Composite probiotic agent and application thereof in treatment of helicobacter pylori infection

Abstract

The invention discloses a composite probiotic agent and application thereof in treatment of helicobacter pylori infection. The compound probiotic agent is prepared from lactobacillus gasseri, lactobacillus rhamnosus and bifidobacterium animalis subsp. Lactis. The compound probiotic agent disclosed by the invention has a high inhibition rate on helicobacter pylori, and can effectively improve gastric tissue damage and systemic inflammatory response caused by helicobacter pylori infection.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a composite probiotic agent and application thereof in treating Helicobacter pylori infection. Background Art

[0002] Helicobacter pylori (Hp), also known as Helicobacter pylori, is a Gram-negative microaerophilic bacillus with a spiral, S-shaped or seagull-shaped body. It was first discovered and isolated by scientists Warren and Marshall from the gastric tissue of patients with chronic gastritis in 1983. Hp can survive in a highly acidic environment and often parasitizes in the gastric mucosal tissue. It is associated with the occurrence of diseases such as chronic gastritis, gastrointestinal ulcers, gastric cancer and gastric mucosa-associated lymphoma. Gastric cancer is the second most deadly cancer in the world, and Hp-induced gastritis is the most powerful single factor causing gastric cancer. Therefore, Hp was listed as a Class I biological carcinogen by the World Health Organization in 2022. Most H. pylori infected people have no obvious symptoms in the early stage. Some may experience symptoms of acute gastritis, including upper abdominal pain, abdominal distension, belching, nausea and vomiting, and loss of appetite. However, almost all patients will experience symptoms of chronic active gastritis to varying degrees. Once infected, H. pylori is extremely difficult to heal on its own, and infected people will suffer from it for life. The transmission routes of H. pylori include oral-oral and fecal-oral transmission, and group clusters of infections often occur. Studies have shown that the infection rate of Helicobacter pylori in my country is as high as 56%. Moreover, due to its subtle characteristics of onset symptoms, it is often neglected by patients and not promptly eradicated. Currently, the diagnosis of Helicobacter pylori mainly includes urea breath test (UBT), fecal Helicobacter pylori antigen detection (HpSA), Helicobacter pylori serological antibody detection and histological examination. Timely inspection and eradication treatment are important intervention methods to prevent the progression of the disease.

[0003] Although CN 2021103471976 has disclosed that multiple strains of composite probiotics have a certain effect on the treatment of diseases caused by Helicobacter pylori, their mechanism of inhibiting Helicobacter pylori and the scientific nature of the strain combination lack effective in vitro and in vivo verification. The effect of composite probiotic preparations directly depends on the types and quantities of the strains contained and the interactions between different strains. Scientific ratios and verification of in vitro and in vivo efficacy are the basis for clarifying their role. Summary of the Invention

[0004] The present invention provides a composite probiotic agent and its use in treating Helicobacter pylori infection to improve the inhibition rate of Helicobacter pylori. The composite probiotic agent of the present invention has a high inhibition rate against Helicobacter pylori and can effectively improve gastric tissue damage and systemic inflammatory response caused by Helicobacter pylori infection.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] In one aspect, the present invention provides a composite probiotic comprising Lactobacillus gasseri, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis.

[0007] In some embodiments, the Lactobacillus gasseri is Lactobacillus gasseri TF08-1, with the deposit number being GDMCC60092.

[0008] In some embodiments, the Lactobacillus rhamnosus is OF44-15Ph10T, with the deposit number being GDMCC60406.

[0009] In some embodiments, the Bifidobacterium animalis subsp. lactis is BGI-N3, with the deposit number being CCTCCM20221585.

[0010] In some embodiments, the composite probiotic is a bacterial solution or bacterial powder; when it is a bacterial solution, the number of viable bacteria in the bacterial solution is at least 1.0×10 8 CFU / mL; in the case of bacterial powder, the number of viable bacteria in the powder should be at least 5.0×10 8 CFU / g.

[0011] In some embodiments, the Lactobacillus gasseri TF08-1, Lactobacillus rhamnosus OF44-15Ph10T and Bifidobacterium animalis subsp. lactis BGI-N3 each account for 10% to 80% in the composite probiotic.

[0012] In some specific embodiments, the proportions of Lactobacillus gasseri TF08-1, Lactobacillus rhamnosus OF44-15Ph10T and Bifidobacterium animalis subsp. lactis BGI-N3 in the composite probiotic are selected from the following combinations:

[0013] 1)TF08-1:OF44-15Ph10T:BGI-N3=1:1:8;

[0014] 2)TF08-1:OF44-15Ph10T:BGI-N3=1:4:5;

[0015] 3)TF08-1:OF44-15Ph10T:BGI-N3=1:8:1;

[0016] 4)TF08-1:OF44-15Ph10T:BGI-N3=2:3:5;

[0017] 5)TF08-1:OF44-15Ph10T:BGI-N3=2:5:3;

[0018] 6)TF08-1:OF44-15Ph10T:BGI-N3=2:1:2;

[0019] 7)TF08-1:OF44-15Ph10T:BGI-N3=2:1:1;

[0020] 8)TF08-1:OF44-15Ph10T:BGI-N3=3:1:1;

[0021] 9)TF08-1:OF44-15Ph10T:BGI-N3=8:1:1;

[0022] 10)TF08-1:OF44-15Ph10T:BGI-N3=33:33:34.

[0023] Another aspect of the present invention provides a method for inhibiting the activity of Helicobacter pylori or urease, which comprises adding the above-mentioned composite probiotic agent to Helicobacter pylori.

[0024] In some embodiments, the methods are for non-diagnostic and / or non-therapeutic purposes.

[0025] Another aspect of the present invention provides the use of the composite probiotic agent in inhibiting the activity of Helicobacter pylori or urease and in preparing an inhibitor for inhibiting the activity of Helicobacter pylori or urease.

[0026] Another aspect of the present invention provides use of the composite probiotic agent in the preparation of a medicament for treating and / or preventing diseases caused by Helicobacter pylori infection.

[0027] In some embodiments, the disease caused by Helicobacter pylori infection is a gastrointestinal disease or an inflammatory response.

[0028] In some specific embodiments, the gastrointestinal disease or inflammatory response is systemic inflammatory response syndrome, gastritis, gastric tissue damage or gastric mucosal atrophy; the gastritis is, for example, acute gastritis or chronic gastritis.

[0029] Another aspect of the present invention provides use of the composite probiotic agent in the preparation of a medicament for regulating serum proinflammatory factors and / or serum proinflammatory factor levels.

[0030] In some embodiments, the serum anti-inflammatory factor is IL-10; the serum pro-inflammatory factor is TNF-α, IL-1β or IL-6.

[0031] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0032] The reagents and raw materials used in the present invention are commercially available.

[0033] The positive progress effect of the present invention is:

[0034] The beneficial effects of compound probiotics in inhibiting Helicobacter pylori infection are specifically manifested in the following aspects: (1) In in vitro experiments, compound probiotics can effectively inhibit Helicobacter pylori and Helicobacter pylori urease, and their inhibitory effects are significantly better than those of single-component bacterial agents and compound bacterial agents of any two strains; (2) In in vivo experiments, the use of compound probiotics to treat mice infected with Helicobacter pylori reduced the Helicobacter pylori load in their stomachs, and effectively improved the gastric tissue damage and systemic inflammatory response caused by Helicobacter pylori infection; (3) Compound probiotics will not cause Helicobacter pylori to develop drug resistance, solving the problem that conventional antibiotic therapy easily causes Helicobacter pylori to develop drug resistance.

[0035] Biomaterial deposit information

[0036] The animal Bifidobacterium subsp. lactis BGI-N3 of the present invention was deposited in the China Center for Type Culture Collection on October 17, 2022, with the address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a postal code of 430072, and the deposit number being CCTCC M20221585. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the gastric infection and damage conditions of each group of mice in Example 27 of this application.

[0038] Figure 2 These are the serum cytokine levels of each group of mice in Example 27 of this application. DETAILED DESCRIPTION

[0039] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0040] The main experimental consumables involved in the following examples are as follows:

[0041] MRS broth medium: 10.0 g of peptone, 10.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 5.0 g of sodium acetate, 2.0 g of diammonium hydrogen citrate, 1.0 mL of Tween-80, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate heptahydrate, and 1000 mL of distilled water. After mixing the above components, adjust the pH to 6.2-6.4, and sterilize in an autoclave at 121°C for 15 min.

[0042] TPY liquid medium: 10 g of hydrolyzed casein, 5 g of phytone, 2 g of yeast powder, 5 g of glucose, 2 g of dipotassium hydrogen phosphate, 0.5 g of magnesium chloride, 0.25 g of zinc sulfate, 0.15 g of calcium chloride, 0.1 mg of ferric chloride, 0.5 g of cysteine-hydrochloric acid, and 1 mL of Tween-80. After mixing the above components, adjust the pH to 6.5±0.2, and sterilize in an autoclave at 121°C for 15 min.

[0043] MRS agar medium: Based on the formula of MRS broth medium, add 20g / L agar, adjust the pH to 6.2-6.4, and place it in a high pressure sterilizer at 121℃ for 15 minutes.

[0044] TPY agar medium: Based on the formula of TPY liquid medium, 20 g / L agar was added, the pH was adjusted to 6.2-6.4, and the medium was sterilized in an autoclave at 121°C for 15 min.

[0045] GSSA-Hp selective Columbia blood plate: 12.0 g of tryptic peptone, 5.0 g of animal tissue protein digest, 3.0 g of yeast extract, 3.0 g of beef extract, 1.0 g of corn starch, 5.0 g of sodium chloride, 13.5 g of agar, 0.5 mg of polymyxin B, 2 mg of nalidixic acid, 2.5 mg of amphotericin, 200 mg of bacitracin, 6 mg of vancomycin, and 1000 mL of distilled water. After mixing the above components, adjust the pH to 7.2-7.4, place in an autoclave and sterilize at 121°C for 15 min. After the culture medium cools to about 50°C, add 70 mL of sterile defibrinated sheep blood and mix well.

[0046] Urea-phenol red solution: Dissolve 10 g of urea and 0.006 g of phenol red, mix well, adjust the pH of the solution to 6.5, and make up to 50 mL. The ratios of urea and phenol red are 20% and 0.012%, respectively.

[0047] The Helicobacter pylori strain used in the experiment was H. pylori ATCC 43504, obtained from the American Type Culture Collection (ATCC). Elisa assay kits for TNF-α, IL-1β, IL-6, and IL-10 were purchased from Nanjing Jiancheng Biotechnology Co., Ltd. All other experimental materials and equipment were commonly used in microbiology laboratories.

[0048] Example 1

[0049] In this example, 71 strains of Lactobacillus and Bifidobacterium preserved in our laboratory were screened to obtain probiotic strains with excellent Helicobacter pylori inhibition efficacy. The strain names are detailed in Table 1. After thawing, the frozen tubes of each strain were inoculated into MRS liquid medium or TPY liquid medium at a mass ratio of 1%, and incubated anaerobically at 37°C for 24 hours to obtain a fermentation broth. The fermentation broth was centrifuged at 10,000 rpm for 10 minutes to remove the bacteria, and then filtered through a 0.22 μm aqueous filter membrane to obtain 71 cell-free fermentation supernatants (CFS). The antibacterial activity of CFS against Helicobacter pylori was determined using a microplate antibacterial assay: Helicobacter pylori ATCC43504 strain was inoculated on GSSA-Hp selective Columbia blood agar plates and revived for 3-4 days. After the incubation period, the bacteria were scraped and the concentration was adjusted to 5.0×10 7 Take 50 μL of Helicobacter pylori suspension after adjusting the bacterial concentration and place it in a 96-well plate with 150 μL of sample. Mix well and culture at 37°C for 18 hours before measuring OD 600 nm value, the results are recorded as the test group data, blank MRS liquid culture medium is used as the control group instead of the sample to perform the test, and the results are recorded as the control group data. The Helicobacter pylori inhibition rate is calculated according to the following formula: Helicobacter pylori inhibition rate (%) = [(OD 对照组 -OD 试验组 ) / OD 对照组 ]×100, the above experiment was repeated 3 times, and the results were expressed as mean ± standard deviation.

[0050] Table 1: Inhibitory effect of different strains on the growth of Helicobacter pylori

[0051]

[0052]

[0053]

[0054] The test results are shown in Table 1. Different letter headers in each group of data indicate statistical differences (p<0.05). The inhibition rates of each strain on the growth and reproduction of Helicobacter pylori ranged from 55.26% to 92.44%, among which the inhibition rate of Lactobacillus gesellschaft TF08-1 was 92.44±3.51%, the inhibition rate of Lactobacillus rhamnosus OF44-15Ph10T was 91.74±2.46%, and the inhibition rate of Bifidobacterium animalis subspecies lactis BGI-N3 was 88.34±2.64%, which were significantly better than other strains and showed stronger antibacterial ability against Helicobacter pylori.

[0055] Example 2

[0056] Urease is one of the important physiological enzymes of Helicobacter pylori, which can hydrolyze urea in gastric juice into carbon dioxide and ammonia, thereby neutralizing gastric acid and protecting Helicobacter pylori from the corrosion of gastric acid. This example intends to screen out strains with strong urease inhibition ability against Helicobacter pylori from 71 strains of lactobacillus and bifidobacterium preserved in this laboratory. The names of the strains are detailed in Table 2. The test sample preparation method is the same as Example 1. Take 40 μL of Helicobacter pylori suspension after adjusting the bacterial concentration, place it in a 96-well plate with 10 μL of test sample, and place it in a microaerobic environment for co-cultivation for 48 hours. After the cultivation is completed, add 150 μL of urea-phenol red solution, shake and measure the absorbance at a wavelength of 550nm, record it as the test group data, and use blank MRS liquid culture medium instead of sample for control test, record it as the control group data. The urease activity inhibition rate is calculated according to the following formula: Urease activity inhibition rate (%) = [(OD 对照组 -OD 试验组 ) / OD 对照组 ] × 100. The experiment was repeated three times, and the results are expressed as mean ± standard deviation.

[0057] Table 2: Urease inhibition ability of different strains against Helicobacter pylori

[0058]

[0059]

[0060]

[0061] The test results are shown in Table 2. Different letters in the shoulder of each group of data indicate statistical differences (p<0.05). The inhibition rate of each strain on Helicobacter pylori urease activity is concentrated between 51.35% and 80.66%, among which the inhibition rate of Lactobacillus gesellschaft TF08-1 is 80.66±4.55%, the inhibition rate of Lactobacillus rhamnosus OF44-15Ph10T is 78.25±2.41%, and the inhibition rate of Bifidobacterium animalis subspecies lactis BGI-N3 is 76.64±3.15%, which is significantly better than other strains and shows stronger Helicobacter pylori urease inhibition ability.

[0062] Example 3

[0063] A single-component probiotic for inhibiting Helicobacter pylori comprises 100 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1 to obtain the single-component probiotic.

[0064] Example 4

[0065] A single-component probiotic for inhibiting Helicobacter pylori comprises 100 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T to obtain the single-component probiotic.

[0066] Example 5

[0067] A single-component probiotic for inhibiting Helicobacter pylori comprises 100 parts of freeze-dried bacterial powder of Bifidobacterium animalis subspecies lactis BGI-N3 to obtain the single-component probiotic.

[0068] Example 6

[0069] A composite probiotic for inhibiting Helicobacter pylori comprises 75 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1 and 25 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0070] Example 7

[0071] A composite probiotic for inhibiting Helicobacter pylori comprises 50 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1 and 50 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0072] Example 8

[0073] A composite probiotic for inhibiting Helicobacter pylori comprises 25 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1 and 75 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0074] Example 9

[0075] A composite probiotic for inhibiting Helicobacter pylori comprises 75 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1 and 25 parts of freeze-dried bacterial powder of Bifidobacterium animalis subspecies lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0076] Example 10

[0077] A composite probiotic for inhibiting Helicobacter pylori comprises 50 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1 and 50 parts of freeze-dried bacterial powder of Bifidobacterium animalis subspecies lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0078] Example 11

[0079] A composite probiotic for inhibiting Helicobacter pylori comprises 25 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1 and 75 parts of freeze-dried bacterial powder of Bifidobacterium animalis subspecies lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0080] Example 12

[0081] A composite probiotic for inhibiting Helicobacter pylori comprises 75 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T and 25 parts of freeze-dried bacterial powder of Bifidobacterium animalis subspecies lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0082] Example 13

[0083] A composite probiotic for inhibiting Helicobacter pylori comprises 50 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T and 50 parts of freeze-dried bacterial powder of Bifidobacterium animalis subspecies lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0084] Example 14

[0085] A composite probiotic for inhibiting Helicobacter pylori comprises 25 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T and 75 parts of freeze-dried bacterial powder of Bifidobacterium animalis subspecies lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0086] Example 15

[0087] A composite probiotic for inhibiting Helicobacter pylori comprises 10 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 10 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 80 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0088] Example 16

[0089] A composite probiotic for inhibiting Helicobacter pylori comprises 10 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 40 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 50 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0090] Example 17

[0091] A composite probiotic for inhibiting Helicobacter pylori comprises 10 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 80 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 10 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0092] Example 18

[0093] A composite probiotic for inhibiting Helicobacter pylori comprises 20 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 30 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 50 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0094] Example 19

[0095] A composite probiotic for inhibiting Helicobacter pylori comprises 20 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 50 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 30 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0096] Example 20

[0097] A composite probiotic for inhibiting Helicobacter pylori comprises 40 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 20 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 40 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0098] Example 21

[0099] A composite probiotic for inhibiting Helicobacter pylori comprises 50 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 25 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 25 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0100] Example 22

[0101] A composite probiotic for inhibiting Helicobacter pylori comprises 60 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 20 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 20 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0102] Example 23

[0103] A composite probiotic for inhibiting Helicobacter pylori comprises 80 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 10 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 10 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above components are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0104] Example 24

[0105] A composite probiotic for inhibiting Helicobacter pylori comprises 33 parts of freeze-dried bacterial powder of Lactobacillus gasseri TF08-1, 33 parts of freeze-dried bacterial powder of Lactobacillus rhamnosus OF44-15Ph10T, and 34 parts of freeze-dried bacterial powder of Bifidobacterium animalis subsp. lactis BGI-N3. The above ingredients are placed in a blender and stirred at a speed of 100 rpm for 10 minutes to obtain the composite probiotic.

[0106] Example 25: Helicobacter pylori inhibition ability of single-component and composite-component probiotics

[0107] This example aimed to evaluate the ability of different probiotic compositions to inhibit the growth and reproduction of Helicobacter pylori. The probiotic compositions of Examples 3 to 24 were inoculated at 1% by mass into MRS liquid culture medium and incubated anaerobically at 37°C for 24 hours to obtain a fermentation broth. The fermentation broth processing, Helicobacter pylori culture, antibacterial activity assay, and data processing were identical to those described in Example 1.

[0108] Table 3: Inhibitory effect of different test samples on the growth of Helicobacter pylori

[0109] Test samples Antibacterial rate (%) Test samples Antibacterial rate (%) Negative control group 0 Example 14 <![CDATA[92.74±1.24 ab ]]> Example 3 <![CDATA[92.44±3.51 ab ]]> Example 15 <![CDATA[96.74±2.24 c ]]> Example 4 <![CDATA[91.74±2.46 ab ]]> Example 16 <![CDATA[96.36±1.25 c ]]> Example 5 <![CDATA[88.34±2.64 a ]]> Example 17 <![CDATA[97.97±1.19 c ]]> Example 6 <![CDATA[93.44±2.36 b ]]> Example 18 <![CDATA[97.95±1.58 c ]]> Example 7 <![CDATA[92.52±1.73 ab ]]> Example 19 <![CDATA[96.42±2.25 c ]]> Example 8 <![CDATA[92.55±2.32 ab ]]> Example 20 <![CDATA[96.26±2.15 c ]]> Example 9 <![CDATA[94.57±2.17 bc ]]> Example 21 <![CDATA[98.86±1.03 c ]]> Example 10 <![CDATA[92.19±1.16 ab ]]> Example 22 <![CDATA[97.54±2.05 c ]]> Example 11 <![CDATA[93.26±1.47 b ]]> Example 23 <![CDATA[96.73±2.53 c ]]> Example 12 <![CDATA[92.54±2.15 ab ]]> Example 24 <![CDATA[97.36±2.11 c ]]> Example 13 <![CDATA[93.64±2.23 b ]]>

[0110] The test results are shown in Table 3. Different letters in the shoulder of each group of data indicate statistical differences (p<0.05). The single-component probiotics prepared in Examples 3 to 5 have an inhibition rate on the growth and reproduction of Helicobacter pylori of 88.34% to 92.44%. The two-component probiotics prepared in Examples 6 to 14 have an inhibition rate on the growth and reproduction of Helicobacter pylori of 92.19% to 94.57%. The composite probiotics prepared in Examples 15 to 24 have an inhibition rate on the growth and reproduction of Helicobacter pylori of 96.26% to 98.86%. The above experimental data show that the composite probiotics prepared under suitable compounding conditions of the three strains can significantly inhibit the growth and reproduction of Helicobacter pylori, and the inhibition effect is better than that of the single-component and two-component strains, showing stronger antibacterial ability.

[0111] Example 26: Helicobacter pylori urease inhibition ability of single-component and composite-component probiotics

[0112] This example aimed to evaluate the urease inhibition ability of different probiotic compositions against Helicobacter pylori. The probiotic compositions from Examples 3 to 24 were inoculated at 1% by mass into MRS liquid culture medium and incubated anaerobically at 37°C for 24 hours to obtain fermentation broth. The fermentation broth processing, Helicobacter pylori culture, urease inhibition assay, and data processing were the same as those described in Example 2.

[0113] Table 4: Urease inhibition ability of different test samples against Helicobacter pylori

[0114]

[0115]

[0116] The test results are shown in Table 4. Different letters in the shoulder of each group of data indicate statistical differences (p<0.05). The single-component probiotics prepared in Examples 3 to 5 have an inhibition rate of Helicobacter pylori urease of 76.64% to 80.66%, the two-component probiotics prepared in Examples 6 to 14 have an inhibition rate of Helicobacter pylori urease of 82.41% to 85.25%, and the composite probiotics prepared in Examples 15 to 24 have an inhibition rate of Helicobacter pylori urease of 88.14% to 92.57%. The above experimental data show that under suitable compounding conditions, the composite probiotics prepared by the three strains can significantly inhibit the urease activity of Helicobacter pylori, and the inhibition effect is better than that of the single-component and two-component strains, showing better urease activity inhibition ability.

[0117] Example 27: Animal Experimental Verification of the Efficacy of the Composite Probiotic for Treating Helicobacter pylori Infection

[0118] A total of 32 6-week-old SPF-grade C57BL / 6 male mice were used to carry out animal experiments on the treatment of Helicobacter pylori infection with compound probiotics. They were divided into 4 groups: (1) normal control group (N=8): the mice were not modeled and were continuously fed with sterile saline for 40 days, once a day, 0.5 mL each time; (2) model control group (N=8): the mice were modeled for 12 days, and after the modeling was completed, they were gavaged with sterile saline for 28 days, once a day, 0.5 mL each time; (3) ) Positive drug intervention group (N=8): The mice were modeled for 12 days, and then the mice were treated with triple antibiotic therapy for 14 days, followed by gavage with sterile saline for 14 days, once a day, 0.5 mL each time; (4) Composite probiotic intervention group (N=8): The mice were modeled for 12 days, and then the mice were fed with composite probiotics for 28 days, once a day, after the modeling was completed, wherein the ratio of the components of the composite probiotics was 2:1:1, and the number of live bacteria fed daily was 1 billion CFU.

[0119] During the above-mentioned grouping and intervention process, the H. pylori-infected mouse model was established as follows: first, an antibiotic mixed solution (containing 10 mg / mL ampicillin, 2 mg / mL gentamicin, and 10 mg / mL azithromycin) was gavaged for 3 consecutive days to eliminate the gastric flora, and then H. pylori bacterial solution was gavaged at a dose of 100 million / day / mouse, every other day, for a total of 5 times to establish a Helicobacter pylori infection model; the antibiotic triple therapy included omeprazole 0.12 mg / mL, amoxicillin 6 mg / mL, and clarithromycin 3 mg / mL, twice a day, 0.5 mL each time. After the experiment, the corresponding Elisa test kits were used to determine the levels of serum TNF-α, IL-1β, IL-6, and IL-10. The GSSA-Hp selective Columbia blood plate was used to count the Hp load in the stomach. HE staining was used for histopathological analysis of the gastric tissue. The pathological sections were scored according to the severity of five indicators, including Hp number, activity, chronic inflammatory response, atrophy, and intestinal metaplasia area (normal: 0 points, mild: 1 point, moderate: 2 points, severe: 3 points).

[0120] The experimental results are attached. Figure 1 、 2, the "*" marked in the chart indicates that the group is different from the model control group (p < 0.05), the "**" marked indicates that the group is significantly different from the model control group (p < 0.01), and the "***" marked indicates that the group is extremely significantly different from the model control group (p < 0.001). Compared with the normal control group, the mice after modeling showed increased gastric Hp load, gastric tissue damage, increased levels of pro-inflammatory factors such as serum TNF-Alpha, IL-1Beta, and IL-6, and decreased levels of anti-inflammatory factors IL-10. The above results show that after modeling, Helicobacter pylori successfully survived and colonized in the stomach of mice, causing gastric tissue lesions and causing systemic inflammatory responses. After 28 days of intervention with a composite probiotic, the gastric Hp load of infected mice was extremely significantly reduced. Compared with the model control group, the gastric Hp load of mice after intervention was reduced by 99.78% (p < 0.001) (see Appendix for details). Figure 1 A), showing a strong ability to eliminate Helicobacter pylori. Compared with the model control group, after intervention with the compound probiotics, the gastric tissue pathology score of infected mice was significantly reduced from 8.67±1.63 to 5.67±1.03 (p<0.01) (see Appendix for details). Figure 1 B, C). In addition, serum TNF-α, IL-1β, IL-6 and other pro-inflammatory factors showed varying degrees of decrease (see Figure 2 A, B, C), serum anti-inflammatory factor IL-10 level increased significantly (see Appendix for details). Figure 2 D), and compared to conventional triple antibiotic therapy, the compound probiotic not only effectively inhibits Helicobacter pylori but also significantly improves the body's inflammatory response, resulting in a superior therapeutic effect compared to antibiotic therapy. These results demonstrate that after H. pylori infection, intervention with a compound probiotic can significantly reduce post-infection H. pylori load, improve gastric tissue damage, and mitigate the systemic inflammatory response caused by the infection, demonstrating excellent efficacy in treating H. pylori infection and the gastric inflammatory lesions it causes.

Claims

1. A composite probiotic, characterized in that: The composite probiotic comprises Lactobacillus gasseri, Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis.

2. The composite probiotic according to claim 1, characterized in that The Lactobacillus gasseri is Lactobacillus gasseri TF08-1, with the deposit number GDMCC60092; And / or, the Lactobacillus rhamnosus is OF44-15Ph10T, with the deposit number GDMCC60406; And / or, the Bifidobacterium animalis subspecies lactis is BGI-N3, and the deposit number is CCTCC M20221585.

3. The composite probiotic according to claim 1 or 2, characterized in that The composite probiotic agent is a bacterial liquid or bacterial powder; when it is a bacterial liquid, the number of live bacteria in the bacterial liquid is at least 1.0×10 8 CFU / mL; in the case of bacterial powder, the number of viable bacteria in the powder should be at least 5.0×10 8 CFU / g.

4. The composite probiotic according to any one of claims 2 to 3, characterized in that The proportions of the Lactobacillus gasseri TF08-1, Lactobacillus rhamnosus OF44-15Ph10T and Bifidobacterium animalis subspecies lactis BGI-N3 in the composite probiotic are all 10% to 80%.

5. The composite probiotic according to any one of claims 2 to 4, characterized in that: The proportions of Lactobacillus gasseri TF08-1, Lactobacillus rhamnosus OF44-15Ph10T and Bifidobacterium animalis subspecies lactis BGI-N3 in the composite probiotic are selected from the following combinations: 1)TF08-1:OF44-15Ph10T:BGI-N3=1:1:8; 2)TF08-1:OF44-15Ph10T:BGI-N3=1:4:5; 3)TF08-1:OF44-15Ph10T:BGI-N3=1:8:1; 4)TF08-1:OF44-15Ph10T:BGI-N3=2:3:5; 5)TF08-1:OF44-15Ph10T:BGI-N3=2:5:3; 6)TF08-1:OF44-15Ph10T:BGI-N3=2:1:2; 7)TF08-1:OF44-15Ph10T:BGI-N3=2:1:1; 8)TF08-1:OF44-15Ph10T:BGI-N3=3:1:1; 9)TF08-1:OF44-15Ph10T:BGI-N3=8:1:1; 10)TF08-1:OF44-15Ph10T:BGI-N3=33:33:

34.

6. A method for inhibiting Helicobacter pylori or urease activity, characterized in that: The method comprises adding the composite probiotic according to any one of claims 1 to 5 to Helicobacter pylori, and the method is preferably for non-diagnostic and / or non-therapeutic purposes.

7. Use of the composite probiotic according to any one of claims 1 to 5 in inhibiting Helicobacter pylori or urease activity and in preparing an inhibitor, wherein the inhibitor is used to inhibit Helicobacter pylori or urease activity.

8. Use of the composite probiotic according to any one of claims 1 to 5 in the preparation of a medicament for treating and / or preventing diseases caused by Helicobacter pylori infection.

9. The use according to claim 8, characterized in that The disease caused by Helicobacter pylori infection is a gastrointestinal disease or an inflammatory reaction; Preferably, the gastrointestinal disease or inflammatory response is systemic inflammatory response syndrome, gastritis, gastric tissue damage or gastric mucosal atrophy; the gastritis is, for example, acute gastritis or chronic gastritis.

10. Use of the composite probiotic according to any one of claims 1 to 5 in the preparation of a medicament for regulating serum proinflammatory factors and / or serum proinflammatory factor levels; Preferably, the serum anti-inflammatory factor is IL-10; the serum pro-inflammatory factor is TNF-α, IL-1β or IL-6.

Images