Screening and application of salt-resistant lactobacillus plantarum with multiple uric acid reducing functions

Abstract

The invention discloses screening and application of salt-resistant lactobacillus plantarum with multiple uric acid reducing functions. Samples are screened and collected in soil, sauced beans, fermented flour paste, fresh milk, pickled vegetables and the like, a strain obtained through screening is identified as Lactobacillus plantarum WQ47 and is named as Lactobacillus plantarum WQ47, the strain has high inosine and guanosine degradation capacity, the degradation rate is 99.9% or above, the degradation rate is 99.0% or above, and therefore the serum uric acid level can be effectively reduced. Besides, the strain WQ47 is cultured in an MRS lactic acid bacteria culture medium to a fermentation supernatant, a bacterial suspension and a cell-free extract in the middle and later periods of logarithmic growth, the xanthine oxidase inhibition rates are 44.9%, 33.4% and 62.8% respectively, uric acid generation can be effectively inhibited, purine generation can be reduced by degrading inosine and guanosine and inhibiting the activity of xanthine oxidase, and the strain WQ47 can be used for preparing a new strain for preventing and treating uric acid generation. The traditional Chinese medicines jointly achieve the effects of relieving and treating the hyperuricemia. Moreover, the strain WQ47 screened by the invention can well survive in an environment with high content of salt (NaCl) of 20% (mass fraction), can be widely applied to fermented food, pickled food, functional food and the like, and has a better application effect on preventing, relieving and treating hyperuricemia, gout and complications of the hyperuricemia and the gout.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering, and particularly relates to the screening and application of a salt-tolerant Lactobacillus plantarum strain having multiple uric acid-lowering functions. Background Art

[0002] Hyperuricemia is a metabolic disease caused by abnormally elevated blood uric acid levels due to purine metabolism disorders. Disorders in human purine metabolism or excessive intake of purine nucleotide foods can cause abnormal increases in serum uric acid concentrations. Currently, the treatment and prevention measures for HUA mainly use two approaches: exogenous drug effects and limiting the intake of purine substances in the diet. Most clinical uric acid-lowering drugs rely on xanthine oxidase (XO) inhibitors (allopurinol) and uricosuric drugs (benzbromarone). Although uric acid-lowering drugs have obvious efficacy, long-term use may cause adverse reactions in the human body. Therefore, it is urgent to find new technologies to reduce uric acid in the blood of HUA people. Therefore, it is of great significance to explore a new, low-toxic and food-borne HUA treatment and prevention approach. At present, the treatment and prevention measures for HUA are limited, and probiotic intervention therapy has gradually been recognized by people because of its regulatory effect on host metabolic disorders and intestinal flora imbalance, thus becoming one of the important methods for treating metabolic diseases.

[0003] The current methods for separating and screening probiotics often use dilution coating method, plate streaking method, molecular biology method and high-throughput screening technology. Lactic acid bacteria are usually separated and identified by colony morphology, physiological and biochemical tests, etc., and the strain category is determined based on 16S rDNA gene sequence and molecular identification to obtain the target strain. The ability to degrade inosine and guanosine in vitro was determined by high performance liquid chromatography. Studies have shown that some strains can reduce the absorption of nucleosides by the intestinal epithelium by degrading or absorbing nucleosides, thereby reducing the production of uric acid. Since the precursor substances of uric acid in the process of production are xanthine, hypoxanthine, inosine / guanosine, nucleotides, etc., their contents are related to the final uric acid content in the blood. Nucleoside uptake is the main way for purine and pyrimidine to enter intestinal epithelial cells, so premature degradation of nucleosides is an effective way for strains to regulate blood uric acid.

[0004] Xanthine oxidase is a key enzyme in the purine catabolism process, which can catalyze hypoxanthine and xanthine to directly generate uric acid. Therefore, when the xanthine oxidase activity in the body is abnormally active, it will lead to the generation of a large amount of uric acid, thereby causing hyperuricemia. Therefore, xanthine oxidase inhibitors are also an important target for the current research on the effect of lowering uric acid. Patent CN116218746A (a plant lactobacillus with uric acid lowering effect) reported that plant lactobacillus Lactobacillus plantarum NML21 was isolated from yak yogurt samples in Nimalong Village, Amuquhu Town, Xiahe County, Gannan Tibetan Autonomous Prefecture, Gansu Province, with degradation rates of inosine and guanosine of 96.57% and 98.33% respectively, and the inhibition rate of the cell-free extract of the strain on xanthine oxidase was 32.80% respectively.

[0005] Most traditional fermentations are open mixed bacteria natural fermentation systems. Harmful microorganisms in the environment will enter the fermentation system. High concentrations of salt can inhibit the growth of spoilage bacteria in some foods, enhance food safety, and maintain or even enhance the competitiveness and functional advantages of functional strains. In some fermented foods, pickled foods, functional foods, etc., there will inevitably be a high concentration of salt environment. Therefore, it is of great application value to breed functional probiotic strains that tolerate high concentrations of salt environments. It is worth noting that in our study, we found that the probiotic strains with the function of lowering uric acid were activated in MRS liquid culture medium for several generations, and the environment of tolerance to high concentrations of salt (NaCl) was used as the screening and directed evolution conditions to screen out an excellent strain WQ47 that can tolerate a high salt culture environment containing 20% ​​(mass fraction). At present, the strains that have been screened are mostly manifested in one aspect, such as resistance or probiotic characteristics. Therefore, the strains screened in this application have both specific functions and excellent tolerance, that is, excellent and unique probiotic strains that have both uric acid lowering functions and tolerance to high salt containing 20% ​​(mass fraction), which have important value and significance. Patent CN111269864 A (a salt-tolerant plant lactobacillus C4 and its application) uses MRS agar medium to screen salt-tolerant plants from the rice grass naturally grown on the Langtoushan beach in Changle, Fuzhou City, Fujian Province. Lactobacillus C4 can grow in a 10% NaCl salt concentration. Chen Yi, Lin Kaicheng, Lin Qin, etc., in "Isolation and Identification of Salt-Tolerant Lactobacillus in Rice Grass and Its Effect on the Quality of Rice Grass Silage" ( Grassland News , 2020,28(2)) reported the screening of lactic acid bacteria with a salt tolerance of 10%, and the isolation and identification of the lactic acid bacteria and the study of their influence on the quality of rice grass silage. A probiotic strain screened by the present invention can grow in a 20% salt environment. The samples of the strain screened by the present invention were collected from soil, soybeans, noodle sauce, fresh milk, pickles, etc., and named Lactobacillus plantarumWQ47. The strain has a strong ability to degrade inosine and guanosine, and xanthine oxidase inhibition, thereby being able to inhibit uric acid production, and jointly play a role in alleviating and treating hyperuricemia, and has a good ability to tolerate salt, survives well in a high salt environment of 20%, can be widely used in fermented foods, pickled foods, functional foods and other products, and has important value and significance for preventing, alleviating and treating hyperuricemia and gout and their complications. Summary of the invention

[0006] The first object of the present invention is to provide a screening method for screening Lactobacillus plantarum.

[0007] The second object of the present invention is to provide the use of the plant lactobacillus in the preparation of fermented food, pickled food, functional food and the like.

[0008] The purpose of the present invention is achieved through the following technical solutions: A plant lactobacillus strain named Lactobacillus plantarum and a method for culturing the plant lactobacillus include the following specific steps: screening, separating and identifying the plant lactobacillus strain from soil, soybean paste, flour paste, fresh milk and pickled vegetables, inoculating the strain into a culture medium and culturing the strain under certain temperature conditions.

[0009] Furthermore, samples for screening bacterial strains come from soil in cattle and sheep farms; homemade soybean paste and noodle sauce; pickle and kimchi workshops; pickles and kimchi homemade at home or purchased in supermarkets, etc.

[0010] Furthermore, the culture medium is MRS lactic acid bacteria broth culture medium.

[0011] Furthermore, the culture temperature is 28-37°C.

[0012] Furthermore, the culture time is 24-48 hours.

[0013] Furthermore, the initial pH of the culture medium is 6-7.

[0014] Furthermore, the culture rotation speed is 50-200r / min.

[0015] Furthermore, the screening method of plant lactobacillus is as follows: 100uL of the treated sample is spread on a solid plate for culture, a typical single colony on the plate is picked, and pure colonies are isolated after streaking and purification on the solid plate for 2-3 generations. DNA is extracted using a bacterial DNA extraction kit, and the supernatant is used as a DNA template; PCR amplification is performed using universal primers 27F / 1492R, and the amplified product is sequenced for 16S rDNA.

[0016] Furthermore, the PCR amplification reaction system was: 25uL 2×Rapid Taq Master Mix, 2uL forward primer 27F, 2uL reverse primer 1492R, 1uL DNA, and 20uL double distilled water.

[0017] Furthermore, the PCR reaction program was: 1 pre-denaturation at 95.0°C: 3 min; 2 denaturation at 95.0°C: 15 s; 3 annealing at 54.2°C: 15 s; 4 extension at 72°C: 45 s, 30 cycles; 5 final extension at 72°C: 5 min, (2-4 cycles, 30-35 times).

[0018] Further, the method for determining the degradation rate of inosine of plant lactobacillus is as follows: the plant lactobacillus is inoculated into the MRS medium with a pH of 6-7 according to the inoculum amount of 0.5-2% (v / v), cultivated at a temperature of 28-37°C and a speed of 50-200r / min for 24-48 hours, and continuously subcultured 2-4 times; a neutral potassium phosphate solution of inosine is prepared and placed in a 4°C refrigerator for standby use. 1-2mL of bacterial solution is centrifuged at 3500-5000r / min for 10-15min at 4°C, the supernatant is poured out, the bacterial cells are collected, and after PBS cleaning, the bacterial cells are collected again, the inosine reaction solution is added, and the inosine reaction solution is cultivated at a temperature of 28-37°C for 30-60min, the resulting bacterial solution is subjected to termination reaction in a 100°C water bath for 5-10min, and centrifuged at 12000r / min for 2-3min; subsequently, a 0.22μm filter membrane is used for filtration, and the filtrate is used for high performance liquid chromatography detection.

[0019] Furthermore, the preparation method of the inosine neutral potassium phosphate solution is: weigh 1.5-2.5g / L of K3PO4, dissolve it in deionized water, and make up the volume with a 1L volumetric flask; then adjust the pH to 6-7 with phosphoric acid to obtain a neutral potassium phosphate buffer solution, dissolve an appropriate amount of inosine with the potassium phosphate buffer, and make up the volume with a 100mL volumetric flask. The prepared solution is the inosine neutral potassium phosphate solution.

[0020] Furthermore, the inosine content was determined by high performance liquid chromatography, the retention time of inosine was determined by external standard method, and the standard curve was prepared with the concentration as the abscissa and the peak area as the ordinate; the chromatographic conditions were: liquid chromatograph LC-20A; the reverse chromatographic column was Agilent-C18 (4.6mm x 250mm); mobile phase was V (pure water): V (chromatographic grade methanol) = 90:10; flow rate was 1mL / min, injection volume was 20μL, measurement wavelength was 254nm, column temperature was 30℃.

[0021] Further, the method for determining the degradation rate of guanosine of plant lactobacillus is as follows: the plant lactobacillus is inoculated into the MRS medium with a pH of 6-7 according to the inoculum amount of 0.5-2% (v / v), and cultured at 28-37°C at 50-200r / min for 24-48 hours, and continuously subcultured 2-4 times; a guanosine neutral potassium phosphate solution is prepared and placed in a 4°C refrigerator for standby use. 1-2mL of bacterial solution is centrifuged at 3500-5000r / min for 10-15min at 4°C, the supernatant is poured out, the bacterial cells are collected, and after PBS cleaning, the bacterial cells are collected again, inosine reaction solution is added, and cultured at 28-37°C for 30-60min, the resulting bacterial solution is subjected to termination reaction in a 100°C water bath for 5-10min, and centrifuged at 12000r / min for 2-3min; subsequently, a 0.22μm filter membrane is used for filtration, and the filtrate is used for high performance liquid chromatography detection.

[0022] Furthermore, the preparation method of the guanosine neutral potassium phosphate solution is as follows: weigh 1.5-2.5 g / L of K3PO4, dissolve it in deionized water, and use a 1L volumetric flask to make up the volume; then use phosphoric acid to adjust the pH to 6-7 to obtain a neutral potassium phosphate buffer solution. Use potassium phosphate buffer to dissolve an appropriate amount of guanosine, use a 100 mL volumetric flask to make up the volume, and the prepared solution is the guanosine neutral potassium phosphate solution.

[0023] Furthermore, the guanosine content was determined by high performance liquid chromatography, and the retention time of guanosine was determined by external standard method. The concentration was used as the abscissa and the peak area was used as the ordinate to prepare the standard curve. The chromatographic conditions were: liquid chromatograph LC-20A; the reverse chromatographic column was Agilent-C 18 (4.6mm x 250mm); mobile phase was V (pure water): V (chromatographic grade methanol) = 90:10; flow rate was 1mL / min, injection volume was 20μL, measurement wavelength was 254nm, column temperature was 30℃.

[0024] Furthermore, the method for determining the salt tolerance of Lactobacillus plantarum is as follows: Lactobacillus plantarum WQ47 cultured to the logarithmic phase is inoculated into MRS liquid culture medium containing different NaCl at an inoculation amount of 1-3%, and then transferred to an incubator for culture. At a certain time, appropriate dilution gradient bacterial liquid is taken for plate coating, and culture is performed. After a certain period of time, the survival rate was calculated and the salt tolerance was evaluated based on the survival rate.

[0025] Furthermore, the salt content of the culture medium is 8-20%.

[0026] Further, the inhibitory effect of the lactic acid bacteria on the activity of xanthine oxidase: prepare MRS fermentation supernatant, bacterial suspension, cell-free extract, evaluate the reaction system according to the inhibitory ability of xanthine oxidase, take 1.5mL xanthine substrate solution (0.20g / L), 1.5mL extract solution, and then add 2mL xanthine oxidase solution (0.20U / mL) pre-warmed at 37°C for 20min. Start the reaction and record the change of absorbance value during the reaction time under ultraviolet conditions of wavelength 295nm. Replace the xanthine oxidase solution with sterilized PBS buffer in the control sample. Since xanthine has an absorbance value at a wavelength of 295nm, the absorbance value without sample and enzyme must be deducted. Three parallel experiments are performed for each sample. Allopurinol is used as a positive control.

[0027] Furthermore, the preparation of MRS fermentation supernatant: 10 mL of lactic acid bacteria culture fermented in MRS lactic acid bacteria medium to logarithmic phase was centrifuged at 6000 r / min for 10 min, and the supernatant was collected as MRS fermentation supernatant.

[0028] Further, bacterial suspension preparation: 10 mL of lactic acid bacteria culture fermented in MRS lactic acid bacteria medium to the logarithmic phase was centrifuged at 6000 r / min for 10 min, and the resulting bacterial precipitate was collected. The bacterial precipitate was washed three times with sterile PBS buffer and then resuspended in PBS to adjust the OD 600 Make it 1.0 and incubate at 37°C for 12 hours to obtain a bacterial suspension.

[0029] Further, cell-free extract preparation: the concentration of 1×10 9 The cell suspension of CFU / mL was treated with ultrasonic power of 300W for 5s, and then ultrasonically disrupted in an ice bath for 10min at an interval of 5s. The ultrasonic bacterial suspension was then centrifuged at 8000r / min for 5min, and the supernatant was filtered through a 0.22μm microporous water filter to obtain a cell-free extract.

[0030] A product containing plant lactobacillus is prepared by fermentation with the plant lactobacillus.

[0031] Furthermore, the product includes applications in fermented foods, pickled foods, functional foods, etc. Beneficial Effects of the Invention

[0032] The plant lactobacillus (Lactobacillus plantarum) WQ47 screened by the present invention is a beneficial active microorganism screened and separated from soil, soybeans, noodle sauce, fresh milk, and pickled vegetables. The strain has a strong ability to degrade inosine and guanosine, and the degradation rates are respectively 99.9% and 99.0% or more, thereby being able to effectively reduce serum uric acid levels. In addition, the strain WQ47 is cultured in an MRS lactic acid bacteria culture medium to the fermentation supernatant, bacterial suspension, and cell-free extract of the middle and late logarithmic growth, and the inhibition rates of xanthine oxidase are 44.9%, 33.4%, and 62.8%, respectively, which can effectively inhibit uric acid production. The strain WQ47 can reduce purine production by degrading inosine and guanosine and inhibiting the activity of xanthine oxidase, and together play a role in alleviating and treating hyperuricemia.

[0033] The plant lactobacillus WQ47 screened by the present invention not only has the function of lowering uric acid, but can also survive well in an environment with a high salt (NaCl) content of 20% (mass fraction), can be widely used in fermented foods, pickled foods, functional foods and other products, and has important value and significance for preventing, alleviating and treating hyperuricemia, gout and their complications. DETAILED DESCRIPTION

[0034] The following examples are used to further illustrate the present invention, but are not intended to limit the scope of the present invention.

[0035] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The experimental methods in the following examples where specific experimental conditions are not specified are usually based on conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0036] Example 1 Screening and Identification of Lactobacillus plantarum Take samples such as soil, soybeans, noodle sauce, fresh milk, pickles, etc., dilute and spread them, and culture them in MRS medium at 28-37°C, 50-200r / min, and an initial pH of 6-7 for 20-28 hours under shaking conditions, and continuously subculture for 2-4 times for use. Pick the milky white, round, neatly edged, opaque, and smooth colonies on the MRS lactic acid bacteria medium solid plate, streak and purify them on the solid plate for 2-4 generations to obtain pure colonies.

[0037] DNA was extracted using a bacterial DNA extraction kit and used as a template. PCR amplification was performed using universal primers 27F / 1492R. The PCR amplification reaction system was: 25uL 2×Rapid Taq Master Mix, 2uL forward primer 27F, 2uL reverse primer 1492R, 1uL DNA, and 20uL double distilled water.

[0038] The PCR reaction program is: 1. Pre-denaturation at 95.0°C: 3 min; 2. Denaturation at 95.0°C: 15 s; 3. Annealing at 54.2°C: 15 s; 4. Extension at 72°C: 45 s, 30 cycles; 5. Final extension at 72°C: 5 min, (2-4 cycles 30-35 times) The PCR product after the above-mentioned verification is sent to Sangon Biotech Co., Ltd. for sequencing, and the 16SrDNA sequence obtained is BLASTed on NCBI, and the sequencing information of known strains is compared for homology to identify the strain. The strain screened by the present invention is identified as plant lactobacillus (Lactobacillus plantarum). The present invention names this strain as plant lactobacillus Lactobacillus plantarumWQ47.

[0039] Example 2 Lactobacillus plantarum WQ47 reduces serum uric acid levels by degrading inosine Lactic acid bacteria can competitively reduce the absorption of nucleosides by the intestinal epithelium by degrading or absorbing uric acid precursors such as nucleosides, purines, and purine-related metabolites, thereby reducing the production of uric acid to assist in the treatment of hyperuricemia. Some lactic acid bacteria can degrade inosine in the intestine into purine bases that are not easily absorbed and directly excrete them from the body, thereby avoiding the absorption of inosine by the intestine and reducing the chance of the liver converting it into uric acid.

[0040] Plant lactobacillus inosine degradation rate assay method: above-mentioned plant lactobacillus is inoculated into the MRS culture medium of pH 6-7 according to the inoculum size of 0.5-2% (v / v), cultivates 24-48 hours under 50-200r / min rotating speed at 28-37 ℃ of temperature, and is continuously passed down 2-4 times; prepares inosine neutral potassium phosphate solution, is placed in 4 ℃ of refrigerators for standby use. Get 1-2mL bacterial liquid and pour out supernatant at 4 ℃ of following 3500-5000r / min centrifugal 10-15min, collect bacterial body, after PBS cleaning, collect bacterial body again, add inosine reaction solution, cultivate 30-60min under 28-37 ℃ of temperature, the gained bacterial liquid is carried out termination reaction in 100 ℃ of water baths 5-10min, centrifugal 2-3min at 12000r / min; subsequently, adopt 0.22μm filter membrane to filter, and the filtrate is used for high performance liquid chromatography detection.

[0041] The preparation method of inosine neutral potassium phosphate solution is as follows: weigh 1.5-2.5g / L K3PO4, dissolve it in deionized water, and use a 1L volumetric flask to make up the volume; then use phosphoric acid to adjust the pH of the buffer to 6-7 to obtain a neutral potassium phosphate buffer solution. Use potassium phosphate buffer to dissolve an appropriate amount of inosine, use a 100mL volumetric flask to make up the volume, and the prepared solution is the inosine neutral potassium phosphate solution.

[0042] The inosine content was determined by high performance liquid chromatography, and the retention time of inosine was determined by external standard method. The concentration was used as the horizontal axis and the peak area was used as the vertical axis to make a standard curve. The chromatographic conditions were: liquid chromatograph LC-20A; the reverse chromatographic column was Agilent-C 18 (4.6mm x 250mm); mobile phase was V (pure water): V (chromatographic grade methanol) = 90:10; flow rate was 1mL / min, injection volume was 20μL, measurement wavelength was 254nm, column temperature was 30℃.

[0043] Example 3 Lactobacillus plantarum WQ47 reduces serum uric acid levels by degrading guanosine Studies have found that lactic acid bacteria can competitively reduce the absorption of nucleosides by the intestinal epithelium by degrading or absorbing uric acid precursors such as nucleosides, purines, and purine-related metabolites, thereby reducing the production of uric acid and assisting in the treatment of hyperuricemia. Some lactic acid bacteria can degrade guanosine in the intestine into purine bases that are not easily absorbed and directly excrete them from the body, thereby avoiding the absorption of guanosine by the intestine and reducing the chance of the liver converting it into uric acid.

[0044] The method for measuring the degradation rate of guanosine of plant lactobacillus is as follows: the plant lactobacillus is inoculated into the MRS medium with a pH of 6-7 according to the inoculum amount of 0.5-2% (v / v), and the culture is carried out at a temperature of 28-37°C and a rotation speed of 50-200r / min for 24-48 hours, and the culture is continuously transferred 2-4 times; a guanosine neutral potassium phosphate solution is prepared and placed in a 4°C refrigerator for standby use. 1-2mL of bacterial solution is taken and centrifuged at 3500-5000r / min for 10-15min at 4°C, the supernatant is poured out, the bacterial body is collected, and after PBS cleaning, the bacterial body is collected again, the inosine reaction solution is added, and the culture is carried out at a temperature of 28-37°C for 30-60min, the obtained bacterial solution is subjected to termination reaction in a 100°C water bath for 5-10min, and the reaction is carried out at a centrifugal speed of 12000r / min for 2-3min; subsequently, a 0.22μm filter membrane is used for filtering, and the filtrate is used for high performance liquid chromatography detection.

[0045] Furthermore, the preparation method of the guanosine neutral potassium phosphate solution is as follows: weigh 1.5-2.5 g / L of K3PO4, dissolve it in deionized water, and use a 1L volumetric flask to make up the volume; then use phosphoric acid to adjust the pH of the buffer solution to 6-7 to obtain a neutral potassium phosphate buffer solution. Use potassium phosphate buffer to dissolve an appropriate amount of guanosine, use a 100 mL volumetric flask to make up the volume, and the prepared solution is the guanosine neutral potassium phosphate solution.

[0046] Furthermore, the guanosine content was determined by high performance liquid chromatography, and the retention time of guanosine was determined by external standard method. The concentration was used as the abscissa and the peak area was used as the ordinate to prepare the standard curve. The chromatographic conditions were: liquid chromatograph LC-20A; the reverse chromatographic column was Agilent-C 18 (4.6mm x 250mm); mobile phase was V (pure water): V (chromatographic grade methanol) = 90:10; flow rate was 1mL / min, injection volume was 20μL, measurement wavelength was 254nm, column temperature was 30℃.

[0047] Example 4 Inhibitory effect of lactic acid bacteria on xanthine oxidase activity (1) Preparation of MRS fermentation supernatant: 10 mL of lactic acid bacteria culture cultured in MRS lactic acid bacteria medium to the logarithmic phase was centrifuged at 6000 r / min for 10 min, and the supernatant was collected as the MRS fermentation supernatant.

[0048] (2) Preparation of bacterial suspension: Collect the bacterial precipitate obtained in (1), wash it three times with sterile PBS buffer, and then resuspend it in PBS to adjust the OD 600 Make it 1.0 and incubate at 37°C for 12 hours to obtain a bacterial suspension.

[0049] (3) Preparation of cell-free extract: The concentration of 1×10 9 The cell suspension of CFU / mL was treated with ultrasonic power of 300W for 5s, and then ultrasonically disrupted in an ice bath for 10min at an interval of 5s. The ultrasonic bacterial suspension was then centrifuged at 8000r / min for 5min, and the supernatant was filtered through a 0.22μm microporous water filter to obtain a cell-free extract.

[0050] According to the inhibition ability of xanthine oxidase, the reaction system was evaluated to determine the inhibition rate of strain fermentation supernatant, bacterial suspension, and cell-free extract on xanthine oxidase. Take 1.5mL of xanthine substrate solution (0.20g / L), 1.5mL of extract solution and mix well, then add 2mL of xanthine oxidase solution (0.20U / mL) that was pre-warmed at 37°C for 20min. Start the reaction and record the change of absorbance value during the reaction time under ultraviolet conditions at a wavelength of 295nm. In the control sample, the xanthine oxidase solution was replaced with sterilized PBS buffer. The activity of xanthine oxidase is expressed by the inhibition rate of xanthine oxidase. Since xanthine has an absorbance value at a wavelength of 295nm, the absorbance value without sample and enzyme must be deducted. Three parallel experiments were performed for each sample. The reaction solution with a wavelength of 295nm was measured with an enzyme marker. With allopurinol as the positive control, the calculation formula for the xanthine oxidase inhibition rate is as follows: Inhibition rate (%) = [1-(CD) / (AB)] Wherein: A is the absorbance with xanthine oxidase but without sample; B is the absorbance with sample but without xanthine oxidase; C is the absorbance with xanthine oxidase and sample; D is the absorbance with sample but without xanthine oxidase.

[0051] Example 5 Application of Lactobacillus plantarum WQ47 in fermented food Prepare wheat flour, wherein the particle size D50 of the tempered wheat flour is 56 μm, the mass proportion of damaged starch in the tempered wheat flour is 22%, and the peak viscosity of the tempered wheat flour after gelatinization is 1860 cp; add the soybeans that have been removed and selected into clean water of 2-3 times their mass, soak for 6-8 hours, take out and steam into a paste; pour out the soybeans and spread them out for cooling for later use. Then mix the soybeans and flour (3:1) evenly, cultivate them at 28-38°C, 70-95% relative humidity and 20-50Hz wind frequency for 35 hours to make koji, and make koji material, wherein the mass ratio of koji to cooked beans is 0.03:10. The fermented sauce embryo is prepared by adding watermelon pulp (1:4) and salt (1:3) to the koji material, wherein the brine temperature is 30-40 DEG C, and the mixture is sent to a fermentation tank for fermentation for 70 days, wherein the salt content in the brine is controlled at 20-25 g / 100 mL; the mixture is stirred evenly, and the tank is turned over on the second day, and the mixture is stirred once every other day thereafter; after the salting is completed, 0.5-3% (v / v) of plant lactobacillus by weight of soybean is inoculated, the mixture is covered with a lid, the tank mouth is sealed, and the mixture is fermented for 60 days to obtain the finished product.

[0052] Example 6 Application of Lactobacillus plantarumWQ47 in Pickling Sauerkraut Soak dehydrated cabbage and radish in 95°C hot water for 2-5 minutes, put them in warm water for thorough washing, drain them after washing, cut them into blocks or strips, add 4-7% edible salt and 1-5% sucrose, stir for 3-5 minutes, let stand for 10-15 minutes, add 2-5% chopped pepper and 5-10% garlic after standing, stir again for 2-5 minutes, let stand for 10-15 minutes, after standing, inoculate 0.5-3% Lactobacillus plantarum (v / v), and stir thoroughly for 3-5 minutes; after stirring, the vegetables are put into a fermentation tank and pressed tightly, and then 1-2% edible white vinegar is added to the fermentation tank in proportion, and then 5-10% clean water is added to the fermentation tank, and then the tank is sealed and placed at a temperature of 18-22°C, and fermented in a clean and dry room for 3-5 days to obtain the finished kimchi.

[0053] Example 7 Application of Lactobacillus plantarum WQ47 in functional foods Lactobacillus plantarum was inoculated into an MRS medium with a pH of 6-7 at an inoculum size of 0.5-2% (v / v), and cultured at a temperature of 28-37° C. and a rotation speed of 50-200 r / min for 24-48 hours to separate and extract the bacteria, and then the probiotic bacteria were concentrated 25 times, quickly frozen into pellets, freeze-dried and pulverized to obtain Lactobacillus plantarum powder; inulin was crushed to 200 mesh for standby use. 4-6 parts of plant lactobacillus powder, 4-6 parts of inulin, 15-18 parts of oligofructose powder, 4-5 parts of polydextrose and 25-30 parts of resistant dextrin are prepared in proportion, mixed evenly and passed through a 80-85 mesh sieve; the sieved raw materials are poured into a mixer at a speed of 800-900 r / h and a mixing time of 25-35 min to mix thoroughly; the mixed raw materials are poured into a filling device for aseptic filling to obtain a uric acid-lowering probiotic solid beverage.

[0054] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Screening of a salt-tolerant Lactobacillus plantarum strain with multiple uric acid-lowering functions, characterized in that: A strain with uric acid-lowering function was isolated and identified as Lactobacillus plantarum ( Lactobacillus plantarum )WQ47; this strain has a strong ability to degrade inosine and guanosine; in addition, the strain can also reduce purine production by inhibiting the activity of xanthine oxidase. The fermentation supernatant, bacterial suspension and cell-free extract of strain WQ47 cultured in MRS lactic acid bacteria medium to the logarithmic phase have a high inhibition rate on xanthine oxidase; and the screened strain WQ47 can survive well in an environment with a higher concentration of NaCl, which is conducive to its wide application in products such as the prevention, relief and treatment of hyperuricemia, gout and their complications.

2. As claimed in claim 1, characterized in that Samples for screening strains were collected from the soil of cattle farms and sheep farms; homemade soybean paste and noodle sauce; pickled vegetables homemade or purchased from supermarkets, etc.

3. As claimed in claim 1, characterized in that Lactobacillus plantarum The WQ47 strain can effectively degrade inosine and guanosine, with degradation rates of 99.9% and over 99.0%, respectively, thereby effectively reducing serum uric acid levels and having the effect of alleviating and treating hyperuricemia.

4. As claimed in claim 1, characterized in that When strain WQ47 was cultured in MRS lactic acid bacteria medium to the middle and late logarithmic growth stage, the fermentation supernatant, bacterial suspension and cell-free extract were able to reduce purine production by effectively inhibiting the activity of xanthine oxidase. The inhibition rates of xanthine oxidase were 44.9%, 33.4% and 62.8%, respectively, thereby inhibiting uric acid production; together with its ability to degrade inosine and guanosine, it can alleviate and treat hyperuricemia.

5. As claimed in claim 1, characterized in that The screened strain WQ47 can survive in an environment with a high salt (NaCl) content of 20% (mass fraction).

6. As claimed in claim 1, characterized in that Lactobacillus plantarum WQ47 can be widely used in fermented foods, pickled foods, functional foods, etc., and has a good application effect on the prevention, relief and treatment of hyperuricemia, gout and their complications.