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Probing the location of casein fractions in the casein micelle using enzymes and enzyme–dextran conjugates

Published online by Cambridge University Press:  01 June 2009

Brian Chaplin  and
Margaret L. Green
Brian Chaplin
Affiliation:
National Institute for Research in Dairying, Shinfield, Reading, RG2 9 AT, UK
Margaret L. Green
Affiliation:
National Institute for Research in Dairying, Shinfield, Reading, RG2 9 AT, UK
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Summary

The rates of milk clotting and of formation of para-κ-casein in milk, colloidal phosphate-free milk and isolated κ-casein by pepsin and by its soluble size-fractionated conjugates with dextran were determined. Milk clotting with all the enzyme derivatives was dependent on the rate of the enzymic phase and required essentially complete κ-casein hydrolysis at 30 °C and throughout the range of pH 5·6–6·7. κ-Casein hydrolysis by pepsin at pH 6·6 was fastest in milk and slowest in isolated κ-casein, but the rate decreased as the enzyme size increased, especially with milk. When corrected for the changes in pepsin activity, the rates of κ-casein hydrolysis in all substrates were identical at 30 and 5 °C, but increased with decrease in pH, especially with the larger enzyme conjugates. Hydrolysis of the C-terminal bonds of β-and κ-casein in native and disrupted casein micelles by carboxypeptidase A and soluble conjugates of it were also investigated. κ-Casein was hydrolysed much faster, and β-casein slightly faster, in native than in disrupted micelles by the native enzyme. Increase in the size of carboxypeptidase A increased the rate of hydrolysis of κ-casein in disrupted micelles and also induced lag periods before hydrolysis commenced, especially with disrupted micelles. The results are compatible with a model for the casein micelle in which the κcasein is on the outside and the casein components are in a more ordered arrangement than in the casein complexes formed on micelle disruption. They also indicate that immobilized coagulants would be unable to clot milk.

Type
Original Articles
Information
Journal of Dairy Research , Volume 49 , Issue 4 , November 1982 , pp. 631 - 643
Copyright
Copyright © Proprietors of Journal of Dairy Research 1982

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References

REFERENCES

Ali, A. E., Andrews, A. T. & Cheeseman, G. C. 1980 Factors influencing casein distribution in cold-stored milk and their effects on cheese-making parameters. Journal of Dairy Research 47 383–391CrossRefGoogle Scholar
Arima, S., Shimazaki, K., Yamazumi, T. & Kanamaru, Y. 1974 Studies on the preparation of insoluble derivatives of rennin (coupling to Sepharose and aminoethylcellulose). Japanese Journal of Dairy Science 23 A83–A87Google Scholar
Beeby, R. 1979 The proteolysis of casein by immobilized preparations of α-chymotrypsin, chymosin and a fungal protease. New Zealand Journal of Dairy Science and Technology 14 1–11Google Scholar
Berridge, N. J. 1952 Some observations on the determination of the activity of rennet. Analyst 77 57–62CrossRefGoogle Scholar
Brown, R. J. & Swaisgood, H. W. 1975 Preparation of an active form of immobilized rennin. Journal of Dairy Science 58 796Google Scholar
Chaplin, B. 1980 A study of milk casein micelles using conjugated enzymes. Thesis, University of ReadingGoogle Scholar
Chaplin, B. & Green, M. L. 1980 Determination of the proportion of κ-casein hydrolysed by rennet on coagulation of skim-milk. Journal of Dairy Research 47 351–358CrossRefGoogle Scholar
Chaplin, B. & Green, M. L. 1981 The rate of hydrolysis of κ-casein in various complexes by pepsin and soluble dextran-pepsin conjugates. Netherlands Milk and Dairy Journal 35 377–380Google Scholar
Chaplin, B. & Green, M. L. 1982 Preparation and characterization of soluble conjugates of defined molecular sizes prepared by linkage of pepsin and carboxypeptidase A to dextrans. Biotechnology and Bioengineering, in press.CrossRefGoogle ScholarPubMed
Chervenka, C. H. 1970 A Manual of Methods for the Analytical Centrifuge pp. 83–86. Palo Alto, Calif., USA: Beckman Instruments Inc.Google Scholar
Chow, R. B. & Kassell, B. 1968 Bovine pepsinogen and pepsin. I. Isolation, purification and some properties of the pepsinogen. Journal of Biological Chemistry 243 1718–1724CrossRefGoogle ScholarPubMed
Cooke, R. D. & Caygill, J. C. 1974 The possible utilisation of plant proteases in cheesemaking. Tropical Science 16 149–153Google Scholar
Creamer, L. K. & Berry, G. P. 1975 A study of the properties of dissociated bovine casein micelles. Journal of Dairy Research 42 169–183CrossRefGoogle Scholar
Creamer, L. K., Berry, G. P. & Mills, O. E. 1977 A study of the dissociation of β-casein from the bovine casein micelle at low temperature. New Zealand Journal of Dairy Science and Technology 12 58–66Google Scholar
Davies, D. T. & Law, A. J. R. 1980 The content and composition of protein in creamery milks in south-west Scotland, Journal of Dairy Research 47 83–90CrossRefGoogle Scholar
Dickson, I. R. & Perkins, D. J. 1971 Studies on the interactions between purified bovine caseins and alkaline-earth-metal ions. Biochemical Journal 124 235–240CrossRefGoogle ScholarPubMed
Downey, W. K. & Murphy, R. F. 1970 The temperature-dependent dissociation of β-casein from bovine casein micelles and complexes. Journal of Dairy Research 37 361–372CrossRefGoogle Scholar
Foltmann, B. 1959 On the enzymatic and the coagulation stages of the rennetting process. 15th International Dairy Congress, London 2 655–661Google Scholar
Fox, P. F. & Guiney, J. 1973 Casein micelle structure: susceptibility of various casein systems to proteolysis. Journal of Dairy Research 40 229–234CrossRefGoogle ScholarPubMed
Green, M. L. 1969 Simple methods for the purification of crude κ-casein and β-casein by treatment with calcium phosphate gel. Journal of Dairy Research 36 353–357CrossRefGoogle Scholar
Green, M. L. 1982 Effect on the composition and properties of casein micelles of interaction with ionic materials. Journal of Dairy Research 49 87–98CrossRefGoogle Scholar
Green, M. L. & Crutchfield, G. 1969 Studies on the preparation of water-insoluble derivatives of rennin and chymotrypsin and their use in the hydrolysis of casein and the clotting of milk. Biochemical Journal 115 183–190.CrossRefGoogle Scholar
Green, M. L. & Marshall, R. J. 1977 The acceleration by cationic materials of the coagulation of casein micelles by rennet. Journal of Dairy Research 44 521–531CrossRefGoogle Scholar
Grosclaude, F., Mahé, M-F. & Ribadeau-Dumas, B. 1973 Primary structure of bovine αs1,-casein and β-casein: correction. European Journal of Biochemistry 40 323–324CrossRefGoogle Scholar
Hicks, C L., Ferrier, L. K., Olson, N. F. & Richardson, T. 1975 Immobilized pepsin treatment of skim milk and skim milk fractions. Journal of Dairy Science 58 19–24CrossRefGoogle Scholar
Jenness, R. & Koops, J. 1962 Preparation and properties of a salt solution which simulates milk ultrafiltrate. Netherlands Milk and Dairy Journal 16 153–164Google Scholar
Kalab, M., Emmons, D. B. & Sargant, A. G. 1976 Milk gel structure. V. Microstructure of yoghurt as related to the heating of milk. Milchwissenschaft 31 402–408Google Scholar
Lin, S. H. C., Leong, S. L., Dewan, R. K., Bloomfield, V. A. & Morr, C. V. 1972 Effect of calcium ion on the structure of native bovine casein micelles. Biochemistry 11 1818–1821CrossRefGoogle ScholarPubMed
McGann, T. C. A., Donnelly, W. J., Kearney, R. D. & Buchheim, W. 1980 Composition and size distribution of bovine casein micelles. Biochimica el Biophysica Acta 630 261–270CrossRefGoogle ScholarPubMed
Mercier, J-C., Brignon, G. & Ribadeau-Dumas, B.. 1973 Primary structure of bovine κ-casein B. Complete sequence. European Journal of Biochemistry 35 222–235CrossRefGoogle ScholarPubMed
Morr, C. V. 1967 Effect of oxalate and urea upon ultracentrifugation properties of raw and heated skimmilk casein micelles. Journal of Dairy Science 50 1744–1751CrossRefGoogle Scholar
Morr, C. V., Josephson, R. V., Jenness, R. & Manning, P. B. 1971 Composition and properties of submicellar casein complexes in colloidal phosphate-free skimmilk. Journal of Dairy Science 54 1555–1563CrossRefGoogle Scholar
Neurath, H. 1955 Carboxypeptidase and procarboxypeptidase. Methods in Enzymology 2 77–83CrossRefGoogle Scholar
Ohmiya, K., Tanimura, S., Kobayashi, T. & Shimizu, S. 1979 Application of immobilized alkaline protease to cheese-making. Journal of Food Science 44 1584–1588CrossRefGoogle Scholar
Payens, T. A. J. 1979 Casein micelles: the colloid-chemical approach. Journal of Dairy Research 46 291–306CrossRefGoogle ScholarPubMed
Pyne, G. T. 1962 Reviews of the progress of Dairy Science. Section C. Dairy Chemistry. Some aspects of the physical chemistry of the salts of milk. Journal of Dairy Reseach 29 101–130CrossRefGoogle Scholar
Ribadeau Dumas, B. 1968 Simultaneous determination of αs1-, β- and κ-caseins in whole casein by using carboxypeptidase A. Biochimica el Biophysica Acta 168 274–281CrossRefGoogle Scholar
Ribadeau Dumas, B. & Garnier, J. 1970 Structure of the casein micelle: The accessibility of the subunits to various reagents. Journal of Dairy Research 37 269–278CrossRefGoogle Scholar
Rowland, S. J. 1938 The determination of the nitrogen distribution in milk. Journal of Dairy Research 9 42–46.CrossRefGoogle Scholar
Sachse, H. & Langhammer, G. 1981 Investigations on immobilization of milk-clotting enzymes of different origin. I. Immobilization of calf rennet. Nahrung 25 281–291CrossRefGoogle Scholar
Schmidt, D. G. 1980 Colloidal aspects of casein. Netherlands Milk and Dairy Journal 34 42–64Google Scholar
Schmidt, D. G., Walstra, P. & Buchheim, W. 1973 The size distribution of casein micelles in cow's milk. Netherlands Milk and Dairy Journal 27 128–142Google Scholar
Shindo, K., Sakurada, K., Niki, R. & Arima, S. 1980 Studies on immobilized chymosin. 5. Experiments in cheesemaking with immobilized chymosin. Milchwissenschaft 35 527–530Google Scholar
Slattery, C. W. & Evard, R. 1973 A model for the formation and structure of casein micelles from subunits of variable composition. Biochimica el Biophysica Acta 317 529–538CrossRefGoogle Scholar
Tanford, C. 1961 Physical Chemistry of Macromolecules Chapter 6, New York: John Wiley & Sons Inc.Google Scholar
Taylor, M. J., Olson, N. F. & Richardson, T. 1979 Coagulation of skimmilk with immobilised proteases. Process Biochemistry 14(2) 10 12–14 16 26Google Scholar
Thomas, M. A. W. 1973 The accessibility and lability of the rennin-sensitive bond of bovine κ-casein. Netherlands Milk and Dairy Journal 27 273Google Scholar
Vrbeean, H. J. 1979 The association of bovine SH-α-casein at pH 7·0. Journal of Dairy Research 46 271–276.Google Scholar
Walstra, P. 1979 The voluminosity of bovine casein micelles and some of its implications. Journal of Dairy Research 46, 317–323CrossRefGoogle ScholarPubMed
Walstra, P., Bloomfield, V. A., Wei, G. J. & Jenness, R. 1981 Effect of chymosin action on the hydrodynamie diameter of casein micelles. Biochimica el Biophysica Acta 669 258–259CrossRefGoogle ScholarPubMed
Wei, G. J. & Deal, W. C. 1978 A simple ‘width’ method for easy determination of diffusion coefficients with maximum utilization of data. Analytical Biochemistry 87 433–446CrossRefGoogle ScholarPubMed
Zittle, C. A. & Custer, J. H. 1963 Purification and some of the properties of αs-casein and κ-casein. Journal of Dairy Science 46 1183–1188CrossRefGoogle Scholar