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Modified Procedure of Starch Gel Electrophoresis for β-Casein Phenotyping
1284
JOUR,NAL OF DAI}~Y SCIENCE
Hence, by using tributyriu as the substrate under the optimal conditions of the test described above, we have been able for the first time to demonstrate lipolytie activity in the following lactic acid bacteria: Lactobacillus
bulgaricus~ L. helveticus, L. ~a,ctis~ L. pIantarum, L. brevis~ L. viridescens, Leuconostoe mesenteroides~ and Pediocoecus cerevisiae. We have also been able to confirm the presence of lipolytic activity in L. easel, as reported by Peterson and Johnson (7). Details of this and further work on the lipolytie activity of the lactobaeilli using the p H star assay system will be reported later. A. OTERHOLM and Z. J. ORDAL Department of Food Science University o~ ~llinois, Urbana Acknowledgment
The authoTs express their appreciation to Dr. M. Elizabeth Sharpe for providing many of the lactobacilll cultures and to Dr. L. D. Witter and Dr. M. Elizabeth Sharpe for helpful suggestions during the course of this study. This work was supported by Training Grant T1-ES-25 from the U.S. Public Health Service.
(2)
(3) (4)
(5) (6) (7)
(8)
(9) (10) (11)
References
(1) Alford, 3". A., and Stein/e, E. ]966. Improved Plating Methoil for the Determlnation of Microbial Lipolysis. Abstrs. 66th
(12)
Annual Meeting, American Society for Microbiology. A80 : 14. Los Angeles, California. Anderson, 3". A. 1934. An Agar Plate Method for the Detection and Enumeration of Lipolytie Microorganisms. Abst. J. Bacteriol., 27:69. Eijkman, C. 1901. ~3ber Enzyme yon Baeterien mid Sehimmel pilzen. Abteilung Lipasem ZbL Bakt. I - O. 29:847. E1Sadek, G. M., and Richards, T. 1955. The Effects of the Heat Treatment on the Lipolytic Flora of Cream. J. Dairy Research, 22 : 295. Fryer, T. F., Lawrence, R. C., and Reiter, B. Personal Communication. Jensen, L. B., and Grettie, D. P. 1937. Action of Microorganisms on Fat. Food Research, 2 : 97. Petersou, M. H., and Johnson, M. J. 1949. Delayed Hydrolysis of Butterfat by Certain Lactebacilli and Micrococci Isolated from Cheese. J. Dairy Sol., 32:701. Rath, S. 1961. Ein Versuch zur Verbesserung tier Methodik zur quantitativen Ermittlung lipolytischer Keime. Milchwirtsch. Ber., 11;97. Rottem, S., and Razin, S. 1961. Lipase Activity of Mycoplasma. 5. Gem Microbiol., 37:123. Turner, R.. It. 1929. The Action of Bacteria on Fat. J. Infectious Diseases, 44:126. Wolf, J. 1940. Observations on Tributyrin Agar. Prec. Soc. Agr. Bacteriol. Abst. 48-50. Wolf, J. 1941. A Note on the Lipase of Some Lactic Acid Organisms. Prec. See. Agr. Bacteriol. Abst.
Modified Procedure of Starch Gel Electrophoresis for 13-Casein Phenotyping Peterson and Kopfler (2) have recently shown that one of the genetic variants of the caseins of cow's milk, fi-casein A, which forms a single band on gel electrophoresis in alkaline buffer systems, can be resolved into three distinct entities by gel eleetrophoresis at low pH. Existing data on fl-casein phenotypes will, therefore, require revision and, in future, phenotyping in alkaline gel media will have to be supplemented by a second electrophoretic run at low pH. Peterson and I~opfler (2) used vertical polyaerylamide gels for this purpose. With the modifications described below, the technique of horizontal starch gel electrophoresis (1) is equally suitable. I t can be used without isolation of the caseins from the milk samples. P H 1.7--buffer. Dilute 30 ml formic acid (98/ 100%) and 120 ml glacial acetic acid with H : 0 to one liter. This buffer is used to make the gel and to charge the apparatus. Milk samples. Dilute whole milk with an equal volume of a solution of 100 g urea in 100 ml
I-I~O. Warm the urea solution if crystals have formed on storage. Samples of total casein (dry). Dissolve 10 mg in 1 ml of the p H 1.7--buffer to which urea has been added at the rate of 6 g per 10 ml. Starch gels. Prepare these as described (1), but omit the 2-mercaptoethanol, and reduce the urea concentration from 40 to 24 g per 80 ml of buffer. Gels are best made in the morning for overnight runs. Eleetrophoresis. Apply a constant 220 v, corresponding to about 16 mA per gel for approximately 16 hr at four degrees.. A suitable dye, such as methyl red, when added to one of the samples, should migrate a distance of about 11 cm from the line of application. Results obtained with five milk samples of different fl-casein phenotype are shown in figure 1, to illustrate the resolution attainable by the modified technique. As the nomenclature proposals of Peterson and ]t~opfler (2) are unlikely 5. DAIRY SCIENCE VOL. 49, NO. 10
TECENICXL ~OTES
1285
FIG. 1. l%solution of ~-caseins ($) by starch gel electrophores[s at low pH. Whole milk applied at O. The bottom application consists of an artificial mixture of caseins. to prove aceeptable~ the variants of fl-easein A are here designated A1, A2, A3, in descending order of mobility. Like the polyacrylamide procedure, starch gel electrophoresis at low p H fails to resolve the variants of asl-casein ; nor does it permit differentiation of the variants of fl-lactoglobulin which form the leading band on the starch gel. Studies of the occurrence, and frequency of occurrence, of the fl-casein phenotypes in various breeds of cattle will require the examination of large numbers of milk samples. The modified starch gel procedure is capable of handling more samples per working day than the vertical polyacrylamide gel technique, and requires f a r less costly equipment.
Acknowledgement The technical assistance of Miss J. E. D. Worth is gratefully acknowledged. R. Aschaffenburg National Institute for Research in Dairying Shinfield, Reading, England References (1) Aschaffenburg, 1% and Thymann, Mariann. 1965. Simultaneous Phenotyping Procedure for the Principal Proteins of Cow's Milk. J. Dairy Sci., 48:1524. (2) Peterson, R. F., and Kopfler, F. C. 1966. Detection of New Types of fl-Casein by Polyaerylamide Gel Electrophoresis at Acid p i t : A Proposed Nomenclature. Bioehem. Bio* phys. Research Cmnmun., 22:388.
Objective Evaluation of Body in Ice Cream A person judging ice cream for body and texture must have the accepted ideal firmly in mind. Unintentional bias on the p a r t of the judge may occur because of the subjective method of evaluating ice cream. To insure an objective judgment an instrument should be J . DAIRY SCIENCE -~OL. 49, NO. 10
used, because it will give a completely unbiased evaluation. Szezesniak (2) has developed a system for the classification of textural characteristics of food whereby they are grouped into three main classes: mechanical, geometrical, and other
JOUR,NAL OF DAI}~Y SCIENCE
Hence, by using tributyriu as the substrate under the optimal conditions of the test described above, we have been able for the first time to demonstrate lipolytie activity in the following lactic acid bacteria: Lactobacillus
bulgaricus~ L. helveticus, L. ~a,ctis~ L. pIantarum, L. brevis~ L. viridescens, Leuconostoe mesenteroides~ and Pediocoecus cerevisiae. We have also been able to confirm the presence of lipolytic activity in L. easel, as reported by Peterson and Johnson (7). Details of this and further work on the lipolytie activity of the lactobaeilli using the p H star assay system will be reported later. A. OTERHOLM and Z. J. ORDAL Department of Food Science University o~ ~llinois, Urbana Acknowledgment
The authoTs express their appreciation to Dr. M. Elizabeth Sharpe for providing many of the lactobacilll cultures and to Dr. L. D. Witter and Dr. M. Elizabeth Sharpe for helpful suggestions during the course of this study. This work was supported by Training Grant T1-ES-25 from the U.S. Public Health Service.
(2)
(3) (4)
(5) (6) (7)
(8)
(9) (10) (11)
References
(1) Alford, 3". A., and Stein/e, E. ]966. Improved Plating Methoil for the Determlnation of Microbial Lipolysis. Abstrs. 66th
(12)
Annual Meeting, American Society for Microbiology. A80 : 14. Los Angeles, California. Anderson, 3". A. 1934. An Agar Plate Method for the Detection and Enumeration of Lipolytie Microorganisms. Abst. J. Bacteriol., 27:69. Eijkman, C. 1901. ~3ber Enzyme yon Baeterien mid Sehimmel pilzen. Abteilung Lipasem ZbL Bakt. I - O. 29:847. E1Sadek, G. M., and Richards, T. 1955. The Effects of the Heat Treatment on the Lipolytic Flora of Cream. J. Dairy Research, 22 : 295. Fryer, T. F., Lawrence, R. C., and Reiter, B. Personal Communication. Jensen, L. B., and Grettie, D. P. 1937. Action of Microorganisms on Fat. Food Research, 2 : 97. Petersou, M. H., and Johnson, M. J. 1949. Delayed Hydrolysis of Butterfat by Certain Lactebacilli and Micrococci Isolated from Cheese. J. Dairy Sol., 32:701. Rath, S. 1961. Ein Versuch zur Verbesserung tier Methodik zur quantitativen Ermittlung lipolytischer Keime. Milchwirtsch. Ber., 11;97. Rottem, S., and Razin, S. 1961. Lipase Activity of Mycoplasma. 5. Gem Microbiol., 37:123. Turner, R.. It. 1929. The Action of Bacteria on Fat. J. Infectious Diseases, 44:126. Wolf, J. 1940. Observations on Tributyrin Agar. Prec. Soc. Agr. Bacteriol. Abst. 48-50. Wolf, J. 1941. A Note on the Lipase of Some Lactic Acid Organisms. Prec. See. Agr. Bacteriol. Abst.
Modified Procedure of Starch Gel Electrophoresis for 13-Casein Phenotyping Peterson and Kopfler (2) have recently shown that one of the genetic variants of the caseins of cow's milk, fi-casein A, which forms a single band on gel electrophoresis in alkaline buffer systems, can be resolved into three distinct entities by gel eleetrophoresis at low pH. Existing data on fl-casein phenotypes will, therefore, require revision and, in future, phenotyping in alkaline gel media will have to be supplemented by a second electrophoretic run at low pH. Peterson and I~opfler (2) used vertical polyaerylamide gels for this purpose. With the modifications described below, the technique of horizontal starch gel electrophoresis (1) is equally suitable. I t can be used without isolation of the caseins from the milk samples. P H 1.7--buffer. Dilute 30 ml formic acid (98/ 100%) and 120 ml glacial acetic acid with H : 0 to one liter. This buffer is used to make the gel and to charge the apparatus. Milk samples. Dilute whole milk with an equal volume of a solution of 100 g urea in 100 ml
I-I~O. Warm the urea solution if crystals have formed on storage. Samples of total casein (dry). Dissolve 10 mg in 1 ml of the p H 1.7--buffer to which urea has been added at the rate of 6 g per 10 ml. Starch gels. Prepare these as described (1), but omit the 2-mercaptoethanol, and reduce the urea concentration from 40 to 24 g per 80 ml of buffer. Gels are best made in the morning for overnight runs. Eleetrophoresis. Apply a constant 220 v, corresponding to about 16 mA per gel for approximately 16 hr at four degrees.. A suitable dye, such as methyl red, when added to one of the samples, should migrate a distance of about 11 cm from the line of application. Results obtained with five milk samples of different fl-casein phenotype are shown in figure 1, to illustrate the resolution attainable by the modified technique. As the nomenclature proposals of Peterson and ]t~opfler (2) are unlikely 5. DAIRY SCIENCE VOL. 49, NO. 10
TECENICXL ~OTES
1285
FIG. 1. l%solution of ~-caseins ($) by starch gel electrophores[s at low pH. Whole milk applied at O. The bottom application consists of an artificial mixture of caseins. to prove aceeptable~ the variants of fl-easein A are here designated A1, A2, A3, in descending order of mobility. Like the polyacrylamide procedure, starch gel electrophoresis at low p H fails to resolve the variants of asl-casein ; nor does it permit differentiation of the variants of fl-lactoglobulin which form the leading band on the starch gel. Studies of the occurrence, and frequency of occurrence, of the fl-casein phenotypes in various breeds of cattle will require the examination of large numbers of milk samples. The modified starch gel procedure is capable of handling more samples per working day than the vertical polyacrylamide gel technique, and requires f a r less costly equipment.
Acknowledgement The technical assistance of Miss J. E. D. Worth is gratefully acknowledged. R. Aschaffenburg National Institute for Research in Dairying Shinfield, Reading, England References (1) Aschaffenburg, 1% and Thymann, Mariann. 1965. Simultaneous Phenotyping Procedure for the Principal Proteins of Cow's Milk. J. Dairy Sci., 48:1524. (2) Peterson, R. F., and Kopfler, F. C. 1966. Detection of New Types of fl-Casein by Polyaerylamide Gel Electrophoresis at Acid p i t : A Proposed Nomenclature. Bioehem. Bio* phys. Research Cmnmun., 22:388.
Objective Evaluation of Body in Ice Cream A person judging ice cream for body and texture must have the accepted ideal firmly in mind. Unintentional bias on the p a r t of the judge may occur because of the subjective method of evaluating ice cream. To insure an objective judgment an instrument should be J . DAIRY SCIENCE -~OL. 49, NO. 10
used, because it will give a completely unbiased evaluation. Szezesniak (2) has developed a system for the classification of textural characteristics of food whereby they are grouped into three main classes: mechanical, geometrical, and other