Enterococcus faecalis FC-1 helpful for reducing blood fat and application of enterococcus faecalis FC-1

Abstract

The invention belongs to the technical field of microorganisms, and provides enterococcus faecalis FC-1 helpful for reducing blood fat and application of the enterococcus faecalis FC-1, the enterococcus faecalis FC-1 is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.33967. The enterococcus faecalis FC-1 can be used for reducing blood fat. In a model experiment of a golden hamster fed with a high-fat and high-cholesterol feed, the strain shows the capabilities of retarding weight gain and fat deposition, and reducing blood fat and liver lipid level; after intervention, the liver index of the hamster is reduced by 24.74%, the triglyceride level in serum is reduced by 3.93 times, the total cholesterol level is reduced by 2.05 times, the low-density lipoprotein cholesterol level is reduced by 3.02 times, and the triglyceride level, the total cholesterol level and the low-density lipoprotein cholesterol level in the liver are respectively reduced by 34.44%, 58.85% and 40.76%. The strain can be used for preparing medicines and health-care products for losing weight, treating fatty liver and reducing blood fat.

Description

Technical Field

[0001] This application belongs to the field of microorganisms. Specifically, this application provides an Enterococcus faecalis FC-1 that helps reduce blood lipids and its applications. Background Art

[0002] In recent years, with the in-depth study of the gut microbiome, the application of probiotics in the prevention and treatment of metabolic diseases has attracted much attention. Enterococcus faecalis, as a resident gut flora, has become a potential probiotic to be developed due to its unique acid and bile salt tolerance characteristics and potential metabolic regulatory functions. However, there are still many technical bottlenecks for Enterococcus faecalis strains with clear blood lipid-lowering functions in the prior art: First, the survival rate of most wild-type Enterococcus faecalis in the simulated gastric acid environment of pH 2.5 is less than 30%, resulting in the difficulty of reaching the functional dose of viable bacteria; Second, the bile salt-tolerant strains screened traditionally often accompany the expression of virulence genes, facing challenges in clinical safety; In addition, most reported strains such as E. faecalis EF-2001 only focus on the improvement of serum lipids, and the improvement effect on liver lipid deposition is limited. The current solutions have significant limitations: Although genetic modification technology can improve safety, it faces strict regulatory restrictions; Although microencapsulation technology can improve the survival rate of bacteria, it will increase production costs and affect the metabolic activity of bacteria. It is worth noting that recent studies have found that there is significant strain specificity in the regulation of liver lipid metabolism by Enterococcus faecalis. The improvement rate of common commercial strains such as E. faecalis Symbioflor 1 on liver steatosis is less than 10%. In addition, most commercially available probiotic preparations use complex flora (such as Bifidobacterium + Lactobacillus), and the market for single-strain applications of Enterococcus faecalis remains to be developed. Summary of the Invention

[0003] To solve the above technical problems, the present invention isolated and obtained a novel Enterococcus faecalis FC-1, which has three technical advantages: (1) Outstanding tolerance to extreme environments, with a survival rate of 86.1% at pH 3.0 for 3 h and 94.1% at 0.3% bile salt treatment for 3 h; (2) Significant lipid-lowering effect. Animal experiments showed that this strain could significantly relieve weight gain and reduce blood lipid levels, specifically manifested as a 2.05-fold decrease in serum total cholesterol level, a 3.93-fold decrease in triglyceride level, and a 3.02-fold decrease in low-density lipoprotein cholesterol level; (3) Good liver protection effect. Animal experiments showed that this strain could significantly reduce liver fat deposition, and the levels of triglyceride, total cholesterol, and low-density lipoprotein cholesterol in the liver decreased by 34.44%, 58.85%, and 40.76% respectively. This strain provides a solution for the development of single-component strain blood lipid-lowering probiotic preparations.

[0004] In view of the deficiencies of the prior art, the present invention provides a strain of Enterococcus faecalis FC-1, which has probiotic functions, acid resistance, bile salt resistance, and excellent characteristics of reducing blood lipid.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: On the one hand, the present application provides an Enterococcus faecalis FC-1 that helps reduce blood lipid. The Enterococcus faecalis FC-1 is deposited in the China General Microbiological Culture Collection Center, and the deposit number is: CGMCC No. 33968.

[0006] On the other hand, the present application provides a probiotic preparation, which contains the above-mentioned Enterococcus faecalis FC-1.

[0007] Further, the probiotic preparation is an orally administered freeze-dried bacterial powder or bacterial liquid.

[0008] Further, the preparation further contains excipient components selected from one or more of freeze-drying protectants, fillers, solvents, suspending agents, antioxidants, osmotic pressure regulators, and pH regulators.

[0009] On the other hand, the present application provides the use of the above-mentioned Enterococcus faecalis FC-1 or probiotic preparation in the preparation of a drug for slowing down weight gain or losing weight.

[0010] On the other hand, the present application provides the use of the above-mentioned Enterococcus faecalis FC-1 or probiotic preparation in the preparation of a health product that helps control body fat.

[0011] On the other hand, the present application provides the use of the above-mentioned Enterococcus faecalis FC-1 or probiotic preparation in the preparation of a drug for reducing blood lipid.

[0012] On the other hand, the present application provides the use of the above-mentioned Enterococcus faecalis FC-1 or probiotic preparation in the preparation of a health product that helps maintain a healthy blood lipid level.

[0013] Further, the drug for reducing blood lipid is a drug for reducing the levels of serum triglyceride, total cholesterol, and / or low-density lipoprotein cholesterol.

[0014] On the other hand, the present application provides the use of the above-mentioned Enterococcus faecalis FC-1 or probiotic preparation in the preparation of a drug for improving liver fat deposition.

[0015] The above-mentioned drugs and health products can be prepared into various dosage forms in the fields of drugs and health products, including but not limited to tablets, powders, capsules, oral liquids, etc. Preparation methods such as freeze-drying, granulation, tabletting, coating, etc. can use methods known in the art that can be used for microbial preparations. Due to its excellent acid and bile salt tolerance, the strain of the present application is particularly suitable for preparing oral medicaments or health products.

[0016] In addition to the above-mentioned strain, the above-mentioned drugs and health products may contain excipients known in the fields of drugs and health products, including but not limited to: solvents, cosolvents, osmotic pressure regulators, pH regulators, surfactants, fillers, lubricants, binders, antioxidants, preservatives, taste masking agents, flavoring agents, coating materials, capsule materials, etc.; they may also include traditional Chinese and Western medicines, health products, and food ingredients that are helpful for the uses of drugs and health products.

[0017] The present invention discloses a strain of Enterococcus faecalis FC-1, which is isolated from healthy golden hamsters fed with buffalo milk for a long time at the SPF level. This strain has a variety of probiotic effects, has good effects of reducing blood lipid and protecting the liver, and can significantly reduce the increase in triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels and body weight induced by a high-fat and high-cholesterol diet in serum and liver. At the same time, this strain also has extremely strong acid and bile salt tolerance characteristics and is suitable for colonizing in the human gastrointestinal tract. Therefore, the Enterococcus faecalis FC-1 strain has important application value in the fields of alleviating weight gain, liver fat deposition, reducing blood lipid, and reducing liver lipid indexes.

[0018] Strain preservation information: The Enterococcus faecalis FC-1 strain is preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is: March 25, 2025. The classification name is: Enterococcus faecalis. The preservation number is: CGMCC No. 33968. Description of the drawings

[0019] Figure 1 It is the colony morphology of the Enterococcus faecalis FC-1 strain.

[0020] Figure 2 It is the microscopic examination image of the bacteria of the Enterococcus faecalis FC-1 strain after Gram staining.

[0021] Figure 3Results of the catalase-negative experiment for Enterococcus faecalis strain FC-1

[0022] Figure 4 Agarose gel electrophoresis pattern of the PCR amplification product of Enterococcus faecalis strain FC-1

[0023] Figure 5 Acid tolerance test of Enterococcus faecalis strain FC-1

[0024] Figure 6 Bile salt tolerance test of Enterococcus faecalis strain FC-1

[0025] Figure 7 Effect of Enterococcus faecalis strain FC-1 on the body weight change of golden hamsters fed a high-fat and high-cholesterol diet

[0026] Figure 8 Effect of Enterococcus faecalis strain FC-1 on the food intake change of golden hamsters fed a high-fat and high-cholesterol diet

[0027] Figure 9 Effect of Enterococcus faecalis strain FC-1 on the blood lipid change of golden hamsters fed a high-fat and high-cholesterol diet. A represents triglyceride level; B represents total cholesterol level; C represents low-density lipoprotein cholesterol level

[0028] Figure 10 Anatomical diagram of the liver of golden hamsters fed a high-fat and high-cholesterol diet intervened by Enterococcus faecalis strain FC-1

[0029] Figure 11 Results of the liver index of golden hamsters fed a high-fat and high-cholesterol diet intervened by Enterococcus faecalis strain FC-1

[0030] Figure 12 HE staining diagram (first row) and Oil Red O (second row) staining diagram of the liver of golden hamsters fed a high-fat and high-cholesterol diet intervened by Enterococcus faecalis strain FC-1

[0031] Figure 13To observe the changes in liver lipid indexes of golden hamsters with high-fat and high-cholesterol diet intervened by Enterococcus faecalis FC-1 strain. A represents the triglyceride level; B represents the total cholesterol level; C represents the low-density lipoprotein cholesterol level. Detailed implementation manners

[0032] The present invention will be further described below in conjunction with embodiments. The following embodiments are helpful for better understanding the present invention, but do not limit the present invention. The experimental methods in the following embodiments are all conventional experimental methods unless otherwise specified.

[0033] The test strain used in the present invention: Enterococcus faecalis FC-1 strain was isolated from healthy golden hamsters fed with buffalo milk for a long time at the SPF level.

[0034] All the culture media used in the present invention are purchased from Qingdao Haibo Biotechnology Co., Ltd. Other reagents, unless otherwise specified, can be obtained from commercial channels.

[0035] Example 1: Screening and identification of Enterococcus faecalis FC-1 strain Screening of Enterococcus faecalis FC-1 strain: Sample collection: The samples were derived from healthy golden hamsters fed with buffalo milk for a long time at the SPF level. Take 1 g of fecal sample and add 9 mL of sterile normal saline (0.85% NaCl), vortex and mix for 1 min.

[0036] Isolation and purification of the strain: Take 1 mL of the supernatant and perform 10-fold serial dilution (10 -1 to 10 -6 ), and then filter the impurities with a 0.45 μm filter membrane. Take 10 -4 , 10 -5 , 10 -6 gradient concentrations and coat the plates. Under sterile conditions, coat the samples on brain heart infusion (BHI) agar plates and incubate aerobically at 37°C for 48 h. Select colonies that are round, smooth, grayish-white, 1-2 mm in diameter, and have neat edges for repeated streak culture to obtain single colonies ( Figure 1 ). The whole process of isolation and purification is in a sterile environment.

[0037] BHI agar medium: 12.5 g of bovine brain infusion powder, 5.0 g of bovine heart infusion powder, 10.0 g of peptone, 2.0 g of glucose, 5.0 g of sodium chloride, 2.5 g of disodium hydrogen phosphate, 15.0 g of agar, made up to 1 L with distilled water, pH 7.4 ± 0.2.

[0038] Identification of Enterococcus faecalis FC-1 strain: Enterococcus faecalis appears as off-white, circular colonies with a diameter of 1-2 mm and neat edges on BHI agar medium. Strains that meet the above morphology are selected and inoculated into BHI liquid medium. After culturing for 24 hours, a small amount of bacterial liquid is taken for Gram staining and the morphology of the strain is observed under an optical microscope. Gram staining is positive and the colonies are arranged in pairs or short chains ( Figure 2 Pick a portion of the isolated and purified single colonies and place them in a 3% H2O2 solution. Let it stand for 30 seconds until there are no bubbles ( Figure 3 ), the catalase test result was negative, which was consistent with the catalase test result of Enterococcus faecalis, and it could be further confirmed that the isolated colonies were Enterococcus faecalis.

[0039] Suspected single colonies were added to 20 µL of sterile saline, then denatured at 95°C for 5 min to prepare template DNA. Finally, the sequence of the specific gene was amplified using PCR. The PCR reaction system (25 µL) included: (2×) Taqmix 12.5 µL; ddH2O 9.5 µL; primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2) 1 µL each; and template DNA 1 µL. The reaction conditions were set to 95°C pre-denaturation (5 min), followed by 30 cycles of: 94°C denaturation (30 s), 55°C annealing (30 s), 72°C extension (90 s), and finally 72°C extension (10 min). The amplified PCR product was detected by 1.0% agarose gel electrophoresis to confirm that the target DNA fragment had been successfully amplified ( Figure 4 The PCR product was then sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing, yielding the 16S rRNA sequence of Enterococcus faecalis FC-1 (SEQ ID NO. 3). The sequence data was then aligned with known sequences of Enterococcus faecalis. The bacterial species was determined through alignment analysis.

[0040] The measured 16S rRNA sequence of Enterococcus faecalis FC-1 is SEQ ID NO.3:

[0041] Example 2: Acid Tolerance Experiment of Enterococcus faecalis FC-1 Strain Pick a single colony from the BHI agar medium and inoculate it into 5 mL of BHI liquid medium. Incubate at 37°C for 12 - 16 h, adjust the bacterial suspension to 10 8 CFU / mL. Take 1 mL of the bacterial suspension and centrifuge it at 5000 rpm for 5 min. Discard the supernatant, and add 9 mL of PBS with pH 2.0, pH 3.0, pH 4.0, pH 5.0, and pH 6.0 respectively. Incubate at 37°C for 0 h and 3 h, then spread it on the BHI agar medium and calculate the survival rate ( Figure 5 ).

[0042] Survival rate (%) = A3 / A0 × 100%. A3 is the count at 3 h, and A0 is the count at 0 h.

[0043] Example 3: Bile Salt Tolerance Experiment of Enterococcus faecalis FC-1 Strain Pick a single colony from the BHI agar medium and inoculate it into 5 mL of BHI liquid medium. Incubate at 37°C for 12 - 16 h, adjust the bacterial suspension to 10 8 CFU / mL. Take 1 mL of the bacterial suspension and centrifuge it at 5000 rpm for 5 min. Discard the supernatant, and add 9 mL of PBS / BHI containing bile salts (final concentrations of bile salts are 0.1%, 0.15%, 0.2%, 0.25%, and 0.3% respectively). Incubate at 37°C for 0 h and 3 h, then spread it on the BHI agar medium and calculate the survival rate ( Figure 6 ).

[0044] Survival rate (%) = A3 / A0 × 100%. A3 is the count at 3 h, and A0 is the count at 0 h.

[0045] Example 4: Effect of Enterococcus faecalis FC-1 Strain on the Body Weight of Golden Hamsters Fed with High-Fat and High-Cholesterol Diet Preparation of Bacterial Suspension: The activated Enterococcus faecalis FC-1 strain (activation conditions: 10% (v / v), inoculated into BHI liquid medium, cultured at 37°C for 16 h until the quantity reaches 1×109 CFU / mL) is resuspended in 1 mL of PBS buffer (0.1 M, pH 6.0) to obtain a bacterial suspension with a concentration of 1×108 CFU / mL.

[0046] Grouping of Animal Experiments: Male golden hamsters weighing 110 ± 10 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Jing) 2021-0006). The laboratory conditions were 22℃ ± 2℃ and a 12 h light-dark cycle. The animals had free access to food, and the food formula is shown in Table 1. After one week of adaptive feeding, the experiment was conducted for a total of 10 weeks. In this experiment, the treatment of animals complied with the standard regulations of the Laboratory Animal Committee.

[0047] Table 1: Feed formula (example)

[0048] The golden hamsters were randomly divided into 3 groups (10 in each group), namely the control group (NC group), the high-fat model group (HFD group), and the high-fat FC-1 treatment group (HFD+FC-1 group). The relevant treatments for each group of animals are shown in Table 2.

[0049] Table 2: Grouping and treatment of experimental animals

[0050] Measurement indicators: The body weight and food intake of the hamsters were recorded every three days until the end of the experiment.

[0051] Measurement results: At the end of the experiment, all hamsters survived, and no obvious abnormalities were found during daily observation, indicating good basic safety under experimental conditions. The average body weight of golden hamsters in the HFD group was 207.3 g, the average body weight of the NC group was 185.3 g, and the average body weight of the HFD+FC-1 treatment group was 181 g. The average body weight of the HFD group was extremely significantly higher than that of the HFD+FC-1 treatment group ( Figure 7 ). There were no significant differences in the food intake among the three groups during the experiment ( Figure 8 ).

[0052] Example 5: Regulation of blood lipids in golden hamsters with high-fat and high-cholesterol diet by Enterococcus faecalis FC-1 strain Preparation of bacterial suspension: The same as in Example 4.

[0053] Grouping of animal experiments: The same as in Example 4.

[0054] Measurement indicators: After the experiment, the golden hamsters were euthanized by inhaling carbon dioxide after 12 h of fasting with free access to water. Blood samples of the hamsters were collected. After standing at room temperature for 45 min, they were centrifuged at 3,000 rpm for 10 min at 4 °C in a high-speed low-temperature centrifuge, and the supernatant was taken to obtain serum. An automatic biochemical analyzer (company: URIT, model: URIT-8021AVet) was used to detect the levels of TG, TC and LDL-C (company: URIT, product numbers: U82885040, U82785040, U83085045) in the serum.

[0055] Measurement results: The results were as Figure 9 shown. At the end of the experiment, the levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the serum of golden hamsters in the HFD group were 26.05 mmol / L, 22.58 mmol / L and 11.46 mmol / L, respectively. After the intervention with Enterococcus faecalis FC-1 strain, the levels of triglyceride, total cholesterol and low-density lipoprotein cholesterol in the serum of golden hamsters decreased to 6.63 mmol / L, 11.02 mmol / L and 3.79 mmol / L, respectively. It was indicated that the intervention with Enterococcus faecalis FC-1 strain significantly improved the abnormal blood lipid levels induced by high-fat diet.

[0056] Example 6: Improvement effect of Enterococcus faecalis FC-1 strain on liver fat deposition in golden hamsters fed with high-fat and high-cholesterol diet Preparation of bacterial suspension: The same as in Example 4.

[0057] Grouping of animal experiments: The same as in Example 4.

[0058] Measurement indexes: After the experiment, the golden hamsters were euthanized by inhaling carbon dioxide after 12 h of fasting with free access to water. The dissection instruments were pre-cooled and disinfected in advance. An incision was made along the midline of the abdomen of the hamster to expose the abdominal cavity, the liver was completely taken out, the blood stains on the surface were rinsed with normal saline (0.9% NaCl), photographed and recorded, and the liver index was calculated by weighing.

[0059]

[0060] A part of the intact liver tissue was cut and placed in a clean 2 mL centrifuge tube for the detection of lipid indicators, and another part was fixed in 4% paraformaldehyde for 24 h. After fixation, the liver tissue was dehydrated, cleared, embedded, and sectioned to make paraffin sections, followed by HE staining. The specific steps are shown in Table 3. The liver tissue was embedded in OCT and frozen sections were made at -20 °C, followed by Oil Red O staining. The specific steps are as follows: rinsing with 60% isopropanol for 30 s, staining with 0.5% isopropanol for 15 min, counterstaining the nucleus with hematoxylin for 1 min, and mounting with glycerin jelly.

[0061] Table 3: Steps of HE staining

[0062] Measurement results: At the end of the experiment, the liver of golden hamsters in the NC group showed a normal dark red color, with a uniform texture on the cut surface and sharp edges. The liver of golden hamsters in the HFD group was significantly enlarged, with a yellowish-white surface, increased capsule tension, and a greasy cut surface. After intervention with Enterococcus faecalis FC-1 strain, the liver color of golden hamsters returned to the transitional state of yellowish-red, and the greasy feeling on the cut surface was significantly improved ( Figure 10 ), and the liver index decreased by 24.74% ( Figure 11 ).

[0063] Observation of sections under an optical microscope showed that the hepatic lobule structure in the NC group was intact, the hepatocytes were arranged neatly, the cytoplasm was uniform, and only occasional tiny lipid droplet vacuoles were seen. The HFD group showed typical pathological changes of fatty liver, with disordered hepatic cord structure, enlarged hepatocyte volume, and round vacuoles of different sizes visible in the cytoplasm, and a significant increase in lipid droplets. The hepatic tissue structure in the HFD+FC-1 group was significantly improved, the arrangement of hepatocytes tended to be regular, the inflammatory infiltration was reduced, and the lipid droplet deposition was significantly reduced, and most of them were small lipid droplets. It shows that the intervention of Enterococcus faecalis FC-1 strain significantly improves the hepatic lipid deposition induced by a high-fat and high-cholesterol diet ( Figure 12 ).

[0064] Example 7: Regulation of hepatic lipid indicators in golden hamsters with a high-fat and high-cholesterol diet by Enterococcus faecalis FC-1 strain Preparation of bacterial suspension: The same as in Example 4.

[0065] Grouping of animal experiments: The same as in Example 4.

[0066] Measurement indicators: After the experiment, the golden hamsters were euthanized by carbon dioxide inhalation after 12 h of fasting with free access to water. 50 mg of liver tissue was placed in 450 μL of PBS (pH 7.4), homogenized at 4°C and 12,000 rpm for 1 min, and the supernatant was taken after centrifugation at 4°C and 3,000 rpm for 10 min. The levels of TG, TC, and LDL-C in the liver were detected using a complete kit (Company: Nanjing Jiancheng, Catalog No.: A110-1-1, A111-1-1, A113-1-1).

[0067] Measurement results: The results were as Figure 13 shown. At the end of the experiment, the triglyceride level in the liver of golden hamsters in the HFD group was 1.08 mmol / gprot, the total cholesterol level was 5.39 mmol / gprot, and the low-density lipoprotein cholesterol level was 0.90 mmol / gprot. After intervention with Enterococcus faecalis strain FC-1, the levels of triglyceride, total cholesterol, and low-density lipoprotein cholesterol in the liver of golden hamsters decreased by 34.44%, 58.85%, and 40.76%, respectively. This indicates that the intervention of Enterococcus faecalis strain FC-1 significantly improved the abnormal liver lipid levels induced by a high-fat and high-cholesterol diet.

Claims

1. An Enterococcus faecalis FC-1 that helps reduce blood lipids, characterized in that, The Enterococcus faecalis FC-1 is deposited in the China General Microbiological Culture Collection Center with the deposit number: CGMCC No. 33968.

2. Use of the Enterococcus faecalis FC-1 according to claim 1 in the preparation of a drug for slowing down weight gain or losing weight.

3. Use of the Enterococcus faecalis FC-1 according to claim 1 in the preparation of a health product for helping to control body fat.

4. Use of the Enterococcus faecalis FC-1 according to claim 1 in the preparation of a drug for reducing blood lipid.

5. Use of the Enterococcus faecalis FC-1 according to claim 1 in the preparation of a health product for helping to maintain a healthy blood lipid level.

6. According to the use described in claim 4, the drug for reducing blood lipid is a drug for reducing the levels of serum triglyceride, total cholesterol and / or low-density lipoprotein cholesterol.

7. Use of the Enterococcus faecalis FC-1 according to claim 1 in the preparation of a drug for improving liver fat deposition.

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