Lactobacillus plantarum with antibacterial effect and application thereof

Abstract

The invention relates to the field of microorganisms, and discloses lactobacillus plantarum with an antibacterial effect and application of the lactobacillus plantarum, and the preservation number of the lactobacillus plantarum is CGMCC (China General Microbiological Culture Collection Center) No.33151. The strain provided by the invention has relatively strong cholate resistance, gastric acid resistance, acid production and H2O2 production capabilities, and has a relatively strong bacteriostatic effect on common pathogenic bacteria such as staphylococcus aureus, candida albicans, escherichia coli, salmonella and helicobacter pylori; the compound can inhibit the activity of urease, interfere helicobacter pylori colonization and the like to inhibit helicobacter pylori infection or reduce the symptoms of helicobacter pylori infection and reduce the disease degree of mice with colitis, can be used for preparing microbial preparations, and has the potential of preparing drugs for preventing and / or treating diseases caused by helicobacter pylori infection.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of plant lactobacillus with antibacterial efficacy and application thereof. Background Art

[0002] Helicobacter pylori ( Helicobacter pylori H. pylori) is a Gram-negative, microaerophilic bacterium that generally colonizes between the gastric mucus layer and the gastric antrum mucosal epithelial cells and is a Class I carcinogen. People infected with Helicobacter pylori will develop gastrointestinal diseases of varying degrees, 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 disadvantages such as unstable viable counts and cannot stably function in the human body.

[0004] Therefore, there is an urgent need to breed strains with excellent fermentation characteristics and capable of stably surviving 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 the present invention is to overcome the problems existing in the prior art and provide a plant lactobacillus with antibacterial efficacy and application thereof.

[0006] In order to achieve the above object, the present invention provides a plant lactobacillus strain Lactobacillus plants The deposit number of the plant lactobacillus is CGMCC No.33151.

[0007] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the aforementioned Lactobacillus plantarum.

[0008] The third aspect of the present invention provides the use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for inhibiting the colonization of pathogenic bacteria.

[0009] A fourth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for inhibiting the reproduction of pathogenic bacteria.

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

[0011] A sixth aspect of the present invention provides the use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for preventing and / or treating gastrointestinal diseases.

[0012] Through the above technical solution, the beneficial effects obtained by the present invention include at least: (1) The strain provided by the present invention can reduce the degree of colon disease in mice and improve the health of mice by inhibiting weight loss, increasing stool viscosity, inhibiting bloody stool symptoms, 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); (2) The strain provided by the present invention can inhibit the activity of urease, prevent Helicobacter pylori colonization, and inhibit Helicobacter pylori infection or reduce the symptoms of Helicobacter pylori infection; (3) The strain provided by the present invention has strong ability to produce acid and H2O2, and has strong antibacterial effect on common pathogenic bacteria such as Helicobacter pylori, Staphylococcus aureus, Candida albicans, Escherichia coli, Atopobium and Salmonella; (4) The strain provided by the present invention does not cause cell hemolysis, is sensitive to common antibiotics, has biosafety, and can be used to prepare drugs for preventing and / or treating colitis.

[0013] Biological Deposit The strain provided by the present invention is classified and named as Lactobacillus plantarum Lactobacillus plantarum , was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (abbreviated as CGMCC) on December 23, 2024. Its deposit number is CGMCC No.33151, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0014] The strain provided by the present invention is classified and named as Lactobacillus plantarum Lactobacillus plantarum , was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (abbreviated as CGMCC) on December 23, 2024. Its deposit number is CGMCC No.33152, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a colony morphology diagram of the strain CCNH185 provided by the present invention; Figure 2 It is the Gram microscopic image of the strain CCNH185 provided by the present invention; Figure 3 These are the results of H&E staining of colon tissues of mice in the control group, model group, and CCNH185 group. DETAILED DESCRIPTION

[0016] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] The inventor of the present invention accidentally isolated a strain of Lactobacillus plantarum from sour mare's milk that is resistant to bile salts and gastric acid and produces high amounts of lactic acid and hydrogen peroxide. Lactobacillus plantarum Its colony morphology and Gram microscopic examination are shown in Figure 1 and Figure 2 Further studies have shown that the strain provided by the present invention can not only specifically inhibit the reproduction of pathogens, but is also sensitive to commonly used antibiotics and has the potential to be applied to organisms.

[0018] Based on the above findings, the present invention provides a plant lactobacillus strain in the first aspect. Lactobacillus plantarum The deposit number of the plant lactobacillus is CGMCC No.33151.

[0019] "CCNH185" is the number given to the strain by the inventor of the present invention during the strain screening process, and "CGMCC No.33151" is the preservation number of the strain. The two represent the same strain and can be used interchangeably in the following text.

[0020] A second aspect of the present invention provides a bacterial agent, wherein the bacterial agent contains the aforementioned Lactobacillus plantarum.

[0021] The present invention has no particular limitation on the type of the bacterial agent, which can be any type of bacterial agent currently available in the art. According to a preferred embodiment of the present invention, the bacterial agent is selected from at least one of a liquid bacterial agent, a concentrated bacterial agent and a solid bacterial agent.

[0022] In the present invention, the bacterial agent may further contain auxiliary materials, and the auxiliary materials may be selected from protective agents (for example, freeze-dried protective agents, such as skim milk powder, maltodextrin, trehalose, dextran, corn oil and glycerol, etc.) and / or buffers (for example, buffers remaining in the process of preparing solid bacterial agents, etc.). Preferably, the auxiliary materials are selected from glycerol and / or corn oil.

[0023] Preferably, relative to 1 mL of auxiliary material, the viable count of the plant lactobacillus is 10 4 -10 12 CFU.

[0024] The plant lactobacillus provided by the present invention can produce a large number of live cells of plant lactobacillus through liquid culture. There is no special requirement for the culture method, as long as the plant lactobacillus can be proliferated. For example, the live cells of plant lactobacillus can be inoculated into a culture medium at an inoculum amount of 1-5 vol%, and cultured at a temperature of 30-40° C. for 9-40 hours to obtain a culture solution. The culture medium can be any culture medium suitable for culturing plant lactobacillus known in the art, for example, an MRS culture medium.

[0025] The present invention can further separate the live cells of Lactobacillus plantarum in the above-mentioned culture solution. The separation method is not particularly limited as long as the cells can be enriched from the culture solution. For example, it can be achieved by centrifugation and / or filtration. The conditions of the centrifugation and the filtration can be well-known conditions, and the present invention will not be repeated here.

[0026] The third aspect of the present invention provides the use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for inhibiting the colonization of pathogenic bacteria.

[0027] A fourth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for inhibiting the reproduction of pathogenic bacteria.

[0028] According to the use of the third and fourth aspects of the present invention, the pathogenic bacteria is preferably Escherichia coli ( Escherichia coli ), Staphylococcus aureus ( Staphylococcus aureus ),salmonella( Salmonella )、Candida albicans( Candida albicans )、Gardnerella ( Gardnerella vaginalis ) 、 Atopobium Atopobium minutum ) and Helicobacter pylori ( Helicobacter pylori ) at least one of the following.

[0029] According to the present invention, the pathogenic bacteria is more preferably Atopobium.

[0030] According to some embodiments of the present invention, the Salmonella preferably includes Salmonella typhimurium ( Salmonella typhimurium ) and Salmonella Enteritidis ( Salmonella enteritidis ).

[0031] The fifth aspect of the present invention provides the use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for inhibiting urease activity.

[0032] A sixth aspect of the present invention provides use of the aforementioned Lactobacillus plantarum in the preparation of a medicament for preventing and / or treating gastrointestinal diseases.

[0033] Preferably, the gastrointestinal disease is colitis, more preferably ulcerative colitis.

[0034] The present invention also provides a method for culturing plant lactobacillus, the method comprising: inoculating the plant lactobacillus as described above into an MRS medium, wherein the composition of the MRS medium comprises: relative to 1L of the medium, 10-20g of peptone, 10-30g of glucose, 1-7g of yeast powder, 0.5-3g of dipotassium hydrogen phosphate, 0.5-3g of diammonium hydrogen citrate, 0.5-1.5g of Tween-80, 0.02-0.3g of magnesium sulfate, and 0.005-0.1g of manganese sulfate; based on the volume of the culture medium, the number of viable bacteria inoculated with the plant lactobacillus can be 10 1 -10 7 CFU / mL.

[0035] The plant lactobacillus provided by the present invention can be cultured in any commonly used culture medium in the art, but the inventors of the present invention have found in research that when the plant lactobacillus provided by the present invention is cultured using the MRS culture medium as described above, the plant lactobacillus provided by the present invention can complete growth and reproduction in a shorter time, and the number of live bacteria when growth stops and the bacterial mud finally separated are more than those of the MRS culture conventionally used in the art.

[0036] The present invention also provides a method for preparing a freeze-dried powder, the method comprising: adding a protective agent to the bacterial mud of Lactobacillus plantarum provided by the present invention, and then freeze-drying at a temperature of -90°C to -70°C.

[0037] According to the present invention, based on the total weight of the mixture obtained after mixing, the protective agent may include 1-20wt% skimmed milk powder, 1-20wt% trehalose, 0.5-10wt% sodium glutamate, 0.1-10wt% glycerol, 1-10wt% glucose and 1-10wt% maltodextrin.

[0038] The present invention also provides a method for strain embedding, the method comprising: treating the plant lactobacillus sludge provided by the present invention with calcium chloride and polymethyl methacrylate in sequence. Wherein, CaCl2 and / or polymethyl methacrylate are preferably provided in liquid form, wherein the content of CaCl2 is preferably 5-20M, and the content of polymethyl methacrylate is preferably 0.5-5mg / mL.

[0039] According to the present invention, the embedding method may further include: performing solid-liquid separation on the mixture of the CaCl2 solution and the bacterial mud, and then adding the separated solid to the polymethyl methacrylate solution for solid-liquid separation. In order to obtain a better embedding effect, the bacterial mud is added to the polymethyl methacrylate solution for solid-liquid separation for more than 1 times, preferably 3-10 times.

[0040] The present invention also provides a composite bacterial agent, which comprises the plant lactobacillus with a preservation number of CGMCC No. 33151 and the plant lactobacillus with a preservation number of CCGMCC No. 33152 provided by the present invention.

[0041] As shown in the application document with application number 202510222543.6, Lactobacillus plantarum with a deposit number of CGMCC No. 33152 also has the effects of antibacterial (especially inhibiting Atopobium), inhibiting the reproduction of pathogens, and reducing the expression of pro-inflammatory factors, and also has biosafety and antibiotic sensitivity. Therefore, the Lactobacillus plantarum with a deposit number of CCGMCC No. 33151 provided by the present invention is particularly suitable for compounding with Lactobacillus plantarum with a deposit number of CGMCC No. 33152 to prepare a composite bacterial agent, so as to prepare a drug for preventing and / or treating vaginitis and / or diseases caused by Atopobium.

[0042] In the composite bacterial agent provided by the present invention, the two strains can be packaged independently or mixed, but in order to maintain the activity of the strains to the greatest extent to exert the effect, they are preferably packaged independently.

[0043] When a patient uses a medicine containing the composite bacterial agent provided by the present invention, the medicines containing the two strains can be used simultaneously or sequentially. However, in order to maximize the effect, it is preferably used sequentially, but the order of sequential use is not limited.

[0044] The present invention will be described in detail below through examples.

[0045] Unless otherwise specified in the following examples and comparative examples, the reagents and materials used are commercial products purchased from regular chemical or biological reagent and material suppliers.

[0046] Escherichia coli ( Escherichia coli ), numbered (1) ATCC 25922, purchased from the American Type Culture Collection, (2) CICC 10421, purchased from the China Industrial Microbiological Culture Collection; Staphylococcus aureus ( Staphylococcus aureus ), numbered CMCC(B)26001 and CMCC(B)26003, both purchased from the Chinese Medical Bacteria Collection Center; Salmonella, (1) Salmonella typhimurium ( Salmonella typhimurium ), numbered ATCC 14028, purchased from the American Type Culture Collection; (2) Salmonella Enteritidis ( Salmonella enteritidis ), numbered CVCC 3378, purchased from the National Veterinary Microbiological Bacteria (Virus) Collection Center; Candida albicans ( Candida albicans ), numbered (1) CICC 1965, purchased from China Industrial Microbiological Culture Collection, (2) ATCC10231, purchased from American Type Culture Collection; Gardnerella Gardnerella vaginalis ), numbered ATCC 14018, purchased from the American Type Culture Collection; Atopobium Atopobium minutum ), numbered ATCC 33267, purchased from the American Type Culture Collection; Helicobacter pylori ( Helicobacter pylori ), No. ATCC 43504, purchased from the American Type Culture Collection; MRS liquid medium: peptone 10g, beef extract powder 10g, yeast extract powder 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween-80 1mL, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g, deionized water 1L, pH 6.5, autoclave; add agar 18g before volume adjustment to obtain MRS solid medium; Brain heart infusion broth (BHI) solid agar plate: purchased from Qingdao Haibo Biotechnology Co., Ltd., catalog number HB8297-1; Gastric mucosal cells GES-1 were purchased from Shanghai Stemcell Biotechnology Co., Ltd., with the catalog number STM-CL-5009; High glucose medium (DMEM) and fetal bovine serum (FBS) were purchased from Merck, Darmstadt, Germany, with catalog numbers D1145 and 12103C, respectively; PBS buffer was purchased from Solebol, catalog number P1003; Reference strains: (1) Lactobacillus plantarum, with a deposit number of CGMCC No. 15013, which can be found in CN109423467B, hereinafter referred to as Lactobacillus plantarum L-1; (2) Lactobacillus rhamnosus ( Lactobacillus buckthorn ), numbered ATCC 53103, purchased from the American Type Culture Collection, and referred to as LGG.

[0047] Example 1 Weigh 10g of sour mare's milk sample and add it to 90mL of sterile water, 180r / min, shake for 20min, dilute step by step, select the appropriate gradient to be coated on a plate containing MRS solid culture medium, and culture at 37°C for 48h. Use an inoculation loop to pick a single colony with a smooth surface, milky white round or oval, then streak it on MRS solid culture medium, culture at 37°C for 48h, and repeat the purification for 6 generations. Randomly select a single colony with a milky white, smooth and round or oval colony on the solid plate, use an inoculation loop to pick the purified single colony, inoculate it in MRS liquid culture medium, culture it at 37°C for 24h, and pass it 3 times with a 2vol% inoculum, and freeze it.

[0048] The strain CCNH185 and LGG glycerol tubes were inoculated into fresh MRS liquid medium at 2 vol% and cultured overnight at 37°C for 18 h. 600 The activated bacterial solution was normalized and inoculated into fresh MRS liquid culture medium at an inoculum size of 2 vol%, and cultured overnight at 37° C. for 18 h to obtain the expanded culture solution of Lactobacillus plantarum CCNH185 and the expanded culture solution of LGG.

[0049] Measure the OD of Lactobacillus plantarum CCNH185 and LGG culture solutions 600 The values ​​were 9.59±0.03 and 8.13±0.02 respectively. The growth performance of Lactobacillus plantarum CCNH185 was better than that of LGG.

[0050] Example 2 Take the culture liquid of Lactobacillus plantarum CCNH185 and LGG, and refer to T / CNHFA435-2024 "Test Method for Gastric Juice Tolerance of Probiotics" to evaluate the gastric acid resistance and bile salt resistance of the strains. In the gastric acid resistance test, the pH is 3 and the culture is 2h; in the bile salt resistance test, the bile salt content is 0.2wt% and the culture is 4h; OD 600 Measure the absorbance of the bacterial solution before and after the experiment.

[0051] Survival rate: (OD before experiment 600 Value - OD after experiment 600 value) / OD before the experiment 600 value × 100%.

[0052] The gastric acid resistance survival rates of Lactobacillus plantarum CCNH185 and LGG were 93.55% and 93.33%, and the bile salt resistance survival rates were 71.29% and 53.13%, respectively. It can be found that the gastric acid resistance and bile salt resistance of Lactobacillus plantarum CCNH185 are better than those of LGG.

[0053] Example 3 The activated Lactobacillus plantarum CCNH185 and LGG bacterial liquid were inoculated at 2 vol% in 30 mL of fresh MRS liquid culture medium, cultured at 37°C for 18 h, and then centrifuged to obtain the supernatant, which was passed through a membrane (with a pore size of 0.22 μm) and the lactic acid production was detected by high performance liquid chromatography HPLC.

[0054] The lactic acid production of strain CCNH185 was 18.67 g / L, and 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 could metabolize and decompose more sugars during the cultivation process and reduce the sugar content in the environment.

[0055] Example 4 Take the cultured bacterial solution, wash it twice with saline, and resuspend it in 0.1M PBS buffer until the OD600 of the bacterial suspension is 1. Pipette 100 μL of the bacterial suspension into the ELISA plate and detect the initial OD 600 The bacterial solution was allowed to stand for 4 hours, and 100 μL of the upper bacterial solution was taken to the ELISA plate to detect the OD value after standing. 600 The value was recorded as A1, and the self-aggregation rate of the strain was calculated; 900 μL of the above bacterial suspension was taken, 180 μL of dodecane was added, vortexed until mixed evenly, and the mixture was allowed to stand for 20 minutes to separate, and the lower aqueous phase was taken to measure its OD 600 value, recorded as A2, to calculate the hydrophobicity of the strain; The self-aggregation rate or hydrophobicity rate of the strain = (A0-A1 or A2) / A0×100%.

[0056] The self-aggregation rates of strains CCNH185 and LGG were 60.26% and 54.55%, and the hydrophobicity rates were 46.74% and 43.85%, respectively. The two data combined show that Lactobacillus plantarum CCNH185 has a good adhesion effect, can bind to intestinal epithelial cells and colonize in the intestine, thereby inhibiting the colonization of pathogens in the gastrointestinal tract.

[0057] Example 5 Weigh 44 g of Columbia blood agar medium powder (purchased from Qingdao Haibo Biological, catalog number HB9295), add 1000 mL of water, sterilize at 121°C for 15 min, and then add 10 vol% sterile defibrinated sheep blood to obtain Columbia blood agar plate. Inoculate 10 μL of the culture solution on the Columbia blood agar plate. In addition, use Escherichia coli ATCC 25922 and Staphylococcus aureus CMCC 26001 with the same number of viable cells as control strains.

[0058] The results showed that Lactobacillus plantarum CCNH185 and Escherichia coli ATCC 25922 had no hemolysis, while Staphylococcus aureus CMCC 26001 was completely hemolyzed, with β-hemolysis zones around white colonies, indicating that the strain CCNH185 provided by the present invention would not cause hemolysis when applied to an organism, and thus has the possibility of in vivo application.

[0059] Example 6 The 4-aminoacylantipyrine method was used to detect the H2O2 production capacity. 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 values ​​at different concentrations were detected. 505 Measure the absorbance of the culture medium and establish a standard curve of absorbance-concentration. Take 30 mL of the culture medium, centrifuge and take the supernatant, measure its absorbance, and calculate the H2O2 yield based on the standard curve of absorbance-concentration.

[0060] The H2O2 production of Lactobacillus plantarum CCNH185 and LGG were 4.38 μg / mL and 4.31 μg / mL, respectively, indicating that both strains can non-specifically inhibit the growth of pathogenic bacteria by producing H2O2.

[0061] Example 7 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 in LB liquid medium, Candida albicans (CICC 1965 and ATCC 10231) were inoculated in YPD solid medium, Gardnerella (ATCC 14018) and Atopobium (ATCC 33267) were seeded in blood agar plates, and Helicobacter pylori (ATCC 43504) was inoculated in BHI solid agar plates. The above strains were used as indicator strains. Except for Gardnerella anaerobic and Helicobacter pylori microaerophilic culture, all other indicator strains were cultured aerobically. After culturing and activation at 37°C for 24 h (Helicobacter pylori culture for 72 h), they were transferred to the corresponding fresh liquid culture medium and cultured until the viable count reached 10 5 -10 7 CFU / mL, dilute to 10 5 CFU / mL was used as the indicator bacterial solution. The activated bacterial solution of Lactobacillus plantarum CCNH185 and LGG was inoculated into 30 mL of fresh MRS liquid medium at a 2 vol% inoculation amount, incubated at 37°C for 18 h, and then centrifuged to obtain the supernatant, which was then processed through a membrane (the membrane pore size was 0.22 μm).

[0062] Three experimental groups were set up: S1: 100 μL fermentation supernatant, 100 μL indicator bacteria; S2: 50 μL fermentation supernatant, 150 μL indicator bacteria; S3: 25 μL fermentation supernatant, 175 μL indicator bacteria; 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 (content 100 μg / mL) as a positive control group, with 3 replicates in each group. Measure the OD of the experimental group and the negative control group 600 The values ​​are A and A0 respectively, and 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: Inhibition rate = (A0-A) / A0×100%.

[0063] Table 1

[0064] Under the same culture system, the inhibition rate of strain CCNH185 against all pathogenic bacteria was higher than that of strain LGG, especially the inhibition rate against Helicobacter pylori was significantly higher than that of strain LGG.

[0065] Example 8 Take the culture solution and dilute it with MRS medium until the viable count is 1.5×10 5 CFU / mL.

[0066] Prepare antibiotic samples with different initial concentrations (INC) according to Table 2, dilute the antibiotics by double dilution method and measure the growth of the strains in them until the strains grow. At this time, the antibiotic concentration is the minimum inhibitory concentration (MIC). Compare the MIC with the breakpoint value in the General Rules for Probiotics for Food (T / CIFST 009-2022) to determine its antibiotic sensitivity. If the MIC is less than the judgment concentration (breakpoint value), it means that the strain is highly sensitive to the antibiotic, and a low concentration of antibiotics can inhibit the growth of the strain; if the MIC is greater than the breakpoint value, it means that the strain is highly resistant to the antibiotic, and a higher concentration of antibiotics is required to inhibit the growth of the strain. Among them, nr means not required. The test results (INC, MIC) of the strain in different antibiotics and the corresponding breakpoint values ​​are shown in Table 2. MRS liquid culture medium was used as a blank control, and the aqueous solution was used as a negative control.

[0067] Table 2

[0068] As can be seen from Table 2, overall, CCNH185 is highly sensitive to all antibiotics (lower antibiotic content can inhibit its growth). This result shows that Lactobacillus plantarum CCNH185 is safe and can be used to develop probiotic additives or as a food fermentation strain.

[0069] Example 9 The in vitro anti-Helicobacter pylori ability of CCNH185 was determined by the Oxford cup method. CCNH185 and Lactobacillus plantarum L-1 bacterial suspensions were prepared according to the methods of Examples 1 and 4 and stored at 4°C for later use.

[0070] The cultured Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 were centrifuged at 4000 rpm and 4°C for 10 minutes, the supernatant was collected, and stored at 4°C for later use.

[0071] Metronidazole (5 mg / mL) was used as the positive control, and sterile water was used as the negative control (the diameter of the Oxford cup was 7.8 mm).

[0072] The number of viable bacteria in 100uL is 1×10 8 The suspension of CFU / mL Helicobacter pylori ATCC43504 was evenly spread on a BHI solid agar plate without antibiotics, and then the solid plate was punched, and 100 μL of bacterial suspension and supernatant of Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 were added to the wells, respectively. Then, the plates were cultured at 37°C in a microaerobic environment for 72 hours, and the diameter of the inhibition zone (in mm) was measured using a vernier caliper. Each sample was replicated three times. The results are shown in Table 3.

[0073] Table 3

[0074] Note: In different groups of data under the same parameter, the same letters indicate no significant difference between the groups ( P >0.05), different letters indicate significant differences among groups ( P <0.05).

[0075] As shown in Table 3, the diameters of the inhibition zones of the bacterial suspension and supernatant of Lactobacillus plantarum CCNH185 are significantly larger than those of Lactobacillus plantarum L-1, indicating that Lactobacillus plantarum CCNH185 has good anti-Helicobacter pylori ability and has the potential to protect gastrointestinal health by resisting Helicobacter pylori.

[0076] Example 10 Prepare Helicobacter pylori suspension according to the method of Examples 1 and 4, take 40 μL of Helicobacter pylori suspension and add it to 10 μL of Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 with equal viable counts and the supernatant prepared from the fermentation broth with equal viable counts, mix them evenly, add 10 μL of sterile MRS liquid culture medium to 40 μL of Helicobacter pylori suspension as a control group. Place the mixed solution in a 96-well plate and culture it under microaerobic conditions at 37°C for 48 hours, add 50 μL of the mixed solution to each well of the 96-well plate containing 150 μL of urease-phenol red reagent, shake and mix well, and measure OD with a microplate reader. 561 The inhibition rate of urease activity was calculated using the following formula.

[0077] Inhibition rate (%) = (OD 561 - Experimental group OD 561 ) / control group OD 561 ×100%.

[0078] The urease inhibition rates of the fermentation broth and fermentation supernatant of strain CCNH185 were 70.21% and 65.93%, respectively, and the urease inhibition rates of the fermentation broth and fermentation supernatant of Lactobacillus plantarum L-1 were 67.84% and 54.33%, respectively, indicating that the urease activity inhibition rate of the strain provided by the present invention is higher than that of the control strain X1.

[0079] Embodiment 11 Pre-culture of gastric mucosal cells GES-1: gastric mucosal cells GES-1 were inoculated in a sterile 96-well plate, and cultured overnight at 37°C and 5 vol% CO2 using 90 vol% DMEM + 10 vol% FBS as the culture medium. The cultured monolayer cells were then washed three times with sterile PBS liquid for later use.

[0080] Simultaneous intervention test: 1 mL of the experimental bacterial suspension of Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 was added to 1 mL of 10 8 CFU / mL of Helicobacter pylori suspension, and then add 1 mL of gastric mucosal GES-1 cells and culture at 37°C and 5 vol% CO2 for 3 hours.

[0081] Post-intervention test: 1 mL of activated 1×10 9 Helicobacter pylori with CFU / mL was added to the washed gastric mucosal GES-1 cells and co-cultured for 3 hours. The cells were washed three times with sterile PBS solution and 1 mL of bacterial suspension of Lactobacillus plantarum CCNH185 and Lactobacillus plantarum L-1 were added and co-cultured for 2 hours.

[0082] After the intervention test and post-intervention test were completed, the cells were washed three times with sterile PBS solution to remove the non-adherent Helicobacter pylori and the urease produced during the test. After adding urease-phenol red reagent and culturing for 2 hours, the OD was measured with a microplate reader. 561 The absorbance value was used to indirectly characterize the ability of lactobacillus to inhibit the adhesion of Helicobacter pylori cells by taking the activity of urease produced by Helicobacter pylori adhered to the gastric mucosal GES-1 cells as a parameter. The control group used sterile MRS liquid culture medium instead of Lactobacillus plantarum suspension, and the inhibition rate of the strain on urease activity was calculated according to the formula in Example 10. The inhibition rate of lactobacillus on urease activity was the inhibition rate on the adhesion of Helicobacter pylori cells.

[0083] In the simultaneous intervention test group, the inhibition rates of urease activity in the Lactobacillus plantarum CCNH185 group and the Lactobacillus plantarum L-1 group were 79%±4% and 34%±10%, respectively. In the post-intervention test group, the inhibition rates of urease activity in the Lactobacillus plantarum CCNH185 group and the Lactobacillus plantarum L-1 group were 66%±7% and 45%±12%, respectively, indicating that Lactobacillus plantarum CCNH185 has the potential to treat / prevent Helicobacter pylori infection by preventing Helicobacter pylori from adhering to cells.

[0084] Example 12 1. Preparation of fermented seed liquid 1 mL of the Lactobacillus plantarum CCNH185 glycerol tube was inoculated into a 100 mL shake flask containing 30 mL of MRS liquid culture medium and cultured at 37°C for 16 h to obtain activated primary seeds; the activated primary seeds were then inoculated into secondary shake flasks at a 3 vol% inoculation rate for expansion culture, and the secondary seed shake flasks were cultured at 37°C for 12 h before use.

[0085] 2. Fermentation medium Basic MRS liquid culture medium: 1L deionized water, 10g peptone, 10g beef extract powder, 5g yeast extract powder, 2g diammonium hydrogen 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.

[0086] Optimized MRS liquid culture medium: 1L deionized water, 14g peptone, 26g glucose monohydrate, 4g yeast extract powder, 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.

[0087] 3. Strain fermentation The secondary seeds were inoculated with 10 vol% inoculation rate in the basic MRS liquid medium and the optimized MRS liquid medium for fermentation. The fermentation system was 4 L and the culture temperature was 37 °C. After inoculation, the pH of the fermentation liquid was controlled to 6 by feeding with a peristaltic pump. The fermentation liquid OD was sampled every 2-3 h. 600 , when OD 600 When the fermentation becomes stable or slow in growth, the fermentation liquid is stopped, and the fermentation liquid is collected for later use. The number of live bacteria in the fermentation liquid fermented with the optimized medium becomes stable at 8.1×10 10 CFU / mL, while the number of viable bacteria in the basal culture medium tended to stabilize at 12 h, with the number of viable bacteria being only 2.4×10 9 CFU / mL.

[0088] 4. Preparation of freeze-dried powder The fermentation liquid was centrifuged to collect bacterial sludge, and the bacterial sludge yields of the basic and optimized culture media were calculated to be 1.4% and 3.5%, respectively. A protective agent was added to the bacterial sludge, and the composition of the protective agent was: 6wt% skim milk powder, 4.8wt% trehalose, 1.7wt% sodium glutamate, 0.5wt% glycerol, 4.0wt% glucose, and 5.5wt% maltodextrin, based on the total weight of the mixture obtained after mixing. After the protective agent and the bacterial sludge were stirred evenly, they were frozen at -80°C and freeze-dried to obtain freeze-dried bacterial powder (control group) obtained by culturing with the basic culture medium and freeze-dried bacterial powder (optimized group) obtained by culturing with the optimized culture medium.

[0089] Take 5 g of freeze-dried bacterial powder from the control group and the optimization group, add them into 95 mL of sterile water, mix them thoroughly and perform 10-fold gradient dilution. Select 100 μL of the appropriate gradient dilution solution and spread it on MRS solid culture medium. After culturing at 37 °C for 48 h, count the colonies.

[0090] The highest number of viable bacteria in the optimized group was 1.2×10 12 CFU / g culture medium, while the highest number of viable bacteria in the control group was only 6.3×10 11 CFU / g culture medium. The results show that the optimized MRS culture medium can significantly improve the subsequent fermentation effect of bacterial powder.

[0091] Example 13 Take 3mL of CCNH185 activated bacterial solution, centrifuge at 4000rpm, 4°C for 10min, collect bacterial sludge, and wash it three times with sterile saline to obtain bacterial sludge. Take 10mL of CaCl2 solution (12.5M) to resuspend the bacterial sludge, shake for 20min, centrifuge and discard the aqueous phase, add 10mL of polymethyl methacrylate solution (Eudragit L 100-55, purchased from Merck, Darmstadt, Germany, product number GF93887601) with a content of 1.6mg / mL, shake for 30min, centrifuge and discard the aqueous phase. Repeat this step 5 times to complete the strain embedding.

[0092] According to the method of Example 2, the bile salt resistance of the strains that were not embedded (control group) and the strains that were embedded (embedded group) was compared, the number of live bacteria at 0, 3 and 6 hours of the experiment was recorded, 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 embedding group at 3 hours and 6 hours were 91.3% and 29.57%, respectively. The bile salt survival rate of the strains after embedding was greatly improved, proving that the embedding method provides a guarantee for stabilizing the number of live bacteria in the product, extending the shelf life, and providing a high survival rate of the strains after the product enters the human body.

[0093] Embodiment 14 After one week of adaptive feeding, male mice of C57BL / 6 strain, aged 7-9 weeks, drank 1.5wt% dextran sulfate sodium (DSS) aqueous solution for 7 days to induce the construction of colitis mouse model. When mice showed weight loss, loose stools, diarrhea, bloody stools or fecal occult blood and ulcers, DSS modeling was considered successful. The mice were randomly divided into three groups, with 10 mice in each group, and fed for a total of 28 days.

[0094] (1) Control group: The mice were given sterile water and gavaged with PBS buffer once a day.

[0095] (2) Model group: The mice drank DSS aqueous solution and were gavaged with PBS buffer once a day.

[0096] (3) CCNH185 group: Drink DSS aqueous solution and gavage CCNH185 dissolved in PBS buffer once a day. The drinking amount of strain solution was 10 9 CFU / kg mouse body weight.

[0097] The volume of the liquid administered intragastrically every day was v=10 mL / kg mouse body weight.

[0098] During the experiment, the weight, stool status (stool consistency) and blood in the stool of the mice were recorded. The weight of the mice was used to calculate the percentage of weight loss at the end of the experiment according to the following formula.

[0099] Percentage of weight loss: (mouse weight at the beginning of the experiment - mouse weight at the end of the experiment) / mouse weight at the beginning of the experiment × 100%; The supernatant of mouse feces was tested using o-tolidine according to conventional methods. The more blood the feces contained, the darker the blue of the supernatant.

[0100] After the experiment, samples of mouse blood, feces, and colon tissue and its contents were collected for subsequent testing.

[0101] Test methods for detection indicators: (1) Mouse body weight: weigh and record the body weight once a week.

[0102] (2) Evaluate the disease activity index (DAI) of mice based on body weight, stool status, and blood in stool, referring to Table 4. The lower the score, the healthier the mouse.

[0103] Table 4

[0104] (3) Length of mouse colon tissue: After the experiment, the mice were killed, the colon tissue was collected and its length was measured.

[0105] (4) Intestinal tissue pathology: After 28 days of intervention, the mice were killed, and the middle part of the colon (about 0.5-1 cm) was cut. After rapid rinsing with saline, the colon tissue was fixed overnight in a universal tissue fixative. After complete fixation, the colon tissue was routinely dehydrated, embedded, sliced, and stained with H&E. The degree of colon tissue inflammation, crypt damage, depth of inflammation, area of ​​inflammatory infiltration, and depth of lesions were observed under a microscope.

[0106] (5) Inflammatory factors: The relative expression levels of mRNA 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.

[0107] Experimental results: (1) At the end of the experiment, the 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 weight of mice in the model group was significantly reduced, indicating that the colitis mouse model was successfully established. After CCNH185 treatment, the weight of mice was not significantly different from that of the control group, indicating that this strain can slow down the weight loss of mice caused by the DSS diet.

[0108] (2) During the experiment, mice in both the model group and CCNH185 group showed a significant weight loss, and their feces showed a trend from normal to loose stools. On the 10th day, the weight of the mice reached the lowest point, the feces were loose, the fecal occult blood was visible to the naked eye, and the DAI score reached the highest point. 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.

[0109] (3) Colon tissue length After the experiment, the colon tissue length of the mice in the control group was 8.22±1.03cm, the colon tissue length of the mice in the model group was 6.10±0.8cm, and the colon tissue length of the mice in the CCNH185 group was 7.85±1.26cm. The results showed that after the intervention treatment of strain CCNH185, the colon length was restored, which means that CCNH185 can alleviate the symptoms of colon shortening.

[0110] (4) Intestinal tissue pathology The results of H&E staining of the colon tissues of mice in the control group, model group, and CCNH185 group showed that Figure 3 As shown in the figure, after inducing colitis in mice, the colon tissue of the mice showed obvious lesions, thickening of the peripheral muscle layer, reduction of crypt structure, partial mucosal erosion, and decrease of goblet cells, but the above symptoms were milder in the CCNH185 group.

[0111] (5) Inflammatory factors The relative expression levels of mRNA of inflammatory factors are shown in Table 5.

[0112] Table 5

[0113] Note: In different groups of data under the same parameter, the same letters indicate no significant difference between the groups ( P >0.05), different letters indicate significant differences among groups ( P <0.05).

[0114] The results showed that after DSS modeling, the relative expression level of mRNA of proinflammatory factors in mouse colon tissue increased significantly, while the CCNH185 strain could inhibit the increase in the relative expression level of mRNA of proinflammatory factors IL-6, IL-1β and TNF-α, and promote the increase in mRNA expression level of two anti-inflammatory factors IL-10 and TGF-β.

[0115] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

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

2. A bacterial agent, characterized in that The bacterial agent contains the Lactobacillus plantarum according to claim 1.

3. The bacterial agent according to claim 2, wherein The bacterial agent also contains auxiliary materials, and the auxiliary materials are selected from glycerol and / or corn oil.

4. The bacterial agent according to claim 3, wherein Relative to 1mL of auxiliary material, the viable count of the plant lactobacillus is 10 4 -10 12 CFU.

5. Use of the plant lactobacillus according to claim 1 in the preparation of a medicament for inhibiting colonization of pathogenic bacteria.

6. Use of the plant lactobacillus according to claim 1 in preparing a medicament for inhibiting the reproduction of pathogenic bacteria.

7. The use according to claim 5 or 6, wherein: The pathogenic bacteria are Escherichia coli, Staphylococcus aureus, Salmonella, Candida albicans, Gardnerella 、 At least one of Atopobium and Helicobacter pylori.

8. Use of the plant lactobacillus according to claim 1 in the preparation of a medicament for inhibiting urease activity.

9. Use of the plant lactobacillus according to claim 1 in the preparation of a medicament for preventing and / or treating gastrointestinal diseases.

10. The use according to claim 9, wherein the gastrointestinal disease is colitis.

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