Enterococcus faecalis EF-1 capable of reducing blood lipid, microbial preparation and application of enterococcus faecalis EF-1

Abstract

The invention relates to the technical field of microorganisms, in particular to enterococcus faecalis EF-1 capable of reducing blood lipid, a microbial preparation and application of the enterococcus faecalis EF-1. The enterococcus faecalis EF-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 27, 2024, and the preservation number is CGMCC No.32097. The enterococcus faecalis EF-1 is named as enterococcus faecalis EF-1. The enterococcus faecalis EF-1 has the functions of: (1) relieving the weight increase speed of animals, reducing body weight gain, and preventing, relieving or treating obesity; (2) the contents of triglyceride, cholesterol and low-density lipoprotein cholesterol in liver and serum are reduced; (3) the liver function is protected, and the contents of ALT and AST in serum and liver are reduced; (4) liver lipid deposition and lesion are relieved; (5) relieving adipose tissue hypertrophy; (6) preventing, relieving or treating metabolic syndrome; (7) reducing blood sugar, improving insulin resistance and relieving diabetes mellitus; 8, increasing the content of short-chain fatty acid; and (9) regulating intestinal flora.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a glucose-lipid-reducing Enterococcus faecalis EF-1, a microbial preparation and application thereof. Background Art

[0002] With the changes in lifestyle in modern society, the incidence of obesity, diabetes and related metabolic syndrome has increased year by year and has become a global health problem. Obesity is not only a problem of weight gain, but is also closely related to a variety of chronic diseases such as cardiovascular disease and liver disease. Diabetes, especially type 2 diabetes, is a common metabolic disease in life. In patients with type 2 diabetes and prediabetes, lipid metabolism is often accompanied by abnormalities, leading to elevated blood lipids, complications such as hyperlipidemia, fatty liver, and hypertension, which endanger the health of patients. As a metabolic disease characterized by high blood sugar, the pathogenesis of diabetes involves insulin resistance and insulin secretion defects. As the main metabolic organ of the human body, the liver plays a core role in glucose and lipid metabolism. Abnormally elevated lipid levels in serum and liver are closely related to the occurrence and development of a variety of metabolic diseases. Therefore, finding methods that can regulate liver and serum lipid levels is of great significance for the prevention and treatment of these diseases.

[0003] Probiotics have attracted extensive attention due to their regulatory effects on intestinal microecology and potential benefits to host health. Enterococcus faecalis ), as part of the human intestinal microbiota, has been studied for its history of use in traditional fermented foods and its potential health benefits. However, not all strains of Enterococcus faecalis have the same probiotic properties. In order to achieve better preventive and therapeutic effects, it is often necessary to use a combination of multiple Enterococci or to combine Enterococcus faecalis with other probiotics, and some strains may be associated with safety issues. Therefore, screening Enterococcus faecalis strains with specific health benefits and conducting a comprehensive safety assessment are crucial for the development of new probiotic products. Summary of the invention

[0004] In view of the current lack of Enterococcus faecalis strains that have the functions of lowering blood sugar and lipid, absorbing free fatty acids, increasing the abundance of bacteria producing short-chain fatty acids, and increasing the concentration of short-chain fatty acids in the cecum, and the technical problem that compounding is often required to obtain the corresponding functions, the present invention provides a blood sugar and lipid lowering Enterococcus faecalis EF-1, a microbial preparation and its application.

[0005] The technical solution of the present invention is as follows: In the first aspect, the present invention provides a lipid-lowering Enterococcus faecalis EF-1, which has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee on September 27, 2024, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC No. 32097, and is classified as Enterococcus faecalis Enterococcus faecalis .

[0006] In a second aspect, the present invention provides a microbial preparation comprising the above-mentioned Enterococcus faecalis EF-1.

[0007] Furthermore, the content of Enterococcus faecalis EF-1 in the microbial preparation was 1.0×10 7 ~1.0×10 10 CFU / mL.

[0008] In a third aspect, the present invention provides a use of the above-mentioned Enterococcus faecalis EF-1 or a microbial preparation thereof in the preparation of a functional product for hypoglycemic lipids.

[0009] Furthermore, functional products for lowering glucose and lipid levels can be used to (1) prevent, alleviate or treat obesity and lipid metabolism disorders, (2) prevent, alleviate or treat insulin resistance and diabetes, (3) prevent, alleviate or treat hyperlipidemia, (4) alleviate liver fat deposition and adipose tissue lesions, and (5) improve intestinal flora and increase the relative abundance of bacterial genera that produce short-chain fatty acids.

[0010] Further, short-chain fatty acid-producing bacteria include Bacteroides ( Bacteroides ), Faecalibacterium ( Faecalibaculum )、Desulfovibrio spp.( Desulfovibrio ) and Lachnospiraceae ( Lachnoclostridium ).

[0011] Furthermore, the functional product of glucose-lowering lipids is food (including items for human and / or pet consumption), food additives, feed, feed additives or medicines.

[0012] The beneficial effects of the present invention are: The Enterococcus faecalis EF-1 provided by the present invention is safe and sensitive to a variety of antibiotics, and can reach the gastrointestinal tract of animals to exert its effects, including the following functions: ① Alleviate the weight gain rate of animals, reduce body weight gain, prevent, relieve or treat obesity; ② Reduce the levels of triglycerides, cholesterol and low-density lipoprotein cholesterol in the liver and serum; ③ Protect liver function and reduce the levels of ALT and AST in serum and liver; ④ Alleviate liver lipid deposition and lesions; ⑤ Alleviate adipose tissue hypertrophy; ⑥ Prevent, relieve or treat metabolic syndrome; ⑦ Lower blood sugar, improve insulin resistance, and relieve diabetes; ⑧ Increase the content of short-chain fatty acids; ⑨ Regulate intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 This is the colony morphology of Enterococcus faecalis EF-1.

[0015] Figure 2 This is a microscopic image of Enterococcus faecalis EF-1.

[0016] Figure 3 It is a bar graph of the bacterial survival rate of Enterococcus faecalis EF-1 in Example 1 at different time periods in artificial gastrointestinal fluid.

[0017] Figure 4 1 is the bile salt hydrolase activity in Example 1, (A) is a photograph of filter paper on an MRS plate, (B) is a photograph of filter paper on a sodium glycocholate plate, and (C) is a photograph of filter paper on a sodium taurocholate plate.

[0018] Figure 5 These are photographs of the results of the hemolytic test in Example 1, (A) is a photograph of the results of the hemolytic test of Enterococcus faecalis EF-1, and (B) is a photograph of the results of the hemolytic test of Escherichia coli K88.

[0019] Figure 6 This is a graph of glucose consumption of HepG2 cells in Example 2.

[0020] Figure 7 3 is a graph showing the weight changes of mice in different groups in Example 3.

[0021] Figure 8 It is a bar graph of organ indexes of different groups of mice in Example 3.

[0022] Fig. 9 It is the staining picture of liver tissue of different groups of mice in Example 3.

[0023] Fig.10 This is the staining image of adipose tissue of different groups of mice in Example 3.

[0024] Fig.11 The figures are histograms of serum indicators of mice in different groups in Example 3, (A) is a histogram of serum total cholesterol, (B) is a histogram of serum triglycerides, (C) is a histogram of serum low-density lipoprotein cholesterol, (D) is a histogram of serum glucose, (E) is a histogram of serum alanine aminotransferase, and (F) is a histogram of serum aspartate aminotransferase.

[0025] Fig.12 These are the bar graphs of liver indicators of different groups of mice in Example 3, (A) is a bar graph of liver total cholesterol, (B) is a bar graph of liver triglycerides, (C) is a bar graph of liver high-density lipoprotein cholesterol, (D) is a bar graph of liver low-density lipoprotein cholesterol, (E) is a bar graph of liver very low-density lipoprotein cholesterol, (F) is a bar graph of liver glucose, (G) is a bar graph of liver alanine aminotransferase, and (H) is a bar graph of liver aspartate aminotransferase.

[0026] Fig.13 3. It is a graph of the content of short-chain fatty acids in the cecum of mice in different groups in Example 3, (A) is a bar graph of short-chain fatty acid composition, (B) is a bar graph of acetic acid concentration, (C) is a bar graph of propionic acid concentration, (D) is a bar graph of butyric acid concentration, (E) is a bar graph of isobutyric acid concentration, (F) is a bar graph of valeric acid concentration, (G) is a bar graph of isovaleric acid concentration, (H) is a bar graph of hexanoic acid concentration, (I) is a bar graph of heptanoic acid concentration, (J) is a bar graph of octanoic acid concentration, (K) is a bar graph of nonanoic acid concentration, and (L) is a bar graph of decanoic acid concentration.

[0027] Fig.14 This is the PCoA analysis diagram of the feces of different groups of mice in Example 3.

[0028] Fig.15 It is a bar chart showing the composition of the intestinal flora at the phylum level of different groups of mice in Example 3.

[0029] Fig.16 It is a bar graph of the relative abundance percentage and ratio of Firmicutes and Bacteroidetes in the intestines of different groups of mice in Example 3.

[0030] Fig.17 This is a heat map of the intestinal colony composition at the genus level in different groups of mice in Example 3. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0032] Example 1 (1) Isolation, identification and preservation of strains: In November 2023, a new probiotic strain was isolated from the feces of healthy dogs in Changping District, Beijing. Figure 1 , Figure 2 The strain was identified as Enterococcus faecalis by comparative analysis of 16S rRNA gene sequences. Enterococcus faecium , and named Enterococcus faecalis EF-1.

[0033] On September 27, 2024, Enterococcus faecalis EF-1 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No.32097 and the classification name Enterococcus faecalis Enterococcus faecium .

[0034] (2) Artificial gastrointestinal fluid test: The second generation activated Enterococcus faecalis EF-1 solution was adjusted to 10% EF-1 concentration with 0.01M PBS (pH 7.2). 9 CFU / mL. First, EF-1 bacterial solution was added to artificial gastric juice (0.03 g pepsin dissolved in 0.01 L 0.2 M PBS buffer, pH = 3) at a ratio of 10% (volume ratio), and then cultured at 37°C for 3 hours. Samples were taken at 0, 1.5, and 3 hours of culture and diluted to 10 -6 , 10 -7 , 10 -8 , and each dilution was counted three times. Next, 1 mL of artificial gastric juice containing Enterococcus faecalis EF-1 was taken and transferred to artificial intestinal juice (0.01 g trypsin dissolved in 0.01 L of 0.2 M PBS buffer, pH = 8), and cultured at 37 ° C for 8 h. At 0, 2, 4, and 8 h of culture, samples were also taken and diluted to 10 -6 , 10 -7 , 10 -8 Counting was repeated 3 times for each dilution. Figure 3As shown, W1.5h and W3h represent the bacterial survival rates of the strains after being treated in artificial gastric juice for 1.5h and 3h, respectively, and C0h, C2h, C4h, and C8h represent the bacterial survival rates of the strains treated in artificial intestinal juice for 0, 2, 4, and 8h, respectively. It can be seen that Enterococcus faecalis EF-1 proliferated in artificial gastric juice and artificial intestinal juice, and the survival rate was above 100%, indicating that Enterococcus faecalis EF-1 can fully play a role in gastrointestinal juice.

[0035] (3) Cholesterol-lowering trial: The second-generation activated Enterococcus faecalis EF-1 bacterial solution was inoculated into MRS-CHOL medium (1L MRS-CHOL medium containing 0.1g cholesterol and 0.2g ox bile salt) at a volume ratio of 2.0% and cultured at a constant temperature of 37°C. After 48 hours of culture, 0.50mL of the bacterial solution was mixed with 4.50mL of anhydrous ethanol, allowed to stand for 10min, and centrifuged at 3000g / min for 15min. Then 0.50mL of the supernatant was taken, 0.20mL of o-phthalaldehyde and 4.30mL of mixed acid were added, and the mixture was fully shaken and mixed to prepare the experimental group; MRS-CHOL medium without inoculation of Enterococcus faecalis EF-1 was used instead of the bacterial solution, and the control group was prepared according to the same steps.

[0036] After the experimental group and the control group were left to stand for 30 min, the 550nm The absorbance was measured at 40 °C and the cholesterol degradation rate was calculated. Cholesterol degradation rate = (CA) / C × 100%, where C is the OD of the control group. 550nm The cholesterol content corresponding to the value, A is the OD of the experimental group 550nm The cholesterol content corresponding to the value.

[0037] After calculation, the degradation rate of EF-1 cholesterol was 46.47%.

[0038] (4) Bile salt hydrolase activity test: Sterile filter paper was placed on MRS plate, sodium glycocholate plate (MRS plate + 0.35% (w / v) CaCl 2 and 0.5% (w / v) sodium glycocholate hydrate) and sodium taurocholate plates (MRS plates + 0.35% (w / v) CaCl 2 and 0.5% (w / v) sodium taurocholate hydrate), and then inoculated 20 μL of activated Enterococcus faecalis EF-1 bacterial solution onto the sterile filter paper of the sodium glycocholate plate and the sodium taurocholate plate, respectively, and the three plates were cultured under anaerobic conditions at 37°C for 72 h.

[0039] By observing the area around the filter paper in the three plates (e.g. Figure 4As shown in the figure, white particles appeared around the filter paper inoculated with the Enterococcus faecalis EF-1 bacterial solution. This phenomenon indicates that Enterococcus faecalis EF-1 has bile salt hydrolase activity and can hydrolyze sodium taurocholate and sodium glycocholate into free bile salts when cultured in a medium containing sodium taurocholate and sodium glycocholate.

[0040] (5) α-glucosidase inhibition test: First, the second-generation activated Enterococcus faecalis EF-1 bacterial solution was inoculated into MRS medium at a volume ratio of 2.0% and cultured at 37°C for 24 hours. Subsequently, 50 μL of the cultured bacterial solution was taken out and added to 100 μL of 0.1M phosphate buffer (pH adjusted to 6.9) containing α-glucosidase prepared in advance, and pre-incubated in a 96-well plate at 25°C for 10 minutes. After the pre-incubation, 50 μL of 5mM p-nitrophenyl-α-D-pyranoglucoside solution was added to each well. Then, these reaction mixtures were incubated at 25°C for another 5 minutes, and the absorbance at 405 nm was measured by a microplate reader. As a control, the same experimental steps were performed using 50 μL of 0.1M phosphate buffer (pH 6.9) instead of the bacterial solution.

[0041] After calculation, the inhibition rate of Enterococcus faecalis EF-1 on α-glucosidase was 38.51%, indicating that Enterococcus faecalis EF-1 has the potential to lower blood sugar and thus can play a role in alleviating diabetes.

[0042] (6) Fatty acid absorption test: First, after the second generation of activation culture of Enterococcus faecalis EF-1, the concentration of the bacterial solution was adjusted to OD 600nm =0.7. Then, the adjusted bacterial solution was inoculated into a medium containing 0.5% Brij 58 and 0.25mmol / L sodium palmitate at a volume ratio of 2.0%, and cultured at 37°C for 24h. After the culture, the fatty acid concentration in the culture medium was measured using a free fatty acid determination kit, and it was calculated that the absorption rate of free fatty acids by Enterococcus faecalis EF-1 reached 59%.

[0043] (7) Hemolytic test Hemolysis can be divided into three main types: type A (α), type B (β) and type C (γ). Specifically, type A hemolysis forms a grass-green hemolysis ring around the colonies; type B hemolysis forms a 2-4 mm wide transparent hemolysis ring around the colonies; type C hemolysis does not cause hemolysis, that is, no hemolysis ring appears.

[0044] Hemolytic tests were performed on Enterococcus faecalis EF-1 and the control strain Escherichia coli K88. The results are as follows: Figure 5As shown, no hemolytic ring was observed around the colony of the Enterococcus faecalis EF-1 of the present invention, that is, it showed a negative reaction in the hemolysis test, which was γ hemolysis, indicating that the strain did not have hemolytic characteristics, and therefore had a higher safety. Escherichia coli K88 was β hemolysis.

[0045] (8) Antibiotic susceptibility test: The sensitivity of Enterococcus faecalis EF-1 to antibiotics was tested by the drug sensitivity paper agar diffusion method. The specific steps are as follows: The second generation of activated culture of Enterococcus faecalis EF-1 was inoculated into MRS medium at an inoculum size of 2.0% (v / v), cultured at 37°C for 6 h, 100 μL of the bacterial solution was aspirated to spread on the plate, and drug-susceptibility papers containing different antibiotics (kanamycin 30 μg / tablet, tetracycline 30 μg / tablet, clindamycin 2 μg / tablet, gentamicin 10 μg / tablet, erythromycin 15 μg / tablet, chloramphenicol 30 μg / tablet, amcarcillin 10 μg / tablet) and positive drug-susceptibility sheets were placed on the plates and cultured at 37°C for 24 h to observe the sensitivity of Enterococcus faecalis EF-1 to drugs.

[0046] The results are shown in Table 1. "S" indicates that there is an antibiotic sensitivity zone, and the bacteria are sensitive to antibiotics; "R" indicates that there is no antibiotic sensitivity zone, and the bacteria are insensitive to antibiotics and have drug resistance. It can be seen that Enterococcus faecalis EF-1 is sensitive to all the commonly used antibiotics tested and has no drug resistance.

[0047] Table 1 Antibiotic sensitivity test results of Enterococcus faecalis EF-1

[0048] Example 2 Glucose absorption test of HepG2 cells Insulin resistance is usually manifested as a weakened response to insulin in diabetic patients, which makes insulin unable to effectively lower blood sugar levels, thereby causing blood sugar concentrations to rise, which is a key factor in the development of type 2 diabetes. The liver is a core organ responsible for multiple energy metabolic processes in the human body, including carbohydrate metabolism. The HepG2 cell line is a liver cancer cell line that mimics the function of normal liver cells, so it is often used to study blood sugar lowering activity. In order to determine whether Enterococcus faecalis EF-1 has the function of improving the insulin resistance of HepG2 cells, the glucose content of insulin-resistant HepG2 cells before and after the addition of Enterococcus faecalis EF-1 was measured.

[0049] Construction of HepG2 insulin resistance model (IR-HepG2): HepG2 cells were subcultured for at least 5 generations, digested with 0.25% trypsin, and repeatedly pipetted into single cell suspension with serum-free DMEM medium. The cell concentration was adjusted to 5 × 10 4 The cells were inoculated into 96-well plates and incubated at 37°C and 5% CO 2Incubate in the incubator for 12 h. After the incubation, carefully remove the supernatant, wash twice with serum-free DMEM medium, and add 100 μL of 10% ethanol prepared with DMEM medium. -6 mol / L insulin was added to a 96-well plate and induced for 24 h as the model group. The glucose content in the cell supernatant was determined using a glucose oxidase kit.

[0050] The activated Enterococcus faecalis EF-1 was centrifuged at 4000 g for 5 min, the cells were collected, washed three times with serum-free DMEM medium, and the cell concentration was adjusted with DMEM complete medium to obtain a concentration of 1×10 8 CFU / mL of the low-dose group and the concentration was 1×10 9 According to the established HepG2 insulin resistance model, the supernatant was discarded, and 100 μL of low-dose group and high-dose group bacterial solutions were added to the 96-well plate, with 6 replicates for each concentration, and DMEM complete medium was used as the control group. The plates were incubated at 37°C and 5% CO 2 After incubation for 6 h, the cells were centrifuged at 3000 g for 5 min, the precipitate was discarded, the supernatant was collected, and the glucose content in the cell supernatant was detected.

[0051] The results are as follows Figure 6 As shown, insulin treatment significantly reduced the glucose consumption of HepG2 cells. After being treated with Enterococcus faecalis EF-1 (low-dose group and high-dose group), the cells' glucose consumption increased significantly. The glucose consumption capacity of IR-HepG2 cells in the high-dose group increased by 24.63%, and that in the low-dose group increased by 15.40%. This indicates that Enterococcus faecalis EF-1 can enhance the sensitivity of HepG2 cells to insulin and has a positive effect on improving insulin resistance and diabetes.

[0052] Example 3 Effects of Enterococcus faecalis EF-1 on Alleviating Obesity and Lowering Lipids (1) Culture of Enterococcus faecalis EF-1: After the second generation of activated Enterococcus faecalis EF-1 was cultured in MRS medium, it was inoculated into MRS medium at a volume ratio of 2% and cultured at 37°C for 24 hours. After the culture was completed, the cells were collected by centrifugation at 4°C and 5000g / min for 10 minutes, and then washed twice with 0.9% saline. Finally, the viable bacterial concentration of Enterococcus faecalis EF-1 was adjusted to 1×10 by plate viable bacterial count method. 10 CFU / mL, and obtain the Enterococcus faecalis microbial preparation.

[0053] (2) Experimental feeding and grouping: Thirty-two healthy 7-week-old C57BL / 6J male mice were randomly assigned to four experimental groups: control group (CT group), high-fat diet group (HFD group), atorvastatin calcium positive control group (PC group) and Enterococcus faecalis EF-1 group (EF-1 group), each group containing 8 mice, and every two mice shared a cage. After a 7-day adaptation period, the CT group was given regular feed, while the HFD group, PC group and EF-1 group were given high-fat feed D12492. The CT group and HFD group were given 200 μL of sterile 0.9% saline every day, the PC group was given an equal volume of atorvastatin calcium (dose of 30 mg / kg), and the Enterococcus faecalis EF-1 group was given 200 μL of the Enterococcus faecalis EF-1 microbial preparation (viable count of 2×10) prepared in step (1) by oral gavage. 9 CFU), and then continuously administered orally for 16 weeks. During the experiment, the mice were kept in an environment with a temperature control of 22±2℃ and had free access to water and food. At the end of the experiment, the mice were fasted for 16h, then their body weight was measured, blood was collected, and they were killed by cervical dislocation, and finally their organs were separated for analysis.

[0054] (3) Weight changes: like Figure 7 As shown in the figure, the weight changes of mice reflected the effects of different diets and intervention measures. Compared with the CT group, the weight of mice in the HFD group increased significantly. The weight gain rate of mice in the EF-1 group and the PC group, which received the intervention of Enterococcus faecalis EF-1, was significantly slowed down. Compared with atorvastatin calcium used in the PC group, Enterococcus faecalis EF-1 was more effective in slowing down the weight gain of mice, especially during the 6th to 16th week of the experiment, the weight gain rate of mice treated with Enterococcus faecalis EF-1 was significantly lower than that of mice in the PC group, indicating that the intervention effect of the EF-1 group was better than that of the PC group as a positive control.

[0055] (4) Mouse organ index: The liver, white adipose tissue, kidney and spleen of each group of mice were taken, rinsed with saline, dried with filter paper and weighed, and the organ index was calculated according to the formula: organ index = (organ weight / body weight) × 100%.

[0056] The results are as follows Figure 8 As shown, the white fat weight of mice treated with Enterococcus faecalis EF-1 and atorvastatin calcium was significantly reduced (P<0.05). At the same time, the organ indexes of liver, kidney and spleen were not significantly different from those of mice in the high-fat diet group (HFD group), indicating that the intervention of atorvastatin calcium and Enterococcus faecalis EF-1 may help to alleviate the increase of adipose tissue.

[0057] (5) Liver lesions: The liver tissues of mice in each group were fixed with 4% tissue fixative, dehydrated, transparent, wax-impregnated, embedded, and sectioned, and then stained with Oil Red O (ORO) and HE. Finally, the changes in tissue morphology were observed under an optical microscope.

[0058] like Fig. 9 As shown in the figure, the liver cells of mice in the CT group did not show abnormalities such as fatty degeneration, the liver tissue structure appeared clear and complete, the liver lobule structure remained normal, the cell boundaries were clear, and the cell nucleus was located in the center. In contrast, the liver tissue of mice in the HFD group showed vesicular fatty degeneration, increased fat content in the liver, a large number of fat vacuoles, outflow of cell contents, cell swelling, and damaged cell wall integrity. The degree of fatty degeneration of the liver tissue of mice in the PC group and EF-1 group was significantly reduced compared with the HFD group, the fat vacuoles in the liver cells became smaller and the number decreased, and the morphology of liver cells was closer to that of the CT group.

[0059] (6) Fatty lesions: The diameter of fat vesicles is an intuitive indicator of the obesity degree of mice. The abdominal fat of each group of mice was fixed with 4% tissue fixative, and pathological HE staining sections were made to observe tissue changes.

[0060] like Fig.10 As shown, compared with that in the CT group, the diameter of the fat vacuoles in the adipose tissue of the HFD group mice was significantly increased, while the diameter of the fat vacuoles in the adipose tissue of the PC and EF-1 group mice was reduced, among which the effect in the EF-1 group was more significant, and the morphology of the fat vacuoles was more similar to that in the CT group.

[0061] (7) Serum and liver index testing: Long-term intake of a high-fat diet will accelerate the body's fat decomposition process and inhibit lipid synthesis, thereby producing excessive fatty acids, which in turn leads to increased levels of triglycerides (TG) and total cholesterol (TC) in the serum. In the obese state, the activity of liver lipase in the body increases, accelerating the clearance of high-density lipoprotein cholesterol (HDL-C), resulting in a decrease in its content, while the content of low-density lipoprotein cholesterol (LDL-C) increases relatively. As the main detoxification and lipid metabolism organ in the human body, the functional status of the liver can be assessed by the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), the levels of which can indicate the degree of liver abnormality. When liver cells undergo necrosis, alanine aminotransferase and aspartate aminotransferase are released into the blood, resulting in increased activity of these enzymes in the serum, and the degree of increase is proportional to the severity of liver tissue damage.

[0062] According to the instructions of the kit, the levels of total cholesterol, triglyceride, low-density lipoprotein cholesterol, glucose, alanine aminotransferase and aspartate aminotransferase in the serum of each group of mice were measured.

[0063] The results are as follows Fig.11 As shown in the results, compared with the CT group, the HFD group had significantly higher levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, and glucose (P<0.05). Compared with the HFD group, the EF-1 group had significantly lower levels of triglycerides and glucose (P<0.05); lower levels of total cholesterol, low-density lipoprotein cholesterol, alanine aminotransferase, and aspartate aminotransferase. Compared with the PC group, the EF-1 group had significantly lower levels of glucose (P<0.05); lower levels of total cholesterol, triglycerides, low-density lipoprotein cholesterol, alanine aminotransferase, and aspartate aminotransferase. These data indicate that Enterococcus faecalis EF-1 is superior to the positive control atorvastatin calcium in lipid regulation, glucose reduction, and protection of liver function.

[0064] According to the instructions of the kit, triglycerides, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, glucose, alanine aminotransferase, and aspartate aminotransferase in the liver of each group of mice were measured.

[0065] The results are as follows Fig.12 As shown in the results, compared with the CT group, the levels of triglyceride, low-density lipoprotein cholesterol, and alanine aminotransferase in the HFD group were significantly increased (P<0.05); the level of very low-density lipoprotein cholesterol was significantly decreased (P<0.05). Compared with the HFD group, the levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol, alanine aminotransferase, and aspartate aminotransferase in the EF-1 group were significantly decreased (P<0.05); the level of glucose was decreased; and the level of very low-density lipoprotein cholesterol was significantly increased. Compared with the PC group, the levels of low-density lipoprotein cholesterol, alanine aminotransferase, and aspartate aminotransferase in the EF-1 group were significantly decreased (P<0.05); the levels of triglyceride, total cholesterol, and glucose were decreased; and the level of very low-density lipoprotein cholesterol was significantly increased. These data indicate that the comprehensive effect of Enterococcus faecalis EF-1 in reducing liver lipid accumulation, lowering blood sugar, and protecting liver function is better than that of the positive control atorvastatin calcium.

[0066] (8) Changes in short-chain fatty acids: Short-chain fatty acids, mainly including acetate, propionate and butyrate, are metabolites produced by intestinal flora fermenting undigested dietary fiber. Total short-chain fatty acids (SCFA) in the cecum can affect the host's metabolic state by regulating plasma lipid levels; they can act as signal regulators to target and regulate intracellular lipid metabolism, which helps treat metabolic diseases such as obesity and malnutrition. Short-chain fatty acids have a protective effect on liver function and can improve liver fatty degeneration and abnormal lipid metabolism.

[0067] like Fig.13 As shown in the figure, the total SCFA concentration was 1.91 μg / mg in the CT group, which decreased to 1.06 μg / mg in the HFD group and increased to 1.30 μg / mg after the intervention of E. faecalis EF-1. Compared with the HFD group, the contents of acetic acid, propionic acid, isobutyric acid, isovaleric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid and decanoic acid in the cecum of the EF-1 group mice were increased.

[0068] (9) Changes in fecal flora: The PCoA analysis results of each group of mice are shown in Fig.14 As shown in the figure, compared with the CT group, the HFD group was separated from the CT group, indicating that feeding a high-fat diet caused changes in the intestinal flora. There were significant differences between the EF-1 group and the HFD group, indicating that Enterococcus faecalis EF-1 has a significant regulatory effect on the intestinal flora.

[0069] Analyze the intestinal flora structure. Fig.15 The middle stacked columns show the top 10 taxa at the relative abundance level at the intestinal phylum level in each group of mice.

[0070] Fig.16 The following Table 2 compares the Firmicutes in the intestines of different groups of mice ( Firmicutes ), Bacteroidetes ( Bacteroidota ) Relative abundance and ratio of bacterial flora, Firmicutes / Bacteroidetes ( F / B ) is the main feature of the intestinal flora structure of obesity and cardiovascular diseases. Compared with the CT group, the ratio of Firmicutes / Bacteroidetes in the intestines of mice in the HFD group was significantly increased; the ratio of Firmicutes / Bacteroidetes in the intestines of mice in the HFD group was 2.8, and the ratio of Firmicutes / Bacteroidetes in the intestines of mice in the EF-1 group was 2.43, indicating that the ratio of Firmicutes / Bacteroidetes in the intestines of mice was significantly reduced after the intervention of Enterococcus faecalis EF-1, indicating that Enterococcus faecalis EF-1 can alleviate the disorder of intestinal flora structure caused by high-fat diet by regulating intestinal flora.

[0071] Table 2 Relative abundance and ratio of Firmicutes and Bacteroidetes in the intestines of mice in each group

[0072] At the genus level, the structure of the intestinal flora was analyzed, e.g. Fig.17 As shown, the color gradient from blue to red indicates that the abundance of bacterial genera in different samples ranges from low to high, and the colors correspond to the legend. At the genus level, compared with the HFD group, the genus Bacteroides ( Bacteroides ), Faecalibacterium ( Faecalibaculum )、Desulfovibrio spp.( Desulfovibrio ) and Lachnospiraceae ( Lachnoclostridium) increased, and the intestinal flora structure was similar to that of the PC group, indicating that Enterococcus faecalis EF-1 can regulate intestinal flora by increasing the relative abundance of bacteria producing short-chain fatty acids, thereby reducing body weight and regulating lipid metabolism.

[0073] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention.

Claims

1. A lipid-lowering Enterococcus faecalis EF-1, characterized in that: Enterococcus faecalis EF-1 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 27, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.32097 and the classification name is Enterococcus faecalis Enterococcus faecalis .

2. A microbial preparation comprising the Enterococcus faecalis EF-1 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The content of Enterococcus faecalis EF-1 in the microbial preparation was 1.0×10 7 ~1.0×10 10 CFU / mL.

4. Use of the Enterococcus faecalis EF-1 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of a functional product for reducing glucose lipids.

5. The use according to claim 4, characterized in that Functional products for lowering glucose and lipid levels can be used to (1) prevent, alleviate or treat obesity and lipid metabolism disorders, (2) prevent, alleviate or treat insulin resistance and diabetes, (3) prevent, alleviate or treat hyperlipidemia, (4) alleviate liver fat deposition and adipose tissue lesions, and (5) improve intestinal flora and increase the relative abundance of bacteria that produce short-chain fatty acids.

6. The use according to claim 5, characterized in that Bacterial genera that produce short-chain fatty acids include Bacteroides, Faecalibacterium, Desulfovibrio, and Lachnospira.

7. The use according to claim 4, characterized in that The functional products of glucose-lowering lipids are foods, food additives, feeds, feed additives or medicines.

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