Strong-adhesion plant lactobacillus DACN962 and application thereof in inhibition of helicobacter pylori

Abstract

The invention discloses a high-adhesion plant lactobacillus DACN962 strain and an application of the high-adhesion plant lactobacillus DACN962 strain in inhibition of helicobacter pylori. The plant lactobacillus DACN962 provided by the invention has a remarkable effect in prevention of helicobacter pylori colonization, and the DACN962 can compete for inhibiting the helicobacter pylori from being adhered to GES-1 cells in vitro and can be up to 53.55 + / -1.54%; in-vitro exclusion inhibition of helicobacter pylori adhesion to GES-1 cells can reach up to 82.98 + / -2.37%; the diameter of the inhibition zone of the freeze-dried supernatant of the plant lactobacillus DACN962 can reach 22.69 + / -0.76 mm, and the diameter of the inhibition zone of the fermentation liquor (containing thalli) can reach 15.33 + / -0.82 mm. The DACN962 is good in safety, has high adhesiveness, can compete / exclude and inhibit adhesion of helicobacter pylori, has helicobacter pylori resistance, is sensitive to various antibiotics, and is safe and harmless to a human body.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Lactobacillus plantarum DACN962 with strong adhesion and application thereof in inhibiting Helicobacter pylori. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative, microaerophilic bacterium that generally colonizes between the gastric mucus layer and the gastric antral mucosal epithelial cells. As one of the most prevalent pathogens worldwide, infection with Helicobacter pylori can cause many gastrointestinal diseases, such as chronic active gastritis, gastroduodenal ulcers, gastric mucosa-associated lymphoid tissue lymphoma, gastric cancer and other digestive system diseases. Since Hp is resistant to some antibiotics and is often accompanied by many side effects, it has brought serious problems to medical science. Therefore, it is urgent to find a solution to eradicate Hp colonization in the gastrointestinal tract and reduce the incidence of diseases such as gastrointestinal inflammation.

[0003] Studies have shown that the adhesion of Hp is the main factor for its colonization in gastric mucosal tissue and its pathogenicity. Reducing the adhesion of Hp is an important mechanism to protect the stomach and duodenal lumen from inflammation and prevent atrophic gastritis, which can reduce its pathogenicity. Lactobacillus, as a type of probiotic, has the characteristics of strong activity, acid resistance, regulation of intestinal flora and high safety, and has attracted much attention in the clinical treatment of pathogen infections. Some metabolites secreted by lactobacilli can exert anti-adhesion activity. Probiotics can compete or exclude Hp through their own strong adhesion, thereby inhibiting the colonization of Hp. Summary of the invention

[0004] The purpose of the present invention is to solve the above problems and provide a strain of Lactobacillus plantarum DACN962 with strong adhesion and its application in inhibiting Helicobacter pylori.

[0005] In order to achieve its purpose, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides a strain of Lactobacillus plantarum DACN962, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M20241458.

[0007] A second aspect of the present invention provides a bacterial agent, the active ingredient of which comprises the above-mentioned Lactobacillus plantarum DACN962 or its fermentation liquid.

[0008] Preferably, the bacterial agent is a freeze-dried powder.

[0009] The third aspect of the present invention provides a method for preparing the above-mentioned bacterial agent, which is obtained by inoculating the above-mentioned Lactobacillus plantarum DACN962 into a culture medium for cultivation.

[0010] The preparation method of the fermentation liquid is as follows: after activating the Lactobacillus plantarum DACN962 strain, the strain is cultured in a liquid culture medium at 35-37° C. for 12-24 hours to obtain the fermentation liquid of Lactobacillus plantarum DACN962; preferably, the liquid culture medium is an MRS liquid culture medium;

[0011] The preparation method of the freeze-dried powder comprises the following steps: filtering the prepared Lactobacillus plantarum DACN962 fermentation liquid through a filter membrane to obtain a cell-free fermentation supernatant, and performing rotary evaporation and freeze-drying on the cell-free fermentation supernatant to prepare the freeze-dried powder.

[0012] The fourth aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum DACN962 or the above-mentioned bacterial agent in a product for any of the following purposes:

[0013] (1) Competitive / exclusion inhibition of Helicobacter pylori adhesion;

[0014] (2) Prevent Helicobacter pylori infection;

[0015] (3) Inhibit the growth of Helicobacter pylori.

[0016] The fifth aspect of the present invention provides the use of the above-mentioned Lactobacillus plantarum DACN962 or the above-mentioned bacterial agent in the preparation of a medicament for preventing and treating diseases caused by Helicobacter pylori.

[0017] The sixth aspect of the present invention provides a product, comprising the above-mentioned Lactobacillus plantarum DACN962 or the above-mentioned bacterial agent.

[0018] Preferably, the product is selected from food, food additives, health products, feed, feed additives, reagents or medicines.

[0019] The product is used for any of the following purposes:

[0020] (1) Competition / exclusion inhibition of Helicobacter pylori adhesion;

[0021] (2) Prevent Helicobacter pylori infection;

[0022] (3) Inhibit the growth of Helicobacter pylori;

[0023] (4) Used for preventing and treating diseases caused by Helicobacter pylori.

[0024] The beneficial effects of the present invention are:

[0025] The plant lactobacillus DACN962 provided by the present invention has a significant effect in preventing Helicobacter pylori colonization. The research results show that the plant lactobacillus DACN962 can competitively inhibit the adhesion of Helicobacter pylori to GES-1 cells by up to 53.55±1.54% in vitro; the in vitro exclusion inhibition of Helicobacter pylori adhesion to GES-1 cells can be up to 82.98±2.37%; the diameter of the inhibition zone of the freeze-dried supernatant of plant lactobacillus DACN962 can reach 22.69±0.76mm, and the diameter of the inhibition zone of the fermentation liquid (containing bacteria) can reach 15.33±0.82mm.

[0026] The plant lactobacillus DACN962 screened by the present invention has good safety, high adhesion, can competitively / exclude the adhesion of Helicobacter pylori, has the ability to resist Helicobacter pylori, is sensitive to multiple antibiotics, and is safe and harmless to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The colony morphology (A) and Gram staining results (B) of Lactobacillus plantarum DACN962.

[0028] Figure 2 The hemolytic test results of Lactobacillus plantarum DACN962 (A) and Staphylococcus aureus (B).

[0029] Figure 3 This is the result of competitive inhibition of Helicobacter pylori adhesion by Lactobacillus plantarum DACN962.

[0030] Figure 4 The results of exclusion and inhibition of Helicobacter pylori adhesion by Lactobacillus plantarum DACN962.

[0031] Figure 5 These are the electron microscopic morphological results of competitive exclusion of Helicobacter pylori adhesion to GES-1 cells by Lactobacillus plantarum DACN962.

[0032] All experiments were repeated three times, and the results are expressed as mean ± standard deviation. IBM SPSS Statistics24 and GraphPadPrism 9.5 software were used for data analysis and plotting. Differences between groups were estimated by one-way analysis of variance, followed by post hoc multiple comparisons. Values ​​were statistically significant at p < 0.05. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0034] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0035] Example 1 Identification and in vitro adhesion assay of Lactobacillus plantarum DACN962

[0036] 1 Experimental Materials

[0037] The strains used in the experiment were from the strain resource bank of Suo Huayi Laboratory of Southwest University.

[0038] 2 Experimental methods

[0039] 2.1 Morphological observation of strains

[0040] The strains preserved in the strain resource bank were activated by culturing in MRS liquid medium at 37°C for 24 h. The bacterial liquid after two generations of activation was picked up with an inoculation loop and streaked on MRS solid medium plates for separation. After culturing at 37°C for 48 h, the colony morphology was observed and a single colony was picked for morphological observation by Gram staining.

[0041] 2.2 PCR amplification of 16S rDNA sequences

[0042] The DNA of the purified strain was extracted using a bacterial genomic DNA extraction kit. PCR amplification was performed using a 25 μL reaction system, and agarose gel electrophoresis was used for detection after the reaction. Qualified samples were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the sequencing results were analyzed for homology comparison using the BLAST program in NCBI.

[0043] 2.3 Evaluation of indirect adhesion of lactic acid bacteria

[0044] The self-aggregation ability, hydrophobicity and adhesion ability of different strains are strain-specific. Hydrophobicity and self-aggregation ability are considered to be one of the important factors affecting the two-phase reaction and are related to various adhesion phenomena.

[0045] 2.3.1 Self-aggregation assay

[0046] Lactobacillus was cultured in MRS liquid medium at 37°C for 24 h, the fermentation broth was centrifuged at 8000 r / min for 5 min to collect the bacterial precipitate, and the OD was adjusted with phosphate buffered saline (PBS). 600nm To about 0.50±0.05. Incubate at 37℃, measure the absorbance of the upper solution at 600nm at 0h, 6h, and 24h, and record them. Calculate the bacterial self-aggregation rate according to the following formula:

[0047]

[0048] Where: A0 is the absorbance value at 0h; A t is the absorbance value of th.

[0049] 2.3.2 Hydrophobicity determination

[0050] Lactobacillus was cultured in MRS liquid medium at 37°C for 24 h, the fermentation broth was centrifuged at 8000 r / min for 5 min to collect the bacterial precipitate, washed twice with PBS, and the bacterial solution concentration was adjusted to 10 8 CFU / mL. Take 2mL of the adjusted bacterial suspension and mix it with 2mL of xylene, and let it stand for 30 minutes. Take the lower aqueous phase, measure the absorbance value at a wavelength of 600nm, and record it. Calculate the hydrophobicity of the strain according to the following formula:

[0051]

[0052] Where: A0 is the absorbance of the water phase at a wavelength of 600 nm before mixing with xylene; A t The absorbance value of the aqueous phase at a wavelength of 600 nm after mixing with xylene and standing for 30 minutes

[0053] 2.4 Evaluation of direct adhesion of lactic acid bacteria

[0054] The lactic acid bacteria solution after activation for 3 generations was aspirated, and the cells were collected by centrifugation for 10 min, resuspended in PBS, and washed twice with PBS to adjust the OD 600nm To about 1.00. The washed cells were resuspended in an equal volume of 2×CFDA, SE labeling working solution, incubated at 37°C in the dark, centrifuged for 10 min, washed twice with PBS, and resuspended in serum-free and dual antibody-free RPMI-1640 medium.

[0055] GES-1 cells were inoculated in a 96-well plate for overnight culture, and fluorescently labeled lactic acid bacteria strains were added to the 96-well cell culture plate. After lactic acid bacteria and GES-1 cells were incubated at 37°C, they were washed twice with PBS to remove non-adherent lactic acid bacteria; after trypsin digestion for 2 minutes, the digestion reaction was terminated with RPMI-1640 medium. The relative fluorescence intensity (RFU) of the cell suspension was measured using a multifunctional microplate reader.

[0056] The relative adhesion rate was calculated according to the following formula:

[0057]

[0058] Where: RFU H is the fluorescence value of the Helicobacter pylori group (after removing the blank); RFU L is the fluorescence value of the lactic acid bacteria group (after removing the blank).

[0059] 2.5 Safety evaluation of strains

[0060] 2.5.1 Evaluation of hemolytic activity of strains

[0061] Lactobacillus plantarum DACN962 was activated in MRS medium at 37℃ for 24h, and hemolytic Staphylococcus aureus was activated in LB medium at 37℃ for 24h as a positive control for hemolysis. The culture solution of the strain was streaked on a Columbia blood agar medium plate containing 5% sterile defibrinated sheep blood using a sterilized inoculation loop, and cultured at 37℃ for 48h. The hemolysis zone was observed and photographed for record.

[0062] 2.5.2 Evaluation of strain antimicrobial susceptibility

[0063] Adjust the concentration of activated bacterial solution to 1×10 7 CFU / mL, take 100 μL in a 5mL sterile centrifuge tube, add 4mL MRS agar semi-solid medium (containing 0.75% agar) and mix well, and quickly pour it on the MRS agar solid plate. After solidification, use sterile tweezers to pick up 20 different drug-sensitive paper discs (Bickman Biology, BKMAM) and place them on the plate, with three drug-sensitive paper discs (triangular) on each plate. In order to minimize the error, the measuring instrument uses a vernier caliper, with the center of the inhibition zone as the center of the circle, and measures the distance between two points on the circumference of the circle passing through the center of the inhibition zone, which is used as the diameter of the inhibition zone, and the diameter of the inhibition zone is used to represent the size of the inhibition zone.

[0064] 2.6 Statistical analysis

[0065] Each experiment was performed three times in parallel. The experimental data were expressed as "mean ± standard deviation" and variance analysis was performed using SPSS24. P < 0.05 indicated statistical significance.

[0066] 3 Experimental results and analysis

[0067] 3.1 Identification of strains

[0068] After the 16S rDNA PCR amplification of strain DACN962 was successful, sequencing was completed by Bioengineering (Shanghai) Co., Ltd. After BLAST comparison, the sequence of the 16S rDNA gene amplification product of DACN962 (as shown in SEQ ID NO.1) was found to have a 100% similarity with the 16S rDNA sequence of Lactobacillus plantarum in the database, and strain DACN962 was identified as Lactobacillus plantarum. Figure 1 The colony morphology of Lactobacillus plantarum DACN962 grown on MRS solid plates and its morphology observed under a microscope after Gram staining.

[0069] 3.2 Results of indirect adhesion rate of strains

[0070] 3.2.1 Strain self-aggregation

[0071] Seven different lactobacilli with high self-aggregation rates were screened from our laboratory for comparison. As shown in Table 1, the 6h self-aggregation ability of the 7 different lactobacilli ranged from 25.84±2.26% to 70.70±3.53%, with significant differences (P<0.05), and the 24h self-aggregation ability of the 7 different lactobacilli was basically above 75%. Among the 4 plant lactobacilli, DACN962 had the highest 6h self-aggregation ability of 70.70±3.53% and 24h self-aggregation ability of 90.01±5.95%, and it had the strongest self-aggregation ability. Adherence to epithelial cells and subsequent colonization in the gastrointestinal tract is a characteristic that gives probiotics a huge advantage and helps them compete and proliferate effectively in the intestine. The ability to automatically aggregate is related to adhesion, and probiotics need to aggregate to form a sufficiently large biomass. Studies have shown that the self-aggregation ability of strains is positively correlated with their surface adhesion ability, so strains with higher self-aggregation ability represent strong adhesion ability.

[0072] Table 1 Self-aggregation ability of different lactobacilli

[0073]

[0074] 3.2.2 Strain hydrophobicity

[0075] As shown in Table 2, the surface hydrophobicity of the six different lactobacilli ranged from 0.40±0.30% to 8.47±0.13%, with significant differences (P<0.05). Among the four plant lactobacilli, the hydrophobicity of DACN962 was the highest at 8.47±0.13%. Hydrophobicity is considered to be one of the important factors affecting the two-phase reaction and is related to a variety of adhesion phenomena. Studies have shown that hydrophobicity determines the adhesion ability of bacteria, which is the key to whether probiotics can reproduce in the intestines of animals. The higher the hydrophobicity of the strain, the better its adhesion ability in the gastrointestinal tract. Among the several lactobacilli tested, DNCN962 has the best hydrophobicity.

[0076] Table 2 Hydrophobicity of different lactobacilli

[0077]

[0078] 3.3 Evaluation of hemolytic activity of strains

[0079] Detect whether Lactobacillus plantarum DACN962 is hemolytic. Figure 2 It can be observed that Lactobacillus plantarum DACN962 ( Figure 2 A) No transparent hemolytic ring was produced on the blood plate, indicating that the strain was non-hemolytic. On the contrary, the positive control hemolytic Staphylococcus aureus ( Figure 2 B) An obvious transparent hemolytic ring appeared, which preliminarily determined that Lactobacillus plantarum DACN962 was safe.

[0080] 3.4 Antibiotic resistance experiment

[0081] This experiment uses the drug sensitivity paper method to detect the sensitivity of Lactobacillus plantarum DACN962 to 20 antibiotics, which is the most widely used drug sensitivity test method in clinical practice. The drug sensitivity of probiotic strains to multiple antibiotics is judged by referring to the latest NCCLS standards provided by WHO. If a probiotic is sensitive to a certain antibiotic, it is represented by S; if it is resistant, it is represented by R; if it is intermediate, it is represented by I. By comparing the average value of the diameter of the inhibition zone produced by the resistance of Lactobacillus plantarum DACN962 to these 20 antibiotics determined in the experiment with the NCCLS drug sensitivity test standard, the drug sensitivity results of Lactobacillus plantarum DACN962 to these 20 antibiotics are shown in Table 3:

[0082] Table 3 Resistance results of Lactobacillus plantarum DACN962 to 20 antibiotics

[0083]

[0084]

[0085] Note: “—” indicates insensitive, S indicates sensitive, R indicates resistant, and I indicates moderately sensitive.

[0086] The standard for evaluating whether a probiotic can be safely used in food is whether it has drug resistance. The strain DACN962 has a certain degree of resistance to antibiotics such as lincomycin and gentamicin, and is sensitive to eight of the most commonly used antibiotics in clinical practice, including tetracycline, minocycline, doxycycline, penicillin, cefazolin, ceftriaxone, ampicillin, and piperacillin. It is moderately sensitive to cephalexin, ceftazidime, cefoperazone, cefuroxime sodium, erythromycin, etc., and there is no risk of transmitting resistance genes for commonly used antibiotics in clinical practice to intestinal microorganisms.

[0087] The deposit information of Lactobacillus plantarum DACN962 is as follows:

[0088] The strain DACN962 was sent to the China Center for Type Culture Collection (CCTCC) for preservation in July 2024. The address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The preservation date is July 3, 2024; the preservation number is CCTCC NO: M20241458; the classification name is: Lactobacillus plantarum DACN962 Lactiplantibacillus plantarum DACN962.

[0089] Example 2 Competition / exclusion inhibition of Hp adhesion and growth by Lactobacillus plantarum DACN962

[0090] 1 Experimental Materials

[0091] The experimental strain is Lactobacillus plantarum DNCN962, and its deposit number is CCTCC NO: M20241458.

[0092] Helicobacter pylori strain SS1 was purchased from Guangdong Microbiological Culture Collection Center.

[0093] 2 Experimental methods

[0094] 2.1 Strain competition / exclusion inhibition of Helicobacter pylori adhesion assay

[0095] The washed cells were resuspended in an equal volume of 2×CFDA, SE labeling working solution, incubated at 37°C in the dark, centrifuged for 10 minutes, washed twice with PBS, and resuspended in RPMI-1640 medium without serum and dual antibodies. The lactobacillus and labeled Helicobacter pylori suspensions were prepared in RPMI 1640 cell culture medium without serum and dual antibodies, and stored at 4°C for later use; GES-1 cells were plated in 24-well plates before inoculation of bacteria, and the experiment was carried out in four groups:

[0096] 1. Control group: GES-1 cells were cultured normally without treatment; 2. Helicobacter pylori SS1 infection group: Helicobacter pylori SS1 was added and incubated with cells; 3. Competition group: Lactobacillus resuspension and Helicobacter pylori SS1 were added and incubated at the same time. 4. Exclusion group: Lactobacillus resuspension was added and incubated first, and then Helicobacter pylori SS1 was added and incubated. After incubation for 2 hours, the non-adherent lactic acid bacteria were removed by washing twice with PBS; after digestion with trypsin, the digestion reaction was terminated with RPMI-1640 medium. The relative fluorescence intensity (RFU) of the cell suspension was measured with a multifunctional microplate reader.

[0097] The competitive adhesion rate was calculated according to the following formula:

[0098]

[0099] Wherein: A0 is the fluorescence value of the infection group (after removing the blank); A1 is the fluorescence value of the competition / exclusion group (after removing the blank).

[0100] 2.2 Determination of the cross-agglutination ability between Lactobacillus plantarum DACN962 and Helicobacter pylori

[0101] Lactobacillus was cultured in MRS liquid medium at 37°C for 24 h. H. pylori was activated for 2 generations on CAB plates and then collected and washed with PBS. The OD 600nm =0.50±0.05, bacterial concentration is about 10 7 -10 8CFU / mL, equal amounts of lactic acid bacteria and H. pylori were mixed, shaken thoroughly for 5 min, and cultured at 37°C. The absorbance of the upper solution at a wavelength of 600 nm was measured and recorded after 2 h.

[0102]

[0103] Where: A H A is the absorbance value of Helicobacter pylori group at a wavelength of 600 nm; L A is the absorbance value of the lactic acid bacteria group at a wavelength of 600 nm; mix It is the absorbance value at a wavelength of 600nm after the two bacteria are mixed.

[0104] 2.3 SEM electron microscopy of Lactobacillus plantarum DACN962 inhibiting Hp adhesion to GES-1 cells

[0105] First, place the sterilized coverslip Place in a 24-well plate, then dot GES-1 cells in the well plate (2×105 / well), add Lactobacillus resuspension and Helicobacter pylori SS1 (MOI=100:1) and incubate for 2 hours. After the culture is completed, remove the cover glass from the culture medium and gently wash it 3 times with sterile PBS to remove the unadhered strains. After washing, place the cover glass in 2.5% glutaraldehyde fixative at 4°C for more than 8 hours, remove the cover glass and wash it with PBS 3 to 5 times, and use gradient ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100% (v / v)) for graded dehydration. Finally, the glass slide is dried and sprayed with gold for scanning electron microscopy observation.

[0106] 2.4 Determination of the inhibition of Helicobacter pylori growth by Lactobacillus plantarum DACN962

[0107] The agar diffusion method was used to measure the antibacterial activity of the strain against Helicobacter pylori. MRS liquid culture medium was used as a blank control. 100 μL of Helicobacter pylori bacterial suspension was evenly spread on a Columbia blood agar plate without antibiotics, and 100 μL of the test liquid was added to each well (the test liquids were freeze-dried supernatant of lactic acid bacteria fermentation, lactic acid bacteria suspension and blank control). The plate with the fermentation liquid was placed at 37°C and cultured in a microaerobic environment for 72 to 96 hours. After the culture was completed, the diameter of the inhibition zone was measured with a vernier caliper.

[0108] The preparation method of DACN962 fermentation freeze-dried supernatant is as follows: take DACN962 bacteria and inoculate it into MRS liquid culture medium and culture it at 37°C for 18 hours. The fermentation broth is filtered through a filter membrane to obtain a cell-free fermentation supernatant. The cell-free fermentation supernatant is subjected to rotary evaporation and then freeze-dried to prepare a freeze-dried powder. The freeze-dried powder is added to sterile water to prepare a freeze-dried supernatant with a concentration of 400 mg / ml.

[0109] 3 Experimental results and analysis

[0110] 3.1 Results of competitive inhibition of Helicobacter pylori adhesion by Lactobacillus plantarum DACN962

[0111] The study showed that Lactobacillus DACN962 could inhibit the adhesion of Hp to gastric epithelial cells GES-1, and cFDA fluorescently labeled Hp was used to evaluate whether Lactobacillus could inhibit the colonization of Hp on gastric mucosal cells. Figure 3 It can be seen that the in vitro competitive adhesion rate of lactic acid bacteria in inhibiting Hp adhesion to GES-1 cells determined by our laboratory is between 15% and 54%, among which the in vitro competitive inhibition of Helicobacter pylori adhesion to GES-1 cells by Lactobacillus plantarum DACN962 can be as high as 53.55±1.54%, showing a good effect in competitively inhibiting Hp adhesion.

[0112] 3.2 Results of the exclusion inhibition assay of Helicobacter pylori adhesion by Lactobacillus plantarum DACN962

[0113] cFDA fluorescently labeled Hp was used to evaluate whether Lactobacillus could inhibit Hp colonization on gastric mucosal cells. Figure 4 It can be seen that the competitive adhesion rate of lactic acid bacteria in vitro exclusion inhibition of Hp adhesion to GES-1 cells determined in this laboratory is between 49% and 83%, among which the in vitro exclusion inhibition of Helicobacter pylori adhesion to GES-1 cells by Lactobacillus plantarum DACN962 can be as high as 82.98±2.37%, which has a good exclusion inhibition effect on Hp adhesion.

[0114] 3.3 Results of the coaggregation ability test between Lactobacillus plantarum DACN962 and Hp

[0115] The study selected lactobacilli from the strain collection of the research group for the in vitro coaggregation test of Hp. As shown in Table 4, except for a few strains that did not have coaggregation, the plant lactobacilli that were screened out had a high coaggregation with Helicobacter pylori, among which the coaggregation rate of 2h was mainly between 10% and 51%, the coaggregation rate of 4h could reach 39-70%, the coaggregation rate of 6h could reach 52-81%, and the coaggregation rate of 24h could reach up to 93.5%. The aggregation ability of probiotics and pathogens also showed strain specificity. Among the tested lactobacilli strains, only the coaggregation ability of plant lactobacilli species was evaluated this time. Among them, the coaggregation rate of plant lactobacilli DACN962 reached 51.38±6.12% at 2h and 93.50±0.41% at 24h, which is a representative strain with coaggregation effect.

[0116] Table 4 Results of the interactive agglutination ability of Lactobacillus plantarum and Hp

[0117]

[0118] 3.4 SEM results of Lactobacillus plantarum DACN962 inhibiting Hp adhesion to GES-1 cells

[0119] In order to study the morphological changes of Hp after interaction with gastric epithelial cells in vitro and the morphology of epithelial cells after intervention with lactic acid bacteria, GES-1 cells were tested by electron microscopy. Figure 5 It can be seen that the surface structure of normal cells not infected by Hp is intact and there is no bacterial adhesion. The surface of cells in the infected group is invaded by Hp, and the spirochetes adhere to the epithelial surface. In the lactobacillus intervention group, the adhesion of spirochetes Hp to cells is significantly reduced, and the rod-shaped bacteria adhere to the epithelial surface.

[0120] 3.5 Inhibition zone results of Lactobacillus plantarum DACN962 against Hp

[0121] The lactic acid bacteria screened in this laboratory were subjected to antibacterial tests one by one, and the lactic acid bacteria with the function of inhibiting Helicobacter pylori were screened by comparing the diameters of the antibacterial circles. This test finally screened out 7 lactic acid bacteria with good antibacterial effects, and their antibacterial results are shown in Table 5.

[0122] Table 5 Inhibition zone data of lactobacillus

[0123]

[0124] As shown in Table 5, the diameters of the inhibition zones of the freeze-dried supernatants of the seven lactobacilli are between 12 and 22 mm, and the diameters of the inhibition zones of the fermentation broth are between 10 and 15 mm. The diameter of the inhibition zone of the freeze-dried supernatant of Lactobacillus plantarum DACN962 can reach 22.69 ± 0.76 mm, and the diameter of the inhibition zone of the fermentation broth (fermented at 37 ° C for 18 h after inoculation, including bacteria) can reach 15.33 ± 0.82 mm, which has a good antibacterial effect.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described with reference to the preferred embodiments of the present invention, it should be understood by those skilled in the art that various changes may be made in form and details without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A strain of Lactobacillus plantarum DACN962, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20241458.

2. A bacterial agent, the active ingredient of which comprises the plant lactobacillus DACN962 or its fermentation liquid according to claim 1.

3. The bacterial agent according to claim 2, characterized in that: The bacterial agent is freeze-dried powder.

4. A method for preparing the bacterial agent according to claim 2 or 3, characterized in that: The lactobacillus plantarum DACN962 according to claim 1 is inoculated into a culture medium and cultured to obtain the product.

5. The preparation method according to claim 4, characterized in that: The preparation method of the fermentation liquid is as follows: after activating the Lactobacillus plantarum DACN962 strain, the strain is cultured in a liquid culture medium at 35-37° C. for 12-24 hours to obtain the fermentation liquid of Lactobacillus plantarum DACN962; preferably, the liquid culture medium is an MRS liquid culture medium; The preparation method of the freeze-dried powder comprises the following steps: filtering the prepared Lactobacillus plantarum DACN962 fermentation liquid through a filter membrane to obtain a cell-free fermentation supernatant, and performing rotary evaporation and freeze-drying on the cell-free fermentation supernatant to prepare the freeze-dried powder.

6. Use of the plant lactobacillus DACN962 according to claim 1 or the bacterial agent according to claim 2 in any of the following products: (1) Competitive / exclusion inhibition of Helicobacter pylori adhesion; (2) Prevent Helicobacter pylori infection; (3) Inhibit the growth of Helicobacter pylori.

7. Use of the Lactobacillus plantarum DACN962 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a medicament for preventing and treating diseases caused by Helicobacter pylori.

8. A product, characterized in that: It includes the plant lactobacillus DACN962 described in claim 1 or the bacterial agent described in claim 2.

9. The product according to claim 8, characterized in that: The product is selected from food, food additives, health products, feed, feed additives, reagents or medicines.

10. The product according to claim 8, characterized in that: The product is used for any of the following purposes: (1) Competition / exclusion inhibition of Helicobacter pylori adhesion; (2) Prevent Helicobacter pylori infection; (3) Inhibit the growth of Helicobacter pylori; (4) Used for preventing and treating diseases caused by Helicobacter pylori.

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