Lactococcus garviae LG3092 producing bacteriocin GarQ and efficiently antagonizing multi-drug resistant helicobacter pylori and application thereof

Abstract

This invention discloses a strain of *Lactococcus gasseri* LG3092 that produces the bacteriocin GarQ and effectively antagonizes multidrug-resistant *Helicobacter pylori*, and its applications. *Lactococcus gasseri* LG3092 was deposited at the Guangdong Provincial Microbial Culture Collection Center on October 30, 2023, with accession number GDMCC No: 63939. Studies have shown that LG3092 has the following beneficial effects: (1) it can produce the bacteriocin GarQ, effectively inhibiting the growth of *Helicobacter pylori* and other common pathogenic bacteria; (2) it has a high survival rate in the stomach, can colonize the stomach, and can compete with pathogenic bacteria for binding sites, effectively reducing the infection rate of *Helicobacter pylori* in the stomach; (3) it can improve the pathological state of the stomach and alleviate inflammation. Therefore, *Lactococcus gasseri* LG3092 has great application potential in the preparation of products for the prevention or treatment of *H. pylori*, especially multidrug-resistant *H. pylori* infection.

Description

Technical Field

[0001] This invention belongs to the fields of microbial technology and pharmaceutical technology, specifically relating to a strain of Lactococcus gasseri LG3092 that produces the bacteriocin GarQ and effectively antagonizes multidrug-resistant Helicobacter pylori and its application. Background Technology

[0002] Helicobacter pylori (Hp) is a Gram-negative, microaerophilic, spiral-shaped pathogenic microorganism that can cause various gastric diseases such as functional dyspepsia, chronic gastritis, and peptic ulcers. In 1994, Hp was classified as a Group 1 carcinogen by the World Health Organization. Research data shows that over 90% of gastric cancer cases can be attributed to Hp infection, and gastric cancer caused by Hp infection is particularly prevalent in East Asia, Eastern Europe, Central America, and South America. As the largest country in East Asia, my country experiences severe impacts from both Hp infection and gastric cancer. Therefore, a series of studies are urgently needed to prevent and treat Hp infection, maintain gastric microbial homeostasis, and restore gastric inflammation caused by infection.

[0003] The Maastricht / Florence Consensus, published in 2011, states that all patients diagnosed with *Helicobacter pylori* (Hp) infection should undergo eradication therapy. Currently, first-line treatment for Hp infection in adults primarily involves 14 days of bismuth-free quadruple therapy (PPI, clarithromycin, amoxicillin, metronidazole) and 10 days of bismuth-containing quadruple therapy (PPI, bismuth, tetracycline, metronidazole). However, with the widespread use of antibiotics in clinical practice, side effects such as bacterial resistance, patient compliance, and gut microbiota dysbiosis have gradually emerged, severely impacting the efficacy of antibiotics against Hp and consequently posing greater challenges to Hp prevention and treatment. Therefore, finding a new, non-antibiotic, individualized treatment regimen that antagonizes multidrug-resistant Hp and can regulate the patient's gut microbiota and repair gastric damage is of great significance.

[0004] In recent years, an increasing number of reports have indicated that certain probiotic preparations can inhibit *Helicobacter pylori* (Hp) colonization in the stomach, demonstrating good preventative and therapeutic effects against Hp infection. *Lactococcus garvieae* is a Gram-positive facultative anaerobic bacterium belonging to the phylum Firmicutes and the genus *Lactococcus*, capable of fermenting carbohydrates to produce acid. As a novel probiotic, *Lactococcus garvieae* has been reported to enhance immunity and optimize gut microbiota; however, there are currently no reports on its ability to antagonize *Hp* and other common pathogens in the stomach, thereby balancing the gastric microecological environment and reducing inflammation and damage caused by *Hp*.

[0005] Bacteriocins are a class of antibacterial proteins or polypeptides produced by certain bacteria through ribosome synthesis. Unlike antibiotics, which have a broad spectrum of activity, most bacteriocins only inhibit closely related bacteria. Furthermore, most bacteriocins are non-toxic, leave no residue, do not induce drug resistance, are easily hydrolyzed by some proteases in the human digestive system, and do not pollute the environment. Therefore, bacteriocins can reduce or even replace the use of antibiotics, making them popular in the medical and agricultural industries. In particular, bacteriocins produced by lactic acid bacteria (LAB) have shown the greatest application potential in recent years. This is because LABs that produce bacteriocins, in addition to their antibacterial function, can be directly used as fermentation agents and biological protection cultures in industry, facilitating their widespread application in industrial and biopharmaceutical sectors. Garvicin Q (GarQ) is reportedly a LAB-derived bacteriocin that has attracted more attention. Based on its protein structure, it can be classified as a subclass IId bacteriocin. This class of bacteriocins has a relatively broad antibacterial spectrum, including Listeria, Enterococcus, Pediococcus, Lactobacillus, and Lactococcus.

[0006] Currently, no studies have clearly indicated that Lactococcus gasseri can solve the problem of multidrug-resistant Helicobacter pylori infection. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of Lactococcus garvieae LG3092 and its application in the preparation of products for the prevention or treatment of Helicobacter pylori infection. Lactococcus garvieae LG3092 can effectively prevent and treat gastric inflammation caused by Helicobacter pylori infection by antagonizing the growth of multidrug-resistant Helicobacter pylori.

[0008] To achieve the above objectives, the present invention adopts the following technical measures:

[0009] The first objective of this invention is to provide *Lactococcus garvieae* LG3092, with accession number GDMCC No: 63939. *Lactococcus garvieae* LG3092 was deposited on October 30, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 63939.

[0010] A second objective of this invention is to provide the use of the aforementioned Lactococcus gasseri LG3092 in the preparation of products for the prevention and / or treatment of Helicobacter pylori infection.

[0011] Preferably, the Helicobacter pylori is a multidrug-resistant Helicobacter pylori. This includes the preparation of inhibitors of Helicobacter pylori, especially multidrug-resistant Helicobacter pylori, using Lactococcus gasseri LG3092, or the preparation of therapeutic or preventative drugs for Helicobacter pylori infection, especially multidrug-resistant Helicobacter pylori.

[0012] A third objective of this invention is to provide the application of the aforementioned Lactococcus gasseri LG3092 in the preparation of products that regulate the gastric microbial environment and bacterial growth cycle.

[0013] Preferably, the growth cycle of the bacterial community is the growth cycle of Salmonella Typhimurium, Listeria monocytogenes, Enterococcus faecalis, Lactococcus gasseri, Lactobacillus paracasei, or Lactobacillus plantarum.

[0014] A fourth objective of this invention is to provide the application of the aforementioned Lactococcus gasseri LG3092 in the production of class IId bacteriocin GarQ, the amino acid sequence of which is shown in SEQ ID NO.3.

[0015] The Lactococcus gasseri LG3092 described in this invention produces the class IId bacteriocin GarQ, which regulates the gastric microbial environment. It also has the effect of highly antagonizing the Hp standard strain ATCC 43504 and the multidrug-resistant Helicobacter pylori GZ6B5 (GDMCC No: 61571) isolated in this experiment.

[0016] The Lactococcus gasseri LG3092 described in this invention has a high survival rate in the stomach, can colonize the stomach, and can compete with pathogenic bacteria for binding sites in the body, effectively reducing the infection rate of Helicobacter pylori in the stomach.

[0017] The Lactococcus gasseri LG3092 described in this invention can improve the pathological state of the stomach and relieve gastric inflammation.

[0018] The Lactococcus gasseri LG3092 described in this invention can not only compete with Helicobacter pylori for binding sites on gastric epithelial cells in the stomach, but also produce the active antibacterial substance GarQ to inhibit the growth of other digestive tract pathogens.

[0019] The fifth objective of this invention is to provide a product for preventing and / or treating Helicobacter pylori infection and / or regulating the gastric microbial environment and bacterial growth cycle, which contains live Lactococcus gasseri LG3092, fermentation supernatant or bacterial fragments as active ingredients.

[0020] The sixth object of the present invention is to provide a target nucleotide sequence that specifically recognizes the above-mentioned Lactococcus gasseri LG3092, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0021] The seventh object of the present invention is to provide a primer set for identifying the above-mentioned Lactococcus gasseri LG3092, namely: 5'-GCGGTTCAGAAAGCCTATGA-3' and 5'-CGATGCCACATCATTGAAAC-3'.

[0022] The eighth objective of this invention is to provide a method for identifying the above-mentioned Lactococcus gasseri LG3092, which involves using the above-mentioned primer set as amplification primers to perform a PCR reaction to amplify the test bacteria. If a 525 bp product is amplified, it is Lactococcus gasseri LG3092; if no 525 bp product is amplified, it is not Lactococcus gasseri LG3092.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] 1. The probiotics in this invention are sourced from Meizhou City, Guangdong Province, China, a world-renowned longevity town, and are excellent strains from local origin.

[0025] 2. Compared with traditional antibiotics for treating Helicobacter pylori, this invention has no toxic side effects on the ecological environment, no residue risk, and is green and safe.

[0026] 3. This invention has a good effect against Helicobacter pylori, especially multidrug-resistant Helicobacter pylori, specifically manifested in:

[0027] (1) The probiotics involved in this invention can produce a variety of active antibacterial substances such as organic acids and type IId bacteriocins, which can effectively inhibit the growth of Helicobacter pylori and other pathogens in the stomach;

[0028] (2) It has a high survival rate in the stomach, can colonize the stomach, and can compete with pathogenic bacteria for binding sites in the body, effectively reducing the infection rate of Helicobacter pylori in the stomach;

[0029] (3) It can improve the pathological state of the stomach caused by Helicobacter pylori and relieve gastric inflammation;

[0030] (4) It can regulate the gastric microbial environment and reduce the side effects of digestive dysfunction and decreased immunity caused by dysbiosis during other drug treatments.

[0031] This patent addresses the globally prevalent problem of multidrug-resistant *Helicobacter pylori* (Hp) by developing a strain, *Lactococcus gasseri* LG3092, which efficiently secretes the class IId bacteriocin GarQ, regulates the gastric microecological environment, and exhibits excellent anti-Hp activity, especially against multidrug-resistant Hp, both in vitro and in vivo. Due to its selective and highly effective bactericidal action and biosafety, this strain represents a highly attractive alternative and has the potential to be developed into novel antibacterial drugs for the prevention and treatment of gastric diseases caused by Hp. Therefore, *Lactococcus gasseri* LG3092 shows great promise in the preparation of drugs for the prevention or treatment of Hp, particularly multidrug-resistant Hp infections.

[0032] Lactococcus garvieae LG3092 was deposited on October 30, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 63939. Attached Figure Description

[0033] Figure 1 The inhibitory effects of Lactococcus gasseri LG3092 and other commercial lactic acid bacteria strains on the standard strain of Helicobacter pylori were observed.

[0034] Figure 2 It is the inhibitory effect of Lactococcus gasseri LG3092 on multidrug-resistant Helicobacter pylori.

[0035] Figure 3 This study investigated the effects of Lactococcus gasseri LG3092 and other commercial lactic acid bacteria strains on the amelioration of inflammatory factors induced by Helicobacter pylori-associated toxins. Figure caption: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0036] Figure 4 This refers to the alleviating effect of Lactococcus gassifolius LG3092 on gastric pathological conditions.

[0037] Figure 5 This is a diagram of the structural domains of GarQ, a type IId bacteriocin produced by Lactococcus gasseri LG3092.

[0038] Figure 6 This is an electrophoresis diagram of the recombinant protein GarQ, where M: protein molecular weight marker, 1: purified protein.

[0039] Figure 7 This describes the growth-inhibiting effect of recombinant protein GarQ on several common lactic acid bacteria and foodborne pathogens.

[0040] Figure 8 This describes the effect of Lactococcus gasseri LG3092 on improving gastric flora imbalance caused by Hp infection.

[0041] Figure 9 This is the molecular target verification of Lactococcus gasseri LG3092. Figure caption: M is DNA marker, 1 is the electrophoresis image of the PCR product of Lactococcus gasseri LG3092 molecular target sequence (SEQ ID NO.2) amplified by specific primer sets 3092F and 3092R, and 2-89 are the electrophoresis images of the PCR products of other lactic acid bacteria amplified by molecular target sequences.

[0042] Figure 10 This is a schematic diagram of the real-time Ct value and the bacterial concentration standard curve of the qPCR method for quantitative detection of Lactococcus gasseri LG3092 molecular target.

[0043] Figure 11 This is a comparison chart of the results of PMA-qPCR and regular qPCR detection of Lactococcus gasseri LG3092. Detailed Implementation

[0044] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0045] The present invention will be further described below with reference to specific embodiments.

[0046] The culture media involved in the following examples are as follows:

[0047] MRS agar plates (g / L): peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 g / L, K2PO4·3H2O 2.0 g / L, sodium acetate 5.0 g / L, triamine citrate 2.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L, agar 20 g / L, with water as the solvent. The preparation method is to mix all the components evenly and sterilize at 115℃ for 20 min.

[0048] MRS broth medium (g / L): peptone 10.0 g / L, beef extract 5.0 g / L, yeast extract 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 g / L, K2PO4·3H2O 2.0 g / L, sodium acetate 5.0 g / L, triamine citrate 2.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L, with water as the solvent. The preparation method is to mix all the components evenly and sterilize at 115℃ for 20 min.

[0049] Selective culture medium for Helicobacter pylori: 2.02 g / L yeast extract, 10.52 g / L tryptone, 1.56 g / L glucose, 0.10 g / L sodium bisulfite, 10.0 g / L soluble starch, 5.0 g / L sodium chloride, and 13.5 g / L agar. Dissolve the above components in sterile water according to their respective concentrations, sterilize at 121°C for 20 min, then add 100 mL / L fetal bovine serum and 10 mL / L selective additive. The selective additive consists of: vancomycin 0.01 g / L, amphotericin B 0.005 g / L, trimethoprim 0.02 g / L, and cefsulfuron-methyl 0.01 g / L. Mix thoroughly and prepare plates.

[0050] Example 1: Isolation and identification of Lactococcus gasseri LG3092

[0051] 1.1. Isolation and preservation of Lactococcus gasseri LG3092

[0052] Fecal samples were collected from healthy centenarians in Jiaoling County, Meizhou City, Guangdong Province, China, a world-renowned longevity village. Under sterile conditions, 0.1 g of fecal matter was added to 10 mL of MRS broth medium, shaken to mix, and then incubated anaerobically at 37°C for 24 h. 0.5 mL of the bacterial culture was then incubated in physiological saline for 10... -1 Up to 10 -8 Gradient dilution. Select 10 -6 10 -7 10 -8 Three gradient bacterial suspensions were each aspirated at 100 μL and spread evenly onto MRS agar plates, then incubated anaerobically at 37°C for 48 h. Morphologically typical colonies from the plates were picked and streaked onto MRS agar medium for purification. After purification, single colonies were inoculated into MRS liquid medium and incubated anaerobically at 37°C for 48 h. The culture was then stored in 30% glycerol at -80°C.

[0053] 1.2. Identification of Lactococcus gasseri LG3092

[0054] Bacterial DNA was extracted using a bacterial DNA extraction kit (Mabio, CHINA), followed by PCR amplification using a 2×PCR mix (Dongshengbio, CHINA). Universal primers for the 16S rRNA gene were used for PCR amplification. The upstream primer sequence was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 5); the downstream primer sequence was 1492R: 5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID NO. 6). PCR reaction conditions were as follows: pre-denaturation at 95℃ for 5 min; 35 cycles of 95℃ for 30 s, 56℃ for 30 s, and 72℃ for 1 min 30 s; followed by annealing and extension at 72℃ for 10 min. The PCR products were recovered by gel extraction and then sequenced using Sanger sequencing. The obtained 16S rRNA gene sequence is shown in SEQ ID NO. 1. The sequence was compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov), and the results showed that it had the highest homology with Lactococcus garvieae. It was named Lactococcus garvieae LG3092 and was deposited on October 30, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 63939.

[0055] Example 2: Cultivation of Lactococcus gasseri LG3092 and preparation of its fermentation broth

[0056] 2.1. Resuscitation and culture of Lactococcus gasseri LG3092

[0057] Lactococcus gasseri LG3092 was inoculated from glycerol tubes onto MRS agar plates and anaerobically cultured at 37°C for 48 h. Colony morphology was then observed. Lactococcus gasseri colonies are small, smooth and glossy, spherical or oval-shaped with regular edges, and milky white and opaque. In liquid culture, they form pairs or short chains, do not form spores, are non-motile, and lack capsules.

[0058] A single colony of *Lactococcus gasseri* LG3092 was inoculated into MRS broth medium and anaerobically cultured at 37°C for 48 h to generate a growth curve. It was found that the bacterium reached its stationary phase after 12 h of culture at 37°C, exhibiting heterofermentation, producing acid and gas from glucose.

[0059] 2.2. Preparation of fermentation broth of Lactococcus gasseri LG3092

[0060] Single colonies of *Lactococcus gasseri* LG3092 were picked and inoculated into MRS broth medium and anaerobically cultured at 37°C for 48 h. The fermentation supernatant was obtained by centrifugation at 10000 × g, 4°C for 15 min, which was the fermentation supernatant of *Lactococcus gasseri* LG3092. This supernatant was filtered through a 0.22 μm filter membrane to obtain cell-free supernatant from the fermentation of *Lactococcus gasseri* LG3092 and stored at -80°C for later use.

[0061] Example 3: Evaluation of the in vitro antibacterial activity of Lactococcus gasseri LG3092 against Helicobacter pylori

[0062] 3.1. Culture of Helicobacter pylori

[0063] Helicobacter pylori ATCC 43504 was purchased from the Guangdong Provincial Microbial Culture Collection Center, and the strain was isolated from a patient with chronic gastritis in Australia. Helicobacter pylori GZ6B5 (GDMCC No: 61571) (Chinese Patent ZL202110612046.9) was isolated from a gastric mucosal tissue sample of a patient with chronic gastritis in Guangdong, my country. The strain was resistant to five antibiotics: amoxicillin, metronidazole, clarithromycin, levofloxacin, and tetracycline.

[0064] Activate the culture medium from the preservation tube, and spread 100 μL of the preservation solution onto a Helicobacter pylori selective culture medium plate. Incubate at 37°C in a microaerophilic environment (5% O2, 10% CO2, 85% N2) for 5-7 days. Select morphologically typical colonies from the plate and streak them onto a Helicobacter pylori selective culture plate for purification, repeating the purification process twice. Scrape the purified Helicobacter pylori using an inoculation loop and resuspend them in physiological saline.

[0065] 3.2. Evaluation of the in vitro antibacterial activity of Lactococcus gasseri LG3092 fermentation broth against Helicobacter pylori

[0066] Adjust the suspension of Helicobacter pylori (ATCC 43504 and GZ6B5) to achieve a bacterial count of 1×10⁻⁶. 8 CFU / mL, 100 μL of bacterial suspension was added to a selective culture medium plate for Helicobacter pylori and spread. Sterilized Oxford cups were placed on the plate, and 100 μL of lactic acid bacteria fermentation supernatant was added to each Oxford cup (the preparation method can refer to the fermentation broth of Lactococcus gasseri LG3092). After refrigerating for 6 h, the culture plates were transferred to a microtrophic environment (5% O2, 10% CO2, 85% N2) at 37℃ and incubated for 72 h. The size of the inhibition zone was observed and measured.

[0067] The fermentation broths of Lactococcus garvieae LG3092 and commercially available strains Lactobacillus Johnsonii MH-68 and Lacticaseibacillus paracasei Shirota were compared to show their inhibitory effects on Helicobacter pylori ATCC43504 in vitro. The results after 72 h of culture were as follows: Figure 1 As shown, the inhibitory effect of Lactococcus garvieae LG3092 on Helicobacter pylori ATCC 43504 was higher than that of the two commercially available strains (Lactobacillus Johnsonii MH-68 inhibition zone diameter 22.0 ± 0.1 mm, Lacticaseibacillus paracasei Shirota inhibition zone diameter 24.0 ± 0.1 mm, and Lactococcus garvieae LG3092 inhibition zone diameter 28.0 ± 0.1 mm, p < 0.01).

[0068] The inhibitory effect of the fermentation broth of *Lactococcus garvieae* LG3092 on *Helicobacter pylori* GZ6B5 was detected in vitro. Results were obtained after 72 h of culture. Figure 2 As shown, Lactococcus garvieae LG3092 has a good inhibitory effect on GZ6B5, with a corresponding inhibition zone diameter of 24.0 ± 0.1 mm.

[0069] Example 4: Evaluation of the anti-Helicobacter pylori activity of Lactococcus gasseri LG3092 in animals

[0070] 4.1. Laboratory animals and their housing conditions

[0071] Twenty C57BL / 6 mice aged 6-8 weeks were used in each group for the experiment. Animals were acclimatized to the animal facility for 5 days prior to the experiment. The experimental animals and the animal facility should comply with relevant national regulations, and standard formulated feed was used; water was not restricted.

[0072] 4.2. Experimental Grouping

[0073] This experiment consisted of eight groups: Group 1 was the normal control group (NC); Group 2 was the Lactococcus garvieae LG3092 group administered by gavage (LG3092); Group 3 was the Helicobacter pylori SS1 group (SS1 was an animal domestication strain of ATCC 43504, purchased from Guangdong Provincial Microbial Culture Collection Center) (SS1); Group 4 was the Helicobacter pylori GZ6B5 group (GZ6B5); Group 5 was the LG3092 prophylaxis group (SS1+LG3092); Group 6 was the LG3092 prophylaxis group (GZ6B5+LG3092); Group 7 was the Lactobacillus paracasei Shirota prophylaxis group (SS1+Shirota); and Group 8 was the Lactobacillus paracasei Shirota prophylaxis group (GZ6B5+Shirota). The SS1+Shirota group and the GZ6B5+Shirota group were the positive control groups for the SS1+LG3092 group and the GZ6B5+LG3092 group, respectively.

[0074] 4.3. Experimental Procedure

[0075] Before the formal commencement of the experiment, all six groups of mice were administered antibiotics (ampicillin 10 mg / mL, gentamicin 1.2 mg / mL, azithromycin 10 mg / mL, mixed antibiotic 0.3 mL / mouse, once daily) via gavage for 3 consecutive days. Then, starting from day 4, a proton pump inhibitor (omeprazole prepared as a 5 mg / mL solution, administered once daily at a rat body weight of 30 mg / kg) was administered before each gavage of the bacterial suspension. For the SS1, GZ6B5, SS1+LG3092, GZ6B5+LG3092, SS1+Shirota, and GZ6B5+Shirota groups, each mouse received a concentration of 1×10⁻⁶ mg / mL. 9 500 μL of CFU / mL Helicobacter pylori suspension was administered by gavage every other day. The SS1+ LG3092 group, GZ6B5+ LG3092 group, SS1+ Shirota group, and GZ6B5+ Shirota group received daily gavage at a concentration of 1×10⁻⁶ CFU / mL before Hp administration. 9 500 μL of CFU / mL LG3092 or Shirota bacterial suspension was administered by gavage to the SS1 and GZ6B5 groups using the same volume of physiological saline. In the NC group, both Hp and Lactobacillus bacterial suspensions were replaced with the same volume of physiological saline during gavage. The LG3092 group received a daily gavage concentration of 1×10⁻⁶ CFU / mL LG3092 or Shirota bacterial suspension. 9 500 μL of LG3092 bacterial suspension with CFU / mL was added, followed by replacing the Hp bacterial suspension with an equal volume of physiological saline.

[0076] After the feeding period, the experimental animals were euthanized by neck dislocation, and their stomachs were dissected using sterile, enzyme-free scalpels and scissors. The stomach tissue was divided into two parts: one part was fixed in 4% paraformaldehyde, and the other part was quickly transferred to a -80°C freezer for subsequent experiments.

[0077] 4.4. Inhibitory effect of Lactococcus gasseri LG3092 on Helicobacter pylori in animal stomach

[0078] A novel quantitative detection method for Helicobacter pylori-specific targets was employed. The qPCR amplification primers for quantitative detection of Helicobacter pylori are shown in Table 1, and the specific procedures are as follows:

[0079] Table 1 Primers for qPCR amplification reaction for quantitative detection of Hp

[0080]

[0081] ① Template DNA preparation: 0.1 g of mouse gastric tissue was taken and gastric mucosal microbial DNA was extracted using the QIAamp® PowerFecal® Pro DNA Kit as the template to be tested;

[0082] ② qPCR detection system and amplification procedure:

[0083] The qPCR reaction system for Hp was prepared as follows: 2×SYBR Green Premix, 5 μL; Hp-F (10 μmol / L), 0.5 μL; Hp-R (10 μmol / L), 0.5 μL; template DNA, 1 μL; ddH2O, 3 μL.

[0084] The following is the qPCR amplification program: 95℃, 30s; 95℃, 5s, 60℃, 30s (40 cycles).

[0085] ③ Reading qPCR results:

[0086] The system was amplified and detected using a Roche LightCycler® 96 quantitative PCR instrument, and the amplification results were read using the LightCycler® 96 SW 1.1 software. If a fluorescence signal was generated in the blank control (where no fluorescence signal was observed), it indicates the presence of *Helicobacter pylori* (Hp); if no fluorescence signal was generated, the sample did not contain Hp.

[0087] Apply physiological saline to a concentration of 1×10 9 Hp ATCC 43504 CFU / mL was diluted 10-fold to obtain a concentration of 10. 9 10 8 10 7 10 6 105 10 4 10 3 10 2 10 1 DNA was extracted from pure cultures of the strain at CFU / mL for different concentrations, and Hp quantification was performed on the samples according to the qPCR protocol described above. Each concentration sample was tested in triplicate.

[0088] Plotting a standard curve: The standard curve for quantitative detection of *Helicobacter pylori* (Hp) is obtained by plotting the logarithmic number of colony-forming units (log CFU) at different dilution gradients in the sample on the x-axis and the real-time Ct value of the corresponding qPCR on the y-axis. The fitted standard curve for *Helicobacter pylori* is y = -2.044x + 35.36, with a correlation coefficient R0. 2 It is 0.9813.

[0089] The results showed that the average Ct value of the gastric tissue of mice in the 0.1 g SS1 group was 28.69, indicating that the colonization amount of SS1 in the mouse gastric tissue was 10. 3.3 The average Ct value of the gastric tissue of mice in the 0.1g GZ6B5 group was 29.28, indicating that the colonization level of the multidrug-resistant strain GZ6B5 in the mouse gastric tissue was 10. 3 The average Ct value of the gastric tissue of mice in the 0.1g SS1+ LG3092 group was 30.6, indicating that the colonization of SS1 in the mouse gastric tissue decreased to 10. 2.3 The average Ct value of gastric tissue in mice in the 0.1g GZ6B5+LG3092 group was 30.56, indicating that the colonization level of multidrug-resistant *Helicobacter pylori* in the stomach of this group decreased to 10%. 2.3 CFU / g. In summary, Lactococcus gasseri LG3092 has a strong scavenging effect on Hp and multidrug-resistant Hp in mouse gastric tissue.

[0090] 4.5. The allergic effect of Lactococcus gasseri LG3092 on Hp-associated gastritis

[0091] 4.5.1. The effect of Lactococcus gasseri LG3092 on improving the imbalance of inflammatory factors induced by Hp-associated toxins.

[0092] Total RNA was extracted from gastric tissue using the HiPure Total RNA Mini Kit and reverse transcribed into cDNA using Evo M-MLV reverse transcription reagent premix. qPCR was then performed using SYBR Premix Ex Taq II to analyze the expression of pro-inflammatory factors IL-1β, IL-8, and TNF-α in mouse gastric tissue. The primers used are shown in Table 2.

[0093] Table 2 Primers for qPCR detection of pro-inflammatory factors in mice

[0094]

[0095] The 10 μL reaction system contained 5 μL of LSYBR Premix Ex Taq II (2x), 0.5 μL of forward and reverse primers, 1 μL of cDNA, and the remainder was added to a final volume of sterile, enzyme-free water. The reaction program was: 95℃ pre-denaturation for 1 min, 95℃ denaturation for 5 s, 60℃ extension for 1 min, for 40 cycles. The Ct values ​​of the internal reference gene and the target gene for each group were analyzed using Formula 2. -△△Ct Calculate the relative expression levels of IL-1β, IL-8, and TNF-α.

[0096] The results are as follows Figure 3 As shown, compared with the NC group, the levels of pro-inflammatory factors IL-1β, IL-8 and TNF-α in the stomach of mice in the SS1 group and GZ6B5 group all showed an upward trend, especially TNF-α, which was significantly upregulated (SS1 group was upregulated by 1.62 times, p < 0.05). Following gavage administration of LG3092 bacterial suspension, gastric pro-inflammatory factors IL-1β, IL-8, and TNF-α returned to normal levels, showing a significant downward trend compared to the SS1 and GZ6B5 groups: in the SS1+ LG3092 group, IL-1β decreased from 1.21 to 0.44 (p < 0.05), IL-8 decreased from 1.29 to 0.15 (p < 0.01), and TNF-α decreased from 2.62 to 0.85 (p < 0.01); in the SGZ6B5+ LG3092 group, IL-1β decreased from 1.15 to 0.34 (p < 0.01), IL-8 decreased from 1.05 to 0.14 (p < 0.001), and TNF-α decreased from 3.15 to 0.48 (p < 0.05). In the group that received Shirota via gavage, IL-8 and TNF-α pro-inflammatory factors were also downregulated to normal levels, but Lactococcus gasseri LG3092 showed better regulatory effects.

[0097] 4.5.2. Preparation of Specimen Sections

[0098] Gastric tissue fixed in 4% paraformaldehyde for more than 48 hours was taken, rinsed with running water overnight, and then paraffin embedded the following day: 70% ethanol for 1 hour → 80% ethanol for 1 hour → 90% ethanol for 1 hour → 95% ethanol for 1 hour (Ⅰ) → 95% ethanol for 1 hour (Ⅱ) → 100% ethanol for 1 hour (Ⅰ) → 100% ethanol for 1 hour (Ⅱ) → n-Butanol for 1 hour → Xylene for 30 minutes → Paraffin dissolution for 1.5 hours (Ⅰ) → Paraffin dissolution for 2 hours (Ⅱ) → Embedding.

[0099] Standard gastric tissue paraffin sections, 5 μm thick, were prepared and air-dried overnight at room temperature, then sealed and stored in a 4°C refrigerator for later use.

[0100] 4.5.3. H&E staining

[0101] Dewax the paraffin sections from step 4.5.2 above and stain with hematoxylin for 8 min. Wash away excess stain with distilled water and separate the sections with 1% hydrochloric acid-alcohol solution (prepared with 70% ethanol) for about 25 s (monitor under a microscope until the cell nuclei and chromatin are clearly visible). Immediately rinse with running water for about 15 s until the cell nuclei turn blue. Rinse briefly with double-distilled water. Then stain with 1% eosin solution for 2 min. Dehydrate the sections sequentially with 70%, 85%, 95%, and 100% ethanol, each step requiring 3 min. After clearing the sections with xylene (twice), wipe away excess xylene, add an appropriate amount of neutral resin, quickly cover with a coverslip, and observe and photograph the sections under a light microscope.

[0102] 4.5.4. Results

[0103] like Figure 4 As shown, in the NC group and LG3092 group, the gastric mucosal epithelial cells were neatly arranged, smooth, intact, and full, with normal lamina propria morphology and no inflammatory cell infiltration. After Hp infection, the mucosa in the SS1 group and GZ6B5 group atrophied and partially sloughed off, showing brush borders and unnamed grooves, with obvious inflammatory cell infiltration in the gastric lamina propria, and the inflammation in the GZ6B5 group was more severe. The mucosa in the SS1+LG3092 group and GZ6B5+LG3092 group did not show atrophy, sloughing, brush borders, or other pathological morphologies, and the inflammatory cell infiltration was also alleviated, with almost no obvious inflammatory response.

[0104] Example 5: Discovery and evaluation of functional active substances in Lactococcus gasseri LG3092

[0105] 5.1. Genomic mining and bioinformatics analysis of functional active substances from Lactococcus gasseri LG3092

[0106] The whole genome of *Lactococcus garvieae* LG3092 was sequenced using Illumina Nextseq 550 next-generation sequencing and Nanopore MinION next-generation sequencing platforms. The bacterial genomic DNA extraction method was the same as before. Next-generation sequencing used the AMT RapidDNA-Seq Kit for Illumina (CISTRO, CHINA) for library construction and the High Output v2.5 kit (Illumina, USA) for sequencing. Next-generation sequencing used the Rapid Barcoding Sequencing Kit (Nanopore, UK) for library construction and the R9.4.1 microarray (Nanopore, UK) for sequencing. The sequenced data were quality controlled using Trimmomatic (v0.39) and Filtlong (v0.2.0) software, and then assembled using Unicycler (v0.4.8) software. The assembled *Lactococcus garvieae* LG3092 genome was evaluated for genome quality control using Quast (v5.0.2) software. The genome of LG3092 was functionally annotated using Prokka (v1.11).

[0107] Sequencing revealed that the genome data of Lactococcus gasseri LG3092 contained the gene sequence of the bacteriocin Garvicin Q (GarQ). This gene has a GarQ-Core functional domain and a Lactococcin functional domain, with GarQ-Core being the core functional domain of a class II bacteriocin. Figure 5 Therefore, it is speculated that GarQ is a class IId bacteriocin with antibacterial activity, with a molecular weight (Mw) of 7586.48 Da and an isoelectric point of 6.54. The amino acid sequence of GarQ is shown in SEQ ID NO.3, consisting of 70 amino acids, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0108] 5.2. Heterologous expression and purification of bacteriocin GarQ

[0109] 5.2.1. Construction of the bacteriocin GarQ recombinant expression vector

[0110] Primers were designed for the nucleotide sequence SEQ ID NO.4 of GarQ. The primer sequence is GarQ-F: 5'-AAAAAA CATATG AAAAAATTAAATTATGAAGTGC-3' (SEQ ID NO. 17) and GarQ-R: 5'-GTGGTG CTCGAGATGTTGGGGACCAAAACCAG-3' (SEQ ID NO.18) corresponds to the restriction sites of NdeI and XhoI, respectively. The target gene GarQ was extracted by PCR using the above primers. The PCR product was purified using the Cycle Pure Kit (Omega) according to the manufacturer's instructions. The reaction system and conditions are as follows:

[0111] The reaction system was prepared as follows: Q5® High-Fidelity DNA Polymerase, 25 μL; DMSO, 1.5 μL; GarQ-F (10 μmol / L), 1.5 μL; GarQ-R (10 μmol / L), 1.5 μL; template DNA, 1 μL; ddH2O, 19.5 μL.

[0112] The following is the PCR amplification program: 98℃, 30 s; 98℃, 10 s, 72℃, 2 min (30 cycles); 72℃, 10 min.

[0113] The empty vector plasmid PET-30a and the purified PCR product were double-digested with enzymes and reacted at 37℃ for 1 h. The enzyme digestion system was as follows: target gene GarQ / empty vector plasmid PET-30a, 500 ng; FastDigest XhoI, 1 μL; FastDigest NdeI, 1 μL; 10 × Buffer, 2 μL; ddH2O, up to 20 μL.

[0114] The target gene and empty vector plasmid, after double enzyme digestion, were subjected to agarose gel electrophoresis. Bands of the corresponding fragment sizes were recovered from the gel and purified using the Gel Extraction Kit according to the manufacturer's instructions. The purified target gene fragment and PET-30a fragment were ligated at a molar ratio of 2:1 to 6:1, with 5 μL of T4 ligase added to a total volume of 10 μL. The reaction was carried out overnight at 16°C. The ligation product was used to transform DH5α to construct a recombinant strain using the heat shock method. The recombinant strain was plated on LB agar plates (containing 50 μg / mL kanamycin) and incubated overnight at 37°C. Positive colonies were picked, and PCR detection and sequencing were performed to verify the sequence against the gene nucleotide sequence. Mutation-free recombinant plasmids were then transformed into the BL21 expression strain using the heat shock method.

[0115] 5.2.2. Heterologous expression and purification of bacteriocin GarQ

[0116] The positive clone BL21 expressing strain was inoculated into LB liquid medium containing 50 μg / mL kanamycin and a final concentration of 0.5 mmol / L IPTG, and cultured at 37°C with shaking for 4 h to induce fusion protein expression. The culture was centrifuged at 1000×g for 5 min, the supernatant was discarded, and the cell pellet was resuspended in lysis buffer (1 mol / L NaCl, 50 mmol / L NaH2PO4, 70 mmol / L Na2HPO4 and 50 mmol / L imidazole, pH=7.8). The cells were intermittently disrupted by sonication (400 W, 4 sec operation, 8 sec interval, 20 min total). The cell lysate was centrifuged at low temperature and high speed (4°C, 10000 r / min, 20 min). The supernatant was collected and filtered through a 0.22 μm sterile filter for sterilization.

[0117] Using a low-pressure chromatography system, the supernatant was loaded at a flow rate of 0.5 mL / min onto a Ni-TED-FF affinity chromatography column pre-equilibrated with Ni-TED Binding-Buffer (20 mmol / L Tris-HCl, 0.15 mol / L NaCl, 6 mol / L urea, pH 8.0). The column was first washed with Ni-TED Binding-Buffer at a flow rate of 0.5 mL / min, then washed with Ni-TED Washing-Buffer (20 mmol / L Tris-HCl, 8 mmol / L imidazole, 0.15 mol / L NaCl, 6 mol / L urea, pH 8.0) at a flow rate of 1 mL / min, and finally eluted with Ni-TED Elution-Buffer (20 mmol / L Tris-HCl, 150 mmol / L imidazole, 0.15 mol / L NaCl, 6 mol / L urea, pH 8.0) at a flow rate of 1 mL / min. The eluent was collected and added to a dialysis bag for overnight dialysis. The dialysis sample was concentrated with PEG20000 to obtain recombinant bacteriocin GarQ.

[0118] The protein was analyzed by 12% SDS-PAGE, and its molecular weight was 9.38 kDa, with a purity greater than 90%. Figure 6 This improved the activity and yield of GarQ.

[0119] 5.3. Evaluation of the antibacterial effect of recombinant bacteriocin GarQ

[0120] The following bacteria were identified as being in logarithmic growth phase: *Salmonella typhimurium* (ATCC 14028), *Listeria monocytogenes* (ATCC 19115), *Enterococcus faecalis* (ATCC 29212), *Enterobacter sakazakii* (ATCC 29544), *Bacillus subtilis* (ATCC 6633), *Bacillus mycoides* (ATCC 10206), *Escherichia coli* (ATCC 25922), *Staphylococcus aureus* (ATCC 29213), *Lactococcus garvieae* (ATCC 43921), and *Lactobacillus paracasei*. Shirota and Lactiplantibacillus plantarum (isolated strain LP1Z) were inoculated at a 10% (v / v) in nutrient broth (Huankai Microbiology, Guangdong, China, 22010D1) and MRS broth (Huankai Microbiology, Guangdong, China, C27312G1), respectively, with 1 mg / mL or 0.5 mg / mL of recombinant bacteriocin GarQ added. The cultures were incubated aerobicly or anaerobic at 37°C with shaking for 48 h. The absorbance (OD) of the cultures was measured at different time points (0, 2, 4, 6, 8, 10, 12, 24, and 48 h). 600 And determine the growth status of bacteria.

[0121] The results showed that recombinant bacteriocin GarQ, at concentrations of 1 mg / mL and 0.5 mg / mL, had inhibitory effects on Salmonella Typhimurium, Listeria monocytogenes, Enterococcus faecalis, Lactococcus gasseri, Lactobacillus paracasei, and Lactobacillus plantarum, but no inhibitory activity against Cronobacter sakazakii, Bacillus subtilis, Bacillus flavus, Escherichia coli, and Staphylococcus aureus. Figure 7 The bacteriocin GarQ inhibits bacteria not by directly killing them, but by regulating their growth cycle and delaying the logarithmic growth phase. Therefore, it is speculated that GarQ will hardly cause dysbiosis and may even regulate the composition of the gastrointestinal microbiota to some extent, maintaining microecological balance and alleviating side effects such as gastrointestinal dysfunction and digestive discomfort caused by Hp eradication therapy.

[0122] Example 6: Lactococcus gasseri LG3092 inhibits the growth of gastritis-related microorganisms and regulates the gastric microbial environment in vivo.

[0123] 6.1. DNA extraction from gastric tissue and 16S rRNA gene amplicon sequencing

[0124] 0.1 g of mouse gastric tissue was collected, and microbial DNA was extracted from the gastric mucosa using the QIAamp® PowerFecal® Pro DNA Kit. The v3-v4 variable region of the bacterial 16S rRNA gene was amplified using a PCR thermal cycler. The purified product was quantified using a NanoDrop2000 spectrophotometer and a quantum fluorescence meter. A library was constructed, and sequencing was performed using an Illumina MiSeq PE300 platform.

[0125] The sequences were merged, quality filtered, and denoised. After denoising and leveling, the ASV table was obtained from the Silva 16S rRNA gene database. Based on the ASV table, the samples were analyzed. The analysis included Alpha diversity analysis and Beta diversity analysis. Multivariate principal coordinate analysis (PCOoA) and unweighted group average clustering analysis were used to explore the differences in microbial community abundance at the phylum and genus levels among the sample groups. LEfse analysis was used to analyze the differences between groups. Linear discriminant analysis (LDA) was used to estimate the effect of species abundance in each group on the difference effect, and to identify communities or species with significant differences in the intergroup division.

[0126] 6.2. Results Analysis

[0127] The results are as follows Figure 8 As shown, the effect of Lactococcus garvieae LG3092 intervention on bacterial community composition was identified by 16S rRNA gene amplicon pyrosequencing. Figure 8 A reflects α diversity through different indices. Compared with the control group (NC group), Helicobacter pylori SS1 and multidrug-resistant Helicobacter pylori GZ6B5 infection can reduce gastric α diversity to some extent. After intervention with Lactococcus gasseri LG3092, α diversity showed an upward trend. In particular, the Shannon index showed a significant difference in the upward adjustment between the GZ6B5_LG3092 group and the control group and the GZ6B5 group (p < 0.05). Figure 8 Principal coordinate analysis (PCoA) showed that the gastric flora composition of mice under the four different treatments exhibited significant aggregation, and the LG3092 intervention group tended to change towards the Control group, indicating that the gastric flora structure of mice supplemented with Lactococcus gasseri LG3092 was more normal. Figure 8At the phylum and genus levels, *Helicobacter pylori* (Hp) infection increased the relative abundance of *Proteobacteria* and *Bacteroidota*, and decreased the relative abundance of *Firmicutes*. After LG3092 intervention, the gut microbiota structure in infected mice became more similar to the normal state. At the genus level, compared with the control group, *Hp* infection significantly reduced *Lactobacillus* and significantly increased *Muribaculaceae*, *Lachnospiraceae*, and *Staphylococcus*. After LG3092 intervention, compared with the infection group, the abundance of *Lactobacillus* increased, while the abundance of *Lachnospiraceae* and *Staphylococcus* decreased. *Staphylococcus*, which can induce inflammatory responses, is one of the bacteria, besides *Hp*, closely related to the development of gastric diseases and capable of producing N-nitroso compounds; it is generally found in individuals with low risk of gastric cancer. The results indicate that LG3092 supplementation can alleviate *Hp*-induced gastric microbiota dysbiosis in mice and inhibit the growth of gastric inflammation-related bacteria.

[0128] Example 7: Safety evaluation of the genome and phenotype of Lactococcus gasseri LG3092

[0129] 7.1. Genomic characteristics and safety assessment of Lactococcus gasseri LG3092

[0130] The whole genome of *Lactococcus garvieae* LG3092 was sequenced using an Illumina Nextseq 550 second-generation sequencing system and a Nanopore MinION third-generation sequencing platform. The bacterial genomic DNA extraction and sequencing methods were the same as in step 5.1 of Example 5. Virulence genes and drug resistance genes were identified and annotated using Abricate (v0.8.13) software.

[0131] Sequencing revealed that the genome of *Lactococcus garvieae* LG3092 is 1.95 Mb in size with a GC ratio of 38.2%. The genome contains 1967 CDS regions, 0 repeat regions, 47 tRNAs, and 4 rRNAs, but no plasmids. Prokka annotation revealed 1395 functional coding genes and 572 hypothetical proteins in the *Lactococcus garvieae* LG3092 genome.

[0132] Using Abricate software to compare the bacteria with VFDB (Virulence Factor Database), ARG-Annot (Antibiotic Resistance Gene-ANNOTation), CARD (the Comprehensive Antibiotic Research Database), and Resfinder databases, no virulence genes or drug resistance genes were found.

[0133] 7.2. Antibiotic susceptibility of Lactococcus gasseri LG3092

[0134] According to the standards of the European Food Safety Authority (EFSA), the susceptibility of *Lactococcus gasseri* LG3092 to eight antibiotics was determined using the microbroth dilution method. These eight antibiotics were: ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. Suspensions of *Lactococcus gasseri* grown to the logarithmic growth phase were adjusted to 0.5 McFarland turbidity, and then different concentrations of antibiotic diluent (from 0.5 to 64 μg / mL) were added. The strains were then anaerobically incubated at 37°C for 48 h. After 48 h, the minimum inhibitory concentration (MIC) of each antibiotic was recorded. Based on the bacterial resistance criteria provided by EFSA, the strains were determined to be sensitive (S), intermediated (I), or resistant (R) to the antibiotics.

[0135] Table 3. Antibiotic sensitivity of Lactococcus garvieae LG3092 to different antibiotics.

[0136]

[0137] As shown in Table 3, the MICs of Lactococcus garvieae LG3092 against ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol are 0.5 μg / mL, 2 μg / mL, 64 μg / mL, 64 μg / mL, 16 μg / mL, 0.5 μg / mL, 2 μg / mL, and 1 μg / mL, respectively. This means that the bacterium is sensitive to the six antibiotics specified by EFSA.

[0138] 7.3. Hemolysis test of Lactococcus gasseri LG3092

[0139] Under aseptic conditions, the target lactobacillus was inoculated onto blood agar plates using an inoculation loop and anaerobically cultured at 37°C for 48 h. Hemolysis was then observed. After 48 h, no hemolysis was observed around the colonies of *Lactococcus garvieae* LG3092, while a clear hemolytic zone was observed around the colonies of the positive control *Staphylococcus aureus* ATCC 25923, suggesting that *Lactococcus garvieae* LG3092 does not pose a risk of hemolysis.

[0140] 7.4. Safety evaluation of Lactococcus gasseri LG3092 in mice

[0141] Mice were administered Lactococcus gasseri LG3092 via gavage, following the same method as in Example 4. The study found that no adverse symptoms such as death, weight loss, or diarrhea occurred in the mice one week after gavage. Pathological examination of the stomach revealed no significant inflammatory cell infiltration, and qPCR detection did not detect elevated inflammatory factors in the stomach tissue, suggesting that Lactococcus gasseri LG3092 is safe for use in mice.

[0142] Example 8: Specific molecular target recognition of Lactococcus gasseri LG3092

[0143] 8.1. Discovery of specific molecular targets in Lactococcus gasseri LG3092

[0144] Pan-genome analysis was performed on the whole genomes of *Lactococcus garvieae* LG3092, 97 other *Lactococcus garvieae* isolates from the NCBI database, and 22 other *Lactococcus garvieae* isolates from our team using Prokka (v1.11) and Roary (v3.11.2) software. After obtaining the core genome, Gubbins (v2.4.1) was used to identify genes containing high base substitution densities. Specific sequences of *Lactococcus garvieae* LG3092, distinct from other lactic acid bacteria, were obtained based on the pan-genome analysis. Primers were designed for these specific sequences using Oligo (v7) software, yielding the specific molecular target sequence SEQ ID NO.2 for recognizing this bacterium.

[0145] 8.2. Validation of the effectiveness of Lactococcus gasseri LG3092 in recognizing specific targets

[0146] The effectiveness of the specific molecular recognition target sequence of *Lactococcus garvieae* LG3092 was verified using polymerase chain reaction (PCR) and agarose gel electrophoresis. The template was bacterial DNA, extracted using the same method as before. The amplification primer sequences were: 3092F: 5'-GCGTTCAGAAAGCCTATGA -3' and 3092R: 5'-CGATGCCACATCATTGAAAC -3'.

[0147] The PCR reaction system was prepared as follows: 2×PCR Mix, 12.5 μL; 3092F (10 μmol / L), 1 μL; 3092R (10 μmol / L), 1 μL; template DNA (2.5 ng / μL), 1 μL; ddH2O, 9.5 μL;

[0148] The following are the PCR reaction conditions: 95℃, 7 min; 95℃, 30 s, 68℃, 30 s, 72℃, 30 s, 30 cycles; 72℃, 10 min; 4℃, ∞.

[0149] After PCR, 5-10 μL of the PCR product was subjected to 1.5% agarose gel electrophoresis. If Lactococcus garvieae LG3092 could form a single specific band at 525 bp, while other lactic acid bacteria could not form a single band at 525 bp, it indicates that this target pair has good efficacy in recognizing Lactococcus garvieae LG3092.

[0150] As attached Figure 9 As shown in Table 4, except for the DNA of *Lactococcus garvieae* LG3092, which produced a specific 525 bp amplification product after amplification using primers 3092F (5'-GCGGTTCAGAAAGCCTATGA-3') and 3092R (5'-CGATGCCACATCATTGAAAC-3'), no specific amplification products were observed for the other lactic acid bacteria isolates. This result suggests that the molecular target sequence can be detected by amplification using primers 3092F and 3092R, yielding a single band that can specifically distinguish *Lactococcus garvieae* LG3092 from other lactic acid bacteria.

[0151] Table 4. Amplification results of the target detected by LG3092 for different strains.

[0152]

[0153]

[0154]

[0155] 8.3. Quantitative real-time PCR detection of the effectiveness of Lactococcus gasseri LG3092 in recognizing specific molecular targets

[0156] The effectiveness and specificity of primers 3092F and 3092R were further verified using quantitative real-time PCR. First, *Lactococcus gasseri* LG3092 was cultured in MRS medium and the viable count was adjusted to 10⁻⁶. 9 CFU / mL, diluted 10-fold with physiological saline to obtain a concentration of 10. 3 10 4 10 5 10 6 10 7 10 8 10 9 DNA was extracted from a pure culture of the strain at CFU / mL to serve as a qPCR standard. Each template was tested in triplicate.

[0157] The qPCR reaction system was prepared as follows: 2×SYBR Green Premix, 5 μL; 3092F (10 μmol / L), 0.5 μL; 3092R (10 μmol / L), 0.5 μL; template DNA, 1 μL; ddH2O, 3 μL.

[0158] The following is the qPCR amplification program: 95℃, 30 s; 95℃, 5 s, 68℃, 30 s (40 cycles).

[0159] Figure 10 A and Figure 10 B is a schematic diagram of the real-time Ct values ​​of the qPCR quantitative detection method using primers 3092F and 3092R. Figure 10 A represents a bacterial concentration of 10. 2 -10 9 CFU / mL Figure 10 B represents a bacterial concentration of 10. 4 -10 9 CFU / mL. Results showed that when the bacterial concentration was ≥10... 4 At CFU / mL, the Ct fluorescence curve is relatively stable, therefore the detection limit of this primer is 10. 4 CFU / mL.

[0160] Figure 10C represents the standard curve for this primer. Plotting Ct from qPCR on the ordinate and the logarithm of the concentration of the pure culture of the standard strain on the abscissa, the fitted standard curve is y = -2.5024x + 29.394, R² = 0.9891. Therefore, these two target primers have good specificity and can be used specifically for the detection of this bacterium.

[0161] 8.4. Detection of viable bacterial count using PMA (propidium azidobromide) real-time PCR.

[0162] Lactococcus gasseri LG3092 was cultured to the stationary phase. A certain amount of the bacteria was then inactivated by heating at 95°C for 10 min. The absence of bacterial growth was detected using the 10 µL plate culture method. Two 500 µL aliquots of the inactivated bacteria were placed in clean centrifuge tubes. One aliquot was not treated with PMA, and the other was treated with 50 µmol / L PMA. Similarly, two aliquots of live bacteria were prepared and treated using the same method.

[0163] The specific procedure for the PMA reaction is as follows: Dissolve the PMA dye in DMSO to prepare a 20 mmol / L stock solution and store it at -20°C in the dark. Add 500 µL of the sample to be tested to a 1.5 mL transparent centrifuge tube, then add 1.25 µL of 20 mmol / L PMA (final concentration 50 µmol / L), shake up and down 3-4 times, and react on a shaker in the dark for 10 min. Then, place the centrifuge tube flat on ice and irradiate it with a 650 W halogen lamp for 10 min, with a distance of 20 cm between the centrifuge tube and the halogen lamp bulb. After the reaction, centrifuge the tube at 8000 rpm for 10 min, collect the bacterial cells, and extract DNA using a bacterial DNA extraction kit.

[0164] Two types of samples were simultaneously detected using PMA-qPCR and conventional qPCR, respectively. The qPCR reaction system and amplification program shown in 8.3 were used. The viable bacterial count of the samples was calculated using a standard curve. The detection results of the two molecular biological methods were compared. Each experiment was performed in duplicate.

[0165] The results are as follows Figure 11 The results showed that the number of viable bacteria in the live bacteria + PMA-qPCR treatment group and the groups amplified by ordinary qPCR (live bacteria-qPCR and dead bacteria-qPCR) were basically the same, indicating that ordinary qPCR could not accurately distinguish between live and dead bacteria. However, there was a significant difference between the live bacteria + PMA-qPCR group and the dead bacteria + PMA-qPCR group. The detection line of the Lactococcus gasseri LG3092 specific primer was 10. 4 CFU / mL, including 10 CFU / mL for dead bacteria + PMA-qPCR detection. 4 CFU / mL indicates that the PMA method can detect 10 4The results indicate that PMA-qPCR can selectively detect viable bacteria above the detection limit for Lactococcus gasseri LG3092.

[0166] Example 9: Evaluation of the probiotic properties of Lactococcus gasseri LG3092

[0167] 9.1. Tolerance test of Lactococcus gasseri LG3092 to artificial gastric juice

[0168] Adjust the pH of PBS (pH 7.4) buffer to 3.0 with 0.1 mol / L HCl, add pepsin (3.0 g / L), dissolve, and filter through a 0.22 μm sterile filter to prepare simulated gastric juice, which should be prepared fresh for use. Inoculate the prepared bacterial culture at a volume ratio of 2% into 10 mL of MRS liquid medium, and incubate under anaerobic conditions at 37℃ for 18–24 h. Collect the bacterial cells by centrifugation at 4000×g for 10 min, wash the cells three times with PBS, and adjust the bacterial concentration to 10% with PBS. 8 -10 9 The concentration of Lactococcus garvieae was between CFU / mL. 1 mL of Lactococcus garvieae resuspended in PBS was added to 5 mL of simulated gastric fluid, along with 1.5 mL of 0.5% (w / v) NaCl. The mixture was quickly placed in a 37°C incubator and incubated for 0 h and 3 h. After each incubation, the culture was serially diluted 1:10 with sterile physiological saline. 100 μL of each diluted culture was then evenly spread onto MRS solid medium using a disposable spreader. The medium was incubated at 37°C under anaerobic conditions for 48 h. Viable Lactococcus counts were then performed, and the survival rate of Lactococcus garvieae LG3092 at pH 3.0 for 3 h was calculated using the formula: Survival rate (%) = (Number of surviving Lactococcus at 3 h / Number of surviving Lactococcus at 0 h) × 100%. The calculated survival rate of Lactococcus garvieae LG3092 in simulated gastric fluid for 3 h was 75.82%.

[0169] 9.2. Determination of the gastric adhesion and colonization ability of Lactococcus gasseri LG3092

[0170] 9.2.1. Determination of in vitro cell adhesion of Lactococcus gasseri LG3092

[0171] The adhesion and colonization ability of *Lactococcus garvieae* LG3092 was assessed by co-culturing human gastric mucosal epithelial GES-1 cells with bacteria. *Lactococcus garvieae* cells were collected, washed twice with PBS, and resuspended in physiological saline. GES-1 cells were pre-coated into 6-well plates, with two sampling time points: 0 h and 3 h. The adhesion and colonization ability of LG3092 at different sampling time points was assessed in triplicate. The culture medium in the wells was aspirated and discarded, and the cells were washed twice with PBS. *Lactococcus garvieae* LG3092 bacterial suspension was added to the wells, adjusting the cell-to-bacterial ratio to 1:10. The cell culture volume was 2 mL, and the plates were incubated at 37°C in a 5% CO2 incubator. Samples were taken at 0 h and 3 h for cell counting.

[0172] Upon reaching the sampling time point, the culture medium in the corresponding wells was aspirated, and the sample was washed twice with PBS. 1 mL of trypsin was added to each well for 4 min of digestion. After digestion, 1 mL of physiological saline was added. The mixture in each well was serially diluted to an appropriate concentration, and 100 μL was plated onto MRS plates and incubated at 37°C in an anaerobic environment. Colony counting was performed after 48 h. Adhesion rate (%) = (Number of viable Lactococcus at 3 h / Number of viable Lactococcus at 0 h) × 100%. The experiment showed that the adhesion rate of *Lactococcus garvieae* LG3092 was 72.56% after 3 h.

[0173] 9.2.2. Determination of the colonization ability of Lactococcus gasseri LG3092 in mouse stomach

[0174] 0.1 g of gastric tissue was taken from mice in the GZ6B5+LG3092 group, SS1+LG3092 group, and LG3092 group, and was analyzed using QIAamp. ® PowerFecal ® Microbial DNA was extracted using the Pro DNA Kit. Quantification was performed using the standard curve and method shown in step 8.3 of Example 8. The standard curve was y = -2.5024x + 29.394, with R² = 0.9891.

[0175] The qPCR primers for Lactococcus gasseri LG3092 were specific molecular target primers, with primer sequences of 3092F: 5'-GCGTTCAGAAAGCCTATGA-3' (SEQ ID NO.19) and 3092R: 5'-CGATGCCACATCATTGAAAC-3' (SEQ ID NO.20). The results indicated that the average Ct value of the gastric tissue of mice in the 0.1 g LG3092 group was 16.13, therefore, the colonization level of LG3092 in the mouse gastric tissue was 10-1. 6.3 CFU / g; In the SS1+LG3092 group, the average Ct value of 0.1 g mouse gastric tissue was 16.63, indicating that the colonization amount of LG3092 in the mouse gastric tissue was 10. 6.1 CFU / g; In the GZ6B5+LG3092 group, the average Ct value of 0.1g of mouse gastric tissue was 19.38, indicating that the colonization amount of LG3092 in the mouse gastric tissue was 10. 5.0 CFU / g.

[0176] The above results suggest that Lactococcus garvieae LG3092 has good potential for gastric colonization in both normal vivo and Hp infection models, and therefore has good application prospects.

[0177] While preferred embodiments of the present invention have been disclosed above, they are not intended to limit the invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0178] SEQ ID NO.1 (16S rRNA gene sequence of Lactococcus gasseri LG3092)

[0179]

[0180] SEQ ID NO.2 (Specific molecular target sequence of Lactococcus garvieae LG3092)

[0181] GCGGTTCAGAAAGCCTATGA TCATGTGAACTACATCTACGATCAGTGTGTTGACAAGGTAAGTTATGCCATTACCGAAAATAATGGGAGATTGTCAAATGAGTTAGATGAGTTCTATAATCGTGATTTTTCAACACAACTGCAGTTTAGGCTAGAAGAAAAGAATGCCGCTGGGAATCCATTTGTTAAACGGATATTTAAATCTGACATCTTTACACAAGTCGGACAAAAGGTCATAGAAAATACAGTTGGTGAGGCTGGTGCTAACGGACTAAAAGTTTTTTCTGGTAGTAATGTGCATGAGTGGGTGCTTAATATAGGACATCACTTTGGCAAAAGCTTTAAACCCTGGGAAGCAGTGAAATGGACTAAGGGAATAAATTTTGCTGGAAAGGCTGTAGGTATCTTCGGGGTGGTACTGTCTGTGGGTATGCAGGCGAAAGAAGATGTGGACTCTGACAATCGAGAAAAAGAGATGCGTGTCAGCCGTGAAAAACTTCGAGCCG GTTTCAATGATGTGGCATCG

[0182] SEQ ID NO.3 (Amino acid sequence of GarQ)

[0183] MKKLNYEVLSDEELQKIDGSGTPLFYGANGYLTRENGKYVYRVTKDPVSAVFGVISNGWGSAGAGFGPQH

[0184] SEQ ID NO.4 (Nucleotide sequence of GarQ)

[0185] GTGAAAAAATTAAATTATGAAGTGCTTTCAGATGAAGAGTTACAAAAAATTGATGGTAGTGGTACACCTCTATTTTATGGTGCTAATGGTTACTTAACAAGAGAAAACGGAAAATATGTATATCGAGTTACAAAGGATCCAGTTAGTGCAGTCTTTGGAGTTATCTCAAACGGTTGGGGAAGCGCTGGGGCTGGTTTTGGTCCCCAACATTAA。

Claims

1. Lactococcus gasseri ( Lactococcus garvieae LG3092, with accession number GDMCC No: 63939.

2. The use of Lactococcus gasseri LG3092 as described in claim 1 in the preparation of medicaments for the prevention and / or treatment of Helicobacter pylori infection.

3. The application according to claim 2, characterized in that, The Helicobacter pylori mentioned is a multidrug-resistant Helicobacter pylori.

4. The application of Lactococcus gasseri LG3092 as described in claim 1 in the production of class IId bacteriocin GarQ, wherein the amino acid sequence of the bacteriocin GarQ is shown in SEQ ID NO.3, and the bacteriocin has an inhibitory effect on Helicobacter pylori, Salmonella typhimurium, Listeria monocytogenes, Enterococcus faecalis, Lactococcus gasseri, Lactobacillus paracasei, and Lactobacillus plantarum.

5. A medicine for the prevention and / or treatment of Helicobacter pylori infection, characterized in that, The active ingredient is a live bacterium containing Lactococcus gasseri LG3092 as described in claim 1.

6. A method for identifying *Lactococcus gasseri* LG3092 as described in claim 1, characterized in that, The test bacteria were amplified by PCR using primer sets 5'-GCGGTTCAGAAAGCCTATGA-3' and 5'-CGATGCCACATCATTGAAAC-3'. If a 525 bp product was amplified, it was identified as Lactococcus gasseri LG3092; otherwise, it was not Lactococcus gasseri LG3092. The nucleotide sequence of the 525 bp product is shown in SEQ ID NO.2.

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