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Detection of Cow Milk in Goat Milk by Polyacrylamide Gel Electrophoresis
Detection of Cow Milk in Goat Milk by Polyacrylamide Gel Electrophoresis M U C I O M. F U R T A D O 1 Michigan State University Department of Food Science and Human Nutrition East Lansing, MI 48824
milk. According to Aschaffenburg and Dance (1) this technique was capable of detecting as little as 1% cow milk in goat milk. The same method, in horizontal plates of polyacrylamide gel, was employed by Portmann (7), who developed equations to evaluate adulteration of goat milk cheese by addition of cow milk to goat milk. The purpose of this study will be to characterize cow milk in pasteurized goat milk by discontinuous polyacrylamide gel electrophoresis.
ABSTRACT
Pasteurized goat milk was adulterated with increasing proportions of cow milk and submitted to polyacrylamide gel electrophoresis. A frontal band, missing from the pattern of genuine goat milk and possessing the same electrophoretic mobility as bovine ~sl-casein, was expressed. The area of this zone was directly proportional to the amount of cow milk added to the goat milk. INTRODUCTION
In most countries where goat milk is produced either for direct consumption or for cheese making, it commands a considerably higher price than cow milk. For economic as well as ethical reasons it is, therefore, desirable to ascertain that goat milk offered for sale is free from admixtures with cow milk. Many different methods have been used to detect cow milk added to goat milk. Historically a method based on the relationship between soluble and insoluble volatile acids in goat milk constituted the method of choice (3). A serological method applicable only to milk was introduced by Solberg and Hadland (10) in 1953. KuzdzalSavoie and Kuzdzal (5) suggested a method based on the absence of ~3-carotene in goat milk, which was incapable of detecting the addition of cow skim milk. More recently, methods have been developed based on the electrophoretic mobility of milk proteins; the asl-caseins of cow milk have higher electrophoretic mobilities in urea-containing alkaline gel media than the fastest-moving casein constituent of goat milk, so that the presence of bovine asl-casein manifests itself in the appearance of a frontal zone missing from the pattern of genuine goat
Received October 14, 1982. 11nstituto de Laticinios Candido Tostes--EPAMIG 36100 -- Juiz de Fora-MG-Brazil. 1983 J Dairy Sci 66:1822-1824
METHODS AND M A T E R I A L S Goat Milk
Goat's milk from a mixed herd was used. The milk was pasteurized at 65°C for 30 min and cooled to 40°C. Then, 5, 15, 30, and 50% (vol/vol) of pasteurized cow milk were added, followed by centrifugation at 10,000 x g for 10 min to separate the fat. Preparation of Caseins
Caseins were precipitated from skim milk by adjusting pH to 4.6 with N He1. The precipitated casein was washed four times with distilled water, resuspended in water b y adjusting the pH to 7.5 with N NaOH, reprecipitated, washed, and dried. Gel Electrophoresis
Electrophoresis in 9% polyacrylamide gels was in a vertical water-cooled Bio-Rad Model 150 A electrophoresis cell with a method essentially similar to that of Ornstein (6) and Davis (4). Two drops of 2-mercaptoethanol were added to the samples during the extraction with 7 M urea solution. Gels were stained for protein with Coomassie brilliant blue G 250 (,04%) for 15 h and electrolytically destained in 4% acetic acid for 60 min.
1822
ADULTERATION OF GOAT MILK WITH COW MILK Densitometry
Gels were scanned at 550 nm with a Beckman DU Spectrophotometer Model 2400 equipped with a gel scanner Model 2520 and a Photometer 252 (Gilford Instrument Laboratories). The system was summoned to an ItP Integrator, Model 3380-S, and relative protein concentrations were calculated from integration signs on the densitograms.
1823
can see that goat casein (A) does not present a major band in the domain of bovine asl-casein (F). However, with increased adulteration of goat milk with cow milk, stepwise from 5 to 50% (densitograms B, C, D, and E), a frontal band with the same mobility of bovine O~sl-
RESULTS A N D DISCUSSION
Figure 1 shows the densitograms of the caseins of pure cow (F) and goat (A) milk as well as those of goat milk following the addition of 5 (B), 15 (C), 30 (D), and 50% (E) of cow milk. In densitogram A goat casein presents two strong bands in the 3-casein region, amounting to about 81% of the total area of the densitogram. From densitogram A (100% goat milk) compared with densitogram F (100% cow milk), ~-caseins from both goat and cow milk have similar electrophoretic mobilities. Also, 3-casein is the major component of caprine caseins. A similar result was reported by Zittle and Custer (11), who observed that caprine casein contains two major ~-casein components with electrophoretic mobilities in alkaline buffers similar to mobility of bovine 3-casein. These results were confirmed further by Richardson and Creamer (8), who found that the molecular weight of each caprine /3-casein was about 24,500, both caseins similar to bovine ~-casein in molecular weight and net charge. In Figure 1, caprine 0~sl-casein has a lower electrophoretic mobility than bovine &sl-casein. In both densitograms A and F O~si-casein is the leading band, but it shows up in much smaller proportion (16.9%) and lower mobility in A than in F, which represents bovine caseins. In the latter, &sl-casein represents 54.4% of all bands. According to Richardson and Creamer (9) less than 25% of the goat casein is the 0~si-component. Assenat (2) studied the electrophoretic mobility of caprine and bovine caseins and found that the relative mobility of bovine ~sl-casein was higher than that of caprine ~sl-casein. The absence in goat casein of any electrophoretic component with the mobility of bovine 0~sl-casein makes it possible to detect adulteration of goat's milk with cow's milk. By observing Figure 1, densitograms A and F, one
r
+
E
F
I I
Figure 1. Densitometric patterns of discontinuous polyacrylamide gel e]ecrrophoresis of pure goat's (A) and cow's (F) milk and goat's milk adulterated with 5 (B), 15 (C), 30 (D) and 50% of cow's milk. Journal of Dairy Science Vol. 66, No. 9, 1983
1824
FURTADO
TABLE 1. Relationship between the percentage of cow milk added to goat milk and percentage area of the frontal band in the densitograms. Densitogram (Figure 1)
Area of frontal band
Cow milk added (%)
A B
1.209 1.777
C
7.797
D E F
15.480 20.440 41.610
0 5 15 30 50 100
casein b e c a m e increasingly a p p a r e n t , its area being directly proportional to the amount of c o w ' s m i l k a d d e d t o t h e g o a t ' s milk. In T a b l e 1 t h e p e r c e n t a g e area of t h e b a n d s h o w i n g u p in this region o f t h e c a p r i n e casein e l e c t r o p h e r o g r a m s is r e l a t e d t o t h e a m o u n t of c o w milk a d d e d to t h e g o a t milk. Regression analysis of t h e s e d a t a revealed a close c o r r e l a t i o n (.996) b e t w e e n p e r c e n t a g e o f a d d e d c o w m i l k a n d area o f t h e b a n d s h o w i n g u p in t h a t region. C o w milk (5% or higher) in p a s t e u r i z e d g o a t milk m a y b e d e t e c t e d b y t h e e l e c t r o p h o r e t i c r e s o l u t i o n o f t h e casein f r a c t i o n .
ACKNOWLEDGMENTS
T h e a u t h o r is i n d e b t e d t o J. R. B r u n n e r o f Michigan S t a t e University for his valuable discussion o f these results.
Journal of Dairy Science Vol. 66, No. 9, 1983
REFERENCES
1 Aschaffenburg, R., and J. E. Dance. 1968. Detection of cow's milk in goat's milk by gel electrophoresis. J. Dairy Res. 35:383. 2 Assenat, L. 1967. Contribution a l'etude d'une methode d'identification des laits et fromagcs au moyen de l'electrophorese sur gel de polyacrymalide. Le Lait 47: 393. 3 Chollet, A., and A. Camus. 1937. Etude de la matiere grasse du lair de chevre, son application eventuelle a' la recherche du melange du lait de chevre et du lair de vache. Ann. Falsif. Fraudes 337:405. 4 Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. New York Acad. Sci. 121:404. 5 Kuzdzal-Savoie, S., and W. Kuzdzal. 1959. La recherche du lait de vache ajoute au lait de chevre: application au cas du fromage. Ann. Technol. Agric. 8:131. 6 Ornstein, L. 1964. Disc electrophoresis. I. Background and theory. Ann. New York Acad. Sci. 121:321. 7 Porlxnann, A., and A. Pierre. 1970. Emploi de 1'electrophorese in gel de polyacrylamide pour mettre en evidence et doser le lait de vache ajoute au lait de chevre: application au cas des dromages. Ann. Technol. Agric. 19:107. 8 Richardson, B. C., and L. K. Creamer. 1974. Comparative micelle structure, lII. The isolation and chemical characterization of caprine ~3-casein and #2-casein. Biochim. Biophys. Acta 365:133. 9 Richardson, B. C., L. K. Creamer, K. N. Pearce, and R. E. Mumford. 1974. Comparative micelle structure. II. Structure and composition of casein micelles in ovine and caprine milk as compared with those in bovine milk. J. Dairy Res. 41:239. i 0 Solberg, P., and G. Hadland. 1953. Serological detection of cow's milk added to milk from goat. Proc. Xlll Int. Dairy Congr. Iih1287. 11 Zittle, C. A., and J. H. Custer. 1966. Identification of the g-casein among the components of whole goat casein. J. Dairy Sci. 49:788.
milk. According to Aschaffenburg and Dance (1) this technique was capable of detecting as little as 1% cow milk in goat milk. The same method, in horizontal plates of polyacrylamide gel, was employed by Portmann (7), who developed equations to evaluate adulteration of goat milk cheese by addition of cow milk to goat milk. The purpose of this study will be to characterize cow milk in pasteurized goat milk by discontinuous polyacrylamide gel electrophoresis.
ABSTRACT
Pasteurized goat milk was adulterated with increasing proportions of cow milk and submitted to polyacrylamide gel electrophoresis. A frontal band, missing from the pattern of genuine goat milk and possessing the same electrophoretic mobility as bovine ~sl-casein, was expressed. The area of this zone was directly proportional to the amount of cow milk added to the goat milk. INTRODUCTION
In most countries where goat milk is produced either for direct consumption or for cheese making, it commands a considerably higher price than cow milk. For economic as well as ethical reasons it is, therefore, desirable to ascertain that goat milk offered for sale is free from admixtures with cow milk. Many different methods have been used to detect cow milk added to goat milk. Historically a method based on the relationship between soluble and insoluble volatile acids in goat milk constituted the method of choice (3). A serological method applicable only to milk was introduced by Solberg and Hadland (10) in 1953. KuzdzalSavoie and Kuzdzal (5) suggested a method based on the absence of ~3-carotene in goat milk, which was incapable of detecting the addition of cow skim milk. More recently, methods have been developed based on the electrophoretic mobility of milk proteins; the asl-caseins of cow milk have higher electrophoretic mobilities in urea-containing alkaline gel media than the fastest-moving casein constituent of goat milk, so that the presence of bovine asl-casein manifests itself in the appearance of a frontal zone missing from the pattern of genuine goat
Received October 14, 1982. 11nstituto de Laticinios Candido Tostes--EPAMIG 36100 -- Juiz de Fora-MG-Brazil. 1983 J Dairy Sci 66:1822-1824
METHODS AND M A T E R I A L S Goat Milk
Goat's milk from a mixed herd was used. The milk was pasteurized at 65°C for 30 min and cooled to 40°C. Then, 5, 15, 30, and 50% (vol/vol) of pasteurized cow milk were added, followed by centrifugation at 10,000 x g for 10 min to separate the fat. Preparation of Caseins
Caseins were precipitated from skim milk by adjusting pH to 4.6 with N He1. The precipitated casein was washed four times with distilled water, resuspended in water b y adjusting the pH to 7.5 with N NaOH, reprecipitated, washed, and dried. Gel Electrophoresis
Electrophoresis in 9% polyacrylamide gels was in a vertical water-cooled Bio-Rad Model 150 A electrophoresis cell with a method essentially similar to that of Ornstein (6) and Davis (4). Two drops of 2-mercaptoethanol were added to the samples during the extraction with 7 M urea solution. Gels were stained for protein with Coomassie brilliant blue G 250 (,04%) for 15 h and electrolytically destained in 4% acetic acid for 60 min.
1822
ADULTERATION OF GOAT MILK WITH COW MILK Densitometry
Gels were scanned at 550 nm with a Beckman DU Spectrophotometer Model 2400 equipped with a gel scanner Model 2520 and a Photometer 252 (Gilford Instrument Laboratories). The system was summoned to an ItP Integrator, Model 3380-S, and relative protein concentrations were calculated from integration signs on the densitograms.
1823
can see that goat casein (A) does not present a major band in the domain of bovine asl-casein (F). However, with increased adulteration of goat milk with cow milk, stepwise from 5 to 50% (densitograms B, C, D, and E), a frontal band with the same mobility of bovine O~sl-
RESULTS A N D DISCUSSION
Figure 1 shows the densitograms of the caseins of pure cow (F) and goat (A) milk as well as those of goat milk following the addition of 5 (B), 15 (C), 30 (D), and 50% (E) of cow milk. In densitogram A goat casein presents two strong bands in the 3-casein region, amounting to about 81% of the total area of the densitogram. From densitogram A (100% goat milk) compared with densitogram F (100% cow milk), ~-caseins from both goat and cow milk have similar electrophoretic mobilities. Also, 3-casein is the major component of caprine caseins. A similar result was reported by Zittle and Custer (11), who observed that caprine casein contains two major ~-casein components with electrophoretic mobilities in alkaline buffers similar to mobility of bovine 3-casein. These results were confirmed further by Richardson and Creamer (8), who found that the molecular weight of each caprine /3-casein was about 24,500, both caseins similar to bovine ~-casein in molecular weight and net charge. In Figure 1, caprine 0~sl-casein has a lower electrophoretic mobility than bovine &sl-casein. In both densitograms A and F O~si-casein is the leading band, but it shows up in much smaller proportion (16.9%) and lower mobility in A than in F, which represents bovine caseins. In the latter, &sl-casein represents 54.4% of all bands. According to Richardson and Creamer (9) less than 25% of the goat casein is the 0~si-component. Assenat (2) studied the electrophoretic mobility of caprine and bovine caseins and found that the relative mobility of bovine ~sl-casein was higher than that of caprine ~sl-casein. The absence in goat casein of any electrophoretic component with the mobility of bovine 0~sl-casein makes it possible to detect adulteration of goat's milk with cow's milk. By observing Figure 1, densitograms A and F, one
r
+
E
F
I I
Figure 1. Densitometric patterns of discontinuous polyacrylamide gel e]ecrrophoresis of pure goat's (A) and cow's (F) milk and goat's milk adulterated with 5 (B), 15 (C), 30 (D) and 50% of cow's milk. Journal of Dairy Science Vol. 66, No. 9, 1983
1824
FURTADO
TABLE 1. Relationship between the percentage of cow milk added to goat milk and percentage area of the frontal band in the densitograms. Densitogram (Figure 1)
Area of frontal band
Cow milk added (%)
A B
1.209 1.777
C
7.797
D E F
15.480 20.440 41.610
0 5 15 30 50 100
casein b e c a m e increasingly a p p a r e n t , its area being directly proportional to the amount of c o w ' s m i l k a d d e d t o t h e g o a t ' s milk. In T a b l e 1 t h e p e r c e n t a g e area of t h e b a n d s h o w i n g u p in this region o f t h e c a p r i n e casein e l e c t r o p h e r o g r a m s is r e l a t e d t o t h e a m o u n t of c o w milk a d d e d to t h e g o a t milk. Regression analysis of t h e s e d a t a revealed a close c o r r e l a t i o n (.996) b e t w e e n p e r c e n t a g e o f a d d e d c o w m i l k a n d area o f t h e b a n d s h o w i n g u p in t h a t region. C o w milk (5% or higher) in p a s t e u r i z e d g o a t milk m a y b e d e t e c t e d b y t h e e l e c t r o p h o r e t i c r e s o l u t i o n o f t h e casein f r a c t i o n .
ACKNOWLEDGMENTS
T h e a u t h o r is i n d e b t e d t o J. R. B r u n n e r o f Michigan S t a t e University for his valuable discussion o f these results.
Journal of Dairy Science Vol. 66, No. 9, 1983
REFERENCES
1 Aschaffenburg, R., and J. E. Dance. 1968. Detection of cow's milk in goat's milk by gel electrophoresis. J. Dairy Res. 35:383. 2 Assenat, L. 1967. Contribution a l'etude d'une methode d'identification des laits et fromagcs au moyen de l'electrophorese sur gel de polyacrymalide. Le Lait 47: 393. 3 Chollet, A., and A. Camus. 1937. Etude de la matiere grasse du lair de chevre, son application eventuelle a' la recherche du melange du lait de chevre et du lair de vache. Ann. Falsif. Fraudes 337:405. 4 Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. New York Acad. Sci. 121:404. 5 Kuzdzal-Savoie, S., and W. Kuzdzal. 1959. La recherche du lait de vache ajoute au lait de chevre: application au cas du fromage. Ann. Technol. Agric. 8:131. 6 Ornstein, L. 1964. Disc electrophoresis. I. Background and theory. Ann. New York Acad. Sci. 121:321. 7 Porlxnann, A., and A. Pierre. 1970. Emploi de 1'electrophorese in gel de polyacrylamide pour mettre en evidence et doser le lait de vache ajoute au lait de chevre: application au cas des dromages. Ann. Technol. Agric. 19:107. 8 Richardson, B. C., and L. K. Creamer. 1974. Comparative micelle structure, lII. The isolation and chemical characterization of caprine ~3-casein and #2-casein. Biochim. Biophys. Acta 365:133. 9 Richardson, B. C., L. K. Creamer, K. N. Pearce, and R. E. Mumford. 1974. Comparative micelle structure. II. Structure and composition of casein micelles in ovine and caprine milk as compared with those in bovine milk. J. Dairy Res. 41:239. i 0 Solberg, P., and G. Hadland. 1953. Serological detection of cow's milk added to milk from goat. Proc. Xlll Int. Dairy Congr. Iih1287. 11 Zittle, C. A., and J. H. Custer. 1966. Identification of the g-casein among the components of whole goat casein. J. Dairy Sci. 49:788.