Fermentation enzymatic soybean meal and preparation method and application thereof

Abstract

The present application relates to a kind of fermented enzymatic soybean meal and its preparation method and application, belong to the field of feed biotechnology processing.The preparation method includes: soybean meal raw material is carried out warm water soaking and ultrasonic treatment physical pretreatment;After pretreatment, inoculate Bacillus natto and carry out first-stage solid fermentation;After cooling, inoculate Lactobacillus plantarum and carry out second-stage solid fermentation until pH drops to 4.0-4.5;After heating, first join acidic protease and carry out first-step enzymolysis, then join neutral protease and carry out second-step enzymolysis;Drying is finished product.The present application is effectively degraded trypsin inhibitor and soybean antigen protein in soybean meal by physical pretreatment combined with segmented temperature control fermentation and step-by-step enzymolysis process, significantly improve the content of small peptide, while avoiding the loss of nutrients caused by heat treatment, product palatability is good.The fermented enzymatic soybean meal prepared by the present application can be applied to piglets, calves, aquatic animals and pet feed as high-quality protein raw material.

Description

Technical Field

[0001] This invention relates to the field of feed biotechnology processing, and more particularly to a fermented enzymatic hydrolysis of soybean meal, its preparation method, and its application. Background Technology

[0002] Soybean meal is a byproduct of soybean oil extraction through pressing or leaching. It contains 40%–50% crude protein and is rich in various essential amino acids, making it the most widely used and consumed plant protein raw material in the feed industry. my country is the world's largest producer and consumer of soybean meal, with an annual output exceeding 70 million tons. The safe and efficient utilization of soybean meal is directly related to the security of feed resource supply and the economic benefits of the livestock industry. However, soybean meal naturally contains various anti-nutritional factors such as trypsin inhibitors and soybean antigens, which limit its full potential as feed. Therefore, appropriate processing of soybean meal to eliminate or neutralize these anti-nutritional factors is an important research direction in the field of feed processing technology.

[0003] Currently, the industrial approach to eliminating anti-nutritional factors in soybean meal mainly employs three technical routes: heat treatment, mechanical-physical methods, and biological methods. Heat treatment methods, such as puffing and baking, can denature and inactivate most anti-nutritional factors, but these methods can easily cause Maillard reactions, leading to the loss of essential amino acids such as lysine. Furthermore, excessive heat treatment can cause excessive denaturation of proteins, reducing their digestibility and utilization.

[0004] Mechanical and physical methods, such as crushing and extrusion, break down the cell walls and protein aggregation structures of soybean meal through physical force, but they do not directly destroy the chemical structure of nutritional factors, thus limiting their effectiveness.

[0005] Biological methods include microbial fermentation and enzymatic hydrolysis. Microbial fermentation involves inoculating probiotics to ferment and utilize the enzymes produced by microbial metabolism to degrade anti-nutritional factors. Enzymatic hydrolysis involves directly adding exogenous protease preparations for enzymatic hydrolysis. However, these methods still have shortcomings: the optimal growth temperatures of different functional strains in microbial fermentation vary, making it difficult to maintain constant temperature fermentation throughout the process, resulting in limited fermentation efficiency; the action targets of a single protease system in enzymatic hydrolysis are limited, and the enzymatic hydrolysis process easily produces bitter substances; when microbial fermentation and enzymatic hydrolysis are combined, the acid produced during fermentation can easily lead to a decrease in the system pH, which will inhibit the activity of exogenous proteases and affect the enzymatic hydrolysis efficiency. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, this invention provides a fermented enzymatic hydrolyzed soybean meal, its preparation method, and its application. This method, through physical pretreatment combined with segmented temperature-controlled fermentation and stepwise enzymatic hydrolysis, can effectively degrade trypsin inhibitors and antigenic proteins in soybean meal, significantly increasing the content of small peptides, while avoiding nutrient loss caused by heat treatment. The prepared fermented enzymatic hydrolyzed soybean meal exhibits good palatability and digestibility.

[0007] The objective of this invention can be achieved through the following technical solutions: In one aspect, a method for preparing fermented enzymatically hydrolyzed soybean meal is provided, comprising the following steps: (1) Physical pretreatment: Soak the soybean meal raw material in warm water first, and then treat it with ultrasound; (2) First stage of fermentation: The pretreated soybean meal is inoculated with Bacillus natto and fermented in solid state at 37-40℃; (3) Second stage fermentation: After the first stage fermentation is completed, the temperature is lowered to 28-32℃, Lactobacillus plantarum is inoculated, and solid-state fermentation continues until the pH of the material drops to 4.0-4.5; (4) Stepwise enzymatic hydrolysis: After the second stage of fermentation is completed, the temperature is raised, acidic protease is added first for the first step of enzymatic hydrolysis, and then neutral protease is added for the second step of enzymatic hydrolysis. (5) Drying: Dry the enzymatically hydrolyzed material to obtain fermented enzymatically hydrolyzed soybean meal.

[0008] Physical pretreatment fully exposes the proteins, creating conditions for subsequent degradation; during the first stage of fermentation, the protease secreted by Bacillus natto initially degrades large protein molecules, reducing the burden of subsequent enzymatic hydrolysis; during the second stage of fermentation, Lactobacillus plantarum produces acid, lowering the pH of the material to 4.0-4.5, providing a suitable environment for acidic proteases; stepwise enzymatic hydrolysis allows the two proteases to efficiently hydrolyze residual antigen proteins and trypsin inhibitors successively under their respective optimal pH conditions.

[0009] Further, in step (1), the temperature of the warm water soaking is 50-60℃ and the time is 30-60min; the power of the ultrasonic treatment is 200-400W, the frequency is 20-40kHz, and the time is 10-30min.

[0010] Furthermore, in step (1), the soybean meal raw material is pretreated by adding water to adjust the moisture content to 40%-45%.

[0011] Further, in step (2), the inoculation amount of Bacillus natto is 5%-8% of the soybean meal mass, and the fermentation time is 36-48h; during the fermentation process, turning or forced ventilation is carried out to control the material temperature to not exceed 42℃.

[0012] Furthermore, in step (3), the inoculation amount of Lactobacillus plantarum is 2%-4% of the soybean meal mass, and the fermentation time is 18-30h.

[0013] Furthermore, in step (4), the specific operation of the stepwise enzymatic hydrolysis is as follows: (a) First step of enzymatic hydrolysis: Add acidic protease and hydrolyze at 45-55℃ for 6-8 hours; (b) Second enzymatic hydrolysis: After the first enzymatic hydrolysis is completed, add alkaline solution to adjust the pH to 7.0-7.5, add neutral protease, and continue enzymatic hydrolysis at 45-55℃ for 6-8 hours.

[0014] Further, in step (4), the amount of acidic protease added is 3750-5000 U / g soybean meal, and the amount of neutral protease added is 3600-5000 U / g soybean meal.

[0015] Furthermore, in step (5), the drying is low-temperature drying below 60°C until the moisture content of the material is ≤10%.

[0016] In a second aspect, a fermented enzymatically hydrolyzed soybean meal is provided, which is prepared by the fermented enzymatically hydrolyzed soybean meal preparation method described in the first aspect.

[0017] Thirdly, the application of the fermented enzymatically hydrolyzed soybean meal described in the second aspect as a protein raw material for animal feed, wherein the animal is a young mammal, aquatic animal, or pet.

[0018] The fermented enzymatically hydrolyzed soybean meal prepared by this invention can be used as a high-quality plant protein raw material in animal feed. The animals include, but are not limited to: young mammals, such as piglets, calves, lambs, and puppies; aquatic animals, such as fish, shrimp, and crabs; and pets, such as dogs and cats.

[0019] The beneficial effects of this invention are as follows: (1) The present invention adopts a combined physical pretreatment of soaking in warm water and then ultrasonic treatment, and the temperature is controlled below 60℃ throughout the process, which can avoid the loss of nutrients caused by heat treatment and improve the enzymatic hydrolysis performance of soybean meal.

[0020] First, the soybean meal is soaked in warm water to allow it to fully absorb water and swell, loosening its protein structure. Then, it undergoes ultrasonic treatment, using the physical disruption of cell walls by ultrasound to further break down the cell walls and protein aggregates. The synergistic effect of these two processes effectively exposes the proteins encapsulated within the cell walls, increasing the likelihood of protease contact with the substrate during subsequent fermentation and enzymatic hydrolysis. This provides more cleavage sites, thereby improving hydrolysis efficiency.

[0021] Compared with Comparative Examples 1, 2, and 3, the lysine content of the embodiments of the present invention is 5.9-6.1 g / 100 g protein, and the degradation rate of trypsin inhibitor is over 93%.

[0022] (2) The present invention adopts a staged temperature-controlled fermentation strategy of first high-temperature aerobic fermentation of Bacillus natto, and then microaerobic fermentation of Lactobacillus plantarum after cooling, which can effectively improve the problem that it is difficult to take into account the suitable temperature requirements of different strains during the whole constant temperature fermentation.

[0023] The fermentation process was divided into two temperature stages: the first stage involved aerobic fermentation using Bacillus natto at 37-40℃, allowing the Bacillus natto to metabolize efficiently and secrete large amounts of proteases, initially degrading macromolecular antigenic proteins and trypsin inhibitors in the soybean meal. After the first stage fermentation, the temperature was lowered to 28-32℃ before inoculation with Lactobacillus plantarum for the second stage fermentation, allowing Lactobacillus plantarum to proliferate effectively and produce acid at the same suitable temperature. Simultaneously, the oxygen consumed by Bacillus natto during the first aerobic fermentation stage created favorable conditions for the microaerobic fermentation of Lactobacillus plantarum in the second stage. This strategy of fermenting before inoculation also avoided the inhibition of Bacillus natto's enzyme activity caused by premature acid production and pH drop. Through this staged temperature control strategy, the two functional bacteria completed their main functions within their respective suitable temperature windows, helping to avoid the problem of limited activity of one strain due to unsuitable temperature during constant-temperature fermentation.

[0024] Compared with Comparative Examples 4 and 5, the pH of the embodiments of the present invention can be reduced to 4.0-4.5, and the degradation rate of trypsin inhibitors reaches more than 93%.

[0025] (3) The stepwise enzymatic hydrolysis strategy of first acidic protease and then neutral protease can effectively improve the problem of acid production during fermentation inhibiting the activity of exogenous protease, while allowing both proteases to exert high catalytic efficiency under their respective suitable pH conditions.

[0026] After the second stage of fermentation, the acidic environment of pH 4.0-4.5 naturally formed by *Lactobacillus plantarum* fermentation is utilized directly to add acidic protease for the first step of enzymatic hydrolysis. This pH condition is within the effective activity range of the acidic protease, requiring virtually no external acid adjustment and reducing the use of chemical reagents. After the acidic protease hydrolysis is complete, alkali solution is added to adjust the pH to 7.0-7.5, and neutral protease is added for the second step of enzymatic hydrolysis, allowing the neutral protease to exert high catalytic efficiency under its suitable pH conditions. Through this stepwise enzymatic hydrolysis strategy, the two proteases achieve efficient enzymatic hydrolysis sequentially under their respective suitable pH conditions, effectively hydrolyzing the antigenic epitopes of β-conglycinin and the peptide bond structures of trypsin inhibitors.

[0027] Compared with Comparative Examples 6, 7, and 8, the small peptide content of the embodiments of the present invention is 24.8%-27.6%, the degradation rate of β-conglycinin is 92.1%-95.3%, and the product has no bitter taste. Detailed Implementation

[0028] To further illustrate the technical means and effects of the present invention in achieving the intended purpose, the following detailed description of the specific implementation methods, structures, features and effects of the present invention, in conjunction with preferred embodiments, is provided below.

[0029] Sources of bacterial strains and enzyme preparations in the examples Bacillus subtilis natto and Lactobacillus plantarum are available from Cangzhou Wangfa Biotechnology Research Institute Co., Ltd. The products are Bacillus subtilis natto raw powder (live bacteria content ≥2×10⁻⁶). 9 CFU / g) and Lactobacillus plantarum progeny powder (live bacteria content ≥1×10⁻⁶) 10 CFU / g).

[0030] Acidic protease (CAS No. 9025-49-4, solid feed grade, enzyme activity ≥60,000 U / g) and neutral protease (CAS No. 9068-59-1, solid feed grade, enzyme activity ≥60,000 U / g) can be purchased from Ningxia Xiasheng Industrial Group Co., Ltd., with product codes SDG-2422 and SDG-2456 respectively.

[0031] Seed liquid preparation in the examples Preparation of Bacillus natto seed culture: Inoculate Bacillus natto original bacterial powder at a ratio of 1:10 into sterilized LB liquid medium and culture with shaking at 30-32℃ and 150 rpm for 13-16 hours to obtain Bacillus natto seed culture (with a viable count of approximately 1×10⁻⁶). 8 (CFU / mL).

[0032] Preparation of *Lactobacillus plantarum* seed culture: *Lactobacillus plantarum* original bacterial powder was inoculated into sterilized MRS liquid medium at a ratio of 1:10 and incubated statically at 36-37℃ for 16-20 hours to obtain *Lactobacillus plantarum* seed culture (with a viable count of approximately 1×10⁻⁶). 8 (CFU / mL).

[0033] Example 1 Take 100 parts of soybean meal, grind it through a 40-mesh sieve, and adjust the moisture content to 42% with water. Soak it in a 55℃ water bath for 45 minutes, then perform ultrasonic treatment at 300W power and 30kHz frequency for 20 minutes. Cool the material to 38℃, inoculate with 6.5 parts of Bacillus natto seed liquid (inoculation amount 6.5%), with a material-to-water ratio of 1:1, and ferment in a solid state at 38℃ for 42 hours, turning the material every 6 hours to control the temperature below 42℃. After the first stage of fermentation, cool to 30℃, inoculate with 3 parts of Lactobacillus plantarum seed liquid (inoculation amount 3%), and continue fermentation for 24 hours until the pH of the material drops to 4.2. Raise the temperature to 50℃, add 4500U / g soybean meal of acidic protease, and enzymatically hydrolyze at 50℃ for 7 hours. After the first stage of enzymatic hydrolysis, add alkali solution to adjust the pH to 7.2, add 4200U / g soybean meal of neutral protease, and continue enzymatic hydrolysis at 50℃ for 7 hours. After enzymatic hydrolysis, the soybean meal is dried at a temperature below 60°C until the moisture content is ≤10%.

[0034] Example 2 Take 100 parts of soybean meal, grind it through a 40-mesh sieve, and adjust the moisture content to 40%. Soak it in a 50℃ water bath for 60 minutes, then perform ultrasonic treatment at 200W power and 20kHz frequency for 30 minutes. Cool the material to 37℃, inoculate it with 5 parts of Bacillus natto seed liquid (inoculation amount 5%), with a material-to-water ratio of 1:1, and ferment it in a solid state at 37℃ for 48 hours, turning it every 4 hours to control the material temperature to not exceed 42℃. After the first stage of fermentation, cool it to 28℃, inoculate it with 2 parts of Lactobacillus plantarum seed liquid (inoculation amount 2%), and continue fermentation for 30 hours until the pH of the material drops to 4.5. Raise the temperature to 45℃, add 3750U / g soybean meal of acidic protease, and enzymatically hydrolyze it at 45℃ for 8 hours. After the first stage of enzymatic hydrolysis, add alkali solution to adjust the pH to 7.0, add 3600U / g soybean meal of neutral protease, and continue enzymatic hydrolysis at 45℃ for 8 hours. After enzymatic hydrolysis, the soybean meal is dried at a temperature below 60°C until the moisture content is ≤10%.

[0035] Example 3 Take 100 parts of soybean meal, grind it through a 40-mesh sieve, and adjust the moisture content to 45% with water. Soak it in a 60℃ water bath for 30 minutes, then perform ultrasonic treatment at 400W power and 40kHz frequency for 10 minutes. Cool the material to 40℃, inoculate with 8 parts of Bacillus natto seed liquid (8% inoculum), with a material-to-water ratio of 1:1, and ferment in a solid state at 40℃ for 36 hours, turning the material every 6 hours to control the temperature not to exceed 42℃. After the first stage of fermentation, cool to 32℃, inoculate with 4 parts of Lactobacillus plantarum seed liquid (4% inoculum), and continue fermentation for 18 hours until the pH of the material drops to 4.0. Raise the temperature to 55℃, add 5000U / g soybean meal of acidic protease, and enzymatically hydrolyze at 55℃ for 6 hours. After the first stage of enzymatic hydrolysis, add alkali solution to adjust the pH to 7.5, add 5000U / g soybean meal of neutral protease, and continue enzymatic hydrolysis at 55℃ for 6 hours. After enzymatic hydrolysis, the soybean meal is dried at a temperature below 60°C until the moisture content is ≤10%.

[0036] The fermented enzymatically hydrolyzed soybean meal of this invention can be used as a functional protein source in piglet feed. It is recommended to add 10%-25% of the soybean meal. Adding fermented enzymatically hydrolyzed soybean meal to piglet diets can improve the daily weight gain and feed conversion rate of piglets, reduce the frequency of diarrhea, and improve the growth performance and immune function of piglets. Farmers can adjust the amount of soybean meal according to the age and health status of the piglets.

[0037] Application Example 1: Application of Fermented Enzymatic Hydrolyzed Soybean Meal in Piglet Feed The fermented enzymatically hydrolyzed soybean meal prepared in Example 1 was added to the basal diet of piglets at a ratio of 15% (by mass), mixed evenly, and formulated into a complete feed for feeding weaned piglets. The basal diet uses corn and wheat bran as the main energy sources and contains appropriate amounts of fishmeal, premixed feed, and other ingredients, so that the crude protein content of the final complete feed is not less than 18%.

[0038] Application Example 2: Application of Fermented Enzymatic Hydrolyzed Soybean Meal in Piglet Feed The fermented enzymatically hydrolyzed soybean meal prepared in Example 2 was added to the basal diet of piglets at a ratio of 10% (by mass percentage), mixed evenly, and formulated into a complete feed.

[0039] Application Example 3: Application of Fermented Enzymatic Hydrolyzed Soybean Meal in Piglet Feed The fermented enzymatically hydrolyzed soybean meal prepared in Example 3 was added to the basal diet of piglets at a ratio of 20% (by mass percentage), mixed evenly, and formulated into a complete feed.

[0040] The fermented enzymatically hydrolyzed soybean meal of this invention can be used as a functional additive in aquatic feed and pet food, with a recommended addition ratio of 2%-5%. When it is necessary to replace fishmeal or milk protein as the main protein source, the addition ratio can be appropriately increased according to the actual formula requirements (up to 15%-30% in aquatic feed and 12%-15% in pet food), but the amino acid balance needs to be adjusted accordingly.

[0041] Application Example 4: Application of Fermented Enzymatic Hydrolyzed Soybean Meal in Aquatic Feed The fermented enzymatically hydrolyzed soybean meal prepared in Example 1 was added to the compound feed of aquatic animals, such as shrimp, crab, and fish, at a ratio of 2%-5%, and fed after thorough mixing. Fermented enzymatically hydrolyzed soybean meal is characterized by strong hydrophilicity and high water retention, which has an appealing effect on aquatic animals and can improve the palatability and stability of the feed in water.

[0042] Application Example 5: Application of Fermented Enzymatic Hydrolyzed Soybean Meal in Pet Food The fermented enzymatically hydrolyzed soybean meal prepared according to any one of Examples 1-3 is added to the daily diet of pets, such as dogs and cats, at a ratio of 2%-5%, and mixed with other raw materials (meat, grains, vegetables, etc.) to make extruded pelleted food or wet food. Fermented enzymatically hydrolyzed soybean meal is rich in functional small peptides and probiotic metabolites, which are beneficial to the nutritional balance of the pet's intestines and can improve digestive and absorptive functions.

[0043] Comparative Example 1 Based on Example 1, heat treatment (160℃, 30min) was used instead of warm water soaking and ultrasonic treatment for physical pretreatment, and the rest was the same as in Example 1.

[0044] Comparative Example 2 Based on Example 1, only warm water soaking was used for physical pretreatment, and the rest was the same as in Example 1.

[0045] Comparative Example 3 Based on Example 1, only ultrasonic treatment was used for physical pretreatment, and the rest was the same as in Example 1.

[0046] Comparative Example 4 Based on Example 1, Bacillus natto seed liquid (inoculation amount 6.5%) was first inoculated and solid-state fermented at 38°C for 42 hours; after fermentation, Lactobacillus plantarum seed liquid (inoculation amount 3%) was inoculated at 38°C and fermented for another 24 hours, with the rest being the same as in Example 1.

[0047] Comparative Example 5 Based on Example 1, Bacillus natto seed liquid (6.5% inoculation amount) and Lactobacillus plantarum seed liquid (3% inoculation amount) were simultaneously inoculated and solid-state fermented at 38°C for 42 hours; then the temperature was lowered to 30°C and fermentation continued for 24 hours, with the rest being the same as in Example 1.

[0048] Comparative Example 6 Based on Example 1, acidic protease (4500 U / g soybean meal) and neutral protease (420 U / g soybean meal) were added simultaneously, and the enzymes were enzymatically hydrolyzed at 50°C for 14 hours. The rest of the process was the same as in Example 1.

[0049] Comparative Example 7 Based on Example 1, only acidic protease (4500 U / g soybean meal) was added, and the mixture was enzymatically hydrolyzed at 50°C for 7 hours. The rest of the process was the same as in Example 1.

[0050] Comparative Example 8 Based on Example 1, an alkaline solution was added to adjust the pH to 7.2, and only neutral protease (4200 U / g soybean meal) was added. The mixture was enzymatically hydrolyzed at 50°C for 7 hours, with the rest being the same as in Example 1.

[0051] Comparative Example 9 Based on Example 1, the order of physical pretreatment was adjusted to first perform ultrasonic treatment, then soak in warm water, and the rest was the same as in Example 1.

[0052] Comparative Example 10 Based on Example 1, fermentation was terminated when the pH of the second stage fermentation material dropped to 5.0, and the rest was the same as in Example 1.

[0053] Comparative Example 11 Based on Example 1, the order of stepwise enzymatic hydrolysis was adjusted so that neutral protease was added first for the first step of enzymatic hydrolysis, and then acidic protease was added for the second step of enzymatic hydrolysis, with the rest being the same as in Example 1.

[0054] Performance testing I. Testing Methods (1) Detection of anti-nutritional factor content The trypsin inhibitor content was determined according to GB / T 46002-2025 standard; the contents of glycinin and β-conglycinin were determined using an indirect competitive ELISA kit. The degradation rate was calculated using the following formula: Degradation rate (%) = (content of anti-nutritional factors in untreated soybean meal - content of anti-nutritional factors in fermented and enzymatically hydrolyzed soybean meal) / content of anti-nutritional factors in untreated soybean meal × 100%.

[0055] (2) Detection of protein degradation degree The content of small peptides and the molecular weight distribution of proteins were determined by high-performance gel filtration chromatography (HPLC-GFC) according to Appendix A of GB / T 22492-2008.

[0056] The chromatographic conditions were as follows: TSK-GEL G2000SWXL column (300mm×7.8mm, 5μm), mobile phase of acetonitrile + water + trifluoroacetic acid, detection wavelength of 220nm, flow rate of 0.5mL / min, injection volume of 20μL, and column temperature of room temperature.

[0057] Small peptides are defined as peptides with a molecular weight of less than 5 kDa.

[0058] The molecular weight distribution of proteins was determined using 30 kDa as the criterion for whether macromolecular antigen proteins were sufficiently degraded. When the proportion of proteins with a molecular weight ≥ 30 kDa decreased significantly, it indicated that glycinin and β-conglycinin had been effectively degraded.

[0059] The lysine content was determined according to GB / T 18246-2019 standard, using acid hydrolysis combined with an automatic amino acid analyzer.

[0060] (3) Detection of fermentation product characteristics pH values ​​were determined using a pH meter. Ten samples were taken and mixed with 90 parts deionized water. After stirring thoroughly, the mixture was allowed to stand, and the pH value of the supernatant was measured using a pH meter.

[0061] (4) Sensory quality evaluation Take samples and observe their sensory characteristics such as color, odor, and texture.

[0062] Evaluation criteria: The color is light yellow to light brown and uniform; the odor is a mild sour fragrance, without moldy, rotten, or obvious bitter taste; the texture is powdery, without lumps or mold.

[0063] II. Test Results Table 1. Test results of Examples 1-3

[0064] Table 2-1 Test results of Comparative Examples 1-5

[0065] Table 2-2 Test results of Comparative Example 6-11

[0066] Table 1 shows that the degradation rates of trypsin inhibitors, 90.8%-94.1% of daidzein, and 92.1%-95.3% of β-congaidzein in the fermented enzymatically hydrolyzed soybean meal prepared in Examples 1-3 were high, indicating that the present invention has a highly efficient degradation effect on the main anti-nutritional factors in soybean meal. The content of small peptides was 24.8%-27.6%, the proportion of proteins with a molecular weight ≥30KDa was no higher than 18%, and the lysine content was 5.9-6.1 g / 100g protein, indicating that while large protein molecules were fully degraded, nutrient loss caused by heat treatment was effectively avoided. The pH was 4.0-4.5, the product was light yellow, had a mild sour aroma, no bitterness, and good sensory quality.

[0067] Tables 2-1 and 2-2 show that Comparative Example 1, which used heat treatment instead of warm water soaking and ultrasonic treatment, had lower degradation rates of all anti-nutritional factors than Example 1, and its lysine content decreased to 4.7 g / 100 g protein. The product was dark brown and had a burnt smell. Comparative Example 2, which only used warm water soaking, and Comparative Example 3, which only used ultrasonic treatment, had lower levels of all indicators than Example 1, indicating a synergistic effect between warm water soaking and ultrasonic treatment. Comparative Example 4, under a constant temperature of 38°C throughout the process, had a pH that decreased to 5.1, and all degradation indicators were lower than those of Example 1, indicating that segmented temperature-controlled fermentation is key to ensuring effective acid production and subsequent enzymatic hydrolysis efficiency of *Lactobacillus plantarum*.

[0068] In Comparative Example 5, simultaneous inoculation led to premature acid production by acid-producing bacteria, inhibiting the activity of enzyme-producing bacteria; its trypsin inhibitor degradation rate was 70.6%. In Comparative Example 6, during simultaneous compound enzymatic hydrolysis, the activity of neutral protease was inhibited under acidic conditions, and all indicators were lower than in Example 1. Comparative Example 7 used only acidic protease, and Comparative Example 8 used only neutral protease; their degradation effects were lower than in Example 1, and the products all had a bitter taste. Comparative Example 9 used a treatment method of first ultrasonication and then warm water soaking; the trypsin inhibitor degradation rate was 86.7%, the β-conglycin degradation rate was 81.5%, and the small peptide content was 21.8%. In Comparative Example 10, the second stage of fermentation was terminated when the pH was controlled at 5.0; its trypsin inhibitor degradation rate was 78.8%, the β-conglycin degradation rate was 75.2%, and the small peptide content was 20.8%. Comparative Example 11 had its enzymatic hydrolysis sequence adjusted to neutral protease first, followed by acidic protease. The degradation rate of trypsin inhibitor was 82.3%, the degradation rate of β-conglycinin was 76.8%, the content of small peptides was 21.5%, and the product had a slightly bitter taste.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing fermented enzymatically hydrolyzed soybean meal, characterized in that, Includes the following steps: (1) Physical pretreatment: Soak the soybean meal raw material in warm water first, and then treat it with ultrasound; (2) First stage fermentation: The pretreated soybean meal is inoculated with Bacillus natto and fermented in solid state at 37-40℃; (3) Second stage fermentation: After the first stage fermentation is completed, the temperature is lowered to 28-32℃, Lactobacillus plantarum is inoculated, and solid-state fermentation continues until the pH of the material drops to 4.0-4.5; (4) Stepwise enzymatic hydrolysis: After the second stage of fermentation is completed, the temperature is raised, acidic protease is added first for the first step of enzymatic hydrolysis, and then neutral protease is added for the second step of enzymatic hydrolysis. (5) Drying: Dry the enzymatically hydrolyzed material to obtain fermented enzymatically hydrolyzed soybean meal.

2. The method for preparing fermented enzymatically hydrolyzed soybean meal according to claim 1, characterized in that, In step (1), the temperature of the warm water soak is 50-60℃ and the time is 30-60min; the power of the ultrasonic treatment is 200-400W, the frequency is 20-40kHz, and the time is 10-30min.

3. The method for preparing fermented enzymatically hydrolyzed soybean meal according to claim 1, characterized in that, In step (1), the soybean meal raw material is pretreated by adding water to adjust the moisture content to 40%-45%.

4. The method for preparing fermented enzymatically hydrolyzed soybean meal according to claim 1, characterized in that, In step (2), the inoculation amount of Bacillus natto is 5%-8% of the soybean meal mass, and the fermentation time is 36-48h; during the fermentation process, turning or forced ventilation is carried out to control the material temperature to not exceed 42℃.

5. The method for preparing fermented enzymatically hydrolyzed soybean meal according to claim 1, characterized in that, In step (3), the inoculation amount of Lactobacillus plantarum is 2%-4% of the soybean meal mass, and the fermentation time is 18-30h.

6. The method for preparing fermented enzymatically hydrolyzed soybean meal according to claim 1, characterized in that, In step (4), the specific operation of the stepwise enzymatic hydrolysis is as follows: (a) First step of enzymatic hydrolysis: Add acidic protease and hydrolyze at 45-55℃ for 6-8 hours; (b) Second enzymatic hydrolysis: After the first enzymatic hydrolysis is completed, add alkaline solution to adjust the pH to 7.0-7.5, add neutral protease, and continue enzymatic hydrolysis at 45-55℃ for 6-8 hours.

7. The method for preparing fermented enzymatically hydrolyzed soybean meal according to claim 6, characterized in that, In step (4), the amount of acidic protease added is 3750-5000 U / g soybean meal, and the amount of neutral protease added is 3600-5000 U / g soybean meal.

8. The method for preparing fermented enzymatically hydrolyzed soybean meal according to claim 1, characterized in that, In step (5), the drying is low-temperature drying below 60°C until the moisture content of the material is ≤10%.

9. A fermented enzymatically hydrolyzed soybean meal, characterized in that, It is prepared by the fermentation and enzymatic hydrolysis method of any one of claims 1-8.

10. The application of the fermented enzymatically hydrolyzed soybean meal according to claim 9 as a protein raw material for animal feed, wherein the animal is a young mammal, aquatic animal, or pet.

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