Chinese Royal cheese, an ancient and attractive dairy product now in China, is made from milk coagulated with glutinous rice wine. In this paper, it was mainly studied on the proteolytic property toward proteins of bovine milk including caseins (CN) and whey proteins and the cleavage bond on the kappa-CN of rennet-like enzyme purified from glutinous rice wine by ion-exchange chromatography. Compared with whey protein, the rennet-like protease has substrate specificity toward CN but with different hydrolysis degrees among kappa-, alpha-, and beta-CN, and the alpha-CN was almost completely degraded, whereas kappa- and beta-CN partly showed hydrolysis in 12 h. The analysis for enzyme digestion by electrospray tandem mass spectrometry, Q-TOF2, and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry revealed that the cleavage of protease from glutinous rice wine on kappa-CN mainly happens at the Thr94-Met95 bond, which is different from the most chymosin-sensitive bond, Phe105-Met106.
The rennet of glutinous rice wine (wine rennet) is an exclusive clotting agent for Chinese Royal cheese production. Some characterizations are reported herein in an attempt to provide evidence about the use of the protease as either a rennet substitute or an accelerator in cheese making and ripening. The results showed that wine rennet was a monomeric and unglycosylated protease. The N-sequencing indicated a high degree of similarity to other fungal rennets. The cleavage sites of wine rennet on oxidized insulin B chain identified by HPLC-mass spectrometry included Gln(4)-His(5), Ala(14)-Leu(15), Leu(15)-Tyr(16), Tyr(16)-Leu(17), and Phe(24)-Phe(25) at pH 6.5, which were similar to those observed for Mucor rennet, but different from calf chymosin except for Leu(15)-Tyr(16). A comparison study of the kinetic properties of wine rennet on bovine caseins with that of rennets from calf and Mucor miehei by gel electrophoresis showed that these rennets had similar coagulation efficiency but different reaction rates. Wine rennet exhibited a higher degree of degradation than the calf and Mucor enzymes at pH 6.5 and 40 degrees C. Therefore, wine rennet would be an adjunct for calf rennet or an accelerator in cheese making.
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