Aquatic product source Weissella cibaria and application thereof

Abstract

The invention relates to an aquatic product source Weissella cibaria, the Weissella cibaria is named Weissella cibaria Gut 2, and is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO.65901, the preservation address is the 5th floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation date is February 18, 2025. The aquatic product source Weissella cibaria is named as Weissella cibaria Gut 2, and the Weissella cibaria is preserved in the Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO.65901. The aquatic product source Weissella cibaria can generate bacteriocin with broad-spectrum antibacterial activity, the bacteriocin has a remarkable inhibition effect on various food-borne pathogenic bacteria, due to the characteristics, the aquatic product source Weissella cibaria can become a potential antibiotic substitute for controlling the food-borne pathogenic bacteria, and a promising application scheme is provided for prolonging the shelf life of food.

Description

Technical Field

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

[0002] With the increasing abuse of antibiotics, the problem of drug resistance has become more serious, and traditional antibiotics have encountered bottlenecks in dealing with drug-resistant bacteria. Therefore, there is an urgent need to develop a new type of antibacterial agent that is not easy to induce bacterial resistance. Bacteriocins are a class of natural peptide antibacterial agents that have attracted much attention because of their non-specific target action mechanism against pathogens.

[0003] Although there are a lot of research literature on bacteriocins from microorganisms such as Lactobacillus, Enterococcus and Leuconostoc, there is still a lack of research on bacteriocins produced by Weissella, especially Weissella cibaria. In view of this, developing and utilizing the ability of Weissella cibaria to produce bacteriocins can provide an important resource for the development of new antimicrobial agents.

[0004] Weissella sinusitis is a lactic acid bacterium that is widely found in plants, vegetables, soil, water, feces, breast milk, animal skin and milk, and mucous membranes (oral cavity, gastrointestinal tract, and vagina) of humans and animals. In recent years, Weissella sinusitis has attracted much attention due to its potential probiotic properties and antibacterial potential. Studies have shown that this strain can produce exopolysaccharides and bacteriocins, and these metabolites show great application prospects in the fields of food preservation and biomedicine. Therefore, in-depth research and development of Weissella sinusitis can provide innovative solutions for the food and pharmaceutical industries. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a strain of aquatic-derived Weissella sinusoidalis and application thereof.

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

[0007] The present invention provides a strain of aquatic Weissella cibaria, which is named as Weissella cibaria Gut 2, preserved in Guangdong Microbiological Culture Collection Center, with a preservation number of GDMCCNO.65901, a preservation address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation date of February 18, 2025.

[0008] The invention proposes a strain of aquatic Weissella sinusitis, which can produce bacteriocins with broad-spectrum antibacterial activity. The bacteriocin has a significant inhibitory effect on a variety of foodborne pathogens. These characteristics make it a potential antibiotic substitute for controlling foodborne pathogens and provide a promising application solution for extending the shelf life of food.

[0009] Preferably, the 16S rDNA of Weissella cibaria 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, opaque, small and convex, with a flat edge; the microscopic examination result of the bacteria is long rod-shaped.

[0011] On the other hand, the present invention provides an application of the aquatic-source Weissella cibaria as described in any one of the above technical solutions in the preparation of an antibacterial agent.

[0012] The present invention screens Weissella cibaria capable of producing broad-spectrum bacteriocin from perch, which can be used for the preparation of antibacterial agents and applied in the food industry.

[0013] Preferably, the antibacterial agent is used for preventing, improving or treating diseases related to Gram-negative bacteria and / or Gram-positive bacteria infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the interference exclusion result of the bacteriocin of Weissella cibaria Gut 2 of the present invention. Among them, A is the exclusion of organic acid interference (pH 4 and 5 are adjusted by HCl to adjust the fermentation broth to pH = 4 and 5, and pH4,5HCl is to adjust HCl to pH = 4 and 5), and B is the exclusion of hydrogen peroxide interference;

[0015] Figure 2 It is the biocompatibility of Weissella cibaria Gut 2 of the present invention;

[0016] Figure 3 It is the morphological characteristics of Weissella cibaria Gut 2 of the present invention. Among them, A is the colony morphology on the front and back, and B is the morphology of the bacteria under scanning electron microscopy;

[0017] Figure 4 It is the gel electrophoresis pattern of the PCR amplification product of Weissella cibaria Gut 2 of the present invention;

[0018] Figure 5 It is the phylogenetic tree of Weissella cibaria Gut 2 of the present invention constructed based on the 16S rDNA gene sequence;

[0019] Figure 6 It is the bacteriocin inhibition spectrum of Weissella cibaria Gut 2 of the present invention;

[0020] Figure 7 It is the anti-biofilm activity of the bacteriocin of Weissella cibaria Gut 2 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] The exemplary embodiments of the present invention will be described in detail below. This detailed description should not be construed as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0022] In the embodiment of the present invention, Weissella cibaria Gut 2 with the preservation number of GDMCC NO.65901 is used, hereinafter referred to as "Weissella cibaria Gut 2" or "strain Gut 2" for short.

[0023] Example 1 Isolation, Identification, and Preservation of Weissella cibaria Gut 2

[0024] 1. Isolation of Weissella cibaria Gut 2

[0025] Taking perch in Fujian Province as the isolation source, the specific isolation method is as follows:

[0026] Collect fish intestines from perch, 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 intestinal mucus, add it to 900 μL of 0.9% NaCl, gently pipette 5 - 6 times, and dilute it in a 10-fold serial gradient. Operate according to the above steps to obtain dilutions of 10 -3 ~10 -8 . Put the pre-sterilized and solidified MRS agar medium into the microwave oven to heat until it completely melts, and pour it into plates for standby. Pipette 10 μL of the above-mentioned liquid with different concentrations and well-mixed onto the plate surface, and gently spread it with a sterilized and cooled spreading rod. After completion, invert it 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 to 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 or 5 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 for strains producing antibacterial substances by the agar diffusion method, and name it Gut 2.

[0027] Figure 1 The results show that as the pH increases, the antibacterial activity of the supernatant of the fermentation broth of strain Gut 2 shows a downward trend, but the change in pH does not cause it to lose its 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.

[0028] Meanwhile, the biocompatibility of strain Gut 2 was verified by blood agar plate. It can be seen from Figure 2 that it does not produce a hemolytic zone in the blood plate, has good biocompatibility, and can become a potential bacteriocin candidate producer.

[0029] 2. Identification of Weissella cibaria Gut 2

[0030] 2.1 Morphological identification

[0031] Take out the glycerol tube containing strain Gut 2, 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, dehydrate it with gradient ethanol, air-dry it naturally, and then sputter gold for 30 s and observe it with a scanning electron microscope.

[0032] The results are as Figure 3 shown. Under the optical microscope, the colonies of strain Gut 2 are round, about 0.6 mm in diameter, grayish-white, opaque, small and convex, with a flat edge ( Figure 3 A); under the scanning electron microscope, its cells are long rod-shaped ( Figure 3 B).

[0033] 2.2 Molecular biology identification

[0034] Strain Gut 2 was identified by DNA sequencing technology. Take 20 μL of the strain Gut 2 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.

[0035] DNA extraction: The genomic DNA of strain Gut 2 was extracted with reference to the operation instructions of the OMEGA Biotek nucleic acid extraction and purification kit, and the concentration and purity of the extracted DNA were detected by measuring the OD260 / 280 value with a micro nucleic acid and protein analyzer.

[0036] PCR amplification: The universal primers for bacterial identification, 27F (5’-AGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’), were selected to identify the species of strain Gut 2.

[0037] Gel electrophoresis: After PCR, prepare a 1% agarose gel, set the electrophoresis voltage at 110 V and the current at 100 mA, and observe the results after 20 min.

[0038] Purification and recovery: Carefully cut the electrophoresis 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.

[0039] The BLAST alignment results showed that the E value (Expect) of strain Gut 2 and Weissella cibaria was 0% and the identity was 100%, indicating good homology between the two. Strain Gut 2 belongs to the genus Weissella cibaria ( Figure 4 and Figure 5 ).

[0040] The PCR product was purified and recovered using the kit, and the full 16S rDNA sequence was sequenced by Tsingke Biotechnology Co., Ltd. The 16S rDNA sequence of strain Gut 2 was 1495 bp in total, and the sequencing results were shown as SEQ ID NO.1.

[0041] 3. Preservation of Weissella cibaria Gut 2

[0042] The strain Gut 2 was classified and named as Weissella cibaria, preserved in the Guangdong Provincial Microbial Culture Collection Center, with the preservation number GDMCC NO.65901, the preservation address being the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the preservation date being February 18, 2025.

[0043] Example 2 Performance determination of Weissella cibaria Gut 2

[0044] Perform performance determination on Weissella cibaria Gut 2 screened in Example 1.

[0045] 1. Antibacterial activity determination of Weissella cibaria Gut 2

[0046] Weissella cibaria Gut 2 was inoculated into MRS liquid medium and cultured 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. The agar diffusion method was used to evaluate the antibacterial effect of Weissella cibaria Gut 2 as an antibacterial agent against indicator bacteria.

[0047] Figure 6 The results showed that the bacteriocin produced in the supernatant of the fermentation broth of Weissella cibaria Gut 2 exhibited good antibacterial activity against 7 Gram-negative bacteria, namely Vibrio alginolyticus 2512, Vibrio alginolyticus TJ-2, Vibrio alginolyticus TJ-11, Vibrio parahaemolyticus 2503, Vibrio harveyi 2510, Escherichia coli K88, and Pseudomonas aeruginosa PAO1. At the same time, it also showed good antibacterial activity against 5 Gram-positive bacteria, namely Listeria monocytogenes 19115, Staphylococcus aureus 6538, Bacillus cereus 63302, Enterococcus faecium 29122, and methicillin-resistant Staphylococcus aureus 43300.

[0048] 2. Determination of the anti-biofilm activity of Weissella cibaria Gut 2

[0049] Determined by crystal violet (CV) staining, cover glasses 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 Weissella cibaria Gut 2 fermentation broth was added, and incubated at 30 °C for 24 h. Each well was gently washed 3 times with phosphate buffer solution (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 600 nm was measured using a microplate reader (ThermoFisher Scientific, Waltham, MA, USA), and observed with an optical microscope.

[0050] Figure 7 The results showed that the bacteriocin produced by Weissella cibaria Gut 2 not only inhibited planktonic bacteria but also biofilm bacteria.

[0051] 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 described in the foregoing embodiments, or perform equivalent replacements for 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 Weissella cibaria isolated from aquatic products, characterized in that, The named Weissella cibaria Gut 2 was deposited in the Guangdong Microbial Culture Collection Center, with the deposit number of GDMCC NO. 65901, the deposit address being the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou, and the deposit date being February 18, 2025.

2. The Weissella cibaria of claim 1, characterized in that, The 16S rDNA of the Weissella cibaria is as shown in SEQ ID NO.

1.

3. The Weissella cibaria of claim 1, characterized in that, The morphological characteristics of the Weissella cibaria include: on MRS medium, the colonies are round, grayish-white, opaque, small and convex, with a smooth edge; the microscopic examination result of the bacteria is rod-shaped.

4. Application of the aquatic-source Weissella cibaria according to any one of claims 1-3 in the preparation of an antibacterial agent.

5. The application according to claim 4, wherein The antibacterial agent is used for preventing, improving or treating diseases related to Gram-negative bacteria and / or Gram-positive bacteria infections.

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