Antibacterial peptide prepared from copper-loaded lactobacillus plantarum

Abstract

The invention provides an antibacterial peptide prepared by utilizing copper-loaded lactobacillus plantarum, which is prepared by inoculating high-copper-loaded duck-origin lactobacillus plantarum into a culture medium for fermentation and then using fermentation liquor, and the preservation number of the copper-loaded duck-origin lactobacillus plantarum is CGMCC (China General Microbiological Culture Collection Center) NO.32835. The lactobacillus plantarum obtained through domestication can tolerate high-concentration copper ions, the lactobacillus plantarum obtained through domestication and breeding can stably grow, and metabiotics generated through fermentation of the lactobacillus plantarum contain small peptides, small molecular organic acids, digestive enzymes and the like. Due to the fact that the antibiotic peptide rich in the afterbiogen has an obvious inhibiting effect on harmful bacteria of waterfowls, the prepared antibiotic peptide preparation effectively improves the antibacterial activity, and as a feed additive, the antibiotic peptide preparation is beneficial to elimination of inflammatory factors of intestinal tracts of poultry, maintenance of intestinal health, increase of the utilization rate of feed and improvement of the product quality.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioactive product preparation, and particularly relates to an antibacterial peptide prepared by using copper-loaded lactobacillus plantarum. BACKGROUND

[0002] Lactobacillus plantarum (Lactobacillus plantarum) is a kind of probiotics widely used in medicine, food and animal husbandry fields. Many studies have shown that Lactobacillus plantarum can produce probiotic effects on the host by maintaining the balance of intestinal flora, reducing intestinal inflammatory factors, promoting the digestion and absorption of nutrients and improving intestinal microbial metabolism. Lactobacillus plantarum Because Lactobacillus plantarum itself and its metabolites (postbiotics) have the functions of inhibiting harmful bacteria, maintaining intestinal health and promoting growth and development, they are applied as probiotic preparations in poultry production. In addition, the live Lactobacillus plantarum preparation is easily affected by various physical and chemical factors during preservation, preparation and transportation, causing bloating and inactivation. The above phenomena often lead to insufficient effect of adding probiotic agents in feed. Therefore, selecting specific strains to prepare high-temperature-resistant and high-activity postbiotics and developing new microecological preparations have important economic value and market prospects for poultry industry development.

[0003] In poultry breeding, pathogenic microbial infection is one of the important factors leading to poultry disease and death. People have always chosen to use antibiotics to treat pathogenic microbial infection, but bacterial resistance caused by the abuse of antibiotics has brought new threats to human food safety and public health.

[0004] Antibacterial peptides are small molecular polypeptides with broad-spectrum resistance to pathogenic microorganisms, which can quickly resist the invasion of pathogens and are an important part of innate immunity. Therefore, antibacterial peptides are currently a promising substitute for antibiotics.

[0005] SUMMARY The purpose of the present application is to provide an antibacterial peptide prepared by using copper-loaded Lactobacillus plantarum, which has a bacteriostatic effect and can be used as a feed additive for poultry.

[0006] The present application first provides an antibacterial peptide, which is prepared from the postbiotic preparation of copper duck-derived Lactobacillus plantarum (Lactobacillus plantarum) with the preservation number of CGMCC No.32835.

[0007] The amino acid sequences of the antibacterial peptide are as follows: Lactobacillus plantarum ​​VLTLGGGALIRHWF (SEQ ID NO:1), ATTGAIALGATAAKA (SEQ ID NO:2), SLLLSTALLPMLSGKA (SEQ ID NO:3), TAVGLVACSKQSANQQ (SEQ ID NO:4); The application provides a use of the antibacterial peptide, which is used for preparing an antibacterial product. The antibacterial product is used for resisting common disease microorganisms such as Riemerella anatipestifer, Salmonella or Escherichia coli in meat duck breeding. The antibacterial product is preferably a feed additive.

[0008] The application further provides a polypeptide composition containing one or more polypeptides with the sequences of SEQ ID NO:1-4.

[0009] Further, the polypeptide composition further contains a polypeptide with the sequence of AVGLLLATGQAANA (SEQ ID NO:5).

[0010] The antibacterial peptide provided by the application is obtained by separation and purification from a duck-derived plantarum postbiotic after domestication, and has obvious bacteriostatic activity on common duck harmful bacteria (Riemerella anatipestifer, Salmonella and Escherichia coli). BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 : Inhibition zone diagram of mixed peptides of duck-derived plantarum before and after domestication and breeding on Escherichia coli and Staphylococcus aureus; Figure 2 : Inhibition effect diagram of mixed peptides of duck-derived plantarum before and after domestication and breeding on Riemerella anatipestifer, Escherichia coli and Salmonella; Figure 3 : Amino acid sequence diagram of mixed peptides of duck-derived plantarum after domestication and breeding; Figure 4 : Structure diagram of mixed peptides produced by duck-derived plantarum after domestication and breeding; Figure 5 : Secondary mass spectrum diagram of the amino acid sequence (SEQ ID NO:1); Figure 6 : Secondary mass spectrum diagram of the amino acid sequence (SEQ ID NO:2); Figure 7 : Secondary mass spectrum diagram of the amino acid sequence (SEQ ID NO:3); Figure 8 : Secondary mass spectrum diagram of the amino acid sequence (SEQ ID NO:4); Figure 9Amino acid sequence (SEQ ID NO: 1~4) 3D structure diagram. DETAILED DESCRIPTION

[0012] The application screens or obtains a duck-derived Lactobacillus plantarum (Lactobacillus plantarum) with copper ion enrichment. Lactobacillus plantarum The Lactobacillus plantarum with copper ion enrichment with the preservation number of CGMCC No.32835 was preserved in the General Microbiological Center of the Chinese Microbial Culture Collection Committee of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 1, Xili, Beichen West Road, Chaoyang District, Beijing on November 28, 2024.

[0013] The strain is obtained by intensive domestication and culture screening from the duck-derived Lactobacillus plantarum with the preservation number of CGMCC No.26622. Lactobacillus plantarum The Lactobacillus plantarum with the preservation number of CGMCC No.26622 was preserved in the General Microbiological Center of the Chinese Microbial Culture Collection Committee of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 1, Xili, Beichen West Road, Chaoyang District, Beijing on February 21, 2023. The duck-derived Lactobacillus plantarum obtained by selection can greatly enhance the tolerance of copper ion loading, and has good adaptability and carrying capacity for high-concentration Cu 2+ The domesticated strain has the characteristics of copper loading, which lays a good foundation for the production of high-copper duck-derived Lactobacillus plantarum feed additives. In addition, the strain has the function of inhibiting harmful bacteria such as Riemerella anatipestifer, Escherichia coli, Staphylococcus aureus and Salmonella, and the postbiotics produced has stronger antibacterial property; at the same time, after multi-generation adversity domestication, the strain morphology and characteristics change, the activity is stronger, the metabolic products (postbiotics) are more abundant, and the growth and development of poultry and the intestinal health probiotic effect are better. The application is to separate the antibacterial peptide with antibacterial effect from the metabolic products (postbiotics) of the strain.

[0014] The application will be described in detail below in combination with specific examples and drawings.

[0015] Example 1: Preparation of the mixed peptide of the duck-derived Lactobacillus plantarum with copper ion with the preservation number of CGMCC No.32835 and identification of antibacterial effect The extraction steps of the mixed peptides and proteins are as follows: duck-derived Lactobacillus plantarum with preservation number CGMCC No. 26622 (strain before breeding) and copper ion-containing duck-derived Lactobacillus plantarum with preservation number CGMCC No. 32835 (strain after breeding) are respectively inoculated into 15 mL MRS liquid medium, and then cultured at 37°C and 180 r / min in a constant temperature shaker for 24 hours. Then, the supernatant is collected by centrifugation at 4°C and 6000 r / min for 15 min, and then filtered through a 0.22 μm sterile filter membrane. The collected fermentation supernatant is added to a 15 mL ultrafiltration tube with a 3 ku ultrafiltration membrane, and then centrifuged at 4°C and 6500 r / min for 40 min. The concentrated solution is collected and diluted in MRS medium, so that the total amount of the protein solution is consistent with the initial fermentation volume.

[0016] 1) Oxford cup antibacterial effect identification The mixed peptides and extracellular proteins extracted from duck-derived Lactobacillus plantarum before and after domestication and breeding are used to determine the influence of the samples before and after domestication on the antibacterial activity of Escherichia coli and Staphylococcus aureus by using the Oxford cup method, with 6 repeats in each group.

[0017] Table 1: Antibacterial circle effect table of mixed peptides and proteins prepared by duck-derived Lactobacillus plantarum before and after domestication and breeding

[0018] As shown by the results in Table 1, the mixed peptides and proteins produced by the duck-derived Lactobacillus plantarum with high copper loading have better antibacterial activity on Escherichia coli. Figure 1 As shown by the results in Table 2, the mixed peptides and proteins produced by the duck-derived Lactobacillus plantarum with high copper loading have better antibacterial activity on duck harmful bacteria (Riemerella anatipestifer, Escherichia coli and Salmonella).

[0019] 2) Antibacterial curve The above extraction liquid is mixed with broth medium containing harmful bacteria at a certain proportion (the ratio of supernatant to harmful bacteria is 1:4), and then placed in a microbial growth analyzer (enzyme label instrument) to determine the 24h growth curve.

[0020] Table 2: Antibacterial curve effect table of mixed peptides and proteins prepared by duck-derived Lactobacillus plantarum before and after domestication and breeding

[0021] As shown by the results in Table 2, the mixed peptides and proteins produced by the duck-derived Lactobacillus plantarum with high copper loading have better antibacterial activity on duck harmful bacteria (Riemerella anatipestifer, Escherichia coli and Salmonella). Figure 2The bacteriostatic curve results show that when the addition amount ratio of the produced mixed peptide and protein solution to the harmful bacteria is 1:4, the mixed peptide and protein produced by the duck-derived Lactobacillus plantarum before and after domestication and breeding have significant bacteriostatic effect, but the mixed peptide and protein produced by the duck-derived Lactobacillus plantarum after domestication and breeding have better bacteriostatic effect.

[0022] Example 2: Sequence analysis and effect identification of duck-derived Lactobacillus plantarum after domestication and breeding 1) Sequence analysis of duck-derived Lactobacillus plantarum after domestication and breeding The commercial company was commissioned to analyze the protein Shotgun of duck-derived Lactobacillus plantarum and high-load copper ion duck-derived Lactobacillus plantarum. The test method is as follows: add an appropriate amount of 0.1% TFA to the sample, mix well, centrifuge at 20000 g for 5 min, take the supernatant, transfer to a 10 KD ultrafiltration centrifuge tube, centrifuge at 12000 g for 15 min. Add 200 μL of 0.1% TFA and centrifuge at 12000 g for 15 min, repeat 2 times, collect the filtrate, desalt using C18 StageTip, and vacuum dry. After drying, the peptide segments are reconstituted with 0.1% FA, and the peptide segment concentration is determined for LC-MS analysis. An appropriate amount of peptide segments from each sample is used for chromatographic separation using a nanoflow rate Easy nLC1200 chromatographic system (Thermo Scientific). Buffer: after sample injection to the Trap Column, gradient separation is performed on the chromatographic analysis column at a flow rate of 300 nl / min. After peptide separation, Q-Exactive HF-X mass spectrometer (Thermo Scientific) is used for DDA (data-dependent acquisition) mass spectrometry analysis. Peptide secondary mass spectrometry analysis collection: after each full scan, 20 secondary mass spectra of the highest intensity parent ions (MS2 scan) are collected.

[0023] The literature reports that the antibacterial peptide generally has specific amino acids such as lysine, arginine, tryptophan and glycine; the related main amino acid sequences of the antibacterial peptide of the present application are: VLTLGGGALIRHWF (SEQ ID NO: 1), ATTGAIALGATAAKA (SEQ ID NO: 2), SLLLSTALLPMLSGKA (SEQ ID NO: 3), TAVGLVACSKQSANQQ (SEQ ID NO: 4), AVGLLLATGQAANA (SEQ ID NO: 5); wherein the sequences of the antibacterial peptides common to the strains before and after domestication and breeding are SEQ ID NO: 1-4 sequences ( Figures 5 to 9), the unique amino acid sequence of the domesticated and selected duck-derived Lactobacillus plantarum is AVGLLLATGQAANA (SEQ ID NO: 5).

[0024] The amino acid sequence secondary mass spectrum and 3D structure of the unique small peptide amino acid sequence AVGLLLATGQAANA of the domesticated and selected high-yield antibacterial peptide copper-loaded duck-derived Lactobacillus plantarum probiotic are shown in FIGS. 1-4. Figures 3 to 4 The amino acid sequence secondary mass spectrum of the amino acid sequences (SEQ ID NO: 1-4) is shown in FIGS. 1-4, and the 3D structure is shown in FIGS. 1-4. Figures 5 to 8 The amino acid sequence secondary mass spectrum of the amino acid sequences (SEQ ID NO: 1-4) is shown in FIGS. 1-4, and the 3D structure is shown in FIGS. 1-4. Figure 9 The amino acid sequence secondary mass spectrum of the amino acid sequences (SEQ ID NO: 1-4) is shown in FIGS. 1-4, and the 3D structure is shown in FIGS. 1-4.

[0025] 2) Analysis of the bacteriostatic effect of small peptides The company was commissioned to artificially synthesize small peptides with sequences SEQ ID NO: 1-5, and then diluted with MRS medium to match the initial fermentation liquid in terms of total liquid volume. The same amount of polypeptide compositions of SEQ ID NO: 1-4 and SEQ ID NO: 1-5 were taken to test the inhibition of duck harmful bacteria (Escherichia coli, Riemerella anatipestifer, Salmonella) growth, and to verify the in vitro bacteriostatic activity of artificially synthesized peptides on harmful bacteria. The medium without added bacteria was set as the negative control, the medium with added bacteria but without antibacterial peptides was set as the positive control, and the medium with added antibacterial peptides was set as the test group. The absorbance was measured at 600 nm after 24 h of incubation at 37°C, and the inhibition rate was calculated.

[0026] Table 3: Inhibition rate of synthetic small peptides on harmful bacteria

[0027] The results in Table 3 show that the antibacterial peptides screened from the duck-derived Lactobacillus plantarum probiotic before and after domestication have obvious bacteriostatic effect on duck harmful bacteria; compared with the bacteriostatic effect of antibacterial peptides produced by duck-derived Lactobacillus plantarum before domestication, the bacteriostatic effect of antibacterial peptides produced by duck-derived Lactobacillus plantarum after domestication is better.

[0028] 3) Analysis results of related bacteriostatic proteins of the domesticated and selected duck-derived Lactobacillus plantarum Table 4: Analysis table of bacteriostatic proteins of the domesticated and selected high-yield antibacterial peptide copper-loaded duck-derived Lactobacillus plantarum

[0029] The data collected by mass spectrometry was searched by MaxQuant 2.4.14.0, and the proteins with high reliability and related to the antibacterial effect were detected as shown in Table 4. In addition to Cell wall-binding protein YocH, the other four related proteins can degrade or hydrolyze the peptidoglycan of bacterial cell wall, or synergize with bacteriocins to achieve the effect of inhibiting bacteria. YocH is a related protein produced after domestication and breeding, which is a cell wall-binding protein belonging to the bacterial surface protein family, and affects cell division and morphology by binding to the peptidoglycan in the cell wall. At present, the related function is not shown in the database search, and the direct antibacterial function related research is less, and it may also play a role through cell wall modification or synergism with other antibacterial molecules.

Claims

1. An antibacterial peptide, characterized in that, The antibacterial peptide is prepared from a postbiotic preparation of Lactobacillus plantarum of a copper duck source with a preservation number of CGMCC No.32835.

2. The antimicrobial peptide of claim 1, wherein, The antibacterial peptide has an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

3. Use of the antibacterial peptide in claim 1 in the preparation of an antibacterial product.

4. An antimicrobial article, characterized by, The antibacterial product comprises the antibacterial peptide in claim 1.

5. The antimicrobial article of claim 4, wherein, The antibacterial product is a feed additive.

6. A polypeptide composition, characterized in that, The polypeptide composition comprises one or more of the antibacterial peptides in claim 2.

7. The polypeptide composition of claim 6, wherein The polypeptide composition comprises a polypeptide with an amino acid sequence of SEQ ID NO:5.

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