Core microbiome of chinese fermentation starter, construction method therefor, and use thereof

Abstract

Provided are a core microbiome of a Chinese fermentation starter, a construction method therefor, and a use thereof, relating to the field of microorganisms of traditional brewed food. According to the provided construction method for the core microbiome, from the perspectives of taxonomy and functions, the composition of the core microbiome of a yellow rice wine fermentation starter is comprehensively defined on the system level, and a core microbiome for producing an artificially fermented Chinese fermentation starter is constructed on the basis of the composition; and the core microbiome is used for preparation of the artificially fermented Chinese fermentation starter, and is used for production of traditional fermented food, such that the problem of unstable batch-to-batch quality control of the fermented food caused by the traditional Chinese fermentation starter can be effectively solved, and the core microbiome has important significance for realizing controllable production of the fermented food.

Description

A core microbial group of yeast for wine production and its construction method and application Technical Field

[0001] The invention relates to a yeast-making core microbial group and a construction method and application thereof, and belongs to the field of traditional brewing food microorganisms. Background Art

[0002] Wine yeast, also known as Xiaoqu, Baiyao, or Jiubing, is a saccharifying and fermenting agent unique to southern my country. It is typically used as a starter in rice wine production in traditional artisanal rice wine. As the saying goes, "Good wine requires good koji." Wine yeast not only provides a microbial ecosystem composed of diverse microorganisms and hydrolytic enzymes for rice wine fermentation, but also provides the substrate for the formation of the rice wine flavor metabolic network. However, current wine yeast production is still based on inoculation of mother koji and natural fermentation. This means that the yeast produced in a given year is sealed in a wine jar, with some used as a saccharifying and fermenting agent for winter wine production that year and some retained the following summer as the mother koji for a new batch of yeast. This traditional, handcrafted production method, based on experience and inheritance, not only has low production capacity but also relies heavily on the quality of the previous year's yeast. This makes it difficult to ensure a stable microbial community and standardized yeast quality during production, hindering the rice wine industry's transition toward mechanization, modernization, and standardization.

[0003] Achieving control over microbial function, production processes, and product quality within traditional fermentation ecosystems is a common challenge facing the traditional fermented food industry. Currently, two main approaches are being adopted to improve the quality stability of traditional yeast for fermentation: 1. Fermentation process optimization. Solid-state fermentation is susceptible to parameters such as temperature and humidity, oxygen content, fermentation time, and stacking thickness, and the development of modern intelligent manufacturing equipment and procedures is still in its infancy. 2. Functional microbial selection and enhanced inoculation. The functions of individual microorganisms are clear but limited, overlooking the role of microbial interactions in stabilizing the dynamic succession and metabolic processes of complex communities.

[0004] In reality, only a limited number of microorganisms in natural microbiota can drive the fermentation process. Uncovering the composition of these core microorganisms and constructing synthetic microbial communities can help simplify the fermentation system and improve fermentation controllability. This has been demonstrated in fermented foods such as light-flavored baijiu (Chinese liquor), sake, vinegar, and cheese. While the physicochemical properties, metabolic characteristics, microbial community structure, and environmental drivers of community succession in yeast (Jiuyao)—a solid-state fermentation system composed of multiple microorganisms and environmental variables—have been well characterized, the composition, identification, construction methods, and application effects of the yeast core microbiome remain unclear, hindering the transition from natural to artificial fermentation in yeast production. Therefore, comprehensively identifying core microorganisms from both taxonomic and functional perspectives is crucial for rationally constructing multi-species fermentation cultures for yeast and ensuring the stability of the yeast fermentation process and product quality.

[0005] Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of existing rice wine yeast production processes and provide a core microbial community for rice wine yeast and a method for constructing the same. The core microbial community construction method provided by the present invention is based on third-generation sequencing technology. The composition of the core microbial community is comprehensively determined by comprehensively analyzing the distribution frequency, relative abundance, co-occurrence, and contribution of microorganisms to rice wine yeast hydrolase activity. A synthetic microbial community is then constructed for use in rice wine yeast, rice wine yeast, and handmade rice wine production. This method ensures the saccharification and fermentation functions of the rice wine yeast without adversely affecting the rice wine flavor.

[0007] The present invention provides a microbial group comprising the strains shown in (a) to (e):

[0008] (a) Pediococcus pentosaceus, wherein the Pediococcus pentosaceus X14 was deposited in the China Center for Type Culture Collection on November 13, 2023, with a deposit number of CCTCC NO: M20232209;

[0009] (b) Saccharomycopsis fibuligera, wherein the Saccharomycopsis fibuligera CY2111 was deposited in the China Center for Type Culture Collection on August 15, 2022, with the deposit number CCTCC NO: M20221276;

[0010] (c) Saccharomyces cerevisiae, wherein Saccharomyces cerevisiae 23-1-35 was deposited in the China Center for Type Culture Collection on October 9, 2023, with the deposit number CCTCC NO: M20231847;

[0011] (d) Mucor indicus, wherein the Mucor indicus SYH-1 was deposited in the China Center for Type Culture Collection on September 26, 2023, with the deposit number CCTCC NO: M20231800;

[0012] (e) Rhizopus microsporus enzyme, the microsporus enzyme SM4 has been deposited in the China Center for Type Culture Collection on November 13, 2023, with a deposit number of CCTCC NO: M20232210.

[0013] The present invention also provides a microbial composition, comprising: Pediococcus pentosaceus, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Mucor indica and microsporin, and the order of magnitude ratio of each strain is 10000:100:10:1:10.

[0014] The present invention also provides a method for determining and constructing the core microbial group of rice wine yeast, which specifically comprises the following steps:

[0015] S1 Analysis of microbial community structure: Yeast samples produced in the same factory were collected for four consecutive years, and the genomic DNA of each sample was extracted. PCR amplification and high-throughput amplicon sequencing were performed on bacteria and fungi respectively. The sequencing sequences were clustered and species annotated, and microorganisms and pathogens with an occurrence frequency of less than 50% and an average relative abundance of less than 1% were excluded; the pathogens included but were not limited to Cronobacter, Enterobacter, and Klebsiella.

[0016] Analysis of the contribution of S2 microorganisms to yeast enzyme activity: Calculate the Spearman correlation between the relative abundance of microorganisms and the yeast saccharification power, liquefaction power, and acid protease activity, and exclude microorganisms whose relative abundance is negatively correlated with enzyme activity.

[0017] S3 Microbial co-occurrence network analysis: Calculate the Spearman correlation between the relative abundances of microorganisms, and evaluate the role of the species it represents in maintaining microbial interactions and community structure stability based on the connectivity, closeness centrality, and betweenness centrality of the nodes in the co-occurrence network.

[0018] S4 Determination of core microorganisms: The potential core microbial group of yeast yeast is determined based on the distribution of microorganisms in different samples, their contribution to yeast yeast enzyme activity, and their role in the construction and stabilization of microbial communities.

[0019] S5 core microbiome construction: The theoretical inoculum size of each core microorganism was determined based on the biomass and relative abundance obtained by RT-qPCR. The bacterial suspension / spore liquid of the core microorganisms were mixed according to the volume ratio to obtain a synthetic microbial community for artificial yeast fermentation.

[0020] The present invention also provides application of the microbial group in yeast production.

[0021] In one embodiment, the application is to add the microbial group to the raw materials for preparing yeast for wine.

[0022] In one embodiment, the application is to replace the mother yeast microorganism of traditional wine yeast with the microbial group.

[0023] In one embodiment, the inoculation method of the microorganisms includes but is not limited to inoculating a mixed bacterial solution, or inoculating the bacterial solution of each microorganism after pure culture separately; there is no obvious inhibitory effect when the strains of the synthetic microbial community are mixed and inoculated, and the original physiological activity can be maintained under 20% to 40% moisture conditions.

[0024] In one embodiment, the inoculation amount of Pediococcus pentosaceus X14 is 1.0×10 9-9.9×10 9 CFU / kg indica rice flour; Optionally, the method is to inoculate the Pediococcus pentosaceus X14 into MRS liquid culture medium, culture at 37°C, 150r / min shaking for 24h, take the bacterial solution, centrifuge, wash, resuspend, and actually dilute to 2.65×10 9 CFU / mL, the inoculation volume was 1000 μL / kg indica rice flour.

[0025] In one embodiment, the theoretical inoculation amount of the capsule-coated yeast CY2111 is 1.0×10 8 -9.9×10 8 CFU / kg indica rice flour; Optionally, the method is to inoculate the capsule-coated yeast CY2111 into YPD liquid culture medium, culture at 28°C, 150r / min shaking for 24h, take the bacterial solution, centrifuge, wash, resuspend, and actually dilute to 9.70×10 8 CFU / mL, the inoculation volume was 1000 μL / kg indica rice flour.

[0026] In one embodiment, the theoretical inoculation amount of the cerevisiae 23-1-35 is 1.0×10 5 -9.9×10 5 CFU / kg indica rice flour; Optionally, the method is to inoculate the brewer's yeast 23-1-35 into YPD liquid culture medium, culture at 28°C, 150r / min shaking for 24h, take the bacterial solution, centrifuge, wash, resuspend, and actually dilute to 9.70×10 8 CFU / mL, and the inoculation volume was 1 μL / kg indica rice flour.

[0027] In one embodiment, the theoretical inoculum size of the Mucor indica SYH-1 is 1.0×10 4 -9.9×10 4 CFU / kg indica rice flour; Optionally, the method is to inoculate the Indian mold SYH into PDA solid culture medium, culture it at 28 ° C for 72 hours, scrape the spores, centrifuge, wash, and resuspend them, and then dilute them to 4.45×10 6 CFU / mL, and the inoculation volume was 49 μL / kg indica rice flour.

[0028] In one implementation method, the theoretical inoculation amount of the microsporogenase SM4 is 1.0×10 6 -9.9×10 6 CFU / kg indica rice flour; Optionally, the method is to inoculate the Indian mold SYH into PDA solid culture medium, culture it at 28 ° C for 72 hours, scrape the spores, centrifuge, wash, and resuspend them, and then dilute them to 4.45×10 6CFU / mL, the inoculation volume was 1000 μL / kg indica rice flour.

[0029] In one embodiment, 3.05 mL of a mixed bacterial solution containing the microbial group is inoculated per 1 kg of indica rice flour.

[0030] In one embodiment, the order of magnitude ratio of Pediococcus pentosaceus X14, Saccharomyces cerevisiae CY2111, Saccharomyces cerevisiae 23-1-35, Mucor indica SYH-1, and Microsporum root enzyme SM4 in the mixed bacterial solution is 10 5 :10 3 :10:1:100.

[0031] The present invention provides a microbial preparation containing the core microbial group.

[0032] In one embodiment, in the microbial preparation, the order of magnitude ratio of the Pediococcus pentosaceus X14, Saccharomyces cytogenes CY2111, Saccharomyces cerevisiae 23-1-35, Mucor indica SYH-1, and Microsporum root enzyme SM4 is 10 5 :10 3 :10:1:100.

[0033] In one embodiment, the microbial preparation is a liquid preparation, including but not limited to a mixed bacterial suspension or a spore suspension.

[0034] In one embodiment, the microbial preparation is a solid preparation, including but not limited to a live bacterial preparation prepared by using a porous substance as an adsorbent to adsorb bacterial suspension and spore suspension.

[0035] The present invention also provides the application of the core microbial group in stabilizing the yeast fermentation process and product quality.

[0036] The present invention also provides a method for preparing artificially fermented yeast for wine making, wherein the production of the artificially fermented yeast for wine making is carried out during the dog days of summer, and specifically comprises the following steps:

[0037] S1: Mix 1 kg of indica rice flour, 8.25 g of Polygonum hydropiper powder, and 555 mL of water.

[0038] S2 Pile Dust: Beat with a mallet until the mixture forms larger lumps;

[0039] S3 Shaping: Flatten the surface of the mixture, divide it into cubes with a side length of about 2 cm, and roll them into balls to make yeast blocks;

[0040] S4 inoculation: transfer the yeast block prepared in step S3 to a bamboo basket and spray 3.05 mL of the microbial preparation to evenly distribute it on the surface of the yeast block;

[0041] S5 fermentation: The first fermentation is carried out in a closed vat. The cover is opened for cooling and ventilation in the later stage depending on the fermentation situation. This process lasts for 25-35 hours. The second fermentation is carried out in a closed workshop. During this period, the medicine is regularly turned over and the doors and windows are opened for cooling and ventilation depending on the fermentation situation. This process lasts for about 15-18 hours.

[0042] S6 Shade drying: Dry the yeast in the sun for a while every morning, and dry it in the shade in a ventilated room for the rest of the day; the whole process lasts for about 3-4 days until the moisture content of the yeast is lower than 13%.

[0043] In one embodiment, the early morning is 6:00-9:00 am.

[0044] In one embodiment, the artificially fermented yeast is prepared in summer.

[0045] The present invention also provides application of the artificial fermented yeast in traditional fermented foods.

[0046] In one implementation method, the traditional fermented foods include but are not limited to handmade yellow rice wine, cooking wine, shochu, sweet fermented glutinous rice, millet wine, vinegar, etc. Beneficial effects

[0047] 1. The present invention establishes a method for determining the core microorganisms in a complex fermentation system from the system biology level. Starting from the taxonomic and functional perspectives, the core microbial composition of yeast was comprehensively determined, and a synthetic microbial community was constructed for yeast fermentation. Compared with traditional yeast, there were no significant differences in the biomass of bacteria and fungi during the fermentation process, the composition and relative abundance of the core microbial community, the physical and chemical indicators of yeast, and the activity of key enzymes, thus realizing the rational construction of a multi-strain fermentation agent for yeast. The method of the present invention provides a reference for achieving the controllable production of traditional fermented foods and has great scientific significance and application value.

[0048] 2. The present invention uses artificial fermentation yeast instead of traditional yeast to ferment rice wine starter and handmade yellow rice wine in sequence. The fermentation performance of the control group (traditional yeast) and the experimental group (artificial fermentation yeast) of rice wine starter is basically the same, and after the fermentation, there is no significant difference in the alcohol content, reducing sugar, acidity, and ammonia nitrogen between the two groups, and both meet the empirical standards for rice wine starter; after the fermentation, the two groups of yellow rice wine have no significant difference in basic physical and chemical indicators, flavor substances such as organic acids and amino acids, and the contents of volatile flavor substances such as alcohols, esters, and aldehydes, and both meet GB / T 17946-2008, among which the content of higher alcohols is reduced by 9.64%, and the content of esters is increased by 19.42%, indicating that the artificial starter constructed based on core microorganisms can effectively reproduce the flavor characteristics of handmade yellow rice wine and improve the comfort of yellow rice wine after drinking.

[0049] 3. The artificially fermented yeast provided by the present invention can also be applied to the production of cooking wine, shochu, sweet fermented glutinous rice, millet wine, vinegar, etc., to stabilize the production process, reduce the quality difference between different batches, and improve product quality.

[0050] Biomaterial Deposit

[0051] Pediococcus pentosaceus X14, classified as Pediococcus pentosaceus X14, was deposited in the China Center for Type Culture Collection on November 13, 2023, with the deposit number CCTCC NO: M 20232209, and the deposit address is Wuhan University, Wuhan, China.

[0052] Saccharomycopsis fibuligera CY2111, classified and named Saccharomycopsis fibuligera CY2111, was deposited in the China Center for Type Culture Collection on August 15, 2022, with the deposit number CCTCC NO: M 20221276, and the deposit address is Wuhan University, Wuhan, China.

[0053] Saccharomyces cerevisiae 23-1-35, classified as Saccharomyces cerevisiae 23-1-35, was deposited in the China Center for Type Culture Collection on October 9, 2023, with the deposit number CCTCC NO: M 20231847, and the deposit address is Wuhan University, Wuhan, China.

[0054] Mucor indicus SYH-1, classified as Mucor indicus SYH-1, was deposited in the China Center for Type Culture Collection on September 26, 2023, with the deposit number CCTCC NO: M20231800, and the deposit address is Wuhan University, Wuhan, China.

[0055] Rhizopus microsporus SM4, classified and named as Rhizopus microsporus SM4, was deposited in the China Center for Type Culture Collection on November 13, 2023, with the deposit number CCTCC NO: M20232210, and the deposit address is Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows the distribution of microorganisms in traditional wine yeast. (a) Bacterial and (b) fungal community structure; (c) bacterial and (d) fungal distribution in the sample.

[0057] Figure 2 is a heat map showing the correlation between the relative abundance of microorganisms and the saccharification power, liquefaction power, and acid protease activity of yeast.

[0058] Figure 3 shows the co-occurrence network based on the Spearman correlation of the relative abundance of microorganisms.

[0059] Figure 4 shows the comparison of temperature and humidity changes during yeast fermentation in (a) the control group and (b) the experimental group.

[0060] Figure 5 shows the comparison of microbial diversity during yeast production between the control and experimental groups. (a) Bacterial and (b) fungal community structure; (c) bacterial and (d) fungal biomass.

[0061] Figure 6 shows the changes in alcohol content, reducing sugar, acidity and ammonia nitrogen content during the fermentation process.

[0062] Figure 7 shows the results of OPLS-DA prediction of sample grouping information and screening of differential volatile flavor substances between groups. DETAILED DESCRIPTION

[0063] The alcohol content, total acidity, and ammonia nitrogen content were determined in accordance with GB / T 13662-2018 Yellow Wine, and the reducing sugar content was determined using the DNS colorimetric method.

[0064] The saccharification capacity, liquefaction capacity, acid protease activity and fermentation capacity tests are all based on GB 1886.174-2016 "National Food Safety Standard Food Additives Enzyme Preparations for Food Industry".

[0065] Organic acids and amino acids were determined by high performance liquid chromatography (HPLC).

[0066] Volatile flavor compounds were determined by headspace solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS).

[0067] Example 1 Determination of the core microbial group of rice wine yeast

[0068] (1) Analysis of the microbial community structure of traditional wine yeast

[0069] Normally fermented yeast samples were selected from the factory in 2019, 2020, 2021, and 2022, and total DNA was extracted using the Power Soil DNA kit. For bacterial communities, the 16S rDNA gene was amplified using universal primers 27F (5'-AGRGTTYGATYMTGGCTCAG-3') and 1492R (5'-RGYTACCTTGTTACGACTT-3'). For fungal communities, ITS sequences were amplified using ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and ITS4R (5'-TCCTCCGCTTATTGATATGC-3'). Amplicon sequencing was performed on the PacBio Sequel platform.

[0070] QIIME2 was used for bioinformatics processing of amplicon sequencing data. After quality filtering, denoising, merging, and dechimerization, bacterial sequences were clustered into operational taxonomic units (OTUs) using UCLUST with a similarity threshold of 0.97. Fungal sequences were semi-parametrically clustered into amplicon sequence variants (ASVs) using the DADA2 plugin. Representative sequences were then annotated to the species level using the Basic Query Tool for Local Similarity (BLAST) with a confidence threshold of 0.9, referencing the Silva bacterial database and the Unite fungal database.

[0071] Prior to visualization, microorganisms that appeared in less than 50% of the samples and had an average relative abundance of less than 1% were first merged into "others." As shown in Figure 1, Pediococcus pentosaceus was present in all 12 samples and was dominant in the 2021 samples. Regarding fungi, Saccharomyces cerevisiae was present in every sample and was dominant. Furthermore, although Saccharomyces cerevisiae, Mucor indica, Microsporum rhizogenes, and Rhizogenes pauciradiata had relatively low relative abundances, they were present in more than 10 / 12 samples. Yeasts generally considered to produce flavor, such as Issachus orientalis and Wickham's yeast, showed significant differences in their distribution between samples from different years. From a taxonomic perspective, we primarily considered distribution frequency rather than relative abundance, as some microorganisms with lower abundances can also be extremely critical.

[0072] (2) Analysis of the contribution of microorganisms to yeast enzyme activity

[0073] During the fermentation process of yeast, microbial metabolism produces a variety of functional enzymes, which affects the quality of yeast. Table 1 shows that the enzyme activity of yeast in different years varies greatly, and the enzyme activity of yeast in 2020 and 2021 is generally low. The spearman correlation between the relative abundance of microorganisms and enzyme activity is shown in Figure 2. The main ones that are significantly positively correlated with enzyme activity are the capsule-coated yeast and the Enterobacteriaceae microorganisms represented by Cronobacter and Enterobacter. Considering that Enterobacter is a pathogenic bacteria and is basically only detected in samples from 2022, it is not included in the core microbiome. In addition, Pediococcus pentosaceus, Saccharomyces cerevisiae, Mucor indica, and microsporin enzymes are positively correlated with saccharification power and liquefaction power, while oligosporin enzymes are negatively correlated with the activities of various enzymes, but the correlations are not very high.

[0074] Table 1 Enzyme activity of traditional yeast

[0075] (3) Microbial co-occurrence network analysis

[0076] As shown in Figure 3, the microbial co-occurrence network based on the spearman correlation coefficient (r>0.5, p<0.05) includes 34 valid nodes and 288 edges, which can reveal potential microbial interspecies interactions. The key nodes often represent key species that may play an important role in maintaining the stability of the microbial community structure. In the present invention, we use Gephi software to calculate the weighted connectivity, closeness centrality and betweenness centrality of the nodes, and use this as a basis to identify potential core microorganisms. In particular, weighted connectivity is defined as the sum of the weights of all edges connected to a node (i.e., the absolute value of the spearman correlation coefficient); closeness centrality is defined as the inverse of the sum of the distances from a node to all other nodes; betweenness centrality measures the extent to which a node is between other node pairs. As shown in Table 2, the closeness centrality of Saccharomyces cerevisiae is as high as 0.70, indicating that it is close to other nodes and is at the center of the network. The betweenness centrality of Trichoderma indica is as high as 200.50, indicating that it plays a key role in the "communication" of other nodes. It is worth noting that nodes with high betweenness centrality do not necessarily have high connectivity.

[0077] Table 2 Top 10 microorganisms ranked by the sum of the three parameters

[0078] Based on the distribution of microorganisms in different samples, their contribution to yeast enzyme activity, and their stabilizing effects on the microbial community, Pediococcus pentosaceus, Saccharomyces cerevisiae, Mucor indicus, and Microsporum spp. were ultimately identified as the potential core microbiome of yeast. Pediococcus pentosaceus, as a lactic acid bacterium, provides an acidic environment for yeast fermentation and promotes the formation of flavor compounds. Saccharomyces cerevisiae is responsible for ethanol production. Saccharomyces cerevisiae and filamentous fungi secrete enzymes to provide small molecule substrates for the growth of other microorganisms. Overall, these five microorganisms, each with their own unique role, are essential for the formation of the yeast microbial ecosystem.

[0079] Example 2: Construction of the core microbial community of rice wine yeast

[0080] (1) Analysis of microbial biomass of traditional yeast

[0081] RT-qPCR reactions were performed using a series of single-copy plasmid standards containing the target fragment at known concentrations as templates. After the reaction, a standard curve was plotted with the cycle threshold value (Ct) as the horizontal axis and the logarithm of the standard concentration as the vertical axis. The concentration range of the bacterial standard was 3.76×10 2 -6.34×10 6 copies / μL, the standard curve is y=-0.302x+10.573, R 2 =0.9977, the amplification efficiency is 100.45%; the concentration range of fungal standards is 1.54×10 3 -9.06×10 6 copies / μL, the standard curve is y=-0.3014x+10.697, R 2 =0.9986, and the amplification efficiency was 100.17%. It can be seen that the linear fit of the bacterial and fungal standard curves is good, and the amplification efficiency is in the range of 90%-110%, which can be used for the quantification of bacterial and fungal biomass in wine yeast.

[0082] Total DNA from the 2022 traditional liquor yeast was extracted using a modified CTAB method. OD260 / OD280 and OD230 / OD260 were measured to assess DNA quality and concentration. The bacterial 16S rRNA gene was amplified using upstream primer P1 (5'-CCTACGGGAGGCAGCAG-3') and downstream primer P2 (5'-ATTACCGCGGCTGCTGG-3'). The fungal 18S rRNA gene was amplified using upstream primer Y1 (5'-GCGGTAATTCCAGCTCCAATA-3') and downstream primer Y2 (5'-GCCACAAGGACTCAAGGTTAG-3'). The bacterial and fungal biomasses in the 2022 liquor yeast were calculated based on the standard curve to be 5.62 × 10 8and 3.61×10 7 CFU / g.

[0083] (2) Rational construction of the core microbiome

[0084] The production of traditional yeast is usually to use the yeast produced in the previous year as the mother yeast. Specifically, the microorganisms in the yeast produced in 2022 are inoculated onto the surface of the yeast produced in 2023. In order to maximize the reproduction of the microorganisms and functions of artificial fermentation yeast, in this invention, we determined the theoretical inoculation amount of each core microorganism in the synthetic microbial community based on the biomass and relative abundance of microorganisms in the yeast produced in 2022. Taking Pediococcus pentosaceus as an example, its theoretical inoculation amount (2.65×10 9 CFU / kg indica rice flour) = bacterial biomass (5.62×10 8 CFU / g)×mother koji inoculum amount (28 g / kg indica rice flour)×average relative abundance (16.83%).

[0085] First, mix 5g of wine medicine powder with 50mL of sterile saline, shake well to make the stock solution, and then dilute the stock solution in a gradient manner. Take 10 -3 -10 -5 100 μL of the dilution was spread onto MRS, YPD, and PDA plates. The MRS plates were incubated at 37°C for 48 hours, and the YPD and PDA plates were incubated at 28°C for 48 hours. Single colonies were isolated and purified, and DNA was extracted using the SDS-CTAB method. After amplification, bacteria were identified using 16sDNA, and fungi were identified using ITS. Five core microbial strains with outstanding overall performance were selected based on their acid production, amylase production, and acid protease production. Among them, Pediococcus pentosaceus X14 had average acid production, while Saccharomyces cyperus CY2111 and Microsporum rhizogenes SM4 had the highest saccharifying and liquefying enzyme activities. Mucor indica SYH-1 had the highest amylase activity, and Saccharomyces cerevisiae 23-1-35 had the lowest alcohol-to-ester ratio per unit alcohol content.

[0086] The core microorganisms were inoculated at the same order of magnitude as the theoretical inoculum volume. The Pediococcus pentosaceus culture solution was expanded in MRS liquid medium and diluted to 2.65×10 9 Similarly, the bacterial suspensions of the five core microorganisms were diluted to the concentrations shown in Table 3, and then further mixed according to the volume ratios shown in Table 3 to obtain a synthetic microbial community for artificial fermentation yeast production. That is, for 1 kg of indica rice flour raw material, a total of 3.05 mL of bacterial suspension was required to be sprayed.

[0087] Table 3 Specific composition of the core microbiome

[0088] Example 3: Preparation method of artificial fermented wine yeast

[0089] 1. Preparation of core microbial group bacterial solution: Prepare 3.05 mL of mixed bacterial solution according to the rational construction of the core microbial group in Example 2 (2).

[0090] 2. The core microbial group bacterial liquid is used to prepare artificial fermentation wine yeast, the specific steps are as follows:

[0091] S1: Mix 1 kg of indica rice flour, 8.25 g of Polygonum hydropiper powder, and 555 mL of water.

[0092] S2 Pile powder: Beat the mixture of S1 with a mallet to make the mixture into larger lumps.

[0093] S3 Shaping: Flatten the surface of the block mixture prepared in S2, divide it into cubes with a side length of about 2 cm, and roll them into balls.

[0094] S4 inoculation: Transfer the yeast blocks shaped in S3 to a bamboo basket and spray 3.05mL of bacterial solution to evenly distribute it on the surface of the new yeast.

[0095] S5 Fermentation: The yeast block inoculated in S4 is fermented. The first fermentation is carried out in a sealed vat. The lid is opened for cooling and ventilation depending on the fermentation progress. This process lasts 25-35 hours. The second fermentation is carried out in a sealed workshop. The yeast is regularly turned over and doors and windows are opened for cooling and ventilation depending on the fermentation progress. This process lasts about 15-18 hours. The temperature is controlled below 38°C throughout the process.

[0096] S6 Shade Drying: Place the yeast fermented in S5 on a straw mat to dry in the sun; every morning (6:00-9:00 am) transfer the yeast to the sun for drying, and the rest of the time dry it in the shade in a ventilated workshop; the whole process lasts for about 3-4 days until the moisture content of the yeast is lower than 13%.

[0097] Example 4: Application of synthetic core microbial communities in artificial fermentation of wine yeast production

[0098] Artificial fermentation yeast was prepared according to the method of Example 3, and relevant indicators were tested.

[0099] (1) Temperature and humidity monitoring during the main fermentation process of artificial fermented yeast

[0100] Considering the characteristics of natural fermentation of yeast, its quality is inevitably affected by environmental factors. Furthermore, the microenvironment is also affected by fermentation feedback. Therefore, we used an electronic thermo-hygrometer to monitor the changes in microenvironmental temperature and humidity during the main fermentation process (step S5, pre-fermentation and post-fermentation). Figure 4(a) shows that the temperature and humidity during both the pre-fermentation and post-fermentation periods initially increase and then decrease. Pre-fermentation takes place in a vat, which is covered and wrapped in sacks to maintain heat and moisture. This allows filamentous fungi to rapidly grow and occupy a dominant ecological niche. The ambient temperature and humidity reach their first peak (34.7°C, 93.6%) at the 23rd hour. Subsequently, the sacks and vat lids are removed, and the temperature and humidity subsequently decrease. Returning to room temperature marks the end of pre-fermentation. The yeast is then transferred to a sealed workshop for post-fermentation. The vigorous bacterial growth and metabolism cause the temperature and humidity to reach their second peak (38.1°C, 84.5%) at the 35th hour. During this period, the yeast is turned over every two hours to ensure the temperature does not exceed 38°C. Near the end of post-fermentation, doors and windows are opened for ventilation and dehumidification.

[0101] (2) Analysis of microbial diversity in the production process of artificial fermentation yeast

[0102] Although artificially fermented yeast is inoculated with specific microorganisms, its microbial community structure is inevitably affected by internal and external environmental factors, such as workshop temperature and humidity, bioheat, and microbial interactions. Similar to traditional yeast, the production of artificially fermented yeast can also be divided into three stages: pre-fermentation, post-fermentation, and shade drying. Since the colonization and succession of microorganisms in yeast is a relatively long process, we used third-generation amplicon sequencing technology and RT-qPCR to analyze the community structure and biomass of artificially fermented yeast microorganisms at the beginning of pre-fermentation (entry into the tank), the end of pre-fermentation (pre-fermentation), the end of post-fermentation (post-fermentation), and after the completion of shade drying (shade drying). The results are shown in Figure 5.

[0103] The initial bacterial community of artificially fermented yeast is dominated by Pediococcus pentosaceus, with some Enterobacteriaceae present at very low abundance. As fermentation progresses, bacterial species diversity increases significantly due to colonization by environmental microorganisms. At the end of post-fermentation, Weissella sinusoidalis, a probiotic lactic acid bacterium widely found in daqu, kimchi, sourdough, and fermented meat products, was detected in samples, likely originating from the post-fermentation workshop. During the shade-drying period, the bacterial community composition stabilized, with only minor fluctuations in relative abundance. Throughout the production process, the fungal community of artificially fermented yeast was dominated by Saccharomyces cerevisiae, Saccharomyces cerevisiae, Mucor indica, Microsporum spp., and Rhizobacter spp., with Saccharomyces cerevisiae consistently dominating. Other microorganisms, such as Candida tropicalis and Fusarium, were present at very low relative abundance at the beginning of fermentation, but as fermentation progressed, they were eliminated by the yeast's rich internal environment of ethanol, organic acids, sesquiterpenes, and flavonoids. It is worth noting that we did not artificially inoculate Rhizopus oligomyces, but it still entered from the workshop environment and participated in the entire production process of yeast and the succession of microbial communities, indicating that in actual open fermentation, the synthetic core microbial community is also regulated by the environment, and some microorganisms from the environment improve the function of artificially fermented yeast and may shape the terroir characteristics of the product.

[0104] The changes in biomass indicate that the pre-fermentation and post-fermentation periods are the stages of large-scale microbial proliferation. During the shade-drying period, due to the decrease in the moisture content of the yeast, the activity of the microorganisms decreased and they were basically dead or dormant. After the production was completed, the bacterial and fungal biomass of the artificial fermented yeast were 4.72×10 9 CFU / g, 8.49×10 8 CFU / g.

[0105] (3) Analysis of physical and chemical indicators and enzyme activity after the completion of artificial fermentation yeast production

[0106] The physical and chemical properties of yeast are important indicators of its quality. Moisture content is determined by drying to constant weight. Bioheat generated by microbial metabolism promotes water evaporation, and the moisture content of artificially fermented yeast decreases continuously from approximately 40.42±1.33% at the beginning of fermentation to 19.46±1.36% at the end. After air-drying, the moisture content reaches 12.70±0.08%, at which point most microbial metabolism is inhibited, facilitating the long-term storage of the yeast. Acidity is determined by titration. The presence of acidic substances not only provides a microenvironment for yeast fermentation and inhibits the growth of other harmful microorganisms, but also enhances the flavor of rice wine during brewing. The warm and moist environment during the pre-fermentation phase promotes the growth and reproduction of acid-producing microorganisms, leading to the rapid accumulation of organic acids. During the post-fermentation phase and air-drying phase, the organic acid content decreases due to volatilization and esterification reactions, ultimately stabilizing at 9.90±1.82 g / kg.

[0107] Enzyme activity is the most intuitive indicator for evaluating the performance of yeast as a saccharifying and fermenting agent. Saccharifying enzymes, amylases, and liquefying enzymes are collectively referred to as starch hydrolases. Their synergistic effect is crucial for improving raw material utilization and wine yield. Saccharifying enzymes accumulate continuously throughout the production process, reaching a saccharifying enzyme activity of 343.28±32.27 U / g in artificially fermented yeast after air-drying. Meanwhile, accumulation of amylases and liquefying enzymes occurs primarily during the main fermentation phase, with some activity being lost during the air-drying phase. Acidic protease activity increases continuously during the main fermentation phase and remains at 234.58±28.08 U / g after air-drying. Its hydrolysis products, including amino acids and peptides, are essential nutrients for other microorganisms and also serve as precursors for flavor development in rice wine brewing. Fermentation capacity refers to the ability of yeast to ferment sugars to produce alcohol. The fermentation capacity of artificially fermented yeast increases rapidly during the pre-fermentation phase and then stabilizes between 3.2-3.5 U / g.

[0108] In general, all indicators of the artificial fermentation yeast based on core microbiome inoculation meet the factory's empirical standards and can theoretically be used as a saccharification and fermentation agent in handmade rice wine production.

[0109] Comparative Example 1: Mother koji used in traditional yeast production

[0110] (1) Temperature and humidity monitoring during the main fermentation process of traditional yeast

[0111] The specific implementation method is the same as Example 4, except that mother koji powder is used instead of synthetic microbial community inoculation; the mother koji powder is obtained from the wine yeast produced by Guyuelongshan in 2022, and contains the five core microorganisms in the embodiment (Pediococcus pentosaceus, Saccharomyces cerevisiae, Saccharomyces cerevisiae, Mucor indicus, Microsporum enzyme) and Enterobacter, Rhizobacter pauciphyllolyticus, etc.

[0112] The microenvironmental temperature and humidity trends for traditional and artificially fermented yeast are similar, with peaks in both the pre-fermentation and post-fermentation stages. As shown in Figure 4(b), the first peak occurs at the 17th hour (34.5°C, 92.7%), followed by a decrease in temperature and humidity due to ventilation. The second peak occurs at the 33rd hour (37.7°C, 84.9%), followed by a continuous decrease. However, the fermentation of artificially fermented yeast lags slightly behind that of traditional yeast, with the first peak occurring six hours later, but the time difference between the two peaks is smaller than that of traditional yeast.

[0113] (2) Analysis of microbial diversity in the traditional yeast production process

[0114] Similar to artificially fermented yeast, the initial bacterial community of traditional yeast is dominated by Pediococcus pentosaceus. As shown in Figure 5, as fermentation progresses, the relative abundance of Pediococcus pentosaceus decreases significantly, while the bacterial species diversity increases significantly. The fungal community is also composed of Saccharomyces cerevisiae, Saccharomyces cerevisiae, Mucor indica, Microsporin, and Rhizobacterium pauciflorum. Among them, Saccharomyces cerevisiae has always occupied an absolute dominant position throughout the production process of traditional yeast. After production is completed, the difference in the microbial community structure between traditional yeast and artificially fermented yeast is mainly reflected in the composition of non-core microorganisms such as Enterobacteriaceae, while the composition and relative abundance of the core microbial group are basically the same. This indicates from the perspective of microbiology that artificially fermented yeast can replace traditional yeast as a saccharification and fermentation agent.

[0115] The variation trend of biomass is basically the same as that of artificial fermentation yeast. The bacterial and fungal biomass of traditional yeast can reach 2.52×10 10 CFU / g, 5.41×10 9 CFU / g, although slightly higher than that of artificially fermented yeast, there is no statistically significant difference.

[0116] (3) Analysis of physical and chemical indicators and enzyme activity after traditional yeast production

[0117] The changing trends of various physical and chemical indicators and enzyme activities during the production of traditional yeast were consistent with those of artificially fermented yeast. After the drying process, except that the fermentation capacity of artificially fermented yeast was significantly higher than that of traditional yeast (p=0.047), there were no significant differences in the physical and chemical indicators and other enzyme activities between the two. This theoretically confirms that artificially fermented yeast can be used as a saccharification and fermentation agent in the production of handmade rice wine.

[0118] Table 4 Physical and chemical indicators and enzyme activities of artificially fermented yeast and traditional yeast

[0119] Example 5: Artificial fermentation yeast is used in the production of handmade yellow rice wine

[0120] (1) Production and physical and chemical index analysis of rice wine starter

[0121] The production of rice wine yeast using the artificial fermentation rice wine yeast prepared in Example 3 as a saccharification starter specifically includes the following steps:

[0122] S1 Rice Soaking: Soak 1 kg of glutinous rice in sufficient tap water at room temperature for 48 hours.

[0123] S2 Washing rice: After pouring out the soaking water, rinse with tap water until the discharged water is colorless.

[0124] S3 Steaming rice: Steam the drained rice for 40 minutes until the rice is cooked but not mushy, and there is no undercooked rice.

[0125] S4: Rinse the steamed rice directly with cold boiled water, quickly cool the rice to 30-35℃, and drain the water quickly.

[0126] S5: Place rice and 5g of wine powder in a beaker and mix well.

[0127] S6: Build a nest for saccharification: Pile the rice evenly around the rice, use a sterilized spoon to dig an inverted cone with a diameter of about 5cm at the bottom of the cone, and ferment it in a 28℃ incubator for 48h.

[0128] S7 flushing the cylinder: When the height of the nest liquid reaches about 4 / 5 of the height of the cone, add 1.1L of water and 153g of raw malt koji and stir evenly.

[0129] S8 fermentation: The temperature of the pre-fermentation is set at 28℃ and lasts for 3 days, during which the soil is stirred twice a day; the temperature of the post-fermentation is set at 15℃ and lasts for 12 days, during which the soil is stirred once every five days.

[0130] The microorganisms in rice wine starter primarily originate from the yeast, with smaller amounts derived from malt koji, raw materials, and the environment. The fermentation of rice wine starter is essentially a process of enriching functional microorganisms, particularly yeast, whose quality directly impacts the quality of handmade rice wine and reflects the quality of the yeast. As shown in Figure 6, the fermentation of rice wine starter can be divided into three stages: the nest saccharification phase, the pre-fermentation phase, and the post-fermentation phase. The nest saccharification phase, which occurs 48 hours after material placement, is a solid-state fermentation phase in which amylases and proteases introduced by the yeast play a major role. During this phase, the alcohol content, reducing sugars, total acidity, and ammoniacal nitrogen content of the nest solution all increase. Because starch hydrolysis far outstrips reducing sugar consumption, reducing sugars accumulate rapidly, reaching a peak of 106.71±10.55 g / L at the end of the nest saccharification phase. When the height of the broiler reaches 4 / 5, water and malt koji are added to flush the vat, and the fermentation is switched to semi-solid state. The yeast grows and reproduces in large quantities, consuming a large amount of reducing sugar, and the alcohol content rises rapidly. At the end of the pre-fermentation, the alcohol content of the rice wine starter in the experimental group reaches 16.33±1.47%vol. At the same time, the acidity and ammonia nitrogen drop significantly within one day after flushing the vat, and then gradually increase, which promotes the flavor formation of the rice wine starter and inhibits the growth of miscellaneous bacteria. The temperature of the post-fermentation is controlled at 15℃. During this stage, the microbial metabolism is weakened, and various indicators are basically stable, with a slight decrease in acidity.

[0131] (2) Analysis of physical and chemical indicators of handmade rice wine

[0132] The production of handmade yellow rice wine uses the experimental group rice wine starter prepared in part (1) of this embodiment as the starter, and specifically includes the following steps:

[0133] S1 Rice Soaking: Soak 1 kg of glutinous rice in sufficient tap water at room temperature for 48 hours.

[0134] S2 Steaming rice: Drain the soaking water and steam the rice for 40 minutes, until the rice is cooked but not mushy, and there is no undercooked rice.

[0135] S3: Add cooled cooked rice, 1L of water, 153g of raw malt koji, and 114g of rice wine starter into a beaker and mix well.

[0136] S4 fermentation: The temperature of the pre-fermentation was set at 28°C and lasted for 5 days, during which the soil was stirred twice a day; the temperature of the post-fermentation was set at 15°C and lasted for 15 days.

[0137] The conversion of fermentable sugars into alcohol is a key process in the production of all alcoholic beverages. Therefore, alcohol content is the most intuitive indicator of the fermentation process's effectiveness. After fermentation, the experimental group's handmade rice wine achieved an alcohol content of 15.97 ± 0.40% vol. Furthermore, reducing sugar, acidity, and ammoniacal nitrogen levels were at 10.36 ± 0.98, 7.00 ± 0.19, and 0.63 ± 0.03 g / L, respectively. All physical and chemical parameters met the requirements of GB / T 13662-2018.

[0138] (3) Flavor analysis of handmade rice wine

[0139] The flavor of rice wine mainly focuses on taste and smell. Organic acids and free amino acids are the main components of taste characteristics, while volatile components are important components of aroma characteristics.

[0140] Organic acids in rice wine are primarily derived from raw materials (soaked rice and koji), microbial metabolism, and the oxidation of alcohols and aldehydes. They play a crucial role in maintaining the sensory, nutritional, and safety properties of rice wine. In this study, we determined the composition and content of organic acids using HPLC. The HPLC conditions were: a Waters E2695 liquid chromatography system, an SB-C18 Analytical column (4.6 mm × 250 mm, 5 μm), a mobile phase of 0.02 mol / L Na₂HPO₄, pH 2.70, an injection volume of 10 μL, a flow rate of 0.8 mL / min, a column temperature of 30°C, and a UV detector at 210 nm. As shown in Table 5, lactic acid and acetic acid constituted the majority of the organic acids in the experimental group, accounting for 71.83% of the total organic acid content and playing a crucial buffering role in the wine's flavor. Pyruvate, on the other hand, is a precursor to various metabolites. Malic acid, citric acid, and succinic acid participate in the tricarboxylic acid cycle and therefore do not accumulate in significant amounts.

[0141] Furthermore, the free amino acids in rice wine are not only an important nitrogen source for brewing microorganisms but also key precursors for the synthesis of various flavor compounds. Rice wine has the highest amino acid content of any wine and boasts a rich variety. High-quality rice wine contains over 20 amino acids, including eight essential amino acids. We also determined its composition and content by HPLC. The specific HPLC conditions were: liquid phase system Agilent 1100, OPA and FMOC pre-column derivatization, gradient elution on the chromatographic column ODS HYPERSIL (4.6 mm × 250 mm, 5 μm), mobile phase A (6.5 g crystalline sodium acetate dissolved in 1 L water, 200 μL triethylamine added, pH adjusted to 7.20 with acetic acid, 5 mL tetrahydrofuran added), mobile phase B (6.5 g crystalline sodium acetate dissolved in 500 mL water, pH adjusted to 7.20 with acetic acid, 400 mL acetonitrile and 400 mL methanol added), injection volume 10 μL, flow rate 1.0 mL / min, column temperature 40°C, detector UV 338 nm and 262 nm (Pro, Hypro). The results are shown in Table 6. The total amino acid content of the handmade rice wine in the experimental group fluctuated between 3.58 and 4.49 g / L, and 17 amino acids were detected (the standard product only contained 17 amino acids, and the sample may contain more types of amino acids that were not quantified). The contents of umami, sweet, bitter, and sour amino acids were 0.57, 1.12, 1.95, and 0.02 g / L, respectively.

[0142] The volatile flavor substances of rice wine mainly include alcohols, aldehydes, acids, esters, phenols, etc. The present invention uses HS-SPME-GC / MS to detect 41 volatile flavor substances in the experimental group of rice wine samples, including 8 alcohols, 15 esters, 6 acids, 8 aldehydes and 4 phenols. The specific parameters are: internal standard 0.1018 mg / L 2-octanol, rotation speed 200 rpm, water bath adsorption at 50°C for 50 minutes, desorption at 250°C for 5 minutes; GC chromatographic column TG-WAXMS (30m×0.25mm, 0.25μm), GC inlet temperature 250°C, GC temperature program, 40°C hold for 3 minutes, heating at 6°C / min to 100°C, heating at 10°C / min to 230°C, and holding for 7 minutes. The GC carrier gas was high-purity helium (>99.999%), splitless, at a flow rate of 1.0 mL / min. The MS ionization mode was EI, the MS emission current was 50 μA, and the MS electron energy was 70 eV. The MS ion source temperature was 230°C, the MS transfer line temperature was 250°C, and the MS scan range was 33-400 amu. Table 7 shows that the total amounts of volatile flavor compounds for alcohols, esters, acids, aldehydes, and phenols fluctuated within the ranges of 326.18-490.68, 67.75-87.19, 4.52-5.18, 29.66-32.25, and 0.21-0.35 mg / L, respectively. Among them, phenylethyl alcohol, isopentanol, n-propanol, and isobutanol constitute the majority of the higher alcohols. While an appropriate amount of higher alcohols can impart a harmonious body and a mellow taste, an excessive amount can add off-flavors and reduce the aftertaste. On the other hand, ethyl ester compounds represented by ethyl acetate and ethyl lactate contribute a pleasant fruity aroma to rice wine.

[0143] In general, the flavor of handmade rice wine produced with artificial fermented yeast is overall harmonious and meets the quality standards of handmade rice wine.

[0144] Comparative Example 2: Traditional yeast used in handmade rice wine production

[0145] (1) Analysis of the physical and chemical indicators of rice wine starter

[0146] The specific implementation method is the same as Example 5, except that the traditional yeast in Comparative Example 1 is used instead of the artificial fermentation yeast as the saccharification and fermentation agent.

[0147] As shown in Figure 6, the changing trends of the physical and chemical indicators of the two groups of rice wine starters were the same. During the nesting and saccharification period, the alcohol content, reducing sugar, total acid and ammonia nitrogen contents of the rice wine starter nest liquid of the control group increased, and the reducing sugar content also reached the peak of the entire production process (112.91±4.03g / L) at 48h. Then the vat was flushed and entered the pre-fermentation period. The reducing sugar content dropped rapidly and the alcohol content rose rapidly. At the end of the pre-fermentation, the alcohol content of the control group reached 17.77±1.11%vol. In addition, the acidity and ammonia nitrogen dropped significantly within one day after flushing, and then gradually increased. All indicators were basically stable during the post-fermentation period.

[0148] In general, the fermentation performance of rice wine starter inoculated with traditional wine yeast and artificially fermented wine yeast is basically the same, and there is no significant difference in various indicators after the fermentation is completed, and all meet the empirical standards of rice wine starter. From the perspective of production practice, it is confirmed that the artificially fermented wine starter inoculated with the core microbiome is no different from the traditional wine starter.

[0149] (2) Analysis of physical and chemical indicators of handmade rice wine

[0150] After fermentation, the alcohol content of the control group's handmade rice wine reached 16.37 ± 0.51% vol, slightly higher than that of the experimental group. This may be due to the higher biomass of Saccharomyces cerevisiae in the traditional yeast. Furthermore, reducing sugar, acidity, and ammoniacal nitrogen levels were at 9.72 ± 0.32, 6.81 ± 0.46, and 0.60 ± 0.07 g / L, respectively. Overall, there were no statistically significant differences in the physical and chemical properties of the two groups of rice wine samples, and all met the requirements of GB / T 17946-2008.

[0151] (3) Flavor analysis of handmade rice wine

[0152] The organic acid composition and content of the control group's rice wine are shown in Table 5. Again, lactic acid and acetic acid constitute the majority of the organic acids, accounting for 70.14% of the total organic acid content. The total organic acid content of the control group was slightly higher than that of the experimental group, but the difference was not significant. The main difference was in the lactic acid and succinic acid content, which may have contributed to the slightly less mellow aftertaste of the experimental group's rice wine.

[0153] Table 5 Organic acid content of handmade rice wine (g / L)

[0154] The control group's handmade rice wine also contained 17 amino acids, with total amounts ranging from 3.62 to 4.11 g / L. Umami, sweetness, bitterness, and sourness were detected at 0.57, 1.24, 2.07, and 0.02 g / L, respectively. These concentrations were not significantly different from those in the experimental group. This suggests that the balance of bitterness, sweetness, sourness, astringency, and umami in the two groups of rice wines was essentially the same.

[0155] Table 6 Amino acid content of handmade yellow rice wine (g / L)

[0156] In the control group of rice wine samples, 41 volatile flavor compounds were also detected. The total amounts of volatile alcohols, esters, acids, aldehydes, and phenols fluctuated within the ranges of 441.65-514.97, 65.73-79.89, 4.99-5.50, 29.92-31.45, and 0.37-0.93 mg / L, respectively. Table 7 suggests that artificial fermentation yeast does not significantly affect the flavor of rice wine.

[0157] Furthermore, as shown in Figure 7, OPLS-DA (a supervised discriminant analysis statistical method) was used to predict sample grouping information and screen for differentially expressed volatile flavor compounds between groups. Both R2X and R2Y were greater than 0.5, indicating a good explanatory power of the established model. The lack of separation between the two sample groups on the x-axis in the score plot indicates that the volatile flavor profiles of the experimental and control handmade rice wines are difficult to distinguish. No differentially expressed metabolites were identified based on the criteria of VIP > 1 and P < 0.05, further confirming that the volatile flavor profiles of the two groups of rice wines are essentially identical.

[0158] Table 7 Volatile flavor compound content of handmade rice wine (μg / L)

[0159] Example 6: Artificial fermentation yeast is used in sweet wine production

[0160] The production of sweet fermented glutinous rice uses the artificial fermented yeast in Example 3 as a saccharification starter, and specifically includes the following steps:

[0161] S1 Rice Soaking: Soak 1 kg of glutinous rice in sufficient tap water at room temperature for 48 hours.

[0162] S2 Washing rice: After pouring out the soaking water, rinse with tap water until the discharged water is colorless.

[0163] S3 Steaming rice: Steam the drained rice for 40 minutes until the rice is cooked but not mushy, and there is no undercooked rice.

[0164] S4: Rinse the steamed rice directly with cold boiled water, quickly cool the rice to 30-35℃, and drain the water quickly.

[0165] S5: Place the cooked rice and 5 g of artificial fermented wine powder in a beaker and mix well.

[0166] S6: Build the nest and saccharify: Pile the rice evenly on all sides, use a sterilized spoon to dig an inverted cone, and ferment in a closed container at a constant temperature of 28℃ for 48-72h.

[0167] The resulting sweet fermented glutinous rice from different batches all had a uniform milky white color, intact grains, a moderately sweet and sour taste, and a soft mellow flavor. The alcohol content was 5.35±0.17%vol, and the reducing sugar, acidity, and ammoniacal nitrogen contents were 106.71±10.55, 7.73±0.46, and 0.50±0.04g / L, respectively. The quality of the rice wine was stable with minimal inter-batch variation.

[0168] Example 7: Artificial fermentation yeast is used in rice wine production

[0169] The production of rice wine uses the artificial fermentation yeast in Example 3 as a saccharification starter, and specifically comprises the following steps:

[0170] S1 Rice Soaking: Soak 1 kg of glutinous rice in sufficient tap water at room temperature for 48 hours.

[0171] S2 Washing rice: After pouring out the soaking water, rinse with tap water until the discharged water is colorless.

[0172] S3 Steaming rice: Steam the drained rice for 40 minutes until the rice is cooked but not mushy, and there is no undercooked rice.

[0173] S4: Rinse the steamed rice directly with cold boiled water, quickly cool the rice to 30-35℃, and drain the water quickly.

[0174] S5: Place the cooked rice and 5 g of artificial fermented wine powder in a beaker and mix well.

[0175] S6: Build a nest for saccharification: Pile the rice evenly around the surface, use a sterilized spoon to dig an inverted cone with a diameter of about 5 cm at the bottom of the cone, and ferment in a 28°C incubator for 48 hours.

[0176] S7 flushing: When the height of the nest liquid reaches about 4 / 5 of the cone height, add 1.1L of water and stir evenly.

[0177] S8 fermentation: The pre-fermentation temperature is set at 28°C and lasts for 4-6 days; the post-fermentation temperature is set at 15°C and lasts for 15-20 days; after the fermentation is completed, the wine is squeezed and filtered to obtain the rice wine.

[0178] The rice wine obtained from different batches had stable quality, with rich and pure rice and wine aroma, appropriate sourness and sweetness, soft and refreshing body, and harmonious flavor. The alcohol content was 7.58±0.41%vol, and the reducing sugar content, acidity, and ammonia nitrogen content were 5.62±0.73, 2.25±0.13, and 0.37±0.04g / L, respectively.

[0179] Example 8: Artificial fermentation yeast is used in the production of raw cooking wine

[0180] The production of the original brewing cooking wine is based on the handmade yellow rice wine prepared in Example 5, supplemented with 15% (v / w) of an extract of spice powders such as pepper, star anise, cumin, laurel, cloves, and cinnamon, and 4% (w / w) of salt; different batches of original brewing cooking wine obtained after pressing and decocting have stable quality, a uniform brown-red color, appropriate sour, sweet, salty and fresh flavors, a unique aroma of spices, a strong wine aroma, and harmonious components of the wine body.

[0181] Example 9: Application of artificial fermented yeast in the production of yellow rice wine distilled liquor

[0182] The production of yellow rice wine distilled liquor uses the handmade yellow rice wine prepared in Example 5 as the base wine, and adopts a pot distiller to carry out double batch intermittent distillation, wherein the first distillation is a crude distillation, which realizes the transformation of yellow rice wine (alcohol content 15.86-16.88% vol) into a crude distillate liquid (alcohol content 52% vol). The two batches of crude distillate are combined and poured into a rectifying pot. The second distillation is a rectification, and the volume of the distillate of the first 1% of the filling volume of the rectifying pot is the head of the liquor, and the volume to 40% vol of alcohol content is the heart of the liquor, and the alcohol content of 40% vol to 10% vol is the tail of the liquor, and the rest is waste liquid. The yellow rice wine distilled liquor only intercepts the heart of the liquor, and does not recover the distillate. Under relatively stable heating power and wine outlet temperature, the sensory quality and volatile flavor profile of the yellow rice wine distilled liquor obtained from different batches are basically stable, and the alcohol content is in the range of 71.50-73.00% vol.

[0183] Example 10: Application of Artificial Fermentation Wine Yeast in Millet Yellow Wine Production

[0184] Millet yellow wine was produced in the same manner as in Example 5, except that millet of the same mass was used instead of glutinous rice. The results showed that the millet yellow wine obtained from different batches had stable quality, with an alcohol content of 15.02 ± 0.26% vol, and reducing sugar content, acidity, and ammoniacal nitrogen content of 12.63 ± 0.95 g / L, 4.71 ± 0.33 g / L, and 0.75 ± 0.14 g / L, respectively.

[0185] Example 11: Application of Artificially Fermented Wine Yeast in Vinegar Production

[0186] S1 Fermentation preparation: select 10 kg of high-quality glutinous rice, soak the rice for 24 hours, then drain the rice until there is no white pulp, steam it, drain it and cool it to 25-30°C; before putting the glutinous rice into the vat, mix it with 0.15-0.2 kg of the artificial fermentation yeast prepared in Example 3, and saccharify it at low temperature for 72-96 hours.

[0187] S2 alcohol fermentation: After saccharification, add 3kg of water and 0.6kg of malt koji, and ferment at 28℃ for 144-168h to obtain mature mash.

[0188] S3: Put 15kg of bran into the fermentation tank and spread it flat. Put the fermented mash into the tank and stir it evenly. Take 0.5kg of rice husk and spread it evenly on the upper layer of the tank. Then take 0.5kg of fermented vinegar mash, stir it evenly, cover it with 0.5kg of rice husk, and spread it evenly to complete the mash making.

[0189] S4 acetic acid fermentation: stir the mash every 24 hours, add rice husks after each stir to keep it warm and moist. No more rice husks are added from the 11th day of fermentation. Stir the mash to cool the temperature. After 20 days, when the acidity no longer increases, add 0.4 kg of salt and seal it for 45 days.

[0190] S5 Vinegar Infusion and Decocting: The aged mash was subjected to a cyclic leaching method. Sugar was added to the resulting vinegar juice, and after clarification, the vinegar was decocted. After the product temperature dropped to 75-80°C, it was canned and sealed for storage. The total acid content of the resulting vinegar from different batches was 5.41±0.29g / 100mL, the amino acid nitrogen content was 1.08±0.13g / 100mL, and the soluble salt-free solids content was 1.35±0.29g / 100mL.

[0191] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A microbial group, characterized in that Contains the strains shown in (a) to (e): (a) Pediococcus pentosaceus X14, deposited in the China Center for Type Culture Collection on November 13, 2023, with the deposit number CCTCC NO: M 20232209; (b) Saccharomycopsis fibuligera CY2111, deposited in the China Center for Type Culture Collection on August 15, 2022, with the accession number CCTCC NO: M 20221276; (c) Saccharomyces cerevisiae 23-1-35, deposited in the China Center for Type Culture Collection on October 9, 2023, with the deposit number CCTCC NO: M 20231847; (d) Mucor indicus SYH-1, deposited in the China Center for Type Culture Collection on September 26, 2023, with the deposit number CCTCC NO: M 20231800; (e) Rhizopus microsporus SM4 was deposited in the China Center for Type Culture Collection on November 13, 2023, with the deposit number being CCTCC NO: M 20232210.

2. A composition comprising the microbial group according to claim 1.

3. The composition according to claim 2, characterized in that The composition is a microbial preparation.

4. The composition according to claim 2, characterized in that The order of magnitude ratio of the Pediococcus pentosaceus X14, Saccharomyces cytogenes CY2111, Saccharomyces cerevisiae 23-1-35, Mucor indica SYH-1, and Microsporum root enzyme SM4 is 10 5 :10 3 :10:1:

100.

5. The composition according to claim 3, characterized in that The microbial preparation is a liquid preparation, including but not limited to a mixed bacterial suspension or a spore suspension.

6. The composition according to claim 3, characterized in that The microbial preparation is a solid preparation, including but not limited to a live bacterial preparation prepared by using a porous substance as an adsorbent to adsorb bacterial suspension and spore suspension.

7. The composition according to any one of claims 3 to 6, characterized in that The viable bacterial counts of Pediococcus pentosaceus X14, Saccharomyces cytogenes CY2111, Saccharomyces cerevisiae 23-1-35, Mucor indica SYH-1 and Microsporum rhizogenes SM4 in the microbial preparations were 1.0×10 9 -9.9×10 9 , 1.0×10 8 -9.9×10 8 , 1.0×10 5 -9.9×10 5 , 1.0×10 5 -9.9×10 5 , 1.0×10 6 -9.9×10 6 CFU / mL or CFU / g range.

8. A method for preparing artificially fermented yeast for wine making, characterized in that: include: S1. Mixing flour: Evenly mix the indica rice flour, hot polygonum tiliaceum powder and appropriate amount of water; S2, pile powder: Beat with a mallet until the mixture prepared in S1 becomes larger lumps; S3, shaping: flatten the surface of the mixture prepared in S2, divide it into cubes, and roll it into balls; S4, inoculation: transfer the yeast block prepared in S3 to a bamboo basket, and spray the composition according to any one of claims 3 to 6 on the surface of the yeast block; S5, Fermentation: Ferment the yeast block inoculated in S4; the first fermentation is carried out in a sealed vat, and the cover is opened for cooling and ventilation depending on the fermentation situation. This process lasts for 25-35 hours; the second fermentation is carried out in a sealed workshop, during which the yeast is regularly turned and the doors and windows are opened for cooling and ventilation depending on the fermentation situation. This process lasts for 15-18 hours; the temperature is controlled at ≤38℃ throughout the process; S6. Dry in the shade: air it in the sun for a period of time every day, and dry it in the shade indoors for the rest of the time; the whole process lasts for 3-4 days until the moisture content of the yeast is lower than 13%.

9. Artificially fermented rice wine yeast prepared by the method according to claim 8.

10. A method for constructing a core microbial community of rice wine yeast, characterized in that: The following steps are involved: S1: Determine the composition of the core microbiome based on the microbial community structure in the mother song, the contribution of microorganisms to enzyme activity, and the microbial co-occurrence network; S2: Determine the theoretical inoculum size of each core microorganism; S3: After expansion, centrifugation, washing and resuspension, the bacterial suspension / spore liquid of each core microorganism is mixed according to the volume ratio to obtain a synthetic microbial community for artificial wine yeast fermentation.

11. The method for constructing a core microbial group according to claim 10, characterized in that: The core microorganisms are all separated and screened from traditional wine yeast.

12. Use of the microbial group according to claim 1 or the composition according to any one of claims 2 to 7 in stabilizing the yeast fermentation process and product quality.

13. Use of the artificially fermented yeast for wine making according to claim 9 in traditional fermented foods.

14. The use according to claim 13, characterized in that Traditional fermented foods include but are not limited to handmade rice wine, cooking wine, shochu, sweet rice wine, millet wine, and vinegar.

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