Phytobacterium plantarum KSFY202408 and application thereof

Abstract

The invention provides a plant lactobacillus KSFY202408 strain and application thereof, and belongs to the technical field of microorganisms. The preservation number of the plant lactobacillus KSFY202408 provided by the invention is CGMCC (China General Microbiological Culture Collection Center) No. 31102. The survival rate of the strain in artificial gastric juice with the pH value of 3.0 can reach 55.2%, the survival rate of the strain in artificial gastric juice with the pH value of 2.5 can reach 33.2%, the activity of a helicobacter pylori strain can be remarkably inhibited, formation of a helicobacter pylori thallus biological membrane can be inhibited, and the activity of helicobacter pylori metabolite urease can be reduced. Meanwhile, the bacterial strain can remarkably protect cell membrane integrity and cell viability of gastric mucosa epithelial cells, and has important significance on prevention or treatment of gastritis, gastric ulcer and other digestive tract diseases caused by helicobacter pylori.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus plantarum KSFY202408 and an application thereof. Background Art

[0002] Currently, people all over the world are infected with Helicobacter pylori ( Helicobacter pylori , H. pylori ) and the infection rate is high, with a detection rate of 70% to 90% in developing countries and 25% to 50% in developed countries. The infection rate of Helicobacter pylori is closely related to sanitary conditions, and children are more susceptible to infection and can survive in the human body for a long time. Its incidence rate increases with age and is related to socioeconomic conditions, race and other factors. Long-term infection with Helicobacter pylori can cause chronic gastritis and eventually develop into gastric ulcers and gastric cancer. The clinical symptoms caused by Helicobacter pylori infection are determined by several factors such as the type of Helicobacter pylori, the degree of inflammation and the colonization density of Helicobacter pylori. Its pathogenicity is closely related to its physiological characteristics of motility, acid resistance, adhesion, production of virulence factors, and production of antioxidant enzymes.

[0003] Probiotics have the functions of inhibiting the growth of pathogenic bacteria, adjusting the balance of microecology in the body, promoting digestion and absorption, regulating the immune system, and treating diseases caused by pathogenic bacteria, such as acute diarrheal infection and colitis. In clinical practice, a variety of probiotic preparations have been used to treat diseases caused by gastrointestinal pathogenic bacteria. Recently, many researchers have explored non-antibiotic preparations such as probiotics, antioxidants, and plant extracts (such as tea extracts and apple peel extracts) to improve or perfect the existing anti-Helicobacter pylori infection treatment regimen. Probiotics have significant advantages in improving the eradication rate of Helicobacter pylori in drug treatment and reducing the adverse reactions of antibiotics. Clinical trials of lactic acid bacteria combined with triple therapy for Helicobacter pylori infection have shown that it is better than triple therapy alone. Therefore, it is of great significance to develop probiotics that can significantly inhibit Helicobacter pylori. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a Lactobacillus plantarum KSFY202408 and its application. The Lactobacillus plantarum KSFY202408 provided by the present invention can survive in gastric juice, significantly inhibit Helicobacter pylori, and protect gastric mucosal epithelial cells, which is of great significance for preventing or treating gastritis, gastric ulcer and other digestive tract diseases caused by Helicobacter pylori.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: A strain of Lactobacillus plantarum KSFY202408, wherein the classification of the Lactobacillus plantarum KSFY202408 is named Lactiplantibacillus plantarumThe deposit number is CGMCC No.31102, and it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 27, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The present invention also provides an application of Lactobacillus plantarum KSFY202408 described in the above technical scheme in preparing a product for preventing or treating gastric mucosal damage, gastritis or gastric ulcer.

[0007] In the present invention, the Lactobacillus plantarum KSFY202408 can protect the integrity and cell activity of gastric mucosal epithelial cells.

[0008] The present invention also provides an application of the Lactobacillus plantarum KSFY202408 described in the above technical solution in preparing a product for inhibiting Helicobacter pylori.

[0009] In the present invention, the Lactobacillus plantarum KSFY202408 can inhibit the growth of Helicobacter pylori, destroy the formation of Helicobacter pylori biofilm, and reduce the urease activity of Helicobacter pylori.

[0010] The present invention also provides a Helicobacter pylori inhibitor, which contains live bacterial cells of Lactobacillus plantarum KSFY202408 and / or fermentation metabolites described in the above technical solution.

[0011] The present invention also provides a use of the Helicobacter pylori inhibitor described in the above technical solution in the preparation of a product for preventing or treating gastric mucosal damage, gastritis or gastric ulcer.

[0012] Beneficial technical effect: The present invention provides a plant lactobacillus KSFY202408 and its application. The preservation number of the plant lactobacillus KSFY202408 provided by the present invention is CGMCC No.31102. The survival rate of the strain in artificial gastric juice with a pH of 3.0 can reach 55.2%, and the survival rate in artificial gastric juice with a pH of 2.5 can reach 33.2%. It can significantly inhibit the activity of Helicobacter pylori strains, inhibit the formation of Helicobacter pylori bacterial biofilm, and reduce the activity of urease, a metabolite of Helicobacter pylori. At the same time, the strain can significantly protect the cell membrane integrity and cell activity of gastric mucosal epithelial cells, which is of great significance for preventing or treating digestive tract diseases such as gastritis and gastric ulcer caused by Helicobacter pylori. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Effects on gastric mucosal epithelial cell membrane damage; NC: blank control group; H. pylori: Helicobacter pylori; MRS-broth: negative control; PBS: negative control; KSFY202408: Helicobacter pylori + Lactobacillus plantarum KSFY202408; LGG: Helicobacter pylori + Lactobacillus rhamnosus; Figure 2 for the effects on gastric mucosal epithelial cells, where NC: blank control group; H. pylori : Helicobacter pylori; MRS-broth: negative control; PBS: negative control; KSFY202408: Helicobacter pylori + Lactobacillus plantarum KSFY202408; LGG: Helicobacter pylori + Lactobacillus rhamnosus. DETAILED DESCRIPTION

[0014] In order to better understand the present invention, the content of the present invention is further explained in conjunction with the following examples, but the content of the present invention is not limited to the following examples. The materials, reagents, etc. used in the examples and test examples of the present invention, unless otherwise specified, can be obtained from commercial channels; the methods used in the examples and test examples of the present invention, unless otherwise specified, are conventional methods.

[0015] Example 1 Isolation and Identification of Lactobacillus plantarum KSFY202408 1.1 Experimental Materials Naturally fermented yogurt was collected from Kashgar City, Xinjiang Uygur Autonomous Region. 40 mL of yogurt was taken out and placed in a sterile centrifuge tube, which was placed in a food sampling box and stored in a 4°C refrigerator in the laboratory for later use.

[0016] 1.2 Isolation and purification of strains Take 1 mL of yogurt sample and dilute it 10 times to 10 with sterile saline. -6 , then take 10 -4 , 10 -5 , 10 -6 100 μL of the bacterial solution of 3 gradients was spread on MRS-Agar plates and cultured at 37℃ for 24h~48h, and the colony morphology was observed and recorded. The colonies with different morphologies on the plate were picked for MRS-Agar plate streaking separation. After culturing at 37℃ for 48h, the single colonies with different morphologies on the plate were picked again for MRS-Agar plate streaking separation. This was repeated 2 to 3 times until a pure single colony with consistent morphology was obtained, which was numbered KSFY202408.

[0017] 1.3 Identification of strains The purified suspected target strain was inoculated into MRS-broth and cultured at 37℃ for 18h~24h. DNA was extracted using the Thermo Fisher Scientific (item number: K0512) bacterial genomic DNA extraction kit. The extracted DNA was numbered and stored in a -20℃ freezer for later use.

[0018] The extracted DNA was subjected to PCR amplification. The amplification system was: 1 μL of upstream primer 27F (5'-AGA GTT TGA TCCTGGCTC AG-3', SEQ ID NO.1), 1 μL of downstream primer 1495R (5'-CTA CGG CTA CCTTGT TAC GA-3', SEQ ID NO.2), 12.5 μL of 2×Taq plus Buffer, 1 μL of template DNA, and the system was supplemented to 25 μL with sterile dd H2O. Sterile ultrapure water was used to replace template DNA as a negative control. The amplification conditions were: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, 29 cycles, and 72°C for 5 min.

[0019] Take 5 μL of amplified product for agarose gel electrophoresis detection, the agarose concentration is 1.5%, the electrophoresis conditions are 110V, 45min. The successfully detected PCR product was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequenced sequence was compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0020] The 16S rDNA species analysis showed that the strain numbered KSFY202408 was Lactobacillus plantarum, which was classified as Lactiplantibacillus plantarum , was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on June 27, 2024, with the deposit number CGMCC No.31102, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0021] The sequencing result of the PCR amplification product of KSFY202408 is shown in SEQ ID NO.3:

[0022] Example 2 2.1 Materials and Equipment As shown in Table 1 and Table 2.

[0023] Table 1 Materials and reagents name Manufacturer Ethanol Chongqing Wansheng Chuandong Chemical Co., Ltd. MRS Beijing Road and Bridge COLUMBIA-agar blood agar Beijing Solable BHI Qingdao Hibo Biotech NaCl Beijing Solable Pepsin Beijing Solable Urease Beijing Solable Crystal violet stain Beijing Solable PBS buffer Beijing Solable Fetal bovine serum Evergreen Penicillin-Streptomycin Evergreen RPMI-1640 Beijing Solable Acridine Orange (AO)-EB fluorescent dye Coolaber Calcein-AM / PI live and dead cell fluorescent double staining solution Shanghai Yisheng Biotechnology Table 2 Instruments and equipment name Model / Article No. factory Constant temperature incubator IS-RDV1 CRYSTAL Three-gas incubator AW2009G ELECTROTEK Multifunctional ELISA reader LUX Thermo Fisher CO2 Incubator 160i Thermo Fisher Inverted fluorescence microscope Ix53 Olympus 2.1 Bacterial recovery The sample tubes of Lactobacillus plantarum KSFY202408 and Lactobacillus rhamnosus subsp. (ATCC53103, purchased from Beijing Biological Collection Center, Beijing, China) frozen at -80°C were dissolved in a 37°C water bath and inoculated into 5 mL of MRS-broth medium at a volume ratio of 10% under sterile conditions. The culture was kept at 37°C and 100 rpm for 24 h to 48 h, and the culture was stopped when the liquid became turbid. Helicobacter pylori ( H. pylori ) (ATCC 43504, purchased from Ningbo Mingzhou Biotechnology Co., Ltd., Zhejiang, China) was spread on COLUMBIA-agar blood plates containing 7% defibrinated sterile sheep blood and cultured in a 37°C tri-gas incubator (O2:CO2:N2=5:10:85) for 72 h; BHI-broth was used to prepare the Helicobacter pylori suspension according to a 2% inoculation amount and cultured in a 37°C tri-gas incubator.

[0024] The experiments in Sections 2.2 to 2.8 were repeated three times, and the results were analyzed statistically and for significance using GraphPad, One-way ANOVA (Tukey's multiple comparisons), and t-test.

[0025] 2.2 In vitro acid resistance test 1x10 8 CFU / mL of Lactobacillus plantarum KSFY202408 was inoculated into artificial gastric juice (NaCl 0.2%, pepsin 0.30%) at pH 3.0 and 2.5 after filtration and sterilization at a ratio of 10%, and then placed at 37°C with constant temperature shaking at 50rpm. Samples were taken after 1 hour, and the number of viable bacteria was determined by plate counting method to observe the acid resistance activity of Lactobacillus plantarum KSFY202408. Sterile water was used as negative control, and Lactobacillus rhamnosus was used as positive control.

[0026] The results are shown in Table 3. As can be seen from Table 3, the survival rate of Lactobacillus plantarum KSFY202408 in artificial gastric juice with pH = 3.0 for 3 hours was 55.2%, and the survival rate in artificial gastric juice with pH = 2.5 for 3 hours was 33.2%, which was slightly higher than that of Lactobacillus rhamnosus, but the difference was not significant, indicating that Lactobacillus plantarum KSFY202408 has the ability to survive in the stomach and may be used to treat gastric disorders.

[0027] Table 3 In vitro acid resistance test results 2.3 Sensitivity to Helicobacter pylori The KB method was used to detect the sensitivity of Lactobacillus plantarum KSFY202408 to Helicobacter pylori. 600nm ≈1 of Helicobacter pylori suspension, streak the surface of COLUMBIA-agar blood plate, and incubate it upside down in a 37℃ three-gas incubator. 600nm ≈1) was pasted on the surface of the plate, left at room temperature for 3-5 minutes, incubated upside down in a 37°C three-gas incubator for 72 hours, and the diameter of the inhibition zone (mm) was measured. Sterile MRS-broth medium and PBS were used as controls.

[0028] The results are shown in Table 4. As can be seen from Table 4, fresh MRS-broth medium as a negative control can also inhibit the growth of Helicobacter pylori, and the inhibition zone is about 8mm~9mm. PBS as a negative control has no ability to inhibit the growth of Helicobacter pylori, and no inhibition zone appears. Both Lactobacillus plantarum KSFY202408 and Lactobacillus rhamnosus can inhibit the growth of Helicobacter pylori, and the inhibition zone is about 14~17mm. Lactobacillus plantarum KSFY202408 (16.93±0.12) can achieve similar inhibition ability as Lactobacillus rhamnosus (16.67±0.46).

[0029] Table 4 Sensitivity to Helicobacter pylori Note: The diameter of the inhibition zone is expressed as x ̅±s, and the diameter of the drug-sensitive paper is 6 mm; *p<0.05 indicates a significant difference between the MRS-broth and the antibacterial zone.

[0030] 2.4 Effect on Helicobacter pylori urease activity The urease activity of the sample was determined by colorimetry. 600nmKSFY202408 with a volume ratio of ≈1 and Helicobacter pylori suspension were mixed at a ratio of 1:4, and BHI liquid culture medium without Helicobacter pylori was used as a control, and incubated in a three-gas incubator at 37°C for 72 hours. 50 μL of the mixed culture solution was added to 1 mL of GDH working solution and mixed, and the initial absorbance A1 at 20 seconds and the absorbance A2 at 5 minutes and 20 seconds were recorded at a wavelength of 340 nm, and ΔA=A1-A2 was calculated.

[0031] Urease activity is crucial for the survival of Helicobacter pylori in vitro at acidic pH and gastric mucosal colonization in animal models. The effect of Lactobacillus plantarum KSFY202408 on the activity of Helicobacter pylori was detected by detecting urease activity. The results are shown in Table 5. No obvious inhibitory effect was found in PBS and MRS-broth; after 72 hours of co-culture of Lactobacillus plantarum KSFY202408 and Lactobacillus rhamnosus with Helicobacter pylori, the activity of uromycin was reduced. From the data results, the urease activity of Lactobacillus plantarum KSFY202408 group and Lactobacillus rhamnosus group was significantly different from that of Helicobacter pylori group, while there was no significant difference between Lactobacillus plantarum KSFY202408 and Lactobacillus rhamnosus.

[0032] Table 5 Effect on urease activity Note: * p<0.05, ** p<0.01 indicates H. pylori Significant and extremely significant.

[0033] 2.5 Effects on Helicobacter pylori biofilm formation Helicobacter pylori biofilm construction: Add OD 600nm ≈1 Helicobacter pylori suspension 2mL / well, placed in a three-gas incubator at 37℃ and cultured for 24h.

[0034] The inhibitory effect on biofilm was detected by crystal violet staining. After 24 h of culture, the cells were washed three times with sterile PBS and the Helicobacter pylori that was not attached to the biofilm culture plate was discarded. 600nm ≈1 KSFY202408 bacterial suspension was added to the biofilm culture plate at a dose of 2 mL / well and cultured in a three-gas incubator at 37°C for 48 h. After 48 h, the culture medium was removed and washed three times with sterile PBS. 0.1% crystal violet stain was added, incubated for 15 min, rinsed with running water, and 1 mL of 95% ethanol was added to dissolve the biofilm attached to the slide, and the absorbance of the dissolved solution was detected at 595 nm.

[0035] The formation of biofilm helps Helicobacter pylori to develop resistance to antibiotics and immune defense mechanisms. Using static culture of Helicobacter pylori, it will attach and grow on the crawling plate to form plaques. The effect of Lactobacillus plantarum KSFY202408 on the total amount of Helicobacter pylori biofilm generation was determined by crystal violet staining. The results are shown in Table 6: Fresh MRS-broth as a negative control has an inhibitory effect on biofilm formation, with an inhibition rate of 20%, and PBS as a negative control has no inhibitory ability. The inhibition rates of Lactobacillus plantarum KSFY202408 and Lactobacillus rhamnosus on Helicobacter pylori biofilm formation were 57.3% and 60.5%, respectively.

[0036] Table 6 Effects on Helicobacter pylori biofilm formation Note: * p<0.05 indicates a significant difference from MRS-broth.

[0037] 2.6 Effects on GES-1 cell membrane integrity GES-1 cell culture: Gastric epithelial cells GES-1 were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The culture medium was RPMI-1640 culture medium containing 10% fetal bovine serum and 1% double antibody (penicillin-streptomycin). They were cultured in a 5% CO2 incubator and passaged every other day. The cells used in the experiment were in the logarithmic growth phase.

[0038] Add a concentration of 1×10 5 2 mL of GES-1 cell suspension with a concentration of 1 cell / mL was cultured for 24 h. The integrity of GES-1 cell membrane was observed by acridine orange (AO)-EB fluorescence staining.

[0039] Add Lactobacillus plantarum KSFY202408 and Helicobacter pylori suspension (OD 600nm ≈1, volume ratio was 1:4, KSFY202408 group), Helicobacter pylori + Lactobacillus rhamnosus (OD 600nm The volume ratio was 1:4 (all ≈ 1, LGG group) MRS-broth and PBS were incubated with GES-1 cells at 37°C and 5% CO2 for 6 h, the culture medium was discarded, the slides were rinsed 3 times with sterile PBS, fixed with 2.5% glutaraldehyde at room temperature for 2 h, rinsed 3 times with sterile PBS, stained with 0.01% (w / v) AO-EB at room temperature in the dark for 10 min, rinsed 3 times with sterile PBS, the slides were mounted and fixed on slides, and observed under a fluorescence microscope.

[0040] The results are as follows Figure 1 As shown by Figure 1It can be seen that 6 hours after Helicobacter pylori infected GES-1 cells, the cells showed elongation, deformation, vacuolation and a large number of hummingbird-like morphological changes, and the orange-red fluorescence color development effect was significant; MRS-broth had a partial inhibitory effect on the cell membrane integrity of GES-1 cells after Helicobacter pylori infection, and the orange-red fluorescence color development intensity decreased, which was related to the inhibitory effect of MRS-broth on the activity of Helicobacter pylori, while PBS had no inhibitory effect; Lactobacillus plantarum KSFY202408 and Lactobacillus rhamnosus could significantly alleviate the morphological changes of GES-1 cells caused by Helicobacter pylori after co-culture with Helicobacter pylori.

[0041] 2.7 Effect of Helicobacter pylori inhibition on GES-1 activity The cell survival rate was observed by Calcein-AM / PI live-dead cell fluorescence double staining. The cells with a fusion rate of 80% were added with Lactobacillus plantarum KSFY202408 and Helicobacter pylori suspension (1:4) according to the grouping, and incubated with GES-1 cells in a 37°C, 5% CO2 incubator for 6 hours. The culture medium was discarded, and the cells were washed 3 times with sterile 1×Assay Buffer. 1 mL of Calcein-AM / PI (10 μL: 5 μL) staining solution was added to the cell culture plate, and the cells were stained in the dark for 15 minutes at room temperature and observed under a fluorescence microscope.

[0042] The results are as follows Figure 2 As shown, after 6 hours of co-culture with GES-1, Helicobacter pylori can increase the mortality rate of GES-1 cells; MRS-broth, Lactobacillus plantarum KSFY202408 and Lactobacillus rhamnosus intervention can significantly reduce the mortality rate of GES-1 cells, with the effect of Lactobacillus plantarum KSFY202408 being more significant.

[0043] In summary, KSFY202408 of Lactobacillus plantarum and its metabolites can effectively inhibit the damage and pro-apoptosis effects of Helicobacter pylori on gastric mucosal epithelial cells by inhibiting the formation of Helicobacter pylori biofilm and reducing the activity of Helicobacter pylori, thereby effectively inhibiting the occurrence of Helicobacter pylori and protecting the function of gastric mucosal epithelial cells.

[0044] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus plantarum KSFY202408, It is characterized in that The classification of the plant lactobacillus KSFY202408 is named Lactiplantibacillus plantarum The deposit number is CGMCC No.31102, and it was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on June 27, 2024. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. Use of the Lactobacillus plantarum KSFY202408 according to claim 1 in preparing a product for preventing or treating gastric mucosal damage, gastritis or gastric ulcer.

3. The use according to claim 2, It is characterized in that The Lactobacillus plantarum KSFY202408 can protect the integrity and cell activity of gastric mucosal epithelial cells.

4. Use of the Lactobacillus plantarum KSFY202408 according to claim 1 in preparing a product for inhibiting Helicobacter pylori.

5. The use according to claim 4, It is characterized in that The Lactobacillus plantarum KSFY202408 can inhibit the growth of Helicobacter pylori, destroy the formation of Helicobacter pylori biofilm, and reduce the urease activity of Helicobacter pylori.

6. A Helicobacter pylori inhibitor, It is characterized in that The Helicobacter pylori inhibitor comprises live bacterial cells of Lactobacillus plantarum KSFY202408 described in claim 1, and / or fermentation metabolites.

7. Use of the Helicobacter pylori inhibitor according to claim 6 in the preparation of a product for preventing or treating gastric mucosal damage, gastritis or gastric ulcer.

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