Selection And Characterization Of ?-glucosidase Producing Yeast Strains

Yeast strains play a leading role in winemaking.?-glucosidase from yeast strains which had obvious advantage in alcoholic fermentation and releasing aroma components simultaneously could break down aroma precursors of grape juice and wine that had important effect on improving the aroma of wine.In recent years,as people grow the demands for wine,wine quality had become the focus of attention.But now commercial saccharomyces cerevisiaes were used in the fermentation of wine,which lead to a consistent trend in the flavor of the wine,so it was important to select native yeasts with?-glucosidase activity for China's wine industry.In this study,the p-nitrophenyl-?-D-glucoside(pNPG)was used to select the yeast strains with(3-D-glucosidase,then the enzyme activity was analyzed quantitatively.The cellular location of ?-D-glucosidase in yeast strains was studied by breaking the cell wall and detecting the enzyme activity.To improve the capacity of producing enzyme,salicin and esculin were used to domesticate yeast strains as inductors.The yeast strains with higher enzyme activity,compared with commercial yeasts,were selected to study the enzymatic properties,including pH,temperature,metal ions,glucose and alcohol's effect on enzyme activity.Km and Vmax,which use pNPG as substrate were calculated according to Lineweaver-Burk plot.The high yield yeast strains were used to make wine and to analyze the physical and chemical indicators and the the content of aroma.The results of the study were as follows:210 strains which can produce ?-D-glucosidase were selected among the 236 native yeast strains.Eight selected strains of Saccharomyces cerevisiae,whose enzyme activity rang from 0.0075 U/ml to 0.0099 U/ml,showed that the activity of extracellular enzyme was maximum,followed by intracellular enzyme,the enzyme activity of the cell wall was minimal.Only S4 strain can be induced by salicin.However,S1 and S4 strains could be induced by esculin.The ?-glucosidase enzyme optimum pH of 3 native yeast strains and 5 commercial yeasts were 5.0.They were relatively stable at pH4?7.The optimal temperature was 500C.Temperature-stability analysis when insulated in 20? for 1 h revealed that the enzyme obtained maximum relative activity above 90%.These enzymes were inhibited by glucose and still kept higher enzyme activity at presence 12%ethanol.Metal ions had different effects on the activity of enzymes from different sources and showed different degrees of activation or inhibition.The results of Km values and Vmax values indicated that the enzyme from 3 native yeast strains had a better affinity for pNPG and showed higher catalytic rate.By the experiment of fermentation of Cabernet Sauvignon,we found that the detection results of the physical and chemical indicators were similar among native yeast strains and commercial yeast strains.The contents of nerol and geraniol of Cabernet Sauvignon dry red wine brewed by S1 and C3 commercial yeast strains were similar,but the contents of nerol and geraniol of S4 and C3 were higher than the others.However,for the aroma contents of alcohols,esters and organic acids,compared with commercial yeast strains,the level of C3 was higher or similar but S1 and S4 generated contents lower.The sensory tasting results of S1 and C3 fermented Cabernet Sauvignon wine were better.In the dry white experiments of Chardonnay,we found that the detection results of the physical and chemical indicators were similar among three native yeast strains and CY3079,but better than Angel yeast.In the aroma,the contents of geraniol which was detected only one kind of terpene aroma were not significant difference in all yeast strains.For the aroma contents of alcohols,esters and organic acids,in S1 and C3 levels were significantly lower than CY3079,but were above Angel yeast.The contents were similar between in S4 and CY3079.The sensory tasting results of S4 fermented Chardonnay wine was better.