Screening Of Antifreeze Yeasts And Study Of Their Metabolites

Yeast metabolism has a decisive influence on the quality of related foods.During food processing,yeasts are often required to withstand cold stress;however,at present,there are few studies on the effects of low temperature stress on yeast metabolism.In this paper,freeze-thaw method was used to enrich antifreeze yeast.The anti-frozen yeast was isolated and purified by coating plate method and plate striation method.25 strains of yeasts with antifreeze properties were obtained.The growth and fermentation characteristics of these yeast strains were analyzed.Yeast gas production test results showed that strains J3,J7,J8,J9,J12,J15,J18,J25 had better gas production capabilities,and strain J8 had the best gas production capability;8 strains were analyzed by 5.8S rDNA sequencing and phylogenetic analysis.The yeasts were all identified as Wickerhamomyces anomalus.The strain J8 was further used to study the effect of cold stress treatment(4?)on the yeast fermentation capacity.The results showed that strain J8 significantly reduced the yeast's fermentation capacity in the short-term(0.5 h)cold stress condition;when the cold stress was 4 hours,the yeast's fermentation capacity has been restored,but when the cold stress exceeds 24 hours,the yeast's fermentation capacity continues to decrease.The SPME-GC/MS method was used to determine the effects of the volatile metabolites of eight antifreeze yeasts under cold stress(4?)conditions.A total of 43 major metabolites were detected,including 15 alcohols,7 esters,7 aldehydes,3 ketones,8 hydrocarbons,2 phenols,and 1 other substance.The results of principal component analysis showed that cold stress had the most significant effect on the volatile metabolites of strain J7,and strain J8 was the most stable to cold stress.Under the condition of cold stress at different time,T-test was performed on the peak area data of volatile metabolites of yeast.The results showed that the content of volatile metabolites increased significantly(P<0.05)during the cold stress period of 72 hours.2,4-Bis(1,1-dimethylethyl)phenol and 2-methyl-1-(1,1-dimethylethyl)-2-methyl-1,3-propanediyl propionate;Significant(P<0.05)reduction was octanal.They may be related characteristic metabolites under the cold stress of Wickerhamomyces anomalus.The Pearson correlation analysis was performed on the peak area data of volatile metabolites.The results show that octanal has a strong correlation with 4-fluorenone and 2-ethylhexanol,2,4-bis(1,1-dimethylethyl)phenol and propionate-2-methyl-1-(1,1-Dimethylethyl)-2-methyl-1,3-propanediyl ester has a strong correlation.Eight strains of antifreeze Wickhamomyces anomalus strains were derivatized by methoxamine and BSTFA-TMS,and 8 strains non-volatile metabolites of cold tolerance(4?)were tested by GC/MS.A total of 41 metabolites were detected.Among them,there are 5 kinds of amines,7 kinds of acids,5 kinds of alcohols,1 kind of hydrocarbons,18 kinds of sugars,and 5 other kinds,which reflect the complex carbohydrate metabolism ability of Wickerhamomyces anomalus.The results of principal component analysis showed that cold stress had the most obvious effect on strains J9,J12,and strain J8 had the most stable metabolic profile in cold stress.The results of principal component analysis showed that cold stress had the most significant effect on strains J9,J12,and strain J8 had the most stable metabolic profile in cold stress.Through T-test of peak area data of yeast metabolites at different times of cold stress,yeast was found to accumulate glycerol in the short-term cold stress(24h),and accumulated erythritol and squalene in cold stress for 48h;,squalene,erythritol,D-galactose were synthesized in cold stress for 72 h.Glycerol,squalene,erythritol,and D-galactose may be the cold stress characteristic metabolites of Wickerhamomyces anomalus.Pearson correlation analysis of non-volatile metabolite peak area data showed that meso-erythritol,D-galactose,and squalene have strong correlations,sucrose and N-ethylacetamide have strong correlations.This study can provide a theoretical reference for the cold stress mechanism of yeast and the formation mechanism of the taste quality of yeast fermented food.