Marine plant lactobacillus rhamnosus antibacterial peptide amp-ouc329 and application thereof

Abstract

The application provides an antibacterial peptide AMP-OUC329 of marine plant Lactobacillus and application thereof, the antibacterial peptide is derived from marine plant Lactobacillus Bac-329, the amino acid sequence is VSGFETLDEDAKKLSNN, the molecular weight is 1866.5 Da, and the antibacterial peptide has thermal stability and acid stability.The application extracts the metabolite of Bac-329 strain by culture, obtains the crude extract of antibacterial peptide, uses the antibacterial activity of water product spoilage bacteria Shewanella and the damage of bacterial microstructure as directional screening index, separates by Sephadex G-25 gel filtration chromatography and TSKgel G2000SWxl reverse phase high performance liquid chromatography, optimizes the separation and purification conditions of antibacterial peptide, and the obtained antibacterial peptide has strong inhibition activity on Shewanella and precise target.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, specifically relating to an antimicrobial peptide AMP-OUC329 from the marine plant Lactobacillus and its applications. Background Technology

[0002] Contamination by spoilage bacteria during the storage and transportation of aquatic products has long been a technical challenge. This not only leads to a significant decline in the sensory quality and nutritional value of the products but can also trigger the proliferation of foodborne pathogens, posing serious food safety risks. Currently, chemical preservatives are widely used in industry to inhibit microbial growth and reproduction. However, these chemical preservatives bring potential health risks such as drug resistance and can no longer meet the modern consumer demand for natural and safe foods. Therefore, developing efficient and non-toxic biological preservatives has become an important research direction in the field of food science.

[0003] Antimicrobial peptides, as a class of antimicrobial peptides produced by microbial metabolism, possess significant advantages such as high efficiency in antibacterial activity, safety and non-toxicity, and easy degradation by proteases, showing broad application prospects in the field of aquatic product preservation. Studies have shown that antimicrobial peptides exert their antibacterial effects through multiple mechanisms, including disrupting cell membrane integrity, inhibiting protein synthesis, and interfering with DNA replication, and are less likely to induce bacterial resistance. *Lactobacillus lactis* Bac-329, derived from the intestines of cold-water marine fish, exhibits strong low-temperature resistance and rapid growth under low-temperature conditions. Its antimicrobial peptides differ significantly from existing lactic acid bacteria bacteriocins in molecular structure and antibacterial mechanism. Marine microbial antimicrobial peptides provide an important research direction and application potential for the development of novel marine microbial-derived biopreservatives. Summary of the Invention

[0004] The technical problem to be solved: The purpose of this invention is to provide an antimicrobial peptide AMP-OUC329 from *Lactiplantibacillus* and its application in the control of *Shewanella*, a dominant spoilage bacterium in aquatic products. This antimicrobial peptide is derived from *Lactiplantibacillus* Bac-329, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 28326, classified as *Lactiplantibacillus plantarum*, with a deposit date of September 1, 2023. Its amino acid sequence is Val-Ser-Gly-Phe-Glu-Thr-Leu-Asp-Glu-Asp-Ala-Lys-Lys-Leu-Ser-Asn-Asn, with a molecular weight of 1866.5 Da, and it exhibits both thermal and acid stability. This invention obtains a crude extract of antimicrobial peptides by culturing Bac-329 strain and extracting its metabolites. Using the antimicrobial activity against Shewanella, a spoilage bacterium of aquatic products, and the damage to the bacterial microstructure as targeted screening indicators, the antimicrobial peptides are separated by Sephadex G-25 gel filtration chromatography and TSKgel G2000SWxl reversed-phase high-performance liquid chromatography. The separation and purification conditions of the antimicrobial peptides are optimized, and the obtained antimicrobial peptides have strong inhibitory activity against Shewanella and precise target.

[0005] Technical solution: A marine plant Lactobacillus antimicrobial peptide AMP-OUC329, wherein the amino acid sequence of the antimicrobial peptide AMP-OUC329 is VSGFETLDEDAKKLSNN, the molecular weight is 1866.5 Da, and the secondary structure is mainly α-helix and random coil.

[0006] Furthermore, a method for preparing the antimicrobial peptide AMP-OUC329 from the marine plant *Lactobacillus lactis* includes the following steps:

[0007] S1. Culture of marine plant lactobacillus: After activating Bac-329 of plant lactobacillus, it was inoculated into MRS broth medium and cultured to obtain fermentation broth. The fermentation broth was heated at 60-80℃ for 25-35 min and then cooled to room temperature.

[0008] S2. Preparation of crude antimicrobial peptide extract: The pH of the fermentation broth obtained in S1 was adjusted to 6.0-7.0 with NaOH solution. After centrifugation, the supernatant was discarded. The cells were washed twice with phosphate buffer. The cells were resuspended in 0.1 mol / L NaCl solution. The pH of the solution was adjusted to 2.0-3.0 with phosphate. The precipitate was discarded by centrifugation. The supernatant was filtered through a filter membrane, dialyzed, concentrated and lyophilized to obtain crude antimicrobial peptide extract.

[0009] S3. Purification of antimicrobial peptides: After redissolving the crude antimicrobial peptide extract, it is separated and purified by gel filtration column and reversed-phase high-performance liquid chromatography. The antimicrobial activity against Shewanella, a spoilage bacterium of aquatic products, and the damage to the bacterial microstructure are used as targeted screening indicators to collect specific active antimicrobial components.

[0010] S4. Freeze-drying: After concentrating and freeze-drying the specific active antibacterial components, the antimicrobial peptide AMP-OUC329 is obtained.

[0011] Furthermore, in step S1, the marine plant lactobacillus Bac-329 was derived from the intestines of marine cold-water fish, deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 28326 and deposit date of September 1, 2023.

[0012] Furthermore, the inoculation amount in step S1 is 2%; the culture conditions are 24h, 35-40℃.

[0013] Furthermore, in step S2, the pH of the phosphate buffer solution is 7.2-7.4; the filter membrane is a 0.2 μm filter membrane; and the dialysis bag has a molecular weight of 1000 Da.

[0014] Furthermore, in step S3, the gel filter column is a Sephadex G-25 gel filter column; the separation and purification conditions of the reversed-phase high-performance liquid chromatography are as follows: TSKgel G2000SWxl separation column, mobile phase A is acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B is ultrapure water containing 0.1% trifluoroacetic acid, the volume ratio of mobile phase A to mobile phase B is 9:1, and equilibration and gradient elution are performed at a flow rate of 0.5 mL / min.

[0015] The application of the above-mentioned marine plant Lactobacillus antimicrobial peptide AMP-OUC329 in inhibiting Shewanella, the dominant spoilage bacterium in aquatic products.

[0016] Beneficial effects

[0017] The antimicrobial peptide AMP-OUC329 obtained by this invention overcomes the limitation of traditional lactic acid bacteria bacteriocins having poor inhibitory effects on Gram-negative bacteria. In the preparation process, Gram-negative bacteria Shewanella were used as screening indicators. The selected Shewanella was a dominant spoilage bacterium isolated from spoiled aquatic products, which is highly targeted. Experiments have shown that the antimicrobial peptide has thermal stability and acid stability.

[0018] The antimicrobial peptide AMP-OUC329 and Ca obtained by this invention 2+ and Mg 2+They have a strong interaction, and by binding with calcium and magnesium ions, they disrupt the aggregation state of bacterial lipopolysaccharides and teichoic acid, affecting the stability of the bacterial cell wall. This makes the target more precise and significantly improves the antibacterial effect. Attached Figure Description

[0019] Figure 1 Schematic diagram of high performance liquid chromatography separation and purification of antimicrobial peptide AMP-OUC329;

[0020] Figure 2 The UV circular dichroism chromatogram of the antimicrobial peptide AMP-OUC329;

[0021] Figure 3 The diagram shows the binding isotherm and thermodynamic parameters of the antimicrobial peptide AMP-OUC329 with CaCl2, where a is the binding isotherm and b is the thermodynamic parameter.

[0022] Figure 4 The diagram shows the binding isotherm and thermodynamic parameters of the antimicrobial peptide AMP-OUC329 with MgCl2, where a is the binding isotherm and b is the thermodynamic parameter.

[0023] Figure 5 The effect of the antimicrobial peptide AMP-OUC329 on the cell membrane of Shewanella was detected by flow cytometry. In this study, a represents untreated Shewanella, and b represents Shewanella treated with the antimicrobial peptide AMP-OUC329.

[0024] Figure 6 To investigate the effect of the antimicrobial peptide AMP-OUC329 on the cell structure of Shewanella using transmission electron microscopy, a represents untreated Shewanella; b represents Shewanella treated with the antimicrobial peptide AMP-OUC329; and c is a magnified view of b. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments:

[0026] Example 1: Preparation and purification of the antimicrobial peptide AMP-OUC329 from the marine plant Lactobacillus.

[0027] S1. Culture of marine plant lactobacillus: After activation, Bac-329 of marine lactobacillus was inoculated into MRS broth medium at an inoculation amount of 2% and cultured at 37℃ for 24 h to obtain fermentation broth. The fermentation broth was heated at 70℃ for 30 min and then cooled to room temperature.

[0028] S2. Preparation of crude antimicrobial peptide extract: The pH of the fermentation broth was adjusted to 6.0 with NaOH solution. After centrifugation, the supernatant was discarded. The cells were washed twice with phosphate buffer (pH 7.2-7.4). The cells were resuspended in 0.1 mol / L NaCl solution. The pH of the solution was adjusted to 2.0 with phosphate. After centrifugation, the precipitate was discarded. The supernatant was filtered through a 0.2 μm filter membrane, dialyzed through a dialysis bag with a molecular weight of 1000 Da, concentrated and lyophilized to obtain crude antimicrobial peptide extract.

[0029] S3. Purification of antimicrobial peptides: After redissolving the crude antimicrobial peptide extract, it was filtered through a Sephadex G-25 gel filtration column, followed by high-performance liquid chromatography (HPLC) separation using a TSKgel G2000SWxl column (e.g., Figure 1 The freeze-dried concentrate (as shown) has only one UV absorption peak at a wavelength of 220 nm, indicating that it is a single component. It is named lactobacillus Bac-329. The antibacterial activity against Shewanella, a spoilage bacterium of aquatic products, and the damage to the bacterial microstructure are used as targeted screening indicators to collect specific active antibacterial components.

[0030] S4. Freeze-drying: After concentrating and freeze-drying the specific active antibacterial components, the antimicrobial peptide AMP-OUC329 is obtained.

[0031] Secondary mass spectrometry analysis of the antimicrobial peptide AMP-OUC329 revealed its amino acid sequence to be VSGFETLDEDAKKLSNN, with the amino acid sequence from N-terminus to C-terminus being Val-Ser-Gly-Phe-Glu-Thr-Leu-Asp-Glu-Asp-Ala-Lys-Lys-Leu-Ser-Asn-Asn. The molecular weight was 1866.5 Da. Figure 2 It can be seen that the secondary structure of antimicrobial peptides is mainly composed of α-helices and random coils.

[0032] Example 2: The interaction between the antimicrobial peptide AMP-OUC329 and Ca 2+ Mg 2+ Interaction Research

[0033] Freshly prepared 1 mg / mL CaCl2 or MgCl2 solution was added dropwise to 30 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (pH 7.0) containing antimicrobial peptide AMP-OUC329 (0.01-0.05 mg / mL) and vortexed. Ethanol was added to the mixture to ensure a final concentration of 80%. The mixture was allowed to stand overnight to precipitate the complex. After centrifugation at 10,000 × g for 30 min, the precipitate was collected, lyophilized, and measured using an AA6800 flame atomic absorption spectrophotometer.

[0034] In the isothermal titration calorimetry experiment, the calorimeter was filled with 300 μL of 10 mM N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid buffer containing 2.5 mM antimicrobial peptide AMP-OUC329. The syringe was loaded with 60 μL of 10 mM N-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid buffer containing CaCl2 or MgCl2. The initial injection volume was 0.4 μL. Then, at a constant temperature of 25℃, 2 μL was injected every 150 s to ensure complete equilibrium of calcium and magnesium ions. Based on this, the interaction between the antimicrobial peptide AMP-OUC329 and CaCl2 was investigated. 2+ Mg 2+ The interaction effect.

[0035] Figure 3 , Figure 4 The figures show the corrected heat rate-time plots obtained by titrating Bac-329 into buffer solutions containing calcium chloride or magnesium chloride, respectively. The changes in entropy and enthalpy of the reaction are mainly driven by entropy, which is primarily affected by ion interactions.

[0036] The results showed that the antimicrobial peptide AMP-OUC329 and Ca 2+ and Mg 2+ They have a very strong interaction; by binding with calcium and magnesium ions, they can disrupt the aggregation state of lipopolysaccharide and teichoic acid, significantly affecting the stability of bacterial cell walls.

[0037] Example 3: Antimicrobial effect of antimicrobial peptide AMP-OUC329 on Shewanella.

[0038] Shewanella bacteria were inoculated into fresh LB broth at a concentration of 0.1%, and the antimicrobial peptide AMP-OUC329 at a concentration of 0.01-0.05 mg / mL was added to the bacterial suspension. The samples were incubated at 28°C for 4 h, centrifuged, resuspended, stained with propidium iodide, washed, centrifuged, and resuspended. The inhibitory effect of the antimicrobial peptide AMP-OUC329 on the bacterial cell membrane was detected by flow cytometry.

[0039] Figure 5 It can be seen that, in the control group, unstained Shewanella accounted for 83.90% of all cells. Figure 5 a), while after AMP-OUC329 treatment, 99.34% of Shewanella bacteria were stained ( Figure 5 (b) indicates that AMP-OUC329 has a strong inhibitory effect on Shewanella.

[0040] Shewanella bacteria were inoculated into fresh LB broth at a concentration of 0.1%, and the antimicrobial peptide AMP-OUC329 at a concentration of 0.01-0.05 mg / mL was added to the bacterial suspension. After incubation at 28°C for 4 h, the precipitate was collected by centrifugation. Glutaraldehyde fixative was added to the precipitate, and the suspension was fixed at 4°C for 2-4 h. The supernatant was then discarded by centrifugation, and the precipitate was washed three times with 0.1 mol / L PB (phosphate buffer). The resulting bacterial precipitate was suspended in agarose. Different samples were embedded and polymerized into resin blocks, which were then soaked in an ethanol solution containing 2% uranium acetate and stained in the dark for 8 min. The blocks were washed three times with 70% ethanol and stained in a solution containing 2.6% lead citrate for 8 min. After air drying at room temperature, the images were observed and analyzed using a transmission electron microscope to determine the inhibitory effect of the antimicrobial peptide AMP-OUC329 on the bacterial cell membrane.

[0041] Figure 6 The results showed that bacteria treated with the antimicrobial peptide AMP-OUC329 underwent visible deformation, including indentation, outer membrane damage, bacterial rupture and the production of visible fragments, cell wall disorder, and separation of inner and outer membranes, which had a significant destructive effect on bacterial morphology and structure, demonstrating strong antibacterial activity.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide AMP-OUC329 from the marine plant Lactobacillus, characterized in that: The amino acid sequence of the antimicrobial peptide AMP-OUC329 is VSGFETLDEDAKKLSNN.

2. The method for preparing the marine plant *Lactobacillus lactis* antimicrobial peptide AMP-OUC329 according to claim 1, characterized in that, Includes the following steps: S1. Culture of marine plant lactobacillus: After activating Lactiplantibacillus plantarum Bac-329, it was inoculated into MRS broth medium and cultured to obtain fermentation broth. The fermentation broth was heated at 60-80℃ for 25-35 min and then cooled to room temperature. S2. Preparation of crude antimicrobial peptide extract: The pH of the fermentation broth obtained in S1 was adjusted to 6.0-7.0 with NaOH solution. After centrifugation, the supernatant was discarded. The cells were washed with phosphate buffer and resuspended in 0.1 mol / L NaCl solution. The pH of the solution was adjusted to 2.0-3.0 with phosphate. After centrifugation, the precipitate was discarded. The supernatant was filtered through a filter membrane, dialyzed, concentrated, and freeze-dried to obtain crude antimicrobial peptide extract. S3. Purification of antimicrobial peptides: After redissolving the crude antimicrobial peptide extract, it is separated and purified by gel filtration column and reversed-phase high-performance liquid chromatography. The antimicrobial activity against Shewanella, a spoilage bacterium of aquatic products, and the damage to the bacterial microstructure are used as targeted screening indicators to collect specific active antimicrobial components. S4. Freeze-drying: After concentrating and freeze-drying the specific active antibacterial components, the antimicrobial peptide AMP-OUC329 is obtained; In step S1, the marine plant lactobacillus Bac-329 was derived from the intestine of a marine cold-water fish and deposited at the China General Microbiological Culture Collection Center (CGMCC) in Beijing, China, with accession number CGMCC No. 28326 and accession date of September 1, 2023. The inoculation amount in step S1 is 2%; the culture conditions are 24h, 35-40℃. In step S2, the pH of the phosphate buffer solution is 7.2-7.4; the filter membrane is a 0.2 μm filter membrane; and the dialysis bag has a molecular weight of 1000 Da. In step S3, the gel filtration column is a Sephadex G-25 gel filtration column; the separation and purification conditions of the reversed-phase high-performance liquid chromatography are as follows: TSKgel G2000SWxl separation column, mobile phase A is acetonitrile containing 0.1% trifluoroacetic acid, mobile phase B is ultrapure water containing 0.1% trifluoroacetic acid, the volume ratio of mobile phase A to mobile phase B is 9:1, and equilibration and gradient elution are performed at a flow rate of 0.5 mL / min.

3. The application of the marine plant Lactobacillus antimicrobial peptide AMP-OUC329 according to claim 1 in inhibiting Shewanella, the dominant spoilage bacterium of aquatic products.

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