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Identification of peptides in milk as a result of proteolysis at different levels of somatic cell counts using LC MALDI MS/MS detection

Published online by Cambridge University Press:  29 January 2008

Anna Wedholm ,
Hanne S Møller ,
Helena Lindmark-Månsson ,
Morten D Rasmussen ,
Anders Andrén  and
Lotte B Larsen
Anna Wedholm*
Affiliation:
Department of Food Science, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden
Hanne S Møller
Affiliation:
Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark
Helena Lindmark-Månsson
Affiliation:
Swedish Dairy Association, SE-223 70 Lund, Sweden
Morten D Rasmussen
Affiliation:
Department of Animal Health, Welfare and Nutrition, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark
Anders Andrén
Affiliation:
Department of Food Science, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden
Lotte B Larsen
Affiliation:
Department of Food Science, Danish Institute of Agricultural Sciences, Research Centre Foulum, DK-8830 Tjele, Denmark
*
*For correspondence; e-mail: Anna.Wedholm@lmv.slu.se
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Abstract

The somatic cell count (SCC) in milk is associated with increasing proteolytic degradation of caseins and it has been suggested that enzymes derived from somatic cells contribute to a lower yield and poorer quality of cheese. It is essential to increase the knowledge on naturally occurring milk proteinase activities to better understand how to improve the technological quality of milk. The aim of this work was to identify peptides actually present in milk as a result of proteolysis at different levels of SCC and to assign these peptides to potential responsible proteases where possible. Peptide fractions were prepared from acid whey by ultrafiltration at a molecular cut-off value of 10 000 Da. The peptides were separated using capillary reversed phase high performance liquid chromatography (RP-HPLC) and identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Peptides identified ranged in mass from 1023 to 2000 Da, and originated from αS1-, αS2- or β-casein. Possible responsible proteases that could be suggested when examining the peptide cleavage sites included plasmin, cathepsin B, D and leukocyte elastase. The results indicated that plasmin was primarily responsible for the observed proteolysis in milk at low cell count, whereas the cathepsins and elastase became implicated at elevated cell count. Specificity and activity of cathepsins and elastase has earlier mainly been studied in model systems, whereas less is known about their activities in milk itself. This is also the first indication of involvement of elastase in milk proteolysis through the unequivocal determination of cleavage sites.

Keywords

Caseincathepsinelastaseplasmin

Information

Type
Research Article
Information
Journal of Dairy Research , Volume 75 , Issue 1 , February 2008 , pp. 76 - 83
Copyright
Copyright © Proprietors of Journal of Dairy Research 2008

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References

Akers, RM 2002 Lactation and the mammary gland. Iowa, Iowa State PressGoogle Scholar
Azzara, CD & Dimick, PS 1985 Lipoprotein lipase activity of milk from cows with prolonged subclinical mastitis. Journal of Dairy Science 68 3171–3175CrossRefGoogle ScholarPubMed
Christensen, S & Sottrup-Jensen, L 1994 Characterization of two serpins from bovine plasma and milk. Biochemical Journal 303 383–390CrossRefGoogle ScholarPubMed
Considine, T, Healy, A, Kelly, AL & McSweeney, PLH 1999 Proteolytic specificity of elastase on bovine β-casein. Food Chemistry 66 463–470CrossRefGoogle Scholar
Considine, T, Healy, A, Kelly, AL & McSweeney, PLH 2000 Proteolytic specificity of elastase on bovine αS1-casein. Food Chemistry 69 19–26CrossRefGoogle Scholar
Considine, T, Geary, S, Kelly, AL & McSweeney, PLH 2002a Proteolytic specificity of cathepsin G on bovine αS1- and β-caseins. Food Chemistry 76 59–67CrossRefGoogle Scholar
Considine, T, Mc Sweeney, PLH & Kelly, AL 2002b The effect of lysosomal proteinases and plasmin on the rennet coagulation properties of skim milk. Milchwissenschaft 57(8) 425–428Google Scholar
Considine, T, Healy, A, Kelly, AL & McSweeney, PLH 2004 Hydrolysis of bovine caseins by cathepsin B, a cysteine proteinase indigenous to milk. International Dairy Journal 14 117–124CrossRefGoogle Scholar
Cooney, S, Tiernan, D, Joyce, P & Kelly, A L 2000 Effect of somatic cell count and polymorphonuclear leucocyte content of milk on composition and proteolysis during ripening of Swiss-type cheese. Journal of Dairy Research 67 301–307CrossRefGoogle ScholarPubMed
Creamer, LK & Olson, NF 1982 Rheological evaluation of matured cheddar cheese. Journal of Food Science 47 631–636, 646CrossRefGoogle Scholar
Fox, PF & Guiney, J 1973 Casein micelle structure: susceptibility of various casein systems to proteolysis. Journal of Dairy Research 40 229–234CrossRefGoogle ScholarPubMed
Fox, PF 1992 Proteinases. In Advanced Dairy Chemistry Vol 1: Proteins, pp. 310–320 (Eds PF, Fox). London: Elsevier Applied ScienceGoogle Scholar
Fox, PF & McSweeney, PLH 1998 Enzymology of milk and milk products. In Dairy chemistry and biochemistry, pp. 317–346 (Eds Fox, PF & McSweeney, PLH). London: Blackie Academic and ProfessionalGoogle Scholar
Grandison, AS & Ford, GD 1986 Effects of variations in somatic cell count on the rennet coagulation properties of milk and on the yield, composition and quality of cheddar cheese. Journal of Dairy Research 53 645–655CrossRefGoogle Scholar
Haddadi, K, Prin-Mathieu, C, Moussaoui, F, Faure, GC, Vangroenweghe, F, Burvenich, C & Le Roux, Y 2006 Polymorphonuclear neutrophils and Escherichia coli proteases involved in proteolysis of casein during experimental E. coli mastitis. International Dairy Journal 16 639–647CrossRefGoogle Scholar
Hurley, MJ, Larsen, LB, Kelly, AL & McSweeney, PLH 2000 The milk acid proteinase cathepsin D: a review. International Dairy Journal 10 673–681CrossRefGoogle Scholar
Kaminogawa, S & Yamauchi, K 1972 Acid protease of bovine milk. Agricultural and Biological Chemistry 36(13) 2351–2356CrossRefGoogle Scholar
Kelly, AL, O'Flaherty, F & Fox, PF 2006 Indigenous proteolytic enzymes in milk: A brief overview of the present state of knowledge. International Dairy Journal 16 563–572CrossRefGoogle Scholar
Kirschke, H & Barrett, AJ 1987 Chemistry of lysosomal proteinases. In Lysosomes: Their role in protein breakdown, pp. 193–238 (Eds Glaumann, F & Ballard, J). New York: Academic PressGoogle Scholar
Lane, CN & Fox, PF 1999 The individual or combined action of chymosin and plasmin on sodium caseinate or β-casein in solution: effect of NaCl and pH. Lait 79 423–434CrossRefGoogle Scholar
Larsen, LB, Boisen, A & Petersen, TE 1993 Procathepsin D cannot autoactivate to cathepsin D at acid pH. FEBS Letters 319 54–58CrossRefGoogle ScholarPubMed
Larsen, LB, Benfeldt, C, Rasmussen, LK & Petersen, TE 1996 Bovine milk procathepsin D and cathepsin D: coagulation and milk protein degradation. Journal of Dairy Research 63 119–130CrossRefGoogle ScholarPubMed
Larsen, LB, Rasmussen, MD, Bjerring, M & Nielsen, JH 2004 Proteases and protein degradation in milk from cows infected with Streptococcus uberis. International Dairy Journal 14 899–907CrossRefGoogle Scholar
Larsen, LB, McSweeney, PLH, Hayes, MG, Andersen, JB, Ingvartsen, KL & Kelly, AL 2006 Variation in activity and heterogeneity of bovine milk proteases with stage of lactation and somatic cell count. International Dairy Journal 16 1–8CrossRefGoogle Scholar
Le Bars, D & Gripon, JC 1989 Specificity of plasmin towards bovine αS2-casein. Journal of Dairy Research 56 817–821CrossRefGoogle Scholar
Le Bars, D & Gripon, JC 1993 Hydrolysis of αS1-casein by bovine plasmin. Lait 73 337–344CrossRefGoogle Scholar
Leitner, G, Shoshani, E, Krifucks, O, Chaffer, M & Saran, A 2000 Milk leucocyte population patterns in bovine udder infection of different aetiology. Journal of Veterinary Medicine B 47 581–589CrossRefGoogle ScholarPubMed
Lindmark-Månsson, H, Timgren, A, Aldén, G & Paulsson, M 2005 Two-dimentional gel electrophoresis of proteins and peptides in bovine milk. International Dairy Journal 15 111–121CrossRefGoogle Scholar
Magboul, AAA, Larsen, LB, McSweeney, PLH & Kelly, AL 2001 Cysteine protease activity in bovine milk. International Dairy Journal 11 865–872CrossRefGoogle Scholar
Marino, R, Considine, T, Sevi, A, McSweeney, PLH & Kelly, AL 2005 Contribution of proteolytic activity associated with somatic cells in milk to cheese ripening. International Dairy Journal 15 1026–1033CrossRefGoogle Scholar
McSweeney, PLH, Fox, PF & Olson, NF 1995 Proteolysis of bovine caseins by cathepsin-D; Preliminary observations and comparison with chymosin. International Dairy Journal 5 321–336CrossRefGoogle Scholar
Moussaoui, F, Laurent, F, Girardet, JM, Humbert, G, Gaillard, J-L & Le Roux, Y 2003 Characterization and proteolytic origins of specific peptides appearing during lipopolysaccharide experimental mastitis. Journal of Dairy Science 86 1163–1170CrossRefGoogle ScholarPubMed
Nabhan, MA, Girardet, J-M, Campagna, S, Gaillard, J-L & Le Roux, Y 2004 Isolation and characterisation of copolymers of β-lactoglobulin, α-lactalbumin, κ-casein and αS1-casein generated by pressurisation and thermal treatment of raw milk. Journal of Dairy Science 87 3614–3622Google Scholar
Owen, CA & Campbell, EJ 1999 The cell biology of leukocyte-mediated proteolysis. Journal of Leukocyte Biology 65 137–150CrossRefGoogle ScholarPubMed
Prin-Mathieu, C, Le Roux, Y, Faure, GC, Laurent, F, Béné, MC & Moussaoui, F 2002 Enzymatic activities of bovine peripheral blood leucocytes and milk polymorphonuclear neutrophils during intramammary inflammation caused by lipopolysaccharide. Clinical and Diagnostic Laboratory Immunology 9 812–817Google ScholarPubMed
Roepstorff, P & Fohlmann, J 1984 Proposal for a common nomenclature for sequence ions in mass-spectra of peptides. Biomed. Mass Spectrosc. 11 601. Scopes RK 1974 Measurement of protein by spectrophotometry at 205 nm. Analytical Biochemistry 59 277–282Google Scholar
Sordillo, LM, Shafer-Weaver, K & DeRosa, D 1997 Immunobiology of the mammary gland. Journal of Dairy Science 80 1851–1865CrossRefGoogle ScholarPubMed
Travis, J 1988 Structure, function and control of neutrophil proteinases. The American Journal of Medicine 84(6A) 37–42CrossRefGoogle ScholarPubMed
Verdi, RJ, Barbano, DM, Dellavalle, ME & Senyk, GF 1987 Variability in true protein, casein, nonprotein nitrogen, and proteolysis in high and low somatic-cell milks. Journal of Dairy Science 70 230–242Google Scholar
Weinstein, MJ & Doolittle, RF 1972 Differential specificities of thrombin, plasmin and trypsin with regard to synthetic and natural substrates and inhibitors. Biochimica et biophysica acta 258 577–590CrossRefGoogle ScholarPubMed