Tibet naturally-fermented yak milk source lactobacillus reuteri SAU-R-8 for producing exopolysaccharides and application of lactobacillus reuteri SAU-R-8 in reducing uric acid

Abstract

The invention relates to lactobacillus reuteri SAU-R-8 which is derived from Tibet naturally-fermented sour yak milk and is used for producing exopolysaccharides and application of the lactobacillus reuteri SAU-R-8 in reducing uric acid, and belongs to the technical field of microorganisms. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.30373, and the preservation date is April 19, 2024. The lactobacillus reuteri SAU-R-8 provided by the invention is screened from yak yogurt naturally fermented from herdsmen in the Sankarst region of Tibet, the yield of exopolysaccharides in an MRS culture medium reaches 318.57 mg / L, nucleoside substances such as inosine and guanosine can be efficiently degraded, purine substances such as guanine can be efficiently absorbed, the activity of xanthine oxidase can be inhibited, and the lactobacillus reuteri SAU-R-8 can be used for preparing the yak yogurt. The method can be further applied to product development for relieving or treating hyperuricemia.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Lactobacillus mucosae rhamnosus SAU-R-8 derived from naturally fermented Tibetan sour yak milk that produces exopolysaccharides and its application in reducing uric acid. Background Art

[0002] Hyperuricemia is a chronic metabolic disease in which the serum uric acid level exceeds the normal value. In China, the incidence of hyperuricemia has reached 13.3%, which means that hundreds of millions of people are suffering from hyperuricemia. Its onset is closely related to improper diet and the occurrence of other metabolic diseases. Hyperuricemia has become the second most common metabolic disease after diabetes, and the treatment of hyperuricemia has attracted more and more attention. At present, the main drugs used to treat hyperuricemia are xanthine oxidase inhibitors and urate-excreting drugs, but side effects such as allopurinol hypersensitivity syndrome and liver and kidney damage are likely to occur after long-term medication. In recent years, relevant studies have found that probiotics have a relieving effect on hyperuricemia. Lactobacillus gasseri PA-3 can extracellularly degrade nucleosides, absorb nucleosides into the bacteria, reduce the absorption of nucleoside substances in the intestine, and achieve the effect of reducing blood uric acid. Lactobacillus brevis DM9218 can reduce hyperuricemia in mice by improving the intestinal microbiota disorder and mucosal barrier function in fructose-induced hyperuricemic mice.

[0003] Lactobacillus mucosae rhamnosus is a lactic acid bacterium that has been reported to be almost naturally present in the intestines of all vertebrates and mammals. It has a strong adhesion ability to the intestinal mucosa, can improve the distribution of intestinal flora, antagonize the colonization of harmful bacteria, and regulate human immunity. The probiotic preparation of Lactobacillus mucosae rhamnosus can improve human functions and enhance immunity, thus promoting human health. In 2003, China approved Lactobacillus mucosae rhamnosus as a microbial strain for human health products, and this bacterium is already a recognized probiotic lactic acid bacterium in the world, with high theoretical research and production application value. Lactobacillus mucosae rhamnosus is mainly colonized in the intestines of humans, rodents, pigs, and chickens. Therefore, most of the strains are derived from the intestines or milk of humans, livestock, and poultry, and there are few isolations from fermented foods.

[0004] Exopolysaccharides (EPS) produced by lactic acid bacteria are the general term for the mucus or capsular polysaccharides secreted by lactic acid bacteria outside the cell wall during growth and metabolism. EPS has functional properties such as gelling, stabilizing, thickening, and water-holding, and is widely used in the food industry to improve the texture, sensory quality, nutritional properties, and stability of products. In addition, EPS is beneficial to health, can be used to prevent human diseases, and has functions such as anti-cancer, immunomodulation, antiviral, hypoglycemic, and cholesterol-lowering effects. There are few reports in the prior art on the effect of Lactobacillus reuteri isolated from fermented yogurt on reducing uric acid. Screening and exploring food-derived Lactobacillus reuteri that produces exopolysaccharides contribute to the further application of probiotic strains in the food industry. Summary of the Invention

[0005] The object of the present invention is to provide a strain of Lactobacillus reuteri SAU-R-8 isolated from naturally fermented Tibetan sour yak milk that produces exopolysaccharides, which can be used for reducing uric acid. Limosilactobacillus reuteri

[0006] To achieve the above object of the invention, one of the technical solutions provided by the present invention is a strain of Lactobacillus reuteri SAU-R-8 isolated from naturally fermented Tibetan sour yak milk that produces exopolysaccharides. Limosilactobacillus reuteri This strain was deposited at the China General Microbiological Culture Collection Center on April 19, 2024. Address: No. 1, Beichen West Road, Chaoyang District, Beijing, Postcode: 100101. The registration number of the deposit center is CGMCC No. 30373.

[0007] The strain of Lactobacillus reuteri SAU-R-8 isolated from naturally fermented Tibetan sour yak milk that produces exopolysaccharides Limosilactobacillus reuteri has an exopolysaccharide production of 318.57 mg / L in MRS medium.

[0008] Another technical solution provided by the present invention is the application of the above-mentioned Lactobacillus reuteri SAU-R-8 in reducing uric acid, especially in the degradation of nucleosides, absorption of purines, and inhibition of xanthine oxidase.

[0009] Lactobacillus reuteri SAU-R-8 provided by the present invention has excellent uric acid-lowering effect in a short time. After being treated with strain SAU-R-8 for 2 h, the degradation rates of inosine and guanosine with a concentration of 1.26 mmol / L are both 100%. The absorption rates of guanine, xanthine, and hypoxanthine with a concentration of 0.02 g / L are 11.17%, 6.29%, and 10.99% respectively. The inhibition rate of the strain metabolites on xanthine oxidase reaches 27.73%, providing substantial application value and great significance in the development of products for relieving or treating hyperuricemia. Brief Description of the Drawings

[0010] Figure 1 is the colony morphology and microscopic morphology of Lactobacillus mucosae SAU-R-8. Limosilactobacillus reuteri ).

[0011] Figure 2 is the phylogenetic tree of Lactobacillus mucosae SAU-R-8. Limosilactobacillus reuteri ). Specific implementation

[0012] The following examples will further illustrate the present invention in conjunction with the accompanying drawings.

[0013] Example 1: Isolation of strains Natural fermented yak yogurt was collected from the Shigatse area of Tibet, placed in an ice box and taken back to the laboratory for storage at -80°C. The sample was serially diluted with sterile normal saline, and 1 mL of an appropriate dilution was placed in a sterilized petri dish, and a modified MRS medium supplemented with vancomycin (50 μg / mL) was poured. An equal amount of sterile normal saline was used as a blank control, and anaerobic culture was carried out at 45°C for 48 h. Colonies with a calcium dissolution zone and typical colony characteristics of lactic acid bacteria on the MRS plate were picked, and multiple streak purifications were carried out. The purified strains were numbered and stored at -80°C in 30% ( v / v ).

[0014] Example 2: Identification of strain SAU-R-8 (1) Gram staining, catalase test Gram staining: Colonies on the plate were picked for smearing, fixation, primary staining with crystal violet, mordanting, decolorization, and counterstaining with safranin, drying, and microscopic examination; Catalase test: The test bacteria were first exposed to air for 30 min, and a small amount of 3% H2O2 was aspirated with a capillary tube and dropped on the colonies growing on the plate surface. If no bubbles were produced, it was catalase negative.

[0015] (2) Observation of colony morphology, microscopic morphology of bacteria, and physiological and biochemical identification The colony morphological characteristics of the strain were observed with the naked eye, and the observation results are shown in Figure 1 a. The strain was round, semi-transparent, with a smooth and moist surface, regular edges, and a slightly raised center. After Gram staining, the cell morphological characteristics of the strain were observed under an oil immersion lens of an optical microscope, and the observation results are shown in Figure 1 b. Its cell morphology was rod-shaped, Gram-positive, and catalase-negative.

[0016] (3) 16S rDNA sequence analysis and identification The genomic DNA of the strain was extracted and used as a template to amplify the 16S rDNA fragment with the universal primers 27F and 1492R. The 16S rDNA sequencing results were compared with the sequences in the gene bank, and combined with morphological, physiological and biochemical indexes. Finally, the strain SAU-R-8 was identified as Lactobacillus mucosae Roy Limosilactobacillus reuteri ); High similarity sequences were selected on NCBI, and the phylogenetic distances of the sequences were calculated using MEGA-X to construct a phylogenetic tree, see Figure 2 .

[0017] Example 3: Determination of exopolysaccharide production of strain SAU-R-8 The strain obtained in Example 1 was statically cultured in MRS broth medium for 12 h to make its concentration reach 10 9 CFU / mL. It was inoculated into MRS liquid medium at an inoculation amount of 2% and cultured at 37 °C for 24 h. The culture solution was collected, boiled in a water bath for 10 min, cooled and centrifuged (8000 r / min, 4 °C, 20 min) to remove the bacteria. The supernatant was concentrated under reduced pressure to 1 / 3 volume, and trichloroacetic acid was added to a final concentration of 4% (12%). It was kept at 4 °C for about 16 h, and centrifuged (8000 r / min, 4 °C, 20 min) to remove the protein precipitate. 3-4 times the volume of precooled absolute ethanol of the supernatant was added and stored overnight in a refrigerator at 4 °C. Centrifugation (8000 r / min, 4 °C, 20 min) was used to obtain the polysaccharide precipitate. The precipitate was washed with 80% ethanol, and the precipitate was dissolved in ultrapure water and transferred to a dialysis bag (8000-14000 Da) and dialyzed with ultrapure water in a refrigerator at 4 °C for 3 d, changing the water every 4 h. After dialysis, vacuum freeze-drying was carried out to obtain EPS.

[0018] Making a glucose standard curve: First, accurately weigh 50 mg of anhydrous glucose, add an appropriate amount of deionized water to make a 0.1 mg / mL glucose solution. Respectively pipette 0.2, 0.4, 0.6, 0.8, 1.0 mL of the standard solution into test tubes, and each was made up to 100 mL with distilled water. At the same time, 1.0 mL of distilled water was added to another test tube as a blank control. 1 mL of freshly prepared 6% phenol solution was added to the above test tubes, shaken and mixed evenly, then 5 mL of concentrated sulfuric acid was added, mixed evenly, left to stand at room temperature for 30 min, and then the absorbance value at 490 nm was measured. The glucose content was used as the abscissa and the absorbance value as the ordinate to draw the glucose standard curve.

[0019] The regression equation of the glucose standard curve is: y = 0.0108x + 0.0081 (R 2 = 0.9994).

[0020] Weigh 1 mg of EPS sample and dissolve it in 10 mL of distilled water. Pipette 1 mL of the sample solution and replace the glucose solution in the above protocol, measure its absorbance at 490 nm, and then calculate the total amount of EPS contained in the fermentation product of strain SAU-R-8 according to the standard curve.

[0021] Strain SAU-R-8 was fermented in MRS medium for 24 h, and its EPS production was 318.57 mg / L.

[0022] Example 4: Determination of uric acid-lowering ability of strain SAU-R-8 Identification of nucleoside degradation ability of strain SAU-R-8 Take the strain obtained in Example 1, activate it to obtain 1×10 9 CFU / mL, centrifuge at 12000 r / min for 2 min, discard the supernatant, collect the cells, wash the cells twice with sterile normal saline (0.85% NaCl solution), add 1.0 mL of 1.26 mmol / L inosine-guanosine buffer to the washed cells, mix well, and incubate at 37°C anaerobically at 160 rpm / min for 1 h. After incubation, centrifuge at 4°C and 12000 r / min for 2 min, take 810 μL of the supernatant, add 90 μL of the reaction terminator 0.1 mol / L perchloric acid solution, mix well, then centrifuge at 4°C and 12000 r / min for 2 min, and filter the supernatant through a 0.22 μm filter membrane for high performance liquid chromatography detection.

[0023] The liquid phase determination method is as follows: chromatographic column: reverse phase C18 column, mobile phase: 20 mmol / L KH2PO4 (pH 3.0): methanol = 90:10; detection wavelength 254 nm; column temperature 25°C; elution time 10 min; volume flow rate 1 mL / min.

[0024] Degradation rate (%) = (1 - peak area of inosine or guanosine in the supernatant / peak area of the original inosine or guanosine in the supernatant) × 100% The degradation rates of inosine and guanosine by strain SAU-R-8 were both 100%.

[0025] Identification of purine absorption ability of strain SAU-R-8 Take the strain obtained in Example 1, activate it to obtain 1×10 9CFU / mL, centrifuge at 12,000 r / min for 2 min, discard the supernatant, collect the bacterial cells, wash the bacterial cells twice with sterile normal saline (0.85% NaCl solution), add 1.0 mL of 0.02 g / L guanine - hypoxanthine - xanthine buffer to the washed bacterial cells, mix well, incubate anaerobically at 37°C at 160 rpm / min for 1 h. After incubation, centrifuge at 4°C and 12,000 r / min for 2 min, take 810 μL of the supernatant, add 90 μL of the reaction terminator 0.1 mol / L perchloric acid solution, mix well, then centrifuge at 4°C and 12,000 r / min for 2 min, filter the supernatant through a 0.22 μm filter membrane and use it for high - performance liquid chromatography detection. The detection method is the same as that for nucleoside detection.

[0026] Degradation rate (%) = (1 - peak area of purine in the supernatant / peak area of original purine in the supernatant) × 100% The absorption rates of strain SAU - R - 8 for guanine, xanthine, and hypoxanthine are 11.17%, 6.29%, and 10.99% respectively.

[0027] Identification of the inhibitory ability of strain SAU - R - 8 on xanthine oxidase Take the strain obtained in Example 1, centrifuge at 12,000 r / min for 2 min to collect the bacterial cells, wash the bacterial cells 1 - 2 times with sterile PBS (pH 7.4), then resuspend in PBS, adjust the bacterial liquid concentration to 1×10 8 CFU / mL, incubate at 37°C for 12 h, centrifuge at 12,000 r / min for 2 min, collect the supernatant, filter through a 0.22 μm microfiltration membrane to obtain the cell metabolites. According to Table 1, add PBS (pH 7.5), sample solution, 0.2 U / mL XOD in sequence, incubate at 37°C for 10 min, then add 100 μL of 1.2 mmol / L substrate xanthine, incubate at 37°C for 10 min, and record the absorbance value at 295 nm. Use 0.04 mg / mL allopurinol as the positive control.

[0028] Table 1 Enzyme reaction system Group PBS Sample Xanthine oxidase Xanthine Positive 100 0 50 50 Blank 150 0 0 50 Allopurinol 50 50 50 50 Strain 0 100 50 50 Inhibition rate of XOD (%) = (1 - (A1 - A0) / (A2 - A0)) × 100%, where A0 is the blank, A1 is the sample / control experimental group, and A2 is the positive experimental group The inhibition rate of strain SAU - R - 8 on xanthine oxidase reaches 27.73%.

[0029] The above examples are only descriptions of the embodiments of the present invention, rather than limitations on the scope of the present invention. For those skilled in the art, the above description can be improved or deformed, but all such improvements or deformations shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Lactobacillus mucosae rogosae producing extracellular polysaccharide from naturally fermented Tibetan sour yak milk ( Limosilactobacillus reuteri ), characterized in that It is preserved in the China General Microbiological Culture Collection Center, the preservation location is No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 30373, and the preservation date is April 19, 2024.

2. The Lactobacillus reuteri mucilage according to claim 1, characterized in that, The extracellular polysaccharide production of Lactobacillus reuteri Limosilactobacillus reuteri SAU-R-8 reaches 318.57 mg / L.

3. The application of Limosilactobacillus reuteri SAU-R-8 as described in claim 1 in reducing uric acid.

4. The application according to claim 3, wherein It is the application in degrading nucleosides, absorbing purines, and inhibiting xanthine oxidase.

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