A probiotic combination for inhibiting Helicobacter pylori and its application

Abstract

The present invention provides a probiotic combination for inhibiting Helicobacter pylori, Lactobacillus rhamnosus INM3105, which was deposited in Guangdong Microbial Culture Collection Center on April 11, 2024, with a deposit number of GDMCC NO: 64513. Lactobacillus plantarum INM3106 was deposited in Guangdong Microbial Culture Collection Center on April 11, 2024, with a deposit number of GDMCC NO: 64514. The advantages are: the peptidoglycan extracted from Lactobacillus rhamnosus and Lactobacillus plantarum can effectively inhibit Helicobacter pylori. The ability of the peptidoglycan ratio of the two strains to inhibit Helicobacter pylori is further improved than that of a single strain of the same concentration.

Description

Technical Field

[0001] The invention relates to a probiotic combination for inhibiting Helicobacter pylori and application thereof. Background Art

[0002] Peptidoglycan, also known as mucocomplex and murein, is a multilayered macromolecular structure composed of disaccharide units, tetrapeptide tails and peptide bridges. It is a macromolecule formed by cross-linking heteropolysaccharides alternately connected by N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAMA) with peptides of different compositions. Peptidoglycan is the main component of the cell walls of many bacteria. It is also a good inducer. The polysaccharides of lactic acid bacteria and the biosynthesis methods of peptidoglycans of different types of bacteria are also different, which is the direct reason for the complexity of the peptidoglycan structure. The complex structure and various functions of peptidoglycan are enduring research hotspots in the fields of drug development, biological signals and biomaterials.

[0003] Helicobacter pylori is a Gram-negative spiral bacterium found in the gastric mucosa. It grows in a microaerobic environment, is oxidase and catalase positive, has a smooth cell wall and 1-5 flagella, the latter of which are sheathed and have a spherical end.

[0004] Helicobacter pylori is a microaerophilic bacterium that parasitizes the gastric mucosa of the human body. Infection can cause digestive tract diseases such as chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoma, and even gastric cancer. Helicobacter pylori is a spiral, S-shaped Gram-negative bacillus in the human body. The common transmission route is oral-oral or fecal-oral between people. The main modes of transmission include unhygienic eating habits and endoscopes that are not thoroughly disinfected in hospitals. Most patients do not have any symptoms in the early stages of infection, and a small number of patients will experience symptoms of acute gastritis such as upper abdominal pain, bloating, nausea, vomiting, and loss of appetite. As the disease progresses, most patients have typical symptoms such as chronic upper abdominal pain, fullness, discomfort, acid reflux, belching, and morning nausea.

[0005] During the treatment process, the use of a large amount of antibiotics can cause gastrointestinal side effects, which can easily cause gastrointestinal discomfort symptoms, generally manifested as abdominal pain, diarrhea, nausea, etc. Some patients will be allergic to proton pump inhibitors and need to stop using them. In addition, long-term use may cause abnormal liver and kidney function, bone marrow suppression, etc., and blood routine and blood biochemistry tests need to be monitored during medication. Some patients may also experience constipation and changes in stool color, such as black stool, which are often related to the use of bismuth. It is also necessary to rule out changes in the condition and gastrointestinal bleeding.

[0006] Some patients may experience effects on the central nervous system after using anti-Helicobacter pylori drug treatment. Central nervous system side effects generally manifest as symptoms such as excessive anxiety, excitement, and headache.

[0007] Since Helicobacter pylori is a persistent infection and can still be infected by other carriers even after being cured, both in life and in medicine, there is a need for a product that is safe, has no side effects, and can suppress Helicobacter pylori for a long time.

[0008] Patent: CN202011412221.1 A probiotic composition and application for inhibiting Helicobacter pylori. This invention relates to a probiotic composition and application for inhibiting Helicobacter pylori, and belongs to the field of food and pharmaceutical technology. The probiotic composition of the present invention includes 4 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 levels of virulence factors CagA, VacA and Ure and aqueous solution, but does not involve peptidoglycan as the main antibacterial substance.

[0009] Patent: CN202111636673.2 A plant lactobacillus for resisting Helicobacter pylori infection and its application. This invention relates to a plant lactobacillus for resisting Helicobacter pylori infection and its application. The plant lactobacillus for resisting Helicobacter pylori infection inhibits the proliferation of Helicobacter pylori and reduces its adhesion to gastric epithelial cells, creating conditions for eradicating Helicobacter pylori. However, it does not involve peptidoglycan as the main antibacterial substance.

[0010] Patent: CN202210639204.4 A screening method for functional probiotics based on inhibiting lipopolysaccharide production. This invention relates to a screening method for functional probiotics based on inhibiting lipopolysaccharide production, the screening method comprising testing the ability of candidate probiotics to inhibit lipopolysaccharide production, including screening for inhibition of Helicobacter pylori lipopolysaccharide.

[0011] The above patents all use probiotics to inhibit Helicobacter pylori, but the inhibitory components are still unknown and the inhibitory effect is not good. Summary of the invention

[0012] In view of the above problems, the present invention provides a probiotic combination for inhibiting Helicobacter pylori and application thereof.

[0013] The invention aims to achieve the following objectives: a probiotic combination for inhibiting Helicobacter pylori,

[0014] Lacticaseibacillus rhamnosus INM3105 was deposited in Guangdong Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCC NO: 64513;

[0015] Lactiplantibacillus plantarum INM3106 was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCC NO:64514.

[0016] Furthermore, the above-mentioned Lactobacillus rhamnosus and Lactobacillus plantarum are mixed in a ratio of 1:1.

[0017] Application of a probiotic combination for inhibiting Helicobacter pylori.

[0018] The invention has the advantages that the peptidoglycan extracted from Lactobacillus rhamnosus and Lactobacillus plantarum can effectively inhibit Helicobacter pylori. The ability of the peptidoglycan of the two strains to inhibit Helicobacter pylori is further improved than that of a single strain with the same concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the inhibition zone diagram of Helicobacter pylori inhibition by NM2101 strain, NM2102 strain, NM2109 strain and blank group.

[0020] Figure 2 Figure 2 shows the inhibition of Helicobacter pylori adhesion to gastric mucosal epithelial cells by peptidoglycan from NM2101, NM2102 and NM2109 strains

[0021] Figure 3 SDS-PAGE results of peptidoglycan extracted from strains NM2101 and NM2102.

[0022] Figure 4 This is a picture of the NM2101 strain observed under a microscope after Gram staining.

[0023] Figure 5 This is a picture of the NM2102 strain observed under a microscope after Gram staining.

[0024] Figure 6 The combination of strains NM2101 and NM2102 inhibits the adsorption rate of Helicobacter pylori to gastric mucosal epithelial cells.

[0025] Figure 7 This is a graph showing the inhibition rate of Helicobacter pylori before and after the mixed inactivation of strains NM2101 and NM2102 (1:1). DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments:

[0027] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0028] Example 1, see attached Figure 1-7 , a probiotic combination that inhibits Helicobacter pylori,

[0029] Lactobacillus rhamnosus INM3105 was deposited in Guangdong Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCCNO:64513;

[0030] Lactiplantibacillus plantarum INM3106 was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCC NO:64514.

[0031] A probiotic combination for inhibiting Helicobacter pylori, wherein the above-mentioned Lactobacillus rhamnosus and Lactobacillus plantarum are mixed in a ratio of 1:1.

[0032] Example 2, see attached Figure 1-7 , preparation method of Lactobacillus rhamnosus and Lactobacillus plantarum INM3106

[0033] 1. Processing of raw materials

[0034] Take yogurt and dilute it to 10% with sterile saline. -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 concentration, made into bacterial dilutions, took 0.1 mL of each dilution and spread it evenly on an MRS solid culture medium plate containing 0.015% bromocresol purple, cultured at 37°C for 48 h, selected single colonies with yellow color, raised colonies, smooth surface and neat colony edges, streaked the colonies on MRS solid culture medium for secondary purification, cultured at 37°C for 48 h, picked single colonies for glycerol preservation, and named them NM2101 to NM2109 respectively.

[0035] 2. Preliminary screening of bacterial strains

[0036] The 9 strains were inoculated into MRS liquid culture medium at 1% inoculum and cultured at 37°C for 24 h. Then, the 9 strains were inoculated into MRS culture medium at 1% inoculum and cultured at 37°C for 24 h. The final cell density reached 10 6 cfu / mL.

[0037] Screening of Helicobacter pylori inhibition culture medium (g / L): Contains 5% sheep blood, 23g / L special peptone, 1.0g / L soluble starch, 5.0g / L sodium chloride, and 10.0g / L agar. Use a hole puncher to make a 7mm hole, add 100μL of sample, and culture at 30℃ for 48h. The results are as follows Figure 1 The Helicobacter pylori inhibition zone was detected using a vernier caliper. The results are shown in Table 1.

[0038] Table 1 Results of bacterial species inhibiting Helicobacter pylori

[0039]

[0040]

[0041] The strains NM2101, NM2104 and NM2109 with strong antibacterial ability were selected for rescreening.

[0042] 3. Strain rescreening

[0043] The lactic acid bacteria strains NM2101, NM2102 and NM2109 selected initially were reactivated and cultured to extract peptidoglycan.

[0044] Peptidoglycan extraction: The three strains were inoculated into 50 mL MRS medium at a 1% inoculation rate and cultured at 37°C for 24 h. The culture was centrifuged (4°C, 8000 r / min) for 5 min, and the cells were collected and washed twice with sterile deionized water. Then, 10% trichloroacetic acid (TCA) was added to the bacteria at a ratio of 1:10, and the mixture was shaken and stirred in 100°C boiling water for 20 min. After cooling, the mixture was centrifuged at 6000 r / min and 4°C for 10 min. The precipitate was washed with sterile deionized water, and then 2 volumes of chloroform:methanol (1:2, V / V) were added and shaken for 6 h. The mixture was centrifuged at 6000 r / min and 4°C for 10 min. The precipitate was washed again and dissolved in 0.1 mol / L pH 7.6 Tris-HCl buffer containing 3 mg / mL trypsin and 10 mg / mL neutral protease. The mixture was shaken on a shaker at 37°C and 150 r / min overnight, and then centrifuged at 8000 r / min for 20 min to collect the precipitate, wash it, and freeze-dry it in a freeze dryer to obtain a powdered peptidoglycan extract.

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

[0046] Peptidoglycan purity detection: prepare a polyacrylamide separation gel with a concentration of 12.5% ​​and a concentrated gel with a concentration of 5%; take the sample and mix it with 5×SDS loading buffer at a ratio of 5:1, denature it in a boiling water bath for 10 minutes, and cool it; perform electrophoresis in an electrophoresis tank, the concentrated gel is 80V, 30min; the separation gel is 120V, 70min; after the electrophoresis, the gel is stained with Coomassie Brilliant Blue R-250 for 2-3h; after decolorization, the gel is imaged using a gel imaging system.

[0047] Gastric mucosal epithelial cell adhesion test: Take the subcultured gastric mucosal epithelial cells and adjust the cell suspension concentration to 1×10 8 cfu / mL, inoculated into 12-well plates, each strain was replicated three times, and peptidoglycan of equal concentration of 2.0 (mg / mL) was added respectively. After reacting for 2 hours, the well plates were washed twice with buffer, and then the number of Helicobacter pylori on gastric mucosal cells was detected by Gram staining. The results are shown in Table 2.

[0048] Table 2 Peptidoglycan inhibits the adsorption rate of Helicobacter pylori to gastric mucosal epithelial cells

[0049] Strain name Peptidoglycan yield (%) Inhibition rate (%) NM2101 8.8±0.32 80±14.1 NM2102 9.5±0.14 60±12.4 NM2109 7.4±0.23 25±7.1

[0050] From the results in Table 2, it can be seen that the peptidoglycan of NM2101 and NM2102 can effectively inhibit the adhesion of Helicobacter pylori to gastric mucosal epithelial cells. The results of microscopic observation are as follows: Figure 2 . Figure 3 The results of SDS-PAGE on the peptidoglycan extracted from strains NM2101 and NM2102 show that the molecular weight of the peptidoglycan of the two strains is less than 10KDa, and the bands are relatively single, indicating that the peptidoglycan extracted from the two strains has a high purity.

[0051] As the target strain for rescreening, the morphology of the two strains on the solid MRS plate medium was observed. The strain NM2101 was a round-ended straight bacillus, generally less than 1.5 μm in width, Gram-positive, and non-spore-forming. It was facultatively anaerobic, with a surface colony diameter of about 3 mm, raised, round, smooth, dense, and white. The results of observation under the microscope are as follows Figure 4 shown.

[0052] Strain NM2102 is 0.9-1.2μm×3.0-8.0μm, single, paired or short chain. Usually lacks flagella, but can move. Gram-positive, non-spore-forming. Facultative anaerobic, surface colony diameter is about 3mm, raised, round, smooth, dense, white, occasionally light yellow or dark yellow, the results of observation under the microscope are as follows Figure 5 shown.

[0053] The NM2101 obtained by the above screening was subjected to 16SrDNA sequencing and identification. After comparative analysis, the 16S region sequence obtained from the strain NM2101 of the present invention and that of Lacticaseibacillus rhamnosus reached a similarity of 100.00%, and the strain was determined to be Lacticaseibacillus rhamnosus.

[0054] The NM2102 obtained by the above screening was subjected to 16SrDNA sequencing and identification, and the 16S region sequence obtained by comparative analysis of the NM2102 strain of the present invention and Lactiplantibacillus plantarum had a similarity of 99.93%, so the strain was confirmed to be Lactiplantibacillus plantarum.

[0055] Both strains can be used in the food fermentation industry and are named Lactobacillus rhamnosus INM3105 and Lactobacillus plantarum INM3106.

[0056] Lacticaseibacillus rhamnosus INM3105 was deposited in Guangdong Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCC NO: 64513;

[0057] Lactiplantibacillus plantarum INM3106 was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCC NO:64514.

[0058] Therefore, in this application, INM3105 and NM2101 refer to the same strain, and INM3106 and NM2102 refer to the same strain. The strains INM3105 and INM3106 of the present invention were identified by gene, and their gene sequences are as follows:

[0059] >INM3105

[0060]

[0061] >INM3106

[0062]

[0063] Example 3, see attached Figure 6 Different peptidoglycan concentrations of INM3105 and INM3106 have an inhibitory effect on Helicobacter pylori

[0064] Peptidoglycan was extracted according to the method of Example 2, and the concentration of peptidoglycan was compounded according to different ratios, with the final concentration of 2 mg / mL. Peptidoglycan extracted from single bacteria of NM3105 and INM3106 was used as a control bacteria to detect the inhibition of Helicobacter pylori adsorption rate on gastric mucosal epithelial cells. The results are as follows: Figure 6 As shown in the figure, the inhibitory rates of peptidoglycans of different concentrations of INM3105 and INM3106 on Helicobacter pylori are different.

[0065] Depend on Figure 6 The results show that the peptidoglycans of INM3105 and INM3106 can be compounded to significantly increase the inhibition rate of Helicobacter pylori. When the peptidoglycans of INM3105 and INM3106 were mixed at a ratio of 1:1, the inhibition rate reached more than 95%. Compared with the mixing ratio of 1:1, increasing the peptidoglycan content of a single strain significantly affected the inhibition rate, causing it to decrease. This shows that the peptidoglycan mixing ratio of INM3105 and INM3106 is 1:1, which is the best ratio.

[0066] Example 4, see attached Figure 7 , the inhibition rate of Helicobacter pylori before and after the mixed inactivation of INM3105 and INM3106 (1:1)

[0067] Since the extraction rate of peptidoglycan is low and it is not conducive to industrial application, the present embodiment adopts the bacterial method to compare the inhibition of Helicobacter pylori adsorption rate to gastric mucosal epithelial cells. Two strains of bacteria were inoculated with 1% inoculum into 50mL MRS medium and cultured at 37℃ for 24h. After centrifugation (4℃, 8000r / min) for 5min, the bacterial cells were collected and washed twice by centrifugation with sterile deionized water. Both were at the same OD 600 The mixture was mixed at a ratio of 1:1 under the same conditions and divided into two equal parts. The number of colonies in each part was 1.0×10 8 CFU / mL, one portion was inactivated at 60℃ and the other was not inactivated; the inhibition rate of Helicobacter pylori adsorption to gastric mucosal epithelial cells was detected.

[0068] Depend on Figure 5 The results showed that there was no significant difference in the adsorption rate of Helicobacter pylori on gastric mucosal epithelial cells before and after inactivation when INM3105 and INM3106 (1:1) were mixed. This indicates that the mixed bacteria of INM3105 and INM3106 can inhibit Helicobacter pylori both under live bacteria and sterilized conditions, providing a broader prospect for the application of its preparations.

[0069] Example 5, INM3105 and INM3106 (1:1) mixed in rice ball application

[0070] INM3105 and INM3106 (1:1) were mixed at a concentration of 10 8 CFU / mL was used to make rice balls, which were placed at 4°C for 3 days. Another group was inoculated with NM2103 as a control group. The number of two probiotics was detected using MRS medium. Lactobacillus plantarum was yellow and Lactobacillus rhamnosus was white. The rice products were filtered out, and the inhibition of Helicobacter pylori adsorption to gastric mucosal epithelial cells was detected according to the method in Example 1 during the fermentation process. At the same time, professional appraisers were asked to conduct sensory evaluation of the flavor of the rice balls, and the results were recorded in Table 3.

[0071] Table 3 Probiotics in rice balls, inhibition rate detection and sensory evaluation results

[0072]

[0073]

[0074] Note: Each sensory evaluation item has a full score of 10 points, with a scoring interval of 0.1, and the comprehensive score is the sum of the values ​​of each item.

[0075] From the results in Table 3, we can see that 10 8 CFU / mL of INM3105 and INM3106 (1:1) can not only significantly increase the rice balls' inhibition rate of Helicobacter pylori adsorption on gastric mucosal epithelial cells; in addition, it can also improve the sensory evaluation of rice balls, increase the aroma of rice balls, and enhance the flavor and taste of rice balls, making the rice balls fragrant, sweet and sour.

[0076] Example 6, INM3105 and INM3106 (1:1) mixed in whole grain bread application

[0077] INM3105 and INM3106 (1:1) were mixed at a concentration of 10 8 CFU / mL was used to make whole grain flour seeds, and another group was inoculated with NM2103 as a control group. During the storage of the flour seeds, the two probiotics were detected according to Example 5, and then bread was made according to the national standard GB / T14612-2002 method.

[0078] The method in Example 1 was used to detect the inhibition rate of Helicobacter pylori adsorption to gastric mucosal epithelial cells by whole-grain bread. Professional tasters were asked to conduct sensory evaluation on the flavor of the whole-grain bread. The results are recorded in Table 4.

[0079] Table 4 Probiotics in whole grain bread, inhibition rate detection and sensory evaluation results

[0080]

[0081]

[0082] Note: Each sensory evaluation item has a full score of 10 points, with a scoring interval of 0.1, and the comprehensive score is the sum of the values ​​of each item.

[0083] From the results in Table 4, it can be seen that the colony concentration of INM3105 and INM3106 (1:1) in whole grains is 10 8 CFU / mL in making whole grain sourdough, which can not only significantly increase the whole grain bread's inhibition rate of Helicobacter pylori adsorption to gastric mucosal epithelial cells, but also reduce the inhibition rate relative to Example 5, which may be due to the excessively high baking temperature during the bread making process, resulting in the inhibition rate being affected; in addition, it can also improve the sensory evaluation of whole grain bread, increase the aroma of whole grain bread, and improve the flavor and taste of whole grain bread, making the whole grain bread fragrant, sweet and sour.

[0084] Example 7, INM3105 and INM3106 (1:1) mixed in milk powder

[0085] INM3105 and INM3106 (1:1) were mixed at a concentration of 10 8 CFU / mL was added to the milk powder, another group of NM2103 was inoculated as the control group, and the 37°C accelerated test was used for verification. During the storage of the milk powder, the two probiotics were detected according to Example 5. And they were mixed according to the number of probiotics, and the number of probiotics used to make the milk powder was used to detect the adsorption rate of Helicobacter pylori on gastric mucosal epithelial cells by the method in Example 1. At the same time, professional appraisers were invited to conduct sensory evaluation on the flavor of the milk powder, and the results were recorded in Table 5.

[0086] Table 5 Probiotics in milk powder, inhibition rate detection and sensory evaluation results

[0087]

[0088]

[0089] Note: Each sensory evaluation item has a full score of 10 points, with a scoring interval of 0.1, and the comprehensive score is the sum of the values ​​of each item.

[0090] The results in Table 5 show that in whole grains, 10 8CFU / mL INM3105 and INM3106 (1:1) can significantly increase the rate of milk powder inhibiting the adsorption of Helicobacter pylori to gastric mucosal epithelial cells; but compared with the control group, the total bacterial count of the probiotics INM3105 and INM3106 (1:1) was significantly higher than that of the control group, indicating that the lactic acid bacteria in the control group would have obvious colony loss under dry conditions. However, the two groups of data did not significantly improve the sensory evaluation of milk powder.

[0091] Based on the results of the above embodiments, the combination of Lactobacillus rhamnosus and Lactiplantibacillus plantarum provided by the present invention is applied to rice ball making, whole grain bread and milk powder. On the basis of not changing the existing process, the inhibition rate of Helicobacter pylori adsorption on gastric mucosal epithelial cells of the relevant products can be significantly improved, and the aroma and taste of the product can be improved, thereby significantly improving the product quality.

[0092] Application of a probiotic combination for inhibiting Helicobacter pylori.

[0093] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

[0094]

[0095]

[0096]

[0097]

Claims

1. A probiotic combination for inhibiting Helicobacter pylori, characterized in that: Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus )INM3105, deposited in Guangdong Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCC NO:64513; Lactobacillus plantarum ( Lactiplantibacillus plantarum ) INM3106 was deposited in Guangdong Provincial Microbiological Culture Collection Center on April 11, 2024, with the deposit number: GDMCC NO:64514.

2. The probiotic combination for inhibiting Helicobacter pylori according to claim 1, characterized in that: The Lactobacillus rhamnosus and Lactobacillus plantarum are mixed in a ratio of 1:1.

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