Leuconostoc lactis and application thereof

Abstract

The invention relates to a strain of leuconostoc lactis, which is named as leuconostoc lactis Gill 3, and is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC NO.65904, the preservation address is building 5, building 59, No. 100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation date is February 18, 2025. The leuconostoc lactis can generate antibacterial substances with narrow-spectrum activity, the antibacterial range is more accurate, the leuconostoc lactis has a remarkable antibacterial effect on common gram-positive bacteria and partial negative pathogenic bacteria in various foods, but the leuconostoc lactis has weak resistance to other indicator bacteria or has no antibacterial activity; meanwhile, the antibacterial substance not only has an inhibition effect on bacterial planktonic cells, but also can resist the formation of bacterial biofilms. Therefore, the leuconostoc lactis disclosed by the invention can be used for prolonging the shelf life of food, and even can be developed into a probiotic.

Description

Technical Field

[0001] The present invention relates to the technical field of food microbiology, and particularly to a Leuconostoc lactis and its application. Background Art

[0002] With the large-scale use of antibiotics, the emergence of drug-resistant bacteria has become a major challenge in the global public health field. To address this issue, there is an urgent need to find a new antibacterial strategy to slow down the spread of antibiotic resistance.

[0003] In this context, bacteriocins, as a natural antibacterial substance, have attracted extensive attention. Bacteriocins are produced by microorganisms and have the characteristics of strong selectivity, low toxicity, and high antibacterial efficiency. Different from traditional antibiotics, bacteria can kill target bacteria by directly acting on the bacterial cell membrane, inhibiting cell wall synthesis, or interfering with protein synthesis, thereby reducing the generation of bacterial drug resistance. Moreover, bacteriocins have low cross-resistance, which makes them show higher application potential in antibacterial therapy.

[0004] Leuconostoc lactis, as a common probiotic, not only plays an important role in intestinal health, immune regulation, etc., but also can produce bacteriocins with broad antibacterial activity. Therefore, in-depth research and development of Leuconostoc lactis can provide innovative solutions for the food and pharmaceutical industries. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a Leuconostoc lactis and its application.

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

[0007] On the one hand, the present invention provides a Leuconostoc lactis named Leuconostoc lactis Gill 3, which is deposited in the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC NO.65904, the deposit address being the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the deposit date being February 18, 2025.

[0008] A strain of Leuconostoc lactis proposed by the present invention can produce an antibacterial substance (bacteriocin) with narrow-spectrum activity, and the antibacterial range is more precise. It has a significant antibacterial effect on Gram-positive bacteria and some negative pathogenic bacteria common in various foods, but has weak antibacterial activity or no antibacterial activity against other indicator bacteria. At the same time, this antibacterial substance can not only inhibit the planktonic cells of bacteria, but also resist the formation of bacterial biofilms. Therefore, the Leuconostoc lactis of the present invention can be used to extend the shelf life of foods and even develop into a probiotic.

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

[0010] Preferably, the morphological characteristics of the Leuconostoc lactis include: on MRS medium, the colonies are round, white and opaque, with a moist and flat surface; the microscopic examination result of the bacteria is short rod-shaped.

[0011] On the other hand, the present invention provides an application of the Leuconostoc lactis according to any one of the above technical solutions in the preparation of antibacterial products.

[0012] The Leuconostoc lactis provided by the present invention is applied in the preparation of antibacterial products, and can be specifically used for the preparation of food preservatives, antibacterial drugs, etc., with broad application prospects.

[0013] Preferably, the objects of action of the antibacterial products include Gram-negative bacteria and Gram-positive bacteria.

[0014] More preferably, the Gram-negative bacteria include Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive bacteria include Staphylococcus aureus, Bacillus cereus and Enterococcus faecalis. Description of the Drawings

[0015] Figure 1 It is the interference exclusion result of the antibacterial substance produced by the Leuconostoc lactis Gill 3 of the present invention. Among them, A is the exclusion of organic acid interference (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 the exclusion of hydrogen peroxide interference;

[0016] Figure 2 It is the biocompatibility of the Leuconostoc lactis Gill 3 of the present invention;

[0017] Figure 3 It is the morphological characteristics of the Leuconostoc lactis Gill 3 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;

[0018] Figure 4 It is the gel electrophoresis diagram of the PCR amplification product of the Leuconostoc lactis Gill 3 of the present invention;

[0019] Figure 5 It is the phylogenetic tree of the Leuconostoc lactis Gill 3 of the present invention constructed based on the 16S rDNA gene sequence;

[0020] Figure 6 It is the bacteriocin antibacterial spectrum of the Leuconostoc lactis Gill 3 of the present invention;

[0021] Figure 7For the bacteriocin anti-biofilm activity of Leuconostoc lactis Gill 3 of the present invention. Detailed implementation manners

[0022] To better understand the above technical solutions, the exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Although the exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present invention can be understood more clearly and thoroughly, and the scope of the present invention can be completely conveyed to those skilled in the art.

[0023] In the embodiments of the present invention, based on Leuconostoc lactis Gill 3 with the preservation number of GDMCC NO.65904, abbreviated as "Leuconostoc lactis Gill 3" or "strain Gill 3".

[0024] Example 1 Isolation, identification and preservation of Leuconostoc lactis Gill 3

[0025] 1. Isolation of Leuconostoc lactis Gill 3

[0026] Taking perch in Fujian Province as the isolation source, the specific isolation method is as follows:

[0027] Collect fish gills from perch, seal them in sterile bags respectively, and store them in a 4°C refrigerator. Put the sample into the ultra-clean bench and irradiate it with ultraviolet light for 30 min in advance. In the ultra-clean bench, take 100 μL of fish gill liquid, add it to 900 μL of 0.9% NaCl, gently pipette 5 - 6 times, and dilute it in a 10-fold serial gradient to obtain fish gill dilutions of 10 -3 ~10 -8 . Put the pre-sterilized and solidified MRS agar medium into the microwave oven to heat until completely melted, pour the plate for standby. Pipette 10 μL of the above-mentioned diluted liquid with different concentrations and well-mixed and drop it on the surface of the plate, gently spread it with a sterilized and cooled spreading rod. After finishing, invert it and culture it at 37°C until obvious single colonies can be observed and take it out. Pick the single colonies and place them in MRS liquid medium, statically culture them at 37°C for 24 h, centrifuge and take the supernatant, and conduct acid action exclusion test and hydrogen peroxide action exclusion experiment (exclude the interference of acid by adjusting the pH of the fermentation broth to 4, 5, 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 Gill 3.

[0028] Figure 1The results of A showed that with the increase of pH, the antibacterial activity of the fermentation supernatant of strain Gill 3 showed a downward trend, but the change in pH did not cause it to lose its antibacterial effect. Combining with Figure 1 the results of B, it can be seen that the addition of catalase had almost no effect on the antibacterial effect of the strain fermentation broth, which indicated that the antibacterial substances in the strain fermentation broth were not organic acids and hydrogen peroxide antibacterial substances, but bacteriocins. That is, the strain Gill 3 of the present invention belongs to the bacteriocin-producing strain.

[0029] Meanwhile, the biocompatibility of strain Gill 3 was verified by blood agar plate. Figure 2 The results showed that it had good biocompatibility, making it a potential candidate producer of probiotics and bacteriocins.

[0030] 2. Identification of Leuconostoc lactis Gill 3

[0031] 2.1 Morphological identification

[0032] Take out the glycerol tube containing the above-mentioned strain Gill 3, mix it with 6 mL of LB liquid medium, place it in a 37°C incubator 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 place it in a 37°C incubator 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 ethanol gradient, air dry it naturally, and then 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 Gill 3 were round, about 0.5 mm in diameter, white and opaque, with a moist and flat surface ( Figure 3 A); under the scanning electron microscope, its cells were short rod-shaped ( Figure 3 B).

[0034] 2.2 Molecular biology identification

[0035] Select the above-screened strain Gill 3 and identify it by DNA sequencing technology. Take 20 μL of the strain Gill 3 from the cryopreservation tube and inoculate it into 2 mL of MRS liquid medium, place it in a 37°C incubator for static culture for 24 h, then transfer it to 20 - 30 mL of MRS medium, and place it in a 37°C incubator for static culture for 24 h until the logarithmic growth phase.

[0036] DNA extraction: Refer to the operation instructions of the OMEGA Biotek nucleic acid extraction and purification kit to extract the genomic DNA of strain Gill 3, 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 in one group.

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

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

[0039] Purification and recovery: Carefully cut the PCR product electrophoresis band containing the target DNA, purify the PCR product according to the operation instructions of the gel recovery kit, 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 show that the E value (Expect) of strain Gill 3 and Leuconostoc lactis is 0% and the identity is 100%, indicating that the two have good homology, and it is clear that strain Gill 3 belongs to the genus Leuconostoc lactis ( Figure 4 and Figure 5 ).

[0041] Purify and recover the PCR product using the kit, and perform full-length sequencing of 16S rDNA by Tsingke Biological Co., Ltd. The 16S rDNA sequence of strain Gill 3 is 1442bp in total, and the sequencing result is shown in SEQ ID NO.1.

[0042] 3. Preservation of Leuconostoc lactis Gill 3

[0043] The taxonomic name of strain Gill 3 is Leuconostoc lactis, which has been deposited in the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC NO.65904, 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.

[0044] Example 2 Performance determination of Leuconostoc lactis Gill 3

[0045] The Lactobacillus gasseri Gill 3 screened in Example 1 was subjected to performance determination.

[0046] 1. Determination of the antibacterial activity of Lactobacillus gasseri Gill 3

[0047] Lactobacillus gasseri Gill 3 was inoculated into MRS liquid medium and statically cultured and fermented at 37 °C for 24 h. The supernatant was obtained by centrifugation. 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 Lactobacillus gasseri Gill 3 on indicator bacteria when applied to antibacterial products.

[0048] The results are as Figure 6 shown. The supernatant of the fermentation broth of Lactobacillus gasseri Gill 3 has antibacterial activity against foodborne pathogenic bacteria such as Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus, and Enterococcus faecium, but has no antibacterial activity against Listeria monocytogenes and methicillin-resistant Staphylococcus aureus. The above results indicate that the bacteriocin produced by Lactobacillus gasseri Gill 3 in the fermentation broth has narrow-spectrum antibacterial activity.

[0049] 2. Determination of the anti-biofilm activity of Lactobacillus gasseri Gill 3

[0050] Determined by crystal violet (CV) staining, cover glasses were added to six-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 Leuconostoc gilvus Gill 3 was added, and incubated at 30 °C for 24 h. Each well was gently washed 3 times with phosphate buffered 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 7 shown, indicating that the bacteriocin produced by Leuconostoc gilvus Gill 3 can not only inhibit the growth of planktonic cells, but also has anti-biofilm activity.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit 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 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 Leuconostoc lactis strain, characterized in that, The described Leuconostoclactis is named Leuconostoclactis Gill 3, and is deposited in Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO.65904. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit date is February 18, 2025.

2. The Leuconostoc lactis according to claim 1, wherein The 16S rDNA of the Leuconostoc lactis is shown as SEQ ID NO.

1.

3. The Leuconostoc lactis according to claim 1, wherein, The morphological characteristics of the lacteal candidiasis include: on the MRS culture medium, the colonies are round, white and opaque, with a moist and flat surface; and the bacteria are short rod-shaped according to microscopic examination results.

4. Use of the Leuconostoc lactis according to any one of claims 1 to 3 in the preparation of antibacterial products.

5. The application according to claim 4, characterized in that, The objects of action of the antibacterial product include Gram-negative bacteria and Gram-positive bacteria.

6. The application according to claim 5, characterized in that, The Gram-negative bacteria include Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa, and the Gram-positive bacteria include Staphylococcus aureus, Bacillus cereus and Enterococcus faecalis.

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