Application of binding cholic acid TrpCA and producing strain thereof in treatment and prevention of metabolic diseases

Abstract

The invention provides application of combined cholic acid TrpCA and producing bacteria thereof in treating and preventing metabolic diseases. The present invention relates to the use of tryptophan-binding cholic acid or a producing bacterium thereof in the preparation of a composition for preventing and / or treating a metabolic disease or for promoting the production of GLP-1 and / or insulin. The inventor finds for the first time that tryptophan-bound cholic acid can remarkably promote secretion of GLP-1 on an in-vivo animal model and an in-vitro cell model, the effects of reducing weight, reducing blood sugar and relieving the symptoms of the non-alcoholic fatty liver disease are achieved, and the same metabolism improvement effect can also be achieved through planting of probiotics capable of generating cholic acid TrpCA.

Description

Technical Field

[0001] The present invention relates to the field of microbiology, and in particular to the use of tryptophan-bound bile acid and bacteria producing the same in the treatment and prevention of metabolic-related diseases. Background Art

[0002] Metabolic disease is a general term for diseases that occur due to metabolic disorders in the body. Metabolic diseases are usually caused by an imbalance of carbohydrates, lipids, proteins, vitamins, electrolytes, water, etc. Examples of metabolic diseases include obesity, diabetes, hyperlipidemia, arteriosclerosis, hypertension, non-alcoholic fatty liver disease, etc. With the development of society, the continuous improvement of people's living standards and the adjustment of dietary structure, the diet has become more abundant, resulting in an increasing incidence of metabolic diseases, among which obesity, hyperglycemia and hyperlipidemia are the most concerned. Obesity is an inducing factor for a series of diseases, such as hypertension, diabetes, coronary heart disease, gallbladder disease, osteoarthritis, and sleep apnea. Weight loss, blood sugar lowering, and lipid-lowering drugs have become one of the focuses of drug research.

[0003] There remains a need in the art for active ingredients capable of preventing and treating metabolic diseases. Summary of the invention

[0004] Tryptophan-bound bile acid (also known as bile acid TrpCA or TRP-CA) is a newly discovered amino acid-bound bile acid produced by bacteria in recent years. It is widely present in the human population, but its physiological function has not been reported. The inventors' research results show that tryptophan-bound bile acid can significantly promote the secretion of GLP-1 in in vivo animal models and in vitro cell models, play a role in weight loss, lowering blood sugar, and relieving the symptoms of non-alcoholic fatty liver disease, and probiotic colonization that can produce bile acid TrpCA can also play the same metabolic improvement role.

[0005] After extensive and in-depth research and experiments, the inventors found that tryptophan-bound bile acid has the effect of preventing and treating metabolic-related diseases (including obesity, diabetes, and fatty liver). Through in vitro experiments, strains that can produce tryptophan-bound bile acid were screened, and the animal Bifidobacterium lactis subsp. lactis (B. animalis subsp. lactis) that can produce tryptophan-bound bile acid was colonized in mice fed a high-fat diet. It was found that the colonization of the strain can also reduce blood sugar by producing tryptophan-bound bile acid, effectively alleviating diabetes. On this basis, the present invention was completed.

[0006] In one aspect, provided is the use of tryptophan-bound bile acid or its producing bacteria in the preparation of a pharmaceutical composition or a food composition, wherein the pharmaceutical composition is used to prevent and / or treat metabolic diseases, or to promote the production of GLP-1 and / or insulin, and the food composition is used to maintain healthy blood sugar levels, maintain healthy blood lipids and / or control body fat.

[0007] In one aspect, a method for treating and / or preventing a metabolic disease in a subject, or a method for promoting the production of GLP-1 and / or insulin in a subject is provided, the method comprising administering tryptophan-bound bile acid or a bacteria producing the same, or a pharmaceutical composition comprising the tryptophan-bound bile acid or a bacteria producing the same, to the subject.

[0008] In one aspect, a method for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat in a subject is provided, the method comprising administering to the subject tryptophan-bound bile acid or a bacteria producing the same or a food composition comprising the same.

[0009] In one aspect, tryptophan-bound bile acid or its producing bacteria or a pharmaceutical composition comprising tryptophan-bound bile acid or its producing bacteria is provided, for use in treating and / or preventing metabolic diseases in a subject, or in promoting the production of GLP-1 and / or insulin in a subject.

[0010] In one aspect, tryptophan-bound bile acid or its producing bacteria or a food composition comprising tryptophan-bound bile acid or its producing bacteria is provided, which is used for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat in a subject.

[0011] In one embodiment of the above aspects, the subject is a mammal, preferably a human.

[0012] In one embodiment of the above aspects, the tryptophan-binding bile acid-producing bacteria are one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucosus, Lactobacillus euchromaticus, Lactobacillus unisorbitus, Lactobacillus plantarum, Bifidobacterium and Lactococcus.

[0013] In one embodiment of the above aspects, the tryptophan-binding bile acid-producing bacteria are one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis and Lactococcus lactis.

[0014] In one embodiment of the above aspects, the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus. In one embodiment of the above aspects, the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis.

[0015] In one embodiment of the above aspects, the metabolic disease is a GLP-1 related metabolic disease, preferably selected from diabetes or a disease related to diabetes.

[0016] In one embodiment of the above aspects, the diabetes is selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obese diabetes, autoimmune diabetes, and borderline diabetes.

[0017] In one embodiment of the above aspects, the disease associated with diabetes is selected from the group consisting of obesity, obesity-related disorders, metabolic syndrome, diabetic neuropathy, kidney disease such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorders, non-alcoholic steatohepatitis, prediabetes, hyperlipidemia, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, increased blood fatty acid or glycerol levels, impaired wound healing, glucose intolerance, increased fasting glucose and dyslipidemia, pancreatic beta cell deficiency, enteroendocrine cell deficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis and stroke.

[0018] In one embodiment of the above aspects, obesity is selected from the group consisting of symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity.

[0019] In one embodiment of the above aspects, the disease associated with obesity is selected from the group consisting of: glucose tolerance, abnormal lipid metabolism, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity hypoventilation syndrome and visceral obesity syndrome.

[0020] In one embodiment of the above aspects, the food composition further comprises a food acceptable carrier, diluent or excipient.

[0021] In one embodiment of the above aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0022] In one embodiment of the above aspects, the pharmaceutical or food composition further comprises probiotics and / or prebiotics.

[0023] In one embodiment of the above aspects, the composition is an oral composition, preferably selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions.

[0024] In one aspect of the above-mentioned aspects, there is provided a composition for preventing and / or treating metabolic diseases, comprising tryptophan-bound bile acid or its producing bacteria and another active ingredient for preventing and / or treating metabolic diseases. In one embodiment, another active ingredient is one or more of biguanides, sulfonylureas, glinides, alpha-glucosidase inhibitors, thiazolidinediones and GLP-1 drugs. In one embodiment, the composition is an oral composition. In one embodiment, the oral composition is selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions.

[0025] In one embodiment of the above aspects, the food composition further comprises a food acceptable carrier, diluent or excipient.

[0026] In one embodiment of the above aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

[0027] In one embodiment of the above aspects, the pharmaceutical or food composition further comprises probiotics and / or prebiotics.

[0028] In one embodiment of the above aspects, the composition is an oral composition, preferably selected from granules, capsules, tablets, powders, oral solutions, suspensions and emulsions. In one embodiment, a unit dose of the composition comprises 1x10-1x10 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL tryptophan-binding bile acid-producing bacteria.

[0029] In one aspect, a method for producing tryptophan-bound bile acid is provided, comprising culturing a probiotic bacterium in the presence of bile acid and tryptophan, wherein the probiotic bacterium is one or more of the genera Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucosus, Lactobacillus euchromaticus, Lactobacillus unisorans, Lactobacillus plantarum, Bifidobacterium, and Lactococcus.

[0030] In one embodiment, the probiotic is one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus muciniphila, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Phytobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0031] In one embodiment, the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus. In one embodiment, the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis.

[0032] In one embodiment, the use of probiotics in producing tryptophan-bound bile acid, wherein the probiotics are one or more of Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucosus, Lactobacillus spp., Lactobacillus unisorans, Lactobacillus plantarum, Bifidobacterium and Lactococcus.

[0033] In one embodiment, the probiotic is one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus muciniphila, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Phytobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0034] In one embodiment, the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus. In one embodiment, the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis.

[0035] In another aspect, a method for screening a probiotic that promotes GLP-1 and / or insulin production is provided, comprising:

[0036] (1) culturing probiotics in the presence of bile acid and tryptophan to obtain a culture supernatant;

[0037] (2) determining the content of tryptophan-bound bile acid in the culture supernatant; and

[0038] (3) Select probiotics that produce tryptophan-conjugated bile acid.

[0039] In one embodiment, the probiotic is one or more of Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucinus, Lactobacillus euchromaticus, Lactobacillus unitarianis, Lactobacillus plantarum, Bifidobacterium, and Lactococcus.

[0040] In one embodiment, the probiotic is one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus muciniphila, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Phytobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0041] In one embodiment, the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus. In one embodiment, the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis.

[0042] In one embodiment, the method comprises mixing the probiotic culture solution with methanol, and detecting the content of tryptophan-bound bile acid by mass spectrometry.

[0043] In each of the above embodiments, the tryptophan-bound bile acid has the following structure:

[0044]

[0045] Advantages of the present invention include:

[0046] 1. The inventors discovered for the first time that tryptophan-conjugated bile acid can significantly promote the secretion of GLP-1 in in vivo animal models and in vitro cell models, play a role in weight loss, lowering blood sugar, and alleviating the symptoms of non-alcoholic fatty liver disease, and that the colonization of probiotics that can produce bile acid TrpCA can also play the same metabolic improvement role.

[0047] 2. The inventors provide a method for screening probiotics that promote GLP-1 production based on the production of tryptophan-bound bile acid.

[0048] 3. The inventors have found that Bifidobacterium (eg, Bifidobacterium animalis) can produce tryptophan-bound bile acid at a relatively high yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The tryptophan-conjugated bile acid structure is shown.

[0050] Figure 2 The effect of tryptophan-conjugated bile acid on the body weight of mice fed a high-fat diet was shown. The statistical method was a two-way repeated measures analysis of variance combined with an unpaired T test. *P<0.05, **P<0.01.

[0051] Figure 3 Shown is the effect of tryptophan-conjugated bile acid on glucose tolerance in mice fed a high-fat diet. Figure 3 The graph above shows how blood sugar levels change over time. Figure 3 The figure below shows the AOC (area under the curve) of the control group and the treatment group. The statistical method was two-way repeated measures analysis of variance combined with unpaired T test. *P<0.05, **P<0.01, ***P<0.001.

[0052] Figure 4 The effect of tryptophan-conjugated bile acid on serum AST and ALT in mice fed a high-fat diet is shown. The statistical method is a two-tailed unpaired T test. *P<0.05, ***P<0.001.

[0053] Figure 5 The figure shows the changes in the content of tryptophan-conjugated bile acid in the ileum of mice after oral administration of tryptophan-conjugated bile acid (10 mg / kg) for one day. The statistical method was two-tailed unpaired T test. ***P<0.001.

[0054] Figure 6 The effect of Bifidobacterium animalis subsp. lactis on glucose tolerance in mice fed a high-fat diet is shown. The statistical method was two-way repeated measures ANOVA combined with unpaired T test. *P<0.05, **P<0.01. Figure 6 The graph above shows how blood sugar levels change over time. Figure 6The lower graph shows the AOC (area under the curve) of the control and treated groups. DETAILED DESCRIPTION

[0055] In the present application, the term "tryptophan-bound bile acid" refers to a conjugate formed by the reaction of the amino group of tryptophan with the carboxyl group at position 24 of bile acid to form an amide bond. The structure of tryptophan-bound bile acid is as follows Figure 1 As shown in. Reports on tryptophan-bound bile acid can be found in, for example, Nature. 2024 Feb; 626(7998): 419-426. doi: 10.1038 / s41586-023-06906-8. Epub 2023 Dec 5.

[0056] In the present application, the term "tryptophan-bound bile acid producing bacteria" refers to probiotics that can produce tryptophan-bound bile acid in the presence of bile acid, a substrate for tryptophan-bound bile acid, and tryptophan. Examples of tryptophan-bound bile acid producing bacteria include, but are not limited to, Streptococcus, Pediococcus, Lactobacillus, Lactobacillus, Lactobacillus, Lactobacillus, Lactobacillus, Bifidobacterium, and Lactococcus. Tryptophan-bound bile acid producing bacteria can be one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis. More specifically, Streptococcus salivarius can be Streptococcus salivarius thermophilus subspecies. Bifidobacterium animalis can be Bifidobacterium animalis lactis subspecies.

[0057] In the present application, "glucagon-like peptide-1 (GLP-1)" is a hormone mainly produced by intestinal L cells. GLP-1 plays a hypoglycemic role by promoting insulin secretion. It can also reduce weight and lower blood lipids by suppressing appetite and promoting energy metabolism.

[0058] In the present application, the term "metabolic disease" generally refers to a disease or condition that destroys the normal metabolic process in the body, making it impossible for the body to normally utilize and / or store energy. Metabolic disease can be any metabolic disease that benefits from regulating (e.g., increasing) GLP-1 activity, or any metabolic disease that benefits from exciting GLP-1 receptor activity. Metabolic disease can be a metabolic disease related to GLP-1, including diabetes or a disease related to diabetes. Diabetes includes, but is not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, obesity diabetes, autoimmune diabetes and / or critical diabetes. Diseases associated with diabetes include, but are not limited to, obesity, obesity-related disorders, metabolic syndrome, diabetic neuropathy, kidney disease such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorders, nonalcoholic steatohepatitis, prediabetes, hyperlipidemia, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, elevated blood fatty acid or glycerol levels, impaired wound healing, glucose intolerance, increased fasting glucose and dyslipidemia, pancreatic beta cell deficiency, enteroendocrine cell deficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis and / or stroke. Obesity includes, but is not limited to, symptomatic obesity, simple obesity, childhood obesity, morbid obesity and abdominal obesity. Diseases associated with obesity include, but are not limited to, glucose tolerance, abnormal lipid metabolism, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity hypoventilation syndrome and visceral obesity syndrome.

[0059] In this application, the term "prevention" refers to the delay of onset of one or more symptoms of a specific disease, disorder or condition, and / or the reduction of frequency and / or severity. The term "treatment" refers to any administration of a therapy that partially or completely relieves, improves, alleviates, inhibits one or more symptoms, features and / or causes of a specific disease, disorder and / or condition, delays its onset, reduces its severity, and / or reduces its incidence.

[0060] In the present application, the term "subject" refers to an individual receiving the administration of the provided treatment. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal, such as a mammal experiencing or susceptible to metabolic diseases as described herein. In some embodiments, the animal is a vertebrate, such as a mammal, such as a non-human primate (particularly a higher primate), a sheep, a dog, a rodent (e.g., a mouse or a rat), a guinea pig, a goat, a pig, a cat, a rabbit, or a cow. In some embodiments, the subject is a person.

[0061] In the present application, "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level can be determined according to factors including the following: severity of the disease, drug activity, patient age, weight, health status, sex, patient sensitivity to the drug, drug administration time, administration route and release rate, treatment period, drugs used in combination with the composition or simultaneously, and other factors known in the medical field. The pharmaceutical composition according to an example can be used as a single therapeutic agent, or in combination with other therapeutic agents, and can be used sequentially or simultaneously with conventional therapeutic agents. In addition, it can be used alone or multiple times. Considering all factors, it is important to use an amount that can achieve maximum efficacy with a minimum amount without side effects.

[0062] Treatment

[0063] The present invention provides the use of tryptophan-bound bile acid in the treatment and prevention of metabolic-related diseases. When a subject ingests a high-fat diet, tryptophan-bound bile acid has the ability to inhibit the subject's weight gain. The inventors found that C57BL / 6J mice treated with tryptophan-bound bile acid and fed with a high-fat diet that can cause obesity had less weight gain than untreated controls, and helped improve blood sugar and fatty liver symptoms. Therefore, tryptophan-bound bile acid can be used to prevent and treat obesity and diseases caused by obesity such as diabetes, fatty liver, etc. The present invention also provides the use of tryptophan-bound bile acid-producing bacteria in the treatment and prevention of metabolic-related diseases. In vitro detection of the following bacterial species including Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium spp. and Lactococcus spp. can all produce tryptophan-bound bile acid. Therefore, those skilled in the art can expect that these strains tested in vitro can improve the glucose metabolism disorders induced by a high-fat diet. The inventors also demonstrated in a mouse model that colonization with animal Bifidobacterium lactis subsp. lactis can significantly improve the glucose metabolism disorder induced by a high-fat diet.

[0064] Therefore, the present invention provides a method for treating and / or preventing metabolic diseases in a subject, which comprises administering tryptophan-bound bile acid or its producing bacteria to the subject. The present invention also provides a method for promoting the production of GLP-1 and / or insulin in a subject.

[0065] The type of tryptophan-bound bile acid producing bacteria is not particularly limited, as long as it is a probiotic that can produce tryptophan-bound bile acid. Tryptophan-bound bile acid producing bacteria can include Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucus, Lactobacillus genus, Lactobacillus genus, Lactobacillus plantarum, Bifidobacterium and Lactococcus. For example, tryptophan-bound bile acid producing bacteria is one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus mucus reuteri, Lactobacillus sake, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis and Lactococcus lactis. Streptococcus salivarius can be Streptococcus salivarius thermophilus subspecies. Animal Bifidobacterium can be Bifidobacterium animalis lactis subspecies. Those skilled in the art can easily select tryptophan-bound bile acid producing bacteria suitable for mammals (particularly humans) to be administered according to the teachings of the present invention.

[0066] The method of the present invention can be applied to any mammalian subject, but is preferably applied to human subjects. As described above, the metabolic disease treated by the method is a metabolic disease associated with GLP-1 or obesity, preferably selected from diabetes or a disease associated with diabetes, such as various specific diseases described herein.

[0067] Health care methods

[0068] The inventors have found that tryptophan-bound bile acid or its producing bacteria or a composition comprising tryptophan-bound bile acid or its producing bacteria can be used to maintain healthy blood sugar levels, maintain healthy blood lipids and / or control body fat. The present invention provides a method for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat, which comprises administering tryptophan-bound bile acid or its producing bacteria or a composition comprising tryptophan-bound bile acid or its producing bacteria to a subject. In the health care method herein, the subject may be a healthy subject.

[0069] The type of tryptophan-bound bile acid producing bacteria is not particularly limited, as long as it is a probiotic that can produce tryptophan-bound bile acid. Tryptophan-bound bile acid producing bacteria can include Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucus, Lactobacillus genus, Lactobacillus genus, Lactobacillus plantarum, Bifidobacterium and Lactococcus. For example, tryptophan-bound bile acid producing bacteria is one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus mucus reuteri, Lactobacillus sake, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis and Lactococcus lactis. Streptococcus salivarius can be Streptococcus salivarius thermophilus subspecies. Bifidobacterium animalis can be Bifidobacterium animalis lactis subspecies.

[0070] The health care method of the present invention can be applied to any mammalian subject, but is preferably applied to human subjects who need to maintain healthy blood sugar levels, maintain healthy blood lipids and / or control body fat.

[0071] Composition

[0072] The composition administered to the subject may be a food composition, such as a beverage, a dairy product, a solid food, but is not limited thereto. The food composition may also contain additional probiotics, prebiotics and / or food acceptable carriers, diluents or excipients. The composition administered to the subject may also be a pharmaceutical composition. The pharmaceutical composition may also contain another active ingredient for preventing and / or treating metabolic diseases, preferably, another active ingredient is one or more of biguanides, sulfonylureas, glinides, α-glucosidase inhibitors, thiazolidinediones and GLP-1 drugs. The composition herein may be an oral composition, preferably selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions. The tryptophan-bound bile acid-producing bacteria in the composition should be a therapeutic or preventive effective amount. Those skilled in the art can routinely determine the therapeutic or preventive effective amount of probiotics, such as tryptophan-bound bile acid-producing bacteria. For example, the unit dose of the tryptophan-bound bile acid-producing bacteria in the composition contains 1x10-1x10 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL tryptophan-binding bile acid-producing bacteria.

[0073] Some non-limiting examples of foods to be used with the methods and compositions described herein include: popsicles, cheese, cream, chocolate, milk, etc. In other embodiments, the food may be fruit juice, refreshing drinks, tea drinks, beverage products, jelly drinks, and functional drinks; alcoholic beverages such as beer; carbohydrate-containing foods such as rice foods, noodles, bread, and pasta; paste products such as fish, ham, sausage, and seafood paste products; retort pouch products such as curry, foods coated with thick starch sauces, and Chinese soups; soups; dairy products such as milk, dairy drinks, ice cream, and yogurt; fermented products such as fermented soy milk, fermented drinks, and pickles; bean products; various confectionery products, including biscuits, cookies, etc., candy, chewing gum, soft candy, cold desserts (including jelly, cream caramel, and frozen desserts); instant foods such as instant soups and instant bean soups; and the like. In one embodiment, the food for administration is frozen. In some embodiments, the food product can be or include one or more of the following: bars, candies, baked goods, cereals, salty snacks, pasta, chocolate and other solid foods, as well as liquid or semi-solid foods (including yogurt, soups and stews) and beverages (such as smoothies, milkshakes, juices and other carbonated or non-carbonated beverages).

[0074] Oral compositions may include an inert diluent or an edible carrier. For oral therapeutic administration, the active compound may be blended with an excipient and used in the form of a tablet, lozenge, pastille, lozenge or capsule (e.g., gelatin capsule). Oral compositions may also be prepared by combining the compositions of the present disclosure with food. In some embodiments, probiotics may be formulated in food.

[0075] In some embodiments, the composition is administered to the subject in a single dose. In some embodiments, the composition is administered to the subject in multiple doses. In some embodiments, the dosage of the composition is administered to the subject twice a day, daily, weekly, or monthly.

[0076] In some embodiments, a unit dose comprises 10 to 10 15 , 10 2 Up to 10 14 , 10 3 Up to 10 13 , 10 4 Up to 10 12 , 10 5 Up to 10 11 , 10 6 Up to 10 10 , 10 2 Up to 10 7 , 10 2 Up to 10 8or 10 2 Up to 10 9 Colony forming units (CFU). In some embodiments, the probiotics may be living cells. In some embodiments, the composition includes a cell culture of the probiotics and / or its culture supernatant and / or a powder formed therefrom and / or is prepared by using a cell culture of the probiotics and / or its culture supernatant and / or a powder formed therefrom.

[0077] Pharmaceutical compositions are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include oral administration. Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and books in the Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) series. Oral compositions generally include an inert diluent or an edible carrier. For example, oral formulations may be or include syrups, liquids, tablets, lozenges, soft candies, capsules (e.g., gelatin capsules), powders, gels, films, etc.

[0078] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil can be used as carriers, excipients or diluents of the pharmaceutical composition of the present invention.

[0079] In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and spices, etc. The pharmaceutical composition of the present invention can be produced as an enteric coating preparation by various well-known methods, so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass through the stomach smoothly without being destroyed by gastric acid.

[0080] The pharmaceutical composition of the present invention can be made into enteric-coated tablets for oral use. The term "enteric coating" in the present application includes all coatings allowed for use in conventional drugs, which are not degraded by gastric acid but can be fully decomposed in the small intestine and quickly release the microorganisms of the present invention. The enteric coating of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as HCl solution with pH=1, and preferably decomposed within 1.0 hour in synthetic intestinal fluid such as buffer with pH=7.0.

[0081] The enteric coating of the present invention is coated with about 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The thickness of the enteric coating of the present invention is 5-100 μm, and the ideal thickness is 20-80 μm. The enteric coating components are conventional polymers known to the public.

[0082] Example

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0084] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0085] Example 1: Effects of tryptophan-bound bile acid on glucose tolerance-related indices, body weight, body fat and liver function in obese mice

[0086] Materials: blood glucose test strips (Roche), blood glucose meter (Roche), active GLP-1 detection kit (Merck), insulin detection kit (Huamei Biotechnology), serum aspartate aminotransferase AST assay kit (Sinosino Biotechnology), serum alanine aminotransferase ALT assay kit (Sinosino Biotechnology).

[0087] Establishment of mouse model: C57BL / 6 mice (purchased from Jicui Yaokang), temperature 20-24°C, constant humidity 50-60%, light 12 hours (8:00-20:00), soundproof, free food and water, experiment after one week of adaptation. C57BL / 6 mice were fed a high-fat diet with tryptophan-conjugated bile acid by gavage for 8 weeks, with a dose of 10 mg / kg body weight, three times a week. Obesity model of mice induced by high-fat diet was obtained.

[0088] Methods: Male C57 mice aged 6-8 weeks, weighing 20-22 g, fed a high-fat diet were divided into the following groups according to their weight: (1) control group: saline containing 1% DMSO, without tryptophan-conjugated bile acid; (2) tryptophan-conjugated bile acid treatment group (i.e., experimental group): 6 mice per group, administered for 8 consecutive weeks (10 mg / kg, three times a week). For the detection of mouse small intestinal contents, another 6 mice were included. One week after the mice were gavaged with 10 mg / kg tryptophan-conjugated bile acid, the mice were killed, and the small intestine segments of the mice were cut off and the contents were taken out. The mouse small intestinal contents were extracted with 20 times methanol, and the concentration of tryptophan-conjugated bile acid in the mouse small intestinal contents was detected by mass spectrometry.

[0089] The two groups were given an equal amount of normal saline. The two groups of mice were fed a high-fat diet for 8 weeks, and their body weight was monitored weekly. A glucose tolerance test was performed in the second week of treatment (i.e., mice were gavaged with glucose, and GLP-1 and insulin levels were tested in mice at different time points). In Table 2, 0 minutes, i.e., before glucose gavage, and 15 minutes, i.e., 15 minutes after glucose gavage, the levels of GLP-1 and insulin secretion in the two time periods were tested. The blood glucose of mice was measured using a blood glucose meter at 0 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes. GLP-1 and insulin secretion levels were tested using an active GLP-1 detection kit (Merck) and an insulin detection kit (Huamei Bio) according to the kit instructions. After the last dose (week 8), body fat was detected by nuclear magnetic resonance (to measure fat and muscle content), blood was collected and centrifuged at 3000 rpm at 4°C, and serum aspartate aminotransferase (also known as aspartate aminotransferase) AST assay kit (Sino-Biologics) and serum alanine aminotransferase ALT assay kit (Sino-Biologics) were used to measure serum ALT and AST according to the instructions of the kit.

[0090] Results: Compared with the control group, the tryptophan-bound bile acid treatment group showed a trend of change from the first week, and a significant difference appeared in the sixth week, which showed that tryptophan-bound bile acid had a significant effect on weight control and could significantly reduce body fat; it could also improve glucose tolerance induced by a high-fat diet, among which promoting the secretion of glucose-stimulating GLP-1 and insulin was the reason for improving glucose tolerance; it also reduced the levels of serum AST and ALT, key indicators of liver function. Figure 2-4 , Table 1-2.

[0091] Figure 2 The effect of tryptophan-conjugated bile acid on the body weight of mice fed a high-fat diet was shown. The statistical method was a two-way repeated measures analysis of variance combined with an unpaired T test. *P<0.05, **P<0.01.

[0092] Figure 3 Shown is the effect of tryptophan-conjugated bile acid on glucose tolerance in mice fed a high-fat diet. Figure 3 The graph above shows how blood sugar levels change over time. Figure 3 The figure below shows the blood glucose AOC (area under the curve) of the control group and the treatment group. The statistical method was two-way repeated measures analysis of variance combined with unpaired T test. *P<0.05, **P<0.01, ***P<0.001.

[0093] Figure 4 The effect of tryptophan-conjugated bile acid on serum AST and ALT in mice fed a high-fat diet is shown. The statistical method is a two-tailed unpaired T test.

[0094] Figure 5The concentration of tryptophan-conjugated bile acid in the small intestine of mice one week after oral administration of 10 mg / kg tryptophan-conjugated bile acid to mice is shown.

[0095] Table 1 Effects of tryptophan-conjugated bile acid on body fat in mice fed a high-fat diet after continuous administration for 8 weeks (statistical method: two-tailed unpaired T test)

[0096] Grouping Fat content (g) Muscle content (g) Control group 10.1 19.3 Experimental Group 5.5 19.5 p-value 0.001 0.800

[0097] Table 2 Effects of tryptophan-bound bile acid on glucose tolerance test of mice fed a high-fat diet and glucose-stimulated GLP-1 and insulin after continuous administration for 2 weeks

[0098] (The statistical method was two-way repeated measures analysis of variance combined with unpaired T test)

[0099]

[0100]

[0101] Example 2: Effect of tryptophan-bound bile acid on GLUTag in intestinal L cell line

[0102] Materials: Mouse L cells (GLUTag cells, from DJ Drucker laboratory), cultured in high glucose medium, active GLP-1 detection kit (Merck).

[0103] Methods: After L cells were cultured adherently in DMEM high-glucose medium containing 10% fetal bovine serum to a density of 80%, 50 mM tryptophan-bound bile acid was diluted using the above medium to different final concentrations (0, 1, 10 or 50 μM) of tryptophan-bound bile acid sample group and 1‰ DMSO solvent control group. The mouse L cell samples with different concentrations of tryptophan-bound bile acid were allowed to stand for one hour, and the supernatant was extracted and the GLP-1 active detection kit (Merck) was used to detect the GLP-1 level in the supernatant.

[0104] Results: Compared with the control group, tryptophan-bound bile acid increased the level of GLP-1 in the cell supernatant in a dose-dependent manner. When the concentration was 10 μM, it had a significant promoting effect (Table 3). This result is consistent with the in vivo effect data of animals ( Figure 5 ) are consistent.

[0105] Table 3 Effect of tryptophan-bound bile acid on GLP-1 secretion by GLUTag of intestinal L cell line (statistical method: one-way ANOVA Tukey post hoc test)

[0106]

[0107] Example 3: Detection of tryptophan-bound bile acid production by various probiotics

[0108] Materials: The strains used are shown in Table 4. All strains were isolated conventionally without screening. Methods: When the OD value of each bacterial culture (MRS medium, anaerobic culture at 37°C) was 0.6, the substrates 1mM bile acid and 1mM tryptophan were added. After further culture for 24 hours, 50ul of the bacterial solution was added with 50ul of methanol for oscillation and mixing, centrifuged at 12000rpm, and the supernatant was passed through a membrane (0.22μm pore size water filter membrane), and the content of tryptophan-bound bile acid was detected by mass spectrometry. Mass spectrometry detection conditions: TrpCA was quantified using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. The LC-MS / MS system consisted of an Acquity ultra-high performance liquid chromatography system (Waters Corporation, Milford, USA) connected to a Sciex 5500 triple quadrupole linear ion trap mass spectrometer (AB Sciex, Framingham, MA, USA). The chromatographic column was ACQUITY UPLC CSH C18 (2.1×100mm, 1.7μm, Waters), the column temperature was 40℃, and the flow rate was 0.25mL / min. The injection volume was 5μL, the mobile phase A was 0.1% formic acid in water, and the mobile phase B was 0.1% formic acid in acetonitrile. TrpCA was detected in MRM mode under negative ion conditions. Metabolite separation was performed by gradient elution: 0-1min, 40% B; 1-6min, 40-100% B; 6-7min, 100% B; 7-8min, 40% B. A mixed working standard solution of different concentrations was obtained by gradient dilution (0.1, 1, 10, 100, 1000nM), and a standard curve was constructed. CA-d4 (200nM) was used as the internal standard for content correction. The LC-MS / MS control system used Analyst 1.6.2 version, and the quantitative analysis used MultiQuant software (version 3.0.1).

[0109] Results: All the bacterial species listed in Table 4 can synthesize tryptophan-conjugated bile acid, among which Bifidobacterium animalis subsp. lactis has the highest yield.

[0110] Table 4 Detection of tryptophan-bound bile acid synthesized by various probiotics

[0111]

[0112] Example 4: Effects of Bifidobacterium lactis subsp. animalis on glucose tolerance-related indices in hyperglycemic mice

[0113] Materials: The strain tested was Bifidobacterium animalis subsp. lactis. C57BL / 6 mice were purchased from Jicui Yaokang, and the temperature was 20-24°C, the humidity was 50-60%, the light was 12 hours (8:00-20:00), and the mice were soundproofed. The mice were allowed to eat and drink freely. The experiment was conducted after one week of adaptation. The blood glucose meter was a product of Roche, Germany.

[0114] C57BL / 6 mice were gavaged with the tested strain at a bacterial volume of 2x10 8 CFU / each, once a week. The bacterial solution needs to be cultured in advance, activated weekly to ensure freshness, and the concentration is measured separately.

[0115] Methods: After 1 week of adaptation, 6-8 week old male C57BL / 6 mice (weight 20-22 g) were divided into two groups according to their body weight: (1) control group, (2) animal Bifidobacterium lactis subsp. lactis colonization group. Each group had 7 mice, and they were gavaged once a week for 2 consecutive weeks. The control group was given an equal amount of normal saline. After 2 weeks, a glucose tolerance test was performed. Blood glucose was measured at different time points (0 min, 30 min, 60 min, 90 min and 120 min) after glucose gavage, and the area under the curve (AOC) was calculated. GLP-1 and insulin secretion levels were detected using an active GLP-1 detection kit (Merck) and an insulin detection kit (Huamei Biotechnology) according to the kit instructions.

[0116] Results: Colonization of mice with Bifidobacterium animalis subsp. lactis significantly improved glucose tolerance and promoted the secretion of glucose-stimulated GLP-1 and insulin. Figure 6 The effect of Bifidobacterium animalis subsp. lactis on glucose tolerance in mice fed a high-fat diet is shown. The statistical method was two-way repeated measures ANOVA combined with unpaired T test. *P<0.05, **P<0.01. Figure 6 The graph above shows how blood sugar levels change over time. Figure 6 The lower graph shows the AOC (area under the curve) of the control and treated groups.

[0117] Table 5 Effects of animal Bifidobacterium lactis subsp. lactis on glucose-promoting GLP-1 and insulin in mice fed a high-fat diet (statistical method: two-way repeated measures ANOVA combined with unpaired T test)

[0118]

[0119] Although the present invention has been described with reference to illustrative embodiments, it will be appreciated by those skilled in the art that various other changes, omissions and / or additions may be made and that elements of the described embodiments may be substituted with substantial equivalents without departing from the spirit and scope of the present invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from the scope of the present invention. Therefore, it is not intended herein to limit the present invention to the specific disclosed embodiments for carrying out the present invention, but rather it is intended that the present invention will include all embodiments within the scope of the appended claims.

Claims

1. Use of tryptophan-bound bile acid or its producing bacteria in the preparation of a pharmaceutical composition or a food composition, wherein the pharmaceutical composition is used to prevent and / or treat metabolic diseases, or to promote the production of GLP-1 and / or insulin, and the food composition is used to maintain healthy blood sugar levels, maintain healthy blood lipids and / or control body fat; the tryptophan-bound bile acid preferably has the following structure:

2. The use according to claim 1, wherein the tryptophan-binding bile acid-producing bacteria are one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucosus, Lactobacillus eutropha, Lactobacillus unisorans, Lactobacillus plantarum, Bifidobacterium and Lactococcus.

3. The use according to claim 1 or 2, wherein the tryptophan-binding bile acid-producing bacteria are one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus mucosus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis and Lactococcus lactis.

4. The use according to claim 3, wherein the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus; and the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis.

5. The use according to any one of claims 1 to 4, wherein the metabolic disease is a GLP-1-related metabolic disease, preferably selected from diabetes or a disease associated with diabetes; Preferably, the diabetes is selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obesity diabetes, autoimmune diabetes and borderline diabetes; Preferably, the disease associated with diabetes is selected from the group consisting of obesity, obesity-related disorders, metabolic syndrome, diabetic neuropathy, kidney disease such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorders, nonalcoholic steatohepatitis, prediabetes, hyperlipidemia, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, elevated blood fatty acid or glycerol levels, impaired wound healing, glucose intolerance, increased fasting glucose and dyslipidemia, pancreatic beta cell deficiency, enteroendocrine cell deficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis and stroke; Preferably, obesity is selected from the group consisting of symptomatic obesity, simple obesity, childhood obesity, morbid obesity and abdominal obesity; Preferably, the disease associated with obesity is selected from the group consisting of glucose tolerance, abnormal lipid metabolism, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity hypoventilation syndrome and visceral obesity syndrome.

6. The use according to any one of claims 1 to 5, wherein the food composition further comprises a food-acceptable carrier, diluent or excipient, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient; Preferably, the food composition or pharmaceutical composition further comprises probiotics and / or prebiotics; Preferably, the composition is an oral composition, preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral solutions, suspensions and emulsions; Preferably, a unit dose of the composition comprises 1x10-1x10 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL tryptophan-binding bile acid-producing bacteria.

7. A composition for preventing and / or treating metabolic diseases, comprising tryptophan-bound bile acid or its producing bacteria and another active ingredient for preventing and / or treating metabolic diseases, preferably, the other active ingredient is one or more of biguanides, sulfonylureas, glinides, α-glucosidase inhibitors, thiazolidinediones and GLP-1 drugs; preferably, the composition is an oral composition, preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral solutions, suspensions and emulsions; Preferably, the composition is a food composition or a pharmaceutical composition; Preferably, the composition further comprises probiotics, prebiotics and / or food or pharmaceutically acceptable carriers, diluents or excipients; Preferably, the composition is an oral composition, preferably selected from granules, capsules, tablets, powders, oral solutions, suspensions and emulsions.

8. A method for producing tryptophan-bound bile acid, comprising culturing a probiotic in a culture medium in the presence of bile acid and tryptophan, wherein the probiotic is one or more of the genera Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucosus, Lactobacillus euchromaticus, Lactobacillus unisorans, Lactobacillus plantarum, Bifidobacterium, and Lactococcus; Preferably, the probiotics are one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus mucosa reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis and Lactococcus lactis; Preferably, the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus; and the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis.

9. Use of probiotics in producing tryptophan-bound bile acid, wherein the probiotics are one or more of Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucosus, Lactobacillus spp., Lactobacillus spp., Lactobacillus plantarum, Bifidobacterium and Lactococcus; Preferably, the probiotics are one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus mucosa reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis and Lactococcus lactis; Preferably, the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus; the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis; Preferably, the tryptophan-bound bile acid has the following structure:

10. A method for screening probiotics that promote GLP-1 and / or insulin production, comprising: (1) culturing probiotics in the presence of bile acid and tryptophan to obtain a culture supernatant; (2) determining the content of tryptophan-bound bile acid in the culture supernatant; and (3) Selecting probiotics that produce tryptophan-conjugated bile acid; Preferably, the probiotics are one or more of Streptococcus, Pediococcus, Lactobacillus, Lactobacillus mucosa, Lactobacillus spp., Lactobacillus spp., Lactobacillus plantarum, Bifidobacterium and Lactococcus; Preferably, the probiotics are one or more of the following species: Streptococcus salivarius, Pediococcus acidilactici, Lactobacillus gasseri, Lactobacillus mucosa reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis and Lactococcus lactis; Preferably, the Streptococcus salivarius is Streptococcus salivarius subsp. thermophilus; the Bifidobacterium animalis is Bifidobacterium animalis subsp. lactis; Preferably, the method comprises mixing the probiotic culture solution with methanol, and detecting the content of tryptophan-bound bile acid by mass spectrometry; Preferably, the tryptophan-bound bile acid has the following structure:

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