Study On The Flavor Formation And Functional Microorganism Of Shaoxing Mechanized Huangjiu

Mechanized Huangjiu is Chinese rice wine that is produced by Mechanization production line.Multi-microorganisms participate in the saccharification and fermentation processes of mechanized Huangjiu in large fermentation tanks with temperature regulating device.The relationship between the formation of flavor and microbial metabolism was studied to reveal the main function microorganisms and metabolic pathways associated with the specific flavor substances in Huangjiu,which is of great significance in guiding the production of Huangjiu.At present,there have been many researches about the evolution of microorganisms and the dynamics of flavor components,but the relationships between the formation of specific flavor and the main function microorganisms are still unknown.The main contents and conclusions are as followed:1.A total of 94 flavor components,including 18 amino acids,8 organic acids,6 polyphenols and 62 volatile components,were analyzed by gas chromatography-mass spectrometry and high performance liquid chromatography.The determination results showed that the flavor components were mainly produced in the pre-fermentation,and the concentration of amino acids and higher alcohols,volatile phenols,esters decreased from 312 h.In the fermentation process,the organic acids were mainly lactic acid,57.57%~73.17% of the total organic acids,and the amino acids were mainly bitter amino acids,42.39%~53.29% of the total amino acids,additionally,?-aminobutyric acid(GABA)reached 202.56±10.11 mg/L in the end.In aspect of the polyphenols,the concentration of catechin was the highest during prefermentation,while the concentration of epicatechin was highest in the post-fermentation.Additionally,the esters are mainly ethyl lactate and ethyl acetate;the higher alcohols are mainly phenylethyl alcohol,isopropanol,isobutanol,3-methithipropanol,butane-2,3-diol;the volatile phenols were mainly 4-ethylenol,4-ethylprocrefol and 4-ethylphenol.2.According to the tendency of flavor changes and the control point of the fermentation process,mechanized Huangjiu samples which were fermented at 24 h,72 h,120 h and 312 h were chosen to analyze by metagenomic sequencing technology,then 105794 unigenes were obtained in gene catalogue.The taxonomy prediction results showed the dominant microorganisms were mainly Saccharopolyspora,Saccharomyces,Aspergillus,Staphylococcus,Lactobacillus,Streptomyces,Actinopolyspora,Amycolatopsis,Lactococcus,Saccharomonospora,whose relative abundance was between 81.53% and 83.72%.The functional databases annotation results showed the main metabolic pathways of mechanized Huangjiu were carbohydrate metabolism and amino acid metabolism,energy metabolism,which evidence the material basis of the flavor formation in Huangjiu at the level of genes.3.To establish the metabolic network of main flavor components,enzymes that were related to enzymatic reaction or metabolic pathways of the main flavor components in mechanized Huangjiu were collected,and encoded genes of these enzymes in gene catalogue were predicted at the genera level by taxonomy annotation.223 enzymes were sorted out,including degradation of amylase and cellulose,formation of flavor(amino acids,alcohols,acids,esters,and phenols),and 139 enzymes and 69 genera were found in sample gene catalogue.From the aspect of analysis of metabolic pathways related to flavor formation,the formation of lactic acid,acetic acid,ethanol and 2,3-butanediol were related to carbohydrate metabolism;the formation of high alcohols was related to carbohydrate metabolism and amino acid metabolism;the formation of fatty acids,the precursor of esters,was associated with lipid metabolism and amino acid metabolism;the formation of phenolic acids,such as ferulic acid,4-coumric acid,2-methoxy-4-vinylphenol,was related to amino acid metabolism and amino acid metabolism.Prediction results of function microorganism participating in flavor formation revealed that 6 genera are the most closely related to the flavor of mechanized Huangjiu,including Saccharomyces,Aspergillus,Saccharopolyspora,Staphylococcus,Lactobacillus,Lactococcus.The number of functional enzymes annotated in Aspergillus was the most,while the absolute abundance of these enzymes was low.Additionally,prediction results showed that Actinopolyspora,Saccharopolyspora,Lactobacillus,Lactococcus,Aspergillus and Saccharomyces could produce GABA.4.To further study the relationship between microorganisms and flavor formation in mechanized Huangjiu,the verifying experiment of the function microorganisms producing GABA was conducted as a representative.7 strains of Lactobacillus,6 strains of Aspergillus and 6 strains of Saccharomyces with GABA-producing ability were screened out from Huangjiu-derived microorganisms.It was found that strains from this 3 genra could produce GABA in fermentation condition simulating Huangjiu production.Additionally,the highest GABA yield was 37.46±0.97 mg/L by a Saccharomyces cerevisiae(S2).This project mainly studied the dynamics of the main flavor components in mechanized Huangjiu,then established the network relationship between specific flavor and the main function microorganism in fermentation process of Shaoxing mechanized Huangjiu by metagenomic sequencing and annotation.Lastly,the functional microorganisms producing GABA derived from process of Huangjiu were screened out for verifying metagenomic prediction results.