CN115161246B

CN115161246B - Saccharopolyspora rosea strain capable of producing saccharifying enzyme and liquefying enzyme at high yield and application of strain

Info

Publication number: CN115161246B
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: 2023-08-22
Anticipated expiration: 2042-08-15
Also published as: CN115161246A

Abstract

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

Description

Saccharopolyspora rosea strain capable of producing saccharifying enzyme and liquefying enzyme at high yield and application of strain

Technical Field

The invention relates to a roseose polyporus strain with high yield of saccharifying enzyme and liquefying enzyme and application thereof, belonging to the field of fermentation engineering and biotechnology.

Background

Raw wheat starter is one of the most common saccharifying agents in 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 fermented products (generally wines). The raw wheat starter provides various enzymes for fermented foods, mainly amylase, protease, lipase and the like, promotes liquefaction and decomposition of high molecular substances such as starch, protein, fat and the like contained in raw materials, and simultaneously, various metabolites formed in the starter making process, and substances such as color, aroma and the like generated by interaction of the metabolites, so that the brewed foods are endowed with unique styles. Amylase in raw wheat starter plays a vital role in the utilization of raw materials in brewed foods, and two key enzymes for hydrolyzing starch are saccharifying enzyme and liquefying enzyme, so saccharifying enzyme and liquefying enzyme in raw wheat starter are also key indexes for characterizing the quality of wheat starter.

Metagenomic sequencing demonstrated that saccharopolyspora is a core microorganism in yellow wine wheat starter and fermentation process, and especially saccharopolyspora shawl, saccharopolyspora johnsonii, saccharopolyspora rosea and saccharopolyspora jiangcha in wheat starter are dominant abundant in actinomycetes. Functional predictions also indicate that the genus Saccharopolyspora is closely related to the flavor substances during the fermentation of food products, mainly related to the sources of amino acids (aspartic acid, threonine or methionine) fatty acids (caprylic acid, capric acid, lauric acid), and alcohol, ester synthesis. Therefore, increasing the abundance of saccharopolyspora in wheat starter is of great importance for improving the flavor and quality of fermented foods.

At present, the existing production of raw wheat starter mainly utilizes environmental-species microorganisms, belongs to open culture, has few excellent strains in starter and lower enzyme activity, and causes large starter consumption during brewing, thus increasing the production cost of enterprises. A strain of roseose polyporus which can be used for making raw wheat starter is screened in the early stage of a laboratory, but the strain has the defects of low activity of hydrolyzed amylase, low utilization rate of raw materials and the like.

Meanwhile, the content of higher alcohol in the yellow wine brewed by the prior art is higher (560 mg/L-620 mg/L), which can cause adverse reactions such as dizziness, nausea and the like after drinking, so that the content of higher alcohol is controlled in the process of fermenting the yellow wine. In addition, the yellow wine brewed by the prior art has higher protease due to the addition of the cooked wheat starter, 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 a main reason for causing the bitter and astringent taste of the yellow wine. At present, the alcoholic strength of the yellow wine brewed by yellow wine enterprises is generally between 17% vol and 18% vol, a novel wheat starter is to be developed to replace the traditional wheat starter, and the alcoholic strength is close to that of the original factory at first.

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

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) with the preservation number of M20221039 in the 7 th month of 2022.

The Saccharopolyspora rosea (Saccharopolyspora rosea) A22 is a mutant strain obtained by carrying out composite mutation on an original strain of Saccharopolyspora rosea and screening, and 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 Saccharopolyspora roseosa A22 or fermentation liquor thereof or the Saccharopolyspora roseosa A22 cell lysate.

In one embodiment of the present invention, the microbial agent has a content of Saccharopolyspora rosea A22 of at least 1.0X10 ⁶ spore/mL。

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

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

The invention also provides a method for producing inoculated raw wheat starter by fermenting the multispora rosea (Saccharopolyspora rosea) A22, which comprises the following steps: mixing activated Saccharopolyspora rosea A22 seed solution with clear water of mixed yeast, and adjusting the concentration of the bacteria to 10 ⁴ ～10 ⁸ CFU/mL, the bacterial suspension is obtained, the bacterial suspension is inoculated into crushed wheat according to the addition amount of 20% -26% (v/m), and the stirring is uniform, particularly, the generation of starch clusters is avoided, and the subsequent generation of bad yeast is prevented.

Placing the mixed wheat starter in starter basket (length: width: height=60×42×26cm) for fermenting for 6-7d, wherein starter room temperature is 45deg.C, and humidity is 95%.

In one embodiment of the invention, the preparation method of the raw wheat starter comprises the steps of mixing activated Saccharopolyspora rosea A22 seed liquid with clear water of starter propagation, and adjusting the concentration of the bacteria to 10 ⁴ ～10 ⁸ CFU/mL, the bacterial suspension is obtained, the bacterial suspension is inoculated into crushed wheat according to the addition amount of 20% -26% (v/m), and after being stirred uniformly, the mixed scattered yeast is subjected to the preparation of the bulk yeast.

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

In one embodiment of the invention, the technical scheme is adopted, the highest temperature reaches 52-54 ℃ in the process of preparing the inoculated raw wheat starter for brewing food, the growth of high-temperature-resistant mixed bacteria is inhibited, the high-temperature-resistant roseose polyporus is propagated in a 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 number of spores are generated, and the starter flavor is normal.

In one embodiment of the invention, the conditions of the fermentation are: culturing at 45 deg.c and 95% humidity for 6-7d, and stoving after white hypha grows inside wheat starter.

The invention also provides application of the Saccharopolyspora rosea A22 or the metabolite thereof in improving enzyme activities of saccharifying enzyme and liquefying enzyme in the field of fermentation.

In one embodiment of the invention, the metabolites include, but are not limited to, the supernatant of the Saccharopolyspora rosea A22 from which the somatic cells were removed after fermentation.

In one embodiment of the invention, the field of fermentation includes preparing a brew, a fermented food, an alcoholic beverage or a condiment.

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

The invention also provides a preparation method of the inoculated raw wheat starter which can be applied to the preparation process of various starter prepared brewing foods such as yellow wine, white wine, red yeast yellow wine, cooking wine, vinegar, soy sauce and the like and corresponding starter thereof.

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

In one embodiment of the invention, the brew includes, but is not limited to: yellow wine, chinese liquor, sake or rice wine, soy sauce, table vinegar, sweet rice wine or cooking wine.

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

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

The invention also provides application of the Saccharopolyspora rosea A22 or the microbial agent or the raw wheat starter in preparing fermented food.

In one embodiment of the invention, the fermented food product includes, but is not limited to, alcoholic beverages, soy products, fermented vegetables, condiments.

The invention also provides application of the Saccharopolyspora rosea A22 or the microbial agent in extracting the saccharification force and the liquefaction force of Gao Shengmai yeast.

The invention also provides an application of the Saccharopolyspora rosea A22 or the microbial agent or the raw wheat starter in improving the alcoholic strength of yellow wine, or in reducing the content of higher alcohols in yellow wine or in reducing the content of bitter amino acids in yellow wine.

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

the raw wheat starter is directly added into a raw material containing yeast to ferment to prepare the raw wheat starter instead of the traditional raw wheat starter and cooked wheat starter formula of a factory; such wines include, but are not limited to, yellow wine, white wine, sake and sake.

Advantageous effects

(1) The invention takes the existing roseosporus roseus in the laboratory as a starting strain, and the mutant strain roseosporus roseus (CCTCC NO: M20221039) is obtained through composite mutation screening, and has high saccharifying enzyme and liquefying enzyme.

(2) The invention provides a roseose polyporus strain A22, which has saccharifying enzyme activity of 1200-1400U/g and liquefying enzyme activity of 1.3-1.6U/g in the preparation of a wheat starter fermenting agent by solid state fermentation, wherein the saccharifying enzyme activity of the strain is improved by 29% and the liquefying enzyme activity is improved by 49% compared with that of an outgoing strain (roseose polyporus F2014).

(3) The mutant strain roseoglycospora A22 has better genetic stability after the strain is subjected to slant culture and passage to 6 generations.

(4) The alcoholic strength (18%vol) of the yellow wine prepared by inoculating the raw wheat starter fermentation by using the strain roses saccharopolyspora spinosa A22 is higher than the alcoholic strength (17.8%vol) of the mechanized yellow wine (raw wheat starter + cooked wheat starter fermentation), meanwhile, compared with the mechanical yellow wine, the method can reduce the total alcohol content in the yellow wine by 12.7%, reduce the content of bitter amino acid by 28%, and improve the drinking comfort of the yellow wine on the basis of not changing the whole flavor of the yellow wine. Meanwhile, the bitter amino acid content of the yellow wine prepared by inoculating the raw wheat starter and fermenting by adopting the Saccharopolyspora rosea A22 is also reduced.

(5) The novel roseose polyporus strain prepared by adopting the strain disclosed by the invention is inoculated with raw wheat starter, is a functional microorganism reinforcement on common raw wheat starter, and has higher saccharifying enzyme and liquefying enzyme than the common raw wheat starter. The process is an innovation of the traditional starter making process, changes the process of using the traditional mechanized production of yellow wine by matching the cooked wheat starter and the raw wheat starter in factories, completely uses the reinforced raw wheat starter to produce the yellow wine, can reduce the starter quantity for producing the manual yellow wine from 16-17% to 12-13%, and satisfies the national standard requirement of the yellow wine in physicochemical index. As the wheat starter consumption is reduced, the flavor and appearance of the brewed yellow wine are improved, the color is yellow, bright and transparent, the flavor is elegant and fine, the brewing production cost is saved, and the economic benefit of enterprises is improved.

(6) The mutant strain roses saccharopolyspora A22 obtained by the invention can be used for manufacturing bulk yeast, can be applied to the production of block yeast, reduces the fermentation time of the block yeast, is not limited by seasons, can be produced throughout the year, and improves the production efficiency.

(7) The raw wheat starter produced by the invention not only can be applied to yellow wine, but also can be applied to the production of starter-method brewed foods such as white wine, soy sauce, vinegar, rice wine, cooking wine, red yeast rice wine and the like, and has wide application prospect.

Preservation of biological materials

A strain of Saccharopolyspora rosea (Saccharopolyspora rosea) A22, taxonomically named Saccharopolyspora rosea A22 Saccharopolyspora rosea A, was deposited in China center for type culture Collection, with a deposit number of CCTCC NO: M20221039, and a deposit address of Wuhan university, china, 7 months, 6 days.

Drawings

Fig. 1: a mortality curve (A) and a positive mutation rate curve (B) of ARTP mutagenesis.

Fig. 2: basic physicochemical index change in yellow wine brewing process in the process of inoculating the wheat starter with the roseose polyporus; wherein, the alcohol degree is A; reducing sugar B; c titratable acid; and D amino acid nitrogen.

Fig. 3: the mutagenized strain is used for preparing saccharifying enzyme (A) and liquefying enzyme activity (B) of inoculated raw wheat starter.

Fig. 4: basic physicochemical index change in yellow wine brewing process in the process of inoculating the wheat starter with the roseose polyporus; wherein, A is total acid; nitrogen in amino acid form B; c, alcohol content; d reducing sugar.

Fig. 5: comparison of the mutagenized strains with the starting strain saccharifying enzyme (A) and liquefying enzyme (B).

Detailed Description

The red or yellow skin soft wheat referred to in the examples below was purchased from tin-free pooled food trade and yeast deposited at the university of Jiangnan traditional brewing food research center.

The term "liquefying enzyme" as used herein refers mainly to the enzymatic activity of amylase.

The mutagenized starting strain involved in the following examples was Saccharopolyspora rosea F2014, sacctccc NO: m2020952 is described in the application publication No. CN 113249268A.

The following examples relate to the following media:

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

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

YPD medium: 10g/L yeast extract, 20g/L peptone, 20g/L glucose, 1000mL of water, 2% agar, and sterilizing at 115℃for 20min, and cooling.

The detection method involved in the following examples is as follows:

and (3) enzyme activity detection:

the alpha-amylase and the saccharifying enzyme in the self-elevating Wen Maiqu are detected by adopting national standard GB 1886.174-2016.

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

And (3) detecting physical and chemical indexes of the yellow rice wine:

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

Example 1: preparation of mutant Saccharopolyspora rosea (Saccharopolyspora rosea) strains

1. Preparation of Saccharopolyspora rosea mutant strains

The original starting strain of the laboratory, namely Saccharopolyspora rosea F2014 (CCTCC NO: M2020952), is subjected to constant-pressure room-temperature plasma.

(1) Preparation of Saccharopolyspora rosea spore suspension

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

Preparation of spore suspension: washing activated slant strain with sterile physiological saline containing 0.05% Tween, transferring into a triangular flask containing 30mL sterile water, adding 10-15 glass beads with diameter of 5mm into the bottom of the triangular flask, mixing with spores, sealing, shaking at 180r/min in a shaker at 37deg.C for 30min, thoroughly scattering spores, filtering with single-layer sterilized cotton gauze to remove mycelium, separating precipitated spores with a centrifuge, and removing supernatant. 40mL of the eluate was added, and the centrifugation was repeated 3 times. The concentration is (0.8-1.2) x 10 ⁶ Spores/mL of mould spore suspension.

(2) Determination of plasma mutagenesis treatment time

Mixing the thallus suspension with 10% (v/v) glycerol at a ratio of 1:1, uniformly coating 10 μL of spore suspension on the surface of a slide in an ultra-clean workbench, drying with sterile air to obtain a bacterial film, and placing a sample applicator into a mutagenesis device by tweezers. 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 180s for mutagenesis. After mutagenesis, 1mL of sterile physiological saline is added in advance into an EP tube, the treated slide is transferred into the EP tube, shake is carried out for 5min, and bacterial suspension is formed by elution. After the collected bacterial liquid is properly diluted, 100 mu L of diluted liquid is coated on a Gaoshi first plate, and the bacterial liquid is cultured for 120 hours at the constant temperature of 37 ℃, wherein each group is three times in parallel. The colony count was recorded and the lethality of mutagenesis was calculated at different ARTP treatment times to generate a lethality curve. Taking the colony morphology of the original strain as a reference, observing colonies growing on the plate, and picking the strain with better growth to a fresh Gao's first plate. The growth of colonies on the plate and the size of transparent circles around the colonies were observed, strains with hydrolytic circles were selected, the sizes of hydrolytic circles and colony diameters were recorded, forward mutant strains were selected by the ratio of the hydrolytic circle diameters to the colony diameters of enzyme activity indexes (Enzymatic activity index, EI), and forward mutation rate curves were drawn. As can be seen from FIG. 1, when the mutagenesis time was 120s, the mortality was 85% and exceeded 150s, the mortality was higher than 90%, and when the mutagenesis time was 120s, the positive mutation rate was highest, and in order to select a preferred mutagenesis strain, we selected the mutagenesis time with the mortality higher than 80% and the positive mutation rate was highest, and therefore 120s was selected as the optimal mutagenesis time.

(3) Screening of Positive mutant Strain after Normal temperature isobaric plasma mutagenesis

Plate primary screening: according to the ratio of the diameter of a hydrolysis ring to the diameter of a colony of the strain 26 EI values which are larger than those of the original strain are initially screened according to the enzyme activity index (Enzymatic activity index, EI), and then the strain is subjected to preparation of inoculated wheat starter and experimental evaluation of yellow wine brewing, namely 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.

2. Screening of mutant Saccharopolyspora rosea strains

(1) Preparation of wheat starter by inoculation of saccharopolyspora

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, a 74) and the mixed clear water are mixed to adjust the concentration of bacteria to 10 ⁴ ～10 ⁸ CFU/mL, the bacterial suspension is obtained, the bacterial suspension is inoculated into crushed wheat according to the addition amount of 20% -26% (v/m), and the stirring is uniform, particularly, the generation of starch clusters is avoided, and the subsequent generation of bad yeast is prevented. Placing the mixed wheat starter in starter basket (length: width: height=60×42×26cm), placing in starter room, fermenting at 45deg.C and humidity of 95% for 6-7d to obtain starter, respectively: the saccharopolyspora is inoculated with raw wheat starter.

(2) Preparation of yellow wine

1) Activating and culturing yeast:

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

2) And (3) preparation of the yeast:

600g of steamed rice is taken, 1600mL of clear water, 60g of wheat starter in a factory and 800U/g of rice saccharifying enzyme (purchased from Jiangsu Mi biosciences and technologies Co., ltd.) are added for saccharification, the saccharification temperature is controlled to be 55-65 ℃, the time is 3-4 hours, after the saccharification is finished, the appearance sugar degree is not lower than 12 DEG Bx, the temperature is 115 ℃ for sterilization for 15 minutes, the temperature is cooled to 24-31 ℃ after sterilization, the yeast culture solution prepared in the step (1) is inoculated according to the inoculation amount of 5% (v/v), the culture temperature is 28 ℃, the culture time is 18-24 hours under the condition of 150r/min, and the yeast is obtained after the culture is mature.

3) Fermenting yellow rice wine:

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

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

Table 2: blanking table (2L fermentation system) of wheat starter fermentation yellow wine inoculated with saccharopolyspora

As shown in FIG. 2, the physical and chemical indexes of the yellow wine brewed by inoculating raw wheat starter prepared by using different mutagenesis strains are different by taking mechanical yellow wine as a control. According to the comprehensive index, the Saccharopolyspora rosea mutant strains A58, A54, A48, A22, A45 and A44 are obtained through screening.

Example 2: application of mutant strain A22 of Saccharopolyspora rosea (Saccharopolyspora rosea) in improving enzyme activities of inoculated wheat-yeast saccharifying enzyme and liquefying enzyme

The method comprises the following specific steps:

(1) Preparation of roseose polyporus fungus liquid

Inoculating Saccharopolyspora rosea F2014, saccharopolyspora rosea A22, mutagenized strains A58, A54, A48, A45 and A44 into actinomycetes culture medium, culturing at 37 ℃ for 96 hours, picking colonies from a flat plate, inoculating into actinomycetes liquid culture medium, and culturing at 37 ℃ for 48 hours in a shaking table at 150r/min to obtain seed solutions;

then respectively transferring the seed solution into actinomycete liquid culture medium according to the inoculum size of 3% (v/v), and shake culturing at 37deg.C and 150r/min for 96 hr to obtain Saccharopolyspora rosea liquid.

(2) Crushing the red skin or yellow skin soft wheat produced in the current year after sieving treatment, wherein the water content of the wheat is 13% +/-1.5% (mass fraction), and each wheat is crushed into 3-5 pieces and is plum blossom-shaped;

(3) Mixing the polysaccharide-rose fungus solution prepared in the step (1) with water according to the inoculation amount of 3-5% (v/m), and adjusting the final concentration of the fungus solution to 10 ⁴ ～10 ⁶ Spoe/mL to obtain 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 taking no white heart caking formed by water absorption of starch clusters as a reference group, stirring with water without bacterial liquid, then placing a starter frame at 45 ℃ and fermenting for 120-122h under the humidity condition of 95%, finally obtaining finished starter prepared by adopting different roseofloxacin, and detecting the enzyme activities of saccharifying enzyme and liquefying enzyme in the finished starter.

The results are shown in FIG. 3, and the results show that the activity of saccharifying enzyme and liquefying enzyme in the finished yeast prepared by using the Saccharopolyspora rosea A22 in the mutant strains A58, A54, A48, A22, A45 and A44 obtained by screening in the embodiment 1 is higher than that of other strains.

(5) The mutant strain Saccharopolyspora rosea A22 and the original strain Saccharopolyspora rosea F2014 are subjected to preparation of finished yeast according to the methods of the steps (1) - (4), and the enzyme activity effects of saccharifying enzyme and liquefying enzyme in the obtained finished yeast are compared, so that the results are shown in figure 5.

The result shows that compared with the original strain of the Saccharopolyspora rosea F2014, the mutant strain of the Saccharopolyspora rosea A22 has 29 percent of improvement and 47 percent of improvement on the liquefying enzyme, and finally, the mutant strain of the Saccharopolyspora rosea A22 has 1372.85U/g+/-55.54U/g and 1.53U/g+/-0.09U/g of liquefying enzyme, so that compared with the original strain, both the saccharifying enzyme and the liquefying enzyme are improved.

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

The saccharopolyspora spinosa prepared in example 2 is used for inoculating raw wheat Qu Niangzao yellow wine respectively, and the specific steps are as follows:

1. preparation of yellow wine

(1) Activating and culturing yeast:

(2) And (3) preparation of the yeast:

(3) Fermenting yellow rice wine:

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

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

Table 4: blanking table (2L fermentation system) of wheat starter fermentation yellow wine inoculated with saccharopolyspora

Respectively preparing mechanical yellow wine and yellow wine prepared by inoculating wheat starter with saccharopolyspora.

2. Effect characterization

The results show that:

(1) At the end of post fermentation, the alcoholic strength of the adopted roseopolyspora A58, A54, A48, A22, A45 and A44 is higher than that of a control mechanical yellow wine group (16%vol), namely A58 (16.3%vol), A54 (16.6%vol), A48 (16%vol), A22 (17.3%vol), A45 (16.4%vol) and A44 (16.1%vol), and the total acid, reducing sugar and amino state contents are all in the range of GB/T13662-2018 required by the national standard of yellow wine.

(2) The physical and chemical indexes of the yellow wine brewed by each strain are shown in figure 4, and the result shows that the total acid control group (yellow wine brewed by the traditional process) has the highest content of 6.67+/-0.13 (g/L), and the total acid content of A22 is 6.07+/-0.14 (g/L) which is obviously different from the control group (P < 0.05). The maximum content of the amino nitrogen in the control group is 1.13+/-0.03 (g/L), the mutant strains A22, A48, A58 and A44 have no obvious difference (P > 0.05), and the content of the amino nitrogen in the yellow wine is 0.81-0.86 (g/L).

(3) The alcohol content is 17.37 plus or minus 0.37 (% vol) compared with that of a control mechanical yellow wine group, the alcohol content of the mutant strain A22 is 18.63 plus or minus 0.52 (% vol) higher than that of the control group (yellow wine brewed by the traditional process), and the alcohol content of the mutant strain A44 is 16.16 plus or minus 0.59 (% vol) at the minimum.

(4) The reducing sugar content of the control group is 9.82+/-0.97 (g/L), and the reducing sugar content of the A44 mutant strain is 6.35+/-0.25 (g/L) compared with other mutant strains (P < 0.05), wherein the reducing sugar content of the yellow wine brewed by inoculating the raw wheat starter by 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 brewing is optimal by combining with the basic physicochemical index of the yellow wine.

3. Mutant strain Saccharopolyspora rosea A22 genetic stability

The high-yield amylase mutant saccharopolyspora obtained by screening is subjected to subculture on a first plate culture medium of Gao's type I for 6 generations, each generation is subjected to solid state fermentation for 120 hours, and the enzyme production capacity of each generation strain is detected, and the results are shown in Table 5.

The solid state fermentation is as follows: mixing activated Saccharopolyspora rosea seed solution with clear water of mixed starter, and adjusting the concentration of the strain to 10 ⁴ ～10 ⁸ CFU/mL, the bacterial suspension is obtained, the bacterial suspension is inoculated into crushed wheat according to the addition amount of 20% -26% (v/m), and the stirring is uniform, particularly, the generation of starch clusters is avoided, and the subsequent generation of bad yeast is prevented. Placing the mixed wheat starter in starter basket (length: width: height=60×42×26cm), and fermenting at 45deg.C and humidity of 95% for 6-7d to obtain the final starter.

Table 5: mutant strain A22 passage stability experiment

The results show that the saccharification power and the liquefying power of the Saccharopolyspora rosea A22 after 6 passages are not obviously changed, the saccharifying enzyme is maintained at 1286-1372U/g, the activity of the liquefying enzyme is maintained at 1.43-1.51U/g, and the activity of the enzyme is relatively stable, so that the Saccharopolyspora rosea A22 strain is selected to be preserved in China center for type culture collection.

4. The yellow wine is prepared by adopting the roseose polyporus A22 according to the method of the step 1, meanwhile, the yellow wine brewed by the traditional mechanical wheat starter is used as a reference, and the physicochemical index and the volatile flavor substances of the yellow wine prepared after fermentation are measured, and the results are shown in the table 6.

Table 6: physical and chemical index measurement result of wheat starter inoculated with saccharopolyspora A22 to brew yellow wine and conventional mechanical wheat starter brewed yellow wine

The results show that:

(1) As can be seen from Table 6, there is a significant difference between the inoculation of P.roseoflorium A22 with raw wheat starter and the co-fermentation of Saccharomyces cerevisiae. The alcohol degree is an visual index for reflecting whether the brewing is normally performed, and from table 6, it can be seen that the alcohol degree of the yellow wine brewed by inoculating the raw wheat starter with A22 is obviously higher than that of the traditional yellow wine (18.35%vol vs 17.41%vol), which shows that the wheat starter of the strain A22 produces a large amount of amylase to hydrolyze starch for yeast growth and reproduction and ethanol production.

(2) The flavor of the yellow wine is that of the soul of the yellow wine. Different strains also have differences in flavor due to differences in metabolism. Table 6 shows that the flavor of the yellow wine brewed by the A22 wheat starter is different from that of the traditional yellow wine.

The higher alcohol content in the yellow wine is a main wind substance forming the yellow wine, but the excessive content can cause 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. 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 the control group, and the higher alcohol content is reduced by 12.7% compared with the control group, thus the drinking comfort of the yellow wine can be improved by inoculating the raw wheat starter with A22 to ferment the yellow wine.

(3) Bitter amino acid: because cooked wheat starter is added in the traditional mechanized yellow wine fermentation, 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, thus impeding the development of yellow wine enterprises. Compared with the traditional yellow wine, the content of bitter amino acid in the yellow wine brewed by adopting the A22 wheat starter is reduced by 28%, the bitter taste of the mechanized yellow wine is improved, and the sensory experience of the yellow wine is improved.

Example 4: production process of inoculating raw wheat starter (block starter) with roses sugar and multiple spore bacteria

(1) Inoculating the roseosporus roseus A22 into an actinomycete culture medium, culturing at a constant temperature of 37 ℃ for 96 hours, then picking a bacterial colony from a flat plate, inoculating into an actinomycete liquid culture medium, and culturing at a shaking table of 150r/min for 48 hours at the temperature of 37 ℃ to obtain seed liquid; then inoculating the seed solution into actinomycete liquid culture medium according to the inoculum size of 3% (v/v), and culturing in shaking table at 37 deg.C and 150r/min for 96 hr to obtain Saccharopolyspora rosea liquid.

(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 taking no need of generating white heart caking formed by water absorption of starch clusters, and pressing the uniformly stirred yeast into yeast blocks with specific shapes by adopting a mechanical yeast making machine, wherein the length of the yeast blocks is 25 (+ -1) cm, the width of the yeast blocks is 15 (+ -1) cm, and the height of the yeast blocks is 6 (+ -1) cm. And standing the pressed yeast blocks for 30-40min, placing the yeast blocks according to a T shape to ensure air flow between adjacent yeast blocks, then conveying the yeast blocks into a yeast room, fermenting at 45 ℃ under the humidity condition of 95% for 120-122h, and finally obtaining the finished product yeast blocks.

Example 5: application of Saccharopolyspora rosea A22 inoculated raw wheat Qu Zai red rice yellow wine

The method for activating yeast and preparing yeast and yeast wine is the same as that of example 3, blanking wheat is shown in Table 7, blanking is carried out according to the raw material table of Table 7, wherein the following roseose polyporus frondosus inoculated raw wheat starter is prepared by adopting the process conditions of example 2.

Table 7: beer blanking table (5L fermentation system)

The preparation method of the red yeast rice in the batching table comprises the following steps:

selecting clean rice, soaking the rice for 48h, steaming with a rice steaming cabinet, cooling to room temperature, washing cultured Monascus spores from the inclined plane with 0.1% sterile warm water, and mixing according to 10 ⁶ Mixing the spores/g rice with rice, and culturing in a yeast room when the rice is completely reddish; the yeast needs to be turned over uninterruptedly in the fermentation process of the yeast room, the temperature of the product is kept at 28-35 ℃, after the culture for 5-7 d, hypha in rice is propagated vigorously, the yeast is red outside and inside, the yeast is moved outside, and the finished product of the red yeast is obtained after drying.

After blanking is completed, fermenting for 5 days at the temperature of 28 (+ -2) DEG C, and starting a first harrow after fermenting for 10-12 hours, wherein the first harrow is started every 8 hours; fermenting at 15 (+ -2) deg.C for 15 days, and raking every 24h after the start of the post-fermentation. The physical and chemical index measurement of the red rice yellow wine after fermentation is carried out, and the result shows that 8, the alcoholic strength of the brewed red rice yellow wine is 16.72+/-0.23%vol, and the lovastatin 55.65 +/-1.43 mg/L.

Table 8: physicochemical index of roseose polyporus inoculation raw wheat Qu Niangzao red rice yellow wine

Example 6: application of roseose polyporus A22 inoculated raw wheat starter in soy sauce brewing

The following polysaccharide-containing fungus is inoculated with raw wheat starter and prepared by the process conditions of example 2.

Selecting full and clean soybeans, washing, soaking in water for 5-10 h, and pressurizing and steaming after the soybeans fully absorb water and expand and drain water; cooling the hot beans to about 70-80 ℃ after taking out of the pot, adding 30-40% of flour (based on the mass of the soybeans), stirring uniformly, continuing to cool to 30-40 ℃, then adding 2% -5% of roseose polyporus to inoculate raw wheat starter, stirring uniformly, keeping the temperature of the product to about 28-30 ℃ for fermentation, and obtaining soy sauce starter after 72-96 hours due to the fact that the starter needs to be continuously turned, ventilated and cooled during fermentation due to heat production of microbial metabolism; the prepared soy sauce koji is prepared according to the following weight ratio of 1:1.5 (m/v) adding brine, stirring uniformly to prepare fermented grains, fermenting for 3-6 months, and turning over the sauce every 15 days; squeezing soy sauce grains after fermentation, filtering, removing impurities, and sterilizing to obtain the final product soy sauce. The physical and chemical indexes of the finished soy sauce are shown in the following table.

Table 9: physicochemical index of soy sauce brewed by inoculating raw wheat starter with roseose polyporus roseus A22

Example 7: application of roseose polyporus A22 inoculated raw wheat starter in vinegar brewing

Selecting materials: selecting 200kg of high-quality glutinous rice, adding 400kg of water, soaking the rice for 24-48 hours, then pouring the rice until white pulp is absent, draining, steaming and cooling to room temperature;

mixing materials and putting into a cylinder: 3-4 kg of wine is mixed before the glutinous rice is put into a jar, and saccharification is carried out at a low temperature for 72-96 h;

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

preparing fermented grains: adding 300kg of bran into a fermentation tank, flattening, pumping fermented grains which are fermented and ripe into the tank, uniformly stirring, uniformly spreading 10kg of rice hulls on the upper layer in the tank, uniformly stirring 10kg of fermented grains which are fermented and ripe, covering 10kg of rice hulls, and uniformly scattering to finish the preparation of the grains;

acetic acid fermentation: turning over the fermented grains after each fermentation time of 24 hours, adding rice hulls for heat preservation and moisture preservation after each fermentation time, adding no rice hulls after 11 days of fermentation, cooling the fermented grains at the temperature, adding 8kg of salt when the acidity is detected to be no longer increased after 18-20 days, and sealing for 30-45 days;

spraying vinegar and decocting vinegar: taking aged vinegar grains, adding stir-fried rice color according to a proportion, soaking for a plurality of hours, adopting a cover sprinkling method to circularly soak and sprinkle, adding sugar into the obtained vinegar juice for blending, clarifying, decocting the vinegar, cooling the temperature of the product to 75-80 ℃, canning and sealing for storage. The physicochemical index of vinegar brewed by inoculating wheat starter with saccharopolyspora is shown as 10.

Table 10: physicochemical index of vinegar brewed by inoculating raw wheat starter with roseose polyporus roseus A22

Example 8: application of Saccharopolyspora rosea A22 inoculated raw wheat starter in sake brewing

The following polysaccharide-forming fungus inoculated raw wheat starter is prepared by adopting the optimal process conditions of the example 2.

(1) Polishing: the polishing machine is utilized to remove 20 to 25 percent of the mass of the raw rice. The broken rice grains are as few as possible, the polished rice has a shape similar to that of the brown rice, and germs and furrows of the raw material rice are completely removed;

(2) Soaking rice: the rice soaking time is 24-48h under the room temperature condition;

(3) Steaming rice: the rice has distinct particles, hard outside and soft inside, no white core in the inside, looseness and no paste, is cooked without being rotten, is uniform and consistent, and the rice yield of the glutinous rice is generally about 140-150%;

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

Table 11:300L sake fermentation system batching table

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

Table 12: physicochemical index of clear wine brewed by saccharopolyspora fresh wheat starter

Example 9: application of roseose polyporus roseus A22 inoculated raw wheat starter in white spirit brewing

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

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

(3) And (3) stewing: after the primary steaming is finished, adding warm water at 40-50 ℃ and above 13-15cm above the grain surface, and stewing the grain for 20-30 min, and then draining the water;

(4) And (3) re-steaming: adding the rice and the glutinous rice which are well moistened in the step (1) into the raw grains which are steamed for the first time, fully and uniformly stirring, and then putting the mixture into a grain steaming pot for re-steaming for 60-70 min;

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

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

(7) Taking out of the pool and picking up wine: and taking the fermented grains out of the pit, and distilling off the wine by using a steamer filling pot, wherein the wine flowing speed is 2.0-2.5 kg/min, and the wine flowing temperature is 25-30 ℃.

The white spirit yield of the wheat starter inoculated with saccharopolyspora and prepared by the process is 30-35%, the 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 is 5.12-6.33 g/L, and the solid content is 0.5-0.63 g/L; the brewed white wine is colorless or yellowish transparent, has outstanding ester fragrance and burnt fragrance, is clean and cool in mouth, soft and moist in sweetness , dense in multi-grain puffing fragrance, natural in multi-fragrance fusion and mellow in wine body.

Example 10: application of roseose polyporus 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 blanking is based on raw rice, the rice is 140-150%, the water is 125%, the yeast is 11-12%, and 13-16% of the roseose polyporus prepared in example 2 is added for inoculating raw wheat starter.

Fermenting for 5 days at the temperature of 28 (+ -2) DEG C before fermentation, starting to start raking after 10-12 hours after fermentation, and starting raking every 8 hours after raking; fermenting for 15d at 15 (+ -2) deg.C, and raking every 48h after the start of the post-fermentation. After fermentation, the liquid seasoning for cooking is obtained by squeezing, decocting, ageing, adding edible salt and optionally adding plant spice.

The cooking wine which is fermented by inoculating the raw wheat starter with the saccharopolyspora spinosa has the special mellow flavor of yellow wine or the special flavor of plant spices, the flavors are coordinated and harmonious, the content of amino nitrogen is 0.5-0.8 g/L, the alcoholicity is 15-17% vol, the total acid is 4-6 g/L, and the standard of brewing cooking wine is met.

Example 11: application of roseose polyporus A22 inoculated raw wheat starter in sweet rice brewing

The following polysaccharide-forming fungus A22 is inoculated with raw wheat starter and prepared by adopting the optimal process conditions of the example 2.

Soaking rice at normal temperature for about 24-48h to fully absorb water; draining the soaked rice, and cooking for 30-40min under normal pressure; cooling the cooked rice to about 25-30 ℃, transferring the cooked rice to a fermentation tank, adding 0.5-0.8% of roseosaccharopolyspora in proportion, inoculating raw wheat starter, inoculating 0.8-1.5% of saccharomyces cerevisiae, uniformly stirring, adding drinking water with the mass 1-1.5 times of that of the raw rice, and fermenting at the constant temperature of 28 (+ -2) ℃ for 48-72 h. The brewing accuracy of the obtained sweet rice is 3-5% (v/v), the fragrance is coordinated, the sweet rice is rich, the texture is uniform, and the taste is sour and sweet and palatable.

Example 12: application of roseose polyporus A22 inoculated raw wheat starter in rice wine

Soaking rice at normal temperature for about 24-48h to fully absorb water; draining the soaked rice, and cooking for 30-40min under normal pressure; cooling the cooked rice to about 25-30 ℃, transferring the cooked rice to a fermentation tank, adding 5-10% of rose sugar polyporus to inoculate raw wheat starter in proportion for saccharification, adding rice after saccharification is finished, inoculating 5-10% of inoculation saccharomyces cerevisiae, uniformly stirring, adding drinking water with the mass 1-1.5 times of that of the original rice, fermenting for 3-5 days at 28-30 ℃, reducing the temperature to 10-15 ℃ for fermenting for 10-15 days, and squeezing and filtering to obtain rice wine after fermentation is finished. The rice wine has the alcohol content of 5-10% (v/v), is rich in rice fragrance and fruit fragrance, and has rich fragrance and uniform texture.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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. Saccharopolyspora rosea (L.) KuntzeSaccharopolyspora rosea) A22, which has been preserved in China Center for Type Culture Collection (CCTCC) at the date of 2022, 7 and 6NO:M 20221039。

2. A microbial agent comprising the Saccharopolyspora rosea A22 or a fermentation broth thereof according to claim 1.

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

4. A raw wheat starter is characterized in that wheat is crushed, the suspension of Saccharopolyspora rosea A22 in claim 1 is added into the crushed wheat according to a certain adding amount, and the mixture is stirred uniformly and fermented in a starter room for 6-7d to prepare the raw wheat starter.

5. The raw wheat starter according to claim 4, wherein the concentration of the suspension of Saccharopolyspora rosea A22 is 10 ⁴ ~10 ⁸ CFU/mL, inoculating the Saccharopolyspora rosea A22 suspension into crushed wheat according to the addition amount of 20% -26%, and fermenting after uniformly stirring.

6. Use of a roseopolyspora a22 according to claim 1 or a microbial agent according to claim 2 or 3 or a raw wheat starter according to claim 4 or 5 for the preparation of a fermented food.

7. The use according to claim 6, wherein the fermented food product is an alcoholic beverage, a bean product, a fermented vegetable or a flavouring.

8. Use of the polysaccharide of roseosporus a22 of claim 1 or the microbial agent of claim 2 for extracting the saccharification and liquefaction of Gao Shengmai koji.

9. Use of the polysaccharide-forming fungus a22 of claim 1 or the microbial agent of claim 2 or the raw wheat starter of claim 4 or 5 for increasing the alcoholic strength of yellow wine, reducing the higher alcohol content in yellow wine, or reducing the bitter amino acid content in yellow wine.

10. A method for brewing yellow wine, characterized in that the raw wheat starter according to claim 4 or 5 is directly added into a raw material containing yeast for fermentation.