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Non- Saccharomyces yeast (NSY) co-fermentation with Saccharomyces cerevisiae has been widely used to produce high-quality fermented alcoholic beverages. In this study, we evaluated the feasibility of 24 NSY strains belonging to 13 different genera co-fermentation with S. cerevisiae jiangnan1# in huangjiu brewing. Different fermentation trials were set up and the effects on huangjiu fermentation were also analyzed. Results indicated that simultaneous inoculation could realize the application of NSY, while the appropriate proportion of S. cerevisiae and NSY (1:1 or 1:10) showed differential effects. Finally, three types of NSY strains (Zygosaccharomyces rouxii Non-Sc11, Kloeckera apiculata Non-Sc17, and Candida tropicalis Non-Sc21) with high ethanol content (>16% vol), moderate concentration of higher alcohols (<400 mg/L, 14% vol), higher production of esters (P < 0.05), and lower organic acid (P < 0.05) were obtained ultimately. The sensory evaluation demonstrated that using no less than one kind of the three NSY strains in simultaneous fermentation with S. cerevisiae jiangnan1# (1:1) resulted in sensory diversity of new huangjiu styles. In addition, the method for evaluation and application of NSY in co-fermentation with S. cerevisiae jiangnan1# in huangjiu brewing had been established for the first time, which could be directly applied in industrial production of huangjiu. [Display omitted] • This is the first report on the use of non- Saccharomyces yeast for huangjiu co-fermentation. • Simultaneous inoculation of non- Saccharomyces yeast and S. cerevisiae is established. • The feasibility of 24 non- Saccharomyces yeast belonging to 13 different genera is evaluated. • Using three non- Saccharomyces yeast results in sensory diversity of new huangjiu styles. • Three strains of non- Saccharomyces yeast with potential industrial application in huangjiu beverage. [ABSTRACT FROM AUTHOR]

As the core raw material of huangjiu (Chinese rice wine) production and brewing, changes of physichemical factors during the fermentation process significantly affect the succession of the microbial community of raw wheat qu (RWQ), also playing important role in huangjiu quality. In this report we analyzed the community structure and driving force RWQ microorganisms with different processes (manual qu , MAQ; mechanical qu , MEQ), and evaluating the flavor of huangjiu. The production procedure was divided into three stages. Dominant microorganisms were Aspergillus, Alternaria , Epicoccus , Bacillus, Pantoea, Saccharopolyspora and Staphylococcus. In early stage, microbial community of MAQ was significantly correlated with temperature and water content, oxygen in middle stage, while no significant driving force in the last stage. Water content and oxygen affected MEQ community significantly in early stage, while in the final stage, fungal community was affected by both temperature and water content, and bacterial community was significantly correlated with oxygen. Glucoamylase activity reached its maximum at 50 °C, while the α-amylase activity decreased with the rise of temperature. The highest amylase activity was obtained with 19% initial water addition. Amylase activity showed positive correlation with the thickness of RWQ. The total amount of organic acids was higher in mechanical huangjiu at the end of pre-fermentation. Contents of n-hexanol, ethyl isovalerate, ethyl hexanoate and guaiacol in manual huangjiu were significantly higher than those in mechanical one, while the content of 4-vinylguaiacol was lower. [Display omitted] • The microbial community succession of raw wheat qu was identified. • The driving factor was verified by single-factor experiment. • Microbial structure tends to be identical in different part of raw wheat qu. • Driving force of microbiota in raw wheat qu are temperature, water content and oxygen. [ABSTRACT FROM AUTHOR]

[Display omitted] • P. pentosaceus , W. paramesenteroides , R. emersonii, B. spectabilis were dominant in Daqu. • Four Saccharopolyspora species were dominant in wheat Qu. • Flavor metabolic pathways in Qu microbiota were reconstructed. • The dominant microbes in Qu may not be the main functional microbes. Daqu and wheat Qu are saccharification and fermenting agents in Chinese huangjiu and baijiu production. This study aimed to investigate the difference between Daqu and wheat Qu in physicochemical indices, microbial communities, functional genes, and the metabolic network of key microbes responsible for flavor synthesis by whole-metagenome sequencing and metabolite analysis. Herein, physicochemical indices indicated that compared with wheat Qu , Daqu exhibited higher protease and cellulase activity and acidity, and lower glucoamylase and amylase enzyme activity. Metagenomic sequencing reveals that although Daqu and wheat Qu community composition have significant differences at species level, they have similar functional genes. Daqu were enriched in Pediococcus pentosaceus , Weissella paramesenteroides , Rasamsonia emersonii and Byssochlamys spectabilis (22.48% of the total abundance), while wheat Qu harbored greater abundances of Saccharopolyspora (54.78%, Saccharopolyspora rectivirgula , Saccharopolyspora shandongensis , Saccharopolyspora hirsuta , Saccharopolyspora spinose , and Saccharopolyspora erythraea). From a functional perspective, the important functions of Daqu and wheat Qu are both amino acid metabolism and carbohydrate metabolism. Meanwhile, a combined analysis among microbiota, functional genes, and dominant flavors indicated S. shandongensis , S. rectivirgula , and S. spinose might be the main contributor to the synthesis of flavor compounds in wheat Qu , while R. emersonii , W. paramesenteroides , Leuconostoc citreum , Leuconostoc mesenteroides , Weissella cibaria and P. pentosaceus may make the greatest contribution to flavor compounds synthesis in Daqu. This study reveals the microbial and functional dissimilarities of Daqu and wheat Qu , and helps elucidating different metabolic roles of microbes during flavor formation. [ABSTRACT FROM AUTHOR]

Aromatic alcohols (including β-phenylethanol, tyrosol and tryptophol) could contribute positively to the aroma flavor of huangjiu at an appropriate amount. In this study, it was found that the aromatic alcohols in huangjiu were predominantly synthesized by Saccharomyces cerevisiae HJ01. The Ehrlich pathway and shikimate pathway, including 31 genes, are responsible for aromatic alcohol biosynthesis in S. cerevisiae. During the first 120 h of huangjiu fermentation, the genes GAP1 HJ01 (permease), BAT2 HJ01 (transaminase), PDC1 HJ01 and PDC5 HJ01 (pyruvate decarboxylase), and ADH1 HJ01 (alcohol dehydrogenase) formed integrated Ehrlich pathway are responsible for aromatic alcohol biosynthesis. In S. cerevisiae HJ01, the mutations in the promoter region of genes GAP1 HJ01, BAT2 HJ01, PDC1 HJ01, and PDC5 HJ01 involved in the Ehrlich pathway increased the promoter strength and transcriptional levels of related genes. The addition of 20 U/g acid protease could substantially increase the expression levels of genes involved in the Ehrlich pathway, and therefore there were 34.7% increasing for the concentration of total aromatic alcohols. • Aromatic alcohols in huangjiu are synthesized by Saccharomyces cerevisiae. • Aromatic alcohols in huangjiu are mainly synthesized through the Ehrlich pathway. • Key genes and promoter mutations in Ehrlich pathway were identified. • Acid protease enhanced expression level of key genes. [ABSTRACT FROM AUTHOR]