TW201200142A - Lactobacillus isolates having anti-inflammatory activities and uses of the same - Google Patents

Abstract

Disclosed herein are two Lactobacillus isolates having anti-inflammatory activities and beneficial probiotic properties, i.e., Lactobacillus sakei GMNL-76 and Lactobacillus reuteri GMNL-89, which were deposited in the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) under accession numbers BCRC 910355 and BCRC 910340 and in the China Center for Type Culture Collection (CCTCC) under accession numbers CCTCC M 207153 and CCTCC M 207154, respectively. The two Lactobacillus isolates and their sub-cultured offspring can be used in the preparation of a variety of food products, and in the manufacture of pharmaceutical compositions for treating and/or alleviating diseases associated with inflammation, such as rheumatoid arthritis.

Description

201200142 VI. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to two strains having anti-inflammatory activity (ant i _ i nf 1 ammat〇ry act j V iti eS) and beneficial beneficial probiotic properties. Lactobacillus isolate {Lactobacinus iscAotes) 'Ishibited, /> I lactobacillus (j^ctobacillus sakef) GMNL-76 and Lactobacillus reuteri rei/ier7·) qjnl-89, respectively, with the accession number BCRC 910355 and BCRC 910340 are deposited with the Center for Bioresource Conservation and Research (BCRC) of the Food Industry Development Research Institute, (FIRDI), and deposited with the China Type Culture Collection (CCTCC) under the registration numbers CCTCCM 207153 and CCTCC Μ 207154. These two strains of Lactobacillus and their sub-cultured offspring can be used to prepare a variety of food products' and for the manufacture of diseases associated with the treatment and/or alleviation of inflammation. (treating and/or alleviating diseases associated with inflammation) [pharmaceutical compositions such as rheumatoid arthritis]. [Prior Art] Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes swelling of joints such as hands, feet, wrists, elbows, knees, and joints. Swelling), deformity, pain, atrophy, and stiffness. Chronic arthritis will gradually erode normal joint bone cells for a long time, causing joint deformation and eventually loss of function. In addition, rheumatoid arthritis can also invade parts other than the joints, such as glands (such as salivary gland, lacrimal gland, and lymph gland), organs (such as the liver, Spleen, heart and lungs, etc. or systems (such as the circulatory system and the nervous system), which in turn cause rheumatoid vasculitis, pleuropulmonary manifestations [including interstitial fi bros is, P1 europu 1 monary nodu 1 es, pneumon itis and arteritis, and Felty's syndrome. 201200142 The age of onset of rheumatoid arthritis mainly falls between 5 years of age, while the incidence of women is 2 to 4 times that of men. To date, the exact cause of rheumatoid arthritis is still unknown, but previous studies have shown: genetic predis〇 siti〇n, envirOmnental trigger, autoimmune events (aut〇immune events) ), chronic inflammation, cell trafficking, and treatment may be associated with immunopathogenesis of rheumatoid arthritis. The main lesion of rheumatoid arthritis is Syn〇vial tissue. There are no more than 3 layers of synovial cells in normal joints, but there is a significant increase in the joints of arthritic patients. Rheumatoid arthritis may be caused by a patient's immune cells that abnormally attack their own collagen type II (CII) of cartilage and synovial tissue. Patients with rheumatoid arthritis can be detected with collagen type II-specific autoantibodies and/or rheumatoid factor. The rheumatoid factor is an autoantibody derived from a defect in glycosylation in the Fc domain, of which IgM is most common and IgG and IgA are also found. Rheumatoid factor can be detected in approximately 80% of patients with rheumatoid arthritis, and the immune complexes formed by binding to the original antigens are deposited in the synovium, cartilage or blood vessels. Causes lesions. It is known that cytokines involve many important biological processes (bi〇i〇gicai processes)' including: inflamination, tissue repair, cell growth, fibrosis, jk tube Angiogenesis, and an immune response. Therefore, cytokines play an important role in autoimmune diseases. In M. Feldmanneia A (1996), such as /7"./Shi 7" Ru Ren, 14:397-440 in this retrospective paper, Μ·Feldmann et al. analyzed the synovial tissue of rheumatoid arthritis ( The expression and regulation of cytokines in synovial tissue explores the pathogenesis of cytokines, which are classified into the following four categories: (1) proinflammatory cytokines; (2) immunomodulation Sex cytokines (i deleted unoregulatory cytokines); (3) chemotactic cytokines (chemotactic 201200142 cytokines), and (4) mitogenic cytokines (mitogenic cytokines) and the like. The inflammatory cytokine is a group of T-helper 1 cells (Thl cells (Thl. 6115)) that can regulate delayed allergic reactions (1邳6(147?〇hypersensitivity) The molecules of the reaction include: interleukin-1 (IL-1), interferon-r (IFN-r), tumor necrosis factor-α (TNF-α), and granules. The granulocyte-macrophage colony stimulating factor (GM-CSF), etc. The cartiiage destruction observed in rheumatic diseases has been generally ought to be Caused by the activity of matrix metalloproteinases (MMPs), macromolecular and fibroblasts that are activated and activated by pro-inflammatory cytokines such as IL-1 or TNF-α. (fibroblasts) are produced. M. Feldmann et al. further mention that IL-1 or TNF-a is injected into col lagen-immunized mice or rats. Or local injection of IFN-τ into Dijon Collagen immunization (c〇i lagen type ΙΙ-inmunized) in the soles of mice accelerates the development of arthritis and increases the severity of the disease (M. Feldmarm ei a/. (1996), supra). Immunoregulatory cytokines [also known as anti-inflammatory cytokines] are a group of T-helper 2 cells (Th2 cel Is) Molecules capable of inhibiting inflammatory responses, including IL-4, IL-10, IL-13, and Transforming Growth Factor-/3 (TGF-cold), etc. in G. Garcia ei 5 (1999) , c?/ As in the toilet 13:315-324, G. Garcia et al. mentioned that TGF-/3 and IL-10, two immunoregulatory cytokines, not only inhibit pre-inflammatory cells that induce MMPs. Hormone production also induces the production of natural inhibitors of MMPs [ie, tissue inhibitors of matrix metalloproteinases (TIMPs)]. G. Garcia et al. further mentioned that IL-10 is widely known to inhibit the production of IFN-7 from Th1 cells and inhibit the production of various cytokines from other white blood group (ieuk〇cyte populations). IL-10 inhibits the production of il-1, IL-6, TNF-α, IL-8 and G-CSF (granulocyte-colony stimulating factor) from macrophages and inhibits more The nucleated cells (p〇lymorph〇nuciear ceus) produce IL-1, TNF, IL-8, macrophage inflammatory protein ία (macr〇phage infiammat〇ry protein 1 α, MIPloO and macrophage inflammatory protein 1/5 ( Macr〇phage inflammatory protein 10, MIP1/?), and most of these cytokines and chemokines are involved in the pathological process of arthritis (G. Garcia ei a) (1999), same as above. These findings show that proinflammatory cytokines associated with Th1 cells (such as TNF-α and IFN-r) promote inflammation and worsen rheumatoid arthritis In addition to inhibiting the production of pro-inflammatory cytokines, immunoregulatory cytokines (such as IL-10 and IL-4) associated with Th2 cells can also reduce cartilage destruction by inducing the production of TIMPs. The drug can effectively cure rheumatoid Arthritis, so the drugs used clinically can only focus on preventing inflammation and destruction of joints and other tissues or organs, repairing damaged joints to relieve pain, and maintaining joint function and preventing deformation. The treatment mode can be divided into Drug therapy, surgery, rehabilitation, exercise therapy, diet control, etc. Drug treatment is mainly for the treatment of inflammatory symptoms, and the drugs commonly used to treat rheumatoid arthritis can be summarized into the following five categories (Xie Tiaoyang and Chen Peiyu (2〇〇1), “Treatment of Rheumatoid Arthritis”, Journal of Pharmaceutical Sciences, 17(4): 109-116): 1. Biological response modifier (bi〇l〇gicai response m〇difiers) , wake up s): for example, etanercept (trade name EnbreO and infliximab (trade name is Remicade*), they are anti-TNF agents' Specific binding to TNF-α: to inhibit the biological activity of TNF-α, thereby reducing the inflammatory phenomenon caused by TNF-α; 2. Nonster〇idal anti_inflammat〇ry dnjgs, NSAIDs: for example Aspirin and ibuprofen (ibupr〇fen), these drugs have anti-inflammatory and analgesic effects, so they can be used to alleviate long-term or short-term pain and inflammation in patients with rheumatoid arthritis; Type anti-rheumatic drugs ((}丨56 batch 6-1110(^7丨呢如_|:丨1^11贴'(^(11*呢3, DMARDs): eg efeminomide (commodity) Named Arava·), methotrexate, penicillamine, hydroxychloroquine, and gold compound, which are suitable for rheumatoid arthritis patients who do not respond to NSAIDs. . DMARDs delay the disease process by regulating abnormal immune responses that cause rheumatoid arthritis, and the use of such drugs for treatment must be monitored by a physician to avoid side effects; 4. Cortic〇steroids: These drugs are very effective in treating rheumatoid arthritis, but they have many side effects. Corticosteroids are usually administered in oral or injectable dose forms, but frequent injections of corticosteroids can cause softmoons. Therefore, the number of injections is limited to one to two times a year. Currently, oral prednisone is used. Prednisone) is the most commonly prescribed prescription for physicians; and 5. Others: for example, hyaluronic acid [such as sodium hyaluronate (trade name: Hyalgan*)] and its derivatives [such as Hylan G_F 2 〇 (trade name is Synvisc*)]. Hyaluronic acid is the main component of articular canilage and synovial fluid. Therefore, intra-articular injection of hyaluronic acid can stimulate the proliferation of chondrocytes to protect and lubricate joints and reduce inflammation. . The selection of drugs for the treatment of rheumatoid arthritis is generally based on NSAIDs with weaker efficacy and higher safety as the first line of treatment. If the treatment effect is not significant, then the effect is stronger. DMARDs are used as a second line. When neither of these drugs fails to achieve the desired effect, corticosteroids or surgery are considered. Although the drug has the effect of relieving pain, stiffness and inflammation, long-term use may cause insomnia (ins〇mnia) 'sonitus, edema, stomach ulcers, stomach bleeding (gastroirhagia), gastric perforation Side effects such as (gastrobrosia), skin sensitivity, albuminuria, diabetes, hypertensi〇n, osteoporosis, cataract, and resistance to infection, Blood biochemistry and serum tests are required during the treatment to ensure the effectiveness of the treatment and to avoid the above-mentioned side effects. To date, there has not been a drug which can effectively treat or alleviate rheumatoid arthritis without causing undesirable side effects. Food and Agriculture organization of the 201200142

United Nations, FA0) and the World Health Organization (WHO) define probiotics as: a living microorganism that, when administered in an appropriate amount, confers a health benefit to the host ( Guidelines for the Evaluation of Probiotics in Food, Joint FA0/WH0 Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food, April 30 and May 1, 2002). There are many types of microorganisms currently available as probiotics, such as Lactobacillus, Jifidobacteriuni, Chrysanthemum, Enterococcus (and, Yeasts, Streptococcus, etc.). Etc. Especially the species of the first two genus.

Eli Metchnikoff proposed in 1908 that harmful putrefying bacteria present in the gastrointestinal tract (GI tract) are inhibited or antagonized by acid-producing (^7i 1908) , The Prolongation Of Life, Ed. P. Chalmers Mitchell, GP Putnam, s Sons, The Knickerbocker Press, Your Steps. 'A lot of research and clinical trials have confirmed that there is an important correlation between lactobacilli and health. Lactobacillus is a group of Gram-positive anaerobic bacteria (gram-p0SHive facultative anaerobe). They are ubiquitous in the human gastrointestinal tract and vagina, they are able to ferment sugars and use lactic acid as the main metabolite. The efficacy of Lactobacillus has been found to include: (1) improving the bacterial phase balance of the intestinal microflora; (2) preventing diarrhea; (3) reducing colon cancer (c〇i〇n cancer) Risk; (4) stimulate the normal development and function of the gastrointestinal epithelial mucosal system; (5) produce various vitamins ( Vitamins) and nutrients; and (6) prevention and treatment of vaginosis.

Based on the beneficial probiotic properties of Lactobacillus 'Researchers in the field are working to develop a Lactobacillus isolate having a desired biological activity. In this respect, reference can be made, for example, to TW 5881〇9 to disclose Lactobacillus rhamnosus/* such as gamma^5) Tcell-Ι and its use. TW 1241912 reveals 6 strains of novel acid-tolerant and gallbladder-resistant Lactobacillus strains with abiity to lower and assimilate cholesterol, which are 201200142 and Γ?Γ21Ή, B21T6, C21T1, X21B7 and B38T38 and Lactobacillus acidophilus CLΒ6Τ7. TW 1283268 discloses Lactobacillus rhamnosus r/zs/amse) GM-020 and its use for the treatment of obesity. TW 1284149 discloses the use of Lactobacillus paracacia paracas'e/) GMNL-32 and its treatment for allergy-related diseases. In addition, TW200611973 reveals Lactobacillus fermentum / e / we / 7 plus ") GM-090 can effectively stimulate the secretion of scare 7 and / or treatment of allergies 0 In addition 'Jae-Seon So et al reported: Lactobacillus lactis (Zacto Plus Oral administration of £7/7/iAS case/) inhibits collagen-induced arthritis (c〇llagen-in (juced arthritis, CIA) and reduces paw swelling, lymphocyte infiltration, and cartilage tissue Destruction of cartilage tissue. Lactobacillus casei reduces type I collagen-reactive pre-inflammatory molecules through CD4+ T cells (IL-1 cold, IL-2, IL-6, IL-12, IL- 17, IFN-r, TNF-α and Cox-2). Lactobacillus lactis administration also reduces NF- /c B to nuclear translocation and Cl I-reactive Thl-type IgG isotype (CII- Reactive Thl-type IgG isotypes) IgG2a and

IgG2b' simultaneously rises to regulate immune il-10 levels (jae-Se〇n Soeia (2008), shot / side 7" / 7 〇, 45 (9): 2690-2699; Epub 2008 Feb 19). If rheumatoid arthritis can achieve therapeutic effects by taking Lactobacillus isolates, it should provide patients with a safe and inexpensive drug. To this end, the Applicant screened two Lactobacillus isolates from native adult gastrointestinal tract samples, which are phyl 〇 tical 是 是 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 〇 Stimulation produces large amounts of IL_1〇 and a relatively small amount of anti-inflammatory activity of ifn_T and/or TNF-α and is therefore expected to be useful in the treatment of diseases associated with inflammation, including, but not limited to, rheumatoid arthritis. SUMMARY OF THE INVENTION In a first aspect, the present invention provides an isolate having a anti-inflammatory activity of Lactobacillus species (10) and a detailed knowledge, wherein the isolate is: 201200142 ( 0 — selected from the following deposited strains: (4) Lactobacillus sakae (Lactokcz·//ez·) GMNL-76, which is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute under the registration number BCRC 910355, and It is deposited with the China Type Culture Collection under the registration number CCTCC Μ 207153; and (b) Lactobacillus reuteri (10) gmnL-89, which is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Research Institute under the registration number BCRC 910340. And is deposited with the China Type Culture Collection under the registration number CCTCC Μ 207154; or (11) the subcultured offspring of the deposited strain in subparagraph (i) (sub_cukured 〇ffspring). In the first aspect The invention provides a food product (f〇〇d pr〇duct) comprising a isolate of Lactobacillus species as described above and an edible material. In one aspect, the present invention provides a pharmaceutical composition useful for treating and/or alleviating a disease associated with inflammation, which comprises an isolate of a milk-sucking species as described above. In a fourth aspect, the present invention provides A method for treating a disease associated with inflammation of a subject comprising administering to an individual in need of such treatment an isolate of Lactobacillus species as described above. The above and other objects of the present invention The features, advantages and advantages of the invention will become apparent from the following detailed description and the accompanying drawings. It is quoted that the previous publication does not constitute the following recognition: in Taiwan or any of its side towels, the former publication forms part of the general knowledge in the art. For the purposes of this specification, It is clearly understood that the text "comprising" means that "including but not limited to" and "text" including (_prises) have a corresponding meaning. All technical and scientific terms used herein have the meaning as commonly understood by those skilled in the art to which the invention pertains. ... lyophilic Μ妓 龄 龄 龄 system system system system system system system system system system system system system system system system system system system system system system system system system system system system system system system system system system However, 201200142 is currently not clinically effective in the treatment of rheumatoid arthritis, and long-term use of these drugs can cause undesired adverse side effects. In addition, previous studies indicate: pre-inflammatory properties associated with Th1 cells Cytokines such as IFN-γ and TNF-α promote inflammation and exacerbate the condition of rheumatoid arthritis, while immunoregulatory cytokines associated with Th2 cells (such as 11^1〇 and 11^4) It can inhibit the formation of proinflammatory cytokines and reduce the cartilage destruction by inducing the production of TIMPs. Therefore, it is inferred that the treatment of rheumatoid arthritis may be achieved by regulating the secretion of immunoregulatory cytokines and proinflammatory cytokines. Lactic acid bacteria 'In particular, bacterial strains 〇f the genus (10)) are well-known and widely used probiotic microbes, which have been proven to have many beneficial hosts ( Host) health effects, such as: improving the bacterial phase balance of the intestinal microbiota, preventing diarrhea, reducing the risk of colon cancer (c〇i〇n cancer), stimulating the normal development and function of the gastrointestinal epithelial mucosal system, Produce a variety of vitamins and nutrients and prevent and treat vaginosis. Therefore, the applicant has attempted to find a Lactobacillus isolate having advantageous anti-inflammatory activity from among lactic acid bacteria regarded as probiotic microorganisms for the treatment of rheumatoid arthritis. Therefore, the applicant used the gastro〇intestinal traet specimens from healthy adults in Taiwan as the isolated source of the Lactobacillus isolate, and the obtained isolates were separately from the monocytes of the experimental animals. Co-culture to stimulate monocytes to secrete cytokines' and then use IL-10 and IFN-γ as screening markers, and screened to stimulate monocytes to secrete large amounts of IL-1 〇 and less Two strains of Lactobacillus isolates GMNL-76 and GMNL-89 of lFN-γ and/or TNF-α, which were individually identified and assigned to Lactobaci Uus sakei as anti-royce milk Lactobacillus reuteri ° Lactobacillus sphaeroides GMNL-76 and Lactobacillus reuteri GMNL-89 have been deposited in Taiwan on June 14, 2007 and November 14, 2006 under the registration numbers BCRC 910355 and BCRC 910340 respectively. Food Industry Research and Development Institute (F1RDI) Biosource Collection and Research Center (BCRC) (300 Hsinchu Food Road 33 No. 1, Taiwan). The two isolates are also £12 201200142. According to the Budapest Treaty, they were deposited with the China Center for Type Culture Collection (China Center) on November 19, 2007 under the registration numbers CCTCC Μ 207153 and CCTCC Μ 207154 respectively. For Type Culture Collection (CCTCC) (Wuhan, Wuhan University, 430072, People's Republic of China) When the Lactobacillus sphaeroides GMNL-76 and Lactobacillus reuteri GMNL-89 of the present invention are fed to a collagen-induced joint In rats with collagen-induced arthritis, an increase in IL-10 in the serum of rats and a decrease in IFN-γ and TNF-α were observed. This shows the Lactobacillus isolates GMNL-76 and GMNL- of the present invention. 89 can make the joint inflammation of the rat no longer deteriorate. Compared to the conventional strains Lactobacillus flavus BCRC 12933 and BCRC 17500 and Lactobacillus reuteri BCRC 14625, BCRC 16090, BCRC 16091, BCRC 17476 and BCRC 17478 'Lactobacillus sphaeroides GMNL-76 and Lactobacillus reuteri GMNL The ability of -89 to stimulate the secretion of IL-10 by monocytes in vitro is better than that of the conventional strains of their respective species. Based on the above advantageous biological activities, the two strains of Lactobacillus isolates of the present invention or their subcultured progeny are expected to have potential for treating a disease associated with inflammation. Accordingly, the present invention provides a pharmaceutical composition for treating a disease associated with inflammation comprising: (i) at least one deposited strain selected from the group consisting of: (4) Lactobacillus sphaeroides GMNL-76' No. BCRC 910355 is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute, and is deposited with the China Type Culture Collection under the registration number CCTCCM207153; and (b) Lactobacillus reuteri GMNL-89, which is hosted No. BCRC 910340 is deposited with the Bioresource Conservation and Research Center of the Food Industry Development Institute and is deposited with the China Center for Type Culture Collection under the registration number CCTCCM207154; or (ii) the host strain of the deposited strain in subparagraph (i) Generation of offspring. The invention also provides a method for treating a disease associated with inflammation in a subject, comprising administering to an individual in need of such treatment - a milk thistle isolate as described above or a subcultured progeny thereof. According to the present invention, the disease associated with inflammation is selected from the group consisting of rheumatoid arthritis, osteoarthritis (oste〇arthritis), juvenile rheumatoid arthritis (4) such as heart rfiemnatoid arthritis) 'Ankylosing spondylitis (_1〇_ sp〇ndyMs), spondylitis 13 201200142 (spondylitis), psoriatic arthritis, cr〇hn's disease, ulcerative colitis Psoriasis (Eveline Trachsel ei <2/. (2007), Arthritis Research & Therapy, 9(1): R9, Published online 2007 January 29. doi: 10.1186/ar2115; Suchita Nadkami et al. (2007) , The Journal of Experimental Medicine, 204: 33-39; Richard 0 Williams et al. (2007), Current 7: 412-417). In a preferred embodiment of the invention, the disease associated with inflammation is rheumatoid arthritis. The pharmaceutical composition according to the present invention can be formulated into a pharmaceutically acceptable vehicle by using the technique known to those skilled in the art, and then preparing the above-mentioned Lactobacillus isolate or its subcultured progeny. A dosage form suitable for oral administration is prepared. In a preferred embodiment of the invention, the pharmaceutical composition is in a dosage form selected from the group consisting of a solution (s〇luti〇n), a suspension (susPension), an emulsion, and a powder ( Powder) ' tablet, pill, syrup, lozenge, tablet (tr〇che), chewing gum, capsule, thick Slurry and the like. As used herein, the term "pharmaceutically acceptable carrier," means a carrier that, when administered, does not cause an allergic reaction or other undesired effect in the administered subject. In the present invention, the sigma pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of a solvent emulsifier, a SUSpending agent, a decomposer, and a binder ( Binding agent)' exdpient, stabmzing g chelate & chelating agent, diluent, gelling agent, preservative, lubricant Gubricant In addition, the Lactobacillus sphaeroides GMNL-76 and the Lactobacillus reuteri GMNL-89 of the present invention are also developed, which have bile-tolerant and gastric acid-tolerant properties, and thus are suitable as gastrointestinal tract probiotics. For example, a first-class detached plant or its subcultured offspring can be used as a food additive component. (IV) i - can be added by the S-known method during the preparation of the raw material, or added after the fermentation process without participating in the fermentation, and with any Edible materials are formulated for Human and non-human __ money food products. This invention also provides a food product comprising - a Lactobacillus isolate as described above or a subcultured progeny thereof and an edible material. Edible materials of the present invention include, but are not limited to, fluid milk products such as milk (miik), concentrated milk, fermented milk, such as yogurt (y0gurt), g 〇〇ur milk, yoghurt yoghurt, lactic acid bacteria-fermented beverages; milk powder; ice cream; milk Cream cheeses; diy cheeses; soybean milk, fermented SOybean miik; vegetable-fruit juices; fruit juices, sports drinks (Sp0rts drinks); desserts (confectionery); fruit; jellys; candies; infant formuias; heath foods; animal feeds; dietary supplements, and the like. Further, the food product according to the present invention can be manufactured into an instant brewed food (instant £6〇(1) containing a lyophilized or spray-dried bacterial powder which can be directly consumed. Forms. For the preparation of food products, see, for example, 1^6,872,565 82,1; 8, 7,244,424 B2, US 7,270,994 B2, US 7,172,777 B2 and US 6,872,411 B. The food product according to the invention may further comprise at least one probiotic microorganism (probiotic microbes). As used herein, 'probiotic microorganisms, or probiotics, can be exchanged for use' and mean the preparation of active microorganisms (uve microorganisms). When ingested by a human or animal, 'the microorganisms can remain and survive in the gastrointestinal tract' and can provide prevention and treatment of the disease. Probiotic microorganisms suitable for use in the present invention include, but are not limited to, : Lactobacillus species (LactokcW/us less.), Bifidobacterium species (tolerance), Staphylococcus species, yeast (yeasts) and their Preferably, the Lactobacillus species is selected from the group consisting of Lactobacillus acidophilus, Lactobacillus lactis (LactobacWus hc, Lactobacillus Hus hdveticm, short milk) Lactobacmus brevis, Lactobacillus casei (Z^GamόααΥ/w·? coyez·), Lactobacillus plantarum 2CZ·//(10)p/ as you tear), Lactobacillus salivarius iWvank), Lactobacillus bifidum/(d) , Bulgarian Lactobacillus bulgaricusy Lactobacillus rhamnosus, Lactobaci Uus gasseri, 15 201200142 and combinations thereof. Preferably, the Bifidobacterium species is It is selected from the group consisting of: double-forked double-formed MBifidobacterium bifidum), long-type double-bacteria (Bifld〇bacterium l〇ngumy infant-type double-bacteria (and dirty/«/如怡), short type Bifidobacterium 6reve), young bismuth ac/o/asce ribs. $), Bifidobacterium 丨actis, and their tactics. Preferably, the Streptococcus species is selected from The following group Group: Streptococcus thermophilus (•S^e; plus ccwwi recognize mw卬/»7 milk), Streptococcus mutans (&reptoCOCC(10)(6)), Streptococcus cremoris, Streptococcus diacetyhatis ) to reverse their combination. Preferably, the yeast is selected from the group consisting of Candida albicans (CoTitZ/c/a Τ Τ), Flora yeast (iSaccZ/iWOwy/cas, Saccharomyces cerevisiae) (''''''''''' The illustrations are not to be construed as limiting the implementation of the invention. Example 1 - Preliminary screening of Lactobacillus isolates with anti-inflammatory activity Experimental materials and methods: A. Source and preparation of test strains: In February, the applicant obtained a number of healthy adult gastrointestinal tract samples from the China Medical University Hospital (Taichung City, Taiwan) and isolated more than 400 strains of Lactobacillus isolates from these samples. For probiotics having potential for the treatment of rheumatoid arthritis, Applicants have performed anti-inflammatory activity assays on these Lactobacillus isolates and used IL-10 as a screening marker. Each test strain was inoculated to Bactobacillus MRS meat. Tang Pei The base (Bact〇MRS BR〇TH) (DIFCO, Cat. No. 0881) was then anaerobicly cultured at 37 ° C for 12 to 18 hours. The resulting bacterial culture was 4, 〇〇〇φΐη Centrifuge for 15 minutes, remove the supernatant 201200142 solution, and the precipitate (1) (IX) phosphate buffered saline (PBS) was washed a total of 3 times 'and then dispersed in ιχ pBS The formed bacterial solution was adjusted to a concentration of 1~5xl09 cells/mL in IX PBS (the number of bacteria was counted by 〇D6G(), 〇D60〇=1.〇〇= 5.〇xl08 cells/mL) It is used as a stock solution (st〇ck solution) for 10-fold serial dilution, and produces 1〇8, 1〇7, 1〇6, ι〇5, ι〇4, ι〇3, 〇2, ι〇ι cells/mL of test bacterial solution for subsequent experiments. B. Preparation of mouse spleen monocytes: sacrificed at C〇2 for 6 to 8 weeks of age Female BALB/c mice's spleens were removed and ground with a sterilized glass rod. The spleen tissue after grinding was compared to Ficoll-PaqueTM PLUS (17-1441-03, Amersham Biosciences) in a volume ratio of 1:1 (v lume rati〇) by density gradient centrifugation (Density

Gradient centrifbgation) (720 g X 20 min at 4 ° C). Thereafter, the red blood cells were removed, and the thus administered mouse spleen spleen cells were adjusted to a cell concentration of 4χ106 cells in RPMI 1640 medium (RPMI 1640 medium) containing 10% fetal bovine serum (FBS). mL spare. C. Evaluation of Lactobacillus isolates stimulating secretion of il-10 by spleen mononuclear cells in mice: Add spleen mononuclear cell samples prepared in the above B items to each well of the 96 well plate. And divided into 3 groups to be separately added: (1) test group, 20 μί of the test bacterial liquid prepared in the above item A (lxlO7 cells/mL); (2) normal control (normal control), 20 pL containing 10% FBS RPMI 1640 medium; and (3) positive control, 20"T, lipopolysaccharides (LPS) [E. coli co" serotype 055: B5, Sigma]. After incubation in an incubator (37 ° C, 5% CO 2 ) for 24 hours, the culture solution in each well was centrifuged '1 μ μ μ μ from the collected supernatant for use with Mouse IL-10 OptEIATM Set (BD Biosciences, Cat. No. 555252) enzyme-linked immunosorbent assay (ELISA). The experiments in each group were repeated twice. The concentration of IL-10 was measured by the ELIS A test. The 〇D4〇5 value is substituted into the following formula to calculate and is in ELISA unit (%) Indicates: ELISA unit (%) = [(A-C)/(B - C)] X100 where: A = 405D405 value of Lactobacillus isolate 17 201200142 B==odd value of positive control group c == normal control Group ΟΕ) 4 () 5 numerical results: Two numbers in all test ages '46 strains of spleen mononuclear cells secreted IL-10, their experimental results are shown in Table 1., Table 1 .Evaluation of the secretion of IL-10 by spleen mononuclear cells from the isolates of Lactobacillus isolates ______ Test strain number ELISA Unit (%) Experiment 1 Experiment 2 ----nMNL-11 74.4616 69.7510 95.3230 104.7443 --^ nMNL-19 75.8075 64.3674 -^nMNL-22 71.0969 112.1467 -^?SlNL-27 155.2153 157.2342 ^^7^INL-28 266.2517 277.0188 ^^GMNL-32 147.8129 152.5236 -^^nMNL-33 151.1777 143.1023 ^nMNL-34 30.7201 34.757740 "^7iMNL-35 61.6756 34.0848 -^^iiMNL-36 34.0848 36.7766 ^ilMNL-37 45.5249 39.4683 one ^nMNL-38 36.1036 33.4118 ----i^MNL-39 44.8520 44.1790 ----nMNL-41 87.9206 95.3230 - ---fiMNL-42 98.6878 95.3230 ^nMNL-43 157.9071 151.8506 ^^nMNL-44 69.75101 54.9462 -^^nMNL-45 273 .6541 186.8439 -^^MNL-47 31.3930 28.7012 ----GMNL-50 223.1830 168.001 ----nMNL-55 174.7308 161.9448 -^TiMNL-57-I 148.4859 153.8694 •^?iMNL-57-II 230.5855 268.2705 -- ---nMNL-67 242.6985 215.7806 ---"llMNL-68 114.1655 91.9583 -^^OMNL-69 128.9704 113.4926 A GMNL-76 167.3284 146.4670

S 18 201200142 GMNL-78 198.9569 221.1642 _ GMNL-84 73.7887 75.1346 GMNL-87 32.7389 34.7577 GMNL-88 40.8143 46.8708 . _ GMNL-89 54.2732 1 54.2732 GMNL-94 237.3149 225.2019 GMNL-97 "166.6555 176.7497 GMNL-101 355.7537 395.4576 _ GMNL -126 151.1777 141.7564 GMNL-127 196.9381 H 178.7685 GMNL-128 682.1332 450.6393 GMNL-130 498.4186 293.8425 GMNL-131 341.6218 — 480.9219 _ GMNL-132 435.1615 361.8102 GMNL-133 302.5909 268.9435 GMNL-138 672.0390 446.6016 GMNL-139 400.8412 403.5330 GMNL-161 304.6097 339.6030 According to the experimental results shown in Table 1, and considering the growth rate, the number of bacteria and the practicality of the 46 strains, the applicant selected GMNL_19, gmnl_76, GMNL_78 'GMNL-89 ' GMNL_94 'GMNL-101 and GMNL-161 for further sputum testing to assess our ability to stimulate IL-10 and IFN-γ by human peripherai blood mononuclear cells (human PBMCs) Example 2. Lactobacillus isolates stimulate human PBMCs to secrete IL1 (^IFN々) Estimated experimental materials and methods: A. Preparation of human peripheral blood mononuclear cells: Human whke blood cell concentrate (Taiwan Blood Donation Center) and Fic〇U_paqUeTM plus (17-1441- 03, Amersham

Biosciences) was subjected to density gradient centrifugation (72 〇 g x 2 〇 min, substituting) in a volume ratio of 1:1. Thereafter, the 'red blood cells were removed' and the human PBMCs thus obtained were adjusted to a cell concentration of 4χΐ〇6 cells/ RPMI 1640 medium containing 10% FBS. Evaluation of B. Lactobacillus isolates stimulating IL-10 secretion by human PBMCs: Lactobacillus isolates GMNL-19, GMNL-76, GMNL-78, GMNL-89, GMNL-94, 201200142 gmnl-ioi and GMNL-161 stimulate humans The ability of the four halls to divide the account is generally as described in the "Experimental Materials and Methods," item C "Evaluation of the secretion of IL-10 by mouse spleen monocytes stimulated by the Lactobacillus isolate" of Example 1. The procedure was evaluated, except that a test broth having a concentration of 1×10 〇8 cells/mL was used as a test group, and a sample of human PBMCs prepared in the above item A of 1〇〇jjL was used. And Human a few _1 ptmATM (bd

Biosciences, Cat. No. 555157). Evaluation of C. lactis isolates stimulating secretion of IFN-γ by human pbmCs: Lactobacillus isolates GMNL-19, GMNL-76, GMNL-78, GMNL-89, GMNL-94, GMNL-101, and GMNL-161 stimulate human PBMCs The ability to secrete IFN-[gamma] is analyzed as described below. Each well of the 96 well plate was added with 1 sample of the human PBMCs prepared in the above item a and divided into two groups to be separately added: (1) Test group, 2〇 & Example “ "Experiment Materials and Methods" Item A "Source and preparation of test strains, test bacterial liquid prepared in the test (1χ1〇7 cells/mL), and (2) normal control, 20 μί of 10% RPMI 1640 medium of FBS. After culture for 24 hours in an incubator (37 ° C ' 5 ° / 〇 C02), the culture solution in each well was centrifuged and taken out from the collected supernatant for 1 〇〇. Use Bd

IFN-γ ELISA of OptEIATM Human IFN-γ ELISA Kit II (BD Biosciences, Cat. No. 550612). In addition, 'human PBMCs samples were added at a ratio of 4 pg/mL 106 cells/mL to 10 pg/mL of lentil lectin (P/ziweo/wi vw/gans agglutinin, PHA) (Sigma, Cat. No, L2769). The cultivation lasted for 48 hours. After centrifugation (720 g x 20 min at 4 ° C), the supernatant was stored in a -80 ° C refrigerator for use. When subjected to IFN-γ ELISA analysis, the PHA-treated supernatant (100) Μί) was used as an internal positive control.

100-fold diluted 100 times with a coating buffer (0.1 Μ Na2HP〇4, 0.77 mM NaN3, pH 9.0) using a microdispenser (jL mouse anti-human IFN-γ (mouse anti) -human IFN-γ, BD PharmingenTM, Cat. No. 551221) was added to each well of a 96 well plate (Nunc-ImmunoTM 96 MicroWellTM Plates, MaxiSorp, Cat. No. 442404) at room temperature (25 The plate was shaken at 40 rpm for 1 hour at ° C., and then the plate was placed at 4 ° C overnight. S 20 201200142 After that, the plate was warmed and the liquid in each well was removed, and then washed. Washing buffer (PBS containing 0.05 ° / Tween 20) to clean each well 2 times (3 minutes each time), then add 200 μιη blocking buffer (containing 3%) to each well Bovine Serum Albumin (BSA) in PBS], and left at room temperature for 2 hours, then remove the blocking buffer and wash twice with washing buffer. Add to each well and react overnight at 4 ° C. After that, remove the liquid from each well to clean The solution was washed twice, and then diluted with a diluent buffer (PBS containing 1% BSA) 2000 times of 1 μg biotin mouse anti-human IFN-γ (biotin mouse anti-human IFN) - γ, BD PharmingenTM, Cat. No. 554550) was added to each well and allowed to react at room temperature for 2 hours. After washing twice with washing buffer, 1 稀释 diluted 2000 times with dilution buffer was added. 〇pL streptavidin-Alkaline phophatase (Streptavidin-AP, BD PharmingenTM, Cat·

No. 554065) ' and reacted at room temperature for 1 hour. After removing the liquid in each well, it was washed 4 times with a washing buffer, and then 1 〇〇 pL of freshly prepared p-Nitrophenylphosphate (pNPP) solution was added to each well. The plate was placed in the dark at room temperature for a color reaction for 30 minutes. Thereafter, the absorbance of each well at a wavelength of 405 nm (〇D4Q5) was read by an ELISA Microplate Reader (Bio-Rad, Model 550). The experiments of each group were repeated twice. The concentration of IFN-γ was calculated by substituting the 〇D4〇5 value measured by the ELISA test into the following formula and expressed in ELISA units (%):

ELISA unit (%) = [(A-C)/(B - C)] xlOO where: A = 405D405 value of Lactobacillus isolate B = 〇D405 value of internal positive control group C = 〇D405 of normal control group Numerical results: Lactobacillus isolates GMNL-19, GMNL-76, GMNL-78, GMNL-89, GMNL_94, GMNL-101 and GMNL-161 stimulate the secretion of IL-10 and IFN-γ by human peripheral blood mononuclear cells. They are shown in Table 2 and Table 3, respectively. 21 201200142 Table 2. Efficacy of Lactobacillus isolates to stimulate secretion of human peripheral blood mononuclear cells. Evaluation of Lactobacillus isolate number ELISA Unit (%) Experiment 1 Experiment 2 GMNL-19 26.5578 27.5374 UMNL-76 56.3810 57.7959 GMNL- 78 50.1769 56.0544 GMNL-89 104.3810 118.3129 GMNL-94 45.1701 47.5646 GMNL-101 54.7483 54.3129 GMNL-161 44.4082 52.5714 Table 3: Lactobacillus isolates stimulate human peripheral blood mononuclear cells to secrete Ν Ν γ γ Efficacy evaluation Lactobacillus isolate number ELISA Unit (%) Experiment 1 Experiment 2 GMNL-19 137.2893 124.8361 GMNL-76 92.9073 103.6751 _ GMNL-78 36.1657 35.3230 GMNL-89 64.6302 59.9485 GMNL-94 105.2669 89.9111 GMNL-101 152.9260 157.6077 GMNL-161 205.0796 215.0047 It can be seen from Table 2 Among all Lactobacillus isolates, Lactobacillus isolates (}1^^_76 and GMNL-89 stimulated the highest number of IL_1〇 secreted by human peripheral blood mononuclear cells, indicating that these two isolates are stimulating human peripheral blood mononuclear cells. The ability of cells to secrete IL_1〇 is optimal. See also from Table 3, in all Lactobacillus isolates, milk Bacterial isolates (}]^^_78 stimulated human peripheral blood mononuclear cells to secrete Π? Ν γ, the lowest number, while GMNL_89 and gmnl_76, this shows that the ability of Lactobacillus isolate GMNL_78 to stimulate human peripheral blood mononuclear cells to secrete IFN_y It is the worst, and the Lactobacillus isolates GMNL-89 and GMNL-76 are also poor in stimulating the secretion of IFN-γ by human peripheral blood mononuclear cells. Pre-inflammatory cytokines such as IFN-γ and TNF- are known. α) promotes inflammation and worsens the condition of rheumatoid arthritis, and immunoregulatory cytokines such as IL_1〇 can inhibit the production of proinflammatory cytokines. The applicant concludes that if it can stimulate humans Or animal monocytes secrete more immunoregulatory cytokines and fewer pro-inflammatory cytokines, which should be beneficial to improve the condition of humans or animals with RA. According to Table 2 and Table 3 As a result, the applicants considered that the Lactobacillus isolates GMNL-76 and GMNL-89 were the most promising strains of the strain 22 201200142, and the following animal tests were carried out. Evaluation of therapeutic efficacy of L-89 on rats with collagen-induced Example 3. Lactobacillus isolate gmnL-76 and GMN arthritis Experimental materials and methods: A. Experimental animals: obtained from the National Experimental Research Institute Male LEW/S_ar丨 rats (6-week old 'body weight of approximately 200 to 250 g) from Animal Center _〇η&1 AppUed Ubomtories Mtional Labomtory Animal Center) were used in the following experiments. All experimental animals were housed in an independent air-conditioned animal room with light and dark for 2 hours, room temperature at 25 ± 1〇c and humidity maintained at 60 ± 5%, and the water and feed were adequately supply. Animals were given a period of one week to adapt to the environment and the experiment was started after the animals were stabilized. The handling of the experimental animals and all experimental procedures were carried out in accordance with the standard regulations of the Laboratory Animals Committee. B. Preparation of test strain: Lactobacillus isolates GMNL-76 and GMNL-89 were separately inoculated into Bact 0 Lactobacillus MRS broth medium (DIFCO, Cat. No. 0881) at 37. (: Anaerobic culture to saturated growth state. After 15 minutes of centrifugation at 3,000 g, the precipitate was washed twice with 2 mL and 1 mL of IX PBS (ρΗ7·2), and then 1 PBS was added. The concentration of the measured bacteria solution was measured by OD coffee, and the results were all about 1.0x1010 cells/mL. The bacterial solution (IX PBS) was stored frozen at -8 〇. (: Next standby. C. Arthritis induction: The induction of arthritis is carried out with reference to the method described in Y. Kameyama ei α/. (2004), βο/ie, 3 5:948-956' and slightly modified. Bovine π-type collagen (bovine [ >^11(;〇113吕邱' is abbreviated as €:11) (8丨 layer 1113- into 1£11^11, 匚31.>1〇.(:1188) dissolved in 0.05 river acetic acid solution Prepare a CII solution with a concentration of 2 mg/mL. Add 100 μL of Complete Freund's Adjuvant (CFA) to 100 μL of CII solution to prepare a CII emulsion. After that, 200 pL of CII will be prepared. The emulsion was injected subcutaneously into the tail of the rat to make the first immunization' and boosted again after 21 days of immunization. D. Feeding of Lactobacillus isolate: Rat They were randomly divided into 4 groups (n = 6 per group) including 3 experimental groups [GMNL-76 group, 23 201200142 GMNL-89 group and placebo group] and a control group. Rats in each of the other groups were induced to develop arthritis according to the method described in the above-mentioned item C "Induction of arthritis." For the GMNL-76 group and the GMNL-89 group, from the 7th day after the booster immunization The rat was prepared by the preparation of the above-mentioned formula S (test cell preparation) (1. 〇χΐ. cells/day/day). As for the placebo group and the control group, they were countered by meals. Permeate water. After 8 weeks of continuous feeding, 'the blood of the rat was collected by orbitalbleeding and centrifuged' and the resulting serum was used to determine IL-i〇, iFN-γ and TNF-α. In addition, each group of rats subjected to ocular blood sampling was further used for the following arthritis evaluation. E. Determination of the dominance of IL-10, IFN-γ, and TNF-α:

The concentration measurement of IL-10 is generally an operation procedure described in "Experimental Materials and Methods," item c "Evaluation of IL-10 secretion from mouse spleen mononuclear cells by the Lactobacillus isolates" of Example 1. To make an estimate, but use the rat serum obtained in item D above and Rat iL-i〇〇ptEiATM

Set (BD Bioscience PharMingen, San Diego, CA, Cat. No. 2611KI).

The concentration measurement of IFN-γ is generally evaluated by referring to the procedure described in "Experimental Materials and Methods," item c "Evaluation of Lactobacillus isolates stimulating secretion of IFN-γ by human PBMCs" of Example 2, but Use the rat serum obtained in item D above and the Rat IFN_y 〇ptEIATM Set (BD)

Bioscience PharMingen, San Diego, CA) 浓度 TNF-α concentration determination is generally referred to the "Experimental Materials and Methods," c-item 'Lactobacillus isolates of Example i to stimulate the secretion of IL-10 by mouse spleen monocytes The evaluation procedure described in the ', the evaluation procedure described above was used, but the rat serum obtained in item D above and the BD 〇ptEIA Rat TNF-α ELISA Kit (BD Bioscience PharMingen, San Diego, CA) were used. The measured OD4 〇 5 values are then based on a correlation curve previously determined with respect to their own 8-values of IL-lO'IFN-γ or TNF-α standards having different known concentrations. It is expressed as a concentration (pg/mL). F. Eva丨uation of arthritis: Each group of rats that were subjected to ocular blood collection was used for the following arthritis assessment, and the joints of the rats concerned. The inflammation assessment was carried out in accordance with the method described in Y. Kameyama ei d (2004), 35: 948-956. Briefly, changes in redness, swelling, and the like of the four paw joints of the rats were recorded, s 24 201200142 And use the following five kinds of scores as the difference Basis: scored 〇, did not show any arthritis symptoms, 1 point, 1 toe swelling; score 2, more than 3 toe swelling; score 3, the entire sole of the foot swelling; and score 4, all The joints were severely swollen and could not walk normally and were butterfly-like. G•Statistical analysis method: This example used SPSS statistical software (version 10.0) for statistical analysis. The total number of samples of experimental animals was less than 30′. The experimental data were analyzed by n〇nparametric statistical methods [also known as Mann-Wittney u test (Μαηη_·^γ u test)] to evaluate the control group, GMNL_76 group and GMNL_89 group. Differences in the sample were compared with the placebo group. The experimental data were expressed as mean soil deviation values (statistical significance, 0.05).Results: Serum of each group of rats after 8 consecutive weeks of feeding The concentrations of iL-1, lFN-γ, and TNF-a in the respective are shown in Table 4 and Figures 1 to 3. In addition, the scoring results of the arthritis evaluation are also summarized in Table 4. Table 4 .1L_10, Li-Li's Concentration Analysis Project in Rat Serum Group placebo group GMNL-76 GMNL-89 IL-10 (pg/mL) 4.56±5.50 6.22+4.811 11.78+3.747 12.44+5.232 IFN-γ (pg/mL) 40.60±4.243 46.78±52.64 12.20± 10.923 25.00+ 26.482 TNF-a (pg/mL) 3.32+1.757* 39.80+1.998 9.44±3.988* 12.20+6.643* Score of Guanlangyan degree 0* 12.33311.155 11.67+2.563 13.42±2.155 1. All data is in Mann - Mann-Whitney U test for analysis. 2: The score of the degree of arthritis is expressed as the sum of the scores of the four paw joints of the rat. 3: * indicates p < 0.05 compared to placebo. From Table 4 and Figure 1 to Figure 3, it can be seen that the rats in the GMNL-76 group and the GMNL-89 group were fed with the lactobacillus isolates GMNL-76 and GMNL-89, respectively, and their serum IFN-γ and TNF. The -α concentration was significantly lower than the placebo group, while the il-10 concentration was significantly increased. Therefore, the applicant's inference: Lactobacillus isolates GMNL-76 and GMNL-89 can reduce the secretion of TNF-a in addition to IFN-γ secreted by Th1 cells, and reduce the amount of TNF-a secreted. 25 201200142 The amount of protease (MMPs) secreted, so that the cartilage in the joint tissue will not continue to be destroyed. In addition, the Lactobacillus isolates GMNL-76 and GMNL-89 can also increase the secretion of the immunoregulatory cytokine IL-10, and the inflammation is controlled. Therefore, although the joint damage caused by arthritis has not recovered, by feeding the Lactobacillus isolates GMNL-76 and GMNL-89 of the present invention, lFN-γ and TNF- associated with inflammation in the serum of rats. There has been a decrease in alpha, and there is an increase in IL-10 associated with anti-inflammatory, which means that the arthritic condition of the rat can be alleviated and no longer deteriorates. Example 4 Characterization of Lactobacillus isolates GMNL-76 and GMNL-89 In order to confirm the strains of the Lactobacillus isolates GMNL-76 and GMNL-89 selected in the above examples, the following preliminary tests were carried out. , 16SrDNA sequence analysis and random amplified Random Amplified Polymorphic DNA (RAPD) analysis. Experimental materials and methods: A. Preliminary test: Preliminary tests were carried out on Lactobacillus isolates GMNL-76 and GMNL-89. The test items included gram staining, morphological observation, hydrogen peroxide. Enzyme reaction, mobility, growth under aerobic and anaerobic conditions, etc. B. 16S rDNA sequence analysis: Lactobacillus isolates GMNL-76 and GMNL-89 were separately inoculated into 1 mL of Lactobacillus Bactobacterium MRS broth (DIFCO, Cat. No. 0881) under sterile conditions. Incubate overnight at 37 °C. Thereafter, the bacterial solution was centrifuged at 13, 〇〇〇 φιη for 丨 minute, and then the supernatant was removed. The remaining precipitate was added to 2 centrifugation ddH20 to fully disperse the cells, and the supernatant was removed by centrifugation at i3,000 rpm for 1 minute, and this step was repeated several times. Finally, the precipitate obtained by centrifugation was dispersed by 2 〇〇... dclH2〇, which contained genomic DNA of the Lactobacillus isolate. The resulting genomic DNA was used as a template and was designed using a set of (10) rDNA for Lactobacillus published in 5> § M. Yeung (2002), j, and Du (8) (6). The primer having the nucleotide sequence shown below was subjected to polymerase chain reaction (PCR) using the primers 26 201200142 and 536R primers of the primer platform below using the reaction conditions shown in Table 5 below. PAF primer 5'-agagtttgatcctggctcag-3' (SEQ ID NO: 1) 536R primer 5'-gtattaccgcggctgctg-3' (SEQ ID NO: 2) Table 5. Reaction conditions of PCR Content volume (μ基因 Lactobacillus isolate genome) DNA 0.5 PAF primer (ΙΟμΜ) 1 536R primer (1〇μΜ) 1 dNTPs (2.5 mM) 1 10X Ex Taq buffer (1〇χ Ex Taq buffer) (TaKaRa) 5 TaKaRa Ex TaqTM (TaKaRa) (5 υ/ Ίί) 0.2 Water 41.3 Operating conditions: denaturation at 94 ° C for 30 seconds, primer anneaiing at 51 C for 3 sec seconds, at 72. Elongation) lasted 30 seconds 'total 30 cycles. After Yu Yuancheng PCR' by ι·8% agarose gel electrophoresis (agar〇se gei eiectr0ph〇resis) to discern whether it has a size The product was amplified by 500 bi^ PCR, and the confirmed PCR product was purified from the gel. The purified PCR product was commissioned by Kiron Meikes Biotechnology Co., Ltd. (Taiwan) for sequencing. The resulting sequencing result is the use of nucleotides in the NCBI website. BLAST (nucleotide-nucleotide BLAST) software for alignment analysis C. Random Amplified Polymorphic DNA (RAPD) analysis: Identification of Lactobacillus isolates by 16S rDNA sequence analysis (}_1^76 After the GMNL_89 species, the genomic DNA of the Lactobacillus isolates GMNL_76 and GMNL_89 obtained in the above B items was used as a template, and one was selected and published in 乂DE ANGELIS“ (2〇〇1), 27 201200142

Applied and Environmental Microbiology, 67: 20-mer primer 10-c primer with the ~f face shown as the nucleotide sequence of Lac P2 for PCR reaction using the reaction conditions shown in Table 6 below .

LacP2 primer 5^atgtaacgcc-3' (SEQ ID NO: 3) Table 6. Reaction conditions for PCR Content volume &L) Genomic DNA of Lactobacillus isolate 1 LacP2 primer (1 μμΜ) 0.5 dNTPs (2.5 mM) 0.5 10X Ex Taq Buffer (TaKaRa) 2.5 TaKaRa Ex TaqTM (TaKaRa) (5 υ/μΕ) 0.2 Water 20.3 Operating Conditions: Denaturation at 93 ° C for 1 minute, at The primer adhesion was carried out at 36 ° C for 1 minute, and the elongation reaction was carried out at 74 ° C for 1 minute for a total of 35 cycles. After the completion of the PCR, the amplified product was subjected to electrophoresis using a 1.8% agarose gel, followed by staining, and then observed under ultraviolet light and photographed. In this experiment, seven Lactobacillus strains purchased from the Center for Biological Resource Conservation and Research (FCRC) of the Food Industry Development Research Institute (FIRDI) were used as comparative strains for comparative analysis. Lactobacillus Shakespeare BCRC 12933 (corresponding to ATCC 31063, isolated source is sauerkraut); 2. Lactobacillus sinensis succulent subspecies like moxibustion (4) λ 17500 (corresponding to LMG 17302, isolated source is raw sausage); 3. Lactobacillus reuteri (Zactokci/ZiAs rewfcn·) BCRC 14625 (corresponding to ATCC 23272 and DSM20016, isolated from human feces); 4. Lactobacillus reuteri (Zac7/Shao reMteri) BCRC 16090 [corresponds to DSM 20015, isolated source is manure]; 28 201200142 5. Lactobacillus reuteri BCRC 16091 (corresponding to DSM 2〇〇53, isolated source is human feces); 6. L. reuteri BCRC 17476 (corresponding to JCM ι〇8ι, isolated source is chicken intestine); and 7. L. reuteri (X(10) is (10)·//(10)BCRC 17478 [corresponding to JCM 2762, the source of separation is fermented molasses] 〇- Result: Lactobacillus isolate GMNL Characterization of -76: (i) According to the preliminary test results, the isolate is Gram-positive bacillus 'not motility, not catalase, no oxidase, under aerobic and anaerobic conditions Both can be grown; (9) The 16S rDNA sequence analysis results of this isolate are shown in Figure 4, and after comparison with the gene database in the NCm website, the 16S rDNA sequence of this isolate was found (SEQ ID NO: 4) The sequence similarity with the (10) rDNA of Lactobacillus sakazaki ([(10) with (10)·//(10) as (4) is the highest; and ((1)) The results of the rapd analysis of this isolate are shown in Figure 5. As can be seen from Figure 5, this isolate The gene fingerprint of the PCR product is different from that of the other two conventional strains of Lactobacillus sakazaki. The result of the above-mentioned clock is that the Lactobacillus isolate of the present invention is considered to be a new one. Lactobacilhis sakei isolate. 2. Characterization of Lactobacillus isolate gm]sjl_89: (1) According to the preliminary test results, the isolate is a Gram-positive bacterium, not motility, and does not have a enzyme. No oxidase, in both aerobic and anaerobic environments Growth; (8) 16S rDNA; f; column analysis results of this isolate are shown in Figure 6, and the 16S _α sequence (sequence identification number.5) of the mixed data in the website. ) The highest similarity with Roy's milk (4) _; and the results of RAPD analysis of the detached stalks are shown in circle 7. As can be seen from Fig. 7, the genetic fingerprint of the PCR product of this isolate is different from that of the other five conventional Lactobacillus reuteri strains 29 201200142. Based on the above identification results, the Lactobacillus isolate GMNL-89 of the present invention is considered to be a novel isolate of Lactobacillus reuteri rewien·). The Lactobacillus sinensis idez·) GMNL-76 and the GMNL-89 of L. reuteri have been deposited in the registration number BCRC 910355 and BCRC 91034 on June 14, 2007 and November 14, 2006, respectively. Center for the Conservation and Research of Biological Resources (FCRC) of the Food Industry Development Research Institute (FIRDI) (300 Food Road, No. 33, Hsinchu City, Taiwan). The two isolates were also deposited with the China Center for Type Culture Collection (CCTCC) under the Budapest Treaty on November 19, 2007, under the registration numbers CCTCC Μ 207153 and CCTCC Μ 207154 (Wuhan, Wuhan). University, 430072, People's Republic of China). Example 5. Evaluation of Lactobacillus isolates GMNL-76 and GMNL-89 and conventional strains in stimulating the secretion of IL-10 by mouse spleen monocytes

In order to demonstrate that IL-10, which is capable of stimulating the secretion of more monocytes, is a biological activity peculiar to the Lactobacillus isolates GMNL-76 and GMNL-89, in the following examples, they were taken with 7 conventional strains ( Also known as Lactobacillus freundii BCRC from the Food Industry Development Institute

12933 was compared to BCRC 17500 and Lactobacillus reuteri BCRC 14625, BCRC 16090, BCRC 1609, BCRC 17476 and BCRC 17478). Experimental Materials and Methods: The concentration determination of IL-10 is generally an evaluation of the secretion of IL-10 by mouse spleen mononuclear cells stimulated by the Lactobacillus isolate of "Experimental Materials and Methods" of Example 1. The procedure described was used for evaluation ' and experiments were carried out using the Lactobacillus isolates GMNL-76 and GMNL-89 of the present invention and the above 7 conventional strains. The measured 〇〇4〇5 values are then based on the correlation & line (a>rrelation) previously made with IL-10 standards of different known concentrations relative to their own 〇D4〇5 values. Curve) is expressed as a concentration (pg/mL). The experiment of each strain was repeated twice, and the experimental data was expressed as the mean soil deviation value. Results·· Table 7 below shows the results of GMNL-76, GMNL-89, and each comparative strain stimulating IL-10 secretion by mouse spleen monocytes. 201200142 Table 7. Lactobacillus isolates GMNL-76 and GMNL-89 and Comparison of the efficacy of strains in stimulating the secretion of IL-10 by spleen monocytes in mice. Strain number IL-10 (pg/mL) GMNL-76 274.164±9.173 GMNL-89 311.07±11.79 BCRC 14625 231.52±11.78 BCRC 16090 155.35±12.81 BCRC 16091 160.874±6.13 BCRC 17476 292.86124.97 BCRC 17478 232.269±16.385 BCRC 12933 250.996±3.672 BCRC 17500 266.958il2.759 It can be seen from Table 7 that among all the test strains, the Lactobacillus isolate GMNL-89 of the present invention is known from the prior art. The strain BCRC 17476 has the best ability to stimulate the spleen monocyte fraction & IL_1〇 in mice, and the GMNL-76 times the experimental results shown in Table 7 again demonstrate the Lactobacillus isolate GMNL-89 of the present invention and GMNL-76 is not identical to the conventional strains of their respective species. Example 6. Resistance to sputum and gallate tolerance of Lactobacillus sphaeroides GMNL-76 and Lactobacillus reuteri GMNL-89 to confirm the Lactobacillus sphaeroides GMNL-76 and Lactobacillus reuteri of the present invention Whether gmNL-89 survives the harsh conditions of the digestive tract after ingestion, an experiment that simulates the continuous digestion of the human digestive tract is performed. Experimental material: L MRS broth (MRSbroth) (pH=3): Dissolve 55 g of MRS powder (BD, Cat. Να 288130) in 1 L of reverse osmosis water and mix well, then 丨M HC1 The pH was adjusted to 3 ' and then sterilized at 12 rc for 15 minutes' after cooling. 2. mrs broth medium containing 0.2% (w/v) bovine bile: 31 201200142 Dissolve 55 g of MRS powder in 1 L of reverse osmosis water and mix well, then sterilize at 121 ° (: 15) After a minute, the temperature was lowered to about 45 ° C, 2 g of ox gall powder (Sigma) was added and mixed well to obtain a MRS broth medium containing 0.2% (w/v) bovine bile. Then filter it with a filter membrane with a pore size of 0.45 μm. 3. MRS agar plate: Dissolve 55 g of MRS powder in 1 L of reverse osmosis water, and add 15 g after complete dissolution. Agarose powder' then poured the resulting mixture into a 1 l serum bottle and sterilized at 121 ° C for 15 minutes. After that, the temperature was lowered to 45 ° C under aseptic conditions. Next, add about 15 to 20 mL of MRS agar solution to each petri dish, and wait for cooling and solidification. Experimental method: A. Acid tolerance test: 3 mL is implemented Example 1 "Experimental Materials and Methods, 'A" Test The test bacterial solution obtained from the source and preparation of the strain was inoculated into 27 mL of MRS broth medium (pH==3) and thoroughly mixed, and the resulting culture was cultured at 37 ° C for 3 hours. After that, the culture was pulled out and the serial dilution plate (10-9~10-ι) was sterilized with water to detect the number of surviving bacteria. B. Bile salt tolerance test: After the end of the acid resistance test of item A above, 'the remaining 29 mL of the culture was centrifuged at 4, rpm for 15 minutes, the supernatant was removed and 30 mL of sterile water was added to disperse the cells. Thereafter, 4 Centrifuge at rpm for 15 minutes and remove the supernatant to wash away the acidic mrs broth medium. After that, add 30 mL of %^ containing 〇.2./〇(w/v) bovine bile. The broth medium was used to disperse the cells, and then the culture formed was placed at 37. The culture was carried out for 3 hours under the armpits. Thereafter, the culture was taken out and serially diluted with sterilized water (1〇·9~) 1〇-1) to detect the number of surviving bacteria.. Mining: Lactobacillus sphaeroides GMNL-76 and Lactobacillus reuteri GMNL_89 after simulation The growth after continuous '/shortage treatment of the human digestive tract is shown in Table 8 below. Cell concentration (CFU/mL) of the I-__ bacterial solution in the human bacterium of the genus Dictyozoa 32 201200142 Cultured in MRS broth medium (pH = 3) for 3 hours in MRS broth medium containing 0.2% bovine bile for 3 hours before the numbering treatment * GMNL-76 3.76x108 1.83x105 6.3x105 GMNL-89 k · iL 1.9x109 6.83x108 1.2x109: The isolates were previously cultured in MRS broth medium (pH=3) for 3 hours. As can be seen from Table 8, when the two Lactobacillus isolates of the present invention were cultured with MRS broth medium of ΡΗ=3 for 3 hours, the viable count of Lactobacillus sphaeroides GMNL-76 decreased as compared with that before treatment. 99.95〇/〇' and the viable count of L. reuteri GMNL-89 decreased by about 64%. Then, the two Lactobacillus isolates were cultured in MRS broth containing 0.2% bovine bile for another 3 hours, and the number of viable cells did not decrease, and the survival rates of both were extremely excellent. This result shows that Lactobacillus sphaeroides GMNL-76 and Lactobacillus reuteri GMNL-89 can overcome the pressure of the human digestive tract, and can effectively reach the intestine and colonize after ingestion (c〇l〇nizati〇n ) in the intestines. All documents and patent documents cited in the present specification are incorporated herein by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail. While the invention has been described with reference to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows that rats with collagen-induced arthritis were collected and continuously incubated after 8 weeks of continuous feeding of the Lactobacillus isolates GMNL-76 and GMNL-89 of the present invention. As a result of the ELISA test, rats without arthritis and fed with reverse osmosis water and rats with arthritis but fed with reverse osmosis water were used as a control group and a placebo group, respectively; The results of ELISAs of iFN-γ were collected in rats subjected to collagen-induced arthritis after continuous feeding of the M. lactis isolates GMNL-76 and GMNL-89 of the present invention for 8 weeks. Rats without arthritis and treated with reverse osmotic water by silver and rats with arthritis but fed with reverse osmosis water were used as control group and placebo group respectively; 33 201200142 Figure 3 shows collagen with The rats of the induced arthritis were collected after continuous feeding of the Lactobacillus isolates GMNL-76 of the present invention and GMNL-89 for 8 weeks, and the results of the ΤΝΡ-α ELISA test were carried out without arthritis. Being fed Rats with reverse osmosis water and rats with arthritis but fed with reverse osmosis water were used as a control group and a placebo group, respectively; Figure 4 shows the 16S rDNA nucleotides of the Lactobacillus isolate GMNL-76 of the present invention. Sequence; Figure 5 shows the polymorphism of the Lactobacillus isolate GMNL-76 of the present invention and two conventional genomic DNAs of Lactobacillus sakazaki BCRC 12933 and BCRC 17500 as templates and using LacP2 primers for random amplification. DNA (Random Amplified Polymorphic DNA, RAPD) analysis, followed by electrophoresis on a 1.8% agarose gel, and the obtained photographic results were observed under ultraviolet light, wherein in the region (A), the diameter M1: DNA ladder mark ( DNALadder) (100-3000 bp), diameter 1: GMNL-76; in the (B) region, diameter M2: DNA ladder marker (100-3000 bp), diameter 2: BCRC 12933; and in the (C) region, diameter M3: DNA ladder marker (100-3000 bp), diameter 3: BCRC 17500; Figure 6 shows the 16S rDNA nucleotide sequence of the Lactobacillus isolate GMNL-89 of the present invention; and Figure 7 shows the Lactobacillus of the present invention, respectively The isolate GMNL-89 and 5 known Lactobacillus reuteri BCRC 14625, BCRC 16090, BCRC 1609 The genomic DNA of BCRC 17476 and BCRC 17478 was used as a template and the Lac P2 primer was used for RAPD analysis, followed by 1.8 °/. After electrophoresis on an agarose gel, the obtained photographic results were observed under ultraviolet light, wherein in the region (A), the diameter M1: DNA ladder mark (100-3000 bp), diameter 1: GMNL-89; B) In the region, 'M2: DNA ladder marker (100-3000 bp), diameter 2: BCRC 14625; in the region (C), diameter M3: DNA ladder marker (100-3000 bp), diameter 3: BCRC 16090; In the (D) region, the diameter M4: DNA ladder marker (100-3000 bp), diameter 4: BCRC 16091; in the (E) region, the diameter M5: DNA ladder marker (l〇〇_3〇〇〇bp) , Path 5: BCRC 17476; and in the (F) region, diameter M6: DNA ladder mark (100-3000 bp), diameter 6: BCRC 17478. [Main component symbol description] (none)

S 34 201200142 Sequence Listing &lt;110&gt; Jingyue Biotechnology Co., Ltd. <120> Lactobacillus isolate having anti-inflammatory activity and use thereof &lt;130> GMNL-76 and GMNL-89 &lt;160&gt; 5 &lt;170&gt ; Patentln version 3.4 &lt;210&gt; 1 &lt;211> 20 &lt;212&gt; DNA <213> Artificial &lt;220> &lt;223> PAF primer for 16S rDNA for PCR amplification of Lactobacillus &lt;400&gt; Agagtttgat cctggctcag &lt;210> &lt;211&gt;&lt;212><213&gt;&lt;220>&lt;223>&lt;400&gt; 2 18

DNA artificial 536R primer for 16S rDNA for PCR amplification of Lactobacillus 2 gtattaccgc ggctgctg &lt;210> 3 &lt;211&gt; 10 &lt;212> DNA &lt;213>Artifical &lt;220> &lt;223&gt; PCR-amplified 10-member primer for Lactobacillus gene &lt;400> 3 atgtaacgcc &lt;210&gt; 4 201200142

&lt;211&gt; 535 &lt;212> DNA &lt;213> Lactobacillus sakae sa々e/) GMNL-76 &lt;400> 4 gggggggggt gctatacatg caagtcgaac gcactctcgt ttagattgaa ggagcttgct 60 cctgattgat aaacatttga gtgagtggcg gacgggtgag taacacgtgg gtaacctgcc 120 ctaaagtggg ggataacatt tggaaacaga tgctaatacc gcataaaacc taacaccgca 180 tggtgtaggg ttgaaagatg gtttcggcta tcactttagg atggacccgc ggtgcattag 240 ttagttggtg aggtaaaggc tcaccaagac cgtgatgcat agccgacctg agagggtaat 300 cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct 360 tccacaatgg acgaaagtct gatggagcaa cgccgcgtga gtgaagaagg ttttcggatc 420 gtaaaactct gttgttggag aagaatgtat ctgatagtaa ctgatcaggt agtgacggta 480 tccaaccaga aagccacggc taactacgtg ccagcagccg ggggtaatac taaaa 535

&lt;210&gt; 5 &lt;211&gt; 522 &lt;212> DNA &lt;213&gt; Lactobacillus reuteri (25 〇 63 &lt;7/&quot; 51 corpse 7_/) 6-station muscle-89 &lt;400&gt ; 5 ggcttgggat accgtcactg cgtgaacagt tactctcacg cacgttcttc tccaacaaca 60 gagctttacg agccgaaacc cttcttcact cacgcggtgt tgctccatca ggcttgcgcc 120 cattgtggaa gattccctac tgctgcctcc cgtaggagta tggaccgtgt ctcagttcca 180 ttgtggccga tcagtctctc aactcggcta tgcatcatcg ccttggtaag ccgttacctt 240 accaactagc taatgcaccg caggtccatc ccagagtgat agccaaagcc atctttcaaa 300 caaaagccat gtggcttttg ttgttatgcg gtattagcat ctgtttccaa atgttatccc 360 ccgctccggg gcaggttacc tacgtgttac tcacccgtcc gccactcact ggtgatccat 420 cgtcaatcag gtgcaagcac catcaatcag ttgggccagt gcgtacgact tgcatgtatt 480 aggcacaccg ccggcgttca tcctgagcca gaacgaactc tc 522

S 2

Claims (1)

201200142 VII. Patent application scope: 1'-A strain of Lactobacillus species ("full s(4)) with anti-inflammatory activity, wherein the isolate is Lactobacillus serrata (Zac plus c///(10)(10) 鲶〇GMNL 76 It is deposited at the Health (4) Source Conservation and Research Center of the Food and Environmental Development Research Institute under the registration number BCRC 55, and is deposited with the China Center for Type Culture Collection under the registration number CCTCCM207153. 2. - Can be used for treatment and / Or a pharmaceutical composition for alleviating the disease associated with inflammation, comprising: - Shak's nipple HGMNL-76, deposited at the Center for Bioresource Conservation and Research of the Food Industry Development Institute under the accession number BCRC91〇355, and It is deposited in the Chinese typical culture collection with the deposit _$CCTCC 207 207153. 3. The pharmaceutical composition of claim 2, wherein the disease is rheumatoid arthritis 0 4. For example, claim 2 The pharmaceutical composition of the invention, wherein the pharmaceutical composition is in a dosage form for oral administration. The pharmaceutical composition of the fourth aspect of the patent application, wherein the dosage form is selected from the following Group.} gluten, suspension, emulsion, powder, key, pellet, syrup, mouth-containing key, tablet, chewing gum, thick paste and capsule. A 6..: species as in the scope of the patent application An isolate of the Lactobacillus species supplies a use for the preparation of a pharmaceutical composition for treating and/or alleviating a disease associated with inflammation. 201200142 7. The use of the sixth aspect of the patent application, wherein the 3H disease is Rheumatoid arthritis. 8. A dosage form for the administration of Cap 6, wherein the pharmaceutical composition is in an oral form for 9. Chewing gum, thick paste and capsules. Rotating agent, pill, syrup, mouth A food product containing a lot of money, a tablet, and a food product, which comprises an isolated strain and an edible material, such as the Lactobacillus species of claim 1 of the patent application. The food product further comprises at least one probiotic microorganism selected from the group consisting of milk (four) genus 10 (10) such as 勤 (4), BifMacte Hum sp' 鏠 鏠 屣 ( s nostalgia c〇_(four), yeast (yeasts), and their groups 0 12· The food product of claim 5, wherein the Lactobacillus species is selected from the group consisting of: Lactobacillus acidophilus (10), Lactobacillus hctis, Swiss milk Lactobacillus helveticus~, Lactobacillus helveticus (Lacic^c/Z/ws, Lactobacillus lactis cayez'), Lactobacillus salvumius, Lactobacillus salivcnius, Lactobacillus faecalis LaciobaciUus b (/idus), Lactobacilhis bulgaricus, Lactobacillus Uus caucasicus, Lactobacilltis rhamnosus, such as 201200142 Lactobacillus as_yen·, and The combination. 13. The food product according to claim 5, wherein the Bifidobacterium species is selected from the group consisting of: Bifidobacterium longum (7) ww), Bifidobacterium longum ), Bi々d〇bacterium infantis, Bifidobacterium breve, Bifidobacterium adolescentis, Bifd〇ljacterium !actis In contrast to their papers, the food product of claim 5, wherein the Streptococcus species is selected from the group consisting of Streptococcus thermophilus (and reptococc (10) 汍 (10)), lactic acid Streptococcus cerevisiae OSirepiococcus / aciis), Streptococcus lactis (such as OCOCC WiS er (9) ^ 汾), S. cerevisiae (S^e/Jiococci^i^acei^/cato), and combinations thereof. 15. The food product of claim 5, wherein the yeast is selected from the group consisting of: Candida cerevisiae y?ore «imM5), Saccharomyces cerevisiae Like a note), and a combination of them. 16. The food product of claim 10, wherein the edible material is selected from the group consisting of fluid dairy, fermented dairy, milk powder, ice cream, cheese, cheese, soy milk, fermented soy milk, and juice-killing , juices, sports drinks, desserts, fruits; East, sweets, baby food, health foods, animal feed and dietary supplements. 17. A food product as claimed in claim 1 (), which is in the form of a ready-to-eat food product.