CN100396770C - Novel microorganism Lactobacillus rhamnosus GM-020 and application thereof in treating obesity - Google Patents

Abstract

The invention provides a separated microorganism Lactobacillus rhamnosus GM-020, and the Lactobacillus rhamnosus GM-020 is found to be effective in treating obesity and complications thereof. The invention also provides the use of Lactobacillus rhamnosus GM-020 for the treatment of obesity and its complications.

Description

Novel microorganism Lactobacillus rhamnosus GM-020 and its use in treating obesity

technical field

The present invention mainly relates to a new strain of microorganism Lactobacillus rhamnosus GM-020 and its use for treating obesity or its complications.

Background technique

Obesity is usually excess body fat due to altered physiological or biochemical function, and it can significantly impair health. Fats generally include neutral lipids, phospholipids and cholesterol. Weight gain depends on a person's energy intake being greater than energy expenditure. There are two types of obesity: (a) simple obesity and (b) second obesity. Simple obesity can be divided into idiopathic obesity and acquired obesity, which may account for more than 95% of obesity. Congenital obesity is caused by a large number of fat cells and is common in childhood obesity. Acquired obesity results from adipocytes of larger size and is common in obesity in adulthood. Secondary obesity, also known as symptomatic obesity, is usually caused by endocrine or metabolic diseases. Obesity is associated with several chronic diseases such as diabetes, cardiopathy, hypertension, stroke, biliary calculus, gout and certain cancers.

There are currently five strategies for treating obesity: diet reduction, exercise, behavioral therapy, drug therapy, and therapeutic operation. Depending on the patient's health risk factors and the speed and effect of weight loss, these strategies can be selected or combined to treat obese patients. The speed and effect of weight loss are affected by multiple factors such as age, height, family history and risk factors. Impact. Mechanisms of drug therapy include appetite suppression, increased energy expenditure, stimulation of fat mobilization, decreased triglyceride synthesis, and inhibition of fat absorption. Examples of such drugs are phenylpropanolamine (PPA), orlistat (Xenical ^™ ) and sibutramine (Reductil ^™ ). However, it is a new trend to treat obesity by natural substances instead of drugs.

In the prior art, it was found that some microorganisms could be used to treat obesity, or its complications. For example, Lactobacillus gasseri (Lactobacillus gasseri) SBT0270 was found to have the ability to reduce cholesterol concentrations associated with deconjugation of bile acids ((Usman, B. and Hosono, A. (2001), "Lactobacillus gasseri (Lactobacillus gasseri) SBT0270 induces a hypocholesterolemic effect in rats fed a cholesterol-enriched diet" (Hypocholesterolemic effect of Lactobacillus gasseri SBT0270 in rats fed a cholesterol-enriched diet), J. Dairy Res. 68: 617-624. Treatment of Obesity The mechanism is: by Lactobacillus gasseri (L. gasseri) to absorb bile acids from the form and excreted in the feces (because the excreted bile acids cannot be recycled back to the liver, and new ones need to be synthesized from cholesterol bile acids) to reduce the solubility of the decomposed bile acids. In addition, it was found that Lactobacillus gasseri (L. gasseri) can combine with bile acids and cholesterol to achieve excretion.

Lactobacillus rhamnosus is considered a potential probiotic lactic acid bacterium with immune-enhancing properties. The safety assessment of Lactobacillus rhamnosus has also been investigated. Zhou et al. revealed hematological parameters (red blood cell and platelet counts, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration), different white blood cell counts, blood biochemistry in mice administered Lactobacillus rhamnosus. (total plasma protein, albumin, cholesterol, and glucose), mucosal histology (epithelial cell height, mucosal thickness, and villi height), and bacterial contamination to extraintestinal tissues (blood, liver, spleen, kidney, and mesenteric lymph nodes) displacement, showing a profile similar to that of control mice (Zhou, J.S., Shu, Q., Rutherfurd, K.J., Prasad, J., Birtles, M.J., Gopal, P.K. and Gill, H.S. (2000), "BALB/ Safety assessment of potential probiotic lactic acid bacteria strains Lactobacillus rhamnosus HN001, Lactobacillus acidophilus HN017, and Bifidella HN019 in mice" Safety assessment of potential probiotic lactic acidbacterial strains Lactobacillus rhamnosus HN001 , Lb. acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice), International Journal of Food Microbiology56: 87-96). In addition, Agerholm-Larsen et al. revealed that administration of yogurt fermented by Lactobacillus rhamnosus did not change low-density lipoprotein (LDL)-cholesterol, but only significantly reduced systolic blood pressure (Agerholm-Larsen, L., Raben , A., Haulrik N., Hansen, A.S., Manders, M., and Astrup A. (2000), "Effect of 8 week intake of probiotic dairy products on risk factors for cardiovascular disease" probiotic milk products onrisk factors for cardiovascular diseases), Eur J Clin Nutr.54(4):288-97). Thus, it can be shown that the known strains of Lactobacillus rhamnosus do not alter plasma total cholesterol and LDL-cholesterol. In addition, no change in body weight was observed when known strains of Lactobacillus rhamnosus were administered.

Contents of the invention

The present invention provides a new isolated microorganism, Lactobacillus rhamnosus (Lactobacillus rhamnosus) GM-020, which is preserved in China Center for Type Culture Collection with the preservation number CCTCC M 203098.

In another aspect, the present invention provides a composition comprising Lactobacillus rhamnosus GM-020.

In another aspect, the present invention provides the application of Lactobacillus rhamnosus strain GM-020 in the preparation of medicines for treating obesity and its complications.

In another aspect, the present invention provides the application of Lactobacillus rhamnosus strain GM-020 and black fungus in the preparation of medicines for treating obesity and its complications.

In another aspect, the present invention provides a method for treating obesity and its complications in a subject, the method comprising administering to the subject a composition comprising the microorganism Lactobacillus rhamnosus GM-020; wherein The complication is preferably selected from the group consisting of hypercholesterolemia, atherosclerosis and coronary heart disease.

In another aspect, the present invention provides a method for treating obesity and its complications in a subject, the method comprising administering to the subject a method comprising microorganism Lactobacillus rhamnosus GM-020 and black fungus ( Auricularia polytricha); wherein the complication is preferably selected from the group consisting of hypercholesterolemia, atherosclerosis, coronary heart disease, fatty liver and diabetes.

Description of drawings

Figure 1 illustrates a 1000X microscopic view of GM-020.

Figure 2 illustrates the cell wall proteins of GM-020 and known Lactobacillus rhamnosus strains; wherein M represents protein molecular weight; Lane 1 represents Lactobacillus rhamnosus (commodity Antibiophilus); Lane 2 represents Lactobacillus rhamnosus GM -020; Lane 3 represents Lactobacillus rhamnosus ATCC9595; Lane 4 represents Lactobacillus rhamnosus ATCC10940; and Lane 5 represents Lactobacillus rhamnosus ATCC14029.

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

Fig. 4 illustrates the difference in body weight of the animal model treated by GM-020 or/and black fungus (wood ear) according to Example 5; where ^** represents p<0.01; ^a represents negative control; ^b represents positive control; ^c represents 1X black fungus; ^d represents 10X black fungus; ^e represents GM-020; ^f represents 1X black fungus combined with GM-020; and ^g represents 10X black fungus combined with GM-020.

Figure 5 illustrates the difference in lipid tissue weight around the testes of the animal model treated with GM-020 or/and black fungus according to Example 5; where ^** represents p<0.01; ^a represents a negative control; ^b represents a positive control ^c represents 1X black fungus; ^d represents 10X black fungus; ^e represents GM-020; ^f represents 1X black fungus combined with GM-020; and ^g represents 10X black fungus combined with GM-020.

Figure 6 illustrates the difference in lipid tissue weight around the kidneys of animal models treated with GM-020 or/and black fungus according to Example 5; where ^** represents p<0.01; ^a represents a negative control; ^b represents a positive control ^d represents 10X black ^fungus ; ^e represents GM-020; ^f represents 1X black fungus combined with GM-020; and ^g represents 10X black fungus combined with GM-020.

Figure 7 illustrates the difference between the serum concentrations of total cholesterol before (CHOL_0) and after treatment (CHOL_4) according to Example 6; wherein ^* represents p<0.1; ^** represents p<0.05; ^*** represents p<0.01 .

Figure 8 illustrates the serum concentration of LDL-C before treatment (LDL-C_0) and after treatment (LDL-C_4) according to Example 6; where ^* represents p<0.1; ^** represents p<0.05; ^*** represents p<0.01.

Figure 9 illustrates the difference in serum concentration of LDL-C before treatment (LDL-C_0) and after treatment (LDL-C_4) according to Example 6; wherein ^* represents p<0.1; ^** represents p<0.05; ^{** *} represents p<0.01.

Figure 10 illustrates the difference in serum concentrations of LDL-C/HDL-C before treatment (LDL-C/HDL-C_0) and after treatment (LDL-C/HDL-C_4) according to Example 6; where ^* represents p <0.1; ^** represents p<0.05; ^*** represents p<0.01.

Detailed ways

The invention provides a new strain of microorganism Lactobacillus rhamnosus GM-020, which can treat obesity. On December 18, 2003, the strain GM-020 was deposited in the China Center for Type Culture Collection with the accession number CCTCC M 203098.

Lactobacillus rhamnosus GM-020 was isolated from human stomach.

The eubacterial characteristics of Lactobacillus rhamnosus GM-020 are shown below:

(a) Morphological features

(1) Shape and size of cells: When cells were cultured overnight in MRS medium at 37°C, bacilli could be observed through a microscope, which were rod-shaped with rounded edges.

(2) Mobility: inactive

(3) Flagella: None

(4) Sporulation: no sporulation

(5) Gram stain: positive

(b) Culture characteristics:

0881)(如表1中所示)，最终pH值6.5±0.2(1) Medium: MRS Medium (

0881) (as shown in Table 1), the final pH value is 6.5 ± 0.2

Table 1

Component g/L Proteose peptone 10.0 Beef Extract 10.0 Yeast Extract 5.0 Dextrose 20.0 Polysorbate 80 (Polysorbate80) 1.0 ammonium citrate 2.0 Sodium acetate 5.0 Magnesium Sulfate 0.1 Manganese sulfate 0.05 Dipotassium Phosphate 2.0

(2) Culture conditions: anaerobic culture (anaerobic culture) or aerobic culture (aerobic culture) at 37°C

(c) Physiological characteristics:

(1) Catalase: Negative

(2) Oxidase: Negative

(3) API 50CHL test: Use the API 50CHL system to identify Lactobacillus. The character of the lactobacillus can be determined by assaying a series of enzyme reactions. The results of the API 50 CHL test for GM-020 are listed in Table 2:

Table 2

enzyme reaction enzyme reaction enzyme reaction Glycerin - Mannitol + D-Tagatos + Erythritol - Sorbitol + 5-ketogluconic acid - D-arabinose - α-Methyl-D-glucoside + 2-ketogluconic acid - L-Arabinose - N-acetylglucosamine + Gluconic acid - Ribose + Laetrile + L-Arabitol - D-xylose - Arbutin (Arbutine) + D-Arabitol - L-xylose - Esculine + L-fucose - Side calendula alcohol (Adonitol) - Salicin + D-fucose - β-Methyl-xyloside - Cellobiose + D-Lyxose - D-glucose + Galactose + Anisyl Niuline (inuline) - D-Fructose + Lactose + Sucrose - D-Mannose + α-Methyl-D-mannoside - Glycogene - L-Sorbose + Melezitose + Xylitol - D + D-Raffinose (D-Raffinose) - β Gentiobiose - Dulcitol - Methadone (Amidon) - D-Turanose - Inositol - Maltose - Melibiose - trehalose -

(d) Genetic characteristics:

The 16S rDNA sequence analysis of GM-020, as shown in SEQ ID NO: 1, was determined by the Institute of Food Industry Development, Hsinchu, Taiwan. The results showed that GM-020 could be highly homologous to other Lactobacillus rhamnosus strains, with more than 99% similarity.

(e) Cell wall protein of GM-020:

The cell wall proteins of GM-020 showed a specific pattern when compared with other known L. rhamnosus strains. Figure 2 shows the SDS-PAGE pattern of the cell wall proteins of GM-020.

The total protein of GM-020 can be subjected to 2-D electrophoresis, and the pattern shown in Figure 3 is specific.

(f) Standardized detection system used to identify GM-020:

A standard assay system that can be used to identify microorganisms is disclosed in U.S. Patent Application Serial No. 10/446,781, filed May 29, 2003, using test cell lines cultured and unincubated with a given microorganism Differences in gene expression were used as markers for identification. The genes tested are listed in Table 3.

table 3

Gene Signal transducer and activator of transcription 3 c-rel Growth-associated protein 43 N-myc IGF-binding protein 1 IL-16 Lymphotoxin α (formerly TNF-β) interferon-inducible protein 9-27 connective tissue growth factor

Interleukin 10 receptor Calpamodulin mRNA Ubiquitin crosslinking enzyme (UbcH8) mRNA, complex (comp) Modern human (Homo sapiens) protein kinase A binding protein AKAP110 mRNA, complete cds Pyruvate dehydrogenase kinase, isoenzyme 4 Pyridoxal (pyridoxine, vitamin B6) kinase MAP Kinase Interacting Serine/Threonine Kinase 1 leukocyte tyrosine kinase Neurotrophic tyrosine kinase, receptor, type 3 (TrkC) Pyruvate dehydrogenase kinase isoenzyme 3 (PDK3) mRNA, complete cds Human diacylglycerol kinase ζ (diacylglycerol kinase zeta) mRNA, complete cds Protein kinase C, alpha Deoxyguanosine kinase Adenosine kinase Topoisomerase (DNA) IIβ (180kD) IkB kinase β subunit (beta subunit) mRNA stat5a (signal transducer and activator of transcription 5A) Colony-stimulating factor 1 (M-CSF) HGF IL-1 receptor type 1 Interleukin 7 Metallothionein I-B gene Interleukin 6 (B cell stimulating factor 2) Inducible small molecule cytokine A2 (Small inducible cytokine A2) (monocyte chemoattractant protein 1) Proteasome 26S subunit, ATPase, 3

Ubiquitin crosslinker E2A (RAD6 homologue) thymidine kinase 1, soluble tyrosine kinase 2 serine/threonine protein-kinase Transforming growth factor beta-activated kinase 1 Tyrosine kinase 3 associated with Fms creatine kinase B protein serine/threonine kinase stk2 Stress-activated protein kinase 3 (SAPK3) mRNA. Human adenylate kinase 2 (adk2) mRNA, complete cds RAC-alpha serine/threonine kinase hexokinase 1 CDC28 protein kinase 2 (CKS2) mRNA superoxide dismutase 2, mitochondria tumor necrosis factor receptor 2 growth associated protein 43 Genes related to p53 CD30 Metallothionein-III interleukin-4 receptor Interferon-γ-inducible protein IP-30 precursor Interferon-alpha/beta receptor alpha chain precursor early growth response protein 1 Interleukin-13 receptor mRNA, complete cds Protease inhibitor 12 (PI12; neuroserpin)

Serine/threonine protein kinase KKIALRE Phosphorylase kinase, β serine/threonine kinase 9 Serine/Threonine Kinase 10 Protein kinase mitogen-activated 8 (MAP kinase) Focal adhesion kinase (FAK) mRNA, complete cds human activated p21cdc42Hs kinase (ack) mRNA, complete cds Human integrin-linked kinase (ILK) mRNA, complete cds Human guanylate kinase (GUK1) mRNA, complete cds BMK1α kinase mRNA, complete cds Flavin-containing monooxygenase 5 (FMO5) mRNA pyruvate kinase, liver Transcription elongation factor S-II, hS-II-T Nitric oxide synthase 3 (endothelial cells) Bcl2, p53 binding protein Bbp/53BP2 (BBP/53BP2) mRNA, Telomeric DNA sequence stat-like protein (Fe65) mRNA, complete cds IL-5 receptor a EGF FGF2 Interleukin 2 receptor gamma chain C-C chemokine receptor type 2 Guanylate-binding protein 1, interferon-inducible, 67kD Mitochondrial processing peptidase β-subunit syntaxin 8

Cytokine inhibits anti-inflammatory drug-binding protein 1 (p38MAP kinase) protein tyrosine kinase 6 branched-chain alpha-ketoacid dehydrogenase kinase serine/threonine kinase 2 Protein kinase, mitogen-activated 4 (MAP kinase 4; p63) (PRKM4) mRNA Tyrosine-protein kinase SYK Human putative serine/threonine protein kinase PRK (prk) mRNA, complete cds Adenylate kinase isoenzyme 1 hematopoietic kinase Glycerol Kinase Tyrosine-protein kinase CSK General transcription factor IIB Interleukin 6 signaling (gp130, oncostatin M receptor) Caspase-8 (apoptotic caspase mch5(mach-α-1)) Tumor protein p53 Transforming growth factor, beta receptor III (beta glycans, 3 IFN-g Transforming growth factor, β3 TGF-β protein superfamily, complete Fibroblast growth factor 7 (keratinocyte growth factor) Inducible small molecule cytokine A4 (homologous to mouse Mip-1b) hepatocyte growth factor activator inh ubiquitin carboxyl-terminal hydrolase isoenzyme 11 Serine/Threonine Kinase 11 (Pigmented Polyposis Syndrome Cyclin-dependent kinase inhibitor 3 (CDK2-related dual-specificity phosphatase)

Pyruvate dehydrogenase kinase, isoenzyme 3 Carnitine palmitoyltransferase I, liver Protein Kinase Mitogen Activator 7 (MAP Kinase) Human protein tyrosine kinase mRNA, complete cds protein tyrosine kinase 7 Lymphocyte specific protein tyrosine kinase ribosomal protein s6 kinase src-like kinase (slk) mRNA, complete cds nucleoside diphosphate kinase a cyclin-dependent kinase 2 STAT-1α/β Angiopoietin-1 Phospholipase C STAT2 (signal transducer and activator of transcription 2) c-src tyrosine kinase IL-15 TGFb receptor-associated protein 1 Annexin V (lipocertin V; Intrin II) interferon regulatory factor 5 Interferon-γ receptor alpha chain precursor Transforming growth factor, beta receptor II (70-80kD) Modern human caspase activator 1 (Apaf-1) Ubiquitin cross-linking enzyme E2B (RAD6 homologue) MAP/ERK Kinase Kinase 3 Phosphorylase Kinase, Alpha 2 (Liver), Glycostoria IX

serine/threonine kinase 4 Glucosamine-6-phosphate deaminase Mevalonate kinase (Mevalonate kinase) Glucokinase (hexokinase 4, maturity onset diabetes of the young 2) Deoxycytidine kinase Urokinase-type plasminogen activator Human mitochondrial creatine kinase (CKMT) gene, complete cds Choline kinase 53K isoform of human phosphatidyl ino-4-phosphate 5-kinase (PIPK) type II mRNA, complete cds Leukocyte adhesion protein beta subunit c-tos Phosphoenolpyruvate carboxykinase Apoptotic cysteine protease mch4 monocyte chemoattractant protein 1 Transcription factor AP-4 (activation enhancer-binding protein Interleukin-1 receptor, type 1 precursor Caspase-10 (human apoptotic caspase mch4) human kinase (TTK) mRNA, complete cds beta-2 adrenergic receptor Estrogen sulfotransferase (ste) Signal transducer and activator of transcription 6, induced by interleukin-4 protein kinase clk1 Interleukin-8 precursor Modern human FAST kinase mRNA Interferon-γ receptor alpha chain precursor

Insulin-like growth factor I receptor precursor Mitochondrial transcription termination factor Signal transducer and activator of transcription 3 (acute-ph Modern human protein kinase CK1 mRNA MAP kinase-activated protein kinase protein kinase clk3 INF-b General transcription factor IIB Sis, PDGF B chain β-actin Glutathione S-transferase M1 IL-1b MAP/ERK Kinase Kinase 3 INF-b EGR-1 Glutathione S-transferase 12 (microsomal)

The standard assay system used to identify GM-020 uses the Hep G2 cell line as the test cell line. The set of genes listed in Table 4 can differ significantly when the expression patterns of Hep G2 cell lines cultured with or without GM-020 are compared.

Table 4

Gene Interleukin 10 receptor IL-16 EGF Lymphotoxin alpha (formerly tumor necrosis factor beta) Interferon regulatory factor 5 Fibroblast growth factor 7 (keratinocyte growth factor) Proteosome 26S subunit, ATPase, 3 Calpamodulin mRNA Ubiquitin carboxyl-terminal hydrolase isozyme L1 Hexokinase 1 53K isoform of human phosphatidylinositol-4-phosphate 5-kinase (PIPK) type II mRNA, complete cds Mitochondrial transcription terminal factor STAT-1α/β Urokinase-type plasminogen activator Human adenylate kinase 2 (adk2) mRNA, complete cds Protein kinase C, alpha Proto-oncogene tyrosine-protein kinase FES/FPS Human mitochondrial creatine kinase (CKMT) gene, complete cds protein tyrosine kinase 6 serine/threonine kinase 9 IkB kinase β subunit mRNA Caspase-8 (apoptotic caspase mch5(mach-α-1)) Bcl2, p53 binding protein Bbp/53BP2 (BBP/53BP2) mRNA,

Growth-associated protein 43 protein kinase clk3 Tumor necrosis factor inducible protein TSG-6 precursor Insulin-like growth factor I receptor precursor monocyte chemoattractant protein 1 Leukocyte adhesion protein beta subunit Sis, PDGF B chain Humig mRNA CD27L receptor precursor Retinoic acid (retinoic acid) and interferon-inducible 58K protein RI IRF-1

The present invention provides a method for treating obesity and its complications in a subject, the method comprising administering to the subject a composition comprising GM-020.

In the present invention, it was surprisingly found that the strain GM-020 has the ability to inhibit the subject's weight gain even if the subject eats high-energy food, and can suppress the body weight. In one embodiment of the present invention, ICR mice treated with strain GM-020 and fed a high-energy diet that can cause obesity can maintain body weight without any loss compared with untreated controls. Increase.

It was also found in the present invention that strain GM-020 is effective in modulating the cholesterol to lipoprotein ratio. In an embodiment according to the present invention, in obese mice and hamsters fed a cholesterol-rich diet that can cause hypercholesterolemia, treatment with strain GM-020 can reduce the levels of total cholesterol in their serum and liver. concentration. It also lowers the serum concentration of low-density lipoprotein-cholesterol (LDL-C). In addition, it can reduce the ratio of LDL-C to high-density lipoprotein cholesterol (HDL-C) (LDL-C/HDL-C) in serum. In summary, strain GM-020 is effective in treating hypercholesterolemia and reducing the incidence of atherosclerosis and coronary heart disease. That is, strain GM-020 can be used to prepare a composition for treating obesity and its complications (such as hypercholesterolemia) and reducing the incidence of atherosclerosis and coronary heart disease.

The present invention also provides a method for treating obesity and its complications in a subject, the method comprising administering to the subject a composition comprising the microorganism Lactobacillus rhamnosus GM-020 and black fungus strain; Wherein the complication is preferably selected from the group consisting of hypercholesterolemia, atherosclerosis, coronary heart disease, fatty liver, and diabetes.

In the present invention it was found that strain GM-020 in combination with black fungus (Auricularia nigra) provides a surprising effect in the treatment of obesity compared to treatment with black fungus or strain GM-020 alone.

Black fungus, commonly known as fungus, tree ear (tree ear), etc., is a colloid, odorless, edible fungus. It is closely related to the black fungus grown in Asia and has been consumed for a long time. Black fungus is found wild on conifers and sometimes on deciduous trees in spring and fall. Black fungus is usually dried for consumption. When the dried black fungus is eaten, the edible fiber can be extended about 8 times to 10 times after absorbing water. Consumers can feel full and eat less. In addition, it has been reported that polysaccharides in black fungus have the effect of lowering the serum concentration of total cholesterol in rabbits.

In an embodiment according to the present invention, the body weight of obese animal models treated with the combination of black fungus and GM-020 can be reduced; on the contrary, administration of black fungus or GM-020 alone can only inhibit the weight gain.

It was found in the present invention that the combination of black fungus and strain GM-020 has the ability to reduce the concentration of total cholesterol and triglyceride in the serum and liver of this animal model compared to the control group. In summary, the combination of black fungus and strain GM-020 can be used to prepare a composition for treating hypercholesterolemia, fatty liver, diabetes and reducing the incidence of atherosclerosis and coronary heart disease.

According to an embodiment of the present invention, the effects of treating obesity and hypercholesterolemia and reducing the incidence of atherosclerosis and coronary heart disease are directly proportional to the dose of black fungus. Normally, the recommended daily dose (1X) of black fungus for adults is 6gx 0.0026x surface area. In an embodiment of the present invention, compared with 1X black fungus, 10X black fungus has a better effect.

According to the present invention, only strain GM-020, and the combination of GM-020 and black fungus will not cause damage to liver and kidney. In animal models, liver function was normal when the amounts of glutamicoxaloacetic transaminase (GOT), glutamic pyruvictransaminase (GPT), uric acid, and creatinine were monitored. It shows that the administration of strain GM-020 alone, or the combination of strain GM-020 and black fungus is a safe way to treat obesity and its complications. It is also found in the present invention that after treatment with the combination of GM-020 and black fungus, the triglyceride in serum is reduced.

The following examples are given for the purpose of illustration only and are not intended to limit the scope of the invention.

Example

Example 1: Isolation of Lactobacillus rhamnosus GM-020

0881)中。将包含该组织的培养基平放于乳杆菌的选择性琼脂上且在37℃下培育一天。选择生长于该培养板上的单个集落并使其接受革兰氏染色。接着选择革兰氏阳氏细菌。克隆一个株，该株称为鼠李糖乳杆菌GM-020。A piece of gastric tissue taken out by an endoscope (endoscope) was cultured in 2 mL of Lactobacillus MRS medium (

0881). The medium containing the tissue was plated on Lactobacillus selective agar and incubated at 37°C for one day. Single colonies growing on this plate were selected and subjected to Gram staining. Gram positive bacteria are then selected. One strain was cloned, which was called Lactobacillus rhamnosus GM-020.

The cell wall protein extraction and analysis of embodiment 2GM-020

)中的嗜温乳杆菌的24小时大的细胞，将其于含0.1M CaCl₂的0.05M Tris-HCl(pH7.5)中冲洗两次，且再次悬浮于A₆₀₀为10.0的1ml相同的缓冲剂中。在8,000×g下进行离心作用5分钟后，通过含0.01M EDTA、0.01M NaCl，和2％(wt/vol)SDS的1.0ml的萃取缓冲剂(pH 8.0)，可从这些球粒(pellet)来萃取细胞壁蛋白。在室温下，将悬浮液储存60分钟，在100℃下加热5分钟并在4℃下以11,600×g进行离心作用10分钟。通过12％SDS-PAGE来分析上层清液且以Comassie蓝色来对其染色。harvested from MRS medium (

24-hour-old cells of mesophilic Lactobacillus ) in ), washed twice in 0.05M Tris-HCl (pH 7.5) containing 0.1M CaCl ₂ , and resuspended in 1 ml of the same A ₆₀₀ of 10.0 in the buffer. After centrifugation at 8,000 × g for 5 minutes, the pellets can be extracted by passing through 1.0 ml of extraction buffer (pH 8.0) containing 0.01M EDTA, 0.01M NaCl, and 2% (wt/vol) SDS. ) to extract cell wall proteins. The suspension was stored at room temperature for 60 minutes, heated at 100°C for 5 minutes and centrifuged at 11,600 xg for 10 minutes at 4°C. Supernatants were analyzed by 12% SDS-PAGE and stained with Comassie blue.

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

Along with 0.5 mL lysis buffer C (7M urea, 4% CHAPS, 2M thiourea, 40 mM Tris, 0.5% IPG buffer and 5 mM TBP) and 100 L of glass beads, 10 mg GM-020 was added. The solution was then subjected to sonication and centrifugation at 10,000 rpm for 30 minutes. Total protein can be determined by the Bio-rad PlusOne ^™ protein assay followed by 2-D electrophoresis at pH 3 to 10 IEF. Electrophoresis as programmed in Table 5 was performed through a 10% gel at 40 mA/gel for 4 hours. The gel was then fixed by 10% methanol and 7% acetic acid for 30 minutes. After fixation, the gel was stained by sypro ruby stain for 5 hours and then destained with 10% methanol and 7% acetic acid for 6 hours. The result is shown in Figure 3.

table 5

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

Embodiment 4 is used to identify the standardized detection system of GM-020

0881)中，将株GM-020的细胞培育到静止相。在3,000g下进行离心作用15分钟后，通过2mL和1mL的1X的PBS(磷酸缓冲盐水，pH7.2)来冲洗该培养物，并接着将其悬浮于1mL的PBS(1X)中，其中细胞浓度被调节到1×10⁹CFU/mL。将这些培养物储存在-20℃下。 Preparation of GM-020 for standardized detection system: at 37°C Lactobacillus MRS medium (

0881), cells of strain GM-020 were grown to stationary phase. After centrifugation at 3,000 g for 15 minutes, the culture was washed by 2 mL and 1 mL of 1X PBS (phosphate-buffered saline, pH 7.2), and then suspended in 1 mL of PBS (1X), where the cells The concentration was adjusted to 1×10 ⁹ CFU/mL. These cultures were stored at -20°C.

Stimulation : Hep G2 cells were refreshed by adding fresh medium and incubating for 16 hours. Subsequently, these cells were divided into two groups, and one group was used for culture with lactic acid bacteria and the other group was used for culture without lactic acid bacteria. When the cell concentration reached 1×10 ⁷ /10 mL, the cells were stimulated with or without 1× ^{10 7 GM} -020 for 24 hours. After stimulation, the cells were harvested, washed twice by PBS, and used for RNA isolation.

Gaithersburg，Md.)，从细胞萃取RNA。将8L RNA(10μg)与2L低聚dT(12-18mer，0.1g/L)充分混合并在70℃下保持10分钟，且接着通过冰来冷却2分钟。在黑暗中，将RNA与反转录标记混合液和3L Cy5-dUTP(1mM)、2L SuperScriptII(200U/L)和RNasin(1L)混合。在42℃下将混合物培育2小时以用于反转录，且通过添加1.5L的20mM EDTA来终止反应。标记之后，通过NaOH处理来移除RNA，并通过HCl来将其中和。通过STRATAGENE^TMPCR纯化试剂盒来迅速地纯化cDNA。 RNA isolation and labeling (labelling): according to the manufacturer's instructions by using Trizol reagent (Life

Gaithersburg, Md.), RNA extraction from cells. 8 L of RNA (10 μg) was mixed well with 2 L of oligo dT (12-18mer, 0.1 g/L) and kept at 70° C. for 10 minutes, and then cooled by ice for 2 minutes. In the dark, RNA was mixed with reverse transcription labeling mix and 3L Cy5-dUTP (1mM), 2L SuperScriptII (200U/L) and RNasin (1L). The mixture was incubated for 2 hours at 42°C for reverse transcription, and the reaction was stopped by adding 1.5 L of 20 mM EDTA. After labeling, RNA was removed by NaOH treatment and neutralized by HCl. The cDNA was rapidly purified by the STRATAGENE ^™ PCR Purification Kit.

Inc.，Corning，N.Y.)上。印刷(printing)之后，这些微阵列为在室温下被储存于一干燥器中的载玻片容器中的在300mJoulesand下的紫外线交联。表3中列出了这些基因。 Microarray Fabrication: Hundreds of selected genes were amplified by polymerase chain reaction and quantified by 260nm spectrophotometry. All purified PCR products were adjusted to a concentration of 0.1 μg/μL in 50% DMSO and spotted in duplicate on UltraGAPSTM ^TM coated slides (

Inc., Corning, NY). After printing, the microarrays were UV crosslinked at 300 mJoulesand stored in a desiccator at room temperature in slide containers. These genes are listed in Table 3.

Microarray hybridization: Denature fluorescently labeled cDNA in hybridization solution (5xSSC, 0.1% SDS and 25% formamide) at 100°C for 5 minutes, cool to ambient temperature and store on glass slides . Hybridization was performed at 42°C for 18 hours. After hybridization, the slides were washed sequentially in low-stringency (1xSSC and 0.1% SDS), medium-stringency (0.1xSSC and 0.1% SDS), high-stringency (0.1xSSC) buffers, And finally it was dried by compressed _N2 .

4000B扫描仪(Axon

Inc.)上立即扫描经N2干燥的载玻片。可获得原始的荧光资料(10-nm分辨率)，且在Microsoft Excel^TM中来执行随后的处理和资料可视化。为比较独立杂交实验的结果，局部背景信号(local backgroundsignal)被从各个独立点的杂交信号中减去，并接着除以管家基因β-肌动蛋白(housekeeping gene β-actin)。以双份的平均值来代表各个基因的最后表达。接着可获得经GM-020以及未经GM-020培养的Hep G2细胞的基因表达图谱(expression profile)。在经GM-020培养的Hep G2中选择一群相比于未经该细菌培养的Hep G2被向上或向下调整超过2倍(fold)的基因。结果如表4中所示。 Signal detection and data analysis: for each slide with the same laser power and photomultiplier sensitivity level (sensitivity level), in

4000B scanner (Axon

Inc.) and immediately scan the N2-dried slides. Raw fluorescence data (10-nm resolution) were obtained, and subsequent processing and data visualization were performed in Microsoft Excel ^™ . To compare the results of independent hybridization experiments, the local background signal was subtracted from the hybridization signal of each independent spot and then divided by the housekeeping gene β-actin. The final expression of each gene is represented as the average of duplicates. Then the gene expression profile (expression profile) of Hep G2 cells cultured with GM-020 and without GM-020 can be obtained. In Hep G2 cultured with GM-020, a group of genes that were up- or down-regulated more than 2-fold (fold) was selected compared to Hep G2 cultured without the bacteria. The results are shown in Table 4.

Example 5 GM-020 for the treatment of obesity

Animal model: Male ICR mice can be purchased from the Taiwan Experimental Animal Center and raised at a temperature of 25±1°C and a humidity of 60±5% for 12 hours in the light alone and in the dark for 12 hours. Adequate food and water. These mice were divided into two groups. One group was the normal control group and the other was the high energy group. The normal control group was fed a normal diet and the high energy group was fed a high energy diet containing 48% dry feed, 8% corn oil and 44% concentrated milk. The body weight of the mice was measured weekly. The high energy group was further divided according to treatment into the groups listed below: (a) 1X black fungus, (b) 1X black fungus combined with GM-020, (c) GM-020, (d) 10X Black fungus, (e) 10X black fungus combined with GM-020, (f) negative control treated with normal saline, and (g) positive control treated with PPA. Table 6 shows the difference in body weight before and after being fed with a high-energy diet, where ^** represents p<0.01; ^a represents a negative control; ^b represents a positive control; ^c represents 1X black fungus; ^d represents 10X black fungus Fungus; ^e represents GM-020; ^f represents black fungus combined with GM-020 1X; and ^g represents black fungus combined with GM-020 10X.

Table 6

Group weight(%) normal control 12.25±1.80 negative control 20.93±2.27 positive control 20.43±1.35 1X black fungus 22.45±1.46 10X black fungus 18.45±1.03 GM-020 19.23±1.75 1X black fungus combined with GM-020 21.37±1.05 10X black fungus combined with GM-020 22.78±0.67 P value 0.000^{**a, b, c, d, e, f, g}

The data can be analyzed by Kruskal Wallis H test, and the normal control group is used as the baseline of Dunnett test. After 4 weeks, the average body weight of the high energy group was significantly higher than that of the normal control group (p<0.01).

Treatment with GM-020 and/or black fungus: The normal control group was fed with normal diet continuously. Apply the treatment twice a day. Each dose of PPA is 4.875mg. In a 1X diet of black fungus, there was 15.6 mg of black fungus in a 3 g diet, and in a 1OX diet of black fungus, there was 156 mg of black fungus in a 3 g diet. The dose of GM-020 was 10 ⁹ CFU/mL.

Body Weight Differences: After 4 weeks of treatment, blood samples were collected from the tail for biochemical assays. Data were analyzed by Kruskal Wallis H test, and the normal control group was used as the baseline by Dunnett's test. The results are shown in Figure 4.

After 1 week of treatment, the body weight of the 10X black fungus group combined with GM-020 was significantly reduced compared with the negative control group. After 2 weeks, the body weight of the positive control group, the 1X black fungus group combined with GM-020, and the 10X black fungus group combined with GM-020 were significantly reduced compared with the negative control group. After 3 weeks, the positive control group, the black fungus group of 1X, the GM-020 group, the black fungus group of 1X combined with GM-020, and the black fungus group of 10X combined with GM-020, compared with the body weight of the negative control group significantly reduced. These results demonstrate that GM-020 and black fungus are effective in treating obesity.

Difference in weight of lipid tissue around testis: Rats were sacrificed, and lipid tissue around testis was extracted and weighed. The data were analyzed by the Kruskal Wallis H test, and the data of the normal control group were used as the baseline for the Dunnett test. The results are shown in FIG. 5 .

According to Fig. 5, only the positive control group and the 10X black fungus group combined with GM-020 showed a significant reduction compared with the negative control group.

Difference in lipid weight around the kidney: The mice were sacrificed, and the lipid tissue around the kidney was extracted and weighed. The Kruskal Wallis H test was used to analyze the data, and the data of the normal control group was used as the baseline of the Dunnett test. The results are shown in FIG. 6 .

According to Figure 6, the 1X black fungus group, the 10X black fungus group, the 1X black fungus group combined with GM-020, and the 10X black fungus group combined with GM-020 had a significant reduction compared to the negative control group. On the other hand, the positive control group, GM-020 group, and negative control group showed little difference.

Serum concentrations of lipid metabolites: Blood samples were collected from the tail for biochemical assays. Blood samples were left at room temperature for 1 hour and centrifuged at 2,500 rpm for 10 minutes. Take the upper serum for testing.

台湾)来检定总胆固醇(CHOL)的浓度，并通过Autoanalyzer Hitachi^TM 7150来量测吸收作用。根据选择性地抑制方法和酶测定(

日本)来检定HDL-C和LDL-C的浓度，并通过Autoanalyzer Hitachi^TM 7150来量测吸收作用。By TRIGLYCERIDES GPO LIQUID REAGENT ^TM ( Taiwan) to test the concentration of triglyceride (TG) in each group, and the absorption was measured by Autoanalyzer Hitachi ^TM 7150. Via CHOLESTEROL LIQUID REAGENT ^TM (

Taiwan) to determine the concentration of total cholesterol (CHOL), and the absorption was measured by Autoanalyzer Hitachi ^TM 7150. According to the selective inhibition method and enzyme assay (

Japan) to test the concentration of HDL-C and LDL-C, and to measure the absorption by Autoanalyzer Hitachi ^TM 7150.

The data were analyzed by the Kruskal Wallis H test, and the data of the normal control group were used as the baseline for the Dunnett test. The results are shown in Table 7: where ^** represents p<0.01; ^a represents negative control; ^b represents positive control; ^c represents 1X ^black fungus; ^d represents 10X black fungus; ^e represents GM-020; 020 combined 1X black fungus; and ^g represents 10X black fungus combined with GM-020.

Table 7

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 positive control 219.08±11.22 75.83±9.93 123.19±4.20 10.04±0.93 1X black fungus 182.50±8.24 82.08±7.32 100.57±3.58 12.39±1.03 10X black fungus 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 1X black fungus combined with GM-020 190.08±4.85 55.75±4.38 105.38±3.10 10.83±1.51 10X black fungus combined with GM-020 164.20±8.64 57.30±4.61 97.93±5.42 8.04±0.94 P value 0.000^**a,c,d,f,g 0.000 0.000^{**a, c, d, f, g} 0.014^*a

The 1X black fungus group combined with GM-020 and the 10X black fungus group combined with GM-020 had lower serum concentrations of triglycerides than the negative control group. On the other hand, the 1X black fungus group, the 10X black fungus group and the GM-020 group had little difference compared with the negative control group. In addition, the 1X black fungus group, the 10X black fungus group, the 1X black fungus group combined with GM-020, and the 10X black fungus group combined with GM-020 had lower serum total cholesterol and HDL-C concentration. With regard to the concentration of LDL-C, the groups other than that of the normal control group showed little difference.

Liver concentrations of fat metabolites; rats were sacrificed and right lobe livers were taken. The fat is extracted according to known methods.

For each sample, the concentrations of triglyceride (TG) and total cholesterol (CHOL) can be assayed as described above.

The data were analyzed by the Kruskal Wallis H test, and the data of the normal control group were used as the baseline of the Dunnett test. The results are shown in Table 8: where ^** represents p<0.01; ^a represents negative control; ^b represents positive control; ^c represents 1X ^black fungus; ^d represents 10X black fungus; ^e represents GM-020; 020 combined 1X black fungus; and ^g represents 10X black fungus combined with GM-020.

Table 8

Group CHOL TG 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 1x black fungus 20.75±0.85 85.50±3.76 10X black fungus 22.00±0.72 84.83±8.56 GM-020 19.44±0.85 88.56±6.41 1X black fungus combined with GM-020 21.25±0.70 83.25±6.27 10X black fungus combined with GM-020 20.90±0.81 77.50±7.82 P value 0.000^**c,e,f,g 0.004^{*a, c, d, e, f, g}

Each of the 1X black fungus group, the GM-020 group, the 1X black fungus combined with GM-020, and the 10X black fungus combined with GM-020 group had lower serum concentrations of total cholesterol. In terms of triglycerides, each of the 1X black fungus group, the 10X black fungus group, the 1X black fungus group combined with GM-020, and the 10X black fungus group combined with GM-020 were significantly reduced .

Liver and Kidney Function Analysis: Blood samples were processed as described above.

日本)来检定肌酸酐的浓度，并通过Autoanalyzer Hitachi^TM 7150来量测吸收作用。通过GOT(ASAT)IFCC mod^TM(

德国)来检定GOT的浓度，且通过Autoanalyzer Hitachi^TM 7150来量测吸收作用。通过GPT(ALAT)IFCCmod^TM(

德国)来检定GPT的浓度，且通过AutoanalyzerHitachi^TM 7150来量测吸收作用。根据尿酸酶-过氧化物酶方法(日本)来检定尿酸(UA)的浓度，且通过Autoanalyzer Hitachi^TM7150来量测吸收作用。As far as each sample is concerned, according to the method of Jaffe reaction (

Japan) to test the concentration of creatinine, and by Autoanalyzer ^Hitachi 7150 to measure the absorption. Through GOT(ASAT)IFCC mod ^TM (

Germany) to determine the concentration of GOT, and by Autoanalyzer ^Hitachi 7150 to measure the absorption. Through GPT(ALAT)IFCCmod ^TM (

Germany) to determine the concentration of GPT, and by Autoanalyzer ^Hitachi 7150 to measure the absorption. According to the uricase-peroxidase method ( Japan) to test the concentration of uric acid (UA), and by Autoanalyzer ^Hitachi 7150 to measure the absorption.

The data were analyzed by the Kruskal Wallis H test, and the data of the normal control group were used as the baseline for the Dunnett test. The results are shown in Table 9: where ^** stands for p<0.01; ^a stands for negative control; ^b stands for positive control; ^c stands for 1X ^black fungus; ^d stands for 10X black fungus; ^e stands for GM-020; -020 combined 1X black fungus; and ^g represents 10X black fungus combined with GM-020.

Table 9

Group GOT GPT Creatinine UA 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 1x black fungus 107.00±14.77 37.33±2.77 0.47±0.02 5.20±0.91 10X black fungus 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 1X black fungus combined with GM-020 150.00±21.71 44.91±3.93 0.45±0.02 2.12±0.48 10X black fungus combined with GM-020 76.40±13.5 37.50±3.95 0.39±0.03 2.10±0.36 P value 0.072 0.002^{**b, c, e, f, g} 0.002^**g 0.006

The differences in results among these groups were not significant. Indices indicating liver and kidney function were not affected after treatment with GM-020 and/or black fungus.

Example 6: GM-020 for the treatment of hypercholesterolemia

Animal model: Male hamsters were purchased from the Taiwan Experimental Animal Center, and were reared in the light alone for 12 hours and in the dark for 12 hours at a temperature of 25±1° C. and a humidity of 60±5%. Adequate food and water. These mice were divided into two groups: one was a normal control (NC) group and the other was a cholesterol-enriched diet. The normal control group was fed a normal diet and the cholesterol-rich diet group was fed a diet containing 24% protein, 14% fat, 2% cholesterol, 48% carbohydrates, 6% cellulose and 6% minerals and vitamins A mixture of diets rich in 2% cholesterol.

Treatment by GM-020: The normal control group was continuously fed with a normal diet. Apply the treatment twice a day. The group of cholesterol-rich diets was further divided according to treatment into the groups listed below: (a) Lactobacillus gasseri, (b) GM-020, (c) Lactobacillus sporogenes (L. sporogenes), and ( d) Negative control (Control) treated with saline. Each dose was 10 ⁹ CFU/mL.

Serum Concentrations of Lipid Metabolites: Before and after treatment, blood samples were collected from periorbital veins for biochemical assays. Blood samples were left at room temperature for 1 hour and centrifuged at 2,500 rpm for 10 minutes. The supernatant serum was taken for testing.

For each sample, the concentrations of total cholesterol (CHOL), HDL-C, LDL-C, and triglycerides (TG) can be assayed as described above. The data were analyzed by the Kruskal Wallis H test, and the data of the normal control group were used as the baseline for the Dunnett test. Table 10 shows the difference in fat content before and after being fed a diet rich in cholesterol, where ^* represents p<0.1; ^** represents p<0.05; ^*** represents p<0.01; ^a represents negative control; ^b stands for Lactobacillus gasseri; ^c stands for GM-020; ^d stands for Lactobacillus sporogenes.

Table 10:

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 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 sporogenes 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 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***}

According to the results, after being fed with a cholesterol-rich diet for 4 weeks, the group of the cholesterol-rich diet exhibited a significant increase in CHOL, HDL-C, LDL-C and TG compared with the normal control group, and these are rich in Dietary subgroups of cholesterol exhibited little difference among each other.

In terms of TG, after 4 weeks of treatment, all of these cholesterol-enriched diet subgroups exhibited significantly larger TG than the normal control group.

In terms of CHOL, after 4 weeks of treatment, the L. gasseri group, L. sporogenes group and the negative control group exhibited significantly larger CHOL than the normal control group. On the other hand, the GM-020 group showed little increase compared to the normal control group. The difference between (CHOL_0 before treatment) and after treatment (CHOL_4) is shown in Figure 7. GM-020 was shown to have the ability to lower the serum concentration of total cholesterol.

In terms of HDL-C, the Lactobacillus sporogenes group exhibited significantly lower HDL-C than the normal control group after 4 weeks of treatment. However, Lactobacillus gasseri group, GM-020 group and negative control group showed little difference.

For LDL-C, serum concentrations are shown in Figure 8. The GM-020 group exhibited significantly reduced LDL-C compared to the normal control group (p<0.1). Furthermore, after 4 weeks of treatment, the Lactobacillus gasseri group and the Lactobacillus sporogenes group exhibited significantly (p<0.05) lower LDL-C than the normal control group (see Figure 9). GM-020 was shown to have the ability to lower the serum concentration of total cholesterol.

As far as LDL-C/HDL/C is concerned, the ratios are shown in Table 11 and Figure 10, where ^* represents p<0.1; ^** represents p<0.05; ^*** represents p<0.01; ^a represents negative control; ^b represents Lactobacillus gasseri; ^c represents GM-020; ^d represents Lactobacillus sporogenes. Data can be analyzed by the Kruskal Wallis H test, and the data from the normal control group is used as the baseline for Dunnett's test.

Table 11

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 Lactobacillus gasseri 1.27±0.13 2.08±0.27 GM-020 0.90±0.13 1.20±0.27 Lactobacillus sporogenes 1.46±0.42 2.44±0.14 P value 0.003^a** 0.000^{a, c***}

The GM-020 group showed a significant decrease compared to the normal control group (p<0.001). It shows that GM-020 has the ability to reduce LDL-C/HDL-C.

While the embodiment of the present invention has been illustrated and described, various modifications and improvements will occur to those skilled in the art. It is not intended to limit the invention to the particular embodiments as illustrated and all modifications which do not depart from the spirit and scope of the invention are intended to be within the purview as defined in the appended claims.

Claims (8)

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.

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