Marine plant lactobacillus antibacterial peptide AMP-OUC329 and application thereof

Abstract

The invention provides a marine plant lactobacillus antibacterial peptide AMP-OUC329 and application thereof, the antibacterial peptide is derived from marine plant lactobacillus Bac-329, the amino acid sequence of the antibacterial peptide is VSGFETLDEDAKKLSNN, the molecular weight of the antibacterial peptide is 1866.5 Da, and the antibacterial peptide has thermal stability and acid stability. According to the method, a Bac-329 strain is cultured, metabolites of the Bac-329 strain are extracted, an antibacterial peptide crude extracting solution is obtained, the antibacterial activity and bacterial microstructure damage to aquatic product putrefying bacteria shewanella are taken as directional screening indexes, Sephadex G-25 gel filtration chromatography and TSKgel G2000SWxl reversed-phase high-performance liquid chromatography separation are performed, antibacterial peptide separation and purification conditions are optimized, and the antibacterial peptide is obtained. The obtained antibacterial peptide has strong inhibitory activity on shewanella and accurate target spot.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a marine Lactiplantibacillus plantarum antibacterial peptide AMP-OUC329 and its application. Background Art

[0002] During the storage and transportation of aquatic products, the contamination of spoilage bacteria is a long-existing technical problem, which not only leads to a significant decline in the sensory quality and nutritional value of the products, but also may cause the proliferation of foodborne pathogenic bacteria, posing a serious food safety hazard. At present, chemical preservatives are commonly used in the industry to inhibit the growth and reproduction of microorganisms. However, chemical preservatives bring potential health risks such as drug resistance and can no longer meet the needs of modern consumers for natural and safe foods. Therefore, the development of efficient and non-toxic biological preservatives has become an important research direction in the field of food science.

[0003] As a kind of antibacterial peptide substance produced by microbial metabolism, antibacterial peptides have significant advantages such as high-efficiency antibacterial activity, safety and non-toxicity, and being easily degraded by proteases, showing broad application prospects in the field of aquatic product preservation. Research shows that antibacterial peptides can play an antibacterial role through various mechanisms such as destroying the integrity of cell membranes, inhibiting protein synthesis, and interfering with DNA replication, and are not easily induced to produce bacterial drug resistance. Marine Lactiplantibacillus plantarum Bac-329 is derived from the intestine of marine cold-water fish, has strong cold tolerance, can grow rapidly under low-temperature conditions, and the antibacterial peptide it produces has significant differences in molecular structure and antibacterial mechanism from existing lactic acid bacteria bacteriocins. Marine microbial antibacterial peptides provide an important research direction and application potential for the development of new marine microorganism-derived biological preservatives. Summary of the Invention

[0004] Technical problem to be solved: The object of the present invention is to provide an antibacterial peptide AMP-OUC329 of Lactiplantibacillus plantarum from the ocean and its application in controlling Shewanella, a dominant spoilage bacterium in aquatic products. The antibacterial peptide is derived from Lactiplantibacillus plantarum Bac-329, which is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 28326, and the taxonomic name is Lactiplantibacillus plantarum. The preservation date is 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 has thermal stability and acid stability. In the present invention, the Bac-329 strain is cultured and its metabolites are extracted to obtain a crude extract of the antibacterial peptide. Taking the antibacterial activity against Shewanella, a spoilage bacterium in aquatic products, and the damage to the bacterial microstructure as the directional screening indexes, through Sephadex G-25 gel filtration chromatography and TSKgel G2000SWxl reversed-phase high-performance liquid chromatography separation, the separation and purification conditions of the antibacterial peptide are optimized, and the obtained antibacterial peptide has strong inhibitory activity against Shewanella and accurate targets.

[0005] Technical solution: An antibacterial peptide AMP-OUC329 of Lactiplantibacillus plantarum from the ocean, the amino acid sequence of the antibacterial peptide AMP-OUC329 is VSGFETLDEDAKKLSNN, with a molecular weight of 1866.5 Da, and the secondary structure is mainly α-helix and random coil.

[0006] Furthermore, a preparation method of an antibacterial peptide AMP-OUC329 of Lactiplantibacillus plantarum from the ocean, comprising the following steps: S1. Culture of Lactiplantibacillus plantarum from the ocean: After activating Lactiplantibacillus plantarum Bac-329, it is inoculated into MRS broth medium for culture to obtain a fermentation broth. The fermentation broth is heated at 60 - 80 °C for 25 - 35 min and cooled to room temperature; S2. Preparation of crude extract of antibacterial peptide: The pH value of the fermentation broth obtained in S1 is adjusted to 6.0 - 7.0 with NaOH solution, centrifuged, and the supernatant is removed. The cells are washed twice with phosphate buffer solution, resuspended in 0.1 mol / L NaCl solution, the pH value of the solution is adjusted to 2.0 - 3.0 with phosphoric acid, centrifuged to discard the precipitate, and the supernatant is filtered through a filter membrane, dialyzed, concentrated and freeze-dried to obtain a crude extract of the antibacterial peptide; S3. Purification of antibacterial peptide: After the crude extract of the antibacterial peptide is redissolved, it is separated and purified by a gel filtration column and a reversed-phase high-performance liquid chromatograph. Taking the antibacterial activity against Shewanella, a spoilage bacterium in aquatic products, and the damage to the bacterial microstructure as the directional screening indexes, specific active antibacterial components are collected; S4. Lyophilization: After concentrating and lyophilizing the specific active antibacterial component, the antibacterial peptide AMP-OUC329 is obtained.

[0007] Further, the Lactiplantibacillus maritimus Bac-329 in step S1 is derived from the intestine of marine cold-water fish, and is preserved in the China General Microbiological Culture Collection Center, with the preservation address being Beijing, China, the preservation number being CGMCC No. 28326, and the preservation date being September 1, 2023.

[0008] Further, the inoculation amount in step S1 is 2%; the culture conditions are 24 h, 35 - 40 °C.

[0009] Further, 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; the molecular weight cut-off of the dialysis bag is 1000 Da.

[0010] Further, in step S3, the gel filtration column is a Sephadex G-25 gel filtration column; the separation and purification conditions of the reverse-phase high performance liquid chromatography are: a 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 and B is 9:1, and equilibration and gradient elution are carried out at a flow rate of 0.5 mL / min.

[0011] Application of the above-mentioned Lactiplantibacillus maritimus antibacterial peptide AMP-OUC329 in inhibiting Shewanella, the dominant spoilage bacterium in aquatic products.

[0012] Beneficial effects

[0013] The Lactiplantibacillus maritimus antibacterial peptide AMP-OUC329 obtained in the present invention breaks through the limitation that traditional lactic acid bacteria bacteriocins have poor inhibitory effects on Gram-negative bacteria. During the preparation process, Shewanella, a Gram-negative bacterium, is used as the screening index, and the selected Shewanella is the dominant spoilage bacterium isolated from spoiled aquatic products, with strong pertinence. Experiments prove that this antibacterial peptide has thermal stability and acid stability; The antibacterial peptide AMP-OUC329 prepared in the present invention has extremely strong interactions with Ca 2+ and Mg 2+ By binding with calcium and magnesium ions, the aggregation state of bacterial lipopolysaccharide and teichoic acid is disrupted, affecting the stability of the bacterial cell wall, and the action target is more precise, and the antibacterial effect is significantly improved. Description of the drawings

[0014] Figure 1 It is a schematic diagram of the high performance liquid chromatography separation and purification of the antibacterial peptide AMP-OUC329; Figure 2It is the ultraviolet circular dichroism spectrum of the antimicrobial peptide AMP-OUC329; Figure 3 It is the binding isotherm and thermodynamic parameter diagram of the antimicrobial peptide AMP-OUC329 and CaCl2. Among them, a is the binding isotherm; b is the thermodynamic parameter; Figure 4 It is the binding isotherm and thermodynamic parameter diagram of the antimicrobial peptide AMP-OUC329 and MgCl2. Among them, a is the binding isotherm; b is the thermodynamic parameter; Figure 5 It is the detection of the effect of the antimicrobial peptide AMP-OUC329 on the cell membrane of Shewanella by flow cytometry. Among them, a is the untreated Shewanella; b is the Shewanella treated with the antimicrobial peptide AMP-OUC329; Figure 6 It is the detection of the effect of the antimicrobial peptide AMP-OUC329 on the cell structure of Shewanella by transmission electron microscopy. Among them, a is the untreated Shewanella; b is the Shewanella treated with the antimicrobial peptide AMP-OUC329, and c is a single enlarged view of b. Detailed implementation mode

[0015] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 Preparation and purification of the antimicrobial peptide AMP-OUC329 from Lactiplantibacillus plantarum subsp. plantarum isolated from marine plants S1. Culture of Lactiplantibacillus plantarum subsp. plantarum isolated from marine plants: After activating Lactiplantibacillus plantarum Bac-329, it was inoculated into MRS broth medium with an inoculation amount of 2% and cultured at 37 °C for 24 h to obtain a fermentation broth. The fermentation broth was heated at 70 °C for 30 min and cooled to room temperature; S2. Preparation of the crude extract of the antimicrobial peptide: The pH value of the fermentation broth was adjusted to 6.0 with NaOH solution, and the supernatant was removed after centrifugation. The cells were washed twice with phosphate buffer (pH 7.2 - 7.4), and the cells were resuspended in 0.1 mol / L NaCl solution. The pH value of the solution was adjusted to 2.0 with phosphoric acid, and the precipitate was discarded after centrifugation. The supernatant was filtered through a 0.2 μm filter membrane and dialyzed with a dialysis bag with a molecular weight of 1000 Da, and then concentrated and freeze-dried to obtain the crude extract of the antimicrobial peptide; S3. Purification of the antimicrobial peptide: After the crude extract of the antimicrobial peptide was redissolved, it was passed through a Sephadex G-25 gel filtration column, and then a TSKgel G2000SWxl separation column was selected for high performance liquid chromatography separation (such as Figure 1As shown in the figure, the freeze-dried concentrate has only one ultraviolet absorption peak at a wavelength of 220 nm, indicating that it is a single component, which is named bacteriocin Bac-329. Using the antibacterial activity against Shewanella, a spoilage bacterium of aquatic products, and the damage to bacterial microstructure as the directional screening indicators, specific active antibacterial components were collected; S4. Freeze-drying: After concentrating and freeze-drying the specific active antibacterial components, the antibacterial peptide AMP-OUC329 is obtained.

[0016] The antibacterial peptide was detected by secondary mass spectrometry. The results showed that the amino acid sequence of the antibacterial peptide AMP-OUC329 is VSGFETLDEDAKKLSNN. From the N-terminus to the C-terminus, its amino acid sequence is Val-Ser-Gly-Phe-Glu-Thr-Leu-Asp-Glu-Asp-Ala-Lys-Lys-Leu-Ser-Asn-Asn, and the molecular weight is 1866.5 Da. It consists of Figure 2 It can be seen that the secondary structure of the antibacterial peptide is mainly α-helix and random coil.

[0017] Example 2 Interaction study of antibacterial peptide AMP-OUC329 with Ca 2+ and Mg 2+ 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 antibacterial peptide AMP-OUC329 (0.01 - 0.05 mg / mL) and vortexed. Ethanol was added to the above mixture to ensure its final concentration was 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, freeze-dried, and measured using an AA6800 flame atomic absorption spectrophotometer.

[0018] 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 antibacterial peptide AMP-OUC329, and 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 for titration was 0.4 μL, and then 2 μL was injected every 150 s at a constant temperature of 25 °C to ensure complete equilibrium of calcium and magnesium ions. On this basis, the interaction effect between antibacterial peptide AMP-OUC329 and Ca 2+ and Mg 2+ was explored.

[0019] Figure 3 , Figure 4Separate displays show that when Bac-329 is dropped into a buffer solution containing calcium chloride or magnesium chloride, titration is carried out to obtain a corrected heat rate-time graph, where the changes in entropy and enthalpy of the reaction are mainly entropy-driven, and entropy is mainly affected by ionic interactions.

[0020] The results show that the antimicrobial peptide AMP-OUC329 has a very strong interaction with Ca 2+ and Mg 2+ and can disrupt the aggregation state of lipopolysaccharide and teichoic acid by binding to calcium and magnesium ions, significantly affecting the stability of the bacterial cell wall.

[0021] Example 3 Antibacterial effect of antimicrobial peptide AMP-OUC329 against Shewanella Shewanella was inoculated into fresh LB broth at a concentration of 0.1%, and 0.01 - 0.05 mg / mL of the antimicrobial peptide AMP-OUC329 was added to the bacterial suspension. After the above samples were incubated at 28 °C for 4 h, they were centrifuged and resuspended. Finally, after staining with propidium iodide, washing, centrifuging and resuspending, the inhibitory effect of the antimicrobial peptide AMP-OUC329 on the bacterial cell membrane was detected by flow cytometry.

[0022] Figure 5 It can be seen that unstained Shewanella in the control group accounted for 83.90% of all cells ( Figure 5 a), while after treatment with AMP-OUC329, 99.34% of Shewanella was stained ( Figure 5 b), indicating that AMP-OUC329 has a strong inhibitory effect on Shewanella.

[0023] Shewanella was inoculated into fresh LB broth at a concentration of 0.1%, and 0.01 - 0.05 mg / mL of the antimicrobial peptide AMP-OUC329 was added to the bacterial suspension. After the above samples were incubated at 28 °C for 4 h, the precipitate was collected by centrifugation. Glutaraldehyde fixative was added to the precipitate and fixed at 4 °C for 2 - 4 h. Then, the supernatant was discarded by centrifugation and washed 3 times repeatedly with 0.1 mol / L PB (phosphate buffer). The obtained bacterial cell precipitate was suspended in agarose, and different samples were embedded and polymerized into resin blocks. They were stained in the dark for 8 min in an ethanol solution containing 2% uranyl acetate, washed 3 times with 70% ethanol, stained for 8 min in a solution containing 2.6% lead citrate, air-dried at room temperature, and observed with a transmission electron microscope and images were collected for analyzing the inhibitory effect of the antimicrobial peptide AMP-OUC329 on the bacterial cell membrane.

[0024] Figure 6It is shown that the bacteria treated with the antimicrobial peptide AMP-OUC329 undergo visible deformation, including indentation, outer membrane damage, bacterial cracking and generation of visible debris, cell wall disorder, separation of the inner and outer membranes, etc., which have a significant destructive effect on the morphology and structure of the bacteria, indicating strong antibacterial activity.

[0025] As mentioned above, it is only a preferred embodiment of the present invention and does not impose any formal restrictions on the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, make many possible changes and modifications to the technical solution of the present invention by using the methods and technical content disclosed above, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

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

2. The method for preparing the marine plant lactobacillus antimicrobial peptide AMP-OUC329 according to claim 1, characterized in that: The following steps are involved: S1. Cultivation of marine Lactobacillus plantarum: Activate Lactobacillus plantarum Bac-329 and inoculate it into MRS broth medium to obtain a fermentation liquid, heat the fermentation liquid at 60-80°C for 25-35 min, and cool it to room temperature; S2. Preparation of crude antimicrobial peptide extract: The fermentation broth obtained in S1 was adjusted to pH 6.0-7.0 with NaOH solution, the supernatant was removed after centrifugation, the cells were washed with phosphate buffer, the cells were resuspended in 0.1 mol / L NaCl solution, the pH value of the solution was adjusted to 2.0-3.0 with phosphoric acid, the precipitate was discarded by centrifugation, the supernatant was filtered through a filter membrane, dialyzed, concentrated and freeze-dried to obtain a crude antimicrobial peptide extract; S3. Purification of antimicrobial peptides: After the crude antimicrobial peptide extracts are redissolved, they are separated and purified by gel filtration column and reversed-phase high performance liquid chromatography, and the antibacterial activity against Shewanella, aquatic product spoilage bacteria, and the damage to bacterial microstructure are used as directional screening indicators to collect specific active antimicrobial components; S4. Freeze drying: After the specific active antimicrobial components are concentrated and freeze dried, the antimicrobial peptide AMP-OUC329 is obtained.

3. The method for preparing the marine plant lactobacillus antimicrobial peptide AMP-OUC329 according to claim 2, characterized in that: The marine plant lactobacillus Bac-329 in step S1 is derived from the intestines of marine cold-water fish and is deposited in the China General Microbiological Culture Collection Center, with a deposit address in Beijing, China, a deposit number of CGMCC No. 28326, and a deposit date of September 1, 2023.

4. The method for preparing the marine plant lactobacillus antimicrobial peptide AMP-OUC329 according to claim 2, characterized in that: The inoculation amount in step S1 is 2%; the culture conditions are 24h, 35-40°C.

5. The method for preparing the marine plant lactobacillus antimicrobial peptide AMP-OUC329 according to claim 2, characterized in that: The pH of the phosphate buffer in step S2 is 7.2-7.4; the filter membrane is a 0.2 μm filter membrane; and the molecular weight of the dialysis bag is 1000 Da.

6. The method for preparing the marine plant lactobacillus antimicrobial peptide AMP-OUC329 according to claim 2, characterized in that: The gel filtration column in step S3 is a Sephadex G-25 gel filtration column; the separation and purification conditions of the reversed-phase high performance liquid chromatography are: a TSKgel G2000SWxl separation column, mobile phase A liquid is acetonitrile containing 0.1% trifluoroacetic acid, liquid B is ultrapure water containing 0.1% trifluoroacetic acid, the volume ratio of liquid A to liquid B is 9:1, and equilibrium and gradient elution are performed at a flow rate of 0.5 mL / min.

7. Use of the marine plant lactobacillus antimicrobial peptide AMP-OUC329 according to claim 1 in inhibiting Shewanella, a dominant spoilage bacterium in aquatic products.

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