Microbial Community Structure And Environmental Source Of Strong-Flavor Daqu Before And After Production Resumption

As the common fermentation agent in Chinese Baijiu brewing,the functional microorganisms,enzymes,flavor precursors and raw materials in strong-flavor daqu are critical to Baijiu quality.There are two types of production methods for strong-flavor daqu,including manual and mechanical process,whose differences between each other greatly impact the final quality of Baijiu.Mechanical process has been used in strong-flavor daqu production due to its ability to meet manufacture requirements.However,it is unclear how the mechanical environment affects daqu microbiata construction and cause differences in the physicochemical properties in daqu before and after production resumption(continuous production and resumed production).In this study,daqu quality evaluation method was established according to correlation analysis.SMRT sequencing was applied to study fermentation and environmental microecology of mechanically strong-flavor daqu before and after production resumption.Finally,we used Source track method to analyze the interaction between environment and daqu microbiota.Main results of the study are listed as follows:1.Quality evaluation method for strong-flavor daqu was established.Strong-flavor daqu was selected for simulated solid-state fermentation experiments.Physicochemical,microbiological and flavor indexes of daqu and Baijiu were measured respectively.By analyzing the partial least squares regression(PLSR)correlation between strong-flavor daqu and Baijiu indicators,a total of 15 indicators were selected to evaluate the quality of strongflavor daqu,including 3 flavor indicators(phenols,ketones,esters),5 physicochemical indicators(water content,amino nitrogen,amylase activity,reducing sugar content,acid protease activity)and 7 microbial indicators(Kroppenstedtia,Others＿bacterium,Pediococcus,Weissella,Pichia,Lichtheimia,Rhizomucor).Continuous-producing daqu had more indicators to meet the standards of superior and first grade,with lower water and ketone content,and higher abundance of esters,Pichia and Rhizomucor.2.Continuous-producing mechanically strong-flavor daqu and environmental microbiota was studied.Dominant genera during fermentation process were Acetobacter,Weissella,lactic acid bacteria(Latilactobacillus,Companilactobacillus,Lactiplantibacillus,Lactococcus),Leuconostoc,Staphylococcus,Thermoactinomyces,Pichia,Saccharomycopsis,Wickerhamomyces,Rhizomucor and Rhizopus.Ecological environment suitable for daqu fermentation was created during the long-term continuous production process by adding mature daqu powder during daqu brick making and sprinkling it in the workshop,many functional microorganisms(such as Acetobacter,lactic acid bacteria and yeast)were widely distributed in the production environment,indoor air,tube and equipment surfaces.Daqu microbiota construction was affected by both deterministic(56.63%/44.09%)and stochastic processes(43.37%/55.91%).Meanwhile,Water content and temperature were significant driving factors for continuous-producing daqu succession(r>0.36,P<0.05).3.Resumed-producing mechanically strong-flavor daqu and environmental microbiota was studied.Compared with continuous-producing daqu,the temperature was higher during the warming period(0～8 d)in resumed-producing daqu,whose water content,glucoamylase/amylase activity were higher during the cooling period(after 8 d).Dominant genera during fermentation process were Leuconostoc,Staphylococcus,Ralstonia,lactic acid bacteria,Kroppenstedtia,Weissella,Thermoactinomyces,Pichia,Wickerhamomyces,Candida,Saccharomyces,Saccharomycopsis,Thermoascus,Rhizopus and Thermomyces.Bacterial community in daqu brick process and environment samples gradually evolved in the direction favorable to daqu fermentation,similar to those durong continuous-producing period.Daqu microbiota construction was mostly affected by stochastic processes(82.46%/72.46%).Water content was significant driving factors for resumed-producing daqu succession,while temperature was only significantly positively correlated with the fungal community(r>0.32,P<0.05).4.Source of differential microbiota in mechanically strong-flavor daqu before and after production resumption was further studied.When production resumed after three-day shutdown,the microbial diversity and structure during daqu fermentation process changed significantly.For the whole fermentation process,40 and 23 differential bacteria,7 and 13 differential fungi were detected in mechanically strong-flavor daqu with different production methods in winter and spring,respectively.Under continuous production,bacteria and fungi in milled wheat mainly came from raw wheat and bucket elevator tube(85.25%～93.75%).Bacteria in moistened wheat,mixture wheat and 0 d daqu mainly came from mature daqu powder and air in daqu brick processing room(70.16%～92.29%),while fungi mainly came from flour and surface of grain-running machine(78.09%～93.85%).Bacteria and fungi in finished crank mainly came from mature daqu powder and grain rinsing conveyor(58.88%/93.25%).Under resumed production condition,the bacteria and fungi in milled wheat,moistened wheat,mixture wheat and 0 d daqu were mainly from the bucket elevator tube,raw wheat and silo wheat(82.34%～96.93%).The bacteria in the finished daqu were mainly from mature daqu powder(87.70%),while the fungi were mainly from mature daqu powder and the air in daqu brick processing/fermentation room(89.93%).This project firstly established the quality evaluation for strong-flavor daqu method based on physicochemical,microbial and flavor indexes,providing a theoretical reference for the quality assessment and classification of daqu.The differences in the physicochemical properties and microbiota of mechanically strong-flavor daqu before and after production resumption were analyzed by SMRT sequencing.The influence of the environmental microbiota composition on the differences in daqu before and after production resumption were elucidated,which provided a theoretical reference for daqu production process and workshop environment improvement during the production resumption,and laid good foundation for the production of high-quality daqu and Baijiu.