Preparation method of liquid fermentation soybean peptide and application of liquid fermentation soybean peptide in feed

Abstract

The invention belongs to the field of functional feed additives, and provides a preparation method of liquid fermentation soybean peptide and application of the liquid fermentation soybean peptide in feed. A six-step process of pre-dissolving, directional enzymolysis, gamma-glutamyl, cascade tangential flow filtration and steady-state treatment is adopted. Narrow molecular weight distribution is realized through compounding of neutral endo-protease and exo-aminopeptidase; gamma-glutamyltranspeptidase is introduced to enhance the flavor activity; a target molecular weight segment is accurately enriched through a two-stage membrane; mild enzyme deactivation and sterilization are performed to retain active peptides; the pH is adjusted to 3.5-5.5, so that residual enzyme activity and microbial growth are inhibited, and the shelf life stability of the product is improved. According to the process, highly controllable distribution and functional modification of 60-90% of peptide with the molecular weight of 500-3000Da, 30-70% of gamma-glutamyl peptide and 70-100% of peptide with the molecular weight of less than 1000Da are realized, and the contradiction of low-viscosity processing, mild enzyme deactivation, shelf stability, tangential flow grading flux and pollution resistance under the condition of relatively high solid content is solved; the palatability and the flavor stability of the feed are remarkably improved, and the application value in pig feed is wide.

Description

Technical Field

[0001] This invention belongs to the field of functional feed additives and provides a method for preparing liquid fermented soybean peptides and their application in feed. Background Technology

[0002] In modern livestock farming, young animals, especially weaned piglets, face multiple challenges such as incomplete digestive system development, immune stress, and decreased feed intake. Efficient utilization of protein nutrition and maintenance of digestive health are key factors determining farming efficiency. Traditional plant protein sources, such as soybean meal, while high in protein, contain anti-nutritional factors like trypsin inhibitors and phytic acid. Large molecular weight allergenic proteins, such as glycinin and β-conglycinin, can easily induce intestinal immune responses, leading to diarrhea and growth retardation. Small molecule bioactive peptides, especially oligopeptides with a molecular weight of 500-3000 Da, are ideal alternatives to traditional protein sources due to their direct absorption without digestion, low immunogenicity, and ability to promote intestinal mucosal repair. Furthermore, γ-glutamyl peptides, as modified peptides with unique umami and flavor-enhancing functions, can significantly improve feed palatability and increase animal feed intake, making them particularly important in liquid feeding systems. Therefore, developing a soybean bioactive peptide product that has a narrow molecular weight distribution, high γ-glutamyl peptide content, low anti-nutritional factors, stable flavor, and is suitable for liquid addition processes is of great practical significance and application value for improving the nutritional health of young animals and promoting the feed industry towards precision nutrition.

[0003] Currently, the preparation of soybean peptides mainly relies on acid / alkali hydrolysis or single protease hydrolysis processes. For example, Chinese patent CN119662760A discloses a method for preparing and applying hydrolyzed soybean meal rich in peptide zinc, but it has shortcomings such as a wide molecular weight distribution, insufficient proportion of ultra-small peptides less than 1000 Da, the production of bitter peptides during hydrolysis, and the inability to achieve γ-glutamylation functional modification, resulting in poor palatability and limited functionality of the product. For example, Chinese patent CN105614818A discloses a compound enzyme-directed hydrolysis technology, but it does not involve precise molecular weight classification and stabilization treatment after hydrolysis. The ratio of residual large molecular peptides (>3000 Da) and excessively small molecules (free amino acids, dipeptides) in the hydrolysate is unbalanced. The former may cause allergies, while the latter has limited flavor contribution and is prone to Maillard reaction during storage, leading to browning. More importantly, existing processes generally neglect the functional modification step of γ-glutamylation. γ-glutamyl transpeptidase can introduce γ-glutamyl groups at the N-terminus of peptides to form γ-glutamyl peptides, significantly enhancing umami, reducing bitterness, and improving thermal stability. However, this reaction requires precise control of pH, temperature, and donor concentration, and gentle enzyme inactivation is necessary after the reaction to preserve the active peptide structure. Existing enzyme inactivation processes often use high-temperature (above 95°C) long-term treatment, which destroys the active conformation and flavor of the peptide. In addition, although tangential flow membrane separation technology can achieve molecular weight fractionation, single-stage filtration is difficult to obtain narrowly distributed target peptides, while two-stage or multi-stage filtration faces industrial bottlenecks such as severe membrane fouling, rapid flux decline, and frequent cleaning. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing soybean peptides by liquid fermentation and their application in feed, and to solve the problems of existing technologies, such as the contradiction between maintaining a low viscosity processing window under high solid content conditions and enriching the target narrow molecular weight / high γ-glutamyl peptide, the contradiction between mild enzyme inactivation / sterilization to preserve flavor active peptides and shelf-life microbial and enzymatic stability, and the conflict between the throughput robustness of tangential flow bi-stage fractionation and high selectivity against contamination.

[0005] This invention employs a six-step synergistic process: pre-dissolution, targeted enzymatic hydrolysis, γ-glutamylation, cascade fractionation, and stabilization. Pre-dissolution exposes enzyme cleavage sites, and neutral endopeptidase and exopeptidase work together to achieve narrow-distribution hydrolysis. γ-glutamylation enhances umami flavor through functional modification of the hydrolysis products and inhibits browning through steric hindrance. The cascaded double-membrane fractionation first retains large molecules, and the second precisely enriches the target window of 500-1500 Da, avoiding insufficient selectivity in single-stage processes and throughput loss in multi-stage processes. Mild enzyme inactivation preserves the active peptide structure, and stabilization adjusts acidity to inhibit residual enzyme activity and strengthens microbial control. Each step is interconnected, breaking through the limitations of traditional single-step optimization, and achieving multi-objective synergy of high-solids-content and high-efficiency processing, dual preservation of flavor and activity, and stable industrial operation.

[0006] A method for preparing soybean peptides by liquid fermentation includes the following steps: S1. Pre-dissolution treatment: Soybean protein raw materials are pre-dissolved at 50-70℃ and pH 6.0-8.0 to obtain a pre-dissolved solution; S2. Targeted enzymatic hydrolysis: Neutral endopeptidase and exopeptidase are added to the pre-dissolved solution, and enzymatic hydrolysis is carried out at 40-55℃ and pH 6.0-8.0 to obtain hydrolysate; S3. γ-Glutamylation reaction: γ-glutamyl transpeptidase and donor are added to the hydrolysate, and the reaction is carried out at 35-50℃ and pH 7.0-9.0 to obtain the γ-glutamylation product. S4. Enzyme inactivation and sterilization: Treat at 65-85℃ for 15-30 minutes to obtain sterilization solution; S5. Cascaded tangential flow filtration: First, a membrane with a molecular weight cutoff of 2000-5000 Da is used for the first stage of filtration and the first permeate is collected. Then, a membrane with a molecular weight cutoff of 500-1500 Da is used for the second stage of filtration of the first permeate. The second stage permeate or retentate is collected according to the target molecular weight window as the target fractionation to obtain the fractionated liquid. S6. Stabilize the formula by adjusting the pH of the fractionation solution to 3.5-5.5 to obtain the liquid fermented soybean peptide product.

[0007] Furthermore, in S3, the temperature is 35-50℃, the pH is 7.0-9.0, the amount of γ-glutamyl transpeptidase is 15-35 U / g protein, and the donor is L-glutamine and / or reduced glutathione.

[0008] Furthermore, the mass ratio of neutral endopeptidase to exopeptidase in S2 is 1:1 to 4:1, and the mass ratio of enzyme to substrate is 1.0-2.5%.

[0009] Furthermore, the pre-dissolution conditions for S1 are 60-65℃, pH 6.8-7.2, solid content of the liquid material is 8-12%, and the treatment time is 30-60 minutes.

[0010] Furthermore, the soybean protein raw materials are soybean protein concentrate and / or soybean meal.

[0011] Furthermore, in S5: When the molecular weight cutoff of the second-stage membrane is 1000-1500 Da, the second-stage permeate is collected. When the molecular weight cutoff of the second-stage membrane is 500-800 Da, the second-stage retentate is collected.

[0012] Furthermore, the operating parameters of S5 are: transmembrane pressure 0.08-0.15MPa, tangential flow velocity 1.5-2.2m / s, and reflux ratio 3-6.

[0013] Furthermore, in step S3, when the donor is L-glutamine, the molar ratio of the donor to the peptide in the hydrolysate is 0.08-0.12.

[0014] Furthermore, the mass ratio of enzyme to substrate in step S2 is 1.5-2.0%.

[0015] Furthermore, in step S5, the transmembrane pressure is 0.10-0.12 MPa, the tangential flow rate is 1.6-1.9 m / s, the reflux ratio is 4-5, the volume concentration factor is 2-4, the feed temperature is 25-35℃, and the permeate from the second-stage filtration is subjected to constant volume replacement of 2-4 replacement volumes.

[0016] Furthermore, the solid content of the liquid in step S1 is 9-11%.

[0017] Furthermore, in the liquid fermented soybean peptide product, peptides with a molecular weight of less than 1000 Da account for 85-95% of the total peptide mass.

[0018] Furthermore, the liquid fermented soybean peptide product contains lactic acid and / or citric acid as acidifying components, and the acidifying components account for 0.05-0.30% of the total product mass.

[0019] Furthermore, in the liquid fermented soybean peptide product, the trypsin inhibitor activity is ≤10 TIU / g, the phytic acid content is ≤0.05% (determined according to GB / T 5009.153-2016), and allergenic proteins such as soybean globulin and β-conglycinin are completely hydrolyzed into small molecule peptides.

[0020] As a concept of this invention, a six-step synergistic process—pre-dissolution, directed enzymatic hydrolysis, γ-glutamylation, cascade tangential filtration, and stabilization—is primarily used to enhance the molecular weight controllability, functionalization modification level, and shelf stability of soybean peptide products. Pre-dissolution treatment promotes the depolymerization and conformational unfolding of soybean protein through gentle heating and pH adjustment, fully exposing internal enzymatic cleavage sites and laying the structural foundation for subsequent directed enzymatic hydrolysis. This avoids the problems of low hydrolysis efficiency and uncontrolled molecular weight distribution caused by substrate steric hindrance in direct enzymatic hydrolysis. Directed enzymatic hydrolysis employs the synergistic action of a neutral endopeptidase and an exopeptidase. The former preferentially cleaves internal peptide bonds to form medium-length oligopeptides, while the latter progressively trims amino acids from the peptide chain ends to the target molecular weight window. A gradient regulation of the two enzymes' mass ratio from 1:1 to 4:1 achieves a precise balance between hydrolysis depth and molecular weight distribution, increasing the proportion of target peptides in the 500-3000 Da range by 30-50% compared to a single protease system. The γ-glutamylation reaction introduces γ-glutamyl transpeptidase and L-glutamine or reduced glutathione as donors. Under weakly alkaline conditions (pH 7.0-9.0) and mild temperatures (35-50℃), it catalyzes the transfer of the γ-glutamyl group from the donor to the N-terminal amino group of the hydrolyzed peptide, forming a γ-glutamyl peptide with unique umami flavor and antioxidant activity. This modification not only significantly improves the flavor by masking beany taste and enzymatic bitterness, but also inhibits Maillard browning of the peptide chain during storage through N-terminal protection, thus improving shelf-life stability. Cascaded tangential flow filtration employs a first-stage membrane with a molecular weight cutoff of 2000-5000 Da to rapidly remove residual large-molecule proteins and high-molecular-weight peptides. A second-stage membrane with a molecular weight cutoff of 500-1500 Da then precisely enriches the first permeate. This dual-stage design avoids the loss of target peptides or the introduction of impurity peptides caused by the difficulty in selecting the molecular weight cutoff in single-stage filtration, while also overcoming the cumulative flux decay problem of multi-stage filtration. A balance between high flux and anti-fouling is achieved through an optimized combination of transmembrane pressure (0.08-0.15 MPa), tangential flow rate (1.5-2.2 m / s), and reflux ratio (3-6). Mild enzyme inactivation and sterilization at 65-85℃ for 15-30 minutes effectively inactivates residual proteases and γ-glutamyl transferase, preventing further hydrolysis and modification reactions, while preserving the heat-sensitive active structure of γ-glutamyl peptides to avoid high-temperature damage. Formula stabilization further inhibits residual enzyme activity by adjusting the pH to a weakly acidic range of 3.5-5.5, and extends the product shelf life by utilizing the antibacterial effect of the low pH environment, thus achieving a triple synergy of enzymatic stability, microbial control, and flavor preservation.

[0021] The present invention also discloses a liquid fermented soybean peptide product prepared by the above method, wherein peptides with a molecular weight of 500-3000 Da account for 60-90% of the total peptides by mass, γ-glutamyl peptide accounts for 30-70% of the total peptides by mass, and peptides with a molecular weight of less than 1000 Da account for 70-100% of the total peptides by mass, and the product pH is 3.5-5.5.

[0022] Furthermore, peptides with a molecular weight of 500-3000 Da account for 75-82% of the total peptides by mass, and γ-glutamyl peptide accounts for 40-60% of the total peptides by mass.

[0023] Furthermore, peptides with a molecular weight of less than 1000 Da account for 80-100% of the total peptide mass in the product.

[0024] The present invention also discloses the use of liquid fermented soybean peptides in pig feed to improve the palatability and / or flavor stability of the feed. The application method is wet mixing or dilution via a water line system for feeding. The equivalent addition amount is 0.05-0.50%, based on peptide solids and dietary dry matter.

[0025] Furthermore, in the aforementioned applications, the product is suitable for liquid addition systems of complete feed, concentrated feed, or premixed feed; in complete feed, it is uniformly sprayed onto the surface of pelleted feed using a liquid spraying device, with an addition amount of 0.1-0.3%; in concentrated feed, the addition amount is 0.3-0.5%.

[0026] As another aspect of this invention, the liquid fermented soybean peptide product prepared by the above method is designed primarily to enhance the palatability and flavor stability of feed. It achieves multiple optimizations in nutrient absorption, immune protection, and sensory quality through highly controllable molecular weight distribution and functional modification levels. The core feature of the product is that peptides with a molecular weight of 500-3000 Da account for 60-90% of the total peptide mass. Oligopeptides in this molecular weight range have advantages such as direct absorption via the small peptide transport system without digestion, faster absorption rate than free amino acids, and low immunogenicity, making them less likely to induce allergic reactions. Compared to traditional soybean protein sources, they can significantly reduce gastrointestinal stress and diarrhea incidence in weaned piglets. Furthermore, peptides with a molecular weight less than 1000 Da account for 70-100% of the total peptide mass. This ultra-small peptide portion is mainly composed of dipeptides to pentapeptides, exhibiting extremely rapid absorption and low metabolic burden, making it particularly suitable for rapid nutritional supplementation in young animals and under stress conditions. Simultaneously, it avoids the risks of increased osmotic pressure and metabolic disorders caused by excessively high proportions of free amino acids. The high level of functionalization modification, with γ-glutamyl peptide accounting for 30-70% of the total peptide mass, is the key innovation of this product. γ-glutamyl peptide not only possesses a strong umami and flavor-enhancing effect, significantly improving feed palatability and increasing animal feed intake by 10-20%, but its N-terminal γ-glutamyl group's steric hindrance and antioxidant properties also effectively inhibit Maillard reactions, oxidative degradation, and microbial contamination of the peptide chain during storage and liquid feeding systems, solving the industry pain points of short shelf life and rapid flavor decay in liquid feed. The product's weakly acidic pH design of 4.2-4.5 further enhances microbial control and enzymatic stability. This pH range falls within the inhibition range of most spoilage bacteria and pathogens, while also strongly inhibiting residual protease activity, ensuring product quality stability during room temperature storage and water-line dilution feeding. In pig feed, it can be added by wet mixing or diluted and fed through a water line system. An equivalent addition of 0.05-0.50% (based on peptide solids and dietary dry matter) can produce significant effects. Compared with traditional soybean meal or fishmeal, the addition amount is reduced by 80-90%, which greatly reduces feed costs and environmental nitrogen emissions. While improving breeding efficiency, it promotes the transformation of the feed industry towards precision nutrition and sustainable development.

[0027] Beneficial technical effects 1. Achieving synergistic optimization of narrow molecular weight distribution and high γ-glutamyl peptide enrichment. By precisely controlling the mass ratio of neutral endopeptidase to exopeptidase (1:1 to 4:1) and optimizing enzymatic hydrolysis conditions (40-55℃, pH 6.0-8.0, enzyme to substrate mass ratio 1.0-2.5%), the hydrolysate is enriched into a narrow distribution of 500-3000 Da (accounting for 60-90% of the total peptides). Then, γ-glutamyl transferase catalyzes the process (15-35 U / g protein, 35-50℃, pH 7.0-9.0) to introduce γ-glutamyl transferase at the N-terminus of the hydrolysate. -Glutamyl groups form highly functionalized modified products with γ-glutamyl peptides accounting for 30-70% of the total peptides, significantly improving the umami and flavor characteristics of the product, reducing bitterness and beany taste. This solves the problems of wide molecular weight distribution (often covering 200-10000 Da and above) and single flavor in traditional enzymatic hydrolysis processes. While ensuring rapid absorption and utilization, it also imparts excellent palatability, allowing the addition of only 0.05-0.50% to liquid feed to significantly increase animal feed intake by 10-20%.

[0028] 2. Mild enzyme inactivation and sterilization preserves flavor and the structure and function of active peptides. A mild enzyme inactivation and sterilization process is employed, using a treatment temperature of 65-85℃ for 15-30 minutes. Compared to traditional long-term high-temperature treatment above 95℃, this method effectively inactivates residual proteases (residual enzyme activity <5%) and γ-glutamyl transpeptidase, preventing further hydrolysis and modification reactions that could lead to molecular weight drift and product quality fluctuations. It also maximizes the preservation of the heat-sensitive active structure and umami characteristics of γ-glutamyl peptides, avoiding high-temperature-induced peptide chain breakage, side-chain oxidation, and Maillard reactions. Subsequent formulation stabilization adjusts the pH to a slightly acidic range of 3.5-5.5, further providing dual protection against enzyme inhibition and microbial control. This ensures that the γ-glutamyl peptide content remains at ≥90% and microbial indicators meet feed-grade standards (total bacterial count <10%) during 6-12 months of storage at room temperature. 4 With CFU / mL and coliform count <30MPN / 100mL, it solves the industry pain points of short shelf life and rapid flavor decay of traditional liquid protein sources.

[0029] 3. Cascaded tangential flow two-stage stage achieves highly selective enrichment and maintains fouling-resistant flux. A first-stage membrane with a molecular weight cutoff of 2000-5000 Da is used to rapidly remove large molecular weight protein residues (>5000 Da) and high molecular weight peptides, with a permeability of ≥85%. A second-stage membrane with a molecular weight cutoff of 500-1500 Da is then used to precisely enrich peptides within the target window of 500-3000 Da from the first permeate (the second permeate is collected). This two-stage tandem design avoids the difficulties in selecting the molecular weight cutoff that arises with single-stage filtration (e.g., using only a 3000 Da membrane results in the loss of small peptides in the 500-2000 Da range, while using only a 1000 Da membrane results in the loss of small peptides in the 2000-3000 Da range). This method overcomes the problems of flux accumulation and membrane fouling caused by impurities in 0-Da peptides, which can lead to loss of target peptides or insufficient selectivity. It also overcomes the problems of flux accumulation and membrane fouling caused by multi-stage filtration (≥3 stages). Through the optimized combination of transmembrane pressure 0.08-0.15MPa, tangential flow rate 1.5-2.2m / s and reflux ratio 3-6, and constant volume replacement of 2-4 liquid replacement volumes, it achieves a stable flux of ≥50L / m²·h and continuous operation for ≥8 hours without shutdown for cleaning. The recovery rate of target peptides with molecular weight of 500-3000Da is ≥75%, meeting the needs of continuous industrial production.

[0030] 4. Deeply removes anti-nutritional factors and allergenic proteins to ensure safety. Pre-dissolving treatment (50-70℃, pH 6.0-8.0) combined with targeted enzymatic hydrolysis (neutral endo- and exopeptidase synergistic, 40-55℃, pH 6.0-8.0, enzyme to substrate ratio 1.0-2.5%) achieves complete hydrolysis of large molecular weight allergenic proteins such as soybean globulin and β-conglycinin into small molecular weight peptides (molecular weight <3000Da). This eliminates the risk of intestinal allergic reactions and immune stress induced by traditional soybean meal in weaned piglets. Simultaneously, the enzymatic hydrolysis process destroys the conformation of trypsin inhibitors and... Subsequent gentle heating (65-85℃, 15-30 minutes) reduces trypsin inhibitor activity to ≤10TIU / g (compared to 40-60TIU / g in traditional soybean meal), and phytic acid content is reduced to ≤0.05% (compared to 1.0-1.5% in traditional soybean meal) through hydrolysis and membrane fractionation. This significantly improves the bioavailability of protein and minerals (calcium, phosphorus, zinc, etc.) in the feed, and solves the prominent problems of high anti-nutritional factors and poor safety of plant protein sources. It is particularly suitable for young animals with fragile digestive systems and high-stress breeding environments.

[0031] 5. Adaptable to liquid feeding systems for precise nutrition and environmentally friendly emission reduction. Designed in liquid form (solid content 8-15%, pH 3.5-5.5, viscosity <50 mPa·s), the product is naturally compatible with the liquid feeding systems and water-line feeding devices widely used in modern pig farms. It can be added directly by wet mixing or diluted through a water-line system without additional crushing and mixing processes. The equivalent addition amount of only 0.05-0.50% (based on peptide solids and dietary dry matter) can provide high-density small peptide nutrition and flavor enhancement effects. Compared with the traditional addition amount of soybean meal or fishmeal (usually 10-25%), it reduces the amount by 80-90%, significantly reducing feed raw material costs by 15-20% and nitrogen emission load (reducing fecal nitrogen content by 20-30%). At the same time, the liquid feeding method reduces dust generation, improves the breeding environment and workers' working conditions, and responds to green breeding and environmental protection policies while improving breeding efficiency. It promotes the transformation of the feed industry towards precision nutrition, low carbon emission reduction and sustainable development, and has significant economic and social benefits. Attached Figure Description

[0032] Figure 1 This invention illustrates the effect of the molecular weight cutoff of the first-stage membrane on the molecular weight distribution of the product.

[0033] Figure 2 The effect of pH on the shelf-life retention rate and microbial stability of the product of this invention;

[0034] Figure 3 This invention relates to the effect of γ-glutamylation reaction temperature on γ-glutamyl peptide content and antioxidant activity.

[0035] Figure 4 The graphs are high-performance liquid chromatography-tandem mass spectrometry molecular weight distribution histograms and log-normal fitting curves of Example 1 of the present invention, with the horizontal axis representing molecular weight Da.

[0036] Figure 5 This is an XPS comparison image of the dry film of liquid fermented soybean peptides and the dry film of unfermented soybean protein concentrate in Example 1 of the present invention. Detailed Implementation

[0037] 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.

[0038] Example 1

[0039] This embodiment provides a method for preparing soybean peptides through liquid fermentation, comprising the following steps: S1. Pre-dissolution treatment: Soy protein concentrate is pre-dissolved at 60℃ and pH 7.0, with a solid content of 10% and a treatment time of 45 minutes to obtain a pre-dissolved solution; S2. Targeted enzymatic hydrolysis: Neutral endopeptidase and exopeptidase are added to the pre-dissolved solution in this embodiment. The mass ratio of the two enzymes is 2:1, and the mass ratio of enzyme to substrate is 1.8%. Enzymatic hydrolysis is carried out at 48°C and pH 7.0 to obtain hydrolysate. S3. γ-Glutony reaction: γ-glutamyl transpeptidase and donor L-glutamine were added to the hydrolysate of this embodiment. The amount of γ-glutamyl transpeptidase was 25 U / g protein, and the molar ratio of donor to peptide in hydrolysate was 0.10. The reaction was carried out at 42°C and pH 8.0 for 1.0 hour to obtain γ-glutamylation product. S4. Enzyme inactivation and sterilization: The solution was treated at 75°C for 22 minutes to obtain a sterilization solution; S5. Cascaded tangential flow filtration: First, a membrane with a molecular weight cutoff of 3000 Da is used for the first stage of filtration and the first permeate is collected. Then, a membrane with a molecular weight cutoff of 1200 Da is used for the second stage of filtration of the first permeate in this embodiment. The second stage permeate is collected as the target fractionation. The operating parameters are transmembrane pressure 0.12 MPa, tangential flow velocity 1.8 m / s, and reflux ratio 4 to obtain the fractionation solution. S6. Formula stabilization: Adjust the pH of the fractionation liquid in this embodiment to 4.3, add lactic acid as an acidifying component, the acidifying component accounts for 0.15% of the total mass of the product, and obtain the liquid fermented soybean peptide product.

[0040] The liquid fermented soybean peptide product prepared in this embodiment has the following composition: peptides with a molecular weight of 500-3000 Da account for 78% of the total peptides by mass; γ-glutamyl peptide accounts for 48% of the total peptides by mass; peptides with a molecular weight of less than 1000 Da account for 88% of the total peptides by mass; and the product pH is 4.3. The product has a trypsin inhibitor activity of 6 TIU / g, a phytic acid content of 0.03%, and allergenic proteins such as glycinin and β-conglycinin are completely hydrolyzed into small molecule peptides.

[0041] The product in this embodiment is suitable for improving the palatability and flavor stability of pig feed. It can be applied by wet mixing or dilution via a water line system, with an equivalent addition of 0.20% based on peptide solids and dietary dry matter. This product is also suitable for liquid additive systems in complete compound feeds, applied evenly to the surface of pelleted feed using a liquid spraying device, at an addition rate of 0.2%.

[0042] Features of Example 1: This example employs moderate process parameters, with all conditions within a relatively stable technical range, resulting in good process stability, high product quality consistency, and suitability for large-scale production. The pre-dissolving stage utilizes 60℃ neutral conditions and a 10% solids content, ensuring sufficient dissolution of soybean protein while avoiding protein denaturation caused by excessively high temperatures. The enzymatic hydrolysis process uses 48℃ and neutral pH, combined with a 2:1 enzyme ratio and 1.8% enzyme dosage, achieving synergistic effects of endonucleases and exonucleases to obtain an ideal degree of hydrolysis. The γ-glutamylation reaction is carried out at 42℃ and pH 8.0, with an enzyme dosage of 25 U / g and L-glutamine as the donor, ensuring stable modification efficiency and controllable costs. The membrane filtration system employs a combination of 3000Da and 1000Da cutoff values ​​to precisely control the molecular weight distribution of the product, resulting in 78% of peptides in the 500-3000Da range and 88% of small molecule peptides (<1000Da). It also achieves a 48% γ-glutamyl peptide content, resulting in a good balance of nutritional functionality. The product's pH of 4.3, combined with an appropriate amount of lactic acid, ensures microbial stability and shelf life. The overall process parameters are mild and moderate, requiring conventional equipment, and are easy to operate with controllable production costs, making it an ideal solution for industrial-scale mass production. Application scenarios: This product is particularly suitable for daily feed addition in large-scale commercial pig farms. Precise addition is achieved through an automated liquid spraying system, significantly improving the palatability of complete compound feed, reducing weaning stress in piglets, increasing feed intake and daily weight gain, and is suitable for standardized production processes in the nursery and finishing pig stages.

[0043] Example 2

[0044] This embodiment provides a method for preparing soybean peptides through liquid fermentation, comprising the following steps: S1. Pre-dissolving treatment: Soybean meal is pre-dissolved at 55℃ and pH 6.5, with a solid content of 9% in the liquid and a treatment time of 40 minutes to obtain a pre-dissolved liquid. S2. Targeted enzymatic hydrolysis: Neutral endopeptidase and exopeptidase were added to the pre-dissolved solution in this embodiment. The mass ratio of the two enzymes was 1.5:1, and the mass ratio of enzyme to substrate was 1.5%. Enzymatic hydrolysis was carried out at 42°C and pH 6.5 to obtain hydrolysate. S3. γ-Glutonylation reaction: γ-glutamyl transferase and a donor were added to the hydrolysate of this embodiment. The donor was a mixture of L-glutamine and reduced glutathione in a mass ratio of 1:1. The amount of γ-glutamyl transferase was 18 U / g protein. The molar ratio of the donor to the peptide in the hydrolysate was 0.10. The reaction was carried out at 37°C and pH 7.5 for 0.8 hours to obtain the γ-glutamylated product. S4. Enzyme inactivation and sterilization: Treat at 70℃ for 20 minutes to obtain sterilization solution; S5. Cascaded tangential flow filtration: First, a membrane with a molecular weight cutoff of 2500 Da is used for the first stage of filtration and the first permeate is collected. Then, a membrane with a molecular weight cutoff of 650 Da is used for the second stage of filtration of the first permeate in this embodiment. The second stage retentate is collected as the target fractionation. The operating parameters are transmembrane pressure 0.09 MPa, tangential flow rate 1.7 m / s, and reflux ratio 5 to obtain the fractionation solution. S6. Formula stabilization: Adjust the pH of the fractionation liquid in this embodiment to 4.4, add citric acid as an acidifying component, the acidifying component accounts for 0.10% of the total mass of the product, and obtain the liquid fermented soybean peptide product.

[0045] The liquid fermented soybean peptide product prepared in this embodiment has the following composition: peptides with a molecular weight of 500-3000 Da account for 76% of the total peptides by mass; γ-glutamyl peptide accounts for 42% of the total peptides by mass; peptides with a molecular weight of less than 1000 Da account for 86% of the total peptides by mass; and the product pH is 4.4. The product has a trypsin inhibitor activity of 8 TIU / g, a phytic acid content of 0.04%, and allergenic proteins such as glycinin and β-conglycinin are completely hydrolyzed into small molecule peptides.

[0046] The product described in this embodiment is suitable for improving the palatability and flavor stability of pig feed. It can be applied by wet mixing or dilution via a water line system, with an equivalent addition amount of 0.15%, calculated as peptide solids and dietary dry matter. This product is also suitable for liquid addition systems of concentrated feeds, with an addition amount of 0.35%.

[0047] Example 2 Features: This example employs a mild, low-temperature processing route, emphasizing energy conservation, environmental protection, and protection of heat-sensitive components. Overall energy consumption is low, and the product possesses unique functional characteristics. The pre-dissolving stage utilizes a low temperature of 55°C, combined with weakly acidic conditions (pH 6.5) and a low solid content of 9%, reducing energy consumption while minimizing the risk of protein denaturation and preserving the natural active ingredients of soy protein. The enzymatic hydrolysis process selects a mild temperature of 42°C and a weakly acidic environment of pH 6.5, along with an enzyme ratio of 1.5:1 and an enzyme dosage of 1.5%. Although the hydrolysis rate is relatively slow, it better preserves the bioactivity of functional peptides and avoids nutritional value loss due to excessive hydrolysis. The γ-glutamylation reaction is the technical highlight of this embodiment. It utilizes a low temperature of 37°C and near-neutral conditions (pH 7.5), with a moderate enzyme dosage of 18 U / g. Most uniquely, it employs a composite donor system of L-glutamine and reduced glutathione, simultaneously introducing multiple modified structures such as γ-glutamyl-glutamine and γ-glutamyl-cysteine-glycine, significantly enhancing the product's antioxidant capacity and immunomodulatory function. Membrane filtration uses smaller molecular weight cutoffs of 2500 Da and 800 Da, favoring the enrichment of small-molecule bioactive peptides, with peptides smaller than 1000 Da accounting for 86%. Sterilization is performed under mild conditions of 70°C, coupled with low filtration operating parameters (transmembrane pressure 0.09 MPa, tangential flow rate 1.7 m / s), further embodying the gentle processing concept and maximizing the preservation of the product's bioactivity. Application scenarios: Due to its mild processing characteristics, compound γ-glutamyl modification and strong antioxidant function, the product in this embodiment is particularly suitable for nutritional support of animals in stress-sensitive periods, such as early weaned piglets, pigs in the regrouping stress period, feeding under summer heat stress conditions, and sows in special physiological stages such as peripartum period. It can be diluted and fed through a water line system to reduce intestinal irritation, improve digestion and absorption rate, and enhance the body's antioxidant and immune capabilities.

[0048] Example 3

[0049] This embodiment provides a method for preparing soybean peptides through liquid fermentation, comprising the following steps: S1. Pre-dissolution treatment: The mixture of soybean protein concentrate and soybean meal (mass ratio 1:1) is pre-dissolved at 65℃ and pH 7.2. The solid content of the liquid is 11%, and the treatment time is 50 minutes to obtain the pre-dissolved liquid. S2. Targeted enzymatic hydrolysis: Neutral endopeptidase and exopeptidase were added to the pre-dissolved solution in this embodiment. The mass ratio of the two enzymes was 3:1, and the mass ratio of enzyme to substrate was 2.0%. Enzymatic hydrolysis was carried out at 52°C and pH 7.5 to obtain hydrolysate. S3. γ-Glutonylation reaction: γ-glutamyl transferase and donor reduced glutathione were added to the hydrolysate of this embodiment. The amount of γ-glutamyl transferase was 32 U / g protein, and the molar ratio of donor to peptide in hydrolysate was 0.10. The reaction was carried out at 45°C and pH 8.5 for 0.9 hours to obtain the γ-glutamylated product. S4. Enzyme inactivation and sterilization: Treat at 80℃ for 25 minutes to obtain sterilization solution; S5. Cascaded tangential flow filtration: First, a membrane with a molecular weight cutoff of 3500 Da is used for the first stage of filtration and the first permeate is collected. Then, a membrane with a molecular weight cutoff of 1400 Da is used for the second stage of filtration of the first permeate in this embodiment. The second stage permeate is collected as the target fractionation. The operating parameters are transmembrane pressure 0.10 MPa, tangential flow velocity 1.7 m / s, and reflux ratio 4 to obtain the fractionation solution. S6. Formula stabilization: Adjust the fractionation liquid of this embodiment to pH 4.2, add a mixture of lactic acid and citric acid (mass ratio 1:1) as an acidification component, the acidification component accounts for 0.25% of the total mass of the product, and obtain liquid fermented soybean peptide product.

[0050] The liquid fermented soybean peptide product prepared in this embodiment has the following composition: peptides with a molecular weight of 500-3000 Da account for 81% of the total peptides by mass; γ-glutamyl peptide accounts for 58% of the total peptides by mass; peptides with a molecular weight of less than 1000 Da account for 92% of the total peptides by mass; and the product pH is 4.2. The product has a trypsin inhibitor activity of 5 TIU / g, a phytic acid content of 0.02%, and allergenic proteins such as glycinin and β-conglycinin are completely hydrolyzed into small molecule peptides.

[0051] The product in this embodiment is suitable for improving the palatability and flavor stability of pig feed. It can be applied by wet mixing or dilution via a water line system, with an equivalent addition of 0.30% based on peptide solids and dietary dry matter. This product is also suitable for liquid additive systems in complete compound feeds, applied evenly to the surface of pelleted feed using a liquid spraying device, at an addition rate of 0.3%.

[0052] Example 3 Features: This example employs a highly efficient and enhanced process strategy, aiming to maximize protein conversion rate, γ-glutamyl modification degree, and product functionality, making it suitable for applications with higher performance requirements. In terms of raw material selection, a 1:1 blend of soybean protein concentrate and soybean meal is used, utilizing both the high protein content of the concentrate and the economic efficiency of soybean meal. The pre-dissolving stage employs a relatively high temperature of 65℃, a slightly alkaline pH of 7.2, and a high solids content of 11%, significantly improving protein dissolution efficiency and the processing capacity of subsequent steps, while shortening the pre-dissolution time. The enzymatic hydrolysis process uses a relatively high temperature of 52℃ and a slightly alkaline pH of 7.5. A high enzyme ratio of 3:1 enhances the rapid degradation effect of endonucleases, and a relatively high enzyme dosage of 2.0% ensures complete protein hydrolysis, resulting in a higher degree of hydrolysis and free amino acid content. The γ-glutamylation reaction is the core advantage of this embodiment. A highly active environment for transpeptidase was created using alkaline conditions (pH 8.5, close to the enzyme's optimal temperature) at 45°C. A high enzyme dosage of 32 U / g, combined with reduced glutathione as the specific donor, achieved a γ-glutamyl peptide content as high as 58%, with enrichment of the γ-glutamyl-cysteine ​​peptide, significantly enhancing the product's antioxidant, immunomodulatory, and heavy metal chelating functions. Membrane filtration employs a large cutoff combination of 3500 Da and 1400 Da, removing large molecular weight proteins while retaining some functional oligopeptides, resulting in an 81% proportion of 500-3000 Da peptides and a high proportion of small molecule peptides (<1000 Da) of 92%, achieving excellent molecular weight distribution. Sterilization at 80°C ensured adequate microbial control, and a composite acidification system (lactic acid + citric acid) combined with pH 4.2 further enhanced the product's long-term stability and shelf life. Although the overall process is slightly more expensive, the product boasts superior performance, thoroughly removing anti-nutritional factors (trypsin inhibitor only 5 TIU / g, phytic acid 0.02%) and completely eliminating allergens, making it an ideal solution for high-end functional products. Application Scenarios: Due to its high γ-glutamyl peptide content, strong antioxidant function, and excellent nutritional value, the product in this embodiment is particularly suitable for nutritional fortification of pigs in high-intensity production periods, such as fast-growing fattening pigs, high-producing sows, and boars. It can be precisely added to the surface of pelleted feed through a liquid spraying system, significantly improving feed flavor stability and shelf life, enhancing animal stress resistance and immune function, and improving production and reproductive performance. It is suitable for high-end commercial fattening pigs and breeding pigs, as well as export-oriented high-quality pork production systems.

[0053] Example 4

[0054] This embodiment provides a method for preparing soybean peptides through liquid fermentation, comprising the following steps: S1. Pre-dissolution treatment: Soy protein concentrate is pre-dissolved at 62℃ and pH 7.0, with a solid content of 8% and a treatment time of 45 minutes to obtain a pre-dissolved solution. S2. Targeted enzymatic hydrolysis: Neutral endopeptidase and exopeptidase were added to the pre-dissolved solution in this embodiment. The mass ratio of the two enzymes was 2.5:1, and the mass ratio of enzyme to substrate was 2.5%. Enzymatic hydrolysis was carried out at 50°C and pH 7.2 to obtain hydrolysate. S3. γ-Glutony reaction: γ-glutamyl transpeptidase and donor L-glutamine were added to the hydrolysate of this embodiment. The amount of γ-glutamyl transpeptidase was 28 U / g protein, and the molar ratio of donor to peptide in hydrolysate was 0.10. The reaction was carried out at 35°C and pH 9.0 for 1.0 hour to obtain γ-glutamylation product. S4. Enzyme inactivation and sterilization: Treat at 75℃ for 25 minutes to obtain sterilization solution; S5. Cascaded tangential flow filtration: First, a membrane with a molecular weight cutoff of 2800 Da is used for the first stage of filtration and the first permeate is collected. Then, a membrane with a molecular weight cutoff of 1500 Da is used for the second stage of filtration of the first permeate in this embodiment. The second stage permeate is collected as the target fractionation. The operating parameters are transmembrane pressure 0.11 MPa, tangential flow velocity 1.9 m / s, and reflux ratio 5 to obtain the fractionation solution. S6. Stabilize the formula: Adjust the pH of the fractionation liquid in this embodiment to 3.5, add lactic acid as an acidifying component, the acidifying component accounts for 0.18% of the total mass of the product, and obtain the liquid fermented soybean peptide product.

[0055] The liquid fermented soybean peptide product prepared in this embodiment has the following composition: peptides with a molecular weight of 500-3000 Da account for 70% of the total peptides by mass; γ-glutamyl peptide accounts for 55% of the total peptides by mass; peptides with a molecular weight of less than 1000 Da account for 90% of the total peptides by mass; and the product pH is 3.5. The product has a trypsin inhibitor activity of 7 TIU / g, a phytic acid content of 0.04%, and allergenic proteins such as glycinin and β-conglycinin are completely hydrolyzed into small molecule peptides.

[0056] The product in this embodiment is suitable for improving the palatability and flavor stability of pig feed. It can be applied by wet mixing or dilution via a water line system, with an equivalent addition amount of 0.12%, calculated as peptide solids and dietary dry matter. This product is also suitable for liquid additive systems in complete compound feeds, applied evenly to the surface of pelleted feed using a liquid spraying device, at an addition amount of 0.12%.

[0057] Example 4 Features: This example employs a unique, high-performance process configuration. Through a special combination of parameters, it achieves outstanding advantages in microbial stability, storage performance, and specific functionality, making it suitable for applications with specific requirements for product shelf life and stability. The pre-dissolution stage uses a low solids content of 8% combined with a moderate temperature (62°C) and neutral pH (7.0). Although the concentration is low, sufficient dissolution can still be achieved by extending the pre-dissolution time. This low-concentration treatment facilitates the operation of subsequent processes and improves equipment cleanliness. The enzymatic hydrolysis process uses a high enzyme dosage of 2.5%, combined with an enzyme ratio of 2.5:1 and suitable temperature and pH conditions (50°C, pH 7.2), ensuring the most complete protein degradation and the highest free peptide content, providing abundant substrates for the subsequent γ-glutamylation reaction. The γ-glutamylation reaction is the core technical feature of this embodiment. Utilizing a low-temperature condition of 35°C combined with a strongly alkaline environment of pH 9.0, this seemingly contradictory parameter combination actually achieves unique technical effects: the low temperature helps protect the bioactivity of heat-sensitive peptides, while the high pH significantly enhances the catalytic efficiency of γ-glutamyl transpeptidase. The synergistic effect of these two factors preserves the active ingredients while achieving effective modification. Simultaneously, the low temperature reduces non-enzymatic browning such as the Maillard reaction, which is beneficial to the stability of product color and flavor. With an enzyme dosage of 28 U / g and L-glutamine as the donor, a stable γ-glutamyl modification effect can be obtained under these specific conditions for 1.0 hour. Membrane filtration employs a combination of the widest retention windows of 2800 Da and 1500 Da, precisely retaining peptide components of the target molecular weight range while effectively removing large protein molecules and extremely small free amino acids, resulting in highly pure functional peptides. The final product is adjusted to a relatively strong acidity of pH 3.5 and formulated with an appropriate amount of lactic acid as a natural preservative, ensuring excellent microbial stability and an ultra-long shelf life. This makes it particularly suitable for applications requiring long-term storage or use in high-temperature and high-humidity environments. The product contains 70% peptides with a molecular weight of 500-3000 Da, 90% small molecule peptides (<1000 Da), and 55% γ-glutamyl peptide. Although the overall modification degree is relatively low, the modified peptides obtained under low-temperature conditions exhibit higher bioactivity retention. Applications: Due to its strong acidity and resulting superior microbial stability and long shelf life, this product is particularly suitable for applications requiring long-term storage, such as export feed additives (long sea freight times), applications in tropical pig farms (high-temperature and high-humidity environments), and premixed and concentrated feed products requiring extended shelf life. Meanwhile, the low-temperature γ-glutamylation process retains more of the bioactivity of heat-sensitive functional peptides, giving the product unique advantages in anti-oxidation and immune regulation. It is suitable for functional feed additives and high-end pig farming with special nutritional needs, such as SPF-grade breeding pig farms, laboratory animal farming, and organic pork production systems, which have extremely high requirements for product quality and safety.

[0058] Comparative Example 1: It is basically the same as Example 1, except that the pre-dissolution temperature is 45°C, while the amount of other components and preparation conditions remain unchanged.

[0059] Comparative Example 2: It is basically the same as Example 1, except that the pre-dissolution temperature is 75°C, while the amount of other components and preparation conditions remain unchanged.

[0060] Comparative Example 3: It is basically the same as Example 1, except that only neutral endopeptide protease is used for enzymatic hydrolysis in step S2, the mass ratio of enzyme to substrate is 1.8%, and other preparation conditions remain unchanged.

[0061] Comparative Example 4: Basically the same as Example 1, except that the mass ratio of neutral endopeptidase to exopeptidase in step S2 is 1:1, and other preparation conditions remain unchanged.

[0062] Comparative Example 5: It is basically the same as Example 1, except that γ-glutamyl transpeptidase is not added in step S3, and enzyme inactivation and sterilization are performed directly. Other preparation conditions remain unchanged.

[0063] Comparative Example 6: Basically the same as Example 1, except that the amount of γ-glutamyl transpeptidase used in step S3 is 10 U / g protein, and other preparation conditions remain unchanged.

[0064] Comparative Example 7: Basically the same as Example 1, except that the amount of γ-glutamyl transpeptidase used in step S3 is 40 U / g protein, and other preparation conditions remain unchanged.

[0065] Comparative Example 8: It is basically the same as Example 1, except that the reaction temperature in step S3 is 30°C, while other preparation conditions remain unchanged.

[0066] Comparative Example 9: Basically the same as Example 1, except that the enzyme inactivation and sterilization temperature in step S4 is 60°C, while other preparation conditions remain unchanged.

[0067] Comparative Example 10: Basically the same as Example 1, except that in step S5 only a single-stage filtration was performed using a membrane with a molecular weight cutoff of 3000 Da and the permeate was collected as the product, while other preparation conditions remained unchanged.

[0068] Comparative Example 11: It is basically the same as Example 1, except that the first-stage filtration in step S5 uses a membrane with a molecular weight cutoff of 8000 Da, while other preparation conditions remain unchanged.

[0069] Comparative Example 12: Basically the same as Example 1, except that the transmembrane pressure in step S5 is 0.05 MPa, and other preparation conditions remain unchanged.

[0070] Comparative Example 13: Basically the same as Example 1, except that in step S6 the fractionation solution was adjusted to pH 5.8, while other preparation conditions remained unchanged.

[0071] Performance testing: Experimental Scheme 1: Determination of Molecular Weight Distribution Test Subject: Liquid fermented soybean peptide product. Test Objective: To accurately determine the mass percentage distribution of peptides in different molecular weight ranges within the product, and to verify the enrichment effect of the two-stage membrane fractionation process on the target molecular weight range (500-3000 Da). Test Principle: High-performance gel permeation chromatography (HPLC) is used. Based on the differences in retention time of peptide molecules in the chromatographic column, combined with a standard peptide molecular weight calibration curve, accurate quantitative analysis of molecular weight is achieved. Experimental Method: 10 mL of liquid fermented soybean peptide sample was filtered through a 0.45 μm filter membrane and injected. Chromatographic conditions: TSKgel G2000SWXL column (300 mm × 7.8 mm, 5 μm), mobile phase: 0.1 mol / L phosphate buffer (pH 7.0) and acetonitrile (75:25 v / v), flow rate: 0.5 mL / min, column temperature: 30℃, detection wavelength: 220 nm, injection volume: 20 μL. Calibration curves were established using molecular weight standards (1000Da, 2000Da, 3000Da, and 5000Da bovine serum albumin-derived peptides). The percentage of different molecular weight segments was calculated by integrating the peak areas. Key parameters: column selectivity, mobile phase composition, detection wavelength 220nm (peptide bond absorption), and injection volume 20μL. Data processing: The mass percentage of each molecular weight segment in the total peptides was calculated using the peak area percentage method. The determination was repeated three times, and the average value was taken. The standard deviation should be ≤3%.

[0072] Experimental Scheme 2: Determination of γ-glutamyl peptide content Test Subject: Liquid fermented soybean peptide product. Test Objective: To quantitatively determine the content of γ-glutamyl peptide in the product and verify the modification efficiency of the γ-glutamylation reaction and the product's functionality. Test Principle: High-performance liquid chromatography (HPLC) combined with specific hydrolysis of γ-glutamyl peptide was used. The content of γ-glutamyl peptide was indirectly calculated by measuring the difference in free glutamate content before and after hydrolysis. Experimental Method: 2 mL of sample was divided into two portions. One portion was directly hydrolyzed with acid (6 mol / L hydrochloric acid, 110℃, sealed hydrolysis for 22 hours). The other portion was first specifically hydrolyzed with γ-glutamyl transpeptidase (100 U / mL, 37℃, pH 8.0, reaction for 4 hours to release γ-glutamyl residues) and then hydrolyzed with acid. Both hydrolysates were deacidified by rotary evaporation, dissolved and diluted to volume with 0.02 mol / L hydrochloric acid, filtered through a 0.22 μm filter membrane, and the glutamate content was determined using an amino acid analyzer or HPLC. Chromatographic conditions: C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: o-phthalaldehyde (OPA) pre-column derivatization; flow rate: 1.0 mL / min; excitation wavelength: 340 nm; emission wavelength: 450 nm. Standards: Refer to GB 5009.124-2016 "Determination of Amino Acids in Food" or similar standards. Key parameters: γ-glutamyl transferase-specific hydrolysis conditions (100 U / mL, 37℃, pH 8.0, 4 h); acid hydrolysis conditions (6 mol / L HCl, 110℃, 22 h); OPA derivatization. Data processing: γ-glutamyl peptide content (%) = (glutamate content in the enzymatic hydrolysis group - glutamate content in the control group) / total peptide content × 100%, repeated 3 times, relative standard deviation ≤ 5%.

[0073] Experimental scheme 3: Evaluation of product shelf-life stability Test Subject: Liquid fermented soybean peptide product. Test Objective: To evaluate the stability of the product's physicochemical, microbiological, and sensory qualities under ambient temperature storage conditions, and to verify the effectiveness of formula stabilization technology in ensuring shelf life. Test Principle: By simulating actual storage conditions, the changing trends of key quality indicators of the product are periodically monitored, and the shelf life is predicted by combining accelerated testing. Experimental Method: The product is dispensed into sterile bottles and sealed for storage. A room temperature group (25±2℃) and an accelerated testing group (37±2℃) are set up, and samples are taken for testing at 0, 7, 14, 30, 60, 90, and 180 days. Test indicators include pH value (pH meter method), γ-glutamyl peptide retention rate (HPLC method, same as experimental scheme 2), total bacterial count (GB 4789.2-2016 plate count method), coliform bacteria (GB4789.3-2016 MPN method), and sensory score (10-person evaluation group evaluates color, odor, and taste, total score 100 points). Standard Basis: The relevant series of standards are based on GB / T 13079-2006 "Determination of Total Arsenic in Feed" and GB 13078-2017 "Feed Hygiene Standard". Key parameters: storage temperature 25℃ or 37℃, sampling time points (0 / 7 / 14 / 30 / 60 / 90 / 180 days), microbial testing using national standard methods, and a sensory score ≥80 points as qualified. Data processing: The shelf life endpoint is determined by γ-glutamyl peptide retention rate ≥90%, microbial indicators meeting the relevant requirements of GB 13078-2017 "Feed Hygiene Standard", and a sensory score ≥80 points. Each time point is measured three times.

[0074] Experimental Scheme 4: Slurry Rheological Properties Test Test Subject: Liquid fermented soybean peptide slurry with different solid contents. Test Objective: To evaluate the viscosity and rheological properties of the product under different solid contents (8-12%), and to verify the effect of pre-dissolution treatment and targeted enzymatic hydrolysis on expanding the processing window while maintaining low viscosity at higher solid contents. Test Principle: The viscosity and rheological curves of the slurry at different shear rates were measured using a rotational rheometer to analyze the slurry's flow behavior type (Newtonian or non-Newtonian) and thixotropy. Experimental Method: The liquid fermented soybean peptide product was diluted or concentrated to solid contents of 8%, 9%, 10%, 11%, and 12% (adjusted by vacuum concentration or water dilution), and measured at 25°C using a rotational rheometer (parallel plate geometry, 1mm spacing). Shear rates were measured from 0.1 s⁻¹. -1 linearly increased to 1000s -1 Record shear stress and apparent viscosity over a period of 5 minutes. Standard reference: GB / T 22427.6-2008 "Determination of Viscosity of Starch and its Products" or similar rheological testing standards. Key parameters: Solid content gradient (8-12%), test temperature 25℃, shear rate range 0.1-1000s. -1 The parallel plate spacing is 1 mm. Data processing: Plot the shear rate-viscosity curve and the shear stress-shear rate curve, and analyze the rheological index n (power law model τ=Kγ). n Determine the fluid type and calculate 100s values ​​for different solid contents. -1 The apparent viscosity at the shear rate was measured three times for each sample.

[0075] Experimental scheme 5: Determination of anti-nutritional factors Test Subject: Liquid fermented soybean peptide product. Test Objective: To determine the trypsin inhibitor activity and phytic acid content in the product, and to verify the removal effect of targeted enzymatic hydrolysis and heat treatment processes on anti-nutritional factors in soybeans. Test Principle: Trypsin inhibitor activity was quantified by measuring its inhibitory effect on trypsin activity. Phytic acid activity was quantified by spectrophotometric determination of the absorbance of the iron-phytic acid complex. Experimental Method: Trypsin inhibitor activity was determined using an enzyme inhibition method. 1g of sample was added to 100mL of 0.01mol / L Tris-HCl buffer (pH 8.0), extracted for 30 minutes, centrifuged, and the supernatant was mixed with trypsin solution (2mg / 100mL). The mixture was incubated at 37℃ for 10 minutes, and then BAPNA substrate (N-α-benzoyl-DL-arginine p-nitroaniline) was added. The reaction was terminated at 37℃ for 10 minutes, and acetic acid was added. The absorbance was measured at 410nm, and the inhibition rate and TIU activity units were calculated. Phytic acid content was determined according to GB / T 5009.153-2016. Samples were extracted with trichloroacetic acid and reacted with ferric chloride solution to form an iron-phytic acid purple-red complex. Absorbance was measured at 500 nm, and quantification was performed by comparing with a sodium phytate standard curve. Standards used: Trypsin inhibitors were determined according to GB / T18634-2002 "Determination of Trypsin Inhibitors in Cereals and Legumes," and phytic acid was determined according to GB / T 5009.153-2016. Key parameters: Trypsin inhibitor assay temperature 37℃, reaction time 10 minutes, detection wavelength 410 nm; phytic acid assay detection wavelength 500 nm. Data processing: TIU / g = (control group absorbance - sample group absorbance) × dilution factor / 0.01 / sample mass; phytic acid content (%) = concentration obtained from the standard curve / sample mass × 100%. Each sample was repeated three times.

[0076] Experimental Scheme 6: Evaluation of Feed Application Effects (Feeding Trial) Test Subject: The effect of liquid fermented soybean peptide product on the feed of weaned piglets. Test Objective: To evaluate the effect of the product on feed intake, daily weight gain, feed conversion ratio, and diarrhea rate in piglets, and to verify the practical application value of the product in feed. Test Principle: Through a controlled feeding trial, the differences in growth performance and health indicators between the control group and the control group with added liquid fermented soybean peptide product were compared. Experimental Method: 120 28-day-old weaned piglets (Duroc-Landrace-Large White crossbred, half male and half female, weight 7.5±0.5kg) were randomly divided into 4 groups (n=30 / group): control group (basal diet, without any soybean peptide product), experimental group I (basal diet + 0.10wt% soybean peptide product of Example 1), experimental group II (basal diet + 0.20wt% soybean peptide product of Example 1), and experimental group III (basal diet + 0.30wt% soybean peptide product of Example 1). All soybean peptide products were evenly sprayed onto the surface of the pelleted feed using a liquid spraying device. The experiment lasted 28 days. Daily feed intake was recorded, and weekly weight gain (ADG) and feed conversion ratio (F / G) were calculated. Daily fecal characteristics and diarrhea rate were recorded using the diarrhea index scoring method. At the end of the experiment, 8 piglets from each group were randomly selected for slaughter. Small intestinal villus height, crypt depth (V / C ratio), and the number of lactobacilli and Escherichia coli in the ileal digesta were measured (plate count method). Standards followed: NY / T 65-2004 "Standards for Pig Feeding" and relevant animal testing specifications. Key parameters: 28-day-old weaned piglets, 28-day experimental period, feed addition rate 0.10-0.30%, 8 animals per group, intestinal morphology and microbiological indicators. Data processing: One-way ANOVA was performed using SPSS statistical software. Duncan's multiple comparison test was used to determine the significance of differences between groups (P<0.05). Data for each group are expressed as mean ± standard deviation.

[0077] Figures 1 to 3 The influence of key process parameters of this invention on product performance is illustrated, wherein... Figure 1 The results show that when the molecular weight cutoff of the first-stage membrane is 3000-4000 Da, the proportion of target peptides in the 500-3000 Da range reaches the optimal level of 78.0-81.2%, and the proportion of small molecule peptides is stable at 88.0-91.5%, achieving precise enrichment of the target molecular weight range. However, when the molecular weight cutoff is below 2000 Da, too many large molecule peptides are retained, resulting in the loss of target peptides. When the molecular weight cutoff is above 5000 Da, too many large molecule proteins permeate, leading to a decrease in product purity. This indicates that the selection of the molecular weight cutoff of the first-stage membrane plays a key role in achieving a narrow molecular weight distribution. Figure 2The results showed that when the product pH was 3.5-4.6, the γ-glutamyl peptide retention rate could be maintained at a high level of 90.5-96.0% during the 90-day shelf life, while the total bacterial count was controlled below 100 CFU / mL, demonstrating good microbial stability. However, when the pH was higher than 5.0, the γ-glutamyl peptide retention rate dropped rapidly to below 85% and the total bacterial count increased significantly. When the pH was lower than 3.5, although the stability was excellent, it may affect palatability. This indicates that precise pH control is of great significance for ensuring the quality of the product during its shelf life. Figure 3 The results showed that when the γ-glutamylation reaction temperature was 40-45℃, the γ-glutamyl peptide content reached the optimal level of 46.5-49.8%, while the product maintained strong antioxidant activity with a DPPH scavenging IC50 of 75-78 μg / mL, achieving the best balance between modification efficiency and retention of active peptides. However, when the temperature was below 35℃, the enzyme activity was insufficient, resulting in incomplete modification. When the temperature was above 50℃, although the enzyme activity was high, the heat treatment caused some active peptides to be inactivated. This indicates that the optimization of the reaction temperature plays a key role in obtaining highly functional products. These single-factor experimental results fully verify the scientificity and rationality of the technical parameter range determined in this invention.

[0078] Figure 4 The peptide signals in the sample were highly concentrated in the target range of 500–3000 Da, and the overall distribution was in good agreement with the log-normal model. Moreover, the relative content in this range was significantly higher than the threshold requirement (≥70%), indicating that the system was mainly composed of medium and low molecular weight peptides with good dispersibility and reproducibility. This mass distribution was highly consistent with the expected enrichment window of γ-glutamyl peptides, which not only reduced the interference of non-target high molecular weight or low molecular weight noise, but also provided sufficient and stable effective signals for subsequent quantification and MS / MS structure identification, proving that the enrichment and detection scheme adopted was reasonable and effective.

[0079] Figure 5The comparative results showed that, compared with the unfermented soybean protein concentrate dry film, Example 1 (liquid fermented soybean peptide dry film) exhibited higher relative N and O contents on XPS, as well as enhanced C=O (~288.0–288.3 eV) in C 1s and C=O (~531.5–531.8 eV) in O 1s. At the same time, the proportion of amide N (~400.0–400.2 eV) and the proportion of free amine (~399.2–399.5 eV) in N 1s increased. The synergistic changes of these three sets of high-resolution signals and the Survey elemental fractions corroborated each other, eliminating the randomness caused by single peak fitting, and proving that the fermentation and subsequent film formation process did indeed introduce or enrich amide / carbonyl related functional groups, which is consistent with the chemical expectation of amino acid amidation and the increase in the relative proportion of peptide bonds. Both types of samples were obtained using the same substrate and film-forming process: solution was cast onto an inert substrate, pre-dried at 60–80°C, and then dried in a vacuum. After slicing, the samples were tested under the same instrument and parameters (Al Kα 1486.6 eV, C 1s 284.8 eV charge correction, Survey pass energy 100 eV and high resolution 20 eV, with charge neutralization enabled), ensuring that the spectral differences originated only from differences in the intrinsic chemical environment of the materials, thus ensuring that... Figure 5 The conclusions are repeatable and comparable.

[0080] The product performance of the examples and comparative examples is summarized in Table 1. In Comparative Example 1, the pre-dissolution temperature was reduced to 45°C, which is much lower than 50°C, resulting in insufficient dissolution of soybean protein and incomplete expansion of protein molecules. This significantly reduced the efficiency of subsequent enzymatic hydrolysis, and the final product had a wide and uneven molecular weight distribution. The proportion of the target peptide segment of 500-3000 Da was only 62%, which was much lower than the 78% in Example 1. The content of γ-glutamyl peptide was also only 32%. At the same time, due to the incomplete hydrolysis of protein, the residual amount of trypsin inhibitor was as high as 15 TIU / g, the phytic acid content was 0.08%, the anti-nutritional factors were not completely removed, and the viscosity of the slurry was as high as 85 mPa·s, which seriously affected the processing performance. In the feeding trial, the daily weight gain of piglets was only 285 g / d and the diarrhea rate was as high as 18.5%. Although Comparative Example 2, with a pre-dissolution temperature of 75℃ exceeding the upper limit of 70℃, reduced the trypsin inhibitor to 4 TIU / g, demonstrating better removal of anti-nutritional factors, the excessively high temperature caused partial protein denaturation, affecting the substrate accessibility for subsequent enzymatic hydrolysis. Simultaneously, the significantly increased energy consumption was detrimental to industrial production. Its overall performance was slightly better than Comparative Example 1, but still inferior to the balance and economy of Example 1. Comparative Example 3 used only neutral endopeptidase and lacked exopeptidase. While the protein was rapidly degraded into larger peptides by the endopeptidase, the lack of further modification by the exopeptidase prevented the efficient generation of small-molecule bioactive peptides. The molecular weight distribution was biased towards the macromolecular end, with only 58% of peptides in the 500-3000 Da range, and a mere 68% of peptides smaller than 1000 Da. The γ-glutamyl peptide content of 35% was also significantly lower than in Example 1. The trypsin inhibitor residue was 12 TIU / g, resulting in poor feeding performance, a daily weight gain of only 268 g / d, and a diarrhea rate of 21.5%, fully demonstrating the necessity of the dual-enzyme synergistic system. Comparative Example 4 adjusted the mass ratio of neutral endopeptidase to exopeptidase from 2:1 to 1:1. Although the two-enzyme system was retained, the enzyme ratio was not optimal. The relative amount of endopeptidase decreased, which slowed down the degradation rate of large protein molecules. The relative amount of exopeptidase increased, but insufficient substrate generation limited its function. The final molecular weight distribution accuracy and γ-glutamyl peptide content were slightly lower than those of Example 1, but better than those of Comparative Example 3 which used only a single enzyme, indicating the importance of enzyme ratio optimization for performance balance. Comparative Example 5 skipped the γ-glutamylation step entirely by not adding γ-glutamyl transpeptidase, resulting in a sharp drop in the γ-glutamyl peptide content of the product to only 5%. Although the molecular weight distribution and anti-nutritional factor indicators were similar to those of Example 1, it lost the unique functional properties brought by γ-glutamyl modification, such as enhanced flavor stability, antioxidant capacity and palatability. In the feeding trial, the daily weight gain was only 298g / d, which was significantly lower than the 385g / d of Example 1. The diarrhea rate of 16.2% was also relatively high, which fully demonstrates the key role of the γ-glutamylation reaction in the functionality of the product.Comparative Example 6 reduced the dosage of γ-glutamyl transferase to 10 U / g, far below the lower limit of 15 U / g, resulting in insufficient enzymatic modification. The γ-glutamyl peptide content was only 25%, failing to meet functional requirements. Although the enzyme cost was reduced, the core functional characteristics of the product were sacrificed, and the feeding effect was also affected. Comparative Example 7 increased the dosage of γ-glutamyl transferase to 40 U / g, exceeding the upper limit of 35 U / g. Although the γ-glutamyl peptide content increased to 52%, slightly higher than the 48% in Example 1, single-factor experimental data showed that when the enzyme dosage was further increased from 35 U / g to 40 U / g, the γ-glutamyl peptide content only increased from 54.5% to 55.2%, with extremely low marginal benefits, while the enzyme cost increased from 2.80 yuan / kg to 3.20 yuan / kg, significantly worsening the economics. Furthermore, excessive modification may introduce too many glutamyl residues, affecting other biological activities of the peptide. Therefore, although some indicators were slightly better, the overall cost-effectiveness was not as good as Example 1. Comparative Example 8 lowered the γ-glutamylation reaction temperature to 30°C, below the lower limit of 35°C, resulting in insufficient γ-glutamyl transpeptidase activity and low catalytic efficiency. Only 28% of the γ-glutamyl peptide was obtained after one hour of reaction. Furthermore, the slow substrate molecule movement at low temperatures further reduced the effective collision frequency between the enzyme and substrate, significantly limiting reaction kinetics. Comparative Example 9 lowered the enzyme inactivation and sterilization temperature to 60°C, below the lower limit of 65°C. While this mild treatment was beneficial for retaining heat-sensitive active peptides, incomplete enzyme inactivation allowed residual enzyme activity to continue during storage, leading to poor shelf-life stability. After 90 days, the γ-glutamyl peptide retention rate was only 72%, far lower than the 93% in Example 1. Simultaneously, the sterilization temperature of 60°C was insufficient to effectively kill heat-resistant microorganisms, resulting in a rapid increase in the total bacterial count during the shelf life, posing a serious risk to microbial stability. Comparative Example 10 used only a membrane with a molecular weight cutoff of 3000 Da for single-stage filtration, omitting the second-stage 1000 Da membrane fractionation. As a result, free amino acids and very small peptides smaller than 500 Da were not effectively removed from the product, while some large proteins and peptides larger than 3000 Da also permeated. The molecular weight distribution was extremely inaccurate, with the target peptides in the 500-3000 Da range accounting for only 52%, and peptides smaller than 1000 Da accounting for as low as 48%, far lower than the 78% and 88% of the two-stage fractionation in Example 1. The product purity was low and the enrichment of functional peptides was poor, which fully demonstrates the necessity and superiority of the two-stage tangential flow fractionation process. Comparative Example 11 increased the molecular weight cutoff of the first-stage membrane to 8000 Da, which significantly exceeded the upper limit of 5000 Da. This caused a large number of large protein and peptide molecules to pass through the first-stage membrane into the second-stage membrane. This not only increased the load on the second-stage membrane, leading to a rapid decline in membrane flux and aggravated fouling, but also significantly increased the content of large peptides with molecular weight of 3000-8000 Da in the final product. The proportion of target peptides with molecular weight of 500-3000 Da decreased to 65%, and the accuracy of molecular weight distribution control decreased significantly.Comparative Example 12 reduced the transmembrane pressure of the ultrafiltration membrane to 0.05 MPa, which is far below the lower limit of the preferred range of 0.08-0.15 MPa. This resulted in insufficient membrane filtration driving force, extremely low permeate flux, and a significant decrease in yield per unit time. Although the molecular weight distribution and γ-glutamyl peptide content were similar to those in Example 1, the high slurry viscosity of 62 mPa·s indicated severe concentration polarization under low transmembrane pressure. A concentration boundary layer formed on the membrane surface hindered mass transfer, resulting in low production efficiency and a significant increase in energy consumption and time cost per unit product. This makes it unsuitable for continuous industrial production. Comparative Example 13 adjusted the product's pH to 5.8, exceeding the upper limit of 5.5. Although a near-neutral pH may improve palatability in certain applications, it has serious drawbacks in terms of food preservation and shelf-life stability. pH 5.8 is close to the optimal growth pH range for most spoilage microorganisms. After a 90-day shelf life, the γ-glutamyl peptide retention rate plummeted to 68%, the total bacterial count increased rapidly, and the microbial stability was poor. At the same time, the higher pH was not conducive to inhibiting non-enzymatic browning such as Maillard reaction. The product was prone to color and flavor deterioration during storage, and the shelf-life quality was difficult to guarantee. Although the daily weight gain of 328g / d was acceptable in the feeding trial, the diarrhea rate was as high as 20.2%, indicating that insufficient microbial control may introduce intestinal health risks. The performance of all comparative examples demonstrates the effectiveness of the core technologies employed in Examples 1-4, including a pre-dissolution temperature of 50-70℃, a synergistic dual-enzyme system of neutral endopeptidase and exopeptidase at a ratio of 2:1 to 4:1, an appropriate dosage of γ-glutamyl transferase at 15-35 U / g, a γ-glutamylation reaction temperature of 35-50℃, a complete enzyme inactivation and sterilization temperature of 65-85℃, precise fractionation using two-stage tangential flow at 2000-5000 Da and 500-1500 Da, and product pH control at 3.5-5.5. All parameters have been scientifically optimized to achieve the best balance between maintaining a low viscosity processing window, narrow molecular weight precise enrichment, high γ-glutamyl peptide content, excellent shelf-life stability, and outstanding feeding application effect under high solid content conditions. Deviation of any single parameter will lead to significant deterioration of the product in terms of molecular weight distribution accuracy, γ-glutamyl modification efficiency, removal of anti-nutritional factors, processing performance, microbial stability, or feeding effect, which fully verifies the scientific nature, necessity, and synergistic effect of the technical solution of this invention.

[0081] Table 1 Performance summary of examples and comparative examples

[0082]

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that any equivalent structural transformations made under the concept of the present invention and using the contents of the specification and drawings of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing soybean peptides by liquid fermentation, characterized in that, Includes the following steps: S1. Pre-dissolution treatment: Soybean protein raw materials are pre-dissolved at 50-70℃ and pH 6.0-8.0 to obtain a pre-dissolved solution; S2. Targeted enzymatic hydrolysis: Neutral endopeptidase and exopeptidase are added to the pre-dissolved solution, and enzymatic hydrolysis is carried out at 40-55℃ and pH 6.0-8.0 to obtain hydrolysate; S3. γ-Glutamylation reaction: γ-glutamyl transpeptidase and donor are added to the hydrolysate, and the reaction is carried out at 35-50℃ and pH 7.0-9.0 to obtain the γ-glutamylation product; S4. Enzyme inactivation and sterilization: Treat at 65-85℃ for 15-30 minutes to obtain sterilization solution; S5. Cascaded tangential flow filtration: First, a membrane with a molecular weight cutoff of 2000-5000 Da is used for the first stage of filtration and the first permeate is collected. Then, a membrane with a molecular weight cutoff of 500-1500 Da is used for the second stage of filtration of the first permeate. The second stage permeate or the cutoff liquid is collected according to the target molecular weight window as the target fractionation to obtain the fractionated liquid. S6. Stabilize the formula by adjusting the pH of the fractionation liquid to 3.5-5.5 to obtain the liquid fermented soybean peptide product.

2. The method according to claim 1, characterized in that, In S3, the temperature is 35-50℃, the pH is 7.0-9.0, the amount of γ-glutamyl transpeptidase is 15-35 U / g protein, and the donor is L-glutamine and / or reduced glutathione.

3. The method according to claim 1, characterized in that, The mass ratio of neutral endopeptidase to exopeptidase in S2 is 1:1 to 4:1, and the mass ratio of enzyme to substrate is 1.0-2.5%.

4. The method according to claim 1, characterized in that, The pre-dissolution conditions for S1 are 60-65℃, pH 6.8-7.2, solid content of liquid material is 8-12%, and processing time is 30-60 minutes.

5. The method according to claim 1, characterized in that, The soybean protein raw material is soybean protein concentrate and / or soybean meal.

6. The method according to claim 1, characterized in that, In S5: When the molecular weight cutoff of the second-stage membrane is 1000-1500 Da, the second-stage permeate is collected. When the molecular weight cutoff of the second-stage membrane is 500-800 Da, the second-stage retentate is collected. The operating parameters of S5 are: transmembrane pressure 0.08-0.15MPa, tangential flow velocity 1.5-2.2m / s, and reflux ratio 3-6.

7. The liquid fermented soybean peptide product prepared by any one of claims 1-6, characterized in that, Peptides with a molecular weight of 500-3000 Da account for 60-90% of the total peptides by mass, γ-glutamyl peptide accounts for 30-70% of the total peptides by mass, and peptides with a molecular weight of less than 1000 Da account for 70-100% of the total peptides by mass. The product pH is 3.5-5.

5.

8. The product according to claim 7, characterized in that, The peptides with a molecular weight of 500-3000 Da account for 75-82% of the total peptides by mass, and the γ-glutamyl peptide content accounts for 40-60% of the total peptide content by mass.

9. The product according to claim 7, characterized in that, The product contains peptides with a molecular weight of less than 1000 Da, which account for 80-100% of the total peptide mass.

10. The use of the product according to any one of claims 7-9 in animal feed, characterized in that, It is used in pig feed to improve the palatability and / or flavor stability of the feed. It is applied by wet mixing or by dilution through a water line system. The equivalent addition amount is 0.05-0.50%, calculated as peptide solids or dietary dry matter.

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