An anti-helicobacter pylori probiotic composition for repairing gastric mucosa and modulating intestinal flora

Abstract

The present application relates to the technical field of probiotic composition, and discloses an anti-helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora, which comprises 35-45 parts of Lactobacillus plantarum R202448 freeze-dried powder, 20-30 parts of Weizmannia coagulans BHE26 freeze-dried powder, 15-25 parts of Lactobacillus paracasei SMN-LBK freeze-dried powder, 10-15 parts of Lactobacillus acidophilus NCFM freeze-dried powder, 5-10 parts of prebiotic compound, 2-5 parts of gastric mucosa repair enhancer and 5-10 parts of auxiliary materials. Different probiotics are fermented separately in the present application, so as to avoid the nutritional competition and metabolic antagonism of mixed fermentation, and at the same time, a compatible metabolic environment between strains is constructed in advance through metabolic adaptation liquid, so as to ensure that the synergistic effect of each strain after compounding is not weakened.

Description

Technical Field

[0001] This invention relates to the field of probiotic composition technology, and more specifically, to an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora. Background Technology

[0002] Helicobacter pylori (Hp) is a prevalent gastrointestinal pathogen worldwide, with infection rates as high as 70-90% in developing countries and 25-50% in developed countries. Children are particularly susceptible, and the bacteria can colonize the body for a long time, with the incidence rate gradually increasing with age. Long-term Hp infection can lead to chronic gastritis, which can progress to peptic ulcers and, in severe cases, induce gastric cancer, making it one of the core causes of harm to human gastrointestinal health.

[0003] Current treatments for *Helicobacter pylori* (Hp) primarily rely on triple or quadruple therapy with antibiotics. However, these regimens have significant drawbacks: long-term, inappropriate use of antibiotics can easily lead to gastrointestinal microecological disorders and related enteritis; Hp resistance is increasing year by year, causing treatment relapses; and their applicability to asymptomatic patients and children is limited, leaving hidden dangers for Hp transmission. Therefore, exploring non-antibiotic interventions (such as dietary therapy and probiotic preparations) has become an important research direction for the prevention and treatment of Hp infection.

[0004] Probiotics are widely used in the intervention of gastrointestinal diseases because they have functions such as inhibiting pathogenic bacteria, regulating the balance of the intestinal microecology, and enhancing the body's immunity. However, existing probiotic preparations have the following drawbacks: (1) The effect of a single strain is limited, while the fermentation of a multi-strain combination involves nutrient competition and metabolic antagonism, which makes the enhancement of its effect not obvious; (2) Probiotics lose a lot of activity when exposed to the acidic environment of the stomach (pH 1-4), and the survival rate of conventional preparations is only 30-50%, and they lack the ability to repair the gastric mucosa. Therefore, the present invention proposes an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora, which has important practical significance. Summary of the Invention

[0005] In view of this, the present invention proposes an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora, aiming to solve at least one of the problems in the above-mentioned background art.

[0006] This invention proposes an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora, comprising the following components in parts by weight: The product contains 35-45 parts of *Lactobacillus plantarum* R202448 lyophilized powder, 20-30 parts of *Weizmannii coagulans* BHE26 lyophilized powder, 15-25 parts of *Lactobacillus paracasei* SMN-LBK lyophilized powder, 10-15 parts of *Lactobacillus acidophilus* NCFM lyophilized powder, 5-10 parts of prebiotic complex, 2-5 parts of gastric mucosal repair and enhancement agent, and 5-10 parts of excipients.

[0007] Furthermore, the preparation method of the *Lactobacillus plantarum* R202448 lyophilized powder is as follows: Add 0.08% volume of mucin and 0.02% volume of sialic acid to MRS liquid medium, mix and inoculate with Lactobacillus plantarum R202448 at an inoculation rate of 2%, culture at 37℃ and 180 r / min for 20 h, and subculture twice to obtain R202448 targeted activation seed liquid. 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant and 0.05% volume of mucin were added to MRS liquid medium. After mixing, the R202448 targeted activation seed liquid was inoculated at an inoculum rate of 3%. Fermentation was carried out at 37℃ and 180 r / min for 12 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain the R202448 fermentation broth. The R202448 fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Bacillus plantarum R202448 bacterial sludge. The Bacillus plantarum R202448 bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Bacillus plantarum R202448 freeze-dried powder.

[0008] Furthermore, the method for preparing the lyophilized powder of *Weizmannia coagulans* BHE26 is as follows: After adding 0.05% volume of glutamine to GMRS liquid medium, Weizmannii coagulans BHE26 was inoculated at an inoculum rate of 2% and cultured at 37℃ and 180 r / min for 24 h. After two subcultures, activated seed culture of BHE26 was obtained. 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant and 0.03% volume of glutamine were added to GMRS liquid medium. After mixing, the BHE26 activated seed culture was inoculated at an inoculum rate of 3%. Fermentation was carried out at 37℃ and 180 r / min for 16 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain BHE26 fermentation broth. The BHE26 fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain BHE26 coagulated sludge. The BHE26 coagulated sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain BHE26 freeze-dried powder.

[0009] Furthermore, the preparation method of the Lactobacillus paracasei SMN-LBK lyophilized powder is as follows: Add 0.1% volume of fructooligosaccharide to MRS liquid medium, mix, and inoculate with Lactobacillus paracasei SMN-LBK at an inoculum rate of 2%. Culture at 37℃ and 160r / min for 18h, and subculture twice to obtain SMN-LBK activated seed culture. 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant and 0.08% volume of fructooligosaccharide were added to MRS liquid medium. After mixing, the SMN-LBK activated seed culture was inoculated at an inoculum of 2.5%. Fermentation was carried out at 37℃ and 180 r / min for 14 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain SMN-LBK fermentation broth. The SMN-LBK fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Lactobacillus paracasei SMN-LBK bacterial sludge. The Lactobacillus paracasei SMN-LBK bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Lactobacillus paracasei SMN-LBK freeze-dried powder.

[0010] Furthermore, the preparation method of the Lactobacillus acidophilus NCFM lyophilized powder is as follows: Add 0.03% (v / v) of vitamin B complex to MRS liquid medium, mix, and inoculate with Lactobacillus acidophilus NCFM at an inoculum rate of 2%. Culture at 37℃ and 160 r / min for 22 h, and subculture twice to obtain activated NCFM seed culture. Add 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant and 0.02% volume of B vitamins to MRS liquid medium, mix and inoculate the NCFM activated seed culture at an inoculum of 2.5%, and then ferment at 37℃ and 180 r / min for 18 h. After fermentation, add 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C to the fermentation system and incubate at 30℃ for 30 min to obtain NCFM fermentation broth. The NCFM fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Lactobacillus acidophilus NCFM bacterial sludge. The Lactobacillus acidophilus NCFM bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Lactobacillus acidophilus NCFM freeze-dried powder.

[0011] Furthermore, the preparation method of the metabolic adaptor solution is as follows: The R202448 activated seed solution, BHE26 activated seed solution, SMN-LBK activated seed solution and NCFM activated seed solution were mixed in a volume ratio of 1:1:1:1 and incubated at 37°C for 4 hours. The supernatant was collected, filtered through a 0.22 μm filter membrane, and the filtrate was used to obtain the metabolic adaptation solution.

[0012] Furthermore, the preparation method of the composite lyophilization protectant is as follows: Water, hydroxyethyl starch, glucosamine, glutathione, and trehalose are mixed and dissolved in a mass ratio of 32:4:2:1:1.

[0013] Furthermore, the gradient freeze-drying specifically involves: Add the corresponding bacterial sludge to the preheated 30℃ composite freeze-drying protectant at a ratio of 1:2 (w / v), stir at 30 r / min for 10 min, then stir at 50 r / min for 10 min to obtain a mixed solution; The mixed solution was frozen at -10°C for 1 hour, then the temperature was lowered to -30°C and frozen for 2 hours, and then the temperature was lowered to -40°C and frozen for 2 hours to obtain pre-frozen bacterial cells; The pre-frozen bacterial cells were freeze-dried at a vacuum of 10 Pa and a temperature of -20°C for 8 hours, then the temperature was raised to 0°C and freeze-dried for 4 hours, and then the temperature was raised to 25°C and freeze-dried for 2 hours.

[0014] A method for preparing a probiotic composition for further mucosal and intestinal flora regulation against Helicobacter pylori, characterized in that, The prebiotic is composed of fructooligosaccharides, stachyose and gentiosaccharides in a mass ratio of 2:1:1. The gastric mucosa repair enhancer is composed of glutamine, carboxymethyl chitosan and β-glucan in a mass ratio of 2:1:1. The excipients are a compound of maltodextrin, microcrystalline cellulose and magnesium stearate in a mass ratio of 5:3:1.

[0015] Furthermore, the preparation method of the anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora is specifically as follows: Take Lactobacillus plantarum R202448 lyophilized, Weizmannii coagulans BHE26 lyophilized powder, Lactobacillus paracasei SMN-LBK lyophilized powder and Lactobacillus acidophilus NCFM lyophilized powder, mix them at 15℃ and 30r / min for 10min to obtain probiotic compound lyophilized powder. The probiotic compound freeze-dried powder was mixed with the prebiotic and gastric mucosa repair and enhancement agent at 15°C and a rotation speed of 30 r / min for 8 min to obtain a mixture. The mixture was mixed with 10% Eudragit S100 coating solution at a ratio of 1:0.3 (w / v) and then subjected to fluidized bed coating treatment to obtain pH-sensitive coated particles. The pH-sensitive coated granules were mixed with excipients and filled into empty capsules. The capsules were then placed at 25°C and 30% relative humidity for 48 hours to obtain an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora.

[0016] The present invention also provides an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora, prepared by the method described above.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention provides specific culture media for Lactobacillus plantarum R202448, Weizmannii coagulans BHE26, Lactobacillus paracasei SMN-LBK, and Lactobacillus acidophilus NCFM to enhance the colonization ability of the strains. In addition, they are fermented separately to avoid nutrient competition and metabolic antagonism in mixed fermentation. At the same time, a metabolically compatible metabolic environment is pre-constructed through a metabolic adaptation solution to ensure that the synergistic effect of each strain is not weakened after compounding.

[0018] 2. The outer layer of this invention uses a pH-sensitive coating material to achieve targeted release of probiotics into the intestine. At the same time, a gastric mucosa repair and enhancement agent is mixed into the coating solution. When the composition of this invention is in the stomach, the gastric mucosa repair and enhancement agent is leaked through the pores of the coating material, thereby achieving gastric mucosa repair. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0020] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] This invention provides a method for preparing an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora, comprising the following components in parts by weight: The product contains 35-45 parts of *Lactobacillus plantarum* R202448 lyophilized powder, 20-30 parts of *Weizmannii coagulans* BHE26 lyophilized powder, 15-25 parts of *Lactobacillus paracasei* SMN-LBK lyophilized powder, 10-15 parts of *Lactobacillus acidophilus* NCFM lyophilized powder, 5-10 parts of prebiotic complex, 2-5 parts of gastric mucosal repair and enhancement agent, and 5-10 parts of excipients.

[0025] Preferably, the composition includes the following components in parts by weight: 40 parts of *Lactobacillus plantarum* R202448 lyophilized powder, 25 parts of *Weizmannii coagulans* BHE26 lyophilized powder, 20 parts of *Lactobacillus paracasei* SMN-LBK lyophilized powder, 13 parts of *Lactobacillus acidophilus* NCFM lyophilized powder, 8 parts of prebiotic complex, 3 parts of gastric mucosal repair and enhancement agent, and 8 parts of excipients.

[0026] Understandably, *Lactobacillus plantarum* R202448, by inhibiting *H. pylori* biofilm formation and reducing *H. pylori* activity, can effectively inhibit the damage and apoptosis-promoting effects of *H. pylori* on gastric mucosal epithelial cells, thereby effectively inhibiting the occurrence of *H. pylori* and protecting the function of gastric mucosal epithelial cells. *Weizmannii coagulans* BHE26 focuses on gastric mucosal repair, synergistically enhancing the dual effects of anti-*H. pylori* and mucosal protection with R202448. *Lactobacillus paracasei* SMN-LBK and *Lactobacillus acidophilus* NCFM focus on intestinal flora regulation; the former can inhibit foodborne pathogens, while the latter has outstanding resistance to gastric juices. Through a cross-feeding mechanism, the two form metabolic complementarity with the first two strains, proliferating in the intestine and optimizing the microecological balance, reducing indirect damage to the stomach from intestinal inflammatory factors.

[0027] In this invention, the method for preparing the freeze-dried Lactobacillus plantarum R202448 powder is as follows: Add 0.08% volume of mucin and 0.02% volume of sialic acid to MRS liquid medium, mix and inoculate with Lactobacillus plantarum R202448 at an inoculation rate of 2%, culture at 37℃ and 180 r / min for 20 h, and subculture twice to obtain R202448 targeted activation seed liquid. 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant and 0.05% volume of mucin were added to MRS liquid medium. After mixing, the R202448 targeted activation seed liquid was inoculated at an inoculum rate of 3%. Fermentation was carried out at 37℃ and 180 r / min for 12 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain the R202448 fermentation broth. The R202448 fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Bacillus plantarum R202448 bacterial sludge. The Bacillus plantarum R202448 bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Bacillus plantarum R202448 freeze-dried powder.

[0028] Specifically, the *Lactobacillus plantarum* R202448 has the accession number CGMCCNO:31101.

[0029] Specifically, during the R202448 targeted activation of the seed culture fermentation process, the pH value of the fermentation system was monitored in real time, and the pH was stabilized at 5.8-6.2 by adding 1 mol / L sodium lactate solution.

[0030] Understandably, in preparing the seed culture, this invention added 0.08% by volume of mucin and 0.02% by volume of sialic acid to the MRS liquid culture medium. Mucin, as a core component of the gastric mucus layer, can simulate the physical barrier properties of mucus, providing a growth environment similar to that in vivo for *Lactobacillus plantarum* R202448 and inducing the strain to express adhesion factors targeting gastric mucosal epithelial cells. Sialic acid, as a natural component of the mucus layer, can further optimize the realism of the simulated environment, working synergistically with mucin to help the strain adapt to the physiological conditions of the stomach in advance, while enhancing the strain's ability to recognize receptors on the gastric mucosal surface, ultimately significantly improving the gastric mucosal adhesion rate of R202448.

[0031] Understandably, during fermentation, this invention adds tryptone, yeast extract, metabolic adaptor solution, and mucin to the MRS liquid culture medium. Tryptone provides abundant peptides and amino acids, while yeast extract contains vitamins, growth factors, and other nutrients. Together, they meet the nutritional needs of the strain during fermentation. The 20% metabolic adaptor solution contains metabolites compatible with each strain, allowing R202448 to adapt to the bacterial community metabolic environment during subsequent compounding, avoiding metabolic antagonism between strains and ensuring synergistic efficacy. The 0.05% mucin maintains the gastric mucosa simulated environment during the activation phase, sustaining the continuous expression of the strain's adhesion factors and ensuring that it retains strong gastric mucosal colonization ability and anti-Hp activity after fermentation.

[0032] In this invention, the method for preparing the freeze-dried powder of *Weizmannii coagulates* BHE26 is as follows: After adding 0.05% volume of glutamine to GMRS liquid medium, Weizmannii coagulans BHE26 was inoculated at an inoculum rate of 2% and cultured at 37℃ and 180 r / min for 24 h. After two subcultures, activated seed culture of BHE26 was obtained. 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant and 0.03% volume of glutamine were added to GMRS liquid medium. After mixing, the BHE26 activated seed culture was inoculated at an inoculum rate of 3%. Fermentation was carried out at 37℃ and 180 r / min for 16 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain BHE26 fermentation broth. The BHE26 fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain BHE26 coagulated sludge. The BHE26 coagulated sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain BHE26 freeze-dried powder.

[0033] Specifically, the Weizmann's coagulation bacterium BHE26 is designated CGMCC No. 30414.

[0034] Specifically, during the fermentation of the BHE26 activated seed culture, the pH value of the fermentation system was monitored in real time, and the pH was stabilized at 6.0-6.4 by adding 1 mol / L sodium lactate solution.

[0035] It is understandable that, in preparing the seed culture, the addition of 0.05% glutamine to the GMRS liquid culture medium can enhance the expression of mucosal repair-related genes of Weizmannii coagulans BHE26 and improve its subsequent repair function against the gastric mucosa.

[0036] In this invention, the method for preparing the Lactobacillus paracasei SMN-LBK lyophilized powder is as follows: Add 0.1% volume of fructooligosaccharide to MRS liquid medium, mix, and inoculate with Lactobacillus paracasei SMN-LBK at an inoculum rate of 2%. Culture at 37℃ and 160r / min for 18h, and subculture twice to obtain SMN-LBK activated seed culture. 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant and 0.08% volume of fructooligosaccharide were added to MRS liquid medium. After mixing, the SMN-LBK activated seed culture was inoculated at an inoculum of 2.5%. Fermentation was carried out at 37℃ and 180 r / min for 14 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain SMN-LBK fermentation broth. The SMN-LBK fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Lactobacillus paracasei SMN-LBK bacterial sludge. The Lactobacillus paracasei SMN-LBK bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Lactobacillus paracasei SMN-LBK freeze-dried powder.

[0037] Specifically, the Lactobacillus paracasei SMN-LBK has the accession number CCTCC No. M2017429.

[0038] Specifically, during the fermentation of SMN-LBK activated seed liquid, the pH value of the fermentation system was monitored in real time, and the pH was stabilized at 5.9-6.3 by adding 1 mol / L sodium lactate solution. During this period, a mixed gas of 5% CO2 and 95% N2 was introduced every 3 hours.

[0039] It is understandable that adding 0.1% fructooligosaccharides to MRS liquid culture medium during seed culture preparation can target and nourish Lactobacillus paracasei SMN-LBK, thereby enhancing its activation and proliferation efficiency and subsequent intestinal colonization and microbiota regulation functions.

[0040] In this invention, the method for preparing the Lactobacillus acidophilus NCFM lyophilized powder is as follows: Add 0.03% (v / v) of vitamin B complex to MRS liquid medium, mix, and inoculate with Lactobacillus acidophilus NCFM at an inoculum rate of 2%. Culture at 37℃ and 160 r / min for 22 h, and subculture twice to obtain activated NCFM seed culture. Add 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant and 0.02% volume of B vitamins to MRS liquid medium, mix and inoculate the NCFM activated seed culture at an inoculum of 2.5%, and then ferment at 37℃ and 180 r / min for 18 h. After fermentation, add 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C to the fermentation system and incubate at 30℃ for 30 min to obtain NCFM fermentation broth. The NCFM fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Lactobacillus acidophilus NCFM bacterial sludge. The Lactobacillus acidophilus NCFM bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Lactobacillus acidophilus NCFM freeze-dried powder.

[0041] Specifically, the Lactobacillus acidophilus NCFM accession number is: ATCC SD5221; Specifically, during the fermentation process of NCFM-activated seed liquid, the pH value of the fermentation system was monitored in real time, and the pH was stabilized at 6.1-6.5 by adding 1 mol / L sodium lactate solution. During this period, a mixed gas of 5% CO2 and 95% N2 was introduced every 3 hours.

[0042] Specifically, the B vitamins mentioned can be purchased directly from commercially available B vitamin tablets.

[0043] It is understandable that adding 0.03% B vitamins to MRS liquid culture medium can serve as a key coenzyme in the NCFM metabolic process of Lactobacillus acidophilus, participating in the strain's energy metabolism and substance synthesis, thereby improving its proliferation efficiency during the activation phase and enhancing the strain's resistance to gastrointestinal fluids.

[0044] In this invention, the preparation method of the metabolic adaptor solution is as follows: The R202448 activated seed solution, BHE26 activated seed solution, SMN-LBK activated seed solution and NCFM activated seed solution were mixed in a volume ratio of 1:1:1:1 and incubated at 37°C for 4 hours. The supernatant was collected, filtered through a 0.22 μm filter membrane, and the filtrate was used to obtain the metabolic adaptation solution.

[0045] Understandably, the metabolic adaptation solution contains compatible metabolites of various strains. During the individual fermentation of each strain, the metabolic adaptation solution can provide a "pre-adapted" microbial community metabolic environment for independent fermentation of a single strain, eliminating the potential problems of nutrient competition and metabolic antagonism when multiple strains are combined in the future. At the same time, the common metabolic components in the adaptation solution can help strains optimize their metabolic pathways, improve proliferation efficiency and functional stability, and ensure that each strain can maintain good synergistic compatibility after independent fermentation.

[0046] In this invention, the preparation method of the composite lyophilization protectant is as follows: Water, hydroxyethyl starch, glucosamine, glutathione, and trehalose are mixed and dissolved in a mass ratio of 32:4:2:1:1.

[0047] In this invention, the gradient freeze-drying specifically refers to: Add the corresponding bacterial sludge to the preheated 30℃ composite freeze-drying protectant at a ratio of 1:2 (w / v), stir at 30 r / min for 10 min, then stir at 50 r / min for 10 min to obtain a mixed solution; The mixed solution was frozen at -10°C for 1 hour, then the temperature was lowered to -30°C and frozen for 2 hours, and then the temperature was lowered to -40°C and frozen for 2 hours to obtain pre-frozen bacterial cells; The pre-frozen bacterial cells were freeze-dried at a vacuum of 10 Pa and a temperature of -20°C for 8 hours, then the temperature was raised to 0°C and freeze-dried for 4 hours, and then the temperature was raised to 25°C and freeze-dried for 2 hours.

[0048] Understandably, pre-freezing is performed using a gradual cooling mode to avoid rapid freezing forming large ice crystals that puncture the bacterial cell membrane. Subsequent staged vacuum freeze-drying (-20℃ sublimation drying to remove free water, -0℃ desorption drying to remove bound water, and -25℃ removal of residual moisture) thoroughly reduces the moisture content of the freeze-dried powder and minimizes the loss of active bacterial components during the freeze-drying process.

[0049] In this invention, the prebiotic is composed of fructooligosaccharides, stachyose and gentiosaccharides in a mass ratio of 2:1:1. The gastric mucosa repair enhancer is composed of glutamine, carboxymethyl chitosan and β-glucan in a mass ratio of 2:1:1. The excipients are a compound of maltodextrin, microcrystalline cellulose and magnesium stearate in a mass ratio of 5:3:1.

[0050] Understandably, prebiotics, as the "exclusive nutrient source" for probiotics, have targeted nourishing properties: fructooligosaccharides can precisely promote the proliferation of *Lactobacillus plantarum* R202448 and *Weizmannii coagulans* BHE26, strengthening their colonization ability in the gastric mucosa and the upper part of the intestine; stachyose specifically nourishes *Lactobacillus paracasei* SMN-LBK, helping it to quickly form a dominant flora in the intestine and inhibit the growth of harmful bacteria; gentian oligosaccharides not only enhance the colonization stability of *Lactobacillus acidophilus* NCFM, but also work synergistically with compound freeze-drying protectants to improve the freeze-drying survival rate of strains, while improving the intestinal nutrient metabolic environment, providing support for the function of probiotics. The synergistic effect of these three can significantly improve the survival time and functional efficiency of each strain in vivo, maintaining the durability of the microecological balance.

[0051] Understandably, in the gastric mucosal repair enhancer: glutamine, as a small molecule amino acid, can penetrate the coating layer and be rapidly absorbed by gastric mucosal epithelial cells, directly promoting the proliferation and repair of damaged cells and accelerating mucosal healing; carboxymethyl chitosan can slowly leak through the coating pores, forming a protective film on the gastric mucosal surface that mimics the mucus layer, isolating Hp invasion and gastric acid stimulation, and reducing further mucosal damage; β-glucan can enhance the tight junction function of the gastric mucosal barrier, reduce the penetration of inflammatory factors, and at the same time help regulate the body's local immune response, alleviating gastric inflammation. All three do not rely on probiotic release; they can exert their repairing effect in the stomach simply through the slow-release leakage of the coating.

[0052] Understandably, excipients primarily support formulation formation and stability: maltodextrin, as a filler, can adjust the flowability and compressibility of materials, making the mixture easier to form through a capsule filling machine, while reducing the material's hygroscopicity and extending the product's shelf life; microcrystalline cellulose has both filling and flow-aiding effects, improving mixing uniformity, avoiding capsule filling dosage deviations caused by material clumping, and ensuring consistency in the number of live bacteria and the content of functional ingredients in each capsule; magnesium stearate, as a lubricant, can reduce friction between materials and equipment, preventing sticking during filling and improving production efficiency.

[0053] In this invention, the preparation method of the anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora is specifically as follows: Take Lactobacillus plantarum R202448 lyophilized, Weizmannii coagulans BHE26 lyophilized powder, Lactobacillus paracasei SMN-LBK lyophilized powder and Lactobacillus acidophilus NCFM lyophilized powder, mix them at 15℃ and 30r / min for 10min to obtain probiotic compound lyophilized powder. The probiotic compound freeze-dried powder was mixed with the prebiotic and gastric mucosa repair and enhancement agent at 15°C and a rotation speed of 30 r / min for 8 min to obtain a mixture. The mixture was mixed with 10% Eudragit S100 coating solution at a ratio of 1:0.3 (w / v) and then subjected to fluidized bed coating treatment to obtain pH-sensitive coated particles. The pH-sensitive coated granules were mixed with excipients and filled into empty capsules. The capsules were then placed at 25°C and 30% relative humidity for 48 hours to obtain an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora.

[0054] Specifically, the inlet air temperature of the fluidized bed coating treatment is preferably 40°C, the outlet air temperature is preferably 30°C, and the atomization pressure is preferably 0.3 MPa.

[0055] Understandably, the Eudragit S100 coating does not dissolve in the stomach at pH < 5. However, the coating layer has a porous structure, allowing carboxymethyl chitosan in the gastric mucosal repair enhancer to leak through the coating pores and form a protective film on the gastric mucosa surface that mimics the mucus layer, directly isolating it from Hp invasion and gastric acid stimulation. Glutamine, a small molecule amino acid, can penetrate the coating layer and be rapidly absorbed by gastric mucosal epithelial cells, promoting cell proliferation and damage repair. β-glucan can enhance the tight junction function of the gastric mucosal barrier, reduce the penetration of inflammatory factors, and assist in the repair of damaged mucosa.

[0056] The water used in the embodiments of this invention is sterile water.

[0057] Example 1 Raw material preparation: Preparation of probiotic freeze-dried powder: S1. Add 0.08% volume of mucin and 0.02% volume of sialic acid to MRS liquid culture medium, mix and inoculate with Lactobacillus plantarum R202448 at an inoculation rate of 2%, culture at 37℃ and 180r / min for 20h, and subculture twice to obtain R202448 targeted activation seed liquid. After adding 0.05% volume of glutamine to GMRS liquid medium, Weizmannii coagulans BHE26 was inoculated at an inoculum rate of 2% and cultured at 37℃ and 180 r / min for 24 h. After two subcultures, activated seed culture of BHE26 was obtained. Add 0.1% volume of fructooligosaccharide to MRS liquid medium, mix, and inoculate with Lactobacillus paracasei SMN-LBK at an inoculum rate of 2%. Culture at 37℃ and 160r / min for 18h, and subculture twice to obtain SMN-LBK activated seed culture. Add 0.03% (v / v) of vitamin B complex to MRS liquid medium, mix, and inoculate with Lactobacillus acidophilus NCFM at an inoculum rate of 2%. Culture at 37℃ and 160 r / min for 22 h, and subculture twice to obtain activated NCFM seed culture. S2. Take the R202448 activated seed solution, BHE26 activated seed solution, SMN-LBK activated seed solution and NCFM activated seed solution and mix them in a volume ratio of 1:1:1:1. Incubate at 37°C for 4 hours, collect the supernatant, filter it through a 0.22μm filter membrane, and take the filtrate to obtain the metabolic adaptation solution. S3. Add 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant and 0.05% volume of mucin to MRS liquid medium, mix and inoculate the R202448 targeted activation seed liquid at an inoculation rate of 3%, and then ferment at 37℃ and 180r / min for 12h. During the fermentation, monitor the pH in real time and stabilize the pH at 5.8-6.2 by adding 1mol / L sodium lactate solution. After the fermentation is completed, add 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C to the fermentation system and incubate at 30℃ for 30min to obtain R202448 fermentation broth. 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant, and 0.03% volume of glutamine were added to GMRS liquid medium. After mixing, the BHE26 activated seed culture was inoculated at a 3% inoculum. Fermentation was carried out at 37℃ and 180 r / min for 16 h. During the fermentation, the pH was monitored in real time, and the pH was stabilized at 6.0-6.4 by adding 1 mol / L sodium lactate solution. After the fermentation was completed, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and the mixture was incubated at 30℃ for 30 min to obtain the BHE26 fermentation broth. 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant, and 0.08% volume of fructooligosaccharide were added to MRS liquid medium. After mixing, the SMN-LBK activated seed culture was inoculated at an inoculum rate of 2.5%. Fermentation was carried out at 37℃ and 180 r / min for 14 h. During the fermentation, the pH was monitored in real time, and the pH was stabilized at 5.9-6.3 by adding 1 mol / L sodium lactate solution. At the same time, a mixture of 5% CO2 and 95% N2 gas was introduced every 3 h. After the fermentation was completed, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and the mixture was incubated at 30℃ for 30 min to obtain the SMN-LBK fermentation broth. 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant, and 0.02% volume of B vitamins were added to MRS liquid medium. After mixing, the NCFM activated seed culture was inoculated at an inoculum rate of 2.5%. Fermentation was carried out at 37℃ and 180 r / min for 18 h. During the fermentation, the pH was monitored in real time, and the pH was stabilized at 6.1-6.5 by adding 1 mol / L sodium lactate solution. At the same time, a mixture of 5% CO2 and 95% N2 gas was introduced every 3 h. After the fermentation was completed, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and the mixture was incubated at 30℃ for 30 min to obtain the NCFM fermentation broth. S4. Sterile water, hydroxyethyl starch, glucosamine, glutathione, and trehalose are mixed and dissolved in a mass ratio of 32:4:2:1:1 to obtain a composite freeze-drying protectant. The four fermentation broths were centrifuged at 8000 r / min for 15 min at 4℃, the precipitates were collected, and the precipitates were washed twice with physiological saline to obtain the corresponding bacterial sludge. The bacterial sludge of the corresponding bacterial strains was added to the composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v). The mixture was stirred at 30 r / min for 10 min and then at 50 r / min for 10 min to obtain the mixed solution of the corresponding bacterial strains. The mixed solutions of the corresponding bacterial strains were frozen at -10℃ for 1 hour, then the temperature was lowered to -30℃ for 2 hours, and then the temperature was lowered to -40℃ for 2 hours to obtain pre-frozen bacterial cells. Four types of pre-frozen bacterial cells were freeze-dried at a vacuum of 10 Pa and -20°C for 8 hours, then freeze-dried at 0°C for 4 hours, and then freeze-dried at 25°C for 2 hours to obtain freeze-dried powders of four probiotics.

[0058] Prebiotics: are a blend of fructooligosaccharides, stachyose and gentiosaccharides in a mass ratio of 2:1:1.

[0059] Gastric mucosal repair enhancer: composed of glutamine, carboxymethyl chitosan and β-glucan in a mass ratio of 2:1:1; Excipients: Composed of maltodextrin, microcrystalline cellulose and magnesium stearate in a mass ratio of 5:3:1.

[0060] Preparation method: S1. Take 35 parts of freeze-dried Lactobacillus plantarum R202448, 20 parts of freeze-dried Weizmannii coagulans BHE26, 15 parts of freeze-dried Lactobacillus paracasei SMN-LBK, and 10 parts of freeze-dried Lactobacillus acidophilus NCFM. Mix them at 15℃ and 30r / min for 10min to obtain probiotic compound freeze-dried powder. S2. Mix the probiotic compound freeze-dried powder with 5 parts of prebiotics and 2 parts of gastric mucosa repair and enhancement agent at 15°C and 30 r / min for 8 min to obtain the mixture. The mixture was mixed with 10% Eudragit S100 coating solution at a ratio of 1:0.3 (w / v) and then subjected to fluidized bed coating treatment, wherein the inlet air temperature was 40℃, the outlet air temperature was 30℃, and the atomization pressure was 0.3MPa, to obtain pH-sensitive coated particles. The pH-sensitive coated granules were mixed with 5 parts of excipients and filled into empty capsules. The mixture was then placed at 25°C and 30% relative humidity for 48 hours to obtain an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora.

[0061] Example 2 The only difference from Example 1 is that the mass fraction of each component is as follows: 40 parts of *Lactobacillus plantarum* R202448 lyophilized powder, 25 parts of *Weizmannii coagulans* BHE26 lyophilized powder, 20 parts of *Lactobacillus paracasei* SMN-LBK lyophilized powder, 13 parts of *Lactobacillus acidophilus* NCFM lyophilized powder, 8 parts of prebiotic complex, 3 parts of gastric mucosal repair enhancer, and 8 parts of excipients. Example 3 The only difference from Example 1 is that the mass fraction of each component is 45 parts of *Lactobacillus plantarum* R202448 lyophilized powder, 30 parts of *Weizmannii coagulans* BHE26 lyophilized powder, 25 parts of *Lactobacillus paracasei* SMN-LBK lyophilized powder, 15 parts of *Lactobacillus acidophilus* NCFM lyophilized powder, 10 parts of prebiotic complex, 5 parts of gastric mucosal repair and enhancement agent, and 10 parts of excipients.

[0062] Effect test Test sample: The anti-Helicobacter pylori probiotic composition prepared in Example 2 of this invention for repairing gastric mucosa and regulating intestinal flora was tested and found to contain 5.2 × 10⁻⁶ live bacteria per capsule. 9 CFU Control samples: Control Example 1 (the difference from the test sample is that it was coated), Control Example 2 (a single Lactobacillus plantarum R202448 preparation, prepared by activating, fermenting, freeze-drying and coating Lactobacillus plantarum R202448 according to the method in Example 2 of this invention), and positive control drug (amoxicillin clavulanate potassium dispersible tablets).

[0063] Test strains and cells: Helicobacter pylori Sydney strain SS1 and human gastric mucosal epithelial cells GES-1.

[0064] Laboratory animals: SPF grade C57BL / 6 mice, male, 6-8 weeks old, weighing 18-22g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Key reagents: artificial gastric juice (pH 2.5, containing 0.3% pepsin), MRS medium, Hp medium (Columbia blood agar), cell damage repair detection kit, inflammatory factor ELISA kit (TNF-α, IL-6), etc.

[0065] Test Example 1 (Intragastric Survival Rate Validation Experiment) Sample preparation: Grind and pulverize the samples from Example 2, Control Example 1, and Control Example 2 respectively, take 1g of each, add 10mL of sterile physiological saline, and prepare bacterial suspensions (the initial viable count is uniformly adjusted to 5×10⁻⁶). 9 (CFU / mL) Simulated gastric environment treatment: Take 2 mL of each bacterial suspension, add 8 mL of artificial gastric fluid (pH 2.5, preheated at 37℃), and incubate in a shaker at 37℃ and 100 r / min. Take samples at 0 h, 1 h, 2 h, and 3 h respectively. Viable count: Immediately after sampling, the sample was serially diluted with sterile PBS (pH 7.4) and the viable count was determined by plate counting method (MRS medium, anaerobic culture at 37℃ for 48h). The survival rate was calculated (survival rate = viable count at each time point / viable count at 0h × 100%).

[0066] The test results are shown in Table 1: Table 1 Results of intragastric survival test

[0067] As can be seen, both Example 2 and Control Example 2 of this invention used Eudragit S100 coating, and the gastric survival rate was significantly higher than that of the uncoated version of Control Example 1 (P<0.01). Among them, the survival rate of the test sample after treatment with artificial gastric fluid (pH2.5) for 3 hours reached 72.6±3.8%, which was slightly higher than that of the single R202448 preparation of Control Example 2 (66.8±3.5%). This is because the metabolites of multiple strains synergistically enhanced the acid resistance stability of the bacteria.

[0068] Test Example 2 (Helicobacter pylori (Hp) Inhibition Rate Validation Experiment) (1) Determination of inhibition zone diameter Hp culture: Hp SS1 was inoculated onto Columbia blood agar medium and cultured at 37°C for 72 h in a microaerophilic (5% O2 + 10% CO2 + 85% N2) medium to prepare 1×10⁻⁶ Hp culture medium. 8 CFU / mL bacterial suspension; Sample suspension preparation: Grind the samples from Example 2, Control Example 1, and Control Example 2 separately (passing through a 100-mesh sieve). Take 1g of each sample and add 10mL of sterile PBS (pH 7.0) containing 0.05% Tween 80. Vortex for 10min, then sonicate for 5min to prepare a homogeneous suspension (the viable bacteria concentration was adjusted to 5×10⁻⁶). 9 CFU / mL, no stratification after standing at room temperature for 30 min); Plate preparation: Spread 200 μL of Hp bacterial suspension evenly on Columbia blood agar plates, place Oxford cups (6 mm in diameter), and add 200 μL each of the above sample suspension, positive control drug (amoxicillin clavulanate potassium, 100 μg / mL), and sterile PBS (containing 0.05% Tween 80, blank control); Culture and measurement: Incubate at 37℃ for 48 hours under microaerophilic conditions. Measure the diameter of the inhibition zone (mm) with calipers. Measure each plate three times and take the average value. (Note: Coated particles will not penetrate the agar; only probiotic metabolites diffuse to form the inhibition zone, which does not affect the result determination.)

[0069] (2) Determination of Hp biofilm inhibition rate Biofilm formation: Hp SS1 bacterial suspension (1×10⁻⁶) 7 Add 100 μL of (CFU / mL) to each well of a 96-well plate and incubate at 37°C for 48 h in a microaerophilic environment to form a biofilm. Sample intervention: Discard the supernatant and add the above sample suspension (final concentration after gradient dilution 5×10⁻⁶). 7 (CFU / mL), blank control added with sterile PBS containing 0.05% Tween 80, and cultured for another 24 h; Detection: Crystal violet staining method was used, and the OD570nm value was measured by microplate reader. The biofilm inhibition rate was calculated (inhibition rate = (OD value of blank group - OD value of sample group) / OD value of blank group × 100%).

[0070] The test results are shown in Table 2: Table 2 Results of Helicobacter pylori (Hp) inhibition rate test

[0071] As can be seen, the inhibition zone diameter (19.6±0.8 mm) and biofilm inhibition rate (98.3±1.2%) of the sample in Example 2 of this invention against *Hp SS1* were significantly higher than those of the single R202448 formulation in Control Example 2 (17.3±0.7 mm, 88.6±1.9%), and the MIC value was lower (2.5×10⁻⁶). 6(CFU / mL); while the test results of the sample in Example 2 were not much different from those of the control sample in Example 1, because the coating is mainly to cope with the strong acidic environment of gastric acid. After grinding, the active ingredients such as probiotics and prebiotics inside the test sample were similar to those in the uncoated sample.

[0072] Test Example 3 (Experiment to Verify the Effect of Gastric Mucosal Repair) (1) Cell model experiment (GES-1 cell damage repair) Cell culture: GES-1 cells were seeded in 6-well plates in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2 until confluence reached 80%. Cell damage: Add Hp toxin VacA (final concentration 2 μg / mL), incubate for 6 h to establish a gastric mucosal cell damage model; Sample extraction preparation: The samples from Example 2 and Control Example 2 were ground into powders, and 1 g of each was added to 10 mL of sterile physiological saline. The mixture was shaken at 37°C and 100 rpm for 24 h, then centrifuged at 4°C and 8000 rpm for 15 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain an extract containing metabolites (final concentration of 1 × 10⁻⁶ for both samples). 7 (CFU / mL corresponds to the concentration of metabolites) Sample intervention: Discard the culture medium containing toxins, add the above extract to each sample, and add physiological saline without sample to the blank control group. Incubate for 24 hours. Detection indicators: cell viability (CCK-8 assay); scratch healing rate (scratch method, measuring scratch width at 0h and 24h).

[0073] The test results are shown in Table 3: Table 3 Results of GES-1 cell damage repair test

[0074] (2) Animal model experiment (repair of gastric mucosa in Hp-infected mice) Animal grouping: Sixty C57BL / 6 mice were randomly divided into 6 groups (n=10): blank control group (no Hp infection, saline), model control group (Hp infection, saline), and low-dose sample group of Example 2 (Hp infection, 1×10⁻⁶). 9 CFU / animal / day), Example 2 sample high-dose group (Hp infection, 5×10 9 CFU / animal / day), Control Group 2 (Hp infection, 5×10 9 CFU / animal / day), positive control group (Hp infection, amoxicillin clavulanate potassium 100mg / kg / day); Hp infection modeling: Except for the blank control group, mice in the other groups were orally administered Hp SS1 bacterial suspension (1×10⁻⁶). 8CFU / 0.2mL), once daily for 7 consecutive days, infection was confirmed 14 days post-infection (positive Hp culture in gastric tissue). Drug intervention: After successful modeling, mice in each group were administered the drug orally by gavage (coated samples were administered directly by gavage without dispersion; the coating layer dissolved in the intestine after digestion to release the strain), once daily for 21 consecutive days. Detection indicators: gastric mucosal injury index (observation of gastric mucosal erosion and ulceration after dissection, scored according to Guth criteria); pathological histological score (HE staining, observation of mucosal epithelial integrity and degree of inflammatory cell infiltration); inflammatory factor levels (ELISA method to detect TNF-α and IL-6 content in gastric tissue).

[0075] The test results are shown in Table 4: Table 4 Results of gastric mucosal repair test in Hp-infected mice

[0076] As shown in Tables 3 and 4, in the cell model, the extract of the sample from Example 2 of this invention significantly improved the survival rate (89.3±4.1%) and scratch healing rate (85.7±3.2%) of VacA-damaged GES-1 cells, which were significantly higher than those of the single R202448 preparation in Control Example 2 (68.9±4.3%, 65.2±3.1%, respectively). In the animal model, the coated sample could release the bacterial strain normally in the intestine after oral gavage. The gastric mucosal damage index, pathological score, and inflammatory factor levels in the high-dose group of Example 2 of this invention were significantly lower than those in the single R202448 group in Control Example 2, approaching the effect of the positive control drug.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the specific implementation of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A probiotic composition for repairing gastric mucosa and regulating intestinal flora against Helicobacter pylori, characterized in that, The components include the following parts by mass: The product contains 35-45 parts of *Lactobacillus plantarum* R202448 lyophilized powder, 20-30 parts of *Weizmannii coagulans* BHE26 lyophilized powder, 15-25 parts of *Lactobacillus paracasei* SMN-LBK lyophilized powder, 10-15 parts of *Lactobacillus acidophilus* NCFM lyophilized powder, 5-10 parts of prebiotic complex, 2-5 parts of gastric mucosal repair and enhancement agent, and 5-10 parts of excipients.

2. The anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to claim 1, characterized in that, The preparation method of the freeze-dried Lactobacillus plantarum R202448 powder is as follows: Add 0.08% volume of mucin and 0.02% volume of sialic acid to MRS liquid medium, mix and inoculate with Lactobacillus plantarum R202448 at an inoculation rate of 2%, culture at 37℃ and 180 r / min for 20 h, and subculture twice to obtain R202448 targeted activation seed liquid. 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant and 0.05% volume of mucin were added to MRS liquid medium. After mixing, the R202448 targeted activation seed liquid was inoculated at an inoculum rate of 3%. Fermentation was carried out at 37℃ and 180 r / min for 12 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain the R202448 fermentation broth. The R202448 fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Bacillus plantarum R202448 bacterial sludge. The Bacillus plantarum R202448 bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Bacillus plantarum R202448 freeze-dried powder.

3. The anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to claim 1, characterized in that, The method for preparing the freeze-dried powder of *Weizmannii coagulatedis* BHE26 is as follows: After adding 0.05% volume of glutamine to GMRS liquid medium, Weizmannii coagulans BHE26 was inoculated at an inoculum rate of 2% and cultured at 37℃ and 180 r / min for 24 h. After two subcultures, activated seed culture of BHE26 was obtained. 1% volume of tryptone, 0.5% volume of yeast extract, 20% volume of metabolic adaptant and 0.03% volume of glutamine were added to GMRS liquid medium. After mixing, the BHE26 activated seed culture was inoculated at an inoculum rate of 3%. Fermentation was carried out at 37℃ and 180 r / min for 16 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain BHE26 fermentation broth. The BHE26 fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain BHE26 coagulated sludge. The BHE26 coagulated sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain BHE26 freeze-dried powder.

4. The anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to claim 1, characterized in that, The preparation method of the Lactobacillus paracasei SMN-LBK lyophilized powder is as follows: Add 0.1% volume of fructooligosaccharide to MRS liquid medium, mix, and inoculate with Lactobacillus paracasei SMN-LBK at an inoculum rate of 2%. Culture at 37℃ and 160r / min for 18h, and subculture twice to obtain SMN-LBK activated seed culture. 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant and 0.08% volume of fructooligosaccharide were added to MRS liquid medium. After mixing, the SMN-LBK activated seed culture was inoculated at an inoculum of 2.5%. Fermentation was carried out at 37℃ and 180 r / min for 14 h. After fermentation, 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C were added to the fermentation system and mixed. The mixture was then incubated at 30℃ for 30 min to obtain SMN-LBK fermentation broth. The SMN-LBK fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Lactobacillus paracasei SMN-LBK bacterial sludge. The Lactobacillus paracasei SMN-LBK bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Lactobacillus paracasei SMN-LBK freeze-dried powder.

5. The method for preparing an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to claim 1, characterized in that, The method for preparing the Lactobacillus acidophilus NCFM lyophilized powder is as follows: Add 0.03% (v / v) of vitamin B complex to MRS liquid medium, mix, and inoculate with Lactobacillus acidophilus NCFM at an inoculum rate of 2%. Culture at 37℃ and 160 r / min for 22 h, and subculture twice to obtain activated NCFM seed culture. Add 1% volume of tryptone, 0.3% volume of yeast extract, 15% volume of metabolic adaptant and 0.02% volume of B vitamins to MRS liquid medium, mix and inoculate the NCFM activated seed culture at an inoculum of 2.5%, and then ferment at 37℃ and 180 r / min for 18 h. After fermentation, add 0.1% L-cysteine ​​hydrochloride and 0.05% vitamin C to the fermentation system and incubate at 30℃ for 30 min to obtain NCFM fermentation broth. The NCFM fermentation broth was centrifuged at 8000 r / min for 15 min at 4℃, the precipitate was collected, and washed twice with physiological saline to obtain Lactobacillus acidophilus NCFM bacterial sludge. The Lactobacillus acidophilus NCFM bacterial sludge was added to a composite freeze-drying protectant preheated to 30℃ at a ratio of 1:2 (w / v) and subjected to gradient freeze-drying to obtain Lactobacillus acidophilus NCFM freeze-dried powder.

6. The method for preparing the anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to any one of claims 1-5, characterized in that, The preparation method of the metabolic adaptor solution is as follows: The R202448 activated seed solution, BHE26 activated seed solution, SMN-LBK activated seed solution and NCFM activated seed solution were mixed in a volume ratio of 1:1:1:1 and incubated at 37°C for 4 hours. The supernatant was collected, filtered through a 0.22 μm filter membrane, and the filtrate was used to obtain the metabolic adaptation solution.

7. The method for preparing the anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to any one of claims 1-5, characterized in that, The preparation method of the composite freeze-drying protectant is as follows: Water, hydroxyethyl starch, glucosamine, glutathione, and trehalose are mixed and dissolved in a mass ratio of 32:4:2:1:

1.

8. A method for preparing an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to any one of claims 1-5, characterized in that, The gradient freeze-drying specifically refers to: Add the corresponding bacterial sludge to the preheated 30℃ composite freeze-drying protectant at a ratio of 1:2 (w / v), stir at 30 r / min for 10 min, then stir at 50 r / min for 10 min to obtain a mixed solution; The mixed solution was frozen at -10°C for 1 hour, then the temperature was lowered to -30°C and frozen for 2 hours, and then the temperature was lowered to -40°C and frozen for 2 hours to obtain pre-frozen bacterial cells; The pre-frozen bacterial cells were freeze-dried at a vacuum of 10 Pa and a temperature of -20°C for 8 hours, then the temperature was raised to 0°C and freeze-dried for 4 hours, and then the temperature was raised to 25°C and freeze-dried for 2 hours.

9. The method for preparing the anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to claim 8, characterized in that, The prebiotic is composed of fructooligosaccharides, stachyose and gentiosaccharides in a mass ratio of 2:1:

1. The gastric mucosa repair enhancer is composed of glutamine, carboxymethyl chitosan and β-glucan in a mass ratio of 2:1:

1. The excipients are compounded from maltodextrin, microcrystalline cellulose and magnesium stearate in a mass ratio of 5:3:

1.

10. The anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora according to claim 1, characterized in that, The specific preparation method of the anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora is as follows: Take Lactobacillus plantarum R202448 lyophilized, Weizmannii coagulans BHE26 lyophilized powder, Lactobacillus paracasei SMN-LBK lyophilized powder and Lactobacillus acidophilus NCFM lyophilized powder, mix them at 15℃ and 30r / min for 10min to obtain probiotic compound lyophilized powder. The probiotic compound freeze-dried powder was mixed with the prebiotic and gastric mucosa repair and enhancement agent at 15°C and a rotation speed of 30 r / min for 8 min to obtain a mixture. The mixture was mixed with 10% Eudragit S100 coating solution at a ratio of 1:0.3 (w / v) and then subjected to fluidized bed coating treatment to obtain pH-sensitive coated particles. The pH-sensitive coated granules were mixed with excipients and filled into empty capsules. The capsules were then placed at 25°C and 30% relative humidity for 48 hours to obtain an anti-Helicobacter pylori probiotic composition for repairing gastric mucosa and regulating intestinal flora.

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