Czech J. Food Sci., 2002, 20(2):53-62 | DOI: 10.17221/3510-CJFS
Optimisation of indirect competitive ELISAs of a-, b-, and k-caseins for the recognition of thermal and proteolytic treatment of milk and milk products
- Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, Czech Republic
Polyclonal antibodies were raised against six immunogens (three native and three thermally treated casein fractions: a+b-casein, k-casein and whole casein). Using these antibodies the procedures of an indirect competitive enzyme immunoassay (ELISA) were constructed, optimised and characterised for determination of each immunogen. It was found that ELISA of caseins is very specific without any interferences of whey proteins and with proportionally low cross-interactions between caseins. Detection limits for a-, b-, and k-caseins and whole casein were 110, 49, 58 and 505 ng/ml, respectively. The coefficient of variation was lower than 12% in intra-assay and lower than 9% in inter-assay. The developed ELISA format was used to study changes in casein immunoreactivity during heat-treatment and proteolytic hydrolysis. Heating below 100°C did not change the milk immunoreactivity but heating above 100°C caused its significant changes. Depending on type of proteolytic treatment (with enzyme preparation Pancreatin or with microbial cultures of Lactobacillus helveticus and Lactococcus) a decrease and an increase in casein immunoreactivity were observed. While Pancreatin reduced the casein immunoreactivity substantially (5-1000 times in dependence on the casein type), the more gentle proteolysis by bacteria caused not only its reduction (even 100 times at k-casein) but also its increase (1.5 times at a-casein).
Keywords: a-casein; b-casein; k-casein; casein hydrolysates; antibody; immunoassay; ELISA
Published: April 30, 2002 Show citation
ACS | AIP | APA | ASA | Harvard | Chicago | IEEE | ISO690 | MLA | NLM | Turabian | Vancouver |
References
- ADDEO F., GARRO G., INTORCIA N., PELLEGRINO L., RESMINI P., CHIANESE L. (1995): Gel-electrophoresis and immuno-blotting for the detection of casein proteolysis in cheese. J. Dairy Res., 62: 297-309.
Go to original source...
Go to PubMed...
- ANGUITA G., MARTÍN R., GARCÍA T., MORALES P., HAZA A.I., GONZÁLEZ I., SANZ B., HERNÁNDEZ P.E. (1995): Indirect ELISA for detection of cows' milk in ewes' and goats' milks using a monoclonal antibody against bovine β-casein. J. Dairy Res., 62: 655-659.
Go to original source...
- ANGUITA G., MARTÍN R., GARCÍA T., MORALES P., HAZA A.I., GONZÁLEZ I., SANZ B., HERNÁNDEZ P.E. (1996): Immunological characterization of bovine casein fractionated by fast protein liquid chromatography (FPLC). Milchwissenschaft, 51: 21-25.
- ANGUITA G., MARTÍN R., GARCÍA T., MORALES P., HAZA A.I., GONZÁLEZ I., SANZ B., HERNÁNDEZ P.E. (1997): A competitive Enzyme-Linked Immunosorbent Assay for detection of bovine milk in ovine and caprine milk and cheese using a monoclonal antibody against bovine β-casein. J. Food Protect., 60: 64-66.
Go to original source...
Go to PubMed...
- BITRI L., ROLLAND M.P., BESANCON P. (1993): Immunological detection of bovine caseinomacropeptide in ovine and caprine dairy products. Milchwissenschaft, 48: 367-370.
- BOBE G., BEITZ D.C., FREEMAN A.E., LINDBERG G.L. (1998): Separation and quantification of bovine milk proteins by reversed-phase High-Performance Liquid Chromatography. J. Agr. Food Chem., 46: 458-463.
Go to original source...
Go to PubMed...
- BORDIN G., RAPOSO F.C., DE LA CALLE B., RODRIGUEZ A.R. (2001): Identification and quantification of major bovine milk proteins by liquid chromatography. J. Chromatogr. A., 928: 63-76.
Go to original source...
Go to PubMed...
- CALVO M.M., LAW A.J.R., LEAVER J. (1995): Heat-induced interactions between serum albumin, immunoglobulin, and kappa-casein inhibit the primary phase of renneting. J. Ag. Food Chem., 43: 2823-2827.
Go to original source...
- CARRETERO C., TRUJILLO A.J., MORMUR M., PLA R., GUAMIS B. (1994): Electrophoretic study of casein breakdown during ripening of goat's milk cheese. J. Agr. Food Chem., 42: 1546-1550.
Go to original source...
- CORREDIG M., DALGLEISH D.G. (1999): The mechanisms of the heat-induced interaction of whey proteins with casein micelles in milk. Int. Dairy J., 9: 233-236.
Go to original source...
- CREAMER L.K., MATHESON A.R. (1980): Effect of heat treatment on the proteins of pasteurized skim milk. N.Z. J. Dairy Sci. Technol., 15: 37-49.
- CROW V.L., MARTLEY F.G., COOLBEAR T., ROUNDHILL S.J. (1995): The influence of phage-assisted lysis of Lactococcus lactis subsp. lactis ML8 on cheddar cheese ripening. Int. Dairy J., 5: 451-472.
Go to original source...
- ČURDA L., BELHÁČOVÁ L., UHROVÁ M., ŠTĚTINA J., FUKAL L. (1997): Assessment of heat-induced denaturation of whey proteins. J. Chromatogr. A, 772: 231-234.
Go to original source...
- DALGLEISH D. G. (1990): Denaturation and aggregation of serum proteins and caseins in heated milk. J. Agr. Food Chem., 38: 1995-1999.
Go to original source...
- DALGLEISH D.G., HORNE D.S., LAW A.J.R. (1989): Size-related differences in bovine casein micelles. Biochim. Biophys. Acta, 991: 383-387.
Go to original source...
- FUKAL L. (1995): Immunoassays - Food Analysis. In: The Encyclopedia of Analytical Science. Acad. Press Ltd., London: 2093-2101.
- GROEN A.F., VANDERVEGT R., VANBOEKEL M.A.J.S., DEROUW O.L.A.M., VOS H. (1994): Case-study on individual animal variation in milk protein composition as estimated by High-Pressure-Liquid-Chromatography. Neth. Milk Dairy J., 48: 201-212.
- GUZMAN-GONZALEZ M., MORAIS F., AMIGO L. (2000): Influence if skimmed milk concentrate replacement by dry dairy products in a low-fat set-type yoghurt model system. Use of caseinates, co-precipitate and blended dairy powders. J. Sci. Food Agric., 80: 433-438.
Go to original source...
- HILL A.R. (1989): The β-lactoglobulin-κ-casein complex. Can. Inst. Food Sci. Technol. J., 22: 120-123.
Go to original source...
- HURLEY W.L., ZOU S.X., MAGURA C.E., JIMENEZFLORES R. (1993): Heating skim milk alters the migration of immunoreactive milk proteins in acrylamide gels. J. Agr. Food Chem., 41: 280-282.
Go to original source...
- KARASOVÁ L., RAUCH P., FUKAL L. (1999): Construction of competitive enzyme immunoassay for determination of α-lactalbumin and β-lactoglobulins of cow's milk. Czech J. Food Sci., 17: 5-14.
Go to original source...
- LAW A.J.R., HORNE D.S., BANKS J.M., LEAVER J. (1994): Heat-induced changes in the whey proteins and caseins. Milchwissenschaft, 49: 125-129.
- LEARY H.L. (1992): Nonclinical testing of formulas containing hydrolyzed milk protein. J. Pediatr., 121: S42-S46, Part 2, Suppl. S.
Go to original source...
Go to PubMed...
- LEVIEUX D., VENIEN A. (1994): Rapid, sensitive, 2-site ELISA for detection of cows' milk in goats' or ewes' milk using monoclonal antibodies. J. Dairy Res., 61: 91-99.
Go to original source...
- LOPEZ-FANDINO R., CORZO N., VILLAMIEL M., DELGADO T., OLANO A., RAMOS M. (1993): Assessment of quality of commercial UHT milks by chromatographic and electrophoretic methods. J. Food Protect., 56: 263-269.
Go to original source...
Go to PubMed...
- MARSHALL R.G. (1986): Increasing cheese yield by high heat treatment of milk. J. Dairy Res., 53: 313-319.
Go to original source...
- NOH B.S., RICHARDSON T. (1989): Incorporation of radiolabeled whey proteins into casein micelles by heat processing. J. Dairy Sci., 72: 1724-1731.
Go to original source...
- PEDERSEN B. (1994): Removing bitterness from protein hydrolysates. Food Technol., 48: 96-100.
- PICARD C., PLARD I., RONGDAUXGAIDA D., COLLIN J.C. (1994): Detection of proteolysis in raw-milk stored at low-temperature by an inhibition ELISA. J. Dairy Res., 61: 395-404.
Go to original source...
- PICARD C., PLARD I., COLLIN J.C. (1996): Application of the inhibition ELISA method to the study of proteolysis caused by heat-resistant Pseudomonas proteinases specific towards κ-casein in heated milk. Milchwissenschaft, 51: 438-442.
- PLEBANI A., RESTANI P., NASELLI A., GALLI C.L., MEINI A., CAVAGNI G., UGAZIO A.G., POIESI C. (1997): Monoclonal and polyclonal antibodies against casein components of cow milk for evaluation of residual antigenic activity in "hypoallergenic" infant formulas. Clin. Exp. Allergy, 27: 949-956.
Go to original source...
Go to PubMed...
- RICHTER W., KRAUSE I., GRAF CH., SPERRER I., SCHWARZER CH., KLOSTERMEYER H. (1997): An indirect competitive ELISA for the detection of cows' milk and caseinate in goats' and ewes' milk and cheese using polyclonal antibodies against bovine γ-caseins. Z. Lebensm. Unters. Forsch., 204: 21-26.
Go to original source...
- RODRÍGEZ E., MARTÍN R., GARCÍA T., GONZÁLEZ I., MORALES P., SANZ B., HERNÁNDEZ P.E. (1993): Detection of cows' milk in ewes' milk and cheese by a sandwich Enzyme-LinkedImmunosorbent-Assay (ELISA). J. Sci. Food Agric., 61: 175- 180.
Go to original source...
- ROLLAND M.P., BITRI L., BESANCON P. (1993): Polyclonal antibodies with predetermined specifity against bovine αS1-casein: application to the detection of bovine milk in ovine milk and cheese. J. Dairy Res., 60: 413-420.
Go to original source...
Go to PubMed...
- SIEMENSMA A.D., WEIJER W.J., BAK H.J. (1993): The importance of peptide lengths in hypoalergenic infant formulas. Trends Food Sci. Technol., 4: 16-21.
Go to original source...
- SPUERGIN P., WALTER M., SCHILTZ E., DEICHMANN K., FORSTER J., MUELLER H. (1997): Allergenicity of alpha-caseins from cow, sheep and goat. Allergy, 52: 293-298.
Go to original source...
Go to PubMed...
- VISSER S. (1993): Proteolytic enzymes and their relation to cheese ripening and flavour - an overview. J. Dairy Sci., 76: 329-350.
Go to original source...
This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY NC 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.