Saccharomycopsis fibuligera strain, microbial agent, fermentation product, preparation and use

Abstract

A Saccharomycopsis fibuligera strain, a microbial agent, a fermentation product, preparation and a use. The Saccharomycopsis fibuligera strain is preserved in the China Center for Type Culture Collection on 25 September 2023, with the preservation number of CCTCC NO: M20231788. The Saccharomycopsis fibuligera strain can be used to produce multiple enzymes required by fermentation, such as amylase, protease, and cellulase, wherein the amylase has the activities of pullulanase and raw starch amylase, can well adapt to special environmental conditions for fermentation, and has high environmental tolerance and high aroma production capability, thereby improving the quality of fermentation products, and reducing investment costs in fermentation production.

Description

A yeast strain, bacterial agent, fermentation product, and preparation and application thereof

[0001] Related applications

[0002] This application claims priority to the prior application document with application number 202410253232.1 filed with the State Intellectual Property Office of China on March 5, 2024, and incorporates its entire contents into this document. Technical Field

[0003] The present invention belongs to the field of food microorganisms, and in particular relates to a Saccharomyces cerevisiae strain, a bacterial agent, a fermentation product and a preparation and application thereof. Background Art

[0004] Saccharomycopsis fibuligera, belonging to the order Saccharomycetales, family Saccharomycopsidaceae, and genus Saccharomycopsis, is a dimorphic yeast that produces ascospores and pseudohyphae. It can be found in various starchy substrates, such as various daqu (Chinese koji) and fermented grains (Zijiu).

[0005] Saccharomyces punctatus is an aroma-producing yeast that produces a variety of flavor compounds, including esters, lactones, alcohols, organic acids, and aldehydes and ketones. Furthermore, Saccharomyces punctatus secretes a rich enzyme system, including amylase, β-glucosidase, protease, and lipase. These enzymes effectively hydrolyze starch, protein, and cellulose in fermentation substrates, helping to improve raw material utilization. Furthermore, the yeast can utilize a wide variety of substrates from a wide range of sources, giving it significant application value and potential in the fermentation field.

[0006] Chinese patent application CN108251317A discloses a strain of Saccharomyces fulvidraco and its applications. The strain has the ability to degrade starch, protein, fat, cellulose, and lignin, and is well adapted to the high humidity and acidity of distiller's grains. However, the patent does not disclose the production and activity of multiple enzymes, nor does it disclose research on its aroma production properties and other environmental tolerances, such as resistance to high temperatures, high sugars, and ethanol. Nor does it disclose research on the application of this strain in koji production and fermentation.

[0007] Chinese patent application CN107189952A discloses a strain of Saccharomyces fuscae, which exhibits amylase activity and possesses a well-developed mycelium. This strain is used in the production of sesame-flavored medium- and high-temperature Daqu (Daiqu), thereby improving the utilization of the raw material. However, the patent does not address the strain's tolerance, substrate utilization characteristics, enzyme activity determination beyond α-amylase, or flavor profile. Summary of the Invention

[0008] The technical problem solved by the present invention is as follows: In the prior art, when the Saccharomyces cerevisiae strain ferments koji with different substrates, the growth of the strain is easily affected by environmental stress conditions and changes, because its fermentation environment is an extreme environment with high temperature, low pH, high ethanol and hypertonicity, resulting in a decrease in the aroma production ability of the Saccharomyces cerevisiae strain, a decline in the quality of the fermentation product, and an increase in the cost of fermentation production investment.

[0009] To address the problems of the prior art, the present invention provides a Saccharomyces cingulosus strain, a microbial inoculum, a fermentation product, and its preparation and application. This Saccharomyces cingulosus strain produces multiple enzymes required for fermentation, such as pullulanase and amylase, and exhibits high environmental tolerance and aroma production, thereby improving the quality of fermented products and reducing the cost of fermentation production.

[0010] Specifically, the present invention provides the following technical solutions:

[0011] Technical solution 1: A Saccharomycopsis fibuligera yeast strain, characterized in that the Saccharomycopsis fibuligera yeast strain is a Saccharomycopsis fibuligera AMCC32244 strain with pullulanase production function, which was deposited in the China Center for Type Culture Collection on September 25, 2023, with the preservation number: CCTCC NO: M 20231788.

[0012] Technical Solution 2: The yeast strain of the Acidiflora punctata complex according to Technical Solution 1 is characterized in that the ITS rDNA gene sequence of the Acidiflora punctata complex is shown as SEQ ID NO.1.

[0013] Technical Solution 3: The Saccharomyces cerevisiae strain according to Technical Solution 1 or 2 is characterized in that the Saccharomyces cerevisiae strain has high temperature resistance, and / or low pH resistance, and / or high sugar resistance, and / or ethanol resistance, and / or carbon source utilization characteristics.

[0014] Technical Solution 4: The Saccharomyces cerevisiae strain according to any one of Technical Solutions 1-3, characterized in that the carbon source material includes one or a combination of xylose and pullulan.

[0015] Technical Solution 5: The Saccharomyces cerevisiae strain according to any one of Technical Solutions 1-4 is characterized in that the carbon source material also includes one or more substances selected from the group consisting of arabinose, fructose, glucose, melibiose, cellobiose, maltose, trehalose, sucrose, melezitose, stachyose, raffinose, inulin and soluble starch.

[0016] Technical Solution 6: A fermentation preparation method for a spore-forming yeast agent, characterized in that the method comprises the following steps: cultivating the spore-forming yeast strain described in any one of Technical Solutions 1-5.

[0017] Technical Solution 7: The preparation method according to Technical Solution 6 is characterized in that the preparation method comprises the following steps:

[0018] (1) amplifying and culturing the yeast strain of the cystis fusoni described in any one of technical solutions 1-5;

[0019] (2) adding the product obtained in step (1) into a liquid culture medium and fermenting and culturing the product at 28-32° C., preferably, the liquid culture medium is a YPD liquid culture medium.

[0020] Technical Solution 8: A yeast agent of Capsular spores, characterized in that it is obtained by the fermentation preparation method described in Technical Solution 6 or 7.

[0021] Technical Solution 9: A first fermented product, characterized in that the first fermented product is obtained by the following steps:

[0022] (1) Cultivating the Saccharomyces cerevisiae strain described in any one of Technical Solutions 1-5 or the Saccharomyces cerevisiae inoculum described in Technical Solution 8 to obtain a seed solution containing Saccharomyces cerevisiae, preferably, the culture medium used during the cultivation is YPD liquid medium;

[0023] (2) The seed liquid containing the spore yeast obtained in step (1) is inoculated into a bran solid fermentation medium for cultivation to obtain a first fermentation product, wherein, based on the dry weight of the bran, 5 mL to 20 mL of the spore yeast seed liquid is inoculated into every 100 g of the bran.

[0024] Technical solution 10: The first fermentation product according to technical solution 9 is characterized in that the number of viable bacteria of Saccharomyces cerevisiae AMCC32244 in the first fermentation product is greater than or equal to 6.10×10 9 CFU / g, preferably greater than or equal to 1.31×10 10 , more preferably, greater than or equal to 4.70×10 10 CFU / g.

[0025] Technical Solution 11: The first fermented product according to Technical Solution 9 or 10, characterized in that the first fermented product contains at least one or more substances selected from the group consisting of high-temperature α-amylase, medium-temperature α-amylase, glucoamylase, pullulanase and raw amylase;

[0026] More preferably, the protein composition further contains one or two or more substances selected from the group consisting of acidic proteases, neutral proteases, alkaline proteases, cellulases, and lipases.

[0027] Technical Solution 12: The first fermentation product according to Technical Solution 11 is characterized in that, measured in enzyme activity U / g, the high-temperature α-amylase activity is greater than or equal to 39,900; and / or the medium-temperature α-amylase activity is greater than or equal to 79,600; and / or the glucoamylase activity is greater than or equal to 82,500; and / or the pullulanase activity is greater than or equal to 57; and / or the raw amylase activity is greater than or equal to 4,550; and / or the acid protease activity is greater than or equal to 30; and / or the neutral protease activity is greater than or equal to 160; and / or the alkaline protease activity is greater than or equal to 110; and / or the cellulase activity is greater than or equal to 360; and / or the lipase activity is greater than or equal to 7.

[0028] Technical Solution 13: The method for preparing the first fermented product according to any one of Technical Solutions 9 to 12, characterized in that it comprises:

[0029] (1) Cultivating the Saccharomyces cerevisiae strain described in any one of Technical Solutions 1-5 or the Saccharomyces cerevisiae inoculum described in Technical Solution 8 to obtain a seed solution containing Saccharomyces cerevisiae, preferably, the culture medium used during the cultivation is YPD liquid medium;

[0030] (2) The seed liquid containing the spore yeast obtained in step (1) is inoculated into a bran solid fermentation medium for cultivation to obtain a first fermentation product, wherein, based on the dry weight of the bran, 5 mL to 20 mL of the spore yeast seed liquid is inoculated into every 100 g of the bran.

[0031] Technical Solution 14: According to the method for preparing the first fermented product according to Technical Solution 13, 5 mL-16 mL of the seed liquid of Saccharomyces cerevisiae is inoculated into every 100 g of bran, based on the dry weight of the bran.

[0032] Technical Solution 15: The method for preparing the first fermentation product according to Technical Solution 14 or 15 is characterized in that the culture temperature is 28-32°C and the culture time is 3-4 days.

[0033] Technical Solution 16: A second fermentation product, characterized in that the second fermentation product is obtained by the following steps:

[0034] (1) Cultivating the Saccharomyces cerevisiae strain described in any one of technical solutions 1 to 5 or the Saccharomyces cerevisiae agent described in technical solution 8;

[0035] (2) inoculating the Saccharomyces cerevisiae strain or the Saccharomyces cerevisiae inoculum of step (1) into a bran solid fermentation medium for culturing to obtain a first fermentation product, wherein, based on the dry weight of the bran, 5 mL to 20 mL of the Saccharomyces cerevisiae seed liquid is inoculated into every 100 g of the bran;

[0036] (3) The first fermentation product obtained in step (2) is added to a starchy substrate liquid fermentation medium, preferably an unliquefied yellow corn flour liquid fermentation medium, for culturing to obtain a second fermentation product.

[0037] Technical Solution 17: The second fermented product according to Technical Solution 16, characterized in that the second fermented product contains at least one or two or more substances selected from the group consisting of γ-nonalactone, phenylethanol, isoamyl alcohol, trans-4-decenoic acid ethyl ester, 4-vinyl-2-methoxy-phenol, β-ionone, ethyl palmitate and dihydro-β-ionone;

[0038] More preferably, the composition further contains one or more substances selected from the group consisting of ethyl hexanoate, ethyl octanoate, n-hexanol, n-octanol, phenylethyl acetate, ethyl phenylacetate, 1-octen-3-ol, 4-ethylguaiacol, ethyl heptanoate, ethyl decanoate, acetophenone, phenylacetaldehyde, and ethyl sorbate.

[0039] Technical Solution 18: The second fermentation product according to Technical Solution 17 is characterized in that the second fermentation product contains at least: γ-nonalactone greater than or equal to 5.80 mg / L, and / or trans-4-decenoic acid ethyl ester greater than or equal to 3.00 mg / L, and / or 4-vinyl-2-methoxyphenol greater than or equal to 1.20 mg / L, and / or β-ionone greater than or equal to 0.14 mg / L, and / or dihydro-β-ionone greater than or equal to 0.07 mg / L, and / or phenylethanol greater than or equal to 36.00 mg / L, and / or isopentanol greater than or equal to 25.00 mg / L, and / or ethyl hexanoate greater than or equal to 3.00 g / L, and / or ethyl octanoate greater than or equal to 1.30 mg / L, and / or Ethyl palmitate is greater than or equal to 1.10 mg / L, and / or n-hexanol is greater than or equal to 1.10 mg / L, and / or n-octanol is greater than or equal to 0.64 mg / L, and / or phenylethyl acetate is greater than or equal to 0.53 mg / L, and / or ethyl phenylacetate is greater than or equal to 0.47 mg / L, and / or 1-octen-3-ol is greater than or equal to 0.45 mg / L, and / or 4-ethylguaiacol is greater than or equal to 0.26 mg / L, and / or ethyl heptanoate is greater than or equal to 0.17 mg / L, and / or ethyl decanoate is greater than or equal to 0.15 mg / L, and / or acetophenone is greater than or equal to 0.12 mg / L, and / or phenylacetaldehyde is greater than or equal to 0.07 mg / L, and / or ethyl sorbate is greater than or equal to 0.04 mg / L.

[0040] Technical Solution 19: The second fermented product according to any one of Technical Solutions 16-18 is characterized in that the ethanol content per liter of the second fermented product is greater than or equal to 9400 mg, calculated in mg by mass of components contained per liter of the second fermented product.

[0041] Technical Solution 20: The method for preparing the second fermentation product according to any one of Technical Solutions 16 to 19, characterized in that it comprises:

[0042] (1) Cultivating the Saccharomyces cerevisiae strain described in any one of technical solutions 1 to 5 or the Saccharomyces cerevisiae agent described in technical solution 8;

[0043] (2) inoculating the Saccharomyces cerevisiae strain or the Saccharomyces cerevisiae agent of step (1) into a solid-state fermentation medium for gluten to obtain a first fermentation product;

[0044] (3) adding the first fermentation product obtained in step (2) to a starchy substrate liquid fermentation medium, preferably an unliquefied yellow corn flour liquid fermentation medium, and culturing the culture medium to obtain a second fermentation product.

[0045] Technical Solution 21: The preparation method according to Technical Solution 20, wherein the amount of the first fermented product added in step (3) is 5%-10% based on the dry weight of yellow corn flour.

[0046] Technical Solution 22: The preparation method according to Technical Solution 20 or 21 is characterized in that the culture temperature is 28-32°C and the culture time is 3-4 days.

[0047] Technical Solution 23: A method for using the Saccharomyces cerevisiae strain described in any one of Technical Solutions 1-5 or the Saccharomyces cerevisiae yeast agent described in Technical Solution 8 for starchy substrate fermentation, characterized in that the method comprises the following steps: culturing the Saccharomyces cerevisiae strain described in any one of Technical Solutions 1-5 or the Saccharomyces cerevisiae yeast agent described in Technical Solution 8.

[0048] Technical Solution 24: A related product fermented with starch as raw material, which is obtained by adding the Saccharomyces cerevisiae strain described in any one of Technical Solutions 1-5 or the Saccharomyces cerevisiae agent described in Technical Solution 8 to the starch raw material and fermenting.

[0049] Technical Solution 25: Use of the Saccharomyces cerevisiae strain described in any one of Technical Solutions 1-5 or the Saccharomyces cerevisiae agent described in Technical Solution 8 in producing aromatic substances in starchy substrate fermentation.

[0050] Technical Solution 26: Use of the first fermentation product prepared by the method for preparing the first fermentation product described in any one of Technical Solutions 9-12 or any one of Technical Solutions 13-15 in producing aromatic substances in starch substrate fermentation.

[0051] Beneficial effects of the present invention:

[0052] 1. The Saccharomyces glutinosus AMCC32244 strain of the present invention has the ability to produce pullulanase and raw amylase, and the pullulanase activity reaches 57.31 U / g, and the amylase activity reaches 4557.62 U / g. It also has the ability to produce high-temperature α-amylase, medium-temperature α-amylase, glucoamylase, cellulase (FPA enzyme), neutral protease, acid protease, alkaline protease, lipase, pullulanase and raw amylase, and the enzyme activity is high. It can fully decompose rich macromolecules such as crude starch, crude fat, crude protein, and crude fiber in various substrates such as wheat koji into small molecules that are beneficial to microbial absorption, thereby improving substrate utilization efficiency.

[0053] 2. The present invention's Saccharomyces cerevisiae AMCC32244 strain can grow at a temperature of 40°C, a pH of 3, an ethanol content of 8% (v / v) and a glucose content of 60% (w / v), and the viable count after bran fermentation is 4.78×10 10 CFU / g, which can better adapt to the special environmental conditions of fermentation.

[0054] 3. The Saccharomyces cingulosus AMCC32244 strain of the present invention can assimilate sugars such as xylose, arabinose, fructose, glucose, melibiose, cellobiose, maltose, trehalose, sucrose, melezitose, stachyose, inulin, pullulan, soluble starch and raw yellow corn flour, and can be used for fermentation and hydrolysis of related substrates.

[0055] 4. The Saccharomyces fulvidraco strain AMCC32244 of the present invention is used for fermentation of unliquefied yellow corn flour substrate, which can produce a rich variety of flavor substances with high content, and the ethanol content reaches 9434 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows the colony morphology of Saccharomyces cerevisiae;

[0057] FIG2 shows a microscopic observation of Saccharomyces cerevisiae;

[0058] FIG3 shows the appearance of bran after culturing for 72 hours.

[0059] Culture collection information

[0060] The Saccharomycopsis fibuligera AMCC32244 strain provided by the present invention was deposited in the China Center for Type Culture Collection on September 25, 2023, with a deposit number of CCTCC NO: M 20231788, and a deposit address of Wuhan University, Wuhan, China, with a postal code of 430072; and a telephone number of 027-68754052. DETAILED DESCRIPTION

[0061] The present invention provides a Saccharomycopsis fibuligera strain, which is the Saccharomycopsis fibuligera AMCC32244 strain. The Saccharomycopsis fibuligera AMCC32244 strain was deposited with the China Center for Type Culture Collection in October 2023 with a deposit number of CCTCC NO: M 20231788. The Saccharomycopsis fibuligera AMCC32244 strain provided by the present invention was isolated from rice wine. The colonies are round, white, dry and opaque, with hyphae on the surface and edges. The colonies are tightly bound to the culture medium and are difficult to pick. Microscopic observation reveals that single cells are ovoid, colorless, and transparent, measuring 3.0 × 7.3 μm. They reproduce by multipolar budding, with enlarged daughter cells remaining attached to the mother cell and continuing to bud, connecting to form long chains with branches, ultimately forming dendritic mycelium. The junctions between cells are constricted and devoid of transverse septa, resulting in a root-shaped, node-like structure with distinct pseudohyphae characteristics. The ITS rDNA sequence of the Saccharomyces cereus strain is shown in SEQ ID NO. 1.

[0062] The Saccharomycopsis fibuligera AMCC32244 strain provided by the present invention has the ability to produce multiple enzymes required for fermentation, such as pullulanase and amylase; can fully decompose macromolecular substances into small molecules that can be absorbed by microorganisms, thereby improving substrate utilization efficiency; and has high environmental tolerance and high aroma production ability, thereby improving the quality of fermentation products and reducing the cost of fermentation production investment.

[0063] In some specific embodiments, the raw materials used to prepare the YPD solid culture medium of the present invention include: 5-10 g yeast extract powder, 15-20 g glucose, 15-20 g peptone, 15-20 g agar and 950-1000 mL water.

[0064] In some specific embodiments, the raw materials used to prepare the YPD liquid culture medium of the present invention include: 5-10 g of yeast extract powder, 15-20 g of glucose, 15-20 g of peptone, and 950-1000 mL.

[0065] In some specific embodiments, the raw materials used to prepare the carbon source culture medium of the present invention include: 0.67-0.7 g of YNB culture medium, 1-3 g of carbon source substrate, and 95-100 mL of water. The carbon source substrate is one or more substances selected from the group consisting of glucose, xylose, arabinose, galactose, lactose, maltose, melibiose, sucrose, trehalose, cellobiose, melezitose, raffinose, soluble starch, and yellow corn flour.

[0066] The YNB medium comprises, by weight, 4900-5100 parts of ammonium sulfate, 1-3 parts of inositol, 0.2-0.5 parts of niacin, 0.2-0.5 parts of thiamine hydrochloride, 0.02-0.05 parts of copper sulfate, 950-1050 parts of potassium dihydrogen phosphate, 0.4-0.6 parts of boric acid, 0.3-0.5 parts of pyridoxine hydrochloride, 0.3-0.5 parts of calcium pantothenate, and 0.3-0. 5 parts, magnesium sulfate 490-510 parts, manganese sulfate 0.3-0.5 parts, zinc sulfate 0.3-0.5 parts, ferric chloride 0.1-0.3 parts, riboflavin 0.1-0.3 parts, calcium chloride 95-105 parts, potassium iodide 0.05-0.15 parts, sodium molybdate 0.1-0.3 parts, biotin 0.001-0.003 parts, folic acid 0.001-0.003 parts and sodium chloride 95-105 parts.

[0067] Preferably, after mixing the YNB medium with water and adjusting the volume to 1 L, the nutrient composition per L includes: 4900-5100 mg of ammonium sulfate, 1-3 mg of inositol, 0.2-0.5 mg of niacin, 0.2-0.5 mg of thiamine hydrochloride, 0.02-0.05 mg of copper sulfate, 950-1050 mg of potassium dihydrogen phosphate, 0.4-0.6 mg of boric acid, 0.3-0.5 mg of pyridoxine hydrochloride, 0.3-0.5 mg of calcium pantothenate, and 0.2-0.5 mg of p-aminophenyl. Formic acid 0.3-0.5mg, magnesium sulfate 490-510mg, manganese sulfate 0.3-0.5mg, zinc sulfate 0.3-0.5mg, ferric chloride 0.1-0.3mg, riboflavin 0.1-0.3mg, calcium chloride 95-105mg, potassium iodide 0.05-0.15mg, sodium molybdate 0.1-0.3mg, biotin 0.001-0.003mg, folic acid 0.001-0.003mg and sodium chloride 95-105mg.

[0068] In some specific embodiments, the raw materials used for preparing the bran solid-state fermentation medium of the present invention include: 10-20g bran, 0.05-0.07g potassium dihydrogen phosphate, 0.01-0.03g magnesium sulfate heptahydrate, 0.9-0.95g ammonium sulfate and 5-15mL water.

[0069] In some specific embodiments, the raw materials used to prepare the yellow corn flour liquid fermentation medium of the present invention include: 45-55g yellow corn flour, 0.1-0.5g urea, 0.1-0.5g magnesium sulfate heptahydrate, 0.1-0.15mL 1.5% dilute sulfuric acid and 950-1000mL water.

[0070] In some specific embodiments, the raw materials used in preparing YPD solid culture medium, YPD liquid culture medium, and high-glucose-tolerant culture medium include yeast peptone and yeast extract powder. Yeast peptone and yeast extract powder are primarily present in the culture medium as organic nitrogen sources, providing the nitrogen necessary for microbial growth during fermentation. Common organic nitrogen sources such as yeast extract powder and yeast peptone decompose in the culture medium, releasing amino acids and small peptides, which serve as nitrogen sources for microbial growth. In other words, when yeast peptone and yeast extract powder are used as common organic nitrogen sources in preparing culture medium, the present invention does not specifically limit their sources; they can be commercially available or prepared by conventional methods. Commercially available yeast peptone or prepared by conventional methods with a total nitrogen content of 12.5% ​​or greater and an amino nitrogen content of 2.5% or greater can be used in the present invention. Commercially available yeast extract powder with a total nitrogen content of 10.0% or greater and an amino nitrogen content of 5.0% or greater can be used in the present invention.

[0071] Preferably, in some specific embodiments, the yeast extract further comprises, based on the weight of the yeast extract, 2-2.5 ppm of vitamin B1, 37-40 ppm of vitamin B2, 113-116 ppm of vitamin B5, 15-20 ppm of vitamin B6, 6-10 ppm of vitamin B7, 25-28 ppm of vitamin B9, 3205-3210 ppm of choline, 1575-1580 ppm of inositol, 325-330 ppm of niacin, and 2-4 ug of vitamin B12 per 100 g of yeast extract.

[0072] and / or based on the weight of the yeast extract powder, potassium is 31910-31912 mg / kg, sodium is 5735-5740 mg / kg, calcium is 354-357 mg / kg, magnesium is 2670-2675 mg / kg, zinc is 77-83 mg / kg and iron is 77-83 mg / kg.

[0073] And / or based on the weight of the yeast extract powder, the yeast extract powder comprises: 30.3-40.85% free amino acids and 51-70.5% hydrolyzed amino acids.

[0074] The free amino acid content includes, based on the weight of the yeast extract powder, 1-2% of free aspartic acid, 2-3% of free threonine, 1.5-2% of free serine, 6.5-7% of free glutamic acid, 1-1.5% of free glycine, 4-5% of free alanine, 0.1-0.15% of free cysteine, 2-3% of free valine, 0.5-1% of free methionine, 2-2.5% of free isoleucine, 3.3-3.7% of leucine, 0.5-1% of free tyrosine, 1.5-2% of free phenylalanine, 2-2.5% of free lysine, 0.1-1% of free histidine, 1.5-2.5% of free arginine and 0.5-1% of free proline.

[0075] The hydrolyzed amino acid content includes, based on the weight of the yeast extract powder, 6-6.5% hydrolyzed aspartic acid, 2-3% hydrolyzed threonine, 2-3% hydrolyzed serine, 10-15% hydrolyzed glutamic acid, 2-3% hydrolyzed glycine, 5-6% hydrolyzed alanine, 0.5-1% hydrolyzed cysteine, 3-4% hydrolyzed valine, 0.5-1% hydrolyzed methionine, 3-4% hydrolyzed isoleucine, 4-5% hydrolyzed leucine, 1-2% hydrolyzed tyrosine, 2-3% hydrolyzed phenylalanine, 4-5% hydrolyzed lysine, 1-2% hydrolyzed histidine, 3-4% hydrolyzed arginine and 2-3% hydrolyzed proline.

[0076] In some specific embodiments, the present invention provides a first fermentation product, characterized in that the first fermentation product is obtained by the following steps:

[0077] (1) cultivating the yeast strain of Capsella fusca or the yeast agent of Capsella fusca;

[0078] (2) inoculating the Saccharomyces cerevisiae strain or the Saccharomyces cerevisiae agent of step (1) into a solid fermentation medium for gluten to obtain a first fermentation product.

[0079] Preferably, in some embodiments, the yeast Aspergillus flavus is Aspergillus flavus AMCC32244.

[0080] Preferably, in some specific embodiments, the content of total reducing sugars in the first fermentation product is greater than or equal to 10 mg / g, and / or the content of amino nitrogen in the first fermentation product is greater than or equal to 16 mg / g. More preferably, the content of total reducing sugars is 10-15 mg / g, and / or the content of amino nitrogen is 16-20 mg / g.

[0081] In some embodiments, the present invention provides a second fermentation product, which is obtained by the following steps:

[0082] (1) cultivating the yeast strain of any one of claims 1 to 5 or the yeast agent of claim 8;

[0083] (2) inoculating the Saccharomyces cerevisiae strain or the Saccharomyces cerevisiae inoculum of step (1) into a solid-state fermentation medium for culturing to obtain a first fermentation product, wherein, based on the dry weight of the bran, 9 mL to 20 mL of the Saccharomyces cerevisiae seed liquid is inoculated into every 100 g of the bran;

[0084] (3) The first fermentation product obtained in step (2) is added to a starchy substrate liquid fermentation medium, preferably an unliquefied yellow corn flour liquid fermentation medium, for culturing to obtain a second fermentation product.

[0085] Preferably, in some specific embodiments, the second fermentation product contains at least one or more substances selected from the group consisting of γ-nonalactone, phenylethanol, isoamyl alcohol, trans-4-decenoic acid ethyl ester, 4-vinyl-2-methoxy-phenol, β-ionone, ethyl palmitate and dihydro-β-ionone;

[0086] More preferably, the composition further contains one or more substances selected from the group consisting of ethyl hexanoate, ethyl octanoate, n-hexanol, n-octanol, phenylethyl acetate, ethyl phenylacetate, 1-octen-3-ol, 4-ethylguaiacol, ethyl heptanoate, ethyl decanoate, acetophenone, phenylacetaldehyde, and ethyl sorbate.

[0087] Preferably, in some specific embodiments, the second fermentation product contains at least: γ-nonalactone greater than or equal to 5.80 mg / L, and / or trans-4-decenoic acid ethyl ester greater than or equal to 3.00 mg / L, and / or 4-vinyl-2-methoxyphenol greater than or equal to 1.20 mg / L, and / or β-ionone greater than or equal to 0.14 mg / L, and / or dihydro-β-ionone greater than or equal to 0.07 mg / L, and / or phenylethyl alcohol greater than or equal to 36.00 mg / L, and / or isopentanol greater than or equal to 25.00 mg / L, and / or ethyl hexanoate greater than or equal to 3.00 g / L, and / or ethyl octanoate greater than or equal to 1.30 mg / L, and / or ethyl palmitate greater than or equal to 1. =1.10 mg / L, and / or n-hexanol is greater than or equal to 1.10 mg / L, and / or n-octanol is greater than or equal to 0.64 mg / L, and / or phenylethyl acetate is greater than or equal to 0.53 mg / L, and / or ethyl phenylacetate is greater than or equal to 0.47 mg / L, and / or 1-octen-3-ol is greater than or equal to 0.45 mg / L, and / or 4-ethylguaiacol is greater than or equal to 0.26 mg / L, and / or ethyl heptanoate is greater than or equal to 0.17 mg / L, and / or ethyl decanoate is greater than or equal to 0.15 mg / L, and / or acetophenone is greater than or equal to 0.12 mg / L, and / or phenylacetaldehyde is greater than or equal to 0.07 mg / L, and / or ethyl sorbate is greater than or equal to 0.04 mg / L.

[0088] More preferably, in some specific embodiments, the second fermentation product contains at least: γ-nonalactone greater than or equal to 5.80-10.00 mg / L, and / or trans-4-decenoic acid ethyl ester greater than or equal to 3.00-5.00 mg / L, and / or 4-vinyl-2-methoxyphenol greater than or equal to 1.20-5.00 mg / L, and / or β-ionone greater than or equal to 0.14-0.5 mg / L, and / or dihydro-β-ionone greater than or equal to 0.07-0.10 mg / L, and / or phenylethyl alcohol greater than or equal to 36.00-40.00 mg / L, and / or isopentanol greater than or equal to 25.00-30.00 mg / L, and / or ethyl hexanoate greater than or equal to 3.00-5.00 g / L, and / or ethyl octanoate greater than or equal to 1.30-2.00 mg / L, and / or ethyl palmitate greater than or equal to 1 .10-2.00 mg / L, and / or n-hexanol greater than or equal to 1.10-2.00 mg / L, and / or n-octanol greater than or equal to 0.64-1.00 mg / L, and / or phenylethyl acetate greater than or equal to 0.53-0.60 mg / L, and / or ethyl phenylacetate greater than or equal to 0.47-0.8 mg / L, and / or 1-octen-3-ol greater than or equal to 0.45-0.8 mg / L, and / or 4 - Ethylguaiacol greater than or equal to 0.26-0.5 mg / L, and / or ethyl heptanoate greater than or equal to 0.17-0.3 mg / L, and / or ethyl decanoate greater than or equal to 0.15-0.3 mg / L, and / or acetophenone greater than or equal to 0.12-0.3 mg / L, and / or phenylacetaldehyde greater than or equal to 0.07-0.1 mg / L, and / or ethyl sorbate greater than or equal to 0.04-0.08 mg / L.

[0089] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0090] Unless otherwise specified, the various reagents / instruments used in the examples and comparative examples of the present invention are conventional commercially available products. The experimental materials and instrument information used in the present invention are shown in Table 1 below:

[0091] Table 1. Source information of reagents and instruments used in the examples

[0092] The culture medium components involved in the embodiment are as follows:

[0093] (1) YPD solid medium: Yeast extract powder 10 g, glucose 20 g, peptone 20 g, agar 20 g, and water 1000 mL were mixed and sterilized at 115°C for 20 min to obtain YPD solid medium.

[0094] (2) YPD liquid medium (seed liquid medium): Yeast extract powder 10 g, glucose 20 g, peptone 20 g, and water 1000 mL were mixed and sterilized at 115°C for 20 min to obtain YPD liquid medium.

[0095] (3) Tolerance culture medium:

[0096] ①High temperature resistant culture medium: YPD liquid culture medium;

[0097] ② Low pH-tolerant culture medium: The pH of YPD liquid culture medium was adjusted to 4.0, 3.5, 3.0, and 2.5 with hydrochloric acid, and sterilized at 115°C for 20 min to obtain low pH-tolerant culture medium with pH of 4.0, 3.5, 3.0, and 2.5, respectively.

[0098] ③ High-glucose-tolerant culture medium: Dissolve 20 g, 30 g, 40 g, and 50 g of glucose with 1 g of yeast extract powder, 2 g of peptone, and 100 mL of water, respectively, and sterilize at 115°C for 20 min to obtain high-glucose-tolerant culture media containing glucose concentrations of 20% (w / v), 30% (w / v), 40% (w / v), and 50% (w / v), respectively, where w refers to the mass of glucose and v refers to the volume of water.

[0099] ④ Ethanol-resistant culture medium: Mix 6 mL, 8 mL, 10 mL, and 12 mL of anhydrous ethanol and YPD liquid culture medium, respectively, and adjust the volume to 100 mL using YPD liquid culture medium. Filter and sterilize to obtain ethanol-resistant culture medium containing anhydrous ethanol concentrations of 6% (v / v), 8% (v / v), 10% (v / v), and 12% (v / v), respectively.

[0100] (4) Carbon source medium: Dissolve 0.67 g YNB, 2 g carbon source (glucose, xylose, arabinose, galactose, lactose, maltose, melibiose, sucrose, trehalose, cellobiose, melezitose, raffinose, soluble starch, yellow corn flour) and 100 mL water, and filter sterilize.

[0101] (5) Wheat bran solid fermentation medium: 10 mL of water, 20 g of wheat bran, 0.06 g of potassium dihydrogen phosphate, 0.02 g of magnesium sulfate heptahydrate, and 0.92 g of ammonium sulfate were mixed and sterilized at 115°C for 20 min.

[0102] (6) Yellow corn flour liquid fermentation medium: 50 g of yellow corn flour, 0.5 g of urea, 0.4 g of magnesium sulfate heptahydrate, 0.1 mL of 1.5% dilute sulfuric acid, and 1000 mL of water were sterilized under ultraviolet light for 2 h and then dry-heat sterilized at 105°C for 2 h.

[0103] The trace elements contained in the yeast extract powder (Model: FM888) used in the examples are as follows: vitamin B1 2.3 ppm, vitamin B2 38.8 ppm, vitamin B5 115.0 ppm, vitamin B6 18.0 ppm, vitamin B7 7.9 ppm, vitamin B9 26.7 ppm, vitamin B12 2.3 (ug / 100g), choline 3206.0 ppm, inositol 1577.7 ppm, and niacin 328.0 ppm.

[0104] The trace elements contained in the yeast extract powder (model: FM888) used in the examples are as follows: potassium 31911.66 mg / kg, sodium 5738.91 mg / kg, calcium 355.25 mg / kg, magnesium 2673.59 mg / kg, zinc 80.94 mg / kg, and iron 80.21 mg / kg.

[0105] The yeast extract powder (model: FM888) used in the examples contained 35.1% free amino acids and 61.21% hydrolyzed amino acids.

[0106] The free amino acid content is specifically as follows: based on the weight of the yeast extract powder, free aspartic acid 1.6%, free threonine 2.1%, free serine 1.7%, free glutamic acid 6.7%, free glycine 1.2%, free alanine 4.2%, free cysteine ​​0.1%, free valine 2.7%, free methionine 0.8%, free isoleucine 2.2%, leucine 3.5%, free tyrosine 0.9%, free phenylalanine 1.8%, free lysine 2.3%, free histidine 0.5%, free arginine 2.0%, and free proline 0.8%.

[0107] The hydrolyzed amino acid content is specifically as follows: based on the weight of the yeast extract powder, the following: hydrolyzed aspartic acid 6.23%, hydrolyzed threonine 2.71%, hydrolyzed serine 2.73%, hydrolyzed glutamic acid 12.33%, hydrolyzed glycine 2.74%, hydrolyzed alanine 5.17%, hydrolyzed cysteine ​​0.61%, hydrolyzed valine 3.84%, hydrolyzed methionine 0.84%, hydrolyzed isoleucine 3.65%, hydrolyzed leucine 4.72%, hydrolyzed tyrosine 1.65%, hydrolyzed phenylalanine 2.68%, hydrolyzed lysine 4.63%, hydrolyzed histidine 1.19%, hydrolyzed arginine 3.30%, and hydrolyzed proline 2.19%.

[0108] Example 1 Isolation and Identification of Yeast Strains in the Present Invention

[0109] The rice wine sample (the rice wine sample was homemade by a farmer in Heping Village, Geputan Town, Yunmeng County, Xiaogan City) was diluted 10-fold with sterile water to prepare 10-5 , 10 -6 The bacterial suspension was spread on YPD solid medium and cultured at 30℃ for 24h. A single colony was picked, streaked and purified, inoculated on YPD slant medium, and stored at 4℃.

[0110] The purified strain was streaked onto YPD solid culture medium and cultured at 30°C for 24 h before observing the colony morphology. A single colony was picked and inoculated into 5 mL YPD liquid culture medium and cultured at 180 rpm and 30°C for 24 h. 10 μL was aspirated onto a glass slide and the bacterial morphology was observed under an optical microscope at a magnification of 400 times.

[0111] A strain was obtained. Its colonies were round, white, dry, and opaque, with hyphae-like surfaces and edges. The colonies were tightly bound to the culture medium and difficult to pick. Microscopic observation revealed single, ovoid, colorless, and transparent cells measuring 3.0 × 7.3 μm. The strain reproduced by multipolar budding, with grown daughter cells not separating from the mother cell and continuing to bud, connecting to form long chains with branches, ultimately forming dendritic mycelium. The junctions between cells were constricted and devoid of septa, and the hyphae were node-like, exhibiting distinct pseudohyphae characteristics. The yeast strain genome was extracted and ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO.2) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO.3) were used as primers. The PCR program was 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 45 s, 72°C extension for 90 s, 30 cycles, and a final extension at 72°C for 10 min to amplify the yeast ITS rDNA sequence. After detection by 1% gel electrophoresis and sequencing, the sequence was compared with the sequence on GenBank by BLast analysis. The sequence similarity was greater than 99%, indicating that it was the same species. The ITS rDNA sequence gene sequence SEQ ID NO.1 of the strain was determined to be as follows:

[0112] Combined with morphological analysis and molecular identification, the strain was identified as the Saccharomycopsis fibuligera AMCC32244 strain, which was deposited in the China Center for Type Culture Collection on September 25, 2023, with the deposit number: CCTCC NO: M 20231788. Figure 1 shows the colony morphology of the Saccharomycopsis fibuligera AMCC32244 strain. Figure 2 shows a microscopic observation of the Saccharomycopsis fibuligera AMCC32244 strain.

[0113] Example 2 Tolerance test of Saccharomyces cerevisiae AMCC32244 strain

[0114] (1) Seed solution preparation: 50 μL of bacterial solution in a glycerol tube was added to a test tube containing 5 mL of YPD liquid medium and cultured at 180 rpm and 30°C for 24 h.

[0115] (2) Tolerance culture:

[0116] ① High temperature resistance: AMCC32244 strain was inoculated into 96-well culture plates containing 200 mL YPD liquid medium at a 1% (v / v) inoculum. The culture temperatures were 30°C, 37°C, 40°C, and 42°C, respectively, and the rotation speed was 180 rpm. After 48 h of culture, the OD was measured. 600nm .

[0117] ② Low pH tolerance: AMCC32244 strain was inoculated into 200 μL of the corresponding low pH tolerant liquid medium at a 1% (v / v) inoculum in a 96-well culture plate, cultured at 180 rpm and 30°C for 48 h, and then the OD was measured. 600nm .

[0118] ③ High glucose tolerance: AMCC32244 strain was inoculated into 200 μL of the corresponding high glucose tolerance liquid medium at a 1% (v / v) inoculum in a 96-well culture plate, cultured at 180 rpm and 30°C for 48 h, and then the OD was measured. 600nm .

[0119] ④ Ethanol resistance: Inoculate the AMCC32244 strain at a 1% (v / v) inoculum into a 96-well culture plate containing 200 μL of the corresponding ethanol-resistant liquid medium, culture at 180 rpm and 30°C for 48 h, and then measure the OD 600nm .

[0120] The above culture conditions are all set to 3 wells of inoculated liquid, and 1 well of blank culture medium is used as blank control. The OD value of the inoculated well after 48h of bacterial growth is600nm Subtracted blank OD 600nm express.

[0121] Table 2 (A). Tolerance of Saccharomyces cerevisiae AMCC32244 strain

[0122] As shown in Table 2(A), the results indicate that the Saccharomyces cerevisiae AMCC32244 strain can grow at a temperature of 40°C, a pH of 3, an ethanol content of 8% (v / v), and a glucose content of 60% (w / v), further demonstrating that the strain has a good ability to adapt to high temperature, acidity, high ethanol, and high sugar content during fermentation.

[0123] Example 3 Analysis of Carbon Source Utilization Characteristics of Saccharomyces cerevisiae AMCC32244 Strain

[0124] (1) Seed solution preparation: Take 50 μL of glycerol tube bacterial solution and add it to a test tube containing 5 mL of YPD liquid culture medium. Incubate at 180 rpm and 30°C for 24 h.

[0125] (2) Resuspending the seed solution: Centrifuge the seed solution at 12,000 rpm for 5 min, remove the supernatant, and resuspend with 5 mL of sterile pure water. Centrifuge again to remove the supernatant, and then add 5 mL of sterile pure water to wash away the original culture medium.

[0126] (3) Different carbon source assimilation test: Different carbon source culture media were added to 96 deep-well plates, with 4 wells for each culture medium. Among them, 3 wells were filled with resuspended seed solution, and 1 well was used as a blank control. The plates were covered with sealing film and placed in a microplate constant temperature shaker at 200 rpm and 30°C. After culturing for 48 h, the plates were taken out and the OD was measured using a microplate reader. 600nm The bacterial growth OD is the OD of the inoculated well after 48 hours. 600nm Subtracted blank OD 600nm It indicates that a value of 0.1 is considered growth.

[0127] Table 2 (B) Carbon source utilization of Saccharomyces cerevisiae AMCC32244 strain

[0128] Note: “+” indicates positive; “-” indicates negative

[0129] As shown in Table 2(B), the results showed that the Saccharomyces cerevisiae AMCC32244 strain could utilize 15 carbon sources, including xylose, arabinose, fructose, glucose, melibiose, cellobiose, maltose, trehalose, sucrose, melezitose, stachyose, raffinose, inulin, pullulan and soluble starch, which indicated that the strain had a broad spectrum of substrate applicability and had the potential to be used in the fermentation of various raw materials.

[0130] Example 4 Solid-state fermentation test of wheat bran using Saccharomyces cerevisiae AMCC32244 strain

[0131] (1) Preparation of primary seed solution: 50 μL of bacterial solution in a glycerol tube was added to a test tube containing 5 mL of YPD liquid medium and cultured at 180 rpm and 30°C for 24 h.

[0132] (2) Preparation of secondary seed solution: 50 μL of the primary seed solution of S. fulvidraco AMCC32244 was added to test tubes containing 5 mL of YPD liquid medium and cultured at 180 rpm and 30°C for 24 h.

[0133] (3) Solid-state fermentation: 3.6 mL of seed liquid was added to 31 g of bran koji solid-state fermentation medium and cultured at 30°C for 72 h, with stirring once a day.

[0134] (4) Viable cell count: After stirring evenly every 24 hours, samples were taken to measure the viable cell count. After dilution, 0.1% Lu's alkaline methylene blue was added for staining for 3 minutes. The sample was then added to a hemocytometer and counted under a microscope. Figure 3 shows the appearance of bran after 72 hours of culture. The bran color showed obvious white mycelium on the bran surface. The bran odor had a fruity and floral aroma.

[0135] Table 3. Number of viable bacteria in solid-state fermentation of yeast AMCC32244

[0136] As shown in Table 3, the results showed that Saccharomyces cerevisiae AMCC32244 grew well under the conditions of bran fermentation and could adapt well to the bran fermentation environment.

[0137] (5) Enzyme activity detection

[0138] ① Preparation of crude enzyme solution: After fermentation, the solid fermentation product was vacuum freeze-dried, crushed and mixed, and 1.0 g (accurate to 0.001 g) of sample was weighed and added to a 150 mL conical flask containing 50 mL of buffer corresponding to the enzyme reaction pH and glass beads. Oscillate on an oscillator at 150 rpm for 40 min, aspirate an appropriate amount of the suspension and centrifuge at 8000 rpm for 2 min; the supernatant after centrifugation is the crude enzyme solution.

[0139] ② Enzyme activity determination

[0140] High-temperature α-amylase activity determination: Dilute the crude enzyme solution with sodium acetate buffer at pH=6.0 and pour it into a centrifuge tube. Place it in a water bath at 70°C for 5 minutes. Separately, take 0.9 mL of soluble starch solution with a concentration of 1% (w / v) and put it into a 2 mL centrifuge tube. Place it in a water bath at 70°C for 5 minutes. Take 0.1 mL of the diluted enzyme solution and add it to the preheated starch solution. After shaking to mix, place it in a 70°C water bath for 30 minutes. The control group used inactivated enzyme solution instead of enzyme solution. Then take 0.1 mL of the reaction solution and pour it into a 2 mL centrifuge tube. Then add 0.1 mL of DNS solution and boil it quickly for 5 minutes. After cooling, add 0.3 mL of deionized water and mix. Use blank to adjust to zero and measure OD 540nm The activity unit of high-temperature α-amylase is defined as: 1 g of solid fermentation product hydrolyzes soluble starch solution to produce 1 μg of maltose in 1 min at 70°C and pH 6.0, which is 1 unit of enzyme activity, expressed in U / g.

[0141] Determination of medium-temperature α-amylase activity: Dilute the crude enzyme solution with sodium acetate buffer at pH=6.0 and pour it into a centrifuge tube. Place it in a water bath at 60°C for 5 minutes. Separately, take 0.9 mL of soluble starch solution with a concentration of 1% (w / v) and put it into a 2 mL centrifuge tube. Place it in a water bath at 60°C for 5 minutes. Take 0.1 mL of the diluted enzyme solution and add it to the preheated starch solution. After shaking to mix, place it in a 60°C water bath for 30 minutes. The control group used inactivated enzyme solution instead of enzyme solution. Then take 0.1 mL of the reaction solution and pour it into a 2 mL centrifuge tube. Then add 0.1 mL of DNS solution and boil it quickly for 5 minutes. After cooling, add 0.3 mL of deionized water and mix. Use blank to adjust to zero and measure OD 540nm The activity unit of mesophilic α-amylase is defined as the amount of maltose produced by hydrolyzing 1 μg of soluble starch solution in 1 g of solid fermentation product at 60°C and pH 6.0 for 1 min, expressed as U / g.

[0142] Determination of saccharifying enzyme activity: Dilute the crude enzyme solution with sodium acetate buffer at pH=4.6 and pour it into a centrifuge tube. Place it in a water bath at 40°C for 5 minutes. Also, take 0.9 mL of soluble starch solution with a concentration of 2% (w / v) and put it into a 2 mL centrifuge tube. Place it in a water bath at 40°C for 5 minutes. Take 0.1 mL of the diluted enzyme solution and add it to the preheated starch solution. After shaking to mix, place it in a 50°C water bath for 20 minutes. The control group uses inactivated enzyme solution instead of enzyme solution. Then take 0.1 mL of the reaction solution and pour it into a 2 mL centrifuge tube. Then add 0.1 mL of DNS solution and boil it quickly for 5 minutes. After cooling, add 0.3 mL of deionized water and mix. Use the control group to zero and measure OD 540nmThe saccharifying enzyme activity unit is defined as the amount of enzyme required to hydrolyze soluble starch in 1 g of solid fermentation product to produce 1 μg of glucose in 1 min at 40°C and pH 4.6. This is one enzyme activity unit, expressed in U / g.

[0143] Cellulase (FPA) activity assay: Reference standard QB / T 2583-2003. One FPA activity unit is defined as the amount of enzyme required to degrade filter paper (1 g) of sample in 1 minute to produce 1 μg of glucose at 50°C and pH 4.8. This unit is expressed in U / g.

[0144] Neutral protease activity assay: Reference standard GB / T 23527-2009. One unit of neutral protease activity is defined as the amount of enzyme required to hydrolyze casein from 1 g of sample to produce 1 μg of tyrosine in 1 minute at 40°C and pH 7.5. This unit is expressed in U / g.

[0145] Acidic protease activity assay: Reference standard GB / T 23527-2009. One unit of acidic protease activity is defined as the amount of enzyme required to hydrolyze casein from 1 g of sample to produce 1 μg of tyrosine in 1 minute at 40°C and pH 3.0. This unit is expressed in U / g.

[0146] Alkaline protease activity assay: Reference standard GB / T 23527-2009. One unit of alkaline protease activity is defined as the amount of enzyme required to hydrolyze casein from 1 g of sample to produce 1 μg of tyrosine in 1 minute at 40°C and pH 10.5. This unit is expressed in U / g.

[0147] Lipase activity assay: Refer to GB / T 23535. One unit of lipase activity is defined as the amount of enzyme required to hydrolyze 1 g of sample to produce 1 μmol of titratable fatty acid in 1 min at 40°C, expressed in U / g.

[0148] Pullulanase activity assay: Reference standard GB 1886.174-2016. One unit of pullulanase activity is defined as the amount by which 1 g of solid fermentation product hydrolyzes pullulan to produce 1 μg of glucose in 1 min at 60°C and pH 4.5. The activity is expressed in U / g.

[0149] Determination of raw starch enzyme activity: Dilute the crude enzyme solution appropriately and pour it into a centrifuge tube. Place it in a water bath at 50°C for 5 minutes. Also, take 0.9 mL of 1% (w / v) yellow corn flour solution and put it into a 2 mL centrifuge tube. Place it in a water bath at 50°C for 5 minutes. Take 0.1 mL of the diluted enzyme solution and add it to the preheated yellow corn flour solution. Shake to mix and place it in a 50°C water bath for 30 minutes. The control group uses inactivated enzyme solution instead of enzyme solution. Then centrifuge the reaction solution to obtain the supernatant. Take 0.1 mL of the supernatant and pour it into a 2 mL centrifuge tube. Then add 0.1 mL of DNS solution and boil it quickly for 5 minutes. After cooling, add 0.3 mL of deionized water and mix. Use the control group to zero and measure OD 540nm The activity unit of raw amylase is defined as: 1 g of solid fermentation product hydrolyzes yellow corn flour to produce 1 μg of glucose in 1 min at 50°C and pH 5.0, which is 1 unit of enzyme activity, expressed in U / g.

[0150] Table 4. Enzyme activity of Saccharomyces cerevisiae AMCC32244

[0151] As shown in Table 4, the results show that the Saccharomyces cingulans strain AMCC32244 produces a wide variety of enzymes with high enzyme activity, including a raw amylase activity of 4557.62 U / g. Raw amylases are enzymes that exhibit strong hydrolysis activity on raw starch granules that have not undergone cooking and gelatinization. This allows the traditional starch gelatinization, liquefaction, and saccharification processes to be combined into a single step for direct saccharification. Furthermore, this is the first time that a Saccharomyces cingulans strain has been found to produce pullulanase. Pullulanase is a starch debranching enzyme that efficiently cleaves α-1,6-glycosidic bonds at branch sites in polyglucose substrates. This enzyme is often used in combination with saccharifying enzymes in the starch saccharification process. The synergistic effect of these two enzymes effectively improves starch utilization and saccharification yield, while significantly shortening saccharification time. This demonstrates the high application value of the Saccharomyces cingulans strain AMCC32244 in reducing fermentation production inputs and improving production efficiency.

[0152] (6) Detection of small molecule nutrients in bran

[0153] Determination of total reducing sugar: Pour 0.1 mL of the crude enzyme solution of Example 4 (5) ① into a 2 mL centrifuge tube, then add 0.1 mL of DNS solution and boil rapidly for 5 min. After cooling, add 0.3 mL of deionized water and mix well, then measure the OD value. 540nmGlucose was used as the standard curve. 1g of glucose dried to constant weight was added with deionized water to a volume of 1L as the mother liquor. The mother liquor was mixed with deionized water in proportion to prepare 0g / L, 0.2g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.8g / L, and 1g / L standard curve working solutions. 0.1mL of the working solution was poured into a 2mL centrifuge tube, followed by adding 0.1mL of DNS solution and rapidly boiling for 5min. After cooling, 0.3mL of deionized water was added and mixed, and then the OD was measured. 540nm . And calculate the content according to the absorbance value: total reducing sugar content (mg / g) = (sample OD 540nm The value corresponds to the weight of glucose in the standard curve × dilution factor) / sample weight.

[0154] Determination of free amino acids: Determined according to the method of standard GB / T 18246-2019.

[0155] Table 5. Detection of small molecule nutrients in bran

[0156] As shown in Table 5, the strain of Saccharomyces cerevisiae AMCC32244 can fully decompose the abundant macromolecular substances in bran into small molecules that can be absorbed by microorganisms, thereby improving the efficiency of substrate utilization.

[0157] Example 5: Liquid fermentation test of starchy raw materials

[0158] (1) Preparation of primary seed solution: Take 50 μL of glycerol tube bacterial solution of Saccharomyces cerevisiae AMCC32244 and add it into test tubes containing 5 mL of YPD liquid culture medium. Incubate at 180 rpm and 30°C for 24 h.

[0159] (2) Preparation of secondary seed solution: 50 μL of the primary seed solution of S. fulvidraco AMCC32244 was added into test tubes containing 5 mL of YPD liquid culture medium and cultured at 180 rpm and 30°C for 24 h.

[0160] (3) Solid-state fermentation: 3.6 mL of the secondary seed solution was added to 31 g of bran koji solid-state fermentation medium and cultured at 30°C for 72 h, with stirring once a day.

[0161] (4) Liquid fermentation: 8 g of the fermentation product obtained in step (3) was inoculated into 151 g of yellow corn flour liquid fermentation medium and cultured at 30°C for 72 h.

[0162] (5) After the fermentation is completed, the fermentation supernatant is collected by centrifugation at 5000 rpm for 2 min.

[0163] (6) Ethanol content analysis: The ethanol content was detected according to the method of reference standard Q / YB.J19.266-2022, and the ethanol content was 9434 mg / L.

[0164] (7) Flavor Analysis: After fermentation, the supernatant was collected by centrifugation at 8000 rpm for 5 min. After filtration through 0.22 μm, 5 mL of the filtrate was added with 1.5 g of sodium chloride and 10 μL of internal standard solution (2 g / L tetramethyl dipentanol), mixed, and equilibrated at 50°C for 30 min. Volatile component analysis was performed. Gas chromatography conditions: Column: DB-HeavyWAX, 30 m × 0.25 mm × 0.25 μm, inlet temperature: 250°C, carrier gas: He; temperature program: 40°C for 3 min, then increase to 200°C at a rate of 4°C / min, hold for 0 min; then increase to 250°C at a rate of 10°C / min, hold for 3 min. The results are shown in Table 6 below.

[0165] Table 6. Flavor analysis

[0166] Note: Only substances with a matching factor > 85 are selected for display; \ indicates an unknown threshold.

[0167] It should be noted that only flavor substances with an OVA greater than 1 in the samples are presented here. Generally, an aroma activity value (OVA) greater than 1 means that the odor emitted by the substance reaches the threshold range that can be smelled by humans and can be smelled.

[0168] As shown in Table 6, fermentation produced fruity and floral aromas such as phenylethanol, isopentanol, 4-vinyl-2-methoxyphenol, β-ionone, and dihydro-β-ionone; and waxy and creamy aromas such as γ-nonalactone, ethyl palmitate, and ethyl trans-4-decenoate. The OVA values ​​of all these substances were greater than 1, while OVA values ​​of potentially unpleasant flavor substances such as 3-methylthiopropanol, octanoic acid, and butyric acid were less than 1. These results demonstrate that the Saccharomyces cerevisiae strain AMCC32244 has excellent aroma-producing properties and could be used to enhance the flavor of starch-based fermentation products, such as wine, pasta, vinegar, and fermented beverages.

[0169] Furthermore, as shown in Table 6, volatile flavor compounds also include antioxidant and anti-inflammatory compounds. For example, 4-vinyl-2-methoxyphenol exhibits anti-inflammatory activity; 4-ethylguaiacol is a free radical scavenger with antioxidant, disease prevention, immune-enhancing, antibacterial, and anti-infective properties; β-ionone exhibits anti-cancer, antibacterial, anti-inflammatory, antimicrobial, and lipid-lowering properties, among other health benefits; octanoic acid exhibits antibacterial, cholesterol-lowering, and cardiovascular health benefits; butyric acid promotes mesenteric lymph node development, regulates anti-inflammatory signaling pathways, and improves intestinal epithelial barrier function; hexanoic acid exhibits antibacterial, anti-inflammatory, and antioxidant properties; and heptanoic acid exhibits multiple biological activities, including antioxidant, anti-inflammatory, and antimicrobial activities. These results suggest that the yeast strain AMCC32244 can enhance the antioxidant and anti-inflammatory activities of fermented products.

[0170] The above embodiments are only for further explanation and understanding of the technical solutions of the present invention, and are not intended to limit the present invention. Any non-prominent substantial features and non-significant improvements made by those skilled in the art on this basis should fall within the scope of protection of the present invention.

Claims

1. A yeast strain of Saccharomyces cerevisiae, characterized in that: The Saccharomycopsis fibuligera yeast strain is a Saccharomycopsis fibuligera AMCC32244 strain with the function of producing pullulanase, which was deposited in the China Center for Type Culture Collection on September 25, 2023, with the deposit number: CCTCC NO: M 20231788.

2. The yeast strain of Saccharomyces cerevisiae according to claim 1, characterized in that The ITS rDNA gene sequence of the Saccharomyces cerevisiae strain is shown in SEQ ID NO.

1.

3. The yeast strain of claim 1 or 2, characterized in that The Saccharomyces fulvidraco yeast strain has the characteristics of high temperature resistance, and / or low pH resistance, and / or high sugar resistance, and / or ethanol resistance, and / or carbon source utilization.

4. The yeast strain according to any one of claims 1 to 3, characterized in that The carbon source material includes one or a combination of xylose and pullulan.

5. The yeast strain according to any one of claims 1 to 4, characterized in that The carbon source material further includes one or more materials selected from the group consisting of arabinose, fructose, glucose, melibiose, cellobiose, maltose, trehalose, sucrose, melezitose, stachyose, raffinose, inulin and soluble starch.

6. A fermentation method for preparing a yeast agent of the cystic spores, characterized in that: The method comprises the following steps: culturing the Saccharomyces cerevisiae strain according to any one of claims 1 to 5.

7. The preparation method according to claim 6, characterized in that The preparation method comprises the following steps: (1) amplifying and culturing the yeast strain of any one of claims 1 to 5; (2) adding the product obtained in step (1) into a liquid culture medium and fermenting and culturing the product at 28-32° C., preferably, the liquid culture medium is a YPD liquid culture medium.

8. A yeast agent of Capsularia multiflora, characterized in that: The product is obtained by the fermentation preparation method according to claim 6 or 7.

9. A first fermented product, characterized in that The first fermentation product is obtained by the following steps: (1) culturing the Saccharomyces cerevisiae strain according to any one of claims 1 to 5 or the Saccharomyces cerevisiae inoculum according to claim 8 to obtain a seed solution containing Saccharomyces cerevisiae, preferably, the culture medium used during the culture is YPD liquid medium; (2) The seed liquid containing the spore yeast obtained in step (1) is inoculated into a bran solid fermentation medium for cultivation to obtain a first fermentation product, wherein, based on the dry weight of the bran, 5 mL to 20 mL of the spore yeast seed liquid is inoculated into every 100 g of the bran.

10. The first fermented product according to claim 9, characterized in that The number of viable bacteria of Saccharomyces cerevisiae AMCC32244 in the first fermentation product is greater than or equal to 6.10×10 9 CFU / g, preferably greater than or equal to 1.31×10 10 , more preferably, greater than or equal to 4.70×10 10 CFU / g.

11. The first fermented product according to claim 9 or 10, characterized in that The first fermented product contains at least one or more substances selected from the group consisting of high-temperature α-amylase, medium-temperature α-amylase, glucoamylase, pullulanase and raw amylase; More preferably, the protein composition further contains one or two or more substances selected from the group consisting of acidic proteases, neutral proteases, alkaline proteases, cellulases, and lipases.

12. The first fermented product according to claim 11, characterized in that Calculated in enzyme activity U / g, the high-temperature α-amylase activity is greater than or equal to 39900; and / or the medium-temperature α-amylase activity is greater than or equal to 79600; and / or the glucoamylase activity is greater than or equal to 82500; and / or the pullulanase activity is greater than or equal to 57; and / or the raw amylase activity is greater than or equal to 4550; and / or the acid protease activity is greater than or equal to 30; and / or the neutral protease activity is greater than or equal to 160; and / or the alkaline protease activity is greater than or equal to 110; and / or the cellulase activity is greater than or equal to 360; and / or the lipase activity is greater than or equal to 7.

13. The method for preparing the first fermented product according to any one of claims 9 to 12, characterized in that: include: (1) culturing the Saccharomyces cerevisiae strain according to any one of claims 1 to 5 or the Saccharomyces cerevisiae inoculum according to claim 8 to obtain a seed solution containing Saccharomyces cerevisiae, preferably, the culture medium used during the culture is YPD liquid medium; (2) The seed liquid containing the spore yeast obtained in step (1) is inoculated into a bran solid fermentation medium for cultivation to obtain a first fermentation product, wherein, based on the dry weight of the bran, 5 mL to 20 mL of the spore yeast seed liquid is inoculated into every 100 g of the bran.

14. The method for preparing the first fermented product according to claim 13, wherein: Based on the dry weight of the bran, 5mL-16mL of the seed liquid of the yeast Saccharomyces cerevisiae is inoculated into every 100g of the bran.

15. The method for preparing the first fermented product according to claim 14 or 15, characterized in that: The culture temperature is 28-32°C and the culture time is 3-4 days.

16. A second fermented product, characterized in that The second fermentation product is obtained by the following steps: (1) cultivating the yeast strain of any one of claims 1 to 5 or the yeast agent of claim 8; (2) inoculating the Saccharomyces cerevisiae strain or the Saccharomyces cerevisiae inoculum of step (1) into a bran solid fermentation medium for culturing to obtain a first fermentation product, wherein, based on the dry weight of the bran, 5 mL to 20 mL of the Saccharomyces cerevisiae seed liquid is inoculated into every 100 g of the bran; (3) The first fermentation product obtained in step (2) is added to a starchy substrate liquid fermentation medium, preferably an unliquefied yellow corn flour liquid fermentation medium, for culturing to obtain a second fermentation product.

17. The second fermentation product according to claim 16, characterized in that The second fermented product contains at least one or more substances selected from the group consisting of γ-nonalactone, phenylethanol, isoamyl alcohol, trans-4-decenoic acid ethyl ester, 4-vinyl-2-methoxy-phenol, β-ionone, ethyl palmitate and dihydro-β-ionone; More preferably, the composition further contains one or more substances selected from the group consisting of ethyl hexanoate, ethyl octanoate, n-hexanol, n-octanol, phenylethyl acetate, ethyl phenylacetate, 1-octen-3-ol, 4-ethylguaiacol, ethyl heptanoate, ethyl decanoate, acetophenone, phenylacetaldehyde, and ethyl sorbate.

18. The second fermentation product according to claim 17, characterized in that The second fermentation product contains at least: γ-nonalactone greater than or equal to 5.80 mg / L, and / or trans-4-decenoic acid ethyl ester greater than or equal to 3.00 mg / L, and / or 4-vinyl-2-methoxyphenol greater than or equal to 1.20 mg / L, and / or β-ionone greater than or equal to 0.14 mg / L, and / or dihydro-β-ionone greater than or equal to 0.07 mg / L, and / or phenylethyl alcohol greater than or equal to 36.00 mg / L, and / or isopentanol greater than or equal to 25.00 mg / L, and / or ethyl hexanoate greater than or equal to 3.00 g / L, and / or ethyl octanoate greater than or equal to 1.30 mg / L, and / or ethyl palmitate greater than or equal to 1.10 mg / L, and / or n-hexanol is greater than or equal to 1.10 mg / L, and / or n-octanol is greater than or equal to 0.64 mg / L, and / or phenylethyl acetate is greater than or equal to 0.53 mg / L, and / or ethyl phenylacetate is greater than or equal to 0.47 mg / L, and / or 1-octen-3-ol is greater than or equal to 0.45 mg / L, and / or 4-ethylguaiacol is greater than or equal to 0.26 mg / L, and / or ethyl heptanoate is greater than or equal to 0.17 mg / L, and / or ethyl decanoate is greater than or equal to 0.15 mg / L, and / or acetophenone is greater than or equal to 0.12 mg / L, and / or phenylacetaldehyde is greater than or equal to 0.07 mg / L, and / or ethyl sorbate is greater than or equal to 0.04 mg / L.

19. The second fermentation product according to any one of claims 16 to 18, characterized in that Calculated in mg by mass of components contained in each liter of the second fermentation product, the ethanol content in each liter of the second fermentation product is greater than or equal to 9400 mg.

20. The method for preparing the second fermentation product according to any one of claims 16 to 19, characterized in that: include: (1) cultivating the yeast strain of any one of claims 1 to 5 or the yeast agent of claim 8; (2) inoculating the Saccharomyces cerevisiae strain or the Saccharomyces cerevisiae agent of step (1) into a solid-state fermentation medium for gluten to obtain a first fermentation product; (3) adding the first fermentation product obtained in step (2) to a starchy substrate liquid fermentation medium, preferably an unliquefied yellow corn flour liquid fermentation medium, and culturing the culture medium to obtain a second fermentation product.

21. The preparation method according to claim 20, wherein The amount of the first fermented product added in step (3) is 5%-10% based on the dry weight of the yellow corn flour.

22. The preparation method according to claim 20 or 21, characterized in that The culture temperature is 28-32°C and the culture time is 3-4 days.

23. A method for using the Saccharomyces cerevisiae strain according to any one of claims 1 to 5 or the Saccharomyces cerevisiae inoculum according to claim 8 for fermentation on starchy substrates, characterized in that: The method comprises the following steps: culturing the Saccharomyces cerevisiae strain according to any one of claims 1 to 5 or the Saccharomyces cerevisiae agent according to claim 8.

24. A related product fermented with starch as raw material, obtained by adding the Saccharomyces cerevisiae strain according to any one of claims 1 to 5 or the Saccharomyces cerevisiae inoculum according to claim 8 to the starch raw material for fermentation.

25. Use of the Saccharomyces cerevisiae strain according to any one of claims 1 to 5 or the Saccharomyces cerevisiae inoculum according to claim 8 in producing aromatic substances in starch substrate fermentation.

26. Use of the first fermentation product produced by the method for producing a first fermentation product according to any one of claims 9 to 12 or any one of claims 13 to 15 in producing aromatic substances in starch substrate fermentation.