A strain of *Lactobacillus plantarum* producing antimicrobial peptides and its applications

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Lactobacillus plantarum* that produces antimicrobial peptides and its applications. The *Lactobacillus plantarum* is specifically *Lactobacillus plantarum* (…). Lactobacillus plants Lactobacillus plantarum AF-6, with accession number CGMCC No. 33716, is a product of this invention. It generates a variety of antimicrobial peptides with broad-spectrum antibacterial activity, inhibiting spoilage bacteria such as Escherichia coli, Bacillus subtilis, Salmonella, Micrococcus luteus, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus. This provides a more reliable and effective option for food safety preservation.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Lactobacillus plantarum* that produces antimicrobial peptides and its applications. Background Art

[0002] Food safety has become a global concern. Despite the rapid development of the food industry, food spoilage and pathogen contamination remain critical issues that urgently need to be addressed. Currently, chemical preservatives or antibiotics are commonly used to inhibit the growth of harmful microorganisms in food; however, long-term or excessive use may pose potential health risks and exacerbate bacterial resistance. Therefore, finding safe, effective, and non-toxic natural antimicrobial substances has become an important research direction in the fields of food safety and public health. Antimicrobial peptides, due to their broad-spectrum antimicrobial activity and unique mechanism of action, are widely considered ideal natural products to replace traditional preservatives and antibiotics.

[0003] Fermented foods are recognized as a treasure trove of microorganisms and are frequently used to isolate bacteria that produce antimicrobial peptides, especially GRAS-certified lactic acid bacteria. These bacteria, with their abundant resources, ease of cultivation, ability to synthesize antimicrobial peptides, and excellent probiotic and antibacterial properties, have become a major research hotspot in the field of antimicrobial peptide mining and development. However, traditional antimicrobial peptide mining techniques are typically time-consuming, labor-intensive, and inefficient. Therefore, supported by large-scale biological datasets, a virtual mining technique integrating mass spectrometry, machine learning, and molecular dynamics simulations has enabled the rapid prediction and identification of candidate antimicrobial peptides. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to discover lactic acid bacteria that possess both safety and antibacterial properties, and further analyze their antibacterial mechanisms, thereby providing a more reliable and effective option for safe preservation in the food industry.

[0005] To achieve its objective, the present invention employs the following technical solution:

[0006] One of the technical solutions provided by this invention is a strain of *Lactobacillus plantarum* capable of producing antimicrobial peptides, specifically *Lactobacillus plantarum* (…). Lactobacillus plantarum AF-6, this strain was deposited on March 5, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33716.

[0007] The *Lactobacillus plantarum* AF-6 provided by this invention was isolated from Shanxi aged vinegar. It is Gram-positive, and the colonies are milky white, smooth, with obvious protrusions and neat edges. Under scanning electron microscopy, it is rod-shaped and has no spores or flagella.

[0008] The second technical solution provided by this invention is the application of *Lactobacillus plantarum* AF-6 described in the first technical solution, particularly its application in antibacterial and / or preservative purposes; *Lactobacillus plantarum* AF-6 can inhibit putrefactive bacteria including *Escherichia coli*, *Bacillus subtilis*, *Salmonella*, *Micrococcus luteus*, *Pseudomonas aeruginosa*, *Bacillus cereus*, and *Staphylococcus aureus*.

[0009] The third technical solution provided by this invention is the application of Lactobacillus plantarum AF-6 in the production of antimicrobial peptides. This invention extracts multiple antimicrobial peptides from the fermentation broth of Lactobacillus plantarum AF-6, as detailed below:

[0010] AMP-112, amino acid sequence: AKLEEVGGVVTLK (SEQ ID NO.1);

[0011] AMP-175, amino acid sequence: VILQQQEEEEQTIGGIVIANNAK (SEQ ID NO.2);

[0012] AMP-270, amino acid sequence: SIMGVMSLGVGK (SEQ ID NO.3);

[0013] AMP-338, amino acid sequence: DATSVIADGQLITVDSR (SEQ ID NO.4);

[0014] AMP-365, amino acid sequence: AAVEEGFVAGGGTALINVIK (SEQ ID NO.5);

[0015] AMP-463, amino acid sequence: VIELPAGVEVSQAGEVVTVK (SEQ ID NO.6).

[0016] The fourth technical solution provided by the present invention is the application of the antimicrobial peptide described in the third technical solution, especially its application in antibacterial and / or preservative purposes.

[0017] The beneficial effects of this invention are:

[0018] (1) The plant lactobacillus AF-6 of the present invention has broad-spectrum antibacterial activity and can inhibit putrefactive bacteria such as Escherichia coli, Bacillus subtilis, Salmonella, Micrococcus luteus, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus.

[0019] (2) The Lactobacillus plantarum AF-6 of the present invention has good biosafety, does not contain active antibiotic resistance genes and virulence factor genes, is non-hemolytic and sensitive to a variety of antibiotics.

[0020] (3) The plant lactobacillus AF-6 of the present invention has good acid resistance and bile salt resistance.

[0021] (4) The peptides identified in Lactobacillus plantarum AF-6 of the present invention are mainly composed of short chain peptides with a length of 7 to 28 amino acids and a molecular weight between 955.16 and 2947.13 Da. More than 90% of the peptides have positively charged amino acids at the C-terminus, and 48.94% of the peptides contain hydrophobic amino acids, thus possessing strong antibacterial activity.

[0022] (5) The virtual screening strategy for peptides in Lactobacillus plantarum AF-6 of the present invention overcomes the limitations of traditional peptide screening methods that are time-consuming and labor-intensive, and fills an important gap in the current methods for extracting antimicrobial peptides from microbial sources. Attached Figure Description

[0023] Figure 1 The study demonstrated broad-spectrum antibacterial activity in four strains.

[0024] Figure 2 Colony morphology of Lactobacillus plantarum AF-6 was observed under a microscope (×100) and a scanning electron microscope (1 μm).

[0025] Figure 3 Phylogenetic tree of Lactobacillus plantarum AF-6.

[0026] Figure 4 Evaluation of the hemolytic activity of Lactobacillus plantarum AF-6.

[0027] Figure 5 The effect of extracting antimicrobial peptides from Lactobacillus plantarum AF-6 using ammonium sulfate with different saturations.

[0028] Figure 6 Basic information on all peptides in the fermentation extract of Lactobacillus plantarum AF-6

[0029] Among them, (A) amino acid length, (B) hydrophobicity distribution, (C) N-terminal amino acid residues, C-terminal amino acid residues distribution, and (D) molecular weight.

[0030] Figure 7 The structure predicted for AlphaFold2 (green) corresponds to the structure obtained from a MD simulation running for 100 ns (red).

[0031] Figure 8 The number of hydrogen bonds in the six peptides.

[0032] Figure 9 Rg represents the radius of gyration of the six peptides.

[0033] Figure 10 The variation of the root mean square deviation (RMSD) of the six peptides is shown.

[0034] The Lactobacillus plantarum provided by this invention ( Lactobacillus plantarum AF-6, this strain was deposited on March 5, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 33716. Detailed Implementation

[0035] The present invention will now be described through specific embodiments. Unless otherwise specified, all technical means used in this invention are methods well known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting, of the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the invention, are also within the scope of protection of this invention.

[0036] The present invention will be further described below with reference to specific embodiments.

[0037] The main culture media involved in the examples are as follows:

[0038] MRS broth medium: 10 g peptone, 5 g beef meal, 4 g yeast powder, 20 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, pH 6.2±0.2, add distilled water to a final volume of 1 L, sterilize at 115℃ for 30 min;

[0039] LB agar medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 20 g agar powder, adjust pH to 7.1±0.1, add distilled water to a final volume of 1 L, sterilize at 121℃ for 20 min;

[0040] The main indicator bacteria are as follows:

[0041] The standard indicator pathogens used for antibacterial activity assays include Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Salmonella typhimurium, as shown in Table 1.

[0042] Example 1: Screening and Identification of Antimicrobial Peptide-Producing Lactic Acid Bacteria

[0043] 1. The applicant previously isolated 202 strains of lactic acid bacteria from traditional Chinese pickles, chili sauce, and vinegar. The activated lactic acid bacteria strains were inoculated into MRS liquid medium and cultured at 37°C for 36 h. The supernatant was collected by centrifugation, and the antibacterial activity was determined by agar diffusion method.

[0044] The specific procedure is as follows: Add the standard indicator bacteria (1.0 × 10⁻⁶) 6 The mixture (CFU / mL, as shown in Table 1) was mixed with LB agar medium, and 200 μL of supernatant was added to the Oxford cup (8 mm in diameter). The mixture was then incubated at 37°C for 12 h, and the diameter of the inhibition zone was measured to assess the antibacterial activity.

[0045] A preliminary evaluation of the potential antibacterial activity of 202 lactic acid bacteria strains revealed that 4 strains exhibited significant broad-spectrum antibacterial activity (antibacterial results are shown in Table 2 and...). Figure 1 As shown in the figure, it can effectively inhibit the growth of a variety of Gram-positive and Gram-negative bacteria. Among them, the inhibition zone diameter of AF-1, AF-4 and AF-26 strains is between 15-20 mm for most indicator bacteria, and the inhibition zone diameter is less than 15 mm for some indicator bacteria. In contrast, the inhibition zone diameter of AF-6 strain is between 20-28 mm for most indicator bacteria, showing broad-spectrum and strong antibacterial ability. Therefore, strain AF-6 was selected as the subject of further research.

[0046] Table 1 Standard Indicator Bacteria

[0047]

[0048] Table 2 Broad-spectrum antibacterial activity of four strains

[0049]

[0050] 2. After screening out strain AF-6, which exhibited the best antibacterial effect, its antibacterial compounds were further analyzed. The pH of the fermentation supernatant of strain AF-6 was adjusted to 6.0 using sodium hydroxide to eliminate the potential antibacterial effect of organic acids. Subsequently, the fermentation supernatant was treated with catalase, trypsin, and pepsin, respectively, as follows:

[0051] Catalase treatment method: Catalase was added to the fermentation supernatant at a final concentration of 10 mg / mL to eliminate the influence of hydrogen peroxide;

[0052] Treatment of pepsin and trypsin: Pepsin and trypsin were added to the fermentation supernatant at a final concentration of 1 mg / mL. The pH of the supernatant was adjusted to the optimal pH of 2.0 for pepsin and 8.0 for trypsin. The fermentation broths treated with enzymes were incubated in a water bath at 37℃ for 2 h. Then the pH of the supernatant was adjusted back to the original pH value.

[0053] After the above treatment, the inhibition zone experiment was carried out using the same method as described above, with the untreated fermentation supernatant as a blank control. The results are shown in Table 3.

[0054] Table 3 shows that after pH adjustment or catalase treatment, the antibacterial activity of strain AF-6 decreased slightly but not significantly, indicating that organic acids have little effect on antibacterial activity and that hydrogen peroxide is not the main antibacterial component in the fermentation supernatant of strain AF-6. However, after treatment with trypsin or pepsin, the antibacterial activity decreased significantly. This suggests that the main antibacterial component in the fermentation supernatant of strain AF-6 is protein-like, and it is speculated that there may be protein-like antibacterial peptides in the fermentation supernatant of strain AF-6. Further analysis using protein precipitation and mass spectrometry will be conducted to clarify their properties.

[0055] Table 3. Analysis of antimicrobial substances in Lactobacillus plantarum AF-6

[0056]

[0057] 3. Based on the morphological characteristics and phylogenetic analysis of the strain, strain AF-6 was identified.

[0058] Strains AF-6 were cultured at 37°C for 36 h, and colony morphology was observed. Gram staining and scanning electron microscopy (SUM3800, Japan) were used to observe bacterial cell morphology. Species identification was further performed by 16S rRNA gene amplification and sequencing.

[0059] Morphological characteristics of strain AF-6 as follows Figure 2 As shown, the bacteria form smooth, round, white colonies. The strain is Gram-positive and appears rod-shaped under a scanning electron microscope, without spores or flagella. The 16S rRNA gene of strain AF-6 was amplified and sequenced, yielding a 1442 bp sequence fragment (SEQ ID NO.7).

[0060] SEQ ID NO.7:

[0061]

[0062] BLAST analysis showed that this strain was related to Lactobacillus plantarum The sequences are highly similar (≥99%), and a phylogenetic tree is constructed as follows: Figure 3 As shown. Therefore, based on its biochemical, morphological, and molecular characteristics, this strain was identified as Lactobacillus. Plantarum Therefore, it was named: Lactobacillus plantarum ( Lactobacillus plantarum AF-6.

[0063] Example 2: Safety and Probiotic Characteristics Analysis of Lactobacillus plantarum AF-6

[0064] 1. Genome sequencing

[0065] Genomic DNA was extracted and purified from *Lactobacillus plantarum* AF-6 cultured to the exponential growth stage using the PureLink® Genomic DNA Kit and QuickDNA Miniprep Plus Kit (Tiangen Biotech, China). Whole genome sequencing was performed using the PacBio Sequel II platform (Pacific Biosciences, USA).

[0066] The whole genome sequencing results of *Lactobacillus plantarum* AF-6 are shown in Table 4. Its genome size is 2,981,934 bp, with an average G+C content of 44.87%. A total of 2,892 ORFs were identified, with a total coding gene length of 2,504,751 bp and an average length of 866 bp, accounting for 84.00% of the total genome length. There are 65 tRNA genes, as well as 6 genes encoding 5S rRNA, 5 genes encoding 16S rRNA, and 5 genes encoding 23S rRNA.

[0067] According to VFDB analysis, the virulence-related sequences in the *Lactobacillus plantarum* AF-6 genome showed low homology (all similarities were below 77%) with known virulence genes in the VFDB database, and no high-confidence virulence factor genes were detected. CARD analysis showed that, after applying stringent screening criteria (coverage >60%, identity >90%), no known resistance genes were detected. Comprehensive analysis indicates that *Lactobacillus plantarum* AF-6 lacks active antibiotic resistance genes and virulence factor genes, further supporting its good safety profile.

[0068] Table 4. Genomic characteristics of Lactobacillus plantarum AF-6

[0069]

[0070] 2. Hemolytic

[0071] Activated cultures of *Lactobacillus plantarum* AF-6 strain were inoculated onto Columbia blood agar plates and incubated at 37°C for 24 hours. The presence of hemolytic zones around the colonies was observed. *Staphylococcus aureus* ATCC 25923, which produces β-hemolysis, was used as a positive control.

[0072] The results are as follows Figure 4 The results showed that Staphylococcus aureus exhibited significant β-hemolytic activity, while no such phenomenon was observed around Lactobacillus plantarum AF-6 colonies, indicating that this strain does not produce hemolysin, has no hemolytic ability, and is non-pathogenic.

[0073] 3. Antibiotic sensitivity

[0074] 100 μL of the fermentation supernatant of *Lactobacillus plantarum* AF-6 was spread onto MRS solid medium, and drug susceptibility test discs were attached to it. The medium was incubated at 37°C for 36 h, and the diameter of the inhibition zone was measured. Drug susceptibility was classified as resistant (R, ≤12 mm), moderately sensitive (MS, 12-17 mm), or sensitive (S, ≥17 mm).

[0075] The results are shown in Table 5. Lactobacillus plantarum AF-6 showed sensitivity or moderate sensitivity to a variety of antibiotics, including chloramphenicol, ceftriaxone, cefotaxime, nitrofurantoin, rifampin, ampicillin, erythromycin, tetracycline, and doxycycline. It also showed resistance to some antibiotics, including gentamicin, neomycin, vancomycin, and ofloxacin. However, the inhibition zone diameters of neomycin, ofloxacin, and gentamicin were 9.89 mm, 11.75 mm, and 10.58 mm, respectively, which were close to moderate sensitivity.

[0076] Table 5. Antibiotic susceptibility of Lactobacillus plantarum AF-6

[0077]

[0078] 4. Acid resistance analysis

[0079] Lactobacillus plantarum AF-6 was added to MRS liquid medium at pH 3.0 at a 2% inoculum size and cultured at 37°C for 3 hours. Untreated MRS liquid medium was used as a negative control. After stress, the OD of the bacterial culture under different conditions was measured. 600 The values ​​are compared to their growth status. The formula for calculating the survival rate of the strain is as follows:

[0080] Strain survival rate (%) = (OD of bacterial solution in acidic medium) 600 Value / OD of bacterial culture in blank medium 600 Value) × 100%

[0081] The calculation results show that after Lactobacillus plantarum AF-6 was cultured in MRS liquid medium at pH 3.0 for 3 h, the cell OD value of Lactobacillus plantarum AF-6 was significantly reduced.600 The value was 0.163, indicating that the bacterial OD in the blank culture medium was... 600 The value was 0.204, indicating a survival rate of 80%. Currently, most Lactobacillus plantarum strains have an acid resistance survival rate of 50%-80% under similar conditions, suggesting that this strain has good acid resistance.

[0082] 5. Bile salt tolerance analysis

[0083] Lactobacillus plantarum AF-6 was inoculated at a rate of 2% into MRS liquid medium containing 0.3% porcine bile salts and cultured at 37°C for 3 h. MRS liquid medium without bile salts was used as a negative control. After stress, the OD values ​​of the bacterial culture under different conditions were measured. 600 The values ​​are compared to their growth status. The formula for calculating the survival rate of the strain is as follows:

[0084] Strain survival rate (%) = (OD of bacterial solution in bile salt medium) 600 Value / OD of bacterial culture in blank medium 600 Value) × 100%

[0085] The calculation results showed that after Lactobacillus plantarum AF-6 was cultured in MRS liquid medium containing 0.3% porcine bile salts for 3 h, the bacterial OD value of Lactobacillus plantarum AF-6 was significantly reduced. 600 The value reached 0.618, while the bacterial OD in the blank culture medium was... 600 The value was only 0.204. The strain in the bile salt medium showed stronger survival ability, with a survival rate of 303%. It is believed that it can promote growth by utilizing bile salt in a short period of time, indicating that the strain has strong bile salt tolerance.

[0086] In summary, Lactobacillus plantarum AF-6 did not exhibit high-confidence virulence factor genes or known resistance genes, did not show hemolytic activity, was sensitive to multiple antibiotics, and had strong acid and bile salt resistance. It possesses good biosafety and probiotic properties, making it suitable for the discovery of antimicrobial peptides.

[0087] Example 3: Extraction and identification of potential antimicrobial peptides from Lactobacillus plantarum AF-6

[0088] 1. Extraction of antimicrobial peptides from Lactobacillus plantarum AF-6

[0089] Lactobacillus plantarum AF-6 was cultured for 36 h, and the cell-free supernatant was collected by centrifugation. The supernatant was then fractionated by precipitation with 60%-100% saturated ammonium sulfate, cultured overnight at 4°C, and the precipitate was collected by centrifugation. The precipitate was reconstituted with sterile water and its antibacterial activity against Escherichia coli was evaluated using the agar diffusion method mentioned above.

[0090] The results are as follows Figure 5As shown, the maximum diameter of the inhibition zone against Escherichia coli was observed in the precipitate with a saturation of 90%, indicating that ammonium sulfate with a saturation of 90% has the best extraction effect on antimicrobial peptides.

[0091] 2. LC-MS / MS analysis

[0092] The peptides precipitated with 90% ammonium sulfate were fully identified using LC-MS / MS. Peptide sequencing was performed using a nanoElute ultra-high performance liquid chromatography system and a hybrid timsTOF Pro2 mass spectrometer (Brook Dalton, Germany), equipped with a column (15 cm × 75 μm, 1.6 μm particle size) (IonOpticks, Australia). Mobile phases A and B were water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid, respectively, with gradient elution. Relevant parameters were analyzed using mass spectrometry. The global false detection rate (FDR) threshold for peptide identification at both protein and precursor ion levels was set to 0.01. All other parameters were set to their default values.

[0093] Based on LC-MS / MS sequencing and database search, a total of 481 peptides were identified, ranging in length from 7 to 28 amino acids. Figure 6 (Middle A), with a molecular weight between 955.1 and 2947.13 Da. Figure 6 (D). Most peptides (90.02%, 433) had molecular weights between 1 and 2 kDa, 6.44% (31) were less than 1 kDa, and 3.53% (17) were between 2 and 3 kDa. These results indicate that the peptides produced by *Lactobacillus plantarum* AF-6 are mainly short-chain peptides with molecular weights between 1 and 2 kDa. Further analysis of the amino acid composition of the identified peptides revealed the presence of 20 different amino acids, with over 90% of the peptides having a positively charged amino acid (Lys, Arg) at the C-terminus. Figure 6 (C). It is well known that this physicochemical property enhances electrostatic interactions with bacterial membranes, promoting membrane disruption and thus improving antibacterial activity. Furthermore, 48.94% of the peptides contain hydrophobic amino acids, including Ala, Ile, Leu, Val, Trp, Phe, Gly, Pro, and Met. Further analysis showed that 57.59% of the peptides consist of 40-60% hydrophobic residues. Figure 6 (B) Studies have shown that peptides rich in hydrophobic amino acids have stronger antibacterial activity. Therefore, the high proportion of hydrophobic amino acids in these peptides is likely to help improve the antibacterial activity of Lactobacillus plantarum AF-6 fermentation extract.

[0094] 3. Virtual screening of antimicrobial peptides

[0095] Three machine learning methods—random forest (RF), support vector machine (SVM), and artificial neural network (ANN)—were used to predict the 481 peptides identified by LC-MS / MS sequencing and database search. Peptides with a confidence level greater than 0.5 were considered potential antimicrobial peptides. The 3D structure of the peptides was predicted using AlphaFold2 and visualized using PyMOL. Then, a 100 ns MD simulation was performed using the AMBER99SB force field in GROMACS software. After the simulation, virtual screening of antimicrobial peptides was conducted based on trajectory analysis of root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond interactions.

[0096] After machine learning screening, 147 peptides met the criteria (prediction confidence greater than 0.5) and were selected for further analysis. AlphaFold2 was used to predict the structure and perform MD simulations on these 147 peptides. Of the predicted 3D structures, 55 peptides contained α-helical components, 6 showed β-sheet components, and 86 showed irregular coiled conformations. The structural stability of peptides containing intact α-helical or β-sheet regions was assessed through 100 ns MD simulations. Key indicators commonly used to assess peptide structural stability and conformational changes include RMSD, hydrogen bonds, and Rg. When the fluctuations in RMSD and Rg values ​​and the number of hydrogen bonds did not change significantly, it indicated that the system had stabilized. However, MD simulation results showed that most peptides exhibited high instability during the simulation, with significant fluctuations in RMSD, unstable Rg curves, and significant changes in the number of hydrogen bonds, exhibiting widespread conformational fluctuations and skeletal distortions. This indicates that their initial secondary structures were relatively unstable and tended to transition to a disordered state. Ultimately, as shown... Figure 7 , Figure 8 , Figure 9 as well as Figure 10 As shown in Table 6, six peptides with relatively stable RMSD, Rg, and hydrogen bond distributions and which fully or partially retained their secondary structures were selected as potential antimicrobial peptides (named AMP-112, AMP-175, AMP-270, AMP-338, AMP-365, and AMP-463).

[0097] Meanwhile, the physicochemical properties of the six peptides revealed that their molecular weights ranged from 1178.48 to 2524.81 Da, and their hydrophobicity ranged from 39% to 55%. Among them, AMP-270 was the shortest, containing 12 amino acid residues (SIMGVMSLGVGK) with a molecular weight of 1178.48 Da, while AMP-175 (VILQQQEEEEQTIGGIVIANNAK) was the longest, containing 23 residues. It has been reported that shorter peptides with smaller molecular weights are more likely to penetrate cell membranes to exert inhibitory effects and have low synthesis costs, which can provide convenience for application.

[0098] Sequence alignment analysis revealed that all identified peptides contained hydrophobic amino acid residues, with the main components being Val (18 residues, 27.27%), Gly (15 residues, 22.73%), Ala (12 residues, 18.18%), Ile (10 residues, 15.15%), and Leu (7 residues, 10.61%). Therefore, these peptides not only exhibit a high proportion of hydrophobic amino acid residues at the sequence level but also demonstrate significant structural stability in simulations, particularly by maintaining intact or partially preserved α-helical or β-sheet conformations, thus possessing potential antibacterial activity.

[0099] In addition, a comparison with the APD and DRAMP databases confirmed that the above six peptides are not disclosed in the prior art.

[0100] Table 6 Potential antimicrobial peptides and their sequences

[0101]

[0102] Example 5: Chemical Synthesis and Activity Verification of Potential AMPs

[0103] The peptides listed in Table 6 were chemically synthesized by Nanjing Taiyuan Biotechnology Co., Ltd. (Nanjing, China) using a solid-phase synthesis method. The purity (≥95%) of the synthesized peptides was confirmed by high-performance liquid chromatography (HPLC), and the molecular weight was determined by mass spectrometry (MS). The synthesized peptides were stored in powder form at -80°C. Before use, the peptide powder was resuspended in deionized water, and the antibacterial activity of the peptides against *Escherichia coli* and *Staphylococcus aureus* was verified using the agar diffusion method mentioned above (replacing the bacterial culture supernatant with the peptide solution, with a peptide concentration of 30 mg / mL).

[0104] The antibacterial results are shown in Table 7. All six potential antimicrobial peptides exhibited varying degrees of antimicrobial activity against *Escherichia coli*, but some showed no inhibitory effect against *Staphylococcus aureus*, suggesting they primarily act on Gram-negative bacteria. AMP-338, AMP-175, and AMP-365 showed stronger antimicrobial activity compared to the other three peptides, with AMP-338 exhibiting the best antimicrobial effect, inhibiting bacteria up to 17.58 mm in diameter. In conclusion, *Lactobacillus plantarum* AF-6 can produce antimicrobial peptides, and this strain primarily exerts its antimicrobial effect through these peptides.

[0105] Table 7. Antimicrobial activity of six antimicrobial peptides against Escherichia coli and Staphylococcus aureus

[0106]

[0107] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various changes, modifications, substitutions and variations in form and detail to these embodiments without departing from the spirit and principles of the present invention. The scope of the present invention is defined by the claims and their equivalents.

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, Specifically, Lactobacillus plantarum ( Lactobacillus plantarum AF-6, accession number: CGMCC No. 33716.

2. The application of Lactobacillus plantarum AF-6 according to claim 1, characterized in that, This is for use in the preparation of antibacterial and / or preservative products; the product has an inhibitory effect on Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Staphylococcus capitulata, Bacillus cereus, Salmonella typhimurium, Pseudomonas aeruginosa, Serratia marcescens, and Clostridium perfringens.

3. The antimicrobial peptide produced from *Lactobacillus plantarum* AF-6 according to claim 1, characterized in that, Specifically as follows: AMP-338, amino acid sequence: DATSVIADGQLITVDSR, SEQ ID NO.

4.

4. The use of the antimicrobial peptide according to claim 3 in the preparation of antibacterial and / or preservative products, characterized in that, The antimicrobial peptide has an inhibitory effect on Escherichia coli and Staphylococcus aureus.

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