| The unique high-temperature brewing process of Maotai-flavor liquor makes the brewing microorganisms form unique physiological metabolic characteristics and mechanism,and produce special liquor flavor.Bacillus amyloliquefaciens is a functional bacteria which can produce flavor in the brewing system of Maotai-flavor liquor,but its molecular mechanism is not clear.Studying the brewing characteristics and metabolic mechanism of Bacillus amyloliquefaciens JP-1 at different temperatures will help to analyze the brewing mechanism of Maotai-flavor liquor,and improve the theoretical system of Maotai-flavor liquor brewing,which has profound significance for the brewing of Maotai-flavor liquor.In this paper,one strain of Bacillus amyloliquefaciens JP-1,which was screened from the cellars of a Maotai-flavor winery in Guizhou,was selected as the research object.By studying the fermentation characteristics and flavor changes of Bacillus amyloliquefaciens JP-1 at different temperatures,combined with transcriptomics technology,analyze the effect of temperature on flavor metabolism of Bacillus amyloliquefaciens JP-1.The main research contents and conclusions are as follows:1.The physicochemical index and enzyme activity of Bacillus amylolyticus JP-1were determined under high temperature(55℃,45℃)and medium low temperature(37℃).The results showed that at 37℃,the biomass of Bacillus amyloliquefaciens JP-1 is 8.13×10~9cfu/ml,the p H value and moisture content of the samples were 6.14±0.00,41.56±0.737%,which were significantly higher than45℃and 55℃(p<0.05);at 45℃,the acid protease activity,amylase activity,reducing sugar and amino acid nitrogen content of the samples were 257.44±3.947U/g,26.83±0.57 U/g,1.83±0.131 g/100g,0.39±0.013 g/100g,both significantly higher than 37℃and 55℃(p<0.05);at 55℃,the content of starch,crude protein,total acid and free amino acid of the samples were 19.78±0.333%and 7.75±0.133g/100g,0.27±0.008g/g,0.136%,all significantly higher than 37℃and 45℃(p<0.05).2.The volatile flavor compounds of Bacillus amylolyticus JP-1 were analyzed by HS-SPME-GC-MS at high temperature(55℃,45℃)and medium low temperature(37℃).PCA analysis revealed that there were significant differences in the composition and content of volatile flavor compounds of the samples under different temperature conditions.The results show that high temperature conditions are beneficial to increase the relative content of alcohols and pyrazines and reduce the relative content of ketones.Transcriptome data analysis found that under high temperature stress,the down-regulated expression of genes encodingα-acetolactate decarboxylase and 2,3-butanediol dehydrogenase made the relative percentages of acetoin and 2,3-butanedione significant decreases,which in turn leads to a decrease in the content of ketones;the up-regulation of the gene encoding threonine dehydrogenase increases the ability of Bacillus amylolyticus JP-1 to produce2,5-dimethylpyrazine,which in turn led to an increase in the content of pyrazines.3.Illumina Hi Seq TM 2500 was used to sequence the transcriptome of Bacillus amyloliquefaciens JP-1 under high temperature(55℃,45℃)and low temperature(37℃)conditions,and the differential expression analysis of the sequencing results.There are 4,922 differentially expressed genes(DEGs)among the three combinations,with 207 up-regulated expressions at 45℃-VS-37℃and 236 down-regulated expressions;1,130 up-regulated expressions and 1,203 down-regulated expressions at55℃-VS-37℃;1,006 expressions were up-regulated and 1,140 expressions were down-regulated at 55℃-VS-45℃.4.Differentially expressed genes were subjected to significant enrichment analysis of GO function,and the results showed that the differential genes were mainly enriched in proteolysis and redox in biological processes after Bacillus amyloliquefaciens JP-1 was subjected to high temperature(45℃,55℃)stress.In the classification of cell components,most genes were enriched in the plasma membrane and actin cytoskeleton.Among the molecular functional components,more genes were enriched in transferase activity and catalytic activity.KEGG significant enrichment analysis of differentially expressed genes,the results showed that the high temperature of 55℃has a significant effect on the energy metabolism,carbohydrate metabolism,amino acid metabolism and other metabolic pathways of Bacillus amyloliquefaciens JP-1.5.Combined with related literatures,20 candidate genes related to Maotai-flavor were preliminarily screened from 45℃-VS-37℃,55℃-VS-37℃and 55℃-VS-45℃.These genes include asparagine synthetase gene(D2M30＿1151),α-acetolactate decarboxylase gene(D2M30＿3775),3D-(3,5/4)-trihydroxycyclohexane-1,2-dione acyl hydrolase gene(D2M30＿4154),Ketol acid reductoisomerase gene(D2M30＿2954),acetylglutamate kinase gene(D2M30＿1184),threonine synthase gene(D2M30＿3364),sucrose 6-fructose transferase gene(D2M30＿4251)etc.6.Five genes(D2M30＿1151、D2M30＿3775、D2M30＿2950、D2M30＿1184、D2M30＿4154)were selected from the candidate genes of Maotai-flavor,which were verified by fluorescence quantitative PCR.The results showed that compared with37℃,the D2M30＿1151 and D2M30＿3775 genes were down-regulated to varying degrees at 45℃and 55℃;the D2M30＿2950 gene was up-regulated at 45℃and down-regulated at 55℃;Under the high temperature of 55℃,they are all down-regulated to varying degrees.The expression trend is basically consistent with the transcriptome sequencing results,which proves that the sequencing results are reliable,and the selected genes can be studied in more depth. |
