Application of bifidobacterium longum fungicide in preparation of medicine for preventing diabetes or regulating blood sugar

Abstract

The invention discloses an application of a bifidobacterium longum microbial agent in preparation of drugs for preventing diabetes or regulating blood sugar. The bifidobacterium longum microbial inoculum is a selenium-rich bifidobacterium longum microbial inoculum. The bifidobacterium longum microbial agent can protect pancreas islet beta cells from STZ-induced damage, maintain the number and quality of the pancreas islet beta cells and prevent the occurrence of diabetes mellitus; the bifidobacterium longum microbial inoculum can significantly reduce hyperglycemia induced by streptozotocin STZ, alleviate polydipsia and polyphagia phenomena caused by diabetes, improve mouse abdominal sugar tolerance, improve the serum insulin content of diabetic mice, and enhance the metabolic regulation of endogenous insulin of the mice. The selenium-rich bifidobacterium longum agent disclosed by the invention has a better curative effect than bifidobacterium longum. The selenium-rich bifidobacterium longum agent has good safety, and is proved to belong to an actual non-toxic grade through a rat acute oral toxicity test, and is suitable for being used as food, a health-care product or an auxiliary medicine for a long time.

Description

Technical Field

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

[0002] Diabetes is a metabolic disorder syndrome with high blood sugar as the main symptom. With the change of dietary structure, the global prevalence rate has increased year by year, which seriously endangers human health. The main clinical treatment methods are oral hypoglycemic drugs and exogenous insulin supplementation, but they cannot accurately regulate blood sugar, and pancreatic islet damage is irreversible. Existing treatment methods can only improve the patient's quality of life, but cannot cure diabetes. Therefore, protecting pancreatic islet cells from damage and strengthening the metabolic regulation of endogenous insulin are important means to delay the course of diabetes and improve the prognosis.

[0003] In recent years, research on the involvement of intestinal flora in metabolic diseases has provided new ideas for the intervention of diabetes. As the main component of intestinal flora, Bifidobacterium longum plays a vital role in maintaining intestinal homeostasis and body metabolism. Studies in recent years have shown that the composition and structure of the intestinal flora of diabetic patients are quite different from those of healthy people, and the progression of diabetes is related to the imbalance of Bifidobacterium longum. Bifidobacterium longum can control blood sugar by maintaining the balance of intestinal microecology and reducing oxidative stress, and has a good history of safe use and compliance. It has been included in China's "List of Bacteria that can be used in Food" and the EU's qualified presumption of safety (QPS) list.

[0004] Selenium is an essential micronutrient for human and animal health and actively participates in various physiological activities through a variety of selenoproteins. It has been 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 kidney damage in diabetic nephropathy by changing lipid content, restoring lipid ordered structure and membrane dynamics.

[0005] Therefore, it is very important to develop products of Bifidobacterium longum, selenium-enriched Bifidobacterium longum and their mixtures for use as medicines or dietary supplements to protect pancreatic cells from damage, regulate blood sugar, and prevent the occurrence and development of diabetes. 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 selenium-enriched Bifidobacterium longum agent and the use of the Bifidobacterium longum agent in the prevention of diabetes and blood sugar regulation drugs. The probiotics are selenium-enriched Bifidobacterium longum or Bifidobacterium longum agents, which can effectively resist STZ-induced hyperglycemia, delay the onset of diabetes, improve oral glucose tolerance in mice, maintain the quality of pancreatic beta cells and the islet niche, and at the same time alleviate the kidney damage caused by diabetes to a certain extent, and can also increase the abundance of beneficial bacteria in the intestinal microecology.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present application provides the use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing diabetes or regulating blood sugar;

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

[0010] The first aspect of the present application provides a use of a Bifidobacterium longum agent in the preparation of a drug for preventing diabetes or regulating blood sugar. The selenium-enriched Bifidobacterium longum agent is prepared by fermenting Bifidobacterium longum in a culture medium containing selenium, and the strain number of Bifidobacterium longum is CICC6188.

[0011] Furthermore, the Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent.

[0012] Furthermore, the long Bifidobacterium agent significantly reduced hyperglycemia induced by streptozotocin (STZ), alleviated the polydipsia and polyphagia caused by diabetes, improved the intraperitoneal glucose tolerance of mice, increased the serum insulin content of diabetic mice, and strengthened the metabolic regulation of endogenous insulin in mice.

[0013] Furthermore, Bifidobacterium longum maintains the number of pancreatic β-cell pools, reduces changes in the pancreatic niche, effectively protects pancreatic cell function, and prevents the occurrence of diabetes.

[0014] Furthermore, Bifidobacterium longum can alleviate kidney damage caused by diabetes and prevent the occurrence of diabetic complications; it can also increase the abundance of beneficial bacteria in the intestinal microecology.

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

[0016] a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter;

[0017] b. Sterilize at 121°C for 20 minutes and then cool to room temperature;

[0018] c. Add 30mL-180mL of seed solution as 1%-6% inoculum;

[0019] d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5;

[0020] e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L;

[0021] f. The fermentation was carried out for 8-10 hours, during which nitrogen was passed to drive out the air, and 30% glucose was added at a constant rate of 50 mL / h to obtain a selenium-enriched Bifidobacterium longum agent.

[0022] Furthermore, in step (e), the method for preparing the nano-selenium solution comprises the following steps:

[0023] (1) Add stabilizer to a beaker;

[0024] (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;

[0025] (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;

[0026] (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.

[0027] Furthermore, the selenium-enriched Bifidobacterium longum bacterial 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 bacterial agent is 0.5-2.9 μg / g, and the number of active colonies of selenium-enriched thermophilic Streptococcus is 1×10 9 Up to 5×10 10 CFU / g.

[0028] The second aspect of the present application provides a composition for treating diabetes, comprising a selenium-enriched Bifidobacterium longum agent and a Bifidobacterium longum agent and at least one blood sugar-lowering drug or a drug for improving pancreatic islet function.

[0029] Beneficial effects: The selenium-enriched Bifidobacterium longum agent and the Bifidobacterium longum agent of the present invention are used in the preparation of drugs for preventing diabetes or regulating blood sugar. The selenium-enriched Bifidobacterium longum agent and the Bifidobacterium longum agent of the present invention have good safety, no obvious toxicity, and are suitable for long-term use as food, health products or auxiliary drugs. The selenium-enriched Bifidobacterium longum agent has a better therapeutic effect than the Bifidobacterium longum agent. The selenium-enriched Bifidobacterium longum agent has good safety, and the acute oral toxicity test in rats proves that it is actually non-toxic.

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

[0031] Diabetes prevention and blood sugar regulation: Selenium-enriched Bifidobacterium longum preparations and Bifidobacterium longum agents can effectively resist hyperglycemia induced by streptozotocin (STZ), prevent the occurrence of diabetes and regulate blood sugar levels.

[0032] Delaying the progression of diabetes: This preparation can delay the onset of diabetes, improve the oral glucose tolerance of mice, and play a positive role in the long-term management of diabetes.

[0033] Protection of pancreatic beta cells: Selenium-enriched Bifidobacterium longum preparations and Bifidobacterium longum agents help maintain the quality and quantity of pancreatic beta cells, protect pancreatic cells from damage, prevent the occurrence of diabetes, and are crucial for improving the prognosis of diabetes.

[0034] Relief of renal damage: To a certain extent, this preparation can relieve renal damage caused by diabetes and reduce the risk of diabetic complications.

[0035] Balance of intestinal microecology: Selenium-enriched Bifidobacterium longum preparations and Bifidobacterium longum agents can increase the abundance of beneficial bacteria in the intestinal microecology, promote intestinal health, and thus have a positive impact on the body's overall metabolic health.

[0036] Safety: Bifidobacterium longum 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 Bifidobacterium longum has very good safety, and the acute oral toxicity test in rats proves that it is actually non-toxic.

[0037] Wide application: The preparation can be applied to a variety of product forms such as food, health products and medicines, providing diverse options for the prevention and treatment of diabetes.

[0038] Scientific basis: The present invention provides a scientific basis for developing diabetes-related products using selenium-enriched Bifidobacterium longum and Bifidobacterium longum, and provides theoretical support for the development of future drugs or dietary supplements.

[0039] Experimental Validation: Through a series of experimental models and evaluation methods, the beneficial effects of the present invention have been scientifically validated, enhancing its potential for application in the field of diabetes prevention and treatment.

[0040] Economic benefits: The development of diabetes prevention and treatment products based on selenium-enriched Bifidobacterium longum and Bifidobacterium longum is expected to bring significant economic benefits while improving the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the test examples of the present application, the drawings required for use in the test examples or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some test examples of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 It is a display diagram of the random blood sugar changes of mice after STZ modeling with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0043] Figure 2 It is a display diagram of the fasting blood glucose of mice after STZ modeling with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0044] Figure 3 It is a graph showing the weight changes of mice after STZ modeling with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0045] Figure 4 It is a line graph showing the weight change of mice during STZ modeling with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0046] Figure 5 It is a bar chart showing the statistical changes in the body weight of mice during the STZ modeling period with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0047] Figure 6 It is a graph showing the daily food intake of mice after STZ modeling by the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0048] Figure 7 It is a display diagram of the daily water intake of mice after STZ modeling by the selenium-enriched Bifidobacterium longum of the present invention and Bifidobacterium longum.

[0049] Figure 8 The diagram is a display of the intraperitoneal glucose tolerance of mice after STZ modeling with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0050] 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 Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0051] Fig.10 It is a display diagram of the selenium-enriched Bifidobacterium longum and the serum insulin level of mice after STZ modeling with Bifidobacterium longum of the present invention.

[0052] Fig.11 The figure shows the serum glycosylated hemoglobin levels of mice after STZ modeling with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0053] Fig.12 The figure shows the serum urea nitrogen content of mice after STZ modeling of the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0054] Fig.13 The figure shows the serum creatinine content of mice after STZ modeling with the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0055] 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 Bifidobacterium longum and Bifidobacterium longum of the present invention.

[0056] Fig.15 It is a display diagram of the statistics of the percentage of pancreatic β cells of mice after STZ modeling by the selenium-enriched Bifidobacterium longum and Bifidobacterium longum of the present invention. DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the test examples of the present invention to clearly and completely describe the technical solutions in the test examples of the present invention. Obviously, the test examples described are only part of the test examples of the present invention, not all of the test examples. Based on the test examples in the present invention, all other test examples obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] 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.

[0059] 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.

[0060] It should be understood that in the various test examples 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 test examples of the present application.

[0061] The terms used in the test examples of this application are only for the purpose of describing specific test examples, and are not intended to limit this application. The singular forms "a", "said" and "the" used in the test examples of this application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0062] The weight of the relevant components mentioned in the test example description of this application can not only refer to the specific content of each component, but also indicate 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 test example description of this application, it is within the scope disclosed in the test example description of this application. Specifically, the mass described in the test example description of this application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0063] 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 technical features indicated. For example, without departing from the scope of the test examples of this 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.

[0064] Unless otherwise specified, the reagents and materials used in the present invention are prepared by conventional methods or obtained from commercial channels.

[0065] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual points, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0066] The selenium-enriched Bifidobacterium longum agent is prepared by fermenting Bifidobacterium longum in a culture medium containing selenium. The strain number of Bifidobacterium longum is CICC6188.

[0067] In a first aspect, the present application provides a use of a Bifidobacterium longum bacterial agent in the preparation of a drug for preventing diabetes or regulating blood sugar;

[0068] A second aspect of the embodiments of the present application provides a composition for treating diabetes.

[0069] The invention discloses an application of a Bifidobacterium longum bacterial agent in preparing a medicine for preventing diabetes or regulating blood sugar.

[0070] In some embodiments, the Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent.

[0071] Bifidobacterium longum significantly reduced hyperglycemia induced by streptozotocin (STZ), alleviated the polydipsia and polyphagia caused by diabetes, improved the intraperitoneal glucose tolerance of mice, increased the serum insulin content of diabetic mice, and strengthened the metabolic regulation of endogenous insulin in mice.

[0072] Bifidobacterium longum maintains the number of pancreatic β-cell pools, reduces changes in the pancreatic niche, and effectively protects pancreatic cell function to prevent the occurrence of diabetes.

[0073] Bifidobacterium longum can relieve kidney damage caused by diabetes and prevent the occurrence of diabetic complications; it can also increase the abundance of beneficial bacteria in the intestinal microecology.

[0074] In some embodiments, the preparation method of the selenium-enriched Bifidobacterium longum agent comprises the following steps:

[0075] a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter;

[0076] b. Sterilize at 121°C for 20 minutes and then cool to room temperature;

[0077] c. Add 30mL-180mL of seed solution as 1%-6% inoculum;

[0078] d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5;

[0079] e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L;

[0080] f. Ferment for 8-10 hours, during which nitrogen is passed to drive out air, and 30% glucose is added at a constant rate of 50 mL / h to obtain a selenium-enriched long bifidobacterium agent. In terms of feeding strategy, the present invention adopts a constant rate feeding method to ensure a uniform supply of nutrients throughout the fermentation process and avoid bacterial growth inhibition or waste due to insufficient or excessive nutrition.

[0081] In some embodiments, the culture medium formulation used in the fermentation process comprises the following components:

[0082]

[0083] During the fermentation process, the selection of the culture medium is crucial. The culture medium formula used in the present invention is specially designed according to the physiological needs of Bifidobacterium longum to ensure that the bacteria can grow and reproduce under optimal conditions. In addition, the steps of pH control, temperature regulation, nitrogen gas introduction, etc. during the fermentation process are all for creating a suitable anaerobic environment, thereby promoting the growth of Bifidobacterium longum and the absorption of selenium.

[0084] In some embodiments, in step (e), the method for preparing the nano-selenium solution comprises the following steps:

[0085] (1) Add stabilizer to a beaker;

[0086] (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;

[0087] (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;

[0088] (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.

[0089] In some embodiments, in step (1), the stabilizer is epigallocatechin gallate (EGCG).

[0090] In some embodiments, in step (1), the stabilizer is chitosan.

[0091] In some embodiments, in step (1), the stabilizer is gum arabic.

[0092] In some embodiments, in step (1), the stabilizer is bovine serum albumin (BSA).

[0093] In some embodiments, in step (1), the stabilizer is sodium hyaluronate.

[0094] In some embodiments, in step (2), the reducing agent is reduced glutathione GSH.

[0095] In some embodiments, in step (2), the reducing agent is vitamin C (Vc).

[0096] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.1 mM to 40 mM (for EGCG).

[0097] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.01% to 0.7% (for chitosan).

[0098] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.04% to 0.4% (for gum arabic).

[0099] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.4 mg / ml to 60 mg / ml (for BSA).

[0100] In some embodiments, in step (1), the concentration of the stabilizer ranges from 0.02% to 0.25% (for sodium hyaluronate).

[0101] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-50 mM (GSH).

[0102] In some embodiments, in step (2), the final concentration of the reducing agent in the reaction system is 5 mM-100 mM (Vc).

[0103] In step (3), the final concentration of sodium selenite in the reaction system is 5 mM.

[0104] In some embodiments, in step (1), the molecular weight of the stabilizer is in the range of 100 KD - 1000 KD (for chitosan).

[0105] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200 KD to 2000 KD (for gum arabic).

[0106] In some embodiments, in step (1), the molecular weight of the stabilizer ranges from 200 KD to 2000 KD (for sodium hyaluronate).

[0107] In step (4), in the step of adjusting the pH, the pH value ranges from 5.0 to 9.0.

[0108] 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.

[0109] The selenium-enriched long bifidobacterium bacterial 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 bacterial agent is 0.5-2.9 μg / g, and the number of active colonies of selenium-enriched thermophilic streptococci is 1×10 9 Up to 5×10 10 CFU / g.

[0110] In some embodiments, the selenium-enriched Bifidobacterium longum agent is in the form of a tablet.

[0111] In some embodiments, the selenium-enriched Bifidobacterium longum agent is a freeze-dried powder.

[0112] In some embodiments, the selenium-enriched Bifidobacterium longum bacterial agent is in the form of a capsule.

[0113] In some embodiments, the selenium-enriched Bifidobacterium longum agent is a dairy product.

[0114] In some embodiments, the selenium-enriched Bifidobacterium longum agent is stirred yogurt.

[0115] In some embodiments, the selenium-enriched Bifidobacterium longum bacterial agent is in the form of solidified yogurt.

[0116] The composition for treating diabetes of the present invention comprises a selenium-enriched Bifidobacterium longum agent and a Bifidobacterium longum agent and at least one blood sugar lowering drug or a drug for improving pancreatic islet function.

[0117] Example 1

[0118] C57BL / 6J male mice were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.; experimental mouse maintenance feed was purchased from Synergy Bio; streptozotocin (STZ) was purchased from Sigma, USA; trisodium citrate dihydrate and citric acid monohydrate were purchased from Nanjing Chemical Reagent Co., Ltd.; D(+)-glucose was purchased from Sinopharm Chemical Reagent Co., Ltd.; mouse insulin Elisa kit was purchased from Wuhan Yunclone Technology Co., Ltd.; mouse glycosylated hemoglobin Elisa kit was purchased from Nanjing Jiancheng Bioengineering Institute; blood glucose meter and blood glucose test strips were purchased from Roche; mouse insulin antibody was purchased from Abcam, USA; rabbit glucagon antibody was purchased from Cell Signal Technology, USA; Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 594-labeled goat anti-mouse IgG were purchased from Wuhan Sevier Biotechnology Co., Ltd.; immunohistochemistry pen, immunohistochemistry humidification box, and goat serum were purchased from Beijing Solebao Company.

[0119] Preparation and administration of Bifidobacterium longum and selenium-enriched Bifidobacterium longum

[0120] The probiotic freeze-dried powder was resuspended in PBS buffer to obtain a probiotic agent. The number of viable bacteria per daily dose was CFU, the mice were divided into 4 groups, namely the control group, the model group, the model group treated with Bifidobacterium longum, and the model group treated with selenium-enriched Bifidobacterium longum, 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.

[0121] Test Example 1

[0122] Effects of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on random blood glucose changes in mice after STZ modeling

[0123] Model construction: SPF mice were used, the relative humidity of the animal room was 55±5%, the temperature was 22±2℃, the mice were free to drink water and feed, and 4-week-old C57BL / 6J male mice were adaptively fed for one week before the probiotics were administered for 4 weeks. Multiple injections of low-dose STZ (35mg / kg, continuous modeling for 5 days) were used to induce diabetes. 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.

[0124] Monitor the random blood glucose of mice every week: 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.

[0125] Depend on Figure 1 It can be seen that probiotics had no significant effect on the blood sugar of mice when administered orally, and the mice were in a healthy state. After STZ-induced diabetes, the random blood sugar of mice increased over time, with the model group increasing the fastest. After 8 weeks of modeling, the average blood sugar was higher than 30mmol / L. Preventive administration of probiotics Bifidobacterium longum and selenium-enriched Bifidobacterium longum can significantly alleviate the hyperglycemia symptoms of mice and delay the increase of blood sugar. Among them, the blood sugar lowering rate of selenium-enriched Bifidobacterium longum was significantly better than that of Bifidobacterium longum, and the average blood sugar was lower than 20mmol / L after 8 weeks of modeling.

[0126] Test Example 2

[0127] Effect of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on fasting blood glucose in mice at 4 weeks after STZ modeling

[0128] Since random blood glucose levels fluctuate due to the daily food intake and activities of mice, the fasting blood glucose of mice was tested in the fourth week after modeling to determine the progression of diabetes in mice. The fasting blood glucose of mice was measured and recorded after fasting for 12 hours but not water. Figure 2 It 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 the other groups. Bifidobacterium longum and selenium-enriched Bifidobacterium longum 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 selenium-enriched Bifidobacterium longum was similar to that of the control group, indicating that selenium-enriched Bifidobacterium longum has a good effect on regulating blood sugar.

[0129] Test Example 3

[0130] Effects of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on body weight changes in mice after STZ modeling

[0131] Depend on Figure 3It can be seen that there was no significant effect on the weight of mice during the oral administration of probiotics. The weight of mice increased steadily and there was no significant difference with the control group. It can be inferred that the dosage was not significantly toxic. After STZ-induced diabetes, the weight of mice showed a significant downward trend, among which the model group mice lost the most weight and lasted for 3 weeks. The weight of mice intervened by probiotics was less, among which the weight of mice in the selenium-enriched Bifidobacterium longum group was basically stable and slowly increased, and the mice were in better condition, indicating that they can better resist the weight changes caused by diabetes.

[0132] Test Example 4

[0133] Effects of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on body weight changes in mice during STZ modeling

[0134] 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 citrate buffer continued to increase, while the weight of the modeling group decreased. Selenium-enriched Bifidobacterium longum can effectively resist the damage and weight changes caused by STZ modeling.

[0135] Test Example 5

[0136] Food intake and water intake of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum in mice after STZ modeling

[0137] 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 a phenotype of polydipsia and polyphagia, and both Bifidobacterium longum and selenium-enriched Bifidobacterium longum could significantly alleviate the above symptoms of the mice, and the diabetic symptoms of the mice were milder.

[0138] Test Example 6

[0139] Effect of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on peritoneal glucose tolerance in mice after STZ modeling

[0140] 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 8It can be seen that at 0 minutes after fasting, the blood glucose level of mice in the model group was the highest, and the blood glucose levels of mice in the long bifidobacterium and selenium-enriched long bifidobacterium probiotic groups were between the control group and the model group. The blood glucose level of mice in the control group reached a peak at 15 minutes, and then continued to decline. At 120 minutes after the injection of glucose, the blood glucose level was maintained at about 10mmol / L. The blood glucose level of mice in the model group was always at a high level, and it was still greater than 20mmol / L 120 minutes after the injection of glucose. The blood glucose level of mice in the long bifidobacterium and selenium-enriched long bifidobacterium probiotic groups decreased over time, among which the blood glucose level of mice in the long bifidobacterium and selenium-enriched long bifidobacterium probiotic groups decreased more significantly, and was close to that of the control group 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 administration of selenium-enriched Bifidobacterium longum could effectively maintain pancreatic islet β function and improve glucose responsiveness.

[0141] Test Example 7

[0142] Changes of serum insulin levels in mice with Bifidobacterium longum and Selenium-enriched Bifidobacterium longum after STZ modeling

[0143] 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 of the model group decreased by more than 50%, the insulin content of the mice in the Bifidobacterium longum and selenium-enriched Bifidobacterium longum probiotic groups was higher than that in the model group, and the insulin level of the selenium-enriched Bifidobacterium longum group was higher than that of Bifidobacterium longum, indicating that selenium-enriched Bifidobacterium longum can effectively resist STZ-induced β-cell damage, maintain the body's insulin synthesis and secretion, and can effectively regulate blood sugar.

[0144] Test Example 8

[0145] Effects of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on the changes of glycosylated hemoglobin levels in mice after STZ modeling

[0146] Depend on Fig.11 It can be seen that 8 weeks after modeling, the level of glycated hemoglobin in mouse serum was detected by enzyme-linked immunosorbent assay according to the instructions. The glycated hemoglobin content of mice in the model group was as high as 10mmol / L, while that of mice in the selenium-enriched Bifidobacterium longum group was only about 5mmol / L, indicating that selenium-enriched Bifidobacterium longum 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.

[0147] Test Example 9

[0148] Effects of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on early diabetic kidney injury in mice after STZ modeling

[0149] 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 the mice in the model group increased, indicating that the mice had renal damage caused by diabetes. Intervention with Bifidobacterium longum and selenium-enriched Bifidobacterium longum probiotics can effectively improve the early diabetic kidney damage in mice and prevent the occurrence of this complication.

[0150] Test Example 10

[0151] Effects of Bifidobacterium longum and Selenium-enriched Bifidobacterium longum on pancreatic islet ecology in mice after STZ modeling

[0152] In order to observe the changes in the pancreatic islets in situ of mice, the pancreas of mice was paraffin-embedded and sliced, and immunofluorescence double 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 immunohistochemical cylinder, 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 immunohistochemical cylinder 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.

[0153] Depend on Fig.14 It can be seen that more than 90% of the pancreatic islets in the control group were β cells, and α cells were distributed around the islet cells, while the model group lost a large number of β cells, the proportion of α cells increased significantly, and the location of α cells moved to the center of the islets. The administration of Bifidobacterium longum and selenium-enriched Bifidobacterium longum probiotics can effectively maintain the normal ecological niche of the mouse β cell pool and pancreatic islets, and prevent the occurrence and development of diabetes. Fig.15 Statistical results show that selenium-enriched Bifidobacterium longum has better effects as mentioned above and a higher percentage of β cells.

[0154] Test Example 11

[0155] Acute oral toxicity test of selenium-enriched Bifidobacterium longum

[0156] 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.

[0157] 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.

[0158] Main instruments: JJ10 electronic balance, T1000 electronic balance.

[0159] Test method: The acute toxicity test in rats was carried out using the limit method.

[0160] 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 nano-selenium-enriched Bifidobacterium longum on the body weight and mortality of rats are shown in Table 1.

[0161] Table 1 Effects of selenium-enriched Bifidobacterium longum on acute oral toxicity in rats

[0162]

[0163] After oral administration of nano-selenium-enriched Bifidobacterium longum to animals, no obvious signs of poisoning were observed, and there was no significant effect on the weight gain of animals. No animals died during the observation period. Acute oral toxicity test of nano-selenium-enriched Bifidobacterium longum 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.

[0164] In summary, Bifidobacterium longum and selenium-enriched Bifidobacterium longum can effectively resist STZ-induced diabetes, significantly improve the symptoms of high blood sugar levels in mice, and reduce the "three more and one less" phenotype of mice. Effectively maintain the number and function of pancreatic beta cells, and the mouse pancreatic islet glucose responsiveness is better. At the same time, it can improve the insulin level of mice, reduce the level of glycosylated hemoglobin in mice, delay the development of diabetes, and reduce the early diabetic kidney damage in mice. As a commonly used dairy fermentation bacteria, a large amount of research data in various fields show that Bifidobacterium longum is safe. And Bifidobacterium longum has a good history of safe use and compliance, and has been included in China's "List of Food Bacteria" and the EU Safety Qualification (QPS) list, indicating that its safety is recognized. The data of the present invention show that there is no obvious fluctuation in the blood sugar of mice when probiotics are gavaged, and the random blood sugar value is less than 11.1mmol / L, which is in the normal range. At the same time, the weight of mice increases steadily during gavage, and there is no significant difference with the control group. It can be inferred that the dosage has no obvious toxicity. The results of the acute oral toxicity test of selenium-enriched Bifidobacterium longum also showed that the acute oral toxicity test of nano-selenium-enriched Bifidobacterium longum in female and male rats LD 50 Greater than 10000 mg / kg·bw, it is practically non-toxic.

[0165] In addition, Lin et al. continuously administered 3×10 10 The CFU selenium-enriched Bifidobacterium longum preparation was subjected to a safety study for 8 weeks, and the results showed that no toxicity and adverse reactions would occur. This dosage was 100 times the dosage of the present invention, which proved that the dosage used in the present invention had good safety. This shows that the selenium-enriched Bifidobacterium longum preparation in the present invention can effectively slow down the progression of diabetes to a certain extent without adverse reactions, and has a good blood sugar regulating effect.

[0166] It should be noted that compared with the diabetic model group mice, both Bifidobacterium longum and Selenium-enriched Bifidobacterium longum have the effect of regulating blood sugar. At the same dosage concentration, Selenium-enriched Bifidobacterium longum has a stronger effect on alleviating STZ-induced hyperglycemia than Bifidobacterium longum, showing good anti-diabetic efficacy. It can effectively delay the occurrence and development of diabetes, improve oral glucose tolerance in mice, maintain the quality of pancreatic β cells and pancreatic islet niche, and show good pancreatic β cell protection. At the same time, it alleviates diabetic kidney damage to a certain extent. In addition, compared with the role of related Selenium-enriched Thermophilus Streptococcus in regulating blood sugar, Selenium-enriched Bifidobacterium longum showed a stronger hypoglycemic effect. At the 8th week after modeling, the average blood sugar of mice in the Selenium-enriched Thermophilus Streptococcus group was 22.61mmol / L, while the average blood sugar of mice in the Selenium-enriched Bifidobacterium longum group was 20.41mmol / L.

[0167] It can be known from the above technical scheme that the present invention provides the application of selenium-enriched long bifidobacterium preparation and long bifidobacterium preparation in preventing diabetes and regulating blood sugar, which can significantly reduce STZ-induced hyperglycemia, alleviate the polydipsia and polyphagia caused by diabetes, improve mouse peritoneal glucose tolerance, improve the serum insulin content of diabetic mice, and strengthen the endogenous insulin metabolism regulation of mice. Maintain the number of pancreatic islet β cell pools at the same time, reduce the change of islet niche, and effectively protect the function of islet cells. And to a certain extent, alleviate the renal damage caused by diabetes, it is also possible to increase the abundance of beneficial bacteria in the intestinal microecology, and have the potential for application in preventing diabetes and regulating blood sugar. In summary, this is to develop drugs or dietary supplements using selenium-enriched long bifidobacterium and long bifidobacterium preparations to protect islet cells from damage and prevent the occurrence and development of diabetes and provide theoretical reference and guiding basis.

[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 by the above test examples, and the above test examples 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 Bifidobacterium longum bacterial 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 Bifidobacterium longum bacterial agent is a selenium-enriched Bifidobacterium longum bacterial agent.

3. The use according to claim 2, characterized in that: The Bifidobacterium longum bacterial agent significantly reduces the hyperglycemia induced by streptozotocin (STZ), alleviates the polydipsia and polyphagia caused by diabetes, improves the intraperitoneal glucose tolerance of mice, increases the serum insulin content of diabetic mice, and strengthens the metabolic regulation of endogenous insulin in mice.

4. The use according to claim 3, characterized in that: The Bifidobacterium longum bacterial agent maintains the number of pancreatic beta cell pools, reduces changes in the pancreatic islet niche, effectively protects pancreatic islet cell functions, and prevents the occurrence of diabetes.

5. The use according to claim 3, characterized in that: The Bifidobacterium longum bacterial agent can alleviate kidney damage caused by diabetes and prevent the occurrence of diabetic complications; it can also increase the abundance of beneficial bacteria in the intestinal microecology.

6. The use according to claim 2, characterized in that: The preparation method of the selenium-enriched Bifidobacterium longum agent comprises the following steps: a. Prepare 3L culture medium, adjust pH to 5.5-7.0, and load into 5L fermenter; b. Sterilize at 121°C for 20 minutes and then cool to room temperature; c. Add 30mL-180mL of seed solution as 1%-6% inoculum; d. Use 20% NaOH solution to control the fermentation pH to 5.5-6.5; e. Add the filtered and sterilized nano-selenium solution to a final concentration of 20-50 mg / L; f. The fermentation was carried out for 8-10 hours, during which nitrogen was passed to drive out the air, and 30% glucose was added at a constant rate of 50 mL / h to obtain a selenium-enriched Bifidobacterium longum agent.

7. The use according to claim 6, characterized in that: In step (e), the method for preparing the nano-selenium solution comprises the following steps: (1) Add stabilizer to a beaker; (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; (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.

8. The use according to claim 7, characterized in that: The selenium-enriched long bifidobacterium bacterial 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 bacterial 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.

9. A composition for treating diabetes, characterized in that: The invention comprises the selenium-enriched Bifidobacterium longum agent and the Bifidobacterium longum agent as claimed in claim 1 and at least one blood sugar lowering drug or a drug for improving pancreatic islet function.

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