Lactobacillus rhamnosus and Helicobacter pylori-inhibiting compositions

Abstract

This application discloses Lactobacillus rhamnosus and a composition for inhibiting Helicobacter pylori, which comprises the Lactobacillus rhamnosus. The composition further comprises hyaluronic acid or a salt thereof. The composition can significantly reduce the colonization rate of Helicobacter pylori in the stomach, alleviate the inflammatory response and gastrointestinal symptoms (GSRS score) caused by H. pylori infection, increase the negative conversion rate and efficacy in H. pylori-infected patients, increase the abundance of beneficial bacteria in the patient's intestines, and improve the patient's gastrointestinal health.

Description

[Technical Field]

[0001] The present application relates to the technical field of food and health foods, and in particular to compositions that inhibit Lactobacillus rhamnosus and Helicobacter pylori. [Background technology]

[0002] Helicobacter pylori (H. pylori) is a microaerophilic, spiral-shaped, Gram-negative bacillus. H. pylori adheres to and colonizes the gastric mucosa via adhesins, invading the body's defense system and causing gastrointestinal diseases such as gastritis, as well as non-digestive diseases such as ischemic cardiovascular disease, cerebrovascular disease, and cerebral hemorrhage, through the direct action of its toxins and the indirect action of inducing an inflammatory response. More importantly, H. pylori infection induces the Correa cascade, leading to the progression of gastric lesions from nonatrophic gastritis to atrophic gastritis, intestinal metaplasia, and, in severe cases, gastric cancer. Because H. pylori does not heal spontaneously and is extremely dangerous, it is classified as a Class I carcinogen by the International Agency for Research on Cancer. Antibiotic therapy is a viable treatment option for patients with symptomatic infection. However, due to the increasing global antibiotic resistance and the frequent occurrence of other side effects, H. pylori eradication rates continue to decline.

[0003] In recent years, new approaches to combat H. pylori using non-antibiotics, such as probiotics, prebiotics, plant extracts, bioactive proteins, and polysaccharides, have been explored to improve conventional treatments and provide new effective treatments. Probiotics have been widely studied due to their excellent biosafety, resistance to harsh gastrointestinal environments, and antagonistic activity against H. pylori. Currently, the most widely studied anti-H. pylori probiotic strains include Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus bulgaricus, and bifidobacteria. Probiotics play an important role in the auxiliary prevention of gastrointestinal diseases, minimize the use of antibiotics and their side effects as auxiliary therapy, strengthen the mucosal barrier function, reduce inflammatory responses, and improve the clinical symptoms and compliance of patients during treatment. They can directly or indirectly improve the eradication rate of H. pylori, which is of great significance for the prevention and treatment of H. pylori and related diseases.

[0004] Chinese Patent CN108741090A discloses a complex probiotic food for inhibiting H. pylori, including prebiotics, bifidobacteria, Lactobacillus acidophilus, Lactobacillus paracasei, and Lactobacillus rhamnosus, which can be used to prevent H. pylori infection. Patent WO2021 / 238890A1 discloses Lactobacillus rhamnosus, which inhibits H. pylori and has an H. pylori removal rate of 61.54%. Patent WO2004 / 031368 discloses a Lactobacillus reuteri strain for treating or preventing H. pylori-associated inflammation, and Patent WO2013027087A1 discloses a Lactobacillus reuteri strain that does not produce reuterin but can inhibit H. pylori growth in vitro. Chinese Patent CN102174450A relates to an acid-resistant Lactobacillus plantarum that inhibits the growth and urease activity of H. pylori and has the effect of preventing or alleviating H. pylori infection in mice. Patent WO2020 / 083983A1 relates to a Lactobacillus acidophilus that can synergize with antibiotics to kill H. pylori. However, neither of them reported the removal rate of H. pylori.

[0005] [DISCLOSURE OF THE INVENTION] Chinese Patent ZL2020113384895 is the applicant's previous research results, and discloses a composition containing hyaluronic acid and its salts for anti-H. pylori gastrointestinal infection activity, with a molecular weight range of 800-2000 kDa, preferably 1000-2000 kDa. Animal experiments were mainly used to verify the effectiveness, including the effect of HA on the amount of H. pylori colonization in the mouse stomach, but the H. pylori elimination rate was not taken into consideration.

[0006] In the prior art, there has been no research on the synergistic effect of hyaluronic acid and probiotics against H. pylori, and anti-H. pylori probiotics and hyaluronic acid have mainly been screened and verified through in vitro antibacterial tests, with little verification of anti-H. pylori efficacy through animal or human experiments, and a lack of data on the colonization and elimination rates of H. pylori. In light of this, the present application proposes a combination of hyaluronic acid and probiotics, and verifies the anti-H. pylori effect through animal and human experiments.

[0007] Specifically, this application adopts the following technical solutions: 1. Lactobacillus rhamnosus, deposited at the Guangdong Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO. 62419. 2. The Lactobacillus rhamnosus according to Item 1, whose 16s rRNA gene sequence is set forth in SEQ ID NO: 1. 3. Lactobacillus rhamnosus according to paragraph 1, for use in inhibiting Helicobacter pylori. 4. A composition for inhibiting Helicobacter pylori, comprising hyaluronic acid or a salt thereof and Lactobacillus rhamnosus. 5. The composition according to Item 4, wherein the salt of hyaluronic acid is one or more of sodium salt, potassium salt, magnesium salt, calcium salt, zinc salt, and bismuth salt of hyaluronic acid, and preferably sodium salt of hyaluronic acid. 6. The composition according to item 4 or 5, wherein the average molecular weight of the hyaluronic acid or its salt is 1200 to 2000 kDa, preferably 1400 to 1800 kDa. 7. The composition is capable of providing 80 mg / day or more of hyaluronic acid or a salt thereof; or The viable cell count of the Lactobacillus rhamnosus was 1 x 10 8 cfu / day / or more, Preferably, the composition is capable of providing 100 mg / day or more of hyaluronic acid or a salt thereof; or The viable cell count of the Lactobacillus rhamnosus was 1 x 10 9 cfu / day / or more, More preferably, the dosage of the hyaluronic acid or its salt is 100 mg / day / person or more; or The dose of Lactobacillus rhamnosus is such that the viable cell count is 1 x 10 10 Item 7. The composition according to any one of Items 4 to 6, wherein the Lactobacillus rhamnosus is present in an amount of at least cfu / day / day. 8. The food product further contains an additive, and preferably the additive is inulin, galactooligosaccharide, isomaltooligosaccharide, maltodextrin, erythritol, xylose, arabinose, rhamnose, galactose, fucose, mannose, fructose, sorbose, or glutamic acid. Item 8. The composition according to any one of Items 4 to 7, comprising one or more of cocoa, glycerol, galactitol, sorbitol, xylitol, mannitol, lactose, maltitol, lactitol, sucrose, trehalose, raffinose, stachyose, oligofructose, oligoxylose, mannooligosaccharides, β-glucan, hydroxyethyl starch, indigestible dextrin, whey protein, collagen, skim milk, pectin, and gelatin. 9. The composition according to any one of items 4 to 8, wherein the composition can be in the form of a pill, granule, powder, tablet, capsule, or emulsion. 10. The composition according to any one of Items 4 to 9, wherein the Lactobacillus rhamnosus is the Lactobacillus rhamnosus according to any one of Items 1 to 3. 11. Use of Lactobacillus rhamnosus according to any one of Items 1 to 3 or a composition according to any one of Items 4 to 10 in the preparation of a food or health food that inhibits Helicobacter pylori. 12. Use of Lactobacillus rhamnosus in combination with hyaluronic acid or a salt thereof in the preparation of a food or health food that inhibits Helicobacter pylori. 13. The dosage of the hyaluronic acid or its salt is 80 mg / day / person or more, or The dose of Lactobacillus rhamnosus is 1 x 10 viable cells. 8 cfu / day / or more, Preferably, the dosage of the hyaluronic acid or its salt is 100 mg / day / person or more; or The dose of Lactobacillus rhamnosus is 1 x 10 viable cells. 9 cfu / day / or more, More preferably, the dosage of the hyaluronic acid or its salt is 100 mg / day / person or more; or The dose of Lactobacillus rhamnosus is 1 x 10 viable cells. 10 The use according to paragraph 12, wherein the Lactobacillus rhamnosus has a concentration of cfu / day / or more. 14. The use according to item 12 or 13, wherein the salt of hyaluronic acid is one or more of sodium salt, potassium salt, magnesium salt, calcium salt, zinc salt, and bismuth salt of hyaluronic acid. 15. The use according to any one of items 12 to 14, wherein the average molecular weight of the hyaluronic acid or its salt is 1200 to 2000 kDa, more preferably 1400 to 1800 kDa. 16. The use according to any one of items 11 to 15, wherein the Lactobacillus rhamnosus is the Lactobacillus rhamnosus according to any one of items 1 to 3.

[0008] [Effects of the invention] 1. Oral administration of the composition of hyaluronic acid or its salt and Lactobacillus rhamnosus provided by the present application significantly reduces the colonization rate of Helicobacter pylori in the stomach to normal levels, significantly improves the inflammatory response of the gastric mucosa caused by H. pylori infection and the gastrointestinal symptoms of the subjects (GSRS score), with the negative conversion rate and efficacy rate of H. pylori-infected patients reaching 70.59% and 82.35%, respectively, and also increases the abundance of beneficial intestinal flora, helping to improve the gastrointestinal health of patients. 2. The hyaluronic acid and Lactobacillus rhamnosus composition provided in this application provides a new and effective treatment for the prevention of H. pylori infection in susceptible populations, the prevention of recurrence in patients after H. pylori eradication, and the treatment of patients with H. pylori infection who do not have clinical symptoms. 3. The composition of hyaluronic acid or its salt and Lactobacillus rhamnosus provided in the present application, which synergistically inhibits H. pylori, significantly reduces the colonization rate of Helicobacter pylori in the stomach and reduces the inflammatory response and gastrointestinal symptoms (GSRS) caused by H. pylori infection. score), increase the negative conversion rate and efficacy rate of H. pylori-infected patients, increase the abundance of beneficial bacteria in the patient's intestines, and improve the patient's gastrointestinal health. [Brief explanation of the drawings]

[0009] The accompanying drawings are used for better understanding of the present application and shall not constitute undue limitations to the present application. [Figure 1] This figure shows the effects of various treatments on the amount of H. pylori colonization in the mouse stomach. In the figure, * indicates a significant difference (p<0.05) between the intervention group and the model group, ** indicates a significant difference (p<0.01) between the intervention group and the model group, *** indicates a highly significant difference (p<0.001) between the intervention group and the model group, & indicates a significant difference (p<0.05) between the HA group, CCFM1259 group, and the combination group, and ## indicates a significant difference (p<0.01) between the blank group and the model group. [Figure 2] FIG. 1 shows the results of HE staining (200×) of mouse gastric mucosa (A: blank group, B: model group, C: HA group, D: CCFM1259 group, E: HA+CCFM1259 combined group). [Figure 3] This figure shows the effect of the combined use of different molecular weight HA and Lactobacillus rhamnosus on the amount of H. pylori colonization in mouse stomachs. In the figure, * indicates a significant difference (p<0.05) between the intervention group and the model group, ** indicates a significant difference (p<0.01) between the intervention group and the model group, *** indicates a highly significant difference (p<0.001) between the intervention group and the model group, and ## indicates a significant difference (p<0.01) between the blank group and the model group. [Figure 4] FIG. 1 shows the change in GSRS after intervention in each group (* indicates a significant difference compared to before intervention; p<0.05). [Figure 5] Figure 1 shows the results of the subjects' gut microbiota at the phylum level (A) and genus level (B). DETAILED DESCRIPTION OF THE INVENTION

[0010]

[0033] Exemplary embodiments of the present application will be described below. These embodiments include various details of the embodiments of the present application for ease of understanding, and should be considered as examples only. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, in the following description, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted.

[0011] This application provides a Lactobacillus rhamnosus strain deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO. 62419, deposit address Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province, China, postal code 510070, date of deposit May 17, 2022. The name and annotated identifying characteristics of the biological material are: Lactobacillus rhamnosus CCFM1259.

[0012] The term "Lactobacillus rhamnosus" conforms to the general definition in the art. It belongs to the Lactobacillus genus, is one of the normal bacterial flora of the human body, and is present in the oral cavity and intestines, mainly in the intestines. Lactobacillus rhamnosus is an anaerobic, acid-resistant, non-spore-forming probiotic, and is also a Gram-positive bacterium, as it is purple in Gram staining. The main effects of Lactobacillus rhamnosus include strengthening the gastrointestinal mucosal barrier, regulating the body's immune system, antagonizing pathogenic bacteria, promoting digestion, lowering blood lipids, and protecting the liver. It is a non-toxic, side-effect-free probiotic. Its functional characteristics mainly include regulating the intestinal flora, preventing and treating diarrhea, and excreting toxins. and strengthening the body's immune system.

[0013] The Lactobacillus rhamnosus provided in this application is inoculated onto an MRS solid plate and incubated at 37°C for 48 hours. The colonies are round, have clean edges, a smooth and convex surface, a uniform texture, and are milky white in color.

[0014]

[0015] The Lactobacillus rhamnosus provided in the present application can be used to inhibit Helicobacter pylori.

[0016] The present application further provides a composition for inhibiting Helicobacter pylori, comprising hyaluronic acid or a salt thereof and Lactobacillus rhamnosus.

[0017] The viable cell count of the Lactobacillus rhamnosus was 1 x 10 8 CFU / g or 1 x 10 8 CFU / ml or more, and preferably the viable cell count of the Lactobacillus rhamnosus is 1×10 9 CFU / g or 1 x 10 9 CFU / ml or more, and more preferably, the viable cell count of the Lactobacillus rhamnosus is 1×10 10 CFU / g or 1 x 10 10 CFU / ml or more, and more preferably, the viable cell count of the Lactobacillus rhamnosus is 1×10 11 CFU / g or 1 x 10 11 CFU / ml or more.

[0018] The term "hyaluronic acid" conforms to the common definition in the art and refers to a biopolymeric material composed of linearly linked repeating units of N-acetyl-D-glucosamine and D-glucuronic acid, also known as hyaluronan or hyaluronic acid. Hyaluronic acid is an acidic mucopolysaccharide whose unique molecular structure and physical and chemical properties enable it to perform many important functions in the body. The term "hyaluronic acid" or its salts includes hyaluronic acid itself, its salts, or combinations thereof. Examples of hyaluronic acid salts include, but are not limited to, inorganic salts such as sodium hyaluronate, potassium hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc hyaluronate, bismuth hyaluronate, and cobalt hyaluronate, and organic salts such as tetrabutylammonium hyaluronate. In this application, hyaluronic acid itself or its salts may be used alone, or two or more types of hyaluronic acid or their salts may be used in combination. For example, the composition may contain hyaluronic acid salt type 1, hyaluronic acid salt type 2, hyaluronic acid salt type 3, hyaluronic acid salt type 4, hyaluronic acid salt type 5, or hyaluronic acid salt type 6. The salt is selected from sodium salts, potassium salts, magnesium salts, calcium salts, zinc salts, and bismuth salts.

[0019] Although the hyaluronic acid or its salt described in this application is not limited, in a preferred embodiment, the hyaluronic acid salt is a water-soluble salt of hyaluronic acid, more preferably sodium hyaluronate, zinc hyaluronate, magnesium hyaluronate, or potassium hyaluronate. In a specific embodiment, the hyaluronic acid salt is sodium hyaluronate.

[0020] In a preferred embodiment, the average molecular weight of the sodium hyaluronate is 1200 to 2000 kDa, for example, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, and any molecular weight therebetween, preferably 1400 to 1800 kDa.

[0021] In a preferred embodiment, in the anti-Helicobacter pylori composition provided herein, the viable cell count of the Lactobacillus rhamnosus is 1×10 8 cfu / day / person (one person is calculated as 60 kg) or more, and those skilled in the art should understand that although a person's body weight is usually calculated as 60 kg, if the person's body weight is not 60 kg, it can be converted according to this standard. In a preferred embodiment, in the anti-Helicobacter pylori composition provided in the present application, the viable cell count of the Lactobacillus rhamnosus is 1 x 10 9 cfu / day / person or more, and those skilled in the art will understand that although calculations are usually based on a person's weight being 60 kg, conversions can be made according to this standard if the person's weight is not 60 kg.

[0022] In a preferred embodiment, in the anti-Helicobacter pylori composition provided herein, the dosage of the hyaluronic acid or its salt is 80 mg / day / person or more, and those skilled in the art should understand that this is usually calculated based on a person's body weight of 60 kg.

[0023] In a preferred embodiment, the anti-Helicobacter pylori composition provided in the present application The viable cell count of Lactobacillus rhamnosus was 1 x 10 10 cfu / day / person or more, and those skilled in the art will understand that this is usually calculated based on a human body weight of 60 kg.

[0024] In a preferred embodiment, in the anti-Helicobacter pylori composition provided herein, the dosage of the hyaluronic acid or its salt is 100 mg / day / person or more, and those skilled in the art should understand that this is usually calculated based on a person's body weight of 60 kg.

[0025] In a preferred embodiment, the composition further comprises an additive, and the additive of the present application may be a suitable solvent, propellant, solubilizer, co-solvent, emulsifier, colorant, binder, disintegrant, filler, lubricant, wetting agent, osmolality adjuster, stabilizer, glidant, flavoring agent, preservative, suspension aid, coating agent, fragrance, anti-adherent agent, integrating agent, penetration enhancer, pH adjuster, buffer, plasticizer, surfactant, foaming agent, anti-foaming agent, thickener, inclusion agent, humectant, absorbent, diluent, flocculating and deflocculating agent, filter aid, release retardant, etc.

[0026] In a further preferred embodiment, the excipient is one or more of inulin, galactooligosaccharides, isomaltooligosaccharides, or maltodextrin.

[0027] The compositions of the present application can be prepared by conventional methods, and one or more diluents or carriers can be added, for example, in the form of oral preparations such as pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0028] The present application further provides the use of a combination of Lactobacillus rhamnosus and hyaluronic acid or a salt thereof in the preparation of a food or health product that inhibits Helicobacter pylori.

[0029] The combination may be a mixture of Lactobacillus rhamnosus and hyaluronic acid or a salt thereof, or one of Lactobacillus rhamnosus and hyaluronic acid or a salt thereof may be designated as Agent A and the other as Agent B, and A may be used first and then B, or B may be used first and then A, or A and B may be used simultaneously.

[0030] In a preferred embodiment, in the above use, the dosage of the hyaluronic acid or its salt is 80 mg / day / person or more, and those skilled in the art should understand that the body weight of a person is usually calculated as 60 kg.

[0031] In a preferred embodiment, in the use, the dosage of the Lactobacillus rhamnosus is such that the viable cell count is 1×10 8 cfu / day / or more of Lactobacillus rhamnosus, and those skilled in the art will understand that this is typically calculated based on a person's body weight of 60 kg.

[0032] In a preferred embodiment, in the use, the dosage of the Lactobacillus rhamnosus is such that the viable cell count is 1×10 9 cfu / day / or more of Lactobacillus rhamnosus, and those skilled in the art will understand that this is usually calculated based on a person's body weight of 60 kg.

[0033] In a preferred embodiment, in the above-mentioned use, the dosage of the hyaluronic acid or its salt is 100 mg / day or more of the hyaluronic acid or its salt, and those skilled in the art should understand that the normal human body weight is calculated as 60 kg.

[0034] In a preferred embodiment, in the use, the dosage of the Lactobacillus rhamnosus is such that the viable cell count is 1×10 10 cfu / day / or more of Lactobacillus rhamnosus, and those skilled in the art will understand that this is usually calculated based on a person's body weight of 60 kg.

[0035] In a preferred embodiment, the salt of hyaluronic acid is any one or more of sodium, potassium, magnesium, calcium, zinc, and bismuth salts of hyaluronic acid.

[0036] In a preferred embodiment, the average molecular weight of the hyaluronic acid or its salt is 1200 to 2000 kDa, for example, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, and any molecular weight therebetween, preferably 1400 to 1800 kDa.

[0037] The Lactobacillus rhamnosus strain CCFM1259 provided in this application has a good inhibitory effect against Helicobacter pylori, and its antibacterial effect is stronger than that of other strains such as Lactobacillus paracasei TY-O1 and Lactobacillus plantarum SN-L3.The inhibition zone of CCFM1259 is 0.76 mm and 1.67 mm larger than those of the other two strains, respectively, and is superior to other Lactobacillus rhamnosus strains such as Lactobacillus rhamnosus TY-O4 and Lactobacillus rhamnosus FB-O1. In addition, the Lactobacillus rhamnosus CCFM1259 provided in the present application has a better effect on reducing the adhesion rate of H. pylori than Lactobacillus paracasei TY-O1 and Lactobacillus plantarum SN-L3, and the adhesion rate of CCFM1259 is 4.86% and 8.08% lower than those of the other two strains, respectively, and is lower than those of other Lactobacillus rhamnosus strains such as Lactobacillus rhamnosus TY-O4 and Lactobacillus rhamnosus FB-O1.

[0038] The composition provided herein, which inhibits Helicobacter pylori and contains hyaluronic acid or its salt and Lactobacillus rhamnosus powder, is a combination of hyaluronic acid or its salt and Lactobacillus rhamnosus powder, and has a better effect in reducing H. pylori colonization than hyaluronic acid or its salt alone or Lactobacillus rhamnosus alone. Experimental data from the present application shows that the effect of the composition of the present application is significantly different (p<0.05) from the effects of hyaluronic acid or its salt alone and Lactobacillus rhamnosus alone. This indicates that the composition of the present application has a synergistic effect in reducing H. pylori colonization.

[0039] The composition provided herein, which inhibits Helicobacter pylori and contains hyaluronic acid or its salt and Lactobacillus rhamnosus powder, better improves gastric pathology, and the improvement effect of the gastric mucosa in mice treated with the composition of the present application is more significant than that of hyaluronic acid or its salt alone or Lactobacillus rhamnosus alone, with essentially no inflammatory cell infiltration, further demonstrating that the composition of the present application has a synergistic effect in antagonizing the inflammatory response caused by H. pylori infection.

[0040] Furthermore, clinical trials were conducted to evaluate the anti-Helicobacter pylori effects of the composition of the present application. By administering a placebo and a composition of hyaluronic acid or its salt and Lactobacillus rhamnosus powder provided by the present application to subjects, the negative conversion rate and efficacy rate of the combination group increased by 125.88% and 119.60%, respectively, compared to the placebo group. There was no significant change in gastrointestinal symptoms in the placebo group before and after treatment (p>0.05). However, after treatment with the composition, the subjects' gastrointestinal symptoms significantly improved (GSRS score, p<0.05), and the negative conversion rate and efficacy increased several-fold. This indicates that the composition has a significant effect in antagonizing gastric H. pylori infection. Furthermore, the composition of the present application also has the effect of improving the composition and diversity of the intestinal microbiota, reducing the proportion of pathogenic bacteria in the intestine and increasing the abundance (abundance) of beneficial bacteria such as Lactobacillus and Bifidobacterium, thereby contributing to maintaining patients' gastrointestinal health. [Example]

[0041] Animal study details: 1) Origin of the animals used 4-week-old, male C57BL / 6 mice. 2) Information about the facility where the animals will be kept Jiangsu Provincial Institute of Schistosomiasis Control and Prevention. 3) Source of sodium hyaluronate (HA) BLOOMAGE BIOTECHNOLOGY CORPORATION LIMITED, Food grade sodium hyaluronate and related products. 4) Source of probiotics Lactobacillus rhamnosus CCFM1259, deposited in the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC NO. 62419. 5) Methods of anesthesia and euthanasia of laboratory animals After the last intragastric administration, the mice were fasted for 24 hours, anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution, blood was collected from the mouse's eyeball, and finally the mice were sacrificed by cervical dislocation. The stomachs were immediately dissected and incised along the greater curvature of the stomach to collect the complete stomach tissue (including the antrum, body, etc.), half of which was used for pathological section examination, and the other half was used for examination of related indicators such as pylorus colonization and immune factors.

[0042] Example 1 Screening of lactic acid bacteria strains that are antagonistic to H. pylori (1) Experimental materials and methods The Helicobacter pylori strain used was H. pylori SS from the NTCC China Center for Type Culture Collection. When using H. pylori, a small amount of the bacterial solution in the bacterial storage tube was dipped into an inoculation loop and smeared onto a Columbia blood agar plate (containing 7.5% sterile defibrinated sheep blood) in a three-gas incubator (85% N, 10% CO, 5% O) at 37°C for 3 days. A single colony on the surface of the plate was picked and inoculated into liquid BHI medium (containing 5% fetal bovine serum). After incubation for 4 days, a 2% inoculum was inoculated into fresh BHI medium and incubated for 4 days for future use.

[0043] Human gastric adenocarcinoma cells (AGS) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Before use, AGS cells were resuspended in 10% DMSO-containing fetal bovine serum and then frozen in liquid nitrogen after gradient cooling. To use AGS cells, 10 mL of F-12 medium containing 5% fetal bovine serum was added to a petri dish. The cryopreservation tube was centrifuged, the supernatant discarded, and the cells were resuspended in culture medium. The cells were then dispersed evenly in a petri dish and cultured in a 37°C incubator containing 5% CO2. Once the cells had grown to a monolayer, they were passaged at a ratio of 1:3. After two passages, the cells were reserved for future use.

[0044] Lactobacillus samples were collected from fermented foods, dairy products, or human saliva and feces and purchased from the Guangdong Provincial Microbial Culture Collection Center and Biobw Strain Bank, respectively. When using Lactobacillus, they were first streaked onto MRS plates and cultured at 37°C for 2 days. Single colonies were picked and cultured in MRS liquid tubes for 18 hours, then inoculated into fresh MRS culture medium at a 2% inoculum size and cultured for 18 hours for later use.

[0045] (2) Measurement of the inhibitory effect of lactobacillus on H. pylori proliferation: The concentration of activated second-generation H. pylori suspension was 1 × 10 8 CFU / mL, and 100 μL of the suspension was evenly spread on a Columbia blood agar plate. Once the surface of the plate was dry, an Oxford cup was placed on the plate and gently pressed down to distribute the Lactobacillus suspension (1 × 10 8 A blank control (100 μL of MRS liquid medium, pH 6.2) was prepared by adding 150 μL of each medium (CFU / mL). The blank control was also prepared by adding 100 μL of MRS liquid medium, pH 6.2. The incubation was continued for 4 h at 4°C. After the diffusion was completed, the cells were placed upright in a 3-gas incubator and cultured for 72 hours, and the antibacterial effect was observed, and the diameter of the inhibition zone was measured using a vernier caliper.

[0046] (3) Measurement of the inhibitory ability of lactobacilli against H. pylori adhesion to AGS cells: AGS cells were cultured in a 96-well plate (2 × 10 4Cells were seeded at 1000 x 1000 cells / well and cultured overnight. After the cells became attached, they were washed three times with PBS to remove dead cells. Next, H. pylori and lactobacilli resuspended in F-12 medium (serum-free) were added at a multiplicity of infection (MOI) of 100 and co-cultured for 2 hours. Subsequently, the cells were washed three times with PBS to remove unattached lactobacilli and H. pylori. Finally, 200 μL of urease reagent (0.9% NaCl, 14 μg / mL phenol red, 20 mmol / L urea, pH 6.8) was added and the cells were cultured for 3 hours. The absorbance at 550 nm was measured.

[0047] [Table 1]

[0048] (4) Inhibitory effect of lactobacillus on H. pylori growth In the experiment, 30 Lactobacillus strains were used as starting strains. As shown in Table 1, the size of the inhibition zones against H. pylori was compared. The inhibition zones of the 30 strains ranged from 8.27 to 17.51 ​​mm, and six of them had inhibition zones larger than 15 mm against H. pylori, demonstrating stronger antibacterial effects than the other strains. The six strains are ranked according to the size of their inhibition zones as follows: CCFM1259, FB-O1, NJ-F3, TY-O1, and SN. -L3, TY-O4.

[0049] (5) Inhibitory effect of Lactobacillus on the adhesion of H. pylori to AGS cells In this experiment, we compared the inhibitory effects of 30 lactobacilli on H. pylori adhesion to AGS cells. As shown in Table 1, the results showed that the ability of different lactobacilli to reduce H. pylori adhesion varied significantly. After treatment with different lactobacilli, the H. pylori adhesion rates ranged from 66.53% to 113.36%, with five of the strains showing a corresponding H. pylori adhesion rate of less than 80%. The five strains, ranked in descending order by their ability to inhibit H. pylori adhesion, are as follows: FB-A3, CCFM1259, FB-O2, TY-O1, and SN-L3.

[0050] (6) Selection of Lactobacillus strains antagonistic to H. pylori Considering the ability of lactobacilli to inhibit H. pylori growth and its adhesion to AGS cells, three strains demonstrated excellent efficacy in suppressing H. pylori growth while reducing adhesion rates. Ranked in order of adhesion inhibition ability, the three strains were Lactobacillus rhamnosus CCFM1259, Lactobacillus paracasei TY-O1, and Lactobacillus plantarum SN-L3. The inhibition zone of CCFM1259 was 0.76 mm and 1.67 mm larger than the other two strains, respectively, and the adhesion rates were 4.86% and 8.08% lower, respectively. Therefore, Lactobacillus rhamnosus CCFM1259 was selected as the lactobacillus strain antagonistic to H. pylori in this experiment.

[0051] Example 2: Evaluation of the efficacy of a combination of Lactobacillus rhamnosus and HA against Helicobacter pylori in animals After one week of normal feeding, several C57BL / 6 mice were randomly divided into five groups (n = 10): blank group, model group, and intervention group. The intervention group was initially treated with HA (HA with an average molecular weight of 1400 kDa), Lactobacillus rhamnosus CCFM1259 (1 × 10 9 CFU / mL), and the mixture of both were administered intragastrically, followed by BHI-resuspended H. pylori (1 × 10 9 The mice were intragastrically administered with an equal volume of PBS, followed by an equal volume of BHI intragastrically one hour later. The experimental mice were intragastrically administered once every other day. The weight of the experimental mice was measured periodically, and after the experiment, the amount of Helicobacter pylori colonization in the stomach, serum anti-Helicobacter pylori antibody and cytokine (IL-8 and TNF-α) levels were measured, and histopathological sections of the gastric antrum and gastric body were prepared. The mice were provided with regular drinking water and standard chow.

[0052] The specific treatment plan for the experimental animals is shown in Table 2.

[0053] [Table 2]

[0054] (1) Weight changes of mice during the experiment As can be seen from Table 3, the mouse weight was maintained at approximately 20 g during the experiment, and there was no significant difference in mouse weight between the groups. There was also no obvious change in the mouse weight before and after the intervention between the groups. In other words, H. pylori infection and the combined prescription intervention did not significantly affect the mouse weight.

[0055] [Table 3]

[0056] (2) The effect of different treatments on the colonization level of H. pylori The change in H. pylori colonization in the mouse stomach is the most direct indicator of the effectiveness of the intervention regimen against H. pylori infection. As shown in Figure 1, the H. pylori content in the stomachs of mice in the model group was approximately 236-fold higher than that in the blank group, and the H. pylori colonization in the model group significantly increased (p<0.01), indicating successful model construction. After treatment with HA (p<0.01), Lactobacillus rhamnosus CCFM1259 (p<0.05), and the combined regimen (p<0.001), the H. pylori colonization in the stomachs of infected mice significantly decreased, with the mean logarithmic values ​​of H. pylori colonization decreasing by approximately 34.75%, 29.33%, and 52.64%, respectively, compared to the model group. Furthermore, the effect of the combination group was significantly greater than that of the HA group and CCFM1259 group (p<0.05), with the mean Log values ​​of H. Pylori colonization in the combination group decreasing by 27.41% and 32.97% compared with the HA group and CCFM1259 group, respectively. These results suggest that the combined treatment is more effective than HA alone or Lactobacillus rhamnosus CCFM1259 alone, and that Lactobacillus rhamnosus CCFM1259 and HA have a synergistic effect in reducing H. Pylori colonization.

[0057] (3) Improvement of gastric lesions through various treatments The results of HE staining of gastric mucosa from mice in different treatment groups are shown in Figure 2. After H. pylori infection, infiltration of inflammatory cells (lymphocytes and eosinophils) in the gastric lamina propria of mice significantly increased (Figure 2B). Treatment with the HA group (Figure 2C), Lactobacillus rhamnosus CCFM1259 group (Figure 2D), and the combined formula (Figure 4E) significantly improved the inflammatory response caused by H. pylori infection. Compared with the HA group (Figure 2C) and Lactobacillus rhamnosus CCFM1259 group (Figure 2D), the combined formula (Figure 2E) significantly improved the gastric mucosa of mice, with essentially no inflammatory cell infiltration. This further demonstrated that Lactobacillus rhamnosus CCFM1259 and HA have a synergistic effect in antagonizing the inflammatory response caused by H. pylori infection.

[0058] The results of this example demonstrate that HA, Lactobacillus rhamnosus CCFM1259, and the combined formula all significantly reduced H. pylori colonization in the mouse stomach and significantly ameliorated the inflammatory response caused by H. pylori infection. The combined formula was more effective at reducing H. pylori colonization and improving the gastric mucosa of mice than HA alone or probiotics alone. Little inflammatory cell infiltration was observed in the gastric lamina propria of mice, demonstrating that Lactobacillus rhamnosus CCFM1259 and HA have a synergistic effect in antagonizing the inflammatory response caused by H. pylori infection and reducing H. pylori colonization in the mouse stomach.

[0059] Example 3: Evaluation of the effect of Lactobacillus rhamnosus in combination with HA of different molecular weights on in vivo infection with Helicobacter pylori After one week of normal feeding, several C57BL / 6 mice were randomly divided into seven groups (n = 10): blank group, model group, and intervention group. The intervention group received HA with average molecular weights of 1200 kDa, 1400 kDa, 1600 kDa, 1800 kDa, and 2000 kDa and Lactobacillus rhamnosus CCFM1259 (1 × 10 9 The BHI-resuspended H. pylori (1 × 10 CFU / mL) was mixed uniformly at a volume ratio of 1:1 and formulated into a composition. The first intragastric administration was performed, and then, at intervals of 1 hour, the BHI-resuspended H. pylori (1 × 10 9 The mice in the blank and model groups were first intragastrically administered an equal volume of PBS, followed by an equal volume of BHI intragastrically one hour later. The experimental mice were intragastrically administered once every other day. The weight changes of the experimental mice were measured periodically, and the amount of Helicobacter pylori colonization in the mouse stomach was measured after the experiment. The mice were given regular drinking water and standard chow.

[0060] The specific treatment plan for the experimental animals is shown in Table 4.

[0061] [Table 4]

[0062] (1) Survival rate and weight change of mice during the experiment As can be seen from Table 5, the mouse weight was maintained at approximately 20 g during the experiment, and there was no significant difference in mouse weight between the groups. There was also no obvious change in the mouse weight before and after the intervention between the groups. In other words, H. pylori infection and the combined prescription intervention did not significantly affect the mouse weight.

[0063] [Table 5]

[0064] (2) Effect of the combined use of HA with different molecular weights and Lactobacillus rhamnosus on the colonization of H. pylori As can be seen in Figure 3, the H. pylori colonization load in the model group was significantly increased (p<0.01) compared with the blank group, with an average of approximately 155-fold higher than that of the blank group, indicating successful model construction. Treatment with HA with average molecular weights of 1200 kDa, 1400 kDa, 1600 kDa, 1800 kDa, and 2000 kDa in combination with Lactobacillus rhamnosus CCFM1259 significantly reduced the H. pylori colonization load in the mouse stomach (p<0.05). Compared with the model group, the mean Log values ​​of H. pylori colonization load were reduced by 31.93%, 47.83%, 53.92%, 49.16%, and 34.76%, respectively. In particular, when HA with average molecular weights of 1400kDa, 1600kDa, and 1800kDa was combined with Lactobacillus rhamnosus CCFM1259, the amount of H. pylori colonization in the stomach was significantly reduced (p<0.001), and the effect was similar to that of the blank group.

[0065] As can be seen from the results of this example, compared with the model group, after intragastric administration of a mixture of HA with an average molecular weight of 1400-1800 kDa and Lactobacillus rhamnosus CCFM1259 to mice infected with H. pylori, the amount of H. pylori colonization in the stomach was significantly reduced (p<0.01), and the average Log value of H. pylori colonization was reduced by 47.83%-53.92%, which was significantly greater than the effect of the 1200 kDa and 2000 kDa HA mixtures and was close to the normal level.

[0066] Example 4 Clinical trial evaluation of the efficacy of hyaluronic acid-Lactobacillus rhamnosus composition against Helicobacter pylori 4.1 Study population For the clinical study, 40 patients (28-65 years old, half male and half female) who tested positive for Helicobacter pylori infection were recruited. The diagnostic criteria were: 14 The results were based on a C breath test, rapid urease test, or histological examination. Volunteers were required to have no history of anti-Helicobacter pylori drug treatment, to have not taken antibiotics or probiotic products in the month prior to study participation, to have not undergone gastrointestinal surgery, to take the products strictly as required during the study period, and not to take antibiotics or other probiotic products.

[0067] 4.2 Clinical trial design (1) Experimental products The probiotic complex product, hyaluronic acid product, and placebo product used in the study were all food-grade products and could be ingested directly or with warm water (below 37°C). They were provided by the Food Biotechnology Center, School of Food Science, Jiangnan University. All products were powders with identical appearance and packaging. A: HA-Probiotic Complex Product: [1] Lactobacillus rhamnosus freeze-dried powder (1 x 10 11[1] Sodium hyaluronate with an average molecular weight of 1600 kDa 100 mg / packet; [2] Inulin 300 mg / packet; [3] Galactooligosaccharides 150 mg / packet; [4] Isomaltooligosaccharides 80 mg / packet; [5] Maltodextrin 1320 mg / packet. B: Placebo product: [1] Inulin 300 mg / packet; [2] Galactooligosaccharides 150 mg / packet; [3] Isomaltooligosaccharides 80 mg / packet; [4] Maltodextrin 1470 mg / packet. (2) Experimental design and subject grouping The experimental design was a double-blind, parallel, randomized controlled trial. Forty subjects were recruited and then assigned to groups. A random number sequence was generated by computer software, and 40 patients with Helicobacter pylori infection were randomly assigned to two groups: a placebo group and a hyaluronic acid-probiotic combination group, each with 20 patients. Each group took the bacterial powder twice daily. (The appearance and packaging of the products in the placebo and combination groups were the same, except for the different ingredients, and there were no significant differences.) (3) Research Cycle Because it takes time for probiotics to exert their physiological properties, the study duration was 10 weeks, which included a 2-week experimental preparation period, a 4-week probiotic intake period, and a 4-week follow-up observation period. (4) Determination of the index 14 C breath test, scale index (GSRS table), distribution of intestinal flora.

[0068] 4.3 Clinical trial results (1) Basic information on clinical subjects

[0069] [Table 6]

[0070] A total of 40 eligible H. pylori-positive patients participated in the clinical trial, of which 7 dropped out or were lost to follow-up. Table 6 shows basic information such as gender and age for the two participant groups. There were no significant differences in the baseline conditions of the two subject groups.

[0071] (2) Different intervention methods 14 Effect on C-exhaled breath values 14 Measurement of the C breath value can characterize the patient's H. pylori infection status. According to the experimental results in Tables 7 and 8, after taking the combination formula, the negative conversion rate and efficacy rate of Helicobacter pylori patients reached 70.59% and 82.35%, respectively, which was an increase of 125.88% and 119.60% compared with the placebo group.

[0072] [Table 7]

[0073] [Table 8]

[0074] (3) The effect of different intervention methods on subjects' gastrointestinal symptoms The GSRS is an important indicator of the severity and frequency of gastrointestinal symptoms in clinical patients. The lower the score, the milder the symptoms. The subjects answered the questionnaire one day before and one day after the experiment. According to the statistical results in Figure 4, there was no significant change in gastrointestinal adverse symptoms before and after the intervention in the placebo group (p>0.05), but in the HA-probiotic combination group, the gastrointestinal symptoms of the subjects were significantly improved after the intervention (p<0.05).

[0075] (4) The effects of different intervention methods on the composition and diversity of the subjects' gut microbiota Compared with the placebo group, the composition of the subjects' gut microbiota was altered after the HA-probiotic intervention. Phylum-level analysis: As shown in Figure 5A, the intestinal microbiota was mainly composed of Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria. Compared with the placebo group, the proportion of Actinobacteria in the fecal intestinal microbiota increased in the combination group. Genus-level analysis: Furthermore, the fecal microbiota of the two groups was analyzed at the genus level. The results are shown in Figure 5B. Compared with the placebo group, the combination group had a lower abundance of microbiota, such as Streptococcus, Ruminococcus, and the conditional pathogens Escherichia coli and Shigella, and an increased abundance of beneficial microbiota, such as Lactobacillus and Bifidobacterium.

[0076] The results of the above examples show that after treatment with the HA and Lactobacillus rhamnosus prebiotic composition, the subjects' gastrointestinal symptoms significantly improved (GSRS score, p<0.05), and the negative conversion rate and efficacy of the patients increased several-fold. This indicates that the composition of the present application has a significant effect in combating gastric H. pylori infection. After treatment with the combined product, the negative conversion rate and efficacy rate of H. pylori patients reached 70.59% and 82.35%, respectively, which were 2.126- and 2.120-fold higher than those of the placebo group. Furthermore, the combined treatment group also improved the composition and diversity of the intestinal microbiota, reduced the proportion of enteric pathogenic bacteria, and increased the abundance of beneficial bacteria such as Lactobacillus and Bifidobacterium, helping to maintain patients' gastrointestinal health.

[0077] Although the present application has been described above by way of the embodiments of the present application, the present application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely examples and guidelines, and are not intended to be limiting. Those skilled in the art can make many variations under the teachings of this specification without departing from the scope of protection of the claims of this application, and all of them are protected by this application.

Claims

1. Lactobacillus rhamnosus, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO. 62419.

2. 10. Use of Lactobacillus rhamnosus according to claim 1 in inhibiting Helicobacter pylori.

3. A composition for inhibiting Helicobacter pylori, comprising hyaluronic acid or a salt thereof and Lactobacillus rhamnosus.

4. the salt of hyaluronic acid is one or more of sodium salt, potassium salt, magnesium salt, calcium salt, zinc salt, and bismuth salt of hyaluronic acid; The composition according to claim 3, wherein the average molecular weight of the hyaluronic acid or its salt is preferably 1200 to 2000 kDa, more preferably 1400 to 1800 kDa.

5. The composition can provide 80 mg / day / person (calculated based on a person weighing 60 kg) or more of hyaluronic acid or a salt thereof; or The viable cell count of the Lactobacillus rhamnosus was 1 x 10 ８ cfu / day / person (calculated as 60 kg per person) or more of Lactobacillus rhamnosus, Preferably, the composition can provide 100 mg / day / person (calculated based on a person weighing 60 kg) or more of hyaluronic acid or a salt thereof; or The viable cell count of the Lactobacillus rhamnosus was 1 x 10 ９ The composition according to any one of claims 3 to 4, characterized in that the Lactobacillus rhamnosus is present in an amount of at least cfu / day / person (calculated assuming one person weighs 60 kg).

6. The composition according to any one of claims 3 to 5, further comprising an additive, preferably the additive comprising one or more of inulin, galactooligosaccharides, isomaltooligosaccharides, maltodextrin, erythritol, xylose, arabinose, rhamnose, galactose, fucose, mannose, fructose, sorbose, glucose, glycerol, galactitol, sorbitol, xylitol, mannitol, lactose, maltitol, lactitol, sucrose, trehalose, raffinose, stachyose, oligofructose, oligoxylose, mannooligosaccharides, β-glucan, hydroxyethyl starch, indigestible dextrin, whey protein, collagen, skim milk, pectin, and gelatin.

7. The composition according to any one of claims 3 to 6, characterized in that the Lactobacillus rhamnosus is the Lactobacillus rhamnosus according to any one of claims 1 to 2.

8. Use of Lactobacillus rhamnosus according to any one of claims 1 to 2 or a composition according to any one of claims 3 to 7 in the preparation of a food or health food that inhibits Helicobacter pylori.

9. Use of a combination of Lactobacillus rhamnosus and hyaluronic acid or a salt thereof in the preparation of a food or health food that inhibits Helicobacter pylori.

10. The dosage of the hyaluronic acid or its salt is such that the composition can supply 80 mg / day / person (calculated assuming one person weighs 60 kg) or more of hyaluronic acid or its salt. or The dosage of Lactobacillus rhamnosus is such that the number of viable bacteria is 1 x 10 ８ cfu / day / person (calculated as 60 kg per person) or more of Lactobacillus rhamnosus, Preferably, the dosage of the hyaluronic acid or its salt is such that the composition can provide 100 mg / day / person (calculated assuming one person weighs 60 kg) or more of hyaluronic acid or its salt, or The dosage of Lactobacillus rhamnosus is such that the number of viable bacteria is 1 x 10 ９ cfu / day / person (calculated as 60 kg per person) or more of Lactobacillus rhamnosus, Preferably, the salt of hyaluronic acid is one or more of sodium salt, potassium salt, magnesium salt, calcium salt, zinc salt, and bismuth salt of hyaluronic acid, and the average molecular weight of the hyaluronic acid or its salt is 1200 to 2000 kDa, more preferably 1400 to 1800 kDa. More preferably, the use according to claim 9, characterized in that the Lactobacillus rhamnosus is the Lactobacillus rhamnosus according to any one of claims 1 to 2.

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