Study On Microbial Community Structure In The Brewing System Of Guangdong Hakka Rice Wine And Its Influence On Formation Of Flavor Substances

As an important branch of yellow Rice Wine in China,Guangdong Hakka Rice Wine(GHRW)has gained great popularity in Lingnan and Hakka regions.However,There is limited research on microbial community in the brewing technology of GHRW and its effect on the formation of flavor substances.In this study,the high-throughput sequencing technology was used to comprehensively study the microbial community structure and its variation in the fermentation with Qu and fermentation process of GHRW,and the flavor substances in the brewing process were also analyzed.Moreover,effect of red Qu on the quality of GHRW was investigated.The correlation between microorganisms,microflora structure and flavor substance were analyzed by partial least-squares regression analysis.In addition,the functional gene expression of microorganism were analyzed by the macro transcriptome sequencing.Results obtained in the present work were great significance for analyzing the fermentation theory and flavor formation mechanism of GHRW.The main results were as follows:(1)Microbial community structure of Qu used in the brewing of GHRW was analyzed.The fungi in the red Qu belonged to 10 genera and 10 species.The dominant fungus in the red Qu was Monascus purpureus(96.189%).The fungi identified belonged to 9 genera and 9 species,and the dominant fungi in the wheat Qu were Rhizopus microsporus(83.224%),Saccharomycopsis fibuligera(6.538%)and Rhizopus arrhizus(1.762%).The fungi in distiller's yeast belonged to 5 genera and 6 species,and the dominant fungi were Rhizopus arrhizus,(48.385%),Saccharomycopsis fibuligera(32.980%)and Rhizopus microsporus(7.208%).All the bacteria in red Qu belonged to 17 genera and 16 species,and the predominant bacteria were Burkholderia gladioli(51.524%),Pantoea dispersa(36.933%),Serratia marcescens(3.130%)and Pediococcus pentosaceus(1.666%).The bacteria in the wheat Qu belonged to 16 genera and 14 species,and its predominant bacteria were Weissella confusa(41.410%),Bacillus velezensis(21.013%)and Pediococcus pentosaceus(15.737%).As for bacteria in distiller's yeast,they belonged to 18 genera and15 species,and Pediococcus pentosaceus(79.589%),Weissella confuse(4.979%),Pantoea dispersa(3.148%)and Burkholderia gladioli(2.687%)were the predominant bacteria.(2)The change rule of microbial community structure in GHRW was discovered.The predominant fungus in the early brewing process of GHRW was Saccharomycopsis fibuligera.The abundances of Rhizopus microspores and Rhizopus arrhizus increased gradually with the brewing process.Pediococcus pentosaceus was the predominant bacteria in the early brewing process of traditional GHRW and GHRW without the addition of red Qu.As for GHRW added with red Qu,Pantoea dispersa,Kozakia baliensis and Pediococcus pentosaceus were the predominant bacteria in the early brewing process and four Lactobacillus including Lactococcus piscium,Lactobacillus sp.NBRC 3954,Lactobacillus pentosus and Lactobacillus fermentum gradually became predominant in the following brewing process.There was no significant difference between the main fungi in the brewing processes of GHRW with or without red Qu,while significant differences were observed in Bacillus sp.BH072 and Lactococcus piscium.(3)The correlation between the microbial community in the brewing process of GHRW was discussed.Results indicated that Saccharomycopsis fibuligera was competitive with all fungi,and had a mutually beneficial relationship with bacteria such as Pediococcus pentosaceus,Lactococcus piscium,Lactobacillus pentosus and Bacillus sp.BH072.Rhizopus microsporus had mutually beneficial relationships with Rhizopus arrhizus,Aspergillus flavus,Microidium phyllanthi and Syncephalastrum racemosum,while had strong competitive relationships with Saccharomycopsis fibuligera,Pediococcus pentosaceus,Lactobacillus pentosus and Lactobacillus fermentum.The existence of Lactobacillus sp.NBRC 3954 promoted the growth of Lactobacillus pentosus and Lactobacillus fermentum,but was antagonistic to the growth of fungi.Lactobacillus fermentum had a favorable positive correlation with Lactobacillus sp.NBRC 3954 and Lactobacillus pentosus.In addition,Lactobacillus had a strong antagonism effect on thegrowth of Aspergillus flavus.(4)The relationship between microbial community and volatile components in the brewing process of GHRW was investigated.Saccharomycopsis fibuligera had strong correlation with benzyl alcohol,1-nonyl alcohol,6-methyl-5-hepten-2-one,1-octanol and1-hexanol in GHRW,but almost no correlation with ester substances.Rhizopus arrhizus was significantly correlated with esters,especially ethyl heptanate,diethyl succinate,ethyl caprylate,2-hydroxy-propionate and isoamyl lactate.Lactobacillus sp.NBRC 3954 had a strong correlation with important flavor esters and alcohols,while Herbaspirillum frisingense was strongly correlated with flavor esters such as diethyl succinate,ethyl benzoate,ethyl caprylate,2-hydroxy-propionate,ethyl lactate,ethyl hexadecanoate and ethyl oleate.Bacillus sp.BH072 was mainly related to flavor substances including phenols and aldehydes.The main flavor substances in GHRW,including 3-methyl-1-butanol,2-methyl-1-propanol and phenethanol,were mainly related to the bacteria such as Lactobacillus sp.NBRC 3954,Lactobacillus fermentum and Lactobacillus pentosus,while had little correlation with fungi.Ethyl lactate and isoamyl lactate were mainly associated with Rhizopus arrhizus and Herbaspirillum frisingense.Ethyl hexadecanoate,ethyl tetradecanoate and ethyl oleate were mainly related to Lactobacillus sp.NBRC 3954,Herbaspirillum frisingense and Bacillus sp.BH072.(5)The influence of red Qu on the flavor and quality of GHRW was studied.The addition of red Qu in the brewing process of GHRW could not only increase the varieties of volatile substances such as alcohols,esters,aldehydes,ketones and acids,but also increased the contents of aldehydes and ketones.The addition of red Qu also had significant influence on the characteristic flavor of GHRW such as isoamyl alcohol,phenyl alcohol,isobutyl acetate,isobutanol,ethyl palmitate and ethyl decanoate.Red Qu was also beneficial for improving the contents of amino acids and polypeptides in GHRW.(6)The variation of function gene expression for microorganisms during the fermentation process of GHRW were preliminarily explored.High-throughput sequencing of macro transcriptome was performed on GHRW fermented with red Qu in the pre-fermentation,the early fermentation and the mid-fermentation.103023 Unigene were assembled and functionally annotated.The different metabolic pathways between sampleswere obtained by enrichment of differential gene KEGG.Because of the differences in gene expression,the early stage of fermentation was beneficial for the formation of butyl aldehyde,n-butyl alcohol,galactinol,melibiitol,epimelibiose,galactosylglycerol,glycerin,D-mannitol,D-sorbitol,inositol,sucrose,stachyose,D-galactose,and not conducive to the formation of 2,3-butanediol,R-2-acetylmethylcarbinol and succinic acid,while the middle stage of fermentation was beneficial for the production of D-glucose and D-lactic acid and not conducive to the formation of acetaldehyde,ethanol,acetic acid and fatty acids.