Brevibacillus brevis YF02 preparation for degrading creatinine and application of brevibacillus brevis YF02 preparation

Abstract

The invention belongs to the technical field of biology, and relates to a brevibacillus brevis YF02 strain for degrading creatinine, which can generate an enzyme for catalytically degrading creatinine. The invention also relates to a brevibacillus brevis YF02 preparation for degrading creatinine, and the brevibacillus brevis YF02 preparation contains bacterial cells and / or crude enzyme of the brevibacillus brevis YF02 strain. Research results show that the brevibacillus brevis YF02 cell for degrading creatinine and the enzyme produced by the brevibacillus brevis YF02 cell can completely degrade 500mg / L of creatinine within 48 hours and 24 hours respectively, and the brevibacillus brevis YF02 cell and the enzyme produced by the brevibacillus brevis YF02 cell have important application prospects in the aspects of reducing the creatinine level of human serum and preventing and treating nephropathy and corresponding complications.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a Bacillus brevis YF02 preparation for biodegrading creatinine and a preparation method and application thereof. Background Art

[0002] Chronic kidney disease (CKD) is a common chronic disease with multiple complications. Due to renal dysfunction, the excretion of harmful substances, especially creatinine, is blocked, which eventually aggravates the condition and leads to uremia, seriously affecting people's health and quality of life. At present, the global prevalence of CKD has increased significantly and has been recognized as an important global public health issue. Serum creatinine level is one of the most critical biomarkers for evaluating renal function, and there is an urgent need to develop methods and technologies for efficiently removing creatinine.

[0003] Creatinine (Creatinine, molecular formula C4H7N3O) is the final product of creatine metabolism in mammals. There are two sources of serum creatinine in the human body: exogenous from the metabolism of meat food and endogenous from the metabolism of muscle tissue. Creatinine is mainly excreted from the body through the kidney system in the form of urine. Under normal circumstances, the production and excretion of creatinine in the human body are basically in a dynamic equilibrium state. When kidney disease leads to renal insufficiency, the creatinine that cannot be excreted accumulates in the body and causes harm.

[0004] At present, the main strategies for managing CKD include drugs, dialysis and kidney transplantation, but they all have the disadvantages of ineffective efficacy and large side effects. For example, although the use of Chinese and Western medicines such as non-steroidal anti-inflammatory drugs and yam polysaccharides has a certain effect, due to the decrease in the patient's glomerular filtration rate, it further leads to the accumulation of drugs or their metabolites, causing a series of adverse reactions, and even worsening renal function in severe cases. In view of the shortcomings of existing CKD treatment strategies, the use of microbial degradation to remove creatinine provides a new idea for the treatment of CKD. Studies have found that some microorganisms such as Pseudomonas putida and Arthrobacter ureafaciens can produce creatinine hydrolases and have the ability to biodegrade creatinine, but the efficiency of creatinine removal is low. Summary of the invention

[0005] One of the purposes of the present invention is to provide a creatinine-degrading Bacillus brevis YF02 strain, in view of the problem that the drugs for reducing human blood creatinine levels in the prior art are not effective and have side effects. The bacterial cells of the strain and the enzymes produced by the strain can both efficiently biodegrade creatinine and have important application prospects in removing creatinine.

[0006] The second object of the present invention is to provide a creatinine-degrading Bacillus brevis YF02 preparation and its application. The Bacillus brevis YF02 preparation is made from the above-mentioned Bacillus brevis YF02 strain for biodegrading creatinine and can efficiently biodegrade creatinine.

[0007] To this end, the first aspect of the present invention provides a creatinine-degrading Brevibacillus brevis YF02 strain, which can produce an enzyme that catalyzes the degradation of creatinine and has a deposit number of CGMCC No.31609.

[0008] In some embodiments of the present invention, the Bacillus brevis YF02 strain has a cell concentration of 2×10 8 / mL, the initial concentration of creatinine of 500 mg / L can be completely degraded and removed within 48 hours.

[0009] In other embodiments of the present invention, the crude enzyme produced by the Bacillus brevis YF02 strain can completely remove creatinine with an initial concentration of 500 mg / L within 24 hours at a protein concentration of 0.3 g / L.

[0010] The second aspect of the present invention provides a creatinine-degrading Bacillus brevis YF02 preparation, which contains bacterial cells and / or crude enzymes of the Bacillus brevis YF02 strain as described in the first aspect of the present invention; preferably, the Bacillus brevis YF02 preparation contains the Bacillus brevis YF02 strain as described in the first aspect of the present invention.

[0011] In some embodiments of the present invention, the creatinine-degrading Bacillus brevis YF02 preparation is a liquid preparation; preferably, in the creatinine-degrading liquid preparation, the cell concentration of Bacillus brevis YF02 strain is (1-10)×10 8 / mL; and / or, in the liquid preparation for degrading creatinine, the protein concentration of the crude enzyme of Bacillus brevis YF02 strain is 0.1-1g / L.

[0012] In other embodiments of the present invention, the creatinine-degrading Bacillus brevis YF02 preparation is a solid powder preparation; preferably, in the creatinine-degrading solid powder preparation, the cell concentration of Bacillus brevis YF02 strain is (1-10)×10 8 / g, more preferably (8-10)×10 8 / g; and / or, in the solid powder preparation for degrading creatinine, the protein content of the crude enzyme of Bacillus brevis YF02 strain is 0.1-1.0 g / kg, more preferably 0.8-1.0 g / kg.

[0013] The third aspect of the present invention provides a method for preparing the creatinine-degrading Bacillus brevis YF02 preparation according to the second aspect of the present invention, comprising:

[0014] Step B, inoculating the fermentation bacteria into a fermentation medium for fermentation culture to obtain a fermentation culture of Bacillus brevis YF02 strain;

[0015] Step C, centrifuging the fermentation culture of Bacillus brevis YF02 to harvest bacterial cells of Bacillus brevis YF02;

[0016] Wherein, the fermentation bacteria are obtained by seed culture of the corresponding Bacillus brevis YF02 strain.

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

[0018] Peptone 5-10g; preferably 8-10g;

[0019] 5-10 g beef extract; preferably 8-10 g; and

[0020] Glucose 5-10g; preferably 8-10g;

[0021] Preferably, the pH value of the fermentation medium is 7-8;

[0022] Further preferably, in step B, the fermentation culture temperature is 30-40°C, preferably 38-40°C.

[0023] According to some embodiments of the present invention, the preparation method further comprises:

[0024] Step K, subjecting the cell suspension of the Bacillus brevis YF02 strain to cell disruption treatment under low temperature conditions to obtain a cell-free disrupted liquid of the Bacillus brevis YF02 strain;

[0025] Step L, centrifuging the cell-free disrupted liquid of Bacillus brevis YF02 strain, and taking the supernatant cell-free extract as the crude enzyme of Bacillus brevis YF02 strain;

[0026] Wherein, the low temperature is 0-4°C.

[0027] The fourth aspect of the present invention provides a use of the Brevibacillus YF02 preparation according to the second aspect of the present invention or the Brevibacillus YF02 preparation prepared by the preparation method according to the third aspect of the present invention in the preparation of a creatinine-lowering agent, comprising:

[0028] Step D, washing the bacterial cells of Bacillus brevis YF02 strain with physiological saline to obtain pure bacterial cells of Bacillus brevis YF02 strain;

[0029] Step E, in a physiological saline system, under low temperature conditions, using ultrasound to disrupt the pure bacterial cells of Bacillus brevis YF02 strain, and taking the supernatant after centrifugation to obtain a cell-free extract as a crude enzyme pure product of Bacillus brevis YF02 strain;

[0030] Step F, freeze-drying the pure bacterial cell product and / or the pure crude enzyme product of Bacillus brevis YF02 strain, and diluting the freeze-dried Bacillus brevis YF02 preparation to prepare a creatinine-degrading agent;

[0031] Wherein, the low temperature is 0-4°C.

[0032] In some embodiments of the present invention, in step F, the freeze-dried Bacillus brevis YF02 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent.

[0033] In other embodiments of the present invention, in step F, the freeze-dried Bacillus brevis YF02 preparation is diluted with edible starch to prepare a solid creatinine-lowering agent.

[0034] In some preferred embodiments of the present invention, the creatinine-lowering agent is an oral preparation.

[0035] Studies have found that the Bacillus brevis YF02 strain for biodegrading creatinine provided by the present invention and the enzyme produced can biodegrade creatinine, and have important application prospects in the efficient removal of creatinine for the treatment of human hypercreatinemia and corresponding complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the present invention easy to understand, the present invention is described below with reference to the accompanying drawings.

[0037] Figure 1 The molecular evolution tree of the screened Bacillus brevis YF02 based on 16S rDNA is shown.

[0038] Figure 2 This is the kinetic curve of creatinine biodegradation by Bacillus brevis YF02.

[0039] Figure 3 This is the kinetic curve of creatinine degradation catalyzed by Bacillus brevis YF02 enzyme.

[0040] Strain preservation

[0041] Brevibacillus brevis was isolated and identified by Beijing Beike Yiran Biotechnology Co., Ltd. and has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Committee (referred to as: CGMCC; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), deposit date: August 5, 2024, deposit number: CGMCC No. 31609. In the present invention, the strain is named Brevibacillus brevis strain YF02, also known as Brevibacillus brevis YF02. DETAILED DESCRIPTION

[0042] To make the present invention easy to understand, the present invention 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 terms used herein are only for describing specific embodiments and are not intended to be limiting.

[0043] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, preferred methods and materials are now described.

[0044] I. Terminology

[0045] The term "bacteria" as used in the present invention refers to living cells and / or dead cells of Bacillus brevis.

[0046] The term "crude enzyme" used in the present invention refers to a cell-free extract obtained by breaking bacterial cells of Bacillus brevis and centrifuging the supernatant.

[0047] The term "pure crude enzyme" used in the present invention refers to a cell-free extract obtained by breaking the pure bacterial cells of Bacillus brevis and then centrifuging the supernatant.

[0048] The term "microbial preparation" used in the present invention refers to various forms of preparations that are made from microorganisms with medical research value using traditional technology or modern biotechnology and act on the prevention (health care), treatment and diagnosis of various physiological symptoms of the human body.

[0049] The term "edible starch" as used in the present invention refers to starch that meets the "National Standard for Edible Starch" (GB 31637-2016 National Food Safety Standard Edible Starch).

[0050] The "water" used in the culture medium or fermentation culture process of the present invention, unless otherwise specified, refers to sterile pure water obtained by filtering through a 0.22μ filter membrane.

[0051] II. Implementation Plan

[0052] As mentioned above, the main strategy for the existing management of CKD includes methods such as drugs, dialysis and kidney replacement, but there is a deficiency that the curative effect of reducing blood creatinine concentration is not obvious and the side effect is large. Although it is found that some microorganisms such as Pseudomonas putida and Arthrobacter ureafaciens can produce reports of creatinine hydrolase degradation of creatinine, the efficiency of these bacteria in removing creatinine is very low. On the basis of long-term microbial research, the inventor successfully screened out a strain of Brevibacillus brevis YF02 (Brevibacillus brevis strain YF02) that can efficiently biodegrade creatinine from the bottom mud of seawater aquaculture ponds, and the strain cell and the enzyme produced can fully degrade the creatinine of initial concentration 500mg / L in 48h and 24h respectively, not only having very important research value, but also having important application prospects in efficient biodegradation and removal of creatinine.

[0053] Therefore, the Bacillus brevis YF02 strain for biodegradation of creatinine according to the first aspect of the present invention can produce an enzyme that catalyzes the degradation of creatinine.

[0054] The inventors first successfully screened a strain of Brevibacillus brevis YF02 from the bottom mud of a seawater aquaculture pond. After extracting genomic DNA, it was determined to be Brevibacillus brevis by PCR amplification and molecular identification based on 16S rDNA sequencing. Based on the above, the strain was identified and named Brevibacillus brevis strain YF02. The strain has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 31609.

[0055] The inventors have found that during fermentation of Bacillus brevis YF02 strain, the bacterial cells produced contain more than one enzyme capable of catalyzing the degradation of creatinine. In the present invention, the mixture of these enzymes is referred to as crude enzyme, or Bacillus brevis YF02 crude enzyme.

[0056] Further research found that the cell-free lysate after bacterial cell disruption was centrifuged and the supernatant cell-free extract was taken as the crude enzyme of Bacillus brevis YF02. It is easy to understand that both the cells and crude enzyme of Bacillus brevis YF02 strain can catalyze the degradation of creatinine.

[0057] The results showed that the YF02 strain of Bacillus brevis had an initial bacterial cell concentration of 2×10 8 / mL, the initial concentration of creatinine of 500 mg / L can be completely degraded and removed within 48 hours.

[0058] The crude enzyme produced by the Bacillus brevis YF02 strain can completely degrade creatinine with an initial concentration of 500 mg / L within 24 hours at a protein concentration of 0.3 g / L.

[0059] Based on the above, the second to fourth aspects of the present invention further provide the use or application of the Bacillus brevis YF02 for biodegrading creatinine described in the first aspect of the present invention.

[0060] Specifically, the second aspect of the present invention provides a creatinine biodegrading Bacillus brevis YF02 preparation, which belongs to a creatinine biodegrading microbial preparation, and contains bacterial cells and / or crude enzymes of the Bacillus brevis YF02 strain as described in the first aspect of the present invention.

[0061] In some preferred embodiments of the present invention, the Bacillus brevis YF02 preparation contains bacterial cells of the Bacillus brevis YF02 strain as described in the first aspect of the present invention.

[0062] According to some embodiments of the present invention, the creatinine biodegrading Bacillus brevis YF02 preparation is a liquid preparation.

[0063] In some embodiments of the present invention, in the liquid preparation for biodegradation of creatinine, the cell concentration of the bacterial cells of the Bacillus brevis YF02 strain is (1-10)×10 8 / mL.

[0064] In other embodiments of the present invention, in the liquid preparation for biodegradation of creatinine, the protein concentration of the crude enzyme of Bacillus brevis YF02 strain is 0.1-1.0 g / L.

[0065] According to other embodiments of the present invention, the creatinine biodegrading Bacillus brevis YF02 preparation is a solid powder preparation.

[0066] In some embodiments of the present invention, in the solid powder preparation for biodegradation of creatinine, the cell concentration of Bacillus brevis YF02 strain is (1-10)×10 8 / g, preferably (8-10)×10 8 / g.

[0067] In other embodiments of the present invention, in the solid powder preparation for biodegradation of creatinine, the protein content of the crude enzyme of Bacillus brevis YF02 strain is 0.1-1.0 g / kg, preferably 0.8-1.0 g / kg.

[0068] The third aspect of the present invention provides a method for preparing the creatinine biodegrading Bacillus brevis YF02 preparation according to the second aspect of the present invention, comprising:

[0069] Step B, inoculating the fermentation bacteria into a fermentation medium, and fermenting and culturing at 30-40° C., preferably 38-40° C., at a shaking speed of 100-300 rpm for 3-5 days to obtain a fermentation culture of Bacillus brevis YF02 strain;

[0070] Step C, centrifuging the fermentation culture of Bacillus brevis YF02 to harvest bacterial cells of Bacillus brevis YF02;

[0071] Wherein, the fermentation bacteria are obtained by seed culture of the corresponding Bacillus brevis YF02 strain.

[0072] As those skilled in the art know, currently, 16S rRNA is commonly used internationally for molecular identification of prokaryotic microorganisms, so 16S rRNA can be used for comparison to obtain homology in similarity comparison. Therefore, the fermentation strain used in the present invention is not limited to the field isolate used in the present invention. 16S rDNA is a DNA sequence corresponding to rRNA encoded on the bacterial genome, and exists in the genome of all prokaryotic microorganisms. Figure 1 The molecular evolution tree of Bacillus brevis YF02 strain based on 16S rDNA is shown.

[0073] In the above step C, the centrifugal separation treatment includes resuspending and washing the precipitate (i.e., bacterial cells of Bacillus brevis YF02 strain) obtained by centrifugal separation of the liquid fermentation culture with physiological saline, and then centrifuging it to obtain bacterial cells of Bacillus brevis YF02 strain.

[0074] The present invention has no particular limitation on the conditions for centrifugal separation in the above step C. In some embodiments of the present invention, for example, the substance to be separated may be centrifuged at 8000-10000 r / min for 10 min.

[0075] According to the method of the present invention, the fermentation culture is a shaking table or fermentation tank fermentation culture of the strain, and the fermentation strain is inoculated into the fermentation medium in the form of seed liquid. The inoculation amount of the seed liquid is 0.1%-1% (v / v); preferably, the inoculation amount of the seed liquid is 0.2%-0.5% (v / v); more preferably, the inoculation amount of the seed liquid is 0.5% (v / v).

[0076] Specifically, the fermentation medium comprises the following components in 1L of water:

[0077] Peptone 5-10g;

[0078] 5-10g beef extract; and

[0079] Glucose 5-10g.

[0080] Preferably, the fermentation medium comprises the following components in 1L of water:

[0081] Peptone 8-10g;

[0082] 8-10g beef extract; and

[0083] Glucose 8-10g.

[0084] In some embodiments of the present invention, the initial pH of the fermentation medium is adjusted to 7-8 using 40% (wt / v) sodium hydroxide solution and 36% (v / v) hydrochloric acid solution.

[0085] According to some embodiments of the present invention, the preparation method of the Bacillus brevis YF02 preparation involved in the present invention also includes a seed culture step A before step B: picking the monoclonal colony of the Bacillus brevis YF02 strain provided by the present invention and inoculating it into 100 mL of fermentation liquid culture medium, and culturing it on a shaking table at a temperature of 38°C and a rotation speed of 200r / min for 3 days to obtain a fermentation strain (seed liquid).

[0086] The inventors studied the effects of different temperatures on the growth of Bacillus brevis YF02 and found that Bacillus brevis YF02 grew faster at 38°C.

[0087] According to some embodiments of the present invention, the preparation method further comprises:

[0088] Step K, subjecting the cell suspension of the Bacillus brevis YF02 strain to cell disruption in an ice-water bath (i.e., an ice-water mixture, 0-4° C.) to obtain a cell-free disruption solution of the Bacillus brevis YF02 strain;

[0089] Step L, centrifuging the cell-free disrupted liquid of Bacillus brevis YF02 strain, and taking the supernatant cell-free extract as the crude enzyme of Bacillus brevis YF02 strain.

[0090] The present invention has no particular limitation on the conditions for centrifugal separation in the above step L. In some embodiments of the present invention, for example, the separation material may be centrifuged at 15000-18000 r / min for 10-20 min.

[0091] The fourth aspect of the present invention provides a use of the creatinine-degrading Bacillus brevis YF02 preparation according to the second aspect of the present invention or the creatinine-degrading Bacillus brevis YF02 preparation prepared by the preparation method according to the third aspect of the present invention in the preparation of a creatinine-lowering agent, which comprises:

[0092] Step D, washing the bacterial cells of Bacillus brevis YF02 strain with physiological saline to obtain pure bacterial cells of Bacillus brevis YF02 strain;

[0093] Step E, in a physiological saline system, at a low temperature of 0°C-4°C, using ultrasonic waves to disrupt the pure bacterial cells of Bacillus brevis YF02 strain, and taking the supernatant after centrifugation to obtain a cell-free extract as a crude enzyme pure product of Bacillus brevis YF02 strain;

[0094] Step F, freeze-drying the pure bacterial cell product and / or the pure crude enzyme product of Bacillus brevis YF02 strain, and diluting the freeze-dried Bacillus brevis YF02 preparation to prepare a creatinine-lowering agent.

[0095] In some embodiments of the present invention, in step F, the freeze-dried Bacillus brevis YF02 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent.

[0096] In other embodiments of the present invention, in step F, the freeze-dried Bacillus brevis YF02 preparation is diluted with edible starch to prepare a solid creatinine-lowering agent.

[0097] In some preferred embodiments of the present invention, the creatinine-lowering agent is an oral preparation.

[0098] III. Related materials and detection methods in the present invention

[0099] 1. Materials

[0100] The seawater aquaculture pond mud for screening bacterial species involved in the present invention was collected by the inventor from a large-scale seawater aquaculture pond in Dalian, Liaoning in 2020.

[0101] 2. Detection method

[0102] (1) The cell concentration in the present invention is determined by the following method:

[0103] To determine the cell concentration of Bacillus brevis YF02, the culture of Bacillus brevis YF02 was diluted a certain multiple with physiological saline, and the cell concentration was directly determined using a flow cytometer (SYSMEX, Germany).

[0104] (2) The creatinine concentration in the present invention is measured by the following method:

[0105] The creatinine concentration was determined by high performance liquid chromatography (Shimadzu LC-20AT), specifically, the chromatographic column was Kromasil C18 (4.6×250 mm, 5 μ particle size); the mobile phase was methanol: 0.5 M ammonium acetate solution (v / v 20 / 80); the ultraviolet detection wavelength was 233 nm; the flow rate was 1 mL / min; the injection volume was 20 μL; and the temperature was 35°C.

[0106] (3) The crude enzyme protein concentration in the present invention is measured by the following method:

[0107] The cell-free extract of Bacillus brevis YF02 was diluted a certain multiple with phosphate buffer solution, and Coomassie Brilliant Blue G-250 dye reagent was added in proportion to react for 10 minutes. The absorbance was measured at 595 nm using a 722S visible spectrophotometer (Shanghai Lingguang), and the protein concentration was calculated using the standard curve method.

[0108] IV. EXAMPLES

[0109] The present invention is described in detail below through specific examples. The experimental methods described below, unless otherwise specified, are all routine laboratory methods. The experimental materials described below, unless otherwise specified, can all be obtained from commercial channels.

[0110] Embodiment 1:

[0111] (1) Prepare the growth medium of Bacillus brevis YF02, the composition of which is (per liter): 10.0 g of peptone, 10.0 g of beef extract, and 10.0 g of glucose. Add 100 ml of the prepared liquid medium into a 500 ml Erlenmeyer flask, sterilize it at high temperature and high pressure (121°C) for 20 minutes, and then sterilize it under ultraviolet light in a clean bench for another 20 minutes.

[0112] (2) Under sterile conditions in a clean bench, 0.5 ml of Bacillus brevis YF02 bacterial solution was inoculated into a liquid culture medium in a triangular flask, and batch culture was carried out at a temperature of 38° C. and a shaking speed of 200 rpm for 3 days. The Bacillus brevis YF02 cells were harvested by centrifugation (8000 rpm, 10 minutes) and the supernatant was discarded.

[0113] Take 20mL of the suspension of Bacillus brevis YF02 cells and add it to a 50mL glass tube and then insert it into ice water. Use an ultrasonic cell disruptor to disrupt the Bacillus brevis YF02 cells. The conditions are: ultrasonic power 400W, interval 2 seconds, ultrasonic oscillation 10 seconds, and disruption time 15 minutes (5 minutes each time). After the cell disruption is completed, the cell disruption solution is centrifuged at 15000 rpm for 20 minutes, and then the supernatant is slowly poured out as a cell-free extract (crude enzyme) of Bacillus brevis YF02.

[0114] (3) According to the different concentrations of creatinine, the cultured Bacillus brevis YF02 cells and crude enzymes were added in a certain proportion as a fast and efficient biocatalyst to achieve the purpose of rapid and efficient degradation and removal of creatinine.

[0115] Figure 1 It showed that the strain we screened was most closely related to Bacillus brevis, so it was named Bacillus brevis YF02 strain.

[0116] Figure 2 It showed that Bacillus brevis YF02 had a cell concentration of 2.0×10 8 / mL, creatinine with an initial concentration of 500 mg / L can be completely degraded within 48 hours, indicating that Bacillus brevis YF02 has a strong biodegradation ability for creatinine.

[0117] Figure 3 The results showed that the cell-free extract (crude enzyme) of Bacillus brevis YF02 could catalyze the degradation of creatinine at a faster rate. At a protein concentration of 0.3 g / L, creatinine with an initial concentration of 500 mg / L could be completely degraded within 24 hours, and it had a higher creatinine degradation rate.

[0118] It should be noted that the embodiments described above are only preferred embodiments of the present invention, which are used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein. On the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A creatinine-degrading Bacillus brevis YF02 strain, which can produce an enzyme that catalyzes the degradation of creatinine and has a deposit number of CGMCC No.31609.

2. The Bacillus brevis YF02 strain according to claim 1, It is characterized in that The Brevibacillus YF02 strain was grown at a cell concentration of 2×10 8 / mL, the initial concentration of creatinine of 500 mg / L can be completely degraded and removed within 48 hours.

3. The Bacillus brevis YF02 strain according to claim 1, It is characterized in that The crude enzyme produced by the Bacillus brevis YF02 strain can completely remove creatinine with an initial concentration of 500 mg / L within 24 hours at a protein concentration of 0.3 g / L.

4. A creatinine-degrading Bacillus brevis YF02 preparation, comprising bacterial cells and / or crude enzymes of the Bacillus brevis YF02 strain as claimed in any one of claims 1 to 3; preferably, the Bacillus brevis preparation comprises the Bacillus brevis YF02 strain as claimed in any one of claims 1 to 3.

5. The Bacillus brevis YF02 preparation according to claim 4, It is characterized in that The creatinine-degrading Bacillus brevis YF02 preparation is a liquid preparation; preferably, in the creatinine-degrading liquid preparation, the cell concentration of Bacillus brevis YF02 strain is (1-10)×10 8 / mL; and / or, in the liquid preparation for degrading creatinine, the protein concentration of the crude enzyme of Bacillus brevis YF02 strain is 0.1-1.0 g / L; Alternatively, the creatinine-degrading Bacillus brevis YF02 preparation is a solid powder preparation; preferably, in the creatinine-degrading solid powder preparation, the cell concentration of Bacillus brevis YF02 strain is (1-10)×10 8 / g, more preferably (8-10)×10 8 / g; and / or, in the solid powder preparation for degrading creatinine, the protein content of the crude enzyme of Bacillus brevis YF02 strain is 0.1-1.0 g / kg, more preferably 0.8-1.0 g / kg.

6. A method for preparing the creatinine-degrading Bacillus brevis YF02 preparation as claimed in any one of claims 4 or 5, wherein include: Step B, inoculating the fermentation bacteria into the culture medium for fermentation culture to obtain the fermentation culture of Bacillus brevis YF02 strain; Step C, centrifuging the fermentation culture of Bacillus brevis YF02 to harvest bacterial cells of Bacillus brevis YF02; Wherein, the fermentation bacteria are obtained by seed culture of the corresponding Bacillus brevis YF02 strain.

7. The preparation method according to claim 6, It is characterized in that The fermentation medium, based on 1L of water, includes the following components in 1L of water: Peptone 5-10g; preferably 8-10g; 5-10g beef extract; preferably 8-10g; as well as Glucose 5-10g; preferably 8-10g; Preferably, the pH value of the fermentation medium is 7-8; Further preferably, in step B, the fermentation culture temperature is 30-40°C, preferably 38-40°C.

8. The preparation method according to claim 6 or 7, It is characterized in that The preparation method further comprises: Step K, subjecting the cell suspension of the Bacillus brevis YF02 strain to cell disruption treatment under low temperature conditions to obtain a cell-free disrupted liquid of the Bacillus brevis YF02 strain; Step L, centrifuging the cell-free disrupted liquid of Bacillus brevis YF02 strain, and taking the supernatant cell-free extract as the crude enzyme of Bacillus brevis YF02 strain; Wherein, the low temperature is 0-4°C.

9. Use of the creatinine-degrading Bacillus brevis YF02 preparation according to claim 4 or 5 or the preparation of Bacillus brevis YF02 prepared by the preparation method according to any one of claims 6 to 8 in the preparation of a creatinine-lowering agent, wherein include: Step D, washing the bacterial cells of Bacillus brevis YF02 strain with physiological saline to obtain pure bacterial cells of Bacillus brevis YF02 strain; Step E, in a physiological saline system, under low temperature conditions, using ultrasound to disrupt the pure bacterial cells of Bacillus brevis YF02 strain, and taking the supernatant after centrifugation to obtain a cell-free extract as a crude enzyme pure product of Bacillus brevis YF02 strain; Step F, freeze-drying the pure bacterial cell product and / or the pure crude enzyme product of Bacillus brevis YF02 strain, and diluting the freeze-dried Bacillus brevis YF02 preparation to prepare a creatinine-degrading agent; Wherein, the low temperature is 0-4°C.

10. The use according to claim 9, It is characterized in that In step F, the freeze-dried Bacillus brevis YF02 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent; Alternatively, in step F, the freeze-dried Bacillus brevis YF02 preparation is diluted with edible starch to prepare a solid creatinine-lowering agent; Preferably, the creatinine-lowering agent is an oral preparation.

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