Molecular Mechanism Of Reducing Higher Alcohols Content In Chinese Rice Wine Through The Addition Of Ammonium Salt

Chinese rice wine(CRW)is a kind of wine made from rice and fermented by Saccharomyces cerevisiae.It has unique flavor and high nutritional value.As by-product of CRW fermentation,higher alcohols give the wine a good taste,but excessive intake will cause dizziness and other adverse symptoms.This paper based on the previous research that nitrogen compensation(diammonium hydrogen phosphate)can reduce the content of higher alcohols in CRW.Saccharomyces cerevisiae was selected as the research object,which was cultured on two kinds of media(adding ammonium salt and non-adding ammonium salt).The fermentation characteristics were analyzed by turbidimetry,gravimetric method and counting method.And the metabolin was determined by activity detection kit.Metabolim including pyruvate,glutamate,glutamine synthase,glutamic dehydrogenase,glutamate synthase,and higher alcohols detected by chemical methods?The differential proteins of Saccharomyces cerevisiae were identified by i TRAQ.The main results are as follows:(1)The growth curve of Saccharomyces cerevisiae was determined by turbidimetry,gravimetric method and counting method.The lag phase of Saccharomyces cerevisiae was in 0-2 hours,logarithmic phase in 2-8 hours and stationary phase in 8-12 hours.The trend of growth curve measured by the three methods was basically the same,and there was a linear relationship among three methods.(2)The detection results of metabolites in the fermentation process of saccharomyces cerevisiae showed that the pyruvate,glutamate and higher alcohols in the addition group and the control group had the same change trend.The content of pyruvate in the added group was lower than that in the unadded group.The content of glutamate was lower in the group with added ammonium salt than in the group without added ammonium salt.In the logarithmic period,the content of higher alcohol in the additive group was lower than that in the non-additive group,and in general,the content decrease in the additive group was greater than that in the non-additive group.During the fermentation the results of enzyme activity test showed that the addition of ammonium salt did not change the activity trend of glutamate synthase(GOGAT)and glutamate dehydrogenase(GDH),but generally reduced the activity of the enzyme.The activity of glutamine synthase(GS)was increased and glutamine synthesis was promoted.(3)Analysis of intracellular proteins of Saccharomyces cerevisiae added ammonium salt and not added in the 8th hour of growth by i TRAQ.It was identified 211 differential proteins,123 up-regulated proteins and 88 down-regulated proteins.In the GO enrichment analysis,the order of decreasing number of functional differential proteins was as follows: Cellular component,Biological processes,and Molecular function.The number of down-regulated proteins in nitrogen utilization differential protein was more than that in up-regulated proteins in biological process.In the Pathway enrichment analysis,the number of differential proteins was ranked as: Metabolism,Genetic information processing,Cellular processes,Environmental Information processing,human diseases.It was identified that the pathways of significant enrichment of differential proteins,glucose metabolism and amino acid metabolism related to higher alcohols metabolism were 15 and 9 respectively.The protein involved in pyruvate metabolism,glycolysis and citrate cycle pathway is down-regulated of protein phosphoenolpyruvate carboxykinase,and its enzyme activity affects the metabolism of three pathways thus affecting the metabolism of higher alcohols.In KOG annotation,the order of differential protein quantity is: Cellular processes and signaling,Metabolism,Information storage and processing,lack of characteristic function.In the metabolism,the ratio of amino acid transport and carbohydrate transport and metabolism which related to the metabolism of higher alcohols was 10.9% and 8.7%.This paper studied the fermentation characteristics,metabolite,enzyme activity and proteome of Saccharomyces cerevisiae under the condition of ammonium salt addition or not,and discussed the molecular mechanism of ammonium salt compensation to reduce the content of higher alcohols.The results provide experimental data to guide the theoretical research on the production of reducing the content of higher alcohols in CRW with ammonium salt compensation.