TW201132286A - Novel lactobacillus strain, composition and use thereof for improving the syndrome of diabetes and complication thereof - Google Patents

Abstract

The invention relates to a composition for improving the syndrome of diabetes and complication thereof. The composition comprises a effevtive amount of Lactobacillus strain which is at least one selected from the group consisting of: Lactobacillus reuteri GMNL-89 strain, Lactobacillus gasseri GMNL-205 strain, Lactobacillus reuteri GMNL-263 strain and a pharmaceutical acceptable vechile. The Lactobacillus gasseri GMNL-205 strain and Lactobacillus reuteri GMNL-263 strain are novel isolated Lactobacillus strains. In addition, the invention also relates to a novel use of the compositon or the Lactobacillus strains for improving the syndrome of diabetes and complication thereof.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the isolation of novel Lactobacillus isolates and to the technical field of improving symptoms and complications associated with diabetes. [Prior Art] Diabetes Mellitus is a multi-pathogenic metabolic disease caused by a generalized metabolic disorder caused by defects in insulin secretion or action. Diabetes is a disease characterized by persistent chronic hyperglycemia, and it also causes metabolic disorders such as protein, fat, water, and electrolytes in the body. Clinically, diabetes is mainly divided into two categories: Type I: Insulin-dependent diabetic mellitus (IDDM)' is generally more common in people under the age of 30, so it used to be called "young-aged diabetes". In fact, any age can happen. Type 1 diabetes is an autoimmune disease in which the autoimmune system attacks and destroys the beta cells of the Islet of Langerhans. The reason is related to the genetic inheritance of an individual, a viral infection in an environmental factor, or a toxic substance destroying the β-cell of the pancreas and an autoimmune antibody forming an anti-β cell, and a cellular immune action attacking the β cell. Eventually, the patient's pancreas is unable to secrete insulin normally, and ketonic acidemia is highly prone to occur, so insulin is required for treatment. Non-insulin dependent diabetic mellitus (NIDDM) mostly occurs after 4 years of age, and most of the patients are obese, formerly known as "adult type diabetes," but it is also possible Occurred in young people, family-type disease is more common, this type of diabetes accounts for more than 95% of the total population of diabetes in Taiwan. This type of diabetes is caused by insulin breakdown 3 and insulin resistance; although some patients have reduced knife clearance of insulin; >, most patients have the ability to secrete temsin. Therefore, most of them rely on diet control and oral hypoglycemic drugs to control blood sugar' without immediate injection of tamsin. In addition, most patients are accompanied by symptoms of insulin resistance (insulinresistanee). The formation of resistance to Tengdao is mainly due to the excessive secretion of hyperi_inamia by the β-cells of the spleen island, which causes the sensitivity of the bone path muscle, adipose tissue and the surrounding tissues of the liver to the temsin. Kiss) lowered. This reduces the rate of tissue lysine and causes hyperglycemia. This type of disease progresses slowly, so it is too early to rely on the symptoms of miscellaneous urine. But often accompanied by diabetes I. Severe blood e (such as muscle infarction, stroke), small blood vessels (such as kidney, omentum and neuropathy) and other chronic complications. In addition, 'in the second face of urinary disease patients' accompanied by Abnormalities in lipid thieves, such as increased concentration of triglyceride (TG) in blood polycondensation, decreased concentration of high-density lipoprotein cholesterol (hdlc), and concentration of healthy self-contained (LDL_C). It can lead to the risk of heart disease in patients with type 2 diabetes. In addition, according to the research, the blood-clearing ability of liver in patients with severe diabetes will drop. When the job is triglyceride, the fate of cholesterol is continuously tested. 'Hepatocytes will occur _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ® is mainly used for the treatment of difficult limbs and noodles. In the treatment of mystery, the main purpose is to raise the insulin, reduce the high dose after eating and improve the anti-resistance of Tengdaosu. Diabetes _ can be divided into 201132286 (1) continued to grow urea (Sulfonylurea): the main mechanism of these drugs to promote the secretion of pancreatic insulin 'in particular, enhance the role of pancreatic β cells in glucose stimulation and release of insulin; The commonly used hypoglycemic drugs are glibenclamide (trade name: euglucon), gram ratio, glipizide (trade name: such as oil), and gliclazide (trade name: diamicron). In addition to the side effects that have been discovered, such as rashes and itching, such drugs have their limitations. If there are severe liver and kidney dysfunction, pregnant women and lactators, and those who are severely allergic to sulfonamides, It is not advisable to use such hypoglycemic drugs. (2) a-Glucosidase inhibitors: the main action of these drugs is to inhibit pancreatic a-amylation g-(a-amylase) and intestinal a- The activity of a-glucosidase, which inhibits the decomposition and absorption of carbohydrates in the intestines, can effectively reduce postprandial blood glucose and insulin concentrations, but the side effects are bloating or occasional diarrhea, abdominal pain and vomiting. (3) 塞. Sitting on a _ class (Thiazolidinedione) derivatives: These drugs are mainly used to increase the nucleus of the nucleoside proliferator-activated receptor _Y (per〇xis〇me pr〇liferatOT activated receptor ( The activity of PPAR)-garmm), which enhances the action of insulin, increases the intracellular glucose transfer proteins GLUT2 and GLUT4, and transports them to cells. The clinically used is troglitazone (tr〇giitazone, trade name: Rezulin), rosiglitazone (r〇siglitaz nickname: avandia), pirinone (pi〇glitazone, trade name: act〇s), etc.; however, it is worth noting that troglitazone has caused Fatal liver toxicity, therefore, was banned two months after listing in the UK (1997 ίο月). In addition, the "Thiaz〇lidinedi〇ne derivatives have also been ordered to be fully recycled and banned in the United States. (4) Double veins (Biguanid is also a derivative of guanidine). At present, the M metf face is mainly in the blood sugar-lowering age of the biguanide. This _ itself does not have the index of insulin in 201132286. The blood glucose machine is converted into the following five points: a. Inhibition is healthy, so it is preferentially used in obese type 2 diabetics, which reduces the eating and weight loss and improves the peripheral effects of insulin, b delays intestinal absorption, and e. Promotes the anaerobic decomposition of glucose in the intestine, thereby increasing the glucose in the intestine, but may produce too much lactate (10)), which is easy to cause lactic acidosis, d-the tranexin on the side of the liver, thus inhibiting the liver The reliance on sugar nascent action, reducing glucose from the liver ώ ώ, e. 舰 肤 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ In addition, the side effects of such hypoglycemic drugs, such as the initial service may have gastrointestinal discomfort, such as anorexia, ah heart, "regional vomiting or abdominal scales, a few people may be secret, and the thief may be inactivated after use. A phenomenon occurs. In addition to being used for the preparation of fermented foods, many studies have also found that Lactobacilli have a variety of beneficial functions, such as] secretion of various enzymes, help food decomposition, improve nutritional value, 2. break down lactose, improve lactose Symptomatic, 3 secreted vitamin b group, 4 maintain normal microbes in the intestine _ 'Suppressing the financial side (four) side, 5. improving the lining or constipation, 6 enhancing the immune system function, 7. improving liver function, reducing liver damage, 8. lowering blood biliary _, 9 · anti-cancer and anti-citation, etc. Because the milk (4) has many beneficial effects on the host, Lactobacillus is also reported to improve the sexually transmitted diseases (4) Wei, and diabetes is being reported. In addition to its order (4) Wiili diet rats can delay the improvement of diabetes. In some literatures or patents, diabetic mice (4) can effectively diagnose diabetes and reduce blood sugar, but the literature or patents that have been published about lactic acid bacteria to improve diabetes are limited to (4) blood sugar value and body weight. , blood amplitude and concentration of bribes. For other complications that may be caused by diabetes, such as body inflammatory response and liver function decline, there is no further improvement in the 201132286 step. In the patent lactic acid research, in addition to the improvement of fat and cholesterol in the blood of the mad sugar value, the improvement of the cap blood hemoglobin, the inflammatory cytokines in the blood, the liver amplitude and the liver function G0T and GPT are all improved. Into the _ step improvement effect. The existing clinical treatment of silk disease Lai (four) Xian is very large. The reduced Lactobacillus is the General Recognized As Safe (GRAS). Therefore, it is the most natural and healthy method to develop products that improve diabetes using Lactobacillus. Applicants in this case are considering the various types of diaper diapers, and the diabetes is a chronic disease that requires patient coordination and long-term treatment and control. Therefore, this financial person hopes to use the local masts to develop products that improve the symptoms of diabetes, which can be easily used by ordinary users or diabetes in daily life, except for the beneficial effects of Lactobacillus itself. Function, no side, no more than the general user or patient can simultaneously improve, control, treat or prevent diabetes-related symptoms such as hyperglycemia and possible complications. Therefore, the present article "New Wei Bacillus, its composition, and the use of fresh disease to improve the disease of the county" was proposed. SUMMARY OF THE INVENTION The object of the present invention is to provide a composition for improving the symptoms of diabetes at least selected from the known strains: Lactobacillus reuteri (10) (four) GMNL_89, and two novel species: reduced milk (four) ( Laet()badllus gasseri) GMNL2G5, recording Lactobacillus reuteri (6) (10) ad fine r ri) at least one of the groups consisting of GMNL-263. The second aspect of the present invention is to provide a weaned (four) isolate which is different from the known seed, and which is a strain of Lactobacillus. Another object of the present invention is to provide a new type of larvae, and two new strains of Lactobacillus new Lactobacillus isolates, which can be used by people with diabetes to change 201132286. Its symptoms of diabetes and its complications. It is still another object of the present invention to provide a composition for improving symptoms of diabetes and its complications, such as foods, drinks, health foods, additives, medical compositions, and the like, which are easy to use in daily life, For general-purpose patients with diabetes, it can be easily taken for long-term use, in order to achieve daily health care, or disease control. A novel Lactobacillus, a composition thereof, and a use thereof, which can achieve the above-mentioned objects, include a Lactobacillus reuteri GMNL-89 having a registration number of bcrc 910340; Lactobacillus gasseri GMNL-205 with accession number BCRC 910451; and Lact〇bad Uus reuteri (10) see-plus, with accession number BCRC 910452. Among them, the Lactobacillus gasseri GMNL-205 and the LaCt〇bacillus reuteri GMNL_263 were identified by the Food Industry Development Research Institute for the identification of bacterial names and the correlation analysis. Latex _ isolate. The novel milk (four) points selected from the present invention and the known Lact〇badllus reuteri GM milk strains are respectively subjected to diabetes animal model analysis to evaluate the mastoid branches with subcutaneous diabetes. The results of the recording and the concurrent work I are not obvious. The two strains of Lactobacillus isolates provided by the present invention can improve the high blood sugar level, the change of the high blood sugar level, the high glycosylated hemoglobin ratio, and the high value in the diabetic subject. Diabetes symptoms such as total cholesterol concentration, high LDL/HDL ratio, high IFN-γ amount, high liver triglyceride lipid concentration and high liver cholesterol concentration' further improve diabetes and related complications. 201132286 [Embodiment]: ^All of the technical ships and scientific terms mentioned in the above-mentioned Japanese versions, unless otherwise defined, are of a meaning that can be understood by those skilled in the art. The present invention provides a composition for improving symptoms of diabetes comprising at least an effective amount selected from the group consisting of: Lactobacillus reuteri (5) Zhiqin_m_) 〇89_, Lactobacillus gassed GMNL - 205 ^ ^m^l^(Lact〇bacillus reuteri) GMNL-263 is a group of pharmacists, and a pharmaceutically acceptable carrier. # Among the Lactobacillus reuteri (10) (10) · fine m / ien ·) GMNL-89 is - the disclosed species _ species characteristics and storage information has been published in the Republic of China patent application "with anti-inflammatory activity of Lactobacillus Separation strain and its use", its public office is: fine 5). The applicant of the case found that it has a novel use or function to improve the symptoms of diabetes by means of a diabetic animal model. It makes Lactobacillus sinensis (kc secret (10)·//(10) brand (four) GMNL_2〇5, and Lactobad-rcuteri GMNL_263 is the novel Lactobacillus isolate isolated in this case. The two isolates are different in the respective ages of the respective species, and the analysis of the two isolates also has the new use or function of improving the symptoms of diabetes and its complications. The above three strains of Lactobacillus 'Analyzed by the animal model of diabetes, it is found that it can effectively improve, reduce, control, treat and prevent high blood sugar level, high blood sugar level change, high glycosylated hemoglobin ratio, high total cholesterol concentration, high in subjects with diabetes. LDL/HDL ratio, high ΙρΝ_γ amount, southern liver diglyceride lipid concentration and high liver cholesterol concentration, thereby improving the condition and related complications. The Lactobacillus isolate also contains its subcultured progeny, or mutant However, it still has the same characteristics as the species, genomic, or use of the invention (for improving the symptoms of diabetes) 201132286. The composition described herein may comprise, but It is not limited to: foods, drinks, health foods, animal drinking water additives, animal feed additives, animal and human medical compositions, food additives, beverage additives, and the like which are applicable to the present invention. The term "improvement" means 'Compared to the use of the lactobacillus of the present invention or a composition thereof, the use of the lactobacillus of the present invention, or a composition thereof, can effectively alleviate, reduce, control, treat or prevent diabetes symptoms and related Complications. The term "diabetes symptoms", pure but not limited to &罹; patients with diabetes, high blood sugar levels, asked for changes in blood glucose levels, high glycosylated hemoglobin ratio, high total cholesterol concentration, high / HDL ratio ' Diabetes symptoms such as lFN-γ $, high liver triglyceride lipid concentration and high cholesterol concentration. Good. Effective amount of thinking can effectively improve, treat, attenuate or eliminate one or more diseases (such as diabetes). An effective amount of a mature age, which may be referred to as "therapeutically effective amount, or "improving an effective amount," D. pharmaceutically acceptable " means that the substance or composition must be associated with the formulation The ingredients are compatible and harmless to the patient. The term "diabetes-related complications," includes 3 kernels not limited to. Diabetic neuropathy (including but not limited to bladder weakness, bloating, constipation, writing, and sputum, Cold and heat sensation), kidney disease (including but not limited to, __, glomerulosclerosis, kidney _, high blood neonatal kidney hardening, glutinous, lining), hair (four), heart _ _ butterfly hair her but Not limited to brain stroke 'myocardial infarction, coronary embolism, long-term sputum ~ agricultural exhaustion, unstable heart rhythm, poor peripheral blood circulation, foot infection, etc.), eye.. occupational lesions, cataracts, glaucoma, amblyopia (visual weakness) ), liver disease (including but not limited to liver 匕 month θ fat liver, non-alcoholic fatty liver, 201132286 - and cirrhosis). The composition of the present invention can be obtained by a technique known to those skilled in the art, and the above-mentioned medicinal carrier is detached from the plant with a pharmaceutically acceptable carrier (acceptable achicie) Prepared into a dosage form suitable for use in the compositions of the invention. Wherein the dosage form includes but is not limited to: solution ' ^L#J(emulsion)' (suspension) > ^^.(powder) ' It(tablet)' pill, mouth containing ingot (l〇 Zenge), tablets, chewing gums, sluffry, and other dosage forms similar or suitable for use in the present invention. The pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of solvents, emulsifjers, SUSpending agents, decomposers, and binding agents. Agent), excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, Surfactant, and other carriers similar or suitable for use in the present invention. In the above composition, a dissolution aid, a buffer, a preservative, a colorant, a flavor, a flavor, and the like which are usually used in the field of one or more preparations may be appropriately added as needed. In another preferred embodiment, the aforementioned composition provided by the present invention may further comprise an edible material to prepare a food product or a health care product. Wherein the edible material comprises, but is not limited to: water, fluid milk products, milk, concentrated milk; fermented milk, such as yogurt, Sour milk, fr〇zen y0gUrt, iactic acid bacteria-fermented beverages; milk powder; ice cream; cream cheeses; (dry cheeses); soybean milk; fermented soy milk (fermented 11 201132286 soybean milk); vegetable-fruit juices; juices; sports drinks; confectionery; (jellys); candies; infant formulas; health foods; animal feeds; Chinese herbals; dietary supplements. Further, the novel strains discovered by the present invention may be contained in a composition together with other conventional strains. Wherein the composition may further comprise at least one conventional probiotic selected from the group consisting of Lactobacillus species (/^Ga (10)·//!^·^·), Keyococcus species (Sire/) Jiococcw·^/?·), Bifidobacterium species?/?·), yeast (yeasts), wherein the Lactobacillus species (/dciokciZ/M·? sp.) includes but is not limited to: lactate milk Bacillus (lactobacUlus 〖actis), Lactobacillus acidophilus (Actobacillus acidophilus), Lactobacillus helveticus, Lactobacillus bifidum (/flci〇kCi7/like 垆 垆, Lactobacillus casei (kcioka7/(10) Lactobacillus paracasei Cheese subspecies (Laci〇^cl7/M:ysubsp, Lactobacillus rhamnosus ([Shen (10)'丨/(10)rh_n(WMiS), Lactobacillus gargle (Z^zcio6aa7/wi(10)m·), Roy's milk Bacillus reM&n·), Lactobacillus fermentum (LactobacillusβεΓηιεηίιιηί), or a paper thereof. The conventional Streptococcus species (*Sire/?iococ(10)s/7.) includes but is not limited to: Sireptococci^/izcto, Streptococcus thermophilus (additional sputum (: 〇 (^ is known as 6/771 〇 fine), Neisseria pallidum («Sirepiococci^cremor/i·), or a combination thereof. Among them, 3 Xuan province knows double; 1 = dry halogen species (β complaints, Shen Ji also cut.) including but not limited to: short bifidobacteria (mail secret (10) mum Wve), Bifidobacterium lactis (administered Must (10) (7) · tear (6) called, long-type bifidobacteria (mail - sink (four) (10) / which (10)), double-forked Bifidobacterium (postal secret (10) en • mail 12 201132286 mail dwm), or a combination thereof. Eight in 43⁄4 know yeast (yeasts) includes, but is not limited to, Saponinella (10) cerm· earn), Candida albicans (c secret also _r), F. cerevisiae ccW (^(10) yZorenriwws), or a combination thereof. In addition, the present invention also provides a method for improving symptoms of diabetes and its complications by administering an effective amount of the composition to a subject suffering from diabetes for improving and reducing π blood sugar level and blood sugar in the body. Value change, glycated hemoglobin ratio, total cholesterol concentration, pediatric _tb case, IFN-γ amount, high lysine triglyceride f concentration, high lyrics and liver function indicators GOT, GPT, thereby improving diabetes and Related complications. In addition, the present invention also provides the aforementioned lactobacillus for the preparation of a symptom of improving diabetes and The method or the use of the composition of the invention. The composition provided by the invention and the method for turning over the urinary tract and its complications, the route of administration can be adjusted according to the needs, and is not particularly limited, and the preferred one is oral. Apply the appropriate dosage form. The invention is exemplified by the following examples, but the invention is not limited by the following examples. The pharmaceuticals and biological materials of the present invention are all obtained in the following, and the following are only examples of available pipelines. Example 1 Selected strains The applicant (Jingyue Biotechnology Co., Ltd.) is a gastrointestinal tract sample of healthy adults provided by the hospital. In the middle, more than 100 isolates were isolated to establish the isolate-separate. From the bacterial material, the high-concentration JL-H and the three Lactobacillus strains were extracted from the bacterial immune system, and the results were analyzed. (4) Analysis of the pattern of diabetes urinary blues. The registration number of the three laps, the storage 13 201132286 曰 and the _ name are shown in the table - the latitude of the milk (LaetGbaemus _) GMNL-89 is a public The original strain of the strain, the characteristics of the strain, the deposit certificate, the survival test report, and the «information, which are included in the towel _ _ application _ please call the public number: 2_215); (10) see the move and the coffee The newly isolated Lactobacillus licheniformis isolate of the present invention. The strain characteristics of the two Lactobacillus strains, the API identification system analysis, and the 16S rDNA analysis 'gene map analysis are described in the second and third sections. Table - The milk of the present invention Rod hiding Hsinchu food IH (Iv) Institute of Health Research in secret and kept

The registration number of the heart (BCRC) and the registration period --_ registration number 窨 in the __

Lactobacillus reuteri GMNL-89 BCRC 910340 Republic of China 95 u 14

Lactobacillus gasseri GMNL-205 BCRC (10) Your country's egg year ^ month% day attachment t ^Lactobacillus reuteri_g^k^l_jCRC91^52 Republic of China November 06, 1998 Annex 2 Example 2 Lactobacillus brevis (Lacii ia7/MS gassm) GMNL-205 2-1. Identification of the scientific name of Lactobacillus gasseri GMNL-205 strain The isolate GMNL-205 was entrusted to the Food Industry Development Research Institute for identification of bacterial names, which are described as follows: Background of isolate GMNL-205: 1·Separation Source: Human gastrointestinal tract 2. Medium: MRS 3. Culture temperature: 37 ° C 4. Pathogenicity: None, please refer to Figure 1 and Table 2. The analysis results show that the isolate GMNL_2〇5 is a Gram-positive bacillus, not The enzyme, oxidase and motility are grown in aerobic environment and anaerobic environment. 14 201132286 In addition, the 16S rDNA partial sequence of the isolate GMNL-205 is shown in SEQ ID No: 1, according to 16S rDNA analysis. The isolate GMNL-205 is similar to Lacioimcii/MS gflssm·, LaciobadZZits ίαίνναηβηώ and car cross, and the similarity is over 99%. Then by API identification system analysis (Table 2), the isolate GMNL-205 is closest to Lactobacillus gargle ( Lacioki Ci'/Zwsgim^r/) Therefore, the above results show that the isolate GMNL-205 is Lactobacillus gasseri. The results of API identification analysis of the second isolate GMNL-205

Lactobacillus Lactobacillus Lactobacillus API Test isolate gasseri taiwanensis johnsonii GMNL-205 BCRC BCRC BCRC 14619T 17474T 17755T Glycerol - - - - Erythritol - - - - D-Arabinose - - Ink - L-Arabinose - - - - D-Ribose - - - Ink D-Xylose - - - - L- Xylose - - - - D-Adonitol - - - - Methyl-3D-Xylopyranoside - - - - D-Galactose + + + + D-Glucose + + + + D-Fructose + + - f + D-Mannose + + + + L-Sorbose - - - - L-Rhamnose - - - - Dulcitol - - - - Inositol - - - - D-Mannitol - - - - D-Sorbitol - - - - Methyl-aD -mannopyranoside - - - - Methyl-a D-glucopyranoside - - - - N-AcetylGlucosamine + + + Amygdalin - + - - Arbutin + + - - Esculin + + + + 15 201132286

Salicin + + - D-Celiobiose + + - _ D-Maltose + + + + D-Lactose + + D-Melibiose - - - D-Saccharose + + + + D-Trehalose + + - Inulin - - A_D-Melezitose - - - D-Raffinose - + Amidon - + + + Glycogen - - - Xylitol - - - _ Gentiobiose + + + + D-Turanose - - - _ D-Lyxose - - - • D-Tagatose - + + • D- Fucose - - - • L-Fucose - - - D-Arabitol - - - L-Arabitol - - - _ Potassium Gluconate - - - _ Potassium 2-Ketogluconate - - - _ Potassium 5-Ketogluconate - - - A total of 49 physiology Biochemical test project: The isolate GMNL-205 is in accordance with the standard g strain~~ 46 42 44

—···················· The genetic map of Lactobacillus gasseri GMNL_2〇5 strain was analyzed. The actual steps are briefly described as follows: A. Preparation of strain template: GMNL-205 was inoculated on MRS culture dish and placed in 37〇c culture. From the MRS culture solitary after 2 days of culture at 37 °C, single-colon colonies were picked and inoculated in a sterile environment to μ cis 16 201132286

Broth, placed at 37 ° C for 16 hours. After centrifuging the bacterial solution at 13 rpm for one minute, the supernatant was removed. Add 200 μM sterile water and mix well with pauet. After centrifugation at i3 rpm for one minute, remove the supernatant' and repeat this step once. After the cleaned pallet is added to 200 μl of sterile water and thoroughly mixed, it can be used as a template for strain MPD experiments. B. RAPD analysis: PCR reaction was carried out using primer Lac P2 (5,-ATg TAA CgC C-3, SEQ ID No: 3). The composition of the PCR mixture is shown in Table 3: Table 3. Composition of PCR mixtrue Template Ιμΐ Lac P2 Ιμΐ dNTPmix 1 μΐ 1 Ox Buffer 2.5 μΐ Ex Taq 0.2 μΐ h2o 19.3 μΐ Total volume 25 μΐ PCR reaction conditions: 10 minutes after the reaction at 95 ° C, start 35 cycles: 93 ° C The denaturation reaction lasted for 1 min at 36 °C for 1 min, 72. (: The Lu extension reaction was carried out for 1 min. Finally, after reacting at 72 ° C for 7 min, the PCR reaction was stopped and placed at 4 ° C. After the completion of the PCR, 6 μl of the PCR product was taken on a 2% agarose gel ( Agarose gel) for electrophoresis, after electrophoresis, and then stained with EtBr, developed under UV light, and analyzed the RAPD spectrum of the single colony for the photograph. C. The experimental results are shown in Figure 2. The gene map unique to GMNL-205 can be used to identify GMNL-205. Example 3 Lactobacillus reuteri GMNL-263 17 201132286 3-1. Name of the strain of Lactobacillus reuteri GMNL-263 strain The identification of the scientific name, the separation of the GMNL-263 commissioned by the Food Industry Development Institute is described as follows: Background of the isolate GMNL-263: 1. Source of separation: human gastrointestinal tract

2. Medium: MRS 3. Culture temperature: 37 ° C 4. Pathogenicity: none

As shown in Figure 3 and Table 4, the analysis results show that the isolate (;}]^]^1^263 is a Gram-positive bacillus, without enzyme, oxidase and motility, in aerobic environment and anaerobic In the environment, the 16S rDNA partial sequence of the isolate GMNL-263 is shown in SEQ ID No: 2. According to the 16S rDNA analysis, the isolate GMNL-263 is most similar to miiert, and the similarity is over 99%. Further analyzed by API identification system (Table 4), isolate GMNL-263

The closest is Lactobacillus reuteri (LaciofeadZ/w·? rewien'). Therefore, the above results show that the isolate GMNL-263 is L. reuteri (LflciofcadZ/ws rewim.). Table 4 Identification results of API GMNL-263 API Test isolate GMNL-263 Lactobacillus reuteri BCRC 14625T Glycerol - - Erythritol - - D-Arabinose - - L-Arabinose + + D-Ribose - + D-Xylose - - L - Xylose - - D-Adonitol - - Methyl-pD-Xylopyranoside - - 18 201132286

D-Galactose + + D-Glucose + + D-Fructose - + D-Mannose - - L-Sorbose - - L-Rhamnose - - Dulcitol - - Inositol - - D-Mannitol - - D-Sorbitol - - Methyl-aD- Mannopyranoside - - Methyl-aD-glucopyranoside - - N-AcetylGlucosamine - - Amygdalin - - Arbutin - - Esculin - - Salicin - - D-Celiobiose - - D-Maltose + + D-Lactose - + D-Melibiose + + D-Saccharose + + D-Trehalose - - Inulin - - D-Melezitose - - D-Raffinose + + Amidon - - Glycogen - - Xylitol - - Gentiobiose - - D-Turanose - - D-Lyxose - - D-Tagatose - - D-Fucose - - L-Fucose - - D-Arabitol - - L-Arabitol - - Potassium Gluconate + + Potassium 2-Ketogluconate - - Potassium 5-Ketogluconate - - The isolate GMNL-205 is in compliance with the standard strain: 46 -:Negative reaction; + : Positive reaction 19 201132286 3-2. Genetic map analysis of Lactobacillus reuteri GMNL 263 strain The present invention is also || analyzed by RAPD 'to analyze the Roy's milk stem _ GMNL_263 _ strain's exhibition diagram 碏' its experiment For the procedure, please refer to the second embodiment 2-2. A to B As shown in the figure, the strains analyzed were L. reuteri GMNL 263 and L. reuteri (10) n experimental results - month shown in Figure 4 'can clearly show that the genetic map of GMNL_89 and gmnl_263 is very different' According to this hiding, Lai and 6 Gu [_263 are both Roy's milk (four) (Iaci〇6aa fine, but these two kinds of bacteria are not the same strain. Example 4 Diabetic animal model 4_1 · Experimental animals from Lesco biotechnology Male SD rats of 5 weeks old were purchased, and the experimental animals were allowed to start the experiment after waiting for one week. The entire experimental rat was housed in an animal room with darkness and light hours for 12 hours and no restriction on eating or drinking, and the room temperature was maintained by air conditioning. c and indoor relative humidity of 55%. The treatment of experimental animals and all experimental procedures in this study were carried out in accordance with the fresh regulations of the Laboratory Animals Committee. 4-2. Diabetes mellitus method is wrong by the following recipe: rat diaper shop material: 咖 咖 Μ Μ Μ Μ Μ Μ Μ cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit cit ^(Streptozotocin,STZ)^ citrate buffer , , , ί to the STZ cold solution, the injection dose is 65 mg / kg. Three days after STZ injection, the blood glucose of the rats was measured. If the recording was higher than 3 GGmg/dLfm, the transfusion was successful. Example 5 Evaluation of the effect of Lactobacillus isolates on the diabetes mellitus and phase markers 201132286 5·1·Experimental flow The rats were randomly divided into six groups, and the treatment methods of the groups and groups were as shown in Table 5. Among them, only the control group did not use STZ for diabetes mellitus. The other six groups were successfully treated with STZ, and each was treated with anti-feed water (RO water, placebo _laeeM) and Weidaosu (Tengdao). Ordinary group), or feeding each Lactobacillus isolate 'after treatment, silver food for one month, the rats sacrificed and collected serum' to detect various biochemical indicators of serum towels to evaluate each Lactobacillus isolate to improve diabetes and related indicators The effect.

The placebo group received intraperitoneal injection of STZ 'RO water 1ml per day'. The Tengdaosu group received intraperitoneal injection of STZ' per day intraperitoneal injection of Insulin7_丨0U/Kg GMNL-89 group GMNL-205 group received intraperitoneal injection of STZ, lactobacillus was isolated daily. Plant GMNL_89 2xl〇9 cfu/* Intravenous injection of STZ, daily feeding of Lactobacillus isolate GMNL_2〇5 2x丨〇9cfu/夭GMNL-263 group with intraperitoneal injection of STZ 'daily feeding Lactobacillus isolate gmnl-263 2χ 1〇9 Cfu/夭1. Weight and blood glucose record During the experiment, mother's blood glucose is measured once in the morning and evening, and the body weight is recorded once a week. 2. Detection of biochemical indicators in serum Jinqing biochemical value: alkaline phosphatase, acridine Alanine aminotransferase, aspartate aminotransferase 'γ-giutamyi transferase (γ-GP), albumin (bilimin) (bilirubin, total) , creatinine, urea nitrogen 'glucose, phosphorous,! Calcium, chloride, 21 201132286 potassium 'sodium, protein, total, triglyceride, total cholesterol' HDL, LDL, C-reactive protein (c-reactive protein) , CRP), glycated hemoglobin (HbAlc). Blood cell classification count: full blood count (CBC). 3. Detection of cytokines: The concentration of lFN-γ in serum was detected by ELISA. 4. Detection of liver lipids: The animals were sacrificed to remove the largest lower right lobe of the liver, and the sputum 25 g liver was placed in two tissue homogenizers (Model: MICROMOT IB/E, PR0XX0N) for 1.5 ml microcentrifugal centrifugation. Add 0.5 ml of extract to each tube (chi〇roform/methanol mixed solution 2:1 v/v), homogenize the machine at a speed of 1 〇, 〇〇〇 rpm for about 20 seconds, then fill the two together. After pouring the liquid microcentrifuge tube into a 15 ml FALCON centrifuge tube, aspirate the lm] chloroform/methanol mixed solution (2: 1 v/v) into the centrifuge tube to clean the residual tissue of the tube wall with the micro-dispenser. After that, pour into the above 15 ml FALCON centrifuge tube, and finally add chloroform/methanol mixed solution (2: 1 v/v) 7 ml (total volume 10 ml). Take 25 μ μl of liver lipid extract, add 250 μM Triton X-100 (Sigma), mix and analyze the total cholesterol and triglyceride in the liver. 5-3. Statistical Methods The statistical method used in the present invention is Student's test. 5_4. Results 赵·Zhaozhong Please refer to Figure 5Α to Figure 5D. After one month of feeding the Lactobacillus isolates, the diabetic rats of the Lactobacillus feeding group had no body weight compared with the placebo group. Significant changes. In the liver, the spleen, and the kidneys, there was no significant difference between the lactation test groups and the placebo group. B. Blood glucose and other ion aspects, as shown in the figure, A and figure, b, in terms of blood sugar levels, the blood glucose changes of diabetic rats with separate food isolates GMNL-205 or GMNL-263 There was a significant reduction in the placebo group. · After transfusion of GMNLI or GMNL-263, it can effectively reduce the high blood sugar level and blood sugar change of diabetic patients. In terms of ion concentration in the blood (please refer to Table 6), there was no significant difference between the Lactobacillus group and the placebo group. Table 6 Feeding GMNL-89, GMNL-205 and GMNL-263 - months later , measurement of ion concentration in blood of diabetic rats

Normal Placebo Insulin Na Nano (mEq/V) 147.267士3.823 140.733土6.381 144.175±1.882 K Potassium (mEq/1) 12.785± 1.548 8.473±0.363 9.53± 1.332 Cl gas (mEq/1) 88.65±3.849 79.4±5.897 79.15± 3.122 Ca Mom (mg/dl) 8.933± 1.305 9.4±0.7 8.825±2.027 P Scale (mg/dl) 26.483±2.27 17.833±3.717 21.9±2.481 GMNL-89 GMNL-205 GMNL-263 Na Nano (mEq/i) 144.013 ±2.446 140.314±2.021 141.171±4.162 K Potassium (mEq/l) 9.298±2.827 10.093±2.367 9.704±2.023 Cl gas (mEq/1) 79.813±4.222 80.8±2.918 83.114±4.819 Ca ^(mg/dl) 10.075±〇 .933 9.343± 1.356 8.514± 1.899 P Lin (mg/dl) 22.038±4.779 18.529±3.528 17.857±4.357

C. For the biochemical value of serum, please refer to Figure 7A to Figure 7C and Table 7. In the serum biochemical value, the glycated hemoglobin in the serum of diabetic rats was isolated after 23 days of the 201132286 strain of GMNL-205. The inferiority of HbAlc) was lower than that of placebo; after the GMNL_89, the total gallemia concentration and LDL/HDLtt_ in the serum of diabetic rats were significantly lower than that of An__; Significant «. In terms of improvement of liver function index, the concentration of GOT and GPT in serum of diabetic rats decreased after one month of _food isolate gmnl_89. After feeding the isolate GMNL-263 for one month, the concentration of GpT in the serum of diabetic rats also decreased compared with the placebo group. Hemoglobin is gradually saccharified by blood glucose in the life cycle of red blood cells (average (3) days). Therefore, in addition to monitoring the blood sugar level and blood sugar change of diabetic patients, (heap hemoglobin Coated h_glGbin) is also widely used as a monitor. The indicator of the patient's blood glucose (four) situation. In addition, many studies have indicated that patients with diabetes have a significant increase in blood biliary concentration in addition to high blood glucose levels, so the chances of developing heart and vascular disease will increase significantly. Based on the above results, it is confirmed that the ship's (four) miscellaneous coffee and coffee - one job will be able to effectively reduce the hemoglobin value of diabetic patients' reflects that long-term use of the Lactobacillus isolate GMNL-2G5 will be effective (four) Lighten the situation. In addition, taking Lactobacillus isolates after reading L-89- will effectively reduce the total curdol concentration in diabetic patients and the related diseases or complications of LDL ancestors. opportunity. In addition, in terms of blood cell sorting (see Table 8), there was no significant difference between the Lactobacillus group and the placebo group. D. For cytokine (cytokine), please refer to Figure 8. In the serum cytokine concentration, the Lactobacillus brevis isolate 24 201132286: GMNL_263 one month later 'Lactobacillus arrogant group compared to the comfort ship group , Lactobacillus silver food group. Can significantly reduce the sputum concentration of sputum in the serum of diabetic rats. The annual smear field indicates that diabetes is closely related to inflammation, which leads to chronic inflammation in diabetic patients. Therefore, the present invention demonstrates that a single leg of the lactobacillus isolate GMNL-263, in addition to effectively regulating the blood glucose level of the patient with sugar 2, can effectively reduce the concentration of inflammatory-related cells in the serum of diabetic patients, Taking these Lactobacillus isolates will effectively improve diabetes, inflammatory response or related complications. φ E. For liver lipids, please refer to Figure 9A. In the concentration of triglyceride in the liver, the diabetic mice of the Lactobacillus brevis isolate GMNL-89 or GMNL-263 have a decreasing trend compared with the control group. . Please refer to Figure 9B. 'In terms of cholesterol concentration in the liver, the mice of the Lactobacillus gallisepticum isolate GMNL-89 have a decreased _ potential; while the mice of the Lactobacillus isolate GMNL-263 are torn. The cholesterol concentration was significantly lower than that of the control group. From the results, it can be seen that (4) the milk (four) urinary tract patients provided by the present invention can effectively reduce the concentration of triglyceride and cholesterol in the liver, thereby reducing the occurrence of non-sexual fatty liver. risks of. The above detailed description is intended to be illustrative of a possible embodiment of the invention, and is not intended to limit the scope of the invention. In the scope of this patent. The technical features disclosed in this case have fully complied with the statutory requirements of the new and progressive. X-specialized articles are submitted in accordance with the law, and the application for invention patents is approved by the responsible office to encourage invention. 25 201132286 <^¥%wsj^^-^^w^Y 堞唼御-?靶呎学丨 £9z-TNWO^,tn0z-JNHO, 68-TNWO^^^-<

GMNL-263 6·286±1.173 5.457士0.562 3.6士0.4 1.986士0.344 554土307.77 199士124.988 0.371士 0.189 9.5士士 11.684 0.051±0.105 I 45.2±12.466 0_429土 0.111 387.571±318.053 1 140.571士 85.893 0.231士0· 177 GMNL-205 6.8 ± 0.245* 5.257 ± 0.346 3.543 ± 0.299 1.929 ± 0.243 609.857 ± 225.069 239_571 ± 105 · 065 0.343 soil 0_ 172 8.9 soil 5.725 0.159 ± 0.241 44.429 ± 6.748 0.414 ± 0 09 519.143 395.062 1 111.714 士 54.227 0.163 soil 0.119 GMNL-89 6.913 ± 1.203 5.95 ± 0.548 3.838 ± 0.283 1.888 ± 0.348 418.875 soil 246.526 159.25 ± 113.235 0.3 soil 0.177 6.4 ± 4A44 1 0.185 ± 0.211 42.825 ± 6.804 1 0.513 ± 0.083 269.75 soil 233.066 85.75 ± 25.789 * 0.149 ± 0.046* Adenosine group 7.25 ± 0·238 5.875 ± 0.287 3.425 ± 0.126 1.425 ± 0.126 * 407 soil 86.76 100.25 ± 40.352 * 0.225 soil 0.096 2 · 15 ± 2 · 456 * 0.01 ± 0 44.025 ± 7.258 0.525 ± 0.05 201.75 soil 41.732* '78±18.493* 0.138±0.023 placebo group 7.2±0.173 | 5.333±0.289 3.6±0.265 2.067±0.153 546.333士81.929 | 235±26.211 0.333士0.231 9.7 67士4.186 0.053士0.04 44.4±9.358 0.433±0.058 393.333士81.402 150±30.199 0.341士 0.233 Control group 4.2±0.089 | 6.7士0_424 4.367土0.288 1.883士 0.147 1 277.5±52.687 丨48.333±7.23 0.15 士0.055 0.1士0 0.01士0 29.017土2.073 0.617 soil 0.041 59.167±11.907 1 73.333±14.459 0.136 ± 0.032 HbAlc glycated hemoglobin (%) Total protein total protein (g/dl) Albumin albumin (g/dl) A/G Ratio white ball than GOT ( Unit) GPT(units) Total Bilirubin Total biliary pigment (mg/dl) γ-GT alcoholic hepatitis (IU/1) CRP (mg/dl) BUN urea protein (mg/dl) Creatinine Creatine dry (mg/dl) Triglyceride Triglyceride (mg/dl0 total Cholesterol Total Cholesterol (mg/dl) LDL/HDL

201132286

埏 骤 骤 窭 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 靶 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 ±2.81 18.657±0.69 575±192.28 77.33±12.46 GMNL-205 8.36±3.33 7.829±0.76 14.47±1.33 47.21±4.34 _ 60.36±1.22 18.5±0.497 610.57±170.68 69.014±10.262 GMNL-89 7.9U2.71 8.03±0.57 14.78士1_52 47.6±4.74 1 1 59.15 士2.09 18.35±0.838 595.88±136.14 65.83±11.28 Insulin group 10.33±2.88* 7.69±0.14 14.55±0.44 46.58±0.86+ 60.58±1.19 18.925±0.591 787.5±78.37* 71.45±8.48 Placebo Group 5.23±1.62 7.57±0.15 1 14.233±0.40 44.77±0.76 59.1±1.1 18.8±0.361 527.67±161.85 59.5±0.85 Control group 9.42±2.94 7.57±0.86 _i 14.33±1.37 46.82±9.30 61.55±6.941 18.983±0.93 623.33±308.21 75.95±9.03 white blood cells WBCClOOO/μΙ) red blood cells RBC (1CT7/W) _1 HGB hemoglobin (g/dl) HCT hematocrit (%) MCV red blood cell average size (fl) MCH red blood cell average hemoglobin (pg) PLT platelets (1000/μ1 Lymph% lymphocytes (%) 201132286 [Simple diagram] Figure 1 shows the micrograph of Lactobacillus delbrueckii (Laciobad/Zw·? gimen·) GMNL-205. Figure 2 shows the gene map of Lactic^ad//i^ gassm· GMNL-205. bump. Wherein Μ represents a molecular marker (Molecular Marker)', respectively, with arrows indicating the molecular weight of each fragment (bp). Figure 2 shows the morphology of GMNL-263 under the microscope. Figure 4 shows Lactobacillus reuteri ([aciofeaa7/i« rewim) GMNL-263 and GMNL-89. The gene map is sigma. Among them, Μ represents the molecular marker (Molecular Marker), and the molecular weight (bp) of each fragment is indicated by an arrow. Figure 5A shows the weekly weight record of each group of diabetic rats; Figure 5B shows the diabetes of each group. The spleen weight measurement results of the atmosphere; Figure 5c shows the weight measurement results of the rats in each group of urinary diseases; Figure 5D shows the kidney weight measurement results of each group of diabetic rats. Figure 6A shows the daily weight of each group of diabetic rats. Figure 6b shows the glycosuria of each group: the blood sugar of the milk; the result of the kg. * indicates that the data is statistically significant compared with the placebo group 〇p<0.05. Figure 7A shows the diabetic rats of each group. Glycated hemoglobin bMe) measurement results; Figure 7B is the total gallbladder concentration measurement results of each group of diabetic rats; Figure 7c is the LDL/HDL ratio analysis results of each group of women with urinary disease. * indicates data and safety The boots (4) are statistically significant (p<0.05) ° The picture shows the inflammatory phase in the serum of each group of diabetic rats. The cytokine (4) 丫; agronomic measurement knot 28 201132286 fruit. * indicates that the data is statistically significant compared with the placebo group (ρ < 0. 05). Figure IX is the liver triglyceride of each group of diabetic rats (liver triglyceride) measurement results; Figure IX B shows the liver cholesterol measurement results of each group of diabetic rats. * indicates that the data is statistically significant compared with the placebo group (p<〇.〇5). Component symbol description]

29

Claims (1)

201132286 VII. Patent Application Range: 1. A composition for improving symptoms of diabetes and its complications, comprising at least one selected from the group consisting of: Lactobacillus reuteri GMNL-89, Gardner's milk One of a group consisting of Lactobacillus gasseri GMNL-205 and Lactobacillus reuteri GMNL-263, and a pharmaceutically acceptable carrier. 2. The composition of claim 1, wherein the Lactobacillus reuteri 1 decoy is reuteri) GMNL-89 is registered as BCRC 910340, Lactobacillus gasseri GMNL The registration number of -205 is bcRC 9HM51, and the registration number of Lactobacillus reuteri GMNL-263 is BCRC 910452. 3. The composition according to claim 1, wherein the diabetes symptom comprises a high blood sugar level, a high blood sugar level change amount, a high glycated hemoglobin ratio, a high total cholesterol concentration, a high LDL/HDlj ratio, a high IFNi amount, High liver triglyceride lipid concentration, high liver cholesterol concentration and improved liver function indicators GOT, GPT. 4_ The composition of claim 1, further comprising at least one probiotic selected from the group consisting of Lactobacillus species (/aci〇Ma7/Wiy W.), Bifidobacterium Species (5 and /〇~1<^6 coffee/«15/?.), Streptococcus species (15/7拗〇?0:1«1>5/?.), yeast (丫63 he ). 5. The composition of claim 1, further comprising an edible material comprising water, fluid dairy, milk, concentrated milk, yogurt, yogurt, cold yoghurt, Lactobacillus fermented beverages, milk powder, ice cream, cheese, dried legumes, soy milk, fermented soy milk, vegetable juices, juices, sports drinks, desserts, jellies, sweets, baby food, health foods, animal feed, Chinese herbal medicines, dietary supplements. 6. A method for improving the symptoms of diabetes and its complications by using an effective amount of the composition described in claim 1 of the patent application 201132286: the scope of claim 1 for the purpose of improving and reducing High blood sugar level, blood sugar value change, glycated hemoglobin ratio, total cholesterol concentration, LDL/HDL _, ΙΡΝ·γ t, occupational _ fat f concentration, and thus improve diabetes and related complications . 7. A novel Lactobacillus isolate 'This isolate is selected from the following deposited strains: (1) Lactobacillus gargle ([(10)-Qingm') GMNL 2〇5 ' is deposited in the development of Hsinchu Food Industry The Center for the Conservation and Research of Biological Resources', the registration number is bcrc • 910451; (2) Lactobacillus reuteri r_r〇GMNL 263, which is deposited at the Bioresource Conservation and Research Center of Hsinchu Food Industry Development Institute, with the registration number bcrc 910452 〇 8. The lactobacillus according to item 7 of the patent application, wherein the lactobacillus can be used to reduce and improve high blood sugar level, high blood sugar level change, high glycosylated hemoglobin ratio, high total cholesterol in a diabetic subject Concentration, high LDL/HDL ratio, high IFN-γ amount, high liver triglyceride concentration and high liver cholesterol concentration, thereby improving diabetes and related complications. 31