| Long fermentation period is an important technological measure for Luzhou-flavor Baijiu to improve the quality of basic liquor.The longer fermentation period,the more aroma components would be produced in the fermented grains.Especially in the late stage of fermentation,with the extension of fermentation time,the aroma components of fermented grains were significantly improved.However,the microbial metabolism mechanism of fermented grains in long fermentation period was not clear,and there have been no relevant research reports.Therefore,this study focuses on the fermented grains from the long fermentation period of Luzhou-flavor Baijiu as the research object.The study simultaneously analyzed the evolutionary changes and driving forces of the microbial communities structure in the fermented grains,as well as the changes in the metabolites,to reveal the relationship between microorganisms and metabolites.The study also constructed a metagenomic and proteomic database of the fermented grains during the long fermentation period.The changes,functional distribution and microbial sources of the proteome of fermented grains were analyzed.The changes of differential proteins in key metabolic pathways and the relationship between key metabolic pathways and the formation of flavor substances were revealed.At the same time,based on the analysis of the change trend of the protein group of fermented grains,antioxidant alcohol-soluble peptides with good activity were prepared for the first time from the protein of distiller’s grains of long fermentation period by enzymatic hydrolysis,and their effects on reducing alcoholic liver injury and stability in Baijiu were investigated.It provided a new technical idea for improving the health quality of Baijiu and applying distiller’s grains to liquor.（1）This study utilized amplicon sequencing to analyze the succession of microbial communities during long fermentation period of Luzhou-flavor Baijiu.Through high-throughput sequencing of three generations of amplicons,336genera and 1139 species of bacteria and 654 genera and 3522 species of fungi were identified in fermented grains.In the first 10 days of fermentation,the bacterial diversity decreased and the dominant flora was formed.After the 68th days,the diversity increased slowly,and increased significantly during the 130th to 220th days.The fungal diversity fluctuated at a low level during the 130th to 220th days,and increased significantly during the 100th to 220th days.In the genus level,Weissella and Bacillus decreased in the first 8 days of fermentation,Acetobacter and Ralstonia increased first and then decreased,and Lactobacillus became the absolute dominant genus after the 10th days of fermentation.The relative abundance of Lactobacillus decreased from 98.68%to 76.99%,while that of Acetobacter,Comamonas,Stenotrophomonas and Bacillus increased,during the 100th to 220th days.In the genus of fungi,Kazachstania and Issatchenkia were the dominant bacteria during the 4th to62th days and the 68th to 100th days,respectively.The relative abundance of Issatchenkia decreased from 48.60%to 0.23%,while the relative abundance of Aspergillus,Cladosporium,Chaetomium,Mortierella and Aureobasidium increased during the 100th to 220th days of fermentation.At the species level,Lactobacillus jinshani was the main dominant bacteria,accounting for 99.00%during the 10th to 26th days.However,during the 100th to 220th days,Lactobacillus jinshani decreased from86.00%to 44.33%,and Lactobacillus homohiochii,Lactobacillus buchneri,Ralstonia pickettii,Acetobacter pasteurianus,Acetobacter malorum,Stenotrophomonas maltophilia increased.In the fungal species,Kazachstania humilis and Issatchenkia orientalis were the main dominant fungi during the 4th to 100th days.During the 100th to 220th days,Kazachstania humilis and Issatchenkia orientalis decreased from 48.60%and 17.33%to 0.23%and 0.08%,respectively,while Pezizaceae others,Cladosporium delicatulum,Aspergillus amstelodami and Aspergillus flavus increased.Through the analysis of driving factors,temperature,moisture,acidity and alcohol had significant effects on the succession of microbial communities in fermented grains during long fermentation period.Most fungal genera were positively correlated.Lactobacillus was negatively correlated with most bacteria and positively correlated with Saccharomyces.（2）Through Untargeted Metabolomics,a total of 1064 metabolites from fermented grains were identified,among which lipids and lipids,organic acids and their derivatives（including amino acids,peptides and their derivatives）,carbohydrates and their derivatives were the main differential metabolites.Through targeted quantitative analysis technology,17 free amino acids in fermented grains were identified,showing an upward trend with fermentation time.143 organic acids and flavor substances were identified.Most acids,esters,and alcohols showed an upward trend with the extension during long fermentation period,but aldehydes first increased and then decreased.In the first 8 days of fermentation,lactic acid rapidly increases,then slowly decreases,and ethyl lactate decreases during the 130th to 220th days.Through the correlation analysis between metabolites and microorganisms,Lactobacillus homohiochii,Lactobacillus buchneri,Aspergillus amstelodami,Aspergillus flavus,etc.were positively correlated with acids and esters.Chryseobacterium bovis was positively correlated with phenylacetic acid and caproic acid.Ralstonia pickettii was positively correlated with the natural strong antioxidant catechins.（3）Through metagenomic sequencing of fermented grains during long fermentation period,a total of 254767 scaffolds were obtained and 189962 genes were annotated,including 362 CAZy library genes,10777 COG library genes,and 12868KEGG library genes.Through the metagenomic database,1525 proteins were identified,including 44 plant proteins and 1460 microbial proteins.The relative abundance of microbial protein accounted for more than 90.00%.It was found that the relative abundance of plant proteins decreased significantly during the 0th to 24th days.During the 0th to 12th days,the fungal protein increased,which was the main component of the fermented grains protein,accounting for 62.29%～71.26%.After the 12th day,the fungal protein decreased rapidly,and the bacterial protein increased rapidly to the main component of the fermented grains protein,accounting for 64.64%～81.28%.Through species annotation,709 proteins were annotated to the species level,and the relative content of Lactobacillus jinshani was the highest.Through functional annotation,the main differential proteins were concentrated in carbohydrate metabolism,energy metabolism,and amino acid metabolism pathways.During the 0th to 12th days,the differential protein expression of fungal proteolytic enzymes and amino acid metabolic pathways was dominant.During the 18th to 220th days,the differential protein expression of bacterial proteolytic enzymes and amino acid metabolic pathways was dominant.The pathway relationship between amino acid metabolism and the formation of key flavor organic acids was preliminarily analyzed by differential protein function analysis.Cysteine,tyrosine,proline and isoleucine were metabolized to pyruvate and acetyl coenzyme A,which were metabolized to acetic acid,butyric acid and caproic acid.Phenylalanine was metabolized to phenylacetaldehyde,phenylethyl alcohol,phenylacetic acid+phenylethyl acetate.In addition,with the extension of fermentation time,lactic acid was converted into pyruvate to generate other acids,and aldehydes were converted into alcohols and acids.（4）Through free radical scavenging activity evaluation,the optimal enzymatic hydrolysis,extraction and purification scheme of antioxidant glycolytic peptides of Baijiu distillers’grains during long fermentation period was established.First,the distiller’s grains were wet crushed,and then amylase 0.2%+cellulase 0.1%+papain0.05%+neutral protease 0.05%were added at the same time.The reaction conditions wer as follows:the ratio of distiller’s grains to solution was 1∶7,and the enzymatic hydrolysis reaction was carried out at 50℃for 3 hours.Afterwards,the enzymatic hydrolysis of distiller’s grains solution was extracted twice with 52%vol ethanol at35℃,decolorized with 1%coconut shell carbon for 10 minutes,adsorbed with LXSM83 macroporous resin,and eluted with 60%vol ethanol.Then,purified antioxidant alcohol-soluble peptides from distiller’s grains were obtained.By combining enzymatic hydrolysis schemes,the extraction amount of alcohol-soluble peptides from distiller’s grains reached 2.12 times that of untreated distiller’s grains.Finally,a grade less than 3 k Da was obtained through ultrafiltration,which was stable in 52%vol ethanol,but the peptides content in 52%vol Baijiu decreased by 33.70%～35.36%,which may be related to the complexity of components in Baijiu.The acute alcoholic liver injury model in mice was evaluated.It was found that the antioxidant alcohol-soluble peptide less than 3 k Da fraction at a dose of 300 mg/kg/body weight could effectively alleviate alcohol-induced oxidative stress,steatosis and lipid peroxidation,protect liver cells,and maintain liver antioxidant enzyme activity and alcohol metabolism function.The effect was close to that of the positive drug group（silymarin 50 mg/kg/body weight）.（5）Gel size exclusion chromatography was used to chromatography of antioxidant alcohol-soluble peptides less than 3 k Da.The fraction F3 with higher antioxidant activity and peptide content was obtained.Among the 7824 peptides sequences identified by peptidomics technology.Three unreported peptides with high antioxidant activity were screened by prediction tools,molecular docking and free radical scavenging.Among them,LLPFYPQG derived from Acidobacteria bacterium,LMFPYPQ derived from Paenibacillus lautus,while AAHVLAAAFL was unknown peptides,with scavenging IC50 values of 0.81 mg/m L,0.69 mg/m L and 0.64 mg/m L for DPPH free radicals,0.31 mg/m L,0.58 mg/m L and 0.48 mg/m L for ABTS free radicals,respectively.They contained more hydrophobic amino acids.They stably bound to Keap1 and MPO through hydrogen bonds and hydrophobic interactions,enhancing antioxidant activity and inhibiting ROS production. |
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