CN100396770C

CN100396770C - Microbial L. rhamnosus GM-020 and its use for treating obesity

Info

Publication number: CN100396770C
Application number: CNB2004100301698A
Authority: CN
Inventors: 许清祥; 苏伟志
Original assignee: JINGYUE BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd
Current assignee: JINGYUE BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd
Priority date: 2004-03-19
Filing date: 2004-03-19
Publication date: 2008-06-25
Anticipated expiration: 2024-03-19
Also published as: CN1670183A

Abstract

The present invention provides a strain of separated microbiological isodulcitol bacterium lacticum GM-020. Obesity and the complication thereof can be effectively treated by the microbiological isodulcitol bacterium lacticum GM-020 by discovering. The present invention also provides the purpose of the microbiological isodulcitol bacterium lacticum GM-020 for treating the obesity and the complication thereof.

Description

New microorganism lactobacillus rhamnosus GM-020 and the fat purposes of treatment thereof

Technical field

The present invention mainly relates to the new microorganism lactobacillus rhamnosus GM-020 of a strain and treatment is fat or the purposes of its complication.

Background technology

Fat normally because the body fat surplus that physiology or biochemical function change cause, and it can significantly impair one's health.Fat generally includes neutral fat, phosphatide and cholesterol.The increase of weight depends on that people's energy absorption is greater than energy expenditure.Two types of fat existence: (a) simple obesity (simple obesity) and (b) Secondary Obesity (second obesity).Simple obesity can be divided into congenital obesity (idiopathic obesity) and acquired character obesity (acquired obesity), can account for to surpass 95% obesity.Congenital obesity is by due to a large amount of adipocytes, and is common in obesity in childhood.The acquired character obesity is by due to the larger sized adipocyte, and is common in the Adulthood obesity.The Secondary Obesity symptomatic obesity that is otherwise known as, it is normally by due to internal secretion or the disease of metabolism.Fat and some chronic diseases are relevant with some cancer such as diabetes, myocardosis (cardiopathy), hypertension, apoplexy, cholelith (biliary calculus), gout.

Five kinds of strategies that treatment is fat are arranged at present: subtract food, exercise, behavior therapy, pharmacological agent and rehabilitation operation (therapeutic operation).Decide on patient's the health risk factor and speed and the effect that loses weight, can select or make up these strategies adiposis patient is treated, speed that it loses weight and effect are subjected to the influence such as a plurality of factors such as age, height, family history and risk factors.The mechanism of pharmacological agent comprises depress appetite, increases energy expenditure, stimulation fat is mobile, triglyceride reducing is synthetic and suppresses fat absorbing.The example of these medicines be Phenylpropanolamine (phenylpropanolamine, PPA), Xenical (orlistat, Xenical ^TM) and Reductil (sibutramine, Reductil ^TM).Yet, by crude substance but not medicine treat obesity and become new trend.

In the prior art, discovery can use some microorganisms to treat obesity, or its complication.For example, find that Lactobacillus gasseri (Lactobacillus gasseri) SBT0270 has the ability ((Usman in conjunction with relevant cholesterol concentration of going that reduces with bile acide, B. and Hosono, A. (2001), " Lactobacillus gasseri (Lactobacillus gasseri) SBT0270 causes the hypocholesterolemic effect of feeding with in the rat of the diet that is rich in cholesterol " (Hypocholesterolemic effect ofLactobacillus gasseri SBT0270 in rats fed a cholesterol-enriched diet), J.Dairy Res.68:617-624).The fat mechanism of treatment is: by being absorbed by Lactobacillus gasseri (L.gasseri) from discharging (because the bile acide of being discharged can not recirculation be got back to liver by the bile acide of form and through ight soil, and need synthesize new bile acide from cholesterol), reduce the solubility of the bile acide of this decomposition.In addition, find that Lactobacillus gasseri (L.gasseri) can make up to reach drainage with bile acide and cholesterol.

Lactobacillus rhamnosus (Lactobacillus rhamnosus) is considered to a kind of potential probiotic lactobacillus with the characteristic that improves immunity.The safety assessment of lactobacillus rhamnosus (Safetyassessment) is also investigated.People such as Zhou have disclosed hematologic parameter (red corpuscle and the platelet count of the mouse of dispensing lactobacillus rhamnosus, hemoglobin concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration (MCHC)), different white blood cell count(WBC)s, haemobiochemistry (blood plasma total protein, albumin, cholesterol, and glucose), mucosal tissue is learned (epithelial cell height, mucosal thickness, and height of naps), and outside enteron aisle, organize (blood, liver, spleen, kidney and mesenteric lymph nodes) the bacterium displacement, showed and similar collection of illustrative plates (the profile) (Zhou of contrast mouse, J.S., Shu, Q., Rutherfurd, K.J., Prasad, J., Birtles, M.J., Gopal, P.K. and Gill, H.S. (2000), " potential probiotic lactobacillus strain lactobacillus rhamnosus HN001 in the BALB/c mouse; Lactobacterium acidophilum HN017; and the safety assessment of Bifidus HN019 " Bifidus (Safety assessment of potential probiotic lactic acidbacterial strains Lactobacillus rhamnosus HN001, Lb.acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice), International Journal ofFood Microbiology56:87-96).In addition, people such as Agerholm-Larsen have disclosed dispensing can not change low-density lipoprotein (LDL)-cholesterol through the yogourt of lactobacillus rhamnosus fermentation, and only significantly reduced systolic pressure (Agerholm-Larsen, L., Raben, A., Haulrik N., Hansen, A.S., Manders, M., with Astrup A. (2000), " taking in of the influence of beneficial 8 weeks of lactogenesis goods " (Effect of 8 week intake of probiotic milk products onrisk factors for cardiovascular diseases), Eur J Clin Nutr.54 (4): 288-97) to the Hazard Factor of cardiovascular disorder.Therefore, provable known lactobacillus rhamnosus strain can not change total plasma cholesterol (totalcholesterol) and LDL-cholesterol.In addition, when the known lactobacillus rhamnosus strain of dispensing, do not observe any weight and change.

Summary of the invention

The invention provides the new separated microorganism of a strain, lactobacillus rhamnosus (Lactobacillusrhamnosus) GM-020, it is preserved in Chinese typical culture collection center with deposit number CCTCC M 203098.

On the other hand, the invention provides the composition that comprises lactobacillus rhamnosus GM-020.

On the other hand, the invention provides lactobacillus rhamnosus bacterial strain GM-020 and be used for the treatment of application in the medicine of obesity and complication thereof in preparation.

On the other hand, the invention provides lactobacillus rhamnosus bacterial strain GM-020 and black fungus and be used for the treatment of application in the medicine of obesity and complication thereof in preparation.

On the other hand, the invention provides a kind of subject's of being used for the treatment of the obesity and the method for complication thereof, this method comprises a kind of composition that comprises microorganism lactobacillus rhamnosus GM-020 of offeing medicine to this subject; Wherein this complication preferably is selected from the group of being made up of hypercholesterolemia, atherosclerosis and coronary heart disease (coronary heart disease).

Another aspect the invention provides a kind of subject's of being used for the treatment of the obesity and the method for complication thereof, and this method comprises this subject is imposed a kind of composition that comprises microorganism lactobacillus rhamnosus GM-020 and black fungus (Auricularia polytricha); Wherein this complication preferably is selected from the group of being made up of hypercholesterolemia, atherosclerosis, coronary heart disease, fatty liver and diabetes.

Description of drawings

Fig. 1 illustrates the 1000X microgram of GM-020.

Fig. 2 illustrates the cell wall protein of GM-020 and known lactobacillus rhamnosus strain; Wherein M represents protein molecular weight; Lactobacillus rhamnosus (commodity Antibiophilus) is represented in road (Lane) 1; Lactobacillus rhamnosus GM-020 is represented in road 2; Lactobacillus rhamnosus ATCC9595 is represented in road 3; Lactobacillus rhamnosus ATCC10940 is represented in road 4; Represent lactobacillus rhamnosus ATCC14029 with road 5.

Fig. 3 illustrates the 2-D total protein electrophoresis of GM-020.

Fig. 4 explanation is passed through GM-020 or/and the body weight difference of the animal model that black fungus (wood ear) is treated according to embodiment 5; Wherein ^*Represent p＜0.01; ^aRepresent negative control; ^bRepresent positive control; ^cRepresent the black fungus of 1X; ^dRepresent the black fungus of 10X; ^eRepresent GM-020; ^fThe black fungus of the 1X of representative and GM-020 combination; And ^gThe black fungus of the 10X of representative and GM-020 combination.

Fig. 5 explanation is passed through GM-020 or/and the difference of the lipid tissue weight around the testis of the animal model that black fungus is treated according to embodiment's 5; Wherein ^*Represent p＜0.01; ^aRepresent negative control; ^bRepresent positive control; ^cRepresent the 1X black fungus; ^dRepresent the 10X black fungus; ^eRepresent GM-020; ^fThe 1X black fungus of representative and GM-020 combination; And ^gThe 10X black fungus of representative and GM-020 combination.

Fig. 6 explanation is passed through GM-020 or/and the difference of the circumrenal lipid tissue weight of the animal model that black fungus is treated according to embodiment's 5; Wherein ^*Represent p＜0.01; ^aRepresent negative control; ^bRepresent positive control; ^cRepresent wooden 1X ear; ^dRepresent the 10X black fungus; ^eRepresent GM-020; ^fThe 1X black fungus of representative and GM-020 combination; And ^gThe 10X black fungus of representative and GM-020 combination.

Difference between the serum-concentration of the total cholesterol of (CHOL_4) after (CHOL_0) and the treatment before the treatment of Fig. 7 explanation according to embodiment 6; Wherein ^*Represent p＜0.1; ^*Represent p＜0.05; ^{* *}Represent p＜0.01.

Fig. 8 explanation is according to the serum-concentration of the LDL-C of (LDL-C_4) after (LDL-C_0) and the treatment before treatment of embodiment 6; Wherein ^*Represent p＜0.1; ^*Represent p＜0.05; ^{* *}Represent p＜0.01.

Fig. 9 explanation is according to the difference of the serum-concentration of the LDL-C of (LDL-C_4) after (LDL-C_0) and the treatment before treatment of embodiment 6; Wherein ^*Represent p＜0.1; ^*Represent p＜0.05; ^{* *}Represent p＜0.01.

Figure 10 explanation is according to the difference of the serum-concentration of the LDL-C/HDL-C of (LDL-C/HDL-C_4) after (LDL-C/HDL-C_0) and the treatment before treatment of embodiment 6; Wherein ^*Represent p＜0.1; ^*Represent p＜0.05; ^{* *}Represent p＜0.01.

Embodiment

The invention provides the new microorganism lactobacillus rhamnosus GM-020 of a strain, it can treat obesity.On December 18th, 2003 this strain GM-020 is preserved in Chinese typical culture collection center with deposit number CCTCC M 203098.

Lactobacillus rhamnosus GM-020 system isolates from people's stomach.

The eubacterium feature of below having showed lactobacillus rhamnosus GM-020:

(a) morphological feature

(1) shape of cell and size: when cell under 37 ℃ in the MRS substratum after the cultivation overnight, can be by microscopic examination to bacillus, it is the staff-like shape with circular edge.

(2) motility: inactive

(3) flagellum: do not have

(4) sporulation: no sporulation

(5) gramstaining: the positive

(b) cultural characteristic:

(1) medium: the MRS substratum (

0881) (as shown in table 1), final pH value 6.5 ± 0.2

Table 1

Component	g/L
Component	g/L	Show peptone (Proteose peptone)	10.0
Beef extract (Beef Extract)	10.0	Show peptone (Proteose peptone)	10.0
Beef extract (Beef Extract)	10.0	Yeast extract (Yeast Extract)	5.0
Dextrose	20.0	Yeast extract (Yeast Extract)	5.0
Dextrose	20.0	Poly-sorbitol ester 80 (Polysorbate 80)	1.0
Ammonium citrate	2.0	Poly-sorbitol ester 80 (Polysorbate 80)	1.0
Ammonium citrate	2.0	Sodium acetate	5.0
Sal epsom	0.1	Sodium acetate	5.0
Sal epsom	0.1	Manganous sulfate	0.05
Dipotassium hydrogen phosphate (Dipotassium Phosphate)	2.0	Manganous sulfate	0.05

(2) culture condition: 37 ℃ of anaerobism are cultivated (anaerobic culture) or aerobic cultivation (aerobicculture)

(c) physiological characteristic:

(1) catalase (Catalase): feminine gender

(2) oxydase: feminine gender

(3) API 50CHL test: use API 50CHL system to discern Bacterium lacticum.Examine and determine by reaction, can determine the characteristic (character) of this Bacterium lacticum a series of enzymes.Listed the result of the API 50 CHL test of GM-020 in the table 2:

Table 2

Enzyme	Reaction	Enzyme	Reaction	Enzyme	Reaction
Enzyme	Reaction	Enzyme	Reaction	Enzyme	Reaction	Glycerine	-	N.F,USP MANNITOL	+	Lattice sugar (D-Tagatos) too	+
Erythritol (Erythritol)	-	Sorbyl alcohol (Sorbitol)	+	5 ketone group gluconic acids	-	Glycerine	-	N.F,USP MANNITOL	+	Lattice sugar (D-Tagatos) too	+
Erythritol (Erythritol)	-	Sorbyl alcohol (Sorbitol)	+	5 ketone group gluconic acids	-	The D-pectinose	-	Alpha-Methyl-D-glucoside	+	2 ketone group gluconic acids	-
L-arabinose	-	The N acetylglucosamine	+	Gluconic acid	-	The D-pectinose	-	Alpha-Methyl-D-glucoside	+	2 ketone group gluconic acids	-
L-arabinose	-	The N acetylglucosamine	+	Gluconic acid	-	Ribose	+	Amygdaloside	+	L-arabinose alcohol	-
The D-wood sugar	-	Arbutin (Arbutine)	+	The D-arabitol	-	Ribose	+	Amygdaloside	+	L-arabinose alcohol	-
The D-wood sugar	-	Arbutin (Arbutine)	+	The D-arabitol	-	The L-wood sugar	-	Vitamin C2 (Esculine)	+	The L-Fucose	-
Pentitol (Adonitol)	-	Saligenin (Salicine)	+	The D-Fucose	-	The L-wood sugar	-	Vitamin C2 (Esculine)	+	The L-Fucose	-
Pentitol (Adonitol)	-	Saligenin (Salicine)	+	The D-Fucose	-	Beta-methyl-xyloside	-	Cellobiose	+	The D-lyxose	-
D-glucose	+	Semi-lactosi	+	O14-Methyldelectine (inuline)	-	Beta-methyl-xyloside	-	Cellobiose	+	The D-lyxose	-
D-glucose	+	Semi-lactosi	+	O14-Methyldelectine (inuline)	-	D-fructose	+	Lactose	+	Sucrose	-
The D-seminose	+	Alpha-Methyl-D-mannoside	-	Glycogen (Glycogene)	-	D-fructose	+	Lactose	+	Sucrose	-
The D-seminose	+	Alpha-Methyl-D-mannoside	-	Glycogen (Glycogene)	-	The L-sorbose	+	Melizitose (Melezitose)	+	Xylitol	-
Rhamnosyl	+	D-raffinose (D-Raffinose)	-	β gentiobiose (β Gentiobiose)	-	The L-sorbose	+	Melizitose (Melezitose)	+	Xylitol	-
Rhamnosyl	+	D-raffinose (D-Raffinose)	-	β gentiobiose (β Gentiobiose)	-	Winged euonymus sugar alcohol (Dulcitol)	-	Methadone (Amidon)	-	D-turanose (Turanose)	-
Inositol (Inositol)	-	Maltose	-	Melibiose	-	Winged euonymus sugar alcohol (Dulcitol)	-	Methadone (Amidon)	-	D-turanose (Turanose)	-
Inositol (Inositol)	-	Maltose	-	Melibiose	-	Trehalose	-

(d) hereditary feature:

Determine the 16S rDNA sequential analysis of GM-020 by Foodstuff Industrial and Development Inst. of Hsin-chu, as shown in SEQ ID NO:1.The result show GM-020 can with other lactobacillus rhamnosus strain height homology, have and surpass 99% similarity.

(e) cell wall protein of GM-020:

When comparing with other known lactobacillus rhamnosus strain, the cell wall protein of GM-020 has shown specific pattern.The SDS-PAGE pattern of having showed the cell wall protein of GM-020 among Fig. 2.

The total protein of GM-020 can stand the 2-D electrophoresis, and the pattern shown in Fig. 3 is specific.

(f) be used to discern the stdn detection system of GM-020:

Be the U.S. patent application case the 10/446th that on May 29th, 2003 was applied for, disclosed the standard detection system that can be used for discerning microorganism in No. 781, this system uses through given microorganism culturing and gene expression difference without the test cell system of given microorganism culturing and is used as the identification mark.Listed the gene of being tested in the table 3.

Table 3

Gene
Gene	Signal transduction and activating transcription factor (Signal transducer and activator of transcription) 3
c-rel
c-rel	Growth associated protein 43
N-myc	Growth associated protein 43
N-myc	Igf binding protein 1
IL-16	Igf binding protein 1
IL-16	Lymphotoxin α (Lymphotoxin α) (previous tumor necrosis factor)
Interferon inducible protein 9-27
Interferon inducible protein 9-27	Connective Tissue Growth Factor

White plain 10 acceptors are situated between
White plain 10 acceptors are situated between	calpamodulin mRNA
Ubiquitin crosslinking enzyme (UbcH8) mRNA, mixture (comp)	calpamodulin mRNA
Ubiquitin crosslinking enzyme (UbcH8) mRNA, mixture (comp)	The conjugated protein AKAP110mRNA of modern's (Homo sapiens) protein kinase A, cds fully
Pyruvic dehydrogenase kinase, isozyme 4
Pyruvic dehydrogenase kinase, isozyme 4	Pyridoxal (pyridoxol (pyridoxine), vitamin B6) kinases
The interactional Serine of map kinase (serine)/Threonine (threonine) kinases 1	Pyridoxal (pyridoxol (pyridoxine), vitamin B6) kinases
	The white corpuscle Tyrosylprotein kinase
The neurotrophy Tyrosylprotein kinase, acceptor, type 3 (TrkC)	The white corpuscle Tyrosylprotein kinase
	Pyruvic dehydrogenase kinase isozyme 3 (PDK3) mRNA, cds fully
People's diacylglycerol kinases ζ (diacylglycerol kinase zeta) mRNA, cds fully
	Protein kinase C, α
Deoxidation guanosine kinases (Deoxyguanosine kinase)	Protein kinase C, α
Deoxidation guanosine kinases (Deoxyguanosine kinase)	E.C. 2.7.1.20
Topoisomerase (DNA) II β (180kD)	E.C. 2.7.1.20
Topoisomerase (DNA) II β (180kD)	The mRNA of IkB kinase beta subunit (beta subunit)
Stat5a (signal transduction and activating transcription factor 5A)	The mRNA of IkB kinase beta subunit (beta subunit)
	Colony-stimulating factor 1 (M-CSF)
HGF	Colony-stimulating factor 1 (M-CSF)
HGF	IL-1 acceptor type 1
Be situated between white plain 7	IL-1 acceptor type 1
Be situated between white plain 7	Metallothionein(MT) I-B gene
White plain 6 (B-cell stimulating factors 2) are situated between	Metallothionein(MT) I-B gene
	Derivable small molecules cytokine A2 (Small inducible cytokine A2) (MCP 1)
Proteoplast (Proteasome) 26S subunit, ATPase, 3

Ubiquitin crosslinking enzyme E2A (RAD6 autoploid)
Ubiquitin crosslinking enzyme E2A (RAD6 autoploid)	Thymidine kinase (thymidine kinase) 1, solubilized
Tyrosylprotein kinase 2	Thymidine kinase (thymidine kinase) 1, solubilized
Tyrosylprotein kinase 2	Serine/threonine protein-kinases
Growth factor beta (Transforming growth factor beta) activated kinases 1 makes the transition	Serine/threonine protein-kinases
	The Tyrosylprotein kinase 3 related with Fms
Creatine kinase B	The Tyrosylprotein kinase 3 related with Fms
Creatine kinase B	Albumen serine/threonine kinase stk2
Stress-activated protein kinase 3 (SAPK3) mRNA.	Albumen serine/threonine kinase stk2
Stress-activated protein kinase 3 (SAPK3) mRNA.	Human adenylate kinases 2 (adk2) mRNA, cds fully
RAC-α serine/threonine kinase	Human adenylate kinases 2 (adk2) mRNA, cds fully
RAC-α serine/threonine kinase	Hexokinase 1
CDC28 protein kinase 2 (CKS2) mRNA	Hexokinase 1
CDC28 protein kinase 2 (CKS2) mRNA	Superoxide-dismutase 2, plastosome
Tumor necrosis factor receptor 2	Superoxide-dismutase 2, plastosome
Tumor necrosis factor receptor 2	Growth associated protein 43
The gene relevant with p53	Growth associated protein 43
The gene relevant with p53	CD30
Metallothionein(MT)-III	CD30
Metallothionein(MT)-III	White plain 4 acceptors are situated between
Gamma-interferon inducible protein ip-30 precursor	White plain 4 acceptors are situated between
Gamma-interferon inducible protein ip-30 precursor	Interferon-' alpha '/beta receptor α chain precursor
Early growth reactive protein 1	Interferon-' alpha '/beta receptor α chain precursor
Early growth reactive protein 1	White plain-13 receptor mrnas, cds fully are situated between
Proteinase inhibitor 12 (PI12; Neuroserpin)

Serine/threonine protein kitase KKIALRE
Serine/threonine protein kitase KKIALRE	Phosphorylase kinase (Phosphorylase kinase), β
Serine/threonine kinase 9	Phosphorylase kinase (Phosphorylase kinase), β
Serine/threonine kinase 9	Serine/threonine kinase 10
Protein kinase silk mitogen activated 8 (Protein kinase mitogen-activated 8) (map kinase)	Serine/threonine kinase 10
	Focal adhesion kinase (focal adhesion kinase, FAK) mRNA, cds fully
People's activation p21cdc42Hs kinases (ack) mRNA, cds fully
	The people integrates plain link kinases (integrin-linked kinase, ILK) mRNA, cds fully
People's guanylate kinase (GUK1) mRNA, cds fully
People's guanylate kinase (GUK1) mRNA, cds fully	BMK1 alpha kinase mRNA, cds fully
Monooxygenase 5 (FMO5) mRNA that contains flavine	BMK1 alpha kinase mRNA, cds fully
Monooxygenase 5 (FMO5) mRNA that contains flavine	Pyruvate kinase, liver
Transcriptional elongation factor S-II, hS-II-T	Pyruvate kinase, liver
Transcriptional elongation factor S-II, hS-II-T	Nitric oxide synthase 3 (endotheliocyte)
Bcl2, the conjugated protein Bbp/53BP2 of p53 (BBP/53BP2) mRNA,	Nitric oxide synthase 3 (endotheliocyte)
	Telomerize (telomeric) dna sequence dna
Class stat albumen (Fe65) mRNA, cds fully	Telomerize (telomeric) dna sequence dna
Class stat albumen (Fe65) mRNA, cds fully	IL-5 acceptor a
EGF	IL-5 acceptor a
EGF	FGF2
White plain 2 receptor y chains are situated between	FGF2
White plain 2 receptor y chains are situated between	C-C Chemokine Receptors type 2
Guanine nucleotide binding protein 1, Interferon, rabbit can be induced, 67kD	C-C Chemokine Receptors type 2
	Plastosome processing peptidase beta subunit
syntaxin 8	Plastosome processing peptidase beta subunit

Cytokine suppresses conjugated protein 1 (the p38MAP kinases) of anti-inflammatory drug
	Protein tyrosine kinase 6
The branched-chain alpha-keto acid dehydrogenase kinases	Protein tyrosine kinase 6
The branched-chain alpha-keto acid dehydrogenase kinases	Serine/threonine kinase 2
Protein kinase, silk mitogen activated 4 (map kinases 4; P63) (PRKM4) mRNA	Serine/threonine kinase 2
	Tyrosine-protein kinase SYK
The people infers (putative) serine/threonine protein kitase PRK (prk) mRNA, fully cds	Tyrosine-protein kinase SYK
	Myokinase isozyme 1
The hematopoietic cell kinases	Myokinase isozyme 1
The hematopoietic cell kinases	Glycerol kinase
Tyrosine-protein kinase C SK	Glycerol kinase
Tyrosine-protein kinase C SK	General transcription factor IIB
White plain 6 signal transductions (gp130, oncostatin M acceptor) are situated between	General transcription factor IIB
	Caspase-8 (apoptosis L-Cysteine HCL Anhydrous mch5 (mach-α-1))
Oncoprotein p53
Oncoprotein p53	The transition somatomedin, beta receptor III (beta glycan, 3
IFN-g
IFN-g	The transition somatomedin, β 3
TGF-β superfamily protein, (complete) fully	The transition somatomedin, β 3
TGF-β superfamily protein, (complete) fully	Fiber mother cell growth factor 7 (keratinocyte growth factor)
Derivable small molecules cytokine A4 (with mouse Mip-1b homology)
	PHGF activates sub-inh
Ubiquitin carboxyl-terminal hydrolase isozyme 11	PHGF activates sub-inh
Ubiquitin carboxyl-terminal hydrolase isozyme 11	Serine/threonine kinase 11 (pigmentation polyp syndromes
Cyclin-dependent kinase (Cyclin-dependent kinase) inhibitor 3 (the dual specificity phosphatase enzyme that CDK2-is relevant)	Serine/threonine kinase 11 (pigmentation polyp syndromes

Pyruvic dehydrogenase kinase, isozyme 3
Pyruvic dehydrogenase kinase, isozyme 3	Carnitine palmitoyl transferase I, liver
Protein kinase silk mitogen activates 7 (map kinases)	Carnitine palmitoyl transferase I, liver
Protein kinase silk mitogen activates 7 (map kinases)	Human protein tyrosine kinases mRNA, cds fully
Protein tyrosine kinase 7	Human protein tyrosine kinases mRNA, cds fully
Protein tyrosine kinase 7	Lymphocyte specific protein Tyrosylprotein kinase
Ribosomal protein s6 kinases	Lymphocyte specific protein Tyrosylprotein kinase
Ribosomal protein s6 kinases	Class src kinases (slk) mRNA, cds fully
Nucleoside diphosphokinase a	Class src kinases (slk) mRNA, cds fully
Nucleoside diphosphokinase a	Plain dependent kinase Enzyme 2 of cycle
STAT-1α/β	Plain dependent kinase Enzyme 2 of cycle
STAT-1α/β	Angiogenine (Angiopoietin)-1
Phospholipase C	Angiogenine (Angiopoietin)-1
Phospholipase C	STAT2 (signal transduction and activating transcription factor 2)
The c-src Tyrosylprotein kinase
The c-src Tyrosylprotein kinase	IL-15
The TGFb receptor-related proteins 1	IL-15
The TGFb receptor-related proteins 1	Annexin (Annexin) V (lipocortin V; Endonexin II)
Interferon regulatory factor 5	Annexin (Annexin) V (lipocortin V; Endonexin II)
Interferon regulatory factor 5	Interferon-receptor alpha chain precursor
The transition somatomedin, beta receptor II (70-80kD)	Interferon-receptor alpha chain precursor
The transition somatomedin, beta receptor II (70-80kD)	Modern's apoptotic proteins enzyme incitant 1 (Apaf-1)
Ubiquitin crosslinking enzyme E2B (RAD6 homologue)	Modern's apoptotic proteins enzyme incitant 1 (Apaf-1)
Ubiquitin crosslinking enzyme E2B (RAD6 homologue)	MAP/ERK kinase kinase 3
Phosphorylase kinase, α 2 (liver), glycogen stores up disease IX	MAP/ERK kinase kinase 3

Serine/threonine kinase 4
Serine/threonine kinase 4	Glucosamine-6-phosphate salt desaminase
Mevalonic kinase (Mevalonate kinase)	Glucosamine-6-phosphate salt desaminase
Mevalonic kinase (Mevalonate kinase)	Glucokinase (hexokinase 4, young maturity-onset diabetes (maturity onset diabetes of the young) 2)
The desoxycytidine kinases
The desoxycytidine kinases	Urokinase type plasminogen incitant
Human mitochondrion creatine kinase (CKMT) gene, cds fully	Urokinase type plasminogen incitant
Human mitochondrion creatine kinase (CKMT) gene, cds fully	Choline kinase
The 53K abnormal shape of human phosphatidyl-4-4-phosphoric acid 5-kinases (PIPK) Type II mRNA, cds fully	Choline kinase
	White corpuscle adhesion protein β subunit
c-tos	White corpuscle adhesion protein β subunit
c-tos	Phosphoenolpyruvate carboxykinase (phosphoenolpyruvate carboxykinase)
Apoptosis L-Cysteine HCL Anhydrous mch4
Apoptosis L-Cysteine HCL Anhydrous mch4	MCP
1	MCP
1	Transcription factor AP-1-4 (the activated reinforced factor-conjugated protein
White plain-1 acceptor of Jie, the Class1 precursor
White plain-1 acceptor of Jie, the Class1 precursor	Caspase-10 (people's apoptosis L-Cysteine HCL Anhydrous mch4)
Human kinase (TTK) mRNA, cds fully
Human kinase (TTK) mRNA, cds fully	Beta-2 adrenergic receptor
Estrin sulfotransferase (ste)	Beta-2 adrenergic receptor
Estrin sulfotransferase (ste)	Signal transduction and activating transcription factor 6, white element-4 is induced by being situated between
Protein kinase clk1
Protein kinase clk1	White plain-8 precursors are situated between
Modern FAST kinases mRNA	White plain-8 precursors are situated between
Modern FAST kinases mRNA	Interferon-receptor alpha chain precursor

The quasi-insulin growthing factor I acceptor precursor
The quasi-insulin growthing factor I acceptor precursor	The plastosome transcription termination factor
Signal transduction and activating transcription factor 3 are (acute-ph	The plastosome transcription termination factor
	Modern's protein kinase C K1mRNA
Through map kinase activated protein kinase	Modern's protein kinase C K1mRNA
Through map kinase activated protein kinase	Protein kinase clk3
INF-b	Protein kinase clk3
INF-b	General transcription factor IIB
Sis, PDGF B chain	General transcription factor IIB
Sis, PDGF B chain	Beta-actin
Gsh (Glutathione) S-transferring enzyme M1	Beta-actin
Gsh (Glutathione) S-transferring enzyme M1	IL-1b
MAP/ERK kinase kinase 3	IL-1b
MAP/ERK kinase kinase 3	INF-b
EGR-1	INF-b
EGR-1	Glutathione S-transferase 12 (microsome)

The standard detection system that is used to discern GM-020 with Hep G2 clone as test cell is.When the expression pattern of the cultivation Hep G2 clone that will have or not have GM-020 compared, this listed in the table 4 group gene can be significantly different.

Table 4

Gene
Gene	White plain 10 acceptors are situated between
IL-16	White plain 10 acceptors are situated between
IL-16	EGF
Lymphotoxin α (previous tumor necrosis factor)	EGF
Lymphotoxin α (previous tumor necrosis factor)	Interferon regulatory factor 5
Fiber mother cell growth factor 7 (keratinocyte growth factor)	Interferon regulatory factor 5
	Proteoplast 26S subunit, ATPase, 3
calpamodulin mRNA	Proteoplast 26S subunit, ATPase, 3
calpamodulin mRNA	Ubiquitin carboxyl-terminal hydrolase isozyme L1
Hexokinase	Ubiquitin carboxyl-terminal hydrolase isozyme L1
Hexokinase	1
The 53K abnormal shape of human phosphatidyl-inositol-4-phosphoric acid salt 5-kinases (PIPK) Type II mRNA, cds fully	1
	Plastosome is transcribed termination factor
STAT-1α/β	Plastosome is transcribed termination factor
STAT-1α/β	Urokinase-type plasminogen incitant
Human adenylate kinases 2 (adk2) mRNA, cds fully	Urokinase-type plasminogen incitant
Human adenylate kinases 2 (adk2) mRNA, cds fully	Protein kinase C, α
Proto-oncogene tyrosine-protein kinase FES/FPS	Protein kinase C, α
Proto-oncogene tyrosine-protein kinase FES/FPS	Human mitochondrion creatine kinase (CKMT) gene, cds fully
Protein tyrosine kinase 6	Human mitochondrion creatine kinase (CKMT) gene, cds fully
Protein tyrosine kinase 6	Serine/threonine kinase 9
IkB kinase beta subunit mRNA	Serine/threonine kinase 9
IkB kinase beta subunit mRNA	Caspase-8 (apoptosis L-Cysteine HCL Anhydrous mch5 (mach-α-1))
The conjugated protein Bbp/53BP2 of Bcl2, p53 (BBP/53BP2) mRNA,

Growth associated protein 43
Growth associated protein 43	Protein kinase clk3
Tumour necrosis factor inducible protein TSG-6 precursor	Protein kinase clk3
Tumour necrosis factor inducible protein TSG-6 precursor	The quasi-insulin growthing factor I acceptor precursor
MCP	The quasi-insulin growthing factor I acceptor precursor
MCP	1
White corpuscle adhesion protein β subunit	1
White corpuscle adhesion protein β subunit	Sis, PDGF B chain
Humig mRNA	Sis, PDGF B chain
Humig mRNA	The CD27L acceptor precursor
Vitamin A acid (retinoic acid) and the derivable 58K albumen of Interferon, rabbit RI	The CD27L acceptor precursor
	IRF-1

The invention provides the obesity among a kind of subject of being used for the treatment of and the method for complication thereof, this method comprises the offer medicine composition of a kind of GM-020 of comprising to this subject.

Surprisingly find in the present invention: even the subject takes in high-octane food, strain GM-020 also has the ability that suppresses this subject's weight increase, and can suppress body weight.In according to one embodiment of the invention, the quilt for the treatment of through strain GM-020 is fed the ICR mouse with the high-octane food that can cause fat, compares with the control group of not receiving treatment, and it can keep body weight not have any increase.

Also find in the present invention: strain GM-020 is effective in the ratio of cholesterol regulating and lipoprotein.In according to one embodiment of the invention, in the obesity mice and hamster of feeding,, can reduce the concentration of total cholesterol in its serum and the liver through strain GM-020 treatment with the food that is rich in cholesterol that can cause hypercholesterolemia.Also can reduce the serum-concentration of low-density lipoprotein-cholesterol (LDL-C).In addition, can reduce LDL-C and highdensity lipoprotein cholesterol (HDL-C) ratio (LDL-C/HDL-C) in the serum.The summary speech, strain GM-020 is being effective aspect treatment hypercholesterolemia and reduction atherosclerosis and the evidence of coronary heart diseases.That is, strain GM-020 can be used to prepare a kind of composition that is used for treating obesity and complication (such as hypercholesterolemia) thereof and reduces atherosclerosis and evidence of coronary heart diseases.

The present invention also provides the obesity among a kind of subject of being used for the treatment of and the method for complication thereof, and this method comprises a kind of composition that comprises microorganism lactobacillus rhamnosus GM-020 and black fungus strain of offeing medicine to the subject; Wherein this complication preferably is selected from the group of being made up of hypercholesterolemia, atherosclerosis, coronary heart disease, fatty liver and diabetes.

Find in the present invention: with the strain GM-020 of black fungus (black fungus) combination when treatment is fat, when treating with black fungus or strain GM-020, it can provide surprising effect than single.

Black fungus, common name auricularia auriculajudae, tree ear (tree ear) etc. are the tasteless edibility fungi of colloid.Its with plant in the black fungus in Asia closely relatedly, and consumed for a long time.Find that black fungus can be wild on the softwood tree and sometimes on the fallen leaves trees in spring and two seasons of autumn.Usually black fungus is dry in order to edible.When the black fungus of edible drying, edible fiber after suction extensible about 8 times to 10 times.The human consumer can feel full and eat less.In addition, according to reports, the saccharan in the black fungus has the effect that reduces the total cholesterol serum-concentration in the rabbit.

In according to one embodiment of the invention, the fat animal model body weight for the treatment of through the combination of black fungus and GM-020 can be reduced; On the contrary, only the dispensing of black fungus or GM-020 only can suppress the increase of body weight.

Find in the present invention: than control group, the combination of black fungus and strain GM-020 has the ability of the concentration of total cholesterol and triglyceride level in the serum that reduces this animal model and the liver.The summary speech, the combination of black fungus and strain GM-020 can be used to prepare a kind of composition that is used for treating hypercholesterolemia, fatty liver, diabetes and reduction atherosclerosis and evidence of coronary heart diseases.

In according to one embodiment of the invention, treatment effect fat and hypercholesterolemia and reduction atherosclerosis and evidence of coronary heart diseases is directly proportional with the dosage of black fungus.Normally, recommended doses every day (1X) of adult black fungus is a 6gx 0.0026x surface-area.In one embodiment of this invention, than the black fungus of 1X, the black fungus of 10X has better effect.

According to the present invention, only the combination of strain GM-020 and GM-020 and black fungus can not damage liver and kidney.In animal model, as monitoring glutamic oxaloacetic transaminase (GOT) (glutamicoxaloacetic transaminase, GOT), gpt (glutamic pyruvictransaminase, GPT), during the amount of uric acid and creatinine (creatinine), liver function is normal, and this combination that can show offer medicine strain GM-020 only or strain GM-020 and black fungus is a kind of secured fashion for the treatment of obesity and complication thereof.Also find in the present invention: after treating through the combination of GM-020 and black fungus, the triglyceride level in the serum is minimized.

Only for illustrative purposes has provided following examples, and do not desire to limit the scope of the invention.

Embodiment

Embodiment 1: the separation of lactobacillus rhamnosus GM-020

A slice stomach-tissue of the people that will take out by endoscope (endoscope) be incubated at 2mL Bacterium lacticum MRS substratum (

0881) in.The substratum that will comprise this tissue lies against on the selectivity agar of Bacterium lacticum and at 37 ℃ and cultivated one day down.Selection grows in the single colony on this culture plate and makes it accept gramstaining.Then select gram Yang Shi bacterium.Clone a strain, this strain is called lactobacillus rhamnosus GM-020.

Cell wall protein extraction and the analysis of embodiment 2GM-020

Be harvested from the MRS substratum (

) in 24 hours big cells having a liking for warm Bacterium lacticum, with it in containing 0.1M CaCl ₂0.05M Tris-HCl (pH7.5) in flushing twice, and be suspended in A once more ₆₀₀Be in the identical buffer reagent of 10.0 1ml.Carry out centrifugation after 5 minutes under 8,000 * g, the extraction buffer reagent (pH 8.0) of the 1.0ml by containing 0.01M EDTA, 0.01M NaCl and 2% (wt/vol) SDS can extract cell wall protein from these spherolites (pellet).At room temperature, suspension is stored 60 minutes, descended heating 5 minutes and under 4 ℃, carried out centrifugation 10 minutes with 11,600 * g at 100 ℃.Analyze supernatant liquid and come its dyeing by 12%SDS-PAGE with the Comassie blueness.

The 2-D total protein electrophoresis of embodiment 3GM-020

Together with 0.5mL dissolving buffer reagent C (7M urea, 4%CHAPS, 2M thiocarbamide, 40mM trihydroxy-aminomethane, 0.5%IPG buffer reagent and 5mM TBP) and 100L granulated glass sphere, add 10mg GM-020.Then make solution accept that ultrasonic degradation is handled and with 10,000rpm carried out centrifugation 30 minutes.Can pass through Bio-rad PlusOne ^TMAlbumen is examined and determine and is measured total protein, and then to make it accept pH be 3 to 10IEF 2-D electrophoresis.By 10% gel and with the 40mA/ gel carry out 4 hours as electrophoresis designed in the table 5.Then by 10% methyl alcohol and 7% acetate with gel sets 30 minutes.After fixing, come this gel was carried out painted 5 hours by sypro ruby stain, and then come this gel was decoloured 6 hours with 10% methyl alcohol and 7% acetate.The result as shown in Figure 3.

Table 5

30V	12hr
30V	12hr	100V	0:10hr
250V	0:10hr	100V	0:10hr
250V	0:10hr	500V	0:10hr
1000V	0:30hr	500V	0:10hr
1000V	0:30hr	4000V	0:30hr
6000V	60KVhr	4000V	0:30hr

Embodiment 4 is used to discern the stdn detection system of GM-020

Be used for the preparation of the GM-020 of stdn detection system:37 ℃ of Bacterium lacticum MRS substratum (

0881) in, static phases is arrived in the cell culture of strain GM-020.3, carry out centrifugation under the 000g after 15 minutes, (phosphate-buffered saline pH7.2) is washed this culture to the PBS of the 1X by 2mL and 1mL, and then it is suspended among the PBS (1X) of 1mL, and wherein cell concn is adjusted to 1 * 10 ⁹CFU/mL.These cultures are stored under-20 ℃.

Stimulate: by adding fresh medium and cultivated 16 hours, renewable (refresh) Hep G2 cell.Subsequently, these cells are divided into two groups, and one group is used for cultivating and another group is used for not cultivating by milk-acid bacteria by milk-acid bacteria.When cell concn reaches 1 * 10 ⁷During/10mL, by 1 * 10 ⁷GM-020 or not by 1 * 10 ⁷GM-020 came irritation cell 24 hours.After stimulating, collect these cells, by PBS flushing twice, and use it for RNA and separate.

RNA separates and mark (labelling):Pass through to use Trizol reagent (Life according to manufacturer's explanation

Gaithersburg, Md.), from cell extraction RNA.With 8L RNA (10 μ g) and the oligomeric dT of 2L (12-18mer, 0.1g/L) thorough mixing and kept 10 minutes down, and then cooling off 2 minutes by ice at 70 ℃.In the dark, RNA and reverse transcription mark mixed solution and 3L Cy5-dUTP (1mM), 2L SuperScriptII (200U/L) and RNasin (1L) are mixed.Under 42 ℃, mixture cultivated 2 hours being used for reverse transcription, and come termination reaction by the 20mM EDTA that adds 1.5L.After the mark, handle to remove RNA by NaOH, and by HCl with its neutralization.Pass through STRATAGENE ^TMThe PCR purification kit comes purifying cDNA promptly.

Microarray is made:By the selected hundreds of gene of PCR amplification, and pass through 260nm spectrophotometer (spectrophotometry) with its quantification.In 50% dimethyl sulfoxide (DMSO), the PCR product that all are purified is adjusted to the concentration of 0.1 μ g/ μ L, and gets (spot) ready in UltraGAPSTM with double (in duplicate) ^TMCoating slide glass (slide) (

Inc., Corning, N.Y.) on.Print (printing) afterwards, these microarraies are at room temperature being stored in the ultraviolet-crosslinkable under 300mJoulesand in the slide receptacle in the moisture eliminator.These genes have been listed in the table 3.

Microarray hybridization:Under 100 ℃, make sex change (denature) 5 minutes in the hybridization solution (5xSSC, 0.1%SDS and 25% methane amide) with fluorescently-labeled cDNA, be cooled to surrounding temperature and it is deposited on the slide glass.Under 42 ℃, hybridized 18 hours.After the hybridization, in low severity (low-stringency) (1xSSC and 0.1%SDS), middle severity (0.1xSSC and 0.1%SDS), high severity (0.1x SSC) buffer reagent, wash these slide glasss successively, and at last by compressed N ₂Be dried.

Signal detection and data analysis:For each slide glass with identical laser power and photomultiplier cell sensitivity level (sensitivity level),

4000B scanner (Axon

Inc.) scan immediately on through N2 exsiccant slide glass.Can obtain primary fluorescence data (10-nm resolving power), and at Microsoft Excel ^TMIn processing and the data carried out subsequently visual.Be the result of more independent hybrid experiment, local background's signal (local backgroundsignal) is deducted from the hybridization signal of each independent point, and then divided by house-keeping gene beta-actin (housekeeping gene β-actin).Represent the last expression of each gene with double mean value.Then can obtain through GM-020 and the Hep G2 gene expression of cells collection of illustrative plates (expression profile) cultivated without GM-020.In the Hep G2 that cultivates through GM-020, select a group to be adjusted the gene that surpasses 2 times (fold) up or down than Hep G2 without this microbial culture.The result is as shown in table 4.

Embodiment 5 is used for the treatment of fat GM-020

Animal model:Male ICR mouse can be available from the Taiwan Experimental Animal Center, and under the humidity of 25 ± 1 ℃ temperature and 60 ± 5%, raises 12 hours in light separately and raised in the dark 12 hours.Abundant supplementing foodstuff and water.These mouse are divided into two groups.One group is that normal control group and another group is the high-energy group.This normal control group fed with normal diet and to this high-energy group feed to contain the high-energy diet of 48% dry feed, 8% Semen Maydis oil and 44% enriching milk.Measure the body weight of mouse weekly.According to treatment the high-energy group further is divided into following listed group: (a) black fungus of 1X, (b) with the black fungus of the 1X of GM-020 combination, (c) GM-020, (d) black fungus of 10X, (e) with the black fungus of the 10X of GM-020 combination, (f) through the negative control of physiological saline treatment and the positive control of (g) treating through PPA.Shown in the table 6 before feeding and the difference of the body weight afterwards, wherein with the high-energy diet ^*Represent p＜0.01; ^aRepresent negative control; ^bRepresent positive control; ^cRepresent the black fungus of 1X; ^dRepresent the black fungus of 10X; ^eRepresent GM-020; ^fThe black fungus of the 1X of representative and GM-020 combination; And ^gThe black fungus of the 10X of representative and GM-020 combination.

Table 6

Group	Weight (%)
Group	Weight (%)	Normal control	12.25±1.80
Negative control	20.93±2.27	Normal control	12.25±1.80
Negative control	20.93±2.27	Positive control	20.43±1.35
The black fungus of 1X	22.45±1.46	Positive control	20.43±1.35
The black fungus of 1X	22.45±1.46	The black fungus of 10X	18.45±1.03
GM-020	19.23±1.75	The black fungus of 10X	18.45±1.03
GM-020	19.23±1.75	Black fungus with the 1X of GM-020 combination	21.37±1.05
Black fungus with the 10X of GM-020 combination	22.78±0.67	Black fungus with the 1X of GM-020 combination	21.37±1.05
Black fungus with the 10X of GM-020 combination	22.78±0.67	The P value	0.000 ^{**a，b，c，d，e，f，g}

Can test analysis of data by Kruskal Wallis H, and with the baseline of normal control group as the Dunnett test.After 4 weeks, the mean body weight of this high-energy group is significantly higher than the mean body weight of this normal control group (p＜0.01).

Treatment with GM-020 and/or black fungus:This normal control group is fed continuously with normal diet.One day twice ground imposes treatment.The dosage of each PPA is 4.875mg.In the meals of the black fungus of 1X, have the black fungus of 15.6mg in the 3g diet, and in the meals of the black fungus of 10X, have the black fungus of 156mg in the 3g diet.The dosage of GM-020 is 10 ⁹CFU/mL.

Body weight difference:After treating for 4 weeks, collect blood sample to be used for the biological chemistry calibrating from afterbody.Test analysis of data by Kruskal Wallis H, and with the baseline of normal control group as the Dunnett test.The results are shown among Fig. 4.

After treating for 1 week, with the black fungus group of the 10X of GM-020 combination than this negative control group, body weight significantly reduces.After 2 weeks, positive controls, with the black fungus group of the 1X of GM-020 combination and with the black fungus group of the 10X of GM-020 combination, significantly reduce than this negative control group body weight.After 3 weeks, the black fungus group of positive controls, 1X, GM-020 group, with the black fungus group of the 1X of GM-020 combination and with the black fungus group of the 10X of GM-020 combination, significantly reduce than this negative control group body weight.It is effective controlling aspect fat that these results prove GM-020 and black fungus.

The difference of the lipid tissue weight around the testis:Mouse is killed, and extract the lipid tissue around the testis and weigh.Test analysis of data by Kruskal Wallis H, and with the data of this normal control group baseline as the Dunnett test.The results are shown among Fig. 5.

According to Fig. 5, only positive controls and with the black fungus group of the 10X of GM-020 combination than this negative control group, shown significant minimizing.

The difference of circumrenal lipid weight:Mouse is killed, and extract this circumrenal lipid tissue and weigh.Test analysis of data with Kruskal Wallis H, and with the data of this normal control group baseline as the Dunnett test.The results are shown among Fig. 6.

According to Fig. 6, the black fungus group of 1X, the black fungus group of 10X, with the black fungus group of the 1X of GM-020 combination and with the black fungus group of the 10X of GM-020 combination than negative control group, have significant minimizing.On the other hand, this positive controls, GM-020 group and negative control group have been showed little difference.

The serum-concentration of metabolism of fat thing:Collect blood sample to be used for the biological chemistry calibrating from afterbody.Blood sample was statically placed in room temperature following 1 hour, and with 2,500rpm carried out centrifugation 10 minutes.Get upper serum and be used for calibrating.

By TRIGLYCERIDES GPO LIQUID REAGENT ^TM( Taiwan) examine and determine the concentration of every group triglyceride level (TG), and pass through Autoanalyzer Hitachi ^TM7150 measure sorption.By CHOLESTEROL LIQUID REAGENT ^TM(

Taiwan) examine and determine the concentration of total cholesterol (CHOL), and pass through Autoanalyzer Hitachi ^TM7150 measure sorption.According to optionally suppress method and enzymatic determination (

Japan) concentration of examining and determine HDL-C and LDL-C, and by Autoanalyzer Hitachi ^TM7150 measure sorption.

Test analysis of data by Kruskal Wallis H, and with the data of this normal control group baseline as the Dunnett test.The results are shown in the table 7: wherein ^*Represent p＜0.01; ^aRepresent negative control; ^bRepresent positive control; ^cRepresent the black fungus of 1X; ^dRepresent the black fungus of 10X; ^eRepresent GM-020; ^fThe black fungus of the 1X of representative and GM-020 combination; And ^gThe black fungus of the 10X of representative and GM-020 combination.

Table 7

Group	CHOL	TG	HDL	LDL
Group	CHOL	TG	HDL	LDL	Negative control	232.00±16.88	86.60±22.40	126.39±7.37	11.21±1.40
Normal control	135.80±6.67	120.00±20.35	86.90±3.14	4.22±0.56	Negative control	232.00±16.88	86.60±22.40	126.39±7.37	11.21±1.40
Normal control	135.80±6.67	120.00±20.35	86.90±3.14	4.22±0.56	Positive control	219.08±11.22	75.83±9.93	123.19±4.20	10.04±0.93
The black fungus of 1X	182.50±8.24	82.08±7.32	100.57±3.58	12.39±1.03	Positive control	219.08±11.22	75.83±9.93	123.19±4.20	10.04±0.93
The black fungus of 1X	182.50±8.24	82.08±7.32	100.57±3.58	12.39±1.03	The black fungus of 10X	176.83±9.84	63.92±4.62	93.51±6.02	9.74±1.07
GM-020	204.75±8.90	96.56±10.20	121.64±8.47	14.04±3.10	The black fungus of 10X	176.83±9.84	63.92±4.62	93.51±6.02	9.74±1.07
GM-020	204.75±8.90	96.56±10.20	121.64±8.47	14.04±3.10	Black fungus with the 1X of GM-020 combination	190.08±4.85	55.75±4.38	105.38±3.10	10.83±1.51
Black fungus with the 10X of GM-020 combination	164.20±8.64	57.30±4.61	97.93±5.42	8.04±0.94	Black fungus with the 1X of GM-020 combination	190.08±4.85	55.75±4.38	105.38±3.10	10.83±1.51
Black fungus with the 10X of GM-020 combination	164.20±8.64	57.30±4.61	97.93±5.42	8.04±0.94	The P value	0.000 ^{**a，c，d，f，g}	0.000	0.000 ^{**a，c，d，f，g}	0.014 ^*a

With the black fungus group of the 1X of GM-020 combination and with the black fungus group of the 10X of GM-020 combination than this negative control group, have the serum-concentration of lower triglyceride level.On the other hand, the black fungus group of 1X, the black fungus group of 10X and GM-020 group have little difference than this negative control group.In addition, the black fungus group of the black fungus group of 1X, 10X, have lower total cholesterol and the serum-concentration of HDL-C with the black fungus group of the 1X of GM-020 combination with the black fungus group of the 10X of GM-020 combination.With regard to the concentration of LDL-C, each group except this normal control group has shown little difference.

The liver concentration of metabolism of fat thing;Kill mouse, and get the lobus dexter liver.Extract fat according to known method.

With regard to each sample, can examine and determine the concentration of triglyceride level (TG) and total cholesterol (CHOL) as mentioned above.

Test analysis of data by Kruskal Wallis H, and with the data of the normal control group baseline as the Dunnett test.The results are shown in the table 8: wherein ^*Represent p＜0.01; ^aRepresent negative control; ^bRepresent positive control; ^cRepresent the black fungus of 1X; ^dRepresent the black fungus of 10X; ^eRepresent GM-020; ^fThe black fungus of the 1X of representative and GM-020 combination; And ^gThe black fungus of the 10X of representative and GM-020 combination.

Table 8

Group	CHOL	TG
Group	CHOL	TG	Negative control	25.75±2.17	135.00±11.92
Normal control	21.80±0.58	65.60±6.56	Negative control	25.75±2.17	135.00±11.92
Normal control	21.80±0.58	65.60±6.56	Positive control	26.75±1.48	103.58±11.41
The black fungus of 1X	20.75±0.85	85.50±3.76	Positive control	26.75±1.48	103.58±11.41
The black fungus of 1X	20.75±0.85	85.50±3.76	The black fungus of 10X	22.00±0.72	84.83±8.56
GM-020	19.44±0.85	88.56±6.41	The black fungus of 10X	22.00±0.72	84.83±8.56
GM-020	19.44±0.85	88.56±6.41	Black fungus with the 1X of GM-020 combination	21.25±0.70	83.25±6.27
Black fungus with the 10X of GM-020 combination	20.90±0.81	77.50±7.82	Black fungus with the 1X of GM-020 combination	21.25±0.70	83.25±6.27
Black fungus with the 10X of GM-020 combination	20.90±0.81	77.50±7.82	The P value	0.000 ^{**c，e，f，g}	0.004 ^{*a，c，d，e，f，g}

The black fungus group of 1X, GM-020 group, with the black fungus of the 1X of GM-020 combination and with the black fungus group of the 10X of GM-020 combination in each all have the serum-concentration of lower total cholesterol.With regard to triglyceride level, the black fungus group of 1X, the black fungus group of 10X, with the black fungus group of the 1X of GM-020 combination and with the black fungus group of the 10X of GM-020 combination in each all significantly reduce.

Liver and renal function analysis:Handle blood sample with aforesaid method.

With regard to each sample, can according to the method for Jaffe reaction (

Japan) concentration of examining and determine creatinine, and by Autoanalyzer Hitachi ^TM7150 measure sorption.By GOT (ASAT) IFCC mod ^TM(

Germany) concentration of examining and determine GOT, and by Autoanalyzer Hitachi ^TM7150 measure sorption.By GPT (ALAT) IFCCmod ^TM(

Germany) examine and determine the concentration of GPT, and pass through AutoanalyzerHitachi ^TM7150 measure sorption.According to uriKoxidase-superoxide enzyme method ( Japan) concentration of examining and determine uric acid (UA), and by Autoanalyzer Hitachi ^TM7150 measure sorption.

Test analysis of data by Kruskal Wallis H, and with the data of this normal control group baseline as the Dunnett test.The results are shown in the table 9: wherein ^*Represent p＜0.01; ^aRepresent negative control; ^bRepresent positive control; ^cRepresent the black fungus of 1X; ^dRepresent the black fungus of 10X; ^eRepresent GM-020; ^fThe black fungus of the 1X of representative and GM-020 combination; And ^gThe black fungus of the 10X of representative and GM-020 combination.

Table 9

Group	GOT	GPT	Creatinine	UA
Group	GOT	GPT	Creatinine	UA	The normal control group	134.00±23.11	72.67±6.98	0.52±0.04	2.66±0.92
Negative control	79.20±6.22	49.00±9.18	0.52±0.02	3.90±1.33	The normal control group	134.00±23.11	72.67±6.98	0.52±0.04	2.66±0.92
Negative control	79.20±6.22	49.00±9.18	0.52±0.02	3.90±1.33	Positive control	117.50±12.95	47.00±6.17	0.56±0.03	2.42±0.50
The black fungus of 1X	107.00±14.77	37.33±2.77	0.47±0.02	5.20±0.91	Positive control	117.50±12.95	47.00±6.17	0.56±0.03	2.42±0.50
The black fungus of 1X	107.00±14.77	37.33±2.77	0.47±0.02	5.20±0.91	The black fungus of 10X	120.00±15.78	57.42±5.68	0.46±0.02	2.88±0.57
GM-020	109.67±17.30	36.22±3.05	0.47±0.03	1.96±0.40	The black fungus of 10X	120.00±15.78	57.42±5.68	0.46±0.02	2.88±0.57
GM-020	109.67±17.30	36.22±3.05	0.47±0.03	1.96±0.40	Black fungus with the 1X of GM-020 combination	150.00±21.71	44.91±3.93	0.45±0.02	2.12±0.48
Black fungus with the 10X of GM-020 combination	76.40±13.5	37.50±3.95	0.39±0.03	2.10±0.36	Black fungus with the 1X of GM-020 combination	150.00±21.71	44.91±3.93	0.45±0.02	2.12±0.48
Black fungus with the 10X of GM-020 combination	76.40±13.5	37.50±3.95	0.39±0.03	2.10±0.36	The P value	0.072	0.002 ^{**b，c，e，f，g}	0.002 ^**g	0.006

The difference of result in these groups is not remarkable.Show that the index of liver and renal function is not affected after GM-020 and/or black fungus treatment.

Embodiment 6: the GM-020 that is used for the treatment of hypercholesterolemia

Animal model:Male hamster system is available from the Taiwan Experimental Animal Center, and under the humidity of 25 ± 1 ℃ temperature and 60 ± 5%, raises 12 hours in light separately and raised in the dark 12 hours.Abundant supplementing foodstuff and water.These mouse are divided into two groups: one group is that normal control (NC) group and another group are the group that is rich in the diet of cholesterol.This normal control group fed with normal diet and to this group that is rich in the diet of cholesterol feed to contain the diet that is rich in 2% cholesterol of 24% albumen, 14% fat, 2% cholesterol, 48% carbohydrate, 6% Mierocrystalline cellulose and 6% mineral substance and vitamine mixture.

Treatment by GM-020:Normal control group is fed continuously with normal diet.One day twice ground imposes treatment.The group that will be rich in the diet of cholesterol according to treatment further is divided into following listed group: (a) Lactobacillus gasseri, (b) GM-020, (c) negative control (Control) of giving birth to spore Bacterium lacticum (L.sporogenes) and (d) handling through physiological saline.Each dosage is 10 ⁹CFU/mL.

The serum-concentration of metabolism of fat thing:Before treatment and after the treatment, collect blood sample to be used for the biological chemistry calibrating from the vein in socket of the eye week.Blood sample was statically placed in room temperature following 1 hour, and with 2,500rpm carried out centrifugation 10 minutes.Get upper serum to be used for calibrating.

With regard to each sample, can examine and determine the concentration of total cholesterol (CHOL), HDL-C, LDL-C and triglyceride level (TG) as mentioned above.Test analysis of data by Kruskal Wallis H, and with the data of this normal control group baseline as the Dunnett test.Shown in the table 10 before feeding and the difference of the lipid content afterwards, wherein with the diet that is rich in cholesterol ^*Represent p＜0.1; ^*Represent p＜0.05; ^{* *}Represent p＜0.01; ^aRepresent negative control; ^bRepresent Lactobacillus gasseri; ^cRepresent GM-020; ^dThe spore Bacterium lacticum is given birth in representative.

Table 10:

	HDL-C_0	HDL-C_4	LDL-C_0	LDL-C_4	CHOL_0	CHOL_4	TG_0	TG_4
	HDL-C_0	HDL-C_4	LDL-C_0	LDL-C_4	CHOL_0	CHOL_4	TG_0	TG_4	NC	117.6±10.8	119.5±11.5	105.4± 8.4	243.6± 25.2	398.8± 31.2	485.5± 70.4	421.0± 59.9	365.8± 65.9
Control	70.5±3.6	64.9±5.0	23.5±1.3	20.8±1.8	104.0±4.1	96.8±5.3	224.0± 23.1	180.7± 14.8	NC	117.6±10.8	119.5±11.5	105.4± 8.4	243.6± 25.2	398.8± 31.2	485.5± 70.4	421.0± 59.9	365.8± 65.9
Control	70.5±3.6	64.9±5.0	23.5±1.3	20.8±1.8	104.0±4.1	96.8±5.3	224.0± 23.1	180.7± 14.8	Lactobacillus gasseri	141.3±10.5	103.1±3.8	179.4± 21.2	211.2± 23.9	531.6± 32.9	653.0± 45.1	585.8± 103.0	822.6± 59.1
GM-020	108.8±14.2	118.7±8.4	106.9±9.0	136.5± 19.8	412.0± 63.0	420.4± 53.0	483.3± 67.5	760.5± 98.7	Lactobacillus gasseri	141.3±10.5	103.1±3.8	179.4± 21.2	211.2± 23.9	531.6± 32.9	653.0± 45.1	585.8± 103.0	822.6± 59.1
GM-020	108.8±14.2	118.7±8.4	106.9±9.0	136.5± 19.8	412.0± 63.0	420.4± 53.0	483.3± 67.5	760.5± 98.7	Give birth to the spore Bacterium lacticum	104.9±23.7	82.8±3.0	127.1±6.6	202.3± 11.6	414.5± 18.1	541.5± 51.9	439.5± 42.0	687.8± 131.0
The P value	0.008 ^a**	0.000 ^d***	0.000 ^a，b***	0.000 ^a，c***	0.000 ^a***	0.000 ^a***	0.008 ^a**	0.000 ^b，c，d***	Give birth to the spore Bacterium lacticum	104.9±23.7	82.8±3.0	127.1±6.6	202.3± 11.6	414.5± 18.1	541.5± 51.9	439.5± 42.0	687.8± 131.0

According to this result, feeding with the diet that is rich in cholesterol after 4 weeks, this is rich in the normal control group of group of the diet of cholesterol, showed that significant CHOL, HDL-C, LDL-C and TG increase, and these child groups that are rich in the diet of cholesterol is being showed little difference each other.

With regard to TG, after treating for 4 weeks, all these child groups that are rich in the diet of cholesterol have been showed than the remarkable bigger TG of this normal control group.

With regard to CHOL, after treating for 4 weeks, Lactobacillus gasseri group, living spore Bacterium lacticum group and negative control group have been showed than the remarkable bigger CHOL of this normal control group.On the other hand, the GM-020 group has been showed the increase very little than this normal control group.Showed the difference between (CHOL_4) after (CHOL_0 before the treatment) and treatment among Fig. 7.Shown that GM-020 has the ability of the serum-concentration of reducing total cholesterol.

With regard to HDL-C, after treating for 4 weeks, give birth to spore Bacterium lacticum group and showed than the remarkable lower HDL-C of this normal control group.Yet Lactobacillus gasseri group, GM-020 group and negative control group have been showed little difference.

With regard to LDL-C, shown serum-concentration among Fig. 8.The GM-020 group has been showed the LDL-C that significantly reduces than this normal control group (p＜0.1).In addition, after treating for 4 weeks, Lactobacillus gasseri group and living spore Bacterium lacticum group have been showed than remarkable (p＜0.05) the lower LDL-C (referring to Fig. 9) of this normal control group.Shown that GM-020 has the ability of the serum-concentration of reducing total cholesterol.

With regard to LDL-C/HDL/C, showed this ratio among table 11 and Figure 10, wherein ^*Represent p＜0.1; ^*Represent p＜0.05; ^{* *}Represent p＜0.01; ^aRepresent negative control; ^bRepresent Lactobacillus gasseri; ^cRepresent GM-020; ^dThe spore Bacterium lacticum is given birth in representative.Can test analysis of data by Kruskal Wallis H, and with the data of this normal control group baseline as the Dunnett test.

Table 11

	LDL-C/HDL-C_0	LDL-C/HDL-C_4
	LDL-C/HDL-C_0	LDL-C/HDL-C_4	NC	0.98±0.07	2.13±0.47
Control	0.33±0.02	0.32±0.01	NC	0.98±0.07	2.13±0.47
Control	0.33±0.02	0.32±0.01	Lactobacillus gasseri	1.27±0.13	2.08±0.27
GM-020	0.90±0.13	1.20±0.27	Lactobacillus gasseri	1.27±0.13	2.08±0.27
GM-020	0.90±0.13	1.20±0.27	Give birth to the spore Bacterium lacticum	1.46±0.42	2.44±0.14
The P value	0.003 ^a**	0.000 ^a，c***	Give birth to the spore Bacterium lacticum	1.46±0.42	2.44±0.14

The GM-020 group has been showed significant reduction than this normal control group (p＜0.001).Shown that GM-020 has the ability that reduces LDL-C/HDL-C.

Though illustrated and described embodiments of the invention, the person of ordinary skill in the field can do various modifications and improvement.And be not intended to the present invention is limited to embodiment as described, and all modifications that do not deviate from the spirit and scope of the present invention all belong to as in the scope that is defined in the claim of enclosing.

Claims

1. the separated microorganism of a strain, lactobacillus rhamnosus (Lactobacillusrhamnosus) GM-020, it is preserved in Chinese typical culture collection center with deposit number CCTCC M 203098.

2. composition that comprises microorganism according to claim 1.

3. lactobacillus rhamnosus bacterial strain GM-020 is used for the treatment of application in the medicine of obesity and complication thereof in preparation, and wherein lactobacillus rhamnosus bacterial strain GM-020 is preserved in Chinese typical culture collection center with deposit number CCTCC M203098.

4. application according to claim 3, wherein said medicine further comprises black fungus.

5. application according to claim 3, wherein said complication is selected from the group of being made up of hypercholesterolemia, atherosclerosis and coronary heart disease.

6. lactobacillus rhamnosus bacterial strain GM-020 and black fungus are used for the treatment of application in the medicine of obesity and complication thereof in preparation, and wherein lactobacillus rhamnosus bacterial strain GM-020 is preserved in Chinese typical culture collection center with deposit number CCTCC M 203098.

7. application according to claim 6, wherein said complication is selected from the group of being made up of hypercholesterolemia, atherosclerosis, coronary heart disease, fatty liver and diabetes.

8. application according to claim 6, wherein said complication is selected from the group of being made up of hypercholesterolemia, atherosclerosis and coronary heart disease.