A new type of fortified wine medicine and its solid state fermentation box

Abstract

The application discloses a novel fortified wine medicine and a solid-state fermentation box thereof, and belongs to the field of microbial technology and wine brewing production. The novel fortified wine medicine is prepared by solid-state fermentation of a functional fortified strain of Saccharomycopsis fibuligera, indica rice and bran as carriers, and addition of Chinese herbal medicines such as polygonum hydropiper, dried tangerine or orange peel and radix codonopsis, so as to improve the enzyme activity of the wine medicine and stabilize the quality of the wine medicine. The method provided by the application is simple and convenient to produce, the quality is stable and uniform, the production efficiency can be improved compared with the traditional process, the whole fermentation time of the medicinal wine is shortened, and the method has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a novel fortified wine starter, particularly a novel fortified wine starter that can be used in the brewing of rice wine, belonging to the field of fermentation engineering. Background Technology

[0002] Wine yeast, also known as koji or white yeast, is mainly used in the production of Shaoxing rice wine. It is a unique saccharification and fermentation agent used in Chinese brewing, with a dual function of simultaneous saccharification and fermentation. Wine yeast contains various brewing microorganisms such as Rhizopus and yeast. During the fermentation process, these microorganisms not only produce various enzymes that promote the saccharification and decomposition of raw materials, but also form abundant metabolites, giving traditional Shaoxing rice wine its rich aroma and full-bodied, mellow taste.

[0003] Recent studies have found that the main dominant fungi in traditional rice wine starter are various Rhizopus, Lysimachia christinae, and Saccharomyces cerevisiae. Among them, Lysimachia christinae is rich in enzymes and can secrete a variety of hydrolytic enzymes such as saccharifying enzymes, amylases, and acidic proteases. It is also the main fungus that produces aroma in the starter and the fermentation liquid during the saccharification period of rice wine starter, and plays an important role in rice wine brewing.

[0004] However, the traditional production of rice wine starter has the following problems: (1) The starter is produced by fermentation in an open environment, which is easily affected by a variety of complex environmental factors such as temperature and humidity, resulting in poor batch stability; (2) The microorganisms in the starter come from the yam powder, which has many uncertain factors and is easily contaminated by harmful bacteria in the environment; (3) Making the starter pills is time-consuming and labor-intensive, and the manual labor intensity is high; (4) The size and compactness of the starter pills are inconsistent, the strains grow inconsistently, and the quality uniformity is poor. Therefore, it is urgent to find a new type of fortified starter to solve the problems of rice wine starter production, which is of great significance for promoting the transformation of rice wine to mechanized production. Summary of the Invention

[0005] Based on the existing problems of seasonal limitations, high labor intensity, small production scale, and unstable quality in the production of Shaoxing rice wine starter, this invention provides a novel fortified starter. It utilizes a standardized production process, employing *Saccharomyces cerevisiae* selected from Shaoxing rice wine starter as the fortifying agent, adding various traditional Chinese herbs, and using rice and wheat bran as carriers to control the fermentation process. The resulting starter exhibits high saccharification and liquefaction power, solving the problems of unstable starter quality, seasonal production limitations, and complex production processes. It also provides guidance for combining modern production equipment with traditional techniques.

[0006] This invention provides a novel fortified wine starter, which is mainly prepared through the following process:

[0007] Step 1: The functional enhancement strain, *Saccharomyces cerevisiae* CY2111, was cultured in solid slant tubes and liquid Erlenmeyer flasks and then used as the enhancement agent.

[0008] Step 2: Grind the Chinese herbal medicine into powder and sieve it to obtain powder. Grind the herbal medicine into powder and sieve it for later use.

[0009] Step 3: Mix indica rice, wheat bran, and Chinese herbal medicine powder to prepare a culture medium. Add the fortifying agent to water and add it to the culture medium along with the herbal medicine powder. After mixing evenly, sieve the mixture and place it in a solid fermentation chamber for cultivation. After fermentation, cool and dehumidify the mixture, ventilate and dry it to obtain the fortified wine yeast.

[0010] To better control the quality of the yeast, the weight proportions of each ingredient are controlled as follows:

[0011] 800-1000 parts of indica rice, 80-300 parts of wheat bran, 8-30 parts of yam powder, 4-10 parts of Polygonum hydropiper, 16-25 parts of dried tangerine peel, 8-15 parts of Codonopsis pilosula, 8-50 parts of fortified microbial agent, and 320-600 parts of water.

[0012] The described capsule-forming yeast CY2111 was deposited at the China Center for Type Culture Collection (CCTCC) on August 15, 2022, with accession number CCTCC NO: M 20221276, and the deposit address is Wuhan University, Wuhan, China.

[0013] Preferably, the indica rice used as the raw material for fortified rice wine yeast can be cooked rice grains, raw rice flour, or cooked rice flour, and its specific processing technology is as follows:

[0014] (1) Cooked rice grains: Remove impurities from the rice, wash it 2-3 times, soak it in cold water for 4-6 hours, drain it for 30-40 minutes, steam it for 40-45 minutes, and then spread the steamed rice out to cool to a temperature of 30-35℃.

[0015] (2) Raw rice flour: After removing impurities from the rice, grind it into powder using a grinder and pass it through a 50-mesh sieve.

[0016] (3) Cooked rice flour: After grinding the rice into powder using a grinder, soak it in 40% to 60% water for 1 to 2 hours, and then steam it in high-temperature steam for 1 to 2 hours.

[0017] Preferably, the bran used as the raw material for making the fortified wine yeast can be raw material or processed by high-temperature steam cooking / high-pressure steam sterilization.

[0018] Preferably, in step one, the specific method for preparing the enhanced bacterial agent is as follows: take the lyophilized tube of *Saccharomyces cerevisiae* and inoculate it onto YPD slant medium, culture it at 28–30°C for 36–48 h, then inoculate it into liquid YPD medium, culture it at 28–30°C for 36–48 h, and then inoculate it into liquid YPD medium at a 1% inoculation rate, culture it at 28–30°C for 36–48 h to obtain the enhanced bacterial agent.

[0019] Preferably, in step two, both the herbal powder and the herbal powder should pass through a 50-mesh sieve.

[0020] Preferably, in step three, the activated yeast fortifying agent is mixed with water to adjust the bacterial concentration to 10. 6 ~10 8 CFU / mL.

[0021] Preferably, the enhanced wine yeast fermentation is controlled as follows: the culture medium is mixed evenly and then piled in a solid fermentation box that has been lined with four layers of gauze to a thickness of 2-3 cm.

[0022] (1) After 8 to 10 hours, this stage is the start-up and heating stage of the wine and medicine. The temperature inside the box rises by 3 to 4°C and the humidity reaches 98% to 100%. When the product temperature rises to 32 to 33°C, open the box lid about 5cm to let out excess moisture and CO2.

[0023] (2) After 10 to 12 hours, when the surface temperature of the wine and yeast rises to 34 to 35°C and the core temperature rises to 36 to 37°C, open the lid of the box about 10 cm and start intermittent ventilation to adjust the surface and internal temperature. Stop ventilation when the temperature drops to 32°C.

[0024] (3) After 12 to 14 hours, fully open the box lid and ventilate continuously, increasing the air volume as much as possible, and control the maximum product temperature at 37 to 38℃.

[0025] (4) In the later stage of ventilation, as the moisture decreases and the temperature drops below 35°C, ventilation can be suspended and the culture can continue for about 30 hours.

[0026] (5) After about 30 hours, the yeast enters the post-fermentation stage. The temperature inside the fermentation tank is maintained at 32-35℃, and this stage is maintained for 40-42 hours.

[0027] Preferably, in step three, the fortified wine yeast is dried at 38°C for 24–30 hours after fermentation.

[0028] The application of the novel wine-making agent prepared by this invention in wine is also within the scope of protection claimed by this invention.

[0029] The wines mentioned include, but are not limited to, yellow wine, sweet rice wine, rice wine, sake, and small-batch white wine.

[0030] The present invention also provides a solid fermentation box, characterized in that it includes: a main fermentation chamber (1), a porous sieve plate (2) at the bottom of the main fermentation chamber, a ventilation opening (3) at the bottom of the box body, an air outlet (4) at the top of the box cover, a window (5) on the left side of the box body, thermometer and hygrometer ports (6) on both sides, and a drain outlet (7) at the bottom of the box body.

[0031] Preferably, the fermentation box is also equipped with a blower, and the ventilation openings of the box body and the air outlets of the box cover are all connected to the blower through air inlet pipes, with an air filter in between.

[0032] Preferably, the air inlet pipe connected to the vent in the solid fermentation box is open to air on the other side, and an air filter is provided in the middle. The circulating air intake can be adjusted by controlling the valve that is open to air.

[0033] The application of the fermentation box provided by this invention in the preparation of yeast is also within the scope of protection of this invention. The yeast includes, but is not limited to, yeast starter, wheat yeast, large yeast, small yeast, rice yeast and bran yeast.

[0034] Beneficial effects

[0035] 1. Using the technical solution of this invention, a novel capsule-encapsulated yeast-enhanced wine yeast is obtained through microbial functional enhancement, which has higher saccharification and liquefaction power compared with traditional wine yeast.

[0036] 2. The process of this invention combines the advantages of small-grain koji and bran koji production, changes the traditional form of wine-making pills, and produces loose koji, which is simple and convenient to produce, with stable and uniform quality, and is suitable for mechanized production.

[0037] 3. The process of this invention is carried out in a solid-state fermentation chamber. Compared with the traditional process, it can improve production efficiency and shorten the entire fermentation time of the yeast. The new fortified yeast can be made and used immediately, reducing storage losses.

[0038] 4. The process of this invention incorporates a variety of traditional Chinese herbs, which contain multiple active ingredients, and when applied to wine production and brewing, they impart health benefits.

[0039] 5. The novel fortified wine yeast produced by this invention can be applied not only to yellow wine, but also to the production of rice wine, medicinal wine, baijiu and other fermented foods, and has broad application prospects.

[0040] 6. The solid-state fermentation box equipment involved in this invention can not only be used to make wine yeast, but also to solid-state fermentation of koji such as daqu, xiaoqu, wheat koji, rice koji, and bran koji, and has a wide range of application prospects. Attached Figure Description

[0041] Figure 1 Primary screening and secondary screening of high-amylase-producing encapsulated yeasts.

[0042] Figure 2 Cell morphology of *Saccharomyces cerevisiae* CY2111 observed under a microscope at 400x magnification.

[0043] Figure 3 Phylogenetic tree of *Saccharomyces cerevisiae* CY2111

[0044] Figure 4 Comparison of solid-state enzyme activity of *Saccharomyces cerevisiae* CY2111 on indica rice and wheat bran

[0045] Figure 5 : Front sectional view of solid fermentation chamber

[0046] Figure 6 Left and right views of the solid-state fermentation chamber. Detailed Implementation

[0047] YPD liquid culture medium: 10g tryptone, 20g glucose, 5g yeast extract, add 1000mL distilled water, pH at rest, sterilize at 115℃ for 20min.

[0048] YPD medium: 10g tryptone, 20g glucose, 5g yeast extract, 20g agar, add 1000mL distilled water, autoclave at 121℃ for 20min.

[0049] Fermentation liquid culture medium: 5g yeast extract, 10g peptone, 10g soluble starch, 1.0g sodium chloride, 0.1g magnesium sulfate, 0.5g potassium dihydrogen phosphate, 0.05g calcium chloride, 1000mL distilled water, natural pH, sterilized at 115℃ for 20min.

[0050] The saccharification and liquefaction power of fortified wine yeast were determined according to the 3,5-dinitrosalicylic acid colorimetric method and GB1886.174-2016.

[0051] Example 1: Screening and identification of high amylase-producing strains in wine yeast

[0052] (1) Sample collection and pretreatment

[0053] Samples of the wine-making agent were collected from four rice wine factories in Shaoxing City, Zhejiang Province, including the Industrial Park, Shenyonghe, Jianhu, and Tapai. The collected agents were placed in sterile sealed bags and stored at 4℃. 2.5g of the agent was weighed into a 50mL centrifuge tube containing 22.5mL of sterile water and incubated on a shaker at 30℃ and 150r / min for 30min to obtain 10... -1 The sample dilution solution.

[0054] (2) Initial screening of the transparent zone of the strain

[0055] Amylase isolation medium: 10g yeast extract, 20g peptone, 20g soluble starch, 18g agar, add 1000mL distilled water, pH at rest, sterilize at 115℃ for 20min.

[0056] The bacterial culture was diluted to different gradients to obtain 10 -2 ~10 -7Take 10 times the sample dilution solution. -3 10 -4 10 -5 Three dilution gradients were used for selective plate spreading. 100 μL of each dilution was spread onto amylase isolation medium and incubated at 28°C for 2–3 days. Diluted iodine solution was then poured onto the plates, and the diameter of the clear zone (D) was measured. h ) and colony diameter (D c ), calculate the diameter ratio (D) h / D c After purifying the colonies by streaking 2-3 times to ensure purity, the bacterial suspension was mixed with 30% glycerol at a 1:1 ratio and stored at -80℃.

[0057] (3) Re-screening of liquid enzyme production by strains

[0058] The strains initially screened for clear zones were aseptically inoculated into YPD liquid medium for activation and subculturing, and cultured at 30℃ and 180 rpm for 24 h on a shaker to obtain the seed culture. The seed fermentation broth was inoculated at a rate of 2% (v / v) into 50 mL / 250 mL of liquid fermentation medium and incubated at 30℃ and 180 rpm for 48 h on a shaker. The fermentation broth was centrifuged at 10000 rpm for 10 min, and the supernatant was used as the crude enzyme solution for enzyme activity determination. The results are as follows: Figure 1 .

[0059] (4) Strain identification

[0060] The genomes of the selected strains were extracted, and ITS amplification and sequencing were performed on the selected strains using universal ITS primers.

[0061] The amplification primer sequences were ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4.

[0062] (5'-TCCTCCGCTTATTGATATGC-3').

[0063] The PCR amplification reaction conditions were as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 45 s, 35 cycles, and a final extension at 72℃ for 10 min. PCR products were detected by 1% agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0064] Sequencing results were aligned to homologous sequences using BLAST in the GenBank nucleic acid database, and phylogenetic analysis was performed. The results are as follows: Figure 3As shown, the nucleotide sequence of strain CY2111 has a homology similarity of more than 99.62% with that of Saccharomycopsis fibuligera (GenBank accession number: MH855892.1), and this strain is named Saccharomycopsis fibuligera CY2111.

[0065] Example 2: Enzyme production capacity analysis of *Saccharomyces cerevisiae* under solid-state fermentation conditions

[0066] (1) Preparation of rice koji and wheat bran koji

[0067] The strains initially screened by the transparent zone were inoculated into malt juice liquid medium, and the quantitative spore suspension or bacterial solution was 1×106 spores / mL, which was used for rice koji and wheat bran koji culture.

[0068] Preparation of rice koji: Take 50g of soaked and drained indica rice and put it into a 250mL Erlenmeyer flask. Add 7mL of distilled water and stir well. Seal the flask with 8 layers of gauze and sterilize at 121℃ for 20min. Then cool to room temperature. Aseptically add 10% seed liquid to the sterilized indica rice, shake and mix it one by one, and place it in an incubator at 30℃ and 80% relative humidity for 72h.

[0069] Preparation of bran koji: Weigh 50g of wheat bran, add 30mL of distilled water and stir well. Shake the Erlenmeyer flask to mix the material and water evenly. Sterilize at 121℃ for 20min and then cool to room temperature.

[0070] (2) Enzyme activity assay

[0071] Weigh 1.00g of rice koji or wheat bran koji (converted to oven-dried koji for calculation), accurate to 0.001g, and place it in a 10mL centrifuge tube sterilized by autoclaving. Add 10mL of acetate-sodium acetate buffer solution (pH 4.6 0.2M) and extract for 1h at 30℃ and 180r / min on a shaker. Centrifuge the extract at 10000r / min and 4℃ for 10min, and take the supernatant as the crude enzyme solution. Measure the saccharifying enzyme and liquefying enzyme activities of the enzyme-producing strains to be tested.

[0072] The results are as follows Figure 4 As shown, the experiment showed that the saccharifying enzyme activity (1130.84±55.18 U / g DW) and liquefying enzyme activity (1.51±0.06 U / g DW) of the cytosolic yeast CY2111 on wheat bran were greater than its amylase activity on indica rice. Traditional wine yeast production uses indica rice as raw material. In order to improve the enzyme activity of the wine yeast, the yeast is fortified by combining indica rice and wheat bran.

[0073] Example 3: Preparation of yeast fortified with sac-coated yeast (cooked rice grains + wheat bran)

[0074] The preparation of a new type of fortified wine yeast includes the following raw materials: 900 parts of indica rice, 180 parts of wheat bran, 27 parts of yam powder, 9 parts of Polygonum hydropiper, 18 parts of dried tangerine peel, 9 parts of Codonopsis pilosula, 45 parts of fortified microbial agent, and 450 parts of water.

[0075] A novel process for manufacturing fortified wine yeast includes the following steps:

[0076] (1) Take the freeze-dried yeast capsules and inoculate them onto YPD slant medium. After culturing at 28℃ for 48h, inoculate them into liquid YPD medium. After culturing at 28℃ for 48h, inoculate them into liquid YPD medium at a 1% inoculation rate and culture at 30℃ for 36h. This is then used as a strengthening agent.

[0077] (2) Remove impurities from the rice, rinse it twice, soak it in cold water for 5 hours and drain it for 30 minutes. Then steam the rice in a rice cooker for 40 minutes. After steaming, make sure the rice is cooked but not mushy and has no filling. Then spread the steamed rice out to cool to 32°C for later use.

[0078] (3) After mixing the wheat bran and water evenly, sterilize them by high-pressure steam at 121°C for 15 minutes.

[0079] (4) Grind the Chinese herbal medicine and the mother's medicine into powder and pass it through a 50-mesh sieve for later use.

[0080] (5) Mix the enhanced bacterial agent prepared in step (1) with water at an inoculum rate of 5%, and adjust the concentration of the bacterial solution to 10. 8 CFU / mL.

[0081] (6) After mixing the wheat bran, Chinese herbal medicine powder, and steamed rice evenly, add the herbal medicine powder and the inoculant and place it in a solid fermentation box lined with four layers of gauze. Spray sterile water to keep it moist, and cover the box for cultivation. After 8 to 10 hours, this stage is the start-up and heating stage of the wine-making process. The temperature inside the box rises by 3 to 4°C, and the humidity reaches 98% to 100%. When the temperature rises to 32 to 33°C, open the box lid about 5 cm to allow excess moisture and CO2 to escape.

[0082] (7) After 10 to 12 hours, when the surface temperature of the wine and yeast rises to 34 to 35°C and the core temperature rises to 36 to 37°C, open the lid of the box about 10 cm and start intermittent ventilation to adjust the surface and internal temperature. Stop ventilation when the temperature drops to 32°C.

[0083] (8) After 12 to 14 hours, fully open the box lid and ventilate continuously, increasing the air volume as much as possible, and control the maximum product temperature at 37 to 38℃.

[0084] (9) In the later stage of ventilation, as the moisture decreases and the temperature drops below 35°C, ventilation can be suspended and the culture can continue for about 30 hours.

[0085] (10) After about 30 hours, the yeast enters the post-fermentation stage. The temperature inside the fermentation tank is maintained at 32-35℃, and this stage is maintained for 40-42 hours.

[0086] (11) After fermentation, the fortified wine yeast is dried at 38°C for 24-36 hours.

[0087] Ultimately, a compound fortified wine yeast made from cooked rice grains and bran was obtained.

[0088] Example 4: Preparation of yeast fortified with sac-forming yeast (raw rice flour + wheat bran)

[0089] The preparation of a new type of fortified wine yeast includes the following raw materials: 900 parts of indica rice, 180 parts of wheat bran, 27 parts of yam powder, 9 parts of Polygonum hydropiper, 18 parts of dried tangerine peel, 9 parts of Codonopsis pilosula, 45 parts of fortified microbial agent, and 450 parts of water.

[0090] A novel process for manufacturing fortified wine yeast includes the following steps:

[0091] (1) Take the freeze-dried yeast capsules and inoculate them onto YPD slant medium. After culturing at 28℃ for 48h, inoculate them into liquid YPD medium. After culturing at 28℃ for 48h, inoculate them into liquid YPD medium at a 1% inoculation rate and culture at 30℃ for 36h. This is then used as a strengthening agent.

[0092] (2) Crush the rice and Chinese herbal medicines and pass them through a 50-mesh sieve. Grind the aged yeast into powder and pass it through a 50-mesh sieve.

[0093] (3) Mix the rice flour, Chinese herbal medicine powder and wheat bran evenly.

[0094] (4) Mix the enhanced bacterial agent prepared in step (1) with water at an inoculum rate of 5%, and adjust the concentration of the bacterial solution to 10. 6 CFU / mL.

[0095] (5) Add the bacterial suspension and the herbal powder to the culture medium at a ratio of 50% and mix evenly. Then pile the mixture into a solid fermentation box that has been lined with four layers of gauze to a thickness of 2.5 cm.

[0096] (6) After 8 hours, this stage is the start-up and heating stage of the wine and medicine. The temperature inside the box rises by 3-4℃, the humidity reaches 98%-100%, and when the product temperature rises to 32-33℃, open the box lid about 5cm to let out excess moisture and CO2.

[0097] (7) After 10 to 12 hours, when the surface temperature of the wine and yeast rises to 34 to 35°C and the core temperature rises to 36 to 37°C, open the lid of the box about 10 cm. The culture medium will form clumps. Start intermittent ventilation to adjust the surface and internal temperature. Stop ventilation when the temperature drops to 32°C.

[0098] (8) After 12 to 14 hours, fully open the box lid and ventilate continuously, increasing the air volume as much as possible, and control the maximum product temperature at 37 to 38℃.

[0099] (9) In the later stage of ventilation, as the moisture decreases and the temperature drops below 35°C, ventilation can be suspended and the culture can continue for about 30 hours.

[0100] (10) After about 30 hours, the yeast enters the post-fermentation stage. The temperature inside the fermentation tank is maintained at 32-35℃, and this stage is maintained for 40-42 hours.

[0101] (11) After fermentation, the fortified wine yeast is dried at 38°C for 24-36 hours.

[0102] Ultimately, a compound fortified wine yeast made from raw rice flour and wheat bran was obtained.

[0103] Comparative Example 1: Preparation of Unfortified Wine Yeast

[0104] The preparation of a new type of fortified wine yeast includes the following raw materials: 900 parts of indica rice, 180 parts of wheat bran, 27 parts of yam powder, 9 parts of Polygonum hydropiper, 18 parts of dried tangerine peel, 9 parts of Codonopsis pilosula, 45 parts of fortified microbial agent, and 450 parts of water.

[0105] The specific process steps are the same as in Example 3. The difference is that the strengthening agent in step (4) is replaced with the same amount of sterile water.

[0106] Comparative Example 2: Preparation of Traditional Wine-Based Pills

[0107] The traditional method of making medicinal pills involves the following ingredients: 100 parts rice flour, 1 part Polygonum hydropiper, and 54 parts water.

[0108] The traditional method of making medicinal wine pills involves grinding, mixing, sifting, pressing, cutting, and then rolling the pills into balls for inoculation. After inoculation, the pills are placed in a fermentation vat and incubated for 30 hours, covered with a lid and burlap sacks to maintain warmth and moisture. As fermentation continues, the burlap sacks and lids are gradually removed. When small white spheres appear on the pills with mycelial growth, they are transferred to bamboo trays and incubated indoors for 48 hours, turning them every 4 hours. After air-drying for 3-4 days, the traditional medicinal wine pills are obtained.

[0109] Example 5: Preparation of yeast fortified with sac-forming yeast (cooked rice flour + wheat bran)

[0110] The preparation of a new type of fortified wine yeast includes the following raw materials: 900 parts of indica rice, 180 parts of wheat bran, 27 parts of yam powder, 9 parts of Polygonum hydropiper, 18 parts of dried tangerine peel, 9 parts of Codonopsis pilosula, 45 parts of fortified microbial agent, and 450 parts of water.

[0111] A novel process for manufacturing fortified wine yeast includes the following steps:

[0112] (1) Take the freeze-dried yeast capsules and inoculate them onto YPD slant medium. After culturing at 28℃ for 48h, inoculate them into liquid YPD medium. After culturing at 28℃ for 48h, inoculate them into liquid YPD medium at a 1% inoculation rate and culture at 30℃ for 36h. This is then used as a strengthening agent.

[0113] (2) Grind the Chinese herbal medicine and the mother's medicine into powder and pass it through a 50-mesh sieve for later use.

[0114] (3) Grind the rice into powder using a grinder and pass it through a 50-mesh sieve. Then soak it in 40% to 60% water for 1 to 2 hours, and then steam it in a high-temperature steamer for 1 to 2 hours.

[0115] (4) Quickly break up the steamed rice flour and mix it evenly with bran and Chinese herbal medicine powder to prepare a culture medium.

[0116] (5) Mix the enhanced bacterial agent prepared in step (1) with water at an inoculum rate of 5%, and adjust the concentration of the bacterial solution to 10. 8 CFU / mL.

[0117] (6) Add the medicated powder and bacterial solution to the culture medium, stir well, and place it in a solid fermentation box lined with four layers of gauze. Cover the box and start the fermentation. After 8 to 10 hours, this is the initial heating stage of the fermentation process. The temperature inside the box rises by 3 to 4°C, and the humidity reaches 98% to 100%. When the temperature rises to 32 to 33°C, open the box lid about 5 cm to allow excess moisture and CO2 to escape.

[0118] (7) After 10 to 12 hours, when the surface temperature of the wine and yeast rises to 34 to 35°C and the core temperature rises to 36 to 37°C, open the lid of the box about 10 cm and start intermittent ventilation to adjust the surface and internal temperature. Stop ventilation when the temperature drops to 32°C.

[0119] (8) After 12 to 14 hours, fully open the box lid and ventilate continuously, increasing the air volume as much as possible, and control the maximum product temperature at 37 to 38℃.

[0120] (9) In the later stage of ventilation, as the moisture decreases and the temperature drops below 35°C, ventilation can be suspended and the culture can continue for about 30 hours.

[0121] (10) After about 30 hours, the yeast enters the post-fermentation stage. The temperature inside the fermentation tank is maintained at 32-35℃, and this stage is maintained for 40-42 hours.

[0122] (11) After fermentation, the fortified wine yeast is dried at 38°C for 24-36 hours.

[0123] Ultimately, a compound fortified wine yeast made from cooked rice flour and wheat bran was obtained.

[0124] The enzyme activity of the wine-derived wines obtained from Examples 3, 4, and 5, and Comparative Example 1, was determined. The average value was taken from three trials. The results are shown in Table 1.

[0125] Table 1. Determination of saccharifying enzyme and liquefying enzyme activities in fortified wine starter.

[0126]

[0127] Note: Different superscripts in the same column indicate significant differences between the data, p < 0.05.

[0128] The results showed that the saccharification power of the fortified yeast was as high as 610.01±83.55 U / g, and the liquefaction power was 1.45±0.18 U / g, while the saccharification power of the unfortified yeast in the control group was 374.41±33.68 U / g, and the liquefaction power was 1.03±0.18 U / g. This indicates that fortification with saccharifying yeast can significantly improve the saccharification and liquefaction power of the yeast.

[0129] Example 6: Application of fortifying yeast in rice wine

[0130] The fortified wine agent described below was prepared in Example 4.

[0131] (1) Yeast activation: The yeast cells in the glycerol tubes were streaked onto plates in a sterile room and incubated at 28°C for 48 hours. Single yeast colonies from the plates were then inoculated into YPD medium in a sterile room under sterile conditions and incubated at 30°C for 36–48 hours, with the temperature controlled between 28–30°C, not exceeding 30°C. This inoculum is then ready for use as YPD culture medium.

[0132] (2) Preparation of yeast starter: First, prepare the saccharification solution. Soak glutinous rice in a rice soaking tank for 30 minutes, drain the rice slurry, and steam the rice for 30-40 minutes. Cool the steamed rice to 60℃ and set aside. Add 2‰ saccharifying enzyme and 1‰ liquefying enzyme, stir evenly, and place in a water bath at 60℃ for saccharification for about 4 hours or more, stirring once every hour to ensure uniform saccharification. Filter the saccharification solution through gauze to obtain a sugar solution, and dilute with water to 13±0.5°Bx (20℃). Pour into bottles, seal with 4 layers of gauze, add sealing film, and sterilize in an autoclave at 121℃ for 20 minutes. Remove and cool for later use. Transfer the cultured bacterial solution to the rice saccharification solution at an inoculation ratio of 5%, and then incubate at a constant temperature for 36-48 hours before use.

[0133] (3) Fermentation of Shaoxing Wine: The ingredients are added according to the Shaoxing wine brewing ingredient list in Table 2. The total fermentation time is approximately 20 days. During fermentation, attention should be paid to stirring. The pre-fermentation temperature should be controlled below 33℃, and the post-fermentation temperature should be controlled at approximately 15±2℃. The pre-fermentation temperature is 28℃, and the total fermentation time is 5 days; stirring is performed twice daily. The post-fermentation temperature is 15℃, and the fermentation time is 15 days; stirring is performed once every 5 days. The physicochemical properties of the fermented Shaoxing wine can reach 15.80±0.17% vol, and the total acid content is 6.50±0.26 g / L, basically meeting the national standards for Shaoxing wine.

[0134] Table 2 Ingredients for Yellow Rice Wine Brewing

[0135]

[0136] Example 7: Application of fortified yeast in sweet rice wine

[0137] Weigh out the glutinous rice according to the specified ratio and wash it thoroughly. Soak it at room temperature for 8-10 hours. Drain the water and steam it under normal pressure for 30-40 minutes until there is no white center in the rice. Spread the rice out to cool to 28-30℃, then add 30%-40% of the weight of raw rice in cooled boiled water to break up the glutinous rice. Mix in 0.2%-0.5% fortified yeast, stir well, and then transfer it to a fermentation container. Gently flatten the rice and make a well 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 large amount of liquid, a harmonious aroma, a moderate sweetness, and an alcohol content of 2-4% (v / v).

[0138] Example 8: Application of fortifying yeast in rice wine

[0139] Weigh out the glutinous rice according to the specified ratio, wash it thoroughly, and soak it at room temperature for 10-12 hours. The rice should be soaked until the grains are intact and can be easily crushed into powder without any hard core. Rinse the soaked rice with clean water and drain. Steam under normal pressure for about 30 minutes until the rice is cooked but not mushy, the grains are soft, and there is no white core. Pour cold boiled water over the steamed rice powder to a temperature of 28-30℃, mix in 0.4-0.6% fortified yeast, and saccharify for 36-48 hours. Once 4 / 5 of the mixture has a sweet liquid, add water at a ratio of 100%-120% of the rice volume and 0.1%-0.25% activated dry yeast. Stir well and allow it to enter the pre-fermentation stage at 28℃ for 4-6 days. After the primary fermentation, allow it to undergo secondary fermentation at a low temperature (15-18℃) for approximately 15-20 days. After fermentation, press, filter, and distill the liquid to obtain rice wine. Rice wine is rich in aroma, with a harmonious fragrance and a rich taste, and an alcohol content of around 10%.

[0140] Example 9: A solid-state fermentation box

[0141] To better support the rice wine fortification process proposed in this invention, a dedicated solid-state fermentation box was designed, comprising a main fermentation chamber (1), a porous sieve plate (2) at the bottom of the main fermentation chamber, a ventilation opening (3) at the bottom of the box, an air outlet (4) at the top of the box cover, a window (5) on the left side of the box, thermometer and hygrometer ports (6) on both sides, and a drain outlet (7) at the bottom of the box. This fermentation box can also be equipped with a blower. The ventilation openings and air outlets on the box cover are connected to the blower via air inlet pipes, with an air filter in the middle. The other side of the air inlet pipe connected to the ventilation opening is connected to the air, with an air filter in the middle. The circulating air intake can be adjusted by controlling the valve connected to the air.

[0142] 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 novel fortified wine yeast, characterized in that, The preparation method is as follows: (1) Take the freeze-dried yeast capsules and inoculate them onto YPD slant medium. After culturing at 28℃ for 48 h, inoculate them into liquid YPD medium. After culturing at 28℃ for 48 h, inoculate them into liquid YPD medium at a 1% inoculation rate and culture at 30℃ for 36 h. This is used as a strengthening agent. (2) Crush the rice and Chinese herbal medicines and pass them through a 50-mesh sieve; grind the herbal medicine into powder and pass it through a 50-mesh sieve. (3) Mix the rice flour, Chinese herbal medicine powder, and wheat bran evenly; (4) Mix the enhanced bacterial agent prepared in step (1) with water at an inoculum rate of 5%, and adjust the concentration of the bacterial solution to 10. 6 CFU / mL; (5) Add the bacterial suspension and the herbal powder to the culture medium at a ratio of 50% and mix evenly. Then pile the mixture into a solid fermentation box that has been lined with four layers of gauze to a thickness of 2.5 cm. (6) After 8 hours, this stage is the start-up and heating stage of the wine and medicine. The temperature inside the box rises by 3~4℃, the humidity reaches 98%~100%, and when the product temperature rises to 32~33℃, open the box lid by 5 cm to let out excess moisture and CO2. (7) After 10-12 hours, when the surface temperature of the wine and yeast rises to 34-35℃ and the core temperature rises to 36-37℃, open the box lid 10 cm. The culture medium will form blocks. Start intermittent ventilation to adjust the surface and internal temperature. Stop ventilation when the temperature drops to 32℃. (8) After 12-14 hours, fully open the box lid and ventilate continuously, increasing the air volume as much as possible, and control the maximum product temperature at 37-38℃; (9) During the later stage of ventilation, as the moisture content decreases and the temperature drops below 35°C, ventilation is suspended and the culture continues for 30 hours. (10) After 30 h, the yeast enters the post-fermentation stage. The temperature inside the fermentation tank is maintained at 32~35℃. This stage is maintained until 40~42 h. (11) After fermentation, the fortified wine yeast is dried at 38℃ for 24~36 h; Ultimately, a compound fortified wine yeast made from raw rice flour and wheat bran was obtained; The solid-state fermentation box includes: a main fermentation chamber with a porous sieve plate at the bottom; a vent at the bottom of the box body; an air outlet at the top of the box cover; a window on the left side of the box body; thermometer and hygrometer ports on both sides; and a drain outlet at the bottom of the box body. It also includes a blower; the vents in the box body and the air outlet on the box cover are connected to the blower via air inlet pipes, with an air filter in between. The other side of the air inlet pipe connected to the vents is open to air, and an air filter is also provided in between. The circulating air intake can be adjusted by controlling the valve connecting it to the air. The weight proportions of each raw material in the new fortified wine yeast are as follows: 900 parts indica rice, 180 parts wheat bran, 27 parts yam powder, 9 parts Polygonum hydropiper, 18 parts dried tangerine peel, 9 parts Codonopsis pilosula, 45 parts fortifying microbial agent, and 450 parts water; Enhanced strain of capsule-coated yeast ( Saccharomycopsis fibuligera CY2111 was deposited at the China Center for Type Culture Collection (CCTCC) on August 15, 2022, at Wuhan University, Wuhan, China, with accession number CCTCC NO: M20221276.

2. The novel fortified wine agent as described in claim 1, characterized in that, In step (11), the fortified yeast is dried at 38°C for 24-30 h after fermentation to achieve a moisture content of 10%-12%.

3. A method for preparing the novel fortified wine agent as described in claim 1, characterized in that, The ingredients include: 900 parts indica rice, 180 parts wheat bran, 27 parts yam powder, 9 parts Polygonum hydropiper, 18 parts dried tangerine peel, 9 parts Codonopsis pilosula, 45 parts fortifying microbial agent, and 450 parts water; the steps are as follows: (1) Take the freeze-dried yeast capsules and inoculate them onto YPD slant medium. After culturing at 28℃ for 48 h, inoculate them into liquid YPD medium. After culturing at 28℃ for 48 h, inoculate them into liquid YPD medium at a 1% inoculation rate and culture at 30℃ for 36 h. This is used as a strengthening agent. (2) Crush the rice and Chinese herbal medicines and pass them through a 50-mesh sieve; grind the herbal medicine into powder and pass it through a 50-mesh sieve. (3) Mix the rice flour, Chinese herbal medicine powder, and wheat bran evenly; (4) Mix the enhanced bacterial agent prepared in step (1) with water at an inoculum rate of 5%, and adjust the concentration of the bacterial solution to 10. 6 CFU / mL; (5) Add the bacterial suspension and the herbal powder to the culture medium at a ratio of 50% and mix evenly. Then pile the mixture into a solid fermentation box that has been lined with four layers of gauze to a thickness of 2.5 cm. (6) After 8 hours, this stage is the start-up and heating stage of the wine and medicine. The temperature inside the box rises by 3~4℃, the humidity reaches 98%~100%, and when the product temperature rises to 32~33℃, open the box lid by 5 cm to let out excess moisture and CO2. (7) After 10-12 hours, when the surface temperature of the wine and yeast rises to 34-35℃ and the core temperature rises to 36-37℃, open the box lid 10 cm. The culture medium will form blocks. Start intermittent ventilation to adjust the surface and internal temperature. Stop ventilation when the temperature drops to 32℃. (8) After 12-14 hours, fully open the box lid and ventilate continuously, increasing the air volume as much as possible, and control the maximum product temperature at 37-38℃; (9) During the later stage of ventilation, as the moisture content decreases and the temperature drops below 35°C, ventilation is suspended and the culture continues for 30 hours. (10) After 30 h, the yeast enters the post-fermentation stage. The temperature inside the fermentation tank is maintained at 32~35℃. This stage is maintained until 40~42 h. (11) After fermentation, the fortified wine yeast is dried at 38℃ for 24~36 h; Ultimately, a compound fortified wine yeast made from raw rice flour and wheat bran was obtained.

4. The application of the novel fortified wine yeast as described in claim 1 in brewing wine.

5. The application according to claim 4, characterized in that, The wines mentioned include, but are not limited to, yellow wine, sweet rice wine, rice wine, sake, and small-batch white wine.

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