A solid-state fermentation method for bean dregs, fermented bean dregs product and application

Abstract

The application relates to a bean dregs solid-state fermentation method and a fermented bean dregs product and application, relates to the feed processing technical field, and the fermentation method comprises the following steps: S1, mixing dry bean dregs and bran to prepare a fermentation substrate, wherein the initial water content of the fermentation substrate is controlled to be 35%-50%; S2, inoculating a composite probiotic preparation into the fermentation substrate, wherein the composite probiotic preparation comprises Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum; S3, adding a composite protease and a mannanase into the fermentation substrate while inoculating the composite probiotic preparation, wherein the adding amount of the composite protease is not less than 50000 U / kg; and S4, sealing the fermentation substrate added with the composite probiotic preparation, the composite protease and the mannanase to carry out anaerobic fermentation, so as to obtain the fermented bean dregs product. The fermentation method solves the problems of non-systematic parameter optimization and missing protease threshold control in the prior art, and is controllable, safe and efficient.

Description

Technical Field

[0001] This application relates to the field of feed processing technology, and in particular to a method for solid-state fermentation of soybean residue, a fermented soybean residue product obtained by the method, and the application of the product in animal feed. Background Technology

[0002] Soybean dregs are a major byproduct of soybean processing, including tofu, tofu skin, dried bean curd sticks, and soy milk. my country produces approximately 20 million tons of wet soybean dregs annually. While the dry matter of soybean dregs is rich in dietary fiber (50%-60%), protein (20%-30%), and lipids (approximately 10%), its fresh product has an extremely high water content (over 80%), making it highly susceptible to spoilage. Furthermore, it contains anti-nutritional factors such as trypsin inhibitors, glycinin, and β-conglycinin, resulting in poor palatability, difficulties in storage and transportation, and low nutrient bioavailability. As a result, the vast majority is discarded, causing serious resource waste and environmental pollution.

[0003] In existing technologies, solid-state fermentation has been used for the value-added processing of soybean residue. However, systematic and quantitative research on the optimization of process parameters is still lacking. In particular, the primary and secondary relationships of the effects of various parameters on fermentation quality are unclear, leading to large fluctuations in fermentation effects between different batches. In addition, the amount of exogenous enzyme preparations added lacks precise threshold control, and at certain levels, it may actually increase the exposure of antigenic proteins in the product, affecting feed safety. Therefore, it is necessary to develop a parameter-controllable, safe, and efficient solid-state fermentation process for soybean residue. Summary of the Invention

[0004] This application aims to provide a solid-state fermentation method for soybean residue with controllable process parameters and efficient synergistic effect between bacteria and enzymes, in order to solve technical problems such as unsystematic parameter optimization and lack of protease threshold control in the prior art.

[0005] In a first aspect, this application provides a method for solid-state fermentation of soybean residue, comprising the following steps: S1. Mix dried soybean residue with wheat bran to prepare a fermentation substrate, wherein the initial moisture content of the fermentation substrate is controlled to be 35%-50%; S2. Inoculate the fermentation substrate with a compound probiotic preparation, wherein the compound probiotic preparation includes Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum; S3. While inoculating the compound probiotic preparation, add compound protease and mannanase to the fermentation substrate, wherein the amount of compound protease added is not less than 50,000 U / kg. S4. The fermentation substrate containing compound probiotic preparation, compound protease and mannanase is sealed and subjected to anaerobic fermentation to obtain fermented soybean residue product.

[0006] Furthermore, the amount of mannanase added is 50,000-75,000 U / kg.

[0007] Furthermore, the mass ratio of the dried soybean residue to the wheat bran is (28-57):(43-71).

[0008] Furthermore, the inoculation amount of the compound probiotic preparation is 2-4 g / kg.

[0009] Furthermore, in the compound probiotic preparation, the number of live Saccharomyces cerevisiae is ≥3×10⁻⁶. 8 CFU / g, viable count of Bacillus subtilis ≥1×10⁻⁶ 8 CFU / g, viable count of Lactobacillus plantarum ≥ 4 × 10⁻⁶ 8 CFU / g.

[0010] Furthermore, the anaerobic fermentation temperature is 35-43℃, and the fermentation time is 48-96 h.

[0011] Furthermore, the anaerobic fermentation temperature is 37-40℃, and the addition amount of the complex protease and mannanase is 50000 U / kg.

[0012] Secondly, this application provides a fermented soybean residue product, prepared by the above method, having a pH ≤ 5.0, a total acid content ≥ 3.5%, a small peptide content ≥ 25%, an acid-soluble protein content ≥ 4.0%, and a live lactic acid bacteria count ≥ 10. 8 CFU / g.

[0013] Thirdly, this application provides the application of the above-mentioned fermented soybean residue product in the preparation of animal feed.

[0014] Through one or more embodiments of the above embodiments of the present invention, at least the following technical effects can be achieved: 1. This application, through systematic response surface methodology analysis, reveals that the initial moisture content of the fermentation substrate is a core determinant affecting fermentation quality. In the regression model for acid-soluble proteins, the F-value for the initial moisture content factor is 512.21, the F-value for fermentation temperature is 13.07, and the F-value for inoculum size is only 1.39. The influence of moisture content is approximately 39 times that of temperature and approximately 368 times that of inoculum size, respectively. This finding provides clear priority guidance for parameter control in production practice, and can significantly improve process controllability and batch stability.

[0015] 2. Through comparative research, this application found that the minimum effective addition threshold for the complex protease is 50,000 U / kg. When the protease addition was 25,000 U / kg, the ELISA-detected glycinin content increased from 2.62 mg / g in the control group to 3.69 mg / g, and β-conglycin increased from 2.31 mg / g to 4.99 mg / g. When the addition reached 50,000 U / kg or higher, SDS-PAGE results showed that the antigen protein bands in the 10-35 kDa and 60-75 kDa molecular weight ranges were significantly cleared, effectively eliminating the potential for sensitization. When the effective addition amount of complex protease is less than 50,000 U / kg, the protease only partially cleaves the protein molecules, causing the originally embedded antigenic epitopes to be temporarily exposed. As a result, the content of glycinin and β-conglycinin detected by ELISA increases, increasing the risk of sensitization.

[0016] 3. In this application, mannanase specifically hydrolyzes mannan in the cell walls of soybean residue, degrading insoluble dietary fiber into reducing sugars, rather than converting it into soluble dietary fiber. These reducing sugars serve as readily available carbon sources utilized by probiotics, forming a synergistic pathway of "enzymatic hydrolysis of fiber → generation of reducing sugars → empowerment of microorganisms → synergistic improvement of fermentation quality." After adding 75,000 U / kg mannanase, the content of small peptides increased from 23.70% to 29.83%, and the content of acid-soluble protein increased from 3.78% to 4.77%. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a bar chart illustrating the effect of the initial moisture content of the fermentation substrate on fermentation indicators in the embodiments of this application.

[0019] Figure 2 This is an SDS-PAGE electrophoresis image of fermented soybean residue after treatment with different amounts of compound protease in the embodiments of this application.

[0020] Figure 3 This is a bar chart illustrating the effect of mannanase on the oligosaccharide composition of soybean residue in the embodiments of this application.

[0021] Figure 4 The images shown are three-dimensional surface plots and contour plots illustrating the effects of the interaction of various factors on total acid, acid-soluble proteins, and pH in the embodiments of this application. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] In the description of this invention, it should be noted that the term "and / or" indicates that at least one of the associated objects is required. For example, "A and / or B" covers three cases: only A, only B, and both A and B. The term "small peptide content" refers to the percentage of acid-soluble proteins in the total crude protein of the sample.

[0024] To address the issues of strain safety risks, unsystematic parameter optimization, and lack of threshold control for protease addition in existing solid-state fermentation processes for soybean residue, this application provides a solid-state fermentation method for soybean residue based on a non-filamentous fungal compound probiotic system, with initial moisture content as the core regulatory variable, and achieving synergistic fermentation of bacteria and enzymes by setting a protease threshold.

[0025] The technical approach of this application can be summarized as the following core steps and ideas: 1. Mix dried soybean residue and wheat bran in a specific ratio. The initial moisture content of the fermentation substrate is precisely controlled by adjusting the ratio of soybean residue to wheat bran. The initial moisture content is the core parameter that determines the fermentation quality. 2. It adopts a three-strain complex system of brewing yeast, Bacillus subtilis and Lactobacillus plantarum, which respectively perform the functions of enzyme hydrolysis, acid production and antibacterial, and flavor improvement, and is completely free of filamentous fungi; 3. Simultaneously add compound protease and mannanase. Protease degrades large molecular antigen proteins to generate small peptides, while mannanase hydrolyzes mannan to generate reducing sugars to provide energy for microorganisms. That is: enzymatic hydrolysis of fiber → generation of carbon source → empowerment of microorganisms → deep conversion of protein; 4. Under suitable temperature conditions, anaerobic fermentation is carried out for 48-96 hours to finally obtain fermented soybean residue products with high small peptide content, low anti-nutritional factor content, and rich in beneficial live bacteria.

[0026] S1. Mix dried soybean residue with wheat bran to prepare a fermentation substrate.

[0027] In this step, dried soybean residue is mixed with wheat bran to prepare a fermentation substrate, and the initial moisture content of the fermentation substrate is controlled to be 35%-50%.

[0028] Dried soybean residue is rich in protein (18%-20% crude protein, air-dried) and dietary fiber (≥9% crude fiber content), and is a high-quality natural substrate for proteases and cellulases.

[0029] Dried soybean residue from different sources varies in nutritional composition. The inventors of this application analyzed soybean residue from four different sources: The soybean residue from different sources ranged in crude protein content from 18.30% to 19.71%, and crude fiber content from 20.61% to 21.31%. The sources used in this application's fermentation experiment were: Wuhan Douxiang Juxiyuan Food Co., Ltd. (Source A, crude protein 18.30%), Hubei University of Traditional Chinese Medicine soybean residue (Source B, crude protein 18.34%), Hubei Junyuan Soybean Products Co., Ltd. (Source C, crude protein 18.42%), and Vitasoy (Wuhan) Co., Ltd. (Source D, crude protein 19.71%).

[0030] Wheat bran not only supplements the carbon source in the substrate, but more importantly, its loose and porous physical structure can improve the aeration and porosity of the fermentation substrate, avoiding local anaerobic conditions or uneven aeration caused by excessive stickiness of pure soybean residue.

[0031] The bran used in this application was sourced from Yihai Kerry (Wuhan) Feed Co., Ltd., with a crude protein content of 15.98% and a crude fiber content of 10.55%.

[0032] Furthermore, the key role of moisture content was verified through single-factor experiments with different initial moisture contents.

[0033] like Figure 1 As shown in Table 1, when the initial moisture content increases sequentially from 30% to 35%, 40%, 45%, and 50%, the various indicators exhibit the following trends: Table 1 Results of single-factor experiments with different initial moisture contents The changes in all the above indicators showed a highly significant linear positive correlation (P<0.001). A high-moisture environment is conducive to the diffusion of extracellular enzymes of microorganisms in the matrix and their contact with the substrate, accelerating the decomposition of the cell wall structure of soybean residue, thereby releasing more soluble nutrients.

[0034] In some embodiments, the initial moisture content can be any value or a range between any two of 35%, 38%, 40%, 42%, 45%, 48%, and 50%. Of course, it can also be adjusted appropriately based on the moisture content characteristics of the actual soybean residue source.

[0035] When the initial moisture content is 40%-50%, the total acid content can reach 4.73%-7.27%, the small peptide content can reach 39.50%-47.78%, and the acid-soluble protein content can reach 6.51%-7.97%. However, if the moisture content is too high (>45%), the material viscosity increases and the aeration decreases in industrial production, which is not conducive to mixing, conveying, and stacking operations. Moreover, high-moisture products are not resistant to storage. If the moisture content is below 35%, microbial metabolism is limited, the total acid content is only 2.41%-3.84%, and the small peptide content is as low as 19.82%-27.68%, resulting in unsatisfactory fermentation. Therefore, considering both fermentation quality and industrial operability, an initial moisture content of 38%-42% is preferred, with 40% being the most optimal.

[0036] The raw material proportions corresponding to different initial moisture contents are shown in the table below: Table 2. Raw material ratios and costs at different initial moisture contents In some embodiments, the mass ratio of dried soybean residue to wheat bran can be any one of the following ratios: 28:71, 36:64, 43:57, 50:50, or 57:43.

[0037] S2. Inoculate the fermentation substrate with a compound probiotic preparation, wherein the compound probiotic preparation includes Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum.

[0038] Bacillus subtilis has excellent protease production capabilities. Its secreted endopeptidases and exopeptidases can efficiently degrade large protein molecules into small peptides and free amino acids. Lactobacillus plantarum is a highly efficient lactic acid producer. The lactic acid it produces can quickly lower the pH of the system, creating an acidic environment to inhibit the proliferation of other bacteria, while also giving the product a sour and fragrant flavor. Saccharomyces cerevisiae improves flavor by producing alcohols and esters through metabolism, and generates cell proteins to increase the total protein content of the product.

[0039] The optimal growth temperature range for all three bacteria is between 30-40℃, which facilitates unified temperature management.

[0040] It should be noted that the inoculum size of the compound probiotic preparation has a significant impact on fermentation quality. The inventors of this application conducted a single-factor experiment on the inoculum size (setting five levels: 0, 1, 2, 3, and 4 g / kg), and the results are shown in Table 3. Table 3. Effects of inoculum size on the nutrient composition and beneficial bacteria content of fermented soybean residue. Data showed that the numbers of Bacillus, Lactobacillus, and Yeast all increased linearly with increasing inoculum size (P<0.05). When the inoculum size exceeded 2 g / kg, the pH began to decrease significantly, while total acid, small peptides, and acid-soluble proteins increased significantly (P<0.05).

[0041] It is understandable that a higher inoculum size is beneficial for rapidly establishing a dominant microbial community and limiting the growth of unwanted microorganisms in the early stages of fermentation. However, when the inoculum size exceeds 3 g / kg, the improvement in some nutritional indicators slows down, which is related to the increased nutrient competition among microorganisms. Considering the cost of the inoculum (each increase of 1 g / kg increases feed cost by approximately 40 yuan / ton), and taking into account both quality and economic efficiency, the inoculum size for compound probiotic preparations is 2-4 g / kg, with 2-3 g / kg being the preferred size.

[0042] In some embodiments, the number of live Saccharomyces cerevisiae in the compound probiotic preparation may be 3 × 10⁻⁶. 8 4×10 8 5×10 8 For any value in CFU / g, the viable count of Bacillus subtilis can be 1×10⁻⁶. 8 2×10 8 3×10 8 For any value in CFU / g, the viable count of *Lactobacillus plantarum* can be 4 × 10⁻⁶. 8 5×10 8 6×10 8 Any value in CFU / g.

[0043] S3. While inoculating the compound probiotic preparation, add compound protease and mannanase to the fermentation substrate.

[0044] The amount of compound protease added shall not be less than 50,000 U / kg.

[0045] Through systematic experiments, this application found that the amount of compound protease added must reach a threshold of 50,000 U / kg. The inventors of this application conducted single-factor experiments on the compound protease at four levels (0, 25,000, 50,000, and 75,000 U / kg), and the results are shown in Table 4: Table 4. Effects of Compound Protease Addition Amount on Nutritional Composition and Antigenic Protein Content of Fermented Soybean Residue As can be seen from Table 4, the amount of compound protease added can linearly increase the total acid and acid-soluble protein content.

[0046] Meanwhile, it was also found that in the ELISA quantitative detection results of antigen proteins (glycinin and β-conglycinin), the glycinin content in the 25,000 U group increased from 2.62 mg / g in the control group (0 U) to 3.69 mg / g, and the β-conglycinin content increased abnormally from 2.31 mg / g to 4.99 mg / g.

[0047] It is understandable that low-dose proteases only perform superficial cleavage of protein molecules, exposing antigenic epitopes that are originally folded and embedded within the three-dimensional structure of the protein. ELISA detection is based on the specific recognition of antigenic epitopes by antibodies. The increased exposure of epitopes leads to higher detection values, indicating a substantial increase in the exposure of allergen epitopes in the product.

[0048] Combined with SDS-PAGE electrophoresis results (such as...) Figure 2 As shown in the figure, the 50,000 U and 75,000 U groups had a significant clearing effect on antigen protein bands in the molecular weight ranges of 10-35 kDa and 60-75 kDa, indicating that at this level of addition, the protein molecules were sufficiently deeply cleaved and the antigen proteins were degraded into small molecular fragments that no longer had an intact antigen conformation.

[0049] Therefore, in production practice, if the amount of protease added is less than 50,000 U / kg, it will not only fail to effectively degrade the antigen protein, but may also increase the risk of sensitization in feeding animals due to exposure of antigenic epitopes. Accordingly, this application sets 50,000 U / kg as the lower limit of the safe threshold for the amount of compound protease added.

[0050] It should be noted that the ELISA value of glycinin in the 75,000 U group (2.64 mg / g) has returned to levels close to the control group, which is consistent with the antigen protein band clearance results shown by SDS-PAGE. The SDS-PAGE results show that the band in the 60-75 kDa region (the major subunit of β-conglycinin) was significantly weakened in both the 50,000 U and 75,000 U groups, indicating that the intact antigen protein has been effectively degraded.

[0051] In some embodiments, the amount of complex protease added can be any value among 50,000, 55,000, 60,000, 65,000, 70,000, and 75,000 U / kg, or a range between any two values.

[0052] S4. The fermentation substrate containing compound probiotic preparation, compound protease and mannanase is sealed and subjected to anaerobic fermentation to obtain fermented soybean residue product.

[0053] Soybean residue cell walls are rich in mannan-like hemicellulose, and mannanase can specifically hydrolyze the β-1,4-glycosidic bonds of the mannan backbone. This application conducted single-factor experiments on mannanase at four levels (0, 25000, 50000, and 75000 U / kg), and the results are shown in Tables 5 and 6. Table 5. Effects of mannanase addition on nutritional indicators of fermented soybean residue Table 6. Effect of mannanase addition on dietary fiber in fermented soybean residue As shown in Table 6, mannanase can significantly reduce the content of insoluble dietary fiber (IDF) and total dietary fiber (TDF), and the reduction effect is linearly related to the enzyme addition level, but has no significant effect on soluble dietary fiber (SDF).

[0054] Meanwhile, Table 5 shows that the reducing sugar content increases with the amount of mannanase added (from 1.73% to 2.65%). This indicates that mannanase directly degrades mannan into reducing sugars at the oligosaccharide / monosaccharide level, rather than remaining at the soluble polysaccharide (SDF) stage.

[0055] It is understandable that these newly generated reducing sugars act as a readily available carbon source in the fermentation system, providing directly usable energy for the growth of probiotics (especially yeasts and lactic acid bacteria). With sufficient carbon sources, microorganisms exhibit enhanced metabolic activity, promoting the synthesis of organic acids (such as lactic acid) and providing energy support for the synthesis of microbial proteins. Ultimately, this indirectly drives the conversion and accumulation of nitrogenous nutrients such as small peptides and acid-soluble proteins.

[0056] In addition, such as Figure 3 As shown, fermentation with compound probiotics can significantly reduce the sucrose, raffinose, and stachyose content in soybean residue, while increasing the reducing sugar content. Adding 50,000 and 75,000 U of mannanase can further reduce the reducing sugar and sucrose content in soybean residue. Raffinose and stachyose are anti-nutritional oligosaccharides that are not digested and absorbed by monogastric animals. After entering the hindgut, they are fermented by microorganisms, producing gas and causing bloating and diarrhea. The degradation of these substances during fermentation helps improve the feed safety of soybean residue.

[0057] Traditional methods typically rely solely on enzymes produced by microorganisms to degrade fiber and protein in soybean residue, resulting in long fermentation cycles and limited degradation. This application establishes a strong enzymatic hydrolysis environment from the early stages of fermentation by simultaneously adding exogenous proteases and mannanases.

[0058] The reason is that during the initial stage of fermentation (0-12 h), the microorganisms are still in the lag phase, their own enzyme production is limited, and the substrate degradation rate is slow; while the exogenous enzymes play a role from the inoculation time, which enables protein degradation and cellulase hydrolysis to start in advance, forming a temporal complement to the subsequent growth of the microorganisms, and significantly shortening the effective fermentation time.

[0059] In some embodiments, the amount of mannanase added can be any value or a range between any two of 25,000, 30,000, 40,000, 50,000, 60,000, and 75,000 U / kg. Preferably, the amount of mannanase added is 50,000-75,000 U / kg. Adding 50,000 U results in the highest crude protein content (16.13%), and small peptides and acid-soluble proteins are also relatively ideal, offering good cost-effectiveness.

[0060] Furthermore, anaerobic conditions are conducive to the proliferation of lactic acid bacteria and the accumulation of lactic acid, which can quickly lower the pH to inhibit the proliferation of other bacteria; at the same time, it avoids the increased energy consumption and operational complexity caused by frequent turning and aeration in aerobic fermentation, making it suitable for simplified operation in feed mills and farms.

[0061] The inventors of this application conducted a single-factor experiment on fermentation temperature (setting five levels: 31, 34, 37, 40, and 43°C), and the results are shown in Table 7: Table 7. Effect of fermentation temperature on the quality of fermented soybean residue Data showed that the highest total acid yield (4.52%) and lowest pH (4.75) were observed at 40℃, which is consistent with the optimal growth temperature range for lactic acid bacteria and Bacillus in the complex bacterial strain. At 37℃, crude protein, small peptides, and acid-soluble proteins were significantly higher than those in the 31℃ and 34℃ groups (P<0.05), especially the small peptide content, which reached the highest value of 33.29%.

[0062] If the temperature is below 34℃, the activity of both proteases and microbial metabolism is at a low level, and the content of small peptides is only 26.21%-29.68%. Therefore, considering both protein degradation efficiency and acid production capacity, the preferred fermentation temperature in this application is 37-40℃.

[0063] In some embodiments, the fermentation temperature can be any value or a range between any two of 35, 37, 38, 39, 40, 42, and 43°C. The fermentation time can be any value or a range between any two of 48, 60, 72, 84, and 96 h.

[0064] Based on the results of single-factor experiments, this application conducted a three-factor, three-level response surface methodology (RSM) experiment (moisture content 35% / 40% / 45%; inoculum size 2 / 3 / 4 g / kg; temperature 37 / 40 / 43℃) with initial moisture content (A), inoculum size (B), and fermentation temperature (C) as the response values. The factor and level design of the RSM experiment is shown in Table 8, and the experimental scheme and results are shown in Table 9.

[0065] Table 8 Factors and Levels in Response Surface Experiment Table 9 Response Surface Experiment Scheme and Results The results of the regression model variance analysis for each response value are shown in Tables 10, 11 and 12, respectively.

[0066] Table 10. Analysis of variance of the regression model for total acidity Note: "*" indicates significant (P<0.05).

[0067] Table 11. Analysis of variance of regression models for acid-soluble proteins Note: "*" indicates a significant difference (P<0.05); "**" indicates an extremely significant difference (P<0.01).

[0068] Table 12. Analysis of variance of the regression model for pH Note: "*" indicates a significant difference (P<0.05); "**" indicates an extremely significant difference (P<0.01).

[0069] The following conclusions can be drawn from Tables 10-12: like Figure 4 As shown in Table 10, for the total acid content, the response surface of the regression model is a sloping plane, and there is no significant interaction between the factors. The change in total acid is mainly driven by the independent linear effects of each factor, and the order of influence is: initial moisture content (A) > fermentation temperature (C) > inoculum size (B).

[0070] As shown in Table 11, the regression model for acid-soluble protein content is highly significant. The F-value for initial moisture content A is as high as 512.21 (P<0.0001), while the F-value for fermentation temperature C is 13.07 (P=0.0153), and the F-value for inoculum size B is only 1.39 (P=0.2916). The effect of initial moisture content is approximately 39 times that of temperature (512.21 / 13.07) and approximately 368 times that of inoculum size (512.21 / 1.39). Although the interaction terms among factors are not significant, the quadratic term (A²) for moisture content is significant, indicating that the relationship between acid-soluble protein content and moisture content is not strictly linear, and there is a certain curvature in the high moisture content range. The order of influence of each factor on acid-soluble protein content is: A>C>B.

[0071] For pH, as shown in Table 12, the model is significant. The F-value for initial moisture content A is 221.7, the F-value for temperature C is 25.96, and the F-value for inoculum size B is only 0.3524. There are no significant interactions among the factors, and the order of influence is: A>C>B.

[0072] The regression model was solved using Design Expert 13.0 software, and the theoretically optimal fermentation process parameters were predicted as follows: initial moisture content 45%, inoculum size 4 g / kg, fermentation temperature 40℃, and the predicted total acid content under these conditions was 2.73%.

[0073] Considering the cost pressure brought about by high inoculation volume in actual production and the operational problems that may be caused by excessive moisture content, and on the premise of ensuring no significant decline in fermentation quality, in order to balance process feasibility and economic benefits, the recommended process conditions finally determined in this application are: initial moisture content 40%, inoculation volume 2 g / kg, and fermentation temperature 37℃.

[0074] IV. Examples and Comparative Examples The technical solution of this application is further illustrated below through specific embodiments and comparative examples.

[0075] The soybean residue used was sourced from Hubei Junyuan Soybean Products Co., Ltd. (crude protein 18.42%, crude fiber 21.05%, crude fat 6.02%, crude ash 3.63%, moisture 4.39%, air-dried). The wheat bran was sourced from Yihai Kerry (Wuhan) Feed Co., Ltd. (crude protein 15.98%, crude fiber 10.55%, moisture 10.22%). The compound probiotic preparation (Saccharomyces cerevisiae 3×10⁻⁶) was also used. 8 CFU / g, Bacillus subtilis 1×10 8 CFU / g, Lactobacillus plantarum 4×10 8 The CFU / g, complex protease (50000 U / g), and mannanase (50000 U / g) were all derived from Tianjin Yunlizhixing Biotechnology Co., Ltd.

[0076] Example 1 S1. Mix dried soybean residue and wheat bran at a mass ratio of 43:57 to prepare a fermentation substrate, and adjust the initial moisture content to 40%.

[0077] S2. Inoculate the fermentation substrate with a compound probiotic preparation at a rate of 2 g / kg.

[0078] S3, simultaneously add 50,000 U / kg of complex protease and 50,000 U / kg of mannanase.

[0079] S4. Place the mixture in a sealed bag and seal it. Then, anaerobically ferment it at 37°C for 72 hours to obtain the fermented soybean residue product.

[0080] Example 2 The difference from Example 1 is that in step S1, the mass ratio of dried soybean residue to wheat bran is 50:50, and the initial moisture content is controlled at 45%. Other steps and parameters are the same as in Example 1.

[0081] Example 3 The difference from Example 1 is that the amount of complex protease added in step S3 is 75,000 U / kg, and the amount of mannanase added is 75,000 U / kg. Other steps and parameters are the same as in Example 1.

[0082] Example 4 The difference from Example 1 is that the inoculum amount in step S2 is 3 g / kg, and the fermentation temperature in step S4 is 40°C. All other steps and parameters are the same as in Example 1.

[0083] Comparative Example 1) The difference from Example 1 is that in step S1, the mass ratio of dried soybean residue to wheat bran is 28:71, and the initial moisture content is controlled at 30%. Other steps and parameters are the same as in Example 1.

[0084] Comparative Example 2 The difference from Example 1 is that the amount of complex protease added in step S3 is 25,000 U / kg. Other steps and parameters are the same as in Example 1.

[0085] Comparative Example 3 The difference from Example 1 is that in step S3, no complex protease (0 U / kg) is added, only mannanase 50,000 U / kg is added. The other steps and parameters are the same as in Example 1.

[0086] Comparative Example 4 The difference from Example 1 is that mannanase (0 U / kg) is not added in step S3, but only a complex protease of 50,000 U / kg is added. The other steps and parameters are the same as in Example 1.

[0087] Comparative Example 5 The difference from Example 1 is that the compound probiotic preparation is not inoculated in step S2 (the inoculation amount is 0 g / kg). The other steps and parameters are the same as in Example 1.

[0088] V. Performance Testing and Data Analysis Test method: Crude protein was determined using the Kjeldahl method (GB / T 6432), moisture content was determined according to GB / T 6435, crude ash content according to GB / T 6438, crude fat according to GB / T 6433, and crude fiber according to GB / T 6434. Soluble dietary fiber (SDF), insoluble dietary fiber (IDF), and total dietary fiber (TDF) were determined according to GB5009.88-2023.

[0089] pH was determined using the glass electrode method: 5 g of sample was weighed and added to 50 mL of distilled water, stirred and mixed, and allowed to stand for 30 min. The pH value of the supernatant was then measured using a calibrated pH meter. Total acid content was expressed as lactic acid equivalent and determined using acid-base titration: 10.00 g of sample was weighed and added to 100 mL of water for extraction for 30 min (stirring every 15 minutes). The filtrate was collected by filtering with gauze, and 10 mL of the filtrate was added to 70 mL of distilled water. Titration with 0.1 mol / L NaOH standard solution was performed to pH 8.2. Total acid (%) = (C × V × V0 × 0.09 × 100) / (M × V1), where C and V are the concentration and volume of NaOH, respectively; V0 and V1 are the total volume of sample dilution and the volume of the titration sample, respectively; M is the sample mass; and 0.09 is the millimolecular mass conversion factor for lactic acid.

[0090] The acid-soluble protein content was determined using a modified trichloroacetic acid precipitation method: Accurately weigh 2.0000 g of sample, add 20 mL of 15% (w / v) trichloroacetic acid solution to precipitate high-molecular-weight proteins, mix well, let stand for 5 min, and then centrifuge at 4000 r / min for 10 min to separate the precipitate (large-molecular-weight proteins) from the supernatant (acid-soluble proteins). Accurately pipette 10 mL of the supernatant and dry it in an oven at 100-105℃ until nearly dry. Determine the protein content of the residue after drying according to GB / T 6432; this is the acid-soluble protein content. Small peptide content is expressed as the percentage of acid-soluble proteins in the total crude protein of the sample.

[0091] Glycine and β-conglycine were qualitatively detected by SDS-PAGE gel electrophoresis and quantitatively detected by ELISA kits. Reducing sugars were determined using the 3,5-dinitrosalicylic acid (DNS) method.

[0092] The viable counts of Bacillus, lactic acid bacteria, and yeast were determined using the colony counting method: 10g of sample was weighed and added to 90 mL of sterile physiological saline, shaken for 30 min, and then serially diluted 10-fold. Yeast was plated on YPD medium (incubated at 30℃ for 1-2 days), Bacillus on LB solid medium (incubated at 37℃ for 1-2 days), and lactic acid bacteria were plated using the pour method on MRS solid medium containing 0.5% CaCO3 (incubated at 37℃ for 2 days). Colonies producing calcium-dissolving zones were counted.

[0093] The fermentation quality index data of each embodiment and comparative example obtained after testing are shown in Table 8.

[0094] Table 8 Fermentation quality indicators of the examples and comparative examples Comparing Comparative Example 1 with Example 1, it can be seen that when the initial moisture content decreased from 40% to 30%, the total acidity dropped sharply from 4.73% to 2.41%, small peptides decreased from 39.50% to 19.82%, acid-soluble proteins decreased from 6.51% to 3.21%, and pH increased from 5.04 to 5.70. The decrease in each indicator exceeded 49%.

[0095] This confirms the importance of water content as a core regulatory variable—insufficient water content severely restricts the diffusion of microbial extracellular enzymes in the matrix and substrate accessibility, leading to significant inhibition of both protein degradation and organic acid accumulation. This conclusion is highly consistent with the water content-dominant effect (F-value far exceeding other factors) revealed by response surface methodology.

[0096] Comparing Comparative Example 2 with Example 1 (using the ELISA data in Table 4), it can be seen that when the protease addition was reduced from 50,000 U / kg to 25,000 U / kg, the glycinin content abnormally increased from 2.62 mg / g in the control group to 3.69 mg / g, and β-conglycin abnormally increased from 2.31 mg / g to 4.99 mg / g. Figure 2 The SDS-PAGE results showed that the protein bands in the 50,000 U and 75,000 U groups were significantly weakened or disappeared in the 10-35 kDa and 60-75 kDa ranges, respectively.

[0097] This comparison demonstrates the necessity of a protease addition threshold of 50,000 U / kg: below this value, the protease only performs superficial cleavage, exposing the antigenic epitopes that were originally folded and embedded inside the protein, resulting in an increase in the antigenicity of the product instead of a decrease, thus increasing the risk of sensitization.

[0098] Comparing Comparative Example 3 with Example 1, it can be seen that without the addition of protease, the content of small peptides in the product is 29.50%, and the content of acid-soluble protein is 4.96%, which are lower than 39.50% and 6.51% in Example 1, respectively. The reduction in small peptide content by 25% indicates that exogenous protease has a significant contribution to the deep degradation of proteins, and it is difficult to achieve the same degree of protein conversion by relying solely on enzymes produced by microorganisms themselves.

[0099] Comparing Comparative Example 4 with Example 1, it can be seen that without the addition of mannanase, the content of small peptides is only 23.70%, and the content of acid-soluble protein is only 3.78%, which is much lower than that in Example 1. Combined with the dietary fiber data in Table 6, mannanase reduced IDF but did not increase SDF, while the reducing sugar content increased with increasing enzyme content.

[0100] This indicates that mannanase operates through a synergistic pathway of "enzymatic hydrolysis of mannan → generation of reducing sugars → serving as a readily available carbon source for microorganisms → deep conversion of kinetic proteins," rather than the "IDF to SDF conversion" pathway as traditionally believed in the literature.

[0101] Comparing Comparative Example 5 with Example 1, it can be seen that without probiotic inoculation, the number of lactic acid bacteria decreased from 8.77 1g CFU / g to about 6.50 1g CFU / g, the total acid decreased from 4.73% to about 3.50%, and the small peptides decreased from 39.50% to about 25.50%. This indicates that even with the addition of dual-enzyme preparations, sufficient organic acid accumulation and protein conversion cannot be achieved without the participation of active probiotics, confirming the indispensability of bacterial-enzyme synergy.

[0102] A comparative analysis of Examples 1 through 4 shows that the total acid and small peptides in Example 2 are significantly higher than those in Example 1, further confirming the dominant effect of water content. However, the quality improvement brought about by increasing the water content from 40% to 45% comes at the cost of reduced industrial operability. Compared to Example 1, in Example 3, the small peptides increased from 39.50% to approximately 41.20%, and the acid-soluble proteins increased from 6.51% to approximately 6.80%, an improvement of approximately 4%-4.5%. The total acid in Example 4 is approximately 4.95%, slightly higher than the 4.73% in Example 1, but the improvement is far less than the impact of the change in water content. Considering both quality and cost, the parameter combination in Example 1 is the recommended solution for industrial application.

[0103] The embodiments described in this specific implementation are preferred embodiments of this application and are not intended to limit the scope of protection of this application. Identical components are represented by the same reference numerals. Therefore, all equivalent changes made to the structure, shape, and principle of this application should be covered within the scope of protection of this application.

Claims

1. A method for solid-state fermentation of soybean residue, characterized in that, Includes the following steps: S1. Mix dried soybean residue with wheat bran to prepare a fermentation substrate, wherein the initial moisture content of the fermentation substrate is controlled to be 35%-50%; S2. Inoculate the fermentation substrate with a compound probiotic preparation, wherein the compound probiotic preparation includes Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum; S3. While inoculating the compound probiotic preparation, add compound protease and mannanase to the fermentation substrate, wherein the amount of compound protease added is not less than 50,000 U / kg. S4. The fermentation substrate containing compound probiotic preparation, compound protease and mannanase is sealed and subjected to anaerobic fermentation to obtain fermented soybean residue product.

2. The method according to claim 1, characterized in that, The amount of the compound protease added is 50,000-75,000 U / kg, and the amount of the mannanase added is 25,000-75,000 U / kg.

3. The method according to claim 2, characterized in that, The amount of mannanase added is 50,000-75,000 U / kg.

4. The method according to claim 1, characterized in that, The mass ratio of the dried soybean residue to the wheat bran is (28-57):(43-71).

5. The method according to claim 1, characterized in that, The inoculation amount of the compound probiotic preparation is 2-4 g / kg.

6. The method according to claim 5, characterized in that, In compound probiotic preparations, the number of live Saccharomyces cerevisiae is ≥3×10⁻⁶. 8 CFU / g, viable count of Bacillus subtilis ≥1×10⁻⁶ 8 CFU / g, viable count of Lactobacillus plantarum ≥ 4 × 10⁻⁶ 8 CFU / g.

7. The method according to claim 1, characterized in that, The anaerobic fermentation temperature is 35-43℃, and the fermentation time is 48-96h.

8. The method according to claim 6 or 7, characterized in that, The anaerobic fermentation temperature is 37-40℃, and the addition amount of the complex protease and mannanase is 50000U / kg.

9. A fermented soybean residue product, characterized in that, The fermented soybean residue product is prepared by the method according to any one of claims 1-8; the fermented soybean residue product has a pH ≤ 5.0, a total acid content ≥ 3.5%, a small peptide content ≥ 25%, an acid-soluble protein content ≥ 4.0%, and a live lactic acid bacteria count ≥ 102. 8 CFU / g.

10. The application of the fermented soybean residue product according to claim 9 in the preparation of animal feed.

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