Bacterial strain with purine reducing function and application thereof

Abstract

The invention belongs to the technical field of functional microorganism screening and application, and particularly relates to a strain with a purine reducing function and application thereof. The lactococcus lactis AMCC 11671 (Lactococcus lactis AMCC 11671) provided by the invention is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number of the lactococcus lactis AMCC 11671 is CCTCC NO: M 2024078. The lactococcus lactis AMCC 11671 provided by the invention has good crude enzyme activity, can effectively slow down the generation and accumulation of uric acid by reducing the purine content and degrading uric acid, has good tolerance to acid and cholate, has the ability to tolerate gastrointestinal tract environment, and has potential of colonization in human gastrointestinal tracts and play roles.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microorganism screening and application, and specifically relates to a strain with purine reduction function and application thereof. Background Art

[0002] Hyperuricemia and gout are caused by purine metabolism disorders and uric acid excretion disorders in the human body, and have become the second largest metabolic disease after diabetes. Purines in the human body come from both endogenous nucleic acid metabolism and exogenous dietary intake, accounting for 70% and 30% respectively. Purines mainly exist in the form of purine nucleosides (including inosine, adenosine, guanosine and xanthine), purine nucleotides (including inosine monophosphate (IMP), xanthine monophosphate (XMP), adenosine monophosphate (AMP) and guanosine monophosphate (GMP)) and purine bases (hypoxanthine, xanthine, adenine and guanine), and their metabolic pathways in the human body include de novo synthesis and salvage synthesis pathways. De novo synthesis is the synthesis of IMP from phosphoribosyl pyrophosphate synthetase (PRPPS) using phosphoribosyl pyrophosphate synthetase (PRPPS) as raw materials, followed by conversion to AMP and GMP. After further metabolism, IMP, GMP and AMP form hypoxanthine, guanine and adenine respectively, and guanine is converted to xanthine by deamination. Under the action of xanthine oxidase (XO), hypoxanthine and xanthine are metabolized to produce uric acid. Salvage synthesis is a secondary synthesis pathway of purine metabolism, which uses free purine or purine nucleosides in the body to produce nucleotides through some simple reactions with low energy consumption. However, with the continuous evolution of humans, urate oxidase is gradually lost, resulting in uric acid being unable to be further oxidized to form allantoin. Therefore, uric acid becomes the final product of purine metabolism in the human body. On the other hand, due to the improvement of quality of life and economic level, people tend to eat a high-purine diet. Long-term intake of high-purine foods causes a large amount of uric acid to accumulate in the body to form hyperuricemia, and severe cases even develop into gout. Gout is a crystal-related arthritis caused by the deposition of monosodium urate in the joints. Gout not only causes arthritis, joint deformities and uric acid urinary stones in patients, but also causes kidney disease and other metabolic syndromes (including hypertension, hyperlipidemia, diabetes and coronary heart disease, etc.). Currently, the three main strategies to relieve and treat hyperuricemia and gout are to reduce purine intake, prevent uric acid synthesis and increase uric acid excretion. Specifically, it can be divided into drug treatment and food prevention. Clinical treatment drugs include allopurinol, febuxostat, benzbromarone and rasburicase.

[0003] Lactobacillus is a probiotic that has a long history of use and is highly recognized by the public. In recent years, some lactobacilli have been reported to have the ability to reduce purine.

[0004] The Chinese patent application with publication number CN112458002A discloses a uric acid-lowering lactic acid bacteria strain, a screening method thereof, and an application in preparing functional yogurt. The strain has antioxidant, anti-inflammatory, immunomodulatory, uric acid-lowering functions and can be used to relieve and treat hyperuricemia.

[0005] The Chinese patent application with publication number CN115725437A discloses a strain of fermentative lactobacillus that efficiently utilizes purine and its application in rice wine brewing. The strain is applied to rice wine brewing, shortening the fermentation period of rice wine and reducing the purine content in the rice wine fermentation liquid by 68.48%.

[0006] The above-mentioned existing technologies show that lactic acid bacteria have great potential in preventing and improving hyperuricemia, and are very likely to become one of the important means of daily prevention and routine auxiliary treatment of hyperuricemia in the future. Therefore, in view of people’s current living and eating habits, in order to more effectively protect people’s health and disease prevention, it is of great social significance to develop low-cost functional lactic acid bacteria for use in functional foods to prevent metabolic rheumatoid diseases. Summary of the invention

[0007] The problem solved by the present invention is that: when hyperuricemia and gout are relieved and treated by drugs in the prior art, different drugs often have different degrees of toxic side effects, which are harmful to the human body. At the same time, when hyperuricemia and gout are relieved and treated by reducing the intake of exogenous purine compounds and reducing the purine level in the body in the prior art, it is difficult to relieve hyperuricemia by controlling diet because the rich nutrients required for the human body to maintain normal physiological metabolism and purine exist in most foods.

[0008] The purpose of the present invention is to provide a strain with the function of reducing purine, and apply it to the production of multifunctional probiotics and health foods to assist in the treatment, relief and improvement of gout and hyperuricemia, so as to achieve the purpose of developing a green, effective and safe new uric acid-lowering technology.

[0009] In a first aspect, the present invention provides a Lactococcus lactis strain, characterized in that the Lactococcus lactis strain is: Lactococcus lactis AMCC 11671 (Lactococcus lactis AMCC 11671), which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2024078.

[0010] Preferably, the degradation rate of purine compounds by Lactococcus lactis AMCC 11671 is greater than 20% by mass;

[0011] Preferably, the degradation rate of purine compounds by Lactococcus lactis AMCC 11671 is 25.06%-93.61%.

[0012] Preferably, the purine compound comprises one or more of inosine, guanosine, hypoxanthine, guanine and xanthine by mass.

[0013] Preferably, by mass, the Lactococcus lactis AMCC 11671 has an inosine degradation rate of more than 90%, and / or a guanosine degradation rate of more than 80%, and / or a hypoxanthine degradation rate of more than 25%, and / or a guanine degradation rate of more than 90%, and / or a xanthine degradation rate of more than 35%.

[0014] Preferably, by mass, the degradation rate of inosine by Lactococcus lactis AMCC 11671 is 92.55-93.61%, and / or the degradation rate of guanosine is 79.32-81.15%, and / or the degradation rate of hypoxanthine is 25.06-26.88%, and / or the degradation rate of guanine is 92.68-93.71%, and / or the degradation rate of xanthine is 32.66-38.61%.

[0015] Preferably, the Lactococcus lactis AMCC 11671 has a uric acid degradation rate of more than 10% by mass; preferably, the Lactococcus lactis AMCC 11671 has a uric acid degradation rate of 9.82-12.77% by mass.

[0016] Preferably, the 16S rDNA gene sequence of the Lactococcus lactis strain is shown as SEQ ID NO.3.

[0017] Preferably, the survival rate of the Lactococcus lactis strain under the condition of pH 1.5-3.5 is greater than or equal to 73%.

[0018] Preferably, the survival rate of the Lactococcus lactis strain in a liquid culture medium with a bile salt concentration of 0.1%-0.5% is greater than or equal to 94%, calculated on the basis of the ratio of bile salt weight to liquid culture medium volume (w / v).

[0019] In a second aspect, the present invention provides a method for preparing a Lactococcus lactis inoculant by fermentation, the method comprising the following steps: culturing the Lactococcus lactis strain according to any one of claims 1 to 9.

[0020] Preferably, the preparation method comprises the following steps:

[0021] (1) amplifying and culturing the Lactococcus lactis strain according to any one of claims 1 to 9;

[0022] (2) Adding the product obtained in step (1) into a liquid culture medium and fermenting and culturing at 30-37°C.

[0023] In a third aspect, the present invention provides a bacterial agent, which is obtained by the fermentation preparation method according to claim 10 or 11.

[0024] In a fourth aspect, the present invention provides a fermented product obtained by culturing the Lactococcus lactis strain according to any one of claims 1 to 9 or the bacterial agent according to claim 12.

[0025] Preferably, the fermentation product contains a xanthine oxidase specific activity of 3.73-4.30 U / g, measured in micromoles of xanthine consumed per milligram of protein in the fermentation product per minute; and / or a urate oxidase specific activity of 7.78-9.22 U / g, measured in micromoles of uric acid consumed per milligram of protein in the fermentation product per minute.

[0026] In a fifth aspect, the present invention also provides the use of the Lactococcus lactis strain or the bacterial agent in reducing the purine content in food.

[0027] Preferably, the food is a food containing yeast protein.

[0028] Beneficial effects of the present invention:

[0029] (1) The Lactococcus lactis AMCC 11671 provided by the present invention is a Lactococcus lactis AMCC 11671 having purine-reducing compounds obtained by screening and separation from tea leaves produced in Guangxi Zhuang Autonomous Region for the first time. It is a food-derived lactic acid bacterium and is suitable for fermentation of various foods.

[0030] (2) The Lactococcus lactis AMCC 11671 provided by the present invention has key enzymes of purine metabolism including xanthine oxidase and urate oxidase, and has good crude enzyme activity.

[0031] (3) The Lactococcus lactis AMCC 11671 provided by the present invention can effectively degrade inosine, guanosine, hypoxanthine, guanine, xanthine and uric acid, and can effectively slow down the generation and accumulation of uric acid by reducing the purine content and degrading uric acid.

[0032] (4) The Lactococcus lactis AMCC 11671 provided by the present invention has a survival rate of 73% after 3 hours at a pH of 1.5, and a survival rate of 94% after 12 hours at a bile salt concentration of 0.5% (w / v). It has good tolerance to acid and bile salts, has the ability to tolerate the gastrointestinal environment, and has the potential to colonize and function in the human gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The Gram staining results of Lactococcus lactis AMCC 11671.

[0034] Figure 2 It is the result of the relative enzyme activity determination of xanthine oxidase and urate oxidase.

[0035] Figure 3 It is the HPLC chromatogram of the degradation of purine compounds and uric acid by the strain of Example 3.

[0036] Strain deposit information

[0037] The Lactococcus lactis AMCC 11671 (Lactococcus lactis AMCC 11671) provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on January 11, 2024, with a deposit number of CCTCCNO: M 2024078, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 027-68754052. DETAILED DESCRIPTION

[0038] The lactococcus lactis AMCC 11671 provided by the present invention is separated, purified and identified from tea produced in the Guangxi Zhuang Autonomous Region. The morphological characteristics of the lactococcus lactis AMCC 11671 strain are as follows: the colony surface is smooth, the colony is milky white, the aerobic mode is facultative anaerobic, the suitable growth temperature is 30-37°C, the suitable growth pH is 5-6, the shape is spherical or oval under a microscope, the Gram staining is positive, and no capsule and spore are produced. The 16S rDNA gene sequence of the lactococcus lactis AMCC 11671 is shown in SEQ ID NO.3. The strain has the ability to degrade purine compounds.

[0039] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0040] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercially available products. The sources of experimental materials and instrument information used in the present invention are shown in Table 1:

[0041] Table 1 Source information of reagents and instruments used in the examples

[0042]

[0043]

[0044]

[0045] The preparation of the culture medium involved in the embodiments of the present invention is as follows:

[0046] (1) MRS solid medium containing 2% CaCO3: 10.0 g of peptone, 8.0 g of beef extract powder, 4.0 g of yeast extract powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate and Tween 80 were mixed to 1 mL, and the volume was made up to 1 L with distilled water. The pH was adjusted to 5.7 ± 0.2. When preparing the solid medium, 15 g of agar and 20 g of calcium carbonate were added, and the mixture was sterilized by high pressure sterilization at 121°C for 15 mins.

[0047] (2) M17 liquid medium: 5.0 g of soytone, 2.5 g of peptone, 2.5 g of casein peptone, 2.5 g of yeast extract powder, 5.0 g of beef extract powder, 5 g of lactose, 0.5 g of sodium ascorbate, 19.0 g of sodium β-glycerophosphate and 0.25 g of magnesium sulfate were mixed, and the volume was made up to 1 L with distilled water. The pH value was 7.2±0.2 at 25°C. When preparing the solid medium, 15 g of agar was added. The mixture was sterilized by high pressure at 121°C for 15 mins. When used, a glucose solution with a final concentration of 1% (w / v) was added.

[0048] Example 1: Isolation, purification and screening of purine-lowering strains

[0049] 1. Isolation and purification of purine-reducing strains

[0050] Take 2g of tea leaves, cut them into pieces, add them to a blue-mouthed bottle containing 20mL of sterile water, and incubate them at 37°C for 2 days to obtain the sample to be separated. Take 100μL of the sample to be separated, add 900μL of sterile water to dilute, shake and mix, and use sterile water to perform a 10-fold gradient dilution to obtain 10 -1 -10 -5 A dilution gradient series was performed, and 200 μL was taken and spread on MRS solid medium containing 2% CaCO3, and placed in a 37°C constant temperature incubator for anaerobic culture for 36 hours. Finally, three candidate strains were isolated from the sample, and according to the results of microscopic examination, the bacterial morphology was found to be spherical, and it was preliminarily identified as Lactococcus lactis. Therefore, the MRS solid medium containing 2% CaCO3 was replaced with M17 liquid medium in subsequent culture. At the same time, the three candidate strains were named: strain AR214, strain AR215 and strain AR216. Strain AR214, strain AR215 and strain AR216 were respectively purified by plate streaking, and single colonies were picked and cultured in M17 liquid culture medium at 37°C for 16 h to obtain a bacterial solution, which was mixed with 30% (W / V) glycerol in a ratio of 1:1 and frozen at -80°C. Among them, 30% (W / V) glycerol was prepared by weighing 30 g glycerol, dissolving it in ultrapure water, and then making up the volume to 100 ml, sterilizing it at 121°C for 15 min, and storing it at room temperature for use.

[0051] 2. Preliminary screening of purine-lowering strains

[0052] 1. In vitro metabolic enzyme activity screening of xanthine oxidase (XOD) and urate oxidase (UOX), the solution used in the reaction system was prepared as follows:

[0053] (1) Tris-HCl solution: Weigh 0.788 g of Tris-HCl solid, dissolve it in an appropriate amount of ultrapure water, and dilute to 50 mL to prepare a 100 mM Tris-HCl solution. Adjust the pH to 8.5 with 4 M HCl and 5 M NaOH.

[0054] (2) Ethylenediaminetetraacetic acid solution (EDTA solution): Weigh 37.2 mg of EDTA solid, dissolve it in an appropriate amount of ultrapure water, and make up to 10 mL to prepare a 10 mM EDTA solution. Adjust the pH to 8.0 with 4 M HCL and 5 M NaOH.

[0055] (3) Uric acid solution: Weigh 33.6 mg of uric acid, dissolve it in an appropriate amount of 50 mM NaOH solution, and then dilute it to 10 mL to prepare a 20 mM uric acid solution.

[0056] (4) Xanthine solution: Weigh 30.4 mg of xanthine, dissolve it in an appropriate amount of 50 mM NaOH solution, and then dilute it to 10 mL to prepare a 20 mM xanthine solution.

[0057] (5) Potassium oxonate solution: Weigh 20 mg of potassium oxonate, dissolve it in an appropriate amount of ultrapure water, and use the water to make up to 5 mL to prepare a 10 mM potassium oxonate solution.

[0058] (6) Nicotinamide adenine dinucleotide solution (NAD solution): Weigh 33.1 mg of NAD solid, dissolve it in an appropriate amount of ultrapure water, and use it to make up to 5 mL to prepare a 100 mM NAD solution.

[0059] (7) Reduced nicotinamide adenine dinucleotide phosphate solution (NADPH solution): Weigh 70.8 mg of NADPH solid, dissolve it in an appropriate amount of ultrapure water, and use it to make up to 1 mL to prepare a 100 mM NADPH solution.

[0060] (8) NaOH solution: Weigh 0.1 g of NaOH solid, dissolve it in an appropriate amount of ultrapure water, and make up to 50 mL to prepare a 50 mM NaOH solution.

[0061] 2. In vitro metabolic enzyme activity screening of xanthine oxidase (XOD) and urate oxidase (UOX), the PBS buffer solution used was prepared as follows:

[0062] PBS buffer solution: Measure 84.5 mL of 1M disodium hydrogen phosphate and 15.5 mL of 1M sodium dihydrogen phosphate solution, mix them evenly and dilute them by half, then adjust the pH to 7.5 with 4M phosphoric acid or 5M NaOH.

[0063] Disodium hydrogen phosphate: Weigh 14.2 g of solid disodium hydrogen phosphate, dissolve it with an appropriate amount of ultrapure water, and dilute to 100 mL to prepare a disodium hydrogen phosphate solution with a mother liquor concentration of 1 M;

[0064] Sodium dihydrogen phosphate: Weigh 15.6 g of sodium dihydrogen phosphate solid, dissolve it in an appropriate amount of ultrapure water, and then dilute to 100 mL to prepare a sodium dihydrogen phosphate solution with a mother liquor concentration of 1 M.

[0065] 3. Screening of in vitro metabolic enzyme activities of xanthine oxidase (XOD) and urate oxidase (UOX). The steps for screening strains with crude enzyme activities of XOD and UOX are as follows:

[0066] (1) Preparation of crude enzyme solution to be tested: strain AR214, strain AR215 and strain AR216 frozen at -80°C were thawed, streaked and cultured in a 37°C constant temperature incubator for 16 hours, and then single colonies were picked in 10 mL of M17 liquid culture medium, and static culture was performed at 37°C to obtain seed solution, and 1 volume % was inoculated in 800 mL of M17 liquid culture medium, and cultured at 37°C for 16 hours. The bacterial solution was centrifuged at 4°C and 12000 r / min for 15 minutes, and the supernatant was removed to retain the bacterial cells. 25 mL of PBS buffer solution with a pH of 7.5 was added to the bacterial cells, and the bacterial cells were resuspended and washed twice, and then resuspended with 25 mL of PBS again, and 2.5 g of quartz sand and 0.5 g of glass beads were added, and the cells were broken for 900 seconds at -30°C using a high-throughput tissue grinder. After the cell lysis is completed, the bacterial solution is centrifuged at 13000r / min and 4°C for 2mins, and the supernatant is the crude enzyme solution to be tested.

[0067] (2) Xanthine oxidase (XOD) enzyme activity assay: The reaction system is shown in Table 2. After the reaction solution is prepared, the crude enzyme solution inactivated in a 90°C water bath for 20 minutes is used as a blank control. 10 μL of the crude enzyme sample solution to be tested and the inactivated crude enzyme sample solution to be tested are added to the wells of the ELISA plate, respectively. Three parallel groups are set up at the same time. Within 5 minutes, 190 μL of the reaction solution is added to each well of the ELISA plate to start the reaction. Among them, the reaction solution is 100 μL of the Tris-HCL solution, 20 μL of the EDTA solution, 2 μL of the potassium oxonate solution, 1 μL of the xanthine solution, 0.2 μL of the NAD solution and 66.8 μL of ultrapure water. The absorbance at 295 nm is measured using an infinite M200 PRO microplate reader to calculate the generation of uric acid.

[0068] Table 2 Xanthine oxidase (XOD) 200 μL reaction system

[0069]

[0070]

[0071] (3) Urate oxidase (UOX) enzyme activity assay: The reaction system is shown in Table 3. The reaction solution is prepared according to Table 3. The crude enzyme solution inactivated in a 90°C water bath for 20 minutes is used as a blank control. 10 μL of the crude enzyme sample solution to be tested and the inactivated crude enzyme sample solution to be tested are added to the wells of the ELISA plate, respectively. Three parallel groups are set up at the same time. 190 μL of the reaction solution is added to each well of the ELISA plate within 5 minutes to start the reaction. Among them, the reaction solution is 100 μL of the Tris-HCL solution, 20 μL of the EDTA solution, 1 μL of the uric acid solution, 0.2 μL of the NADPH solution and 68.8 μL of ultrapure water. The absorbance at 295 nm is measured using the infinite M200 PRO ELISA instrument to calculate the reduction in uric acid.

[0072] Table 3 Urate oxidase (UOX) 200 μL reaction system

[0073]

[0074] (4) Determination of protein content: Use the BCA protein quantification kit to determine the protein content of the crude enzyme solution to be tested, which is used to further calculate the specific enzyme activity. According to the operating instructions of the BCA protein quantification kit, dilute the standard and prepare the BCA working solution. Dilute the sample solution 20 times with PBS buffer solution, and determine the total protein content in the crude enzyme solution to be tested according to the instructions. Perform a linear fit on the concentration and absorbance value of the diluted standard to draw a standard curve. The protein standard curve y = 1.0993x + 0.1697, R2 = 0.9994. According to the protein standard curve, the protein content in the crude enzyme solution to be tested (strain AR214, strain AR215 and strain AR216) was calculated, and the protein contents of strain AR214, strain AR215 and strain AR216 were 4.62 mg / mL, 5.10 mg / mL and 5.62 mg / mL, respectively.

[0075] (5) Determination of whether the crude enzyme solution to be tested has XOD and UOX enzyme activities:

[0076] It should be noted that according to the change of absorbance at the characteristic wavelength of uric acid 295nm, whether the crude enzyme solution to be tested has the enzyme activity of XOD and UOX is analyzed, and the absolute enzyme activity of XOD and UOX of the three strains is calculated by SLOPE function. If uric acid is generated, the crude enzyme solution to be tested contains XOD enzyme activity, which oxidizes xanthine to uric acid; if uric acid is reduced, the crude enzyme solution to be tested has UOX enzyme activity, which oxidizes uric acid to generate allantoin. The absolute enzyme activities of XOD of strain AR214, strain AR215, and strain AR216 are 9.24U / L, 15.3U / L, and 24.17U / L, respectively, and the absolute enzyme activities of UOX are 22.96U / L, 47.43U / L, and 75.87U / L, respectively.

[0077] (6) Calculation of XOD and UOX enzyme specific activity of crude enzyme solution to be tested:

[0078] The XOD and UOX specific enzyme activities of the crude enzyme solutions to be tested (strain AR214, strain AR215 and strain AR216) were calculated. The XOD specific enzyme activity was expressed as the micromoles of xanthine consumed per milligram of protein per minute, and the UOX specific enzyme activity was expressed as the micromoles of uric acid consumed per milligram of protein per minute. The XOD and UOX specific enzyme activity determination results are shown in Figure 2. Figure 2 As shown, the results showed that strain AR214, strain AR215 and strain AR216 all showed different XOD specific enzyme activities and UOX specific enzyme activities. Among them, the XOD specific enzyme activities of strain AR214, strain AR215 and strain AR216 were not much different, and the XOD specific enzyme activity of strain AR216 was slightly higher than that of strain AR214 and strain AR215, while the UOX specific enzyme activity of strain AR216 was the highest, significantly higher than that of the other two strains. Therefore, through the XOD specific enzyme activity and UOX specific enzyme activity, it can be preliminarily judged that strain AR216 has a good ability to degrade uric acid.

[0079] Example 2: Identification of purine-lowering strains

[0080] 1. Molecular identification of purine-lowering strains:

[0081] The preserved strains AR214, AR215 and AR216 were cultured at 37°C for 16 hours using M17 liquid culture medium, and then the genomes of the strains were extracted. The genomic DNAs were amplified by PCR and sequenced using bacterial universal primers 16S rDNA 27F / 1492R. The 16S rDNA sequences of the strains were selected for BLAST comparison analysis in the NCBI database to obtain strains AR214, AR215 and AR216, and the similarity rates with Lactococcus lactis were all 99.93%, indicating that the strains belonged to Lactococcus lactis strains. The 16S rDNA gene sequence of strain AR214 was shown in SEQ ID NO.1, the 16S rDNA gene sequence of strain AR215 was shown in SEQ ID NO.2, and the 16S rDNA gene sequence of strain AR216 was shown in SEQ ID NO.3.

[0082] SEQ ID NO.1:

[0083]

[0084] SEQ ID NO.2:

[0085]

[0086] SEQ ID NO.3:

[0087]

[0088] According to the 16S rDNA sequence identification results of strain AR214, strain AR215 and strain AR216 in Example 1, they are all Lactococcus lactis. Further comprehensive comparison of the specific enzyme activities of two key enzymes (XOD and UOX) in the purine metabolic pathway finally selected strain AR216 as the preferred strain for reducing purine, and its morphological characteristics were identified.

[0089] 2. Identification of morphological characteristics of strain AR216:

[0090] The strain AR216 was streaked on an M17 plate until a single colony grew out, and a single colony was picked for Gram staining and catalase test. Catalase test steps: an appropriate amount of bacteria was picked and applied to a clean glass slide and 3% hydrogen peroxide solution was added. Observe whether bubbles are generated. If bubbles are generated, it is a positive reaction, otherwise it is a negative reaction. The experimental result of the present invention is negative.

[0091] The morphological characteristics of strain AR216 are as follows: the colony texture is smooth, and the individual colonies are milky white; they are spherical or oval under a microscope, Gram-positive, and do not produce capsules or spores.

[0092] Combined with the identification of molecular and morphological characteristics, strain AR216 was identified as Lactococcus lactis AMCC 11671 (Lactococcus lactis AMCC 11671), which was deposited in the China Center for Type Culture Collection (CCTCC) on January 11, 2024, with the deposit number CCTCC NO: M 2024078. The deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: 027-68754052.

[0093] Figure 1 Shown are the Gram stain results of Lactococcus lactis AMCC 11671.

[0094] Example 3: HPLC Verification of Purine Compound Degradation Ability of Lactococcus lactis AMCC 11671

[0095] 1. The purine compound degradation ability of Lactococcus lactis AMCC 11671 was verified by high performance liquid chromatography (HPLC). The solution used in the reaction system was prepared as follows:

[0096] (1) Inosine standard solution: Weigh 53.6 mg of inosine standard, dissolve it in an appropriate amount of ultrapure water, and make up to 10 mL to prepare a 20 mM inosine stock solution.

[0097] (2) Guanosine standard solution: Weigh 56.6 mg of guanosine standard, dissolve it in an appropriate amount of 50 mM NaOH solution, and then dilute it to 10 mL to prepare a 20 mM guanosine stock solution.

[0098] (3) Hypoxanthine standard solution: Weigh 27.2 mg of hypoxanthine standard, dissolve it in an appropriate amount of 50 mM NaOH solution, and then dilute it to 10 mL to prepare a 20 mM hypoxanthine stock solution.

[0099] (4) Guanine standard solution: Weigh 30.2 mg of guanine standard, dissolve it in an appropriate amount of 50 mM NaOH solution, and then dilute it to 10 mL to prepare a 20 mM guanine stock solution.

[0100] (5) Xanthine standard solution: Weigh 30.4 mg of xanthine, dissolve it in an appropriate amount of 50 mM NaOH solution, and then dilute to 10 mL to prepare a 20 mM xanthine stock solution.

[0101] (6) Uric acid standard solution: Weigh 33.6 mg of uric acid, dissolve it in an appropriate amount of 50 mM NaOH solution, and then dilute it to 10 mL to prepare a 20 mM uric acid stock solution.

[0102] (7) NaOH solution: Weigh 0.1 g of NaOH solid, dissolve it in an appropriate amount of ultrapure water, and make up to 50 mL to prepare a 50 mM NaOH solution.

[0103] 2. The purine compound degradation ability of Lactococcus lactis AMCC 11671 was verified by high performance liquid chromatography (HPLC). The PBS buffer solution used was prepared as follows:

[0104] PBS buffer solution: Adjust the pH of disodium hydrogen phosphate solution to 7.5 with sodium dihydrogen phosphate solution to obtain a 100 mM PBS buffer solution, wherein:

[0105] Sodium dihydrogen phosphate: Weigh 1.56 g of sodium dihydrogen phosphate, dissolve it in an appropriate amount of water, and make up to 100 mL to prepare a 100 mM sodium dihydrogen phosphate solution;

[0106] Disodium hydrogen phosphate: Weigh 1.42 g of anhydrous disodium hydrogen phosphate, dissolve it in appropriate amount of water, and make up to 100 mL to prepare a 100 mM disodium hydrogen phosphate solution.

[0107] 3. The purine compound degradation ability of Lactococcus lactis AMCC 11671 was verified by high performance liquid chromatography (HPLC). The mobile phase (sodium dihydrogen phosphate dihydrate solution) used was prepared as follows:

[0108] Sodium dihydrogen phosphate dihydrate: Weigh 1.56 g of sodium dihydrogen phosphate dihydrate, dilute to 1 L with appropriate amount of water, adjust the pH to 4.7 with 4 M phosphoric acid or 5 M NaOH to make a 10 mM sodium dihydrogen phosphate solution, which is the mobile phase.

[0109] 4. The steps of verifying the ability of Lactococcus lactis AMCC 11671 to degrade purine compounds and uric acid by high performance liquid chromatography (HPLC) are as follows:

[0110] It should be noted that nucleosides in food are precursors for the production of purines and are the main source of purines in the body. Purines will accumulate and oxidize to uric acid through a series of metabolic pathways. In order to screen out strains with the function of reducing purine compounds, the metabolic capacity of resting cells for inosine, guanosine, hypoxanthine, guanine, xanthine and uric acid was used as an indicator. The content of inosine, guanosine and purine compounds was detected and analyzed at 25°C in a Waters e2695 liquid chromatograph equipped with a Waters 2998PDA photodiode matrix chromatographic detector to explore the degradation ability of Lactococcus lactis AMCC 11671 for purine compounds. The specific detection process is as follows:

[0111] (1) The configuration of the standard sample is as follows:

[0112] Inosine standard sample: 100 μL of the 20 mM inosine stock solution was taken and mixed with 1 mL of the PBS buffer solution and 900 μL of ultrapure water to obtain 2 mL of inosine standard sample. Take 0 μL, 10 μL, 20 μL, 30 μL, 40 μL, 60 μL, 80 μL, 100 μL, and 120 μL of the 20 mM inosine stock solution and mix with 1 mL of the PBS buffer and 1000 μL, 990 μL, 980 μL, 970 μL, 960 μL, 940 μL, 920 μL, 900 μL, and 880 μL of ultrapure water to obtain inosine standards with concentrations of 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, 600 μM, 800 μM, 1000 μM, and 1200 μM, and use the above samples to make a standard curve.

[0113] The preparation methods of guanosine standard sample, hypoxanthine standard sample, guanine standard sample, xanthine standard sample and uric acid standard sample and their standard curves are the same as those of the inosine standard sample.

[0114] (2) Preparation of the test bacterial solution: Lactococcus lactis AMCC 11671 strain was inoculated into 200 mL of M17 liquid culture medium at 2% by volume, cultured in a 37°C constant temperature incubator for 16 h, and then centrifuged at 4°C, 6000 r / min for 3 mins to collect the bacterial cells. The bacterial cells were washed twice with 25 mL of PBS solution and then resuspended in 5 mL of PBS solution to obtain the test bacterial solution.

[0115] (3) The preparation of the reaction solution is as follows:

[0116] Inosine reaction solution: 1 mL of the test bacterial solution obtained in step 4(2) was mixed with 2 mL of the inosine standard sample obtained in step 4(1) to obtain an inosine reaction solution.

[0117] The preparation methods of the guanosine reaction solution, the hypoxanthine reaction solution, the guanine reaction solution, the xanthine reaction solution and the uric acid reaction solution are the same as those of the inosine reaction solution.

[0118] (4) HPLC determination:

[0119] (4-1) Inosine standard solution, guanosine standard solution, guanine standard solution, hypoxanthine standard solution, xanthine standard solution and uric acid standard solution with final concentrations of 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, 600 μM, 800 μM, 1000 μM and 1200 μM, respectively, were filtered through a 0.22 μm water filter membrane and analyzed according to HPLC chromatographic conditions. The HPLC chromatographic conditions are shown in Table 5.

[0120] (4-2) The reaction solutions in step 4 (3) of this embodiment were placed in a 37°C incubator and incubated at 160 r / min for 60 min. After the incubation, the reaction solution was centrifuged at 4°C and 6000 r / min for 3 min. 2 mL of the supernatant was taken and the enzyme was inactivated in a 90°C water bath for 20 min to terminate the reaction. After cooling, the solution was filtered using a 0.22 μm water filter membrane and then analyzed according to HPLC chromatographic conditions. The HPLC chromatographic conditions are shown in Table 5. The HPLC chromatograms of the degradation of inosine (a), guanosine (b), guanine (c), hypoxanthine (d), xanthine (e) and uric acid (f) by Lactococcus lactis AMCC 11671 are shown in Table 5. Figure 3 shown.

[0121] Table 5 HPLC chromatographic conditions

[0122]

[0123] Linear fitting was performed with the corresponding peak areas of inosine standard solution, guanosine standard solution, guanine standard solution, hypoxanthine standard solution, xanthine standard solution and uric acid standard solution with final concentrations of 0μM, 100μM, 200μM, 300μM, 400μM, 600μM, 800μM, 1000μM and 1200μM, respectively. Specifically, linear fitting was performed with the different concentrations (X) of each standard solution as the abscissa and the corresponding peak area (Y) as the ordinate, and the corresponding standard curve was drawn; wherein,

[0124] The standard curve of inosine is Y = 7017.5X-250106, R2 = 0.9961;

[0125] The standard curve of guanosine is Y = 7444.4X-41998, R2 = 0.9979;

[0126] The standard curve of hypoxanthine is Y = 5365X + 11907, R2 = 0.9973;

[0127] The standard curve of xanthine is Y = 3704.8X + 17196, R2 = 0.9985;

[0128] The standard curve of uric acid is Y = 4640.4X-116892, R2 = 0.9932;

[0129] The standard curve of guanine is Y=4594.6X-80573, R2=0.9940.

[0130] Substitute the peak area in the sample into the standard curve of each purine, calculate the content (μM) of residual purine or uric acid in the reaction solution in step 4 (3) of this embodiment, and calculate the degradation rate of purine compounds and uric acid by each strain using formula 1. Wherein, α represents the degradation rate (%), n represents the content of purine compounds or uric acid in the bacterial solution to be tested (μM), and X represents the content of residual purine compounds or uric acid in the reaction solution in step 4 (3) of this embodiment (μM).

[0131]

[0132] The calculation results of formula 1 show that the degradation rates of inosine, guanosine, hypoxanthine, guanine, xanthine and uric acid by Lactococcus lactis AMCC 11671 are 93.61%, 81.15%, 26.88%, 93.71%, 38.61% and 12.77%, respectively. It can be seen that Lactococcus lactis AMCC 11671 has the ability to degrade the above purine compounds and uric acid to a certain extent. Figure 3 As shown, Figure 3 (a) is a high performance liquid chromatogram of degraded inosine; Figure 3 (b) is a high performance liquid chromatogram of guanosine degradation; Figure 3 (c) is a high performance liquid chromatogram of degradation of guanine; Figure 3 (d) is a high performance liquid chromatogram of degradation of hypoxanthine; Figure 3 (e) is a high performance liquid chromatogram of xanthine degradation; Figure 3(f) in the figure is a high-performance liquid chromatogram of uric acid reduction. Analysis of the high-performance liquid chromatogram shows that hypoxanthine is generated after the reaction of inosine, and hypoxanthine is oxidized to uric acid through a series of XOD, and uric acid is degraded to allantoin through the action of UOX; guanine is generated after the reaction of guanosine, and guanine is oxidized to uric acid through a series of XOD reactions, and uric acid is further degraded to allantoin; and each purine compound is degraded to a certain extent after the reaction; and a certain amount of allantoin is generated, which verifies that Lactococcus lactis AMCC 11671 has a good ability to reduce purines. Based on this, the ability of Lactococcus lactis AMCC 11671 to resist acid and bile salts is further explored.

[0133] Example 4: Acid and bile resistance of Lactococcus lactis AMCC 11671

[0134] 1. Preparation of the test bacterial solution: Inoculate the bacterial solution of Lactococcus lactis AMCC 11671 at a volume % inoculum into a freshly prepared M17 liquid culture medium for activation, and culture at 37° C. until the strain reaches the growth station phase.

[0135] 2. Acid tolerance determination: Use 4mol / L HCl and 5mol / L NaOH to prepare fresh M17 liquid culture medium with pH values ​​of 1.5, 2.5 and 3.5 respectively, take 1mL of the test bacterial solution and centrifuge to collect the bacteria, the centrifugation conditions are 4℃, 8000r / min, 5mins. Wash the bacterial precipitate twice with 1mL 0.9% NaCl. Then transfer the bacteria to 1mL fresh M17 liquid culture medium with pH values ​​of 1.5, 2.5 and 3.5, mix well, and incubate at 37℃ for 3h. Determine the OD600 value of the reaction solution in each experimental group. The survival rate calculation formula is as shown in Formula 2:

[0136]

[0137] In formula 2, A1 represents the OD600 value of the reaction solution of each experimental group after incubation for 3 hours; A0 represents the OD600 value of the bacterial solution to be tested.

[0138] Since the pH of the gastrointestinal tract of the human body after eating is about 3.0, Lactococcus lactis AMCC 11671 must be able to survive in this environment for 1.5-2.0 hours to be able to exert its efficacy. The survival rate of acid-resistant strains of Lactococcus lactis AMCC 11671 is shown in Table 6. The results show that the survival rate of the strain decreases with the decrease of pH. Despite this, the survival rate of Lactococcus lactis AMCC 11671 is still greater than 73% when incubated for 3 hours at pH 1.5, 2.5 and 3.5. The results show that Lactococcus lactis AMCC 11671 can be used as a probiotic and has the potential to colonize in the human gastrointestinal tract.

[0139] Table 6 Survival rate of Lactococcus lactis AMCC 11671 under different pH conditions

[0140]

[0141] 3. Bile salt tolerance test

[0142] Prepare fresh M17 liquid culture medium containing bile salts at final concentrations of 0.1% (w / v), 0.3% (w / v) and 0.5% (w / v), take 1mL of the test bacterial solution and centrifuge to collect the bacteria. The centrifugation conditions are 4°C, 8000r / min, 5mins. Wash the bacterial precipitate twice with 1mL0.9% NaCl. Then transfer the bacteria to 1mL fresh M17 liquid culture medium containing bile salts at concentrations of 0.1% (w / v), 0.3% (w / v), and 0.5% (w / v), and incubate at 37°C for 12h. Determine the OD600 value of each experimental group. The survival rate calculation formula is as follows:

[0143]

[0144] In formula 3, A1 represents the OD600 value of the reaction solution of the experimental group; A0 represents the OD600 value of the bacterial solution to be tested.

[0145] The survival rate of bile salt resistant strains of Lactococcus lactis AMCC 11671 is shown in Table 7. The results show that with the increase of bile salt concentration, the survival rate of Lactococcus lactis AMCC 11671 slightly decreased, but the survival rate remained above 94%. The mass concentration of bile salt in the human small intestine is 0.03%-0.3%, so the above results further confirm that Lactococcus lactis AMCC 11671 has the potential to colonize in the human gastrointestinal tract to a certain extent.

[0146] Table 7 Survival rate of Lactococcus lactis AMCC 11671 under different bile salt concentrations

[0147]

[0148] Application example: Lactococcus lactis AMCC 11671 degrades purine compounds in yeast protein

[0149] 1. Preparation of Purine Standard Solution

[0150] (1) Adenine stock solution (200 mg / L): Accurately weigh 10 mg of adenine standard, dissolve it in ultrapure water and dilute it to 100 mL in a 50 mL volumetric flask, mix well, and if it does not dissolve, use an appropriate amount of sodium hydroxide solution (1 mol / L) to dissolve it to obtain adenine stock solution. Sodium hydroxide solution (1 mol / L): weigh 4.00 g of sodium hydroxide, dissolve it in ultrapure water and dilute it to 100 mL.

[0151] (2) Adenine standard solution: 0.025 mL, 0.05 mL, 0.1 mL, 0.2 mL, 0.5 mL, 1.0 mL and 2.0 mL of the adenine stock solution were taken, respectively, and 5 mL of trifluoroacetic acid and 5 mL of formic acid were added thereto, respectively. The mixture was pyrolyzed in a water bath at 85°C for 15 mins, rapidly cooled and rotary evaporated to dryness at 80°C, reconstituted with 10 mL of mobile phase (as shown in Table 8), centrifuged at 6000 r / min for 5 mins, the supernatant was collected, and filtered through a 0.22 μm organic microporous filter membrane to obtain adenine standard solutions with final concentrations of 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L and 40 mg / L, respectively.

[0152] (3) The preparation methods of guanine stock solution, hypoxanthine stock solution and xanthine stock solution are the same as those of adenine stock solution.

[0153] (4) The preparation methods of guanine standard solution, hypoxanthine standard solution and xanthine standard solution are the same as those of adenine standard solution.

[0154] 2. Preparation of Lactococcus lactis AMCC 11671 bacterial liquid: Lactococcus lactis AMCC 11671 bacterial liquid was inoculated into 100 mL of M17 liquid medium with 2% by volume, cultured in a 37°C constant temperature incubator for 16 h, and then centrifuged at 4°C, 6000 r / min for 3 mins to collect the bacterial cells. The bacterial cells were washed twice with 25 mL of PBS solution and then resuspended in 2 mL of PBS solution. The Lactococcus lactis AMCC 11671 bacterial liquid was ready for use.

[0155] 3. Preparation of yeast protein solution: Weigh 0.2 g of yeast protein powder and dilute to 2 mL with water, place in an ultrasonic tank (40 Hz) and perform ultrasonic extraction for 30 mins to obtain a yeast protein solution.

[0156] 4. Preparation of test solution:

[0157] (1) Preparation of the negative control group test solution: 1 mL of Lactococcus lactis AMCC 11671 bacterial solution and 1 mL of yeast protein solution containing free purine were mixed and placed in a 37°C shaking incubator (160 r / min) for 0 h. 4 mL of trifluoroacetic acid and 4 mL of formic acid were added, mixed and pyrolyzed in an 85°C water bath for 15 mins, quickly cooled and rotary evaporated at 80°C to near dryness, re-dissolved with 10 mL of mobile phase (as shown in Table 8), centrifuged at 10000 r / min for 5 mins, and the supernatant was taken and filtered with a 0.22 μm organic microporous filter membrane to obtain the negative control group test solution.

[0158] (2) Preparation of the test solution of the experimental group: 1 mL of Lactococcus lactis AMCC 11671 bacterial solution and 1 mL of yeast protein solution containing free purine were mixed and placed in a 37°C shaking incubator (160 r / min) for 2 h. 4 mL of trifluoroacetic acid and 4 mL of formic acid were added, mixed and pyrolyzed in an 85°C water bath for 15 mins, quickly cooled and rotary evaporated at 80°C to near dryness, re-dissolved with 10 mL of mobile phase (as shown in Table 8), centrifuged at 10000 r / min for 5 mins, and the supernatant was taken and filtered with a 0.22 μm organic microporous filter membrane to obtain the test solution of the experimental group.

[0159] 5.HPLC determination:

[0160] (1) Adenine standard solution, guanine standard solution, hypoxanthine standard solution and xanthine standard solution with final concentrations of 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L and 40 mg / L, respectively, were loaded and analyzed according to HPLC chromatographic conditions. The HPLC chromatographic conditions are shown in Table 8.

[0161] (2) The negative control group test solution and the experimental group test solution were loaded and analyzed according to HPLC chromatographic conditions. The HPLC chromatographic conditions are shown in Table 8.

[0162] Table 8 HPLC chromatographic conditions

[0163]

[0164] Linear fitting was performed on purine standard solutions with different final concentrations (0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 40 mg / L) and their corresponding peak areas, wherein the purine standard solutions included: adenine standard solution, guanine standard solution, xanthine standard solution and hypoxanthine standard solution. Specifically, linear fitting was performed with the different final concentrations (x) of each purine standard solution as the abscissa and the corresponding peak area as the ordinate y to draw the standard curves of adenine, guanine, xanthine and hypoxanthine, wherein,

[0165] The standard curve of adenine is y = 68858.37x-557445, R2 = 0.9997;

[0166] The standard curve of guanine is y = 65210.21x-471143, R2 = 0.9999;

[0167] The standard curve of xanthine is y = 36749.87x-29420.6, R2 = 0.9999;

[0168] The standard curve of hypoxanthine is y=55911.22x-60074, R2=0.9999.

[0169] The peak area in the sample was substituted into the standard curve of each purine, and the concentration of purine compounds in the negative control group and the experimental group were calculated respectively. The concentration of purine compounds in the test solution of the negative control group and the test solution of the experimental group are shown in Table 8, wherein the purine compounds include: adenine, guanine, xanthine and hypoxanthine.

[0170] Table 8 Concentration of purine compounds in the negative control group test solution and the experimental group test solution

[0171]

[0172] According to the concentration of purine compounds, the content of purine compounds is calculated using Formula 4, where x represents the content of purine compounds (mg / 100g), c represents the concentration of purine compounds (mg / L), V represents the final fixed volume of the yeast protein solution (L), m represents the weighed amount of yeast protein powder (g), and 100 is the unit conversion coefficient.

[0173]

[0174] According to the content of purine compounds, the total content of purine compounds in the yeast protein solution is calculated using Formula 5. As shown in Formula 5, in Formula 5, X represents the total content of purine compounds in the yeast protein solution (mg / 100g), x1 represents the content of adenine in the yeast protein solution, x2 represents the content of guanine in the yeast protein solution, x3 represents the content of xanthine in the yeast protein solution, and x4 represents the content of hypoxanthine in the yeast protein solution (mg / 100g).

[0175] X=x1+x2+x3+x4 Formula 5

[0176] According to the calculation of formula 4 and formula 5, after the reaction of Lactococcus lactis AMCC 11671 with the yeast protein solution for 0 hours, the total purine content in the sample was 2902.37 mg / 100 g, while after the reaction of Lactococcus lactis AMCC 11671 with the yeast protein solution for 2 hours, the total purine content in the sample was 1867.37 mg / 100 g, and the total purine content decreased by 35.66%. It can be seen that Lactococcus lactis AMCC 11671 can reduce the purine content in the yeast protein solution to a certain extent, confirming that Lactococcus lactis AMCC 11671 has the function of reducing the purine content in yeast protein.

[0177] In summary, Lactococcus lactis AMCC 11671 has the ability to degrade purine compounds and has the function of reducing the total purine content in yeast protein. To a certain extent, it can be used as a probiotic to reduce the purine content in food, preferably in yeast-made food.

[0178] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Lactococcus lactis strain, characterized in that The lactococcus lactis strain is: Lactococcus lactis AMCC 11671 (Lactococcus lactis AMCC 11671), which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2024078.

2. The Lactococcus lactis strain according to claim 1, characterized in that The degradation rate of purine compounds by the Lactococcus lactis AMCC 11671 is greater than 20% by mass; Preferably, the degradation rate of purine compounds by Lactococcus lactis AMCC 11671 is 25.06%-93.61%.

3. The Lactococcus lactis strain according to any one of claims 1 to 2, characterized in that The purine compound includes one or more of inosine, guanosine, hypoxanthine, guanine and xanthine by mass.

4. The Lactococcus lactis strain according to any one of claims 1 to 3, characterized in that By mass, the Lactococcus lactis AMCC 11671 has an inosine degradation rate of more than 90%, and / or a guanosine degradation rate of more than 80%, and / or a hypoxanthine degradation rate of more than 25%, and / or a guanine degradation rate of more than 90% and / or a xanthine degradation rate of more than 35%.

5. The Lactococcus lactis strain according to any one of claims 1 to 4, characterized in that By mass, the degradation rate of inosine for Lactococcus lactis AMCC 11671 is 92.55-93.61%, and / or the degradation rate of guanosine is 79.32-81.15%, and / or the degradation rate of hypoxanthine is 25.06-26.88%, and / or the degradation rate of guanine is 92.68-93.71%, and / or the degradation rate of xanthine is 32.66-38.61%.

6. The Lactococcus lactis strain according to any one of claims 1 to 5, characterized in that The Lactococcus lactis AMCC 11671 also has a uric acid degradation rate of more than 10% by mass; preferably, the Lactococcus lactis AMCC 11671 has a uric acid degradation rate of 9.82-12.77% by mass.

7. The Lactococcus lactis strain according to any one of claims 1 to 6, characterized in that The 16S rDNA gene sequence of the Lactococcus lactis strain is shown in SEQ ID NO.

3.

8. The Lactococcus lactis strain according to any one of claims 1 to 7, characterized in that The survival rate of the Lactococcus lactis strain under the condition of pH 1.5-3.5 is greater than or equal to 73%.

9. The Lactococcus lactis strain according to any one of claims 1 to 8, characterized in that Calculated by the ratio of bile salt weight to volume of liquid culture medium (w / v), the survival rate of the Lactococcus lactis strain in the liquid culture medium with a bile salt concentration of 0.1%-0.5% is greater than or equal to 94%.

10. A method for preparing a lactococcus lactis inoculant by fermentation, characterized in that: The method comprises the following steps: culturing the Lactococcus lactis strain according to any one of claims 1 to 9.

11. The preparation method according to claim 10, characterized in that: The preparation method comprises the following steps: (1) amplifying and culturing the Lactococcus lactis strain according to any one of claims 1 to 9; (2) Adding the product obtained in step (1) into a liquid culture medium and fermenting and culturing at 30-37°C.

12. A bacterial agent, characterized in that: The bacterial agent is obtained by the fermentation preparation method according to claim 10 or 11.

13. A fermented product, characterized in that: The fermented product is obtained by culturing the Lactococcus lactis strain according to any one of claims 1 to 9 or fermenting the bacterial agent according to claim 12.

14. The fermentation product according to claim 13, characterized in that The fermentation product contains a xanthine oxidase specific activity of 3.73-4.30 U / g, measured in micromoles of xanthine consumed per milligram of protein in the fermentation product per minute; and / or a urate oxidase specific activity of 7.78-9.22 U / g, measured in micromoles of uric acid consumed per milligram of protein in the fermentation product per minute.

15. Use of the Lactococcus lactis strain according to any one of claims 1 to 9 or the bacterial agent according to claim 12 for reducing the purine content in food.

16. The use according to claim 15, characterized in that The food is a food containing yeast protein.

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