Lactobacillus paracasei CN23, preparation method of microbial inoculum and preparation method of fermented bean curd

Abstract

The invention discloses a lactobacillus paracasei CN23, a preparation method of a microbial agent and a preparation method of fermented bean curd, and relates to the technical field of microorganisms. The invention relates to a lactobacillus paracasei CN23, which is preserved in the China General Microbiological Culture Collection Center on September 2, 2024, and the preservation number is CGMCC NO. 31794. The lactobacillus paracasei CN23 is named as the lactobacillus paracasei CN23. The fermented bean curd prepared by fermenting the acid water with the microbial agent containing the casei paracasei CN23 is higher in total acid content and amino acid nitrogen content, richer and more complex in flavor and rich in layering sense, the total biogenic amine content and the white dot ratio are remarkably reduced, and the fermented bean curd has the advantages of being good in taste and flavor, rich in nutrition, good in taste and rich in flavor. The fermented bean curd has the characteristics of no food safety risk, no preservative and coagulator, and significantly improves the quality of the fermented bean curd.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a strain of Lactobacillus paracasei CN23, a preparation method of a bacterial agent, and a preparation method of fermented bean curd. Background Art

[0002] The traditional fermented bean curd production process involves soaking soybeans in water to swell, grinding them, and filtering the residue. The soy milk is then boiled and a coagulant is added to make tofu. The tofu is then cut into small pieces to form the base, which is then inoculated with specific strains of bacteria, such as Mucor. Under suitable temperature and humidity conditions, the tofu is encased in the mycelium during an initial fermentation phase, where microorganisms and enzymes gradually break down the macromolecules in the base into smaller molecules, developing aromas such as esters. Finally, salt, spices, sugar, chili peppers, red yeast rice, and fermented glutinous rice are added, bottled, and matured for several months. In recent years, the use of acidified water for fermentation has become increasingly popular in the market, replacing coagulants. The acidified water is collected and transferred to a storage tank after fermentation. It is then naturally cooled and fermented for several hours until the pH reaches the desired level for fermentation. This acidified water method combines traditional techniques with modern production, adding unique flavor and quality characteristics to fermented bean curd and expanding the range of coagulant options available. However, natural fermentation of sour water is affected by environmental and climatic fluctuations, resulting in low total acidity, poor stability, and susceptibility to contamination. This leads to inconsistent quality in both the slurry and fermented bean curd, and can also pose food safety risks (such as bloom, mold, and off-flavor). This can also cause fermented bean curd to become sour, produce gas, develop white spots, brown, and produce biogenic amines. Therefore, the development of high-quality strains suitable for sour water fermentation in fermented bean curd production remains a challenge. Summary of the Invention

[0003] The main purpose of this application is to provide a strain of Lactobacillus paracasei CN23 and a preparation method of a bacterial agent and a preparation method of fermented bean curd, aiming to solve the current technical problem of lack of bacterial strains suitable for sour water fermentation in fermented bean curd brewing.

[0004] To achieve the above purpose, the present application proposes a strain of Lactobacillus paracasei CN23 ( Lacticaseibacillus paracasei ), the Lactobacillus paracasei CN23 was deposited in the General Microbiology Center of China Culture Collection Administration on September 2, 2024, with the deposit number CGMCC NO. 31794.

[0005] Optionally, the gene sequence of the Lactobacillus paracasei CN23 is shown in Seq ID NO.1.

[0006] The present application also proposes a method for preparing a bacterial agent, wherein the bacterial agent comprises Lactobacillus paracasei CN23; The preparation method comprises the following steps: The frozen Lactobacillus paracasei CN23 was re-dissolved in a water bath, inoculated into MRS broth, and subcultured to prepare a tertiary seed solution; subjecting the third-grade seed liquid to high-density fermentation culture to obtain a fermentation liquid; Centrifuging the fermentation broth to collect bacterial cells; The bacterial cells and the protective agent are emulsified and mixed, and then vacuum freeze-dried to obtain a bacterial agent.

[0007] Optionally, the step of re-dissolving the frozen and preserved Lactobacillus paracasei CN23 in a water bath, inoculating it into an MRS broth medium, and subculturing it to prepare a tertiary seed solution comprises: The Lactobacillus paracasei CN23 stored in the frozen tube was reconstituted in a water bath at 37° C., inoculated into MRS broth medium, cultured at 37° C. for 18 h-24 h, and then inoculated into MRS broth medium with an inoculum size of 2%-5% for one passage, and cultured to the logarithmic growth phase to obtain a first-level seed solution; The first-level seed solution is inoculated into a triangular flask at an inoculum rate of 2%-5%, and cultured at 37°C for 18h-24h until the logarithmic growth phase to obtain the second-level seed solution; The secondary seed solution is inoculated into a seed tank at an inoculation rate of 2%-5%, and cultured at 37° C. for 18 h-24 h until the logarithmic growth phase to obtain the tertiary seed solution.

[0008] Optionally, the step of subjecting the tertiary seed liquid to high-density fermentation to obtain a fermentation liquid comprises: The third-grade seed liquid is added to a fermentation tank at an inoculation rate of 2%-5%, and high-density fermentation culture is carried out for 18h-24h to obtain a fermentation liquid.

[0009] Optionally, in the step of emulsifying and mixing the bacterial cells and the protective agent, the bacterial cells and the protective agent are emulsified and mixed in a ratio of 1: (1-3); The components of the protective agent include skim milk, trehalose and glycerol.

[0010] The present application also proposes a method for preparing fermented bean curd, which uses the above-mentioned bacterial agent for fermentation, comprising the following steps: After adding glucose or sucrose to the tofu swill, sterilizing and cooling, the bacterial agent is inoculated at an inoculum rate of 0.5wt‰-2.5wt‰, and after static cultivation, acid water with a total acid of ≥1.0g / 100mL and a pH of <4.0 is obtained; Soaking, grinding and boiling soybeans, adding 10% to 30% of the acid water to form a paste, and then squeezing and cutting the paste to obtain tofu white embryos; After pure culture and amplification of the Mucor strain, the Mucor liquid is obtained by filtration; Spraying the Mucor bacterial liquid onto the tofu embryo, and fermenting the tofu embryo to obtain the embryo; After adding the acid water, salt, white wine and water into the blank, the blank is sealed and naturally fermented to obtain fermented bean curd.

[0011] Optionally, the step of adding glucose or sucrose to the tofu swill, sterilizing and cooling the mixture, inoculating the bacterial agent at an inoculum size of 0.5 wt‰ to 2.5 wt‰, and culturing the mixture statically to obtain acid water having a total acid content of ≥1.0 g / 100 mL and a pH value of <4.0 comprises: 1.5wt%-5wt% of glucose or sucrose is added to the tofu swill, and then the mixture is sterilized at 115°C-121°C for 10min-30min. After cooling to 35°C-42°C, the bacterial agent is inoculated at an inoculum amount of 0.5wt‰-2.5wt‰, and statically cultured at 37°C-42°C for 36h-120h to obtain acid water with a total acid of ≥1.0g / 100mL and a pH of <4.0.

[0012] Optionally, in the step of spraying the Mucor bacterial liquid onto the tofu blank and fermenting it to obtain the blank, the fermentation time is 24h-48h.

[0013] Optionally, in the step of adding the acid water, salt, liquor and water to the blank and then sealing it for natural fermentation to obtain fermented bean curd, the amount of acid water added is 0.15vt%-0.45vt%, the amount of salt added is 12wt%-25wt%, and the amount of liquor added is 10vt%-25vt; and the natural fermentation time is 90d-180d.

[0014] This application has at least the following beneficial effects: The Lactobacillus paracasei CN23 of the present application is isolated from a whey sample naturally fermented in Jidam Grassland, Yunnan. The gene sequence is shown in Seq ID NO.1. As a lactic acid bacterium, it can use fermentable carbohydrates to produce a large amount of lactic acid, and lactic acid is one of the important organic acids in the fermentation process of fermented bean curd. The increase in its content can significantly increase the flavor of fermented bean curd. It is used as a strain for sour water fermentation, which has the characteristics of pure fermentation, high total acidity of sour water, strong strain activity and high stability, avoiding the problems of low total acidity, poor stability and easy contamination in natural fermentation of sour water. In addition, the lactic acid bacteria themselves are also beneficial bacteria. During the fermentation process, they will not only not destroy the nutrients in the soy milk, but may also produce some beneficial metabolites, such as B vitamins, thereby increasing the nutritional value of the fermented bean curd product. At the same time, after the fermentation of Lactobacillus paracasei CN23, the digestion of soy protein is improved. The absorption rate may be improved, making it easier for the human body to absorb and utilize. In addition, during the fermentation process of Lactobacillus paracasei CN23, in addition to producing lactic acid, it will also metabolize and generate a variety of flavor substances, such as esters, alcohols, aldehydes, etc. These substances can give fermented bean curd a unique and rich flavor. Compared with the fermented bean curd prepared by the existing acid water slurrying method and the fermented bean curd made by traditional brine, the fermented bean curd prepared by fermenting acid water with a bacterial agent containing Lactobacillus paracasei CN23 in this application has higher total acid content and amino acid nitrogen content, and the flavor is richer, more complex and layered. The total biogenic amine content and white spot ratio are significantly reduced, so that the fermented bean curd has good taste and flavor, no food safety risks, and zero added preservatives and coagulants, which significantly improves the quality of the fermented bean curd. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0016] Figure 1 This is a plate morphology diagram of Lactobacillus paracasei CN23 described in the examples of the present application; Figure 2 This is a microscopic morphology of Lactobacillus paracasei CN23 described in the examples of this application; Figure 3 This is a flow chart of the preparation method of the bacterial agent described in the examples of this application; Figure 4 This is a flow chart of the method for preparing fermented bean curd described in the examples of this application.

[0017] The realization of the objectives, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0019] Description of sequence listing (sequence listing content is provided separately): Seq ID NO.1 shows the gene sequence of Lactobacillus paracasei CN23 in the examples of this application.

[0020] In order to facilitate those skilled in the art to understand the technical solution of the present application, the technical solution of the present application is described in detail below in conjunction with specific implementation methods.

[0021] Example 1 Isolation and Screening of Lactobacillus paracasei CN23 1 Isolation and purification of strains Take 25g of the naturally fermented whey sample collected from the Jidam Grassland in Yunnan, add it to 225mL of sterile physiological saline, and beat it to mix thoroughly to obtain a mixed solution; take 1mL of the mixed solution and perform a 10-fold gradient dilution in sequence, then take 100μL of the appropriate gradient dilution sample and spread it on an MRS agar plate supplemented with 2% calcium carbonate, and incubate it at 37°C for 48h-72h; select colonies with appropriate colony density and different characteristics such as color, size, edge, and gloss from the MRS agar plate and streak them onto a new MRS agar plate, and incubate it at 37°C for 48h-72h; repeat the picking and streaking process 2-3 times until all colonies on the same plate have no obvious differences, which means that a single strain has been purified; A single colony purified more than three times was inoculated into MRS liquid culture medium and cultured at 37°C for 18 h-24 h. Then 600 μL of the culture medium was added with an equal volume of 60% glycerol and stored in a -86°C refrigerator for later use.

[0022] It was observed that the colonies of the obtained target strain on the MRS agar plate supplemented with calcium carbonate were milky white raised colonies with smooth edges and a surrounding calcium-soluble ring, and the strain was Gram-positive.

[0023] It was initially found that the strains with calcilytic circles and Gram-positive staining were likely lactic acid bacteria. The target strain DNA was then extracted using the bacterial genomic DNA rapid extraction kit produced by Sangon Biotech (Shanghai) Co., Ltd., and the 16S rDNA was amplified using the bacterial universal primers 27F / 1492R. The PCR amplification products were sequenced by Sangon Biotech (Shanghai) Co., Ltd., and the measured sequences were compared with NCBI BLAST.

[0024] Through the comparison results, 10 strains of Lactobacillus paracasei were confirmed, and the strain numbers were: CN01, CN04, CN06, CN08, CN11, CN13, CN15, CN17, CN22, and CN23.

[0025] 2. Strain screening The 10 isolated and preserved Lactobacillus paracasei strains were inoculated into yellow swill medium (the mass ratio of yellow swill to glucose was 100:6) and cultured at 37°C for 72 h. The pH value and total acid content of the yellow swill medium were measured and are shown in Table 1.

[0026] Table 1 Acid production results of isolated strains

[0027] As can be seen from Table 1, the strain with the strongest acid production ability in the yellow swill culture medium, namely CN23, was screened out and was deposited in the General Microbiology Center of the China Culture Collection Administration on September 2, 2024, with the deposit number CGMCC NO.31794.

[0028] The plate morphology of Lactobacillus paracasei CN23 is as follows Figure 1 As shown, the microscopic morphology Figure 2 The gene sequence is shown in SeqID NO.1.

[0029] Example 2 A bacterial agent comprising Lactobacillus paracasei CN23, such as Figure 3 As shown, it is prepared by the following steps: 1. Seed solution preparation The frozen tube of Lactobacillus paracasei CN23 stored in a -86°C refrigerator was re-dissolved in a 37°C water bath, then inoculated into MRS broth medium, cultured at 37°C for 21 hours, and then inoculated into MRS broth medium with a 3% inoculum volume for one passage to prepare a first-level seed liquid; after the first-level seed liquid was cultured to the logarithmic growth phase, it was inoculated into a triangular flask with a 3% inoculum volume and cultured at 37°C for 21 hours to prepare a second-level seed liquid; after the second-level seed liquid was cultured to the logarithmic growth phase, it was inoculated into a seed tank with a 3% inoculum volume to prepare a third-level seed liquid.

[0030] 2 High-density fermentation culture After the tertiary seed liquid is cultured to the logarithmic growth phase, it is inoculated into a fermentation tank of the final production scale at a 3% inoculation rate and cultured at 37°C for 21 hours. The fermentation endpoint is determined by spectrophotometry and microscopic observation to ensure that the number and activity of viable bacteria of Lactobacillus paracasei CN23 are at a high level, thereby obtaining a fermentation liquid.

[0031] 3. Preparation of direct-injection inoculant The bacteria were collected from the fermentation broth by centrifugation; The collected bacteria were emulsified and mixed with a pre-prepared protective agent in a ratio of 1:2, and then dried by vacuum freeze drying to obtain bacterial powder; The dried bacterial powder is immediately packaged in aluminum foil bags to obtain a direct-injection bacterial agent; The components of the protective agent include skim milk, trehalose and glycerol.

[0032] Example 3 A fermented bean curd, such as Figure 4 As shown, it is prepared by the following steps: 2 wt% glucose was added to the yellow tofu swill, and then sterilized at 121°C for 15 min. After cooling to 37°C, the bacterial agent of Example 2 was inoculated at an inoculum rate of 1 wt‰ and cultured at 37°C for 48 h to obtain sour water. At the end of fermentation, the sour water had the natural fragrance of normal sour water and no odor. Soaking, grinding and boiling soybeans, adding 15% acid water to make pulp, and then squeezing and cutting to obtain tofu embryos; After pure culture and amplification of the Mucor strain, the Mucor liquid is obtained by filtration; Spraying the Mucor bacterial liquid onto the tofu blank and fermenting it for 48 hours to obtain the blank; The rough material is bottled and poured into a soup, wherein the salt concentration of the soup is 15% (w / v), the concentration of liquor is 15% (v / v), the concentration of acid water is 0.15% (v / v), and the rest is boiled water. After capping, the fermentation is carried out naturally for 180 days to obtain fermented bean curd.

[0033] Example 4 A fermented bean curd, such as Figure 4 As shown, it is prepared by the following steps: 3 wt% glucose was added to the tofu swill, and then sterilized at 115°C for 30 min. After cooling to 37°C, the bacterial agent of Example 2 was inoculated at an inoculum rate of 0.05 wt‰, and statically cultured at 42°C for 36 h to obtain sour water. At the end of fermentation, the sour water had the natural fragrance of normal sour water and no odor. Soaking, grinding and boiling soybeans, adding 12% acid water to make pulp, and then squeezing and cutting to obtain tofu embryos. After pure culture and amplification of the Mucor strain, the Mucor liquid is obtained by filtration; Spraying the Mucor bacterial liquid onto the tofu blank and fermenting it for 48 hours to obtain the blank; The rough material is bottled and poured into a soup, wherein the soup comprises 15% (w / v) salt concentration, 15% (v / v) liquor concentration, 0.25% (v / v) acid water concentration, and the rest is boiled water. After capping, the soup is naturally fermented for 180 days to obtain fermented bean curd.

[0034] Comparative Example 1 The fermented bean curd is prepared by the existing acid water slurry method, namely: The yellow tofu swill is collected into a fermentation tank, cooled naturally, and fermented for more than 5 days. When the pH value suitable for slurrying (pH ≤ 4) is reached, acid water is obtained. Soaking, grinding and boiling soybeans, adding 25% acid water to make pulp, and then squeezing and cutting to obtain tofu embryos; After pure culture and amplification of the Mucor strain, the Mucor liquid is obtained by filtration; Spray the fungus liquid evenly onto the tofu embryo and ferment for 48 hours to obtain the embryo; The raw material is bottled and poured into soup, in which the salt concentration is 15% (w / v), the liquor concentration is 15% (v / v), and the rest is boiled water. After sealing, it is naturally fermented for 180 days to obtain fermented bean curd.

[0035] Comparative Example 2 Compared with Example 3, when the blank is bottled and the soup is poured in, the soup does not contain acid water.

[0036] Comparative Example 3 The traditional fermented bean curd production process uses bittern (MgCl2) to make the paste, namely: Soaking, grinding and boiling soybeans, adding brine to make the pulp, and then squeezing and cutting to obtain tofu embryos; After pure culture and amplification of the Mucor strain, the Mucor liquid is obtained by filtration; Spray the fungus liquid evenly onto the tofu embryo and ferment for 48 hours to obtain the embryo; The raw material is bottled and poured into soup, in which the salt concentration is 15% (w / v), the liquor concentration is 15% (v / v), and the rest is boiled water. After sealing, it is naturally fermented for 180 days to obtain fermented bean curd.

[0037] Experimental example The quality of the fermented bean curds prepared in Examples 3-4 and Comparative Examples 1-3 was tested, and the test results are shown in Table 2 below.

[0038] Table 2 Quality test results of different fermented bean curd samples

[0039] As can be seen from Table 2, in Comparative Example 1, the existing acid water slurrying method is used to prepare fermented bean curd. Due to the natural fermentation of acid water, its total acid value is low, its stability is poor, and it is easy to be contaminated, resulting in unstable slurrying and the quality of the fermented bean curd produced. The total biogenic amine content of the fermented bean curd is very high, and more white spots appear after being stored at room temperature for 12 months. In Comparative Example 2, although the bacterial agent containing Lactobacillus paracasei CN23 in the present application is used for acid water slurrying, no acid water is added during the natural fermentation of the blank, resulting in a significant reduction in its total acid. However, compared with the fermented bean curd prepared in Comparative Example 1 without using the bacterial agent containing Lactobacillus paracasei CN23 to ferment acid water, the total biogenic amine content of the fermented bean curd and the white spot ratio after being stored at room temperature for 12 months in Comparative Example 2 are significantly reduced, indicating that the use of Lactobacillus paracasei CN23 is a good method for preparing fermented bean curd. Sour water fermentation with bacteria CN23 can improve the quality of fermented bean curd and reduce food safety risks; in Comparative Example 3, the traditional fermented bean curd production process is adopted, and brine is used as a coagulant to produce fermented bean curd. Its total acid value is low, and the types of flavor substances are significantly fewer. There are also problems such as high total biogenic amine content of fermented bean curd and a large number of white spots after being stored at room temperature for 12 months. The fermented bean curd prepared in the embodiment of the present application has high total acid content and amino acid nitrogen content, more types of flavor substances, and significantly reduced total biogenic amine content and white spot ratio after being stored at room temperature for 12 months, compared with the fermented bean curd prepared by the existing acid water slurrying method and the fermented bean curd prepared by the traditional brine. This shows that the lactobacillus paracasei CN23 of the present application can be used as a strain for sour water fermentation, thereby achieving the improvement of the quality of fermented bean curd.

[0040] In summary, the Lactobacillus paracasei CN23 of the present application was isolated from a whey sample naturally fermented in Jidam Grassland, Yunnan. The gene sequence is shown in Seq ID NO.1. As a lactic acid bacterium, it can use fermentable carbohydrates to produce a large amount of lactic acid, and lactic acid is one of the important organic acids in the fermentation process of fermented bean curd. The increase in its content can significantly increase the flavor of fermented bean curd. It is used as a strain for sour water fermentation, which has the characteristics of pure fermentation, high total acidity of sour water, strong strain activity and high stability, avoiding the problems of low total acidity, poor stability and easy contamination in natural fermentation of sour water. In addition, the lactic acid bacteria themselves are also beneficial bacteria. During the fermentation process, they will not only not destroy the nutrients in the soy milk, but may also produce some beneficial metabolites, such as B vitamins, thereby increasing the nutritional value of the fermented bean curd product. At the same time, after the fermentation of Lactobacillus paracasei CN23, the digestion of soy protein is improved. The absorption rate may be improved, making it easier for the human body to absorb and utilize. In addition, during the fermentation process of Lactobacillus paracasei CN23, in addition to producing lactic acid, it will also metabolize and generate a variety of flavor substances, such as esters, alcohols, aldehydes, etc. These substances can give fermented bean curd a unique and rich flavor. Compared with the fermented bean curd prepared by the existing acid water slurrying method and the fermented bean curd made by traditional brine, the fermented bean curd prepared by fermenting acid water with a bacterial agent containing Lactobacillus paracasei CN23 in this application has higher total acid content and amino acid nitrogen content, and the flavor is richer, more complex and layered. The total biogenic amine content and white spot ratio are significantly reduced, so that the fermented bean curd has good taste and flavor, no food safety risks, and zero added preservatives and coagulants, which significantly improves the quality of the fermented bean curd.

[0041] The above description is merely an optional embodiment of the present application and does not limit the patent scope of the present application. All equivalent structural transformations made using the contents of the present application specification and drawings under the inventive concept of the present application, or direct / indirect application in other related technical fields are included in the patent protection scope of the present application.

Claims

1. A strain of Lactobacillus paracasei CN23 ( Lacticaseibacillus paracasei ), characterized in that, The Lactobacillus paracasei CN23 was deposited in the General Microbiology Center of the China Culture Collection Administration on September 2, 2024, with the deposit number CGMCC NO. 31794.

2. Lactobacillus paracasei CN23 according to claim 1, is characterized in that, The gene sequence of the Lactobacillus paracasei CN23 is shown in Seq ID NO.

1.

3. A method for preparing a microbial agent, characterized in that: The bacterial agent comprises the Lactobacillus paracasei CN23 according to any one of claims 1-2; The preparation method comprises the following steps: The frozen Lactobacillus paracasei CN23 was re-dissolved in a water bath, inoculated into MRS broth, and subcultured to prepare a tertiary seed solution; subjecting the third-grade seed liquid to high-density fermentation culture to obtain a fermentation liquid; Centrifuging the fermentation broth to collect bacterial cells; The bacterial cells and the protective agent are emulsified and mixed, and then vacuum freeze-dried to obtain a bacterial agent.

4. The method for preparing the microbial agent according to claim 3, characterized in that: The step of re-dissolving the frozen and preserved Lactobacillus paracasei CN23 in a water bath, inoculating the mixture into an MRS broth medium, and subculturing the mixture to prepare a tertiary seed solution comprises: The Lactobacillus paracasei CN23 stored in the frozen tube was reconstituted in a water bath at 37° C., inoculated into MRS broth medium, cultured at 37° C. for 18 h-24 h, and then inoculated into MRS broth medium with an inoculum size of 2%-5% for one passage, and cultured to the logarithmic growth phase to obtain a first-level seed solution; The first-level seed solution is inoculated into a triangular flask at an inoculum rate of 2%-5%, and cultured at 37°C for 18h-24h until the logarithmic growth phase to obtain the second-level seed solution; The secondary seed solution is inoculated into a seed tank at an inoculation rate of 2%-5%, and cultured at 37° C. for 18 h-24 h until the logarithmic growth phase to obtain the tertiary seed solution.

5. The method for preparing the microbial agent according to claim 3, characterized in that: The step of subjecting the third-grade seed liquid to high-density fermentation culture to obtain a fermentation liquid comprises: The third-grade seed liquid is added to a fermentation tank at an inoculation rate of 2%-5%, and high-density fermentation culture is carried out for 18h-24h to obtain a fermentation liquid.

6. The method for preparing the microbial agent according to claim 3, characterized in that: In the step of emulsifying and mixing the bacterial cells and the protective agent, the bacterial cells and the protective agent are emulsified and mixed in a ratio of 1: (1-3); The components of the protective agent include skim milk, trehalose and glycerol.

7. A method for preparing fermented bean curd, characterized in that: Fermentation using the bacterial agent according to any one of claims 3 to 6 comprises the following steps: After adding glucose or sucrose to the tofu swill, sterilizing and cooling, the bacterial agent is inoculated at an inoculum rate of 0.5wt‰-2.5wt‰, and after static cultivation, acid water with a total acid of ≥1.0g / 100mL and a pH of <4.0 is obtained; Soaking, grinding and boiling soybeans, adding 10% to 30% of the acid water to form a paste, and then squeezing and cutting the paste to obtain tofu white embryos; After pure culture and amplification of the Mucor strain, the Mucor liquid is obtained by filtration; Spraying the Mucor bacterial liquid onto the tofu embryo, and fermenting the tofu embryo to obtain the embryo; After adding the acid water, salt, white wine and water into the blank, the blank is sealed and naturally fermented to obtain fermented bean curd.

8. The method for preparing fermented bean curd according to claim 7, wherein: The step of adding glucose or sucrose to the tofu swill, sterilizing and cooling the mixture, then inoculating the bacterial agent at an inoculum amount of 0.5 wt‰ to 2.5 wt‰, and culturing the mixture statically to obtain acid water with a total acid content of ≥1.0 g / 100 mL and a pH value of <4.0 comprises: 1.5wt%-5wt% of glucose or sucrose is added to the tofu swill, and then the mixture is sterilized at 115°C-121°C for 10min-30min. After cooling to 35°C-42°C, the bacterial agent is inoculated at an inoculum amount of 0.5wt‰-2.5wt‰, and statically cultured at 37°C-42°C for 36h-120h to obtain acid water with a total acid of ≥1.0g / 100mL and a pH of <4.

0.

9. The method for preparing fermented bean curd according to claim 7, wherein: In the step of spraying the Mucor bacterial liquid onto the tofu embryo and fermenting the tofu embryo to obtain the embryo, the fermentation time is 24h-48h.

10. The method for preparing fermented bean curd according to claim 7, characterized in that: In the step of adding the acid water, salt, liquor and water to the blank and then sealing it for natural fermentation to obtain fermented bean curd, the amount of acid water added is 0.15vt%-0.45vt%, the amount of salt added is 12wt%-25wt%, and the amount of liquor added is 10vt%-25vt; and the natural fermentation time is 90d-180d.

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