A paracasei, probiotics and application thereof in preparing anti-helicobacter pylori products

Abstract

The application discloses a Lacticaseibacillus paracasei, a probiotic and application of the Lacticaseibacillus paracasei in preparation of anti-Helicobacter pylori products, and belongs to the technical field of probiotics.The application isolates 28 strains of bacteria from infant feces, and the 28 strains of bacteria are identified as including Lacticaseibacillus paracasei, Lactobacillus plantarum and the like through analysis, and the strains are evaluated through in-vitro experiments, and finally a Lacticaseibacillus paracasei FL5 with potential anti-Helicobacter pylori is screened out, and then through in-vitro co-culture experiments, anti-bacterial metabolite quantification, scanning electron microscopy and in-vivo model experiments, the efficacy of the Lacticaseibacillus paracasei FL5 in resisting Helicobacter pylori in the body is verified from aspects of anti-inflammation, anti-oxidation, implantation inhibition ability and flora, and the like, and a new treatment strategy for resisting Helicobacter pylori infection is provided.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of probiotics, in particular to a paracasei, a postbiotic and application of the paracasei and the postbiotic in preparation of an anti-Helicobacter pylori product. BACKGROUND

[0002] Helicobacter pylori (H. pylori) is a microaerophilic spiral-shaped bacterium, which was first isolated from human gastric mucosa by Barry J. Marshall and Robin Warren in 1982. This pathogen colonizes more than half of the world's population. Its etiology is related to chronic gastritis, peptic ulcer, gastric mucosal atrophy, intestinal metaplasia and gastric cancer. In 1994, the International Agency for Research on Cancer (IARC / WHO) listed H. pylori as a class 1 carcinogen. The successful colonization of H. pylori in the gastric wall depends on various virulence factors, including flagella, urease and adhesin, which together mediate its pathogenic mechanism. Among them, urease plays a key role in bacterial survival by hydrolyzing gastric urea to ammonia, thereby neutralizing gastric acid and creating a favorable microenvironment. Genetic studies have confirmed the importance of urease, as H. pylori strains lacking the ureA or ureB gene show impaired gastric colonization in mouse models.

[0003] The current first-line recommended standard treatment for H. pylori is the bismuth-containing quadruple therapy (14-day course), which is a proton pump inhibitor + bismuth + two antibiotics. However, due to widespread antibiotic misuse, antibiotic resistance is rising, which seriously undermines treatment effectiveness, reduces treatment success rates, and exacerbates clinical management challenges. Therefore, there is an urgent need to develop alternative treatments that are both effective and minimally interfere with host physiological functions.

[0004] Probiotic adjunctive therapy has become a promising strategy to improve the eradication of H. pylori. Among probiotics, lactic acid bacteria are generally considered safe and have been widely used in clinical settings for the management of H. pylori. However, the prior art discloses lactic acid bacteria for preventing and treating H. pylori, including Paracasei (such as application number 202210032204.8, entitled "Application of Paracasei nbk-LC16 in improving H. pylori infection and preparing anti-inflammatory stomach protection products"; application number 202311302060.4, entitled "Preparation method and application of Paracasei IOB413 probiotic extract product"; application number 202110066239.9, entitled "Application and product of Paracasei JLPF-176 for inhibiting H. pylori infection"), Lactobacillus plantarum (201110059827.6, Lactobacillus plantarum for resisting H. pylori infection and its use), Lactobacillus rhamnosus (202310284857.X, Lactobacillus rhamnosus for improving H. pylori related gastrointestinal diseases and its application), and Lactobacillus fermentum, etc., but the above-mentioned strains are not ideal for preventing and treating H. pylori, such as low urease activity inhibition rate, poor antibacterial ability to H. pylori, and most of the live bacteria have prevention and treatment effects, and the inactivated strains do not have corresponding prevention and treatment effects, etc., resulting in a lack of specific strains that can effectively antagonize H. pylori.

[0005] Therefore, how to provide a new type of high-efficiency antagonistic H. pylori strain is a technical problem that those skilled in the art urgently need to solve. SUMMARY

[0006] Therefore, the present application provides a Paracasei, a probiotic and its application in preparing an anti-H. pylori product. From the aspects of anti-inflammatory, antioxidant, inhibition of colonization ability and flora, etc., the efficacy of Lacticaseibacillus paracasei FL5 in vivo against H. pylori is verified, providing a new treatment strategy for H. pylori infection.

[0007] The first object of the present application is to provide:

[0008] A Paracasei, which is Lacticaseibacillus paracasei FL5, deposited in the Guangdong Microbial Culture Collection Center on March 6, 2025, with the accession number GDMCC NO: 65986, and the address of the depositary is No. 59, Building 5, 100, Martyrs' Road, Guangzhou.

[0009] Still another object of the present application is to provide the probiotic of the above-mentioned Lactobacillus paracasei, which comprises one or more of the Lactobacillus paracasei FL5 fermentation broth, the Lactobacillus paracasei FL5 fermentation supernatant and the Lactobacillus paracasei FL5 inactivated strain.

[0010] In one embodiment, the Lactobacillus paracasei FL5 fermentation broth is prepared by culturing the Lactobacillus paracasei FL5 in MRS liquid medium to obtain the Lactobacillus paracasei FL5 fermentation broth. The Lactobacillus paracasei FL5 fermentation broth of the present application can also be prepared by culturing in other fermentation medium as long as the medium can provide the necessary nutrients for the proliferation of the Lactobacillus paracasei FL5.

[0011] In one embodiment, the Lactobacillus paracasei FL5 fermentation supernatant is prepared by centrifuging and separating the prepared Lactobacillus paracasei FL5 fermentation broth, collecting the supernatant to obtain the Lactobacillus paracasei FL5 fermentation supernatant.

[0012] In one embodiment, the Lactobacillus paracasei FL5 inactivated strain is prepared by centrifuging and separating the prepared Lactobacillus paracasei FL5 fermentation broth, discarding the supernatant, washing and inactivating the bacterial cells obtained after centrifugation to obtain the Lactobacillus paracasei FL5 inactivated strain.

[0013] Still another object of the present application is to provide the application of the above-mentioned Lactobacillus paracasei or the above-mentioned probiotic of the Lactobacillus paracasei, which is any one of the following:

[0014] (1) the application in preparing a product for inhibiting the urease activity of H. pylori;

[0015] (2) the application in preparing a product for inhibiting the proliferation of H. pylori;

[0016] (3) use in the preparation of a product for inhibiting Helicobacter pylori colonization;

[0017] (4) use in the preparation of a product for preventing and / or treating inflammation caused by Helicobacter pylori;

[0018] (5) use in the preparation of a product for preventing and / or treating oxidative damage caused by Helicobacter pylori;

[0019] (6) use in the preparation of a product for preventing and / or treating dysbiosis caused by Helicobacter pylori.

[0020] Still another object of the present application is to provide an anti-Helicobacter pylori product comprising the Lacticaseibacillus paracasei and / or the postbiotic of the Lacticaseibacillus paracasei.

[0021] In one embodiment, the anti-Helicobacter pylori product further comprises other active ingredients or excipients.

[0022] In one embodiment, the product comprises a food or a drug.

[0023] From the above technical solutions, compared with the prior art, the present application has the following beneficial effects:

[0024] 1) The present application isolates 28 strains of bacteria from infant feces, which are identified as including Lacticaseibacillus paracasei, Lactobacillus plantarum, Pediococcus acidilactici, Bifidobacterium longum, Bifidobacterium breve and Bifidobacterium animalis, and through in vitro experiments, the self-aggregation ability, co-aggregation ability with Helicobacter pylori, cell surface hydrophobicity, urease inhibition and direct antibacterial activity of the strains are evaluated, and finally a Lacticaseibacillus paracasei FL5 strain with potential anti-Helicobacter pylori is selected, and then through in vitro co-culture experiments, antibacterial metabolite quantification and scanning electron microscopy, and in vivo model experiments, the efficacy of Lacticaseibacillus paracasei FL5 against Helicobacter pylori in vivo is verified from the aspects of anti-inflammatory, antioxidant, colonization inhibition ability and flora, etc., thereby providing a new treatment strategy for anti-Helicobacter pylori infection.

[0025] 2) The live Lacticaseibacillus paracasei, the fermentation broth, the supernatant and the inactivated strain of the present application all have a prevention and treatment effect on Helicobacter pylori, and the prevention and treatment effect is better than that of the published strain of the prior art; and it is also emphasized that the inactivated strain of Lacticaseibacillus paracasei FL5 is better than the live bacteria in regulating the inflammatory response after Helicobacter pylori infection.

[0026] 3) In the process of constructing a mouse model, once every other day for a total of 3 weeks of gavage, the construction of a Helicobacter pylori mouse model is carried out, compared with the prior art of once every other day for a total of 3 times, the modeling time is long, the model infection and severity are deeper, compared with other patent research, the degree is deeper, and the index is high in reliability. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0028] Figure 1 Figure 1 is a graph of the urease activity inhibition rate of each component of Lacticaseibacillus paracasei FL5 on Helicobacter pylori.

[0029] Figure 2 Figure 2 is a graph of the bacteriostatic ability determination of each component of Lacticaseibacillus paracasei FL5 on Helicobacter pylori.

[0030] Figure 3 Figure 3 is a scanning electron microscope (SEM) of Lacticaseibacillus paracasei FL5 and Helicobacter pylori.

[0031] Figure 4 Figure 4 is a graph of the body weight of mice in different groups.

[0032] Figure 5 Figure 5 is a graph of the urease activity of the stomach tissue of mice in different groups.

[0033] Figure 6 Figure 6 is a graph of the relative expression amount of Helicobacter pylori virulence genes CagA and VacA in the stomach tissue of mice in different groups.

[0034] Figure 7 Figure 7 is a representative graph of the stomach tissue sections of different groups.

[0035] Figure 8 Figure 8 is a graph of the inflammation factors of the stomach tissue of different groups.

[0036] Figure 9 Figure 9 is a graph of the oxidative stress of the liver of different groups.

[0037] Figure 10 Figure 10 is a PCA graph of the stomach flora of different groups.

[0038] Figure 11Figure 6: LEfSe plot of different groups of gastric bacterial community.

[0039] Figure 12 Figure 7: Bar plot of community composition of different groups.

[0040] Figure 13 Figure 8: Relative expression of claudin 1 and occludin in different groups.

[0041] Figure 14 Figure 9: Relative expression of TLR2 / MyD88 / NF-κB signaling pathway in different groups. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0043] The strains used in the embodiments of the present application are as follows:

[0044] The Helicobacter pylori SS1 (ATCC 43504, GDMCC 1.1820) was obtained from the Guangdong Microbial Culture Collection Center (GDMCC).

[0045] Culture conditions:

[0046] Solid culture, under microaerobic conditions (5% O2, 10% CO2, 85% N2) on Columbia agar supplemented with 7% defibrinated sheep blood at 37°C for 72 hours;

[0047] Liquid culture, using brain heart infusion (BHI) broth containing 10% fetal bovine serum.

[0048] Example 1

[0049] Screening of anti-Helicobacter pylori strains

[0050] 28 strains of lactic acid bacteria (LAB) were screened and isolated from infant feces. The 28 strains were identified by 16S rRNA analysis, including 5 strains of Paracaseicovis casei, 5 strains of Lactobacillus plantarum, 5 strains of Pediococcus acidilactici, 5 strains of Bifidobacterium longum, 1 strain of Bifidobacterium breve and 7 strains of Bifidobacterium animalis, which were named as FL1 / 3 / 5 / 7 / 9 (Paracaseicovis casei), Z2 / 4 / 6 / 8 / 10 (Lactobacillus plantarum), ZPA1 / 2 / 3 / 4 / 5 (Pediococcus acidilactici), BL3 / 4 / 5 / 11 / 22 (Bifidobacterium longum), LE4 (Bifidobacterium breve) and BD3 / 4 / 7 / 8 / 9 / 10 / 12 (Bifidobacterium animalis), respectively.

[0051] In order to evaluate the anti-Helicobacter pylori potential of the 28 strains of lactic acid bacteria, the self-aggregation ability, co-aggregation ability, hydrophobicity, bacteriostatic effect on Helicobacter pylori and urease inhibition activity of different strains were evaluated by in vitro experiments, and strains with anti-Helicobacter pylori potential were screened, and the specific process was as follows:

[0052] (1) Self-aggregation ability determination

[0053] The 28 strains were inoculated in MRS liquid medium (MRS liquid medium: 10 g / L of proteose peptone, 5 g / L of beef extract powder, 20 g / L of glucose, 4 g / L of yeast extract powder, 2 g / L of potassium phosphate dibasic, 2 g / L of triammonium citrate, 5 g / L of sodium acetate, 0.2 g / L of magnesium sulfate, 0.05 g / L of manganese sulfate and 1 g / L of Tween 80), and cultured at 37°C for 24 hours. The absorbance (OD 600 ) of the strains at 0h, 2h, 6h and 24h of the culture was determined, and the corresponding self-aggregation percentage at different culture times was calculated according to the absorbance. Each experiment was repeated for 3 times, and the experimental results were shown in Table 1.

[0054] The determination method of absorbance (OD 600 ): after the strains were cultured for different time periods, centrifugation (4200 rpm, 10 minutes, 25°C) was performed, and the supernatant was discarded. The precipitate was washed twice with phosphate buffer (PBS, pH 7.4±0.02). The cells were resuspended in PBS, and the absorbance at 600 nm was 0.5±0.02 (A0). Then, 4 mL of cell suspension was vortexed for 10 seconds, and the absorbance (OD 600 ) was immediately measured.

[0055] The self-aggregation percentage formula was as follows:

[0056] Self-aggregation ability (%) = (1-A t / A0) × 100%.

[0057] Where A0is the initial absorbance of the strain, Atis the absorbance at 2, 6 or 24 hours.

[0058] Table 10 - Auto-aggregation rate of lactic acid bacteria at 24h

[0059]

[0060] Note: a, b, c, d, e, f, g, h, i, j, k letters indicate significant differences between strains

[0061] Results analysis: As shown in Table 1, the auto-aggregation ability of the strains increased gradually with the incubation time from 2h to 24h. After 24h incubation, the auto-aggregation rate varied from 49.47% to 88.07%. Except for strain FL9, all the tested strains showed auto-aggregation ability more than 50%. The auto-aggregation ability is positively correlated with the adhesion ability in the gastrointestinal tract, which can promote the formation of biofilm and thus reduce the adhesion of pathogenic bacteria to the gastric mucosa.

[0062] (2) Co-aggregation ability determination

[0063] Lactic acid bacteria of ≥3 generations were centrifuged (4200 rpm, 10 min, 25°C), washed with PBS three times, and resuspended in PBS. Similarly, Helicobacter pylori SS1 (≥3 generations) was harvested, washed, and resuspended in artificial gastric juice (0.5% NaCl, 0.3% pepsin, pH 4.0±0.02). The absorbance (OD 600 ) of the lactic acid bacteria and Helicobacter pylori suspensions at 600 nm was adjusted to 0.5±0.02 using a UV spectrophotometer. Equal volumes (2 mL each) of lactic acid bacteria and Helicobacter pylori suspensions were mixed, vortexed for 10 seconds, and incubated statically at 37°C for 24 hours. The absorbance (OD 600 ) of the supernatant was measured at 2, 6, and 24 hours during incubation.

[0064] Co-aggregation rate was calculated as follows:

[0065] Co-aggregation ability (%) = (1-2xA min / (A x +A y )) x 100%.

[0066] Where A x is the initial absorbance of LAB, A y is the initial absorbance of Helicobacter pylori, and A min is the absorbance of the mixture at 2, 6, or 24 hours.

[0067] Table 20 - Co-aggregation rate of lactic acid bacteria at 24h

[0068] Note: abcdefg letters indicate significant differences between strains

[0069] Result analysis: The mechanism of probiotic bacteria to reduce Helicobacter pylori colonization includes specific binding with Helicobacter pylori, strong co-aggregation ability, indicating that the interaction between probiotic bacteria and Helicobacter pylori is strong, which can competitively bind to the specific proteins on the surface of Helicobacter pylori, thereby reducing its binding to gastric mucosa proteins and forming aggregates, excreted from the gastrointestinal tract, reducing the ability of Helicobacter pylori to colonize the gastrointestinal tract.

[0070] As can be seen from Table 2, the co-aggregation ability showed a time-dependent increase within the 24-hour observation period. The initial (2-hour) co-aggregation rate varied between 1.20% and 43.2%, with strain ZPA1 showing the highest co-aggregation ability (43.2%), followed by LE4 (25.6%) and FL5 (20.8%). After 24 hours, the co-aggregation efficiency significantly increased to 68.07%-97.93%, with FL5 reaching the maximum value (97.93%), indicating that Paracasei FL5 has strong specific binding ability with Helicobacter pylori, reducing its colonization in the gastrointestinal tract.

[0071] (3) Hydrophobicity determination

[0072] The surface hydrophobicity of lactic acid bacteria was determined by microbial adhesion to hydrocarbons (MATH). The strains were incubated at 37°C for 24 hours, then centrifuged at 4200 rpm (10 minutes, 25°C). The supernatant was discarded, the cells were washed twice with sterile PBS, and resuspended in PBS to an absorbance of 0.5±0.02 at 600 nm (A0). Three milliliters of bacterial suspension were mixed with one milliliter of xylene (chromatographic grade) and equilibrated for 10 minutes; the mixture was vortexed for 2 minutes and allowed to stand for 20 minutes to allow the phases to separate; the water phase was carefully aspirated and the absorbance (OD 600 ) was measured.

[0073] The cell surface hydrophobicity (%) was calculated as follows:

[0074] Hydrophobicity (%) = (1-A1 / A0) x 100%.

[0075] Where A0 is the initial absorbance of the bacterial suspension and A1 is the absorbance of the water phase after xylene treatment.

[0076] Table 3 Hydrophobicity of strains

[0077] Note: abcdefghi letters indicate significant differences between strains

[0078] Result analysis: LAB cell surface has lipoteichoic acid, peptidoglycan and surface protein, which not only protects the cell itself, but also recognizes and adheres to the body cells, so high hydrophobicity is selected as one of the screening indicators.

[0079] The cell surface hydrophobicity of 28 strains of lactic acid bacteria was between 4.0%-97.2%, and the hydrophobicity of BD4 was the strongest, reaching 97.2%. Bifidobacterium longum BL3, BL4, BL11, animal bifidobacterium BD8, BD9 and paracasei FL5 had relatively strong hydrophobicity, all above 50%; among them, the ability of lactobacillus plantarum, pediococcus acidilactici and bifidobacterium breve was less than 20%.

[0080] (4) Helicobacter pylori inhibition test

[0081] Adjust 28 strains of lactic acid bacteria (LAB) and helicobacter pylori suspension (suspension preparation process of lactic acid bacteria (LAB) and helicobacter pylori in step (2) simultaneously) to 1×10 8 CFU / mL, 100 μL aliquot of helicobacter pylori suspension was evenly coated on Columbia blood agar plate, and sterile Oxford cup (outer diameter 8 mm) was placed sterilely on the agar surface, and pressed gently to ensure contact. Then, 100 μL of LAB suspension was added to each Oxford cup. The plate was incubated at 37℃ for 72 hours under microaerobic conditions (5% O2, 10% CO2, 85% N2), and after incubation, the diameter of the inhibition zone around the Oxford cup was measured with a caliper. MRS medium was used as negative control, and TA triple antibiotic (omeprazole, amoxicillin, clarithromycin) was used as positive control, and the experimental results are shown in Table 4.

[0082] Table 4: Inhibition of helicobacter pylori by strains and determination of urease activity inhibition rate

[0083] Note: abcdefg letters indicate significant differences between strains

[0084] Result analysis: Oxford cup method was used for antibacterial test to determine the size of the inhibition zone, and lactic acid bacteria capable of antagonizing helicobacter pylori were screened, and the inhibition zones of TA and MRS against helicobacter pylori were 25.78 mm and 12.05 mm respectively; among the 28 strains, the diameter of the inhibition zone ranged from 11.83 to 22.00 mm. The inhibition zone of strain FL5 was the largest (22.00 mm), while FL9, LE4, BD7, ZPA4 and FL7 also showed significant antibacterial activity, with inhibition zone diameter exceeding 18 mm.

[0085] (5) Determination of helicobacter pylori urease activity inhibition rate

[0086] Urease activity of H. pylori SS1 was evaluated by phenol red method. Fresh cultures of H. pylori and LAB were washed twice with PBS and adjusted to 1 x 10 8 CFU / mL in BHI medium. Aliquots of 40 μL of H. pylori suspension and 10 μL of LAB suspension were co-cultured in microaerophilic conditions (5% O2, 10% CO2, 85% N2) for 48 h at 37°C in 96-well plates. Control wells contained only H. pylori suspension, MRS medium as negative control, and positive control wells were TA triple antibiotic (Omeprazole, Amoxicillin, Clarithromycin). After incubation, 50 μL of culture supernatant was mixed with 150 μL of urease reaction buffer (0.9% NaCl, 20 mM urea, 14 μg / mL phenol red, pH 6.8 adjusted with HC1), after vortex mixing, the absorbance was measured at 550 nm using a microplate reader, and the results are shown in Table 4.

[0087] Urease inhibition activity = (1 - A1 / A0) x 100%.

[0088] A0 is the absorbance of H. pylori at 550 nm, which was determined to be 1.14, and A1 is the absorbance of co-culture at 550 nm.

[0089] Result analysis: H. pylori mainly relies on its own secreted urease to colonize in the stomach, urease decomposes urea in the stomach and produces ammonia, which increases the pH value of the stomach, neutralizes stomach acid and creates an environment conducive to the growth of H. pylori. Paracasei and Lactobacillus plantarum have strong urease inhibition ability, both greater than 40%, among which Paracasei FL5 has the strongest urease inhibition ability, which is 77.58%. The TA positive control, the urease inhibition rate is 67.38%.

[0090] Based on the above determination results of self-aggregation ability, co-aggregation ability, hydrophobicity, antibacterial effect on H. pylori and urease activity inhibition activity of different strains, principal component analysis was performed, and SPSS analysis was used to obtain the comprehensive score of 28 strains of lactic acid bacteria, and the experimental results are shown in Table 5.

[0091] Table 5 Comprehensive scores and rankings of different lactic acid bacteria

[0092] Result analysis: Lacticaseibacillus paracasei FL5 ranked the highest, and was the most promising anti-Helicobacter pylori strain. The strain was preserved, and the strain name was Lacticaseibacillus paracasei FL5, the taxonomic name was Lacticaseibacillus paracasei, and it belonged to Lacticaseibacillus paracasei. It was preserved in the Guangdong Microbial Culture Collection Center (GDMCC), and the preservation date was 2025.03.06, the preservation number was GDMCC NO: 65986, and the preservation address was No. 59, Building 5, 100, Martyrs' Road, Guangzhou.

[0093] Example 2

[0094] Preparation of Lacticaseibacillus paracasei FL5 fermentation broth, supernatant, live bacteria and inactivated bacteria

[0095] Fermentation broth: Lacticaseibacillus paracasei FL5 strain was cultured in MRS liquid medium at 37°C for 24 h to obtain the strain fermentation broth.

[0096] Supernatant: Then centrifuged at 4200 rpm for 15 min at 4°C to obtain the supernatant.

[0097] Live bacteria: Discard the supernatant, wash the bacteria obtained after centrifugation twice with sterile phosphate buffer solution (PBS, pH 7.2), resuspend in PBS, and adjust to 1×10 8 CFU / mL to obtain FL5 live bacteria.

[0098] Inactivated bacteria: Adjust the 1×10 8 CFU / mL live bacteria to 90°C for 20 min to obtain the inactivated strain.

[0099] Example 3

[0100] In vitro antibacterial activity of Lacticaseibacillus paracasei FL5 and its postbiotic against Helicobacter pylori

[0101] (1) Inhibition rate of Helicobacter pylori urease activity

[0102] The urease activity of Helicobacter pylori SS1 was evaluated by the phenol red method. Fresh culture of Helicobacter pylori was washed twice with PBS and adjusted to 1×10 8CFU / mL. 40 pL of H. pylori suspension and 10 pL of FL5 fermentation broth, supernatant, live bacteria and inactivated bacteria were co-cultured in a 96-well plate under microaerobic conditions (5% O2, 10% CO2, 85% N2) at 37°C for 48 hours. The control wells only contained H. pylori suspension (indicated as HP), MRS liquid medium as negative control, and TA triple antibiotic (omeprazole, amoxicillin, clarithromycin) as positive control. After incubation, 50 pL of culture supernatant was mixed with 150 pL of urease reaction buffer (0.9% NaCl, 20 mM urea, 14 pg / mL phenol red, pH 6.8 adjusted with HC1). After vortex mixing, the absorbance was measured at 550 nm using a microplate reader, and the inhibition rate of urease activity was calculated. The experimental results are shown in Figure 1 .

[0103] Result analysis: Urease can decompose urea to increase the pH of the solution, causing the indicator solution to change color. Its activity can be reflected by measuring the value of the solution at OD=550 nm.

[0104] As can be seen from Figure 1 , after fermentation broth, supernatant, FL5 live bacteria, and inactivated bacteria treatment, the OD values of H. pylori were 0.1297±0.078, 0.1509±0.023, 0.2608±0.088, and 0.4103±0.006, respectively, which were significantly lower than the control group value of 0.8688±0.035 (P<0.01). The inhibition rates were 85.08%, 82.64%, 69.99%, and 52.78%, respectively, indicating that Lactobacillus paracasei FL5 fermentation broth, supernatant, FL5 live bacteria, and inactivated bacteria inhibited the growth of H. pylori by inhibiting urease activity.

[0105] (2) Lactobacillus paracasei FL5 Inhibition Ability on H. pylori Determination

[0106] Adjust FL5 fermentation broth, supernatant, FL5 live bacteria, and inactivated bacteria and H. pylori suspension to 1 x 10 8 CFU / mL. 40 pL of H. pylori suspension and 10 pL of FL5 fermentation broth, supernatant, live bacteria and inactivated bacteria were co-cultured in a 96-well plate under microaerobic conditions (5% O2, 10% CO2, 85% N2) at 37°C for 48 hours. The control wells only contained H. pylori suspension (indicated as HP), MRS liquid medium as negative control, and TA triple antibiotic (omeprazole, amoxicillin, clarithromycin) as positive control. After incubation, 50 pL of culture supernatant was mixed with 150 pL of urease reaction buffer (0.9% NaCl, 20 mM urea, 14 pg / mL phenol red, pH 6.8 adjusted with HC1). After vortex mixing, the absorbance was measured at 550 nm using a microplate reader, and the inhibition rate of urease activity was calculated. The experimental results are shown in

[0107] Results analysis: As shown in Figure 2 Table 1, FL5 fermentation broth showed the strongest inhibitory activity (21.75 mm inhibition zone) compared with the negative (MRS) control, followed by supernatant (19.55 mm) and live bacteria (17.90 mm), and inactivated bacteria also had a certain inhibitory effect.

[0108] (3) Scanning electron microscopy (SEM) of Lacticaseibacillus paracasei FL5 and Helicobacter pylori

[0109] A co-culture system was established between Helicobacter pylori SS1 and Lacticaseibacillus paracasei FL5. Helicobacter pylori SS1 was resuspended in BHI broth, then Lacticaseibacillus paracasei FL5 fermentation broth, supernatant or live / dead cells were added respectively. Then the mixture was incubated at 37°C for 24 hours under microaerobic conditions (85% N2, 10% CO2, 5% O2). The co-cultured cells were collected, centrifuged at 2000 for 5 minutes, fixed in 2.5% (v / v) glutaraldehyde at 4°C for 12 hours, and examined for morphological analysis under scanning electron microscopy (JEOL JSM-IT 2000x resolution), TA triple antibiotic (omeprazole, amoxicillin, clarithromycin) as a positive control, HP as a model group containing only Helicobacter pylori. The experimental results are shown in Figure 3

[0110] Results analysis: Lacticaseibacillus paracasei FL5 supernatant and fermentation broth induced significant structural changes in Helicobacter pylori, including spiral to spherical transformation and cell collapse. Live bacteria showed direct adhesion to Helicobacter pylori, while inactivated bacteria caused moderate cell deformation.

[0111] Example 4

[0112] In vivo antibacterial activity of Lacticaseibacillus paracasei FL5 and its postbiotic against Helicobacter pylori

[0113] (1) Modeling

[0114] ​Animal experiments were approved by the Animal Ethics Committee of Jilin Agricultural University. SPF C57BL / 6J mice (male, 6 weeks old) were purchased from the Experimental Animal Center of Jilin Agricultural University. After 7 days of adaptive feeding, the mice were divided into 5 groups of 6 each: negative control group (NC), model group (HP), live bacteria FL5 group, inactivated bacteria IFL5 group and TA triple antibiotic (omeprazole, amoxicillin, clarithromycin) as positive control. Except for the NC group, the other groups were perfused with normal saline every other day, and the rest of the groups were perfused with Helicobacter pylori solution (300 μL, 1×10 8 CFU / mL) every other day for 3 weeks. To evaluate the colonization of Helicobacter pylori, 3 mice from the group of mice gavaged with Helicobacter pylori were randomly selected, and gastric tissue samples were collected after overnight fasting for routine rapid urease test. The obvious red reaction of urease reagent confirmed the success of the model.

[0115] After the establishment of the Helicobacter pylori infection model, the NC and HP groups were given 300 μL of normal saline daily, the FL5 group was given Lacticaseibacillus paracasei FL5 (300 μL, 1×10 10 CFU / mL) daily, the IFL5 group was given inactivated Lacticaseibacillus paracasei FL5 (300 μL, 1×10 10 CFU / mL) daily, and the TA group was given triple antibiotic (omeprazole 0.02 mg / d, amoxicillin 1 mg / d, clarithromycin 0.5 mg / d, dissolved in 300 μL of normal saline) daily, once a day, gavage for 4 weeks. After the experiment, the mice were fasted for 12 hours and then euthanized, and their blood and tissue samples were collected.

[0116] (2) Changes in body weight of mice

[0117] During the experiment, body weight was measured every four days, and the amount of weight change was recorded, and the experimental results are shown in Figure 4 .

[0118] Results analysis: Body weight analysis showed that the body weight of the model group was significantly lower than that of the NC group, and the body weight of the mice receiving FL5, IFL5 and TA treatment was significantly restored compared with the model group.

[0119] (3) Significant reduction in Helicobacter pylori urease activity in gastric tissue of mice

[0120] Urease activity in the gastric tissue of all experimental mice was measured, and the experimental results are shown in Figure 5 .

[0121] Results analysis: FL5, IFL5 and TA groups significantly reduced urease activity.

[0122] (4) Relative expression of H. pylori virulence genes CagA and VacA in mouse stomach tissue

[0123] The relative expression of H. pylori virulence genes CagA and VacA in the stomach tissue of all experimental mice was determined, and the experimental results are shown in Figure 6

[0124] Results analysis: Compared with the NC group, the relative expression of CagA and VacA in the model group was significantly increased. Compared with the model group, the relative expression of CagA and VacA in the FL5, IFL5 and TA treatment groups was significantly reduced. The significant reduction in urease activity and pathogenic factor expression indicates that the colonization of H. pylori in the mouse stomach tissue was significantly inhibited.

[0125] (5) Histopathological analysis of mice

[0126] After the mice were sacrificed, the stomach tissue was collected and fixed in 4% paraformaldehyde for 24 h, and then the tissue was dehydrated and embedded in paraffin. Sections with a thickness of 4 μm were cut and stained with hematoxylin and eosin (H&E), and HE-stained section images were obtained using a microscope at a magnification of 200x, and the experimental results are shown in Figure 7

[0127] Results analysis: In the normal control group, the stomach tissue structure was complete, the epithelial layer was arranged in order, the inflammatory cell infiltration in the mucosa layer was slight, the mucosal glands were arranged normally and densely, and no obvious inflammatory reaction was observed. In contrast, the model group mice showed significant inflammatory cell infiltration in the submucosal layer, especially neutrophils (black arrows), accompanied by mild gland atrophy, reduced gland density, and obvious hyperemia of the gastric fundus glands (red arrows). FL5, IFL5 and TA treatment all effectively alleviated these pathological changes.

[0128] (6) Changes in cytokine levels in mice

[0129] The mouse stomach tissue was homogenized with an appropriate amount of normal saline, and then centrifuged at 3000 rpm for 10 minutes. The cytokines (TNF-α, IL-1β, IL-10, IL-6, IL-8 and IL-4) and IgG content in the stomach tissue were quantitatively detected by enzyme-linked immunosorbent assay (ELISA), and the experimental results are shown in Figure 8

[0130] ​​​Results analysis: In the model group mice, the levels of TNF-a, IL-1b, IL-6, IgG and IL-8 were significantly increased. However, after treatment with FL5, IFL5 and TA, the expression of these pro-inflammatory factors was significantly reduced. In contrast, H. pylori infection significantly inhibited the production of anti-inflammatory cytokines IL-10 and IL-4, while treatment with FL5, IFL5 effectively reversed this inhibition. These findings indicate that administration of FL5, IFL5 and TA significantly modulated the inflammatory response after H. pylori infection, and IFL5 was more effective than FL5.

[0131] (7) Changes in antioxidant indicators in mouse liver

[0132] Liver tissue was homogenized with physiological saline at a solid-liquid ratio of 1:9 (g:mL) in an ice water bath, and then the supernatant was collected. The activities of glutathione (GSH, A006-1-1, Nanjing Jiancheng Biological Engineering Institute), malondialdehyde (MDA, A003-1), catalase (CAT, A007-1-1) and superoxide dismutase (SOD, A001-3) were determined according to the kit instructions, and the experimental results are shown in Figure 9 .

[0133] Results analysis: As shown in Figure 9 , compared with the NC group, the model group showed significant down-regulation of GSH, CAT and SOD levels, while the MDA level was up-regulated. FL5, IFL5 and TA treatment groups showed varying degrees of recovery in these oxidative stress markers, with FL5 showing the most significant recovery effect. These findings indicate that Paracasei FL5 has strong antioxidant properties and can reduce oxidative damage caused by H. pylori.

[0134] (8) Sequencing analysis of mouse gastric flora

[0135] Genomic DNA was extracted using a commercial DNA extraction kit (Omega Bio-tek, USA). The DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). The V3-V4 hypervariable region of the bacterial 16S rRNA gene was amplified using universal primers 338F and 806R. High-throughput sequencing was performed on the Illumina NextSeq2000 platform. Operational taxonomic units (OTUs) were classified using the RDP classifier against the SILVA 16S rRNA database (v138.2) with a confidence threshold of 70%. Bioinformatics analysis was performed on the MajorBio cloud platform (https: / / cloud.majorbio.com), and the experimental results are shown in Figure 10 , Figure 11 andFigure 12 Results analysis: As shown in

[0136] Results analysis: As shown in Figure 10 PCA analysis showed that there were differences in the composition of gastric microbiota between groups, but the FL5 and IFL5 groups were close to the control group.

[0137] As shown in Figure 11 The difference in bacterial genera between groups was significantly different, and the difference in bacterial genera in the model group was mainly o - Campylobacterales and g - Helicobacter, while the difference in bacterial genera in the FL5 group was o - Lactobacillales, g - Lachnospiraceae_NK4A136_group and g - Limosilactobacillus, which belongs to beneficial bacteria, and the difference in bacterial genera in the IFL5 group was o - Lachnospirales.

[0138] As shown in Figure 12 Compared with the NC group, the abundance of Campylobacterota in the model group was significantly increased, and the abundance of other bacterial phyla was significantly reduced. The abundance of Bacillota, Bacteroidota, Pseudomonadota and Patescibacteria in the Paracasei FL5 group was significantly increased, similar to the NC group. The abundance of Bacteroidota and Patescibacteria in the IFL5 group was significantly increased. The gastric microbiota diversity of the TA group was destroyed, and the microbial structure was relatively simple. The FL5, IFL5 and TA groups significantly down-regulated the abundance of the phylum where Helicobacter pylori was located.

[0139] (9) Lacticaseibacillus paracasei FL5 restores the integrity of the gastric mucosal barrier and inhibits the TLR-2 / NF-κB signaling pathway.

[0140] Further study of the regulatory effect of Helicobacter pylori infection and probiotic intervention on gastric mucosal barrier proteins and TLR2 / NF-κB inflammatory pathways, and the experimental results are shown in Figure 13 and Figure 14

[0141] Results analysis: As shown in Figure 13 ​As shown, compared with the NC group, the HP group exhibited a decreasing trend in the expression of Claudin-1 and Occludin proteins. Both FL5 and IFL5 interventions effectively restored the expression of these tight junction proteins, with efficacy comparable to the antibiotic treatment group. FL5 significantly upregulated the expression of Claudin-1 and Occludin, while IFL5 significantly increased the expression level of Claudin-1. FL5 showed relatively stronger efficacy in promoting barrier restoration.

[0142] like Figure 14 As shown, Helicobacter pylori infection significantly activated the TLR2 / MyD88 / NF-κB inflammatory signaling pathway. Compared with the NC group, the expression levels of TLR2 and its downstream adaptive protein MyD88 were significantly increased in the HP group, while the expression of key inflammatory regulators Iκ-Kα, p-IκB-α, and NF-κB were also significantly increased. IFL5 was more effective than FL5 in inhibiting inflammatory signal transduction.

[0143] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0144] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Paracaseicetobacter casei, characterized in that, The paracasei is Lacticaseibacillus paracasei FL5, deposited with the Guangdong Microbial Culture Collection Center on March 6, 2025, with the accession number GDMCC NO: 65986, and the address of the deposit is No. 59, Building 5, 100, Martyrs' Road, Guangzhou.

2. The probiotic of L. paracasei of claim 1, characterized by the fact that it is a postbiotic, The probiotic is Lacticaseibacillus paracasei FL5 fermentation broth, Lacticaseibacillus paracasei FL5 fermentation supernatant, Lacticaseibacillus paracasei One or more of the inactivated strains of FL5.

3. The postbiotic of Paracaseicolactis according to claim 2, characterized in that, The Lacticaseibacillus paracasei FL5 fermentation broth is prepared by the following method: FL5 is cultured in MRS liquid medium to prepare Lacticaseibacillus paracasei FL5 fermentation broth. Lacticaseibacillus paracasei Lacticaseibacillus paracasei 4. The postbiotic of Paracaseicolactis according to claim 2, characterized in that, The Lacticaseibacillus paracasei FL5 fermentation supernatant is prepared by centrifuging, separating the FL5 fermentation broth prepared in claim 3, and collecting the supernatant. Lacticaseibacillus paracasei FL5 fermentation supernatant is prepared by centrifuging, separating the FL5 fermentation broth prepared in claim 3, and collecting the supernatant. Lacticaseibacillus paracasei FL5 fermentation supernatant is prepared by centrifuging, separating the FL5 fermentation broth prepared in claim 3, and collecting the supernatant.

5. The postbiotic of Paracaseicolactis according to claim 2, characterized in that, The Lacticaseibacillus paracasei The FL5 inactivated strain is prepared by the following method: the FL5 strain prepared in claim 3 is centrifuged and separated, the supernatant is discarded, the bacterial body obtained after centrifugation is washed, inactivated, and the FL5 inactivated strain is prepared. The application is any of the following: The FL5 inactivated strain is prepared by the following method: the FL5 inactivated strain prepared in claim 3 is centrifuged and separated, the supernatant is discarded, the bacterial body obtained after centrifugation is washed, inactivated, and the FL5 inactivated strain is prepared. (1) application in the preparation of products for inhibiting the urease activity of H. pylori; The FL5 inactivated strain is prepared by the following method: the FL5 inactivated strain prepared in claim 3 is centrifuged and separated, 6. Use of the Paracasei subsp. Parietina of claim 1 or of the probiotic of any one of claims 2 to 5, characterized in that, (2) application in the preparation of products for inhibiting the proliferation of H. pylori; (3) application in the preparation of products for inhibiting the colonization of H. pylori; (4) application in the preparation of products for preventing and / or treating inflammation caused by H. pylori; (5) application in the preparation of products for preventing and / or treating oxidative damage caused by H. pylori; (6) application in the preparation of products for preventing and / or treating dysbiosis caused by H. pylori; The product is a drug. The product includes the L. paracasei of claim 1 and / or the probiotic of any one of claims 2-5; the product is a drug. It also includes other active ingredients or adjuvants.

7. A product for use against H. pylori, characterized in that, ​ 8. The product against H. pylori according to claim 7, characterized in that, ​

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