A method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal

Abstract

This invention relates to the field of microbial enzyme fermentation technology, and proposes a method for synergistic microbial enzyme fermentation to remove anti-nutritional factors from soybean meal, comprising the following steps: S1, pretreatment of soybean meal: soybean meal, water, and auxiliary agents are mixed and subjected to steam explosion treatment, wherein the auxiliary agents include citric acid, hydrogen peroxide solution, and polyphenols; S2, microbial enzyme fermentation: the pretreated soybean meal is mixed with molasses and water to obtain raw material, a compound microbial agent and a compound enzyme are added, and fermentation is carried out after stirring evenly; the compound microbial agent includes yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus; the compound enzyme includes acidic protease, pectinase, phytase, glucanase, α-galactosidase, and keratinase; S3, after fermentation, the material is dried to obtain fermented soybean meal. The fermentation method of this invention can achieve a soybean globulin degradation rate of over 79%, increase the acid-soluble protein content to over 26%, significantly reduce the content of anti-nutritional factors, and greatly improve the nutritional value.

Description

Technical Field

[0001] This invention relates to the field of microbial enzyme fermentation technology, and in particular to a method for microbial enzyme co-fermentation to remove anti-nutritional factors from soybean meal. Background Technology

[0002] Soybean meal is a commonly used high-protein feed ingredient in animal feed, but it contains various anti-nutritional factors (such as glycinin, β-conglycinin, phytic acid, and trypsin inhibitors). These components not only affect the digestion and absorption of nutrients by animals but also interfere with the utilization of protein and trace minerals, reducing feed utilization efficiency. Currently, conventional methods such as physical, chemical, or single-microbial fermentation can decompose or reduce some anti-nutritional factors to a certain extent, but the treatment effect is limited, and the problem of residual anti-nutritional factors remains prominent.

[0003] In recent years, co-fermentation of microorganisms and enzymes has been used to improve plant protein raw materials such as soybean meal due to its combination of the advantages of microbial degradation and enzyme-enhanced hydrolysis. Various microbial strains, such as lactic acid bacteria and Bacillus, can secrete acids and some degrading enzymes. Combined with exogenous proteases and phytases, these can accelerate the decomposition of anti-nutritional factors in raw materials such as soybean meal to a certain extent. However, current co-fermentation systems still have shortcomings in the combination of various microorganisms and enzymes and the optimization of process conditions, resulting in a still low removal rate of key anti-nutritional factors, which affects the deep application of soybean meal in high-quality feed. To address the problem of low removal rates of anti-nutritional factors in soybean meal using existing technologies, there is an urgent need to develop an efficient co-fermentation method of microorganisms and enzymes to improve the degradation effect of anti-nutritional factors in soybean meal. Summary of the Invention

[0004] In view of this, the present invention proposes a microbial-enzyme co-fermentation method for removing anti-nutritional factors from soybean meal, in order to solve the problem of low removal rate of anti-nutritional factors in soybean meal in the prior art.

[0005] The technical solution of this invention is achieved as follows: This invention provides a method for synergistic fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal, comprising the following steps:

[0006] S1, Pre-treated soybean meal: Soybean meal, water and additives are mixed and then subjected to steam explosion treatment, wherein the additives include citric acid, hydrogen peroxide solution and polyphenols.

[0007] Specifically, steam explosion, under high temperature and pressure, causes the macromolecular structures (proteins, oligosaccharides) to break and expand, which, combined with adjuvants, can accelerate degradation. Citric acid in the adjuvants provides an acidic environment, promoting partial protein denaturation. Citric acid also synergistically breaks hydrogen bonds and hydrophobic interactions under the high temperature of steam explosion, weakening the tertiary structure of globulins. Furthermore, citric acid can activate some endogenous enzymes (such as phytase). The strong oxidizing properties of hydrogen peroxide can destroy the stable structures of polysulfide bonds and ether bonds within proteins / antinutritional factors (such as soy globulin, raffinose, etc.). Polyphenols form hydrogen bonds or hydrophobic complexes with proteins, promoting conformational changes and further disrupting the structure of antinutritional factors. Polyphenols can also inhibit unwanted microorganisms, creating a favorable environment for subsequent probiotic colonization. In addition, polyphenols have antioxidant properties, preventing nutrient loss due to lipid oxidation during fermentation. The synergistic effect of citric acid, hydrogen peroxide solution, and polyphenols significantly reduces the structural compactness (depolymerization, loosening) of soybean meal, exposing more target sites for subsequent bacterial and enzyme action.

[0008] To avoid residual hydrogen peroxide affecting subsequent fermentation, spraying 50-100 U / kg of food-grade catalase on the material can completely degrade residual hydrogen peroxide within 15-30 minutes.

[0009] S2, Fermentation by bacteria and enzymes: The pretreated soybean meal is mixed with molasses and water to obtain the raw material. Compound bacteria and compound enzymes are added, and the mixture is stirred evenly before fermentation.

[0010] The compound microbial agent includes yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus;

[0011] The complex enzyme includes acidic protease, pectinase, phytase, glucanase, α-galactosidase, and keratinase.

[0012] Bacillus subtilis secretes proteases and cellulases to degrade large protein / polysaccharide molecules, while Lactobacillus and Pediococcus produce lactic acid and oligosaccharide enzymes, which specialize in raffinose, stachyose and other oligosaccharides and improve flavor; yeast works together to break down polysaccharides and synthesize B vitamins, thus enhancing nutrition.

[0013] Pectinase / glucanase disrupts the intercellular layer, promoting the release of contents. The acidic protease in the complex enzyme directly hydrolyzes denatured globulins into polypeptides. *Lactobacillus rhamnosus* and *Pediococcus pentosus* rapidly produce lactic acid, inhibiting contaminating bacteria such as *Escherichia coli*. α-Galactosidase specifically hydrolyzes the α-1,6-galactosidic bonds in raffinose and stachyose; α-galactosidase synergistically enhances protease activity with *Bacillus*. Phytase degrades phytic acid, releasing phosphorus and minerals.

[0014] Synergistic advantages of bacteria and enzymes: Endogenous bacteria producing enzymes + exogenous enzyme enhancement achieves dual protection of self-production and external supplementation, avoiding degradation bottlenecks caused by single bacteria or insufficient enzyme activity.

[0015] S3, after fermentation, the material is dried to obtain fermented soybean meal.

[0016] Based on the above technical solutions, preferably, in step S1, the mass ratio of soybean meal, water, citric acid, hydrogen peroxide solution and polyphenols is 100:10-15:0.5-1.5:0.2-0.6:0.1-0.3, and the mass concentration of hydrogen peroxide solution is 30%.

[0017] Based on the above technical solutions, preferably, the polyphenol is one or a combination of two of tea polyphenols and gallic acid.

[0018] Based on the above technical solutions, preferably, in step S1, during the steam explosion treatment, saturated steam is introduced, and the pressure inside the explosion tank is controlled at 1.5-2.5 MPa and the temperature at 130-150℃, and maintained for 10-15 minutes. After that, the material inside the explosion tank is exploded and released, and the material temperature is reduced to 20-30℃.

[0019] Based on the above technical solutions, preferably, in step S2, the mass ratio of soybean meal, molasses, and compound microbial agent is 100:20:0.4-0.5, and water is added until the total moisture content is 50%-60%.

[0020] Based on the above technical solutions, the preferred method for preparing the compound microbial agent is as follows: yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus are cultured separately and then mixed in a specific ratio. Specifically:

[0021] Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus were inoculated into culture medium and cultured for 24 hours to obtain seed inoculum. Subsequently, the seed inoculum was mixed with yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus in a mass ratio of 1-2:3-5:2-4:2-4 to obtain a compound inoculum. The yeast was cultured at 28-30℃, Bacillus subtilis at 37-42℃, and Lactobacillus rhamnosus and Pediococcus pentosus at 30-37℃, and cultured in a closed system.

[0022] Based on the above technical solutions, preferably, the culture medium comprises the following components in parts by weight: 30-50 parts soybean meal, 10-20 parts wheat bran, 5-15 parts corn flour, 1-5 parts peptone and 10-15 parts glucose, with water added to a solid content of 30wt%-40wt%.

[0023] Based on the above technical solutions, preferably, the effective viable number of yeast cells in the seed inoculant is (1.5-2)×10⁻¹⁰. 8 CFU / g (i.e., the number of viable bacteria per gram of seed inoculum (the solid material after fermentation)), the effective viable count of Bacillus subtilis is (1-1.5)×10⁻⁶.9 The effective viable count of Lactobacillus rhamnosus is (1-1.5) × 10 CFU / g. 9 CFU / g, the effective viable count of Pediococcus pentosaceus is (2-3) × 10⁻⁶. 9 CFU / g.

[0024] Based on the above technical solutions, the preferred mass ratio of soybean meal: acidic protease: pectinase: phytase: glucanase: α-galactosidase: keratinase is 100: 0.1-0.2: 0.2-0.4: 0.1-0.2: 0.25-0.5: 0.1-0.2: 0.05-0.08.

[0025] Based on the above technical solutions, preferably, in step S2 fermentation, a staged fermentation method is adopted:

[0026] First stage: fermentation temperature 30-35℃, O2 concentration 1%-2%, pH value 5.0, fermentation time 12-18h;

[0027] Specifically, the first stage involves microaerobic and suitable temperature conditions; lactic acid bacteria lead the rapid production of acid, lowering the pH and inhibiting other bacteria; α-galactosidase is activated to hydrolyze raffinose and stachyose; yeast assists in enzyme production and provides a carbon source.

[0028] Second stage: Fermentation temperature 37-40℃, O2 concentration 3%-5%, pH value 4.5-5.0, fermentation time 18-36h;

[0029] The second stage involves aerobic activity and warming; Bacillus subtilis becomes active and secretes large amounts of proteases and phytases under aerobic conditions; the acidic environment activates acidic proteases, which efficiently hydrolyze globulins and generate large amounts of small peptides.

[0030] The third stage: fermentation temperature 40-42℃, O2 concentration 1%-2%, pH value 4.5, fermentation time 24-36h.

[0031] The third stage involves high temperature and micro-oxygen; inhibiting miscellaneous bacteria and some dying bacteria, while Bacillus continues to produce enzymes to further degrade residual anti-nutritional factors; and promoting the further conversion of small peptides into smaller molecular peptides or free amino acids.

[0032] During fermentation, the pH value of the material is monitored in real time, and citric acid and dilute 1M NaOH are used to adjust the pH value during fermentation.

[0033] By precisely controlling environmental parameters, the microbial community is guided to evolve along the path of "lactic acid bacteria initiation → Bacillus dominance → high temperature enhancement," thus achieving functional succession.

[0034] The microbial-enzyme co-fermentation method for removing anti-nutritional factors from soybean meal according to the present invention has the following advantages over the prior art:

[0035] (1) Pretreatment provides a basis for bacterial enzyme action: Steam explosion + adjuvants cause cell wall rupture, protein denaturation and oligosaccharide exposure. These effects greatly enhance the enzymatic hydrolysis of complex enzymes and the adsorption capacity of microorganisms, thereby increasing the hydrolysis rate and accelerating fermentation start-up.

[0036] Synergistic effects of microorganisms and enzymes provide the foundation for staged fermentation: the combination of multiple microorganisms and multiple enzymes provides full-spectrum degradation capabilities; in staged fermentation, different microorganisms can fully exert their functions under their optimal conditions, avoiding the inactivation or inhibition of a single microorganism in a complex environment.

[0037] Segmented fermentation extends and amplifies the synergistic effect of steam explosion and bacterial enzymes: staged temperature and oxygen control extends the time window for highly active enzymatic hydrolysis, allowing the pretreated substrates to be continuously and thoroughly degraded; it also promotes the continuous generation and further degradation of small peptides into functional small molecule peptides.

[0038] (2) This invention not only solves the problems of low efficiency and instability of traditional fermented soybean meal, but also embodies the advanced concept of integrating physical, chemical and biological disciplines, and has the potential to become a benchmark technology for high-end fermented soybean meal. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] The keratinase (500,000 u / g), phytase (300,000 u / g), acidic protease (200,000 u / g), galactosidase (100,000 u), and dextranase (30,000 u / g) used in this invention are all from Bestgene. The pectinase (30,000 u) is from Daheng Biotechnology. The soybean meal is grade 45.5 and branded as Nantong Cargill.

[0041] The yeast used in this invention is active dry yeast, branded Keweibo. The *Pediococcus pentosaceus*, *Lactobacillus rhamnosus*, and *Bacillus subtilis* used in this invention were all purchased from Luohe Weikang Biotechnology.

[0042] The activation method for the strains was as follows: The ultra-clean workbench was pre-sterilized for 30 minutes. After turning off the UV lamp, one loop of each strain was picked under an alcohol lamp and inoculated into sterile liquid culture medium (yeast was inoculated into YPD medium, Bacillus subtilis into LB medium, and Lactobacillus rhamnosus and Pediococcus pentosus into MRS medium, and cultured in a closed container). The strains were then activated and cultured in a shaker at 180 rpm for 18 hours (yeast at 29℃, Bacillus subtilis at 40℃, Lactobacillus rhamnosus at 37℃, and Pediococcus pentosus at 35℃) to obtain the activated strains.

[0043] Example 1

[0044] This embodiment describes a microbial-enzyme co-fermentation method for removing anti-nutritional factors from soybean meal, comprising the following steps:

[0045] S1, Pre-treated soybean meal: Mix 10kg soybean meal, 1.2kg water, 0.1kg citric acid, 0.04kg hydrogen peroxide solution (30wt%) and 0.02kg tea polyphenols and then subject to steam explosion treatment.

[0046] During steam explosion treatment, saturated steam is introduced, and the pressure inside the explosion tank is controlled at 2MPa and the temperature at 140℃. This is maintained for 12 minutes, after which the material inside the explosion tank is exploded and released, and the material temperature is reduced to 25℃.

[0047] The pretreated soybean meal is sprayed with food-grade catalase at a rate of 80 U / kg to completely degrade residual hydrogen peroxide and avoid affecting subsequent fermentation.

[0048] S2, bacterial enzyme fermentation:

[0049] S21, the preparation method of the compound microbial agent is as follows: Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus are inoculated into a culture medium (containing 40g soybean meal, 15g wheat bran, 10g corn flour, 3g peptone, and 13g glucose, with water added to a solids content of 35wt%) at 6wt%, and cultured for 24h to obtain the seed microbial agent. The culture temperature for yeast is 29℃, for Bacillus subtilis it is 40℃, for Lactobacillus rhamnosus it is 37℃, and for Pediococcus pentosus it is 35℃.

[0050] In the seed inoculant, the effective viable count of yeast is 1.8 × 10⁻⁶. 8 The effective viable count of Bacillus subtilis (CFU / g) is 1.5 × 10⁻⁶. 9 CFU / g, the effective viable count of Lactobacillus rhamnosus is 1×10⁻⁶. 11 The effective viable count of *Pediococcus pentosaceus* was 2.5 × 10⁻⁶ CFU / g. 9 CFU / g.

[0051] Subsequently, a compound inoculum was obtained by mixing yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus seed inoculum in a mass ratio of 1.5:4:3:4.

[0052] S22, Fermentation: Take 10kg of pretreated soybean meal, 2kg of molasses and 0.045kg of compound microbial agent, add water to the total moisture content to 55%, add 0.015kg of acidic protease, 0.03kg of pectinase, 0.015kg of phytase, 0.04kg of glucanase, 0.015kg of galactosidase and 0.006kg of keratinase, stir evenly and ferment in stages;

[0053] The staged fermentation method is as follows:

[0054] First stage: fermentation temperature 33℃, O2 concentration 1.5%, pH value 5.0, fermentation time 16h;

[0055] Second stage: fermentation temperature 38℃, O2 concentration 4%, pH value 4.5, fermentation time 25h;

[0056] Third stage: fermentation temperature 41℃, O2 concentration 1.5%, pH value 4.5, fermentation time 30h.

[0057] During fermentation, the pH value of the material is monitored in real time, and citric acid and dilute 1M NaOH are used to adjust the pH value during fermentation.

[0058] S3, after fermentation, the material is dried to obtain fermented soybean meal.

[0059] After fermentation, the material enters the drum dryer via an auger with an inlet air temperature of 110℃ and an outlet air temperature of 60℃. After drying, it is conveyed by air into a pulverizer for crushing and then packaged.

[0060] Example 2

[0061] This embodiment describes a microbial-enzyme co-fermentation method for removing anti-nutritional factors from soybean meal, comprising the following steps:

[0062] S1, Pre-treated soybean meal: Mix 10kg soybean meal, 1kg water, 0.05kg citric acid, 0.02kg hydrogen peroxide solution (30wt%) and 0.01kg tea polyphenols and then subject to steam explosion treatment.

[0063] During steam explosion treatment, saturated steam is introduced, and the pressure inside the explosion tank is controlled at 1.5 MPa and the temperature at 130°C for 10 minutes. After that, the material inside the explosion tank is exploded and released, and the material temperature is reduced to 30°C.

[0064] The pretreated soybean meal is sprayed with 50 U / kg of food-grade catalase to completely degrade residual hydrogen peroxide and avoid affecting subsequent fermentation.

[0065] S2, bacterial enzyme fermentation:

[0066] S21, the preparation method of the compound microbial agent is as follows: Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus are inoculated at 6 wt% into a culture medium (the culture medium contains 30 g soybean meal, 10 g wheat bran, 5 g corn flour, 1 g peptone, and 10 g glucose, with water added to a solid content of 30 wt%) and cultured for 24 h to obtain the seed microbial agent. The culture temperature for yeast is 28℃, for Bacillus subtilis it is 37℃, for Lactobacillus rhamnosus it is 35℃, and for Pediococcus pentosus it is 30℃.

[0067] In the seed inoculant, the effective viable count of yeast is 1.5 × 10⁻⁶. 8 CFU / g, the effective viable count of Bacillus subtilis is 1×10⁻⁶. 9 CFU / g, the effective viable count of Lactobacillus rhamnosus is 1×10⁻⁶. 9 CFU / g, the effective viable count of Pediococcus pentosaceus is 2×10⁻⁶. 9 CFU / g.

[0068] Subsequently, a compound inoculum was obtained by mixing yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus seed inoculum in a mass ratio of 1:3:2:2.

[0069] S22, Fermentation: Take 10kg of pretreated soybean meal, 2kg of molasses and 0.04kg of compound microbial agent, add water to the total moisture content to 50%, add 0.01kg of acidic protease, 0.02kg of pectinase, 0.01kg of phytase, 0.025kg of glucanase, 0.01kg of galactosidase and 0.005kg of keratinase, stir evenly and ferment in stages;

[0070] The staged fermentation method is as follows:

[0071] First stage: fermentation temperature 30℃, O2 concentration 1%, pH value 5.0, fermentation time 12h;

[0072] Second stage: fermentation temperature 37℃, O2 concentration 3%, pH value 5.0, fermentation time 36h;

[0073] Third stage: fermentation temperature 40℃, O2 concentration 1%, pH value 4.5, fermentation time 24h.

[0074] During fermentation, the pH value of the material is monitored in real time, and citric acid and dilute 1M NaOH are used to adjust the pH value during fermentation.

[0075] S3, after fermentation, the material is dried to obtain fermented soybean meal.

[0076] After fermentation, the material enters the drum dryer via an auger with an inlet air temperature of 120°C and an outlet air temperature of 70°C. After drying, it is conveyed by air into a pulverizer for crushing and then packaged.

[0077] Example 3

[0078] This embodiment describes a microbial-enzyme co-fermentation method for removing anti-nutritional factors from soybean meal, comprising the following steps:

[0079] S1, Pre-treated soybean meal: Mix 10kg soybean meal, 1.5kg water, 0.15kg citric acid, 0.06kg hydrogen peroxide solution (30wt%) and 0.03kg gallic acid and then subject to steam explosion treatment.

[0080] During steam explosion treatment, saturated steam is introduced, and the pressure inside the explosion tank is controlled at 2.5 MPa and the temperature at 150°C. This is maintained for 15 minutes, after which the material inside the explosion tank is exploded and released, and the material temperature is reduced to 20°C.

[0081] The pretreated soybean meal is sprayed with 100 U / kg of food-grade catalase to completely degrade residual hydrogen peroxide and avoid affecting subsequent fermentation.

[0082] S2, bacterial enzyme fermentation:

[0083] S21, the preparation method of the compound microbial agent is as follows: Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus are inoculated into a culture medium (containing 50g soybean meal, 20g wheat bran, 15g corn flour, 5g peptone, and 15g glucose, with water added to a solid content of 40wt%) at 6wt%, and cultured for 24h to obtain the seed microbial agent. The culture temperature for yeast is 30℃, for Bacillus subtilis it is 42℃, for Lactobacillus rhamnosus it is 30℃, and for Pediococcus pentosus it is 37℃.

[0084] In seed inoculants, the effective viable count of yeast is 2 × 10⁻⁶. 8 The effective viable count of Bacillus subtilis (CFU / g) is 1.5 × 10⁻⁶. 9 The effective viable count of Lactobacillus rhamnosus is 1.5 × 10⁻⁶ CFU / g. 9 CFU / g, the effective viable count of Pediococcus pentosaceus is 3×10⁻⁶. 9 CFU / g.

[0085] Subsequently, a compound inoculant was obtained by mixing yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus seed inoculants in a mass ratio of 2:5:4:4.

[0086] S22, Fermentation: Take 10kg of pretreated soybean meal, 2kg of molasses and 0.05kg of compound microbial agent, add water to the total moisture content to 60%, add 0.02kg of acidic protease, 0.04kg of pectinase, 0.02kg of phytase, 0.05kg of glucanase, 0.02kg of galactosidase and 0.008kg of keratinase, stir evenly and ferment in stages;

[0087] The staged fermentation method is as follows:

[0088] First stage: fermentation temperature 35℃, O2 concentration 2%, pH value 5.0, fermentation time 18h;

[0089] Second stage: fermentation temperature 40℃, O2 concentration 5%, pH value 4.8, fermentation time 18h;

[0090] Third stage: fermentation temperature 42℃, O2 concentration 2%, pH value 4.5, fermentation time 36h.

[0091] During fermentation, the pH value of the material is monitored in real time, and citric acid and dilute 1M NaOH are used to adjust the pH value during fermentation.

[0092] S3, after fermentation, the material is dried to obtain fermented soybean meal.

[0093] After fermentation, the material enters the drum dryer via an auger with an inlet air temperature of 115℃ and an outlet air temperature of 65℃. After drying, it is conveyed by air into a pulverizer for crushing and then packaged.

[0094] Example 4

[0095] This embodiment describes a microbial-enzyme co-fermentation method for removing anti-nutritional factors from soybean meal, comprising the following steps:

[0096] S1, Pre-treated soybean meal: Mix 10kg soybean meal, 1.1kg water, 0.12kg citric acid, 0.03kg hydrogen peroxide solution (30wt%), 0.01kg tea polyphenols, and 0.015kg gallic acid, and then subject the mixture to steam explosion treatment.

[0097] During steam explosion treatment, saturated steam is introduced, and the pressure inside the explosion tank is controlled at 2.2 MPa and the temperature at 135°C for 14 minutes. After that, the material inside the explosion tank is exploded and released, and the material temperature is reduced to 25°C.

[0098] The pretreated soybean meal is sprayed with food-grade catalase at a rate of 80 U / kg to completely degrade residual hydrogen peroxide and avoid affecting subsequent fermentation.

[0099] S2, bacterial enzyme fermentation:

[0100] S21, the preparation method of the compound microbial agent is as follows: Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus are inoculated into a culture medium (containing 45g soybean meal, 18g wheat bran, 14g corn flour, 4g peptone, and 11g glucose, with water added to a solid content of 32wt%) at 6wt%, and cultured for 24h to obtain the seed microbial agent. The culture temperature for yeast is 30℃, for Bacillus subtilis it is 41℃, for Lactobacillus rhamnosus it is 37℃, and for Pediococcus pentosus it is 36℃.

[0101] In seed inoculants, the effective viable count of yeast is 2 × 10⁻⁶. 8 The effective viable count of Bacillus subtilis was 1.4 × 10⁻⁶ CFU / g. 9 The effective viable count of Lactobacillus rhamnosus is 1.2 × 10⁻⁶ CFU / g. 9 The effective viable count of *Pediococcus pentosaceus* was 2.8 × 10⁻⁶ CFU / g. 9 CFU / g.

[0102] Subsequently, a compound inoculant was obtained by mixing yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus seed inoculants in a mass ratio of 2:4.5:3.5:2.5.

[0103] S22, Fermentation: Take 10kg of pretreated soybean meal, 2kg of molasses and 0.05kg of compound microbial agent, add water to the total moisture content to 55%, add 0.01kg of acidic protease, 0.02kg of pectinase, 0.02kg of phytase, 0.03kg of glucanase, 0.02kg of galactosidase and 0.007kg of keratinase, stir evenly and ferment in stages;

[0104] The staged fermentation method is as follows:

[0105] First stage: fermentation temperature 34℃, O2 concentration 2%, pH value 5.0, fermentation time 18h;

[0106] Second stage: fermentation temperature 39℃, O2 concentration 4%, pH value 4.5, fermentation time 30h;

[0107] Third stage: fermentation temperature 41℃, O2 concentration 1%, pH value 4.5, fermentation time 34h.

[0108] During fermentation, the pH value of the material is monitored in real time, and citric acid and dilute 1M NaOH are used to adjust the pH value during fermentation.

[0109] S3, after fermentation, the material is dried to obtain fermented soybean meal.

[0110] After fermentation, the material enters the drum dryer via an auger with an inlet air temperature of 110℃ and an outlet air temperature of 60℃. After drying, it is conveyed by air into a pulverizer for crushing and then packaged.

[0111] Example 5

[0112] This embodiment describes a microbial-enzyme co-fermentation method for removing anti-nutritional factors from soybean meal, comprising the following steps:

[0113] S1, Pre-treated soybean meal: Mix 10kg soybean meal, 1kg water, 0.1kg citric acid, 0.05kg hydrogen peroxide solution (30wt%) and 0.03kg gallic acid and then subject to steam explosion treatment.

[0114] During steam explosion treatment, saturated steam is introduced, and the pressure inside the explosion tank is controlled at 2MPa and the temperature at 135℃. This is maintained for 15 minutes, after which the material inside the explosion tank is exploded and released, and the material temperature is reduced to 20℃.

[0115] The pretreated soybean meal is sprayed with food-grade catalase at a concentration of 90 U / kg to thoroughly degrade residual hydrogen peroxide and prevent it from affecting subsequent fermentation.

[0116] S2, bacterial enzyme fermentation:

[0117] S21, the preparation method of the compound microbial agent is as follows: Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus are inoculated into a culture medium (the culture medium contains 35g soybean meal, 15g wheat bran, 10g corn flour, 3g peptone, and 13g glucose, with water added to a solid content of 40wt%) at 6wt%, and cultured for 24h to obtain the seed microbial agent. The culture temperature for yeast is 30℃, for Bacillus subtilis it is 40℃, for Lactobacillus rhamnosus it is 37℃, and for Pediococcus pentosus it is 37℃.

[0118] In seed inoculants, the effective viable count of yeast is 2 × 10⁻⁶. 8 The effective viable count of Bacillus subtilis (CFU / g) is 1.3 × 10⁻⁶. 9 The effective viable count of Lactobacillus rhamnosus is 1.5 × 10⁻⁶ CFU / g. 9 The effective viable count of *Pediococcus pentosaceus* was 2.5 × 10⁻⁶ CFU / g. 9 CFU / g.

[0119] Subsequently, a compound inoculant was obtained by mixing yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus seed inoculants in a mass ratio of 1:5:2:4.

[0120] S22, Fermentation: Take 10kg of pretreated soybean meal, 2kg of molasses and 0.05kg of compound microbial agent, add water to the total moisture content to 55%, add 0.01kg of acidic protease, 0.04kg of pectinase, 0.02kg of phytase, 0.05kg of glucanase, 0.018kg of galactosidase and 0.008kg of keratinase, stir evenly and ferment in stages;

[0121] The staged fermentation method is as follows:

[0122] First stage: fermentation temperature 35℃, O2 concentration 2%, pH value 5.0, fermentation time 18h;

[0123] Second stage: fermentation temperature 38℃, O2 concentration 3%, pH value 4.5, fermentation time 34h;

[0124] Third stage: fermentation temperature 40℃, O2 concentration 2%, pH value 4.5, fermentation time 30h.

[0125] During fermentation, the pH value of the material is monitored in real time, and citric acid and dilute 1M NaOH are used to adjust the pH value during fermentation.

[0126] S3, after fermentation, the material is dried to obtain fermented soybean meal.

[0127] After fermentation, the material enters the drum dryer via an auger with an inlet air temperature of 110℃ and an outlet air temperature of 70℃. After drying, it is conveyed by air into a pulverizer for crushing and then packaged.

[0128] Comparative Example 1

[0129] Compared with Example 1, in step S1, when pretreating soybean meal, citric acid, hydrogen peroxide solution and tea polyphenols were not added, and the treatment was carried out directly by steam explosion. The remaining steps were the same as in Example 1.

[0130] Comparative Example 2

[0131] Compared with Example 1, in Comparative Example 2, only citric acid was added during the pretreatment of soybean meal in step S1, while the remaining steps were the same as in Example 1.

[0132] Comparative Example 3

[0133] Compared with Example 1, in Comparative Example 3, only citric acid and hydrogen peroxide solution were added during the pretreatment of soybean meal in step S1, while the remaining steps were the same as in Example 1.

[0134] Comparative Example 4

[0135] Compared with Example 1, in Comparative Example 4, the amount of tea polyphenols used in step S1, when pretreating soybean meal, exceeded the limit, specifically 0.05 kg. The remaining steps were the same as in Example 1.

[0136] Comparative Example 5

[0137] Compared with Example 1, Comparative Example 5 did not use staged fermentation, but only the second stage fermentation method of Example 1, with a fermentation temperature of 38°C, an O2 concentration of 4%, a pH value of 4.5, and a fermentation time of 71 hours.

[0138] Comparative Example 6

[0139] Compared with Example 1, Comparative Example 6 did not use staged fermentation, but only the first stage fermentation method of Example 1, with a fermentation temperature of 33°C, an O2 concentration of 1.5%, a pH value of 5.0, and a fermentation time of 71 hours.

[0140] Comparative Example 7

[0141] Compared with Example 1, Comparative Example 7 did not add any additives or perform steam explosion pretreatment, but the remaining steps were the same as in Example 1.

[0142] Detection indicators: The content of crude protein, acid-soluble protein, soy globulin, β-conglycinin, stachyose, and raffinose in the fermented soybean meal of the example and comparative examples.

[0143] Crude protein was detected according to QB / T 6432-2018, and acid-soluble protein was detected according to QB / T 2653-2004. Glycine globulin and β-conglycine globulin were detected using kits from Beijing Longke Ark Co., Ltd. Stachyose and raffinose were detected by HPLC, following the method described in the *Chinese Cereals and Oils Journal*, Vol. 29, No. 12, Dec. 2014. Results are shown in Table 1.

[0144] Table 1 Test Results

[0145]

[0146] As shown in Table 1, compared with the comparative example, the fermentation method of the present invention can achieve a soybean globulin degradation rate of up to 79%, an acid-soluble protein content of up to 26% or more, a significant decrease in the content of anti-nutritional factors, and a substantial increase in nutritional value.

[0147] The comparative examples, with no pretreatment, steam explosion pretreatment without additives, or no staged fermentation, all showed incomplete degradation and poor results, demonstrating the necessity of this proposed method. Specifically, Example 1 and Comparative Examples 1-3 confirmed the synergistic effect of citric acid, hydrogen peroxide solution, and tea polyphenols. Appropriate amounts of polyphenols can form hydrogen bonds or hydrophobic complexes with proteins, promoting conformational changes and further disrupting the structure of anti-nutritional factors. When the amount of polyphenols in Comparative Example 4 exceeded the specified range, the polyphenols formed complexes with proteins and inhibited enzyme activity, thus hindering the degradation of anti-nutritional factors.

[0148] As shown in Example 1 and Comparative Examples 5-6, the three-stage fermentation achieves a more thorough degradation effect through the relay of microbial communities; a single temperature cannot take into account the optimal conditions for all microbial species, resulting in a decrease in efficiency.

[0149] Comparative Example 7 showed the worst results because, during the steam explosion pretreatment, the soybean meal cell walls remained intact, and the proteins were encapsulated in a dense matrix. This prevented exogenous enzymes and cells from effectively contacting the substrate, resulting in extremely low hydrolysis efficiency.

[0150] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal, characterized in that, Includes the following steps: S1, Pre-treated soybean meal: Soybean meal, water, and additives are mixed and then subjected to steam explosion treatment. The additives include citric acid, hydrogen peroxide solution, and polyphenols. The mass ratio of soybean meal, water, citric acid, hydrogen peroxide solution, and polyphenols is 100:10-15:0.5-1.5:0.2-0.6:0.1-0.3, and the mass concentration of hydrogen peroxide solution is 30%. The polyphenols are one or a combination of tea polyphenols and gallic acid. S2, bacterial and enzyme fermentation: pretreated soybean meal is mixed with molasses and water to obtain raw materials, compound bacterial agent and compound enzyme are added, and fermentation is carried out after stirring evenly. The compound microbial agent includes yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus; The complex enzyme includes acidic protease, pectinase, phytase, glucanase, α-galactosidase, and keratinase. During fermentation, a staged fermentation process is adopted: First stage: fermentation temperature 30-35℃, O2 concentration 1%-2%, pH value 5.0, fermentation time 12-18h; Second stage: Fermentation temperature 37-40℃, O2 concentration 3%-5%, pH value 4.5-5.0, fermentation time 18-36h; The third stage: fermentation temperature 40-42℃, O2 concentration 1%-2%, pH value 4.5, fermentation time 24-36h; S3, after fermentation, the material is dried to obtain fermented soybean meal.

2. The method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal as described in claim 1, characterized in that, In step S1, during the steam explosion treatment, saturated steam is introduced, and the pressure inside the explosion tank is controlled at 1.5-2.5 MPa and the temperature at 130-150℃. This is maintained for 10-15 minutes, after which the material inside the explosion tank is exploded and released, and the material temperature is reduced to 20-30℃.

3. The method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal as described in claim 1, characterized in that, In step S2, the mass ratio of soybean meal, molasses, and compound microbial agent is 100:20:0.4-0.5, and water is added until the total moisture content is 50%-60%.

4. The method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal as described in claim 3, characterized in that, The preparation method of the compound microbial agent is as follows: Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus were inoculated into the culture medium and cultured for 24 hours to obtain seed inoculum. Subsequently, the seed inoculum was mixed with yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosus in a mass ratio of 1-2:3-5:2-4:2-4 to obtain a compound inoculum. The yeast was cultured at 28-30℃, and Bacillus subtilis at 37-42℃; Lactobacillus rhamnosus and Pediococcus pentosus were cultured at 30-37℃ in a closed system.

5. The method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal as described in claim 4, characterized in that, The culture medium comprises the following components in parts by weight: 30-50 parts soybean meal, 10-20 parts wheat bran, 5-15 parts corn flour, 1-5 parts peptone and 10-15 parts glucose, with water added to a solids content of 30wt%-40wt%.

6. The method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal as described in claim 4, characterized in that, In the seed inoculant, the effective viable count of yeast is (1.5-2) × 10⁻¹⁰. 8 The effective viable count of Bacillus subtilis is (1-1.5) × 10⁻⁶ CFU / g. 9 The effective viable count of Lactobacillus rhamnosus is (1-1.5) × 10 CFU / g. 9 CFU / g, the effective viable count of Pediococcus pentosaceus is (2-3) × 10⁻⁶. 9 CFU / g.

7. The method for co-fermentation of bacteria and enzymes to remove anti-nutritional factors from soybean meal as described in claim 1, characterized in that, The mass ratio of soybean meal to acidic protease to pectinase to phytase to glucanase to α-galactosidase to keratinase is 100:0.1-0.2:0.2-0.4:0.1-0.2:0.25-0.5:0.1-0.2:0.05-0.08.

Images