Lactic acid bacteria fermented concentrated asparagus extract juice having efficacy in preventing helicobacter pylori infection, and preparation and use thereof

Abstract

The application discloses lactic acid bacteria fermented concentrated asparagus extract juice with Helicobacter pylori infection prevention efficacy and preparation and application thereof, and belongs to the field of food biotechnology and microbial fermentation technology. The fermented concentrated asparagus extract juice is prepared from the following raw materials: concentrated asparagus extract juice 20-50 parts, pure water 50-80 parts, glucose 2-6 parts, white granulated sugar 2-4 parts, sodium isoascorbic acid 0.01-0.1 part and compound lactic acid bacteria 0.001-0.01 part. The fermented concentrated asparagus extract juice is obtained by crushing, extracting and concentrating asparagus as raw material, and fermentation. The antioxidant capacity, Helicobacter pylori inhibition capacity and Helicobacter pylori infection prevention efficacy of the obtained fermented concentrated asparagus extract juice are obviously improved. The asparagus extract juice can be widely applied in the field of preventing and / or treating Helicobacter pylori infection, and is especially applied in the preparation of medicines for preventing and / or treating Helicobacter pylori infection.

Description

Technical Field

[0001] This invention relates to the fields of food biotechnology and microbial fermentation technology, and in particular to a concentrated asparagus extract fermented with lactic acid bacteria that has the effect of preventing Helicobacter pylori infection, as well as its preparation and application. Background Technology

[0002] Asparagus is a perennial herbaceous plant belonging to the genus Asparagus in the family Liliaceae. Its tender stems are rich in various nutrients and functional components, including easily absorbed soluble proteins, sugars, amino acids, microorganisms, and minerals. It is also rich in flavonoids such as rutin and quercetin, saponins, sulfur-containing flavor compounds, selenium, and antioxidants such as glutathione, giving it significant nutritional and pharmacological value. Internationally, asparagus is known as the "King of Vegetables" and is an important resource for the production of health products and pharmaceuticals. Furthermore, some studies have shown that the active ingredients in asparagus have antibacterial, antitumor, lipid-lowering, anti-inflammatory, and immune-boosting effects.

[0003] However, fresh asparagus undergoes vigorous physiological metabolism after harvesting, making it highly susceptible to lignification and deterioration at room temperature. This leads to hardening of the texture and loss of flavor, severely restricting the extension of the industry chain. Currently, asparagus development focuses mainly on canned asparagus and quick-freezing technology, while the development of technology for functional probiotic fermented asparagus juice is relatively limited.

[0004] Probiotic fermentation technology, with its advantages of mild conditions and targeted enrichment of functional factors, has become an important direction for breaking through the bottleneck of high-value processing of asparagus, especially suitable for developing fermented products with both nutritional and health benefits. Probiotics have received widespread attention in the prevention and treatment of chronic diseases due to their safety, effectiveness, and healthy properties. Currently, the use of probiotics and their fermented products is an emerging alternative for the prevention and adjuvant treatment of Helicobacter pylori infection, which can improve the eradication rate of Helicobacter pylori, balance the normal gut microbiota, and reduce antibiotic-related side effects. Therefore, developing new probiotic fermentation technology for asparagus is crucial. Summary of the Invention

[0005] The purpose of this invention is to provide a lactic acid bacteria fermented concentrated asparagus extract with preventive effects against Helicobacter pylori infection, as well as its preparation and application, to solve the problems existing in the prior art. This invention uses asparagus as raw material to develop a probiotic fermented concentrated asparagus extract product, and studies its efficacy in preventing and / or treating Helicobacter pylori infection, aiming to fill the technological gap in functional probiotic fermented asparagus extract products for the prevention of Helicobacter pylori infection, and has potential application prospects.

[0006] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a lactic acid bacteria fermented concentrated asparagus extract, which is made from the following raw materials by weight: 20-50 parts concentrated asparagus extract, 50-80 parts purified water, 2-6 parts glucose, 2-4 parts white sugar, 0.01-0.1 parts sodium isovitamin C and 0.001-0.01 parts compound lactic acid bacteria.

[0007] The second technical solution of the present invention, the method for preparing the concentrated asparagus extract by lactic acid bacteria fermentation, includes the following steps: (1) Select asparagus that is intact, moist at the cut, without dryness or shrinkage, free from disease spots, insect infestation, rot, spoilage, or off-odor. After cleaning, cut the asparagus into 3-5 cm pieces, crush and pulp it, and add pure water at a ratio of 1:1. Soak at a constant temperature of 50-70 ℃ for 2-3 hours. After filtering and extracting the juice, heat at 80-90 ℃ to evaporate and concentrate for 0.5-2 hours to obtain concentrated asparagus extract juice for later use. (2) Lactic acid bacteria fermentation concentrate of asparagus extract, the raw materials include the following components by weight fraction: 20-50 parts of concentrated asparagus extract, 50-80 parts of purified water, 2-6 parts of glucose, 2-4 parts of white sugar, 0.01-0.1 parts of sodium isovitamin C, after stirring evenly, filter, pasteurize, and quickly cool to room temperature; (3) Add 0.001~1 part of compound lactic acid bacteria direct-inoculation starter, with the ratio of live bacteria of Lactobacillus rhamnosus and Lactobacillus plantarum being 1:1, and ferment at 30 ℃-37 ℃ for 24-72 h; Further, the compound lactic acid bacteria direct-inoculation starter culture includes *Lactobacillus rhamnosus* and *Lactobacillus plantarum*; preferably, the *Lactobacillus rhamnosus* specifically is *Lactobacillus rhamnosus* (…). Lacticaseibacillus rhamnosus The strain JGSLR02 was deposited on April 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 34273. The *Lactobacillus plantarum* specifically refers to *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum JGS49, accession number CGMCC NO.28396; Furthermore, the viable count of the compound lactic acid bacteria direct-inoculation starter culture is (1-9) × 10⁻⁶. 10 CFU / g.

[0008] (4) After fermentation, the asparagus extract is sterilized at 85 ℃-121 ℃ for 5-30 min and then aseptically bottled or stored at 4 ℃ to obtain fermented concentrated asparagus extract.

[0009] The third technical solution of the present invention is the application of the lactic acid bacteria fermented and concentrated asparagus extract in the preparation of a drug with the effect of preventing and / or treating Helicobacter pylori infection.

[0010] The fourth technical solution of the present invention is a drug for preventing and / or treating Helicobacter pylori infection, the drug comprising the lactic acid bacteria fermented concentrated asparagus extract; the Helicobacter pylori includes Helicobacter pylori SS1, Helicobacter pylori ATCC43504 and Helicobacter pylori 26695.

[0011] The fifth technical solution of the present invention is the application of the lactic acid bacteria fermented concentrated asparagus extract in the preparation of a drug with the efficacy of preventing and / or treating Escherichia coli, Salmonella typhi and / or Staphylococcus aureus infections.

[0012] Based on the above technical solution, the present invention has the following technical effects: (1) The lactic acid bacteria fermented concentrated asparagus extract provided by this invention does not require the addition of food additives such as flavorings and colorings, nor does it require the use of preservatives such as sodium benzoate and potassium sorbate, thus effectively extending the shelf life and preventing spoilage. At the same time, this process does not require the addition of any enzyme preparations, is simple to operate, and retains the original nutritional components of the asparagus extract to the greatest extent while reducing costs. The finished product has a rich flavor and is sweet and sour.

[0013] (2) Compared with unfermented concentrated asparagus extract, the content of organic acids, polyphenols and flavonoids in fermented concentrated asparagus extract is significantly increased; (3) The lactic acid bacteria fermented concentrated asparagus extract prepared by the present invention has excellent antioxidant capacity and performs well in terms of DPPH free radical, hydroxyl free radical, ABTS free radical scavenging capacity and total reducing capacity.

[0014] (4) The lactic acid bacteria fermented concentrated asparagus extract prepared in this invention can significantly inhibit the urease activity of Helicobacter pylori; the inhibition zone against the three Helicobacter pylori indicator bacteria all exceeded 20 mm, which can significantly inhibit the growth of Helicobacter pylori, and the inhibition rate of urease activity of Helicobacter pylori reached 85.32%; (5) The lactic acid bacteria concentrated asparagus extract prepared in this invention has the effect of preventing Helicobacter pylori infection: After the Helicobacter pylori infection model mice ingested the fermented concentrated asparagus extract, it could significantly reduce the number of Helicobacter pylori in the stomach and the activity of urease, reduce the level of pro-inflammatory cytokines IL-6 and IL-1β in the gastric mucosa, and increase the level of anti-inflammatory factor IL-10. At the same time, it increased the level of superoxide dismutase (SOD) in the gastric mucosa and the content of short chain fatty acids in mouse feces. (6) The manufacturing process of this invention is simple, easy to control, highly standardized, and the product quality is stable, making it easy to achieve large-scale industrial production.

[0015] (7) The present invention discovered a strain of Lactobacillus rhamnosus JGSLR02 that has the effect of preventing Helicobacter pylori infection. After Lactobacillus rhamnosus JGSLR02 and Lactobacillus plantarum JGS49 were mixed and fermented on concentrated asparagus extract, the fermented asparagus extract obtained had significantly improved inhibitory ability against common pathogens and Helicobacter pylori, prevention and treatment efficacy against Helicobacter pylori infection, and antioxidant capacity. Attached Figure Description

[0016] Figure 1 The effects of different fermentation strains on the content of Helicobacter pylori (A) and urease activity (B) in the gastric tissue of mice.

[0017] Figure 2 The effects of different fermentation strains on the histopathology of gastric antrum tissue in the pyloric region of mice.

[0018] Figure 3 The effects of different fermentation strains on cytokines and superoxide dismutase (SOD) in mouse gastric tissue were investigated. A represents IL-1β, B represents IL-6, C represents IL-10, and D represents SOD.

[0019] Figure 4 The effect of different fermentation strains on the content of short-chain fatty acids in mouse feces was investigated. A represents acetic acid, B represents propionic acid, C represents butyric acid, D represents valeric acid, E represents isobutyric acid, and F represents isovaleric acid.

[0020] Figure 5 Image of Gram-stained Lactobacillus rhamnosus JGSLR02.

[0021] Figure 6 NucGreen / EthD-III fluorescence staining-live / dead cell double staining image of Helicobacter pylori after treatment with probiotic supernatant for 24 h.

[0022] Figure 7 The effect of Lactobacillus rhamnosus JGSLR02 on the content of Helicobacter pylori in the gastric tissue of mice.

[0023] Figure 8 The effect of Lactobacillus rhamnosus JGSLR02 on urease activity in the gastric mucosa of mice.

[0024] Figure 9 The effects of Lactobacillus rhamnosus JGSLR02 on the histopathology of gastric antrum tissue in the pylorus region of mice.

[0025] Figure 10 The effects of Lactobacillus rhamnosus JGSLR02 on MPO activity and cytokines in mouse gastric tissue were investigated. In this study, A represents MPO activity in mouse gastric tissue, B represents IL-1β, C represents IL-6, and D represents IL-10.

[0026] Figure 11 The effect of Lactobacillus rhamnosus JGSLR02 on SOD in mouse gastric tissue.

[0027] Figure 12 The effect of Lactobacillus rhamnosus JGSLR02 on apoptosis of mouse gastric epithelial cells.

[0028] Figure 13 The effect of Lactobacillus rhamnosus JGSLR02 on gastric mucosal epithelial repair proteins in mice. Detailed Implementation

[0029] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0030] This invention provides a lactic acid bacteria fermented concentrated asparagus extract, which is made from the following raw materials by weight: 20-50 parts concentrated asparagus extract, 50-80 parts purified water, 2-6 parts glucose, 2-4 parts white sugar, 0.01-0.1 parts sodium isovitamin C, and 0.001-0.01 parts compound lactic acid bacteria.

[0031] In some specific implementation schemes, the method for preparing the concentrated asparagus extract is as follows: Select green asparagus that is intact, with moist cut surfaces without shrinkage, free from disease spots, insect infestation, rot, spoilage, and off-odors. After cleaning, cut the asparagus into small sections, crush and pulp it, and add pure water at a 1:1 mass ratio. Soak the asparagus at a constant temperature of 50-70 ℃ for 2-3 hours. After filtering and extracting the juice, heat and evaporate it at 80-90 ℃ for 0.5-2 hours to obtain the concentrated asparagus extract.

[0032] In some specific implementations, the viable count of the compound lactic acid bacteria is (1-9) × 10⁻⁶. 10 CFU / g, composed of Lactobacillus rhamnosus and Lactobacillus plantarum in a 1:1 ratio of live bacteria; The Lactobacillus rhamnosus is Lactobacillus rhamnosus JGSLR02, with accession number CGMCC No.34273; The Lactobacillus plantarum mentioned is Lactobacillus plantarum JGS49, with the accession number CGMCC NO.28396.

[0033] This invention also provides a method for preparing the lactic acid bacteria fermented concentrated asparagus extract, which includes the following steps: mixing and fermenting each raw material, sterilizing after fermentation, and obtaining the lactic acid bacteria fermented concentrated asparagus extract.

[0034] In some specific implementations, the fermentation conditions are: fermentation at 30-37 ℃ for 24-72 h.

[0035] In some specific implementations, the sterilization conditions are: 85-121 ℃, sterilization for 5-30 min.

[0036] This invention also provides the application of the lactic acid bacteria fermented concentrated asparagus extract in the preparation of a drug with the efficacy of preventing and / or treating Helicobacter pylori infection.

[0037] In some specific implementations, the Helicobacter pylori includes Helicobacter pylori SS1, Helicobacter pylori ATCC43504, and Helicobacter pylori 26695.

[0038] This invention also provides a drug for preventing and / or treating Helicobacter pylori infection, the drug comprising the lactic acid bacteria fermented concentrated asparagus extract; the Helicobacter pylori includes Helicobacter pylori SS1, Helicobacter pylori ATCC43504 and Helicobacter pylori 26695.

[0039] This invention also provides the application of the lactic acid bacteria fermented concentrated asparagus extract in the preparation of a drug with the efficacy of preventing and / or treating Escherichia coli, Salmonella typhi and / or Staphylococcus aureus infections.

[0040] This invention provides a lactic acid bacteria fermented concentrated asparagus extract, which is rich in nutrients, has an excellent flavor, is safe and hygienic, and possesses antioxidant activity. Its efficacy in preventing and treating Helicobacter pylori infection has been verified through in vivo and in vitro tests. This lactic acid bacteria fermented concentrated asparagus extract contains no added flavorings, colorings, or preservatives, retaining the original nutritional components of the asparagus extract and extending its shelf life, making it an excellent health product.

[0041] The *Lactobacillus rhamnosus* involved in this invention and its embodiments is *Lactobacillus rhamnosus* JGSLR02, which was deposited on April 21, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 34273. The *Lactobacillus rhamnosus* JGSLR02 has the following properties: (1) Acid resistance: The survival rate after treatment in a pH 2.5 environment for 3 h was 90.85%; (2) Bile salt tolerance: The survival rate was as high as 93.03% after treatment in an environment with a bile salt concentration of 0.3% for 4 hours; (3) The scavenging rates of DPPH free radicals and hydroxyl free radicals were 49.88% and 58.35%, respectively, and the reducing power was 36.98%; (4) It has strong inhibitory properties against three common foodborne pathogens: Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium. (5) The inhibition zones of the three Helicobacter pylori indicator strains all exceeded 20 mm, which could significantly inhibit the growth of Helicobacter pylori and the inhibition rate of urease activity of Helicobacter pylori reached 82.60%; (6) The adhesion rate to gastric mucin reached 19.58%, and the adhesion rate to human gastric adenocarcinoma cells reached 12.68%; (7) Prevention and treatment effects on mouse models of Helicobacter pylori infection: ① Reduce the activity of Helicobacter pylori in the gastric antrum of a mouse model of Helicobacter pylori infection; ② Reduce the activity of Helicobacter pylori urease in a mouse model of Helicobacter pylori infection; ③ Alleviate gastric mucosal tissue damage in Helicobacter pylori-infected mouse models; ④ Reduce the level of inflammation in the gastric mucosa of mice with Helicobacter pylori infection; ⑤ Reducing gastric epithelial cell apoptosis in a mouse model of Helicobacter pylori infection; ⑥ Promotes the repair of gastric mucosa in Helicobacter pylori-infected mouse models.

[0042] The *Lactobacillus plantarum* involved in this invention and its embodiments is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum JGS49 was deposited on September 8, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.28396. Lactobacillus plantarum JGS49 has been published in Chinese invention patent CN117987298A.

[0043] Example 1: A concentrated asparagus extract fermented with lactic acid bacteria Select green asparagus that is intact, with moist cut surfaces, no signs of drying or shrinkage, no disease spots or insect infestation, no rot or spoilage, and no off-odors. After cleaning, cut the asparagus into 3 cm sections, crush and pulp them, and add pure water at a 1:1 mass ratio. Soak and extract at a constant temperature of 60 ℃ for 3 hours. After filtering and extracting the juice, heat and evaporate at 90 ℃ for 2 hours to obtain concentrated asparagus extract juice for later use.

[0044] Add 25 parts concentrated asparagus extract, 75 parts purified water, 4 parts glucose, 2 parts white sugar, and 0.1 parts sodium isosorbide C according to the following weight ratio. Stir well, filter, sterilize at 95 ℃ for 20 min, quickly cool to room temperature, and add 0.01 parts of compound lactic acid bacteria direct-inoculation starter (1 × 10⁻⁶). 10 CFU / g, Lactobacillus rhamnosus: Lactobacillus plantarum = 1:1 (live bacteria ratio)), mixed well and fermented at 35 ℃ for 48 h; after fermentation, sterilized at 95 ℃ for 20 min and then aseptically bottled or stored at 4 ℃ to obtain fermented concentrated asparagus extract.

[0045] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus JGSLR02, accession number CGMCC No. 34273; *Lactobacillus plantarum* ( Lactiplantibacillus plantarum JGS49, accession number CGMCCNo. 28396.

[0046] Example 2: A concentrated asparagus extract fermented with lactic acid bacteria Select green asparagus that is intact, with moist cut surfaces, no signs of drying or shrinkage, no disease spots or insect infestation, no rot or spoilage, and no off-odors. After cleaning, cut the asparagus into 3 cm sections, crush and pulp them, and add pure water at a 1:1 mass ratio. Soak and extract at a constant temperature of 60 ℃ for 3 hours. After filtering and extracting the juice, heat and evaporate at 90 ℃ for 2 hours to obtain concentrated asparagus extract juice for later use.

[0047] Add 20 parts concentrated asparagus extract, 80 parts purified water, 2 parts glucose, 4 parts white sugar, and 0.05 parts sodium isosorbide C according to the following weight ratio. Stir well, filter, sterilize at 95 ℃ for 30 min, quickly cool to room temperature, and add 0.001 parts of compound lactic acid bacteria direct-inoculation starter (1 × 10⁻⁶). 10 CFU / g, Lactobacillus rhamnosus: Lactobacillus plantarum = 1:1 (live count ratio)), mixed well and fermented at 28 ℃ for 72 h; after fermentation, sterilized at 85 ℃ for 30 min and then aseptically bottled or stored at 4 ℃ to obtain fermented concentrated asparagus extract.

[0048] Example 3: A concentrated asparagus extract fermented with lactic acid bacteria Select green asparagus that is intact, with moist cut surfaces, no signs of drying or shrinkage, no disease spots or insect infestation, no rot or spoilage, and no off-odors. After cleaning, cut the asparagus into 3 cm sections, crush and pulp them, and add pure water at a 1:1 mass ratio. Soak and extract at a constant temperature of 60 ℃ for 3 hours. After filtering and extracting the juice, heat and evaporate at 90 ℃ for 2 hours to obtain concentrated asparagus extract juice for later use.

[0049] Add 35 parts concentrated asparagus extract, 65 parts purified water, 6 parts glucose, 2 parts white sugar, and 0.01 parts sodium isosorbide C according to the following weight ratio. Stir well, filter, sterilize at 95 ℃ for 30 min, quickly cool to room temperature, and add 0.001 parts of compound lactic acid bacteria direct-inoculation starter (1 × 10⁻⁶). 10 CFU / g, Lactobacillus rhamnosus: Lactobacillus plantarum = 1:1 (live bacteria ratio)), mixed well and fermented at 38 ℃ for 24 h; after fermentation, sterilized at 121 ℃ for 5 min and then aseptically bottled or stored at 4 ℃ to obtain fermented concentrated asparagus extract.

[0050] Comparative Example 1 The only difference from Example 1 is that, in the inoculation and fermentation step, 0.01 parts of Lactobacillus rhamnosus JGSLR02 lactic acid bacteria starter (1×10⁻⁶) were added. 10 (CFU / g) was used for direct inoculation fermentation, with other conditions the same as in Example 1.

[0051] Comparative Example 2 The only difference from Example 1 is that, in the inoculation and fermentation step, 0.01 parts of *Lactobacillus plantarum* JGS49 lactic acid bacteria starter (1×10⁻⁶) were added. 10 (CFU / g) was used for direct inoculation fermentation, with other conditions the same as in Example 1.

[0052] Experimental Example 1 The organic acid content and antioxidant activity of the fermented and concentrated asparagus extracts from Examples 1-3 and Comparative Examples 1-2 were determined, and the results are as follows: (1) Effect on organic acid content Table 1. Effects of different fermentation strains on the organic acid content of fermented concentrated asparagus extract (g / L)

[0053] Note: "Unfermented Example 1" refers to a mixture of all raw materials and starter culture that has been sterilized directly without fermentation and then aseptically packaged or refrigerated at 4°C. The same applies below.

[0054] As shown in Table 1, the organic acid content of asparagus extract increased after fermentation, with lactic acid being the main organic acid. No lactic acid was detected in the unfermented Example 1, but after fermentation, the lactic acid content increased dramatically, reaching 15.28 g / L, and the citric acid content was 1.35 g / L. Compared to the comparative example, the overall organic acid content of Example 1 was higher, with a significant increase in lactic acid and citric acid, resulting in a milder and more fragrant sour taste. Studies have shown that organic acids have significant antibacterial effects, promoting digestion, stimulating gastrointestinal motility, and regulating intestinal flora. Therefore, the asparagus extract concentrated by fermentation with compound lactic acid bacteria prepared from *Lactobacillus rhamnosus* JGSLR02 and *Lactobacillus plantarum* JGS49 has the potential to inhibit pathogenic bacteria.

[0055] (2) Effects on antioxidant activity Table 2. Effects of different fermentation strains on the antioxidant activity of fermented and concentrated asparagus extract.

[0056] Oxidative stress refers to a pathological state in which the production and clearance of reactive oxygen species (ROS) in the body are imbalanced, leading to the accumulation of excessive ROS and causing damage to cellular lipids, proteins, and DNA. In Helicobacter pylori infection, the bacteria's metabolites and cell wall components can directly stimulate gastric mucosal cells to produce ROS. Simultaneously, the immune inflammatory response triggered by the infection further promotes ROS release, and long-term oxidative stress exacerbates gastric mucosal damage. Dietary antioxidants can directly remove excess ROS, reduce oxidative stress damage to the gastric mucosa, alleviate inflammatory responses, improve gastrointestinal antioxidant capacity, enhance the body's immune function, and help reduce bacterial colonization, thereby lowering the risk of infection-induced gastric mucosal lesions.

[0057] The antioxidant activity results of the concentrated asparagus extract fermented with lactic acid bacteria are shown in Table 2. The DPPH and hydroxyl radical scavenging rates, ABTS, and FRAP content of the fermented concentrated asparagus extract prepared from *Lactobacillus rhamnosus* JGSLR02 and *Lactobacillus plantarum* JGS49 in the examples were all higher than those in the unfermented examples and the comparative example. Furthermore, the total phenol and total flavonoid content of the compound lactic acid bacteria fermented concentrated asparagus extract prepared in Example 1 was higher, reaching 2.18 mg / mL and 2.06 mg / mL, respectively.

[0058] In conclusion, the asparagus extract concentrated by fermentation with compound lactic acid bacteria prepared from Lactobacillus rhamnosus JGSLR02 and Lactobacillus plantarum JGS49 exhibits excellent antioxidant activity and has the potential to prevent and treat Helicobacter pylori infection.

[0059] Experimental Example 2 To verify the beneficial effects of the present invention, relevant indicators of the final fermentation products of Examples 1-3 and Comparative Examples 1-2 were tested, mainly involving the inhibitory ability of the above fermentation products against pathogenic bacteria, the inhibitory ability against Helicobacter pylori and its urease activity, and the preventive and therapeutic effects against Helicobacter pylori infection in mice. It was found that the mixed fermentation of *Lactobacillus rhamnosus* JGSLR02 and *Lactobacillus plantarum* JGS49 (Example) significantly improved the in vitro inhibition of common pathogenic bacteria and Helicobacter pylori, as well as the prevention of Helicobacter pylori infection in model mice, compared to fermentation of *Lactobacillus rhamnosus* JGSLR02 (Comparative Example 1) and *Lactobacillus plantarum* JGS49 alone (Comparative Example 2). Specific results are as follows: (1) In vitro inhibitory ability against common pathogens and Helicobacter pylori Table 3. Effects of different fermentation strains on the antibacterial activity of fermented concentrated asparagus extract (mm)

[0060] As shown in Table 3, compared with Comparative Examples 1-2, the compound lactic acid bacteria fermented concentrated asparagus extract prepared in Examples 1-3 had the highest inhibition zone diameter against common pathogenic bacteria and Helicobacter pylori, and the best inhibitory effect. It can effectively inhibit the proliferation of common pathogenic bacteria and Helicobacter pylori, indicating that the compound lactic acid bacteria fermented concentrated asparagus extract prepared in the examples has excellent antibacterial activity.

[0061] The comparison results of Examples 1-3 and Comparative Examples 1-2 show that, compared with single-strain fermentation, the mixed-strain fermentation of Lactobacillus rhamnosus JGSLR02 and Lactobacillus plantarum JGS49 of the present invention significantly improved the inhibitory ability against common pathogenic bacteria and Helicobacter pylori. It can be seen that Lactobacillus rhamnosus JGSLR02 and Lactobacillus plantarum JGS49 produced a synergistic effect, which significantly improved the inhibitory effect of fermented and concentrated asparagus extract on pathogenic bacteria.

[0062] The asparagus extract prepared by the compound lactic acid bacteria fermentation in the examples showed strong inhibitory effects against common pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium. This may be due to the action of antibacterial substances such as organic acids and antimicrobial peptides produced during the fermentation process. As shown in Table 3, both Lactobacillus rhamnosus JGSLR02 and Lactobacillus plantarum JGS49, when fermented individually, showed some ability to inhibit Helicobacter pylori. However, after mixed-culture fermentation, the compound lactic acid bacteria fermented asparagus extract prepared in the examples showed a significant improvement in its inhibitory effect on Helicobacter pylori compared to single-strain fermentation, achieving unexpected technical results.

[0063] In summary, the compound lactic acid bacteria fermented concentrated asparagus extract prepared in the examples has a good ability to inhibit foodborne pathogens and Helicobacter pylori.

[0064] (2) Inhibitory effect on Helicobacter pylori urease activity Studies have shown that saponins and flavonoid polyphenols in asparagus can inhibit the urease activity of Helicobacter pylori. Lactic acid bacteria metabolites significantly inhibit the urease activity of Helicobacter pylori through multiple pathways, including further pH reduction, secretion of antimicrobial peptides, competitive inhibition, and gene regulation. The inhibitory ability on the urease activity of Helicobacter pylori is shown in Table 3. The examples showed a reduction of urease activity of Helicobacter pylori by 81.44%–85.32%, which was superior to the unfermented Example 1, Comparative Example 1, and Comparative Example 2. The results indicate that the compound lactic acid bacteria fermented and concentrated asparagus extract prepared in the examples significantly improved the inhibitory ability on the urease activity of Helicobacter pylori compared to unfermented and single-strain fermentation.

[0065] (3) Effects on the activity of Helicobacter pylori and urease in the stomach of mice infected with Helicobacter pylori The results are as follows Figure 1As shown, the model group exhibited the highest Helicobacter pylori colonization activity. The colonization activities of Comparative Examples 1 and 2 were 2.41 and 2.48 Log (CFU / mL), respectively, while the colonization activities of Example 1 and the positive control group were only 1.12 and 0.84 Log (CFU / mL). Example 1 showed little difference from the positive control group and was superior to Comparative Examples 1 and 2. Furthermore, the urease content in mouse gastric tissue was determined by adding a urease indicator to the tissue homogenate and measuring the absorbance of the resulting color at a wavelength of 561 nm. Figure 1 The absorbance of urease in the model group was 4.20, the absorbance of the positive control group was 0.55, and the absorbances of Comparative Example 1, Comparative Example 2, and Example 1 were 2.36, 2.45, and 1.25, respectively. The results of Example 1 were better than those of Comparative Example 1 and Comparative Example 2, indicating that the mixed fermentation of Lactobacillus rhamnosus JGSLR02 and Lactobacillus plantarum JGS49 of the present invention can effectively improve the inhibitory ability of fermented and concentrated asparagus extract on Helicobacter pylori infection in the stomach, and has a certain preventive and alleviating effect on Helicobacter pylori infection.

[0066] (4) Effects on gastric tissue of mice infected with Helicobacter pylori Pathological results of mouse gastric antrum tissue as follows Figure 2 As shown. In the control group, the gastric mucosa of mice was clearly structured, with intact epithelium, tightly arranged mucosal glands, normal morphology, and no obvious inflammatory response. In the model group, the gastric mucosal epithelial cells and some glandular cells were damaged, vacuolation occurred in the lamina propria, the structure was significantly disordered, and there was infiltration of immune cells. In Example 1, the mucosal structure of the intervention group was clear, the epithelial tissue was relatively intact, and the mucosal glands were tightly arranged and normal. However, the gastric tissue in the pathological sections of Comparative Examples 1 and 2 still showed a small amount of inflammatory cell infiltration. This indicates that intragastric administration of the concentrated asparagus extract fermented by mixed bacteria of *Lactobacillus rhamnosus* JGSLR02 and *Lactobacillus plantarum* JGS49 of the present invention has a certain preventive and alleviating effect on *Helicobacter pylori* infection, and may be used as an adjunct therapy and preventive measure to deal with the inflammatory response caused by *Helicobacter pylori* infection.

[0067] (5) Effects on serum cytoinflammatory factors in mice like Figure 3 As shown, in the Helicobacter pylori infection model group, the levels of pro-inflammatory cytokines IL-6 and IL-1β in mouse serum were significantly increased, while gavage administration of concentrated asparagus extract fermented with lactic acid bacteria reduced the levels of pro-inflammatory cytokines and increased the level of anti-inflammatory cytokine IL-10. Furthermore, Example 1 showed better results than Comparative Examples 1 and 2. These results indicate that the concentrated asparagus extract fermented with lactic acid bacteria can reduce the levels of inflammatory factors in mice and decrease serum inflammation levels, thereby exerting an anti-inflammatory effect to prevent and alleviate Helicobacter pylori infection.

[0068] Superoxide dismutase (SOD) is an important antioxidant that protects cells from oxidative damage by scavenging superoxide anion free radicals induced by free radicals. Exogenous SOD can effectively alleviate acute gastric mucosal damage caused by various factors. For example... Figure 3 As shown, the interventions in Example 1, Comparative Example 1, and Comparative Example 2 increased the level of superoxide dismutase (SOD) in the gastric mucosa of mice, and Example 1 was superior to Comparative Example 1 and Comparative Example 2, indicating that the concentrated asparagus extract fermented by lactic acid bacteria has an antioxidant stress effect and can reduce gastric mucosal damage.

[0069] (6) Short-chain fatty acid content in mouse feces Short-chain fatty acids (SCFAs) are saturated fatty acids containing six or fewer carbon atoms. They can interfere with intestinal osmotic pressure and pH balance to protect the gut, promote the expression of antimicrobial peptides in the host, and affect the nutrient uptake and energy production of pathogens. Studies have shown that SCFAs can be absorbed and utilized by intestinal epithelial cells as nutrients, and can regulate the function of innate immune cells (such as macrophages, neutrophils, and dendritic cells) involved in the immune system, as well as the differentiation of T cells and B cells and antigen-specific adaptive immunity.

[0070] like Figure 4 As shown, after Helicobacter pylori infection, the total short-chain fatty acid (SCL) content in the feces of mice in the model group was significantly reduced. After ingestion of concentrated asparagus extract produced by lactic acid bacteria (Example 1), the total SCL content in the feces of mice increased, significantly higher than that in the control group. From the perspective of different SCL contents, ingestion of concentrated asparagus extract produced by lactic acid bacteria fermentation (Example 1) significantly alleviated the reduction in SCL in mouse feces, indicating that concentrated asparagus extract produced by lactic acid bacteria fermentation (Example 1) has a beneficial effect on increasing SCL. Increasing the SCL content can improve the body's immunity, indirectly correct immune-metabolic imbalance, and block the vicious cycle of infection. Furthermore, after ingestion of concentrated asparagus extract produced by fermentation (Example 1), the SCL content was higher than that of concentrated asparagus extract produced by single-strain fermentation in Control Groups 1 and 2.

[0071] Example 4: Relevant performance of Lactobacillus rhamnosus JGSLR02 1. Isolation, screening and identification of Lactobacillus rhamnosus JGSLR02 Obtaining *Lactobacillus rhamnosus* JGSLR02 of this invention: Fecal samples were collected from healthy infants aged 0-24 months in the Nanchang area. The inner wall of the infant feces was collected using a sterile sampler. Samples were sent to the laboratory within 4 hours for subsequent experiments. The samples were serially diluted with physiological saline (10-10). -1 10 -2 10 -3 10 -4 10 -5Afterwards, 100 μL of the serially diluted solution was spread onto MRS solid medium supplemented with 0.04% (w / v) bromocresol purple. The medium was incubated at room temperature for 8 min until the bacterial culture was absorbed. The culture dish was then inverted and incubated at 37 ℃ for 48 h. Colonies with a yellow color ring around them were selected. Strains of different morphologies and sizes were streaked twice, repeating the streaking 2-3 times. The purified strain was then stored in 25% (v / v) glycerol at -80 ℃ for later use. Subsequently, its acid and bile salt tolerance, antioxidant capacity, and inhibition of common pathogens and Helicobacter pylori were measured to obtain a strain with good performance. Finally, DNA extraction, PCR, and 16S rRNA sequencing identified it as *Lactobacillus rhamnosus*. Lacticaseibacillus rhamnosus It was named Lactobacillus rhamnosus (Lactobacillus casei). Lacticaseibacillus rhamnosus JGSLR02. The morphology of JGSLR02 cells is as follows: Figure 5 As shown.

[0072] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus JGSLR02 was deposited on April 21, 2025, at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.34273.

[0073] The 16S rRNA sequence of *Lactobacillus rhamnosus* JGSLR02 described in this invention is as follows (SEQ ID NO.1):

[0074] 2. Acid and bile salt tolerance of Lactobacillus rhamnosus JGSLR02 Lactobacillus rhamnosus JGSLR02 was inoculated into MRS broth medium and cultured at 37 ℃ for 24 hours to obtain bacterial fermentation broth (bacterial suspension). The bacterial suspension was centrifuged at 8000 g for 5 min (4 ℃), the supernatant was discarded and the bacterial cells were collected. The cells were washed twice with sterile PBS and then resuspended in MRS liquid medium at pH 2.5. The suspension was incubated at 37 ℃ for 3 h, and 1 mL of the bacterial suspension was taken at 0, 1, 2, and 3 h respectively and serially diluted 10-fold with 0.9% physiological saline (10... -1 -10 -8 Then, an appropriate concentration was spread onto MRS agar plates and anaerobically incubated at 35°C for 72 hours. After incubation, the viable cell count was determined, with three replicates for each sample. Simultaneously, the acid tolerance of *Lactobacillus rhamnosus* CICC 6144 was determined under the same conditions.

[0075] JGSLR02 bacterial cells were collected and washed according to the above method. The bacterial cells were then resuspended in MRS liquid medium (pH 8.0) containing 0.3% ox bile salts and incubated at 37°C for 3 hours. At 0, 2, and 4 hours, 1 mL of the bacterial suspension was taken and serially diluted 10-fold with 0.9% physiological saline. -1 -10 -7 Then, an appropriate concentration was spread onto MRS agar plates and anaerobically incubated at 35°C for 72 hours. After incubation, the viable bacterial count was determined, with three replicates for each sample. Simultaneously, the bile salt tolerance of *Lactobacillus rhamnosus* CICC 6144 was determined under the same conditions. The survival rate of lactic acid bacteria was calculated using the following formula: ; In the formula, N1 is the number of surviving bacteria after incubation; N0 is the initial number of surviving bacteria.

[0076] Table 4. Acid tolerance of Lactobacillus rhamnosus

[0077] Table 5. Bile salt tolerance of Lactobacillus rhamnosus

[0078] As shown in Table 4, the survival rate of *Lactobacillus rhamnosus* JGSLR02 remained above 90% after treatment in a pH 2.5 environment for 3 h; as shown in Table 5, the survival rate reached over 93% after treatment in an environment with a bile salt concentration of 0.3% for 4 h. The experimental results indicate that *Lactobacillus rhamnosus* JGSLR02 possesses strong acid and bile salt resistance.

[0079] 3. Antioxidant properties of Lactobacillus rhamnosus JGSLR02 The ability of Lactobacillus rhamnosus JGSLR02 to scavenge DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) free radicals, scavenge hydroxyl free radicals, and reduce free radicals was detected, and the results are shown in Table 6.

[0080] Table 6 Antioxidant activity of strains

[0081] The above experimental results show that Lactobacillus rhamnosus JGSLR02 has acid and bile salt resistance and strong antioxidant activity, and can be used to prepare food or health products with anti-aging and antioxidant effects.

[0082] 4. Inhibitory ability of Lactobacillus rhamnosus JGSLR02 against common pathogenic bacteria and Helicobacter pylori. The antibacterial activity of fermentation supernatant of Lactobacillus rhamnosus JGSLR02 against Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Helicobacter pylori was determined by the perforation method.

[0083] Table 7. Inhibitory ability of lactic acid bacteria against common pathogenic bacteria and Helicobacter pylori (mm)

[0084] As shown in Table 7, the fermentation supernatant of *Lactobacillus rhamnosus* JGSLR02 exhibited strong inhibitory effects against three common foodborne pathogens: *Escherichia coli*, *Staphylococcus aureus*, and *Salmonella typhimurium*. Furthermore, the inhibitory effect on *Helicobacter pylori* demonstrated by this invention showed that *Lactobacillus rhamnosus* JGSLR02 exhibited inhibition zones exceeding 20 mm against all three *Helicobacter pylori* indicator strains, significantly inhibiting the growth of *Helicobacter pylori*. This indicates that *Lactobacillus rhamnosus* JGSLR02 possesses a strong inhibitory effect on *Helicobacter pylori*.

[0085] Co-culture analysis with Helicobacter pylori: A co-culture system of Helicobacter pylori with supernatant or bacterial suspension of Lactobacillus rhamnosus was established using microbial co-culture technology. First, Helicobacter pylori 26695 was resuspended in Brucella broth, and supernatant or bacterial suspension of Lactobacillus rhamnosus JGSLR02 and Lactobacillus rhamnosus CICC6144 were added, respectively, and cultured under microaerophilic conditions at 37 °C. During co-culture, the solution was sampled at regular time intervals to assess the survival count of Helicobacter pylori 26695, which was determined by culturing on CAB agar. Simultaneously, Helicobacter pylori was resuspended in supernatant of Lactobacillus rhamnosus JGSLR02 and Lactobacillus rhamnosus CICC6144, respectively. Subsequently, the supernatant was placed at 37 °C to induce Helicobacter pylori into a VBNC (viable but unculturable) state. After 24 h, the number of culturable cells in the induction solution was determined by plate counting. The VBNC state transition of Helicobacter pylori was detected using a LIVE / DEAD staining kit. First, NucGreen / EthD-III was added to an appropriate amount of cell suspension. The cell suspension was then gently mixed and stained in the dark for 15 min. Finally, 10 μL of the mixture was dropped onto a glass slide covered with an 18 mm square coverslip and observed using a Leica DM3000B fluorescence microscope.

[0086] Table 8. Changes in the survival count of Helicobacter pylori during co-culture with probiotic supernatant and probiotic suspension.

[0087] As shown in Table 8, after 24 h of co-culturing with *Helicobacter pylori*, the fermentation supernatant of *Lactobacillus rhamnosus* JGSLR02 reduced the number of *Helicobacter pylori* to 1.95 × 10⁻⁶. 2 CFU / mL, superior to *Lactobacillus rhamnosus* CICC6144. For probiotic cell resuspensions, *Lactobacillus rhamnosus* JGSLR02 cell resuspension reduced *Helicobacter pylori* counts to 3.63 × 10⁻⁶. 3 This indicates that *Lactobacillus rhamnosus* JGSLR02 has a strong ability to inhibit *Helicobacter pylori*. The results of the LIVE / DEAD staining assay are as follows: Figure 6 As shown, after being stressed in the supernatant of Lactobacillus rhamnosus JGSLR02 for 24 h, a large number of Helicobacter pylori cells died (red), and the effect was better than that of Lactobacillus rhamnosus CICC6144.

[0088] 5. Inhibitory effect of Lactobacillus rhamnosus JGSLR02 on urease activity in Helicobacter pylori Method for Assay of Helicobacter pylori Urease Activity Inhibition: Helicobacter pylori bacterial suspension and Lactobacillus rhamnosus JGSLR02 strain resuspension were prepared as described above. 50 μL of each suspension was added to a 96-well plate and incubated at 37 ℃ under microaerophilic conditions for 24 h. Then, 150 μL of urea-phenol red solution (20% urea and 0.012% phenol, pH 6.5) was added to each well to initiate the urease biochemical reaction. Color change was observed after 30 min, and absorbance was measured at 561 nm. A separate culture of Helicobacter pylori was used as a control. The same method was used to determine the inhibitory effect of Lactobacillus rhamnosus CICC 6144 on Helicobacter pylori urease activity. The urease activity inhibition was calculated as follows: ; In the formula, A 0 OD of the control group after 24 hours 561 value, A t The OD of each experimental group after 24 hours 561 value.

[0089] The results of the inhibition of urease activity of Helicobacter pylori by the strain are shown in Table 9. Lactobacillus rhamnosus JGSLR02 reduced the urease activity of Helicobacter pylori by 82.60%, which was better than strain CICC 6144. The results indicate that Lactobacillus rhamnosus JGSLR02, which can inhibit the urease activity of Helicobacter pylori, has important potential in the prevention of pathogen infection.

[0090] 6. The adhesion properties of Lactobacillus rhamnosus JGSLR02 to mucin and human gastric adenocarcinoma (AGS) cells are shown in Table 9.

[0091] Table 9. Urease inhibition properties of the strain, adhesion properties to mucin and human gastric adenocarcinoma (AGS) cells.

[0092] As shown in Table 9, the adhesion rates of Lactobacillus rhamnosus JGSLR02 to gastric mucin and AGS cells were 19.58% and 12.68%, respectively, which were better than those of strain CICC 6144. This indicates that Lactobacillus rhamnosus JGSLR02 has good adhesion properties to the gastric mucosa and has the potential to reduce Helicobacter pylori adhesion.

[0093] Effects of Helicobacter pylori infection on inflammatory factors in human gastric adenocarcinoma (AGS) cells: AGS cells were cultured in microplates until a complete monolayer was formed. Then, Helicobacter pylori 26695 and Lactobacillus rhamnosus JGSLR02 were co-infected in AGS cells at an infection ratio of 1:100 (MOI) and cultured for 12 h. Helicobacter pylori infection alone served as a positive control, and untreated AGS cells served as a negative control. The concentration of IL-8 in the cell supernatant was calculated using an interleukin-8 (IL-8) ELISA kit (Solepro Beijing) according to the standard curve, and the corresponding OD values ​​were calculated according to the manufacturer's instructions. The same method was used to determine the inflammatory factors in AGS cells co-infected with Helicobacter pylori 26695 and Lactobacillus rhamnosus CICC 6144. The results are shown in Table 10.

[0094] Table 10. Interleukin-8 (IL-8) levels in Helicobacter pylori-infected AGS cells treated with Lactobacillus rhamnosus

[0095] Table 10 shows that Helicobacter pylori 26695 increased IL-8 secretion in gastric AGS cells to 108.33 pg / mL, while Lactobacillus rhamnosus JGSLR02 and CICC 6144 reduced IL-8 secretion levels in AGS cells stimulated by Helicobacter pylori, with secretion levels of 72.14 and 82.50 pg / L, respectively. This indicates that Lactobacillus rhamnosus JGSLR02 has a better anti-inflammatory effect.

[0096] 7. The preventive and therapeutic effects of Lactobacillus rhamnosus JGSLR02 on Helicobacter pylori-infected mice. Seventy-eight male C57BL / 6 mice aged 4-6 weeks (20-24 grams) were randomly divided into six groups of 13 mice each. The groups were: blank control group, model group, positive control group, low-dose Lactobacillus rhamnosus group, medium-dose Lactobacillus rhamnosus group, and high-dose Lactobacillus rhamnosus group.

[0097] One week after acclimatization, all mice (n=8 per group) except for the control group (replaced with saline) received Helicobacter pylori solution (10... 8 Modeling was performed by gavage with CFU / mL (20 μL / g) once every other day for four weeks. After this period, mice (n=2) were sacrificed and infection was tested. Four mice were randomly selected for dissection, and gastric tissue was plated. A Helicobacter pylori infection concentration greater than 6.0 Lg (CFU / mL) in the model group indicated successful modeling.

[0098] During the above experimental period: the Lactobacillus rhamnosus group began daily gavage administration of different doses (high dose: 1×10) after the fourth week.9 CFU / mL; Medium dose: 1×10 8 CFU / mL; Low dose: 1×10 7 Resuspend 200 μL of Lactobacillus rhamnosus JGSLR02 bacterial suspension in PBS (CFU / mL) until the end of the experiment.

[0099] The positive control group received a combination of antibiotics (0.125 μg / mL amoxicillin and 0.5 μg / mL metronidazole) by gavage daily for 14 days starting from week 4. Mice in the model group and normal group were administered 200 μL of PBS by gavage daily; the mice were kept in an environment of 23-25 ​​℃, 55-60% humidity, and 12 h of alternating light.

[0100] Mice were observed daily and weighed and recorded weekly. After drug administration was discontinued, mice were euthanized by cervical dislocation at week 9. Serum samples were collected from retroorbital blood by centrifugation (2000 g, 10 min) and stored at -80 ℃. The stomachs of mice were removed, dissected along the greater and lesser curvatures, rinsed with sterile PBS, and the gastric body and antrum were collected and preserved separately. The number of Helicobacter pylori, urease, pathological findings, inflammatory factors, and immunohistochemical markers in the stomach were measured. The results are as follows: (1) Effects of Lactobacillus rhamnosus JGSLR02 on the activity of Helicobacter pylori and urease in the gastric antrum of mice infected with Helicobacter pylori The results are as follows Figure 7 As shown, the model group exhibited the highest Helicobacter pylori colonization activity at 6.87 Log (CFU / mL), while the low-dose and medium-dose groups showed colonization activities of 5.78 and 4.8 Log (CFU / mL), respectively. The high-dose group and the positive control group showed colonization activities of only 1.94 and 0.45 Log (CFU / mL), respectively. Furthermore, the urease content in mouse gastric tissue was determined by adding a urease indicator to the tissue homogenate and measuring the absorbance of the resulting color at a wavelength of 561 nm.

[0101] like Figure 8 The absorbance of urease in the model group was 3.995, while that in the positive control group was 0.63. The absorbances of the low-dose, medium-dose, and high-dose groups were 3.08, 2.24, and 1.90, respectively. Therefore, *Lactobacillus rhamnosus* JGSLR02 can reduce the activity of *Helicobacter pylori* and urease in mice infected with *Helicobacter pylori*, and has a certain preventive and alleviating effect on *Helicobacter pylori* infection.

[0102] (2) Effects of Lactobacillus rhamnosus JGSLR02 on gastric tissue pathology in Helicobacter pylori-infected mice In the pathological examination of the mouse model of Helicobacter pylori infection, in order to assess inflammation and mucosal damage, inflammatory cells in the lamina propria were counted on H&E-stained sections, and the entire mucosal layer was observed regionally.

[0103] Pathological results of mouse gastric antrum tissue as follows Figure 9 As shown in the figure, the gastric mucosa of mice in the control group was clear, with intact epithelium, tightly packed mucosal glands, normal morphology, and no obvious inflammatory response. In the model group, the gastric mucosal epithelial cells and some glandular cells were damaged, vacuolation was observed in the lamina propria, and a large number of neutrophils infiltrated the epithelial and lamina propria layers. Compared with the model group, the gastric mucosa structure in the low-dose and medium-dose groups was clearer and more intact, with a relatively tighter arrangement, a significant reduction in neutrophils in the lamina propria, and the disappearance of neutrophil hemorrhage in the lamina propria and muscularis mucosae; however, slight lymphocyte infiltration was still present in the gastric tissue pathological sections. Furthermore, some erythrocytes appeared in the muscularis mucosa of the positive control group, indicating hemorrhage and ulceration; however, the gastric mucosal tissue damage in the high-dose group recovered after 4 weeks of treatment. Therefore, *Lactobacillus rhamnosus* JGSLR02 can alleviate gastric tissue lesions in mice infected with *Helicobacter pylori*, prevent further lesions of the gastric mucosa, and has a certain preventive effect against *Helicobacter pylori* infection.

[0104] (3) Effects of Lactobacillus rhamnosus JGSLR02 on gastric mucosal inflammation in Helicobacter pylori-infected mice According to the manufacturer's instructions, use an ELISA kit to determine the levels of IL-1β, IL-6, and IL-10 in serum.

[0105] like Figure 10 As shown, the level of myeloperoxidase (MPO) in the gastric mucosa of the model group mice was higher than that in other groups, while the MPO level was lowest in the positive control group. Treatment with different doses of *Lactobacillus rhamnosus* JGSLR02 reduced MPO activity in the gastric mucosa of mice to varying degrees, with the high-dose group showing a greater effect than other dose groups. Furthermore, intervention with *Lactobacillus rhamnosus* JGSLR02 effectively reduced the levels of pro-inflammatory cytokines such as interleukin IL-1β and IL-6, and increased the level of the anti-inflammatory factor IL-10. These results indicate that *Lactobacillus rhamnosus* JGSLR02 can alleviate the inflammatory level of the gastric mucosa in *Helicobacter pylori*-infected mouse model, thereby exerting an anti-inflammatory effect to alleviate the occurrence and development of *Helicobacter pylori* infection.

[0106] like Figure 11 As shown, intervention with Lactobacillus rhamnosus JGSLR02 can increase the level of superoxide dismutase (SOD) in the gastric mucosa of mice, indicating that Lactobacillus rhamnosus JGSLR02 has an antioxidant stress effect.

[0107] (4) The repair effect of Lactobacillus rhamnosus JGSLR02 on gastric mucosa of Helicobacter pylori-infected mice Assessment of gastric epithelial cell apoptosis: Paraffin sections were stained with Ki67 and β-catenin heterologous double-label immunofluorescence. After incubation with primary antibody, Alexa Fluor 488-labeled goat anti-rabbit IgG and CY3-labeled goat anti-rabbit IgG were used as secondary antibodies. Assessment of gastric mucosal repair: Paraffin sections were stained with Occludin and ZO-1 homologous double-label immunofluorescence. After incubation with primary antibody, HRP-labeled goat anti-rabbit IgG and CY3-labeled goat anti-rabbit IgG were used as secondary antibodies. All double staining was enhanced using a tyramine signal amplification system (TSA). After staining, cell nuclei were counterstained with DAPI (4',6-diamidindo-2-phenylindole), and finally, images were observed and captured at the appropriate wavelengths under a fluorescence microscope.

[0108] Inflammation-induced oxidative stress, mediated by reactive oxygen species, can induce changes in mitochondrial membrane potential and permeability. This not only promotes the translocation of apoptosis-related proteins on the mitochondrial membrane but also releases apoptosis factors into the cytoplasm, thereby activating the mitochondrial apoptosis pathway. Ki-67 is a cell proliferation-specific nuclear antigen; β-catenin is a core molecule regulating proliferation and differentiation in the classical Wnt signaling pathway. Therefore, immunofluorescence was used to detect the expression of β-catenin and the proliferating cell nuclear antigen Ki-67 in mouse gastric mucosa. Immunofluorescence staining images of mouse gastric tissue are shown below. Figure 12 The results showed that different dose groups could resist apoptosis to varying degrees, while the number of gastric epithelial cells in the high-dose group JGSLR02 was close to that in the control group, indicating that high-dose Lactobacillus rhamnosus JGSLR02 has good anti-apoptotic ability.

[0109] Zona occludens 1 (ZO-1), a tight junction protein, is an important regulator of the banded region of intercellular tight junctions, playing a crucial role in tight junction formation. Occludin-1 is a membrane-integrated protein that forms and regulates the permeability barrier surrounding tight junctions. ZO-1 protein can interact with Occludin-1 protein to form a complex mediating intercellular tight junctions and signal transduction. Figure 13 As shown, the expression of ZO-1 and Occludin-1 proteins in the gastric tissue of mice treated with medium- and high doses of *Lactobacillus rhamnosus* JGSLR02 was upregulated, and the protein levels tended to normalize. These results indicate that *Lactobacillus rhamnosus* JGSLR02 can restore gastric mucosa damaged by *Helicobacter pylori* by inhibiting gastric epithelial cell apoptosis and promote the expression of gastric mucosal repair proteins in mouse gastric tissue.

[0110] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of a lactic acid bacteria fermented and concentrated asparagus extract in the preparation of a drug with preventive and / or therapeutic effects against Helicobacter pylori infection, characterized in that, By weight, the lactic acid bacteria fermented concentrated asparagus extract is made from the following raw materials: 20-50 parts concentrated asparagus extract, 50-80 parts purified water, 2-6 parts glucose, 2-4 parts white sugar, 0.01-0.1 parts sodium isovitamin C, and 0.001-0.01 parts compound lactic acid bacteria; The Helicobacter pylori species include Helicobacter pylori SS1, Helicobacter pylori ATCC43504 and Helicobacter pylori 26695; The viable count of the compound lactic acid bacteria is (1-9) × 10⁻⁶. 10 CFU / g, composed of Lactobacillus rhamnosus and Lactobacillus plantarum in a 1:1 ratio of live bacteria; The Lactobacillus rhamnosus is Lactobacillus rhamnosus JGSLR02, with accession number CGMCC No.34273; The Lactobacillus plantarum mentioned is Lactobacillus plantarum JGS49, with the accession number CGMCC NO.28396; The method for preparing the concentrated asparagus extract is as follows: Select green asparagus that is intact, with moist cut surfaces without shrinkage, disease spots, insect infestation, rot, spoilage, or off-odors. After cleaning, cut the asparagus into small sections, crush and pulp them, and add pure water at a 1:1 mass ratio. Soak the asparagus at a constant temperature of 50-70 ℃ for 2-3 hours. After filtering and extracting the juice, heat and evaporate it at 80-90 ℃ for 0.5-2 hours to obtain the concentrated asparagus extract.

2. A method for preparing concentrated asparagus extract by lactic acid bacteria fermentation as described in claim 1, characterized in that, Includes the following steps: Mix all the raw materials and ferment them. After fermentation, sterilize them to obtain the lactic acid bacteria fermented concentrated asparagus extract.

3. The preparation method according to claim 2, characterized in that, The fermentation conditions are: 30-37 ℃ for 24-72 h.

4. The production method according to claim 2, characterized by, The sterilization conditions are: 85-121 ℃, sterilization for 5-30 minutes.

5. A medicament for preventing and / or treating Helicobacter pylori infection, characterized by, The drug comprises the lactic acid bacteria fermented concentrated asparagus extract as described in claim 1; the Helicobacter pylori comprises Helicobacter pylori SS1, Helicobacter pylori ATCC43504 and Helicobacter pylori 26695.

6. The use of the lactic acid bacteria fermented concentrated asparagus extract as described in claim 1 in the preparation of a medicament with the efficacy of preventing and / or treating Escherichia coli, Salmonella typhi and / or Staphylococcus aureus infections.

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