Schizosaccharomyces pombe strain capable of producing active dry yeast through high-density culture and application of schizosaccharomyces pombe strain

Abstract

The invention discloses a schizosaccharomyces pombe strain capable of producing active dry yeast through high-density culture and application of the schizosaccharomyces pombe strain, and belongs to the technical field of microorganisms. According to the invention, a strain of schizosaccharomyces pombe JGWN-SP04 capable of culturing and producing active dry yeast at high density is screened from Maotai-flavor fermented grains, the strain is inoculated to a seed solution culture medium according to the inoculum size of 5% (v / v) (1 * 10 < 8 > CFU / mL), the OD600 value can reach 18.73 after the strain is cultured for 36 hours, the strain is inoculated to a fermentation culture medium according to the inoculum size of 8% (v / v) (1 * 10 < 8 > CFU / mL), the OD600 value is increased from 1.0 to 30 after high-density fermentation is carried out for 48 hours, the dry thallus can reach 15g / L, and the strain can be used for producing active dry yeast. Meanwhile, the strain has the characteristics of high temperature resistance and ethanol resistance, and has a good application prospect.

Description

Technical Field

[0001] A strain of *Schizosaccharomyces cerevisiae* capable of high-density cultivation for the production of active dry yeast and its applications belong to the field of microbial technology. Background Technology

[0002] Active dry yeast is a commonly used alcoholic fermentation agent in the brewing industry. In solid-state fermentation of baijiu (Chinese liquor), it is also common to directly inoculate with active dry yeast to increase the number of microorganisms. Currently, the active dry yeast on the market is mainly brewing yeast (Saccharomyces cerevisiae). The production method involves high-density cultivation in fermentation tanks, followed by separation, collection, drying, and compounding of the cells to obtain active dry yeast. Because brewing yeast and similar yeasts have strong growth capabilities and are less prone to contamination under high-density cultivation, they are relatively easy to produce. In recent years, with the slowdown in market economic growth, the changing demographics of consumers, and shifts in consumption concepts, the baijiu market in China has experienced a downward trend. Therefore, reducing baijiu costs and mitigating hangover reactions after consumption are important issues for the baijiu industry in an era of shrinking volume competition. Improving the quality of baijiu, in essence, means improving the quality of the brewing microorganisms.

[0003] Schizosaccharomyces cerevisiae is a common microorganism in soy sauce-flavored baijiu, wine, and fruit wine. It possesses strong acid resistance and exhibits high ethanol production and low fusel oil production, making it a key strain in the brewing of high-quality baijiu. It plays a crucial role in the multi-microbial fermentation process of baijiu. Its growth and metabolic capabilities are directly related to the final quality of the baijiu. Schizosaccharomyces cerevisiae has been found in the mash of baijiu from several high-quality baijiu-producing regions.

[0004] The solid-state fermentation process of baijiu is quite complex, influenced by various factors such as different regional microorganisms, temperature, and humidity. Furthermore, due to the slow growth of *Schizophyllum commune*, it is difficult to stably enrich *Schizophyllum commune* during the enrichment of microorganisms through baijiu starter culture and stacking fermentation. Therefore, it is challenging to achieve the industrial-scale production of active dry yeast products from *Schizophyllum commune*, and also to industrialize the fermentation production of metabolites from a single strain of *Schizophyllum commune*.

[0005] Therefore, to improve the quality of liquor and help distilleries enhance their products, the addition of *Schizosaccharomyces cerevisiae* cells to the baijiu fermentation process is imperative. In conclusion, it is necessary to further develop a *Schizosaccharomyces cerevisiae* strain capable of high-density cultivation to produce active dry yeast, making the industrial production of acid-resistant, high-ethanol-producing, low-fusel oil-producing, and arginine- and urea-degrading active dry yeast possible. Summary of the Invention

[0006] To solve the above-mentioned technical problems, this invention has screened a strain of *Schizosaccharomyces pombe* JGWN-SP04, which can be cultured at high density to produce active dry yeast, from soy sauce-flavored liquor mash. This strain can be cultured at 5% (v / v) (1×10⁻⁶) 8 Inoculate the seed culture medium with an inoculum of CFU / mL, and after 36 h of culture, OD 600 The value can reach 18.73, at 8% (v / v)(1×10) 8 Inoculation with CFU / mL was performed into the fermentation medium, and after 48 hours of high-density fermentation, OD was obtained. 600 The value increased from 1.0 to 30, and the dry bacterial count reached 15 g / L. At the same time, this strain is resistant to high temperature and ethanol, and has good application prospects.

[0007] The first objective of this invention is to provide a strain of *Schizosaccharomyces pombe* JGWN-SP04, which was deposited on July 11, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251563, located at Wuhan University, Wuhan, China.

[0008] In one embodiment of the present invention, the millet wine fission yeast strain is obtained by isolating and screening from soy sauce-flavored liquor mash.

[0009] In one embodiment of the present invention, the 26S rDNA nucleotide sequence of the *Schizosaccharomyces cerevisiae* is as shown in SEQ ID NO. 1.

[0010] A second objective of this invention is to provide a microbial agent containing the aforementioned *Schizosaccharomyces cerevisiae* JGWN-SP04.

[0011] In one embodiment of the present invention, the microbial agent further contains glucose, tryptone, yeast extract, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, anhydrous magnesium sulfate, and anhydrous calcium chloride.

[0012] In one embodiment of the present invention, the following components are used: glucose 50-100 g / L, tryptone 10-30 g / L, yeast extract 10-20 g / L, potassium dihydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 0.5-1.5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, and anhydrous calcium chloride 1-10 g / L.

[0013] A third objective of this invention is to provide a method for high-density culture of the above-mentioned *Schizosaccharomyces cerevisiae*, comprising the steps of:

[0014] (1) Seed culture: Select a single colony and inoculate it into seed culture medium to obtain seed liquid;

[0015] (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 2-10% for fermentation.

[0016] In one embodiment of the present invention, the conditions for seed culture and fermentation culture are 25-35℃ for 36-48 hours.

[0017] In one embodiment of the present invention, the seed culture medium comprises, by mass, 50-100 g / L glucose, 10-30 g / L tryptone, and 10-20 g / L yeast extract.

[0018] In one embodiment of the present invention, the fermentation culture medium comprises, by mass: 10-20 g / L glucose, 10-30 g / L tryptone, 10-20 g / L yeast extract, 1-3 g / L potassium dihydrogen phosphate, 0.5-1.5 g / L dipotassium hydrogen phosphate, 0.5-2 g / L anhydrous magnesium sulfate, and 1-10 g / L anhydrous calcium chloride.

[0019] A fourth objective of this invention is to provide the application of the above-mentioned strains and microbial agents in alcoholic beverage brewing.

[0020] Beneficial effects of the present invention

[0021] (1) In this invention, the seed culture of *Schizosaccharomyces cerevisiae* showed an OD value after 36 hours of cultivation. 600 The value can reach 18.73, and the output is 9 times higher than previously reported.

[0022] (2) In the high-density fermentation of chestnut wine fissilla yeast in this invention, fermentation for 48 hours can reduce the OD of chestnut wine fissilla yeast. 600 The value increased from 1.0 to 30, and the dry cell volume reached 15g / L, with the yield increasing by 20 to 30 times compared to previous reports, laying the foundation for the industrial production of Saccharomyces cerevisiae.

[0023] Preservation of biological materials

[0024] Schizosaccharomyces pombe JGWN-SP04, taxonomically named Schizosaccharomyces pombe JGWN-SP04, was deposited on July 11, 2025, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251563, located at Wuhan University, Wuhan, China. Attached Figure Description

[0025] Figure 1 The graph shows the effect of glucose content on the growth of the strain.

[0026] Figure 2 Figure showing the effect of time on the growth of *Schizosaccharomyces cerevisiae* strain;

[0027] Figure 3 Figure showing the effect of carbon source on the growth of *Schizosaccharomyces cerevisiae* strain;

[0028] Figure 4 Figure showing the effect of nitrogen source on the growth of *Schizosaccharomyces cerevisiae* strain;

[0029] Figure 5 The figure shows the effect of a complex nitrogen source on the growth of the yeast strain *Schizosaccharomyces cerevisiae*. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, where specific conditions are not specified, are generally performed under conventional conditions in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art.

[0031] Raw material source:

[0032] The mash for this sauce-flavored liquor is sourced from a sauce-flavored liquor factory in Maotai Town, Guizhou Province.

[0033] Schizosaccharomyces pombe JGWN-SP01, JGWN-SP02, JGWN-SP03, and JGWN-SP04 were isolated from soy sauce-flavored liquor mash.

[0034] The culture media involved in the following examples are as follows:

[0035] Screening medium: Bengal Red Agar medium: peptone 5g / L, glucose 10g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, Bengal Red 0.033g / L, chloramphenicol 0.1g / L, agar 20g / L.

[0036] YPD medium: glucose 20g / L, tryptone 20g / L, yeast extract 10g / L.

[0037] Malt extract culture medium: 130 g / L malt extract powder, 0.1 g / L chloramphenicol, pH adjusted to 5.6.

[0038] TTC agar alcohol production medium: commercially available

[0039] Seed culture medium: glucose 100g / L, tryptone 20g / L, yeast extract 10g / L, pH natural.

[0040] Fermentation medium: glucose 20 g / L, tryptone 10 g / L, yeast extract 10 g / L, potassium dihydrogen phosphate 3 g / L; dipotassium hydrogen phosphate 1.5 g / L, anhydrous magnesium sulfate 0.5 g / L, anhydrous calcium chloride 5 g / L.

[0041] Example 1: Screening and identification of *Schizosaccharomyces cerevisiae*

[0042] 1. Screening of *Schizosaccharomyces cerevisiae*

[0043] (1) Sample source

[0044] The strain used in this invention was isolated from soy sauce-flavored liquor mash.

[0045] (2) Isolation and purification of strains

[0046] Detailed isolation and purification method: Place 25g of fermented mash in an Erlenmeyer flask containing 225mL of physiological saline. Incubate at 30℃, shake at 180rpm for 120min. Perform 10-fold serial dilutions with sterile physiological saline. Spread 50μL of each dilution onto Bengal red agar medium and incubate at 30℃ for 2 days. After incubation, based on the color and morphology of single colonies on the agar plates, streak different single colonies onto YPD medium for purification. Select single colonies for enrichment culture in malt extract medium. The obtained strains are suspended in PBS containing 20% ​​glycerol and stored at -80℃.

[0047] The purified strain was streaked onto YPD medium and incubated at 30°C. A layer of TTC agar was then placed on top of the streak, and the culture was incubated in the dark for 60 minutes. TTC agar changed from colorless to red under the action of respiratory chain dehydrogenases, thus determining the strain's ethanol production capacity. Eighteen deep red single colonies were selected for enrichment culture and numbered. The resulting strains were then suspended in PBS containing 20% ​​glycerol and stored at -80°C.

[0048] After activating the above 18 single colonies, they were examined under a microscope, and four fission-reproducing yeast strains, JGWN-SP01, JGWN-SP02, JGWN-SP03, and JGWN-SP04, were selected.

[0049] 2. Identification of *Schizosaccharomyces cerevisiae*

[0050] (1) Colony characteristics:

[0051] The colony morphology is milky white to pale yellow, opaque, smooth, hemispherical colonies with a moist and glossy texture and neat edges.

[0052] (2) Bacterial cell characteristics:

[0053] Under an optical microscope, the strain appears pear-shaped or short rod-shaped, often appearing singly or in pairs, without obvious aggregation. It reproduces by fission.

[0054] (3) Growth characteristics:

[0055] The strain JGWN-SP04 is a yeast strain of the wine schizophyte. It grows best at a temperature of 30℃, with the highest and lowest initial growth pH being 2 and 9, respectively, and the optimal initial growth pH being 4.5.

[0056] 3. Preservation of bacterial strains

[0057] The selected *Schizosaccharomyces pombe* JGWN-SP04 was deposited at the China Center for Type Culture Collection (CCTCC). Its taxonomic name is *Schizosaccharomyces pombe* JGWN-SP04, and its accession number is CCTCC NO: M 20251563.

[0058] The *Schizophyllum commune* strains JGWN-SP01, JGWN-SP02, JGWN-SP03, and JGWN-SP04, which were screened concurrently, were used as controls for subsequent experiments.

[0059] Example 2: Performance Characteristics Determination of Schizosaccharomyces cerevisiae (Chestnut Wine Saccharomyces)

[0060] 1. Determination of bacterial growth characteristics

[0061] (1) Experimental methods

[0062] Pick one loopful of a single colony from each strain and inoculate it into 100 mL of YPD medium. Incubate at 30°C and 180 rpm for 36 h with shaking. Measure the OD of the bacterial culture every four hours. 600 Value. Take 30 mL of bacterial culture and place it in a 50 mL centrifuge tube. Centrifuge in a refrigerated centrifuge at 4 °C, discard the supernatant, and measure the mass of the bacterial sludge. Dry the bacterial sludge in an oven at 80 °C until constant weight, and determine the dry weight of the bacterial cells.

[0063] (2) Experimental Results

[0064] Table 1 shows the results of the bacterial growth characteristic determination. It was found that the OD values ​​of most strains were... 600 The OD values ​​were only 2.02–3.36, while those of *Schizosaccharomyces cerevisiae* JGWN-SP04 were much higher. 600 It can reach 6.71, which is 2 to 3 times that of other strains.

[0065] Table 1. Growth assay results of different bacterial strains

[0066] Fungal name <![CDATA[OD 600 ]]> Dry weight (g / L) Saccharomyces cerevisiae JGWN-SP01 3.36 4.5 Saccharomyces cerevisiae JGWN-SP02 2.02 2.5 Saccharomyces cerevisiae JGWN-SP03 2.48 3.1 Saccharomyces cerevisiae JGWN-SP04 6.71 8.7

[0067] 2. Temperature tolerance test of *Schizosaccharomyces cerevisiae*

[0068] (1) Experimental methods

[0069] One loopful of a single colony from each strain was picked from a plate and inoculated into 100 mL of YPD medium. The culture was then incubated on a shaker at 30°C and 180 rpm for 36 h to prepare the seed culture. 5 × 10⁻⁶ cells / mL were then used. 8 CFU-purified *Schizosaccharomyces cerevisiae* seed culture was inoculated into YPD medium and cultured at 20℃, 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃ for 36 h in a shaker at 200 rpm. OD values ​​were then measured. 600 Value. OD 600 A value ≥ 2.0 indicates that the temperature is tolerable.

[0070] (2) Experimental Results

[0071] As shown in Table 2:

[0072] Table 2 Results of temperature tolerance test of strains

[0073] Fungal name Temperature resistance Saccharomyces cerevisiae JGWN-SP01 20～35℃ Saccharomyces cerevisiae JGWN-SP02 25～35℃ Saccharomyces cerevisiae JGWN-SP03 25～30℃ Saccharomyces cerevisiae JGWN-SP04 25～35℃

[0074] 3. pH tolerance test of *Schizosaccharomyces cerevisiae*

[0075] (1) Experimental methods

[0076] One loopful of a single colony from each strain was picked from a plate and inoculated into 100 mL of YPD medium. The culture was then incubated on a shaker at 30°C and 180 rpm for 36 h to prepare the seed culture. 5 × 10⁻⁶ cells / mL were then used. 8 CFU-purified *Schizosaccharomyces cerevisiae* seed culture was inoculated into YPD medium. The pH was adjusted using concentrated phosphoric acid and 10% sodium hydroxide solution. The culture was then incubated at pH 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 for 36 hours using a shaker at 200 rpm. OD values ​​were measured at each culture. 600 Value. OD 600 A value ≥ 1.0 indicates tolerance to that pH.

[0077] (2) Experimental Results

[0078] As shown in Table 3:

[0079] Table 3 Results of pH tolerance test for strains

[0080] Fungal name pH tolerance Saccharomyces cerevisiae JGWN-SP01 2～8 Saccharomyces cerevisiae JGWN-SP02 2～7 Saccharomyces cerevisiae JGWN-SP03 3～9 Saccharomyces cerevisiae JGWN-SP04 2～9

[0081] 4. Glucose tolerance test of *Schizosaccharomyces cerevisiae*

[0082] (1) Experimental methods

[0083] One loopful of a single colony from each strain was picked from a plate and inoculated into 100 mL of YPD medium. The culture was then incubated on a shaker at 30°C and 180 rpm for 36 h to prepare the seed culture. 5 × 10⁻⁶ cells / mL were then used. 8 CFU-purified *Schizosaccharomyces cerevisiae* seed culture was inoculated into YPD medium and cultured for 36 h at 200 rpm under different glucose concentrations (2%, 6%, 10%, 14%, and 18%). OD values ​​were then measured. 600 Value. OD 600 A value ≥ 2.0 indicates tolerance to that concentration of glucose.

[0084] (2) Experimental Results

[0085] As shown in Table 4:

[0086] Table 4 Results of glucose tolerance test for bacterial strains

[0087] Fungal name Glucose tolerance Saccharomyces cerevisiae JGWN-SP01 18％ Saccharomyces cerevisiae JGWN-SP02 18％ Saccharomyces cerevisiae JGWN-SP03 14％ Saccharomyces cerevisiae JGWN-SP04 18％

[0088] 5. Ethanol tolerance test of *Schizosaccharomyces cerevisiae*

[0089] (1) Experimental methods

[0090] One loopful of a single colony from each strain was picked from a plate and inoculated into 100 mL of YPD medium. The culture was then incubated on a shaker at 30°C and 180 rpm for 36 h to prepare the seed culture. 5 × 10⁻⁶ cells / mL were then used. 8 CFU-purified *Schizosaccharomyces cerevisiae* seed culture was inoculated into YPD medium, and ethanol was added to achieve concentrations of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%. Ethanol was sterilized by filtering through a 0.22 μm organic phase filter before use. Each group was tested in triplicate. The medium was incubated at 180 rpm and 30 °C for 24 h, and OD values ​​were measured. 600 Value. OD 600 A value ≥ 1.0 indicates tolerance to that concentration of ethanol.

[0091] (2) Experimental Results

[0092] As shown in Table 5:

[0093] Table 5 Results of ethanol tolerance test for strains

[0094] Fungal name Tolerance to ethanol Saccharomyces cerevisiae JGWN-SP01 8％ Saccharomyces cerevisiae JGWN-SP02 9％ Saccharomyces cerevisiae JGWN-SP03 8％ Saccharomyces cerevisiae JGWN-SP04 10％

[0095] Based on comprehensive consideration, the Schizosaccharomyces cerevisiae SP04 was selected for subsequent experiments.

[0096] Example 3: Optimization of culture conditions for *Schizosaccharomyces cerevisiae*

[0097] I. Optimization of Seed Culture Medium

[0098] 1. Preparation of seed liquid

[0099] The solution consisted of 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, and water as the remaining component. 100 mL of the prepared seed culture medium was sterilized by moist heat at 115°C for 30 min. After cooling to room temperature under sterile conditions, 100 μL of the solution was aspirated using a sterile pipette tip and cryopreserved. The culture was then incubated in a shaker at 30°C and 180 rpm for 36 h.

[0100] 2. Optimization Experimental Methods for Seed Culture Medium

[0101] 100 μL of purified *Schizosaccharomyces cerevisiae* seed culture was inoculated into 100 mL of seed culture medium. The nitrogen source of the medium was adjusted to 20 g / L tryptone and 10 g / L yeast extract. Glucose concentrations were adjusted to 20 g / L, 60 g / L, 100 g / L, 140 g / L, and 180 g / L. The cultures were then incubated at 30 °C and 180 rpm for 36 h, and the OD values ​​were measured. 600 value.

[0102] 3. Experimental Results

[0103] The effect of glucose content on strain growth, such as Figure 1 As shown in the results, the growth of *Schizosaccharomyces cerevisiae* reached its highest OD value when the glucose addition was 100 g / L. 600 It is 20.87, which is higher than the original OD. 600 It increased by 14.

[0104] II. Optimization of Seed Culture Time

[0105] 1. Preparation of seed liquid

[0106] The solution consisted of 100 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, and water as the remaining component. 100 mL of the prepared seed culture medium was sterilized by moist heat at 115°C for 30 min. After cooling to room temperature under sterile conditions, 100 μL of the solution was aspirated into a glycerol tube for cryopreservation using a sterile pipette tip. The culture was then incubated in a shaker at 30°C and 180 rpm for 36 h.

[0107] 2. Optimization of Seed Culture Time Experimental Method

[0108] 100 μL of purified *Schizosaccharomyces cerevisiae* seed culture was inoculated into 100 mL of seed culture medium. The culture was incubated at 30℃ and 180 rpm for 48 h, with enzyme samples taken every 2 h to measure OD. 600 Values ​​and plot growth curves.

[0109] 3. Experimental Results

[0110] The effect of time on the growth of *Schizosaccharomyces cerevisiae* strains, such as Figure 2 As shown in the results, when the culture time is 36 hours, the growth of *Schizosaccharomyces cerevisiae* reaches OD200. 600 It is 18.73.

[0111] III. Optimization of Fermentation Medium for Saccharomyces cerevisiae

[0112] 1. Experimental Methods

[0113] (1) Preparation of seed solution:

[0114] The culture medium consisted of 100 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, and water as the remaining component. 100 mL of the prepared seed culture medium was sterilized by moist heat at 115°C for 30 min. After cooling to room temperature under sterile conditions, a single colony was inoculated onto a loopful of culture dish using a sterile inoculation loop. The culture was then incubated in a shaker at 30°C and 180 rpm for 36 h.

[0115] (2) Fermentation process:

[0116] The prepared seed culture was inoculated into the fermentation medium at an inoculation rate of 5% (v / v) and cultured at 30°C to obtain the fermentation broth. The OD was measured after 48 hours of culture. 600 .

[0117] 2. The fermentation medium from step 1 was optimized using a single-factor optimization method, as follows:

[0118] (1) The specific implementation method is the same as step 1, except that the carbon source in the fermentation culture medium in step (2) of step 1 is adjusted to be: glucose, malt syrup, molasses, sucrose;

[0119] Fermentation was carried out according to the method in step 1, and the results showed that ( Figure 3 The results for glucose, sucrose, and molasses were similar, and glucose was superior to malt syrup. However, sucrose is more expensive, and the pre- and post-treatment of molasses is cumbersome and varies significantly between batches. Considering all factors, glucose was chosen as the carbon source.

[0120] (2) The specific implementation method is the same as step 1, except that the nitrogen source in the fermentation medium in step (2) of step 1 is adjusted to: ammonium sulfate, tryptone, yeast extract powder, urea (concentration of 30g / L);

[0121] Fermentation was carried out according to the method in step 1, and the results showed that ( Figure 4 Tryptone and yeast extract are more effective than urea and ammonium sulfate. Consider using a compound organic nitrogen source for the experiment: the specific implementation method is the same as step 1, except that the nitrogen source in the fermentation medium in step (2) of step 1 is adjusted as follows: 3% tryptone, 2% tryptone + 1% yeast extract, 1% tryptone + 2% yeast extract, and 3% yeast extract are used as nitrogen sources respectively. The results show that ( Figure 5 The optimal nitrogen source was 2% tryptone + 1% yeast extract. The optimized fermentation medium consisted of 20 g / L glucose, 20 g / L tryptone, 10 g / L yeast extract, 3 g / L potassium dihydrogen phosphate, 1.5 g / L dipotassium hydrogen phosphate, 0.5 g / L anhydrous magnesium sulfate, and 5 g / L anhydrous calcium chloride.

[0122] Example 4: Fermentation of Saccharomyces cerevisiae

[0123] (1) Preparation of seed liquid

[0124] Samples of the obtained Schizosaccharomyces pombe strain were taken from cryopreservation tubes, inoculated into seed culture medium, and cultured in a shaking medium at 30℃ (180 rpm) for 1 day. When the culture medium became turbid, it was spread on YPD solid culture medium plates and incubated in a constant temperature incubator at 30℃ for 2 days to activate the strain.

[0125] A single colony was picked and inoculated into an Erlenmeyer flask containing 180 mL of seed culture medium. The flask was then incubated at 30°C in a shaking incubator (180 rpm) for 1.5 days. At this time, the OD value of the bacterial culture was measured. 600 The value was around 15. Microscopic examination of the sample showed no contamination by other microorganisms and more than 80% of the yeast was in the process of fission, so it could be used as a seed culture.

[0126] (2) High-density fermentation

[0127] The cultured seed solution was prepared at a volume ratio of 8% (v / v) (1×10) 8 (CFU / mL) was inoculated into the fermentation medium (5L glass fermenter, 45% loading). The culture conditions were: 30℃, pH 4.5, dissolved oxygen 30%. The feed medium consisted of 50% glucose solution and 5% yeast extract. The initial fermentation speed was 200 rpm, and the aeration rate was 4 L / min. Feeding was started after 20 hours of fermentation, and fermentation was stopped after 48 hours to obtain the cultured fermentation broth.

[0128] The experimental results were obtained by measuring the OD of the fermentation broth after fermentation. 600 Discover the final OD 600 The concentration reached 30.2. The fermentation broth was centrifuged at 4℃ and 7500rpm for 15min, and the liquid phase was removed to obtain bacterial cells. The bacterial cells were added to a mixture of 5% glucose, 4% glycerol, 1% trehalose and 10% skim milk, mixed well, and cultured at 30℃ for 1h. After freeze-drying, 39g of freeze-dried powder was obtained.

[0129] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A strain of *Schizosaccharomyces pombe* JGWN-SP04, characterized in that, The aforementioned *Schizosaccharomyces cerevisiae* strain was deposited at the China Center for Type Culture Collection (CCTCC) on July 11, 2025, with accession number CCTCC NO: M 20251563.

2. A microbial inoculant, characterized in that, The microbial agent contains the millet wine fission yeast JGWN-SP04 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent also contains glucose, tryptone, yeast extract, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, anhydrous magnesium sulfate, and anhydrous calcium chloride.

4. The microbial agent according to claim 3, characterized in that, Glucose 50-100 g / L, tryptone 10-30 g / L, yeast extract 10-20 g / L, potassium dihydrogen phosphate 1-3 g / L, dipotassium hydrogen phosphate 0.5-1.5 g / L, anhydrous magnesium sulfate 0.5-2 g / L, anhydrous calcium chloride 1-10 g / L.

5. The method for high-density culture of *Schizosaccharomyces cerevisiae* according to claim 1, characterized in that, Including the following steps: (1) Seed culture: Select a single colony and inoculate it into seed culture medium to obtain seed liquid; (2) Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculation rate of 2-10% for fermentation.

6. The high-density culture method according to claim 5, characterized in that, The conditions for seed culture and fermentation culture are 25-35℃ for 36-48 hours.

7. The high-density culture method according to claim 5, characterized in that, The seed culture medium comprises, by mass, 50–100 g / L glucose, 10–30 g / L tryptone, and 10–20 g / L yeast extract.

8. The high-density culture method according to claim 5, characterized in that, The fermentation medium comprises, by mass, 10–30 g / L glucose, 10–30 g / L tryptone, 10–20 g / L yeast extract, 1–3 g / L potassium dihydrogen phosphate, 0.5–1.5 g / L dipotassium hydrogen phosphate, 0.5–2 g / L anhydrous magnesium sulfate, and 1–10 g / L anhydrous calcium chloride.

9. The application of the strain according to claim 1 and the microbial agents according to claims 2 to 4 in brewing.

10. The application of the microbial inoculant according to claim 9 in brewing, characterized in that, The alcoholic beverages mentioned are wine, spirits, and fruit wine.

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