A multi-strain combined solid-state fermentation rapeseed meal probiotic agent and a fermentation method thereof

Abstract

The application discloses a multi-strain combined solid-state fermentation rapeseed meal probiotic agent and a fermentation method thereof, adopts Bacillus amyloliquefaciens, Saccharomyces cerevisiae and Bacillus subtilis to combine solid-state fermentation rapeseed meal to reduce the content of anti-nutritional factors such as glucosinolates, and comprises the following steps: pretreatment and sterilization of rapeseed meal raw materials, preparation of fermentation seed liquid, solid-state fermentation of probiotics, and preparation of fermented rapeseed meal products. The application firstly uses Bacillus amyloliquefaciens to solid-state ferment rapeseed meal, and combines with Saccharomyces cerevisiae and Bacillus subtilis to add into rapeseed meal to carry out combined fermentation; compared with other technical solutions, the application can greatly reduce the content of anti-nutritional factor glucosinolate in rapeseed meal, and can reduce the content of anti-nutritional factors tannin, phytic acid and crude fiber in rapeseed meal and improve the content of peptides in the rapeseed meal product, various bioactive substances can be produced in the probiotic fermentation process, and the method has the advantages of low cost, simple operation, short time consumption and high efficiency.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and relates to a probiotic agent for the combined solid-state fermentation of rapeseed meal by multiple strains and its fermentation method. Background Technology

[0002] Rapeseed meal is a byproduct of rapeseed oil production. It typically contains 35%–40% crude protein, has a balanced amino acid composition, and is rich in trace elements, making it a commonly used plant protein feed source in the feed industry. Although rapeseed meal is nutritious, its use is limited by the presence of numerous anti-nutritional factors, such as glucosinolates, tannins, phytic acid, and crude fiber. Glucosinolates are not only toxic themselves, but their main decomposition products, such as isothiocyanates and oxazolidinyl thiones, also pose serious health risks to livestock, causing goiter, gastrointestinal damage, and liver and spleen damage. Phytic acid binds to metal ions in feed, severely impacting animal development. Tannins not only produce a bitter taste but also hinder the digestion and absorption of their own protein. Non-ruminant animals lack the enzymes to degrade crude fiber, resulting in excessive cellulose that cannot be digested, reducing feed efficiency and affecting the absorption and utilization of protein and minerals.

[0003] Rapeseed meal requires detoxification treatment for its application. Common methods for rapeseed meal detoxification include genetic breeding, physicochemical detoxification, and biological detoxification. Microbial fermentation can effectively degrade anti-nutritional factors, while improving feed palatability, increasing the digestibility and utilization of nutrients, and also benefiting animal gut health and enhancing immunity. Therefore, screening for strains with strong detoxification capabilities is of great significance for the biotechnological treatment of rapeseed meal. Currently, there are no reports of solid-state fermentation methods using microbial combinations that simultaneously possess good capabilities in removing glucosinolates, tannins, phytic acid, and crude cellulose, while also increasing peptide content. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, this invention provides a fermentation method for rapeseed meal using a multi-strain combined solid-state fermentation process. The fermentation method of this invention can effectively remove glucosinolates, tannins, phytic acid, and crude cellulose from rapeseed meal, while increasing peptide content.

[0005] This invention also discloses a probiotic preparation for a fermentation method of multi-strain combined solid-state fermentation of rapeseed meal, and the fermented rapeseed meal product prepared by the fermentation method described in this invention.

[0006] Technical Solution: To achieve the above objectives, the present invention provides a multi-strain combined solid-state fermentation method for rapeseed meal, employing Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis for combined solid-state fermentation of rapeseed meal, comprising the following steps:

[0007] (1) Pretreatment and sterilization of rapeseed meal raw material: The rapeseed meal cake is crushed and sieved, the crushed and sieved rapeseed meal is weighed and sterilized to obtain pretreated and sterilized rapeseed meal fermentation raw material.

[0008] (2) Preparation of fermentation seed liquid: Bacillus amyloliquefaciens, Saccharomyces cerevisiae and Bacillus subtilis fermentation seed liquid were cultured by shake flask culture method respectively;

[0009] (3) Solid-state fermentation: Under aseptic conditions, the Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis fermentation seed liquid prepared in step (2) are inoculated into the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1), and mixed evenly for fermentation;

[0010] (4) Preparation of fermented rapeseed meal: After solid-state fermentation in step (3), the fermented rapeseed meal is dried at low temperature to obtain the fermented rapeseed meal product.

[0011] In step (1), the rapeseed meal cake is crushed and passed through a 40-60 mesh sieve. The crushed and sieved rapeseed meal is weighed and sterilized at 121°C with high-pressure steam for 20 minutes.

[0012] Preferably, the rapeseed meal cake is pulverized through a 60-mesh sieve and then sterilized.

[0013] In step (2), the culture medium used for Bacillus amyloliquefaciens and Bacillus subtilis is broth medium, and the culture medium used for Saccharomyces cerevisiae is potato-glucose medium. All of them are liquid culture media, which are sterilized by high pressure steam at 121℃ for 20 minutes and then cooled for later use. The fermentation seed liquid of the three bacteria is cultured at 28-37℃ and shaker speed of 120-200 rpm for 15-20 hours.

[0014] As preferred, the culture temperature of Saccharomyces cerevisiae is 30℃ and the culture speed of shaker is 200 rpm for 15 h; the culture temperature of Bacillus amyloliquefaciens and Bacillus subtilis is 37℃ and the culture speed of shaker is 150 rpm for 15 h.

[0015] In step (3), the Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis fermentation seed liquid are inoculated into the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1). Saccharomyces cerevisiae and Bacillus subtilis are first inoculated, and fermented at 28-37℃ for 24-48 hours. Then, Bacillus amyloliquefaciens is inoculated, and fermented at 28-37℃ for 24-48 hours. The inoculation amount is calculated as the percentage of the mass of the culture medium containing the bacteria (i.e., fermentation liquid) to the mass of rapeseed meal (i.e., the mass of the original rapeseed meal), and the inoculation amount is 5-20%.

[0016] Preferably, in step (3), the pretreated and sterilized rapeseed meal fermentation raw material in step (1) is mixed evenly with sterile water, and the material-to-water mass ratio is 1:0.8-1:1.4, and then Bacillus subtilis and brewer's yeast fermentation liquid are added.

[0017] Preferably, the mass ratio of the inoculation amount of Saccharomyces cerevisiae, Bacillus subtilis and Bacillus amyloliquefaciens in step (3) is 3:2:1, and preferably the total amount of the three bacteria inoculated is 30%.

[0018] The probiotic agent of the fermentation method for multi-strain combined solid-state fermentation of rapeseed meal described in this invention includes Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis.

[0019] The fermented rapeseed meal product obtained by the multi-strain combined solid-state fermentation method of rapeseed meal described in this invention.

[0020] The Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis used in this invention were all provided by Huaiyin Institute of Technology, and other raw materials are commercially available.

[0021] The probiotics in this invention refer to a class of active microorganisms that primarily colonize the host's intestines, alter the composition of the intestinal flora, and are beneficial to the host. They are characterized by resistance to intestinal acidity, safety and non-toxicity, and the production of antibacterial active substances during their growth and metabolism. Probiotics mainly include yeasts and Bacillus. Saccharomyces cerevisiae and its metabolites have functions such as improving the intestinal flora barrier, regulating immune responses, preventing toxins, and inducing apoptosis. Bacillus is a type of spore-forming Gram-positive bacteria with characteristics such as heat resistance, ease of cultivation, and strong vitality. It can produce various nutrients and active factors, and has beneficial functions such as regulating intestinal microecology, improving livestock and poultry growth and production performance, and enhancing the body's immunity. In addition, Bacillus amyloliquefaciens also has biocontrol functions.

[0022] This invention is the first to employ solid-state fermentation of rapeseed meal using Bacillus amyloliquefaciens, combined with stepwise mixing of Bacillus subtilis and Saccharomyces cerevisiae. This probiotic-based solid-state fermentation method significantly degrades glucosinolates and increases peptide content in rapeseed meal, while also effectively degrading tannins, phytic acid, and crude cellulose. In this invention, a one-step fermentation with Bacillus amyloliquefaciens and Saccharomyces cerevisiae, or Bacillus amyloliquefaciens and Bacillus subtilis, does not produce a synergistic effect. Stepwise fermentation, however, exhibits a better synergistic effect, hence the adoption of this stepwise fermentation method.

[0023] In this invention, during the fermentation of rapeseed meal, *Bacillus amyloliquefaciens*, *Bacillus subtilis*, and *Saccharomyces cerevisiae* can produce enzymes that degrade glucosinolates, tannins, phytic acid, and crude cellulose, and can also produce proteases to hydrolyze the protein in rapeseed meal into peptides. Among the three strains, *Bacillus amyloliquefaciens* has the most outstanding ability to degrade glucosinolates and produce peptides from rapeseed meal protein. *Saccharomyces cerevisiae* has a more outstanding ability to degrade phytic acid and tannins than *Bacillus amyloliquefaciens*, and *Bacillus subtilis* has a more outstanding ability to degrade cellulose than *Bacillus amyloliquefaciens*. Adding *Saccharomyces cerevisiae* and *Bacillus subtilis* to the feed for fermentation can compensate for the insufficient ability of *Bacillus amyloliquefaciens* to degrade phytic acid, tannins, and crude fiber. Furthermore, the three strains exhibit a synergistic effect when combined in pairs or as a triad, significantly improving the ability to degrade glucosinolates and produce peptides from rapeseed meal protein.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0025] This invention is the first to use Bacillus amyloliquefaciens for solid-state fermentation of rapeseed meal, combined with Saccharomyces cerevisiae and Bacillus subtilis. Compared with other technical solutions, this invention can significantly reduce the content of anti-nutritional factors glucosinolates, tannins, phytic acid, and crude cellulose in rapeseed meal, while increasing peptide content and producing physiologically active substances. Moreover, it is low in cost, simple to operate, short in time, and highly efficient.

[0026] The product produced by the fermentation method of this invention not only significantly reduces the content of the anti-nutritional factor glucosinolates but also greatly increases the peptide content. The content of anti-nutritional factors tannins, phytic acid, and crude cellulose is also significantly reduced. Compared with rapeseed meal raw material (ordinary rapeseed meal), the product produced by the process of this invention completely removes glucosinolates, reducing their content by 100%; tannin content is reduced by 33.88%; phytic acid content by 37.87%; cellulose content by 38.00%; and peptide content is increased by 297.78%. Therefore, this invention can significantly reduce the glucosinolate content in rapeseed meal, while also reducing the content of tannins, phytic acid, and crude cellulose, thus reducing their toxicity, and significantly increasing peptide content, thereby greatly improving the quality of rapeseed meal. Detailed Implementation

[0027] The present invention will be further described below with reference to the embodiments.

[0028] Example 1

[0029] (1) Pretreatment and sterilization of rapeseed meal raw materials: The rapeseed meal was dried in an oven at 45℃ for 4 hours and then crushed through a 60-mesh sieve. It was then sterilized by high-pressure steam at 121℃ for 20 minutes.

[0030] (2) Preparation of fermentation seed liquid: Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis were fermented and cultured separately using the shake flask method. The fermentation medium used for Bacillus amyloliquefaciens and Bacillus subtilis was broth medium, and the fermentation medium used for Saccharomyces cerevisiae was potato-glucose medium. All were liquid media. After being sterilized by autoclaving at 121℃ for 20 min, the cultures were cooled to room temperature for later use. The three strains were cultured to the logarithmic growth phase in test tubes containing the three media respectively. The test tube liquid was inoculated into the fermentation medium of the three strains at a volume ratio of 1%. The culture temperature of Saccharomyces cerevisiae was 30℃ and the shaking speed was 200 rpm for 15 h. The culture temperature of Bacillus amyloliquefaciens and Bacillus subtilis was 37℃ and the shaking speed was 150 rpm for 15 h. Fermentation seed liquids of the three strains were obtained.

[0031] (3) Three-strain combined solid-state fermentation: Under aseptic conditions, the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1) was mixed evenly with aseptic water, with a material-to-water mass ratio of 1:1.1. Then, brewer's yeast and Bacillus subtilis fermentation seed liquid were added. After fermentation at 37℃ for 48 hours, Bacillus amyloliquefaciens fermentation liquid was inoculated and fermented at 37℃ for 48 hours. The inoculation amount was calculated as the percentage of the mass of the culture medium containing the strains to the mass of the rapeseed meal. The inoculation amounts of the three strains, brewer's yeast, Bacillus subtilis and Bacillus amyloliquefaciens, were 15%, 10% and 5%, respectively.

[0032] (4) Preparation of fermented rapeseed meal: After solid-state fermentation in step (3), the fermented rapeseed meal is dried at low temperature to obtain the fermented rapeseed meal product.

[0033] Comparative Example 1

[0034] (1) Pretreatment and sterilization of rapeseed meal raw materials: The rapeseed meal was dried in an oven at 45℃ for 4 hours and then crushed through a 60-mesh sieve. It was then sterilized by high-pressure steam at 121℃ for 20 minutes.

[0035] (2) Preparation of fermentation seed liquid: Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis were fermented and cultured separately using the shake flask method. The fermentation medium used for Bacillus amyloliquefaciens and Bacillus subtilis was broth medium, and the fermentation medium used for Saccharomyces cerevisiae was potato-glucose medium. All were liquid media. After being sterilized by autoclaving at 121℃ for 20 min, the cultures were cooled to room temperature for later use. The three strains were cultured to the logarithmic growth phase in test tubes containing the three media respectively. The test tube liquid was inoculated into the three fermentation media at a volume ratio of 1%. The culture temperature of Saccharomyces cerevisiae was 30℃ and the shaking speed was 200 rpm for 15 h. The culture temperature of Bacillus amyloliquefaciens and Bacillus subtilis was 37℃ and the shaking speed was 150 rpm for 15 h. Fermentation seed liquids of the three strains were obtained.

[0036] (3) Single-strain solid-state fermentation: Under aseptic conditions, the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1) is mixed evenly with aseptic water, and the material-to-water mass ratio is 1:1.1; the fermentation seed liquid of three strains is inoculated into the rapeseed meal fermentation raw material, and the inoculation amount is calculated as the percentage of the mass of the culture medium containing the strain to the mass of the rapeseed meal. The inoculation amount is 30% for each strain, and single-strain fermentation is carried out at 37°C for 96 hours.

[0037] (4) Preparation of fermented rapeseed meal: After solid-state fermentation in step (3), the fermented rapeseed meal is dried at low temperature to obtain the fermented rapeseed meal product.

[0038] Comparative Example 2

[0039] (1) Pretreatment and sterilization of rapeseed meal raw materials: The rapeseed meal was dried in an oven at 45℃ for 4 hours and then crushed through a 60-mesh sieve. It was then sterilized by high-pressure steam at 121℃ for 20 minutes.

[0040] (2) Preparation of fermentation seed liquid: Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis were fermented and cultured separately using the shake flask method. The fermentation medium used for Bacillus amyloliquefaciens and Bacillus subtilis was broth medium, and the fermentation medium used for Saccharomyces cerevisiae was potato-glucose medium. All were liquid media. After being sterilized by autoclaving at 121℃ for 20 min, the cultures were cooled to room temperature for later use. The three strains were cultured to the logarithmic growth phase in test tubes containing the three media respectively. The test tube liquid was inoculated into the fermentation medium of the three strains at a volume ratio of 1%. The culture temperature of Saccharomyces cerevisiae was 30℃ and the shaking speed was 200 rpm for 15 h. The culture temperature of Bacillus amyloliquefaciens and Bacillus subtilis was 37℃ and the shaking speed was 150 rpm for 15 h. Fermentation seed liquids of the three strains were obtained.

[0041] (3) Solid-state fermentation with two bacteria: Under aseptic conditions, the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1) was mixed evenly with aseptic water at a material-to-water mass ratio of 1:1.1. Saccharomyces cerevisiae and Bacillus amyloliquefaciens were co-fermented. Saccharomyces cerevisiae fermentation seed liquid was first inoculated into the rapeseed meal, fermented at 37°C for 48 hours, and then Bacillus amyloliquefaciens fermentation seed liquid was inoculated, continuing fermentation at 37°C for another 48 hours. The inoculation amount was calculated as the percentage of the culture medium containing the bacteria to the rapeseed meal mass, with inoculation amounts of 22.5% for Saccharomyces cerevisiae and 7.5% for Bacillus amyloliquefaciens. Bacillus subtilis and Bacillus amyloliquefaciens were co-fermented. Bacillus subtilis fermentation seed liquid was first inoculated into the rapeseed meal, fermented at 37°C for 48 hours, and then Bacillus amyloliquefaciens fermentation seed liquid was inoculated, continuing fermentation at 37°C for another 48 hours. The inoculation amount was calculated as the percentage of the culture medium containing the bacteria to the rapeseed meal mass, with inoculation amounts of 20% for Bacillus subtilis and 10% for Bacillus amyloliquefaciens. Saccharomyces cerevisiae and Bacillus subtilis were co-fermented in a one-step process at 37°C for 96 hours. The inoculum amounts of Saccharomyces cerevisiae and Bacillus subtilis were 18% and 12%, respectively.

[0042] (4) Preparation of fermented rapeseed meal: After solid-state fermentation in step (3), the fermented rapeseed meal is dried at low temperature to obtain the fermented rapeseed meal product.

[0043] The degradation rate of anti-nutritional factors and changes in peptide content of fermented rapeseed meal in Example 1 and Comparative Examples 1-2 were tested, and the results are shown in Table 1.

[0044] Table 1. Degradation rate of antinutritional factors and increase rate of peptide content in fermented rapeseed meal in Example 1

[0045]

[0046]

[0047] Note: J: Bacillus amyloliquefaciens, N: Saccharomyces cerevisiae, K: Bacillus subtilis, / indicates not detected.

[0048] As shown in Table 1, the product produced by the process of this invention not only significantly reduces the content of the anti-nutritional factor glucosinolates but also greatly increases the peptide content. The contents of anti-nutritional factors tannins, phytic acid, and crude cellulose are also significantly reduced. Compared with rapeseed meal raw materials (ordinary rapeseed meal), the product produced by fermentation according to this invention completely removes glucosinolates, reducing their content by 100%; tannin content decreases by 33.88%; phytic acid content decreases by 37.78%; cellulose content decreases by 38.00%; and peptide content increases by 297.78%. Therefore, this invention utilizes a highly efficient fermentation process using *Bacillus amyloliquefaciens*, a probiotic, in combination with *Saccharomyces cerevisiae* and *Bacillus subtilis* for effective detoxification and significantly increases peptide content, thereby improving the quality of rapeseed meal.

[0049] Example 2

[0050] (1) Pretreatment and sterilization of rapeseed meal raw materials: The rapeseed meal was dried in an oven at 45℃ for 4 hours and then crushed through a 40-mesh sieve. It was then sterilized by high-pressure steam at 121℃ for 20 minutes.

[0051] (2) Preparation of fermentation seed liquid: Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis were fermented and cultured separately using the shake flask method. The fermentation medium used for Bacillus amyloliquefaciens and Bacillus subtilis was broth medium, and the fermentation medium used for Saccharomyces cerevisiae was potato-glucose medium. All were liquid media. After being sterilized by autoclaving at 121℃ for 20 min, the cultures were cooled to room temperature for later use. The three strains were cultured to the logarithmic growth phase in test tubes containing the three media respectively. The test tube liquid was inoculated into the fermentation medium of the three strains at a volume ratio of 1%. The culture temperature of Saccharomyces cerevisiae was 28℃ and the shaking speed was 200 rpm for 20 h. The culture temperature of Bacillus amyloliquefaciens and Bacillus subtilis was 33℃ and the shaking speed was 200 rpm for 20 h. Fermentation seed liquids of the three strains were obtained.

[0052] (3) Three-strain combined solid-state fermentation: Under aseptic conditions, the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1) was mixed evenly with aseptic water, with a material-to-water mass ratio of 1:0.8. Then, Bacillus subtilis and Saccharomyces cerevisiae fermentation seed liquid were added. After fermentation at 28℃ for 24 hours, Bacillus amyloliquefaciens was inoculated and fermented at 33℃ for 24 hours. The inoculation amount was calculated as the percentage of the mass of the culture medium containing the strains to the mass of the rapeseed meal. The inoculation amounts of the three strains, Saccharomyces cerevisiae, Bacillus subtilis, and Bacillus amyloliquefaciens, were 20%, 5%, and 5%, respectively.

[0053] (4) Preparation of fermented rapeseed meal: After solid-state fermentation in step (3), the fermented rapeseed meal is dried at low temperature to obtain the fermented rapeseed meal product.

[0054] Example 3

[0055] (1) Pretreatment and sterilization of rapeseed meal raw materials: The rapeseed meal was dried in an oven at 45℃ for 4 hours and then crushed through a 60-mesh sieve. It was then sterilized by high-pressure steam at 121℃ for 20 minutes.

[0056] (2) Preparation of fermentation seed liquid: Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis were fermented and cultured separately using the shake flask method. The fermentation medium used for Bacillus amyloliquefaciens and Bacillus subtilis was broth medium, and the fermentation medium used for Saccharomyces cerevisiae was potato-glucose medium. All were liquid media. After being sterilized by autoclaving at 121℃ for 20 min, the cultures were cooled to room temperature for later use. The three strains were cultured to the logarithmic growth phase in test tubes containing the three media respectively. The test tube liquid was inoculated into the fermentation medium of the three strains at a volume ratio of 1%. The culture temperature of Saccharomyces cerevisiae was 37℃ and the shaking speed was 120 rpm for 20 h. The culture temperature of Bacillus amyloliquefaciens and Bacillus subtilis was 30℃ and the shaking speed was 200 rpm for 20 h. Fermentation seed liquids of the three strains were obtained.

[0057] (3) Three-strain combined solid-state fermentation: Under aseptic conditions, the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1) was mixed evenly with aseptic water, with a material-to-water mass ratio of 1:1.4. Then, Bacillus subtilis and Saccharomyces cerevisiae fermentation seed liquid were added. After fermentation at 30°C for 36 hours, Bacillus amyloliquefaciens was inoculated and fermented at 30°C for 36 hours. The inoculation amount was calculated as the percentage of the mass of the culture medium containing the strains to the mass of the rapeseed meal. The inoculation amount of the three strains, Saccharomyces cerevisiae, Bacillus subtilis and Bacillus amyloliquefaciens, was 10%.

[0058] (4) Preparation of fermented rapeseed meal: After solid-state fermentation in step (3), the fermented rapeseed meal is dried at low temperature to obtain the fermented rapeseed meal product.

Claims

1. A fermentation method for rapeseed meal using a combination of Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis in solid-state fermentation, characterized in that... Includes the following steps: (1) Pretreatment and sterilization of rapeseed meal raw materials: The rapeseed meal was dried in an oven at 45℃ for 4 h and then crushed through a 60-mesh sieve and sterilized by high-pressure steam at 121℃ for 20 min. (2) Preparation of fermentation seed liquid: Bacillus amyloliquefaciens, Saccharomyces cerevisiae, and Bacillus subtilis were fermented and cultured separately using the shake flask method. The fermentation medium used for Bacillus amyloliquefaciens and Bacillus subtilis was broth medium, and the fermentation medium used for Saccharomyces cerevisiae was potato-glucose medium. All were liquid media. After being autoclaved at 121℃ for 20 min, they were cooled to room temperature for later use. The three strains were cultured to the logarithmic growth phase in test tubes containing the three media respectively. The test tube liquid was inoculated into the fermentation medium of the three strains at a volume ratio of 1%. The culture temperature of Saccharomyces cerevisiae was 30℃ and the shaking speed was 200 rpm for 15 h. The culture temperature of Bacillus amyloliquefaciens and Bacillus subtilis was 37℃ and the shaking speed was 150 rpm for 15 h. Fermentation seed liquids of the three strains were obtained. (3) Three-strain combined solid-state fermentation: Under aseptic conditions, the rapeseed meal fermentation raw material after pretreatment and sterilization in step (1) was mixed evenly with aseptic water, with a material-to-water mass ratio of 1:1.

1. Then, brewer's yeast and Bacillus subtilis fermentation seed liquid were added. After fermentation at 37℃ for 48 hours, Bacillus amyloliquefaciens fermentation liquid was inoculated and fermented at 37℃ for 48 hours. The inoculation amount was calculated as the percentage of the mass of the culture medium containing the strains to the mass of the rapeseed meal. The inoculation amounts of the three strains, brewer's yeast, Bacillus subtilis, and Bacillus amyloliquefaciens, were 15%, 10%, and 5%, respectively. (4) Preparation of fermented rapeseed meal: After solid-state fermentation in step (3), the fermented rapeseed meal is dried at low temperature to obtain the fermented rapeseed meal product.

2. The fermented rapeseed meal product obtained by the fermentation method of multi-strain combined solid-state fermentation of rapeseed meal according to claim 1.

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