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

[0001] This application is a divisional application of Chinese Patent Application No. 202410620762.5, filed on May 17, 2024, entitled "Application of Conjugated Bile Acid TrpCA and Its Producing Bacteria in the Treatment and Prevention of Metabolic Diseases". Technical Field

[0002] This invention relates to the field of microbiology. Specifically, it relates to the application of tryptophan-binding bile acids and their producing bacteria in the treatment and prevention of metabolic-related diseases. Background Technology

[0003] Metabolic diseases are a general term for diseases caused by metabolic disorders in the body. They are usually caused by imbalances in carbohydrates, lipids, proteins, vitamins, electrolytes, and water. Examples of metabolic diseases include obesity, diabetes, hyperlipidemia, arteriosclerosis, hypertension, and non-alcoholic fatty liver disease. With social development, the continuous improvement of people's living standards and the adjustment of dietary structure, diets are becoming increasingly abundant, leading to a continuous increase in the incidence of metabolic diseases, among which obesity, hyperglycemia, and hyperlipidemia are the most concerning. Obesity is a contributing factor to a series of diseases, such as hypertension, diabetes, coronary heart disease, gallbladder disease, osteoarthritis, and sleep apnea. Weight loss, blood sugar lowering, and blood lipid lowering drugs have become one of the key areas of drug research.

[0004] There is still a need in this field for active ingredients that can prevent and treat metabolic diseases. Summary of the Invention

[0005] Tryptophan-bound bile acid (also known as bile acid TrpCA, Trp-CA, 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 functions have not yet 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, thereby playing a role in weight loss, lowering blood sugar, and alleviating symptoms of non-alcoholic fatty liver disease. Furthermore, the colonization of probiotics that produce bile acid TrpCA can also exert the same metabolic improvement effects.

[0006] Through extensive and in-depth research and experimentation, the inventors discovered that tryptophan-bound bile acids have a preventive and therapeutic effect on metabolic-related diseases (including obesity, diabetes, and fatty liver). In vitro experiments screened strains capable of producing tryptophan-bound bile acids, and mice fed a high-fat diet were colonized with *Bifidobacterium animalis* subsp. *lactis*, which can produce tryptophan-bound bile acids. The colonization of this strain, by producing tryptophan-bound bile acids, also lowered blood sugar, effectively alleviating diabetes. Based on this, the present invention was completed.

[0007] In one aspect, the use of tryptophan-binding cholic acid or its producing bacteria in the preparation of pharmaceutical compositions, food compositions, or health food compositions is provided, wherein the pharmaceutical compositions are used to prevent and / or treat metabolic diseases, or to promote the production of GLP-1 and / or insulin, and the food compositions or health food compositions are used to (help) maintain healthy blood glucose levels, (help) maintain healthy blood lipids, and / or (help) control body fat.

[0008] In one aspect, methods are provided for treating and / or preventing metabolic diseases in subjects, or for promoting the production of GLP-1 and / or insulin in subjects, including administering to subjects tryptophan-binding bile acid or its producing bacteria or a pharmaceutical composition containing tryptophan-binding bile acid or its producing bacteria.

[0009] In one aspect, methods are provided for maintaining healthy blood glucose levels, maintaining healthy blood lipids, and / or controlling body fat in subjects, including administering tryptophan-binding cholic acid or its producing bacteria, or a food composition or health food composition containing tryptophan-binding cholic acid or its producing bacteria to the subjects.

[0010] In one aspect, tryptophan-binding cholic acid or its producing bacteria or a pharmaceutical composition comprising tryptophan-binding cholic acid or its producing bacteria is provided for use in treating and / or preventing metabolic diseases in a subject, or for promoting the production of GLP-1 and / or insulin in a subject.

[0011] On one hand, tryptophan-binding cholic acid or its producing bacteria or food compositions or health food compositions containing tryptophan-binding cholic acid or its producing bacteria are provided for use in subjects to maintain healthy blood glucose levels, maintain healthy blood lipids and / or control body fat.

[0012] In one implementation of the above aspects, the subject is a mammal, preferably a human.

[0013] In one embodiment of the above aspects, the tryptophan-binding bile acid-producing bacteria is one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxobolus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium, and Lactococcus.

[0014] In one embodiment of the above aspects, the tryptophan-binding bile acid-producing bacteria is one or more of the following: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0015] In one embodiment of the above aspects, *Streptococcus salivarius* is the thermophilic subspecies of *Streptococcus salivarius*. In one embodiment of the above aspects, *Bifidobacterium animalis* is the lactobacillus subspecies of *Bifidobacterium animalis*.

[0016] In one embodiment of the above aspects, the metabolic disease is a GLP-1-related metabolic disease, preferably selected from diabetes or diabetes-related diseases.

[0017] In one implementation of the above, diabetes is selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obese diabetes, autoimmune diabetes, and borderline diabetes.

[0018] In one implementation of the above aspects, the diabetes-related diseases are selected from: obesity, obesity-related conditions, metabolic syndrome, diabetic neuropathy, nephropathy such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macrovascular disease, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral circulatory disorders, non-alcoholic 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 β-cell insufficiency, enteroendocrine cell insufficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary artery disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis, and stroke.

[0019] In one implementation of the above aspects, obesity is selected from: symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity.

[0020] In one implementation of the above aspects, obesity-related diseases are selected from: glucose intolerance, lipid metabolism disorders, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity-hypoventilation syndrome, and visceral obesity syndrome.

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

[0022] In one embodiment of the above aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0023] In one embodiment of the foregoing, the pharmaceutical or food composition or health food composition further comprises probiotics and / or prebiotics.

[0024] 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.

[0025] In one aspect of the foregoing, a composition for the prevention and / or treatment of metabolic diseases is provided, comprising tryptophan-binding bile acid or its producing bacteria and another active ingredient for the prevention and / or treatment of metabolic diseases. In one embodiment, the other active ingredient is one or more of biguanides, sulfonylureas, meglitinides, α-glucosidase inhibitors, thiazolidinediones, and GLP-1 inhibitors. In one embodiment, the composition is an oral composition. In one embodiment, the oral composition is selected from granules, capsules, tablets, powders, oral solutions, suspensions, and emulsions.

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

[0027] In one embodiment of the above aspects, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0028] In one embodiment of the foregoing, the pharmaceutical or food composition or health food composition further comprises probiotics and / or prebiotics.

[0029] 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, the unit dose of the composition comprises 1x10⁻¹ to 1x10⁻¹⁰. 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL tryptophan-binding bile acid-producing bacteria.

[0030] In one aspect, a method for producing tryptophan-bound bile acids is provided, comprising culturing probiotics in the presence of bile acids and tryptophan, said probiotics being one or more of the genera Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxobolus, Lactobacillus spp., Lactobacillus synergists, Lactobacillus lactis, Bifidobacterium, and Lactococcus.

[0031] In one embodiment, the probiotic is one or more of the following species: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sacchariformis, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0032] In one embodiment, *Streptococcus salivarius* is the thermophilic subspecies of *Streptococcus salivarius*. In one embodiment, *Bifidobacterium animalis* is the lactobacillus subspecies of *Bifidobacterium animalis*.

[0033] In one embodiment, the use of probiotics in the production of tryptophan-binding bile acids, wherein the probiotics are one or more of the genera Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxoidis, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium, and Lactococcus.

[0034] In one embodiment, the probiotic is one or more of the following species: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sacchariformis, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0035] In one embodiment, *Streptococcus salivarius* is the thermophilic subspecies of *Streptococcus salivarius*. In one embodiment, *Bifidobacterium animalis* is the lactobacillus subspecies of *Bifidobacterium animalis*.

[0036] In another aspect, methods are provided for screening probiotics that promote GLP-1 and / or insulin production, including:

[0037] (1) Culturing probiotics in the presence of bile acids and tryptophan to obtain culture supernatant;

[0038] (2) Determine the content of tryptophan-bound bile acids in the culture supernatant; and

[0039] (3) Select probiotics that produce tryptophan-binding bile acids.

[0040] In one implementation, the probiotic is one or more of the genera Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxobolus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium, and Lactococcus.

[0041] In one embodiment, the probiotic is one or more of the following species: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sacchariformis, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0042] In one embodiment, *Streptococcus salivarius* is the thermophilic subspecies of *Streptococcus salivarius*. In one embodiment, *Bifidobacterium animalis* is the lactobacillus subspecies of *Bifidobacterium animalis*.

[0043] In one embodiment, the method includes mixing a probiotic culture with methanol and detecting the content of tryptophan-binding cholic acid by mass spectrometry.

[0044] In the above embodiments, tryptophan-bound bile acids have the following structures:

[0045] .

[0046] The advantages of this invention include:

[0047] 1. The inventors discovered for the first time that tryptophan-bound bile acids can significantly promote the secretion of GLP-1 in in vivo animal models and in vitro cell models, thereby reducing weight, lowering blood sugar, and alleviating symptoms of non-alcoholic fatty liver disease. Furthermore, the colonization of probiotics that produce bile acid TrpCA can also exert the same metabolic improvement effects.

[0048] 2. The inventors have provided a method for screening probiotics that promote GLP-1 production based on the production of tryptophan-binding cholic acid.

[0049] 3. The inventors discovered that Bifidobacteria (such as Bifidobacterium animalis) can produce tryptophan-bound bile acids in high quantities. Attached Figure Description

[0050] Figure 1 The structural formula of tryptophan-bound bile acid is shown.

[0051] Figure 2 The effect of tryptophan-binding bile acids on body weight in mice fed a high-fat diet was demonstrated. Statistical methods employed included two-way repeated measures ANOVA combined with unpaired t-tests. P<0.05, P<0.01.

[0052] Figure 3 The study demonstrated the effect of tryptophan-bound bile acids on glucose tolerance in mice on a high-fat diet. Figure 3 The graph above shows how blood sugar levels change over time. Figure 3 The figure below shows the area under the curves (AOC) for the control and treatment groups. The statistical method used was two-way repeated measures ANOVA combined with an unpaired t-test. P<0.05, P<0.01, P<0.001.

[0053] Figure 4 The effects of tryptophan-bound bile acids on serum AST and ALT levels in mice fed a high-fat diet were shown. The statistical method used was a two-tailed unpaired t-test. P<0.05, P<0.001.

[0054] Figure 5The changes in tryptophan-binding bile acid content in the ileal contents of mice after one day of gavage administration (10 mg / kg) were shown. The statistical method used was a two-tailed unpaired t-test. P<0.001.

[0055] Figure 6 This study demonstrated the effect of *Bifidobacterium lactis* subsp. *animal* on glucose tolerance in mice fed a high-fat diet. The statistical method employed was two-way repeated measures ANOVA combined with unpaired t-tests. P<0.05, P<0.01. Figure 6 The graph above shows how blood sugar levels change over time. Figure 6 The figure below shows the AOC (area of ​​the curve) for the control group and the treatment group. Detailed Implementation

[0056] In this application, the term "tryptophan-bound bile acid" refers to a compound 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 the figure. Reports on tryptophan-bound bile acids can be found, for example, Nature. 2024 Feb; 626(7998):419-426. doi: 10.1038 / s41586-023-06906-8. Epub 2023 Dec 5.

[0057] In this application, the term "tryptophan-binding bile acid-producing bacteria" refers to probiotics capable of producing tryptophan-binding bile acids in the presence of bile acids and tryptophan, the substrates for tryptophan-binding bile acids. Examples of tryptophan-binding bile acid-producing bacteria include, but are not limited to, *Streptococcus*, *Pediococcus*, *Lactobacillus*, *Lactobacillus mucinus*, *Lactobacillus spp.*, *Lactobacillus assemblica*, *Lactobacillus lactis*, *Bifidobacterium*, and *Lactococcus*. Tryptophan-binding bile acid-producing bacteria can be one or more of the following species: *Streptococcus salivarius*, *Pediococcus lactis*, *Lactobacillus gasseri*, *Lactobacillus reuteri*, *Lactobacillus sakei*, *Lactobacillus acidophilus*, *Lactobacillus assemblica*, *Lactobacillus plantarum*, *Bifidobacterium animalis*, and *Lactococcus lactis*. More specifically, *Streptococcus salivarius* can be *Streptococcus thermophilus* subsp. *salivarius*. *Bifidobacterium animalis* can be *Bifidobacterium animalis* subsp. *lactobacter*.

[0058] In this application, "glucagon-like peptide-1 (GLP-1)" is a hormone mainly produced by intestinal L cells. GLP-1 exerts a hypoglycemic effect by promoting insulin secretion, and can also reduce weight and lower blood lipids by suppressing appetite and promoting energy metabolism.

[0059] In this application, the term "metabolic disease" generally refers to a disease or condition that disrupts normal metabolic processes in the body, preventing the body from properly utilizing and / or storing energy. A metabolic disease can be any metabolic disorder that benefits from regulation (e.g., increased) of GLP-1 activity, or any metabolic disorder that benefits from stimulating GLP-1 receptor activity. Metabolic diseases can be GLP-1-related metabolic disorders, including diabetes or diabetes-related disorders. Diabetes includes, but is not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, obese diabetes, autoimmune diabetes, and / or borderline diabetes. Diseases associated with diabetes include, but are not limited to, obesity, obesity-related conditions, metabolic syndrome, diabetic neuropathy, nephropathy such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macrovascular disease, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral circulatory disorders, non-alcoholic 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 β-cell insufficiency, enteroendocrine cell insufficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary artery 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. Obesity-related diseases include, but are not limited to, glucose intolerance, lipid metabolism disorders, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity-hypopnea syndrome, and visceral obesity syndrome.

[0060] In this application, the term "prevention" means the delay of the onset of one or more symptoms of a particular disease, condition, or disorder, and / or a reduction in their frequency and / or severity. The term "treatment" means any application of a therapy that partially or completely relieves, improves, alleviates, or suppresses one or more symptoms, features, and / or causes of a particular disease, condition, and / or disorder, delays its onset, reduces its severity, and / or reduces its incidence.

[0061] In this application, the term "subject" refers to an individual who receives the administered treatment. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal, such as a mammal that experiences or is susceptible to a metabolic disease as described herein. In some embodiments, the animal is a vertebrate, such as a mammal, such as a non-human primate (especially 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 human.

[0062] In this application, "pharmaceutically effective amount" means an amount sufficient to treat or prevent disease with a reasonable benefit / risk ratio suitable for medical treatment or prevention, and the effective dose level can be determined based on factors including: disease severity, drug activity, patient age, weight, health status, sex, patient sensitivity to the drug, timing of drug administration, route of administration and release rate, duration of treatment, drugs used in combination with or concurrently with the composition, and other factors known in the medical field. A pharmaceutical composition according to one example may be administered as a single therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or concurrently with conventional therapeutic agents. Furthermore, it may be administered alone or multiple times. Taking all factors into account, it is important to administer an amount that achieves maximum efficacy with minimal dosage and without side effects.

[0063] Treatment

[0064] This invention provides the use of tryptophan-bound bile acids in the treatment and prevention of metabolic-related diseases. When subjects consume a high-fat diet, tryptophan-bound bile acids have the ability to inhibit weight gain. The inventors found that C57BL / 6J mice treated with tryptophan-bound bile acids and fed a high-fat diet that could lead to obesity experienced less weight gain compared to an untreated control group, and showed improvement in blood sugar and fatty liver symptoms. Therefore, tryptophan-bound bile acids can be used to prevent and treat obesity and obesity-related diseases such as diabetes and fatty liver. This invention also provides the use of tryptophan-bound bile acid-producing bacteria in the treatment and prevention of metabolic-related diseases. In vitro tests showed that the following bacterial species, including *Streptococcus*, *Pediococcus*, *Lactobacillus*, *Lactobacillus myxitis*, *Lactobacillus spp.*, *Lactobacillus synergists*, *Lactobacillus lactis*, *Bifidobacterium*, and *Lactococcus*, can produce tryptophan-bound bile acids. Therefore, those skilled in the art can expect that these strains tested in vitro can improve glucose metabolism disorders induced by a high-fat diet. The inventors also demonstrated in mouse models that colonization of Bifidobacterium animalis subsp. lactis can significantly improve glucose metabolism disorders induced by a high-fat diet.

[0065] Therefore, the present invention provides a method for treating and / or preventing metabolic diseases in a subject, comprising administering tryptophan-binding 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.

[0066] There is no particular limitation on the type of tryptophan-binding bile acid-producing bacteria; any probiotic capable of producing tryptophan-binding bile acids is acceptable. Tryptophan-binding bile acid-producing bacteria can include *Streptococcus*, *Pediococcus*, *Lactobacillus*, *Lactobacillus mucinus*, *Lactobacillus spp.*, *Lactobacillus assemblica*, *Lactobacillus plantarum*, *Bifidobacterium*, and *Lactococcus*. For example, tryptophan-binding bile acid-producing bacteria can be one or more of the following: *Streptococcus salivarius*, *Pediococcus lactis*, *Lactobacillus gasseri*, *Lactobacillus reuteri*, *Lactobacillus sakei*, *Lactobacillus acidophilus*, *Lactobacillus assemblica*, *Lactobacillus plantarum*, *Bifidobacterium animalis*, and *Lactococcus lactis*. *Streptococcus salivarius* can be *Streptococcus thermophilus* subsp. *salivarius*. *Bifidobacterium animalis* can be *Bifidobacterium animalis* subsp. *lactobacter*. Those skilled in the art can readily select tryptophan-binding bile acid-producing bacteria suitable for administration to mammals (especially humans) according to the teachings of this invention.

[0067] 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 diseases treated by this method are GLP-1-related or obesity-related metabolic diseases, preferably selected from diabetes or diabetes-related diseases, such as the various specific diseases described herein.

[0068] Health care methods

[0069] The inventors have discovered that tryptophan-binding cholic acid or its producing bacteria, or compositions containing tryptophan-binding cholic acid or its producing bacteria, can be used to (help) maintain healthy blood glucose levels, (help) maintain healthy blood lipids, and / or (help) control body fat. This invention provides a method for maintaining healthy blood glucose levels, maintaining healthy blood lipids, and / or controlling body fat, comprising administering tryptophan-binding cholic acid or its producing bacteria, or compositions containing tryptophan-binding cholic acid or its producing bacteria, to a subject. In this health care method, the subject may be a healthy subject.

[0070] There are no specific limitations on the types of tryptophan-binding bile acid-producing bacteria; any probiotic capable of producing tryptophan-binding bile acids is acceptable. Tryptophan-binding bile acid-producing bacteria can include *Streptococcus*, *Pediococcus*, *Lactobacillus*, *Lactobacillus mucinus*, *Lactobacillus spp.*, *Lactobacillus assemblica*, *Lactobacillus plantarum*, *Bifidobacterium*, and *Lactococcus*. For example, tryptophan-binding bile acid-producing bacteria can be one or more of the following species: *Streptococcus salivarius*, *Pediococcus lactis*, *Lactobacillus gasseri*, *Lactobacillus reuteri*, *Lactobacillus sakei*, *Lactobacillus acidophilus*, *Lactobacillus assemblica*, *Lactobacillus plantarum*, *Bifidobacterium animalis*, and *Lactococcus lactis*. *Streptococcus salivarius* can be *Streptococcus thermophilus* subsp. *salivarius*. *Bifidobacterium animalis* can be *Bifidobacterium animalis* subsp. *lactobacter*.

[0071] The health care method of the present invention can be applied to any mammalian subject, but is preferably applied to human subjects. These human subjects need to maintain healthy blood glucose levels, maintain healthy blood lipid levels, and / or control body fat.

[0072] Composition

[0073] The composition administered to the subject may be a food composition or a health food composition, such as a beverage, dairy product, or solid food, but not limited thereto. The food composition or health 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 the prevention and / or treatment of metabolic diseases, preferably one or more of biguanides, sulfonylureas, meglitinides, α-glucosidase inhibitors, thiazolidinediones, and GLP-1 inhibitors. The compositions described herein may be oral compositions, preferably selected from granules, capsules, tablets, powders, oral solutions, suspensions, and emulsions. The tryptophan-binding bile acid-producing bacteria in the composition should be in a therapeutically or preventively effective amount. Those skilled in the art can routinely determine the therapeutically or preventively effective amount of probiotics, such as tryptophan-binding bile acid-producing bacteria. For example, a unit dose of tryptophan-binding bile acid-producing bacteria in the composition may contain 1x10⁻¹ to 1x10⁻¹⁰. 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL tryptophan-binding bile acid-producing bacteria.

[0074] 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, energy drinks, tea drinks, beverage products, jelly drinks, and functional beverages; alcoholic beverages, such as beer; carbohydrate-containing foods, such as rice products, noodles, bread, and pasta; paste products, such as paste products of fish, ham, sausage, and seafood; retortable packaged products, such as curry, foods coated with thick starch sauce, and Chinese soups; soups; dairy products, such as milk, dairy beverages, ice cream, and yogurt; fermented products, such as fermented soy milk, fermented beverages, and pickles; soy products; various pastry and confectionery products, including biscuits, cookies, etc., candies, chewing gum, soft candies, cold desserts (including jellies, caramel cream, and frozen desserts); ready-to-eat foods, such as ready-to-eat soups and ready-to-eat bean soups; and so on. In one embodiment, the food for application is frozen. In some implementations, the food may be or contain one or more of the following: bars, candy, baked goods, cereals, savory snacks, pasta, chocolate and other solid foods, as well as liquid or semi-solid foods (including yogurt, soup and stew) and beverages (such as smoothies, milkshakes, juices and other carbonated or non-carbonated beverages).

[0075] Oral compositions may contain an inert diluent or an edible carrier. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of tablets, lozenges, pasties, tablets, or capsules (e.g., gelatin capsules). Oral compositions may also be prepared by combining the compositions of this disclosure with food. In some embodiments, probiotics may be formulated in food or health food products.

[0076] 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 composition is administered to the subject twice daily, daily, weekly, or monthly.

[0077] In some implementations, the unit dose contains 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 108 Or 10 2 Up to 10 9 One colony-forming unit (CFU). In some embodiments, the probiotics may be live cells. In some embodiments, the composition comprises a cell culture of probiotics and / or its culture supernatant and / or a powder formed therefrom and / or is formulated using a cell culture of probiotics and / or its culture supernatant and / or a powder formed therefrom.

[0078] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include oral administration. Methods for formulating suitable pharmaceutical compositions are known in the art; see, for example, books in Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). Oral compositions generally contain an inert diluent or an edible carrier. For example, oral formulations can be or contain syrups, liquids, tablets, lozenges, gummies, capsules (e.g., gelatin capsules), powders, gels, films, etc.

[0079] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil can all be used as carriers, excipients, or diluents for the pharmaceutical composition in this invention.

[0080] Furthermore, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings. The pharmaceutical compositions of the present invention can be produced as enteric-coated formulations using various known methods, so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.

[0081] The pharmaceutical compositions of the present invention can be formulated into enteric-coated tablets for oral administration. The term "enteric-coated" as used in this application includes all coatings permitted for conventional pharmaceutical use that are not degraded by gastric acid but are readily decomposed in the small intestine, rapidly releasing the microorganisms of the present invention. The enteric-coated tablets of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as an HCl solution with pH=1, and preferably decompose within 1 hour in synthetic intestinal fluid such as a buffer solution with pH=7.0.

[0082] The casings of this invention are coated with approximately 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The casing thickness in this invention is 5-100 μm, ideally 20-80 μm. The casing composition is a conventional polymer known to the public.

[0083] Example

[0084] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0085] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0086] Example 1: Effects of tryptophan-binding bile acids on glucose tolerance-related indicators, body weight, body fat, and liver function in obese model mice.

[0087] Materials: Blood glucose test strips (Roche), blood glucose meter (Roche), active GLP-1 assay kit (Merck), insulin assay kit (Huamei Biotechnology), serum aspartate aminotransferase (AST) assay kit (Zhong Sheng Bei Kong), serum alanine aminotransferase (ALT) assay kit (Zhong Sheng Bei Kong).

[0088] Establishment of the mouse model: C57BL / 6 mice (purchased from Jicui Pharmaceutical) were kept at a temperature of 20-24℃, a constant humidity of 50-60%, and a light exposure of 12 hours (8:00-20:00). They were kept in a soundproof environment with free access to food and water. Experiments were conducted one week after the mice acclimatized to the environment. C57BL / 6 mice were fed a high-fat diet containing tryptophan-binding bile acids via gavage for 8 weeks at a dose of 10 mg / kg body weight, three times a week. This established a high-fat diet-induced mouse obesity model.

[0089] Methods: Male C57 mice aged 6-8 weeks, weighing 20-22g, were fed a high-fat diet and divided into groups according to their weight: (1) Control group: physiological saline containing 1% DMSO, without tryptophan-binding bile acid; (2) Tryptophan-binding bile acid treatment group (i.e., experimental group): 6 mice per group, administered the drug for 8 consecutive weeks (10 mg / kg, three times a week). In order to detect the contents of the small intestine of mice, another 6 mice were also included. One week after the mice were administered 10 mg / kg tryptophan-binding bile acid by gavage, the mice were sacrificed, and the small intestine segments were cut off and the contents were removed. The contents of the small intestine of mice were extracted with 20 times methanol, and the concentration of tryptophan-binding bile acid in the contents of the small intestine of mice was detected by mass spectrometry.

[0090] Both groups were given equal volumes of physiological saline. Mice in both groups were fed a high-fat diet for 8 weeks, with body weight monitored weekly. In the second week of treatment, a glucose tolerance test was performed (i.e., mice were administered glucose by gavage, and GLP-1 and insulin levels were measured at different time points). Table 2 shows the GLP-1 and insulin secretion levels measured at two time points: 0 minutes (before glucose gavage) and 15 minutes (15 minutes after glucose gavage). Blood glucose levels were measured using a glucometer at 0, 15, 30, 60, 90, and 120 minutes. GLP-1 and insulin secretion levels were measured using an active GLP-1 assay kit (Merck) and an insulin assay kit (Huamei Biotechnology) according to the kit instructions. After the last administration (week 8), body fat was measured by nuclear magnetic resonance (NMR) to determine fat and muscle content. Blood was collected, centrifuged at 3000 rpm at 4°C, and serum ALT and AST were measured according to the instructions of the kits using a serum aspartate aminotransferase (AST) assay kit (Zhong Sheng Bei Kong) and a serum alanine aminotransferase (ALT) assay kit (Zhong Sheng Bei Kong).

[0091] Results: Compared with the control group, the tryptophan-binding bile acid treatment group showed a trend of change from week 1, with a significant difference appearing at week 6. This indicates that tryptophan-binding bile acids have a significant effect on weight control and can significantly reduce body fat. Furthermore, it can improve glucose tolerance induced by a high-fat diet, with the promotion of glucose-stimulating GLP-1 and insulin secretion being the reasons for this improvement. It also reduced serum AST and ALT levels, key liver function indicators. (See attached results) Figure 2-4 Table 1-2.

[0092] Figure 2 The effect of tryptophan-binding bile acids on body weight in mice fed a high-fat diet was demonstrated. Statistical methods employed included two-way repeated measures ANOVA combined with unpaired t-tests. P<0.05, P<0.01.

[0093] Figure 3 The study demonstrated the effect of tryptophan-bound bile acids on glucose tolerance in mice on a high-fat diet. Figure 3 The graph above shows how blood sugar levels change over time. Figure 3 The figure below shows the area under the curves (AOC) of blood glucose in the control and treatment groups. The statistical method used was two-way repeated measures ANOVA combined with an unpaired t-test. P<0.05, P<0.01, P<0.001.

[0094] Figure 4The effects of tryptophan-bound bile acids on serum AST and ALT levels in mice fed a high-fat diet were shown. The statistical method used was a two-tailed unpaired t-test.

[0095] Figure 5 The concentration of tryptophan-binding bile acids in the small intestinal contents of mice after one week of gavage with 10 mg / kg tryptophan-binding bile acids is shown.

[0096] Table 1. Effects of tryptophan-binding bile acids on body fat in mice on a high-fat diet after 8 weeks of continuous administration.

[0097] (The statistical method used was a two-tailed unpaired t-test)

[0098]

[0099] Table 2. Effects of tryptophan-binding bile acids on glucose tolerance test GLP-1 and insulin in high-fat diet mice after 2 weeks of continuous administration.

[0100] (The statistical method used was two-way repeated measures ANOVA combined with unpaired t-test.)

[0101]

[0102] Example 2: Effects of tryptophan-bound bile acids on the intestinal L cell line GLUTag

[0103] Materials: Mouse L cells (GLUTag cells, from DJ Drucker's laboratory), cultured in high glucose medium, and an activity GLP-1 assay kit (Merck).

[0104] Methods: L cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum until they reached 80% confluence. Then, 50 mM tryptophan-binding cholic acid was diluted in the same medium to different final concentrations (0, 1, 10, or 50 μM) to create a tryptophan-binding cholic acid sample group and a 1‰ DMSO solvent control group. Mouse L cell samples with different concentrations of tryptophan-binding cholic acid were allowed to stand for one hour, and the supernatant was extracted. The GLP-1 level in the supernatant was detected using an active GLP-1 assay kit (Merck).

[0105] Results: Compared with the control group, tryptophan-bound cholic acid increased the level of GLP-1 in cell supernatant in a dose-dependent manner. A significant promoting effect was observed at a concentration of 10 μM (Table 3). These results are consistent with in vivo efficacy data in animals (…). Figure 5 (Same as above)

[0106] Table 3. Effects of tryptophan-bound bile acids on GLP-1 secretion in the intestinal L cell line GLUTag.

[0107] (The statistical method used was one-way ANOVA and Tukey post-hoc test)

[0108]

[0109] Example 3: Detection of tryptophan-binding bile acid production by various probiotics

[0110] Materials: The bacterial strains used are shown in Table 4. All strains were routinely isolated and did not require screening.

[0111] Methods: When the OD value of each bacterial culture (MRS medium, anaerobic culture at 37℃) reached 0.6, 1 mM bile acid and 1 mM tryptophan were added. After culturing for another 24 hours, 50 μL of bacterial culture was taken, mixed with 50 μL of methanol, centrifuged at 12000 rpm, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. 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) tandem with a Sciex 5500 triple quadrupole linear ion trap mass spectrometer (AB Sciex, Framingham, MA, USA). The chromatographic column was an ACQUITY UPLC CSH C18 (2.1 × 100 mm, 1.7 μm, Waters), with a column temperature of 40 ℃ and a flow rate of 0.25 mL / min. The injection volume was 5 µL, with mobile phase A being 0.1% formic acid aqueous solution and mobile phase B being 0.1% formic acid acetonitrile solution. TrpCA was detected in MRM mode under negative ion conditions. Metabolite separation was performed using gradient elution: 0–1 min, 40% B; 1–6 min, 40–100% B; 6–7 min, 100% B; 7–8 min, 40% B. Different concentrations of mixed working standard solutions were obtained through serial dilutions (0.1, 1, 10, 100, 1000 nM) to construct a standard curve, with CA-d4 (200 nM) used as an internal standard for content correction. The LC-MS / MS control system uses Analyst version 1.6.2, and quantitative analysis uses MultiQuant software (version 3.0.1).

[0112] Results: All the bacterial species listed in Table 4 can synthesize tryptophan-bound bile acids, with Bifidobacterium animalis subsp. lactis having the highest yield.

[0113] Table 4. Detection of tryptophan-binding bile acids synthesized by various probiotics

[0114]

[0115] Example 4: Effects of Bifidobacterium animalis subsp. lactis on glucose tolerance-related indicators in hyperglycemic mice

[0116] Materials: The tested strain was *Bifidobacterium animalis* subsp. *lactis*. C57BL / 6 mice were purchased from Jicui Pharmaceutical Co., Ltd. The mice were kept at 20-24℃, constant humidity 50-60%, with 12 hours of light (8:00-20:00), soundproofed, and allowed free access to food and water. The mice were allowed to acclimatize for one week before the experiment. The blood glucose meter was a product of Roche, Germany.

[0117] C57BL / 6 mice were administered the test strain via gavage, with a bacterial count of 2 x 10⁻⁶. 8 CFU / each, once a week. The bacterial culture needs to be cultured in advance, activated weekly to ensure freshness, and the concentration measured separately.

[0118] Methods: After one week of acclimatization, 6-8 week old male C57BL / 6 mice (weighing 20-22g) were randomly divided into two groups based on their body weight: (1) control group and (2) Bifidobacterium lactis colonization group. Seven mice were in each group and were administered the medication by gavage once a week for two consecutive weeks. The control group was given an equal volume of physiological saline. Two weeks later, a glucose tolerance test was performed. Blood glucose levels were measured at different time points (0 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes) 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 assay kit (Merck) and an insulin assay kit (Huamei Biotechnology) according to the kit instructions.

[0119] Results: Colonization of mice with Bifidobacterium animalis subsp. lactis significantly improved glucose tolerance and promoted the secretion of glucose-stimulating GLP-1 and insulin. Figure 6 This study demonstrated the effect of *Bifidobacterium lactis* subsp. *animal* on glucose tolerance in mice fed a high-fat diet. The statistical method employed was two-way repeated measures ANOVA combined with unpaired t-tests. P<0.05, P<0.01. Figure 6 The graph above shows how blood sugar levels change over time. Figure 6 The figure below shows the AOC (area of ​​the curve) for the control group and the treatment group.

[0120] Table 5. Effects of Bifidobacterium lactis subsp. on glucose-stimulating GLP-1 and insulin in mice fed a high-fat diet.

[0121] (The statistical method used was two-way repeated measures ANOVA combined with unpaired t-test.)

[0122]

[0123] Although the invention has been described with reference to illustrative embodiments, those skilled in the art will understand that various other changes, omissions, and / or additions can be made without departing from the spirit and scope of the invention, and that elements of the described embodiments can be substituted with substantially equivalents. Furthermore, many modifications can be made without departing from the scope of the invention to adapt particular situations or materials to the teachings of the invention. Therefore, this invention is not intended to be limited to the specific embodiments disclosed for carrying out the invention, but rather is intended to encompass all embodiments falling within the scope of the appended claims.

[0124] 1. Use of tryptophan-binding bile acid or its producing bacteria in the preparation of pharmaceutical or food compositions, wherein the pharmaceutical compositions are used for the prevention and / or treatment of metabolic diseases, or for promoting the production of GLP-1 and / or insulin, and the food compositions are used for maintaining healthy blood glucose levels, maintaining healthy blood lipids, and / or controlling body fat; the tryptophan-binding bile acid preferably has the following structure:

[0125] .

[0126] 2. According to the purpose of item 1, the tryptophan-binding bile acid-producing bacteria are one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxoidis, Lactobacillus spp., Lactobacillus syngenes, Lactobacillus lactis, Bifidobacterium, and Lactococcus.

[0127] 3. According to the use of item 1 or 2, the tryptophan-binding bile acid-producing bacteria is one or more of the following: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sacchariformis, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0128] 4. According to the uses in item 3, *Streptococcus salivarius* is *Streptococcus thermophilus* subspecies; *Bifidobacterium animalis* is *Bifidobacterium animalis* subspecies *lactobacterium animalis*.

[0129] 5. According to the use of any one of items 1-4, wherein the metabolic disease is a GLP-1-related metabolic disease, preferably selected from diabetes or diabetes-related diseases;

[0130] Preferably, the diabetes mellitus is selected from type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, obese diabetes mellitus, autoimmune diabetes mellitus, and borderline diabetes mellitus;

[0131] Preferably, the diabetes-related diseases are selected from: obesity, obesity-related conditions, metabolic syndrome, diabetic neuropathy, nephropathy such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macrovascular disease, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral circulatory disorders, non-alcoholic 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 β-cell insufficiency, enteroendocrine cell insufficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis and stroke;

[0132] Preferably, obesity is selected from: symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity;

[0133] Preferably, the obesity-related diseases are selected from: glucose intolerance, lipid metabolism disorders, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity-hypoventilation syndrome, and visceral obesity syndrome.

[0134] 6. According to any one of items 1-5, 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;

[0135] Preferably, the food composition or pharmaceutical composition further comprises probiotics and / or prebiotics;

[0136] Preferably, the composition is an oral composition, and is preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral liquids, suspensions and emulsions;

[0137] Preferably, the unit dose of the composition contains 1x10⁻¹ to 1x10⁻¹ 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL tryptophan-binding bile acid-producing bacteria.

[0138] 7. A composition for the prevention and / or treatment of metabolic diseases, comprising tryptophan-binding bile acid or its producing bacteria and another active ingredient for the prevention and / or treatment of metabolic diseases, preferably, the other active ingredient being one or more of biguanides, sulfonylureas, meglitinides, α-glucosidase inhibitors, thiazolidinediones, and GLP-1 inhibitors; preferably, the composition is an oral composition, preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral solutions, suspensions, and emulsions;

[0139] Preferably, the composition is a food composition or a pharmaceutical composition;

[0140] Preferably, the composition further comprises probiotics, prebiotics and / or food- or pharmaceutically acceptable carriers, diluents or excipients;

[0141] Preferably, the composition is an oral composition, and is preferably selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions.

[0142] 8. A method for producing tryptophan-bound bile acids, comprising culturing probiotics in a culture medium containing bile acids and tryptophan, said probiotics being one or more of the genera Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxobolus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium, and Lactococcus.

[0143] Preferably, the probiotics are one or more of the following: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0144] Preferably, *Streptococcus salivarius* is *Streptococcus thermophilus* subspecies; *Bifidobacterium animalis* is *Bifidobacterium animalis* subspecies *lactobacterium animalis*.

[0145] 9. Use of probiotics in the production of tryptophan-binding bile acids, wherein the probiotics are one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxoidis, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium spp., and Lactococcus spp.

[0146] Preferably, the probiotics are one or more of the following: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0147] Preferably, *Streptococcus salivarius* is *Streptococcus thermophilus* subspecies; *Bifidobacterium animalis* is *Bifidobacterium animalis* subspecies *lactobacterium*.

[0148] Preferably, tryptophan-bound cholic acid has the following structure:

[0149] .

[0150] 10. Methods for screening probiotics that promote GLP-1 and / or insulin production, including:

[0151] (1) Culturing probiotics in the presence of bile acids and tryptophan to obtain culture supernatant;

[0152] (2) Determine the content of tryptophan-bound bile acids in the culture supernatant; and

[0153] (3) Select probiotics that produce tryptophan-binding bile acids;

[0154] Preferably, the probiotics are one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxoidis, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium spp., and Lactococcus spp.

[0155] Preferably, the probiotics are one or more of the following: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis.

[0156] Preferably, *Streptococcus salivarius* is *Streptococcus thermophilus* subspecies; *Bifidobacterium animalis* is *Bifidobacterium animalis* subspecies *lactobacterium*.

[0157] Preferably, the method includes mixing a probiotic culture with methanol and detecting the content of tryptophan-binding cholic acid by mass spectrometry;

[0158] Preferably, tryptophan-bound cholic acid has the following structure:

[0159] .

Claims

1. Use of tryptophan-binding bile acid or its producing bacteria in the preparation of a composition for maintaining healthy blood glucose levels, maintaining healthy blood lipids, and / or controlling body fat; the tryptophan-binding bile acid has the following structure: 。 2. The use according to claim 1, wherein the tryptophan-binding bile acid-producing bacteria is one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxobolus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium spp., and Lactococcus spp.

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

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

5. Use of tryptophan-binding cholic acid or its producing bacteria in the preparation of a pharmaceutical composition for the treatment or prevention of diabetes or diabetes-related diseases; Preferably, the diabetes mellitus is selected from type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, obese diabetes mellitus, autoimmune diabetes mellitus, and borderline diabetes mellitus; Preferably, the diabetes-related diseases are selected from: obesity, obesity-related conditions, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, diabetic cachexia, diabetic dyslipidemia, non-alcoholic steatohepatitis, prediabetes, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, increased fasting glucose, pancreatic β-cell insufficiency, glycosuria, diabetic coronary heart disease, diabetic cerebrovascular disease, and diabetic peripheral vascular disease. Preferably, obesity is selected from: symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity; Preferably, the obesity-related diseases are selected from: glucose intolerance, heart failure, hyperuricemia, fatty liver, obesity-hypoventilation syndrome, and visceral obesity syndrome.

6. The use according to any one of claims 1-5, wherein the composition is a health product composition or a food composition, optionally further comprising a food-acceptable carrier, diluent or excipient, and the pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent or excipient; Preferably, the health product composition, food composition, or pharmaceutical composition further comprises probiotics and / or prebiotics; Preferably, the composition is an oral composition, and is preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral liquids, suspensions and emulsions; Preferably, the unit dose of the composition contains 1x10⁻¹ to 1x10⁻¹ 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 the prevention and / or treatment of metabolic diseases, comprising tryptophan-binding bile acid or its producing bacteria and another active ingredient for the prevention and / or treatment of metabolic diseases, preferably, the other active ingredient being one or more of biguanides, sulfonylureas, meglitinides, α-glucosidase inhibitors, thiazolidinediones, and GLP-1 inhibitors; 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, and is preferably selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions.

8. A method for producing tryptophan-bound bile acids, comprising culturing probiotics in a culture medium containing bile acids and tryptophan, said probiotics being one or more of the genera Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxobolus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium, and Lactococcus. Preferably, the probiotics are one or more of the following: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis. Preferably, *Streptococcus salivarius* is *Streptococcus thermophilus* subspecies; *Bifidobacterium animalis* is *Bifidobacterium animalis* subspecies *lactobacterium animalis*.

9. Use of probiotics in the production of tryptophan-binding bile acids, wherein the probiotics are one or more of the following genera: Streptococcus, Pediococcus, Lactobacillus, Lactobacillus myxobolus, Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Lactobacillus spp., Bifidobacterium spp., and Lactococcus spp. Preferably, the probiotics are one or more of the following: Streptococcus salivarius, Pediococcus lactis, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Bifidobacterium animalis, and Lactococcus lactis. Preferably, *Streptococcus salivarius* is *Streptococcus thermophilus* subspecies; *Bifidobacterium animalis* is *Bifidobacterium animalis* subspecies *lactobacterium*. Preferably, tryptophan-bound cholic acid has the following structure: 。

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