Properties of some animal derived milk coagulating enzymes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University

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Extracts of milk coagulating enzymes were obtained, adult bovine from the abomasums of pasture fed cattle and lamb rennet from the abomasums of partially milk fed lambs slaughtered between three and six months of age. Both rennets were, in the presence of sodium chloride, most stable at 10°C or lower at pH 4.7 for adult bovine and pH 4.1 for lamb. Heat treatment in a sodium chlorine solution (200 mg/ml) at 68°C for 60 minutes destroyed less than 35% of the activity of each rennet. Under the same conditions calf rennet was completely inactivated. The cheesemaking properties of adult bovine and lamb rennets were compared with calf rennet. Adult bovine and calf rennets responded similarly to changes of pH however lamb rennet appeared less active than calf rennet at higher pH's when measured in caseinate solution but appeared more active than calf rennet when measured in milk by a curd tension method. The optimum temperature for milk coagulating activity was 40°C for adult bovine and calf rennets but only 30°C for lamb rennet when measured in caseinate solution. The curd tension of milk coagulated with the three rennets increased with time but both adult bovine and lamb rennets appeared more sensitive to milk calcium levels than calf rennet. Whole casein, αs-, β- and k-caseins were hydrolysed in a similar manner by the three rennets. Adult bovine rennet was the most proteolytic on whole and β- caseins while calf rennet hydrolysed αs- and k-caseins more rapidly than the other rennets. Two pepsins, of similar amino acid compositions, were isolated, one from adult bovine rennet and one from lamb rennet which also contained a rennin. All three enzymes were purified so that lamb rennin and pepsin each produced only a single band on polyacrylamide gel electrophoresis but the adult bovine pepsin produced two bands and appeared to be heterogeneous. Cheese made with adult bovine, lamb or a 50/50 mixture of calf-adult bovine rennets were compared with cheese made with calf rennet and found to be similar in manufacturing characteristics, flavour and body after three and six months storage at 6 or 13°C. Polyacrylaride gel electrophoretograms of these cheese after one to twelve months storage at 6°C showed that the milk coagulant had no effect on the casein degradation products produced in the cheese although the rate of degradation varied slightly. Perchloric acid was found to be a good protein precipitant for quenching casein-rennet reactions and was utilize in a ethod which was developed for assaying milk coagulating activity. Rennet was added to sodium caseinate solution and one minute later the reaction quenched with perchloric acid. The quantity of peptides hydrolysed from the casein was measured at 217 nm and was directly proportional to rennet activity over a limited range of activity. A method for removing mucoproteins, the major impurity in rennet, was developed and a commercial scale plant commissioned. A diethylaminoethyl cellulose based resin, equilibrated with a citrate buffer, retained the rennet enzymes while the mucoproteins passed through uninhibited. The enzymes were eluted with the same buffer made 1.0 M with sodium chloride. The eluent was more active than the original rennet, was crystal clear in appearance and remained so during twelve months storage when normally mucoproteins would have precipitated out of solution to form a cloud in the rennet.
Enzymes, Rennet, Cheese manufacture, Casein