| Maotai-flavor liquor is one of the typical liquor styles in China,which has the characteristics of outstanding Maotai-flavor,long full flavor,elegant and delicate,and empty cup flavor.The pit fermentation was the key step of liquor making,but the relationship between the metabolism of Maotai-flavor substance and specific fermentation microorganism is unclear.The purpose of this study is to carry out the fermented grains in the fourth round of Maotai-flavor liquor pit fermentation through the metagenomics technology,revealed the changes of microorganisms community structure in pit fermentation 0 and 30 days.Additionlly,combined with the results of physicochemical infactors and the results of flavor substances,clarified the influencing factors and the distribution of microbes in fermented grains and the expression of key enzyme genes in the metabolic pathway of different flavor substances in Maotai-flavor liquor,the main results and conclusions as follows:(1)The moisture of fermented grains was 53.25%,p H was 3.65,acidity was 5.44(g / kg),reducing sugar content was 10.81%,total sugar content was 32.37%,amino acid nitrogen content was 26.13(mg / 100ml),crude protein content was 10.87% in pit fermentation 0 day;the moisture of fermented grains changed to 54.45%(P >0.05),p H was reduced to 3.56(P < 0.01),acidity was increased to 6.91(g / kg)(P <0.01),reducing sugar content was reduced to 8.12%(P < 0.05),total sugar content decreased to 23.40%(P < 0.01),amino acid nitrogen content changed to 25.61%(P >0.05),crude protein content was increased to 6.72%(P < 0.01)in pit fermentation 30 days.It was proved that the environment of pit fermentation was moist,acid and the metabolism of nutrients changed greatly.(2)The results of metagenomis sequencing showed that there were 14 bacteriaphylum,4 fungi phylum,409 bacteria genera and 40 fungi genera,among which the dominant bacteria at genus level were Lactococcus,Enterococcus,Bacillus,Lactobacillus,Acetobacter and Mycobacterium,and the dominant fungi were Aspergillus,Moesziomyces,Schizosaccharomyces,Pichia,Bipolaris,Byssochlamys.(3)In the pit fermentation stage,the content of Enterococcus,Lactococcus,Bacillus and Mycobacterium increased from 22.02%,21.61%,11.02% and 5.34% to32.42%,33.85%,14.08% and 8.62%,respectively.The content of Lactobacillus and Acetobacter decreased from 7.95%,2.41% to 0.22% and 0%.The content of Aspergillus,Schizosaccharomyces and Pichia increased from 17.62%,9.47% and9.11% to 19.58%,22.26% and 13.78%,respectively.The content of Byssochlamys,Moesziomyces and Bipolaris decreased from 29.81%,9.21% and 3.92% to 14.64%,0% and 0%,respectively.(4)According to the results of correlation analysis between physical and chemical factors and dominant genus,it was found that p H,total acid,reducing sugar and total sugar had a high correlation with dominant genus(r>0.6),among which p H was significant negatively correlated with Enterococcus and Bacillus(r<0,P<0.05),significant positively correlated with Bipolaris(r>0,P<0.05),very significant positively correlated with Byssochlamys(r>0,P<0.01),acidity was significant positively correlated with Lactococcus and Mycobacterium(r>0,P<0.05),very significant positively correlated with Enterococcus and Bacillus(r>0,P<0.01),significant negatively correlated with Acetobacter and Moesziomyces(r<0,P<0.05),very significant negatively correlated with Lactobacillus(r<0,P<0.01).Reducing sugar and total sugar were very significant positively correlated with Lactobacillus,Acetobacter,Moesziomyces and Bipolaris(r>0,P<0.05).(5)Through the detcetion of volatile flavor substances found that the main flavor substances were alcohols and esters in pit fermentation,the higher contents were ethanol(37.30%),butanol(1.00%),propanol(0.23%),furfural(1.78%),ethyl lactate(17.41%),ethyl palmitate(6.71%),ethyl acetate(5.91%),ethyl linoleic acid(2.26%)and ethyl oleate(1.37%).The detection of free amino acids found that citruline(1.18%),glutate(1.10%),leucine(0.71%),sering(0.43%)and threonine(0.43%)were higher.(6)Based on the analysis of the metagenomics sequencing data and KEGG database,the result found that Carbohydrate metabolism(17.20%)and Amino acid metabolism(15.37%)were higher in microorganisms metabolism function,and revealed that Lactococcus was the main microbe in starch degradation pathway,alpha-amylase was the key enzyme;Enterococcus,Lactococcus,Bacillus and Pantoea were the main microbes in glucose metabolism pathway,6-phospho-beta-glucosidase,glucokinase and alpha-glucosidase were the key enzymes;Unclassified,Acetobacter,Enterococcus,Lactococcus and Bacillus were the mian microbes in glutamate metabolism pathway,glutamine synthetase and glutaminase were the key enzymes;Bacillus,Enterococcus and Lactococcus were the main microbes in citruline metabolism pathway,ornithine carbamoyltransferase,arginine deiminase and acetylornithine deacetylase were the key enzymes;Unclassified,Bacillus,Acetobacter and Enterococcus were the main microbes in leucine metabolism pathway,leucine dehydrogenase and branched-chain amino acid aminotransferase were the key enzymes;Unclassified,Bacillus,Acetobacter and Lactococcus were the main microbes in serine metabolism pathway,serine 0-acetyltransferase and glycine hydroxymethyltransferase were the key enzymes;Lactococcus and Unclassified were the main microbes in 2,3-butanediol metabolism pathway,meso-butanediol dehydrogenase was the key enzyme;Burkholderia and Methylobacterium were the main microbes in furfural metabolism pathway,5-(hydroxymethyl)furfural oxideas was the key enzyme;Acetobacter,Unclassified,Pantoea,Lactococcus and Bacillus were the main microbes in acetate metabolism pathway,acetate kinase,acetyl-Co A synthetase and cysteine synthase were the key enzymes;Lactococcus,Enterococcus and Bacillus were the main microbes in lactate metabolism pathway,L-lactate dehydrogenase and hydroxyacylglutathione hydrolase were the key enzymes;Enterococcus,Bacillus and Acetobacter were the main microbes in ethanol metabolism pathway,alcohol dehydrogenase was the key enzyme.(7)In addition,according to the results,this study established the prediction metabolism network map of flavor substances in Maotai-flavor liquor,and found thatacetyl-Co A and pyruvate were the key compounds in the flavor metabolic network,which provided guidance for the production process of Maotai-flavor liquor.Above all,this study used the metagenomics technology to revealed the changes of the dominant genus,and confirmed that alcohol and ester were main flavor substances,the prediction metabolism network of flavor could be used to analyze the flavor metabolism mechanism of Maotai-flavor liquor,and provided the scientific basis for the in-deepth study on the relationship between flavor substances and microbial metabolism. |
