Lactobacillus grisonii KD9, bacteriocin B-KD9, product and application of lactobacillus grisonii KD9 and bacteriocin B-KD9

Abstract

The invention provides a Lactobacillus grisonii KD9, a bacteriocin B-KD9, a product and application, and the obtained Lactobacillus grisonii KD9 is a lactic acid bacterium with good probiotic characteristics, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M 2024604. The leader peptide-free bacteriocin B-KD9 produced by the lactobacillus garnatae KD9 has a remarkable bactericidal effect on gram-negative bacteria such as vibrio parahaemolyticus and vibrio alginolyticus, can improve the anti-infection effect of the zebra fish on the vibrio parahaemolyticus, and shows good thermal stability and acid-base tolerance at the same time; wide development prospects are realized in the fields of feeds, antibacterial agents, aquaculture, foods and the like.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to liquid Lactobacillus KD9, bacteriocin B-KD9, products and applications. Background Art

[0002] Vibrio parahaemolyticus and Vibrio alginolyticus are halophilic foodborne pathogens that pose a threat to aquaculture and aquatic product safety. If people eat aquatic products contaminated by them, they will suffer from acute gastroenteritis, and even death in severe cases. Fish infected with Vibrio parahaemolyticus and Vibrio alginolyticus will develop skin ulcers, blood spots, protruding eyes, and fin ulceration. The prevention and control of Vibrio parahaemolyticus and Vibrio alginolyticus has become an important research topic in aquaculture. Antibiotics are usually widely used in aquaculture and clinical practice as a prevention and control measure. However, the frequent use of antibiotics in aquaculture increases the risk of the spread of drug-resistant bacteria and drug-resistant genes, and threatens the health of aquatic animals and humans. Therefore, there is an urgent need to develop new and effective alternatives to antibiotics.

[0003] In recent years, the alternatives that have been studied more include vaccines, bacteriophages, probiotics, nanoparticles, plant-derived extracts, bacteriocins, and the combined use of various fungicides. Vaccines have problems such as large vaccination doses and short immunity time; the use of bacteriophages can also lead to drug resistance; nanoparticles and plant-derived antibacterial agents are subject to many restrictions due to their stability and safety. So far, few studies have reported the use of bacteriocins as a means of prevention and control of Vibrio parahaemolyticus and Vibrio alginolyticus, and there have been no reports on their combined use with bacteriocin-producing probiotic lactic acid bacteria.

[0004] Probiotic lactic acid bacteria are a type of Gram-positive bacteria that are widely found in fermented foods, animals, plants, and the human body. They can regulate the intestinal microbial flora and the immune system. Their metabolites, such as polysaccharides, organic acids, and bacteriocins, have antioxidant, anticancer, and antibacterial effects. In recent years, the study and application of probiotic lactic acid bacteria and their metabolites, bacteriocins, as antibacterial substances have become a hot topic. Bacteriocins are widely found in bacteria. They are a class of peptides with antibacterial activity produced by ribosome synthesis during bacterial growth and metabolism. They are an effective alternative to antibiotics. Although bacteriocins have a highly efficient and accurate antibacterial effect on pathogenic bacteria, their thermal stability and acid-base tolerance become key factors in whether they can be used and exert their antibacterial effects. In addition, most of the bacteriocins reported so far have good antibacterial effects on Gram-positive bacteria. For example, Nisin is the only Class I bacteriocin currently commercialized as a food preservative (food additive). It has good antibacterial effects on Gram-positive bacteria such as Listeria monocytogenes and Staphylococcus aureus, but weak antibacterial effects on Gram-negative bacteria. Therefore, finding new bacteriocins with good antibacterial effects on Gram-negative bacteria has become the focus of researchers.

[0005] Bacteriocins can achieve precise sterilization and infection prevention for pathogens and antibiotic-resistant strains. They are safe and reliable, easily digested and absorbed by the human body, and have inhibitory effects on different types of bacteria, fungi and viruses. They have broad application prospects in agriculture, fisheries, food, medicine and other fields, and are being paid attention to by more and more researchers. They can not only inhibit the growth of pathogens in aquaculture disease prevention and control, but also increase the phagocytic activity of macrophages as immunomodulators, and are one of the best alternatives to antibiotics. Leaderless bacteriocins are a type of bacteriocin that is synthesized by ribosomes and is active without any modification after translation. This type of bacteriocin has a simple genetic structure, is easy to express in other microbial cells, and is easy to mass produce through bioengineering. It has a unique antibacterial mechanism and has huge commercial application potential. Summary of the invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a Ghanaian liquid Lactobacillus KD9, a bacteriocin B-KD9, a product and an application. The obtained Ghanaian liquid Lactobacillus KD9 is a lactic acid bacteria with good probiotic properties. The leader-free peptide bacteriocin B-KD9 produced by the liquid Lactobacillus KD9 has a significant bactericidal effect on Gram-negative bacteria such as Vibrio parahaemolyticus and Vibrio alginolyticus, and exhibits good thermal stability and acid-base tolerance. The liquid Lactobacillus KD9 has broad development prospects in the fields of feed, antibacterial drugs, aquaculture, food, etc.

[0007] To achieve the above object, the present invention provides the following technical solution: a liquid Lactobacillus Ghanaiensis KD9, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO:M 2024604.

[0008] Furthermore, the Ghanaian liquid Lactobacillus KD9 produces bacteriocin B-KD9, and the bacteriocin B-KD9 includes 5 peptides, specifically: FTGPDVKFP, PVGLADPDGPA, PDSPLFDKT, FPSLVGRPR, and TDDLLEDLK.

[0009] The present invention also provides the use of liquid Lactobacillus Ghana KD9 as a probiotic in functional food or feed.

[0010] The invention also provides a Ghanaian liquid Lactobacillus KD9 fermentation liquid, which is prepared by fermenting the Ghanaian liquid Lactobacillus KD9.

[0011] The present invention also provides an application of liquid Lactobacillus Ghana KD9 fermentation liquid in the preparation of antibacterial drugs.

[0012] The present invention also provides a bacteriocin B-KD9. The bacteriocin B-KD9 is obtained by centrifuging, extracting, ultrafiltering, concentrating, freeze-drying, separating and purifying the above-mentioned Ghanaian liquid lactobacillus KD9 fermentation supernatant.

[0013] Furthermore, the bacteriocin B-KD9 contains 5 polypeptides, and the amino acid sequences of the 5 polypeptides are specifically: FTGPDVKFP, PVGLADPDGPA, PDSPLFDKT, FPSLVGRPR, and TDDLLEDLK.

[0014] The present invention also provides an application of bacteriocin B-KD9 in the preparation of antibacterial drugs.

[0015] The present invention also provides an antibacterial drug, comprising the above-mentioned bacteriocin B-KD9; or bacteriocin B-KD9 and Ghana liquid Lactobacillus KD9; or Ghana liquid Lactobacillus KD9 fermentation liquid; or Ghana liquid Lactobacillus KD9 fermentation liquid and Ghana liquid Lactobacillus KD9.

[0016] The present invention also provides an aquaculture biological feed, wherein the feed contains the above-mentioned antibacterial drug.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention screened out a lactic acid bacteria with good probiotic properties - Ghana liquid Lactobacillus KD9, which showed good anti-infection effect when applied to zebrafish infected with Vibrio parahaemolyticus. Ghana liquid Lactobacillus KD9 can produce bacteriocin B-KD9, which shows good thermal stability and acid-base tolerance, and exhibits significant antibacterial activity against Gram-negative bacteria, such as Vibrio parahaemolyticus and Vibrio alginolyticus. In addition, bacteriocin B-KD9 also showed good anti-infection effect when applied to zebrafish infected with Vibrio parahaemolyticus. At the same time, the combined use of Ghana liquid Lactobacillus KD9 and bacteriocin B-KD9 enhances the anti-infection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is the colony and cell morphology of Ghana liquid Lactobacillus KD9; Figure 2 Analysis of the hemolytic results of Ghana liquid Lactobacillus KD9; Figure 3 The inhibition zone of Ghana liquid Lactobacillus KD9 against Vibrio parahaemolyticus and Vibrio alginolyticus; Figure 4 is the preparative chromatogram of bacteriocin B-KD9; Figure 5 It is the high performance liquid chromatography analysis diagram of bacteriocin B-KD9; Figure 6 It is the secondary MS / MS map of peptide FTGPDVKFP, the molecular structure of the amino acid sequence, the distribution of amino acid hydrophilicity and hydrophobicity, and the three-dimensional structure prediction map; Figure 7 The secondary MS / MS map of the peptide PVGLADPDGPA, the molecular structure of the amino acid sequence, the distribution of amino acid hydrophilicity and hydrophobicity, and the three-dimensional structure prediction map; Figure 8 It is the secondary MS / MS map of peptide PDSPLFDKT, the molecular structure of amino acid sequence, the distribution of amino acid hydrophilicity and hydrophobicity, and the three-dimensional structure prediction map; Fig. 9 It is the secondary MS / MS map of peptide FPSLVGRPR, the molecular structure of amino acid sequence, the distribution of amino acid hydrophilicity and hydrophobicity, and the three-dimensional structure prediction map; Fig.10It is the secondary MS / MS map of peptide TDDLLEDLK, the molecular structure of the amino acid sequence, the distribution of amino acid hydrophilicity and hydrophobicity, and the three-dimensional structure prediction map; Fig.11 The growth status of Vibrio parahaemolyticus under the action of different concentrations of bacteriocin B-KD9; Fig.12 The growth of Vibrio parahaemolyticus after being treated with different concentrations of bacteriocin B-KD9; Fig.13 The effect of different pH on the antibacterial activity of bacteriocin B-KD9; Fig.14 The effect of different temperatures on the antibacterial activity of bacteriocin B-KD9; Fig.15 The CCD camera was used to photograph the morphology of zebrafish in the control group and the model group; Fig.16 This is a graph showing the efficacy of bacteriocin B-KD9 and (or) Ghana liquid Lactobacillus KD9 in preventing Vibrio parahaemolyticus infection. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail. It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The liquid Lactobacillus KD9 used in the following examples ( Liquorilactobacillus ghanensis Ghana liquid Lactobacillus KD9, a lactic acid bacteria isolated from water kefir grains, has been deposited in the China Center for Type Culture Collection (CCTCC) on April 1, 2024; the deposit address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit number is CCTCC NO:M 2024604. The isolation and identification, whole genome sequencing, bacteriocin B-KD9 preparation and structural identification and application of this strain are as follows: 1. Water kefir grains were stored in the 2-065 laboratory of the School of Food Science and Engineering of Shaanxi University of Science and Technology. The water kefir grains washed with PBS buffer were mixed with sterile saline to make a homogenous solution. The homogenous solution was diluted with sterile saline and then spread on MC, MRS, and Elliker culture media, cultured at 37°C for 48 to 96 hours, and a single colony was picked with an inoculation loop for separation and purification.

[0026] 2. Cultivation characteristics like Figure 1 As shown, colony morphology: the shape is mostly round, convex, with neat edges and milky white (MRS culture medium), bacterial cells: microscopic observation shows that they are short rods, and further observation through scanning electron microscopy shows that the bacteria are short rods with a smooth surface. 3. Gene identification The genome of the strain was used as a template, and PCR amplification was performed using universal primers for identifying bacteria, 27F and 1492R, 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′, 1492R: 5′-GGTTACCTTGTTACGACTT- 3′. The 16srDNA sequence of the strain (such as sequence 1) is: Sequence comparison showed that the 16S rDNA sequence of this strain was similar to that of Ghana liquid Lactobacillus Liquorilactobacillus ghanensis The 16S rDNA sequence identity of the strain was 99.86% (NCBI accession number: AB690235.1). Based on the colony morphology, cell morphology and 16srDNA sequence comparison, the strain was confirmed to be Ghana liquid Lactobacillus and was named: Ghana liquid Lactobacillus KD9 ( Liquorilactobacillus ghanensis Ghana liquid Lactobacillus KD9). 4. Whole genome sequencing of Ghana liquid Lactobacillus KD9 Ghanaian liquid Lactobacillus KD9 was activated three times at a 2% inoculation rate and then cultured at 37°C for 10h (logarithmic phase); centrifuged at 4°C, 14,000 rpm for 5min, the bacteria were collected and washed 2-3 times with sterile water; DNA was extracted using a genome purification kit, the genomic DNA was collected and purified, the DNA was fragmented using Covaris, an Illumina sequencing library was constructed, and the library was processed and sequenced according to the procedure; the DNA was processed into 8-10k fragments using the G-tubes method, the ends were blunted, and the two ends were connected to circular single strands to construct a PacBio sequencing library, which was processed and sequenced according to the procedure.

[0027] The results showed that there was one chromosome and one plasmid in Ghanaian liquid lactobacillus KD9, with a G+C content of 37.15% and a genome of 2584510 bp. The chromosome was 2541967 bp long, with a G+C content of 37.21%. The plasmid (PlasmidA) was 42543 bp long, with a G+C content of 33.15%, and a total of 2371 coding genes. At the same time, the genome of Ghanaian liquid lactobacillus KD9 contained 18 rRNAs, 55 tRNAs and 43 sRNAs.

[0028] Plasmids were identified in the bacterial genome assembly results using PlasFlow (https: / / github.com / smaegol / PlasFlow) software. The plasmid sequences were obtained and annotated using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) software and the PLSDB database (https: / / ccb-microbe.cs.uni-saarland.de / plsdb / ). We identified it as a new plasmid genome.

[0029] 5. Application of Ghana Liquid Lactobacillus KD9 The Ghanaian liquid Lactobacillus KD9 of the present invention was tested for hemolytic activity, survival rate in simulated gastrointestinal fluid in vitro and adhesion ability of Caco-2 cells. Based on the test results, it is shown that the Ghanaian liquid Lactobacillus KD9 of the present invention can be added to functional foods or feeds as a bacteriocin-producing probiotic lactic acid bacteria, as follows: 1) Hemolytic activity test of Ghana liquid Lactobacillus KD9 Some bacteria in nature secrete hemolysin and show certain pathogenicity, so the selected Ghana liquid Lactobacillus KD9 should be non-pathogenic to be used as probiotic lactic acid bacteria. Figure 2 As shown in the figure, a typical transparent ring appeared around the Vibrio parahaemolyticus in the control group, indicating strong pathogenicity. No transparent ring appeared around the Ghana liquid Lactobacillus KD9, indicating no pathogenicity.

[0030] 2) Survival rate of Ghana liquid Lactobacillus KD9 in simulated gastrointestinal fluid in vitro Good probiotic lactic acid bacteria need to have a certain tolerance to gastrointestinal fluid. After Ghana liquid Lactobacillus KD9 was treated with simulated gastric fluid for 3 hours, it was added to simulated intestinal fluid for 4 hours, and the viable bacteria were counted at each step to verify its gastrointestinal fluid tolerance. The survival rate of Ghana liquid Lactobacillus KD9 in simulated gastric fluid was 41.34%, and the final survival rate after 4 hours of artificial intestinal fluid culture was 29.67%. The results showed that Ghana liquid Lactobacillus KD9 showed good gastrointestinal fluid tolerance.

[0031] 3) Adhesion ability of Ghana liquid Lactobacillus KD9 to Caco-2 cells Adhesion ability is an important prerequisite for lactic acid bacteria to colonize and play a role in regulating intestinal flora, resisting pathogens, and improving the body's immunity. The adhesion rate of Ghana liquid Lactobacillus KD9 was 55.90%, showing good adhesion to Caco-2 cells.

[0032] 6. Application of Ghanaian liquid Lactobacillus KD9 fermentation supernatant Cultivation characteristics in MRS liquid medium: After inoculation, the culture was carried out at 37°C for 48 h, and the obtained fermentation broth was centrifuged (4°C, 8000r / min, 15min) to obtain the cell-free fermentation supernatant, such as Figure 3 As shown, the inhibition zone sizes of its fermentation supernatant against Vibrio parahaemolyticus and Vibrio alginolyticus were determined by the Oxford cup method and were 15.49±0.24 mm and 15.61±0.22 mm, respectively. The results showed that its fermentation supernatant had antibacterial activity against Vibrio parahaemolyticus and Vibrio alginolyticus.

[0033] 7. Preparation method of bacteriocin B-KD9 The Ghanaian liquid Lactobacillus KD9 was cultured in MRS liquid medium at 37°C for 48 hours to obtain the fermentation broth, which was centrifuged, mixed with ethyl acetate in a ratio of 1:1 for extraction, and the filtrate with a molecular weight cutoff of <5 kDa was retained using an ultrafiltration device and an ultrafiltration membrane. After concentration, freeze-drying, and separation by preparative chromatography, the chromatographic separation conditions were: absorption wavelength: 220 nm, flow rate: 40 mL / min, preparative chromatographic column 50DAC, mobile phase A, acetonitrile (CAN), mobile phase B: 0.1% trifluoroacetic acid in ultrapure water (TFA-H2O). The gradient elution program was that the acetonitrile concentration increased from 5% to 65%, and the fraction with a retention time of 23.3 min was collected. Bacteriocin B-KD9 with a purity greater than 75% can be obtained, see Figure 4 and 5 . 8. Structural identification of bacteriocin B-KD9 The obtained bacteriocin B-KD9 was analyzed by LC-MS / MS equipped with an online nanospray ion source. The whole system was an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, MA, USA) connected in series with an EASY-nanoLC 1200. The mass spectrometry detection conditions were a scan range of 200-1500 m / z, a column flow rate of 350 nL / min, a column temperature of 40°C, and an electrospray voltage of 2 kV. The gradient started from 2.2% of phase B, and the nonlinear gradient was increased to 50% after 51 minutes, and then rapidly increased to 90% within 3.5 minutes, and maintained at this state for 5.5 minutes. It was identified that bacteriocin B-KD9 contained 5 peptides, and the amino acid sequence (such as sequence 2~6), molecular formula, molecular weight, electrostatic charge number, isoelectric point, and water solubility of the peptides are listed in Table 1: Table 1 Amino acid sequence of bacteriocin B-KD9

[0034] The identified amino acid sequence was compared and analyzed using the APD3 antimicrobial peptide database, NCBI BLAST and UniProt databases. One known peptide and four unknown potential peptides (de novo sequencing) were identified, of which PVGLADPDGPA is part of AP01970 in the APD3 database. Studies have shown that the peptide has antibacterial activity against both Gram-negative and Gram-positive bacteria, and the same peptide was also found in lactic acid bacteria MN047. On the other hand, no similar peptides were found for FTGPDVKFP, PDSPLFDKT, FPSLVGRPR and TDDLLEDLK in the comparison of APD3, NCBI BLAST and UniProt databases, and they are all different from the amino acid sequences of the bacteriocins that have been reported and identified so far, indicating that bacteriocin B-KD9 is a new type of leaderless bacteriocin and contains 5 peptides, which was studied, identified and reported by the inventors for the first time. Its secondary mass spectrum is shown in the figure below. Figure 6 , 7 , 8, 9, and 10.

[0035] 8. Application of bacteriocin B-KD9 Bacteriocin B-KD9 can be used as a food preservative, a functional feed additive, and an antibacterial drug for infections with Vibrio parahaemolyticus and Vibrio alginolyticus.

[0036] Example 1 Chemical Synthesis of Bacteriocin B-KD9 Bacteriocin B-KD9 can also be obtained by chemical synthesis by biotechnology companies based on its amino acid sequence, with a purity greater than 98%.

[0037] Example 2 Determination of Minimum Inhibitory Concentration (MIC) of Bacteriocin B-KD9 against Vibrio parahaemolyticus The crude bacteriocin B-KD9 was prepared into a 160 mg / mL stock solution with ultrapure water, and then the stock solution was diluted twice with LB liquid medium. Different volumes of stock solution were added to 96-well plates, and Vibrio parahaemolyticus was used as the indicator bacteria. The plates were cultured at 37°C for 12 h. As shown in Table 2, the OD at 0.5 mg / mL was 600nm The value was 0.048. With the further increase of bacteriocin B-KD9 concentration, OD 600nm The value tends to be stable. Further observation of the results of the 96-well plate, such as Fig.11 As shown in the figure, 96-well plate numbers 1 to 12 represent concentrations from 32 mg / mL to 0 mg / mL; B, C, and D represent three groups of parallel samples, and F is a control group with only the corresponding concentration of bacteriocin added. The results showed that when the concentration of bacteriocin B-KD was 0.5 mg / mL, the culture medium appeared clear, and the MIC was preliminarily determined to be 0.5 mg / mL. The minimum inhibitory concentration (MIC) of bacteriocin B-KD9 against Vibrio parahaemolyticus was 0.5 mg / mL; Table 2 OD values ​​of Vibrio parahaemolyticus under the action of different concentrations of bacteriocin B-KD9

[0038] Example 3 Determination of the minimum bactericidal concentration (MBC) of bacteriocin B-KD9 against Vibrio parahaemolyticus According to the MIC results, bacteriocin B-KD9 was added to LB liquid culture medium to give final concentrations of 0, 1 / 2MIC, MIC, 2MIC and 4MIC, respectively, and Vibrio parahaemolyticus (1×10 6 CFU / mL), and after culturing for 9 h (37°C, 200 r / min), 100 μl of each was spread on an LB plate and cultured at 37°C for 24 h. The growth of the colonies was observed and photographed using a colony counter. Fig.12 As shown in the figure, the control group of Vibrio parahaemolyticus grew well; after the Vibrio parahaemolyticus was treated with 1 / 2MIC, the colonies growing on the plate were significantly reduced. When the added concentrations were MIC, 2MIC, and 4MIC, no Vibrio parahaemolyticus colonies grew; therefore, it was determined that the MBC and MIC concentrations of bacteriocin B-KD9 against Vibrio parahaemolyticus were consistent, both 0.5 mg / mL.

[0039] Example 4 pH tolerance of bacteriocin B-KD9 The pH values ​​of bacteriocin B-KD9 were adjusted to 2, 4, 6, 8, and 10 with 1 mol / L HCL and NaOH, and the antibacterial activity was determined by the Oxford cup method. Fig.13 As shown, bacteriocin B-KD9 maintained strong antibacterial activity against Vibrio parahaemolyticus in the pH range of 2 to 10, indicating that bacteriocin B-KD9 has good stability to pH. Example 5 Sensitivity of bacteriocin B-KD9 to temperature The bacteriocin B-KD9 was treated at 40℃, 60℃, 80℃, 100℃, and 120℃ for 30 min, and its antibacterial activity was determined by the Oxford cup method. Fig.14 As shown, bacteriocin B-KD9 maintained strong antibacterial activity against Vibrio parahaemolyticus in the range of 40°C to 120°C, indicating that bacteriocin B-KD9 has good tolerance to temperature.

[0040] Example 6 Application of bacteriocin B-KD9 and / or Lactobacillus ghanaiensis KD9 in zebrafish infected with Vibrio parahaemolyticus 4 dpf wild-type AB strain zebrafish were randomly selected and placed in a 6-well plate, with 30 zebrafish in each well (experimental group). Griffonia liquid Lactobacillus KD9 (concentration range: 1.00×10 7 ~1.00×109 CFU / mL), bacteriocin B-KD9 (concentration range: 125-500 μg / mL), bacteriocin B-KD9+Ghana liquid Lactobacillus KD9 (concentration range: 125-500 μg / mL, 1.00×10 7 ~1.00×10 9 CFU / mL), and set up a normal control group and a positive control group (chloramphenicol: 2000 μg / mL). The zebrafish Vibrio parahaemolyticus infection model was established by immersion infection. After 48 hours of treatment, the number of dead tails was recorded, and the number of deaths was counted and photographed under a dissecting microscope to analyze the mortality rate of zebrafish. Fig.15 The CCD camera was used to capture the morphology of zebrafish in the control group and the model group.

[0041] Table 3 Experimental results of the efficacy of bacteriocin B-KD9 and (or) Ghana liquid Lactobacillus KD9 against Vibrio parahaemolyticus infection (n=30)

[0042] Note: Compared with the model control group, * p <0.05,** p <0.01,*** p <0.001 The results are shown in Table 3 and Fig.16 As shown in the figure, the red arrow is the zebrafish liver: Under the experimental conditions: Ghana liquid Lactobacillus KD9 was 1.00×10 7 , 1.00×10 8 and 1.00×10 9 CFU / mL concentration, 16, 14, and 10 zebrafish died, with mortality rates of 53%, 47%, and 33%, respectively. 7 , 1.00×10 8 There was no significant difference in CFU / mL compared with the model control group ( p >0.05), 1.00×10 9 CFU / mL was significantly different from that of the model control group ( p <0.05), indicating that Ghana liquid Lactobacillus KD9 is 1.00×10 7 , 1.00×10 8 CFU / mL concentration was not effective against Vibrio parahaemolyticus infection. 9 CFU / mL concentration, it has anti-Vibrio parahaemolyticus infection efficacy, further proving that the bacteriocin-producing probiotic Ghana liquid Lactobacillus KD9 screened in this study can colonize in zebrafish and exert anti-infection probiotic effects.

[0043] Bacteriocin B-KD9 at concentrations of 125 μg / mL, 250 μg / mL, and 500 μg / mL caused the death of 8, 5, and 2 zebrafish, respectively, with mortality rates of 27%, 17%, and 7%, respectively, which were significantly different from the model control group ( p <0.01& p <0.001& p <0.001), indicating that bacteriocin B-KD9 has anti-Vibrio parahaemolyticus efficacy at concentrations of 125 μg / mL, 250 μg / mL and 500 μg / mL.

[0044] Bacteriocin B-KD9 + Ghana liquid Lactobacillus KD9 at 125 μg / mL + 1.00×10 7 CFU / mL, 250 μg / mL+1.00×10 8 CFU / mL and 500 μg / mL+1.00×10 9 At the CFU / mL concentration, 6, 3, and 0 zebrafish died, respectively, with mortality rates of 20%, 10%, and 0%, respectively, which were significantly different from the model control group ( p <0.001), indicating that bacteriocin B-KD9 + Ghana liquid Lactobacillus KD9 at 125 μg / mL + 1.00×10 7 CFU / mL, 250 μg / mL+1.00×10 8 CFU / mL and 500 μg / mL+1.00×10 9 CFU / mL concentration, and the effect was more significant than that of using Ghana liquid Lactobacillus KD9 or bacteriocin B-KD9 alone. It further proved that the combined use of Ghana liquid Lactobacillus KD9 used in this study and its bacteriocin B-KD9 showed a synergistic effect in inhibiting zebrafish infected with Vibrio parahaemolyticus.

[0045] The present invention can also clone and overexpress the bacteriocin-producing gene encoded by Ghana liquid Lactobacillus KD9 through genetic engineering technology combined with bioinformatics, and use fermentation technology to optimize the production process of KD9, thereby increasing the yield of bacteriocin B-KD9 from multiple dimensions. The present invention conducts in-depth research and analysis on the molecular structure of bacteriocin B-KD9, and studies its structural modification and functional optimization through synthetic biology methods, providing the possibility for developing derivatives with stronger antibacterial activity and better stability.

[0046] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A liquid Lactobacillus KD9, characterized in that It is deposited in China Center for Type Culture Collection with the deposit number CCTCC NO:M 2024604.

2. A liquid Lactobacillus Ghana KD9 according to claim 1, characterized in that, The Ghanaian liquid lactobacillus KD9 produces bacteriocin B-KD9, and the bacteriocin B-KD9 includes 5 peptides, specifically: FTGPDVKFP, PVGLADPDGPA, PDSPLFDKT, FPSLVGRPR, and TDDLLEDLK.

3. Use of the liquid Lactobacillus Ghana KD9 described in claim 1 or 2 as a probiotic in functional food or feed.

4. A liquid Lactobacillus KD9 fermentation liquid, characterized in that: The method is prepared by fermenting the liquid Lactobacillus KD9 of claim 1 or 2.

5. Use of a liquid Lactobacillus Ghana KD9 fermentation liquid according to claim 4 in the preparation of antibacterial drugs.

6. A bacteriocin B-KD9, characterized in that The fermentation supernatant of the Ghanaian Lactobacillus KD9 according to claim 1 is centrifuged, extracted, ultrafiltered, concentrated, freeze-dried, separated and purified to obtain bacteriocin B-KD9.

7. A bacteriocin B-KD9 according to claim 6, characterized in that: The bacteriocin B-KD9 contains 5 polypeptides, and the amino acid sequences of the 5 polypeptides are specifically: FTGPDVKFP, PVGLADPDGPA, PDSPLFDKT, FPSLVGRPR, and TDDLLEDLK.

8. Use of the bacteriocin B-KD9 described in claim 6 or 7 in the preparation of antibacterial drugs.

9. An antibacterial drug, characterized in that: The bacteriocin B-KD9 according to claim 6; Or the bacteriocin B-KD9 described in claim 6 and the liquid Lactobacillus KD9 described in claim 1; Or the Ghana liquid Lactobacillus KD9 fermentation broth according to claim 4; Or the Ghana liquid Lactobacillus KD9 fermentation liquid described in claim 4 and the Ghana liquid Lactobacillus KD9 described in claim 1.

10. An aquaculture biological feed, characterized in that: The feed contains the antibacterial drug according to claim 9.

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