Microbial Interaction And Regulatory Mechanism Of Organic Acids Metabolism During Fermentation Of Shanxi Aged Vinegar

Shanxi aged vinegar(SAV)has a long brewing history and unique brewing technology.In the process of open solid-state brewing,the microbial community continues to succession,completing the decomposition and transformation of substances and the synthesis of flavor substances such as organic acids.Microbial community and its metabolism are the essence of SAV brewing.However,at present,the interaction between microbial communities in this complex ecosystem and their metabolic regulatory mechanisms are still not clear,and the mechanism of traditional vinegar brewing has not been deeply understood,which limits the improvement of the scientific and technological level of the industry.In this study,meta-omics(top-down)method was used to systematically analyze the microbial community composition and dynamic change of metabolic network of SAV fermentation.Additionally,simulated solid-state fermentation(bottom-up)method was used to verify the main microbial functions,and further proposed the interaction of SAV community and the regulatory mechanism of organic acid metabolism.The microorganism in the fermentation process of SAV was detected and analyzed by metagenomics and metatranscriptomics.The results show that the main microorganism in Cupei were Lactobacillus,Acetobacter and Streptococcus at genus level,and the abundance of Lactobacillus acetotolerans,Acetobacter pasteurianus and Lactobacillus helveticus was the highest at the species level.The microbial cooccurrence network of SAV during acetic acid fermentation was constructed based on metatranscriptomics.The results show that Lactobacillus,Acetobacter and Pediococcus present the highest correlation coefficient,which are the most critical functional microorganisms to maintain the ecosystem stability in the fermentation process of SAV.Metabolic network of microbial community in Cupei shows that the species with the most abundant metabolic activities in Cupei are Lactobacillus casei,Wickerhamomyces anomalus and Lactobacillus pentosus.The most common substrates are glucose,fructose,and maltose and the most frequently occurring metabolites are lactic acid,acetic acid and ethanol.Furthermore,the influence value of interaction among species was evaluated by mathematical calculation model,and the key microbial influence network was constructed.It was found that it exists universal competition among microbiota in the ecosystem of SAV fermentation process.Lactobacillus pentosus is the core species in the metabolic interaction network of Cupei,and it is also a key species with significant metabolic influence.Lactic acid,acetic acid,ethanol,2,3-butanedione,butyric acid and propionic acid are potential regulatory substances in microbial metabolic interactions.The distribution analysis of differentially expressed genes in CAZy and KEGG databases shows that during the early-middle and middle-late fermentation of SAV,397(223 up-regulated and 174 down-regulated)and 1468(518 up-regulated and 950 downregulated)differentially expressed genes are respectively enriched in CAZy,mainly in GT family and GH family,and 1080(618 up-regulated,462 down-regulated)and 3418(1239 up-regulated,2179 down-regulated)were respectively enriched in KEGG metabolism pathway,mainly in carbohydrate metabolism,nucleotide metabolism,energy metabolism and amino acid metabolism.As the fermentation progresses,the degradation of cellulose and starch by microorganisms decreases gradually,and the metabolic activities of microorganisms at the middle-late stage of fermentation are more drastic than the changes at the early-middle stage of fermentation.By analyzing the KEGG API interface,the visualization of microbial metagenome-scale metabolic pathway in SAV fermentation process was realized.The results show that the microorganism involves in all the metabolic sub-networks of KEGG global metabolic pathway(map 01100)during the fermentation process,and the expression level of functional genes tends to be balanced during the early and middle fermentation stages.However,the expression levels of most functional genes are significantly decreased while some pathways are more active at the late fermentation stage.Most of the abundant enzymes related to carbohydrate transport system in fermentation are down-regulated at the late stage.Nitrogen metabolism and oxidative phosphorylation are the most active in energy metabolism,especially at the middle and late fermentation stage.Amino acid metabolism is more active at the late fermentation stage,especially the alanine,aspartate and glutamate metabolism(ko00250)and the glycine,serine and threonine metabolism(ko00260).Further analysis of the microorganism distribution in the pathway of differential gene enrichment shows that the microorganism involves in metabolism has significant diversity characteristics,but Lactobacillus and Acetobacter are the main ones,and the succession from Lactobacillus dominant to Acetobacter plays a role together with Lactobacillus.Organic acids are the most important flavor substances in vinegar.Therefore,the metabolic network of main organic acids(taste active value > 1)in SAV was reconstructed by extracting information related to organic acid metabolism from metaomics data.The result shows that the number of enzyme systems involved in the formation of acetic acid in SAV is the largest(11),followed by malate(6)and succinate(5),and are mainly from Acetobacter and Lactobacillus.On day 1 and day 3 of fermentation,metabolism of acetic acid and lactic acid is mainly through acetylphosphate pathway and pyruvate pathway in Lactobacillus,respectively.At later stages of fermentation,the metabolic pathways of acetic acid and lactic acid shift to acetaldehyde and L-lactaldehyde pathway,respectively,with Acetobacter as the core microorganism.The verification of the metabolic activity of organic acid-derived microorganisms shows that the metabolism of lactic acid and acetic acid of SAV is mainly regulated by environmental factors of temperature and acetic acid.The regulatory mechanism was further analyzed by simulated solid-state fermentation.The acid-resistant gene grp E of Acetobacter pasteurianus was recombined and expressed in the temperature-resistant Petiococcus acidilacticii AAF1-5,and was used to the simulated solid-state fermentation of vinegar.At the end of fermentation,the lactic acid content of P.acidilacti-grp E supplemental group was 2.25 g/100 g Cupei,which was 23.63% higher than that of the control group and 37.20% higher than that of the blank group.In addition,the ratio of lactic acid and acetic acid content in P.acidilacti-grp E supplemental group increased by 47.37% compared with P.acidilactici supplemental group and 30.23% compared with blank control group.The results confirm that the temperature tolerance and acid tolerance of the main functional microorganisms,acetic acid bacteria and lactic acid bacteria,is one of the regulation mechanisms of acetic acid and lactic acid metabolism in the fermentation process of SAV.