Weissella sinus producing bacteriocin and application thereof

Abstract

The weissella cibarius is named as weissella cibarius H3 and is preserved in the Guangdong Microbial Culture Collection Center, the preservation number of the weissella cibarius H3 is GDMCC NO.65902, the preservation address of the weissella cibarius H3 is the 5th floor of the building 59, No. 100 courtyard, Xianlie Middle Road, Guangzhou, and the preservation date of the weissella cibarius H3 is February 18, 2025. The Weissella sinus producing bacteriocin disclosed by the invention can produce bacteriocin with broad-spectrum antibacterial activity, and the bacteriocin has an antibacterial effect on various food-borne gram-negative pathogenic bacteria (vibrio alginolyticus, vibrio parahaemolyticus and the like) and gram-positive bacteria (listeria monocytogenes, enterococcus faecalis and the like), and has application potential in the aspects of food preservation and fresh keeping.

Description

Technical Field

[0001] The invention relates to the technical field of food microorganisms, in particular to a strain of Weissella sinusoidalis producing bacteriocin and application thereof. Background Art

[0002] With the widespread use of antibiotics, bacterial resistance has become a major challenge facing global health. Today, traditional antibiotics have gradually lost their effectiveness, and many common infectious diseases have once again become a thorny problem. Against this background, it is urgent to find new antibacterial substances that are less likely to cause bacteria to develop resistance.

[0003] Weissella cibaria is a lactic acid bacterium widely found in nature. In recent years, Weissella cibaria has attracted much attention due to its unique probiotic properties. In addition, Weissella cibaria has the ability to produce a variety of antibacterial substances, including organic acids, hydrogen peroxide, and bacteriocins.

[0004] Bacteriocins are a class of natural peptide antimicrobial substances synthesized and secreted by bacteria. They are highly potential candidates for new antibiotics because they can specifically destroy the biofilm of pathogens and have good biocompatibility. Nowadays, in response to the problem of drug resistance in pathogenic bacteria caused by the abuse of antibiotics, the bacteriocins produced by Weissella sinusitis have high research value and are expected to become a new antimicrobial agent for use in the food and pharmaceutical industries. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a bacteriocin-producing Weissella sinusoidalis and application thereof.

[0006] The technical solution adopted by the present invention is:

[0007] In the first aspect, the present invention provides a bacteriocin-producing Weissella cibaria, wherein the Weissella cibaria is named Weissella cibaria H3, deposited in Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC NO.65902, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a deposit date of February 18, 2025.

[0008] The present invention proposes a bacteriocin-producing Weissella sinusitis, which can produce bacteriocin with broad-spectrum antibacterial activity. The bacteriocin has antibacterial effects on a variety of foodborne Gram-negative pathogens (Vibrio alginolyticus, Vibrio parahaemolyticus, etc.) and Gram-positive bacteria (Listeria, Enterococcus faecalis, etc.), and has application potential in food preservation.

[0009] Preferably, the 16S rDNA of Weissella sinusoidalis is as shown in SEQ ID NO.1.

[0010] Preferably, the morphological characteristics of Weissella cibaria include: on MRS medium, the colonies are round, grayish-white, with a smooth and rounded surface and neat edges; the microscopic examination result of the bacterial cells is rod-shaped.

[0011] In a second aspect, the present invention provides an application of the bacteriocin-producing Weissella cibaria as described in any one of the above technical solutions in the preparation of an antibacterial agent.

[0012] Compared with the existing antibacterial and preservative agents on the market, the Weissella cibaria of the present invention applied to an antibacterial agent can inhibit the growth of almost all pathogenic bacteria and better extend the shelf life of food.

[0013] Preferably, the objects to which the antibacterial agent acts include Gram-negative bacteria and Gram-positive bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the interference exclusion result of the bacteriocin of Weissella cibaria H3 of the present invention. Among them, A is to exclude the interference of organic acids (pH 4, 5, 6 means adjusting the fermentation broth to pH = 4, 5, 6 with HCl, and pH4 HCl means adjusting HCl to pH = 4), and B is to exclude the interference of hydrogen peroxide;

[0015] Figure 2 It is the hemolytic property of Weissella cibaria H3 of the present invention;

[0016] Figure 3 It is the morphological characteristics of Weissella cibaria H3 of the present invention. Among them, A is the colony morphology on the front and back, and B is the cell morphology under scanning electron microscopy;

[0017] Figure 4 It is the gel electrophoresis pattern of the PCR amplification product of Weissella cibaria H3 of the present invention;

[0018] Figure 5 It is the phylogenetic tree of Weissella cibaria H3 of the present invention constructed based on the 16S rDNA gene sequence;

[0019] Figure 6 It is the bacteriocin inhibition spectrum of Weissella cibaria H3 of the present invention;

[0020] Figure 7 It is the anti-biofilm activity of the bacteriocin of Weissella cibaria H3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The following will describe the specific embodiments of the present invention in detail with reference to the drawings, but do not make any limitation to the claims of the present invention.

[0022] In the embodiments of the present invention, Weissella cibaria H3 with the preservation number of GDMCC NO.65902 is used, hereinafter referred to as "Weissella cibaria H3" or "strain H3".

[0023] Example 1

[0024] Isolation, identification and preservation of Weissella cibaria H3 in Example 1

[0025] 1. Isolation of Weissella cibaria H3

[0026] Using sea bass in Fujian Province as the isolation source, the specific isolation method is as follows:

[0027] Collect the head tissues from sea bass, seal them in sterile bags respectively, and store them in a refrigerator at 4°C. Put the samples into the ultra-clean bench and irradiate them with ultraviolet light for 30 minutes in advance. In the ultra-clean bench, take 100 μL of head tissue fluid, add it to 900 μL of sterile 0.9% NaCl, gently pipette 5 - 6 times, and dilute it in a 10-fold serial dilution. Operate according to the above steps to obtain 10 -3 ~10 -8 dilutions of head tissue. Put the pre-sterilized and solidified MRS agar medium into the microwave oven and heat it at medium power until it completely melts, then pour it into plates for standby. Pipette 10 μL of the above-mentioned diluted solutions with different concentrations and mix them evenly, drop them on the surface of the plate, and gently spread them with a sterilized and cooled spreading rod. After that, invert the plate and culture it at 37°C until obvious single colonies can be observed and take it out. Pick a single colony and place it in MRS liquid medium, statically culture it at 37°C for 24 hours, centrifuge and take the supernatant, and conduct an acid effect exclusion test and a hydrogen peroxide effect exclusion experiment (exclude the interference of acid by adjusting the pH of the fermentation broth to 4, 5, and 6 by adding 1 mol / L hydrochloric acid, and further add 10 mg / mL catalase to exclude the interference of hydrogen peroxide). Use Pseudomonas aeruginosa PAO1 as the indicator bacterium to screen the strains producing antibacterial substances by the agar diffusion method, and name it H3.

[0028] Figure 1 The results show that with the increase of pH, the antibacterial activity of the fermentation supernatant of strain H3 shows a downward trend, but the change of pH does not make it lose the antibacterial effect ( Figure 1 A), and the addition of catalase has almost no effect on the antibacterial effect ( Figure 1 B), which indicates that the antibacterial substances in the fermentation broth are not organic acids and hydrogen peroxide antibacterial substances, but bacteriocins.

[0029] At the same time, verify the biocompatibility of strain H3 through a blood agar plate. From Figure 2 it can be seen that it will not cause a hemolysis circle on the blood plate, has good biocompatibility, and can become a potential candidate producer of probiotics and bacteriocins.

[0030] 2. Identification of Weissella cibaria H3

[0031] 2.1 Morphological identification

[0032] Take out the glycerol tube containing strain H3, mix it with 6 mL of LB liquid medium, incubate it at 37 °C for 24 h, then take 1 mL of the bacterial liquid, centrifuge it at 12,000 r / min for 1 min in a centrifuge, take the supernatant, and dilute it successively by 10 -1 ~10 -15 , coat it on MRS solid medium, and incubate it at 37 °C for 24 h. At the same time, take 1 mL of the cultured bacterial liquid, centrifuge it at 12,000 r / min for 1 min in a centrifuge, discard the supernatant, add 1 mL of 2.5% glutaraldehyde to fix it overnight, then dehydrate it with a gradient of ethanol, air-dry it naturally, and observe it with a scanning electron microscope after sputtering gold for 30 s.

[0033] The results are as Figure 3 shown. Under the optical microscope, the colonies of strain H3 are round, with a diameter of about 0.6 mm, grayish-white, with a smooth and round surface and neat edges ( Figure 3 A); under the scanning electron microscope, its cells are long rod-shaped ( Figure 3 B).

[0034] 2.2 Molecular biology identification

[0035] Identify strain H3 by DNA sequencing technology. Take 20 μL of the strain H3 obtained from the second screening from the cryopreservation tube and inoculate it into 2 mL of MRS liquid medium. After static incubation at 37 °C for 24 h, transfer it to 20 - 30 mL of MRS medium and statically incubate it at 37 °C for 24 h until the logarithmic growth phase.

[0036] DNA extraction: Extract the genomic DNA of strain H3 with reference to the operation instructions of the OMEGA Biotek nucleic acid extraction and purification kit, and use a micro nucleic acid and protein analyzer to measure the OD260 / 280 value to detect the concentration and purity of the extracted DNA.

[0037] PCR amplification: Select the universal primers for bacteria 27F (5’-AGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) to identify the species of strain H3.

[0038] Gel electrophoresis: After PCR, prepare a 1% agarose gel, set the electrophoresis voltage to 110 V and the current to 100 mA, and observe the results after 20 min.

[0039] Purification and recovery: Carefully cut the electrophoretic band of the PCR product containing the target DNA. According to the operation instructions of the gel recovery kit, purify the PCR product and then perform direct sequencing on it. Finally, input the obtained 16S rDNA sequence into NCBI (https: / / www.ncbi.nlm.nih.gov / ) for BLAST alignment, and construct a phylogenetic tree using MEGA X 11.

[0040] The BLAST alignment results showed that the E value (Expect) between strain H3 and Weissella cibaria was 0% and the identity was 99.74%, indicating good homology between the two. Strain H3 belongs to the genus Weissella cibaria, as Figure 4 and Figure 5 shown.

[0041] The PCR product was purified and recovered using the kit, and the full 16S rDNA sequence was sequenced by Tsingke Biological Co., Ltd. The 16S rDNA sequence of strain H3 was 1511 bp in total, and the sequencing result was shown as SEQ ID NO.1.

[0042] 3. Preservation of Weissella cibaria H3

[0043] The classification and naming of strain H3 is Weissella cibaria. It is preserved in the Guangdong Provincial Microbial Culture Collection Center, with the preservation number GDMCC NO.65902. The preservation address is on the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the preservation date is February 18, 2025.

[0044] Example 2 Performance determination of Weissella cibaria H3

[0045] The Weissella cibaria H3 screened in Example 1 was subjected to performance determination.

[0046] 1. Antibacterial activity determination of Weissella cibaria H3

[0047] Weissella cibaria H3 was inoculated into MRS liquid medium and statically cultured and fermented at 37 °C for 24 h. After centrifugation, the supernatant was taken. Vibrio alginolyticus 2512, Vibrio alginolyticus TJ-2, Vibrio alginolyticus TJ-11, Vibrio parahaemolyticus 2503, Vibrio harveyi 2510, Escherichia coli K88, Pseudomonas aeruginosa PAO1, Listeria monocytogenes 19115, Staphylococcus aureus 6538, Bacillus cereus 63302, Enterococcus faecium 29122, and methicillin-resistant Staphylococcus aureus (MRSA) 43300 were inoculated into LB solid medium, and the agar diffusion method was used to evaluate the antibacterial effect of Weissella cibaria H3 used as an antibacterial agent against indicator bacteria.

[0048] The results are as Figure 6 shown. The supernatant of the fermentation broth of Weissella cibaria H3 has antibacterial activity against foodborne pathogenic bacteria such as Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Enterococcus faecium, and methicillin-resistant Staphylococcus aureus, indicating that Weissella cibaria H3 can produce bacteriocins with broad-spectrum antibacterial activity.

[0049] 2. Determination of the anti-biofilm activity of Weissella cibaria H3

[0050] Through crystal violet (CV) staining, cover slips were added to 6-well plates. 200 μL of activated Vibrio parahaemolyticus TJ-2 was cultured in LB liquid medium, incubated at 30 °C for 12 h, then the supernatant of the fermentation broth of Weissella cibaria H3 was added, and incubated at 30 °C for 24 h. Each well was gently washed 3 times with phosphate buffer saline (PBS, pH 7.2). The adherent cells were air-dried at 60 °C for 30 min, then stained with 200 μL of 0.1% (w / v) crystal violet at room temperature for 5 min, and each well was gently washed again with PBS (pH 7.4). After staining, the stain was dissolved in glacial acetic acid for 10 min. Subsequently, the absorbance at 595 nm was measured using a microplate reader (ThermoFisher Scientific, Waltham, MA, USA), and observed with an optical microscope.

[0051] The experimental results are as Figure 7As shown, it indicates that the bacteriocin produced by Weissella cibaria H3 not only has an inhibitory effect on planktonic bacteria, but also can inhibit biofilm bacteria.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than limiting them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bacteriocin-producing Weissella sinusoidalis strain, characterized in that: The Weissella cibaria is named Weissella cibaria H3, and is preserved in the Guangdong Provincial Microbiological Culture Collection Center with a preservation number of GDMCCNO.65902. The preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is February 18, 2025.

2. The bacteriocin-producing Weissella sinusitis according to claim 1, characterized in that The 16S rDNA of Weissella sinusoidalis is shown as SEQ ID NO.

1.

3. The bacteriocin-producing Weissella sinusitis according to claim 1, characterized in that The morphological characteristics of Weissella sinusoidalis include: on the MRS culture medium, the colonies are round, grayish white, with a round and smooth surface and neat edges; the bacterial body is long rod-shaped according to microscopic examination results.

4. Use of the bacteriocin-producing Weissella sinusitis according to any one of claims 1 to 3 in the preparation of an antibacterial agent.

5. The use according to claim 4, characterized in that The targets of the antibacterial agent include Gram-negative bacteria and Gram-positive bacteria.

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