阅读说明：本技术 一种能够缓解类风湿性关节炎的罗伊氏乳杆菌组合物 (Lactobacillus reuteri composition capable of relieving rheumatoid arthritis ) 是由 陈卫 杨波 樊哲新 赵建新 张灏 于 2019-10-25 设计创作，主要内容包括：本发明公开了一种能够缓解类风湿性关节炎的罗伊氏乳杆菌组合物,属于食品微生物技术领域。本发明分别将罗伊氏乳杆菌CCFM8631和罗伊氏乳杆菌CCFM14活化、扩大培养,离心,重悬于30％的蔗糖溶液制备成浓缩生物制剂,经平板计数罗伊氏乳杆菌CCFM8631和CCFM14的活菌数均大于10<Sup>10</Sup>CFU/mL。本发明提供的罗伊氏乳杆菌组合物能够显著的缓解II型胶原诱导的类风湿性关节炎,缓解关节发红和肿胀症状,抑制抗II型胶原抗体的水平,降低血清中促炎细胞因子和抑炎因子的含量,降低Th1和Th2细胞相关的效应特征因子的水平,改善肠道功能,具有广泛的市场应用前景。(The invention discloses a lactobacillus reuteri composition capable of relieving rheumatoid arthritis, and belongs to the technical field of food microorganisms. The invention respectively activates, carries out enlarged culture and centrifugation on the lactobacillus reuteri CCFM8631 and the lactobacillus reuteri CCFM14, and then the lactobacillus reuteri CCFM14 is suspended in 30 percent of sucrose solution to prepare a concentrated biological agent, and the viable count of the lactobacillus reuteri CCFM8631 and the viable count of the lactobacillus reuteri CCFM14 are both more than 10 through flat plate counting 10 CFU/mL. The lactobacillus reuteri composition provided by the invention can obviously relieve type II collagen-induced rheumatoid arthritis, relieve redness and swelling of joints, inhibit the level of anti-type II collagen antibody, reduce the content of proinflammatory cytokines and anti-inflammatory factors in serum and reduce the content of proinflammatory cytokines and anti-inflammatory factorsThe levels of the effect characteristic factors related to the Th1 and the Th2 cells improve the intestinal function, and have wide market application prospect.)

1. The application of the Lactobacillus reuteri composition in preparing a rheumatoid arthritis product is characterized in that the Lactobacillus reuteri composition comprises Lactobacillus reuteri (Lactobacillus reuteri) CCFM14 and Lactobacillus reuteri (Lactobacillus reuteri) CCFM 8631.

2. The use according to claim 1, wherein the ratio of viable count of lactobacillus reuteri CCFM14 to lactobacillus reuteri CCFM8631 is 1:2-2: 1.

3. The use according to claim 1 or 2, wherein the concentration of the strains of lactobacillus reuteri CCFM14 and lactobacillus reuteri CCFM8631 is in the range of 10⁵-10¹¹CFU/mL or 10⁵-10¹¹CFU/g。

4. The use of claim 1, wherein the use comprises reducing the levels of pro-inflammatory cytokines IL-1 β, IL-6 and/or TNF α, and/or reducing the levels of anti-II collagen-specific antibodies CII-IgG, CII-IgG1, CII-IgG2a and/or CII-IgG2b, and/or reducing the levels of helper T cell effector factors ifny, IL-4 and/or IL-17A, and/or alleviating weight loss and/or reducing the degree of joint swelling.

5. The use of claim 1, wherein the product comprises a food, pharmaceutical or nutraceutical product.

6. The use according to claim 5, wherein the product is a medicament comprising Lactobacillus reuteri CCFM14 and Lactobacillus reuteri CCFM8631, a pharmaceutical carrier and/or a pharmaceutical excipient.

7. The use of claim 6, wherein the pharmaceutical product is in a dosage form comprising granules, capsules, tablets, pills or oral liquid.

8. The use of claim 5, wherein the product is a food, a fermented dairy product, a fermented soy product, or a fermented fruit and vegetable product, with the Lactobacillus reuteri composition as a starter.

9. The lactobacillus reuteri composition capable of relieving rheumatoid arthritis is characterized by comprising lactobacillus reuteri CCFM14 and lactobacillus reuteri CCFM8631, wherein the ratio of the number of viable bacteria of the lactobacillus reuteri CCFM14 to the number of viable bacteria of the lactobacillus reuteri CCFM8631 is 1:2-2: 1.

10. Use of the lactobacillus reuteri composition according to claim 9 in the pharmaceutical, health care or food field.

Technical Field

The invention relates to a lactobacillus reuteri composition capable of relieving rheumatoid arthritis, and belongs to the technical field of food microorganisms.

Background

Rheumatoid Arthritis (RA) is a chronic and systemic autoimmune disease with symmetric and persistent pain of the facet joints, which is characterized by synovitis and pannus formation in the early stage, then articular cartilage destruction, bone tissue inflammation, later stage joint sclerosis, significant narrowing or disappearance of joint cavities, and finally development of osteoarthritis, joint cavity stenosis or even disappearance, joint deformity and functional disability. Epidemiological investigation shows that about 0.5 to 1 percent of people worldwide suffer from rheumatoid arthritis, and the prevalence rate of China is about 0.32 to 0.36 percent. The incidence of rheumatoid arthritis disease is influenced by various factors such as genetics, living environment, sex, age, etc., wherein the incidence rate of male and female is about 1: 3, the incidence of the elderly is higher than that of the infants, and patients with periodontal diseases are more likely to suffer from RA than healthy oral people.

The exact etiology and pathogenesis of rheumatoid arthritis are not clear, generally, the body is considered to induce self-antigen formation after being stimulated by environmental factors under the genetic background, abnormal development and differentiation of T lymphocytes are activated to generate a large amount of proinflammatory cytokines, and meanwhile, B cells are generated from antibodies to destroy the immune tolerance of the body to trigger autoimmune reaction, so that the rheumatoid arthritis is a chronic inflammatory disease. After stimulation, T lymphocytes differentiate into different subsets of helper T cells, which secrete corresponding characteristic cytokines to exert their corresponding effects. The T cell sub-population related to rheumatoid arthritis is mainly Th1, Th2, Th17 and Treg cells, and the imbalance among the cells has important influence on the pathogenesis and is closely related to the disease degree. The imbalance of Th1/Th2 cells is one of the important causes of RA. Th1 and Th2 cells have different functions, Th1 mainly produces some proinflammatory cytokines such as IL-1 and IFN-gamma, Th2 can secrete IL-4, IL-10 and the like to play an inflammation inhibiting role, and a large amount of Th1 related cytokines are found in joint synovium, which indicates that Th1 cells mediate the pathogenesis of RA. In recent years, Th17/Treg cells are also newly found to be a key cell subset influencing the onset and development of RA, mainly because IL-17A and IL-22 secreted by Th17 cells can aggravate inflammatory response, and TGF beta and IL-10 produced by Treg secretion have the function of inhibiting immune activation. In addition, citrullination is also associated with rheumatoid arthritis pathogenesis. Citrulline is a non-standard amino acid in human body and cannot be synthesized in vivo, but arginine can be converted into citrulline under the action of Peptidyl Arginine Deiminase (PAD), and the PAD is a key catalytic enzyme for citrullination.

Numerous studies have shown that there is a close relationship between intestinal microorganisms and immune responses and rheumatoid arthritis, and the composition of feces and oral flora of patients with rheumatoid arthritis is significantly different from that of healthy people in clinical diagnosis and treatment, but the disordered flora tends to return to normal after drug treatment. It was also demonstrated in animal studies that the pathogenesis of idiopathic rheumatoid arthritis animals depends on the presence of both external and autologous intestinal flora, and that the disease cannot occur in sterile rats. The intestinal tract and intestinal mucosa are used as important immune sites of human bodies to participate in systemic immune response of the whole body, which can affect the characteristics of intestinal mucus layers, induce the development of intestinal lymphoid structures, mediate the production of inflammatory factors and regulate the differentiation of immune cells and the secretion of antibodies, and the immune effector molecules play an important role in the innate immune system through systemic circulation. In addition, intestinal tract bacteria are required to stimulate the development and differentiation of intestinal tract mucosal T lymphocytes. Therefore, the intestinal tract can be used as a target for treating RA, and a certain technical means is used for enabling intestinal flora of disordered rheumatoid arthritis patients to be recovered to be normal, improving local intestinal immune response and further relieving RA-related symptoms.

Currently, the clinical treatment of rheumatoid arthritis generally adopts drug therapy, such as non-steroidal anti-inflammatory drugs, antirheumatic drugs, glucocorticoid, immunosuppressant, novel biological agents and the like. The traditional drug therapy can only relieve pain to a certain extent and delay the disease process, but the gastrointestinal tract can be seriously injured after long-term administration, the novel biological agent has high price and potential risk of infection, and the defects limit the popularization and the use of the drug and can not effectively improve the disease condition. Therefore, there is a need to find a prophylactic or therapeutic approach that does not exhibit the limitations of drug-based therapies, is easy to implement, and is effective in alleviating symptoms and conditions.

Lactic acid bacteria, as a safe and edible food, have been widely used in the field of prevention or treatment of various diseases, such as prevention or treatment of gastrointestinal diseases, neurological diseases, alleviation of heavy metal toxicity, promotion of digestion and absorption, antagonism of infection by pathogenic bacteria, and enhancement of immunity. Therefore, the development of the probiotic product capable of effectively intervening the occurrence and development of the rheumatoid arthritis diseases has important social application value. Although there is a related patent application (publication No. CN104546947A) of lactobacillus crispatus for treating or relieving rheumatoid arthritis, lactobacillus crispatus is an inedible fungus; there are patents on Lactobacillus casei for the relief of rheumatoid arthritis (publication No. CN108926579A), but focus only on the relief of the arthritis-associated disease phenotype and the reduction of proinflammatory cytokines. Currently, in clinical or animal research, lactobacillus rhamnosus L.rhamnosus GG (LGG) is found to be effective in relieving rheumatoid arthritis. However, there is no research report on the occurrence and development of rheumatoid arthritis by lactobacillus reuteri.

Disclosure of Invention

The invention aims to provide a probiotic composition for regulating immune response and relieving rheumatoid arthritis. The lactobacillus reuteri composition can be applied to reducing the weight loss of rats with rheumatoid arthritis, reducing rheumatoid arthritis joint swelling, reducing the level of proinflammatory factors in serum, reducing anti-type II collagen specific antibodies, improving intestinal flora and improving the disease condition of rheumatoid arthritis diseases.

The first object of the invention is to provide the application of a Lactobacillus reuteri composition in preparing a rheumatoid arthritis product, wherein the Lactobacillus reuteri composition comprises Lactobacillus reuteri (Lactobacillus reuteri) CCFM14 and Lactobacillus reuteri (Lactobacillus reuteri) CCFM 8631.

The lactobacillus reuteri CCFM14 is purchased from China industrial microorganism strain preservation management center, is preserved in China industrial microorganism strain preservation management center in 23.05.2007, has the preservation address of No. 6 building China food fermentation industry research institute of No. 24 Hokkaido, Zhonglu, Hanxian, Zhao, Beijing city, and has the preservation number of CICC 6226.

The lactobacillus reuteri CCFM8631 is preserved in the common microorganism center of China microorganism culture preservation management committee on 07.07.2017, the preservation address is the microorganism research institute of China academy of sciences No. 3, West Lu No.1 institute of North Chen, Xilu, Chaoyang, Beijing, the preservation number is CGMCC NO.14394, and the patent application with the publication number of CN107523526A is disclosed.

In one embodiment of the invention, the ratio of the number of viable bacteria of the lactobacillus reuteri CCFM14 to the number of viable bacteria of the lactobacillus reuteri CCFM8631 is 1:2-2: 1.

In one embodiment of the invention, the concentration range of the strains of lactobacillus reuteri CCFM14 and lactobacillus reuteri CCFM8631 is 10⁵-10¹¹CFU/mL or 10⁵-10¹¹CFU/g。

In one embodiment of the invention, the use comprises reducing the level of proinflammatory cytokines IL-1 β, IL-6 and/or TNF α.

In one embodiment of the invention, the use comprises reducing the level of anti-II collagen-specific antibodies CII-IgG, CII-IgG1, CII-IgG2a and/or CII-IgG2 b.

In one embodiment of the invention, the use comprises reducing the levels of the T helper effector factors IFN γ, IL-4 and/or IL-17A.

In one embodiment of the invention, the use comprises reducing weight loss and/or reducing the degree of joint swelling.

In one embodiment of the invention, the product comprises a food, pharmaceutical or nutraceutical product.

In one embodiment of the invention, the product is a medicine, and the medicine comprises lactobacillus reuteri CCFM14 and lactobacillus reuteri CCFM8631, and a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment of the invention, the drug carrier comprises microcapsules, microspheres, nanoparticles, and liposomes.

In one embodiment of the present invention, the pharmaceutical excipients comprise one or more of solvents, propellants, solubilizers, solubilizing agents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, tonicity adjusting agents, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, penetration enhancers, pH adjusting agents, buffers, plasticizers, surfactants, foaming agents, antifoaming agents, thickeners, encapsulation agents, humectants, absorbents, diluents, flocculants and deflocculants, filter aids, excipients, additives, and release retardants.

In one embodiment of the present invention, the dosage form of the pharmaceutical product includes granules, capsules, tablets, pills or oral liquid.

In one embodiment of the invention, the product is food, and the lactobacillus reuteri composition is used as a leavening agent, fermented dairy products, fermented bean products or fermented fruit and vegetable products.

The second purpose of the invention is to provide a lactobacillus reuteri composition capable of relieving rheumatoid arthritis, wherein the lactobacillus reuteri composition comprises lactobacillus reuteri CCFM14 and lactobacillus reuteri CCFM8631, and the ratio of the number of viable bacteria of the lactobacillus reuteri CCFM14 to the number of viable bacteria of the lactobacillus reuteri CCFM8631 is 1:2-2: 1.

The invention also provides a preparation method of the lactobacillus reuteri composition, which is to activate, enlarge and culture the lactobacillus reuteri CCFM8631 and the lactobacillus reuteri CCFM14, centrifuge, and prepare the concentrated biological agent by suspending in 30 percent of sucrose solution.

In one embodiment of the invention, after lactobacillus reuteri CCFM14 and lactobacillus reuteri CCFM8631 are continuously activated for three generations in MRS culture medium respectively, the activated strain is transferred to the MRS culture medium for amplification culture in an inoculation amount (V/V) of 1-5%; respectively centrifuging the expanded cultures of the lactobacillus reuteri CCFM14 and the lactobacillus reuteri CCFM8631 (5000-.

The invention has the beneficial effects that:

the two strains of lactobacillus reuteri have high survival rate in simulated gastric juice and small intestinal juice in vitro, have good adhesion performance to Caco2 cells, and can tolerate extreme environments of intestinal tracts. The prepared lactobacillus reuteri composition can remarkably relieve rheumatoid arthritis, relieve redness and swelling symptoms of joints, inhibit the level of anti-type II collagen antibodies, reduce the content of proinflammatory cytokines in serum (the lactobacillus reuteri composition can respectively reduce the levels of IL-1 beta, IL-6 and TNF alpha in serum by 13.1%, 30.7% and 27.3%), reduce the levels of effect characteristic factors related to Th1 and Th2 cells, improve the composition of intestinal flora, and has wide market application prospect.

Drawings

FIG. 1: effect of lactobacillus reuteri composition on rheumatoid arthritis rats, a: body weight; b: a clinical score; c: the thickness of the joint.

FIG. 2: effect of lactobacillus reuteri composition on pro-inflammatory cytokines in rheumatoid arthritis rat serum, a: IL-1 β; b: IL-6; c: TNF alpha.

FIG. 3: effect of lactobacillus reuteri composition on anti-collagen II specific antibodies in rheumatoid arthritis rat serum, a: anti-CII-IgG; b: CII-IgG 1; c: CII-IgG2 a; d: CII-IgG2 b.

FIG. 4: the effect of the lactobacillus reuteri composition on an auxiliary Th1 cellular effector characteristic factor IFN gamma in the serum of a rat with rheumatoid arthritis.

FIG. 5: the effect of the Lactobacillus reuteri composition on an auxiliary Th2 cellular effector characteristic factor IL-4 in the serum of a rat with rheumatoid arthritis.

FIG. 6: the effect of the Lactobacillus reuteri composition on an auxiliary Th17 cellular effector characteristic factor IL-17A in the serum of a rat with rheumatoid arthritis.

FIG. 7: effect of lactobacillus reuteri composition on relative abundance of rheumatoid arthritis rat intestinal flora S24-7 family.

FIG. 8: effect of lactobacillus reuteri composition on the relative abundance of the enterobacteria Anaeroplasma genus in rats with rheumatoid arthritis.

FIG. 9: effect of lactobacillus reuteri composition on the relative abundance of the intestinal flora Clostridium in rheumatoid arthritis rats.

FIG. 10: effect of lactobacillus reuteri composition on relative abundance of the rheumatoid arthritis rat intestinal flora veillonella acease.

Detailed Description