Bacteria-enzyme synergistic fermentation method for removing antinutritional factors in soybean meal

Abstract

The present invention relates to the technical field of bacterial enzyme fermentation, and provides a bacterial enzyme synergistic fermentation method for removing antinutritional factors in soybean meal, the method comprises the following steps: S1, soybean meal pretreatment: soybean meal, water and an auxiliary agent are mixed and then subjected to steam explosion treatment, and the auxiliary agent comprises citric acid, a hydrogen peroxide solution and polyphenol; s2, bacterial enzyme fermentation: mixing the pretreated soybean meal with molasses and water to obtain a raw material, adding a complex microbial inoculant and a complex enzyme, uniformly stirring, and fermenting; the complex microbial inoculants comprise saccharomycetes, bacillus subtilis, lactobacillus rhamnosus and pediococcus pentosaceus; the compound enzyme comprises acid protease, pectinase, phytase, glucanase, alpha-galactosidase and keratinase; and S3, after fermentation is finished, drying the materials to obtain the fermented soybean meal. According to the fermentation method, the glycinin degradation rate can reach 79% or above, the acid soluble protein content is increased to 26% or above, the anti-nutritional factor content is remarkably reduced, and the nutritional value is greatly increased.

Description

Technical Field

[0001] The present invention relates to the technical field of bacterial enzyme fermentation, in particular to a bacterial enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal. Background Art

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

[0003] In recent years, bacterial-enzyme synergistic fermentation has been used to improve plant protein raw materials such as soybean meal because it has the advantages of both microbial degradation and enzyme-enhanced hydrolysis. Various bacterial species such as lactic acid bacteria and Bacillus can secrete acid and partial degradation enzymes, and in combination with exogenous proteases, phytases, etc., can accelerate the decomposition of anti-nutritional factors in raw materials such as soybean meal to a certain extent. However, in the current common synergistic fermentation system, there are still deficiencies in the matching of various bacteria and enzymes and the optimization of process conditions, resulting in a low removal rate of key anti-nutritional factors, which affects the in-depth application of soybean meal in high-quality feed. In view of the problem of low removal rate of anti-nutritional factors in soybean meal in the existing technology, it is urgent to develop an efficient bacterial-enzyme synergistic fermentation method 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 bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal, so as to solve the problem of low removal rate of anti-nutritional factors in soybean meal in the prior art.

[0005] The technical solution of the present invention is achieved as follows: The present invention provides a bacterial enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal, comprising the following steps: S1, pre-treating soybean meal: mixing soybean meal, water and additives and then performing steam explosion treatment, wherein the additives include citric acid, hydrogen peroxide solution and polyphenols.

[0006] Specifically, steam explosion, under high temperature and pressure, causes macromolecular structures (proteins and oligosaccharides) to break and expand, which, combined with additives, can accelerate degradation. The citric acid in the additives provides an acidic environment, promoting partial protein denaturation. Citric acid also synergizes with the high temperature of steam explosion to disrupt hydrogen bonds and hydrophobic interactions, 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 disrupt stabilizing structures such as polysulfide and ether bonds within proteins and anti-nutritional factors (such as glycinin and raffinose). Polyphenols form hydrogen bonds or hydrophobic complexes with proteins, promoting conformational changes and further disrupting the structure of anti-nutritional factors. Polyphenols also inhibit bacterial growth, creating a favorable environment for subsequent probiotic colonization. Furthermore, their antioxidant properties can prevent nutrient loss caused by lipid oxidation during fermentation. Citric acid, hydrogen peroxide solution, and polyphenols synergistically significantly reduce the structural density of soybean meal (depolymerization and loosening), exposing more targets for subsequent bacterial enzyme action.

[0007] To avoid residual hydrogen peroxide affecting the later fermentation, spraying 50-100 U / kg of food-grade catalase can completely degrade residual hydrogen peroxide within 15-30 minutes.

[0008] S2, bacterial enzyme fermentation: the pretreated soybean meal is mixed with molasses and water to obtain raw materials, composite bacterial agent and composite enzyme are added, and the mixture is stirred evenly and then fermented.

[0009] The composite bacterial agent comprises yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus; The complex enzyme comprises acid protease, pectinase, phytase, glucanase, α-galactosidase and keratinase.

[0010] Bacillus subtilis secretes proteases, cellulases, etc. to degrade large molecular proteins / polysaccharides, while Lactobacillus and Pediococcus produce lactic acid, oligosaccharidases, etc., specializing in oligosaccharides such as raffinose and stachyose and improving flavor; yeast synergistically decomposes polysaccharides and synthesizes B vitamins to enhance nutrition.

[0011] Pectinase / glucanase disrupts the intercellular layer, promoting the release of contents. The acidic protease in the enzyme complex directly hydrolyzes denatured globulins into polypeptides. Lactobacillus rhamnosus and Pediococcus pentosaceus rapidly produce lactic acid, inhibiting contaminants such as Escherichia coli. α-galactosidase specifically hydrolyzes the α-1,6-galactosidic bonds in raffinose and stachyose, synergizing with Bacillus to enhance protease activity. Phytase degrades phytic acid, releasing phosphorus and minerals.

[0012] The synergistic advantage of bacteria and enzymes: endogenous bacterial enzyme production + exogenous enzyme enhancement achieves the dual guarantee of self-production and external supplementation, avoiding the degradation bottleneck caused by a single bacteria or insufficient enzyme activity.

[0013] S3, after the fermentation is completed, the material is dried to obtain fermented soybean meal.

[0014] Based on the above technical solution, preferably, in step S1, the mass ratio of the 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 the hydrogen peroxide solution is 30%.

[0015] On the basis of the above technical solution, preferably, the polyphenol is one or a combination of two of tea polyphenol and gallic acid.

[0016] On the basis of the above technical solution, preferably, in step S1, during the steam explosion treatment, saturated steam is introduced to control the pressure in the explosion tank to 1.5-2.5 MPa and the temperature to 130-150°C for 10-15 minutes, after which the material in the explosion tank is exploded and released, and the temperature of the material is reduced to 20-30°C.

[0017] Based on the above technical solution, preferably, in step S2, the mass ratio of soybean meal, molasses and composite bacterial agent is 100:20:0.4-0.5, and water is added to a total moisture content of 50%-60%.

[0018] Based on the above technical solution, the preferred preparation method of the composite bacterial agent is: yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus are cultured separately and then mixed according to the proportion. Specifically: The activated yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus are respectively inoculated into the culture medium and cultured for 24 hours to obtain a seed agent, and then the yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus seed agents are mixed according to the mass ratio of 1-2:3-5:2-4:2-4 to obtain a composite agent; wherein, the yeast culture temperature is 28-30°C, the Bacillus subtilis culture temperature is 37-42°C; the Lactobacillus rhamnosus culture temperature and the Pediococcus pentosaceus culture temperature are both 30-37°C, and the culture is closed.

[0019] Based on the above technical solution, preferably, the culture medium comprises the following components in parts by weight: 30-50 parts of soybean meal, 10-20 parts of bran, 5-15 parts of corn flour, 1-5 parts of peptone and 10-15 parts of glucose, and water is added to a solid content of 30wt%-40wt%.

[0020] On the basis of the above technical solution, preferably, the effective viable bacteria count of yeast in the seed inoculant is (1.5-2)×10 8 CFU / g (i.e., the number of viable bacteria per gram of seed inoculant (solid material after fermentation)), the effective viable bacteria count of Bacillus subtilis is (1-1.5)×10 9CFU / g, the effective viable count of Lactobacillus rhamnosus is (1-1.5)×10 9 CFU / g, the effective viable count of Pediococcus pentosaceus is (2-3)×10 9 CFU / g.

[0021] Based on the above technical solution, preferably, the mass ratio of soybean meal: acid 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.

[0022] On the basis of the above technical solution, preferably, during the fermentation in step S2, fermentation is carried out in stages: Stage 1: Fermentation temperature 30-35°C, O2 concentration 1%-2%, pH 5.0, fermentation time 12-18h; Specifically, the first stage is micro-oxygen and suitable temperature; lactic acid bacteria are dominant in rapid acid production, which lowers the pH and inhibits miscellaneous bacteria; α-galactosidase is activated to hydrolyze raffinose and stachyose; yeast assists in enzyme production and provides a carbon source.

[0023] The second stage: fermentation temperature 37-40℃, O2 concentration 3%-5%, pH value 4.5-5.0, fermentation time 18-36h; The second stage is aerobic and warming; Bacillus subtilis is active and secretes a large amount of protease, phytase, etc. under aerobic conditions; the acidic environment activates acid protease, efficiently hydrolyzes globulin, and produces a large amount of small peptides.

[0024] The third stage: fermentation temperature 40-42℃, O2 concentration 1%-2%, pH value 4.5, fermentation time 24-36h.

[0025] The third stage is high temperature and micro-oxygen; it inhibits miscellaneous bacteria and some dying bacteria, and Bacillus continues to produce enzymes to further degrade residual anti-nutritional factors; it promotes the further conversion of small peptides into smaller molecular peptides or free amino acids.

[0026] During fermentation, the pH value of the material was monitored in real time, and citric acid and dilute 1 M NaOH were used to adjust the pH value during fermentation.

[0027] Through precise control of environmental parameters, the microbial community is guided to evolve along the path of "lactic acid bacteria start → Bacillus dominate → high temperature strengthen", thus achieving functional relay.

[0028] The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors from soybean meal of the present invention has the following beneficial effects compared with the prior art: (1) Pretreatment provides a basis for the action of bacterial enzymes: steam explosion + additives rupture the cell wall, denature the protein, and expose the oligosaccharides. These effects greatly enhance the enzymatic hydrolysis of the complex enzyme and the adsorption capacity of microorganisms, thereby increasing the enzymatic hydrolysis rate and starting the fermentation faster.

[0029] The synergy of bacteria and enzymes provides the basis for segmented fermentation: the combination of multiple bacterial species and multiple enzymes provides full-spectrum degradation capabilities; in segmented fermentation, different bacterial species can fully exert their functions under their optimal conditions, avoiding the inactivation or inhibition of a single bacterial species in a complex environment.

[0030] Segmented fermentation prolongs and amplifies the synergistic effect of steam explosion and bacterial enzymes: staged temperature and oxygen control prolongs the time window of high-activity enzymatic hydrolysis, allowing the pre-treated exposed substrate to be continuously and thoroughly degraded; it also promotes the continuous generation of small peptides and their further degradation into functional small molecule peptides.

[0031] (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 multidisciplinary integration of physics, chemistry and biology, and has the potential to become a benchmark technology for high-end fermented soybean meal. DETAILED DESCRIPTION

[0032] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] The keratinase (enzyme activity 500,000 U / g), phytase (enzyme activity 300,000 U / g), acid protease (enzyme activity 200,000 U / g), galactosidase (enzyme activity 100,000 U), and glucanase (enzyme activity 30,000 U / g) used in this invention are all from BestJet. The pectinase (enzyme activity 30,000 U) is from Daheng Biotechnology. The soybean meal used is Soybean Meal 45.5, a brand from Nantong Cargill.

[0034] The yeast of the present invention is active dry yeast, and the brand is Keweibo. The Pediococcus pentosaceus, Lactobacillus rhamnosus, and Bacillus subtilis used in the present invention are all purchased from Luohe Weikang Biology.

[0035] The strain activation method is as follows: the clean bench is pre-sterilized for 30 minutes. After turning off the ultraviolet lamp, one loop of the strain is picked up and inoculated into a sterile liquid culture medium under an alcohol lamp (yeast is inoculated in YPD medium, Bacillus subtilis is inoculated in LB medium, Lactobacillus rhamnosus and Pediococcus pentosaceus are inoculated in MRS medium, and cultured in a closed culture medium). The activated culture is carried out in a shaker at 180 r / min for 18 hours (yeast at 29°C, Bacillus subtilis at 40°C, Lactobacillus rhamnosus at 37°C, and Pediococcus pentosaceus at 35°C) to obtain the activated strain.

[0036] Example 1 The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors from soybean meal in this embodiment comprises the following steps: S1, pretreatment of soybean meal: 10 kg soybean meal, 1.2 kg water, 0.1 kg citric acid, 0.04 kg hydrogen peroxide solution (30 wt%) and 0.02 kg tea polyphenols were mixed and then subjected to steam explosion treatment.

[0037] During steam explosion treatment, saturated steam is introduced to control the pressure in the explosion tank to 2MPa and the temperature to 140℃ for 12 minutes. After that, the material in the explosion tank is exploded and released, and the material temperature is reduced to 25℃.

[0038] The pretreated soybean meal is sprayed with 80 U / kg of food-grade catalase to completely degrade residual hydrogen peroxide and avoid affecting subsequent fermentation.

[0039] S2, bacterial enzyme fermentation: S21. A method for preparing a composite bacterial agent is as follows: inoculating activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosaceus at 6 wt% each into a culture medium (the culture medium contains 40 g soybean meal, 15 g bran, 10 g corn flour, 3 g peptone, and 13 g glucose, with water added to a solid content of 35 wt%), and culturing for 24 hours to obtain a seed bacterial agent. The yeast culture temperature is 29°C, the Bacillus subtilis culture temperature is 40°C, the Lactobacillus rhamnosus culture temperature is 37°C, and the Pediococcus pentosaceus culture temperature is 35°C.

[0040] The effective viable count of yeast in the seed inoculant is 1.8×10 8 CFU / g, the effective viable count of Bacillus subtilis is 1.5×10 9 CFU / g, the effective viable count of Lactobacillus rhamnosus is 1×10 11 CFU / g, the effective viable count of Pediococcus pentosaceus was 2.5×10 9 CFU / g.

[0041] Subsequently, yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus seed inoculants were mixed in a mass ratio of 1.5:4:3:4 to obtain a composite inoculum.

[0042] S22, fermentation: taking 10 kg of pretreated soybean meal, 2 kg of molasses and 0.045 kg of composite bacterial agent, adding water to a total moisture content of 55%, adding 0.015 kg of acid protease, 0.03 kg of pectinase, 0.015 kg of phytase, 0.04 kg of glucanase, 0.015 kg of galactosidase and 0.006 kg of keratinase, stirring evenly and fermenting in stages; The staged fermentation method is: Stage 1: fermentation temperature 33°C, O2 concentration 1.5%, pH 5.0, fermentation time 16h; Stage 2: fermentation temperature 38°C, O2 concentration 4%, pH 4.5, fermentation time 25h; The third stage: fermentation temperature 41℃, O2 concentration 1.5%, pH value 4.5, fermentation time 30h.

[0043] During fermentation, the pH value of the material was monitored in real time, and citric acid and dilute 1 M NaOH were used to adjust the pH value during fermentation.

[0044] S3, after the fermentation is completed, the material is dried to obtain fermented soybean meal.

[0045] After the fermentation is completed, the material enters the drum dryer through the auger, with the inlet air temperature of 110℃ and the outlet air temperature of 60℃; after the drying is completed, it is sent to the crusher for crushing and then packaged.

[0046] Example 2 The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors from soybean meal in this embodiment comprises the following steps: S1, pretreatment of soybean meal: 10 kg soybean meal, 1 kg water, 0.05 kg citric acid, 0.02 kg hydrogen peroxide solution (30 wt%) and 0.01 kg tea polyphenols were mixed and then subjected to steam explosion treatment.

[0047] During steam explosion treatment, saturated steam is introduced to control the pressure in the explosion tank to 1.5 MPa and the temperature to 130°C for 10 minutes. After that, the material in the explosion tank is exploded and released, and the material temperature is reduced to 30°C.

[0048] 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.

[0049] S2, bacterial enzyme fermentation: S21. A method for preparing a composite microbial agent is as follows: Activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosaceus are inoculated at 6 wt% into a culture medium (the culture medium contains 30 g soybean meal, 10 g 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 hours to obtain a seed microbial agent. The yeast culture temperature is 28°C, the Bacillus subtilis culture temperature is 37°C, the Lactobacillus rhamnosus culture temperature is 35°C, and the Pediococcus pentosaceus culture temperature is 30°C.

[0050] The effective viable count of yeast in the seed inoculant is 1.5×10 8 CFU / g, the effective viable count of Bacillus subtilis is 1×10 9 CFU / g, the effective viable count of Lactobacillus rhamnosus is 1×10 9 CFU / g, the effective viable count of Pediococcus pentosaceus is 2×10 9 CFU / g.

[0051] Subsequently, yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus seed inoculants were mixed in a mass ratio of 1:3:2:2 to obtain a composite inoculum.

[0052] S22, fermentation: taking 10 kg of pretreated soybean meal, 2 kg of molasses and 0.04 kg of composite bacterial agent, adding water to a total moisture content of 50%, adding 0.01 kg of acid protease, 0.02 kg of pectinase, 0.01 kg of phytase, 0.025 kg of glucanase, 0.01 kg of galactosidase and 0.005 kg of keratinase, stirring evenly and fermenting in stages; The staged fermentation method is: Stage 1: fermentation temperature 30°C, O2 concentration 1%, pH 5.0, fermentation time 12h; Stage 2: Fermentation temperature 37°C, O2 concentration 3%, pH 5.0, fermentation time 36h; The third stage: fermentation temperature 40℃, O2 concentration 1%, pH value 4.5, fermentation time 24h.

[0053] During fermentation, the pH value of the material was monitored in real time, and citric acid and dilute 1 M NaOH were used to adjust the pH value during fermentation.

[0054] S3, after the fermentation is completed, the material is dried to obtain fermented soybean meal.

[0055] After the fermentation is completed, the material enters the drum dryer through the auger, with the air inlet temperature of 120℃ and the air outlet temperature of 70℃; after the drying is completed, it is sent to the crusher for crushing and then packaged.

[0056] Example 3 The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors from soybean meal in this embodiment comprises the following steps: S1, pretreatment of soybean meal: 10 kg soybean meal, 1.5 kg water, 0.15 kg citric acid, 0.06 kg hydrogen peroxide solution (30 wt%) and 0.03 kg gallic acid were mixed and then steam exploded.

[0057] During steam explosion treatment, saturated steam is introduced to control the pressure in the explosion tank to 2.5 MPa and the temperature to 150°C for 15 minutes. After that, the material in the explosion tank is exploded and released, and the material temperature is reduced to 20°C.

[0058] 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.

[0059] S2, bacterial enzyme fermentation: S21. A method for preparing a composite bacterial agent comprises inoculating activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosaceus at 6 wt% each into a culture medium (the culture medium contains 50 g soybean meal, 20 g bran, 15 g corn flour, 5 g peptone, and 15 g glucose, with water added to a solid content of 40 wt%), and culturing for 24 hours to obtain a seed bacterial agent. The yeast culture temperature is 30°C, the Bacillus subtilis culture temperature is 42°C, the Lactobacillus rhamnosus culture temperature is 30°C, and the Pediococcus pentosaceus culture temperature is 37°C.

[0060] The effective viable count of yeast in the seed inoculant is 2×10 8 CFU / g, the effective viable count of Bacillus subtilis is 1.5×10 9 CFU / g, the effective viable count of Lactobacillus rhamnosus is 1.5×10 9 CFU / g, the effective viable count of Pediococcus pentosaceus is 3×10 9 CFU / g.

[0061] Subsequently, yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus seed inoculants were mixed in a mass ratio of 2:5:4:4 to obtain a composite inoculum.

[0062] S22, fermentation: taking 10 kg of pretreated soybean meal, 2 kg of molasses and 0.05 kg of composite bacterial agent, adding water to a total moisture content of 60%, adding 0.02 kg of acid protease, 0.04 kg of pectinase, 0.02 kg of phytase, 0.05 kg of glucanase, 0.02 kg of galactosidase and 0.008 kg of keratinase, stirring evenly and fermenting in stages; The staged fermentation method is: Stage 1: fermentation temperature 35°C, O2 concentration 2%, pH 5.0, fermentation time 18h; The second stage: fermentation temperature 40℃, O2 concentration 5%, pH value 4.8, fermentation time 18h; The third stage: fermentation temperature 42℃, O2 concentration 2%, pH value 4.5, fermentation time 36h.

[0063] During fermentation, the pH value of the material was monitored in real time, and citric acid and dilute 1 M NaOH were used to adjust the pH value during fermentation.

[0064] S3, after the fermentation is completed, the material is dried to obtain fermented soybean meal.

[0065] After the fermentation is completed, the material enters the drum dryer through the auger, with the air inlet temperature of 115℃ and the air outlet temperature of 65℃; after the drying is completed, it is sent to the crusher for crushing and then packaged.

[0066] Example 4 The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors from soybean meal in this embodiment comprises the following steps: S1, pretreatment of soybean meal: 10 kg soybean meal, 1.1 kg water, 0.12 kg citric acid, 0.03 kg hydrogen peroxide solution (30 wt%), 0.01 kg tea polyphenols, and 0.015 kg gallic acid were mixed and then steam exploded.

[0067] During steam explosion treatment, saturated steam is introduced to control the pressure in the explosion tank to 2.2 MPa and the temperature to 135°C for 14 minutes. After that, the material in the explosion tank is exploded and released, and the material temperature is reduced to 25°C.

[0068] The pretreated soybean meal is sprayed with 80 U / kg of food-grade catalase to completely degrade residual hydrogen peroxide and avoid affecting subsequent fermentation.

[0069] S2, bacterial enzyme fermentation: S21. A method for preparing a composite bacterial agent comprises inoculating activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosaceus at 6 wt% each into a culture medium (the culture medium contains 45 g soybean meal, 18 g bran, 14 g corn flour, 4 g peptone, and 11 g glucose, with water added to a solid content of 32 wt%), and culturing for 24 hours to obtain a seed bacterial agent. The yeast culture temperature is 30°C, the Bacillus subtilis culture temperature is 41°C, the Lactobacillus rhamnosus culture temperature is 37°C, and the Pediococcus pentosaceus culture temperature is 36°C.

[0070] The effective viable count of yeast in the seed inoculant is 2×10 8 CFU / g, the effective viable count of Bacillus subtilis is 1.4×10 9 CFU / g, the effective viable count of Lactobacillus rhamnosus was 1.2×10 9CFU / g, the effective viable count of Pediococcus pentosaceus was 2.8×10 9 CFU / g.

[0071] Subsequently, yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus seed inoculants were mixed in a mass ratio of 2:4.5:3.5:2.5 to obtain a composite inoculum.

[0072] S22, fermentation: taking 10 kg of pretreated soybean meal, 2 kg of molasses and 0.05 kg of composite bacterial agent, adding water to a total moisture content of 55%, adding 0.01 kg of acid protease, 0.02 kg of pectinase, 0.02 kg of phytase, 0.03 kg of glucanase, 0.02 kg of galactosidase and 0.007 kg of keratinase, stirring evenly and fermenting in stages; The staged fermentation method is: Stage 1: fermentation temperature 34°C, O2 concentration 2%, pH 5.0, fermentation time 18h; Stage 2: fermentation temperature 39°C, O2 concentration 4%, pH 4.5, fermentation time 30h; The third stage: fermentation temperature 41℃, O2 concentration 1%, pH value 4.5, fermentation time 34h.

[0073] During fermentation, the pH value of the material was monitored in real time, and citric acid and dilute 1 M NaOH were used to adjust the pH value during fermentation.

[0074] S3, after the fermentation is completed, the material is dried to obtain fermented soybean meal.

[0075] After the fermentation is completed, the material enters the drum dryer through the auger, with the inlet air temperature of 110℃ and the outlet air temperature of 60℃; after the drying is completed, it is sent to the crusher for crushing and then packaged.

[0076] Example 5 The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors from soybean meal in this embodiment comprises the following steps: S1, pretreatment of soybean meal: 10 kg soybean meal, 1 kg water, 0.1 kg citric acid, 0.05 kg hydrogen peroxide solution (30 wt%) and 0.03 kg gallic acid were mixed and then steam exploded.

[0077] During steam explosion treatment, saturated steam is introduced to control the pressure in the explosion tank to 2MPa and the temperature to 135℃ for 15min. After that, the material in the explosion tank is exploded and released, and the temperature of the material is reduced to 20℃.

[0078] The pretreated soybean meal is sprayed with 90 U / kg of food-grade catalase to completely degrade residual hydrogen peroxide and avoid affecting subsequent fermentation.

[0079] S2, bacterial enzyme fermentation: S21. A method for preparing a composite bacterial agent is as follows: inoculating activated yeast, Bacillus subtilis, Lactobacillus rhamnosus, and Pediococcus pentosaceus at 6 wt% into a culture medium (the culture medium contains 35 g soybean meal, 15 g bran, 10 g corn flour, 3 g peptone, and 13 g glucose, with water added to a solid content of 40 wt%), and culturing for 24 hours to obtain a seed bacterial agent. The yeast culture temperature is 30°C, the Bacillus subtilis culture temperature is 40°C, the Lactobacillus rhamnosus culture temperature is 37°C, and the Pediococcus pentosaceus culture temperature is 37°C.

[0080] The effective viable count of yeast in the seed inoculant is 2×10 8 CFU / g, the effective viable count of Bacillus subtilis is 1.3×10 9 CFU / g, the effective viable count of Lactobacillus rhamnosus is 1.5×10 9 CFU / g, the effective viable count of Pediococcus pentosaceus was 2.5×10 9 CFU / g.

[0081] Subsequently, yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus seed inoculants were mixed in a mass ratio of 1:5:2:4 to obtain a composite inoculum.

[0082] S22, fermentation: taking 10 kg of pretreated soybean meal, 2 kg of molasses and 0.05 kg of composite bacterial agent, adding water to a total moisture content of 55%, adding 0.01 kg of acid protease, 0.04 kg of pectinase, 0.02 kg of phytase, 0.05 kg of glucanase, 0.018 kg of galactosidase and 0.008 kg of keratinase, stirring evenly and fermenting in stages; The staged fermentation method is: Stage 1: fermentation temperature 35°C, O2 concentration 2%, pH 5.0, fermentation time 18h; Stage 2: Fermentation temperature 38°C, O2 concentration 3%, pH 4.5, fermentation time 34h; The third stage: fermentation temperature 40℃, O2 concentration 2%, pH value 4.5, fermentation time 30h.

[0083] During fermentation, the pH value of the material was monitored in real time, and citric acid and dilute 1 M NaOH were used to adjust the pH value during fermentation.

[0084] S3, after the fermentation is completed, the material is dried to obtain fermented soybean meal.

[0085] After the fermentation is completed, the material enters the drum dryer through the auger, with the air inlet temperature of 110℃ and the air outlet temperature of 70℃; after the drying is completed, it is sent to the crusher for crushing and then packaged.

[0086] Comparative Example 1 Comparative Example 1 Compared with Example 1, in step S1, when pre-treating soybean meal, no citric acid, hydrogen peroxide solution and tea polyphenols were added, and the soybean meal was directly steam exploded. The remaining steps were the same as in Example 1.

[0087] Comparative Example 2 Compared with Example 1, in Comparative Example 2, only citric acid was added during the pretreatment of soybean meal in step S1, and the remaining steps were the same as in Example 1.

[0088] Comparative Example 3 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, and the remaining steps were the same as in Example 1.

[0089] Comparative Example 4 Comparative Example 4 Compared with Example 1, in step S1, when pretreating soybean meal, the amount of tea polyphenols used exceeded the limit range, specifically 0.05 kg, and the remaining steps were the same as Example 1.

[0090] Comparative Example 5 Compared with Example 1, Comparative Example 5 does not adopt staged fermentation, but only adopts the second stage fermentation method of the example, with a fermentation temperature of 38°C, an O2 concentration of 4%, a pH value of 4.5, and a fermentation time of 71h.

[0091] Comparative Example 6 Compared with Example 1, Comparative Example 6 does not adopt staged fermentation, but only adopts the first stage fermentation method of the example, with a fermentation temperature of 33°C, an O2 concentration of 1.5%, a pH value of 5.0, and a fermentation time of 71h.

[0092] Comparative Example 7 Compared with Example 1, Comparative Example 7 did not add any additives and did not perform steam explosion pretreatment. The remaining steps were the same as in Example 1.

[0093] Detection indicators: the contents of crude protein, acid-soluble protein, glycinin, β-conglycinin, stachyose and raffinose in the fermented soybean meal of the examples and comparative examples were detected.

[0094] Crude protein was determined according to QB / T 6432-2018, and acid-soluble protein was determined according to QB / T 2653-2004. Glycinin and β-conglycinin were determined using kits from Beijing Longke Ark Co., Ltd. Stachyose and raffinose were determined by HPLC using the method described in Journal of the Chinese Cereals and Oils Association, Vol. 29, No. 12, December 2014. The results are shown in Table 1.

[0095] Table 1 Test results

[0096] As shown in Table 1, compared with the comparative example, the soybean globulin degradation rate of the fermentation method of the embodiment of the present invention can reach up to 79%, the acid-soluble protein content can be increased to more than 26%, the anti-nutritional factor content is significantly reduced, and the nutritional value is greatly improved.

[0097] The comparative examples without pretreatment, steam explosion pretreatment without additives, or staged fermentation all showed incomplete degradation and poor results, demonstrating the necessity of this approach. Example 1 and Comparative Examples 1-3 demonstrate the synergistic treatment 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 used in Comparative Example 4 exceeded the specified range, the polyphenols formed complexes with proteins and inhibited enzyme activity, hindering the degradation of anti-nutritional factors.

[0098] As shown in Example 1 and Comparative Examples 5-6, the three-stage fermentation achieves a more thorough degradation effect through the relay of the bacterial community; a single temperature cannot take into account the optimal conditions of all bacterial species, resulting in a decrease in efficiency.

[0099] The effect of Comparative Example 7 was the worst. This was because without steam explosion pretreatment, the soybean meal cell wall was intact and the protein was wrapped in a dense matrix. Exogenous enzymes and bacteria could not effectively contact the substrate, resulting in extremely low hydrolysis efficiency.

[0100] 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 in the scope of protection of the present invention.

Claims

1. A bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors from soybean meal, characterized in that: The following steps are involved: S1, pre-treating soybean meal: mixing soybean meal, water, and an additive and then performing a steam explosion treatment, wherein the additive includes citric acid, hydrogen peroxide solution, and polyphenols; the mass ratio of the 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 the hydrogen peroxide solution is 30%; the polyphenols are one or a combination of tea polyphenols and gallic acid; S2, bacterial enzyme fermentation: pre-treated soybean meal is mixed with molasses and water to obtain raw materials, composite bacterial agent and composite enzyme are added, stirred evenly and then fermented; The composite bacterial agent comprises yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus; The complex enzyme comprises acid protease, pectinase, phytase, glucanase, α-galactosidase and keratinase; During fermentation, fermentation is carried out in stages: Stage 1: Fermentation temperature 30-35°C, O2 concentration 1%-2%, pH 5.0, fermentation time 12-18h; The 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 the fermentation is completed, the material is dried to obtain fermented soybean meal.

2. The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal according to claim 1, characterized in that: In step S1, during the steam explosion treatment, saturated steam is introduced to control the pressure in the explosion tank to 1.5-2.5 MPa and the temperature to 130-150°C for 10-15 minutes, after which the material in the explosion tank is exploded and released, and the temperature of the material is reduced to 20-30°C.

3. The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal according to claim 1, characterized in that: In step S2, the mass ratio of soybean meal, molasses, and composite bacterial agent is 100:20:0.4-0.5, and water is added to a total moisture content of 50%-60%.

4. The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal according to claim 3, characterized in that: The preparation method of the composite bacterial agent is as follows: The activated yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus were inoculated into the culture medium respectively and cultured for 24 hours to obtain a seed inoculum, and then the yeast, Bacillus subtilis, Lactobacillus rhamnosus and Pediococcus pentosaceus seed inoculum were mixed in a mass ratio of 1-2:3-5:2-4:2-4 to obtain a composite inoculum; Among them, the culture temperature of yeast is 28-30℃, the culture temperature of Bacillus subtilis is 37-42℃; the culture temperature of Lactobacillus rhamnosus and Pediococcus pentosaceus are both 30-37℃, and they are cultured in a closed environment.

5. The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal according to claim 4, characterized in that: The culture medium comprises the following components in parts by weight: 30-50 parts of soybean meal, 10-20 parts of bran, 5-15 parts of corn flour, 1-5 parts of peptone and 10-15 parts of glucose, and water is added to the culture medium to achieve a solid content of 30wt%-40wt%.

6. The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal according to claim 4, characterized in that: The effective viable count of yeast in the seed inoculant is (1.5-2)×10 8 CFU / g, the effective viable count of Bacillus subtilis is (1-1.5)×10 9 CFU / g, the effective viable count of Lactobacillus rhamnosus is (1-1.5)×10 9 CFU / g, the effective viable count of Pediococcus pentosaceus is (2-3)×10 9 CFU / g.

7. The bacterial-enzyme synergistic fermentation method for removing anti-nutritional factors in soybean meal according to claim 1, characterized in that: The 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.

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