Effect Of Environmental Factors On Microbiota Dynamics During Traditional Solid-state Fermentation Of Daqu Starter For Chinese Qishan Vinegar

Daqu starter is primary liquefying and saccharifying agents for fermentation of traditional Chinese liquor and vinegar,it contains highly complex microbial communities and enzymes.Nowadays,increasing attention is likely to be poured to the intricate relationship between the environmental parameters and the microbial dynamics in various ecosystems.From an ecological point of view,traditional spontaneous solid-state fermentation(SSF)of Daqu,is a dynamic process due to the combined activity of varieties of microbial populations,which were linked to consecutive environmental conditions.Various factors,such as temperature,moisture content,pH and acidity,related to one another determine the succession of the different environmental conditions appearing throughout the SSF process.This study combined molecular techniques to reveal microbiota dynamics during the SSF processes of Qishan Daqu starter differing in temperature regimes with consideration for potential relation to environmental variables.The physicochemical parameters and enzymatic activity of Qishan Daqu attained the common standards;Qishan Daqu contains highly complex microbial communities.The moisture content was <13%,pH value was 6.18-6.57;actidy was 1.38-2.04 mmol 10-1 g-1.The saccharifying and liquefying power were highest in outer part of Daqu,and reached 1697.91 mg glucose g-1 h-1and 0.99 g starch g-1 h-1 respectively.The saccharifying power was lowest in Hong-Xin Daqu and reached 131.65 mg glucose g-1 h-1,the liquefying power was lowest in Huai-Rang Daqu and reached 0.10 g starch g-1 h-1.The microbial diversity of outer part was more complex than the inner part of Daqu,the microbiota of Qing-Cha and Hong-Xin were more complex than that of Huai-Rang Daqu.We performed Illumina MiSeq sequencing combined with PCR-denaturing gradient gel electrophoresis to trace microbial dynamics in both fermentation stages.During the entire SSF process,the pH steadily increased and the acidity steadily declined,the starch content was >60%,and the reducing sugar content dynamic decreased.The enzymatic dynamics of glucoamylase and amylase also showed a similar trend,with the activities clearly increased in the first 7 days and reached the highest of 1812.67 mg glucoseg-1 h-1 and 1.01 g starch g-1 h-1.A succession of microbial assemblages was observed during the Daqu SSF.Lactobacillales and Saccharomycetales dominated the initial stages,with a continuous decline in relative abundance.However,thermotolerant and drought-resistant Bacillales,Eurotiales,and Mucorales were considerably accelerated during the heating stages,and these organisms dominated until the end of fermentation.Enterobacteriales were consistently ubiquitous throughout the process.The effect of bioaugmented inoculation with autochthonous of Bacillus,Pediococcus,Saccharomycopsis and Wickerhamomyces on the microbiota dynamics during traditional solidstate fermentation of Daqu was evaluted by Illumina-based high-throughput sequencing.Highly similar dynamics of physicochemical parameters,enzymatic activities and microbial communities were observed during the traditional and bioaugmented solid-state fermentation processes.No significant difference(P>0.05)in microbial structure was observed between the traditional and bioaugmented fermentation processes.However,significantly higher in acidity and obviously higher in glucoamylase and amylase activities were observed during bioaugmented fermentation process on days 2-10 d.Also,slightly higher in microbial richness was found during the bioaugmented fermentation process after 10 days fermentation.Moreover,a medium temperature SSF(MTSSF)process and a low temperature SSF(LTSSF)process of Daqu starters were performed to investigate the influence of environmental factors on microbiota dynamics by Illumina-based high-throughput sequencing.Similar evolution profiles of environmental parameters,enzymatic activities,microbial amounts,and communities were observed during the MTSSF and LTSSF processes.Nevertheless,(i)in case of MT process relatively higher temperature profile was observed than LT process at the mesophilic,thermophilic and cooling stages;(ii)significantly(P<0.05)higher in pH and lower in titratable acidity were displayed at the mesophilic and thermophilic stages of the MTSSF process compare with the LTSSF process;(iii)significantly(P<0.05)higher in protease activity and amino acid nitrogen content were displayed on day 14 during MTSSF process compare with the LTSSF process;(iv)significantly(P<0.05)higher in glucoamylase activities were noted after the mesophilic stage of the MTSSF process compare with the LTSSF process;(v)higher levels of total bacteria,LAB,Bacillus,and fungi were displayed at the thermophilic stages of the MTSSF process compare with the LTSSF process.Furthermore,the relative abundance of Enterobacteriales in MTSSF process was obviously higher comparatively to the LTSSF process,but the relative abundance of Lactobacillales and Eurotiales in MTSSF process was obviously lower comparatively to the LTSSF process.In addition,lower bacterial richness but higher fungal richness were displayed,markedly differences in bacterial communities but highly similarities in fungal communities were exhibited,during MTSSF process comparatively to the LTSSF process.Our results provide a direct evidence that persistent high temperature at cooling stage might have a deleterious effect on Eurotiales communities.Moisture and acidity showed strongly positively correlation with bacterial and fungal composition at the mesophilic stages,and positively correlated with Rickettsiales,Enterobacteriales,Cyanobacteria,Xanthomonadales,and Lactobacillales.However,core temperature(pile temperature)and pH showed positively correlation with bacterial and fungal composition at cooling and maturation stages,and positively correlated with Bacillales,Saccharomycetales,Mucorales,and Eurotiales.Therefore,it could be more appropriate to consider that a significant proportion of microorganisms growing at thermophilic stages during the SSF process were actually thermotolerant and drought-resistant community,and the microbial communities can adapt to variations in environmental conditions by changes in the thermotolerant and droughtresistant community structure during the SSF process.This study can provide progress in our knowledge of the role of microbiota in contributing to the quality and safety of final products,and will aid in upgrading the vinegar brewing technology and improving the quality and safety of vinegar products.