Cheese whey bacillus YWC-01 preparation for degrading uric acid and application thereof

Abstract

The application belongs to the technical field of biotechnology, and relates to a kind of degradation of uric acid's casein lactobacterium YWC-01 strain, which can produce catalytic degradation of uric acid enzyme.The application also relates to a kind of degradation of uric acid's casein lactobacterium YWC-01 preparation, which contains the bacterial cell and / or crude enzyme of casein lactobacterium YWC-01 strain.The research results show that the casein lactobacterium YWC-01 strain and the enzyme produced by the casein lactobacterium YWC-01 strain provided by the application for biological degradation of uric acid are safe for human body, and can efficiently biodegrade uric acid, can remove all uric acid with initial concentration of 1g / L within 48 hours under the initial concentration of bacterial cell of 8x10 8 / mL; can remove all uric acid with initial concentration of 1g / L within 18 hours under the crude enzyme protein concentration of 0.8g / L.The casein lactobacterium YWC-01 strain and the enzyme produced by the casein lactobacterium YWC-01 strain have important application prospect in preventing and treating gout and corresponding complications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a uric acid-degrading Lactobacillus casei YWC-01 preparation, its preparation method, and its application. Background Technology

[0002] Hyperuricemia (HUA) is defined as a condition where, under normal purine dietary conditions, two separate fasting blood uric acid levels exceed the normal range; specifically, a serum uric acid level greater than 420 μmol / L for adult men and greater than 360 μmol / L for adult women. Based on the saturated solubility of uric acid in the blood, when the concentration exceeds these levels, uric acid easily forms crystals and deposits in tissues, forming tophi (gouty nodules) that cause severe pain. Currently, the number of HUA patients in my country is rapidly increasing, posing a serious threat to people's health.

[0003] In the human body, intermediate products of purine metabolism, such as inosine and guanosine, can be further metabolized into uric acid as the final product. Under normal circumstances, about two-thirds of uric acid is excreted through the kidneys. Most of this uric acid is filtered by the glomeruli and reabsorbed in the proximal convoluted tubule, then excreted in urine through secretion and reabsorption. The remaining one-third is broken down by intestinal bacteria in the intestines into allantoin, which is then excreted in feces. Because humans lack uricase, they cannot break down uric acid into the more soluble allantoin, hindering uric acid excretion. This results in blood uric acid levels being 3-10 times higher than in non-primates, which is one of the important reasons why humans are more susceptible to hyperuricemia (HUA).

[0004] Currently, the treatment or improvement of hyperuricemia (HUA) mainly falls into two categories: drug therapy and dietary intervention. Drug therapy is the primary approach, with commonly used medications including allopurinol, febuxostat, benzbromarone, and uricase (raburicase). These drugs primarily control blood uric acid levels by inhibiting xanthine oxidase activity to reduce uric acid production or by reducing uric acid reabsorption to increase uric acid excretion. The main mechanism of uricase is to further degrade uric acid into more soluble allantoin, making it easier for the kidneys to excrete. While there are reports of *Lactobacillus casei* biodegrading nucleoside analogs such as inosine, there are no studies on its biodegradation of uric acid. Although chemical drugs can play a role in controlling HUA, the associated side effects, such as allergic reactions, liver damage, and abnormal kidney function, cannot be ignored. Dietary intervention mainly aims to alleviate HUA by limiting the intake of high-purine foods such as beer and fructose. While it can control blood uric acid levels to some extent, the effect is not ideal. Therefore, researching safer and more efficient methods to remove uric acid to control HUA has become a key scientific research problem that urgently needs to be solved in the biomedical field. Summary of the Invention

[0005] One of the objectives of this invention is to address the problem that existing drugs or foods for degrading and removing uric acid are not very effective and have side effects. This invention provides a strain of Lactobacillus casei YWC-01 that degrades uric acid. The bacterial cells of this strain and the enzymes it produces are safe for the human body and can efficiently biodegrade uric acid, thus having significant application prospects.

[0006] The second objective of this invention is to provide a *Lactobacillus casei* YWC-01 preparation for degrading uric acid and its application. The *Lactobacillus casei* YWC-01 preparation is made from the above-mentioned *Lactobacillus casei* YWC-01 strain used for biodegrading uric acid and can efficiently biodegrade uric acid.

[0007] Therefore, the first aspect of the present invention provides a strain of Lacticaseibacillus casei YWC-01 that degrades uric acid, which can produce an enzyme that catalyzes the degradation of uric acid, and its accession number is CGMCC No.31534.

[0008] In some embodiments of the present invention, the *Lactobacillus casei* strain YWC-01 was prepared at an initial cell concentration of 8 × 10⁻⁶. 8 At a concentration of 1 g / L, it can completely degrade and remove uric acid with an initial concentration of 1 g / L within 48 hours.

[0009] In other embodiments of the present invention, the crude enzyme produced by the Lactobacillus casei strain YWC-01 can completely remove uric acid with an initial concentration of 1 g / L within 18 hours at a protein concentration of 0.8 g / L.

[0010] A second aspect of the present invention provides a *Lactobacillus casei* YWC-01 preparation for degrading uric acid, which contains bacterial cells and / or crude enzymes of the *Lactobacillus casei* YWC-01 strain as described in the first aspect of the present invention; preferably, the *Lactobacillus casei* YWC-01 preparation contains the *Lactobacillus casei* YWC-01 strain as described in the first aspect of the present invention.

[0011] In some embodiments of the present invention, the uric acid-degrading Lactobacillus casei YWC-01 preparation is a liquid preparation; preferably, in the uric acid-degrading liquid preparation, the bacterial cell concentration of Lactobacillus casei YWC-01 strain is (1-10)×10⁻⁶. 8 / mL; and / or, in the liquid formulation for degrading uric acid, the protein concentration of the crude enzyme of Lactobacillus casei strain YWC-01 is 0.1-1.0 g / L.

[0012] In other embodiments of the present invention, the uric acid-degrading Lactobacillus casei YWC-01 preparation is a solid powder preparation; preferably, in the uric acid-degrading solid powder preparation, the content of Lactobacillus casei YWC-01 strain cells is (1-10)×10⁻⁶. 8 / g, more preferably (8-10)×10 8 / g; and / or, in the solid powder formulation for degrading uric acid, the crude enzyme protein content of Lactobacillus casei strain YWC-01 is 0.1-1.0 g / kg, more preferably 8-10 g / kg.

[0013] A third aspect of the present invention provides a method for preparing a uric acid-degrading Lactobacillus casei YWC-01 formulation as described in the second aspect of the present invention, comprising:

[0014] Step B: Inoculate the fermentation strain into the fermentation medium for fermentation culture to obtain the fermentation culture of Lactobacillus casei strain YWC-01;

[0015] Step C: Centrifuge the fermentation culture of Lactobacillus casei strain YWC-01 to harvest the bacterial cells of Lactobacillus casei strain YWC-01.

[0016] The fermentation strain was obtained from the corresponding Lactobacillus casei strain YWC-01 through seed culture.

[0017] According to the present invention, the fermentation medium comprises, per 1L of water, the following components:

[0018]

[0019] Preferably, the pH value of the fermentation medium is 7-8;

[0020] More preferably, in step B, the fermentation culture temperature is 30-40℃, more preferably 38-40℃.

[0021] According to some embodiments of the present invention, the preparation method further includes:

[0022] Step K: The cell suspension of Lactobacillus casei strain YWC-01 is subjected to cell disruption treatment under low temperature conditions to obtain cell-free lysate of Lactobacillus casei strain YWC-01.

[0023] Step L: Centrifuge the cell-free lysate of Lactobacillus casei strain YWC-01 and take the supernatant cell-free extract as the crude enzyme of Lactobacillus casei strain YWC-01.

[0024] The low temperature is 0-4℃.

[0025] The fourth aspect of this invention provides the use of the uric acid-degrading Lactobacillus casei YWC-01 preparation described in the second aspect of this invention, or the uric acid-degrading Lactobacillus casei YWC-01 preparation prepared by the method described in the third aspect of this invention, in the preparation of uric acid-lowering drugs, comprising:

[0026] Step D: Wash the cells of Lactobacillus casei strain YWC-01 with physiological saline to obtain pure cells of Lactobacillus casei strain YWC-01.

[0027] Step E: In a physiological saline system, under low temperature conditions, the bacterial cell purity of Lactobacillus casei strain YWC-01 is broken by ultrasonication. After centrifugation, the supernatant is taken to obtain cell-free extract as crude enzyme purity of Lactobacillus casei strain YWC-01.

[0028] Step F involves freeze-drying the pure bacterial cells and / or crude enzyme of Lactobacillus casei strain YWC-01, and then diluting the freeze-dried Lactobacillus casei YWC-01 preparation to produce a uric acid degradation agent.

[0029] The low temperature is 0-4℃.

[0030] In some embodiments of the present invention, in step F, the freeze-dried Lactobacillus casei YWC-01 preparation is diluted with physiological saline to prepare a liquid uric acid-lowering agent.

[0031] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Lactobacillus casei YWC-01 preparation to prepare a solid uric acid-lowering agent.

[0032] In some preferred embodiments of the present invention, the uric acid-lowering agent is an oral preparation.

[0033] Studies have found that the Lactobacillus casei strain YWC-01 and the enzymes it produces, which are used to biodegrade uric acid, provided by this invention, are safe for human use and can biodegrade uric acid. They have important application prospects in the efficient removal of uric acid for the treatment of human hyperuricemia and related complications. Attached Figure Description

[0034] To make the present invention easier to understand, it will be described below with reference to the accompanying drawings.

[0035] Figure 1 A molecular phylogenetic tree based on 16S rDNA is shown for screening Lactobacillus casei YWC-01.

[0036] Figure 2 The kinetic curves for the biodegradation of uric acid by Lactobacillus casei YWC-01 are shown.

[0037] Figure 3 The kinetic curves of uric acid degradation catalyzed by Lactobacillus casei YWC-01 enzyme.

[0038] strain preservation

[0039] Lacticaseibacillus casei, isolated and identified by Beijing Beike Yiran Biotechnology Co., Ltd., has been deposited at the China General Microbiological Culture Collection Center (CGMCC; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing) on ​​August 2, 2024, with accession number CGMCC No. 31534. In this invention, this strain is named Lacticaseibacillus casei strain YWC-01, also known as Lacticaseibacillus casei YWC-01. Detailed Implementation

[0040] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0041] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0042] I. Terminology

[0043] In this invention, the term "cell" refers to the live and / or dead cells of Lactobacillus casei.

[0044] In this invention, the term "crude enzyme" refers to the cell-free extract obtained by centrifuging and collecting the supernatant after the bacterial cells of Lactobacillus casei are broken.

[0045] The term "pure crude enzyme" used in this invention refers to a cell-free extract obtained by centrifuging and collecting the supernatant of pure Lactobacillus casei cells after the cells have been broken up.

[0046] The term "microbial preparation" as used in this invention refers to preparations of various forms made from microorganisms with medical research value as raw materials, using traditional or modern biotechnology, and used for the prevention (health care), treatment, and diagnosis of various physiological symptoms in the human body.

[0047] The term "edible starch" as used in this invention refers to starch that meets the "National Standard for Edible Starch" (GB 31637-2016 National Food Safety Standard for Edible Starch).

[0048] The "water" used in the invention for culture media or fermentation processes, unless otherwise specified, refers to sterile pure water obtained by filtration through a 0.22 μm filter membrane.

[0049] II. Implementation Plan

[0050] As mentioned earlier, existing dietary and drug therapies for controlling uric acid in the human body are slow-acting, have limited efficacy, and have side effects. Therefore, the inventors have conducted extensive and in-depth research on the biodegradation of uric acid.

[0051] The inventors noted that although lactic acid bacteria can promote gut health and reduce uric acid accumulation to some extent through a series of metabolic regulation, there are no reports of the strains used directly and efficiently biodegrading uric acid.

[0052] In recent years, research on human microbiome has been a hot topic and frontier in life sciences. Although scholars at home and abroad have screened out microbial strains from traditional foods that can inhibit uric acid synthesis and degrade uric acid precursors such as inosine and guanosine, no evidence has been found that Lactobacillus casei can directly and efficiently biodegrade uric acid.

[0053] The inventors also noted that Lacticaseibacillus casei is a prokaryotic microorganism and one of the probiotic strains approved by the National Health Commission of my country for direct human consumption. It has the ability to promote human intestinal health and improve human immune function and has been widely used in food, fermentation, beer and beverage production. However, no research reports on its biodegradation of uric acid have been found to date.

[0054] Based on long-term research in microorganisms, the inventors successfully screened a strain of *Lactaseibacillus casei* from enzymes. Through further research, the inventors discovered that this strain's cells and the enzymes it produces can completely degrade uric acid at an initial concentration of 1 g / L within 48 hours and 18 hours, respectively. This not only has significant research value but also holds important application prospects for the efficient biodegradation and removal of uric acid. Thus, this invention was obtained.

[0055] Therefore, the Lactobacillus casei strain YWC-01, which relates to the first aspect of the present invention for biodegrading uric acid, is capable of producing an enzyme that catalyzes the degradation of uric acid.

[0056] The inventors first successfully screened a strain of *Lactobacillus casei*, YWC-01, from enzymes. Genomic DNA was extracted, and molecular identification, including PCR amplification and 16S rDNA sequencing, confirmed it to be *Lactobacillus casei*. Based on this, the strain was identified and named *Lactobacillus casei* strain YWC-01. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 31534.

[0057] The inventors have discovered that fermenting Lactobacillus casei strain YWC-01 produces yeast cells containing one or more enzymes that can catalyze the degradation of uric acid. In this invention, the mixture of these enzymes is referred to as crude enzyme, or Lactobacillus casei YWC-01 crude enzyme.

[0058] Further research revealed that centrifugation of the cell-free lysate from *Lactobacillus casei* YWC-01 cells and the resulting cell-free extract were used as the crude enzyme for *Lactobacillus casei* YWC-01. This readily explains why both the cells and the crude enzyme of *Lactobacillus casei* YWC-01 can catalyze the degradation of uric acid.

[0059] The results showed that the *Lactobacillus casei* strain YWC-01, with an initial cell concentration of 8 × 10⁻⁶, was effective. 8 At a concentration of 1 g / L, it can completely degrade and remove uric acid with an initial concentration of 1 g / L within 48 hours.

[0060] The crude enzyme produced by the Lactobacillus casei strain YWC-01 can completely degrade uric acid at an initial concentration of 1 g / L within 18 hours at a protein concentration of 0.8 g / L.

[0061] Based on the above, the second to fourth aspects of the present invention further provide the use or application of Lactobacillus casei for biodegrading uric acid as described in the first aspect of the present invention.

[0062] Specifically, the second aspect of the present invention provides a biodegradable uric acid preparation of Lactobacillus casei YWC-01, which belongs to a biodegradable uric acid microbial preparation, containing bacterial cells and / or crude enzymes of Lactobacillus casei YWC-01 strain as described in the first aspect of the present invention.

[0063] In some preferred embodiments of the present invention, the Lactobacillus casei YWC-01 preparation contains bacterial cells of the Lactobacillus casei YWC-01 strain as described in the first aspect of the present invention.

[0064] According to some embodiments of the present invention, the Lactobacillus casei YWC-01 preparation for biodegrading uric acid is a liquid preparation.

[0065] In some embodiments of the present invention, the bacterial cell concentration of *Lactobacillus casei* strain YWC-01 in the liquid formulation of the biodegradable uric acid is (1-10)×10⁻⁶. 8 / mL.

[0066] In other embodiments of the present invention, the crude enzyme protein concentration of Lactobacillus casei strain YWC-01 in the liquid preparation of biodegradable uric acid is 0.1-1.0 g / L.

[0067] According to other embodiments of the present invention, the biodegradable uric acid Lactobacillus casei YWC-01 preparation is a solid powder preparation.

[0068] In some embodiments of the present invention, the bacterial cell content of *Lactobacillus casei* strain YWC-01 in the biodegradable uric acid solid powder formulation is (1-10)×10⁸ / g, preferably (8-10)×10⁸ / g. 8 / g.

[0069] In other embodiments of the present invention, in the solid powder formulation of biodegradable uric acid, the crude enzyme protein content of Lactobacillus casei strain YWC-01 is 0.1-1.0 g / kg (mg / g), preferably 0.8-1.0 g / kg.

[0070] A third aspect of the present invention provides a method for preparing a biodegradable uric acid Lactobacillus casei YWC-01 formulation as described in the second aspect of the present invention, comprising:

[0071] Step B: Inoculate the fermentation strain into the fermentation medium and allow it to ferment at 30-40℃, preferably 38-40℃, for 3-5 days to obtain the fermentation culture of Lactobacillus casei strain YWC-01.

[0072] Step C: Centrifuge the fermentation culture of Lactobacillus casei strain YWC-01 to harvest the bacterial cells of Lactobacillus casei strain YWC-01.

[0073] The fermentation strain was obtained from the corresponding Lactobacillus casei strain YWC-01 through seed culture.

[0074] As is known to those skilled in the art, 16S rRNA is commonly used internationally for the molecular identification of prokaryotic microorganisms. Therefore, 16S rRNA can be used to compare similarities and obtain homology. Thus, the fermentation strain used in this invention is not limited to the field isolates used in this invention. 16S rDNA is the DNA sequence on the bacterial chromosome that encodes rRNA and exists in the genomes of all prokaryotic microorganisms. Figure 1 A molecular phylogenetic tree based on 16S rDNA of Lactobacillus casei strain YWC-01 is shown.

[0075] In step C above, the centrifugation process includes resuspending and washing the precipitate (i.e., the bacterial cells of Lactobacillus casei strain YWC-01) obtained by centrifugation of the liquid fermentation culture with physiological saline, and then centrifuging it again to obtain the bacterial cells of Lactobacillus casei strain YWC-01.

[0076] The present invention does not impose any particular restrictions on the centrifugal separation conditions in step C above. In some embodiments of the present invention, for example, the analyte can be centrifuged for 10 minutes at 8000-10000 r / min.

[0077] According to the method of the present invention, the fermentation culture is a static culture of the microbial strain or a fermenter fermentation culture, and the fermentation strain is inoculated into the fermentation medium in the form of a seed culture. The inoculation amount of the seed culture is 1%-10% (v / v); preferably, the inoculation amount of the seed culture is 5%-10% (v / v); more preferably, the inoculation amount of the seed culture is 10% (v / v).

[0078] Specifically, the above fermentation medium, calculated per 1L of water, includes the following components in 1L of water:

[0079]

[0080] Preferably, the above fermentation medium, based on 1L of water, includes the following components in 1L of water:

[0081]

[0082] In some embodiments of the present invention, the initial pH of the fermentation medium is adjusted to 7-8 using a 40% (wt / v) sodium hydroxide solution and a 36% (v / v) hydrochloric acid solution.

[0083] According to some embodiments of the present invention, the preparation method of the Lactobacillus casei YWC-01 preparation of the present invention further includes step A, which involves seed culture before step B: picking a single colony of Lactobacillus casei YWC-01 provided by the present invention and inoculating it into 100 mL of fermentation liquid culture medium, and then incubating it at 38°C for 3 days to obtain the fermentation strain (seed liquid).

[0084] The inventors studied the effects of different temperatures on the growth of Lactobacillus casei YWC-01 and found that Lactobacillus casei YWC-01 grew rapidly at a temperature of 38℃.

[0085] According to some embodiments of the present invention, the preparation method further includes:

[0086] Step K: The cell suspension of Lactobacillus casei strain YWC-01 is subjected to cell disruption treatment in an ice-water bath (i.e., ice-water mixture, 0-4℃) to obtain cell-free lysate of Lactobacillus casei strain YWC-01.

[0087] Step L: Centrifuge the cell-free lysate of Lactobacillus casei strain YWC-01 and use the supernatant cell-free extract as the crude enzyme of Lactobacillus casei strain YWC-01.

[0088] The present invention does not impose any particular restrictions on the centrifugal separation conditions in step L above. In some embodiments of the present invention, for example, the analyte can be centrifuged at 15000-18000 r / min for 10-20 min.

[0089] The fourth aspect of this invention provides the use of the Lactobacillus casei YWC-01 preparation for biodegrading uric acid as described in the second aspect of this invention, or the Lactobacillus casei YWC-01 preparation for biodegrading uric acid prepared by the preparation method described in the third aspect of this invention, in the preparation of uric acid-lowering agents, comprising:

[0090] Step D: Wash the cells of Lactobacillus casei strain YWC-01 with physiological saline to obtain pure cells of Lactobacillus casei strain YWC-01.

[0091] Step E: In a physiological saline system, under low temperature conditions of 0℃-4℃, the bacterial cell purity of Lactobacillus casei strain YWC-01 is broken by ultrasonication. After centrifugation, the supernatant is taken to obtain cell-free extract as crude enzyme purity of Lactobacillus casei strain YWC-01.

[0092] Step F involves freeze-drying the pure bacterial cells and / or crude enzyme of Lactobacillus casei strain YWC-01, and then diluting the freeze-dried Lactobacillus casei YWC-01 preparation to produce a uric acid-lowering agent.

[0093] In some embodiments of the present invention, in step F, the freeze-dried Lactobacillus casei YWC-01 preparation is diluted with physiological saline to prepare a liquid uric acid-lowering agent.

[0094] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Lactobacillus casei YWC-01 preparation to prepare a solid uric acid-lowering agent.

[0095] In some preferred embodiments of the present invention, the uric acid-lowering agent is an oral preparation.

[0096] III. Related materials and testing methods in this invention

[0097] 1. Materials

[0098] The enzyme involved in this invention is prepared by fermenting apples with natural bacteria.

[0099] 2. Detection Method

[0100] (1) The cell concentration in this invention is determined using the following method:

[0101] To determine the concentration of Lactobacillus casei YWC-01 cells, Lactobacillus casei YWC-01 culture was diluted with physiological saline and the cell concentration was directly measured using a flow cytometer (SYSMEX, Germany).

[0102] (2) In this invention, the uric acid concentration is determined using the following method:

[0103] Uric acid concentration was determined using a high-performance liquid chromatograph (Shimadzu LC-20AT). The specific chromatographic column was Kromasil C18 (4.6×250 mm, 5 μm particle size); the mobile phase was methanol: 0.5% acetic acid aqueous solution (10:90); the detection wavelength was 283 nm; the flow rate was 1 mL / min; the injection volume was 20 μL; and the temperature was 35 °C.

[0104] (3) The crude enzyme protein concentration in this invention was determined using the following method:

[0105] Cell-free extract of Lactobacillus casei YWC-01 was diluted with phosphate buffer solution and then Coomassie Brilliant Blue G-250 dye reagent was added in proportion and reacted for 10 minutes. The absorbance was measured at 595 nm using a 722S visible spectrophotometer (Shanghai Lingguang). The protein concentration was calculated using the standard curve method.

[0106] III. Examples

[0107] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are standard laboratory methods. Unless otherwise specified, the experimental materials described below are commercially available.

[0108] Example 1:

[0109] (1) Prepare the growth medium for Lactobacillus casei YWC-01, with the following composition (per liter): 10g peptone, 10g beef extract, 10g yeast powder, and 10g glucose, adjusting the initial pH to 7.5. Add 100ml of the prepared liquid culture medium to a 500ml Erlenmeyer flask, sterilize under high temperature and high pressure (115℃) for 30 minutes, and then sterilize again under ultraviolet irradiation in a clean workbench for 20 minutes.

[0110] (2) Under sterile conditions in a clean workbench, 10 ml of Lactobacillus casei YWC-01 bacterial suspension was inoculated into a liquid culture medium in an Erlenmeyer flask. After being incubated at 38°C for 3 days, Lactobacillus casei YWC-01 cells were harvested by centrifugation (8000 rpm, 10 minutes) and discarding the supernatant.

[0111] Add 20 mL of *Lactobacillus casei* YWC-01 cell suspension to a 50 mL glass tube and immerse the tube in ice water. Disrupt the *Lactobacillus casei* YWC-01 cells using an ultrasonic cell disruptor under the following conditions: ultrasonic power 400 W, 2-second intervals, 10-second ultrasonic oscillations, and a disruption time of 15 minutes (5 minutes per cycle). After cell disruption, centrifuge the cell disruption solution at 15,000 rpm for 20 minutes, and then slowly pour off the supernatant as the cell-free extract (crude enzyme) of *Lactobacillus casei* YWC-01.

[0112] (3) Based on different uric acid concentrations, the cultured Lactobacillus casei YWC-01 cells and crude enzymes were added in a certain proportion as a rapid, safe and efficient biocatalyst to achieve the purpose of rapidly and efficiently degrading and removing uric acid.

[0113] Figure 1 The strain we screened was most closely related to Lactobacillus casei, and was therefore named Lactobacillus casei strain YWC-01.

[0114] Figure 2 This indicates that the initial cell concentration of Lactobacillus casei YWC-01 was 8 × 10⁻⁶. 8 At a concentration of 1 g / L, it can completely degrade uric acid at an initial concentration of 1 g / L within 48 hours, indicating that Lactobacillus casei YWC-01 has a strong biodegradation ability for uric acid.

[0115] Figure 3This indicates that the cell-free extract (crude enzyme) of Lactobacillus casei YWC-01 can catalyze the degradation of uric acid at a faster rate. At a protein concentration of 0.8 g / L, it can completely degrade uric acid with an initial concentration of 1 g / L in 18 hours, demonstrating a higher uric acid degradation rate.

[0116] It should be noted that the embodiments described above are merely preferred embodiments of the present invention, used to explain the present invention, and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the terms used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications having the same function.

Claims

1. A kind of dry cheese lactobacillus that degrades uric acid (Lactobacillus casei) Lacticaseibacillus casei ) YWC-01 strain, it can produce the enzyme catalyzing degradation uric acid, and its preservation number is CGMCC No.31534.

2. A preparation of Lactobacillus casei YWC-01 for degrading uric acid, comprising bacterial cells of the Lactobacillus casei YWC-01 strain and / or crude enzyme of the Lactobacillus casei YWC-01 strain according to claim 1. The crude enzyme is obtained by subjecting a cell suspension of the Lactobacillus casei YWC-01 strain to cell disruption treatment under low temperature conditions, and centrifuging the obtained cell-free disruption solution to obtain the supernatant cell-free extract.

3. The Lactobacillus casei YWC-01 preparation according to claim 2, characterized by, The degradation of uric acid in the preparation of cheese lactobacillus YWC-01 is a liquid preparation; in the degradation of uric acid in the liquid preparation, the concentration of the bacterial cells of cheese lactobacillus YWC-01 strain is (1-10) × 10 8 / mL; and / or, in the degradation of uric acid in the liquid preparation, the protein concentration of the crude enzyme of cheese lactobacillus YWC-01 strain is 0.1-1.0 g / L.

4. The Lactobacillus casei YWC-01 preparation according to claim 2, characterized by, The preparation of the Lactobacillus casei YWC-01 for degrading uric acid is a solid powder preparation; in the solid powder preparation for degrading uric acid, the content of the bacterial cells of the Lactobacillus casei YWC-01 strain is (1-10) × 10 8 / g; and / or, in the solid powder preparation for degrading uric acid, the protein content of the crude enzyme of the Lactobacillus casei YWC-01 strain is 0.1-1.0 g / kg.

5. The Lactobacillus casei YWC-01 preparation according to claim 4, characterized by, In the solid powder preparation for degrading uric acid, the content of the bacterial cells of the strain of Lactobacillus casei YWC-01 is (8-10) x 10 8 / g; and / or, in the solid powder preparation for degrading uric acid, the protein content of the crude enzyme of the strain of Lactobacillus casei YWC-01 is 0.8-1.0 g / kg.

6. A preparation method of the Lactobacillus casei YWC-01 for degrading uric acid according to any one of claims 2-5, comprising: Step A: obtaining a fermentation seed of the Lactobacillus casei YWC-01 strain through seed culture; Step B: inoculating the fermentation seed into a fermentation medium to perform fermentation culture, and obtaining a fermentation culture of the Lactobacillus casei YWC-01 strain; Step C: centrifuging the fermentation culture of the Lactobacillus casei YWC-01 strain to harvest bacterial cells of the Lactobacillus casei YWC-01 strain.

7. The preparation method according to claim 6, characterized in that, The fermentation medium comprises the following components in 1L of water: Peptone 5-10 g; Beef extract 5-10 g; Yeast powder 5-10 g; and Glucose 5-10 g. The pH value of the fermentation medium is 7-8.

8. The preparation method according to claim 7, characterized in that, The fermentation medium comprises the following components in 1L of water: Peptone 8-10 g; Beef extract 8-10 g; Yeast powder 8-10 g; and Glucose 8-10 g.

9. The preparation method according to claim 7, characterized in that, In step B, the temperature of the fermentation culture is 30-40℃.

10. The method of claim 9, wherein, In step B, the temperature of the fermentation culture is 38-40℃.

11. The method of any one of claims 6-10, wherein, The preparation method further comprises: Step K: subjecting a cell suspension of the Lactobacillus casei YWC-01 strain to cell disruption treatment under low temperature conditions to obtain a cell-free disruption solution of the Lactobacillus casei YWC-01 strain; Step L: centrifuging the cell-free disruption solution of the Lactobacillus casei YWC-01 strain to obtain a supernatant cell-free extract as a crude enzyme of the Lactobacillus casei YWC-01 strain; wherein the low temperature is 0-4℃.

12. Use of the Lactobacillus casei YWC-01 for degrading uric acid according to any one of claims 2-5 or the preparation method according to any one of claims 6-10 in the preparation of a uric acid-lowering agent, comprising: Step D: washing the bacterial cells of the Lactobacillus casei YWC-01 strain with physiological saline to obtain pure bacterial cells of the Lactobacillus casei YWC-01 strain; Step E: under low temperature conditions, subjecting the pure bacterial cells of the Lactobacillus casei YWC-01 strain to ultrasonic disruption in a physiological saline system, and obtaining a cell-free extract by centrifuging to obtain a crude enzyme pure product of the Lactobacillus casei YWC-01 strain; Step F: freeze-drying the pure bacterial cells and / or the crude enzyme pure product of the Lactobacillus casei YWC-01 strain, and diluting the freeze-dried Lactobacillus casei YWC-01 preparation to prepare a uric acid-lowering agent. The low temperature is 0-4℃.

13. The use according to claim 12, wherein the use is for treating or preventing a disease caused by a pathogenic bacteria. In step F, the freeze-dried preparation of Lactobacillus casei YWC-01 is diluted with physiological saline to form a liquid uric acid-lowering agent. Alternatively, in step F, the freeze-dried preparation of Lactobacillus casei YWC-01 is diluted with edible starch to form a solid uric acid-lowering agent.

14. Use according to claim 13, characterized in that, The uric acid-lowering agent is an oral preparation.

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