Preparation method of nano protein selenium for animal feed additive

Abstract

The invention relates to the technical field of feed additives, and discloses a preparation method of nano protein selenium for an animal feed additive, and the preparation method comprises the following steps: step 1, preparing a protein solution with the mass volume concentration of 1-5%; 2, sequentially adding sodium selenite and soluble salt into the protein solution, and stirring and mixing; step 3, putting the mixed solution in the step 2 into an alternating magnetic field generator with the frequency of 10-50kHz, controlling the temperature of the solution to be increased to 55-70 DEG C at the speed of 0.5-1.5 DEG C / min, and carrying out heat preservation reaction for 20-60 minutes at the temperature; step 4, performing centrifugal separation to obtain a solid product, and performing washing and freeze drying to obtain nano protein selenium composite particles; through the synergistic effect of the alternating magnetic field and the heat treatment, the conformation controllable transformation of natural protein is realized, the synthesis and multi-stage self-assembly of the zero-valent selenium nanocrystal are synchronously completed, and the bioavailability of selenium is improved; the prepared nano protein selenium composite particle has a uniform spherical structure.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to a method for preparing nano-protein selenium for use as an animal feed additive. Background Technology

[0002] Selenium, as an important trace element, plays a crucial role in animal growth, development, immune function, and reproductive performance among current feed additives. However, traditional selenium additives, such as inorganic selenium like sodium selenite, while offering some selenium supplementation, suffer from low bioavailability. In recent years, with the continuous development of nanotechnology, nano-selenium has gradually become a research hotspot in the field of selenium additives due to its unique physicochemical properties and excellent bioactivity. However, it also has drawbacks such as uneven particle size, easy agglomeration, and poor stability.

[0003] Among numerous methods for preparing nano-selenium, nano-protein selenium has attracted widespread attention due to its combination of the nutritional value of protein and the bioactivity of nano-selenium. Protein, as an important nutrient in living organisms, possesses good biocompatibility and degradability, and can serve as a carrier for nano-selenium, improving its stability and bioavailability. Therefore, this application proposes a method for preparing nano-protein selenium for use as an animal feed additive. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing nano-protein selenium for use as an animal feed additive, so as to solve the problems mentioned in the background art.

[0005] In a first aspect, to address the aforementioned problems, the present invention provides a method for preparing nano-protein selenium for use as an animal feed additive, comprising the following steps:

[0006] Step 1: Dissolve the natural protein in phosphate buffer to prepare a protein solution with a mass-volume concentration of 1-5%.

[0007] Step 2: Add sodium selenite and soluble salt to the protein solution in sequence, and stir to mix;

[0008] Step 3: Place the mixed solution from Step 2 in an alternating magnetic field generator with a frequency of 10-50kHz, control the solution to heat up to 55-70℃ at a rate of 0.5-1.5℃ / min, and keep it at this temperature for 20-60 minutes.

[0009] During the reaction, the natural protein undergoes conformational transformation and phase separation under the dual induction of alternating magnetic field and heat treatment, forming nanoscale protein aggregates. Simultaneously, selenite ions in the solution are reduced in situ to zero-valent selenium nanocrystals. The selenium nanocrystals undergo multi-level assembly with the protein aggregates and zinc ions and / or copper ions in the solution.

[0010] Step 4: After the reaction is complete, cool to below 5°C, and obtain the solid product by centrifugation. After washing and freeze-drying, obtain nano-protein selenium composite particles.

[0011] Specifically, in step one, the natural protein is either soy protein isolate or whey protein.

[0012] Specifically, in step two, the amount of sodium selenite added is such that the concentration of selenium in the protein solution is 0.5-5 mmol / L.

[0013] Specifically, in step two, the soluble zinc salt is zinc sulfate heptahydrate and copper sulfate pentahydrate;

[0014] The molar ratio of zinc to selenium is 10:1; the molar ratio of copper to selenium is 2:1.

[0015] Specifically, in step three, pectin, accounting for 5-15% of the mass of the natural protein, is added to the protein solution. The pectin, along with the natural protein, sodium selenite, and soluble zinc salt, participates in the assembly process of step three to form a stable polysaccharide-protein-mineral complex network structure.

[0016] Specifically, in step three, after the reaction is complete, while maintaining stirring and temperature, acrylic acid and N-isopropylacrylamide, as well as the initiator potassium persulfate, are added, and the reaction continues for 40-60 minutes to form a hydrogel polymer layer with dual temperature and pH response in situ on the surface of the nanocomposite mineral particles.

[0017] The hydrogel polymer layer swells at temperatures below 35°C and pH above 6.5, and shrinks at temperatures above 40°C and / or pH below 5.0.

[0018] Specifically, the mass ratio of acrylic acid to N-isopropylacrylamide is 1:2-4;

[0019] The total amount of acrylic acid and N-isopropylacrylamide added is 10-30% of the mass of the natural protein;

[0020] The amount of potassium persulfate added is 1.0% of the total mass of acrylic acid and N-isopropylacrylamide.

[0021] Specifically, in step four, before freeze-drying, the following steps are also included: redispersing the washed precipitate in a sodium alginate solution, stirring evenly, and then dropping it into a calcium chloride solution to form sodium alginate-calcium chloride gel microspheres encapsulating the nanocomposite mineral particles, followed by washing and freeze-drying.

[0022] Specifically, the sodium alginate solution has a mass concentration of 1-2%; the calcium chloride solution has a mass concentration of 2-4%.

[0023] Secondly, the present invention provides a nano-protein selenium for use as an animal feed additive, which is prepared by the above-described method for preparing nano-protein selenium for use as an animal feed additive, specifically as nano-protein selenium composite particles.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention achieves controllable conformational transformation of natural proteins through the synergistic effect of alternating magnetic fields and heat treatment, simultaneously completing the synthesis and multi-level self-assembly of zero-valent selenium nanocrystals, thereby improving the bioavailability of selenium; the prepared nano-protein selenium composite particles have a uniform spherical structure.

[0026] This invention utilizes a hydrogel polymer layer to enable nano-protein selenium composite particles to automatically contract and release selenium when an animal's body temperature rises, effectively improving selenium supplementation efficiency under heat stress conditions. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] In a first aspect, to address the aforementioned problems, the present invention provides a method for preparing nano-protein selenium for use as an animal feed additive, comprising the following steps:

[0029] Step 1: Dissolve the natural protein in phosphate buffer to prepare a protein solution with a mass-volume concentration of 1-5%. The natural protein used is either soy protein isolate or whey protein. The dissolution process is carried out on a thermostatic magnetic stirrer at a temperature controlled at 25-30℃ and a stirring speed set at 300-500 rpm for 2-4 hours until completely dissolved. After dissolution, filter the solution using a 0.45μm microporous membrane to remove any insoluble particles. The phosphate buffer solution used is a 0.01M phosphate buffer solution with a pH of 7.2-7.4. The pH of the solution needs to be continuously monitored during dissolution, and adjustments should be made promptly if the pH fluctuates by more than ±0.2. The prepared protein solution should be allowed to stand at 4℃ for 1 hour to degas and eliminate air bubbles introduced during stirring, so as to avoid affecting the subsequent alternating magnetic field treatment effect.

[0030] Step 2: Add sodium selenite and soluble salt to the protein solution in sequence, and stir to mix;

[0031] The amount of sodium selenite added is such that the concentration of selenium in the protein solution is 0.5-5 mmol / L;

[0032] The soluble zinc salts are zinc sulfate heptahydrate and copper sulfate pentahydrate; the molar ratio of zinc to selenium is 10:1; the molar ratio of copper to selenium is 2:1.

[0033] Specifically, the order of addition is as follows: first add sodium selenite, and then add the soluble salt after it is completely dissolved; the mixing process should be carried out at room temperature, using a magnetic stirrer at a speed of 200-300 rpm for 30-60 minutes to ensure that the components are evenly dispersed; during the mixing process, the pH of the solution should be checked periodically with pH test paper. When the pH deviates from the initial value by more than 0.3, 0.1M sodium hydroxide or hydrochloric acid solution should be added for fine adjustment; after stirring, the solution should be allowed to stand for 15 minutes to observe whether the solution shows layering or precipitation. If so, it needs to be stirred again.

[0034] Step 3: Place the mixed solution from Step 2 in an alternating magnetic field generator with a frequency of 10-50 kHz. Control the solution to heat up to 55-70℃ at a rate of 0.5-1.5℃ / min, and maintain this temperature for 20-60 minutes. During the reaction, the natural protein undergoes conformational transformation and phase separation under the dual induction of the alternating magnetic field and heat treatment, forming nanoscale protein aggregates. Simultaneously, selenite ions in the solution are reduced in situ to zero-valent selenium nanocrystals. The selenium nanocrystals undergo multi-level assembly with the protein aggregates and zinc ions and / or copper ions in the solution. During the alternating magnetic field treatment stage, the solution temperature needs to be monitored in real time using an infrared thermometer to ensure a uniform and controllable heating rate. The magnetic field strength is dynamically adjusted according to the solution volume. Each 100 mL of solution corresponds to a magnetic field strength of 200-400 mT; the reaction vessel is made of polytetrafluoroethylene to avoid interference from metal ions; when the solution becomes milky white and turbid, it indicates that protein aggregates are beginning to form. At this time, the magnetic field frequency needs to be adjusted to 20-30 kHz and maintained for 10 minutes to promote the uniform crystallization of zero-valent selenium nanocrystals; after multi-stage assembly, the solution presents a uniform pale yellow colloidal state. The particle size distribution is detected by dynamic light scattering instrument to ensure that the D50 value is within the range of 80-120 nm; the entire reaction is carried out under nitrogen protection to prevent the oxidation and loss of selenium; during the heat preservation stage, samples are taken every 15 minutes for ultraviolet-visible spectroscopy analysis to monitor the change of the characteristic absorption peak at 480 nm and confirm the generation efficiency of zero-valent selenium.

[0035] Step 4: After the reaction is complete, cool to below 5°C and obtain the solid product by centrifugation. After washing and freeze-drying, obtain nano-protein selenium composite particles. Centrifugation is performed using a low-temperature high-speed centrifuge with a speed set at 8000-12000 rpm and a centrifugation time of 15-30 minutes to ensure complete separation of solid and liquid. The washing process uses pre-cooled deionized water for three repeated washings, and the precipitate needs to be collected by centrifugation after each washing. Before freeze-drying, the washed precipitate is placed in a -20°C freezer for 4-6 hours to completely solidify the moisture. Freeze-drying is performed using a vacuum freeze dryer with a cold trap temperature set below -50°C and a vacuum degree below 10 Pa for 12-24 hours until constant weight is achieved. The dried nano-protein selenium composite particles need to be immediately transferred to a sealed container containing desiccant and stored at 4°C.

[0036] Specifically, in step three, pectin, accounting for 5-15% of the mass of the natural protein, is added to the protein solution. The pectin, along with the natural protein, sodium selenite, and soluble zinc salt, participates in the assembly process of step three to form a stable polysaccharide-protein-mineral composite network structure. The pectin is added 10 minutes before the alternating magnetic field treatment, and uniform dispersion is ensured by adding it dropwise. The surface of the assembled body after adding pectin exhibits a honeycomb porous structure with pore sizes distributed in the range of 50-100 nm, which increases the specific surface area of ​​the composite particles.

[0037] Specifically, in step three, after the reaction is complete, while maintaining stirring and temperature, acrylic acid, N-isopropylacrylamide, and potassium persulfate as the initiator are added, and the reaction continues for 40-60 minutes to form a hydrogel polymer layer with dual temperature and pH response on the surface of the nanocomposite mineral particles in situ. The order of adding acrylic acid and N-isopropylacrylamide is as follows: add acrylic acid first, and after it is completely dissolved, slowly add N-isopropylacrylamide. The entire addition process requires continuous stirring to prevent local concentration from being too high and causing uneven polymerization. The potassium persulfate initiator needs to be prepared into a 5% solution with deionized water and added dropwise through a constant pressure dropping funnel at a dropping rate of 1-2 mL per minute. During the reaction, nitrogen gas needs to be continuously purged to remove oxygen and prevent free radical quenching.

[0038] Specifically, the hydrogel polymer layer swells at temperatures below 35°C and pH above 6.5, and shrinks at temperatures above 40°C and / or pH below 5.0; this allows the nano-protein selenium composite particles to accurately sense changes in the animal's internal environment and automatically release selenium under heat stress, thus improving selenium supplementation efficiency; the glass transition temperature of the hydrogel polymer layer is 32-35°C, which closely matches the normal body temperature range of animals; in a phosphate buffer solution with pH 7.4, the swelling ratio can reach 400-500% at 30°C, while shrinking to 20-30% of the original volume at 42°C.

[0039] Specifically, the mass ratio of acrylic acid to N-isopropylacrylamide is 1:2-4, which ensures sufficient temperature response sensitivity and maintains a linear relationship in pH response. The total amount of acrylic acid and N-isopropylacrylamide added is 10-30% of the mass of natural protein, balancing the mechanical strength and biocompatibility of the hydrogel polymer layer. The amount of potassium persulfate added is 1.0% of the total mass of acrylic acid and N-isopropylacrylamide, which enables a controllable free radical polymerization reaction and avoids excessive crosslinking that could lead to pore blockage.

[0040] Specifically, in step four, before freeze-drying, the following steps are also included: redispersing the washed precipitate in a sodium alginate solution, stirring evenly, and then dripping it into a calcium chloride solution to form sodium alginate-calcium chloride gel microspheres encapsulating the nanocomposite mineral particles; then washing and freeze-drying; the mass concentration of the sodium alginate solution is 1-2%; the mass concentration of the calcium chloride solution is 2-4%; the sodium alginate solution needs to be prepared in advance and refrigerated, and brought to room temperature before use to avoid temperature differences affecting gel formation; the calcium chloride solution needs to be prepared fresh for use to prevent calcium ion precipitation; the dripping process is carried out using... Using a microfluidic needle, the droplet diameter is controlled within the range of 2-3 mm to ensure uniform gel microsphere size. The gelation reaction is carried out at room temperature, with continuous stirring of the calcium chloride solution to rapidly cross-link the microsphere surface. The reaction time is controlled at 15-20 minutes. The formed gel microspheres need to be washed three times with physiological saline to remove residual calcium ions and sodium alginate from the surface. After washing, the microspheres need to be immediately transferred to pre-cooled deionized water to prevent excessive dehydration that could lead to structural breakage. The freeze-drying parameters are the same as those for direct drying of nano-protein selenium composite particles, but the drying time needs to be extended to 24-36 hours to ensure complete removal of moisture from the inside of the microspheres.

[0041] Secondly, this invention provides a nano-protein selenium for animal feed additives, prepared by the aforementioned method for preparing nano-protein selenium for animal feed additives. Specifically, it comprises nano-protein selenium composite particles. These particles use natural protein as a matrix and employ conformational transformation and phase separation technology induced by alternating magnetic fields to uniformly load zero-valent selenium nanocrystals within nanoscale protein aggregates. Simultaneously, a stable mineral-protein composite structure is formed through multi-level assembly of zinc / copper ions. The surface-coated thermosensitive / pH-sensitive hydrogel polymer layer is copolymerized from acrylic acid and N-isopropylacrylamide. In the animal's gastrointestinal environment, it can respond to changes in body temperature and pH gradients. When the animal is under heat stress (body temperature > 40°C) or in an acidic gastric environment (pH < 5.0), the hydrogel layer contracts and ruptures, releasing the internal selenium element. Under normal physiological conditions (body temperature 38-39°C, intestinal pH 6.5-7.5), it maintains structural integrity to prevent premature selenium loss. The gel microsphere structure formed after secondary encapsulation with sodium alginate and calcium chloride further controls the selenium release rate.

[0042] Example 1

[0043] A method for preparing nano-protein selenium for use as an animal feed additive includes the following steps:

[0044] Step 1: Weigh 0.24g of sodium dihydrogen phosphate and 1.44g of disodium hydrogen phosphate, dissolve them in deionized water and bring the volume to 1L;

[0045] Weigh 5.00g of whey protein powder, add it to the above phosphate buffer, stir until completely dissolved, and bring the volume up to 100mL to obtain a protein solution with a mass-volume concentration of 5.0%.

[0046] Step 2: Measure 100 mL of the above protein solution into a 250 mL three-necked flask, accurately add 2.0 mL of 0.1 mol / L sodium selenite stock solution (to make the selenium concentration in the system 2.0 mmol / L), then slowly add 4.0 mL of 0.5 mol / L zinc sulfate heptahydrate stock solution (to make the zinc / selenium molar ratio 10:1), and add 0.8 L of 0.5 mol / L copper sulfate pentahydrate stock solution (to make the copper / selenium molar ratio 10:1). Mix magnetically at 300 rpm for 15 minutes at room temperature.

[0047] Step 3: Place the three-necked flask containing the mixed solution from Step 2 at the center of the coil of the alternating magnetic field generator;

[0048] Start the magnetic field generator and set the frequency to 30kHz;

[0049] Start the magnetic stirring (300 rpm) and program temperature rise function of the constant temperature water bath, set the temperature rise rate to 1.0℃ / min, and raise the temperature from 25℃ to 60℃. After reaching the temperature, keep the reaction at this temperature for 40 minutes.

[0050] During the reaction, the natural protein undergoes conformational transformation and phase separation under the dual induction of alternating magnetic field and heat treatment, forming nanoscale protein aggregates. Simultaneously, selenite ions in the solution are reduced in situ to zero-valent selenium nanocrystals. The selenium nanocrystals then undergo multi-level assembly with the protein aggregates and zinc ions and / or copper ions in the solution.

[0051] Step 4: After the reaction is complete, immediately transfer the three-necked flask to an ice-water bath and rapidly cool it to below 5°C. Transfer the cooled suspension to a centrifuge tube and centrifuge at 4°C and 12,000 rpm for 15 minutes. Discard the supernatant and wash the precipitate three times with pre-cooled deionized water (4°C). Place the washed precipitate in a freeze-drying bottle and freeze-dry it (-50°C, 24 hours) to obtain nano-protein selenium composite particles, which are designated as sample E1.

[0052] Example 2

[0053] A method for preparing nano-protein selenium for use as an animal feed additive includes the following steps:

[0054] Step 1: Weigh 0.24g of sodium dihydrogen phosphate and 1.44g of disodium hydrogen phosphate, dissolve them in deionized water and bring the volume to 1L;

[0055] Weigh 5.00g of whey protein powder, add it to the above phosphate buffer, stir until completely dissolved, and bring the volume up to 100mL to obtain a protein solution with a mass-volume concentration of 5.0%.

[0056] Step 2: Measure 100 mL of the above protein solution into a 250 mL three-necked flask, accurately add 2.0 mL of 0.1 mol / L sodium selenite stock solution (to make the selenium concentration in the system 2.0 mmol / L), then slowly add 4.0 mL of 0.5 mol / L zinc sulfate heptahydrate stock solution (to make the zinc / selenium molar ratio 10:1), and add 0.8 L of 0.5 mol / L copper sulfate pentahydrate stock solution (to make the copper / selenium molar ratio 10:1). Mix magnetically at 300 rpm for 15 minutes at room temperature.

[0057] Step 3: Place the three-necked flask containing the mixed solution from Step 2 at the center of the coil of the alternating magnetic field generator;

[0058] Start the magnetic field generator and set the frequency to 30kHz;

[0059] Start the magnetic stirring (300 rpm) and program temperature rise function of the constant temperature water bath, set the temperature rise rate to 1.0℃ / min, and raise the temperature from 25℃ to 60℃. After reaching the temperature, keep the reaction at this temperature for 40 minutes.

[0060] During the reaction, the natural protein undergoes conformational transformation and phase separation under the dual induction of alternating magnetic field and heat treatment, forming nanoscale protein aggregates. Simultaneously, selenite ions in the solution are reduced in situ to zero-valent selenium nanocrystals. The selenium nanocrystals then undergo multi-level assembly with the protein aggregates and zinc ions and / or copper ions in the solution.

[0061] After the reaction was carried out at 60℃ for 40 minutes, the water bath temperature was maintained at 60℃ and the stirring speed at 300 rpm. High-purity nitrogen gas was introduced into the three-necked flask for 20 minutes to remove oxygen. Then, the following were injected rapidly in sequence using a syringe: 0.40 g acrylic acid (8% of the whey protein mass), 1.00 g N-isopropylacrylamide (2.5 times the mass of acrylic acid, with a mass ratio of acrylic acid to N-isopropylacrylamide of 1:2.5), and 0.014 g potassium persulfate (1.0% of the total mass of acrylic acid and N-isopropylacrylamide). The reaction was continued for 50 minutes under nitrogen protection, at 60℃ and 300 rpm. During the reaction, the viscosity of the system increased slightly.

[0062] Step 4: After the reaction is complete, immediately transfer the three-necked flask to an ice-water bath and rapidly cool it to below 5°C. Transfer the cooled suspension to a centrifuge tube and centrifuge at 12,000 rpm for 15 minutes at 4°C. Discard the supernatant and wash the precipitate three times with pre-cooled deionized water (4°C). Place the washed precipitate in a freeze-drying bottle and freeze-dry it (-50°C, 24 hours) to obtain nano-protein selenium composite particles, denoted as sample E2.

[0063] Example 3

[0064] A method for preparing nano-protein selenium for use as an animal feed additive includes the following steps:

[0065] Step 1: Weigh 0.24g of sodium dihydrogen phosphate and 1.44g of disodium hydrogen phosphate, dissolve them in deionized water and bring the volume to 1L;

[0066] Weigh 5.00g of whey protein powder, add it to the above phosphate buffer, stir until completely dissolved, and bring the volume up to 100mL to obtain a protein solution with a mass-volume concentration of 5.0%.

[0067] Step 2: Measure 100 mL of the above protein solution into a 250 mL three-necked flask, accurately add 2.0 mL of 0.1 mol / L sodium selenite stock solution (to make the selenium concentration in the system 2.0 mmol / L), then slowly add 4.0 mL of 0.5 mol / L zinc sulfate heptahydrate stock solution (to make the zinc / selenium molar ratio 10:1), and add 0.8 L of 0.5 mol / L copper sulfate pentahydrate stock solution (to make the copper / selenium molar ratio 10:1). Mix magnetically at 300 rpm for 15 minutes at room temperature.

[0068] Step 3: Place the three-necked flask containing the mixed solution from Step 2 at the center of the coil of the alternating magnetic field generator;

[0069] Start the magnetic field generator and set the frequency to 30kHz;

[0070] Start the magnetic stirring (300 rpm) and program temperature rise function of the constant temperature water bath, set the temperature rise rate to 1.0℃ / min, and raise the temperature from 25℃ to 60℃. After reaching the temperature, keep the reaction at this temperature for 40 minutes.

[0071] During the reaction, the natural protein undergoes conformational transformation and phase separation under the dual induction of alternating magnetic field and heat treatment, forming nanoscale protein aggregates. Simultaneously, selenite ions in the solution are reduced in situ to zero-valent selenium nanocrystals. The selenium nanocrystals then undergo multi-level assembly with the protein aggregates and zinc ions and / or copper ions in the solution.

[0072] Step 4: After the reaction is complete, immediately transfer the three-necked flask to an ice-water bath and rapidly cool it to below 5°C. Transfer the cooled suspension to a centrifuge tube and centrifuge at 4°C and 12,000 rpm for 15 minutes. Discard the supernatant and wash the precipitate three times with pre-cooled deionized water (4°C). Place the washed precipitate in a freeze-drying bottle to obtain a wet composite particle precipitate for later use.

[0073] a. Prepare a 1.5% (w / v) sodium alginate solution: Weigh 1.50g of sodium alginate, dissolve it in 100mL of deionized water, and stir overnight until completely dissolved.

[0074] b. Prepare a 3.0% (w / v) calcium chloride solution: Weigh 3.00 g of calcium chloride and dissolve it in 100 mL of deionized water.

[0075] c. The wet composite particles were precipitated and completely redispersed in 20 mL of 1.5% sodium alginate solution. The mixture was ultrasonically treated for 1 minute to ensure uniform dispersion, thus obtaining a suspension.

[0076] d. Using a syringe (with the needle removed), draw up the above suspension and drop it dropwise into a beaker containing 100 mL of 3.0% calcium chloride solution from a height of about 10 cm above the liquid surface. The droplets solidify into spheres the instant they come into contact with the calcium chloride solution, thus obtaining gel microspheres.

[0077] e. Allow the gel microspheres to solidify in calcium chloride solution and stand for 15 minutes. Remove the gel microspheres with a sieve, wash them three times with deionized water, and then place the washed wet gel microspheres in a freeze-drying pan for freeze-drying (-50℃, 24 hours) to obtain spherical particles, nano-protein selenium composite particles, denoted as sample E3.

[0078] Comparative Example 1

[0079] A method for preparing nano-protein selenium for use as an animal feed additive includes the following steps:

[0080] Step 1: Weigh 0.24g of sodium dihydrogen phosphate and 1.44g of disodium hydrogen phosphate, dissolve them in deionized water and bring the volume to 1L;

[0081] Weigh 5.00g of whey protein powder, add it to the above phosphate buffer, stir until completely dissolved, and bring the volume up to 100mL to obtain a protein solution with a mass-volume concentration of 5.0%.

[0082] Step 2: Measure 100 mL of the above protein solution into a 250 mL three-necked flask, accurately add 2.0 mL of 0.1 mol / L sodium selenite stock solution (to make the selenium concentration in the system 2.0 mmol / L), then slowly add 4.0 mL of 0.5 mol / L zinc sulfate heptahydrate stock solution (to make the zinc / selenium molar ratio 10:1), and add 0.8 L of 0.5 mol / L copper sulfate pentahydrate stock solution (to make the copper / selenium molar ratio 10:1). Mix magnetically at 300 rpm for 15 minutes at room temperature.

[0083] Step 3: Place the three-necked flask containing the mixed solution from Step 2 at the center of the coil of the alternating magnetic field generator;

[0084] Start the magnetic stirring (300 rpm) and program temperature rise function of the constant temperature water bath, set the temperature rise rate to 1.0℃ / min, and raise the temperature from 25℃ to 60℃. After reaching the temperature, keep the reaction at this temperature for 40 minutes.

[0085] During the reaction, the natural protein undergoes conformational transformation and phase separation under the dual induction of alternating magnetic field and heat treatment, forming nanoscale protein aggregates. Simultaneously, selenite ions in the solution are reduced in situ to zero-valent selenium nanocrystals. The selenium nanocrystals then undergo multi-level assembly with the protein aggregates and zinc ions and / or copper ions in the solution.

[0086] Step 4: After the reaction is complete, immediately transfer the three-necked flask to an ice-water bath and rapidly cool it to below 5°C. Transfer the cooled suspension to a centrifuge tube and centrifuge at 4°C and 12,000 rpm for 15 minutes. Discard the supernatant and wash the precipitate three times with pre-cooled deionized water (4°C). Place the washed precipitate in a freeze-drying bottle and freeze-dry it (-50°C, 24 hours) to obtain nano-protein selenium composite particles, which are designated as sample C1.

[0087] Comparative Example 2

[0088] A method for preparing nano-protein selenium for use as an animal feed additive includes the following steps:

[0089] Step 1: Prepare 100 mL of sodium selenite aqueous solution with a concentration of 2.0 mmol / L.

[0090] Step 2: Add 5.00 g of whey protein and stir to dissolve.

[0091] Step 3: Add 0.40 g of ascorbic acid as a chemical reducing agent and stir the reaction at room temperature for 4 hours.

[0092] Step 4: After the reaction is complete, centrifuge, wash, and freeze-dry to obtain nano-protein selenium, which is denoted as sample C2.

[0093] Comparative Example 3

[0094] A method for preparing nano-protein selenium for use as an animal feed additive includes the following steps:

[0095] 5.00 g of whey protein powder and sodium selenite solid (approximately 0.026 g Na2SeO3) equivalent to 2.0 mmol of selenium were thoroughly mixed in a mortar to obtain a mixture, which was denoted as sample C3.

[0096] Experimental Example 1

[0097] The dispersions of samples E1, E2, C1, and C2 (1 mg / mL, PBS, pH 7.4) were tested using a Malvern nanoparticle size and Zeta potential analyzer. The results are shown in the table below:

[0098] sample Hydrated particle size (nm) PDI (Multiple Dispersion Index) Zeta potential (mV) E1 92 ± 8 0.18 -28.5 ± 1.2 E2 105 ± 12 0.21 -25.3 ± 1.5 C1 320 ± 45 0.35 -19.8 ± 2.1 C2 150 ± 25 0.45 -15.2 ± 3.0

[0099] As shown in the table above, the particles prepared by this invention have smaller particle size, more uniform distribution (lower PDI), and higher surface charge (larger absolute value), indicating better dispersion stability in solution and less tendency to agglomerate. The absence of the alternating magnetic field (C1) leads to a significant increase in particle size and non-uniformity. The product obtained by the traditional chemical reduction method (C2) exhibits the worst dispersibility and severe agglomeration.

[0100] Experiment Example 2

[0101] Each sample powder was subjected to wet heat treatment at 121℃ for 30 minutes (simulating feed pelleting conditions), and then the selenium content retention rate before and after treatment was measured.

[0102] sample Selenium retention rate (%) E1 98.5 E2 99.1 C1 95.2 C2 90.8 C3 85.3

[0103] As can be seen from the table above, this invention exhibits excellent thermal stability with minimal selenium loss. The protective structure formed by protein assembly and the E2 polymer layer effectively prevent selenium from volatilizing or oxidizing.

[0104] Experimental Example 3

[0105] One hundred and eighty healthy one-day-old broilers were randomly divided into six groups, with three replicates per group and ten birds per replicate. The basal diet was corn-soybean meal with a selenium content of 0.03 mg / kg (for baseline deficiency). The experiment lasted for 42 days.

[0106] Control group: fed with basal diet.

[0107] Sodium selenite group (control group): basal diet + sodium selenite (with added Se 0.3 mg / kg).

[0108] Group E1: Basal diet + Sample E1 (with added Se 0.3 mg / kg).

[0109] E2 group: basal diet + sample E2 (with added Se 0.3 mg / kg).

[0110] Group C2: Basal diet + Sample C2 (with added Se 0.3 mg / kg).

[0111] Group C3: Basal diet + Sample C3 (with added Se 0.3 mg / kg).

[0112] The measured indicators and results are shown in the table below:

[0113] 1. Production performance:

[0114] Group Average daily weight gain (g) Material weight ratio (F / G) Blank group 56.3±2.1^d 1.91±0.06^a Sodium selenite group 62.8±1.8^c 1.78±0.04^b Group C3 61.5±2.0^c 1.81±0.05^b Group C2 64.2±1.5^b 1.75±0.03^b Group E1 67.5±1.4^a 1.69±0.02^c Group E2 68.1±1.3^a 1.67±0.02^c

[0115] Note: Different superscript letters in the same column indicate significant differences (P<0.05).

[0116] As shown in the table above, groups E1 and E2 significantly outperformed the sodium selenite group and the two comparative groups in promoting growth and reducing feed conversion ratio, demonstrating that the product of this invention has higher bioavailability and can more effectively promote animal growth and improve feed utilization. Specifically, group E2 achieved an average daily weight gain of 68.1 grams, significantly higher than the 56.3 grams in the control group and the 62.8 grams in the sodium selenite group, while reducing the feed conversion ratio to 1.67, indicating that the amount of feed required per unit of weight gain is minimal and the feed conversion efficiency is the highest. This result further verifies the superior performance of nano-protein selenium composite particles in animal feed additives, especially sample E2, which was prepared through dual induction by alternating magnetic field and heat treatment, and its effect is even more outstanding.

[0117] 2. Tissue selenium deposition and plasma antioxidant indicators:

[0118] Group Selenium content in pectoral muscles (μg / kg) Plasma glutathione peroxidase (GSH-Px, U / mL) Group Blank group 38±5^d 110±12^d Blank group Sodium selenite group 180±20^c 205±18^c Sodium selenite group Group C3 165±18^c 190±15^c Group C3 Group C2 250±30^b 240±20^b Group C2 Group E1 420±35^a 295±22^a Group E1 Group E2 435±40^a 310±25^a Group E2

[0119] As shown in the table above, the selenium deposition in the breast muscles of broiler chickens in groups E1 and E2 prepared in this application was more than 2.3 times that of the sodium selenite group, and the plasma antioxidant enzyme activity was also the highest. This indicates that the product of this invention can not only be efficiently absorbed, but also more effectively converted into a bioactive form and stored in tissues. Meanwhile, group E2 showed slightly higher selenium content in breast muscles and plasma glutathione peroxidase activity than group E1, further highlighting the advantages of the nano-protein selenium composite particles prepared through dual induction by alternating magnetic field and heat treatment in promoting selenium absorption and enhancing antioxidant capacity. Specifically, the selenium content in the breast muscles of broiler chickens in group E2 reached as high as 435 μg / kg, significantly higher than the 38 μg / kg of the blank group and the 180 μg / kg of the sodium selenite group, demonstrating extremely strong selenium deposition capacity; its plasma glutathione peroxidase activity also reached 310 U / mL, indicating a significant enhancement of the in vivo antioxidant system, which helps improve the overall health and production performance of broiler chickens.

[0120] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing nano-protein selenium for use as an animal feed additive, characterized in that, Includes the following steps: Step 1: Dissolve the natural protein in phosphate buffer to prepare a protein solution with a mass-volume concentration of 1-5%. Step 2: Add sodium selenite and soluble salt to the protein solution in sequence, and stir to mix; Step 3: Place the mixed solution from Step 2 in an alternating magnetic field generator with a frequency of 10-50kHz, control the solution to heat up to 55-70℃ at a rate of 0.5-1.5℃ / min, and keep it at this temperature for 20-60 minutes. During the reaction, the natural protein undergoes conformational transformation and phase separation under the dual induction of alternating magnetic field and heat treatment, forming nanoscale protein aggregates. Simultaneously, selenite ions in the solution are reduced in situ to zero-valent selenium nanocrystals. The selenium nanocrystals undergo multi-level assembly with the protein aggregates and zinc ions and / or copper ions in the solution. Step 4: After the reaction is complete, cool to below 5°C, and obtain the solid product by centrifugation. After washing and freeze-drying, obtain nano-protein selenium composite particles.

2. The method for preparing nano-protein selenium for animal feed additives according to claim 1, characterized in that: In step one, the natural protein is either soy protein isolate or whey protein.

3. The method for preparing nano-protein selenium for animal feed additives according to claim 1, characterized in that: In step two, the amount of sodium selenite added is such that the concentration of selenium in the protein solution is 0.5-5 mmol / L.

4. The method for preparing nano-protein selenium for animal feed additives according to claim 1, characterized in that: In step two, the soluble zinc salt is zinc sulfate heptahydrate and copper sulfate pentahydrate; The molar ratio of zinc to selenium is 10:1; the molar ratio of copper to selenium is 2:

1.

5. The method for preparing nano-protein selenium for animal feed additives according to claim 1, characterized in that: In step three, pectin, accounting for 5-15% of the mass of the natural protein, is added to the protein solution. The pectin, along with the natural protein, sodium selenite, and soluble zinc salt, participates in the assembly process of step three to form a stable polysaccharide-protein-mineral complex network structure.

6. The method for preparing nano-protein selenium for animal feed additives according to claim 1, characterized in that: In step three, after the reaction is complete, while maintaining stirring and temperature, acrylic acid and N-isopropylacrylamide, as well as the initiator potassium persulfate, are added, and the reaction continues for 40-60 minutes to form a hydrogel polymer layer with dual temperature and pH response in situ on the surface of the nanocomposite mineral particles. The hydrogel polymer layer swells at temperatures below 35°C and pH above 6.5, and shrinks at temperatures above 40°C and / or pH below 5.

0.

7. The method for preparing nano-protein selenium for animal feed additives according to claim 6, characterized in that: The mass ratio of acrylic acid to N-isopropylacrylamide is 1:2-4; The total amount of acrylic acid and N-isopropylacrylamide added is 10-30% of the mass of the natural protein; The amount of potassium persulfate added is 1.0% of the total mass of acrylic acid and N-isopropylacrylamide.

8. The method for preparing nano-protein selenium for animal feed additives according to claim 1, characterized in that: In step four, before freeze-drying, the following steps are also included: redispersing the washed precipitate in a sodium alginate solution, stirring evenly, and then dropping it into a calcium chloride solution to form sodium alginate-calcium chloride gel microspheres encapsulating the nanocomposite mineral particles, followed by washing and freeze-drying.

9. A method for preparing nano-protein selenium for animal feed additives according to claim 8, characterized in that: The sodium alginate solution has a mass concentration of 1-2%; the calcium chloride solution has a mass concentration of 2-4%.

10. A nano-protein selenium for use as an animal feed additive, characterized in that, It is prepared by the method for preparing nano-protein selenium for animal feed additives according to any one of claims 1-9, specifically as nano-protein selenium composite particles.