Microbial Diversity In The Grape-Wine Natural System And Its Effects On Wine Flavor Compounds

Wine is an economic product with terroir characteristics.Any unnecessary intervention in the production process can reduce the wine’s naturalness.The excessive use of pesticides,SO₂,commercial yeast,sterilization technology and other interventions in the conventional wine production process,wine production gradually industrialized,resulting in reduced naturalness and sensory homogeneity of wine.Wine,as a fermented product,relies heavily on microbes.Grape epidermal microbes are major sources of microorganisms during wine fermentation,and the release of metabolites during fermentation can affect wine composition,flavor,aroma,and quality.Although many studies have been conducted on the microbial diversity of grapes and wine,information on microbial diversity based on the integrated grape-wine production system（grape planting to winemaking is a natural continuum）is still lacking,and the role of natural microbiome on the formation of flavor compounds in the fermentation process is unclear,necessitating further research.Therefore,in this study,the Cabernet Sauvignon from Ningxia’s Qingtongxia,Inner Mongolia’s Wuhai,Shandong’s Penglai,and Shaanxi’s Yangling were selected as the research varieties.Based on the integration of the grape and wine,the microbial diversity during fruit development,spontaneous fermentation and aging was studied by high-throughput sequencing technology,and analyzed the changes of microbial community composition and structure in different regions under natural conditions.Secondly,compared with inoculated commercial yeast,the effects of commercial yeast on natural microbial diversity and wine flavor compounds during fermentation were analyzed.Finally,metagenomics and metabolomic were used to investigate the formation process of flavor compounds in spontaneous fermentation,and to identify the main populations involved in fermentation and their relationships with key flavor compounds.The main results are as follows:1.The epidermal microbial diversity of Cabernet Sauvignon grape was influenced by grape origin and grape development process.Alternaria,Filobasidium,Naganishia,and Golubevia were the most important fungi taxa during the development of Cabernet Sauvignon from Penglai,Wuhai,Qingtongxia and Yangling in 2020 respectively,while Sphingomonas,Chryseobacterium,Bacillus,and Brevundimonas were the most important bacteria taxa respectively;Papiliotrema,Aureobasidium,Alternaria,and Coprinellus were the most important fungi taxa in 2021 respectively,Pseudomonas,Exiguobacterium,Sediminibacterium,and Mesorhizobium were the most important bacteria taxa respectively.According to Shannon index,the diversity of fungi and bacteria in all regions decreased during grape development.The veraison stage was critical,and the relative abundance of many microbial groups has changed significantly.2.Microbial diversity in Cabernet Sauvignon musts from different regions promotes the difference of wine volatile compounds during fermentation.Phenol,ethyl lactate,hexanoic acid,hexyl acetate were the most important compounds in 2020 Penglai,Wuhai,Qingtongxia and Yangling Cabernet Sauvignon natural wines,respectively,1-hexanol,ethyl lactate,diethyl succinate,ethyl caprate were the most important compounds in 2021.Saccharomycetales,Torulaspora,Dothideomycetes,and Capnodiales were the most important fungi taxa in 2020 Penglai,Wuhai,Qingtongxia,and Yangling musts respectively,Alphaproteobacteria,Enterobacteriaceae,Chloroplast,and Brachybacterium were the most important bacteria taxa respectively;Starmerella,Hanseniaspora,Dothideales,and Eurotiales were the most important fungi taxa in 2021 respectively,Alphaproteobacteria,Enterobacteriaceae,Chloroplast,and Brachybacterium were the most important bacteria taxa respectively.The diversity of fungi decreased gradually during the spontaneous fermentation of Cabernet Sauvignon,while the diversity of bacteria exhibited an inconsistent trend.The relative abundance of Saccharomyces increased gradually during fermentation,while that of other fungi taxa decreased.The concentration of fungi was very low and the diversity of bacteria was relatively stable during the aging process,and the relative abundance of acetic acid bacteria decreased gradually.3.The fungi diversity and aroma complexity of spontaneous fermentation were higher than those of inoculated fermentation.Saccharomyces,Hanseniaspora,Filobasidium,Naganishia,Fructobacillus,Massilia,Pantoea,Lactococcus,Pseudomonas,Leuconostoc,Weissella,Gluconobacter,and Lactobacillus were the different species between the two fermentation techniques,in which yeasts（such as Saccharomyces,Starmerella,Hanseniaspora,etc.）were abundant during spontaneous fermentation,while Saccharomyces was the dominant genus in the inoculated fermentation process.A total of 47 different compounds were identified between natural wine and inoculated fermented wine.Among them,phenethyl alcohol,1-pentanol,2,3-butanedione,palmitic acid,tartaric acid and so on were the main flavor compounds in naturally fermented wine,while ethyl acetate,2-methyl-1-propanol,ethyl lactate,shikimic acid,catechin and so on were the main flavor compounds in inoculated wine.4.Fermentation yeast and lactic acid bacteria were important taxa involved in the formation of flavor compounds during natural fermentation of Cabernet Sauvignon.Metagenomic results showed that Saccharomyces,Hanseniaspora,Zygosaccharomyces,Wickerhamiella,Lactobacillus,and Fructobacillus were important genera related to metabolic enzyme genes during fermentation.Enrichment analysis showed that starch and sucrose metabolism,fructose and mannose metabolism,galactose metabolism,amino sugar and nucleotide sugar metabolism,arginine and proline metabolism,butanoate metabolism,histidine metabolism,beta-alanine metabolism,pyruvate metabolism,glycolysis/gluconeogenesis,alanine,aspartate and glutamate metabolism,glyoxylate and dicarboxylate metabolism,biosynthesis of unsaturated fatty acids,fatty acid elongation,fatty acid degradation,tyrosine metabolism,fatty acid biosynthesis,aminoacyl-t RNA biosynthesis were the main metabolic pathways for forming flavor compounds.Zygosaccharomyces rouxii and Saccharomyces cerevisiae were significantly positively correlated with fructose,palmitic acid and mannose;Starmerella bacillaris,Wickerhamiella sorbophila,Hanseniaspora opuntiae,Hanseniaspora valbyensis,Hanseniaspora guilliermondii,and Hanseniaspora uvarum were significantly positively correlated with ethyl gallate,syringic acid and aromadendrin;Streptococcus mutans was significantly positively correlated with palmitic acid,trehalose,caffeic acid and lactose;Lactobacillus plantarum was significantly positively correlated with caffeic acid;Fructobacillus fructosus was significantly positively correlated with ethyl gallate;Enterococcus casseliflavus and Leuconostoc gelidum were significantly positively correlated with ethyl gallate,eugenic acid,and aromadendrin.In conclusion,grape origin has a significant effect on the fruit epidermal microbial community structure and diversity during the development of Cabernet Sauvignon grapes.In the natural fermentation process,grape epidermal microflora can promote the formation of more flavor compounds,especially fermenting yeast and lactic acid bacteria,which can improve the complexity of wine aroma.Therefore,this study can provide a theoretical basis for optimizing the structure and function of wine microbiota and regulating the metabolism of flavor compounds,so as to further improve the characteristics of wine terroir and strengthen the style of the wine region.