Characteristics And Dynamic Of Beta-glucosidase Activity Produced By Wine Related Yeasts During Fermentation

In winemaking,many conditions influence β-glucosidase activity,such as pH,temperature,sugars and ethanol.The study of β-glucosidase activity in grape juice and wine has not been extensively characterized,therefore,their effects on wine flavor are still unclear.A total of 55 wine-related yeasts were qualitatively screened for their β-glucosidase activity by YNB-C agar medium.β-Glucosidase activity of these stains were quantitatively evaluated.The results showed that Clavispora lusitaniae C117 had the highest β-glucosidase activity,152.39 μmol pNP mL-1 h-1,followed by Pichia anomala C84(87.49μmol pNP mL-1 h-1),Pichia anomala C732(80.71 μmol pNP mL-1 h-1)and Metschnikowia pulcherrima F-1-6(60.5l μmol pNP mL-1 h-1).Oxygen significantly stimulated β-glycosidase biosynthesis.C.lusitaniae C117,Hanseniaspora guilliermondii A27-3-4,M.pulcherrima F-1-6 and P.anomala C84 had higher enzyme activity in aerobic condition,displaying a significant decrease during anaerobic condition.In anaerobic condition,β-glucosidase activities of these yeasts were 1.61,0.34,0.27 and 45.9 μmol pNP mL-1 h-1,respectively,accourting for 1.06%,2.00%,0.45%and 52.46%of enzyme activity in aerobic condition.There are four strains had higher β-glucosidase activity in periplasmic space,which are H.guilliermondii A27-3-4,C.lusitaniae C117,M.pulcherrim F-1-6,and P.anomala C84.Using these four strains conducted grape juice fermentations,the results showed that P.anomala C84 had favourable β-glucosidase activity during wine fermentation,other three non-Saccharomyces stains were not significantly different with two Saccharomyces cerevisiae VL1 and RC212.The effects of pH,temperature,sugars and ethanol on β-glucosidase activity were investigated.The β-glucosidase activity at pH5 and 30℃ was 100%.The results showed that the highest β-glucosidase activity of S.cerevisiae was pH 5.0,low pH inhibited the activity ofβ-glucosidase.At pH 4.4,the β-glucosidase of P.anomala C84 had the highest activity.At a lower pH,P.anomala C84 had a higer β-glucosidase activity than S.cerevisiae,showing the relative activity of 64.98%-100.58%in the pH range of 3.4 to 4.0.At 60℃,β-glucosidase of two S.cerevisiae stains RC212 and VL1 had the highest activity for periplasmic space.At 20℃,β-glucosidase activities of RC212 and VL1 were remained 43.64%and 43.74%,respectively.The optimum temperature of β-glucosidase in periplasmic space of P.anomala C84 was 35℃.The effects of sugars on β-glucosidase activity were different.β-Glucosidase of P.anomala C84 was highly inhibited by glucose.The β-glucosidase activity of P.anomala C84 was significantly decreased in presence of 20 g L-1 glucose.In presence of 20 g L-1 and 120 g L-1 glucose,the relative activities were 33.50%and 9.54%of its relative activity,respectively.β-Glucosidase activity was slightly inhibited by fructose.Sucrose had the most slightly inhibition on β-glucosidase activity,in presence of 120 g L-1 sucrose,the relative enzyme activities S.cerevisiae stains RC212,VL1 and P.anomala C84 were 66.12%,74.36%and 67.83%,respectively.Ethanol promoted P-glucosidase activity,particularly in P.anomala C84.Ethanol concentration at 12 and 20%(v/v),the relative enzyme activity of P.anomala C84 were 128.50%and 148.35%,respectively.This effect of ethanol on β-glucosidase activity is important to winemaking.In mixed fermentations of P.anomala C84 with commercial S.cerevisiae VL1,the survival of P.anomala C84 was signaficantly shorter than S.cerevisiae.β-Glucosidase activity in mixed fermentations was not significantly different with S.cerevisiae VL1 pure fermentation.The survival of P.anomala C84 was the major factor affecting β-glucosidase activity in mixed fermentations.Analysis of wine aroma components showed that aroma components of mixed fermentation wines were more complex than pure commercial S.cerevisiae VL1 fermented wine.