A strain with purine-reducing function and its application

Abstract

The present invention belongs to the field of functional microbial screening and application technology, and specifically relates to a strain with the function of reducing purine and its application. The present invention provides Lactococcus lactis AMCC 11671 (Lactococcus lactis AMCC 11671), which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2024078. The Lactococcus lactis AMCC 11671 provided by the present invention has good crude enzyme activity and can effectively slow the production and accumulation of uric acid by reducing purine content and degrading uric acid. At the same time, it has good tolerance to acid and bile salts, has the ability to withstand the gastrointestinal environment, and has the potential to colonize and function in the human gastrointestinal tract.

Description

Technical Field

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

[0002] Hyperuricemia and gout are caused by disorders of purine metabolism and uric acid excretion in the human body, and have become the second most common metabolic disease after diabetes. Purines in the human body are derived 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). Their metabolic pathways in the human body include de novo synthesis and salvage synthesis pathways. De novo synthesis involves the synthesis of IMP from phosphoribosylpyrophosphate synthetase (PRPPS) using ribose phosphate, glutamine, glycine, aspartate, and carbon dioxide as raw materials. This is then converted to AMP and GMP. Further metabolism results in IMP, GMP, and AMP forming hypoxanthine, guanine, and adenine, respectively. Guanine is then deaminated to xanthine. Hypoxanthine and xanthine are metabolized to uric acid by xanthine oxidase (XO). Salvage synthesis is a secondary pathway of purine metabolism, utilizing free purines or purine nucleosides in the body through simple reactions to produce nucleotides with minimal energy consumption. However, with the continuous evolution of humans, the gradual loss of urate oxidase has prevented the further oxidation of uric acid to allantoin. Consequently, uric acid becomes the final product of purine metabolism in the human body. Furthermore, with improvements in quality of life and economic well-being, people are embracing a high-purine diet. Long-term consumption of high-purine foods leads to a significant accumulation of uric acid in the body, resulting in hyperuricemia and, in severe cases, even 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 leads to kidney disease and other metabolic syndromes (including hypertension, hyperlipidemia, diabetes, and coronary heart disease). Currently, the main strategies to alleviate 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] Lactic acid bacteria are a type of 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 purines.

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

[0005] Chinese patent application publication number CN115725437A discloses a strain of fermentative lactobacillus that efficiently utilizes purine and its application in rice wine brewing. The application of this strain in rice wine brewing shortens the fermentation cycle of rice wine and reduces 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, based on people’s current living and eating habits, in order to more effectively protect people’s health and disease prevention, the development of low-cost functional lactic acid bacteria for use in functional foods to prevent metabolic rheumatoid diseases has important social significance. Summary of the Invention

[0007] The present invention addresses the following issues: Prior art methods for alleviating and treating hyperuricemia and gout through medication often suffer from varying degrees of toxic side effects, posing certain risks to the human body. Furthermore, prior art methods for alleviating and treating hyperuricemia and gout by reducing the intake of exogenous purine compounds and lowering purine levels in the body are difficult to achieve through dietary control, as the body requires abundant nutrients necessary for normal physiological metabolism, as well as purines, are present in most foods.

[0008] The purpose of the present invention is to provide a strain with the function of reducing purine, and to apply it to the production of multifunctional probiotics and health foods for auxiliary 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 the 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, based on 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, based on 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 based on the ratio of bile salt weight to volume of liquid culture medium (w / v).

[0019] In a second aspect, the present invention provides a method for preparing a Lactococcus lactis inoculum 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) Add the product obtained in step (1) to a liquid culture medium and ferment and culture 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 enzymatic activity of 3.73-4.30 U / g, measured in micromoles of xanthine consumed per milligram of protein per minute in the fermentation product; and / or a urate oxidase specific enzymatic activity of 7.78-9.22 U / g, measured in micromoles of uric acid consumed per milligram of protein per minute in the fermentation product.

[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 purine-lowering Lactococcus lactis AMCC 11671 obtained by screening and isolating tea leaves from the Guangxi Zhuang Autonomous Region for the first time. It is a food-derived lactic acid bacterium and is suitable for fermenting 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 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 withstand the gastrointestinal environment, and has the potential to colonize and function in the human gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the Gram staining result of Lactococcus lactis AMCC 11671.

[0034] Figure 2 The results of xanthine oxidase and urate oxidase activity determination.

[0035] Figure 3 This is a high performance liquid chromatogram showing the degradation of purine compounds and uric acid by the strain of Example 3.

[0036] Culture collection information

[0037] The 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. The deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: 027-68754052. DETAILED DESCRIPTION

[0038] The Lactococcus lactis AMCC 11671 strain provided by the present invention was isolated, purified, and identified from tea leaves grown in the Guangxi Zhuang Autonomous Region. The strain exhibits morphological characteristics: smooth, milky white colonies, facultative anaerobic growth, a temperature of 30-37°C, and a pH of 5-6. It is spherical or oval under a microscope, Gram-positive, and lacks capsules or spores. The 16S rDNA gene sequence of Lactococcus lactis AMCC 11671 is shown in SEQ ID NO. 3. This 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 commercial 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 adjusted 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. The mixture was sterilized by autoclaving at 121°C for 15 min.

[0047] (2) M17 liquid medium: Mix 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. Add distilled water to make up to 1 L. The pH value was 7.2 ± 0.2 at 25°C. When preparing the solid medium, add 15 g of agar. Autoclave at 121°C for 15 min. When using, add a glucose solution to a final concentration of 1% (w / v).

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

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

[0050] Take 2g of tea leaves, cut them into pieces and add them to a blue-mouth bottle containing 20mL of sterile water. Incubate at 37℃ for 2 days to obtain the sample to be separated. Take 100μL of the sample to be separated and add 900μL of sterile water to dilute it. Shake and mix it. Use sterile water to make a 10-fold gradient dilution to obtain 10 -1 -10 -5 A series of dilutions were performed, and 200 μL of each sample was spread onto MRS solid medium containing 2% CaCO₃ and incubated anaerobically at 37°C for 36 hours. Three candidate strains were ultimately isolated from the sample. Microscopic examination revealed spherical bacterial morphology and preliminary identification as Lactococcus lactis. Therefore, the MRS solid medium containing 2% CaCO₃ was replaced with M17 liquid medium for subsequent incubation. The three candidate strains were named AR214, AR215, and AR216. Strains AR214, AR215, and AR216 were purified by plate streaking, and single colonies were picked and cultured in M17 liquid medium at 37°C for 16 h to obtain a bacterial solution, which was mixed with 30% (W / V) glycerol at a ratio of 1:1 and frozen at -80°C. 30% (W / V) glycerol was prepared by weighing 30 g of glycerol and dissolving it in ultrapure water, then making the volume to 100 ml. The solution was sterilized at 121°C for 15 min and stored at room temperature for use.

[0051] 2. Preliminary Screening of Purine-Degrading Strains

[0052] 1. In vitro metabolic enzyme activity screening of xanthine oxidase (XOD) and urate oxidase (UOX). The solutions used in the reaction system were 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 dilute 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 the volume 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 then use it 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 then 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 then 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 dilute 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) was performed using the following PBS buffer solution:

[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 sodium hydrogen phosphate, dissolve it in an appropriate amount of ultrapure water, and dilute to 100 mL to prepare a sodium 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 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 XOD and UOX enzyme activities are as follows:

[0066] (1) Preparation of crude enzyme solution to be tested: strains AR214, AR215, and AR216 frozen at -80°C were thawed, streaked, and cultured in a 37°C constant temperature incubator for 16 hours. Single colonies were picked and placed in 10 mL of M17 liquid medium. The seed solution was cultured at 37°C to obtain a seed solution. 1% by volume was inoculated into 800 mL of M17 liquid medium. After culture at 37°C for 16 hours, the bacterial solution was centrifuged at 4°C and 12,000 rpm for 15 minutes. The supernatant was removed and the bacterial cells were retained. 25 mL of PBS buffer solution with a pH of 7.5 was added to the bacterial cells, and the cells were resuspended and washed twice. After resuspending with 25 mL of PBS, 2.5 g of quartz sand and 0.5 g of glass beads were added. The cells were lysed 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℃ 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 enzyme labeling 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 enzyme labeling plate to start the reaction. 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 enzyme reader to calculate the production 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 enzyme-labeled 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 enzyme-labeled plate to start the reaction. 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 an infinite M200 PRO enzyme reader to calculate the amount of uric acid reduction.

[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 content of strain AR214, strain AR215 and strain AR216 was 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 changes in absorbance at 295 nm, a wavelength characteristic of uric acid, were used to analyze whether the crude enzyme solution tested possessed XOD and UOX enzyme activities. The absolute enzyme activities of XOD and UOX for each of the three strains were calculated using the SLOPE function. If uric acid was produced, the crude enzyme solution tested contained XOD activity, which oxidized xanthine to uric acid. If uric acid was reduced, the crude enzyme solution tested contained UOX activity, which oxidized uric acid to allantoin. The absolute XOD activities of strains AR214, AR215, and AR216 were 9.24 U / L, 15.3 U / L, and 24.17 U / L, respectively, and the absolute UOX activities were 22.96 U / L, 47.43 U / L, and 75.87 U / L, respectively.

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

[0078] Calculate the XOD and UOX specific enzyme activities of the crude enzyme solution to be tested (strain AR214, strain AR215 and strain AR216). Among them, the XOD specific enzyme activity is expressed as the micromoles of xanthine consumed per milligram of protein per minute, and the UOX specific enzyme activity is expressed as the micromoles of uric acid consumed per milligram of protein per minute. The results of XOD and UOX specific enzyme activity determination are shown in Figure 2. Figure 2 As shown, the results showed that strains AR214, AR215, and AR216 all exhibited varying XOD and UOX specific enzyme activities. The XOD specific enzyme activities of strains AR214, AR215, and AR216 were similar, with AR216 having a slightly higher XOD specific enzyme activity than both strains. However, AR216 had the highest UOX specific enzyme activity, significantly higher than that of the other two strains. Therefore, based on the XOD and UOX specific enzyme activities, we can preliminarily conclude that strain AR216 possesses excellent uric acid degradation capabilities.

[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 in M17 liquid medium for 16 h, and their genomes were extracted. The genomic DNAs were amplified by PCR and sequenced using bacterial universal primers 27F / 1492R for 16S rDNA. The 16S rDNA sequences of the selected strains were compared against the NCBI database by BLAST. The strains AR214, AR215, and AR216 all had a similarity of 99.93% with Lactococcus lactis, indicating that the strains belonged to Lactococcus lactis. The 16S rDNA gene sequence of strain AR214, the 16S rDNA gene sequence of strain AR215, and the 16S rDNA gene sequence of strain AR216 were shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0082] SEQ ID NO.1:

[0083]

[0084] SEQ ID NO.2:

[0085]

[0086] SEQ ID NO.3:

[0087]

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

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

[0090] Strain AR216 was streaked onto an M17 plate until a single colony formed. A single colony was selected for Gram staining and a catalase assay. Catalase assay steps: An appropriate amount of bacteria was spread onto a clean glass slide and a 3% hydrogen peroxide solution was added. Observe for the formation of bubbles; if bubbles were present, the reaction was positive; otherwise, it was negative. The results of the present assay were negative.

[0091] Morphological characteristics of strain AR216: The colony texture is smooth, milky white, and single colonies appear spherical or oval under a microscope, are 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 dilute 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 use it to make up 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 it 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 dilute 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 100mM PBS buffer solution, wherein:

[0105] Sodium dihydrogen phosphate: Weigh 1.56 g of sodium dihydrogen phosphate, dissolve it in water, and dilute 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 an appropriate amount of water, and then dilute 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, and adjust the pH to 4.7 with 4 M phosphoric acid or 5 M NaOH to prepare a 10 mM sodium dihydrogen phosphate solution, which is the mobile phase.

[0109] 4. The steps for 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 to purines and the main source of purines in the body. Purines accumulate and are oxidized to uric acid through a series of metabolic pathways. To screen for strains capable of degrading 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 using a Waters e2695 liquid chromatograph equipped with a Waters 2998PDA photodiode matrix chromatographic detector to explore the ability of Lactococcus lactis AMCC 11671 to degrade 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. 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 were taken in sequence and mixed 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 the above samples were used to prepare 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 was inoculated at a 2% volume ratio into 200 mL of M17 liquid culture medium. The culture was incubated in a 37°C incubator for 16 h, and then centrifuged at 4°C, 6000 rpm, for 3 min to collect the cells. The cells were washed twice with 25 mL of PBS solution and 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) Each reaction solution in step 4 (3) of this example was placed in a 37°C incubator and incubated with shaking at 160 rpm for 60 min. After the incubation, the solution was centrifuged at 4°C and 6000 rpm for 3 min. 2 mL of the supernatant was collected 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 analyzed according to HPLC chromatography conditions. The HPLC chromatography 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 on the 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 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, 600 μM, 800 μM, 1000 μM and 1200 μM, respectively. Specifically, a 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 was Y = 7017.5X-250106, R2 = 0.9961;

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

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

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

[0128] The standard curve for 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 to calculate the residual purine or uric acid content (μM) in the reaction solution in step 4 (3) of this example, 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 purine compound or uric acid content (μM) in the test bacterial solution, and X represents the residual purine compound or uric acid content (μM) in the reaction solution in step 4 (3) of this example.

[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. This shows 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 guanine degradation; 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) is a HPLC chromatogram of uric acid reduction. Analysis of the HPLC chromatogram shows that inosine reacts with hypoxanthine, which undergoes a series of oxidation reactions with XOD to produce uric acid, which is then degraded to allantoin via UOX. Guanosine reacts with guanine, which undergoes a series of XOD reactions to produce uric acid, which is further degraded to allantoin. Furthermore, each purine compound undergoes a certain degree of degradation after the reaction, and a certain amount of allantoin is produced. This confirms that Lactococcus lactis AMCC 11671 has a good purine-reducing ability. Based on this, we further explored the acid and bile salt resistance of Lactococcus lactis AMCC 11671.

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

[0134] 1. Preparation of the test bacterial solution: Lactococcus lactis AMCC 11671 bacterial solution was inoculated at 1% by volume into freshly prepared M17 liquid medium for activation, and cultured at 37° C. until the strain reached the growth plateau phase.

[0135] 2. Acid tolerance assay: Use 4 mol / L HCl and 5 mol / L NaOH to prepare fresh M17 liquid medium with pH values ​​of 1.5, 2.5, and 3.5, respectively. Take 1 mL of the test bacterial solution and centrifuge to collect the bacteria. The centrifugation conditions are 4°C, 8000 r / min, and 5 mins. The bacterial pellet is washed twice with 1 mL of 0.9% NaCl. Then, the bacteria are transferred to 1 mL of fresh M17 liquid medium with pH values ​​of 1.5, 2.5, and 3.5, respectively, and mixed. Incubate at 37°C for 3 hours. Measure the OD600 value of the reaction solution in each experimental group. The survival rate is calculated 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] Because the pH of the human gastrointestinal tract after ingestion is approximately 3.0, Lactococcus lactis AMCC 11671 must survive in this environment for 1.5-2.0 hours to exert its efficacy. Table 6 shows the survival rate of acid-tolerant Lactococcus lactis AMCC 11671. The results show that the survival rate of the strain decreases with decreasing pH. Despite this, the survival rate of Lactococcus lactis AMCC 11671 after incubation for 3 hours at pH 1.5, 2.5, and 3.5 remained greater than 73%. These results indicate that Lactococcus lactis AMCC 11671 can serve as a probiotic and has the potential to colonize 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 1 mL of the test bacterial solution and centrifuge to collect the bacteria. The centrifugation conditions are 4°C, 8000r / min, and 5mins. Wash the bacterial pellet twice with 1 mL of 0.9% NaCl. Then transfer the bacteria to 1 mL of 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 in the experimental group; A0 represents the OD600 value of the bacterial solution to be tested.

[0145] Table 7 shows the survival rate of the bile salt-tolerant Lactococcus lactis AMCC 11671 strain. The results indicate that the survival rate of Lactococcus lactis AMCC 11671 decreased slightly with increasing bile salt concentration, but remained above 94%. The bile salt concentration in the human small intestine ranges from 0.03% to 0.3%. These results further confirm that Lactococcus lactis AMCC 11671 has the potential to colonize the human gastrointestinal tract.

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

[0147]

[0148] Application example: Degradation of purine compounds in yeast protein by Lactococcus lactis AMCC 11671

[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 to 100 mL in a 50 mL volumetric flask. Mix well. If it does not dissolve, add an appropriate amount of sodium hydroxide solution (1 mol / L) to assist dissolution 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 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 respectively drawn, and 5 mL of trifluoroacetic acid and 5 mL of formic acid were added thereto, respectively. The mixture was then pyrolyzed in an 85°C water bath for 15 min. After rapid cooling, the mixture was rotary evaporated to dryness at 80°C, and reconstituted with 10 mL of mobile phase (shown in Table 8). The mixture was centrifuged at 6000 r / min for 5 min. 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 suspension: Inoculate 100 mL of M17 liquid medium at a 2% inoculum volume of the Lactococcus lactis AMCC 11671 bacterial suspension. Incubate in a 37°C constant temperature incubator for 16 hours, then centrifuge at 6000 rpm for 3 minutes at 4°C to collect the cells. Wash the cells twice with 25 mL of PBS solution and resuspend them in 2 mL of PBS solution. The Lactococcus lactis AMCC 11671 bacterial suspension is 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 bath (40 Hz) and extract by ultrasonication for 30 mins to obtain a yeast protein solution.

[0156] 4. Preparation of test solution:

[0157] (1) Preparation of the negative control test solution: 1 mL of Lactococcus lactis AMCC 11671 bacterial suspension and 1 mL of yeast protein solution containing free purines were mixed and placed in a 37°C shaker incubator (160 rpm) for 0 h. 4 mL of trifluoroacetic acid and 4 mL of formic acid were added, mixed, and then pyrolyzed in an 85°C water bath for 15 min. After rapid cooling, the mixture was rotary evaporated at 80°C until nearly dry. The mixture was reconstituted with 10 mL of mobile phase (as shown in Table 8), centrifuged at 10,000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm organic microporous filter membrane to obtain the negative control test solution.

[0158] (2) Preparation of the test solution for the experimental group: 1 mL of Lactococcus lactis AMCC 11671 bacterial suspension and 1 mL of yeast protein solution containing free purines were mixed and incubated in a 37°C shaker incubator (160 rpm) for 2 h. 4 mL of trifluoroacetic acid and 4 mL of formic acid were added, mixed, and then pyrolyzed in an 85°C water bath for 15 min. After rapid cooling, the mixture was rotary evaporated at 80°C to near dryness, reconstituted with 10 mL of mobile phase (as shown in Table 8), centrifuged at 10,000 rpm for 5 min, and the supernatant was collected and filtered through a 0.22 μm organic microporous filter membrane to obtain the test solution for 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 the 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, a linear fitting was performed using the different final concentrations (x) of each purine standard solution as the abscissa and the corresponding peak area as the ordinate y to draw standard curves for adenine, guanine, xanthine, and hypoxanthine, wherein:

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

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

[0167] The standard curve for 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 to calculate the concentration of purine compounds in the negative control group and the experimental group, respectively. The concentrations 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 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 was 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 constant volume of the yeast protein solution (L), m represents the weighed amount of yeast protein powder (g), and 100 is the unit conversion factor.

[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] Calculations using Formulas 4 and 5 show that after 0 hours of reaction between Lactococcus lactis AMCC 11671 and the yeast protein solution, the total purine content in the sample was 2902.37 mg / 100 g. However, after 2 hours of reaction, the total purine content in the sample was 1867.37 mg / 100 g, a decrease of 35.66%. This indicates 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 ability to reduce 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-based foods.

[0178] The above description is merely 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 scope of protection of the present invention.

Claims

1. A strain of Lactococcus lactis ( Lactococcus lactis ) strain, characterized in that The Lactococcus lactis strain is Lactococcus lactis AMCC 11671, which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCCNO: M 2024078.

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

3. The Lactococcus lactis strain according to claim 2, characterized in that Based on mass, the degradation rate of purine compounds by the Lactococcus lactis AMCC 11671 is 25.06%-93.61%.

4. The Lactococcus lactis strain according to claim 2, characterized in that The purine compound includes one or more of inosine, guanosine, hypoxanthine, guanine and xanthine.

5. The Lactococcus lactis strain according to claim 3, characterized in that The purine compound includes one or more of inosine, guanosine, hypoxanthine, guanine and xanthine.

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

7. The Lactococcus lactis strain according to claim 1, 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%.

8. The Lactococcus lactis strain according to claim 1, characterized in that The Lactococcus lactis AMCC 11671 also has a uric acid degradation rate of more than 10% by mass.

9. The Lactococcus lactis strain according to claim 1, characterized in that Calculated by mass, the uric acid degradation rate of the Lactococcus lactis AMCC 11671 is 9.82-12.77%.

10. The Lactococcus lactis strain according to any one of claims 1 to 9, characterized in that The 16S rDNA gene sequence of the Lactococcus lactis strain is shown in SEQ ID NO.

3.

11. The Lactococcus lactis strain according to any one of claims 1 to 9, 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%.

12. The Lactococcus lactis strain according to any one of claims 1 to 9, characterized in that Calculated on the basis of the ratio (w / v) of bile salt weight to volume of liquid culture medium, 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%.

13. A fermentation method for preparing a Lactococcus lactis inoculum, characterized in that: The method comprises the following steps: culturing the Lactococcus lactis strain according to any one of claims 1 to 12.

14. The preparation method according to claim 13, characterized in that The preparation method comprises the following steps: (1) amplifying and culturing the Lactococcus lactis strain; (2) Add the seed solution obtained in step (1) to the liquid culture medium and ferment and culture at 30-37°C.

15. A bacterial agent, characterized in that The bacterial agent is obtained by the fermentation preparation method according to claim 13 or 14.

16. 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 12 or fermenting the bacterial agent according to claim 15, wherein the fermented product contains the Lactococcus lactis cells themselves.

17. The fermentation product according to claim 16, 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 per minute in the fermentation product; 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 per minute in the fermentation product.

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

19. The use according to claim 18, characterized in that The food is a food containing yeast protein.

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