Modulation Of Ester-related Enzymes And Its Correlation With Esters During Winemaking

Esters play an important role in wine fruity and floral traits,increasing concentration of esters during winemaking could enhance wine fruity traits significantly and improve wine sensory quality.Esterase and ester synthase are important factors for esters production during ferementation.In this study,selected Pichia fermentans is applied in modle wine mixed fermentation and intracellular extract of Pichia fermentans is added to modulate the activities of esterase and ester-synthase.To analysis the dynamic change of ester-related enzymes and determine the regulation change of fermentative volatiles,mathematical relationship between activities of esterase 、 ester-synthase and esters contents are established.It is expected to provide theoretical and practical guidance for regulating the formation of esters during winemaking.(1)Winemaking environment adaptability of ester-synthase from intracellular extract of Pichia fermentans.In this study,C2-C10 fatty acid-Co A and ethanol were used as substrates to determine the C2-C10 fatty acid ethyl esters contents catalyzed by ester-synthase from intracellular extract of Pichia fermentans by GC-MS.Acticities of ester-synthase enzymes are obtained according to the production of 1 μg/L corresponding esters.Activities of ester-synthase under different p H value 、 temperature 、 ethanol concentration and glucose concentration are determined.Results show that the optimum p H value is 8,ester-synthase acticity is about 20% at the optimum p H value;the optimum ethanol concentration of ester synthase is 10%-12.5%(v/v).As ethanol concentration is above 12.5%,inhibition of ester synthase activity is increasing with ethanol concentration.The optimum temperature of ester synthase is 20 ℃,and the optimum glucose concentration is 300 g/L.(2)Modulation of ester-related enzymes activities and its effect on the production of esters duing mixed fermentation.Pichia ferementans and Saccharomyce cerevisiae were simultaneously inoculated in modle wine at a ratio of 1:1 for mixed fermentation,with single fermentation of S.cerevisiae as control.Activities of esterase and ester synthase were determined every 24 hours,meanwhile the changes of ester contents are measured by SPME-GC-MS.Compared with single fermentation of S.cerevisiae,mixed fermentation can increase activities of C2,C4,C6 esterase and ester synthase,and has no significant effect on C8 and C10 esterase,but mixed fermentation could inhibit the C8 and C10 ester synthase activities during fermentation process.Accoring to Pearson analysis,activities of esterase and ester synthase have strong positive correlation with acetate(correlation coefficient r is 0.708 and 0.468 respectively)and fatty acid ethyl esters(r=0.545,0.580),which indicated that concentration of esters can increase with activities of ester-related enzymes.P.fermentans mixed fermentation with S.cerevisiae can increase contents of fatty acid ethyl esters and acetate significantly,which is consistent with Pearson analysis.(3)Modulation of ester-related enzyme activities and its effect on ester contents by the addition of intracellular extracts from P.fermentans during winemaking.The intracellular extracts of P.fermentans is added at 0,48 and 120 hour resprctively during Saccharomyces cerevisiae fermentation.Activities of esterase and ester synthase are measured during the fermentation every 24 hour and dynamic changes of ester contents are determined by SPME-GC-MS.Results show that whole ferementation have lasted 144 hours,and accumulated activities of ester-related enzymes are increasing during winemaking.The addition of intracellular extract of P.fermentans can increase C2,C4 and C6 ester syhthase activities and decrease C8 and C10 ester syhthase activities;while it will significantly promote the esterase activities.According to Pearson correlation,ester synthase and fatty acids are weakly positively(r=0.137)correlated,but esterase has strong positive correlation with fatty acids(r=0.601);ester synthase,esterase have strongly positive correlation with acetate(r=0.468,0.708)and fatty acid ethyl esters(r=0.580,0.545).Ester synthase and esterase activity can significantly promote the production of esters,while fatty acid content also affects esterase and ester synthase activities.(4)Effect on production of esters by adding fatty acids during mixed fermentation.In the P.fermentans growth system,medium-chain fatty acids(hexanoic acid,octanoic acid and decanoic acid)are added and different concentrations are set to study the yeast growth kinetics.During P.fermentans and S.cerevisiae mixed fermentation,medium-chain fatty acids are addede at 6 h,48 h and 144 h,and concentration of esters is determined by GC-MS every 24 h.Results show that hexanoic acid inhibited the growth of P.fermentans at high concentration(3000 μg/L),and octanoic acid and decanoic acid could promote growth of P.fermentans.Pearson correlation analysis shows that fatty acids have a strong positive correlation with acetate(r=0.663)and fatty acid ethyl esters(r=0.656).