Dairy Enzymology
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Blackwell Publishing, Ltd. SYMPOSIUM CONTRIBUTION Dairy enzymology LESZEK STEPANIAK Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, Ås, Norway The properties and the significance of the principal indigenous enzymes in milk, milk coagulants, enzymes from dairy microorganisms participating in cheese ripening, and spoilage enzymes are discussed. In particular, the properties of plasmin, lipases, phosphatases, enzymes from somatic cells, enzymes involved in antimicrobial and antiviral systems in milk, enzymes from lactic acid bacteria, propionibacteria, and microorganisms involved in smear- and mould-ripened cheese are reviewed. Some assay methods and the impact of some processing factors on selected enzymes are also discussed. Keywords Dairy microorganisms, Enzymes, Milk. *Author for correspondence: E-mail: [email protected] in wound healing (Parry et al. 2000). Milk contains INTRODUCTION the full plasmin system: plasmin, plasminogen, The aim of this review is to highlight current trends plasmin inhibitors, plasminogen activators of in dairy enzymology and only selected enzymes urokinase (uPA) and tissue (tPA) types, as well as are discussed. The use of recent reviews and papers inhibitors of plasminogen activators. The concen- does not give sufficient recognition to the earlier tration of plasmin in fresh bovine milk is in the works of Professor Fox, in whose honour the range 0.1–0.7 mg/L. The ratio of plasminogen to Symposium was organized, and the works of many plasmin in milk has been reported to range from other researchers. 50 : 1 to 2 : 1. The concentration of plasmin in Basic knowledge of enzymes, their structure blood is c. 200 mg/L. As revealed by comparison and functions in relation to food enzymology, is of the amino acid sequence, immunological and presented by Whitaker et al. (2003). Milk contains biochemical properties, the enzyme long known more than 60 different indigenous enzymes (Fox as alkaline milk proteinase is identical to plasmin 2003). The principal exogenous ‘dairy’ enzymes from bovine blood plasma. Bovine milk apparently are proteinases and lipases of microbial and animal contains both 80- and 85-kDa plasminogens. origin. Among the most significant features of Activation of bovine plasminogen by uPA or indigenous milk enzymes are those used as indices tPA involves cleavage of the Arg557–Ile558 bond. of animal health, the thermal history of milk, dete- Autolytic cleavage at the Lys77–Arg78 bond releases rioration of product quality, and the ability to create the PAP fragment. Human plasmin is activated desirable changes in dairy products and protective by cleavage of the Arg561–Val562 bond (Fox effects (Fox 2003). A comprehensive review by 1989; Benfeldt et al. 1995; Grappin and Beuvier Fox and Wallace (1997) gives an insight into the 1997; Parry et al. 2000; Nielsen 2002; Kelly and complexity and significance of enzymatic reactions McSweeney 2003). In the bovine system, autolytic involved in cheese ripening by indigenous and cleavage of Arg342–Met343 also occurs, resulting exogenous enzymes. in formation of a truncated form of plasmin, called midi-plasmin (see Benfeldt et al. 1995; Kelly and McSweeney 2003 for figures illustrating plasmino- PLASMIN SYSTEM IN MILK gen structures and its activation). Plasminogen is the zymogen of the serine-type Most bacterial species do not possess plasmin proteinase, plasmin. Plasminogen and plasmin activators. However, some invasive human patho- are glycoproteins. Plasminogen consists of an N- gens have evolved nonenzymatic protein activators terminal preactivation peptide (PAP) followed by (e.g. staphylokinase and streptokinase) of human five ‘kringle’ structures and the serine proteinase blood plasminogen. Streptokinase activates plas- *Author for domain. It also contains the only phosphorylated minogen by binding to it, forming a 1 : 1 complexes. correspondence: E-mail: site. Plasmin activation is a key event in the fibri- After binding to plasmin, staphylokinase changes [email protected] nolytic system, which results in the dissolution its specificity (Parry et al. 2000). h.no of blood clots. Plasmin also has a number of other Native plasminogen and plasmin possess a © 2004 Society of physiologically important functions such as activa- specific affinity for lysine residues. This affinity has Dairy Technology tion of some metalloproteinases and participation been ascribed to ‘kringle’ structures. Chromatography Vol 57, No 2/3 May/August 2004 International Journal of Dairy Technology 153 Vol 57, No 2/3 May/August 2004 on lysine–Sepharose was used to isolate plasmino- (Rollema et al. 1983), or a fluorogenic substrate, gen from milk (Benfeldt et al. 1995). such as N-succinyl-l-alanyl-l-phenylalanyl-l-lysyl- In milk, plasmin, plasminogen and plasmino- 7-amido-4-methyl coumarin (Saint-Denis et al. gen activators are associated with the casein 2001), is used to assay plasmin activity. Plasminogen (CN) micelles and are incorporated into rennet- content is measured after addition of urokinase. coagulated cheese. Plasmin inhibitors and inhibi- The sensitivity and accuracy of assay methods tors of plasminogen activators are in the serum and was improved by addition of milk clarifying are lost in whey (Fox and Stepaniak 1993; Grappin agents (Saint-Denis et al. 2001). Fourier-transform and Beuvier 1997). Proteinases from pseudomon- infrared spectroscopy in combination with multi- ads may disrupt casein micelles and release the variate statistical analysis was developed to deter- plasmin system components into whey (Nielsen mine concentrations of plasminogen and plasmin. 2002). Fourier self-deconvolution of subtracted water When assayed on N-succinyl-l-alanyl-l- allowed individual quantification of plasmin and phenylalanyl-l-lysyl-7-amido-4-methyl coumarin, plasminogen added to a protein solution in the inhibition of plasmin activity by whey proteins was concentration range 0.38–1.8 mg/mL (Ozen et al. observed at pH 6.5, while at pH 5.2 in the presence 2003). of 5% NaCl whey proteins apparently increased plasmin activity (Hayes et al. 2002). PROTEINASES FROM SOMATIC CELLS Specificity and significance in dairy products β α Plasmin is most active on - and s2-CNs. It posi- Bovine blood contains several different types of tively influences cheese ripening (Farkye and Fox white blood cells, commonly called somatic cells. 1992). One of most important effects of plasmin Polymorphonuclear leucocytes (PMNs) are the is degradation of β-CN to γ-caseins and proteose predominant somatic cells that enter milk during β γ peptones. The occurrence of -CN f29–209 ( 1-CN), mastitis. Lysosomes of somatic cells contain γ γ γ -CN f106–209 ( 2-CN) and -CN f106–209 aspartyl (acid) proteinases cathepsins D and E, cys- γ ( 3-CN) in urea–polyacrylamide gel electrophoresis teine (thiol) neutral proteinase cathepsin B, L and H (PAGE) patterns is indicative of plasmin activity and neutral serine-type proteinases, cathepsin G and in cheese during ripening (Farkye and Fox 1992; elastase. Cathepsin D, cathepsin G and elastase are Fox and McSweeney 1996; Grappin and Beuvier major proteinases of PMNs. Five molecular forms 1997). Plasmin can also degrade some peptides of cathepsin D were isolated from acid whey, the released by chymosin (Lane and Fox 1999). major forms being 46- and 45-kDa procathepsin D. Activation of plasminogen to plasmin in high Cathepsin G, with a molecular mass of 24–26 kDa, temperature short time (HTST)-pasteurized milk is can occur as three isoforms; the enzyme is present greater than in control milk. Swiss and Cheddar in milk even at low somatic cell counts. PMN elastase cheeses contain 6–13 and 3–4.6 µg plasmin/g, has a molecular mass of 24–30 kDa (McSweeney respectively. Hydrolysis of β-casein to γ-caseins et al. 1995; Chitpinityol and Crabbe 1998; Considine is more rapid in Gouda and Swiss cheeses than et al. 1999, 2000, 2002a,b; Kelly and McSweeney α in Cheddar. Replacement of some whey by water 2003). The specificity of cathepsin D on s1- and would be expected to remove more plasmin inhi- β-CN was similar to that of chymosin. The enzyme bitors from Gouda cheese. Higher pH and higher hydrolysed all casein fractions and was more α β moisture content favours plasmin activity in Gouda active on s1-CN than on -CN and showed poor cheese; the higher cooking temperature used for milk-clotting activity (McSweeney et al. 1995). α Swiss cheese appears to explain the high level of Cathepsin G cleaved s1-casein at a minimum of plasmin activity in these cheeses (Farkye and Fox 16 sites and β-CN at a minimum of 21 sites, some 1992). Changes in viscosity observed in some non- of which were also cleavage sites for chymosin, dairy products supplemented with caseinate were plasmin, elastase, cathepsin B or the cell envelope- attributed to active plasmin. Residual plasmin may associated proteinase (CEP) of Lactococcus cause defects of ultrahigh temperature (UHT)- (Considine et al. 2002a). Elastase cleaved β-casein treated milk (Grappin and Beuvier 1997; Nielsen at several sites. Some of the sites were identical with 2002). Limited hydrolysis of casein in milk by those cleaved by chymosin, plasmin or the CEP of plasmin had no clear effect on the rheology of Lactococcus (Considine et al. 1999). Elastase also α rennet-coagulated gels (Considine et al. 2002b). showed broad specificity on s1-CN. The protein was cleaved at 25 identified sites (Considine et al. Measurement of plasmin and plasminogen 2000). Cathepsin D was active in quarg (Hurley activity in