Method for improving disease-resistant activity by coupling gradient enzymolysis and fermentation

Abstract

The invention provides a method for improving disease-resistant activity through gradient enzymolysis coupling fermentation, and belongs to the technical field of biological fermentation. The method comprises the steps of raw material pretreatment, multi-stage gradient enzymolysis, enzymolysis-fermentation coupling and product post-treatment. The core innovation is that the activity of cellulase, protease and amylase is sequentially activated by precisely regulating and controlling the temperature to be 45-60 DEG C and the gradient change of pH to be 5.5-7.0, and oriented full degradation of raw material macromolecules is achieved; after enzymolysis, directly inoculating lactic acid bacteria-saccharomycetes composite flora for coupled fermentation, and supplementing a nitrogen source in batches. According to the method, the problems of insufficient enzymolysis, low active substance yield and the like of a traditional segmented process are solved, the active substance yield is increased to 2.1-2.4 times of that of the traditional process, the diameter of a pathogenic bacterium inhibition zone of the product reaches 24.8-26.3 mm, and the process is green and efficient, is suitable for industrial production and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of bio-fermentation technology, and more specifically, to a method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation. Background Technology

[0002] Disease-resistant active substances, such as antimicrobial peptides, organic acids, and plant-derived antimicrobial factors, have shown broad application prospects in fields such as green pest and disease control in agriculture, natural food preservation, and anti-infection in biomedicine due to their advantages of high efficiency, environmental friendliness, and low likelihood of inducing drug resistance. They have become one of the current research hotspots in the field of bioengineering. Currently, the mainstream process for the industrial preparation of disease-resistant active substances is still mainly based on enzymatic hydrolysis-fermentation staged processing. The typical process is as follows: first, raw materials rich in dietary fiber, protein, and polysaccharides undergo single or simple mixed enzymatic hydrolysis; then, the hydrolysis products are transferred to specialized fermentation equipment for microbial fermentation; and finally, the active substances are extracted from the fermentation broth.

[0003] However, traditional segmented processes have many insurmountable technical defects that hinder the improvement of product quality and production efficiency: First, enzymatic hydrolysis efficiency is low, and the degradation of macromolecules is insufficient. The optimal operating conditions of different enzymes, such as cellulase, protease, and amylase, vary significantly. For example, the optimal pH for cellulase is mostly 4.8-5.8 and the optimal temperature is 45-50℃, the optimal pH for protease is mostly 6.0-7.0 and the optimal temperature is 50-55℃, and the optimal pH for amylase is mostly 6.5-7.5 and the optimal temperature is 55-60℃. Traditional single-enzyme hydrolysis can only meet the activity requirements of one enzyme, while mixed enzymatic hydrolysis inhibits the activity of most enzymes due to conflicting conditions, making it impossible to achieve comprehensive degradation of dietary fiber, protein, and polysaccharides in raw materials. The production of small molecule nutrients such as glucose, amino acids, and oligosaccharides is low, which directly affects the metabolic efficiency of microorganisms in the subsequent fermentation stage. Second, the process is cumbersome and has a high risk of contamination. Enzymatic hydrolysis and fermentation are carried out in different equipment. During material transfer, not only are nutrients lost, but contamination by other microorganisms is also easily introduced, leading to fermentation failure or decreased product purity. Thirdly, the yield of active substances is low, and disease resistance is weak. Due to insufficient fermentation substrate and interference from other microorganisms, microbial metabolism is incomplete, resulting in limited synthesis of active substances. The product has poor inhibitory effects on pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Penicillium, with inhibition zone diameter typically only 10-12 mm, which is insufficient for practical applications. Fourthly, production energy consumption and costs are high. Segmented processing increases equipment investment, energy consumption, and labor costs, hindering large-scale industrial application. Therefore, a gradient enzymatic hydrolysis coupled with fermentation method is proposed to enhance disease resistance activity. Summary of the Invention

[0004] The purpose of this invention is to address the problems raised in the existing background technology. To achieve the above-mentioned objective, this invention provides the following technical solution: a method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation, comprising the following steps: Step 1, raw material pretreatment: Select raw materials containing dietary fiber, protein, and polysaccharides, pulverize them, sieve them through an 80-100 mesh screen, sterilize them at 121℃ and 0.1MPa for 20-30 minutes, cool them to room temperature, and then add deionized water to prepare a raw material suspension with a solid-liquid ratio of 1:8-1:12; Step 2, Multi-stage gradient enzymatic hydrolysis: Cellulase, protease, and amylase are added sequentially to the raw material suspension, with the addition amounts of cellulase, protease, and amylase being 0.5%-1.0%, 0.3%-0.8%, and 0.2%-0.5% of the dry weight of the raw material, respectively. The activity of each enzyme is activated sequentially by precisely controlling the temperature and pH gradient changes to achieve the directional decomposition of macromolecules in the raw material. The specific control conditions are as follows: initial temperature 45℃, pH 5.5, maintained for 8-12 hours; then the temperature is increased to 52-55℃ at a rate of 1-2℃ / hour, while the pH is adjusted to 6.2-6.5 with dilute hydrochloric acid or sodium hydroxide solution at a rate of 0.1-0.2 pH / hour, maintained for 6-8 hours; finally, the temperature is increased to 60℃ at a rate of 1-2℃ / hour, while the pH is adjusted to 7.0 with dilute hydrochloric acid or sodium hydroxide solution at a rate of 0.1-0.2 pH / hour, maintained for 4-6 hours. Step 3, Enzymatic hydrolysis-fermentation coupling: After the gradient enzymatic hydrolysis is completed, without transferring the material, a complex microbial community of lactic acid bacteria and yeast is directly introduced into the enzymatic hydrolysis system. The inoculation ratio of lactic acid bacteria to yeast in the complex microbial community is 3:1-5:1, and the total inoculation amount is 5%-8% of the volume of the enzymatic hydrolysis system. Maintain a constant temperature of 37°C for fermentation. Add nitrogen source in 3-4 batches through a feeding device, with an interval of 12 hours between each addition. The total amount of nitrogen source added is 2%-5% of the dry weight of the enzymatic hydrolysis system. Fermentation lasts for 48 hours. Step 4, Post-processing of product: The fermentation broth is centrifuged at 8000-10000 r / min for 15-20 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then concentrated under reduced pressure at 50-60℃ to 1 / 5-1 / 3 of the original volume. Finally, spray drying is performed, with the inlet air temperature controlled at 160-180℃ and the outlet air temperature at 80-90℃, to obtain a product with high disease resistance activity.

[0005] As a preferred technical solution of the present invention, in step 1, the raw material is one or more of corn grits, soybean meal, and wheat bran, and the raw material is crushed and passed through a 90-mesh standard sieve.

[0006] As a preferred embodiment of the present invention, in step 2, the cellulase is a neutral cellulase with an enzyme activity ≥ 5000 U / g; the protease is a neutral protease with an enzyme activity ≥ 8000 U / g; and the amylase is an α-amylase with an enzyme activity ≥ 10000 U / g.

[0007] As a preferred technical solution of the present invention, in step 2, during the pH adjustment process, the pH value is sampled and detected every 30 minutes, and the adjustment rate is finely adjusted according to the detection results to ensure that the pH is stable within the target range.

[0008] As a preferred technical solution of the present invention, in step 3, the lactic acid bacteria are Lactobacillus plantarum and the yeast is Saccharomyces cerevisiae.

[0009] As a preferred technical solution of the present invention, in step 3, the nitrogen source is one or more of yeast extract, peptone, and urea, and when a mixed nitrogen source is used, the mass ratio of yeast extract, peptone, and urea is 2:1:1.

[0010] As a preferred technical solution of the present invention, in step 3, the feeding rate of the feeding device is 0.5-1.0 mL / min, and after each feeding is completed, the stirring rate of the fermentation system is maintained at 150-200 r / min for 30 minutes.

[0011] As a preferred technical solution of the present invention, in step 3, the dissolved oxygen content of the fermentation system is maintained at 2-5 mg / L during the fermentation process, which is regulated by the aeration rate of 0.1-0.3 vvm.

[0012] As a preferred technical solution of the present invention, in step 4, the centrifugation process adopts refrigerated centrifugation, and the centrifugation temperature is 4-8℃; the vacuum degree of the reduced pressure concentration is 0.06-0.08MPa.

[0013] As a preferred technical solution of the present invention, in step 4, the feed rate of spray drying is 10-20 mL / min, and the resulting high disease-resistant active product has a water content of ≤5% and a particle size of 100-200 mesh.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention achieves thorough degradation of macromolecules and significantly improves substrate utilization. By precisely controlling the temperature gradient from 45℃ to 60℃ and the pH gradient from 5.5 to 7.0, this invention provides optimal environments for cellulase, protease, and amylase, enabling the efficient sequential activation of these three enzymes. This achieves targeted and stepwise degradation of dietary fiber, protein, and polysaccharides in the raw materials, increasing the production of small-molecule nutrients by 1.8-2.2 times compared to traditional processes. This provides ample and high-quality substrates for subsequent fermentation, ensuring efficient synthesis of active substances from the source.

[0015] This invention achieves high yield of active substances and significantly improves production efficiency. The enzymatic hydrolysis-fermentation coupled system established in this invention eliminates the need for material transfer, allowing the enzymatic hydrolysis products to be directly utilized by microorganisms as fermentation substrates, thus avoiding nutrient loss and contamination by other microorganisms. Simultaneously, lactic acid bacteria and yeast form a synergistic metabolic network. The organic acids produced by lactic acid bacteria optimize the pH of the fermentation system, while the vitamins produced by yeast promote the proliferation of lactic acid bacteria, significantly enhancing microbial metabolic activity and substantially reducing production energy consumption and costs.

[0016] The product of this invention exhibits significantly enhanced disease-resistant activity and a wider range of applications. The product prepared by this invention contains sufficient amounts of active substances such as antimicrobial peptides and organic acids, demonstrating potent inhibitory effects against a variety of pathogenic bacteria. Specifically, the inhibition zone diameter against Staphylococcus aureus reaches 25.6-26.3 mm, and the inhibition zone diameter against Escherichia coli reaches 24.8-25.5 mm, which is 13.6-16.5 mm larger than that of traditional products. This not only meets the needs of agricultural pest and disease control and food preservation, but can also be used in the development of anti-infective products in the biopharmaceutical field, thus broadening its application scenarios.

[0017] This invention features a green and efficient process suitable for industrial production. It achieves integrated continuous operation of enzymatic hydrolysis and fermentation, simplifying production processes, reducing equipment investment and material transfer steps, and lowering the risk of contamination by other microorganisms. Furthermore, the process requires no toxic or harmful chemical reagents, and the fermentation waste liquid can be recycled after simple treatment, meeting green and environmentally friendly production requirements. In addition, all process parameters are precisely controllable, highly stable, and easily automated, making it suitable for large-scale industrial production.

[0018] This invention boasts broad raw material adaptability and high resource utilization. It is compatible with various agricultural waste raw materials such as corn grits, soybean meal, wheat bran, and straw. Through gradient enzymatic hydrolysis, it achieves efficient degradation of macromolecular substances in different raw materials, not only broadening the sources of raw materials but also enhancing the resource utilization value of agricultural waste, thus contributing to the development of circular agriculture. Attached Figure Description

[0019] Figure 1 A data flowchart for the enzymatic hydrolysis stage provided by this invention; Figure 2 A data flow diagram of the microbial community provided by this invention; Figure 3 A flowchart provided for this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 some, not all, of the embodiments of the present invention.

[0021] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0022] Example 1: A method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation, comprising the following steps: Step 1, raw material pretreatment: Select raw materials containing dietary fiber, protein and polysaccharides, crush them, sieve them through an 80-100 mesh, sterilize them at 121℃ and 0.1MPa for 20-30 minutes, cool them to room temperature, and add deionized water to prepare a raw material suspension with a solid-liquid ratio of 1:8-1:12; Step 2, Multi-stage gradient enzymatic hydrolysis: Cellulase, protease, and amylase are added sequentially to the raw material suspension, with the addition amounts of cellulase, protease, and amylase being 0.5%-1.0%, 0.3%-0.8%, and 0.2%-0.5% of the dry weight of the raw material, respectively. The activity of each enzyme is activated sequentially by precisely controlling the temperature and pH gradient changes to achieve the directional decomposition of macromolecules in the raw material. The specific control conditions are as follows: initial temperature 45℃, pH 5.5, maintained for 8-12 hours; then the temperature is increased to 52-55℃ at a rate of 1-2℃ / hour, while the pH is adjusted to 6.2-6.5 with dilute hydrochloric acid or sodium hydroxide solution at a rate of 0.1-0.2 pH / hour, maintained for 6-8 hours; finally, the temperature is increased to 60℃ at a rate of 1-2℃ / hour, while the pH is adjusted to 7.0 with dilute hydrochloric acid or sodium hydroxide solution at a rate of 0.1-0.2 pH / hour, maintained for 4-6 hours. Step 3, Enzymatic hydrolysis-fermentation coupling: After the gradient enzymatic hydrolysis is completed, without transferring the material, a complex microbial community of lactic acid bacteria and yeast is directly introduced into the enzymatic hydrolysis system. The inoculation ratio of lactic acid bacteria to yeast in the complex microbial community is 3:1-5:1, and the total inoculation amount is 5%-8% of the volume of the enzymatic hydrolysis system. Maintain a constant temperature of 37°C for fermentation. Add nitrogen source in 3-4 batches through a feeding device, with an interval of 12 hours between each addition. The total amount of nitrogen source added is 2%-5% of the dry weight of the enzymatic hydrolysis system. Fermentation lasts for 48 hours. Step 4, Post-processing of product: The fermentation broth is centrifuged at 8000-10000 r / min for 15-20 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then concentrated under reduced pressure at 50-60℃ to 1 / 5-1 / 3 of the original volume. Finally, spray drying is performed, with the inlet air temperature controlled at 160-180℃ and the outlet air temperature at 80-90℃, to obtain a product with high disease resistance activity.

[0023] In step 1, the raw material is one or more of corn grits, soybean meal, and wheat bran, and the raw material is crushed and passed through a 90-mesh standard sieve.

[0024] In step 2, the cellulase is a neutral cellulase with an enzyme activity ≥ 5000 U / g; the protease is a neutral protease with an enzyme activity ≥ 8000 U / g; and the amylase is an α-amylase with an enzyme activity ≥ 10000 U / g.

[0025] In step 2, during the pH adjustment process, the pH value is sampled and tested every 30 minutes. The adjustment rate is then fine-tuned based on the test results to ensure that the pH remains stable within the target range.

[0026] In step 3, the lactic acid bacteria are Lactobacillus plantarum and the yeast is Saccharomyces cerevisiae.

[0027] In step 3, the nitrogen source is one or more of yeast extract, peptone, and urea, and when a mixed nitrogen source is used, the mass ratio of yeast extract, peptone, and urea is 2:1:1.

[0028] In step 3, the feeding rate of the feeding device is 0.5-1.0 mL / min. After each feeding, the stirring rate of the fermentation system is maintained at 150-200 r / min for 30 minutes.

[0029] In step 3, the dissolved oxygen content of the fermentation system is maintained at 2-5 mg / L during the fermentation process, which is regulated by the aeration rate of 0.1-0.3 vvm.

[0030] In step 4, the centrifugation process uses refrigerated centrifugation at a temperature of 4-8℃; the vacuum degree for reduced pressure concentration is 0.06-0.08MPa.

[0031] In step 4, the feed rate of spray drying is 10-20 mL / min, and the resulting high disease-resistant active product has a water content of ≤5% and a particle size of 100-200 mesh.

[0032] The working principle of this invention is to achieve the efficient preparation of disease-resistant active substances through the synergistic effect of gradient enzymatic hydrolysis-directed degradation coupled with efficient fermentation synthesis. The specific principle is as follows: Gradient enzymatic hydrolysis stage principle: Different enzymes, such as cellulase, protease, and amylase, have different optimal temperatures and pH values. Cellulase's optimal conditions are 45℃ and pH 5.5, protease's are 52-55℃ and pH 6.2-6.5, and amylase's are 60℃ and pH 7.0. This invention precisely controls the temperature gradient from 45℃ to 60℃ and the pH gradient from 5.5 to 7.0, providing optimal reaction environments for the three enzymes sequentially. This allows them to be activated and efficiently exert their catalytic effects, achieving the targeted and stepwise degradation of dietary fiber, protein, and polysaccharides in the raw materials. This converts macromolecules into small molecule nutrients such as glucose, amino acids, and oligosaccharides, providing sufficient nutrient substrates for subsequent fermentation stages. Simultaneously, the rate of temperature increase and pH adjustment is strictly controlled during the gradient control process to avoid enzyme inactivation due to sudden changes in environmental parameters, ensuring degradation efficiency.

[0033] The principle of the enzymatic hydrolysis-fermentation coupling stage: After gradient enzymatic hydrolysis, the hydrolysis system retains abundant small-molecule nutrients. These nutrients can be directly introduced into a complex microbial community of lactic acid bacteria and yeast without material transfer, establishing a coupled system. Lactic acid bacteria utilize small-molecule sugars to ferment and produce organic acids such as lactic acid and acetic acid, while yeast utilizes nitrogen sources such as amino acids and sugar metabolism to produce active substances such as antimicrobial peptides. Furthermore, the two microbial communities exhibit synergistic effects—the organic acids produced by lactic acid bacteria regulate the pH of the fermentation system, providing a suitable environment for yeast growth and metabolism; and the vitamins and other substances produced by yeast metabolism promote lactic acid bacteria proliferation, further enhancing the efficiency of active substance synthesis. By adding nitrogen sources in batches, the problems of excessively high nitrogen concentration inhibiting microbial growth or insufficient concentration leading to inadequate metabolism caused by single-feeding can be avoided, ensuring the complex microbial community continuously and efficiently produces active substances within the 48-hour fermentation cycle. In addition, parameters such as dissolved oxygen and stirring rate are adjusted during fermentation to further optimize the metabolic environment of the microbial community and improve the yield of active substances.

[0034] The technical solution of the invention consists of a complete working process divided into a raw material pretreatment stage, a multi-stage gradient enzymatic hydrolysis stage, an enzymatic hydrolysis-fermentation coupling stage, and a product post-treatment stage. Each stage is sequentially connected, and the specific process is as follows: Raw material pretreatment stage: First, raw materials containing dietary fiber, protein, and polysaccharides, such as corn grits, soybean meal, and wheat bran, are selected and combined individually or in combination as needed; the raw materials are crushed and passed through an 80-100 mesh sieve to ensure uniform particle size and increase the contact area for subsequent enzymatic hydrolysis reactions; the sieved raw materials are placed in a sterilization device and sterilized at 121℃ and 0.1MPa for 20-30 minutes to kill miscellaneous bacteria in the raw materials and avoid contamination of the product purity during subsequent fermentation; after sterilization, the raw materials are cooled to room temperature and deionized water is added to prepare a raw material suspension with a solid-liquid ratio of 1:8-1:12, providing a uniform reaction system for the enzymatic hydrolysis reaction.

[0035] Multi-stage gradient enzymatic hydrolysis stage: Cellulase, protease, and amylase are added sequentially to the raw material suspension at amounts of 0.5%-1.0%, 0.3%-0.8%, and 0.2%-0.5% of the raw material dry weight, respectively; the temperature and pH control system is activated, maintaining a temperature of 45℃ and a pH of [missing information - likely a specific value]. 5.5, react for 8-12 hours to activate cellulase to degrade dietary fiber; after the first stage, increase the temperature to 52-55℃ at a rate of 1-2℃ / hour, and simultaneously adjust the pH to 6.2-6.5 with dilute hydrochloric acid or sodium hydroxide solution at a rate of 0.1-0.2pH / hour, maintaining the reaction for 6-8 hours to activate protease to degrade protein; after the second stage, continue to increase the temperature to 60℃ at a rate of 1-2℃ / hour, and adjust the pH to 7.0 at a rate of 0.1-0.2pH / hour, maintaining the reaction for 4-6 hours to activate amylase to degrade polysaccharides; throughout the enzymatic hydrolysis process, sample and test the pH value every 30 minutes, and fine-tune the adjustment rate according to the test results to ensure that the parameters of each stage are stable within the target range, ensuring the enzymatic hydrolysis effect.

[0036] Enzymatic hydrolysis-fermentation coupling stage: After the gradient enzymatic hydrolysis reaction is completed, without transferring the hydrolysis products, the lactic acid bacteria *Lactobacillus plantarum* CGMCC 1.3990 and the yeast *Saccharomyces cerevisiae* CGMCC are directly inoculated into the reaction system. 2.1808 complex microbial community, with an inoculation ratio controlled at 3:1-5:1, and a total inoculation amount of 5%-8% of the enzymatic hydrolysis system volume; adjust the reaction system temperature to 37℃ and maintain a constant temperature, start the feeding device, and add nitrogen sources such as yeast extract, peptone, urea, etc., in 3-4 batches, with a total addition amount of 2%-5% of the dry weight of the enzymatic hydrolysis system, with each addition at an interval of 12 hours, and the feeding rate controlled at 0.5-1.0 mL / min. After each feeding, stir at a rate of 150-200 r / min for 30 minutes to ensure uniform dispersion of nitrogen sources; during fermentation, maintain the dissolved oxygen content of the system at 2-5 mg / L by adjusting the aeration rate of 0.1-0.3 vvm to ensure normal growth and metabolism of the complex microbial community, and continue fermentation for 48 hours until the synthesis of active substances reaches its peak.

[0037] Post-processing stage: After fermentation, the fermentation broth is transferred to a centrifuge and centrifuged at 4-8℃ and 8000-10000 r / min for 15-20 minutes to separate and remove the bacterial cells and a small amount of undegraded solid impurities from the fermentation broth. The supernatant is then filtered through a 0.22μm filter membrane to further remove fine impurities and some macromolecular impurities, resulting in a clear crude extract of active substances. The crude extract is then placed in a vacuum concentration device and concentrated to 1 / 5-1 / 3 of its original volume at 50-60℃ and 0.06-0.08MPa to increase the concentration of active substances. Finally, the concentrate is dried using a spray dryer, with the inlet air temperature controlled at 160-180℃, the outlet air temperature at 80-90℃, and the feed rate at 10-20mL / min. After drying, a high-disease-resistant active product with a water content ≤5% and a particle size of 100-200 mesh is obtained, completing the entire preparation process.

[0038] Example 2: A method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation. Raw material pretreatment: Wheat bran is selected as raw material, crushed and passed through a 100-mesh sieve. 80g of the sieved raw material is placed in a fermenter and 800mL of deionized water is added to prepare a raw material suspension. The suspension is then sterilized at 121℃ and 0.1MPa for 20 minutes. Multi-stage gradient enzymatic hydrolysis: 0.4 g cellulase, 0.24 g protease and 0.16 g amylase were added to the sterilized raw material suspension; the initial enzymatic hydrolysis temperature was controlled at 45℃ and pH at 5.5, and maintained for 8 hours; then the temperature was increased to 52℃ at a rate of 1℃ / hour, and the pH was adjusted to 6.2 at a rate of 0.1 pH / hour, and maintained for 6 hours; finally, the temperature was increased to 60℃ at a rate of 1℃ / hour, and the pH was adjusted to 7.0 at a rate of 0.1 pH / hour, and maintained for 4 hours. Enzymatic hydrolysis-fermentation coupling: After the gradient enzymatic hydrolysis is completed, a compound microbial community of lactic acid bacteria and yeast is directly introduced into the fermenter at a ratio of 3:1, with a total inoculation amount of 5% of the volume of the enzymatic hydrolysis system; the fermentation temperature is maintained at a constant 37℃, and urea is added in batches through a feeding device, with a total addition amount of 2% of the dry weight of the raw materials, added in 3 times, with an interval of 12 hours between each addition, and fermentation lasts for 48 hours. Post-processing of the product: After fermentation, the fermentation broth was centrifuged at 8000 r / min for 15 minutes, and the supernatant was filtered through a 0.22 μm filter membrane; it was concentrated under reduced pressure at 50℃ to 1 / 5 of the original volume; spray drying was carried out with an inlet air temperature of 160℃ and an outlet air temperature of 80℃ to obtain a product with high disease resistance activity.

[0039] Testing showed that the yield of active substances in the product prepared in this embodiment was 2.1 times that of the traditional segmented process, and the diameter of the inhibition zone against Escherichia coli was 24.8 mm, indicating significantly enhanced disease resistance.

[0040] Example 3: A method for enhancing disease resistance through gradient enzymatic hydrolysis coupled fermentation, comprising the following steps: Raw material pretreatment: Corn residue, soybean meal and wheat bran were mixed in a mass ratio of 2:1:1 as raw materials. After being crushed, the mixture was passed through a 90-mesh sieve. 120g of the sieved raw material was placed in a fermenter and 1440mL of deionized water was added to prepare a raw material suspension. The suspension was then sterilized at 121℃ and 0.1MPa for 30 minutes. Multi-stage gradient enzymatic hydrolysis: 1.2g cellulase, 0.96g protease and 0.6g amylase were added to the sterilized raw material suspension; the initial enzymatic hydrolysis temperature was controlled at 45℃ and pH at 5.5, and maintained for 12 hours; then the temperature was increased to 55℃ at a rate of 2℃ / hour, and the pH was adjusted to 6.5 at a rate of 0.2pH / hour, and maintained for 8 hours; finally, the temperature was increased to 60℃ at a rate of 2℃ / hour, and the pH was adjusted to 7.0 at a rate of 0.2pH / hour, and maintained for 6 hours. Enzymatic hydrolysis-fermentation coupling: After the gradient enzymatic hydrolysis is completed, a complex microbial community of lactic acid bacteria and yeast is directly introduced into the fermenter at a ratio of 5:1, with a total inoculation amount of 8% of the enzymatic hydrolysis system volume; the fermentation temperature is maintained at a constant 37℃, and a mixed nitrogen source of yeast extract, peptone and urea at a mass ratio of 2:1:1 is added in batches through a feeding device, with a total addition amount of 5% of the dry weight of the raw materials, added in 4 batches at 12-hour intervals, and fermentation lasts for 48 hours; Product post-processing: After fermentation, the fermentation broth was centrifuged at 10,000 r / min for 20 minutes, and the supernatant was filtered through a 0.22 μm filter membrane; it was concentrated under reduced pressure at 60℃ to 1 / 3 of the original volume; spray drying was carried out with an inlet air temperature of 180℃ and an outlet air temperature of 90℃ to obtain a product with high disease resistance activity.

[0041] Testing showed that the yield of active substances in the product prepared in this embodiment was 2.4 times that of the traditional segmented process, and the diameters of the inhibition zones against Staphylococcus aureus and Escherichia coli were 26.3 mm and 25.5 mm, respectively, indicating excellent disease resistance activity.

[0042] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. A method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation, characterized in that, Includes the following steps: Step 1, Raw material pretreatment: Select raw materials containing dietary fiber, protein and polysaccharides, crush them, sieve them through an 80-100 mesh, sterilize them at 121℃ and 0.1MPa for 20-30 minutes, cool them to room temperature and add deionized water to make a raw material suspension with a solid-liquid ratio of 1:8-1:

12. Step 2, Multi-stage gradient enzymatic hydrolysis: Cellulase, protease, and amylase are added sequentially to the raw material suspension, with the addition amounts of cellulase, protease, and amylase being 0.5%-1.0%, 0.3%-0.8%, and 0.2%-0.5% of the dry weight of the raw material, respectively. The activity of each enzyme is activated sequentially by precisely controlling the temperature and pH gradient changes to achieve the directional decomposition of macromolecules in the raw material. The specific control conditions are as follows: initial temperature 45℃, pH 5.5, maintained for 8-12 hours; then the temperature is increased to 52-55℃ at a rate of 1-2℃ / hour, while the pH is adjusted to 6.2-6.5 with dilute hydrochloric acid or sodium hydroxide solution at a rate of 0.1-0.2 pH / hour, maintained for 6-8 hours; finally, the temperature is increased to 60℃ at a rate of 1-2℃ / hour, while the pH is adjusted to 7.0 with dilute hydrochloric acid or sodium hydroxide solution at a rate of 0.1-0.2 pH / hour, maintained for 4-6 hours. Step 3, Enzymatic hydrolysis-fermentation coupling: After the gradient enzymatic hydrolysis is completed, without transferring the material, a complex microbial community of lactic acid bacteria and yeast is directly introduced into the enzymatic hydrolysis system. The inoculation ratio of lactic acid bacteria to yeast in the complex microbial community is 3:1-5:1, and the total inoculation amount is 5%-8% of the volume of the enzymatic hydrolysis system. Maintain a constant temperature of 37°C for fermentation. Add nitrogen source in 3-4 batches through a feeding device, with an interval of 12 hours between each addition. The total amount of nitrogen source added is 2%-5% of the dry weight of the enzymatic hydrolysis system. Fermentation lasts for 48 hours. Step 4, Post-processing of product: The fermentation broth is centrifuged at 8000-10000 r / min for 15-20 minutes. The supernatant is filtered through a 0.22 μm filter membrane and then concentrated under reduced pressure at 50-60℃ to 1 / 5-1 / 3 of the original volume. Finally, spray drying is performed, with the inlet air temperature controlled at 160-180℃ and the outlet air temperature at 80-90℃, to obtain a product with high disease resistance activity.

2. The method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 1, the raw material is one or more of corn grits, soybean meal, and wheat bran, and the raw material is crushed and passed through a 90-mesh standard sieve.

3. The method for enhancing disease resistance through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 2, the cellulase is a neutral cellulase with an enzyme activity ≥ 5000 U / g; the protease is a neutral protease with an enzyme activity ≥ 8000 U / g; and the amylase is an α-amylase with an enzyme activity ≥ 10000 U / g.

4. The method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 2, during the pH adjustment process, the pH value is sampled and tested every 30 minutes. The adjustment rate is then fine-tuned based on the test results to ensure that the pH remains stable within the target range.

5. The method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 3, the lactic acid bacteria are Lactobacillus plantarum and the yeast is Saccharomyces cerevisiae.

6. The method for enhancing disease resistance through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 3, the nitrogen source is one or more of yeast extract, peptone, and urea, and when a mixed nitrogen source is used, the mass ratio of yeast extract, peptone, and urea is 2:1:

1.

7. The method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 3, the feeding rate of the feeding device is 0.5-1.0 mL / min. After each feeding, the stirring rate of the fermentation system is maintained at 150-200 r / min for 30 minutes.

8. The method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 3, the dissolved oxygen content of the fermentation system is maintained at 2-5 mg / L during the fermentation process, which is regulated by the aeration rate of 0.1-0.3 vvm.

9. The method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 4, the centrifugation process uses refrigerated centrifugation at a temperature of 4-8℃; the vacuum degree for reduced pressure concentration is 0.06-0.08MPa.

10. The method for enhancing disease resistance activity through gradient enzymatic hydrolysis coupled fermentation according to claim 1, characterized in that, In step 4, the feed rate of spray drying is 10-20 mL / min, and the resulting high disease-resistant active product has a water content of ≤5% and a particle size of 100-200 mesh.

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