Lactobacillus plantarum for producing antibacterial peptide and application of lactobacillus plantarum

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum for producing antibacterial peptide and application of the lactobacillus plantarum. The lactobacillus plantarum is specifically lactobacillus plantarum AF-6, and the preservation number of the lactobacillus plantarum AF-6 is CGMCC (China General Microbiological Culture Collection Center) No. 33716. The lactobacillus plantarum AF-6 disclosed by the invention can generate various antibacterial peptides, has broad-spectrum antibacterial activity, can play a role in inhibiting putrefying bacteria such as escherichia coli, bacillus subtilis, salmonella, micrococcus luteus, pseudomonas aeruginosa, bacillus cereus and staphylococcus aureus, and can provide a more stable and effective choice for safety and preservation of food industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a plant lactobacillus strain producing antimicrobial peptides and application thereof. Background Art

[0002] Food safety has become a global concern. With the rapid development of the food industry, food spoilage and pathogenic contamination remain key issues that need to be addressed. Currently, chemical preservatives or antibiotics are often used to inhibit the growth of harmful microorganisms in food. However, long-term or excessive use may pose potential hazards to human health and exacerbate the development of bacterial resistance. Therefore, the search for safe, highly effective, and non-toxic natural antimicrobial substances has become an important research direction in the current fields of food safety and public health. Antimicrobial peptides, due to their broad-spectrum antimicrobial activity and unique mechanism of action, are widely considered to be 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. Lactic acid bacteria, in particular, are certified as GRAS (Gradually Antimicrobial-Assured) bacteria. Their abundance, ease of cultivation, ability to synthesize antimicrobial peptides, and excellent probiotic and antimicrobial properties have made them a key research focus in the field of antimicrobial peptide mining and development. However, traditional antimicrobial peptide mining techniques are often time-consuming, labor-intensive, and inefficient. Therefore, with the support of 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] In order to solve the above technical problems, the purpose of the present invention is to discover lactic acid bacteria that are both safe and antibacterial, and further analyze their antibacterial mechanisms, so as to provide a more reliable and effective option for safe preservation in the food industry.

[0005] In order to achieve its purpose, the present invention adopts the following technical solutions: One of the technical solutions provided by the present invention is a strain of Lactobacillus plantarum that can produce antimicrobial peptides, wherein the Lactobacillus plantarum is specifically Lactobacillus plantarum ( Lactobacillus plantarum ) AF-6, which was deposited on March 5, 2025 at the General Microbiology Center of China Culture Collection Administration (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 33716.

[0006] The plant lactobacillus AF-6 provided by the present invention is isolated from Shanxi aged vinegar, is Gram-positive, has milky white colonies with smooth surfaces, obvious protrusions, and neat edges, and is rod-shaped under a scanning electron microscope without spores or flagella.

[0007] The second technical solution provided by the present invention is the application of the Lactobacillus plantarum AF-6 described in the first technical solution, especially in antibacterial and / or antiseptic applications; the Lactobacillus plantarum AF-6 can inhibit spoilage bacteria including Escherichia coli, Bacillus subtilis, Salmonella, Micrococcus luteus, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus.

[0008] The third technical solution provided by the present invention is the use of Lactobacillus plantarum AF-6 in the production of antimicrobial peptides. The present invention extracts multiple antimicrobial peptides from the fermentation broth of Lactobacillus plantarum AF-6, as follows: AMP-112, amino acid sequence: AKLEEVGGVVTLK (SEQ ID NO. 1); AMP-175, amino acid sequence: VILQQQEEEEQTIGGIVIANNAK (SEQ ID NO. 2); AMP-270, amino acid sequence: SIMGVMSLGVGK (SEQ ID NO. 3); AMP-338, amino acid sequence: DATSVIADGQLITVDSR (SEQ ID NO. 4); AMP-365, amino acid sequence: AAVEEGFVAGGGTALINVIK (SEQ ID NO. 5); AMP-463, amino acid sequence: VIELPAGVEVSQAGEVVTVK (SEQ ID NO. 6).

[0009] The fourth technical solution provided by the present invention is the application of the antimicrobial peptide described in the third technical solution, especially in antibacterial and / or antiseptic applications.

[0010] Beneficial effects of the present invention: (1) The plant lactobacillus AF-6 of the present invention has a broad-spectrum antibacterial activity and can inhibit spoilage bacteria such as Escherichia coli, Bacillus subtilis, Salmonella, Micrococcus luteus, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus.

[0011] (2) The plant lactobacillus AF-6 of the present invention has good biosafety, does not contain active antibiotic resistance genes and virulence factor genes, is non-hemolytic and is sensitive to multiple antibiotics.

[0012] (3) The plant lactobacillus AF-6 of the present invention has good acid and bile salt resistance.

[0013] (4) The peptides identified in the 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, which have strong antibacterial activity.

[0014] (5) The virtual screening strategy for peptides in Lactobacillus plantarum AF-6 of the present invention overcomes the time-consuming and labor-intensive limitations of traditional peptide screening methods, and fills an important gap in the current methods for extracting antimicrobial peptides from microbial sources. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It has broad-spectrum antibacterial activity against 4 strains.

[0016] Figure 2 Colony morphology, microscopic (×100) and scanning electron microscopic (1 μm) observations of Lactobacillus plantarum AF-6.

[0017] Figure 3 This is the phylogenetic tree of Lactobacillus plantarum AF-6.

[0018] Figure 4 To evaluate the hemolytic activity of Lactobacillus plantarum AF-6.

[0019] Figure 5 The results show the extraction effect of antimicrobial peptides from Lactobacillus plantarum AF-6 after ammonium sulfate with different saturations.

[0020] Figure 6 The basic information of all peptides in Lactobacillus plantarum AF-6 fermentation extract Among them, (A) amino acid length, (B) hydrophobicity distribution, (C) N-terminal amino acid residue, C-terminal amino acid residue distribution, (D) molecular weight.

[0021] Figure 7 The predicted structure for AlphaFold2 (green) corresponds to the structure obtained from a 100 ns MD simulation (red).

[0022] Figure 8 is the number of hydrogen bonds of the six peptides.

[0023] Figure 9 is the radius of gyration Rg of the six peptides.

[0024] Figure 10 is the change of root mean square deviation (RMSD) of six peptides.

[0025] The present invention provides plant lactobacillus ( Lactobacillus plantarum) AF-6, which was deposited on March 5, 2025 at the General Microbiology Center of China Culture Collection Administration (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No. 33716. DETAILED DESCRIPTION

[0026] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the embodiments are to be understood as illustrative rather than limiting the scope of the present invention, and the spirit and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material components and dosages in these embodiments, without departing from the spirit and scope of the present invention, also fall within the scope of protection of the present invention.

[0027] The present invention will be further described below with reference to specific embodiments. The main culture medium involved in the embodiment is as follows: MRS broth medium: 10 g peptone, 5 g beef powder, 4 g yeast powder, 20 g glucose, 1 mL Tween 80, 2 g potassium dihydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate tribasic, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, pH 6.2 ± 0.2, dilute to 1 L with distilled water, and sterilize at 115°C for 30 min. LB agar medium: 10 g tryptone, 10 g sodium chloride, 5 g yeast extract, 20 g agar powder, adjust the pH to 7.1 ± 0.1, add distilled water to 1 L, and sterilize at 121°C for 20 min. The main indicator bacteria are as follows: The standard indicator pathogens used for antibacterial activity determination include Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium, etc., as shown in Table 1.

[0028] Example 1 Screening and identification of antimicrobial peptide-producing lactic acid bacteria 1. The applicant previously isolated 202 lactic acid bacteria strains from traditional Chinese pickles, chili sauce, and vinegar. These activated lactic acid bacteria strains were inoculated into MRS liquid culture medium and cultured at 37°C for 36 hours. The supernatant was collected by centrifugation and the antibacterial activity was determined using the agar diffusion method.

[0029] The specific operation is as follows: standard indicator bacteria (1.0 × 10 6 CFU / mL, as shown in Table 1) was mixed with LB agar medium, punched with an Oxford cup (8 mm in diameter), 200 μL of supernatant was added, and the cells were cultured at 37°C for 12 h. The diameter of the inhibition zone was measured to evaluate the antibacterial activity.

[0030] After preliminary evaluation of the antibacterial activity of 202 potential lactic acid bacteria strains, it was found that 4 strains showed obvious broad-spectrum antibacterial activity (the antibacterial results are shown in Table 2 and Figure 1 As shown in Figure 2 ), strains AF-1, AF-4, and AF-26 exhibited inhibition zones with diameters ranging from 15 to 20 mm against most indicator bacteria, with some exhibiting inhibition zones with diameters below 15 mm for some. In contrast, strain AF-6 exhibited inhibition zones with diameters ranging from 20 to 28 mm against most indicator bacteria, demonstrating broad-spectrum and potent antibacterial activity. Therefore, strain AF-6 was selected for further study.

[0031] Table 1 Standard indicator bacteria

[0032] Table 2 Broad-spectrum antibacterial activity of four strains

[0033] 2. After identifying the strain AF-6 with the best antibacterial activity, further analysis of its antimicrobial compounds was performed. The pH of the fermentation supernatant of strain AF-6 was adjusted to 6.0 using sodium hydroxide to eliminate the potential antimicrobial effects of organic acids. Subsequently, the fermentation supernatant was treated with catalase, trypsin, and pepsin, respectively, as follows: Catalase treatment: catalase was added to the fermentation supernatant at a final concentration of 10 mg / mL to eliminate the influence of hydrogen peroxide; Pepsin and trypsin treatment: Pepsin and trypsin were added to the fermentation supernatant at a final concentration of 1 mg / mL, respectively. The pH of the supernatant was adjusted to the optimal pH of 2.0 for pepsin and 8.0 for trypsin, respectively. The fermentation broths treated with enzymes were incubated in a 37°C water bath for 2 h, and then the pH of the supernatant was adjusted to the original pH value. After the above treatment, the inhibition zone test was performed using the same method as above, with the untreated fermentation supernatant as the blank control. The results are shown in Table 3.

[0034] As shown in Table 3, the antibacterial activity of strain AF-6 decreased slightly, but not significantly, after pH adjustment or catalase treatment, demonstrating that organic acids have a minimal impact on antibacterial activity and that hydrogen peroxide is not the primary antibacterial component in the fermentation supernatant of strain AF-6. However, treatment with trypsin or pepsin significantly decreased the antibacterial activity, suggesting that the primary antibacterial component in the fermentation supernatant of strain AF-6 is proteinaceous. It is speculated that protein-based antibacterial peptides may be present in the fermentation supernatant of strain AF-6. Their properties will be further clarified through protein precipitation and mass spectrometry.

[0035] Table 3 Analysis of antimicrobial substances of Lactobacillus plantarum AF-6

[0036] 3. Based on the morphological characteristics and phylogenetic analysis of the strain, strain AF-6 was identified.

[0037] Strain AF-6 was cultured at 37°C for 36 hours, and colony morphology was observed. Cell morphology was observed using Gram staining and a scanning electron microscope (SUM3800, Japan). The strain was further identified by 16S rRNA gene amplification and sequencing.

[0038] The morphological characteristics of strain AF-6 are as follows Figure 2 As shown, the strain appears as round, white colonies with smooth edges. It is Gram-positive and exhibits a rod-shaped morphology 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).

[0039] SEQ ID NO.7: BLAST analysis showed that the strain Lactobacillus. plantarum The sequences of Figure 3 Therefore, based on its biochemical, morphological and molecular characteristics, the strain was identified as Lactobacillus. Plantarum , hence the name: Lactobacillus plantarum ( Lactobacillus plantarum )AF-6.

[0040] Example 2 Analysis of the safety and probiotic properties of Lactobacillus plantarum AF-6 1. Genome Sequencing Genomic DNA from Lactobacillus plantarum AF-6 cultured to exponential phase was extracted and purified using the PureLink® Genomic DNA Kit and QuickDNA Miniprep Plus Kit (Tiangen, China). Whole-genome sequencing was performed using the Pacific Biosciences Sequel II platform (PacBio, USA).

[0041] 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, encoding genes totaling 2,504,751 bp in length, with an average length of 866 bp, accounting for 84.00% of the entire genome length. There are 65 tRNA genes, as well as six genes encoding 5S rRNA, five genes encoding 16S rRNA, and five genes encoding 23S rRNA.

[0042] VFDB analysis revealed that virulence-related sequences in the Lactobacillus plantarum AF-6 genome shared low homology with known virulence genes in the VFDB database (similarity below 77%), and no high-confidence virulence factor genes were detected. CARD analysis revealed that no known resistance genes were detected after applying stringent screening criteria (coverage >60% and identity >90%). Comprehensive analysis demonstrated the absence of active antibiotic resistance genes and virulence factor genes in Lactobacillus plantarum AF-6, further supporting its favorable safety profile.

[0043] Table 4 Genomic characteristics of Lactobacillus plantarum AF-6

[0044] 2. Hemolytic An activated culture of Lactobacillus plantarum AF-6 was inoculated onto Columbia blood agar plates and incubated at 37°C for 24 hours. The colonies were observed for the presence of hemolytic zones. Staphylococcus aureus ATCC 25923, which produces β-hemolysis, was used as a positive control.

[0045] The results are as follows Figure 4 The results showed that Staphylococcus aureus exhibited obvious β-hemolysis, while this phenomenon did not occur around the colonies of Lactobacillus plantarum AF-6, indicating that this strain did not produce hemolysin, had no hemolytic ability, and was non-pathogenic.

[0046] 3. Antibiotic sensitivity 100 μL of the fermentation supernatant of Lactobacillus plantarum AF-6 was spread onto MRS solid medium, onto which a drug-susceptibility paper was attached. The culture was incubated at 37°C for 36 hours, and the diameter of the inhibition zone was measured. The drug susceptibility was classified as resistant (R, ≤12 mm), moderately sensitive (MS, 12-17 mm), or sensitive (S, ≥17 mm).

[0047] The results are shown in Table 5. Lactobacillus plantarum AF-6 was sensitive or moderately sensitive to multiple antibiotics, including chloramphenicol, ceftriaxone, cefotaxime, nitrofurantoin, rifampicin, ampicillin, erythromycin, tetracycline, and doxycycline. It was resistant 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.

[0048] Table 5 Antibiotic susceptibility of Lactobacillus plantarum AF-6

[0049] 4. Acid resistance analysis Lactobacillus plantarum AF-6 was added to MRS liquid medium with a pH of 3.0 at a 2% inoculum volume and cultured at 37°C for 3 h. Untreated MRS liquid medium was used as a negative control. After stress, the OD of the bacterial solution under different conditions was measured. 600 The survival rate of the strain was calculated by the following formula: Strain survival rate (%) = (OD of bacterial solution in acid culture medium) 600 Value / OD value of bacterial solution of blank culture medium 600 value) × 100% The calculation results showed that the OD value of Lactobacillus plantarum AF-6 was 0.04 after being cultured in MRS liquid medium with pH 3.0 for 3 h. 600 The value is 0.163, and the bacterial OD in the blank culture medium 600 The value is 0.204, the strain survival rate is 80%, and currently the acid-resistant survival rate of most Lactobacillus plantarum strains under similar conditions is mostly between 50% and 80%, indicating that the strain has good acid resistance.

[0050] 5. Bile Salt Tolerance Analysis Lactobacillus plantarum AF-6 was added to MRS liquid medium containing 0.3% porcine bile salt at a 2% inoculum volume and cultured at 37°C for 3 h. MRS liquid medium without bile salt was used as a negative control. After stress, the OD of the bacterial solution under different conditions was measured. 600 The survival rate of the strain was calculated by the following formula: Strain survival rate (%) = (OD of bacterial solution in bile salt medium) 600 Value / OD value of bacterial solution of blank culture medium 600 value) × 100% The calculation results showed that the OD of Lactobacillus plantarum AF-6 was 0.3% after being cultured in MRS liquid medium containing 0.3% pig bile salt for 3 h. 600 The value reached 0.618, while the OD of the bacteria in the blank culture medium was 600 The value only reached 0.204. The strain in the bile salt-containing medium showed a stronger survival ability, with a survival rate of 303%. It is believed that it can promote growth by utilizing bile salts in a short period of time, indicating that the strain has a strong bile salt tolerance.

[0051] In summary, no high-confidence virulence factor genes and known resistance genes were detected in Lactobacillus plantarum AF-6, and it did not show hemolytic activity. It was sensitive to multiple antibiotics and had strong acid and bile salt resistance. It had good biosafety and probiotic properties and was suitable for the discovery of antimicrobial peptides.

[0052] Example 3 Extraction and identification of potential antimicrobial peptides from Lactobacillus plantarum AF-6 1. Extraction of antimicrobial peptides from Lactobacillus plantarum AF-6 Lactobacillus plantarum AF-6 was cultured for 36 h, and the cell-free supernatant was collected by centrifugation and fractionated by 60%-100% saturation ammonium sulfate precipitation. The supernatant was 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.

[0053] The results are as follows Figure 5 As shown in the figure, 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% had the best extraction effect on antimicrobial peptides.

[0054] 2. LC-MS / MS Analysis Comprehensive identification of peptides from the aforementioned ammonium sulfate precipitation at 90% saturation was performed using LC-MS / MS analysis. Peptide sequencing was performed using a nanoElute ultra-high-performance liquid chromatography system coupled to a hybrid timsTOF Pro2 mass spectrometer (Bruker Daltonics, Germany) equipped with a 15 cm × 75 μm column (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. Mass spectrometry parameters were analyzed. A global false discovery rate (FDR) threshold of 0.01 was set for peptide identification at the protein and precursor ion levels. All other parameters were set to default values.

[0055] 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 A), with molecular weights ranging from 955.1 to 2947.13 Da ( Figure 6 The majority of 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 primarily 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 more than 90% of the peptides possessing positively charged amino acids (Lys, Arg) at the C-terminus ( Figure 6 C). This physicochemical property is known to enhance electrostatic interactions with bacterial membranes, promoting membrane disruption and thus improving antibacterial activity. Furthermore, 48.94% of the peptides contained hydrophobic amino acids, including Ala, Ile, Leu, Val, Trp, Phe, Gly, Pro, and Met. Further analysis revealed that 57.59% of the peptides were composed 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 likely contributes to the antibacterial activity of Lactobacillus plantarum AF-6 fermentation extract.

[0056] 3. Virtual Screening of Antimicrobial Peptides 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 searching. Peptides with a confidence score greater than 0.5 were considered potential antimicrobial peptides. The three-dimensional structures of the peptides were predicted using AlphaFold2 and visualized using PyMOL. MD simulations were then performed for 100 ns using the AMBER99SB force field in GROMACS software. After the simulations, virtual screening of antimicrobial peptides was performed based on trajectory analysis of root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond interactions.

[0057] After machine learning screening, a total of 147 peptides met the criteria, that is, the prediction confidence was greater than 0.5, and were selected for further analysis. AlphaFold2 was used to perform structural prediction and MD simulation on the 147 peptides. In the predicted three-dimensional structures, 55 peptides contained α-helical components, 6 showed β-fold components, and 86 showed irregular coiled conformations. The structural stability of peptides containing complete α-helical or β-fold regions was evaluated through 100ns MD simulation. Among them, the key indicators commonly used to evaluate the structural stability and conformational changes of polypeptides include RMSD, hydrogen bonds and Rg. When the fluctuation amplitude of the RMSD value and the Rg value and the number of hydrogen bonds do not change significantly, it indicates that the system has stabilized. The MD simulation results found that most peptides showed a high degree of instability during the simulation, with obvious RMSD fluctuations, unstable Rg curves, and significant changes in the number of hydrogen bonds. There were extensive conformational fluctuations and backbone distortions, indicating that their initial secondary structures were relatively unstable and tended to transition to a disordered state. Finally, as Figure 7 、 Figure 8 、 Figure 9 as well as Figure 10 As shown, six peptides with relatively stable fluctuation ranges of RMSD, Rg and hydrogen bond distribution and fully or partially retained secondary structures were selected and identified as potential antimicrobial peptides (named AMP-112, AMP-175, AMP-270, AMP-338, AMP-365 and AMP-463), and their sequences are shown in Table 6.

[0058] The physicochemical properties of the six peptides revealed molecular weights ranging from 1178.48 to 2524.81 Da, with hydrophobicity rates ranging from 39% to 55%. AMP-270 is the shortest, containing 12 amino acid residues (SIMGVMSLGVGK) with a molecular weight of 1178.48 Da, while AMP-175 (VILQQQEEEEQTIGGIVIANNAK) is 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 are inexpensive to synthesize, facilitating their application.

[0059] Sequence alignment analysis showed that all identified peptides contained hydrophobic amino acid residues, with the major 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 exhibit significant structural stability in simulations, particularly by maintaining complete or partial α-helical or β-sheet conformations, thus possessing potential antibacterial activity.

[0060] In addition, after comparison with the APD and DRAMP databases, it was confirmed that the above 6 polypeptides were not disclosed in the prior art.

[0061] Table 6 Potential antimicrobial peptides and their sequences

[0062] Example 5 Chemical synthesis and activity verification of potential AMP 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 of the synthesized peptides (≥95%) was confirmed by high-performance liquid chromatography, and the molecular weight was determined by mass spectrometry. The synthesized peptides were stored as powders at -80°C. Before use, the peptide powders were resuspended in deionized water and the antibacterial activity against Escherichia coli and Staphylococcus aureus was verified using the agar diffusion method described above (the culture supernatant was replaced with the peptide solution at a peptide concentration of 30 mg / mL).

[0063] The antibacterial results are shown in Table 7. All six potential antimicrobial peptides exhibited varying degrees of antimicrobial activity against Escherichia coli. However, some showed no inhibitory effect against Staphylococcus aureus, suggesting that they primarily target Gram-negative bacteria. AMP-338, AMP-175, and AMP-365 exhibited stronger antimicrobial activity than the other three antimicrobial peptides, with AMP-338 demonstrating the greatest inhibitory effect, reaching an inhibitory diameter of 17.58 mm. In summary, Lactobacillus plantarum AF-6 can produce antimicrobial peptides, and this strain primarily exerts its antimicrobial activity through these peptides.

[0064] Table 7 Antibacterial activity of six antimicrobial peptides against Escherichia coli and Staphylococcus aureus

[0065] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes, modifications, substitutions and variations in form and details 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 use of Lactobacillus plantarum AF-6 according to claim 1, characterized in that It is used in antibacterial and / or antiseptic applications.

3. The use according to claim 2, characterized in that The plant lactobacillus AF-6 has an inhibitory effect on spoilage bacteria including Escherichia coli, Bacillus subtilis, Salmonella, Micrococcus luteus, Pseudomonas aeruginosa, Bacillus cereus and Staphylococcus aureus.

4. The antimicrobial peptide produced by the Lactobacillus plantarum AF-6 according to claim 1, characterized in that The details are as follows: AMP-112, amino acid sequence: AKLEEVGGVVTLK, shown in SEQ ID NO. 1; AMP-175, amino acid sequence: VILQQQEEEEQTIGGIVIANNAK, shown in SEQ ID NO. 2; AMP-270, amino acid sequence: SIMGVMSLGVGK, shown in SEQ ID NO. 3; AMP-338, amino acid sequence: DATSVIADGQLITVDSR, shown in SEQ ID NO. 4; AMP-365, amino acid sequence: AAVEEGFVAGGGTALINVIK, as shown in SEQ ID NO. 5; AMP-463, the amino acid sequence is: VIELPAGVEVSQAGEVVTVK, as shown in SEQ ID NO.

6.

5. Use of the antimicrobial peptide according to claim 4 in antibacterial and / or antiseptic activities.

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