Application of Lactobacillus paracasei Lc19 in lowering blood sugar

Abstract

This invention provides the use of *Lactobacillus paracasei* or its metabiotics in the preparation of pharmaceutical or food compositions for the generation of tryptophan-binding bile acids. *Lactobacillus paracasei* or its metabiotics may be used for the prevention and / or treatment of metabolic diseases, or for promoting the production of GLP-1 and / or insulin, or for maintaining healthy blood glucose levels, maintaining healthy blood lipids, and / or controlling body fat.

Description

Technical Field

[0001] This invention relates to the field of microbiology. Specifically, this invention relates to the use of *Lactobacillus paracasei* Lc19, its compositions, and related metabiotics in the treatment and prevention of metabolic-related diseases. Background Technology

[0002] Probiotics have undergone years of development and exploration from their initial research to clinical application. The types and mechanisms of action of probiotics have gradually become clearer, and their clinical applications are becoming increasingly widespread, encompassing organ protection, immunity, antibacterial activity, lipid-lowering, blood sugar-lowering, and weight control. In my country, various probiotic preparations have shown promising prospects in the prevention or treatment of diseases; however, most studies at this stage are small-scale, and the molecular mechanisms and target mechanisms by which these probiotics exert their effects are not yet clear.

[0003] Lactobacillus paracasei Lc19 was first disclosed in Chinese patent application CN202010507598.9. In this Chinese patent application, it was named Lactobacillus paracasei Lc19. It not only has significant therapeutic effects on constipation and colitis, but also can target and improve various gut-associated microbiota characteristic of constipation and colitis, increasing the abundance of various beneficial bacteria, decreasing the abundance of various harmful bacteria, maintaining the balance of gut microbiota, and greatly enhancing the ability to restore the health and diversity of the gut microbiota. Lactobacillus paracasei Lc19 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO.17827.

[0004] Chinese patent application CN202010506279.6 discloses yogurt containing Lactobacillus paracasei Lc19, its preparation method, and its application. This yogurt not only has significant therapeutic effects on constipation and colitis, but also can target and improve various characteristic gut-associated flora of constipation and colitis, increase the abundance of various beneficial bacteria, reduce the abundance of various harmful bacteria, maintain the balance of gut flora, and fundamentally restore the health and diversity of gut flora. It has the advantages of convenient consumption, simple treatment, high relief rate, and no toxic side effects.

[0005] Chinese patent application CN202010635622.7 discloses a lactic acid bacteria composition including Lactobacillus paracasei Lc19, which has the effect of regulating the intestinal flora with constipation characteristics.

[0006] Chinese patent application CN202111566851.9 discloses room-temperature yogurt containing xylooligosaccharides and inactivated Lactobacillus paracasei Lc19, which can improve the stability of room-temperature yogurt and reduce water separation.

[0007] There is still a need to further expand the application of Lactobacillus paracasei in existing technologies. Summary of the Invention

[0008] In their research on *Lactobacillus paracasei*, the inventors discovered that *Lactobacillus paracasei* can produce tryptophan-conjugated cholic acids (Trp-CA or TRP-CA) in vivo or in vitro. Surprisingly, the inventors found that tryptophan-conjugated cholic acids can promote the secretion of GLP-1 and insulin, improve glucose tolerance, and reduce serum AST and ALT levels, key liver function indicators.

[0009] In one aspect, the use of *Lactobacillus paracasei* or its metagener in the preparation of pharmaceutical or food compositions for the generation of tryptophan-binding cholic acid is provided.

[0010] In one embodiment, the pharmaceutical composition is used to prevent and / or treat metabolic diseases or to promote the production of GLP-1 and / or insulin.

[0011] In one embodiment, the food composition is used to maintain healthy blood glucose levels, maintain healthy blood lipids, and / or control body fat.

[0012] In one aspect, methods for preventing and / or treating metabolic diseases in a subject are provided, comprising administering to the subject *Lactobacillus paracasei* or its post-generant, or a pharmaceutical composition comprising *Lactobacillus paracasei* or its post-generant. In another aspect, methods for promoting the production of GLP-1 and / or insulin are provided, comprising administering to the subject *Lactobacillus paracasei* or its post-generant, or a pharmaceutical composition comprising *Lactobacillus paracasei* or its post-generant.

[0013] In another aspect, methods are provided for maintaining healthy blood glucose levels, maintaining healthy blood lipids, and / or controlling body fat in subjects, comprising administering to the subjects Lactobacillus paracasei or its post-generic form, or a food composition containing Lactobacillus paracasei or its post-generic form.

[0014] In one embodiment of the above aspects, *Lactobacillus paracasei* is *Lactobacillus paracasei* with accession number CGMCCNO.17827.

[0015] In one embodiment of the above aspects, Lactobacillus paracasei is in powder or culture medium form.

[0016] In one implementation of the above aspects, the metabolic disease is a GLP-1-related metabolic disease.

[0017] In one embodiment of the above, the metabolic disease is selected from diabetes or diabetes-related diseases.

[0018] 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.

[0019] 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.

[0020] In one implementation of the above aspects, obesity is selected from: symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity.

[0021] 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.

[0022] In one embodiment of the above aspects, the pharmaceutical composition is also used to improve constipation and / or improve inflammatory bowel disease.

[0023] In one embodiment of the above aspects, the constipation is functional constipation, and the inflammatory bowel disease is colitis or Crohn's disease.

[0024] In one embodiment of the foregoing, the pharmaceutical composition further comprises probiotics, prebiotics, and / or a pharmaceutically acceptable carrier, diluent, or excipient.

[0025] In one embodiment of the above aspects, the food composition further comprises probiotics, prebiotics, and / or food-acceptable carriers, diluents, or excipients.

[0026] In one embodiment of the above aspects, the pharmaceutical composition is an oral composition, preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral liquids, suspensions, and emulsions.

[0027] In one embodiment of the above aspects, the food composition is liquid, semi-solid, or solid, preferably selected from dairy products, confectionery products, fruit juices, and instant foods.

[0028] In one embodiment of the foregoing, the unit dose of the pharmaceutical composition or food composition comprises 1×10⁻¹⁰ to 1×10⁻¹⁰. 12 cfu / mL, 1×10 4 -1×10 9 cfu / mL or 1×10 5 -1×10 8 cfu / mL live bacteria.

[0029] In another aspect, compositions comprising *Lactobacillus paracasei* for the generation of tryptophan-binding bile acids are provided, which are pharmaceutical compositions for the prevention and / or treatment of metabolic diseases or for promoting the production of GLP-1 and / or insulin, or food compositions for maintaining healthy blood glucose levels, maintaining healthy blood lipids and / or controlling body fat, wherein the pharmaceutical composition further comprises another active ingredient for the prevention and / or treatment of metabolic diseases, or the food composition further comprises another food active ingredient for maintaining healthy blood glucose levels, maintaining healthy blood lipids and / or controlling body fat.

[0030] In one embodiment, *Lactobacillus paracasei* is the *Lactobacillus paracasei* with accession number CGMCC NO.17827.

[0031] In one embodiment, the other active ingredient is one or more of biguanides, sulfonylureas, meglitinides, α-glucosidase inhibitors, thiazolidinediones, and GLP-1 inhibitors.

[0032] In one embodiment, the pharmaceutical composition is an oral composition, preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral solutions, suspensions, and emulsions.

[0033] In one embodiment, the pharmaceutical composition further comprises probiotics, prebiotics, and / or pharmaceutically acceptable carriers, diluents, or excipients.

[0034] In one embodiment, the food composition is liquid, semi-solid, or solid, preferably selected from dairy products, confectionery products, fruit juices, and instant foods.

[0035] In one embodiment, the food composition further comprises probiotics, prebiotics, and / or food-acceptable carriers, diluents, or excipients.

[0036] In another aspect, a method for producing tryptophan-bound bile acids is provided, which includes culturing *Lactobacillus paracasei* in a culture medium containing bile acids and tryptophan to produce tryptophan-bound bile acids.

[0037] In one embodiment, *Lactobacillus paracasei* is the *Lactobacillus paracasei* with accession number CGMCC NO.17827.

[0038] In another aspect, the use of *Lactobacillus paracasei* in the production of tryptophan-binding bile acids is provided. In one embodiment, *Lactobacillus paracasei* is the *Lactobacillus paracasei* with accession number CGMCC NO. 17827.

[0039] In another aspect, a method is provided for screening *Lactobacillus* strains that produce tryptophan-binding bile acids, comprising:

[0040] (1) Culture Lactobacillus strains in a culture medium containing bile acids and tryptophan to obtain culture supernatant;

[0041] (2) Determine the content of tryptophan-binding cholic acid in the culture supernatant; and

[0042] (3) Select Lactobacillus strains that produce tryptophan-binding bile acids.

[0043] In one implementation, the method is a method of screening Lactobacillus strains that promote the production of GLP-1 and / or insulin.

[0044] In one embodiment, the Lactobacillus strain is Lactobacillus paracasei, preferably Lactobacillus paracasei with accession number CGMCCNO.17827.

[0045] The advantages of this invention include:

[0046] 1. The inventors discovered for the first time that *Lactobacillus paracasei* can produce tryptophan-bound bile acids, and that tryptophan-bound bile acids 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.

[0047] 2. The inventors discovered that colonization of Lactobacillus paracasei can also play a role in improving blood glucose metabolism. Attached Figure Description

[0048] Figure 1 The structural formula of tryptophan-bound bile acid is shown.

[0049] Figure 2The effect of tryptophan-binding bile acids on body weight in mice fed a high-fat diet was shown. Statistical analysis was performed using a two-way repeated measures ANOVA combined with an unpaired t-test. *P<0.05, **P<0.01.

[0050] 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. Statistical analysis was performed using two-way repeated measures ANOVA combined with unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001.

[0051] Figure 4 The effects of tryptophan-bound bile acids on serum AST and ALT levels in mice fed a high-fat diet were shown. Statistical analysis was performed using a two-tailed unpaired t-test. *P<0.05, ***P<0.001.

[0052] Figure 5 This study demonstrated the effect of *Lactobacillus paracasei* Lc19 on glucose tolerance in mice fed a high-fat diet. Statistical analysis was performed using two-way repeated measures ANOVA combined with unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001. Figure 5 The graph above shows how blood sugar levels change over time. Figure 5 The figure below shows the AOC (area of ​​the curve) for the control group and the treatment group. Detailed Implementation

[0053] 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. In addition, the incidence of cardiovascular diseases such as arteriosclerosis and coronary heart disease caused by elevated blood lipids is increasing year by year and is showing a trend towards affecting younger people. Weight loss, blood sugar lowering, and blood lipid lowering drugs have become one of the key areas of drug research.

[0054] Tryptophan-bound bile acids are newly discovered amino acid-bound bile acids produced by bacteria in recent years. They are widely present in the human population, but their physiological functions have not yet been reported. The inventors' research results show that tryptophan-bound bile acids can significantly promote GLP-1 secretion in in vivo animal models and in vitro cell models, thereby contributing to weight loss, lowering blood sugar, and alleviating symptoms of non-alcoholic fatty liver disease. Furthermore, colonization by probiotics that produce tryptophan-bound bile acids can also exert the same metabolic improvement effects.

[0055] Through extensive and in-depth research and experimentation, the inventors discovered that tryptophan-binding 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-binding bile acids, and mice fed a high-fat diet were colonized with *Lactobacillus paracasei*, a strain capable of producing tryptophan-binding bile acids. The results showed that colonization of this strain, through the production of tryptophan-binding bile acids, could also lower blood sugar and effectively alleviate diabetes. This invention was completed based on these findings.

[0056] In this application, the term "glucagon-like peptide-1 (GLP-1)" refers to 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.

[0057] 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 cholic acid can be found, for example, Nature. 2024 Feb; 626(7998):419-426. doi:10.1038 / s41586-023-06906-8. Epub 2023 Dec 5.

[0058] In this application, the term "postbiotics" refers to inactivated microorganisms and / or cell components with a clearly defined genetic background that are beneficial to the health of the host, including or excluding their metabolites; excluding chemically synthesized components and viruses / bacteriophages and their products (see China Biofermentation Industry Association Group Standard T / CBFIA 09001-2023). In this application, the postbiotics of *Lactobacillus paracasei* contain tryptophan-bound bile acids.

[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 *Lactobacillus paracasei* or its post-generative agents for the production of tryptophan-bound bile acids. The inventors have discovered that tryptophan-bound bile acids can prevent and / or treat metabolic diseases or promote the production of GLP-1 and / or insulin. Therefore, *Lactobacillus paracasei* or its post-generative agents can be used in pharmaceuticals or pharmaceutical compositions for the prevention and / or treatment of metabolic diseases or for promoting the production of GLP-1 and / or insulin. Additionally, *Lactobacillus paracasei* or its post-generative agents can also be used in foods or food compositions for maintaining healthy blood glucose levels, maintaining healthy blood lipids, and / or controlling body fat.

[0065] In this invention, the treatment method may be a method for preventing and / or treating metabolic diseases in a subject, or it may be a method for promoting GLP-1 and / or insulin production in a subject. The method may include administering *Lactobacillus paracasei* or its post-biotic to the subject, for example, by oral administration of *Lactobacillus paracasei* or its post-biotic. In the treatment method, *Lactobacillus paracasei* or its post-biotic is administered at a dose and for a duration sufficient to allow *Lactobacillus paracasei* to colonize the subject's intestine. Pharmaceutically effective amounts of probiotics and the duration of administration are known to those skilled in the art. For example, a pharmaceutically effective amount may be 10 to 10... 15 10 2Up 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 8 Or 10 2 Up to 10 9 One colony forming unit (CFU).

[0066] The strain of *Lactobacillus paracasei* is not particularly limited, but it is preferred to be *Lactobacillus paracasei* with accession number CGMCC NO.17827. The form of *Lactobacillus paracasei* is not particularly limited and can be in lyophilized powder or culture medium form.

[0067] The method of this invention can be used to treat or prevent metabolic diseases. Metabolic diseases can be GLP-1-related metabolic diseases, preferably selected from diabetes or diabetes-related diseases. Diabetes can be selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obese diabetes, autoimmune diabetes, and borderline diabetes. Diseases related to diabetes can be 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. Obesity can be selected from: symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity. Obesity-related diseases can be selected from: 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.

[0068] The method of the present invention can also be used to improve constipation and / or improve inflammatory bowel disease. For example, the constipation is functional constipation, or the inflammatory bowel disease is colitis or Crohn's disease.

[0069] The method of this 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 metabolic diseases, preferably selected from diabetes or diabetes-related diseases.

[0070] Composition

[0071] The composition administered to the subject may be a food composition, such as a beverage, dairy product, or 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 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 herein may be oral compositions, preferably selected from granules, capsules, tablets, powders, oral solutions, suspensions, and emulsions. The *Lactobacillus paracasei* or its post-biotic in the composition should be in an effective amount. Those skilled in the art can routinely determine the therapeutic or preventative effective amount of probiotics, such as *Lactobacillus paracasei* or its post-biotic. For example, a unit dose of *Lactobacillus paracasei* or its post-biotic in the composition may contain 1×10⁻¹ to 1×10⁻¹⁰. 12 cfu / mL, 1×10 4 -1×10 9 cfu / mL or 1×10 5 -1×10 8 cfu / mL live bacteria.

[0072] 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).

[0073] 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 into food.

[0074] 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.

[0075] 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 10 8Or 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.

[0076] 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, Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and the books in 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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 components can be prepared using conventional polymers known in the art.

[0081] Example

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0083] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0084] Example 1: Effects of tryptophan-binding bile acids on glucose tolerance-related indicators, body weight, body fat, and liver function in obese model mice.

[0085] 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).

[0086] Establishment of the mouse model: C57BL / 6 mice (purchased from Jicui Yaokang) 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 by 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.

[0087] Methods: 6-8 week old male C57 mice fed a high-fat diet, weighing 20-22g, were divided into groups according to their weight: (1) Control group: physiological saline containing 1% DMSO, without tryptophan-bound bile acid; (2) Tryptophan-bound bile acid treatment group (i.e. experimental group): 6 mice in each group, administered for 8 weeks (10mg / kg, three times a week).

[0088] 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 using NMR (nuclear magnetic resonance imaging) to determine fat and muscle content. Blood was collected and centrifuged at 3000 rpm at 4°C. Serum ALT and AST were measured using the serum aspartate aminotransferase (AST) assay kit (Zhong Sheng Bei Kong) and the serum alanine aminotransferase (ALT) assay kit (Zhong Sheng Bei Kong) according to the kit instructions.

[0089] 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 2-5 Table 1-5. Figure 2 The effect of tryptophan-binding bile acids on body weight in mice fed a high-fat diet was shown. Statistical analysis was performed using a two-way repeated measures ANOVA combined with an unpaired t-test. *P<0.05, **P<0.01.

[0090] 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 curve (AOC) for the control and treatment groups. Statistical analysis was performed using two-way repeated measures ANOVA combined with unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001.

[0091] Figure 4 The effects of tryptophan-bound bile acids on serum AST and ALT levels in mice fed a high-fat diet were shown. Statistical analysis was performed using a two-tailed unpaired t-test. *P<0.05, ***P<0.001.

[0092] Figure 5This study demonstrated the effect of *Lactobacillus paracasei* Lc19 on glucose tolerance in mice fed a high-fat diet. Statistical analysis was performed using two-way repeated measures ANOVA combined with unpaired t-tests. *P<0.05, **P<0.01, ***P<0.001. Figure 5 The graph above shows how blood sugar levels change over time. Figure 5 The figure below shows the area of ​​the curve (AOC) for the control group and the treatment group.

[0093] Table 1. Effect of tryptophan-binding bile acids on body fat in mice on a high-fat diet after 8 weeks of continuous administration (statistical method: two-tailed unpaired t-test).

[0094]

[0095] Table 2. Effect of tryptophan-binding bile acids on body weight in mice on a high-fat diet (Statistical method: two-way repeated measures ANOVA combined with unpaired t-test).

[0096]

[0097] Table 3. Effect of tryptophan-binding bile acids on glucose tolerance in mice on a high-fat diet after two weeks of continuous administration (statistical method: two-way repeated measures ANOVA combined with unpaired t-test).

[0098]

[0099] Table 4. Effects of tryptophan-binding bile acids on glucose-stimulating GLP-1 and insulin in high-fat diet mice after two weeks of continuous administration.

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

[0101]

[0102]

[0103] Table 5. Effects of tryptophan-binding bile acids on serum AST and ALT levels in mice on a high-fat diet (statistical method: two-tailed unpaired t-test).

[0104] Grouping AST(U / L) ALT(U / L) control group 220.80 109.44 experimental group 155.25 66.79 p-value 0.001 0.011

[0105] Example 2: Effect of tryptophan-bound bile acids on GLP-1 secreted by the intestinal L cell line GLUTag

[0106] Materials: Mouse L cells (GLUTag cells, from DJDrucker laboratory), cultured in high glucose medium, and GLP-1 activity assay kit (Merck).

[0107] Methods: Mouse L cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum to 80% confluence. Tryptophan-binding cholic acid (TBC) at a concentration of 50 mM was diluted in the same high-glucose medium to prepare TBC sample groups with different final concentrations (0, 1, 10, or 50 μM) and a 1‰ DMSO solvent control group. Mouse L cell samples with different concentrations of TBC 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).

[0108] Results: Compared with the control group, tryptophan-bound bile acids increased the level of GLP-1 in the cell supernatant in a dose-dependent manner. A significant promoting effect was observed at a concentration of 10 μM (Table 6).

[0109] Table 6. Effects of tryptophan-bound bile acids on GLP-1 secretion by the intestinal L cell line GLUTag (Statistical method: one-way ANOVA with Tukey post-hoc test)

[0110]

[0111] Example 3: Detection of tryptophan-binding bile acid production by Lactobacillus paracasei Lc19

[0112] Materials: Lactobacillus paracasei Lc19 (preservation number CGMCC NO.17827), MRS medium.

[0113] Methods: Lactobacillus paracasei Lc19 was cultured until the OD value reached 0.6. 1 mM bile acids and 1 mM tryptophan were added, and the culture was continued for 24 hours. 50 μL of the bacterial culture was then mixed with 50 μL of methanol, centrifuged at 12000 rpm, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. The tryptophan-bound bile acid content was determined by mass spectrometry. Mass spectrometry detection conditions: Tryptophan-bound bile acid (Trp-CA) 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 °C and a flow rate of 0.25 mL / min. The injection volume was 5 μL. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Tryptophan-binding cholic acid (Trp-CA) was detected under negative ion conditions using MRM mode. 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. CA-d4 (200 nM) was 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).

[0114] Results: Lactobacillus paracasei Lc19 can synthesize tryptophan-bound cholic acid, as shown in Table 7.

[0115] Table 7. Detection of tryptophan-binding bile acids synthesized by Lactobacillus paracasei Lc19

[0116]

[0117] Example 4: Effects of Lactobacillus paracasei Lc19 on glucose tolerance-related indicators in hyperglycemic mice

[0118] Materials: The tested bacterial strain was *Lactaseibacillus paracasei* Lc19, and the amount of bacteria administered via gavage was set at 2 × 10⁻⁶. 8 CFU / 30g body weight, once a week. The bacterial culture needs to be cultured in advance, activated weekly to ensure freshness, and the concentration measured separately.

[0119] C57BL / 6 mice were purchased from Jicui Pharmaceutical Co., Ltd. The environment was maintained at 20-24 degrees Celsius, constant humidity at 50-60%, with 12 hours of light exposure (8:00-20:00). The mice were kept in a soundproof environment and allowed free access to food and water. Experiments were conducted one week after the mice acclimatized to the environment. The blood glucose meter used was a product of Roche, Germany.

[0120] Methods: Male C57BL / 6 mice aged 6-8 weeks, weighing 20-22g, were randomly divided into two groups based on their weight: (1) control group and (2) Lactobacillus paracasei Lc19 colonization group, with 9 mice in each group. The mice were administered the medication twice weekly for 2 weeks. The control group received an equal volume of physiological saline. After 2 weeks of high-fat diet feeding, 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. The following experimental group was also included: One week after Lactobacillus paracasei Lc19 gavage, the mice were sacrificed, and the small intestine was cut off and its contents removed. The small intestinal contents were extracted with 20 times methanol, and mass spectrometry was used to detect changes in tryptophan-binding bile acid content in the small intestinal contents.

[0121] Results: Two weeks after colonization with *Lactobacillus paracasei* Lc19, the levels of tryptophan-binding bile acids in mice were increased, and glucose tolerance was significantly improved (see Tables 8-10). Figure 5 .

[0122] Table 8. Detection of tryptophan-binding bile acids in mice colonized with Lactobacillus paracasei Lc19 (statistical method: two-tailed unpaired t-test).

[0123]

[0124] Table 9. Effects of Lactobacillus paracasei Lc19 colonization on glucose tolerance in mice on a high-fat diet (Statistical method: two-way repeated measures ANOVA combined with unpaired t-test).

[0125]

[0126] Table 10. Effects of *Lactobacillus paracasei* Lc19 colonization on glucose-stimulating GLP-1 and insulin levels in mice on a high-fat diet.

[0127] (The statistical method used was two-way repeated measures ANOVA combined with unpaired t-test)

[0128]

[0129]

[0130] 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.

Claims

1. Live Lactobacillus paracasei ( Lacticaseibacillus paracasei The use of Lactobacillus paracasei as an active ingredient in the preparation of pharmaceutical or food compositions, wherein Lactobacillus paracasei is Lactobacillus paracasei with accession number CGMCC NO. 17827; The pharmaceutical composition is used for the prevention and / or treatment of obesity; The food composition described herein is used to maintain healthy blood sugar levels.

2. The use according to claim 1, wherein Lactobacillus paracasei is in powder or culture medium form.

3. The use according to claim 1 or 2, wherein the pharmaceutical composition further comprises probiotics, prebiotics and / or pharmaceutically acceptable carriers, diluents or excipients.

4. The use according to claim 1 or 2, wherein the food composition further comprises probiotics, prebiotics and / or food-acceptable carriers, diluents or excipients.

5. The use according to claim 1 or 2, wherein the pharmaceutical composition is an oral composition.

6. The use according to claim 5, wherein the oral composition is selected from granules, capsules, tablets, powders, oral liquids, suspensions, and emulsions.

7. The use according to claim 1, wherein the food composition is a liquid, semi-solid, or solid.

8. The use according to claim 1, wherein the unit dose of the pharmaceutical composition or food composition comprises 1x10⁻¹ to 1x10⁻¹⁰. 12 cfu / mL live bacteria.

9. The use according to claim 1, wherein the unit dose of the pharmaceutical composition or food composition comprises 1 x 10 4 -1x10 9 cfu / mL live bacteria.

10. The use according to claim 1, wherein the unit dose of the pharmaceutical composition or food composition comprises 1 x 10 5 -1x10 8 cfu / mL live bacteria.

11. The use according to claim 5, wherein the oral composition is a casing tablet.

12. An in vitro method for producing tryptophan-bound bile acids, comprising culturing *Lactobacillus paracasei* in a medium containing bile acids and tryptophan to produce tryptophan-bound bile acids, wherein *Lactobacillus paracasei* is the *Lactobacillus paracasei* with accession number CGMCC NO.17827.

13. The non-therapeutic use of *Lactobacillus paracasei* in the production of tryptophan-binding bile acids from bile acids and tryptophan as substrates, wherein *Lactobacillus paracasei* is the one with accession number CGMCC NO. 17827.

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