CN102115721A - Lactobacillus isolated strains with anti-inflammatory activity and use thereof - Google Patents
Lactobacillus isolated strains with anti-inflammatory activity and use thereof Download PDFInfo
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Abstract
The invention discloses two Lactobacillus isolated strains with anti-inflammatory activity and beneficial probiotic characteristics, namely lactobacillus sakei GMNL-76 and Lactobacillus reuteri GMNL-89, which are respectively preserved at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) with the preservation numbers of BCRC 910355 and BCRC 910340, and preserved at the China Center for Type Culture Collection (CCTCC) with the preservation numbers of CCTCC M 207153 and CCTCC M 207154. The two Lactobacillus isolated strains and subculture progeny thereof can be used for preparing various food products, and drug composites used for curing and/or relieving diseases related to inflammation, such as rheumatoid arthritis.
Description
The application is to be on May 8th, 2008 applying date, and application number is 200810091395.5, and denomination of invention is divided an application for the Chinese patent application of " having lactobacillus separation strains of anti-inflammatory activity and uses thereof ".
Technical field
The invention relates to that two strains have the lactobacillus separation strains of anti-inflammatory activity and useful beneficial natural disposition matter (beneficial probiotic properties), just picogram Bacterium lacticum (Lactobacillus sakei) GMNL-76 and lactobacillus reuteri (Lactobacillus reuteri) GMNL-89, they are preserved in Biological resources preservation and research centre (BCRC) of Foodstuff Industrial and Development Inst. (FIRDI) respectively with deposit number BCRC 910355 and BCRC 910340, and are preserved in Chinese typical culture collection center (CCTCC) with deposit number CCTCC M 207153 and CCTCC M 207154.This two strains lactobacillus separation strains and succeeding transfer culture offspring thereof can be used to prepare various foods prods, and are used to prepare and are used for the treatment of and/or the pharmaceutical composition of the disease (such as rheumatoid arthritis) of alleviation and inflammation-related connection.
Background technology
(rheumatoid arthritis RA) is a kind of chronic autoimmune disease to rheumatoid arthritis, and it can cause swelling, distortion, pain, the atrophy and stiff of joint (for example hand, foot, wrist, elbow, knee and ankle joint).Secular chronic arthritis can eat away normal articular bone cell gradually, causes joint deformity so that last loss of function.In addition, rheumatoid arthritis also can be invaded the position beyond the joint, for example body of gland (such as sialisterium, lachrymal gland and lymph gland etc.), organ (such as liver, spleen, heart and lungs etc.) or system's (such as the recycle system and neural system), and then cause rheumatoid vasculitis, pleura lung sign (pleuropulmonary manifestations) (comprising interstitial fibrosis, pleura lung tubercle, pneumonia and arteritis) and felty's syndrome etc.
The age of onset of rheumatoid arthritis mainly is to drop between 40 to 50 years old, and women's sickness rate is 2 to 4 times of the male sex.So far, the definite pathogenesis of rheumatoid arthritis is still the unknown, but previous studies show that: genetic predisposition, ambient induced, autoimmune phenomena, chronic inflammatory diseases, cell transportation and treatment etc. all may be relevant with the immunopathogenesis of rheumatoid arthritis.
The main focus of rheumatoid arthritis is a synovial tissue.Synovial cell in the natural joint can not surpass 3 layers, but then tangible hyperplasia can occur at arthritic's intraarticular.Rheumatoid arthritis might be Zhao Yin in patient's immunocyte attack undesiredly the joint cartilage of self and the II Collagen Type VI of synovial tissue (collagen type II, CII).Patient with rheumatoid arthritis can be detected to have specific autoantibody of II Collagen Type VI tool and/or Rheumatoid factors, polyclonal.Rheumatoid factors, polyclonal is the autoantibody that is caused by the glycosylation defect in the Fc zone of IgG, wherein see at most with IgM, and IgG, IgA also has discovery.Rheumatoid factors, polyclonal can detect in about 80% patient with rheumatoid arthritis, and formed immunocomplex can be deposited in synovial membrane, cartilage or the blood vessel and causes pathology after it combined with original antigen.
Known cytokine relates to many important biological procedureses, comprising: inflammation, tissue repair, cell growth, fibrosis, blood vessel take place, and immunne response.Therefore, cytokine is played the part of an important role in autoimmune disease.
At M.Feldmann et al. (1996), Annu.Rev.Immunol., in the retrospective paper of this piece of 14:397-440, the pathogenesis of RA is inquired in people such as M.Feldmann cytokine expression and regulation and control in the synovial tissue of analysis classes rheumatic arthritis, and wherein cytokine is divided into following four big classifications: (1) pro-inflammatory cytokine; (2) immunomodulatory cytokine; (3) chemotactic cytokine; And (4) mitogenesis cytokine etc.
Pro-inflammatory cytokine be a group by 1 type t helper cell (being called for short the Th1 cell) produced can the anaphylactoid molecule of control lag type, comprising: interleukin 1 (IL-1), interferon-(IFN-γ), tumor necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony stimutaing factor (GM-CSF) etc.Viewed cartilage destruction has been thought at large that the active of matrix metalloproteinase (MMP) causes in rheumatism, and this matrix metalloproteinase is produced by the scavenger cell and the inoblast that pro-inflammatory cytokine (such as IL-1 or TNF-α) are responded and be activated.People such as M.Feldmann further mention: IL-1 or TNF-α are injected in the mouse or rat body of collagen immunization, or with IFN-γ local injection in the sole of II Collagen Type VI mice immunized, all can quicken arthritic generation and increase severity of disease (M.Feldmann et al. (1996) is with above-mentioned).
Immunomodulatory cytokine (being also referred to as anti-inflammatory cytokines) be a group by 2 type t helper cells (being called for short the Th2 cell) produced can the inflammation-inhibiting reaction molecule, comprising: IL-4, IL-10, IL-13 and transforming growth factor-beta (TGF-β) etc.At G.Garcia et al. (1999), Journal of Autoimmunity, among the 13:315-324, people such as G.Garcia mention: these two kinds of immunomodulatory cytokines of TGF-β and IL-10 not only can suppress to bring out the generation of the pro-inflammatory cytokine of MMP, also can bring out the generation of the natural inhibitor (the tissue depressant TIMP of matrix metalloproteinase just) of MMP.People such as G.Garcia further mention: IL-10 is widely known except meeting to be suppressed to generate the IFN-γ from Th 1 cell, also can suppress to become various cytokine from other white corpuscle all living creatures.IL-10 can suppress to generate IL-1, IL-6, TNF-α, IL-8 and G-C SF (granulocyte colony-stimulating factor) from scavenger cell, and can suppress to generate IL-1, TNF, IL-8, macrophage inflammatory protein matter 1 α (MIP1 α) and macrophage inflammatory protein matter 1 β (MIP1 β) from polymorphonuclear cell, and the great majority among these cytokines and the chemokine relate to arthritic pathologic process (G.Garcia et al. (1999) is with above-mentioned).
These results of study show: the pro-inflammatory cytokine relevant with the Th1 cell (such as TNF-α and IFN-γ) can promote inflammation and make the patient's condition of rheumatoid arthritis more worsen, and the immunomodulatory cytokine (such as IL-10 and IL-4) relevant with the Th2 cell also can reduce cartilage destruction by the generation of inducing TIMP except the generation that can suppress pro-inflammatory cytokine.
Therefore still do not have medicine at present and can cure rheumatoid arthritis effectively, employed clinically medicine only can focus on that the inflammation that prevents joint and other tissue or organ and destruction, the impaired joint of reparation are to ease the pain and to keep the function in joint and prevent to be out of shape etc.The pattern of treatment can be divided into pharmacological agent, operative treatment, rehabilitation, exercise therapy and dietary control etc.Pharmacological agent mainly is to treat at the symptom of inflammation, and the medicine that is usually used in treating rheumatoid arthritis now can be reduced following five big classes and (thanks to transfer and raise and Chen Peiying (2001), " treatment of rheumatoid arthritis ", pharmaceutical journal, 17 (4): 109-116):
1. biological response modifier (BRM): for example (trade name is Enbrel to etanercept
) and the Ying Li Xidan resists, and (trade name is Remicade
), they are anti-TNF medicaments, can be by being bonded to the biological activity that TNF-α suppresses TNF-α specifically, and then alleviate the inflammatory phenomena that TNF-α is caused;
2. NSAID (non-steroidal anti-inflammatory drug) (NSAID): for example acetylsalicylic acid and Ibuprofen BP/EP, this type of medicine has anti-inflammatory and lenitive effect, therefore is available for the pain and the inflammation of the long-term or short-term of rheumatoid arthritis patient;
3. amelioration of disease type antirheumatic (DMARD): for example (trade name is Arava to leflunomide
), ammonia first dish purine, Trolovol, Oxychloroquine and gold compound etc., they are applicable to the responseless patient with rheumatoid arthritis to NSAID.DMARD can cause by adjusting that the abnormal immune of rheumatoid arthritis reacts and delay lysis, and doctor's supervision must be arranged to avoid the generation of side effect when using this type of medicine to treat;
4. reflunomide: this type of medicine is very effective for the treatment rheumatoid arthritis, but can produce many side effects.Reflunomide is normally used with formulation oral or injection, but often the injection reflunomide can injure cartilage, so frequency injection exceeds with annual 1 to 2 time.At present, oral prednisone is the most normal prescription of opening of doctor; And
Other: for example ((trade name is Hyalgan to hyaluronic acid such as hyaluronate sodium
)) and derivative ((trade name is Synvisc such as Hylan G-F 20
)).Hyaluronic acid is the major ingredient of joint cartilage and synovia, and therefore can reach by hyaluronic intra-articular injection stimulates the proliferation of chondrocytes, protection and lubricated joint, and the effectiveness that reduces inflammation.
Selecting for use of the relevant medicine that is used for the treatment of rheumatoid arthritis, the NSAID that the general initial stage can be weak to render a service, security is higher is as the first line medication, if result of treatment is not remarkable, again to render a service stronger DMARD as the second line medication.When this two classes medicine all can't reach desired curative effect, then will consider and use reflunomide or perform a surgical operation.Though medicine have ease the pain, the effect of stiff sense and inflammation, but take side effects such as to produce insomnia, tinnitus, oedema, stomach ulcer, gastrorrhagia, gastric perforation (gastrobrosia), cutaneous sensibility, proteinuria, diabetes, hypertension, osteoporosis, cataract, the reduction of opposing infection ability for a long time, in the process of treatment, need to do termly blood biochemical, serum inspection simultaneously, with the generation of guaranteeing to treat effect and avoid above-mentioned side effect.
So far, a kind of treatment effectively or rheumatoid arthritis and can not produce the medicine of non-required side effect are not arranged as yet.
Food and Agricultural Organization of the United Nations (FAO) and The World Health Organization (WHO) are defined as probiotic bacterium: the microorganism that lives, when it is proper amt and uses, can give host's one health advantages (Guidelines for the Evaluation of Probiotics in Food, Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food, April 30 and May 1,2002).The microorganism that can be used as the probiotic bacterium use at present has numerous species, for example lactobacillus (Lactobacillus), genus bifidobacterium (Bifidobacterium), lactococcus (Lactococcus), enterococcus spp (Enterococcus), yeast, streptococcus (Streptococcus) or the like, the bacterial classification of particularly preceding two Pseudomonas.
Eli Metchnikoff proposed in 1908: the harmful spoilage organism that is present in the gi tract (GI tract) can be suppressed or antagonism (Eli Metchnikoff by the Bacterium lacticum of product acid, 1908, The Prolongation Of Life, Ed.P.Chalmers Mitchell, G.P.Putnam ' s Sons, The Knickerbocker Press, New York ﹠amp; London).Afterwards, there are many research and clinical trial also to confirm to have between Bacterium lacticum and the health important dependency successively.
Bacterium lacticum is the gram-positive facultative anaerobe of a group, and they are prevalent in human gi tract and intravaginal, and they can fermenting carbohydrates and are main metabolites with lactic acid.The found effect of Bacterium lacticum comprises: (1) is promoted the bacterium of intestinal microflora and is balanced each other; (2) prevention diarrhoea; (3) danger of reduction colorectal carcinoma; (4) normal development of mucocutaneous membrane system and function on the stimulating gastrointestinal; (5) produce various VITAMIN and nutrient substance; And (6) prevention and treatment vaginosis.
Based on the useful beneficial natural disposition matter of Bacterium lacticum, those skilled in the art are devoted to develop and have desired bioactive lactobacillus separation strains.In this regard, can referring to, for example: TW 588109 discloses lactobacillus rhamnosus (Lactobacillus rhamnosus) T cell-1 and uses thereof.TW I241912 discloses 6 strains and has the new acidproof and bile tolerance lactobacillus separation strains that reduces with the assimilating cholesterol ability, and they are Lactobacillus gasseri (Lactobacillus gasseri) B21T1, B21T6, C21T1, X21B7 and B38T38 and Lactobacterium acidophilum (L.acidophilus) B6T7.TW I283268 discloses lactobacillus rhamnosus GM-020 and the fat purposes of treatment thereof.TW I284149 discloses lactobacillus paraceasi (Lactobacillus paracasei) GMNL-32 and treats the purposes of irritated relative disease.In addition, TW200611973 discloses secretion and/or the treatment allergy of lactobacillus fermentum (Lactobacillus fermentum) GM-090 energy effective stimulus IFN-γ.
In addition, people such as Jae-Seon So report: the Orally administered destruction that can suppress collagen-induced property sacroiliitis (CIA) and reduce sole swelling, lymphocytic infiltration and cartilaginous tissue of lactobacterium casei (Lactobacillus casei).Lactobacterium casei passes through CD4
+The T cell reduces the short scorching molecule (IL-1 β, IL-2, IL-6, IL-12, IL-17, IFN-γ, TNF-α and Cox-2) of II Collagen Type VI-reactivity.Use lactobacterium casei and also reduce NF-κ B to endonuclear displacement and reactive Th1-type IgG isotype IgG2a of CII-and IgG2b, raise immunity IL-10 level (Jae-Seon So et al. (2008) simultaneously, Mol.Immunol., 45 (9): 2690-2699; Epub 2008 Feb 19).
If rheumatoid arthritis can reach result of treatment by taking lactobacillus separation strains, should can be the patient one drug safety and the medicine that the charge is small are provided.
For reaching this purpose, the applicant filters out two strain lactobacillus separation strains from the one-tenth human gastrointestinal tract sample in native country, they are fastened (phylogenetically) and are different from disclosed bacterial strain in the affiliated separately bacterial classification in kind, and have to stimulate and produce a large amount of IL-10 and the anti-inflammatory activity of more a spot of IFN-γ and/or TNF-α, thereby the quilt expection can be used for treating the disease that joins with inflammation-related, include but not limited to rheumatoid arthritis.
Summary of the invention
So, aspect first, the invention provides a kind of strain isolated with lactobacillus bacterial classification (Lactobacillus sp.) of anti-inflammatory activity, wherein this strain isolated is:
(i) one be selected from following preservation strain:
(a) picogram Bacterium lacticum GMNL-76, it is preserved and the research centre with the Biological resources that deposit number BCRC 910355 is preserved in Foodstuff Industrial and Development Inst., and is preserved in Chinese typical culture collection center with deposit number CCTCC M 207153; And
(b) lactobacillus reuteri GMNL-89, it is preserved and the research centre with the Biological resources that deposit number BCRC 910340 is preserved in Foodstuff Industrial and Development Inst., and is preserved in Chinese typical culture collection center with deposit number C CTCC M 207154; Or
The (ii) succeeding transfer culture offspring of the described preservation strain in (i).
Aspect second, the invention provides a kind of foods prods, it includes as mentioned above the strain isolated and the edibility material of lactobacillus bacterial classification.
Aspect the 3rd, the invention provides and a kind ofly can be used for treating and/or the pharmaceutical composition of the disease of alleviation and inflammation-related connection, it includes as mentioned above the strain isolated of lactobacillus bacterial classification.
Aspect the 4th, the invention provides a kind of method that is used for the treatment of disease individual and the inflammation-related connection, it comprises uses as mentioned above the strain isolated of lactobacillus bacterial classification to the individuality of this treatment of needs.
Description of drawings
The present invention is described in detail below in conjunction with drawings and Examples, so the present invention in above-mentioned and other purpose and feature, can become more obvious by reference following description, appending claims and accompanying drawing, in the accompanying drawing:
Fig. 1 shows to suffer from the arthritic rat of collagen-induced property in the result who is collected serum and carries out the ELISA test of IL-10 after feeding lactobacillus separation strains GMNL-76 of the present invention and GMNL-89 lasted for 8 weeks continuously, wherein do not suffer from sacroiliitis and by feeding with the rat of reverse osmosis water and suffer from sacroiliitis but be to organize in contrast respectively and placebo with the rat of reverse osmosis water by feeding;
Fig. 2 shows that suffer from the arthritic rat of collagen-induced property is being collected serum and is carrying out the result that the ELISA of IFN-γ tests after feeding lactobacillus separation strains GMNL-76 of the present invention and GMNL-89 lasted for 8 weeks continuously, wherein do not suffer from sacroiliitis and by feeding with the rat of reverse osmosis water and suffer from sacroiliitis but be respectively to organize in contrast and placebo with the rat of reverse osmosis water by feeding;
Fig. 3 shows to suffer from the arthritic rat of collagen-induced property in the result who is collected serum and carries out the ELISA test of TNF-α after feeding lactobacillus separation strains GMNL-76 of the present invention and GMNL-89 lasted for 8 weeks continuously, wherein do not suffer from sacroiliitis and by feeding with the rat of reverse osmosis water and suffer from sacroiliitis but be to organize in contrast respectively and placebo with the rat of reverse osmosis water by feeding;
Fig. 4 shows the 16S rDNA nucleotide sequence of lactobacillus separation strains GMNL-76 of the present invention;
Fig. 5 shows respectively with the genomic dna of lactobacillus separation strains GMNL-76 of the present invention and 2 strains picogram Bacterium lacticum BCRC 12933 in the past and BCRC 17500 and analyzes as template and the polymorphic dna (RAPD) that uses Lac P2 primer to carry out random amplification, then after carrying out electrophoresis with 1.8% sepharose, under UV-light, observe resulting photograph result, wherein in (A) district, swimming lane M1:DNA ladder (100-3000bp), swimming lane 1:GMNL-76; In (B) district, swimming lane M2:DNA ladder (100-3000bp), swimming lane 2:BCRC 12933; And in (C) district, swimming lane M3:DNA ladder (100-3000bp), swimming lane 3:BCRC 17500;
Fig. 6 shows the 16S rDNA nucleotide sequence of lactobacillus separation strains GMNL-89 of the present invention; And
Fig. 7 shows that respectively genomic dna with in the past lactobacillus reuteri BCRC 14625, BCRC 16090 of lactobacillus separation strains GMNL-89 of the present invention and 5 strains, BCRC 16091, BCRC 17476 and BCRC 17478 is as template and use the LacP2 primer to carry out RAPD and analyze, then after carrying out electrophoresis with 1.8% sepharose, under UV-light, observe resulting photograph result, wherein in (A) district, swimming lane M1:DNA ladder (100-3000bp), swimming lane 1:GMNL-89; In (B) district, swimming lane M2:DNA ladder (100-3000bp), swimming lane 2:BCRC 14625; In (C) district, swimming lane M3:DNA ladder (100-3000bp), swimming lane 3:BCRC 16090; In (D) district, swimming lane M4:DNA ladder (100-3000bp), swimming lane 4:BCRC 16091; In (E) district, swimming lane M5:DNA ladder (100-3000bp), swimming lane 5:BCRC 17476; And in (F) district, swimming lane M6:DNA ladder (100-3000bp), swimming lane 6:BCRC 17478.
Detailed description of the invention
Rheumatoid arthritis is a kind of chronic disease of general, but at present used clinically medicine also can't be treated rheumatoid arthritis effectively, and this type of medicine of life-time service also can cause non-required adverse side effect.
In addition, previous research is pointed out: the pro-inflammatory cytokine relevant with the Th1 cell (such as IFN-γ and TNF-α) can promote inflammation and make the patient's condition of rheumatoid arthritis more worsen, and the immunomodulatory cytokine (such as IL-10 and IL-4) relevant with the Th2 cell can suppress the generation of pro-inflammatory cytokine and can reduce cartilage destruction by the generation of inducing TIMP.Therefore, inventor's inference: perhaps the treatment of rheumatoid arthritis can reach by the secretory volume of regulation and control immunomodulatory cytokine and pro-inflammatory cytokine.
Milk-acid bacteria, the bacterial strain of lactobacillus particularly, be familiar with and widely used beneficial natural disposition microorganism by the people, they have been proved has many effects that are of value to host health, and for example: normal development and function, the various VITAMIN of generation and nutrient substance and the prevention and the treatment vaginosis etc. of mucocutaneous membrane system on the danger of colorectal carcinoma, the stimulating gastrointestinal are balanced each other, prevented to suffer from diarrhoea, reduce to the bacterium of promoting intestinal microflora.
Therefore, the applicant attempt in the middle of the milk-acid bacteria that is considered to be beneficial natural disposition microorganism, finding out have favourable anti-inflammatory activity lactobacillus separation strains to be used for the treatment of rheumatoid arthritis.
So, the applicant originates as the separation of lactobacillus separation strains with the gi tract sample from Taiwan health adult, and resulting strain isolated carried out common cultivation to stimulate the monocyte secrete cytokines with the monocyte of laboratory animal respectively, use IL-10 and IFN-γ as selection markers then, can stimulate a large amount of IL-10 of monocyte secretion and two strain lactobacillus separation strains GMNL-76 and the GMNL-89 of more a spot of IFN-γ and/or TNF-α and filter out, they are belonged to picogram Bacterium lacticum and lactobacillus reuteri respectively through characterized.
Picogram Bacterium lacticum GMNL-76 and lactobacillus reuteri GMNL-89 have been preserved in (the Food Industry Research and Development Institute of Foodstuff Industrial and Development Inst. in Taiwan with deposit number BCRC 910355 and BCRC 910340 respectively on June 14th, 2007 and on November 14th, 2006, FIRDI) Biological resources are preserved and research centre (Biosource Collection and Research Center, BCRC) (No. 331,300 Xinzhu City food roads).These two strain isolateds are also according to the regulation of budapest treaty, be preserved in (Wuhan, Chinese typical culture collection center (CCTCC) with deposit number CCTCC M 207153 and CCTCC M 207154 respectively on November 19th, 2007, Wuhan University, 430072, the People's Republic of China (PRC)).
When picogram Bacterium lacticum GMNL-76 of the present invention and lactobacillus reuteri GMNL-89 are given when suffering from the arthritic rat of collagen-induced property by feeding, the IL-10 that can be observed in the rat blood serum raises and IFN-γ and TNF-α reduction, and this shows that lactobacillus separation strains GMNL-76 of the present invention and GMNL-89 can make the arthritis situation of rat no longer worsen.
Than bacterial strain picogram Bacterium lacticum BCRC in the past 12933 and BCRC 17500 and lactobacillus reuteri BCRC 14625, BCRC 16090, BCRC 16091, BCRC 17476 and BCRC 17478, the ability that picogram Bacterium lacticum GMNL-76 and lactobacillus reuteri GMNL-89 stimulate monocyte secretion IL-10 in vitro respectively than they separately under the bacterial strain in the past of bacterial classification better.
Based on above-mentioned favourable biological activity, two strain lactobacillus separation strains of the present invention or their succeeding transfer culture offspring are had the potentiality that can be used for treating the disease that joins with inflammation-related by expection.So, the invention provides a kind of pharmaceutical composition that is used for the treatment of the disease that joins with inflammation-related, it includes:
(i) at least aly be selected from following preservation strain:
(a) picogram Bacterium lacticum GMNL-76, it is preserved and the research centre with the Biological resources that deposit number BCRC 910355 is preserved in Foodstuff Industrial and Development Inst., and is preserved in Chinese typical culture collection center with deposit number CCTCC M 207153; And
(b) lactobacillus reuteri GMNL-89, it is preserved and the research centre with the Biological resources that deposit number BCRC 910340 is preserved in Foodstuff Industrial and Development Inst., and is preserved in Chinese typical culture collection center with deposit number CCTCC M 207154; Or
The (ii) succeeding transfer culture offspring of this preservation strain in (i).
The present invention also provides a kind of method individual and the disease inflammation-related connection that is used for the treatment of, and it comprises uses aforesaid lactobacillus strain isolated or its succeeding transfer culture offspring to the individuality of this treatment of needs.
According to the present invention, described disease with the inflammation-related connection is to be selected from the following group that constitutes: rheumatoid arthritis, osteoarthritis, juvenile rheumatoid arthritis, ankylosing spondylitis, spondylitis, psoriatic arthritis, Crohn's disease, ulcerative colitis and psoriatic (Eveline Trachsel et al. (2007), Arthritis Research ﹠amp; Therapy, 9 (1): R9, Published online 2007 January 29.doi:10.1186/ar2115; Suchita Nadkarni et al. (2007), The Journal of Experimental Medicine, 204:33-39; Richard O Williams et al. (2007), Current Opinion in Pharmacology, 7:412-417).In a preferred embodiment of the present invention, described disease with the inflammation-related connection is a rheumatoid arthritis.
Technology according to pharmaceutical composition of the present invention can utilize those skilled in the art to know clearly and know is formulated in above-mentioned lactobacillus separation strains or its succeeding transfer culture offspring in the one pharmaceutically acceptable carrier, makes then and is applicable to Orally administered formulation.In a preferred embodiment of the present invention, described pharmaceutical composition is the formulation that is selected from following a group: solution, suspension, emulsion, powder, tablet, pill, syrup, lozenge, contain ingot, Chewing gum, capsule, paste and analogue.
As used in this article, term " pharmaceutically acceptable carrier " means a kind of when being applied Shi Buhui causes anaphylaxis or other non-required effectiveness in being applied individuality carrier.According to the present invention, described pharmaceutically acceptable carrier can comprise one or more and be selected from following reagent: solvent, emulsifying agent, suspension agent, decomposition agent, tackiness agent, vehicle, stablizer, sequestrant, thinner, jelling agent, sanitas, lubricant and analogue.
In addition, picogram Bacterium lacticum GMNL-76 of the present invention and lactobacillus reuteri GMNL-89 also are found has bile tolerance and stomach juice-resistant characteristic, thereby is applicable to as a kind of gi tract probiotic bacterium.For example, this strain isolated or its succeeding transfer culture offspring can be used as foodstuff additive, add during through existing method, or do not participate in fermentation and in the ferment making process after, add, and be formulated into the foods prods of ingesting for human and non-human animal with any edibility material in feedstock production.
Therefore, the present invention also provides a kind of foods prods, and it includes as mentioned above lactobacillus separation strains or itself or its a succeeding transfer culture offspring and an a kind of edibility material.
Be applicable to that edibility material of the present invention includes but not limited to: liquid milk products, for example breast, concentrated breast; Fermented-milk, for example yogourt, yogurt, frogurt, lactacidase fermenting beverage; Milk powder; Ice-creams; Cheese; Cheese; Soya-bean milk; Fermented soybean milk; Fruit and vegetable juice; Fruit juice; Sports beverages; Dessert; Jelly; Candy; Infant formula powder; Protective foods; Animal-feed; Dietary supplements or the like.
In addition, can be prepared to the instant form that brews food that wherein contains direct-edible freeze-drying or spraying drying thalline powder according to foods prods of the present invention.The preparation of related food product, can referring to, for example US 6,872,565B2, US 7,244,424B2, US 7,270,994B2, US 7,172,777B2 and US 6,872,411B1.
Can further include at least a beneficial natural disposition microorganism according to foods prods of the present invention." beneficial natural disposition microorganism " or terms such as " probiotic bacteriums " can be by exchange uses mutually as used in this article, and mean the prepared product of microorganism alive, after the quilt mankind or animal ingestion, described microorganism can keep and survive among gi tract, and the prevention and the treatment of disease can be provided.
Be applicable to that beneficial natural disposition microorganism of the present invention includes but not limited to: lactobacillus bacterial classification (Lactobacillus sp.), genus bifidobacterium bacterial classification (Bifidobacterium sp.), streptococcus bacterial classification (Streptococcus sp.), yeast, and their combination.
Preferably, described lactobacillus bacterial classification is to be selected from the following group that constitutes: Lactobacterium acidophilum (Lactobacillus acidophilus), lactobacillus lactis (Lactobacillus lactis), lactobacterium helveticus (Lactobacillus helveticus), short lactobacillus (Lactobacillus brevis), lactobacterium casei (Lactobacillus casei), plant lactobacillus (Lactobacillus plantarum), lactobacillus salivarius (Lactobacillus salivarius), bifidus (Lactobacillus bifidus), lactobacillus bulgaricus (Lactobacillus bulgaricus), bacillus caucasicus (Lactobacillus caucasicus), lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus gasseri (Lactobacillus gasseri), and their combination.
Preferably, described genus bifidobacterium bacterial classification is to be selected from the following group that constitutes: bifidumbacterium bifidum (Bifidobacterium bifidum), bifidus longum bb (Bifidobacterium longum), bifidobacteria infantis (Bifidobacterium infantis), bifidobacterium breve (Bifidobacterium breve), bifidobacterium adolescentis (Bifidobacterium adolescentis), lactic acid Bacillus bifidus (Bifidobacterium lactis), and their combination.
Preferably, described streptococcus bacterial classification is to be selected from the following group that constitutes: thermophilus streptococcus (Streptococcus thermophilus), streptococcus acidi lactici (Streptococcus lactis), Streptococcus cremoris (Streptococcus cremoris), di-acetyl suis (Streptococcus diacetylcatis), and their combination.
Preferably, described yeast is to be selected from the following group that constitutes: candida kefyr (Candida Kefyr), not sieve rib sugar yeast (Saccharomyces florentinus), yeast saccharomyces cerevisiae (Saccharomyces cereviseae), and their combination.
The present invention puts up with following embodiment and is described further, but will be appreciated that, these embodiment only are intended for and illustrate, and should not be interpreted as the restriction in the enforcement of the present invention.
Embodiment
Embodiment 1. has the preliminary screening of the lactobacillus separation strains of anti-inflammatory activity
Experiment material and method:
A. the source of test strain and preparation:
The applicant sets up the gi tract sample that hospital (Taichung) obtains numerous health adults in February, 91 via China Medical University, and isolates the lactobacillus separation strains of strain surplus in the of 400 from these samples.Be the probiotic bacterium that searching has the potentiality that can be used for treating rheumatoid arthritis, the applicant carries out the anti-inflammatory activity analysis to these lactobacillus separation strains, and uses IL-10 as selection markers.
With each test strain be seeded to Bacto Bacterium lacticum MRS broth culture (DIFCO, Cat.No.0881) in, cultivated 12 to 18 hours in 37 ℃ of following anaerobism then.Formed bacterial cultures is with 4, and centrifugal 15 minutes of 000rpm falls to remove supernatant liquor, and throw out is amounted to 3 times with 1X phosphate buffer soln (PBS) washing, is suspended in then among the 1X PBS.Formed bacterium liquid is adjusted concentration to 1～5 * 10 with 1X PBS
9Cell/mL is (with OD
600Carry out the bacterium counting number, OD
600=1.00=5.0 * 10
8Cell/mL). and with carry out 10 times of serial dilutions as stoste, generate 10
8, 10
7, 10
6, 10
5, 10
4, 10
3, 10
2, 10
1The test organisms liquid of cell/mL is used for experiment subsequently.
B. the monocytic preparation of mouse spleen:
With CO
2Put to death the female BALB/c mouse in 6～8 ages in week, take out spleen and use glass rod to grind through sterilization.With spleen tissue and the Ficoll-Paque after grinding
TMPLUS (17-1441-03, Amersham Biosciences) carries out density gradient centrifugation (720g * 20min is under 4 ℃) with 1: 1 volume ratio.Afterwards, remove red blood corpuscle, the mouse spleen monocyte that obtains is thus adjusted cell concn to 4 * 10 with RPMI 1640 substratum that contain 10% foetal calf serum (FBS)
6Cell/mL is standby.
C. lactobacillus separation strains stimulates the assessment of mouse spleen monocyte secretion IL-10:
Each hole to the culture plate in 96 holes adds mouse spleen monocyte sample prepared among the above-mentioned B of 100 μ L, and is divided into 3 groups and adds respectively: (1) test group, prepared test organisms liquid (1 * 10 in the above-mentioned A item of 20 μ L
7Cell/mL); (2) normal control group, the RPMI that contains 10%FBS 1640 substratum of 20 μ L; And (3) positive controls, and the lipopolysaccharide of 20 μ L (LPS) (intestinal bacteria (Escherichia coli) serotype O55:B5, Sigma).Incubator (37 ℃, 5% CO
2) in cultivate last 24 hours after, the nutrient solution in each hole is carried out centrifugal, and take out 100 μ L from the supernatant liquor of being gathered in the crops and use Mouse IL-10 OptEIA
TMSet (BD Biosciences, ferment linked immunosorbent assay (ELISA) Cat.No.555252).The experiment of each group is repeated twice.
The concentration of IL-10 is by ELISA being tested measured OD
405The following formula of numerical value substitution calculates, and represents with ELISA unit (%):
ELISA unit (%)=[(A-C)/(B-C)] * 100
Wherein: the OD of A=lactobacillus separation strains
405Numerical value
The OD of B=positive controls
405Numerical value
The OD of C=normal control group
405Numerical value
The result:
In the middle of all test strains, there are 46 strain meetings to stimulate the IL-10 of mouse spleen monocyte secretion comparatively high amts, their experimental result is shown in Table 1.
Table 1. lactobacillus separation strains stimulates the efficacy assessment of mouse spleen monocyte secretion IL-10
| GMNL-28 | 266.2517 | 277.0188 |
| GMNL-32 | 147.8129 | 152.5236 |
| GMNL-33 | 151.1777 | 143.1023 |
| GMNL-34 | 30.7201 | 34.757740 |
| GMNL-35 | 61.6756 | 34.0848 |
| GMNL-36 | 34.0848 | 36.7766 |
| GMNL-37 | 45.5249 | 39.4683 |
| GMNL-38 | 36.1036 | 33.4118 |
| GMNL-39 | 44.8520 | 44.1790 |
| GMNL-41 | 87.9206 | 95.3230 |
| GMNL-42 | 98.6878 | 95.3230 |
| GMNL-43 | 157.9071 | 151.8506 |
| GMNL-44 | 69.75101 | 54.9462 |
| GMNL-45 | 273.6541 | 186.8439 |
| GMNL-47 | 31.3930 | 28.7012 |
| GMNL-50 | 223.1830 | 168.001 |
| GMNL-55 | 174.7308 | 161.9448 |
| GMNL-57-I | 148.4859 | 153.8694 |
| GMNL-57-II | 230.5855 | 268.2705 |
| GMNL-67 | 242.6985 | 215.7806 |
| GMNL-68 | 114.1655 | 91.9583 |
| GMNL-69 | 128.9704 | 113.4926 |
| GMNL-76 | 167.3284 | 146.4670 |
| 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 | 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 | 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 result shown in the table 1, and consider the speed of growth, bacterium number of this 46 strain test strain simultaneously and after the factors such as practicality on the output, the applicant selects GMNL-19, GMNL-76, GMNL-78, GMNL-89, GMNL-94, GMNL-101 and GMNL-161 and does further test, to assess their abilities on stimulation human peripheral blood mononuclearcell secretion IL-10 and IFN-γ.
Embodiment 2. lactobacillus separation strains stimulate the human PBMC to secrete the assessment of IL-10 and IFN-γ ability
Experiment material and method:
A. the preparation of human peripheral blood single nucleus cell:
Learn from else's experience and be up to the standards for human leukocyte concentrated solution of testing usefulness (the blood center is contributed in platform south) and Ficoll-Paque
TMPLUS (17-1441-03, Amersham Biosciences) carries out density gradient centrifugation (720g * 20min is under 4 ℃) with 1: 1 volume ratio.Afterwards, remove red blood corpuscle, the human PBMC who obtains is thus adjusted cell concn to 4 * 10 with RPMI 1640 substratum that contain 10%FB S
6Cell/mL is standby.
B. lactobacillus separation strains stimulates the assessment that the human PBMC secretes IL-10:
The ability that lactobacillus separation strains GMNL-19, GMNL-76, GMNL-78, GMNL-89, GMNL-94, GMNL-101 and GMNL-161 stimulate the human PBMC to secrete IL-10 is assessed with reference to described schedule of operation in the middle of the C " lactobacillus separation strains stimulates the assessment of mouse spleen monocyte secretion IL-10 " of embodiment 1 " experiment material and method " substantially, and difference is: the concentration with 20 μ L is 1 * 10
8The test organisms liquid of cell/mL is as test group, and uses human PBMC's sample prepared in the above-mentioned A item of 100 μ L and Human IL-10 OptEIA
TMSet (BD Biosciences, Cat.No.555157).
C. lactobacillus separation strains stimulates the assessment of human PBMC's secretion of gamma-IFN:
It is according to following described performing an analysis that lactobacillus separation strains GMNL-19, GMNL-76, GMNL-78, GMNL-89, GMNL-94, GMNL-101 and GMNL-161 stimulate the ability of human PBMC's secretion of gamma-IFN.
Each hole to the culture plate in 96 holes adds human PBMC's sample prepared among the above-mentioned A of 100 μ L, and be divided into two groups and add respectively: (1) test group, prepared test organisms liquid (1 * 10 in the middle of the A " source of test strain and preparation " of the embodiment 1 of 20 μ L " experiment material and method "
7Cell/mL); And (2) normal control group, the RPMI that contains 10%FB S 1640 substratum of 20 μ L.Incubator (37 ℃, 5%CO
2) in cultivated 24 hours after, the nutrient solution in each hole is carried out centrifugal, and take out 100 μ L from the supernatant liquor of being gathered in the crops and use BD OptEIA
TMHuman IFN-γ ELISA Kit II (BD Biosciences, IFN-γ ELISA Cat.No.550612).
In addition, with human PBMC's sample with 4 * 10
6Cell/mL adds 10 μ g/mL phaseolus vulgaris agglutinins (PHA), and (Sigma, ratio Cat.No.L2769) is carried out co-cultivation and is lasted 48 hours.Centrifugal (720g * 20min is under 4 ℃) afterwards, it is standby that the supernatant liquor packing is stored in-80 ℃ of refrigerators.When carrying out IFN-γ elisa assay, this is used as interior positive controls (internal positive control) through the supernatant liquor (100 μ L) that PHA handles.
To be cushioned liquid (0.1M Na through bag with micro-dispenser
2HPO
4, 0.77mM NaN
3, pH 9.0) and diluted 1000 times 100 μ L mouse anti human IFN-γ (BD Pharmingen
TM, Cat.No.551221) be added to the culture plate (Nunc-Immuno in one 96 hole
TM96 MicroWell
TMPlates, MaxiSorp in each hole Cat.No.442404), lasts 1 hour with 40rpm this culture plate that vibrates under room temperature (25 ℃), this culture plate is placed under 4 ℃ spend the night then.
Afterwards, this culture plate risen again and remove liquid in each hole, wash each hole 2 times (each 3 minutes) with lavation buffer solution (PBS that contains 0.05%Tween 20) then, then the sealing damping fluid (PBS that contains 3% bovine serum albumin (BSA)) that in every hole, adds 200 μ L, and be statically placed under the room temperature and last 2 hours, remove the sealing damping fluid subsequently and with lavation buffer solution washing 2 times.
The testing sample of 100 μ L is added in each hole, and reaction is spent the night under 4 ℃.Afterwards, remove the liquid in each hole,, will be diluted 2000 times 100 μ L vitamin H mouse Anti-Human IFN-γ (BD Pharmingen through dilution buffer liquid (PBS that contains 1%BSA) then with lavation buffer solution washing 2 times
TM, Cat.No.554550) be added in each hole, and reaction lasts 2 hours under room temperature.After lavation buffer solution washing 2 times, add 100 μ L streptavidin-alkaline phosphatase (Streptavidin-AP, BD Pharmingen through 2000 times of dilution buffer liquid dilutions
TM, Cat.No.554065), and reaction lasts 1 hour under room temperature.After the liquid in removing each hole,, then the freshly prepared p-nitrophenylphosphates of 100 μ L (pNPP) solution is added in each hole with lavation buffer solution washing 4 times.Under room temperature, place the dark place to last 30 minutes culture plate to carry out color reaction.Afterwards, read the light absorption value (OD of each hole under wavelength 405nm with ELISA Microplate Reader (Bio-Rad, Model 550)
405).Each group experiment is repeated twice.
The concentration of IFN-γ is by ELI SA is tested measured OD
405The following formula of numerical value substitution calculates, and represents with ELISA unit (%):
ELISA unit (%)=[(A-C)/(B-C)] * 100
Wherein: the OD of A=lactobacillus separation strains
405Numerical value
The OD of positive controls in the B=
405Numerical value
The OD of C=normal control group
405Numerical value
The result:
The result of lactobacillus separation strains GMNL-19, GMNL-76, GMNL-78, GMNL-89, GMNL-94, GMNL-101 and GMNL-161 stimulation human peripheral blood mononuclearcell secretion IL-10 and IFN-γ is shown in respectively in table 2 and the table 3.
The secretion of table 2. lactobacillus separation strains stimulation human peripheral blood mononuclearcell
The efficacy assessment of IL-10
The secretion of table 3. lactobacillus separation strains stimulation human peripheral blood mononuclearcell
The efficacy assessment of IFN-γ
As seen from Table 2, in all lactobacillus separation strains, the quantity of lactobacillus separation strains GMNL-76 and GMNL-89 stimulation human peripheral blood mononuclearcell secretion IL-10 is the highest, and this shows that these two strain isolateds are best for the ability of stimulation human peripheral blood mononuclearcell secretion IL-10.
In addition as seen from Table 3, in all lactobacillus separation strains, the quantity of lactobacillus separation strains GMNL-78 stimulation human peripheral blood mononuclearcell secretion of gamma-IFN is minimum, and GMNL-89 and GMNL-76 take second place, this shows that lactobacillus separation strains GMNL-78 is the poorest for the ability of stimulation human peripheral blood mononuclearcell secretion of gamma-IFN, and lactobacillus separation strains GMNL-89 and GMNL-76 also are relatively poor for the ability of stimulation human peripheral blood mononuclearcell secretion of gamma-IFN.
Known pro-inflammatory cytokine (such as IFN-γ and TNF-α) can promote inflammation and make the patient's condition of rheumatoid arthritis more worsen, and immunomodulatory cytokine (such as IL-10) can suppress the generation of pro-inflammatory cytokine.The applicant is in view of the above and inference: if can stimulating human or more immunomodulatory cytokine of the monocyte secretion of animal and less pro-inflammatory cytokine, should help improving the mankind that suffer from RA or the patient's condition of animal.And according to the result shown in table 2 and the table 3, the applicant thinks that lactobacillus separation strains GMNL-76 and GMNL-89 are the bacterial strains of tool potentiality to be exploited, and with carry out following animal experiment.
Embodiment 3. lactobacillus separation strains GMNL-76 and GMNL-89 are for the treatment effect assessment that has the arthritic rat of collagen-induced property
Experiment material and method:
A. laboratory animal:
Experiment below the male LEW/SsNNarl rat (in 6 ages in week, body weight is about 200 to 250g) that will derive from national experimental study institute's Experimental Animal Center (National Applied Research Laboratories National Laboratory Animal Center) is used for.It respectively is that 12 hours, room temperature maintain in the Animal House that 25 ± 1 ℃ and humidity maintains 60 ± 5% independent air-conditioning that all laboratory animal are raised in an illumination and dark, and moisture and feed are supplied with fully.Give the animal week age and conform, treat that animal just begins to test after stable.The processing of relevant laboratory animal and all experimental arrangements all carry out according to the standard article standard of the laboratory animal council.
B. the preparation of test strain:
With lactobacillus separation strains GMNL-76 and GMNL-89 be inoculated in respectively Bacto Bacterium lacticum MRS broth culture (DIFCO, Cat.No.0881) in, be cultured to saturated growth conditions in 37 ℃ of following anaerobism.With 3, after centrifugal 15 minutes of the 000g, throw out is used 1X PBS (pH7.2) washing 2 times of 2mL and 1mL respectively, add the PBS of 1mL then, and in OD
600Measure bacterial concentration, the result all reaches 1.0 * 10
10About cell/mL.Bacterium liquid (1X PBS) freezing being stored in-80 ℃ is standby down.
C. arthritic bringing out:
Relevant arthritic bringing out is with reference to Y.Kameyama et al. (2004), and described method is carried out in the middle of the Bone, 35:948-956, and slightly modified.(Sigma-Aldrich Cat.No.C1188) is dissolved in the 0.05M acetum and has the CII solution that concentration is 2mg/mL with preparation with ox II Collagen Type VI (be called for short CII).Add the mode of the complete Freund's adjuvant (CFA) of 100 μ L with the CII solution of 100 μ L and prepare the CII emulsion.Afterwards, the CII emulsion subcutaneous injection of 200 μ L is carried out initial immunity to the rat tail, and booster immunization is once again after 21 days in immunity.
D. the feeding of lactobacillus separation strains:
Rat is divided into 4 groups (every group of n=6) randomly, comprising 3 experimental group (GMNL-76 group, GMNL-89 group and placebo) and a control group.Except control group, the rat of other each group is all brought out arthritic generation according to described method in the middle of the above-mentioned C " arthritic bringing out ".
For GMNL-76 group and GMNL-89 group, prepared bacterium liquid (1.0 * 10 in the middle of the above-mentioned B of rat feeding " preparation of test strain " is given in behind the booster immunization the 7th day
10Cell/mL/ days).As for the rat of placebo and control group, feeding reverse osmosis water.
At continuous feeding after 8 weeks, collect the blood of rat and carry out centrifugally by the eye blood sampling, the serum that obtains thus is used to measure the concentration of IL-10, IFN-γ and TNF-α.In addition, will be through eye blood sampling respectively organize the sacroiliitis assessment of rat below being further used for carrying out.
The concentration determination of E.IL-10, IFN-γ and TNF-α:
The concentration determination of IL-10 is assessed with reference to described schedule of operation in the middle of the C " lactobacillus separation strains stimulates the assessment of mouse spleen monocyte secretion IL-10 " of embodiment 1 " experiment material and method " substantially, but is to use resulting rat blood serum and Rat IL-10 OptEIA among the top D
TMSet (BD Bioscience PharMingen, San Diego, CA, Cat.No.2611KI).
The concentration determination of IFN-γ is assessed with reference to described schedule of operation in the middle of the C " assessment of lactobacillus separation strains stimulating human PBMCs secretion of gamma-IFN " of embodiment 2 " experiment material and method " substantially, but is to use resulting rat blood serum and Rat IFN-γ OptEIA among the top D
TMSet (BD Bioscience PharMingen, San Diego, CA).
The concentration determination of TNF-α is assessed with reference to described schedule of operation in the middle of the C " lactobacillus separation strains stimulates the assessment of mouse spleen monocyte secretion IL-10 " of embodiment 1 " experiment material and method " substantially, but be to use resulting rat blood serum and BD OptEIA Rat TNF-α ELISA Kit (BD Bioscience PharMingen among the top D, San Diego, CA).
Measured thus OD
405Numerical value so respectively according in advance with IL-10, IFN-γ with different concentration known or TNF-α standard substance with respect to they self OD
405The correlation curve that numerical value has been done (correlation curve) and be converted into concentration (pg/mL) and represent.
F. arthritic assessment:
Will through eye blood sampling respectively organize the sacroiliitis assessment of rat below being used to carry out, and the sacroiliitis of relevant rat assessment is with reference to Y.Kameyama et al. (2004), Bone, the central described method of 35:948-956 is carried out.Briefly, the redness in four sole joints of record rat, swelling etc. changes situation, and with the foundation of following 5 kinds of score as difference: score 0 does not show any arthritic symptom; Score 1 has 1 toe swelling; Score 2 has the toe swelling more than 3; Score 3, whole sole swelling; And score 4, the equal serious swelling in all joints can't normally be walked and be and be rolled up shape.
G. statistical analysis technique:
Present embodiment adopts SPSS statistical software (10.0 editions) to carry out statistical study.Because total sample number of laboratory animal is less than 30, resulting experimental data is to analyze with non-parametric statistics method (nonparametric statistical methods) (being also referred to as Mann Whitney U test (Mann-Whitney U test)), with assessment control group, GMNL-76 group and the GMNL-89 group difference on sample distribution respectively and between the placebo.Experimental data is to represent (statistical significance, P＜0.05) with mean value ± deviate.
The result:
Each organizes rat behind the feeding of going through continuous 8 weeks, and the concentration of the IL-10 in the serum, IFN-γ and TNF-α is shown among table 4 and Fig. 1 to Fig. 3 respectively.In addition, the score result of sacroiliitis assessment also is summarized in the table 4.
After table 4. was gone through the feeding in continuous 8 weeks, each organized IL-10, IFN-γ and the concentration of TNF-α and the score result of sacroiliitis assessment in the rat blood serum
1: all data are to analyze with Mann Whitney U test.
2: the score of sacroiliitis degree is to represent with the score summation in four sole joints of rat.
3:
*Compare with placebo in expression p＜0.05.
From table 4 and Fig. 1 to Fig. 3 as seen, the rat of GMNL-76 group and GMNL-89 group is behind difference feeding lactobacillus separation strains GMNL-76 and GMNL-89, IFN-γ in the It serum and TNF-α concentration have significant reduction compared to placebo, and IL-10 concentration then has significant increase.
Therefore, applicant's inference: lactobacillus separation strains GMNL-76 and GMNL-89 are except reducing the secreted IFN-γ of Th1 cell with the phenomenon that reduces inflammation, also can reduce the secretory volume of TNF-α, and then the secretory volume of minimizing matrix metalloproteinase, make that the cartilage in the joint tissue can not continue to be damaged again.In addition, lactobacillus separation strains GMNL-76 and GMNL-89 also can improve the secretory volume of immunomodulatory cytokine IL-10, and allow the situation of inflammation be controlled.Therefore, though the joint damage that sacroiliitis caused can't reverse, but by feeding lactobacillus separation strains GMNL-76 of the present invention and GMNL-89, in the serum of rat, the existing situation that reduces of IFN-γ that is associated with inflammation and TNF-α, the IL-10 that is associated with anti-inflammatory then has the situation of rising, and the sacroiliitis patient's condition of this expression rat can obtain to alleviate and no longer continue and worsen.
The characterized of embodiment 4. lactobacillus separation strains GMNL-76 and GMNL-89
For bacterial classification under the lactobacillus separation strains GMNL-76 that confirms to be filtered out among the embodiment in the above and the GMNL-89, carry out the polymorphic dna analysis of following tentative experiment, 16SrDNA sequential analysis and random amplification.
Experiment material and method:
A. tentative experiment:
Lactobacillus separation strains GMNL-76 and GMNL-89 are carried out tentative experiment, and test subject comprises: gramstaining, morphological observation, catalase reaction, mobility (mobility), the tests such as growing state under aerobic and anaerobic condition.
B.16S rDNA sequential analysis:
Under aseptic condition, with lactobacillus separation strains GMNL-76 and GMNL-89 be inoculated in respectively 1mL Bacto Bacterium lacticum MRS broth culture (DIFCO, Cat.No.0881) in, and in 37 ℃ of following overnight incubation.Afterwards, with 13,000rpm carries out centrifugally lasting 1 minute, removes supernatant liquor then with bacterium liquid.The ddH that adds 200 μ L to the throw out that stays
2O is with abundant suspension thalline, and with 13,000rpm carries out centrifugally removing supernatant liquor after lasting 1 minute, and repeats this step 1 time.At last, with 200 μ L ddH
2O suspends through centrifugal and throw out that obtain, wherein contains the genomic dna of described lactobacillus separation strains.
With resulting genomic dna as masterplate and use one group to be published in P.S.M.Yeung et al. (2002), J.Dairy Sci., among the 85:1039-1051 design at the 16S rDNA of Bacterium lacticum have below shown in the primer of nucleotide sequence to PAF primer and 536R primer, use the reaction conditions shown in the following table 5 to carry out polymerase chain reaction.
PAF primer: 5 '-agagtttgatcctggctcag-3 ' (SEQ ID NO:1)
536R primer: 5 '-gtattaccgcggctgctg-3 ' (SEQ ID NO:2)
The reaction conditions of table 5.PCR
Finish after the PCR, confirm whether to obtain the pcr amplification product that a size is about 500bp by 1.8% agarose gel electrophoresis, and should be through the PCR product of affirmation from gel recovery purifying.This purified PCR product is to entrust basic Long Mikesi biotech inc (Taiwan) to check order, and resulting sequencing result utilizes the Nucleotide-Nucleotide BLAST software in the NCBI website to compare.
C. the polymorphic dna analysis of random amplification:
Confirming with 16S rDNA sequential analysis under lactobacillus separation strains GMNL-76 and the GMNL-89 after the bacterial classification, with the genomic dna of resulting lactobacillus separation strains GMNL-76 and GMNL-89 among the above-mentioned B as masterplate, and select for use one to be published in M.DE ANGELIS et al. (2001), Applied and Environmental Microbiology, the 10-mer primer Lac P2 of nucleotide sequence shown in below the having among the 67:2011-2020, the reaction conditions shown in the table 6 carries out the PCR reaction below using.
Lac P2 primer: 5 '-atgtaacgcc-3 ' (SEQ ID NO:3)
The reaction conditions of table 6.PCR
Finish after the PCR, amplified production carries out electrophoresis with 1.8% sepharose, and dyeing is observed under UV-light then and taken a picture then.
In this experiment, use simultaneously available from preserve in the Biological resources of Foodstuff Industrial and Development Inst. (FIRDI) and the 7 strain lactobacterium strains of research centre (BCRC) as a comparison bacterial strain compare, they are respectively:
1. picogram Bacterium lacticum picogram subspecies (Lactobacillus sakei subsp.sakei) BCRC 12933 (, separate the source and be sauerkraut) corresponding to ATCC 31063;
2. picogram Bacterium lacticum meat subspecies (Lactobacillus sakei subsp.carnosus) BCRC 17500 (, separating the source sausage of making a living) corresponding to LMG 17302;
3. lactobacillus reuteri (Lactobacillus reuteri) BCRC 14625 (, separate the source and be human feces) corresponding to ATCC 23272 and DSM 20016;
4. lactobacillus reuteri (Lactobacillus reuteri) BCRC 16090 (corresponding to DSM 20015, separates the source and is muck (manure);
5. lactobacillus reuteri (Lactobacillus reuteri) BCRC 16091 (, separate the source and be human feces) corresponding to DSM 20053;
6. lactobacillus reuteri (Lactobacillus reuteri) BCRC 17476 (, separate the source and be the chicken intestines) corresponding to JCM 1081; And
7. lactobacillus reuteri (Lactobacillus reuteri) BCRC 17478 (, separating the molasses fermented molasses of source) for fermentation corresponding to JCM 2762.
The result:
1. the characterized of lactobacillus separation strains GMNL-76:
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a mobility, does not have a catalase, does not have an oxydase, all can grow under aerobic and anaerobic environment;
(ii) the 16S rDNA The sequencing results of this strain isolated is shown among Fig. 4, and through with the NCBI website in gene database comparison after, find that the 16S rDNA sequence (SEQ ID NO:4) of this strain isolated is the highest with the sequence similarity degree of the 16S rDNA of picogram Bacterium lacticum; And
(iii) the RAPD analytical results of this strain isolated is shown among Fig. 5.From Fig. 5 as seen, the gene fingerprint that had of the gene fingerprint of the PCR product of this strain isolated and other 2 strains picogram lactobacterium strain in the past is inequality.
Comprehensive above qualification result, lactobacillus separation strains GMNL-76 of the present invention is considered to the new picogram lactobacillus separation strains of a strain.
2. the characterized of lactobacillus separation strains GMNL-89:
(i) according to Preliminary experiment results, this strain isolated is a Gram-positive bacillus, does not have a mobility, does not have a catalase, does not have an oxydase, all can grow under aerobic and anaerobic environment;
(ii) the 16S rDNA The sequencing results of this strain isolated is shown among Fig. 6, and through with the NCBI website in gene database comparison after, find that the 16S rDNA sequence (SEQ ID NO:5) of this strain isolated is the highest with the 16S rDNA sequence similarity degree of lactobacillus reuteri; And
(iii) the RAPD analytical results of this strain isolated is shown among Fig. 7.From Fig. 7 as seen, the gene fingerprint that had of the gene fingerprint of the PCR product of this strain isolated and other 5 strains lactobacillus reuteri bacterial strain in the past is inequality.
Comprehensive above qualification result, lactobacillus separation strains GMNL-89 of the present invention is considered to the new lactobacillus reuteri strain isolated of a strain.
Picogram Bacterium lacticum of the present invention (Lactobacillus sakei) GMNL-76 and lactobacillus reuteri (Lactobacillus reuteri) GMNL-89 are respectively at preserving and research centre (BCRC) (No. 331,300 Xinzhu City food roads) with the Biological resources that deposit number BCRC 910355 and BCRC 910340 are preserved in Foodstuff Industrial and Development Inst. (FIRDI) in June 14 2007 Christian era and on November 14th, 2006.These two strain isolateds are also according to the regulation of budapest treaty, be preserved in (Wuhan, Chinese typical culture collection center (CCTCC) with deposit number CCTCC M 207153 and CCTCC M 207154 respectively on November 19th, 2007, Wuhan University, 430072, the People's Republic of China (PRC)).
In order to prove that can stimulate the more IL-10 of monocyte secretion is lactobacillus separation strains GMNL-76 and the peculiar biological activity of GMNL-89, in the following embodiments, they are used for 7 strains in the past bacterial strain (just available from the picogram Bacterium lacticum BCRC 12933 of Foodstuff Industrial and Development Inst. and BCRC 17500 and lactobacillus reuteri BCRC 14625, BCRC 16090, BCRC 16091, BCRC 17476 and BCRC 17478) compare.
Experiment material and method:
The concentration determination of IL-10 is assessed with reference to described schedule of operation in the middle of the C of embodiment 1 " experiment material and method " " lactobacillus separation strains stimulates the assessment of mouse spleen monocyte secretion IL-10 " substantially, and use lactobacillus separation strains GMNL-76 of the present invention and GMNL-89 and above-mentioned 7 strains in the past bacterial strain experimentize.Measured thus OD
405Numerical value so respectively according in advance with IL-10 standard substance with different concentration known with respect to they self OD
405The correlation curve that numerical value has been done and be converted into concentration (pg/mL) and represent.The experiment of each bacterial strain is repeated 2 times, and experimental data is represented with mean value ± deviate.
The result:
Show following table 7 GMNL-76, GMNL-89 and each compare bacterial strain stimulates the mouse spleen monocyte to secrete the result of IL-10.
Table 7. lactobacillus separation strains GMNL-76 and GMNL-89 and comparison bacterial strain stimulate
The efficacy assessment of mouse spleen monocyte secretion IL-10
| 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 | 292.86±24.97 |
| BCRC 17478 | 232.269±16.385 |
| BCRC 12933 | 250.996±3.672 |
| BCRC 17500 | 266.958±12.759 |
As seen from Table 7, in the middle of all test strains, for the ability the best that stimulates mouse spleen monocyte secretion IL-10, and GMNL-76 takes second place with lactobacillus separation strains GMNL-89 of the present invention and bacterial strain BCRC 17476 in the past.Experimental result shown in the table 7 proves that again the bacterial strain in the past of bacterial classification is inequality under lactobacillus separation strains GMNL-89 of the present invention and GMNL-76 and they are separately.
Acid resistance and the bile salt tolerance test of embodiment 6. picogram Bacterium lacticum GMNL-76 and lactobacillus reuteri GMNL-89
For confirm whether picogram Bacterium lacticum GMNL-76 of the present invention and lactobacillus reuteri GMNL-89 can survive by gastral critical conditions after ingesting under, carry out the experiment of a gastral continuous digestion process of simulating human.
Experiment material:
1.MRS broth culture (pH=3):
(BD Cat.No.288130) is dissolved in the reverse osmosis water of 1L and gives thorough mixing, then with 1M HCl the pH value is adjusted to 3, sterilizes under 121 ℃ then and lasts 15 minutes, and is standby after to be cooled with the MRS powder of 55g.
2. the MRS broth culture that contains 0.2% (w/v) oxgall:
The MRS powder of 55g is dissolved in the reverse osmosis water of 1L and gives thorough mixing, under 121 ℃, sterilize then and last 15 minutes.Afterwards, treat that temperature reduces to about 45 ℃, (ox gall powder Sigma) and give thorough mixing to obtain a MRS broth culture that contains 0.2% (w/v) oxgall, is that the filtering membrane of 0.45 μ m is filtered afterwards standby with an aperture then to add the oxgall powder of 2g.
3.MRS agar culture plate:
The MRS powder of 55g is dissolved in the reverse osmosis water of 1L, adds the agarose powder of 15g after treating to dissolve fully, pour into formed mixture to the serum bottle of 1L then and under 121 ℃, sterilize and last 15 minutes.Afterwards, treat that temperature reduces to 45 ℃, under aseptic technique, the MRS agar-agar soln with about 15 to 20mL is added in each sterile petri dish, and is standby after to be cooled the solidifying.
Experimental technique:
A. acid resisting test:
The resulting test organisms liquid in the middle of the A " source of test strain and preparation " of embodiment 1 " experiment material and method " of 3mL is seeded in the MRS broth culture (pH=3) of 27mL and thorough mixing, connects and place 37 ℃ to cultivate down and last 3 hours formed culture.Afterwards, take out the culture of 1mL and carry out serial dilution with aqua sterilisa and be coated with dish (10
-9～10
-1) to detect the bacterium number of survival.
B. bile salt tolerance test:
After the acid resisting test of above-mentioned A item finished, with 4,000rpm is centrifugal to last 15 minutes with remaining 29mL culture, removes supernatant liquor and adds 30mL sterilized water suspension thalline.Afterwards, with 4,000rpm is centrifugal to last 15 minutes and removes supernatant liquor, whereby eccysis tart MRS broth culture.Afterwards, the MRS broth culture that contains 0.2% (w/v) oxgall that adds 30mL places formed culture 37 ℃ of following cultivations to last 3 hours with the suspension thalline then.Afterwards, take out the culture of 1mL and carry out serial dilution with aqua sterilisa and be coated with dish (10
-9～10
-1) to detect the bacterium number of survival.
The result:
In picogram Bacterium lacticum GMNL-76 and the lactobacillus reuteri GMNL-89 table 8 below being shown in through the growing state after the gastral continuous digestion process of simulating human.
Table 8. picogram Bacterium lacticum GMNL-76 and the lactobacillus reuteri GMNL-89 growing state after the gastral continuous digestion process of process simulating human
*: strain isolated is cultivated with MRS broth culture (pH=3) in advance and is lasted 3 hours.
As seen from Table 8, when cultivating with the MRS broth culture of pH=3 after two strain lactobacillus separation strains of the present invention last 3 hours, the viable count of picogram Bacterium lacticum GMNL-76 descends before handle and reaches approximately 99.95%, and the viable count of lactobacillus reuteri GMNL-89 descends and reaches about 64%.Then, after these two lactobacillus separation strains are cultivated in containing the MRS broth culture of 0.2% oxgall and are lasted other 3 hours, the phenomenon that viable count does not descend, both survival rates are all very superior.This result shows: picogram Bacterium lacticum GMNL-76 and lactobacillus reuteri GMNL-89 can overcome the environmental stress in human digestive road, can effectively arrive in the intestines also to grow in intestines surely after ingesting.
All data in literature quoted from this case specification sheets and patent documentation are merged in this case as the reference data with their integral body.If to some extent when conflicting, the detailed description of this case (comprise be defined in) will be got the upper hand.
Though the present invention is described with reference to above-mentioned certain embodiments, obviously can make a lot of modifications and variations not deviating under the scope and spirit of the present invention.What therefore be intended to is that the present invention only is subjected to the restriction of scope shown in claims.
Claims (17)
1. strain isolated with lactobacillus bacterial classification (Lactobacillus sp.) of anti-inflammatory activity is characterized in that:
The strain isolated of this lactobacillus bacterial classification is:
(i) a kind of preservation strain:
Lactobacillus reuteri (Lactobacillus reuteri) GMNL-89, it is preserved in Chinese typical culture collection center with deposit number CCTCC M 207154; Or
The (ii) succeeding transfer culture offspring of the described preservation strain in (i).
2. one kind can be used for treating and/or the pharmaceutical composition of the disease of alleviation and inflammation-related connection, it is characterized in that:
This pharmaceutical composition includes:
(i) a kind of preservation strain:
Lactobacillus reuteri GMNL-89, it is preserved in Chinese typical culture collection center with deposit number CCTCC M 207154; Or
The (ii) succeeding transfer culture offspring of the described preservation strain in (i).
3. pharmaceutical composition as claimed in claim 2 is characterized in that: described disease is a rheumatoid arthritis.
4. pharmaceutical composition as claimed in claim 2 is characterized in that: this pharmaceutical composition is can supply Orally administered formulation.
5. pharmaceutical composition as claimed in claim 4 is characterized in that: described formulation is selected from the following group that constitutes: solution, suspension, emulsion, powder, tablet, pill, syrup, lozenge, contain ingot, Chewing gum, paste and capsule.
6. according to the strain isolated of the lactobacillus bacterial classification of claim 1, preparation be used for the treatment of and/or the pharmaceutical composition of the disease of alleviation and inflammation-related connection in purposes.
7. purposes as claimed in claim 6 is characterized in that: described disease is a rheumatoid arthritis.
8. purposes as claimed in claim 6 is characterized in that: described pharmaceutical composition is can supply Orally administered formulation.
9. purposes as claimed in claim 8 is characterized in that:
This formulation is selected from the following group that constitutes: solution, suspension, emulsion, powder, tablet, pill, syrup, lozenge, contain ingot, Chewing gum, paste and capsule.
10. a foods prods is characterized in that this foods prods includes strain isolated and a kind of edibility material of the lactobacillus bacterial classification of claim 1.
11. the foods prods as claim 10 is characterized in that:
This foods prods further includes at least a beneficial natural disposition microorganism that is selected from following a group: lactobacillus bacterial classification (Lactobacillus sp.), genus bifidobacterium bacterial classification (Bifidobacterium sp.), streptococcus bacterial classification (Streptococcus sp.), yeast, and their combination.
12. the foods prods as claim 11 is characterized in that:
This lactobacillus bacterial classification is selected from the following group that constitutes: Lactobacterium acidophilum (Lactobacillus acidophilus), lactobacillus lactis (Lactobacillus lactis), lactobacterium helveticus (Lactobacillus helveticus), short lactobacillus (Lactobacillus brevis), lactobacterium casei (Lactobacillus casei), plant lactobacillus (Lactobacillus plantarum), lactobacillus salivarius (Lactobacillus salivarius), bifidus (Lactobacillus bifidus), lactobacillus bulgaricus (Lactobacillus bulgaricus), bacillus caucasicus (Lactobacillus caucasicus), lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus gasseri (Lactobacillus gasseri), and their combination.
13. the foods prods as claim 11 is characterized in that:
Described genus bifidobacterium bacterial classification is selected from the following group that constitutes: bifidumbacterium bifidum (Bifidobacterium bifidum), bifidus longum bb (Bifidobacterium longum), bifidobacteria infantis (Bifidobacterium infantis), bifidobacterium breve (Bifidobacterium breve), bifidobacterium adolescentis (Bifidobacterium adolescentis), lactic acid Bacillus bifidus (Bifidobacterium lactis), and their combination.
14. the foods prods as claim 11 is characterized in that:
Described streptococcus bacterial classification is selected from the following group that constitutes: thermophilus streptococcus (Streptococcus thermophilus), streptococcus acidi lactici (Streptococcus lactis), Streptococcus cremoris (Streptococcus cremoris), di-acetyl suis (Streptococcus diacetylcatis), and their combination.
15. the foods prods as claim 11 is characterized in that:
Described yeast is to be selected from the following group that constitutes: candida kefyr (Candida Kefyr), not sieve rib sugar yeast (Saccharomyces florentinus), yeast saccharomyces cerevisiae (Saccharomyces cereviseae), and their combination.
16. the foods prods as claim 10 is characterized in that:
Described edibility material is selected from the following group that constitutes: liquid milk products, fermented-milk, milk powder, ice-creams, cheese, cheese, soya-bean milk, fermented soybean milk, fruit and vegetable juice, fruit juice, sports beverages, dessert, jelly, candy, infant formula powder, protective foods, animal-feed and dietary supplements.
17. the foods prods as claim 10 is characterized in that: this foods prods is made into the instant form that brews food.
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