A compound probiotic with metabolic regulation function and its application

Abstract

The present invention relates to a compound probiotic with metabolic regulation function, and the compound probiotic comprises Bifidobacterium animalis subsp. with the preservation number of GDMCC No:64543 Bifidobacterium animalis subsp. lactis C-1 strain and Lactobacillus fermentum with the preservation number of CGMCC No.28336 Lactobacillus fermentum GLF-217 strain. There is a potential interaction between the two strains, which can cooperate with each other and synergistically enhance the effect. The compound of the two strains significantly improves the effect in regulating metabolism, specifically manifested in: regulating body weight and body fat level, regulating blood glucose and blood lipid level, improving the level of inflammatory factors, relieving liver injury and improving the degree of intestinal flora disorder, and can provide coping strategies and reference basis for the intervention of various symptoms caused by metabolic disorders.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and relates to a compound probiotic with a metabolic regulation function and its application. Background Art

[0002] High-fat foods refer to foods with a high fat content. Specifically, the oil component is various saturated and unsaturated fatty acids. For example, foods with a high oil content and fried foods, such as walnuts, sesame seeds, peanuts, fried foods, fatty meat, animal offal, and dairy products. Long-term high-fat diet will cause the body to intake excessive calories. When these calories exceed the daily consumption, the excess calories will be converted into fat and stored in the body, resulting in weight gain and obesity. Persistent obesity is likely to cause hyperlipidemia and other complications, such as diabetes, inflammation, lipid metabolism disorders, atherosclerosis, and fatty liver.

[0003] These obesity-related diseases often lead to an increase in the levels of total serum cholesterol (TC) and triglycerides (TG). The increase in the total serum cholesterol level may be related to the decrease in insulin secretion ability. In addition, the increase in triglycerides will increase the risk of diabetes. It will also affect the expression of related inflammatory factors such as TNF-α and IL-6. These two inflammatory factors can promote the synthesis and secretion of liver triglycerides, inhibit the activity of lipoprotein lipase, resulting in lipid metabolism disorders. Lipid metabolism disorders are an important cause of cardiovascular and cerebrovascular diseases and liver damage. These diseases may also accelerate the occurrence and development of atherosclerosis and liver function impairment. After the liver function is damaged, the function of regulating blood sugar and lipid levels becomes weak, resulting in a large accumulation of fat in the liver, seriously damaging liver function, and further inducing the occurrence and development of liver diseases.

[0004] Probiotics are considered a type of bacteria that can play a beneficial role in improving the host intestinal microecological balance, enhancing the host health level and health status. They mainly affect the host health by regulating the intestinal microbiota and participating in the immune regulation of various diseases, thereby improving the gastrointestinal physiological function and the host metabolism. There is also a close relationship between obesity and the intestinal flora. Research shows that the intestinal flora diversity of obese people is usually lower than that of normal weight and body fat people. The intestinal flora may also relieve related symptoms by regulating the intestinal barrier function, reducing the level of inflammatory factors, regulating lipid metabolism, regulating the composition of indigenous symbiotic bacteria, or directly colonizing in the gastrointestinal tract. Therefore, how to provide a product containing probiotics that can efficiently regulate the metabolic level has important significance and application value. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a compound probiotic with a metabolic regulation function and its application.

[0006] To achieve the object of the present invention, the following technical solutions are adopted in the present invention:

[0007] In a first aspect, the present invention provides a compound probiotic having a metabolic regulation effect, and the compound probiotic includes Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis C-1 strain with the preservation number of GDMCC No: 64543 and Lactobacillus fermentum Lactobacillus fermentum GLF-217 strain with the preservation number of CGMCC No. 28336.

[0008] The present invention has developed a completely new probiotic compounding method and a completely new strategy for regulating the metabolic level, that is, Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis C-1 strain and Lactobacillus fermentum Lactobacillus fermentum GLF-217 strain are compounded, and it is found that there is a potential interaction between the two strains, which can cooperate with each other and synergistically enhance the effect. When the amount of bacteria used is the same, compared with the strain intervention method lacking any one of the bacteria, the compounding of the two bacteria significantly improves the effect in regulating metabolism, specifically manifested in: (1) regulating body weight and body fat levels; (2) improving blood glucose and blood lipid levels; (3) improving inflammatory factor levels; (4) relieving liver damage; (5) improving the degree of intestinal flora disorder. At the same time, both bacteria are probiotics, so when used in the preparation of related efficacy products, they have high safety and are not prone to dependence.

[0009] Preferably, the viable count ratio of the C-1 strain to the GLF-217 strain is 1:10 - 10:1, for example, it can be 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 5:1, 6:1, 8:1, 10:1, etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0010] Based on the potential interaction relationship between the two strains, the present invention also finds that when the two strains are compounded and used in the above specific viable count ratio, the efficacy in regulating the metabolic level is more significant.

[0011] In a second aspect, the present invention provides a probiotic agent having a metabolic regulation effect, and the strains in the probiotic agent include the compound probiotic described in the first aspect.

[0012] Preferably, the total viable count in the probiotic agent is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, for example, 1×10 9CFU / mL (CFU / g), 2×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 8×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 5×10 10 CFU / mL (CFU / g), 1×10 11 CFU / mL (CFU / g), etc. Other specific point values within this numerical range can be selected and will not be elaborated one by one here.

[0013] Preferably, the dosage form of the probiotic agent includes solution, lyophilized powder, capsule, tablet or granule.

[0014] Preferably, the probiotic agent further includes a protective agent.

[0015] Preferably, the protective agent includes any one or a combination of at least two of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, arabic gum, sodium alginate, polyvinylpyrrolidone, sorbitol, xylo-oligosaccharide, fructo-oligosaccharide or xylitol.

[0016] Preferably, the dosage form of the probiotic agent is a solution, which is prepared by the following method:

[0017] The C-1 strain and the GLF-217 strain are respectively inoculated into a culture medium and sequentially activated and fermented to obtain a fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain a C-1 bacterial suspension and a GLF-217 bacterial suspension, and the C-1 bacterial suspension and the GLF-217 bacterial suspension are mixed according to the viable count ratio to obtain the probiotic agent.

[0018] Preferably, the dosage form of the probiotic agent is a lyophilized powder, which is prepared by the following method:

[0019] The C-1 strain and the GLF-217 strain are respectively inoculated into a culture medium and sequentially activated and fermented to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and then freeze-dried to obtain C-1 bacterial powder and GLF-217 bacterial powder, and the C-1 bacterial powder and the GLF-217 bacterial powder are mixed according to the viable count ratio to obtain the probiotic agent.

[0020] In a third aspect, the present invention provides an application of the composite probiotic according to the first aspect or the probiotic agent according to the second aspect in the preparation of a preparation for regulating liver metabolism level.

[0021] Preferably, the preparation further includes a pharmaceutically acceptable excipient.

[0022] Preferably, the excipients include any one or a combination of at least two of fillers, pH regulators, antioxidants, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, bacteriostatic agents or buffers.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention has developed a brand-new probiotic compounding method and a brand-new strategy for regulating metabolic levels, that is, compounding Bifidobacterium animalis subsp. Bifidobacterium animalis subsp. lactis C-1 strain and Lactobacillus fermentum Lactobacillus fermentum GLF-217 strain, and it is found that there is a potential interaction between the two strains, which can cooperate with each other and synergistically enhance the effect. When the amount of bacteria used is the same, compared with the strain intervention method lacking any one of the bacteria, the compounding of the two bacteria significantly improves the effect in regulating metabolism, specifically manifested in: (1) regulating body weight and body fat levels; (2) improving blood glucose and blood lipid levels; (3) improving inflammatory factor levels; (4) relieving liver damage; (5) improving the degree of intestinal flora disorder. At the same time, both bacteria are probiotics, so when used in the preparation of related efficacy products, they have high safety and are not prone to dependence.

[0025] The taxonomic name of the C-1 strain involved in the present invention is Bifidobacterium animalis subsp. lactis , the preservation unit is the Guangdong Provincial Microbial Culture Collection Center, the preservation number is GDMCC No: 64543, the preservation date is April 22, 2024, and the preservation address is the 5th floor of Building 59, No. 100, Middle Xianlie Road, Guangzhou;

[0026] The taxonomic name of the Lactobacillus fermentum GLF-217 strain involved in the present invention is Lactobacillus fermentum Lactobacillus fermentum , the preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC No. 28336, the preservation date is September 4, 2023, and the preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a statistical result chart of the measured body weight and total fat content of mice in each group;

[0028] Figure 2 It is a statistical result chart of the levels of TC, HDL-C, GLU, ALT, LDL-C and NEFA in the serum of mice in each group;

[0029] Figure 3It is a statistical result graph of the contents of pro-inflammatory factors TNF-α and IL-6 in the sera of mice in each group;

[0030] Figure 4 It is a statistical result graph of the metabolic levels of adipose tissues of mice in each group. Specific implementation manners

[0031] The technical solution of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0032] The information of the bacterial strains involved below is as follows:

[0033] ① The taxonomic nomenclature of the C-1 strain involved below is Bifidobacterium animalis subsp. lactis , and the deposit number is GDMCC No: 64543;

[0034] ② The taxonomic nomenclature of the GLF-217 strain involved below is Lactobacillus fermentum Lactobacillus fermentum , and the deposit number is CGMCC No. 28336;

[0035] ③ The CICC 24210 strain involved below is Bifidobacterium animalis subsp. lactis CICC 24210.

[0036] The C57BL / 6J male mice (8 weeks old) involved below are from SPF Biotechnology Co., Ltd.

[0037] The culture media and their formulations involved below are as follows:

[0038] MRS culture medium: beef extract 10.0 g, glucose 20.0 g, peptone 10.0 g, yeast extract 5.0 g, sodium chloride 5.0 g, diammonium citrate 2.0 g, dipotassium hydrogen phosphate 2.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.05 g, Tween-80 1.0 g, made up to 1000 mL with distilled water, pH 6.2 - 6.4.

[0039] The main components of the mouse food formula involved below are (by mass percentage):

[0040] Regular feed: corn 43.8%, wheat middlings 12%, wheat 17%, salted soybean meal 7%, Peruvian fish meal 4%, and American muscle 5%; the main nutritional components are: crude protein 19.54%, crude fat 4.43%, crude fiber 2.14%, calcium 1.36%, and phosphorus 0.86%.

[0041] High-fat diet: Maintain basal diet at 48.1%, lard at 15%, sucrose at 20%, casein at 10%, and experimental animal premix at 2%; The main nutritional components are: protein 19.379%, fat 17.133%, crude fiber 3.321%, ash 5.3114%, calcium 1.26735%, and phosphorus 0.82223%.

[0042] The preparation methods of the bacterial suspensions and bacterial powders involved below are as follows: After activating the strains, they are respectively inoculated into the culture medium for cultivation to obtain the culture solution; The culture solution is centrifuged, and the bacterial cells are resuspended to obtain the bacterial suspension, or further, a cryoprotectant is added for freeze-drying to obtain the freeze-dried bacterial powder product. Example 1

[0043] This example explores the effect of the composite probiotics on improving various indicators of high-fat diet mice.

[0044] (1) Animal grouping, modeling, and intervention methods

[0045] C57BL / 6J male mice (8 weeks old) after one week of adaptive feeding are randomly divided into 8 groups: Group S1 (C-1 bacterial suspension), Group S2 (GLF-217 bacterial suspension), Group S3 (C-1 bacterial suspension + GLF-217 bacterial suspension, viable cell ratio 10:1), Group S4 (C-1 bacterial suspension + GLF-217 bacterial suspension, viable cell ratio 1:1), Group S5 (C-1 bacterial suspension + GLF-217 bacterial suspension, viable cell ratio 1:10), Group S6 (CICC 24210 bacterial suspension + GLF-217 bacterial suspension, viable cell ratio 10:1), Group S7 is the model group, and Group S8 is the blank group. There are 8 mice in each group. The total viable bacteria in each of Groups S1-S6 are 1×10 9 CFU / mL.

[0046] Intervention method: During the experiment, mice in each group are allowed to eat and drink freely.

[0047] Mice in Groups S1-S7 are fed a high-fat mouse diet, and mice in Group S8 are fed a normal mouse diet. Groups S1-S6 are respectively gavaged with 200 μL of the corresponding bacterial suspension, and Groups S7-S8 are respectively gavaged with an equal volume of sterile PBS solution, once a day, 200 μL each time, and the experiment lasts for 6 weeks.

[0048] (2) Body weight and fat content levels of mice

[0049] During the experiment, the body weight of mice is monitored every 7 days, and the body weight of mice in each group is statistically analyzed after the experiment ends; After the experiment ends, the mice are sacrificed, and white fat (epididymal fat, perirenal fat, inguinal fat) is collected after dissection for weighing, and the total fat (white fat) content is statistically analyzed. The results are all as Figure 1 shown.

[0050] As can be seen from the statistical results in the figure, at the sixth week, the average body weight (185.74 g) and fat content (11.345 g) of the mice in group S7 (model group) were both higher than those of the mice in group S8 (control group), with the average body weight being 145.68 g and the fat content being 6.239 g, which proved the success of the modeling. After the intervention with probiotics, the body weight and fat content of the mice in groups S3, S4, and S5 were lower than those in group S7, indicating that the composite probiotics involved in the present invention, which contain a combination of two specific bacteria, namely C-1 bacteria and GLF-217 strains, can effectively delay the weight gain and lipid accumulation caused by a high-fat diet.

[0051] (3)Blood glucose and lipid levels

[0052] After the experiment ended, the mouse serum was centrifuged at 571 g for 15 min at 4 °C, and the content levels of total cholesterol (TC), high-density lipoprotein (HDL-C), glucose (GLU), alanine aminotransferase (ALT), low-density lipoprotein (LDL-C), and non-esterified fatty acids (NEFA) in the serum were measured with reference to the instructions of the ELISA kit. The results are as Figure 2 shown.

[0053] Persistent high-fat diet can cause disorders in the blood glucose and lipid metabolism levels, leading to the occurrence of blood lipid disorders such as hyperlipidemia. As Figure 2 can be seen, after 6 weeks of the experiment, the content levels of TC (17.117 mmol / L), ALT (165.631 U / L), NEFA (3.093 mmol / L), LDL-C (7.624 mmol / L), and GLU (8.834 mmol / L) in the serum samples of the mice in group S7 (model group) were all significantly higher than those in group S8 (control group, TC: 4.938 mmol / L; ALT: 70.379 U / L; NEFA: 0.778 mmol / L; LDL-C: 1.966 mmol / L; GLU: 6.678 mmol / L). The HDL-C (3.756 mmol / L) in group S8 (control group) was higher than that in group S7 (model group) (3.064 mmol / L). After the intervention with probiotics, the TC, ALT, NEFA, LDL-C, and GLU in the serum of the mice in groups S3, S4, and S5 were lower than those in group S8, and the HDL-C levels in the three groups of S3, S4, and S5 were much higher than those in the model group and the control group. It can be seen that the composite probiotics involved in the present invention have a better effect on regulating and improving blood lipid and blood glucose levels, and this regulatory effect helps to reduce the risk of coronary heart disease and other atherosclerosis-related diseases.

[0054] (4)Immune level

[0055] After the experiment, mouse serum was taken and centrifuged at 571 g for 15 min at 4°C. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum were measured according to the ELISA kit instructions. The experimental results were analyzed by data processing software, and the results are as Figure 3 shown.

[0056] TNF-α and IL-6 are two important cytokines in the immune response and are two biomarkers related to inflammation. Among them, TNF-α can not only directly drive the expression of inflammatory genes, but also indirectly promote cell death, participate in inflammatory immune responses and disease development, etc.; IL-6 is one of the main mediators of the acute-phase response and participates in the initiation and regulation of inflammatory responses. Persistent high-fat diet often triggers an inflammatory response in adipose tissue, resulting in the excessive release of related pro-inflammatory factors in the serum and a continuous increase in levels. It can be seen from the detection results in the figure that after intervention with probiotics, the levels of TNF-α and IL-6 pro-inflammatory factors in the serum of mice in groups S3, S4, and S5 were significantly lower than those in group S7 (model group), indicating that the composite probiotics provided by the present invention showed effective anti-inflammatory effects, could effectively relieve cell inflammation, enhance the immune defense ability under high-fat diet, and thus reduce the inflammatory level caused by high-fat diet.

[0057] (5)Adipose tissue metabolic level

[0058] After the experiment, the mice were sacrificed and white adipose tissues (epididymal fat, perirenal fat, inguinal fat) were collected. After homogenization, grinding, and centrifugation, the supernatant was collected, and the resistin and leptin indexes were tested according to the kit instructions. The results are as Figure 4 shown.

[0059] Leptin and resistin are two important adipokines. They are produced in adipose tissue and act in the brain to affect metabolic functions. Among them, the role of leptin is to balance energy and regulate appetite, thereby regulating metabolic levels, improving insulin resistance, and enhancing fat metabolism, etc.; resistin is also an adipocytokine closely related to insulin resistance. Persistent high-fat diet usually leads to an increase in resistin levels, which may interfere with the insulin signaling pathway, reduce the sensitivity of mice to insulin, and thus cause metabolic problems such as hyperglycemia and insulin resistance.

[0060] As can be seen from the figure, after induction by a high-fat diet, the resistin level (15.809 μg / L) in the S7 (model group) was significantly higher than that in the S8 (control group) (13.623 μg / L), and the leptin level (6.271 μg / L) in the S7 (model group) was significantly lower than that in the S8 (control group) (7.904 μg / L). After intervention with probiotics, the resistin levels in the three groups of S3, S4, and S5 were significantly lower than those in the model group, and the leptin levels increased. It can be seen that the composite probiotics provided by the present invention have a good effect on regulating the adipose tissue metabolism of a high-fat diet, reducing the fat accumulation in adipose tissue caused by a high-fat diet, thereby improving insulin resistance and increasing the fat metabolism level.

[0061] In summary, the composite probiotics involved in the present invention have excellent metabolic regulation effects. When the C-1 strain and the GLF-217 strain are compounded, it is found that there is a potential interaction between the two strains, which can cooperate with each other and synergistically enhance the effect. When the amount of bacteria used is the same, compared with the strain intervention method lacking any one of the bacteria, the compounding of the two bacteria significantly improves the effect in regulating metabolism, specifically manifested in: (1) regulating body weight and body fat levels; (2) regulating blood glucose and blood lipid levels; (3) improving the levels of inflammatory factors; (4) alleviating liver damage; (5) improving the degree of intestinal flora disorder. At the same time, both bacteria are probiotics, so when they are used to prepare related efficacy products, they have high safety and are not prone to dependence.

[0062] The applicant declares that the present invention uses the above embodiments to illustrate the technical solutions of the present invention, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0063] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0064] In addition, it should be noted that in the above specific embodiments, the various specific technical features described can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A composite probiotic with metabolic regulation effect, characterized in that: The composite probiotics are prepared from Bifidobacterium animalis subsp. lactis (GDMCC No. 64543). Bifidobacterium animalis subsp. lactis ) C-1 strain and Lactobacillus fermentum with a preservation number of CGMCC No. 28336 ( Lactobacillus fermentum ) GLF-217 strain composition.

2. The composite probiotic with metabolic regulation effect according to claim 1, characterized in that: The ratio of the viable counts of the C-1 strain and the GLF-217 strain is 1:10-10:

1.

3. A probiotic with metabolic regulation effect, characterized in that: The strains in the probiotics include the composite probiotics according to claim 1 or 2.

4. The probiotic agent with metabolic regulation effect according to claim 3, characterized in that: The total live bacteria content in the probiotics is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

5. The probiotic agent with metabolic regulation effect according to claim 3, characterized in that: The dosage form of the probiotics includes solution, lyophilized powder, capsule, tablet or granule.

6. The probiotic agent with metabolic regulation effect according to claim 3, characterized in that: The probiotics also include a protective agent.

7. The probiotic agent with metabolic regulation effect according to claim 6, characterized in that: The protective agent includes any one of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinyl pyrrolidone, sorbitol, xylo-oligosaccharide, fructo-oligosaccharide or xylitol, or a combination of at least two thereof.

8. The probiotic agent with metabolic regulation effect according to claim 5, characterized in that: The dosage form of the probiotic is a solution, which is prepared by the following method: The C-1 strain and the GLF-217 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged respectively and resuspended with a solvent to obtain a C-1 bacterial suspension and a GLF-217 bacterial suspension; the C-1 bacterial suspension and the GLF-217 bacterial suspension are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

9. The probiotic agent with metabolic regulation effect according to claim 5, characterized in that: The dosage form of the probiotic is a freeze-dried powder, which is prepared by the following method: The C-1 strain and the GLF-217 strain are inoculated into a culture medium respectively for activation and fermentation culture in sequence to obtain a fermentation broth; the fermentation broth is centrifuged respectively, mixed with a protective agent and freeze-dried to obtain C-1 bacterial powder and GLF-217 bacterial powder; the C-1 bacterial powder and the GLF-217 bacterial powder are mixed according to the ratio of the number of live bacteria to obtain the probiotic agent.

10. Use of the composite probiotic according to claim 1 or 2 or the probiotic according to any one of claims 3 to 9 in preparing a preparation for regulating liver metabolism, characterized in that: The regulating liver metabolism level is any one of or a combination of at least two of delaying weight gain and lipid accumulation caused by a high-fat diet, regulating and improving blood lipid and blood sugar levels, reducing the levels of inflammatory related factors caused by a high-fat diet, or reducing fat accumulation in adipose tissue caused by a high-fat diet to improve insulin resistance, and improving fat metabolism levels.

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