A walnut meal antibacterial peptide and a method for preparing the walnut meal antibacterial peptide by fermentation

Abstract

The application provides a walnut meal antibacterial peptide and a method for preparing the walnut meal antibacterial peptide by fermentation, and the amino acid sequence of the walnut meal antibacterial peptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. The walnut meal antibacterial peptide has excellent antibacterial stability, and shows good structural stability and functional activity reservation characteristics under the conditions of a wide temperature range, a pH gradient, salt ion strength and protease action. The method for preparing the walnut meal antibacterial peptide by fermentation uses mixed fermentation of specific components, and optimizes the fermentation conditions, thereby improving the yield of the walnut meal antibacterial peptide and improving the antibacterial capacity and stability of the walnut meal antibacterial peptide.

Description

Technical Field

[0001] This invention belongs to the field of antimicrobial peptide technology, and in particular relates to an antimicrobial peptide from walnut meal and a method for preparing antimicrobial peptides from walnut meal by fermentation. Background Technology

[0002] Antimicrobial peptides are a class of small-molecule bioactive peptides composed of fewer than 100 amino acids, possessing broad-spectrum antimicrobial properties. Antimicrobial peptides are derived from both natural and synthetic sources. Natural antimicrobial peptides are widely obtained from animals, plants, and microorganisms, and can be classified as cationic or anionic based on their net charge. The unique advantage of antimicrobial peptides lies in their ability to kill bacteria by disrupting the structural integrity of pathogenic cell membranes. This non-specific physical mechanism not only significantly reduces the risk of bacterial resistance but also endows antimicrobial peptides with low toxicity. Therefore, antimicrobial peptides have broad application potential in food preservation, biomedicine, and agricultural pest control.

[0003] Most antimicrobial peptides originate from animals, microorganisms, and plants. Compared to other sources, plant-derived antimicrobial peptides exhibit lower cytotoxicity to humans. Plant-derived antimicrobial peptides are mainly divided into two categories: one is endogenous antimicrobial peptides naturally occurring in the plant matrix, present in all plant organs and secreted immediately upon infection. The other category involves releasing peptides rich in antimicrobial activity from parent proteins through enzymatic hydrolysis, fermentation, and other technologies, such as degrading plant proteins from grains, legumes, nuts, and oilseeds to prepare peptides with high antimicrobial activity.

[0004] Fermentation is an eco-friendly process that does not produce toxic residues and can shorten reaction time and increase the concentration of bioactive peptides. Antimicrobial peptides in fermented plant products range from less than 5 kDa to 30 kDa. Generally, they can be divided into two main categories: (1) ribosomal bacteriocins synthesized by the fermenting agent; and (2) other peptides produced by bacterial hydrolysis of plant proteins during fermentation. However, the properties, gene expression, and enzyme activity regulation of different fermentation strains affect the release of metabolites. Therefore, a suitable fermentation strain is the key and guarantee for obtaining high-quality antimicrobial peptides. Single-strain fermentation (such as lactic acid bacteria, Bacillus, yeast, etc.) or mixed-strain co-fermentation are commonly used.

[0005] Despite ongoing research into plant-derived antimicrobial peptides and the discovery of an increasing number of promising food preservatives, the complexity of food production environments necessitates ever-increasing demands for their stability. Maintaining high antimicrobial activity under high temperatures, over a wide pH range, and in environments with high salt concentrations remains a critical challenge. Therefore, developing more stable plant-derived antimicrobial peptides, elucidating their antimicrobial mechanisms, and overcoming existing limitations are urgent objectives. Summary of the Invention

[0006] In view of this, the present invention aims to propose an antimicrobial peptide from walnut meal and a method for preparing antimicrobial peptide from walnut meal by fermentation, in order to explore the optimal process conditions for preparing antimicrobial peptide from walnut meal by mixed bacterial fermentation, and to further prepare antimicrobial peptide from walnut meal with high antibacterial properties and high environmental stability through separation technology.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0008] In a first aspect, the present invention provides a walnut meal antimicrobial peptide, said walnut meal antimicrobial peptide comprising at least one peptide with an amino acid sequence as shown in SEQ ID NO:1-4:

[0009] SEQ ID NO:1: SNHDQRRGIVRVE;

[0010] SEQ ID NO:2:DDNIAGRVGAGPA;

[0011] SEQ ID NO:3:IRRGDIVAIPAGVAH;

[0012] SEQ ID NO:4: IDLSNHANQLDRR.

[0013] Furthermore, the amino acid sequence of the walnut meal antimicrobial peptide is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0014] In a second aspect, the present invention provides an antibacterial composition comprising the walnut meal antibacterial peptide described in the first aspect.

[0015] Thirdly, the present invention provides a food preservative containing the walnut meal antimicrobial peptide described in the first aspect.

[0016] Fourthly, the present invention provides a method for preparing the walnut meal antimicrobial peptides described in the first aspect by fermentation, the method comprising the following steps:

[0017] S1. Prepare a mixed bacterial suspension, wherein the mixed bacteria include Bacillus subtilis, Lactobacillus reuteri, Kluyveromyces marxi, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus plantarum, Bacillus natto, and Lactobacillus rhamnosus;

[0018] S2. After crushing the walnut cake, mix it thoroughly with distilled water to obtain a walnut cake suspension;

[0019] S3. Inoculate the mixed bacterial suspension obtained in step S1 into the walnut meal suspension obtained in step S2, shake to mix and form a fermentation substrate, shake the fermentation substrate to culture and obtain fermentation broth.

[0020] S4. Centrifuge the fermentation broth obtained in step S3, filter the supernatant, and obtain the walnut meal antimicrobial peptide.

[0021] Furthermore, the method also includes a step of activating the mixed bacteria: after culturing the strains to the logarithmic development phase, they are spread onto agar plates and incubated statically; smooth colonies on the plates are picked and re-inoculated into fresh broth and incubated to the logarithmic development phase for later use; preferably, the method further includes the following steps: taking 100 μL of bacterial suspension cultured to the logarithmic development phase, performing serial dilutions, spreading it onto agar plates for colony counting, and finally diluting all bacterial suspensions to 1–5 × 10⁻⁶. 10 CFU / mL available for use.

[0022] Further, the mixed bacterial suspension comprises the following bacterial suspensions by volume ratio: 31-40 parts of Bacillus subtilis, 11-20 parts of Lactobacillus reuteri, 11-20 parts of Kluyveromyces martensii, 11-20 parts of Lactobacillus acidophilus, 1-10 parts of Lactobacillus fermentum, 1-10 parts of Lactobacillus plantarum, 1-10 parts of Bacillus natto, and 1-10 parts of Lactobacillus rhamnosus; further, the concentration of each bacterial strain in the mixed bacterial suspension is the same;

[0023] Based on the uniform design experiment, the preferred composition was 40 parts of Bacillus subtilis, 11 parts of Lactobacillus reuteri, 11 parts of Kluyveromyces marxi, 20 parts of Lactobacillus acidophilus, 10 parts of Lactobacillus fermentum, 1 part of Lactobacillus plantarum, 10 parts of Bacillus natto, and 1 part of Lactobacillus rhamnosus.

[0024] Furthermore, step S2 also includes the step of crushing the walnut cake meal and then sieving it; preferably, the sieve used for sieving is 60 mesh.

[0025] Furthermore, in step S2, the mass-to-volume ratio of walnut cake meal to distilled water is 1:10 to 20; preferably 1:15.

[0026] Furthermore, step S3 also includes a step of microbial inactivation of the walnut meal suspension before inoculation; preferably, the method of microbial inactivation is to heat the walnut meal suspension in a constant temperature water bath at 85°C for more than 30 minutes.

[0027] Furthermore, the inoculation amount of the mixed bacteria in step S3 is 4.5% to 10.5%; preferably 7.8%.

[0028] Furthermore, in step S3, the fermentation temperature is 34–40°C and the fermentation time is 28–36 h;

[0029] Preferably, the fermentation temperature is 37.5℃ and the fermentation time is 32h.

[0030] Further, the centrifugation temperature is 1-5℃, the centrifugation rate is 5000-10000 rpm, and the centrifugation time is 15-30 min; preferably, the centrifugation temperature is 4℃, the centrifugation rate is 8000 rpm, and the centrifugation time is 20 min.

[0031] Furthermore, the filter membrane used for filtration in step S4 is a 0.22 μm filter membrane.

[0032] Furthermore, the method also includes a step of ultrafiltration of the antimicrobial peptides in walnut meal;

[0033] Preferably, the ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 1 to 10 kDa, and more preferably 3 to 10 kDa.

[0034] Furthermore, the method further includes a step of separating the antimicrobial peptides from the ultrafiltered walnut meal by column chromatography; preferably, the column chromatography is ODS column chromatography; more preferably, the mobile phase volume ratio of the ODS column chromatography is acetonitrile: ultrapure water = 2:8.

[0035] Fifthly, the present invention provides the application of the walnut meal antimicrobial peptide as described in the first aspect, or the antimicrobial composition as described in the second aspect, or the food preservative as described in the third aspect, or the method described in the fourth aspect, in food preservation; preferably in the preservation of fruits and vegetables or fruit and vegetable juices; more preferably in the preservation of tomato juice.

[0036] Compared with existing technologies, the walnut meal antimicrobial peptides and the method for preparing walnut meal antimicrobial peptides by fermentation described in this invention have the following advantages:

[0037] (1) The walnut meal antimicrobial peptides of the present invention have excellent antimicrobial stability. Under a wide range of temperature, pH gradient, salt ion strength and protease action conditions, they exhibit good structural stability and functional activity retention characteristics.

[0038] (2) The method for preparing antimicrobial peptides from walnut meal by fermentation described in this invention uses mixed fermentation of specific components and optimizes the fermentation conditions, thereby improving the yield of antimicrobial peptides from walnut meal and enhancing the antimicrobial activity and stability of the antimicrobial peptides from walnut meal.

[0039] (3) The walnut meal antimicrobial peptides described in this invention can achieve antimicrobial and bactericidal effects by disrupting the integrity of bacterial cell membranes, and have broad application prospects in the field of food preservation. Attached Figure Description

[0040] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0041] Figure 1 A schematic diagram illustrating the growth of the strain used in the preparation of walnut meal fermented by a single organism.

[0042] Figure 2 A schematic diagram showing the peptide yield (A), pH change (B), inhibition rate against E. coli (C), and inhibition rate against S. aureus (D) in the preparation example of single-strain fermented walnut meal.

[0043] Figure 3 A schematic diagram of an antibacterial plate for a single-strain fermented walnut meal preparation example;

[0044] Figure 4 The diagram shows the inhibition rate of the mixed bacteria combination against E. coli (A), the inhibition rate against S. aureus (B), the antibacterial effect of the antimicrobial peptides in walnut meal fermented at different concentrations under the optimal mixed bacteria ratio (C), and the plate validation results (D).

[0045] Figure 5 A schematic diagram showing the effects of inoculum size (A, B, C), temperature (D, E, F), and time (G, H, I) on the peptide yield and inhibition rate of antimicrobial peptides in walnut meal.

[0046] Figure 6 The diagram illustrates the interaction between the yield of antimicrobial peptides in walnut meal and three factors: the interaction between inoculum amount and fermentation temperature (A); the interaction between inoculum amount and fermentation time (B); and the interaction between fermentation temperature and fermentation time (C).

[0047] Figure 7 The diagram shows the inhibition rate (A) of different concentrations of walnut meal peptides on E. coli and S. aureus under optimal fermentation conditions and the results of plate coating verification (B).

[0048] Figure 8 The diagram shows the changes in molecular weight of walnut meal peptides during different fermentation times (A), the antibacterial effects of four molecular weight walnut meal antimicrobial peptides at different concentrations on E. coli (B) and S. aureus (C), and the results of plate validation (D).

[0049] Figure 9 Schematic diagram of the inhibitory effects of different ODS components on E. coli (A) and S. aureus (B) and the results of plate validation (C);

[0050] Figure 10This is a schematic diagram showing the antibacterial stability of antimicrobial peptides 2-8 under different pH ranges (A), temperatures (B), salt ion concentrations (C), and protease concentrations (D).

[0051] Figure 11 A schematic diagram showing the changes in the total number of microbial communities in tomato juice after adding different concentrations of components 2-8;

[0052] Figure 12 Schematic diagram showing the changes in soluble solids content (A) and pH (B) of tomato juice after adding different concentrations of components 2-8;

[0053] Figure 13 The peak shape and time RP-HPLC of the antimicrobial peptides from walnut meal (A) and the schematic diagram of the antimicrobial effects of each component on E. coli (B) and S. aureus (C) are shown.

[0054] Figure 14 The bactericidal effects of each component of RP-HCLP on E. coli and S. aureus are shown in the figure.

[0055] Figure 15 A schematic diagram illustrating peptide protein information for LC-MS / MS identification of walnut meal peptides F2 (A) and F10 (B) fractions;

[0056] Figure 16 Schematic diagram of the antibacterial effect evaluation results of solid-phase synthesized peptides: determination of antibacterial rate of WAP-1 (A), determination of antibacterial rate of WAP-2 (B), determination of antibacterial rate of WAP-3 (C), determination of antibacterial rate of WAP-4 (D), and antibacterial verification of four peptide plates at a concentration of 5 mg / mL (E).

[0057] Figure 17 This diagram illustrates the effect of WAP-2 peptide on bacterial morphology. (A) shows the morphology of the control group E. coli, (C) shows the morphology of the control group S. aureus, (B) shows the morphology of E. coli treated with WAP-2 peptide (2MIC), and (D) shows the morphology of S. aureus treated with WAP-2 peptide (2MIC).

[0058] Figure 18 Surface binding diagrams of WAP-2 peptide with E. coli membrane protein (A), DHFR enzyme (B), and DNA gyrase (C); and surface binding diagrams of WAP-2 peptide with S. aureus membrane protein (D), DHFR enzyme (E), and DNA gyrase (F).

[0059] Figure 19 Schematic diagram of AKP enzyme and ATP leakage caused by WAP-2 peptide in E. coli and S. aureus: E. coli (A, C), S. aureus (B, D);

[0060] Figure 20 This is a schematic diagram showing the stability test results of WAP-2 peptide under different pH ranges (A), temperatures (B), salt ion concentrations (C), and protease concentrations (D). Detailed Implementation

[0061] The present invention and its technical effects will be further elaborated below with reference to specific embodiments, so as to fully understand the purpose, features and effects of the present invention. Unless otherwise specified, the methods described are conventional methods. Unless otherwise specified, the materials described are all available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0062] Example 1: Preparation of antimicrobial peptides from walnut meal by single-strain fermentation

[0063] The main bacterial species used in this embodiment are shown in Table 1.

[0064] Table 1 Main bacterial species

[0065]

[0066]

[0067] 1. Determination of basic components of walnut meal

[0068] The moisture, ash, fat, and protein contents of walnut meal were determined according to GB 5009.3-2016, GB5009.4-2016, GB 5009.6-2016, and GB 5009.5-2016, respectively. The results are shown in Table 2.

[0069] Table 2. Basic Composition of Walnut Meal

[0070] Sample Name Moisture (%) Ash content (%) Fat(%) protein(%) other(%) Walnut meal 3.50±0.03 5.73±0.03 7.45±0.07 37.45±0.07 45.87±0.11

[0071] As shown in Table 2, the protein content of walnut meal is as high as 37.45±0.07%, which can serve as a good nitrogen source for microbial fermentation.

[0072] 2. Process of single-strain fermentation of walnut meal

[0073] L. reuteri, L. fermentum, L. plantarum, L. rhamnosus, L. acidophilus, K. marxianus, B. natto, and B. subtilis were selected as strains for producing antimicrobial peptides from fermented walnut meal. The specific preparation method is as follows:

[0074] Strain activation: After culturing the strains to the logarithmic development phase, they were spread onto agar plates and incubated statically for 24 hours. Smooth colonies were picked from the plates and re-inoculated into fresh broth, incubated to the logarithmic development phase, and then serially diluted (100 μL) onto agar plates for colony counting. Finally, all bacterial suspensions were diluted to 1 × 10⁻⁶. 10 CFU / mL available for use.

[0075] Fermentation: Walnut meal samples were first pulverized and sieved through a 60-mesh standard sieve to obtain uniform powder for later use. Then, walnut meal powder was fully dissolved in distilled water at a mass-to-volume ratio of 1:15 (w / v) to form a homogeneous suspension. This suspension was heated in an 85℃ constant temperature water bath for 30 minutes to effectively inactivate endogenous microorganisms. After the system cooled naturally to room temperature, a pre-prepared target strain was inoculated at an inoculum size of 1.5%, and the mixture was thoroughly shaken to form the fermentation substrate. The fermentation system was transferred to a 37℃ constant temperature incubator and cultured at 180 rpm for 48 hours to ensure sufficient cell growth and metabolic reactions. The total bacterial count, pH, and peptide yield of the walnut meal fermentation broth from different single-strain fermentations were measured every 8 hours.

[0076] End of fermentation: When processing the fermented walnut meal broth, first centrifuge at 8000 rpm for 20 min at 4℃ to collect the cell-free walnut meal fermentation supernatant, and then remove the bacteria by physical filtration using a 0.22 μm filter membrane.

[0077] Fermentation broth samples were collected every 8 hours during the fermentation process. A serial dilution method was used to process the samples; 50 μL of the dilution was aseptically spread evenly on the surface of PCA (Potentially Calculated Plate Count) agar. After incubation, the number of colonies on each plate was counted according to the standard procedure of plate counting. Based on this, a cell growth curve was plotted to investigate the growth dynamics of the cells in the walnut meal fermentation system. The results are as follows: Figure 1 As shown, within 48 hours, all strains entered the typical logarithmic growth phase within 8–32 hours, with bacterial density ranging from 9.28 to 10.49 Log. 10The bacterial density decreased to varying degrees within 40–48 hours, reaching CFU / mL. L. rhamnosus reached the logarithmic phase at 32 hours, with a bacterial density of 9.90 Log. 10 CFU / mL Figure 1 A) L. fermentum reaches the logarithmic phase at 32 hours, with a bacterial density of 9.28 Log. 10 CFU / mL Figure 1 B), L. reuteri reached the logarithmic phase at 32 h, with a bacterial density of 9.48 Log10 CFU / mL (B). Figure 1 C) L. acidophilus reached the logarithmic phase at 32 hours, with a bacterial density of 9.46 Log. 10 CFU / mL Figure 1 D) L. plantarum reaches the logarithmic phase at 32 hours, with a bacterial density of 10.51 Log. 10 CFU / mL Figure 1 E) K. marxianus reaches the logarithmic phase at 24 hours, with a bacterial density of 10.21 Log. 10 CFU / mL Figure 1 F), B. natto reached the logarithmic phase at 24 hours, with a bacterial density of 10.12 Log. 10 CFU / mL Figure 1 G) and B. subtilis reached the logarithmic phase at 24 h, with a bacterial density of 10.49 Log. 10 CFU / mL Figure 1 The above results indicate that walnut meal can serve as a high-quality substrate for fermentation.

[0078] The yield of polypeptides in walnut meal fermentation broth was determined using the biuret method according to Formula 1.

[0079]

[0080] like Figure 2 As shown in Figure A, with the extension of fermentation time, the large molecular proteins in the walnut meal fermentation broth were continuously hydrolyzed into small molecular peptides by bacteria. The eight fermentation strains exhibited similar peptide yield trends throughout the fermentation process. The peptide yield of the fermented walnut meal by the eight strains generally reached its peak between 16 and 32 hours, ranging from 40.11% to 53.32%. Subsequently, the peptide yield of antimicrobial peptides in the walnut meal decreased between 40 and 48 hours. This is because the bacterial density began to decline, and insufficient bacterial activity affected the production of peptides from the walnut meal.

[0081] During fermentation, the pH of the walnut meal fermentation broth was measured according to GB 5009.237—2016. All operations were repeated three times, and the results are as follows. Figure 2As shown in Figure B, the pH value gradually decreased from an initial 6.86 to 4.16–4.82 within 0–32 hours, then rebounded to 4.39–5.08, showing an overall trend of first decreasing and then increasing. The pH fluctuations during fermentation originated from the abundant carbon source in the walnut meal. Bacillus bacteria metabolized carbohydrates to produce organic acids through extracellular enzymes, while the simultaneous anaerobic lactic acid synthesis by lactic acid bacteria and Saccharomyces cerevisiae led to initial acidification. The subsequent pH rebound was related to the release of amino acids and ammonia compounds through bacterial protein hydrolysis via deamination / decarboxylation pathways.

[0082] The antibacterial effects of walnut meal peptides prepared from different fermentation strains were evaluated using the following methods:

[0083] Bacterial strain activation: Using the standard streak plate isolation method, the thawed bacterial culture was inoculated onto the surface of Luria-Bertani (LB) solid agar plates, which were then incubated at 37°C for 24 hours. Typical single colonies were picked from the culture plates using a sterile inoculation loop and transferred to test tubes containing 5 mL of LB liquid medium. The tubes were then shaken at 150 rpm for 24 hours at 37°C to obtain a bacterial suspension in the logarithmic growth phase. 500 μL of this suspension was used as a seed culture and transferred to a fresh 5 mL LB liquid medium. The culture was continued under the same shaking conditions until the bacterial density reached mid-logarithmic growth, at which point the culture was terminated. The culture was then stored at 4°C for subsequent experiments.

[0084] Minimum inhibitory concentration (MIC) determination: E. coli and S. aureus in the logarithmic growth phase were resuspended in LB liquid medium and the bacterial concentration was adjusted to 10. 6 CFU / mL. The antimicrobial peptide was serially diluted twofold, and 100 μL of the antimicrobial peptide solution and 100 μL of the diluted indicator bacterial solution were added to each well. The mixture was incubated at 37°C for 12 h. The absorbance at 600 nm (OD value) was measured using a multiskan MK3 (USA). The MIC value of the antimicrobial peptide was determined when the inhibition rate reached or exceeded 90%. The bacterial inhibition rate was calculated using Equation 2.

[0085]

[0086] The activity was further verified by plate count method. *Escherichia coli* and *Staphylococcus aureus* were cultured with different concentrations of walnut meal fermentation broth at 37℃ and 180 rpm for 2 hours under constant shaking conditions. The *E. coli* and *S. aureus* samples were then diluted to 1×10⁻⁶. 4 CFU / mL. Spread 100 μL of the diluted microorganism onto LB agar plates. Incubate at 37°C for 16–20 h, then calculate the total colony count.

[0087] The results are as follows Figure 2 C and Figure 2 As shown in Figure D, the changes in the antimicrobial activity of the fermentation broth were consistent with the trends in peptide yield and pH. All single-cell fermentation broths showed inhibition rates of over 90% against *E. coli* and *S. aureus* during the logarithmic growth phase, with a MIC of 20 mg / mL. The production of antimicrobial peptides is related to microbial growth and pH. The logarithmic growth phase is the optimal time to obtain high peptide yields, while the optimal pH for antimicrobial peptide production is mostly in the alkaline (5.5–6) or acidic (<5) range.

[0088] The minimum inhibitory concentration (MIC) of the eight single-strain walnut meal fermentation broths was determined to be 20 mg / mL. Plate plating was performed on these strains, with nisin at the same concentration as a positive control. The results are as follows: Figure 3 As shown, the walnut meal fermentation broth using Bacillus subtilis as the fermentation inoculum showed the best sterilization effect, with no colonies on the plates, consistent with the sterilization effect of nisin. The next best were Lactobacillus reuteri, Lactobacillus acidophilus, and Kluyveromyces marxianus, all of which showed significant bactericidal effects against S. aureus at a concentration of 20 mg / mL, with no colony growth on the plates.

[0089] Example 2: Preparation of antimicrobial peptides from walnut meal by mixed-culture fermentation

[0090] The preparation method of antimicrobial peptides from mixed-strain fermented walnut meal is the same as that of antimicrobial peptides from single-strain fermented walnut meal, except that the formula of the mixed-strain strains used is shown in Table 3-4.

[0091] Table 3. Factor 10 Level Uniform Design Table

[0092] Factor Level X1 X2 X3 X4 X5 X6 X7 X8 N1 3 8 8 3 7 8 3 1 N2 1 6 3 9 5 9 6 10 N3 7 2 2 2 3 7 2 7 N4 8 5 9 7 2 10 8 4 N5 6 10 1 5 9 6 10 5 N6 2 3 5 4 4 1 9 3 N7 4 1 10 6 10 5 5 8 N8 9 4 4 10 8 4 4 2 N9 10 7 7 1 6 3 7 9 N10 5 9 6 8 1 2 1 6

[0093] Table 4. Examples of mixed-strain fermentation of walnut meal antimicrobial peptides: Mixed-strain formulation

[0094]

[0095]

[0096] Mixed-strain fermentation can utilize the enzyme systems (such as cellulase, protease, and lipase) secreted by different strains to synergistically degrade complex substrates such as macromolecular proteins, thereby increasing peptide yield and enhancing peptide structural stability. Therefore, to improve the yield of antimicrobial peptides from walnut meal, based on the uniform experimental design in Tables 3 and 4, with peptide yield as the response variable, the final experimental results are shown in Table 5. Analysis using Minitab software yielded the following quadratic polynomial stepwise regression equation:

[0097] Y=36.615+81.71X1-673.43X2X3+117.32X2X5+169.46X3X4-725.18X3X6-433.20X3X7-79.77X7X8+496.7X7 2

[0098] The coefficient of determination R of the regression equation 2 =0.99. The final optimized mixed-culture fermentation volume ratio was: Bacillus subtilis 40 parts, Lactobacillus reuteri 11 parts, Kluyveromyces martensii 11 parts, Lactobacillus acidophilus 20 parts, Lactobacillus fermentum 10 parts, Lactobacillus plantarum 1 part, Bacillus natto 10 parts, and Lactobacillus rhamnosus 1 part. The predicted peptide yield was 65.49%, and the verified peptide yield was 61.53%, only 3.96% different from the predicted value, proving the reliability of the model.

[0099] Table 5. Results of peptide yield in the preparation of antimicrobial peptides from mixed-strain fermented walnut meal.

[0100]

[0101]

[0102] In addition, the antibacterial activity of the mixed-culture fermentation broth was tested, and the results are as follows: Figure 4 As shown in Figures AB, the inhibition rates against *Escherichia coli* and *Staphylococcus aureus* were 92.25% and 91.56%, respectively. The optimal mixed bacterial combination was further validated for its antibacterial activity, and the results are as follows: Figure 4 As shown in C, the inhibition rate against Escherichia coli was 92.25%, and the inhibition rate against Staphylococcus aureus was 91.56%, with a MIC of 10 mg / mL. Furthermore, at a treatment concentration of 20 mg / mL (…), the inhibition rate was significantly higher. Figure 4 D) No colonies grew on the plate. Compared with single-strain fermentation, mixed-strain fermentation significantly improved the yield of antimicrobial peptides and the antibacterial properties of walnut meal.

[0103] Example 3: Process optimization of antimicrobial peptides from mixed-culture fermentation of walnut meal

[0104] 1. Single-factor experiment

[0105] Walnut meal was fermented using a mixed culture blend ratio determined by uniform experiments (40% Bacillus subtilis, 11% Lactobacillus reuteri, 11% Kluyveromyces martensii, 20% Lactobacillus acidophilus, 10% Lactobacillus fermentum, 1% Lactobacillus plantarum, 10% Bacillus natto, and 1% Lactobacillus rhamnosus). The effects of fermentation conditions (fermentation time, fermentation temperature, and bacterial density) on the production of antimicrobial peptides from liquid fermentation of walnut meal were investigated, with peptide yield as the indicator.

[0106] Determination of bacterial density in fermentation broth: The pretreated mixed-culture walnut meal fermentation bottle was fermented in a constant temperature incubator at 37℃ for 32h, and the peptide yield and antibacterial activity against E. coli and S. aureus were measured at mixed-culture inoculation amounts of 0.75%, 1.5%, 7.5%, 15%, and 75%.

[0107] Determination of fermentation temperature: The total bacterial inoculation in the pretreated walnut meal fermentation bottle was 7.5%, and fermentation was carried out for 32 hours. The peptide yield and antibacterial activity against E. coli and S. aureus were measured at fermentation temperatures of 27, 32, 37, 42, and 47℃.

[0108] Determination of fermentation time: The amount of mixed starter culture in the pretreated walnut meal fermentation bottle was 7.5%, and it was placed in a constant temperature incubator at 37℃ for fermentation. The yield of peptides in the walnut meal fermentation broth and its antibacterial activity against E. coli and S. aureus were measured at different time points of 8, 16, 24, 32 and 40 h.

[0109] The results are as follows Figure 5 As shown, the optimal conditions for preparing antimicrobial peptides from walnut meal were determined through single-factor experiments. The inoculation density at the initial stage of fermentation and the temperature and fermentation time during the fermentation process were crucial to the fermentation efficiency. Firstly, the peptide yield gradually increased with increasing inoculum size (p<0.05). When the inoculation size was 7.5%, the yield of antimicrobial peptides from walnut meal reached a peak of 61.39%. With further increases in inoculum size, the peptide content in the walnut meal fermentation broth began to decrease (e.g., ...). Figure 5 (As shown in A). This is because, within a certain range, microorganisms can secrete more proteases to promote the degradation of substrate proteins and produce more peptides. However, when there are too many microorganisms, they will competitively inhibit the biological activity and metabolism of microorganisms, thereby reducing the amount of peptides produced in the fermentation products. Furthermore, its antibacterial properties are consistent with this trend; at an inoculum size of 7.5%, it is effective against E. coli (e.g., *E. coli*). Figure 5 (as shown in B) and S. aureus (as shown in B) Figure 5 The inhibition rates of the inoculum (as shown in C) were 91.27% and 90.48%, respectively, with a MIC of 10 mg / mL. This indicates that an inoculum size of 7.5% is the optimal bacterial density for the production of antimicrobial peptides from walnut meal. Subsequently, the effect of temperature on the fermentation efficiency of antimicrobial peptides from walnut meal was investigated. When the temperature reached 37℃, the antimicrobial peptide content in the fermentation broth was significantly higher than the peptide yield at other temperatures (p<0.05), with a peptide yield of 62.89% (as shown in C). Figure 5 As shown in D), at this time, the inhibitory effect of the walnut meal antimicrobial peptide on the two pathogenic bacteria was MIC 10 mg / mL (as shown in D). Figure 5As shown in Figure EF, this corresponds to the optimal growth temperature for the fermentation strains. When deviating from this optimal temperature, both the peptide production capacity and antibacterial effect of the walnut meal were significantly inhibited. Finally, the effect of fermentation time on the peptide production and antibacterial effect in the walnut meal fermentation broth was investigated (e.g., ...). Figure 5 As shown in the GI (Gross Interaction Index), the peptide yield of antimicrobial peptides from walnut meal gradually increased with fermentation time, reaching a peak of 63.55% at 32 h (p < 0.05). Subsequently, the peptide content in the fermentation broth decreased significantly at 40 h. The antimicrobial activity was positively correlated with the yield of antimicrobial peptides from walnut meal, with the best antimicrobial performance observed at 32 h. The inhibition rate against Saureus was 90.40%, and the inhibition rate against E. coli was 91.49%, with the MIC remaining at 10 mg / mL. Therefore, based on the results of the single-factor experiments on the fermentation process, the optimal fermentation conditions for antimicrobial peptides from walnut meal were an inoculum size of 7.5%, a fermentation temperature of 37℃, and a fermentation time of 32 h.

[0110] 2. Response Surface Optimization Experiment

[0111] Fermentation time, fermentation temperature, and initial cell concentration in the fermentation broth were used as influencing factors, and peptide yield was used as the response value. Response surface methodology was designed and implemented using Design Expert 13.0 software. The experimental factor levels are shown in Table 6, and the specific experimental scheme and results are shown in Table 7.

[0112] Table 6 Factor Level Table

[0113]

[0114] Table 7. Multifactor Experimental Design and Results

[0115]

[0116]

[0117] Table 7 shows that the yield of antimicrobial peptides from walnut meal ranged from 40.67% to 64.52% in the 17 experimental groups. A quadratic stepwise multiple regression was performed on the data, resulting in the following quadratic multinomial regression equation:

[0118] Y=63.93-1.04A+2.69B+1.89C+4.50AB-0.995AC-1.5BC-6.04A 2 -7.81B 2 -8.52C 2

[0119] Table 8. Analysis of Variance Table

[0120]

[0121]

[0122] * p≤0.05, ** p≤0.01, *** p≤0.001

[0123] As shown in Table 8 (Analysis of Variance), the regression model is highly significant (p < 0.0001, R0.0001). 2 =0.99), this model can be used to predict ideal conditions. The regression equation model is highly significant (p<0.0001), and the lack-of-fit test result is 0.4011 (p>0.05). Predictive coefficient R 2 =0.9472, Corrected coefficient of determination R 2 Adj =0.9859, indicating that the model is highly significant. A, B, C, AB, BC, A 2 B 2 and C 2 The coefficients were significant, indicating that inoculation amount, time, and temperature had a significant impact on the yield of antimicrobial peptides in walnut meal. However, the cross-product coefficient of AC was not significant (p = 0.0711). Based on the F-values, the three variables with the greatest impact on the yield of antimicrobial peptides in walnut meal were, in descending order: B (temperature) > C (inoculation amount) > A (time).

[0124] like Figure 6 As shown, with one factor kept constant, the interaction between the other two factors significantly affected the yield of walnut meal peptides (p<0.05). The results indicated that with an inoculum size of 7.80%, a fermentation temperature of 37.47℃, and a fermentation time of 31.85 h, the predicted peptide yield was 64.25%. To account for practical conditions, the fermentation conditions were adjusted to an inoculum size of 7.50%, a fermentation temperature of 37.5℃, and a fermentation time of 32 h. The peptide yield was 64.18%, and the error in the walnut meal peptide yield compared to the predicted value was 0.07%, which is basically consistent with the theoretical prediction, indicating that the model has good reliability.

[0125] The antibacterial activity of walnut meal fermentation broth under optimal fermentation conditions was verified, such as... Figure 7 As shown in Figure A, under the fermentation conditions of 7.5% inoculum, 37.5℃ fermentation temperature, and 32h fermentation time, the MIC value of the walnut meal fermentation broth was 10 mg / mL, exhibiting better inhibitory effects against *S. aureus* (93.05% inhibition rate) and *E. coli* (91.25% inhibition rate). Plate testing was also conducted at the same concentration. Figure 7 B) At a concentration of 10 mg / mL, only a small number of colonies grew, and when the peptide concentration was 20 mg / mL, no colonies grew on the plate.

[0126] 3. Isolation of antimicrobial peptides from walnut meal

[0127] To obtain antimicrobial peptides with higher antibacterial activity from walnut meal fermentation broth, ultrafiltration was employed for separation. Ultrafiltration centrifuge tubes with molecular weight cutoffs of 1 kDa, 3 kDa, and 10 kDa were used to separate the antimicrobial peptides from the walnut meal fermentation broth fermented with mixed cultures for 0-40 hours under optimal fermentation conditions (7.5% inoculum, 37.5℃, and 32 h). The fractions were divided into four different fractions based on molecular weight range: less than 1 kDa (Mw < 1 kDa), between 1 kDa and 3 kDa (1 kDa ≤ Mw < 3 kDa), between 3 kDa and 10 kDa (3 kDa ≤ Mw < 10 kDa), and greater than 10 kDa (Mw ≥ 10 kDa). Each fraction was freeze-dried, and their antimicrobial activity was tested to assess differences in antimicrobial efficacy.

[0128] The results are shown in Table 9. The content of small molecule peptides gradually increased with time. Among them, the content of walnut meal peptides with 3-10 kDa was the highest after 32 hours of fermentation, with a peptide content of 8.17 mg, accounting for 40.85% of the total peptide content after 32 hours.

[0129] Table 9. Colony density, peptide yield, and molecular weight distribution of walnut meal fermentation broth at different fermentation times.

[0130]

[0131] After 32 hours of fermentation, walnut meal peptides at a concentration of 10 mg / mL (3-10 kDa) showed antibacterial effects against *E. coli* and *S. aureus* of 93.63% and 91.55%, respectively, which were superior to walnut meal peptides at concentrations >10 kDa (36.11% and 28.70%), 1-3 kDa (37.92% and 48.60%), and <1 kDa (26.26% and 31.52%). Figure 8 (As shown in AC). This demonstrates that the 3-10 kDa fraction exhibits significantly stronger antibacterial activity against the target strain, showing a clear advantage compared to other molecular weight ranges. Furthermore, under experimental conditions where the concentration of the 3-10 kDa polypeptide mixture reached 10 mg / mL, the number of colonies on E. coli and S. aureus culture plates was significantly lower than that of other molecular weight fractions, with only a small number of colonies growing (e.g., as shown in AC). Figure 8 (as shown in D).

[0132] The extracts separated by ultrafiltration were further separated by ODS column chromatography: acetonitrile / ultrapure water gradient elution was used with mobile phase volume ratios of 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, and 8:2 (v1 / v2), and the resulting groups were 2-8, 3-7, 4-6, 5-5, 6-4, 7-3, and 8-2. To determine the peptide content and antibacterial activity, the obtained fractions were concentrated under vacuum and reconstituted in 1 mL of ultrapure water, and their antibacterial activity was measured.

[0133] Table 10. Peptide content and antibacterial properties of different ODS column chromatography fractions.

[0134] Components Peptide content percentage Peptide content (mg) E. coli inhibition rate (%) S. aureus inhibition rate (%) 2-8 23.8 42.84 91.64 92.35 3-7 15.2 27.36 15.25 86.54 4-6 15.21 27.37 76.98 82.32 5-5 13.03 23.45 63.66 88.31 6-4 14.63 26.34 61.83 78.15 7-3 9.62 17.31 55.13 66.93 8-2 8.42 15.16 61.94 57.09

[0135] The results are shown in Table 10 and... Figure 9 As shown, the peptide content was highest in fractions 2-8 at 23.8%. As the polarity of the mobile phase decreased, the peptide content gradually declined, with fractions 7-3 and 8-2 containing only 9.62% and 8.42% peptides, respectively. Figure 9 As shown in Figures AB, components 2-8 exhibited excellent antibacterial effects among the seven components, showing a good concentration-dependent trend. The minimum inhibitory concentration (MIC) for components 2-8 was reached at 5 mg / mL, with an inhibition rate of 91.64% against *Escherichia coli* and 92.35% against *Staphylococcus aureus*. Plate testing was performed at 5 mg / mL, as shown in Figures AB. Figure 9 As shown in Figure C, only a small number of colonies were observed on the plates containing fractions 2-8. At this concentration, the inhibitory effect on E. coli and S. aureus was superior to that of nisin. Therefore, the peptides with antibacterial activity are mainly concentrated in fractions 2-8.

[0136] The antibacterial stability of fractions 2-8 was tested under different temperature conditions (4–121℃), acidic / alkaline pH environments (pH 3–11), high salt ion concentrations (50–200 mM), and after treatment with different proteases.

[0137] Temperature tolerance test: The antimicrobial peptides of walnut meal components 2-8 were incubated at 4℃, 25℃, 60℃, 80℃, 100℃ and 121℃ for 30 min respectively. After incubation, the temperature was lowered to room temperature and the antimicrobial activity was then measured.

[0138] pH sensitivity experiment: Walnut meal antimicrobial peptide solutions were prepared using phosphate buffered saline (PBS) with different pH values ​​(3, 5, 7, 9, 11), and the antimicrobial properties of antimicrobial peptides 2-8 were determined under different pH conditions.

[0139] Salt ion concentration effect experiment: NaCl and CaCl2 solutions of different concentrations (50mM, 100mM, 150mM, 200mM) were mixed evenly with the walnut meal antimicrobial peptide 2-8 component solution and incubated for 1 hour to determine the antimicrobial activity of the antimicrobial peptide under different salt ion concentrations.

[0140] Protease hydrolysis resistance experiment: Different concentrations (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL) of pepsin, trypsin and neutral protease solutions were prepared and mixed with walnut meal antimicrobial peptide 2-8 component solutions at a 1:1 volume ratio. The solutions were incubated at the optimal reaction temperature of each protease for 2 h, and then the protease was inactivated by heating at 90 °C for 10 min. Finally, the hydrolysis resistance of the antimicrobial peptides under the action of proteases was measured.

[0141] The results are as follows Figure 10 As shown, components 2-8 exhibit strong resistance to acids, alkalis, high temperatures, and salt ion concentrations. Within a pH range of 3-11, their antibacterial activity against *E. coli* and *S. aureus* remains above 99% (e.g., ...). Figure 10 As shown in Figure A). At a high temperature of 121℃, the inhibition rate against E. coli was 96.66%, and the inhibition rate against S. aureus was 95.47% (as shown in Figure A). Figure 10 (As shown in B). Components 2-8 of the antimicrobial peptides were respectively subjected to high concentrations of Na. + Ions and Ca 2+ Treatment with ions (200 mM) still maintained an inhibition rate of over 98% against E. coli and S. aureus (e.g., Figure 10 As shown in C). With increasing concentrations of pepsin, neutral protease, and trypsin, components 2-8 maintained good antibacterial activity (e.g., ...). Figure 10 As shown in D), the inhibition rate against S. aureus remained above 95% at a concentration of 2 mg / mL. The antibacterial activity against E. coli decreased only under pepsin treatment, but the antibacterial performance remained at 93.44%.

[0142] Example 4: Preservative effect of antimicrobial peptides in walnut meal

[0143] Using highly stable walnut meal antimicrobial peptides (components 2-8 in Example 3) as raw materials, their preservative ability in actual production was investigated by adding them to fresh tomato juice. Different concentrations of component 2-8 were used as treatment groups, with untreated tomato juice as a control group. The effect of the antimicrobial peptides on the bacterial community growth of tomato juice was analyzed, and changes in pH and soluble solids were monitored to evaluate the preservation and antibacterial effects of component 2-8 on tomato juice. Furthermore, 16S rRNA gene high-throughput sequencing technology was used to systematically evaluate the regulatory effect of component 2-8 on the microbial community structure of tomato juice, to assess the antibacterial effect of walnut meal antimicrobial peptides as a natural food preservative in fruit juice. 2 kg of fresh tomatoes were taken, washed with sterile water, peeled, chopped, and cored, and then juiced. The resulting tomato juice was stirred evenly, filtered through sterile gauze, and dispensed into 50 mL blue-capped bottles, with each bottle containing 30 mL. Components 2-8 were added to freshly squeezed tomato juice, and their final concentrations were adjusted to 0.25MIC (1.25 mg / mL), 0.5MIC (2.5 mg / mL), and MIC (5 mg / mL). Three parallel samples were set up for each group, and tomato juice without the addition of components 2-8 was used as a blank control. All samples were stored at room temperature.

[0144] The total bacterial count in tomato juice was monitored at 0, 6, 12, 18, 24, 30, 36, 42, 48, 60, and 72 hours, and the results are as follows: Figure 11 As shown, within 48 hours, the total number of microbial communities in the control group (without added antimicrobial peptides 2-8) tomato juice showed a continuous upward trend. After 48 hours, the total number of colonies in the untreated group reached 9.48 Log. 10 CFU / mL, when the storage time exceeded 48 hours, the total number of colonies in the untreated group gradually decreased, reaching 8.57 Log at 72 hours. 10 The CFU / mL concentration was still higher than that of other treatment groups treated with different concentrations of walnut meal antimicrobial peptides. Treatment with walnut meal fractions 2-8 at concentrations of 0.25 MIC, 0.5 MIC, and 1 MIC showed a good dose-dependent inhibitory effect. Bacterial growth in tomato juice samples was significantly inhibited at 0 h, with the total bacterial count in the untreated group being 3.26 Log0.05. 10 CFU / mL, while the total bacterial count in the treatment group was 3.03 Log. 10 CFU / mL, 2.71 Log 10 CFU / mL, 1.13 Log 10 CFU / mL. After 48 hours of storage, the total bacterial count in the 2-8 fraction treatment groups at 0.25 MIC, 0.5 MIC, and 1 MIC was 8.45 Log. 10 CFU / mL, 7.13 Log 10CFU / mL, 4.12 Log 10 Compared to the control group, the total number of CFU / mL decreased by 1.03 Log. 10 CFU / mL, 2.35 Log 10 CFU / mL, 5.36 Log 10 CFU / mL. The 1MIC treatment group showed good overall antibacterial effect. Although the number of bacteria increased after 36 hours, the total number of colonies decreased by 4.55 Log compared with the control group. 10 The results showed that the walnut meal components 2-8 effectively inhibited the growth of bacterial colonies in fresh tomato juice.

[0145] The pH value and soluble solids (TSS) of tomato juice were measured. Figure 12 As shown in Figure A, the TSS content in the untreated group decreased significantly over time, dropping from an initial 9.5% to 6.1% at 72 hours. This TSS depletion is a result of microbial growth and reproduction. The addition of components 2-8 showed varying degrees of TSS retention in the tomato juice. At 72 hours, the TSS levels in the 0.25MIC, 0.5MIC, and 1MIC treatment groups were 7.2%, 7.7%, and 8.1%, respectively. Compared to the initial TSS value, the 1MIC group only showed a 1.4% decrease. Figure 12 As shown in Figure B, the pH value of the untreated tomato juice remained relatively stable at 4.35 during the first 12 hours. Subsequently, the pH value decreased significantly between 12 and 30 hours, reaching 3.84 at 72 hours, representing an 11.72% change from the initial pH. Although the addition of 2-8 components significantly lowered the pH value of the tomato juice during the first 12 hours, the pH fluctuation range was relatively small during the subsequent 12 to 72 hours. At 0.25 MIC, the pH value decreased from 3.99 to 3.80; at 0.5 MIC, the pH value recovered from 3.69 to 3.88; and at 1 MIC, the pH value recovered from 3.33 to 3.46, with a fluctuation range of 3.76% to 4.9%. The pH change was attributed to the alteration of the microbial community structure in the tomato juice by the addition of walnut meal 2-8 components. In conclusion, the antimicrobial peptides of walnut meal 2-8 components have potential in maintaining the quality of tomato juice.

[0146] Example 5: Identification and antibacterial mechanism of antimicrobial peptides in walnut meal

[0147] This experimental example employed reversed-phase high-performance liquid chromatography (RP-HPLC) to purify fractions 2-8 of the walnut meal antimicrobial peptides, and mass spectrometry was used to identify the sequences of the isolated active peptides. Subsequently, computer-aided virtual screening was used to screen for dominant fragments with potentially high antibacterial activity from the identified peptide sequences. Finally, the target antimicrobial peptide molecules were prepared using solid-phase chemical synthesis technology, and their inhibitory effects on *E. coli* and *S. aureus* were further evaluated. Molecular docking technology was used to explore the key binding sites between the walnut meal antimicrobial peptides and bacterial protein targets. Furthermore, the leakage of intracellular ATPases and AKP enzymes in bacteria was measured, and the effects of the walnut meal antimicrobial peptides on the microstructure of *E. coli* and *S. aureus* cells were observed using scanning electron microscopy (SEM), exploring the inhibitory mechanism of the walnut meal antimicrobial peptide monomers on bacteria at the cell membrane level.

[0148] 1. Experimental Methods

[0149] The freeze-dried walnut meal antimicrobial peptides 2-8 were resuspended in ultrapure water to a concentration of 50 mg / mL per bottle and then filtered through a 0.22 μm filter membrane for later use.

[0150] Based on the differences in polarity and hydrophobicity of the compounds, reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate the antimicrobial peptide 2-8 components using gradient elution. The chromatographic conditions are as follows:

[0151] Chromatographic column: Agilent SB-C18 column (9.4×250mm, 5μm);

[0152] Mobile phase: A - ultrapure water (containing 0.1% TFA); B - acetonitrile (containing 0.1% TFA), flow rate: 4 mL / min;

[0153] Gradient elution program: 0-5 min, 5-7% B; 5-11 min, 7-10% B; 11-20 min, 10% B; 21-30 min, 20% B; 30-35 min, 20-85% B; 35-40 min, 85% B; 40-45 min, 85-5% B;

[0154] UV detection wavelength: Due to the molecular structural characteristics of the peptides, dual-channel detection at 220 nm and 280 nm was used. Walnut meal antimicrobial peptides exhibiting absorption difference peaks were collected, freeze-dried, and stored at -20°C.

[0155] Table 11 Response time, peptide content percentage, and antibacterial properties of 10 RP-HPLC components

[0156]

[0157] As shown in Table 11 and Figure 13As shown, ten purified components with different retention properties were finally obtained (e.g. Figure 13 As shown in Figure A), the antibacterial activity of these 10 components was determined, and the peptide content ratio was also measured. Component F10 had the highest peptide content ratio at 16.42%. Components F2 and F10 exhibited excellent antibacterial effects (e.g., ...). Figure 13 (As shown in BC), and exhibited excellent antibacterial activity. At a concentration with a MIC of 0.5 mg / mL, the F10 fraction showed inhibition rates of 91.81% and 92.06% against E. coli and S. aureus, respectively. Next, the F2 fraction showed inhibition rates of 92.12% and 92.52% against E. coli and S. aureus, respectively, at a concentration with a MIC of 1 mg / mL. The MICs for fractions F7-F9 were all 2 mg / mL.

[0158] The bactericidal effect of antimicrobial peptides in walnut meal was further evaluated using the colony-coating method (e.g., ...). Figure 14 As shown in the figure: at a concentration of 0.5 mg / mL, fraction F10 completely killed E. coli and S. aureus, while fraction F2 killed S. aureus but a small amount of E. coli remained alive. Therefore, fractions F2 and F10, prepared by RP-HPLC separation, have good antibacterial properties against E. coli and S. aureus.

[0159] Homology comparison analysis of different peptides was performed using the UniProt database. Subsequently, based on LC-MS / MS identification results, an antimicrobial peptide prediction tool was used to predict the activity of peptide sequences derived from walnut meal. The specific operational procedure is as follows:

[0160] Antimicrobial property prediction: Based on the Antimicrobial Peptide Database (APD) version 3 (http: / / aps.unmc.edu / AP / main.php, last accessed: November 2, 2024), the surface charge density and hydrophobicity parameters of each peptide were calculated.

[0161] Activity screening: Following the method of Heymich et al. (Generation of antimicrobial peptides Leg1 and Leg2 from chickpea storage protein, active against food spoilage bacteria and foodborne pathogens[J].FOOD CHEMISTRY,2021,347:10), candidate peptides with zero net charge and less than 20% hydrophobic amino acid content were eliminated.

[0162] Toxicity assessment: The ToxinPred 3.0 server (https: / / webs.iiitd.edu.in / raghava / toxinpred3 / index.html) was used to predict the toxicity of the initially screened peptides, and peptides with toxicity were screened out.

[0163] Solubility optimization: The water solubility of peptides was predicted using the PepCalc online tool (https: / / www.pepcalc.com / ), and potential aggregated peptides with solubility below the threshold were eliminated.

[0164] The results are as follows Figure 15 As shown, a total of 115 polypeptide sequences were detected in the F2 fraction, of which 32 (27.83%) were traceable to 11S walnut globulin (e.g., Figure 15 (As shown in A). A total of 752 peptides were identified from the F10 fraction of walnut meal antimicrobial peptides, of which 170 peptides originated from 11S walnut meal globulin, accounting for as high as 22.61% (e.g., Figure 15 (As shown in B). These results indicate that walnut meal globulin is an important source of antimicrobial peptides from walnut meal.

[0165] The antibacterial activity of antimicrobial peptides is mainly determined by their charge characteristics and hydrophobicity. During the screening process, peptides with a charge of 0 and a hydrophobic amino acid content of less than 20% were excluded. The peptides identified in F2 and F10 were classified according to their charge, predicted antimicrobial peptide results, and solubility. As shown in Table 12, 25 peptides were ultimately found to not only possess the potential to become antimicrobial peptides but also have excellent water solubility. One peptide with potential antimicrobial activity was screened from the walnut meal antimicrobial peptide F2 fraction. This peptide carries a positive charge and has a high proportion of hydrophobic amino acids (up to 23%). Among the peptides identified in the walnut meal antimicrobial peptide F10 fraction, a total of 24 peptides were identified. Of these, 13 were positively charged, with a net charge value as high as +2.5 and a hydrophobic amino acid content as high as 53%. Eleven peptides exhibited negative charges, with a minimum charge value of -2.5 and a hydrophobic amino acid content as high as 50%.

[0166] Table 12 Peptide profile analysis of antimicrobial peptides F2 and F10 components in walnut meal

[0167]

[0168]

[0169]

[0170] The target polypeptide sequence was modeled in three dimensions using ChemDraw 2D and ChemDraw 3D (Ultra version 14.0) software, and energy minimization was performed using a molecular mechanics optimization algorithm. Subsequently, the following operations were performed using AutoDockTools-1.5.7 software:

[0171] Dehydration treatment of polypeptide molecules and addition of hydrogen atoms;

[0172] Detect and correct the torsion angle of the rotatable key;

[0173] The pretreated peptide molecules are saved as pdbqt format files and used as ligand input files for molecular docking analysis.

[0174] The mechanism of action of antimicrobial peptides involves both membrane disruption and non-membrane disruption mechanisms. The cell membrane is the first barrier against bacteria; therefore, we first selected transmembrane target proteins (PDB: 1BY3 and 7AHL) on the surface of *E. coli* and *S. aureus* to evaluate the binding effect of antimicrobial peptides on these membrane target proteins, eliminating peptides with binding energies <5.5 kcal / mol. Further simulations of the effects of antimicrobial peptides on bacterial intracellular substances were conducted. Four representative bacterial intracellular target proteins of common antimicrobial drugs were screened from the RCSB protein database (https: / / www.rcsb.org / pdb). Specifically, these included DNA gyrase and dihydrofolate reductase (DHFR) of *E. coli* (PDB: 6RKS, 7REG) and DNA gyrase and DHFR of *S. aureus* (PDB: 2XCT, 3FYW), eliminating peptides with binding energies <7 kcal / mol. The target protein was preprocessed using the molecular visualization platform PyMOL: first, symmetrical repeating structures and non-covalently bound small molecule ligands were removed; then, dehydration and addition of polar hydrogen atoms were performed using AutoDockTools-1.5.7 software; finally, the preprocessed target protein was saved as a receptor molecule in pdbqt format. Molecular docking analysis was performed using Autodock Vina, with 20 docking repetitions. The optimal binding mode was screened by comparing the binding free energies of each conformation, and potential antimicrobial peptide sequences were screened based on an absolute binding energy ≤ 7.0 kcal / mol. Further analysis was conducted using PyMOL and LIGPLOT. + The software analyzes the molecular mechanism of action of antimicrobial peptides in walnut meal: PyMOL is used to visualize and analyze the binding pattern between the antimicrobial peptides and target proteins; LIGPLOT is used... + Two-dimensional interaction maps were generated to analyze the hydrogen bond network and hydrophobic interactions between the antimicrobial peptide and the active pocket of the target protein, and to identify the interaction sites of key amino acid residues.

[0175] The results are shown in Table 13. Four novel peptides with high potential inhibitory activity against E. coli and S. aureus were ultimately screened and named WAP-1 (SEQ ID NO:1: SNHDQRRGIVRVE), WAP-2 (SEQ ID NO:2: DDNIAGRVGAGPA), WAP-3 (SEQ ID NO:3: IRRGDIVAIPAGVAH), and WAP-4 (SEQ ID NO:4: IDLSNHANQLDRR). These four peptides were synthesized in a solid-phase manner, and their antibacterial effects were further determined and their antibacterial mechanisms were explored.

[0176] Table 13. Binding results of antimicrobial peptides from walnut meal with target proteins of E. coli and S. aureus.

[0177]

[0178] like Figure 16 As shown in the AD, the antibacterial effect of the WAP-2 peptide was superior to that of the WAP-1, WAP-3, and WAP-4 peptides. Under experimental conditions where the WAP-2 peptide concentration reached 5 mg / mL, its inhibition rates against *E. coli* and *S. aureus* reached 92.01% and 90.74%, respectively. Figure 16 As shown in Figure E, the antimicrobial peptide exhibits significantly superior bactericidal efficacy compared to other solid-phase synthetic peptides, demonstrating better antimicrobial activity. At a final concentration of 5 mg / mL, it achieved bactericidal effect against *S. aureus*, with no colony growth on the plate, while only a small number of colonies were observed on *E. coli* plates. The WAP-1 peptide showed good inhibitory effects against *S. aureus*, with an inhibition rate of 87.35% at 20 mg / mL, but a poor inhibitory effect on *E. coli*, with an inhibition rate of 72.74%. Experimental data indicate that the WAP-2 peptide exhibits excellent antimicrobial activity against both *E. coli* and *S. aureus*, with a minimum inhibitory concentration (MIC) of 5 mg / mL.

[0179] The results showed that WAP-2 peptide had a strong inhibitory effect on E. coli and S. aureus. This phenomenon is closely related to its high content of hydrophobic amino acids. The WAP-2 peptide contained 8% arginine (basic amino acid), 23% alanine and 23% glycine (hydrophobic amino acids), 8% proline, 8% valine, and 8% isoleucine. The presence of glycine and proline promoted the formation of helical twisted structures. These structures not only maintained the stability of the cell membrane surface pores but also promoted the rapid formation of membrane pores, thereby enhancing its destructive effect on bacterial cell walls or membrane structures. Based on this, it is speculated that the inhibitory effect of WAP-2 peptide on the growth of Escherichia coli and Staphylococcus aureus may stem from its disruption of the microbial cell membrane structure, penetrating the cell membrane to enter the cell interior, interacting with key enzymes, and thus interfering with the synthesis of genetic material and important components such as ATP, ultimately achieving its antibacterial activity.

[0180] The effects of WAP-2 peptide on the morphological appearance of pathogenic bacteria were determined. The intervention of this antimicrobial peptide on the cell membrane structure of two pathogenic bacteria was systematically observed using scanning electron microscopy. The results showed that untreated *E. coli* cells maintained typical rod-shaped morphological characteristics (e.g., ...). Figure 17 As shown in Figure A). Similarly, the S. aureus cells in the control group maintained a complete circular structure, and their cell wall surface was smooth and regular (as shown in Figure A). Figure 17 (As shown). After incubation with WAP-2 peptide at a concentration of 2MIC for 1 hour, obvious depressions appeared on the surface of the E. coli and S. aureus bacterial films after interference and disruption by WAP-2 peptide (as shown). Figure 17 (As shown in B and D).

[0181] The antibacterial mechanism of WAP-2 peptide was analyzed using molecular docking simulations, such as... Figure 18 As shown, it binds to bacterial target proteins (membrane proteins, DNA gyrase proteins, and dihydrofolate reductase proteins) through hydrogen bonds and hydrophobic interactions. The leakage of ATP and AKP caused by WAP-2 peptide at different concentrations was measured in pathogenic bacteria, such as... Figure 19 As shown in the AD diagram, the AKP content of untreated *Escherichia coli* and *Staphylococcus aureus* was 0.38 and 0.34 Kings / 100 mL, respectively. However, after treatment with 2 MIC of WAP-2 peptide, the AKP enzyme content was significantly higher than that of the control group, increasing to 1.39 and 2.19 Kings / 100 mL, respectively. This indicates that WAP-2 peptide affects cell wall biosynthesis, leading to cell death (P<0.001). The leakage of bacterial ATP also showed a concentration-dependent effect with WAP-2 peptide. Under treatment with 2 MIC of peptide, the ATPase leakage of *E. coli* and *S. aureus* increased to 1.20 and 2.35 μmol / 100 mL, respectively. 6(P < 0.001). In conclusion, WAP-2 peptide can damage the structure and integrity of the cell membrane, causing leakage of intracellular substances and leading to bacterial death.

[0182] This embodiment determined the effects of temperature, pH, salt ion concentration, and proteolytic enzymes on the antibacterial activity of WAP-2 peptide under different environmental conditions. The antimicrobial peptide WAP-2 maintained good inhibitory activity against E. coli and S. aureus under pH gradient changes (e.g., ...). Figure 20 As shown in Figure A), at pH 3, the retention rates for E. coli and S. aureus were 94.43% and 92.53%, respectively; at pH 11, the retention rate for E. coli was 80.68%, and the retention rate for S. aureus was 89.04%. The thermal stability of WAP-2 peptide is as follows: Figure 20 As shown in Figure B, at a high temperature of 121℃, the inhibition rates of E. coli and S. aureus remained at 90.42% and 95.61%, respectively. The presence of metal cations in the environmental medium (such as Na+) + Ca 2+ This may interfere with the specific binding of antimicrobial peptides to the cell membrane phospholipid bilayer through electrostatic shielding effects. For example... Figure 20 As shown in C, WAP-2 is affected by Ca 2+ The concentration of ions has a significant impact. When CaCl2 is 50 mM, the peptide's inhibition retention rate against E. coli is 76.86%. As CaCl2 concentration decreases, the inhibition rate decreases further. 2+ With increasing concentration, the antibacterial effect was reduced to only 50.99% at 200 mM, but the antibacterial effect against *S. aureus* was well preserved, remaining at 82.02% at 200 mM. Under treatment with 50–100 mM NaCl, the peptide's antibacterial retention rate remained above 86%. After treatment with 200 mM NaCl, the antibacterial activity of WAP-2 peptide against *E. coli* and *S. aureus* remained at 75.78% and 80.83%, respectively. The sensitivity of WAP-2 to high salt ion concentrations is due to the interaction between cations and the carbonyl groups of the peptide backbone, leading to the breakage of the hydrogen bond network, disruption of the secondary structure, and subsequent impact on antimicrobial activity. Therefore, when the addition amount of NaCl and CaCl2 is relatively low (50 mM), WAP-2 peptide exhibits good antimicrobial activity against NaCl. + Ca 2+ Ionic stability. The antibacterial stability of WAP-2 peptide was then assessed using enzymatic hydrolysis environments with different concentrations of proteases. After co-incubation with high concentrations (2 mg / mL) of trypsin, neutral protease, and pepsin, the retention rate of its antibacterial activity against E. coli was measured (e.g., ionic stability). Figure 20(As shown in D), the retention rates were 85.78%, 79.65%, and 89.21%, respectively; while the retention rates for S. aureus were 91.50%, 85.35%, and 94.20%, respectively. The experimental results indicate that the WAP-2 peptide maintains excellent antibacterial activity and stability under the above three protease treatment conditions.

[0183] In summary, this invention utilizes walnut meal, a high-protein industrial byproduct, as raw material and employs mixed-strain fermentation technology to prepare antimicrobial peptides with significant antibacterial effects and high stability. Simultaneously, the bactericidal and preservative effects of the walnut meal-derived antimicrobial peptides in a food system (tomato juice) were evaluated. Furthermore, through virtual screening combined with molecular docking technology, monomeric components with potentially high-efficiency antimicrobial activity were screened from a walnut meal antimicrobial peptide database. Further, combining scanning electron microscopy (SEM) characterization, microbial physicochemical property analysis, and molecular docking technology, the multi-target antimicrobial mechanism of the walnut meal antimicrobial peptides was explored in depth. The walnut meal antimicrobial peptides exhibit excellent antibacterial properties and resistance to environmental interference, opening up new directions for the development of natural food preservatives.

[0184] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A walnut meal antimicrobial peptide, characterized in that, The amino acid sequence of the walnut meal antimicrobial peptide is shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. An antibacterial composition, characterized in that, The antibacterial composition includes the walnut meal antibacterial peptide of claim 1.

3. A food preservative, characterized in that, The food preservative includes the walnut meal antimicrobial peptide as described in claim 1.

4. A method for preparing the walnut meal antimicrobial peptide of claim 1 by fermentation, characterized in that, The method includes the following steps: S1. Prepare a mixed bacterial suspension, wherein the mixed bacteria are Bacillus subtilis, Lactobacillus reuteri, Kluyveromyces martensii, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus plantarum, Bacillus natto, and Lactobacillus rhamnosus, and the mixed bacterial suspension is prepared by volume ratio of Bacillus subtilis 31-40 parts, Lactobacillus reuteri 11-20 parts, Kluyveromyces martensii 11-20 parts, Lactobacillus acidophilus 11-20 parts, Lactobacillus fermentum 1-10 parts, Lactobacillus plantarum 1-10 parts, Bacillus natto 1-10 parts, and Lactobacillus rhamnosus 1-10 parts; S2. After crushing the walnut meal, mix it thoroughly with distilled water to obtain a walnut meal suspension; S3. Inoculate the mixed bacterial suspension obtained in step S1 into the walnut meal suspension obtained in step S2, shake and mix to form a fermentation substrate, shake and culture the fermentation substrate to obtain a fermentation broth. The inoculation amount of mixed bacteria is 4.5~10.5%, the fermentation temperature is 34~40℃, and the fermentation time is 28~36h. S4. Centrifuge the fermentation broth obtained in step S3, filter the supernatant, and obtain the walnut meal antimicrobial peptide.

5. The method according to claim 4, characterized in that, The mixed bacterial suspension consisted of 40 parts by volume of Bacillus subtilis, 11 parts of Lactobacillus reuteri, 11 parts of Kluyveromyces marxi, 20 parts of Lactobacillus acidophilus, 10 parts of Lactobacillus fermentum, 1 part of Lactobacillus plantarum, 10 parts of Bacillus natto, and 1 part of Lactobacillus rhamnosus.

6. The method according to claim 4, characterized in that, In step S3, the inoculation amount of the mixed bacteria is 7.5%.

7. The method according to claim 4, characterized in that, In step S3, the fermentation temperature is 37.5℃ and the fermentation time is 32h.

8. The method according to claim 4, characterized in that, The method also includes a step of ultrafiltration of antimicrobial peptides from walnut meal.

9. The method according to claim 8, characterized in that, The ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 1~10 kDa.

10. The method according to claim 9, characterized in that, The ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of 3~10 kDa.

11. The method according to claim 8, characterized in that, The method also includes a step of separating the antimicrobial peptides from the ultrafiltered walnut meal by column chromatography.

12. The method according to claim 11, characterized in that, The column chromatography was ODS column chromatography.

13. The method according to claim 12, characterized in that, The mobile phase volume ratio for the ODS column chromatography was acetonitrile: ultrapure water = 2:

8.

14. The application of the walnut meal antimicrobial peptide as described in claim 1, the antimicrobial composition as described in claim 2, or the food preservative as described in claim 3 in food preservation.

Images