The enzymatic coagulation of milk involves destabilisation of the casein micelles by limited proteolysis by enzymes in preparations called rennets. It is not at all unusual to find a proteinase that is able to coagulate milk; indeed, in my experience most proteinases are able to coagulate milk. However, most are most unsuitable for cheesemaking.
The characteristics of a good rennet substitute include:
- Proper specificity. The enzyme must cleave kappa-casein at or near its Phe105-Met106 bond.
- Good activity in milk. The enzyme must be active under the conditions of milk (e.g., have good activity at about pH 6.7).
- Easily denatured in whey. Whey is an increasingly important by-product of cheesemaking (indeed, it is sometimes quipped that "cheese is the by-product of whey manufacture!") It would be highly undesirable to have active coagulant in whey products as the enzyme would be concentrated during whey processing and might cause problems when the whey products are used as ingredients (e.g., in infant formulae).
But most of all, the key criterion of a good rennet substitute is:
- High milk clotting to general proteolysis ratio. In effect, this means that the enzyme must be very specific for the Phe-Met bond of kappa-casein but relatively inactive on peptide bonds elsewhere in the casein system. High proteolytic activity during rennet coagulation will lead to reduction in cheese yield as peptides produced by proteolysis would be lost in the whey rather than being incorporated into the curd. Also, excessive proteolysis by residual coagulant in the cheese can lead to bitterness and perhaps other flavour defects during ripening. In practice, this is the most important criterion for a good rennet substitute and the one least likely to be fulfilled by most proteinases.
I will discuss rennet substitutes in a later post, but briefly, the only enzymes that meet the above criteria to a lesser or greater extent are pepsins (now used rarely), some naturally occurring fungal aspartyl proteinases and, in particular, fermentation-produced chymosins (in which the gene for chymosin is cloned and expressed in a host organism).