A method for producing aquatic feed protein source by fixing carbon dioxide, and the aquatic feed protein source and aquatic feed.

Abstract

This invention provides a method for producing aquatic feed protein source by fixing carbon dioxide, as well as the aquatic feed protein source and aquatic feed, comprising the following steps: (1) fermenting and culturing hydroxide bacteria, and separating the solid and liquid of the obtained fermentation broth to obtain a bacterial precipitate; (2) resuspending and breaking the cell wall of the bacterial precipitate, then adding an enzyme for enzymatic hydrolysis, followed by solid-liquid separation, and desalting, concentrating, and drying the obtained supernatant to obtain the enzymatic hydrolysis product; (3) dissolving the enzymatic hydrolysis product in water, adding low-methoxyl pectin, gelatin, and calcium carbonate and mixing evenly to obtain an aqueous phase; mixing ethyl cellulose, emulsifier, and ethyl acetate evenly to obtain an oil phase; mixing the aqueous phase and oil phase evenly to form a water-in-oil emulsion; adding a cycloalkane solution of acetic acid and mixing evenly, reacting, separating the solid and liquid, washing, and freeze-drying to obtain the final product. The aquatic feed protein source of this invention can partially replace fishmeal, can float stably in water, and is not easily disintegrated.

Description

Technical Field

[0001] This invention relates to the field of aquatic feed technology, and in particular to a method for preparing a protein source for aquatic feed by fixing carbon dioxide, as well as the aquatic feed protein source and aquatic feed. Background Technology

[0002] With the rapid development of aquaculture, the supply of fishmeal, a major source of high-quality protein in aquatic feed, has been declining, leading to a continuous rise in prices due to the imbalance between supply and demand. Therefore, developing a high-quality protein source to replace fishmeal in aquatic feed has become an urgent task to ensure the sustainable development of aquaculture.

[0003] Hydrogen-oxidizing bacteria are a type of facultative chemoautotrophic bacteria capable of utilizing H2 as an electron donor and O2 as an electron acceptor while assimilating CO2. In recent years, with the development of renewable energy technologies and biotechnology, the technology of cultivating hydrogen-oxidizing bacteria to produce single-cell protein using carbon dioxide as a carbon source and hydrogen generated from renewable energy sources as an energy source has gradually attracted attention. Single-cell protein produced through microbial cultivation has advantages such as low cost and stable, controllable quality, and has significant application potential. Applying it to the field of aquatic feed can, to some extent, replace fishmeal and alleviate the problem of insufficient protein source supply in aquatic feed.

[0004] However, if the microbial fermentation powder is fed directly to aquatic animals, it will not be easy for them to digest and absorb. Although enzymatic hydrolysis can improve protein utilization, it will easily cause the protein to dissolve rapidly in water and has poor stability in water, making it difficult for aquatic animals to fully consume and utilize it. Summary of the Invention

[0005] The purpose of this invention is to provide a method for producing aquatic feed protein source by fixing carbon dioxide, as well as the aquatic feed protein source and aquatic feed, to solve the problems of poor digestibility and absorption and poor stability in water in the existing aquatic feed protein source.

[0006] This invention provides the following technical solution:

[0007] This invention provides a method for producing aquatic feed protein source by fixing carbon dioxide, comprising the following steps:

[0008] (1) The hydroxyl bacteria were fermented and cultured, and the resulting fermentation broth was separated into solid and liquid components to obtain bacterial precipitate;

[0009] (2) The bacterial precipitate obtained in step (1) is resuspended and cell wall broken, then enzyme is added to it for enzymatic hydrolysis, then solid-liquid separation is performed, and the supernatant is desalted, concentrated and dried to obtain the enzymatic hydrolysis product.

[0010] (3) Dissolve the enzymatic hydrolysis product obtained in step (2) in water, add low-methoxyl pectin, gelatin and calcium carbonate and mix evenly to obtain an aqueous phase; mix ethyl cellulose, emulsifier and ethyl acetate evenly to obtain an oil phase; mix the aqueous phase and oil phase evenly to form a water-in-oil emulsion; add acetic acid cycloalkane solution and mix evenly; after reaction, separate the solid and liquid, wash and freeze dry to obtain aquatic feed protein source.

[0011] In some embodiments, the fermentation culture includes: inoculating hydroxide bacteria into a culture medium and culturing it with shaking at 27-37°C for 2-4 days in a mixed gas of hydrogen, carbon dioxide, and oxygen; wherein the volume ratio of hydrogen, carbon dioxide, and oxygen is (60-70):(10-20):(15-25).

[0012] Optionally, the culture medium comprises: potassium dihydrogen phosphate 2.0-3.0 g / L, disodium hydrogen phosphate dihydrate 2.0-3.0 g / L, ammonium chloride 1-1.5 g / L, magnesium sulfate heptahydrate 0.2-0.5 g / L, sodium bicarbonate 0.5-0.8 g / L, calcium chloride dihydrate 0.01-0.05 g / L, ferric ammonium citrate 0.05-0.1 g / L, trace element solution 1-3 mL / L, and pH 6.8-7.2;

[0013] Optionally, the trace element solution comprises: boric acid 0.5-0.8 g / L, cobalt chloride hexahydrate 0.2-0.8 g / L, zinc sulfate heptahydrate 0.1-0.5 g / L, manganese chloride tetrahydrate 0.01-0.1 g / L, sodium molybdate dihydrate 0.1-0.2 g / L, nickel chloride hexahydrate 0.1-0.2 g / L, and copper sulfate pentahydrate 0.01-0.05 g / L.

[0014] In some embodiments, in step (1), the hydroxide bacteria are Pseudomonas or Hookworm Copper-Loving Bacteria;

[0015] In some embodiments, in step (2), the resuspension is performed using a buffer solution with a pH of 6.5-7.5;

[0016] Optionally, the concentration of the bacterial cell precipitate in the buffer solution is 40-60 g / L;

[0017] Optionally, the cell wall breaking is performed using high-pressure homogenization, with the cells cyclically broken 2-5 times at 60-80 MPa.

[0018] Optionally, the enzyme comprises a protease; the concentration of the protease in the buffer is 150-300 mg / L; preferably, the enzyme comprises a neutral protease.

[0019] Optionally, the enzymatic hydrolysis specifically includes: reacting at a temperature of 40–45°C and a stirring speed of 150–250 rpm for 2–6 hours.

[0020] The buffer solution can be any of Tris-HCl or PBS buffer, and those skilled in the art can choose an existing conventional buffer solution according to the actual situation.

[0021] In some embodiments, in step (3), the weight ratio of the enzymatic hydrolysate, low-methoxyl pectin, gelatin, and calcium carbonate in the aqueous phase is 1:(0.1-0.3):(0.1-0.4):(0.005-0.015); the concentration of the enzymatic hydrolysate is 80-100 g / L.

[0022] In the oil phase, the weight ratio of ethyl cellulose to emulsifier is 1:(0.05-0.15); the mass concentration of the emulsifier is 3-4%.

[0023] The volume ratio of the aqueous phase to the oil phase is 1:(3-5).

[0024] Optionally, in step (3), the emulsifier is one or more of polyethylene glycol dioleate and Span.

[0025] Optionally, the emulsifier is a mixture of polyethylene glycol dioleate and Span in a weight ratio of 1:(0.5-3.5).

[0026] In some embodiments, in step (3), the aqueous phase further contains one or more of propylene glycol alginate and mannitol;

[0027] Optionally, the weight ratio of the propylene glycol alginate to the enzymatic hydrolysis product is (0.02-0.08):1;

[0028] Optionally, the weight ratio of mannitol to the enzymatic hydrolysis product is (0.1-0.2):1.

[0029] In some embodiments, in step (3), the oil phase further contains one or more of β-sitosterol and sitosterol palmitate;

[0030] Optionally, the weight ratio of β-sitosterol to the enzymatic hydrolysis product is (0.01-0.05):1;

[0031] Optionally, the weight ratio of the sitosterol palmitate to the enzymatic hydrolysis product is (0.005-0.02):1.

[0032] In some embodiments, in step (3), the cycloalkane solution of acetic acid is added dropwise to the water-in-oil emulsion at a dropping rate of 1–2 mL / min, while stirring at a speed of 600–700 r / min.

[0033] Optionally, the cycloalkane is selected from cyclohexane, cyclopentane, or mixtures thereof;

[0034] Optionally, the acetic acid in the cycloalkane solution has a mass concentration of 5 wt%-15 wt%;

[0035] Optionally, the molar ratio of acetic acid to calcium carbonate is greater than 2:1;

[0036] Optionally, the molar ratio of acetic acid to calcium carbonate is (3.0-3.5):1.

[0037] In some embodiments, step (3) specifically includes: dissolving the enzymatic hydrolysis product obtained in step (2) in water, adding low-methoxyl pectin, gelatin and calcium carbonate to it and mixing evenly to obtain an aqueous phase; mixing ethyl cellulose, emulsifier and ethyl acetate evenly to obtain an oil phase; mixing the aqueous phase and oil phase evenly to form a water-in-oil emulsion; adding a cycloalkane solution of acetic acid to it and mixing evenly; after the reaction, centrifuging the reaction solution at 8000-12000 r / min for 10-15 min, collecting the precipitate, washing the obtained precipitate with water and anhydrous ethanol 2-3 times respectively, and freeze-drying to obtain an aquatic feed protein source with a microcapsule structure.

[0038] The present invention also provides an aquatic feed protein source prepared according to the above method.

[0039] The present invention also provides an aquatic feed, wherein the aquatic feed comprises the aquatic feed protein source prepared by the above method.

[0040] The above-described solution of the present invention has at least the following beneficial effects:

[0041] (1) The method for producing aquatic feed protein source by fixing carbon dioxide according to the present invention includes the following steps: fermenting and culturing hydroxide bacteria, and separating the solid and liquid of the obtained fermentation broth to obtain a bacterial precipitate; resuspending and breaking the cell wall of the bacterial precipitate, then adding an enzyme for enzymatic hydrolysis, followed by solid-liquid separation, and desalting, concentrating, and drying the obtained supernatant to obtain an enzymatic hydrolysis product; dissolving the enzymatic hydrolysis product in water, adding low-methoxyl pectin, gelatin, and calcium carbonate and mixing evenly to obtain an aqueous phase; mixing ethyl cellulose, emulsifier, and ethyl acetate evenly to obtain an oil phase; mixing the aqueous phase and oil phase evenly to form a water-in-oil emulsion; adding a cycloalkane solution of acetic acid and mixing evenly, reacting, separating the solid and liquid, washing, and freeze-drying to obtain an aquatic feed protein source. The aquatic feed protein source obtained by the method for producing aquatic feed protein source by fixing carbon dioxide according to the present invention can float stably in the water body, is not easy to disintegrate, has good stability in water, and has good digestibility and absorption.

[0042] The enzymatic hydrolysis product is mixed with low-methoxyl pectin, gelatin, and calcium carbonate as the aqueous phase, and the ethyl cellulose, emulsifier, and ethyl acetate are mixed as the oil phase. After mixing the aqueous and oil phases, an emulsion is formed in an oil-in-water emulsion. When a cycloalkane solution of acetic acid is added to the water-in-oil emulsion, acetic acid slowly diffuses from the oil phase into the aqueous phase. Calcium carbonate reacts and decomposes with acetic acid, releasing calcium ions and carbon dioxide gas. The low-methoxyl pectin crosslinks with calcium ions to form an ionic gel network. Simultaneously, gelatin undergoes physical aggregation, intertwining and interpenetrating with the ionic gel network to form an interpenetrating polymer gel network, encapsulating the enzymatic hydrolysis product within it. As cycloalkanes are continuously added, ethyl cellulose precipitates and deposits in ethyl acetate, forming a microcapsule shell structure that encapsulates the gel, thus enabling the aquatic feed protein source to float stably in the water. Carbon dioxide gas released from the decomposition of calcium carbonate continuously escapes during the formation of the gel network and the solidification of the shell, forming micropores that penetrate the gel-shell double-layer coating structure. This microporous structure allows a small amount of small-molecule protein to leak out after the aquatic feed protein source enters the water, achieving the purpose of attracting food. It can also accelerate the rupture of the microcapsule shell structure after the aquatic feed protein source enters the gastrointestinal tract of aquatic animals (e.g., fish), promoting the release of enzymatic hydrolysis products.

[0043] (2) The method for producing aquatic feed protein source by fixing carbon dioxide according to the present invention further contains one or more of propylene glycol alginate and mannitol in the aqueous phase, and one or more of β-sitosterol and sitosterol palmitate in the oil phase.

[0044] The propylene glycol alginate has a hydrophilic polysaccharide backbone, rich in carboxyl groups and numerous hydroxyl groups, enabling it to form hydrogen bonds with low-methoxyl pectin gels and gelatin gels. It inserts into and fills the gaps in the low-methoxyl pectin-gelatin gel network, making the gel structure more compact. The propylene glycol alginate also has hydrophobic propylene glycol side groups, which readily aggregate at the water-oil interface, entangle with the hydrophobic chains of ethyl cellulose, and participate in the formation of a microcapsule shell structure when ethyl cellulose precipitates. This allows ethyl cellulose to bind more uniformly and firmly to the gel surface during curing. The propylene glycol alginate can also reduce the interfacial energy between the two phases, making the gel-shell bilayer coating structure more stable. Furthermore, the propylene glycol alginate contains partially unesterified carboxyl groups, which can compete with low-methoxyl pectin for calcium ion binding, thus slowing down the cross-linking reaction between low-methoxyl pectin and calcium ions to a certain extent, resulting in a more ordered gel network formation.

[0045] The mannitol can protect proteins during freeze-drying, prevent them from denaturing, and fill the gaps in the gel network, preventing the gel network from shrinking excessively during freeze-drying and maintaining the rigidity of the gel network.

[0046] The β-sitosterol, sitosterol palmitate, and ethyl cellulose exhibit good compatibility. β-sitosterol can intercalate between the hydrophobic chains of ethyl cellulose, increasing the interchain spacing and preventing deformation of the ethyl cellulose shell during freeze-drying, thus making the gel-shell bilayer coating structure more stable. Sitosterol palmitate enhances the continuity of the oil phase and works synergistically with β-sitosterol to regulate the ethyl cellulose shell, making the microcapsule shell more continuous and stable. This helps improve the stability of the gel-shell bilayer coating structure during freeze-drying and water floating, promoting the release of aquatic feed protein sources into the gastrointestinal tract of aquatic animals. Detailed Implementation

[0047] 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 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.

[0048] In the following embodiments, the *Pseudomonas* or *Cupriavidus necator* strains are known strains in the prior art. The Latin name of the *Pseudomonas* is *Pseudomonas wadenswilerensis*, for example, *Pseudomonas* strain CCOS 864T can be used; the Latin name of the *Cupriavidus necator* strain is *Cupriavidus necator*, for example, *Cupriavidus necator* strain BNCC357813 can be used.

[0049] Each strain can be activated using known methods of existing technology or methods described in the product instructions before fermentation culture.

[0050] In the following embodiments, the enzyme can be a neutral protease, such as neutral protease 1398, and a neutral protease with a specification of 100,000 U / g can be selected. To achieve the reuse of the enzyme preparation, an immobilized protease with a carrier containing magnetic materials such as iron oxide can be used for enzymatic hydrolysis. After enzymatic hydrolysis, the immobilized protease is separated under an external magnetic field, achieving the purpose of enzyme reuse. The carrier for the immobilized protease can be magnetic chitosan or other magnetic carriers. The immobilized protease can be purchased directly from the market or prepared using conventional techniques in the art; when prepared, the method for preparing magnetic chitosan-immobilized complex protease as described in, for example, patent 201210161902.4 can be used to prepare the immobilized protease required in this embodiment. In the following embodiments, the protease can also be directly added to the resuspended and cell-wall-broken bacterial precipitate for enzymatic hydrolysis. After enzymatic hydrolysis, the protease is removed by methods such as enzyme inactivation. It should be noted that the following embodiments use unimmobilized protease.

[0051] The CAS number of the low-methoxyl pectin is 9000-69-5, which refers to pectin with a degree of esterification (methoxyl content) of less than 50%; the CAS number of the gelatin is 9000-70-8; the CAS number of the calcium carbonate is 471-34-1; the CAS number of the ethyl cellulose is 9004-57-3; the CAS number of the polyethylene glycol dioleate is 9005-07-6; the Span is Span 80 with a CAS number of 1338-43-8; the CAS number of the ethyl acetate is 141-78-6; the CAS number of the propylene glycol alginate is 9005-37-2; the CAS number of the mannitol is 87-78-5; the CAS number of the β-sitosterol is 83-46-5; and the CAS number of the sitosterol palmitate is 2308-85-2.

[0052] Example 1

[0053] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment includes the following steps:

[0054] (1) The hydroxyl bacteria were fermented and cultured, and the resulting fermentation broth was separated into solid and liquid components to obtain bacterial precipitate;

[0055] The fermentation culture includes: inoculating hydroxide bacteria into a culture medium and culturing them in a mixed gas of hydrogen, carbon dioxide, and oxygen under the following conditions: shaking culture at 30 °C, shaking speed of 200 rpm, and culture time of 3 days.

[0056] The volume ratio of hydrogen, carbon dioxide, and oxygen is 65:15:20; the hydroxide-oxidizing bacteria are hookworm copper-eating bacteria; the culture medium can be a suitable culture medium in the prior art, such as DSMZ medium. In this embodiment, the culture medium includes: potassium dihydrogen phosphate 2.0 g / L, disodium hydrogen phosphate dihydrate 2.0 g / L, ammonium chloride 1.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium bicarbonate 0.5 g / L, calcium chloride dihydrate 0.01 g / L, ferric ammonium citrate 0.05 g / L, and trace element solution 1 mL / L, with a pH of 6.8; the trace element solution includes: boric acid 0.5 g / L, cobalt chloride hexahydrate 0.2 g / L, zinc sulfate heptahydrate 0.1 g / L, manganese chloride tetrahydrate 0.05 g / L, sodium molybdate dihydrate 0.1 g / L, nickel chloride hexahydrate 0.1 g / L, and copper sulfate pentahydrate 0.01 g / L.

[0057] (2) The bacterial precipitate obtained in step (1) is resuspended and cell wall broken, then enzyme is added to it for enzymatic hydrolysis, then solid-liquid separation is performed, and the supernatant is desalted, concentrated and dried to obtain the enzymatic hydrolysis product.

[0058] The resuspension was performed using a buffer solution with a pH of 6.8; the concentration of the bacterial cell precipitate in the buffer solution was 40 g / L; the cell disruption was performed using high-pressure homogenization, with three cycles at 80 MPa; the enzyme included a neutral protease, and the concentration of the neutral protease in the buffer solution was 180 mg / L; the enzymatic hydrolysis specifically included: reacting at 40°C and a stirring speed of 150 rpm for 4 h;

[0059] (3) Dissolve the enzymatic hydrolysis product obtained in step (2) in water, add low-methoxyl pectin, gelatin and calcium carbonate and mix evenly to obtain an aqueous phase; mix ethyl cellulose, emulsifier and ethyl acetate evenly to obtain an oil phase; mix the aqueous phase and oil phase evenly to form a water-in-oil emulsion; add acetic acid cycloalkane solution and mix evenly. After reaction, centrifuge the reaction solution at 10000r / min for 15 min, collect the precipitate, wash the obtained precipitate twice with water and then twice with anhydrous ethanol, and freeze-dry to obtain aquatic feed protein source;

[0060] The weight ratio of the enzymatic hydrolysate, low-methoxyl pectin, gelatin, and calcium carbonate is 1:0.1:0.3:0.005; the concentration of the enzymatic hydrolysate in the aqueous phase is 80 g / L. The weight ratio of ethyl cellulose and emulsifier is 1:0.05; the mass concentration of the emulsifier in the oil phase is 3.5%. The volume ratio of the aqueous phase to the oil phase is 1:3.

[0061] The emulsifier is a mixture of polyethylene glycol dioleate and Span in a weight ratio of 1:0.5.

[0062] A cycloalkane solution of acetic acid is added dropwise to the emulsion at a rate of 1 mL / min, while the mixture is stirred at a speed of 600 r / min; wherein the cycloalkane is cyclopentane; the mass concentration of acetic acid in the cycloalkane solution is 10 wt%; and the molar ratio of acetic acid to calcium carbonate is 3:1.

[0063] Example 2

[0064] (1) The hydroxyl bacteria were fermented and cultured, and the resulting fermentation broth was separated into solid and liquid components to obtain bacterial precipitate;

[0065] The fermentation culture includes: inoculating hydroxide bacteria into a culture medium and culturing them in a mixed gas of hydrogen, carbon dioxide, and oxygen under the following conditions: shaking culture at 32 ℃, shaking speed of 220 rpm, and culture time of 3 days.

[0066] The volume ratio of hydrogen, carbon dioxide, and oxygen is 65:15:20; the hydroxide-oxidizing bacteria are hookworm copper-eating bacteria; the culture medium can be a suitable culture medium in the prior art, such as DSMZ medium. In this embodiment, the culture medium includes: potassium dihydrogen phosphate 3.0 g / L, disodium hydrogen phosphate dihydrate 3.0 g / L, ammonium chloride 1.5 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium bicarbonate 0.8 g / L, calcium chloride dihydrate 0.05 g / L, ferric ammonium citrate 0.1 g / L, and trace element solution 3 mL / L, with a pH of 7.2; the trace element solution includes: boric acid 0.8 g / L, cobalt chloride hexahydrate 0.8 g / L, zinc sulfate heptahydrate 0.5 g / L, manganese chloride tetrahydrate 0.1 g / L, sodium molybdate dihydrate 0.2 g / L, nickel chloride hexahydrate 0.2 g / L, and copper sulfate pentahydrate 0.05 g / L.

[0067] (2) The bacterial precipitate obtained in step (1) is resuspended and cell wall broken, then enzyme is added to it for enzymatic hydrolysis, then solid-liquid separation is performed, and the supernatant is desalted, concentrated and dried to obtain the enzymatic hydrolysis product.

[0068] The resuspension was performed using a buffer solution with a pH of 7.2; the concentration of the bacterial cell precipitate in the buffer solution was 50 g / L; the cell disruption was performed using high-pressure homogenization, with four cycles at 70 MPa; the enzyme included a neutral protease, and the concentration of the neutral protease in the buffer solution was 150 mg / L; the enzymatic hydrolysis specifically included reacting at 42 ℃ and a stirring speed of 200 rpm for 2 h;

[0069] (3) Dissolve the enzymatic hydrolysis product obtained in step (2) in water, add low-methoxyl pectin, gelatin and calcium carbonate and mix evenly to obtain an aqueous phase; mix ethyl cellulose, emulsifier and ethyl acetate evenly to obtain an oil phase; mix the aqueous phase and oil phase evenly to form a water-in-oil emulsion; add acetic acid cycloalkane solution and mix evenly. After reaction, centrifuge the reaction solution at 10000r / min for 15 min, collect the precipitate, wash the obtained precipitate twice with water and then twice with anhydrous ethanol, and freeze-dry to obtain aquatic feed protein source;

[0070] The weight ratio of the enzymatic hydrolysate, low-methoxyl pectin, gelatin, and calcium carbonate is 1:0.2:0.1:0.015; the concentration of the enzymatic hydrolysate in the aqueous phase is 100 g / L. The weight ratio of ethyl cellulose and emulsifier is 1:0.10; the mass concentration of the emulsifier in the oil phase is 4%. The volume ratio of the aqueous phase to the oil phase is 1:4.

[0071] The emulsifier is a mixture of polyethylene glycol dioleate and Span in a weight ratio of 1:2.

[0072] A cycloalkane solution of acetic acid is added dropwise to the emulsion at a rate of 2 mL / min, while stirring at a speed of 700 r / min; wherein the cycloalkane is cyclohexane; the mass concentration of acetic acid in the cycloalkane solution is 10 wt%; and the molar ratio of acetic acid to calcium carbonate is 3.5:1.

[0073] Example 3

[0074] (1) The hydroxyl bacteria were fermented and cultured, and the resulting fermentation broth was separated into solid and liquid components to obtain bacterial precipitate;

[0075] The fermentation culture includes: inoculating hydroxide bacteria into a culture medium and culturing them in a mixed gas of hydrogen, carbon dioxide, and oxygen under the following conditions: shaking culture at 27 °C, shaking speed of 180 rpm, and culture time of 4 days.

[0076] The volume ratio of hydrogen, carbon dioxide, and oxygen is 65:15:20; the hydroxide-oxidizing bacteria is hookworm copper-eating bacteria; the culture medium can be a suitable culture medium in the prior art, such as DSMZ medium. In this embodiment, the culture medium comprises: potassium dihydrogen phosphate 2.5 g / L, disodium hydrogen phosphate dihydrate 2.5 g / L, ammonium chloride 1.5 g / L, magnesium sulfate heptahydrate 0.4 g / L, sodium bicarbonate 0.65 g / L, calcium chloride dihydrate 0.03 g / L, ferric ammonium citrate 0.75 g / L, and trace element solution 2 mL / L, with a pH of 7.2; the trace element solution comprises: boric acid 0.65 g / L, cobalt chloride hexahydrate 0.5 g / L, zinc sulfate heptahydrate 0.3 g / L, manganese chloride tetrahydrate 0.075 g / L, sodium molybdate dihydrate 0.15 g / L, nickel chloride hexahydrate 0.15 g / L, and copper sulfate pentahydrate 0.03 g / L.

[0077] (2) The bacterial precipitate obtained in step (1) is resuspended and cell wall broken, then enzyme is added to it for enzymatic hydrolysis, then solid-liquid separation is performed, and the supernatant is desalted, concentrated and dried to obtain the enzymatic hydrolysis product.

[0078] The resuspension was performed using a buffer solution with a pH of 7.2; the concentration of the bacterial cell precipitate in the buffer solution was 60 g / L; the cell wall disruption was performed using high-pressure homogenization, with 5 cycles at 60 MPa; the enzyme included a neutral protease, and the concentration of the neutral protease in the buffer solution was 300 mg / L; the enzymatic hydrolysis specifically included: reacting at 45 ℃ and a stirring speed of 200 rpm for 6 h;

[0079] (3) Dissolve the enzymatic hydrolysis product obtained in step (2) in water, add low-methoxyl pectin, gelatin and calcium carbonate and mix evenly to obtain an aqueous phase; mix ethyl cellulose, emulsifier and ethyl acetate evenly to obtain an oil phase; mix the aqueous phase and oil phase evenly to form a water-in-oil emulsion; add acetic acid cycloalkane solution and mix evenly. After reaction, centrifuge the reaction solution at 10000r / min for 15 min, collect the precipitate, wash the obtained precipitate twice with water and then twice with anhydrous ethanol, and freeze-dry to obtain aquatic feed protein source;

[0080] The weight ratio of the enzymatic hydrolysate, low-methoxyl pectin, gelatin, and calcium carbonate is 1:0.3:0.4:0.010; the concentration of the enzymatic hydrolysate in the aqueous phase is 90 g / L. The weight ratio of ethyl cellulose and emulsifier is 1:0.15; the mass concentration of the emulsifier in the oil phase is 3%. The volume ratio of the aqueous phase to the oil phase is 1:5.

[0081] The emulsifier is a mixture of polyethylene glycol dioleate and Span in a weight ratio of 1:3.5.

[0082] A cycloalkane solution of acetic acid is added dropwise to the emulsion at a rate of 1.5 mL / min while stirring at a speed of 650 r / min; wherein the cycloalkane is selected from a mixture of cyclohexane and cyclopentane; the mass concentration of acetic acid in the cycloalkane solution is 9 wt%; and the molar ratio of acetic acid to calcium carbonate is 3.5:1.

[0083] Example 4

[0084] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment includes the following steps:

[0085] (1) The hydroxyl bacteria were fermented and cultured, and the resulting fermentation broth was separated into solid and liquid components to obtain bacterial precipitate;

[0086] The fermentation culture includes: inoculating hydroxide bacteria into a culture medium and culturing them in a mixed gas of hydrogen, carbon dioxide, and oxygen under the following conditions: shaking culture at 30 °C, shaking speed of 200 rpm, and culture time of 3 days.

[0087] The volume ratio of hydrogen, carbon dioxide, and oxygen is 65:15:20; the hydroxide-oxidizing bacteria are hookworm copper-eating bacteria; the culture medium can be a suitable culture medium in the prior art, such as DSMZ medium. In this embodiment, the culture medium includes: potassium dihydrogen phosphate 2.3 g / L, disodium hydrogen phosphate dihydrate 2.9 g / L, ammonium chloride 1 g / L, magnesium sulfate heptahydrate 0.5 g / L, sodium bicarbonate 0.5 g / L, calcium chloride dihydrate 0.01 g / L, ferric ammonium citrate 0.05 g / L, and trace element solution 1 mL / L, with a pH of 7.0; the trace element solution includes: boric acid 0.6 g / L, cobalt chloride hexahydrate 0.4 g / L, zinc sulfate heptahydrate 0.2 g / L, manganese chloride tetrahydrate 0.06 g / L, sodium molybdate dihydrate 0.06 g / L, nickel chloride hexahydrate 0.04 g / L, and copper sulfate pentahydrate 0.02 g / L.

[0088] (2) The bacterial precipitate obtained in step (1) is resuspended and cell wall broken, then enzyme is added to it for enzymatic hydrolysis, then solid-liquid separation is performed, and the supernatant is desalted, concentrated and dried to obtain the enzymatic hydrolysis product.

[0089] The resuspension was performed using a buffer solution with a pH of 7.0; the concentration of the bacterial cell precipitate in the buffer solution was 50 g / L; the cell disruption was performed using high-pressure homogenization, with three cycles at 80 MPa; the enzyme included a neutral protease, and the concentration of the neutral protease in the buffer solution was 200 mg / L; the enzymatic hydrolysis specifically included: reacting at 42 ℃ and a stirring speed of 150 rpm for 4 h;

[0090] (3) Dissolve the enzymatic hydrolysis product obtained in step (2) in water, add low-methoxyl pectin, gelatin, calcium carbonate, propylene glycol alginate and mannitol and mix evenly to obtain an aqueous phase; mix ethyl cellulose, emulsifier, ethyl acetate, β-sitosterol and sitosterol palmitate evenly to obtain an oil phase; mix the aqueous phase and oil phase evenly to form a water-in-oil emulsion; add acetic acid cycloalkane solution and mix evenly. After reaction, centrifuge the reaction solution at 10000 r / min for 15 min, collect the precipitate, wash the obtained precipitate twice with water and then twice with anhydrous ethanol, and freeze-dry to obtain aquatic feed protein source;

[0091] The weight ratio of the enzymatic hydrolysate, low-methoxyl pectin, gelatin, and calcium carbonate is 1:0.2:0.3:0.010; the concentration of the enzymatic hydrolysate in the aqueous phase is 90 g / L. The weight ratio of ethyl cellulose and emulsifier is 1:0.10; the mass concentration of the emulsifier in the oil phase is 3-4%. The volume ratio of the aqueous phase to the oil phase is 1:4.

[0092] The weight ratio of propylene glycol alginate to the enzymatic hydrolysis product is 0.05:1; the weight ratio of mannitol to the enzymatic hydrolysis product is 0.2:1; the weight ratio of β-sitosterol to the enzymatic hydrolysis product is 0.03:1; and the weight ratio of sitosterol palmitate to the enzymatic hydrolysis product is 0.01:1.

[0093] The emulsifier is a mixture of polyethylene glycol dioleate and Span in a 1:1 weight ratio;

[0094] A cycloalkane solution of acetic acid is added dropwise to the emulsion at a rate of 1.5 mL / min, while the mixture is stirred at a speed of 700 r / min; wherein the cycloalkane is cyclohexane; the mass concentration of acetic acid in the cyclohexane is 12 wt%; and the molar ratio of acetic acid to calcium carbonate is 3:1.

[0095] Example 5

[0096] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that the propylene glycol alginate is not added.

[0097] Example 6

[0098] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that mannitol is not added.

[0099] Example 7

[0100] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that the propylene glycol alginate and mannitol are not added.

[0101] Example 8

[0102] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that the sitosterol palmitate is not added.

[0103] Example 9

[0104] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that the β-sitosterol and sitosterol palmitate are not added.

[0105] Example 10

[0106] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that the propylene glycol alginate, mannitol and sitosterol palmitate are not added.

[0107] Example 11

[0108] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that in step (3), the cycloalkane solution of acetic acid is added to the emulsion at a dropping rate of 20 mL / min, while stirring at a speed of 700 r / min.

[0109] Comparative Example 1

[0110] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that only the low-methoxyl pectin and calcium carbonate are added to the aqueous phase, and the gelatin, propylene glycol alginate and mannitol are not added.

[0111] Comparative Example 2

[0112] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that only the gelatin is added to the aqueous phase, and the low-methoxyl pectin, calcium carbonate, propylene glycol alginate and mannitol are not added.

[0113] Comparative Example 3

[0114] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in Example 4, except that the acetic acid is replaced with hydrochloric acid.

[0115] Comparative Example 4

[0116] The method for producing aquatic feed protein source by fixing carbon dioxide in this embodiment is the same as that in embodiment 4, except that the enzymatic hydrolysis product obtained in step (2) is the aquatic feed protein source.

[0117] Effect Experiment Example

[0118] To verify the technical effectiveness of the feed protein source obtained by the method for producing aquatic feed protein source by fixing carbon dioxide as described in this invention, the following experiments were conducted:

[0119] 1. Dissolution rate

[0120] 1g of aquatic feed protein source samples from Experimental Examples 1-11 and Comparative Examples 1-4 were taken and immersed in beakers containing 400 mL of water at 25 ℃. The water was gently stirred with a magnetic stirrer to maintain a uniform state. The samples were removed from the water at 0.5h, 1h, and 2h, with three replicates for each time period. After filtration through a 200-mesh sieve and drying, the total nitrogen content of each group of aquatic feed protein source before and after immersion in water was determined by the Kjeldahl method, and the dissolution rate was calculated.

[0121] Dissolution rate = (N content in dry matter before soaking - N content in dry matter after soaking) / N content in dry matter before soaking x 100%.

[0122] 2. Apparent digestibility

[0123] First, the aquatic feed protein source obtained in Examples 1-11 and Comparative Examples 1-2 was mixed evenly with corn flour, fish meal, soybean meal, wheat bran, sweet potato, salt, multivitamins, and dicalcium phosphate in a ratio of 12:60:12:8:5:12:0.5:2:2. The mixture was then granulated and dried to obtain aquatic feed, which served as Experimental Examples 1-11 and Comparative Examples 1-2, respectively. An aquatic feed prepared without the aforementioned aquatic feed protein source, but using corn flour, fish meal, soybean meal, wheat bran, sweet potato, salt, multivitamins, and dicalcium phosphate in a ratio of 60:24:8:5:12:0.5:2:2, served as the control group.

[0124] Grass carp were used as the experimental subject. 180 healthy grass carp of similar weight were randomly placed in 18 aquariums, 10 fish per aquarium. Initially, they were fed a basic diet three times a day until full. After one week, they were switched to the aquatic diets of Experimental Example 1-11 and Comparative Example 1-2 (the aquatic diets of Experimental Example 1-11 and Comparative Example 1-2 had 1% exogenous chromium trioxide added during preparation). They were fed twice a day (8:00 and 16:00). After each feeding, feces and leftover food were siphoned from the bottom of the aquarium. Two hours later, feces were gently scooped from the bottom of the aquarium with a small net, and the collected feces samples were promptly transferred to -20℃ for storage. The protein content (N) in the aquatic diets of Experimental Example 1-11 and Comparative Example 1-2 was determined using the Kjeldahl method. d And the cumulative protein content (N) in feces over 7 days f The chromium trioxide content (M) in the aquatic feed of Experimental Examples 1-13 and Comparative Examples 1-5 was determined by atomic absorption spectrometry. d And the cumulative chromium trioxide content (M) in feces over 7 days f ;

[0125] Apparent digestibility = x100%.

[0126] The results of the experiment are as follows:

[0127]

[0128] 3. Appetizing effect

[0129] A rectangular open tank was used as the experimental setup, and the tank was divided into three areas: A, B, and C. Area A was the experimental area, where aquatic feed made from the aquatic feed protein source of Example 4 was fed (see the apparent digestibility experiment for feed formulation). Area C was the control area, where the aquatic feed of the blank group was fed. The inlet pipe was located on the tank wall on the side of areas A and C, and the outlet pipe was located on the tank wall on the side of area B. To prevent interference between the two aquatic feeds, the experimental fish and the tank were washed with tap water and the water in the tank was replaced after each experiment. The interval between feeding the two feeds of Example 4 and the blank group was 24 hours.

[0130] Before the experiment, grass carp were fasted for 24 hours to induce a state of starvation. The inlet and outlet pipes were simultaneously turned on to maintain water flow in the tank. The grass carp were placed in zone B, and for the first 5 minutes, glass partitions were used to separate zones A, B, and C, preventing the fish from entering zones A and C. After 5 minutes, the glass partitions were removed, allowing the fish free access to zones A, B, and C. A data camera was used to record the total number of times the fish entered zones A and C within 30 minutes. The experiment was repeated three times. For each experiment, the feeding attraction index was calculated using the following formula, and the average feeding attraction index was calculated.

[0131] The feeding attraction index is calculated as follows: (Number of times fish entered the experimental area - Number of times fish entered the control area) / Number of times fish entered the control area. A feeding attraction index > 0 indicates that the fish has a feeding attraction effect; a feeding attraction index = 0 indicates that the fish has no feeding attraction or avoidance effect; a feeding attraction index < 0 indicates that the fish has a feeding refusal effect.

[0132] The results of the experiment are as follows:

[0133]

[0134] Based on the above experimental results, it can be seen that the aquatic feed protein source prepared by the method of producing aquatic feed protein source by fixing carbon dioxide according to the present invention, when added to aquatic feed, can have a low solubility, a high apparent digestibility, and a certain feeding attraction effect.

[0135] Based on the results of Example 4 and Comparative Example 1, the interpenetrating network structure of low-methoxyl pectin gel and gelatin gel can form a denser coating on the enzymatic hydrolysis products, significantly reducing the solubility loss rate of aquatic feed protein sources compared to the gel structure of Comparative Example 1. Based on the results of Example 4 and Comparative Example 2, without the use of low-methoxyl pectin and calcium carbonate to form a gel structure and without the formation of a microporous structure, although the solubility loss rate of aquatic feed protein sources was slightly reduced, the apparent digestibility decreased significantly. Therefore, the formation of a microporous structure helps improve the apparent digestibility and palatability of aquatic feed, and has no significant impact on the solubility loss rate. Based on the results of Example 4, Comparative Examples 3 and 11, it is known that adding acetic acid too quickly or using strong acids may exacerbate the generation of carbon dioxide gas, leading to excessively large pores or shell rupture, resulting in excessively rapid solubility loss. Based on the results of Example 4 and Comparative Example 4, the present invention, through the encapsulation of enzymatic hydrolysis products using a gel-cellulose shell microcapsule structure, can effectively slow down protein loss.

[0136] According to the results of Examples 4 and 5-11, the addition of propylene glycol alginate, mannitol, β-sitosterol, and sitosterol palmitate can further improve the stability of the coating structure. Furthermore, when propylene glycol alginate, mannitol, β-sitosterol, and sitosterol palmitate are added simultaneously, the dissolution rate of the aquatic feed protein source can be significantly reduced, and the apparent digestibility of the aquatic feed can be improved. In Example 8, although the short-term dissolution rate of the aquatic feed was not significantly affected without the addition of sitosterol palmitate, the dissolution rate at 2 hours increased significantly, and the apparent digestibility increased. This may be because sitosterol palmitate can fix β-sitosterol. Without the addition of sitosterol palmitate, over time, the small molecule structure of β-sitosterol gradually migrates outward, disrupting the continuity of the ethyl cellulose shell and affecting its hydrophobicity and stability.

[0137] 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 its equivalents are encompassed by this invention.

Claims

1. A method for producing aquatic feed protein source by fixing carbon dioxide, characterized in that, Includes the following steps: (1) The hydroxyl bacteria were fermented and cultured, and the resulting fermentation broth was separated into solid and liquid components to obtain bacterial precipitate; (2) The bacterial precipitate obtained in step (1) is resuspended and cell wall broken, then enzyme is added to it for enzymatic hydrolysis, then solid-liquid separation is performed, and the supernatant is desalted, concentrated and dried to obtain the enzymatic hydrolysis product. (3) Dissolve the enzymatic hydrolysis product obtained in step (2) in water, add low-methoxyl pectin, gelatin and calcium carbonate and mix evenly to obtain an aqueous phase; mix ethyl cellulose, emulsifier and ethyl acetate evenly to obtain an oil phase; mix the aqueous phase and oil phase evenly to form a water-in-oil emulsion; add acetic acid cycloalkane solution and mix evenly; after reaction, separate the solid and liquid, wash and freeze dry to obtain aquatic feed protein source.

2. The method for producing aquatic feed protein source by fixing carbon dioxide according to claim 1, characterized in that, In step (1), the fermentation culture includes: inoculating hydroxide bacteria into the culture medium and culturing it with shaking at 27-37°C for 2-4 days in a mixed gas of hydrogen, carbon dioxide and oxygen; The volume ratio of hydrogen, carbon dioxide, and oxygen is (60-70):(10-20):(15-25).

3. The method for producing aquatic feed protein source by fixing carbon dioxide according to claim 1, characterized in that, In step (2), the resuspension is performed using a buffer solution with a pH of 6.5-7.5; Optionally, the concentration of the bacterial cell precipitate in the buffer solution is 40-60 g / L; Optionally, the cell wall breaking is performed using high-pressure homogenization, with the cells cyclically broken 2-5 times at 60-80 MPa. Optionally, the enzyme comprises a protease; the concentration of the protease in the buffer solution is 150-300 mg / L; Optionally, the enzymatic hydrolysis specifically includes: reacting at a temperature of 40–45°C and a stirring speed of 150–250 rpm for 2–6 hours.

4. The method for producing aquatic feed protein source by fixing carbon dioxide according to claim 1, wherein in step (3), the weight ratio of the enzymatic hydrolysate, low-methoxyl pectin, gelatin, and calcium carbonate in the aqueous phase is 1:(0.1-0.3):(0.1-0.4):(0.005-0.015); and the concentration of the enzymatic hydrolysate is 80-100 g / L; In the oil phase, the weight ratio of ethyl cellulose to emulsifier is 1:(0.05-0.15); the mass concentration of the emulsifier is 3-4%. The volume ratio of the aqueous phase to the oil phase is 1:(3-5).

5. The method for producing aquatic feed protein source by fixing carbon dioxide according to claim 1, characterized in that, In step (3), the emulsifier is one or more of polyethylene glycol dioleate and Span. Optionally, the emulsifier is a mixture of polyethylene glycol dioleate and Span in a weight ratio of 1:(0.5-3.5).

6. The method for producing aquatic feed protein source by fixing carbon dioxide according to claim 1, characterized in that, In step (3), the aqueous phase also contains one or more of propylene glycol alginate and mannitol; Optionally, the weight ratio of the propylene glycol alginate to the enzymatic hydrolysis product is (0.02-0.08):1; Optionally, the weight ratio of mannitol to the enzymatic hydrolysis product is (0.1-0.2):

1.

7. The method for producing aquatic feed protein source by fixing carbon dioxide according to claim 1, characterized in that, In step (3), the oil phase also contains one or more of β-sitosterol and sitosterol palmitate; Optionally, the weight ratio of β-sitosterol to the enzymatic hydrolysis product is (0.01-0.05):1; Optionally, the weight ratio of the sitosterol palmitate to the enzymatic hydrolysis product is (0.005-0.02):

1.

8. The method for producing aquatic feed protein source by fixing carbon dioxide according to claim 1, characterized in that, In step (3), the cycloalkane solution of acetic acid is added dropwise to the water-in-oil emulsion at a rate of 1–2 mL / min, while stirring at a speed of 600–700 r / min. Optionally, the cycloalkane is selected from cyclohexane, cyclopentane, or mixtures thereof; Optionally, the acetic acid in the cycloalkane solution has a mass concentration of 5 wt%-15 wt%; Optionally, the molar ratio of acetic acid to calcium carbonate is (3.0-3.5):

1.

9. The aquatic feed protein source prepared by the method according to any one of claims 1-8.

10. An aquatic feed comprising an aquatic feed protein source prepared by any one of claims 1-8.

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