| Wickerhamomyces anomalus is one of the most important ester-producing strains in baijiu brewing.Because of its ability to secrete a variety of glycosidases and esterases to increase flavor compounds,it is widely used in wine brewing.Esters are important flavor compounds in baijiu,and their content and proportion are important factors to determine the quality of baijiu,especially ethyl acetate,ethyl butyrate and ethyl lactate.Our laboratory found that the addition of 1% lactic acid could significantly induce W.anomalus NCUF307.1 to produce higher content of esters by simulating the brewing process of special-flavor baijiu.Then ethanol,as the main metabolite in the brewing process of baijiu,is also the precursor substance to produce ester compounds.It is unclear what effect its addition may have on the ester production of W.anomalus induced by lactic acid.Therefore,in this paper,W.anomalus NCUF307.1 used in previous experiments was taken as the starting strain to study the effects of different ethanol concentrations on the growth and ester production of W.anomalus NCUF307.1 under lactic acid induction.Then,based on transcripomics,the influence mechanism of ethanol on W.anomalus NCUF307.1induced by lactic acid was analyzed,and the key genes in the metabolic pathway of ester production were identified,so as to elucidate the mechanism of the ethanol and lactic acid induction on ester-producing metabolic pathway of W.anomalus NCUF307.1.The main research results of this paper were as follows:(1)The effects of different ethanol concentrations(3%,6%,9%(v/v))on the growth,fermentation characteristics and volatile compounds production of 1% lactic acid-induced W.anomalus NCUF307.1 were studied.It was found that the addition of ethanol had an inhibitory effect on the growth of lactic acid-induced W.anomalus NCUF307.1.With the increase of ethanol concentration,the growth inhibition effect of W.anomalus NCUF307.1 was stronger.The results of scanning electron microscopy showed that with the increase of ethanol concentration,the cell surface of W.anomalus NCUF307.1 was damaged and wrinkled,and the cells of W.anomalus NCUF307.1 were even deformed,dented and broken under high ethanol concentration,which may also be the reason for the inhibition of yeast growth.At the same time,the addition of ethanol could reduce the reducing sugar availability of W.anomalus NCUF307.1,and the reducing sugar availability decreased with the increase of ethanol concentration.In addition,due to the induction effect of ethanol on esterification,the addition of ethanol could significantly change the aroma production performance of W.anomalus NCUF307.1.And the addition of low and medium concentrations(3% and 6%)of ethanol could promote the ester production,while the addition of 9% ethanol could inhibit the ester production capacity.The reason is that excessively high concentration of ethanol could cause the W.anomalus NCUF307.1 cells to become deformed and inhibited its growth,resulting in the decrease of ester production capacity.(2)Transcriptomic analysis showed that the addition of ethanol had a significant effect on lactic acid induced W.anomalus NCUF307.1,and the number of up-regulated and down-regulated differentially expressed genes increased with the increase of ethanol concentration.The addition of ethanol could initiate the self-repair mechanism of W.anomalus NCUF307.1,resulting in significantly up-regulated genes related to ribosome pathway.With the increase of ethanol concentration,more and more genes related to genetic information processing pathway were significantly up-regulated in response to ethanol,such as RNA polymerase,cell cycle-yeast,meiosis-yeast,and DNA replication.And the addition of ethanol mainly inhibited the pathways related energy metabolism of W.anomalus NCUF307.1,such as TCA cycle,oxidative phosphorylation,thus leading to insufficient energy supply of yeast and affecting its normal growth and metabolism.With the increase of ethanol concentration,the stronger the inhibition effect on these pathways,and thus the stronger the damage to yeast.Meanwhile,ethanol could also reduce the elimination of reactive oxygen species by inhibiting peroxisome in W.anomalus NCUF307.1,thus causing damage to yeast.It was also found by transcriptomics that the addition of 3% and 6% ethanol could promote the expression of genes associated with the synthesis of acetyl-co A and butyl-Co A to provide more substrates for the formation of ethyl esters,such as DLAT、PDHB、PDC、ACACA genes.In addition,different ethanol concentrations could also regulated the metabolism of ethyl esters produced by W.anomalus NCUF307.1 by regulating the expression of genes encoding alcohol acyltransferase,alcohol dehydrogenase and esterase/lipase.For example,the up-regulation of ATF1,ADH2,ADH5,TGL5 and LipA genes may be the reason for promoting the increase of ester compounds content of W.anomalus NCUF307.1 under low and medium concentration of ethanol,while the down-regulation of ATF1,ADH2,TGL3 and LipA genes under 9% ethanol may lead to the decrease of ester compounds content of W.anomalus NCUF307.1.(3)The key genes in the metabolic pathway of ester production in W.anomalus NCUF307.1 were screened from the results of transcriptomic analysis,and the effects of different ethanol concentrations(3,6,9%(v/v))on the production of ethyl acetate,ethyl butyrate and ethyl lactate of W.anomalus NCUF307.1 induced by lactic acid and the expression levels of key genes were further systematically studied by gas chromatography and real-time quantitative PCR.It was found that the addition of 3%and 6% ethanol could significantly promote the production of ethyl acetate,ethyl butyrate and ethyl lactate from W.anomalus NCUF307.1,and the content of these esters increased with the increase of ethanol concentration.However,the content of these ester compounds in the 9% ethanol group was lower than that in the control group.The key regulatory genes in the metabolism pathway of ester production in W.anomalus NCUF307.1 were EAT1,ADH2 and LipA genes.The addition of 3% and 6% ethanol could promote the up-regulation of genes related to the synthesis of acetyl-Co A and butyryl-Co A,which was conducive to the formation of acetyl-Co A and butyryl-Co A,to provide more substrates for the catalytic generation of acetyl-Co A or butyryl-Co A and ethanol to ethyl acetate and ethyl butyrate.Moreover,the addition of 3% and 6% ethanol could promote the up-regulation of the genes(ATF1 and EAT1)regulating alcohol acyltransferase of W.anomalus NCUF307.1,which may increase the activity of alcohol acyltransferase,which was conducive to the formation of ethyl acetate and ethyl butyrate.At the same time,the addition of 3% and 6% ethanol may improve the activity of alcohol dehydrogenase by promoting the up-regulation of genes(ADH1,ADH2 and ADH5)regulating alcohol dehydrogenase in W.anomalus NCUF307.1,which was conducive to catalyzing the formation of acetaldehyde and ethanol into ethyl acetate.In addition,the addition of 3% and 6% ethanol could promote the up-regulation of the genes(TGL5 and LipA)regulating esterase/lipase in W.anomalus NCUF307.1,which may lead to the improvement of the activity of esterase/lipase,which was conducive to catalyzing acetic acid,butyric acid or lactic acid and ethanol to produce ethyl acetate,ethyl butyrate and ethyl lactate.However,the addition of 9% ethanol could inhibit the expression of these genes,thus inhibiting the production of these ester compounds. |
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