A probiotic oil preparation for improving non-alcoholic fatty liver disease, a preparation method and application thereof

Abstract

The present application relates to a kind of probiotic oil agent for improving non-alcoholic fatty liver disease, preparation method and application, the probiotic oil agent includes active ingredient A and active ingredient B, the active ingredient A is mucinophilic Akkermansia Akk11, and the preservation number is CCTCC NO:M2024119;The active ingredient B is Omega-3 fatty acid.The present application is combined with mucinophilic Akkermansia Akk11 and Omega-3 fatty acid, and is prepared into oil agent, and the improvement of NAFLD symptom can play the coordinated synergistic effect, can be used for the prevention and treatment of NAFLD.

Description

Technical Field

[0001] This invention belongs to the field of microbial pharmaceutical technology, specifically relating to a probiotic oil preparation for improving non-alcoholic fatty liver disease, its preparation method, and its application. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease caused by excessive fat accumulation in the liver after excluding other known liver damage factors such as excessive alcohol consumption. It can progress to nonalcoholic steatohepatitis (NASH), liver fibrosis, and even cirrhosis. The occurrence of this disease is closely related to metabolic disorders such as obesity, type 2 diabetes, hyperlipidemia, and metabolic syndrome. Sedentary lifestyle, high-fat and high-sugar diets, and staying up late are also important contributing factors. Unlike alcoholic liver disease, NAFLD patients usually have no long-term history of alcohol consumption, and the onset is insidious, often without obvious symptoms in the early stages; some people only experience fatigue and mild discomfort in the liver area. Currently, the pathogenesis of NAFLD is complex, the specific molecular mechanisms are not fully understood, and there is a lack of specific drug treatments. Therefore, exploring treatment methods and drugs for nonalcoholic fatty liver disease is a current research hotspot.

[0003] Existing methods for improving non-alcoholic fatty liver disease (NAFLD) primarily focus on lifestyle interventions, such as diet control and exercise, but their effectiveness is limited. Clinically, NAFLD treatment mainly involves lipid-lowering, blood sugar-lowering, antioxidant, and hepatoprotective drugs. However, these drugs often have side effects, either exacerbating the liver burden or exhibiting poor efficacy, thus limiting their use. While current food-grade products or plant extracts, such as *Lactobacillus paracasei*, *Lactobacillus plantarum*, DHA, tea polyphenols, and peppermint extract, are free of toxic side effects and can improve NAFLD to some extent, their effects are often limited, leaving significant room for improvement. Given the lack of more and better safer drugs available, discussing the combination of active molecules or drugs to enhance their therapeutic effects is of great significance.

[0004] Therefore, there is an urgent need to develop a composition and formulation that is free of toxic side effects and can synergistically enhance the improvement or treatment effect of non-alcoholic fatty liver disease (NAFLD) to delay or reverse the progression of NAFLD. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies in the prior art and provide a probiotic oil preparation, preparation method and application for improving non-alcoholic fatty liver disease. The combination of Akk11 myxotrophic bacteria and Omega-3 fatty acids can have a synergistic effect on improving NAFLD symptoms and can be used for the prevention and treatment of NAFLD.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A probiotic oil for improving non-alcoholic fatty liver disease includes active ingredient A and active ingredient B. Active ingredient A is Akk11 of Aktmanella myxophilus, with accession number CCTCC NO: M2024119; and active ingredient B is Omega-3 fatty acid.

[0008] As a further technical solution, the viable count of *Akkermansia myxophilus* in each gram of the probiotic oil is ≥1.6 × 10⁻⁶. 9 CFU.

[0009] As a further technical solution, the mass concentration of Omega-3 fatty acids in the probiotic oil is ≥50%.

[0010] As a further technical solution, the Omega-3 fatty acid includes docosahexaenoic acid (DHA), and the mass concentration of docosahexaenoic acid in the Omega-3 fatty acid is 50-99% (preferably 65-99%).

[0011] As a further technical solution, the Omega-3 fatty acid includes docosahexaenoic acid, and the mass concentration of docosahexaenoic acid in the probiotic oil is ≥30%.

[0012] As a further technical solution, the Omega-3 fatty acid also includes eicosapentaenoic acid (EPA), and the concentration of eicosapentaenoic acid in the Omega-3 fatty acid is 0-40% (preferably 0-34%).

[0013] As a further technical solution, the probiotic oil also includes a diluent, which is a vegetable oil.

[0014] As a further technical solution, the vegetable oil includes one or more of peanut oil, walnut oil, olive oil, and corn oil.

[0015] As a further technical solution, the Omega-3 fatty acids are added in the form of fish oil.

[0016] As a further technical solution, the Akkermansia myxophilus is added in the form of Akkermansia myxophilus powder.

[0017] As a further technical solution, the Akkermansia muciniphila powder also includes a protectant.

[0018] As a further technical solution, the protective agent includes one or more of the following: skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol.

[0019] As a further technical solution, the probiotic oil also includes a stabilizer, fumed silica, wherein the mass concentration of fumed silica in the probiotic oil is 1.5-3.0% (preferably 2.0-2.5%).

[0020] As a further technical solution, the preparation method of the probiotic oil includes the following steps:

[0021] Step 1: Adjust the concentration of Omega-3 fatty acids using a diluent, and then slowly add fumed silica to the Omega-3 fatty acids under stirring conditions. Control the addition time of fumed silica to be 2.5-3.5 min (preferably 3 min). After the addition is completed, continue stirring until the mixture is uniform to obtain a suspension.

[0022] Step 2: Add Akkermansia muciniphila to the suspension and stir until homogeneous (avoid high temperature and violent collision during stirring to maintain the activity of probiotics) to obtain the probiotic preparation.

[0023] The application of the probiotic oil in the preparation of drugs to improve non-alcoholic fatty liver disease.

[0024] As a further technical solution, the improvement of non-alcoholic fatty liver disease includes promoting GLP-1 secretion, reducing liver fat accumulation, improving liver steatosis, reducing liver weight, reducing liver white fat weight, reducing fasting blood glucose, reducing liver triglycerides, reducing liver total cholesterol, reducing liver alanine aminotransferase, or reducing liver alkaline sulfatase.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. Akkermansia muciniphila is a common intestinal probiotic with metabolic-improving and anti-inflammatory effects. Omega-3 fatty acids are polyunsaturated fatty acids, recognized as "vascular cleansers," possessing anti-inflammatory and lipid-lowering effects. They can regulate fat metabolism, reduce fat deposition in the liver, and lower the risk of fatty liver by reducing triglyceride levels. However, when used alone, their effect on improving non-alcoholic fatty liver disease is insufficient. This invention is the first to combine a specific strain of Akkermansia muciniphila (Akk11) with Omega-3 fatty acids. Using a NAFLD mouse model, it was demonstrated that the combined application of these two ingredients can effectively stimulate GLP-1 secretion, reduce liver fat deposition, and produce synergistic effects on multiple key metabolic parameters such as white fat weight, fasting blood glucose, liver triglycerides, total cholesterol, and liver function indicators. This can effectively improve NAFLD symptoms and can be used for the prevention and treatment of NAFLD.

[0027] 2. Due to the poor suspension properties of probiotics in Omega-3 fatty acids, when prepared as an oil-based formulation of probiotics and Omega-3 fatty acids, bacterial sedimentation easily occurs, and remixing is impossible, thus affecting the efficacy. To solve this problem, existing technologies typically require diluting Omega-3 fatty acids to a very low concentration with vegetable oil and / or adding medium-chain triglycerides as stabilizers to prevent probiotic sedimentation. Both methods result in a lower concentration of Omega-3 fatty acids (usually below 10%), making it impossible to obtain a high-concentration Omega-3 fatty acid oil-based formulation (Omega-3 fatty acid content ≥50%, DHA ≥25%). This leads to excessive intake of other fats, resulting in increased total cholesterol in the liver, which is detrimental to the treatment of non-alcoholic fatty liver disease. Therefore, this invention develops a novel fumed silica stabilizer for Akkermansia myxophilus oil-based formulations to prevent the precipitation of Akkermansia myxophilus in Omega-3 fatty acid oil-based formulations and improve the suspension stability of Akkermansia myxophilus in the oil-based formulation.

[0028] 3. This invention precisely combines Akk11 bacteria powder with fish oil containing Omega-3 fatty acids and uses fumed silica stabilization technology. This not only maintains the uniform suspension of the bacteria in the Omega-3 fatty acid oil, but also improves its live bacteria stability at room temperature. The fumed silica is stable and does not react with fish oil or bacteria. Moreover, a probiotic oil with good live bacteria stability and high suspension stability can be obtained simply by stirring and mixing under the process parameters defined in this invention, which is convenient for industrial production. Attached Figure Description

[0029] Figure 1 The figure shows the effect of fumed silica on the suspension stability of probiotic oil preparations.

[0030] exist Figure 1 In the diagram, A: Day 0, Comparative Example 1; B: Day 0, Example 1; C: Day 10, Comparative Example 1; D: Day 10, Example 1;

[0031] Figure 2 The effects of each treatment group on the body weight of the model mice;

[0032] Figure 3 The graph shows the effect of each treatment group on GLP-1 levels in model mice;

[0033] Figure 4 The results of oil red staining of the livers of model mice in each treatment group are shown in the figure.

[0034] exist Figure 4In the diagram, A: Model group, H&E staining; B: Model group + fish oil, H&E staining; C: Model group + Akk, H&E staining; D: Model group + fish oil + Akk, H&E staining; E: Model group, with red staining; F: Model group + fish oil, with red staining; G: Model group + Akk, with red staining; H: Model group + fish oil + Akk, with red staining.

[0035] Figure 5 The graph shows the evaluation of liver damage in model mice by each treatment group;

[0036] exist Figure 5 In the above, a: liver weight; b: white fat weight; c: insulin tolerance; d: fasting blood glucose; e: glucose tolerance; f: liver triglycerides; g: liver total cholesterol; h: liver alanine aminotransferase; i: liver alkaline phosphatase. Detailed Implementation

[0037] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] 1. Akkermansia muciniphila Akk11: Its classification name is Akkermansia muciniphila. The depository institution is China Center for Type Culture Collection, the deposit date is January 15, 2024, the deposit number is CCTCC NO: M2024119, and the address is: Wuhan University, Wuhan, China. It has been disclosed in several Chinese patents, including CN202410805093.9 and CN202410620734.3.

[0039] 2. Akk11 myxophilic bacteria powder (live count 1×10⁻⁶) 10 CFU / g): Centrifuge the Akk11 fermentation broth of Akkermansia myxophilus, collect the cells, add a freeze-drying protectant (the freeze-drying protectant is composed of trehalose, sucrose, casein hydrolysate, amino acids, sodium ascorbate, cysteine, and glycerol) to the cells, stir evenly, and freeze-dry to obtain Akk11 myxophilus powder.

[0040] 3. Fish oil (omega-3 content ≥99%, of which DHA content is 65% and EPA content is 30%): Shanghai Aladdin Biochemical Technology Co., Ltd.

[0041] 4. Fish oil base: Dilute fish oil (omega-3 content ≥99%) with peanut oil to a omega-3 content of 50% (of which the DHA content is about 32%).

[0042] 5. Fumed silica: Anhui Shanhe Pharmaceutical Excipients Co., Ltd.;

[0043] 6. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0044] Example 1

[0045] A probiotic oil preparation, the preparation method of which includes the following steps:

[0046] Step 1: First, weigh 10g of Akk11 myxophilic bacteria powder (live count 1×10⁻⁶). 10 85g of fish oil matrix (50% omega-3 content) and 2g of fumed silica were added. Then, under stirring conditions, the fumed silica was slowly added to the fish oil matrix, and the addition rate of the fumed silica was controlled so that the addition was completed within 3 minutes. After the addition was completed, stirring was continued until the mixture was uniform to obtain a suspension.

[0047] Step 2: Add *Akkermansia myxophilus* to the suspension and shake and stir evenly using a shaker. Avoid high temperatures and violent collisions during shaking and stirring to maintain the probiotic activity. The resulting probiotic oil (total weight approximately 97g, live bacteria count approximately 1×10⁻⁶) is obtained. 9 CFU / g).

[0048] Example 2

[0049] A probiotic oil preparation, the preparation method of which includes the following steps: the same as in Example 1, except that the amount of fumed silica added is 1.8g.

[0050] Example 3

[0051] A probiotic oil preparation, the preparation method of which includes the following steps: the same as in Example 1, except that the amount of fumed silica added is 2.2g.

[0052] Comparative Example 1

[0053] A probiotic oil preparation, the preparation method of which includes the following steps: the same as in Example 1, except that no fumed silica is added.

[0054] Example 1: Live bacteria stability test

[0055] The probiotic oil prepared in Example 1 was filled into reagent bottles and stored at -20°C, 4°C, and 25°C for 120 days. Samples were taken on days 0, 15, 30, 60, 90, and 120 to detect the viable count of Akkermansia myxophilus and calculate its viability. At the same time, Akkermansia myxophilus Akk11 powder was used as a control to evaluate the viability retention of Akkermansia myxophilus Akk11 in the oil. The results are shown in Table 1.

[0056] Table 1

[0057]

[0058] As can be seen from the data in Table 1, the viable bacteria rate of the probiotic oil prepared by this invention is comparable to or slightly better than that of the probiotic powder.

[0059] Example 2: Suspension stability test of oil-based formulations

[0060] The probiotic oils prepared in Example 1 and Comparative Example 1 were left to stand at 25 degrees Celsius for 10 days. Photos were taken and the precipitation of the probiotic oils before and after standing was recorded. The results are shown below. Figure 1 .

[0061] As shown in Figure 1:

[0062] 1) Before and after standing, the probiotic oil in Example 1 and Comparative Example 1 did not show any significant change in color;

[0063] 2) In Comparative Example 1 without added fumed silica, the probiotics showed obvious precipitation, with the upper layer being an oil layer without probiotics and the lower layer being an oil layer containing probiotics. In Example 1 with added fumed silica, after standing, the probiotics did not show obvious precipitation, and the probiotics and fumed silica in the probiotic oil remained in a uniform suspension.

[0064] In summary, the fumed silica of this invention can prevent Akk11 bacteria from precipitating in fish oil (omega-3), which is not found in other types of silica.

[0065] Example 3: Evaluation of the effect of animal models on non-alcoholic fatty liver disease

[0066] This study used a high-fat diet-induced non-alcoholic fatty liver disease (NAFLD) mouse model to evaluate efficacy.

[0067] I. Experimental Methods

[0068] Six-week-old male C57BL / 6J mice were fed a high-fat diet (60% fat energy) for 8 weeks to establish a NAFLD model. The mice were then randomly divided into groups of 10 mice each. Each intervention group received the corresponding medication orally via gavage daily for 8 weeks. Body weight, food intake, and fasting blood glucose were monitored during the experiment. After the intervention, serum GLP-1 levels were measured. Liver tissue was weighed, and white adipose tissue was also weighed. Oil Red O staining was used to analyze lipid deposition, and liver triglyceride, total cholesterol, alanine aminotransferase (ALT), and alkaline phosphatase levels were quantitatively measured. Results are shown below. Figure 2-5 .

[0069] Because, under the NAFLD model, Akk11 bacteria, when administered 0.5 × 10⁻⁶, 9 When the dose is above CFU / day, the dosage has almost no effect on the following indicators. However, when the omega-3 dosage is 150-400 mg / kg / day, a dose-dependent relationship occurs. Therefore, this application administers the drugs to each group in the following manner:

[0070] (1) Model group (model mice): No drug was administered, and an equal amount of peanut oil was used to replace omega-3;

[0071] (2) Akk11-only intervention group (model mice + Akk): based on live bacteria count 1×10 9 CFU / daily administration of Akk11, with an equivalent amount of peanut oil replacing omega-3;

[0072] (3) Omega-3 intervention group alone (model mice + fish oil): Omega-3 was administered at 300 mg / kg / day;

[0073] (4) Akk11 + omega-3 combined intervention group (model mice + Akk + fish oil): based on live bacteria count 0.5 × 10 9 CFU / day for Akk11 bacteria, omega-3 at 150 mg / kg / day, and any reduction in omega-3 is made up with an equal amount of peanut oil.

[0074] II. Experimental Results and Analysis

[0075] 1. Effects of Akk11 + fish oil combined intervention on body weight and GLP-1 levels in mouse models

[0076] Figure 2 The results showed that the mice grew normally during the experiment, and there was no significant difference in food intake or weight gain among the groups.

[0077] Figure 3Data showed that, compared with model mice, mice treated with omega-3, Akk11, and Akk11+omega-3 showed significantly increased serum GLP-1 (glucagon-like peptide-1) levels. Furthermore, the combined intervention of Akk11 and fish oil was more effective than Akk11 alone or omega-3 alone. This indicates that Akk11 and omega-3 can synergistically promote GLP-1 secretion, thereby improving glucose and lipid metabolism.

[0078] 2. Effects of Akk11 + fish oil combined intervention on hepatic lipid deposition in mouse models

[0079] Figure 4 Oil Red O staining of the liver showed that lipid deposition in the liver of mice in the Akk11 + fish oil combined intervention group was significantly reduced compared with the model group, the Akk11 alone intervention group, and the omega-3 alone intervention group. The analysis found that the area of ​​lipid deposition in the liver was reduced by up to 48.7%. The results indicate that the probiotic combination can significantly reduce liver fat accumulation and improve liver steatosis.

[0080] 3. Effects of Akk11 + fish oil combined intervention on liver injury in mouse model

[0081] from Figure 5 The presented indicators related to non-alcoholic fatty liver disease (liver weight, white fat weight, insulin level, glucose metabolism indicators, and liver lipid and enzyme indicators, etc.) can be used to deduce:

[0082] 1) Omega-3 intervention improved liver weight, fasting blood glucose, and liver alkaline phosphatase in model mice, but had almost no effect on white fat weight, insulin tolerance, glucose tolerance, liver triglycerides, liver total cholesterol, and liver alanine aminotransferase.

[0083] Akk11 intervention significantly improved liver weight, fasting blood glucose, total liver cholesterol, liver weight alanine aminotransferase, and liver alkaline phosphatase in model mice, with significant differences compared to the model group. However, it had a moderate effect on improving white fat weight, insulin tolerance, glucose tolerance, and liver triglycerides, with no significant difference compared to the model group.

[0084] 2) For white adipose tissue, fasting blood glucose, and liver triglycerides, there was no significant difference between Akk11 intervention alone and omega-3 intervention alone. In addition, for liver weight and liver alkaline phosphatase, the Akk11+omega-3 combined intervention was superior to Akk11 intervention alone and omega-3 intervention alone. This indicates that the Akk11+omega-3 combined intervention has a synergistic effect on indicators such as white adipose tissue, fasting blood glucose, liver triglycerides, liver weight, and liver alkaline phosphatase. Akk11 and omega-3 can synergistically improve white adipose tissue, fasting blood glucose, liver triglycerides, liver weight, and liver alkaline phosphatase in NAFLD model mice.

[0085] 3) Regarding the total cholesterol index in the liver, under omega-3 intervention alone, the cholesterol level increased rather than decreased compared to the model group, but the difference was not significant. This indicates that omega-3 intervention cannot reduce the level of total cholesterol in the liver. However, when Akk11 + omega-3 were combined for intervention, the total cholesterol in the liver was significantly reduced compared to Akk11 intervention alone. This indicates that Akk11 and omega-3 can synergistically reduce the total cholesterol content in the liver of NAFLD model mice.

[0086] 4) Regarding insulin tolerance, glucose tolerance, and liver alanine aminotransferase, the combined intervention of Akk11 and omega-3 was between the effects of Akk11 alone and omega-3 alone. Therefore, it can be demonstrated that the combined intervention of Akk11 and omega-3 can promote the recovery of insulin tolerance, glucose tolerance, and liver alanine aminotransferase in NAFLD model mice.

[0087] In summary, among the nine liver damage indicators caused by non-alcoholic fatty liver disease (NAFLD), the Akk11+omega-3 combined intervention showed a synergistic improvement effect on six indicators: white fat, fasting blood glucose, liver triglycerides, liver weight, liver alkaline phosphatase, and liver total cholesterol. The remaining three indicators, insulin tolerance, glucose tolerance, and liver alanine aminotransferase, showed a certain degree of improvement. Therefore, it can be concluded that the Akk11+omega-3 combined intervention of this invention can have a synergistic therapeutic effect on NAFLD, effectively improve metabolic disorders induced by a high-fat diet in NAFLD mice, and reduce hepatic steatosis and inflammatory damage.

[0088] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A probiotic oil preparation for improving non-alcoholic fatty liver disease, characterized in that, The product includes active ingredient A and active ingredient B, and also includes the stabilizer fumed silica. Active ingredient A is *Akkermansia obliterans* Akk11, with accession number CCTCC NO: M2024119. The *Akkermansia obliterans* is added in the form of *Akkermansia obliterans* powder, and the viable count of *Akkermansia obliterans* in each gram of the probiotic oil is ≥1.6 × 10⁻⁶. 9 CFU; the active ingredient B is an Omega-3 fatty acid, the Omega-3 fatty acid is docosahexaenoic acid, the mass concentration of docosahexaenoic acid in the probiotic oil is ≥30%; the mass concentration of fumed silica in the probiotic oil is 1.5-3.0%.

2. The probiotic oil preparation for improving non-alcoholic fatty liver disease according to claim 1, characterized in that, The docosahexaenoic acid is added in the form of fish oil.

3. The probiotic oil preparation for improving non-alcoholic fatty liver disease according to claim 1, characterized in that, The Akkermansia muciniphila powder also includes a protectant; the protectant includes one or more of the following: skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol.

4. A probiotic oil preparation for improving non-alcoholic fatty liver disease according to claim 1, characterized in that, The probiotic oil also includes a diluent, which is a vegetable oil.

5. The method for preparing the probiotic oil preparation according to any one of claims 1-4, characterized in that, Includes the following steps: Step 1: Adjust the concentration of Omega-3 fatty acids using a diluent, and then add fumed silica to the Omega-3 fatty acids under stirring conditions. Control the addition time of fumed silica to be 2.5-3.5 min. After the addition is completed, continue stirring until the mixture is uniform to obtain a suspension. Step 2: Add Akkermansia myxophilus to the suspension and stir until homogeneous to obtain the probiotic preparation.

6. The use of the probiotic oil preparation according to any one of claims 1-4 in the preparation of a medicament for improving non-alcoholic fatty liver disease.

Images