Primary Proteolysis

Commonly, two mechanisms dominate the primary hydrolysis of casein of semihard cheese, and that is plasmin activity on h-casein at the three sites lys28 - lys29, lys105 - his106, and lys107 -glu108 and chymosin activity on as1-casein at the site phe23 - phe24. The large casein fragments produced are not soluble at pH 4.6 (the isoelectric point of casein). They are successfully analyzed by capillary electrophoresis (CE)—the g-caseins that are equivalent to h-casein (29-209; 106-209; 108-209) and as1-I-casein that is as1-casein (24-199). Danbo

Figure 3 Typical development of pH during ripening of semihard cheese varieties. The Herrgard cheese was ripened with waxed surfaces; the Danbo cheese with a smear surface microflora producing ammonia.

20% has a relatively large content of moisture in fat-free substance (MNFS), and results of both plasmin and chymosin activity is clearly seen already after 24 hr (Fig. 4). The breakdown products of chymosin activity on as1-casein are not seen in the dryer Herr-gard cheese at this early stage of ripening, whereas plasmin is quite active already during cheese production (18). It may be a result of the higher production temperatures used for Herrgard, which partly inactivate chymosin while the activating plasminogen to plasmin is stimulated.

The main calf rennet coagulating enzyme, chymosin, may cleave h-casein at leu192 -tyr193; however, in cheese this is only possible with some special conditions fulfilled, among them a low salt content as in the interior of brine-salted cheeses during the first weeks of ripening (19). If it is produced, this peptide should be further broken down during ripening. The corresponding h-casein fragment (f1-192) has not been identified by analyzing semihard cheese for casein components using CE, and neither have its further breakdown products from plasmin activity.

B. Starter Lactococcus Protease and Peptidase Activities in Cheese

The starter bacteria of the genus Lactococcus have a well-characterized cell envelope-associated protease (CEP) that has been named PrtP and lactocepin (20,21). It hydrolyzes h-casein in milk and case in solution, and some of its genetic variants also hydrolyze as1-casein. In cheese, however, mainly activity on the chymosin- and plasmin-produced peptides have been shown (22). Especially, results of lactocepin activity on the peptides as1-casein (1-23) and h-casein (1-28; 29-107) have been shown in cheese (23,24).

The starter lactococci have a well characterized system of intracellular peptidase enzymes by which they are able to digest the peptides and release all individual amino acids of casein. This system is well known both at a biochemical and at a genetic level (21,25). Several experiments have been made over a long time showing that autolysis increases

Migration time (min)

Figure 4 Casein components in low-fat (20% FDM) Danbo cheese analyzed with capillary electrophoresis (CE) at different ages. CN, casein; g, g-casein; X, unknown.

Migration time (min)

Figure 4 Casein components in low-fat (20% FDM) Danbo cheese analyzed with capillary electrophoresis (CE) at different ages. CN, casein; g, g-casein; X, unknown.

amino acid content in cheese, but the mechanisms of these activities in cheese and the role of all the different peptidases working in cooperation are not well understood. By using strains deficient for some of the peptidases, the complexity can be studied, but the results obtained from these experiments so far are not easily interpreted. Different combinations of aminopeptidases are apparently complementary to each other.

C. Nonstarter Lactobacillus paracasei

The Lb. paracasei have a complex system of peptidases (21,25,26) and may be supplementary to the starter-mediated release of amino acids. The specificity of the peptidases vary, and some Lactobacillus strains are for instance more active on releasing proline than the starter and these may dominate in semihard cheeses with a sweet note (27). The Lactobacillus strains in cheese may also be complementary to the starter bacteria by producing special flavors using a different equipment of enzymes for amino acid catabolism.

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