A composite probiotic containing animal bifidobacterium lactis subspecies C-2 for alleviating or treating hyperlipidemia and its application

Abstract

The present invention relates to a composite probiotic for preventing, alleviating or treating hyperlipidemia, wherein the composite probiotic comprises Bifidobacterium animalis subsp. lactis with a preservation number of GDMCC No: 65455. Bifidobacterium animalum subsp.lactis C‑2 strain and Lactobacillus fermentum with the deposit number CGMCC No.28336 Lactobacillus fermentum GLF-217 strain. There is a potential interaction between the two strains, which can cooperate with each other and enhance synergy. The combination of the two strains significantly improves the effect of preventing, alleviating or treating hyperlipidemia. Specifically, it can significantly reduce body weight and fat content, improve blood sugar and blood lipid levels, regulate immunity and increase antioxidant levels, regulate adipose tissue metabolism, and improve gastrointestinal short-chain fatty acid metabolism.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to a composite probiotic for preventing, alleviating or treating hyperlipidemia and an application thereof. Background Art

[0002] The popularity of high-energy diets has led to a significant increase in the incidence of obesity and hyperlipidemia. Obesity is not just excessive weight gain, but is also accompanied by a series of metabolic abnormalities such as insulin resistance, chronic inflammation, and lipid metabolism disorders. Hyperlipidemia is one of the common complications. Obesity is closely related to hyperlipidemia. High-energy diets are rich in fat and sugar. Excessive intake of these nutrients can cause excessive proliferation of adipose tissue and hypertrophy of adipocytes, leading to adipocyte dysfunction and the release of a large number of proinflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), exacerbating the body's chronic inflammatory response. These proinflammatory factors will further worsen insulin resistance, causing abnormal lipid metabolism in the liver, muscle, and adipose tissue, leading to increased plasma triglycerides and low-density lipoprotein cholesterol (LDL-C) levels, decreased high-density lipoprotein cholesterol (HDL-C) levels, and ultimately hyperlipidemia. Insulin resistance and chronic inflammation are considered to be the bridge between obesity and hyperlipidemia. Insulin resistance increases the lipid output of the liver, and the proportion of fatty acids entering the blood circulation also increases, thereby aggravating the symptoms of hyperlipidemia. At the same time, the dysfunction of adipose tissue caused by a high-energy diet will further aggravate chronic low-grade inflammation and oxidative stress, forming a vicious cycle of metabolic disorder.

[0003] The intestinal microbiota plays a key role in metabolic health, especially in the development of obesity and hyperlipidemia, where changes in the intestinal flora are closely related to these metabolic abnormalities. The composition of the intestinal flora in obese individuals usually shows a significant imbalance, and the diversity of intestinal microorganisms is also significantly reduced. This imbalanced intestinal flora can affect the host's energy acquisition and storage, increase fat accumulation and the occurrence of chronic inflammation. As an effective means of regulating intestinal flora, probiotics can restore the balance of intestinal flora, reduce liver lipid accumulation, lower plasma lipid levels, and improve the body's metabolic health. Therefore, it is crucial to develop a probiotic product that can effectively prevent, alleviate or treat hyperlipidemia. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a composite probiotic for preventing, alleviating or treating hyperlipidemia and its application.

[0005] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a composite probiotic for preventing, alleviating or treating hyperlipidemia, wherein the composite probiotic comprises a Bifidobacterium animalissubsp. lactis C-2 strain with a preservation number of GDMCC No. 65455 and a Lactobacillusfermentum GLF-217 strain with a preservation number of CGMCC No. 28336.

[0007] The present invention develops a new probiotic compounding method and a new strategy for preventing, alleviating or treating hyperlipidemia, that is, compounding Bifidobacterium animalis subsp. lactis C-2 strain and Lactobacillus fermentum GLF-217 strain, and finds 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 preventing, alleviating or treating hyperlipidemia, which is specifically manifested in: (1) significantly reducing body weight and fat content; (2) improving blood sugar and blood lipid levels; (3) regulating immunity and improving antioxidant levels; (4) regulating adipose tissue metabolism levels; (5) improving gastrointestinal short-chain fatty acid metabolism. Therefore, the composite probiotic has a good prospect for preparing a drug for preventing, alleviating or treating hyperlipidemia. 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 generate dependence.

[0008] The preparation method of the composite probiotics can be prepared by conventional technical methods in the art, and can be exemplified by: activating the four strains, inoculating them into a culture medium for cultivation, obtaining a culture solution; centrifuging the culture solution, resuspending the bacteria to obtain a bacterial suspension; and mixing the four bacterial suspensions according to the ratio of the number of live bacteria. Alternatively, a protective agent is further added for freeze drying to obtain a freeze-dried bacterial powder product.

[0009] Preferably, the culture medium comprises MRS medium or TPY medium.

[0010] Preferably, the MRS medium comprises, by concentration, 8-12 g / L of peptone, 8-12 g / L of beef extract, 15-25 g / L of glucose, 1-3 g / L of sodium acetate, 3-7 g / L of yeast powder, 1-3 g / L of diammonium hydrogen citrate, and 1-3 g / L of K 2 PO 4 ·3H 2 O 2-3 g / L, MgSO 4 7H 2O 0.05-0.2 g / L, MnSO 4 0.01-0.1 g / L, Tween 80 0.5-2 mL / L, cysteine ​​hydrochloride 0.1-1 g / L.

[0011] Preferably, the TPY medium comprises, by concentration, 8-12 g / L of hydrolyzed casein, 3-7 g / L of soytone, 1-3 g / L of yeast powder, 3-7 g / L of glucose, 0.1-1 g / L of L-cysteine, 1-4 g / L of dipotassium hydrogen phosphate, 0.1-1 g / L of magnesium chloride, 0.1-1 g / L of zinc sulfate, 0.05-0.5 g / L of calcium chloride, 0.0001-0.01 mg / L of ferric chloride, and 0.5-2 mL / L of Tween 80.

[0012] Preferably, the ratio of the number of viable cells of the C-2 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.

[0013] Based on the potential interaction between the two strains, the present invention also found that when the two strains are used in combination with the above-mentioned specific live bacteria count ratio, the effects in regulating blood sugar and blood lipid levels, regulating body weight and preventing, alleviating or treating hyperlipidemia are more significant.

[0014] In a second aspect, the present invention provides a probiotic for preventing, alleviating or treating hyperlipidemia, wherein the strain in the probiotic includes the composite probiotic described in the first aspect.

[0015] Preferably, the total viable bacteria content in the probiotic is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g, for example, can be 1×10 8 CFU / mL (CFU / g), 2×10 8 CFU / mL (CFU / g), 5×10 8 CFU / mL (CFU / g), 8×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 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.

[0016] Preferably, the dosage form of the probiotic includes solution, lyophilized powder, capsule, tablet or granule.

[0017] Preferably, the probiotic further comprises a lyophilization protectant.

[0018] Preferably, the lyoprotectant 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.

[0019] In a third aspect, the present invention provides 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 glucose and lipid metabolism levels.

[0020] In a fourth aspect, the present invention provides use of the composite probiotic according to the first aspect or the probiotic agent according to the second aspect in preparing a preparation for regulating the intestinal microecological environment.

[0021] In a fifth aspect, the present invention provides use of the composite probiotic according to the first aspect or the probiotic according to the second aspect in the preparation of a preparation for improving the metabolic level of short-chain fatty acids in the gastrointestinal tract.

[0022] Preferably, the preparation further comprises a pharmaceutically acceptable excipient.

[0023] 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.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention develops a new probiotic compounding method and a new strategy for preventing, alleviating or treating hyperlipidemia, that is, compounding Bifidobacterium animalis subsp. lactis C-2 strain and Lactobacillus fermentum GLF-217 strain, and finds 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 preventing, alleviating or treating hyperlipidemia, which is specifically manifested in: (1) significantly reducing body weight and fat content; (2) improving blood sugar and blood lipid levels; (3) regulating immunity and improving antioxidant levels; (4) regulating adipose tissue metabolism levels; (5) improving gastrointestinal short-chain fatty acid metabolism. Therefore, the composite probiotic has a good prospect for preparing a drug for preventing, alleviating or treating hyperlipidemia. 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 generate dependence.

[0026] The C-2 strain involved in the present invention is classified and named Bifidobacterium animalis subsp. lactis, and the depository is Guangdong Microbiological Culture Collection Center, the deposit number is GDMCC No: 65455, the deposit date is November 8, 2024, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0027] The GLF-217 strain involved in the present invention is classified and named as Lactobacillus fermentum, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation number is CGMCC No. 28336, the preservation date is September 4, 2023, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the statistical result graph of weight measurement of mice in each group;

[0029] Figure 2 It is the statistical result graph of TG, HDL-C, ALT, GLU and NEFA levels in the serum of each group of mice;

[0030] Figure 3 This is a statistical graph of the metabolic levels of adipose tissue in each group of mice;

[0031] Figure 4 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;

[0032] Figure 5 This is the statistical result of the MDA and SOD content levels in the liver tissues of each group of mice;

[0033] Figure 6 This is a statistical graph of the short-chain fatty acid content levels in the feces of each group of mice. DETAILED DESCRIPTION

[0034] 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.

[0035] The following bacterial species information is:

[0036] ① The C-2 strain involved in the following examples is classified as Bifidobacterium animalis subsp. lactis, and its deposit number is GDMCC No: 65455;

[0037] ② The GLF-217 strain involved in the following examples is classified as Lactobacillus fermentum, and its deposit number is CGMCC No. 28336;

[0038] ③ The ATCC 27673 strain involved in the following embodiments is the Bifidobacterium animalis subsp. lactis ATCC 27673 strain.

[0039] The C57BL / 6J male mice (8 weeks old) mentioned below were from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0040] The main nutrients in the normal mouse diet mentioned below are (by mass): crude protein 19.54%, crude fat 4.43%, crude fiber 2.14%, calcium 1.36%, and phosphorus 0.86%.

[0041] The main nutrients in the high-fat mouse diet mentioned below are (by mass): protein 19.379%, fat 17.133%, crude fiber 3.321%, ash 5.3114%, calcium 1.26735%, and phosphorus 0.82223%.

[0042] Example

[0043] This example explores the effect of the composite probiotics of the present invention on improving various indicators of mice fed a high-fat diet:

[0044] (1) Experimental animals

[0045] Eight-week-old C57BL / 6J male mice were housed in an environment with a temperature of 22°C, a humidity of 55%, and a lighting of 12 h. They were fed with normal mouse food for one week (with free access to food and water).

[0046] (2) Animal grouping, modeling, and intervention methods

[0047] After one week of adaptive feeding, the mice were randomly divided into 9 groups: S1 group (C-2 bacterial suspension), S2 group (GLF-217 bacterial suspension), S3 group (ATCC 27673 bacterial suspension), S4 group (C-2 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 10:1), S5 group (C-2 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 1:1), S6 group (C-2 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 1:10), S7 group (ATCC 27673 bacterial suspension + GLF-217 bacterial suspension, live bacteria ratio of 10:1), S8 group as model group, S9 group as blank group, with 8 mice in each group, and the total live bacteria in groups S1-S7 were 1×10 9 CFU / mL.

[0048] Intervention methods:

[0049] During the experiment, mice in each group had free access to food and water.

[0050] Mice in groups S1-S8 were fed a high-fat diet, and mice in group S9 were fed a normal diet. Mice in groups S1-S7 were gavaged with 200 μL of the corresponding bacterial suspension, and mice in groups S8-S9 were gavaged with an equal volume of sterile PBS solution, once a day, 200 μL each time, for 6 weeks.

[0051] (3) Mouse weight analysis

[0052] During the experiment, the weight of mice was monitored every 7 days. After the experiment, the weight statistics of mice in each group were as follows: Figure 1 As shown, at the sixth week, the body weight of mice in group S9 was lower than that in group S8, proving that the model was successful. After probiotic intervention, the body weight of mice in groups S4, S5 and S6 were all lower than that in group S8, indicating that the composite probiotics involved in the present invention can effectively delay the lipid accumulation of a high-fat diet.

[0053] (4) Analysis of blood sugar and blood lipid levels

[0054] After the experiment, the mouse serum was collected and centrifuged at 571 g for 15 min at 4°C. The levels of triglyceride (TG), high-density lipoprotein (HDL-C), alanine aminotransferase (ALT), glucose (GLU), and non-esterified fatty acids (NEFA) in the serum were determined according to the instructions of the ELISA kit. The results were as follows: Figure 2 shown.

[0055] Depend on Figure 2 It can be seen that after 6 weeks of experiment, the levels of TG (0.790 mmol / L), ALT (70.470U / L), NEFA (0.797 mmol / L) and GLU (6.360 mmol / L) in the serum of mice in the S9 group were lower than those in the S8 group (14.408 mmol / L), ALT (165.784 U / L), NEFA (3.085 mmol / L) and GLU (8.437 mmol / L); the level of HDL-C (3.818 mmol / L) in the S9 group was higher than that in the S8 group (3.109 mmol / L). After probiotic intervention, the levels of TG, ALT, NEFA and GLU in the serum of mice in the S4, S5 and S6 groups were lower than those in the S8 group, and the levels of HDL-C in the S4, S5 and S6 groups were higher than those in the S8 and S9 groups. It can be seen that long-term high-fat diet can lead to the occurrence of dyslipidemia and other lipid diseases, causing disorders in the levels of indicators such as TG, HDL-C, ALT, NEFA and GLU in the blood, and the composite probiotics involved in the present invention can better regulate and improve blood lipid and blood sugar levels.

[0056] (5) Analysis of adipose tissue metabolic levels

[0057] 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 3 shown.

[0058] A high-fat diet usually leads to an increase in 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. Leptin can improve insulin resistance and fat metabolism, thereby alleviating hyperlipidemia. Figure 3 It can be seen that after induction of high-fat diet, the resistin level of S8 group (15.706 μg / L) was significantly higher than that of S9 group (13.182 μg / L), and the leptin level of S8 group (6.103 μg / L) was significantly lower than that of S9 group (7.605 μg / L). After intervention with probiotics, the resistin levels of S4, S5 and S6 groups were significantly lower than those of S8 group, and the leptin level was increased. It can be seen that the composite probiotics provided by the present invention have a good effect of regulating the metabolism of adipose tissue caused by high-fat diet, reducing the fat accumulation of adipose tissue caused by high-fat diet, thereby improving insulin resistance and fat metabolism, and then alleviating hyperlipidemia.

[0059] (6) Analysis of anti-inflammatory effects

[0060] 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 4 shown.

[0061] Hyperlipidemia caused by a high-fat diet often triggers an inflammatory response in adipose tissue, leading to increased levels of TNF-α and IL-6 pro-inflammatory factors in serum. Figure 4 It can be seen that after probiotic intervention, the levels of TNF-α and IL-6 proinflammatory factors in the serum of mice in groups S4, S5 and S6 were significantly lower than those in group S8, indicating that the composite probiotics provided by the present invention can alleviate cellular inflammation, thereby reducing the level of inflammation caused by a high-fat diet.

[0062] (7) Analysis of antioxidant levels

[0063] The liver tissues of mice in each group were collected and homogenized and centrifuged, and the supernatant was collected. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were tested according to the instructions of the kit. The results are as follows: Figure 5 shown.

[0064] Hyperlipidemia caused by a high-fat diet often triggers adipose tissue peroxidation, leading to increased levels of malondialdehyde (MDA) and decreased superoxide dismutase (SOD) activity in the liver. Figure 5 It can be seen that after probiotic intervention, the MDA levels in the livers of mice in groups S4, S5 and S6 were lower than those in group S8, and the SOD activity was higher than that in group S8, indicating that the composite probiotics involved in the present invention have a good potential to inhibit liver peroxidation and alleviate liver oxidative stress.

[0065] (8) Analysis of short-chain fatty acid metabolism levels

[0066] The feces of mice in each group were collected and freeze-dried for extraction, and the content of short-chain fatty acids (SCFAs) in each group of samples was quantitatively analyzed by gas chromatography. The results are as follows: Figure 6 shown.

[0067] As can be seen from the figure, after probiotic intervention, the levels of propionic acid, isobutyric acid and isovaleric acid in the samples of groups S4, S5 and S6 were higher than those of group S8. Short-chain fatty acids such as propionic acid, isobutyric acid and isovaleric acid play an important role in the regulation of intestinal microbial metabolism and metabolic health. It can be seen that the composite probiotics involved in the present invention can effectively improve the metabolic level of short-chain fatty acids in the gastrointestinal tract of mice, thereby improving the overall metabolism and regulating intestinal health.

[0068] In summary, the composite probiotics involved in the present invention have excellent effects in preventing, alleviating or treating hyperlipidemia, and the C-2 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 preventing, alleviating or treating hyperlipidemia, which is specifically manifested in: (1) significantly reducing body weight and fat content; (2) improving blood sugar and blood lipid levels; (3) regulating immunity and improving antioxidant levels; (4) regulating adipose tissue metabolism levels; (5) improving gastrointestinal short-chain fatty acid metabolism. Therefore, the composite probiotics have a good prospect for preparing drugs for preventing, alleviating or treating hyperlipidemia. 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.

[0069] 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.

[0070] 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.

[0071] 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 for alleviating or treating hyperlipidemia, characterized in that: The compound probiotics are: Bifidobacterium animalis subsp. lactis with a preservation number of GDMCC No: 65455 Bifidobacterium animalissubsp. lactis C-2 strain and Lactobacillus fermentum with a preservation number of CGMCC No. 28336 Lactobacillus fermentum GLF-217 strain.

2. The composite probiotic for alleviating or treating hyperlipidemia according to claim 1, characterized in that: The ratio of the number of viable bacteria of the C-2 strain to that of the GLF-217 strain is 1:10-10:

1.

3. A probiotic for alleviating or treating hyperlipidemia, characterized in that: The strains in the probiotic agent are composed of the composite probiotics according to claim 1 or 2.

4. The probiotic for alleviating or treating hyperlipidemia according to claim 3, characterized in that: The total live bacteria content in the probiotics is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

5. The probiotic for alleviating or treating hyperlipidemia according to claim 3, characterized in that: The dosage form of the probiotic is selected from solution, lyophilized powder, capsule, tablet or granule.

6. The probiotic for alleviating or treating hyperlipidemia according to claim 3, characterized in that: The probiotics also include a lyoprotectant.

7. The probiotic for alleviating or treating hyperlipidemia according to claim 6, characterized in that: The lyoprotectant includes 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.

8. Use of the composite probiotic according to claim 1 or 2 or the probiotic according to any one of claims 3 to 7 in the preparation of a preparation for relieving or treating hyperlipidemia.

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