Low soybean meal type pig feed formula based on enzymolysis protein and preparation method thereof

Abstract

The invention relates to the technical field of pig feed, and discloses a low soybean meal type pig feed formula based on enzymolysis protein and a preparation method of the low soybean meal type pig feed formula based on enzymolysis protein, dephenolized cottonseed meal, corn alcohol lees and the like are used as main raw materials, and the core of the low soybean meal type pig feed formula is that gradient enzymolysis and directional fermentation are dynamically coupled. The probiotics are subjected to specific induction domestication, so that the probiotics can efficiently adapt to a reaction system; in the coupling process, compound cellulase, protease and the like are added step by step according to a time sequence, the pH value of the system is intelligently adjusted by utilizing acid generated by fermentation, and optimal conditions are created for subsequent directional enzymolysis, so that the functional peptide with specific molecular weight is enriched. And finally, a low-temperature keep-alive drying technology is adopted, so that the biological activity of probiotics and functional peptides in the product is completely reserved. According to the method, raw material harmlessness, protein directional conversion and active substance protection are integrated, and high-value utilization of low-value agricultural and sideline products is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of pig feed, in particular to a low-soybean meal pig feed formula based on enzymatic protein and a preparation method thereof. Background Art

[0002] With the continued development of the global livestock industry, the demand for efficient and economical feed protein sources is growing. The rising price and unstable supply of soybean meal, a traditional high-quality protein source, have prompted the industry to actively seek alternative, more cost-effective, low-value plant protein and industrial by-product resources, such as dephenolized cottonseed meal and distillers grains with grains from corn (DDGS). However, the widespread use of these alternative raw materials has always faced severe technical challenges. They generally contain high levels of cellulose, hemicellulose, and anti-nutritional factors such as phytic acid and gossypol. These components not only limit the release and absorption of nutrients in the raw materials but also pose a potential threat to the intestinal health of young animals, resulting in strict restrictions on their inclusion in feed formulas.

[0003] In order to improve the feed value of these low-value raw materials, existing technologies usually use biotechnology methods such as enzymatic hydrolysis or microbial fermentation for pretreatment. However, the currently commonly used process routes have significant limitations and fail to fully realize the potential of biological treatment. The existing enzymatic hydrolysis process is often a relatively extensive degradation process, tending to add multiple proteases into the reaction system at one time. This "one-step" treatment method makes it difficult to accurately control the degradation process, resulting in a broad and random distribution of the peptide spectrum of the final product, and the inability to stably enrich functional small peptides with specific physiological activities.

[0004] On the other hand, although microbial fermentation technology can effectively degrade anti-nutritional factors and produce beneficial metabolites, its industrial application also faces bottlenecks. When conventional commercial probiotics are directly introduced into a complex industrial raw material system, the strains need to go through a long growth lag period to adapt to the new environment, which makes the fermentation process slow, inefficient and has poor stability between batches. More importantly, the existing technology usually performs enzymatic hydrolysis and fermentation as two independent unit operations in series. This process design is not only lengthy, energy-intensive, and requires large equipment footprint, but also fundamentally separates the possible synergistic effect between enzymatic reactions and microbial metabolism.

[0005] Ultimately, after obtaining the enzymatic hydrolysis or fermentation product, the commonly used drying method to obtain a stable powdered product is high-temperature spray drying. Although this method is highly efficient, its intense thermal shock can cause devastating damage to heat-sensitive substances with biological activity in the product. As a result, even if beneficial probiotics are successfully generated or some enzyme activity is retained in the early stages of the process, they will be almost completely inactivated during the final drying step, rendering the final product merely a carrier of nutrients rather than a functional system with biological activity, greatly reducing its application effectiveness. Summary of the Invention

[0006] The present invention aims to solve the technical problems commonly encountered in the prior art when processing low-value plant protein raw materials, such as the separation of enzymatic hydrolysis and fermentation processes, low process efficiency, single product function and difficulty in retaining bioactive substances.

[0007] In a first aspect, the present invention provides a low-soybean meal pig feed formula composition based on enzymatic protein hydrolysis. This composition is an active peptide synbiotic obtained through a specific biomanufacturing process. The composition is prepared from the following raw materials in parts by weight: 60-70 parts dephenolized cottonseed meal, 20-30 parts corn distillers grains, and 10-20 parts animal-derived protein waste. The essential feature of this composition is that it is an organic, active complex comprising functional peptides derived from the degradation of raw material proteins, a complex probiotic community that grows and ultimately survives during the preparation process, and endogenous prebiotics derived from the fiber components of the raw materials themselves.

[0008] As a preferred technical solution, the surviving composite probiotic bacteria group includes high-protease-producing Bacillus subtilis and acid-tolerant Lactobacillus plantarum. The symbiotic combination of these two strains can work synergistically during the preparation process, with Bacillus subtilis assisting in protein degradation and Lactobacillus plantarum regulating the system environment by producing acid.

[0009] As another preferred technical solution, the molecular weight of the functional peptide group is mainly distributed in the range of 500 to 2000 Da. Peptides in this molecular weight range have good water solubility, low antigenicity, and are easily absorbed and utilized by young animals.

[0010] As another preferred technical solution, the endogenous prebiotics are oligosaccharides produced by the degradation of cellulose and hemicellulose in corn ethanol grains and cottonseed meal by exogenous cellulolytic enzymes during the initial stages of the preparation process. These oligosaccharides can directly serve as a carbon source for the complex probiotic bacteria, promoting their growth and colonization within the system.

[0011] A second aspect of the present invention provides a method for preparing the aforementioned composition. The essence of this method lies in integrating multiple previously separate biochemical unit operations into a dynamically coupled, time-controlled continuous process. The process includes the following core steps: probiotic induction and acclimation, gradient enzymatic hydrolysis and coupled fermentation, and low-temperature drying to maintain viability.

[0012] The probiotic induction and acclimation step is an innovative pretreatment link of the present method. Its purpose is to solve the technical problem that when probiotics directly enter a complex mixed raw material system, a long growth hysteresis period will be generated due to environmental stress. The present method takes part of the raw materials, and after preliminary enzymatic hydrolysis, it is made into an "induction culture medium" that simulates the main reaction environment, and the probiotics are pre-cultured in this culture medium. This allows the probiotics to synthesize and express in advance the metabolic enzyme system that can adapt to and efficiently utilize the specific substrate before entering the main reactor, so that it can immediately play a role in the subsequent coupled fermentation stage. In a specific embodiment, this step includes: taking part of the raw materials to make a slurry, adding cellulase, hemicellulase and phytase for preliminary enzymatic hydrolysis, sterilizing the obtained primary hydrolyzate as an induction culture medium, and then inoculating the probiotics for cultivation to obtain a specific acclimated bacterial liquid.

[0013] The gradient enzymatic hydrolysis and coupled fermentation steps are the core technical components of this method. They creatively combine multi-stage protein degradation with microbial directed fermentation within a single reactor, forming a self-regulating and optimized biochemical reaction cascade. This step includes multiple, sequentially occurring phases: In the first gradient stage, under relatively mild conditions (e.g. 40-50°C), the enzymatic breaking of cell walls is mainly carried out, using cellulase, hemicellulase and phytase to break the physical structural barriers of plant raw materials, creating the prerequisites for the subsequent exposure and degradation of proteins.

[0014] In the second gradient stage, after the temperature is raised (e.g., 50-55°C), the first protease (preferably alkaline or neutral protease) and the prepared specific acclimation bacterial solution are added simultaneously. At this point, macroscopic protein degradation and microbial fermentation are initiated simultaneously.

[0015] The third gradient stage exploits a key natural phenomenon in the coupled fermentation process: the growth of Lactobacillus plantarum metabolizes and produces lactic acid, causing the pH of the entire reaction system to naturally and gently decrease. When the pH drops to a specific range (preferably 5.0-6.5), a second protease (preferably an acidic or flavor protease) with higher activity under slightly acidic conditions is immediately added. This design utilizes microbial metabolism to "intelligently" trigger subsequent enzymatic reactions, resulting in precise "directional cleavage" of preformed peptides to enrich for functional peptides of a specific molecular weight.

[0016] The low-temperature, life-preserving drying step is essential to ensuring the ultimate functionality of the composition of the present invention. After the coupled fermentation, the fermentation product is a complex system containing active probiotics and active peptides. To preserve the biological activity of these heat-sensitive components, this method utilizes low-temperature, life-preserving drying techniques, such as vacuum belt drying or freeze drying, to ensure that the material temperature remains below 60°C throughout the drying process. This fundamentally differs from traditional high-temperature spray drying, making the final product a truly "active" synbiotic rather than a simple nutritional powder.

[0017] The present invention provides a low-soybean meal pig feed formula based on enzymatic protein and a preparation method thereof. It has the following beneficial effects: 1. This invention creates a novel "active peptide synbiotic" composition through an integrated biomanufacturing process. This composition organically integrates endogenously generated functional peptides, a viable complex probiotic community, and prebiotics to form a micro-ecological system. Compared with traditional physically mixed products, the components of this composition exhibit natural synergistic effects, enabling more comprehensive and efficient improvements in animal intestinal health and increased nutrient absorption and utilization, thus providing a solid material foundation for the use of low-soybean meal diets. 2. The "dynamic coupling of gradient enzymatic hydrolysis and directed fermentation" process employed in this invention fundamentally solves the problem of separation between enzymatic hydrolysis and fermentation, leading to uncontrolled reactions in traditional processes. By precisely controlling the addition of different enzyme systems in a timed manner and utilizing the fermentation process's own metabolites to intelligently trigger subsequent directed enzymatic hydrolysis, this technology achieves in-depth control over the protein degradation process, enabling efficient enrichment of functional peptides of a specific molecular weight, significantly improving the targeted functionality and quality uniformity of the final product. 3. By introducing an innovative pretreatment step, "reaction system-specific probiotic acclimation," this invention successfully overcomes the technical challenge of long growth lags in complex industrial raw materials. The acclimated bacteria can quickly adapt to the main fermentation system and immediately initiate efficient metabolism. This not only significantly shortens the overall fermentation cycle but also ensures the stability and repeatability of the entire coupled fermentation process, laying the foundation for the industrialized and stable production of this technical solution. 4. This invention achieves truly high-value and harmless integrated utilization of low-value, difficult-to-use agricultural and sideline products, such as dephenolized cottonseed meal and corn distiller's grains. Through the synergistic action of multiple enzyme systems, this method not only efficiently degrades the protein in these products, but also thoroughly decomposes anti-nutritional factors such as phytic acid. Simultaneously, it converts components such as cellulose into usable prebiotics, greatly enhancing the economic value and feeding safety of these inexpensive raw materials. 5. This invention utilizes "low-temperature drying" as a necessary technical step to ensure the functionality of the final product. This step effectively prevents high temperatures from damaging heat-sensitive active substances such as probiotics and functional peptides, ensuring high probiotic survival and the natural spatial structure of the peptides in the final product, making the product a truly "active" preparation. This complete preservation of biological activity is the essential characteristic that distinguishes this product from traditional fermented feed and enzymatic protein powder, and is the fundamental guarantee for its superior physiological functions. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention. Example

[0019] This example describes a method for preparing an active peptide synbiotic.

[0020] Probiotic Acclimation: Weigh 65g of dephenolized cottonseed meal, 25g of corn ethanol grains, and 15g of fish waste and homogenize them. Take 10.5g of this mixture and add 50mL of deionized water to make a slurry. Add complex cellulase, hemicellulase, and phytase, and react at 45°C and 90 rpm for 1.5 hours. Sterilize this primary hydrolyzate at 121°C for 20 minutes and cool to 37°C. Inoculate a 1:1 mixture of Bacillus subtilis and Lactobacillus plantarum activated solution at a 2% (v / v) inoculum and incubate at 37°C and 110 rpm for 10 hours to prepare a specific acclimation solution.

[0021] Gradient enzymatic hydrolysis and coupled fermentation: The remaining 94.5 g of the mixture was mixed with 380 mL of deionized water to prepare the main slurry.

[0022] First gradient: adjust the temperature to 45°C, add complex cellulase, hemicellulase and phytase, and stir the reaction for 1.8 hours.

[0023] Second gradient: Raise the temperature to 52°C, add neutral complex protease, and immediately pump in all the specific acclimated bacterial solution prepared in step 1, and react under this condition for 5 hours.

[0024] Third gradient: When the pH value of the system was monitored to drop to 5.8, flavor protease was added and the reaction was continued at 52°C for 14 hours.

[0025] Low-temperature drying: The final fermentation slurry was quickly cooled to below 20°C and allowed to stand for 1.5 hours. It was then dried in a vacuum belt dryer with the heating plate set at 50°C and a vacuum degree of -0.09 MPa until the product moisture content was less than 8%.

[0026] Finished product processing: The dried material was crushed and passed through a 50-mesh sieve to obtain about 95 g of light yellow powdered active peptide synbiotics. Example

[0027] This example describes a method for preparing an active peptide synbiotic.

[0028] Probiotic acclimation: Weigh 60g of dephenolized cottonseed meal, 20g of corn distiller's grains, and 20g of fish waste and homogenize them. Take 10g of the mixture and add 50mL of deionized water to make a slurry. Add complex cellulase, hemicellulase, and phytase, and react at 40°C and 80 rpm for 2 hours. After sterilizing and cooling the primary hydrolyzate, inoculate the bacterial solution (inoculation volume 2%) and incubate at 37°C and 100 rpm for 8 hours to prepare the specific acclimation solution.

[0029] Gradient enzymatic hydrolysis and coupled fermentation: The remaining 90 g of the mixture was mixed with 360 mL of deionized water to prepare the main slurry.

[0030] First gradient: adjust the temperature to 40°C, add complex cellulase, hemicellulase and phytase, and stir the reaction for 2.0 hours.

[0031] Second gradient: raise the temperature to 50°C, add alkaline protease, and immediately pump in the acclimated bacterial solution. React under this condition for 4.0 hours.

[0032] Third gradient: When the pH value of the system was monitored to drop to 6.5, acidic protease was added and the reaction was continued at 50°C for 12 hours.

[0033] Low-temperature drying: The final fermentation slurry is quickly cooled to below 20°C and allowed to stand for 1.0 hour. It is then dried in a freeze dryer, pre-frozen to -40°C, and vacuum-dried until the moisture content of the product is less than 8%.

[0034] Finished product processing: The dried material was crushed and passed through a 40-mesh sieve to obtain about 91 g of powdered active peptide synbiotics. Example

[0035] This example describes a method for preparing an active peptide synbiotic.

[0036] Probiotic acclimation: Weigh 70g of dephenolized cottonseed meal, 30g of corn distiller's grains, and 10g of fish waste and homogenize them. Take 11g of the mixture and add 44mL of deionized water to make a slurry. Add complex cellulase, hemicellulase, and phytase, and react at 50°C and 100 rpm for 1.5 hours. After sterilizing and cooling the primary hydrolyzate, inoculate the bacterial solution (inoculation volume 3%) and incubate at 37°C and 120 rpm for 12 hours to prepare the specific acclimation solution.

[0037] Gradient enzymatic hydrolysis and coupled fermentation: The remaining 99 g of the mixture was mixed with 396 mL of deionized water to prepare the main slurry.

[0038] First gradient: adjust the temperature to 50°C, add complex cellulase, hemicellulase and phytase, and stir to react for 1.5 hours.

[0039] Second gradient: Raise the temperature to 55°C, add neutral protease, and immediately pump in the acclimated bacterial solution. React under this condition for 6.0 hours.

[0040] Third gradient: When the pH value of the system was monitored to drop to 5.0, flavor protease was added and the reaction was continued at 55°C for 16 hours.

[0041] Low-temperature drying: The final fermentation slurry was quickly cooled to below 20°C and allowed to stand for 2.0 hours. It was then dried in a vacuum belt dryer with the heating plate temperature set at 55°C and the vacuum degree at 0.08 MPa until the product moisture content was less than 8%.

[0042] Finished product processing: The dried material was crushed and passed through a 60-mesh sieve to obtain about 101 g of powdered active peptide synbiotics.

[0043] Comparative Example 1: Compared to Example 1, the difference is that the specific induction and acclimation step of the probiotics is omitted. Specifically, the Bacillus subtilis and Lactobacillus plantarum used for inoculation are activated only in conventional LB and MRS culture media. The activated bacterial liquids are then mixed in a 1:1 ratio and directly used for inoculation in the subsequent steps. All other raw material ratios, enzyme preparation addition amounts, reaction temperature, reaction time, and drying method are exactly the same as in Example 1.

[0044] Comparative Example 2: Compared to Example 1, the difference lies in the replacement of "gradient enzymatic hydrolysis" with "one-step enzyme addition." Specifically, in the gradient enzymatic hydrolysis and coupled fermentation step, the composite cellulase, hemicellulase, phytase, neutral composite protease, and flavor protease, introduced in three steps in Example 1, were added all at once to the main slurry after the temperature was raised to 52°C, and the acclimated bacterial solution was simultaneously inoculated. All other steps, raw material ratios, total reaction time, and drying method were identical to those in Example 1.

[0045] Comparative Example 3: Compared with Example 1, the difference is that the "enzymatic hydrolysis and fermentation coupling" process is changed to a "step-by-step series" process. Specifically: First, without inoculating any bacterial solution, a pure enzymatic hydrolysis reaction was performed in full accordance with the gradient enzymatic hydrolysis steps of Example 1 (the first and third gradients, wherein the pH of the third gradient was artificially adjusted to 5.8 by adding an organic acid before adding the second protease).

[0046] After the enzymatic hydrolysis was completed, the slurry was heated to 85°C and maintained for 20 minutes to completely inactivate all enzyme preparations.

[0047] The enzyme-inactivated slurry was cooled to 37°C and then inoculated with the same specific acclimated bacterial solution as in Example 1 for a separate fermentation step. The fermentation time was the same as the total fermentation time in Example 1. All other raw material ratios, enzyme dosages, and final drying methods were exactly the same as in Example 1.

[0048] Comparative Example 4: Compared to Example 1, the difference lies in replacing the "low-temperature drying" with "conventional high-temperature spray drying." Specifically, the final fermentation slurry obtained from the gradient enzymatic hydrolysis and coupled fermentation steps was dried using a high-temperature spray dryer. The air inlet temperature was set at 180°C, and the air outlet temperature was set at 85°C. All other steps, raw material ratios, enzyme preparation addition, and reaction parameters were identical to those in Example 1.

[0049] Test Example 1: Comparative test of probiotic activity in the final product 1. Experimental Description 1. Experimental Purpose This experiment aims to determine and compare the total number of surviving probiotics in the final products prepared by different drying methods, so as to verify the key role of the low-temperature drying step adopted in the present invention in protecting the biological activity of probiotics.

[0050] 2. Experimental Materials Sample A: final product powder prepared in Example 1.

[0051] Sample B: the final product powder prepared in Comparative Example 4.

[0052] Experimental equipment and reagents: sterile physiological saline, nutrient agar (NA) medium, sterile culture dishes, constant temperature incubator, high-precision electronic balance, pipette, vortex oscillator, clean bench.

[0053] 3. Experimental Procedure Sample preparation: Under sterile conditions, accurately weigh 1.0 g of sample A and sample B, respectively, and place them in test tubes containing 9.0 mL of sterile saline. Vortex and shake for 2 minutes to mix thoroughly to obtain 10 -1 diluent.

[0054] Serial dilution: from 10 -1 Draw 1.0 mL of the diluent and add it to a new test tube containing 9.0 mL of sterile saline. Vortex and mix thoroughly to make 10 -2 Repeat this process to prepare 10 -2 to 10 -9 Serial dilutions of .

[0055] Plate inoculation: Select an appropriate dilution gradient (for example, for sample A, select 10 -7 , 10 -8 , 10 -9 ; Sample B selected 10 -1 , 10 -2 , 10 -3 ), aspirate 100 μL of bacterial suspension from each dilution tube and evenly spread it on a NA medium plate. Set up three replicates for each dilution.

[0056] Incubation and counting: Place all inoculated plates upside down in a 37°C incubator and incubate for 48 hours. After incubation, select plates with 30-300 colonies for colony counting.

[0057] 5. Calculation of results: Based on the number of colonies on the plate, calculate the total number of viable bacteria per gram of sample (CFU / g) according to the following formula: Total viable bacteria = (average number of colonies on the plate × dilution factor) / 0.1 2. Experimental Data Table 1: Comparison of total viable bacteria in products under different drying methods 3. Summary and Analysis The test data in Table 1 show that the composition prepared by the method of Example 1 of the present invention still maintains a very high level of probiotic activity in the final product, with an average total number of viable bacteria reaching 2.1×10 9 In sharp contrast, the total number of viable bacteria in the product prepared by the method of Comparative Example 4 dropped sharply to 1.8×10 3 The difference in CFU / g between the two is nearly a million times. This significant difference intuitively proves that the low-temperature drying step adopted by the present invention is the key and necessary step to achieve the functionality of the final product.

[0058] The underlying mechanism for this result lies in the fact that the present method considers the final drying step as an integral part of the entire biomanufacturing chain, with the core goal being to protect heat-sensitive bioactive substances, such as probiotics, generated and survived during the coupled fermentation stage. The low-temperature vacuum drying technology employed in Example 1 operates by promoting the direct sublimation or evaporation of water under low temperature and high vacuum conditions. Throughout the process, the material temperature is consistently controlled within the microbial tolerance range (below 60°C), effectively avoiding irreversible thermal damage to microbial cell membranes, key enzyme systems, and genetic material, thereby maximizing the preservation of their biological activity.

[0059] In contrast, the conventional high-temperature spray drying used in Comparative Example 4 has an inlet air temperature of up to 180°C and an outlet air temperature of over 80°C. Although the drying efficiency is high, this severe thermal shock is fatal to probiotics. High temperature will instantly destroy the cell structure, leading to protein denaturation and enzyme inactivation, resulting in the death of most probiotics. Therefore, the test results strongly support the innovative concept of the present invention: a true "active peptide synbiotic" product, its value lies not only in containing functional peptides, but also in its "activity". Only by seamlessly integrating the front-end bioconversion process with the back-end active protection process can this core technical concept be transformed from a concept into a final product with practical application value.

[0060] Test Example 2: Comparative test of fermentation process efficiency 1. Experimental Description 1. Experimental Purpose This experiment aims to evaluate and compare the effects of different bacterial pretreatment methods on the efficiency of the coupled fermentation process by real-time monitoring of the dynamic changes in pH value in the fermentation system, thereby verifying the advanced nature of the "probiotic-specific induction and domestication" step in the present invention.

[0061] 2. Experimental Materials Reaction system A: the gradient enzymatic hydrolysis and coupled fermentation system in Example 1.

[0062] Reaction system B: the gradient enzymatic hydrolysis and coupled fermentation system in Comparative Example 1.

[0063] Experimental equipment: constant temperature reactor with online pH monitoring probe, magnetic stirrer, and data recorder.

[0064] 3. Experimental Procedure Parallel start-up: starting the gradient enzymatic hydrolysis and coupled fermentation steps according to the process flow of Example 1 and Comparative Example 1, respectively.

[0065] Timing and Monitoring: The moment the bacterial solution (either the acclimated or activated solution) is inoculated into the two reaction systems is marked as time zero (t=0). From this moment on, the pH value of the two reaction systems is automatically recorded every hour using an online pH probe.

[0066] Data acquisition: Continuously monitor and record pH value changes within 12 hours.

[0067] Data collation: Organize the recorded data into a table and draw a graph showing the pH value changing over time for easy visual comparison.

[0068] 2. Experimental Data Table 2: Changes in pH during coupled fermentation under different bacterial strain treatments 3. Summary and Analysis The data in Table 2 clearly reveal the significant effects of the two different bacterial strain treatment methods on the fermentation kinetics. The pH value of the system using the method of Example 1 of the present invention dropped rapidly and steadily from the beginning of inoculation, and dropped to 5.85 at the 5th hour, showing extremely high fermentation efficiency and almost non-existent hysteresis period. In contrast, the system using the method of Comparative Example 1 showed an extremely slow pH drop in the first 3 to 4 hours, showing obvious growth retardation, and barely reached 5.89 until the 12th hour. This result strongly demonstrates that the probiotic pretreatment step included in the present invention plays a decisive role in improving the efficiency and controllability of the entire coupled fermentation process.

[0069] The mechanism behind this phenomenon lies in the original "probiotic specific induction and domestication" step of the present invention. In Comparative Example 1, conventionally activated probiotics are directly put into an unfamiliar environment composed of a variety of complex raw materials. They need to spend a lot of time to adapt to the new osmotic pressure, nutrients and potential inhibitors, and induce the expression of the corresponding catabolic enzyme system. This process is the growth hysteresis period. The method of the present invention is equivalent to "pre-job training" for microorganisms by allowing probiotics to grow in a "specific induction culture medium" that simulates the main reaction environment in advance. This allows the probiotics to be "trained" before entering the main reactor, the metabolic pathways in their bodies have been activated, and the enzyme system that can efficiently utilize the target substrate has been pre-expressed.

[0070] Therefore, when these "tamed" probiotics are inoculated into the main reaction system, they can immediately start "working", skipping or greatly shortening the adaptation period, and directly entering the efficient logarithmic growth period, quickly utilizing the enzymatic hydrolysis products for metabolism and producing lactic acid, thereby causing a rapid drop in the pH value of the system. This rapid and predictable pH drop is not only a manifestation of high efficiency, but also a key prerequisite for the precise operation of the "gradient enzymatic hydrolysis and coupled fermentation" process of the present invention. It ensures that the subsequent pH-sensitive acid protease can be "activated" within a preset time window, thereby achieving directional shearing of functional peptides. This test fully demonstrates that the induced domestication of probiotics is not an optional optimization, but a core technical cornerstone to ensure the realization of the entire invention concept.

[0071] Test Example 3: Comparative test of functional peptide distribution in products 1. Experimental Description 1. Experimental Purpose This experiment aims to compare the effects of different enzymatic hydrolysis processes in the directional generation and enrichment of specific molecular weight peptides by analyzing the molecular weight distribution of water-soluble peptides in the final product, thereby verifying the superiority of the "gradient enzymatic hydrolysis and directional fermentation coupling" process adopted in the present invention.

[0072] 2. Experimental Materials Sample A: final product powder prepared in Example 1.

[0073] Sample B: the final product powder prepared in Comparative Example 2.

[0074] Sample C: the final product powder prepared in Comparative Example 3.

[0075] Experimental equipment and reagents: size exclusion high performance liquid chromatography system, molecular weight standards (such as cytochrome C, aprotinin, bacitracin, etc.), ultrapure water, 0.22 μm syringe filter, centrifuge.

[0076] 3. Experimental Procedure Sample extraction: Accurately weigh 1.0 g of each sample A, B, and C, add 20 mL of ultrapure water, and vortex for 5 minutes to fully dissolve. Then, centrifuge at 4°C and 10,000 rpm for 15 minutes, and collect the supernatant.

[0077] Sample filtration: The obtained supernatant was filtered through a 0.22 μm syringe filter to remove residual particles to obtain the sample solution to be tested.

[0078] Chromatographic analysis: Chromatographic conditions: A gel exclusion chromatography column suitable for peptide separation was used; the mobile phase was phosphate buffer; the flow rate was 0.5 mL / min; and the detection wavelength was 220 nm.

[0079] Preparation of standard curve: First, inject standards of different molecular weights, record their retention times, and prepare a standard curve of molecular weight logarithm and retention time.

[0080] Sample determination: Inject the sample solution to be tested into the HPLC system for analysis in sequence and record the chromatogram.

[0081] Data processing: Based on the standard curve, the retention time axis of the sample chromatogram was converted to a molecular weight axis. Using integration software, the peak areas of different molecular weight ranges (<500Da, 500-2000Da, >2000Da) were calculated, and their relative percentages of the total peptide peak area were calculated.

[0082] 2. Experimental Data Table 3: Comparison of peptide molecular weight distribution in products from different processes 3. Summary and Analysis The data results in Table 3 clearly demonstrate the decisive influence of different preparation processes on the peptide spectrum of the final product. The product of the method of Example 1 of the present invention has a peptide segment in the target molecular weight range of 500 to 2000 Da accounting for as high as 65.7%, showing a high degree of targeted enrichment effect. In contrast, the peptide spectrum of the product of Comparative Example 2 is chaotically distributed, with the target peptide segment accounting for only 31.3%, and contains a large number of over-degraded small molecules and incompletely degraded macromolecules. Although the product of Comparative Example 3 is better than Comparative Example 2, its target peptide segment accounts for only 48.1%, which is far lower than the method of the present invention.

[0083] This result profoundly reveals the inherent scientific nature and advancement of the "gradient enzymatic hydrolysis and directional fermentation coupling" process of the present invention. The method of the present invention designs the complex protein degradation process into an orderly, multi-stage "biomanufacturing assembly line." The first gradient first clears the physical obstacles for subsequent reactions; the second gradient uses neutral / alkaline proteases to efficiently degrade macroproteins, and the coupled fermentation starts at the same time; the most critical innovation is that the fermentation process naturally and smoothly lowers the pH of the system, which creates an optimal "working environment" for the pH-sensitive acidic / flavor proteases in the third gradient, enabling them to perform precise secondary "trimming" and "directional shearing" of the polypeptides generated in the second gradient, thereby enriching a large number of functional peptides with specific molecular weights.

[0084] The one-step enzyme rule of comparative example 2 completely destroys this timing and environmental control. Multiple enzymes interfere with each other in an unsuitable pH environment, causing the enzymatic hydrolysis process to get out of control, and the result is bound to be random and inefficient. Although comparative example 3 imitates the step-by-step order, it destroys the "soul" of dynamic synergy between enzymatic hydrolysis and fermentation by separating them. Artificial, instantaneous pH adjustment is far inferior to the gradual and uniform microenvironment created by microbial fermentation, resulting in a significant reduction in the efficiency and specificity of subsequent proteases. This test strongly proves that the core advantage of the present invention is not a simple superposition of steps, but its creative coupling of different biochemical reactions into an organic whole that can self-regulate and synergize, thereby achieving precise control of the molecular level structure of the final product.

[0085] Test Example 4: Comparative test of degradation effect of harmful substances in raw materials 1. Experimental Description 1. Experimental Purpose This experiment aims to determine and compare the residual amounts of two key anti-nutritional factors, phytic acid and free gossypol, in the final products after different process treatments, in order to verify the comprehensive effectiveness of the integrated coupling process of the present invention in harmless treatment of raw materials.

[0086] 2. Experimental Materials Sample A: final product powder prepared in Example 1.

[0087] Sample B: the final product powder prepared in Comparative Example 2.

[0088] Sample C: Initial mixed raw materials without any treatment (mixed according to the proportions in Example 1).

[0089] Experimental equipment and reagents: spectrophotometer, centrifuge, water bath, hydrochloric acid, trichloroacetic acid, phenanthroline-iron reagent, aniline, ethanol and other standard reagents for the determination of phytic acid and free gossypol content.

[0090] 3. Experimental Procedure Phytic acid content determination: Extraction: Accurately weigh 2.0 g of samples A, B, and C respectively, extract them with hydrochloric acid solution at a certain temperature by shaking, and then centrifuge to obtain the supernatant.

[0091] Reaction and determination: aspirate the supernatant, add phenanthroline-iron reagent, react for a certain time, and then measure its absorbance at a wavelength of 500nm.

[0092] Calculation: Calculate the phytic acid content (mg / kg) in the sample based on the pre-made phytic acid standard curve.

[0093] Determination of free gossypol content: Extraction: Accurately weigh 1.0 g of each sample A, B, and C, and heat them in a water bath using a solvent containing a specific proportion of aniline.

[0094] Determination: After cooling and filtering the extract, its absorbance was measured at a wavelength of 440 nm.

[0095] Calculation: Calculate the content of free gossypol in the sample (mg / kg) based on the pre-made gossypol standard curve.

[0096] Each parameter of each sample was measured three times in parallel and the average value was taken.

[0097] 2. Experimental Data Table 4: Comparison of degradation effects of different processes on harmful substances in raw materials 3. Summary and Analysis The test data of Table 4 clearly show that the technique of the embodiment of the present invention 1 has extremely outstanding degradation ability to the anti-nutritional factors in the raw material.Compared with untreated raw materials, the phytic acid and free gossypol content in the embodiment 1 product decreased by approximately 94.6% and 92.3% respectively.Although the comparative example 2 technique also has certain degradation effect, the residual amount of phytic acid and free gossypol in its product is significantly higher than that of embodiment 1, and the effect differs greatly.This fully proves the great advantage of the process design adopted by the present invention in raw material harmless treatment.

[0098] This remarkable effect stems from the synergistic mechanism of the present invention's "gradient enzymatic hydrolysis and coupled fermentation" process. In the present method, phytic acid degradation primarily occurs in the first gradient stage. This stage creates the optimal temperature and pH environment for the addition of exogenous phytase, enabling it to efficiently and specifically hydrolyze phytic acid molecules, breaking them down into inositol and inorganic phosphorus, thereby completely eliminating its anti-nutritional effects. Furthermore, throughout the coupled fermentation process, the vigorous metabolic activity of the probiotic community can also assist in the degradation of some free gossypol or reduce its toxicity through methods such as bioadsorption.

[0099] In contrast, the "one-step enzyme addition" method adopted in Comparative Example 2 adds all enzyme preparations at the same time, resulting in the initial environment of the reaction system being suboptimal for multiple enzymes such as phytase. The activity of phytase is affected by unsuitable pH conditions and interactions with other components such as proteases added subsequently, greatly reducing its degradation efficiency. This reveals the subtlety of the process design of the present invention: it is not a simple stacking of enzymes and microorganisms, but through precise timing control and environmental creation, it creates a "stage" that is most conducive to the functioning of each biocatalyst (enzyme or microorganism), maximizing the efficiency of each unit operation, and ultimately achieving the dual goals of efficient protein conversion and complete harmlessness of raw materials.

[0100] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A low soybean meal pig feed formula based on enzymatic protein, characterized in that: The active peptide synbiotic is prepared by coupling fermentation of the following raw materials in parts by weight through gradient enzymatic hydrolysis and directed functional bacterial flora: Dephenolized cottonseed meal: 60-70 parts; Corn distillers grains: 20-30 parts; Animal protein waste: 10-20 parts; The active peptide synbiotics comprises a functional peptide group, a surviving complex probiotic group and endogenous prebiotics.

2. The low soybean meal pig feed formula based on enzymatic protein according to claim 1, characterized in that: The surviving composite probiotic bacteria group comprises high-protease-producing Bacillus subtilis and acid-resistant Lactobacillus plantarum.

3. The low soybean meal pig feed formula based on enzymatic protein according to claim 1, characterized in that: The molecular weight of the functional peptide group is mainly distributed in the range of 500 to 2000 Da.

4. The low soybean meal pig feed formula based on enzymatic protein according to claim 1, characterized in that: The endogenous prebiotics are oligosaccharides generated by enzymatic hydrolysis of cellulose and hemicellulose in the corn alcohol grains and cottonseed meal.

5. A method for preparing a low-soybean meal pig feed formula based on enzymatic protein, which is used to prepare the pig feed formula according to any one of claims 1 to 4, characterized in that: The following steps are involved: a. Probiotic induction and acclimation step: the probiotics used for fermentation are pre-cultured in the primary hydrolyzate prepared from part of the raw materials to obtain a specific acclimated bacterial solution; b. Gradient enzymatic hydrolysis and coupled fermentation step: the remaining raw materials are made into a slurry, and enzyme systems with different functions are added in stages according to a preset time sequence for gradient enzymatic hydrolysis. During the enzymatic hydrolysis process, the specific acclimated bacterial solution obtained in step a is inoculated to couple the enzymatic hydrolysis with the microbial fermentation process; c. Low-temperature drying step: drying the fermentation product obtained in step b under low-temperature conditions to maintain the biological activity of the probiotics.

6. The method according to claim 5, characterized in that The probiotics induction and acclimation step in step a is specifically as follows: Part of the raw materials is prepared into slurry, and cellulase, hemicellulase and phytase are added for preliminary enzymatic hydrolysis. The obtained primary hydrolyzate is sterilized and used as an induction culture medium, and then probiotics are inoculated for cultivation to obtain the specific domesticated bacterial liquid.

7. The method according to claim 5, characterized in that The gradient enzymatic hydrolysis and coupled fermentation steps in step b include the following stages carried out in chronological order: First gradient: at 40-50°C, cellulase, hemicellulase, and phytase were added to break the cell wall; Second gradient: after heating to 50-55°C, add the first protease and simultaneously inoculate the specific acclimated bacterial solution; The third gradient: After the pH of the reaction system naturally drops due to fermentation, the second protease is added for directional shearing.

8. The method according to claim 7, characterized in that The first protease added in the second gradient is alkaline or neutral protease; the second protease added in the third gradient is acidic or flavor protease.

9. The method according to claim 7, characterized in that The condition for adding the second protease into the third gradient is that the pH value of the reaction system naturally drops to a range of 5.0 to 6.

5.

10. The method according to claim 5, characterized in that The low-temperature drying step in step c adopts vacuum belt drying or freeze drying, and the material temperature during the drying process is lower than 60°C.