Study On The Synthesis Mechanism Of Higher Alcohol In The Fermentation Process Of Traditional Light-flavor Baijiu

Higher alcohols are not only an important flavor compounds in Baijiu,but also as a necessary precursor for the formation of ester.Higher alcohols are mainly produced by the pathway of Harris and Ehrlich in microorganisms.Alcohol dehydrogenase(L.ADHs)is the key enzyme in the last step of Ehrlich and Harris pathway,and L.ADHs can catalyze the mutual transformation between aldehyde and alcohol.Based on the above,the variety of higher alcohols,reducing sugar,free amino acids and diversity of L.adh gene was investigated in the fermentation process of traditional Light-flavor Baijiu in this study.Moreover,the correlation analysis was applied to reveal the correlation between higher alcohols and reducing sugar,free amino acids and L.adh gene.The main results were listed as follows:(1)The content of 8 higher alcohols was lowest on 0 th day and increased rapidly in 0-3 d.Propanol was not detected on 0 th day and the other 7 higher alcohols were detected.The order of the cumulative content of higher alcohols on the end of fermentation is: isoamyl alcohol > isobutanol > 2-methylbutanol > propanol > phenylethanol > n-hexanol > n-butanol >n-pentanol.The content of reducing sugar decreased in 0-3 d,stabilized in 3-12 d,increased in 12-21 d and decreased again after 21 th day.The total content of 17 free amino acids increased in 0-10 d,decreased in 10-21 d and increased again in 21-28 d.In addition,the content of Thr,Val,Ile,Leu,Phe,Glu,Ala,Lys,Pro,Asp,Ser,Gly,Cys,Met,Tyr and His increased rapidly in 0-3 d(P < 0.05),and Arg decreased in the fermentation process.(2)Degenerate primers,L.adh-F(ATAGAATACTGCGGCgtntgyca)and L.adh-R(TTTGTACGCTGTTACnccngcrca),were designed to amplify L.adh gene.High throughput sequencing analysis showed that the gene diversity of L.adh gene was the highest on 0 th day,the lowest on 18 th day.The species number of L.adh gene was highest on 15 th day and the lowest on 24 th day.Moreover,there was significant difference in L.adh gene between 0 th day and 3-28 d(P < 0.05).The sum of the top 30 relative abundances of L.adh gene is 75% and the relative abundance of JP97-1 and JP97-2 are higher than others.The relative abundance of JP97-1 was lowest on 0 th day(< 1%)and highest on 7th day(45.9%).The relative abundance of JP97-2 was higher on 0 th day(> 45%)and lowest on 3 th day(22.4%).L.adh gene could be classified to 8 Clusters by phylogenetic analysis.In cluster I,the L.adh gene of JP97-13,JP97-15 and JP97-21 are similar to fungal ADHs genes,which are Aspergillus,Kluyveromyces and Rhizobium respectively,and other are similar to bacteria ADHs genes.Cluster II is similar to Enterobacteriaceae ADHs genes,and Cluster III and Cluster IV are similar to Limosillactobacillus ADHs genes.JP97-1 and JP97-2 are similar to Limosillactobacillus and Enterobacteriaceae ADHs genes respectively.In addition,there was a significant difference in the abundance of L.adh gene of Cluster Ⅲ between 0t h day and 3-28 days and it indicating that the abundance of L.adh gene of Cluster Ⅲ changed significantly in the fermentation process.The co-occurrence analysis of L.adh gene between the same microbial genera were stronger than that between different microbial genera.(3)Reducing sugar in Jiupei was negatively correlated with higher alcohols and isobutanol,propanol,2-methylbutanol,isoamyl alcohol,n-butanol and n-amyl alcohol were significantly negatively correlated with reducing sugar in the fermentation process(P < 0.05).There was a significant positive correlation between free amino acids and some higher alcohols(P < 0.05)and phenylethanol had a significant negative correlation with Arg(P <0.05).In cluster Ⅲ,JP97-6 had significant positively correlation with n-pentanol,JP97-14 had significant positively correlation with propanol,n-butanol,isobutanol,isoamyl alcohol and 2-methyl butanol,JP97-17 had significant positively correlation with n-pentanol and phenylethanol,and JP97-23 had significant positively correlation with isobutanol(P < 0.05).Cluster Ⅳ was significant positively correlated with n-pentanol and isopentanol(P < 0.05).In cluster Ⅱ,JP97-25 was significantly negatively correlated with propanol(P < 0.05).In cluster I,JP97-5 had significant positively correlation with propanol,JP97-15 had significant positively correlation with n-pentanol and phenylethanol,and JP97-30 had significant positively correlation with 8 kinds higher alcohol(P < 0.05).