Lactic acid bacterial bacteriocin and use thereof

Abstract

This invention relates to a lactobacillus bacteriocin and its application, belonging to the field of microbial applications. One objective of this invention is to provide a lactobacillus bacteriocin that discovers potential lactobacillus bacteriocins through some relatively common and simple methods. A second objective is to provide an application of a lactobacillus bacteriocin in the preparation of antibacterial and / or preservative products. A third objective is to provide an antibacterial and / or preservative product. The lactobacillus bacteriocin is lactobacillus bacteriocin A and / or lactobacillus bacteriocin B; the amino acid sequence of lactobacillus bacteriocin A is: RNKMAYNVGKAISRIMRRVR; the amino acid sequence of lactobacillus bacteriocin B is: KKKRGFWYHVGDAVTSFGRGFASAFG. The application of a lactobacillus bacteriocin in the preparation of antibacterial and / or preservative products is also discussed.

Description

Technical Field

[0001] This invention belongs to the field of microbial applications, specifically relating to a lactic acid bacteria bacteriocin and its applications. Background Technology

[0002] Lactic acid bacteria are a general term for non-spore-forming, Gram-positive bacteria that produce lactic acid during the fermentation of glucose or lactose. Lactic acid bacteria have a long history in fermented food production, and antibacterial lactic acid bacteria are widely found in various lactic acid fermented products. Furthermore, many lactic acid bacteria possess probiotic activity, exerting various beneficial functions on human health by regulating the human gut microbiota, inhibiting the growth of intestinal pathogens, and secreting active compounds.

[0003] Lactic acid bacteria bacteriocins are a class of natural proteins or polypeptides with antibacterial activity, produced by the ribosome synthesis mechanism of lactic acid bacteria. Studies have shown that lactic acid bacteria bacteriocins have excellent properties for inhibiting the growth and reproduction of microorganisms. Over the past 30 years, their safety and effectiveness in food applications have been repeatedly proven. Lactic acid bacteria bacteriocins have a certain effect on inhibiting and eliminating pathogenic bacteria in the body, and can prevent food spoilage. Furthermore, many lactic acid bacteria bacteriocins have demonstrated the ability to inhibit human pathogens in the biomedical field. Because lactic acid bacteria bacteriocins are non-toxic and leave no residue, they have great application prospects.

[0004] However, since most existing lactobacillus bacteriocins have a broad antibacterial spectrum, they may disrupt other inherent beneficial bacteria during application. Therefore, there is a need to explore novel lactobacillus bacteriocins with more precise antibacterial spectrums. Developing a rapid and economical method for screening lactobacillus bacteriocins is of positive significance for the application of bacteriocins in the food and biopharmaceutical industries. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a lactic acid bacteria bacteriocin and its application.

[0006] One of the objectives of this invention is to provide a lactic acid bacteria bacteriocin that discovers potential lactic acid bacteria bacteriocins through some common and simple methods.

[0007] A second objective of this invention is to provide an application of lactic acid bacteria bacteriocin in the preparation of antibacterial and / or preservative products.

[0008] A third objective of this invention is to provide an antibacterial and / or preservative product.

[0009] A lactobacillus bacteriocin, comprising lactobacillus bacteriocin A and / or lactobacillus bacteriocin B;

[0010] The amino acid sequence of the lactic acid bacteria bacteriocin A is: RNKMAYNVGKAISRIMRRVR;

[0011] The amino acid sequence of the lactic acid bacteria bacteriocin B is: KKKRGFWYHVGDAVTSFGRGFASAFG.

[0012] The use of a lactic acid bacteria bacteriocin in the preparation of antibacterial and / or preservative products.

[0013] Furthermore, a lactobacillus bacteriocin is used in antibacterial and / or preservative products to inhibit one or more of Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus.

[0014] An antibacterial and / or preservative product comprising the aforementioned lactobacillus bacteriocin.

[0015] The present invention has the following beneficial effects:

[0016] I. The lactobacillus bacteriocin A and lactobacillus bacteriocin B provided by this invention have the advantages of simple structure, safety and low toxicity, fast screening speed, low cost, easy synthesis, and high efficiency in inhibiting Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Micrococcus luteus.

[0017] II. The lactic acid bacteria bacteriocin provided by this invention can be used in the preparation of antibacterial and / or preservative products. Specifically, it can be used to prepare food preservatives, feed additives, antibacterial drugs, etc., and has broad application prospects. Attached Figure Description

[0018] Figure 1 The inhibition zone of a portion of the bacterial supernatant in Example 1 against three indicator bacteria: Escherichia coli, Staphylococcus aureus, and Bacillus cereus.

[0019] Figure 2 This refers to the gene cluster where the lactobacillus bacteriocin A and / or lactobacillus bacteriocin B screened in Example 1 are located. Detailed Implementation

[0020] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0021] Example 1: The method for screening lactobacillus bacteriocin A and lactobacillus bacteriocin B is specifically carried out according to the following steps:

[0022] I. Preliminary screening of lactic acid bacteria bacteriocins:

[0023] ① RAPD-PCR:

[0024] RAPD is a molecular technique based on PCR that allows for the analysis of the entire genome sequence. In this example, the PCR amplification system used was a 50 μL reaction volume. After amplification, 5 μL of the PCR product was separated by 1.0% agarose gel electrophoresis, stained with ethidium bromide, and observed and photographed using a BioRad2 Gel2700™ gel imaging system. The three primer sequences are as follows:

[0025] M13: 5ˊ-GAGGGTGGGCGGTTCT-3ˊ;

[0026] M14: 5ˊ-GAGGGTGGGGCCGTT-3ˊ;

[0027] D8635:5ˊ-GAGCGGCCAAAGGGAGCAGAC-3ˊ;

[0028] Ninety-nine lactic acid bacteria isolates from different sources were inoculated into 1 mL of MRS liquid medium and cultured at 37°C for 24 h. The cultured cells were then transferred to 2 mL grinding tubes, grinding beads were added, and the cells were lysed in a cryo-grinder (20 m / s, 60 s working time, 120 s interval, repeated 3 times). The supernatant was used as the DNA template for PCR amplification.

[0029] I. The PCR reaction uses a 50 μL system: 1 μL template DNA (10 ng), 2 μL M13 primers (both forward and reverse primers are M13, and the concentration of M13 primers is 10 μM), 25 μL 2×Taq PCR MasterMix, and H2O is added to 50 μL. The final concentration of M13 primers is 0.2 μM.

[0030] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 42℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 10 min; 4℃, ∞.

[0031] II. The PCR reaction uses a 50 μL system: 1 μL template DNA (10 ng), 2 μL M14 primers (both forward and reverse primers are M14, and the concentration of M14 primers is 10 μM), 25 μL 2×Taq PCR MasterMix, and H2O is added to 50 μL. The final concentration of M14 primers is 0.2 μM.

[0032] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 42℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 10 min; 4℃, ∞.

[0033] III. The PCR reaction uses a 50 μL system: 1 μL template DNA (10 ng), 2 μL D8635 primers (both forward and reverse primers are D8635, and the concentration of D8635 primers is 10 μM), 25 μL 2×Taq PCR MasterMix, and H2O is added to 50 μL. The final concentration of D8635 primers is 0.2 μM.

[0034] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 42℃ annealing for 30 s, 72℃ extension for 2 min, 30 cycles; 72℃ extension for 10 min; 4℃, ∞.

[0035] ② Oxford cup method for determining the activity of bacterial supernatant:

[0036] The test results in the above steps are as follows: 99 lactic acid bacteria isolates were identified as 58 different species of lactic acid bacteria isolates. The 58 lactic acid bacteria isolates identified as different species were inoculated into 2 mL of MRS liquid medium, cultured at 37℃ for 24 h, centrifuged at 10000 rpm for 2 min, and the supernatant was collected for later use.

[0037] Prepare and sterilize 0.75% LB agar and 1.5% agar in advance; melt the 1.5% agar and pour it into sterile petri dishes, 10 mL / dish, and set aside. After cooling, place an appropriate amount of sterile Oxford cups on the 1.5% agar in each petri dish, with a certain distance between them. Melt the 0.75% LB agar and, when it cools to 50°C, add 0.01% of indicator bacteria such as Escherichia coli, Staphylococcus aureus, or Bacillus cereus, mix well, and pour into petri dishes, 25 mL / dish. After cooling, remove the Oxford cups. Add 100 μL of MRS liquid medium to each control well in the petri dish and 100 μL of lactic acid bacteria supernatant to each sample well. Incubate at 37°C for 16 hours and observe the results. The inhibition zone of the bacterial supernatant is as shown in the image. Figure 1 As shown;

[0038] II. Genomic DNA Extraction:

[0039] ① The genomic DNA of the lactic acid bacteria isolates with antibacterial effects obtained in step one was extracted using phenol and chloroform:

[0040] First, take 2 mL of MRS overnight culture medium (37℃, 48 h) inoculated with lactic acid bacteria isolates, centrifuge at 10000 rpm for 2 min, discard the supernatant, resuspend the precipitate in 250 μL STE buffer, transfer to a 2 mL grinding tube, add grinding beads, and cryogenically lyse the cells in a cryogenic homogenizer (20 m / s, 60 s working time, 120 s interval, repeated 3 times); after centrifuging the lysate at 6000 rpm for 10 min, accurately transfer the supernatant to a new 2 mL centrifuge tube, add 2.5 μL lysozyme (100 mg / mL) and 10 μL RNase (10 mg / mL), mix by inversion, and incubate at 37℃ for 30 min; then add 7.5 μL Proteinase K (20 mg / mL) and 20 μL SDS (10%), mix by inversion, and incubate at 55℃ for 30 min; after incubation, add 750 μL STE buffer. Add buffer and 600 μL of phenol:chloroform:isoamyl alcohol mixture (25:24:1, v / v / v), invert and mix well, let stand at room temperature for 15 min, centrifuge at 10000 rpm for 5 min, and accurately transfer the upper aqueous layer to a new 2 mL centrifuge tube; repeat this operation twice; add 500 μL of chloroform:isoamyl alcohol mixture (24:1, v / v) to the obtained aqueous solution, centrifuge at 10000 rpm for 5 min, and accurately transfer the upper aqueous layer to a new 2 mL centrifuge tube; add 10% (v / v) sodium acetate solution (3M), mix well, let stand for 2 min, add 66% isopropanol solution (v / v), mix well, and let stand for 15 min to allow DNA precipitation; centrifuge at 10000 rpm for 10 min, and discard the supernatant; wash the precipitate particles 3 times with 80% ethanol, discard the supernatant, and air dry; add 50 μL of TE buffer or deionized water to dissolve the DNA, and incubate at 55 °C for 15 min. All centrifugation conditions were performed at 4°C.

[0041] ② The quantity and quality of the separated DNA were detected using a micro-volume UV spectrophotometer at wavelengths of 230 nm, 260 nm, and 280 nm. DNA sample concentrations ranged from 150 ng / μL to 700 ng / μL, with OD 260 / 280 ratios between 1.8 and 2.0, and OD 260 / 230 ratios exceeding 1.5. Verification was performed on a 1% agarose gel.

[0042] III. Metagenomic sequencing:

[0043] DNA from lactic acid bacteria isolates with antibacterial activity was diluted to the same concentration and then mixed in equal proportions to form a total DNA sample of 35 μL for metagenomic sequencing.

[0044] IV. Analysis of potential lactobacillus bacteriocins:

[0045] By predicting metagenomic secondary metabolites using antiSMASH, two potential bacteriocins were identified, such as... Figure 2 As shown. The amino acid sequences are as follows:

[0046] Lactobacillus bacteriocin A: RNKMAYNVGKAISRIMRRVR;

[0047] Lactobacillus bacteriocin B: KKKRGFWYHVGDAVTSFGRGFASAFG;

[0048] Online BlastP alignment analysis (Protein BLAST: search protein databases using a protein query (nih.gov)) revealed no homology between the two bacteriocins and previously identified and reported bacteriocins, indicating that they are novel bacteriocins and represent the inventors' first research, identification, and reporting of this product. Based on the shown amino acid sequences, the two bacteriocins were further synthesized, and the corresponding antimicrobial peptides with a purity >90% were obtained by Sangon Biotech (Shanghai) Co., Ltd.

[0049] Example 2: Antibacterial effect of lactic acid bacteria bacteriocins:

[0050] Lactobacillus bacteriocin A and B were each prepared into 8 mM solutions using 5% DMSO solution, and then serially diluted to 6.25 μM using a 2-fold dilution method. Indicator bacteria included *Escherichia coli*, *Staphylococcus aureus*, *Bacillus cereus*, and *Micrococcus luteus*, each adjusted to a final concentration of 5 × 10⁻⁶. 5 CFU / mL. Samples were spotted using 96-well plates, with 90 μL of indicator bacteria and 10 μL of lactobacillus bacteriocin solution added to each well. The control sample consisted of 90 μL of indicator bacteria and 10 μL of 5% DMSO solution. The minimum inhibitory concentration (MIC) was determined based on the turbidity of the bacterial suspension in each well. The MICs of the two bacteriocins against Staphylococcus aureus, Escherichia coli, Bacillus cereus, and Micrococcus luteus are shown in Table 1.

[0051] Table 1

[0052]

Claims

1. The application of a lactobacillus bacteriocin, characterized in that... The use of a lactobacillus bacterin in the preparation of antibacterial and / or preservative products; wherein the antibacterial and / or preservative products include lactobacillus bacterin; wherein the lactobacillus bacterin is lactobacillus bacterin A or lactobacillus bacterin B; When the lactobacillus bacteriocin is lactobacillus bacteriocin A, the lactobacillus bacteriocin is used to inhibit Escherichia coli in antibacterial and / or preservative products, and the minimum inhibitory concentration of lactobacillus bacteriocin A against Escherichia coli is 12.5 μM. When the lactobacillus bacteriocin is lactobacillus bacteriocin B, the lactobacillus bacteriocin is used in antibacterial and / or preservative products to inhibit Escherichia coli, Staphylococcus aureus, and Bacillus cereus. The minimum inhibitory concentration (MIC) of lactobacillus bacteriocin B against Escherichia coli is 3.125 μM; against Staphylococcus aureus is 12.5 μM; and against Bacillus cereus is 6.25 μM. The amino acid sequence of the lactic acid bacteria bacteriocin A is: RNKMAYNVGKAISRIMRRVR; The amino acid sequence of the lactic acid bacteria bacteriocin B is: KKKRGFWYHVGDAVTSFGRGFASAFG.

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