An auxiliary hypolipidemic composition and use thereof

Abstract

The present application relates to the technical field of functional product, and particularly relates to a composition for assisting in reducing blood lipid and application thereof.The present application provides a composition which is composed of animal bifidobacterium lactis MN-Gup, nannochloropsis oil and phytosterol ester.The composition of the present application can effectively reduce the levels of triglyceride and total cholesterol, and particularly can significantly reduce low-density lipoprotein cholesterol, and simultaneously increase the content of high-density lipoprotein cholesterol, and compared with each single component, the composition has significant advantages, has a significant effect of assisting in reducing blood lipid, and has good safety and industrialization feasibility, thereby providing a basis for development of a product for assisting in reducing blood lipid, and providing a new intervention scheme for blood lipid health management.

Description

Technical Field

[0001] This invention relates to the field of functional product technology, and in particular to an adjunct lipid-lowering composition and its application. Background Technology

[0002] Dyslipidemia is a significant risk factor for cardiovascular disease, and long-term dyslipidemia is closely related to the occurrence and development of atherosclerosis and cerebrovascular diseases. Hyperlipidemia is mainly characterized by elevated serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C), as well as decreased high-density lipoprotein cholesterol (HDL-C).

[0003] Currently, lipid-lowering interventions mainly include drug therapy and dietary regulation. Among them, statins (such as atorvastatin and rosuvastatin) are the most commonly used lipid-lowering drugs in clinical practice, effectively reducing low-density lipoprotein cholesterol (LDL-C) levels. However, long-term use may cause side effects such as liver damage, muscle disease, and increased risk of diabetes, and some people have poor tolerance to them. Therefore, there is an urgent need for safe, effective, and long-term intervention methods.

[0004] Based on safety and edibility considerations, functional components derived from food sources or the microecology have gradually become an important direction in adjuvant lipid-lowering research. Although existing studies have shown that various food-derived or microecological components have lipid-lowering potential to some extent, the target points of a single component are limited, and the effects are greatly affected by individual differences, dosage, and compatibility conditions, resulting in insufficient stability of their efficacy.

[0005] For multi-component compound systems, existing technologies generally suffer from the following problems: (1) The interaction between different active ingredients is not yet clear; (2) There is a lack of scientific basis, and the formulation design relies heavily on experience and judgment, resulting in insufficient reproducibility; (3) There is a lack of in vivo experimental evidence, making it difficult to form a reliable chain of evidence; (4) It is difficult for compound systems to balance safety, efficacy and dosage form compatibility, and there is a lack of industrializable technical paths and quality control standards.

[0006] Therefore, there is an urgent need to provide a multi-component compound formulation based on scientific ratio and synergistic mechanism verification, which can simultaneously improve key indicators such as triglycerides, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in in vivo models, and has the characteristics of high safety and stable process, so as to meet the technical requirements of auxiliary lipid-lowering products in terms of safety, effectiveness and industrialization. Summary of the Invention

[0007] This invention provides an adjunct lipid-lowering composition and its application.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides a composition comprising the following components: Bifidobacterium animalis subsp. lactis (… Bifidobacterium animalis subsp.lactis The product consists of MN-Gup, *Micrococcus pseudocarpa* oil, and phytosterol esters; wherein the ratio of *Bifidobacterium animalis* subsp. *lactocarpa* MN-Gup, *Micrococcus pseudocarpa* oil, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 4-40μg; 49-139μg; the animal Bifidobacterium lactis subsp. ( Bifidobacterium animalis subsp.lactis MN-Gup is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 15578.

[0010] Phytosterol esters, *Micrococcus pluvialis* oil, and probiotics are all known functional ingredients for lipid regulation. For example, probiotics can influence cholesterol homeostasis by regulating intestinal flora balance and improving lipid absorption and metabolism; Omega-3 polyunsaturated fatty acids can reduce very low-density lipoprotein (VLDL) synthesis and promote fatty acid oxidation; phytosterols and their esters can competitively inhibit cholesterol absorption and promote excretion in the intestine. However, the interaction mechanisms between phytosterol esters, *Micrococcus pluvialis* oil, and different probiotics vary. During the research and development process, this invention discovered that when different probiotics with lipid-regulating functions are combined with phytosterol esters and *Micrococcus pluvialis* oil, the different interaction mechanisms between the components lead to significant differences in the lipid-lowering performance of the compositions. Through continuous screening and verification, this invention has discovered that when a specific probiotic—Bifidobacterium animalis subsp. lactis MN-Gup—is combined with phytosterol esters and microalgae oil in a certain ratio, the three components can produce a synergistic effect, significantly improving the overall effect of improving lipid metabolism. The lipid-lowering effect is significantly enhanced compared with that of a single component. It shows good results in reducing triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels, as well as increasing high-density lipoprotein cholesterol (HDL-C) levels. This achieves a synergistic effect of multi-target joint regulation, providing a safe and effective solution for lipid regulation and cardiovascular health.

[0011] Specifically, this invention has found that: when Bifidobacterium animalis subsp. lactis MN-Gup is used alone, it has a certain effect on reducing TC and TG, but the extent is limited; when Micrococcus pseudochlorella oil is used alone, it can reduce TG but its effect on increasing HDL-C is not significant; when phytosterol esters are used alone, they mainly reduce TC but have limited inhibitory effect on LDL-C. However, when Bifidobacterium animalis subsp. lactis MN-Gup, Micrococcus pseudochlorella oil, and phytosterol esters are combined according to the specific ratio described in this invention, compared with using the components alone or using combinations with other ratios, the composition unexpectedly has the effect of significantly reducing TG, TC, and LDL-C and increasing HDL-C, showing a significant synergistic effect.

[0012] In this invention, *Bifidobacterium animalis* subsp. *lactamella* MN-Gup was deposited on April 10, 2018, at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101), with accession number CGMCC No. 15578, and classified as *Bifidobacterium animalis* subsp. *lactamella*. Bifidobacterium animalis subsp.lactis (), has been disclosed in patent application CN111826299A.

[0013] Preferably, the ratio of Bifidobacterium animalis subsp. lactis MN-Gup, Micrococcus pseudocarpa oil, and phytosterol esters in the composition is 2 × 10⁻⁶. 4 CFU: 10-40μg; 49-139μg.

[0014] More preferably, the ratio of *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil, and phytosterol esters in the composition is 2 × 10⁻⁶. 4 CFU: 19.6-40 μg; 49-100 μg. Within this ratio range, the components exhibit a more stable synergistic effect overall.

[0015] More preferably, the ratio of *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil, and phytosterol esters in the composition is 2 × 10⁻⁶. 4 CFU: 36-40μg; 49-59μg.

[0016] More preferably, the ratio of *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil, and phytosterol esters in the composition is 2 × 10⁻⁶. 4 CFU: 40μg: 49μg.

[0017] In the composition described above, the phytosterol ester contains ≥90% by mass of phytosterol esters, and the total mass percentage of phytosterol esters and phytosterols is ≥97%. Preferably, the total sterol ester content of the phytosterol ester is not less than 59.0%. The phytosterol ester is preferably prepared from soybean-derived phytosterols.

[0018] Preferably, the microalgae oil contains ≥12% EPA and ≥20% ALA by mass.

[0019] In the above-described composition, the *Bifidobacterium animalis* subsp. *lactamella* MN-Gup is preferably provided in the form of bacterial powder. The added mass of bacterial powder can be calculated based on its viable bacteria content per unit mass. The *Microcarboxyphylla* oil is preferably provided in the form of *Microcarboxyphylla* oil powder. *Microcarboxyphylla* oil powder is a microcapsule powder made by using *Microcarboxyphylla* oil as the core material and microencapsulation technology. The oil loading rate of *Microcarboxyphylla* oil powder is preferably 30%-50% (more preferably 40%). Optionally, the mass ratio of *Bifidobacterium animalis* subsp. *lactamella* MN-Gup bacterial powder, *Microcarboxyphylla* oil powder, and phytosterol esters is 1:10-100:49-139, more preferably 1:25-100:49-139, more preferably 1:49-100:49-100, more preferably 1:90-100:49-59, and more preferably 1:100:49.

[0020] The compositions described in this invention may also include excipients acceptable in the pharmaceutical or food fields.

[0021] The compositions of the present invention can be liquid formulations or solid formulations.

[0022] The composition of the present invention may also contain an active ingredient with nutritional regulatory activity, and its dosage form may be powder, granules, tablets, capsules or oral liquid; the content of active ingredient contained in a single dose unit in each dosage form may be adjusted according to the differences in blood lipid levels of the target population, so as to achieve the desired physiological regulatory effect by continuously administering multiple dose units.

[0023] Secondly, the present invention provides a method for preparing the above-described composition, the method comprising: mixing Bifidobacterium animalis subsp. lactis MN-Gup, microalgae oil and phytosterol esters.

[0024] Thirdly, the present invention provides the use of the above-described composition in the preparation of products that lower blood lipids or assist in lowering blood lipids.

[0025] Preferably, the product is a health food or a medicine.

[0026] Fourthly, the present invention provides the use of the above-described composition in the preparation of health foods that help maintain healthy blood lipid levels.

[0027] In this invention, the blood lipids are defined as one or more selected from total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C).

[0028] Fifthly, the present invention provides the application of the above-described composition in the preparation of health foods that assist in lowering blood lipids or improving blood lipid metabolism.

[0029] The health foods mentioned include, but are not limited to, functional dairy products, solid beverages, and fortified foods.

[0030] In this invention, the Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil, and phytosterol esters can all be made from food-grade raw materials and can be used in the preparation of health foods.

[0031] In a sixth aspect, the present invention provides the use of the above-described composition in the preparation of lipid-lowering drugs or drugs for treating hyperlipidemia.

[0032] In this invention, the lipid-lowering effect includes any one or more of the following: lowering total cholesterol (TC), lowering triglycerides (TG), lowering low-density lipoprotein cholesterol (LDL-C), and increasing high-density lipoprotein cholesterol (HDL-C).

[0033] In a seventh aspect, the present invention provides the use of the above-described compositions in adjuvant lipid-lowering or maintenance of healthy lipid levels for non-disease treatment purposes.

[0034] Among them, the auxiliary lipid-lowering or maintenance of healthy lipid levels for non-disease treatment purposes includes maintenance of healthy lipid levels for people with high blood lipids and the general population (non-hyperlipidemia patients) who are concerned about cardiovascular health.

[0035] Eighthly, the present invention provides a product comprising the composition described in the first aspect above.

[0036] Preferably, the product is a health food or a medicine.

[0037] The product may use the composition as the active ingredient, or may be formulated with other lipid-lowering or other active ingredients in addition to the composition.

[0038] When the product is a health food, it may also contain excipients permitted in the food industry. Exemplary excipients include: antioxidants (e.g., vitamin E, rosemary extract, tea polyphenols, etc.), emulsifiers (e.g., mono- and diglyceride fatty acid esters, soy lecithin), sweeteners / flavoring agents (e.g., erythritol, steviol glycosides, citric acid, steviol, menthol, etc.), pH adjusters (e.g., sodium citrate, sodium bicarbonate, disodium hydrogen phosphate, citric acid, etc.), and preservatives.

[0039] When the product is a pharmaceutical product, it may also contain excipients permitted in the pharmaceutical field. Exemplary excipients include: antioxidants (e.g., vitamin E, propyl gallate, sodium bisulfite, etc.), emulsifiers (e.g., soybean lecithin, egg yolk lecithin, etc.), sustained-release matrix (e.g., beeswax, stearic acid, etc.), sweeteners / flavoring agents (e.g., erythritol, steviol glycosides, citric acid, steviol, menthol, etc.), pH adjusters (e.g., sodium citrate, sodium bicarbonate, disodium hydrogen phosphate, citric acid, etc.), and preservatives.

[0040] The beneficial effects of this invention include at least the following: The composition provided by this invention contains Bifidobacterium animalis subsp. lactis MN-Gup, microalgae oil, and phytosterol esters. Verification has shown that, within the permissible range of adverse reactions, the composition of this invention can simultaneously and effectively reduce total cholesterol (TC) and triglyceride (TG) levels, particularly significantly reducing low-density lipoprotein cholesterol (LDL-C) while increasing high-density lipoprotein cholesterol (HDL-C) levels. Compared to each individual component, it exhibits significant advantages, demonstrating significant lipid-lowering efficacy and good safety and industrial feasibility. This provides a basis for the development of functional foods and nutritional intervention foods as auxiliary lipid-lowering products, and offers a new intervention strategy for lipid health management. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0042] Figure 1 This is the experimental result of the effect of the sample on the fluorescence intensity of cholesterol in the tail blood vessels of zebrafish in the experimental examples of this invention (reflecting triglyceride levels); compared with the model control group, p<0.05, p<0.01, p<0.001; compared with composition 3, @ p<0.05, @@@ p<0.001; compared with composition 4, # p<0.05, ## p<0.01, ### p<0.001; compared with composition 5, ！ p<0.05, ！！ p<0.01, ！！！ p<0.001; compared with composition 6, Compared with composition 7, & p<0.05, &&& p<0.001.

[0043] Figure 2 This is the experimental result of the effect of the sample on the staining intensity of zebrafish tail blood vessels (reflecting cholesterol levels) in the experimental examples of this invention; compared with the model control group, p<0.05, p<0.01, p<0.001; compared with composition 3, @ p<0.05, @@@ p<0.001; compared with composition 4, # p<0.05, ## p<0.01, ### p<0.001; compared with composition 5, ！ p<0.05, ！！ p<0.01, ！！！ p<0.001; compared with composition 6, Compared with composition 7, & p<0.05, &&& p<0.001.

[0044] Figure 3 These are the experimental results of the effect of the samples on the HDL-C content of zebrafish in the experimental examples of this invention; compared with the model control group, p<0.05, p<0.01, p<0.001; compared with composition 3, @ p<0.05, @@@ p<0.001; compared with composition 4, # p<0.05, ## p<0.01, ### p<0.001; compared with composition 5, ！ p<0.05, ！！ p<0.01, ！！！ p<0.001; compared with composition 6, Compared with composition 7, & p<0.05, &&& p<0.001.

[0045] Figure 4This presents the experimental results of the effect of the samples on the LDL-C content of zebrafish in the experimental examples of this invention. Compared with the model control group, p<0.05, p<0.01, p<0.001; compared with composition 3, @ p<0.05, @@@ p<0.001; compared with composition 4, # p<0.05, ## p<0.01, ### p<0.001; compared with composition 5, ！ p<0.05, ！！ p<0.01, ！！！ p<0.001; compared with composition 6, Compared with composition 7, & p<0.05, &&& p<0.001.

[0046] Figure 1-4 In this study, the dosage for the sample group was 2000 μg / mL; the positive control was atorvastatin 2 μg / mL; the data are mean ± SEM. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available or prepared according to conventional methods in the art.

[0049] The phytosterol esters used in the following examples and comparative examples were purchased from Shaanxi Haisifu Biotechnology Co., Ltd., with the trade name LowChol. The phytosterol esters had a mass percentage content of ≥90%, and the phytosterol esters and phytosterols (total) had a mass percentage content of ≥97%. The microalgae oil powder was donated by Xiaozao Technology (Anji) Co., Ltd. It is a microcapsule powder made by microencapsulation technology with microalgae oil as the core material (EPA mass percentage content ≥12%, ALA mass percentage content ≥20%) and an oil loading rate of 40%.

[0050] Example 1 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 148μg: 1μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was administered as a bacterial powder (live bacteria content 2×10⁻⁶). 4 Provided in CFU / μg form, at which time the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:148:1.

[0051] Example 2 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 130μg: 19μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was in the form of bacterial powder (live bacteria content 2×10⁻⁶). 4 Provided in CFU / μg form, at which time the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:130:19.

[0052] Example 3 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 10μg: 139μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was in the form of bacterial powder (live bacteria content 2×10⁻⁶). 4 Provided in CFU / μg form, the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:10:139.

[0053] Example 4 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 49μg: 100μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was in the form of bacterial powder (live bacteria content 2×10⁻⁶). 4 Provided in CFU / μg form, at which time the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:49:100.

[0054] Example 5 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 74.5 μg: 74.5 μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was present as a bacterial powder (live bacteria content of 2 × 10⁻⁶). 4 Provided in CFU / μg form, at which time the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:74.5:74.5.

[0055] Example 6 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 100μg: 49μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was administered as a bacterial powder (live bacteria content 2×10⁻⁶). 4 Provided in CFU / μg form, at which time the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:100:49.

[0056] Example 7 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 25μg: 124μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was administered as a bacterial powder (live bacteria content 2×10⁻⁶). 4 Provided in CFU / μg form, at which time the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:25:124.

[0057] Example 8 This embodiment provides a composition comprising *Bifidobacterium animalis* subsp. *lactospirum* MN-Gup, *Micrococcus pseudocarpa* oil powder, and phytosterol esters, wherein the ratio of *Bifidobacterium animalis* subsp. *lactospirum* oil powder, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 1μg: 148μg. Among them, *Bifidobacterium animalis* subsp. *lactamase* MN-Gup was administered as a bacterial powder (live bacteria content 2×10⁻⁶). 4 Provided in CFU / μg form, at which time the mass ratio of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder and phytosterol esters is 1:1:148.

[0058] Comparative Example 1 This comparative example provides a composition, which contains Nannochloropsis oculata oil powder and phytosterol esters, and the mass ratio of Nannochloropsis oculata oil powder to phytosterol esters is 100 μg:49 μg.

[0059] Experimental Example In this invention, the lipid-lowering effects of the compositions and each single component in the above examples are verified through zebrafish hyperglycemic and hyperlipidemic model experiments and multi-index statistical analysis. First, the lipid-lowering effects of three single components, namely Bifidobacterium animalis subsp. lactis MN-Gup, phytosterol esters, and Nannochloropsis oculata oil powder, at different doses are tested respectively, and their effects on triglyceride and cholesterol deposition in zebrafish blood vessels are evaluated. The results show that all three can reduce the staining intensity of zebrafish tail blood vessels and cholesterol fluorescence intensity within a certain concentration range, showing a certain lipid-lowering effect. After determining the maximum test concentration (MTC) of each single component, a verification experiment of combining the three components in different ratios (Examples 1-8) is further designed to systematically compare the differences in the effects of different ratios on reducing triglyceride (TG), cholesterol (TC), and regulating the contents of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C).

[0060] The animal experiments of this invention adopt an animal experiment platform certified by AAALAC, and are verified by referring to the pharmacopoeia and nutritional function detection standards, combined with the zebrafish hyperlipidemia model and LDL-C / HDL-C detection kits, to ensure the scientificity and repeatability of the results. The specific experimental methods and experimental results are as follows.

[0061] 1. Zebrafish strain: For the determination of triglyceride content in the tail vein of hyperlipidemic zebrafish, the zebrafish with melanophore allele mutation (albino) is used, and for the determination of other indexes, the wild-type AB strain of zebrafish is used. Among them, the wild-type AB strain and the zebrafish with melanophore allele mutation (albino) are provided by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd. The experimental animal use license number is: SYXK (Zhe) 2022-0004, and the feeding management meets the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethical review number is: IACUC-2025-11799-01.

[0062] 2. Modeling method and principle: Hyperglycemic and hyperlipidemic-induced zebrafish dyslipidemia model A model was established by administering a high-sugar, high-fat diet containing egg yolk powder and glucose to zebrafish. Egg yolk powder is 60% lipids; when fed to zebrafish along with glucose, it causes damage to the vascular endothelium, leading to the deposition of triglycerides and cholesterol on the blood vessel walls. Under this high-sugar, high-fat diet, zebrafish exhibited significantly elevated levels of triglycerides (TG) and total cholesterol (TC), increased low-density lipoprotein cholesterol (LDL-C), and decreased high-density lipoprotein cholesterol (HDL-C), mimicking the lipid metabolism disorder induced by a high-fat diet.

[0063] Zebrafish lipid metabolism pathways are highly homologous to those in humans, and their key genes (such as hmgcra, ldlr, apoa1, cyp7a1a, etc.) are similar to those in mammals, which can be used to assess the effects and mechanisms of lipid-lowering functional components in vivo. The criteria for successful model establishment are: zebrafish tail vessel staining intensity between 0.05–0.65 (TG) or 0.07–0.67 (TC) in normal / model controls, and LDL-C levels between 0.40–0.80 and HDL-C levels between 0.45–0.85 in normal / model controls, indicating a stable and reliable model establishment.

[0064] 3. Determination of the maximum detectable concentration (MTC) of the sample (1) Experimental group A total of 3 samples were tested (Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder, Micrococcus pseudocarpa oil powder, and phytosterol esters), among which Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder was prepared at 5 concentrations (1×10⁻⁶). 3 1×10 4 1×10 5 1×10 6 1×10 7 Five detection concentrations (125, 250, 500, 1000, and 2000 μg / mL) were set up for both *Micrococcus pseudocarpa* oil powder and phytosterol esters, along with a normal control group and a model control group. Thirty albino zebrafish with melanin allele mutations were included in each group, 5 days post-fertilization (5 dpf).

[0065] (2) Experimental methods Zebrafish (albino) with a melanin allele mutation, 5 days post-fertilization (5 dpf), were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker. The experimental system consisted of 30 zebrafish / 25 mL. Test samples were added according to the groups, and the medium was changed daily. Except for the normal control group, all experimental groups were additionally given a high-sugar, high-fat diet in water to establish a high-sugar, high-fat zebrafish model. After treatment at 28℃ for 2 days, the number of zebrafish deaths and toxicity were recorded in each experimental group to determine the MTC of the samples.

[0066] (3) Evaluation indicators Based on the number of zebrafish deaths and toxicity levels in the experimental group, the mean toxicity (MTC) of the samples was determined, with the highest concentration at which no deaths occurred being used as the MTC for that sample. Under the experimental conditions, the MTC of the *Bifidobacterium animalis* subsp. *lactamase* MN-Gup powder sample was 1 × 10⁻⁶. 6 The CFU / mL and the MTC of both the Microsporum pseudochlorella oil powder and phytosterol ester samples were 2000 μg / mL.

[0067] 4. Determination of its efficacy in lowering triglycerides 4.1 Evaluation of the efficacy of single-sample adjuvant triglyceride reduction (1) Experimental group A normal control group, a model control group, a positive control group, and a test sample group were set up. The normal control group was treated with standard dilution water for zebrafish. The model control group was given 1.5% egg yolk powder during the day (11:00-17:00) and 3% glucose after washing off the egg yolk powder at night (17:00-11:00). The positive control group and the test sample group were treated the same as the model control group (with added egg yolk powder and glucose) except for the administration of the drug throughout the day.

[0068] The positive control group was administered atorvastatin at 11.6 μg / mL; the test samples included five concentration gradients of Bifidobacterium animalis subsp. lactis MN-Gup powder, Micrococcus pseudocarpa oil powder, and phytosterol esters, as detailed below: Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder: 1×10 2 CFU / mL, 1×10 3 CFU / mL, 1×10 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 CFU / mL.

[0069] Micrococcus pseudochlorella oil powder: 125μg / mL, 250μg / mL, 500μg / mL, 1000μg / mL, 2000μg / mL.

[0070] Phytosterol esters: 125 μg / mL, 250 μg / mL, 500 μg / mL, 1000 μg / mL, 2000 μg / mL.

[0071] Each group consists of 30 albino zebrafish with a melanin allele mutation.

[0072] (2) Experimental methods Five-day-fever (5dpf) zebrafish were randomly selected and placed in beakers (25 mL system). Except for the normal control group, all other groups were additionally fed a high-sugar, high-fat diet dissolved in water. After treatment at 28℃ for 2 days, the zebrafish were fixed in 4% tissue cell fixative and stained with Oil Red O for overall fat staining. After destaining, 10 zebrafish from each group were randomly selected and photographed under a dissecting microscope. The staining intensity of the zebrafish tail vessels was analyzed using NIS-Elements D3.20 software.

[0073] (3) Evaluation indicators TG levels were expressed as the staining intensity (pixel value) of the tail vessels. The results showed (Table 1): Under the experimental conditions, all groups of Bifidobacterium animalis subsp. lactis MN-Gup powder significantly reduced TG (p<0.05), all groups of Micrococcus pseudocarpa oil powder significantly reduced TG (p<0.001), and phytosterol esters significantly reduced TG at 250 μg / mL (p<0.05) and 500 μg / mL (p<0.01).

[0074] Table 1. Results of the single-sample efficacy evaluation for lowering triglycerides (single sample) (n=10)

[0075] Note: Compared with the model control group, p<0.05, p<0.01, p<0.001.

[0076] 4.2 Evaluation of the efficacy of the composition in assisting the reduction of triglycerides (1) Experimental group The treatment of the normal control group, model control group, and positive control group (atorvastatin, 11.6 μg / mL) was the same as in section 4.1. Based on the results of previous single-sample dose-effect experiments, the total concentration of the formulation was determined to be 150 μg / mL. Therefore, the same concentration as the composition was set in the single samples to ensure the consistency of the total amount (Bifidobacterium animalis subsp. lactis MN-Gup 3×10). 6 CFU / mL (equivalent to a concentration of 150 μg / mL), therefore, in addition to the normal control group, model control group, and positive control group, animal Bifidobacterium lactis subsp. MN-Gup bacterial powder (1×10⁻⁶) was set up. 6 CFU / mL, 3×10 6 The study included single groups of CFU / mL, *Micrococcus pluvialis* oil powder (150 μg / mL), and phytosterol esters (150 μg / mL), as well as composite groups with different mass ratios, including: Composition 1 (i.e., the composition of Example 1 administered in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×104 CFU / mL + Micrococcus pseudocarpa oil powder 148μg / mL + phytosterol ester 1μg / mL; Composition 2 (i.e., the composition of Example 2 administered in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×10 4 CFU / mL + Micrococcus pseudocarpa oil powder 130μg / mL + phytosterol esters 19μg / mL; Composition 3 (i.e., the composition of Example 3 administered in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×10 4 CFU / mL + Micrococcus pseudocarpa oil powder 10μg / mL + phytosterol esters 139μg / mL; Composition 4 (i.e., the composition of Example 4 administered in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×10 4 CFU / mL + Micrococcus pseudocarpa oil powder 49μg / mL + phytosterol esters 100μg / mL; Composition 5 (i.e., the composition of Example 5 administered in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×10 4 CFU / mL + Micrococcus pseudochlorella oil powder 74.5μg / mL + phytosterol esters 74.5μg / mL; Composition 6 (i.e., the composition of Example 6 administered in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×10 4 CFU / mL + Micrococcus pseudocarpa oil powder 100μg / mL + phytosterol esters 49μg / mL; Composition 7 (i.e., the composition of Example 7 given in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×10 4 CFU / mL + Micrococcus pseudochlorella oil powder 25μg / mL + phytosterol esters 124μg / mL; Composition 8 (i.e., the composition of Example 8 given in water): Bifidobacterium animalis subsp. lactis MN-Gup 2×10 4 CFU / mL + Micrococcus pseudochlorella oil powder 1μg / mL + phytosterol ester 148μg / mL.

[0077] Composition 9 (i.e., the composition given in water as in Comparative Example 1): Micrococcus pseudochlorella oil powder 100.7 μg / mL + phytosterol ester 49.3 μg / mL.

[0078] Each group consists of 30 albino zebrafish with a melanin allele mutation.

[0079] (2) Experimental method: Same as described in (2) of 4.1 above.

[0080] (3) Evaluation indicators TG levels were expressed as the staining intensity (pixel value) of the tail vessels. Results showed ( Figure 1 Under the experimental conditions, compositions 1-9 and each single sample group (Bifidobacterium animalis subsp. lactis MN-Gup powder, Micrococcus pseudocarpa oil powder and phytosterol esters) significantly reduced TG (p<0.05). Among them, compositions 3-7 were significantly better than each single component and composition 9 (Micrococcus pseudocarpa oil powder + phytosterol esters) in reducing TG. Compositions 4-6 had the best effect in reducing TG, and composition 6 reduced TG by the largest amount.

[0081] 5. Determination of its effect in assisting in lowering cholesterol 5.1 Evaluation of the efficacy of single-sample adjuvant cholesterol reduction (1) Experimental group: as described in (1) of 4.1 above.

[0082] (2) Experimental method: Wild-type AB strain zebrafish (5 dpf) were randomly selected, with 30 fish per group, and each group was given a water-soluble feed. Except for the normal control group, all other groups were additionally given a high-sugar, high-fat diet in water. After treatment at 28℃ for 1 day, a cholesterol probe was injected intravenously, and after another day of treatment, fluorescence images of the tail vessels were captured. The fluorescence intensity was analyzed using NIS-Elements D3.20 software to reflect TC levels.

[0083] (3) Evaluation indicators: The cholesterol fluorescence intensity of the tail vessels was used as an indicator to represent TC levels. The results showed (Table 2): Under the experimental conditions, all groups of Bifidobacterium animalis subsp. lactis MN-Gup powder significantly reduced TC (p<0.05), all groups of Micrococcus pseudochlorella oil powder significantly reduced TG (p<0.001), and phytosterol esters significantly reduced TC in the range of 125 μg / mL to 1000 μg / mL (p<0.05).

[0084] Table 2. Results of the experiment evaluating the efficacy of single-sample adjuvant cholesterol-lowering therapy (n=10)

[0085] Note: Compared with the model control group, p<0.05, p<0.01, p<0.001.

[0086] 5.2 Evaluation of the efficacy of the composition in assisting cholesterol reduction (1) Experimental group: as described in (1) of 4.2 above.

[0087] (2) Experimental method: Same as described in (2) of 5.1 above.

[0088] (3) Evaluation indicators The cholesterol fluorescence intensity in the tail vessels was used as an indicator to represent TC levels. The results showed ( Figure 2 Under the experimental conditions, compositions 1-9 and each individual sample group (Bifidobacterium animalis subsp. lactis MN-Gup powder, Micrococcus pseudocarpa oil powder, and phytosterol esters) significantly reduced total TC. Compositions 3 and 7 showed better efficacy in reducing TC than Bifidobacterium animalis subsp. lactis MN-Gup powder 1×10⁻⁶. 6 CFU / mL, Micrococcus pseudocarpa oil powder, phytosterol esters and composition 9 (Micrococcus pseudocarpa oil powder + phytosterol esters), and 3×10 of Bifidobacterium animalis subsp. lactis MN-Gup bacterial powder. 6 The CFU / mL efficacy was comparable; compositions 4-6 were all more effective than the individual sample groups in reducing TC, and were also more effective than compositions 1, 2, 8 and 9; composition 6 reduced TC by the greatest extent.

[0089] 6. Effects on LDL-C and HDL-C levels (1) Experimental group: as described in (1) of 4.2 above.

[0090] (2) Experimental methods Wild-type AB strain zebrafish (5 dpf) were randomly selected, with 30 fish per group, and each group was fed a water-soluble diet. Except for the normal control group, all other groups were additionally fed a high-sugar, high-fat diet via water-soluble administration. Six biological replicates were performed. Samples were collected after treatment at 28℃ for 2 days, and the LDL-C and HDL-C content was detected and calculated using LDL-C and HDL-C kits.

[0091] (3) Evaluation indicators The levels of low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in zebrafish were used as indicators. Results showed (Tables 3, 4, and...) Figure 3 , Figure 4 Under the experimental conditions, (1) Compositions 1-9 and each single sample group (Bifidobacterium animalis subsp. lactis MN-Gup powder, Micrococcus pseudocarpa oil powder and phytosterol ester) all have the effect of reducing LDL-C content and increasing HDL-C content; (2) Compositions 3 and 7 have better effects of reducing LDL-C content and increasing HDL-C content than each single sample group and composition 9 (Micrococcus pseudocarpa oil powder + phytosterol ester); (3) Compositions 4-6 have better effects of reducing LDL-C content and increasing HDL-C content than the single sample groups, and are better than composition 1, composition 2, composition 8 and composition 9; (4) Composition 6 has the best effect of reducing LDL-C content and increasing HDL-C content.

[0092] Table 3. Experimental results of the effect of samples on LDL-C content (compositions)

[0093] Note: Compared with the model control group, p<0.05, p<0.01, p<0.001; compared with composition 3, @ p<0.05; compared with composition 4, # p<0.05, ## p<0.01; compared with composition 5, ！ p<0.05, ！！ p<0.01; compared with composition 6, Compared with composition 7, & p<0.05, && p<0.01.

[0094] Table 4. Experimental results of the effect of samples on HDL-C content (compositions)

[0095] Note: Compared with the model control group, p<0.05, p<0.01, p<0.001; compared with composition 3, @ p<0.05; compared with composition 4, # p<0.05, ## p<0.01, ### p<0.001; compared with composition 5, ！ p<0.05, ！！ p<0.01; compared with composition 6, Compared with composition 7, & p<0.05, && p<0.01.

[0096] 7. Statistical methods All data are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 was considered statistically significant. The KS normality test was used to determine the distribution between two groups: if p > 0.05, an independent samples t-test was used; if p < 0.05, a nonparametric test was used.

[0097] The results above show that, compared with each individual component, the composition of the present invention can more effectively reduce serum total cholesterol (TC) and triglyceride (TG) levels at the same dosage, especially significantly reducing low-density lipoprotein cholesterol (LDL-C) while increasing high-density lipoprotein cholesterol (HDL-C) levels, thereby effectively assisting in the function of lowering blood lipids. This provides a clear technical solution and data support for the development of highly effective auxiliary products for lowering blood lipids.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition, characterized in that, The composition is prepared in a ratio of 2×10 4 CFU: 4-40μg; Bifidobacterium lactis subsp. animalis: 49-139μg Bifidobacterium animalis subsp.lactis Composed of MN-Gup, microalgae oil and phytosterol esters; The Bifidobacterium animalis subsp. lactis MN-Gup is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 15578. The *Micrococcus pseudocarpa* oil contains ≥12% EPA and ≥20% ALA by mass. The phytosterol esters contain ≥90% by mass and ≥97% by mass of both phytosterol esters and phytosterols.

2. The composition according to claim 1, characterized in that, In the composition, the ratio of Bifidobacterium animalis subsp. lactis MN-Gup, Micrococcus pseudocarpa oil, and phytosterol esters is 2 × 10⁻⁶. 4 CFU: 19.6-40μg: 49-100μg.

3. The use of the composition according to claim 1 or 2 in the preparation of health food products that help maintain healthy blood lipid levels.

4. The use of the composition according to claim 1 or 2 in the preparation of health foods that assist in lowering blood lipids or improving blood lipid metabolism.

5. The use of the composition according to claim 1 or 2 in the preparation of lipid-lowering drugs or drugs for treating hyperlipidemia.

6. The use of the composition according to claim 1 or 2 in adjunctive lipid-lowering or maintenance of healthy lipid levels for non-disease treatment purposes.

7. A product characterized in that, The product comprises the composition according to claim 1 or 2.

8. The product according to claim 7, characterized in that, The product is either a health food or a medicine.

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