Enterococcus faecalis capable of degrading uric acid and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to enterococcus faecalis capable of degrading uric acid and application of the enterococcus faecalis, the enterococcus faecalis is named as Enterococcus faecalis SX3, and the preservation number of the enterococcus faecalis is CCTCC NO: M 2025248. The enterococcus faecalis has the advantages of being wide in antibacterial spectrum, rapid in growth, high in stress resistance, high in biological safety and the like, and important application value is provided for formulating a scheme for degrading uric acid.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Enterococcus faecalis capable of degrading uric acid and applications thereof. Background Art

[0002] Hyperuricemia is a disorder of uric acid metabolism, primarily caused by abnormal purine metabolism and impaired uric acid excretion. With improvements in living standards and the prevalence of high-purine diets, the incidence of hyperuricemia has been increasing annually, becoming the fourth most common health condition after hypertension, hyperglycemia, and hyperlipidemia. According to statistics, approximately 10% of patients with hyperuricemia will develop gout. Approximately 30% to 40% of uric acid is excreted through the intestines and bile duct. Uric acid in the intestine can be broken down by intestinal flora or directly excreted. Urate oxidase produced by intestinal microorganisms breaks down uric acid into more soluble allantoin or short-chain fatty acids. For example, Chinese invention patent publication number CN114149947A discloses a Lactobacillus plantarum strain that produces urate oxidase and inhibits xanthine oxidase, and its uses. This Lactobacillus plantarum strain can directly degrade uric acid and inhibit xanthine oxidase activity. Furthermore, intestinal microorganisms can also assist in lowering uric acid by interfering with the absorption of purines in food. For example, Chinese invention patent publication number CN118146982A discloses a Bacillus sp. strain with uric acid-lowering and antioxidant capabilities, and the strain has a strong ability to produce uricase.

[0003] In the prior art, commonly used probiotics such as Lactobacillus casei, Lactobacillus gasseri, and Lactobacillus fermentum are used to degrade adenosine, guanosine, adenylic acid, guanylic acid, adenine, and guanine, thereby reducing uric acid production. These lactobacilli have the advantages of no adverse drug reactions, no need for additional dietary restrictions, and high patient compliance in the treatment of hyperuricemia. However, lactobacilli have poor stress resistance and require strict anaerobic storage, which is unfavorable for storage, limiting their use in preventing and treating hyperuricemia. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of the existing technology and provide an Enterococcus faecalis that can degrade uric acid and its application; the Enterococcus faecalis of the present invention has the advantages of a broad antibacterial spectrum, rapid growth, strong stress resistance and high biosafety, and provides important application value for the formulation of a plan for degrading uric acid.

[0005] In order to achieve the above-mentioned purpose, the technical solution designed by the present invention is:

[0006] The invention provides an Enterococcus faecalis capable of degrading uric acid. The Enterococcus faecalis is named Enterococcus faecalis SX3 and has a preservation number of CCTCC NO: M 2025248.

[0007] The present invention also provides an application of the above-mentioned Enterococcus faecalis in the preparation of a drug for degrading uric acid.

[0008] The present invention also provides a use of the above-mentioned Enterococcus faecalis in the preparation of a microbial preparation.

[0009] The present invention also provides a live bacterial powder of Enterococcus faecalis SX3, wherein the live bacterial powder of Enterococcus faecalis SX3 contains the above-mentioned Enterococcus faecalis, and the effective live bacterial count is 10 11 cfu / g.

[0010] Furthermore, the effective viable bacteria count of Enterococcus faecalis SX3 in the live bacteria powder is 1×10 11 -5x10 11 cfu / g.

[0011] The present invention also provides a method for preparing the above-mentioned Enterococcus faecalis SX3 live bacterial powder, comprising the following steps:

[0012] 1) Fermentation strains of Enterococcus faecalis SX3 were obtained by seed culture;

[0013] 2) inoculating the fermentation bacteria into MRS medium and fermenting at 37°C for 24 hours to obtain a fermentation culture of Enterococcus faecalis;

[0014] 3) The fermentation culture of Enterococcus faecalis was centrifuged to harvest the bacterial cells of Enterococcus faecalis, and freeze-dried to obtain Enterococcus faecalis SX3 live bacterial powder, wherein the effective live bacterial count of Enterococcus faecalis in the Enterococcus faecalis SX3 live bacterial powder was 10 11 cfu / g.

[0015] Furthermore, in the Enterococcus faecalis SX3 live bacterial powder, the effective live bacteria count of Enterococcus faecalis is 1x10 11 -5x10 11 cfu / g.

[0016] The present invention also provides a feed for promoting intestinal development of chicks. The feed is prepared by adding the above additive to a basic diet, with 1 to 5 g of the additive added to every kilogram of the basic diet.

[0017] Furthermore, in the feed, the effective viable bacteria count of Enterococcus faecalis SX3 is 10 11 cfu / g.

[0018] Beneficial effects of the present invention:

[0019] (1) The Enterococcus faecalis SX3 of the present invention has the ability to tolerate high concentrations of uric acid and can still grow in MRS medium containing 5 mmol / L;

[0020] (2) The Enterococcus faecalis SX3 of the present invention has the ability to degrade uric acid. Under anaerobic conditions, it can reduce uric acid by approximately 60% after 24 hours.

[0021] (3) The Enterococcus faecalis SX3 of the present invention has the ability to degrade uric acid to produce short-chain fatty acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the alignment result of the genome amplified sequence of Enterococcus faecalis SX3 in the NCBI database.

[0023] Figure 2 This is a picture of Enterococcus faecalis SX cultured on MRS solid medium for 48 hours.

[0024] Figure 3 This is the Gram staining result of Enterococcus faecalis SX3.

[0025] Figure 4 Schematic diagram of the growth curve of Enterococcus faecalis SX3 in MRS liquid medium under anaerobic conditions with and without the addition of uric acid.

[0026] Figure 5 This figure shows the results of short-chain fat production by Enterococcus faecalis SX3 in MRS liquid medium under anaerobic conditions with or without the addition of uric acid for 24 hours.

[0027] Figure 6 This is a bar graph showing the concentration of uric acid in the serum of the control group, model group and experimental group in the broiler breeding experiment.

[0028] Figure 7 These are kidney pathological sections of the control group, model group, and experimental group in the broiler breeding experiment. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to specific embodiments so that those skilled in the art can understand.

[0030] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0031] The preparation method of the MRS solid culture medium in the embodiment of the present invention is as follows:

[0032] Prepare 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, and 15 g agar powder. Make up to 1 L with distilled water, adjust the pH to 7.2 ± 0.1, and sterilize at 121°C under high temperature and high pressure for 15 min.

[0033] The preparation method of the MRS liquid culture medium in the embodiment of the present invention is as follows:

[0034] Prepare 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate. Make up to 1 L with distilled water, adjust the pH to 7.2 ± 0.1, and sterilize at 121°C under high temperature and high pressure for 15 min.

[0035] The preparation method of the LB solid culture medium in the embodiment of the present invention is as follows:

[0036] Add 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 15 g of agar, make up to 1 L with distilled water, and sterilize at 121°C under high temperature and high pressure for 20 min.

[0037] The preparation method of the LB liquid culture medium in the embodiment of the present invention is as follows:

[0038] Add 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride, make up to 1 L with distilled water, and sterilize at 121°C under high temperature and high pressure for 20 min.

[0039] The sources or configuration methods of the reagents in the embodiments of the present invention are as follows:

[0040] NaOH source: Sinopharm Chemical Reagent Co., Ltd.

[0041] Preparation method of MRS culture medium containing uric acid: add the corresponding mass of uric acid to MRS culture medium and sterilize it at 121℃ and high temperature and high pressure for 15 minutes.

[0042] Preparation method of LB culture medium containing uric acid: add the corresponding mass of uric acid to LB culture medium and sterilize it at 121℃ and high temperature and high pressure for 20 minutes.

[0043] The experiments in the examples of the present invention were all repeated at least three times, and the experimental data were statistically analyzed using SPSS 24.0 software. One-way analysis of variance was adopted, and P < 0.05 was considered statistically significant.

[0044] Example 1 Isolation, culture and identification of Enterococcus faecalis SX3

[0045] 1. Sample Collection

[0046] The samples were isolated from the small intestinal digesta of Cobb broiler chickens, and the organisms were separated and purified using MRS medium. The samples were then anaerobically cultured in a constant temperature anaerobic operating box (Shanghai Longyue LAI-3) at 37°C for 48 hours. The anaerobic gas composition was N2:CO2:H2=80:10:10. Single colonies were picked and purified by plate streaking to obtain pure culture of each strain.

[0047] The genomic DNA of the isolated strain was extracted by referring to the bacterial genome advance kit (BL1044A, Biosharp) and detected by 1% agarose gel electrophoresis. No diffusion or tailing phenomenon was found. The quality and concentration of the extracted DNA were detected by UV spectrophotometer.

[0048] DNA samples with an A260 / 280nm ratio between 1.8 and 2.0 and no less than 20 ng / μL were considered qualified.

[0049] 2. Identification of Enterococcus faecalis SX3

[0050] 2.1 16S rDNA PCR amplification

[0051] 16SrDNA universal primers were used to amplify the genomic DNA of qualified strains by PCR. The primers were synthesized by Wuhan Jinkairui Bioengineering Co., Ltd. The specific sequences are as follows:

[0052] Upstream primer 27F: 5′-AGAGTTTGATCATGGCTCAG-3′;

[0053] Downstream primer 1492R: 5′-TAGGGTTACCTTGTTACGACTT-3′.

[0054] PCR amplification system and program settings:

[0055] PCR reaction system: 20 μL system contains 1 μL of strain genomic DNA template, 1 μL of upstream and downstream primers, and 10 μL of Taq enzyme mix (purchased from Nanjing Novozyme Biotechnology Co., Ltd.);

[0056] The PCR reaction procedure is as follows:

[0057] Stage 1: 95℃, 5min

[0058] Stage 2: 95℃, 10s

[0059] 48℃, 15s

[0060] 72℃, 90s

[0061] Stage 3: 72℃, 5min

[0062] Stage 4: 20℃, 10min

[0063] Among them, Stage 2 repeats 35 cycles.

[0064] 2.2 Sequencing and identification of strain 16S rDNA:

[0065]

[0066] The 16S rRNA sequence of SX3 was compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / ), and the bacteria with the highest homology to SX3 was Enterococcus faecalis strain RE25, with a similarity of 100% ( Figure 1 The isolated SX strain was confirmed to belong to Enterococcus faecalis and was named Enterococcus faecalis SX3, or E. faecalis SX3 for short. On February 19, 2025, E. faecalis SX3 was deposited in the China Center for Type Culture Collection at Wuhan University, Wuhan, China, with the deposit number: CCTCC NO: M2025248.

[0067] Example 2 Physiological Characteristics of Enterococcus faecalis SX3 and Its Tolerance to Uric Acid

[0068] 1. Physicochemical properties of Enterococcus faecalis SX3

[0069] 1.1 Colony morphology and characteristics of Enterococcus faecalis SX3

[0070] The optimal growth conditions for the Enterococcus faecalis SX3 isolated in Example 1 were 37°C and anaerobic conditions. The colonies cultured on MRS solid medium for 48 hours were white, spherical, and about 2-3 mm in diameter. Figure 2 ).

[0071] Gram staining results showed that the bacteria were Gram-positive rod-shaped bacteria ( Figure 3 ).

[0072] 1.2 Plotting the growth curve of Enterococcus faecalis SX3

[0073] From the 24-hour growth curve of Enterococcus faecalis SX3 ( Figure 4 ) It can be seen that SX3 enters the logarithmic growth phase at 5 hours and enters the plateau phase at 10 hours. The specific process is as follows:

[0074] 1) Take the SX3 strain out of the -80°C freezer, draw a line on a plate, and incubate the plate in an anaerobic chamber at 37°C for 24 hours.

[0075] 2) Pick a single colony from the plate and inoculate it into 10 mL of MRS medium. Incubate anaerobically at 37°C for 24 hours.

[0076] 3) Prepare three identical anaerobic tubes containing 200 μL of MRS medium and three identical anaerobic tubes containing 200 μL of MRS medium containing 5 mmol / L uric acid as technical replicates. Transfer the cultured bacterial suspension to three 200 μL culture tubes at a 1% inoculum volume.

[0077] All culture solutions were cultured in an anaerobic operating chamber at 37°C, and the OD600nm absorbance was monitored every 10 minutes using a microplate reader.

[0078] The experimental results are as follows Figure 4 As shown: The SX3 strain has the ability to tolerate 5mmol / L uric acid.

[0079] Example 3 Ability of Enterococcus faecalis SX3 to degrade uric acid in different culture media

[0080] 1. Ability of Enterococcus faecalis SX3 to degrade uric acid in LB medium

[0081] 1) Take the SX3 strain out of the -80°C freezer, draw a line on a plate, and incubate the plate in an anaerobic chamber at 37°C for 24 hours.

[0082] 2) Pick a single colony from the plate and inoculate it into 10 mL of LB medium. Incubate anaerobically at 37°C for 24 h.

[0083] 3) Transfer the mixture to LB liquid medium containing 5 mmol / L uric acid at a ratio of 1:50 and measure the uric acid concentration after anaerobic culture for 24 hours

[0084] 2. Ability of Enterococcus faecalis SX3 to degrade uric acid in MRS medium

[0085] 1) Take the SX3 strain out of the -80°C freezer, draw a line on a plate, and incubate the plate in an anaerobic chamber at 37°C for 24 hours.

[0086] 2) Pick a single colony from the plate and inoculate it into 10 mL of MRS medium. Incubate anaerobically at 37°C for 24 hours.

[0087] 3) Transfer the cells to MRS liquid culture medium containing 5 mmol / L uric acid at a ratio of 1:50, and measure the uric acid concentration after anaerobic culture for 24 hours.

[0088] Table 1 Uric acid concentration before and after culture in different culture media

[0089] culture medium LB MRS Uric acid concentration before culture (average value) 5.6mmol / L 5.0mmol / L Uric acid concentration before culture (standard deviation) 0.54mmol / L 0.92mmol / L Uric acid concentration after 24 hours of culture (average value) 2.01mmol / L 0.08mmol / L Uric acid concentration after 24 hours of culture (standard deviation) 1.57mmol / L 0.13mmol / L

[0090] As shown in the experimental results in Table 1, the SX3 strain has the ability to degrade uric acid under anaerobic culture conditions.

[0091] Example 4 Enterococcus faecalis SX3 has the ability to metabolize uric acid to produce short-chain fatty acids

[0092] The concentration of short-chain fatty acids in the culture supernatant of Example 2 was detected by gas chromatography, as follows:

[0093] 1) Extract the culture supernatant with methanol in a ratio of 1:1, and filter the supernatant with a 0.22 μm filter membrane to remove particulate matter.

[0094] 2) Chromatograph and column: Thermo Scientific gas chromatograph Trace 1300 with DB-FFAP (30m×0.25mm×0.25μm) capillary column

[0095] 3) Carrier gas: high-purity helium (He), flow rate 1.0 mL / min.

[0096] 4) Inlet temperature: 250°C.

[0097] 5) Detector: flame ionization detector (FID), temperature 260°C.

[0098] 6) Column temperature program:

[0099] Initial temperature: 50°C, maintain for 2 minutes.

[0100] The temperature was raised to 200°C at a rate of 10°C / min and maintained for 5 min.

[0101] Total running time: about 20 minutes.

[0102] 7) Injection volume: 5 μL, split ratio 10:1.

[0103] 8) Data analysis: Use Thermo Scientific Chromeleon to integrate each fatty acid peak and record the peak area.

[0104] The results are as follows Figure 5 As shown: After 24 hours of culture with the addition of uric acid, the contents of propionic acid and isobutyric acid in the culture supernatant increased significantly.

[0105] Example 5

[0106] The microbial preparation of this embodiment is live Enterococcus faecalis SX3 powder, which is produced by fermentation of the uric acid-degrading Enterococcus faecalis SX in Example 3. The preparation method includes the following steps:

[0107] 1) inoculating the fermentation strain into MRS medium and fermenting and culturing at 37° C. for 24 hours to obtain a fermentation culture of Enterococcus faecalis; wherein the fermentation strain is obtained by seed culture of Enterococcus faecalis SX3 strain;

[0108] 2) The fermentation culture of Enterococcus faecalis was centrifuged to harvest the bacterial cells of Enterococcus faecalis, and freeze-dried to obtain live bacterial powder of Enterococcus faecalis SX3, wherein the effective live bacterial count of Enterococcus faecalis SX3 in the live bacterial powder of Enterococcus faecalis SX3 was 1×10 11 -5x10 11 cfu / g.

[0109] According to actual conditions, the culture medium of the fermentation culture of Enterococcus faecalis can be washed with physiological saline to obtain pure bacterial cells of Enterococcus faecalis.

[0110] Example 6

[0111] A feed for promoting intestinal development of chicks is prepared by adding 1 to 5 g of the additive of Example 5 to each kilogram of basic diet, wherein the content of Enterococcus faecalis SX3 in the feed is 10 11 cfu / kg.

[0112] Example 7 Application of uric acid-degrading feed in broiler production

[0113] The selected broiler models were grouped as follows:

[0114] Control group: fed with basic diet,

[0115] Model group: fed with basal diet supplemented with 10% yeast extract powder,

[0116] Experimental group: The rats were fed the feed of Example 5 supplemented with 10% yeast extract powder, starting from day 1 and continuing to be fed for 21 days. The specific experimental method is as follows:

[0117] 1. Broiler grouping

[0118] A total of 192 one-day-old male white-feathered broiler chickens (weight 40±2g) were randomly divided into a control group (BD) and an experimental group (LS), with 8 replicates in each group and 8 chickens in each replicate. The broiler chickens were raised for 21 days.

[0119] 2. Analysis of uric acid content in broiler serum

[0120] After the three groups of broilers were treated for 21 days, serum was collected and the uric acid content was determined by HPLC.

[0121] like Figure 6 As shown: When broilers were fed a diet containing Enterococcus faecalis SX3, the uric acid concentration in the serum of the broilers was reduced, which was not significantly different from that of the basal diet group.

[0122] 3. Pathological section analysis of broiler kidneys

[0123] After the three groups of broilers were treated for 21 days, they were slaughtered and samples were collected, and AB-PAS staining was performed on the paraffin sections of the kidneys.

[0124] like Figure 7 Figure 3: Feeding broiler chickens with a diet containing Enterococcus faecalis SX3 alleviated kidney damage.

[0125] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without inventiveness, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An Enterococcus faecalis capable of degrading uric acid, characterized by: The Enterococcus faecalis is named Enterococcus faecalis SX3, and its preservation number is CCTCC NO: M 2025248.

2. Use of the Enterococcus faecalis according to claim 1 in the preparation of a medicament for degrading uric acid.

3. Use of the Enterococcus faecalis according to claim 1 in the preparation of a microbial preparation.

4. A live Enterococcus faecalis SX3 bacterial powder, characterized by: The Enterococcus faecalis SX3 live bacterial powder contains the Enterococcus faecalis according to claim 1, and its effective live bacterial count is 10 11 cfu / g.

5. The live Enterococcus faecalis SX3 powder according to claim 4, characterized in that: The effective live bacteria count of Enterococcus faecalis SX3 in the live bacteria powder is 1x10 11 ~5x10 11 cfu / g.

6. A method for preparing the live Enterococcus faecalis SX3 powder according to claim 4, characterized in that: The following steps are involved: 1) Fermentation strains of Enterococcus faecalis SX3 were obtained by seed culture; 2) inoculating the fermentation bacteria into MRS medium and fermenting at 37°C for 24 hours to obtain a fermentation culture of Enterococcus faecalis; 3) The fermentation culture of Enterococcus faecalis was centrifuged to harvest the bacterial cells of Enterococcus faecalis, and freeze-dried to obtain Enterococcus faecalis SX3 live bacterial powder, wherein the effective live bacterial count of Enterococcus faecalis in the Enterococcus faecalis SX3 live bacterial powder was 10 11 cfu / g.

7. The live Enterococcus faecalis SX3 powder according to claim 6, characterized in that: The effective live bacteria count of Enterococcus faecalis SX3 in the live bacteria powder is 1x10 11 ~5x10 11 cfu / g.

8. A feed for promoting intestinal development in chicks, characterized by: The feed is prepared by adding the additive according to claim 4 or 5 to a basic diet, and 1 to 5 g of the additive is added to each kilogram of the basic diet.

9. The feed according to claim 8, characterized in that: In the feed, the effective viable bacteria count of Enterococcus faecalis SX3 is 10 11 cfu / kg.

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