A composition of probiotics and protein and its application against Helicobacter pylori

Abstract

The present invention provides a composition of probiotics and protein and its application against Helicobacter pylori, belonging to the technical field of microbial preparations. The composition of the present invention comprises animal Bifidobacterium Bb‑12, Lactobacillus acidophilus NCFM, egg yolk globulin powder and resveratrol, and the composition has a good ability to inhibit Helicobacter pylori, can significantly eliminate Helicobacter pylori in the human body, and reduce the occurrence of gastric cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preparations, and in particular relates to a composition of probiotics and protein and an application thereof in resisting Helicobacter pylori. Background Art

[0002] Helicobacter pylori is a Gram-negative bacterium, about 2.5-4.0 μm long and 0.5-1.0 μm wide. The bacteria are spiral, S-shaped or seagull-shaped, acid-resistant and carbon dioxide-loving. Warren and Marshall first discovered and isolated Helicobacter pylori from gastric tissue of patients with chronic gastritis in 1983. Existing studies have shown that the colonization of Helicobacter pylori on the gastric mucosa stimulates strong local and systemic inflammatory and immune responses, which together with the toxic substances produced by the bacteria cause peptic ulcers in 10% to 20% of infected individuals and gastric cancer in 1% to 3% of infected individuals (Suerbaum S, Michetti P).

[0003] Helicobacter pylori is contagious and is usually spread through the digestive tract, mainly through the fecal-oral route and the oral-oral route. The fecal-oral route refers to the infected person's feces containing bacteria directly or indirectly contaminating the food, and the eater is infected. The oral-oral route refers to the infected person's saliva containing bacteria contaminating the food or tableware, and the person is infected after eating the food or using the tableware. In addition, there is a special route, namely, medical-induced infection, which is mainly caused by the infected person not being thoroughly sterilized after a digestive endoscopy, which leads to the infection of Helicobacter pylori to others during the examination.

[0004] Nowadays, the commonly used treatment for Helicobacter pylori is "triple therapy", which has further developed into "quadruple therapy". The combination of two antibiotics and a proton pump inhibitor or bismuth citrate is the mainstream formula of "triple" therapy in medicine (taken continuously for one and a half to two weeks). The "quadruple therapy" is to add a dose of bismuth citrate to the two antibacterial drugs and proton pumps and take it continuously for 10-14 days. In in vitro experiments, many antibiotics have a certain inhibitory effect on Helicobacter pylori, but only a few antibiotics can be used for in vivo treatment. The incompleteness and repeated recurrence of the commonly used treatments for Helicobacter pylori, coupled with the limited selection of antibiotics, have led to the easy mutation and drug resistance of Helicobacter pylori strains, and the emergence of super bacteria in the case of poor treatment. This has greatly weakened the effectiveness and sustainability of related therapies in inhibiting Helicobacter pylori, delaying the treatment of related diseases (Gerrits et al. 2006). In fact, in the clinical treatment of diseases, excessive use of antibiotics is not only not beneficial to the human body, but also causes a series of potential harms to the human body. The abuse of antibiotics will affect the distribution and number of normal flora in the human body, may inhibit the growth of probiotics, and even more so, will cause a series of endogenous diseases caused by microbial disorders, such as Clostridium difficile infection or metabolic diseases (Ianiro et al. 2016). Compared with antibiotics, probiotics have obvious advantages in the treatment of Helicobacter pylori. For example, probiotics have the functions of strengthening the biological barrier of gastric mucosa, releasing cytotoxins and producing short-chain fatty acids to resist Helicobacter pylori infection, competitively inhibiting Helicobacter pylori colonization, and balancing the levels of multiple cytokines. Patent CN201910101339.3 discloses a probiotic composition against Helicobacter pylori, which includes Lactobacillus plantarum (CCTCCNO: M2018268) powder and Lactobacillus plantarum metabolite powder as active ingredients. The Lactobacillus plantarum is cultured by expanding the strain, primary seed culture, seed tank culture and fermentation culture to obtain Lactobacillus plantarum fermentation liquid, which can effectively inhibit Helicobacter pylori until Helicobacter pylori is eliminated from the human body, reducing the occurrence of gastric cancer, and is harmless to the human body. Patent CN202010574273.2 provides a composite probiotic composition for inhibiting Helicobacter pylori and its application, which includes Lactobacillus reuteri with a live bacterial count of 1-2×10 10 CFU / g, viable Lactobacillus johnsonii count 3-4×10 10CFU / g, and other components are calculated by weight as follows: 80-160 parts of oligofructose, 400-600 parts of resistant dextrin, 200-400 parts of galacto-oligosaccharide, 8-12 parts of licorice extract, 4-6 parts of strawberry extract, and the remainder of maltodextrin. The total amount of the composition is 2000 parts. It has a strong ability to inhibit Helicobacter pylori. Each added component is conducive to the colonization of probiotics and improves their vitality, can significantly improve the gastric environment, and thus can effectively eradicate Helicobacter pylori. It has great application potential in the treatment of diseases caused by Helicobacter pylori.

[0005] Therefore, there is an urgent need to provide a probiotic product with better anti-Helicobacter pylori effect. Summary of the invention

[0006] In view of the above problems, the present invention provides a composition of probiotics and protein and its application against Helicobacter pylori. The composition of the present invention comprises animal Bifidobacterium Bb-12, Lactobacillus acidophilus NCFM, egg yolk globulin powder and resveratrol, and the composition has a good ability to inhibit Helicobacter pylori, can significantly eliminate Helicobacter pylori in the human body, and reduce the occurrence of gastric cancer.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0008] In one aspect, the present invention provides a composition comprising: Bifidobacterium animalis Bb-12, Lactobacillus acidophilus NCFM and egg yolk globulin powder.

[0009] Specifically, the composition also includes resveratrol.

[0010] Specifically, the viable count of animal Bifidobacterium Bb-12 is 10 6 -10 10 CFU / mL.

[0011] Specifically, the viable count of Lactobacillus acidophilus NCFM is 10 5 -10 9 CFU / mL.

[0012] Specifically, the concentration of the egg yolk globulin powder is 0.1-10 mg / L, preferably 1 mg / L.

[0013] Specifically, the concentration of resveratrol is 5-20 mg / L, preferably 10-15 mg / L.

[0014] In another aspect, the present invention provides use of the above composition in preparing an anti-Helicobacter pylori product.

[0015] More specifically, the product is a health product, food or medicine.

[0016] More specifically, the types of health products include but are not limited to: medicinal liquor, capsules, tablets, granules, tea products, fruit juice, fruit vinegar, oral liquid, soft capsules, granules, fermented dairy products, fermented cereal products, fermented soy products, honey paste, dew, powder, fresh juice, meal replacement powder, etc.

[0017] The health care product also includes health care product additives.

[0018] The health care product additives include, but are not limited to: flavors and fragrances, colorants, sweeteners, acidulants, flavor enhancers, emulsifiers, thickeners, preservatives, antioxidants, nutritional enhancers, etc.

[0019] More specifically, the types of food include but are not limited to: biscuits, dairy products, meal replacements, meat products, sauces, baked goods, yogurt, ice cream, fermented cereal-based products, fruit juice, rice wine, candy, syrup, canned food, pickled products, condiments, soy products, chocolate, fillings, tea products, puffed food, etc.

[0020] The food also includes food additives.

[0021] The food additives include but are not limited to preservatives, acidity regulators, anti-caking agents, defoaming agents, antioxidants, bleaching agents, leavening agents, base materials in gum-based candies, colorants, color protectants, emulsifiers, enzyme preparations, flavor enhancers, flour treatment agents, coating agents, moisture retaining agents, nutritional enhancers, preservatives, stabilizers and coagulants, sweeteners, thickeners, natural food flavors, synthetic food flavors, etc.

[0022] More specifically, the dosage form of the drug includes, but is not limited to, a gastrointestinal dosage form or a parenteral dosage form according to the administration method.

[0023] The dosage forms for administration via the gastrointestinal tract include, but are not limited to, powders, tablets, granules, capsules, solutions, emulsions, suspensions, and oils.

[0024] The non-gastrointestinal administration dosage forms include, but are not limited to, injection dosage forms, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms, and cavity administration dosage forms.

[0025] The injectable dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0026] The respiratory tract administration dosage forms include but are not limited to sprays, aerosols, and powder sprays.

[0027] The skin administration dosage forms include, but are not limited to, external solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0028] The mucosal administration dosage forms include, but are not limited to, eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive sheets and patches.

[0029] The cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops and pills.

[0030] The medicine also includes pharmaceutically acceptable excipients.

[0031] The pharmaceutically acceptable excipients include, but are not limited to, solvents, emulsifiers, disintegrants, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, antibacterial agents, diluents, wetting agents, adhesives, film-forming agents, and the like.

[0032] In yet another aspect, the present invention provides an anti-Helicobacter pylori product, comprising the above composition.

[0033] Specifically, the product is a health product, food or medicine.

[0034] More specifically, the types of health products include but are not limited to: medicinal liquor, capsules, tablets, granules, tea products, fruit juice, fruit vinegar, oral liquid, soft capsules, granules, fermented dairy products, fermented cereal products, fermented soy products, honey paste, dew, powder, fresh juice, meal replacement powder, etc.

[0035] The health care product also includes health care product additives.

[0036] The health care product additives include, but are not limited to: flavors and fragrances, colorants, sweeteners, acidulants, flavor enhancers, emulsifiers, thickeners, preservatives, antioxidants, nutritional enhancers, etc.

[0037] More specifically, the types of food include but are not limited to: biscuits, dairy products, meal replacements, meat products, sauces, baked goods, yogurt, ice cream, fermented cereal-based products, fruit juice, rice wine, candy, syrup, canned food, pickled products, condiments, soy products, chocolate, fillings, tea products, puffed food, etc.

[0038] The food also includes food additives.

[0039] The food additives include but are not limited to preservatives, acidity regulators, anti-caking agents, defoaming agents, antioxidants, bleaching agents, leavening agents, base materials in gum-based candies, colorants, color protectants, emulsifiers, enzyme preparations, flavor enhancers, flour treatment agents, coating agents, moisture retaining agents, nutritional enhancers, preservatives, stabilizers and coagulants, sweeteners, thickeners, natural food flavors, synthetic food flavors, etc.

[0040] More specifically, the dosage form of the drug includes, but is not limited to, a gastrointestinal dosage form or a parenteral dosage form according to the administration method.

[0041] The dosage forms for administration via the gastrointestinal tract include, but are not limited to, powders, tablets, granules, capsules, solutions, emulsions, suspensions, and oils.

[0042] The non-gastrointestinal administration dosage forms include, but are not limited to, injection dosage forms, respiratory tract administration dosage forms, skin administration dosage forms, mucosal administration dosage forms, and cavity administration dosage forms.

[0043] The injectable dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0044] The respiratory tract administration dosage forms include but are not limited to sprays, aerosols, and powder sprays.

[0045] The skin administration dosage forms include, but are not limited to, external solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0046] The mucosal administration dosage forms include, but are not limited to, eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive sheets and patches.

[0047] The cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops and pills.

[0048] The medicine also includes pharmaceutically acceptable excipients.

[0049] The pharmaceutically acceptable excipients include, but are not limited to, solvents, emulsifiers, disintegrants, solubilizers, antioxidants, pH regulators, osmotic pressure regulators, antibacterial agents, diluents, wetting agents, adhesives, film-forming agents, and the like.

[0050] The preparation methods of the drugs, foods and health products described in the present invention can adopt the relevant preparation methods currently available in the art and those developed in the future. It should be clear that the specific preparation method adopted cannot be used as a limitation on the protection scope of this application. Regardless of whether the preparation method currently available or developed in the future is adopted, as long as the above-mentioned microorganisms and resveratrol are included, they are within the scope of protection claimed in this application.

[0051] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0052] The composition of the present invention comprises animal bifidobacterium Bb-12, lactobacillus acidophilus NCFM, egg yolk globulin powder and resveratrol. The composition has good ability to inhibit Helicobacter pylori, can significantly eliminate Helicobacter pylori in the human body, and reduce the occurrence of gastric cancer. DETAILED DESCRIPTION

[0053] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0054] Experimental Materials

[0055] 1. Bifidobacterium animalis Bb-12, purchased from Chr. Hansen.

[0056] 2. Lactobacillus acidophilus NCFM, purchased from Danisco.

[0057] 3. Egg yolk globulin powder: Take egg yolk and add deionized water to obtain egg yolk dilution, let the egg yolk dilution stand for 1-2 hours and take the supernatant; add flocculant to the supernatant and stir evenly; separate the precipitate and permeate, take the permeate for ultrafiltration to obtain a crude IgY solution, perform microfiltration to obtain a concentrated IgY solution, and prepare egg yolk globulin powder after freeze-drying.

[0058] 3. Resveratrol: purchased from Nanjing Hegu Life Biotechnology Co., Ltd.

[0059] 4. Helicobacter pylori (ATCC43504): purchased from Beijing Biobio Biotechnology Co., Ltd.

[0060] 5.BALB / c mice: SPF grade, male, 6-8 weeks old, purchased from Kaixue Biotech (Shanghai) Co., Ltd.

[0061] 6. TSB broth: purchased from Hibo Biotechnology.

[0062] Example 1. An anti-Helicobacter pylori composition

[0063] The number of viable bacteria of animal Bifidobacterium Bb-12 is 10 8 CFU / mL, the number of viable Lactobacillus acidophilus NCFM is 10 7 CFU / mL, the concentration of egg yolk globulin powder is 1mg / L, and the concentration of resveratrol is 12mg / L.

[0064] Example 2. An anti-Helicobacter pylori composition

[0065] The number of viable bacteria of animal Bifidobacterium Bb-12 is 10 8 CFU / mL, the number of viable Lactobacillus acidophilus NCFM is 10 7 CFU / mL, the concentration of egg yolk globulin powder is 1 mg / L.

[0066] Example 3. An anti-Helicobacter pylori composition

[0067] The number of viable bacteria of animal Bifidobacterium Bb-12 is 10 12 CFU / mL, the number of viable Lactobacillus acidophilus NCFM is 10 10 CFU / mL, the concentration of egg yolk globulin powder was 1 mg / L, and the concentration of resveratrol was 30 mg / L.

[0068] Comparative Example 1. An anti-Helicobacter pylori product

[0069] The concentration of resveratrol is 12 mg / L.

[0070] Experimental Example 1. Helicobacter pylori antibacterial detection

[0071] (1) Preparation of bacterial solution: Helicobacter pylori was cultured to the logarithmic phase, 5 mL of bacterial solution was taken, centrifuged at 5000 g for 5 min, and the bacteria were resuspended in TSB medium and the concentration of the bacterial solution was adjusted to 1×10 6 CFU / mL.

[0072] (2) Take a 96-well plate, add 100 μL of the bacterial solution in step (1) above, 95 μL of TSB medium, and 5 μL of the composition of Examples 1-3 and Comparative Example 1 to each well, and add 5 μL of DMSO solution to each well. Set 3 replicates for each treatment. Place the 96-well plate at 37° C. and shake at 150 rpm for 48 h.

[0073] (3) The OD600 value reflects the concentration of the bacterial solution, and the inhibition rate (%) is calculated according to the following formula:

[0074] (OD600 of control bacterial solution - OD600 of treated bacterial solution) / OD600 of control bacterial solution × 100%.

[0075] The test results are shown in Table 1 below.

[0076] Table 1

[0077] Group Antibacterial rate (%) Example 1 72.5 Example 2 57.9 Example 3 73.3 Comparative Example 1 5.2

[0078] It can be seen from the above table that the composition described in the present application can better exert the anti-Helicobacter pylori effect.

[0079] Experimental example 2.

[0080] (1) Model preparation

[0081] 42 mice were selected and given mixed antibiotic solution (ampicillin 10 mg / mL, gentamicin 1.2 mg / mL, azithromycin 10 mg / mL) by gavage, 0.3 mL / mouse, once a day, for 3 days. After gavage of mixed antibiotics for 7 days, mice were gavaged with 0.3 mL of freshly cultured Helicobacter pylori (1×10 9 CFU / mL), once every other day, for a total of 5 times. The mice were fasted for 24 hours before gavage and 2 hours after gavage. Two weeks after the last gavage, 2 mice were randomly selected and killed, and the gastric antrum tissue was taken, RUT reagent was added, and it was left to stand at room temperature. If the reagent turned red within 12 hours, it was positive, otherwise it was negative. At the same time, the gastric antrum-gastric body tissue was taken, fixed with 4% paraformaldehyde, embedded, sliced, and HE stained to determine whether the infection was positive. Both RUT and HE staining results showed positive, indicating that the model was successfully established.

[0082] (2) Grouping and drug administration

[0083] The mice with successful modeling were randomly divided into 4 groups, namely, the model group, the Example 1 group, the Example 2 group, and the comparative example 1 group, with 10 mice in each group. The mice without modeling were the blank control group (10 mice). The administration methods of each group were as follows:

[0084] Blank control group: no intervention measures were taken.

[0085] Model group: intragastric administration of normal saline, 100 mg / (kg·d), for 4 consecutive weeks.

[0086] Group 1 of Example 1: The mice were intragastrically administered with the composition described in Example 1, 100 mg / (kg·d), for 4 consecutive weeks.

[0087] Group 2 of Example 2 was intragastrically administered with the composition described in Example 2, 100 mg / (kg·d), for 4 consecutive weeks.

[0088] Comparative Example 1 group: gavage the composition of Comparative Example 1, 100 mg / (kg·d), for 4 consecutive weeks.

[0089] (3) Helicobacter pylori treatment results

[0090] After 4 weeks of administration, the mice were killed, gastric antrum tissue was taken, RUT reagent was added for staining, and the mixture was left to stand at room temperature. If the reagent turned red within 12 hours, it was positive, otherwise it was negative. Negative results indicated that Helicobacter pylori was completely eradicated. The test results are shown in Table 2 below.

[0091] Table 2

[0092] Group Total (pcs) Number of negative animals Eradication rate (%) Blank control group 10 10 - Model Group 10 0 - Example 1 10 10 100 Example 2 10 8 80 Comparative Example 1 10 0 0

[0093] It can be seen from the contents of Table 2 above that the composition described in the present application also has a significant inhibitory effect on Helicobacter pylori in vivo, can effectively eliminate Helicobacter pylori, and reduce the occurrence of gastric cancer.

[0094] Experimental Example 3. Human Experiment

[0095] In order to verify that the composition described in this application is indeed effective for patients infected with Helicobacter pylori, the composition described in Example 1 was added with 1 mg / mL of strawberry powder, 0.03 mg / mL of fucoidan, 5 mg / mL of dextrin, 0.2 mg / mL of mannitol, 0.2 mg / mL of starch, and 0.5 mg / mL of magnesium stearate, and prepared into tablets, which were orally administered at a rate of 2 g / person / day. The experiments were all conducted with the informed consent of the patients themselves.

[0096] 60 patients who received urea in hospital were selected 14 C] breath test, confirmed as positive for Helicobacter pylori. Age ranged from 30 to 70, with a male to female ratio of 34:26. ​​The above patients were randomly divided into two groups, one of which was the experimental group, which took the composition tablets described in this application orally; the other was the placebo group, which took the medicine without the composition described in Example 1 of this application orally, but with the same other ingredients as the tablets described in this application. After oral administration for 10-15 days, urea [ 14 C] Breath test.

[0097] After oral administration of the composition described in the present application, the experimental group was treated with urea [ 14 C] The breath test showed negative results, while the placebo group was still positive, indicating that the composition described in the present application can be used to treat Helicobacter pylori infection.

[0098] The following is a case in detail: Patient: Kou xx, male, 40 years old, was admitted to Yuci Clinic in Jinshui District, Zhengzhou City through urea[ 14 C] breath test detected C = 80, diagnosed as positive Hp (+), after taking the above tablets for 10 days, urea [ 14 C] The breath test detected C=38, which was diagnosed as positive Hp(-).

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A composition, characterized in that: The composition comprises: animal bifidobacterium Bb-12, lactobacillus acidophilus NCFM, egg yolk globulin powder and resveratrol; The viable count of the animal Bifidobacterium Bb-12 is 10 8 CFU / mL; The viable count of Lactobacillus acidophilus NCFM is 10 7 CFU / mL; The concentration of the egg yolk globulin powder is 1 mg / L; The concentration of resveratrol is 12 mg / L.

2. Use of the composition according to claim 1 in the preparation of an anti-Helicobacter pylori product, wherein the product is a medicine.

3. An anti-Helicobacter pylori product, characterized in that: The product comprises the composition according to claim 1, and the product is a medicine.