Functional Analysis Of Microbial Community Of Zhenjiang Aromatic Vinegar And Construction Of A Defined Starter For Acitic Acid Production

Traditional Chinese solid-state vinegar is made in an open work environment with unsterilized raw materials and multiple microorganisms.The fermentation process of Zhenjiang aromatic vinegar(ZAV)has four stages:(i)the rice starch is decomposed into glucose by jiuqu(starch saccharification,SS);(ii)alcohol production,in which microorganisms in maiqu convert glucose into ethanol and other substances(alcohol fermentation,AF);(iii)solid-state acetic acid fermentation(AAF),which is the main stage of ZAV flavor formation;and(iv)sealed fermentation(SF)is performed to promote the accumulation of flavor substances.To initiate the AAF process and retain the uniformity among different fermentation batches,craftsmen empirically use 7-day-old vinegar culture(termed vinegar Pei)from the last batch as an autochthonous starter culture for the new batch.The structure and succession patterns of bacterial and fungal communities in the whole fermentation process of Zhenjiang aromatic vinegar were investigated by amplicon sequencing analysis to target the key functional microorganisms.We provided a method for synthesizing a defined starter to replace the autochthonous starter used in traditional solid-state cereal vinegar fermentation process,for maintaining vinegar quality.It was the theoretical and practical basis for the innovation of vinegar fermentation technology.In order to identify the primary variables impacting community assembly and functional division of microbial communities,researchers looked at the relationships between microbial communities,environmental conditions,and taste metabolites in different fermentation phases.The sources of fermentation microbiota and the contributions of starters to different fermentation stages were investigated using microbial source tracking methodology.Using metagenomic(MG),metatranscriptomic(MT),and 16 S r RNA gene sequencing,we discovered the important and dominating microbe in the autochthonous vinegar starter.This study used a bottom-up method to create a simpler microbial community that could be used to quickly initiate the AAF process while retaining vinegar quality.The main research content and major results of the research were as follows:(1)The high-throughput sequencing technology was used to investigate the effects of environmental variables and metabolome on microbiome profiles during the ZAV fermentation process.The impact of the traditional vinegar fermentation process on microbiota and flavor compounds was studied scientifically.In terms of the bacterial community,the genera Acetilactobacillus,Acetobacter,Acinetobacter,Aeromonas,Lactobacillus,Pseudomonas,Alternaria,Aspergillus,Fusarium,Rhizopus,Saccharomyces,Trichosporon,Wickerhamomyces,and Xeromyces had the most dominant OTUs with average relative abundance greater than 10% in at least one fermentation stage.The analysis of similarities(ANOSIM)revealed significant differences in the fungal and bacterial community structures during four stages of fermentation.This finding demonstrated that changes in microbiota,flavor metabolites,and environmental factors had stage characteristics,and that the relationship between microorganisms,environmental factors,and flavor metabolites requires further investigation.The high-throughput sequencing technology was used to analyze the effects of environmental variables and metabolome on the profiles of microbiome during fermentation process of ZAV.The effect of traditional vinegar fermentation process on microbiota and flavor compounds was scientifically analyzed.As for bacterial community,the dominant OTUs with average relative abundance over 10% in at least one fermentation stage were assigned to the genera Acetilactobacillus,Acetobacter,Acinetobacter,Aeromonas,Lactobacillus,Pseudomonas,Alternaria,Aspergillus,Fusarium,Rhizopus,Saccharomyces,Trichosporon,Wickerhamomyces and Xeromyces.Analysis of similarities(ANOSIM)revealed significant differences in the fungal and bacterial community structures in four fermentation stages.This result showed that the changes of microbiota,flavor metabolites and environmental factors had stage characteristics,and the relationship between microorganisms,environmental factors and flavor metabolites needs to be discussed in stages.According to the RDA results,the p H value was the key physicochemical factor influencing the microbial community of AF stage,the content of ethanol and reducing sugar were correlated with the microbiota of AAF stage,and the AAF and SF stages were significantly affected by the content of ethanol,p H value and tempereture.These environmental factors drived the succession of high abundance microbial communities in different fermentation stages of Zhenjiang aromatic vinegar,such as Ac.jinshanensis,A.pasteurianus,L.helveticus,Al.alternata and W anomalus.Based on the significant correlations between the abundances of dominant microbial OTUs and different flavor metabolites,our findings revealed that the growth of dominant abundance microorganisms such as L.helveticus,Ac.jinshanensis,As.intermedius,As.oryzae was highly positively correlated with the flavor metabolites,indicating that microbial community succession was important contributor to flavor production during the fermentation processes.Acetic acid fermentation was the main stage for the accumulation of flavor substances among the four fermentation stages,and the total contents of organic acids,amino acids,and volatile metabolites continued to enrich in the sealed fermentation stage.The findings indicate that the sealed fermentation technology has the potential to increase the content of flavor metabolites in vinegar.(2)Source Tracker was used to investigate the sources of microbes in the studied samples in order to determine the effects of starters on fermentation microbiota.Our findings indicated that jiuqu played a role in the early stages of the SS stage.For AF,rice mash provided the majority of the bacteria.The primary contribution of the fungal community in the AAF stage was raw materials.The contribution of seed pei to the bacterial community of vinegar pei increased at first and then decreased during AAF,peaking on day seven.The bacteria and fungi composition in the 7-day-old vinegar culture was the most similar to the seed pei,suggesting that it could be used as the seed pei to initiate the next AAF process.We achieved dominant L.acetotolerans,Ac.jinshanensis,and A.Pasteurianus by effectively proliferating seed pei.(3)To gain a better understanding of the functional roles of microbial taxa,seed pei was subjected to metagenomic(MG)and metatranscriptomic(MT)sequencing.The findings revealed that carbohydrate metabolic pathways and amino acid metabolic pathways were the primary players in seed pei.In the microbial community of Zhenjiang aromatic vinegar,a metabolic network for breakdown and dominant flavor formation was built.Many bacterial genera have been discovered to be involved in the degradation of starch,glucose,and cellulose,as well as the synthesis of flavor compounds such as acetic acid,lactic acid,various amino acids,and acetoin.The entire metabolic network was divided into 21 metabolic function modules based on the classification of key enzymes,such as the lactic acid production module and the acetoin production module.The researchers looked at the relationship between these key enzymes and 38 microorganisms in Acetobacter and Lactobacillus.It was discovered that key enzymes produced by the pasteurianus and L.acetotolerans contributed to the formation of a variety of flavor metabolites in seed pei.(4)Based on metagenomic(MG),metatranscriptomic(MT),and 16 S r RNA gene sequencings,11 bacterial operational taxonomic units(OTUs)with significant metabolic activity(MT/MG ratio >1)and dominance(relative abundance >1%)were targeted in seed pei to construct a simplified microbial community for AAF.The AAF process was divided into three fermentation phases: phase I(1–6 d),phase II(7–9 d),and phase III(10–18 d).Then,we evaluated the successions and interactions of these 11 bacterial OTUs at different AAF stages.Last,a defined starter was constructed with four core species isolated from the autochthonous starter(A.pasteurianus,L.acetotolerans,L.helveticus and Ac.jinshanensis).Then,we evaluated the successions and interactions of these 11 bacterial OTUs at different AAF stages.The 11 vital and dominant OTUs from autochthonous starter were distributed in clusters 1 to 4,showing four different succession patterns.OTUs of Cluster 1 and Cluster 4 were maintained in phase I,increased sharply in phase II,andfluctuated in phase III,and the vital and dominant OTUs were assigned to the species Ac.Jinshanensis and L.acetotolerans.OTUs of Cluster 2showed afluctuation in phase I,a rapid decline in phase II,and afluctuation again in phase III,and the vital and dominant ones were annotated as A.pasteurianus,L.acetotolerans,and L.helveticus.A significant downward trend throughout three phases was observed in the OTUs of cluster 3,and the vital and dominant OTUs belonged to L.helveticus.At the same time,the ecological role of these dominant species in acetic acid fermentation was revealed using cluster analysis and collinearity network analysis.Finally,a defined starter was constructed with 4 core species isolated from the seed pei(A.pasteurianus,L.acetotolerans,L.helveticus,and Ac.jinshanensis).The defined starter culture could rapidly initiate the AAF in a sterile or unsterile environment,and similar dynamics of metabolites(ethanol,titratable acidity,acetic acid,lactic acid,and volatile compounds)and environmental indexes(temperature,p H)of fermentation were observed as compared with that of autochthonous starter(P >0.05).This work provided a method to construct a defined microbiota from a complex system while preserving its metabolic function.