of the main components of the human diet. Milk production varying between 1800 and 12700 kg during a lactations period between 9 and 18 months has been reported [2]. Available information concerning camel milk is mainly limited to data on gross composition. Information on the nutritional quality of camel milk, especially on important minor constituents, such as vitamins, is scarce. The present investigation was undertaken to study the content of the water soluble vitamins C, B2 and some fat soluble vitamins A and E in camel milk.

Material and Methods Milk samples: Camel milk samples were taken at 01 Maisor Camel Farm, which is situated just North of the Equator in Kenya's Laikipia District and at an altitude of between 1767 and 1889 m above sea level. The animals of indigenous breed (Camelus dromedarius) were fed all year around exclusively by grazing. The milk samples were collected from 20 individual camels in two different occasions. The samples were kept refrigerated at 4آ°C and transported to our laboratory within 24 hours. Prior to refrigeration, all the samples for vitamin C determination were stabilised with 10% metaphosphoric acid. Upon arrival, the milk samples were stored at -20آ°C until analysis.

Z. FARAH et al: Vitamin Content of Camel Milk

31

Analytical methods: Both retinol and خ±-tocopherol were determined on the same sample extract on the milk specimen after saponification, but with different HPLC conditions. The deep frozen milk samples were warmed to about 35آ°C and mixed to obtain homogeneous distribution of milk fat. In 30 ml centrifuge tubes with stoppers 5g of milk were mixed with 6 ml of ethanol abs. and 200 mg of ascorbic acid. The tubes were heated to 80آ°C in a water bath for 5 min. under N2 and stirred using a magnetic stirrer. Then I ml of NaOH, 12.5 M was added and the mixture saponified for 20 minutes. After cooled to room temperature, the saponification mixture was extracted with a mixture of 10 ml nhexane and toluene 1:1 by shaking mechanically during 10 minutes at a frequency of250 strokes/min. 6 ml of H2O dest. were added, the tubes inverted several times and centrifuged. The clear organic phase was subjected to HPLC separations. Retinol was determined under the following HPLC condition:
Column: Stationary phase: Mobile phase: Flow: Pressure: Injection: Injection volume: Integrator: Calculation: Standard: Retention time: Run time/sample: Hibar RT (125-4 mm) LiChrosorb Si 60, 51lm, combined with LiChro-CART 4-4 mm as precolumn (MERCK) 2.5070 i-propanol in n-hexane 1.3 ml/min. approx. 32 bar Automatic, autosampler WISP 712^(WATERS) 20-80 III Spectrofluorometer 650-10 LC (PERKIN-ELMER), excitation 330 nm, emission 408 nm SPECTRA-PHYSICS SP 4270 External standard method, peak area O.4آµg of retinol/ml n-hexane containing 0.02070 butyl-hydroxy-toluene (BHT) as antioxidant 4.4 min. 10 min.

خ±-tocopherol was determined under the following HPLC condition:
Column: Stationary phase: Mobile phase: Flow: Pressure: Injector: Injection: Injection volume: Integrator: Calculation: Standard: Retention time: Run time/sample: Hibar RT (125-4 mm) LiChrosorb Si 60, 5 11m, combined with gard column 20-4 mm Si 60, 5 11m (STAGROMA WALLISELLEN, SWITZERLAND) 3070, l,4-dioxane in n-hexane 1.6 ml/min. approx. 50 bar Automatic, autosampler WISP 712^(WATERS) 20-80 III Spectrofluorometer 650-10 LC (PERKIN-ELMER), excitation 295 nm, emission 330 nm SPECTRA-PHYSICS SP 4270 External standard method, peak area I آµg of dl-خ±-tocopherol/ml n-hexane containing 0.01070 BHT as antioxidant 4.7 min.. 15 min

The concentration of vitamin B2 (riboflavin) was estimated by taking advantage of the strong fluorescence of the vitamin. To hydrolyse FAD and protein bindings trichloracetic acid was used as described by RETTENMAIER et al [3]. Vitamin C (ascorbic acid) was measured fluorometrically after it has been oxidized with iodine to dehydroascorbic acid, which is then coden sed with orthophenylenediamine to form a fluorescent quinoxaline. Essentially the same method as described by BRUBACHER and VUILLEUMIER [4] for plasma vitamin C was followed. However, the stabilisation of the vitamin in milk was performed by diluting I volume of milk with I volume of metaphosphoric acid 10070 (w/v).

32

Z. FARAH et al: Vitamin Content of Camel Milk

Results and Discussion The coefficient of variation (CV) calculated on 20 double determinations were found to be آ±4.45OJo for retinol and آ±4.05% for خ±-tocopherol. For riboflavin and ascorbic acid, CV was found to be آ± 2.83% and آ± 1.22% (10 double determinations) respectively. Recoveries of added dl-خ±-tocopherol and riboflavin were 98.1% آ±5.7, n=5 and 94.7% آ± 3.4, n = 6 respectively. The results obtained using the above described method I for vitamin A and E correspond with those of a method which includes exhaustive extraction [5]. However, in cases of very low vitamin A levels (e.g. 0.05 آµg/g) exhaustive extraction led to slightly lower vitamin A values (max. 15%). This might be due to losses during concentration of the large volume associated with the repeated extraction steps. The table shows the vitamin content of 20 individual samples of camel milk as well as the corresponding values in cow milk [6]. The mean values of the vitamins A, B2, E and Care 0.1,0.57,0.56 and 37.4 mg/l respectively. Comprehensive information on vitamin content in milk of the dromedary type camel is not available. The only published work comparable with our findings is the

Table: Vitamin content of camel milk (Figures in parenthesis are the values for cow milk) Vitamin in Sample mg/I No. A B2 E C 1 0.14 0.69 0.39 34.0 2 0.11 0.55 0.49 32.6 3 0.11 0.72 0.60 33.5 4 0.05 0.56 0.21 39.5 5 0.08 0.45 0.27 32.3 6 0.11 0.56 0.64 34.5 7 0.09 0.76 0.48 41.5 8 0.11 0.45 0.36 35.0 9 0.07 0.56 0.40 36.6 10 0.12 0.44 0.56 32.5 11 0.13 0.78 0.91 36.0 12 0.09 0.56 0.57 35.7 13 0.07 0.59 0.40 61.1 14 0.07 0.69 0.91 26.2 15 47.0 16 0.08 0.46 0.53 31.6 17 0.12 0.53 0.75 37.3 18 0.12 0.43 0.69 35.2 19 0.09 0.52 0.72 47.0 20 0.08 0.58 0.73 Mean 0.10 0.57 0.56 37.4 (0.27) (1.56) (0.60) (11.0) Range 0.05-0.14 0.43-0.78 0.21-0.91 26.2-61.1 (0.17-0.38) 1.16-2.02) (0.2-1.0) (3-23)

Z. FARAH et al: Vitamin Content of Camel Milk

33

report of SAWAYA el al [7] who studied the vitamin content of II Saudi dromedaries and found mean values of 0.15,0.42 and 24 mg/kg for vitamin A, 82 and C respectively. No data on the content of vitamin E is given in the report. From the results of the present investigation it can be concluded that camel milk contains less vitamin A and 82 than cow milk while the content of vitamin E is about the same level. The level of vitamin C is in average three times higher than that of cow milk. The availability of a relatively fair amount of vitamin C (average 37.4 mg/l) in camel milk is of significant relevance from the nutritional standpoint in the arid areas where fruits and vegetables containing vitamin C are scarce.
References 1. FAO Production Yearbook, FAO Rome (1978). 2. YAGIL, R. (1982) Camel Milk, FAO Production and Health Paper 26,9. 3. RETTENMAIER, R. and VUILLEUMIER, J. P. (1983) A simple Method for the Determination of Riboflavin in Human Milk, Internal. J. Vit. Nutr. Res. 53, 32-35. 4. BRUBACHER, G. and VUILLEUMIER, J. P. (1974) Vitamin C, in Clinical Biochemistry, Principles and Methods (Curtius, H. Ch., Roth, M., eds.), Vo. II, pp. 989-997, Walter de Gruyter, Berlin, New York. 5. DOSTأپLOVأپ, L., SALMENPERA, L., VأپCLAVINKOVأپ, V., HEINZ-ERIAN, P. and SCHUEP, W. (1988) Vitamins and Minerals, in Pregnancy and Lactation (Berger, H., ed.), Nestle Nutrition Workshop Series, Vol. 16, Nestlأ© Ltd., Vevey-Raven Press, Ltd., New York. 6. Ciba-Geigy AG (1977) Wissenschaftl. Tabellen, p. 211, Ciba-Geigy AG, Basel. 7. SAWAYA, W. N., KHALIL, J. K., AL-SHALAHAT, A. and AL-MOHAMMED, H. (1984), Chemical Composition and Nutritional Quality of Camel Milk, J. of Food Sci. 49, 744-747. Dr. Z. Farah, Laboratory of Dairy Science, Swiss Federal Institute of Technology, ETHZentrum, CH-8092 Zأ¼rich (Switzerland)

pdf

Milk and Meat from the Camel

Handbook on Products and Processing The book is based on results of research work carried out in partnership with universities, camel farmers and pastoralists in Eastern African countries. The first part deals with chemical composition, technological properties, hygienic safetyandqualitycontrolofcamelmilk.Thisisfollowedbythepresentationofprocessing methods for different dairy products which should allow smallholder producers to process milk on farm or in centralised small scale dairy unit. In the second part special knowledge gained in slaughtering, deboning and processing of camels on-site over several years of long-term stays on the Ol Maisor Farm in Kenya is presented. During the development of camel meat products the authors set a great value on the keeping quality of the products as well as creating a wide range of product groups with low and high cost for the local market and a good shelf life. The book presents background information and recipes on the manufacture of camel milk and meat products and it is intended as a practical guide to professionals, government bodies and development agencies interested in building up small scale processing units for camel meat and milk products. It is hoped that it may also serve as a reference for extension personal working with camel herders, food scientists and students. For further information please contact the author: zakaria.farah@ilw.agrl.ethz.ch published: February 2004 232 p., 17 x 24 cm, hardcover, many tables, recipes and 4-coloured figures US/Europe: CHF 47.­/EUR 32.­ (D)/USD 33.50, plus freight and taxes Rest of the world: USD 25.­, plus freight and taxes ISBN 3 7281 2527 X

Zakaria Farah, Albert Fischer (eds.)


Where to order the book The book can be obtained: In West Africa Dr. Bassirou Bonfoh of the Institut du Sahel Bamako Mali bassirou@agrosoc.insah.org

In East Africa Direct link to the online bookshop: here Legacy books, Yaya Centre, 2nd floor P.O. BOX 68077, Nairobi Kenya http://www.legacybookshop.com Tel: 254-2-573993 Fax: 254-2-573993 E-mail: info@legacybookshop.com

In Europe, The Americas and Asia

M

vdf Hochschulverlag AG an der ETH Zürich

R

O

Send your order to: Send your order to: vdf Hochschulverlag AG an der ETH Zürich, ETH Zentrum, CH-8092 Zürich, Tel. +41/1/632 42 42 Fax +41/1/632 12 32 E-Mail: verlag@vdf.ethz.ch F

I/We order from vdf Hochschulverlag AG, ETH Zentrum, CH-8092 Zürich: __ Ex. Milk and Meat from the Camel Zakaria Farah, Albert Fischer (eds.) US/Europe: CHF 47.­/EUR 32.­ (D)/USD 33.50, plus freight and taxes Rest of the world: USD 25.­, plus freight and taxes I will pay By bank transfer (only upon receipt of the invoice)

By credit card No. name address place date/signature VISA American Express Mastercard Expir. date

/

R

E

www.vdf.ethz.ch

D

R

O

Prices are subject to change


TABLE OF CONTENTS Dedication Acknowledgement Preface An introduction to the camel Part A: Camel as a milk animal 1. Milk 1.1 Milk production 1.2 Milk composition 2. Milk products 2.1 Introduction 2.2 Cheese 2.3 Fermented milk 2.4 Butter 3. Methods for quality control 3.1 Introduction 3.2 Determination of milk freshness and hygienic quality 3.3 Milk adulteration 4. Milk Hygiene and Udder Health 4.1 Milk Hygiene 4.2 Udder Health 5. Equipment for small scale milk plants 5.1 Introduction 5.2 Milk collection 5.3 Small-scale milk processing systems 5.4 Equipment requirement and specification Part B: Camel as a meat animal 6. Traditional slaughter, carcass dressing and processing of camels 6.1 Locations for slaughter of camels

6.2 The traditional method of slaughter for camels 6.3 Traditional dressing of the carcass 6.4 Traditional meat products in Africa ands Asia

7. Method for hygienic slaughter of camels 7.1 Requirementstobesatisfiedbyslaughterhouses 7.2 Slaughter procedure 7.3 The dressing percentage of camels 7.4 By-products from slaughter of camels

8.Dressing of the camel carcass 8.1 Requirements to be satisfied by deboning rooms and equipment 8.2 Cutting of the camel into primal cuts 8.3 Deboning of the camel 8.4 Cutting of the camel into retail cuts 8.5 Standardization of processing material 8.6 Percentageofretailcuts,processingmaterial and remaining parts 8.7 Camel meat composition

9.Meat products from camel meat 9.1 Product groups 9.2 Equipment needed for meat products 9.3 Additives and ingredients for meat products 9.4 Casings for meat products 9.5 Recipes for camel meat products

Recommended references About the Authors


pdf

Milk and Meat from the Camel
Handbook on Products and Processing
Zakaria Farah, Albert Fischer (eds.)
The book is based on results of research work carried out in partnership with universities, camel farmers and pastoralists in Eastern African countries. The first part deals with chemical composition, technological properties, hygienic safety and quality control of camel milk. This is followed by the presentation of processing methods for different dairy products which should allow smallholder producers to process milk on farm or in centralised small scale dairy unit. In the second part special knowledge gained in slaughtering, deboning and processing of camels on-site over several years of long-term stays on the Ol Maisor Farm in Kenya is presented. During the development of camel meat products the authors set a great value on the keeping quality of the products as well as creating a wide range of product groups with low and high cost for the local market and a good shelf life. The book presents background information and recipes on the manufacture of camel milk and meat products and it is intended as a practical guide to professionals, government bodies and development agencies interested in building up small scale processing units for camel meat and milk products. It is hoped that it may also serve as a reference for extension personal working with camel herders, food scientists and students. For further information please contact the author: zakaria.farah@ilw.agrl.ethz.ch published: February 2004 232 p., 17 x 24 cm, hardcover, many tables, recipes and 4-coloured figures US/Europe: CHF 47.–/EUR 32.– (D)/USD 33.50, plus freight and taxes Rest of the world: USD 25.–, plus freight and taxes ISBN 3 7281 2527 X

Where to order the book
The book can be obtained: In West Africa Dr. Bassirou Bonfoh of the Institut du Sahel Bamako Mali bassirou@agrosoc.insah.org

In East Africa Direct link to the online bookshop: here Legacy books, Yaya Centre, 2nd floor P.O. BOX 68077, Nairobi Kenya http://www.legacybookshop.com Tel: 254-2-573993 Fax: 254-2-573993 E-mail: info@legacybookshop.com

In Europe, The Americas and Asia

F O R M

I/We order from vdf Hochschulverlag AG, ETH Zentrum, CH-8092 Zأ¼rich:

__ Ex. Milk and Meat from the Camel
Zakaria Farah, Albert Fischer (eds.) US/Europe: CHF 47.–/EUR 32.– (D)/USD 33.50, plus freight and taxes Rest of the world: USD 25.–, plus freight and taxes I will pay By bank transfer (only upon receipt of the invoice) By credit card VISA American Express Mastercard Expir. date / No. name address place date/signature

vdf Hochschulverlag AG an der ETH Zأ¼rich Send your order to: Send your order to: vdf Hochschulverlag AG an der ETH Zأ¼rich, ETH Zentrum, CH-8092 Zأ¼rich, Tel. +41/1/632 42 42 Fax +41/1/632 12 32 E-Mail: verlag@vdf.ethz.ch

www.vdf.ethz.ch

O R D E R

Prices are subject to change

TABLE OF CONTENTS
Dedication Acknowledgement Preface An introduction to the camel Part A: Camel as a milk animal 1. Milk 1.1 Milk production 1.2 Milk composition 2. Milk products 2.1 Introduction 2.2 Cheese 2.3 Fermented milk 2.4 Butter 3. Methods for quality control 3.1 Introduction 3.2 Determination of milk freshness and hygienic quality 3.3 Milk adulteration 4. Milk Hygiene and Udder Health 4.1 Milk Hygiene 4.2 Udder Health 5. Equipment for small scale milk plants 5.1 Introduction 5.2 Milk collection 5.3 Small-scale milk processing systems 5.4 Equipment requirement and specification Part B: Camel as a meat animal 6. Traditional slaughter, carcass dressing and processing of camels 6.1 Locations for slaughter of camels Recommended references About the Authors 6.2 The traditional method of slaughter for camels 6.3 Traditional dressing of the carcass 6.4 Traditional meat products in Africa ands Asia 7. Method for hygienic slaughter of camels 7.1 Requirements to be satisfied by slaughterhouses 7.2 Slaughter procedure 7.3 The dressing percentage of camels 7.4 By-products from slaughter of camels 8. Dressing of the camel carcass 8.1 Requirements to be satisfied by deboning rooms and equipment 8.2 Cutting of the camel into primal cuts 8.3 Deboning of the camel 8.4 Cutting of the camel into retail cuts 8.5 Standardization of processing material 8.6 Percentage of retail cuts, processing material and remaining parts 8.7 Camel meat composition 9. Meat products from camel meat 9.1 Product groups 9.2 Equipment needed for meat products 9.3 Additives and ingredients for meat products 9.4 Casings for meat products 9.5 Recipes for camel meat products

pdf

Milk and Meat from the Camel
Handbook on Products and Processing
Zakaria Farah, Albert Fischer (eds.)
The book is based on results of research work carried out in partnership with universities, camel farmers and pastoralists in Eastern African countries. The first part deals with chemical composition, technological properties, hygienic safety and quality control of camel milk. This is followed by the presentation of processing methods for different dairy products which should allow smallholder producers to process milk on farm or in centralised small scale dairy unit. In the second part special knowledge gained in slaughtering, deboning and processing of camels on-site over several years of long-term stays on the Ol Maisor Farm in Kenya is presented. During the development of camel meat products the authors set a great value on the keeping quality of the products as well as creating a wide range of product groups with low and high cost for the local market and a good shelf life. The book presents background information and recipes on the manufacture of camel milk and meat products and it is intended as a practical guide to professionals, government bodies and development agencies interested in building up small scale processing units for camel meat and milk products. It is hoped that it may also serve as a reference for extension personal working with camel herders, food scientists and students. For further information please contact the author: zakaria.farah@ilw.agrl.ethz.ch published: February 2004 232 p., 17 x 24 cm, hardcover, many tables, recipes and 4-coloured figures US/Europe: CHF 47.–/EUR 32.– (D)/USD 33.50, plus freight and taxes Rest of the world: USD 25.–, plus freight and taxes ISBN 3 7281 2527 X

Where to order the book
The book can be obtained: In West Africa Dr. Bassirou Bonfoh of the Institut du Sahel Bamako Mali bassirou@agrosoc.insah.org

In East Africa Direct link to the online bookshop: here Legacy books, Yaya Centre, 2nd floor P.O. BOX 68077, Nairobi Kenya http://www.legacybookshop.com Tel: 254-2-573993 Fax: 254-2-573993 E-mail: info@legacybookshop.com

In Europe, The Americas and Asia

F O R M

I/We order from vdf Hochschulverlag AG, ETH Zentrum, CH-8092 Zأ¼rich:

__ Ex. Milk and Meat from the Camel
Zakaria Farah, Albert Fischer (eds.) US/Europe: CHF 47.–/EUR 32.– (D)/USD 33.50, plus freight and taxes Rest of the world: USD 25.–, plus freight and taxes I will pay By bank transfer (only upon receipt of the invoice) By credit card VISA American Express Mastercard Expir. date / No. name address place date/signature

vdf Hochschulverlag AG an der ETH Zأ¼rich Send your order to: Send your order to: vdf Hochschulverlag AG an der ETH Zأ¼rich, ETH Zentrum, CH-8092 Zأ¼rich, Tel. +41/1/632 42 42 Fax +41/1/632 12 32 E-Mail: verlag@vdf.ethz.ch

www.vdf.ethz.ch

O R D E R

Prices are subject to change

TABLE OF CONTENTS
Dedication Acknowledgement Preface An introduction to the camel Part A: Camel as a milk animal 1. Milk 1.1 Milk production 1.2 Milk composition 2. Milk products 2.1 Introduction 2.2 Cheese 2.3 Fermented milk 2.4 Butter 3. Methods for quality control 3.1 Introduction 3.2 Determination of milk freshness and hygienic quality 3.3 Milk adulteration 4. Milk Hygiene and Udder Health 4.1 Milk Hygiene 4.2 Udder Health 5. Equipment for small scale milk plants 5.1 Introduction 5.2 Milk collection 5.3 Small-scale milk processing systems 5.4 Equipment requirement and specification Part B: Camel as a meat animal 6. Traditional slaughter, carcass dressing and processing of camels 6.1 Locations for slaughter of camels Recommended references About the Authors 6.2 The traditional method of slaughter for camels 6.3 Traditional dressing of the carcass 6.4 Traditional meat products in Africa ands Asia 7. Method for hygienic slaughter of camels 7.1 Requirements to be satisfied by slaughterhouses 7.2 Slaughter procedure 7.3 The dressing percentage of camels 7.4 By-products from slaughter of camels 8. Dressing of the camel carcass 8.1 Requirements to be satisfied by deboning rooms and equipment 8.2 Cutting of the camel into primal cuts 8.3 Deboning of the camel 8.4 Cutting of the camel into retail cuts 8.5 Standardization of processing material 8.6 Percentage of retail cuts, processing material and remaining parts 8.7 Camel meat composition 9. Meat products from camel meat 9.1 Product groups 9.2 Equipment needed for meat products 9.3 Additives and ingredients for meat products 9.4 Casings for meat products 9.5 Recipes for camel meat products

pdf

Rأ¼egg, Camel milk fat

361

Melting curves of camel milk fat
By M.W. RأœEGG and Z. FARAH Federal Dairy Research Institute, CH-3097 Liebefeld-Bern and Institute of Food Science, Swiss Federal Institute of Technology, CH-8092 Zأ¼rich, Switzerland

1. Introduction A previous study on camel milk butter showed a difference in churning behaviour between camel and bovine cream (1). Camel cream of varying fat contents (20-30 %) was churned at temperatures between 15 and 36آ°C. Highest fat yield (80-85 %) in butter from cream with 22.5 % fat content was obtained at a churning temperature of 25آ°C. This churning temperature was much higher than that commonly used during the manufacture of butter from bovine milk (10-14 آ°C). The reason for the difference in churnabillty was attributed partly to the higher melting point of camel milk fat (1,2), It was therefore considered useful to study the melting properties of the triglycerides of camel milk in more detail and to compare the results with those obtained for bovine milk fat. 2. Materials and methods 2.1 Butter samples Camel milk butter was made by a manual churning procedure described previously (1). Four butter samples from different batches of pooled camel (Camelus dromedanus) milk were used. All butter samples were obtained at a churning temperature of 25آ°C from sour cream (S1, S2) and sweet cream (S3, S4) with 22.5 % fat content. Data related to butter from bovine cream (Simmental cows) were collected during a previous study (4). 2.2 Differential scanning calorimetry Melting thermograms and ratios of solid to liquid in milk fat were determined by differential scanning calorimetry (DSC) as described by RأœEGG et al (3). Butterfat was liquefied at 45-50 آ°C and dehydrated by filtration through a hydrophobic filter. 15 آµI of butterfat was then filled and hermetically sealed in aluminium pans and cooled in the calorimeter from 40 to -50آ°C at a rate of 5 آ°C/min, The melting thermogram was recorded after 10 min equilibration time at -50آ°C by heating at a rate of 5 آ°C/min, The percentage of solid fat was calculated at 10آ°C Intervals from the area fractions under the melting thermograms. The area fractions were weighted with a factor which is proportional to the heat of fusion of the corresponding butter fat fractions (3), Triplicate measurements were made for each of the 4 butterfat samples. For comparison, both the corrected and the uncorrected percent solid values are reported in Table 1.
Fig 1 Differential scanning calorimetry thermogram of camel milk tat (a)
and bovine milk fat (b)

Table 1: Percentages of solid fat as a function of temperature In butterfat prepared from camel milk. Means and standard deviation of 4 samples. Data without standard deviations correspond to predetermined values. Temperature (آ°C) Uncorrected
% Solid fat

Corrected'

x
-25.9 -20.0 -10.0 0.0 10.0 15.0 20.0 30.0 40.0 42.5
1

sx
2.2 1.1

x
1000 99.8 98.1 94.4 87.2 82.6 76.8 49.5 13.0 0.0

sx
-

x
1000 99.7 96.8 91 81.5 77.2 70.7 43.7 11.0 0.0

sx
-

0,1 0,6 09 14 16 23 2,1 1,9 -

0.2 0.9 1.3 2.6 1.9 2.7 2.3 1.7
-

Corrected for the temperature dependence of the heat fusion (3)

3. Results and discussion Fig. 1 shows typical melting thermograms obtained with dehydrated butter prepared from camel and bovine milk. The thermogram for camel butter differed in shape and did not show the peak around 15آ°C that is characteristic of the middle melting fraction of triglycerides (5). The different amounts of low, middle and high melting fractions of triglycerides, which are suggested by the shape of the thermograms, are consistent with the differences in the fatty acid

Milchwissenschaft 46 (6) 1991

362

Rأ¼egg, Camel milk fat

composition. Camel milk fat contains a lower percentage of short-chain fatty acids (C4 to C12 and a somewhat higher percentage of C6 acids (1, 2). Fusion of the camel milk fat started around -26 'C and was complete at about 43 آ°C (Table 1). Mean values and standard deviations of these temperatures are given in Table 1. Corresponding values for bovine milk were -25 and 37 آ°C, respectively, with a standard deviation of 2 'C The average heat of fusion, calculated from the area under the thermograms, was 79.2 J/g, with a standard deviation of 35 J/g, Using the same method, butterfat samples from bovine milk gave values in the range of 70-80 J/g. The percentages of solid fat in the 4 samples of camel milk fat were determined from the melting thermograms and plotted as a function of temperature in Fig. 2. For comparison, a typical curve for butterfat from bovine milk is included. The mean percentages of solid fat and their standard deviations are summarized in table 1.
~

4. References
(1) FARAH, Z, STREIFF, T, BACHMANN, M.R .

Milchwissenschaft 44 412-414 (1989) (21 ABU-LEHIA, IH Food Chemistry 34 261-271 (1989) (31 RأœEGG, M, MOOR, U, BLANC, B. Milchwissenschaft 38601-605 (1983) (4) RأœEGG, M, MOOR, U.: Das Schmelzverhalten van Milchfett und Margarine. Interner Bericht Nr. 4. Federal Dairy Research Institute, Liebefeld-Bern, Switzerland (1983) (5) TIMMS, RE: Aust J. Dairy Technology 35 47-53 (1980)

5. Summary
RأœEGG, M.W., FARAH, Z.: Melting curves of camel milk fat Milchwissenschaft 46 (6) 361-362 (1991) 44 Milk fat (camel milk) Melting behaviour of camel milk fat was studied by differential scanning calorimetry. Melting thermograms were measured in the temperature range of -50 to +50 آ°C. Melting started around -26 آ°C and was complete below 43 آ°C. The average heat of fusion was 79.2 J/g. Compared to bovine milk fat, camel milk fat contains a higher amount of high melting triglycerides and a lower percentage of triglycerides that melt in the medium range around 15 آ°C. Melting curves derived from thermograms explain some of the difficulties encountered when converting camel milk into butter and should allow optimization of the temperature profile used during butter manufacture. RأœEGG, M.W., FARAH, Z.: Schmelzverhalten von Kamelmilchfett Milchwissenschaft 46 (6) 361-362 (1991) 44 Milchfett (Kamelmilch)

100

" '
< 5
U) 60 80

4 0

- s,
2 0

-+S2

-:+:- S3 -B- S4

o

....•.•.... Bovine

-40 -30 -20 -10

0

10

20

30

40

50

Temperature (DC)

Fig 2

Melting curves of camel milk fat compared with typical curve for bovine milk fat Percentage of fat being solid as a function of temperature

Das Schmelzverhalten von Kamelmilchfett wurde mit Hilfe der Differential-Scanning-Kalorimetrie im Temperaturbereich von -50 bis +50 آ°C gemessen. Das Kamelmilchfett begann bei -26 آ°C zu schmelzen und war bei 43 آ°C vollstأ¤ndig verflأ¼ssigt. Die mittlere Schmelzwأ¤rme betrug 79.2 J/g. Im Vergleich zu Kuhmilch enthأ¤lt Kamelmilch grأ¶ssere Anteile an hochschmelzenden Triglyceriden und geringere Anteile an Triglyceriden, die im mittleren Temperaturbereich um 15 آ°C schmelzen. Die Schmelzkurven erklأ¤ren einige Schwierigkeiten bei der Verbutterung von Rahm aus Kamelmilch und kأ¶nnen zur Optimierung des Verbutterungsprozesses dienen.

It is evident from these data that butter from camel milk contains significantly higher percentages of solid fat over the entire melting range relative to butter from bovine milk (Fig. 2). At around 25 آ°C, i.e., the temperature which gave the best churning results, about 55 % of camel milk fat was in crystalline form. Temperatures commonly used in similar procedures for cream from bovine milk are in the range of 10 to 14 آ°C. It is interesting to note that similar percentages of solid fat are found at these temperatures. Acknowledgement The authors thank Miss U. Moor for her careful technical assistance.

RأœEGG, M.W., FARAH, Z. Comportement de fonte de matiere grasse laitiere de chamelle. Milchwissenschaft 46 (6) 361-362 (1991). 44 Matiere grasse laitiere (Ialt de chamelle) RأœEGG, M.W., FARAH, Z.: Caracteristicas de fusion de materia grasa lأ،ctea de camella. Mllchwissenschaft 46 (6) 361-362 (19911. 44 Materia grasa lأ،ctea (Ieche de camella)

Milchwissenschaft 46 (6) 1991

pdf

M. Younan

67

4
Milk Hygiene and Udder Health
4.1 Milk Hygiene
M. Younan

Introduction
The unhygienic handling practices in traditional camel milk production and in the informal camel milk trade represent serious obstacles for the introduction of modern dairy processing and marketing. The successful adaptation of pastoral subsistence production to the needs of an improved camel milk production and marketing system will depend, to a large extent, on safeguarding the milk quality at production, during transport, processing and marketing.

The present status
Camel milk possesses superior keeping quality to cows’ milk due to its high contents of proteins that have inhibitory properties against bacteria. This makes raw camel milk a marketable commodity, even under conditions of high temperatures and very basic hygiene. In Somalia and Kenya, camel milk production areas are often located far from markets. Distances to provincial markets range from 20 to 90 km and may be up to 400 km for distant urban markets. During periods of milk surplus (rainy season) transport on dirt roads is unreliable resulting in breakdowns and delays in milk delivery. Storage in unhygienic containers, mixing of evening and morning milk, pooling of milk from different suppliers, prolonged transport times, high environmental temperatures and road-side selling out of open containers all increase contamination and spoilage of milk. Spoilage does not always equal wastage. Sour camel milk is part of the traditional diet (Somali â€‌Susaâ€‌, Arabic â€‌Al-Garssâ€‌) and sour milk of acceptable quality is sold and consumed. But growth of contaminants in raw camel milk poses a threat to consumer health when milk of poor hygienic quality is sold. Spoilage reduces

68

Milk Hygiene and Udder Health

the market value of the milk causing income losses to producers and vendors. Souring or sour camel milk is also unsuitable for heat treatment in dairy plants. The common practice of smoking traditional milk containers and milking buckets (made from gourds, natural fibres) achieves high temperatures and appears to have a beneficial effect on the keeping quality of milk, although this has not been studied in detail. However, the obvious advantages of plastic containers (cheap, light weight, durable, large volume per container, better suited for transport in vehicles) coupled with the limited availability, high costs and small volumes of traditional containers leads to the increasing use of these containers in the camel milk trade. In Kenya and Somalia, smoking of plastic containers is standard practice to keep the traditional flavour of the milk in accordance with customer preference. Such â€‌cold smokingâ€‌ of plastic containers is unlikely to have a sanitising effect. Plastic jerry-cans of cheap quality (e.g. recycled cooking oil containers) have a fast corroding surface and are very difficult to clean in pastoral areas because of the lack of clean water. The non-availability of safe clean water also implies that the introduction of common hygiene recommendations will be difficult and adapting hygiene practices and guidelines to the pastoral situation remains a challenge.

The hygienic quality of camel milk
The monitoring of hygienic quality of camel milk from pastoral production areas by performing Total Bacteria Counts (TBCs) has serious logistical problems because of the distances to the laboratory. Hence bacterial counts in milk from pastoral regions must be interpreted with caution. The figures presented here almost certainly reflect higher TBCs than originally present in the samples. Spoiled camel milk has been found to have TBCs of 107 cfu/ml to 108 cfu/ ml, although milk with lower TBCs is occasionally perceived as spoiled by organoleptic testing. The results of Coliform Counts (CC) are even more affected by the delays before laboratory testing, CC’s of less than 102 cfu/ml have been found in milk samples from traditional milking buckets. The following is a compilation of examples from camel milk analysis. Total bacteria counts (TBC) in camel milk in Kenya
Milk sample From udders milked directly into clean container From traditional milking bucket From transport container immediately after end of milking From bulk milk stored 24h without cooling From milk purchased – in the production area (less than 24h old milk) – in Nairobi (24h to 36h old milk)
* For comparison: EU-standard for raw cow milk TBC < 10 cfu/ml
5

TBC* (cfu/ml) 102-104 103-104 103-105 105-108 106-107 106-108

M. Younan

69

These results show that good quality raw camel milk is produced but it deteriorates rapidly as it enters the informal marketing chain. Pooling of different raw milk batches and unhygienic plastic containers accelerate spoilage with non8 refrigerated bulk milk reaching a TBC of 10 cfu/ml and turning sour in less than 24 hours at 25آ؛C or in less than 12 hours under hot conditions (35آ؛C). However, provision of clean containers and chilling of raw milk taken from milking buckets after normal (traditional) milking, resulted in TBCs remaining within acceptable limits (≤ 105 cfu/ml) for four days. This milk also tasted fresh for up to four days. The influence of pooling of different camel milk batches along the collection and marketing chain is illustrated by the increase in prevalence of Streptococcus agalactiae, a mastitis pathogen that originates from the udder. This pathogen was found in 50% of transport containers coming from producing herds, in 62% of milk containers sampled at primary collection sites and in 70% of milk containers sampled from an urban market of the same region. Aseptically sampled milk from non-infected bovine udders contains 102 to 103 cfu/ml. The TBCs increase to 105 cfu/ml from cattle with subclinical mastitis. In comparison, mastitic camel milk seems to have a lower pre-secretional bacterial load. Milk from four subclinically infected camel udders (with bacteriologically confirmed Streptococcus agalactiae infections) ranged in TBC from 1.0 to 6.5 x 103 cfu/ml. Under pastoral production conditions, environmental contamination is likely to play a bigger role in the hygiene of raw camel milk than mastitis bacteria. Adulteration of marketed camel milk occurs. Addition of up to 15% water to marketed camel milk has been reported from South Somalia. The quality of the water added to the milk represents an additional hygiene risk. – The specific gravity of camel milk tested in three large commercial herds in Kenya over a two months period varied between 1.026 and 1.029 indicating a difference to the specific gravity of cows milk. The following list of milk hygiene risk factors is based on the current practices of Somali milk producers/traders:
Camel milk production and marketing chain lactating camel ⇓ milker (male) Milk hygiene risk factors unclean udder, subclinical mastitis, zoonotic infections with lactogen transmission unclean hands, personal hygiene and health status, unclean (plastic) milking bucket, unclean milking site no/unclean filtration, unclean storage container (plastic), pooling of fresh and old milk

⇓ milk handler (male/female) ⇓

70

Milk Hygiene and Udder Health

primary milk collector (mostly female)

no/unclean filtration, unclean (plastic) transport container, pooling of milk from different producers, high environmental temperatures during intermediate storage, adding unclean water delayed transport, prolonged exposure to high environmental temperatures additional pooling, exposure to high environmental temperatures, adding unclean water selling from open containers in unclean environment, further exposure to high environmental temperatures, adding unclean water traditional preference for consumption of raw milk

⇓ transporter (male) ⇓ secondary milk collector (mostly female) ⇓ milk vendor (female)

⇓ consumer

How to improve camel milk hygiene
Machine milking and hygienic milk production from camels, in line with both EU and German raw milk standards, have been achieved in a modern commercial intensive stationary camel dairy farm in Dubai. However, this must be regarded as exceptional given the production conditions and economic constraints that normally apply in pastoral camel herds. Optimising milk hygiene under pastoral conditions requires the availability of safe clean water, an unrealistic expectation in most situations. However, the introduction of clean metal containers to producing herds has a measurably positive effect on raw milk quality. Camel milk transported from four producing herds to a local market in metal containers had TBCs between 0.7 x 107 cfu/ml and 7.1 x 107 cfu/ml while the TBCs from duplicates of the same milk transported in plastic containers ranged from 2.7 x 107 cfu/ml to 60 x 107 cfu/ ml. The milk was sampled on arrival at the market and the average transport time between herd and market was 6 h آ± 40 min. Another simple approach to improving camel milk hygiene is the provision of clean gauze for filtration. Pooled camel milk sampled at a primary collection point immediately after it had been filtered through old reused gauze into the transport container ranged in TBC from 107 cfu/ml to 108 cfu/ml. The TBC was reduced to 106 cfu/ml when the same milk was filtered through fresh gauze. Receiving milk directly from producers without pooling of different batches coupled with shorter transport distances may offer hygiene advantages. However, such a system can cover only a very limited milk-production area and is unlikely to provide sufficient quantities of milk for commercially viable processing and marketing.

M. Younan

71

Potentials and constraints for improving camel milk hygiene in pastoral production systems • replacement of cheap plastic with quality steel containers for storage and transport

⇒ problem of increased weight/transport costs;
• filtration of milk with disposable clean gauze/paperfilters

⇒ problem of availability, old filters are often reused;
• rotation of sealed sanitised containers between production areas and dairy processors

⇒ used successfully by some traders who receive milk directly from large herds without any intermediate collection/pooling, difficult to implement in the widespread complex collection and trade chain with repeated pooling and transfer of milk (inconsistent milk containers);
• short heating/flash boiling of raw milk at primary collection sites

⇒ used increasingly by long-distance milk traders, potentially negative effect on valuable milk components (e.g. vitamin C) and on the taste;
• reducing milk temperature at primary/secondary collection sites using solar or gas-powered refrigerators, evaporative cooling from charcoal-walled cooling chambers or simply by wrapping milk containers in a moist cloth, provision of shade/cooling box during vehicle transport; • encouraging the use of quality steel buckets or traditional â€‌smokableâ€‌ milking buckets rather than plastic milking buckets in the producing herds, using only boiled water as hot as possible for final cleaning of storage-containers in milk producing herds,

⇒ costs/availability of heating/firewood;
• training and extension to raise awareness among producers on clean milking and handling practice; • accelerated transport from production to market

⇒ economic constraints to setting up an independent milk transport system.

The introduction of the lactoperoxydase system (LPS) to the camel milk trade may prolong the keeping quality of camel milk. Currently there is no information on whether any significant extra keeping time is gained by the use of LPS in raw camel milk.

Consumer Health
Milk contaminants, including faecal organisms, pose an important threat to consumers of marketed camel milk. Zoonotic risks from camel milk must be considered in view of the traditional preference for raw milk for consumption. Brucellosis prevalence in camels varies widely ranging from 1% to 30% positive reactors in the Rose Bengal Plate Test according to the literature. Brucellosis prevalence seems to be higher in regions where camels are kept under more stationary conditions and close together with other livestock. Many pastoralists rank raw camel milk as safe while raw goats milk is regarded as potentially harmful. This empirical experience could be related to both lower lactogen Brucella excretion rates in camels and a higher Brucella melitensis prevalence in goats.

72

Milk Hygiene and Udder Health

The two most common mastitis pathogens in camels, Streptococcus agalactiae and Staphylococcus aureus (see chapter on mastitis), are both potential human pathogens. While toxin producing Staphylococcus aureus may cause food poisoning, Streptococcus agalactiae is a known cause of human infections, particularly in newborn children. Streptococcus agalactiae isolates from camels seem to be more closely related to the human than to the bovine biotype and may survive for up to 7 days in souring camel milk, showing no significant decline in viable numbers down to a pH of 4.5. Raw camel milk may occasionally play a role in transmission of tuberculosis to humans. Tuberculosis is rare among camels under nomadic conditions, with almost all reports on tuberculosis in camels originating from non-pastoral situations where camels are kept in confinement and/or in close contact with other livestock. Salmonella infections are common in camels, but human Salmonella infections originating from raw camel milk have not been documented. Numerous other zoonotic diseases, including Plague and Rift Valley Fever, have been recorded from camels, but the literature provides no detailed information on lactogen transmission.

The Future
Consumer consciousness about the hygienic quality of camel milk has yet to be developed. Although higher prices for fresh camel milk as compared to souring camel milk is an indication that urban consumers are prepared to pay more for better hygienic quality. In most countries, food safety regulations do not include camel milk but such regulations are necessary and may be enforced in the near future. For modern processing, the heat stability of camel milk proteins and other camel milk constituents are clearly an advantage. Selling heat-treated packed camel milk could provide the solution to hygiene woes and, at the same time, eliminate zoonotic risks for camel milk consumers. The problem faced by any fixed milk processing plant is to ensure an adequate and regular supply of raw milk with acceptable quality from mobile lactating camel herds. The presence of centralised modern milk processing plants may lead to over-concentration of lactating camel herds in a limited grazing area with severe negative impact on the very fragile environment and increased disease prevalence in lactating animals. Decentralised and possibly mobile processing systems relying on simple adapted technology might be more sustainable and should be tested. Another possible solution could be cooling or flash-boiling of camel milk in the production area, coupled with an improved hygiene-conscious collection and transport system. Extension, training and regular monitoring will be important elements in any attempts to improve the hygiene of marketed camel milk.

M. Younan

73

4.2

Udder Health
O. Abdurahman, M. Younan

Introduction
Camels are kept for cultural and food security reasons in large areas of the horn of Africa. They make an important contribution to human survival and utilization of these dry and arid lands. The horn of Africa region has and is undergoing major changes that require new production strategies. These changes mainly limited the free movement of camel herds and affect their husbandry system. They include: • Increasing human population growth, • Frequent drought due to climate change, • Competition for resources, • Insecurity, • Settlement and expanding rural cultivation and increasing sedentarisation of pastoralists to obtain schooling for children and healthcare. The development of camel dairying and the commercialization of milk are elements of the new production system that will have important implications for the future management and husbandry of camels. The commercialization of camel milk is on the increase and milk is now taken regularly to urban centers for sale. Small commercial dairy farms and processing plants are planned in an attempt to supply fresh pasteurized milk to urban consumers. The introduction of modern dairy processing means that mastitis and other production related problems would play a bigger role than in traditional subsistence husbandry systems. There is likely to be an increasing demand for clean milk from consumers and by regulatory agencies (food safety authorities) in the near future. This chapter highlights the main udder infections of camels and describes simple methods for mastitis diagnosis and ways to improve udder health.

Mastitis
Mastitis is defined as inflammation of the mammary gland. It is associated with chemical, physical and most commonly bacteriological changes in the milk, and pathological changes in the mammary tissue. Reports of mastitis in the camel have increased tremendously during the past decade. However, much of the reports are not planned studies, but single or compiled case reports and surveys. Mastitis causes suffering for the animal, reduces milk production and poses public health risk. Mastitis also influences the technological properties of milk.

74

Milk Hygiene and Udder Health

In bovine mastitis the changes related to inflammation of the mammary gland include an increase in the concentration of chloride and sodium, an increase in the pH, a decrease in the dry matter content as well as a decrease in casein concentration and a negative effect on the coagulation of milk casein (delayed rennet coagulation). These changes have deleterious effects on the processing of milk. Mastitis is considered to be a multifactorial disease, closely related to the production system and environment that the animals are kept in. Mastitis risk factors or disease determinants can be classified into three groups: host, pathogen and environmental determinants (including management). Bacteria, which can cause mastitis, are present in air, water and other surfaces in the environment including human and animal skin. Streptococcus agalactiae, other Streptococcus spp., Staphylococcus aureus, coagulase-negative staphylococci, and Escherichia coli are incriminated as the major bacterial causes of mastitis in the camel. There is evidence that Streptococcus agalactiae and Staphylococcus aureus are the two most important mastitis pathogens in camels (Table 4.1).
Table 4.1: Prevalence of Streptococcus agalactiae and Staphylococcus aureus mastitis in different camel populations, n=number of milk samples.

Mastitis pathogen
Country KENYA* (n=1305) SUDAN** (n=757) SUDAN*** (n=391) Streptococcus agalactiae 12.1% Individual herd prevalence 250% 26.7% 17.6% Staphylococcus aureus 10.6% Individual herd prevalence 9–13% 17.0% 5.4%

* Younan et al. (2001), ** Obied et al. (1996), *** Abdurahman et al. (1995)

In Somalia, the prevalence of Streptococcus agalactiae in raw camel milk sampled from producers (n=10), primary collectors (n=37), milk vendors (n=23) and from raw milk batches received at a dairy plant for processing (n=18) was 50%, 62%, 70% and 89%, respectively. These findings indicate a very widespread occurrence of the pathogens in milk-producing camel herds and in the milk collection and distribution systems. Streptococcus agalactiae is the single most important organism affecting the overall productivity of dairy cows. This pathogen causes a 30% decrease in milk yield on an individual animal basis and a 15% decrease on a herd basis. In Sudan and in Kenya subclinical Streptococcus agalactiae infections were found to be the most common intramammary infections in camels. – Staphylococcus aureus has been ranked as the most frequent or second most frequent microorganism isolated from udder infections in camels.

M. Younan

75

Mastitis occurs in clinical or subclinical forms. Clinical mastitis is self-evident and can be detected without special tests. There are changes in the secreted milk (color, consistency, floccules etc.) and/or the udder (red, swollen) and other generalized signs exhibited by the animal (fever, anorexia, deteriorating body condition). Subclinical mastitis, on the other hand, is difficult to diagnose. A camel with subclinical mastitis produces less milk, but does not have a swollen udder or abnormal milk. Infection is present but can only be detected with the help of indirect methods. These include the California Mastitis Test (CMT see section 3.2.9), a simple and rapid test that can be applied in the field. The CMT is particularly useful for subclinical udder infections caused by either one of the two major mastitis pathogens, Streptococcus agalactiae and Staphylococcus aureus. A second method is the direct microscopic somatic cell count (DMSCC) that requires only simple laboratory equipment and produces results on the same day. Subclinical mastitis causes an increase in the total bacterial count in milk. It is a major factor in depressing milk yield and has a much greater impact on the productivity of lactating animals than the sporadic clinical forms of the disease. In a longitudinal study of 207 lactating camels, only 3.4% were affected by clinical mastitis while 21.3% were affected by subclinical mastitis. – Chronic intramammary infection ultimately leads to loss of intact quarters by destruction of the gland tissue. Loss of teats is reported from one third of Gabra and Somali camels in northern Kenya, in a smaller sample in the same area, between 10% and 50% of female camels had less than four intact quarters. Early culling of female camels due to chronic mastitis is reported from Iraq. Control of mastitis has been estimated to result in a 9% increase in milk yield from Somali camels in Kenya. Bacterial mastitis pathogens also represent a potential threat to humans if the milk is consumed raw, a common practice in most camel keeping communities. Mastitis can be prevented or reduced by improving animal health and udder hygiene. Currently there is almost a complete absence of modern mastitis control measures practised by camel keepers. And there is little evidence of effective ethno-veterinary interventions in treating and curing mastitis. Good quality dairy products can only be obtained from healthy camels. Attention must be paid to udder health and hygiene, not only during lactation, but continuously, even when the animal is dry. Animals suffering from any contagious disease, including mastitis should be separated from the healthy animals and milk from diseased camels should be kept separate and disposed of safely.

Management
Management practices prevalent in traditional husbandry systems include tying the teats with soft bark to prevent the calf from suckling and cauterization

76

Milk Hygiene and Udder Health

of the udder skin, that aggravates the existing lesion and leaves behind scar tissue, blind teats and permanent loss of milk production. The traditional practice of tying teats may contribute to the development of mastitis in camels. The effect of the unrestricted (ad lib) suckling of calves when small and the simultaneous and frequent milking (up to six times a day) and their relationship with mastitis is not known. The build up of the keratin barrier inside the teat inbetween milking may be weakened. Further studies are needed to examine this relationship. The udder is a predilection site for tick infestation. It is a good practice to remove ticks even when the animal is not lactating. Tick infestation causes skin lesions, facilitates bacterial entry and leaves behind permanent tissue damage. In a limited study in Kenya, 22% of tick bite lesions were shown to harbour Streptococcus agalactiae. Teat canal blockage with dilatation of the gland is a commonly observed problem in dromedaries. The cause of this blockage is not known and needs further investigation. It is cheaper and easier to prevent mastitis by improving hygienic measures and culling chronically infected camels to eliminate important pathogen reservoirs, than to treat by medication. The cost of treatment includes veterinary fees, medicines, and risk of quackery and loss of milk production. Treatment also contributes to the build up of antibiotic resistance. The teat of the camel udder contains two, sometimes three, separate teat canals, that open independently into the teat sphincter. The separate canals drain separate gland complexes. This implies that for intramammary treatment of mastitis not only must each quarter but also each gland complex be treated separately, that is, one intramammary tube per gland complex. Great caution is necessary when applying intramammary treatment to camels. The teat canal openings are smaller than those of the cow and thus require smaller cannula. Unhygienic and traumatic application of intramammary treatment is very likely to do more harm than good.

Future Research Work
There is limited information or knowledge regarding effective camel mastitis management. Organized problem oriented research is needed to monitor udder health and milk quality of camels. Camel herds should be carefully monitored for a range of quality and disease parameters throughout lactation to establish baseline values and infection information. Both formal and informal methods should be employed to strengthen the understanding and reliability of data collected and to achieve analytical quality. This kind of research must fulfill the needs of the camel owners and the consumers. The cost benefit relationship of mastitis control measures in camels needs to be examined in detail before making treatment recommendations.

pdf

Int. Dairy .Journal 8 (1998) 617-621 آ© 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$-see front matter

PII:S095S-6946(98)00092-2 ELSEVIER

Iso-electric Focusing of Camel Milk Proteins
J. Wangoh1, Z. Farah* and Z. Puhan Laboratory of Dairy Science, Institute of Food Science, Swiss Federal Institute of Technology, Cff-8092, Zurich, Switzerland ABSTRACT (Received 18 November 1997; accepted 27 June 1998)

The procedure for phenotyping of most genetic variants in cow milk was optimised for iso-electric focusing (IEF) of camel milk proteins and milk from individual camels of different breeds was screened. The caseins obtained from IEF bands were also investigated by Nterminal sequencing. Camel milk casein was separated at different pH and the proteins in the whey obtained were then separated by IEF. Above pH 4.3 casein bands were observed in the whey. According to the pattern of protein bands in IEF, the 103 camels screened had one of the three main groups of milk designated aa, ab and, bb. A small number of camels differed from bb milk type by an extra band and this group was designated as bbl. The high frequency of particular milk type in some breeds suggests that their production characteristics could be related to the phenotypes. آ© 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION Genetic polymorphism in milk proteins is due to gene mutation resulting in either substitution or deletion of amino acids sequence along a peptide chain. In over 30 genetic variants identified in bovine milk, gene mutations leading to deletion of amino acids occur in only two cases whereas the more frequent gene mutations caused by substitution of amino acid are responsible for the other variants (Ng-Kwai-Hang and Grosclaude, 1992). Because milk protein genes are inherited in a simple Mendelian fashion, there is considerable interest in using them as markers for milk production and composition traits. It has been reported that خ؛-casein B (خ؛-Cn B) and خ²-lactoglobulin A (خ²-Lg A) are associated with a higher protein content in cow milk. The ratio of casein to whey proteins is increased by the presence of خ²-Lg B and خ؛-Cn B. Favourable effects of خ؛-Cn B, خ±s1-Cn C and خ²-Cn B on rennetability and coagulum properties have also been observed, and even a positive relationship between خ؛-Cn B and cheese yield (Ng-Kwai-Hang and Grosclaude, 1992). Moreover, casein variants that have been associated with higher casein content in milk have also been correlated with higher amounts of the corresponding casein fraction (Jakob, 1994). Studies on goat خ±s1-Cn have also shown convincing evidence that certain alleles of this protein are associated with higher concentration of casein in milk (Ng-KwaiHang and Grosclaude, 1992). The main work on genetic polymorphism using isoelectric focusing (IEF) has been done mainly on cow and to some extent on goat milk. Only fragmentary data are available for genetic polymorphism of milk proteins in other species. Analysis of casein and whey protein fractions from milk of Somali camels by Di Stasio et al.

(1983) reported the occurrence of polymorphism for the supposedly خ²-Lg, but not in the other proteins. Two variants, A and B, of خ±-La were later detected by Conti et al. (1985) in milk from Somali camels. These two forms of خ±-La had similar mobility in PAGE at pH 8.3, but differed in pI due to a difference in amino acid sequence at the N- terminal. An IEF procedure for simultaneous phenotyping of most genetic variants in cow milk was described by Seibert et al. (1985). This procedure was optimised in the present investigation for IEF of camel milk proteins. In addition the occurrence of genetic polymorphism was also investigated by N-terminal sequencing of protein obtained from IEF bands.

MATERIALS AND METHODS Preparation of milk for IEF In Ol Maisor Ranch, Kenya, milk from 103 individual camels was collected. To prevent microbial growth 10 mg 2-bromo-2nitro-1,3 propanediol (Bronopol Tablets, Preservative systems Ltd from Chemgo Organica, Basel, Switzerland) were added to 50 mL milk. The samples were immediately cooled to 4آ°C and transported to the laboratory within 24 h. Milk fat was removed by centrifugation (400g, 4آ°C, 30 min) before freezing or lyophilization. Acid casein was prepared by adding to skim milk 10% acetic acid (10% v/v) allowing to stand 30 min at 35آ°C then adding 10% 1 M NaOAc and adjusting the pH to 4.3 with HCI and shaking gently and allowing to stand for 30 min before centrifugation (20,000g, 5آ°C, 30 min). The supernatant was retained and the casein subsequently washed twice with equal volume of buffer (400 mL water + 20 mL acetic acid (10% v/v) + 20 mL 1 M NaOAc adjusted to pH 4.3) and freeze dried.

1

*Corresponding author. Permanent address: Department of Food Tech. and Nutr., University of Nairobi, P.O. Box 29053, Kabete, Nairobi, Kenya. 617

6l8

J. Wangoh et al. micro-trap and for each sample trap 5 mA and 1 W were applied and the limit voltage was 200 V. Running times were 1-2 h and the sample migrated to the anode. Fractions 3 and 4 were harvested from the micro-traps and made to 2.5 mL using the electro-elution buffer. Small molecular weight products were removed in a pre-packed Sephadex G-25 M PD-10 column (Pharmacia Biotech AG, Switzerland) by elution using the same buffer. The sample was then freeze dried and the dye and SDS removed using Solvent System A according to Koningsberg and Henderson (1983). N-terminal sequence of proteins was then determined.

To prepare whey proteins, the supernatant from the acid casein was centrifuged (20,000g, 5آ°C, 30 min) and whey proteins were precipitated by saturation with ammonium sulphate and left overnight. The precipitate was collected by centrifugation (10,000g, 15آ°C, 15 min) and the sediment of whey proteins was lyophilised. IEF of camel milk proteins Separation of casein and whey protein To 1 M NaOAc was added an equal amount of 10% v/v acetic acid to obtain a buffer of pH 4.40. To this buffer was added either sodium acetate or acetic acid to obtain buffers of pH ranging from 3.3 to 5.2 in steps of 0.1 pH unit. Bulk camel milk was then mixed with the respective buffer (milk : buffer 1: 1) and the pH of the mixture measured. Precipitation was allowed to occur for 1 h at 20آ°C and the mixture was centrifuged at 1000g for 30 min. The whey was centrifuged at 1000g for 1 h at 4آ°C to remove any casein particles before IEF.

N-terminal sequence N-terminal sequencing was by Adman degradation (Matsudaira, 1989) using an automated device.

RESULTS AND DISCUSSION Acid precipitation of camel milk casein To determine the pH at which casein and whey proteins can be separated, casein was precipitated at different pH between 3.55 and 5.30 at 20آ°C. The proteins of whey obtained were then separated by IEF (Fig. f). Above pH 4.3 both casein and whey proteins bands were present in the TEF patterns. Therefore, it was concluded that the best separation of casein and whey proteins of camel milk occurred at pH 4.3. These results show the precipitation of casein in camel milk can not be performed in the same way as in cow milk, in which casein is precipitated by acidification of milk at 20آ°C to pH 4.6 (Eigel et al. 1984). This low pI of camel milk casein has implications on the determination of casein in camel milk. In the literature casein in camel milk has been precipitated at the same pH as casein in cow milk (Farah and Farah-Riesen, 1985; Larsson-Razniciewicz and Mohamed, 1986). In our findings, if camel casein is precipitated at pH 4.6, a proportion of casein will remain in the whey and the non casein nitrogen (NCN) is overestimated leading to the subsequent underestimation of the casein content. This may explain the low casein content in camel milk compared to that of cow milk cited in literature (Bayoumi, 1990; Farah, 1996).

IEF procedure IEF was carried out according to Seibert el al. (1985) except that using a mixture of 1 part Ampholineآ® pH 4-6 and 5 parts Pharmalyteآ® pH 4.2-4.9, (Pharmacia Biotech AG, Switzerland) IEF gels having a pH gradient of 4-6 were prepared. The gels of 0.2 x 10 x 240 mm were run on a 2217 Ultophor Electrofocusing Unit with 2303 Multidrive XL power supply (Pharmacia-LKB, Bromma, Sweden). Pre-focusing conditions of 1500 V, 10 W, 20 mA for 300 Vh and focusing conditions of 3500 V, 25 W, 50 mA for 3500 Vh were used. In cow milk analysis, proteins are solubilised by a 1: 10 dilution of skim milk with sample buffer before IEF. In camel milk, a dilution of 1:4 with sample buffer was found the most suitable. In all cases, 5 آµL samples were applied per gel lane.

Preparative IEF Preparative IEF was carried out as in normal IEF except that the gel thickness and length were 0.1 and 24 cm, respectively. The anode was 0.2 M phosphoric acid and the cathode 0.2 M lysine. Gels were run at 10آ°C on a 2217 Ultophor Electrofocusing Unit with 2303 Multidrive XL power supply (Pharmacia-LKB, Bromma, Sweden). Ten mg Iyophilised or 100 آµL liquid sample was solubilised in 700 آµL sample buffer according to Bjellqvist et al. (1993) containing 0.1 % spermine and 30 آµL mercaptoethanol added. In each case all the sample was applied near the anode and focused at 3000V, 50mA, l0W to 15,000Vh then 3000V, 50mA, 5W to 20,000 Vh.

Milk proteins from individual camels Milk samples from 103 individual camels, belonging to Somali, Turkana, Somali x Turkana, Somali x Pokot and Pakistan breed, were screened by the modified IEF method. According to the pattern of protein bands, each of the camels screened had one of the three main milk types aa, ab or, bb shown in Fig. 2. A small number of camels differed from milk type bb by only an extra band b1 this group was designated as bb1. The major differences in bands between camels are shown in Table 1. The observed frequencies for the three main types of milks are shown in Table 2. The frequencies for the three main types of milk for the camel population sampled were 0.47 for aa, 0.12 for bb, 0.40 for ab. The low frequency for milk type bb can be attributed to the fact that bulls that were carrying either genes of aa or ab sired most of the animals as indicated in Table 3. Turkana breed had a high frequency for aa of 0.82 compared to 0.43 for Somali breed.

Electro-elution and purification of proteins bands from IEF gels Individual bands were cut off from preparative IEF gels and equilibrated for 15 min prior to electro-elution with 200 آµL solution of 0.05 M Tris-HCl, ph 6.8, containing 6 M urea, 30% glycerol, 2 % SDS and 2% DTE followed by a 5 min equilibration in a solution where DTE was substituted with 2.5% iodoacetamide (Gأ¶rg et al 1985). Electro-elution was done using the Little Blue Tankâ„¢ (ISCO, Inc, Nebraska, USA) according to manufacturers' instructions. Tris-HCl 12.5 mM, 0.1 % SDS buffer at pH 8.0 was used in the anode, cathode and the sample

Iso-electric focusing camel milk proteins

619

Fig 1. IEF of whey proteins from pooled camel skim milk (SM) precipitated at different pH. The whey proteins are indicated, the other bands are casein.

Fig. 2. IEF of different types of camel whole milk.

Table 1. Distribution of the IEF Bands in the different Groups of Camel Milk Proteins Bandsa a1 a2 a3 a4 b1 b2 b3 b4 b5 خ²-Cn
a

Table 2. Frequency of Three Main Milk Types in Camel Breeds

Group aa + + + + ab + + + + + + + + + bb bb1

Camel breed Somali x Turkana Somali Turkana Pakistani Somali/Pokot

Number aa 49 30 11 3 6 0.429 0.433 0.818 0.333 0.500

Milk bb 0.143 0.133 0.000 0.333 0.000 ab 0.429 0.433 0.182 0.333 0.500

+

+ + + + +

+ + + + + +

See Fig. 2 for the location of the hands in IEF.

Cross breeding of Turkana and Somali breed resulted in a aa frequency of 0.43 that was very similar to that of the Somali breed.

Milk type bb was not found in Turkana and Somali x Pokot breed while Somali and Somali x Turkana breed had the similar frequency for type bb. It has been observed that Turkana breed had a lower milk yield, a higher content of fat, total solids and lactose in the milk, than the Somali and Somali x Turkana breed (Wangoh, 1997). These characteristics of Turkana breed

620 Table 3. Frequency in Daughters of Bulls Bull identity
a

Number of daughters aa ab 34 26 12 8 6 5 5 0.56 0.46 0.58 0.25 0.17 0.00 0.20

Frequency bb 0.15 bb1 0.03

Possible bull genotype

Unknown 17S Somali 110S Somali Buna Somali Karandil Somali 115S Somali Pakistanb
a

0.26 0.54 0.42 0.50 0.67 0.60 0.20

0.25 0.17 0.20 0.20

0.20 0.40

aa aa ab ab bb bb1 (predominantly)

This is the identity given to the bulls at Ol Maisor Ranch; only bulls with more than five daughters are included in the list, all other bulls had one daughter each. b This represents all the Pakistani camels sampled which are not from the same bull

Table 4. N-Terminal Sequences of Protein Bands Milk type aa Band a1 a2 a3 a4 bb b1 b2 b3 b4 b5 b3 b4 خ²-Cn Sequence Arg, Glu, Met, Tyr, Asp, Leu, Lys Arg, Pro, Lys, Tyr, Pro, Leu, Arg, Tyr, Pro Arg, Met, Lys, Tyr, Pro, Leu, Arg, Tyr, Pro Same a2 Arg, Glu, Lys, Glu, Glu, Phe, Lys, Thr, Ala Arg, Glu, Val, Tyr, GIll Arg, Pro, Lys, Tyr, Pro, Leu, Arg, Tyr, Tyr Arg, Pro, Pro, Gin, Pro, Leu, Arg Same as a2 Same as a2 Arg, Ala, Lys, Glx Arg, Tyr Arg, Glu, Lys, GIll, GIll, Phe, Thr, Lys, Thr Homologue None Human خ±s1-Cn Bovine خ±s1-Cn Pig خ±s1-Cn Human خ±s1-Cn Same as a2 None Human خ±s1-Cn Bovine خ±s1-Cn Pig خ±s1-Cn Human خ±s1-Cn Bovine خ±s1-Cn Pig خ±s1-Cn Human خ±s1-Cn Same as a2 Same as a2 None Rat خ²-Cn
a

Casein Match % 85.2 59.3 85.2 73.7 Same as a2 75 61.5 61.5 72.2 59.3 59.3 68.1 Same as a2 Same as a2 67.5

ab

a

European Mo1ecular Biology Laboratory, Swissprot protein sequence data base searches.

could be related to high frequency for aa and a low one for bb. However, more results are needed to establish a relationship between the protein alleles and milk composition and yield. Band b1 that distinguished bb1 from bb was always present as an extra band and was not found in the other milk types. The frequency for bb1 was 0.33 in the combined bb and bb1 population and 0.04 in the sample population. The bb1 milk type was only found in Somali and Pakistani breed. Its frequency in Somali and Pakistani breed was 0.07 and 0.67, respectively. It is recognised that Pakistani camels yield more milk than all other camel breeds in general. For the native Kenyan camels, Somali camels have the highest milk yield. The reason might be the presence of milk type bb1.

The possible genotype of the bulls could be deduced from the type of milk of the 103 daughters (Table 3). We are aware of the fact that such a deduction normally requires a much larger number of observations. The milk type the 103 daughters suggest that, two bulls were aa, two ab and one bb type. The Pakistani bulls were predominantly bb1. It should be noted that Pakistani camels were imported to Kenya to cross breed with local camels and pass on their renowned milk producing ability. It was also noticed that bulls of genotype aa or ab sired most of the camels. Since this milk-type appears to be related to low milk yield, this trait may not be the overriding criteria on which the selection of bulls for breeding is based. In general, the pastoralists look for, in order of importance, beauty, physical condition, and performance of parents in selecting a male stud for breeding and the stud is used until its reproductive life (Farah, 1996).

Iso-electric focusing of camel milk proteins Preparative IEF, mass determination and N-terminal sequencing The protein patterns obtained by preparative IEF from the four types of milks are the same as shown in Fig. 2. Each of these bands was excised from the preparative gel, electro-eluted and cleaned as described and subsequently the N-terminal sequence of the protein from each band determined. The result of European Molecular Biology Laboratory, Swissprot protein, database searches and the respective sequences are shown in Table 4. In milk type aa, a2 and a4 had the same N-terminal sequence. a3 differed from both a2 and a4 in the N-terminal sequence. The highest homology of the N-terminal sequence of a2, a3 and a4 were found with خ±s1-Cn of human, pig and bovine (Table 4). Therefore it can be assumed that a2, a3 and a4 are N-terminal sequences of camel خ±s1-Cn. The substitution of methionine in a3 for proline in a4 can indicate a genetic variant of خ±s1-Cn. For milk type in bb, the difference of N-terminal sequences between b3 and b5 was found to be the substitution of tyrosine for proline. In b4, proline and lysine substituted the lysine and tyrosine in b3 and b5. b2 had an Nآ·terminal sequence that differed markedly from that of b3,b4 and b5, but it also had some homology to human, bovine and pig خ±s1-Cn. Moreover the خ²-Cn had some homology to rat خ²-Cn. REFERENCES

621

CONCLUSIONS The procedure for simultaneous phenotyping of most genetic variants in cow milk was optimised for IEF of camel milk proteins and milk from individual camels was screened. According to the pattern of milk protein bands in IEF, the camels screened were assigned to different milk types. The high frequency of particular milk types in some breeds suggests that their production characteristics could be related to the types. Studies on a large number of camels are needed, therefore, to establish a relationship between the protein variants and milk composition and yield. This would help the breeders to improve the performance of camels. From the present study, there is some evidence of genetic polymorphism of milk proteins in camel milk. Confirmation of this is needed by complete amino acid sequencing of proteins obtained from IEF bands.

Bayoumi, S. (1990) Studies on composition and rennet coagulation of camel milk. Kieler Milchwissenschaft and Forschungsberichte 42(1), 3-8. Bjellqvist, B., Sanchez. J., Pasquali, C., Ravier F., Paquet, N., Frutiger, S., Hughes. J.G. and Hochstrasser. D. (1993) Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amount of proteins. Electrophoresis 14, 1375-1378. Conti, A., Godovac Zimmermann. J., Napolitano. L. and Liberatori, J. (1985) Identification and characterisation of two lactalbumins from Somali camel milk (Camelus dromedarius). Milchwissenschaft 40. 673-675. Di Stasio. L., Cristofori, F. and Sartore, G. (1983) Phenotypic variations in blood and milk of the Somali camel. Animal Blood Groups Biochemistry and Genetics. 14(3). 225 228. Eigel W. N, Butler, J. E., Ernstrom. C. A ., Farrell, H. M., Harwalkar. V. R., Jenness, R. and Whitney. R. M. (1984) Nomenclature of proteins of cow's milk: fifth revision. Journal of Dairy Science 67(S), 1599-1631. Farah. Z. and Farah-Riesen. M. (1985) Separation and characterisation of major components of camel milk casein. Milchwissenschaft 40. 669-671. Farah. Z. (1996) Camel milk: Properties and Products. SKT Publisher. S1. Gallen. Switzerland. Gأ¶rg, A ., Postel, W. Guenther. S. and Weser, J. (1985) Improved horizontal two-dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying-on transfer to the second dimension. Electrophoresis 6(12), 599آ· 604. Jakob. E. (1994) Genetic polymorphism of milk proteins. Bulletin 298. International Dairy Federation. Brussels, Belgium, pp 1727. Koningsberg, W. H. and Henderson, L. (1983) Removal of sodium dodecyl sulphate from proteins by ion-pair extraction. Methods of Enzymology 91. 254-259. Larsson-Raznikiewicz, M. and Mohamed, M. A. (1986) Analysis of casein content in camel (Camelus dromedarius) milk. Swedish Journal of Agricultural Research 16(1), 13 18. Matsudaira. P. T. (1989) A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press Inc., San Diego, California, USA. Science Pub. Ltd .. England. pp. 405455. Ng-Kwai-Hang. K. G. and Grosclaude. F. (1992) Genetic polymorphism of milk proteins. In Advanced Dairy Chemistry. Vol. 1, proteins. ed. P. F. Fox. Elsevier. Amsterdam. Seibert, B., Erhardt, G. and Senft, B. (1985) Procedure for simultaneous phenotyping of genetic variants in cow's milk by isoelectric focusing. Animal Blood Groups Biochemistry and Genetics 16, 183-191. Wangoh. J. (1997) Chemical and technological properties of camel milk. PhD thesis, ETH Nr. 12295. Swiss fed. Inst. Technol, Zurich, Switzerland.

pdf