Lactobacillus paracasei strain CN23, a preparation method of the strain and a preparation method of fermented bean curd

Abstract

The application discloses a strain of paracaseic lactobacterium CN23, a preparation method of a bacterial agent and a preparation method of fermented bean curd, and relates to the technical field of microorganisms. The strain of paracaseic lactobacterium CN23 is preserved in the China General Microbiological Culture Collection Center on September 2, 2024, and the preservation number is CGMCC NO. 31794. The fermented bean curd prepared by using the bacterial agent containing the paracaseic lactobacterium CN23 has higher total acid content and amino acid nitrogen content, richer, more complex and more layered flavor, and the total biological amine content and white point ratio are significantly reduced, so that the fermented bean curd has good taste and flavor, no food safety risk, zero addition of preservatives and coagulants, and the quality of the fermented bean curd is significantly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a strain of Paracaseolus casei CN23, a preparation method of a microbial agent and a preparation method of fermented bean curd. BACKGROUND

[0002] The traditional production process of fermented bean curd is as follows: soybeans are soaked in water, ground, and the residue is filtered, then the soybean milk is cooked and a coagulant is added to form tofu, the tofu is cut into small pieces as a blank, then a specific strain such as Mucor is inoculated, and the tofu is wrapped by Mucor mycelium under suitable temperature and humidity conditions to form a shape, and then the large molecules in the tofu blank are gradually decomposed into small molecules under the action of microorganisms and enzymes in the later fermentation stage, and esters and other aromas are formed, finally, salt, spices, sugar, chili, red yeast, vinegar and other ingredients are added, and the fermented bean curd is bottled and aged for several months. In recent years, the use of acid water for point slurry to replace coagulants for the production of fermented bean curd has gradually become popular in the market. The production of acid water mainly involves collecting the soybean whey discharged after point slurry and transferring it to a storage tank, allowing it to cool naturally and ferment for several hours until it reaches the desired pH value for point slurry, thus obtaining acid water. This method of using acid water for point slurry is a combination of traditional and modern production methods, adding unique flavors and quality characteristics to fermented bean curd production, and enriching the application options of coagulants in fermented bean curd production. However, the natural fermentation of acid water is affected by environmental and climate fluctuations, and the key technical indicators such as low total acid, poor stability and easy contamination, result in unstable quality of point slurry and fermented bean curd, and may also pose food safety risks (such as mold growth, mold growth and the presence of odors), leading to problems such as acidification, gas production, white spots, browning and biological amines in fermented bean curd. Therefore, in the field of fermented bean curd brewing, high-quality strains suitable for acid water fermentation still need to be further developed. SUMMARY

[0003] The main purpose of the present application is to provide a strain of Paracaseolus casei CN23, a preparation method of a microbial agent and a preparation method of fermented bean curd, aiming to solve the technical problem of the lack of strains suitable for acid water fermentation in the field of fermented bean curd brewing.

[0004] To achieve the above-mentioned purpose, the present application provides a strain of Paracaseolus casei CN23, Lacticaseibacillus paracasei The Paracaseolus casei CN23 was deposited with the China General Microbiological Culture Collection Center on September 2, 2024, and the deposit number is CGMCC NO. 31794.

[0005] Alternatively, the gene sequence of the Paracaseolus casei CN23 is shown in Seq ID NO. 1.

[0006] The present application also provides a preparation method of a microbial agent, wherein the microbial agent comprises the Paracaseolus casei CN23;

[0007] The preparation method comprises the following steps:

[0008] After the freeze-preserved Paracaseic lactis CN23 is rehydrated in a water bath, it is inoculated into MRS broth medium, and after subculture, a third-level seed solution is prepared;

[0009] The third-level seed solution is subjected to high-density fermentation culture to obtain a fermentation liquor;

[0010] The fermentation liquor is subjected to centrifugation to collect bacterial cells;

[0011] After the bacterial cells are mixed with a protective agent, vacuum freeze-drying is performed to obtain a bacterial agent.

[0012] Optionally, the step of rehydrating the freeze-preserved Paracaseic lactis CN23 in a water bath, inoculating it into MRS broth medium, and after subculture, preparing a third-level seed solution comprises the following steps:

[0013] After the freeze-preserved Paracaseic lactis CN23 in a freeze-preservation tube is rehydrated in a water bath at 37 DEG C, it is inoculated into MRS broth medium, cultured at 37 DEG C for 18-24 hours, inoculated into MRS broth medium at a 2%-5% inoculation amount for subculture once, and cultured to the logarithmic growth phase to obtain a first-level seed solution;

[0014] The first-level seed solution is inoculated into a triangular flask at a 2%-5% inoculation amount, cultured at 37 DEG C for 18-24 hours, and after the logarithmic growth phase is reached, a second-level seed solution is obtained;

[0015] The second-level seed solution is inoculated into a seed tank at a 2%-5% inoculation amount, cultured at 37 DEG C for 18-24 hours to the logarithmic growth phase to obtain a third-level seed solution.

[0016] Optionally, the step of subjecting the third-level seed solution to high-density fermentation culture to obtain a fermentation liquor comprises the following steps:

[0017] The third-level seed solution is inoculated into a fermentation tank at a 2%-5% inoculation amount, subjected to high-density fermentation culture for 18-24 hours to obtain a fermentation liquor.

[0018] Optionally, in the step of mixing the bacterial cells with the protective agent, the bacterial cells and the protective agent are mixed at a ratio of 1: (1-3);

[0019] The components of the protective agent include skimmed milk, trehalose, and glycerol.

[0020] The application further provides a preparation method of fermented bean curd, which is fermented by using the bacterial agent, and comprises the following steps:

[0021] After adding glucose or sucrose in the bean curd yellow water, sterilization, cooling, and then inoculating the bacterial agent at an inoculation amount of 0.5wt‰-2.5wt‰, the acid water with total acid ≥1.0g / 100 mL and pH<4.0 is obtained after static culture;

[0022] After soaking, pulping and boiling the soybeans, the acid water is added at 10vt%-30vt% to point the pulp, and then the soybean white embryo is obtained after pressing and cutting;

[0023] After pure culture and scale-up culture of the Mucor strain, the Mucor strain liquid is obtained by filtration;

[0024] The Mucor strain liquid is sprayed on the soybean white embryo, and the rough product is obtained after fermentation;

[0025] The acid water, salt, liquor and water are added to the rough product, and the fermented bean curd is obtained after natural fermentation.

[0026] Optionally, the step of adding glucose or sucrose in the bean curd yellow water, sterilization, cooling, and then inoculating the bacterial agent at an inoculation amount of 0.5wt‰-2.5wt‰, the acid water with total acid ≥1.0g / 100 mL and pH<4.0 is obtained after static culture, comprises:

[0027] 1.5wt%-5wt% of glucose or sucrose is added in the bean curd yellow water, and then sterilized at 115℃-121℃ for 10min-30min, and cooled to 35℃-42℃, and then the bacterial agent is inoculated at an inoculation amount of 0.5wt‰-2.5wt‰, and static culture is carried out at 37℃-42℃ for 36h-120h, and the acid water with total acid ≥1.0g / 100 mL and pH<4.0 is obtained.

[0028] Optionally, in the step of spraying the Mucor strain liquid on the soybean white embryo, and obtaining the rough product after fermentation, the fermentation time is 24h-48h.

[0029] Optionally, in the step of adding the acid water, salt, liquor and water in the rough product, and obtaining the fermented bean curd after natural fermentation, the addition amount of the acid water is 0.15vt%-0.45vt%, the addition amount of the salt is 12wt%-25wt%, and the addition amount of the liquor is 10vt%-25vt%; and the natural fermentation time is 90d-180d.

[0030] The present application at least has the following beneficial effects:

[0031] The paracasei CN23 of the application is isolated from whey samples naturally fermented in Jidamu grassland in Yunnan, and the genetic sequence is shown in Seq ID NO. 1. As a kind of lactic acid bacteria, it can produce a large amount of lactic acid by using fermentable carbohydrates, and lactic acid is one of the important organic acids in the fermentation process of fermented bean curd. The increase of its content can significantly increase the flavor of fermented bean curd. As a strain for acid water fermentation, it has the characteristics of pure strain fermentation, high total acid of acid water, strong strain activity and high stability, avoiding the problems of low total acid, poor stability and easy pollution existing in natural fermentation of acid water. Moreover, lactic acid bacteria itself is also a beneficial bacteria. In the fermentation process of bean curd, not only the nutritional ingredients in the bean curd will not be destroyed, but also some beneficial metabolites such as vitamin B family will be produced, thereby increasing the nutritional value of fermented bean curd product. At the same time, after the fermentation of paracasei CN23, the digestion and absorption rate of soybean protein may be improved, which is more easily absorbed and utilized by human body. In addition to lactic acid, a variety of flavor substances such as esters, alcohols and aldehydes will be metabolized during the fermentation process of paracasei CN23. These substances can endow fermented bean curd with unique and rich flavor. Compared with fermented bean curd prepared by using existing acid water point paste method and fermented bean curd prepared by using traditional salt brine, the fermented bean curd prepared by using the bacterial agent containing paracasei CN23 has higher total acid content and amino acid nitrogen content, richer, more complex and layered flavor, and significantly reduced total biogenic amine content and white spot ratio, so that the fermented bean curd has the characteristics of good taste and flavor, no food safety risk, zero addition of preservative and coagulant, and significantly improved quality of fermented bean curd. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the drawings shown.

[0033] Figure 1 The plate morphology diagram of paracasei CN23 described in the embodiments of the present application;

[0034] Figure 2 The microscopic morphology diagram of paracasei CN23 described in the embodiments of the present application;

[0035] Figure 3 The preparation method flow chart of the bacterial agent described in the embodiments of the present application;

[0036] Figure 4 The preparation method flow chart of the fermented bean curd described in the embodiments of the present application.

[0037] The objectives, functional features and advantages of the present application will be further described with reference to the embodiments in combination with the accompanying drawings. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of protection of the present application.

[0039] SEQUENCE LISTING (SEQUENCE LIST CONTENT IS PROVIDED SEPARATELY):

[0040] Seq ID NO. 1 shown is the gene sequence of the Paracaseolus casei CN23 in the embodiments of the present application.

[0041] In order to facilitate the understanding of the technical solutions of the present application by those skilled in the art, the technical solutions described in the present application will be described in detail below in combination with specific embodiments.

[0042] Embodiment 1: Isolation and screening of Paracaseolus casei CN23

[0043] 1. Isolation and purification of the strain

[0044] Take 25 g of the whey sample naturally fermented in the Jidamu grassland of Yunnan collected, add to 225 mL of sterile normal saline, homogenize and mix well to obtain a mixed solution; take 1 mL of the mixed solution, perform 10-fold gradient dilution in sequence, and then take 100 μL of the appropriate gradient dilution sample and spread on a MRS agar plate added with 2% calcium carbonate, and culture at 37°C for 48-72 h; select colonies with different color, size, edge, luster and other properties from the MRS agar plate, and streak on a new MRS agar plate, and culture at 37°C for 48-72 h; repeat the selection and streaking for 2-3 times until all the colonies on the same plate have no obvious difference, and it is considered that a single strain is purified;

[0045] Inoculate the single colony purified for more than 3 times in a MRS liquid culture medium, and culture at 37°C for 18-24 h, then take 600 μL of the culture solution, add an equal volume of 60% glycerol, and store in a-86°C refrigerator for standby.

[0046] According to observation, the obtained target strain has a milk-white raised colony on the MRS agar plate added with calcium carbonate, with a smooth edge and a calcium-dissolving ring around, and is a gram-positive strain.

[0047] The strains preliminarily obtained with the calcium-dissolving ring and positive Gram staining can be lactic acid bacteria, then the bacterial genomic DNA rapid extraction kit of Shenguo Bioengineering (Shanghai) Co., Ltd. is used to extract the DNA of the target strain, the universal bacterial primer 27F / 1492R is used to amplify 16S rDNA, and the PCR amplification product is sequenced by Shenguo Bioengineering (Shanghai) Co., Ltd. The measured sequence is compared with NCBI BLAST.

[0048] Through the comparison result, 10 strains of Paracaseolus caseicus are obtained, and the strain numbers are: CN01, CN04, CN06, CN08, CN11, CN13, CN15, CN17, CN22 and CN23.

[0049] 2 Strain screening

[0050] The above-mentioned 10 strains of Paracaseolus caseicus are inoculated into yellow water culture medium (the mass ratio of yellow water to glucose is 100:6), and cultured at 37°C for 72h. The pH value and total acid content in the yellow water culture medium are shown in Table 1.

[0051] Table 1 Acid production results of isolated strains

[0052]

[0053] As shown in Table 1, the strain CN23 with the strongest acid production ability in the yellow water culture medium is screened out, which is preserved in the China General Microbiological Culture Collection Center on September 2, 2024, and the preservation number is CGMCC NO.31794.

[0054] The plate morphology of Paracaseolus caseicus CN23 is shown in Figure 1 , the microscopic morphology is shown in Figure 2 , and the gene sequence is shown in SeqID NO.1.

[0055] Example 2

[0056] A microbial agent containing Paracaseolus caseicus CN23, as shown in Figure 3 , is prepared by the following steps:

[0057] 1 Seed liquid preparation

[0058] The frozen tube of Paracaseicilus casei CN23 preserved in the refrigerator at -86℃ is thawed in a 37℃ water bath, inoculated into MRS broth culture medium, and cultured at 37℃ for 21h. The first-stage seed liquid is prepared by inoculating 3% of the first-stage seed liquid into MRS broth culture medium and culturing at 37℃ for 21h. The second-stage seed liquid is prepared by inoculating 3% of the first-stage seed liquid into a triangular flask and culturing at 37℃ for 21h when the first-stage seed liquid reaches the logarithmic growth phase. The third-stage seed liquid is prepared by inoculating 3% of the second-stage seed liquid into a seed tank.

[0059] 2 High-density fermentation culture

[0060] The third-stage seed liquid is inoculated into a final production scale fermentation tank at 3% of the inoculation amount, and cultured at 37℃ for 21h. The end of fermentation is determined by spectrophotometry and microscopic observation, and the viable cell count and activity of Paracaseicilus casei CN23 are ensured to be at a high level, and the fermentation broth is obtained.

[0061] 3 Preparation of direct-inoculation microbial agent

[0062] The bacterial cells are collected from the fermentation broth by centrifugation;

[0063] The collected bacterial cells and the prepared protective agent are emulsified and mixed at a ratio of 1:2, and then dried by vacuum freeze-drying to obtain bacterial powder.

[0064] The dried bacterial powder is immediately packaged in an aluminum foil bag to obtain the direct-inoculation microbial agent.

[0065] The components of the protective agent include skimmed milk, trehalose, and glycerol.

[0066] Example 3

[0067] A fermented bean curd, as shown in Figure 4 is prepared by the following steps:

[0068] 2wt% of glucose is added to the bean curd yellow hogwash, which is sterilized at 121℃ for 15min, and then cooled to 37℃. The microbial agent in Example 2 is inoculated at an inoculation amount of 1wt‰, and the mixture is cultured at 37℃ for 48h to obtain acid water. The acid water has a natural fragrance of normal acid soup without any odor at the end of fermentation.

[0069] After the soybeans are soaked, ground, and cooked, 15vt% of the acid water is added for point-pasting. After pressing and cutting, the white embryo of bean curd is obtained.

[0070] The Mucor strain is purified and scaled up, and then filtered to obtain a Mucor bacterial liquid.

[0071] Spray the Mucor strain liquid onto the tofu white embryo, and after 48 h of fermentation, obtain the roughcast;

[0072] Put the roughcast into a bottle, pour in the soup juice, and seal the bottle. The salt concentration in the soup juice is 15% (w / v), the baijiu concentration is 15% (v / v), the acid water concentration is 0.15% (v / v), and the rest is cold boiled water. After natural fermentation for 180 d, obtain the fermented bean curd.

[0073] Example 4

[0074] A fermented bean curd, as shown in Figure 4 is prepared by the following steps:

[0075] Add 3 wt% of glucose to the tofu yellow water, sterilize at 115°C for 30 min, cool to 37°C, and then inoculate the bacterial agent in Example 2 at an inoculation amount of 0.05 wt‰, and incubate at 42°C for 36 h to obtain acid water. The acid water has a natural fragrance of normal acid slurry water and no odor at the end of fermentation;

[0076] After soaking, grinding, and cooking the soybeans, add 12 vt% of acid water to point the slurry, and then press and carve the roughcast to obtain the tofu white embryo;

[0077] After pure culture and scale-up culture of the Mucor strain, filter to obtain a Mucor strain liquid;

[0078] Spray the Mucor strain liquid onto the tofu white embryo, and after 48 h of fermentation, obtain the roughcast;

[0079] Put the roughcast into a bottle, pour in the soup juice, and seal the bottle. The salt concentration in the soup juice is 15% (w / v), the baijiu concentration is 15% (v / v), the acid water concentration is 0.25% (v / v), and the rest is cold boiled water. After natural fermentation for 180 d, obtain the fermented bean curd.

[0080] Comparative Example 1

[0081] Prepare the fermented bean curd by using the existing acid water point slurry method, namely:

[0082] Collect the tofu yellow water into a fermentation tank, naturally cool and reduce the temperature, and ferment and culture for more than 5 d to reach the pH value (pH≤4) required for point slurry, and then obtain the acid water;

[0083] After soaking, grinding, and cooking the soybeans, add 25 vt% of acid water to point the slurry, and then press and carve the roughcast to obtain the tofu white embryo;

[0084] After pure culture and scale-up culture of the Mucor strain, filter to obtain a Mucor strain liquid;

[0085] Spray the Mucor strain liquid uniformly onto the tofu white embryo, and after 48 h of fermentation, obtain the roughcast;

[0086] The blank is bottled and filled with soup juice, the salt concentration in the soup juice is 15% (w / v), the concentration of baijiu is 15% (v / v), and the rest is cold boiled water. After sealing, natural fermentation is carried out for 180 days to obtain fermented bean curd.

[0087] Comparative Example 2

[0088] Compared with Example 3, when the blank is bottled and filled with soup juice, the soup juice does not contain sour water.

[0089] Comparative Example 3

[0090] Using the traditional fermented bean curd making process, salt brine (MgCl2) is used to point the pulp, that is:

[0091] After soaking, grinding and cooking the soybeans, add salt brine to point the pulp, then press and carve the blank to obtain tofu white embryo;

[0092] After pure culture and scale-up culture of the mucor strain, the mucor strain liquid is obtained by filtration;

[0093] The mucor strain liquid is uniformly sprayed on the tofu white embryo, and fermented for 48 h to obtain the blank;

[0094] The blank is bottled and filled with soup juice, the salt concentration in the soup juice is 15% (w / v), the concentration of baijiu is 15% (v / v), and the rest is cold boiled water. After sealing, natural fermentation is carried out for 180 days to obtain fermented bean curd.

[0095] Experimental Example

[0096] The fermented bean curds prepared in Examples 3-4 and Comparative Examples 1-3 are respectively subjected to quality detection, and the detection results are shown in Table 2.

[0097] Table 2 Quality detection results of different fermented bean curd samples

[0098]

[0099] As can be seen from Table 2, the fermented bean curd in Comparative Example 1 was prepared by using the existing acid water point slurry method, and due to the natural fermentation of the acid water, the total acid value was low, the stability was poor, and the product was easily contaminated, resulting in unstable quality of the point slurry and the produced fermented bean curd, and the total biogenic amine content of the fermented bean curd was very high, and after 12 months of storage at room temperature, many white spots appeared; in Comparative Example 2, although the acid water was inoculated with the bacterial agent containing Paracaseicoccus CN23 in the present application, but since no acid water was added during the natural fermentation of the blank, the total acid was greatly reduced, but compared with the fermented bean curd prepared by not using the bacterial agent containing Paracaseicoccus CN23 to ferment the acid water in Comparative Example 1, the total biogenic amine content and the white spot ratio after 12 months of storage at room temperature were greatly reduced, indicating that the use of Paracaseicoccus CN23 to ferment the acid water can improve the quality of the fermented bean curd and reduce the food safety risk; in Comparative Example 3, the traditional fermented bean curd making process was used to produce fermented bean curd by using salt brine as a coagulant, and the total acid value was low, and the types of flavor substances were significantly less, and the total biogenic amine content of the fermented bean curd was also very high, and after 12 months of storage at room temperature, many white spots appeared, while the fermented bean curd prepared in the present application had higher 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 12 months of storage at room temperature, indicating that Paracaseicoccus CN23 in the present application can be used as a bacterial strain for acid water fermentation, thereby improving the quality of fermented bean curd.

[0100] In summary, the Paracaseicillus casei CN23 of the application is isolated from whey samples naturally fermented in the Ji Dam grassland of Yunnan, and the genetic sequence is shown in Seq ID NO. 1. As a lactic acid bacteria, it can produce a large amount of lactic acid by utilizing fermentable carbohydrates, and lactic acid is one of the important organic acids in the fermentation process of sufu, and its increase can significantly increase the flavor of sufu. As a strain for acid water fermentation, it has the characteristics of pure strain fermentation, high total acid of acid water, strong strain activity and high stability, avoiding the problems of low total acid, poor stability and easy pollution existing in natural fermentation of acid water. Moreover, lactic acid bacteria are also beneficial bacteria, which not only do not destroy the nutritional ingredients in soy milk during fermentation, but also may produce some beneficial metabolites such as vitamin B family, thereby increasing the nutritional value of sufu products. At the same time, after fermentation by Paracaseicillus casei CN23, the digestion and absorption rate of soybean protein may be improved, which is more easily absorbed and utilized by the human body. In addition, in the fermentation process of Paracaseicillus casei CN23, lactic acid is produced, and a variety of flavor substances such as esters, alcohols and aldehydes are also metabolized and generated. These substances can endow sufu with unique and rich flavor. Compared with sufu prepared by using existing acid water point paste method and sufu prepared by using traditional salt brine, the sufu prepared by using the bacterial agent containing Paracaseicillus casei CN23 for fermentation of acid water has higher total acid content and amino acid nitrogen content, and has more mellow, complex and hierarchical flavor. The total biogenic amine content and white point ratio are significantly reduced, so that the sufu has good taste and flavor, no food safety risk, zero addition of preservatives and coagulants, and the quality of sufu is significantly improved.

[0101] The above only describes optional embodiments of the application, and does not limit the patent scope of the application. Any equivalent structural transformation based on the content of the application specification and drawings, or direct / indirect application in other related technical fields is included in the patent protection scope of the application.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei CN23, characterized in that, The *Lactobacillus paracasei* CN23 was deposited on September 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 31794; the *Lactobacillus paracasei* CN23 was isolated from whey samples naturally fermented in the Jidamu Grassland of Yunnan Province.

2. A method for preparing a microbial agent, characterized in that, The microbial agent comprises Lactobacillus paracasei CN23 as described in claim 1; The preparation method includes the following steps: The frozen Lactobacillus paracasei CN23 was reconstituted in a water bath and inoculated into MRS broth medium. After subculturing, a tertiary seed culture was prepared. The three-stage seed culture was subjected to high-density fermentation to obtain a fermentation broth; The fermentation broth was centrifuged to collect the bacterial cells; After emulsifying and mixing the bacterial cells with the protectant, the mixture is then freeze-dried under vacuum to obtain the bacterial agent.

3. The method for preparing the microbial agent according to claim 2, characterized in that, The step of reconstituted the cryopreserved *Lactobacillus paracasei* CN23 in a water bath, inoculated it into MRS broth medium, and after subculturing, to prepare a tertiary seed culture includes: The Lactobacillus paracasei CN23 preserved in cryopreservation tubes was reconstituted in a water bath at 37°C and then inoculated into MRS broth medium. After culturing at 37°C for 18-24 hours, it was subcultured once in MRS broth medium at an inoculation rate of 2%-5% and cultured until the logarithmic growth phase to obtain the primary seed culture. The primary seed culture was inoculated into Erlenmeyer flasks at an inoculation rate of 2%-5% and cultured at 37°C for 18-24 hours until the logarithmic growth phase was reached, thus obtaining the secondary seed culture. The secondary seed solution was inoculated into the seed tank at an inoculation rate of 2%-5%, and cultured at 37°C for 18-24 hours until the logarithmic growth phase to obtain the tertiary seed solution.

4. The method for preparing the microbial agent according to claim 2, characterized in that, The step of performing high-density fermentation culture of the three-stage seed liquid to obtain fermentation broth includes: The third-level seed liquid was inoculated into the fermenter at an inoculation rate of 2%-5% and fermented at high density for 18-24 hours to obtain the fermentation broth.

5. The method for preparing the microbial agent according to claim 2, characterized in that, In the step of emulsifying and mixing the bacterial cells with the protective agent, the bacterial cells and the protective agent are emulsified and mixed in a ratio of 1:(1-3); The protective agent comprises skim milk, trehalose, and glycerin.

6. A method for preparing fermented bean curd, characterized in that, Fermentation using the microbial agent prepared by any one of claims 2-5 includes the following steps: After adding glucose or sucrose to tofu swill, sterilize and cool it, then inoculate it with the bacterial agent at an inoculation rate of 0.5wt‰-2.5wt‰. After static culture, acidic water with total acid ≥1.0g / 100mL and pH <4.0 is obtained. Soaking, grinding, and boiling soybeans, then adding 10%-30% of the acidic water for coagulation, followed by pressing and shaping to obtain tofu embryos; Mucor ( Mucor After pure culture and scale-up culture, the strain of Mucor was filtered to obtain Mucor liquid; The Mucor liquid is sprayed onto the tofu embryo, and after fermentation, the tofu embryo is obtained. After adding the acidic water, salt, liquor, and water to the raw material, it is sealed and allowed to ferment naturally to obtain fermented bean curd.

7. The method for preparing fermented bean curd according to claim 6, characterized in that, The steps of adding glucose or sucrose to tofu swill, sterilizing and cooling it, then inoculating it with the bacterial agent at an inoculation rate of 0.5wt‰-2.5wt‰, and allowing it to stand for incubation to obtain acidic water with a total acidity ≥1.0g / 100mL and pH <4.0 include: Add 1.5wt%-5wt% glucose or sucrose to the yellow rice water of tofu, then sterilize it at 115℃-121℃ for 10min-30min. After cooling to 35℃-42℃, inoculate it with the bacterial agent at an inoculation rate of 0.5wt‰-2.5wt‰ and incubate it at 37℃-42℃ for 36h-120h to obtain acidic water with a total acidity ≥1.0g / 100mL and pH<4.

0.

8. The method for preparing fermented bean curd according to claim 6, characterized in that, In the step of spraying the Mucor mycelium solution onto the tofu embryo and obtaining the raw tofu after fermentation, the fermentation time is 24h-48h.

9. The method for preparing fermented bean curd according to claim 6, characterized in that, In the step of adding the acidic water, salt, liquor and water to the raw material and then sealing it for natural fermentation to obtain fermented bean curd, the amount of acidic water added is 0.15vt%-0.45vt, the amount of salt added is 12wt%-25wt%, and the amount of liquor added is 10vt%-25vt; the natural fermentation time is 90d-180d.

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