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CN119144507A - Bacillus brevis YF02 preparation for degrading creatinine and application thereof - Google Patents

Bacillus brevis YF02 preparation for degrading creatinine and application thereof Download PDF

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CN119144507A
CN119144507A CN202411503417.XA CN202411503417A CN119144507A CN 119144507 A CN119144507 A CN 119144507A CN 202411503417 A CN202411503417 A CN 202411503417A CN 119144507 A CN119144507 A CN 119144507A
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bacillus pumilus
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creatinine
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闫海
吴安情
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Beijing Beike Yiran Biotechnology Co ltd
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Beijing Beike Yiran Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of biology, and relates to a Bacillus pumilus YF02 strain for degrading creatinine, which can produce enzymes for catalyzing and degrading creatinine. The invention also relates to a preparation of the Bacillus pumilus YF02 for degrading creatinine, wherein the preparation of the Bacillus pumilus YF02 contains bacterial cells and/or crude enzyme of the strain of the Bacillus pumilus YF 02. Research results show that the Bacillus pumilus YF02 cells for degrading creatinine and the enzymes produced by the same can degrade 500mg/L of creatinine within 48 hours and 24 hours respectively, and have important application prospects in the aspects of reducing human creatinine level, preventing and treating nephropathy and corresponding complications.

Description

Bacillus brevis YF02 preparation for degrading creatinine and application thereof
Technical Field
The invention belongs to the technical field of biology, and relates to a bacillus pumilus YF02 preparation for biologically degrading creatinine, and a preparation method and application thereof.
Background
Chronic Kidney Disease (CKD) is a ubiquitous chronic disease accompanied by various complications, and due to renal dysfunction, the excretion of harmful substances, particularly creatinine, is blocked, and finally, the condition is aggravated to cause uremia, so that the health and the quality of life of people are seriously affected. Currently, the prevalence of CKD worldwide has increased significantly, and has been recognized as a globally significant public health problem. Serum creatinine levels are one of the most critical biomarkers for assessing renal function, and there is a strong need to develop methods and techniques for efficient creatinine removal.
Creatinine (CREATININE, molecular formula C 4H7N3 O) is the end product of creatine metabolism in mammals, and serum creatinine sources in humans are divided into two types, exogenous sources from the metabolism of meat foods, endogenous sources from the metabolism of muscle tissues, and creatinine excretion is mainly excreted in urine excreted through the renal system. Under normal conditions, the production and excretion of creatinine in the human body is basically in a dynamic balance state, and when renal dysfunction is caused by kidney lesions, the accumulation of non-excreted creatinine in the body causes harm.
At present, main strategies for managing the CKD comprise medicines, dialysis and kidney replacement, but the main strategies have the defects of unobvious curative effect and large side effect, such as that although the treatment by adopting Chinese and western medicines such as non-steroidal anti-inflammatory medicines, chinese yam polysaccharide and the like has a certain effect, the glomerular filtration rate of patients is reduced, the accumulation of medicines or metabolites thereof is further caused, a series of adverse reactions are caused, and the serious renal function is even deteriorated. Aiming at the defects of the existing CKD treatment strategy, the novel thought is provided for treating the CKD by adopting microbial degradation to remove creatinine. It has been found that some microorganisms such as Pseudomonas putida (Pseudomonas putida) and Arthrobacter urealyticus (Arthrobacter ureafaciens) can produce creatinine hydrolase and the like, and have the ability to biodegrade creatinine, but have low creatinine removal efficiency.
Disclosure of Invention
One of the purposes of the invention is to provide a Bacillus pumilus YF02 strain for degrading creatinine, which has the advantages that bacterial cells of the strain and enzymes produced by the bacterial cells can be used for efficiently biodegrading creatinine and has important application prospect in creatinine removal, aiming at the problems of insignificant effect and side effects of medicines for reducing the human creatinine level in the prior art.
The second object of the invention is to provide a preparation of Bacillus pumilus YF02 for degrading creatinine and application thereof, wherein the preparation of Bacillus pumilus YF02 is prepared from the strain of Bacillus pumilus YF02 for degrading creatinine, and can be used for efficiently biodegrading creatinine.
To this end, the first aspect of the present invention provides a strain of Bacillus pumilus (Brevibacillus brevis) YF02 for degrading creatinine, which is capable of producing an enzyme for catalyzing the degradation of creatinine, and which has a preservation number of CGMCC No.31609.
In some embodiments of the invention, the Bacillus pumilus YF02 strain was able to degrade all creatinine removal at an initial concentration of 500mg/L for 48 hours at a cell concentration of 2X 10 8/mL.
In other embodiments of the invention, the crude enzyme produced by the Bacillus pumilus YF02 strain is capable of removing all creatinine at an initial concentration of 500mg/L in 24 hours at a protein concentration of 0.3 g/L.
In a second aspect, the invention provides a preparation of Bacillus pumilus YF02 for degrading creatinine, which contains bacterial cells and/or crude enzymes of the strain of Bacillus pumilus YF02 according to the first aspect, preferably, the preparation of Bacillus pumilus YF02 contains the strain of Bacillus pumilus YF02 according to the first aspect.
In some embodiments of the invention, the creatinine-degrading Bacillus pumilus YF02 preparation is a liquid preparation, preferably, the cell concentration of the Bacillus pumilus YF02 strain is (1-10) x 10 8/mL in the creatinine-degrading liquid preparation, and/or the protein concentration of the crude enzyme of the Bacillus pumilus YF02 strain is 0.1-1g/L in the creatinine-degrading liquid preparation.
In other embodiments of the present invention, the creatinine-degrading Bacillus pumilus YF02 preparation is a solid powder preparation, preferably, the cell concentration of the strain of Bacillus pumilus YF02 in the creatinine-degrading solid powder preparation is (1-10) ×10 8/g, more preferably (8-10) ×10 8/g, and/or the protein content of the crude enzyme of the strain of Bacillus pumilus YF02 in the creatinine-degrading solid powder preparation is 0.1-1.0g/kg, more preferably 0.8-1.0g/kg.
In a third aspect, the invention provides a preparation method of a preparation of the creatinine degrading bacillus pumilus YF02, which comprises the following steps:
Step B, inoculating a fermentation strain into a fermentation medium for fermentation culture to obtain a fermentation culture of the Bacillus pumilus YF02 strain;
Step C, performing centrifugal separation treatment on the fermentation culture of the Bacillus pumilus YF02 strain, and harvesting bacterial cells of the Bacillus pumilus YF02 strain;
wherein, the fermentation strain is obtained by seed culture of a corresponding Bacillus pumilus YF02 strain.
According to the invention, the fermentation medium comprises the following components in 1L of water, calculated as 1L of water:
5-10g of peptone, preferably 8-10g;
Beef extract 5-10g, preferably 8-10g, and
Glucose 5-10g, preferably 8-10g;
Preferably, the pH of the fermentation medium is 7-8;
further preferably, in step B, the temperature of the fermentation culture is 30-40 ℃, preferably 38-40 ℃.
According to some embodiments of the invention, the method further comprises:
Step K, performing cell disruption treatment on the cell suspension of the Bacillus pumilus YF02 strain at a low temperature to obtain a cell-free disruption solution of the Bacillus pumilus YF02 strain;
Step L, performing centrifugal separation on a cell-free disrupted solution of the Bacillus pumilus YF02 strain, and taking a supernatant cell-free extract as crude enzyme of the Bacillus pumilus YF02 strain;
Wherein the low temperature is 0-4 ℃.
According to a fourth aspect of the present invention, there is provided an application of the Bacillus pumilus YF02 preparation according to the second aspect of the present invention or the Bacillus pumilus YF02 preparation prepared by the preparation method according to the third aspect of the present invention in the preparation of creatinine-reducing agents, comprising:
Step D, washing bacterial cells of the Bacillus pumilus YF02 strain by using physiological saline to obtain bacterial cell pure products of the Bacillus pumilus YF02 strain;
Step E, in a physiological saline system, under the low-temperature condition, crushing a bacterial cell pure product of the Bacillus pumilus YF02 strain by adopting ultrasonic waves, and taking supernatant after centrifugal treatment to obtain a cell-free extract as a crude enzyme pure product of the Bacillus pumilus YF02 strain;
step F, freeze-drying a bacterial cell pure product and/or a crude enzyme pure product of the Bacillus pumilus YF02 strain, and diluting a freeze-dried Bacillus pumilus YF02 preparation to prepare a medicament for degrading creatinine;
Wherein the low temperature is 0-4 ℃.
In some embodiments of the invention, in step F, the lyophilized preparation of bacillus pumilus YF02 is diluted with physiological saline to form a liquid creatinine lowering agent.
In other embodiments of the present invention, in step F, the lyophilized preparation of bacillus pumilus YF02 is diluted with an edible starch to form a solid creatinine-lowering agent.
In some preferred embodiments of the invention, the creatinine-lowering agent is an oral formulation.
The research shows that the Bacillus pumilus YF02 strain for biologically degrading creatinine and the generated enzyme can be used for biologically degrading creatinine, and have important application prospects in the aspect of efficiently removing creatinine for treating human hypercreatinine blood and corresponding complications.
Drawings
In order that the invention may be readily understood, the invention will be described with reference to the accompanying drawings.
FIG. 1 shows a molecular evolution tree based on 16S rDNA for screening Bacillus pumilus YF 02.
FIG. 2 is a kinetic profile of Bacillus pumilus YF02 for the biodegradation of creatinine.
FIG. 3 is a kinetic profile of the enzymatic degradation of creatinine by Bacillus pumilus YF 02.
Preservation of bacterial species
Brevibacillus brevis (Brevibacillus brevis) separated and identified by Beijing Beike Yi Xie Bio-technology Co., ltd, has been preserved in China general microbiological culture Collection center (CGMCC; address: north Star, national academy of sciences of China, no.3, chaoyang area North Star, beijing, with a preservation date of 2024, 8 months, 5 days, and a preservation number of CGMCC No. 31609). The strain is named as a Bacillus pumilus YF02 strain (Brevibacillus brevis strain YF) and is also named as Bacillus pumilus YF02.
Detailed Description
In order that the invention may be readily understood, the invention will be described in detail. Before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Unless defined otherwise, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. 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, the preferred methods and materials are now described.
I. Terminology
The term "cell" as used herein refers to a live cell and/or dead cell of Brevibacillus brevis.
The term "crude enzyme" as used herein refers to a cell-free extract obtained by disrupting bacterial cells of Brevibacillus brevis and then centrifuging the resulting supernatant.
The term "crude enzyme purified product" as used herein refers to a cell-free extract obtained by disrupting a bacterial cell purified product of Brevibacillus brevis and then centrifuging the disrupted product to obtain a supernatant.
The term "microbial preparation" in the present invention refers to a preparation which is produced by using a microorganism having medical research value as a raw material and is used for preventing (health care), treating and diagnosing various physiological symptoms of human body by using a traditional technology or a modern biotechnology.
The term "edible starch" as used herein refers to starch which meets the national standard of edible starch (GB 31637-2016 national standard of food safety edible starch).
The term "water" used in the culture medium or the fermentation culture process of the invention refers to sterile pure water obtained by filtration through a 0.22. Mu. Filter, unless otherwise specified.
II. Description of the embodiments
As described above, the main strategies for the existing CKD management include methods of medicine, dialysis, kidney replacement, etc., but have the disadvantages of insignificant therapeutic effects and large side effects in reducing the concentration of creatinine. Although some microorganisms such as Pseudomonas putida (Pseudomonas putida) and Arthrobacter urealyticum (Arthrobacter ureafaciens) have been found to produce reports of creatinine hydrolase degradation of creatinine, these bacteria have very low creatinine removal efficiency. Based on the long-term study of microorganisms, the inventor successfully screens out a strain of Brevibacillus brevis YF02 (Brevibacillus brevis strain YF 02) capable of efficiently biodegrading creatinine from bottom mud of a mariculture pond, cells of the strain and generated enzymes can degrade creatinine with initial concentration of 500mg/L in 48 hours and 24 hours respectively, so that the strain has very important research value and important application prospect in the aspect of efficiently biodegrading and removing the creatinine.
Thus, the strain YF02 of Brevibacillus for biodegradation of creatinine according to the first aspect of the present invention is capable of producing an enzyme that catalyzes the degradation of creatinine.
The inventor firstly successfully screens out a Brevibacillus brevis YF02 strain from bottom mud of a mariculture pond. The strain was identified as Brevibacillus brevis by PCR amplification and molecular identification based on 16S rDNA sequencing by extracting genomic DNA, and was identified and designated as Brevibacillus brevis YF02 strain (Brevibacillus brevis strain YF 02) based on the above. The strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.31609.
The present inventors have found that, when Bacillus pumilus YF02 strain is fermented, bacterial cells produced during the fermentation culture contain one or more enzymes capable of catalyzing and degrading creatinine, and that a mixture of these enzymes is referred to as crude enzyme or Bacillus pumilus YF02 crude enzyme in the present invention.
Further research shows that the cell-free disrupted solution after bacterial cell disruption is subjected to centrifugal separation, and the supernatant cell-free extract is taken as crude enzyme of Bacillus pumilus YF 02. It is thus readily understood that both the cells of the strain Bacillus pumilus YF02 and the crude enzyme are capable of catalyzing the degradation of creatinine.
The research result shows that the Bacillus pumilus YF02 strain can degrade and remove creatinine with the initial concentration of 500mg/L in 48 hours under the initial concentration of 2X 10 8/mL of bacterial cells.
Crude enzyme produced by the Bacillus pumilus YF02 strain can degrade creatinine with an initial concentration of 500mg/L in 24 hours at a protein concentration of 0.3 g/L.
Based on the above, the second to fourth aspects of the present invention further provide the use or application of the Bacillus pumilus YF02 for the biodegradation of creatinine according to the first aspect of the present invention.
Specifically, the second aspect of the invention provides a preparation of Bacillus pumilus YF02 for biodegradation of creatinine, which belongs to a microbial preparation for biodegradation of creatinine, and comprises bacterial cells and/or crude enzymes of the strain of Bacillus pumilus YF02 according to the first aspect of the invention.
In some preferred embodiments of the invention, the preparation of Bacillus pumilus YF02 comprises bacterial cells of the strain Bacillus pumilus YF02 according to the first aspect of the invention.
According to some embodiments of the invention, the creatinine-biodegradable bacillus pumilus YF02 formulation is a liquid formulation.
In some embodiments of the invention, the concentration of cells of strain YF02 of Brevibacillus brevis in the liquid formulation for biologically degrading creatinine is (1-10). Times.10 8/mL.
In other embodiments of the invention, the crude enzyme of strain YF02 of Brevibacillus brevis has a protein concentration of 0.1-1.0g/L in the liquid formulation of biodegradable creatinine.
According to other embodiments of the present invention, the creatinine-biodegradable Bacillus pumilus YF02 formulation is a solid powder formulation.
In some embodiments of the present invention, in the creatinine biodegradable solid powder formulation, the cell concentration of Bacillus pumilus YF02 strain is (1-10) ×10 8/g, preferably (8-10) ×10 8/g.
In other embodiments of the present invention, the crude enzyme of strain YF02 of Brevibacillus brevis has a protein content of 0.1-1.0g/kg, preferably 0.8-1.0g/kg in the solid powder formulation for biologically degrading creatinine.
In a third aspect, the present invention provides a method for preparing a preparation of Bacillus pumilus YF02 for biologically degrading creatinine, which comprises:
Step B, inoculating a fermentation strain into a fermentation medium, and carrying out fermentation culture for 3-5 days at 30-40 ℃, preferably 38-40 ℃ and a rotation speed of a shaking table of 100-300 rpm to obtain a fermentation culture of the Bacillus pumilus YF02 strain;
Step C, performing centrifugal separation treatment on the fermentation culture of the Bacillus pumilus YF02 strain, and harvesting bacterial cells of the Bacillus pumilus YF02 strain;
wherein, the fermentation strain is obtained by seed culture of a corresponding Bacillus pumilus YF02 strain.
As known to those skilled in the art, at present, 16S rRNA is commonly used internationally for molecular identification of prokaryotic microorganisms, and thus 16S rRNA can be used for comparison of similarity to obtain homology. The fermentation strain used in the present invention is not limited to the field isolate used in the present invention, and 16S rDNA is a DNA sequence corresponding to the coding rRNA on the bacterial genome, and is present in the genome of all prokaryotic microorganisms. FIG. 1 shows the molecular evolution tree of Bacillus pumilus YF02 strain based on 16S rDNA.
In the step C, the centrifugal separation treatment includes subjecting the liquid fermentation culture to centrifugal separation treatment to obtain a precipitate (namely, bacterial cells of strain YF02 of bacillus pumilus), re-suspending and washing the precipitate with physiological saline, and then subjecting the precipitate to centrifugal separation treatment to obtain bacterial cells of strain YF02 of bacillus pumilus.
The conditions for the centrifugation in the above step C are not particularly limited in the present invention, and in some embodiments of the present invention, for example, the to-be-separated substance may be centrifuged at 8000-10000r/min for 10min.
According to the method of the invention, the fermentation culture is a shaking table or a fermentation tank for fermentation culture of strains, and the fermentation strains are inoculated into a fermentation culture medium in the form of seed liquid. The seed liquid is inoculated in an amount of 0.1 to 1% (v/v), preferably 0.2 to 0.5% (v/v), and more preferably 0.5% (v/v).
Specifically, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
5-10g of peptone;
Beef extract 5-10g, and
Glucose 5-10g.
Preferably, the fermentation medium comprises the following components in 1L of water, based on 1L of water:
8-10g of peptone;
beef extract 8-10g, and
Glucose 8-10g.
In some embodiments of the 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.
According to some embodiments of the invention, the preparation method of the preparation of the Bacillus pumilus YF02 also comprises a step A of seed culture before the step B, wherein the step A is to pick up a monoclonal colony of the Bacillus pumilus YF02 strain provided by the invention, inoculate the monoclonal colony into 100mL fermentation liquid culture medium, and shake culture the colony at the temperature of 38 ℃ and the rotating speed of 200r/min for 3 days to obtain a fermentation strain (seed liquid).
The inventors studied the effect of different temperatures on the growth of Bacillus pumilus YF02 and found that Bacillus pumilus YF02 grows fast at a temperature of 38 ℃.
According to some embodiments of the invention, the method further comprises:
Step K, performing cell disruption treatment on the cell suspension of the Bacillus pumilus YF02 strain in an ice water bath (namely an ice water mixture at 0-4 ℃) to obtain a cell-free disruption solution of the Bacillus pumilus YF02 strain;
And step L, carrying out centrifugal separation on the cell-free disruption solution of the Bacillus pumilus YF02 strain, and taking the supernatant cell-free extraction solution as crude enzyme of the Bacillus pumilus YF02 strain.
The conditions for the centrifugation in the above step L are not particularly limited in the present invention, and in some embodiments of the present invention, for example, the to-be-separated substance may be centrifuged at 15000-18000r/min for 10-20min.
According to a fourth aspect of the present invention, there is provided an application of the creatinine-biodegradable Bacillus pumilus YF02 preparation according to the second aspect of the present invention or the creatinine-biodegradable Bacillus pumilus YF02 preparation prepared by the preparation method according to the third aspect of the present invention in the preparation of creatinine-reducing agents, which comprises:
Step D, washing bacterial cells of the Bacillus pumilus YF02 strain by using physiological saline to obtain bacterial cell pure products of the Bacillus pumilus YF02 strain;
E, in a physiological saline system, under the low temperature condition of 0-4 ℃, crushing a bacterial cell pure product of the Bacillus pumilus YF02 strain by adopting ultrasonic waves, and taking a supernatant after centrifugal treatment to obtain a cell-free extract as a crude enzyme pure product of the Bacillus pumilus YF02 strain;
And F, freeze-drying a bacterial cell pure product and/or a crude enzyme pure product of the Bacillus pumilus YF02 strain, and diluting a freeze-dried Bacillus pumilus YF02 preparation to prepare the creatinine-reducing medicament.
In some embodiments of the invention, in step F, the lyophilized preparation of bacillus pumilus YF02 is diluted with physiological saline to form a liquid creatinine lowering agent.
In other embodiments of the present invention, in step F, the lyophilized preparation of bacillus pumilus YF02 is diluted with an edible starch to form a solid creatinine-lowering agent.
In some preferred embodiments of the invention, the creatinine-lowering agent is an oral formulation.
III, related materials and detection methods in the present invention
1. Material
The invention relates to a seawater culture pond bottom mud for screening strains, which is collected in a Liaoning large-scale seawater culture pond in 2020 by the inventor.
2. Detection method
(1) The cell concentration in the invention is determined by the following method:
Measurement of Brevibacillus YF02 cell concentration the cell concentration of the Brevibacillus YF02 culture was measured directly by flow cytometry (SYSMEX, germany) after dilution by physiological saline solution by a certain multiple.
(2) The creatinine concentration in the invention is determined by the following method:
The creatinine concentration was determined by high performance liquid chromatography (Shimadzu LC-20 AT), specifically a column Kromasil C18 (4.6X250 mm, 5. Mu. Particle size), a mobile phase methanol 0.5M ammonium acetate solution (v/v 20/80), an ultraviolet detection wavelength of 233nm, a flow rate of 1mL/min, a sample injection amount of 20. Mu.L, and a temperature of 35 ℃.
(3) The concentration of crude enzyme protein in the invention is determined by the following method:
Taking a cell-free extract of Brevibacillus brevis YF02, diluting the extract by a certain multiple by a phosphate buffer solution, adding a coomassie brilliant blue G-250 dye reagent proportionally, reacting for 10 minutes, measuring absorbance at 595nm by using a 722S visible spectrophotometer (Shanghai-Shang-Shanghai-Shangen), and calculating protein concentration by using a standard curve method.
IV, examples
The present invention will be specifically described below by way of specific examples. The experimental methods described below, unless otherwise specified, are all laboratory routine methods. The experimental materials described below, unless otherwise specified, are commercially available.
Example 1:
(1) A growth medium for Bacillus pumilus YF02 was prepared, and the medium consisted of (per liter) peptone 10.0g, beef extract 10.0g, and glucose 10.0g. 100 ml of the prepared liquid medium was added to a 500 ml Erlenmeyer flask, sterilized at high temperature and high pressure (121 ℃) for 20 minutes, and then sterilized under ultraviolet irradiation in a clean bench for 20 minutes.
(2) Inoculating 0.5 ml of Bacillus pumilus YF02 bacterial liquid in a triangular flask liquid culture medium under the aseptic condition in a clean workbench, carrying out batch culture for 3 days at the temperature of 38 ℃ and the rotation speed of a shaking table of 200 rpm, and obtaining the Bacillus pumilus YF02 cells by a method of pouring out supernatant after centrifugation (8000 rpm, 10 minutes).
And adding 20mL of the Bacillus pumilus YF02 cell suspension into a 50mL glass tube, inserting into ice water, and crushing the Bacillus pumilus YF02 cells by using an ultrasonic cell crusher, wherein the conditions are that the ultrasonic power is 400W, the interval is 2 seconds, the ultrasonic vibration is 10 seconds, and the crushing time is 15 minutes (5 minutes each time). After completion of cell disruption, the cell disruption solution was centrifuged at 15000 rpm for 20 minutes, and then the supernatant was slowly poured out as a cell-free extract (crude enzyme) of Brevibacillus brevis YF 02.
(3) According to different concentrations of creatinine, the Bacillus pumilus YF02 cells and crude enzyme prepared by culture are used as a quick and efficient biocatalyst, and are added according to a certain proportion, so that the purpose of quick and efficient degradation and removal of creatinine is achieved.
FIG. 1 shows that our strain screened was closest to Brevibacillus brevis and was therefore designated Brevibacillus brevis YF02 strain.
FIG. 2 shows that Bacillus pumilus YF02 can completely degrade creatinine with an initial concentration of 500mg/L in 48 hours at a cell concentration of 2.0X10 8/mL, indicating that Bacillus pumilus YF02 has a strong biodegradability for creatinine.
FIG. 3 shows that the cell-free extract (crude enzyme) of Brevibacillus brevis YF02 can catalyze and degrade creatinine at a faster rate, and at a protein concentration of 0.3g/L, creatinine with an initial concentration of 500mg/L can be completely degraded in 24 hours, so that the creatinine degradation rate is higher.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are used for explaining the present invention, not to be construed as limiting the present invention. The invention has been described with reference to exemplary embodiments, but it is understood that the words which have been used are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined in the appended claims, and the invention may be modified without departing from the scope and spirit of the invention. Although the invention is described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, as the invention extends to all other means and applications which perform the same function.

Claims (10)

1. A Bacillus pumilus YF02 strain for degrading creatinine can produce an enzyme for catalyzing and degrading creatinine, and the preservation number of the strain is CGMCC No.31609.
2. The strain of Brevibacillus brevis YF02 as claimed in claim 1, wherein the strain of Brevibacillus brevis YF02 can degrade and remove creatinine having an initial concentration of 500mg/L at a cell concentration of 2X 10 8/mL in 48 hours.
3. The strain of Brevibacillus brevis YF02 as claimed in claim 1, wherein the crude enzyme produced by the strain of Brevibacillus brevis YF02 can completely remove creatinine having an initial concentration of 500mg/L in 24 hours at a protein concentration of 0.3 g/L.
4. A preparation of Bacillus pumilus YF02 for degrading creatinine, which comprises the bacterial cell and/or crude enzyme of the strain of Bacillus pumilus YF02 described in any one of claims 1 to 3, preferably, the preparation of Bacillus pumilus YF02 described in any one of claims 1 to 3.
5. The preparation of Bacillus pumilus YF02 according to claim 4,
The preparation of the Bacillus pumilus YF02 for degrading the creatinine is a liquid preparation, preferably, the cell concentration of the Bacillus pumilus YF02 strain in the liquid preparation for degrading the creatinine is (1-10) multiplied by 10 8/mL, and/or the protein concentration of crude enzyme of the Bacillus pumilus YF02 strain in the liquid preparation for degrading the creatinine is 0.1-1.0g/L;
Or the creatinine-degrading solid powder preparation of Bacillus pumilus YF02 strain, preferably, the cell concentration of the strain of Bacillus pumilus YF02 is (1-10) x 10 8/g, more preferably (8-10) x 10 8/g, and/or the protein content of crude enzyme of the strain of Bacillus pumilus YF02 is 0.1-1.0g/kg, more preferably 0.8-1.0g/kg.
6. A method of preparing the creatinine-degrading bacillus pumilus YF02 formulation of any one of claims 4 or 5, comprising:
Step B, inoculating a fermentation strain into a culture medium for fermentation culture to obtain a fermentation culture of the Bacillus pumilus YF02 strain;
Step C, performing centrifugal separation treatment on the fermentation culture of the Bacillus pumilus YF02 strain, and harvesting bacterial cells of the Bacillus pumilus YF02 strain;
wherein, the fermentation strain is obtained by seed culture of a corresponding Bacillus pumilus YF02 strain.
7. The method of claim 6, wherein the fermentation medium comprises the following components in 1L of water, based on 1L of water:
5-10g of peptone, preferably 8-10g;
Beef extract 5-10g, preferably 8-10g, and
Glucose 5-10g, preferably 8-10g;
Preferably, the pH of the fermentation medium is 7-8;
further preferably, in step B, the temperature of the fermentation culture is 30-40 ℃, preferably 38-40 ℃.
8. The production method according to claim 6 or 7, characterized in that the production method further comprises:
Step K, performing cell disruption treatment on the cell suspension of the Bacillus pumilus YF02 strain at a low temperature to obtain a cell-free disruption solution of the Bacillus pumilus YF02 strain;
Step L, performing centrifugal separation on a cell-free disrupted solution of the Bacillus pumilus YF02 strain, and taking a supernatant cell-free extract as crude enzyme of the Bacillus pumilus YF02 strain;
Wherein the low temperature is 0-4 ℃.
9. Use of a formulation of bacillus pumilus YF02 for the degradation of creatinine according to claim 4 or 5 or a formulation of bacillus pumilus YF02 prepared by the preparation method according to any one of claims 6 to 8 for the preparation of a creatinine-lowering agent, comprising:
Step D, washing bacterial cells of the Bacillus pumilus YF02 strain by using physiological saline to obtain bacterial cell pure products of the Bacillus pumilus YF02 strain;
Step E, in a physiological saline system, under the low-temperature condition, crushing a bacterial cell pure product of the Bacillus pumilus YF02 strain by adopting ultrasonic waves, and taking supernatant after centrifugal treatment to obtain a cell-free extract as a crude enzyme pure product of the Bacillus pumilus YF02 strain;
step F, freeze-drying a bacterial cell pure product and/or a crude enzyme pure product of the Bacillus pumilus YF02 strain, and diluting a freeze-dried Bacillus pumilus YF02 preparation to prepare a medicament for degrading creatinine;
Wherein the low temperature is 0-4 ℃.
10. The use according to claim 9, wherein,
In the step F, the lyophilized preparation of the Bacillus pumilus YF02 is diluted by using physiological saline to prepare a liquid creatinine-reducing medicament;
Or in the step F, edible starch is used for diluting the freeze-dried preparation of the bacillus pumilus YF02 to prepare a solid creatinine-reducing medicament;
Preferably, the creatinine-lowering agent is an oral formulation.
CN202411503417.XA 2024-10-25 2024-10-25 Bacillus brevis YF02 preparation for degrading creatinine and application thereof Pending CN119144507A (en)

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