Preparation of probiotic fermented composition of medicinal and edible Chinese herbs against helicobacter pylori

Abstract

This invention provides a method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori, comprising: preparing a fermentation culture broth of traditional Chinese medicine, dissolving probiotics, expanding the probiotic culture, preparing the fermentation broth of medicinal and edible herbal probiotics for combating Helicobacter pylori, and preparing the fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori. It also provides an application, wherein the fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori is used to prepare a drug for improving oral Helicobacter pylori infection. The fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori of this invention achieves the purpose of targeted anti-Helicobacter pylori infection by enriching characteristic metabolite groups with anti-HP activity and reducing the toxic side effects of traditional Chinese medicine extracts.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to the preparation method and application of a fermentation composition of medicinal and edible herbal probiotics that combats Helicobacter pylori. Background Technology

[0002] Helicobacter pylori (Hp) is a Gram-negative microaerophilic bacterium that colonizes the gastric mucosa. It primarily enters the stomach through the oral route and specifically adheres to the surface of gastric epithelial cells. Once colonized, it is difficult for the body's immune system to spontaneously clear it, leading to active inflammation of the gastric mucosa. Epidemiological data shows that Hp is one of the most prevalent chronic bacterial pathogens in the population and is closely related to the occurrence of chronic gastritis, peptic ulcers, and gastric cancer. Approximately 80% of patients with chronic gastritis are infected with Hp, far higher than in the general population. Unlike general gastritis, the causative factor of Hp-positive chronic gastritis is clearly Hp infection. This bacterium can remain latent in the gastric mucosa for a long time, continuously inducing chronic inflammatory responses and increasing the risk of cardiovascular disease by interfering with lipid metabolism. The World Health Organization (WHO) has classified Hp as a Group 1 carcinogen, confirming its leading role in the development of gastric cancer. Helicobacter pylori (Hp) infection can gradually progress from active inflammation to chronic gastritis, chronic atrophic gastritis, intestinal metaplasia, and dysplasia, ultimately leading to serious diseases such as gastric cancer, posing a significant threat to health. Due to the highly insidious nature of Hp, if it cannot be completely eradicated, it can easily cause recurrent episodes of chronic gastritis, forming a malignant pathological chain of "infection-inflammation-precancerous lesions," severely restricting patients' quality of life and increasing the medical burden.

[0003] *Helicobacter pylori* (Hp) is significantly distributed in saliva, dental plaque, periodontal pockets, and oral mucosa. The failure rate of Hp eradication in the stomach is significantly higher in oral Hp-positive patients than in oral Hp-negative patients. The presence of oral Hp can cause local symptoms such as halitosis, oral ulcers, and chronic pharyngitis. More importantly, oral Hp exhibits high infectivity, transmissibility, and drug resistance. The oral environment provides a relatively stable microecological niche for Hp, and the biofilm structure on the tooth surface protects Hp from host immune clearance. Oral Hp can be transmitted between people through saliva, utensils, and kissing, making it a significant source of clustered infections within families. The oral biofilm structure makes Hp highly resistant to conventional antibiotics, hindering drug penetration and effectiveness.

[0004] Current clinical *Helicobacter pylori* (Hp) eradication regimens are centered on a "quadruple therapy" in the stomach (proton pump inhibitor + bismuth agent + two antibiotics), but this approach has significant drawbacks: antibiotic resistance is increasing year by year (clarithromycin resistance rate >20%, metronidazole resistance rate >40%), leading to an eradication rate below 80%; long-term use of broad-spectrum antibiotics causes adverse reactions such as intestinal flora imbalance and secondary infections; more importantly, existing regimens mainly focus on gastric eradication, while seriously neglecting the crucial role of the oral cavity as an important reservoir and transmission route for Hp. After gastric Hp eradication treatment, if oral Hp is not simultaneously eradicated, it can recolonize in the gastric mucosa through continuous swallowing, causing infection "re-entry".

[0005] In addition, currently, in the field of probiotics against Helicobacter pylori, Novozymes of Denmark mainly monopolizes the core patent of Lactobacillus reuteri DSM17648 (Pylopass®). This strain is excreted from the body through physical copolymerization of Hp, but it cannot eradicate the infection. Domestic patents are mostly focused on the antibacterial application of single strains or complex bacteria such as Bifidobacterium animalis and Weizmannii coagulans, lacking breakthrough solutions. In the field of single-herb anti-Hp in traditional Chinese medicine, active ingredients such as Coptis chinensis (berberine), Scutellaria baicalensis (baicalin), and patchouli alcohol have been widely studied, but patents mostly focus on the in vitro antibacterial activity of extracts, which suffer from technical bottlenecks such as low bioavailability, bitter and cold nature that can damage the stomach, and difficulty in quality control. In the field of compound anti-Hp in traditional Chinese medicine, existing patents are mainly oral preparations or oral care products composed of licorice, dandelion, and Corydalis yanhusuo, which have certain safety advantages, but the curative effect is slow, cannot eradicate Hp alone, and still face the problem of drug resistance when used in combination with antibiotics. Crucially, the technology of "probiotic fermentation of traditional Chinese medicine" is currently in a patent vacuum period. Existing technologies have failed to address core challenges such as low dissolution rates of active ingredients in traditional Chinese medicine, unclear synergistic mechanisms between probiotics and traditional Chinese medicine, and a lack of standardized quality control. Simultaneously, due to the lack of a simultaneous eradication strategy for oral *H. pylori*, the "oral-gastric" transmission cycle persists. *H. pylori* eradicated in the stomach can re-infect via oral reflux, becoming a structural reason for the persistently high recurrence rate. Therefore, establishing effective intervention methods for oral *H. pylori* and achieving synergistic prevention and control between the "oral" and "gastric" systems is a good approach and a key breakthrough for solving recurrent *H. pylori* infections and improving eradication rates. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing a fermented composition of medicinal and edible herbal probiotics that is effective against Helicobacter pylori by addressing the shortcomings of the prior art. This fermented composition of medicinal and edible herbal probiotics that is effective against Helicobacter pylori by enriching characteristic metabolite groups with anti-HP activity and reducing the toxic side effects of traditional Chinese medicine extracts.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a fermented composition of medicinal and edible herbal probiotics that combats Helicobacter pylori, the method being as follows: S1. Licorice, houttuynia cordata, purslane, dandelion, poria cocos, honeysuckle, mulberry, mint and ginger are mixed to obtain a medicinal and edible herbal composition. After being pulverized, the mixture is soaked in water and extracted at 100°C. After filtration, filtrate a is obtained. Water is added to the remaining residue and extracted at 100°C. After filtration, filtrate b is obtained. The filtrates are combined, concentrated under reduced pressure, a carbon source is added, and the pH is adjusted to 7.0. After sterilization, a fermentation culture broth for traditional Chinese medicine is obtained. S2, Probiotic Dissolution: S201. Add Lactobacillus plantarum CICC 22195 to sterile physiological saline, shake to dissolve, and obtain Lactobacillus plantarum suspension. S202. Add Lactobacillus reuteri CICC 6118 to sterile physiological saline, shake to dissolve, and obtain Lactobacillus reuteri bacterial suspension. S203. Add Lactococcus lactis CICC 20090 to sterile physiological saline, shake to dissolve, and obtain Lactococcus lactis suspension. S204. Add Bifidobacterium animalis CICC 21711 to sterile physiological saline, shake to dissolve, and obtain a suspension of Bifidobacterium animalis. S3, Probiotic culture expansion: S301. The *Lactobacillus plantarum* suspension obtained in S201 is inoculated into MRS liquid culture medium at a volume ratio of 1%:1. After mixing evenly, the medium is sealed and incubated at 30℃ for 24 hours to obtain expanded culture of *Lactobacillus plantarum*. S302. The Lactobacillus reuteri suspension obtained in S202 was inoculated into MRS liquid medium at a volume ratio of 1%:1. After mixing evenly, the medium was sealed and incubated at 30°C for 24 hours to obtain the expanded Lactobacillus reuteri suspension. S303. The lactococcus lactis suspension obtained in S203 is inoculated into MRS liquid culture medium at a volume ratio of 1%:1. After mixing evenly, the medium is sealed and incubated at 30℃ for 24 hours to obtain the expanded culture of lactococcus lactis suspension. S304. The animal bifidobacterium suspension obtained in S204 was inoculated into MRS liquid medium at a volume ratio of 1%:1. After mixing evenly, the medium was sealed and incubated at 30°C for 24 hours to obtain the expanded animal bifidobacterium suspension. S305. The expanded culture of Lactobacillus plantarum obtained in S301, the expanded culture of Lactobacillus reuteri obtained in S302, the expanded culture of Lactococcus lactis obtained in S303, and the expanded culture of Bifidobacterium animalis obtained in S304 are mixed to obtain an expanded culture probiotic suspension. This invention utilizes the extracellular enzyme system of probiotics to catalyze the in vitro directed transformation of large molecules in traditional Chinese medicine (such as polysaccharides, proteins, and glycosides) into easily absorbed forms such as small-molecule active aglycones and oligosaccharides, significantly improving bioavailability and pharmacological activity. *Lactobacillus plantarum* efficiently hydrolyzes flavonoid glycosides with β-glucosidase to generate highly active aglycones; *Bifidobacterium animalis* converts saponins into aglycones using α-galactosidase and specific glycosidases; *Lactobacillus reuteri* uses glycoside hydrolases and transferases to convert oligosaccharides into functional oligosaccharides and short-chain fatty acids; and *Lactococcus lactis* degrades proteins through the PrtP extracellular protease system to produce active peptides, ultimately collectively enhancing the bioavailability and pharmacological activity of the traditional Chinese medicine components. S4. The probiotic suspension obtained in S305 was inoculated into the traditional Chinese medicine fermentation culture broth obtained in S1 at a volume ratio of 1%:1. The broth was cultured at 30℃ for 48 hours to obtain the probiotic fermentation broth of traditional Chinese medicine with anti-Helicobacter pylori properties. In this invention, the organic acids and esters produced by fermentation metabolism can significantly improve the bitter taste of traditional Chinese medicine and enhance patient compliance. Lactobacillus plantarum is the main producer of organic acids such as lactic acid, acetic acid, and malic acid, as well as floral and fruity esters such as ethyl phenylacetate and ethyl acetate. Bifidobacterium animalis produces short-chain fatty acids such as acetic acid, propionic acid, and butyric acid through "bifidogenic diversion" metabolism, which imparts a mild sour aroma. Lactobacillus reuteri converts phenylalanine to produce characteristic organic acids such as indole-3-lactic acid and 4-hydroxyphenyllactic acid, and participates in the metabolism of terpene precursors. Lactococcus lactis synthesizes creamy and nutty flavor substances such as diacetyl, acetaldehyde, and 2,3-butanediol. In the compound fermentation system, glycyrrhizin and flavonoids are hydrolyzed by glycosidases to produce glycyrrhetinic acid, isoflavone aglycones, and esters such as isobutyl formate; houttuynia cordata volatile oil and decanoyl acetaldehyde are converted into decyl acetate and other mild aroma components under the action of esterases; purslane polysaccharides are degraded to produce oligosaccharides, lactic acid, and 1-octen-3-ol and other green aroma substances; dandelion inulin is converted by bifidobacteria to produce fructose, acetic acid, and terpene alcohols; and poria cocos triterpenes and polysaccharides are hydrolyzed to release poria cocos... The compound system contains roasted aromas such as linguic acid, lactic acid, and benzaldehyde; honeysuckle chlorogenic acid ester bonds break to generate quinic acid, caffeic acid, and phenethyl acetate; mulberry anthocyanins and sugars co-ferment to produce sweet aromas such as lactic acid, acetic acid, phenylethyl alcohol, and furfural; menthol and menthone are modified by oxidoreductases to form cooling derivatives such as menthol esters and citronellol; gingerol and gingerol are transformed into spicy flavors such as gingerone, gingerol, and ginger acetate under the action of proteases and esterases. The final composite system will form a three-dimensional flavor structure with a sour base, a sweet aftertaste, a spicy essence, and a refreshing quality, combining the original flavor of traditional Chinese medicine with the mellow aroma of fermentation. S5. The fermentation broth of medicinal and edible probiotics against Helicobacter pylori obtained in S4 is centrifuged, ultrafiltered, and aseptically dispensed to obtain a fermentation composition of medicinal and edible probiotics against Helicobacter pylori.

[0008] Preferably, the medicinal and edible herbal composition described in S1 is made from the following raw materials in parts by weight: 10 parts licorice, 25 parts houttuynia cordata, 15 parts purslane, 15 parts dandelion, 15 parts poria cocos, 15 parts honeysuckle, 15 parts mulberry, 10 parts peppermint, and 5 parts ginger.

[0009] Preferably, the mass ratio of the medicinal and edible herbal composition to water in S1 is 1:8; when water is added to the remaining filter residue, the mass ratio of the remaining filter residue to water is 1:10.

[0010] Preferably, the carbon source in S1 is maltose; the final concentration of the carbon source in the traditional Chinese medicine fermentation culture medium is 10 mg / mL.

[0011] Preferably, the sterilization conditions in S1 are: 121°C for 30 minutes.

[0012] Preferably, the MRS liquid culture medium described in S301 to S304 is prepared by adding the following raw materials to distilled water at a final concentration, adjusting the pH to 7.0, sterilizing at 121°C for 30 minutes, and then cooling to 28°C to 40°C: glucose 20g / L, peptone 10g / L, yeast powder 4g / L, beef powder 8g / L, ammonium citrate 2g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, and polysorbate-80 1g / L.

[0013] Preferably, the probiotic suspension in S305 is composed of a mixture of expanded cultured Lactobacillus plantarum suspension, expanded cultured Lactobacillus reuteri suspension, expanded cultured Lactococcus lactis suspension, and expanded cultured Bifidobacterium animalis suspension in a volume ratio of 1:1:1:1.

[0014] The present invention also provides the application of the anti-Helicobacter pylori medicinal and edible herbal probiotic fermentation composition prepared by the above preparation method, wherein the anti-Helicobacter pylori medicinal and edible herbal probiotic fermentation composition is used to prepare a drug for improving oral Helicobacter pylori.

[0015] Preferably, the dosage form of the drug includes capsules, tablets, or granules.

[0016] Compared with the prior art, the present invention has the following advantages: 1. The medicinal and edible herbal probiotic fermentation composition of the present invention, which is effective against Helicobacter pylori, reduces the toxic side effects of the herbal extract by enriching characteristic metabolite groups with anti-HP activity, thereby achieving the purpose of targeted anti-Helicobacter pylori infection.

[0017] 2. The raw materials of the probiotic fermented traditional Chinese medicine composition of the present invention include water extracts of medicinal and edible herbs, probiotic liquid and its characteristic metabolites such as organic acids and polysaccharides. This preparation strategy combines the advantages of traditional Chinese medicine theory and modern biotransformation technology: on the one hand, it completely preserves the full component matrix of the water extract of traditional Chinese medicine, maintaining the multi-target synergistic characteristics of the compound decoction; on the other hand, it utilizes the extracellular enzyme system of probiotics to catalyze the in vitro directional transformation of large molecules of traditional Chinese medicine (such as polysaccharides, proteins, and glycosides) to generate easily absorbed forms such as small molecule active aglycones and oligosaccharides, significantly improving bioavailability and pharmacological activity. During the fermentation process, the probiotic metabolism produces abundant antibacterial substances. At the same time, the flavor substances such as organic acids and esters produced by fermentation metabolism can significantly improve the bitter taste of traditional Chinese medicine and improve patient compliance. The composition described in this invention can be applied to the development of multiple dosage forms: for Helicobacter pylori infection in the stomach, oral preparations such as capsules, tablets, granules, and oral liquids can be prepared; for Helicobacter pylori infection in the oral cavity, topical preparations such as sprays, mouthwashes, and periodontal gels can be developed to achieve synergistic prevention and control of "oral-gastric" infection.

[0018] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This figure shows the determination of the inhibition rate of Escherichia coli by the traditional Chinese medicine fermentation culture medium (TCM) before and after probiotic fermentation and the fermented composition of medicinal and edible traditional Chinese medicine probiotics (TMB) against Helicobacter pylori in Example 2 of this invention, using gradient dilutions. In the figure, ns indicates no significant difference, and **** indicates a significant difference at the P<0.0001 level.

[0020] Figure 2 This invention relates to Example 3, which describes the determination of the inhibition rate of Helicobacter pylori against Helicobacter pylori using gradient dilutions of the traditional Chinese medicine fermentation culture medium (TCM) before and after probiotic fermentation and the anti-Helicobacter pylori traditional Chinese medicine probiotic fermentation composition (TMB). In the figure, ns indicates no significant difference, * indicates P<0.01, ** indicates P<0.005, *** indicates P<0.001, and **** indicates significant difference at the P<0.0001 level.

[0021] Figure 3 The results are obtained by scanning electron microscopy observation of the effects of the blank control group (A), the TCM treatment group (B) before and after probiotic fermentation, and the TMB treatment group (C) on the morphology of Helicobacter pylori in Example 4 of the present invention.

[0022] Figure 4 This figure shows the acute toxicity test (72h cumulative mortality rate) of the traditional Chinese medicine fermentation culture medium (TCM) before and after probiotic fermentation and the anti-Helicobacter pylori traditional Chinese medicine probiotic fermentation composition (TMB) against Caenorhabditis elegans in Example 5 of the present invention against Caenorhabditis elegans. In the figure, ns indicates no significant difference, *** indicates a significant difference at the P<0.001 level, and **** indicates a significant difference at the P<0.0001 level. Detailed Implementation

[0023] Example 1

[0024] The preparation method of the medicinal and edible herbal probiotic fermentation composition for combating Helicobacter pylori in this embodiment is as follows: S1. Licorice root, houttuynia cordata, purslane, dandelion, poria cocos, honeysuckle, mulberry, mint, and ginger are mixed to obtain a medicinal and edible herbal composition. After pulverizing, the mixture is soaked in water and extracted at 100℃ for 30 minutes. Filtrate a is obtained by filtration. Water is added to the remaining residue, and the mixture is extracted at 100℃ for 30 minutes. Filtrate b is obtained by filtration. The filtrates are combined, concentrated under reduced pressure to 500 mL, a carbon source (maltose) is added, and the pH is adjusted to 7.0. After sterilization at 121℃ for 30 minutes, the temperature was lowered to room temperature to obtain the traditional Chinese medicine fermentation culture medium, labeled TCM, for later use; the final concentration of maltose in the traditional Chinese medicine fermentation culture medium was 10 mg / mL; the mass ratio of the medicinal and edible traditional Chinese medicine composition to water was 1:8; when water was added to the remaining filter residue, the mass ratio of the remaining filter residue to water was 1:10; the medicinal and edible traditional Chinese medicine composition was made from the following raw materials in parts by weight: licorice 10g, houttuynia cordata 25g, purslane 15g, dandelion 15g, poria cocos 15g, honeysuckle 15g, mulberry 15g, peppermint 10g, and ginger 5g; S2, Probiotic Dissolution: S201. Add Lactobacillus plantarum (CICC 22195) to sterile physiological saline, shake to dissolve, and obtain Lactobacillus plantarum suspension; S202. Add Lactobacillus reuteri (CICC 6118) to sterile physiological saline, shake to dissolve, and obtain Lactobacillus reuteri bacterial suspension; S203. Add Lactococcus lactis (CICC 20090) to sterile physiological saline, shake to dissolve, and obtain Lactococcus lactis suspension; S204. Add Bifidobacterium animalis (CICC 21711) to sterile physiological saline, shake to dissolve, and obtain a suspension of Bifidobacterium animalis. The *Lactobacillus plantarum* (CICC 22195), *Lactobacillus reuteri* (CICC 6118), *Lactococcus lactis* (CICC 20090), and *Bifidobacterium animalis* (CICC 21711) used in this embodiment were commercially available from the China Industrial Microbial Culture Collection Center (CICC) (Beijing, China), with *Bifidobacterium animalis* being *Bifidobacterium animalis* CICC21711. S3, Probiotic culture expansion: S301. The *Lactobacillus plantarum* suspension obtained in S201 is inoculated into MRS liquid culture medium at a volume ratio of 1%:1. After mixing evenly, the medium is sealed and incubated at 30℃ for 24 hours to obtain expanded culture of *Lactobacillus plantarum*. S302. The Lactobacillus reuteri suspension obtained in S202 was inoculated into MRS liquid medium at a volume ratio of 1%:1. After mixing evenly, the medium was sealed and incubated at 30°C for 24 hours to obtain the expanded Lactobacillus reuteri suspension. S303. The lactococcus lactis suspension obtained in S203 is inoculated into MRS liquid culture medium at a volume ratio of 1%:1. After mixing evenly, the medium is sealed and incubated at 30℃ for 24 hours to obtain the expanded culture of lactococcus lactis suspension. S304. The animal bifidobacterium suspension obtained in S204 was inoculated into MRS liquid medium at a volume ratio of 1%:1. After mixing evenly, the medium was sealed and incubated at 30°C for 24 hours to obtain the expanded animal bifidobacterium suspension. S305. Mix equal volumes of the expanded culture of Lactobacillus plantarum obtained in S301, the expanded culture of Lactobacillus reuteri obtained in S302, the expanded culture of Lactococcus lactis obtained in S303, and the expanded culture of Bifidobacterium animalis obtained in S304 to obtain an expanded culture probiotic suspension. S4. The probiotic suspension obtained in S305 was inoculated into the traditional Chinese medicine fermentation culture broth obtained in S1 at a volume ratio of 1%:1. The broth was cultured at 30℃ for 48 hours to obtain the probiotic fermentation broth of traditional Chinese medicine with anti-Helicobacter pylori properties. The MRS liquid culture medium described in S301 to S304 is prepared by adding the following raw materials to distilled water to adjust the pH to 7.0, sterilizing at 121℃ for 30 min, and then cooling to 28℃ to 40℃: glucose 20g / L, peptone 10g / L, yeast powder 4g / L, beef powder 8g / L, ammonium citrate 2g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, and polysorbate-80 1g / L; S5. The fermentation broth of medicinal and edible probiotics against Helicobacter pylori obtained in S4 is centrifuged at 12000 rpm for 10 min, ultrafiltered, and aseptically dispensed to obtain the fermentation composition of medicinal and edible probiotics against Helicobacter pylori, designated as TMB.

[0025] This embodiment also provides the application of the anti-Helicobacter pylori medicinal and edible herbal probiotic fermentation composition prepared by the above preparation method. The anti-Helicobacter pylori medicinal and edible herbal probiotic fermentation composition is used to prepare a drug to improve oral Helicobacter pylori; the dosage form of the drug includes capsules, tablets or granules. Example 2

[0026] This embodiment demonstrates the anti-Escherichia coli proliferation effect of the medicinal and edible herbal probiotic fermentation composition (TMB) prepared in Example 1 against Helicobacter pylori, with the traditional Chinese medicine fermentation culture broth (TCM) prepared in step S1 of Example 1 serving as a control.

[0027] The anti-e.g., antiproliferative activity of the TCM and TMB compositions prepared by the above method was determined using the micro-broth dilution method. E. coli were inoculated into LB liquid medium and cultured in a shaker at 37°C and 180 r / min until the strain entered the logarithmic growth phase. The bacterial concentration was finally adjusted to 2 × 10⁻⁶ using LB medium. 6 CFU / mL was used as the stock solution for the tested Escherichia coli for later use.

[0028] A 96-well microplate was used. The experimental groups were as follows: ① Blank control group: 200 µL LB medium; ② Negative control group: 100 µL E. coli culture + 100 µL LB medium; ③ Positive control group: 100 µL E. coli culture + 100 µL kanamycin sulfate (final concentration 50 µg / mL); ④ Sample control group: 100 µL LB medium + 100 µL test sample (diluted 4, 8, 16, and 32 times with TCM or TMB, respectively); ⑤ Experimental group: 100 µL E. coli culture + 100 µL test sample (diluted 0, 4, 8, 16, and 32 times with TCM or TMB, respectively). Each group had 5 replicates. The total volume per well was 200 µL, and the plates were incubated at 37℃ for 24 h. After incubation, the absorbance was measured at 600 nm using a microplate reader, the data were recorded, and the inhibition rate was calculated. The statistical results are shown below. Figure 1 As shown. To eliminate the interference of the sample's own color on the absorbance detection and to ensure the accuracy and comparability of experimental data, this study subtracted the background absorbance value of the control group from the absorbance value of the experimental group to obtain the corrected absorbance, and calculated the antibacterial rate based on this (antibacterial rate = [1 - (corrected absorbance of experimental group / absorbance of negative control group - absorbance of blank control group)] × 100%).

[0029] The results showed that the fermented probiotic composition of traditional Chinese medicine (TCM) with anti-Helicobacter pylori properties (TMB) exhibited significant antibacterial activity against Escherichia coli (inhibition rate 98%, P<0.01), while the TCM fermentation broth at the same concentration showed no significant antibacterial effect (inhibition rate <5%). TMB maintained significant antibacterial activity at a 16-fold dilution (inhibition rate 98%, P<0.05), and the inhibition rate was 25% at a 32-fold dilution, showing a clear dose-response relationship. TCM at the same dilutions showed no significant antibacterial activity (inhibition rate <5%). These results confirm that the probiotic fermentation process can significantly activate or enrich the antibacterial active ingredients in TCM, producing a synergistic effect. Example 3

[0030] This embodiment demonstrates the anti-Helicobacter pylori proliferation effect of the medicinal and edible herbal probiotic fermentation composition (TMB) prepared in Example 1, with the traditional Chinese medicine fermentation culture broth (TCM) prepared in step S1 of Example 1 before fermentation serving as a control.

[0031] The concentration of the glycerol-preserved bacterial culture was determined using the McFarland turbidimetric method. The culture was diluted to a McFarland turbidimetric level (approximately 1.5 × 10⁻⁶) with BHI medium (tryptone 10 g / L, bovine heart extract 17.5 g / L, glucose 2 g / L, sodium chloride 5 g / L, disodium hydrogen phosphate 2.5 g / L, pH 7.4, 10% fetal bovine serum) to a McFarland turbidimetric level of 0.5 (approximately 1.5 × 10⁻⁶). 8 Prepare a working bacterial solution (CFU / mL) for later use.

[0032] A 96-well microplate was used, and the experimental groups were as follows: ① Blank control group: 200µL BHI medium; ② Negative control group: 100µL working bacterial solution + 100µL BHI medium; ③ Positive control group: 100µL BHI medium + 100µL working bacterial solution + quadruple antibiotics (clarithromycin 4µg / mL + amoxicillin 40µg / mL + omeprazole 20µg / mL + bismuth potassium citrate 5µg / mL); ④ Sample control group: 100µL working bacterial solution + 100µL test sample (diluted 0, 4, 8, 16, and 32 times with TCM or TMB, respectively); ⑤ Experimental group: 100µL working bacterial solution + 100µL test sample (diluted 0, 4, 8, 16, and 32 times with TCM or TMB, respectively), with 5 replicates per group. The total volume of each well was 200 µL, and the cultures were incubated at 37°C in a tri-gas incubator (microaerobic conditions: 5% O2, 10% CO2, 85% N2) for 72 h. After incubation, the viability of Helicobacter pylori was determined using the MT colorimetric method: 20 μL of MT solution (5 mg / mL) was added to each well, mixed well, and incubated at 37°C for another 4 h. The absorbance (OD) at 570 nm was then measured using a microplate reader. 570 Record the data and calculate the antibacterial rate.

[0033] Statistical results are as follows Figure 2 As shown. To eliminate the interference of the sample's own color on the absorbance detection and to ensure the accuracy and comparability of experimental data, this study subtracted the background absorbance value of the control group from the absorbance value of the experimental group to obtain the corrected absorbance, and calculated the antibacterial rate based on this (antibacterial rate = [1 - (corrected absorbance of experimental group / absorbance of negative control group - absorbance of blank control group)] × 100%).

[0034] The results showed that the fermented composition of medicinal and edible herbal probiotics (TMB) and the same concentration of traditional Chinese medicine fermentation broth (TCM) exhibited significant activity against Helicobacter pylori, with an inhibition rate of 96%, which was basically consistent with the positive control. The test results for Helicobacter pylori after sample dilution are as follows: Figure 2 As shown, the pure Chinese medicine liquid (TCM) rapidly lost its inhibitory activity as the dilution ratio increased, while the fermented Chinese medicine liquid TMB, after being diluted 4 times, achieved a 95% inhibition rate against Helicobacter pylori, and after being diluted 8 times, it still maintained considerable activity (60% inhibition rate), showing a significantly better sustained anti-Helicobacter pylori efficacy than the pure Chinese medicine liquid. Example 4

[0035] This example illustrates the effects of the fermented medicinal and edible herbal probiotic composition (TMB) prepared in Example 1 on the morphology of Helicobacter pylori before and after fermentation, specifically the effects of TCM and TMB on the morphology of Helicobacter pylori.

[0036] Take Helicobacter pylori in the logarithmic growth phase, and measure it at 0.5 McFarland turbidity (approximately 1.5 × 10⁻⁶). 8 CFU / mL was inoculated into BHI liquid medium (containing 10% fetal bovine serum), and the experimental groups were as follows: ① Blank control group: BHI medium + bacterial suspension; ② TCM treatment group: BHI medium + bacterial suspension + TCM fermentation culture medium (final concentration was 16 times diluted from the mother liquor); ③ TMB treatment group: BHI medium + bacterial suspension + TMB fermentation composition of medicinal and edible traditional Chinese medicine (final concentration was 16 times diluted from the mother liquor), and cultured in a 37℃ three-gas incubator (microaerobic conditions: 5% O2, 10% CO2, 85% N2) for 72 h.

[0037] After cultivation, the bacterial pellets of each group were collected by centrifugation at 7000 rpm for 10 min at 4 °C. The pellets were washed three times (5 mL each time) with 0.01 M PBS (pH 7.2-7.4) to remove residual culture medium and drugs. Subsequently, the pellets were fixed at 4 °C for 24 h with a 2.5% glutaraldehyde-paraformaldehyde mixed fixative (prepared with 2.5% glutaraldehyde + 2% paraformaldehyde + PBS).

[0038] After fixation, the bacterial cells were washed three times with PBS and dehydrated with ethanol in the following gradient: 30% ethanol for 15 min → 50% ethanol for 15 min → 70% ethanol for 15 min → 85% ethanol for 15 min → 95% ethanol for 15 min → 100% ethanol twice (20 min each time). Subsequently, the ethanol was replaced twice with tert-butanol (20 min each time), pre-frozen at -20℃, and then freeze-dried for 24 h.

[0039] The dried sample was sputter-coated with gold (approximately 10 nm thick) and observed under a scanning electron microscope (SEM, model: Hitachi SU8010) with an accelerating voltage of 5-10 kV to acquire typical field-of-view images.

[0040] like Figure 3 As shown, the blank control group ( Figure 3 In sample A), the surface of Helicobacter pylori is smooth and the cells are plump; after TCM treatment, the cells ( Figure 3 The morphology of Hp (Chemical B) cells was not significantly different from that of the blank control group, and the surface was intact, suggesting that a 16-fold dilution of the TCM fermentation broth had no significant effect on the morphology of Hp; TMB treatment of the cells (Chemical B) Figure 3 After C), obvious morphological damage occurs, manifested as: surface wrinkling and roughness, local membrane structure damage, and some bacteria becoming spherical or lysed fragments. This result directly confirms that the fermented composition of medicinal and edible herbal probiotics (TMB) against Helicobacter pylori can exert its antibacterial effect by disrupting the cell wall / membrane integrity of Helicobacter pylori, while the same concentration of traditional Chinese medicine fermentation culture medium (TCM) has no such effect, suggesting that the fermentation process significantly enhances the activity of traditional Chinese medicine against Helicobacter pylori. Example 5

[0041] This embodiment examines the safety of the TCM and TMB compositions of the anti-Helicobacter pylori probiotic fermentation composition (TMB) prepared in Example 1 before and after fermentation. The following efficacy tests were conducted using a Caenorhabditis elegans model.

[0042] Caenorhabditis elegans (N2 Bristol wild type) was inoculated with Escherichia coli OP50 as a food source on nematode growth medium (NGM) plates and cultured in a constant temperature incubator at 20±0.5℃. The nematodes used in the experiment were synchronized L4 stage larvae, at which stage the nematodes' digestive system is fully developed, sensitive to drug stimulation, and easy to observe for survival. The experimental groups and treatments are as follows: ① Blank control group: K solution (32 mM KCl, 51 mM NaCl, pH 7.0); ② Experimental group (TMB, TCM): TMB, a fermented combination of medicinal and edible traditional Chinese medicine probiotics against Helicobacter pylori, and TCM fermentation culture broth, diluted 16 times with K solution; ③ Positive control group: Qingyoule (commercially available anti-Helicobacter pylori functional food), diluted 16 times with K solution.

[0043] A 96-well microplate was used, with 200 μL of the corresponding group's solution added to each well. Ten L4-stage synchronized nematodes were then placed in each well, with three replicates per group. The microplates were incubated in a constant temperature incubator at 20±0.5℃ in the dark. The number of dead nematodes was observed and recorded under a stereomicroscope every 24 h, with the experimental period lasting 72 h. Mortality was determined by: no response to mechanical stimulation of platinum wire, body rigidity, and the appearance of granular structures or signs of dissolution within the nematodes.

[0044] The results are as follows Figure 4 As shown, the TCM group exhibited the highest toxicity, with a 72-hour mortality rate of 99%. The TMB group showed significantly improved safety, with a 72-hour mortality rate dropping to 0, and no nematode deaths were detected. The mortality rate in the positive control group was 3%.

[0045] The results confirm that probiotic fermentation can significantly reduce the acute toxicity of traditional Chinese medicine raw materials, expand the scope of safe use, and provide reliable safety data support for the development of functional foods.

[0046] In summary, this invention employs synergistic fermentation with four specific probiotic strains, utilizing targeted biotransformation through microbial enzyme systems to construct a three-pronged technical strategy of "fermentation enhancement, structural optimization, and synergistic intervention." Fermentation enhancement manifests in the hydrolysis of highly polar flavonoid glycosides into lipid-soluble active aglycones, while simultaneously modifying irritating triterpenes. Structural optimization improves the safety of the components, transforming them from bitter and cold, potentially stomach-damaging, to mild and safe. Synergistic intervention, through the multi-target synergistic interaction of metabolites such as organic acids and lactobacillus with the transformed traditional Chinese medicine components, achieves multiple technical effects—enhanced anti-Helicobacter pylori activity, reduced toxicity, and gastric mucosal protection—while avoiding antibiotic resistance. This provides a safe and effective non-antibiotic alternative for the prevention and treatment of Helicobacter pylori infection and related diseases.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori, characterized in that, The method is as follows: S1. Licorice, houttuynia cordata, purslane, dandelion, poria cocos, honeysuckle, mulberry, mint and ginger are mixed to obtain a medicinal and edible herbal composition. After being pulverized, the mixture is soaked in water and extracted at 100°C. After filtration, filtrate a is obtained. Water is added to the remaining residue and extracted at 100°C. After filtration, filtrate b is obtained. The filtrates are combined, concentrated under reduced pressure, a carbon source is added, and the pH is adjusted to 7.

0. After sterilization, a fermentation culture broth for traditional Chinese medicine is obtained. S2, Probiotic Dissolution: S201. Add Lactobacillus plantarum CICC 22195 to sterile physiological saline, shake to dissolve, and obtain Lactobacillus plantarum suspension. S202. Add Lactobacillus reuteri CICC 6118 to sterile physiological saline, shake to dissolve, and obtain Lactobacillus reuteri bacterial suspension. S203. Add Lactococcus lactis CICC 20090 to sterile physiological saline, shake to dissolve, and obtain Lactococcus lactis suspension. S204. Add Bifidobacterium animalis CICC 21711 to sterile physiological saline, shake to dissolve, and obtain a suspension of Bifidobacterium animalis. S3, Probiotic culture expansion: S301. The *Lactobacillus plantarum* suspension obtained in S201 is inoculated into MRS liquid culture medium at a volume ratio of 1%:

1. After mixing evenly, the medium is sealed and incubated at 30℃ for 24 hours to obtain expanded culture of *Lactobacillus plantarum*. S302. The Lactobacillus reuteri suspension obtained in S202 was inoculated into MRS liquid medium at a volume ratio of 1%:

1. After mixing evenly, the medium was sealed and incubated at 30°C for 24 hours to obtain the expanded Lactobacillus reuteri suspension. S303. The lactococcus lactis suspension obtained in S203 is inoculated into MRS liquid culture medium at a volume ratio of 1%:

1. After mixing evenly, the medium is sealed and incubated at 30℃ for 24 hours to obtain the expanded culture of lactococcus lactis suspension. S304. The animal bifidobacterium suspension obtained in S204 was inoculated into MRS liquid medium at a volume ratio of 1%:

1. After mixing evenly, the medium was sealed and incubated at 30°C for 24 hours to obtain the expanded animal bifidobacterium suspension. S305. The expanded culture of Lactobacillus plantarum obtained in S301, the expanded culture of Lactobacillus reuteri obtained in S302, the expanded culture of Lactococcus lactis obtained in S303, and the expanded culture of Bifidobacterium animalis obtained in S304 are mixed to obtain an expanded culture probiotic suspension. S4. The probiotic suspension obtained in S305 was inoculated into the traditional Chinese medicine fermentation culture broth obtained in S1 at a volume ratio of 1%:

1. The broth was cultured at 30℃ for 48 hours to obtain the probiotic fermentation broth of traditional Chinese medicine with anti-Helicobacter pylori properties. S5. The fermentation broth of medicinal and edible probiotics against Helicobacter pylori obtained in S4 is centrifuged, ultrafiltered, and aseptically dispensed to obtain a fermentation composition of medicinal and edible probiotics against Helicobacter pylori.

2. The method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori according to claim 1, characterized in that, The medicinal and edible herbal composition described in S1 is made from the following raw materials in parts by weight: 10 parts licorice, 25 parts houttuynia cordata, 15 parts purslane, 15 parts dandelion, 15 parts poria cocos, 15 parts honeysuckle, 15 parts mulberry, 10 parts peppermint, and 5 parts ginger.

3. The method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori according to claim 1, characterized in that, The mass ratio of the medicinal and edible herbal composition to water in S1 is 1:8; when water is added to the remaining filter residue, the mass ratio of the remaining filter residue to water is 1:

10.

4. The method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori according to claim 1, characterized in that, The carbon source mentioned in S1 is maltose; the final concentration of the carbon source in the fermentation culture medium of the traditional Chinese medicine is 10 mg / mL.

5. The method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori according to claim 1, characterized in that, The sterilization conditions in S1 are: 121℃ for 30 minutes.

6. The method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori according to claim 1, characterized in that, The MRS liquid culture medium described in S301 to S304 is prepared by adding the following raw materials to distilled water at a final concentration, adjusting the pH to 7.0, sterilizing at 121℃ for 30 min, and then cooling to 28℃ to 40℃: glucose 20g / L, peptone 10g / L, yeast powder 4g / L, beef powder 8g / L, ammonium citrate 2g / L, sodium acetate 5g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, and polysorbate-80 1g / L.

7. The method for preparing a fermented composition of medicinal and edible herbal probiotics for combating Helicobacter pylori according to claim 1, characterized in that, The probiotic suspension for expanded culture described in S305 is composed of a mixture of expanded cultured Lactobacillus plantarum suspension, expanded cultured Lactobacillus reuteri suspension, expanded cultured Lactococcus lactis suspension, and expanded cultured Bifidobacterium animalis suspension in a volume ratio of 1:1:1:

1.

8. The application of a fermented composition of medicinal and edible herbal probiotics prepared by the preparation method according to any one of claims 1-7, characterized in that, The fermented composition of medicinal and edible herbal probiotics for treating Helicobacter pylori is used to prepare a drug for improving oral Helicobacter pylori.

9. The application according to claim 8, characterized in that, The dosage form of the drug includes capsules, tablets, or granules.

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