| Soy sauce aroma type baijiu(also known as Maotai-flavor baijiu)is one of the most popular types of baijiu in China.Traditionally,it is mainly produced in Southwest China.However,in recent decades,some other regions in China,such as Tianjin,have also been able to produce high-quality soy sauce aroma type baijiu,but their microbial flora characteristics during fermentation are still unclear.Among them,lactic acid bacteria are the dominant bacteria in the fermentation process of baijiu,and lactic acid bacteria have received a lot of attention due to their probiotic properties.However,the tolerance mechanism of lactic acid bacteria cells to alcohol stress is not well understood at home and abroad.In addition,with the continuous improvement of people’s living standard,people have more and more opportunities to drink alcohol and the amount of alcohol consumed,but due to excessive and long-term alcohol consumption,it may cause alcoholic liver disease.With people’s increasing attention to health and the rapid rise of intestinal microbial research,more and more researchers have begun to focus on the role of probiotics(lactic acid bacteria)in preventing,alleviating or treating specific diseases,while little research has been reported on the use of probiotics(lactic acid bacteria)for alcohol detoxification.In this study,the microbial community structure of fermented grains in different rounds of Lutaichun soy sauce aroma type baijiu produced in Tianjin was first investigated using high-throughput sequencing technology.The results showed that lactic acid bacteria were the main bacteria in the process of baijiu fermentation.However,as the number of repeated fermentation rounds increased,the proportion of lactic acid bacteria decreased.Lactobacillus(71.29%)were the main bacteria at the genus level.While yeast was the main fungus in the fermentation process of baijiu,with the increase of rounds,the proportion of yeast first fluctuated up and then decreased,and at the genus level was dominated by the genus Issatchenkia(31.10%).The canonical correspondence analysis between physicochemical factors and microorganisms during the fermentation of baijiu showed that the Lactobacillus in bacteria,Issatchenkia,Saccharomyces,Byssochlamys,Torulaspora,Pichia in fungi were positively correlated with the alcohol content of fermented grains.The PLS analysis of volatile flavour compounds in baijiu fermentation showed that Lactobacillus of bacteria,Issatchenkia,Saccharomyces,Torulaspora,Pichia of fungi were more correlated with Ethyl Acetate,1-Butanol,3-methyl-,acetate,Pentanoic acid,ethyl ester,Octanoic acid,ethyl ester,Butanoic acid,3-hydroxy-,ethyl ester,Butanedioic acid,diethyl ester,Hexadecanoic acid,ethyl ester,Acetic acid,2-phenylethyl ester,Ethyl Oleate,Linoleic acid ethyl ester,Ethanol,1-Butanol,3-methyl-,1-Hexanol;among the bacteria,Weissella and Pediococcus and among the fungi,Saccharomycopsis,Thermomyces,Wickerhamomyces,Candida were more correlated with Pyrazine,tetramethyl-in the fermentation of baijiu;while other Bacillus,Monascus,unclassified＿o＿＿Saccharomycetales and Schizosaccharomyces played an important role in the formation of Hexanoic acid,ethyl ester,Dodecanoic acid,ethyl ester,2-Furanmethanol,2-Butanone,3-hydroxy-,Furfural,Benzaldehyde and Pyrazines during the fermentation of baijiu.Secondly,two strains of lactic acid bacteria,Lactiplantibacillus pentosus LTJ12 and Pediococcus acidilactici LTJ28,were selected from 48 strains of lactic acid bacteria isolated from fermented grains for their ability to resist alcohol stress.Alcohol stress affected the growth,cell membrane integrity,cell morphological structure,surface physicochemical properties,key enzyme activity,cell membrane fatty acids,and intracellular and extracellular metabolism of lactic acid bacteria to different degrees by comparing tolerant and less tolerant strains of the same genus,and the effects were different for tolerant and less tolerant strains of the same genus.Again,the probiotic bacteria were investigated in depth for alcohol tolerance as well as functional mechanisms of alcohol metabolism using histological techniques.By genomic analysis,the strain LTJ12 and LTJ28 have multiple genes that may be related to alcohol metabolism and probiotic-related genes,such as alcohol dehydrogenase,bile salt hydrolase,and extracellular polysaccharide biosynthesis protein.Further,after conducting the comparative genomics analysis,it was found that strain LTJ12 has more functional genes than other L.pentosus strains that are mainly related to carbohydrate transport and metabolism,transcription,replication,recombination and repair,signal transduction mechanisms,defense mechanisms and cell wall/membrane/envelope biogenesis.Similarly,strain LTJ28 has more functional genes than other P.acidilactici,mainly related to replication,recombination and repair,intracellular trafficking,secretion,and vesicular transport.These unique functional genes may enhance the ethanol tolerance and alcohol metabolism of the strain.Subsequent transcriptomic analysis of the more similar lactic acid bacteria under alcohol stress showed that for Lactiplantibacillus,the differentially significant genes of 8%alcohol-treated strain LTJ12 were mainly involved in starch and sucrose metabolism,ribosome,citrate cycle(tca cycle),pyruvate metabolism,purine metabolism,galactose metabolism compared to the alcohol-free treatment;while strain LTJ30,which is more similar to LTJ12 but less alcohol tolerant,differentially significant genes were mainly involved in ribosome,glycolysis / gluconeogenesis,pentose phosphate pathway,aminoacyl-trna biosynthesis,etc.For P.acidilactici,the differentially significant genes of8% alcohol-treated strain LTJ28 were mainly involved in phosphotransferase system(PTS)compared with the alcohol-free treatment;while the differentially significant genes of strain LTJ32 which is more similar to LTJ28 but less alcohol tolerant were mainly involved in aminoacyl-t RNA biosynthesis and ribosome.Then,by cloning key genes from probiotic bacteria into E.coli,it was found that the relative alcohol tolerance of all four recombinant strains overexpressing the alcohol dehydrogenase(gene0155),glucosamine--fructose-6-phosphate aminotransferase(gene0768,glm S),glutamine amidotransferase(gene1427)and nucleoside-diphosphate kinase(gene1774,ndk)genes were higher than that of the empty vector strain E.coli p ET28 a control,and the selected above key genes played an important role in the alcohol tolerance mechanism.Finally,acute alcoholism and chronic alcoholic liver injury mouse models were established to explore the functional mechanism of probiotics on host alcohol metabolism and liver protection.Through pharmacological experiments,the results showed that the strain LTJ12,LTJ28,LTJ30 and LTJ32 could prevent drunkenness and sober up,and had a good protective effect on alcoholic liver injury.The above strains can reduce the drunken rate and mortality of drinking mice,shorten the sobriety time(shortened from 302 min in the model group to 137-188 min),reduce the content of ethanol in serum(decreased from1.86 mg/m L in the model group to 1.34-1.50 mg/m L),reduce the activity or content of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),triglyceride(TG)and malondialdehyde(MDA)and increase the activity or content of superoxide dismutase(SOD),glutathione(GSH)and nitric oxide(NO),making the serum and liver biochemical indexes tend to be normal.And lactic acid bacteria also improved the activity of alcohol dehydrogenase(ADH)and acetaldehyde dehydrogenase(ALDH)of liver.Probiotic intervention basically reversed the changes in m RNA expression levels of genes related to ethanol/drug metabolism(CYP2E1,ADH1 and ALDH2),antioxidant markers(SOD2 and CAT),cellular inflammation,apoptosis and proliferation(SIRT1,SMYD3 and BRD4),lipid metabolism(SREBP1 and FASn).In addition,we have found that the regulatory effect of probiotics may be related to the SMYD3/BRD4 pathway,which needs further detailed research.Besides,probiotics increased the abundance of gut microbes,restored the imbalance of intestinal flora caused by alcohol consumption,and regulated the changes in intestinal short-chain fatty acid content caused by chronic alcohol.To the best of our knowledge,this study is the first to reveal the microbial succession of fermented grains in different rounds of soy sauce aroma type baijiu in Tianjin,and has the benefit of explaining the deep molecular mechanism in the process of baijiu fermentation.In addition,the obtained lactic acid bacteria strains with high alcohol tolerance and their in-depth exploration of alcohol stress response mechanisms and their functional mechanisms of alcohol metabolism provide theoretical basis for improving the industrial processing performance of probiotics and are beneficial to the development of new products such as active probiotic alcoholic beverages and new antialcoholism health products based on probiotics and intestinal microecological regulation mechanism,which have important industrial development prospects. |
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