Lactococcus formosanus strain with helicobacter pylori co-aggregation capability and application of lactococcus formosanus strain

Abstract

The invention discloses lactococcus formosanus with coaggregation capacity with helicobacter pylori and application of the lactococcus formosanus, and belongs to the technical field of microorganisms and the technical field of medicines. The lactococcus formosanus CCFM1461 screened by the invention has relatively strong gastric environment tolerance and acid production capacity, the copolymerization rate of the lactococcus formosanus CCFM1461 and helicobacter pylori within 120 minutes can reach 69.94%, the adhesion of the helicobacter pylori in AGS cells can be reduced, and the adhesion rate of the helicobacter pylori is reduced by 43.79%; the strain also has strong co-aggregation ability with other pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Shigella, Salmonella typhimurium and Pseudomonas fluorescens), and has huge application prospects in the aspects of removal of helicobacter pylori, preparation of a helicobacter pylori adhesion scavenger and the like.

Description

Technical Field

[0001] The present invention relates to a Lactococcus taiwanensis strain with the ability to co-aggregate with Helicobacter pylori and its application, belonging to the fields of microbial technology and pharmaceutical technology. Background Art

[0002] Helicobacter pylori (Hp) is a spiral or S-shaped microaerophilic Gram-negative bacterium with strong acid tolerance, which can colonize on the surface of human gastric mucosa and the duodenum. In 1994, Helicobacter pylori was classified as a Group I carcinogen by the World Health Organization. Helicobacter pylori can survive in the human body for a long time and is one of the most important epidemic pathogenic bacteria in the world at present. The detection rate of Helicobacter pylori positive has exceeded 50% globally. The infection rate of Helicobacter pylori increases with age and is related to factors such as socioeconomic and health status and race. After the body is infected with Helicobacter pylori, it can cause related diseases such as acute gastritis, peptic gastric ulcer, duodenal bulb ulcer and gastric cancer, seriously affecting people's physical health and quality of life.

[0003] Currently, the treatment of Helicobacter pylori mainly uses triple or quadruple therapies combined with antibiotics. However, due to the frequent use of antibiotics, drug-resistant Helicobacter pylori strains have emerged. At the same time, during the use of triple or quadruple therapies for patients infected with Helicobacter pylori, serious adverse reactions (such as abdominal pain, nausea, diarrhea, etc.) often occur, causing great harm to patients. Therefore, probiotics have been proposed for use in the treatment of Helicobacter pylori. A large number of in vivo and clinical studies have proved that certain probiotics have the effect of preventing and / or treating Helicobacter pylori infection. The Chinese patent application document with the publication number CN111607538A discloses the application of a Lactobacillus rhamnosus CCFM1119 strain in the prevention and / or treatment of Helicobacter pylori, proving that Lactobacillus rhamnosus CCFM1119 can significantly reduce the colonization amount of Helicobacter pylori in patients with Helicobacter pylori, relieve the gastrointestinal adverse symptoms of patients, and significantly improve the eradication rate of Helicobacter pylori in patients with Helicobacter pylori.

[0004] Coaggregation refers to the process in which two types of cells adhere to each other to form aggregates. When at least two different cell types adhere to each other and form coaggregates, this process is called coaggregation. The coaggregation of probiotics and Helicobacter pylori masks the adhesion-related substances on the surface of Helicobacter pylori cells with probiotic cells, preventing Helicobacter pylori cells from binding to gastric epithelial cells, thereby reducing the host inflammatory response caused by the binding of Helicobacter pylori to gastric epithelial cells. The coaggregates of probiotics and Helicobacter pylori are excreted through the gastrointestinal tract, reducing the Helicobacter pylori load in the host body. Therefore, appropriate probiotic intervention can reduce the Helicobacter pylori load in the stomach by forming coaggregates with Helicobacter pylori, and may even eradicate Helicobacter pylori in the host stomach. Lactobacillus mucosae DSM17648 (PylopassTM) is the most widely used probiotic with the ability to coaggregate Helicobacter pylori at present. Clinical trials have shown that the inactivated bacterial powder can still reduce the Helicobacter pylori load in patients. Subsequently, the patent document CN103648511A publicly disclosed a method for improving the formation of coaggregates between DSM17648 and Helicobacter pylori, making its coaggregation effect better.

[0005] The Chinese invention patent application document with the publication number CN103648511A also reported many lactobacilli with coaggregation effects on Helicobacter pylori, including Lactobacillus helveticus, Lactiplantibacillus plantarum, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus amylophilus, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus mucosae, etc. However, the coaggregation study of Lactococcus and Helicobacter pylori is relatively less. Most of the studies on probiotics inhibiting Helicobacter pylori mainly focus on the inhibitory effect on single Helicobacter pylori. However, the research on the ability to simultaneously inhibit multiple pathogenic bacteria is still relatively lacking. Although existing studies have shown that certain probiotics have good inhibitory effects on Helicobacter pylori, the exploration of their comprehensive antibacterial effects in multiple pathogenic bacteria populations has not received enough attention.

[0006] Lactococcus taiwanensis is a Gram-positive, catalase-negative, non-motile facultative anaerobe belonging to the genus Lactococcus. It is usually spherical or oval in shape and often arranged in chains or symmetrically. Lactococcus taiwanensis was first isolated and identified from milk and is widely used in dairy fermentation, especially in the production of cheese and yogurt. It has certain probiotic properties, which are helpful for intestinal health and enhancing immunity.

[0007] Therefore, obtaining a Lactococcus that can form coaggregates with Helicobacter pylori to effectively inhibit the colonization of Helicobacter pylori and also has a certain inhibitory effect on other common pathogenic bacteria has become the focus and difficulty of the research. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a Lactococcus taiwanensis CCFM1461 that can effectively form a co-aggregated bacterial body with Helicobacter pylori and its application. The preservation number is GDMCC No: 65679, and the preservation date is December 26, 2024.

[0009] In one embodiment, the Lactococcus taiwanensis CCFM1461 is derived from a Yunnan milk cake sample, and the 16S rDNA sequence of this strain is as shown in SEQ ID NO.1.

[0010] In one embodiment, the colonies of the Lactococcus taiwanensis CCFM1461 on MRS solid medium are milky white, round and convex, with a smooth and semi-transparent surface, and the diameter is 1 - 2 mm.

[0011] The present invention provides a composition containing the Lactococcus taiwanensis CCFM1461.

[0012] In one embodiment, the composition contains live cells of the Lactococcus taiwanensis CCFM1461.

[0013] In one embodiment, the number of the Lactococcus taiwanensis CCFM1461 ≥ 1×10 7 CFU / mL.

[0014] In one embodiment, the composition includes but is not limited to food, medicine, or health products.

[0015] The present invention also provides a starter containing the Lactococcus taiwanensis CCFM1461.

[0016] In one embodiment, the preparation method of the starter is as follows: culture the Lactococcus taiwanensis CCFM1461 in a medium at 37°C for 48 h to obtain a culture solution; centrifuge the culture solution to obtain bacterial cells; wash the bacterial cells 3 times with physiological saline and then resuspend them with a lyoprotectant to obtain a resuspended solution; optionally, freeze-dry the resuspended solution by vacuum freezing.

[0017] In one embodiment, the mass ratio of the lyoprotectant to the bacterial cells is 2:1.

[0018] In one embodiment, the lyoprotectant contains 130 g / L of skim milk powder.

[0019] In one embodiment, the medium contains skim milk, glucose, tryptone, and yeast extract.

[0020] In one embodiment, the pH of the medium is 6.8.

[0021] In one embodiment, the medicine contains the aforementioned Lactococcus Taiwanense CCFM1461, a drug carrier and / or a pharmaceutical excipient.

[0022] In one embodiment, the drug carrier comprises microcapsules, microspheres, nanoparticles and / or liposomes.

[0023] In one embodiment, the pharmaceutical excipient comprises an excipient and / or an additive.

[0024] In one embodiment, the excipient comprises a binder, a filler, a disintegrant and / or a lubricant.

[0025] In one embodiment, the additive comprises a solubilizer, a co-solvent, a latent solvent and / or a preservative.

[0026] In one embodiment, the dosage form of the drug is powder, granules, capsules, tablets, pills or oral liquid.

[0027] The present invention also provides a microbial preparation containing the formosan lactococcus CCFM1461.

[0028] In one embodiment, the content of Lactococcus Taiwanis in the microbial preparation is at least 5×10 9 CFU / mL or 5×10 9 CFU / g.

[0029] In one embodiment, the microbial preparation is in solid, liquid or powder form.

[0030] The present invention also provides a use of the above-mentioned Taiwan Lactococcus CCFM1461 in preparing a medicine for preventing and / or treating Helicobacter pylori infection.

[0031] In one embodiment, the drug contains the Taiwan Lactococcus CCFM1461, a drug carrier and / or a pharmaceutical excipient.

[0032] The present invention also provides the use of the above-mentioned Taiwan lactococcus CCFM1461 and Helicobacter pylori for the purpose of preventing and treating diseases through copolymerization.

[0033] The present invention also provides an inhibitor for reducing the load of pathogenic bacteria, wherein the inhibitor contains the Taiwan lactococcus (Lactococcus taiwanensis) CCFM1461.

[0034] In one embodiment, the pathogenic bacteria include, but are not limited to, one or more of Helicobacter pylori, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shigella, and Pseudomonas fluorescens.

[0035] Beneficial effects

[0036] 1. The present invention provides a strain of Lactococcus taiwanensis CCFM1461, which can specifically bind to the free Helicobacter pylori existing in the stomach and prevent it from continuing to grow and form a pathogenic biofilm colonizing in the stomach. Specifically, it is embodied in:

[0037] (1) It can co-aggregate with Helicobacter pylori in artificial gastric juice (pH = 3), and the co-aggregation rate can reach 69.93% after 120 min, forming visible flocculent co-aggregates to the naked eye.

[0038] (2) It has strong self-aggregation ability, and the self-aggregation rate reaches 78.43% after 120 min.

[0039] (3) It can effectively inhibit the adhesion of Helicobacter pylori to AGS (gastric adenocarcinoma) cells, and the inhibition rate of adhesion reaches 46.73%.

[0040] 2. The Lactococcus taiwanensis CCFM1461 provided by the present invention has good in vitro acid tolerance. The survival rate reaches 91.66% after 3 h and 64.68% after 6 h in artificial gastric juice (pH = 3); and Lactococcus taiwanensis CCFM1461 can grow in MRS medium at pH = 3, and the OD 600 reaches 0.24 after 24 h. In addition, Lactococcus taiwanensis CCFM1461 also has strong acid production ability, and the pH of the fermentation supernatant reaches 3.83 after 18 h.

[0041] 3. The Lactococcus taiwanensis provided by the present invention also has strong co-aggregation effects with pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Shigella, Salmonella typhimurium, and Pseudomonas fluorescens. The co-aggregation rates reach 59.39%, 34.67%, 28.63%, 40.32%, and 52.20% respectively after 120 min.

[0042] It can be seen that Lactococcus taiwanensis CCFM1461 has great application prospects in inhibiting Helicobacter pylori and preparing Helicobacter pylori inhibitors.

[0043] Biological material preservation

[0044] A strain of Lactococcus taiwanensis CCFM1461, taxonomically named Lactococcus taiwanensis, has been deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCCNO: 65679. The deposit address is Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : Growth curve of Lactococcus formosanus.

[0046] Figure 2 :Colony morphological characteristics of Taiwan Lactococcus CCFM1461.

[0047] Figure 3 :Verification of the co-aggregation of strains with Helicobacter pylori; among them, (A) Differences in the co-aggregation of different lactococci with Helicobacter pylori (B) Macroscopic view of the co-aggregation of Taiwan Lactococcus and Helicobacter pylori.

[0048] Figure 4 : Differences in self-aggregation ability among different lactococci.

[0049] Figure 5 :The ability of different Taiwan lactococci to inhibit the adhesion of Helicobacter pylori.

[0050] Figure 6 : Growth of different Taiwan lactococci in MRS medium (pH=3) and survival rates in artificial gastric juice (pH=3).

[0051] Figure 7 : Acid production capacity of different Formosan lactococci.

[0052] Figure 8 : Coaggregation ability of different Taiwan lactococci with other pathogenic bacteria.

[0053] Figure 9 :Analysis of functional substances in the co-aggregation of Taiwan Lactococcus CCFM1461 and Helicobacter pylori, including the effects of different inactivation conditions and removal of surface proteins on the co-aggregation of Taiwan Lactococcus CCFM1461 and Helicobacter pylori, the differences in surface protein concentrations of different Taiwan Lactococci and surface protein analysis of Taiwan Lactococcus CCFM1461. DETAILED DESCRIPTION

[0054] The Helicobacter pylori involved in the following examples is Helicobacter pylori SS1 from the National Collection of Type Cultures (NTCC); the NaCl involved in the following examples is purchased from the China National Pharmaceutical Corporation; the Columbia medium involved in the following examples is purchased from OXOID Company in the UK; the sterile defibrinated sheep blood involved in the following examples is purchased from Hangzhou Xinrui Company; the BHI liquid medium involved in the following examples is purchased from Qingdao Haibo Company; the fetal bovine serum involved in the following examples is purchased from Nanjing Senbeijia Company; the F12 liquid medium and fetal bovine serum involved in the following examples are purchased from Gibco Company in the United States; the phenol red and urea involved in the following examples are purchased from Macklin Company; the pepsinogen involved in the following examples is purchased from Shanghai Sangon Biotech Co., Ltd.

[0055] The Lactococcus strains involved in the following examples are: Lactococcus taiwanensis CCFM1461, Lactococcus taiwanensis FDRB31B5, Lactococcus lactis FDRB37B13, Lactococcus lactis FDRB37B11, Lactococcus cremoris FDQL61B3, and Lactococcus cremoris FDRB37B7. All of them are strains screened during the same period and are preserved in the self-owned strain library of the Biotechnology Center of the School of Food Science and Technology, Jiangnan University. The positive control strain, Lactobacillus mucosae DSM17648, involved in the following examples is a commercial strain from Shanghai Vita Medical Technology Co., Ltd. The other pathogenic bacteria involved in the following examples are: Escherichia coli CICC24187 (standard strain, published in the literature "Research on Standard Strains for the Detection of Diarrheagenic Escherichia coli [J]. Food and Fermentation Industries, 2020"), Staphylococcus aureus ATCC6538P (standard strain, published in the literature A concise synthesis and antimicrobial activities of 3-polyamino-23,24-bisnorcholanes as steroid-polyamine conjugates), Salmonella typhimurium TA100 (standard strain, published in the literature "Development of Ready-to-Use Standard Strains of Salmonella typhimurium TA97, TA98, TA100, and TA102"), Shigella sp. ATCC12022 (standard strain, published in the literature "Establishment of a Visual Turbidity / Fluorescence Loop-Mediated Isothermal Amplification Method for Shigella"), Pseudomonas fluorescens AS1.55 (standard strain, published in the literature "Preparation and characterization of chitosan / pullulan film loading carvacrol for targeted antibacterial packaging of chilled meat"), and all are preserved in the self-owned strain library of the Biotechnology Center of the School of Food Science and Technology, Jiangnan University. Lactococcus taiwanensis CCFM1461, Lactococcus taiwanensis FDRB31B5, Lactococcus lactis FDRB37B13, Lactococcus lactis FDRB37B11, Lactococcus cremoris FDQL61B3, and Lactococcus cremoris FDRB37B7 are all strains screened by the inventor team during the same period and are currently preserved in the self-owned strain library of the Biotechnology Center of the School of Food Science and Technology, Jiangnan University.

[0056] The culture media involved in the following examples are as follows:

[0057] MRS liquid medium (g / L): Peptone 10 g / L, Beef extract 10 g / L, Glucose 20 g / L, Sodium acetate anhydrous 2 g / L, Yeast extract 5 g / L, Di-ammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.25 g / L, Tween 80 1 mL / L; pH is 6.8.

[0058] MRS solid medium: Add agar 20 g / L based on mMRS liquid medium.

[0059] Columbia blood agar plate: Dissolve 39 g of solid powder of Columbia medium in 1 L of water, sterilize at 121 °C for 15 min, and add 7.5% (v / v) sterile defibrinated sheep blood after cooling to 55 °C - 60 °C, then mix well and pour plates.

[0060] BHI liquid medium (g / L): Tryptone 10.0 g / L, Beef heart infusion powder 17.5 g / L, Sodium chloride 5.0 g / L, Glucose 2.0 g / L, Disodium hydrogen phosphate (12H2O) 2.5 g / L, pH value 7.4 ± 0.2.

[0061] LB liquid medium (g / L): Peptone 10 g / L, Yeast extract 5 g / L, Sodium chloride 5 g / L, pH is 6.8.

[0062] LB solid medium: Add agar 20 g / L based on LB liquid medium.

[0063] Artificial gastric juice (g / L): Pepsin (porcine pepsin) 3 g / L, Sodium chloride 5 g / L, adjust pH value to 3.0 ± 0.2.

[0064] The detection methods involved in the following examples are as follows:

[0065] Detection method for viable count: Adopt the national standard "GB 4789.35-2016 National Food Safety Standard Food Microbiology Examination Detection of Lactic Acid Bacteria".

[0066] The detection and calculation method of copolymerization rate refers to the paper "In vitro screening of lactobacilli with antagonistic activity against Helicobacter pylori from traditionally fermented foods", and the specific steps are as follows:

[0067] Measure the OD values of Lactococcus and the mixed bacterial liquid before / after reaction (0 min / T min) respectively, and calculate the copolymerization rate according to the following formula: Copolymerization rate (%) = [(OD600 Lactococcus + OD 600 Helicobacter pylori) - 2×OD 600 Mixture] / (OD 600 Lactobacillus + OD 600 Helicobacter pylori) × 100.

[0068] The preparation method of Helicobacter pylori bacteria involved in the following examples is as follows:

[0069] After streaking Helicobacter pylori SS1 on Columbia blood agar plates, culture it in a triple gas incubator at 37°C (85% N2, 10% CO2, 5% O2) for 3 days to obtain single colonies; pick single colonies and inoculate them into BHI medium containing 5% (v / v) fetal bovine serum, and culture in a triple gas incubator at 37°C (85% N2, 10% CO2, 5% O2) for 4 days to obtain a seed solution; inoculate the seed solution into BHI medium at an inoculation amount of 2% (v / v), and culture in a triple gas incubator at 37°C (85% N2, 10% CO2, 5% O2) for 4 days to obtain Helicobacter pylori bacterial liquid; centrifuge the Helicobacter pylori bacterial liquid at 8000g for 10 min to obtain Helicobacter pylori bacteria;

[0070] Among them, Columbia blood agar plate: Dissolve 39 g of Columbia medium solid powder in 1 L of water, sterilize at 121°C for 15 min, and add 7.5% (v / v) sterile defibrinated sheep blood after cooling to 55°C - 60°C, mix well and pour plates.

[0071] The preparation method of Lactococcus lactis subsp. taiwanensis bacteria involved in the following examples is as follows:

[0072] Streak Lactococcus lactis subsp. taiwanensis on mMRS solid medium and culture it under aerobic conditions at 37°C for 48 h to obtain single colonies; pick single colonies and inoculate them into 5 mL of mMRS liquid medium, culture under aerobic conditions at 37°C for 18 h for activation, and activate continuously for two generations to obtain an activated solution; inoculate the activated solution into mMRS liquid medium at an inoculation amount of 2% (v / v), and culture under aerobic conditions at 37°C for 18 h to obtain bacterial liquid; centrifuge the bacterial liquid at 5000 r / min for 15 min, discard the upper layer of the medium to obtain Lactococcus lactis subsp. taiwanensis bacteria.

[0073] The preparation method of other pathogenic bacteria (Escherichia coli, Salmonella, Staphylococcus aureus, Shigella, and Pseudomonas fluorescens) bacteria involved in the following examples is as follows:

[0074] Escherichia coli CICC 24187, Salmonella TA100, Staphylococcus aureus ATCC 6538P, Shigella ATCC 12022, and Pseudomonas fluorescens AS1.55 were streaked on LB solid medium and cultured under aerobic conditions at 37 °C for 48 h to obtain single colonies; single colonies were picked and inoculated into 5 mL of LB liquid medium and cultured under aerobic conditions at 37 °C for 18 h for activation. After two consecutive generations of activation, an activated solution was obtained; the activated solution was inoculated into LB liquid medium at an inoculation amount of 2% (v / v) and cultured under aerobic conditions at 37 °C for 18 h to obtain a bacterial solution; the bacterial solution was centrifuged at 5000 r / min for 15 min, and the upper layer of the culture medium was discarded to obtain pathogenic bacteria cells.

[0075] Example 1: Screening, strain identification, and culture of Lactococcus taiwanensis

[0076] 1. Screening

[0077] Taking Yunnan milk cake as a sample, after pretreatment of the sample, the sample was mixed evenly, 0.5 mL of the sample was taken and added to 4.5 mL of 0.9% normal saline for gradient dilution. A suitable gradient dilution solution was selected and spread on MRS solid medium, cultured at 37 °C for 48 h, typical colonies were picked and streaked and purified on an MRS plate, single colonies were picked and transferred to liquid MRS liquid medium for enrichment, and preserved with 30% glycerol to obtain strain CCFM1461.

[0078] 2. Identification

[0079] The genome of strain CCFM1461 was extracted, and the 16S rDNA of strain CCFM1461 was amplified and sequenced (completed by Sangon Biotech (Shanghai) Co., Ltd.). This sequence was compared in GenBank, and the homology with the genus Lactococcus was 99%; the results showed that the strain was Lactococcus taiwanensis, named Lactococcus taiwanensis CCFM1461. The 16S rDNA sequence of this strain is shown in SEQ ID NO.1.

[0080] 3. Culture

[0081] The obtained Lactococcus taiwanensis was inoculated on mMRS solid medium and anaerobically cultured at 37 °C for 48 h, and then the colony morphology was observed and recorded ( Figure 2 ). The colonies were milky white, round and raised, with a smooth and semi-transparent surface, and the diameter was 1 - 2 mm. Before and after culture, the pH of the blank culture solution and the culture solution with added bacteria was measured by a pH meter, and it was found that Lactococcus taiwanensis produced acid during the culture process. The Lactococcus taiwanensis strain was inoculated into mMRS liquid medium and placed in a microplate reader and aerobically cultured at 37 °C for 48 h. During the culture process, the OD of the culture solution was measured at intervals of 1 h 600, it was found that the growth stationary phase of Lactococcus taiwanensis was reached after 18 - 24 h of cultivation, and the results were as Figure 1 shown.

[0082] Example 2: Verification of the co - aggregation effect between strains and Helicobacter pylori

[0083] The Lactococcus taiwanensis screened in Example 1, the commercial strain Limosilactobacillus reuteri DSM17648, Lactococcus lactis subsp. Lactis, and Lactococcus cremoris were verified for their co - aggregation effect with Helicobacter pylori under the same conditions. The specific steps are as follows:

[0084] (1) Cultivation of Helicobacter pylori:

[0085] Helicobacter pylori SS1 was streaked on Columbia blood agar plates (Columbia blood agar plates: 39 g of Columbia medium solid powder was dissolved in 1 L of water, sterilized at 121 °C for 15 min, and after cooling to 55 °C - 60 °C, 7.5% (v / v) sterile defibrinated sheep blood was added, mixed well and poured into plates), and then cultured in a triple - gas incubator at 37 °C (85% N2, 10% CO2, 5% O2) for 3 days to obtain single colonies; single colonies were picked and inoculated into BHI liquid medium containing 5% (v / v) fetal bovine serum, and cultured in a triple - gas incubator at 37 °C (85% N2, 10% CO2, 5% O2) for 4 days to obtain a seed solution; the seed solution was inoculated into BHI liquid medium at an inoculation amount of 2% (v / v) and cultured in a triple - gas incubator at 37 °C (85% N2, 10% CO2, 5% O2) for 4 days to obtain a Helicobacter pylori bacterial solution; the Helicobacter pylori bacterial solution was centrifuged at 8000 g for 5 min to obtain Helicobacter pylori cells, and the concentration of the bacterial solution was adjusted to OD 600 = 0.5 with artificial gastric juice (pH = 3) to standardize the bacterial solution concentration (1×10 7 ~1×10 8 CFU / mL) to obtain a Helicobacter pylori bacterial suspension.

[0086] (2) Cultivation of Lactococcus:

[0087] Streak the Lactococcus to be tested on MRS solid medium and culture it at 37 °C for 48 h to obtain single colonies; pick the single colonies and inoculate them into MRS liquid medium, culture them at 37 °C for 18 h for activation, and activate them continuously for two generations to obtain an activated solution; inoculate the activated solution into MRS liquid medium at an inoculation amount of 2% (v / v) and culture it at 37 °C for 18 h to obtain a bacterial solution; centrifuge the bacterial solution at 8000 g for 5 min to obtain Lactococcus cells, and adjust the concentration of the bacterial solution with PBS (pH = 7.42) to OD 600 = 0.5 to standardize the concentration of the bacterial solution (10 7 ~10 8 CFU / mL).

[0088] (3) Culture of Limosilactobacillus reuteri DSM17648:

[0089] Dissolve the purchased Limosilactobacillus reuteri DSM17648 bacterial powder in PBS buffer and adjust the concentration to OD 600 = 0.5.

[0090] (3) Take 2 mL of Helicobacter pylori with adjusted bacterial concentration and mix it with 2 mL of different Lactococcus bacterial suspensions in equal volume ratio, and shake well for 30 s to mix evenly, so that the number ratio of Helicobacter pylori and the strain to be tested after mixing is 1:1. Incubate statically at room temperature of 37 °C for 120 min. Measure the OD value of the upper liquid of the mixed bacterial solution of Lactococcus and Helicobacter pylori respectively, and calculate the coaggregation rate. The results are as Figure 3 shown in Table 1.

[0091] Table 1 Coaggregation rate of the mixed bacterial solution of different species of Lactococcus and Helicobacter pylori after reaction

[0092]

[0093] The results show that using the commercially available Limosilactobacillus reuteri DSM17648 as the control strain, the coaggregation ability of 3 species of Lactococcus (Lactococcus lactis, Lactococcus cremoris, and Lactococcus taiwanensis) with Helicobacter pylori was evaluated. Among them, Lactococcus taiwanensis showed the strongest coaggregation index( Figure 3 A). Further study on the inter-strain differences in the coaggregation ability of Lactococcus taiwanensis and Helicobacter pylori. The results show that the coaggregation characteristics of Lactococcus taiwanensis and Helicobacter pylori have significant inter-strain differences, and Lactococcus taiwanensis CCFM1461 shows a stronger coaggregation index, and the coaggregation rate with Helicobacter pylori reaches 69.94%( Figure 3 A). Figure 3Figure B shows the macroscopic manifestation after Lactococcus taiwanensis CCFM1461 was co-incubated with Helicobacter pylori for 120 min. Through co-aggregation, the two formed white flocculent precipitates and sedimentation occurred, and the absorbance of the upper layer solution decreased. However, this phenomenon did not occur in Lactococcus taiwanensis FDRB31B5. Therefore, Lactococcus taiwanensis CCFM1461 is a rare probiotic with the specific ability to aggregate Helicobacter pylori.

[0094] Example 3: Screening of the auto-aggregation ability of different Lactococcus strains

[0095] The Lactococcus strains to be tested were streaked on MRS solid medium and cultured at 37 °C for 48 h to obtain single colonies. Single colonies were picked and inoculated into MRS liquid medium, and cultured at 37 °C for 18 h for activation. After continuous activation for two generations, an activated solution was obtained. The activated solution was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and cultured at 37 °C for 18 h to obtain a bacterial solution. The bacterial solution was centrifuged at 8000 g for 10 min to obtain Lactococcus cells, and the concentration of the bacterial solution was adjusted to OD 600 = 0.5 with PBS (pH = 7.42).

[0096] Take 2 mL of the Lactococcus suspension with the adjusted bacterial concentration, and shake it thoroughly for 30 s to mix evenly. Incubate statically at room temperature of 37 °C for 120 min. Measure the OD value of the upper layer liquid after the reaction of different Lactococcus bacterial solutions for 120 min, and calculate the auto-aggregation rate. The results are as Figure 4 shown in Table 2.

[0097] Auto-aggregation rate (%) = (OD 600 initial - OD 600 120 min) / OD 600 initial × 100.

[0098] Table 2 Auto-aggregation rates of different Lactococcus strains

[0099]

[0100] The results showed that using the commercially available Limosilactobacillus reuteri DSM17648 as a control strain, the auto-aggregation abilities of three Lactococcus strains (Lactococcus lactis, Lactococcus cremoris, and Lactococcus taiwanensis) were evaluated. Among them, Lactococcus taiwanensis showed the strongest auto-aggregation ability against Helicobacter pylori. Further study on the inter-strain differences in the auto-aggregation ability of Lactococcus taiwanensis was carried out. The results showed that there were significant inter-strain differences in the auto-aggregation ability of Lactococcus taiwanensis, and Lactococcus taiwanensis CCFM1461 showed a stronger auto-aggregation ability, with the auto-aggregation rate reaching 78.43% ( Figure 4 ), and the auto-aggregation ability of the strain is related to its own adhesion ability. Therefore, Lactococcus taiwanensis CCFM1461 is a rare probiotic with specific adhesion and the ability to effectively antagonize Helicobacter pylori.

[0101] Example 4: Effect of Lactococcus taiwanensis on the adhesion of Helicobacter pylori

[0102] The cultivation of Lactococcus taiwanensis and Helicobacter pylori was the same as in Example 2.

[0103] Human gastric adenocarcinoma cells (AGS cells) were resuspended in F12 medium containing 5% (v / v) fetal bovine serum and added to a 96-well plate (2×10 4 cells / well), and cultured at 37 °C and 5% CO2 for 12 - 16 h. After the AGS cells were adherent, the AGS cells were washed 3 times with PBS to remove dead cells; 0.2 mL of Helicobacter pylori resuspension (resuspended in F12 medium) was added to the washed AGS cells, and cultured in an incubator at 37 °C and 5% CO2 for 2 h, then washed 3 times with PBS to remove unadsorbed Helicobacter pylori, and AGS cells infected with Helicobacter pylori were obtained; 0.2 mL of different Lactococcus taiwanensis resuspensions were added to the AGS cells infected with Helicobacter pylori respectively, and cultured in an incubator at 37 °C and 5% CO2 for 2 h to obtain AGS cells infected with Helicobacter pylori treated with different Lactococcus taiwanensis; the AGS cells infected only with Helicobacter pylori were used as a control (Hp group) and cultured for 2 h under the same conditions; after washing 3 times with PBS respectively, 200 μL of urease reagent (9 g / L NaCl, 14 μg / mL phenol red, 20 mM urea, pH 6.8) was added respectively, and cultured in an incubator at 37 °C and 5% CO2 for 2 h; the absorbance of the culture solutions of different experimental groups at a wavelength of 550 nm was measured by an enzyme-linked immunosorbent assay reader, and the adhesion rate measured by subtracting the absorbance of the blank group from the absorbance of the model group was 100%; the difference obtained by subtracting the absorbance of the blank group from the absorbance of the other groups was compared with the difference obtained by subtracting the absorbance of the blank group from the absorbance of the model group to obtain the relative adhesion rate. The results are as Figure 5 shown in Table 3.

[0104] Table 3 Effect of different Lactococcus taiwanensis on the adhesion of Helicobacter pylori to AGS cells

[0105]

[0106] The results showed that after treatment with different Lactococcus taiwanensis, the adhesion ability of Helicobacter pylori to AGS cells decreased significantly. However, compared with Lactococcus taiwanensis FDRB31B5, the decline trend of Lactococcus taiwanensis CCFM1461 was more significant, and it could reduce the adhesion rate of Helicobacter pylori to AGS cells by 46.73% ( Figure 5 ). Therefore, Lactococcus taiwanensis CCFM1461 with strong co-aggregation ability can effectively inhibit the adhesion of Helicobacter pylori to gastric epithelium and thus achieve the effect of antagonizing Helicobacter pylori.

[0107] Example 5: Tolerance of Different Lactococcus taiwanensis Strains to the Gastric Environment

[0108] (1) Cultivation of Lactococcus taiwanensis

[0109] Streak the Lactococcus taiwanensis strain to be tested on MRS solid medium and culture it at 37 °C for 48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium, and culture them at 37 °C for 18 h for activation. Activate for two consecutive generations to obtain an activated solution; inoculate the activated solution into MRS liquid medium at an inoculation amount of 2% (v / v) and culture it at 37 °C for 18 h to obtain a bacterial solution; centrifuge the bacterial solution at 8000 g for 5 min to obtain Lactococcus taiwanensis cells.

[0110] (1) Growth Ability of Lactococcus taiwanensis in MRS Medium with pH = 3

[0111] Suspend the cells in an equal volume of MRS liquid medium with pH = 3 and culture them statically at 37 °C. Samples are taken at 0 h, 3 h, 6 h, 12 h, and 24 h respectively, and the OD 600 value of the bacterial solution is measured at different times.

[0112] (2) Survival Rate of Lactococcus taiwanensis in Artificial Gastric Juice with pH = 3

[0113] Suspend the cells in an equal volume of MRS liquid medium with pH = 3 and culture them statically at a constant temperature of 37 °C. Samples are taken at 0 h, 3 h, and 6 h respectively, and counted by the coating method, with the viable cell count at 0 h as the control.

[0114] Survival rate (%) = (N1 / N2) × 100, where N1 represents the viable cell count in the strain system after treatment, and N2 represents the initial viable cell count in the strain system, that is, the viable cell count measured at 0 h.

[0115] Table 4 Survival Rate of Lactococcus taiwanensis in Artificial Gastric Juice with pH = 3

[0116]

[0117] The Figure 6 results show that both strains of Lactococcus taiwanensis can grow to varying degrees in MRS medium with pH = 3. Among them, Lactococcus taiwanensis CCFM1461 has a faster growth rate, and over time, the difference in the OD 600 value between the bacterial solution of Lactococcus taiwanensis CCFM1461 and Lactococcus taiwanensis FDRB31B5 gradually increases. After culturing for 24 h, the OD 600 value of the bacterial solution of Lactococcus taiwanensis CCFM1461 can reach 0.24, while the OD 600The value is 0.16. Secondly, the survival rates of Lactococcus taiwanensis CCFM1461 after being cultured in artificial gastric juice for 3 h and 6 h were as high as 91.67% and 64.68% respectively, which were significantly higher than those of Lactococcus taiwanensis FDRB31B5. It can be seen that Lactococcus taiwanensis CCFM1461 has strong tolerance to the gastric environment and can effectively play its probiotic role in the gastric environment.

[0118] Example 6: Acid production ability of Lactococcus taiwanensis

[0119] (1) Cultivation of Lactococcus taiwanensis: The cultivation method was the same as that in Example 5. The collected bacterial cells were suspended in an equal volume of MRS liquid medium and cultured statically at 37 °C for 24 h. Samples were taken every 2 h and the pH values of the bacterial liquid were measured at different times. The results are as Figure 7 shown in Table 5.

[0120] Table 5 Acid production ability of different Lactococcus taiwanensis

[0121]

[0122]

[0123] The results showed that the pH values of the MRS medium gradually decreased for both strains of Lactococcus taiwanensis. This indicates that both strains of Lactococcus taiwanensis have different acid production abilities. According to Figure 7 the results, the pH decrease rate of the culture supernatant of Lactococcus taiwanensis CCFM1461 was significantly higher than that of Lactococcus taiwanensis FDRB31B5, and the pH of the 18 h fermentation supernatant reached 3.83. This shows that Lactococcus taiwanensis CCFM1461 not only has strong acid tolerance but also strong acid production ability, which helps to inhibit the growth of Helicobacter pylori.

[0124] Example 7: Co-aggregation ability of Lactococcus taiwanensis with other pathogenic bacteria

[0125] (1) Cultivation of Lactococcus taiwanensis: The cultivation method of the bacterial cells was the same as that in Example 5.

[0126] (2) Cultivation of pathogenic bacteria:

[0127] After streaking Escherichia coli CICC24178, Staphylococcus aureus ATCC6538P, Shigella ATCC12022, Salmonella typhimurium TA100, and Pseudomonas fluorescens AS1.55 on LB solid plates respectively, they were cultured at 37 °C for 24 h to obtain single colonies; single colonies were picked and inoculated into LB liquid medium, and cultured at 37 °C for 18 h for activation. They were continuously activated for two generations to obtain activation solutions.

[0128] (3) Take appropriate amounts of the adjusted bacterial concentrations (OD 600 = 0.5 to standardize the bacterial liquid concentration (107 ~10 8 CFU / mL) of Escherichia coli, Staphylococcus aureus, Shigella, Salmonella typhimurium, and Pseudomonas fluorescens were mixed with 2 mL of different suspensions of Lactococcus taiwanensis in equal volume ratios and shaken vigorously for 30 s to mix evenly. Under the condition of 37 °C at room temperature, the mixture was incubated statically for 120 min. The OD values of the upper liquid of the reaction mixture of Lactococcus taiwanensis and pathogenic bacteria after 120 min were measured respectively, and the coaggregation rate was calculated. The results are as Figure 8 shown in Table 6.

[0129] Table 6 Coaggregation rates of different Lactococcus taiwanensis with other pathogenic bacteria

[0130]

[0131]

[0132] The results showed that using commercially available Lactobacillus reuteri DSM17648 as the control strain, the coaggregation ability of two different Lactococcus taiwanensis with other pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Shigella, Salmonella typhimurium, and Pseudomonas fluorescens) was evaluated. As Figure 8 shown, the coaggregation ability of Lactococcus taiwanensis CCFM1461 with different pathogenic bacteria was different. The coaggregation rates after 120 min reached 59.39%, 34.67%, 28.63%, 40.32%, and 52.20% respectively. Among them, the coaggregation with Escherichia coli and Pseudomonas fluorescens was stronger, while that with Shigella was weaker. But they were all generally lower than the coaggregation with Helicobacter pylori. This indicates that the coaggregation of Lactococcus taiwanensis CCFM1461 with pathogenic bacteria mainly binds to Helicobacter pylori. On the other hand, whether it is Helicobacter pylori or other pathogenic bacteria, the coaggregation rate of Lactococcus taiwanensis CCFM1461 with them was significantly higher than that of Lactobacillus reuteri DSM17648 and Lactococcus taiwanensis FDRB31B5. The coaggregation of DSM17648 and FDRB31B5 with pathogenic bacteria was different. This shows that Lactococcus taiwanensis CCFM1461 not only has a strong coaggregation effect with Helicobacter pylori, but also has a strong coaggregation effect with other pathogenic bacteria.

[0133] Example 8: Effect of heat-inactivated Lactococcus taiwanensis on coaggregation

[0134] (1) Culture of Helicobacter pylori: The culture method was the same as in Example 2.

[0135] (2) Culture of Lactococcus taiwanensis: The culture method was the same as in Example 5. The obtained Lactococcus taiwanensis cells were adjusted to an OD 600 of 0.5 with PBS (pH = 7.42).

[0136] (3) Influence of Different Inactivation Conditions on the Coaggregation of Lactococcus taiwanensis CCFM1461

[0137] Take the Lactococcus taiwanensis with the adjusted bacterial concentration in step (2) and place them in a metal bath at 65 °C and 95 °C for 30 min and 10 min respectively. Mix 2 mL of the Helicobacter pylori suspension with the adjusted bacterial concentration (OD 600 = 0.5) with 2 mL of the Lactococcus suspension treated at different temperatures in equal volume ratio, and shake well for 30 s to mix evenly. Incubate statically at 37 °C at room temperature for 120 min. Measure the OD value of the supernatant liquid of the mixed bacterial suspension of Lactococcus and Helicobacter pylori respectively, and calculate the coaggregation rate.

[0138] Table 7 Influence of Different Inactivation Conditions on the Coaggregation of Lactococcus taiwanensis and Helicobacter pylori

[0139]

[0140] The results show that ( Figure 9 ), the coaggregation of Lactococcus taiwanensis CCFM1461 with Helicobacter pylori decreased significantly after inactivation treatment at different temperatures. Among them, the coaggregation rate decreased to 44.50% after pasteurization (65 °C, 30 min); the coaggregation rate decreased to 33.11% after high-temperature short-time sterilization (95 °C, 10 min). This shows that the activity of the key functional substances for the coaggregation of Lactococcus taiwanensis CCFM1461 and Helicobacter pylori is negatively correlated with temperature and is not heat-resistant. It can be speculated that the key functional substances for the coaggregation of Lactococcus taiwanensis CCFM1461 and Helicobacter pylori may be related to protein substances.

[0141] Example 9: Influence of the Surface Protein of Lactococcus taiwanensis on Coaggregation

[0142] (1) Cultivation of Lactococcus taiwanensis CCFM1461:

[0143] Streak Lactococcus taiwanensis CCFM1461 on MRS solid medium and culture at 37 °C for 48 h to obtain single colonies; pick single colonies and inoculate them into MRS liquid medium, culture at 37 °C for 18 h for activation, and activate continuously for two generations to obtain an activated solution; inoculate the activated solution into MRS liquid medium at an inoculation amount of 2% (v / v) and culture at 37 °C for 18 h to obtain a bacterial solution; centrifuge the bacterial solution at 8000 g for 5 min to prepare Lactococcus taiwanensis cells.

[0144] (2) Cultivation of Helicobacter pylori:

[0145] After streaking Helicobacter pylori on Columbia blood agar plates, culture them in a triple-gas incubator at 37°C (85% N2, 10% CO2, 5% O2) for 3 days to obtain single colonies; pick a single colony and inoculate it into BHI medium containing 5% (v / v) fetal bovine serum, and culture it in a triple-gas incubator at 37°C (85% N2, 10% CO2, 5% O2) for 4 days to obtain a seed solution; inoculate the seed solution into BHI liquid medium at an inoculation amount of 2% (v / v), and culture it in a triple-gas incubator at 37°C (85% N2, 10% CO2, 5% O2) for 4 days to obtain Helicobacter pylori bacterial solution; centrifuge the Helicobacter pylori bacterial solution at 8000g for 5 min to obtain Helicobacter pylori cells, and adjust the concentration of the bacterial solution to OD 600 = 0.5 with artificial gastric juice (pH = 3) to obtain a Helicobacter pylori bacterial suspension.

[0146] (3) Copolymerization effect of LiCl on removing surface proteins of Lactococcus taiwanensis CCFM1461

[0147] Take the cultured Lactococcus taiwanensis CCFM1461 in step (1), treat the lactococcus with 5 mol / L LiCl at 1 / 20 volume of the initial culture solution for 30 min (37°C), centrifuge and discard the supernatant, wash the bacterial cell suspension twice with PBS, and prepare a bacterial solution concentration of OD 600 = 0.5 of Lactococcus taiwanensis CCFM1461 bacterial suspension. Mix 2 mL of each of the Helicobacter pylori bacterial suspension obtained in step (2) with the Lactococcus taiwanensis CCFM1461 bacterial suspension obtained above, shake and mix well, and incubate at room temperature for 120 min. Measure the OD values of lactococcus and the mixed bacterial solution before / after the reaction (0 min / T min) respectively and calculate the copolymerization rate.

[0148] (4) Extraction and determination of surface proteins of Lactococcus taiwanensis CCFM1461

[0149] Treat Lactococcus taiwanensis with 5 mol / L LiCl in step (3) for 30 min (37°C), collect the supernatant after centrifugation, filter it through a 0.22 μm sterile filter membrane, and dialyze it overnight at 4°C to obtain a crude extract of the surface protein on the cell wall of lactococcus. Place the dialyzed crude protein extract in a petri dish, wrap it with plastic wrap and puncture holes for vacuum freeze-drying, then weigh the mass of the protein freeze-dried powder and store it in a -80°C refrigerator for long-term use. Use a BCA protein concentration determination kit to measure the protein concentration of the crude extract of the surface protein on the cell wall of lactococcus, and the results are shown in Table 9 and Figure 9 as shown. And collect the surface proteins into clean test tubes, mix 10 ul of 2×SDS Gel-Loading Buffer with 10 ul of the protein supernatant and boil, and analyze the molecular weight of the extract by SDS-PAGE electrophoresis experiment with a 10% concentration, and the sample loading amount is 20 μL.

[0150] Table 8 Effect of removing surface proteins on the coaggregation rate of Lactococcus taiwanensis and Helicobacter pylori

[0151]

[0152]

[0153] Table 9 Content of different surface proteins of Lactococcus taiwanensis

[0154]

[0155] The results showed ( Figure 9 ) that the coaggregation of Lactococcus taiwanensis CCFM1461 and Helicobacter pylori was significantly reduced to 32.57% after treatment with 5 mol / L LiCl. This indicates that the main functional substance for the coaggregation of Lactococcus taiwanensis CCFM1461 and Helicobacter pylori is the surface protein on the cell wall. The surface protein concentration (4.07 mg / ml) on the cell surface of Lactococcus taiwanensis CCFM1461 was significantly higher than that of Lactococcus taiwanensis FDRB31B5 (3.48 mg / ml). There were a large number of protein bands in the range of 25 - 71 kDa for Lactococcus taiwanensis CCFM1461, while no obvious bands were observed for Lactococcus taiwanensis FDRB31B5. Bacterial surface proteins are mainly distributed in the range of 25 - 71 kDa. It can be thus explained that Lactococcus taiwanensis CCFM1461 has a strong ability to coaggregate with Helicobacter pylori because there are a large number of 25 - 71 kDa surface proteins on the bacterial surface.

[0156] Example 10: Application of Lactococcus taiwanensis CCFM1461

[0157] Lactococcus taiwanensis CCFM1461 can be used to prepare bacterial powder, and the specific preparation process of the bacterial powder is as follows:

[0158] Lactococcus taiwanensis CCFM1461 was streaked on MRS solid medium and cultured at 37 °C for 48 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium and cultured at 37 °C for 18 h for activation. After continuous activation for two generations, an activated solution was obtained; the activated solution was inoculated into the medium at an inoculation amount of 2% (v / v) and cultured at 37 °C for 18 h to obtain a bacterial solution; the bacterial solution was centrifuged at 8000 g for 5 min to obtain bacterial sludge; the bacterial sludge was washed 3 times with physiological saline and then resuspended with a protective agent to a concentration of 1×10 10 CFU / mL to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37 °C for 60 min and then freeze-dried to obtain Lactococcus taiwanensis CCFM1461 bacterial powder;

[0159] Among them, the preparation method of the culture medium is as follows: Dissolve 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and then adjust its pH to 6.8 to obtain the culture medium; The components of the cryoprotectant include: 130 g / L skim milk powder.

[0160] Example 11: Application of Lactococcus taiwanensis CCFM1461

[0161] Lactococcus taiwanensis CCFM1461 can be used to prepare capsule products. The specific preparation process of the capsule products is as follows:

[0162] Lactococcus taiwanensis CCFM1461 is streaked on MRS solid medium and cultured at 37 °C for 48 h to obtain single colonies; Pick the single colonies and inoculate them into MRS liquid medium, and culture them at 37 °C for 18 h for activation. Activate continuously for two generations to obtain the activation solution; Inoculate the activation solution into the culture medium at an inoculation amount of 2% (v / v) and culture it at 37 °C for 18 h to obtain the bacterial solution; Centrifuge the bacterial solution at 8000 g for 5 min to obtain the bacterial sludge; Wash the bacterial sludge 3 times with physiological saline and then resuspend it with the cryoprotectant to a concentration of 1×10 10 CFU / mL to obtain the bacterial suspension; Add the bacterial suspension to a sodium alginate solution with a concentration of 30 g / L until the concentration reaches 2×10 9 CFU / mL, and then stir well to make the cells of Lactococcus taiwanensis CCFM1461 evenly dispersed in the sodium alginate solution to obtain the mixed solution; Extrude the mixed solution into a calcium chloride solution with a concentration of 20 g / L to form gel particles; After the formed gel particles are allowed to stand and solidify for 30 min, filter and collect the gel particles; Freeze-dry the collected gel particles for 48 h to obtain the powder; Load the powder into medicinal capsules to obtain the capsule products;

[0163] Among them, the preparation method of the culture medium is as follows: Dissolve 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and then adjust its pH to 6.8 to obtain the culture medium.

[0164] Example 12: Application of Lactococcus taiwanensis CCFM1461

[0165] Lactococcus taiwanensis CCFM1461 can be used to prepare tablets. The specific preparation process of the tablets is as follows:

[0166] Lactococcus taiwanensis CCFM1461 was streaked on MRS solid medium and cultured at 37 °C for 48 h to obtain single colonies; single colonies were picked and inoculated into MRS liquid medium, cultured at 37 °C for 18 h for activation, and continuously activated for two generations to obtain an activated solution; the activated solution was inoculated into the medium at an inoculation amount of 2% (v / v) and cultured at 37 °C for 18 h to obtain a bacterial solution; the bacterial solution was centrifuged at 8000 g for 5 min to obtain bacterial sludge; the bacterial sludge was washed 3 times with physiological saline and then resuspended with a protective agent to a concentration of 1×10 10 CFU / mL to obtain a bacterial suspension; the bacterial suspension was pre-cultured at 37 °C for 60 min and then freeze-dried to obtain Lactococcus taiwanensis CCFM1461 bacterial powder;

[0167] Among them, the preparation method of the medium is as follows: 10% enzymatically hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract were dissolved with 87.7% water by the total weight of the medium, and then its pH was adjusted to 6.8 to obtain the medium;

[0168] The components of the protective agent include: 130 g / L skim milk powder.

[0169] Weigh 25.7 parts by weight of Lactococcus taiwanensis CCFM1461 bacterial powder, 55.0 parts by weight of starch, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of sodium carboxymethyl starch, 0.8 parts by weight of talc powder, 1.0 part by weight of sucrose and 1.0 part by weight of water to obtain raw materials; mix the raw materials to obtain wet granules; press the wet granules with a tablet press of Zhongnan Pharmaceutical Machinery Factory and then dry them with a small drug dryer of Qingzhou Yikang Traditional Chinese Medicine Machinery Co., Ltd. to obtain tablets.

[0170] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Lactococcus taiwanensis CCFM1461 was deposited at the Guangdong Provincial Culture Collection of Microorganisms on December 26, 2024, with the deposit number GDMCC No: 65679.

2. A composition comprising Lactococcus taiwanensis CCFM1461 as claimed in claim 1.

3. The composition according to claim 2, wherein The composition contains live cells of Lactococcus taiwanensis CCFM1461.

4. The composition according to claim 2 or 3, characterized in that, The quantity of the Lactococcus taiwanensis CCFM1461 ≥ 1×10 7 CFU / mL.

5. The composition according to any one of claims 2 to 4, characterized in that, The composition includes, but is not limited to, food, medicine, or health products.

6. The composition according to claim 5, wherein The medicine contains Lactococcus taiwanensis CCFM1461, as well as a pharmaceutical carrier and / or pharmaceutical excipients.

7. A microbial preparation containing Lactococcus taiwanensis CCFM1461 as claimed in claim 1.

8. Use of Lactococcus taiwanensis CCFM1461 as claimed in claim 1 in the preparation of a drug for preventing and / or treating Helicobacter pylori infection.

9. An inhibitor for reducing the load of pathogenic bacteria, characterized in that, The inhibitor contains Lactococcus taiwanensis CCFM1461; the pathogenic bacteria include, but are not limited to, one or more of Helicobacter pylori, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shigella, and Pseudomonas fluorescens.

10. Use of Lactococcus taiwanensis CCFM1461 as claimed in claim 1 in inhibiting one or more pathogenic bacteria among Helicobacter pylori, Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shigella, and Pseudomonas fluorescens in an in vitro environment.

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