CN115161246A

CN115161246A - Saccharopolyspora rosea strain for high yield of saccharifying enzyme and liquefying enzyme and application thereof

Info

Publication number: CN115161246A
Application number: CN202210973187.8A
Authority: CN
Inventors: 毛健; 刘双平; 马东林; 刘彩霞
Original assignee: Industrial Technology Research Institute Of Jiangnan University Shaoxing; Jiangnan University
Current assignee: Industrial Technology Research Institute Of Jiangnan University Shaoxing; Jiangnan University
Priority date: 2022-08-15
Filing date: 2022-08-15
Publication date: 2022-10-11
Anticipated expiration: 2042-08-15
Also published as: CN115161246B

Abstract

The invention discloses a saccharopolyspora rosea strain for high yield of saccharifying enzyme and liquefying enzyme and application thereof, belonging to the field of fermentation engineering and biotechnology. The invention obtains the saccharopolyspora rosea A22 with high yields of saccharifying enzyme and liquefying enzyme by plasma mutagenesis screening at normal temperature and normal pressure, the saccharifying enzyme activity is 1372.85U/g +/-55.54U/g, the liquefying enzyme activity is 1.53U/g +/-0.09U/g, compared with the original strain, the saccharifying enzyme activity is improved by 29%, and the liquefying enzyme activity is improved by 47%. The inoculated raw wheat starter prepared by the mutagenic strain A22 with high enzyme yield is an enhancement of the traditional raw wheat starter, can be used for producing brewed foods prepared by various starter methods such as white spirit, yellow wine, rice wine, cooking wine, soy sauce, vinegar and the like, and has wide application prospect in the foods brewed by the starter method.

Description

Saccharopolyspora rosea strain for high yield of saccharifying enzyme and liquefying enzyme and application thereof

Technical Field

The invention relates to a saccharopolyspora rosea strain for high yield of saccharifying enzyme and liquefying enzyme and application thereof, belonging to the field of fermentation engineering and biotechnology.

Background

The raw wheat koji is one of the most common saccharifying agents in the traditional brewed foods in China, not only plays a role in providing saccharifying enzyme and liquefying enzyme, but also has obvious influence on the flavor characteristics of a fermented product (generally wine). The raw wheat koji provides various enzymes, mainly amylase, protease, lipase and the like, for fermented food, promotes liquefaction and decomposition of high molecular substances such as starch, protein, fat and the like contained in raw materials, simultaneously forms various metabolites in the koji making process, and gives substances such as color, aroma and the like generated by interaction of the metabolites to the brewed food, thereby endowing the brewed food with unique style. Amylase in the raw wheat koji plays a crucial role in the utilization of raw materials in brewing food, and two key enzymes for hydrolyzing starch are saccharifying enzyme and liquefying enzyme, so the saccharifying enzyme and the liquefying enzyme in the raw wheat koji are also key indexes for representing the quality of the wheat koji.

Macro-genome sequencing proves that saccharopolyspora is a core microorganism in the process of fermenting wheat starter and yellow rice wine, and especially saccharopolyspora fulgidae, saccharopolyspora holtziae, saccharopolyspora rosea and saccharopolyspora Jiangxi in wheat starter occupy main abundance in actinomycetes. Functional prediction also indicates that saccharopolyspora is closely related to flavor substances in food fermentation processes, mainly related to sources of amino acids (aspartic acid, threonine or methionine) fatty acids (caprylic acid, capric acid, lauric acid), and alcohol and ester substance synthesis. Therefore, the improvement of the abundance of saccharopolyspora in the wheat koji has important significance for improving the flavor and the quality of the fermented food.

At present, the existing production of raw wheat koji mainly utilizes microorganisms of environmental species, belongs to open culture, has few excellent strains in the koji and lower enzyme activity, leads to large amount of koji used during brewing, increases the production cost of enterprises, and in addition, the excessive use of the wheat koji during production easily causes the flavor of brewed food to be poor and the color and luster to be darker, thereby influencing the quality of the brewed food. One roseosaccharomyces roseus capable of being used for making raw wheat starter is screened in the early stage of a laboratory, but the roseosaccharomyces roseus has the defects of low activity of hydrolyzed amylase, low utilization rate of raw materials and the like.

Meanwhile, the yellow wine brewed by the prior art has high content of higher alcohol (560 mg/L-620 mg/L), and causes adverse reactions such as dizziness, nausea and the like after drinking, so the content of the higher alcohol is controlled in the fermentation process of the yellow wine. In addition, the yellow wine brewed by the prior art is added with the cooked wheat starter, the cooked wheat starter has high protease, and can hydrolyze raw materials to generate a large amount of amino acids including some bitter amino acids (2.5 g/L-3 g/L), which is also the main reason for causing the yellow wine to be bitter and heavy. At present, the alcoholic strength of yellow wine brewed by yellow wine enterprises is generally between 17 percent vol and 18 percent vol, and the alcoholic strength is close to that of the original factory firstly to develop a novel wheat koji to replace the traditional wheat koji.

Therefore, aiming at the problems existing in the yellow wine enterprises at present, the strain with high amylase yield and good genetic stability is bred and inoculated into the raw wheat starter, and the method has important significance for improving the flavor of the yellow wine and improving the utilization rate of other raw materials for brewing food.

Disclosure of Invention

The invention provides a strain of Saccharopolyspora rosea (Saccharopolyspora rosea) A22 which is preserved in China Center for Type Culture Collection (CCTCC) at 7-6 months in 2022 with the preservation number of CCTCC NO: M20221039.

The Saccharopolyspora rosea (Saccharomyces rosea) A22 is a mutagenized strain obtained by carrying out compound mutagenesis on an original strain of the Saccharopolyspora rosea and screening, the strain can be stably passaged, and compared with the original strain, the production level of saccharifying enzyme and liquefying enzyme can be obviously improved.

The invention also provides a microbial agent which contains the roseopolyspora rosea A22 or fermentation liquor thereof, or cell lysate of the roseopolyspora rosea A22.

In one embodiment of the present invention, the content of saccharopolyspora rosea a22 in the microbial agent is at least 1.0 × 10 ⁶ spore/mL。

The invention also provides raw wheat starter which is prepared by crushing wheat, adding the saccharopolyspora rosea A22 bacterial suspension into the crushed wheat according to a certain addition amount, uniformly stirring, and fermenting in a starter house for 6-7 days.

In one embodiment of the invention, the concentration of the saccharopolyspora rosea A22 bacterial suspension is 10 ⁴ ～10 ⁸ CFU/mL, inoculating the saccharopolyspora rosea A22 bacterial suspension into the crushed wheat according to the addition amount of 20-26% (v/m), and fermenting after uniformly stirring.

The invention also provides a method for producing inoculated wheat starter by fermenting the Saccharopolyspora rosea (Saccharomyces polyspora rosea) A22, which comprises the following steps: mixing activated Saccharopolyspora rosea A22 seed solution with clear water mixed with yeast, adjusting bacteria concentration to 10 ⁴ ～10 ⁸ CFU/mL to obtain bacterial suspension, inoculating the bacterial suspension into the crushed wheat according to the addition of 20-26% (v/m), uniformly stirring, and particularly paying attention to not generate starch lumps to prevent bad koji from being generated subsequently.

Fermenting the mixed wheat yeast in yeast basket (length: width: height =60 × 42 × 26cm) for 6-7 days at 45 deg.C and 95% humidity.

In one embodiment of the present invention, the raw wheat koji is prepared by mixing live wheat kojiMixing the seed solution of Saccharopolyspora rosea A22 with clear water mixed with yeast, and adjusting the concentration of bacteria to 10 ⁴ ～10 ⁸ CFU/mL to obtain bacterial suspension, inoculating the bacterial suspension into pulverized wheat according to the addition amount of 20-26% (v/m), stirring uniformly, and making the bulk koji with the above mixed bulk koji.

In one embodiment of the present invention, the preparation method of the lump koji comprises: and (3) pressing the scattered yeast after uniformly stirring into yeast blocks with specific shapes by using a mechanical yeast making machine, wherein the yeast blocks are 25 (+ -1) cm in length, 15 (+ -1) cm in width and 6 (+ -1) cm in height. And (3) standing the pressed koji blocks for 30-40min, placing the koji blocks according to a T shape to ensure air flow between adjacent koji blocks, then conveying the koji blocks into a koji room, fermenting for 120-122h under the conditions of 45 ℃ and 95% humidity, and finally obtaining finished product koji blocks.

In an embodiment of the invention, by adopting the technical scheme, in the process of manufacturing the inoculated raw wheat starter for brewing food, the highest temperature reaches 52-54 ℃, the growth of the non-high temperature resistant mixed bacteria is inhibited, the high temperature resistant saccharopolyspora rosea is propagated in large quantity at the temperature, the enzyme production is strong, the saccharifying enzyme and the liquefying enzyme are high, the interior of the wheat starter is white, a large amount of spores are generated, and the flavor of the starter is normal.

In one embodiment of the invention, the fermentation conditions are: culturing at 45 deg.C under 95% humidity for 6-7 days, taking out of the yeast room after the wheat yeast grows with white hypha, and oven drying for use.

The invention also provides application of the roseosaccharomyces roseus A22 or the metabolite thereof in improving the enzyme activity of saccharifying enzyme and liquefying enzyme in the field of fermentation.

In one embodiment of the present invention, the metabolite includes, but is not limited to, the supernatant from which the somatic cells are removed after fermentation of the saccharopolyspora rosea a 22.

In one embodiment of the invention, the field of fermentation comprises the preparation of a brewed food, a fermented food, an alcoholic drink or a seasoning.

The invention also provides application of the saccharopolyspora rosea A22 in preparation of inoculated raw wheat starter.

The invention also provides the application of the inoculated raw wheat koji in the preparation of brewed foods prepared by various koji methods such as yellow wine, white spirit, red yeast yellow wine, cooking wine, vinegar, soy sauce and the like and the preparation process of corresponding koji.

The invention also provides application of the raw wheat koji in preparing brewed foods, alcoholic beverages or seasonings.

In one embodiment of the invention, the brewed food product includes, but is not limited to: yellow wine, white spirit, sake or rice wine, soy sauce, vinegar, sweet fermented rice or cooking wine.

In one embodiment of the present invention, the alcoholic beverage includes, but is not limited to: yellow wine, chinese liquor, sake or rice wine.

In one embodiment of the present invention, the flavoring includes, but is not limited to: cooking wine, soy sauce and vinegar.

The invention also provides the application of the roseosaccharomyces A22 or the microbial agent or the raw wheat koji in preparing fermented food.

In one embodiment of the present invention, the fermented food includes, but is not limited to, alcoholic beverages, bean products, fermented vegetables, seasonings.

The invention also provides application of the roseosacrata bacteria A22 or the microbial agent in improving the saccharifying power and liquefying power of raw wheat koji.

The invention also provides the application of the roselle polyspora A22 or the microbial agent or the raw wheat starter in improving the alcoholic strength of yellow wine, reducing the content of higher alcohol in the yellow wine or reducing the content of bitter amino acid in the yellow wine.

The invention also provides a brewing method of the wine, which comprises the following steps:

the raw wheat koji is used for replacing the traditional formula of raw wheat koji and cooked wheat koji in a factory, and is directly added into a raw material containing yeast for fermentation preparation; the wine includes, but is not limited to, yellow wine, white spirit, sake and sake.

Advantageous effects

(1) The present invention uses the existing roseopolyspora rosea in a laboratory as a starting strain, and obtains a mutant strain roseopolyspora rosea (CCTCC NO: M20221039) through composite mutagenesis and screening, wherein the mutant strain has high saccharifying enzyme and liquefying enzyme.

(2) The invention provides a mutant strain A22 of Saccharopolyspora rosea, wherein the saccharifying enzyme activity of the strain in a wheat starter prepared by solid-state fermentation is 1200-1400U/g, the liquefying enzyme activity is 1.3-1.6U/g, the saccharifying enzyme activity is improved by 29% compared with that of a starting strain (Saccharopolyspora rosea F2014), and the liquefying enzyme activity is improved by 49%.

(3) After the mutagenic strain saccharopolyspora rosea A22 is cultured on a slant and subcultured to 6 generations, the strain has better genetic stability.

(4) The alcoholic strength (18 percent vol) of the yellow wine prepared by inoculating the raw wheat starter and fermented by the strain of the saccharopolyspora rosea A22 is higher than that (17.8 percent vol) of the mechanized yellow wine (raw wheat starter and cooked wheat starter fermented), and meanwhile, compared with the mechanical yellow wine, the method can reduce the total high-grade alcohol content in the yellow wine by 12.7 percent and the content of bitter amino acid by 28 percent, and can improve the drinking comfort of the yellow wine on the basis of not changing the integral flavor of the yellow wine. Meanwhile, the bitter amino acid content of the yellow wine fermented by inoculating the raw wheat starter prepared by the roseospora A22 is also reduced.

(5) The novel saccharopolyspora rosea inoculated raw wheat starter prepared by the strain is a functional microorganism enhancement of common raw wheat starters, and has higher saccharifying enzyme and liquefying enzyme than the common raw wheat starters. The process of the invention is an innovation of the traditional yeast making process, changes the process of matching the cooked wheat yeast and the raw wheat yeast in a factory to use the traditional mechanized production process of the yellow wine, completely uses the strengthened raw wheat yeast to produce the yellow wine, can reduce the yeast consumption for the manual production of the yellow wine from 16-17 percent to 12-13 percent, and meets the national standard requirements of the yellow wine on the physicochemical indexes. Due to the reduction of the using amount of the wheat starter, the flavor and the appearance of the brewed yellow wine are improved, the color is yellow, bright and transparent, the flavor is elegant and delicate, the brewing production cost is saved, and the economic benefit of an enterprise is improved.

(6) The mutant strain saccharopolyspora rosea A22 obtained by the invention can be used for not only making loose koji, but also producing the lump koji, reducing the fermentation time of the lump koji, being not limited by seasons, being capable of producing the lump koji throughout the year and improving the production efficiency.

(7) The raw wheat starter produced by the invention can be applied to yellow wine, and also can be applied to the production of food brewed by a starter method such as white spirit, soy sauce, vinegar, rice wine, cooking wine, red yeast rice wine and the like, and has wide application prospect.

Biological material preservation

A strain of Saccharopolyspora rosea (Saccharopolyspora rosea) A22, which is classified and named as Saccharopolyspora rosea A22, has been preserved in China center for type culture Collection (CCTCC NO: M20221039) at 7.6.2022, and has a preservation address of Wuhan university, wuhan China.

Drawings

FIG. 1: lethality curves (a) and positive mutation rate curves (B) for ARTP mutagenesis.

FIG. 2: basic physicochemical index change of the raw wheat starter inoculated with polyspora rosea in the process of brewing yellow wine; wherein, the alcohol degree is A; b, reducing sugar; c titratable acid; d amino acid nitrogen.

FIG. 3: the mutant strain is used for preparing saccharifying enzyme (A) inoculated with raw wheat starter and liquefying enzyme activity (B).

FIG. 4: basic physicochemical index change of the raw wheat starter inoculated with polyspora rosea in the process of brewing yellow wine; wherein, A is total acid; b amino acid nitrogen; c, alcohol content; d reducing sugar.

FIG. 5: and (3) comparing the mutant strain with the original strain, namely saccharifying enzyme (A) and liquefying enzyme (B).

Detailed Description

The soft red-skinned or yellow-skinned wheat referred to in the following examples was purchased from the traditional brewed food research center of Wuxi Convergence Tongliao and Saccharomyces cerevisiae, university in south of the Yangtze river.

The "liquefying enzyme" mainly refers to the enzyme activity of amylase.

The mutagenesis starting strain related in the following examples is saccharopolyspora rosea F2014, and is saccharopolyspora rosea CCTCC NO: m2020952, disclosed in application publication No. CN 113249268A.

The media involved in the following examples are as follows:

actinomycete liquid culture medium: 1.0g/L potassium nitrate, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.5g/L sodium chloride, 20.0g/L soluble starch and pH value of 7.2-7.4 (25 ℃).

Actinomycetes culture medium: 1.0g/L potassium nitrate, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate, 0.01g/L ferrous sulfate, 0.5g/L sodium chloride, 20.0g/L soluble starch, 15.0g/L agar, and pH value of 7.2-7.4 (25 ℃).

YPD medium: 10g/L yeast extract powder, 20g/L peptone, 20g/L glucose, adding water to 1000mL,2% agar, autoclaving at 115 deg.C for 20min, and cooling.

The detection methods referred to in the following examples are as follows:

and (3) enzyme activity detection:

the national standard GB1886.174-2016 is adopted to detect alpha-amylase and saccharifying enzyme in self-warming wheat koji.

The water content is measured by using a national standard GB5009.3-2016, and the temperature and the humidity are measured by using a hygrothermograph.

Detecting physical and chemical indexes of the yellow wine:

the alcoholic strength is measured according to the national standard GB13662-2018, the reducing sugar is measured by a3, 5-dinitrosalicylic acid method, organic acids and free amino acids in the yellow wine are detected by a high performance liquid chromatography instrument, and flavor substances in the yellow wine are detected by a gas chromatography-mass spectrometer.

Example 1: preparation of mutagenized Strain of Polycospora rosea (Saccharomyces rosea)

1. Preparation of mutant strains of Saccharopolyspora rosea

The original starting strain of Saccharopolyspora rosea F2014 (CCTCC NO: M2020952) in a laboratory is subjected to plasma at the room temperature and the normal pressure.

(1) Preparation of a Maliosporium roseum spore suspension

Activating strains: transferring the slant strain F2014 preserved in a refrigerator at 4 ℃ to a fresh slant, and carrying out biological culture at 37 ℃ for 8-10 days until white spores grow on the slant.

Preparation of spore suspension: will be provided withWashing the activated slant strain with sterile physiological saline containing 0.05% Tween, transferring to a triangular flask filled with 30mL sterile water, adding 10-15 glass beads with the diameter of 5mm at the bottom of the triangular flask, mixing with spores, sealing, shaking for 30min at 180r/min in a shaking table at 37 ℃, fully scattering the spores, filtering with a single-layer sterile cotton gauze to remove hyphae, separating the precipitated spores with a centrifuge, and removing supernatant. A further 40mL of eluate was added and the centrifugation was repeated 3 times. The concentration is (0.8-1.2) × 10 ⁶ spores/mL of a mold spore suspension.

(2) Determination of plasma mutagenesis treatment time

Mixing the thallus suspension with 10% (v/v) glycerol according to the proportion of 1. Under the conditions that the working gas flow is 10L/min, the distance between a plasma emission source and a sample is 2mm, and the operating temperature is 23.0-35.0 ℃, the mutagenesis time is set to be 0s, 30s, 60s, 90s, 120s, 150s and 180 s. After mutagenesis, 1mL of sterile physiological saline is added into an EP tube in advance, the treated slide is transferred into the EP tube and shaken for 5min, and the slide is eluted to form bacterial suspension. After the collected bacterial liquid is properly diluted, 100 mu L of the diluted liquid is taken to coat a Gao's first plate and cultured at the constant temperature of 37 ℃ for 120h, and each group comprises three parallel plates. The colony number is recorded, and the lethality rate of the mutagenesis under different ARTP treatment time is calculated to prepare a lethality rate curve. Observing colonies growing on the plate by taking the colony morphology of the original strain as a reference, and picking the strain with better growth to a fresh Gao's first plate. Observing the growth condition of colonies on the plate and the size of a transparent ring around the colonies, selecting strains with hydrolysis rings, recording the diameters of the hydrolysis rings and the diameters of the colonies, selecting forward mutant strains according to the ratio of the enzyme activity index (EI) to the diameters of the hydrolysis rings and the colonies, and drawing a forward mutation rate curve. As can be seen from FIG. 1, when the mutagenesis time was 120s, the mortality was 85%, and when it exceeded 150s, the mortality was higher than 90%, and further, when the mutagenesis time was 120s, the positive mutation rate was the highest, and in order to select a better mutagenized strain, we selected the mutagenesis time with the mortality higher than 80% and the positive mutation rate was the highest, and therefore 120s was selected as the optimal mutagenesis time.

(3) Screening of positive mutant strain after normal-temperature isobaric plasma mutagenesis

Primary screening by a flat plate: 26 mutant strains with EI values higher than that of the original strain are preliminarily screened according to the ratio of the EI hydrolysis circle diameter to the colony diameter, inoculated with raw wheat starter and subjected to yellow wine brewing experimental evaluation, wherein the mutant strains are Saccharopolyspora rosea A21, A22, A23, A31, A36, A37, A42, A44, A45, A46, A47, A48, A49, A51, A52, A54, A55, A56, A58, A59, A60, A61, A68, A69, A73 and A74 respectively.

2. Screening of mutant strains of Saccharopolyspora rosea

(1) Preparation of saccharopolyspora inocula inoculated raw wheat starter

Respectively mixing activated Saccharopolyspora rosea seed liquid (Saccharopolyspora rosea A21, A22, A23, A31, A36, A37, A42, A44, A45, A46, A47, A48, A49, A51, A52, A54, A55, A56, A58, A59, A60, A61, A68, A69, A73, A74) with clear water mixed with koji, adjusting bacteria concentration to 10 ⁴ ～10 ⁸ CFU/mL to obtain bacterial suspension, inoculating the bacterial suspension into the crushed wheat according to the addition of 20-26% (v/m), uniformly stirring, and particularly paying attention to not generate starch lumps to prevent bad koji from being generated subsequently. Placing the mixed wheat koji into a koji basket (length: width: height =60 × 42 × 26cm) and placing in a koji room, fermenting at 45 ℃ and 95% humidity for 6-7 days to respectively obtain finished koji, namely: inoculating saccharopolyspora to raw wheat starter.

(2) Preparation of yellow wine

1) And (3) yeast activation culture:

inoculating Saccharomyces cerevisiae into YPD culture medium, and performing activation culture at 28 deg.C and 150r/min for 24 hr to obtain yeast culture solution;

2) Preparing yeast wine:

taking 600g of steamed rice, adding 1600mL of clear water, 60g of factory raw wheat starter and 800U/g of rice saccharifying enzyme (purchased from Jiangsu Banaba Biotechnology Co., ltd.) for saccharification, controlling the saccharifying temperature at 55-65 ℃ for 3-4 hours, sterilizing at 115 ℃ for 15 minutes after the saccharifying is finished and the apparent sugar degree is not lower than 12 DEG Bx, cooling to 24-31 ℃ after the sterilizing, inoculating the yeast culture solution prepared in the step (1) according to the inoculation amount of 5% (v/v), culturing at 28 ℃ for 18-24 hours under the condition of 150r/min, and obtaining the yeast after the yeast is cultured and matured.

3) Yellow wine fermentation:

the blanking tables are shown in table 1 and table 2, and the blanking is carried out according to the raw material tables of table 1 and table 2 respectively

Table 1: mechanical yellow wine fermented mash blanking meter (2L fermentation system)

Table 2: saccharopolyspora inoculation raw wheat starter fermentation yellow wine blanking table (2L fermentation system)

The results are shown in fig. 2, and the physical and chemical indexes of the yellow wine brewed by inoculating raw wheat starter prepared by different mutant strains by using mechanical yellow wine as a control have differences. And screening to obtain mutant strains A58, A54, A48, A22, A45 and A44 of the saccharopolyspora rosea according to comprehensive indexes.

Example 2: application of mutant strain A22 of Saccharopolyspora rosea (Saccharomyces rosea) in improving enzyme activity of inoculated raw wheat starter saccharifying enzyme and liquefying enzyme

The method comprises the following specific steps:

(1) Preparation of Saccharopolyspora rosea liquid

Respectively inoculating saccharopolyspora rosea F2014, saccharopolyspora rosea A22, mutant strains A58, A54, A48, A45 and A44 into an actinomycetes culture medium, culturing at a constant temperature of 37 ℃ for 96 hours, then picking colonies from a flat plate, inoculating into an actinomycetes liquid culture medium, and performing shake culture at a temperature of 37 ℃ and 150r/min for 48 hours to respectively prepare seed solutions;

then, the seed liquid is respectively transferred into an actinomycete liquid culture medium according to the inoculation amount of 3% (v/v), and shaking culture is carried out for 96 hours at 37 ℃ and 150r/min to obtain the polyspora rosea liquid.

(2) Crushing red-skin or yellow-skin soft wheat produced in the current year after sieving treatment, wherein the moisture content of the wheat is 13 +/-1.5 percent (mass fraction), and each wheat flour is crushed to 3-5 pieces in a plum blossom shape during crushing;

(3) Mixing the saccharopolyspora rosea bacterial liquid prepared in the step (1) with water according to the inoculation amount of 3-5% (v/m), and adjusting the final concentration of the bacterial liquid to 10 ⁴ ～10 ⁶ Performing spore/mL to obtain a bacterial suspension;

(4) Adding the bacterial suspension obtained in the step (3) into the crushed wheat obtained in the step (2) according to the amount of 20-26% (v/m), uniformly stirring, particularly paying special attention to not generate white-heart cakes formed by water absorption of starch clusters, simultaneously mixing yeast with water without bacteria liquid as a control group, then placing a yeast frame under the conditions of 45 ℃ and 95% humidity for fermentation for 120-122h, finally obtaining finished yeast prepared by adopting different polyspora rosea, and detecting the enzyme activity of saccharifying enzyme and liquefying enzyme in the finished yeast.

As shown in FIG. 3, it was found that the activities of the saccharifying enzyme and liquefying enzyme in the finished koji prepared by using the saccharopolyspora rosea A22 were higher in the mutant strains A58, A54, A48, A22, A45 and A44 of the saccharopolyspora rosea obtained by screening in example 1 than in the other strains.

(5) And (3) preparing a finished product koji from the mutant strain Saccharopolyspora rosea A22 and the original strain Saccharopolyspora rosea F2014 according to the methods in the steps (1) to (4), and comparing the enzyme activity effects of the saccharifying enzyme and the liquefying enzyme in the obtained koji, wherein the result is shown in figure 5.

The result shows that compared with the original strain, namely the roseopolyspora rosea A22, the saccharifying enzyme is improved by 29 percent, the liquefying enzyme is improved by 47 percent, and finally, the saccharifying enzyme of the mutagenized roseopolyspora rosea A22 is 1372.85U/g +/-55.54U/g, and the liquefying enzyme is 1.53U/g +/-0.09U/g, so that compared with the original strain, the saccharifying enzyme and the liquefying enzyme are both improved.

Example 3: application of Saccharopolyspora rosea A22 in improving alcohol content of yellow wine and reducing content of higher alcohol and bitter amino acid in yellow wine

Yellow wine is brewed by respectively adopting saccharopolyspora prepared in example 2 to inoculate raw wheat starter, and the specific steps are as follows:

1. preparation of yellow wine

(1) And (3) yeast activation culture:

(2) Preparing yeast wine:

taking 600g of steamed rice, adding 1600mL of clear water, 60g of raw wheat starter and 800U/g of rice saccharifying enzyme (purchased from Jiangsu Musca Biotech Co., ltd.) for saccharification, controlling the saccharifying temperature to be 55-65 ℃ and the time to be 3-4 hours, sterilizing at 115 ℃ for 15 minutes after the saccharifying is finished and the apparent sugar degree is not lower than 12 DEG Bx, cooling to 24-31 ℃ after the sterilizing, inoculating the yeast culture solution prepared in the step (1) according to the inoculation amount of 5% (v/v), culturing at the culturing temperature of 28 ℃ for 18-24 hours under the condition of 150r/min, and obtaining the yeast wine.

(3) Yellow wine fermentation:

the blanking tables are shown in the table 3 and the table 4, and the blanking is carried out according to the raw material tables of the table 3 and the table 4 respectively

Table 3: mechanical yellow wine fermented mash blanking meter (2L fermentation system)

Table 4: saccharopolyspora inoculation raw wheat starter fermentation yellow wine blanking table (2L fermentation system)

Respectively preparing the mechanical yellow wine and inoculating saccharopolyspora into raw wheat starter to prepare the yellow wine.

2. Effect characterization

The results show that:

(1) The alcoholic strength at the end of post-fermentation using the roseopolyspora rosea a58, a54, a48, a22, a45, a44 was higher than the control mechanical yellow wine group (16 vol%), respectively a58 (16.3 vol%), a54 (16.6 vol%), a48 (16 vol%), a22 (17.3 vol%), a45 (16.4 vol%), a44 (16.1 vol%), while the total acid, reducing sugar and amino-state contents were all within the range of the yellow wine national standard GB/T13662-2018.

(2) The physical and chemical indexes of the yellow wine brewed by each strain are shown in figure 4 by adopting the raw wheat starter to carry out a yellow wine fermentation experiment, and the result shows that the content of total acid control group (the yellow wine brewed by adopting the traditional process) is 6.67 +/-0.13 (g/L) at most, the content of total acid of A22 is 6.07 +/-0.14 (g/L), and the content is obviously different from that of a control group (P is less than 0.05). The content of amino nitrogen in the control group is 1.13 +/-0.03 (g/L) at most, the mutation strains A22, A48, A58 and A44 have no remarkable difference (P is more than 0.05), and the content of amino nitrogen in yellow wine is 0.81-0.86 (g/L).

(3) The alcoholicity of the mutant strain A22 is higher than that of the control group (yellow wine brewed by adopting the traditional process) by comparing 17.37 +/-0.37 (% vol) of the alcoholicity of the control mechanical yellow wine group, and the alcoholicity of the mutant strain A44 is at least 16.16 +/-0.59 (% vol).

(4) The reducing sugar content of a control group 9.82 +/-0.97 (g/L) has a significant difference (P < 0.05) from other mutant strains, wherein the reducing sugar content of the A44 mutant strain is 6.35 +/-0.25 (g/L), and the reducing sugar content of yellow wine brewed by inoculating wheat starter for the other mutant strains is lower than 5g/L, which indicates that the reducing sugar in the yellow wine is basically completely consumed by microorganisms.

In conclusion, the comprehensive index of the mutagenized strain A22 after wine brewing is optimal by combining the basic physicochemical index of the yellow wine.

3. Genetic stability of mutant strain Saccharopolyspora rosea A22

The screened high-yield amylase mutant saccharopolyspora is subcultured on a Gao's No. one plate medium for 6 generations, solid state fermentation is carried out for 120 hours for each generation, the enzyme production capacity of strains of each generation is detected, and the result is shown in Table 5.

The solid state fermentation was as follows: will be provided withMixing the activated Saccharopolyspora rosea seed liquid with clear water mixed with yeast, adjusting bacteria concentration to 10 ⁴ ～10 ⁸ CFU/mL to obtain a bacterial suspension, inoculating the bacterial suspension into the crushed wheat according to the addition of 20-26% (v/m), uniformly stirring, and particularly paying attention to not generate starch lumps to prevent bad koji from being generated subsequently. Placing the mixed wheat koji into a koji basket (length: width: height =60 × 42 × 26cm) and placing in a koji room, fermenting at 45 deg.C and 95% humidity for 6-7 days to obtain the final product koji.

Table 5: passage stability test of mutant Strain A22

The result shows that the saccharifying ability and liquefying ability of the saccharopolyspora rosea A22 after 6 passages are not obviously changed, the saccharifying enzyme is maintained at 1286-1372U/g, the liquefying enzyme activity is maintained at 1.43-1.51U/g, and the enzyme activity is relatively stable, so the strain saccharopolyspora rosea A22 is selected and stored in the China center for type culture collection.

4. The preparation of the yellow wine was carried out according to the method of step 1 using saccharopolyspora rosea a22, and the physicochemical indices and volatile flavor substances of the yellow wine prepared after fermentation were measured using the yellow wine brewed by traditional mechanical malt as a control, and the results are shown in table 6.

Table 6: measurement results of physical and chemical indexes of yellow wine brewed by inoculating saccharopolyspora A22 with wheat starter and yellow wine brewed by traditional mechanical wheat starter

The results show that:

(1) As can be seen from Table 6, there is a clear difference between the inoculation of raw wheat koji with Saccharopolyspora rosea A22 and the co-fermentation with Saccharomyces cerevisiae. The alcoholic strength is an intuitive index reflecting whether the wine making is normally carried out, and from Table 6, it can be seen that the alcoholic strength of the yellow wine brewed by inoculating the A22 wheat starter is obviously higher than that of the traditional yellow wine (18.35 vol vs 17.41 vol%), which shows that the strain A22 wheat starter produces a large amount of amylase hydrolyzed starch for yeast growth and propagation and ethanol production.

(2) The flavor of yellow wine is the soul of yellow wine. Different strains produce different flavors due to the difference in metabolism. Table 6 shows the difference in flavor between the yellow wine brewed with a22 wheat koji and the conventional yellow wine.

The high alcohol content in the yellow wine is a main material of the yellow wine, but the high alcohol content can cause adverse reactions such as dizziness, nausea and the like after drinking, so the high alcohol content is controlled in the fermentation process of the yellow wine. Table 6 shows that the total higher alcohol content of the yellow wine brewed by inoculating the raw wheat starter with A22 is lower than that of a control group, and the higher alcohol content is reduced by 12.7 percent compared with the control group, which indicates that the yellow wine fermented by inoculating the raw wheat starter with A22 can improve the drinking comfort of the yellow wine.

(3) Bitter amino acids: the traditional mechanized yellow wine fermentation is added with cooked wheat koji, so that a large amount of bitter amino acids including His, arg, val, phe, ile, leu and Lys are generated, and the bitter amino acids aggravate the bitter taste of the yellow wine and hinder the development of yellow wine enterprises. Compared with the traditional yellow wine, the content of bitter amino acids in the yellow wine brewed by adopting the A22 wheat koji is reduced by 28 percent, the bitter taste of the mechanized yellow wine is improved, and the sensory experience of the yellow wine is improved.

Example 4: production process for inoculating raw wheat starter (block starter) with polyspora rosea

(1) Inoculating saccharopolyspora rosea A22 into an actinomycete culture medium, culturing for 96h at a constant temperature of 37 ℃, then selecting a bacterial colony from a flat plate, inoculating into an actinomycete liquid culture medium, and culturing for 48h at a temperature of 37 ℃ and at a speed of 150r/min in a shaking table to prepare a seed liquid; then, the seed solution is transferred into an actinomycete liquid culture medium according to the inoculation amount of 3% (v/v) for amplification culture, and shaking culture is carried out for 96 hours at 37 ℃ and 150r/min to obtain the saccharopolyspora rosea liquid.

(3) Mixing the polyspora rosea bacterial liquid prepared in the step (1) with water according to the inoculation amount of 3-5% (v/m), and adjusting the final concentration of the bacterial liquid to 10 ⁴ ～10 ⁶ Performing spore/mL to obtain a bacterial suspension;

(4) Adding the bacterial suspension obtained in the step (3) into the crushed wheat obtained in the step (2) according to the amount of 20-26% (v/m), uniformly stirring, particularly paying special attention to not generating white heart cakes formed by water absorption of starch mass, pressing the uniformly stirred koji into koji blocks with a specific shape by using a mechanical koji making machine, wherein the length of the koji blocks is 25 (+ -1) cm, the width is 15 (+ -1) cm, and the height is 6 (+ -1) cm. And (3) standing the pressed koji blocks for 30-40min, placing the koji blocks in a T shape to ensure air flow between adjacent koji blocks, then conveying the koji blocks into a koji room, fermenting for 120-122h under the conditions of 45 ℃ and 95% humidity, and finally obtaining finished product koji blocks.

Example 5: application of saccharopolyspora rosea A22 inoculated raw wheat starter in red yeast rice yellow wine

The yeast activation and yeast preparation method is the same as example 3, the wheat is shown in table 7, and the wheat is blanked according to the raw material table in table 7, wherein the raw wheat yeast inoculated with the saccharopolyspora rosea is prepared by adopting the process conditions of example 2.

Table 7: fermented mash blanking meter (5L fermentation system)

The preparation method of the red yeast rice in the ingredient list comprises the following steps:

selecting clean rice, soaking rice for 48h, steaming with rice steaming cabinet, cooling to room temperature, washing cultured Monascus ruber spores from inclined plane with 0.1% sterile water for spitting out according to the ratio of 10 ⁶ Mixing the spores/g rice into the cooked rice, and culturing in a yeast room when the cooked rice grains are all reddish; continuously turning over the koji in the fermentation process of the koji room, maintaining the temperature of the product at 28-35 ℃, culturing for 5-7 d, enabling the hypha in the rice to be propagated vigorously and turn red inside and outside, moving the koji to the outside, and drying to obtain the finished productRed Rice.

After blanking is finished, fermentation is carried out, the primary fermentation is carried out for 5 days at the temperature of 28 +/-2 ℃, harrowing is started after fermentation is started for 10-12 hours, and harrowing is started every 8 hours after harrowing is started; the post-fermentation is carried out for 15 days at 15 (+ -2) DEG C, and the rake is opened every 24h after the post-fermentation is started. The physical and chemical index measurement of the fermented red yeast yellow wine shows that 8, the alcohol content of the brewed red yeast yellow wine is 16.72 +/-0.23 percent vol and 55.65 +/-1.43 mg/L lovastatin.

Table 8: physical and chemical indexes of red yeast rice yellow wine brewed by inoculating raw wheat starter with polyspora rosea

Example 6: application of polyspora rosea A22 inoculated raw wheat starter in soy sauce brewing

The raw wheat koji inoculated with the saccharopolyspora rosea is prepared by adopting the process conditions of the example 2.

Selecting full and clean soybeans, cleaning, adding water, soaking for 5-10 h, draining water after the soybeans completely absorb water and swell, and then pressing and steaming; after the hot beans are taken out of the pot, cooling to about 70-80 ℃, adding 30-40% of flour (based on the mass of the soybeans), uniformly stirring, continuously cooling to 30-40 ℃, then adding 2-5% of polyspora rosea to inoculate raw wheat starter, uniformly stirring, keeping the product temperature at about 28-30 ℃ for fermentation, continuously turning over the starter to ventilate for cooling due to heat generated by microbial metabolism in the middle of fermentation, and obtaining soy sauce starter after 72-96 hours; mixing the prepared soy sauce koji according to the proportion of 1:1.5 (m/v) adding saline water, stirring uniformly to prepare fermented grains, fermenting for 3-6 months, and turning over the sauce once every 15 days; squeezing the soy sauce mash after fermentation is finished, filtering to remove impurities and sterilizing to obtain the finished soy sauce. The physicochemical indexes of the finished soy sauce are shown in the following table.

Table 9: physical and chemical indexes of brewing soy sauce by inoculating raw wheat starter to saccharopolyspora rosea A22

Example 7: application of saccharopolyspora rosea A22 inoculated raw wheat starter in vinegar brewing

Selecting materials: selecting high-quality glutinous rice 200kg, adding water 400kg, soaking rice for 24-48h, then pouring rice until no white pulp exists, draining, steaming, and cooling to room temperature;

stirring materials into a cylinder: before glutinous rice is put into a jar, 3-4 kg of vinum agent is mixed, and low-temperature saccharification is carried out for 72-96 h;

alcohol fermentation: adding 60kg of water after saccharification is finished, adding 12kg of saccharopolyspora rosea inoculated wheat starter (the saccharopolyspora rosea inoculated wheat starter is prepared by adopting the process conditions of the embodiment 2), and fermenting at 28 ℃ for 168-192 h to obtain mature fermented grains;

preparing fermented grains: putting 300kg of bran into a fermentation tank, spreading, putting fermented grains into the tank, uniformly stirring, uniformly spreading 10kg of rice hulls on the upper layer of the tank, uniformly stirring 10kg of fermented vinegar grains, covering the rice hulls with the mixture, and uniformly scattering the mixture to finish fermented grains preparation;

acetic acid fermentation: turning over the fermented grains after every 24 hours of fermentation, adding rice husks for heat preservation and moisture preservation after each turn over of the fermented grains is finished, adding no rice husks from the 11 th day of fermentation, cooling the fermented grains at a warm temperature, adding 8kg of salt when acidity is detected after 18-20 days and does not rise any more, and sealing for 30-45 days;

pouring vinegar and frying vinegar: adding stir-fried rice color into the aged vinegar grains according to a proportion, soaking for several hours, circularly soaking and pouring by adopting a pouring method, adding sugar into the obtained vinegar juice for blending, clarifying, then decocting the vinegar, canning, sealing and storing after the temperature of the product is reduced to 75-80 ℃. Physical and chemical indexes of vinegar brewed by inoculating saccharopolyspora with raw wheat starter are shown as 10.

Table 10: physical and chemical indexes for brewing vinegar by inoculating raw wheat starter to saccharopolyspora rosea A22

Example 8: application of polyspora rosea A22 inoculated raw malt yeast in sake brewing

The raw wheat koji inoculated with the saccharopolyspora rosea is prepared by adopting the optimal process conditions of the embodiment 2.

(1) Polishing: removing 20-25% of the raw material rice by a polishing machine. The broken rice grains are as few as possible, the polished rice is similar to the brown rice in shape, and germs and ditches of the raw material rice are completely removed;

(2) Rice soaking: soaking rice for 24-48h at room temperature;

(3) And (3) steaming rice: the rice has clear grains, hard outside and soft inside, no white core inside, looseness, no paste, uniform and consistent cooking, and the rice yield of the glutinous rice is generally about 140 to 150 percent;

(4) Blanking: the temperature of the rice is cooled to 25-30 ℃ during the blanking, wherein the composition of the rice, the rice-koji, the yeast and the water is shown in the table 11.

Table 11:300L sake fermentation system ingredient list

(5) Fermentation: the initial fermentation temperature is controlled at 28-30 deg.C for 3 days, then the fermentation is carried out at 20 deg.C for 22 days, the total fermentation time is 25 days, and the physicochemical index of the sake is measured at the end of the fermentation, as shown in Table 12.

Table 12: physicochemical index of sake brewed by saccharopolyspora raw malt

Example 9: application of polyspora rosea A22 inoculated raw wheat starter in liquor brewing

(1) Weighing 45-50% of sorghum, 8-10% of wheat, 8-10% of corn, 8-10% of glutinous rice and 20-25% of rice according to a proportion, soaking in hot water (55-65 ℃) for 24-48h, steaming rice chaff (25-30% of the total weight of grains) at 90-100 ℃ for 40-50min, and spreading for cooling for later use;

(2) Primary steaming: draining surface water of the soaked corns and sorghum wheat, and carrying out primary steaming for 40-60 min;

(3) Water stewing: after the primary steaming is finished, adding warm water with the temperature of 40-50 ℃, sealing the grain for 20-30 min when the water surface is 13-15cm higher than the grain surface, and then draining the water;

(4) Re-steaming: adding the rice and the sticky rice which are moistened in the step (1) into the primarily steamed raw grain, fully and uniformly stirring the mixture, and then putting the mixture into a grain steaming pot for re-steaming for 60-70 min;

(5) Cooling and bending: cooling the re-steamed fermented grains to 30-35 ℃, adding the rice chaff treated in the step (1) and 20-28% of white spirit Daqu, adding 8-10% of polyspora rosea to inoculate raw wheat starter (calculated according to the weight of grains), uniformly stirring, and fermenting in a cellar;

(6) Sealing the cellar for fermentation: covering the fermented grains after the fermented grains are put into a pit pool, wherein the covered grains are at least 40cm higher than the ground, sealing the pit by using pit mud, the thickness of the pit mud is 15-20 cm, and fermenting for 45-70 d;

(7) Taking out of the pool and liquor taking: taking the fermented grains out of the cellar, and distilling the fermented grains in a steamer to obtain liquor, wherein the liquor flowing speed is 2.0-2.5 kg/min, and the liquor flowing temperature is 25-30 ℃.

The liquor yield of the white spirit prepared by inoculating saccharopolyspora into raw wheat starter by applying the process is 30-35%, the high-quality product rate is 90-93%, the total acid content is 1.12-1.24 g/L, the total ester content is 15.8-16.3 g/L, the ethyl caproate content is 5.12-6.33 g/L, and the solid content is 0.5-0.63 g/L; the brewed white spirit is colorless or yellowish and transparent, has prominent ester fragrance and burnt fragrance, is clean and cool in mouth, is sweet, and is 373050, has a rich grain swelling fragrance, and naturally fuses the fragrance, and has a mellow wine body.

Example 10: application of saccharopolyspora rosea A22 inoculated raw wheat starter in cooking wine brewing

Soaking clean glutinous rice in water for 24-48h, steaming, cooling the steamed rice to 30-35 ℃ for blanking, wherein the raw rice is used as a reference, the rice is 140-150%, the water is 125%, the yeast is 11-12%, and 13-16% of roseosacrasis prepared in example 2 is added to inoculate raw wheat starter.

Fermenting for 5 days at 28 +/-2 ℃ before fermentation, starting raking after 10-12 h, and starting raking every 8h after head raking; fermenting at 15 +/-2 ℃ for 15 days, and harrowing every 48 hours after the post-fermentation is started. After fermentation, the liquid seasoning for cooking is prepared by squeezing, wine decocting, aging, adding edible salt and optionally adding plant spices.

The cooking wine fermented by inoculating the raw wheat starter with saccharopolyspora polyspora has the specific mellow fragrance of yellow wine or the specific fragrance of plant spices, the fragrances are coordinated and harmonious, the content of amino nitrogen is 0.5-0.8 g/L, the alcoholic strength is 15-17 vol%, the total acid content is 4-6 g/L, and the standard for brewing the cooking wine is met.

Example 11: application of polyspora rosea A22 inoculated raw wheat starter in sweet rice wine

The raw wheat koji inoculated with the saccharopolyspora rosea A22 is prepared by adopting the optimal process conditions of the example 2.

Soaking rice at room temperature for about 24-48h until water is absorbed sufficiently; draining the soaked rice, and cooking for 30-40min under normal pressure; after the rice is steamed, the rice is cooled to about 25 to 30 ℃, the rice is transferred into a fermentation tank, 0.5 to 0.8 percent of rosepolyspora rosea is added according to the proportion to inoculate raw wheat starter, 0.8 to 1.5 percent of saccharomyces cerevisiae is inoculated, the mixture is stirred uniformly, drinking water with the mass of 1 to 1.5 times of that of the raw rice is added, and the mixture is fermented for 48 to 72 hours at the constant temperature of 28 +/-2 ℃. The brewing precision of the sweet rice is 3-5% (v/v), the fragrance is harmonious and strong, the texture is uniform, and the taste is sour, sweet and palatable.

Example 12: application of polyspora rosea A22 inoculated raw wheat starter in rice wine

The polyspora rosea inoculated raw wheat koji was prepared by the process conditions of example 2.

Soaking rice at room temperature for about 24-48h until water is absorbed sufficiently; draining the soaked rice, and cooking for 30-40min under normal pressure; the rice is cooled to about 25-30 ℃ after being steamed, is transferred to a fermentation tank, 5% -10% of polyspora rosea is added according to the proportion to inoculate raw wheat starter for saccharification, the rice is supplemented after the saccharification is finished, 5% -10% of inoculated saccharomyces cerevisiae is inoculated, the stirring is uniform, drinking water with the mass of 1-1.5 times of that of the original rice is added, the rice is fermented for 3-5 days at the temperature of 28-30 ℃, the temperature is reduced to 10-15 ℃ for fermentation for 10-15 days, and after the fermentation is finished, the rice wine is obtained through squeezing and filtering. The alcohol content of the rice wine is 5-10% (v/v), the rice wine is rich in rice aroma and fruit aroma, and has harmonious aroma, rich flavor and uniform texture.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A strain of Saccharopolyspora rosea (Saccharopolyspora rosea) A22 has been preserved in China center for type culture Collection in 2022, 7 and 6 days, with the preservation number of CCTCC NO: M20221039.

2. A microbial agent comprising the Saccharopolyspora rosea A22 or a fermentation solution thereof according to claim 1, or comprising a cell lysate of the Saccharopolyspora rosea A22 according to claim 1.

3. The microbial agent according to claim 2, wherein the content of saccharopolyspora rosea a22 in the microbial agent is at least 1.0 x 10 ⁶ spore/mL。

4. The raw wheat starter is characterized in that the raw wheat starter is prepared by crushing wheat, adding the saccharopolyspora rosea A22 bacterial suspension of claim 1 into the crushed wheat according to a certain addition amount, uniformly stirring, and fermenting for 6-7 days in a starter house.

5. The raw wheat starter as claimed in claim 4 wherein the concentration of the polyspora rosea A22 bacterial suspension is 10 ⁴ ～10 ⁸ CFU/mL, inoculating the saccharopolyspora rosea A22 bacterial suspension into the powder according to the addition amount of 20-26 percentStirring pulverized wheat uniformly, and fermenting.

6. Use of the saccharopolyspora rosea a22 of claim 1 or the microbial agent of claim 2 or 3 or the raw malt of claim 4 or 5 for the preparation of fermented food.

7. The use of claim 6, wherein said fermented food products include but are not limited to alcoholic beverages, soy products, fermented vegetables, spices.

8. Use of the polyspora rosea a22 of claim 1 or the microbial agent of claim 2 for improving the saccharifying power and liquefying power of raw wheat koji.

9. Use of the saccharopolyspora rosea a22 as claimed in claim 1 or the microbial agent as claimed in claim 2 or the raw wheat koji as claimed in claim 4 or 5 for improving the alcoholic strength of yellow wine, or for reducing the content of higher alcohols in yellow wine, or for reducing the content of bitter amino acids in yellow wine.

10. A method for brewing a wine, comprising directly adding the fermentation product according to claim 4 or 5 to a starting material containing a yeast, and fermenting the mixture; the wine includes but is not limited to yellow wine, white spirit, sake and rice wine.