Bacillus velezensis yf01 preparation for degrading creatinine and application thereof

Abstract

The application belongs to the technical field of biology and relates to a Bysinum perfringens YF01 strain capable of producing an enzyme for catalyzing degradation of creatinine. The application also relates to a Bysinum perfringens YF01 preparation for degrading creatinine, which contains bacterial cells and / or crude enzymes of the Bysinum perfringens YF01 strain. Research results show that the Bysinum perfringens YF01 cell and the enzyme produced by the Bysinum perfringens YF01 cell can respectively degrade 500 mg / L of creatinine within 48 hours and 24 hours, and have important application prospects in reducing human blood creatinine levels and preventing and treating kidney diseases and corresponding complications.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a Bacillus vesiculosus YF01 preparation for biodegrading creatinine, its preparation method and application. Background Technology

[0002] Chronic kidney disease (CKD) is a prevalent chronic disease with multiple complications. Due to impaired kidney function, the excretion of harmful substances, particularly creatinine, is hindered, ultimately leading to uremia and severely impacting people's health and quality of life. Currently, the global prevalence of CKD is rising significantly and is recognized as a major global public health issue. Serum creatinine levels are one of the most critical biomarkers for assessing kidney function, highlighting the urgent need to develop efficient methods and technologies for creatinine removal.

[0003] Creatinine (molecular formula C4H7N3O) is the final product of creatine metabolism in mammals. In humans, serum creatinine comes from two sources: exogenous creatinine from the metabolism of meat, and endogenous creatinine from the metabolism of muscle tissue. Creatinine is primarily excreted through the kidneys in the form of urine. Under normal circumstances, the production and excretion of creatinine in the human body are in a state of dynamic equilibrium. However, when kidney disease leads to renal insufficiency, the creatinine that cannot be excreted accumulates in the body, causing harm.

[0004] Currently, the main strategies for managing CKD include medication, dialysis, and kidney transplantation. However, all of these have drawbacks such as limited efficacy and significant side effects. While treatment with traditional Chinese medicine and Western medicine, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and yam polysaccharides, has shown some effectiveness, the reduced glomerular filtration rate in patients leads to the accumulation of drugs or their metabolites, causing a series of adverse reactions and, in severe cases, even deterioration of kidney function. To address the shortcomings of existing CKD treatment strategies, the use of microbial degradation to remove creatinine offers a new approach to CKD treatment. Studies have found that some microorganisms, such as *Pseudomonas putida* and *Arthrobacter ureafaciens*, can produce creatinine hydrolases and possess the ability to biodegrade creatinine, but their creatinine removal efficiency is low. Summary of the Invention

[0005] One of the objectives of this invention is to address the problem that existing drugs for lowering human serum creatinine levels are not very effective and have side effects. This invention provides a strain of Bacillus vesiculus YF01 that degrades creatinine. The bacterial cells of this strain and the enzymes it produces can efficiently biodegrade creatinine, and it has important application prospects in the removal of creatinine.

[0006] The second objective of this invention is to provide a Bacillus vesiculus YF01 preparation for degrading creatinine and its application. The Bacillus vesiculus YF01 preparation is made from the aforementioned Bacillus vesiculus YF01 strain used for biodegrading creatinine and can efficiently biodegrade creatinine.

[0007] Therefore, the first aspect of the present invention provides a strain of Bacillus velezensis YF01 that degrades creatinine, which is capable of producing an enzyme that catalyzes the degradation of creatinine, and its accession number is CGMCC No. 31608.

[0008] In some embodiments of the present invention, the Bacillus belye YF01 strain at a cell concentration of 1×10⁻⁶ 8 At a concentration of 500 mg / L, creatinine can be completely degraded and removed within 48 hours.

[0009] In other embodiments of the present invention, the crude enzyme produced by the Bacillus vesiculosus strain YF01 can completely remove creatinine at an initial concentration of 500 mg / L within 24 hours at a protein concentration of 0.2 g / L.

[0010] A second aspect of the present invention provides a Bacillus vesiculus YF01 preparation for degrading creatinine, which contains bacterial cells and / or crude enzymes of the Bacillus vesiculus YF01 strain as described in the first aspect of the present invention; preferably, the Bacillus vesiculus YF01 preparation contains the Bacillus vesiculus YF01 strain as described in the first aspect of the present invention.

[0011] According to some embodiments of the present invention, the Bacillus vesiculosus YF01 preparation for degrading creatinine is a liquid preparation.

[0012] Preferably, in the liquid formulation for degrading creatinine, the cell concentration of Bacillus vesiculosus strain YF01 is (1-10)×10⁻⁶. 8 / mL.

[0013] Preferably, in the liquid formulation for degrading creatinine, the protein concentration of the crude enzyme of Bacillus vesiculosus strain YF01 is 0.1-1 g / L.

[0014] In some other embodiments of the present invention, the Bacillus vesiculosus YF01 formulation that degrades creatinine is a solid powder formulation.

[0015] Preferably, in the solid powder formulation for degrading creatinine, the cell concentration of Bacillus belye YF01 strain is (1-10)×10⁻⁶. 8 / g, more preferably (8-10)×10 8 / g.

[0016] Preferably, in the solid powder formulation for degrading creatinine, the crude enzyme protein content of Bacillus vesiculosus strain YF01 is 0.1-1.0 g / kg, more preferably 0.8-1.0 g / kg.

[0017] A third aspect of the present invention provides a method for preparing a creatinine-degrading Bacillus vesiculosus YF01 formulation as described in the second aspect of the present invention, comprising:

[0018] Step B: Inoculate the fermentation strain into the fermentation medium for fermentation culture to obtain the fermentation culture of Bacillus belyss YF01 strain;

[0019] Step C: Centrifuge the fermentation culture of Bacillus belye YF01 strain to harvest the bacterial cells of Bacillus belye YF01 strain;

[0020] The fermentation strain was obtained from the corresponding Bacillus berberis strain YF01 through seed culture.

[0021] According to the present invention, the fermentation medium comprises, per 1L of water, the following components:

[0022] 5-10g of peptone; preferably 8-10g;

[0023] 5-10g of beef extract; preferably 8-10g; and

[0024] 5-10g of glucose; preferably 8-10g;

[0025] Preferably, the pH value of the fermentation medium is 7-8;

[0026] More preferably, in step B, the fermentation culture temperature is 30-40℃, more preferably 38-40℃.

[0027] According to some embodiments of the present invention, the preparation method further includes:

[0028] Step K: The cell suspension of Bacillus vesiculus YF01 strain is subjected to cell disruption treatment under low temperature conditions to obtain cell-free lysate of Bacillus vesiculus YF01 strain.

[0029] Step L: Centrifuge the cell-free lysate of Bacillus belysium YF01 strain and take the supernatant cell-free extract as the crude enzyme of Bacillus belysium YF01 strain.

[0030] The low temperature is 0-4℃.

[0031] The fourth aspect of this invention provides the use of the *Bacillus vesiculosus* YF01 preparation described in the second aspect of this invention, or the *Bacillus vesiculosus* YF01 preparation prepared by the method described in the third aspect of this invention, in the preparation of a creatinine-lowering agent, comprising:

[0032] Step D: Wash the cells of Bacillus vesiculosus strain YF01 with physiological saline to obtain pure cells of Bacillus vesiculosus strain YF01.

[0033] Step E: In a physiological saline system, under low temperature conditions, the pure bacterial cells of Bacillus vesiculosus strain YF01 are broken by ultrasonication. After centrifugation, the supernatant is taken to obtain a cell-free extract as the crude enzyme pure product of Bacillus vesiculosus strain YF01.

[0034] Step F involves freeze-drying the pure bacterial cells and / or crude enzyme of Bacillus vesiculus strain YF01, and then diluting the freeze-dried Bacillus vesiculus strain YF01 to prepare a drug for degrading creatinine.

[0035] The low temperature is 0-4℃.

[0036] In some embodiments of the present invention, in step F, the freeze-dried Bacillus vesiculosus YF01 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent.

[0037] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Bacillus vesiculosus YF01 preparation to prepare a solid creatinine-lowering agent.

[0038] Preferably, the creatinine-lowering agent is an oral preparation.

[0039] The inventors have discovered that the Bacillus vesiculosus strain YF01 and the enzymes produced by it, which are used to biodegrade creatinine, are both capable of biodegrading creatinine and have significant application prospects in the efficient removal of creatinine for the treatment of hypercreatinineemia and related complications in humans. Attached Figure Description

[0040] To make the present invention easier to understand, it will be described below with reference to the accompanying drawings.

[0041] Figure 1 A molecular phylogenetic tree based on 16S rDNA is shown for screening Bacillus belyssus YF01.

[0042] Figure 2 The kinetic curves of creatinine biodegradation by Bacillus belyssus YF01 cells.

[0043] Figure 3The kinetic curve of creatinine degradation catalyzed by crude enzyme of Bacillus belyssus YF01.

[0044] strain preservation

[0045] Bacillus velezensis, 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 5, 2024, with accession number CGMCC No. 31608. In this invention, this strain is named Bacillus velezensis YF01, also known as Bacillus velezensis YF01. Detailed Implementation

[0046] 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.

[0047] 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.

[0048] I. Terminology

[0049] In this invention, the term "bacterial body" refers to the live and / or dead cells of Bacillus belye.

[0050] 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 Bacillus belye are broken.

[0051] The term "pure crude enzyme" used in this invention refers to a cell-free extract obtained by centrifuging and collecting the supernatant of Bacillus belye after the pure bacterial cells are broken.

[0052] 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.

[0053] 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).

[0054] The term "cell suspension" as used in this invention refers to a liquid obtained by adding sterile physiological saline or pH buffer to a microbial fermentation culture after centrifugation to remove the supernatant, thereby suspending the precipitated microbial cells.

[0055] 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.

[0056] II. Implementation Plan

[0057] As mentioned earlier, existing strategies for managing CKD include medication, dialysis, and kidney transplantation, but these methods have limitations in reducing serum creatinine levels, often exhibiting limited efficacy and significant side effects. While some microorganisms, such as *Pseudomonas putida* and *Arthrobacter ureafaciens*, have been reported to produce creatinine-degrading enzymes, their creatinine removal efficiency is very low. Based on long-term microbiological research, the inventors have successfully screened a highly efficient *Bacillus velezensis* strain YF01 from the sediment of marine aquaculture ponds. This strain's cells and the enzymes it produces can completely degrade an initial concentration of 500 mg / L of creatinine within 48 hours and 24 hours, respectively. This not only has significant research value but also holds important application prospects for the efficient biodegradation and removal of creatinine.

[0058] Therefore, the first aspect of the present invention provides a strain of Bacillus vesiculus YF01 for the biodegradation of creatinine. Studies have found that the strain of Bacillus vesiculus YF01 can produce one or more enzymes that can catalyze the degradation of creatinine.

[0059] The inventors first successfully screened a strain of Bacillus velezensis YF01 from the bottom sediment of a marine aquaculture pond. After extracting genomic DNA, molecular identification via PCR amplification and 16S rDNA sequencing confirmed it to be Bacillus velezensis. Based on the above, this strain was identified and named Bacillus velezensis YF01. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCCNo. 31608.

[0060] The inventors have discovered that fermentation culture of Bacillus vesiculosus strain YF01 produces bacterial cells containing one or more enzymes capable of catalyzing the degradation of creatinine. In this invention, the mixture of these enzymes is referred to as crude enzyme, or Bacillus vesiculosus strain YF01 crude enzyme.

[0061] Further research revealed that centrifugation of the cell-free lysate after bacterial cell disruption, and the collection of the supernatant cell-free extract, served as the crude enzyme for *Bacillus belyssiensis* YF01. This readily explains why both the cells and the crude enzyme of *Bacillus belyssiensis* YF01 strain can catalyze the degradation of creatinine.

[0062] The results showed that the *Bacillus belyssioides* strain YF01, with an initial cell concentration of 1×10⁻⁶, was effective. 8 At a concentration of 500 mg / L, creatinine can be completely degraded and removed within 48 hours.

[0063] The results also showed that the crude enzyme produced by the Bacillus vesiculosus strain YF01 could completely degrade creatinine at an initial concentration of 500 mg / L within 24 hours at a protein concentration of 0.2 g / L.

[0064] The second aspect of the present invention provides a Bacillus vesiculus YF01 preparation for biodegrading creatinine, which belongs to a type of microbial preparation for biodegrading creatinine, and contains bacterial cells and / or crude enzymes of the Bacillus vesiculus YF01 strain as provided in the first aspect of the present invention.

[0065] In some preferred embodiments of the present invention, the Bacillus vesiculus YF01 preparation contains bacterial cells of the Bacillus vesiculus YF01 strain as described in the first aspect of the present invention.

[0066] According to some embodiments of the present invention, the Bacillus vesiculosus YF01 preparation for biodegrading creatinine is a liquid preparation.

[0067] For example, in some embodiments of the present invention, the cell concentration of Bacillus vesiculosus strain YF01 in the liquid formulation of the biodegradable creatinine is (1-10)×10⁻⁶. 8 / mL.

[0068] For example, in other embodiments of the present invention, the crude enzyme protein concentration of Bacillus vesiculosus strain YF01 in the liquid formulation of the biodegradable creatinine is 0.1-1.0 g / L.

[0069] According to other embodiments of the present invention, the Bacillus vesiculosus YF01 preparation for biodegrading creatinine is a solid powder preparation.

[0070] For example, in some embodiments of the present invention, the cell concentration of Bacillus vesiculosus strain YF01 in the biodegradable creatinine solid powder formulation is (1-10)×10⁻⁶. 8 / g, preferably (8-10)×10 8 / g.

[0071] For example, in other embodiments of the present invention, in the solid powder formulation of the biodegradable creatinine, the crude enzyme protein content of Bacillus vesiculosus strain YF01 is 0.1-1.0 g / kg, preferably 0.8-1.0 g / kg.

[0072] A third aspect of the present invention provides a method for preparing a Bacillus vesiculosus YF01 formulation of biodegradable creatinine as described in the second aspect of the present invention, comprising:

[0073] Step B: Inoculate the fermentation strain into the fermentation medium and ferment at 30-40℃, preferably 38-40℃, at a shaking speed of 100-300 rpm for 3-5 days to obtain the fermentation culture of Bacillus belyss YF01 strain.

[0074] Step C: Centrifuge the fermentation culture of Bacillus belye YF01 strain to harvest the bacterial cells of Bacillus belye YF01 strain;

[0075] The fermentation strain was obtained from the corresponding Bacillus berberis strain YF01 through seed culture.

[0076] 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 for comparison to 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 genome that encodes rRNA and exists in the genomes of all prokaryotic microorganisms. Figure 1 A molecular phylogenetic tree based on 16S rDNA of Bacillus belysium strain YF01 is shown.

[0077] In step C above, the centrifugation process includes centrifuging the liquid fermentation culture to obtain the precipitate (i.e., the bacterial cells of Bacillus vesiculosus strain YF01), washing it with physiological saline, and then centrifuging it again to obtain the bacterial cells of Bacillus vesiculosus strain YF01.

[0078] The present invention does not impose any particular restrictions on the centrifugal separation conditions in step C above. For example, in some cases, the analyte can be centrifuged for 10 minutes at 8000-10000 r / min.

[0079] According to the method of the present invention, the fermentation culture is a shaker or fermenter fermentation culture of the microbial strain, 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 0.1%-1% (v / v); preferably, the inoculation amount of the seed culture is 0.2%-0.5% (v / v); more preferably, the inoculation amount of the seed culture is 0.5% (v / v).

[0080] In some specific embodiments of the present invention, the above-mentioned fermentation culture medium, based on 1L of water, includes the following components in 1L of water:

[0081] 5-10g of peptone;

[0082] 5-10g of beef extract; and

[0083] 5-10g of glucose.

[0084] Preferably, the above fermentation medium, based on 1L of water, includes the following components in 1L of water:

[0085] 8-10g of peptone;

[0086] 8-10g of beef extract; and

[0087] 8-10g of glucose.

[0088] More preferably, 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.

[0089] According to some embodiments of the present invention, the preparation method of the Bacillus vesiculus YF01 preparation involved in the present invention further includes step A, which involves seed culture before step B: picking a single colony of Bacillus vesiculus YF01 strain provided by the present invention and inoculating it into 100 mL of fermentation liquid culture medium, and then culturing it in a shaker at 38°C and 200 r / min for 3 days to obtain the fermentation strain (seed liquid).

[0090] The inventors studied the effects of different temperatures on the growth of Bacillus belyss YF01 and found that Bacillus belyss YF01 grew rapidly at a temperature of 38℃.

[0091] According to some specific embodiments of the present invention, the preparation method further includes:

[0092] Step K: The cell suspension of Bacillus belyss YF01 strain is subjected to cell disruption treatment in an ice-water bath (i.e., ice-water mixture, 0-4℃) to obtain cell-free lysate of Bacillus belyss YF01 strain.

[0093] Step L: Centrifuge the cell-free lysate of Bacillus vesiculus strain YF01 and use the supernatant cell-free extract as the crude enzyme of Bacillus vesiculus strain YF01.

[0094] The present invention does not impose any particular restrictions on the centrifugal separation conditions in step L above. For example, in some cases, the analyte can be centrifuged at 15,000-18,000 r / min for 10-20 min.

[0095] The fourth aspect of this invention provides the use of the *Bacillus vesiculosus* YF01 preparation for biodegrading creatinine as described in the second aspect of this invention, or the *Bacillus vesiculosus* YF01 preparation for biodegrading creatinine prepared by the method described in the third aspect of this invention, in the preparation of a creatinine-lowering agent, comprising:

[0096] Step D: Wash the cells of Bacillus vesiculosus strain YF01 with physiological saline to obtain pure cells of Bacillus vesiculosus strain YF01.

[0097] Step E: In a physiological saline system, under low temperature conditions of 0℃-4℃, the bacterial cell purity of Bacillus belysium strain YF01 is broken by ultrasonication. After centrifugation, the supernatant is taken to obtain cell-free extract as crude enzyme purity of Bacillus belysium strain YF01.

[0098] Step F involves freeze-drying the pure bacterial cells and / or crude enzymes of Bacillus vesiculosus strain YF01, and then diluting the freeze-dried Bacillus vesiculosus strain YF01 to prepare a drug for lowering creatinine.

[0099] In some embodiments of the present invention, in step F, the freeze-dried Bacillus vesiculosus YF01 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent.

[0100] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Bacillus vesiculosus YF01 preparation to prepare a solid creatinine-lowering agent.

[0101] Particularly preferred is that the creatinine-lowering agent is an oral preparation.

[0102] III. Related materials and testing methods in this invention

[0103] 1. Materials

[0104] The marine aquaculture pond bottom mud used for screening bacterial strains in this invention was collected by the inventor in a large-scale marine aquaculture pond in Dalian, Liaoning Province in 2020.

[0105] 2. Detection Method

[0106] (1) The cell concentration in this invention is determined using the following method:

[0107] To determine the concentration of Bacillus belyss YF01 cells, Bacillus belyss YF01 culture was diluted with physiological saline and the cell concentration was directly measured using a flow cytometer (SYSMEX, Germany).

[0108] (2) The creatinine concentration in this invention is determined using the following method:

[0109] Creatinine 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 M ammonium acetate solution (v / v 20 / 80); the UV detection wavelength was 233 nm; the flow rate was 1 mL / min; the injection volume was 20 μL; and the temperature was 35 °C.

[0110] (3) The crude enzyme protein concentration in this invention was determined using the following method:

[0111] Cell-free extract of Bacillus vesiculosus YF01 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.

[0112] IV. Examples

[0113] 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.

[0114] Example 1:

[0115] (1) Prepare the growth medium for Bacillus belyssus YF01, with the following composition (per liter): 10.0 g peptone, 10.0 g beef extract, and 10.0 g glucose. Add 100 ml of the prepared liquid culture medium to a 500 ml Erlenmeyer flask, sterilize under high temperature and high pressure (121℃) for 20 minutes, and then sterilize again under ultraviolet irradiation in a clean workbench for 20 minutes.

[0116] (2) Under sterile conditions in a clean workbench, 0.5 ml of Bacillus belyss YF01 bacterial suspension was inoculated into a triangular flask of liquid culture medium. After batch culture for 3 days at a temperature of 38°C and a shaking speed of 200 rpm, Bacillus belyss YF01 cells were harvested by centrifugation (8000 rpm, 10 minutes) and the supernatant was discarded.

[0117] Add 20 mL of *Bacillus belyssae* YF01 cell suspension to a 50 mL glass tube and immerse the tube in ice water. Disrupt the *Bacillus belyssae* YF01 cells using an ultrasonic cell disruptor under the following conditions: ultrasonic power 400 W, 2-second intervals, 10-second ultrasonic oscillation, 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, then slowly pour off the supernatant as the cell-free extract (crude enzyme) of *Bacillus belyssae* YF01.

[0118] (3) Based on the different concentrations of creatinine, the cultured Bacillus vesiculosus YF01 cells and crude enzymes were added in a certain proportion as a rapid and efficient biocatalyst to achieve the purpose of rapidly and efficiently degrading and removing creatinine.

[0119] Figure 1 The strain we screened was most closely related to Bacillus belyes, so it was named Bacillus belyes YF01 strain.

[0120] Figure 2 This indicates that Bacillus belye YF01 at a cell concentration of 1.0 × 10⁻⁶ cells... 8 At a concentration of 500 mg / L, creatinine can be completely degraded within 48 hours, indicating that Bacillus belyeis YF01 has a strong biodegradation ability for creatinine.

[0121] Figure 3 This indicates that the cell-free extract (crude enzyme) of Bacillus belyss YF01 can catalyze the degradation of creatinine at a faster rate. At a protein concentration of 0.2 g / L, it can completely degrade creatinine with an initial concentration of 500 mg / L in 24 hours, demonstrating a higher creatinine degradation rate.

[0122] 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 Bacillus velezensis YF01 strain that degrades creatinine, which produces an enzyme that catalyzes the degradation of creatinine, and its accession number is CGMCC No. 31608.

2. The Bacillus belyssus YF01 according to claim 1, characterized in that, The Bacillus berberis YF01 was at a cell concentration of 1×10⁻⁶. 8 At a concentration of 500 mg / L, creatinine can be completely degraded and removed within 48 hours.

3. The Bacillus belyssus YF01 according to claim 1, characterized in that, The crude enzyme produced by the Bacillus vesiculosus YF01 can completely remove creatinine at an initial concentration of 500 mg / L within 24 hours at a protein concentration of 0.2 g / L.

4. A creatinine-degrading Bacillus vesiculus YF01 preparation, comprising Bacillus vesiculus YF01 cells and / or crude enzymes as described in any one of claims 1-3.

5. The Bacillus belyss YF01 preparation according to claim 4, characterized in that, The creatinine-degrading Bacillus vesiculus YF01 preparation is a liquid preparation; in the creatinine-degrading liquid preparation, the cell concentration of Bacillus vesiculus YF01 is (1-10)×10⁻⁶. 8 / mL; and / or, in the liquid formulation for degrading creatinine, the protein concentration of the crude enzyme of Bacillus vesiculosus YF01 is 0.1-1.0 g / L.

6. The Bacillus belyss YF01 preparation according to claim 4, characterized in that, The creatinine-degrading Bacillus vesiculosus YF01 preparation is a solid powder formulation; in the creatinine-degrading solid powder formulation, the cell concentration of Bacillus vesiculosus YF01 is (1-10)×10⁻⁶. 8 / g; and / or, in the solid powder formulation for degrading creatinine, the protein content of the crude enzyme of Bacillus vesiculosus YF01 is 0.1-1.0 g / kg.

7. The Bacillus belyss YF01 preparation according to claim 6, characterized in that, In the solid powder formulation for degrading creatinine, the cell concentration of Bacillus belye YF01 is (8-10)×10⁻⁶. 8 / g; and / or, in the solid powder formulation for degrading creatinine, the protein content of the crude enzyme of Bacillus vesiculus YF01 is 0.8-1.0 g / kg.

8. A method for preparing a creatinine-degrading Bacillus vesiculosus YF01 formulation as described in any one of claims 4-7, comprising: Step B: Inoculate the fermentation strain into the culture medium for fermentation culture to obtain the fermentation culture of Bacillus belyssus YF01; Step C: Centrifuge the fermentation culture of Bacillus belye YF01 to harvest the bacterial cells of Bacillus belye YF01; The fermentation strain is obtained from the corresponding Bacillus berberis YF01 through seed culture.

9. The preparation method according to claim 8, characterized in that, The culture medium, calculated per 1L of water, comprises the following components per 1L of water: 5-10g of peptone; 5-10g of beef extract; as well as 5-10g of glucose; The pH value of the fermentation medium is 7-8.

10. The preparation method according to claim 9, characterized in that, The culture medium, calculated per 1L of water, comprises the following components per 1L of water: 8-10g of peptone; 8-10g of beef extract; as well as 8-10g of glucose; In step B, the fermentation culture temperature is 30-40℃.

11. The preparation method according to claim 10, characterized in that, In step B, the fermentation culture temperature is 38-40℃.

12. The preparation method according to any one of claims 8-11, characterized in that, The preparation method further includes: Step K: The cell suspension of Bacillus belyss YF01 is subjected to cell disruption treatment under low temperature conditions to obtain cell-free lysate of Bacillus belyss YF01. Step L: Centrifuge the cell-free lysate of Bacillus belyss YF01 and take the supernatant cell-free extract as the crude enzyme of Bacillus belyss YF01. The low temperature is 0-4℃.

13. The use of a *Bacillus vesiculosus* YF01 preparation for degrading creatinine as described in any one of claims 4-7, or a *Bacillus vesiculosus* YF01 preparation prepared by any one of claims 8-11, in the preparation of a creatinine-lowering agent, comprising: Step D: Wash the cells of Bacillus belyssus YF01 with physiological saline to obtain pure Bacillus belyssus YF01 cells; Step E: In a physiological saline system, under low temperature conditions, the bacterial cell purity of Bacillus belysium YF01 is broken by ultrasonication. After centrifugation, the supernatant is taken to obtain a cell-free extract as the crude enzyme purity of Bacillus belysium YF01. Step F involves freeze-drying the pure bacterial cells and / or crude enzymes of Bacillus belye YF01, and then diluting the freeze-dried Bacillus belye YF01 preparation to produce a drug for degrading creatinine. The low temperature is 0-4℃.

14. The application according to claim 13, characterized in that, In step F, the freeze-dried Bacillus belye YF01 preparation is diluted with physiological saline to prepare a liquid creatinine-lowering agent; Alternatively, in step F, edible starch is used to dilute the freeze-dried Bacillus vesiculosus YF01 preparation to prepare a solid creatinine-lowering agent.

15. The application according to claim 14, characterized in that, The creatinine-lowering drug is an oral preparation.

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