Strong-flavor Baijiu Research Articles

Differences in microbial communities among different types of zaopei and their effects on quality and flavor of baijiu

Three types of zaopei (fermented grain) of xiaoqu light-flavor baijiu (XQZP), daqu light-flavor baijiu (DQZP), and strong-flavor baijiu (SFZP) at the end of fermentation and their dominant lactic acid bacteria were systematically compared and analyzed in this study. The results showed that these three types of zaopei differed significantly in acidity, reducing sugar content, and ethanol content, and that the main factors influencing their microbial community were acidity and lactic acid. The diversity and contents of flavor substances were significantly higher in SFZP than in DQZP and XQZP. Additionally, there was a strong correlation between dominant lactic acid bacteria and flavor substances in all three zaopei, but the correlation between fungi and flavor substances was higher than that between bacteria and flavor substances. Differential gene analysis revealed that the microbial activities followed the order of SFZP > DQZP > XQZP. The KEGG enrichment analysis indicated that the differential genes from different zaopei were enriched in different metabolic pathways. Furthermore, various microorganisms in 3 types of zaopei contained different functional genes, of which fungi mainly contained genes responsible for the synthesis of ethanol and acetic acid, while lactic acid bacteria mainly contained genes responsible for the synthesis of lactic acid. In XQZP, L. helveticus was dominant lactic acid bacteria prominent in acetic acid tolerance and lactic acid production; in DQZP, L. acetotolerans was remarkable in its tolerance to lactic acid, acetic acid, ethanol and lactic acid production; and in SFZP, A. jinshanensis was superior in acetic acid tolerance and production. Taken together, this study reveals the mechanism underlying flavor differences among three types of baijiu and provides valuable references for the development and utilization of baijiu microbial resources.

Caproiciproducens converts lactic acid into caproic acid during Chinese strong-flavor Baijiu brewing

Strong-flavor Baijiu, a type of Chinese liquor, is produced through anaerobic solid-state fermentation in a sealed mud pit. Ethyl caproate, the characteristic flavor compound of strong-flavor Baijiu, is influenced by caproic acid-producing bacteria in the pit mud. To better understand the formation of caproic acid, this study investigated the microbial composition and physicochemical parameters of pit mud from different layers (top, middle, and bottom) in Hubei and Sichuan provinces, China. The results revealed that Caproiciproducens plays a key role in caproic acid production by using lactic acid as a substrate, with its abundance increasing with the depth of the pit mud. A strain Caproiciproducens sp. R1, isolated from the pit mud, was shown to produce caproic acid from lactic acid within an initial pH range of 5.5–9.0 and lactic acid concentrations of 1 %–5 % (m/v). In addition, inoculation of strain R1 into Huangshui (a lactic acid-rich liquid from Baijiu production) resulted in 40.72 mM caproic acid production. This study demonstrates that Caproiciproducens plays a crucial role in caproic acid production from lactic acid during the fermentation process of strong-flavor Baijiu.

Metagenomics-based insights into the microbial community dynamics and flavor development potentiality of artificial and natural pit mud

Strong-flavor Baijiu (SFB) production has relied on pit mud (PM) as a starter culture. The maturation time of natural PM (NPM) is about 30 years, so artificial PM (APM) with a shorter maturation time has attracted widespread attention. This study reveals the microbial and functional dissimilarities of APM and NPM, and helps to elucidate the different metabolic roles of microbes during substrate degradation and flavor formation. Significant differences in the microbial community were observed between APM and NPM, manifesting as variations in the abundance of core microorganisms. Total of 187 high-quality metagenome-assembled genomes (MAGs) were obtained based on the metagenomic binning technology, mainly including Firmicutes (n = 106), Bacteroidota (n = 15) and Chloroflexota (n = 14). Furthermore, the relative concentration of flavor compounds in 4-year APM was similar to those in 30-year NPM, but different from those in 100-year NPMs. Methanosarcina, Methanobacterium, Methanoculleus, Anaerolineae bacterium and Aminobacterium were the key bacteria responsible for the flavor differences. From a functional perspective, amino acid and carbohydrate metabolism were key functions of PM microbial, and showed differences between APM and NPM. Finally, substrate degradation and flavor generation pathways were found to exist in multiple microorganisms. Combine the relative abundance of microorganisms with the absolute abundance of enzymes, Clostridium, Lactobacillus, Petrimonas, Methanoculleus, Prevotella, Methanobacterium, Methanosarcina, Methanothrix, Proteiniphilum, Bellilinea, Anaerolinea, Anaeromassilibacillus, Syntrophomonas and Brevefilum were identified as the key microorganisms in APM and NPM.

Evolution of self-assembled amphiphilic colloidal particles in strong-flavor Chinese baijiu

This study proposed the evolution of self-assembled amphiphilic colloidal particles in Strong-Flavor (SF) Baijiu based on Ostwald ripening for the first time. The evolution process occurs in two stages: disordered amphiphilic molecules self-assemble into small colloidal particles and subsequently undergo Oswald ripening to form larger hydrophobic particles. Microscopic observations revealed the average size of oil-like spherical colloidal particles in Baijiu increased from 1.86 μm to 2.96 μm while the number of particles decreased by 39.50% during the 16-year cellaring process of SF Baijiu, consistent with the particle size trend observed via laser scattering. During fusion process, the charge-to-mass ratio of positively charged colloidal particles decreased, leading ζ-potential decreased from 23.7 mV to 4.66 mV within 16 years of storage. The electrochemical impedance spectroscopy approach tracked the unidirectional variation in the dielectric constant during evolution of SF Baijiu, reflecting the gradual expansion of colloidal particles, which aligns with the evolution trend observed in molecular dynamics simulations. By integrating direct microscopic observations of amphiphilic colloidal particles with electrochemical techniques, the evolution of Baijiu samples is capable to be evaluated in-situ, laying the foundation for intelligent Baijiu aging monitoring technology.

Characteristics and Functions of Dominant Yeasts Together with Their Applications during Strong-Flavor Baijiu Brewing.

Yeasts are pivotal brewing microbes that are associated with the flavor and quality of Chinese baijiu, yet research on dominant yeasts in strong-flavor baijiu brewing remains limited. In this study, Saccharomyces cerevisiae, Pichia kudriavzevii, and Kazachstania bulderi were identified as predominated yeasts in strong-flavor baijiu. Each strain showed distinct characteristics in ethanol resistance, thermal tolerance, and lactic acid tolerance, severally. S. cerevisiae FJ1-2 excelled in ethanol and ethyl ester production, P. kudriavzevii FJ1-1 in ethyl acetate, and K. bulderi FJ1-3 in lactic acid generation. Subsequently, the reinforced Fuqu of each yeast were severally prepared for application in baijiu brewing to verify their functions. Results revealed that the relative abundance of fortified yeast in each group rose. Pichia, Kazachstania, and Saccharomyces emerged as the core microbe for each group, respectively, by co-occurrence network analysis, influencing the microbiota to regulate flavor substances. In short, P. kudriavzevii FJ1-1 enhanced ethyl acetate. K. bulderi FJ1-3 improved ethyl caproate production and decreased levels of ethyl acetate and higher alcohols by modulating yeast community between Pichia and Saccharomyces. This is a systematic endeavor to study the functions of yeasts of strong-flavor baijiu, providing a solid basis for improving baijiu quality.

Identifying distinct markers in two Sorghum varieties for baijiu fermentation using untargeted metabolomics and molecular network approaches

The quality of strong-flavor Baijiu, a prominent Chinese liquor, is intricately tied to the choice of sorghum variety used in fermentation. However, a significant gap remains in our understanding of how glutinous and non-glutinous sorghum varieties comprehensively impact Baijiu flavor formation through fermentation metabolites. This study employed untargeted metabolomics combined with feature-based molecular networking (FBMN) to explore the unique metabolic characteristics of these two sorghum varieties during fermentation. FBMN analysis revealed 267 metabolites within both types of fermented sorghum (Zaopei) in the cellar. Further multidimensional statistical analyses highlighted sphingolipids, 2,5-diketopiperazines, and methionine derivatives as critical markers for quality control. These findings represent a significant advancement in our understanding and provide valuable insights for regulating the quality of Baijiu flavors.

Heterogenetic mechanism in multidimensional pit mud with different fermentation years: From microbial structure dynamic succession to metabolism phenotypes

Pit mud (PM) is fermenting agents in the strong-flavor baijiu (SFB) production. In this paper, the discrepancies in fermentation parameters, microbial community succession patterns and metabolic phenotypes were compared in multidimensional PMs. The results showed that pyruvic acid, succinic acid, S-Acetyldihydrolipoamide-E, glycerol and glyceric acid were the key metabolites responsible for the metabolic differences between the 2-, 30-,100- and 300-year multidimensional PMs, while the butanoic acid, heptyl, heptanoic acid, heptanoic acid ethyl ester, hexanoic acid and octanoic acid were the key differential flavor compounds in the 2-, 30-,100- and 300-year multidimensional PMs. Concurrently, the diversity and abundance of microbial community also exhibited significant differences between the new and old multidimensional PMs, the assembly pattern of bacterial communities changed from deterministic to stochasticity from lower (bottom of the pit and under the huangshui fluid) to upper PM (up the huangshui fluid and top of the pit). Key microorganisms related to the succession process of the lower PM were Clostridium, Methanobacterium, Petrimonas, Lactobacillus, Methanobrevibacter, Bellilinea, Longilinea, Bacillus. In contrast, the upper PM were Caproicibacter, Longilinea, Lactobacillus, Proteinphilum, Methanobrevibacter, Methanobacterium, Methanobacteriaceae, Petrimonas, Bellilinea and Atopobium. Redundancy analysis (RDA) indicated that the key environmental factors regulating the succession of microbial in upper PM were lactic acid, moisture, pH and available phosphorus. In contrast, the lower was lactic acid, acetic acid and ammonia N. Based on these results, heterogeneous mechanisms between new and old multidimensional PMs were explored, providing a theoretical support for improving the quality of new PM.

Relationship between microbial protein and amino acid metabolism in fermented grains of long fermentation period strong-flavor Baijiu

Relationship between microbial protein and amino acid metabolism in fermented grains of long fermentation period strong-flavor Baijiu

Succession of microbial community structure in fermented grains during the fermentation of strong-flavor Baijiu and its impact on the metabolism of acids, alcohols, and esters

Succession of microbial community structure in fermented grains during the fermentation of strong-flavor Baijiu and its impact on the metabolism of acids, alcohols, and esters

Targeted microbial collaboration to enhance key flavor metabolites by inoculating Clostridium tyrobutyricum and Saccharomyces cerevisiae in the strong-flavor Baijiu simulated fermentation system

Ethyl hexanoate and ethyl butyrate are indispensable flavor metabolites in strong-flavor Baijiu (SFB), but batch production instability in fermenting grains can reduce the quality of distilled Baijiu. Biofortification of the fermentation process by designing a targeted microbial collaboration pattern is an effective method to stabilize the quality of Baijiu. In this study, we explored the metabolism under co-culture liquid fermentation with Clostridium tyrobutyricum DB041 and Saccharomyces cerevisiae YS219 and investigated the effects of inoculation with two functional microorganisms on physicochemical factors, flavor metabolites, and microbial communities in solid-state simulated fermentation of SFB for the first time. The headspace solid-phase microextraction-gas chromatography-mass spectrometry results showed that ethyl butyrate and ethyl hexanoate significantly increased in fermented grain. High-throughput sequencing analysis showed that Pediococcus, Lactobacillus, Weissella, Clostridium_sensu_stricto_12, and Saccharomyces emerged as the dominant microorganisms at the end of fermentation. Co-occurrence analysis showed that ethyl hexanoate and ethyl butyrate were significantly correlated (|r| > 0.5, P < 0.05) with a cluster of interactions dominated by lactic acid bacteria (Pediococcus, Lactobacillus, Weissella, and Lactococcus), which was driven by the functional C. tyrobutyricum and S. cerevisiae. Mantel test showed that moisture and reducing sugars were the main physicochemical factor affecting microbial collaboration (|r| > 0.7, P < 0.05). Taken together, the collaborative microbial pattern of inoculation with C. tyrobutyricum and S. cerevisiae showed positive results in enhancing typical flavor metabolites and the synergistic effects of microorganisms in SFB.

Optimizing Baijiu fermentation with high-yield ethyl caproate-producing Candida parapsilosis strain

Ethyl caproate is an important flavor substance of Baijiu. It is effective to increase the content of ethyl caproate to improve the quality of strong-flavor Baijiu. In this study, the high-yielding ethyl caproate Candida parapsilosis strain F-5 was isolated from pit mud. Under optimized fermentation conditions, F-5 achieved an ethyl caproate yield of 53.69 mg/L. A novel strategy was employed by adding caproic acid and F-5 directly to the fermented grain. The application of F-5 in the fermentation of Xiaoqu and strong-flavor Baijiu significantly increased the ethyl caproate content by 24.86% and 17.45%, respectively. Transcriptomic analysis showed that Lip2 and esterase genes in the lipase synthesis pathway, and Fas1, Fas2, Eht1, and Faa4 genes in the acyltransferase pathway were significantly upregulated. These findings suggest that strain F-5 has the potential to contribute to the development of artificial pit mud and improve the flavor quality of strong-flavor Baijiu.

Characterizing the Contribution of Functional Microbiota Cultures in Pit Mud to the Metabolite Profiles of Fermented Grains.

Elevating the flavor profile of strong flavors Baijiu has always been a focal point in the industry, and pit mud (PM) serves as a crucial flavor contributor in the fermentation process of the fermented grains (FG). This study investigated the influence of wheat flour and bran (MC and FC) as PM culture enrichment media on the microbiota and metabolites of FG, aiming to inform strategies for improving strong-flavor Baijiu flavor. Results showed that adding PM cultures to FG significantly altered its properties: FC enhanced starch degradation to 51.46% and elevated reducing sugar content to 1.60%, while MC increased acidity to 2.11 mmol/10 g. PM cultures also elevated FG's ester content, with increases of 0.36 times for MC-FG60d and 1.48 times for FC-FG60d compared to controls, and ethyl hexanoate rising by 0.91 times and 1.39 times, respectively. Microbial analysis revealed that Lactobacillus constituted over 95% of the Abundant bacteria community, with Kroppenstedtia or Bacillus being predominant among Rare bacteria. Abundant fungi included Rasamsonia, Pichia, and Thermomyces, while Rare fungi consisted of Rhizopus and Malassezia. Metagenomic analysis revealed bacterial dominance, primarily consisting of Lactobacillus and Acetilactobacillus (98.80-99.40%), with metabolic function predictions highlighting genes related to metabolism, especially in MC-FG60d. Predictions from PICRUSt2 suggested control over starch, cellulose degradation, and the TCA cycle by fungal subgroups, while Abundant fungi and bacteria regulated ethanol and lactic acid production. This study highlights the importance of PM cultures in the fermentation process of FG, which is significant for brewing high-quality, strong-flavor Baijiu.

Analysis of differences in microorganisms and aroma profiles between normal and off-flavor pit mud in Chinese strong-flavor Baijiu

The unique cellar fermentation process of CFSB is the reason for its characteristic “cellar aroma” flavor. The types, abundance, community structure and metabolic activity of microorganisms in the pit mud directly affect the microbial balance in the white spirit production environment, promoting the formation of typical aromas and influencing the quality of CFSB. During the production process, the production of off-flavor in the cellar may occur. The aim of this study is to elucidate the differences in microbiota and flavor between normal pit mud and abnormal pit mud (pit mud with off-flavor). A total of 46 major volatile compounds were identified, and 24 bacterial genera and 21 fungal genera were screened. The esters, acids, and alcohols in the abnormal pit mud were lower than those in the normal pit mud, while the aldehydes were higher. Indole, 3-methyl-, which have been proven to be responsible for the muddy and musty flavors, were detected in both types of pit mud, and for the first time, high levels of 4-Methylanisole were detected in the pit mud. The microbial composition of the two types of pit mud showed significant differences in the bacterial genera of Sporosarcina, Lactobacillus, Garciella, Anaerosalibacter, Lentimicrobium, HN-HF0106, Petrimonas, Clostridium_sensu_stricto_12 and Bacillus, and the fungal genera of Millerozyma, Penicillium, Mortierella, Monascus, Saccharomyces, Issatchenkia, Pithoascus, Pseudallescheria, and Wickerhamomyces. While we speculate that Sporosarcina is the predominant bacterial genus responsible for the imbalance of microbiota in pit mud.

Response and assembly of abundant and rare taxa in Zaopei under different combination patterns of Daqu and pit mud: from microbial ecology to Baijiu brewing microecosystem

The quality and aroma of strong-flavor Baijiu are mainly dependent on Daqu, pit mud (PM), and the interaction of both. However, little is known about how their combination patterns affect the microbiome and metabolome of Zaopei, especially the metabolic function of rare taxa. Here, an experiment on industrial size was designed to assess the effects of 6 combinations (3 kinds of Daqu × 2 kinds of PM) on the composition and assembly of different taxa, as well as the flavor profile. The results showed that Zaopei’s microbiota was composed of a few abundant taxa and enormous rare taxa, and rare bacterial and abundant fungal subcommunities were significantly affected by combination patterns. The assembly processes of abundant/rare taxa and bacterial/fungal communities were distinct, and environmental changes mediated the balance between stochastic and deterministic processes in rare bacteria assembly. Furthermore, specific combination patterns improved the flavor quality of Zaopei by enhancing the interspecies interaction, which was closely related to rare taxa, especially rare bacteria. These findings highlighted that rare bacteria might be the keystone in involving community interaction and maintaining metabolic function, which provided a scientific foundation for better understanding and regulating the brewing microbiota from the viewpoint of microbial ecology.

Hydrogen bonding dominated self-assembly mechanism of amphiphilic molecules in Chinese Baijiu

Molecular mechanisms underlying the aging of Chinese Baijiu remained elusive. This study proposed the self-assembly behavior of amphiphilic molecules dominated by hydrogen bonds in Chinese Baijiu for the first time. The self-assembly degree of amphiphilic clusters gradually intensifies with the prolonged storage time of Baijiu, comprehensively characterized at both micro and macro levels. The results indicated that the blue-shift of the Raman hydrogen bond vibrational peak (about 11 cm−1 and 7 cm−1, respectively), the increase in viscosity (5.71% and 2.22%, respectively), and the rise in dielectric constant (95.63% and 94.99%, respectively) during the 17-year cellaring process of Strong-flavor Baijiu and Jiang-flavor Baijiu were consistent with the evolutionary trends observed in molecular dynamics simulations. The essential driving factors of cluster structure alteration of amphiphilic aroma substances in Chinese Baijiu during cellaring were demonstrated from molecular level. This study provided a research approach to comprehending the aging mechanism of Chinese Baijiu from the micro level.

Illuminating the Characteristics and Assembly of Prokaryotic Communities across a pH Gradient in Pit Muds for the Production of Chinese Strong-Flavor Baijiu.

Pit mud (PM), as an important source of microorganisms, is necessary for Chinese strong-flavor baijiu (CSFB) production. Although it has been revealed that the PM prokaryotic community diversities are influenced by its quality, product area, ages, etc., the characteristics and assembly process of the prokaryotic community in PMs across a pH gradient are still unclear. In this study, the regular changes of α- and β-diversities of the prokaryotic community across a pH gradient in PMs were revealed, which could be divided into "stable", "relatively stable", and "drastically changed" periods. A total of 27 phyla, 53 classes, and 381 genera were observed in all given samples, dominated by Firmicutes, Bacteroidetes, Proteobacteria, Lactobacillus, Caproiciproducens, Proteiniphilum, etc. Meanwhile, the complexity of the network structure of the prokaryotic microbial communities is significantly influenced by pH. The community assembly was jointly shaped by deterministic and stochastic processes, with stochastic process contributing more. This study was a specialized report on elucidating the characteristics and assembly of PM prokaryotic communities across a pH gradient, and revealed that the diversity and structure of PM prokaryotic communities could be predictable, to some degree, which could contribute to expanding our understanding of prokaryotic communities in PM.

Seasonal dynamics of the microbial community in a strong-flavor baijiu fermentation.

The microbial community plays a crucial role in Chinese strong-flavor baijiu (SFB) fermentation. However, the seasonal dynamics of the microbial community in the SFB fermentation system and its contribution to the unique flavor of SFB have not been fully elucidated. In this study, we investigated the seasonal dynamics of the microbial community through 16S rRNA and ITS gene sequencing. The results revealed significant temporal dynamics of microbial communities and environmental variables throughout the four seasons. The influence of seasons on fungal communities was found to be more significant than on bacterial communities. The diversity of bacteria was higher during the winter and summer, whereas fungal diversity was more prominent in summer and autumn. Stochastic processes maintained their dominance in microbial assembly throughout all four seasons but the significance of heterogeneous selection increased during summer for both bacteria and fungi, whereas homogeneous selection became more pronounced during winter for fungi. The pH and environmental temperature were important drivers of microbial community assembly across different seasons, primarily impacting the core genera responsible for the production of major volatile flavor compounds (VFCs), especially ethyl caproate. These findings provide new insights into the impact of seasons on microbial communities and hold promise for improving the quality-control measures for SFB brewed in different seasons. © 2024 Society of Chemical Industry.

Mucorpepsin from Rhizomucor pusillus relates the quality of medium-temperature Daqu

Medium-temperature Daqu acts as a fermentation starter for strong-flavor Baijiu. However, there is limited research available in terms of its quality attributes and formation mechanisms. In this study, the distinct characteristics of different quality medium-temperature Daqu were examined using the novel Qu-omics techniques. Multivariate statistical analyses indicated that the high-quality strong-flavor medium-temperature Daqu had increased acidity and enzyme activity, with the most significant variation observed in acidic protease activity. We revealed for the first time that mucorpepsin derived from Rhizomucor pusillus was the primary source of the acidic protease in high-quality Daqu using in-depth analysis. Volatile composition analysis revealed a positive correlation between nitrogenous and acidic substances with the predominant bacterial genera of Daqu, while a negative correlation was observed with the dominant fungal genera. The principal source of acidic compounds in Daqu was revealed to be lactic acid bacteria, and the inter-group differences in aromatic compounds were extremely significant (p < 0.001). The levels of phenylmethanol, phenylethanol, benzaldehyde, phenol, and 4-ethylguaiacol were significantly higher in high-quality Daqu compared to abnormal Daqu, while notably lower levels of phenylacetaldehyde, (±)-1-phenyl-2-propanol, phenylacetone, and other off-flavor substances were detected in high-quality Daqu than in abnormal Daqu (p < 0.05).

Analysis of ester-producing performance in high-yield ethyl hexanoate yeast and the effect on metabolites in bio- enhanced Daqu, the starter for Baijiu and other traditional fermented foods.

Ethyl hexanoate, one of the key flavor compounds in strong-flavor Baijiu. To improve the content of ethyl hexanoate in strong-flavor Baijiu, a functional strain with high yield of ethyl hexanoate was screened and its ester-producing performance was studied. Upon identification, the strain was classified as Candida sp. and designated as ZY002. Under optimal fermentation conditions, the content of ethyl hexanoate synthesized by ZY002 can be as high as 170.56mg L-1. A fermentation test was carried out using the ZY002 strain bioaugmented Daqu to verify the role of the strain applied to Baijiu brewing. It was found that strain ZY002 could not only improve the moisture and alcohol contents of fermented grains but also diminish the presence of reducing sugar and crude starch. Furthermore, it notably amplified the abundance of flavor compounds. In this study, Candida sp. ZY002 with a high yield of ethyl hexanoate provided high-quality strain resources for the actual industrial production of Baijiu.

Effects of Saccharomyces cerevisiae on microbial community and flavor metabolites in solid-state fermentation of strong-flavor Baijiu

Targeted biofortification is an effective way to regulate the microbial community succession and the production of flavor metabolites in the Baijiu fermentation. However, the high organic acid present in the Baijiu brewing process causes the gradual succession of exogenous functional microorganisms to be less dominant, ultimately resulting in defective flavor substances. In this study, a strain of Saccharomyces cerevisiae YS219 with high production of ethyl hexanoate and excellent organic acid tolerance was excavated, and the effects of this strain on the microbial composition and flavor metabolites during the fermentation of strong-flavor Baijiu were investigated. The microbial community was analyzed using the Illumina Miseq Pe300 Sequencing Platform, and at the genus level, Saccharomyces, Lactobacillus, and Pediococcus became the dominant genera after biofortification. A total of 77 flavor substances were detected in the biofortified fermented grains, and the contents of total esters and alcohols increased significantly. Among them, ethanol and ethyl hexanoate were significantly increased. Mantel test showed significant positive correlation between lactic acid and Pediococcus. Moreover, lactic acid, acetic acid, and ethanol were negatively correlated with most of the fermenting microorganisms. Following inoculation with S. cerevisiae YS219, Saccharomyces and Pediococcus exhibited a positively significant link with ethyl hexanoate, according to Spearman's correlation analysis. Taken together, the results demonstrate that the addition of S. cerevisiae with excellent organic acid tolerance and high ethyl hexanoate production helps to promote the change of microbial community and the elevation of ethanol and ethyl hexanoate.