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Veal rennet now plays a minor role in United States cheese production, reflecting an inadequate and continually decreasing supply. Rennet has been supplanted principally by rennet-pepsin blends which also usually contain bovine pepsin, and by fermentation-derived rennets. The description, classification, and specification of rennets and swine pepsin are included in the First Supplement to the Food Chemicals Codex, 2nd ed. A wide array of techniques for differentiating milk clotting enzymes has been published; however, none of these techniques is employed interlaboratory. All commercially marketed milk clotting enzyme preparations incorporating various rennets or rennet-pepsin blends generally exhibit satisfactory performance during the cheesemaking process. Cheese yields with various commercial milk clotting enzyme preparations are comparable. Utilization of fermentation-derived rennets in long-hold cheese is limited by their alleged implication in the protein related defects of short acid body and astringent or bitter flavor. The greater thermostability of Mucor-derived rennets combined with the relatively high carryover into the whey poses concern for the whey processing industries.

Effects on Cheddar cheese ripening and quality were compared for five milk clotting enzymes including crude and purified enzymes from Bacillus subtilis K-26, calf rennet, and 50-50 mixtures of the purified bacterial enzymes and calf rennet or swine pepsin. During cheese manufacture, acid development was relatively faster, protein and fat losses were higher, and cheese yields were lower when crude bacterial enzymes were used. Yields of cheese were comparable with purified enzyme in 50:50 combination with calf rennet. The initial soluble nitrogen contents and the maturity indices of bacterial enzyme cheese were higher than for the control cheese. Also the α s - and β -casein fractions were degraded more in bacterial enzyme cheese. Organoleptic evaluations indicated that cheese made with a 50:50 combination of purified bacterial enzyme and calf rennet was as acceptable as control cheese with calf rennet.

Four commercial sterile milk-based dietetic products were inoculated aseptically with small concentrations of a commericial milk clotting enzyme derived from Mucor miehei and incubated at 30 C for 32 wk. One of the products, Sustacal, exhibited no observable changes in body and texture during storage. Enzyme concentrations of 1 x 10(-4) chymosin units per milliliter or higher induced undesirable changes in the body of the other three products, Enfamil Ready-to-Use, Enfamil Concentrate, and Metrecal Shape. None of the products was affected visibly by enzyme concentrations of 1 x 10(-5) chymosin units per milliliter or less. In-can sterilization of Enfamil Ready-to-Use at 126.1 C for 4.5 min and Enfamil Concentrate at 126.7 C for 4.23 min completely destroyed all measurable enzyme activity at concentrations (up to 5.5 x 10(-2) chymosin units per milliliter) in these experiments. However, 1 to 3 h between formulation and sterilization allowed time for the enzyme at concentrations of 1 x 10(-2) chymosin units per milliliter or more to cause coagulation during heat sterilization.

An assay technique for quantifying milk-clotting enzymes is described. By the technique, enzyme concentrations as small as 2×10 −5 rennet units/ml can be determined accurately in milk solutions in as little as 2h. The technique can be used to assay for enzymes in milk and whey solutions without prior removal of casein. The technique should find application in detecting enzymes in a variety of systems commonly encountered in studies of milk and cheese products.

Firmness of milk gels formed with rennet, a mixture of rennet and porcine pepsin (Chymoset), and proteases from Mucor miehei and Mucor pusillus were measured with an apparatus which imposes an oscillatory motion to milk during clotting. This causes the clotting milk to create an oscillating pressure against a sensing diaphragm; the extent of pressure varies directly with clot firmness. No differences in rates of firming were significant between gels formed by the various enzymes in milk at pH 6.5 and 30C and at pH 6.7 and 34C. Gels formed by Mucor proteases at the extremes of experimental conditions (pH 6.7, 30C and pH 6.5, 34C) firmed less rapidly than gels formed by the other enzymes. Adding calcium chloride to milk reduced clotting time and increased rates of firming of gels formed by all enzymes tested. Clotting times of milk treated with rennet did not vary markedly at various seasons, but rate of firming of clots formed from milk in June was much slower than clots formed at other periods of the year. The time required for initial clot formation and subsequent rate of firming were affected differently by deviations in type of enzyme, pH, and temperature of milk, calcium 2+ concentration, season of year, and interactions of these factors.

The enzyme complex from a mutant of Bacillus subtilis revealed less proteolytic activity on milk proteins when pH of the milk was lowered by addition of acid. Nonprotein N in whey was much lower when milk was acidified before addition of milk clotting enzyme. Canadian Cheddar cheese was made from milk adjusted to pH 6.63 to 5.86 with additions of Bacillus subtilis proteases as coagulants. During manufacture the firmest curd resulted from the most acid milk. Protein recovery was greatest in cheese from milk acidified to the lowest pH. Cheese yields were significantly increased by acidification of milk. The best cheese was made from milk acidified to pH 6.3 to 6.0.