A method for producing single-cell protein using hydrogen-oxidizing bacteria

Abstract

This invention provides a method for producing single-cell protein using hydroxyl bacteria, comprising the following steps: Hydroxyl bacteria are inoculated into a fermentation medium, and a mixture of hydrogen, oxygen, and carbon dioxide is introduced for fermentation culture, allowing the hydroxyl bacteria to synthesize single-cell protein and obtain a fermentation broth; the fermentation broth is separated into solid and liquid components to obtain crude single-cell protein; the crude single-cell protein is dispersed in a resuspension solvent for cell disruption; immobilized nuclease is added to the disruption solution; after reaction, the immobilized nuclease is recovered using magnetic separation; the pH of the disruption solution is adjusted to 4.5-5.5 to cause protein flocculation and precipitation; solid-liquid separation, washing, and drying are performed to obtain single-cell protein. This method for producing single-cell protein using hydroxyl bacteria achieves mild, efficient, and low-cost nucleic acid removal with good selectivity and minimal protein loss, effectively improving product quality.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for producing single-cell proteins using hydroxyl bacteria. Background Technology

[0002] Hydrogen-oxidizing bacteria (HOB) are a type of chemoautotrophic microorganism that can utilize H2 as an electron donor and O2 as an electron acceptor to fix CO2 and absorb nitrogen to synthesize microbial protein. HOB is one of the fastest-growing microorganisms, with high cell yield and advantages such as high protein content and a balanced amino acid composition, comparable to fishmeal and soy protein, showing great potential in single-cell protein production.

[0003] Nucleic acid content is a crucial quality indicator for single-cell protein produced by microorganisms, typically accounting for 5%-18% of its dry weight. When animals or humans ingest single-cell protein, the nucleic acids are metabolized into uric acid. Excessive uric acid can lead to hyperuricemia, increasing the risk of diseases such as gout and kidney stones. Single-cell protein derived from hydroxyl bacteria contains a high level of nucleic acid, posing certain health risks when used as feed or food ingredients. Therefore, reducing the nucleic acid content in single-cell protein derived from hydroxyl bacteria is a key technological challenge in this field.

[0004] Existing nucleic acid removal technologies mostly involve post-processing after fermentation. Since nucleic acids are long-chain macromolecules, they are easily adsorbed and physically entangled with proteins. Conventional methods have poor selectivity and are prone to causing a large loss of protein during processing. Introducing chemical reagents also increases the safety risks of the product. How to improve the quality and yield of single-cell proteins remains a challenge in the current technological field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing single-cell protein using hydroxyl bacteria, in order to solve the problems of high nucleic acid content and significant protein loss during nucleic acid removal in existing methods for producing single-cell protein using hydroxyl bacteria.

[0006] This invention provides the following technical solution: A method for producing single-cell protein using hydroxyl bacteria includes the following steps: (1) Hydroxyhydric bacteria are inoculated into a fermentation medium, and a mixture of hydrogen, oxygen and carbon dioxide is introduced to carry out fermentation culture, so that the hydroxyhydric bacteria can synthesize single-cell protein and obtain fermentation broth; (2) Separate the solid and liquid components of the fermentation broth obtained in step (1) to obtain crude single-cell protein; (3) Disperse the crude single-cell protein obtained in step (2) in a resuspension solvent to break down the cells. Add immobilized nuclease to the lysate. After the reaction, use magnetic separation to recover the immobilized nuclease. Adjust the pH of the lysate to 4.5-5.5 to cause the protein to flocculate and precipitate. Separate the solid and liquid, wash and dry to obtain single-cell protein. The immobilized nuclease has an iron oxide core and a polymer layer as its shell, with the nuclease coupled to the polymer layer.

[0007] Preferably, in step (3), the immobilized nuclease is obtained by the following method: first, the iron oxide microparticles are modified with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, and then prepolymerized with divinylbenzene and styrene to form a protective layer; then, it is polymerized with glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline and vinylimidazole to form a core-shell structure carrier; then, it is coupled with the nuclease to obtain the immobilized nuclease.

[0008] Preferably, in step (3), the immobilized nuclease is obtained by the following method: (a) Take iron oxide particles, disperse them in an ethanol solution, add vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, stir in the dark at 20-30℃ for 1-2 hours, magnetically separate, wash and dry to obtain modified iron oxide; (b) Take the modified iron(III) oxide from step (a) and add it to an ethanol solution containing divinylbenzene and styrene. Stir for 1-3 hours at 20-30°C, protected from light and by an inert gas. Then add azobisisobutyronitrile and carry out a prepolymerization reaction at 60-65°C for 2-4 hours to obtain a prepolymer solution. (c) Add polyvinylpyrrolidone to the prepolymer solution in step (b), mix well, then add glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile. Stir and react at 60-65℃ for 4-6 hours, then magnetically separate, wash, and dry to obtain a core-shell structured carrier. (d) Take the core-shell structured carrier from step (c), disperse it in a buffer solution with a pH of 7.5-8.5, stir for 1-2 hours, add nuclease, react at 35-40°C for 4-12 hours, add lysine, continue the reaction for 0.5-1.5 hours, magnetically separate, wash, and dry to obtain immobilized nuclease.

[0009] Preferably, in step (a), the concentration of the iron oxide particles in the ethanol solution is 10-20 g / L; the weight ratio of the iron oxide particles, vinylphosphonic acid, and p-[(4-vinylphenyl)methyl]phosphonic acid is 100:(10-20):(5-15). Optionally, in step (b), the concentration of the modified iron oxide in the ethanol solution is 8-12 g / L; the weight ratio of the modified iron oxide, divinylbenzene, styrene, and azobisisobutyronitrile is 100:(50-60):(20-30):(1.5-2.5). Optionally, in step (c), the mass concentration of polyvinylpyrrolidone in the prepolymer solution is 2-6 wt%; the weight ratio of the modified iron oxide, the glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile is 100:(40-50):(15-25):(10-15):(1.0-2.0). Optionally, in step (d), the concentration of the core-shell structured vector in the buffer solution is 20-25 g / L; the buffer solution is any one of tris(hydroxymethyl)aminomethane buffer, phosphate buffer, citrate buffer, and acetic acid buffer; the concentration of the nuclease in the buffer solution is 0.5-2.5 g / L; and the concentration of lysine in the buffer solution is 10-20 g / L.

[0010] Preferably, step (c) further includes the addition of 1,3-divinyl-2-imidazolinone; the weight ratio of the modified iron oxide to the 1,3-divinyl-2-imidazolinone is 100:(3-5).

[0011] Preferably, in step (3), the concentration of the crude single-cell protein in the resuspension solvent is 50-80 g / L; Optionally, the resuspension solvent has a pH of 7.5-8.0 and includes buffer solution, sodium chloride, and magnesium chloride; Optionally, the buffer solution is any one of tris(hydroxymethyl)aminomethane buffer, phosphate buffer, citrate buffer, and acetate buffer; Optionally, the concentration of the buffer solution is 20-30 mmol / L; the concentration of the sodium chloride is 3-8 g / L; and the concentration of the magnesium chloride is 0.35-0.45 g / L. Optionally, the resuspension solvent further contains an auxiliary agent; the auxiliary agent is glycerol; the volume fraction of glycerol in the resuspension solvent is 3-5%.

[0012] Preferably, in step (3), the cell disruption is performed by high-pressure homogenization or ultrasonic disruption; Optionally, the high-pressure homogenization crushing is performed by cycling 3-5 times at 60-80 MPa; Optionally, the ultrasonic fragmentation is performed at a power of 200-300W for 10-15 minutes.

[0013] Preferably, in step (3), the immobilized nuclease is hydrolyzed at 25-37°C for 1-2 hours; Optionally, the amount of the immobilized nuclease in the disruption solution is 30-50 g / L.

[0014] Preferably, in step (1), the fermentation culture is specifically carried out by shaking culture at 25-30°C for 72-96 hours in a mixed gas with a volume ratio of hydrogen, oxygen and carbon dioxide of 10:(1.4-2.5):(1.4-2.5).

[0015] Preferably, in step (1), the fermentation medium comprises: 2.8-3.2 g / L dipotassium hydrogen phosphate, 1.1-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L ammonium chloride, 0.1-0.2 g / L sodium chloride, 0.15-0.30 g / L magnesium sulfate heptahydrate, 0.04-0.08 g / L ferric ammonium citrate, 0.01-0.02 g / L calcium chloride dihydrate, and 3-5 ml / L trace component solution; the pH value is 6.8-7.2.

[0016] Preferably, the trace component solution comprises: boric acid 30-70 mg / L, manganese chloride tetrahydrate 30-70 mg / L, nickel chloride hexahydrate 30-70 mg / L, cobalt chloride hexahydrate 30-70 mg / L, ammonium molybdate dihydrate 30-70 mg / L, ethylenediaminetetraacetic acid 1-5 mg / L, and selenite 3-7 mg / L.

[0017] Preferably, in step (2) and / or step (3), the solid-liquid separation is centrifugal separation.

[0018] Preferably, the hydroxide-oxidizing bacteria is one of the following: Hydrogenophaga laconesensis, Hydrogenophaga pseudoflava, Hydrogenophaga flava, or Copper-loving bacteria. Preferably, the hydroxide-oxidizing bacteria is Cupriavidus necator or recombinant Cupriavidus necator.

[0019] Preferably, the nuclease is Benzonase.

[0020] The above-described solution of the present invention has at least the following beneficial effects: (1) The method for producing single-cell protein using hydroxide bacteria of the present invention includes the following steps: inoculating hydroxide bacteria into a fermentation culture medium, introducing a mixed gas of hydrogen, oxygen and carbon dioxide, and fermenting the culture to allow the hydroxide bacteria to synthesize single-cell protein and obtain a fermentation broth; separating the solid and liquid components of the fermentation broth to obtain crude single-cell protein; dispersing the crude single-cell protein in a resuspension solvent to break down the cells, adding immobilized nuclease to the broken broth, and after the reaction, recovering the immobilized nuclease by magnetic separation; adjusting the pH of the broken broth to 4.5-5.5 to cause protein flocculation and precipitation; and then separating the solid and liquid components, washing, and drying to obtain single-cell protein. The method for producing single-cell protein using hydroxide bacteria of the present invention specifically removes nucleic acids through immobilized nucleases and causes protein flocculation and precipitation through isoelectric point precipitation. Enzymatic hydrolysis breaks down large nucleic acid molecules into small nucleic acid products, which are highly water-soluble and do not precipitate with the protein but remain in the supernatant. This achieves mild, efficient, and low-cost nucleic acid removal with good selectivity and minimal protein loss, effectively improving product quality and enabling the recovery and reuse of nucleases.

[0021] (2) The method for producing single-cell protein using hydroxyl bacteria described in this invention employs an immobilized nuclease with iron oxide as the core and a polymer layer as the shell, on which the nuclease is coupled. First, the iron oxide particles are modified with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, and then prepolymerized with divinylbenzene and styrene to form a protective layer; then, it is polymerized with glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, and vinylimidazole to form a core-shell structure carrier; then, it is coupled with the nuclease to obtain the immobilized nuclease. In this process, vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid are coordinated with the iron oxide microparticles, causing their terminal alkenyl groups to be distributed on the surface of the iron oxide microparticles. These alkenyl groups can then undergo prepolymerization with divinylbenzene and styrene to form a protective layer, preventing oxidation of the iron oxide and shell shedding, thus enhancing the stability of the core-shell structure. The numerous active ends remaining after prepolymerization then undergo polymerization with glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, and vinylimidazole to form a shell. The glycidyl methacrylate introduces epoxy groups, which then interact with nucleases... The amino group reacts to couple the nuclease; the 2-methacryloyloxyethylphosphorylcholine polymerizes to form a hydrophilic layer, which reduces non-specific adsorption and protects the enzyme conformation. As an amphoteric monomer, it can act as a charge buffer. The vinylimidazole has a certain pH responsiveness and can generate an electrostatic attraction effect on the nuclease under neutral or weakly alkaline conditions, increasing the enzyme loading. During nucleic acid hydrolysis, it enriches the nuclease on the surface of the immobilized nuclease, increasing the local substrate concentration and thus improving the enzymatic hydrolysis efficiency. In addition, the vinylimidazole can also improve the stability of the core-shell structure carrier and extend the lifespan of the immobilized nuclease.

[0022] (3) In the method for producing single-cell protein using hydroxyl bacteria described in this invention, in step (b), the modified iron oxide is first added to an ethanol solution containing divinylbenzene and styrene, stirred at room temperature for 1-3 hours, and then azobisisobutyronitrile is added to react. Since divinylbenzene and styrene are hydrophobic, they will accumulate on the surface of the modified iron oxide after stirring in the ethanol solution, and then, during prepolymerization, divinylbenzene and styrene will polymerize in situ on the surface of the modified iron oxide to form a protective layer. In step (c), polyvinylpyrrolidone is first added, which can be directionally adsorbed on the hydrophobic surface, and then glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, and vinylimidazole are added to ensure uniform growth of the shell layer. In step (d), the core-shell structure carrier is first fully swollen, then the nuclease is added to carry out a coupling reaction, and then lysine is added for end capping to prevent epoxy residue. Detailed Implementation

[0023] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention. Specifically, the CAS number of the vinylphosphonic acid is 1746-03-8; the CAS number of p-[(4-vinylphenyl)methyl]phosphonic acid is 53459-43-1; the CAS number of the divinylbenzene is 1321-74-0; the CAS number of the styrene is 100-42-5; the CAS number of the glycidyl methacrylate is 106-91-2; the CAS number of 2-methacryloyloxyethylphosphonic choline is 67881-98-5; the CAS number of the vinylimidazole is 1072-63-5; and the CAS number of 1,3-divinyl-2-imidazolinone is 13811-50-2.

[0024] Example 1 The method for producing single-cell protein using a mixed bacterial community in this embodiment includes the following steps: (1) Hydroxyhydric bacteria are inoculated into a fermentation medium, and a mixture of hydrogen, oxygen and carbon dioxide is introduced to carry out fermentation culture, so that the hydroxyhydric bacteria can synthesize single-cell protein and obtain fermentation broth; Specifically, the fermentation culture is carried out by shaking culture at 28°C for 72 hours in a mixed gas with a volume ratio of hydrogen, oxygen and carbon dioxide of 10:1.4:1.4.

[0025] The fermentation medium comprises: 2.8 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium chloride, 0.15 g / L sodium chloride, 0.15 g / L magnesium sulfate heptahydrate, 0.08 g / L ferric ammonium citrate, 0.01 g / L calcium chloride dihydrate, and 3 ml / L trace component solution; the pH value is 7.0.

[0026] The trace component solution comprises: boric acid 30 mg / L, manganese chloride tetrahydrate 70 mg / L, nickel chloride hexahydrate 70 mg / L, cobalt chloride hexahydrate 30 mg / L, ammonium molybdate dihydrate 70 mg / L, ethylenediaminetetraacetic acid 3 mg / L, and selenite 5 mg / L. The hydroxylating bacteria is *Cupriavidus necator*.

[0027] (2) Centrifuge the fermentation broth obtained in step (1) to obtain crude single-cell protein; (3) The crude single-cell protein obtained in step (2) is dispersed in a resuspension solvent and the cells are broken by high pressure homogenization. Immobilized nuclease is added to the lysate and hydrolyzed at 25°C for 2 hours. After the reaction, the immobilized nuclease is recovered by magnetic separation. The pH of the lysate is then adjusted to 4.5 to cause the protein to flocculate and precipitate. The protein is then separated by centrifugation, washed, and dried to obtain single-cell protein. The concentration of the crude single-cell protein in the resuspension solvent is 70 g / L; the resuspension solvent has a pH of 7.5 and includes buffer, sodium chloride, magnesium chloride, and an auxiliary agent; the buffer is acetate buffer with a concentration of 25 mmol / L; the concentration of sodium chloride is 5 g / L; the concentration of magnesium chloride is 0.45 g / L; the auxiliary agent is glycerol with a volume fraction of 3% in the resuspension solvent. The high-pressure homogenization is performed at 70 MPa for 3 cycles. The amount of immobilized nuclease in the lysis buffer is 40 g / L.

[0028] In this embodiment, the immobilized nuclease uses iron oxide (Fe3O4) as the core and a polymer layer as the shell. The nuclease is coupled to the polymer layer and is obtained by the following method: First, the iron oxide particles are modified with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, and then prepolymerized with divinylbenzene and styrene to form a protective layer; then, it is polymerized with glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, and vinylimidazole to form a core-shell structure carrier; finally, it is coupled with the nuclease to obtain the immobilized nuclease.

[0029] In this embodiment, the immobilized nuclease is obtained by the following method: (a) Take iron oxide particles, disperse them in an ethanol solution, add vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, stir at 20°C in the dark for 2 hours, magnetically separate, wash and dry to obtain modified iron oxide; The concentration of the iron(III) oxide microparticles in the ethanol solution is 15 g / L; the weight ratio of the iron(III) oxide microparticles, vinylphosphonic acid, and p-[(4-vinylphenyl)methyl]phosphonic acid is 100:10:15; the iron(III) oxide microparticles are obtained by co-precipitation of ferrous chloride, ferric chloride, and sodium hydroxide. The ethanol solution is a 95% (v / v) ethanol solution.

[0030] (b) Take the modified iron(III) oxide from step (a) and add it to an ethanol solution containing divinylbenzene and styrene. Stir for 1 hour at 30°C, in the dark, under inert gas protection. Then add azobisisobutyronitrile and carry out a prepolymerization reaction at 60°C for 4 hours to obtain a prepolymer solution. The modified ferric oxide has a concentration of 12 g / L in the ethanol solution; the weight ratio of the modified ferric oxide, divinylbenzene, styrene, and azobisisobutyronitrile is 100:55:25:1.5; and the ethanol solution is an 80% ethanol solution by volume.

[0031] (c) Add polyvinylpyrrolidone to the prepolymer solution in step (b), mix well, then add glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile. Stir and react at 62°C for 6 hours, then perform magnetic separation, washing, and drying to obtain a core-shell structured carrier. The polyvinylpyrrolidone in the prepolymer solution has a mass concentration of 2 wt%; the weight ratio of the modified iron oxide, glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile is 100:50:15:10:2.0. (d) Take the core-shell structured carrier from step (c), disperse it in a buffer solution with a pH of 7.5, stir for 1 h, add nuclease to it, react at 38 °C for 12 h, add lysine to it, continue the reaction for 1.5 h, magnetically separate, wash and dry to obtain immobilized nuclease.

[0032] The nuclease is Benzonase. The concentration of the core-shell structured carrier in the buffer solution is 25 g / L; the buffer solution is acetate buffer; the concentration of the nuclease in the buffer solution is 2.5 g / L; and the concentration of lysine in the buffer solution is 15 g / L.

[0033] Example 2 The method for producing single-cell protein using a mixed bacterial community in this embodiment includes the following steps: (1) Hydroxyhydric bacteria are inoculated into a fermentation medium, and a mixture of hydrogen, oxygen and carbon dioxide is introduced to carry out fermentation culture, so that the hydroxyhydric bacteria can synthesize single-cell protein and obtain fermentation broth; Specifically, the fermentation culture is carried out by shaking at 30°C for 96 hours in a mixed gas of hydrogen, oxygen and carbon dioxide in a volume ratio of 10:2.5:2.5.

[0034] The fermentation medium comprises: 3.0 g / L dipotassium hydrogen phosphate, 1.1 g / L potassium dihydrogen phosphate, 0.8 g / L ammonium chloride, 0.2 g / L sodium chloride, 0.20 g / L magnesium sulfate heptahydrate, 0.04 g / L ferric ammonium citrate, 0.02 g / L calcium chloride dihydrate, and 5 ml / L trace component solution; the pH value is 6.8.

[0035] The trace component solution comprises: boric acid 70 mg / L, manganese chloride tetrahydrate 70 mg / L, nickel chloride hexahydrate 30 mg / L, cobalt chloride hexahydrate 50 mg / L, ammonium molybdate dihydrate 30 mg / L, ethylenediaminetetraacetic acid 2 mg / L, and selenite 3 mg / L. The hydroxylating bacteria is *Cupriavidus necator*.

[0036] (2) Centrifuge the fermentation broth obtained in step (1) to obtain crude single-cell protein; (3) Disperse the crude single-cell protein obtained in step (2) in a resuspension solvent, and use ultrasonic disruption to disrupt the bacterial cells. Add immobilized nuclease to the disruption solution and hydrolyze at 30°C for 1 hour. After the reaction, use magnetic separation to recover the immobilized nuclease. Then adjust the pH of the disruption solution to 5.0 to cause the protein to flocculate and precipitate. Centrifuge, wash, and dry to obtain single-cell protein. The concentration of the crude single-cell protein in the resuspension solvent is 80 g / L; the resuspension solvent has a pH of 7.8 and includes buffer, sodium chloride, magnesium chloride, and an auxiliary agent; the buffer is citrate buffer with a concentration of 20 mmol / L; the concentration of sodium chloride is 8 g / L; the concentration of magnesium chloride is 0.35 g / L; the auxiliary agent is glycerol with a volume fraction of 4% in the resuspension solvent. The ultrasonic disruption is performed at 200 W for 13 min. The amount of immobilized nuclease in the disruption solution is 30 g / L.

[0037] In this embodiment, the immobilized nuclease uses iron oxide (Fe3O4) as the core and a polymer layer as the shell. The nuclease is coupled to the polymer layer and is obtained by the following method: First, the iron oxide particles are modified with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, and then prepolymerized with divinylbenzene and styrene to form a protective layer; then, it is polymerized with glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, and vinylimidazole to form a core-shell structure carrier; finally, it is coupled with the nuclease to obtain the immobilized nuclease.

[0038] In this embodiment, the immobilized nuclease is obtained by the following method: (a) Take iron oxide particles, disperse them in an ethanol solution, add vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, stir at 25°C in the dark for 1 h, magnetically separate, wash and dry to obtain modified iron oxide; The concentration of the iron oxide microparticles in the ethanol solution is 10 g / L; the weight ratio of the iron oxide microparticles, vinylphosphonic acid, and p-[(4-vinylphenyl)methyl]phosphonic acid is 100:20:10; the iron oxide microparticles are obtained by co-precipitation of ferrous chloride, ferric chloride, and sodium hydroxide. The ethanol solution is a 95% (v / v) ethanol solution.

[0039] (b) Take the modified iron(III) oxide from step (a) and add it to an ethanol solution containing divinylbenzene and styrene. Stir for 2 hours at 20°C, protected from light and under inert gas. Then add azobisisobutyronitrile and carry out a prepolymerization reaction at 62°C for 3 hours to obtain a prepolymer solution. The modified ferric oxide has a concentration of 8 g / L in the ethanol solution; the weight ratio of the modified ferric oxide, divinylbenzene, styrene, and azobisisobutyronitrile is 100:60:20:2.0; and the ethanol solution is an 80% ethanol solution by volume.

[0040] (c) Add polyvinylpyrrolidone to the prepolymer solution in step (b), mix well, then add glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile. Stir and react at 60°C for 5 hours, then perform magnetic separation, washing, and drying to obtain a core-shell structured carrier. The polyvinylpyrrolidone in the prepolymer solution has a mass concentration of 6 wt%; the weight ratio of the modified iron oxide, glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile is 100:45:25:12:1.0. (d) Take the core-shell structured carrier from step (c), disperse it in a buffer solution with a pH of 8.0, stir for 2 hours, add nuclease to it, react at 40°C for 4 hours, add lysine to it, continue the reaction for 1 hour, magnetically separate, wash and dry to obtain immobilized nuclease.

[0041] The nuclease is Benzonase. The concentration of the core-shell structured carrier in the buffer solution is 20 g / L; the buffer solution is citrate buffer; the concentration of the nuclease in the buffer solution is 1.5 g / L; and the concentration of lysine in the buffer solution is 10 g / L.

[0042] Example 3 The method for producing single-cell protein using a mixed bacterial community in this embodiment includes the following steps: (1) Hydroxyhydric bacteria are inoculated into a fermentation medium, and a mixture of hydrogen, oxygen and carbon dioxide is introduced to carry out fermentation culture, so that the hydroxyhydric bacteria can synthesize single-cell protein and obtain fermentation broth; Specifically, the fermentation culture is carried out by shaking culture at 25°C for 84 hours in a mixed gas with a volume ratio of hydrogen, oxygen and carbon dioxide of 10:2.0:2.0.

[0043] The fermentation medium comprises: 3.2 g / L dipotassium hydrogen phosphate, 1.1 g / L potassium dihydrogen phosphate, 1.2 g / L ammonium chloride, 0.1 g / L sodium chloride, 0.30 g / L magnesium sulfate heptahydrate, 0.06 g / L ferric ammonium citrate, 0.02 g / L calcium chloride dihydrate, and 4 ml / L trace component solution; the pH value is 7.2.

[0044] The trace component solution comprises: boric acid 50 mg / L, manganese chloride tetrahydrate 30 mg / L, nickel chloride hexahydrate 50 mg / L, cobalt chloride hexahydrate 70 mg / L, ammonium molybdate dihydrate 50 mg / L, ethylenediaminetetraacetic acid 5 mg / L, and selenite 7 mg / L. The hydroxylating bacteria is *Cupriavidus necator*.

[0045] (2) Centrifuge the fermentation broth obtained in step (1) to obtain crude single-cell protein; (3) Disperse the crude single-cell protein obtained in step (2) in a resuspension solvent, and use ultrasonic disruption to disrupt the bacterial cells. Add immobilized nuclease to the disruption solution and hydrolyze at 37°C for 1.5 h. After the reaction, use magnetic separation to recover the immobilized nuclease, and then adjust the pH of the disruption solution to 5.5 to cause the protein to flocculate and precipitate. Centrifuge, wash, and dry to obtain single-cell protein. The concentration of the crude single-cell protein in the resuspension solvent is 50 g / L; the resuspension solvent has a pH of 8.0 and includes buffer, sodium chloride, magnesium chloride, and an auxiliary agent; the buffer is phosphate buffer with a concentration of 30 mmol / L; the concentration of sodium chloride is 3 g / L; the concentration of magnesium chloride is 0.40 g / L; the auxiliary agent is glycerol with a volume fraction of 5% in the resuspension solvent. The ultrasonic disruption is performed at 300 W for 10 min. The amount of immobilized nuclease in the disruption solution is 50 g / L.

[0046] In this embodiment, the immobilized nuclease uses iron oxide (Fe3O4) as the core and a polymer layer as the shell. The nuclease is coupled to the polymer layer and is obtained by the following method: First, the iron oxide particles are modified with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, and then prepolymerized with divinylbenzene and styrene to form a protective layer; then, it is polymerized with glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, and vinylimidazole to form a core-shell structure carrier; finally, it is coupled with the nuclease to obtain the immobilized nuclease.

[0047] In this embodiment, the immobilized nuclease is obtained by the following method: (a) Take iron oxide particles, disperse them in an ethanol solution, add vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, stir at 30°C in the dark for 1.5 h, magnetically separate, wash and dry to obtain modified iron oxide; The concentration of the iron(III) oxide particles in the ethanol solution is 20 g / L; the weight ratio of the iron(III) oxide particles, vinylphosphonic acid, and p-[(4-vinylphenyl)methyl]phosphonic acid is 100:15:5; the iron(III) oxide particles are obtained by co-precipitation of ferrous chloride, ferric chloride, and sodium hydroxide. The ethanol solution is a 95% (v / v) ethanol solution.

[0048] (b) Take the modified iron(III) oxide from step (a) and add it to an ethanol solution containing divinylbenzene and styrene. Stir for 3 hours at 25°C, protected from light and under inert gas. Then add azobisisobutyronitrile and carry out a prepolymerization reaction at 65°C for 2 hours to obtain a prepolymer solution. The modified ferric oxide has a concentration of 10 g / L in the ethanol solution; the weight ratio of the modified ferric oxide, divinylbenzene, styrene, and azobisisobutyronitrile is 100:50:30:2.5; and the ethanol solution is an 80% ethanol solution by volume.

[0049] (c) Add polyvinylpyrrolidone to the prepolymer solution in step (b), mix well, then add glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile. Stir and react at 65°C for 4 hours, then perform magnetic separation, washing, and drying to obtain a core-shell structured carrier. The polyvinylpyrrolidone in the prepolymer solution has a mass concentration of 4 wt%; the weight ratio of the modified iron oxide, glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile is 100:40:20:15:1.5. (d) Take the core-shell structured carrier from step (c), disperse it in a buffer solution with a pH of 8.5, stir for 1.5 h, add nuclease to it, react at 35 °C for 8 h, add lysine to it, continue the reaction for 0.5 h, magnetically separate, wash and dry to obtain immobilized nuclease.

[0050] The nuclease is Benzonase. The concentration of the core-shell structured carrier in the buffer solution is 23 g / L; the buffer solution is phosphate buffer; the concentration of the nuclease in the buffer solution is 0.5 g / L; and the concentration of lysine in the buffer solution is 20 g / L.

[0051] Example 4 The method for producing single-cell protein using a mixed bacterial community in this embodiment includes the following steps: (1) Hydroxyhydric bacteria are inoculated into a fermentation medium, and a mixture of hydrogen, oxygen and carbon dioxide is introduced to carry out fermentation culture, so that the hydroxyhydric bacteria can synthesize single-cell protein and obtain fermentation broth; Specifically, the fermentation culture is carried out by shaking culture at 28°C for 84 hours in a mixed gas with a volume ratio of hydrogen, oxygen and carbon dioxide of 10:2.0:2.0.

[0052] The fermentation medium comprises: 3.0 g / L dipotassium hydrogen phosphate, 1.2 g / L potassium dihydrogen phosphate, 1.0 g / L ammonium chloride, 0.15 g / L sodium chloride, 0.25 g / L magnesium sulfate heptahydrate, 0.06 g / L ferric ammonium citrate, 0.02 g / L calcium chloride dihydrate, and 4 ml / L trace component solution; the pH value is 7.0.

[0053] The trace component solution comprises: boric acid 50 mg / L, manganese chloride tetrahydrate 50 mg / L, nickel chloride hexahydrate 50 mg / L, cobalt chloride hexahydrate 50 mg / L, ammonium molybdate dihydrate 50 mg / L, ethylenediaminetetraacetic acid 3 mg / L, and selenite 5 mg / L. The hydroxylating bacteria is *Cupriavidus necator*.

[0054] (2) Centrifuge the fermentation broth obtained in step (1) to obtain crude single-cell protein; (3) Disperse the crude single-cell protein obtained in step (2) in a resuspension solvent, and use high pressure homogenization to break down the cells. Add immobilized nuclease to the lysate and hydrolyze at 30°C for 1 hour. After the reaction, use magnetic separation to recover the immobilized nuclease. Then adjust the pH of the lysate to 5.0 to cause the protein to flocculate and precipitate. Centrifuge, wash, and dry to obtain single-cell protein. The concentration of the crude single-cell protein in the resuspension solvent is 65 g / L; the resuspension solvent has a pH of 7.8 and includes buffer, sodium chloride, magnesium chloride, and an auxiliary agent; the buffer is tris(hydroxymethyl)aminomethane buffer with a concentration of 25 mmol / L; the concentration of sodium chloride is 5 g / L; the concentration of magnesium chloride is 0.40 g / L; the auxiliary agent is glycerol with a volume fraction of 4% in the resuspension solvent. The high-pressure homogenization is performed at 70 MPa for 4 cycles. The amount of immobilized nuclease in the lysis buffer is 40 g / L.

[0055] In this embodiment, the immobilized nuclease uses iron oxide (Fe3O4) as the core and a polymer layer as the shell. The nuclease is coupled to the polymer layer and is obtained by the following method: First, the iron oxide particles are modified with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, and then prepolymerized with divinylbenzene and styrene to form a protective layer; then, it is polymerized with glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, and vinylimidazole to form a core-shell structure carrier; finally, it is coupled with the nuclease to obtain the immobilized nuclease.

[0056] In this embodiment, the immobilized nuclease is obtained by the following method: (a) Take iron oxide particles, disperse them in an ethanol solution, add vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, stir at 25°C in the dark for 1.5 h, magnetically separate, wash and dry to obtain modified iron oxide; The concentration of the iron oxide microparticles in the ethanol solution is 15 g / L; the weight ratio of the iron oxide microparticles, vinylphosphonic acid, and p-[(4-vinylphenyl)methyl]phosphonic acid is 100:15:10; the iron oxide microparticles are obtained by co-precipitation of ferrous chloride, ferric chloride, and sodium hydroxide. The ethanol solution is a 95% (v / v) ethanol solution.

[0057] (b) Take the modified iron(III) oxide from step (a) and add it to an ethanol solution containing divinylbenzene and styrene. Stir for 2 hours at 25°C, protected from light and under inert gas. Then add azobisisobutyronitrile and carry out a prepolymerization reaction at 65°C for 3 hours to obtain a prepolymer solution. The modified ferric oxide has a concentration of 10 g / L in the ethanol solution; the weight ratio of the modified ferric oxide, divinylbenzene, styrene, and azobisisobutyronitrile is 100:55:25:2; and the ethanol solution is an 80% ethanol solution by volume.

[0058] (c) Add polyvinylpyrrolidone to the prepolymer solution in step (b), mix well, then add glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile. Stir and react at 65°C for 5 hours, then perform magnetic separation, washing, and drying to obtain a core-shell structured carrier. The polyvinylpyrrolidone in the prepolymer solution has a mass concentration of 4 wt%; the weight ratio of the modified iron oxide, glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile is 100:45:20:12:1.5. (d) Take the core-shell structured carrier from step (c), disperse it in a buffer solution with a pH of 8.0, stir for 1.5 h, add nuclease to it, react at 38 °C for 8 h, add lysine to it, continue the reaction for 1 h, magnetically separate, wash and dry to obtain immobilized nuclease.

[0059] The nuclease is Benzonase. The concentration of the core-shell structured carrier in the buffer solution is 23 g / L; the buffer solution is tris(hydroxymethyl)aminomethane buffer; the concentration of the nuclease in the buffer solution is 1.5 g / L; and the concentration of lysine in the buffer solution is 15 g / L.

[0060] Example 5 The method for producing single-cell protein using mixed bacterial communities in this embodiment is basically the same as that in Example 4, except that step (c) also includes the addition of 1,3-divinyl-2-imidazolinone; the weight ratio of the modified iron oxide and the 1,3-divinyl-2-imidazolinone is 100:5.

[0061] Example 6 The method for producing single-cell protein using mixed bacterial communities in this embodiment is basically the same as that in Example 4, except that step (c) includes the addition of 1,3-divinyl-2-imidazolinone; the weight ratio of the modified iron oxide to the 1,3-divinyl-2-imidazolinone is 100:3.

[0062] Example 7 The method for producing single-cell protein using mixed bacterial communities in this embodiment is basically the same as that in Example 4, except that step (c) includes the addition of 1,3-divinyl-2-imidazolinone; the weight ratio of the modified iron oxide to the 1,3-divinyl-2-imidazolinone is 100:4.

[0063] Comparative Example 1 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0064] In step (a) of the method for preparing the immobilized nuclease in this comparative example, vinylphosphonic acid is not added.

[0065] Comparative Example 2 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0066] In step (a) of the method for preparing the immobilized nuclease in this comparative example, p-[(4-vinylphenyl)methyl]phosphonic acid is not added.

[0067] Comparative Example 3 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0068] The preparation method of the immobilized nuclease in this comparative example does not include step (a); that is, the iron oxide microparticles are not modified, and iron oxide microparticles are used directly in step (b).

[0069] Comparative Example 4 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0070] In step (b) of the method for preparing the immobilized nuclease in this comparative example, divinylbenzene is not added.

[0071] Comparative Example 5 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0072] In step (b) of the method for preparing the immobilized nuclease in this comparative example, no styrene is added.

[0073] Comparative Example 6 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0074] The preparation method of the immobilized nuclease in this comparative example did not involve a prepolymerization reaction. Specifically, step (c) includes: taking the modified iron(III) oxide from step (a), adding it to an ethanol solution containing divinylbenzene and styrene, and stirring for 2 hours at 25°C under light-proof and inert gas protection; then adding polyvinylpyrrolidone, mixing evenly, and then adding glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile, stirring the reaction at 65°C for 5 hours, followed by magnetic separation, washing, and drying to obtain a core-shell structured carrier.

[0075] Comparative Example 7 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0076] The method for preparing the immobilized nuclease in this comparative example did not include any monomers for prepolymerization, nor did it involve a prepolymerization reaction. Specifically, step (c) includes: taking the modified iron(III) oxide from step (a), adding polyvinylpyrrolidone, mixing thoroughly, then adding glycidyl methacrylate, 2-methacryloyloxyethylphosphonic choline, vinylimidazole, and azobisisobutyronitrile, stirring at 65°C for 5 hours, followed by magnetic separation, washing, and drying to obtain a core-shell structured carrier.

[0077] Comparative Example 8 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0078] In the preparation method of the immobilized nuclease in this comparative example, step (c) does not involve the addition of 2-methacryloyloxyethylphosphorylcholine.

[0079] Comparative Example 9 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0080] In the preparation method of the immobilized nuclease in this comparative example, vinylimidazole is not added in step (c).

[0081] Comparative Example 10 The method for producing single-cell protein using mixed bacterial communities in this comparative example is basically the same as that in Example 4, except that the immobilized nuclease used in step (3) is different.

[0082] In the preparation method of the immobilized nuclease in this comparative example, polyvinylpyrrolidone was not added in step (c).

[0083] Effect Experiment Example To verify the technical effectiveness of the method for producing single-cell protein using hydroxyl bacteria described in this invention, the following experiments were conducted: 1. Nucleic acid removal efficiency test The products obtained in Examples 1-7 and Comparative Examples 1-10 were used to measure the nucleic acid concentration and calculate the nucleic acid removal rate using ultraviolet spectrophotometry.

[0084] 2. Protein yield Take the crude single-cell protein obtained in step (2) of Examples 1-7 and Comparative Examples 1-10, dry it, and use the Kjeldahl method to determine the crude protein content of the dried bacterial cells, weigh it, and calculate the total protein. Take the single-cell protein obtained in step (3) of Examples 1-7 and Comparative Examples 1-10, determine the crude protein content, weigh it, calculate the amount of protein after purification, and calculate the protein yield.

[0085] 3. Stability of immobilized nucleases The immobilized nucleases prepared in Examples 1-7 and Comparative Examples 1-10 were used as substrate solutions. The immobilized nucleases were added to the solution and reacted at 30°C for 15 min. The immobilized enzymes were then separated and collected. Nucleic acid precipitant (0.25% ammonium molybdate-2.5% perchloric acid) was added to the reaction solution. After centrifugation in an ice-water bath for 10 min, the supernatant was collected and diluted with pure water. The absorbance of the supernatant was measured by ultraviolet spectrophotometry, and the nucleic acid hydrolysis rate was calculated. The initial relative enzyme activity was defined as 100% based on the nucleic acid hydrolysis rate of the first batch. The collected immobilized enzymes were washed three times with buffer solution. Then, the same batch of lysate was used as substrate solution, and the above operation was repeated 10 times. The nucleic acid hydrolysis rate of the immobilized nuclease after 10 reuses was calculated, and the relative enzyme activity was calculated.

[0086] The test results of Examples 1-7 and Comparative Examples 1-10 are shown in Table 1.

[0087] Table 1 Test Results Based on the results of Examples 1-7 and Comparative Examples 1-10, it can be seen that the method for producing single-cell protein using hydroxyl bacteria described in this invention can effectively reduce the nucleic acid content of single-cell protein and minimize protein loss.

[0088] Based on the results of Examples 4 and 5-7, although the addition of 1,3-divinyl-2-imidazolinone slightly decreased the protein yield, it improved the nucleic acid removal rate and the relative enzyme activity for reuse. This indicates that 1,3-divinyl-2-imidazolinone participates in free radical copolymerization and cross-linking, improving the regulation of the polymer layer, thereby enhancing the nucleic acid hydrolysis effect and effectively improving the reusability stability of the immobilized nuclease.

[0089] Based on the results of Example 4 and Comparative Examples 1-3, it is evident that the immobilized nuclease obtained by modifying the iron oxide microparticles with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid exhibits better nucleic acid hydrolysis efficiency, protein yield, and reusable relative enzyme activity. Comparative Examples 1-2 (without vinylphosphonic acid or p-[(4-vinylphenyl)methyl]phosphonic acid) and Comparative Example 3 (without modification) all showed varying degrees of decrease in nucleic acid removal rate, protein yield, and reusable relative enzyme activity. Among these, the reusable stability of the immobilized nuclease decreased most significantly. Therefore, modification of the iron oxide microparticles plays a crucial role in improving the stability of the immobilized nuclease.

[0090] Based on the results of Example 4 and Comparative Examples 4-7, it is evident that prepolymerization with divinylbenzene and styrene to form a protective layer plays a crucial role in improving the stability of the immobilized enzyme. Comparative Examples 4 and 5, which used divinylbenzene or styrene alone, were less effective than Example 4, which used both in combination. The combined use of both synergistically enhances coordination bonding and surface alkenyl group density, thereby more effectively anchoring the subsequent polymer layer and improving the stability of the immobilized enzyme.

[0091] Based on the results of Example 4 and Comparative Examples 8-9, it is evident that the polymerization reaction using 2-methacryloyloxyethyl phosphorylcholine and vinylimidazole plays a crucial role in the construction and effectiveness of immobilized nucleases, improving enzyme activity and reducing non-specific adsorption. Comparative Example 8, lacking 2-methacryloyloxyethyl phosphorylcholine, exhibited low nuclease hydrolysis efficiency and a certain degree of non-specific adsorption, resulting in reduced protein yield. Comparative Example 9, lacking vinylimidazole, showed decreased enzyme hydrolysis efficiency and reduced stability of the immobilized enzyme. This may be because vinylimidazole possesses a certain degree of hydrophilicity and exhibits π-π conjugation with divinylbenzene and styrene, serving as a transition monomer to improve the compatibility of 2-methacryloyloxyethyl phosphorylcholine with divinylbenzene and styrene.

[0092] Based on the results of Example 4 and Comparative Example 10, it can be seen that polyvinylpyrrolidone, as a steric stabilizer, can prevent the aggregation of carrier particles during polymerization and ensure the dispersibility and uniformity of immobilized nucleases; in Comparative Example 10 without the addition of polyvinylpyrrolidone, the repeatability stability and nucleic acid removal rate of the immobilized nuclease both decreased.

[0093] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or equivalent to the scope of this invention should be considered to fall within the protection scope of this invention.

Claims

1. A method for producing single-cell protein using hydroxyl bacteria, characterized in that, Includes the following steps: (1) Hydroxyhydric bacteria are inoculated into a fermentation medium, and a mixture of hydrogen, oxygen and carbon dioxide is introduced to carry out fermentation culture, so that the hydroxyhydric bacteria can synthesize single-cell protein and obtain fermentation broth; (2) Separate the solid and liquid components of the fermentation broth obtained in step (1) to obtain crude single-cell protein; (3) Disperse the crude single-cell protein obtained in step (2) in a resuspension solvent to break down the cells. Add immobilized nuclease to the lysate. After the reaction, use magnetic separation to recover the immobilized nuclease. Adjust the pH of the lysate to 4.5-5.5 to cause the protein to flocculate and precipitate. Separate the solid and liquid, wash and dry to obtain single-cell protein. The immobilized nuclease has an iron oxide core and a polymer layer as its shell, with the nuclease coupled to the polymer layer.

2. The method for producing single-cell protein using hydroxyl bacteria according to claim 1, characterized in that, In step (3), the immobilized nuclease is obtained by the following method: first, the iron oxide microparticles are modified with vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, and then prepolymerized with divinylbenzene and styrene to form a protective layer; then, it is polymerized with glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine and vinylimidazole to form a core-shell structure carrier; then, it is coupled with the nuclease to obtain the immobilized nuclease.

3. The method for producing single-cell protein using hydroxyl bacteria according to claim 2, characterized in that, In step (3), the immobilized nuclease is obtained by the following method: (a) Take iron oxide particles, disperse them in an ethanol solution, add vinylphosphonic acid and p-[(4-vinylphenyl)methyl]phosphonic acid, stir in the dark at 20-30℃ for 1-2 hours, magnetically separate, wash and dry to obtain modified iron oxide; (b) Take the modified iron(III) oxide from step (a) and add it to an ethanol solution containing divinylbenzene and styrene. Stir for 1-3 hours at 20-30°C, protected from light and by an inert gas. Then add azobisisobutyronitrile and carry out a prepolymerization reaction at 60-65°C for 2-4 hours to obtain a prepolymer solution. (c) Add polyvinylpyrrolidone to the prepolymer solution in step (b), mix well, then add glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile. Stir and react at 60-65℃ for 4-6 hours, then magnetically separate, wash, and dry to obtain a core-shell structured carrier. (d) Take the core-shell structured carrier from step (c), disperse it in a buffer solution with a pH of 7.5-8.5, stir for 1-2 hours, add nuclease, react at 35-40°C for 4-12 hours, add lysine, continue the reaction for 0.5-1.5 hours, magnetically separate, wash, and dry to obtain immobilized nuclease.

4. The method for producing single-cell protein using hydroxyl bacteria according to claim 3, characterized in that, In step (a), the concentration of the iron oxide particles in the ethanol solution is 10-20 g / L; the weight ratio of the iron oxide particles, vinylphosphonic acid, and p-[(4-vinylphenyl)methyl]phosphonic acid is 100:(10-20):(5-15). Optionally, in step (b), the concentration of the modified iron oxide in the ethanol solution is 8-12 g / L; the weight ratio of the modified iron oxide, divinylbenzene, styrene, and azobisisobutyronitrile is 100:(50-60):(20-30):(1.5-2.5). Optionally, in step (c), the mass concentration of polyvinylpyrrolidone in the prepolymer solution is 2-6 wt%; the weight ratio of the modified iron oxide, the glycidyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, vinylimidazole, and azobisisobutyronitrile is 100:(40-50):(15-25):(10-15):(1.0-2.0). Optionally, in step (d), the concentration of the core-shell structured vector in the buffer solution is 20-25 g / L; the buffer solution is any one of tris(hydroxymethyl)aminomethane buffer, phosphate buffer, citrate buffer, and acetic acid buffer; the concentration of the nuclease in the buffer solution is 0.5-2.5 g / L; and the concentration of lysine in the buffer solution is 10-20 g / L.

5. The method for producing single-cell protein using hydroxyl bacteria according to claim 1, characterized in that, In step (3), the concentration of the crude single-cell protein in the resuspension solvent is 50-80 g / L; Optionally, the resuspension solvent has a pH of 7.5-8.0 and includes buffer solution, sodium chloride, and magnesium chloride; Optionally, the buffer solution is any one of tris(hydroxymethyl)aminomethane buffer, phosphate buffer, citrate buffer, and acetate buffer; Optionally, the concentration of the buffer solution is 20-30 mmol / L; the concentration of the sodium chloride is 3-8 g / L; and the concentration of the magnesium chloride is 0.35-0.45 g / L. Optionally, the resuspension solvent further contains an auxiliary agent; the auxiliary agent is glycerol; the volume fraction of glycerol in the resuspension solvent is 3-5%.

6. The method for producing single-cell protein using hydroxide bacteria according to claim 1, characterized in that, In step (3), the cell disruption is performed by high-pressure homogenization or ultrasonic disruption; Optionally, the high-pressure homogenization crushing is performed by cycling 3-5 times at 60-80 MPa; Optionally, the ultrasonic fragmentation is performed at a power of 200-300W for 10-15 minutes.

7. The method for producing single-cell protein using hydroxide bacteria according to claim 1, characterized in that, In step (3), the immobilized nuclease is hydrolyzed at 25-37°C for 1-2 hours; Optionally, the amount of the immobilized nuclease in the disruption solution is 30-50 g / L.

8. The method for producing single-cell protein using hydroxide bacteria according to claim 1, characterized in that, In step (1), the fermentation culture specifically includes: in a mixed gas with a volume ratio of hydrogen, oxygen and carbon dioxide of 10:(1.4-2.5):(1.4-2.5), the mixture is shaken at 25-30℃ for 72-96h.

9. The method for producing single-cell protein using hydroxyl bacteria according to claim 1, characterized in that, In step (1), the fermentation medium comprises: 2.8-3.2 g / L dipotassium hydrogen phosphate, 1.1-1.2 g / L potassium dihydrogen phosphate, 0.8-1.2 g / L ammonium chloride, 0.1-0.2 g / L sodium chloride, 0.15-0.30 g / L magnesium sulfate heptahydrate, 0.04-0.08 g / L ferric ammonium citrate, 0.01-0.02 g / L calcium chloride dihydrate, and 3-5 ml / L trace component solution; the pH value is 6.8-7.

2.

10. The method for producing single-cell protein using hydroxyl bacteria according to claim 9, characterized in that, The trace component solution includes: boric acid 30-70 mg / L, manganese chloride tetrahydrate 30-70 mg / L, nickel chloride hexahydrate 30-70 mg / L, cobalt chloride hexahydrate 30-70 mg / L, ammonium molybdate dihydrate 30-70 mg / L, ethylenediaminetetraacetic acid 1-5 mg / L, and selenite acid 3-7 mg / L.

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