Breeding and application of a high amylase-producing strain

Abstract

The application discloses a high amylase-producing strain and application thereof, and belongs to the field of microorganisms. The application screens a high amylase-producing Mucor indicus SYH-1# and uses the high amylase-producing characteristic of the Mucor indicus SYH-1# to prepare a strong extension wine medicine, so as to solve the problem that the original process cannot be directly applied to the brewing of yellow rice wine due to the insufficient amylase activity. The application improves the amylase activity, the paste starch amylase activity and the fermentation power of the strong extension wine medicine, thereby stably improving the quality of the wine medicine. The strain can also be applied to the preparation of wheat koji, bran koji and soy sauce koji, and the fermentation of fermented foods such as rice wine, yellow rice wine, vinegar, soy sauce and fermented bean curd, so as to improve the yield and quality.

Description

Technical Field

[0001] This invention relates to the selection and application of a high amylase-producing strain, belonging to the field of microbiology. Background Technology

[0002] Raw amylase is a class of amylases, including α-amylase, β-amylase, glucoamylase, and debranching enzymes. Raw amylase refers to enzymes that directly hydrolyze ungelatinized starch granules. Raw amylase can directly break down raw starch into glucose, combining the traditional starch gelatinization, liquefaction, and saccharification processes into a single, direct saccharification step.

[0003] Raw amylase first adsorbs onto the surface of starch granules, hydrolyzing them and creating a porous structure. It then enters the starch granules through these channels, hydrolyzing the starch from within. The exoglucanase's glutamate activity creates numerous tiny pores on the starch surface, making them sharp and deep, while the endoglucanase's α-amylase enlarges these pores. The combined action of these two enzymes catalyzes the continuous release of glucose from the starch granules through these pores. Amylase can also break down starch in yeast into fermentable sugars such as glucose and maltose. These sugars are essential nutrients for yeast fermentation, promoting its growth and reproduction.

[0004] Amylase activity is one of the important indicators of yeast quality. Yeast, also known as koji or white yeast, is made from raw rice flour, Polygonum hydropiper, and mother koji powder, and is a key saccharification and fermentation agent for handmade rice wine. Yeast contains various brewing microorganisms such as Rhizopus, Mucor, and yeast. Therefore, screening for high amylase-producing strains from yeast is a good option. Current research reports that Aspergillus niger and Bacillus amyloliquefaciens have high amylase activity, but they are not suitable for use in yeast production. Mucor indicus, on the other hand, is a dicrystalline, highly safe, non-pathogenic fungus. It is widely found in starch-based fermented foods and has important industrial applications. While its abundance and quantity in yeast are relatively low, it has the ability to decompose raw starch, produce ethanol, and generate certain characteristic flavor compounds, such as 2-phenylethanol, isoamyl alcohol, ethyl oleate, and limonene.

[0005] In existing technologies, using *Mucor indicus* with an abundance of over 98% in the production of Fangxian rice wine has improved the stability and yield of the wine. In the study of yeast propagation, the gelatinization amylase activity of the propagated yeast was increased to 616.01±83.55 U / g by strengthening the inoculation with *Saccharomyces cerevisiae*. However, because its raw amylase activity is low, microorganisms in the yeast that cannot utilize starch, such as brewing yeast, non-brewing yeast, and lactic acid bacteria, will grow and reproduce poorly. This results in the yeast being unsuitable for direct use in rice wine brewing. Therefore, there is an urgent need to select and breed high-amylase-producing strains to solve the problem of low raw amylase activity in yeast. Summary of the Invention

[0006] In order to further optimize the quality of expanded fermentation yeast, strengthen the control of the fermentation process, and solve the problem of insufficient activity of raw amylase.

[0007] This invention provides a strain of *Mucor* in India, which has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231800, deposit date of September 26, 2023, and deposit address of Wuhan University, Wuhan, Hubei Province.

[0008] The present invention also provides a microbial preparation containing the aforementioned *Rhizopus indicus*.

[0009] In one embodiment, the microbial preparation is a live bacterial preparation obtained from Mucor acinosa through production and propagation.

[0010] In one embodiment, the microbial preparation is a liquid preparation, including but not limited to spore suspensions.

[0011] In one embodiment, the microorganism is a solid preparation, including but not limited to a live bacterial preparation made by using porous materials as adsorbents (such as peat moss or stone) to adsorb bacterial cells from fermentation broth.

[0012] In one embodiment, the content of *Rhizopus ininus* in the microbial preparation is ≥1×10⁻⁶. 7 spores / g or ≥1×10 7 spores / mL.

[0013] This invention provides a method for preparing enhanced amylase-active fermented wine starter, which involves culturing *Rhizopus indicus* SYH-1#, which produces high amylase activity, as an enhanced inoculum for fermentation and expansion of the wine starter.

[0014] In one embodiment, the culture is carried out under the following conditions;

[0015] (1) Ferment in a closed environment for the first 12 hours, so that the temperature reaches 30-35℃ and the humidity reaches 90-95%;

[0016] (2) From the 13th to the 20th hour, when the core temperature rises to 35℃, turn on the ventilation to keep the core temperature at 35-37℃; turn the core appropriately.

[0017] (3) From the 21st to the 25th hour, ventilate intermittently and keep the temperature at 30±5℃;

[0018] (4) From the 26th to the 48th hour, the temperature was lowered and kept warm, so that the temperature was 25±5℃;

[0019] In one embodiment, after the fermentation in step (4) is completed, the product is dried at 38±1℃ until the moisture content is between 8 and 10% to obtain the finished fortified wine yeast.

[0020] In one embodiment, the enhanced culture of the wine-drug specifically includes the following steps:

[0021] S1: Use raw indica rice flour and wheat bran as raw materials, and grind Polygonum hydropiper and mother koji powder into powder and sift for later use;

[0022] S2: Mix raw indica rice flour, wheat bran, mother koji powder, and Polygonum hydropiper in a certain proportion to prepare a culture medium. Add the Indian mold strengthening agent to the culture medium, mix evenly, sieve and put it into a solid fermentation box for cultivation. After fermentation, cool down, dehumidify and ventilate to dry to obtain the fortified raw amylase expanded wine yeast.

[0023] In one embodiment, the weight proportions of each raw material are as follows:

[0024] 1000 parts rice flour, 300 parts wheat bran, 10 parts mother koji powder, 7.5 parts Polygonum hydropiper, and Rhizopus indicum fungicide (spore concentration 1×10⁻⁶). 7 100 parts (CFU / mL) and 500 parts water. The mother koji powder and Polygonum hydropiper powder should be sieved through a 50-mesh sieve.

[0025] In one embodiment, the bran used as a raw material for fortified wine yeast production can be raw material or treated with high-pressure steam sterilization.

[0026] In one embodiment, the mother koji powder includes, but is not limited to, brewing koji obtained from within China, such as Shaoxing koji from Zhejiang, Fengwo koji from Xiaogan, Xinhua koji from Hunan, Guizhou koji, Xiamen white koji, Ningbo white koji, Sichuan koji, and Suzhou sweet wine koji.

[0027] In one embodiment, the starter culture powder is obtained from a Shaoxing rice wine factory, and the total bacterial count in the starter culture powder is approximately 1.5 × 10⁻⁶. 8 CFU / g, containing microorganisms including but not limited to Pediococcus pentosaceus, Weissella esculenta, and Weissella fusionis; total fungal count is approximately 4.5 × 10⁻⁶. 7 CFU / g contains microorganisms including but not limited to *Saccharomyces cerevisiae*, ...Rhizopus microsporum*, and *Mucor*.

[0028] This invention also provides the application of Rhizopus in India CCTCC NO: M 20231800 or its metabolites in improving the activity of raw amylase and gelatinizing amylase in the fermentation field.

[0029] In one embodiment, the fermentation field includes, but is not limited to, yeast, raw wheat koji, cooked wheat koji, bran koji, fermented bean curd koji, and daqu (a type of starter culture).

[0030] In one embodiment, the Indian Mucor-enhanced culture yeast is used in fermented foods including but not limited to: rice wine, raw rice wine, cooking wine, sweet rice wine or rice wine, vinegar, soy sauce, and baijiu (Chinese liquor).

[0031] Beneficial effects:

[0032] (1) This invention provides a high amylase-producing Rhizopus ininus SYH-1# screened from wine yeast, which can be applied to the production of wheat koji, bran koji, soy sauce koji, fermented bean curd and other fermented foods (yellow wine, rice wine, soy sauce, vinegar, etc.), improving the utilization rate and quality of raw materials, and saving energy and resources.

[0033] (2) The *Rhizopus indicus* SYH-1# obtained by screening in this invention has the characteristic of high amylase production and can be used to strengthen the cultivation of yeast starter. When *Rhizopus indicus* SYH-1# is applied to strengthen the cultivation of yeast starter, the raw amylase activity of the yeast starter can reach 160.18±7.42 U / g, and the gelatinized amylase activity can reach 706.94±19.33 U / g. The strengthened yeast starter produced can be used in the brewing of fermented foods such as rice wine, rice wine, and vinegar. It also provides the possibility for the brewing of raw rice wine and can be used directly to replace cooked wheat koji in rice wine brewing.

[0034] (3) This invention provides a method for preparing fortified yeast using *Rhizopus indicus* SYH-1#. The fortified yeast prepared by this method can significantly increase the yield of rice wine (1.92±0.02), which is higher than the yield of Fangxian rice wine brewed using *Rhizopus indicus* yeast (1.87±0.05). Compared with rice wine brewed without fortified yeast, using *Rhizopus indicus* SYH-1# fortified yeast can significantly increase the volatile aroma substances in rice wine.

[0035] Preservation of biological materials

[0036] Mucor indicus SYH-1#, classified as Mucor indicus SYH-1#, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231800, deposited on September 26, 2023, at Wuhan University, Wuhan, Hubei Province. Attached Figure Description

[0037] Figure 1 Amylase activity and liquefaction power in solid-state fermentation.

[0038] Figure 2 : Colony and microscopic images of Mucor SYH-1# from India; from left to right: colony morphology, 160x microscopic image, and 400x microscopic image.

[0039] Figure 3Phylogenetic tree of *Mucor syH-1#*.

[0040] Figure 4 : Volatile flavor compounds in rice wine. Detailed Implementation

[0041] (I) Technical Terminology:

[0042] Wine starter: The "wine starter" mentioned in this invention refers to a saccharifying and fermenting agent used in brewing, possessing saccharification and fermentation functions. It can also be called koji, white yeast, or wine cake. In some embodiments of this invention, the wine starter mainly contains microorganisms such as *Pediococcus pentosaceus*, *Cytomyces cladosporioides*, *Rhizopus microsporus*, *Mucor*, and *Saccharomyces cerevisiae*. In some embodiments of this invention, the *Mucor* SYH-1# wine starter is a wine starter with increased *Mucor* SYH-1# cell count, which can be used in the fermentation process of alcoholic beverages such as rice wine, sweet rice wine, and rice wine, or fermented condiments such as vinegar and soy sauce.

[0043] Enhanced microbial agents: The "enhanced microbial agents" mentioned in this invention refer to microbial preparations containing beneficial functional microbial strains used in the original system (liquid, semi-solid, or solid environment). The enhanced microbial agents are added to the system in a certain proportion to exert a specific effect.

[0044] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0045] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the distribution of selectable values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every value between these two endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer.

[0046] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0047] In this invention, if the unit for a data range is only followed by the right endpoint, it indicates that the units for the left and right endpoints are the same. For example, 20–200 rpm means that the units for the left endpoint “20” and the right endpoint “200” are both rpm. The “data” in “data range” can be any quantitative value, such as a number, percentage, or proportion. “Data range” can broadly include quantitative ranges such as percentage intervals, proportion intervals, and ratio intervals.

[0048] In this invention, when defining particle size using sieve mesh number, for example, using 50 mesh means that it can pass through a 50-mesh sieve.

[0049] In this invention, the term "room temperature" generally refers to 4℃~35℃, preferably 20℃±5℃; in some embodiments of this invention, room temperature refers to 20℃~25℃.

[0050] (II) Culture medium:

[0051] Enrichment medium (g / L): glucose 20.0, peptone 20.0, soluble starch 5.0, NaCl 10.0, pH 7.0, sterilized at 121℃ for 20 min.

[0052] Screening medium (g / L): raw indica rice flour 20.0, NaNO3 3.0, K2HPO4 1.0, MgSO4·7H2O 0.5, KCl 0.5, FeSO4·7H2O 0.01, agar 15.0, pH 5.5; the indica rice flour needs to be dry heat sterilized at 160℃ for 2h. After the other components are cooled to 45℃, they are aseptically added to the sterilized medium, quickly shaken to mix, poured into plates, cooled and solidified for later use.

[0053] Potato glucose agar (PDA) medium: 200 g / L potato (peeled) to make an extract, 20 g / L glucose, and 15 g / L agar (solid).

[0054] Liquid fermentation medium (g / L): The liquid fermentation medium is a selection medium without added agar. Fermentation conditions are 30℃, 200 rpm. Sterilize at 121℃ for 20 min.

[0055] Simulated solid culture medium: 30g of indica rice, 9g of wheat bran, and 0.22g of Polygonum hydropiper, sterilized by dry heat at 160℃ for 2 hours.

[0056] (III) Detection Methods:

[0057] Assay for raw amylase activity:

[0058] Accurately pipette 25 mL of 2% rice flour suspension (ultrasonically dissolved) into a 50 mL colorimetric tube; add 5 mL of acetate-sodium acetate buffer solution (pH = 4.6), shake well, and preheat in a 40℃ constant temperature water bath shaker for 5 min; then add 2 mL of fermentation supernatant, shake immediately, and start timing; react at 40℃ and 160 r / min for 1 h, stirring every 20 min. After the reaction, add 0.2 mL of 20% (w / v) NaOH to terminate the reaction. Take 1 mL of the reaction solution and immediately add 1 mL of DNS solution, shake well. Boil in a boiling water bath for 5 min, and immediately cool in ice water. Under the same conditions, the blank group is prepared by first adding 0.2 mL of 20% (w / v) NaOH, followed by 2 mL of fermentation supernatant. Add distilled water to the above reaction system to make up to 10 mL, mix well, and measure the absorbance at 540 nm using a 1 cm cuvette. Calculate the amount of reducing sugar produced using the glucose standard curve.

[0059] Accurately weigh 1.00g of solid fermentation koji (convert to oven-dry koji for calculation), place it in a 50mL autoclaved centrifuge tube, add 18mL of deionized water and 2mL of acetate-sodium acetate buffer solution, and extract in a 40℃ constant temperature water bath for 1h. Then, sift the extract through a 10000r·min cycle. -1 Centrifuge at 4℃ for 10 min, and take the supernatant as crude enzyme solution. Determine the raw amylase activity of the enzyme-producing strains to be tested.

[0060] Enzyme activity (U) is defined as the amount of enzyme required to release 1 mg of reducing sugar (equivalent to glucose) from 1 mL of fermentation broth or 1 g of oven-dried koji at pH 4.0 and 40℃ by hydrolyzing raw (rice, cassava, corn, etc.) starch or gelatinized starch for 1 hour. The enzyme activity unit for crude enzyme solution is U / mL, and the enzyme activity unit for koji is U / g.

[0061] Determination of gelatinizing amylase activity:

[0062] Except for replacing the reaction substrate, raw indica rice flour, with gelatinized soluble starch, the method is the same as that for determining the activity of raw amylase.

[0063] Raw starch digestion ability (RDA) is calculated as follows: RDA = B / A × 100%; where B is the activity of enzymes that degrade raw starch and A is the activity of enzymes that degrade gelatinized starch.

[0064] Morphological and molecular biological identification of raw amylase strains:

[0065] Mold morphology observation: The strain was picked up with an inoculation loop and spotted onto the center of a PDA plate. It was incubated at 28°C for 3–5 days. Colony morphology was observed, and the colony shape, color, and edge condition were recorded. A small amount of mycelium with spores was picked up from the edge of the colony with an inoculation loop and placed in a clean glass slide containing lactic acid, carbolic acid, and cotton blue staining solution. The mycelium was spread out, and a coverslip was placed on top. Observation was performed under a microscope, and the morphology of spores, sporangia, and mycelium was recorded.

[0066] Genomic DNA was extracted from the strain using the CTAB method, and identification was performed using ITS universal primers.

[0067] The amplification primer sequences were ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system consisted of 12.5 μL of 2×San Taq PCR Mix, 1 μL each of ITS1 and ITS4, 1 μL of DNA template, and ddH2O to a final volume of 25 μL. The PCR amplification conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 15 s, 72℃ extension for 45 s, for 35 cycles, followed by a final extension at 72℃ for 10 min. The concentration and quality of the PCR amplification products were assessed by agarose gel electrophoresis. PCR amplification products with correct band size and appropriate concentration were sent to Shanghai Sangon Biotech for DNA sequencing. The obtained microbial sequences were compared for homology with the NCBI database using BLAST. The known sequence with the highest homology match to the tested strain was selected, and the species of the microorganism was determined based on the comparison results. The phylogenetic tree of the strain was constructed using MEGA11.0 software to study the phylogenetic relationship between the tested strain and other bacterial species for species identification.

[0068] Physicochemical index determination: The liquefaction power and fermentation power of the expanded culture wine yeast were determined according to the method in QBT4257-2011. Acidic protease activity was determined according to the method in GB1886.174-2016. Volatile flavor compounds were quantitatively determined using headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS).

[0069] Yield: The yield of rice wine is usually expressed as the percentage of total liquid produced to the weight of glutinous rice raw material. For example, if the total liquid weight of 1 kg of glutinous rice is 2 kg, the yield is 2.00.

[0070] Vinegar yield: Calculated according to the following formula:

[0071] X = (X1 × M) ÷ (3.5 × m);

[0072] In the formula: X - vinegar yield when converted to vinegar with an acidity of 3.5 g / 100 mL, kg / kg; X1 - total acid content in the sample (calculated as acetic acid), g / 100 g; M - mass of mature vinegar mash, kg; m - total mass of all dry materials added before fermentation, kg.

[0073] Example 1: Screening of high amylase-producing strains in wine yeast

[0074] 1. Plate screening

[0075] Weigh 2g of the wine-making agent and place it in a 100mL Erlenmeyer flask. Add 25mL of enrichment medium and then incubate at 30℃ and 200r / min for 2h. Perform serial dilutions (10⁻⁶ ppm) of the enriched culture. -3 10 -4 10 -5 100 μL of the diluted solution was spread onto a plate selection medium with rice flour as the sole carbon source and incubated at 30°C for 3 days. Colony growth was observed, and strains with different colony morphologies were selected and isolated and purified into single colonies. The purified single colonies were then temporarily stored on PDA slant solid medium at 4°C.

[0076] After initial screening using agar plates, eight morphologically slightly different bacterial strains and eight mold strains were identified. Strains from the plates were collected using sterile toothpicks and inoculated onto screening medium. After incubation at 30°C for 2 days, 0.05% dilute iodine solution was added. The diameter of the clear zone (Dh) around the colony and the colony diameter (Dc) were measured using calipers, and their ratio was calculated. Colonies with larger diameters were identified as molds. Colonies with higher ratios were selected as the initial screening strains. A larger clear zone on the plate indicates more starch breakdown and a stronger amylase-producing ability in the strain.

[0077] Table 1. Initial screening results of amylase-producing bacteria

[0078]

[0079]

[0080] 2. Fermentation and secondary screening of strains

[0081] Four bacterial strains with a Dh / Dc ratio > 1.8 and eight mold strains with a Dh / Dc ratio > 1.00, totaling 12 strains, were selected. Single colonies or spores were inoculated into liquid fermentation medium to evaluate their enzyme production capacity under liquid conditions. Their starch degradation capacity was also calculated.

[0082] The initially screened strains were inoculated into fermentation medium and cultured at 30℃ with shaking at 200 rpm for 3 days. All strains were then adjusted to the same concentration (1×10⁻⁶). 7(CFU / mL) Take 10 mL of culture medium, centrifuge at 12,000 r / min for 5 min, collect the supernatant, and determine the activity of raw amylase and gelatinized amylase in the fermentation supernatant. Three replicates were performed for each strain, and the average value was calculated. As shown in Table 2, strains with raw amylase activity greater than 100 U / mL and 7 strains with RDA > 30% were selected for solid-state enzyme production evaluation.

[0083] Table 2 Results of secondary screening of amylase-producing bacteria

[0084]

[0085] Single-strain solid-state fermentation screening: 50% sterile water was added to the simulated solid-state fermentation medium, and the inoculum concentration was 1×10⁻⁵ at a rate of 5%. 7 The CFU / mL seed culture was mixed thoroughly with a sterilized spatula, sealed with film, and placed in an incubator at 30℃ and 80% relative humidity for 3 days. After 18 hours of incubation, once the mycelium had formed clumps, the first shake was performed. After 28 hours, shake and inverted culture were performed, followed by horizontal culture after 40 hours. The fermented koji were then placed in koji trays and dried in an oven at 38℃ for 12 hours. Subsequently, the amylase activity and liquefaction power were measured. Three replicates were performed for each strain, and the average value was calculated. Results are as follows: Figure 1 As shown, strain SYH-1# exhibited the highest amylase activity and liquefaction power during solid-state fermentation, at 227.29 U / g and 0.9186 U / g, respectively.

[0086] Example 2: Identification of high amylase-producing strains

[0087] The strain SYH-1# screened in Example 1 was inoculated onto PDA solid agar plates and its colony morphology was observed. The results are as follows: Figure 2 As shown. The colonies are loose, appearing as white or grayish-white flocculent particles with a loose texture and neat edges. The hyphae can penetrate the culture medium. In later stages, black spores appear at the top of the well-developed hyphae. Its microscopic characteristics include erect, highly branched conidiophores, large, spherical sporangia containing a large number of spores at the apex. It is preliminarily determined that this strain belongs to the genus Mucor.

[0088] Total DNA was extracted from the strain and used as a template to amplify its ITS1–ITS4 region. The amplified sequence of the strain was determined, and the results were analyzed using BLAST in the NCBI database. The results showed that the ITS nucleotide sequence exhibited 98% homology with *Mucorindicus* in the GenBank database. Their molecular phylogenetic tree is shown below. Figure 3 As shown. Based on morphological analysis of the strain, SYH-1# was identified as *Mucor indicus*.

[0089] Example 3: Preparation of Mucor microbial preparations

[0090] Mucor syH-1# was isolated and purified from glycerol tubes, and then aseptically inoculated onto PDA solid agar plates for activation and subculturing. Mycelia were picked and inoculated onto PDA liquid agar plates and cultured at 30°C for 48 h to obtain a spore concentration of 1×10⁻⁶. 7 CFU / mL spore suspension.

[0091] Example 4: Enhanced inoculation process for producing expanded wine yeast from Mucor spp.

[0092] Preparation of strain seed culture: *Rhizopus indusia* SYH-1# was isolated and purified from glycerol tubes, and activated under aseptic conditions by inoculation onto PDA solid agar plates. Spores were then collected and inoculated onto PDA liquid agar plates and cultured at 30℃ for 48 h. The spore concentration was adjusted to 1×10⁻⁶. 7 CFU / mL as a bacterial agent.

[0093] Table 3. Raw material ratio for enhanced culture and expansion of winemaking agents

[0094]

[0095] According to the raw material ratio in Table 3, mix rice flour, wheat bran, and four-fifths of the total amount of yeast starter powder (obtained from Shaoxing Rice Wine Factory) evenly to obtain a mixed culture medium. Then, slowly add water to the mixed culture medium while stirring, stirring clockwise until well mixed, and pass the clump of culture medium through a 10-mesh sieve. Line a solid fermentation box with four layers of gauze (or two layers of coarse gauze steamer cloth), pile the evenly mixed raw materials into the solid fermentation box to a thickness of 2-3 cm, and then evenly sprinkle the remaining one-fifth of the yeast starter powder on the surface. Cover the box and start fermentation. Place thermometers and hygrometers around the yeast starter and at the center (center and surface) to record the fermentation temperature.

[0096] The solid-state fermentation process is as follows:

[0097] (1) The first 12 hours are the pre-fermentation heating stage, during which microorganisms grow and generate heat. Fermentation takes place in the fermentation box with the temperature gradually rising to 30-35℃ and the humidity reaching 90-95%. High humidity is conducive to the spread and growth of microorganisms.

[0098] (2) The 13th to 20th hour is the continuous ventilation and temperature control stage. When the core temperature rises to 35℃, continuous ventilation is turned on. Under good ventilation control, the core temperature is maintained at 35-37℃. If the temperature is too high, the core needs to be turned over. This is to cool down the core, as excessively high temperatures will inhibit fungal growth, and to increase the oxygen content in the core chamber, as well as to remove excess moisture. At this time, the humidity on the surface of the core drops sharply due to the water absorption process of microbial growth.

[0099] (3) The period from 21 to 25 hours is the intermittent ventilation and temperature control stage, which requires manual control to maintain the temperature at 30±5℃ and the humidity gradually increases.

[0100] (4) The 26th to 48th hour is the post-fermentation cooling and heat preservation stage, with a temperature of 25±5℃. During this stage, the temperature and humidity are relatively stable, and the microorganisms are in the metabolic enzyme production stage.

[0101] (5) After fermentation, place it in an oven at 38°C for about 12 hours to dry until the moisture content is between 8 and 10%, which is the finished product of the fermentation starter. Then pack it in a self-sealing bag and store it in a cold storage at 4°C for later use.

[0102] Comparative Example 1: Unvaccinated booster culture of alcoholic beverages

[0103] The specific implementation method is the same as in Example 4, except that the Mucor SYH-1# bacterial solution is replaced with the same volume of sterile water. The results are shown in Table 4.

[0104] Comparative Example 2: Enhanced Rhizopus indicus JM25 culture wine yeast

[0105] The specific implementation method is the same as in Example 4, except that Rhizopus indicus SYH-1# is replaced with Rhizopus indicus JM25 (published in the paper "Research on Core Microorganisms and Controlled Solid-State Fermentation of Wine Fermentation Agents"), and the same volume and concentration of bacterial solution are used to expand the wine fermentation agent.

[0106] The physicochemical properties of the above-mentioned expanded culture starter are shown in Table 4. Compared with the unfortified expanded culture starter of Comparative Example 1, the raw amylase activity of the fortified starter prepared in Example 4 was 160.18 ± 7.42 U / g, an increase of approximately 314.6%. Furthermore, the gelatinization amylase activity increased by approximately 57.03%, while liquefaction power, acidic protease power, and fermentation power also significantly improved. Compared with Comparative Example 2, the raw amylase activity increased by 181.3%, and the gelatinization amylase activity increased by 20.15%.

[0107] Table 4 Physicochemical Indicators of Fortified Wine Agents

[0108]

[0109]

[0110] Note: Different superscripts in the same column indicate significant differences between the data, p < 0.05, and the same applies below.

[0111] Example 5: Enhanced application of Rhizopus in India SYH-1# cultured yeast in rice wine

[0112] The fortified wine starter was prepared according to the method in Example 4 and used in the brewing of rice wine. The specific steps are as follows:

[0113] (1) Soaking rice: Soak 10kg of glutinous rice in sufficient deionized water at room temperature for 2-3 days.

[0114] (2) Washing rice: After slowly pouring out the rice slurry, rinse it with tap water and let it stand until the water drains.

[0115] (3) Steaming rice: Steam the rice in a rice steamer for 30 minutes until the rice is cooked but not mushy and has no filling inside.

[0116] (4) Spreading the rice: Cool the steamed rice to 30℃~35℃.

[0117] (5) Mixing of ingredients according to the new formula: Since the yeast prepared in Example 4 has high activity of raw amylase and gelatinized amylase, the use of cooked wheat koji (which provides high saccharification power) required for brewing rice wine is omitted. Ingredients are added according to the ingredient list in Table 5, mixed evenly, and then placed in a constant temperature fermentation tank for fermentation. The total fermentation time is 20 days. During the fermentation process, attention should be paid to stirring and ruffling. The pre-fermentation temperature is controlled at 30±2℃, and the pre-fermentation lasts for 5 days, with stirring once in the morning and once in the evening. The post-fermentation temperature is 15±2℃, and the fermentation lasts for 15 days, with stirring once every 5 days.

[0118] Table 5 Ingredients for Yellow Rice Wine Brewing

[0119]

[0120] Comparative Example 3: Yellow Rice Wine Brewed Without Fortification and Amplification of Wine Fermentation Agents

[0121] The specific implementation method is the same as in Example 5, except that the wine yeast prepared in Comparative Example 1 is used to replace the fortified Indian Mucor SYH-1# wine yeast.

[0122] Comparative Example 4: Yellow Rice Wine Brewing with Enhanced Mucor JM25 Cultured Yeast

[0123] The specific implementation method is the same as in Example 5, except that the wine yeast prepared in Comparative Example 2 is used to replace the fortified Indian Mucor SYH-1# wine yeast.

[0124] The physicochemical properties of the fermented rice wine are shown in Table 6. The results show that the rice wine in Comparative Example 3 is prone to spoilage under the same conditions. This may be due to insufficient carbon source in the raw materials, leading to poor yeast growth. Example 5, with its enhanced inoculation of *Rhizopus indicus* SYH-1#, not only produces amylase in the yeast starter, breaking down starch to produce glucose and providing a good energy source for yeast growth, but also produces ethanol and various flavor compounds during the rice wine fermentation process, such as... Figure 4As shown, this significantly increased the levels of volatile flavor compounds in rice wine, including esters (ethyl acetate, ethyl propionate, ethyl lactate, etc.), alcohols (n-propanol, isobutanol, 2-phenylethanol, etc.), and aldehydes (furfural, cinnamaldehyde, vanillin, etc.). The resulting rice wine had a higher alcohol content and lower sugar content; the higher yield indicates full utilization of the raw materials. The physicochemical properties of the rice wine met the requirements of the national standard GB / T13662-2018 for rice wine, and it possessed a rich aroma, balanced body, and smooth taste.

[0125] Table 6 Physicochemical Indicators of Shaoxing Wine

[0126]

[0127] Example 6: Enhancing the application of Rhizopus ininus SYH-1# yeast in raw rice wine

[0128] Raw material: 10kg of broken glutinous rice (passed through a 20-mesh sieve): It must be white, fresh, free of mold and impurities.

[0129] Soaking Rice and Mixing with Yeast: Soak broken glutinous rice in a vat for 2 days, drain, and transfer to a clean large vat. Add 6% of the weight of *Rhizopus indicus* SYH-1# fortified yeast and mix well. Then, make a funnel-shaped indentation in the center, incubate for saccharification, and add water at a grain-to-water ratio of 1:1.1. Simultaneously add 15.6% wheat koji and a compound enzyme, and stir well. The compound enzyme consists of α-amylase and amylase glucoside enzyme, with each enzyme added at 0.1‰ of the fermentation system weight. The α-amylase was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an enzyme activity concentration ≥3500 U / mL. The amylase glucoside enzyme was purchased from... Beijing Innocare Technology Co., Ltd. The enzyme activity concentration is ≥100000U / mL.

[0130] Fermentation: The total fermentation time is 20 days. During the fermentation process, pay attention to stirring and raking. The temperature for the first fermentation is controlled at 30±2℃, and the first fermentation lasts for 5 days, with stirring once in the morning and once in the evening. The temperature for the second fermentation is 15±2℃, and the fermentation lasts for 15 days, with stirring once every 5 days.

[0131] Due to the high amylase activity of *Rhizopus indicus*, this process eliminates the need for large amounts of enzyme preparations to decompose the raw starch, allowing for the brewing of raw rice wine. The resulting rice wine is sweet, refreshing, and has a unique style. It is a dry rice wine with an alcohol content of 12.5% ​​vol, total acidity of 5.15 ± 0.30 g / L, and total sugar of 6.70 ± 0.20 g / L.

[0132] Example 7: Enhancing the application of Rhizopus ininus SYH-1# yeast in sweet rice wine

[0133] Soak an appropriate amount of glutinous rice at room temperature for 24 hours; drain the water and steam under normal pressure for 30-40 minutes until the rice has no white center; spread the rice out to cool, then pour in 30%-40% of the weight of raw rice in cooled boiled water to loosen the rice. Lower the rice temperature to 28-30℃ and mix in 0.5%-1% fortified yeast. Mix well, then transfer to a fermentation container, gently flatten it, and make a small indentation in the center. Ferment at a constant temperature of 28-30℃ in a sealed container for 48-72 hours. The resulting sweet rice wine is milky white, has good fermentation, a harmonious aroma, is sweet, has an alcohol content of approximately 3.0% vol, and a total sugar content of 110±5.80 g / L. Using the yeast from Comparative Example 1 will not allow the sweet rice wine to ferment successfully, leading to spoilage. The alcohol yield of the fortified Indian Rhizopus yeast was 1.60%, while the alcohol content of the wine made using Comparative Example 2, Indian Rhizopus JM25 fortified yeast, was 2.5% vol, the total sugar content was 102±8.40 g / L, and the alcohol yield was 1.53%. The alcohol content, total sugar content, and alcohol yield were all lower than those of the sweet wine fermented with fortified Indian Rhizopus SYH-1# yeast.

[0134] Example 8: Enhancing the application of Rhizopus ininus SYH-1# yeast in rice wine

[0135] Soak an appropriate amount of glutinous rice at room temperature for 24 hours; the rice grains should be intact and easily crumble into powder when pinched, with no hard core. Rinse the soaked rice with clean water and drain. Steam under normal pressure for about 30-40 minutes until the rice is cooked but not mushy, the grains are fluffy, and there is no white core inside. Cool the steamed rice powder with cold boiled water to 28-30℃, mix in 0.5%-1% of the weight of glutinous rice in fortified yeast, and saccharify for 36-48 hours. After 4 / 5 of the liquid in the yeast has become sweet, add water at 100%-120% of the rice weight, stir well, and then start the pre-fermentation at 28℃ for 4-6 days. After the main fermentation is completed, carry out low-temperature (15℃) post-fermentation, with a fermentation cycle of about 15-20 days. After fermentation, press, filter, and distill to obtain rice wine. Rice wine is rich in aroma, harmonious in flavor, and has a rich taste. The alcohol content is 12.0% vol, and the yield is 1.85%, which is high. The alcohol content of the wine made using the fortified yeast of Rhizopus in India JM25 (Comparative Example 2) was 10.5% vol, with a yield of 1.76%.

[0136] Example 9: Enhancing the application of Rhizopus ininus SYH-1# yeast in vinegar

[0137] S1. Fermentation preparation: Select 10kg of high-quality glutinous rice, soak the rice for 24 hours, then drain the rice until there is no white slurry, steam it, rinse it with water and cool it to 25-30℃; before putting the glutinous rice into the vat, mix in 0.15-0.2kg of enhanced Indian Mucor SYH-1# yeast, and saccharify at low temperature for 72-96 hours.

[0138] S2. Alcoholic fermentation: After saccharification, add 3 kg of water and 0.6 kg of wheat koji, and ferment at 28℃ for 144-168 hours to obtain mature mash;

[0139] S3. Making mash: Put 15kg of wheat bran into the fermentation tank and spread it evenly. Put the fermented mash into the tank and stir it evenly. Take 0.5kg of rice husks and spread them 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 husks, and spread it evenly to complete the making mash.

[0140] S4. Acetic acid fermentation: Turn the mash every 24 hours of fermentation. After each turning, add rice husks to keep it warm and moist. Stop adding rice husks from the 11th day of fermentation. Turn the mash to cool the product temperature. After 20 days, when the acidity no longer rises, add 0.4 kg of salt and seal for 45 days.

[0141] S5. Vinegar Leaching and Decoction: The vinegar mash after aging is leached using a cyclic leaching method. Sugar is added to the resulting vinegar juice for blending. After clarification, the vinegar is decocted. Once the temperature drops to 75–80℃, it is bottled and sealed for storage. The total acidity of vinegar brewed using *Rhizopus indicus* enhanced culture is 5.25±0.42 g / 100 mL, amino acid nitrogen is 1.08±0.16 g / 100 mL, soluble non-salt solids are 1.38±0.32 g / 100 mL, and the vinegar yield is 3.12 kg / kg.

[0142] Comparative Example 5: Preparation of Vinegar from Rhizopus indicus JM25 as a Starter

[0143] Vinegar was brewed using the same method as in Example 9, except that the *Rhizopus indicus* JM25 starter prepared in Comparative Example 2 was used instead of the fortified *Rhizopus indicus* SYH-1# starter. The results showed that the prepared vinegar had a total acidity of 4.86±0.65 g / 100 mL, amino acid nitrogen of 0.85±0.32 g / 100 mL, soluble non-salt solids of 1.20±0.28 g / 100 mL, and a vinegar yield of 2.96 kg / kg. Therefore, applying *Rhizopus indicus* to the solid-state vinegar fermentation process can improve the quality and yield of vinegar, reduce production costs, and to some extent enhance the taste of the vinegar.

[0144] Example 10: Process for producing raw wheat koji from Rhizopus indicus SYH-1#

[0145] Raw wheat koji accounts for more than 1 / 10 of the total raw wheat koji used in the brewing process of Shaoxing wine. The raw amylase activity of the factory-made koji is low, approximately 40-50 U / g, meaning the raw starch it contains is difficult to completely break down, resulting in low raw material utilization. Inoculating with *Rhizopus indicus* SYH-1# can increase the raw amylase activity and improve the utilization rate of the raw material. The specific steps are as follows:

[0146] (1) Preparation of seed liquid of Mucor SYH-1# in India, same as in Example 4.

[0147] (2) After properly crushing 50kg of wheat, spray it with water. When crushing, make sure that each grain of wheat is crushed into 3 to 5 pieces.

[0148] (3) Add the Indian Mucor SYH-1# seed liquid obtained in step (1) to the crushed wheat obtained in step (2) at a rate of 20% (v / m), stir evenly, and pay special attention to avoid the formation of white core clumps formed by starch clumps absorbing water. Cultivate in a solid fermentation chamber with a stack thickness of 10cm. The koji cultivation process is divided into four stages: the start-up heating stage, the self-heating fermentation stage, the cooling stage, and the drying stage. The koji block after drying is the finished koji. The fermentation process is controlled as follows:

[0149] S1. The first 0-24 hours is the start-up and heating stage: the start-up temperature of the curing room is 25℃, and the temperature of the core of the curing room slowly increases from room temperature to 30℃ within 12-24 hours. During this stage, the relative humidity of the curing room is maintained at 95±2%.

[0150] S2. The 25th to 72nd hour is the self-heating fermentation stage: the temperature of the koji block rises from 30℃ to 40℃; when the temperature detection device detects that the temperature of the koji core is higher than that of the koji surface (temperature difference greater than 3℃), the temperature control device is turned off, and the temperature is maintained only by the biological heat of microbial growth. When the temperature is too high, the ventilation is automatically adjusted to a high level. When the temperature is too low, the ventilation is at a low level or there is no ventilation. The relative humidity is 95% to 99%. This stage is maintained for 24 to 48 hours.

[0151] S3. The 73rd to 90th hour is the cooling stage: that is, the temperature slowly decreases. The temperature of the briquette block starts to decrease from 35 to 40°C and slowly drops to about 28 to 30°C within 18 hours; while maintaining the relative humidity below 85%.

[0152] S4. The drying stage is from 91 to 120 hours: When the relative humidity gradually decreases to 65%, maintain this condition for about 12 hours, then take out the finished koji and dry it at 38℃ to complete the koji drying. The amylase activity of the prepared wheat koji is 148.82±12.50U / g.

[0153] Comparative Example 6: Production Process of Rhizopus ininus JM25 Wheat Koji

[0154] The specific implementation method is the same as in Example 10, except that the same volume and concentration of *Rhizopus indicus* SYH-1# was replaced with *Rhizopus indicus* JM25 bacterial culture. The results showed that the amylase activity of the wheat koji prepared from *Rhizopus indicus* JM25 was 77.50 ± 4.18 U / g.

[0155] Example 11: Process for producing bran koji from Mucor SYH-1#

[0156] After dry heat sterilization of 1 kg of wheat bran, add approximately 50% water and inoculate with 5% by weight of *Rhizopus indicus* SYH-1# bacterial culture (preparation method as in Example 4). In a solid-state fermentation chamber, maintain the temperature at 30±5℃ and ferment for 60 hours to obtain the koji. The raw amylase activity of the obtained wheat bran koji is 105.60±15.20 U / g.

[0157] Comparative Example 7: Production Process of Rhizopus ininus JM25 Bran Koji

[0158] The specific implementation method is the same as in Example 11, except that the *Rhizopus indicus* SYH-1# is replaced with *Rhizopus indicus* JM25 bacterial culture of the same volume and concentration. The results showed that the raw amylase activity of the *Rhizopus indicus* JM25 bran koji was 40.60 ± 2.85 U / g.

[0159] Example 12: Enhanced process for producing soy sauce koji using Rhizopus indicus SYH-1#

[0160] S1. Starter culture: Mix wheat bran, soybean meal, flour, and water in a large Erlenmeyer flask at a mass ratio of 8:1:1:10. Spread the mixture evenly, seal with cotton plugs, sterilize, and then shake to loosen the starter culture. After cooling, inoculate the sterilized flask with *Aspergillus oryzae* (CCTCC NO: M 2015201) and *Rhizopus indicus* SYH-1# bacterial solution (1×10⁻⁶ concentration) under aseptic conditions. 7 Incubate the culture medium (CFU / mL) at a 1:3 ratio, upright in a 30°C incubator. After 15–24 hours, white colonies will appear on the medium, and when the medium begins to form a cake, gently shake the Erlenmeyer flask to break up the clumps and spread them evenly at the bottom. After 24–48 hours of incubation, the mycelium will have multiplied extensively, and the medium will have formed a cake. At this point, invert the flask and incubate for approximately 72 hours.

[0161] S2. Soy sauce koji preparation: Select 20kg of plump and clean soybeans, wash them, soak them in water for 5-10 hours, and drain them after they have fully absorbed water and expanded. Then steam them under pressure until cooked. After the hot soybeans are taken out of the steamer, cool them to about 70-80℃, add 30-40% flour (based on the weight of soybeans), stir well, continue to cool to 30℃, then add 5% koji starter, stir well, and maintain the temperature at about 28-30℃ for fermentation. Ferment the koji in a ventilated fermentation incubator. After 72-96 hours, you will get soy sauce koji.

[0162] The quality of the soy sauce koji was tested, and the results showed that the activity of raw amylase in the soy sauce koji was 126.18±10.22U / g, and the activity of acidic protease was 75.76±6.48U / g.

[0163] Comparative Example 8: Preparation of Soy Sauce Koji from Rhizopus ininus JM25

[0164] The specific implementation method is the same as in Example 12, except that the *Rhizopus indicus* SYH-1# is replaced with *Rhizopus indicus* JM25 of the same volume and concentration.

[0165] The amylase activity of the soy sauce koji obtained in Comparative Example 8 was 105.24±9.58 U / g, and the acidic protease activity was 60.76±4.95 U / g. The addition of *Rhizopus ininus* SYH-1# resulted in more complete starch saccharification and liquefaction reactions, and increased acidic protease activity. The functional microorganisms (molds, yeasts, and lactic acid bacteria) in the koji grew well, which had a significant impact on the color, aroma, flavor, and body of the soy sauce in the later stages.

[0166] Example 13: Process for making fermented bean curd from Mucor SYH-1#

[0167] S1. Preparation of raw soy milk: Soak soybeans until they swell, then remove them, grind them into a paste, filter them through a cloth to obtain raw soy milk.

[0168] S2. Preparation of tofu blocks: After mixing evenly, boil for 20 minutes, let stand to remove residue, slowly add 0.96% glacial acetic acid to the soy milk for coagulation, and keep warm at 80℃ for 30 minutes. Press, cut into blocks, and obtain tofu blocks;

[0169] S3. Preparation of fermented bean curd blanks: A suspension of *Rhizopus indicus* SYH-1# was prepared (1×10⁻⁶). 7 The tofu blanks were sprayed with CFU / mL until they were not dripping wet, and then fermented at a temperature of 25-32℃ and a humidity of 80-90% for 35-40 hours. Mold mycelium was generated on the surface of the tofu blanks, and the raw tofu blanks were obtained.

[0170] S4. Pickling of fermented bean curd blanks: Smooth the mycelium on the surface of the fermented bean curd blanks, add 5% salt, and pickle.

[0171] The quality of fermented bean curd was tested and analyzed. The results showed that the amino acid nitrogen content of fermented bean curd made with Rhizopus ininus SYH-1# was 0.50±0.12g / 100g and the water-soluble protein content was 1.65±0.16g / 100g.

[0172] Comparative Example 9: Process for preparing fermented bean curd using Mucor JM25

[0173] The specific implementation method is the same as in Example 13, except that the *Rhizopus indicus* SYH-1# is replaced with *Rhizopus indicus* JM25 of the same volume and concentration.

[0174] The fermented bean curd from Comparative Example 9 had an amino acid nitrogen content of 0.41±0.09 g / 100g and a water-soluble protein content of 1.32±0.15 g / 100g. The fermented bean curd prepared from *Rhizopus indicus* SYH-1# exhibited better stability during fermentation, a more delicious flavor, and superior quality.

[0175] 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 type of Indian Mucor ( Mucor indicus SYH-1# has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20231800, deposited on September 26, 2023, at Wuhan University, Wuhan, Hubei Province.

2. A microbial preparation containing *Rhizopus indicus* SYH-1# as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation is a live bacterial preparation obtained from Mucor acinosa through production and propagation.

4. The microbial preparation according to claim 3, characterized in that, The microbial preparation is a liquid preparation, including but not limited to spore suspension.

5. The microbial preparation according to claim 3, characterized in that, The microorganisms are solid preparations, including but not limited to live bacterial preparations made by using porous materials as adsorbents to adsorb bacterial cells from fermentation broth.

6. The microbial preparation according to any one of claims 3 to 5, characterized in that, The content of *Rhizopus ininus* in the microbial preparation is ≥1×10⁻⁶. 7 spores / g or ≥1×10 7 spores / mL.

7. A method for enhancing the activity of raw amylase in wine yeast, characterized in that, The Indian Mucor SYH-1# fermentation and expansion yeast as described in claim 1 is applied.

8. A method for preparing fortified wine yeast, characterized in that, Includes the following steps: S1: Using raw indica rice flour and wheat bran as raw materials, grind the raw materials into powder and sift them for later use; S2: Mix the raw materials with the mother koji powder and the Indian mold SYH-1# as described in claim 1, and culture them under certain conditions to obtain a fortified wine starter with improved raw amylase activity.

9. The method according to claim 8, characterized in that, Cultivate under the following conditions; (1) Ferment in a sealed environment for the first 12 hours, so that the temperature reaches 30~35 ℃ and the humidity reaches 90-95%; (2) From the 13th to the 20th hour, when the core temperature rises to 35 ℃, turn on the ventilation to keep the core temperature at 35~37 ℃; turn the core appropriately; (3) From the 21st to the 25th hour, ventilate intermittently and maintain the temperature at 30±5 ℃; (4) From 26 to 48 hours, cool down and keep warm to maintain a temperature of 25±5℃.

10. The method according to claim 9, characterized in that, After fermentation in step (4), the product is dried at 38±1℃ until the moisture content is between 8 and 10% to obtain the finished fortified wine yeast.

11. The Indian Mucor SYH-1# fortified wine starter prepared by the method described in any one of claims 8 to 10.

12. The application of the Indian mold SYH-1# as described in claim 1 in improving the activity of raw amylase and gelatinized amylase in the fermentation field; the fermentation field includes the preparation of wine yeast, raw wheat koji, cooked wheat koji, bran koji, fermented bean curd koji or daqu.

13. The application of the Indian mold SYH-1# as described in claim 1 in the production of fermented foods.

14. The application according to claim 13, characterized in that, The fermented foods include: yellow rice wine, cooking wine, white wine, sweet rice wine, rice wine, vinegar or soy sauce.

15. The application according to claim 13, characterized in that, The fermented food includes raw rice wine.

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