Application of streptococcus thermophilus microbial inoculum in preparation of medicine for preventing diabetes or regulating blood sugar

Abstract

The invention discloses an application of a streptococcus thermophilus microbial inoculum in preparation of a medicine for preventing diabetes or regulating blood sugar. The streptococcus thermophilus microbial inoculum is a selenium-rich streptococcus thermophilus microbial inoculum. The streptococcus thermophilus agent can be applied to effectively resisting hyperglycemia induced by streptozotocin STZ, and the progress of diabetes mellitus is delayed. The selenium-rich streptococcus thermophilus microbial inoculum and the streptococcus thermophilus microbial inoculum can protect pancreas islet beta cells from STZ-induced damage, maintain the number and quality of the pancreas islet beta cells and prevent diabetes. The selenium-rich streptococcus thermophilus microbial inoculum and the streptococcus thermophilus microbial inoculum can reduce serum urea nitrogen and serum creatinine levels, relieve early diabetic kidney injury and prevent diabetic complications. The invention relates to a selenium-rich streptococcus thermophilus agent and application of the streptococcus thermophilus agent in preparation of drugs for preventing diabetes or regulating blood sugar. The selenium-rich streptococcus thermophilus agent and the streptococcus thermophilus agent have good safety, and the rat acute oral toxicity test proves that the selenium-rich streptococcus thermophilus agent and the streptococcus thermophilus agent belong to the actual non-toxic level.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial application, and in particular to application of a thermophilic streptococcus agent in the preparation of a drug for preventing diabetes or regulating blood sugar. Background Art

[0002] With the rapid development of modern society, lifestyles, including diet, have undergone tremendous changes. Excessive intake of salt, sugar, and fat in the diet has led to a continuous increase in the incidence of chronic metabolic diseases such as obesity and diabetes. Diabetes is a metabolic disease characterized by high blood sugar and disorders of water, electrolyte, and protein metabolism. Currently, there are more than 400 million diabetes patients in the world, of which Chinese diabetes patients account for the total number of diabetes patients in the world. Therefore, the research on effective prevention and treatment methods for diabetes is of great urgency and clinical value. First-line therapeutic drugs are mainly divided into insulin secretion promoters, insulin sensitizers and other types, but the damage to pancreatic beta cells is irreversible, so diabetes cannot be cured fundamentally. Therefore, protecting pancreatic cells from damage is an urgent problem to be solved.

[0003] With people's attention to intestinal flora in recent years, studies have shown that the composition and structure of the intestinal flora of diabetic patients are quite different from those of healthy people. The proportion of Firmicutes in diabetic patients is relatively low, while the proportion of Bacteroidetes and Proteus is relatively high. The intestinal flora plays an important role in human health and has long been closely related to the functions of digesting complex polysaccharides, regulating bile acid metabolism, and producing vitamins. An imbalance in intestinal flora can cause abnormalities in physiological processes such as energy metabolism, digestion and absorption, and immune regulation, leading to the occurrence of diseases. Therefore, people continue to try to artificially intervene in the types and quantities of intestinal flora through daily diet and the use of live bacteria to prevent or treat diseases.

[0004] As an aerobic Gram-positive bacterium, Streptococcus thermophilus is widely used in the fermentation production of dairy products due to its low production cost and stable fermentation. With the in-depth research in recent years, Streptococcus thermophilus has shown other biological functions besides fermentation. Studies have shown that Streptococcus thermophilus can significantly improve the intraperitoneal glucose tolerance of diabetic mouse models. Reduced insulin sensitivity is a typical feature of diabetes. Previous studies have confirmed that glucose tolerance is positively correlated with insulin sensitivity, indicating that Streptococcus thermophilus can improve the sensitivity of target organ insulin receptors to insulin to a certain extent. At the same time, selenium is an essential micronutrient for human and animal health, and actively participates in various physiological activities through a variety of selenoproteins. It is reported that selenium supplementation can reduce plasma glucose levels in diabetic rats and diabetic patients. Selenium has also been shown to continuously improve glucose homeostasis in diabetic patients to regulate important metabolic processes such as glycolysis and gluconeogenesis. Appropriate doses of selenium can also improve renal damage in diabetic nephropathy by changing lipid content, restoring lipid ordered structure and membrane dynamics.

[0005] In the existing biotechnology field, thermophilic streptococci are mainly used in dairy fermentation and dairy composite bacterial fermentation, and the ratio of probiotic species and core compound is adjusted to achieve better fermentation effect. Studies have shown that thermophilic streptococci have a certain enrichment effect on selenium, and selenium can be used for nutritional fortification to make various selenium-enriched probiotic products, so that they have the dual effects of probiotics and selenium at the same time, and the development prospects are broad, but the development of related invention patents for selenium-enriched thermophilic streptococci is currently limited. Therefore, it is urgent to develop thermophilic streptococci, selenium-enriched thermophilic streptococci and their mixtures for use as medicines or dietary supplements to protect pancreatic islet cells from damage, regulate blood sugar, and prevent the occurrence and development of diabetes. There is an urgent need to provide a selenium-enriched thermophilic streptococcus agent and its preparation method and its application in drugs for preventing diabetes and regulating blood sugar. Summary of the invention

[0006] In order to solve the problems of the prior art, the purpose of the present invention is to provide a use of a thermophilic streptococcus agent in the preparation of a drug for preventing diabetes or regulating blood sugar.

[0007] In order to achieve the above-mentioned purpose, the present invention adopts the following technical scheme: In the first aspect, the present application provides a use of a thermophilic Streptococcus agent in the preparation of a drug for preventing diabetes or regulating blood sugar.

[0008] In a second aspect, the present application provides a drug for treating diabetes.

[0009] In a third aspect, the present application provides a composition for treating diabetes.

[0010] The first aspect of the present application provides a use of a thermophilic Streptococcus agent in the preparation of a drug for preventing diabetes or regulating blood sugar, wherein the thermophilic Streptococcus is from the China Industrial Microbiological Culture Collection Administration Center, with the strain number: CICC6038.

[0011] Furthermore, thermophilic Streptococcus agents can effectively resist the hyperglycemia induced by streptozotocin (STZ) and delay the progression of diabetes.

[0012] Furthermore, the thermophilic Streptococcus agent can protect pancreatic β cells from STZ-induced damage, maintain the quantity and quality of pancreatic β cells, and prevent the occurrence of diabetes.

[0013] Furthermore, thermophilic streptococci can reduce early diabetic kidney damage and prevent the occurrence of diabetic complications by lowering serum urea nitrogen and serum creatinine levels.

[0014] Furthermore, the thermophilic Streptococcus agent can improve the glucose tolerance of mice and maintain the quantity and quality of pancreatic β cells; it can relieve symptoms such as polydipsia, polyphagia, and weight loss caused by diabetes.

[0015] Furthermore, the preparation method of the selenium-enriched thermophilic streptococcus agent comprises the following steps:

[0016] a. Prepare 3L of conventional MRS medium, adjust the pH to 6.4-7.2, load into a 5L fermenter, sterilize at 121℃ for 20 minutes, and cool to room temperature;

[0017] b. Set the fermentation parameters of the fermentation tank: the fermentation temperature is set to 38℃-45℃, the stirring speed is set to 180rpm-280rpm, and the pH is set to 6.4-7.2;

[0018] c. Add 30-180 mL of the overnight activated Streptococcus thermophilus seed solution to a 3000 mL MRS fermenter as an inoculum of 1%-6%, and add the nano-selenium solution that has been sterilized by filtration through a 0.22 μm filter membrane to a final concentration of 20-50 mg / L;

[0019] d. The culture temperature was set to 38°C-45°C, the initial speed was set to 180rpm-280rpm, the ventilation volume was 3LPM, and fermentation began. After the dissolved oxygen rebounded, 30% glucose was fed at a constant rate, with a flow rate of 3ml / min;

[0020] e. After 5 hours of fermentation, the rotation speed is reduced to any value of 100rpm-150rpm, the ventilation volume is reduced to 1.5LPM, and the fermentation is stopped after 8-10 hours of fermentation. When the dissolved oxygen in the fermentation tank rebounds, feeding is started, and 30% glucose is fed at a constant rate of 3ml / min; a selenium-rich thermophilic streptococcus agent is obtained.

[0021] Furthermore, in step (c), the method for preparing the nano-selenium solution comprises the following steps:

[0022] (1) Add stabilizer to a beaker;

[0023] (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved;

[0024] (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring;

[0025] (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

[0026] Furthermore, the selenium-enriched thermophilic streptococcus agent is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body, wherein the content of nano-selenium in the agent is 0.5-2.9 micrograms / gram, and the number of active colonies of the selenium-enriched thermophilic streptococcus is 1×10 9 Up to 5×10 10 CFU / g.

[0027] The second aspect of the present application provides a drug for treating diabetes, comprising a thermophilic Streptococcus agent or a selenium-enriched thermophilic Streptococcus agent as an active ingredient.

[0028] The third aspect of the present application provides a composition for treating diabetes, comprising a thermophilic Streptococcus agent and a selenium-enriched thermophilic Streptococcus agent.

[0029] Beneficial effects: The selenium-enriched thermophilic streptococcus agent and the thermophilic streptococcus agent of the present invention are used in the preparation of drugs for preventing diabetes or regulating blood sugar. The selenium-enriched thermophilic streptococcus agent and the thermophilic streptococcus agent have good safety, and the acute oral toxicity test of rats proves that they are practically non-toxic and are suitable for long-term use as food, health products or auxiliary preparations.

[0030] Compared with the prior art, the selenium-enriched thermophilic streptococcus agent and the thermophilic streptococcus agent of the present invention have the following advantages:

[0031] (1) Effective prevention of diabetes: The application of selenium-rich thermophilic streptococci and thermophilic streptococci can effectively resist STZ-induced hyperglycemia, significantly reduce the incidence of diabetes, and delay the progression of diabetes.

[0032] Improved glucose tolerance: The preparation was able to improve the intraperitoneal glucose tolerance of mice, indicating that it helps to improve the body's absorption and utilization of glucose, thereby lowering blood sugar levels.

[0033] (2) Maintain pancreatic β-cell function: Selenium-enriched thermophilic streptococci and thermophilic streptococci can protect pancreatic β-cells from STZ-induced damage and maintain the quantity and quality of pancreatic β-cells, which is essential for the synthesis and secretion of insulin and helps maintain normal blood sugar levels. Relieve symptoms of diabetes: The preparation can effectively relieve symptoms such as polydipsia, polyphagia, and weight loss caused by diabetes, and improve the quality of life of patients.

[0034] (3) Protect kidney function: By lowering serum urea nitrogen and serum creatinine levels, selenium-enriched thermophilic streptococci and thermophilic streptococci can reduce early diabetic kidney damage and prevent the occurrence of diabetic complications.

[0035] (4) Convenience: The preparation can be conveniently taken in the form of probiotic tablets, probiotic powder, probiotic capsules, etc., and is easy for patients to use in daily life. The strain is expanded, selenium-enriched, and freeze-dried to prepare active bacterial powder, which can be used as a common food, health food, or auxiliary medicine to prevent and treat diabetes.

[0036] (5) Safety: Thermophilic Streptococcus has a good history of safe use and compliance, and has been included in China's "List of Bacteria that Can Be Used in Food" and the EU Safety Qualification (QPS) list, indicating that its safety is recognized. Selenium-enriched thermophilic Streptococcus has a very good safety profile, and the acute oral toxicity test in rats has proven that it is practically non-toxic. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 It is a display diagram of the random blood sugar changes of mice after STZ modeling with the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0039] Figure 2 It is a graph showing the fasting blood glucose of mice after STZ modeling by the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0040] Figure 3 It is a graph showing the changes in body weight of mice after STZ modeling using the selenium-enriched thermophilic streptococci and the thermophilic streptococcus agent of the present invention.

[0041] Figure 4 It is a line graph showing the changes in mouse body weight during STZ modeling with the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0042] Figure 5 The figure is a bar chart showing the weight change of mice during STZ modeling by using the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0043] Figure 6 It is a graph showing the daily food intake of mice after STZ modeling by the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0044] Figure 7 It is a graph showing the daily water intake of mice after STZ modeling by the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0045] Figure 8The figure shows the intraperitoneal glucose tolerance of mice after STZ modeling using the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0046] Fig. 9 The figure is a statistical display of the area under the peritoneal glucose tolerance curve of mice after STZ modeling of the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0047] Fig.10 The graph shows the serum insulin level of mice after STZ modeling by the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0048] Fig.11 The graph shows the serum glycosylated hemoglobin level of mice after STZ modeling by the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0049] Fig.12 The figure shows the serum urea nitrogen content of mice after STZ modeling by the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0050] Fig.13 The graph shows the serum creatinine content of mice after STZ modeling using the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0051] Fig.14 The diagram shows the immunofluorescence staining of insulin and glucagon in the pancreatic paraffin sections of mice after STZ modeling using the selenium-enriched thermophilic streptococcus and the thermophilic streptococcus agent of the present invention.

[0052] Fig.15 It is a display diagram of the statistics of the percentage of pancreatic β cells in mice after STZ modeling by the selenium-enriched thermophilic streptococci and the thermophilic streptococci agent of the present invention. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0055] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0056] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0057] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "said" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0058] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass described in the embodiment description of the present application can be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0059] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0060] Unless otherwise specified, the reagents and materials used in the present invention are prepared by conventional methods or obtained from commercial channels.

[0061] The data were statistically analyzed using GraphPad Prism9 software, and the analysis methods used were two-way ANOVA, one-way ANOVA, and t-test. # represents a significant difference compared with the control group; * represents a significant difference compared with the model group; + represents a significant difference between the selenium-enriched thermophilic Streptococcus group and the thermophilic Streptococcus group. # / * / + represents p < 0.05, ## / ** / ++ represents p less than 0.01, and ### / *** / +++ represents p less than 0.001.

[0062] The first aspect of the embodiment of the present application provides a use of a thermophilic Streptococcus agent in the preparation of a drug for preventing diabetes or regulating blood sugar, wherein the thermophilic Streptococcus is from the China Industrial Microbiological Culture Collection Administration Center, with a strain number of CICC6038.

[0063] Selenium-enriched thermophilic Streptococcus agents and thermophilic Streptococcus agents can effectively resist hyperglycemia induced by streptozotocin (STZ) and delay the progression of diabetes.

[0064] Selenium-enriched thermophilic Streptococcus agent and thermophilic Streptococcus agent can protect pancreatic β cells from STZ-induced damage, maintain the quantity and quality of pancreatic β cells, and prevent the occurrence of diabetes.

[0065] Selenium-enriched thermophilic streptococci can reduce serum urea nitrogen and serum creatinine levels, alleviate early diabetic kidney damage and prevent the occurrence of diabetic complications.

[0066] Selenium-enriched thermophilic Streptococcus can improve the glucose tolerance of mice and maintain the quantity and quality of pancreatic β cells; it can relieve symptoms such as polydipsia, polyphagia, and weight loss caused by diabetes.

[0067] The preparation method of the selenium-enriched thermophilic streptococcus agent comprises the following steps:

[0068] a. Prepare 3L of MRS medium, adjust the pH to 6.4-7.2, load into a 5L fermenter, sterilize at 121°C for 20 minutes, and cool to room temperature;

[0069] b. Set the fermentation parameters of the fermentation tank: the fermentation temperature is set to 38℃-45℃, the stirring speed is set to 180rpm-280rpm, and the pH is set to 6.4-7.2;

[0070] c. Add 30-180 mL of the overnight activated Streptococcus thermophilus seed solution to a 3000 mL MRS fermenter as an inoculum of 1%-6%, and add the nano-selenium solution that has been sterilized by filtration through a 0.22 μm filter membrane to a final concentration of 20-50 mg / L;

[0071] d. The culture temperature was set to 38°C-45°C, the initial speed was set to 180rpm-280rpm, the ventilation volume was 3LPM, and fermentation began. After the dissolved oxygen rebounded, 30% glucose was fed at a constant rate, with a flow rate of 3ml / min;

[0072] e. After 5 hours of fermentation, the rotation speed is reduced to any value of 100rpm-150rpm, the ventilation volume is reduced to 1.5LPM, and the fermentation is stopped after 8-10 hours of fermentation. When the dissolved oxygen in the fermentation tank rebounds, feeding is started, and 30% glucose is fed at a constant rate of 3ml / min; a selenium-rich thermophilic streptococcus agent is obtained.

[0073] In some embodiments, a method for preparing a nano-selenium solution comprises the following steps: (1) adding a stabilizer to a beaker;

[0074] (2) slowly adding the aqueous solution containing the reducing agent into the beaker, and maintaining magnetic stirring during the addition process until the stabilizer is completely dissolved;

[0075] (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring;

[0076] (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

[0077] In some embodiments, in step (1), the stabilizer is epigallocatechin gallate (EGCG).

[0078] In some embodiments, in step (1), the stabilizer is chitosan.

[0079] In some embodiments, in step (1), the stabilizer is gum arabic.

[0080] In some embodiments, in step (1), the stabilizer is bovine serum albumin (BSA).

[0081] In some embodiments, in step (1), the stabilizer is sodium hyaluronate.

[0082] In some embodiments, in step (2), the reducing agent is reduced glutathione GSH.

[0083] In some embodiments, in step (2), the reducing agent is vitamin C (Vc).

[0084] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.1 mM to 40 mM (for EGCG).

[0085] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.01% to 0.7% (for chitosan).

[0086] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.04% to 0.4% (for gum arabic).

[0087] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.4 mg / ml to 60 mg / ml (for BSA).

[0088] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.02% to 0.25% (for sodium hyaluronate).

[0089] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-50 mM (GSH).

[0090] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-100 mM (Vc).

[0091] In step (3), the final concentration of sodium selenite in the reaction system is 5 mM.

[0092] In some embodiments, in step (1), the molecular weight of the stabilizer is in the range of 100 KD - 1000 KD (for chitosan).

[0093] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200KD to 2000KD (for gum arabic).

[0094] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200KD to 2000KD (for sodium hyaluronate).

[0095] In step (4), in the step of adjusting the pH, the pH value ranges from 5.0 to 9.0.

[0096] In some embodiments, the peak value of the particle size distribution of the nano-selenium prepared by the above steps can be precisely controlled within a range of 20 nm-60 nm, and the particle size is mainly distributed within a range of 10 nm-150 nm.

[0097] The selenium-enriched thermophilic streptococcus agent is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body. The content of nano-selenium in the agent is 0.5-2.9 micrograms / gram, and the number of active colonies of selenium-enriched thermophilic streptococci is 1×10 9 Up to 5×10 10 CFU / g.

[0098] In some embodiments, the selenium-enriched thermophilic Streptococcus agent is in the form of a tablet.

[0099] In some embodiments, the selenium-enriched thermophilic Streptococcus agent is a lyophilized powder.

[0100] In some embodiments, the selenium-enriched thermophilic Streptococcus agent is a capsule.

[0101] In some embodiments, the selenium-enriched thermophilic Streptococcus agent is a dairy product.

[0102] In some embodiments, the selenium-enriched thermophilic Streptococcus agent is stirred yogurt.

[0103] In some embodiments, the selenium-enriched thermophilic Streptococcus agent is in the form of coagulated yogurt.

[0104] A second aspect of an embodiment of the present application provides a drug for treating diabetes, comprising a thermophilic Streptococcus agent or a selenium-enriched thermophilic Streptococcus agent as an active ingredient.

[0105] In some embodiments, the pharmaceutical dosage form is an oral solution.

[0106] In some embodiments, the pharmaceutical dosage form is a lyophilized powder.

[0107] In some embodiments, the pharmaceutical dosage form is a capsule.

[0108] In some embodiments, the pharmaceutical dosage form is made of a selenium-enriched Bifidobacterium longum bacterial agent and excipients acceptable to mammals.

[0109] The third aspect of the embodiment of the present application provides a composition for treating diabetes, comprising a thermophilic streptococcus agent and a selenium-enriched thermophilic streptococcus agent. The selenium-enriched thermophilic streptococcus agent has good safety, and the acute oral toxicity test in rats proves that it is practically non-toxic.

[0110] Example 1

[0111] The selenium-rich thermophilic streptococcus agent of the present invention and the use of the thermophilic streptococcus agent in the preparation of a pharmaceutical preparation for preventing diabetes or regulating blood sugar;

[0112] The preparation is used in effectively resisting hyperglycemia induced by streptozotocin (STZ) and delaying the progression of diabetes.

[0113] The preparation is used in protecting pancreatic beta cells from STZ-induced damage, maintaining the quantity and quality of pancreatic beta cells, and preventing the occurrence of diabetes.

[0114] The preparation can reduce serum urea nitrogen and serum creatinine levels, and the selenium-enriched thermophilic streptococcus agent can alleviate early diabetic kidney damage and prevent the occurrence of diabetic complications.

[0115] The preparation can improve the glucose tolerance of mice and maintain the quantity and quality of pancreatic beta cells; it can relieve symptoms such as polydipsia, polyphagia, and weight loss caused by diabetes.

[0116] The invention discloses a method for preparing a selenium-enriched thermophilic streptococcus agent, comprising the following steps: (a) culturing thermophilic streptococci in a culture medium containing selenium; (b) collecting the cultured thermophilic streptococci; (c) preparing the collected thermophilic streptococci into active bacterial powder by a freeze-drying process; and (d) packaging the active bacterial powder into at least one of the following forms: probiotic tablets, probiotic powder, probiotic capsules, probiotic dairy products, probiotic stirred yogurt or solidified yogurt.

[0117] The invention discloses a method for preventing diabetes and regulating blood sugar by using a selenium-rich thermophilic streptococcus agent and a thermophilic streptococcus agent, comprising the following steps: administering the preparation in a daily dose for 4 consecutive weeks.

[0118] The composition for treating diabetes of the present invention is characterized by comprising selenium-enriched thermophilic streptococcus agent and thermophilic streptococcus agent.

[0119] Example 2

[0120] Preparation and administration of thermophilic streptococci and selenium-enriched thermophilic streptococci

[0121] 1. Resuspend the thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus probiotic freeze-dried powder in PBS buffer to obtain thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus probiotic agents. The number of viable bacteria per daily dose is 0.5-10×10 8 CFU, the mice were divided into 4 groups, namely the control group, the model group, the model group treated with thermophilic Streptococcus, and the model group treated with selenium-enriched thermophilic Streptococcus, with 8 mice in each group. Specifically: the control group and the model group were gavaged with 100 μl PBS daily, and the probiotic group was gavaged with 100 μl of the corresponding bacterial suspension daily, and the administration lasted for 4 weeks.

[0122] Test Example 1

[0123] Effects of Streptococcus thermophilus and selenium-enriched Streptococcus thermophilus on random blood glucose changes in mice after STZ modeling

[0124] Model construction: 4-week-old healthy SPF-grade C57BL / 6J male mice bred by Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (License No.: SCXK (Su) 2020-0009) were selected. The experimental animals were raised in the Pharmaceutical Animal Experiment Center of China Pharmaceutical University, and the experimental animal use license number is SYXK (Su) 2023-0019. The relative humidity of the animal room environment was 55±5%, and the temperature was 22±2℃. The mice were free to drink water and feed. After the mice were adaptively fed for 1 week, probiotics were administered for 4 weeks. In the 5th week, multiple injections of small doses of STZ (35 mg / kg, continuous modeling for 5 days) were intraperitoneally injected to induce diabetes. The control group was given citric acid / sodium citrate buffer. The model was successfully established when the random blood glucose was ≥11.1mmol / L for two consecutive times or the fasting blood glucose was ≥7mmol / L for two consecutive times.

[0125] Monitor the random blood glucose of mice weekly: When the mice are in a quiet state, cut their tails 1-2mm, and use a blood glucose meter to measure the random blood glucose of the mice and record it.

[0126] Depend on Figure 1 It can be seen that there was no obvious fluctuation in the blood sugar of mice when probiotics were gavaged, and it was within the normal range. After STZ-induced diabetes, the random blood sugar of mice increased rapidly, and the model group increased the fastest. After 8 weeks of modeling, the average blood sugar of the control group was 8.27mmol / L, the model group was as high as 32.85mmol / L, the average blood sugar of the thermophilic streptococcus intervention group was 27.67mmol / L, and the average blood sugar of the selenium-enriched thermophilic streptococcus intervention group was 22.61mmol / L, indicating that the preventive administration of probiotics can significantly alleviate the symptoms of hyperglycemia in mice and delay the increase of blood sugar. Among them, the blood sugar lowering rate of selenium-enriched thermophilic streptococcus is significantly better than that of thermophilic streptococcus. Figure 1 Shows 3×10 8 Results of CFU dosing, other 0.5-10×10 8 CFU dosage and Figure 1 The results were highly similar.

[0127] Test Example 2

[0128] Effect of thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus on fasting blood glucose in mice at 4 weeks after STZ modeling

[0129] Since random blood glucose fluctuates due to the influence of daily food intake and activities of mice, in order to more accurately judge the progress of diabetes in mice, the fasting blood glucose of mice was tested in the fourth week after modeling. Specifically: the fasting blood glucose of mice was measured and recorded after fasting for 12 hours but not water. Figure 2It can be seen that the blood sugar of the mice in the control group was less than 7mmol / L, and the blood sugar of the model group was significantly higher than that of other groups. Thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus can prevent the occurrence of hyperglycemia and diabetes to a certain extent. It is worth mentioning that the fasting blood sugar of mice intervened by probiotics is similar to that of the control group, indicating that it has a good effect on regulating blood sugar.

[0130] Test Example 3

[0131] Effects of Streptococcus thermophilus and selenium-enriched Streptococcus thermophilus on body weight changes in mice after STZ modeling

[0132] Depend on Figure 3 It can be seen that the thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus probiotics had no significant effect on the weight of mice during intragastric administration, and the weight of mice increased steadily, and there was no significant difference with the control group. It can be inferred that the dosage had no obvious toxicity, and the safety of probiotics as food, health products or auxiliary medicines in the present invention was good. After STZ induced diabetes, the weight of mice showed a significant downward trend, among which the weight of mice in the model group decreased the most and continued to decrease for 3 weeks. The weight of mice intervened by probiotics was less, and the weight of mice was steadily and slowly restored after modeling was completed, and the mice were in a better state, indicating that it can better alleviate the phenomenon of weight loss in the diabetic state.

[0133] Test Example 4

[0134] Effects of Streptococcus thermophilus and selenium-enriched Streptococcus thermophilus on body weight changes in mice during STZ modeling

[0135] Depend on Figure 4 , Figure 5 It can be seen that during the 5-day STZ modeling, the weight of the mice in the control group given citric acid / sodium citrate buffer continued to increase, while the weight of the modeling group decreased. Selenium-enriched thermophilic Streptococcus can effectively resist the damage and weight changes caused by STZ modeling.

[0136] Test Example 5

[0137] Food intake and water intake of mice with thermophilic streptococci and selenium-enriched thermophilic streptococci after STZ modeling

[0138] Metabolic cages were used to detect the food and water intake of mice after the onset of diabetes. Figure 6 Figure 7 It can be seen that the mice in the model group showed polydipsia and polyphagia phenotypes, and both thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus could significantly alleviate the above symptoms of mice. The diabetic symptoms of mice were milder, and the effect of selenium-enriched thermophilic Streptococcus was better.

[0139] Test Example 6

[0140] Effect of Streptococcus thermophilus and Selenium-enriched Streptococcus thermophilus on intraperitoneal glucose tolerance in mice after STZ modeling

[0141] In order to evaluate the pancreatic β-cell function of mice and their responsiveness to glucose, an intraperitoneal glucose tolerance test was performed: the mice were fasted for 14-16 hours, but not water, and 2 g / kg D-glucose was injected intraperitoneally. The blood glucose levels of the mice were tested at 0, 15, 30, 60, 90, and 120 hours. Figure 8 It can be seen that at 0 minutes after fasting, the blood glucose level of mice in the model group was the highest, the blood glucose levels of mice in the thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus probiotic groups were between the control group and the model group, and the blood glucose of mice in the control group and the selenium-enriched thermophilic Streptococcus intervention group reached a peak at 15 minutes and then continued to decline. The blood glucose level of mice in the model group remained at a high level and was still greater than 20mmol / L 120 minutes after the injection of glucose. Fig. 9 The area under the curve of intraperitoneal glucose tolerance was statistically analyzed. The results showed that the glucose tolerance of mice was significantly impaired after modeling, but the administration of selenium-enriched thermophilic Streptococcus could effectively improve the glucose responsiveness of pancreatic β cells.

[0142] Test Example 7

[0143] Changes of serum insulin levels in mice induced by Streptococcus thermophilus and Selenium-enriched Streptococcus thermophilus after STZ modeling

[0144] After STZ modeling, the pancreatic β cells of mice are severely damaged, resulting in insufficient insulin secretion and increased blood sugar in the mice. Therefore, the serum insulin level of mice is tested to evaluate the progress of diabetes. After STZ modeling, blood is collected from the orbital venous plexus of mice. The blood is allowed to stand at room temperature for 30 minutes at 4°C, then centrifuged at 3000rpm for 20 minutes, and the supernatant is taken to obtain serum. The serum insulin content is tested by enzyme-linked immunosorbent assay according to the instructions. Fig.10 It can be seen that compared with the control group mice, the serum insulin content in the model group decreased by more than 50%, and the insulin content in the thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus probiotic groups was slightly higher than that in the model group, but there was no significant difference.

[0145] Test Example 8

[0146] Effects of thermophilic streptococci and selenium-enriched thermophilic streptococci on the changes of glycosylated hemoglobin levels in mice after STZ modeling

[0147] Depend on Fig.11 It can be seen that 8 weeks after modeling, the level of glycosylated hemoglobin in mouse serum was detected by enzyme-linked immunosorbent assay according to the instructions. The glycosylated hemoglobin content of mice in the model group was as high as 10mmol / L, while that of mice in the selenium-enriched thermophilic streptococcus group was only about 5mmol / L, indicating that selenium-enriched thermophilic streptococcus can better control the average blood sugar level of diabetic mice within 8 weeks after the onset of the disease, and effectively alleviate the occurrence and development of diabetes.

[0148] Test Example 9

[0149] Effects of Streptococcus thermophilus and selenium-enriched Streptococcus thermophilus on early diabetic kidney injury in mice after STZ modeling

[0150] Eight weeks after modeling, serum urea nitrogen and serum creatinine levels were detected to reflect the early stage of diabetic kidney damage in mice. Fig.12 , Fig.13 It can be seen that compared with the control group, the serum urea nitrogen and serum creatinine levels of mice in the model group increased, indicating that the mice had kidney damage caused by diabetes. The intervention of thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus probiotics can effectively reduce the levels of serum urea nitrogen and serum creatinine in mice, improve early diabetic kidney damage in mice, and prevent the occurrence of this complication.

[0151] Test Example 10

[0152] Effects of Streptococcus thermophilus and selenium-enriched Streptococcus thermophilus on pancreatic islet ecology in mice after STZ modeling

[0153] In order to observe the changes in the islet niche of mice, the pancreas of mice was paraffin-embedded and sliced, and immunofluorescence staining of insulin and glucagon was performed. Specifically, baking: the experimental slices were placed in an oven for 0.5h at a temperature of 60°C to melt the paraffin on the surface of the slices, which was conducive to subsequent dewaxing and prevented from peeling. Dewaxing: the warm slices were transferred to the fume hood, 100ml of xylene solution was added to the immunohistochemistry tank, and the slices were completely immersed for 10min, and dewaxed twice to fully dissolve the paraffin. Washing: 100ml of anhydrous ethanol was added to the immunohistochemistry tank to dissolve the residual xylene, soak the slices for 5min, and wash twice. Water attachment: prepare ethanol solutions with a concentration gradient, respectively: 95%, 90%, 80%, 60% and 30%, 100ml each, soak the slices for 3min. Then soak the slices in double distilled water for 3min. Membrane permeation: soak the slices in membrane permeation solution for 15min to increase the permeability of the cell membrane. Wash with double distilled water for 3min. Antigen repair: EDTA buffer system and microwave repair were used. Prepare 120ml EDTA antigen repair solution, pour it into the immunohistochemistry cylinder to immerse the tissue, and heat it in a microwave oven to a slight boil, about 95°C. Take it out after 15 minutes and cool it naturally to room temperature. Wash with double distilled water for 5 minutes and PBS buffer for 5 minutes. Blocking: Prepare 10% goat serum with PBS buffer, draw circles around the pancreatic tissue with a hydrophobic pen, place the slices in the immunohistochemical humidified box, add 100μl 10% goat serum to each tissue, and block at room temperature for 2h. Primary antibody incubation: Prepare the primary antibody working solution as required according to the instructions of each antibody, discard the blocking solution, add 80-100μl antibody working solution to each tissue, and place the immunohistochemical humidified box at 4°C for incubation overnight. Secondary antibody incubation: Wash the slices with PBS buffer × 3, each time, each time for 10 minutes. Prepare the secondary antibody working solution as required according to the instructions of the secondary antibody, and discard the primary antibody. Protect from light, add 100 μl of secondary antibody working solution to each tissue, incubate the secondary antibody at room temperature, and incubate at room temperature for 3 hours. Sealing: Protect from light, dry the slices slightly, add 10 μl of anti-fluorescence quenching sealing agent containing DAPI to each tissue, and cover with a coverslip.

[0154] Depend on Fig.14 It can be seen that more than 90% of the pancreatic islets of the control mice were β cells, and α cells were distributed around the islet cells. However, after modeling, the pancreatic β cells of the mice were lost in large quantities; the proportion of α cells increased significantly, and the position of α cells moved to the center of the islets, indicating that STZ damaged the normal ecological niche of the islets. The administration of thermophilic Streptococcus and selenium-enriched thermophilic Streptococcus probiotics can effectively maintain the number of mouse β cells, and α cells are still basically distributed around the islets. At the same time, Fig.15 The statistical results show that the selenium-enriched thermophilic streptococcus has better effects and a higher percentage of β cells. The above results indicate that thermophilic streptococcus and selenium-enriched thermophilic streptococcus probiotics can protect mouse pancreatic β cells from damage by STZ, and therefore have the effect of preventing the occurrence of diabetes.

[0155] Test Example 11

[0156] Acute oral toxicity test of selenium-enriched thermophilic streptococci

[0157] Experimental animals: 20 healthy adult SPF SD rats, half male and half female, with a weight of 187-205g for females and 206-220g for males, provided by Nanjing Medical University, production license number SCXK (Su) 2021-0001, certificate number: 202335660. The experimental animals were raised in the barrier system of this center at a temperature of 20-26°C and a relative humidity of 40%-70%, and the experimental animal use license number is SYXK (Su) 2020-0006. The experimental mouse maintenance feed was provided by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd., production license: Su Feed Certificate (2019) 01008.

[0158] Dose selection and test substance administration method: The acute toxicity test of rats was carried out using the limit method, and the dose was 10000 mg / kg·bw. The animals were quarantined for 3 days. The animals were fasted for 16 hours before the test and had free access to water. The test sample was prepared with purified water at a concentration of 500 mg / mL. The gavage volume was 20 mL / kg·bw, and the sample was given once. The dose of the test substance was 10000 mg / kg·bw. After sampling, the animals were observed for 14 consecutive days, and the symptoms of poisoning and death were recorded. The animals were weighed once on the 0th, 7th, and 14th days. At the end of the observation period, the animals underwent gross anatomical examination.

[0159] Main instruments: JJ10 electronic balance, T1000 electronic balance.

[0160] Test method: The acute toxicity test in rats was carried out using the limit method.

[0161] During the experimental observation period, the animals in each group had normal diet and activities, grew well, and no poisoning symptoms or deaths were observed. At the end of the observation period, no abnormal pathological changes were observed in the gross autopsy of the animals. The effects of selenium-enriched thermophilic streptococci on the body weight and mortality of rats are shown in Table 1.

[0162] Table 1 Effects of selenium-enriched thermophilic Streptococcus on acute oral toxicity in rats

[0163]

[0164] After oral administration of selenium-enriched thermophilic streptococci to animals, no obvious signs of poisoning were observed, no significant effect on the weight gain of animals, and no animal deaths occurred during the observation period. Acute oral toxicity test of selenium-enriched thermophilic streptococci in female and male rats LD 50 Greater than 10000 mg / kg·bw, according to the acute toxicity dose classification standard, the sample is actually non-toxic.

[0165] In summary, there was no significant fluctuation in the blood glucose of mice when probiotics were administered orally, and the random blood glucose value was less than 11.1mmol / L, which was within the normal range. At the same time, the weight of mice increased steadily during the administration, and there was no significant difference with the control group. It can be inferred that the dosage was not significantly toxic. In addition, selenium-enriched thermophilic streptococci and thermophilic streptococci can effectively resist STZ-induced diabetes, significantly improve the symptoms of high blood sugar levels in mice, and alleviate the phenotypes of polydipsia, polyphagia, and weight loss in mice. The experimental data showed that the average blood glucose of the control group was 8.27mmol / L, the model group was as high as 32.85mmol / L, the average blood glucose of the thermophilic streptococcus intervention group was 27.67mmol / L, and the average blood glucose of the selenium-enriched thermophilic streptococcus intervention group was 22.61mmol / L. At the same time, it can effectively maintain the number and function of pancreatic β cells, and the pancreatic glucose responsiveness is better. In the glucose tolerance test, the blood glucose of the control group mice and the selenium-enriched thermophilic streptococcus intervention group mice reached a peak at 15 minutes and then continued to decline. The blood glucose level of mice in the model group remained at a high level, still greater than 20mmol / L 120 minutes after glucose injection, while the blood glucose level of mice in the selenium-enriched thermophilic streptococcus intervention group dropped to 15mmol / L. The β-cell percentage statistics showed that the β-cell ratio in the model group was only 20%, and the β-cell percentage could be maintained at 40-50% after the intervention of selenium-enriched thermophilic streptococcus and thermophilic streptococcus probiotics. Probiotics can also increase the insulin level of mice, maintain the body's insulin synthesis and secretion, reduce the level of glycosylated hemoglobin in mice, delay the development of diabetes, and reduce early diabetic kidney damage in mice.

[0166] As a commonly used fermentation bacteria for dairy products, a large number of studies have shown that thermophilic Streptococcus has good safety. 10 The CFU selenium-enriched probiotic preparation will not produce toxicity and adverse reactions. This dosage is 100 times the dosage of the present invention, indicating that the selenium-enriched thermophilic streptococcus agent of the present invention can effectively slow down the progression of diabetes to a certain extent without adverse reactions, and has a good blood sugar regulation effect. The results of the acute oral toxicity test of selenium-enriched thermophilic streptococcus also showed that the acute oral toxicity test of selenium-enriched thermophilic streptococcus in female and male rats LD 50 Greater than 10000 mg / kg·bw, it is practically non-toxic.

[0167] The present invention is applied in delaying the progression of diabetes induced by streptozotocin. Compared with the model group mice, the selenium-enriched probiotic can effectively resist hyperglycemia and β-cell damage induced by streptozotocin, delay the progression of diabetes, and improve the glucose tolerance of mice, maintain the quality of pancreatic β-cells and the islet niche, and alleviate the kidney damage caused by diabetes to a certain extent. In summary, selenium-enriched thermophilic streptococci and thermophilic streptococci can effectively resist streptozotocin-induced islet damage, providing a basis for the development of diabetes prevention and treatment related foods, health products or auxiliary preparations using selenium-enriched thermophilic streptococci and thermophilic streptococci.

[0168] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. Use of a thermophilic streptococcus agent in the preparation of a drug for preventing diabetes or regulating blood sugar.

2. The use according to claim 1, characterized in that: The thermophilic streptococcus agent is a selenium-enriched thermophilic streptococcus agent.

3. The use according to claim 2, characterized in that: The thermophilic streptococcus agent can effectively resist hyperglycemia induced by streptozotocin (STZ) and delay the progression of diabetes.

4. The use according to claim 1, characterized in that: The thermophilic streptococcus agent can protect pancreatic beta cells from STZ-induced damage, maintain the quantity and quality of pancreatic beta cells, and prevent the occurrence of diabetes.

5. The use according to claim 2 or 3, characterized in that: The thermophilic streptococcus agent can reduce serum urea nitrogen and serum creatinine levels, alleviate early diabetic kidney damage, and prevent the occurrence of diabetic complications.

6. The use according to claim 2 or 3, characterized in that: The thermophilic streptococcus agent can improve the glucose tolerance of mice and maintain the quantity and quality of pancreatic beta cells; and can alleviate symptoms such as polydipsia, polyphagia, and weight loss caused by diabetes.

7. The use according to claim 2, characterized in that: The preparation method of the selenium-enriched thermophilic streptococcus agent comprises the following steps: a. Prepare 3 L of conventional MRS medium, adjust the pH to 6.4-7.2, load into a 5 L fermenter, sterilize at 121°C for 20 minutes, and cool to room temperature; b. Set the fermentation parameters of the fermentation tank: the fermentation temperature is set to 38℃-45℃, the stirring speed is set to 180 rpm-280rpm, and the pH is set to 6.4-7.2; c. Add 30 mL-180 mL of the thermophilic Streptococcus seed solution activated overnight into a 3000 mL MRS fermenter as an inoculum of 1%-6%, and add the nano-selenium solution that has been sterilized by filtration through a 0.22 mm filter membrane to a final concentration of 20-50 mg / L; d. The culture temperature was set to 38°C-45°C, the initial speed was set to 180 rpm-280 rpm, the ventilation volume was 3 LPM, and fermentation began. After the dissolved oxygen rebounded, 30% glucose was fed at a constant rate, with a flow rate of 3 ml / min; e. After 5 hours of fermentation, the rotation speed was reduced to any value of 100 rpm-150 rpm, the ventilation volume was reduced to 1.5 LPM, and the fermentation was stopped after 8-10 hours of fermentation. When the dissolved oxygen in the fermentation tank rebounded, feeding began, and 30% glucose was fed at a constant rate of 3 ml / min; a selenium-rich thermophilic streptococcus inoculum was obtained.

8. The use according to claim 7, characterized in that: In step (c), the method for preparing the nano-selenium solution comprises the following steps: (1) Add stabilizer to the beaker; (2) Slowly add the aqueous solution containing the reducing agent into the beaker and maintain magnetic stirring during the addition process until the stabilizer is completely dissolved; (3) slowly adding the sodium selenite aqueous solution to the solution obtained in step (2), during which the solution in step (2) is under magnetic stirring; (4) After the sodium selenite aqueous solution is added, the pH is immediately adjusted to a predetermined range using a sodium hydroxide solution, and then the magnetic stirring is continued for 30 minutes to obtain a red nano-selenium solution.

9. The use according to claim 2, characterized in that: The selenium-enriched thermophilic streptococcus agent is prepared by a biological fermentation process and contains organic selenium that can be absorbed by the body, wherein the content of nano-selenium in the agent is 0.5-2.9 micrograms / gram, and the number of active colonies of the selenium-enriched thermophilic streptococcus is 1×10 9 Up to 5×10 10 CFU / g.

10. A drug for treating diabetes, characterized in that The invention comprises the thermophilic streptococcus agent according to claim 1 or the selenium-enriched thermophilic streptococcus agent according to claim 2 as an active ingredient.

11. A composition for treating diabetes, characterized in that: It comprises the thermophilic streptococcus agent according to claim 1 and the selenium-enriched thermophilic streptococcus agent according to claim 2.

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