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Comparison of methods for analysis of proteolysis by plasmin in milk

Published online by Cambridge University Press:  17 March 2011

Lucy M Chove ,
Alistair S Grandison  and
Michael J Lewis
Lucy M Chove
Affiliation:
Department of Food Science and Technology, Sokoine University of Agriculture, P.O. Box 3006, Morogoro, Tanzania
Alistair S Grandison*
Affiliation:
Department of Food and Nutritional Sciences, University of Reading, P.O. Box 226, Reading RG6 6AP, UK
Michael J Lewis
Affiliation:
Department of Food and Nutritional Sciences, University of Reading, P.O. Box 226, Reading RG6 6AP, UK
*
*For correspondence; e-mail: [email protected]
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Abstract

Sensitive methods that are currently used to monitor proteolysis by plasmin in milk are limited due to their high cost and lack of standardisation for quality assurance in the various dairy laboratories. In this study, four methods, trinitrobenzene sulphonic acid (TNBS), reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis and fluorescamine, were selected to assess their suitability for the detection of proteolysis in milk by plasmin. Commercial UHT milk was incubated with plasmin at 37°C for one week. Clarification was achieved by isoelectric precipitation (pH 4·6 soluble extracts) or 6% (final concentration) trichloroacetic acid (TCA). The pH 4·6 and 6% TCA soluble extracts of milk showed high correlations (R2 > 0·93) by the TNBS, fluorescamine and RP-HPLC methods, confirming increased proteolysis during storage. For gel electrophoresis, extensive proteolysis was confirmed by the disappearance of α- and β-casein bands on the seventh day, which was more evident in the highest plasmin concentration. This was accompanied by the appearance of α- and β-casein proteolysis products with higher intensities than on previous days, implying that more products had been formed as a result of casein breakdown. The fluorescamine method had a lower detection limit compared with the other methods, whereas gel electrophoresis was the best qualitative method for monitoring β-casein proteolysis products. Although HPLC was the most sensitive, the TNBS method is recommended for use in routine laboratory analysis on the basis of its accuracy, reliability and simplicity.

Keywords

plasminproteolysisTNBSRP-HPLCfluorescamineelectrophoresis
Type
Research Article
Information
Journal of Dairy Research , Volume 78 , Issue 2 , May 2011 , pp. 184 - 190
Copyright
Copyright © Proprietors of Journal of Dairy Research 2011

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References

Andrews, AT 1978 The Composition, Structure and Origin of Protease peptone Component 8F of Bovine Milk. European Journal of Biochemistry 90 67–71CrossRefGoogle ScholarPubMed
Bastian, ED & Brown, RJ 1996 Plasmin in milk and dairy products: an update. International Dairy Journal 6 435–457CrossRefGoogle Scholar
Beeby, R 1980 The use of fluorescamine at pH 6·0 to follow the action of chymosin on κ-casein and to estimate this protein in milk. New Zealand Journal of Dairy Science and Technology 15 99–108Google Scholar
Bio-Rad Labs, Mini-PROTEAN III electrophoresis cell. Instruction manual. Richmond, California, USAGoogle Scholar
Borda, D, Loey, AV, Smout, C & Hendrickx, M 2004 Mathematical models for combined high pressure and thermal plasmin inactivation kinetics in two model systems. Journal of Dairy Science 87 4042–4049CrossRefGoogle ScholarPubMed
Castel, JV, Cervere, M & Marco, R 1979 A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A re-examination of the conditions of reaction. Journal of Analytical Biochemistry 99 379–391CrossRefGoogle Scholar
Chism, GW, Huang, XL & Marshall, JA 1979 Sensitive assay for proteases in sterile milk. Journal of Dairy Science 62 1798–1800CrossRefGoogle Scholar
Crudden, A & Kelly, AL 2003 Studies of plasmin activity in whey. International Dairy Journal 13 987–993CrossRefGoogle Scholar
Datta, N & Deeth, HC 2001 Age gelation of UHT milk. Food and Bioproduct processing 79 197–210CrossRefGoogle Scholar
Datta, N & Deeth, HC 2003 Diagnosing the cause of proteolysis in UHT milk. Lebensmittel-Wissenschaft und -Technologie 36 173–182CrossRefGoogle Scholar
Fairbairn, DJ & Law, BA 1986 Proteinases of psychrotrophic bacteria: their production, properties effects and control. Journal of Dairy Research 53 139–177CrossRefGoogle ScholarPubMed
Kelly, AL & Mcsweeney, PLH 2003 Indigeneous proteinases in milk. In Advanced Dairy Chemistry Volume 1 Proteins: 3rd edition Part A (Eds Fox, P & , PLH , Mcsweeney). New York, Kluwer academic/Plenum Publishers pages: 495–544CrossRefGoogle Scholar
Kwan, KKH, Nakai, S & Skura, BJ 1983 Comparison of four methods for determining protease activity in milk. Journal of Food Science 48 1418–1421CrossRefGoogle Scholar
Law, BA, Andrews, AT & Sharpe, ME 1977 Gelation of ultra-high temperature-sterilised milk by proteinases from a strain of Pseudomonas fluorescens isolated from milk. Journal of Dairy Research 44 145–148CrossRefGoogle Scholar
Le, TX, Datta, N & Deeth, HC 2006 A sensitive HPLC method for measuring bacterial proteolysis and proteinase activity in UHT milk. Food Research International 39 823–830CrossRefGoogle Scholar
Lopez-fandino, R, Olano, R, San jose, C & Ramos, M 1993 Application of reverse phase HPLC to the study of proteolysis in UHT milk. Journal of Dairy Research 60 111–116CrossRefGoogle Scholar
Ma, Y & Barbano, D 2003 Effect of CO2 addition to raw milk as affected by the action of plasmin. Journal of Dairy Science 88 1616–163CrossRefGoogle Scholar
Mara, O, Roupie, C, Duffy, YA & Kelly, AL 1998 The Curd-forming Properties of Milk as affected by the action of plasmin. International Dairy Journal 8 807–812CrossRefGoogle Scholar
Mckellar, RC 1981 Development of off-flavour in Ultra-High Temperature and Pasteurised Milk as a function of proteolysis. Journal of Dairy Science 64 2138–2145CrossRefGoogle Scholar
Mulvihill, DM & Donovan, M 1987 Whey proteins and their thermal denaturation - A review. Irish Journal of Food Sceince and Technology 11 43–75Google Scholar
Nielsen, SS 2002 Plasmin system: characteristic, roles, and relationships. Journal of Agriculture and Food Chemistry 50 6628–6634CrossRefGoogle Scholar
Pereda, J, Ferragut, V, Buffa, M, Guamis, B & Trujillo, AJ 2008 Proteolysis of ultra-high pressure homogenised treated milk during refrigerated storage. Food Chemistry 111 696–702CrossRefGoogle Scholar
Richardson, BC 1983 The proteinases of bovine milk and the effect of pasteurisation on their activity. New Zealand Journal of Dairy Science and Technology 18 233–245Google Scholar
Schwabe, C 1973 A fluorescent assay for proteolytic enzymes. Analytical Biochemistry 53 484–490CrossRefGoogle ScholarPubMed
Snoeren, THM & Riel, JAM 1979 The properties of αs2 group casein. Zuivelzitch 71 766–768Google Scholar
Walstra, P & Jennes, R (Eds) (1984) Dairy chemisty and physics. New York, WileyGoogle Scholar