Study On Microbial Diversity Of Chinese Baijiu Daqu

Chinese Baijiu is one of the world’s six major distilled spirits,with unique strains,solid-state Daqu-making,solid-state fermentation,solidstate distillation,and storage in clay jars,resulting in a unique taste and rich flavor profile.The microbes of Daqu mainly come from natural inoculation,however,the microbial composition of the matured Daqu has not been well studied,there have been few research on the microbial succession in different sections of Daqu during the Daqu fermentation process.The quality evaluation of Daqu is primarily based on physicochemical and sensory properties,with no microbiological indicators.Therefore,this study focused on the microbial diversity of Daqu,the microbial communities and physicochemical characteristics of different sections of Daqu during the fermentation process,as well as the correlation of Daqu with various quality.The primary results obtained were listed as follows:(1)By collecting 296 Daqu samples from 52 different distilleries in10 provinces,including 103 low-temperature Daqu,92 mediumtemperature Daqu,and 101 high-temperature Daqu,which revealed 310 bacterial species such as Staphylococcus gallinarum,Serratia marcescens,Pantoea agglomerans,Lentibacillus massiliensis,and Bacillus licheniformis,as well as 86 fungal species including Lichtheimia ramosa,Saccharomycopsis fibuligera,and Pichia kudriavzevii etc.by using the Pac Bio single-molecule real-time sequencing,reflecting the complexity and diversity of the microbial species in traditional Chinese Daqu.The results also indicated that the microbial communities of Daqu from different types and regions differed greatly,with the similarity of bacterial communities among different types of Daqu ranging from 4.55% to11.99%.In different regions,the similarity of communities in lowtemperature Daqu,medium-temperature Daqu,and high-temperature Daqu was only 9.61% to 22.02%,2.1% to 18.13%,and 2.63% to 14.04%,respectively.A number of enriched fungi and bacteria were found among the three different types of Daqu and the same type Daqu from different places using LEf Se(Linear discriminant analysis effect size)and indicator species analysis,which could be used as indicators to identify different sources of Daqu.Furthermore,the correlation between Daqu’s dominant fungi and bacteria was calculated to reveal potential microbial co-existence or co-exclusion,providing important support for future research on the microbial diversity on Daqu.(2)In order to further investigate the composition and succession of microbial community during daqu fermentation,utilizing culturedependent and Pac Bio single molecules real-time sequencing,and the correlation between microorganisms and their physicochemical properties,the results revealed that the microbial community structure and diversity in different sections of medium-temperature Daqu had significant succession during the fermentation process,which primarily reflected differences between the surface and interior of Daqu.With the exception for Bacillus,the quantity of culturable bacteria and fungi in different sections of Daqu during the fermentation process generally increased,then decreased,and then increased again.Moisture,starch,acidity,and fermentation power gradually decreased during the fermentation process,whereas the liquefaction and protease activity gradually increased.At the early stage of fermentation,the differences of microorganisms in different sections of Daqu were limited,with the predominant microbes being Weissella confusa and Pichia kudriavzevii.In the middle and late stages of Daqu fermentation,the bacteria of Daqu surface were dominated by lactic acid bacteria,such as Weissella confusa and Pediococcus pentosaceus,the main fungi were Pichia kudriavzevii and Lichtheimia ramosa;while inside Daqu,the main bacteria included Bacillus,such as Bacillus licheniformis and Bacillus sp.,and the dominant fungi were Thermoascus aurantiacus and Thermomyces lanuginosus.The results showed that the number of cultivable microorganisms and microbial community on the Daqu surface could effectively reflect the information of the entire Daqu.According to the heat-map correlation revealed that Weissella confusa was negatively correlated with the majority of bacteria and fungi,while other bacteria were positively correlated with most fungi.Redundancy analysis(RDA)also revealed significant correlations between different fermentation periods,dominating microbes,and physicochemical properties.This study explored the microbial community structure succession in different sections of Daqu during the fermentation process,which helps control microorganisms during the Daqu fermentation process and achieve more stable Baijiu.(3)By comparing the physicochemical characteristics of qualified Daqu(QD)and inferior Daqu(ID),combining with the culture-dependent and Pac Bio single molecules real-time sequencing,and metabolome analysis to evaluate their impact on the Baijiu fermentation,the results indicated that the glucoamylase and α-amylase activity of QD were significantly higher than those of ID,with an average value approximately3.17 and 2.51 times than those of ID,respectively.The number of yeasts and the relative abundance of functional microorganisms,particularly Bacillus licheniformis,Saccharomycopsis fibuligera and Lichtheimia ramosa,were significantly higher in QD than in ID.The laboratory-scale fermentation trial showed that the relative abundance of amylaseproducing microorganisms in the QD fermentation group was higher than that in the ID fermentation group,indicating QD had higher fermentation efficiency.Specifically,the QD fermentation group experienced greater weight loss(10.7% vs 5.4%)and higher moisture(70% vs 63.7%)compared to the ID fermentation group.The QD fermentation group produced more glycerol,acetic acid,ethanol,and some other important volatile flavor substances.The above results indicate that Daqu quality is determined by,and can be evaluated based on,its microbial community.This study is the first large-scale analysis of high-,medium-,and lowtemperature Daqu samples from typical representative regions across the country using third-generation high-throughput sequencing,revealing the complexity and diversity of different types of Daqu at the species level.The results of the microbial succession and physicochemical analysis at different sections of the medium-temperature Daqu showed the variability during the Daqu fermentation process.The microbial and physicochemical differences of Daqu with different quality grades suggested that microbial indicators should be considered as indicators for judging Daqu quality grades.This study provides a foundation for the microbial community construction of Daqu fermentation and the evaluation of Daqu quality,and provides reliable ideas for the development of intelligent and standardized production of Daqu and Baijiu.