关键词: 微生物蛋白酶；蛋白水解特性；水解位点；抗氧化；降血糖；金属螯合

Abstract: In this study, we firstly determined the optimal hydrolysis temperature and pH of the protease from Bacillus methanolicus LB-1 (protease LB-1), and then explored the proteolytic characteristics by continuously monitoring the changes in hydrolysis pH and oxidation-reduction potential (ORP) as well as using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Bioactivities of casein hydrolysates obtained with the protease including scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical, inhibition effects on α-amylase and α-glucosidase, and metal ion-chelating activity were determined. The results showed that the optimal hydrolysis temperature and pH of protease LB-1 were 52 ℃ and 6.0–7.0, respectively. Casein was more effectively hydrolyzed than whey protein by the protease, and the hydrolysis extent of individual caseins followed the decreasing order: κ-casein > α-casein > β-casein. The hydrolysis sites of κ-casein were mainly Lys21-Ile22 and Lys112-Asn113. The highest scavenging rates of DPPH and ABTS cation radicals by the casein hydrolysate were 75.4% and 48.45%, the highest inhibition rates of α-amylase and α-glucosidase were 80.89% and 93.12%, and the highest chelating rates of calcium and zinc ions were 63.13% and 35.31%, respectively, indicating that the casein hydrolysate had potent antioxidant capacity, hypoglycemic effect and metal ion-chelating ability. Therefore, the protease isolated from B. methanolicus LB-1 could be potentially used in functional cheese processing and the development of bioactive peptides of dairy origin.

Key words: microbial protease; proteolytic properties; hydrolysis site; antioxidant activity; antidiabetic activity; metal-chelating ability