Lactococcus lactis Jlus 880314 and application thereof

Abstract

The invention belongs to the field of microorganisms, and particularly relates to lactococcus lactis Jlus 880314 and application thereof. The strain is classified and named as Lactococcus lactis, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation number of the strain is CGMCC No. 33961. Time-of-flight mass spectrometry analysis shows that nisin exists in a metabolite of the strain, and the strain can be used for producing nisin. The compound shows an excellent and wide antibacterial function in antibacterial experiments, can be used for preparing antibacterial drugs, particularly has a remarkable inhibiting effect on growth of gram-negative bacteria and fungi, can be used for preparing drugs for relieving or treating enteritis, or preparing drugs, nourishment or health care products for regulating gastrointestinal tract micro-ecological balance of people or animals, and can be used for preparing medicines for relieving or treating enteritis. The compound can also be used for preparing antibacterial food additives and / or animal feed additives. A hemolysis experiment result shows that hemolysis is avoided, toxic and side effects are small, and wide development prospects and application values are achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to a strain of lactococcus lactis Jlus 880314 and its applications. Background Technology

[0002] Foodborne pathogens are pathogenic bacteria that can cause food poisoning or spread through food, and are a significant source of food safety problems. Every year, a large number of people worldwide fall ill from consuming food contaminated with foodborne pathogens (such as Salmonella, Escherichia coli, and Staphylococcus aureus). Meanwhile, food spoilage caused by bacterial or mold contamination is also a serious issue. Approximately one-third of the world's food is wasted annually due to microbial contamination, and some of these microorganisms can produce toxins that directly threaten human health.

[0003] Antibiotics and chemical preservatives are currently widely used to inhibit the growth of these microorganisms. However, chemical preservatives pose safety risks and may adversely affect the flavor and quality of food. Overuse of antibiotics may accelerate the formation of antibiotic-resistant bacteria. Furthermore, the use of these chemicals may affect the activity of some beneficial bacteria in food and even the fermentation process of fermented foods.

[0004] Nisin is a polypeptide composed of 34 amino acid residues. It inhibits most Gram-positive bacteria and has a strong inhibitory effect on Bacillus spores; moreover, it is unlikely to induce resistance and is globally recognized as a safe and harmless natural food preservative and antibacterial agent. Under the physiological pH conditions and enzymatic action of the human body, it can be quickly hydrolyzed into amino acids, without altering the normal flora in the human gut or causing resistance problems as seen with other antibiotics. Furthermore, it does not exhibit cross-resistance with other antibiotics, making it a highly effective, non-toxic, safe, and side-effect-free natural food preservative. While existing nisin formulations have excellent antibacterial effects against Gram-positive bacteria, their antibacterial effect against Gram-negative bacteria is usually not prominent. Summary of the Invention

[0005] This invention aims to develop a novel lactococcus lactis strain resistant to Gram-negative bacteria, promote the development of the natural food preservative industry, and explore its new uses. It provides a strain of lactococcus lactis Jlus 880314 and its related applications.

[0006] On one hand, this invention provides a strain of *Lactococcus lactis* Jlus 880314, classified as *Lactococcus lactis*, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is March 24, 2025, and the accession number is CGMCC No. 33961. Its 16S rDNA sequence is shown in SEQ ID No. 1.

[0007] Furthermore, the Lactococcus lactis Jlus 880314 provided by this invention was isolated and screened from naturally fermented cheese, and obtained through a series of screening experiments after ultraviolet radiation mutagenesis.

[0008] In another aspect, the present invention also provides a microbial agent, wherein the active ingredient of the microbial agent is the lactococcus lactis Jlus880314.

[0009] Furthermore, the microbial agent also includes acceptable excipients; the excipients may function as excipients, act as carriers, improve stability, solubilize, aid solubility, or provide sustained release.

[0010] On the other hand, Lactococcus lactis Jlus 880314 can be used to produce nisin, therefore the present invention also provides the use of said Lactococcus lactis Jlus 880314 or said bacterial agent in the preparation of nisin.

[0011] In another aspect, the present invention also provides the use of the lactococcus lactis Jlus 880314 or the bacterial agent in the preparation of antimicrobial drugs, particularly showing a significant inhibitory effect on the growth of Gram-negative bacteria and / or fungi.

[0012] Furthermore, the Gram-negative bacteria include Escherichia coli and / or Salmonella; the fungi are one or more of Staphylococcus aureus, Trichoderma pinkis, and Penicillium oxalate.

[0013] Bacterial or fungal infections often lead to enteritis or gastrointestinal microecological disorders. Lactococcus lactis Jlus 880314 has a significant inhibitory effect on the growth of Gram-positive bacteria, Gram-negative bacteria, and fungi. Therefore, based on the antibacterial function of Lactococcus lactis Jlus 880314, this invention further provides the use of Lactococcus lactis Jlus 880314 or the bacterial agent in the preparation of drugs for relieving or treating enteritis, especially enteritis caused by Gram-negative bacteria and / or fungi, or for the preparation of drugs, nutritional products, or health products for regulating the gastrointestinal microecological balance of humans or animals, especially gastrointestinal microecological imbalance caused by Gram-negative bacteria and / or fungi.

[0014] Based on the antibacterial function of Lactococcus lactis Jlus 880314, the present invention further provides the application of Lactococcus lactis Jlus 880314 or the bacterial agent in the preparation of antibacterial food additives and / or animal feed additives.

[0015] The beneficial effects of this invention are:

[0016] Time-of-flight mass spectrometry analysis showed that the metabolites of *Lactococcus lactis* Jlus 880314 provided in this invention contain nisin, which can be used to produce nisin. *Lactococcus lactis* Jlus 880314 provided in this invention exhibited excellent and broad-spectrum antibacterial activity in antibacterial experiments, effectively inhibiting the growth of bacteria and fungi such as *Escherichia coli*, *Staphylococcus aureus*, *Salmonella*, *Botrytis cinerea*, *Trichoderma pinki*, and *Penicillium oxalate*, especially showing significant inhibitory effects on the growth of Gram-negative bacteria and fungi. The hemolysis experiment showed γ-hemolysis, i.e., no hemolysis, indicating low toxicity and demonstrating its safety. It can be used to prepare antibacterial drugs, nutritional products, or health products, and can also be used to prepare antibacterial food additives and / or animal feed additives. Therefore, *Lactococcus lactis* Jlus 880314 provided in this invention has broad development prospects and application value. Attached Figure Description

[0017] Figure 1 This is a microscopic image of the *Lactococcus lactis* strain Jlus 880314 of the present invention.

[0018] Figure 2 This is a mass flight spectrum of the metabolites of Lactococcus lactis Jlus 880314, which is the subject of this invention.

[0019] Figure 3 This relates to the effect of the present invention, Example 3, on the growth of Staphylococcus aureus;

[0020] Figure 4 The effect of this invention on the growth of Escherichia coli in Example 3 of the present invention;

[0021] Figure 5 This relates to the effect of Salmonella growth on Example 3 of the present invention;

[0022] Figure 6 This relates to the impact of *Botrytis cinerea* growth on Example 4 of the present invention.

[0023] Figure 7 The effect of this invention on the growth of *Trichoderma pinkis* in Example 4 of this invention;

[0024] Figure 8 This is the effect of Penicillium oxalate on the growth of Example 4 of the present invention. Detailed Implementation

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Example 1

[0028] The isolation, screening, identification, and safety assessment of Lactococcus lactis Jlus 880314 are as follows:

[0029] I. Separation and Screening:

[0030] Natural cheese samples were diluted with physiological saline and spread onto MRS plates containing calcium carbonate, then incubated at 37°C for 48 h. Single colonies with distinct calcium dissolution zones and different morphologies were then picked from the plates and incubated in MRS liquid medium at 37°C for 24 h. This process of streaking on solid medium followed by incubation of single colonies in liquid medium was repeated three times. The purified bacterial suspensions were then subjected to Gram staining and microscopic examination, as well as catalase assays. Gram-positive spherical and catalase-negative bacteria were selected for further experiments. Each bacterial suspension was irradiated with 254 nm UV light for 30 s and then spread onto MRS plates containing calcium carbonate. Colonies with distinct calcium dissolution zones and larger morphologies were picked and incubated in MRS liquid medium at 37°C for 24 h. This process of streaking on solid medium followed by incubation of single colonies in liquid medium was repeated three times. Finally, a lactococcus lactis strain Jlus 880314 producing lactic acid styramine was obtained.

[0031] Preservation instructions for the obtained Lactococcus lactis Jlus 880314:

[0032] Strain name: Lactococcus lactis;

[0033] Latin name: Lactococcus lactis;

[0034] Referenced biological material (strain): Jlus 880314;

[0035] Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures;

[0036] The abbreviation for the depository institution is CGMCC.

[0037] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing;

[0038] Deposit date: March 24, 2025;

[0039] CGMCC Registration Number: 33961;

[0040] II. Strain Identification:

[0041] After activating the frozen glycerol-preserved bacteria three times with MRS medium, they were isolated on MRS solid plates using the four-zone streak method. The plates were incubated upside down at 37°C for 24 h. The colony morphology was observed, and colonies were picked for Gram staining and microscopic observation.

[0042] The results showed that the strain presented as white, round colonies with smooth and regular edges and surfaces. Gram staining was positive. The strain was spherical, appearing singly, in pairs, or in chains, and did not produce spores. Figure 1 This is a microscopic image of the strain.

[0043] DNA was extracted from the strain using a DNA kit and amplified by PCR, followed by 16S rDNA sequencing. The sequencing results identified the strain as *Lactococcus lactis*, and its 16S rDNA sequence is shown in SEQ ID No. 1.

[0044] The culture temperature for Lactococcus lactis Jlus 880314 was 37℃; natural pH; culture medium composition: 10 g peptone, 10 g beef extract, 5 g yeast extract, 20 g glucose, 5 g sodium acetate, 2 g diammonium citrate, 1 mL Tween 80, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, 2 g dipotassium hydrogen phosphate, 15 g agar, and 1 L distilled water.

[0045] III. Safety assessment of the strain:

[0046] The activated Lactococcus lactis Jlus 880314 bacterial culture was inoculated onto the surface of MRS solid medium containing 5% defibrinated sheep blood. After incubation at 37°C upside down for 24 h, the hemolysis of the plates was observed.

[0047] The results showed that the Lactococcus lactis Jlus 880314 exhibited γ-hemolysis (i.e., no hemolysis) in blood agar culture medium, demonstrating its biocompatibility.

[0048] Example 2

[0049] Detection of nisin production by time-of-flight mass spectrometry:

[0050] Activated Lactococcus lactis Jlus 880314 was inoculated into whey and cultured at 37°C for 48 h. 5 g of the supernatant was collected, and 10 mL of formic acid solution (0.1%) and 20 mL of n-hexane were added. The mixture was shaken for 5 min, centrifuged for 2 min, and the upper n-hexane layer was removed. Then, 40 mL of a solution containing 0.1% formic acid and 20% acetonitrile was added to the lower sample. The mixture was sonicated for 10 min, centrifuged at 8000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane for testing.

[0051] A C18 column was used for the test; the test parameters were as follows: mobile phase A was 0.1% formic acid aqueous solution, mobile phase B was acetonitrile, A:B ratio was 4:1; column temperature was 30℃; injection volume was 10 μL; flow rate was 0.2 mL / min. Ionization was performed using ESI; scanning mode was positive ion scanning; detection mode was multiple reaction monitoring; curtain gas was 10.0 psi; nebulizer gas was 40.0 psi; heating gas was 10.0 psi; spray voltage was 5000 V; desolventizing temperature was 400℃.

[0052] Partial spectra of the flight mass spectrometry are shown below. Figure 2 A high-intensity peak was observed at a mass-to-charge ratio of 834.6163, corresponding to the tetraprotonated structure of the Z configuration of nisin, i.e., [Nisin + 4H]. 4+ This result indicates that Lactococcus lactis Jlus 880314 can produce nisin through its own metabolism, which can be used to prepare food preservatives and food additives.

[0053] Example 3

[0054] Inhibition test of bacterial growth by Lactococcus lactis Jlus 880314:

[0055] I. Source of experimental bacteria:

[0056] The Staphylococcus aureus used in the experiment was obtained from the China Industrial Microbial Culture Collection Center, number CICC 10001; Escherichia coli was obtained from Jilin University, number ATCCO157: H7; and Salmonella enterica was obtained from the United States Culture Collection Center, number ATCC 13076.

[0057] II. Effects of Lactococcus lactis Jlus 880314 on bacterial growth:

[0058] After thawing the frozen bacterial culture, streaks were applied to solid culture medium and incubated at 37°C for 24 h. Single colonies were then picked and cultured in liquid medium until the logarithmic growth phase. Finally, each harmful bacterium was diluted to 10⁻¹⁰ using TSB medium.6 CFU / mL. Lactococcus lactis Jlus 880314 was inoculated into MRS medium and cultured at 37℃ for 48 h. The supernatant was filtered through a 0.22 μm filter to obtain sterile fermentation broth. An equal volume of harmful bacteria dilution and Lactococcus lactis Jlus 880314 fermentation broth were mixed and cultured at 37℃ for 24 h. Absorbance values ​​were measured every 0.5 h to plot the growth curve of the harmful bacteria. The control group consisted of MRS medium without Lactococcus lactis Jlus 880314, and the same procedure was performed to compare the inhibitory effect of Lactococcus lactis Jlus 880314 on harmful bacteria.

[0059] Figures 3 to 5 The growth curves of Staphylococcus aureus, Escherichia coli, and Salmonella under different treatments are shown. In the control group, all three bacteria experienced a brief lag phase before rapidly proliferating and entering a stationary phase after 14 hours. However, the bacteria treated with the fermentation broth of *Lactococcus lactis* Jlus 880314 showed no significant proliferation within 24 hours, exhibiting almost 100% inhibition of the growth of all three bacteria, demonstrating excellent antibacterial activity. Nisin has excellent effects against Gram-positive bacteria, but its effect on Gram-negative bacteria is usually not prominent. However, *Lactococcus lactis* Jlus 880314 produces a large amount of lactic acid during its metabolism, significantly inhibiting the growth of Gram-negative bacteria. Furthermore, the acidic environment is conducive to the protonation of nisin, further enhancing its antibacterial activity. Therefore, Lactococcus lactis Jlus 880314 has broad antibacterial activity against a variety of bacteria, especially Gram-negative bacteria, and can be used to prepare antibacterial agents that inhibit Gram-negative bacteria. It can also be used alone to inhibit Gram-negative bacteria.

[0060] Example 4

[0061] Inhibition test of Lactococcus lactis Jlus 880314 on fungal growth:

[0062] I. Source of experimental fungi

[0063] The *Botrytis cinerea* used in the experiment was obtained from the Shanghai Microbiology Preservation Center, with the number AS3.3789; *Trichothecium roseum* was isolated from cherry tomatoes; and *Penicillium oxalicum* was obtained from spoiled citrus fruits.

[0064] II. Effects of Lactococcus lactis Jlus 880314 on fungal growth:

[0065] Frozen fungal spore suspensions were thawed, spread on solid culture media, and activated by incubation at 25°C. *Botrytis cinerea* was cultured for 5 days, *Trichoderma pinkis* for 3 days, and *Penicillium oxalate* for 24 hours. *Lactococcus lactis* Jlus 880314 was inoculated into MRS medium and incubated at 37°C for 48 hours. The supernatant was filtered through a 0.22 μm filter to obtain sterile fermentation broth. Before solidification, an equal volume of *Lactococcus lactis* Jlus 880314 fermentation broth was mixed with sterilized PDA medium (supplemented with 1.5% agar), and the medium was then poured off. After activation, small agar blocks of each fungus were punched into PDA plates containing the fermentation broth and cultured. The control group consisted of MRS medium without *Lactococcus lactis* Jlus 880314 inoculation and underwent the same procedure. The hyphal expansion of each group of fungi was observed, and the inhibitory effect of Lactococcus lactis Jlus 880314 on putrefactive fungi was compared.

[0066] Because different fungi have significantly different growth rates, the observation time also varies. Figures 6 to 8 The images show the hyphal expansion of various fungi after growth. After 8 days of culture, *Botrytis cinerea* in the control group grew to a diameter of approximately 45 mm, while *Lactococcus lactis* fermentation broth treatment resulted in a growth of only 26 mm, an inhibition rate of 52.3%. *Trichoderma pinki*, which grew to 44 mm in the control group after 5 days of culture, showed no growth at all after treatment with *Lactococcus lactis* fermentation broth, an inhibition rate of 100%. Simultaneously, *Lactococcus lactis* Jlus 880314 reduced the growth range of *Penicillium oxalate* from 28 mm to 22 mm within 5 days, exhibiting an inhibition rate of 31.6%. In summary, *Lactococcus lactis* Jlus 880314 also demonstrates a significant inhibitory effect on the growth of various fungi that can cause food spoilage. The above results indicate that Lactococcus lactis Jlus 880314 can metabolize and produce nisin, which exhibits excellent antibacterial activity against a variety of pathogenic bacteria and putrefactive fungi. It can be used to prepare antibacterial drugs or food additives to extend the shelf life of food.

[0067] Although the present invention has been described above with general descriptions and specific embodiments, these are only some examples and do not represent all embodiments of the present invention. Modifications or improvements can be made to the present invention, which will be obvious to those skilled in the art. Therefore, any modifications or improvements made based on the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Lactococcus lactis Jlus 880314, characterized in that: The strain is classified and named Lactococcus lactis. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 24, 2025, and the accession number is CGMCC No. 33961.

2. A microbial agent, characterized in that: The active ingredient of the bacterial agent is Lactococcus lactis Jlus880314 as described in claim 1.

3. The use of Lactococcus lactis Jlus 880314 according to claim 1 or the bacterial agent according to claim 2 in the preparation of nisin.

4. The application of Lactococcus lactis Jlus 880314 according to claim 1 or the bacterial agent according to claim 2 in the preparation of antibacterial drugs, wherein the antibacterial species are Gram-negative bacteria and / or fungi.

5. The application according to claim 4, characterized in that: The Gram-negative bacteria are Escherichia coli and / or Salmonella; the fungi are one or more of Botrytis cinerea, Trichoderma pinkis, and Penicillium oxalate.

6. The use of Lactococcus lactis Jlus 880314 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a medicament for relieving or treating enteritis.

7. The application according to claim 6, characterized in that: The aforementioned enteritis is enteritis caused by Gram-negative bacteria and / or fungi.

8. The use of Lactococcus lactis Jlus 880314 according to claim 1 or the bacterial agent according to claim 2 in the preparation of drugs, nutritional products or health products for treating gastrointestinal microecological imbalance in humans or animals.

9. The application according to claim 8, characterized in that: The aforementioned gastrointestinal microecological imbalance is caused by Gram-negative bacteria and / or fungi.

10. The use of Lactococcus lactis Jlus 880314 according to claim 1 or the microbial agent according to claim 2 in the preparation of antibacterial food additives and / or animal feed additives.

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