Application of retort bottom water in selective enrichment of saccharomycetes and / or maintenance of stability of microbial community structure in white spirit brewing

Abstract

The invention discloses application of retort bottom water in selective enrichment of saccharomycetes and / or maintenance of stability of a Baijiu brewing microbial community structure, and belongs to the technical field of microbial culture and Baijiu byproduct reutilization. The byproduct retort bottom water generated in the fermentation process of Maotai-flavor liquor is used as the culture medium to be applied to selective enrichment of saccharomycetes and / or maintenance of stability of a microbial community structure in liquor brewing, and the culture medium can more effectively screen out the saccharomycetes strain which is resistant to acid and hyperosmosis and adapts to the specific micro-ecology; the strain has good enrichment capacity on various saccharomycetes related to brewing, such as pichia pastoris, trichosporon, saccharomyces cerevisiae, Wickerhamomyces and saccharomyces cerevisiae; and non-main brewing microorganisms in the high-temperature yeast can be inhibited, and the community stability of the brewing microorganisms in the high-temperature yeast can be maintained. Therefore, the retort bottom water can be recycled, a more appropriate culture medium can be provided for microbiological research and culture of high-temperature yeast for making hard liquor, and the method has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial culture and recycling of by-products of baijiu (Chinese liquor), and more specifically, to the application of still water in the selective enrichment of yeast and / or maintenance of the stability of the microbial community structure in baijiu brewing. Background Technology

[0002] The wastewater generated during the brewing process of baijiu mainly includes still water, fermentation wastewater (also known as yellow water), cooling water, water used for cleaning the site, and water used for washing bottles. Sauce-flavor baijiu is distilled in seven different stages. Still water generally occurs in later stages and specifically refers to the high-concentration organic liquid dripping from the bottom of the still after the distillation process is complete. This liquid contains a large amount of acids, esters, and solids, as well as small amounts of aldehydes and alcohols, directly resulting in relatively high concentrations of COD, BOD, ammonia nitrogen, total phosphorus, and total nitrogen, far exceeding wastewater discharge standards.

[0003] In production, the water from the bottom of the steaming pot often drips onto the membrane and is collected, then discarded as solid waste, failing to be valued and effectively utilized, resulting in the waste of its valuable components. If this byproduct were to be utilized as a resource, its high COD value and complex composition would become major obstacles to its reuse. Therefore, the treatment and resource utilization of the water from the bottom of the steaming pot has become one of the industry's challenges.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide the application of still water in the selective enrichment of yeast and / or maintenance of the stability of the microbial community structure in baijiu brewing. The method provided by this invention can not only make use of still water as a resource, but also selectively enrich yeast in the baijiu fermentation process and maintain the stability of the microbial community structure in baijiu brewing through the culture medium prepared by still water.

[0006] This invention is implemented as follows: In a first aspect, the present invention provides the application of the bottom water of the still in the preparation of a functional culture medium, which includes a culture medium for selectively enriching and culturing yeasts in the fermentation of baijiu.

[0007] Secondly, the present invention provides a culture medium for selectively enriching yeast and / or maintaining the stability of the microbial community structure in Baijiu brewing, the main active ingredient of which is the bottom water from the fermentation process of Maotai-flavor Baijiu.

[0008] Thirdly, the present invention provides a method for selectively enriching yeast and / or maintaining the stability of the microbial community structure in Baijiu brewing using the bottom water of the still, comprising: inoculating the diluted Daqu solution into the above-mentioned culture medium for cultivation; the cultivation conditions are: 25~30℃, 160~200 rpm / min.

[0009] The present invention has the following beneficial effects: This invention utilizes the still water, a byproduct of the fermentation process of Maotai-flavor liquor, for selective enrichment of yeast and / or maintenance of the stability of the microbial community structure in liquor brewing. By processing and preparing a culture medium, this medium can more effectively screen for yeast strains that are acid-resistant, osmotic-pressure-resistant, and adapted to this specific microecology. It exhibits excellent enrichment capabilities for various brewing-related yeasts, including Pichia pastoris, Mycosaccharomyces cerevisiae, Saccharomyces cerevisiae, Wickham's yeast, and Saccharomyces cerevisiae. Furthermore, it can inhibit non-primary brewing microorganisms in high-temperature koji (fermentation starter culture) and maintain the community stability of brewing microorganisms in high-temperature koji. Therefore, this invention can utilize the still water as a resource and provide a more suitable culture medium for microbial research and cultivation in high-temperature koji, demonstrating promising application prospects. Attached Figure Description

[0010] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 The results of enrichment of bacterial community structure in high-temperature Daqu (a type of starter culture) in Example 5; Figure 2 Analysis of the enrichment results of different culture media on the bacterial community structure of high-temperature Daqu in Example 5; Figure 3 The number of culturable yeast cells in high-temperature Daqu enriched with different concentrations of still water in Example 6; Figure 4 The enrichment results of different concentrations of steaming bottom water on the high-temperature Daqu fungal community structure in Example 7; Figure 5 The enrichment results of Pichia pastoris under different culture conditions in Example 7; Figure 6 The enrichment results of *Saccharomyces cerevisiae* under different culture conditions in Example 7; Figure 7 The enrichment results of *Wickhamia spp.* under different culture conditions in Example 7; Figure 8 The enrichment results of *Mycosaccharomyces* genus under different culture conditions in Example 7; Figure 9 The growth status of the three yeast strains in Example 8 is shown in two solid culture media. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0013] In the research of microorganisms in the Baijiu brewing industry, the isolation, purification, and cultivation of yeasts in high-temperature Daqu (a type of starter culture) and fermented mash are crucial steps. Baijiu fermentation is a complex process involving a mixture of multiple microbial species. Isolation and purification can identify the core microorganisms that dominate saccharification, fermentation, and aroma production. The isolated pure strains can be used to study symbiotic, antagonistic, or synergistic relationships among microorganisms, revealing the microbiological basis of flavor compound formation. After clarifying the growth characteristics and metabolic patterns of key strains, their functions can be optimized by controlling process conditions (such as temperature, moisture, and acidity), improving fermentation controllability. Furthermore, strains with high enzyme activity (such as amylase and esterase), high-temperature resistance, and ethanol resistance can be isolated and screened for use in strengthening Daqu or artificial inoculation fermentation. Simultaneously, the cultured strains can serve as seed banks for designing and synthesizing microbial communities, promoting the upgrading of traditional brewing towards precision and standardization. In addition, the Baijiu brewing environment is an important source of extreme microorganisms (resistant to high temperatures, high acids, and high ethanol), potentially leading to the isolation of new strains with unique enzyme systems or metabolic pathways for application in other industrial fields.

[0014] In the brewing of Maotai-flavor baijiu, important ester-producing yeasts mainly include *Pichia pastoris*, *Hansenula*, *Wickhamia*, *Zygosaccharomyces*, and *Candida*. They produce abundant esters, which are key to the main aroma of baijiu. In addition, some non-traditional yeasts, such as *Myriosporum*, have been found to synthesize ethanol and ethyl acetate, and *Saccharomyces cerevisiae* plays an auxiliary role in baijiu flavor formation and fermentation. Currently, the yeast culture medium commonly used in most studies is YPD, which requires a large amount of chemical reagents and protein resources (such as yeast extract and peptone), resulting in high costs. Furthermore, its composition differs significantly from the actual environment of baijiu brewing. Although many yeast enrichment media involving high-temperature koji and fermented mash add substances such as sorghum juice and distillate to simulate the microecological conditions of the brewing environment, they cannot effectively simulate the selection pressures of high lactic acid, high acetic acid, amino acids, low pH, high nitrogen, and high phosphorus in the brewing environment during the screening process.

[0015] Marine Bacillus ( ) Oceanobacillus ), Croponstead spp. Kroppenstedtia ), Bacillus spp. ( Bacillus ) and Mycobacterium spp. ( VirgibacillusMicroorganisms (MICs) are beneficial microorganisms that contribute rich flavor precursors to the mash during the fermentation process of Maotai-flavor liquor. Baijiu is created by a complex and sophisticated microbial ecosystem. Only when various microorganisms work synergistically in specific proportions and sequences can a rich and harmonious typical style be produced, ensuring a stable community and authentic flavor. For commercial production, consistency in quality between each batch or bottle is crucial. A stable microbial community structure is the fundamental biological basis for ensuring consistent quality across batches. Community imbalance leads to flavor deviations and a decrease in the yield of high-quality products. Therefore, maintaining a stable microbial community structure in baijiu brewing is essential to ensuring the complexity, layering, and overall robustness of the flavor profile.

[0016] The still water from the fermentation process of Maotai-flavor baijiu (Chinese liquor) is rich in substances produced by multiple rounds of fermentation, such as amino acids, organic acids, unutilized sugars, and various aromatic precursors, making it a complex system. Due to its high acidity and high organic content, it acts as a natural barrier, aiding in the screening of microorganisms adapted to the unique brewing environment of Maotai-flavor baijiu. Therefore, the inventors proposed using the still water to screen and enrich yeasts used in baijiu fermentation. However, direct use would affect the selective enrichment effect. Therefore, the inventors treated the collected still water in a specific way and used it as a culture medium for the selective enrichment of brewing yeasts from high-temperature daqu (a type of starter culture). Subsequent verification experiments demonstrated that, compared to the commonly used YPD medium, the treated still water was more effective in screening out acid-resistant, high-osmotic-pressure-resistant yeast strains adapted to this specific microecology.

[0017] The inventors also discovered that the water at the bottom of the still, as a culture medium, can maintain the stability of the community structure of high-temperature Daqu brewing bacteria. In the later stage of laboratory simulated fermentation, the above-mentioned fermentation products can be added to the fermentation embryo in the middle stage of fermentation as an enhancer to play a reinforcing role and guide its microbial community to develop towards the ideal structure.

[0018] Compared to synthetic culture media, the water at the bottom of the still comes from the fermentation process, avoiding the potential impact of exogenous additives on the microbial community structure and more closely resembling the microecological conditions for yeast growth and reproduction during the fermentation of the mash.

[0019] Based on this, the present invention proposes the application of the bottom water of the still in the preparation of functional culture media, which includes a culture medium for selectively enriching and culturing yeasts in the fermentation of baijiu, and a culture medium for maintaining the stability of the microbial community structure in baijiu brewing.

[0020] In some embodiments, the yeasts mentioned above include Pichia pastoris, Mycosaccharomyces cerevisiae, Saccharomyces cerevisiae, Wickham yeast, and Saccharomyces cerevisiae.

[0021] Verification showed that, compared to YPD medium, the enrichment of yeast in the bottom water medium can effectively avoid contamination of the solid medium with filamentous fungi during subsequent isolation and screening. In contrast, the contamination of the solid medium with filamentous fungi during yeast isolation and screening after enrichment in YPD medium resulted in poor yeast isolation performance.

[0022] In some embodiments, the above-mentioned microorganisms for brewing baijiu include Bacillus marineus, Croppensteinella, Bacillus, and Mycobacterium.

[0023] In some embodiments, the water at the bottom of the still is derived from the fermentation process of soy sauce-flavored baijiu.

[0024] Meanwhile, the present invention also provides a culture medium for selectively enriching yeast and / or maintaining the stability of the microbial community structure in Baijiu brewing, the main active ingredient of which is the bottom water from the fermentation process of Maotai-flavor Baijiu.

[0025] In some embodiments, the proportion of bottom water added to the culture medium is 10% to 100%. Optionally, the proportion of bottom water added can be 10%, 25%, 50%, 75%, and 100%, more preferably, it is 25% to 75%, and more preferably, it is 75%.

[0026] During the research, it was found that the composition of the still water from different batches and production sites of Maotai-flavor liquor did not differ significantly. However, different concentrations of still water culture media prepared with this still water had a selective enrichment function for different types of yeast. When the addition ratio of still water was 10% to 100%, it could more effectively selectively enrich the brewing-related yeasts required in the fermentation process of Maotai-flavor liquor.

[0027] In some embodiments, the method for preparing the culture medium includes: collecting the distillate of the mash that naturally settles and drips from the still, collecting the supernatant after centrifugation to obtain the still bottom water stock solution; diluting the still bottom water stock solution with pure water in a certain proportion to obtain the still bottom water diluted solution, then sterilizing it, and cooling it to obtain the culture medium.

[0028] The distillate is centrifuged in the above process to remove insoluble substances mixed in during collection and to avoid experimental errors caused by the inhomogeneity of the sample itself; dilution to different concentrations is to enrich or screen different types of yeast.

[0029] The culture medium process of this invention is simple, requiring only three main steps: simple pretreatment, dilution, and sterilization. It does not require the addition of complex nutrients. It can not only reuse waste water from the still, reducing carbon emissions and providing significant economic and environmental benefits, but also provide new culture media and cultivation methods for the cultivation and screening of brewing microorganisms in high-temperature Daqu, thereby improving the stability, yield, and uniqueness and consistency of the flavor quality of the brewed wine.

[0030] Using the above-mentioned culture medium containing the bottom water of the still, yeast can be selectively enriched and / or the microbial community structure of baijiu brewing can be maintained stably. The method includes: inoculating the diluted daqu solution into the above-mentioned culture medium for cultivation.

[0031] In some embodiments, the above-mentioned culture conditions are: 25~30℃, 160~200 rpm / min.

[0032] In some embodiments, the culture medium conditions are as follows: sugar content 6°Bx~25°Bx, pH 3.70~3.75, nitrate 3 mg / L~12.5 mg / L, ammonia nitrogen 300 mg / L~1600 mg / L, chemical oxygen demand 63000 mg / L-260000 mg / L, total phosphorus 2400 mg / L-9700 mg / L, total nitrogen 1200 mg / L-5000 mg / L, lactic acid 25000 mg / L-100000 mg / L, acetic acid 5000 mg / L-20000 mg / L, and amino acids 7600 mg / L-30000 mg / L.

[0033] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0034] Example 1 This embodiment provides a culture medium containing still water and its preparation method. The still water is the water produced after seven rounds of distillation of a sauce-flavored baijiu. Details are as follows: The culture medium consists of raw water from the bottom of the still and pure water, with a volume ratio of raw water to pure water of 1:3.

[0035] The preparation method of the above culture medium is as follows: (1) Lay the food film flat on the ground, and use a crane to suspend the still directly above the food film after the distillation is completed. Collect the fermented mash in the still by letting it settle and drip naturally.

[0036] (2) The collected water from the bottom of the steamer was placed in a 50 mL centrifuge tube and processed in a high-speed centrifuge at 10000 rpm / min for 10 min. The supernatant was collected to obtain the original water from the bottom of the steamer.

[0037] (3) Dilute the original water from the bottom of the steamer with pure water at different volume ratios, mix them evenly, and obtain the diluted water from the bottom of the steamer.

[0038] (4) The obtained dilute solution from the bottom of the steamer was sterilized by high-pressure steam (121℃, 20 min). The sterilized culture medium was cooled to room temperature to obtain the culture medium.

[0039] Example 2 This embodiment provides a culture medium containing steamed water and its preparation method. The difference between this embodiment and Example 1 is that the volume ratio of the steamed water stock solution to pure water is 1:1.

[0040] Example 3 This embodiment provides a culture medium containing steamed water and its preparation method. The difference between this embodiment and Example 1 is that the volume ratio of the steamed water stock solution to pure water is 3:1.

[0041] Example 4 This embodiment provides a culture medium containing steamed water and its preparation method. The difference between this embodiment and Example 1 is that the volume ratio of the steamed water stock solution to pure water is 1:0.

[0042] The culture media prepared in Examples 1-4 were tested for relevant indicators. Soluble solids were measured using a saccharimeter, pH was measured using a pH meter, nitrate was measured using thymol spectrometry, ammonia nitrogen was measured using salicylic acid spectrophotometry, chemical oxygen demand was measured using rapid digestion spectrophotometry, total phosphorus was measured using ammonium molybdate spectrophotometry, total nitrogen was measured using alkaline potassium persulfate digestion ultraviolet spectrophotometry, lactic acid was measured using liquid chromatography, acetic acid was measured using liquid chromatography, and amino acids were measured using an automated amino acid analyzer.

[0043] The composition of the steaming bottom water culture medium in Examples 1-4 is shown in Table 1: Table 1. Composition information of the bottom water culture medium.

[0044] Example 5 The effectiveness of the culture media in Examples 1-4 in maintaining the stability of the bacterial community structure in high-temperature Daqu (a type of starter culture) was verified as follows: (1) Preparation of Daqu dilution: Weigh 10 g of high-temperature Daqu and add it to a 250 mL conical flask containing 90 mL of sterile water. Place it on a shaker at 180 rpm / min and shake well to obtain a Daqu dilution diluted 10 times as a microbial inoculation mother liquor.

[0045] (2) Negative control - 0% pure water culture medium: accurately measure 50 mL of pure water and add it to a 150 mL conical flask, seal it with sealing film, sterilize it by high pressure steam at 121℃ for 20 min, and then cool it to room temperature for later use.

[0046] (3) Positive control - YPD medium: Prepared according to the formula of yeast extract 10 g / L, glucose 20 g / L, peptone 20 g / L, chloramphenicol 0.1 g / L, add pure water to 50 mL into a 150 mL Erlenmeyer flask, seal with sealing film, and autoclave at 121℃ for 20 min, then cool to room temperature for use.

[0047] (4) Take 50 mL of the culture medium prepared in Examples 1-4 and inoculate it with the microbial inoculation stock solution prepared in step (1) in the negative control and positive control respectively. Use a pipette to draw 500 μL for each group. All operations are completed in a sterile laminar flow hood.

[0048] (5) Incubate at 28℃ and 180rpm / min in a constant temperature shaker for 3 days.

[0049] (6) Total DNA was extracted from the culture products using the EZNATMMag-Bind Soil DNA Kit.

[0050] Microbial data were sequenced using the Illumina MiSeq platform. For bacteria, the V3-V4 region of the 16S rRNA gene was amplified and sequenced using universal primers 341 F (5'-CCTACGGGNGGCWGCAG-3', SEQ ID NO: 1) and 805 R (5'-GACTACHVGGGTATCTAATCC-3', SEQ ID NO: 2). For fungi, the ITS1-ITS2 region was amplified and sequenced using universal primers ITS1 F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 3) and ITS2 R (5'-GCTGCGTTCTTCATCGATGC-3', SEQ ID NO: 4). OTU clustering analysis was performed based on sequence similarity (97%) to obtain OTU data.

[0051] At the genus level, calculate the relative abundance of bacteria and fungi. The method for calculating the relative abundance of bacteria / fungi is as follows: The relative abundance of a bacterial or fungal taxonomic unit (genus) = (number of valid sequences of that taxonomic unit / total number of valid sequences of all bacterial or fungal taxonomic units in the sample) × 100% OTU clustering: a technique for studying microbial diversity that simplifies data analysis and species annotation by grouping similar sequences into the same OTU.

[0052] The results are shown in Table 2: Table 2. Statistical table of relative abundance of bacterial genera in each treatment group after high-temperature Daqu enrichment culture.

[0053] This shows that the high-temperature Daqu dilution solution mainly contains Bacillus spp. (Marine Bacillus) Oceanobacillus ), Croponstead spp. Kroppenstedtia ), Bacillus spp. Bacillus ) and Mycobacterium spp. ( Virgibacillus() is the dominant bacterial genus. After enrichment culture in different types of culture media for 3 days, the bacterial community structure in high-temperature Daqu (a type of starter culture) showed significant differences.

[0054] The 0% culture medium (i.e., the negative control group) was mainly enriched with Acinetobacter spp. after 3 days. Acinetobacter ), Enterobacteriaceae ( Enterobacter ), Sphingomyelin spp. ( Sphingobacterium ), Masseilles ( Massilia ), Cloacibacterium The bacterial community composition differs significantly from that in high-temperature Daqu (a type of starter culture).

[0055] In YPD medium (positive control group), after 3 days of enrichment culture, the bacteria were mainly Enterobacteriaceae (…). Enterobacter ), Pseudomonas spp. Pseudomonas ), Sphingomyelin spp. ( Sphingobacterium ) and Stenotrophomonas spp. Stenotrophomonas ) is the dominant bacterial genus, and its bacterial community composition differs significantly from that in high-temperature Daqu (a type of starter culture).

[0056] The enrichment results of the 25%, 50%, 75%, and 100% concentrations of steaming bottom water culture medium (Examples 1-4) after 3 days of enrichment culture are as follows: Figure 1 As shown: the bacteria are mainly of the genus Bacillus (Marine Bacillus). Oceanobacillus ), Croponstead spp. Kroppenstedtia ), Bacillus spp. ( Bacillus ) and Mycobacterium spp. ( Virgibacillus ) is the dominant bacterial genus, and its composition is similar to that of the bacterial community in high-temperature Daqu (a type of starter culture).

[0057] like Figure 2 As shown, PCA cluster analysis of the enrichment results of different experimental groups revealed that the bacterial community structure of the 0% and YPD enrichment cultures differed significantly from that of the high-temperature Daqu (a type of starter culture). However, the bacterial enrichment results of different concentrations of the bottom water culture medium maintained similarities to the bacterial community structure of the initial high-temperature Daqu, mainly consisting of *Bacillus* spp. (…). Oceanobacillus ), Croponstead spp. Kroppenstedtia ), Bacillus spp. ( Bacillus ) and Mycobacterium spp. ( Virgibacillus The above-mentioned bacterial genera are the core functional brewing microorganisms in the fermentation process of Maotai-flavor liquor, imparting rich flavor precursors to the mash. Therefore, the bottom water culture medium can be used to stabilize the community structure of high-temperature Daqu brewing bacteria and screen brewing microorganisms.

[0058] Example 6 This embodiment verifies the effectiveness of the culture media from Examples 1-4 in enriching yeast in high-temperature Daqu (a type of starter culture). The specific details are as follows: The preparation of Daqu dilution, liquid culture medium of different concentrations of still water, YPD culture medium, and microbial inoculation methods are the same as in Example 5. The enrichment culture steps are as follows: all culture mediums inoculated with Daqu dilution are placed in a constant temperature shaking shaker at 28°C and 180 rpm / min for shaking culture. After 72 h of culture, the number of culturable yeast cells is detected.

[0059] The number of culturable yeast cells was determined using the microbial plate count method. Fermentation broth from all treatment groups after enrichment culture was collected and diluted to 10⁻⁶ using a serial dilution method. -3 10 -4 and 10 -5 After determining the concentration, use a pipette to add 100 μL of the diluted solution to WL solid medium, spread it evenly using a sterile spreader, invert the plate and incubate it in a 28℃ constant temperature incubator for 3 days, and then count the yeast cells.

[0060] Test results as follows Figure 3 As shown, after 72 hours of enrichment culture, the number of culturable yeast cells was statistically analyzed using the microbial plate count method. The results showed that the yeast concentration obtained from enrichment in 0% pure water medium was 8.67 × 10⁻⁶. 4 The concentration of yeast obtained by enrichment in a medium containing 25% steaming bottom water was 2.04 × 10⁻⁶ CFU / mL. 9 The concentration of yeast obtained by enrichment in a medium containing 50% steaming bottom water was 1.37 × 10⁻⁶ CFU / mL. 9 The concentration of yeast enriched in a 75% still water medium was 1.30 × 10⁻⁶ CFU / mL. 8 The yeast concentration obtained by enrichment in 100% still water medium was 18.33 CFU / mL, and the yeast concentration obtained by enrichment in YPD medium was 1.09 × 10⁻⁶ CFU / mL. 8 CFU / mL. Compared with two control groups, 0% pure water culture medium and YPD culture medium, three different concentrations of water at the bottom of the still (25%, 50%, and 75%) were found to have the function of enriching yeast in high-temperature Daqu (a type of starter culture).

[0061] Example 7 This embodiment verifies the effect of the culture medium prepared in Examples 1-4 on the targeted enrichment of functional yeasts in high-temperature Daqu (a type of starter culture). The specific details are as follows: The methods for preparing Daqu dilution solution, liquid culture medium of different concentrations of still water, YPD culture medium, microbial inoculation, and enrichment culture are the same as in Example 5.

[0062] Total DNA was extracted from samples using the EZNATMMag-Bind Soil DNA Kit. Microbial data were sequenced using the Illumina MiSeq platform. Fungal pairs were amplified and sequenced in the ITS1-ITS2 region using universal primers ITS1 F (5'-CTTGGTCATTTAGAGGAAGTAA-3', SEQ ID NO: 3) and ITS2 R (5'-GCTGCGTTCTTCATCGATGC-3', SEQ ID NO: 4). OTU clustering analysis was performed based on sequence similarity (97%) to obtain OTU data.

[0063] At the genus level, calculate the relative abundance of fungi. The calculation method is as follows: Relative abundance of fungal taxonomic units (genus) = (Number of valid sequences of that taxonomic unit / Total number of valid sequences of all fungal taxonomic units in the sample) × 100% OTU clustering: a technique for studying microbial diversity that simplifies data analysis and species annotation by grouping similar sequences into the same OTU.

[0064] Analysis of relative abundance results of fungi, as follows Figure 4 As shown, 0-3d represents the fungal community structure after sterilization of pure water culture medium, inoculated with Daqu dilution, and cultured on a shaker for 3 days; 25-3d represents the fungal community structure after sterilization of the original water from the bottom of the still and pure water at a ratio of 1:3, inoculated with Daqu dilution, and cultured on a shaker for 3 days; 50-3d represents the fungal community structure after sterilization of the original water from the bottom of the still and pure water at a ratio of 1:1, inoculated with Daqu dilution, and cultured on a shaker for 3 days; 75-3d represents the fungal community structure after sterilization of the original water from the bottom of the still and pure water at a ratio of 3:1, inoculated with Daqu dilution, and cultured on a shaker for 3 days; 100-3d represents the fungal community structure after sterilization of the original water from the bottom of the still, inoculated with Daqu dilution, and cultured on a shaker for 3 days; YPD-3d represents the fungal community structure after sterilization of YPD culture medium, inoculated with Daqu dilution, and cultured on a shaker for 3 days.

[0065] The relative abundance of yeast species in each treatment group is shown in Table 3 and Figure 5-8 As shown: Table 3. Statistical table of relative abundance of yeast species in each treatment group after high-temperature Daqu enrichment culture.

[0066] Relative abundance: The proportion of the number of DNA sequences of a particular microorganism (such as bacteria or fungi) in a sample to the total number of DNA sequences of all microorganisms in the sample. This proportion is usually expressed as a percentage. In this paper, the relative abundance of fungi is the proportion of the number of DNA sequences of a particular fungal genus to the total number of DNA sequences of all fungal genera in the sample.

[0067] As shown in Table 3, compared with the initial fungal community structure in the Daqu dilution solution, after 72 h of cultivation, the enrichment results of the fungal community structure in the high-temperature Daqu varied among all treatment groups. Compared with the initial fungal community structure in the DQ group, the 75-3d treatment group was mainly enriched with Pichia pastoris (…). Pichia Its relative abundance increased from 11.59% to 51.07%.

[0068] The 50-3d treatment group was mainly enriched with *Pichia pastoris* and *Saccharomyces cerevisiae*. Cyberlindnera ) and Wickham yeast ( Wickerhamomyces The relative abundance of *Pichia pastoris* increased from 11.59% to 24.68%; the relative abundance of *Cerberlindnerella sebrindrome* increased from 0.75% to 22.90%; and the relative abundance of *Wickhamia spp.* increased from 3.95% to 44.50%.

[0069] The 25-3d treatment group was mainly enriched with *Pichia* and *Mycosaccharomyces* genera. Trichosporon The relative abundance of *Pichia pastoris* increased from 11.59% to 39.20%; the relative abundance of *Mycosaccharomyces* increased from 1.57% to 25.36%; the relative abundance of *Pichia pastoris* increased from 0.75% to 18.59%; and the relative abundance of *Pichia pastoris* increased from 3.95% to 10.33%.

[0070] like Figure 5 As shown, compared with the 0% and YPD control media, Pichia pastoris was mainly enriched in the 25%, 50% and 75% concentrations of steaming bottom water media, and the 75% concentration of steaming bottom water media yielded the best enrichment results. Therefore, the 75% concentration of steaming bottom water media is the best enrichment condition for Pichia pastoris.

[0071] like Figure 6 As shown, *Saccharomyces cerevisiae* was enriched in 0%, 25%, 50%, and YPD media. Compared to the initial relative abundance of 0.75%, the 25% and 50% concentrations of the simmering water medium increased the abundance to 18.59% and 22.90%, respectively, achieving effective enrichment of *Saccharomyces cerevisiae*. Therefore, 50% concentration of the simmering water medium is the optimal enrichment condition for *Saccharomyces cerevisiae*.

[0072] like Figure 7As shown, *Wickham's yeast* was enriched in 0%, 25%, and 50% media. The 50% concentration of the bottom water medium showed the best enrichment effect, increasing the relative abundance from 3.95% to 44.50%, higher than the 4.45% of 0% and 0.87% of YPD. Therefore, 50% concentration of the bottom water medium is the optimal enrichment condition for *Wickham's yeast*.

[0073] like Figure 8 As shown, *Mycosaccharomyces* was enriched in 0%, 25%, and YPD media. The 25% concentration of steam-bottom water medium showed better enrichment of *Mycosaccharomyces* than 0%, with the relative abundance of the genus increasing from the initial 1.57% to 25.36%. Although lower than the 38.62% in YPD medium, it still demonstrated effective enrichment ability for *Mycosaccharomyces*. Therefore, 25% concentration of steam-bottom water medium is the optimal enrichment condition for *Mycosaccharomyces*.

[0074] Example 8 This embodiment provides a steam-bottom water solid culture medium and its preparation method, as detailed below: (1) Culture medium diluted 10 times with the water at the bottom of the steamer Measure 20 mL of the original still water solution and add 80 mL of pure water, mix well to prepare a 5-fold diluted still water solution. Measure 100 mL of pure water and add 3 g of agar powder, mix well to prepare an agar solution. Place the 5-fold still water solution and the agar solution separately in an autoclave and sterilize at 121°C for 20 minutes. After sterilization, while still at high temperature, pour the agar solution into the still water solution in a clean bench and mix well to prepare a 10-fold diluted still water solid culture medium. Pour this medium into sterile petri dishes with a diameter of 90 mm, about 15-20 mL per dish, cover the dishes, and allow them to cool and solidify naturally to obtain the 10-fold still water solution solid culture medium plates of this invention.

[0075] (2) Other dilution ratios of the bottom water culture medium are prepared in the same way.

[0076] Example 9 This embodiment verifies the effectiveness of the steam-bottom water solid culture medium in Example 8 for yeast plate plating detection, as detailed below: The experimental strains used in this experiment were selected from the group's own strain library, including pure cultures of Pichia pastoris (strain number: XJ-YPPMA001-1), Pichia kudriazwei (strain number: XJ-YPPKU001-1), and Saccharomyces cerevisiae (strain number: XJ-YSSCE001-1).

[0077] WL solid medium: Commercial medium from Shanghai Bowei Microbial Technology Co., Ltd. was used.

[0078] The dilution-spread plate method was used. The suspensions of the three yeasts were diluted to 10⁻⁶ using a serial dilution method. -6 and 10 -7 To determine the concentration, 100 μL of the diluted solution was spread onto both WL and the culture medium plates of this invention, using a sterile spreader to spread evenly. After incubating the plates upside down in a 28°C incubator for 48-72 h, the colonies on the plates were counted.

[0079] The experimental results are shown in Table 4 and Figure 9 As shown: Table 4. Yeast count results for different types of solid culture media

[0080] Experimental data showed that *Pichia gondii*, *Pichia kudriazines*, and *Saccharomyces cerevisiae* all grew well on a culture medium diluted 50 times with water from the bottom of the still, with yeast colony counts of 9.30 × 10⁻⁶. 8 CFU / mL, 8.30×10 8 CFU / mL and 1.20×10 9 CFU / mL, which is different from 9.15 × 10⁻⁶ on WL medium. 8 CFU / mL, 8.20×10 8 CFU / mL and 1.52×10 9 There was no significant difference in the number of colonies at CFU / mL. Furthermore, Figure 9 The results showed that the three yeast strains exhibited consistent growth in both WL solid medium and 50-fold diluted still water solid medium, demonstrating that the medium of this invention can effectively support the growth of these yeast strains and fully meet the requirements for isolation and culture. Therefore, a 50-fold dilution of still water concentration is the optimal condition for isolating and culturing yeast strains.

[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of steaming bottom water in the preparation of functional culture media, characterized in that, The culture medium includes a medium for selectively enriching and culturing yeasts used in the fermentation of baijiu (Chinese liquor).

2. The application according to claim 1, characterized in that, The yeasts include Pichia pastoris, Mycosaccharomyces cerevisiae, Saccharomyces cerevisiae, Wickham yeast, and Saccharomyces cerevisiae.

3. The application according to claim 1, characterized in that, The culture medium also includes a culture medium for maintaining the stability of the microbial community structure in baijiu brewing.

4. The application according to claim 3, characterized in that, The microorganisms used in the brewing of baijiu include marine Bacillus, Croponstead Bacillus, Bacillus, and Mycobacterium.

5. The application according to any one of claims 1 to 4, characterized in that, The water at the bottom of the still comes from the fermentation process of soy sauce-flavored baijiu.

6. A culture medium for selectively enriching yeast and / or maintaining the stability of the microbial community structure in Baijiu brewing, characterized in that, Its main active ingredient is the water from the bottom of the still during the fermentation process of soy sauce-flavored liquor.

7. The culture medium according to claim 6, characterized in that, The proportion of water added to the culture medium is 10% to 100%.

8. The culture medium according to claim 7, characterized in that, The method for preparing the culture medium includes: Collect the distillate of the mash that naturally settles and drips into the still, centrifuge it and collect the supernatant to obtain the still bottom water stock solution; dilute the still bottom water stock solution with pure water in a certain proportion to obtain the still bottom water diluted solution, then sterilize it and cool it to obtain the culture medium.

9. A method for selectively enriching yeast and / or maintaining the stability of the microbial community structure in Baijiu brewing using water from the bottom of the still, characterized in that, include: The diluted Daqu solution is inoculated into the culture medium described in any one of claims 6 to 8 and cultured thereon. The culture conditions are: 25~30℃, 160~200 rpm / min.

10. The method according to claim 9, characterized in that, The culture medium has a sugar content of 6°Bx to 25°Bx and a pH of 3.70 to 3.75, and contains nitrates of 3 mg / L to 12.5 mg / L, ammonia nitrogen of 300 mg / L to 1600 mg / L, chemical oxygen demand of 63000 mg / L to 260000 mg / L, total phosphorus of 2400 mg / L to 9700 mg / L, total nitrogen of 1200 mg / L to 5000 mg / L, lactic acid of 25000 mg / L to 100000 mg / L, acetic acid of 5000 mg / L to 20000 mg / L, and amino acids of 7600 mg / L to 30000 mg / L.

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