Probiotic against helicobacter pylori infection and use thereof

Abstract

This invention belongs to the field of probiotic technology, specifically relating to a probiotic for combating Helicobacter pylori infection and its application. The probiotic is Limosilactobacillus reuteri ZKAW05, derived from Tibetan yak milk residue, with the preservation number GDMCC NO: 65682. This probiotic exhibits good acid resistance, bile salt resistance, and Helicobacter pylori inhibition. After being cultured in an inducible culture medium, it is used to prepare probiotic powders, capsules, or tablets for combating Helicobacter pylori infection. It can maintain good probiotic activity in the gastrointestinal tract and is used to prevent or alleviate Helicobacter pylori infection in the oral cavity, gastrointestinal tract, and other sites.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to a probiotic that combats Helicobacter pylori infection and its application. Background Technology

[0002] Helicobacter pylori infection, a widespread chronic bacterial infection, is a significant cause of chronic active gastritis, peptic ulcers, and gastric mucosa-associated lymphoid tissue lymphoma, and is a recognized key risk factor in the development of gastric cancer. Currently, the problem of Helicobacter pylori resistance caused by antibiotic treatment is becoming increasingly serious, leading to a continuous decline in the eradication rate of traditional therapies. At the same time, the series of side effects of antibiotic therapy, such as intestinal flora imbalance, nausea, and diarrhea, not only reduce patient adherence but may also cause new health problems. This treatment dilemma has prompted the research and industry communities to continuously explore safer, gentler alternative or adjuvant treatment strategies that are less likely to induce drug resistance.

[0003] Probiotics are considered a highly promising antibacterial pathway due to their inherent functions in regulating the balance of the gut microbiota, inhibiting pathogen colonization, and enhancing the host's immune barrier. However, not all lactic acid bacteria or probiotics possess direct and highly effective anti-Helicobacter pylori activity. Common probiotic strains generally have low survival rates in the highly acidic environment of the stomach, making it difficult to ensure a sufficient number of viable bacteria reach and colonize the intestinal or gastric mucosal ecological sites. Therefore, they cannot fully exert their expected biological antagonistic and immunomodulatory effects, failing to meet the specific clinical needs for assisting in the eradication of Helicobacter pylori.

[0004] Therefore, there is an urgent need to find a new type of natural active probiotic that can effectively intervene in Helicobacter pylori infection, tolerate gastrointestinal environmental stress, and have good colonization ability. Developing new probiotic products is of great significance and application prospects for breaking through existing treatment bottlenecks, enriching biological therapy options, and improving public health. Summary of the Invention

[0005] In response to the above situation, the present invention provides a probiotic that resists Helicobacter pylori infection and its application. The probiotic strain with antibacterial properties is isolated and screened from Tibetan milk residue, and induced in a modified culture medium to prepare a probiotic product with high efficacy against Helicobacter pylori.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a probiotic for combating Helicobacter pylori infection. The probiotic is Limosilactobacillus reuteri ZKAW05, isolated from Tibetan yak milk residue, and deposited on December 26, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Its taxonomic name is Limosilactobacillus reuteri, and its viability test result is positive. The accession number is GDMCC NO: 65682.

[0008] Furthermore, the method for isolating and identifying the probiotics that resist Helicobacter pylori infection is as follows:

[0009] S1: Mix Tibetan yak milk residue and sterile physiological saline at a mass-to-volume ratio of 1:9 to obtain a milk residue suspension. Dilute the milk residue suspension using a 10-fold serial dilution method and spread it on MRS solid medium containing 1% CaCO3 and 10 ppm actinomycete ketone. Incubate anaerobicly at 37°C for 48 h. Pick single colonies with clear zones and draw lines on the MRS solid medium for purification. Repeat the operation until the colony growth characteristics on the MRS solid medium are consistent to obtain purified colonies with different characteristics.

[0010] S2: Select purified colonies with different characteristics, perform Gram staining and observe the bacterial cells to distinguish Gram-positive strains. Further perform catalase experiment on the Gram-positive strains to screen out Gram-positive and catalase-negative strains, i.e. lactic acid bacteria characteristic strains.

[0011] S3: Take the characteristic strains of lactic acid bacteria and inoculate them into MRS liquid medium. After activation for 3 generations, centrifuge and discard the supernatant to obtain bacterial sludge. Add sterile physiological saline to the bacterial sludge and wash to obtain the test strains. Identify the genus of the test strains to obtain the lactic acid bacteria strains.

[0012] S4: 16S rDNA sequencing, sequence alignment, and homology analysis were performed on the Lactobacillus strain to obtain a novel Lactobacillus strain, Limosilactobacillus reuteri ZKAW05, which is a probiotic that resists Helicobacter pylori infection.

[0013] This invention provides the application of probiotics that combat Helicobacter pylori infection in the preparation of antibacterial products, wherein the antibacterial products are probiotic powders, probiotic capsules, or probiotic tablets.

[0014] Furthermore, the probiotics that resist Helicobacter pylori infection are used in the preparation of antibacterial products in the form of probiotic freeze-dried powder, which is prepared by culturing probiotics in an induction medium and then freeze-drying them.

[0015] Furthermore, the induction medium comprises the following raw materials: glycerol, monigrin, L-cysteine, taurine, lactoferrin, and MRS broth medium, wherein the mass ratio of glycerol, monigrin, L-cysteine, taurine, lactoferrin, and MRS broth medium is 2:0.13:0.5:1:1.5:1000.

[0016] Furthermore, the preparation method of the probiotic freeze-dried powder is as follows:

[0017] Y1: The probiotics against Helicobacter pylori infection obtained after isolation and identification were streaked onto MRS agar medium using an inoculation loop. Single colonies were obtained by culturing and then picked and inoculated into MRS broth medium for activation culture for 18 h to obtain activated bacterial solution.

[0018] Y2: The activated bacterial solution was transferred to fresh MRS broth medium at a 1% inoculum and cultured to the logarithmic growth phase for amplification to obtain a seed culture. The seed culture was then inoculated into an induction medium at a 4% inoculum for anaerobic culture. Glycerol in the induction medium served as a substrate for the probiotics to efficiently synthesize antibacterial substances such as reuterin. Mangiferin served as an inducer for its synthesis pathway. L-cysteine ​​helped promote the anaerobic growth of probiotics and served as a precursor for bacteriocin synthesis. Bovine bile salts and lactoferrin were designed to induce the stress tolerance mechanism of the strain in advance, so that the probiotics could better survive and function in the gastrointestinal tract, thus obtaining the induction solution.

[0019] Y3: Centrifuge the induction solution at 4℃ and 8000 rpm for 10 min, discard the supernatant and collect the precipitate. Add skim milk and sodium glutamate as freeze-drying protectants to the precipitate for freeze-drying to obtain probiotic freeze-dried powder.

[0020] Furthermore, the mass fractions of skim milk and monosodium glutamate in the probiotic freeze-dried powder are 5% and 1%, respectively, and the number of live probiotics in the probiotic freeze-dried powder is 300 billion CFU / g.

[0021] Furthermore, the probiotic powder comprises the following raw materials in parts by weight: 0.3 parts of lyophilized probiotic powder, 7 parts of maltodextrin, 6 parts of fructooligosaccharides, 0.2 parts of sucrose, 0.1 parts of citric acid, and 0.05 parts of silicon dioxide. The specific preparation method is as follows: 7 parts of maltodextrin are pulverized and sieved, and then premixed with 0.3 parts of lyophilized probiotic powder to obtain a premix. 6 parts of fructooligosaccharides and 0.2 parts of sucrose are pulverized and sieved, and then mixed with the premix for 5 min. Then, 0.1 parts of citric acid and 0.05 parts of silicon dioxide are added and mixed for another 10 min to obtain the probiotic powder.

[0022] Furthermore, the probiotic capsule is composed of capsule powder and hydroxypropyl methylcellulose enteric-coated capsule shell. The capsule powder comprises the following raw materials in parts by weight: 1 part of probiotic freeze-dried powder, 1 part of lactoferrin, 2.5 parts of fructooligosaccharide, 15 parts of maltodextrin, 1.4 parts of microcrystalline cellulose, and 0.1 parts of silica. The specific preparation method is as follows: 2.5 parts of fructooligosaccharide and 15 parts of maltodextrin are pulverized and sieved, and then mixed with 1 part of probiotic freeze-dried powder for 5 min. 1 part of lactoferrin and 1.4 parts of microcrystalline cellulose are added and the mixture is continued to be mixed for 10 min to obtain a preliminary mixture. The preliminary mixture and 0.1 parts of silica are mixed for 5 min and then filled into hydroxypropyl methylcellulose enteric-coated capsule shells to obtain probiotic capsules.

[0023] Furthermore, the probiotic tablets comprise the following ingredients in parts by weight: 0.2 parts lyophilized probiotic powder, 8 parts sorbitol, 3 parts isomaltitol, 2 parts xylitol, 0.05 parts natural menthol, 0.4 parts citric acid, 0.2 parts malic acid, 0.1 parts sweet orange flavor powder, and 0.1 parts magnesium stearate. The specific preparation method is as follows:

[0024] Step 1: Mix 3 parts isomaltitol, 2 parts xylitol, 0.4 parts citric acid and 0.2 parts malic acid, then pulverize and sieve to obtain premix A. Pulverize and sieve 8 parts sorbitol and mix it evenly with 0.2 parts probiotic freeze-dried powder to obtain premix B.

[0025] Step 2: Mix premix A and premix B for 10 min, add 0.05 parts of natural menthol and 0.1 parts of sweet orange flavor powder and mix for 5 min, then add 0.1 parts of magnesium stearate and continue mixing for 3 min to obtain a mixed powder;

[0026] Step 3: Place the mixed powder into a tablet press and compress it into tablets to obtain probiotic tablets.

[0027] The beneficial effects achieved by this invention are as follows:

[0028] The probiotic ZKAW05 (Limosilactobacillus reuteri ZKAW05) provided by this invention, which combats Helicobacter pylori infection, is derived from Tibetan yak milk residue. After rigorous isolation, screening, and identification, it exhibits excellent anti-Helicobacter pylori activity, effectively inhibiting the growth and colonization of the pathogen, providing a new biological solution for the prevention and adjuvant treatment of related infections. Furthermore, culturing it in an inducible culture medium not only provides nutrients for bacterial growth but also effectively stimulates the metabolic potential of the probiotic ZKAW05 through specific components, prompting the probiotic to synthesize large amounts of antibacterial substances with direct inhibitory effects, such as reuterin. Simultaneously, it induces a stress tolerance mechanism in response to the gastrointestinal environment, resulting in a strain with stronger resistance to gastric acid and bile and a higher colonization survival rate, thus greatly enhancing its actual efficacy against pathogens in the complex in vivo environment. In addition, ZKAW05 also exhibits good gastrointestinal tolerance, resisting the stress of gastric acid and bile salts, maintaining a high survival rate in the digestive tract, which facilitates its successful arrival at the site of action and the exertion of its therapeutic effect. Based on ZKAW05 probiotics, various dosage forms such as powders, capsules, and lozenges have been developed. These products are convenient to use, have a wide range of applications, and can effectively prevent and inhibit Helicobacter pylori infection in the oral cavity, stomach, and other parts of the body. They provide efficient, safe, and stable probiotic products for daily health care and adjunctive intervention. Attached Figure Description

[0029] Figure 1 Results of microscopic morphological investigation of Limosilactobacillus reuteri ZKAW05;

[0030] Figure 2 Gel electrophoresis results of PCR amplification products of DNA extracted from Limosilactobacillus reuteri ZKAW05;

[0031] Figure 3 Results of the antibacterial activity study of Limosilactobacillus reuteri ZKAW05 against Helicobacter pylori;

[0032] Figure 4 The results of the hemolytic safety study of Limosilactobacillus reuteri ZKAW05. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0035] Unless otherwise specified, all methods used in the following examples are conventional. Unless otherwise specified, all materials used in the following examples are new materials purchased from the market, and all quantities are by weight. The induction culture media used in the following examples and comparative examples were prepared from the following raw materials by weight: 20 parts glycerol, 1.3 parts mangiferin, 5 parts L-cysteine, 10 parts ox bile salts, 15 parts lactoferrin, and 10,000 parts MRS broth medium. The probiotic freeze-dried powder used contains 5% and 1% by mass of skim milk and monosodium glutamate, respectively, and the number of live probiotics in the probiotic freeze-dried powder is 300 billion CFU / g. The hydroxypropyl methylcellulose enteric-coated capsule shell used is enteric-coated No. 1.

[0036] Example 1: Isolation and identification of Limosilactobacillus reuteri ZKAW05

[0037] S1: Take 10 parts of Tibetan yak milk residue and 90 parts of sterile physiological saline, mix and stir evenly to obtain milk residue suspension. Dilute the milk residue suspension with a 10-fold serial dilution method. After dilution to 1000 times, obtain the diluted solution. Spread the diluted solution on MRS solid medium containing 1% CaCO3 and 10 ppm actinomycete ketone. Incubate anaerobicly at 37°C for 48 h. Pick single colonies with clear zones and streak them on MRS solid medium for purification. Repeat the operation until the colony growth characteristics on MRS solid medium are consistent to obtain purified colonies with different characteristics.

[0038] S2: Select purified colonies with different characteristics, perform Gram staining, and observe the size, shape, and arrangement of the bacteria to distinguish Gram-positive strains. Further perform catalase experiments on the Gram-positive strains to screen out Gram-positive and catalase-negative strains, which are temporarily identified as lactic acid bacteria strains.

[0039] S3: The characteristic strains of lactic acid bacteria were inoculated into MRS liquid medium and activated for 3 generations. After centrifugation at 4℃ and 8000rpm for 10 min, the supernatant was discarded to obtain bacterial sludge. Sterile physiological saline was added to the bacterial sludge for washing to obtain the test strains. The test strains were subjected to catalase test, acid tolerance test and salt tolerance test to identify the genus. The pH of the acid tolerance test was 3.0 and the salt tolerance test was 3 g / L porcine bile salt. A total of 11 rod-shaped lactic acid bacteria strains were screened.

[0040] S4: The 11 selected rod-shaped lactic acid bacteria strains were subjected to Helicobacter pylori inhibition zone investigation, and 4 strains with anti-Helicobacter pylori were selected. The 4 selected strains were subjected to 16S rDNA sequencing, sequence alignment and homology analysis, and a novel lactic acid bacteria strain, Limosilactobacillus reuteri ZKAW05, was obtained, which is a probiotic against Helicobacter pylori infection.

[0041] Example 2: 16S rDNA sequencing of Lactobacillus strains

[0042] R1: The 11 strains of Lactobacillus rod-shaped bacteria obtained in step S3 of Example 1 were activated and cultured to obtain activated bacterial suspensions. The activated bacterial suspensions were centrifuged at 10,000 rpm for 1 min, the supernatant was discarded, and bacterial sludge was obtained. DNA was extracted from each bacterial sludge sample using a DNA extraction kit.

[0043] R2: PCR amplification was performed on the extracted DNA from each sample. Primers 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3', amplifying a fragment of approximately 1500 bp. The reaction mixture consisted of: 0.2 μL each of forward and reverse primers, 5 μL of 10×PCR Buffer, 1 μL of dNTP Mix, 3 μL of MgCl2, 0.2 μL of Tap enzyme, 1 μL of template DNA, and 39.4 μL of ultrapure water. The amplification program was: 95℃ for 5 min; 95℃ for 30 s; 55℃ for 30 s; 72℃ for 2 min, 35 cycles; 72℃ for 10 min, 4℃ incubation, yielding the PCR amplification product.

[0044] R3: The PCR amplification products were detected by gel electrophoresis and homology sequencing was performed to determine the corresponding probiotic species, resulting in a novel probiotic strain with anti-Helicobacter pylori function, namely Limosilactobacillus reuteri ZKAW05.

[0045] Example 3: This example provides a probiotic freeze-dried powder, which comprises the following raw materials in parts by weight:

[0046] Y1: Limosilactobacillus reuteri ZKAW05 was streaked onto MRS agar medium using an inoculation loop, placed in an anaerobic culture jar, and cultured anaerobically at 37℃ for 48 h to obtain a single colony. A single colony was picked and inoculated into 50 mL of MRS broth medium, and activated anaerobically at 37℃ for 18 h to obtain an activated bacterial solution.

[0047] Y2: The activated bacterial solution was transferred to fresh MRS broth medium at an inoculation rate of 1%, and anaerobic cultured at 37°C for 12 h. The OD600 of the culture medium was 0.94, indicating that it was in the logarithmic growth phase, thus obtaining the seed culture. The seed culture was then inoculated into the induction medium at an inoculation rate of 4% and anaerobic cultured for 36 h. After two subcultures, the induction solution was obtained.

[0048] Y3: Centrifuge the induction solution at 4℃ and 8000 rpm for 10 min, discard the supernatant and collect the precipitate. Resuspend the precipitate in 0.05M pH 7.0 PBS buffer pre-cooled at 4℃, wash, centrifuge, discard the supernatant, and add skim milk and sodium glutamate as freeze-drying protectants to the precipitate for freeze-drying to obtain probiotic freeze-dried powder.

[0049] Example 4: This example provides the application of probiotics against Helicobacter pylori infection in the preparation of probiotic powder. The probiotic powder comprises the following raw materials in parts by weight: 0.3 parts of lyophilized probiotic powder, 7 parts of maltodextrin, 6 parts of fructooligosaccharides, 0.2 parts of sucrose, 0.1 parts of citric acid, and 0.05 parts of silicon dioxide. The specific preparation method is as follows:

[0050] Weigh 7 parts of maltodextrin, pulverize it and pass it through an 80-mesh sieve, then premix it with 0.3 parts of freeze-dried probiotic powder for 3 minutes to obtain a premix. Pulverize 6 parts of fructooligosaccharide and 0.2 parts of sucrose, pass them through an 80-mesh sieve, and mix them with the premix for 5 minutes. Then add 0.1 parts of citric acid and 0.05 parts of silicon dioxide and continue mixing for 10 minutes to obtain a dry powder. Package the dry powder to obtain probiotic powder.

[0051] Example 5: This example provides the application of probiotics against Helicobacter pylori infection in the preparation of probiotic capsules. The probiotic capsules are composed of capsule powder and hydroxypropyl methylcellulose enteric-coated capsule shells. The capsule powder comprises the following raw materials in parts by weight: 1 part lyophilized probiotic powder, 1 part lactoferrin, 2.5 parts fructooligosaccharides, 15 parts maltodextrin, 1.4 parts microcrystalline cellulose, and 0.1 parts silica. The specific preparation method is as follows:

[0052] 2.5 parts of fructooligosaccharide and 15 parts of maltodextrin were pulverized and passed through an 80-mesh sieve. They were then mixed with 1 part of lyophilized probiotic powder for 5 minutes. 1 part of lactoferrin and 1.4 parts of microcrystalline cellulose were added and the mixture was continued for 10 minutes to obtain a preliminary mixture. The preliminary mixture was then mixed with 0.1 parts of silica for 5 minutes. The mixture was then filled into hydroxypropyl methylcellulose enteric-coated capsule shells at a ratio of 0.35 g / capsule to obtain probiotic capsules.

[0053] Example 6: This example provides the application of probiotics against Helicobacter pylori infection in the preparation of probiotic lozenges. The probiotic lozenges comprise the following raw materials in parts by weight: 0.2 parts of freeze-dried probiotic powder, 8 parts of sorbitol, 3 parts of isomaltitol, 2 parts of xylitol, 0.05 parts of natural menthol, 0.4 parts of citric acid, 0.2 parts of malic acid, 0.1 parts of sweet orange flavor powder, and 0.1 parts of magnesium stearate. The specific preparation method is as follows:

[0054] Step 1: Mix 3 parts isomaltitol, 2 parts xylitol, 0.4 parts citric acid and 0.2 parts malic acid, then pulverize and pass through an 80-mesh sieve to obtain premix A. Pulverize 8 parts sorbitol and pass through an 80-mesh sieve, then mix with 0.2 parts probiotic freeze-dried powder for 5 min to obtain premix B.

[0055] Step 2: Mix premix A and premix B for 10 min, add 0.05 parts of natural menthol and 0.1 parts of sweet orange flavor powder and mix for 5 min, then add 0.1 parts of magnesium stearate and continue mixing for 3 min to obtain a mixed powder;

[0056] Step 3: Place the mixed powder in a tablet press and compress it into tablets at a specification of 0.5 g / tablet to obtain probiotic tablets.

[0057] Morphological examination of probiotics: The morphological characteristics of Limosilactobacillus reuteri ZKAW05 were observed using optical microscopy and SEM (scanning electron microscopy). The results are shown in the figure. Figure 1 .

[0058] Gel electrophoresis detection: The PCR amplification products of Limosilactobacillus reuteri ZKAW05 were analyzed by gel electrophoresis. The standard reference was DNA Marker, and the negative control was no PCR amplification product. The results are shown in the figure. Figure 2 .

[0059] Physiological and biochemical tests of probiotics:

[0060] Acid tolerance test: After activating the isolated Limosilactobacillus reuteri ZKAW05 for 2 generations, single colonies were picked and inoculated into MRS broth medium and cultured for 24 h as seed culture. The seed culture was then inoculated into MRS broth medium sterilized at 115℃ for 30 min and pH 3 at a 2% inoculation rate and incubated at 37℃. The absorbance at 600 nm was measured at 0 h and 3 h to evaluate the acid tolerance of the probiotic. The results are shown in Table 1.

[0061] Salt tolerance test: Limosilactobacillus reuteri ZKAW05 seed culture was inoculated at a rate of 2% into MRS broth medium containing 3 g / L porcine bile salts after being autoclaved at 115℃ for 30 min. The medium was incubated at 37℃ for 4 h. The absorbance at 600 nm was measured at 0 h and 4 h to evaluate the salt tolerance of the probiotic. The results are shown in Table 1.

[0062] Physiological and biochemical tests were performed on Limosilactobacillus reuteri ZKAW05 according to the "Manual of Systematic Identification of Common Bacteria", including sugar fermentation test, indole test, methyl red test, catalase test, citrate test, hydrogen sulfide test, starch hydrolysis test and nitrate reduction test. The results are shown in Table 1.

[0063] Antibacterial activity of probiotics was investigated: Using Helicobacter pylori as an indicator bacterium, 2% of the Limosilactobacillus reuteri ZKAW05 seed culture was inoculated into fresh MRS broth and incubated at 37°C for 24 h. The probiotic concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL, indicator bacteria were incubated overnight in nutrient broth at 37°C with shaking, and the concentration was adjusted to 1×10⁻⁶. 7 CFU / mL, the indicator bacterial suspension was evenly spread onto NA plates with wells in Oxford cups, and 200 μL of probiotic suspension was added to each well. The plates were incubated at 37°C for 24 h, and the diameter of the inhibition zone was observed and recorded. Results are shown below. Figure 3 .

[0064] Hemolytic activity test: Using Staphylococcus aureus as the standard reference strain, the control (Staphylococcus aureus) and ZKAW05 (Limosilactobacillus reuteri ZKAW05) were streaked on blood agar plates and incubated at 37°C for 24 h. The hemolytic activity of the probiotics was observed and determined. The results are shown in […]. Figure 4 .

[0065] Table 1. Biochemical reaction identification results of ZKAW05

[0066]

[0067] Figure 1 The results showed that the Gram cells of Limosilactobacillus reuteri ZKAW05 were rod-shaped, and scanning electron microscopy revealed that the cells were rod-shaped with a length of 2-3 μm and a width of 0.5-1 μm.

[0068] Figure 2 The results showed that the molecular weight of the PCR amplification product of Limosilactobacillus reuteri ZKAW05 was approximately 1500 bp, indicating that the PCR amplification was successful.

[0069] Figure 3 The results showed that no Helicobacter pylori colonies formed in the Oxford cup wells containing Limosilactobacillus reuteri ZKAW05 bacterial suspension, indicating that the probiotics had good inhibitory effect on Helicobacter pylori.

[0070] Figure 4 The results showed that Staphylococcus aureus, as a control, exhibited strong hemolysis on blood agar plates, with clear hemolytic rings appearing around the formed colonies, while no clear rings were observed around Limosilactobacillus reuteri ZKAW05 colonies, indicating good biocompatibility and safety.

[0071] Table 1 shows that Limosilactobacillus reuteri ZKAW05 exhibits good acid and bile salt tolerance. The survival rate was 107% after 3 hours at pH 3 and 44% after 4 hours in a high-salt environment, demonstrating strong acid and bile salt tolerance, making it suitable for oral administration. It also maintains bacterial activity in the acidic environment of the stomach. Furthermore, the catalase test result in the biochemical assay was negative, indicating that Limosilactobacillus reuteri ZKAW05 does not produce catalase, a key characteristic of Lactobacillus.

[0072] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0073] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A freeze-dried powder of probiotic bacteria against Helicobacter pylori infection, characterized in that, The probiotic in the probiotic freeze-dried powder is Limosilactobacillus reuteri ZKAW05, and the preservation number is GDMCC NO: 65682. The probiotic freeze-dried powder is prepared by inducing the culture medium, and freeze-drying the probiotic; The inducing culture medium comprises the following raw materials in a mass ratio: 2:0.13:0.5:1:1.5:1000 of glycerol, formononetin, L-cysteine, bovine bile salt, lactoferrin and MRS broth medium; The specific preparation method of the probiotic freeze-dried powder is as follows: Y1: Activating the probiotic to obtain an activated bacterial solution; Y2: The activated bacterial solution is amplified and inoculated into the inducing culture medium to obtain an induced liquid; Y3: The induced liquid is centrifuged to collect the precipitate, and defatted milk and sodium glutamate are added to the precipitate and freeze-dried to obtain the probiotic freeze-dried powder.

2. Use of the freeze-dried probiotic powder against H. pylori infection according to claim 1 for the preparation of an antibacterial product, characterized by the fact that, The antibacterial product is a probiotic granule, a probiotic capsule or a probiotic tablet, and the antibacterial product is a product against Helicobacter pylori.

3. Use according to claim 2, characterized in that, The probiotic tablet comprises the following raw materials in parts by weight: 0.2 parts of the probiotic freeze-dried powder of claim 1, 8 parts of sorbitol, 3 parts of isomalt, 2 parts of xylitol, 0.05 parts of natural menthol, 0.4 parts of citric acid, 0.2 parts of malic acid, 0.1 parts of sweet orange essence powder and 0.1 parts of magnesium stearate, and the specific preparation method is as follows: Step 1: Mix isomalt, xylitol, citric acid and malic acid, crush and sieve to obtain premix A, and mix the crushed sorbitol with the probiotic freeze-dried powder of claim 1 to obtain premix B; Step 2: Mix premix A and premix B, and then add natural menthol, sweet orange essence powder and magnesium stearate in sequence to obtain a mixed powder; Step 3: The mixed powder is tableted to obtain the probiotic tablet.

4. The probiotic lyophilized powder according to claim 1 or the use according to any of claims 2-3, characterized in that, The number of viable probiotic bacteria in the probiotic freeze-dried powder is 300 billion CFU / g.

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