Fermented soybean antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses a fermented soybean antibacterial peptide and a preparation method and application thereof. The preparation method comprises the following steps: fermenting soybean raw materials in the presence of four probiotics including lactobacillus plantarum, lactobacillus casei, streptococcus thermophilus and bifidobacterium; the antibacterial peptide which has a remarkable inhibiting effect on escherichia coli and staphylococcus aureus and has an effect of promoting wound healing can be obtained through separation and purification from fermentation liquor, sequencing analysis shows that the sequence of the antibacterial peptide is FLVPPQESQK, and the antibacterial peptide further has good thermal stability, acid-base stability and ionic strength stability and can be used for preparing the antibacterial peptide. The method is suitable for various practical application scenes of medical supplies, cosmetics and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of polypeptides, and particularly relates to a fermented soybean bacteriostatic peptide as well as a preparation method and application thereof. BACKGROUND

[0002] After being damaged by external factors such as mechanical, chemical or thermal factors, the skin barrier function of the wound is damaged, and it is extremely easy to become a channel for pathogenic microorganisms to invade. Once infected, not only does it significantly delay the wound healing process, but it can also cause local swelling, exudation, tissue necrosis, and even serious systemic infections such as cellulitis, lymphangitis, sepsis, etc. Especially in diabetic patients, the elderly and immunocompromised populations, wound infection is more likely to recur, forming a chronic non-healing wound, which seriously affects the patient's quality of life and significantly increases the consumption of medical resources and economic burden.

[0003] At present, synthetic antibacterial drugs (such as antibiotics) are often used in clinical practice to control wound infection, which has a certain effect, but long-term use has led to an increasingly prominent problem of bacterial drug resistance, and is also accompanied by various adverse reactions such as liver and kidney toxicity, allergic reactions, etc. Therefore, the development of a natural source, high safety, and good antibacterial performance of alternative products has become an important research direction in the field of wound infection prevention and control.

[0004] In recent years, research has shown that most grains do not have obvious antibacterial activity, but after microbial fermentation or enzymatic treatment, the proteins can be hydrolyzed to produce specific low molecular weight peptide segments, some of which have significant antibacterial activity. This kind of natural bacteriostatic peptide not only has broad-spectrum antibacterial properties and low drug resistance risk, but also has good biocompatibility and safety, and is therefore considered an ideal antibiotic substitute. However, the types of plant-derived bacteriostatic peptides currently reported are still limited, and their antibacterial activity and stability differ, and they cannot yet meet the needs of multiple scene applications. Therefore, it is urgent to develop new plant bacteriostatic peptides with clear activity and scalable preparation to meet the needs of safe, efficient, and low-toxicity infection prevention and control.

[0005] "Fermented soybean protein peptide enhances the immune function and anti-fatigue ability of mice", Fang Lei et al. used soybean protein isolate as raw material, and after enzymatic hydrolysis, microbial fermentation, and then separation and purification, obtained fermented soybean protein peptide. The effect of fermented soybean protein peptide on the immune function and anti-fatigue ability of mice was studied. The results showed that appropriate intake of fermented soybean protein peptide had the effect of enhancing the immune function and anti-fatigue ability of the body. This study did not develop a fermented soybean protein peptide with antibacterial activity.

[0006] In the paper "Preparation of Small Peptides from Soybean Residue by Solid-State Fermentation of Paecilomyces pupae and Its Physicochemical Properties", Zhu Yunlan et al. used small peptide content as an indicator and carried out solid-state fermentation of soybean residue using Paecilomyces pupae. They optimized the conditions for preparing small peptides from soybean residue using single-factor experiments and orthogonal experiments, and determined the relative molecular mass, apparent viscosity, solubility, emulsifying properties and emulsification stability, and antioxidant properties of the small peptides from soybean residue. The results showed that the optimal conditions for fermenting soybean residue peptides were an inoculum size of 10% and fermentation at 22℃ for 15 days, under which the peptide content reached 15.35%. Most of the soybean residue peptides had a relative molecular mass below 15 kDa, exhibiting good solubility unaffected by pH, with a nitrogen solubility index remaining relatively stable at around 95%. The apparent viscosity of the soybean residue peptides was unaffected by solution concentration, showing minimal variation and remaining at 9.8 mPa·s, indicating good flowability. The soybean residue peptides possessed certain emulsifying properties and emulsion stability, which increased with increasing solution concentration. Fermented soybean residue peptides exhibited better antioxidant capacity than unfermented soybean residue, showing greater resistance to DPPH free radicals and ABTS. + The free radical scavenging rates increased by 2.72 times and 1.74 times, respectively. This study did not develop fermented soybean residue peptides with antibacterial activity, and the use of *Paecilomyces pupae* for solid-state fermentation resulted in high costs, making large-scale production impossible.

[0007] "Optimization of Enzymatic Hydrolysis for the Preparation of Flavorful Soy Peptides Using Response Surface Methodology" by Zhang Lihua explored the optimal preparation process for flavorful soybean peptides. Using soy protein isolate as raw material, and considering both the degree of hydrolysis and sensory evaluation, the study determined that a combination of flavor protease and alkaline protease yielded the best enzymatic hydrolysis results. Response surface methodology was then used to optimize the hydrolysis conditions. Results showed that the flavorful soybean peptides prepared under the following conditions—a flavor protease to alkaline protease ratio of 0.8:1.0 (enzyme activity ratio), a hydrolysis time of 2.5 h, a hydrolysis temperature of 50℃, a pH of 7.5, and an enzyme dosage of 2167 U / g—exhibited the best flavor and achieved the highest sensory score of 5.38. However, this study employed an enzymatic hydrolysis process for soybean peptide preparation, which is costly and only investigated the flavor of soybean peptides; it did not develop soybean peptides with antibacterial activity.

[0008] In their paper "Soybean Antimicrobial Peptides, Their Preparation Method, and Preservation of Oyster Meat," Liu Tianqi et al. used a complex enzyme (at least two of papain, trypsin, and chymotrypsin) to enzymatically hydrolyze a mixture of soybean meal and water, inactivating the enzyme and preparing enzymatic hydrolysate. They collected the hydrolysate and retained peptides with molecular weights between 1 kDa and 10 kDa to prepare soybean antimicrobial peptides. While this method is effective, it may have limitations in terms of controllability of the enzymatic hydrolysis process, enzyme cost, separation and purification efficiency, activity, and safety, especially in large-scale production where it may face further challenges.

[0009] "A Preliminary Study on the Preparation of Antibacterial Liquid from Soybean Meal by Aspergillus oryzae Fermentation and Its Antibacterial Mechanism" by Hu Yalan used Aspergillus oryzae as the fermentation strain and soybean meal as the raw material, employing a liquid fermentation method. The optimized conditions were: a material-to-liquid ratio of 1:9, an initial fermentation pH of 4.5, an Aspergillus oryzae addition of 0.7‰, a glucose addition of 2.5%, and constant temperature fermentation at 37℃ for 42 hours. The antibacterial liquid from the soybean meal was fractionated using 80% ethanol solution. The H-4-Q fraction showed the strongest antibacterial effect, and 65 antimicrobial peptides were identified within it. Although Aspergillus oryzae is an edible fungus, it may pose a risk of contamination with secondary metabolites (such as aflatoxin), and a single strain does not possess the synergistic metabolic advantages of probiotic-based fermentation. The products are mainly protein hydrolysates and lack the bacteriocin components unique to probiotics.

[0010] In her study, "Isolation, Extraction, and Antibacterial Properties of Basic Polypeptides from Soybean Meal," Dai Zengying extracted soybean globulin using the isoelectric point method, purified it via dextran gel chromatography, and then extracted basic soybean globulin peptides from the purified protein by disrupting disulfide bonds with β-mercaptoethanol, demonstrating their antibacterial properties. However, this method is complex and uses a chemical reducing agent (β-mercaptoethanol), which is not conducive to large-scale production and food applications. Summary of the Invention

[0011] In view of the above-mentioned technical problems existing in the prior art, the purpose of the present invention is to provide a fermented soybean antimicrobial peptide, its preparation method and application. The antimicrobial peptide provided by the present invention has a variety of plant-derived sources, which simultaneously has high activity against Escherichia coli and Staphylococcus aureus and promotes wound healing.

[0012] This invention provides a method for preparing the fermented soybean antimicrobial peptide, comprising the following steps:

[0013] A fermented soybean antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO:1, specifically FLVPPQESQK.

[0014] The method for preparing fermented soybean antimicrobial peptides includes the following steps:

[0015] S1: Ferment soybean raw materials under the action of probiotics to obtain fermentation liquid, and then sterilize the fermentation liquid;

[0016] S2: Enriching components with a value less than 3 kDa from the fermentation broth;

[0017] S3: The components with less than 3kDa are separated by semi-preparative liquid chromatography using a C8 column, and the components with antibacterial activity are collected.

[0018] S4: Mass spectrometry sequencing was performed on the antibacterial component to obtain fermented soybean antibacterial peptides.

[0019] Furthermore, in step S1, the probiotics include Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, and Bifidobacterium, and fermentation is carried out in the presence of these four probiotics.

[0020] Furthermore, the bacterial concentration of the four probiotic strains was (1–10) × 10⁻⁶. 8 The CFU / mL concentration and the inoculum size are 0.5%-1.5%, preferably 1%.

[0021] Further, in step S1, the soybean raw material is pulverized soybean powder, and during the fermentation, the mass ratio of soybean raw material to water in the system is 1:(12-18).

[0022] Furthermore, in step S1, the fermentation temperature is 34–40°C, and the fermentation time is 10–20 days.

[0023] Further, in step S3, the chromatographic column used for semi-preparative liquid phase separation includes a Sepax Bio-C8(2) chromatographic column with dimensions of 30×250mm and 8μm.

[0024] Furthermore, the eluent separated by the C8 column includes mobile phase A and mobile phase B. Mobile phase A is an acetonitrile solution containing 0.1% TFA by volume, and mobile phase B is an ultrapure aqueous solution containing 0.1% TFA by volume. The eluent flow rate is 10 mL / min, and the detection wavelength of the ultraviolet detector is 280 nm.

[0025] A linear gradient elution method was used, 0–60 min, 0%–65% A.

[0026] Furthermore, in step S3, the component with antibacterial activity is a component with antibacterial activity >40%, and the inhibited bacteria are Escherichia coli and / or Staphylococcus aureus.

[0027] The present invention also provides an antibacterial agent comprising an antibacterial peptide with an amino acid sequence as shown in SEQ ID NO:1.

[0028] This application uses soybeans as raw material and employs a compound fermentation technology, along with methods such as ultrafiltration and semi-preparative liquid chromatography, to prepare specific soybean antimicrobial peptides. Compared with existing technologies, these peptides offer the following advantages:

[0029] (1) It has good antibacterial effect and promotes wound healing: Experiments have shown that the antibacterial peptide FLVPPQESQK has a significant inhibitory effect on Escherichia coli and Staphylococcus aureus and promotes wound healing.

[0030] (2) The product is safe and stable, and suitable for large-scale promotion: The antimicrobial peptide FLVPPQESQK obtained in this invention has excellent blood compatibility, is not toxic to mouse fibroblasts L929, and has good thermal stability, acid-base stability and ionic strength stability, making it suitable for various practical application scenarios such as pharmaceuticals and cosmetics. Attached Figure Description

[0031] Figure 1 Chromatograms of semi-preparative liquid phase separation and purification of antibacterial components in fermented soybean broth;

[0032] Figure 2 Electron micrographs of Escherichia coli and Staphylococcus aureus before and after FLVPPQESQK treatment;

[0033] Figure 3. Hemolysis rate of 0.1% Triton × 100, FLVPPQESQK (SBAP1), and soybean fermentation broth (FS);

[0034] Figure 4 The toxicity of FLVPPQESQK to mouse fibroblast L929 cells after 24 and 48 hours of culture;

[0035] Figure 5 Thermal stability of FLVPPQESQK;

[0036] Figure 6 Acid and base stability of FLVPPQESQK;

[0037] Figure 7 Ion strength stability of FLVPPQESQK;

[0038] Figure 8 Representative images of zebrafish tail fin regeneration in different treatment groups;

[0039] Figure 9 Quantitative analysis of zebrafish tail fin regeneration. Detailed Implementation

[0040] This invention provides a method for preparing the fermented soybean antimicrobial peptide, comprising the following steps:

[0041] (1) Ferment soybean raw material under the action of probiotics to obtain fermentation liquid, sterilize fermentation liquid, centrifuge, and collect supernatant;

[0042] (2) By using ultrafiltration technology, components with a value of less than 3 kDa are enriched from the supernatant of the fermentation broth;

[0043] (3) The components with less than 3 kDa were separated by semi-preparative liquid chromatography on a C8 column and the components with antibacterial activity were collected.

[0044] (4) Mass spectrometry sequencing was performed on the antibacterial active fraction to obtain fermented soybean antibacterial peptides.

[0045] This invention involves fermenting soybean raw materials with Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, and Bifidobacterium to obtain a fermentation broth.

[0046] In this invention, the soybean raw material is preferably pulverized soybeans. The soybean powder is preferably prepared by screening and washing soybeans, followed by drying and pulverizing. The particle size of the soybean powder is preferably 10-60 mesh. This invention does not impose any special restrictions on the type of soybean used; any soybean variety well-known in the art can be used. It is preferable to sterilize the soybean powder before fermentation. The sterilization method used in this invention is high-pressure steam sterilization.

[0047] In this invention, the probiotics are preferably four types: *Lactobacillus plantarum*, *Lactobacillus casei*, *Streptococcus thermophilus*, and *Bifidobacterium*. The preferred bacterial concentration of the four probiotic strains is (1–10) × 10⁻⁶. 8 The CFU / mL concentration and the inoculum concentration in the fermentation broth were both 0.5% to 1.5%, more preferably 1%.

[0048] In this invention, the fermentation temperature is 34–40°C, more preferably 35–38°C, and most preferably 37°C. The fermentation time is preferably 10–20 days, and most preferably 14 days. During the fermentation period, the mass ratio of soybean raw material to water in the system is preferably 1:(12–18), and most preferably 1:15.

[0049] This invention was tested using a three-factor, three-level orthogonal experiment. The results showed that different combinations of fermentation conditions had a significant impact on the antibacterial activity of the fermentation broth. The best antibacterial effect could be achieved when the inoculation ratio of each strain was 1%, the fermentation temperature was 37℃, and the mass ratio of soybean raw material to water in the system was 1:15.

[0050] After obtaining the fermentation broth, the present invention enriches components with less than 3 kDa from the fermentation broth.

[0051] In this invention, the enrichment method is preferably carried out by ultrafiltration. Specifically, after centrifugation, the supernatant of the fermentation broth is collected, the filtrate is collected by passing it through an ultrafiltration tube with a molecular weight cutoff of 3 kDa, and the powder is collected by freeze drying.

[0052] In this invention, the fraction with a concentration less than 3 kDa is preferably prepared using pure water as the loading solution. The concentration of the loading solution is preferably 0.1 g / mL, and it is filtered through a 0.22 μm membrane filter. The antimicrobial peptides are further separated and purified using a semi-preparative liquid chromatography (HPLC) system and a C8 column. Mobile phases A (ACN solution containing 0.1% (v / v) TFA) and B (ultrapure water containing 0.1% (v / v) TFA) are used as mobile phases, with an injection volume of 5 mL, a mobile phase flow rate of 10 mL / min, and a UV detector wavelength of 280 nm. The sample is eluted using a gradient with the following parameters: the volume percentage of mobile phase A in the eluent increases uniformly from 0% to 65% within 60 min. The purified fractions are collected and concentrated in a rotary evaporator at 45 °C and 0.1 MPa pressure, followed by lyophilization. The antimicrobial activity of each fraction is measured. In this embodiment of the invention, components with an antibacterial rate of not less than 50% are collected and then subjected to mass spectrometry to obtain soybean antibacterial peptides.

[0053] In this invention, the antimicrobial activity and stability of the prepared antimicrobial peptide were tested. The results showed that the antimicrobial peptide with the amino acid sequence shown in SEQ ID NO:1 achieved an inhibition rate of over 50% against *Escherichia coli* and *Staphylococcus aureus*, and exhibited certain thermal stability, acid-base stability, and ionic strength stability. Furthermore, using a zebrafish model with a severed tail as a model, the antimicrobial peptide dissolved in culture water showed a wound-healing effect.

[0054] The following detailed description of the preparation method of fermented soybean antimicrobial peptide provided by the present invention, with reference to the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] Screening method for probiotic fermentation of soybeans.

[0057] 1) Soybean pretreatment: After screening and washing, soybeans are thoroughly dried, mechanically crushed, and then mixed with water at a mass-volume ratio of 1:12-1:18. The mixture is then sterilized by high-temperature steam to obtain sterilized soybean liquid.

[0058] 2) Probiotic strain activation method: *Lactobacillus plantarum*, *Lactobacillus casei*, *Streptococcus thermophilus*, and *Bifidobacterium* were inoculated into sterilized MRS medium and incubated at 37±1℃ for 24 h for activation. *Bifidobacterium* activation required strict anaerobic conditions. After incubation, the bacterial suspension was collected by aseptic centrifugation (5000 r / min, 10 min), resuspended in sterile physiological saline, and the concentration of the bacterial suspension was adjusted to 1.0 × 10⁻⁶. 8 CFU / mL, ready for subsequent inoculation and fermentation.

[0059] 3) Fermentation method: The concentration is 1×10 8 Simultaneously inoculate *Lactobacillus plantarum*, *Lactobacillus casei*, *Streptococcus thermophilus*, and *Bifidobacterium* at CFU / mL into the sterile soybean broth from step 1), using inoculation amounts of 0.8%–1.2%. The inoculation amounts of the four probiotics are the same. Fermentation is carried out at 35–39°C and 180 rpm for 14 days, followed by sterilization. The fermentation broth is centrifuged at 8000 rpm for 10 min, and the supernatant is collected and then freeze-dried to obtain the fermented soybean product. Specific parameter combinations are shown in Table 1.

[0060] Table 1. Factor Levels in the Orthogonal Experiment of Probiotic Fermented Soybeans (L9(3)) 3 )

[0061]

[0062] Evaluation method for antibacterial activity of fermentation broth: Antibacterial activity was tested using the broth microdilution method and determined in sterile 96-well microplates. Example 1: Freeze-dried soybean fermentation products prepared under different conditions were diluted and dispersed with sterile physiological saline to prepare sample solutions with a concentration of 1.0 mg / mL. 100 μL of the sample solution was added to each well of the microplate, along with 100 μL of *Escherichia coli* and *Staphylococcus aureus* cultured to the logarithmic growth phase (concentration approximately 1 × 10⁻⁶). 7 CFU / mL), incubated at 37℃ for 12 h, and the absorbance value OD was measured. 600 .

[0063] Antibacterial activity (%) = (Ac-As) / Ac×100 (1)

[0064] Where Ac is the absorbance OD of the negative control. 600 The average value refers to the negative control, which is the bacteria treated with sterile physiological saline; while As is the absorbance (OD) of the bacteria treated with the sample solution. 600 The average value was calculated. All experiments were performed in triplicate, and the antibacterial test results are shown in Tables 2 and 3.

[0065] Table 2. Orthogonal analysis table of antibacterial activity (Escherichia coli)

[0066]

[0067]

[0068] Table 3. Orthogonal analysis table of antibacterial activity (Staphylococcus aureus)

[0069]

[0070] The results showed that the best antibacterial effect was achieved when the inoculation ratio of each strain was 1%, the fermentation temperature was 37℃, and the material-to-liquid ratio (soybean flour:water) was 1:18. However, an excessively high water ratio would increase the cost of subsequent separation and purification. Therefore, considering all factors, the material-to-liquid ratio was set at 1:15.

[0071] Example 2

[0072] A method for preparing antibacterial components in fermented soybean liquid.

[0073] 1) Soybean pretreatment: After screening and washing, soybeans are thoroughly dried, mechanically crushed, and then mixed with water at a mass-volume ratio of 1:15. After high-temperature steam sterilization, sterilized soybean liquid is obtained.

[0074] 2) Probiotic strain activation method: *Lactobacillus plantarum*, *Lactobacillus casei*, *Streptococcus thermophilus*, and *Bifidobacterium* were inoculated into sterilized MRS medium and incubated at 37°C for 24 hours for activation. *Bifidobacterium* activation required strict anaerobic conditions. After incubation, the bacterial suspension was collected by aseptic centrifugation (5000 rpm, 10 min), resuspended in sterile physiological saline, and the concentration of the bacterial suspension was adjusted to 1.0 × 10⁻⁶. 8 CFU / mL, ready for subsequent inoculation and fermentation.

[0075] 3) Fermentation method: The concentration is 1×10 8 Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, and Bifidobacterium were inoculated at 1% of each concentration into sterile soybean broth with a solid-liquid ratio of 1:15. Fermentation was carried out at 37°C and 180 rpm for 14 days, followed by sterilization. The fermentation broth was centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected.

[0076] 4) Ultrafiltration enrichment: The supernatant of the above fermentation broth is passed through an ultrafiltration tube with a molecular weight cutoff of 3kDa, the filtrate is collected, and the powder is collected by freeze drying.

[0077] 5) Separation of antibacterial components: The powder obtained after ultrafiltration was dissolved in ultrapure water at a concentration of 0.1 g / mL and filtered through a 0.22 μm membrane filter. The antimicrobial peptides were further separated and purified using a semi-preparative liquid chromatography system and a C8 column. The column used for semi-preparative liquid chromatography included a Sepax Bio-C8(2) column with dimensions of 30 × 250 mm and 8 μm. Mobile phase A (ACN solution containing 0.1% (v / v) TFA) and mobile phase B (ultrapure water containing 0.1% (v / v) TFA) were used as mobile phases. The injection volume was 5 mL, the mobile phase flow rate was 10 mL / min, and the UV detector wavelength was 280 nm. The sample was eluted using the following gradient parameters: the volume percentage of mobile phase A in the eluent increased uniformly from 0% to 65% within 60 min. The separation results are as follows: Figure 1 As shown, the various purified samples are collected, which is to say, the samples obtained are... Figure 1 The peak components F1-F7 were collected and concentrated by rotary evaporator at 45℃ and 0.1MPa pressure, and then freeze-dried.

[0078] Finally, the antibacterial activity of each component was measured according to the method for evaluating antibacterial activity in Example 1. The results showed that the component with the strongest antibacterial activity was F5, with antibacterial activities of 48.13% and 59.45% against Escherichia coli and Staphylococcus aureus, respectively.

[0079] Example 3

[0080] The F5 fraction collected in Example 2 was sequenced using the following method: LC-MS / MS was used to identify the peptide sequences in the purified fraction. The F5 fraction collected in Example 2 was first pre-enriched using a Zorbax 300SB-C8 peptide enrichment column, and then separated using an RP-C8 column.

[0081] The pre-enrichment steps for the F5 fraction collected in Example 2 using a Zorbax 300SB-C8 peptide enrichment column are as follows: The F5 fraction was dissolved in an aqueous solution containing 0.1% formic acid and loaded onto a pre-equilibrated Zorbax 300SB-C8 column (equilibration solution was 0.1% formic acid aqueous solution, equilibration volume was 3-5 column volumes). After loading the sample onto the column, the column was further washed with the same equilibration solution for 1-3 column volumes to remove unbound impurities and salts. Subsequently, the column was eluted using an acetonitrile solution containing 0.1% formic acid as the eluent, with an eluent volume of 1-2 column volumes, thereby eluting the peptides bound to the stationary phase. The eluent was collected to obtain the pre-enriched fraction.

[0082] The pre-enriched fraction obtained from the elution was injected into an RP-C8 column for separation. The chromatographic conditions for separation using the RP-C8 column were as follows: during liquid chromatography separation, mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was an acetonitrile solution containing 0.1% formic acid (acetonitrile volume fraction 84%). An RP-C8 column (0.15 mm × 150 mm, Column Technology Inc.) was used, and pre-column equilibration was performed under 95% phase A conditions. Samples were loaded via an autosampler, and the liquid chromatography gradient program was set as follows: 0–50 min: linear gradient of mobile phase B increased from 4% to 50%; 50–54 min: linear gradient of mobile phase B increased from 50% to 100%; 54–60 min: mobile phase B was maintained at 100%.

[0083] F5 fraction was separated by an RP-C8 column and then directly analyzed by mass spectrometry. The mass spectrometry conditions were as follows: a QExactive HF-X mass spectrometer (Thermo Fisher Scientific) was used. The total analysis time was 60 min, and positive ion mode was used for detection. The mass-to-charge ratio (m / z) was acquired by sequentially acquiring 10 fragment ion spectra (MS / MS, MS / MS) after each full MS scan. 2 (scan) to obtain accurate mass spectrometry information of peptides and their fragments.

[0084] Mass spectrometry analysis showed that a total of 16 peptides were obtained from the F5 fraction (Table 4). Among them, the peptide sequence with the highest score was FLVPPQESQK, with a molecular weight of 1172.35 Da and a content of 18.92% in the F5 fraction. It was not identified in the AMP database (Antimicrobial Peptide Database) and belongs to a novel antimicrobial peptide.

[0085] Table 4. Peptide sequences detected by mass spectrometry

[0086]

[0087]

[0088] Example 4

[0089] The peptide FLVPPQESQK isolated and identified in Example 3 was prepared using a chemical synthesis method, and its antibacterial activity was tested.

[0090] In the preparation of pure peptide FLVPPQESQK using chemical synthesis methods, the amino acids are sequentially coupled according to the order KQSEQPPVLF. Each amino acid has an N-terminus with an Fmoc protecting group, denoted as Fmoc-AA. The specific synthesis method is as follows:

[0091] 1. Activation of CTC resin: Weigh 3.0g of blank CTC resin into a clean and dry reaction tube, add dichloromethane to swell, then dry under vacuum, add 10% acetyl chloride in dichloromethane solution, activate at room temperature for 30min, dry under vacuum, and wash three times with dichloromethane.

[0092] 2. Amino acid activation: First, add 2.5 mmol of amino acids, then add 15-20 mL of DMF to dissolve the amino acids. Next, add 2.5 mmol of HOBT, stir to dissolve, and quickly place in ice water to cool. Then, add 2.5 mmol of DIC dropwise. After the addition is complete, activate for 10 min.

[0093] 3. Reaction: Add the activated amino acid solution from step 2 to the reactor containing CTC resin from step 1. Add a small amount of DMF so that the DMF level in the reactor is approximately twice the height of the CTC resin. React at room temperature for 1 hour, and take a sample to determine if the reaction is complete. The determination method is as follows: Take a small amount of resin, rinse it into a test tube with anhydrous ethanol, and then wash it three times in the order of ethanol-DMF-ethanol. Add solutions A, B, and C in sequence, shake well, and heat at 115℃ for 3 minutes. Solution A is an ethanol solution containing 80% phenol by mass, solution B is redistilled pyridine, and solution C is 5% ninhydrin. The color should be colorless or pale yellow.

[0094] 4. Removal of the Fmoc protecting group at the N-terminus of the amino acid: After the reaction in step 3 is completed, pour out the reaction solution and add three times the volume of the resin in a 2% DBU DMF solution to the reactor. React at room temperature for 30 minutes, and take a sample to determine whether the reaction is complete. The determination method is as follows: Take a small amount of resin, rinse it into a test tube with anhydrous ethanol, and then wash it three times in the order of ethanol-DMF-ethanol. Add solutions A, B, and C in sequence, shake well, and heat at 115℃ for 3 minutes. Solution A is an ethanol solution containing 80% phenol by mass, solution B is redistilled pyridine, and solution C is 5% ninhydrin. The color should be deep blue.

[0095] 5. Washing: Wash 5 times with 2 column volumes of DMF.

[0096] 6. Continue synthesizing the next amino acid, repeating the above steps until the complete amino acid sequence is synthesized, thus finally obtaining the polypeptide fragment loaded on CTC resin.

[0097] 7. Peptide cleavage: The CTC resin containing the peptides obtained in step 6 was cleaved using a 1:1 volume ratio mixture of trifluoroacetic acid and dichloromethane. The mixture was stirred at room temperature for 60 min to release the peptide backbone. The lysate was collected by filtration, and the residue on the CTC resin was eluted with dichloromethane. The lysate filtrate and dichloromethane eluate were combined, and 5 volumes of anhydrous methyl tert-butyl ether were added to precipitate the crude peptide. After centrifugation, washing, and lyophilization, the crude peptide was obtained.

[0098] 8. Purification of the peptide: The crude product obtained in step 7 was further purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic column used was an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was acetonitrile solution containing 0.1% TFA. The liquid phase gradient program was set as follows: 0–30 min: linearly increasing from 5% B to 60% B, flow rate was 1.0 mL / min, detection wavelength was 214 nm, gradient elution was used to collect the target peak, and after lyophilization, the pure FLVPPQESQK peptide was obtained.

[0099] The synthesized peptide FLVPPQESQK was diluted to 1 mg / mL with sterile physiological saline, and its antibacterial activity was tested using the method described in Example 1. The results showed that the synthesized peptide FLVPPQESQK exhibited antibacterial activities of 58.75% and 67.48% against Escherichia coli and Staphylococcus aureus, respectively.

[0100] Example 5

[0101] The effect of antimicrobial peptides on bacterial morphology (SEM) was investigated using the following steps:

[0102] 1) Take Escherichia coli and Staphylococcus aureus separately into sterile LB medium and incubate them in a shaker at 37°C for 14 hours to reach the logarithmic growth phase, so that the bacterial concentration is approximately 1×10⁻⁶. 7 CFU / mL, collect bacterial cells by centrifugation.

[0103] 2) The experimental group was treated with FLVPPQESQK (using the pure FLVPPQESQK peptide synthesized in Example 4) at a final concentration of 4.0 mg / mL for 8 h. The blank control group received no treatment. After centrifugation at 3000 r / min for 10 min, the bacteria were washed twice with 0.1 M, pH 7.2 PBS, then 2.5% glutaraldehyde was added, and the bacteria were stored in the dark for 4 h, followed by washing three times with PBS. The bacteria were then dehydrated with ethanol solutions of 20%, 50%, 75%, and 95% gradients, with fresh tert-butanol replacing the ethanol. The lyophilized bacterial cells were analyzed by scanning electron microscopy.

[0104] like Figure 2 As shown, the bacterial cells in the untreated group (FLVPPQESQK group) exhibited typical and regular morphology, smooth and plump surfaces, and uniform size. In contrast, the FLVPPQESQK-treated group (i.e.,...) Figure 2After 8 hours of treatment with the 4MIC group, significant changes in bacterial cell morphology were observed. Some bacterial cells even ruptured, revealing numerous fragments. This indicates that FLVPPQESQK exhibits good antibacterial activity against both Escherichia coli and Staphylococcus aureus, and that its antibacterial effect is exerted by affecting the structural integrity of the bacterial cell wall.

[0105] Example 6

[0106] Blood compatibility assay of the antimicrobial peptide: To evaluate the blood compatibility of the pure FLVPPQESQK peptide synthesized in Example 4, a hemolytic test was performed.

[0107] Fresh pig blood was used in the experiment and treated with 3.2% sodium citrate solution at a volume ratio of 9:1 for anticoagulation. The anticoagulated blood was centrifuged at 3000 r / min for 10 min, the plasma was discarded, and the red blood cell pellet was retained. After washing three times with PBS buffer, a 2% red blood cell suspension was prepared.

[0108] The sample solution was either a 1.0 mg / mL FLVPPQESQK aqueous solution (SBAP1) or a 20 mg / mL soybean compound fermentation broth (FS). The 20 mg / mL soybean compound fermentation broth (FS) was prepared by reconstituted with pure water from the freeze-dried product after final fermentation under the conditions of Example 1 (1% inoculation ratio of each strain, fermentation temperature of 37°C, and material-to-liquid ratio (soybean flour:water) 1:18).

[0109] The PBS group was used as a negative control solution (0% hemolysis), and the 0.1% Triton X-100 group was used as a positive control solution (100% hemolysis).

[0110] The above sample group solutions, negative control solutions, and positive control solutions were mixed with 2% erythrocyte suspension at a ratio of 1:1 (v / v) and incubated in a 37℃ water bath for 1 h. After incubation, the mixture was centrifuged at 3000 r / min for 10 min, and the supernatant was collected into a 96-well plate. The absorbance was measured at 540 nm. The hemolysis rate was calculated according to formula (2), as follows:

[0111] Hemolysis rate (%) = (ODt - OD0) / (OD1 - OD0) × 100% (2)

[0112] Wherein, ODt is the absorbance of the sample group at 540 nm after solution treatment, OD0 is the absorbance of the PBS group at 540 nm after treatment, and OD1 is the absorbance of the 0.1% Triton×100 group at 540 nm after treatment.

[0113] The above-mentioned 0.1% Triton X-100 group was treated with a 0.1% Triton X-100 solution, which was prepared by adding 10 mL of Triton X-100 to 100 mL of PBS to prepare a 10% Triton X-100 stock solution, and then diluting it 100 times with PBS to obtain a 0.1% Triton X-100 solution.

[0114] Hemolysis rate is one of the important indicators for evaluating the blood compatibility of materials, reflecting the material's impact on the integrity of red blood cell membranes. Ideally, biomaterials should not cause red blood cell rupture and hemoglobin release upon contact with blood. According to ISO 10993-4, a hemolysis rate below 5% is considered acceptable blood compatibility, and below 2% is considered high compatibility. The color of the red blood cell suspension and the hemolysis rate for each group are shown below. Figure 3 As shown, the PBS group and the FLVPPQESQK group showed no hemolytic activity, while the 0.1% Triton X-100 group exhibited significant hemolytic activity. Quantitative analysis revealed that the hemolysis rate of FLVPPQESQK was less than 1%, indicating good blood compatibility, while the acidic conditions (pH < 5) of the soybean fermentation broth disrupted the erythrocyte membrane, causing nonspecific hemolysis.

[0115] Example 7

[0116] Antimicrobial peptide cytotoxicity assay

[0117] The cytotoxicity of the samples to cells was determined using the CCK-8 assay. Mouse fibroblast L929 cells, containing 5000 cells per 100 μL, were seeded in wells, with at least three replicates per sample. After the cell aggregation reached 70%, 200 μL of fresh culture medium (the fresh medium refers to the complete culture medium used for L929 cell culture) containing the pure FLVPPQESQK peptide synthesized in Example 4 was added, and the cells were co-cultured for 24 and 48 h. The concentration gradient of the FLVPPQESQK peptide was set at 0.5-8 mg / mL. After culturing, 10 μL of CCK-8 solution was added (the final concentration of CCK-8 in the wells was 4.76% (v / v)), and the cells were stored at 37°C in the dark for 30 min. The absorbance was measured at 450 nm using a microplate reader to assess cell viability. Cell viability was calculated according to formula (3), as follows:

[0118] Cell viability (%) = (As-Ab) / (Ac-Ab) × 100% (3)

[0119] Wherein, As is the absorbance of the experimental wells containing cells, drugs, and CCK-8 solution; Ac is the absorbance of the negative control wells containing cells and CCK-8 solution, but no drugs; and Ab is the absorbance of the blank wells containing only culture medium and CCK-8 solution, but no cells or drugs.

[0120] The results show that: Figure 4 As shown, compared with the blank group, the peptide FLVPPQESQK samples in the concentration range of 0.5-8 mg / mL did not produce cytotoxicity and even promoted cell proliferation. With the extension of culture time, no toxicity was observed and the survival rate increased instead, further verifying the good biocompatibility.

[0121] Example 8

[0122] The stability of the antimicrobial peptide was determined using the pure FLVPPQESQK peptide synthesized in Example 4:

[0123] A 1 mg / mL FLVPPQESQK solution was prepared using sterile water to obtain an antimicrobial peptide solution, and its stability was investigated.

[0124] 1) Thermal stability: The antimicrobial peptide solution was treated at -20, 4, 20, 40, 60, and 80℃ for 30 min, respectively, and then rapidly cooled to room temperature to obtain the heat-treated sample solutions. The untreated antimicrobial peptide solution was used as the control solution.

[0125] 2) Acid-base stability: The pH of the antimicrobial peptide solution was adjusted to 2.0, 4.0, 6.0, 8.0, and 10.0 using 0.1 mol / L NaCO3 and HCl, respectively. After standing for 1 hour, the pH was adjusted back to 7.0 to obtain the pH-adjusted sample solutions. The antimicrobial peptide solution without pH adjustment was used as the control solution.

[0126] 3) Ionic strength stability: Salt solutions of 5%, 10%, and 15% by mass were added to the antimicrobial peptide solution at a volume ratio of 1:1 to obtain sample solutions treated with the salt solutions. A 1:1 volume mixture of antimicrobial peptide solution and sterile water was used as the control solution.

[0127] Take 100 μL of the antimicrobial peptide solution from the above sample group or control group, and 100 μL of diluted Staphylococcus aureus (Staphylococcus aureus concentration approximately 1 × 10⁻⁶). 7 The antimicrobial peptide solution (CFU / mL) was incubated in a 96-well plate at 37°C for 12 hours, and the absorbance was measured at 600 nm using a microplate reader. The antimicrobial activity of the antimicrobial peptide solution in each treatment group was calculated according to the method in Example 1 and formula (1), and the relative antimicrobial activity of the antimicrobial peptide solution in each treatment group was calculated according to formula (4).

[0128] R = A1 / A0 × 100% (4)

[0129] In the formula: A0 represents the antibacterial activity of the control group; A1 represents the antibacterial activity of the sample group.

[0130] The test results are as follows: Figures 5-7 As shown.

[0131] Thermal stability: The relative antibacterial activity of FLVPPQESQK was 82.56% at -20℃, 86.24% at 20℃, and decreased to 45.93% at 80℃. It can be seen that high temperature has a certain impact on the antibacterial activity of soybean antimicrobial peptides. High temperature will cause the antimicrobial peptides to denature and lose their ability to bind to bacterial cell membranes, resulting in a significant decrease in antimicrobial activity. In order to maintain its activity, it should be stored at the lowest possible temperature during use.

[0132] Acid-base stability: The relative antibacterial activity of FLVPPQESQK initially increases and then decreases with increasing pH. The relative antibacterial activity is low in acidic and alkaline environments, with the best relative antibacterial activity (89.16%) at pH 6.0. This may be because excessively acidic and alkaline environments alter the structure of the antimicrobial peptide, leading to a decrease in its antibacterial activity.

[0133] Ionic strength stability: The salt solution environment has a certain impact on the antibacterial effect of FLVPPQESQK. With increasing ion concentration, the relative antibacterial activity decreases. In high concentrations of KCl, NaCl, and MgCl2, it only maintains a certain level of antibacterial activity, with the soybean antibacterial peptide showing the highest sensitivity to MgCl2 solution. Salt ions affect the electrostatic interaction between the antibacterial peptide and the bacterial cell membrane, reducing the binding capacity of the antibacterial peptide to the cell membrane, thus altering its antibacterial effect. Furthermore, salt ions can change the spatial conformation of the antibacterial peptide, preventing the active site from interacting with the bacterial cell membrane.

[0134] Example 9: Zebrafish Wound Healing Experiment

[0135] After tail amputation, the fish were divided into 5 groups. A vertical incision was made along the longitudinal axis of the fish in the middle of the tail, bisecting the tail and cutting along a direction parallel to the longitudinal axis of the fish to form an approximately right-angled incision. Sample solutions of FLVPPQESQK at concentrations of 20, 40, and 80 μg / ml were prepared, and a 20 μg / ml silver sulfadiazine solution was prepared as a positive control. The treated zebrafish were placed in the sample solutions of different concentrations or the positive control solutions. Each incision was photographed and recorded under a camera. The healing of the fish tails was observed and recorded every 3 days during the experiment. All experiments were conducted in accordance with the guidelines of the Experimental Animal Welfare and Ethics Committee of Zhejiang University of Technology and met the ethical principles of animal experiments (230508001F).

[0136] The results showed that the regeneration of the caudal fin was as follows: Figures 8-9As shown, on day 6 of treatment, a concentration of 80 μg / mL FLVPPQESQK significantly promoted caudal fin regeneration; and on day 9 after treatment with concentrations of 5 μg / mL and 40 μg / mL, the regeneration rate of the caudal fin was also significantly improved. These results indicate that FLVPPQESQK can effectively accelerate the wound healing process of the zebrafish caudal fin in a time- and dose-dependent manner.

Claims

1. A fermented soybean antibacterial peptide, characterized in that, The amino acid sequence is shown in SEQ ID NO:

1.

2. The method for preparing fermented soybean antimicrobial peptides as described in claim 1, characterized in that, Includes the following steps: S1: Ferment soybean raw materials under the action of probiotics to obtain fermentation liquid, and then sterilize the fermentation liquid; S2: Enriching components with a value less than 3 kDa from the fermentation broth; S3: The components with less than 3kDa are separated by semi-preparative liquid chromatography using a C8 column, and the components with antibacterial activity are collected. S4: Mass spectrometry sequencing was performed on the antibacterial component to obtain fermented soybean antibacterial peptides.

3. The method for preparing fermented soybean antimicrobial peptides as described in claim 1, characterized in that, In step S1, the probiotics include Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, and Bifidobacterium, and fermentation is carried out in the presence of these four probiotics.

4. The method for preparing fermented soybean antimicrobial peptides as described in claim 3, characterized in that, The bacterial concentrations of the four probiotic strains were all (1–10) × 10⁻⁶. 8 CFU / mL, and the inoculum volume of the bacterial suspension was 0.5%-1.5%.

5. The method for preparing fermented soybean antimicrobial peptides as described in claim 1, characterized in that, In step S1, the soybean raw material is crushed soybean powder, and during the fermentation, the mass ratio of soybean raw material to water in the system is 1:(12-18).

6. The method for preparing fermented soybean antimicrobial peptides as described in claim 1, characterized in that, In step S1, the fermentation temperature is 34–40°C, and the fermentation time is 10–20 days.

7. The method for preparing fermented soybean antimicrobial peptides as described in claim 1, characterized in that, In step S3, the chromatographic column used for semi-preparative liquid phase separation includes a Sepax Bio-C8(2) chromatographic column with dimensions of 30×250mm and 8 μm. The eluent for separation using the C8 column includes mobile phase A and mobile phase B. Mobile phase A is an acetonitrile solution containing 0.1% TFA by volume, and mobile phase B is an ultrapure aqueous solution containing 0.1% TFA by volume. The eluent flow rate is 10 mL / min, and the UV detector wavelength is 280 nm. A linear gradient elution method was used, 0–60 min, 0%–65% A.

8. The method for preparing fermented soybean antimicrobial peptides as described in claim 1, characterized in that, In step S3, the antibacterial component is a component with antibacterial activity >40%, and the inhibited bacteria are Escherichia coli and / or Staphylococcus aureus.

9. An antibacterial agent, characterized in that... Includes the antimicrobial peptide of claim 1.

10. The application of the fermented soybean antimicrobial peptide as described in claim 1 in promoting wound healing.

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