Application of Lactobacillus paracasei IOB413 metagen in prevention of Helicobacter pylori infection

Abstract

The invention provides an application of a lactobacillus paracasei IOB413 metagen in prevention of helicobacter pylori infection, and relates to the technical field of microorganisms. The lactobacillus paracasei IOB413 metaplasm can competitively occupy gastric epithelial cell adhesion sites to directly block the initial colonization of helicobacter pylori, so that infection preposed interception is realized, and the lactobacillus paracasei IOB413 metaplasm is suitable for early prevention of high-incidence people. The compound has dual mechanisms of site blocking and urease inhibition, and can cooperate with antibiotics to improve the eradication rate and reduce the drug resistance. Animal experiments show that the traditional Chinese medicine composition can reduce gastric mucosa proinflammatory factors in a dose-dependent manner, improve the activity of antioxidant enzymes, reduce lipid peroxidation products, relieve mucosa lesion, promote repair and reduce the risk of complications; moreover, gastrointestinal hormone disorder can be reversed, the digestive function is maintained, and related discomfort is relieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and particularly relates to application of a Paracaseiclovagus IOB413 probiotic in preventing Helicobacter pylori infection. BACKGROUND

[0002] Helicobacter pylori (Hp) infection is one of the main pathogenic causes of peptic ulcer, which can specifically adhere to the surface of human gastric mucosal epithelial cells, thereby causing gastric infection. According to the report of the World Gastroenterology Organization, at least 50% of the global population is infected with Hp, and the Hp infection rate of the population in China is as high as 60%. Studies have found that Hp is related to the onset of most gastric diseases, such as peptic ulcer, chronic gastritis, atrophic gastritis, gastric cancer, etc., and the World Health Organization has listed it as a class I carcinogen. Hp infection can induce the increase of the levels of pro-inflammatory factors and chemotactic factors in the stomach, thereby causing serious oxidative stress and inflammatory response. Clinically, probiotics combined with antibiotics have great advantages and prospects in the treatment of gastrointestinal diseases caused by Hp positive.

[0003] As a first-line solution to eradicate Hp, the traditional triple therapy is two antibiotics plus a standard dose of proton pump inhibitor, and the four-drug therapy containing bismuth is more commonly used in clinical practice, but both methods can cause gastrointestinal flora disorder and drug adverse reactions, of which the most serious is antibiotic-associated diarrhea in the elderly. In addition, with the emergence of Hp drug resistance, the eradication rate has also decreased. Some studies have shown that probiotic preparations combined with antibacterial drugs can effectively alleviate the clinical symptoms of Hp infection patients, improve the Hp eradication effect, and reduce the incidence of drug adverse reactions; Some studies have shown that probiotic preparations as an adjunct cannot improve the eradication rate of Hp, and even cannot reduce adverse reactions. The commonly used probiotic preparations in clinical practice include Lactobacillus acidophilus, Bifidobacterium triple viable bacteria, Bacillus subtilis double viable bacteria, and Bacillus licheniformis, etc. However, they are all used for adjunctive therapy after Hp infection, and do not consider preventing Hp infection from the perspective of prevention. Therefore, it is urgent to develop a probiotic that can effectively prevent Helicobacter pylori infection. SUMMARY

[0004] In view of the technical problems existing in the prior art, the application aims to provide application of a Paracaseiclovagus IOB413 probiotic in preventing Helicobacter pylori infection.

[0005] Paracaseiclovagus IOB413 (Lactobacillus paracasei IOB413) Lacticaseibacillus paracasei) is a naturally fermented sourdough strain independently screened from the homes of residents in Tianjin. This strain has been preserved and tested for physical and chemical indicators. On March 19, 2021, a bacterial identification test was conducted at the China Food Fermentation Industry Research Institute Co., Ltd. The colonies are white, round, moist, opaque, and have neat edges. They are classified and named Lactobacillus paracasei ( Lacticaseibacillus paracasei ), was deposited on June 29, 2018 in the General Microbiology Center of China Culture Collection Administration, referred to as CGMCC, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No.16022.

[0006] One of the purposes of the present invention is to provide an application of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a medicament for preventing Helicobacter pylori infection. The deposit number of the Lactobacillus paracasei IOB413 is CGMCC No.16022.

[0007] Preferably, the drug for preventing Helicobacter pylori infection comprises Lactobacillus paracasei IOB413 postbiotics and pharmaceutically or food acceptable excipients.

[0008] Preferably, the preparation method of the Lactobacillus paracasei IOB413 postbiotic comprises: The strain in the cryopreserved tube is inoculated into a slant culture medium, cultured at 36.5-37.5°C for 18-24 hours to obtain a slant strain; the slant strain is inoculated into a liquid culture medium containing 2%-4% of, but not limited to, soybeans and yam, cultured in a sealed container at 34-38°C for 18-22 hours to obtain a seed solution; The seed liquid is inoculated into a culture medium including but not limited to soybeans and yam at a material-liquid ratio of 1:1.5, and is cultured in a closed state at 36±2°C for 24-48 hours. After fermentation, the seed liquid is inactivated at 80-95°C, and after drying to a moisture content of ≤10%, the seed liquid is crushed to obtain the postbiotics of Lactobacillus paracasei IOB413.

[0009] Preferably, the slant culture medium comprises 4.0-7.0 g of peptone, 4.0-7.0 g of beef extract, 5.0-7.0 g of yeast powder, 13.0-18.0 g of glucose, 0.8-1.0 mL of Tween 80, 1.5-2.0 g of dipotassium hydrogen phosphate, 3.0-4.0 g of sodium acetate, 1.0-1.2 g of triammonium citrate, 0.1-0.2 g of magnesium sulfate, 0.03-0.05 g of manganese sulfate, 12-15.0 g of agar, pH = 6.2 ± 0.2, and the amount of water added is 1-1.2 L.

[0010] The second object of the present invention is to provide an application of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a health food for preventing gastric mucosal damage caused by Helicobacter pylori.

[0011] The third object of the present invention is to provide an application of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a probiotic preparation for preventing gastric mucosal damage caused by Helicobacter pylori.

[0012] Beneficial effects of the present invention: The present invention provides an application of a postbiotic of Lactobacillus paracasei IOB413 in preventing Helicobacter pylori infection. The postbiotic of Lactobacillus paracasei IOB413 of the present invention can directly block the initial colonization of Helicobacter pylori by competitively occupying the adhesion sites of gastric epithelial cells, thereby achieving pre-infection interception and being suitable for early prevention in high-risk populations. It has a dual mechanism of site blocking and urease inhibition, and can synergize with antibiotics to improve the eradication rate and reduce drug resistance. Animal experiments have shown that it can dose-dependently reduce pro-inflammatory factors in the gastric mucosa, enhance the activity of antioxidant enzymes, reduce lipid peroxidation products, alleviate mucosal damage, promote repair, and reduce the risk of complications; it can also reverse gastrointestinal hormone disorders, maintain digestive function, and relieve related discomfort. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the Hp adhesion after treatment with Lactobacillus paracasei IOB413; Figure 2 is the Hp adhesion capacity of Lactobacillus paracasei IOB413 after postbiotic treatment; Figure 3 is the adhesion of Hp after genistein treatment; Figure 4 The results of gastric mucosal biopsy pathology test; Figure 5 The results of the rapid urease test were used to evaluate the infection of Helicobacter pylori in the gastric tissues of each group; Figure 6 The results of flat plate coating; Figure 7 is the Hp colony count; Figure 8 This is the result of hematoxylin-eosin (H&E) staining of gastric mucosal tissue; Figure 9 is the tumor necrosis gene-α level in gastric mucosal supernatant; Figure 10 is the interleukin-6 level in gastric mucosal supernatant; Figure 11 is the interleukin-8 level in gastric mucosal supernatant; Figure 12 is the interleukin-1β level in gastric mucosal supernatant; Figure 13 is the cyclooxygenase-2 level in gastric mucosal supernatant; Figure 14is the monocyte chemoattractant protein-1 level in gastric mucosal supernatant; Figure 15 is the serum motilin level; Figure 16 is the serum gastrin level; Figure 17 is the superoxide dismutase content in mouse gastric mucosal tissue; Figure 18 is the glutathione peroxidase content in mouse gastric mucosal tissue; Figure 19 MDA content in gastric mucosal tissue of mice.

[0014] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei ) is a naturally fermented sourdough strain independently screened from the homes of residents in Tianjin. This strain has been preserved and tested for physical and chemical indicators. On March 19, 2021, a bacterial identification test was conducted at the China Food Fermentation Industry Research Institute Co., Ltd. The colonies are white, round, moist, opaque, and have neat edges. They are classified and named Lactobacillus paracasei ( Lacticaseibacillus paracasei ), was deposited on June 29, 2018 in the General Microbiology Center of China Culture Collection Administration, referred to as CGMCC, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No.16022. DETAILED DESCRIPTION

[0015] The present invention provides an application of a Lactobacillus paracasei IOB413 postbiotic in preventing Helicobacter pylori infection.

[0016] According to a first aspect of the present invention, there is provided a use of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a medicament for preventing Helicobacter pylori infection, wherein the deposit number of the Lactobacillus paracasei IOB413 is CGMCC No.16022.

[0017] In this study, once H. pylori has initially adhered and established a stable colonization, its binding to host cells likely involves more complex molecular interactions, making it difficult to effectively eliminate it through probiotic-mediated physical site competition alone. Lactobacillus paracasei IOB413 can effectively block H. pylori colonization in the gastric epithelium or mucosa by pre-competitively occupying surface adhesion sites on GES-1 cells (a normal human gastric epithelial cell line) and inhibiting urease activity.

[0018] In a preferred embodiment of the present invention, the drug for preventing Helicobacter pylori infection comprises a Lactobacillus paracasei IOB413 postbiotic and pharmaceutically or food-acceptable excipients.

[0019] In a preferred embodiment of the present invention, the preparation method of Lactobacillus paracasei IOB413 postbiotics comprises: The bacterial strain in the cryopreserved tube is inoculated into a slant culture medium, cultured at 36.5-37.5°C for 18-24 hours to obtain a slant culture; the slant culture is inoculated into a liquid culture medium containing 2%-4% of a substrate including but not limited to soybeans, yam, etc., cultured in a sealed static manner at 34-38°C for 18-22 hours to obtain a seed solution; The seed liquid is inoculated into a culture medium including but not limited to soybeans, yam and the like at a material-liquid ratio of 1:1.5, cultured in a closed state at 36±2°C for 24-48 hours, inactivated at 80-95°C after fermentation, dried for 20-24 hours and then crushed to obtain the postbiotics of Lactobacillus paracasei IOB413.

[0020] In a preferred embodiment of the present invention, the slant culture medium comprises 4.0-7.0 g of peptone, 4.0-7.0 g of beef extract, 5.0-7.0 g of yeast powder, 13.0-18.0 g of glucose, 0.8-1.0 mL of Tween 80, 1.5-2.0 g of dipotassium hydrogen phosphate, 3.0-4.0 g of sodium acetate, 1.0-1.2 g of triammonium citrate, 0.1-0.2 g of magnesium sulfate, 0.03-0.05 g of manganese sulfate, 12-15.0 g of agar, pH = 6.2 ± 0.2, and the amount of water added is 1-1.2 L.

[0021] According to a second aspect of the present invention, there is provided a use of a Lactobacillus paracasei IOB413 postbiotic in the preparation of a health food for preventing gastric mucosal damage caused by Helicobacter pylori.

[0022] According to a third aspect of the present invention, there is provided a use of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a probiotic preparation for preventing gastric mucosal damage caused by Helicobacter pylori.

[0023] Example 1 The present embodiment is a method for determining the Hp adhesion capacity of Lactobacillus paracasei 10B413, Lactobacillus paracasei 10B413 postbiotics and genistein, comprising the following steps: Preparation of Lactobacillus paracasei 10B413 bacterial powder and Lactobacillus paracasei 10B413 postbiotics: Preparation of Lactobacillus paracasei 10B413 bacterial powder: Strain activation: The bacteria in the cryopreserved tube were inoculated into a slant culture medium and cultured at 37°C for 20 hours. The activated colonies were white, round, with a moist surface, opaque, and neat edges. Microscopic examination showed that the bacteria were rod-shaped, uniform, and robust. After activation, the unfermented sample of Lactobacillus paracasei IOB413 was harvested.

[0024] The slant medium uses a modified MRS medium, and the formula is: 5.0 g of protein peptone, 5.0 g of beef extract, 6.0 g of yeast powder, 15.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of potassium phosphate, 4.0 g of sodium acetate, 1.0 g of triammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15.0 g of agar, pH = 6.2, and the water amount is 1 L.

[0025] The strain is inoculated in the MRS medium at 1:20, 37℃, static, anaerobic, and cultured for 36 h to obtain the primary seed liquid, wherein the MRS medium formula is 5 g of protein peptone, 15 g of glucose, 7 g of beef extract powder, 5 g of sodium acetate, and 1000 ml of distilled water is used for dissolving and configuration, the above components are mixed, and the solution pH is adjusted to 6.5, and then 121℃ sterilization is performed for 25 min.

[0026] The primary seed is inoculated in the unoptimized medium at 1:25, 37℃, static, anaerobic, and cultured for 36 h to obtain the secondary seed liquid; Fermentation: the cultured secondary seed liquid is inoculated into the sterilized fermentation liquid medium at an inoculation amount of 1:50, the fermentation tank is stirred at a speed of 30 r / min, 37℃, anaerobic culture is performed for 12 h to obtain the fermentation liquid; wherein the fermentation liquid medium is: 5 g of protein peptone, 15 g of glucose, 7 g of beef extract powder, 5 g of sodium acetate, and 1000 ml of distilled water is used for dissolving and configuration, the above components are mixed, and the solution pH is adjusted to 6.2, and then 121℃ sterilization is performed for 15 min.

[0027] Bacterial powder: the fermentation liquid medium is centrifuged, and the bacterial slurry is collected; the bacterial slurry is freeze-dried to obtain the Lactobacillus paracasei IOB413 bacterial powder.

[0028] Preparation of Lactobacillus paracasei IOB413 postbiotic: Strain activation: the strain in the frozen tube is inoculated into the slant medium and placed in a 37℃ culture, and the activation time is 20 h; the activated colony is white, round, surface wet, opaque, edge neat, and the bacterial body is rod-shaped, individual uniform and robust; the Lactobacillus paracasei IOB413 unfermented sample is harvested after activation.

[0029] The slant medium uses a modified MRS medium, and the formula is: 5.0 g of protein peptone, 5.0 g of beef extract, 6.0 g of yeast powder, 15.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of potassium phosphate, 4.0 g of sodium acetate, 1.0 g of triammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15.0 g of agar, pH = 6.2, and the water amount is 1 L.

[0030] Take a loop of fresh slant culture medium and inoculate it into 25 mL of liquid culture medium containing 2% soybean powder. Incubate it in a sealed container at 36°C for 20 h to obtain seed solution.

[0031] The seed liquid with the best fermentation conditions was inoculated into soybean powder at a material-liquid ratio of 1:1.5, and cultured in a closed state at 36°C for 24 hours. After fermentation, it was inactivated at 85°C, dried to a moisture content of 8%, and crushed to obtain the postbiotics of Lactobacillus paracasei IOB413.

[0032] Adjust the GES-1 cell concentration to 5.0 × 10 3 cells / mL, seeded in 96-well plates, cultured in a 37°C, 5% CO2 incubator for 24 h, and washed the GES-1 cells cultured to a monolayer with PBS three times to obtain a monolayer GES-1 cell suspension.

[0033] The Hp standard strain and Lactobacillus paracasei IOB413 were resuspended in RPMI1640 solution, and the concentration of the bacterial suspension was adjusted to 1.5×10 7 CFU / mL, and obtain Hp bacterial suspension and Lactobacillus paracasei IOB413 suspension.

[0034] Blank group: 200 μL of RPMI1640 solution was added to the monolayer GES-1 cell suspension; the cells were washed three times with PBS, and 200 μL of urea-phenol red reagent was added and incubated for 1 hour. The absorbance at a wavelength of 550 nm was measured using a microplate reader.

[0035] Helicobacter pylori group: 200 μL of RPMI1640 was added to the monolayer GES-1 cell suspension, washed with PBS after 2 h, and 50 μL of H. pylori suspension and 150 μL of RPMI1640 were added. The cells were cultured in a 37°C, 5% CO2 incubator for 2 h; washed three times with PBS, 200 μL of urea-phenol red reagent was added, and cultured for 1 h. The absorbance at a wavelength of 550 nm was measured using a microplate reader.

[0036] IOB413 pretreatment group: 50ul of Lactobacillus paracasei IOB413 suspension and 150ul of RPMI1640 were added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. The cells were then washed three times with PBS to clean the unadsorbed probiotics. 50μL of Hp bacterial suspension and 150μL of RPMI1640 were then added, and the cells were cultured in a 37°C, 5% CO2 incubator for another 2 hours. The cells were washed three times with PBS, 200μL of urea-phenol red reagent was added, and the cells were cultured for 1 hour. The absorbance at a wavelength of 550nm was measured using a microplate reader.

[0037] IOB413 postbiotic pretreatment group: 50 μL of Lactobacillus paracasei IOB413 postbiotic solution and 150 μL of RPMI1640 solution were added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 2 hours. The cells were then washed three times with PBS, and 50 μL of Hp bacterial suspension and 150 ul of RPMI1640 solution were added, and the cells were continued to be cultured in a 37°C, 5% CO2 incubator for 2 hours. The cells were washed three times with PBS, and 200 μL of urea-phenol red reagent was added to culture for 1 hour. The absorbance at a wavelength of 550 nm was measured using a microplate reader.

[0038] Genistein pretreatment group: 50 μL of genistein solution and 150 μL of RPMI1640 solution were added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator for 2 h. The cells were then washed three times with PBS, and 50 μL of Hp bacterial suspension and 150 ul of RPMI1640 solution were added, and the cells were continued to be cultured in a 37°C, 5% CO2 incubator for 2 h. The cells were washed three times with PBS, and 200 μL of urea-phenol red reagent was added to culture for 1 h. The absorbance at a wavelength of 550 nm was measured using a microplate reader.

[0039] IOB413 post-treatment group: 50 μL of H. pylori suspension and 150 μL of RPMI1640 were added to each well and incubated at 37°C in a 5% CO2 incubator for 2 h. The wells were then washed three times with PBS to remove any unadsorbed H. pylori. 50 μL of Lactobacillus paracasei IOB413 suspension and 150 μL of RPMI1640 were then added and incubated for another 2 h. The wells were then washed three times with PBS and incubated for 1 h with 200 μL of urea-phenol red reagent. The absorbance at 550 nm was then measured using a microplate reader.

[0040] IOB413 postbiotic treatment group: 50 μL of H. pylori suspension and 150 μL of RPMI1640 were added to each well and incubated at 37°C in a 5% CO2 incubator for 2 hours. The cells were then washed three times with PBS to remove any unadsorbed H. pylori. 50 μL of the Lactobacillus paracasei IOB413 postbiotic solution and 150 μL of RPMI1640 were then added and incubated for another 2 hours. The cells were then washed three times with PBS and incubated for 1 hour with 200 μL of urea-phenol red reagent. The absorbance at 550 nm was measured using a microplate reader.

[0041] Genistein post-treatment group: 50 μL of H. pylori suspension and 150 μL of RPMI1640 were added to each well and incubated at 37°C in a 5% CO2 incubator for 2 h. The wells were then washed three times with PBS to remove any unadsorbed H. pylori. 50 μL of genistein solution and 150 μL of RPMI1640 were then added and incubated for another 2 h. The wells were then washed three times with PBS and incubated for 1 h with 200 μL of urea-phenol red reagent. The absorbance at 550 nm was then measured using a microplate reader.

[0042] The formula for calculating Hp adhesion is as follows: Hp adhesion force = [OD 实验组 -OD 空白组 ] / [OD 阳性组 -OD 空白组 ] Wherein, OD represents the absorbance, the experimental group includes IOB413 pretreatment group, IOB413 postbiotic pretreatment group, IOB413 post-treatment group and IOB413 postbiotic post-treatment group; the positive group is Hp group.

[0043] As shown in Figure 1 , Figure 2 and Figure 3 , after GES-1 cells were pretreated with IOB413 probiotics, postbiotics and genistein, the adhesion force of Helicobacter pylori showed a significant downward trend. Taking the adhesion force of the Hp positive control group as 100% as the benchmark, the adhesion force of the Paracasei group, the postbiotic group and the genistein group decreased to 60.8% ± 4.2%, 48.8% ± 2.7% and 72.8% ± 4.9% respectively, showing excellent inhibitory effect. Statistical analysis further confirmed that compared with the Hp positive group, the adhesion force of the three pretreatment groups had statistically significant difference (P<0.001). According to this phenomenon, it is speculated that the potential mechanism is that probiotics and their metabolites can effectively block the adhesion process of Hp by competitively occupying the adhesion sites on the surface of GES-1 cells, thereby achieving the effect of preventing Hp infection.

[0044] Helicobacter pylori was co-incubated with GES-1 cells for 2 hours, and then Paracasei IOB413, postbiotics and genistein were used for 2 hours of intervention after the adhesion sites on the gastric epithelial cells were fully occupied. As shown in Figure 1 , Figure 2 and Figure 3 , the Hp adhesion force of each post-treatment group was significantly higher than that of the corresponding pretreatment group (P<0.001). In the post-treatment group, the IOB413 postbiotic post-treatment group with the strongest inhibitory effect only reduced the Hp adhesion force to 92.2% ± 3.1%. The pretreatment and post-treatment groups were in sharp contrast. When Paracasei IOB413 or Paracasei IOB413 postbiotics preferentially occupied the adhesion sites, the Hp adhesion force was significantly reduced, among which the Hp adhesion force of the Paracasei IOB413 postbiotic pretreatment group was about 48%; while the post-treatment group could not achieve the same inhibitory effect. This phenomenon shows that once Hp completes the initial adhesion and forms stable colonization, the combination with host cells may involve more complex molecular interaction mechanisms, which is difficult to achieve effective clearance by probiotics.

[0045] Example 2 This example is a study on the in vivo inhibitory effect of Paracasei IOB413 postbiotic powder on Helicobacter pylori, including: 1.1 Experimental materials Experimental animals SPF level healthy male CBLB / A mice (rearing temperature about 20-22℃, humidity about 60%-70%, purchased from SPFBEIFU (Beijing, China), license number SCXK (Beijing) 2024-0001. 12 hours of light per day, 12 hours of darkness, all animals free to eat and drink water).

[0046] Bacteria removal: the pre-prepared antibiotic mixture for removing bacteria was used for oral gavage operation on Hp infected SPF Kunming mice, 1 mL per SPF Kunming mouse, fasting for 6 hours before gavage, fasting and water deprivation for 2 hours after gavage.

[0047] Establishment of animal model 1.2 Experimental method 1.2.1 Grouping The SPF CBLB / A mice were divided into 8 groups, 8 mice in each group, namely blank control group, model group, protection 1 group, protection 2 group, protection 3 group, treatment 1 group, treatment 2 group and treatment 3 group. Before modeling, the protection group was given probiotic gavage of Paracasei IOB413 for 20 days (once a day), and the rest of the groups were normally fed and watered.

[0048] 1.2.2 Modeling At the beginning of modeling, except for the blank control group, the rest of the 7 groups were subjected to bacteria removal, and then Hp suspension gavage operation was performed. Fasting for 6 hours before gavage, fasting and water deprivation for 2 hours after gavage, and then resuming open diet and water; at the same time, the blank control group was given 2.0m1 saline gavage with the same type of SPF CBLB / A mice, fasting for 6 hours before gavage, fasting and water deprivation for 2 hours after gavage, and then resuming open diet and water; gavage once every other day, a total of 4 times (i.e. gavage operation on days 1, 3, 5 and 7), after completing all the modeling related operations, the rest of the groups were normally raised for 7 days, the treatment group was given probiotic gavage of Paracasei IOB413 (once a day), and after continuous gavage for one week, all the mice were treated.

[0049] First, the mice were anesthetized with chloral hydrate (0.5mL / 100g) in proportion to their body weight, the chest was opened after anesthesia, and the heart blood was collected with a disposable vacuum negative pressure blood collection tube without anticoagulant, then centrifuged (3000rpm, 20min), the supernatant was taken, and the supernatant was taken again (i.e. heart serum) for determination of plasma gastrointestinal hormone levels and serum factor levels; then the abdomen was opened, and several pieces of mouse antral and gastric body mucosa were taken for antioxidant capacity determination, rapid urease test, colony count experiment and gastric mucosa histopathological detection, etc., and finally the mice were sacrificed for recovery.

[0050] The specific sample gavage doses of each group of mice are shown in Table 1: Table 1 Specific sample gavage doses of each group of mice

[0051] Note: The test sample is dissolved in 8 mL / kg (body weight) of 0.85% normal saline, and then gavaged.

[0052] 1.3 Index detection results 1.3.1 Hp colonization of mouse gastric mucosa To detect the effect of postbiotics of different concentrations on the inhibition of Helicobacter pylori in vivo, we established a CBLB / A mouse model infected with Helicobacter pylori. After the modeling was completed, the gastric tissue was collected, and the gastric mucosa was detected by histopathology, as shown in Figure 4 compared with the control group, the gastric mucosa of mice infected with Helicobacter pylori showed damage and hyperemia, and after intervention with postbiotics of different concentrations, the size and severity of gastric mucosa lesions decreased.

[0053] In addition, the infection of Helicobacter pylori in the gastric tissue of each group was evaluated by rapid urease test. Yellow negative (-), red or pink positive (+). As shown in Figure 5 all samples of the model group were positive, indicating high urease activity and bacterial colonization level, and the bacteriostatic efficiency of the protection group and the treatment group showed a gradient dependence. The urease absorbance value of the high concentration protection group was significantly lower than that of the model group, and the inhibition efficiency of the protection group was better than that of the treatment group under the same concentration.

[0054] As shown in Figure 6 Plate culture results showed that compared with the blank control group, a large number of Hp colonies appeared in the plate culture of gastric tissue of Hp infected mice. As shown in the statistics of Hp colony number Figure 7 Postbiotic intervention showed a gradient dependence, reducing Hp colonies, and preventive intervention was significantly better than therapeutic administration.

[0055] This indicates that postbiotic IOB413 can effectively eradicate Helicobacter pylori infection in vivo. These in vivo study results indicate that postbiotics have the potential to be an effective urease inhibitor, effectively hindering the colonization of Helicobacter pylori on the gastric epithelium or mucosa layer by inhibiting urease activity. Also suggests that preventive high-dose postbiotic intervention may work through a dual mechanism - both reducing H. pylori urease activity to reduce gastric mucosa damage and enhancing mucosal barrier function to synergistically promote tissue repair.

[0056] 1.3.2 ELISA determination of mouse inflammatory factors After Helicobacter pylori infection, the microenvironment of gastric mucosa tissue showed characteristics of inflammatory imbalance. As shown in Figure 8Hematoxylin and eosin (H&E) staining revealed intact gastric mucosal structure in the blank control group (control), with orderly arranged epithelial cells and densely distributed glands, and no inflammatory cell infiltration was observed. The model group (Hp group) exhibited pathological changes, with inflammatory cell infiltration visible in the lamina propria and submucosal layers. Microscopic observations revealed a dose-response relationship between the different intervention groups. In the protection group, compared with the model group (Hp group), the number of inflammatory cell infiltrates in the lamina propria of the gastric mucosa in the high-dose protection group 3 was reduced, demonstrating robust anti-inflammatory activity. This group exhibited superior anti-inflammatory efficacy compared to the protection groups 1 and 2. This suggests that, under the preventive intervention strategy, the inhibitory effect of postbiotics on Hp infection-induced gastric mucosal inflammation exhibited a dose-dependent enhancement. Similar dose-response trends were observed in treatment groups 1, 2, and 3 (including low, medium, and high doses). Notably, at the same dose level, the protection group exhibited generally superior anti-inflammatory efficacy compared to the treatment group, suggesting that the preventive intervention strategy has a superior inflammatory modulating effect.

[0057] like Figures 9-14 As shown, Figure 9 The level of tumor necrosis gene-α (TNF-α) in gastric mucosal supernatant, Figure 10 The level of interleukin-6 (IL-6) in gastric mucosal supernatant, Figure 11 The level of interleukin-8 (IL-8) in gastric mucosal supernatant, Figure 12 The level of interleukin-1β (IL-1β) in gastric mucosal supernatant, Figure 13 The cyclooxygenase-2 level in gastric mucosal supernatant, Figure 14 The levels of monocyte chemoattractant protein-1 in the gastric mucosal supernatant were significantly altered in the model group (Hp group). ELISA assays showed that Hp infection strongly activated proinflammatory signaling pathways, as evidenced by significantly increased secretion of proinflammatory cytokines TNF-α, IL-6, IL-8, and IL-1β compared with the control group (p < 0.001). Simultaneously, gene expression of the inflammatory-related factors monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) was significantly upregulated (p < 0.01), suggesting a synergistic effect between the inflammatory cascade and the imbalance of mucosal repair. At the same concentration, the decrease in TNF-α, IL-1β, IL-8, and IL-1β levels in the preventive intervention group was significantly greater than that in the therapeutic intervention group (p < 0.05).

[0058] In addition, serological tests suggest that Helicobacter pylori infection can be accompanied by gastrointestinal hormone imbalance—the expression of motilin (which regulates gastrointestinal motility) is suppressed, while gastrin (which promotes gastric acid secretion) is abnormally elevated, suggesting that inflammatory response and secretory-motor dysfunction synergistically exacerbate tympanic membrane damage. Postbiotic intervention significantly reversed the above pathological changes in a dose-dependent manner: Figure 14-16 As shown in FIG. 6, Figure 15 As serum motilin levels, Figure 16 As serum gastrin levels, the protective group 3 (high dose) not only effectively inhibited the expression of pro-inflammatory factors and COX-2 (p<0.01), but also simultaneously restored motilin levels and reduced gastrin secretion (p<0.01), and the comprehensive regulation effect was significantly better than that of the protective group 1 and the protective group 2 (medium and low dose groups). Notably, the mucosa protective effect of the prophylactic administration group was better than that of the same dose treatment group, indicating that the probiotic can inhibit the inflammatory cascade through early regulation of the signaling pathway and repair the gastrointestinal hormone homeostasis, thereby playing a multi-target mucosa repair function.

[0059] 1.3.3 Determination of oxidative stress factors in mouse gastric mucosa tissue As a key antioxidant enzyme, superoxide dismutase (SOD) plays a protective role in gastric mucosa by scavenging active oxygen free radicals. As shown in FIG. 7, the SOD content in mouse gastric mucosa tissue was determined, and the SOD activity in the gastric tissue of the model group was significantly lower than that of the blank control group (P<0.001), while the SOD activity of each dose intervention group showed a dose-dependent recovery. Notably, the protective group showed more significant enzyme activity improvement than the treatment group (P>0.001), suggesting that the prophylactic intervention strategy can more effectively maintain the function of the SOD system. Figure 17 The presence of Helicobacter pylori virulence factors can trigger excessive production of oxygen free radicals in cells, leading to increased lipid peroxidation levels and damaged cell membrane structure, ultimately causing gastric mucosa damage. Malondialdehyde (MDA) is an indicator for assessing oxidative stress, and its level is positively correlated with the degree of oxidative stress, while glutathione peroxidase (GSH-Px) is negatively correlated with the degree of oxidative stress. As shown in FIG. 8, the glutathione peroxidase content in mouse gastric mucosa tissue was determined, and the GSH-Px level in the gastric tissue of the model group was significantly reduced (P<0.001). After intervention treatment, the GSH-Px activity of each experimental group showed a gradient recovery, and the prophylactic intervention was more effective than the therapeutic intervention (P>0.001). As shown in FIG. 9, the malondialdehyde content in mouse gastric mucosa tissue was determined, and the MDA content of the model group was significantly higher than that of the control group (P>0.001). After intervention treatment, the MDA content of each dose (low, medium, and high) of the protective group showed a gradient downward trend. This result further confirmed the advantage of prophylactic intervention in inhibiting lipid peroxidation. The probiotic can effectively restore the activity of antioxidant enzymes such as SOD and GSH-Px in a dose-dependent manner, while significantly reducing the accumulation of peroxidation products such as MDA, and the prophylactic administration strategy shows a more optimal intervention effect in improving oxidative stress damage, which provides key experimental evidence for elucidating the gastric mucosa protection mechanism of the probiotic.

[0060] Figure 18 Figure 19 ​​​

[0061] In summary, Lactobacillus paracasei IOB413 and its postbiotics competitively occupy adhesion sites on the surface of gastric epithelial cells (GES-1), directly blocking the initial colonization of Helicobacter pylori (Hp), thereby preventing infection at the source. Unlike existing probiotics, which are only used for post-infection adjuvant treatment, this invention is the first to achieve pre-emptive intervention for Hp infection, making it particularly suitable for early prevention in populations at high risk of Hp.

[0062] like Figure 5 Rapid urease tests have shown that the Lactobacillus paracasei IOB413 postbiotic not only inhibits H. pylori adhesion through physical site competition but also significantly reduces H. pylori urease activity, diminishing its survival advantage in the stomach. This dual mechanism of site blocking combined with urease inhibition can synergize with traditional antibiotic therapy to improve H. pylori eradication rates and reduce the risk of drug resistance.

[0063] Animal experiments have confirmed that Lactobacillus paracasei IOB413 postbiotics can reduce the levels of proinflammatory factors in the gastric mucosa in a dose-dependent manner, while also increasing the activity of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase, and reducing the accumulation of malondialdehyde, a lipid peroxidation product. This effect can alleviate mucosal damage caused by H. pylori infection, promote gastric mucosal repair, and reduce the risk of complications such as chronic gastritis and gastric ulcers.

[0064] Lactobacillus paracasei IOB413 postbiotics can reverse the gastrointestinal hormone disorders caused by Hp infection, restore serum motilin levels and reduce gastrin secretion, thereby maintaining normal digestive function and alleviating symptoms such as abdominal distension and indigestion associated with Hp infection.

Claims

1. An application of a Lactobacillus paracasei IOB413 postbiotic in the preparation of a medicine for preventing Helicobacter pylori infection, characterized in that: The deposit number of the Lactobacillus paracasei IOB413 is CGMCC No.16022.

2. The use according to claim 1, characterized in that The medicine for preventing Helicobacter pylori infection comprises Lactobacillus paracasei IOB413 postbiotics and pharmaceutically or food-acceptable excipients.

3. The use according to claim 2, characterized in that The preparation method of the Lactobacillus paracasei IOB413 postbiotic comprises: The strain in the cryopreserved tube is inoculated into a slant culture medium, cultured at 36.5-37.5°C for 18-24 hours to obtain a slant strain; the slant strain is inoculated into a liquid culture medium containing 2%-4% of, but not limited to, soybeans and yam, cultured in a sealed container at 34-38°C for 18-22 hours to obtain a seed solution; The seed liquid is inoculated into a culture medium including but not limited to soybeans and yam at a material-liquid ratio of 1:1.5, and is cultured in a closed state at 36±2°C for 24-48 hours. After fermentation, the seed liquid is inactivated at 80-95°C, and after drying to a moisture content of ≤10%, the seed liquid is crushed to obtain the postbiotics of Lactobacillus paracasei IOB413.

4. The use according to claim 3, characterized in that The slant culture medium includes 4.0-7.0 g of peptone, 4.0-7.0 g of beef extract, 5.0-7.0 g of yeast powder, 13.0-18.0 g of glucose, 0.8-1.0 mL of Tween 80, 1.5-2.0 g of dipotassium hydrogen phosphate, 3.0-4.0 g of sodium acetate, 1.0-1.2 g of triammonium citrate, 0.1-0.2 g of magnesium sulfate, 0.03-0.05 g of manganese sulfate, 12-15.0 g of agar, pH=6.2±0.2, and the amount of water added is 1-1.2 L.

5. Use of a Lactobacillus paracasei IOB413 postbiotic in the preparation of a health food for preventing gastric mucosal damage caused by Helicobacter pylori.

6. Use of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a probiotic for preventing gastric mucosal damage caused by Helicobacter pylori.

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