Study On Core Microbiota Analysis And Controlled Solid Fermentation Of Jiuyao

Jiuyao is a small saccharifying and spontaneously fermenting starter for traditional Shaoxing huangjiu brewing.The microbial community of jiuyao has an important influence on the quality and flavor of manual huangjiu.However,the contribution of core functional microorganisms to the fermentation of huangjiu yeast starter is unclear,and the traditional production of jiuyao makes it difficult to ensure the stability of microflora and the quality of jiuyao.Therefore,this study first took traditional jiuyao as the research object and analyzed the core microbiota of jiuyao and its influence on the fermentation of manual huangjiu yeast starter based on single-molecule real-time（SMRT）sequencing.Then,strains with high starch-degrading abilities were successfully isolated.Further,this study provided a steady and controlled fermentation process for jiuyao by fortification.Finally,the applicable feasibility in the huangjiu brewing of jiuyao was evaluated.Taken together,it provided a scientific basis and theoretical guidance to improve the traditional handcrafting of jiuyao,develop modern intelligent manufacturing procedures,scale up the production of jiuyao under controllable fermentation,and stabilize jiuyao quality.The main research results were as follows:（1）SMRT sequencing was used to reveal the core microbial community of jiuyao and its influence on the fermentation process of manual huangjiu yeast starter.Microbial network analysis and molecular ecological network analysis（MENA）were applied to profile the microbial diversity of jiuyao collected from various production workshops in Shaoxing City.A core community consisting of six fungal species and three bacterial species was found,including Rhizopus microsporus,Rhizopus arrhizus,Saccharomycopsis fibuligera,Saccharomyces cerevisiae,Pichia kudriavzevii,Wickerhamomyces anomalus,Pediococcus pentosaceus,Weissella confusa,and Weissella cibaria.Saccharomycopsis fibuligera and Rhizopus microsporus were revealed as the core functional species related to starch-degrading enzyme activities.The glucoamylase activity increased significantly during the first 72 h fermentation of Lin-fan yeast starter,and reached the peak value of 104.54±14.05 U·m L^-1 at 72h of fermentation,which indicated that the core microorganisms continuously secreted saccharifying enzyme in the early solid-state fermentation and late semi-solid fermentation of Lin-fan yeast starter.（2）Based on the analysis of the core saccharifying microbiota,48 strains with the ability to hydrolyze starch were isolated and screened from jiuyao samples.The ability to produce starch-degrading enzymes in liquid-state fermentation（LSF）and solid-state fermentation（SSF）was evaluated.Two strains with high starch-degrading abilities were successfully isolated and identified as Saccharomycopsis fibuligera CY2111 and Rhizopus microsporus SM4.Strain CY2111 exhibited higher enzyme activities in SSF using wheat bran as substrates,which revealed activities for glucoamylase activity(1130.84±55.18 U·g^-1)andα-amylase activity(1.51±0.06 U·g^-1),and the strain SM4 showed the highest glucoamylase activity(1338.30±22.44 U·g^-1)andα-amylase activity(1.69±0.33 U·g^-1).（3）The controllable solid-state fermentation environment and conditions were simulated and optimized to improve the jiuyao glucoamylase activity andα-amylase activity by fortifying techniques.Further,the specific control and conditions were determined in scale-up fermentation.The results showed that both wheat bran and rice were selected as the substrate for sanqu preparation.The glucoamylase activity(374.41±33.68 U·g^-1)andα-amylase activity(1.03±0.18 U·g^-1)of sanqu were significantly higher than traditional jiuyao sphere(262.59±75.24 U·g^-1,0.73±0.14 U·g^-1).Finally,the optimum processing parameters of jiuyao fermentation were summarized as inoculation amounts of 5%,28℃with 95%of humidity in Qianjiao,32℃with 80%of humidity in Houjiao,water contents of 50%,stack thickness of 3cm,and fermentation time of 54 h.The glucoamylase andα-amylase activities of jiuyao increased by 207.82%and 215.22%than traditional jiuyao under the optimum conditions,respectively.The optimized fermentation conditions were applied to scale-up preparation.The stack thickness was reduced to 2.5 cm,and the added proportion of wheat bran was rised to35%.Finally,the jiuyao was attained with glucoamylase activity of 1480.13±49.89 U·g^-1 andα-amylase activity of 1.33±0.08 U·g^-1,respectively.And the acid protease activity and fermentative activity of jiuyao met the requirements for huangjiu brewing.The core fungal community of jiuyao under controlled SSF remained stable.It is dominated by R.microsporus,R.arrhizus,and S.fibuligera.（4）The new Lin-fan yeast starter（NLF）and new yeast starter（NYS）brewed with jiuyao were applied in huangjiu brewing,and their influences on huangjiu quality were explored.The alcohol content of huangjiu brewed with new Lin-fan yeast starter（LHJ）could reach19.40±0.17%vol when the ratio of jiuyao to raw wheat was 0.15:1,and fermented at a 1:1inoculation ratio of NLF and activated yeast,which improved raw material utilisation.The alcohol content of huangjiu brewed with new yeast starter（NHJ）（17.47±0.06%vol）was significantly higher than that of mechanized huangjiu（MHJ）,which indicated that NYS had higher fermentation activity than Su-niang Yeast Starter（SYS）.The flavor of huangjiu brewed with different yeast starter was measured and analyzed.Their organic acid contents and amino acid contents were in accordance with the national standard.There were more kinds of volatile aroma substances in LHJ,such as ethyl propionate,vanillin,and 2,5-dimethylpyrazine.PLS-DA analysis showed that the flavor profiles of NHJ and Traditional Manual Huangjiu（THJ）were similar,with the largest contributions from esters and alcohols,suggesting that the jiuyao under controlled fermentation has the potential to be applied in mechanical huangjiu brewing,and the flavor profile was similar to that of traditional manual huangjiu.In summary,this study revealed the core saccharifying microorganisms of jiuyao,explored its influence on the Lin-fan yeast starter brewing,and optimized the controllable solid-state fermentation process of jiuyao.It provided clues for biofortification with specific functional strains to improve the quality of jiuyao and huangjiu.It would provide a foundation to develop a modern production for jiuyao manufacturing on an industrial scale for stability and efficiency.