Probiotic composition for inhibiting helicobacter pylori and use thereof

Abstract

The present invention provides a probiotic composition for inhibiting Helicobacter pylori. Lacticaseibacillus rhamnosus was preserved in Guangdong Microbial Culture Collection Center on 11 April 2024, and the accession number is GDMCC NO: 64513. Lactiplantibacillusplantarum was preserved in Guangdong Microbial Culture Collection Center on 11 April 2024, and the accession number is GDMCC NO: 64514. The advantage is: peptidoglycans extracted from the Lacticaseibacillus rhamnosus and the Lactiplantibacillusplantarum can effectively inhibit Helicobacter pylori. The combination of peptidoglycans from the two strains exhibits an enhanced ability to inhibit Helicobacter pylori compared with the same concentration of peptidoglycan from a single strain.

Description

A probiotic composition for inhibiting helicobacter pylori and application thereof TECHNICAL FIELD

[0001] The present application relates to a probiotic composition for inhibiting helicobacter pylori and application thereof. BACKGROUND

[0002] Peptidoglycan, also known as mucopolysaccharide, cell wall. It is a multi-layer network macromolecular structure polymerized by double sugar units, tetrapeptide tail and peptide bridge. The heteropolysaccharide alternately connected by N-acetylglucosamine and N-acetylmuramic acid is cross-linked with different composition of peptides to form a macromolecule. Peptidoglycan is the main component of the cell wall of many bacteria. It is also a good inducer. The polysaccharide of lactic acid bacteria and the biosynthesis mode of peptidoglycan of different types of bacteria are different, which is the direct reason for the complex structure of peptidoglycan. The complex structure and the multiple functions of peptidoglycan are the research hotspots in the fields of drug development, biological signals and biological materials for a long time.

[0003] Helicobacter pylori is a gram-negative spiral bacillus found on gastric mucosa, grows in microaerobic environment, is positive for oxidase and catalase, has smooth cell wall and 1-5 flagella, and the latter is wrapped in sheath and the tip is spherical.

[0004] Helicobacter pylori is a microaerophilic bacterium that parasitizes in the gastric mucosa of human body. After infection, it can cause chronic gastritis, peptic ulcer, gastric mucosa-related lymphoma and even gastric cancer and other digestive tract diseases.

[0005] Helicobacter pylori is a gram-negative bacillus in the human body, which is spiral and S-shaped. The common transmission route is oral-oral or fecal-oral between people, and the main transmission modes include unhygienic eating habits, incomplete endoscopic disinfection in hospitals, etc. Most patients have no symptoms in the early stage of infection, and a small number of patients may have acute gastritis symptoms such as upper abdominal pain, abdominal distension, nausea, vomiting, loss of appetite, etc. With the progression of the disease, the typical symptoms of most patients are chronic upper abdominal pain, fullness, discomfort, acid reflux, belching, morning nausea, etc.

[0006] During the treatment process, due to the use of a large amount of antibiotics, gastrointestinal-related side effects may easily occur, which are generally manifested as abdominal pain, diarrhea, nausea, etc. Some patients may have allergic reactions to proton pump inhibitors and need to stop using them. In addition, long-term use may cause liver and kidney function abnormalities, bone marrow suppression, etc. Blood routine, blood biochemistry and other examinations need to be monitored during the medication process. Some patients may also have constipation, and the color of their stool may change, such as black stool, which is often related to the use of bismuth agents. It is necessary to rule out changes in disease conditions and digestive tract bleeding.

[0007] Some patients may have central nervous system side effects after using anti-Helicobacter pylori drugs, which can affect the central nervous system and cause central nervous system side effects, which are usually manifested as excessive anxiety, excitement, headache and other symptoms.

[0008] Since Helicobacter pylori is a persistent infection, it can still be infected by other carriers after recovery, so a safe, side-effect-free and long-term Helicobacter pylori inhibiting product is needed in life and medicine.

[0009] Patent: CN202011412221.1 A probiotic composition for inhibiting Helicobacter pylori and application. The present invention relates to a probiotic composition for inhibiting Helicobacter pylori and application, belonging to the technical field of food and drug. The probiotic composition comprises four strains of probiotics: Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri and Lactobacillus salivarius. The probiotic composition has a synergistic inhibitory effect on Helicobacter pylori, can significantly inhibit the growth of Helicobacter pylori, reduce the gene expression level of virulence factors and water solution, but does not involve peptidoglycan as the main bacteriostatic substance.

[0010] Patent: CN202111636673.2 Anti-Helicobacter pylori infection Lactobacillus plantarum and its application. The present invention relates to an anti-Helicobacter pylori infection Lactobacillus plantarum and its application, which inhibits the proliferation of Helicobacter pylori and reduces its adhesion to gastric epithelial cells, creating conditions for eradicating Helicobacter pylori. But it does not involve peptidoglycan as the main bacteriostatic substance.

[0011] Patent: CN202210639204.4 A screening method for functional probiotics based on inhibition of lipopolysaccharide production. The present invention relates to a screening method for functional probiotics based on inhibition of lipopolysaccharide production, which comprises testing the ability of candidate probiotics to inhibit the production of lipopolysaccharide, including screening for Helicobacter pylori lipopolysaccharide.

[0012] The above patents all inhibit Helicobacter pylori by probiotics, but the inhibitory components are still unknown and the inhibitory effect is not good SUMMARY

[0013] The present invention is directed to the above problems, and provides a probiotic composition for inhibiting Helicobacter pylori and its application.

[0014] The purpose of the invention is achieved by the following scheme: a probiotic composition for inhibiting Helicobacter pylori,

[0015] Lactobacillus rhamnosus INM3105, deposited in Guangdong Microbial Culture Collection Center on April 11, 2024, with the accession number GDMCC NO:64513;

[0016] Lactobacillus rhamnosus INM3106, deposited on April 11, 2024, in the Guangdong Microbial Culture Collection Center, with the accession number: GDMCC NO: 64514.

[0017] Further, the above-mentioned Lactobacillus rhamnosus and Lactobacillus plantarum are mixed at a ratio of 1:1.

[0018] Application of a probiotic combination for inhibiting Helicobacter pylori.

[0019] Advantages of the present application: the extracted peptidoglycan of Lactobacillus rhamnosus and Lactobacillus plantarum can effectively inhibit Helicobacter pylori. The peptidoglycan of the two strains in combination can further enhance the inhibitory ability on Helicobacter pylori compared to the same concentration of a single strain. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a diagram of the inhibition zone of NM2101 strain, NM2102 strain, NM2109 strain and blank group on Helicobacter pylori.

[0021] Figure 2 is a diagram of the inhibition of peptidoglycan of NM2101 strain, NM2102 strain and NM2109 strain on the adhesion of Helicobacter pylori to gastric mucosa epithelial cells.

[0022] Figure 3 is a diagram of the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis of the extracted peptidoglycan of strains NM2101 and NM2102.

[0023] Figure 4 is a diagram of the observation under a microscope after Gram staining of NM2101 strain.

[0024] Figure 5 is a diagram of the observation under a microscope after Gram staining of NM2102 strain.

[0025] Figure 6 is a diagram of the adhesion rate of strains NM2101 and NM2102 in combination to Helicobacter pylori to gastric mucosa epithelial cells.

[0026] Figure 7 is a diagram of the inhibition rate of strains NM2101 and NM2102 in combination (1:1) on Helicobacter pylori before and after inactivation. DETAILED DESCRIPTION

[0027] The present application is further described below in conjunction with specific embodiments:

[0028] In the following implementation examples, the experimental methods used are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0029] Example 1, with reference to Figures 1-7, a probiotic combination for inhibiting Helicobacter pylori,

[0030] Lactobacillus rhamnosus INM3105, deposited on April 11, 2024, in the Guangdong Microbial Culture Collection Center, with the accession number GDMCC NO: 64513;

[0031] Lactobacillus plantarum INM3106, deposited on April 11, 2024, in the Guangdong Microbial Culture Collection Center, with the accession number GDMCC NO: 64514.

[0032] A probiotic combination for inhibiting Helicobacter pylori, the Lactobacillus rhamnosus and the Lactobacillus plantarum are mixed at a ratio of 1:1.

[0033] Example 2, with reference to Figures 1-7, method for preparing Lactobacillus rhamnosus and Lactobacillus plantarum INM3106

[0034] 1. Treatment of raw materials

[0035] Yogurt was taken and diluted with sterile physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 concentration to prepare a bacterial diluent. 0.1 mL of each concentration of the diluent was uniformly coated on MRS solid medium containing 0.015% bromocresol purple, and cultured at 37°C for 48h. Single colonies with yellow color, raised colonies, smooth surface, and neat colony edges were selected, and the colonies were streaked on MRS solid medium for secondary purification, and cultured at 37°C for 48h. Single colonies were picked and preserved in glycerol, and named NM2101-NM2109 in order.

[0036] 2. Primary screening of strains

[0037] The 9 strains were inoculated into MRS liquid medium at a 1% inoculum, and cultured at 37°C for 24h. Then, they were inoculated into MRS medium at a 1% inoculum, and statically cultured at 37°C for 24h, with a final concentration of 10 6 cfu / mL.

[0038] The medium for screening Helicobacter pylori inhibition was prepared as follows (g / L): 5% sheep blood, special proteose peptone 23 g / L, soluble starch 1.0 g / L, sodium chloride 5.0 g / L, and agar 10.0 g / L. A puncher was used to make a 7 mm aperture, and 100 μL of the sample was added. The culture was incubated at 30°C for 48h. The results are shown in Figure 1. The Helicobacter pylori inhibition zone was measured using a vernier caliper, and the results are shown in Table 1.

[0039] Table 1. Results of strains inhibiting Helicobacter pylori

[0040] Strain name pH Total acid (g / 100g) Inhibition diameter (mm) NM2101 5.23 30.41 4.96 NM2102 4.21 66.51 3.80 NM2103 4.10 85.4- NM2104 5.03 42.7- NM2105 4.84 52.4- NM2106 4.78 58.8- NM2107 5.05 42.4- NM2108 4.92 50.3- NM2109 4.28 62.31 1.77

[0041] The strains NM2101, NM2104 and NM2109 with strong inhibition ability were selected for re-screening.

[0042] 3. Strain re-screening

[0043] The strains NM2101, NM2102 and NM2109 selected by preliminary screening were reactivated and cultured, and the peptidoglycan was extracted.

[0044] Peptidoglycan extraction: three strains were inoculated into 50 mL MRS medium at an inoculation amount of 1%, and cultured at 37°C for 24 h, then centrifuged (4°C, 8000 r / min) for 5 min, and the bacterial bodies were collected and washed twice with sterile deionized water. Then 10% trichloroacetic acid (TCA) was added to the bacterial bodies at a ratio of 1:10, shaken well, and then placed in boiling water at 100°C for 20 min, cooled, and centrifuged at 6000 r / min, 4°C for 10 min. The precipitate was washed with sterile deionized water, and 2 volumes of chloroform:methanol (1:2, V / V) were added, shaken for 6 h, and centrifuged at 6000 r / min, 4°C for 10 min. The precipitate was washed again and dissolved in 0.1 mol / L pH7.6 tris(hydroxymethyl) aminomethane hydrochloride buffer containing 3 mg / mL trypsin and 10 mg / mL neutral protease, 37°C, 150 r / min shaking bed overnight, then centrifuged at 8000 r / min for 20 min to collect the precipitate and wash it, then freeze-dried by freeze dryer to obtain powder-like peptidoglycan extract.

[0045] The calculation formula of the yield of peptidoglycan (%) is [peptidoglycan weight (mg) / collected bacterial body weight (mg)] x 100%

[0046] Peptidoglycan purity detection: 12.5% polyacrylamide separating gel and 5% concentrated gel were prepared; sample was mixed with 5x sodium dodecyl sulfate loading buffer at a ratio of 5:1, denatured in boiling water bath for 10 min, and cooled; electrophoresis was performed in an electrophoresis tank, 80 V for concentrated gel for 30 min, and 120 V for separating gel for 70 min; after electrophoresis, the gel was stained with coomassie brilliant blue R-250 for 2-3 h; after decolorization, imaging was performed by gel imaging system.

[0047] Gastric mucosa epithelial cell adhesion detection: gastric mucosa epithelial cells were taken from subcultured cells, cell suspension concentration was adjusted to 1´10 8 cfu / mL, inoculated into 12-well plates, 3 parallel for each strain, and equal concentration 2.0 (mg / mL) peptidoglycan was added, after 2 h reaction, the well plate was washed twice with buffer, and gram staining method was used to detect the number of H. pylori on gastric mucosa cells. The results are shown in Table 2.

[0048] Table 2 Peptidoglycan inhibits the adhesion rate of H. pylori to gastric mucosa epithelial cells

[0049] Strain name Peptidoglycan yield (%) Inhibition rate (%) NM2101 18.8±0.3 80±14.1 NM2102 29.5±0.1 60±12.4 NM2109 7.4±0.2 25±7.1

[0050] As can be seen from the results in Table 2, the peptidoglycan of NM2101 and NM2102 can effectively inhibit the adhesion of H. pylori to gastric mucosa epithelial cells, and the microscopic observation results are shown in Figure 2. Figure 3 shows the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis of the peptidoglycan extracted from strains NM2101 and NM2102, and the molecular weight of the peptidoglycan of the two strains is less than 10 KDa, and the relative band is single, indicating that the purity of the extracted peptidoglycan of the two strains is high.

[0051] As a re-screening target strain, the morphology of the two strains on solid MRS plate medium was observed, strain NM2101 was round-ended straight rod bacteria, the general width was less than 1.5 μm, gram-positive, and no spores were produced. It was facultative anaerobic, and the surface colony diameter was about 3 mm, raised, round, smooth surface, fine, and white in color. The microscopic observation results are shown in Figure 4.

[0052] Strain NM2102 was 0.9-1.2 μm x 3.0-8.0 μm, single, paired or short chain. It usually lacked flagella, but could move. It was gram-positive, and no spores were produced. It was facultative anaerobic, and the surface colony diameter was about 3 mm, raised, round, smooth surface, fine, and white in color, occasionally light yellow or dark yellow, and the microscopic observation results are shown in Figure 5.

[0053] The NM2101 screened above is subjected to 16S rDNA sequencing identification, and through comparative analysis, the 16S region sequence of the NM2101 strain of the application reaches 100.00% similarity with Lactobacillus rhamnosus, and it can be determined that the strain is Lactobacillus rhamnosus.

[0054] The NM2102 screened above is subjected to 16S rDNA sequencing identification, and through comparative analysis, the 16S region sequence of the NM2102 strain of the application reaches 99.93% similarity with Lactobacillus plantarum. It can be determined that the strain is Lactobacillus plantarum.

[0055] Both strains can be applied to the food fermentation industry, and are named Lactobacillus rhamnosus INM3105 and Lactobacillus plantarum INM3106,

[0056] Lactobacillus rhamnosus INM3105, on April 11, 2024, was preserved in the Guangdong Microbial Culture Collection Center, with the preservation number being GDMCC NO:64513;

[0057] Lactobacillus plantarum INM3106, on April 11, 2024, was preserved in the Guangdong Microbial Culture Collection Center, with the preservation number being GDMCC NO:64514.

[0058] Therefore, INM3105 and NM2101 refer to the same strain in this application, and INM3106 and NM2102 refer to the same strain.

[0059] Example 3, referring to Figure 6, different peptidoglycan concentrations of INM3105 and INM3106 for inhibiting H. pylori

[0060] According to the method of Example 2, peptidoglycan is extracted, and the concentration of peptidoglycan is compounded according to different proportions, and the final concentration is 2 mg / mL. NM3105 and INM3106 single bacterial body peptidoglycan are extracted as control bacteria, and the adsorption rate of H. pylori on gastric mucosa epithelial cells is detected, and the results are shown in Figure 6. The peptidoglycan of INM3105 and INM3106 of different concentrations has different inhibition rates on H. pylori.

[0061] As can be seen from the results of Figure 6, the peptidoglycan of INM3105 and INM3106 can be compounded, which can significantly increase the inhibition rate on H. pylori. Among them, when the mixing ratio of the peptidoglycan of INM3105 and INM3106 is 1:1, the inhibition rate is highest, reaching more than 95%, and compared with the mixing ratio of 1:1, increasing the content of peptidoglycan of a single strain significantly affects the inhibition rate, making the inhibition rate decrease. This shows that the mixing ratio of the peptidoglycan of INM3105 and INM3106 is 1:1, which is the best ratio.

[0062] Example 4, reference to Figure 7, the inhibition rate of H. pylori before and after inactivation of INM3105 mixed with INM3106 (1:1)

[0063] Since the extraction rate of single extracted peptidoglycan is low and not conducive to industrial application, this example uses the bacterial body method to compare the inhibition rate of H. pylori on gastric mucosa epithelial cell adsorption. Two strains of bacteria were inoculated into 50 mL of MRS medium at a 1% inoculation amount and cultured at 37°C for 24 h, then centrifuged (4°C, 8000 r / min) for 5 min, and the bacterial bodies were collected and washed twice with sterile deionized water. Both were mixed at 1:1 under the same OD 600 conditions, divided into two equal parts, and the number of colonies in each part was 1.0´10 8 CFU / mL, one part was inactivated at 60°C and the other part was not inactivated; the inhibition rate of H. pylori on gastric mucosa epithelial cell adsorption was detected.

[0064] As can be seen from the results of Figure 5, there is no significant difference in the inhibition rate of H. pylori on gastric mucosa epithelial cell adsorption before and after inactivation of INM3105 mixed with INM3106 (1:1), which shows that mixed bacteria of INM3105 and INM3106 can inhibit H. pylori under both live and sterilized conditions, providing a broader prospect for the application of its preparation.

[0065] Example 5, INM3105 mixed with INM3106 (1:1) in rice ball application

[0066] INM3105 and INM3106 (1:1) were mixed at a concentration of 10 8 CFU / mL to make rice balls, which were placed at 4°C for 3 days, and another group of NM2103 was inoculated as a control group. MRS medium was used for detection of the number of two probiotics, Lactobacillus plantarum was yellow, and Lactobacillus rhamnosus was white. The rice product was filtered out, and the inhibition rate of H. pylori on gastric mucosa epithelial cell adsorption was detected during fermentation according to the method in Example 1. At the same time, professional evaluators were invited to evaluate the flavor of the rice balls, and the results are recorded in Table 3.

[0067] Table 3 Results of probiotic bacteria, inhibition rate detection and sensory evaluation of rice balls

[0068]

[0069] Note: The sensory evaluation is scored on a scale of 0.1, with a total score of 10 points, and the comprehensive score is the sum of each item

[0070] As can be seen from the results of Table 3, the inoculation of 10 8INM3105 and INM3106 (1:1) at a concentration of 10

[0071] INM3105 and INM3106 (1:1) at a concentration of 10 8 CFU / mL were added to the whole grain flour, and another group was inoculated with NM2103 as a control group. The two probiotics were detected according to Example 5 during the preservation of the flour, and the whole grain bread was prepared according to the method of GB / T14612-2002.

[0072] The inhibition rate of the whole grain bread on the adhesion of H. pylori to gastric mucosal epithelial cells was detected according to the method of Example 1, and the flavor of the whole grain bread was evaluated by a professional evaluator. The results are shown in Table 4.

[0073] Table 4 Results of probiotic bacteria detection, inhibition rate detection and sensory evaluation of whole grain bread

[0074]

[0075] Note: The sensory evaluation is scored on a scale of 0.1, with a total score of 10. The overall score is the sum of the individual scores.

[0076] According to the results in Table 4, INM3105 and INM3106 (1:1) at a concentration of 10 8 CFU / mL were added to the whole grain flour, and another group was inoculated with NM2103 as a control group. The two probiotics were detected according to Example 5 during the preservation of the flour, and the whole grain bread was prepared according to the method of GB / T14612-2002.

[0077] INM3105 and INM3106 (1:1) at a concentration of 10 8 CFU / mL were added to the whole grain flour, and another group was inoculated with NM2103 as a control group. The two probiotics were detected according to Example 5 during the preservation of the flour, and the whole grain bread was prepared according to the method of GB / T14612-2002.

[0078] Table 5 Results of probiotic bacteria detection, inhibition rate detection and sensory evaluation of milk powder

[0079]

[0080] Note: Sensory evaluation, each assessment full score 10 points, 0.1 as the score interval, the comprehensive score is the sum of each item

[0081] From the results of Table 5, it can be seen that the inoculation of 10 8 CFU / mL INM3105 and INM3106 (1:1) to make milk powder can significantly increase the inhibition of H. pylori on gastric mucosal epithelial cell adsorption rate; but relative to the control group, the total number of probiotics INM3105 and INM3106 (1:1) added is significantly higher than that of the control group, which shows that the lactic acid bacteria in the control group will have obvious colony loss under dry conditions. But the two groups of data did not significantly improve the sensory evaluation of milk powder.

[0082] In summary, the above examples show that the combination of L. rhamnosus and P. plantarum provided by the present application can be used in rice balls, whole grain bread and milk powder. Without changing the existing process, it can significantly improve the inhibition of H. pylori on gastric mucosal epithelial cell adsorption rate, and also improve the aroma and taste of the product, significantly improving the product quality.

[0083] A probiotic combination for inhibiting H. pylori.

[0084] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the scope of the patent. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, several modifications and improvements belong to the protection scope of the present application. Therefore, the protection scope of the present application patent should be subject to the appended claims.

Claims

1. A probiotic composition for inhibiting Helicobacter pylori, characterized in that: Lactobacillus rhamnosus, deposited in the Guangdong Microbial Culture Collection Center on April 11, 2024, with a preservation number of GDMCC NO: 64513; Lactobacillus plantarum, deposited in the Guangdong Microbial Culture Collection Center on April 11, 2024, with a preservation number of GDMCC NO: 64514.

2. The probiotic composition for inhibiting H. pylori according to claim 1, wherein: Lactobacillus rhamnosus and Lactobacillus plantarum are mixed at a ratio of 1:

1. 3.The use of the probiotic composition of claim 1 for inhibiting Helicobacter pylori.

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