Lactobacillus plantarum with antibacterial efficacy and application thereof

Abstract

The present application relates to the field of microorganisms, and discloses a lactobacillus plantarum with antibacterial efficacy and application thereof, wherein the lactobacillus plantarum has a preservation number of CGMCC No.33151.The strain provided by the present application has strong abilities of resisting bile salt, resisting gastric acid, producing acid and producing H2O2, and has strong bacteriostatic effects on common pathogenic bacteria such as staphylococcus aureus, candida albicans, escherichia coli, salmonella and helicobacter pylori, and can inhibit the activity of urease, interfere with the colonization of helicobacter pylori and the like, so as to inhibit helicobacter pylori infection, reduce the symptoms of helicobacter pylori infection and reduce the disease degree of colonitis mice, and can be used for preparing a microbial preparation and has potential for preparing a medicine for preventing and / or treating diseases caused by helicobacter pylori infection.

Description

Technical Field

[0001] This invention relates to the field of microbiology, specifically to a strain of *Lactobacillus plantarum* with antibacterial properties and its applications. Background Technology

[0002] Helicobacter pylori ( Helicobacter pylori Helicobacter pylori (Hp) is a Gram-negative, microaerophilic bacterium that typically colonizes the gastric mucus layer and the epithelial cells of the gastric antrum mucosa. It is classified as a Group 1 carcinogen. Individuals infected with Helicobacter pylori may develop varying degrees of gastrointestinal diseases, such as indigestion, chronic gastritis, colitis, peptic ulcers, and even gastric malignancies.

[0003] Current research has confirmed that microorganisms have significant effects in alleviating and preventing Helicobacter pylori and the gastrointestinal diseases it causes. Compared with traditional drug treatments, microorganisms have no obvious side effects on the body and do not cause complications. They can also produce beneficial effects on the human body by regulating the intestinal flora and producing short-chain fatty acids. However, existing microorganisms have drawbacks such as unstable viable cell counts, which prevents them from stably exerting their effects in the human body.

[0004] Therefore, it is urgent to select strains with excellent fermentation characteristics that can survive stably in the human body in order to prepare drugs for inhibiting Helicobacter pylori and preventing and / or treating gastrointestinal diseases caused by Helicobacter pylori. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a strain of *Lactobacillus plantarum* with antibacterial effects and its applications.

[0006] To achieve the above objectives, the first aspect of the present invention provides a strain of *Lactobacillus plantarum*. Lactobacillus plants The preservation number of the Lactobacillus plantarum is CGMCC No. 33151.

[0007] A second aspect of the present invention provides a microbial agent containing *Lactobacillus plantarum* as described above.

[0008] A third aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for inhibiting the colonization of pathogenic bacteria.

[0009] The fourth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for inhibiting the growth of pathogenic bacteria.

[0010] The fifth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for inhibiting urease activity.

[0011] The sixth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of medicaments for the prevention and / or treatment of gastrointestinal diseases.

[0012] The beneficial effects obtained by the present invention through the above technical solution include at least the following:

[0013] (1) The strain provided by the present invention can reduce the severity of colon disease and improve the health of mice by inhibiting weight loss, increasing fecal viscosity, inhibiting hematochezia, inhibiting the expression of inflammatory factors (TNF-α, IL-1β and IL-6) and increasing the expression of anti-inflammatory factors (IL-10 and TGF-β);

[0014] (2) The strains provided by this invention can inhibit the activity of urease, prevent Helicobacter pylori colonization, and thus inhibit Helicobacter pylori infection or reduce the symptoms of Helicobacter pylori infection;

[0015] (3) The strain provided by the present invention has a strong ability to produce acid and H2O2, and has a strong inhibitory effect on common pathogens such as Helicobacter pylori, Staphylococcus aureus, Candida albicans, Escherichia coli, Atobococcus and Salmonella.

[0016] (4) The strains provided by the present invention do not cause cell hemolysis, are sensitive to common antibiotics, have biosafety, and can be used to prepare drugs for the prevention and / or treatment of colitis.

[0017] Biological Preservation

[0018] The strain provided by this invention is classified and named *Lactobacillus plantarum*. Lactobacillus plantarum It was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33151 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0019] The strain provided by this invention is classified and named *Lactobacillus plantarum*. Lactobacillus plantarum It was deposited on December 23, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33152 and address at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of strain CCNH185 provided by the present invention;

[0021] Figure 2 This is a Gram micrograph of the strain CCNH185 provided by this invention;

[0022] Figure 3 The results are H&E staining of colon tissues from mice in the control group, model group, and CCNH185 group. Detailed Implementation

[0023] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0024] The inventors of this invention accidentally isolated a strain of *Lactobacillus plantarum* that is resistant to bile salts and gastric acid and produces high levels of lactic acid and hydrogen peroxide from fermented mare's milk. Lactobacillus plantarum Its colony morphology and Gram microscopic images are as follows: Figure 1 and Figure 2 As shown. Further research indicates that the strain provided by this invention can not only specifically inhibit pathogen reproduction, but is also sensitive to commonly used antibiotics, and has the potential for application in organisms.

[0025] Based on the above findings, the first aspect of this invention provides a strain of *Lactobacillus plantarum*. Lactobacillus plantarum The preservation number of the Lactobacillus plantarum is CGMCC No. 33151.

[0026] "CCNH185" is the strain number assigned by the inventors during the strain screening process, and "CGMCC No.33151" is the preservation number of the strain. Both represent the same strain and can be used interchangeably in the following text.

[0027] A second aspect of the present invention provides a microbial agent containing *Lactobacillus plantarum* as described above.

[0028] The present invention does not impose any particular limitation on the type of microbial agent, and it can be any type of microbial agent existing in the art. According to a preferred embodiment of the present invention, the microbial agent is selected from at least one of liquid microbial agents, concentrated microbial agents, and solid microbial agents.

[0029] In this invention, the bacterial agent may also contain excipients, which may be selected from protectants (e.g., freeze-drying protectants, such as skim milk powder, maltodextrin, trehalose, dextran, corn oil, and glycerol) and / or buffers (e.g., buffer solutions remaining during the preparation of solid bacterial agents). Preferably, the excipients are selected from glycerol and / or corn oil.

[0030] Preferably, the viable count of the *Lactobacillus plantarum* is 10 relative to 1 mL of excipient. 4 -10 12 CFU.

[0031] The *Lactobacillus plantarum* provided by this invention can produce a large number of live *Lactobacillus plantarum* cells through liquid culture. The culture method is not particularly demanding, as long as it enables the *Lactobacillus plantarum* to proliferate. For example, live *Lactobacillus plantarum* cells can be inoculated into a culture medium at an inoculum volume of 1-5 vol%, and cultured at 30-40°C for 9-40 hours to obtain a culture solution. The culture medium can be any culture medium suitable for *Lactobacillus plantarum* culture known in the art, such as MRS medium.

[0032] This invention can further isolate live cells of Lactobacillus plantarum from the above-mentioned culture medium. The method of separation is not particularly limited, as long as it can enrich the cells from the culture medium. For example, it can be achieved by centrifugation and / or filtration. The conditions for centrifugation and filtration can be known conditions, which will not be elaborated here.

[0033] A third aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for inhibiting the colonization of pathogenic bacteria.

[0034] The fourth aspect of the present invention provides the use of *Lactobacillus plantarum* in the preparation of a medicament for inhibiting the growth of pathogenic bacteria, as described above.

[0035] According to the application described in the third and fourth aspects of the present invention, the pathogenic bacterium is preferably *Escherichia coli* (Escherichia coli). Escherichia coli Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella Candida albicans ( Candida albicans Gardnerella vaginalis Gardnerella vaginalis ) 、 Atobococcus ( Atopobium minutum ) and Helicobacter pylori ( Helicobacter pylori At least one of the following.

[0036] According to the present invention, the pathogenic bacterium is more preferably Atoborobacter.

[0037] According to some embodiments of the present invention, the Salmonella preferably includes Salmonella Typhimurium (Salmonella Typhimurium). Salmonella typhimurium ) and Salmonella enteritidis ( Salmonella enteritidis ).

[0038] The fifth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of a medicament for inhibiting urease activity.

[0039] The sixth aspect of the present invention provides the use of *Lactobacillus plantarum* as described above in the preparation of medicaments for the prevention and / or treatment of gastrointestinal diseases.

[0040] Preferably, the gastrointestinal disease is colitis, more preferably ulcerative colitis.

[0041] This invention also provides a method for culturing *Lactobacillus plantarum*, the method comprising: inoculating *Lactobacillus plantarum* into MRS medium as described above, wherein the composition of the MRS medium comprises, relative to 1L of medium, 10-20g peptone, 10-30g glucose, 1-7g yeast extract, 0.5-3g dipotassium hydrogen phosphate, 0.5-3g diammonium hydrogen citrate, 0.5-1.5g Tween-80, 0.02-0.3g magnesium sulfate, and 0.005-0.1g manganese sulfate; the viable count of *Lactobacillus plantarum* inoculated with the medium volume can be 10... 1 -10 7 CFU / mL.

[0042] The *Lactobacillus plantarum* provided by this invention can be cultured in any commonly used culture medium in the art. However, the inventors of this invention have discovered in their research that when the *Lactobacillus plantarum* provided by this invention is cultured in the MRS medium as described above, the *Lactobacillus plantarum* provided by this invention can complete its growth and reproduction in a shorter time, and the number of viable bacteria at the point of cessation of growth and the final isolated bacterial sludge are both greater than those cultured in the MRS medium conventionally used in the art.

[0043] The present invention also provides a method for preparing freeze-dried powder, the method comprising: adding a protective agent to the Lactobacillus plantarum sludge provided by the present invention, and then freeze-drying at a temperature of -90°C to -70°C.

[0044] According to the present invention, the protective agent may include 1-20 wt% skim milk powder, 1-20 wt% trehalose, 0.5-10 wt% monosodium glutamate, 0.1-10 wt% glycerol, 1-10 wt% glucose, and 1-10 wt% maltodextrin, based on the total weight of the mixture obtained after mixing.

[0045] The present invention also provides a method for embedding bacterial strains, the method comprising: treating the *Lactobacillus plantarum* sludge provided by the present invention sequentially with calcium chloride and polymethyl methacrylate. Preferably, CaCl2 and / or polymethyl methacrylate are provided in liquid form, wherein the content of CaCl2 is preferably 5-20 M, and the content of polymethyl methacrylate is preferably 0.5-5 mg / mL.

[0046] According to the present invention, the encapsulation method may further include: performing solid-liquid separation on the mixture of CaCl2 solution and bacterial sludge, and then adding the separated solid to a polymethyl methacrylate solution for further solid-liquid separation. In order to obtain a better encapsulation effect, the number of times the bacterial sludge is added to the polymethyl methacrylate solution for solid-liquid separation may be greater than 1, preferably 3-10 times.

[0047] The present invention also provides a compound microbial agent, which includes *Lactobacillus plantarum* with accession number CGMCC No. 33151 and accession number CCGMCC No. 33152 provided by the present invention.

[0048] As described in the application document with application number 202510222543.6, *Lactobacillus plantarum* with accession number CGMCC No. 33152 also has antibacterial (especially inhibiting *Atopobacterium*), inhibiting the reproduction of pathogenic bacteria and reducing the expression of pro-inflammatory factors, and also has biosafety and antibiotic sensitivity. Therefore, *Lactobacillus plantarum* with accession number CGMCC No. 33151 provided by this invention is particularly suitable for compounding with *Lactobacillus plantarum* with accession number CGMCC No. 33152 to prepare a compound bacterial agent for the preparation of drugs for the prevention and / or treatment of vaginitis and / or diseases caused by *Atopobacterium*.

[0049] In the above-mentioned compound microbial agent provided by the present invention, the two strains can be packaged independently or mixed. However, in order to maximize the preservation of the activity of the strains and exert their effects, it is preferable to package them independently.

[0050] When a patient uses a drug containing the above-mentioned compound bacterial agent provided by the present invention, the drugs containing the two strains can be used simultaneously or sequentially. However, in order to maximize the effect, it is preferred to use them sequentially, but the order of use is not limited.

[0051] The present invention will be described in detail below through embodiments.

[0052] Unless otherwise specified, all reagents and materials used in the following examples and comparative examples are commercially available products purchased from legitimate chemical or biological reagent and material suppliers.

[0053] Escherichia coli ( Escherichia coli (1) ATCC 25922, purchased from the American Center for Type Culture Collection; (2) CICC 10421, purchased from the China Industrial Microbial Culture Collection Center.

[0054] Staphylococcus aureus ( Staphylococcus aureus The items, numbered CMCC(B)26001 and CMCC(B)26003, were purchased from the China Medical Bacteriological Culture Collection Center.

[0055] Salmonella, (1) Salmonella typhimurium ( Salmonella typhimurium (2) Salmonella enteritidis (ATCC 14028), purchased from the American Center for Type Culture Collection; Salmonella enteritidis(Item number CVCC 3378), purchased from the National Veterinary Microbial Culture Collection Center;

[0056] Candida albicans ( Candida albicans (1) CICC 1965, purchased from China Industrial Microbial Culture Collection Center; (2) ATCC10231, purchased from the American Type Culture Collection Center.

[0057] Gardnerella vaginalis ( Gardnerella vaginalis (ATCC 14018), purchased from the American Center for Type Culture Collection;

[0058] Atobococcus ( Atopobium minutum (ATCC 33267), purchased from the American Center for Type Culture Collection;

[0059] Helicobacter pylori ( Helicobacter pylori (ATCC 43504), purchased from the American Center for Type Culture Collection;

[0060] MRS liquid medium: 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 20g glucose, 1mL Tween-80, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1L deionized water, pH 6.5, autoclaved; add 18g agar before final volume adjustment to obtain MRS solid medium;

[0061] Brain Heart Infusion Broth (BHI) Solid Agar Plates: Purchased from Qingdao Haibo Biotechnology Co., Ltd., catalog number HB8297-1;

[0062] GES-1 gastric mucosal cells were purchased from Stemer (Shanghai) Biotechnology Co., Ltd., catalog number STM-CL-5009.

[0063] High glucose medium (DMEM) and fetal bovine serum (FBS) were purchased from Merck Darmstadt, Germany, with catalog numbers D1145 and 12103C, respectively.

[0064] PBS buffer was purchased from Solarbio, product number P1003;

[0065] Reference strains: (1) *Lactobacillus plantarum*, preservation number CGMCC No. 15013, this strain can be referenced by CN109423467B, hereinafter referred to as *Lactobacillus plantarum* L-1; (2) *Lactobacillus rhamnosus* ( Lactobacillus buckthorn (ATCC 53103), purchased from the American Center for Type Culture Collection, abbreviated as LGG.

[0066] Example 1

[0067] Weigh 10g of fermented mare's milk sample and add it to 90mL of sterile water. Shake at 180 rpm for 20 min, then serially dilute. Select an appropriate gradient and spread the diluted sample onto MRS solid medium plates. Incubate at 37°C for 48 h. Use an inoculation loop to pick up smooth, milky-white, round or oval single colonies and streak them onto MRS solid medium. Incubate at 37°C for 48 h. Repeat this purification process for 6 generations. Randomly select milky-white, smooth, round or oval single colonies from the solid plate. Use an inoculation loop to pick up the purified single colonies and inoculate them into MRS liquid medium. Incubate at 37°C for 24 h. Subculture three times at 2 vol% inoculum and then freeze.

[0068] Strains CCNH185 and LGG glycerol tubes were inoculated into fresh MRS liquid medium at a 2 vol% inoculum and incubated overnight at 37°C for 18 h. OD 600 Normalization was performed to obtain activated bacterial solutions, which were then inoculated into fresh MRS liquid medium at an inoculum of 2 vol% and cultured overnight at 37°C for 18 h to obtain expanded bacterial solutions of Lactobacillus plantarum CCNH185 and LGG.

[0069] Measurement of OD values ​​of Lactobacillus plantarum CCNH185 and LGG culture broths 600 The growth performance of Lactobacillus plantarum CCNH185 was better than that of LGG, with values ​​of 9.59±0.03 and 8.13±0.02, respectively.

[0070] Example 2

[0071] The gastric acid resistance and bile salt resistance of *Lactobacillus plantarum* CCNH185 and LGG were evaluated according to T / CNHFA435-2024, "Test Method for Gastric Juice Tolerance of Probiotics". In the gastric acid resistance test, the pH was 3 and the incubation time was 2 hours; in the bile salt resistance test, the bile salt content was 0.2 wt% and the incubation time was 4 hours. OD... 600 Measure the absorbance of the bacterial solution before and after the experiment.

[0072] Survival rate: (OD before experiment) 600 Value - OD after experiment 600 Value) / OD before experiment 600 Value × 100%.

[0073] The gastric acid resistance survival rates of Lactobacillus plantarum CCNH185 and LGG were 93.55% and 93.33%, respectively, and the bile salt resistance survival rates were 71.29% and 53.13%, respectively. It can be found that Lactobacillus plantarum CCNH185 has better gastric acid resistance and bile salt resistance than LGG.

[0074] Example 3

[0075] Activated Lactobacillus plantarum CCNH185 and LGG bacterial cultures were inoculated into 30 mL of fresh MRS liquid medium at a 2 vol% inoculum. After incubation at 37°C for 18 h, the supernatant was collected by centrifugation and filtered through a membrane (membrane pore size of 0.22 μm). The lactic acid yield was then detected by high performance liquid chromatography (HPLC).

[0076] The lactic acid production of strain CCNH185 was 18.67 g / L, while that of LGG was 15.1 g / L. The lactic acid production performance of strain CCNH185 was better than that of LGG, indicating that strain CCNH185 can metabolize and decompose more sugars during the culture process, thereby reducing the sugar content in the environment.

[0077] Example 4

[0078] Take the expanded bacterial culture, wash twice with physiological saline, and resuspend in 0.1M PBS buffer to make the bacterial suspension OD600=1. Pipette 100μL of the bacterial suspension into an ELISA plate and measure the initial OD. 600 The value is recorded as A0; after the bacterial culture is allowed to stand for 4 hours, 100 μL of the upper bacterial culture is taken and transferred to an ELISA plate to detect its OD value after standing. 600 The value is denoted as A1, and the self-aggregation rate of the strain is calculated. 900 μL of the above bacterial suspension is taken, 180 μL of dodecane is added, and the mixture is vortexed until homogeneous. After standing for 20 min to separate into layers, the lower aqueous phase is taken and its OD value is measured. 600 The value is denoted as A2, and the hydrophobicity of the strain is calculated.

[0079] Strains’ self-aggregation rate or hydrophobicity = (A0 - A1 or A2) / A0 × 100%.

[0080] The self-aggregation rates of strains CCNH185 and LGG were 60.26% and 54.55%, respectively, and their hydrophobicities were 46.74% and 43.85%, respectively. These two data combined indicate that *Lactobacillus plantarum* CCNH185 has excellent adhesion properties, enabling it to bind to intestinal epithelial cells and colonize the intestine, thereby inhibiting the colonization of pathogens in the gastrointestinal tract.

[0081] Example 5

[0082] Weigh 44g of Columbia blood agar medium powder (purchased from Qingdao Haibo Biotechnology, catalog number HB9295), add 1000mL of water, sterilize at 121°C for 15min, then add 10vol% sterile defibrinated sheep blood to obtain Columbia blood agar plates. Inoculate 10μL of the expanded bacterial culture into the above Columbia blood agar plates. In addition, use Escherichia coli ATCC 25922 and Staphylococcus aureus CMCC 26001 with equal viable counts as control strains.

[0083] The results showed that Lactobacillus plantarum CCNH185 and Escherichia coli ATCC 25922 did not exhibit hemolysis, while Staphylococcus aureus CMCC 26001 showed complete hemolysis with β-hemolysis zones around the white colonies. This indicates that the strain CCNH185 provided by this invention will not cause hemolysis when applied to organisms, thus making it possible for in vivo application.

[0084] Example 6

[0085] The H₂O₂ production capacity was determined using the 4-aminoacylantipyrine method. H₂O₂ solutions with concentrations of 0, 0.5, 1, 1.5, 2, 5, 8, 10, 25, and 50 μg / mL were prepared, and the OD (exponential growth rate) at different concentrations was measured. 505 To determine the absorbance, a standard curve of absorbance versus concentration was established. 30 mL of the cultured bacterial solution was centrifuged, the supernatant was collected, and its absorbance was measured. The H2O2 yield was calculated based on the absorbance-concentration standard curve.

[0086] The H2O2 production of Lactobacillus plantarum CCNH185 and LGG was 4.38 μg / mL and 4.31 μg / mL, respectively, indicating that both strains can non-specifically inhibit the growth of pathogenic bacteria by generating H2O2.

[0087] Example 7

[0088] Equal amounts of Escherichia coli (CICC 10421 and ATCC 25922), Staphylococcus aureus (CMCC(B)26001 and CMCC(B)26003), and Salmonella (ATCC 14028 and CVCC 3378) were inoculated into LB liquid medium, respectively. Candida albicans (CICC 1965 and ATCC 10231) were inoculated into YPD solid medium. Gardnerella vaginalis (ATCC 14018) and Atobococcus aureus (ATCC 33267) were inoculated into blood agar plates. Helicobacter pylori (ATCC 43504) was inoculated into BHI solid agar plates. The above-mentioned strains were used as indicator strains. Except for Gardnerella vaginalis (anaerobic) and Helicobacter pylori (microaerophilic), all other indicator strains were cultured aerobically. After incubation at 37°C for 24 hours (72 hours for Helicobacter pylori), they were transferred to the corresponding fresh liquid culture medium and cultured until the viable count reached 10^6. 5 -10 7 CFU / mL, diluted to 10 with the appropriate culture medium. 5 CFU / mL was used as the indicator culture. Activated cultures of Lactobacillus plantarum CCNH185 and LGG were inoculated into 30 mL of fresh MRS liquid medium at a 2 vol% inoculum. After incubation at 37°C for 18 h, the supernatant was collected by centrifugation and filtered through a membrane (membrane pore size 0.22 μm).

[0089] Three experimental groups were set up: S1: 100 μL fermentation supernatant, 100 μL indicator culture; S2: 50 μL fermentation supernatant, 150 μL indicator culture; S3: 25 μL fermentation supernatant, 175 μL indicator culture. The fermentation supernatant was replaced with an equal volume of sterile MRS liquid culture medium as a negative control group, and the fermentation supernatant was replaced with 10 mL of kanamycin (100 μg / mL) as a positive control group. Each group was repeated in triplicate. The OD values ​​of the experimental groups and the negative control group were measured. 600 The values ​​are A and A0, respectively. The inhibition rate is calculated according to the following formula. The data of Lactobacillus plantarum CCNH185 and LGG are shown in Table 1, where the unit of inhibition rate is %, and the calculation formula is as follows:

[0090] Inhibition rate = (A0-A) / A0×100%.

[0091] Table 1

[0092]

[0093] Under the same culture system, strain CCNH185 showed a higher inhibition rate against all pathogenic bacteria than strain LGG, especially against Helicobacter pylori.

[0094] Example 8

[0095] Take the expanded bacterial culture and dilute it with MRS medium to a viable count of 1.5 × 10⁻⁶. 5 CFU / mL.

[0096] Antibiotic samples with different initial concentrations (INC) were prepared according to Table 2. The antibiotics were diluted using the two-fold dilution method, and the growth of the bacterial strains was measured until growth was observed. This concentration is the minimum inhibitory concentration (MIC). The MIC was compared with the breakpoint value in the "General Rules for Probiotics for Food Use" (T / CIFST 009-2022) to determine antibiotic sensitivity. A MIC lower than the cutoff concentration (breakpoint value) indicates that the strain is highly sensitive to the antibiotic, and low concentrations of antibiotic are sufficient to inhibit its growth. A MIC higher than the breakpoint value indicates that the strain is highly resistant to the antibiotic, and higher concentrations of antibiotic are required to inhibit its growth. Here, nr indicates no resistance. The test results (INC, MIC) of the strains in different antibiotics and the corresponding breakpoint values ​​are shown in Table 2. MRS liquid medium was used as a blank control, and aqueous solution was used as a negative control.

[0097] Table 2

[0098]

[0099] As shown in Table 2, CCNH185 exhibits strong sensitivity to all antibiotics (its growth can be inhibited with relatively low antibiotic concentrations). This result indicates that Lactobacillus plantarum CCNH185 is safe and can be used to develop probiotic additives or as a food fermentation strain.

[0100] Example 9

[0101] The in vitro anti-Helicobacter pylori activity of CCNH185 was determined using the Oxford cup method. Suspensions of CCNH185 and Lactobacillus plantarum L-1 were prepared according to the methods in Examples 1 and 4 and stored at 4°C for later use.

[0102] The cultured Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 were centrifuged at 4000 rpm and 4℃ for 10 minutes to collect the supernatant, which was then stored at 4℃ for later use.

[0103] Metronidazole (5 mg / mL) was used as a positive control, and sterile water was used as a negative control (Oxford cup diameter was 7.8 mm).

[0104] Take 100uL of live bacteria, the number is 1×10 8 A suspension of CFU / mL Helicobacter pylori ATCC43504 was evenly spread onto antibiotic-free BHI solid agar plates. The solid plates were then perforated, and 100 μL of Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 bacterial suspensions and supernatants were added to the wells, respectively. The plates were then incubated at 37°C under a microaerophilic environment for 72 hours. The diameter of the inhibition zone (in mm) was measured using calipers. Each sample was tested in triplicate, and the results are shown in Table 3.

[0105] Table 3

[0106]

[0107] Note: In different groups of data with the same parameters, the same letter indicates that there is no significant difference between the groups. P >0.05), different letters indicate significant differences between groups ( P <0.05).

[0108] As shown in Table 3, the inhibition zones of both the bacterial suspension and supernatant of Lactobacillus plantarum CCNH185 were significantly larger than those of Lactobacillus plantarum L-1, indicating that Lactobacillus plantarum CCNH185 has a good ability to fight Helicobacter pylori and has the potential to protect gastrointestinal health by fighting Helicobacter pylori.

[0109] Example 10

[0110] Helicobacter pylori suspensions were prepared according to the methods in Examples 1 and 4. 40 μL of the Helicobacter pylori suspension was added to 10 μL of Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 culture medium with equal viable cell counts, and to the supernatant prepared from fermentation broth with equal viable cell counts. The mixtures were thoroughly mixed. 10 μL of sterile MRS liquid culture medium was added to the 40 μL Helicobacter pylori suspension as a control group. The mixtures were placed in 96-well plates and incubated at 37°C under microaerophilic conditions for 48 h. 50 μL of the mixture was added to each well of the 96-well plate containing 150 μL of urease-phenol red reagent, and after shaking to mix, the OD was measured using a microplate reader. 561 The value is used to calculate the inhibition rate of urease activity using the following formula.

[0111] Inhibition rate (%) = (OD of control group) 561 -Experimental group OD 561 ) / Control group OD 561 ×100%.

[0112] The fermentation broth and fermentation supernatant of strain CCNH185 showed inhibition rates of 70.21% and 65.93% on urease, respectively, while the fermentation broth and fermentation supernatant of Lactobacillus plantarum L-1 showed inhibition rates of 67.84% and 54.33% on urease, respectively. This indicates that the strain provided by the present invention has a higher inhibition rate on urease activity than the control strain X1.

[0113] Example 11

[0114] Pre-culture of GES-1 gastric mucosal cells: GES-1 gastric mucosal cells were seeded in sterile 96-well plates and cultured overnight at 37°C and 5 vol% CO2 using 90 vol% DMEM + 10 vol% FBS as the medium. The cultured monolayer cells were then washed three times with sterile PBS before use.

[0115] Simultaneous intervention test: 1 mL of *Lactobacillus plantarum* CCNH185 and *Lactobacillus plantarum* L-1 bacterial suspensions were added to 1 mL of a 102 solution. 8 The sample was placed in a suspension of Helicobacter pylori at CFU / mL, and then 1 mL of gastric mucosal GES-1 cells was added. The mixture was then cultured at 37°C and 5 vol% CO2 for 3 h.

[0116] Post-intervention test: 1 mL of the activated solution with a concentration of 1×10⁻⁶ was used. 9 After adding CFU / mL of Helicobacter pylori to washed gastric mucosa GES-1 cells and co-culturing for 3 hours, the cells were washed three times with sterile PBS solution and then co-cultured for 2 hours with 1 mL of Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 bacterial suspensions, respectively.

[0117] Simultaneously, after the culture for both the intervention and post-intervention tests was completed, the cells were washed three times with sterile PBS solution to remove any unadhered Helicobacter pylori and urease produced during the testing period. After adding urease-phenol red reagent and culturing for 2 hours, the OD was measured using a microplate reader. 561 The absorbance value, with the activity of urease produced by Helicobacter pylori adhering to GES-1 cells of the gastric mucosa as a parameter, indirectly characterizes the ability of lactobacillus to inhibit the adhesion of Helicobacter pylori cells. The control group was replaced with sterile MRS liquid culture medium instead of Lactobacillus plantarum suspension. The inhibition rate of urease activity of the strain was calculated according to the formula in Example 10. The inhibition rate of urease activity of lactobacillus is the inhibition rate of Helicobacter pylori cell adhesion.

[0118] In the intervention test group, the inhibition rates of urease activity by *Lactobacillus plantarum* CCNH185 group and *Lactobacillus plantarum* L-1 group were 79%±4% and 34%±10%, respectively. In the post-intervention test group, the inhibition rates of urease activity by *Lactobacillus plantarum* CCNH185 group and *Lactobacillus plantarum* L-1 group were 66%±7% and 45%±12%, respectively. This indicates that *Lactobacillus plantarum* CCNH185 has the potential to treat / prevent *Helicobacter pylori* infection by preventing *Helicobacter pylori* from adhering to cells.

[0119] Example 12

[0120] 1. Preparation of Fermentation Seed Liquid

[0121] 1 mL of Lactobacillus plantarum CCNH185 glycerol was inoculated into a 100 mL shake flask containing 30 mL of MRS liquid medium and incubated at 37°C for 16 h to obtain activated primary seed culture. The activated primary seed culture was then inoculated into a secondary shake flask at a 3 vol% inoculation rate for further expansion. The secondary seed culture was then incubated at 37°C for 12 h before use.

[0122] 2. Fermentation medium

[0123] Basic MRS liquid culture medium: 1L deionized water, 10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 20g glucose monohydrate, 1mL Tween-80, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate, and 0.25g manganese sulfate monohydrate.

[0124] Optimized MRS liquid culture medium: 1L deionized water, 14g peptone, 26g glucose monohydrate, 4g yeast extract, 1.5g dipotassium hydrogen phosphate, 1.6g diammonium hydrogen citrate, 1.5mL Tween-80, 0.25g magnesium sulfate heptahydrate, and 0.05g manganese sulfate monohydrate.

[0125] 3. Fermentation by bacterial strain

[0126] Secondary seed culture was inoculated at a 10 vol% in both basal and optimized MRS liquid media for fermentation. The fermentation system was 4 L in both media, and the culture temperature was 37 °C. After inoculation, the pH of the fermentation broth was controlled to 6 using a peristaltic pump. Oddi concentration (OD) of the fermentation broth was measured every 2-3 hours. 600 When OD 600 Fermentation was stopped once the count stabilized or growth slowed, and the fermentation broth was collected for later use. A viable count was performed on the fermentation broth at the time of shutdown. The viable count of the fermentation broth fermented on the optimized culture medium stabilized at 8.1 × 10⁻⁶ after 10 hours. 10 The CFU / mL count was high, while the viable count in the basal medium stabilized at only 2.4 × 10⁻⁶ after 12 hours. 9 CFU / mL.

[0127] 4. Preparation of freeze-dried powder

[0128] The fermentation broth was centrifuged to collect the mycelial sludge, and the yields of mycelial sludge in the basal and optimized media were calculated to be 1.4% and 3.5%, respectively. A protectant was added to the mycelial sludge, and based on the total weight of the mixture, the protectant consisted of: 6 wt% skim milk powder, 4.8 wt% trehalose, 1.7 wt% monosodium glutamate, 0.5 wt% glycerol, 4.0 wt% glucose, and 5.5 wt% maltodextrin. After thoroughly mixing the protectant and the mycelial sludge, the mixture was frozen at -80°C and then freeze-dried to obtain freeze-dried mycelial powder cultured in the basal medium (control group) and freeze-dried mycelial powder cultured in the optimized medium (optimized group).

[0129] Take 5g of freeze-dried bacterial powder from the control group and the optimized group, add it to 95mL of sterile water, mix thoroughly, and then perform a 10-fold serial dilution. Select 100μL of the appropriate serial dilution and spread it on MRS solid medium. After incubation at 37°C for 48h, colony counting is performed.

[0130] The highest viable count in the optimized group was 1.2 × 10⁻⁶. 12 The CFU / g culture medium was used, while the highest viable count in the control group was only 6.3 × 10⁻⁶. 11 CFU / g medium. These results show that the optimized MRS medium significantly improves the subsequent fermentation effect of the mycelium.

[0131] Example 13

[0132] Take 3 mL of activated CCNH185 bacterial suspension, centrifuge at 4000 rpm and 4°C for 10 min, collect the bacterial sludge, and wash three times with sterile physiological saline to obtain the bacterial sludge. Resuspend the bacterial sludge in 10 mL of CaCl2 solution (12.5 M), shake for 20 min, centrifuge and discard the aqueous phase, then add 10 mL of polymethyl methacrylate solution (Eudragit L 100-55, purchased from Merck Darmstadt, Germany, catalog number GF93887601, concentration 1.6 mg / mL), shake for 30 min, centrifuge, and discard the aqueous phase. Repeat this procedure 5 times to complete the bacterial encapsulation.

[0133] Following the method described in Example 2, the bile salt tolerance of unencapsulated strains (control group) and encapsulated strains (encapsulated group) was compared. The viable cell counts were recorded at 0, 3, and 6 hours, and the survival rate of the strains relative to 0 hours was calculated. The survival rates of the control group at 3 hours and 6 hours were 78.73% and 0.006%, respectively, while the survival rates of the encapsulated group at 3 hours and 6 hours were 91.3% and 29.57%, respectively. The bile salt survival rate of the encapsulated strains was significantly improved, demonstrating that this encapsulation method provides a guarantee for stabilizing the viable cell count of the product, extending shelf life, and ensuring high survival rates of the strains after the product enters the human body.

[0134] Example 14

[0135] C57BL / 6 strain male mice aged 7-9 weeks were acclimatized for one week and then given a 1.5wt% sodium dextran sulfate (DSS) aqueous solution for 7 days to induce a colitis mouse model. When mice showed symptoms such as weight loss, loose stools, diarrhea, bloody stools or fecal occult blood and ulcers, the DSS model was considered to be successfully established. The mice were randomly divided into three groups of 10 mice each and fed for a total of 28 days.

[0136] (1) Control group: Drink sterile water and be given PBS buffer by gavage once a day.

[0137] (2) Model group: Drink DSS aqueous solution and be given PBS buffer by gavage once a day.

[0138] (3) CCNH185 group: The group was given DSS aqueous solution and CCNH185 strain dissolved in PBS buffer was administered by gavage once a day. The amount of strain solution consumed was 10 ml per day, based on the number of viable bacteria. 9 CFU / kg mouse body weight.

[0139] The daily volume of fluid administered via gavage was v = 10 mL / kg of mouse body weight.

[0140] During the experiment, the mice's weight, fecal condition (fecal viscosity), and blood in the stool were recorded. The percentage of weight loss at the end of the experiment was calculated using the following formula based on the mice's weight.

[0141] Percentage loss in body weight: (mouse weight at the start of the experiment - mouse weight at the end of the experiment) / mouse weight at the start of the experiment × 100%;

[0142] The supernatant of mouse feces was tested using o-toluidine according to standard methods. The more blood in the feces, the darker the blue color of the supernatant.

[0143] After the experiment, samples of mouse blood, feces, and colon tissue and its contents were collected for subsequent testing.

[0144] Test methods for detection indicators:

[0145] (1) Mouse weight: Weigh and record the weight of the mice once a week.

[0146] (2) Based on body weight, fecal condition and blood in stool, the Disease Activity Index (DAI) of mice was assessed according to Table 4. The lower the score, the healthier the mouse.

[0147] Table 4

[0148]

[0149] (3) Length of mouse colon tissue: After the experiment, the mice were sacrificed, the colon tissue was collected and its length was measured.

[0150] (4) Intestinal histopathology: 28 days after intervention, mice were euthanized, and a 0.5-1 cm section of the middle part of the colon was cut off. After being quickly rinsed with physiological saline, the colon tissue was fixed overnight in a general tissue fixation solution. After complete fixation, the colon tissue was routinely dehydrated, embedded, sectioned, and stained with H&E. The degree of inflammation, crypt damage, depth of inflammation, area of ​​inflammatory infiltration, and depth of lesions in the colon tissue were observed under a microscope.

[0151] (5) Inflammatory factors: The relative mRNA expression levels of pro-inflammatory factors (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6)), anti-inflammatory factors interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) were detected using a kit.

[0152] Experimental results:

[0153] (1) At the end of the experiment, the body weights of mice in the control group, model group, and CCNH185 group were 28.39±1.11g, 25.67±1.9g, and 28.5±1.48g, respectively. Compared with mice fed a normal diet, the body weight of mice in the model group was significantly reduced, indicating that the colitis mouse model was successfully established. After CCNH185 treatment, there was no significant difference in body weight between the mice and the control group, indicating that this strain can alleviate the body weight loss in mice induced by the DSS diet.

[0154] (2) During the experiment, mice in both the model group and the CCNH185 group showed a significant decrease in body weight and a trend of stool changing from normal to loose stool. On day 10, the mice reached their lowest body weight, loose stool, visible fecal occult blood, and highest DAI score. After the experiment, the DAI scores of the control group, model group, and CCNH185 group were 1.33±0.58, 9.33±0.58, and 4.67±0.58, respectively. Although the health status of the CCNH185 group was lower than that of the control group, the health status of the CCNH185 group was better than that of the model group.

[0155] (3) Colon tissue length

[0156] After the experiment, the colonic tissue length of mice in the control group was 8.22±1.03 cm, that in the model group was 6.10±0.8 cm, and that in the CCNH185 group was 7.85±1.26 cm. These results indicate that colonic length was restored after intervention with strain CCNH185, meaning that CCNH185 can alleviate the symptoms of colonic shortening.

[0157] (4) Intestinal histopathology

[0158] The H&E staining results of colon tissues from control group, model group, and CCNH185 group mice showed the following: Figure 3 As shown, after inducing colitis in mice, the colon tissue of mice showed obvious lesions, thickening of the peripheral muscle layer, shrinkage of crypt structures, partial mucosal erosion, and reduction of goblet cells, but the CCNH185 group had milder symptoms.

[0159] (5) Inflammatory factors

[0160] The relative expression levels of inflammatory cytokines mRNA are shown in Table 5.

[0161] Table 5

[0162]

[0163] Note: In different groups of data with the same parameters, the same letter indicates that there is no significant difference between the groups. P >0.05), different letters indicate significant differences between groups ( P <0.05).

[0164] The results showed that after DSS modeling, the relative expression levels of pro-inflammatory factors mRNA in mouse colon tissue increased significantly, while the CCNH185 strain could inhibit the increase in the relative expression levels of IL-6, IL-1β and TNF-α pro-inflammatory factor mRNA, and promote the increase in the expression levels of IL-10 and TGF-β anti-inflammatory factors mRNA.

[0165] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A Lactobacillus plantarum strain Lactobacillus plantarum characterized in that, The preservation number of the Lactobacillus plantarum is CGMCC No. 33151.

2. An inoculant characterized in that, The bacterial agent contains the Lactobacillus plantarum of claim 1.

3. The microbial agent of claim 2, wherein, The bacterial agent further contains an auxiliary material selected from glycerol and / or corn oil.

4. The microbial agent of claim 3, wherein, The viable cell number of the Lactobacillus plantarum was 10 4 -10 12 CFU per 1 mL of adjuvant.

5. Use of Lactobacillus plantarum as claimed in claim 1 for the preparation of a medicament for inhibiting colonization of pathogenic bacteria, characterized in that, The pathogenic bacteria are at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Candida albicans, Gardnerella, Atopobium and Helicobacter pylori.

6. Use of Lactobacillus plantarum as claimed in claim 1 for the preparation of a medicament for inhibiting the multiplication of pathogenic bacteria, characterized in that, The pathogenic bacteria are at least one of Escherichia coli, Staphylococcus aureus, Salmonella, Candida albicans, Gardnerella, Atopobium and Helicobacter pylori.

7. Use of the Lactobacillus plantarum of claim 1 in the preparation of a medicament for preventing and / or treating colitis.

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