A probiotic composition for the benefit of blood lipid health

Abstract

The present application relates to the field of microbial preparation, in particular to a probiotic composition beneficial to blood lipid health. The present application combines Bifidobacterium lactis MN-Gup, Bifidobacterium lactis M8, Lactobacillus plantarum LP6 and Lactobacillus casei PC01, and through animal experiment verification, can obviously reduce triglyceride and cholesterol in serum, and thus can be used for preventing and improving corresponding types of hyperlipidemia.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial preparations, in particular to a probiotic composition for blood lipid health. BACKGROUND

[0002] In recent decades, with the improvement of living standards, cardiovascular and cerebrovascular diseases such as coronary heart disease [1,2] , atherosclerosis [3,4] , etc. have become a common disease that seriously threatens human health. Among them, the mortality of atherosclerotic cardiovascular disease is particularly high. Hyperlipidemia is a major risk factor for atherosclerotic cardiovascular disease, which is usually manifested as abnormal lipid levels, including low high-density lipoprotein cholesterol (HDL-C) levels, high total cholesterol (TC), serum triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) levels, etc. [5] In recent years, it has been found that intestinal flora disorder is also associated with the occurrence of hyperlipidemia.

[0003] At present, the methods for clinically controlling cholesterol include drug therapy and dietary intake intervention. Commonly used cholesterol regulating drugs, such as statins, clofibrate, niacin, and bile acid integrating resins, etc. are not used to treat high cholesterol, but to control cholesterol in the normal range for a long time. These drugs have relatively high treatment costs and have varying degrees of side effects, such as gastrointestinal diseases such as nausea and diarrhea, liver function damage, rhabdomyolysis, etc. [6] In contrast, consuming functional probiotics may be a more ideal method for long-term control of cholesterol levels. Probiotics can increase high-density lipoprotein, reduce low-density lipoprotein and total cholesterol; and have the advantages of low price, balanced nutrition, and small side effects, while improving intestinal flora [7,8] , and thus more and more attention has been paid by domestic and foreign researchers, and has become a hot research topic at present, with great market development potential [9,10] .

[0004] The research on probiotics that help maintain healthy blood lipid levels mostly focuses on single strains (CN202210166383.4) and in vitro cholesterol absorption (CN201580074875.9). Similarly, the research on probiotics that help reduce serum triglycerides also mostly focuses on single strains (CN202110145017.6) and in vitro triglyceride degradation (CN201310751034.X), lacking animal experiment and human clinical data support. However, the intestine is the main site for probiotics to improve blood lipid levels, and the environment is relatively complex. If a probiotic composition containing multiple strains is used to achieve synergistic effects, it will have more advantages. However, it is difficult to confirm its actual effect in the intestine based on in vitro experiments.

[0005] The references are as follows:

[0006] 1. Jimenez-Torres, J., et al., Mediterranean Diet Reduces Atherosclerosis Progression in Coronary Heart Disease: An Analysis of the CORDIOPREV Randomized Controlled Trial. Stroke, 2021. 52(11): p. 3440-3449.

[0007] 2. Wu, H. and J. Chiou, Potential Benefits of Probiotics and Prebiotics for Coronary Heart Disease and Stroke. Nutrients, 2021. 13(8): p. 2787.

[0008] 3. Soehnlein, O. and P. Libby, Targeting inflammation in atherosclerosis-from experimental insights to the clinic. Nat Rev Drug Discov, 2021. 20(8): p. 589-610.

[0009] 4. Ahmed, S. and J. D. Spence, Sex differences in the intestinal microbiome: interactions with risk factors for atherosclerosis and cardiovascular disease. Biol Sex Differ, 2021. 12(1): p. 35.

[0010] 5. Hurtubise, J., et al., The Different Facets of Dyslipidemia and Hypertension in Atherosclerosis. Curr Atheroscler Rep, 2016. 18(12): p. 82.

[0011] 6. Cicero, A. F. G. and A. Colletti, An update on the safety of nutraceuticals and effects on lipid parameters. Expert Opinion on Drug Safety, 2018. 17(3): p. 303-313.

[0012] 7. Wu, Y., et al., Effect of probiotic Lactobacillus on lipid profile: A systematic review and meta-analysis of randomized, controlled trials. PLoS One, 2017. 12(6): p. e0178868.

[0013] 8. Hunter, P. M. and R. A. Hegele, Functional foods and dietary supplements for the management of dyslipidaemia. Nat Rev Endocrinol, 2017. 13(5): p. 278-288.

[0014] 9. Vourakis, M., G. Mayer, and G. Rousseau, The Role of Gut Microbiota on Cholesterol Metabolism in Atherosclerosis. Int J Mol Sci, 2021. 22(15): p. 8074.

[0015] 10. Anselmi, G., et al., Gut Microbiota and Cardiovascular Diseases: A Critical Review. Cardiol Rev, 2021. 29(4): p. 195-204. SUMMARY

[0016] In previous studies, the inventors have found that the fermented yogurt of Bifidobacterium lactis MN-Gup can significantly inhibit the weight gain of obese mice, reduce the body fat rate, and reduce the total cholesterol in the blood. MN-Gup has a significant effect on preventing obesity in animal experiments (CN202010899880.6), but whether MN-Gup can improve hypertriglyceridemia and / or hypercholesterolemia caused by high-fat diet has not been clearly effective.

[0017] The present application unexpectedly found that the combination of Bifidobacterium lactis MN-Gup, Bifidobacterium lactis M8, Lactobacillus plantarum LP6, and at least one selected from Lactobacillus paracasei PC-01 and Lactobacillus casei Zhang can significantly reduce the triglycerides and / or cholesterol in the serum, and thus can be used for preventing and improving hypertriglyceridemia and / or hypercholesterolemia.

[0018] Accordingly, the present application first provides a probiotic composition comprising:

[0019] The first strain is Bifidobacterium lactis MN-Gup, with a preservation number of CGMCC No. 15578;

[0020] The second strain is Bifidobacterium lactis M8, with a preservation number of CGMCC No. 16070;

[0021] The third strain is Lactobacillus plantarum LP6, with a preservation number of CGMCC No. 13458; and

[0022] The fourth strain is at least one selected from the following strains:

[0023] a) Lactobacillus paracasei PC-01, with a preservation number of CGMCC No. 17537;

[0024] b) Lactobacillus casei Zhang, with a preservation number of CGMCC No. 5469;

[0025] CGMCC No. 5469;

[0026] Among them, the number of viable bacteria of the first strain accounts for greater than or equal to 40% based on the total number of viable bacteria of the probiotic composition, and the total number of viable bacteria of the third strain and the fourth strain accounts for greater than or equal to 30%.

[0027] In the present application, the Bifidobacterium lactis MN-Gup has been disclosed in CN113207960A, which has been preserved in the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Xiliucheng West Road, Chaoyang District, Beijing, on April 10, 2018, with a classification name of Bifidobacterium animalis subsp. lactis and a preservation number of CGMCC No. 15578. In the present application, the Bifidobacterium animalis subsp. lactis MN-Gup is simply referred to as Bifidobacterium lactis MN-Gup.

[0028] The Bifidobacterium lactis M8 has been disclosed in CN110157650A, which has been preserved in the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Xiliucheng West Road, Chaoyang District, Beijing, on July 9, 2018, with a classification name of Bifidobacterium lactis and a preservation number of CGMCC No. 16070.

[0029] The Lactobacillus plantarum LP6 has been disclosed in CN107964520A, which has been preserved in the China General Microbiological Culture Collection Center, No. 3, Yuanzhuangxili, Beichen, Chaoyang District, Beijing, China on September 25, 2017, and classified as Lactobacillus plantarum, with the preservation number of CGMCC No. 13458.

[0030] The Lactobacillus paracasei PC-01 has been disclosed in CN110257297A, which has been preserved in the China General Microbiological Culture Collection Center, No. 3, Yuanzhuangxili, Beichen, Chaoyang District, Beijing, China on March 18, 2019, and classified as Lactobacillus paracasei, with the preservation number of CGMCC No. 17537.

[0031] The Lactobacillus casei Zhang has been disclosed in CN102851222A, which has been preserved in the China General Microbiological Culture Collection Center, No. 3, Yuanzhuangxili, Beichen, Chaoyang District, Beijing, China on November 18, 2011, and classified as Lactobacillus casei, with the preservation number of CGMCC No. 5469.

[0032] When the fourth strain in the present application is Lactobacillus paracasei PC-01 or Lactobacillus casei Zhang, the obtained probiotic composition composition has a better synergistic effect. When the fourth strain is a mixture of Lactobacillus paracasei PC-01 and Lactobacillus casei Zhang, it also has a considerable effect.

[0033] As a preferred, the number of live bacteria of the first strain accounts for more than 50±10% of the total number of live bacteria of the probiotic composition.

[0034] As a preferred solution, the ratio of viable cell number of the third strain and the fourth strain is 1:(0.5-3); more preferably, 1:(0.5-1.5). When the ratio of the third strain and the fourth strain meets the above relationship, the composition has more optimal performance in all aspects.

[0035] As a preferred solution, the ratio of viable cell number of the second strain, the third strain and the fourth strain is (0.8-1.2):(1.5-2.5):(1.5-2.5).

[0036] More preferably, the ratio of viable cell number of the first strain, the second strain, the third strain and the fourth strain is (4-6):(0.8-1.2):(1.5-2.5):(1.5-2.5).

[0037] It is also found that, by optimizing the amount of each component in the above manner, the synergistic effect of the composition can be more effectively exerted.

[0038] As a preferred solution, the probiotic composition contains the following components in terms of viable cell number: 4-6 parts of Bifidobacterium lactis MN-Gup, 0.8-1.2 parts of Bifidobacterium lactis M8, 1.5-2.5 parts of Lactobacillus plantarum LP6, and 1.5-2.5 parts of Lactobacillus casei Zhang. When the above formulation is used, the serum cholesterol and triglyceride can be significantly reduced, and thus the composition can be used for preventing, alleviating and improving high cholesterol and high triglyceride symptoms.

[0039] As a preferred solution, the probiotic composition contains the following components in terms of viable cell number: 4-6 parts of Bifidobacterium lactis MN-Gup, 0.8-1.2 parts of Bifidobacterium lactis M8, 1.5-2.5 parts of Lactobacillus plantarum LP6, and 1.5-2.5 parts of Lactobacillus paracasei PC-01. When the above formulation is used, the serum triglyceride can be significantly reduced, and thus the composition is more suitable for preventing, alleviating and improving high triglyceride symptoms.

[0040] In specific implementation, the probiotic composition can be a freeze-dried powder, and the specific amount of the freeze-dried powder can be calculated according to the number of viable cells in the freeze-dried powder, so that the number of viable cells is within the range defined in the present application.

[0041] The present application also provides a probiotic agent containing the probiotic composition.

[0042] As a preference, in the probiotic agent, the total number of viable bacteria of the probiotic composition is greater than or equal to 5.0 x 10 10 cfu / gram.

[0043] The present application also provides the use of the probiotic composition or the probiotic agent in preventing, alleviating or improving hyperlipidemia.

[0044] Further, the present application also provides the use of the probiotic composition or the probiotic agent in at least one of the following aspects:

[0045] (1) preventing, alleviating or improving hypercholesterolemia;

[0046] (2) preventing, alleviating or improving hypertriglyceridemia.

[0047] The present application also provides the use of the probiotic composition or the probiotic agent in the preparation of a health food for maintaining a healthy level of blood lipid or in the preparation of a medicine for preventing, alleviating or improving hyperlipidemia.

[0048] As a preference, the medicine is applied to preventing, alleviating or improving hyperlipidemia.

[0049] More preferably, the medicine has at least one of the following uses:

[0050] (1) preventing, alleviating or improving hypercholesterolemia;

[0051] (2) preventing, alleviating or improving hypertriglyceridemia.

[0052] The present application also provides a health food for maintaining a healthy level of blood lipid or a medicine for preventing, alleviating or improving hyperlipidemia, which contains the probiotic composition or the probiotic agent.

[0053] As a preference, the health food for maintaining a healthy level of blood lipid further contains an edible carrier selected from one or more of milk powder, malt dextrin, oligosaccharide, dietary fiber, fruit juice powder, sugar alcohol, starch and silicon dioxide.

[0054] As a preference, the medicine for preventing, alleviating or improving hyperlipidemia further contains an edible carrier selected from one or more of milk powder, malt dextrin, oligosaccharide, dietary fiber, fruit juice powder, sugar alcohol, starch and silicon dioxide.

[0055] By adding oligosaccharide, the probiotic property of the probiotic composition can be improved, and the intestinal flora can be improved and the growth of beneficial bacteria can be promoted.

[0056] The beneficial effects of the present application are as follows:

[0057] The present application can be used for preventing and improving corresponding types of hyperlipidemia by combining Bifidobacterium lactis MN-Gup, Bifidobacterium lactis M8, Lactobacillus plantarum LP6, Lactobacillus casei Zhang / Paracasei PC-01, and verifying through animal experiments, which can significantly reduce triglyceride and cholesterol in serum. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The figure is the effect of the probiotic composition of Example 3 in the present application on serum TC of SD rats; in the figure, *P<0.05 compared with the model control group, **P<0.01 compared with the model control group.

[0059] Figure 2 The figure is the effect of the probiotic composition of Example 3 in the present application on serum TG of SD rats; in the figure, **P<0.01 compared with the model control group.

[0060] Figure 3 The figure is the effect of the probiotic composition of Example 3 in the present application on serum HDL-C of SD rats.

[0061] Figure 4 The figure is the effect of the probiotic composition of Example 3 in the present application on serum LDL-C of SD rats; in the figure, **P<0.01 compared with the model control group. DETAILED DESCRIPTION

[0062] The following examples are used to illustrate the present application, but not to limit the scope of the present application.

[0063] In the following examples, the Bifidobacterium lactis MN-Gup has been disclosed in CN113207960A, which was deposited in the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 1, Yabian West Road, Chaoyang District, Beijing, China on April 10, 2018, with a postal code of 100101, and was classified as Bifidobacterium animalis subsp. lactis, with a preservation number of CGMCC No. 15578. In the present application, Bifidobacterium animalis subsp. lactis MN-Gup is referred to as Bifidobacterium lactis MN-Gup.

[0064] The Bifidobacterium lactis M8 has been disclosed in CN110157650A, which has been preserved in the China General Microbiological Culture Collection Center, No. 3, Yikhinxi Road, Beijing Chaoyang District, on July 9, 2018, and classified as Bifidobacterium lactis, with a preservation number of CGMCC No. 16070.

[0065] The Lactobacillus plantarum LP6 has been disclosed in CN107964520A, which has been preserved in the China General Microbiological Culture Collection Center, No. 3, Yikhinxi Road, Beijing Chaoyang District, on September 25, 2017, and classified as Lactobacillus plantarum, with a preservation number of CGMCC No. 13458.

[0066] The Lactobacillus paracasei PC-01 has been disclosed in CN110257297A, which has been preserved in the China General Microbiological Culture Collection Center, No. 3, Yikhinxi Road, Beijing Chaoyang District, on March 18, 2019, and classified as Lactobacillus paracasei, with a preservation number of CGMCC No. 17537.

[0067] The Lactobacillus casei Zhang has been disclosed in CN102851222A, which has been preserved in the China General Microbiological Culture Collection Center, No. 3, Yikhinxi Road, Beijing Chaoyang District, on November 18, 2011, and classified as Lactobacillus casei, with a preservation number of CGMCC No. 5469.

[0068] The Bifidobacterium lactis Bb-12 is obtained through a commercial channel.

[0069] In the following examples, the probiotic composition is a combination of freeze-dried powders of the corresponding strains. Those skilled in the art can calculate the specific amount of the freeze-dried powder according to the number of viable bacteria in the freeze-dried powder, so that the number of viable bacteria is within the range defined in the examples of the present application.

[0070] Unless otherwise specified, the techniques and conditions in the examples were carried out according to the techniques and conditions described in the literature or according to the manufacturer's instructions. Unless otherwise specified, the reagents and instruments used were conventional products that can be purchased from a regular channel.

[0071] Example 1

[0072] This example provides a probiotic composition, the formulation of which is as follows in terms of the number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 1 part of Bifidobacterium lactis M8, 1 part of Lactobacillus plantarum LP6, and 3 parts of Lactobacillus casei Zhang.

[0073] This example further provides a solid beverage containing the probiotic composition described above, the formulation of which is as follows in terms of weight ratio: 0.4 parts of the probiotic composition (total number of viable bacteria: 5.0 x 10 10 cfu / g), 0.7 parts of xylo-oligosaccharide, 0.5 parts of maltodextrin, and 0.2 parts of fruit powder.

[0074] Example 2

[0075] This example provides a probiotic composition, the formulation of which is as follows in terms of the number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 1 part of Bifidobacterium lactis M8, 3 parts of Lactobacillus plantarum LP6, and 1 part of Lactobacillus casei Zhang.

[0076] This example further provides a solid beverage containing the probiotic composition described above, the formulation of which is as follows in terms of weight ratio: 0.4 parts of the probiotic composition (total number of viable bacteria: 5.0 x 10 10 cfu / g), 0.8 parts of xylo-oligosaccharide, 0.6 parts of galacto-oligosaccharide, and 0.2 parts of fruit powder.

[0077] Example 3

[0078] The present embodiment provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 1 part of Bifidobacterium lactis M8, 2 parts of Lactobacillus plantarum LP6, and 2 parts of Lactobacillus casei Zhang.

[0079] The present embodiment further provides a probiotic capsule containing the probiotic composition described above, the formulation of which is as follows in terms of weight ratio: 0.4 parts of the probiotic composition (total number of viable bacteria is 5.0 x 10 10 cfu / gram), 0.7 parts of xylooligosaccharide.

[0080] Example 4

[0081] The present embodiment provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 2 parts of Bifidobacterium lactis M8, 1 part of Lactobacillus plantarum LP6, and 2 parts of Lactobacillus casei Zhang.

[0082] The present embodiment further provides a probiotic capsule containing the probiotic composition described above, the formulation of which is as follows in terms of weight ratio: 0.4 parts of the probiotic composition (total number of viable bacteria is 5.0 x 10 10 cfu / gram), 0.2 parts of xylooligosaccharide, and 0.1 parts of galactooligosaccharide.

[0083] Example 5

[0084] The present embodiment provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 2 parts of Bifidobacterium lactis M8, 2 parts of Lactobacillus plantarum LP6, and 1 part of Lactobacillus casei Zhang.

[0085] The present embodiment further provides a probiotic capsule containing the probiotic composition described above, the formulation of which is as follows in terms of weight ratio: 0.4 parts of the probiotic composition (total number of viable bacteria is 5.0 x 10 10 cfu / gram), 0.6 parts of galactooligosaccharide, and 0.6 parts of erythritol.

[0086] Example 6

[0087] The present example provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 1 part of Bifidobacterium lactis M8, 1 part of Lactobacillus plantarum LP6, and 3 parts of Lactobacillus paracasei PC-01.

[0088] The present example further provides a solid beverage containing the probiotic composition described above, the formulation of which is as follows in terms of weight ratio: 0.4 parts of the probiotic composition (total number of viable bacteria is 5.0 x 10 10 cfu / gram), 0.7 parts of xylooligosaccharide, 0.6 parts of maltodextrin, and 0.3 parts of fruit powder.

[0089] Example 7

[0090] The present example provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 1 part of Bifidobacterium lactis M8, 3 parts of Lactobacillus plantarum LP6, and 1 part of Lactobacillus paracasei PC-01.

[0091] The present example further provides a solid beverage containing the probiotic composition described above, the formulation of which is as follows in terms of weight ratio: 0.4 parts of the probiotic composition (total number of viable bacteria is 5.0 x 10 10 cfu / gram), 0.8 parts of xylooligosaccharide, 1 part of galactooligosaccharide, and 0.3 parts of fruit powder.

[0092] Example 8

[0093] The present example provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 1 part of Bifidobacterium lactis M8, 2 parts of Lactobacillus plantarum LP6, and 2 parts of Lactobacillus paracasei PC-01.

[0094] The present example further provides a probiotic capsule containing the probiotic composition described above, which is formulated as follows in terms of weight ratio: the probiotic composition (total number of viable bacteria 5.0 x 10 10 cfu / g) 0.4 parts, xylooligosaccharide 0.5 parts.

[0095] Example 9

[0096] The present example provides a probiotic composition, which is formulated as follows in terms of parts of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 2 parts of Bifidobacterium lactis M8, 1 part of Lactobacillus plantarum LP6, and 2 parts of Lactobacillus paracasei PC-01.

[0097] The present example further provides a probiotic capsule containing the probiotic composition described above, which is formulated as follows in terms of weight ratio: the probiotic composition (total number of viable bacteria 5.0 x 10 10 cfu / g) 0.4 parts, xylooligosaccharide 0.2 parts, and galactooligosaccharide 0.1 parts.

[0098] Example 10

[0099] The present example provides a probiotic composition, which is formulated as follows in terms of parts of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 2 parts of Bifidobacterium lactis M8, 2 parts of Lactobacillus plantarum LP6, and 1 part of Lactobacillus paracasei PC-01.

[0100] The present example further provides a probiotic capsule containing the probiotic composition described above, which is formulated as follows in terms of weight ratio: the probiotic composition (total number of viable bacteria 5.0 x 10 10 cfu / g) 0.4 parts, galactooligosaccharide 0.5 parts, and erythritol 0.5 parts.

[0101] Comparative Example 1

[0102] The present comparative example provides a probiotic composition, which is different from Example 4 only in that Bifidobacterium lactis MN-Gup is replaced by an equal number of viable bacteria of Bifidobacterium lactis Bb-12.

[0103] Comparative Example 2

[0104] This comparative example provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 3 parts of Bifidobacterium lactis M8, 1 part of Lactobacillus plantarum LP6, and 1 part of Lactobacillus casei Zhang.

[0105] Comparative Example 3

[0106] This comparative example provides a probiotic composition, the formulation of which is as follows in terms of parts by number of viable bacteria: 5 parts of Bifidobacterium lactis MN-Gup, 3 parts of Bifidobacterium lactis M8, 1 part of Lactobacillus plantarum LP6, and 1 part of Lactobacillus paracasei PC-01.

[0107] Experimental Example

[0108] I. Efficacy Verification Experiment

[0109] 1. Experimental Method

[0110] 1) In vitro experiment:

[0111] The glycerol bacterial solution of the test probiotic composition and the single strain control group was inoculated into 5 mL of sterilized MRS or TPY liquid medium (115°C, 15 min) at a 2% inoculation amount, activated under anaerobic conditions for 18 h, and the method was continued for 2 generations. The strain of the third generation of activation was used for testing. The activated bacterial solution was diluted 100 times, and the cell counting plate was used for strain counting.

[0112] The activated probiotic composition is inoculated into 5 mL pH 2.0, pH 3.0 artificial gastric juice and bile salt MRS or TPY medium, and the inoculated culture is incubated at 37°C under anaerobic conditions for 4 h. Samples are taken at 0 and 4 h of incubation, respectively, for dilution, and appropriate dilution gradients are selected for viable count and 4 h survival rate calculation. The pH 2.0 artificial gastric juice is prepared by dissolving NaCI 0.2%, pepsin 0.35% in distilled water, and adjusting the pH to 2.0 with 1 mol / L HCI, and filtering to remove bacteria; the pH 3.0 artificial gastric juice is prepared by dissolving NaCI 0.2%, pepsin 0.35% in distilled water, and adjusting the pH to 3.0 with 1 mol / L HCI, and filtering to remove bacteria; the bile salt MRS medium is prepared by adding bile salt to MRS liquid medium to a final concentration of 0.3%, and sterilizing at 115°C for 15 min.

[0113] The o-phthalaldehyde method is used for experimental analysis. The probiotic composition is inoculated into liquid MRS-cholesterol or TPY-cholesterol medium (liquid medium is added with bile salt and 10 g / L cholesterols solution to a final concentration of 0.3% bile salt and 100 ug / mL cholesterols, sterilized at 115°C for 15 min) at an inoculation amount of 2%, and incubated at 37°C under anaerobic conditions for 18 h, with un-inoculated medium as a control. The cholesterols content is calculated according to the cholesterols standard curve. The un-inoculated medium is used as a blank control, and the cholesterols degradation rate of the strain is calculated according to the following formula:

[0114] Cholesterol degradation rate (%) = (C-A) / C x 100; where:

[0115] A is the cholesterols content of the fermentation supernatant / pg

[0116] C is the cholesterols content of the blank control / pg

[0117] 2) Animal experiment:

[0118] SPF male SD rats provided by Sibeifeng (Beijing) Biotechnology Co., Ltd. [License No.: SCXK (Jing) 2019-0010, Experimental Animal Quality Certificate No.: No. 110324210106509617] are selected. After purchase, they are adapted to the environment for 7 days, and at the end of the adaptation period, the body weight is 180-220 g. The animals are randomly divided into 6 groups.

[0119] Animal feeding management conditions: model feed: add 20.0% sucrose, 15% lard, 1.2% cholesterol, 0.2% sodium cholate, appropriate casein, calcium hydrogen phosphate, stone powder, etc. in the maintenance feed. Except for crude fat, the moisture, crude protein, crude fat, crude fiber, crude ash, calcium, phosphorus, calcium: phosphorus of the model feed all meet the national standard of maintenance feed. The model feed and maintenance feed are provided by Beijing Huafukang Biotechnology Co., Ltd. [License No.: SCXK(Jing)2019-0008]. Experimental animal use license No.: SYXK(Jing)2018-0007, free intake of drinking water, animal room temperature 20-26℃, humidity 40%-70%.

[0120] The recommended consumption of probiotic powder formula is 500 billion cfu / g / day. This experiment sets 6 groups (as shown in Table 1), including low, medium and high dose groups of probiotic composition, model control group, positive control group and blank control group. The low, medium and high doses are 5 times, 10 times and 30 times of the recommended human intake, i.e. 0.083g / kg·BW, 0.167g / kg·BW and 0.500g / kg·BW. Accurately weigh 0.415g, 0.835g and 2.500g of the test sample, respectively, and dilute to 50mL with deionized water to prepare low, medium and high dose groups. The model control group is given deionized water. The test animals are given gavage at 10mL / kg·BW, once a day. The gavage amount is adjusted according to the body weight every week, and the sample is given continuously for 30 days, and necessary extension for 45 days.

[0121] The body weight of each group of experimental rats is weighed once a week. After 7 days of model feed for the model group, the blank control group and the model group are not fasted for blood sampling (intraocular canthus). After blood sampling, the serum is separated by centrifugation, and the levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) in the serum are determined. According to the TC level, the model group is randomly divided into model control group, positive control group and low, medium and high dose groups. There is no significant difference in TC, TG, LDL-C and HDL-C between the model group (model control group, positive control group and three dose groups). After grouping, the three dose groups are given test samples every day, the positive control group is given positive drug, the blank control group and the model control group are given deionized water, the blank control group is continuously given maintenance feed, and the model control group and the three dose groups are continuously given model feed. The body weight is weighed regularly, and after 30 and 45 days of test, the blood is sampled without fasting, and the serum is separated as soon as possible after blood sampling. The serum TC, TG, LDL-C and HDL-C are determined.

[0122] Table 1 Experimental grouping

[0123]

[0124] 2. Experimental results

[0125] 1) In vitro experiment results

[0126] The cholesterol removal rate results of the single strain control group are shown in Table 2 below.

[0127] Table 2

[0128]

[0129] The results of the probiotic compositions in each example and comparative example are shown in Table 3 below.

[0130] Table 3

[0131]

[0132]

[0133] From the table, in terms of cholesterol removal rate, pH 2.0 survival rate, pH 3.0 survival rate, and bile salt survival rate, the probiotic compositions in Examples 3 and 8 are the optimal formulations.

[0134] 2) Animal experiment results

[0135] The following mainly reflects the effect data of the animal experiment of the probiotic compositions in Examples 3 and 8.

[0136] Table 4-1 Blood lipid levels of Example 3 SD rats before administration

[0137]

[0138] *P < 0.05 compared with the blank control group, **P < 0.01 compared with the blank control group.

[0139] As can be seen from Table 4-1, after 7 days of modeling according to the probiotic composition in Example 3, statistical analysis showed that the model group (model control group, positive control group, and formulation 3 dose group) had higher TC, TG, and LDL-C in serum compared with the blank control group, and the differences were all extremely significant (P < 0.01), and the difference in serum HDL-C was not significant (P > 0.05), indicating that the model was successfully constructed.

[0140] Table 4-2 Blood lipid levels of Example 8 SD rats before administration

[0141]

[0142]

[0143] **P < 0.01 compared with the blank control group.

[0144] As shown in Table 4-2, after 7 days of modeling with the probiotic composition in Example 8, statistical analysis showed that the serum TC, TG and LDL-C of the model group (model control group, positive control group and three dose groups) were higher than those of the blank control group, and the differences were significant (P<0.01), and the difference in serum HDL-C was not significant (P>0.05), indicating that the model was established.

[0145] Table 5-1 Effect of probiotic agent formulation in Example 3 on the body weight of SD rats

[0146]

[0147] ##Compared with the model control group, P<0.01.

[0148] As shown in Table 5-1, after 30 days of oral administration of different doses of probiotic composition in Example 3 to SD rats, statistical analysis showed that the body weight of SD rats in each dose group and the positive control group had no significant difference compared with the model control group (P>0.05).

[0149] Table 5-2 Effect of probiotic agent formulation in Example 8 on the body weight of SD rats

[0150]

[0151]

[0152] ## Compared with the model control group, P<0.01.

[0153] As shown in Table 5-2, after 45 days of oral administration of different doses of probiotic composition in Example 8 to SD rats, statistical analysis showed that the body weight of SD rats in each dose group and the positive control group had no significant difference compared with the model control group (P>0.05).

[0154] As Figure 1 shown, after 30 days of oral administration of different doses of probiotic composition in Example 3 to SD rats, statistical analysis showed that the serum TC of the low and medium dose groups was significantly lower than that of the model control group, and the difference was statistically significant (P<0.05), and the TC of the positive control group was significantly lower than that of the model control group, and the difference was statistically significant (P<0.05), and there was no significant difference between the low and medium dose groups and the positive control group (P>0.05). It is proved that the low and medium dose groups effectively reduce the content of serum TC, and the effect is consistent with that of the positive control drug.

[0155] Table 6 Effect of Example 8 on the serum TC of SD rats

[0156]

[0157] #P < 0.05 vs. model control group, ##P < 0.01 vs. model control group.

[0158] As shown in Table 6, the SD rats were orally administered with the probiotic composition in Example 8 in different dosages for 45 days. Statistical analysis showed that, for the subjects administered for 30 days and 45 days, the serum TC in each dosage group had no statistically significant difference (P > 0.05) compared with the model control group. The serum TC in the positive control group was significantly lower than that in the model control group, and the difference was statistically significant (P < 0.05).

[0159] As shown in Table 6, the SD rats were orally administered with the probiotic composition in Example 8 in different dosages for 45 days. Statistical analysis showed that, for the subjects administered for 30 days and 45 days, the serum TC in each dosage group had no statistically significant difference (P > 0.05) compared with the model control group. The serum TC in the positive control group was significantly lower than that in the model control group, and the difference was statistically significant (P < 0.05). Figure 2 As shown in Table 6, the SD rats were orally administered with the probiotic composition in Example 8 in different dosages for 45 days. Statistical analysis showed that, for the subjects administered for 30 days and 45 days, the serum TC in each dosage group had no statistically significant difference (P > 0.05) compared with the model control group. The serum TC in the positive control group was significantly lower than that in the model control group, and the difference was statistically significant (P < 0.05).

[0160] Table 7 Effect of Example 8 on serum TG of SD rats

[0161]

[0162] #P < 0.05 vs. model control group, ##P < 0.01 vs. model control group.

[0163] As shown in Table 7, the SD rats were orally administered with the probiotic composition in Example 8 in different dosages for 45 days. Statistical analysis showed that, for the subjects administered for 30 days, the serum TG in each dosage group had no statistically significant difference (P > 0.05) compared with the model control group. For the subjects administered for 45 days, the serum TG in the medium and high dosage groups was significantly lower than that in the model control group, and the difference was statistically significant (P < 0.05). For the subjects administered for 30 days and 45 days, the serum TG in the positive control group was significantly lower than that in the model control group, and the difference was statistically significant (P < 0.05).

[0164] As shown in Table 7, the SD rats were orally administered with the probiotic composition in Example 8 in different dosages for 45 days. Statistical analysis showed that, for the subjects administered for 30 days, the serum TG in each dosage group had no statistically significant difference (P > 0.05) compared with the model control group. For the subjects administered for 45 days, the serum TG in the medium and high dosage groups was significantly lower than that in the model control group, and the difference was statistically significant (P < 0.05). For the subjects administered for 30 days and 45 days, the serum TG in the positive control group was significantly lower than that in the model control group, and the difference was statistically significant (P < 0.05). Figure 3As can be seen from Table 8, the probiotic composition in Example 8 was orally administered to SD rats at different doses for 45 days, and statistical analysis showed that, after 30 days and 45 days of administration, the serum HDL-C of each dose group had no statistically significant difference compared with the model control group (P>0.05), and the serum HDL-C of the positive control group had no statistically significant difference compared with the model control group (P>0.05).

[0165] Table 8 Effect of Example 8 on serum HDL-C of SD rats

[0166]

[0167]

[0168] As can be seen from Table 8, the probiotic composition in Example 8 was orally administered to SD rats at different doses for 45 days, and statistical analysis showed that, after 30 days and 45 days of administration, the serum HDL-C of each dose group had no statistically significant difference compared with the model control group (P>0.05), and the serum HDL-C of the positive control group had no statistically significant difference compared with the model control group (P>0.05).

[0169] As Figure 4 As can be seen from Table 8, the probiotic composition in Example 8 was orally administered to SD rats at different doses for 45 days, and statistical analysis showed that, after 30 days and 45 days of administration, the serum HDL-C of each dose group had no statistically significant difference compared with the model control group (P>0.05), and the serum HDL-C of the positive control group had no statistically significant difference compared with the model control group (P>0.05).

[0170] Table 9 Effect of Example 8 on serum LDL-C of SD rats

[0171]

[0172] ## Compared with the model control group, P<0.01.

[0173] As can be seen from Table 8, the probiotic composition in Example 8 was orally administered to SD rats at different doses for 45 days, and statistical analysis showed that, after 30 days and 45 days of administration, the serum HDL-C of each dose group had no statistically significant difference compared with the model control group (P>0.05), and the serum HDL-C of the positive control group had no statistically significant difference compared with the model control group (P>0.05).

[0174] Based on the above experimental results, after modeling for 7 days, statistical analysis showed that the serum TC, TG and LDL-C of the model group (model control group, positive control group and formula 6 dose groups) were higher than those of the blank control group, and the differences were extremely significant (P<0.01), and the difference in serum HDL-C was not significant (P>0.05), indicating that the model was successfully constructed. After orally administering the probiotic composition to SD rats at different doses for 30 days and / or 45 days, statistical analysis showed that the body weight of each dose group and the positive control group had no significant difference compared with the model control group (P>0.05). The probiotic composition in Example 3 can significantly reduce cholesterol and triglycerides, and can be used to prevent and improve high cholesterol and high triglyceride symptoms.

[0175] After 45 days, the serum TG of the medium and high dose groups was significantly lower than that of the model control group, and the difference was statistically significant (P<0.05). The probiotic composition in Example 8 can significantly reduce serum triglyceride levels and can be used to prevent and improve high triglyceride symptoms.

[0176] Although the present application has been described in detail above with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application.

Claims

1. A probiotic composition, characterized in that, It consists of the following probiotics: The first strain: Bifidobacterium lactis MN-Gup, with accession number CGMCCNo.15578; The second strain: Bifidobacterium lactis M8, with accession number CGMCC No.16070; The third strain: *Lactobacillus plantarum* LP6, with accession number CGMCC No. 13458; and, The fourth strain: Lactobacillus paracasei PC-01, with accession number CGMCC No. 17537; Wherein, based on the total number of live bacteria in the probiotic composition, the proportion of live bacteria in the first strain is greater than or equal to 40%, the total proportion of the third strain and the fourth strain is greater than or equal to 30%, and the ratio of live bacteria in the first strain, the second strain, the third strain and the fourth strain is (4-6):(0.8-1.2):(1.5-2.5):(1.5-2.5); The total number of live bacteria in the probiotic composition is greater than or equal to 5.0 × 10⁻⁶. 10 cfu / gram.

2. The probiotic composition according to claim 1, characterized in that, Based on the total number of live bacteria in the probiotic composition, the percentage of live bacteria in the first strain is 50 ± 10%.

3. A probiotic preparation, characterized in that, It contains the probiotic composition as described in claim 1 or 2.

4. The use of the probiotic composition of claim 1 or 2 or the probiotic agent of claim 3 in the preparation of health foods that help maintain healthy blood lipid levels or in the preparation of medicines for the prevention, relief or improvement of hyperlipidemia.

5. The application according to claim 4, characterized in that, The medicine used to prevent, alleviate or improve hyperlipidemia has at least one of the following uses: (1) Prevent, alleviate or improve symptoms of high cholesterol; (2) Prevent, relieve or improve symptoms of high triglycerides.

6. A health food product that helps maintain healthy blood lipid levels or a medicine for preventing, alleviating, or improving hyperlipidemia, characterized in that, It contains the probiotic composition of claim 1 or 2 or the probiotic agent of claim 3.

7. The health food product according to claim 6 that helps maintain healthy blood lipid levels, characterized in that, It also contains an edible carrier, which is selected from one or more of the following: milk powder, maltodextrin, oligosaccharides, dietary fiber, fruit juice powder, sugar alcohol, starch, and silicon dioxide.

8. The medicament for preventing, alleviating, or improving hyperlipidemia according to claim 6, characterized in that, It also contains an edible carrier, which is selected from one or more of the following: milk powder, maltodextrin, oligosaccharides, dietary fiber, fruit juice powder, sugar alcohol, starch, and silicon dioxide.

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