Analysis of flavor substances in Shaoxing traditional hand-made Jiafan wine with different amounts of ZaoShao liquor.

BACKGROUND: Adding ZaoShao liquor (high-concentration liquor) is one of the most important steps in the brewing process of Shaoxing Jiafan wine, a product protected by Chinese geographical indications. The focus of this study is the effect of different additive amounts of liquor on the flavor of end products. RESULTS: In this study, four kinds of Shaoxing Jiafan wine were brewed by changing the amount of ZaoShao liquor. Headspace solid-phase microextraction and gas chromatography-mass spectrometry were used to detect the flavor substances of four kinds of Jiafan wine. The difference in flavor of four kinds of Jiafan wine was evaluated by electronic nose analysis technology and verified by sensory evaluation. Finally, the reliability of the experimental results was verified through an aroma reconstruction experiment of rice wine. In this study, the differences in flavoring substances under different amounts of ZaoShao liquor were verified from various angles. The results showed that the flavors of the four kinds of wines were significantly different. CONCLUSION: The composition of flavor substances in Shaoxing rice wine varies with the amount of ZaoShao liquor. This study provided a scientific basis for the improvement of production technology of Shaoxing wine. © 2024 Society of Chemical Industry.

Environmental Factors Affecting the Diversity and Composition of Environmental Microorganisms in the Shaoxing Rice Wine Producing Area.

Shaoxing rice wine is a notable exemplar of Chinese rice wine. Its superior quality is strongly correlated with the indigenous natural environment. The results indicated that Firmicutes (75%), Actinobacteria (15%), Proteobacteria (5%), and Bacteroidetes (3%) comprised the prevailing bacterial groups. Among the main bacterial genera, Lactobacillus was the most abundant, accounting for 49.4%, followed by Lactococcus (11.9%), Saccharopolyspora (13.1%), Leuconostoc (4.1%), and Thermoactinomyces (1.1%). The dominant fungal phyla were Ascomycota and Zygomycota. Among the dominant genera, Saccharomyces (59.3%) prevailed as the most abundant, followed by Saccharomycopsis (10.7%), Aspergillus (7.1%), Thermomyces (6.2%), Rhizopus (4.9%), Rhizomucor (2.2%), and Mucor (1.3%). The findings demonstrate that the structure of the bacterial and fungal communities remains stable in the environment, with their diversity strongly influenced by climatic conditions. The continuous fluctuations in environmental factors, such as temperature, air pressure, humidity, rainfall, and light, significantly impact the composition and diversity of microbial populations, particularly the dominant bacterial community.

Analysis on driving factors of microbial community succession in Jiuyao of Shaoxing Huangjiu (Chinese yellow rice wine).

The microbial ecosystem of fermented food is greatly disturbed by human activities.Jiuyao is important saccharification starter for brewing huangjiu. The interaction between environmental factors and microorganisms significantly affected the microbial community structure at different stages of Jiuyao manufacturing. This study combined environmental factor analysis and high-throughput sequencing technology to comprehensively analyze the specific changes of microbial community and environmental factors in each fermentation stage of Jiuyao production and their correlation. The results showed that the activities of liquefaction enzyme, glycosylation enzyme and acid protease reached the highest value on the 8 th day (192 h) after the beginning of fermentation, and the cellulase activity reached the highest value at the end of fermentation. Pediococcus（37.5 %-58.2 %）, Weissella（9.2 %-27.0 %） and Pelomonas（0.1 %-12.1 %） were the main microbial genera in the genus bacteria, and Saccharomycopsis（37.1 %-52.0 %）, Rhizopus（12.5 %-31.0 %） and Saccharomyces（4.0 %-20.5 %） were the main microbial genera in the genus fungi. The results of correlation analysis showed that the microbial communities in Jiuyao were closely related to environmental factors. Most microbial communities were positively correlated with temperature, but negatively correlated with ambient humidity, CO2 concentration, acidity and water content of Jiuyao. In addition, the transcription levels of enzymes related to microbial glucose metabolism in Jiuyao were higher in the late stage of Jiuyao fermentation. Interestingly, these enzymes had high transcription levels in fungi such as Saccharomycopsis, Rhizopus and Saccharomyces, as well as in bacteria such as Pediococcus and Lactobacillus. This study provides a reference for revealing the succession rule of microbial community structure caused by environmental factors during the preparation of Jiuyao in Shaoxing Huangjiu.

Characterization of different grades of Jiuqu hongmei tea based on flavor profiles using HS-SPME-GC-MS combined with E-nose and E-tongue.

In order to distinguish different grades of Jiuqu hongmei tea (black tea), four different grades of Jiuqu hongmei tea were used as materials in this study: Super Grade (SuG), First Grade (FG), Second Grade (SG), and Third Grade (TG). HS-SPME-GC-MS combined with electronic nose (E-nose) and electronic tongue (E-tongue) technology was used to detect and analyze tea samples. The results showed that 162 volatile substances were identified, mainly alcohols, followed by hydrocarbons, aldehydes, ketones and esters. Twenty-nine volatile compounds were found in all grades of tea samples. The results of heat map analysis showed that the relative contents of five volatile compounds in different grades of Jiuqu hongmei tea were positively correlated with the grades of Jiuqu hongmei tea. By orthogonal partial least squares discriminant analysis (OPLS-DA), 35 different compounds of SuG and FG, 30 different compounds of SG and TG, 34 different compounds of FG and SG were found. Overall, the results indicated that there were significant differences in volatile compounds among different grades of Jiuqu hongmei tea, and the use of HS-SPME-GC-MS combined with E-nose and E-tongue could provide a scientific reference method as an effective tool for detecting flavor characteristics of other types of black tea grades.

Screening and application of purine degrading Limosilactobacillus fermentum LF-1 from Huangjiu fermentation broth.

BACKGROUND: As the important building blocks of nucleic acids, purines are alkaloids and responsible for hyperuricemia and gout. The purine content in Huangjiu is higher, and mainly exists in the form of free bases, which is easier to be absorbed by human body. However, the currently available reports on purine in Huangjiu mainly focus on detection methods and content survey. No studies on the regulation of the purine content in Huangjiu have been reported. RESULTS: Eighty-four strains, with the degradation capacity of purine, were screened from the fermentation broth of Huangjiu. In detail, the isolated lactic acid bacteria (LAB) strain 75 # showed the strongest degradation ability of guanosine, inosine and four purines, which reduce their levels by 83.4% (guanosine), 97.4% (inosine), 95.1% (adenine), 95.0% (guanine), 94.9% (hypoxanthine) and 65.9% (xanthine), respectively. Subsequently, the LAB strain 75# was identified to be Limosilactobacillus fermentum by 16S rRNA gene sequencing, which was named as Limosilactobacillus fermentum LF-1 and applied to the fermentation of Huangjiu in the laboratory. Compared with the fermentation broth of Huangjiu without adding L. fermentum LF-1, the content of purine compounds in the fermentation broth inoculated with L. fermentum LF-1 was reduced by 64.7%. In addition, the fermented Huangjiu had richer flavor compounds, and the physicochemical indices were in accordance with the national standard of Chinese Huangjiu. CONCLUSION: The screened strain L. fermentum LF-1 may be a promising probiotic for the development of a novel that can efficiently degrade purine in Huangjiu. © 2023 Society of Chemical Industry.

Highly sensitive electrochemical detection of carbendazim residues in water by synergistic enhancement of nitrogen-doped carbon nanohorns and polyethyleneimine modified carbon nanotubes.

Carbendazim (CBZ) can protect crops from pathogens, but it is also easy to cause pesticide residues, threatening human health. In our work, an electrochemical sensor based on nitrogen-doped carbon nanohorns (N-CNHs) and polyethyleneimine-modified carbon nanotubes (PEI-CNTs) was developed for the detection of CBZ content in water. The results showed that N-doping provided the CN bonds for CNHs and improved the electrochemical reaction performance of N-CNHs surface. With the participation of PEI, the surface of CNTs was positively charged and contained a large number of NH bonds, which not only promoted the electrostatic assembly of N-CNHs and PEI-CNTs but also was beneficial to further enriching CBZ. After further ultrasound-assisted assembly of N-CNHs and PEI-CNTs, the electron transfer capacity, electrochemical active surface area, and catalytic activity of N-CNHs/PEI-CNTs were significantly improved. The sensor performed a wider linear range (15 nmol/L ~ 70 µmol/L), low detection limit (4 nmol/L) and satisfactory recovery (87.33 % ~ 117.67 %) under the optimal conditions. In addition, the sensor had good anti-interference, reproducibility, and stability. Our work provided a new strategy for quantification of CBZ in environment.

The way of Qu-making significantly affected the volatile flavor compounds in Huangjiu (Chinese rice wine) during different brewing stages.

The volatile flavor compounds of Huangjiu (Chinese rice wine) brewed from different raw materials were obviously different, but there were few studies on the volatile flavor compounds of Huangjiu brewed from different wheat Qu at different brewing stages. In this paper, headspace-solid phase microextraction combined with gas chromatography-mass spectrometry, combined with principal component analysis and sensory evaluation, was used to determine the volatile flavor compounds in Huangjiu brewed from wheat Qu made by hand and wheat Qu made by mechanical. The results showed that there were significant differences in the contents and types of volatile flavor substances in Huangjiu brewed from different wheat Qu at fermentation stages, and the prefermentation and postfermentation Huangjiu samples could be well distinguished from each other. Compared with the Huangjiu brewed from wheat Qu made by mechanical, the Huangjiu brewed from wheat Qu made by hand has stronger aroma and better taste.

Metabolites comparison in post-fermentation stage of manual (mechanized) Chinese Huangjiu (yellow rice wine) based on GC-MS metabolomics.

In order to understand the differences of metabolites and their key metabolic pathways between traditional manual and mechanized Huangjiu, gas chromatography-mass spectrometry (GC-MS) combined with non targeted metabolomics was used to track and monitor Huangjiu in the whole post-fermentation stage. The results showed that 25 metabolites and 14 metabolites were identified as differential metabolites in manual and mechanized Huangjiu, respectively (VIP > 1, P < 0.05); three metabolic pathways had significant effects on differential metabolites (-log (P) > 1, impact > 0.01). Compared with the two kinds of Huangjiu, 21 kinds of metabolites were identified as differential metabolites (VIP > 1, P < 0.05); four metabolic pathways had significant effects on differential metabolites (-log (P) > 1, impact > 0.01). This study is helpful to gain insight into the underlying mechanism of flavor formation during the post-fermentation process of Huangjiu and provide a theoretical basis for the industrial development.

Identification and expression of bifunctional acid urea-degrading enzyme/urethanase from Enterobacter sp. R-SYB082 and its application in degradation of ethyl carbamate in Chinese rice wine (Huangjiu).

BACKGROUND: Ethyl carbamate (EC) is a potential carcinogen existing in fermented foods such as Chinese rice wine (Huangjiu). Since urea is an important precursor of EC, the degradation of urea could be an effective way to reduce EC in foods. RESULTS: In this study, an Enterobacter sp. R-SYB082 with acid urea degradation characteristics was obtained through microbial screening. Further research isolated a new acid urea-degrading enzyme from R-SYB082 strain - ureidoglycolate amidohydrolase (UAH) - which could degrade EC directly. The cloning and expression of UAH in Escherichia coli BL21 (DE3) suggested that the activity of urea-degrading enzyme reached 3560 U L-1 , while urethanase activity reached 2883 U L-1 in the optimal fermentation condition. The enzyme had the dual ability of degrading substrate urea and product EC. The removal rate of EC in Chinese rice wine could reach 90.7%. CONCLUSION: This study provided a new method for the integrated control of EC in Chinese rice wine and other fermented foods. © 2022 Society of Chemical Industry.

Ultrasound-assisted aqueous two-phase extraction of total flavonoids from Tremella fuciformis and antioxidant activity of extracted flavonoids.

Here, the extraction of total flavonoids from Tremella fuciformis (TF) by aqueous two-phase extraction combined with ultrasound is presented. The extraction was optimized via a single-variable approach using the variables of mass fraction of ethanol and NaH2PO4, ultrasonication time, and TF sample amount. Response surface methodology (RSM) was then applied to determine the best conditions for extraction using the following three key parameters: ultrasonication time, ethanol mass fraction, and NaH2PO4 mass fraction. The optimum conditions were as follows: an aqueous two-phase system of ethanol/NaH2PO4, an ethanol mass fraction of 23%, a NaH2PO4 mass fraction of 27.18%, and an extraction time of 8.25 min. Under these conditions, the maximum extraction rate of total flavonoids was 0.158 mg/g. The scavenging rates for superoxide anion and hydroxyl radical, as well as the antioxidant activity of total flavonoids in TF, were studied. Total flavonoids from TF showed some scavenging ability, but the scavenging effects were lower than those of vitamin C (Vc) at the same concentrations. Nevertheless, ultrasound-assisted two-phase extraction was considered an efficient and rapid method that can be used to extract total flavonoids from TF.

The sensory and flavor characteristics of Shaoxing Huangjiu (Chinese rice wine) were significantly influenced by micro-oxygen and electric field.

In order to improve the high cost of equipment and difficult management caused by the natural aging of Chinese rice wine (Huangjiu), micro-oxygen (MO) and electric field (PEF) technology are used to accelerate the aging of Huangjiu. The results showed that micro-oxygen and electric field have a significant effect on the sensory characteristics and flavor characteristics of Huangjiu. Compared with the naturally aged Huangjiu, the flavor compounds of Huangjiu treated with micro-oxygen and electric field increase significantly. Based on principal component analysis, Huangjiu processed at 0.35 mg L/day or 0.5 mg L/day combined electric field exhibited similar flavor to the natural aged Huangjiu, which was highly associated with long-chain fatty acid ethyl esters (C13-C18). Moreover, partial least squares regression demonstrated that sensory attributes of cereal aroma and astringency were highlighted after aging time, while fruit aroma, continuation, and full body were dominant after micro-oxygen and electric field treatment. Micro-oxygen and electric field effectively enhanced the quality of Huangjiu, which could be applied in other alcoholic beverages.

Study on relationship between bacterial diversity and quality of Huangjiu (Chinese Rice Wine) fermentation.

Huangjiu (Chinese rice wine) is brewed in an open environment, where bacteria play an important role during the fermentation process. In this study, bacterial community structure and composition changes in the fermented mash liquid of mechanized Huangjiu, well-fermented manual Huangjiu (wines of good qualities), and poorly fermented manual Huangjiu (wines of poor qualities: spoilage, high acidity, low alcohol content) in different fermentation stages from Guyuelongshan Shaoxing Huangjiu company were analyzed via metagenomic sequencing. And bacterial metabolic difference was analyzed via gene prediction of metabolic pathway enzymes. The results showed that the bacterial diversity degree was abundant, and the number of bacterial species in every sample was approximately 200-400. Lactic acid bacteria (LAB) dominated the bacterial community of Huangjiu fermentation, and lactobacillus was predominant species in well-fermented Huangjiu while Lactobacillus brevis had an absolute dominance in spoilage Huangjiu. Further, gene prediction revealed that transformation of malate to pyruvate and lactate anabolism was more active in mash liquid of well-fermented manual Huangjiu, while acetate accumulation was stronger in mash liquid of poorly fermented manual Huangjiu, which explained acidity excess reason in poorly fermented Huangjiu at gene level.

Isolation and identification of the bitter compound from Huangjiu.

The strategy of taste-guided assisted by solvent extraction, solid-phase extraction and semipreparative HPLC were applied to isolate the main nonvolatile bitter components from mechanized Huangjiu. The potential fraction was identified by amino acid analysis and ultra-performance liquid chromatography-quadrupole-time-of-flight-MS/MS. Bitter pyroglutamate peptide Pyr-LFNPSTNPWHSP (PGP) was successfully identified from Huangjiu for the first time. Quantitative analysis showed that PGP contents ranged from below the limit of quantitation to 32.97 mg/L, among mechanized Huangjiu had higher contents than manual and commercial Huangjiu. The formation of PGP mainly occurred in the primary fermentation and it was stable in Huangjiu. Moreover, the PGP content of the Huangjiu brewed using raw wheat Qu was 112.6% higher than that using cooked wheat Qu, but presented subtle change with the increase of raw wheat Qu. The results revealed that PGP contributed the bitterness to Huangjiu, which may offer a possibility to reduce the bitterness of Huangjiu.

Profiling the key metabolites produced during the modern brewing process of Chinese rice wine.

The study quantitatively profiled 83 low-molecular-weight metabolites in the categories of alcohols, aldehydes, amino acids, esters, fatty acids, organic acids, and reducing sugars produced during the advanced brewing process of Chinese rice wine, using multiply chromatography and mass spectrum. In the primary fermentation, vigorous metabolisms were demonstrated by the production of ethanol at the level of 14% by volume, and the consumption of reducing sugars from the maximum level of 100 g/L to 20 g/L. Meantime, more than 70% of the contents of organic acids, fatty acids, higher alcohols, and aldehydes were formed. The metabolisms slowed down in the secondary fermentation, whereas 60% of the contents of amino acids and esters were accumulated in this stage. The nutrients, such as amino acids, organic acids, and reducing sugars reached 10 g/L, 5 g/L, and 3 g/L at the end of brewing, respectively. In terms of flavor and taste attributes to the brewed rice wine, the organoleptic activity value (i.e. the ratio of content to threshold value) was above 1 for 17 compounds, including six organic acids, namely acetic acid, citric acid, lactic acid, malic acid, succinic acid, and tartaric acid, four amino acids, namely cysteine, aspartic acid, glutamic acid, and lysine, three higher alcohols namely isoamyl alcohol, isobutanol, and phenethyl alcohol, three esters, namely ethyl acetate, ethyl butyrate, and ethyl hexanoate, and an aldehyde, namely benzaldehyde.

Identification of colloidal haze protein in Chinese rice wine (Shaoxing Huangjiu) mainly by matrix-assisted laser ionization time-of-flight mass spectrometry.

As one of the three most famous brewed wines in the world, Chinese rice wine is made from rice and husked millet, containing 14 percent to 20 percent alcohol. Highly original, yellow wine brewing techniques are regarded as the model of the wine brewing industry in Asia. Shaoxing Huangjiu is produced in Zhejiang province and remains the oldest and most representative Chinese rice wine. During storage, Shaoxing Huangjiu is susceptible to environmental disturbance and produces colloidal haze to result in turbidity. In this study, the main composition and source of colloidal haze protein in Shaoxing Huangjiu were analyzed by two-dimensional electrophoresis and matrix-assisted laser ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS). The results showed that the proteins in colloidal haze mainly consisted of oat protein b1, oat-like protein, di-amylase inhibitor, pathogenesis-related protein, pathogenesis-related protein-4, chitinase II derived from wheat and oat-like protein, and beta-amylase derived from rice. The amino acid composition and secondary structure of haze protein and supernatant protein in Huangjiu were further explored by high-performance liquid chromatography and Fourier transform infrared spectroscopy. The study has broadened knowledge of the main composition and source of colloidal haze protein in Shaoxing Huangjiu. The corresponding results indicated that the amino acid composition from colloidal haze had the main characteristics of high hydrophobicity and low water solubility.

Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.

Adaptive Evolution Relieves Nitrogen Catabolite Repression and Decreases Urea Accumulation in Cultures of the Chinese Rice Wine Yeast Strain Saccharomyces cerevisiae XZ-11.

Urea is the major precursor of ethyl carbamate in Chinese rice wine. Although efforts have been made to decrease urea accumulation, few methods can be applied to industrial food production due to potential safety concerns. In this study, adaptive laboratory evolution (ALE) followed by high-throughput screening was used to identify low urea-accumulating strains derived from the industrial Chinese rice wine yeast strain Saccharomyces cerevisiae XZ-11. Three evolved strains were obtained that had 47.9%, 16.6%, and 12.4% lower urea concentrations than the wild-type strain. Comparative genomics analysis revealed that genes involved in carbon and nitrogen metabolism evolved quickly. Transcription levels of genes involved in urea metabolism were dramatically upregulated after ALE. This work describes a novel and safe strategy to improve nitrogen utilization of industrial yeast strains involved in food fermentation. The identified genomic variations may also help direct rational genetic engineering of nitrogen metabolism processes to achieve other goals.

Mutant Potential Ubiquitination Sites in Dur3p Enhance the Urea and Ethyl Carbamate Reduction in a Model Rice Wine System.

Ubiquitination can significantly affect the endocytosis and degradation of plasma membrane proteins. Here, the ubiquitination of a Saccharomyces cerevisiae urea plasma membrane transporter (Dur3p) was altered. Two potential ubiquitination sites, lysine residues K556 and K571, of Dur3p were predicted and replaced by arginine, and the effects of these mutations on urea utilization and formation under different nitrogen conditions were investigated. Compared with Dur3p, the Dur3pK556R mutant showed a 20.1% decrease in ubiquitination level in yeast nitrogen base medium containing urea and glutamine. It also exhibited a >75.8% decrease in urea formation in yeast extract-peptone-dextrose medium and 41.3 and 55.4% decreases in urea and ethyl carbamate formation (a known carcinogen), respectively, in a model rice wine system. The results presented here show that the mutation of Dur3p ubiquitination sites could significantly affect urea utilization and formation. Modifying the ubiquitination of specific transporters might have promising applications in rationally engineering S. cerevisiae strains to efficiently use specific nitrogen sources.

Genome-wide mapping of nucleosome positions in Saccharomyces cerevisiae in response to different nitrogen conditions.

Well-organized chromatin is involved in a number of various transcriptional regulation and gene expression. We used genome-wide mapping of nucleosomes in response to different nitrogen conditions to determine both nucleosome profiles and gene expression events in Saccharomyces cerevisiae. Nitrogen conditions influence general nucleosome profiles and the expression of nitrogen catabolite repression (NCR) sensitive genes. The nucleosome occupancy of TATA-containing genes was higher compared to TATA-less genes. TATA-less genes in high or low nucleosome occupancy, showed a significant change in gene coding regions when shifting cells from glutamine to proline as the sole nitrogen resource. Furthermore, a correlation between the expression of nucleosome occupancy induced NCR sensitive genes or TATA containing genes in NCR sensitive genes, and nucleosome prediction were found when cells were cultured in proline or shifting from glutamine to proline as the sole nitrogen source compared to glutamine. These results also showed that variation of nucleosome occupancy accompany with chromatin-dependent transcription factor could influence the expression of a series of genes involved in the specific regulation of nitrogen utilization.

Metagenomic sequencing reveals the relationship between microbiota composition and quality of Chinese Rice Wine.

Chinese Rice Wine (CRW) is a common alcoholic beverage in China. To investigate the influence of microbial composition on the quality of CRW, high throughput sequencing was performed for 110 wine samples on bacterial 16S rRNA gene and fungal Internal Transcribed Spacer II (ITS2). Bioinformatic analyses demonstrated that the quality of yeast starter and final wine correlated with microbial taxonomic composition, which was exemplified by our finding that wine spoilage resulted from a high proportion of genus Lactobacillus. Subsequently, based on Lactobacillus abundance of an early stage, a model was constructed to predict final wine quality. In addition, three batches of 20 representative wine samples selected from a pool of 110 samples were further analyzed in metagenomics. The results revealed that wine spoilage was due to rapid growth of Lactobacillus brevis at the early stage of fermentation. Gene functional analysis indicated the importance of some pathways such as synthesis of biotin, malolactic fermentation and production of short-chain fatty acid. These results led to a conclusion that metabolisms of microbes influence the wine quality. Thus, nurturing of beneficial microbes and inhibition of undesired ones are both important for the mechanized brewery.