| Most of the grape-growing areas in China affected by the monsoon climate were hot and rainy in the grape ripening season,making it difficult for grapes to be entirely ripened,and making fungal diseases serious in vineyards.The lack of maturity of grape materials resulted in the wine produced under the traditional winemaking technology lacking the typical flower and fruit fragrance.Fungal diseases in vineyards led to an growing number of harmful microorganisms on the surface of berries,increasing risk of microbial disease in wine.Based on the above discussion,this thesis focused on mixed fermentation using the killer Saccharomyces cerevisiae and the selected Pichia fermentans.The fermentation trials were carried out to explore the yeast growth,the change of glycosidase activity,and the generating law of the varietal aroma component during fermentation.And the correlation model between the activity of glycosidase and yeast growth as well as the varietal aroma components were established to reveal the effect of glycosidase on aroma enhancement.The fermentation system of‘Ecolly’and‘Riesling’grapes was used to evaluate the effect of mixed fermentation on aroma enhancement,the main results were as follows:(1)The changes laws of yeast growth during mixed fermentation.Yeast biomasss and colony count in killer group and non-killer group treatment were monitored with microscope and WLN plate to build the growth kinetics model and observe the dynamic changes of different yeast strain.The results indicated that the yeast growth rules of the killler group treatment were similar to those of the non-killer group in synthetic must fermentation.The maximum biomass in mixed fermentation was 1.5×10~8 cells/mL,slightly lower than that of pure fermentation by S.cerevisiae(2.0×10~8 cells/mL).The P.fermentans of the co-inoculation treatment only existed in the three days before during fermentation,whereas that of sequential inoculation existed in the whole fermentation process.The growth of P.fermentans could reduce the biomass of S.cerevisiae,which did not affect its dominant position.By comparing the growth of P.fermentans in the two groups,the killer S.cerevisiae showed almost no killer abilities on the P.fermentans strain.(2)The changes of glycosidase activity during mixed fermentation and its correlation with yeast biomass.In this study,the activities of 5 glycosidase in 3 tested strains were located and the activity of 5 glycosidase in 2 extracellular sites was determined during fermentation by using p NP method.Then the correlation between the glycosidase accumulation activity and yeast biomass during fermentation in synthetic must medium was established by Pearson correlation analysis.The results showed that the activity of glucosidase in 3 strains was mainly expressed asβ-D-glucosidase,the enzyme activity of P.fermentans existed in extracellular supernatant(the activity was about 8.99 m U/mL),then the enzyme activity of 2 strains of S.cerevisiae existed in intact cells and intracellular supernatant.The activity of glycosidase in the killer group was slightly higher than that in the non-killer group.In synthetic must fermentation,the activity of glycosidase only existed on the surface of yeast cells,while in fermentation of‘Ecolly’grape juice,β-D-glucosidase(GLU),β-D-galactosidase(GAL)andβ-D-xylosidase(XYL)all existed in the extracellular supernatant and intact cells,andα-L-rhamnosidase(RHA)andα-L-arabinase(ARA)only existed in the extracellular supernatant.The activity of glycosidase in the mixed fermentation reached the highest in exponential phase of yeast growth,the activity of GLU could reach 6 m U/mL in synthetic must fermentation whereas GLU activity was 5 m U/mL in must fermentation.In addition,co-inoculation treatment could improve the glycosidase activities,however sequential inoculation treatment could maintain the activities of GLU,GAL,ARA enzyme.And the correlation analysis revealed that the cumulative activity of each glycosidase during mixed fermentation of synthetic must was significantly positively correlated with the yeast biomass,especially the GLU activity accumulation in each treatment had the strongest correlation with yeast growth,their correlation coefficient were between 0.845～0.968.(3)The production of varietal aroma components and its correlation with glycosidase activity during mixed fermentation.The varietal aroma components were detected and quantified during mixed fermentation of‘Ecolly’must with Solid Phase Microextraction-Gas Chromatography-Mass Spectrometer to studied their generation laws.The correlation between varietal aroma components and the cumulative activity of glycosidase was established by Pearson correlation analysis.The results in the killer group were as follows:the total content of C6 compounds decreased rapidly during fermentation,while the total content of terpenes and C13 compounds increased gradually.In particular,the contents of linalool,farnesol andβ-damascenone were significantly different in different treatments,and the contents of farnesol andβ-damascenone in co-inoculation treatment were 6.0 and 1.3 times higher than that in wine fermented with S.cerevisiae,respectively.Correlation analysis showed that the production of C6 compounds was significantly negatively correlated with the cumulative activity of glycosidases,with the correlation coefficients between -0.673～-0.846,whereas the production of most terpenes and C13 compounds was significantly positively correlated with the cumulative activity of glycosidases,with the correlation coefficients between 0.495～0.908,0.666～0.894,respectively.(4)Evaluation of mixed fermentation on aroma enhancement.The aroma components and aroma characteristics of the final wine samples of‘Ecolly’and‘Riesling’were analyzed,and the effect on aroma enhancement of mixed fermentation with killer S.cerevisiae and P.fermentans was evaluated.The results showed that most varietal aroma contents were increased in the killer group treatment,and that the contents of higher alcohol,acetate,medium chain fatty acid ethyl ester and ethyl lactate were also increased.And mixed fermentation treatment of the killer group could specifically produce farnesol.In addition,the contents of ethyl butyrate and middle chain fatty acid were increased in‘Ecolly’wine sample of killer group,while the contents of isobutyric acid and phenylethanol were significantly increased in the‘Riesling’wine sample of the same group.Moreover,mixed fermentation treatment increased the content of most terpenes and C13 compounds in wine samples of‘Ecolly’and‘Riesling’,but the fermentative components of wine varied with the grape varieties.In‘Ecolly’wine samples,the contents of medium chain fatty acid,acetate and long chain fatty acid ethyl ester were increased by mixed fermentation.Besides,mixed fermentation also increased the content of higher alcohols,short chain fatty acid ethyl esters and other esters of‘Riesling’wine except the components mentioned in‘Ecolly’wine.Similarly,the wine fermented with sequential inoculation was more inclined to produce phenylethyl compounds.The content of phenylethyl alcohol in‘Ecolly’wine was twice as much as that in the control,while the content of phenylethyl acetate and phenylethyl alcohol in‘Riesling’wine was about 40 times and 1.3 times respectively as that in the control wine.The result of sensory analysis showed that the floral and fruity aromas of wine samples were significantly enhanced by the treatment of killer group.Co-inoculation treatment enhanced the citrus,tropical fruit and floral characteristics of the‘Ecolly’wine sample,and reduced the green smell of wine.In‘Riesling’wine sample,Co-inoculation mainly enhanced the fruity characteristics of wine,while sequential inoculation enhanced the floral characteristics of wine,but mixed fermentation also brought green flavor and perfume/spice characteristics to wine. |
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