An antibacterial peptide prepared by using lactobacillus plantarum carrying copper

Abstract

The application provides an antibacterial peptide prepared by using copper-loaded lactobacillus plantarum, which is prepared by inoculating high-copper-loaded duck-derived lactobacillus plantarum into a culture medium for fermentation and then using the fermentation liquor, and the preservation number of the copper-loaded duck-derived lactobacillus plantarum is CGMCC NO.32835. The lactobacillus plantarum obtained by domestication can tolerate high-concentration copper ions, and the lactobacillus plantarum obtained by domestication and selection can stably grow, and the postbiotic produced by fermentation contains small peptides, small-molecule organic acids, digestive enzymes and the like. Since the postbiotic is rich in antibacterial peptides which have obvious inhibitory effect on harmful bacteria of waterfowl, the prepared antibacterial peptide preparation effectively improves the antibacterial activity, and as a feed additive, is beneficial to eliminating poultry intestinal inflammatory factors, maintaining intestinal health, improving feed utilization, and improving product quality.

Description

Technical Field

[0001] This invention belongs to the field of bioactive product preparation technology, specifically relating to an antimicrobial peptide prepared using copper-loaded Lactobacillus plantarum. Background Technology

[0002] Lactobacillus plantarum ( Lactobacillus plantarum Lactobacillus plantarum is a probiotic widely used in medicine, food, and animal husbandry. Many studies have shown that Lactobacillus plantarum can exert beneficial 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.

[0003] Lactobacillus plantarum and its metabolites (metogenic agents) are used as probiotic preparations in livestock and poultry production due to their functions in inhibiting harmful bacteria, maintaining intestinal health, and promoting growth and development. However, live Lactobacillus plantarum preparations are easily affected by various physical and chemical factors during storage, preparation, and transportation, leading to bloating and inactivation. These phenomena often result in insufficient effectiveness of adding probiotics to feed. Therefore, selecting specific strains to prepare heat-resistant, highly active metabiotics and developing novel microecological preparations has significant economic value and market prospects for the development of the poultry industry.

[0004] Pathogenic microbial infections in poultry farming are a major factor leading to disease and mortality in poultry. For a long time, antibiotics have been the primary treatment for these infections; however, the overuse of antibiotics has led to bacterial resistance, posing new threats to human food safety and public health.

[0005] Antimicrobial peptides are small molecule polypeptides with broad-spectrum resistance to pathogenic microorganisms, capable of rapidly resisting pathogen invasion and forming an important component of innate immunity. Therefore, antimicrobial peptides are currently considered a promising alternative to antibiotics. Summary of the Invention

[0006] The purpose of this invention is to provide an antimicrobial peptide prepared using copper-loaded Lactobacillus plantarum. The prepared antimicrobial peptide has antibacterial effects and can be used as a feed additive for poultry.

[0007] This invention first provides an antimicrobial peptide, wherein the antimicrobial peptide is derived from *Lactobacillus plantarum* (CGMCC No. 32835). Lactobacillus plantarum Prepared from post-biotic formulations;

[0008] Furthermore, the amino acid sequences of the aforementioned antimicrobial peptides are as follows:

[0009] VLTLGGGALIRHWF (SEQ ID NO:1), ATTGAIALGATAAKA (SEQ ID NO:2), SLLSTALLPMLSGKA (SEQ ID NO:3), TAVGLVACSKQSANQQ (SEQ ID NO:4);

[0010] This invention provides one use of the antimicrobial peptide, which is its application in the preparation of antimicrobial products;

[0011] The antibacterial product is used to antagonize common pathogenic microorganisms in duck farming, such as Riemerella anatipestifer, Salmonella or Escherichia coli.

[0012] The antibacterial product is preferably a feed additive.

[0013] The present invention also provides a polypeptide composition comprising one or more of the polypeptides having the sequences SEQ ID NO:1-4 described above.

[0014] Furthermore, the polypeptide composition also comprises a polypeptide with the sequence AVGLLLATGQAANA (SEQ ID NO:5).

[0015] The antimicrobial peptides provided by this invention are isolated and purified from domesticated duck-derived Lactobacillus plantarum metagener, and have significant antimicrobial activity against common harmful duck bacteria (Riemerella anatipestifer, Salmonella and Escherichia coli). Attached Figure Description

[0016] Figure 1 : Inhibition zones of mixed peptides of duck-derived Lactobacillus plantarum before and after domestication and breeding against Escherichia coli and Staphylococcus aureus;

[0017] Figure 2 : Inhibitory effect of mixed peptides of duck-derived Lactobacillus plantarum before and after domestication and breeding on Rheumatoid arthritis, Escherichia coli and Salmonella;

[0018] Figure 3 : A mixed peptide amino acid sequence diagram of duck-derived Lactobacillus plantarum after domestication and selective breeding;

[0019] Figure 4 : Structure diagram of mixed peptides produced by duck-derived Lactobacillus plantarum after domestication and selective breeding;

[0020] Figure 5 : Secondary mass spectrum of amino acid sequence (SEQ ID NO:1);

[0021] Figure 6 : Secondary mass spectrum of amino acid sequence (SEQ ID NO:2);

[0022] Figure 7 : Secondary mass spectrum of amino acid sequence (SEQ ID NO:3);

[0023] Figure 8 : Secondary mass spectrum of amino acid sequence (SEQ ID NO:4);

[0024] Figure 9 : 3D structural diagram of amino acid sequence (SEQ ID NO:1~4). Detailed Implementation

[0025] This invention screened or obtained a duck-derived Lactobacillus plantarum rich in copper ions ( Lactobacillus plantarum The specimen was deposited on November 28, 2024, at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.32835.

[0026] This strain was obtained from *Lactobacillus plantarum* of duck origin (accession number CGMCC No. 26622) through intensive domestication culture screening. *Lactobacillus plantarum* ( Lactobacillus plantarum The specimen was deposited on February 21, 2023, at the China General Microbiological Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 26622.

[0027] The selected duck-derived *Lactobacillus plantarum* strain exhibits significantly enhanced tolerance to copper ions, particularly high concentrations of Cu. 2+ The ion loading exhibits excellent adaptability and carrying capacity. This domesticated bacterium possesses copper-loading characteristics, laying a solid foundation for the production of high-copper-loaded duck-derived *Lactobacillus plantarum* feed additives. Furthermore, this strain inhibits harmful bacteria such as *Riemerella anatipestifer*, *Escherichia coli*, *Staphylococcus aureus*, and *Salmonella*, and its metabolites possess stronger antibacterial activity. Simultaneously, through multiple generations of stress-resistant domestication, the strain's morphology and characteristics have changed, resulting in stronger activity and a richer production of metabolites (metoelastomers), leading to better probiotic effects on poultry growth, development, and intestinal health. This invention isolates antimicrobial peptides with antibacterial effects from the metabolites (metoelastomers) of this strain.

[0028] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings.

[0029] Example 1: Preparation of a mixed peptide containing copper ions from *Lactobacillus plantarum* (CGMCC No. 32835) and identification of its antibacterial effect.

[0030] The extraction steps for mixed peptides and proteins are as follows: *Lactobacillus plantarum* strain CGMCC No. 26622 (pre-selection strain) and *Lactobacillus plantarum* strain CGMCC No. 32835 (selection strain) containing copper ions were inoculated into 15 mL of MRS liquid medium. After incubation at 37 ℃ and 180 r / min for 24 h, the mixture was centrifuged at 4 ℃ and 6000 r / min for 15 min. The supernatant was collected and filtered through a 0.22 μm sterile filter membrane, and the filtrate was collected. The collected fermentation supernatant was added to a 15 mL ultrafiltration tube with a 3 kDa ultrafiltration membrane and centrifuged at 4 ℃ and 6500 r / min for 40 min. The concentrate was collected and diluted in MRS medium until the total protein volume was consistent with the initial fermentation broth volume.

[0031] 1) Identification of the antibacterial effect of Oxford cup

[0032] Mixed peptides and extracellular proteins extracted from duck-derived Lactobacillus plantarum before and after domestication and breeding were used in 6 replicates per group. The Oxford cup method was used to determine the effect of the samples before and after domestication on the antibacterial activity of Escherichia coli and Staphylococcus aureus.

[0033] Table 1: Antimicrobial zone effects of mixed peptides and proteins prepared from duck-derived Lactobacillus plantarum before and after domestication and selection.

[0034]

[0035] The results in Table 1 indicate that the mixed peptides and proteins produced by *Lactobacillus plantarum* with high loading on copper duck strains exhibit better antibacterial activity against *Escherichia coli*. Figure 1 The results showed that the mixed peptides containing high-copper-loaded Lactobacillus plantarum exhibited better antibacterial activity against Escherichia coli and Staphylococcus aureus, with a more pronounced inhibition zone effect.

[0036] 2) Antibacterial curve

[0037] The above extract was mixed with broth culture medium containing harmful bacteria in a certain ratio (supernatant: harmful bacteria ratio of 1:4), and the mixture was placed in a microbial growth analyzer (ELISA reader) for 24-hour growth curve determination.

[0038] Table 2: Antibacterial curves of mixed peptides and proteins prepared from duck-derived Lactobacillus plantarum before and after domestication and selection.

[0039]

[0040] The results in Table 2 show that the mixed peptides and proteins produced by highly copper-loaded duck-derived Lactobacillus plantarum have better antibacterial activity against harmful duck bacteria (Riemerella anatipestifer, Escherichia coli, and Salmonella). Figure 2The antibacterial curve results showed that when the ratio of the mixed peptide and protein solution to the harmful bacteria was 1:4, both the mixed peptide and protein produced by *Lactobacillus plantarum* from ducks before and after domestication and selection had significant antibacterial effects, but the mixed peptide and protein produced by *Lactobacillus plantarum* from ducks after domestication and selection had better antibacterial effects.

[0041] Example 2: Analysis of small peptide sequences and effect identification of duck-derived Lactobacillus plantarum metagenerants before and after domestication and breeding

[0042] 1) Sequence analysis of metagenic small peptides from selected duck-derived Lactobacillus plantarum

[0043] A commercial company was commissioned to perform shotgun protein identification analysis on *Lactobacillus plantarum* and *Lactobacillus plantarum* with high copper ion loading. The experimental method was as follows: 0.1% TFA was added to the sample, mixed, centrifuged at 20000 g for 5 min, and the supernatant was transferred to a 10 KD ultrafiltration centrifuge tube and centrifuged at 12000 g for 15 min. 200 µL of 0.1% TFA was added, and the mixture was centrifuged at 12000 g for 15 min. This process was repeated twice. The filtrate was collected, desalted using a C18 StageTip, and vacuum dried. The dried peptides were reconstituted with 0.1% TFA, and their concentrations were determined for LC-MS analysis. A suitable amount of peptide from each sample was used for chromatographic separation using a nano-flow rate Easy nLC1200 chromatography system (Thermo Scientific). Buffer: After the sample was injected into the trap column, it underwent gradient separation through the analytical column at a flow rate of 300 nL / min. After peptide separation, DDA (data-dependent acquisition) mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer (Thermo Scientific). Secondary mass spectrometry analysis of peptides involved acquiring secondary mass spectra (MS2 scans) of the 20 highest-intensity precursor ions after each full scan.

[0044] Literature reports that antimicrobial peptides generally contain specific amino acids such as lysine, arginine, tryptophan, and glycine; the main amino acid sequences of the antimicrobial peptides of this invention were determined to be: 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); among which, the sequences of the antimicrobial peptides common to the strains before and after domestication and selection are SEQ ID NO:1 to 4 (…). Figures 5-9The unique amino acid sequence of the domesticated and selected duck-derived Lactobacillus plantarum is AVGLLLATGQAANA (SEQ ID NO:5).

[0045] Among them, the amino acid sequence of the high-yielding antimicrobial peptide copper-loaded duck-derived *Lactobacillus plantarum* metagenic micropeptide AVGLLLATGQAANA, obtained through domestication and selection, is shown in the secondary mass spectrum and 3D structure as follows: Figures 3-4 As shown. The secondary mass spectra of the amino acid sequences (SEQ ID NO: 1~4) are as follows. Figures 5-8 As shown, and the 3D structure as follows Figure 9 As shown.

[0046] 2) Analysis of the antibacterial effect of small peptides

[0047] After the company synthesized the small peptides with sequences SEQ ID NO:1-5, they were diluted with MRS medium to ensure the total liquid volume was consistent with the initial fermentation broth. Equal amounts of the peptide compositions SEQ ID NO:1-4 and SEQ ID NO:1-5 were then tested against harmful bacteria to inhibit the growth of duck harmful bacteria (Escherichia coli, Riemerella anatipestifer, and Salmonella), verifying the in vitro antibacterial activity of the synthetic peptides against harmful bacteria. A culture medium without added bacteria served as a negative control, a medium with added bacteria but without the antimicrobial peptide served as a positive control, and a medium with the added antimicrobial peptide served as the experimental group. The culture was incubated at 37℃ for 24 hours, and the absorbance was measured at 600 nm to calculate the inhibition rate.

[0048] Table 3: Inhibition rate of synthetic small peptides against harmful bacteria

[0049]

[0050] The results in Table 3 show that the antimicrobial peptides screened from the postbiotics of *Lactobacillus plantarum* derived from ducks before and after domestication have significant antimicrobial effects against harmful bacteria in ducks. Compared with the antimicrobial effect of the antimicrobial peptides produced by *Lactobacillus plantarum* derived from ducks before domestication, the antimicrobial effect of the antimicrobial peptides produced by *Lactobacillus plantarum* derived from ducks after domestication and selection is better.

[0051] 3) Analysis results of relevant antimicrobial proteins in domesticated and selected duck-derived Lactobacillus plantarum

[0052] Table 4: Analysis of Antimicrobial Proteins in High-Yielding Antimicrobial Peptide-Loaded Copper-Loaded Duck-Derived Lactobacillus Plantarum from Domestication and Selection

[0053]

[0054] The mass spectrometry data, after being searched using MaxQuant 2.4.14.0 mass spectrometry database software, identified proteins with high reliability and associated with antibacterial activity, as shown in Table 4. Except for the cell wall-binding protein YocH, the other four related proteins can degrade or hydrolyze bacterial cell wall peptidoglycan, or synergize with bacteriocins to achieve antibacterial effects. YocH, a cell wall-binding protein produced through domestication and selection, belongs to the bacterial surface protein family and affects cell division and morphology by binding to peptidoglycan in the cell wall. Currently, no related functions are found in the search database, and there is limited research on its direct antibacterial function. It may also exert its effects through cell wall modification or synergistically with other antibacterial molecules.

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide is prepared from a metabiotic preparation of *Lactobacillus plantarum* of copper duck origin with accession number CGMCC No. 32835; the amino acid sequence of the antimicrobial peptide is SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:

4.

2. The application of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial products, wherein the antimicrobial products are used to antagonize Riemerella anatipestifer, Salmonella or Escherichia coli.

3. An antibacterial product, characterized in that, The antimicrobial product contains the antimicrobial peptide as described in claim 1.

4. The antibacterial product as described in claim 3, characterized in that, The antibacterial product is a feed additive.

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