Method for preparing active polypeptides based on plant stem cell-induced expression and directional fermentation of flora

Abstract

The present application relates to the field of bioengineering and microbial fermentation technology, and discloses a preparation method of active polypeptide based on plant stem cell induction expression and directional fermentation of flora. 35-45 parts of ginseng stem cell slurry are induced and cultured to express precursor proteins, and after physical cell wall breaking treatment, 4-6 parts of glucose, 2-4 parts of yeast extract powder, 1-3 parts of soybean polypeptide powder, 1-3 parts of a composite inorganic salt composed of potassium dihydrogen phosphate and magnesium sulfate, and the remaining amount of sterile deionized water are supplemented to prepare 100 parts by weight of the directional fermentation basic nutrient medium. The present application endows the active polypeptide with excellent target fragment yield and phase anti-aggregation stability, which highly meets the development needs of high-value plant-derived biological materials.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and microbial fermentation technology, specifically, to a method for preparing active peptides based on plant stem cell-induced expression and microbial community-directed fermentation. Background Technology

[0002] With the development of the biomanufacturing industry, obtaining highly active plant-derived peptides has become an important means of functional development. In complex application systems, plant peptides not only need to possess high specific bioactivity, but also excellent sensory qualities and molecular stability to ensure efficient absorption at the terminal.

[0003] However, in attempts to improve yield, traditional plant extraction and extensive fermentation processes often encounter obstacles from plant tissues that hinder the full release of precursor proteins. Non-directional crude enzymatic hydrolysis easily leads to disordered molecular weight distribution, making it difficult to effectively enrich the target fragments. Furthermore, these multiphase biotransformation systems have a narrow processing window: improper hydrolysis control during the early macromolecular shearing stage can easily cause excessive breakage of fragile active peptide chains; and without specific transpeptide modification in the later stages, short-chain hydrophobic ends tend to remain in large quantities, resulting in a product with a pronounced bitter taste and solution phase instability. These problems—limited precursor release, excessive degradation of the effective backbone, and sensory and physicochemical deterioration—constrain their application in high-end functional applications. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for preparing active peptides based on plant stem cell-induced expression and microbial community-directed fermentation, solving a common technical problem faced in the existing fields of bioengineering and microbial fermentation technology.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: This invention provides a method for preparing active peptides based on plant stem cell-induced expression and microbial-directed fermentation, comprising the following steps: S1, by weight, 35-45 parts of ginseng stem cell plasma are induced and cultured to express precursor proteins. After cell wall disruption, 4-6 parts of glucose, 2-4 parts of yeast extract powder, 1-3 parts of soybean polypeptide powder, 1-3 parts of a complex inorganic salt composed of potassium dihydrogen phosphate and magnesium sulfate, and the remainder of sterile deionized water are added to prepare 100 parts by weight of directional fermentation basic nutrient substrate and then sterilized. S2 introduces 3%-5% (v / v) of Bacillus subtilis seed liquid into the basic nutrient substrate and carries out the first stage of aerobic fermentation under constant temperature conditions of 35-39℃. The sterile air ventilation rate, main stirring speed and continuous fermentation time are controlled. The secreted extracellular neutral protease is used to cleave the macromolecular precursor protein into short-chain polypeptides. After the first stage of fermentation in S3, an environmental change was carried out by lowering the system temperature and reducing the stirring speed to maintain a micro-aerobic environment, and simultaneously inoculating 4%-6% of Lactobacillus plantarum seed liquid. The pH value of the fermentation broth was kept constant by the feed system, and continuous fermentation was carried out. Free aminopeptidase was used to finely remove and debitter the hydrophobic amino acids at the ends of the peptides. After S4 fermentation, the feed liquid is heated to inactivate the bacteria, centrifuged to remove bacterial residue, and the supernatant is collected. This supernatant is then pumped into a first-stage ultrafiltration membrane system with a molecular weight cutoff of 8000 Da for impurity removal and filtration to retain undegraded macromolecular proteins and enzymes. Subsequently, the collected permeate is introduced into a second-stage membrane system with a molecular weight cutoff of 1000 Da, and fractional permeate purification is performed under transmembrane pressure difference. The final permeate rich in the target fragment is collected and freeze-dried under vacuum to obtain the active polypeptide powder.

[0007] As an excellent embodiment of the method for preparing active polypeptides based on plant stem cell induced expression and microbial community directed fermentation according to the present invention, wherein: in step S1, the amount of ginseng stem cell slurry added is 40 parts, the amount of glucose added is 5 parts, the amount of yeast extract powder added is 3 parts; the amount of soybean polypeptide powder added is 2 parts; the amount of the complex inorganic salt composed of potassium dihydrogen phosphate and magnesium sulfate added is 2 parts, and the amount of sterile deionized water added is 48 parts; wherein, the mass ratio of glucose to yeast extract powder is 5:3.

[0008] As an excellent embodiment of the method for preparing active polypeptides based on plant stem cell induced expression and microbial community directed fermentation according to the present invention, wherein: the volume fraction of Bacillus subtilis seed liquid in step S2 is 4%; the constant temperature of the first stage aerobic fermentation is 37℃; the controlled sterile air ventilation rate is 0.8-1.2 vvm; the main stirring speed is 200-300 rpm; and the continuous fermentation is 14-18 hours.

[0009] As an excellent embodiment of the method for preparing active polypeptides based on plant stem cell induced expression and microbial community directed fermentation according to the present invention, the method includes: controlling the sterile air ventilation rate to be 1.0 vvm; the main stirring speed to be 250 rpm; and continuous fermentation for 16 hours; wherein the ventilation rate and stirring speed are 1.0 vvm and 250 rpm.

[0010] As an excellent embodiment of the method for preparing active polypeptides based on plant stem cell induced expression and microbial community directed fermentation according to the present invention, wherein: the micro-oxygen environment temperature in step S3 is 30-34℃; the stirring speed is 50-70 rpm; the liquid fraction of Lactobacillus plantarum seed is 5%; the pH value of the fermentation broth is constant at 6.0-6.5; and the continuous fermentation time is 20-28 hours.

[0011] As an excellent embodiment of the method for preparing active peptides based on plant stem cell induced expression and microbial community directed fermentation according to the present invention, wherein: the micro-aerobic environment temperature in step S3 is 32℃; the stirring speed is 60 rpm; the pH value of the fermentation broth is constant at 6.2; and the continuous fermentation time is 24 hours.

[0012] As an excellent embodiment of the method for preparing active polypeptides based on plant stem cell induced expression and microbial community directed fermentation according to the present invention, wherein: the heating inactivation temperature in step S4 is 80-90℃; and the transmembrane pressure difference is 0.20-0.30MPa.

[0013] As an excellent embodiment of the method for preparing active polypeptides based on plant stem cell induced expression and microbial community directed fermentation according to the present invention, wherein: the heating inactivation temperature in step S4 is 85℃; and the transmembrane pressure difference is 0.25MPa.

[0014] As an excellent embodiment of the method for preparing active polypeptides based on plant stem cell induced expression and microbial community directed fermentation described in this invention, in step S4, the specific process conditions for vacuum freeze drying are as follows: pre-freezing temperature is -50℃, cold trap desorption temperature is -60℃, system vacuum degree is controlled at 10 Pa, and continuous drying treatment is performed for 24 hours.

[0015] This invention also provides an active polypeptide based on plant stem cell-induced expression and microbial-directed fermentation, prepared by the process described in steps S1 to S4 above. The active polypeptide uses a complex system of macromolecular precursor protein induced by ginseng stem cells and soybean polypeptide as the substrate backbone. It undergoes enzymatic degradation and fine modification by two bacteria in aerobic and microaerobic environments, and its molecular weight is mainly distributed below 1000 Da. The hydrophobic amino acid residues at the end of the polypeptide molecular chain are directionally removed, forming a low bitterness characteristic, and it exists in a small molecule free peptide phase with high anti-agglomeration stability.

[0016] The beneficial effects of this invention are as follows: Based on aerobic coarse shearing by Bacillus subtilis and supplemented by microaerobic peptide modification by Lactobacillus plantarum, a dual-strain fermentation system significantly improves the yield of target fragments and sensory quality of the product while maintaining the specific biological activity of the peptides. It overcomes the limitations of traditional processes that easily lead to excessive degradation of the peptide backbone and bitterness caused by hydrophobic end residues. This allows active peptides to achieve efficient hydrolysis of large molecular precursors in the aerobic stage and exhibit precise end-cutting and phase buffering capabilities in a constant microaerobic environment, broadening their application window in high-end nutritional products. Furthermore, the gentle differential pressure ultrafiltration purification effectively reduces concentration polarization and impurity penetration at the membrane surface, and the optimized parameter settings preserve the structural integrity of small molecule peptides, resulting in better physical anti-agglomeration stability and overall flavor performance in rehydration and practical applications. Attached Figure Description

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

[0018] Figure 1a The graph shows the effect of quantitative ratio of glucose / yeast extract on absorbance values.

[0019] Figure 1b The optimization diagram of the effect of quantitative ratio of glucose / yeast extract on the extreme value of extracellular neutral protease activity.

[0020] Figure 1c An optimization diagram of the offset of the hydrogen ion concentration index for the quantitative ratio of glucose / yeast extract.

[0021] Figure 1d The graph shows the optimization of the actual yield of small molecule active peptides by quantitatively combining glucose / yeast extract.

[0022] Figure 2 This is a diagram showing the optimized ratio of soybean polypeptide powder usage.

[0023] Figure 3 This is a diagram showing the optimized ratio of compound inorganic salts.

[0024] Figure 4a This is a graph showing the optimization of aeration rate / stirring speed on the absorbance value of the cells during aerobic fermentation.

[0025] Figure 4b The graph shows the optimization of aeration rate / stirring speed for neutral protease production rate during aerobic fermentation.

[0026] Figure 4c This is a graph showing the optimization of the effect of aeration rate / stirring speed on the dissolved oxygen level of the fermentation broth during aerobic fermentation.

[0027] Figure 5 An optimized timeline for the transition from aerobic to microaerobic stages.

[0028] Figure 6 This diagram shows the process optimization for the isothermal metabolic temperature of the second-stage micro-oxygen transpeptide modification.

[0029] Figure 7 This is a process optimization diagram for the second stage of micro-oxygen transpeptide modification with added acid-base control.

[0030] Figure 8 This is a process optimization diagram for ultrafiltration purification of active peptides based on transmembrane pressure difference.

[0031] Figure 9 This is a flowchart illustrating the preparation of bioactive peptides based on plant stem cell-induced expression and microbial-directed fermentation. Detailed Implementation

[0032] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0033] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "example" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the invention. The appearance of an embodiment in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that mutually excludes other embodiments.

[0035] Example 1 This embodiment aims to explore a method for preparing active peptides that combines high expression of precursor-targeted peptides with directed relay enzymatic hydrolysis, in order to solve the technical bottlenecks in the preparation of traditional plant peptides, such as low expression of active ingredients and the tendency of exogenous crude enzymatic hydrolysis to produce terminal bitterness and wide molecular weight distribution.

[0036] 1.1 Screening and Establishment of Core Fermentation Substrates To construct a targeted transformation and highly active peptide fermentation system, it is first necessary to identify the basic raw materials that serve as the core substrates and active precursors for microbial relay enzymatic hydrolysis. This stage aims to evaluate the synthesis and transformation efficiency, enzymatic response rate, and retention capacity of secondary active metabolites of precursor proteins from different types of plant stem cells, conventional plant tissues, and industrially extracted proteins during environmental stress induction and dual-strain targeted fermentation, thereby screening for fermentation substrates with better induction response potential and biochemical transformation potential.

[0037] Experimental groups: Ginseng stem cell pulp; Centella asiatica stem cell pulp; Saussurea involucrata stem cell pulp; Dendrobium officinale stem cell pulp; conventional ginseng root tissue pulverized pulp; conventional Panax notoginseng root tissue pulverized pulp; soybean protein isolate suspension; corn germ protein suspension; wheat gluten protein suspension; pea protein suspension; rice protein suspension; whey protein suspension; yeast extract suspension. According to GB5009.5-2016 National Food Safety Standard, the increase in target protein of each substrate was measured after 48 hours of environmental stress induction to assess the material's response to the induction signal and its synthesis. According to GB / T22492-2008 National Standard for Soybean Peptide Powder, the final degree of hydrolysis of each material after dual-strain fermentation was determined using the pH-stat method to determine the depth of substrate degradation and transformation by the microbial community. Referring to the relevant evaluation specifications in QB / T2730-2005, the free radical scavenging rate of each product was measured after fermentation to assess the physiological activity level of the peptide system conferred by the substrate. According to the size exclusion chromatography method in GB / T22492-2008, the proportion of small molecule peptides in each group of products was determined to measure the fineness of the overall material being directionally sheared.

[0038] Among them, whey protein suspension and yeast extract suspension showed slightly higher final hydrolysis degrees, but their target protein increment data were relatively low, making it difficult to meet the process requirements for the storage of precursor substances for the target product. Various conventional plant tissue pulverized slurries were constrained by dense cell walls and natural tissue fiber networks, resulting in significant resistance to the entry of exogenous microorganisms and enzyme systems, leading to a shallow overall hydrolysis and transformation depth, and the proportion of small molecule peptides did not reach the ideal range. The free radical scavenging rate of fermentation products obtained from rice protein suspension and wheat gluten protein suspension was low, and the apparent functional activity indicators were unsatisfactory. Although soybean protein isolate suspension and pea protein suspension could achieve a relatively ideal small molecule peptide conversion ratio, their target protein increment and product antioxidant capacity were limited, failing to effectively enrich physiologically active ingredients. While Centella asiatica stem cell slurry and Saussurea involucrata stem cell slurry showed good retention levels of natural active ingredients and a high free radical scavenging rate, the efficiency of complete fragmentation of large molecule peptides was slightly insufficient, resulting in generally average final hydrolysis degrees.

[0039] Ginseng stem cell plasma exhibits a significant increase in targeted protein levels while demonstrating outstanding free radical scavenging efficiency. Its overall fermentation and transformation performance is excellent, exhibiting a balanced characteristic without any obvious weaknesses; therefore, it has been established as the core substrate for the fermentation process.

[0040] 1.2 Screening and Establishment of Primary Carbon Source Fermentation Adaptability After establishing ginseng stem cell plasma as the core fermentation raw material, an appropriate amount of primary carbon source must be added to the system in order to construct a complete fermentation nutrient system and ensure the smooth initiation of the dual-strain enzymatic hydrolysis. This stage aims to evaluate the comprehensive performance of different carbon sources in terms of fermentation initiation rate, enzyme induction activity, and final peptide production efficiency, thereby screening out primary carbon sources with better fermentation conversion potential.

[0041] Experimental groups: glucose; fructose; sucrose; maltose; soluble starch; dextrin; lactose; glycerol; cane molasses. Twelve hours before the first stage of aerobic fermentation, the absorbance of the bacterial cells was measured at regular intervals, and the time required for the system to reach its logarithmic phase peak was recorded to assess the driving effect of each carbon source on the fermentation initiation rate. According to industry standards, the peak activity of neutral protease in the fermentation broth at the end of aerobic fermentation was measured to assess the inducing effect of carbon sources on the enzyme production level of Bacillus subtilis. The final degree of hydrolysis after dual-strain fermentation was determined using the pH-stat method in GB / T 22492-2008 to determine the depth of substrate decomposition and transformation by the microbial community. The actual yield of small molecule active peptides (molecular weight less than 1000 Da) in the final lyophilized powder product was determined using the high-performance liquid chromatography method in GB / T 22492-2008 to measure the overall conversion efficiency of the material under directional shearing.

[0042] Among them, the soluble starch, dextrin, and lactose groups had slow fermentation initiation due to limited transmembrane transport and utilization efficiency, with the time required to reach the logarithmic phase peak exceeding 10 hours, and low enzyme production and final degree of hydrolysis. The glycerol and sugarcane molasses groups were affected by assimilation rate and steric hindrance from impurities, resulting in shallower conversion depth. The maltose group performed better in both the neutral protease activity peak and the final degree of hydrolysis, exhibiting good slow-release enzymatic hydrolysis characteristics, but its fermentation initiation was slightly slower, and the actual yield of small molecule active peptides was second only.

[0043] In comparison, the glucose group exhibited excellent bioavailability and a yield of 82.8% for its small molecule active peptides, demonstrating superior data. Although the glucose group was slightly lower than the maltose group in terms of enzyme production and degree of hydrolysis, its overall metabolism was smooth, and the system showed balanced performance without any obvious shortcomings. Therefore, glucose was established as an excellent primary carbon source for this process.

[0044] 1.3 Screening and Establishment of Primary Nitrogen Source Fermentation Adaptability After establishing ginseng stem cell plasma and glucose as the core substrate and excellent carbon source, respectively, the fermentation system also needs to be supplemented with a suitable primary nitrogen source. This stage aims to evaluate the actual biochemical performance of different nitrogen sources in promoting cell proliferation, inducing extracellular protease secretion, and regulating the final peptide yield, thereby screening out a primary nitrogen source with better compatibility for the dual-strain fermentation system.

[0045] Experimental groups: yeast extract powder; peptone; soybean meal powder; urea; ammonium sulfate; sodium nitrate; corn steep liquor powder; beef extract powder; casein hydrolysate. Samples were taken periodically during fermentation, and the maximum cell biomass of the fermentation broth was determined spectrophotometrically to assess the supporting capacity of nitrogen sources for cell population expansion. The maximum extracellular total protease activity at mid-fermentation was determined using the Folin-phenol method to investigate the activation level of different nitrogen sources on the enzyme degradation capacity of the fermentation population. The final degree of hydrolysis was determined according to GB / T 22492-2008 to assess the chain scission depth of macromolecular precursor proteins. The actual yield of small-molecule active peptides in the final product was determined using high-performance liquid chromatography (HPLC) to measure the conversion yield of the finished peptides.

[0046] Among the groups using inorganic nitrogen sources such as urea, ammonium sulfate, and sodium nitrate, the biochemical indicators were generally low, with the maximum bacterial biomass not exceeding 2.00, indicating a slow assimilation and utilization efficiency of inorganic small-molecule nitrogen by the inoculated heterotrophic bacterial community. The soybean meal powder and corn steep liquor powder groups, due to their high content of crude fiber and insoluble macromolecules, had limited enzymatic hydrolysis depth. The beef extract powder and casein hydrolysate groups showed relatively stable overall indicators, but the overall effect was not optimal.

[0047] Yeast extract powder can provide abundant B vitamins and growth factors such as nucleotides. Its cell biomass reaches an extreme value of 4.25, and the actual yield of small molecule active peptides reaches 82.5%. It shows balanced performance in various indicators and has no obvious conversion shortcomings. It can well meet the high-efficiency conversion of high-activity peptides. Therefore, yeast extract powder is established as an excellent primary nitrogen source for this process.

[0048] 1.4 Screening and Establishment of Adaptability of Peptide-Assisted Substrate Fermentation After establishing the basic ratio of ginseng stem cell wall-breaking pulp, glucose, and yeast extract powder, in order to enhance the terminal modification ability of *Lactobacillus plantarum* in the second stage under microaerobic conditions and effectively remove bitter terminal amino acids from the product through transpeptidation, an appropriate amount of low-molecular-weight transpeptidation auxiliary substrate needs to be introduced into the system. This stage aims to evaluate the effects of different peptide substrates on the induction activity of aminopeptidase secretion, the debittering modification efficiency of the product, and the yield of physiological activity, thereby screening for substrates with better auxiliary modification effects on the two-strain relay system.

[0049] Experimental groups: soybean peptide powder; corn peptide powder; wheat oligopeptide powder; collagen peptide powder; pea peptide powder; rice peptide powder; whey peptide powder; peanut peptide powder; walnut peptide powder. The extreme values ​​of aminopeptidase activity in the fermentation broth were determined spectrophotometrically to assess the activation level of the substrate on the terminal modification enzyme system of *Lactobacillus plantarum*. Following GB / T 22492-2008 standard, an electronic tongue system combined with sensory quantitative evaluation was used to determine the removal rate of bitter peptides after enzymatic hydrolysis of the fermentation broth, to examine the actual effect of transpeptide modification on taste improvement. According to QB / T 2730-2005 standard, the free radical scavenging rate of the final freeze-dried product was determined to examine the synergistic activation level of the auxiliary substrate on the overall antioxidant function system. The actual yield of small molecule active peptides in the finished product was determined by high-performance liquid chromatography.

[0050] The measured data for various indicators of collagen peptide powder, rice peptide powder, and corn peptide powder were relatively low, with their peak aminopeptidase activity all failing to reach 130.0 U / mL, corresponding to a bitter peptide removal rate of less than 75.0%. They also showed difficulty in effectively binding to the transpeptidation sites of *Lactobacillus plantarum*. The wheat oligopeptide powder group performed better in terms of peak aminopeptidase activity, reaching 152.8 U / mL, exhibiting a better tendency to induce enzymatic reactions.

[0051] Soybean peptide powder exhibits good compatibility in peptide end-modification characteristics and has a balanced overall biochemical performance, achieving a relatively stable balance between taste optimization and active peptide yield. Therefore, soybean peptide powder is established as an excellent transpeptide auxiliary substrate for this process.

[0052] 1.5 Screening and Establishment of Adaptability to Inorganic Salt Fermentation After establishing the basic carbon-nitrogen and excipient ratios of ginseng stem cell wall-breaking pulp, glucose, yeast extract powder, and soybean polypeptide powder, an appropriate amount of inorganic salt ions needs to be introduced into the system. The complex inorganic salt system not only maintains the osmotic pressure balance inside and outside the microbial cells and buffers pH fluctuations, but specific metal cations and phosphate ions are also essential cofactors and activators for the synthesis of extracellular proteases and aminopeptidases by Bacillus subtilis and Lactobacillus plantarum. This stage aims to evaluate the effects of different inorganic salt formulations on the microenvironment buffering capacity, the degree of improvement in key enzyme catalytic activity, and the final yield of active products, thereby screening out the complex inorganic salt combination with the best ion-promoting effect for this fermentation process.

[0053] Experimental groups: 1:1 potassium dihydrogen phosphate and magnesium sulfate; 1:1 sodium dihydrogen phosphate and magnesium sulfate; 1:1 dipotassium hydrogen phosphate and magnesium chloride; 1:1 potassium dihydrogen phosphate and calcium chloride; 1:1 magnesium sulfate and ferric sulfate; 1:1 potassium chloride and magnesium sulfate; 1:1 disodium hydrogen phosphate and calcium chloride; 1:1 zinc sulfate and potassium dihydrogen phosphate; 1:1 sodium chloride and potassium dihydrogen phosphate. Maintaining a constant ratio of other raw materials in the fermentation substrate (i.e., 40 parts ginseng stem cell wall-breaking pulp, 5 parts glucose, 3 parts yeast extract powder, and 2 parts soybean polypeptide powder), two parts of each of the above different 1:1 weight ratio inorganic salt combinations were added for fermentation. At the end of the first stage of aerobic fermentation, the pH deviation at the fermentation endpoint (i.e., the absolute value of the difference from the initial pH of 6.8) was measured using a precision pH meter to assess the buffering capacity of inorganic salts on acidic metabolic products. The extreme activity of Bacillus subtilis neutral protease during fermentation was determined using the Folin-phenol method to investigate the activation effect of different salt ions on key enzymes. According to GB / T 22492-2008 standard, the final degree of hydrolysis after dual-strain fermentation was determined using the pH-stat method to assess the promoting effect of the ion system on the entire directional cleavage process. The actual yield of small-molecule active peptides in the finished powder was determined using high-performance liquid chromatography (HPLC).

[0054] Among them, the 1:1 magnesium sulfate and ferric sulfate, 1:1 potassium chloride and magnesium sulfate, and 1:1 sodium chloride and potassium dihydrogen phosphate groups all showed significant pH shifts at the fermentation endpoint (greater than 0.65). The 1:1 potassium dihydrogen phosphate and calcium chloride group performed well in the final degree of hydrolysis determination, reaching 39.4%, showing a positive modifying effect of calcium ions on the catalytic stability of some proteases. However, due to the lack of crucial assistance from magnesium ions, the system's pH buffering capacity and final active peptide yield were insufficient.

[0055] The 1:1 potassium dihydrogen phosphate and magnesium sulfate group showed a fermentation endpoint pH shift of only 0.32, maintaining a stable near-neutral metabolic microenvironment for the microbial community. Simultaneously, this group achieved an actual yield of 82.8% for small molecule bioactive peptides, ranking among the top performers in the data. Although the extreme value of neutral protease activity was slightly lower than some calcium salt groups, the system demonstrated balanced transformation performance without significant weaknesses through a stable microecology and appropriate activation by divalent metal ions. Therefore, the 1:1 potassium dihydrogen phosphate and magnesium sulfate combination was established as an excellent composite inorganic salt.

[0056] Example 2 Referring to Figures 1-3, this is the second embodiment of the present invention. This embodiment aims to quantitatively optimize the proportions of the selected superior raw materials through systematic experimental design. Although the basic transformation functions of each individual component have been confirmed, the activity and yield of the final polypeptide product are not simply the sum of the effects of each component, but depend on the dynamic balance between cell metabolism and proliferation and the efficiency of exogenous enzymatic hydrolysis during the two-strain fermentation, as well as the synergistic regulatory effect of the concentration and proportion of each component on the dynamics of this microenvironment.

[0057] 2.1 Quantitative optimization of the ratio of glucose to yeast extract powder Using a final basic fermentation system of 100 parts by weight as a baseline, this study aimed to investigate the growth kinetics, extracellular enzyme production efficiency, and microenvironment buffering boundaries of *Bacillus subtilis* and *Lactobacillus plantarum* during a two-strain fermentation process under different carbon and nitrogen concentrations and ratios. It also aimed to provide experimental data to support the establishment of a highly efficient biochemical nutrient range for continuous directional enzymatic hydrolysis and peptide conversion, demonstrating the synergistic regulatory effect of rapid energy supply metabolism from primary carbon sources and the supplementation of growth factors and buffering of the acid-base microenvironment by primary nitrogen sources. A full cross-matrix experiment was conducted, with primary carbon source (glucose) ranging from 3.0% to 10.0% in 1.0% steps and primary nitrogen source (yeast extract powder) ranging from 1.5% to 5.0% in 0.5% steps. A systematic evaluation was performed on four core indicators: cell absorbance (OD600), maximum extracellular neutral protease activity (NPAP), hydrogen ion concentration index shift, and actual yield of small molecule active peptides (SMPY) during fermentation, to establish suitable process ratios.

[0058] Test results are as follows Figure 1aAs shown in Figure -d, the absorbance value (OD600) of the fermentation broth cells, when the amount of yeast extract powder is fixed, exhibits a non-linear trend of first increasing and then decreasing with the increase of glucose. When the amount of glucose is fixed, it also shows a trend of first gradually increasing and then stabilizing with the increase of yeast extract powder. The optimal value range is concentrated in the range of 5.0%–7.0% glucose and 3.0%–3.5% yeast extract powder. Excessive carbon-nitrogen ratios lead to hyperosmolar inhibition and organic acid accumulation, resulting in a significant decline in biomass in the high-concentration range. The maximum extracellular neutral protease activity (NPAP) shows a positive response with the increase of yeast extract powder, but continued excessive glucose accumulation will limit the synthesis and extracellular release of neutral protease due to premature acidification of the system and the inhibitory effect of carbon source decomposition metabolism. The higher activity range is distributed in the region of 5.0%–6.0% glucose and 3.0%–3.5% yeast extract powder. The hydrogen ion concentration index offset of the fermentation system showed a continuous upward trend with the increase of glucose dosage, while it showed a significant decrease with the increase of yeast extract powder dosage, gradually stabilizing when the proportion reached around 3.0%. The region with small offset and good acid-base buffering appeared in the range of medium-low glucose and medium-high yeast extract powder. The actual yield of small molecule active peptides (SMPY) showed the result of the synergy of multiple biochemical stages. Its value was not a linear accumulation of a single substrate, but was highly dependent on the coarse cleavage efficiency of the neutral protease in the first stage and the connection balance of the terminal transpeptidation and debittering modification of aminopeptidase in the microaerobic environment of the second stage. The high yield region and the medium carbon-nitrogen balance zone (5.0%~6.0% glucose, 3.0%~3.5% yeast extract powder) highly overlapped, while the low yield region fell into the nutrient-deficient zone and the metabolic inhibition zone of high acid and high osmotic pressure.

[0059] By systematically normalizing the data of the four key process indicators mentioned above, and then combining them with biochemical reaction kinetic feedback for multidimensional weighted calculations, the aim was to simulate and find the comprehensive equilibrium point of continuous multi-microbial community proliferation, catalytic enzyme library construction, microenvironmental acid-base stability, and terminal active peptide enrichment. The theoretical equilibrium point of the comprehensive physiological transformation performance of this peptide fermentation system was found to be glucose: yeast extract powder = 5.24%: 3.12%. Considering the material weighing system's requirements for industrial-scale fermentation, the uniformity of mass transfer and mixing in large tanks, the factors for compensating for sterilization heat loss, and the robust requirements for continuous multi-batch production, a fixed ratio close to the theoretical point was finally selected as the process formulation: glucose 5.0%, yeast extract powder 3.0%.

[0060] 2.2 Optimization of the dosage ratio of transpeptide co-substrate (soybean peptide powder) To investigate the dynamic balance between the efficiency of peptide terminological modification and the mass transfer rheological resistance of the fermentation system at different concentrations of the transpeptide helper substrate (soybean peptide powder), and to provide data support for ensuring efficient chain scission and enrichment of short-chain bioactive peptides in the second-stage microaerobic transpeptide fermentation, a single-factor gradient experiment with soybean peptide powder was set up, ranging from 0.5% to 4.0% in 0.5% increments, for a total of 8 experimental groups. The absorbance (OD600) of the bacterial cells during the peak fermentation period was determined by spectrophotometry at a wavelength of 600 nm to monitor the direct impact of substrate concentration changes on the basic proliferation dynamics of heterotrophic bacteria. The extreme extracellular aminopeptidase activity (APAP) during the peak microaerobic fermentation period was determined using the standard Folin-Ciocalteu method to evaluate the secretion-inducing effect of different excipient concentrations on the terminal transpeptide-modified enzyme system of *Lactobacillus plantarum*. An electronic tongue system combined with sensory quantitative evaluation was used to determine the removal rate of bitter peptides after enzymatic modification to examine the actual effect of transpeptide modification on improving the product's taste. The actual yield (SMPY) of small molecule active peptides (molecular weight less than 1000 Da) in the final product powder was determined by high performance liquid chromatography to comprehensively evaluate the actual peptide enrichment efficiency of different ratios in the dual-strain relay catalysis system.

[0061] Test results are as follows Figure 2 As shown, the extracellular aminopeptidase activity and bitter peptide removal rate were both low in the low addition range, failing to form a sufficient transpeptide modification donor substrate library and not meeting the basic conversion requirements of targeted excision and deep modification of terminal amino acids during fermentation. In the high addition range, with the continuous increase of soybean peptide powder mass fraction, the bitter peptide removal rate and the actual yield of small molecule active peptides did not show a significant increase, and even showed a slight decline. Excessive mass fraction of exogenous peptide solid content leads to high flow viscosity of fermentation materials, which easily generates additional shear resistance and dissolved oxygen mass transfer obstacles within the material system, spatially inhibiting the effective catalytic collision between exogenous enzymes and substrates, resulting in a decrease in subsequent conversion rate, and also unnecessarily increasing the raw material cost of bio-fermentation preparation.

[0062] By normalizing the four core performance indicators and performing weighted fitting analysis, the theoretical comprehensive equilibrium point for peptide conversion performance was calculated to be 2.06%. Considering the accuracy tolerance of the weighing system during industrial-scale fermentation, the energy consumption limitations of material mixing and mass transfer, and the robustness of continuous production across multiple batches, a value close to this theoretical equilibrium point and easily operable and controllable was ultimately selected, namely, the determined dosage of soybean peptide powder is 2.0%.

[0063] 2.3 Optimization of the dosage ratio of composite inorganic salts To investigate the balance between the acid-base buffering homeostasis of the fermentation system and the catalytic activity of core metalloenzymes under different concentrations of a compound inorganic salt (composed of potassium dihydrogen phosphate and magnesium sulfate in a 1:1 weight ratio), and to provide experimental data to support the avoidance of metabolic inhibition of Bacillus subtilis and Lactobacillus plantarum by hyperosmotic salt precipitation, this study aimed to demonstrate the synergistic regulatory effect of phosphate ions providing a strong buffer and magnesium ions activating the catalytic center of key enzymes. A single-factor gradient experiment with the compound inorganic salt was set up, ranging from 0.5% to 4.0% in 0.5% steps, for a total of 8 experimental groups.

[0064] Fermentation was carried out under conditions where the optimized organic ratios of base slurry, glucose, yeast extract powder, and soybean peptide powder were kept constant. An online pH meter was used to monitor the pH shift at the end of the first fermentation stage (i.e., the absolute value of the difference between the final pH and the initial pH of 6.8) to assess the buffering capacity of inorganic salt concentration on metabolic acidic products. The peak extracellular neutral protease activity (NPAP) at the fermentation peak was determined using the standard Folin-phenol method to assess the activation performance of key degradative enzymes in Bacillus subtilis under different ionic intensities. The actual yield (SMPY) of small molecule active peptides in the final product powder was determined by high-performance liquid chromatography to investigate the effect of the ionic microenvironment on the overall directional chain scission yield.

[0065] Test results are as follows Figure 3 As shown, in the low addition range (0.5%~1.0%), due to the lack of sufficient phosphate ions as a buffer and insufficient concentration of free magnesium ion cofactor, the pH shift at the fermentation endpoint increased significantly. The rapid acidification of the system's metabolism hindered cell secretion, resulting in low neutral protease activity and low yield of small molecule peptides. With increasing mass fraction of the complex inorganic salts, all parameters showed a positive response. Specifically, when the mass fraction reached 2.0%, the actual yield of small molecule active peptides (SMPY) reached its extreme value of 84.8%, and its pH shift at the fermentation endpoint was controlled at a relatively optimal level of 0.28. Although the extreme protease activity and pH shift at 2.5% were slightly better than at 2.0%, the two sets of indicators were very close. However, in the high addition range (above 3.0%), the high concentration of dissociated inorganic cations increased the exogenous osmotic pressure of the fermentation system, triggering severe cell osmotic stress and even local micro-salting-out effects, leading to changes in enzyme catalytic conformation and a significant convergence and decline in peptide conversion rate.

[0066] By normalizing and weighting the three core physicochemical performance indicators mentioned above, a theoretical comprehensive equilibrium point of 2.08% was finally calculated, which balances buffer stability and high yield performance. Considering the control threshold requirements for conductivity monitoring during large-volume liquid preparation in industrial-scale fermentation systems and the robustness of multi-batch production, a fixed-point value that is close to this theoretical equilibrium point and easy to operate and control was finally selected, namely, the determined dosage of the compound inorganic salt is 2.0%.

[0067] Example 3 Refer to Figure 4- Figure 8 This is the third embodiment of the present invention. This embodiment aims to scale up the previously determined core raw materials and optimized matrix ratios to a pilot-scale operation. Considering that in continuous industrial-scale production, large-volume fermenters are prone to oxygen transfer limitations and nutrient gradient shearing, this will lead to significant differences between the heterotrophic bacterial proliferation and metabolic rates, the synthesis and release of extracellular degradation enzymes, and the biochemical processes of microaerobic peptide modification in the dual-strain fermentation system and the static microscopic results in the laboratory. Therefore, it is necessary to conduct pilot-scale experiments to finely modify and optimize core engineering parameters such as aeration rate, stirring speed, and the process window for the transition from aerobic to microaerobic stages during fermentation. This ensures that under large-scale production conditions, both healthy growth and metabolism of the bacterial community can be maintained, and deep enzymatic hydrolysis can be achieved, thereby comprehensively guaranteeing high conversion reproducibility and batch consistency of the active peptide product in large-scale industrial production.

[0068] 3.1 Preparation of Ginseng Stem Cell Cell Wall-Breaking Pulp Suspension The raw materials were pretreated using a phased nutrient buffering and gentle shearing method. 48.0 parts by weight of sterile deionized water were added to a sterile preparation vessel, and stirring was started to maintain surface flow. 5.0 parts by weight of glucose and 2.0 parts by weight of a complex inorganic salt were slowly added until completely dissolved to establish initial osmotic pressure balance and improve the cell microenvironment. Maintaining a low shear state, 40.0 parts by weight of ginseng stem cell aggregates were uniformly added to ensure thorough infiltration and dispersion, initially constructing a cell suspension system. Next, 3.0 parts by weight of yeast extract powder and 2.0 parts by weight of soybean polypeptide powder were added sequentially, utilizing good flowability to prevent localized aggregation of cell aggregates. Finally, the homogenization and disruption system was activated, using mechanical shear force to fully disperse the aggregates and effectively break down the cell walls, promoting the release of intracellular active precursor substances. After all materials were added, the rotation speed was adjusted for continuous homogenization for 20 minutes to achieve microscopic uniformity and stability, completing the preparation of the ginseng stem cell cell suspension.

[0069] 3.2 Optimization of the coupling between aerobic fermentation aeration rate and stirring speed The gas-liquid mass transfer efficiency and fluid physical shear force in the pilot-scale fermenter are core engineering parameters affecting heterotrophic metabolism, biomass expansion, and target enzyme secretion in Bacillus subtilis. The core aerobic hydrolytic strain used in this experiment is Bacillus subtilis (… Bacillus subtilisThe strain, derived from the China General Microbiological Culture Collection Center (CGMCC), underwent three generations of adaptive shake-flask passages before being introduced into the pilot-scale fermentation system. It was further screened using the casein agar plate clear zone method, confirming its stable and excellent extracellular neutral protease secretion efficiency. To investigate the performance boundaries of gas-liquid interface mass transfer efficiency and hydrodynamic shear network under different process input conditions during viable cell amplification and continuous enzyme production, and to provide comparative data for establishing the dissolved oxygen steady-state and mechanical shear control range of the fermentation system, this study aimed to demonstrate the synergistic regulatory effect of aeration supply reducing oxygen mass transfer resistance and mechanical stirring constructing gas-liquid contact surface area. A matrix experiment was conducted with aeration rates ranging from 0.6 vvm to 2.0 vvm in 0.2 vvm increments and stirring speeds ranging from 150 rpm to 500 rpm in 50 rpm increments. The optimal process parameters were determined by evaluating factors such as dissolved oxygen maintenance level in the fermentation broth, cell absorbance (OD600), and neutral protease production rate.

[0070] Test results are as follows Figure 4a As shown in -c, the dissolved oxygen level in the fermentation broth, when the stirring speed is constant, shows a continuous upward trend with increasing aeration rate. When the aeration rate is constant, the dissolved oxygen level in the fermentation broth also shows an overall upward trend with increasing stirring speed, gradually leveling off after the aeration rate exceeds 1.6 vvm and the stirring speed exceeds 400 rpm. The relatively high value region appears in the range of high aeration rates and high stirring speeds. The cell absorbance value (OD600) shows a non-linear parabolic trend of first increasing and then decreasing with increasing aeration rate or stirring speed, exhibiting a significant relatively high value band and tending to converge when the aeration rate reaches 1.0 vvm and the stirring speed reaches approximately 250 rpm. The neutral protease production rate, when the aeration rate is constant, shows a trend of first increasing and then decreasing with increasing stirring speed; the enzyme production rate also shows similar fluctuations with increasing aeration rate. The low-value region, where the proliferation and yield decrease significantly due to increased fluid shear resistance and foaming-induced damage to live bacteria and the structure of free enzyme proteins, appears in the process range of high aeration rate and high stirring speed.

[0071] By normalizing the data of the three key indicators mentioned above and then performing weighted calculations, the comprehensive biochemical equilibrium point under the combined effects of gas-liquid mass transfer, oxygen supply, and fluid dynamic shear was simulated and sought. The theoretical equilibrium point of the fermentation process was obtained as an aeration rate of 1.02 vvm and a stirring speed of 254.6 rpm. Considering the adjustment tolerance of the gas supply valve in industrial-scale fermenters, the control accuracy of the variable frequency motor, and the robustness of continuous operation across multiple batches, the fixed-point parameters closest to the theoretical point were finally selected as the determined operating process, namely an aeration rate of 1.0 vvm and a stirring speed of 250 rpm.

[0072] 3.3 Optimization of the timing of the transition from aerobic to microaerobic stages The timing of the transition from aerobic hydrolysis to microaerobic transpeptidation is a key pilot-scale process parameter for regulating peptide chain scission depth and improving taste. If the transition is too early, insufficient hydrolysis of large protein molecules will result in a lack of sufficient substrate library for the transpeptidation reaction; if the transition is too late, Bacillus subtilis is prone to enter the apoptosis phase and undergo cell autolysis, and the acidic intermediate metabolites accumulated in the later stage will severely disrupt the microenvironment, thereby inhibiting the subsequent colonization and enzymatic response of Lactobacillus plantarum. A single-factor time gradient experiment was set up from 8.0 hours to 22.0 hours with a step size of 2.0 hours.

[0073] After transpeptide fermentation, the product was placed in a tank and inactivated at 85°C for 15 minutes. The supernatant was collected by centrifugation. The extreme values ​​of extracellular aminopeptidase activity (APAP, U / mL) during the transition between the two stages were measured. The removal rate (%) of bitter peptides after relay enzymatic hydrolysis was determined using an electronic tongue system combined with sensory quantitative evaluation. The actual yield (SMPY, %) of small molecule active peptides (molecular weight less than 1000 Da) in the final product powder was determined by high performance liquid chromatography.

[0074] Test results are as follows Figure 5 As shown, during the low aerobic duration (8.0 h ~ 12.0 h), insufficient hydrolysis time by neutral proteases failed to provide sufficient transpeptidation substrate for subsequent *Lactobacillus plantarum*, resulting in low levels of various indicators in the final product. With prolonged aerobic operation, the hydrolysis depth and transpeptidation response gradually increased, reaching their maximum when the aerobic duration was extended to 18.0 hours. However, the removal rate of bitter peptides and the actual yield of small-molecule active peptides did not reach their maximum values; instead, they decreased. Excessive aerobic fermentation led to over-hydrolysis of some peptides and premature accumulation of organic acids, resulting in slight acidic flocculation and loss of some peptides before ultrafiltration.

[0075] In contrast, when the aerobic duration was controlled at 16.0 hours, although the extreme value of extracellular aminopeptidase activity was slightly lower than that at 18.0 hours, this time point provided the optimal balance window between substrate concentration and microenvironment pH. This moderate transition state ensured the specificity and efficiency of the transpeptide reaction, so that at 16.0 hours, both the removal rate of bitter peptides and the actual yield of small molecule active peptides, which reflect the core value of the final product, reached the extreme values ​​of the entire experimental group.

[0076] To achieve a reasonable engineering balance between substrate hydrolysis depth in the aerobic stage, transpeptidase expression activation in the microaerobic stage, improvement of overall product taste, and small molecule enrichment yield, and to avoid sacrificing end-product yield in pursuit of a single enzyme activity peak, the above three performance indicators were normalized and weighted fitting analysis was performed. The theoretical comprehensive equilibrium point was calculated to be 15.68 hours. Considering the response delay of cooling medium switching in large-volume fermenters during industrial-scale pilot continuous production and the process robustness of continuous and stable operation across multiple batches, an integer time node that is close to the theoretical equilibrium point and easy to accurately measure in actual scheduling was finally selected, namely, the transition time node from aerobic fermentation to microaerobic fermentation, which was determined to be 16.0 hours.

[0077] 3.4 Process Optimization of Isothermal Metabolism Temperature for Second-Stage Micro-Oxygen Peptide Modification The core microaerobic transpeptidase strain used in this experiment was *Lactobacillus plantarum* (…). Lactobacillus plantarum This strain, originating from the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC 1.2437, exhibits a high-activity broad-spectrum extracellular aminopeptidase (especially leucine aminopeptidase and proline peptidase) under microaerobic stress. These enzyme systems possess specific affinity and cleavage activity for exposed hydrophobic amino acids (such as leucine, proline, and phenylalanine—core bittering groups) at the polypeptide chain ends, thereby achieving targeted cleavage and debittering of free ends without disrupting the backbone of small polypeptides.

[0078] To ensure the stability of fermentation efficiency, the preparation parameters for the above-mentioned *Lactobacillus plantarum* seed culture were set as follows: the frozen strain was inoculated into MRS broth medium and activated statically at 37°C for 24 hours; then, it was transferred to fresh MRS liquid medium at a 3% inoculation rate and cultured statically at 37°C for 16-18 hours until the absorbance (OD600) of the bacterial solution at 600 nm wavelength reached 1.2-1.5, thus obtaining the desired seed culture. To investigate the performance boundaries of *Lactobacillus plantarum* growth metabolism and extracellular aminopeptidase spatial conformation during long-cycle transpeptidation modification under different temperature input conditions, and to provide comparative data for establishing the thermodynamics and enzyme activity stability range of the fermentation system, demonstrating the synergistic regulatory effect of isothermal metabolic temperature on the metabolic kinetics of live bacteria and enzymatic fine cleavage debittering, a single-factor gradient experiment of microaerobic fermentation temperature was set up from 26.0°C to 40.0°C with a step size of 2.0°C, for a total of 8 experimental groups.

[0079] After 16.0 hours of aerobic fermentation, a stage shear program was initiated, reducing the stirring speed to 60 rpm for oxygen-limited maintenance (dissolved oxygen level below 10%), and simultaneously adding 5% (v / v) of *Lactobacillus plantarum* seed culture. Each experimental group underwent continuous microaerobic transpeptidation fermentation for 24 hours under corresponding isothermal conditions. During this period, the feed-feed control system of the pilot-scale fermenter automatically titrated an appropriate amount of alkali solution to maintain the pH of the fermentation broth at a constant level of 6.2. The optimal process parameters were determined by evaluating factors such as the peak extracellular aminopeptidase activity (U / mL) during microaerobic fermentation, the removal rate of bitter peptides in the final product (%), and the actual yield of small molecule active peptides (%).

[0080] Test results are as follows Figure 6 As shown, the extreme value of extracellular aminopeptidase activity initially increased and then decreased with increasing microaerobic fermentation temperature, reaching a relatively high value around 34.0℃. The removal rate of bitter peptides and the actual yield of small molecule active peptides also showed a non-linear parabolic trend with increasing temperature, but these two core indicators characterizing the final product quality showed significant convergence and stabilization at 32.0℃, indicating a relatively excellent level. None of the three indicators reached their extreme values ​​simultaneously at the same temperature.

[0081] While the relatively high initial thermodynamics at 34.0℃ stimulated vigorous metabolism in the early stages of *Lactobacillus plantarum* in the short term and increased the short-term secretion concentration of free enzymes, it also increased the probability of thermal decay and degradation of the higher-order structure of aminopeptidase during the continuous fermentation cycle of up to 24 hours, thus affecting the overall cumulative catalytic efficiency. In contrast, 32.0℃ provided a milder and more stable microenvironment for enzyme structure, ensuring that the transpeptidase system continued to play a stable terminal cleavage role throughout the later stages of microaerobic fermentation. The low-value region, where excessive heat stress led to premature death of *Lactobacillus plantarum* and thermal denaturation and inactivation of the higher-order structure of free aminopeptidase, resulting in a significant decrease in debittering performance and actual yield of small molecule peptides, appeared in the high-temperature (38.0℃~40.0℃) process range.

[0082] By normalizing the data of the three key indicators mentioned above and then performing weighted calculations, a comprehensive biochemical equilibrium point was simulated to find the interaction between thermodynamic metabolism, extracellular enzyme structural stability, and peptide enrichment and conversion rate. The theoretical equilibrium point of the fermentation preparation process was found to be 32.18℃. Considering the temperature control tolerance, thermal inertia delay, and physical robustness of continuous operation in multiple batches of the industrial-scale fermenter jacketed circulating cooling water system, a fixed-point parameter close to the theoretical point was finally selected as the determined operating process, that is, the isothermal metabolic temperature for the second-stage micro-oxygen transpeptide modification was determined to be 32.0℃.

[0083] 3.5 Optimization of process parameters for the second-stage micro-oxygen transpeptide modification with controlled pH level Using 100 parts by weight of directional fermentation basal nutrient substrate and the liquid from the first stage of excellent aerobic fermentation (16.0 hours) as a baseline, this study investigated the physicochemical boundaries of different acid-base microenvironments on the long-term transpeptidation modification of *Lactobacillus plantarum*. This experiment aimed to evaluate the synergistic regulatory effect of fed-batch controlled pH on viable bacterial metabolism, aminopeptidase conformation, and peptide dissociation state. A single-factor constant pH gradient experiment was set up from pH 5.3 to pH 7.4 (step size 0.3), with a total of 8 experimental groups. The stage shear program was initiated, the temperature was adjusted to the established 32.0℃, and the stirring was reduced to 60 rpm (oxygen-limited maintenance, dissolved oxygen <10%). A 5% (v / v) *Lactobacillus plantarum* seed culture was inoculated. Transpeptidation was continued for 24 hours, during which a 5% concentration of sodium hydroxide or hydrochloric acid solution was fed through a DCS system to strictly maintain a constant pH. After fermentation, inactivation was performed at 85℃, and the peak value of aminopeptidase activity (U / mL), the removal rate of bitter peptides (%), and the actual yield of small molecule active peptides (%) were measured.

[0084] Test results are as follows Figure 7 As shown, aminopeptidase activity initially increased and then decreased with increasing pH, reaching a relatively high value near pH 6.5 (P5 group). However, the bitterness removal rate and the actual yield of small molecules, which characterize the final quality, tended to be excellent at pH 6.2 (86.2% and 84.6%, respectively).

[0085] While the slightly acidic environment of pH 6.5 stimulates a large amount of aminopeptidase secretion, it alters the charge distribution of some bitter peptide terminal hydrophobic amino acids, reducing substrate-specific affinity and easily triggering minor side reactions during the heat inactivation stage, leading to a slight decrease in debittering rate and effective yield. In contrast, although pH 6.2 has a slightly lower amount of free enzyme, it provides a more balanced ionic microenvironment for enzyme catalysis and moderate peptide charge dissociation, ensuring the specificity of terminal modification and the anti-agglomeration stability of the peptide phase. Excessive acidity (<5.6) or alkalinity (>7.1) stress both disrupt the enzyme protein conformation and induce peptide isoelectric point aggregation, resulting in a significant decrease in yield.

[0086] By normalizing and weighting the above indicators, the theoretical equilibrium point was calculated to be pH 6.24. Considering the titration hysteresis effect of industrial-scale feed-batch pumps and the physical robustness of continuous operation across multiple batches, the final pH setting parameter for feed-batch control of micro-oxygen transpeptide modification was determined to be pH 6.2.

[0087] 3.6 Optimization of the transmembrane pressure difference (TMP) step in the ultrafiltration purification of active peptides Using the two-stage fermentation supernatant (after inactivation and centrifugation to remove residue) as a baseline, this study investigated the physicochemical boundaries of physical operating pressure on peptide permeation efficiency and membrane module antifouling performance during the ultrafiltration purification stage. A suitable transmembrane pressure gradient (TMP) can effectively overcome fluid resistance, while excessively high pressure gradients can easily form concentration polarization gel layers, leading to membrane flux decline and hindering the permeation of small molecule peptides. Therefore, a single-factor TMP gradient experiment was set up from 0.10 MPa to 0.45 MPa (in 0.05 MPa steps), with a total of 8 experimental groups.

[0088] An ultrafiltration membrane system with a molecular weight cutoff of 8000 Da was used, maintaining a constant feed inlet temperature (25℃) and cross-flow velocity at the membrane surface for 6 hours. The average membrane flux (L / (m²·h)) and membrane flux retention rate (%, ratio of the initial instantaneous flux at the end of operation) were calculated according to the relevant specifications of "Test Methods for Hollow Fiber Ultrafiltration Membranes" (HY / T 050-1999). The permeability (%) of small molecule active peptides was determined by high performance liquid chromatography (HPLC) according to "Soybean Peptide Powder" (GB / T 22492-2008).

[0089] Test results are as follows Figure 8 As shown, the purification indicators exhibit a non-linear, dislocation-like response to the increase of TMP. In the low pressure differential range, due to the small fluid shear and filtration driving forces, a dense adhesion layer is not easily formed on the membrane surface, and the membrane flux retention rate is above 90%; however, limited by the low physical driving force, the average membrane flux and peptide permeability of the system do not reach ideal levels. With increasing TMP, the separation driving force strengthens. When TMP reaches 0.30 MPa, the sufficient initial driving force causes the average membrane flux to reach a relatively high value for the entire group, but the peptide permeability and membrane flux retention rate of this group decline. In contrast, at 0.25 MPa, although the average membrane flux is slightly lower than at 0.30 MPa, this pressure differential effectively slows down the formation rate of the concentration polarization gel layer induced by impurities. This relatively balanced hydrodynamic boundary allows the peptide permeability and membrane flux retention rate to reach excellent levels at 0.25 MPa. Entering the high pressure differential range (0.35~0.45MPa), the forced pressurization causes an irreversible dense fouling layer to form instantly on the membrane surface, generating additional steric hindrance effect, and causing a significant convergence of various production indicators.

[0090] Normalized weighted fitting analysis of the above indicators yielded a theoretical comprehensive equilibrium point of 0.246 MPa for the separation system. Considering the pressure pulse fluctuation tolerance of the feed pump in industrial ultrafiltration equipment and the robustness requirements for continuous operation, a fixed-point parameter was finally selected: the determined operating transmembrane pressure difference (TMP) of the ultrafiltration system in the retention and purification stage is 0.25 MPa.

[0091] Example 4 refer to Figure 9This embodiment aims to fully demonstrate the entire process of preparing ginseng-derived active peptides under a series of suitable process parameters determined in the early stage, and to comprehensively test the yield of active fragments, sensory debittering effect, and physicochemical stability after rehydration of the final peptide product. To reflect the synergistic effect of the dual-strain relay and parameter coupling in the optimized scheme, a comparative example was set up for parallel testing.

[0092] The preparation process was strictly performed according to the optimized parameters. First, 100 parts by weight of ginseng stem cell-directed fermentation nutrient substrate were prepared and sterilized. Bacillus subtilis seed culture was inoculated at a 4% (v / v) fraction, initiating the first stage of aerobic fermentation. The temperature was maintained at 37℃, the sterile air flow rate was maintained at 1.0 vvm, and the main stirring speed was set to 250 rpm. The culture was carried out for 16.0 hours to allow for coarse shearing of precursor proteins. Subsequently, a shear program was initiated, lowering the temperature to 32.0℃ and the stirring speed to 60 rpm to maintain a microaerobic environment. Simultaneously, 5% (v / v) of screened and validated Lactobacillus plantarum (CGMCC source) seed culture was inoculated. The pH of the fermentation broth was maintained at 6.2 using a fed-batch system, and microaerobic transpeptide modification fermentation was continuously carried out for 24 hours. After inactivation at 85℃ and centrifugation, the supernatant was collected and pumped into an ultrafiltration membrane system with a molecular weight cutoff of 8000 Da for purification and separation at a transmembrane pressure difference (TMP) of 0.25 MPa. The permeate was collected and freeze-dried for 24 hours under the conditions of pre-freezing temperature of -50℃, cold trap desorption temperature of -60℃, and system vacuum degree controlled at 10 Pa, finally obtaining the active peptide powder.

[0093] To verify the comprehensive performance of the product obtained by this process, the sample from Example 4 was selected, and comparative examples 1 (using traditional single-strain fermentation, with only Bacillus subtilis inoculated for continuous aerobic fermentation for 40 hours, without a microaerobic peptide modification stage) and 2 (using dual-strain relay fermentation, but without temperature control at 37°C and pH feed control throughout the microaerobic stage, and ultrafiltration purification TMP set to conventional high pressure of 0.40 MPa) were tested for comparison. According to relevant national standards and industry specifications, the actual yield (%) of small molecule active peptides (<1000 Da), the bitterness sensory response value established based on the electronic tongue system (dimensionless, the lower the value, the weaker the bitterness), and the transmittance (%,) of the solution after the powder was rehydrated at 5% concentration and left to stand at room temperature for 24 hours were measured at a wavelength of 600 nm to characterize anti-agglomeration stability. The compiled data comparison results are shown in the table below: Under a dynamically optimized process, the aerobic coarse shearing of Bacillus subtilis and the microaerobic fine modification of Lactobacillus plantarum formed a synergistic effect. Suitable temperature (32.0℃) and a constant ionic microenvironment (pH 6.2) ensured efficient catalysis by aminopeptidase and stability of the peptide phase, resulting in excellent yields of small peptides in the final product and a significant reduction in bitterness response. Simultaneously, a gentle purification pressure difference of 0.25 MPa effectively retained large molecular weight proteins that easily cause turbidity, and the rehydration transmittance was excellent.

[0094] In contrast, Comparative Example 1, lacking end-cut modification with *Lactobacillus plantarum*, exposed a large number of hydrophobic amino acid residues, resulting in a significantly higher bitterness response value and incomplete macromolecular degradation, leading to a lower target yield. Comparative Example 2, due to the lack of buffering intervention for thermodynamics and acid-base stress during the microaerobic stage, experienced partial enzyme inactivation and peptide isoelectric point aggregation. Furthermore, the excessively high ultrafiltration pressure differential caused concentration polarization, reducing the effective permeability and causing some impurities to penetrate, resulting in poor rehydration stability. Overall, Example 4 demonstrates a synergistic effect between various physicochemical and biochemical indicators, achieving a good engineering balance between efficient substrate conversion, improved sensory flavor, and product physical phase stability.

[0095] In summary, the dual-strain fermentation system based on aerobic coarse shearing by Bacillus subtilis and supplemented by microaerobic transpeptide modification by Lactobacillus plantarum significantly improved the yield of target fragments and sensory quality of the product while maintaining the specific bioactivity of peptides. It overcomes the limitations of traditional processes that easily lead to excessive degradation of the peptide backbone and bitterness caused by hydrophobic end residues. This allows active peptides to achieve efficient hydrolysis of large molecular precursors in the aerobic stage and exhibit precise end-cutting and phase buffering capabilities in a constant microaerobic environment, broadening their application window in high-end nutritional products. Furthermore, the gentle differential pressure ultrafiltration purification effectively mitigates concentration polarization and impurity penetration at the membrane surface, and the optimized parameter settings preserve the structural integrity of small molecule peptides, resulting in better physical anti-agglomeration stability and overall flavor performance in rehydration and practical applications.

[0096] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing bioactive peptides based on plant stem cell-induced expression and microbial-directed fermentation, characterized in that... Includes the following steps: S1, by weight, 35-45 parts of ginseng stem cell plasma are induced and cultured to express precursor proteins. After cell wall disruption, 4-6 parts of glucose, 2-4 parts of yeast extract powder, 1-3 parts of soybean polypeptide powder, 1-3 parts of a complex inorganic salt composed of potassium dihydrogen phosphate and magnesium sulfate, and the remainder of sterile deionized water are added to prepare 100 parts by weight of directional fermentation basic nutrient substrate and then sterilized. S2 introduces 3%-5% (v / v) of Bacillus subtilis seed liquid into the basic nutrient substrate and carries out the first stage of aerobic fermentation under constant temperature conditions of 35-39℃. The sterile air ventilation rate, main stirring speed and continuous fermentation time are controlled. The secreted extracellular neutral protease is used to cleave the macromolecular precursor protein into short-chain polypeptides. After the first stage of fermentation of S3 is completed, an environmental shear is carried out, the system temperature is lowered, the stirring speed is reduced to maintain a micro-aerobic environment, and 4%-6% volume fraction of Lactobacillus plantarum seed liquid is simultaneously inoculated. The pH of the fermentation broth is kept constant by a feedstock system, and continuous fermentation is carried out. Free aminopeptidase is used to precisely remove and debitter the hydrophobic amino acids at the ends of the peptides. After S4 fermentation is completed, the liquid is heated to inactivate the bacteria, and centrifuged to remove the bacterial residue and obtain the supernatant. The permeate was pumped into a first-stage ultrafiltration membrane system with a molecular weight cutoff of 8000 Da for impurity removal and filtration to retain undegraded macromolecular proteins and enzymes. The collected permeate was then introduced into a second-stage membrane system with a molecular weight cutoff of 1000 Da for fractional permeation purification under transmembrane pressure difference. The final permeate rich in the target fragment was collected and freeze-dried under vacuum to obtain the active peptide powder.

2. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 1, characterized in that, In step S1, the amount of ginseng stem cell paste added is 40 parts, the amount of glucose added is 5 parts, the amount of yeast extract powder added is 3 parts, the amount of soybean polypeptide powder added is 2 parts, the amount of the complex inorganic salt composed of potassium dihydrogen phosphate and magnesium sulfate added is 2 parts, and the amount of sterile deionized water added is 48 parts; wherein, the mass ratio of glucose to yeast extract powder is 5:

3.

3. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 1, characterized in that, In step S2, the volume fraction of the Bacillus subtilis seed liquid is 4%; the constant temperature for the first stage of aerobic fermentation is 37°C; the controlled sterile air ventilation rate is 0.8-1.2 vvm; the main stirring speed is 200-300 rpm; and the continuous fermentation lasts for 14-18 hours.

4. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 3, characterized in that, The sterile air ventilation rate is controlled at 1.0 vvm; the main stirring speed is 250 rpm; and the continuous fermentation lasts for 16 hours. The aeration rate is 1.0 rpm and the stirring speed is 250 rpm.

5. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 1, characterized in that, In step S3, the micro-aerobic environment temperature is 30-34℃; the stirring speed is 50-70 rpm; the liquid fraction of Lactobacillus plantarum seed is 5%; the pH value of the fermentation broth is constant at 6.0-6.5; and the continuous fermentation time is 20-28 hours.

6. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 5, characterized in that, In step S3, the micro-aerobic environment temperature is 32℃; the stirring speed is 60 rpm; the pH value of the fermentation broth is constant at 6.2; and the continuous fermentation time is 24 hours.

7. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 1, characterized in that, The heating inactivation temperature in step S4 is 80-90℃; the transmembrane pressure difference is 0.20-0.30MPa.

8. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 7, characterized in that, The heating inactivation temperature in step S4 is 85°C; the transmembrane pressure difference is 0.25 MPa.

9. The method for preparing active polypeptides based on plant stem cell-induced expression and microbial-directed fermentation according to claim 1, characterized in that, In step S4, the specific process conditions for vacuum freeze drying are as follows: pre-freezing temperature is -50℃, cold trap desorption temperature is -60℃, system vacuum degree is controlled at 10 Pa, and continuous drying treatment is carried out for 24 hours.

10. An active polypeptide prepared based on plant stem cell-induced expression and microbial-directed fermentation, characterized in that, Prepared by the method according to any one of claims 1 to 9, the active polypeptide uses a complex system composed of a macromolecular precursor protein induced by ginseng stem cells and soybean polypeptide as the substrate backbone, and undergoes enzymatic degradation and fine modification by aerobic and microaerobic environments, with its molecular weight mainly distributed below 1000 Da; the hydrophobic amino acid residues at the end of the polypeptide molecular chain are directionally cleaved to form a low bitterness characteristic, and exist in a small molecule free peptide phase with high anti-agglomeration stability.

Images