Lactococcus lactis subsp. lactis VB346 for purine degradation and its application

Abstract

The present invention relates to the field of microorganisms. The present invention discloses a Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp.Lactis )VB346, was deposited in the China Center for Type Culture Collection on September 20, 2024, with a deposit number of CCTCC NO: M 20242013. The Lactococcus lactis subspecies lactis VB346 of the present invention has the ability to degrade purine nucleosides, can decompose purine nucleosides in ingested food in the intestine, thereby reducing the absorption of purine nucleosides by the small intestine, and has the ability to degrade bilirubin. At the same time, it has the acid resistance required for survival in the stomach, the bile salt tolerance required for survival in the intestine, and the ability to survive stably in the intestine, and has great application prospects in the preparation of microbial agents, health products or foods.

Description

Technical Field

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

[0002] Purine is a precursor to uric acid, primarily existing in the body as purine nucleotides and purine nucleosides. It further forms hypoxanthine, which is then oxidized with xanthine to produce uric acid. Excessive purine intake or purine metabolism disorders can increase uric acid levels in the blood, leading to the metabolic disease hyperuricemia. Elevated uric acid crystals can deposit in the joints, renal pelvis, ureters, and renal interstitium, causing gout, urate nephropathy, and uric acid urinary stones. In severe cases, it can damage joints and lead to renal insufficiency. Furthermore, hyperuricemia is often complicated by hypertension, non-alcoholic fatty liver disease, cardiovascular disease, and diabetes.

[0003] Currently, drugs for treating hyperuricemia fall into three main categories: those that inhibit uric acid production, those that promote uric acid excretion, and those that activate urate oxidase. Commonly used drugs, such as allopurinol and febuxostat, primarily reduce uric acid production by inhibiting xanthine oxidase, while benzbromarone and probenecid primarily alleviate hyperuricemia by promoting uric acid excretion. However, these drugs are commonly associated with allergic reactions and toxic side effects. In addition to medications, the treatment and prevention of hyperuricemia also requires controlling the intake of high-purine foods, such as beer, meat, animal offal, and seafood. However, this dietary restriction makes it difficult to maintain a balanced diet and can lead to a decline in quality of life due to the inability to enjoy the pleasure of food. 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. Milk )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 being CCTCC NO: M 20242013.

[0006] According to an embodiment of the present invention, the strain has the ability to degrade purine nucleosides. The strain can decompose purine nucleosides in 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, the strain has the ability to degrade bilirubin.

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

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

[0011] According to the embodiment of the present invention, the strain exhibits relatively strong survival ability in simulated intestinal fluid, has the ability to survive stably in the intestine, and exerts a probiotic effect.

[0012] According to an embodiment of the present invention, when Lactococcus lactis subsp. lactis VB346 functions, it may exist in the form of living cells or in the form of non-living cells.

[0013] According to an embodiment of the present invention, the living cells refer to cells with the ability to metabolize, reproduce or replicate, and the non-living cells refer to cells without the ability to metabolize, reproduce and replicate, including but not limited to dried bacteria (such as freeze-dried powder).

[0014] The present invention provides a microbial agent, wherein the microbial agent contains the aforementioned Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346.

[0015] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 :Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) Bilirubin degradation activity of strain VB346;

[0017] Figure 2 :Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) VB346 strain acid resistance test results;

[0018] Figure 3 :Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) VB346 strain bile salt tolerance test results, where different percentages represent the amount of bile salt added;

[0019] Figure 4 :Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) Results of the test on the tolerance of VB346 strain to artificial intestinal fluid. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0021] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0022] In this document, the terms “contain”, “include” or “include” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0023] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0024] As used herein, "prevent" and "prevent" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," Lactococcus lactis subsp. lactis or a product containing the same may be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have yet been made.

[0025] As used herein, the terms "treat" and "alleviate" refer to methods used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive 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 covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0026] In this article, "acceptable in health foods" refers to substances or compositions that can be consumed by humans, which may be adjusted according to the health food requirements of different countries.

[0027] As used herein, "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, "pharmaceutically acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0028] As used herein, the term "pharmaceutically acceptable carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, which are known to those skilled in the art. Except where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is encompassed.

[0029] In this article, the term "pharmaceutically acceptable excipients" may include sugars, including monosaccharides or polysaccharides, such as lactose, sucrose, mannitol and sorbitol; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinyl pyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, 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 cereal hydrolyzed solids and other non-toxic compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, etc., which are commonly used excipients in pharmaceutical preparations.

[0030] According to a specific embodiment of the present invention, the present invention proposes a Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. lactis ) VB346. According to an embodiment of the present invention, the Lactococcus lactis subsp. lactis has a deposit number of 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 bilirubin degradation ability, gastric acid resistance, choline resistance, and intestinal tolerance, and has great application prospects in the preparation of pharmaceuticals.

[0031] In this article, "Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346” and “Lactococcus lactis subsp. lactis VB346”, “VB346” and “strain VB346” are synonymous.

[0032] 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.

[0033] It should be noted that “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.

[0034] 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.

[0035] 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.; it can also be a solid microbial agent, including but not limited to freeze-dried powder, etc.

[0036] According to an embodiment of the present invention, the Lactococcus lactis subsp. lactis VB346 exists in the form of living cells and / or non-living cells.

[0037] As used herein, "living cells" refer to cells that have the ability to metabolize, reproduce or replicate.

[0038] For example, the living cells may be immobilized cells. Herein, "immobilized cells" refer to Lactococcus lactis subsp. lactis VB346 immobilized on a carrier, which can carry out life activities such as growth, development, reproduction, inheritance and metabolism within a certain spatial range.

[0039] In this article, "non-viable cells" refer to cells that do not have the ability to metabolize, reproduce and replicate, including but not limited to dried bacteria. Exemplarily, the microbial agent is a freeze-dried powder.

[0040] As a specific embodiment, the Lactococcus lactis subspecies lactis VB346 exists in the form of living cells, dry bacteria, immobilized cells or any other forms.

[0041] As a specific embodiment, the dry bacteria are obtained by freeze-drying the Lactococcus lactis subspecies lactis VB346.

[0042] According to an embodiment of the present invention, the present invention provides a medicine, health product, or food containing Lactococcus lactis subsp. lactis VB346. It should be noted that the medicine, health product, or food of the present application can be various preparations of active Lactococcus lactis subsp. lactis VB346 alone, or can be used in combination with other active ingredients, as long as they do not affect the activity of each other; further, the best situation for the medicine, health product, or food is that the active ingredients can have functional complementarity or a promoting effect. For example, Lactococcus lactis subsp. lactis VB346 can be combined with other probiotics to form a composite probiotic tablet to achieve better or more active functions. Specifically, it can be determined according to the activity and components of the composite probiotic tablet, which is not limited here. Optionally, the medicine, health product, or food further includes a carrier or excipient acceptable in the development of the medicine, health product, or food. It should be noted that when preparing a live bacterial preparation, it is usually necessary to add a carrier or excipient, as long as the added carrier or excipient does not inhibit each other or have adverse side effects with Lactococcus lactis subsp. lactis VB346. Medicines, health products or foods can be in the form of powders, tablets, drinks or capsules.

[0043] In this article, hyperuricemia refers to a condition characterized by elevated uric acid levels in the blood, typically exceeding the normal range (typically >7.0 mg / dL in men and >6.0 mg / dL in women). Uric acid is the end product of purine metabolism and is primarily excreted through the kidneys. Hyperuricemia itself typically has no obvious symptoms, but persistently elevated uric acid levels may lead to the following diseases and symptoms: gout, uric acid stones, and kidney damage.

[0044] For the purposes of this article, hyperbilirubinemia refers to elevated blood levels of bilirubin. 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. However, abnormalities in bilirubin production, metabolism, or excretion can lead to elevated blood levels of bilirubin, resulting in hyperbilirubinemia. Hyperbilirubinemia may contribute to the following conditions and symptoms: obstructive jaundice, hepatocellular jaundice, liver cancer, pancreatic cancer, cholelithiasis, bile duct cancer, and hepatitis.

[0045] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0046] Example 1: Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )Isolation, screening, purification and identification of VB346 strain.

[0047] 1. Isolation of lactic acid bacteria

[0048] The strain was isolated from the intestines of healthy humans. A gradient dilution of the suspension was prepared using sterile saline solution. 100 μL of each dilution was spread onto MRS agar plates and incubated anaerobically at 37°C for 48 hours. Suspected lactic acid bacteria colonies were streaked and purified, and DNA was extracted. 16S rDNA was amplified by PCR using specific primers, and then sequenced. The corresponding strains were stored in glycerol tubes. Based on the 16S rDNA identification results, six Lactococcus lactis strains were selected and numbered 000567, 000609, 000630, VB346, 000679, and 000695.

[0049] 2. Screening of lactic acid bacteria strains

[0050] Preparation of MRS-purine medium: Weigh 80 mg of adenine (Shanghai MacLean Biochemical Technology Co., Ltd.) and dissolve it in 200 mL of pure water to make an adenine solution. Weigh 80 mg of hypoxanthine (Shanghai MacLean Biochemical Technology Co., Ltd.) and dissolve it in 200 mL of pure water to make a hypoxanthine solution. Weigh 80 mg of xanthine (Shanghai MacLean Biochemical Technology Co., Ltd.) and add 1 mL of sodium hydroxide solution (pH 13) and sonicate to make a xanthine solution. Weigh 80 mg of guanine (Shanghai MacLean Biochemical Technology Co., Ltd.) and add 1 mL of sodium hydroxide solution (pH approximately 13) and sonicate to make a guanine solution. Weigh 54 g of MRS finished medium (Guangdong Huankai Microbiology Technology Co., Ltd.) and add 200 mL each of adenine and hypoxanthine solutions, 1 mL of xanthine solution, and 1 mL of guanine solution. Dose the medium to 1 L with pure water until turbidity is eliminated. The final concentration of all four purines is 80 μg / mL. The final pH of the medium is 6.6. Sterilize the medium at 121°C for 15 minutes and set aside.

[0051] Strain culture: Thaw the glycerol tubes of the six different Lactococcus lactis strains isolated above at room temperature and inoculate them onto MRS slants. Incubate anaerobically at 37°C for 1 day to obtain a slant lawn. Scrape a 2 cm × 2 cm section of the lawn into 20 mL of MRS-purine medium and incubate anaerobically at 37°C for 20-24 hours.

[0052] Detection of free purine: Take 1 mL of cultured bacterial liquid, centrifuge at 14000 rpm for 10 minutes, take the supernatant, and detect the free purine concentration by HPLC.

[0053] Total purine concentration detection: Take the remaining bacterial liquid and centrifuge it at 3500 rpm for 10 minutes. Take 5 mL of the supernatant and mix it with 2.5 mL of trifluoroacetic acid and 2.5 mL of formic acid. Place it in a 90°C water bath and boil it for 12 minutes. Cool it to room temperature in an ice bath. Neutralize it with 15 mol / L potassium hydroxide (weigh 42 g of potassium hydroxide and add 30 mL of pure water to dissolve it) to pH = 7.0. 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.

[0054] The degradation rates of adenine, hypoxanthine, xanthine and guanine were calculated according to the following formulas:

[0055] Degradation rate = (C0-C1)÷C0×100%, where C0 is the concentration of total purine and C1 is the concentration of free purine (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]

[0059] Table 2 Degradation rates of four purines by different Lactococcus lactis strains

[0060]

[0061] The degradation rates of four purines by different Lactococcus lactis strains were tested, among which strain VB346 had a 100% degradation rate for adenine.

[0062] 3. Strain identification

[0063] The strain was sequenced by sequencing the Lactococcus lactis subsp. lactis VB346 obtained in Example 1, and the 16S rRNA sequence of the strain was compared with the homology of the strain in GenBank, and was identified as Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ), named Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346 was deposited on September 20, 2024, at the China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC NO: M 20242013. Its 16S rRNA sequence information is as follows:

[0064]

[0065] Example 2: Bilirubin-lowering properties of Lactococcus lactis subsp. lactis VB346

[0066] Weigh 50 mg of bilirubin (Shanghai Yuanye Biotechnology Co., Ltd.) and add 50 mL of DMSO (Shenggong Bioengineering Co., Ltd.). Dissolve the solution by ultrasonication until clear and 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 finished BHI medium, dissolve it in 1 L of pure water, stir it evenly, and then divide it into 250 mL shake flasks, fill it with 100 mL, and sterilize it by autoclaving at 121°C for 15 minutes. Before use, add 2 mL of 1 mg / mL bilirubin to each bottle) and add 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 extract and separate using a separatory funnel. Air-dry the resulting chloroform-urobilinogen solution until the chloroform is completely evaporated. Redissolve the extract in 600 μL of deionized water, and transfer 400 μL of the reconstituted material to a new centrifuge tube. Add 10 μL of 10% povidone-iodine solution (Sangon Biotechnology Co., Ltd.: weigh 1 g of povidone-iodine powder, add 10 mL of pure water, and sonicate to dissolve). Then, add 10 μL of 100 mM cysteine solution (Sangon Biotechnology Co., Ltd.: weigh 0.242 g of cysteine powder, add 20 mL of pure water, and sonicate to dissolve). Finally, add 400 μL of 545 mM zinc acetate-methanol solution (zinc acetate: Xilong Scientific Co., Ltd.; methanol: JTBaker; weigh 1.2 g of zinc acetate dihydrate in 10 mL of methanol and sonicate to dissolve). 100 µL of the final reaction solution was added to a 96-well plate for detection by microplate reader (excitation wavelength: 495 nm, emission wavelength: 525 nm). Three replicates were taken for each sample. BHI medium without bilirubin was used as a blank control, and urobilinogen (Aladdin, 0.1 mL of 25 g / L urobilinogen standard was taken and diluted to 5 mL with deionized water) was used as a positive control. The ratio of the test group to the blank group indicated the bilirubin-degrading activity of the strain.

[0067] Table 3: Bilirubin degradation activity of VB346

[0068]

[0069] The results are shown in Table 3 and Figure 1 As shown, Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346 has the ability to degrade bilirubin.

[0070] Example 3: Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis) Tolerance of VB346 in gastric juice, choline system and intestinal juice

[0071] 1. Gastric acid resistance test

[0072] Prepare 1% sodium chloride solution, adjust the pH to 1, 2, 3, and 4 respectively, then package and sterilize at 121°C for 30 min; prepare 3% pepsin, filter with a 0.22 μm filter membrane, and mix evenly at a ratio of 3% pepsin: 1% sodium chloride solution = 1:9.

[0073] Take the frozen tubes of the strain and spread the stock solution on MRS solid medium (add 5.4% MRS broth, 2% agar, deionized water, and autoclave at 121°C for 15 min), culture anaerobically at 37°C for 24 hours, scrape the bacterial lawn, inoculate with 20% glycerol to prepare the bacterial suspension, and dilute to a viable count of 10 9 ~ 10 10 CFU / mL. Take 10 μL of bacterial suspension and add 990 μL of simulated gastric fluid. Mix well and apply to MRS solid medium at 0 h, 2 h, and 4 h. After anaerobic incubation at 37°C for 1-2 days, count the number of viable bacteria and calculate the change in the number of live bacteria and the mortality rate of the strain at different pH levels.

[0074] Table 4: Live bacterial count and mortality of VB346 in simulated gastric fluids with different pH values

[0075]

[0076] Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) The gastric acid resistance of VB346 is shown in Table 4 and Figure 2 As shown in Figure 2, although the strong acidic environment of pH = 1 and pH = 2 has a greater impact on its survival ( Figure 2 The two lines overlap), and the pH = 3 condition can maintain 3 × 10 8 The number of viable bacteria above 1 × 10 CFU / mL was maintained at 1 × 10 8 The number of viable bacteria in CFU / mL. It can be seen that the lactococcus lactis subsp. lactis of the present invention ( Lactococcus lactis subsp. Lactis ) VB346 has the ability to resist the acidic environment of the human body under most conditions.

[0077] 2. Choline resistance test

[0078] Prepare a 1% sodium chloride solution and add taurine at concentrations of 0, 0.033%, 0.066%, 0.11%, 0.22%, and 0.33%. Autoclave at 121°C for 30 min. Prepare a 1% trypsin solution and filter through a 0.22 μm filter. Mix thoroughly at a ratio of 1% trypsin to 1% saline at each choline concentration (1:9).

[0079] Take the frozen tubes of the strain and spread the stock solution on MRS solid medium (add 5.4% MRS broth, 2% agar, deionized water, and autoclave at 121°C for 15 min), culture anaerobically at 37°C for 24 hours, scrape the bacterial lawn, inoculate with 20% glycerol to prepare the bacterial suspension, and dilute to a viable count of 10 9 ~ 10 10 CFU / mL. Take 10 μL of bacterial suspension and add 990 μL of choline solution. Mix well and apply to MRS solid medium at 0 h, 2 h, and 4 h. After anaerobic incubation at 37°C for 1-2 days, count the number of viable bacteria and calculate the change in the number of live bacteria and the mortality rate of the strain at different choline concentrations.

[0080] Table 5: Viable bacteria count and mortality of VB346 at different choline concentrations

[0081]

[0082] The concentration of choline in the human small intestine ranges from 0.03% to 0.3%. Lactic acid bacteria that can grow and metabolize in normal physiological choline concentrations are considered to be able to survive intestinal transit. The results are shown in Table 5 and Figure 3 As shown, when bile salt concentration is ≤0.3%, the Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) The viable count of VB346 was maintained at 4 × 10 7 CFU / mL and above, indicating that the present invention is lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) VB346 can tolerate normal bile concentrations in the human body and has the bile salt tolerance required for survival in the intestine.

[0083] 3. Tolerance of Lactococcus lactis subsp. lactis VB346 to artificial intestinal fluid

[0084] Take the frozen tubes of the strain and spread the stock solution on MRS solid medium (add 5.4% MRS broth, 2% agar, deionized water, and autoclave at 121°C for 15 min), culture anaerobically at 37°C for 24 hours, scrape the bacterial lawn, inoculate with 20% glycerol to prepare the bacterial suspension, and dilute to a viable count of 10 9 ~ 10 10CFU / mL. Add 10 μL of bacterial suspension to 990 μL of artificial intestinal fluid (Coolaber, measured pH = 7.9), mix thoroughly, and apply to MRS solid medium at 0, 2, and 4 hours. After anaerobic incubation at 37°C for 1 to 2 days, count the cells and calculate the change in viable bacterial count and mortality of the strain in artificial simulated intestinal fluid.

[0085] Table 6: Live bacterial count and mortality of VB346 in simulated intestinal fluid

[0086]

[0087] The results are shown in Table 6 and Figure 4 As shown, in the simulated intestinal fluid environment, Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. lactis VB346 showed a relatively strong survival ability, with the viable cell count at 2 × 10 8 CFU / mL or more, indicating that the present invention lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ) VB346 can survive stably in the intestine and play a prebiotic role.

[0088] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and variations without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by 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. A microbial agent, characterized in that: Including the Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis )VB346.

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