Application of Lactobacillus paracasei Lc19 in blood sugar reduction

Abstract

There is provided the use of Lactobacillus paracasei or a afterbiogen in the preparation of a pharmaceutical composition or a food composition for the production of tryptophan-binding cholic acid. The Lactobacillus paracasei or the afterbiogen thereof can be used to prevent and / or treat metabolic diseases or to promote the production of GLP-1 and / or insulin, or to maintain blood glucose healthy levels, to maintain blood lipid health and / or to control fat in the body.

Description

Technical Field

[0001] The present invention relates to the field of microbiology, and in particular to the application of Lactobacillus paracasei Lc19, a composition thereof and related postbiotics in the treatment and prevention of metabolism-related diseases. Background Art

[0002] Probiotics have gone through years of development and exploration from development to clinical application. The categories and mechanisms of action of probiotics have gradually become clear, and their clinical applications have become more extensive, covering organ protection, immunity, antibacterial, lowering blood lipids, lowering blood sugar, and weight control. In my country, a variety of probiotic preparations have shown good prospects in preventing or treating diseases, but most of the current research is small-scale, and there is no clear molecular mechanism or target of action for how these probiotics work.

[0003] Lactobacillus paracasei Lc19 was first disclosed in Chinese patent application CN202010507598.9. In this Chinese patent application, it was named Lactobacillus paracasei Lc19, which not only has significant therapeutic effects on constipation and colitis, but also can target and improve a variety of intestinal flora characteristic of constipation and colitis, increase the abundance of a variety of beneficial bacteria, reduce the abundance of a variety of harmful bacteria, maintain the balance of intestinal flora, and greatly improve the ability to restore the health and diversity of intestinal flora. Lactobacillus paracasei Lc19 has been deposited in the China General Microbiological Culture Collection Center, and its preservation number is CGMCC NO.17827.

[0004] Chinese patent application CN202010506279.6 discloses a yogurt including Lactobacillus paracasei Lc19, and a preparation method and application thereof. The yogurt contains Lactobacillus paracasei Lc19. The yogurt not only has significant therapeutic effects on constipation and colitis, but also can target and improve a variety of intestinal-related flora characteristic of constipation and colitis, increase the abundance of a variety of beneficial bacteria, reduce the abundance of a variety of harmful bacteria, maintain the balance of intestinal flora, and fundamentally restore the health and diversity of intestinal flora. It has the advantages of easy 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 function of regulating 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 precipitation.

[0007] There is still a need to further expand the application of Lactobacillus paracasei in the prior art. Summary of the invention

[0008] The inventors found that Lactobacillus paracasei can produce tryptophan-conjugated cholic acid (cholic acids conjugated to tryptophan, TrpCA or TRP-CA) in vivo or in vitro during the study of Lactobacillus paracasei. Surprisingly, the inventors found that tryptophan-conjugated cholic acid can promote the secretion of GLP-1 and insulin, improve glucose tolerance, and reduce the serum AST and ALT levels of key indicators of liver function.

[0009] In one aspect, provided is a use of Lactobacillus paracasei or a postbiotic thereof in the preparation of a pharmaceutical composition or a food composition for producing tryptophan-bound bile acid.

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

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

[0012] In one aspect, a method for preventing and / or treating a metabolic disease in a subject is provided, comprising administering Lactobacillus paracasei or its postbiotics, or a pharmaceutical composition comprising Lactobacillus paracasei or its postbiotics to the subject. In another aspect, a method for promoting the production of GLP-1 and / or insulin is provided, comprising administering Lactobacillus paracasei or its postbiotics, or a pharmaceutical composition comprising Lactobacillus paracasei or its postbiotics to the subject.

[0013] In yet another aspect, a method for maintaining healthy blood glucose levels, maintaining healthy blood lipids and / or controlling body fat in a subject is provided, comprising administering to the subject Lactobacillus paracasei or a postbiotic thereof, or a food composition comprising Lactobacillus paracasei or a postbiotic thereof.

[0014] In one embodiment of the above aspect, the Lactobacillus paracasei is the Lactobacillus paracasei with the deposit number CGMCCNO.17827.

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

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

[0017] In one embodiment of the above aspects, the metabolic disease is selected from diabetes or a disease associated with diabetes.

[0018] In one embodiment of the above aspects, the diabetes is selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obese diabetes, autoimmune diabetes, and borderline diabetes.

[0019] In one embodiment of the above aspects, the disease associated with diabetes is selected from the group consisting of obesity, obesity-related disorders, metabolic syndrome, diabetic neuropathy, kidney disease such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorders, non-alcoholic steatohepatitis, prediabetes, hyperlipidemia, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, increased blood fatty acid or glycerol levels, impaired wound healing, glucose intolerance, increased fasting glucose and dyslipidemia, pancreatic beta cell deficiency, enteroendocrine cell deficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis and stroke.

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

[0021] In one embodiment of the above aspects, the disease associated with obesity is selected from the group consisting of: glucose tolerance, abnormal lipid metabolism, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity hypoventilation syndrome and visceral obesity syndrome.

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

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

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

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

[0028] In one embodiment of the above aspects, a unit dose of the pharmaceutical composition or food composition comprises 1×10-1×10 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL viable bacteria.

[0029] In another aspect, a composition comprising Lactobacillus paracasei for producing tryptophan-bound bile acid is provided, which is a pharmaceutical composition for preventing and / or treating metabolic diseases or for promoting the production of GLP-1 and / or insulin, or a food composition for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat. The pharmaceutical composition also contains another active ingredient for preventing and / or treating metabolic diseases, or the food composition also contains another food active ingredient for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat.

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

[0031] In one embodiment, the other active ingredient is one or more of a biguanide, a sulfonylurea, a meglitinide, an alpha-glucosidase inhibitor, a thiazolidinedione, and a GLP-1.

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

[0033] In one embodiment, the pharmaceutical composition further comprises a probiotic, a prebiotic and / or a pharmaceutically acceptable carrier, diluent or excipient.

[0034] In one embodiment, the food composition is liquid, semisolid or solid, preferably selected from dairy products, confectionery products, juices and ready-to-eat foods.

[0035] In one embodiment, the food composition further comprises a probiotic, a prebiotic and / or a food acceptable carrier, diluent or excipient.

[0036] In another aspect, a method of producing tryptophan-bound bile acid is provided, comprising culturing Lactobacillus paracasei in a culture medium in the presence of bile acid and tryptophan to produce tryptophan-bound bile acid.

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

[0038] In another aspect, a use of Lactobacillus paracasei in producing tryptophan-bound bile acid is provided. In one embodiment, the Lactobacillus paracasei is the Lactobacillus paracasei with a deposit number of CGMCC NO.17827.

[0039] In another aspect, a method for screening a Lactobacillus strain that produces tryptophan-bound bile acid is provided, comprising:

[0040] (1) culturing a Lactobacillus strain in a culture medium in the presence of bile acid and tryptophan to obtain a culture supernatant;

[0041] (2) determining the content of tryptophan-bound bile acid in the culture supernatant; and

[0042] (3) Selecting a Lactobacillus strain that produces tryptophan-binding bile acid.

[0043] In one embodiment, the method is a method of screening a Lactobacillus strain that promotes the production of GLP-1 and / or insulin.

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

[0045] Advantages of the present invention include:

[0046] 1. The inventors discovered for the first time that Lactobacillus paracasei can produce tryptophan-bound bile acid, and that tryptophan-bound bile acid 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 the symptoms of non-alcoholic fatty liver disease.

[0047] 2. The inventors found that colonization of Lactobacillus paracasei can also improve blood sugar metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0049] Figure 2The effect of tryptophan-conjugated bile acid on the body weight of mice fed a high-fat diet was shown. The statistical method was a two-way repeated measures analysis of variance combined with an unpaired T test. *P<0.05, **P<0.01.

[0050] Figure 3 Shown is the effect of tryptophan-conjugated bile acid on glucose tolerance in mice fed a high-fat diet. Figure 3 The graph above shows how blood sugar levels change over time. Figure 3 The figure below shows the AOC (area under the curve) of the control group and the treatment group. The statistical method was two-way repeated measures analysis of variance combined with unpaired T test. *P<0.05, **P<0.01, ***P<0.001.

[0051] Figure 4 The effect of tryptophan-conjugated bile acid on serum AST and ALT in mice fed a high-fat diet is shown. The statistical method is a two-tailed unpaired T test. *P<0.05, ***P<0.001.

[0052] Figure 5 The effect of Lactobacillus paracasei Lc19 on glucose tolerance in mice fed a high-fat diet is shown. The statistical method was two-way repeated measures analysis of variance combined with unpaired T test. *P<0.05, **P<0.01, ***P<0.001. Figure 5 The graph above shows how blood sugar levels change over time. Figure 5 The lower graph shows the AOC (area under the curve) of the control and treated groups. DETAILED DESCRIPTION

[0053] Metabolic disease is a general term for diseases that occur due to metabolic disorders in the body. Metabolic diseases are usually caused by an imbalance of carbohydrates, lipids, proteins, vitamins, electrolytes, water, etc. Examples of metabolic diseases include obesity, diabetes, hyperlipidemia, atherosclerosis, hypertension, non-alcoholic fatty liver disease, etc. With the development of society, the continuous improvement of people's living standards and the adjustment of dietary structure, the diet has become more abundant, resulting in an increasing incidence of metabolic diseases, among which obesity, hyperglycemia and hyperlipidemia are the most concerned. Obesity is an inducing factor for a series of diseases, such as hypertension, diabetes, coronary heart disease, gallbladder disease, osteoarthritis, and sleep apnea. In addition, the incidence of cardiovascular diseases such as atherosclerosis and coronary heart disease caused by increased blood lipids has increased year by year, and there is a trend of younger age. Weight loss, blood sugar lowering, and blood lipid lowering drugs have become one of the focuses of drug research.

[0054] Tryptophan-bound bile acid is an amino acid-bound bile acid produced by bacteria that has been newly discovered in recent years. It is common in the human population, but its physiological function has not been reported. The inventors' research results show that tryptophan-bound bile acid can significantly promote the secretion of GLP-1 in in vivo animal models and in vitro cell models, play a role in weight loss, lowering blood sugar, and relieving the symptoms of non-alcoholic fatty liver disease, and that colonization of probiotics that can produce tryptophan-bound bile acid can also play the same metabolic improvement role.

[0055] After extensive and in-depth research and experiments, the inventors found that tryptophan-bound bile acid has the effect of preventing and treating metabolic-related diseases (including obesity, diabetes, and fatty liver). Through in vitro experiments, strains that can produce tryptophan-bound bile acid were screened, and Lactobacillus paracasei that can produce tryptophan-bound bile acid was colonized in mice fed a high-fat diet. It was found that the colonization of the strain can also reduce blood sugar by producing tryptophan-bound bile acid, effectively alleviating diabetes. The present invention was completed on this basis.

[0056] In the present application, the term "glucagon-like peptide-1 (GLP-1)" is a hormone mainly produced by intestinal L cells. GLP-1 plays a hypoglycemic role by promoting insulin secretion. It can also reduce weight and lower blood lipids by suppressing appetite and promoting energy metabolism.

[0057] In the present application, the term "tryptophan-bound bile acid" refers to a conjugate formed by the reaction of the amino group of tryptophan with the carboxyl group at position 24 of bile acid to form an amide bond. The structure of tryptophan-bound bile acid is as follows Figure 1 As shown in. Reports on tryptophan-bound bile acid can be found in, for example, Nature. 2024 Feb; 626(7998): 419-426. doi: 10.1038 / s41586-023-06906-8. Epub 2023 Dec 5.

[0058] In this application, the term "postbiotics" refers to inactivated microorganisms and / or bacterial components with a clear genetic background that are beneficial to the health of the host, including or excluding products of their metabolites; chemically synthesized components and viruses / bacteriophages and their products are excluded (see China Bio-fermentation Industry Association Group Standard T / CBFIA09001-2023). In this application, the postbiotics of Lactobacillus paracasei include tryptophan-bound bile acid.

[0059] In the present application, the term "metabolic disease" generally refers to a disease or condition that destroys the normal metabolic process in the body, making it impossible for the body to normally utilize and / or store energy. Metabolic disease can be any metabolic disease that benefits from regulating (e.g., increasing) GLP-1 activity, or any metabolic disease that benefits from exciting GLP-1 receptor activity. Metabolic disease can be a metabolic disease related to GLP-1, including diabetes or a disease related to diabetes. Diabetes includes, but is not limited to, type 1 diabetes, type 2 diabetes, gestational diabetes, obesity diabetes, autoimmune diabetes and / or critical diabetes. Diseases associated with diabetes include, but are not limited to, obesity, obesity-related disorders, metabolic syndrome, diabetic neuropathy, kidney disease such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorders, nonalcoholic steatohepatitis, prediabetes, hyperlipidemia, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, elevated blood fatty acid or glycerol levels, impaired wound healing, glucose intolerance, increased fasting glucose and dyslipidemia, pancreatic beta cell deficiency, enteroendocrine cell deficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis and / or stroke. Obesity includes, but is not limited to, symptomatic obesity, simple obesity, childhood obesity, morbid obesity and abdominal obesity. Diseases associated with obesity include, but are not limited to, glucose tolerance, abnormal lipid metabolism, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity hypoventilation syndrome and visceral obesity syndrome.

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

[0061] In the present application, the term "subject" refers to an individual receiving the administration of the provided treatment. In some embodiments, the subject is an animal. In some embodiments, the subject is a mammal, such as a mammal experiencing or susceptible to metabolic diseases as described herein. In some embodiments, the animal is a vertebrate, such as a mammal, such as a non-human primate (particularly a higher primate), a sheep, a dog, a rodent (e.g., a mouse or a rat), a guinea pig, a goat, a pig, a cat, a rabbit, or a cow. In some embodiments, the subject is a person.

[0062] In the present application, "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level can be determined according to factors including the following: severity of the disease, drug activity, patient age, weight, health status, sex, patient sensitivity to the drug, drug administration time, administration route and release rate, treatment period, drugs used in combination with the composition or simultaneously, and other factors known in the medical field. The pharmaceutical composition according to an example can be used as a single therapeutic agent, or in combination with other therapeutic agents, and can be used sequentially or simultaneously with conventional therapeutic agents. In addition, it can be used alone or multiple times. Considering all factors, it is important to use an amount that can achieve maximum efficacy with a minimum amount without side effects.

[0063] Treatment

[0064] The present invention provides the use of Lactobacillus paracasei or its postbiotics for generating tryptophan-bound bile acid. The inventors found that tryptophan-bound bile acid can prevent and / or treat metabolic diseases or promote the generation of GLP-1 and / or insulin. Therefore, Lactobacillus paracasei or its postbiotics can be used in medicines or pharmaceutical compositions for preventing and / or treating metabolic diseases or for promoting the generation of GLP-1 and / or insulin. In addition, Lactobacillus paracasei or its postbiotics can also be used in foods or food compositions for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat.

[0065] In the present invention, the treatment method can be a method for preventing and / or treating a metabolic disease in a subject, or it can also be a method for promoting GLP-1 and / or insulin production in a subject. The method may include administering Lactobacillus paracasei or its postbiotics to the subject, for example, by orally administering Lactobacillus paracasei or its postbiotics. In the treatment method, Lactobacillus paracasei or its postbiotics is administered at a certain dose for a certain period of time, and the dose and time period are sufficient to allow Lactobacillus paracasei to colonize in the intestine of the subject. The pharmaceutically effective amount of probiotics and the administration time period are known to those skilled in the art. For example, the pharmaceutically effective amount can 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 colony forming units (CFU).

[0066] The strain of Lactobacillus paracasei is not particularly limited, but it is preferred that Lactobacillus paracasei is Lactobacillus paracasei with a deposit number of CGMCC NO. 17827. The form of Lactobacillus paracasei is not particularly limited, and may be in the form of a lyophilized powder or a culture solution.

[0067] The method of the present invention can be used to treat or prevent metabolic diseases. The metabolic disease can be a metabolic disease associated with GLP-1, preferably selected from diabetes or a disease associated with diabetes. Diabetes can be selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obesity diabetes, autoimmune diabetes and critical type diabetes. Diseases associated with diabetes can be selected from the group consisting of obesity, obesity-related conditions, metabolic syndrome, diabetic neuropathy, nephropathy such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorders, nonalcoholic steatohepatitis, prediabetes, hyperlipidemia, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, elevated blood fatty acid or glycerol levels, impaired wound healing, glucose intolerance, increased fasting glucose and dyslipidemia, pancreatic β cell deficiency, enteroendocrine cell deficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis, and stroke. Obesity can be selected from the group consisting of symptomatic obesity, simple obesity, childhood obesity, morbid obesity, and abdominal obesity. The diseases associated with obesity may be selected from the group consisting of glucose tolerance, abnormal lipid metabolism, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity hypoventilation syndrome and visceral obesity syndrome.

[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 the present invention can be applied to any mammalian subject, but is preferably applied to human subjects. As mentioned above, the metabolic disease treated by the method is a metabolic disease associated with GLP-1, preferably selected from diabetes or a disease associated with diabetes.

[0070] Composition

[0071] The composition administered to the subject may be a food composition, such as a beverage, a dairy product, a solid food, but is not limited thereto. The food composition may also include 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 include another active ingredient for preventing and / or treating metabolic diseases, preferably, another active ingredient is one or more of biguanides, sulfonylureas, glinides, α-glucosidase inhibitors, thiazolidinediones and GLP-1 drugs. The composition herein may be an oral composition, preferably selected from granules, capsules, tablets, powders, oral liquids, suspensions and emulsions. The Lactobacillus paracasei or its postbiotic in the composition should be an effective amount. Those skilled in the art can routinely determine the effective amount of probiotics, such as the treatment or prevention of Lactobacillus paracasei or its postbiotic. For example, the unit dose of Lactobacillus paracasei or its postbiotic in the composition contains 1x10-1x10 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL viable 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, refreshing drinks, tea drinks, beverage products, jelly drinks, and functional drinks; alcoholic beverages such as beer; carbohydrate-containing foods such as rice foods, noodles, bread, and pasta; paste products such as fish, ham, sausage, and seafood paste products; retort pouch products such as curry, foods coated with thick starch sauces, and Chinese soups; soups; dairy products such as milk, dairy drinks, ice cream, and yogurt; fermented products such as fermented soy milk, fermented drinks, and pickles; bean products; various confectionery products, including biscuits, cookies, etc., candy, chewing gum, soft candy, cold desserts (including jelly, cream caramel, and frozen desserts); instant foods such as instant soups and instant bean soups; and the like. In one embodiment, the food for administration is frozen. In some embodiments, the food product can be or include one or more of the following: bars, candies, baked goods, cereals, salty snacks, pasta, chocolate and other solid foods, as well as liquid or semi-solid foods (including yogurt, soups and stews) and beverages (such as smoothies, milkshakes, juices and other carbonated or non-carbonated beverages).

[0073] Oral compositions may include an inert diluent or an edible carrier. For oral therapeutic administration, the active compound may be blended with an excipient and used in the form of a tablet, lozenge, pastille, lozenge or capsule (e.g., gelatin capsule). Oral compositions may also be prepared by combining the compositions of the present disclosure with food. In some embodiments, probiotics may be formulated in food.

[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 dosage of the composition is administered to the subject twice a day, daily, weekly, or monthly.

[0075] In some embodiments, a unit dose comprises 10 to 10 15 , 10 2 Up to 10 14 , 10 3 Up to 10 13 , 10 4 Up to 10 12 , 10 5 Up to 10 11 , 10 6 Up to 10 10 , 10 2 Up to 10 7 , 10 2 Up to 10 8or 10 2 Up to 10 9 Colony forming units (CFU). In some embodiments, the probiotics may be living cells. In some embodiments, the composition includes a cell culture of the probiotics and / or its culture supernatant and / or a powder formed therefrom and / or is prepared by using a cell culture of the probiotics and / or its culture supernatant and / or a powder formed therefrom.

[0076] Pharmaceutical compositions are generally formulated to be compatible with their intended route of administration. Examples of routes of administration include oral administration. Methods for preparing suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st edition, 2005; and books in the Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY) series. Oral compositions generally include an inert diluent or an edible carrier. For example, oral formulations may be or include syrups, liquids, tablets, lozenges, soft candies, capsules (e.g., gelatin capsules), powders, gels, films, etc.

[0077] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinyl pyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil can be used as carriers, excipients or diluents of the pharmaceutical composition of the present invention.

[0078] In addition, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners and spices, etc. The pharmaceutical composition of the present invention can be produced as an enteric coating preparation by various well-known methods, so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass through the stomach smoothly without being destroyed by gastric acid.

[0079] The pharmaceutical composition of the present invention can be made into enteric-coated tablets for oral use. The term "enteric coating" in the present application includes all coatings allowed for use in conventional drugs, which are not degraded by gastric acid but can be fully decomposed in the small intestine and quickly release the microorganisms of the present invention. The enteric coating of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as HCl solution with pH=1, and preferably decomposed within 1.0 hour in synthetic intestinal fluid such as buffer with pH=7.0.

[0080] The enteric coating of the present invention is coated with about 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The thickness of the enteric coating of the present invention is 5-100 μm, and the ideal thickness is 20-80 μm. The enteric coating components 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, the materials and reagents used in the following examples can be obtained from commercial sources.

[0084] Example 1: Effects of tryptophan-bound bile acid on glucose tolerance-related indices, body weight, body fat and liver function in obese mice

[0085] Materials: blood glucose test strips (Roche), blood glucose meter (Roche), active GLP-1 detection kit (Merck), insulin detection kit (Huamei Biotechnology), serum aspartate aminotransferase AST assay kit (Sinosino Biotechnology), serum alanine aminotransferase ALT assay kit (Sinosino Biotechnology).

[0086] Establishment of mouse model: C57BL / 6 mice (purchased from Jicui Yaokang), temperature 20-24°C, constant humidity 50-60%, light 12 hours (8:00-20:00), soundproof, free food and water, experiment after one week of adaptation. C57BL / 6 mice were fed a high-fat diet by gavage for 8 weeks, with a dose of 10 mg / kg body weight, three times a week. Obesity model of mice induced by high-fat diet was obtained.

[0087] Methods: Male C57 mice aged 6-8 weeks, weighing 20-22 g, were fed a high-fat diet and were divided into the following groups according to their body weight: (1) control group: physiological saline containing 1% DMSO without tryptophan-conjugated bile acid; (2) tryptophan-conjugated bile acid treatment group (i.e., experimental group): 6 mice in each group, and the drug was administered continuously for 8 weeks (10 mg / kg, three times a week).

[0088] The two groups were given an equal amount of normal saline. The two groups of mice were fed a high-fat diet for 8 weeks, and their body weight was monitored weekly. A glucose tolerance test was performed in the second week of treatment (i.e., mice were gavaged with glucose, and GLP-1 and insulin levels were tested in mice at different time points). In Table 2, 0 minutes, i.e., before glucose gavage, and 15 minutes, i.e., 15 minutes after glucose gavage, the levels of GLP-1 and insulin secretion in the two time periods were tested. The blood glucose of mice was measured using a blood glucose meter at 0 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes. GLP-1 and insulin secretion levels were detected using an active GLP-1 detection kit (Merck) and an insulin detection kit (Huamei Bio) according to the kit instructions. After the last administration (week 8), body fat was detected by nuclear magnetic resonance (determination of fat content and muscle content), blood was collected, and centrifuged at 3000rpm at 4 degrees Celsius. Serum ALT and AST were measured using a serum aspartate aminotransferase (AST) assay kit (Sino-Biologics Beijing Controls) and a serum alanine aminotransferase (ALT) assay kit (Sino-Biologics Beijing Controls) according to the instructions of the kits.

[0089] Results: Compared with the control group, the tryptophan-bound bile acid treatment group showed a trend of change from the first week, and a significant difference appeared in the sixth week, which showed that tryptophan-bound bile acid had a significant effect on weight control and could significantly reduce body fat; it could also improve glucose tolerance induced by a high-fat diet, among which promoting the secretion of glucose-stimulating GLP-1 and insulin was the reason for improving glucose tolerance; it also reduced the levels of serum AST and ALT, key indicators of liver function. Figure 2-5 , Tables 1-5. Figure 2 The effect of tryptophan-conjugated bile acid on the body weight of mice fed a high-fat diet was shown. The statistical method was a two-way repeated measures analysis of variance combined with an unpaired T test. *P<0.05, **P<0.01.

[0090] Figure 3 Shown is the effect of tryptophan-conjugated bile acid on glucose tolerance in mice fed a high-fat diet. Figure 3 The graph above shows how blood sugar levels change over time. Figure 3 The figure below shows the AOC (area under the curve) of the control group and the treatment group. The statistical method was two-way repeated measures analysis of variance combined with unpaired T test. *P<0.05, **P<0.01, ***P<0.001.

[0091] Figure 4 The effect of tryptophan-conjugated bile acid on serum AST and ALT in mice fed a high-fat diet is shown. The statistical method is a two-tailed unpaired T test. *P<0.05, ***P<0.001.

[0092] Figure 5The effect of Lactobacillus paracasei Lc19 on glucose tolerance in mice fed a high-fat diet is shown. The statistical method was two-way repeated measures analysis of variance combined with unpaired T test. *P<0.05, **P<0.01, ***P<0.001. Figure 5 The graph above shows how blood sugar levels change over time. Figure 5 The lower graph shows the AOC (area under the curve) for the control and treatment groups.

[0093] Table 1 Effects of tryptophan-conjugated bile acid on body fat in mice fed a high-fat diet after continuous administration for 8 weeks (statistical method: two-tailed unpaired T test)

[0094]

[0095] Table 2 Effects of tryptophan-conjugated bile acid on the body weight of mice fed a high-fat diet (statistical method: two-way repeated measures analysis of variance combined with unpaired T test)

[0096]

[0097] Table 3 Effects of tryptophan-bound bile acid on glucose tolerance in mice fed a high-fat diet after two weeks of continuous administration (statistical method: two-way repeated measures analysis of variance combined with unpaired T test)

[0098]

[0099] Table 4 Effects of tryptophan-bound bile acid on glucose-stimulated GLP-1 and insulin in mice fed a high-fat diet after two weeks of continuous administration

[0100] (The statistical method was two-way repeated measures analysis of variance combined with unpaired T test)

[0101]

[0102]

[0103] Table 5 Effect of tryptophan-bound bile acid on serum AST and ALT in mice fed 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 acid on GLP-1 secretion by GLUTag in intestinal L cell line

[0106] Materials: Mouse L cells (GLUTag cells, from DJ Drucker laboratory), cultured in high glucose medium, active GLP-1 detection kit (Merck).

[0107] Methods: After culturing mouse L cells to 80% density in DMEM high glucose medium containing 10% fetal bovine serum, 50 mM tryptophan-bound bile acid was diluted using the above high glucose medium to different final concentrations (0, 1, 10 or 50 μM) of tryptophan-bound bile acid sample group and 1‰ DMSO solvent control group. The mouse L cell samples with different concentrations of tryptophan-bound bile acid were allowed to stand for one hour, and the supernatant was extracted and the GLP-1 active detection kit (Merck) was used to detect the GLP-1 level in the supernatant.

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

[0109] Table 6 Effect of tryptophan-bound bile acid on GLP-1 secretion by GLUTag of intestinal L cell line (statistical method: one-way ANOVA Tukey post hoc test)

[0110]

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

[0112] Materials: Lactobacillus paracasei Lc19 (Lactobacillus paracasei with the deposit number of CGMCC NO.17827), MRS medium.

[0113] Methods: When Lactobacillus paracasei Lc19 was cultured to an OD value of 0.6, 1 mM bile acid and 1 mM tryptophan were added. After 24 hours of continuous culture, 50 ul of bacterial culture was added with 50 ul of methanol and mixed, centrifuged at 12000 rpm, and the supernatant was filtered through a membrane (0.22 μm pore size water filter membrane). The content of tryptophan-conjugated bile acid was detected by mass spectrometry. Mass spectrometry detection conditions: Tryptophan-conjugated bile acid (TrpCA) was quantified using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. The LC-MS / MS system consisted of an Acquity ultra-high performance liquid chromatography system (Waters Corporation, Milford, USA) connected to a Sciex 5500 triple quadrupole linear ion trap mass spectrometer (AB Sciex, Framingham, MA, USA). The chromatographic column was ACQUITY UPLC CSH C18 (2.1×100mm, 1.7μm, Waters), the column temperature was 40℃, and the flow rate was 0.25mL / min. The injection volume was 5μL, the mobile phase A was 0.1% formic acid in water, and the mobile phase B was 0.1% formic acid in acetonitrile. Tryptophan-conjugated bile acid (TrpCA) was detected in MRM mode under negative ion conditions. Metabolite separation was performed by gradient elution: 0-1min, 40% B; 1-6min, 40-100% B; 6-7min, 100% B; 7-8min, 40% B. A mixed working standard solution of different concentrations was obtained by gradient dilution (0.1, 1, 10, 100, 1000nM), and a standard curve was constructed. CA-d4 (200nM) was used as the internal standard for content correction. The LC-MS / MS control system used Analyst 1.6.2 version, and the quantitative analysis used MultiQuant software (version 3.0.1).

[0114] Results: Lactobacillus paracasei Lc19 could synthesize tryptophan-conjugated bile acid, as shown in Table 7.

[0115] Table 7. Detection of tryptophan-bound bile acid synthesis by Lactobacillus paracasei Lc19

[0116]

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

[0118] Materials: The tested strain was Lactobacillus paracasei Lc19, and the amount of bacteria administered was 2×10 8 CFU / 30g body weight, once a week. The bacterial solution needs to be cultured in advance, activated weekly to ensure freshness, and the concentration is measured separately.

[0119] C57BL / 6 mice were purchased from Jicui Yaokang, and the temperature was 20-24 degrees Celsius, the humidity was 50-60%, the light was on for 12 hours (8:00-20:00), and the mice were soundproofed. The mice were allowed to eat and drink freely. The experiment was conducted after one week of adaptation. The blood glucose meter was a product of Roche, Germany.

[0120] Methods: Male C57BL / 6 mice aged 6-8 weeks, weighing 20-22 g, were divided into two groups according to their weight: (1) control group and (2) Lactobacillus paracasei Lc19 colonization group, with 9 mice in each group. The mice were gavaged twice a week for 2 consecutive weeks. The control group was given an equal amount of normal saline. A glucose tolerance test was performed 2 weeks later. Blood glucose was measured at different time points (0 min, 30 min, 60 min, 90 min and 120 min) after glucose gavage, and the area under the curve (AOC) was calculated. GLP-1 and insulin secretion levels were detected using an active GLP-1 detection kit (Merck) and an insulin detection kit (Huamei Biotechnology) according to the kit instructions. The following experimental groups were also included: One week after gavage with Lactobacillus paracasei Lc19, the mice were killed, and the small intestine segments of the mice were cut off and the contents were taken out. The small intestinal contents of the mice were extracted with 20 times methanol, and the changes in the content of tryptophan-conjugated bile acid in the small intestinal contents of the mice were detected by mass spectrometry.

[0121] Results: Two weeks of colonization with Lactobacillus paracasei Lc19 increased the content of tryptophan-bound bile acid in mice and significantly improved glucose tolerance, as shown in Tables 8-10. Figure 5 .

[0122] Table 8 Detection of tryptophan-bound bile acid in mice colonized with Lactobacillus paracasei Lc19 (statistical method is two-tailed unpaired T test)

[0123]

[0124] Table 9 Effects of Lactobacillus paracasei Lc19 colonization on glucose tolerance in mice fed 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-stimulated GLP-1 and insulin in mice fed a high-fat diet

[0127] (The statistical method was two-way repeated measures analysis of variance combined with unpaired T test)

[0128]

[0129] Although the present invention has been described with reference to illustrative embodiments, it will be appreciated by those skilled in the art that various other changes, omissions and / or additions may be made and that elements of the described embodiments may be substituted with substantial equivalents without departing from the spirit and scope of the present invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the present invention without departing from the scope of the present invention. Therefore, it is not intended herein to limit the present invention to the specific disclosed embodiments for carrying out the present invention, but rather it is intended that the present invention will include all embodiments within the scope of the appended claims.

Claims

1. Use of Lactobacillus paracasei or its postbiotics in preparing a pharmaceutical composition or a food composition, wherein the pharmaceutical composition or the food composition is used to generate tryptophan-bound bile acid.

2. The use according to claim 1, wherein the pharmaceutical composition prevents and / or treats metabolic diseases or is used to promote the production of GLP-1 and / or insulin, and the food composition is used to maintain healthy blood sugar levels, maintain healthy blood lipids and / or control body fat.

3. according to the purposes described in claim 1 or 2, wherein Lactobacillus paracasei is the Lactobacillus paracasei of deposit number CGMCC NO.17827; Preferably, Lactobacillus paracasei is in powder or culture fluid form.

4. The use according to any one of claims 1 to 3, wherein the metabolic disease is a GLP-1-related metabolic disease, preferably selected from diabetes or a disease associated with diabetes; Preferably, the diabetes is selected from type 1 diabetes, type 2 diabetes, gestational diabetes, obesity diabetes, autoimmune diabetes and borderline diabetes; Preferably, the disease associated with diabetes is selected from the group consisting of obesity, obesity-related disorders, metabolic syndrome, diabetic neuropathy, kidney disease such as diabetic nephropathy, diabetic retinopathy, diabetic cardiomyopathy, cataracts, macroangiopathy, osteopenia, hyperosmolar diabetic coma, diabetic gangrene, xerostomia, hearing loss, diabetic cachexia, delayed wound healing, diabetic dyslipidemia, peripheral blood circulation disorders, nonalcoholic steatohepatitis, prediabetes, hyperlipidemia, fatty liver disease, impaired fasting glucose, hyperglycemia, impaired glucose tolerance, insulin resistance, hyperinsulinemia, elevated blood fatty acid or glycerol levels, impaired wound healing, glucose intolerance, increased fasting glucose and dyslipidemia, pancreatic beta cell deficiency, enteroendocrine cell deficiency, glycosuria, metabolic acidosis, cataracts, diabetic coronary heart disease, diabetic cerebrovascular disease, diabetic peripheral vascular disease, metabolic syndrome, atherosclerosis and stroke; Preferably, obesity is selected from the group consisting of symptomatic obesity, simple obesity, childhood obesity, morbid obesity and abdominal obesity; Preferably, the disease associated with obesity is selected from the group consisting of glucose tolerance, abnormal lipid metabolism, hyperlipidemia, hypertension, heart failure, hyperuricemia, gout, fatty liver, coronary heart disease, cerebral infarction, sleep apnea syndrome, obesity hypoventilation syndrome and visceral obesity syndrome.

5. The use according to any one of claims 1 to 4, wherein the pharmaceutical composition or food composition is also used to improve constipation and / or improve inflammatory bowel disease, Preferably, constipation is functional constipation, and the inflammatory bowel disease is colitis or Crohn's disease; Preferably, the pharmaceutical composition further comprises probiotics, prebiotics and / or pharmaceutically acceptable carriers, diluents or excipients; Preferably, the food composition further comprises probiotics, prebiotics and / or food acceptable carriers, diluents or excipients; Preferably, the pharmaceutical composition is an oral composition, preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral solutions, suspensions and emulsions; Preferably, the food composition is liquid, semisolid or solid, preferably selected from dairy products, confectionery products, juices and ready meals; Preferably, a unit dose of the pharmaceutical composition or food composition contains 1×10-1×10 12 cfu / mL, 1x10 4 -1x10 9 cfu / mL or 1x10 5 -1x10 8 cfu / mL viable bacteria.

6. A composition comprising Lactobacillus paracasei for producing tryptophan-bound bile acid, which is a pharmaceutical composition for preventing and / or treating metabolic diseases or for promoting the production of GLP-1 and / or insulin, or a food composition for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat, wherein the pharmaceutical composition further comprises another active ingredient for preventing and / or treating metabolic diseases, or the food composition further comprises another food active ingredient for maintaining healthy blood sugar levels, maintaining healthy blood lipids and / or controlling body fat; Preferably, the Lactobacillus paracasei is the Lactobacillus paracasei with the deposit number CGMCC NO.17827; Preferably, the other active ingredient is one or more of a biguanide, a sulfonylurea, a glinide, an α-glucosidase inhibitor, a thiazolidinedione, and a GLP-1 drug; Preferably, the pharmaceutical composition is an oral composition, preferably selected from enteric-coated tablets, granules, capsules, tablets, powders, oral solutions, suspensions and emulsions; Preferably, the pharmaceutical composition further comprises probiotics, prebiotics and / or pharmaceutically acceptable carriers, diluents or excipients; Preferably, the food composition is liquid, semisolid or solid, preferably selected from dairy products, confectionery products, juices and ready meals; Preferably, the food composition further comprises probiotics, prebiotics and / or food acceptable carriers, diluents or excipients.

7. A method for producing tryptophan-bound bile acid, comprising culturing Lactobacillus paracasei in a culture medium in the presence of bile acid and tryptophan to produce tryptophan-bound bile acid. Preferably, the Lactobacillus paracasei is the Lactobacillus paracasei with the deposit number CGMCCNO.17827.

8. Use of Lactobacillus paracasei in producing tryptophan-bound bile acid, preferably, the Lactobacillus paracasei is the Lactobacillus paracasei with the deposit number CGMCC NO.17827.

9. A method for screening a Lactobacillus strain that produces tryptophan-binding bile acid, comprising: (1) culturing a Lactobacillus strain in a culture medium in the presence of bile acid and tryptophan to obtain a culture supernatant; (2) determining the content of tryptophan-bound bile acid in the culture supernatant; and (3) selecting a Lactobacillus strain that produces tryptophan-binding bile acid; Preferably, the method is a method for screening a Lactobacillus strain that promotes the production of GLP-1 and / or insulin; Preferably, the Lactobacillus strain is Lactobacillus paracasei, preferably Lactobacillus paracasei with the deposit number CGMCCNO.17827.

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