Functional fermentation product preparation process based on directional conversion of bean dregs

Abstract

The invention relates to the technical field of biological fermentation, and discloses a functional fermentation product preparation process based on directional conversion of bean dregs, and the preparation process comprises the following steps: firstly, performing autolysis pretreatment on the bean dregs through an osmotic pressure regulator, activating an endogenous enzyme system, and releasing soluble nutrient substances; then, in the solid state fermentation stage, a composite stress factor composed of short-chain inulin and sorbitol is added, aspergillus oryzae is induced to form a loose and porous hypha network skeleton, and the ventilation and mass transfer efficiency of a fermentation substrate is remarkably improved. Then, regulating and controlling a cooling time sequence, adding L-glutamic acid as a metabolism inducer, and accurately receiving a metabolism path of bacillus megaterium to realize directional enrichment of functional products. The method effectively solves the problem that the physical structure is not matched with microbial metabolism in solid fermentation of the bean dregs, and improves the yield of functional fermentation products and the resource utilization value of the bean dregs.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, specifically to a process for preparing functional fermentation products based on the directional conversion of soybean residue. Background Technology

[0002] Soybean residue, a major byproduct of soybean product processing, is produced in large quantities and is rich in protein, dietary fiber, and various bioactive substances, possessing high potential for resource utilization. Microbial solid-state fermentation is one of the important ways to enhance the added value of soybean residue and develop functional products.

[0003] However, the physical properties of soybean residue itself pose a significant obstacle to its efficient solid-state fermentation. Fresh soybean residue has a high water content, small particles, and a viscous texture, making it prone to compaction during fermentation, forming a dense matrix structure. This structure severely hinders gas exchange and conduction within the material, leading to insufficient oxygen supply and the accumulation of carbon dioxide and heat, which is particularly detrimental to the growth and metabolism of aerobic microorganisms such as Aspergillus oryzae and Bacillus natto. The low mass transfer efficiency within the material also results in uneven distribution of nutrients, limiting the fermentation conversion efficiency.

[0004] Existing technologies for improving the physical structure of soybean residue typically involve adding loosening agents such as rice husks and wheat bran. While this method can increase the porosity of the matrix to some extent, the introduction of exogenous additives dilutes the original nutrients in the soybean residue, reduces the product yield per unit volume, and may introduce contaminating bacteria, increasing the difficulty of subsequent separation and purification.

[0005] Therefore, this invention proposes a process for preparing functional fermentation products based on the directional conversion of soybean residue to address the shortcomings of existing technologies. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a process for preparing functional fermentation products based on the directional conversion of soybean residue, which solves the problems of dense matrix structure and low material mass transfer and aeration efficiency that are common in existing solid-state fermentation processes for soybean residue.

[0007] To address the above problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a composite stress factor composition for regulating the hyphal network structure of Aspergillus oryzae, employing the following technical solution: A composite stress factor composition for regulating the mycelial network structure of Aspergillus oryzae, the composite stress factor composition being composed of short-chain inulin and sorbitol, wherein the weight ratio of short-chain inulin to sorbitol is 1.5:1 to 3:1.

[0008] By adopting the above technical solution, the composite stress factor composition provided by this invention can precisely regulate the growth morphology of Aspergillus oryzae mycelia during the solid-state fermentation stage through the synergistic effect of nutrient stress and osmotic stress. The specific mechanism is as follows: Construction of dual coercion: Nutritional stress: The short-chain inulin in the composition is a carbon source that can be degraded and utilized by Aspergillus oryzae, but its utilization rate is lower than that of simple sugars such as glucose. This puts Aspergillus oryzae in a mild state of nutrient restriction during the early stages of growth, i.e., nutrient stress.

[0009] Osmotic stress: Sorbitol in the composition is a sugar alcohol that is not easily metabolized by microorganisms. It acts as an osmotic regulator in the fermentation substrate, increasing the osmotic pressure of the culture environment. This high osmotic pressure environment constitutes osmotic stress.

[0010] Induction of mycelial network morphology: Under the above dual stress environment, the growth strategy of Aspergillus oryzae changes; in order to find sufficient nutrients and adapt to high osmotic pressure, the mycelium no longer tends to form a dense mycelial moss, but instead transforms into an exploratory extension growth mode; the mycelium grows sparsely, branches more, and extends into three-dimensional space, eventually forming a three-dimensional network structure with strong physical support and mutual cross-linking between soybean residue particles.

[0011] Technical Achievements: This three-dimensional network structure is a loose and porous mycelial network framework. The formation of this framework fundamentally improves the physical properties of the solid-state fermentation substrate, significantly increasing its porosity and gas permeability. This provides sufficient oxygen and a large attachment surface area for the subsequent growth of Bacillus natto, which is a prerequisite for achieving efficient metabolic docking and directional fermentation of the two microorganisms.

[0012] Preferably, the weight ratio of the short-chain inulin to the sorbitol is 3:1 or 1.5:1.

[0013] By adopting the above technical solution, the two specific weight ratios are an optimized ratio that balances nutrient stress and osmotic stress, which can more effectively induce the formation of a mycelial network with a stable structure and high porosity.

[0014] Preferably, the degree of polymerization of the short-chain inulin is 2 to 9.

[0015] By employing the above-mentioned technical solution, limiting the degree of polymerization of short-chain inulin is crucial to ensuring its effectiveness as a slow-release carbon source. If the polymerization degree is too low, the short-chain inulin is easily metabolized rapidly, losing its nutrient stress-relieving effect; if the polymerization degree is too high, the short-chain inulin is difficult to degrade and cannot provide the energy required for the basic growth of *Aspergillus oryzae*. Inulin with a polymerization degree of 2–9 provides moderate nutrient stress, which is the technical guarantee for achieving the target network structure.

[0016] Secondly, the present invention provides a process for preparing functional fermentation products, which adopts the following technical solution: A process for preparing a functional fermentation product, the process comprising the following steps: Step S1, Osmotic pressure induced autolysis: Add an osmotic pressure regulator to the soybean residue slurry and react under preset conditions to activate the endogenous enzyme system of soybean residue and release soluble substances; Step S2, compound stress-induced solid-state fermentation: Aspergillus oryzae is inoculated into the material treated in step S1, and compound stress factors are added for solid-state culture to form a loose and porous mycelial network skeleton. Step S3, Metabolic docking and sequential fermentation: The soybean residue koji obtained in step S2 is cooled and a metabolic inducer is added, and then the temperature is raised and Bacillus natto is inoculated for directional fermentation.

[0017] By adopting the above technical solution, this invention systematically solves the technical problem of mismatch between physical structure and microbial metabolism in solid-state fermentation of soybean residue through three stages of orderly regulation, achieving targeted enrichment of the final functional products. Its innovative principle is embodied in the following process: Phase 1: Biological Pretreatment of the Substrate (Step S1) A high-osmotic-pressure environment is created by introducing a high concentration of osmotic pressure regulator into the soybean residue slurry. This environment induces osmotic stress in the plant cells and endogenous microorganisms within the soybean residue system, activating their own hydrolytic enzyme systems such as proteases and cellulases. These enzyme systems degrade macromolecules such as proteins and cellulose in the soybean residue in situ, converting insoluble macromolecules into soluble proteins, peptides, oligosaccharides, and other small molecule nutrients. This step is a mild autolysis process, providing a sufficient and readily available nutrient basis for the subsequent rapid growth of Aspergillus oryzae.

[0018] The second stage: physical reconstruction of the fermentation microenvironment (step S2). After the nutrients are fully released, Aspergillus oryzae is inoculated and a complex stress factor is introduced. As described in the first aspect, the complex stress factor regulates the Aspergillus oryzae hyphae to form a three-dimensional network skeleton structure. This structure reshapes the physical morphology of the soybean residue substrate, transforming it from a dense paste into a loose and porous solid material. This structural transformation improves the oxygen holding capacity and gas exchange efficiency of the entire fermentation system, providing a suitable physical microenvironment for the subsequent reproduction and metabolism of Bacillus natto, which requires oxygen.

[0019] Phase 3: Precise Targeting of Metabolic Flow (Step S3) This phase is crucial for the targeted synthesis of the product. First, cooling is used to temporarily suppress the metabolic activity of *Aspergillus oryzae*, preventing it from competing with the subsequently inoculated *Bacillus natto* for nutrients. Simultaneously, L-glutamic acid is added at low temperature as a metabolic inducer. L-glutamic acid is a key precursor for the synthesis of γ-polyglutamic acid (γ-PGA) by *Bacillus natto*; the low-temperature addition ensures that L-glutamic acid can fully penetrate and distribute within the porous koji residue. Subsequently, the temperature is raised to the optimal growth temperature for *Bacillus natto*, and inoculation is carried out. At this point, *Bacillus natto* grows in an environment with a loose, porous structure, containing available nutrients and specific metabolic precursors, and its metabolism is converted to the synthetic pathway of the target product, thereby achieving the accumulation of functional products.

[0020] In summary, this invention organically combines enzymatic hydrolysis at the biochemical level, reconstruction at the physical structural level, and temporal regulation at the microbial metabolic level to form a complete targeted transformation technology system.

[0021] Preferably, the osmotic pressure regulator in step S1 is composed of erythritol and xylitol, and the amount of the osmotic pressure regulator added is 5% to 10% of the dry weight of soybean residue.

[0022] By employing the above technical solutions, erythritol and xylitol, as small-molecule sugar alcohols, can efficiently generate osmotic pressure and are chemically stable, not participating in subsequent metabolic reactions, thus avoiding the generation of byproducts. An addition amount of 5%–10% is the optimized range to ensure effective activation of endogenous enzyme systems without excessively inhibiting microbial activity.

[0023] Preferably, the composite stress factor in step S2 is a composite stress factor composition, and the amount of the composite stress factor composition added is 2.0% to 4.5% of the dry weight of the material.

[0024] By adopting the above technical solution, the composite stress factor composition provided by this invention is applied to the process, which is the core technology for realizing the reconstruction of the physical structure of the fermentation substrate; the addition amount of 2.0% to 4.5% is the optimal concentration range for effectively controlling the mycelial morphology.

[0025] Preferably, the cooling process in step S3 involves lowering the fermentation temperature to 15°C–20°C and maintaining it for 8–12 hours.

[0026] By adopting the above technical solution, a temperature of 15℃~20℃ can effectively inhibit the main metabolic activities of Aspergillus oryzae, while a maintenance time of 8 hours to 12 hours provides sufficient guarantee for the uniform diffusion and penetration of the metabolic inducer, which is a key process parameter for achieving orderly separation of the metabolic activities of the two microorganisms.

[0027] Preferably, while the fermentation temperature is lowered, L-glutamic acid, as a metabolic inducer, is added to the fermentation material, wherein the amount of L-glutamic acid added is 50 mg / kg to 200 mg / kg of the dry weight of the material.

[0028] By adopting the above technical solution, the material basis and timing of the metabolic inducer were clarified, realizing a "precursor feeding" strategy for the γ-PGA synthesis pathway. The addition amount of 50 mg / kg to 200 mg / kg is based on precise calculations of the metabolic flux of Bacillus natto, enabling efficient conversion of precursor materials.

[0029] Preferably, before step S1, a pretreatment step of fresh soybean residue is included, wherein the pretreatment includes correcting the moisture content of the soybean residue to 73% to 77% and adjusting the pH value to 6.0 to 6.5.

[0030] By adopting the above-mentioned technical solution and standardizing the raw materials, the stability and repeatability of the entire process are ensured. This moisture content and pH range provide the most suitable starting conditions for the activation of endogenous enzymes in step S1.

[0031] Preferably, in step S2, after adjusting the moisture content of the material to 55%–65%, the inoculation concentration is 1.0 × 10⁻⁶. 7 spores / mL~1.0×10 8 A suspension of Aspergillus oryzae spores / mL.

[0032] By adopting the above technical solution, a moisture content of 55%–65% is the optimal water activity range for solid-state fermentation. This range satisfies the growth requirements of Aspergillus oryzae while avoiding substrate compaction and poor aeration caused by excessive moisture. This inoculation concentration range ensures that the inoculum can germinate rapidly and evenly occupy the entire fermentation substrate.

[0033] This invention provides a process for preparing functional fermentation products based on the directional conversion of soybean residue. It has the following beneficial effects: This invention induces Aspergillus oryzae mycelia to form a loose and porous three-dimensional network framework by adding a composite stress factor composed of short-chain inulin and sorbitol during the Aspergillus oryzae fermentation stage. This framework structure fundamentally changes the dense physical morphology of traditional soybean residue solid fermentation substrate, significantly increases the porosity and gas permeability of the material, and provides an excellent growth microenvironment for subsequent aerobic fermentation of Bacillus natto. The process of this invention includes a cooling-heating time-controlled stage in which L-glutamic acid, as a metabolic precursor, is precisely added. This operation sequentially separates the growth stage of Aspergillus oryzae from the metabolic stage of Bacillus natto in time, and through a precursor feeding strategy, effectively directs the metabolic flow of Bacillus natto to the synthesis of target products such as γ-polyglutamic acid, significantly improving the yield and purity of functional products. This invention employs an osmotic pressure-induced autolysis step to pretreat soybean residue raw materials; it utilizes osmotic pressure regulators such as erythritol and xylitol to activate the endogenous enzyme system of soybean residue, achieving gentle degradation of macromolecules such as proteins and cellulose; compared with traditional heat treatment or external enzyme preparation methods, this step has milder processing conditions, lower costs, and can effectively release the nutrients bound to the soybean residue itself, thus improving the utilization efficiency of the raw materials. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the osmotic pressure autolysis effect verification of the present invention; Figure 2 This is a schematic diagram illustrating the physical properties of the biological skeleton of the present invention; Figure 3 This is a schematic diagram illustrating the time-sequential synergistic fermentation effect verification of the present invention; Figure 4 This is a schematic diagram illustrating the comprehensive functional characteristics evaluation of the final product of this invention. Detailed Implementation

[0035] The technical solutions in 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 some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0037] Soybean residue: derived from the wet processing of non-GMO soybeans (variety: Zhonghuang 13) in tofu production, used within 4 hours after production, with an initial moisture content of 75-85% (w / w); Aspergillus oryzae: strain number ATCC42149; Bacillus natto: commercially available Bacillus natto strain; Erythritol: food grade, CAS number: 149-32-6; Xylitol: food grade, CAS number: 87-99-0; Sorbitol: food grade, CAS number: 50-70-4; Short-chain inulin: food grade, CAS number: 9005-80-5, which is a fructopolymer. The sugar mixture consists of linear chains of D-fructose units linked by β-(2→1) glycosidic bonds, typically ending with a glucose unit linked by an α-(1→2) glycosidic bond; the degree of polymerization (DP) of the short-chain inulin used in this invention ranges from 2 to 9; L-glutamic acid: food grade, CAS number: 56-86-0; rice husk powder: industrial grade, particle size specification is 60-80 mesh; wheat bran: food grade; potato dextrose agar medium: commercially available microbial culture medium grade; beef extract peptone medium: composed of beef extract, peptone and sodium chloride, all of which are commercially available microbial culture medium grades.

[0038] Preparation example: Preparation Example 1: Preparation of the osmotic pressure regulating composition ORC-1 This preparation example provides an osmotic pressure regulator composition ORC-1, comprising the following steps: Weigh out food-grade erythritol and food-grade xylitol, and mix them in a mass ratio of 1:2; pulverize the mixture using a pulverizer so that it passes through an 80-mesh sieve; seal and store the resulting powder under dry conditions for later use.

[0039] Preparation Example 2: Preparation of the osmotic pressure regulating composition ORC-2 This preparation example provides an osmotic pressure regulator composition ORC-2, comprising the following steps: Weigh out food-grade erythritol and food-grade xylitol, and mix them in a 1:1 mass ratio; pulverize the mixture using a pulverizer so that it passes through an 80-mesh sieve; seal and store the resulting powder under dry conditions for later use.

[0040] Preparation Example 3: Preparation of the osmotic pressure regulating composition ORC-3 This preparation example provides an osmotic pressure regulator composition ORC-3, comprising the following steps: Weigh out food-grade erythritol and food-grade xylitol, and mix them in a mass ratio of 2:1; pulverize the mixture using a pulverizer so that it passes through an 80-mesh sieve; seal and store the resulting powder under dry conditions for later use.

[0041] Preparation Example 4: Preparation of the composite stress factor composition CSC-1 This preparation example provides a composite stress factor composition CSC-1, comprising the following steps: Weigh out short-chain inulin with a degree of polymerization (DP) of 2 to 9 and food-grade sorbitol, and mix them in a mass ratio of 3:1; pulverize the mixture using a pulverizer so that it passes through an 80-mesh sieve; seal and store the resulting powder under dry conditions for later use.

[0042] Preparation Example 5: Preparation of the composite stress factor composition CSC-2 This preparation example provides a composite stress factor composition CSC-2, comprising the following steps: Weigh out short-chain inulin with a degree of polymerization (DP) of 2 to 9 and food-grade sorbitol, and mix them at a mass ratio of 1.5:1; pulverize the mixture using a pulverizer so that it passes through an 80-mesh sieve; seal and store the resulting powder under dry conditions for later use.

[0043] Preparation Example 6: Preparation of Metabolic Inducer Solution (MIS) This preparation example provides a metabolism inducer solution (MIS), including the following steps: Accurately weigh food-grade L-glutamic acid, dissolve and dilute it with sterile purified water to prepare a stock solution with a concentration of 1.0 g / L. After sterilizing the stock solution by filtration through a 0.22 μm filter membrane, seal and store it under sterile conditions at 4°C for later use. Example

[0044] Example 1: This example provides a method for preparing functional fermentation products based on the directional conversion of soybean residue, including the following steps: (1) Raw material pretreatment: Take fresh soybean residue, add purified water to adjust the slurry and correct the water content to 75% (w / w), and use citric acid to adjust the pH value to 6.2; let the slurry stand at 22℃ for 1.5 hours to activate it.

[0045] (2) Osmotic pressure induced autolysis: 8% (w / w) of the osmotic pressure regulator composition ORC-2 prepared in Preparation Example 2 was added to the pretreated soybean residue slurry by dry matter weight; the reaction was carried out at 30°C and 80 rpm for 3.0 hours.

[0046] (3) Compound stress-induced solid-state fermentation: Rice husk powder was added to the autolyzed liquid to adjust the moisture content of the material to 60% (w / w); after the material temperature was cooled to 32℃, Aspergillus oryzae spore suspension (1.0×10⁻⁶) was inoculated. 7 The inoculum was 2.5% (v / w) of the wet weight of the material; at the same time, 3% (w / w) of the composite stress factor composition CSC-1 prepared in Preparation Example 4 was added by dry matter weight; the material was cultured at 30°C and 90% relative humidity for 30 hours.

[0047] (4) Metabolic docking and sequential fermentation: The fermentation temperature was lowered to 18°C ​​within 1.5 hours; at the same time, the metabolic inducer solution MIS prepared in Preparation Example 6 was added by atomized spray at a rate of 125 mg / kg based on dry matter weight; the culture was maintained at 18°C ​​for 10 hours; then, the fermentation temperature was raised back to 40°C within 1.5 hours, and Bacillus natto seed liquid was inoculated at a rate of 4% (v / w) of the wet weight of the material; fermentation was carried out at 40°C and 95% relative humidity for 20 hours.

[0048] (5) Post-processing: The fermentation product was inactivated by treating it at 70°C for 45 minutes, and then vacuum freeze-dried until the moisture content was less than 10% (w / w); the dried material was crushed and passed through a 100-mesh sieve to obtain the final product.

[0049] Example 2: This example provides a method for preparing functional fermentation products based on the directional conversion of soybean residue, including the following steps: (1) Raw material pretreatment: Take fresh soybean residue, add purified water to adjust the slurry and correct the water content to 73% (w / w), and use citric acid to adjust the pH value to 6.0; let the slurry stand at 20℃ for 2.0 hours to activate.

[0050] (2) Osmotic pressure induced autolysis: 5% (w / w) of the osmotic pressure regulator composition ORC-1 prepared in Preparation Example 1 was added to the pretreated soybean residue slurry by dry matter weight; the reaction was carried out at 25°C and 50 rpm for 4.0 hours.

[0051] (3) Compound stress-induced solid-state fermentation: Wheat bran was added to the autolysis-treated liquid to adjust the moisture content of the material to 55% (w / w); after the material temperature was cooled to 30℃, Aspergillus oryzae spore suspension (1.0×10⁻⁶) was inoculated. 8 The inoculum was 2% (v / w) of the wet weight of the material; at the same time, 2.0% (w / w) of the composite stress factor composition CSC-2 prepared in Preparation Example 5 was added; the material was cultured at 28°C and 85% relative humidity for 36 hours.

[0052] (4) Metabolic docking and sequential fermentation: The fermentation temperature was lowered to 20°C within 2.0 hours; at the same time, 50 mg / kg of the metabolic inducer solution MIS prepared in Preparation Example 6 was added by atomized spraying; and the culture was maintained at 20°C for 8 hours. Subsequently, the fermentation temperature was raised back to 37°C within 2.0 hours, and Bacillus natto seed culture was inoculated at an inoculation amount of 3% (v / w) of the wet weight of the material; fermentation was carried out at 37°C and 90% relative humidity for 24 hours.

[0053] (5) Post-processing: The fermentation product was inactivated by treating it at 65°C for 60 minutes, and then vacuum freeze-dried until the moisture content was less than 10% (w / w). The dried material was crushed and passed through a 120-mesh sieve to obtain the final product.

[0054] Example 3: This example provides a method for preparing functional fermentation products based on the directional conversion of soybean residue, including the following steps: (1) Raw material pretreatment: Take fresh soybean residue, add purified water to adjust the slurry and correct the water content to 77% (w / w), and use sodium bicarbonate to adjust the pH value to 6.5; let the slurry stand at 25℃ for 1.0 hour to activate it.

[0055] (2) Osmotic pressure induced autolysis: 10% (w / w) of the osmotic pressure regulator composition ORC-3 prepared in Preparation Example 3 was added to the pretreated soybean residue slurry by dry matter weight; the reaction was carried out at 35°C and 100 rpm for 2.0 hours.

[0056] (3) Compound stress-induced solid-state fermentation: Rice husk powder was added to the autolyzed liquid to adjust the moisture content of the material to 65% (w / w); after the material temperature was cooled to 34℃, Aspergillus oryzae spore suspension (1.0×10⁻⁶) was inoculated. 7 The inoculum was 3% (v / w) of the wet weight of the material; at the same time, 4.5% (w / w) of the composite stress factor composition CSC-1 prepared in Preparation Example 4 was added by dry weight; the material was cultured at 32°C and 95% relative humidity for 24 hours.

[0057] (4) Metabolic docking and sequential fermentation: The fermentation temperature was lowered to 15°C within 1.0 hour; at the same time, 200 mg / kg of the metabolic inducer solution MIS prepared in Preparation Example 6 was added by atomized spraying; the culture was maintained at 15°C for 12 hours; then, the fermentation temperature was raised back to 42°C within 1.0 hour, and Bacillus natto seed liquid was inoculated at 5% (v / w) of the wet weight of the material; fermentation was carried out at 42°C and 98% relative humidity for 18 hours.

[0058] (5) Post-processing: The fermentation product was inactivated by treating it at 75°C for 30 minutes, and then vacuum freeze-dried until the moisture content was less than 10% (w / w); the dried material was crushed and passed through a 100-mesh sieve to obtain the final product.

[0059] Comparative example: Comparative Example 1: This comparative example provides a method for preparing fermented soybean residue products using a traditional sequential fermentation method, including the following steps: (1) Take fresh soybean residue, add purified water to adjust the slurry to a moisture content of 60% (w / w), add wheat bran and then perform moist heat sterilization at 121℃ for 20 minutes.

[0060] (2) After the material temperature is cooled to 32°C, inoculate with Aspergillus oryzae spore suspension and culture at 30°C for 30 hours.

[0061] (3) After the Aspergillus oryzae culture is completed, the fermentation temperature is raised to 40°C and Bacillus natto seed liquid is inoculated and fermented at 40°C for 20 hours.

[0062] (4) The subsequent inactivation, drying and pulverization steps are the same as in Example 1.

[0063] Comparative Example 2: Compared with Example 1, the difference is that step (2) does not use osmotic pressure-induced autolysis, but instead the soybean residue slurry pretreated in step (1) is subjected to moist heat sterilization at 121°C for 20 minutes. The remaining steps are the same as in Example 1.

[0064] Comparative Example 3: The difference from Example 1 is that in step (3), no compound stress factor composition CSC-1 is added during solid-state fermentation with Aspergillus oryzae. The remaining steps are the same as in Example 1.

[0065] Comparative Example 4: Compared with Example 1, the difference is that in step (4), after the Aspergillus oryzae fermentation is completed, the "V"-shaped temperature curve treatment and the addition of metabolic inducer solution MIS are not used. Instead, the fermentation temperature is directly raised to 40°C and Bacillus natto is inoculated for fermentation. The remaining steps are the same as in Example 1.

[0066] Test example: Test Example 1: Verification of Osmotic Autolysis Effect This test case aims to verify the actual effect of the osmotic pressure-induced autolysis step used in this invention on the release of cell contents from soybean residue.

[0067] Sample preparation: Sample S1 (Sample of the present invention): Take the slurry after the osmotic pressure induced autolysis reaction in step (2) of Example 1, centrifuge it at 10000g for 10 minutes, and take its supernatant for subsequent testing.

[0068] Sample S2 (control sample): Take the slurry after the raw material pretreatment in step (1) of Example 1 (i.e., the composition ORC-2 without the addition of osmotic pressure regulator), and treat it for the same time (3.0 hours) under the same temperature and stirring conditions (30℃, 80rpm) as Sample S1; after treatment, centrifuge it at 10000g for 10 minutes, and take its supernatant for subsequent detection.

[0069] The experimental steps are as follows: (1) Take the supernatant of samples S1 and S2 and measure the conductivity at 25°C using a calibrated laboratory conductivity meter.

[0070] (2) The soluble total sugar content in the supernatant was determined by the phenol-sulfuric acid method. After the sample solution was diluted, phenol solution and concentrated sulfuric acid were added in sequence, and the absorbance was measured at a wavelength of 490 nm. The content was calculated by the glucose standard curve.

[0071] (3) The content of soluble protein in the supernatant was determined by the Coomassie Brilliant Blue G-250 method; the sample solution was taken, Coomassie Brilliant Blue G-250 reagent was added, the absorbance was measured at a wavelength of 595 nm, and the content was calculated by the bovine serum albumin standard curve.

[0072] For each of the above indicators, the measurements were repeated three times for each sample.

[0073] The experimental data are shown in Table 1: Table 1: Effects of osmotic autolysis on key parameters of soybean residue slurry supernatant Conclusion: Refer to Appendix Figure 1 The data in Table 1 show that the conductivity, total soluble sugar content, and soluble protein content of the supernatant of sample S1 after osmotic pressure treatment were all higher than those of the untreated control sample S2.

[0074] The increase in conductivity reflects the change in cell membrane permeability of soybean residue cells under high osmotic pressure, leading to the release of intracellular electrolytes. The increase in total soluble sugar and soluble protein content indicates that osmotic stress activates endogenous hydrolytic enzymes in soybean residue, which degrade the cell wall and macromolecules such as proteins and polysaccharides stored intracellularly.

[0075] The results confirm that the osmotic pressure-induced autolysis step can effectively release soluble nutrients from soybean residue; these released small molecule carbon and nitrogen sources can provide directly usable substrates for subsequent Aspergillus oryzae fermentation, thus creating conditions for the start-up of the fermentation process.

[0076] Test Example 2: Characterization of the physical properties of the biological skeleton This test case aims to verify the effects of combined stress factors on the physical structure of soybean residue fermented by Aspergillus oryzae.

[0077] Sample preparation: Sample S3 (Sample of the present invention): Wet koji after the solid-state fermentation of Aspergillus oryzae in step (3) of Example 1 was completed.

[0078] Sample S4 (control sample): Wet koji from the solid-state fermentation of Aspergillus oryzae in the control example 3 was taken.

[0079] The two wet koji samples were vacuum freeze-dried to constant weight, pulverized, and passed through a 60-mesh sieve for subsequent testing.

[0080] The experimental steps are as follows: (1) Weigh a certain mass (m) of freeze-dried sample, put it into a 10mL graduated cylinder, record its apparent volume (V), and calculate the bulk density using the formula ρ=m / V.

[0081] (2) Fill 10.0g of freeze-dried sample into a glass tube with an inner diameter of 2.0cm, ensuring that the material layer height is consistent; apply a constant nitrogen pressure of 5kPa at one end of the material layer and record the time required for 100mL of nitrogen to pass through the material layer.

[0082] (3) Weigh 2.0g of freeze-dried sample and soak it in 50mL of purified water for 2 hours; take out the sample and place it on a nylon mesh cloth and centrifuge it at 1000g for 10 minutes; weigh the wet weight of the sample after centrifugation and calculate its water holding capacity (g water / g dry matter).

[0083] All of the above indicators were measured in three parallel trials.

[0084] The experimental data are shown in Table 2: Conclusion: Refer to Appendix Figure 2 The data in Table 2 show that sample S3, which has the added composite stress factor, has a lower bulk density than control sample S4, a shorter gas permeation time, and higher water retention.

[0085] This result is attributed to the regulation of Aspergillus oryzae growth morphology by combined stress (short-chain inulin and sorbitol). Stress induces mycelial branching, forming a three-dimensional network structure among soybean residue particles. This network structure increases the porosity of the substrate, leading to a decrease in bulk density and providing channels for gas exchange, thus shortening the gas permeation time. Simultaneously, this porous structure enhances the water-holding capacity of the substrate through capillary action.

[0086] The formation of this physical structure creates favorable conditions for the subsequent growth of Bacillus natto. Improved substrate permeability facilitates oxygen supply in the later stages of fermentation, while enhanced water retention helps maintain the humidity of the fermentation system. Therefore, the application of complex stress factors is a crucial step in constructing a solid substrate suitable for sequential fermentation.

[0087] Test Example 3: Validation of the effect of time-sequential synergistic fermentation This test case aims to evaluate the impact of the timing synergy strategy employed in this invention on the content of key functional ingredients in the final product.

[0088] Sample preparation: Sample S5 (Sample of the present invention): The dried powder product finally obtained in Example 1.

[0089] Sample S6 (control sample): The dried powder product finally obtained from Comparative Example 4.

[0090] The experimental steps are as follows: (1) The activity of nattokinase was determined by the fibrin plate method. The sample powder was weighed, extracted with phosphate buffer (pH 7.4) and centrifuged. The supernatant was added to the fibrin plate. After incubation at 37°C for 18 hours, the diameter of the lysate zone was measured. The enzyme activity (FU / g dry matter) was calculated by the nattokinase standard curve.

[0091] (2) The content of γ-polyglutamic acid (γ-PGA) was determined by high performance liquid chromatography. The sample powder was weighed and hydrolyzed with hydrochloric acid at 110℃ for 18 hours to degrade γ-PGA into glutamic acid monomers. The hydrolysate was derivatized and then subjected to chromatographic analysis. The content of γ-PGA (mg / g dry matter) was calculated by converting the glutamic acid standard curve and stoichiometric relationship.

[0092] All of the above indicators were measured in three parallel trials.

[0093] The experimental data are shown in Table 3: Table 3: Impact of Time-Sequence Coordinated Regulation on the Functional Indicators of the Final Product Conclusion: Refer to Appendix Figure 3 The data in Table 3 show that the nattokinase activity and γ-PGA content of sample S5 were both higher than those of control sample S6.

[0094] This result is related to the use of a V-shaped temperature profile and the addition of a metabolic inducer. The cooling treatment after the Aspergillus oryzae fermentation stage inhibited the metabolic activity of Aspergillus oryzae and may have promoted the release of intracellular substances, providing more usable nutrients for the subsequent growth of Bacillus natto. The subsequent heating step created suitable conditions for the growth and metabolism of Bacillus natto, achieving an orderly connection between the fermentation processes of the two microorganisms.

[0095] Meanwhile, the exogenously added L-glutamic acid, as a direct precursor for γ-PGA synthesis, is utilized by Bacillus natto to promote γ-PGA synthesis; therefore, through the combination of metabolic docking and precursor supply, the temporal synergistic strategy of the present invention increases the content of these two functional components in the final product.

[0096] Test Example 4: Evaluation of the overall functional characteristics of the final product This test case aims to comprehensively evaluate the performance of final products prepared by different processes in terms of key functional characteristics.

[0097] Sample preparation: The dried powder products finally obtained in Examples 1-3 and Comparative Examples 1-4 were numbered E1, E2, E3 and CE1, CE2, CE3, CE4 respectively; Fresh soybean residue without any treatment was freeze-dried and pulverized as a raw material control, numbered Raw.

[0098] The experimental steps are as follows: (1) Weigh 1.0g of each sample powder, add 50mL of phosphate buffer (pH7.0) and extract by shaking at 40℃ for 2 hours; centrifuge and collect the supernatant for later use.

[0099] (2) Take the supernatant and mix it with DPPH ethanol solution. After reacting in the dark for 30 minutes, measure the absorbance at a wavelength of 517 nm and calculate the DPPH free radical scavenging rate.

[0100] (3) Take the supernatant and add it to the Fenton reaction system. After the reaction, add the colorimetric reagent and measure the absorbance at a wavelength of 510 nm to calculate the hydroxyl radical scavenging rate.

[0101] (4) Take the supernatant and react it with ninhydrin reagent in a boiling water bath for 15 minutes. After cooling and making up to volume, measure the absorbance at a wavelength of 570 nm. Calculate the free amino acid content (mg / g dry matter) by using the leucine standard curve.

[0102] All of the above indicators were measured in three parallel trials.

[0103] The experimental data are shown in Table 4: Table 4: Comparison of comprehensive functional characteristics of different samples Conclusion: Refer to Appendix Figure 4 As shown in Table 4, the example samples (E1-E3) were higher than all comparative samples (CE1-CE4) and raw materials (Raw) in terms of DPPH radical scavenging rate, hydroxyl radical scavenging rate and free amino acid content.

[0104] Comparison with raw materials and conventionally fermented samples (CE1) confirms the effectiveness of the overall process of this invention in improving the functional properties of soybean residue; the data from each comparative sample further elucidates the role of key steps in this invention: The indicators of sample CE2 (lacking osmotic autolysis) were lower than those of E1, indicating that autolysis pretreatment is the basis for subsequent efficient biotransformation.

[0105] The results for sample CE3 (lacking combined stress) were lower than those for E1, indicating that the physical structure of the fermentation substrate constructed by combined stress improved the microbial metabolic environment, thereby affecting the accumulation of functional substances.

[0106] The decrease in the CE4 (lacking temporal synergy) index of the sample demonstrates the direct role of metabolic docking and precursor supply in the synthesis of specific functional products.

[0107] The data from Examples E1, E2, and E3 collectively demonstrate that, within the parameter range described in this invention, the technical solution can stably produce products with enhanced functional properties. These results collectively illustrate that this invention achieves deep biotransformation and enrichment of functional components in soybean residue through a combination of steps such as osmotic autolysis, combined stress, and synergistic metabolic timing.

[0108] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A functional fermentation product, characterized in that, The raw material comprises the following components: bean dregs; an osmotic pressure regulator composition; a complex stress factor composition; a metabolic inducer solution; aspergillus oryzae; bacillus natto.

2. The functional fermentation product according to claim 1, characterized in that, The osmotic pressure regulator composition consists of erythritol and xylitol, the mass ratio of the erythritol to the xylitol being 1:2-2:1; the complex stress factor composition consists of short-chain inulin with a polymerization degree of 2-9 and sorbitol, the mass ratio of the short-chain inulin to the sorbitol being 1.5:1-3:

1.

3. The functional fermentation product of claim 1, wherein, The metabolic inducer solution is an aqueous L-glutamic acid solution with a concentration of 0.5 g / L-1.5 g / L.

4. A process for the preparation of a functional fermentation product based on the directed conversion of bean dregs, for use in the functional fermentation product according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: S1, osmotic pressure-induced autolysis: adding an osmotic pressure regulator to a bean dregs slurry, and performing a reaction under preset conditions to activate endogenous enzymes in the bean dregs and release soluble substances; S2, complex stress-induced solid-state fermentation: inoculating aspergillus oryzae into the material treated in step S1, and adding a complex stress factor to perform solid-state culture to form a loose and porous mycelial network skeleton; S3, metabolic docking and time-series fermentation: performing a temperature reduction treatment on the bean dregs koji obtained in step S2 and adding a metabolic inducer, and then performing a temperature increase and inoculating bacillus natto for directional fermentation.

5. The process for the preparation of a functional fermentation product based on the oriented conversion of bean dregs according to claim 4, characterized in that, The osmotic pressure regulator in step S1 consists of erythritol and xylitol, the mass ratio of the erythritol to the xylitol being 1:2-2:1, and the addition amount of the osmotic pressure regulator being 5%-10% of the dry weight of the bean dregs.

6. The process for the preparation of a functional fermentation product based on the oriented conversion of bean dregs according to claim 4, characterized in that, The complex stress factor in step S2 is a complex stress factor composition consisting of short-chain inulin with a polymerization degree of 2-9 and sorbitol, the mass ratio of the short-chain inulin to the sorbitol being 1.5:1-3:1, and the addition amount of the complex stress factor composition being 2.0%-4.5% of the dry weight of the material.

7. The process for preparing a functional fermentation product based on the oriented conversion of bean dregs according to claim 4, characterized in that, The temperature reduction treatment in step S3 is to reduce the fermentation temperature to 15°C-20°C and maintain for 8 hours-12 hours.

8. The process for the preparation of a functional fermentation product based on the oriented conversion of bean dregs according to claim 7, characterized in that, While the fermentation temperature is reduced, L-glutamic acid as a metabolic inducer is added to the fermentation material, and the addition amount of the L-glutamic acid is 50 mg / kg-200 mg / kg of the dry weight of the material.

9. The process for preparing a functional fermentation product based on the oriented conversion of bean dregs according to claim 4, characterized in that, Before step S1, a step of pretreating fresh bean dregs is further included, and the pretreatment comprises correcting the water content of the bean dregs to 73%-77% and adjusting the pH value to 6.0-6.

5.

10. The process for preparing a functional fermentation product based on the oriented conversion of bean dregs according to claim 4, characterized in that, In step S2, after the moisture content of the material is adjusted to 55% to 65%, a suspension of Aspergillus oryzae spores having a concentration of 1.0 x 10 7 spores / mL to 1.0 x 10 8 spores / mL is inoculated.

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