Compound probiotics with metabolic regulation effect and application thereof

Abstract

The invention relates to a compound probiotic with a metabolic regulation effect. The compound probiotic comprises a Bifidobacterium animalis subsp. Lactis C-1 strain with the preservation number of GDMCC (China General Microbiological Culture Collection Center) No: 64543, and a Lactobacillus fermentum GLF-217 strain with the preservation number of CGMCC No: 28336. The invention further relates to a preparation method of the compound probiotic. The two strains have potential interaction and can be mutually matched and synergistically interacted, and the effects of compounding the two strains in the aspect of regulating metabolism are remarkably improved, specifically, the effects of regulating the body weight and body fat level, regulating the blood sugar and blood fat level, improving the inflammatory factor level, relieving liver injury and improving the intestinal flora disorder degree. And coping strategies and reference bases can be provided for intervention of various symptoms caused by metabolic disorders.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to a composite probiotic with metabolic regulating effect and application thereof. Background Art

[0002] High-fat foods refer to foods with high fat content, which are specifically manifested in various saturated and unsaturated fatty acids in the oil component. For example, foods with high oil content and fried foods, such as walnuts, sesame seeds, peanuts, fried foods, fatty meat, animal offal and cream products, etc. Long-term high-fat diet will cause the body to take in too many calories. When these calories exceed the daily consumption, the excess calories will be converted into fat and stored in the body, leading to weight gain and obesity. Continuous obesity is prone to hyperlipidemia and other complications, such as diabetes, inflammation, lipid metabolism disorders, atherosclerosis and fatty liver.

[0003] This type of obesity-related disease often leads to increased serum total cholesterol (TC) levels and triglyceride (TG) levels. The increase in serum total cholesterol levels may be related to reduced insulin secretion capacity. In addition, increased triglycerides increase the risk of diabetes. It also affects 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 and inhibit the activity of lipoprotein lipase, thereby leading to lipid metabolism disorders. Lipid metabolism disorders are an important cause of cardiovascular and cerebrovascular diseases and liver damage. This type of disease may also accelerate the occurrence and development of atherosclerosis and liver function damage. When liver function is damaged, the function of regulating blood sugar and blood lipid levels becomes weak, resulting in a large amount of fat accumulation in the liver, seriously damaging liver function, and further inducing the occurrence and development of liver disease.

[0004] Probiotics are considered to be a type of bacteria that can play a beneficial role in improving the balance of the host's intestinal microecology, improving the host's health level and health status, etc. They mainly affect the host's health and metabolism by regulating the intestinal microbiota and participating in the immune regulation of various diseases, thereby improving the gastrointestinal physiology. There is also a close relationship between obesity and intestinal flora. Studies have shown that the diversity of intestinal flora in obese people is usually lower than that in people with normal weight and body fat. The intestinal flora may also relieve related symptoms by regulating intestinal barrier function, reducing the level of inflammatory factors, regulating lipid metabolism, regulating the composition of native symbiotic bacteria, or by directly colonizing the gastrointestinal tract. Therefore, how to provide a product containing probiotics that can efficiently regulate metabolic levels is of great significance and application value. Summary of the invention

[0005] In view of the deficiencies of the prior art, the object of the present invention is to provide a composite probiotic with metabolic regulating effect and application thereof.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a composite probiotic having a metabolic regulating effect, wherein the composite probiotic comprises Bifidobacterium animalis subsp. lactis with a preservation number of GDMCC No: 64543. Bifidobacterium animalum subsp. milk C-1 strain and Lactobacillus fermentum with a deposit number of CGMCC No. 28336 Lactobacillus leaven GLF-217 strain.

[0007] The present invention has developed a new probiotic compounding method and a new strategy for regulating metabolic levels, namely, Bifidobacterium animalis subsp. Bifidobacterium animalum subsp. milk C-1 strain and Lactobacillus fermentum Lactobacillus fermentum The GLF-217 strain was compounded and it was found that there was a potential interaction between the two strains, which could cooperate with each other and enhance synergy. When the amount of bacteria was the same, compared with the intervention method without any one of the bacteria, the effect of the combination of the two bacteria in regulating metabolism was significantly improved, specifically in the following aspects: (1) regulating body weight and body fat levels; (2) improving blood sugar and blood lipid levels; (3) improving inflammatory factor levels; (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 functional products, they are highly safe and not easy to become dependent on.

[0008] Preferably, the ratio of the number of viable cells of the C-1 strain and 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 they will not be described here one by one.

[0009] Based on the potential interaction between the two strains, the present invention also found that when the two strains are used in combination at the above-mentioned specific ratio of viable bacteria count, the effect in regulating metabolic levels is more significant.

[0010] In a second aspect, the present invention provides a probiotic with metabolic regulation effect, wherein the strain in the probiotic includes the composite probiotic described in the first aspect.

[0011] Preferably, the total viable bacteria content in the probiotic is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g, for example 1×10 9 CFU / mL (CFU / g), 2×109 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 described here one by one.

[0012] Preferably, the dosage form of the probiotic includes solution, lyophilized powder, capsule, tablet or granule.

[0013] Preferably, the probiotic further comprises a protective agent.

[0014] Preferably, the protective agent comprises any one of skim milk, sucrose, lactose, trehalose, dextran, gelatin, dextrin, gum arabic, sodium alginate, polyvinyl pyrrolidone, sorbitol, xylo-oligosaccharides, fructo-oligosaccharides or xylitol, or a combination of at least two thereof.

[0015] Preferably, the probiotic is in the form of 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.

[0016] Preferably, the probiotic is in the form of 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.

[0017] In a third aspect, the present invention provides a use 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 levels.

[0018] Preferably, the preparation further comprises a pharmaceutically acceptable excipient.

[0019] Preferably, the auxiliary materials 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, antibacterial agents or buffers.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention has developed a new probiotic compounding method and a new strategy for regulating metabolic levels, namely, Bifidobacterium animalis subsp. Bifidobacterium animalum subsp. milk C-1 strain and Lactobacillus fermentum Lactobacillus fermentum The GLF-217 strain was compounded and it was found that there was a potential interaction between the two strains, which could cooperate with each other and enhance synergy. When the amount of bacteria was the same, compared with the intervention method without any one of the bacteria, the effect of the combination of the two bacteria in regulating metabolism was significantly improved, specifically in the following aspects: (1) regulating body weight and body fat levels; (2) improving blood sugar and blood lipid levels; (3) improving inflammatory factor levels; (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 functional products, they are highly safe and not easy to become dependent on.

[0021] The classification of the C-1 strain involved in the present invention is named Bifidobacterium animalum subsp. milk The depository is Guangdong Microbiological Culture Collection Center, the deposit number is GDMCC No: 64543, the deposit date is April 22, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; The fermented lactobacillus GLF-217 strain involved in the present invention is classified and named as fermented lactobacillus Lactobacillus fermentum The depository is the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit number is CGMCC No. 28336, the deposit date is September 4, 2023, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a statistical graph of the body weight and total fat content of each group of mice; Figure 2 It is the statistical result diagram of TC, HDL-C, GLU, ALT, LDL-C and NEFA levels in the serum of each group of mice; Figure 3 This is a statistical result chart of the levels of pro-inflammatory factors TNF-α and IL-6 in the serum of each group of mice; Figure 4 This is a statistical graph of the metabolic levels of adipose tissue in each group of mice. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0024] The following bacterial species information is: ① The classification and nomenclature of the C-1 strains involved below are Bifidobacterium animalum subsp. milk , the deposit number is GDMCC No: 64543; ② The GLF-217 strain involved in the following classification is named Lactobacillus fermentum Lactobacillus leaven , the deposit number is CGMCC No. 28336; ③ The CICC 24210 strain involved in the following is Bifidobacterium animalis subspecies lactis CICC 24210.

[0025] The C57BL / 6J male mice (8 weeks old) mentioned below were from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0026] The culture medium and its formula involved below are: MRS 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 hydrogen 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, distilled water to 1000 mL, pH 6.2-6.4.

[0027] The main ingredients of the following mouse feed formula are (mass percentage): Ordinary feed: corn 43.8%, second-grade flour 12%, wheat 17%, salted soybean meal 7%, Peruvian fish meal 4% and American chicken 5%; the main nutrients are: crude protein 19.54%, crude fat 4.43%, crude fiber 2.14%, calcium 1.36% and phosphorus 0.86%.

[0028] High-fat feed: maintenance base feed 48.1%, lard 15%, sucrose 20%, casein 10%, experimental animal premix 2%; the main nutrients are: protein 19.379%, fat 17.133%, crude fiber 3.321%, ash 5.3114%, calcium 1.26735%, phosphorus 0.82223%.

[0029] The preparation method of bacterial suspension and bacterial powder involved below is: after activating the strain, inoculate it into culture medium for cultivation to obtain culture solution; centrifuge the culture solution, resuspend the bacteria to obtain bacterial suspension, or further add protective agent for freeze drying to obtain freeze-dried bacterial powder product. Example 1

[0030] This example explores the effect of compound probiotics on improving various indicators of mice fed a high-fat diet.

[0031] (1) Animal grouping, modeling, and intervention methods After one week of adaptive feeding, C57BL / 6J male mice (8 weeks old) were randomly divided into 8 groups: S1 group (C-1 bacterial suspension), S2 group (GLF-217 bacterial suspension), S3 group (C-1 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 10:1), S4 group (C-1 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 1:1), S5 group (C-1 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 1:10), S6 group (CICC 24210 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 10:1), S7 group as model group, S8 group as blank group, each group had 8 mice, and the total live bacteria in groups S1-S6 were 1×10 9 CFU / mL.

[0032] Intervention method: During the experiment, mice in each group had free access to food and water.

[0033] Mice in groups S1-S7 were fed a high-fat diet, and mice in group S8 were fed a normal diet. Mice in groups S1-S6 were gavaged with 200 μL of the corresponding bacterial suspension, and mice in groups S7-S8 were gavaged with an equal volume of sterile PBS solution, once a day, 200 μL each time, for 6 weeks.

[0034] (2) Mouse body weight and fat content levels During the experiment, the weight of mice was monitored every 7 days, and the weight of mice in each group was counted after the experiment. After the experiment, the mice were killed, and white fat (epididymal fat, perirenal fat, and inguinal fat) was collected and weighed after dissection, and the total fat (white fat) content was counted. The results were as follows Figure 1 shown.

[0035] From the statistical results in the figure, it can be seen that in the sixth week, the average weight (185.74g) and fat content (11.345g) of mice in the S7 (model group) group were higher than the average weight (145.68g) and fat content (6.239g) of mice in the S8 (control group), proving that the model was successful; and after probiotic intervention, the weight and fat content of mice in the S3, S4 and S5 groups were lower than those in the S7 group, indicating that the composite probiotics comprising two specific bacterial combinations of C-1 bacteria and GLF-217 strains involved in the present invention can effectively delay weight gain and lipid accumulation caused by a high-fat diet.

[0036] (3) Blood sugar and blood lipid levels After the experiment, the mouse serum was collected and centrifuged at 571 g for 15 min at 4°C. The 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 determined according to the instructions of the ELISA kit. The results are as follows: Figure 2 shown.

[0037] Continuous high-fat diet will cause blood sugar and lipid metabolism disorder, leading to the occurrence of dyslipidemia such as hyperlipidemia. Figure 2 It can be seen that after 6 weeks of experiments, the 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 mice in the S7 (model group) group were significantly increased compared with those in the S8 group (control group, TC: 4.938 mmol / L; ALT: 70.379 U / L; NEFA: 0.778mmol / L; LDL-C: 1.966 mmol / L; GLU: 6.678mmol / L), and the HDL-C (3.756mmol / L) in the S8 (control group) group was higher than that in the S7 (model group) group (3.064mmol / L). After probiotic intervention, the TC, ALT, NEFA, LDL-C and GLU in the serum of mice in the S3, S4 and S5 groups were lower than those in the S8 group, and the HDL-C levels in the S3, S4 and S5 groups 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 of regulating and improving blood lipid and blood sugar levels, and this regulatory effect helps to reduce the risk of coronary heart disease and other atherosclerosis-related diseases.

[0038] (4) Immune level After the experiment, the mouse serum was centrifuged at 571 g for 15 min at 4°C, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the serum were determined according to the instructions of the ELISA kit. The experimental results were analyzed by data processing software. Figure 3 shown.

[0039] TNF-α and IL-6 are two important cytokines in the immune response and 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 response and disease development, etc.; IL-6 is one of the main mediators of acute phase response and participates in the initiation and regulation of inflammatory response. Continuous high-fat diet often triggers inflammatory response in adipose tissue, resulting in excessive release of related proinflammatory factors in serum, and the level continues to rise. It can be seen from the test results in the figure that after probiotic intervention, the content levels of TNF-α and IL-6 proinflammatory factors in the serum of mice in the S3, S4 and S5 groups were significantly lower than those in the S7 (model group), indicating that the composite probiotics provided by the present invention show effective anti-inflammatory effects, which can effectively relieve cell inflammation and enhance the immune defense ability under high-fat diet, thereby reducing the level of inflammation caused by high-fat diet.

[0040] (5) Adipose tissue metabolic level After the experiment, the mice were killed and white fat (epididymal fat, perirenal fat, and inguinal fat) was collected. The supernatant was collected after homogenization and centrifugation. Resistin and leptin were tested according to the instructions of the kit. The results are as follows: Figure 4 shown.

[0041] Leptin and resistin are two important adipokines, which are produced in adipose tissue and act in the brain to affect metabolic function. Among them, the role of leptin is to balance energy and regulate appetite, thereby regulating metabolic levels, improving insulin resistance and improving fat metabolism; resistin is also an adipokine closely related to insulin resistance. Continuous high-fat diet usually leads to increased resistin levels, which may interfere with the insulin signaling pathway and reduce the sensitivity of mice to insulin, thereby causing metabolic problems such as hyperglycemia and insulin resistance.

[0042] As can be seen from the figure, after induction of high-fat diet, the resistin level of S7 (model group) group (15.809 μg / L) was significantly higher than that of S8 (control group) group (13.623 μg / L), and the leptin level of S7 (model group) group (6.271 μg / L) was significantly lower than that of S8 (control group) group (7.904 μg / L). After intervention with probiotics, the resistin levels of S3, S4 and S5 groups were significantly lower than those of the model group, and the leptin levels were increased. It can be seen that the composite probiotics provided by the present invention have a good effect on regulating the metabolism of adipose tissue in a high-fat diet, reducing fat accumulation in adipose tissue caused by a high-fat diet, thereby improving insulin resistance and improving fat metabolism.

[0043] In summary, the composite probiotics involved in the present invention have excellent metabolic regulation effects, and the C-1 strain and the 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 synergize. When the amount of bacteria is the same, compared with the intervention method of lacking any one of the bacteria, the compounding of the two bacteria significantly improves the effect of regulating metabolism, which is specifically manifested in: (1) regulating body weight and body fat levels; (2) regulating blood sugar and blood lipid levels; (3) improving the level 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 are highly safe and not easy to produce dependence.

[0044] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0045] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0046] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

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

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-5 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 agent according to any one of claims 3 to 9 in preparing a preparation for regulating liver metabolism level.

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