Lactococcus lactis subsp. Lactis VB346 capable of efficiently degrading purine and application thereof

Abstract

The invention relates to the field of microorganisms, and discloses a Lactococcus lactis subsp. Lactis VB346 which is preserved in the China Center for Type Culture Collection on September 20, 2024, and the preservation number is CCTCC NO: M 20242013. The invention further discloses a preparation method of the Lactococcus lactis subsp. Lactis VB346. The lactococcus lactis subsp. Lactis VB346 disclosed by the invention has the capability of efficiently degrading purine nucleoside, and can decompose purine nucleoside in ingested food in intestinal tracts, so that the absorption amount of purine nucleoside by small intestines is reduced. The polypeptide has the ability of degrading bilirubin, and can be used for treating or improving hyperbilirubinemia, or improving liver and gall injury caused by hyperbilirubin; meanwhile, the acid resistance required by survival in the stomach, the cholate tolerance required by survival in the intestinal tract and the stable survival ability in the intestinal tract are achieved, and the bacillus subtilis has huge application prospects in preparation of drugs, microbial agents, health care products or food.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to Lactococcus lactis subspecies VB346 and applications thereof. Background Art

[0002] Purine is a precursor of uric acid, which mainly exists in the form of purine nucleotides and purine nucleosides in the body, and further forms hypoxanthine and xanthine, which are oxidized and metabolized into uric acid. When the human body consumes too much purine or purine metabolism is disordered, the uric acid level in the blood will increase, causing the metabolic disease hyperuricemia. Increased blood uric acid will crystallize and deposit in the joints, renal pelvis, ureters, renal interstitium and other parts, causing gout, urate nephropathy, uric acid urinary stones and other diseases. In severe cases, it will damage the joints and cause renal insufficiency. In addition, hyperuricemia is often complicated by hypertension, non-alcoholic fatty liver disease, cardiovascular disease, and diabetes.

[0003] At present, drugs for treating hyperuricemia are mainly divided into three categories: drugs that inhibit uric acid production, drugs that promote uric acid excretion, and drugs that promote uric acid oxidase. Commonly used drugs in clinical practice, such as allopurinol and febuxostat, mainly reduce the production of uric acid by inhibiting xanthine oxidase, and benzbromarone and probenecid mainly relieve hyperuricemia by promoting uric acid excretion. However, drugs generally have problems such as allergic reactions and toxic side effects. In addition to drugs, the treatment and prevention of hyperuricemia also requires controlling the intake of high-purine foods, such as beer, meat, animal offal, and seafood, but controlling the diet will make it difficult to maintain the balance of nutrients provided by food, and people will not be able to enjoy the pleasure brought by food, resulting in a decline in quality of life. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0005] To this end, the first aspect of the present invention provides a Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346, the Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346 was deposited in the China Center for Type Culture Collection on September 20, 2024, with the deposit number CCTCC NO: M 20242013.

[0006] According to the embodiment of the present invention, the strain has the ability to efficiently degrade purine nucleosides. The strain can decompose the purine nucleosides in the ingested food in the intestine, thereby reducing the absorption of purine nucleosides by the small intestine.

[0007] In a preferred embodiment of the present invention, the purine nucleoside is preferably adenine.

[0008] According to an embodiment of the present invention, this strain has the ability to degrade bilirubin and can be used to treat or improve hyperbilirubinemia or to improve liver and gallbladder damage caused by hyperbilirubin.

[0009] According to an embodiment of the present invention, this strain has good tolerance within the normal acidic range of the human stomach and has the acid tolerance ability required for survival in the stomach.

[0010] According to an embodiment of the present invention, this strain has good tolerance within the normal concentration range of small intestine choline and has the bile salt tolerance ability required for survival in the intestine.

[0011] According to an embodiment of the present invention, this strain shows relatively strong survival ability in simulated intestinal fluid and has the ability to stably survive in the intestine to play a probiotic role.

[0012] The second aspect of the present invention provides the use of the Lactococcus lactis subsp. lactis as described above in the preparation of a medicament for preventing or treating hyperuricemia and / or hyperbilirubinemia.

[0013] According to an embodiment of the present invention, the medicament further contains excipients and / or carriers.

[0014] According to an embodiment of the present invention, the excipients include at least one selected from binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, sustained-release agents, etc.

[0015] According to an embodiment of the present invention, the carriers include at least one selected from saccharides, cellulose and its derivatives, calcium phosphates, alkaline earth metal salts of stearic acid, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, hydrolyzed cereal solids, etc.

[0016] According to an embodiment of the present invention, the dosage form of the medicament includes at least one selected from oral liquids, powders, granules, capsules, tablets, dripping pills, etc.

[0017] According to an embodiment of the present invention, when Lactococcus lactis subsp. lactis VB346 exerts its effect, it can exist in the form of live cells or in the form of non-live cells.

[0018] According to an embodiment of the present invention, the live cells refer to cells with the ability of metabolism, reproduction or replication, and the non-live cells refer to cells without the ability of metabolism, reproduction and replication, including but not limited to dried bacterial cells (such as freeze-dried powder).

[0019] The present invention provides a microbial agent, and the microbial agent contains the aforementioned Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) VB346. The microbial agent of the present invention is used for preventing or treating hyperuricemia and / or hyperbilirubinemia.

[0020] The present invention also provides the use of the foregoing Lactococcus lactis subsp. lactis VB346 in the preparation of health products and foods for improving hyperuricemia and / or hyperbilirubinemia.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Bilirubin degradation activity of Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) strain VB346; Figure 2 : Detection result graph of acid resistance of Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) strain VB346; Figure 3 : Detection result graph of bile salt tolerance of Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) strain VB346, where different percentages are the amounts of bile salt added; Figure 4 : Detection result graph of the tolerance of Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) strain VB346 to artificial intestinal fluid. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The embodiments of the present invention will be described in detail below, and the examples of the embodiments are shown in the drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0024] It should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise specified, the meaning of "a plurality" is two or more.

[0025] In this article, the terms "containing", "comprising" or "including" are open expressions, that is, including the content specified by the present invention, but not excluding other aspects of the content.

[0026] In this article, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where the events or conditions occur and the cases where the events or conditions do not occur.

[0027] In this text, "prevention" and "prevention of" are used interchangeably. These terms refer to methods of obtaining a beneficial or desired result, including but not limited to prophylactic benefits. To obtain a "prophylactic benefit", Lactococcus lactis subsp. lactis or a product containing it may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even if the diagnosis of the disease may not have been made.

[0028] In this text, the terms "treatment" and "alleviation" both refer to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein encompasses diseases in mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease. "Treatment" as used herein encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the disease of the individual, including but not limited to administering a drug containing the compound described herein to an individual in need.

[0029] In this text, "acceptable in health foods" refers to substances or compositions that can be consumed by humans and can be adjusted according to the requirements of health foods in different countries.

[0030] In this text, "pharmaceutically acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with the other components of the formulation and / or the mammal being treated therewith. Preferably, "pharmaceutically acceptable" as used in the present invention refers to those approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopeias for use in animals, particularly in humans.

[0031] In this text, the term "pharmaceutically acceptable carrier" includes any solvent, drug stabilizer, or composition thereof, which are known to those skilled in the art. Its use in a therapeutic or pharmaceutical composition is encompassed, except in cases where any conventional carrier is incompatible with the active ingredient.

[0032] In this text, the term "pharmaceutically acceptable excipients" may include saccharides, which include monosaccharides or polysaccharides, such as lactose, sucrose, mannitol, and sorbitol; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal salts of stearic acid, such as magnesium stearate and calcium stearate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; nonionic surfactants, cationic surfactants, anionic surfactants; ethylene glycol polymers; fatty alcohols; and hydrolyzed cereal solids, as well as other non-toxic and compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, and other excipients commonly used in pharmaceutical preparations.

[0033] According to a specific embodiment of the present invention, the present invention provides a Lactococcus lactis subsp. lactis Lactococcus lactis subsp. Lactis VB346. According to the examples of the present invention, the preservation number of the Lactococcus lactis subsp. lactis is CCTCC NO: M 20242013. The Lactococcus lactis subsp. lactis of the present invention has a significant ability to degrade purine nucleosides and can be used to prepare drugs for maintaining intestinal health. It also has strong abilities to degrade bilirubin, resist gastric acid, resist choline, and tolerate the intestine, showing great application prospects in the preparation of pharmaceuticals.

[0034] In this text, "Lactococcus lactis subsp. lactis Lactococcus lactis subsp. Lactis VB346", "Lactococcus lactis subsp. lactis VB346", "VB346", and "strain VB346" are synonymous.

[0035] According to a specific embodiment of the present invention, the present invention provides a fermentation supernatant, which contains Lactococcus lactis subsp. lactis VB346 and / or metabolites of Lactococcus lactis subsp. lactis VB346.

[0036] It should be noted that the "fermentation supernatant" refers to the supernatant after centrifugation of the fermentation broth, which may contain Lactococcus lactis subsp. lactis VB346, or metabolites of Lactococcus lactis subsp. lactis VB346, or both Lactococcus lactis subsp. lactis VB346 and its metabolites.

[0037] According to a specific embodiment of the present invention, the present invention provides a microbial agent, which contains the aforementioned Lactococcus lactis subsp. lactis VB346 and / or the aforementioned fermentation supernatant.

[0038] It should be noted that the microbial agent of the present invention can be a liquid microbial agent, including but not limited to fermentation broth, etc.; or a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0039] According to an embodiment of the present invention, Lactococcus lactis subsp. lactis VB346 exists in the form of live cells and / or inactivated cells.

[0040] As used herein, "live cells" refer to cells having the ability to metabolize, reproduce, or replicate.

[0041] Exemplarily, the live cells may be immobilized cells. As used herein, "immobilized cells" refer to Lactococcus lactis subsp. lactis VB346 immobilized on a carrier and capable of carrying out life activities such as growth, development, reproduction, inheritance, and metabolism within a certain spatial range.

[0042] As used herein, "inactivated cells" refer to cells that do not have the ability to metabolize, reproduce, and replicate, including but not limited to dried bacterial cells. Exemplarily, the microbial inoculant is freeze-dried powder.

[0043] As a specific embodiment, Lactococcus lactis subsp. lactis VB346 exists in the form of live cells, dried bacterial cells, immobilized cells, or in any other form.

[0044] As a specific embodiment, the dried bacterial cells are obtained by freeze-drying Lactococcus lactis subsp. lactis VB346.

[0045] According to an embodiment of the present invention, the present invention provides a drug, health product, or food containing Lactococcus lactis subsp. lactis VB346. It should be noted that the drug, health product, or food of the present application may be various preparations of separate active Lactococcus lactis subsp. lactis VB346, or may be used in combination with other active ingredients, as long as they do not affect each other's activity; further, the best case is that the active ingredients can have complementary functions or promoting effects on each other. For example, Lactococcus lactis subsp. lactis VB346 can be combined with other probiotics to form a compound probiotic tablet to achieve better or more active functions. Specifically, it can be determined according to the activity and components of the compound probiotic tablet, which is not limited herein. Optionally, the drug, health product, or food further includes a carrier or excipient acceptable in drug, health product, or food development. It should be noted that when preparing live bacterial preparations, carriers or excipients are usually added, as long as the added carriers or excipients do not inhibit each other or have adverse side effects with Lactococcus lactis subsp. lactis VB346. The drug, health product, or food can be in the form of powder, tablet, beverage, or capsule.

[0046] In this article, hyperuricemia refers to a state of elevated uric acid concentration in the blood, usually serum uric acid levels exceeding the normal range (usually >7.0 mg / dL for men and >6.0 mg / dL for women). Uric acid is the final product of purine metabolism and is mainly excreted through the kidneys. Hyperuricemia itself usually has no obvious symptoms, but if uric acid levels continue to rise, it may cause the following diseases and symptoms: gout, uric acid stones, and kidney damage.

[0047] In this article, hyperbilirubinemia refers to a condition in which the level of bilirubin in the blood is elevated. Bilirubin is a yellow pigment produced during the breakdown of red blood cells. Normally, it is metabolized in the liver and converted into bile, which is then excreted into the intestines. When there is an abnormality in the production, metabolism, or excretion of bilirubin, the level of bilirubin in the blood increases, leading to hyperbilirubinemia. Hyperbilirubinemia may cause the following diseases and symptoms: obstructive jaundice, hepatocellular jaundice, liver cancer, pancreatic head cancer, cholelithiasis, bile duct cancer, hepatitis, etc.

[0048] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0049] Example 1: Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )Isolation, screening, purification and identification of VB346 strain.

[0050] 1. Isolation of lactic acid bacteria The strain was collected and isolated from the intestine of a healthy human. Different gradients of dilution suspensions were prepared by gradient dilution with sterile saline solution. 100 μL of each dilution was spread on MRS agar plates and cultured anaerobically at 37°C for 48 h. Suspected lactic acid bacteria colonies were picked and streaked for purification and DNA was extracted. 16S rDNA was sequenced after PCR amplification with specific primers, and the corresponding strains were stored in glycerol tubes. Six strains of Lactococcus lactis were selected based on the 16S rDNA identification results, numbered 000567, 000609, 000630, VB346, 000679 and 000695.

[0051] 2. Screening of lactic acid bacteria strains Preparation of MRS-purine medium: Weigh 80 mg of adenine (Shanghai Macklin Biochemical Co., Ltd.), dissolve it in 200 mL of pure water to make an adenine solution; weigh 80 mg of hypoxanthine (Shanghai Macklin Biochemical Co., Ltd.), dissolve it in 200 mL of pure water to make a hypoxanthine solution; weigh 80 mg of xanthine (Shanghai Macklin Biochemical Co., Ltd.), add 1 mL of sodium hydroxide solution with pH = 13, and dissolve it by ultrasonic to make a xanthine solution; weigh 80 mg of guanine (Shanghai Macklin Biochemical Co., Ltd.), add 1 mL of sodium hydroxide solution with pH around 13, and dissolve it by ultrasonic to make a guanine solution. Weigh 54 g of MRS finished medium (Guangdong Huankai Microbial Science and Technology Co., Ltd.), add 200 mL each of adenine and hypoxanthine solutions, 1 mL of xanthine solution, and 1 mL of guanine solution, make up the volume to 1 L with pure water. There is no turbidity. The final concentration of the four purines is 80 μg / mL, the final pH of the medium is 6.6, sterilize at 121 °C for 15 minutes, and keep it for use.

[0052] Strain culture: Take the glycerol tubes of the six different Lactococcus lactis strains isolated above, melt them at room temperature, and then inoculate them on MRS slants. Incubate anaerobically at 37 °C for 1 day to obtain slant bacterial colonies. Scrape a 2 cm × 2 cm sized bacterial colony into 20 mL of MRS-purine medium, and incubate anaerobically at 37 °C for 20 - 24 hours.

[0053] Detection of free purines: Take 1 mL of the cultured bacterial liquid, centrifuge it at 14000 rpm for 10 minutes, take the supernatant, and detect the concentration of free purines by HPLC method.

[0054] Detection of total purine concentration: Take the remaining bacterial liquid, centrifuge it at 3500 rpm for 10 min, take 5 mL of the supernatant, mix it with 2.5 mL of trifluoroacetic acid and 2.5 mL of formic acid, place it in a water bath at 90 °C and boil for 12 min, cool it to room temperature in an ice bath, neutralize it to pH = 7.0 with 15 mol / L potassium hydroxide (weigh 42 g of potassium hydroxide, add 30 mL of pure water to dissolve), then adjust the pH to 3.0 with phosphoric acid, centrifuge it at 14000 rpm for 10 minutes, take the supernatant, and detect the total purine concentration by HPLC method.

[0055] Calculate the degradation rates of adenine, hypoxanthine, xanthine, and guanine respectively according to the following formula: Degradation rate = (C 0 - C 1 ) ÷ C 0 × 100%, where C 0 is the concentration of total purines, and C 1 is the concentration of free purines (the concentration unit is g / L).

[0056] HPLC detection conditions: Ultimate AQ - C18 (5 μm, 4.6×250 mm); flow rate: 1.0 mL / min; column temperature: 40°C; injection volume: 5 μL, 13 min; mobile phase A: 0.1% formic acid solution, mobile phase B: acetonitrile.

[0057] Table 1: HPLC Mobile Phase A: Mobile Phase B (V / V)

[0058] Table 2 Degradation Rates of Four Purines by Different Lactococcus lactis Strains

[0059] The degradation rates of four purines by different Lactococcus lactis strains were tested. Among them, strain VB346 had a 100% degradation rate for adenine.

[0060] 3. Strain Identification The Lactococcus lactis subsp. VB346 screened in Example 1 was sequenced, and the molecular biological identification 16S rRNA sequence of the strain was compared with the strains in GenBank. It was identified as Lactococcus lactis subsp. ( Lactococcus lactis subsp. Lactis ), named Lactococcus lactis subsp. ( Lactococcus lactis subsp. Lactis ) VB346. It was deposited at the China Center for Type Culture Collection on September 20, 2024. The deposit address is Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The deposit number is CCTCC NO: M 20242013. The 16S rRNA sequence information is as follows: Example 2: Characteristics of Lactococcus lactis subsp. lactis VB346 in reducing bilirubin Weigh 50 mg of bilirubin (Shanghai Yuanye Biotechnology Co., Ltd.), add 50 mL of DMSO (Sangon Biotech Co., Ltd.), and dissolve it ultrasonically until it becomes clear. Filter it through a 0.22 μm organic membrane. Take the bacterial culture inoculated in BHI medium (Qingdao Haibo Biotechnology Co., Ltd. Weigh 38.5 g of the finished BHI medium, dissolve it in 1 L of pure water, stir evenly and then dispense it into 250 mL shake flasks, with a filling volume of 100 mL, autoclave at 121 °C for 15 minutes, and add 2 mL of 1 mg / mL bilirubin to each bottle before use), and centrifuge at 3260 × g Centrifuge for 6 minutes. Filter the supernatant through a 0.22 μm filter. Add chloroform (5 mL, Shanghai Lingfeng Chemical Reagent Co., Ltd.) to the supernatant and perform extraction and separation using a separatory funnel. Dry the obtained chloroform-urobilinogen solution until the chloroform completely evaporates. Redissolve the extract with 600 μL of deionized water, and pipette 400 μL of the redissolved solution into a new centrifuge tube. Add 10 μL of 10% povidone iodine solution (Sangon Biotech Co., Ltd. Weigh 1 g of povidone iodine powder, add 10 mL of pure water, and dissolve it ultrasonically), then add 10 μL of 100 mM cysteine solution (Sangon Biotech Co., Ltd. Weigh 0.242 g of cysteine powder, add 20 mL of pure water, and dissolve it ultrasonically), and finally add 400 μL of 545 mM zinc acetate methanol solution (zinc acetate: Xilong Scientific Co., Ltd.; methanol: J.T. Baker; Weigh 1.2 g of zinc acetate dihydrate into 10 mL of methanol solution and dissolve it ultrasonically). Take 100 μL of the final reaction solution and add it to a 96-well plate for detection with an enzyme-labeled instrument (the excitation wavelength of the enzyme-labeled instrument is 495 nm, and the emission wavelength is 525 nm). Take three replicates for each sample, use the BHI medium without bilirubin as the blank control, and use urobilinogen (Aladdin. Take 0.1 mL of 25 g / L urobilinogen standard product and make up the volume to 5 mL with deionized water) as the positive control, and express the activity of the strain in degrading bilirubin as the ratio of the detection group to the blank group.

[0061] Table 3: Bilirubin degradation activity of VB346

[0062] The results are shown in Table 3 and Figure 1 as follows. Lactococcus lactis subsp. lactis( Lactococcus lactis subsp. Lactis ) VB346 has the ability to degrade bilirubin.

[0063] Example 3: Tolerance of Lactococcus lactis subsp. lactis( Lactococcus lactis subsp. Lactis ) VB346 in gastric juice, choline system and intestinal juice 1. Gastric acid tolerance test Prepare 1% sodium chloride solution, adjust the pH to 1, 2, 3, and 4 respectively, then dispense and sterilize at 121°C for 30 min; prepare 3% pepsin, filter it with a 0.22 μm filter membrane, and mix evenly according to the ratio of 3% pepsin: 1% sodium chloride solution = 1:9.

[0064] Take the strain cryotube and spread the original solution on MRS solid medium (added with 5.4% MRS broth, 2% agar, prepared with deionized water, autoclaved at 121°C for 15 min), anaerobically culture at 37°C for 24 hours, scrape the bacterial lawn, inoculate it into 20% glycerol to prepare a bacterial suspension, and dilute it to a viable cell count of 10 9 ~ 10 10 CFU / mL. Take 10 μL of the bacterial suspension and add it to 990 μL of simulated gastric juice, mix evenly, and then spread it on MRS solid medium at 0 h, 2 h, and 4 h respectively. After anaerobic culture at 37°C for 1 - 2 days, count and calculate the change in the viable cell count and mortality rate of the strain at different pH values.

[0065] Table 4: Viable cell count and mortality rate of VB346 in simulated gastric juice at different pH values

[0066] Lactococcus lactis subsp. ( Lactococcus lactis subsp. Lactis ) The gastric acid tolerance of VB346 is shown in Table 4 and Figure 2 As shown, although the strong acidic environments of pH = 1 and pH = 2 have a greater impact on its survival ( Figure 2 the two lines coincide in this case), the viable cell count can remain above 3 × 10 8 CFU / mL after treatment for 0 h under the condition of pH = 3, and the viable cell count of the strain can remain at 1 × 10 8 CFU / mL after treatment for 2 h under the condition of pH = 4. It can be seen that Lactococcus lactis subsp. ( Lactococcus lactis subsp. Lactis ) VB346 of the present invention has the ability to resist the acidic environment of the human body under most conditions.

[0067] 2. Choline tolerance test Prepare 1% sodium chloride solution, add bovine choline at concentrations of 0, 0.033%, 0.066%, 0.11%, 0.22%, and 0.33% respectively, and autoclave at 121°C for 30 min; prepare 1% trypsin, filter it with a 0.22 μm filter membrane. Mix evenly according to the ratio of 1% trypsin: 1% physiological saline with each choline concentration = 1:9.

[0068] Take the strain cryotubes and spread the original liquid onto MRS solid medium (added with 5.4% MRS broth, 2% agar, prepared with deionized water, autoclaved at 121 °C for 15 min), incubate anaerobically at 37 °C for 24 hours, scrape the bacterial lawn, inoculate into 20% glycerol to prepare a bacterial suspension, and dilute to a viable cell count of 10 9 ~ 10 10 CFU / mL. Take 10 μL of the bacterial suspension and add it to 990 μL of choline solution. After mixing evenly, spread it onto MRS solid medium at 0 h, 2 h, and 4 h respectively, and perform counting and calculate the changes in viable cell count and mortality rate of this strain at different choline concentrations after anaerobic incubation at 37 °C for 1 - 2 days.

[0069] Table 5: Viable cell count and mortality rate of VB346 at different choline concentrations

[0070] The concentration range of choline in the human small intestine is 0.03% - 0.3%. Lactic acid bacteria that can grow and metabolize at normal physiological choline concentrations are considered to be able to survive during intestinal transit. The results are shown in Table 5 and Figure 3 as follows. When the bile salt concentration ≤ 0.3%, after treatment for 2 h, the viable cell count of Lactococcus lactis subsp. lactis Lactococcus lactis subsp. Lactis ) VB346 remains at 4 × 10 7 CFU / mL and above, indicating that Lactococcus lactis subsp. lactis Lactococcus lactis subsp. Lactis ) VB346 of the present invention can tolerate normal human bile concentrations and has the bile salt tolerance ability required for survival in the intestine.

[0071] 3. Tolerance of Lactococcus lactis subsp. lactis VB346 to artificial intestinal fluid Take the strain cryotubes and spread the original liquid onto MRS solid medium (added with 5.4% MRS broth, 2% agar, prepared with deionized water, autoclaved at 121 °C for 15 min), incubate anaerobically at 37 °C for 24 hours, scrape the bacterial lawn, inoculate into 20% glycerol to prepare a bacterial suspension, and dilute to a viable cell count of 10 9 ~ 10 10 CFU / mL. Take 10 μL of the bacterial suspension and add it to 990 μL of artificial intestinal fluid (Coolaber, measured pH = 7.9). After mixing evenly, spread it onto MRS solid medium at 0 h, 2 h, and 4 h respectively, and perform counting and calculate the changes in viable cell count and mortality rate of this strain in artificial simulated intestinal fluid after anaerobic incubation at 37 °C for 1 - 2 days.

[0072] Table 6: Viable cell count and mortality rate of VB346 in simulated intestinal fluid

[0073] The results are shown in Table 6 and Figure 4 as follows. In the simulated intestinal fluid environment, Lactococcus lactis subsp. lactis Lactococcus lactis subsp. Lactis ) VB346 showed relatively strong survival ability, and the viable cell count was above 2 × 10 8 CFU / mL, indicating that Lactococcus lactis subsp. lactis Lactococcus lactis subsp. Lactis ) VB346 of the present invention can stably survive in the intestine and play a probiotic role.

[0074] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0075] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and controls can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) VB346, characterized in that, The Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346 deposit number is CCTCCNO: M 20242013.

2. Use of the Lactococcus lactis subspecies VB346 described in claim 1 in a drug for preventing or treating hyperuricemia and / or hyperbilirubinemia.

3. The use according to claim 2, characterized in that: The medicament further contains excipients and / or carriers.

4. The use according to claim 3, characterized in that: The excipient comprises at least one selected from a binder, a disintegrant, a lubricant, a glidant, a stabilizer, a filler, a diluent, and a sustained-release agent.

5. The use according to claim 3, characterized in that: The carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, stearic acid alkaline earth metal salts, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and cereal hydrolyzed solids.

6. The use according to claim 2, characterized in that: The dosage form of the drug includes at least one selected from oral liquid, powder, granule, capsule, tablet, and pill.

7. A microbial agent, characterized in that: The lactococcus lactis subsp. lactis according to claim 1 Lactococcus lactis subsp. Lactis )VB346.

8. Use of the Lactococcus lactis subspecies VB346 according to claim 1 in the preparation of health products or foods, wherein the health products or foods are used to improve hyperuricemia and / or hyperbilirubinemia.

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