| The proteinase of lactic acid bacteria(LAB) is a key enzyme in the proteolytic system as it primarily degrades casein for the rapid growth of LAB in milk. LAB proteinases are known to contribute positively to the texture and flavor formation of many fermented dairy products. In the study, LAB strains with potential technological characteristic for improving gel quality were screened out. Proteinases of the selected strains were characterized and under research for contribution to yogurts production and curd qualities. The work aimed to select exellent yoghurt start cultures through studying the characteristics of LAB proteinase, as to provide theoretical basis for the production of high quality fermented milk.Among the total 123 LAB strains, 69 strains were found to ferment yogurt with firm curd. According to the proteolysis of the 69 LABs, 12 strains were screened out. The proteolysis activity and proteinase activity of 12 selections were evaluated in this study, and it was indicated that different strains of the same species showed hydrolysis ability to different extent. All the selections showed excellence in firmness, consistency, cohesiveness, viscosity and little difference in the particle size. It was found that moderate degree of proteolysis was beneficial to the increase of gel hardness; apparent viscosity of fermented milk was reduced with the increase of proteolysis; the granularity of fermented milk was not significantly affected by protein hydrolysis. Then proteinases of the 12 selctions were determined for their influence on milk gel acidified by GDL. As texture analysis, 3 strains were selected and were identified as Lactobacillus delbrueckii subsp. bulgaricus CH9-3 4.0, SH3 4.3 and SB5 by 16 S r DNA gene sequence analysis and API identification profile.In order to purify proteinase from the selections above, purification conditions were structured and optimized. The crud proteinases of the LAB were obtained by treating the cell with lysozyme, and concentrated by ultra-filtration. The concentrated crud enzyme were loaded on a DEAE ion-exchange column, and then a Hitrap Butyl hydrophobic interaction column. Enzyme obtained from the final chromatograph operation showed single protein band by SDS-PAGE for proteinase from Lactobacillus delbrueckii subsp. bulgaricus CH9-3 4.0, SH3 4.3 and SB5 and their molecular weight were about 40 k Da. Proteinases from strains of Lactobacillus delbrueckii subsp. bulgaricus CH9-3 4.0, SH3 4.3 and SB5 were purified up to 42.8, 32.6 and 31.9 fold with specific activityof 54.4, 80.3 and 61.1 U/mg protein, and the recoveries were 46.6%, 39.3% and 40.4%, respectively.The enzymatic properties of the proteinases from Lactobacillus delbrueckii subsp. bulgaricus CH9-3 4.0, SH3 4.3 and SB5 were characterized. The result showed that optimal activities of the purified enzymes occurred at p H 6.0 and 40℃, and the purified enzymes were stable at p H 5.5~7.0 at 4~50℃. The proteinases were activated by Fe2+ and inhibited by Ba2+, Zn2+, Mn2+, Sn2+, Fe3+ and Cu2+. They were ametallopeptidase and serine proteinase, which were inhibited by EDTA and PMSF.The effects of proteinases from Lactobacillus delbrueckii subsp. bulgaricus on the quality of yoghurt gel were investigated through the addition of purified proteinases with activity of 20 U/g protein. It was found that the proteinase of LAB could significantly(P<0.05) increase the gel hardness, consistency and apparent viscosity of yogurt. The particle size of the aggregates of yoghurt gel sample were significantly(P<0.05) increased, on the other hand the negative charge of the surface of the milk protein were significantly(P<0.05) decreased, when treated by Lactobacillus delbrueckii subsp. bulgaricus proteinase. Hydrolysis by the purified proteinase significantly(P<0.05) increased free Ca2+ level, and significant(P<0.05) decreased the casein bound Ca2+ levels within milk gel networks. Proteinase treatment presented yogurt sample with small holes, uniform and closely connected structure. The effects of proteinase from Lactobacillus delbrueckii subsp. bulgaricus on the rheological properties of yogurt were investigated. It was found that the purified proteinase of Lactobacillus delbrueckii subsp. bulgaricus SH3 4.3 increased viscosity and shear stress on the sample gel, while the proteinase of Lactobacillus delbrueckii subsp. bulgaricus CH9-3 4.0 increased strength of the sample and give yogurt a higher elastic modulus(G’). |
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