Lactobacillus paracasei, metagen and application of lactobacillus paracasei and metagen in preparation of helicobacter pylori resistant product

Abstract

The invention discloses a lactobacillus paracasei, a metagen and application of the lactobacillus paracasei and the metagen in preparation of an anti-helicobacter pylori product. Belongs to the technical field of probiotics. According to the application of the lactobacillus paracasei FL5, 28 strains are separated from excrement of infants, the 28 strains comprise lactobacillus paracasei, lactobacillus plantarum and the like through analysis and identification, and the strains are evaluated through an in-vitro experiment, so that the lactobacillus paracasei FL5 with potential helicobacter pylori resistance is finally screened out, and the lactobacillus paracasei FL5 has the potential helicobacter pylori resistance. According to the method, the helicobacter pylori resisting effect of the Lacticasei paracasei FL5 in vivo is verified from multiple aspects such as inflammation resistance, oxidation resistance, colonization inhibition capacity and flora through an in-vitro co-culture experiment, antibacterial metabolite quantification, a scanning electron microscope and an in-vivo model experiment, and a new treatment strategy is provided for resisting helicobacter pylori infection.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and more particularly to Lactobacillus paracasei, postbiotics and applications thereof in preparing an anti-Helicobacter pylori product. Background Art

[0002] Helicobacter pylori (H. pylori) is a microaerophilic, spiral-shaped bacterium first isolated from the human gastric mucosa in 1982 by Barry J. Marshall and Robin Warren. This pathogen colonizes over half of the world's population. Its etiology has been linked to chronic gastritis, peptic ulcers, gastric mucosal atrophy, intestinal metaplasia, and gastric cancer. In 1994, the International Agency for Research on Cancer (IARC / WHO) designated H. pylori a Group 1 carcinogen. Successful colonization of the gastric wall by H. pylori depends on multiple virulence factors, including flagella, urease, and adhesins, which together mediate its pathogenesis. 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 genes exhibit impaired gastric colonization in mouse models.

[0003] The current recommended first-line standard treatment for Helicobacter pylori is bismuth-based quadruple therapy (14-day course), consisting of a proton pump inhibitor, bismuth, and two antibiotics. However, widespread antibiotic overuse and increasing antibiotic resistance have severely compromised treatment efficacy, reduced success rates, and exacerbated clinical management challenges. Therefore, there is an urgent need to develop alternative treatments that are both effective and minimally disruptive to host physiology.

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

[0005] Therefore, how to provide a new strain that is highly effective in antagonizing Helicobacter pylori is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a Lactobacillus paracasei, a postbiotic and their use in the preparation of an anti-Helicobacter pylori product, and verifies the in vivo anti-Helicobacter pylori efficacy of Lacticaseibacillus paracasei FL5 from multiple aspects such as anti-inflammatory, antioxidant, and inhibition of colonization and flora, providing a new treatment strategy for Helicobacter pylori infection.

[0007] The first purpose of this application is to provide:

[0008] The invention discloses a Lactobacillus paracasei, which is Lacticaseibacillus paracasei FL5, deposited in Guangdong Provincial Microbiological Culture Collection Center, with a deposit date of 2025.03.06, a deposit number of GDMCC NO: 65986, and a deposit address of 5th floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] Another object of the present application is to provide: a postbiotic of the above-mentioned Lactobacillus paracasei, wherein the postbiotic comprises one or more of Lacticaseibacillus paracasei FL5 fermentation broth, Lacticaseibacillus paracasei FL5 fermentation supernatant, and Lacticaseibacillus paracasei FL5 inactivated strain.

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

[0011] In one embodiment, the Lacticaseibacillus paracasei FL5 fermentation supernatant is prepared by the following method: centrifuging and separating the prepared Lacticaseibacillus paracasei FL5 fermentation broth, collecting the supernatant, and preparing the Lacticaseibacillus paracasei FL5 fermentation supernatant.

[0012] In one embodiment, the inactivated Lacticaseibacillus paracasei FL5 strain is prepared by the following method: centrifuging and separating the prepared Lacticaseibacillus paracasei FL5 fermentation broth, discarding the supernatant, washing and inactivating the bacterial cells obtained after centrifugation, and preparing the inactivated Lacticaseibacillus paracasei FL5 strain.

[0013] Another object of the present application is to provide: an application of the above-mentioned Lactobacillus paracasei or a postbiotic of the above-mentioned Lactobacillus paracasei, wherein the application is any one of the following:

[0014] (1) Application in the preparation of products for inhibiting Helicobacter pylori urease activity;

[0015] (2) Application in the preparation of products for inhibiting the proliferation of Helicobacter pylori;

[0016] (3) Application in the preparation of products for inhibiting Helicobacter pylori colonization;

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

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

[0019] (6) Use in the preparation of products for preventing and / or treating dysbacteriosis caused by Helicobacter pylori.

[0020] Another object of the present application is to provide: an anti-Helicobacter pylori product comprising the Lactobacillus paracasei and / or the postbiotics of the Lactobacillus paracasei.

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

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

[0023] It can be seen from the above technical solutions that compared with the prior art, the present invention has the following beneficial effects:

[0024] 1) In this application, 28 bacterial strains were isolated from infant feces, which were analyzed and identified as including Lactobacillus paracasei, Lactobacillus plantarum, Pediococcus acidilactici, Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium animalis. In vitro experiments were conducted to evaluate the strains' self-aggregation ability, coaggregation ability with Helicobacter pylori, cell surface hydrophobicity, urease inhibition, and direct antibacterial activity. Finally, a strain, Lacticaseibacillus paracasei FL5, was screened out as having potential anti-Helicobacter pylori activity. Subsequently, in vitro co-culture experiments, antimicrobial metabolite quantification and scanning electron microscopy, as well as in vivo model experiments, verified the in vivo anti-Helicobacter pylori efficacy of Lacticaseibacillus paracasei FL5 from multiple aspects, including anti-inflammatory, antioxidant, colonization inhibition, and microbial flora, providing a new therapeutic strategy for Helicobacter pylori infection.

[0025] 2) The live bacteria, fermentation broth, supernatant and inactivated strain of Lactobacillus paracasei of the present application all have preventive and therapeutic effects on Helicobacter pylori, and the preventive and therapeutic effects are better than those of the disclosed strains in the prior art; and it should also be emphasized that the inactivated strain of Lacticaseibacillus paracasei FL5 is more effective than the live bacteria in regulating the inflammatory response after Helicobacter pylori infection.

[0026] 3) In the process of constructing the mouse model, this application used oral gavage once every other day for a total of 3 weeks to construct the Helicobacter pylori mouse model. Compared with the existing technology of once every other day for a total of 3 times, the modeling time is longer, the model infection and severity are more serious, and the research level is deeper than other patents, and the indicators are more credible. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

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

[0029] Figure 2 Figure 2: The antibacterial ability test of each component of Lacticaseibacillus paracasei FL5 against Helicobacter pylori.

[0030] Figure 3 For: Scanning electron microscope (SEM) of Lacticaseibacillus paracasei FL5 and Helicobacter pylori.

[0031] Figure 4 Figure 2: Body weight of mice in different groups.

[0032] Figure 5 Figure 2: Urease activity in gastric tissue of mice in different groups.

[0033] Figure 6 Figure 2: Relative expression of Helicobacter pylori virulence genes CagA and VacA in gastric tissues of mice in different groups.

[0034] Figure 7 Figure 2: Representative images of gastric tissue sections in different groups.

[0035] Figure 8 Figure 2: Inflammatory factors in gastric tissues of different groups.

[0036] Figure 9 Figure 4: Liver oxidative stress in different groups.

[0037] Figure 10 Figure 2: PCA diagram of gastric flora in different groups.

[0038] Figure 11Figure 2: LEfSe diagram of gastric flora in different groups.

[0039] Figure 12 It is a bar chart showing the composition of different groups of communities.

[0040] Figure 13 Figure 2: Relative expression of claudin 1 and occludin in different groups.

[0041] Figure 14 Figure 2: Relative expression of TLR2 / MyD88 / NF-κB signaling pathway in different groups. DETAILED DESCRIPTION

[0042] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0043] The strains used in the examples of this application are as follows:

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

[0045] Culture conditions:

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

[0047] For liquid culture, use brain heart infusion (BHI) broth containing 10% fetal bovine serum.

[0048] Example 1

[0049] Screening of strains resistant to Helicobacter pylori

[0050] Lactic acid bacteria were screened and isolated from infant feces, and 28 lactic acid bacteria (LAB) strains were isolated and identified by 16S rRNA analysis, including 5 strains of Lactobacillus paracasei, 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 (Lactobacillus paracasei), 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).

[0051] To evaluate the anti-Helicobacter pylori potential of the 28 lactic acid bacteria isolated above, in vitro experiments were conducted to evaluate the self-aggregation ability, co-aggregation ability, hydrophobicity, antibacterial effect on Helicobacter pylori, and urease inhibitory activity of different strains. Strains with anti-Helicobacter pylori potential were screened. The specific process is as follows:

[0052] (1) Determination of self-aggregation ability

[0053] Twenty-eight strains were inoculated into MRS liquid medium (MRS liquid medium: peptone 10 g / L, beef extract powder 5 g / L, glucose 20 g / L, yeast extract powder 4 g / L, potassium dihydrogen phosphate 2 g / L, ammonium citrate tribasic 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L and Tween 80 1 g / L), and cultured at 37°C for 24 h. During the culture process, the absorbance (OD) of the strains at 0 h, 2 h, 6 h and 24 h were measured. 600 ), the corresponding self-aggregation percentages at different incubation times were calculated based on the absorbance, and each experiment was repeated 3 times. The experimental results are shown in Table 1.

[0054] Absorbance (OD 600 ) determination method: after culturing the strain for different time periods, centrifuge (4200 rpm, 10 minutes, 25°C), discard the supernatant, and wash the precipitate twice with phosphate buffered saline (PBS, pH 7.4±0.02); resuspend the cells in PBS to a value of 0.5±0.02 (A0) at 600 nm; then, take 4 mL of the cell suspension, vortex for 10 seconds, and immediately measure the absorbance (OD 600 ).

[0055] The formula for the percentage of self-aggregation is as follows:

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

[0057] Where A0 is the initial absorbance of the strain, and At is the absorbance at 2, 6, or 24 hours.

[0058] Table 1 Self-aggregation rate of lactic acid bacteria from 0 to 24 hours

[0059]

[0060] Note: abcdefghijk letters indicate significant differences among strains

[0061] Results: Table 1 shows that the self-aggregation ability of the strains gradually increased as the incubation time increased from 2 to 24 hours. After 24 hours of incubation, the self-aggregation rate ranged from 49.47% to 88.07%. With the exception of strain FL9, all tested strains exhibited a self-aggregation ability exceeding 50%. This self-aggregation ability is positively correlated with the adhesion ability in the gastrointestinal tract, promoting biofilm formation and thus reducing the adhesion of pathogens to the gastric mucosa.

[0062] (2) Coaggregation ability determination

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

[0064] The coaggregation rate was calculated as follows:

[0065] Coaggregation ability (%) = (1-2×A min / (A x +A y ))×100%.

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

[0067] Table 2 Coaggregation rate of lactic acid bacteria from 0 to 24 hours

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

[0069] Analysis of results: The mechanism by which probiotics reduce the colonization of Helicobacter pylori includes specific binding to Helicobacter pylori and strong co-aggregation ability, indicating that the interaction between probiotics and Helicobacter pylori is strong, and they can competitively bind to the surface specific proteins of Helicobacter pylori, thereby reducing its binding to gastric mucosal proteins and forming aggregates, which are excreted from the gastrointestinal tract and reduce the ability of Helicobacter pylori to colonize the gastrointestinal tract.

[0070] Table 2 shows that the coaggregation ability increased in a time-dependent manner over the 24-hour observation period. Initial (2-hour) coaggregation rates ranged from 1.20% to 43.2%, with strain ZPA1 exhibiting the highest coaggregation ability (43.2%), followed by LE4 (25.6%) and FL5 (20.8%). After 24 hours, the coaggregation efficiency significantly increased to 68.07%–97.93%, with FL5 reaching the highest level (97.93%). This indicates that Lactobacillus paracasei FL5 has a strong ability to specifically bind to H. pylori, reducing its gastrointestinal colonization.

[0071] (3) Hydrophobicity determination

[0072] The surface hydrophobicity of lactic acid bacteria was determined by microbial adhesion to hydrocarbons (MATH). The strains were cultured at 37°C for 24 hours and then centrifuged at 4200 rpm (10 minutes at 25°C). The supernatant was discarded, and the cells were washed twice with sterile PBS and resuspended in PBS to an absorbance of 0.5 ± 0.02 at 600 nm (A0). 3 ml of the bacterial suspension was mixed with 1 ml 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 phase separation; the aqueous phase was carefully aspirated and the absorbance (OD) was measured. 600 ).

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

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

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

[0076] Table 3 Hydrophobicity of strains

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

[0078] Result analysis: There are lipoteichoic acid, peptidoglycan and surface proteins on the surface of LAB cells, which not only protect the cells themselves but also can recognize and adhere to somatic cells. Therefore, high hydrophobicity was selected as one of the screening indicators.

[0079] The cell surface hydrophobicity of the 28 lactic acid bacteria strains ranged from 4.0% to 97.2%, with BD4 having the highest hydrophobicity at 97.2%. Bifidobacterium longum BL3, BL4, BL11, Bifidobacterium animalis BD8, BD9, and Lactobacillus paracasei FL5 were also highly hydrophobic, all exceeding 50%. Lactobacillus plantarum, Pediococcus acidilactici, and Bifidobacterium breve had hydrophobicity levels below 20%.

[0080] (4) Helicobacter pylori inhibition test

[0081] Adjust the suspension of 28 strains of lactic acid bacteria (LAB) and Helicobacter pylori (prepared in the same way as the suspension of lactic acid bacteria (LAB) and Helicobacter pylori in step (2)) to 1×10 8 To measure the CFU / mL of H. pylori suspension, a 100 μL aliquot was evenly spread onto Columbia blood agar plates. A sterile Oxford cup (outer diameter 8 mm) was aseptically placed on the agar surface, gently pressing to ensure contact. Subsequently, 100 μL of the H. pylori suspension was added to each Oxford cup. The plates were incubated at 37°C under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) for 72 hours. After incubation, the diameter of the inhibition zone around the Oxford cup was measured with a caliper. MRS medium was used as a negative control, and a triple antibiotic combination (omeprazole, amoxicillin, and clarithromycin) was used as a positive control. The results are shown in Table 4.

[0082] Table 4: Determination of the antibacterial effect of the strains on Helicobacter pylori and the inhibition rate of urease activity

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

[0084] Results: Antibacterial activity was assessed using the Oxford cup method to determine the size of the inhibition zone and screen for lactic acid bacteria with antagonistic activity against Helicobacter pylori. The inhibition zones of TA and MRS against Helicobacter pylori were 25.78 mm and 12.05 mm, respectively. Among the 28 strains tested, the inhibition zone diameters ranged from 11.83 to 22.00 mm. Strain FL5 exhibited the largest inhibition zone (22.00 mm), while FL9, LE4, BD7, ZPA4, and FL7 also exhibited significant antibacterial activity, with inhibition zone diameters exceeding 18 mm.

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

[0086] The urease activity of H. pylori SS1 was assessed by the phenol red method. Fresh cultures of H. pylori and LAB were washed twice with PBS and adjusted to 1 × 10 8 Aliquots of 40 μL of H. pylori suspension and 10 μL of LAB suspension were co-cultured in 96-well plates under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) at 37°C for 48 hours. Control wells contained only H. pylori suspension, MRS medium served as a negative control, and positive control wells contained a TA triple antibiotic combination (omeprazole, amoxicillin, and 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, adjusted to pH 6.8 with HCl). After vortexing, absorbance was measured at 550 nm using a microplate reader. The results are shown in Table 4.

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

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

[0089] Results: H. pylori colonizes the stomach primarily through the enzyme urease, which breaks down urea in the stomach and produces ammonia. Ammonia increases the pH in the stomach, neutralizing gastric acid and creating an environment favorable for H. pylori growth. Lactobacillus paracasei and Lactobacillus plantarum exhibited strong urease inhibition, both exceeding 40%. L. paracasei FL5 exhibited the strongest urease inhibition, at 77.58%. The TA positive control exhibited a urease inhibition rate of 67.38%.

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

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

[0092] Result analysis: Lactobacillus paracasei FL5 ranked the highest and was the most promising anti-Helicobacter pylori strain. It was deposited and named Lacticaseibacillus paracasei FL5. Its taxonomic name is Lacticaseibacillus paracasei, belonging to Lactobacillus paracasei. It was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) with a deposit date of March 6, 2025, and a deposit number of GDMCC NO: 65986. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle 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 fermentation broth.

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

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

[0098] Sterilization: Adjust to 1×10 8 CFU / mL of live bacteria were placed at 90°C for 20 min to obtain inactivated strains.

[0099] Example 3

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

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

[0102] The urease activity of Helicobacter pylori SS1 was assessed by the phenol red method. Fresh cultures of Helicobacter pylori were washed twice with PBS and adjusted to 1 × 10 8CFU / mL. 40 μL of Helicobacter pylori suspension and 10 μL 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 well contained only Helicobacter pylori suspension (indicated by HP), MRS liquid culture medium served as a negative control, and TA triple antibiotics (omeprazole, amoxicillin, clarithromycin) served as a positive control. 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 HCl). 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 the figure. Figure 1 shown.

[0103] Result analysis: Urease can decompose urea to increase the pH of the solution and change the color of the indicator solution. Its activity can be reflected by measuring the value of the solution at 0D=550nm.

[0104] from Figure 1 It can be seen that after treatment with fermentation broth, supernatant, FL5 live bacteria and inactivated bacteria, the OD values ​​of Helicobacter 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), and the inhibition rates were 85.08%, 82.64%, 69.99% and 52.78%, respectively, indicating that Lactobacillus casei FL5 fermentation broth, supernatant, FL5 live bacteria and inactivated bacteria inhibited the growth of Helicobacter pylori by inhibiting uricase activity.

[0105] (2) Determination of the inhibitory ability of Lacticaseibacillus paracasei FL5 against Helicobacter pylori

[0106] The FL5 fermentation broth, supernatant, FL5 live bacteria, inactivated bacteria and Helicobacter pylori suspension were adjusted to 1×10 8 CFU / mL. 100 μL of the H. pylori suspension was evenly spread on Columbia blood agar plates. A sterile Oxford cup (outer diameter 8 mm) was aseptically placed on the agar surface and gently pressed to ensure contact. Subsequently, 100 μL of fermentation broth, supernatant, live FL5 bacteria, and inactivated bacteria were added to the Oxford cup, respectively. The plates were incubated at 37°C under microaerobic conditions (5% O2, 10% CO2, 85% N2) for 72 hours. After incubation, the diameter of the inhibition zone around the Oxford cup was measured with a caliper. MRS liquid medium was used as a negative control, and TA triple antibiotic combination (omeprazole, amoxicillin, and clarithromycin) was used as a positive control.

[0107] Result analysis: Figure 2 As shown, compared with the negative (MRS) control, FL5 fermentation broth showed the strongest inhibitory activity (21.75 mm inhibition zone), 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 Lactobacillus paracasei FL5. Helicobacter pylori SS1 was resuspended in BHI broth, and then Lactaseibacillus paracasei FL5 fermentation broth, supernatant, or live / dead cells were added. The mixture was then 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 morphological analysis was performed under a scanning electron microscope (JEOL JSM-IT 2000x resolution). TA triple antibiotics (omeprazole, amoxicillin, clarithromycin) served as a positive control, and HP was the model group containing only Helicobacter pylori. The experimental results are shown in Figure 5. Figure 3 shown.

[0110] Results: Supernatant and fermentation broth of Lacticaseibacillus paracasei FL5 induced significant structural changes in H. pylori, including helical-to-spherical transformation and cell collapse. Live bacteria exhibited direct adhesion to H. pylori, while inactivated bacteria caused moderate cell deformation.

[0111] Example 4

[0112] In vivo antibacterial activity of Lacticaseibacillus paracasei FL5 and its postbiotics 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 five groups, each with 6 mice: a negative control group (NC), a model group (HP), a live bacteria FL5 group, an inactivated bacteria IFL5 group, and a TA triple antibiotic combination (omeprazole, amoxicillin, and clarithromycin) as a positive control. Except for the NC group, the other groups were perfused with normal saline every other day. The remaining groups were perfused with Helicobacter pylori solution (300 μL, 1 × 10 8 CFU / mL) for 3 weeks. To assess H. pylori colonization, three mice were randomly selected from the mice that had been orally inoculated with H. pylori. After an overnight fast, gastric tissue samples were collected for a standard rapid urease test. A distinct red reaction with the urease reagent confirmed successful establishment of the model.

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

[0116] (2) Changes in mouse weight

[0117] During the experiment, body weight was measured every four days and the weight change was recorded. The experimental results are as follows: Figure 4 shown.

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

[0119] (3) The urease activity of Helicobacter pylori in mouse gastric tissue was significantly reduced

[0120] The urease activity in the gastric tissues of all experimental mice was measured, and the experimental results were as follows: Figure 5 shown.

[0121] Result analysis: The urease activity was significantly reduced in the FL5, IFL5 and TA groups.

[0122] (4) Relative expression levels of Helicobacter pylori virulence genes CagA and VacA in mouse gastric tissue

[0123] The relative expression levels of Helicobacter pylori virulence genes CagA and VacA in the gastric tissues of all experimental mice were determined. Figure 6 shown.

[0124] Results: Compared with the NC group, the relative expression levels of CagA and VacA in the model group were significantly increased. Compared with the model group, the relative expression levels of CagA and VacA in the FL5, IFL5, and TA treatment groups were significantly decreased. The significant decreases in urease activity and virulence factor expression indicate that H. pylori colonization in mouse gastric tissue was significantly inhibited.

[0125] (5) Mouse histopathological analysis

[0126] After killing the mice, gastric tissue was collected and fixed in 4% paraformaldehyde for 24 h. The tissue was dehydrated and then embedded in paraffin. 4 μm sections were cut and stained with hematoxylin and eosin (H&E). Images of the HE-stained sections were obtained at a magnification of 200 times using a microscope. The experimental results are shown in Figure 2. Figure 7 shown.

[0127] Results: In the normal control group, gastric tissue architecture was intact, with an orderly arrangement of the epithelial layer, mild inflammatory cell infiltration in the mucosal layer, and normal, densely arranged mucosal glands, with no overt inflammatory response. In contrast, mice in the model group displayed significant inflammatory cell infiltration, particularly neutrophils, in the submucosal layer (black arrows), accompanied by mild glandular atrophy, decreased glandular density, and marked congestion of the fundic glands (red arrows). Treatment with FL5, IFL5, and TA effectively alleviated these pathological changes.

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

[0129] Mouse gastric tissue was homogenized with an appropriate amount of physiological saline and then centrifuged at 3000 rpm for 10 min. Cytokines (TNF-α, IL-1β, IL-10, IL-6, IL-8, and IL-4) and IgG levels in gastric tissue were quantitatively detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The experimental results are shown in Figure 2. Figure 8 shown.

[0130] Analysis of results: In model mice, levels of TNF-α, IL-1β, IL-6, IgG, and IL-8 were significantly elevated. However, treatment with FL5, IFL5, and TA significantly reduced the expression of these proinflammatory cytokines. Conversely, H. pylori infection significantly inhibited the production of anti-inflammatory cytokines IL-10 and IL-4, while treatment with FL5 and IFL5 effectively reversed this inhibitory effect. These findings suggest that the administration of FL5, IFL5, and TA all significantly modulates the inflammatory response after H. pylori infection, with IFL5 having a superior effect than FL5.

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

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

[0133] Result analysis: Figure 9 As shown, compared with the NC group, the model group showed significant downregulation of GSH, CAT, and SOD levels, while MDA levels were upregulated. The FL5, IFL5, and TA treatment groups showed varying degrees of recovery in these oxidative stress markers, with FL5 demonstrating the most significant recovery effect. These findings suggest that Lactobacillus paracasei FL5 possesses potent antioxidant properties and is able to mitigate H. pylori-induced oxidative damage.

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

[0135] Genomic DNA was extracted using a commercial DNA extraction kit (Omega Bio-tek, USA). DNA concentration and purity were determined using a NanoDrop2000 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 the table below. Figure 10 、 Figure 11 and Figure 12 shown.

[0136] Result analysis: Figure 10 As shown in Figure 3, PCA analysis showed that there were differences in the gastric flora composition among the groups, but the FL5 and IFL5 groups were close to the control group.

[0137] like Figure 11 As shown, the differential bacterial genera between the groups were significantly different. The differential bacterial genera in the model group were mainly o - Campylobacterales and g - Helicobacter, while the differential bacterial genus in group FL5. was o - Lactobacillales, g - Lachnospiraceae_NK4A136_group and g - Limosilactobacillus, a beneficial bacteria, the differential bacterial genus of the IFL5 group is o - Lachnospirales.

[0138] like Figure 12 As shown, compared with the NC group, the abundance of Campylobacterota in the model group increased significantly, while the abundance of other bacterial phyla decreased significantly. The abundance of Bacillota, Bacteroidota, Pseudomonadota, and Patescibacteria increased significantly in the FL5 group, similar to the NC group. The abundance of Bacteroidota and Patescibacteria increased significantly in the IFL5 group. In contrast, the gastric microbiota diversity in the TA group was disrupted, resulting in a relatively homogenous structure. The abundance of the phylum containing Helicobacter pylori was significantly decreased in the FL5, IFL5, and TA groups.

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

[0140] We further studied the regulatory effects of Helicobacter pylori infection and probiotic intervention on gastric mucosal barrier proteins and TLR2 / NF-κB inflammatory pathways. Figure 13 and Figure 14 shown.

[0141] Result analysis: Figure 13As shown, compared with the NC group, the HP group showed a trend of decreased 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 that of the antibiotic-treated group. FL5 significantly upregulated the expression of claudin-1 and occludin, while IFL5 significantly increased the expression level of claudin-1. FL5 showed a relatively stronger efficacy in promoting barrier recovery.

[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 HP group showed significantly increased expression of TLR2 and its downstream adaptor protein MyD88, as well as increased expression of key inflammatory regulators Iκ-Kα, p-IκB-α, and NF-κB. IFL5 was more effective than FL5 in inhibiting inflammatory signaling.

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

[0144] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one 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 present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Lactobacillus paracasei, characterized in that The Lactobacillus paracasei is Lacticaseibacillus paracasei FL5, which is deposited in Guangdong Provincial Microbial Culture Collection Center with a deposit date of 2025.03.06, a deposit number of GDMCC NO: 65986, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The postbiotic of Lactobacillus paracasei according to claim 1, wherein The postbiotics include one or more of Lacticaseibacillus paracasei FL5 fermentation broth, Lacticaseibacillus paracasei FL5 fermentation supernatant, and Lacticaseibacillus paracasei FL5 inactivated strains.

3. The postbiotic of Lactobacillus paracasei according to claim 2, wherein The Lacticaseibacillus paracasei FL5 fermentation liquid is prepared by the following method: Lacticaseibacillus paracasei FL5 is cultured in MRS liquid culture medium to prepare the Lacticaseibacillus paracasei FL5 fermentation liquid.

4. The postbiotic of Lactobacillus paracasei according to claim 2, wherein The Lacticaseibacillus paracasei FL5 fermentation supernatant is prepared by the following method: centrifuging and separating the Lacticaseibacillus paracasei FL5 fermentation broth prepared according to claim 3, collecting the supernatant, and preparing the Lacticaseibacillus paracasei FL5 fermentation supernatant.

5. The postbiotic of Lactobacillus paracasei according to claim 2, wherein The Lacticaseibacillus paracasei FL5 inactivated strain is prepared by the following method: centrifuging and separating the Lacticaseibacillus paracasei FL5 fermentation broth prepared according to claim 3, discarding the supernatant, washing and inactivating the bacterial cells obtained after centrifugation, and preparing the Lacticaseibacillus paracasei FL5 inactivated strain.

6. The application of the postbiotic of the Lactobacillus paracasei described in claim 1 or any of claims 2-5, characterized in that, The application is any of the following: (1) Application in the preparation of products for inhibiting Helicobacter pylori urease activity; (2) Application in the preparation of products for inhibiting the proliferation of Helicobacter pylori; (3) Application in the preparation of products for inhibiting Helicobacter pylori colonization; (4) Use in the preparation of products for preventing and / or treating inflammation caused by Helicobacter pylori; (5) Application in the preparation of products for preventing and / or treating oxidative damage caused by Helicobacter pylori; (6) Use in the preparation of products for preventing and / or treating dysbacteriosis caused by Helicobacter pylori.

7. An anti-Helicobacter pylori product, characterized in that: The invention comprises the postbiotics of the Lactobacillus paracasei according to claim 1 and / or the Lactobacillus paracasei according to any one of claims 2 to 5.

8. The anti-Helicobacter pylori product according to claim 7, characterized in that It also includes other active ingredients or excipients.

9. The anti-Helicobacter pylori product according to claim 7, characterized in that The product includes a food or a medicine.

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