Characterization Of Milk Clotting Proteases From Lactobacillus Spp.

In recent years, more and more functional products during the fermentation have beenstudied. Lactobacilli are used as the starter culture for the manufacture of a variety offermented dairy products because of the hydrolysis of cell-envelope proteinases （CEPs）and peptidases, which play key roles in texture and flavor formation in production ofcheese. So far, there’s a little related research about the application of Lactobacilli CEPsin yogurt manufacture. This paper studied the milk clotting activity andcharacterizations of Lactobacilli CEPs, and the application in yogurt.In this study, three steps were used to screen strains with efficient production ofprotease among44Lactobacilli spp. First of all,18strains were selected through theratio between the clear zone and the colony on a skim milk flat, then with the proteinanalysis of content and avtivity,6strains were selected. Texture Analyzer was used inthe clotting test to show the functional properties of yogurt with6different CEPs from6strains above.The CEPs of Lactobacillus delbrueckii subsp. bulgaricus MYC canimprove hardness by34%, consistency by32.5%,cohesiveness by43.1%, and index ofviscosity by44.5%.In order to purify CEPs from MYC, gel filtration and ion exchange chromatographymethod were combined after concentration fermentation liquid by ultrafiltration. Thespecific activity rose from0.805U/mg to1.288U/mg, getting a1.6times ofpurification, enzyme recovery at the level of51.63%.This protease molecular weight isabout51kDa by SDS-PAGE. In the clotting test, the pure CEPs of MYC can improvehardness by34%, consistency by32.5%, cohesiveness by43.1%, index of viscosity by44.5%.The maximum protein activity levels were observed in the middle of the stationaryphase, was reached at pH7.0and37°C. The protein activity was stable from4℃to35℃，with the range of pH from5.0-7.0, it was completely inactivated by heating for1h at65°C. The low concentration of Ca²⁺、Fe²⁺、Zn²⁺could improve the protease activity,the high concentration of Ca²⁺、Zn²⁺would significantly inhibit the activity, the proteaseactivity were inhibited by Cu²⁺significantly.