Cyclic nils sp. yl01 preparation for biodegradation of homocysteine and application thereof

Abstract

This invention belongs to the field of biotechnology and relates to a *N. circumscribed circumscribed filamentum* strain YL01 for biodegrading homocysteine, which produces an enzyme that catalyzes the biodegradation of homocysteine. This invention also relates to a *N. circumscribed filamentum* YL01 preparation for biodegrading homocysteine, which contains bacterial cells of the aforementioned *N. circumscribed filamentum* strain YL01 and / or crude enzyme. Research results show that the *N. circumscribed filamentum* strain YL01 and its produced enzyme provided by this invention are safe for human use and can efficiently biodegrade homocysteine, showing significant application potential in the treatment of hyperhomocysteinemia and related complications in humans.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a Cyclone circumstantiae YL01 preparation for biodegrading homocysteine, its preparation method and application. Background Technology

[0002] The incidence of cardiovascular and cerebrovascular diseases is increasing year by year, becoming a research hotspot in recent years. The main causes are atherosclerosis, thrombosis and the plaques formed by them, which obstruct blood flow and eventually lead to myocardial infarction or cerebral infarction. It has now become the disease with the highest mortality rate in my country and even the world.

[0003] Homocysteine ​​[L-4,4'-disulfide bis(2-aminobutyric acid, molecular formula: C8H16N2O4S2, molecular weight: 268.35], also known as L-homocysteine, is a sulfur-containing non-protein amino acid and an important intermediate in the biosynthesis of cysteine ​​and methionine. Abnormal metabolism of homocysteine ​​in the human body is closely related to a variety of diseases, especially hyperhomocysteinemia, which is a major risk factor for cardiovascular and cerebrovascular diseases. When the level of homocysteine ​​in plasma is higher than 15 µmol / L, it will cause harm to the human body.

[0004] Currently, there are two main methods for preventing and treating hyperhomocysteinemia: dietary therapy and drug therapy. Dietary therapy mainly involves supplementing with nutrients such as folic acid, vitamin B6, and vitamin B12, while reducing the intake of methionine-rich foods such as meat and cheese. Drug therapy mainly involves taking medications such as nebivolol and rosiglitazone, and injecting cobalamin, but these often come with adverse reactions such as headache, fatigue, low blood pressure, and bradycardia. Therefore, existing foods or drugs for controlling homocysteine ​​levels are not very effective and have side effects. Thus, it is necessary to conduct in-depth research on more effective treatments or methods for hyperhomocysteinemia. Summary of the Invention

[0005] One of the objectives of this invention is to address the problem that existing food or drug treatments for controlling homocysteine ​​are not very effective and have side effects. This invention provides a strain of Cynocysteine-degrading Neisseria circumvallate YL01, which is safe for human use and can efficiently biodegrade homocysteine. It has important application prospects in the removal of homocysteine.

[0006] The second objective of this invention is to provide a Cyclone circumvallate YL01 preparation for biodegrading homocysteine ​​and its application. The Cyclone circumvallate YL01 preparation is made from the Cyclone circumvallate YL01 strain used for biodegrading homocysteine ​​and can efficiently biodegrade homocysteine.

[0007] Therefore, the first aspect of the present invention provides a strain of Niallia circulans YL01 that biodegrades homocysteine, which is capable of producing an enzyme that catalyzes the degradation of homocysteine, and its accession number is CGMCCNo.37025.

[0008] The second aspect of the present invention provides a biodegradable homocysteine ​​preparation of *Nyctalida circumflex* YL01 containing bacterial cells and / or crude enzymes of *Nyctalida circumflex* YL01 strain as described in the first aspect of the present invention.

[0009] According to some embodiments of the present invention, the biodegradable homocysteine-containing cyclonitrogen YL01 preparation is a liquid preparation.

[0010] Preferably, in the liquid formulation of biodegradable homocysteine, the cell concentration of *Nyctalobacterium circumflexum* strain YL01 is (2-5) × 10⁻⁶. 7 / mL.

[0011] Preferably, in the liquid formulation of biodegradable homocysteine, the protein concentration of the crude enzyme of Niemon Circulatinga YL01 strain is 3-10 mg / mL.

[0012] In some other embodiments of the present invention, the biodegradable homocysteine-containing circotropic Niell YL01 formulation is a solid powder formulation.

[0013] Preferably, in the solid powder formulation of biodegradable homocysteine, the cell concentration of *Nyctalobacterium circumflexum* strain YL01 is (1-6) × 10⁻⁶. 7 / g, more preferably (2-5)×10 7 / g.

[0014] Preferably, in the solid powder formulation of biodegradable homocysteine, the protein content of the crude enzyme of Niemon Circulatinga YL01 strain is 1-10 mg / g, more preferably 5-10 mg / g.

[0015] A third aspect of the present invention provides a method for preparing a biodegradable homocysteine-containing Niacinib YL01 formulation as described in the second aspect of the present invention, comprising:

[0016] Step B: Inoculate the fermentation strain into the fermentation medium for fermentation culture to obtain the fermentation culture of Nyctalobacterium rings YL01.

[0017] Step C: Centrifuge the fermentation culture of Nyctalobacterium circumflexum strain YL01 to harvest the bacterial cells of Nyctalobacterium circumflexum strain YL01.

[0018] The fermentation strain was obtained from the corresponding Cynodon yunnanensis strain YL01 through seed culture.

[0019] According to the present invention, the fermentation medium comprises, per 1L of water, the following components:

[0020] 5-10g of peptone; preferably 8-10g;

[0021] 5-10g of yeast powder; preferably 8-10g; and

[0022] 3-8 g of glucose; preferably 4-6 g;

[0023] Preferably, the pH value of the fermentation medium is 6-7;

[0024] More preferably, in step B, the fermentation culture temperature is 18-40℃, more preferably 30-37℃.

[0025] According to some embodiments of the present invention, the preparation method further includes:

[0026] Step K: The cell suspension of the Cyclone circumvallate strain YL01 is subjected to cell disruption treatment under low temperature conditions to obtain cell-free lysate of Cyclone circumvallate strain YL01.

[0027] Step L: Centrifuge the cell-free lysate of Nyctalobacterium circumflexum strain YL01 and take the supernatant cell-free extract as the crude enzyme of Nyctalobacterium circumflexum strain YL01.

[0028] The low temperature is 0-4℃.

[0029] The fourth aspect of this invention provides the use of the *Nyctalida circinata* YL01 preparation described in the second aspect of this invention, or the *Nyctalida circinata* YL01 preparation prepared by the method described in the third aspect of this invention, in the preparation of homocysteine-lowering agents, comprising:

[0030] Step D: Wash the cells of N. circumferentiale YL01 strain with physiological saline to obtain pure cells of N. circumferentiale YL01 strain.

[0031] Step E: In a physiological saline system, under low temperature conditions, the cell purity of N. circumferentiale YL01 strain is broken by ultrasonication. After centrifugation, the supernatant is taken to obtain cell-free extract as crude enzyme purity of N. circumferentiale YL01 strain.

[0032] Step F involves freeze-drying the pure bacterial cells and / or crude enzyme of Nyctalidae strain YL01, and then diluting the freeze-dried Nyctalidae strain YL01 to prepare a homocysteine-lowering agent.

[0033] The low temperature is 0-4℃.

[0034] In some embodiments of the present invention, in step F, the freeze-dried *Niella circinata* YL01 preparation is diluted with physiological saline to prepare a liquid homocysteine-lowering agent; preferably, the liquid homocysteine-lowering agent is an oral preparation.

[0035] In other embodiments of the present invention, in step F, the freeze-dried *Nyctalida circinata* YL01 preparation is diluted with edible starch to prepare a solid homocysteine-lowering agent; preferably, the solid homocysteine-lowering agent is an oral preparation.

[0036] The inventors have discovered that the Cyclone circumvallate strain YL01 and the enzyme it produces, which are used to biodegrade homocysteine, provided by this invention, are safe for human use and can biodegrade homocysteine. They have significant application prospects in the efficient removal of homocysteine ​​for the treatment of hyperhomocysteinemia and related complications in humans. Attached Figure Description

[0037] To facilitate understanding of the present invention, a more detailed description will be provided below with reference to the accompanying drawings, during which the above and other objects, features, and advantages of the present invention will become more apparent. The accompanying drawings are provided to further illustrate the present invention and form part of the specification. They are used together with the embodiments of the present invention to explain the invention and do not constitute a limitation thereof.

[0038] Figure 1 A molecular phylogenetic tree based on 16S rDNA was used to screen for Circulating Nielzulbium YL01.

[0039] Figure 2 The kinetic curves for the biodegradation of homocysteine ​​by *Nyctalobacterium circumflexum* YL01 are shown.

[0040] Figure 3 The kinetic curves for the degradation of homocysteine ​​by the enzyme YL01 of *Nyctalobacterium circumflexum*.

[0041] strain preservation

[0042] The bacterial strain provided in this invention is classified and named *Niallia circulans*. It was isolated and identified by Beijing Yiran Biotechnology Co., Ltd., and has been deposited at the China General Microbiological Culture Collection Center (CGMCC; address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing) on ​​December 11, 2025, with accession number CGMCC No. 37025. In this invention, this strain is named *Niallia circulans* strain YL01, also known as *Niallia circulans* YL01. Detailed Implementation

[0043] 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.

[0044] 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.

[0045] In this invention, the range of dosage concentration, temperature or other physical or chemical properties or characteristics, unless otherwise specified, covers or includes the upper and lower limits of that range.

[0046] I. Terminology

[0047] In this invention, the term "cell" refers to the live and / or dead cells of *Nyctalus circinus*.

[0048] 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 *Nyctalus circinus* are broken.

[0049] The term "pure crude enzyme" used in this invention refers to a cell-free extract obtained by centrifuging and collecting the supernatant of pure Cynozobia circulatory bacteria cells after the cells are broken.

[0050] The term "microbial preparation" as used in this invention refers to preparations of various forms made from microorganisms with medicinal 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.

[0051] 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).

[0052] The term "cell suspension" as used in this invention refers to the liquid obtained by adding approximately 50 mL of sterile physiological saline to a microbial fermentation culture after centrifugation to remove the supernatant, thereby suspending the precipitated microbial cells.

[0053] 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.

[0054] II. Implementation Plan

[0055] As mentioned above, existing drugs are not very effective in controlling homocysteine ​​levels in the human body and have toxic side effects. Therefore, the inventors have conducted extensive and in-depth research on the biodegradation of homocysteine.

[0056] The inventors noted that although lactic acid bacteria can promote gut health and reduce homocysteine ​​accumulation to some extent through a series of metabolic regulation, there are no reports of the strains used directly and efficiently biodegrading homocysteine.

[0057] The inventors also noted that Niallia circulans is a prokaryotic microorganism mainly used in the industrial production of thermostable α-amylase and β-glucanase, and has potential applications in biotechnology and industry. However, no research reports on its biodegradation of homocysteine ​​have been found to date.

[0058] Based on long-term research in microorganisms, the inventors have successfully screened a highly efficient pure strain of microorganism capable of biodegrading homocysteine ​​from the intestinal flora of healthy humans. This strain and its produced enzymes are both highly efficient at biodegrading homocysteine, possessing significant research value and promising application prospects in the efficient biodegradation and removal of homocysteine. This invention is thus derived.

[0059] Therefore, the first aspect of the present invention provides a Cyclone circumvallate strain YL01 for biodegrading homocysteine. Studies have found that the Cyclone circumvallate strain YL01 can produce one or more enzymes that can catalyze the biodegradation of homocysteine.

[0060] The inventors first successfully screened a strain of *Niallia circulans* YL01 from the gut microbiota of healthy humans. Genomic DNA was extracted, and molecular identification, including PCR amplification and 16S rDNA sequencing, confirmed it to be *Niallia circulans*. Based on this, the strain was identified and named *Niallia circulansstrain YL01*. This strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 37025.

[0061] The inventors have discovered that fermentation culture of Nyctalobacterium circumflexum strain YL01 produces bacterial cells containing one or more enzymes capable of catalyzing the biodegradation of homocysteine. In this invention, the mixture of these enzymes is referred to as crude enzyme, or Nyctalobacterium circumflexum strain YL01 crude enzyme.

[0062] 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 *Nyctalobacterium circumflexum* YL01. This readily explains why both the cells and the crude enzyme of *Nyctalobacterium circumflexum* YL01 strain can catalyze the biodegradation of homocysteine.

[0063] The results showed that, along with the initial concentration of *Nyctalobacterium circumflex* strain YL01 from 1.0 × 10⁻⁶ cells, the bacterial growth rate increased. 6 / mL grew to 3.2×10 7 / mL, can remove 86% of the initial concentration of 500 mg / L homocysteine ​​within 120 hours.

[0064] The results also showed that the crude enzyme protein concentration of the Cyclone circumvallate strain YL01 was 3.1 mg / mL, which was able to degrade 73% of the homocysteine ​​at an initial concentration of 500 mg / L within 24 hours.

[0065] Based on the above, the second to fourth aspects of the present invention further provide the use or application of the Cyclone circinus bacteria described in the first aspect of the present invention for the biodegradation of homocysteine.

[0066] Specifically, the second aspect of the present invention provides a biodegradable homocysteine ​​preparation of *Nyctalus circinus* YL01, which belongs to a biodegradable homocysteine ​​microbial preparation, containing bacterial cells and / or crude enzymes of *Nyctalus circinus* YL01 strain as provided in the first aspect of the present invention.

[0067] In some preferred embodiments of the present invention, the Cyclone circumflex YL01 preparation contains bacterial cells of the Cyclone circumflex YL01 strain as described in the first aspect of the present invention.

[0068] According to some embodiments of the present invention, the biodegradable homocysteine-containing cyclonitrogen YL01 preparation is a liquid preparation.

[0069] For example, in some embodiments of the present invention, the cell concentration of *Nyctalobacterium circumflexum* strain YL01 in the liquid formulation of the biodegradable homocysteine ​​is (2-5) × 10⁻⁶. 7 / mL.

[0070] For example, in other embodiments of the invention, the crude enzyme protein concentration of Niemon Circulatinga YL01 strain is 3-10 mg / mL in the liquid formulation of the biodegradable homocysteine.

[0071] According to other embodiments of the present invention, the biodegradable homocysteine-containing circotropic Nyctalidobacterium YL01 formulation is a solid powder formulation.

[0072] For example, in some embodiments of the present invention, the cell concentration of *Nyctalobacterium circumflexum* strain YL01 in the biodegradable homocysteine ​​solid powder formulation is (1-6) × 10⁻⁶. 7 / g, preferably (2-5)×10 7 / g.

[0073] For example, in other embodiments of the present invention, the protein content of the crude enzyme of Niemon Circulatinga YL01 strain in the solid powder formulation of the biodegraded homocysteine ​​is 1-10 mg / g, preferably 5-10 mg / g.

[0074] A third aspect of the present invention provides a method for preparing a biodegradable homocysteine-containing Niacinib YL01 formulation as described in the second aspect of the present invention, comprising:

[0075] Step B: Inoculate the fermentation strain into the fermentation medium and ferment at 18-40℃, preferably 30-37℃, at a shaking speed of 100-300 rpm for 3-5 days to obtain the fermentation culture of Cynodon yunnanensis strain YL01.

[0076] Step C: Centrifuge the fermentation culture of Nyctalobacterium circumflexum strain YL01 to harvest the bacterial cells of Nyctalobacterium circumflexum strain YL01.

[0077] The fermentation strain was obtained from the corresponding Cynodon yunnanensis strain YL01 through seed culture.

[0078] 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 Nyctalobacterium circumflexum strain YL01 is shown.

[0079] In step C above, the centrifugation process includes resuspending and washing the precipitate (i.e., the cells of Nyctalobacterium circumflexum YL01 strain) obtained by centrifugation of the liquid fermentation culture with physiological saline, and then centrifuging it again to obtain the cells of Nyctalobacterium circumflexum YL01 strain.

[0080] 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 min at 8000-10000 r / min.

[0081] 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 liquid. The inoculation amount of the seed liquid is 0.1%-1% (v / v), preferably 0.2%-0.5% (v / v), and more preferably 0.5% (v / v).

[0082] 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:

[0083] 5-10g of peptone;

[0084] 5-10g of yeast powder; and

[0085] 3-8g of glucose.

[0086] Preferably, the above fermentation medium, based on 1L of water, includes the following components in 1L of water:

[0087] 8-10g of peptone;

[0088] 8-10g of yeast powder; and

[0089] 4-6 g of glucose.

[0090] More preferably, the initial pH of the fermentation medium is adjusted to 6-7 using a 40% (wt / v) sodium hydroxide solution and a 36% (v / v) hydrochloric acid solution.

[0091] According to some embodiments of the present invention, the preparation method of the *Nyctalobacterium circumflexum* YL01 preparation of the present invention further includes step A, which involves seed culture before step B: picking a single colony of *Nyctalobacterium circumflexum* YL01 provided by the present invention and inoculating it into 100 mL of fermentation liquid culture medium, and then culturing it in a shaker at 30°C and 200 r / min for 5 days to obtain the fermentation strain (seed liquid).

[0092] The inventors studied the effects of different temperatures on the growth of *Nyctalus circinus* YL01 and found that *Nyctalus circinus* YL01 grows rapidly at a temperature of 30℃.

[0093] According to some specific embodiments of the present invention, the preparation method further includes:

[0094] Step K: The cell suspension of the Cyclone circumvallate strain YL01 is subjected to cell disruption treatment in an ice-water bath (i.e., an ice-water mixture, 0-4°C) to obtain a cell-free lysate of Cyclone circumvallate strain YL01.

[0095] Step L: Centrifuge the cell-free lysate of Nyctalobacterium circumflexum strain YL01 and use the supernatant cell-free extract as the crude enzyme of Nyctalobacterium circumflexum strain YL01.

[0096] 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.

[0097] The fourth aspect of this invention provides the use of the *Nyctalida circinata* YL01 preparation of biodegradable homocysteine ​​as described in the second aspect of this invention, or the *Nyctalida circinata* YL01 preparation of biodegradable homocysteine ​​prepared by the preparation method described in the third aspect of this invention, in the preparation of homocysteine-lowering agents, comprising:

[0098] Step D: Wash the cells of N. circumferentiale YL01 strain with physiological saline to obtain pure cells of N. circumferentiale YL01 strain.

[0099] Step E: In a physiological saline system, under low temperature conditions of 0-4℃, the pure bacterial cells of Nyctalobacterium circumflexum strain YL01 are broken by ultrasonication. After centrifugation, the supernatant is taken to obtain a cell-free extract as the crude enzyme pure product of Nyctalobacterium circumflexum strain YL01.

[0100] Step F involves freeze-drying the pure bacterial cells and / or crude enzymes of Nyctalidae strain YL01, and then diluting the freeze-dried Nyctalidae strain YL01 to prepare a homocysteine-lowering agent.

[0101] In some embodiments of the present invention, in step F, the freeze-dried Niacinib YL01 preparation is diluted with physiological saline to prepare a liquid homocysteine-lowering agent.

[0102] In some other embodiments of the present invention, in step F, edible starch is used to dilute the freeze-dried Niacinib YL01 preparation to prepare a solid homocysteine-lowering agent.

[0103] In some preferred embodiments of the present invention, the homocysteine-lowering agent is an oral formulation.

[0104] III. Related materials and testing methods in this invention

[0105] 1. Materials

[0106] The healthy human intestinal fecal bacteria capsule samples involved in this invention were purchased from Beijing Fumate Biotechnology Co., Ltd., and were licensed for use in scientific research activities.

[0107] 2. Detection Method

[0108] (1) The cell concentration in this invention is determined using the following method:

[0109] To determine the concentration of N. circumferentiale YL01 cells, N. circumferentiale YL01 culture was diluted with physiological saline and the cell concentration was directly measured using a flow cytometer (SYSMEX, Germany).

[0110] (2) The homocysteine ​​concentration in this invention is determined using the following method:

[0111] The method for determining homocysteine ​​concentration involves dissolving homocysteine ​​in liquid culture medium in pure aqueous solution according to the specified ratio, reacting it with chloroformic acid-9-fluorenyl methyl ester derivatizing reagent for 10 minutes, centrifuging, taking an appropriate amount of supernatant, and determining the homocysteine ​​concentration in liquid culture medium using a RID-20A high performance liquid chromatograph (Shimadzu).

[0112] (3) The crude enzyme protein concentration in this invention is determined using the following method:

[0113] Cell-free extract of Cynozobia circinata YL01 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.

[0114] IV. Examples

[0115] 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.

[0116] Example 1: Preparation of Cyclone Nyctaginis strain YL01 bacterial cells and crude enzymes

[0117] (1) Prepare the growth medium for Neisseria circularis YL01, which consists of (per liter): 10.0 g peptone, 10.0 g yeast extract, and 5.0 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 °C) for 20 minutes, and then sterilize again under ultraviolet irradiation in a clean workbench for 20 minutes.

[0118] (2) Under sterile conditions in a clean workbench, 1 ml of Nycticebus circumflexus YL01 bacterial suspension was inoculated into a triangular flask of liquid culture medium. After batch culture for 5 days at a temperature of 30℃ and a shaking speed of 200 rpm, Nycticebus circumflexus YL01 cells were harvested by centrifugation (8000 rpm, 10 minutes) and discarding the supernatant.

[0119] Figure 1 The strain we screened was most closely related to *Nyctalida circinata*, and was therefore named *Nyctalida circinata* strain YL01.

[0120] (3) Take 20 mL of N. circumferential cytokine YL01 cell suspension and add it to a 50 mL glass tube. Then, insert the tube into ice water and use an ultrasonic cell disruptor to disrupt the N. circumferential cytokine YL01 cells. The conditions are: ultrasonic power 400W, interval of 2 seconds, ultrasonic oscillation for 10 seconds, and disruption time 15 minutes (5 minutes each time). After cell disruption, centrifuge the cell disruption solution at 15000 rpm for 20 minutes, and then slowly pour out the supernatant as the cell-free extract (crude enzyme) of N. circumferential cytokine YL01.

[0121] Example 2: Degradation of homocysteine ​​by Cynozobacterium circumflexure YL01 strain cells and crude enzymes

[0122] Based on different homocysteine ​​concentrations, cultured Cynozobia cyclicis YL01 cells and crude enzymes were added in a certain proportion as a rapid, safe, and efficient biocatalyst to achieve rapid and efficient degradation and removal of homocysteine. The results are as follows: Figure 2 and Figure 3 As shown.

[0123] Figure 2 This indicates that within 120 hours, the concentration of *Nyctalobacterium circumflex* YL01 cells decreased from 1.0 × 10⁻⁶. 6 / mL grew to 3.2×10 7 The concentration of homocysteine ​​was reduced by 86% to an initial concentration of 500 mg / L by 1 mL / mL, indicating that *Nyctalobacterium circumflexum* YL01 has a strong biodegradation ability for homocysteine.

[0124] Figure 3 The results indicate that the cell-free extract (crude enzyme) of Niacinus circinus YL01 can catalyze the degradation of homocysteine ​​at a relatively fast rate. At a protein concentration of 3.1 mg / mL, it can degrade 73% of homocysteine ​​at an initial concentration of 500 mg / L in 120 hours, demonstrating a high homocysteine ​​degradation rate.

[0125] 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 strain of Niallia circulans YL01 that biodegrades homocysteine, which produces an enzyme that catalyzes the degradation of homocysteine, and its preservation number is CGMCC No.37025.

2. A formulation of *Nyctalus circumflexus* YL01 for the biodegradation of homocysteine, comprising bacterial cells and / or crude enzymes of the *Nyctalus circumflexus* YL01 strain as described in claim 1.

3. The Cyclone circumvallate YL01 preparation according to claim 2, characterized in that, The *Nyctalida circophylla* YL01 preparation for biodegrading homocysteine ​​is a liquid preparation; preferably, in the liquid preparation for biodegrading homocysteine, the cell concentration of *Nyctalida circophylla* YL01 strain is (2-5) × 10⁻⁶. 7 / mL; and / or, in the liquid formulation of biodegradable homocysteine, the protein concentration of the crude enzyme of Niemon Circulatinga YL01 strain is 3-10 mg / mL.

4. The *Nyctalobacterium circumflexum* YL01 preparation according to claim 2, characterized in that, The biodegradable homocysteine-containing Nyctalobacterium circumflexures YL01 formulation is a solid powder formulation; preferably, in the biodegradable homocysteine-containing solid powder formulation, the cell concentration of Nyctalobacterium circumflexures YL01 strain is (1-6)×10⁻⁶. 7 g, more preferably (2-5)×10 7 / g; and / or, in the solid powder formulation of biodegradable homocysteine, the protein content of the crude enzyme of Nyctalobacterium circumflexum YL01 strain is 1-10 mg / g, more preferably 5-10 mg / g.

5. A method for preparing a biodegradable homocysteine-containing Nigelella circinata YL01 formulation as described in any one of claims 2-4, comprising: Step B: Inoculate the fermentation strain into the culture medium for fermentation culture to obtain the fermentation culture of Nyctalobacterium rings YL01. Step C: Centrifuge the fermentation culture of Nyctalobacterium circumflexum strain YL01 to harvest the bacterial cells of Nyctalobacterium circumflexum strain YL01. The fermentation strain was obtained from the corresponding Cynodon yunnanensis strain YL01 through seed culture.

6. The preparation method according to claim 5, characterized in that, The fermentation medium, calculated per liter of water, comprises the following components per liter of water: 5-10g of peptone; preferably 8-10g; 5-10g of yeast powder; preferably 8-10g; as well as 3-8 g of glucose; preferably 4-6 g; Preferably, the pH value of the fermentation medium is 6-7; More preferably, in step B, the fermentation culture temperature is 18-40℃, more preferably 30-37℃.

7. The preparation method according to claim 5 or 6, characterized in that, The preparation method further includes: Step K: The cell suspension of the Cyclone circumvallate strain YL01 is subjected to cell disruption treatment under low temperature conditions to obtain cell-free lysate of Cyclone circumvallate strain YL01. Step L: Centrifuge the cell-free lysate of Nyctalobacterium circumflexum strain YL01 and take the supernatant cell-free extract as the crude enzyme of Nyctalobacterium circumflexum strain YL01. The low temperature is 0-4℃.

8. The use of a *Nyctalida circinata* YL01 preparation of biodegradable homocysteine ​​as described in any one of claims 2-4, or a *Nyctalida circinata* YL01 preparation obtained by the preparation method as described in any one of claims 5-7, in the preparation of homocysteine-lowering agents, comprising: Step D: Wash the cells of N. circumferentiale YL01 strain with physiological saline to obtain pure cells of N. circumferentiale YL01 strain. Step E: In a physiological saline system, under low temperature conditions, the cell purity of N. circumferentiale YL01 strain is broken by ultrasonication. After centrifugation, the supernatant is taken to obtain cell-free extract as crude enzyme purity of N. circumferentiale YL01 strain. Step F involves freeze-drying the pure bacterial cells and / or crude enzyme of Nyctalidae strain YL01, and then diluting the freeze-dried Nyctalidae strain YL01 to prepare an agent for degrading homocysteine. The low temperature is 0-4℃.

9. The application according to claim 8, characterized in that, In step F, the freeze-dried Niellium circumcision YL01 preparation is diluted with physiological saline to prepare a liquid homocysteine-lowering agent; preferably, the liquid homocysteine-lowering agent is an oral preparation.

10. The application according to claim 8, characterized in that, In step F, the freeze-dried Niellium circumcisionum YL01 preparation is diluted with edible starch to prepare a solid homocysteine-degrading agent; preferably, the solid homocysteine-degrading agent is an oral preparation.

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