A Highly Environmentally Stress-Tolerant Capsular Coated Yeast and Its Application in the Production of Aged Vinegar

Abstract

This invention relates to a highly stress-tolerant cyst-forming yeast and its application in aged vinegar production, belonging to the field of vinegar brewing technology. To screen for ester-producing yeast strains with high stress tolerance and apply them to the aged vinegar brewing process, this invention isolated a yeast strain ZLCY-93 from the fermentation mash and vinegar mash of Shanxi aged vinegar. This strain exhibits high enzyme production, high ester production capacity, stronger adaptability to high temperature, high acidity, high osmotic pressure, and high concentration of ethanol during fermentation, good fermentation performance, and outstanding aroma production. Morphological and molecular biological analysis identified this yeast strain as a cyst-forming yeast. This invention combines yeast ZLCY-93 with practical experience in aged vinegar production to create bran koji, which, when applied to aged vinegar production, can shorten the fermentation cycle, improve raw material utilization, promote flavor compound formation, and enhance product quality.

Description

Technical Field

[0001] This invention belongs to the field of vinegar brewing technology, specifically relating to a highly environmentally resistant vesicle-coated yeast and its application in the production of aged vinegar. Background Technology

[0002] Aged vinegar is made primarily from sorghum and wheat bran, supplemented with rice husks and millet husks, and using barley and peas as raw materials to create a saccharification and fermentation agent. After concentrated alcoholic fermentation, it undergoes high-temperature solid-state acetic acid fermentation, followed by processes such as smoking, vinegar extraction, and aging. During concentrated alcoholic fermentation, the initial sugar content reaches approximately 30%, and the ethanol concentration can reach 7% vol to 9% vol. In the acetic acid fermentation stage, the peak temperature can reach 45–50℃. During the brewing process, the initial pH is around 6.0, and the pH continuously decreases during fermentation, reaching a minimum of around 3.4.

[0003] Yeast is a crucial fungus in the brewing process of aged vinegar, directly or indirectly related to alcoholic fermentation, the formation of higher alcohols and esters, and the production of organic acids, significantly impacting the product's flavor. Among them, *Saccharomycopsis fibuligera*, also known as *Saccharomycopsis fibuligera*, is a dimorphic yeast capable of producing ascospores. It possesses the ability to produce flavor compounds such as acids, alcohols, and esters, as well as amylase, saccharifying enzymes, β-glucosidase, and acidic proteases. In the field of fermented foods, *Saccharomycopsis fibuligera* enriches aroma and enhances sensory quality. Furthermore, during fermentation, it degrades macromolecular substrates, providing nutrients for the growth of fermenting microorganisms such as *Saccharomycopsis*.

[0004] Yeasts can generally grow and ferment in a temperature range of 4–40℃, with an optimal temperature of 28–30℃ and an optimal fermentation temperature of 30–33℃. They are highly resistant to low temperatures but have poor tolerance to high temperatures. The optimal pH for growth and reproduction is 4.5–5.5, and they can also grow at pH 3.5–4, but growth is slower. Below pH 3.5, growth is significantly inhibited. Different yeasts have varying tolerances to alcohol. Generally, alcohol yeasts can ferment to 12% vol–14% vol alcohol concentrations, while other types of yeasts generally experience inhibited germination at 8.5% vol alcohol concentrations and completely cease reproduction at 10% vol alcohol concentrations. Different yeasts also have varying tolerances to osmotic pressure. They can generally grow in sugar solutions containing 10%–25% sugar, but higher concentrations result in high osmotic pressure, which is unfavorable for yeast reproduction.

[0005] The high concentration of ethanol, extreme temperatures, low pH, and high osmotic pressure during the brewing process of aged vinegar all significantly impact the proliferation and fermentation activity of yeast, hindering yeast growth and fermentation, and affecting the formation of flavor compounds and the fermentation cycle. Therefore, screening for ester-producing yeast strains with high stress tolerance and applying them to the aged vinegar brewing process is the microbiological basis for improving the production process conditions of aged vinegar, and also an effective method to improve the controllability and stability of flavor compound content. Summary of the Invention

[0006] In order to screen for ester-producing yeast strains with high tolerance to stress and use them in the aged vinegar brewing process, this invention provides a highly environmentally resistant blastocyst yeast. This blastocyst yeast is combined with the actual production of aged vinegar to make bran koji, which can be applied to the production of aged vinegar, shortening the fermentation cycle of aged vinegar, improving the utilization rate of raw materials, promoting the generation of flavor substances, and improving product quality.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first objective of this invention is to provide a *Saccharomycopsis fibuligera* ZLCY-93 strain, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28347, deposited on September 4, 2023, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] The second objective of this invention is to provide the application of the aforementioned ZLCY-93 sac-forming yeast in the brewing of aged vinegar.

[0010] A third objective of this invention is to provide a bran koji prepared from the aforementioned ZLCY-93 sac-forming yeast.

[0011] The fourth objective of this invention is to provide a method for preparing the above-mentioned bran koji, specifically comprising adding 40% to 80% water by weight of the bran to moisten it; steaming the material at 100 to 120°C for 15 to 20 minutes and cooling it to 28 to 32°C; inoculating it with 2% to 5% ZLCY-93 bacterial solution by weight of the bran and culturing it in a koji tank.

[0012] In one embodiment of the present invention, the fermentation process involves spreading the material evenly to maintain a consistent density, with a thickness of 20-30 cm. The material is then statically incubated for 8-12 hours at a product temperature of 26-30°C, a room temperature of 28-30°C, and a relative humidity of 90%-92%. When the product temperature rises to 32-36°C, a fan is turned on for ventilation, maintaining a product temperature of 30-32°C, a room temperature of 28-30°C, and a relative humidity of 92%-94%. Incubation continues for another 5-8 hours, followed by turning the material once, maintaining a room temperature of 28-30°C and a relative humidity of 90%-92%, and continuing incubation for another 5-8 hours. When the white mycelium coverage on the surface of the bran reaches 60%-70%, the incubation is terminated. The number of viable yeast cells is counted using a hemocytometer, and the count is not less than 1.0 × 10⁻⁶. 9 per g.

[0013] In one embodiment of the present invention, the ZLCY-93 bacterial culture is prepared as follows: The ZLCY-93 glycerol tube yeast strain, preserved at -80℃, is activated twice on PDA solid medium at a temperature of 30-32℃ for later use; two loops of bacterial cells are evenly scraped onto a plate and cultured in wheat bran juice liquid medium at 30-32℃ and 120-150 r / min for 16-20 h to obtain primary seed culture; the primary seed culture is inoculated into wheat bran juice liquid medium at an inoculation rate of 2%-5% and cultured in a constant temperature incubator at 30-32℃ for 14-16 h to obtain secondary seed culture for later use.

[0014] In one embodiment of the present invention, the preparation method of the above-mentioned bran juice liquid culture medium is as follows: 100g of bran is boiled in 500mL of water for 20min, cooled and filtered through gauze, 10g of glucose is added, water is added to 1L, and sterilized at 121℃ for 15min.

[0015] The fifth objective of this invention is to provide the application of the above-mentioned bran koji in the brewing process of aged vinegar.

[0016] In one embodiment of the present invention, the application is to use bran koji in the alcoholic fermentation stage or the acetic acid fermentation stage of aged vinegar brewing, or to use it in both the fermentation stage and the acetic acid fermentation stage simultaneously.

[0017] In one embodiment of the present invention, the specific method for using bran koji in the alcoholic fermentation stage of aged vinegar brewing is as follows: after saccharification by extrusion, sorghum is added to water at a temperature of 60-65°C, with a material-to-water mass ratio of 1:4.0-4.5. At the same time, Aspergillus niger AS3.4309 bran koji is added at a rate of 10%-15% of the sorghum mass, while stirring. After the addition is completed, the mixture is kept at 60-65°C for 20-30 minutes. The temperature is then lowered to 28-30°C, and 15%-35% of the sorghum mass of Daqu (a type of starter culture) and 10%-15% of the sorghum mass of ZLCY-93 bran koji are added. The fermentation temperature is controlled at 30-34°C. The mixture is fermented open for the first 3 days and then sealed for 4-7 days to obtain the mash.

[0018] In one embodiment of the present invention, the specific method for using wheat bran koji in the acetic acid fermentation stage of aged vinegar brewing is as follows: after the alcoholic fermentation is completed, auxiliary materials are mixed into the mash to form new mash; wherein, the auxiliary materials include wheat bran, rice husks, and paddy husks, and the addition amounts are 90%–110%, 80%–100%, and 30%–50% of the sorghum mass, respectively; and the moisture content of the new mash is 61%–68%, and the alcohol content is 4.0–4.5% vol; ZLCY-93 wheat bran koji is evenly covered on the new mash. On the surface of the mash, add ZLCY-93 bran koji at a rate of 10%–20% of the bran mass in the new mash. After 4–8 hours, ignite the mash with a ignition rate of 10%–15% of the new mash mass to initiate the acetic acid fermentation stage. Turn the mash daily. During fermentation days 1–3, control the temperature at 38–42℃. During fermentation days 4–6, control the temperature at 42–48℃. During fermentation days 7–8, control the temperature at 35–38℃. The acetic acid fermentation stage ends when the alcohol content of the vinegar mash is ≤0.2% vol.

[0019] In one embodiment of the present invention, the specific method for simultaneously using bran koji in the fermentation stage and the acetic acid fermentation stage is as follows: after saccharification by extrusion, sorghum is added to water at a temperature of 60-65°C, with a material-to-water mass ratio of 1:4.0-4.5. Simultaneously, Aspergillus niger AS3.4309 bran koji is added at a rate of 10%-15% of the sorghum mass, while stirring continuously. After the addition is complete, the mixture is kept at 60-65°C for 20-30 minutes; then cooled to 28-30°C, and 15%-35% of the sorghum mass of Daqu (a type of starter culture) and 10%-15% of the sorghum mass of ZLCY-93 bran koji are added. The fermentation temperature is controlled at 30-34°C. The mixture is left open for the first 3 days of fermentation, followed by sealed fermentation for 4-7 days to obtain mash. Auxiliary materials are then mixed into the mash to form new mash. These auxiliary materials include wheat bran, rice husks, and rice... The husks are added at amounts of 90%–110%, 80%–100%, and 30%–50% of the sorghum mass, respectively; the moisture content of the new mash is 61%–68%, and the alcohol content is 4.0%–4.5% vol. ZLCY-93 bran koji is evenly covered on the surface of the new mash, with the amount of ZLCY-93 bran koji added being 6%–10% of the bran mass in the new mash. After 4–8 hours, the mash is heated, with the amount of heating being 10%–15% of the new mash mass, and it enters the acetic acid fermentation stage. The mash is turned daily. During the first 1–3 days of fermentation, the temperature is controlled at 38–42℃. During the 4th–5th days of fermentation, the temperature is controlled at 42–48℃. During the 6th–8th days of fermentation, the temperature is controlled at 35–38℃. When the alcohol content of the vinegar mash is ≤0.2% vol, the acetic acid fermentation stage ends, and the mash enters the smoking, vinegar extraction, and aging stages.

[0020] The sixth object of the present invention is to provide an aged vinegar brewed from the above-mentioned bran koji.

[0021] The beneficial effects of this invention are:

[0022] This invention isolated and screened four high-ester-producing yeast strains from the fermentation mash and vinegar mash of Shanxi aged vinegar. The esterification, amylase, and protease production capabilities of these four strains were tested, and yeast ZLCY-93 with high enzyme production capacity was obtained. Environmental stress tests revealed that yeast ZLCY-93 can tolerate an acidic environment of pH=2, a high temperature of 52℃, an initial alcohol content of 12% vol, and an initial sugar content of 600 g / L, exhibiting extremely strong stress resistance. It demonstrates greater adaptability to the gradually developing high temperature and high acidity during fermentation, the high osmotic pressure under concentrated fermentation, and the high concentration of ethanol during metabolite accumulation, which is beneficial for maintaining good fermentation activity and improving fermentation efficiency.

[0023] The capsule-forming yeast can metabolize and produce amylase, saccharifying enzyme, and acidic protease, which are then secreted extracellularly. This promotes the decomposition and utilization of materials in the fermentation system, provides nutrition for other microorganisms involved in the fermentation process, such as brewer's yeast, and is beneficial to the reproduction of fermentation microbial communities and the progress of the fermentation process. Furthermore, it can synergistically interact with other microorganisms, which is conducive to the formation of flavor compounds.

[0024] The selected ZLCY-93 blastocyst yeast was prepared into bran koji and applied in the production of aged vinegar. Combined with various fermentation processes, it fully demonstrated the advantage of ZLCY-93 blastocyst yeast in high tolerance to environmental stress.

[0025] High osmotic pressure stress mainly occurs in the early stages of fermentation. In the concentrated alcoholic fermentation process of aged vinegar, the initial glucose concentration is around 30%. The high osmotic pressure of this glucose concentration has a significant negative impact on cell growth, thus requiring brewing yeast strains with high osmotic tolerance. Angel yeast exhibits high osmotic stress tolerance and fermentation alcohol production capacity. Therefore, in the alcoholic fermentation stage of aged vinegar production, in addition to adding Aspergillus niger bran koji and Daqu as starter cultures, Angel yeast is usually added to promote alcoholic fermentation. ZLCY-93 has even stronger osmotic tolerance. Application results of ZLCY-93 bran koji in alcoholic fermentation show that, with the same Daqu usage and a reduced Aspergillus niger bran koji usage, without adding Angel yeast, the alcohol content of the mature mash is no lower than that using Angel yeast, and the mash flavor is even better.

[0026] When ZLCY-93 bran koji is used in alcoholic fermentation, it is more conducive to the reproduction and fermentation of brewing yeast in the koji, shortening the alcoholic fermentation cycle and promoting the formation of flavor compounds. During the sealing fermentation stage of alcoholic fermentation, the pH value can reach as low as pH 3.4 or even lower. ZLCY-93 yeast still has the ability to reproduce at pH 2.0, so it can continue to play a role in the subsequent acetic acid fermentation stage.

[0027] During the high-temperature solid-state fermentation stage of aged vinegar, the peak temperature can reach 48-50℃, while ZLCY-93 can withstand temperatures up to 52℃. ZLCY-93 yeast is applied during the acetic acid fermentation stage. Its secreted hydrolytic enzymes utilize the newly added bran and other nutrients from the mixing stage, improving raw material utilization and reducing the turbidity of the freshly extracted vinegar. Simultaneously, it can utilize small-molecule acids such as acetic acid and lactic acid in the environment to react with ethanol to produce flavor compounds such as ethyl acetate and ethyl lactate, which is beneficial for improving the quality of aged vinegar. By controlling the amount of ZLCY-93 yeast added, the content of major esters in aged vinegar, such as ethyl acetate and ethyl lactate, can also be regulated.

[0028] The ZLCY-93 bran koji prepared by this invention can be used alone in the alcoholic fermentation stage, shortening the alcoholic fermentation cycle and increasing the total ester content of the final fresh vinegar by 20.35% and the total acid content by 10.18%. When used alone in the acetic acid fermentation stage, it increases the total ester content of the final fresh vinegar by 29.87% and the total acid content by 18.16%. If used simultaneously in both alcoholic and acetic acid fermentation stages, the total ester content of the final fresh vinegar increases by 43.72% and the total acid content by 19.56%.

[0029] When the ZLCY-93 bran koji prepared by this invention is applied to the fermentation process of aged vinegar, the aged vinegar obtained by the aging process can achieve the same quality as the newly fermented vinegar obtained without using ZLCY-93 bran koji, and the aging time is shortened by about 1 / 3, which helps to improve the production efficiency of aged vinegar. Attached Figure Description

[0030] Figure 1 The image shows the results of the amylase and protease production abilities of the cladomycin ZLCY-93; among them, Figure 1 In the figure, A represents the results of the amylase production capacity assay for ZLCY-93, a yeast with a cladocysteine ​​membrane. Figure 1 In the figure, B represents the results of protease production ability detection of ZLCY-93, a type of yeast with a cladostomata membrane.

[0031] Figure 2 The results of alcohol, pH, temperature, and sugar tolerance tests for ZLCY-93, a yeast with a capsid membrane.

[0032] Figure 3 Image showing the colony morphology of ZLCY-93, a yeast with a capillary membrane, on a PDA plate;

[0033] Figure 4 This is a microscopic observation of ZLCY-93, a yeast with a capsid membrane. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, and instruments used are all conventional materials, reagents, and instruments in the art, which can be obtained commercially by those skilled in the art.

[0035] The specific preparation method of ZLCY-93 bran koji in this invention is as follows:

[0036] ① Moistening: Add water to wheat bran to moisten the material. The amount of water should be 40% to 80% of the weight of wheat bran. Add water while stirring to allow the material to fully absorb the water and moisten it.

[0037] ② Steaming: Temperature 100~120℃, time 15~20min;

[0038] ③ Cooling: Cool the temperature to 28-32℃;

[0039] ④ Inoculation: The inoculation amount of ZLCY-93 bacterial solution is 2% to 5% based on the weight of wheat bran;

[0040] ⑤ Fermentation in the Fermentation Tank: After the material enters the fermentation tank, spread it evenly to maintain a consistent density, with a thickness of 20-30 cm. After inoculation, the material temperature is generally around 26-30℃, maintaining a room temperature of 28-30℃ and a relative humidity of 90%-92%. Let it stand for about 8-12 hours. When the material temperature rises to 32-36℃, turn on the fan for ventilation to maintain the material temperature at 30-32℃, maintaining a room temperature of 28-30℃ and a relative humidity of 92%-94%. Continue fermentation for 5-8 hours, then turn the fermentation koji once, maintaining a room temperature of 28-30℃ and a relative humidity of 90%-92%. Continue fermentation for 5-8 hours, until the ZLCY-93 bran koji has a light fruity aroma and the white mycelium coverage on the bran surface reaches 60%-70%, at which point the fermentation is complete. Use the hemocytometer to count the yeast cells; the viable yeast count should not be less than 1.0 × 10⁻⁶. 9 pcs / g;

[0041] ⑥ Storage: Transport the koji to a temporary storage warehouse for later use. Spread the koji out to cool and prevent backfire. Turn the koji if necessary.

[0042] The method for preparing ZLCY-93 bacterial culture is as follows:

[0043] The yeast strain ZLCY-93, preserved at -80℃, was activated twice on PDA solid medium and cultured at 30-32℃ for later use. Two loops of the bacterial cells were evenly scraped onto wheat bran juice liquid medium and cultured at 30-32℃ and 120-150 r / min for 16-20 h to obtain primary seed culture. The primary seed culture was inoculated into wheat bran juice liquid medium at an inoculation rate of 2%-5% and cultured in a constant temperature incubator at 30-32℃ for 14-16 h to obtain secondary seed culture for later use.

[0044] The above-mentioned bran juice liquid culture medium is prepared as follows: 100g of bran is boiled in 500mL of water for 20 minutes, cooled and filtered through gauze, 10g of glucose is added, water is added to 1L, and sterilized at 121℃ for 15 minutes.

[0045] The ZLCY-93 bacterial culture can also be replaced by inoculum cultured on sterilized wheat bran medium. When using inoculum for inoculation, the inoculation amount is 3% to 10% based on the weight of wheat bran.

[0046] The method for using the ZLCY-93 bran koji obtained by the above preparation method in the alcoholic fermentation of aged vinegar is as follows:

[0047] Sorghum is extruded and saccharified before being added to water heated to 60-65℃, with a material-to-water ratio of 1:4.0-4.5. Simultaneously, Aspergillus niger AS3.4309 bran koji is added at a rate of 10%-15% of the sorghum mass, while stirring continuously. After the addition is complete, the mixture is kept at 60-65℃ for 20-30 minutes and then rapidly cooled to 28-30℃. 15%-35% of the sorghum mass of Daqu (a type of starter culture) and 10%-15% of ZLCY-93 bran koji are added. The mixture is fermented open for the first 3 days and then sealed for 4-7 days. During the fermentation process, the temperature is controlled at 30-34℃. After fermentation, the mash is obtained. Add auxiliary materials to the mash to make new mash. The auxiliary materials include wheat bran, rice husks, and rice husks, with addition amounts of 90%–110%, 80%–100%, and 30%–50% of the sorghum mass, respectively. The new mash has a moisture content of 61%–68% and an alcohol content of 4.0%–4.5% vol. After mixing, the new mash is heated for 3–4 hours, with the amount of heated mash being 10%–15% of its mass. This initiates the acetic acid fermentation stage. The mash is turned daily. During fermentation days 1–3, the temperature is controlled at 38–42℃. During fermentation days 4–6, the temperature is controlled at 42–48℃. During fermentation days 7–10, the temperature is controlled at 35–40℃. When the alcohol content of the vinegar mash is ≤0.2% vol, the acetic acid fermentation stage ends, and the mash enters the smoking, vinegar extraction, and aging stages.

[0048] The method for using the ZLCY-93 bran koji obtained by the above preparation method in acetic acid fermentation during the brewing of aged vinegar is as follows:

[0049] Sorghum is saccharified by extrusion and then added to water heated to 60-65℃, with a material-to-water ratio of 1:4.0-4.5. Simultaneously, Aspergillus niger AS3.4309 bran koji is added, with the amount of bran koji being 20%-25% of the sorghum mass, while stirring. After the addition is complete, the temperature is maintained at 60-65℃ for 20-30 minutes. The temperature is then rapidly reduced to 28-30℃, and 15%-35% of the sorghum mass of Daqu (a type of starter culture) and 0.1%-0.2% of Angel Yeast are added. Fermentation is carried out open for the first 3 days, followed by sealed fermentation for 6-9 days. During the fermentation stage, the temperature is controlled at 30-34℃. After fermentation, the mash is obtained. Add auxiliary materials to the mash to make new mash; the auxiliary materials include wheat bran, rice husks and rice husks, with the addition amounts being 90%–110%, 80%–100%, and 30%–50% of the sorghum mass, respectively; and the moisture content of the new mash is 61%–68%, and the alcohol content is 4.0–4.5% vol. Evenly cover the surface of the new mash with ZLCY-93 bran koji, with the amount of ZLCY-93 bran koji added being 10% to 20% of the weight of the bran in the new mash. After 4 to 8 hours, ignite the mash, with the amount of ignition being 10% to 15% of the weight of the new mash, and enter the acetic acid fermentation stage. Turn the mash daily. During the first 1 to 3 days of fermentation, control the temperature at 38 to 42℃. During the 4th to 6th days of fermentation, control the temperature at 42 to 48℃. During the 7th to 8th days of fermentation, control the temperature at 35 to 38℃. When the alcohol content of the vinegar mash is ≤0.2% vol, the acetic acid fermentation stage ends, and the mash enters the smoking, vinegar extraction, and aging stages.

[0050] The method for simultaneously using the ZLCY-93 bran koji obtained by the above preparation method in the alcoholic fermentation and acetic acid fermentation of aged vinegar is as follows:

[0051] Sorghum is extruded and saccharified before being added to water heated to 60-65℃, with a material-to-water ratio of 1:4.0-4.5. Simultaneously, Aspergillus niger AS3.4309 bran koji is added at a rate of 10%-15% of the sorghum mass, while stirring continuously. After the addition is complete, the mixture is kept at 60-65℃ for 20-30 minutes and then rapidly cooled to 28-30℃. 15%-35% of the sorghum mass of Daqu (a type of starter culture) and 10%-15% of ZLCY-93 bran koji are added. The mixture is fermented open for the first 3 days and then sealed for 4-7 days. During the fermentation process, the temperature is controlled at 30-34℃. After fermentation, the mash is obtained. Add auxiliary materials to the mash to make new mash; the auxiliary materials include wheat bran, rice husks and rice husks, with the addition amounts being 90%–110%, 80%–100%, and 30%–50% of the sorghum mass, respectively; and the moisture content of the new mash is 61–68%, and the alcohol content is 4.0–4.5% vol. The surface of the mixed new mash is covered with a layer of ZLCY-93 bran koji, with the amount of ZLCY-93 bran koji added being 6% to 10% of the bran mass in the new mash. After 4 to 8 hours, the mash is heated, with the amount of heat added being 10% to 15% of the mass of the acetic acid fermentation mash. The acetic acid fermentation stage begins, with the mash being turned daily. During the first 1 to 3 days of fermentation, the temperature is controlled at 38 to 42℃. During the 4th to 5th days of fermentation, the temperature is controlled at 42 to 48℃. During the 6th to 8th days of fermentation, the temperature is controlled at 35 to 38℃. When the alcohol content of the vinegar mash is ≤0.2% vol, the acetic acid fermentation stage ends, and the mash enters the smoking, vinegar extraction, and aging stages.

[0052] Example 1: Screening, performance testing and identification of ZLCY-93, a highly environmentally resistant encapsulated yeast.

[0053] (I) Screening of ZLCY-93, a yeast with highly resistant environmental stress clasped capsules

[0054] (1) Screening of high ester-producing yeasts

[0055] Samples of fermented mash from Shanxi aged vinegar were collected on days 1, 3, 5, 7, 9, 11, 13, and 15 of alcoholic fermentation and on days 0, 2, 4, 6, 8, and 10 of acetic acid fermentation. Yeasts in the samples were isolated and purified using the traditional dilution plate coating method and PDA medium. The purified yeasts were then screened for high-ester-producing yeasts using the clear zone method, and a secondary screening of high-ester-producing yeasts was conducted by fermenting sorghum juice medium to determine the ester content.

[0056] The culture medium preparation method used in the above-mentioned clear zone method is as follows:

[0057] The PDA liquid culture medium and emulsion were mixed at a volume ratio of 9:1, and 1.8% agar was added and stirred until homogeneous. The culture medium was obtained at the natural pH. The emulsion was prepared by mixing polyvinyl alcohol (PVA) solution and glyceryl tartrate at a volume ratio of 9:1 and shaking thoroughly. The polyvinyl alcohol (PVA) solution was prepared by adding 3g of polyvinyl alcohol (PVA) to 100mL of distilled water and heating until fully dissolved.

[0058] The preparation method of the above-mentioned sorghum juice culture medium is as follows:

[0059] Add 100g of puffed sorghum to 900ml of water, then add 0.3g of saccharifying enzyme (the enzyme activity of saccharifying enzyme is 50000U / g), incubate at 60℃ for 1h, cool to room temperature, filter to obtain filtrate, and sterilize at 121℃ for 20min for later use.

[0060] The specific method for determining the ester production performance of the strain using sorghum juice culture medium is as follows: Activated yeast is inoculated into sorghum juice culture medium at an inoculation rate of 2%, cultured at 30℃ and 120r / min for 24h, and then placed at 30℃ for static culture for 48h; after fermentation, the total ester content of the fermentation broth is determined by the saponification reflux method.

[0061] This invention uses the transparent zone method to initially screen purified yeast for high ester-producing yeasts, and then uses sorghum juice culture medium fermentation to determine the ester content to further screen the yeasts obtained from the initial screening. Finally, four yeast strains with high ester-producing performance were selected, namely yeasts ZLCY-73, ZLCY-74, ZLCY-93 and ZLCY1088.

[0062] (2) Detection of the ability to produce esterase, amylase and protease

[0063] The esterification, amylase, and protease production capabilities of the four high-ester-producing yeast strains obtained above were detected using the clear zone method. The yeast strains were spotted onto various enzyme-producing media and incubated at 30°C for 48 hours. Enzyme production capability was expressed as the ratio of the clear zone diameter to the colony diameter. Simultaneously, the saccharifying enzyme activity of the four high-ester-producing yeast strains was detected. The saccharifying enzyme activity detection method used was as follows: activated yeast was inoculated at a 2% inoculum into sterilized bran medium and incubated at 30°C for 24 hours. The saccharification activity was then detected according to the saccharification power detection method in GB / T 4257-2008 "General Analytical Methods for Brewing Daqu".

[0064] The culture medium used for detecting the esterase production ability of the strains using the transparent zone method is the same as the culture medium used for the initial screening of high ester-producing yeasts in (1) above.

[0065] The culture medium used to detect the amylase production capacity of the strain using the clear zone method was formulated as follows: 1% soluble starch, 1% peptone, 0.5% glucose, 0.5% sodium chloride, 0.5% beef extract, 2% agar, and the remainder being water.

[0066] The culture medium formula used to detect the protease production ability of the strain using the clear zone method is as follows: 0.3% beef extract, 1% peptone, 0.5% sodium chloride, 1.5% skim milk powder, 2% agar, and the remainder is water;

[0067] The formula for the bran culture medium used in the above method for detecting saccharifying enzyme activity is: 50% bran and 50% water.

[0068] The test results are shown in Table 1. The test results for the amylase-producing and protease-producing abilities of yeast ZLCY-93 are as follows: Figure 1 As shown.

[0069] Table 1. Results of enzyme production capacity assay for four high-ester-producing yeast strains.

[0070]

[0071]

[0072] As shown in Table 1, compared with the other three yeast strains, yeast ZLCY-93 has more outstanding esterase production capacity, amylase production capacity and saccharification ability. Therefore, this invention will focus on studying the environmental stress resistance and other properties of yeast ZLCY-93.

[0073] (II) Performance Testing of ZLCY-93, a Highly Environmentally Stress-Resistant Encapsulated Yeast

[0074] (1) Test of the environmental stress tolerance of yeast ZLCY-93

[0075] The alcohol, pH, temperature, and sugar tolerance of yeast ZLCY-93 were determined. Specifically, activated yeast ZLCY-93 was inoculated at a 2% inoculum into YPD liquid medium with different alcohol concentrations (4% vol, 6% vol, 8% vol, 10% vol, 12% vol), different initial sugar concentrations (200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L), and different pH gradients (2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.5). After incubation at different temperatures (32℃, 36℃, 40℃, 44℃, 48℃, 52℃) and 120 r / min for 20 h, the OD was measured. 600nm Bacterial cell concentration. Tolerance test results are shown below. Figure 2 ,Depend on Figure 2It is known that yeast ZLCY-93 can tolerate an initial alcohol content of 12% vol, an acidic environment of pH 2.0, a high temperature of 52℃, and an initial sugar content of 600 g / L.

[0076] (2) Fermentation performance test of yeast ZLCY-93

[0077] The acid-producing and ethanol-producing capabilities of yeast ZLCY-93 were determined by fermentation in sorghum juice medium. Specifically, activated yeast ZLCY-93 was inoculated into sorghum juice medium at a 2% inoculum rate and cultured at 30℃ and 120 r / min for 24 h, followed by static culture at 30℃ for 48 h. After fermentation, the total acid content of the fermentation broth was determined by acid-base titration to evaluate the acid-producing ability of the strain. The ethanol-producing ability of the strain was evaluated by measuring the alcohol content of the fermentation broth. The alcohol content was determined as follows: after fermentation in the sorghum juice medium, 100 g of culture was added to 100 mL of distilled water, 100 mL of the solution was distilled off, and the alcohol content was measured using an alcohol meter.

[0078] The preparation method of sorghum juice culture medium is as follows:

[0079] Add 100g of puffed sorghum to 900ml of water, then add 0.3g of saccharifying enzyme (the enzyme activity of saccharifying enzyme is 50000U / g), incubate at 60℃ for 1h, cool to room temperature, filter to obtain filtrate, and sterilize at 121℃ for 20min for later use.

[0080] The fermentation experiment on sorghum juice culture medium showed that the alcohol production of yeast ZLCY-93 was 5.50±0.23% vol, and the acid production was 0.16±0.05 g / 100 mL.

[0081] (3) Detection of the types and contents of volatile aroma components produced by yeast ZLCY-93

[0082] The activated yeast ZLCY-93 was inoculated into sorghum juice culture medium at a 2% inoculation rate and cultured at 30℃ and 120 r / min for 24 h, and then placed at 30℃ for static culture for 48 h. After fermentation, the types and contents of volatile aroma components in the fermentation broth were systematically determined by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0083] The specific method of headspace-solid phase microextraction-gas chromatography-mass spectrometry is as follows:

[0084] Sample preparation: Take 3g of fermentation broth sample and place it in a 20mL headspace vial. Add 6mL of deionized water and 2.5g of sodium chloride, and seal with a cap with a silicone rubber septum.

[0085] GC parameters: HP-5MS column; injection port temperature 250℃; auxiliary heater on 250℃; temperature program: initial temperature 32℃, hold for 5 min, increase to 180℃ at 5℃ / min, then increase to 240℃ at 12℃ / min, hold for 10 min; carrier gas: high-purity helium, flow rate 0.8 mL / min, splitless.

[0086] MS parameters: EI ion source, electron energy 70 eV; ion source temperature 200℃; scan type MS1 scan, scan range 33–450; scan time 300 ms; step size 0.1; threshold 30.

[0087] The HS-SPME-GC-MS detection results are shown in Table 2. As shown in Table 2, yeast ZLCY-93 mainly produces 8 kinds of alcohols, 1 kind of aldehyde, and 23 kinds of esters after fermentation in sorghum juice medium.

[0088] Table 2. HS-SPME-GC-MS results of fermentation broth obtained from yeast ZLCY-93 fermented on sorghum juice medium.

[0089]

[0090]

[0091] (III) Identification of ZLCY-93, a yeast with highly resistant environmental stress clasped capsules

[0092] (1) Morphological identification

[0093] Yeast strain ZLCY-93 was inoculated onto PDA plates and cultured. Colony morphology was observed. The colony morphology characteristics of yeast ZLCY-93 are shown in [reference needed]. Figure 3 ,Depend on Figure 3 It was observed that single colonies of strain ZLCY-93 were white, regularly round, dry, and opaque, with fine hairs covering the surface and edges. The colonies were tightly bound to the culture medium and difficult to pick, with a diameter of 4–6 mm. The morphological characteristics of strain ZLCY-93 were observed under a microscope (see...). Figure 4 ),Depend on Figure 4 It can be seen that strain ZLCY-93 has yeast-like budding cells and filamentous cells, which is consistent with the morphological characteristics of yeast.

[0094] (2) Molecular biological identification

[0095] The ITS region of the DNA fragment of strain ZLCY-93 was amplified using primer pairs ITS1 (SEQ ID NO.2) and ITS4 (SEQ ID NO.3), and the obtained ITS sequence fragment is shown in SEQ ID NO.1. Alignment of the ITS sequence in GenBank revealed that the ITS sequence of strain ZLCY-93 showed the highest similarity to that of *Saccharomycopsis fibuligera*. Based on morphological characteristics, strain ZLCY-93 was identified as a cladomorph yeast.

[0096] SEQ ID NO.1:

[0097] ;

[0098] SEQ ID NO.2:

[0099] TCCGTAGGTGAACCTGCGG;

[0100] SEQ ID NO.3:

[0101] TCCTCCGCTTATTGATATGC.

[0102] The strain ZLCY-93 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.28347, on September 4, 2023. The address of the depository is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0103] Example 2: Application of ZLCY-93, a highly environmentally tolerant yeast with a capsid membrane, in the alcoholic fermentation stage of aged vinegar brewing.

[0104] (1) Prepare ZLCY-93 bran koji by processing ZLCY-93 sac-forming yeast.

[0105] Add 60% water (by weight of wheat bran) while stirring to ensure the material is fully moistened; steam at 110℃ for 18 minutes; allow to cool to 30℃; inoculate with ZLCY-93 bacterial solution at a rate of 3% (by weight of wheat bran); transfer the material to the koji-making tank, spreading it evenly to maintain a consistent density and a thickness of 25cm; after inoculation, maintain the temperature at approximately 26℃~30℃, keep the room temperature at 28℃~30℃, and the relative humidity at 90%~92%; allow to stand for 10 hours; wait for the temperature to rise. When the temperature reaches 32℃~36℃, turn on the fan for ventilation to maintain the product temperature at 30℃~32℃, room temperature at 28℃~30℃, and relative humidity at 92%~94%. Continue culturing for 6 hours, then turn the koji once, maintaining room temperature at 28℃~30℃ and relative humidity at 90%~92%. Continue culturing for 7 hours, until the ZLCY-93 bran koji has a light fruity aroma and the white mycelium coverage on the bran surface reaches 60%~70%, then the culturing is complete. Use the hemocytometer to count the yeast cells; the viable yeast count should not be less than 1.0×10⁻⁶. 9 Items / g; transport to the temporary storage warehouse for koji, spread out the koji to dry, prevent backfire, and turn the koji if necessary.

[0106] The preparation method of ZLCY-93 bacterial culture is as follows: The yeast strain ZLCY-93 glycerol tube, which is stored at -80℃, is activated twice on PDA solid medium and cultured at 30℃ for later use; two loops of bacterial cells are evenly scraped onto a plate and cultured in wheat bran juice liquid medium at 30℃ and 130 r / min for 18 h to obtain the primary seed culture; the primary seed culture is inoculated into wheat bran juice liquid medium at an inoculation rate of 3% and cultured in a constant temperature incubator at 30℃ for 15 h to obtain the secondary seed culture for later use.

[0107] The above-mentioned bran juice liquid culture medium is prepared as follows: 100g of bran is boiled in 500mL of water for 20 minutes, cooled and filtered through gauze, 10g of glucose is added, water is added to 1L, and sterilized at 121℃ for 15 minutes.

[0108] (2) Use the ZLCY-93 bran koji prepared in step (1) for alcoholic fermentation in the brewing of aged vinegar.

[0109] Sorghum was saccharified by extrusion and added to water at 62°C with a material-to-water ratio of 1:4.2. Simultaneously, Aspergillus niger AS3.4309 bran koji was added at a rate of 12% of the sorghum mass, while stirring. After the addition was completed, the mixture was kept at 62°C for 25 minutes. The temperature was then rapidly reduced to 29°C, and 20% of the sorghum mass of Daqu (a type of starter culture) and 12% of the sorghum mass of ZLCY-93 bran koji were added. The mixture was fermented in an open container for the first 3 days, followed by sealed fermentation for 5 days to obtain the mash. During the fermentation process, the temperature was controlled at 32°C. After alcoholic fermentation, auxiliary materials are mixed in to form new mash. These auxiliary materials include wheat bran, rice husks, and paddy husks, added at amounts of 110%, 90%, and 40% of the sorghum mass, respectively. The new mash has a moisture content of 65% and an alcohol content of 4.2% vol. 3.5 hours after mixing, the mash is heated, with the amount heated being 12% of the new mash mass, to enter the acetic acid fermentation stage. The mash is turned daily. During fermentation days 1-3, the temperature is controlled at 40℃; during fermentation days 4-6, the temperature is controlled at 45℃; and during fermentation days 7-10, the temperature is controlled at 37℃. When the alcohol content of the vinegar mash is ≤0.2% vol, the acetic acid fermentation stage ends, and the mash enters the smoking, vinegar extraction, and aging stages.

[0110] The control group differed from Example 2 in that the amount of Aspergillus niger AS3.4309 bran koji added was 24% of the sorghum weight, ZLCY-93 bran koji was not used, and 0.2% of Angel brewing yeast by weight of sorghum was added.

[0111] The mash obtained in Example 2 and the control group was tested, and the test results are shown in Table 3. The fresh vinegar obtained in Example 2 and the control group was tested, and the test results are shown in Table 4.

[0112] Table 3. Results of mash detection obtained in Example 2 and the control group.

[0113]

[0114] Table 4. Results of fresh lye detection obtained in Example 2 and the control group.

[0115]

[0116] As shown in Tables 3 and 4, when ZLCY-93 bran koji is applied to the alcoholic fermentation stage of aged vinegar brewing, with the amount of Daqu (fermentation starter) remaining unchanged, the amount of Aspergillus niger bran koji is reduced, and Angel yeast is not added, the alcohol content of the mature mash is not lower than that of the control group using Angel yeast. It also shortens the alcoholic fermentation cycle. In addition, the mash obtained can be used in the subsequent processes of aged vinegar brewing to obtain new vinegar with better flavor. Compared with the control group, the total ester content of the new vinegar obtained in Example 2 is increased by 20.35%, and the total acid content is increased by 10.18%.

[0117] Example 3: Application of ZLCY-93, a highly environmentally tolerant, vesicle-coated yeast, in the acetic acid fermentation stage of aged vinegar brewing.

[0118] The ZLCY-93 bran koji prepared in step (1) of Example 2 was used for acetic acid fermentation in the brewing of aged vinegar. The specific method is as follows:

[0119] Sorghum, after being extruded and saccharified, is added to water heated to 62°C at a material-to-water ratio of 1:4.2. Simultaneously, Aspergillus niger AS3.4309 bran koji is added at 24% of the sorghum weight, stirring constantly. After addition, the mixture is kept at 62°C for 25 minutes, then rapidly cooled to 29°C. At the same time, 20% of the sorghum weight of Daqu (a type of starter culture) and 0.2% of the sorghum weight of Angel Yeast are added for alcoholic fermentation to obtain mash. The mash is fermented open for the first 3 days, followed by sealed fermentation for 7 days, with the temperature controlled at 32°C during this period. After alcoholic fermentation, auxiliary materials are mixed in to form new mash. These auxiliary materials include wheat bran, rice husks, and paddy husks, added at 100%, 90%, and 40% of the sorghum weight, respectively. The new mash has a moisture content of 65% and an alcohol content of 4.2% vol. Evenly cover the surface of the new mash with ZLCY-93 bran koji, with the amount of ZLCY-93 bran koji added being 10% of the weight of the bran used for mixing the mash. After 6 hours, ignite the mash with fire, with the amount of fire being 12% of the weight of the acetic acid fermentation mash. The mash enters the acetic acid fermentation stage, and is turned daily. During the first 1-3 days of fermentation, the temperature is controlled at 40℃, during the 4th-6th days of fermentation, the temperature is controlled at 45℃, and during the 7th-10th days of fermentation, the temperature is controlled at 37℃. When the alcohol content of the vinegar mash is ≤0.2% vol, the acetic acid fermentation stage ends and vinegar mash is obtained, which then enters the smoking, vinegar extraction, and aging stages.

[0120] The control group differed from Example 3 in that the amount of bran used during mixing was 110% of the sorghum mass, and the new mash was heated 3.5 hours after mixing, without adding ZLCY-93 bran starter before heating.

[0121] The vinegar mash obtained in Example 3 and the control group was tested, and the test results are shown in Table 5. The fresh vinegar obtained in Example 3 and the control group was tested, and the test results are shown in Table 6.

[0122] Table 5. Detection results of vinegar mash obtained in Example 3 and the control group.

[0123]

[0124] Table 6. Results of fresh lye detection obtained in Example 3 and the control group.

[0125]

[0126] As shown in Tables 5 and 6, applying ZLCY-93 bran koji to the acetic acid fermentation stage of aged vinegar brewing can shorten the acetic acid fermentation cycle and obtain new vinegar with better flavor. Compared with the control group, the total ester content of the new vinegar obtained in Example 3 increased by 29.87% and the total acid content increased by 18.16%.

[0127] Example 4: Application of ZLCY-93, a type of yeast with a vegetative membrane, in the alcoholic and acetic acid fermentation stages of aged vinegar brewing.

[0128] The alcoholic fermentation process is the same as the alcoholic fermentation stage in Example 2. After the alcoholic fermentation is completed, auxiliary materials are mixed in to make new mash. The auxiliary materials include wheat bran, rice husks, and paddy husks, which are added at 100%, 90%, and 40% of the sorghum mass, respectively. The moisture content of the new mash is 65%, and the alcohol content is 4.2% vol. A layer of ZLCY-93 bran koji is covered on the surface of the mixed new mash. The amount of ZLCY-93 bran koji added is 6% of the bran koji mass. After 6 hours, the mash is heated, and the amount of heat is 12% of the mass of the acetic acid fermentation mash. The acetic acid fermentation stage begins. The mash is turned daily. During the first 1-3 days of fermentation, the temperature is controlled at 40℃. During the 4-5 days of fermentation, the temperature is controlled at 45℃. During the 6-8 days of fermentation, the temperature is controlled at 37℃. When the alcohol content of the vinegar mash is ≤0.2% vol, the acetic acid fermentation stage ends, and the mash enters the smoking, vinegar extraction, and aging stages.

[0129] The difference between the control group and Example 4 is that, during the alcoholic fermentation stage, the amount of Aspergillus niger AS3.4309 bran koji added was 24% of the sorghum mass, ZLCY-93 bran koji was not used, and 0.2% of Angel brewing yeast by weight of sorghum was added. During the acetic acid fermentation stage, the amount of bran used during mixing was 110% of the sorghum mass, and the new mash was heated 3.5 hours after mixing, and ZLCY-93 bran koji was not added before heating.

[0130] The vinegar mash obtained in Example 4 and the control group was tested, and the test results are shown in Table 7. The fresh vinegar obtained in Example 4 and the control group was tested, and the test results are shown in Table 8.

[0131] Table 7. Detection results of vinegar mash obtained in Example 4 and the control group.

[0132]

[0133] Table 8. Results of fresh lye detection obtained in Example 4 and the control group.

[0134]

[0135] Tables 7 and 8 show that applying ZLCY-93 bran koji to the alcoholic and acetic acid fermentation stages of aged vinegar brewing, while keeping the amount of Daqu (fermentation starter) constant and reducing the amount of Aspergillus niger bran koji, and without adding Angel yeast during alcoholic fermentation, resulted in an alcohol content in the mature mash that was no lower than the control group using Angel yeast. Furthermore, it shortened the cycles of alcoholic and acetic acid fermentation, leading to aged vinegar with superior flavor. Compared to the control group, the total ester content in the newly extracted vinegar increased by 43.72%, and the total acid content increased by 19.56%.

[0136] This invention utilizes the screened sac-forming yeast ZLCY-93 to prepare bran koji for application in aged vinegar production, fully leveraging the advantages of the fermentation process. High osmotic stress mainly occurs in the early stages of fermentation. In the concentrated alcoholic fermentation process of aged vinegar, the initial glucose concentration is around 30%. The high osmotic pressure of this glucose concentration has a significant negative impact on cell growth, thus requiring brewing yeast strains with high osmotic tolerance. Angel yeast exhibits high osmotic stress tolerance and fermentation alcohol production capacity. Therefore, in the alcoholic fermentation stage of aged vinegar production, in addition to adding Aspergillus niger bran koji and Daqu (a type of starter culture) as fermenting agents, Angel yeast is usually added to promote alcoholic fermentation. ZLCY-93 has even stronger osmotic tolerance. Application results of ZLCY-93 bran koji in alcoholic fermentation show that, with the same amount of Daqu used, reducing the amount of Aspergillus niger bran koji, and without adding Angel yeast, the alcohol content of the mature mash is no lower than that using Angel yeast, and the mash flavor is even better. ZLCY-93 bran koji, when used in alcoholic fermentation, is more conducive to the reproduction and fermentation of brewing yeast in the koji, shortening the alcoholic fermentation cycle while also promoting the formation of flavor compounds. During the sealing fermentation stage of alcoholic fermentation, the pH value can reach as low as pH 3.4 or even lower, but ZLCY-93 yeast still has the ability to reproduce at pH 2.0, thus continuing to play a role in the subsequent acetic acid fermentation stage. In the high-temperature solid-state fermentation stage of aged vinegar, the peak temperature can reach 48-50℃, and ZLCY-93 can withstand temperatures up to 52℃. The ZLCY-93 yeast starter is applied during the acetic acid fermentation stage. Its secreted hydrolytic enzymes facilitate the decomposition and utilization of nutrients such as newly added bran during the mixing stage, improving raw material utilization and reducing the turbidity of the newly extracted vinegar. Simultaneously, it can utilize small-molecule acids such as acetic acid and lactic acid in the environment to react with ethanol to produce flavor compounds such as ethyl acetate and ethyl lactate, which is beneficial for improving the quality of aged vinegar. By controlling the amount of ZLCY-93 yeast starter added, the content of major esters such as ethyl acetate and ethyl lactate in aged vinegar can also be regulated. When the ZLCY-93 yeast starter prepared in this invention is applied to the aged vinegar fermentation process, compared with newly extracted vinegar obtained without ZLCY-93 yeast starter, it achieves the same quality through aging, but the aging time is shortened by about one-third, thus improving the production efficiency of aged vinegar.

[0137] 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 defined by the claims.

Claims

1. Capsule-forming yeast ( Saccharomycopsis fibuligera ZLCY-93, accession number CGMCC NO.28347.

2. The application of the capsule-forming yeast ZLCY-93 as described in claim 1 in the brewing of aged vinegar.

3. A type of bran koji prepared from the ZLCY-93 blastocystis described in claim 1.

4. A method for preparing the bran koji according to claim 3, characterized in that, Add 40% to 80% water by weight of the wheat bran to moisten it; steam the material at 100 to 120°C for 15 to 20 minutes, and cool it to 28 to 32°C; inoculate 2% to 5% ZLCY-93 bacterial solution by weight of wheat bran and carry out fermentation in a fermentation tank.

5. The application of the bran koji as described in claim 3 in the brewing process of aged vinegar.

6. The application according to claim 5, characterized in that, Bran koji can be used in the alcoholic fermentation stage or the acetic acid fermentation stage of aged vinegar brewing, or both stages.

7. The application according to claim 6, characterized in that, The specific method for using bran koji in the alcoholic fermentation stage of aged vinegar brewing is as follows: After sorghum is saccharified by extrusion, it is added to water at a temperature of 60-65℃, with a material-to-water mass ratio of 1:4.0-4.

5. At the same time, bran koji prepared by Aspergillus niger AS3.4309 is added at a rate of 10%-15% of the sorghum mass, while stirring. After the addition is completed, the mixture is kept at 60-65℃ for 20-30 minutes. The temperature is then lowered to 28-30℃, and 15%-35% of the sorghum mass of Daqu (a type of starter culture) and 10%-15% of the sorghum mass of bran koji prepared by ZLCY-93 are added. The fermentation temperature is controlled at 30-34℃. The mixture is left open for fermentation for the first 3 days, and then sealed for fermentation for 4-7 days to obtain the mash.

8. The application according to claim 6, characterized in that, The specific method for using wheat bran koji in the acetic acid fermentation stage of aged vinegar brewing is as follows: After the alcoholic fermentation is completed, auxiliary materials are mixed into the mash to make new mash. These auxiliary materials include wheat bran, rice husks, and paddy husks, added at amounts of 90%–110%, 80%–100%, and 30%–50% of the sorghum mass, respectively. The new mash has a moisture content of 61%–68% and an alcohol content of 4.0–4.5% vol. Wheat bran koji prepared by ZLCY-93 is evenly spread on the surface of the new mash, with the amount added being 10%–20% of the wheat bran mass. After 4–8 hours, the mash is heated, with the amount heated being 10%–15% of the new mash mass, thus entering the acetic acid fermentation stage. The mash is turned daily. During fermentation days 1–3, the temperature is controlled at 38–42℃; during days 4–6, the temperature is controlled at 42–48℃; and during days 7–8… d. The temperature is controlled at 35~38℃. When the alcohol content of the vinegar mash is ≤0.2%vol, the acetic acid fermentation stage ends.

9. The application according to claim 6, characterized in that, The specific method for simultaneously using bran koji in the fermentation and acetic acid fermentation stages is as follows: After sorghum is extruded and saccharified, it is added to water at a temperature of 60-65℃, with a material-to-water mass ratio of 1:4.0-4.

5. Simultaneously, bran koji prepared from Aspergillus niger AS3.4309 is added at a rate of 10%-15% of the sorghum mass, while stirring. After the addition is complete, the mixture is kept at 60-65℃ for 20-30 minutes. The temperature is then lowered to 28-30℃, and 15%-35% of the sorghum mass of Daqu (a type of starter culture) and 10%-15% of the sorghum mass of bran koji prepared from ZLCY-93 are added. The fermentation temperature is controlled at 30-34℃. For the first 3 days of fermentation, the mixture is left open, followed by sealed fermentation for 4-7 days. d. Obtain the mash; mix the mash with auxiliary materials to make new mash; the auxiliary materials include wheat bran, rice husks, and paddy husks, with addition amounts of 90%~110%, 80%~100%, and 30%~50% of the sorghum mass, respectively; and the moisture content of the new mash is 61%~68%, and the alcohol content is 4.0~4.5% vol; evenly cover the surface of the new mash with bran koji prepared by ZLCY-93, with the addition amount of bran koji prepared by ZLCY-93 being 6%~10% of the wheat bran mass in the new mash; after 4~8 hours, heat the mash, with the amount of heat being 10%~15% of the new mash mass, to enter the acetic acid fermentation stage, turning the mash daily, and fermenting for 1~3 days. d. The temperature is controlled at 38~42℃. On the 4th to 5th day of fermentation, the temperature is controlled at 42~48℃. On the 6th to 8th day of fermentation, the temperature is controlled at 35~38℃. When the alcohol content of the vinegar mash is ≤0.2%vol, the acetic acid fermentation stage ends and the fermentation enters the smoking, vinegar leaching and aging stage.

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