A biological nano-selenium feed additive, a preparation method and application thereof

Abstract

The application belongs to the technical field of nano selenium preparation and application, and specifically discloses a kind of biological nano selenium feed additive and its preparation method and application.The composition includes: 5~15 parts of biological nano selenium, 20~60 parts of hydroxypropyl-beta-cyclodextrin, 10~30 parts of L-methionine, 30~80 parts of betaine, 300~600 parts of microcrystalline cellulose, 20~50 parts of sodium butyrate.The application first mixes biological nano selenium dispersion liquid and L-methionine to obtain the dispersion liquid of the two complexes;Then add hydroxypropyl-beta-cyclodextrin for mixing, then add betaine and sodium butyrate, finally add microcrystalline cellulose, mix evenly and dry, to obtain biological nano selenium feed additive.The biological nano selenium feed additive prepared by the application has good dispersibility, no agglomeration, is beneficial to biological absorption, and promotes the synthesis of organic selenium.

Description

Technical Field

[0001] This invention relates to the field of nano-selenium preparation and application technology, and in particular to a biological nano-selenium feed additive and its preparation method and application. Background Technology

[0002] In the natural environment, selenium (Se) exists in various oxidized states and forms, such as ionic selenites and selenates, solid selenium (selenium nanoparticles), and biogenic selenium (selenomethionine, selenocysteine, etc.). Selenium is an essential trace element for animals and humans, primarily exerting its important role through selenoproteins. Approximately one in seven people worldwide suffers from selenium deficiency, which is associated with various human diseases, including Kashin-Beck disease, Keshan disease, and myxedema-related endemic cretinism. Statistics show that over 70% of our population is affected by selenium deficiency, and over 70 million people are at risk of selenium deficiency-related health problems. Selenium plays multiple roles in the body, but its antioxidant effect is most prominent, protecting the body from oxidative damage. GPx and other selenium-containing enzymes are key antioxidants, helping to neutralize highly reactive free radicals. Compared to other forms of selenium, nano-selenium has higher bioavailability, stronger antioxidant capacity, and lower toxicity. Furthermore, biogenic nano-selenium derived from probiotics exhibits stronger antioxidant stress resistance than chemically synthesized nano-selenium.

[0003] Fish is a unique source of selenium for humans because it contains a higher amount of selenium compared to many other foods. Furthermore, the selenium in fish exists primarily in an organic form, which can be effectively absorbed and retained by the human body. Farming selenium-enriched fish may be a safe and effective solution to address selenium deficiency in humans.

[0004] Currently, some studies have incorporated bio-derived nano-selenium into fish feed for the cultivation of selenium-enriched fish, but the following problems still exist: poor dispersion stability and easy aggregation and failure: nano-selenium has high surface energy, and it is prone to agglomeration into large particles during feed mixing or before entering the intestines of aquatic animals, resulting in a significant decrease in bioavailability; rapid release and short absorption window: common fish species have simple digestive tracts, and nano-selenium is rapidly and concentratedly released and quickly excreted after entering the intestines, making it impossible to achieve continuous selenium absorption. At the same time, excessively rapid selenium release may have a momentary impact on the intestinal microbiota, which is detrimental to health; low organic selenium conversion efficiency: the conversion mechanism is simple, and the proportion of organic selenium is not ideal.

[0005] Therefore, how to provide a bio-based nano-selenium feed additive, its preparation method and application, improve the utilization rate of bio-derived nano-selenium, and improve the conversion efficiency of organic selenium in organisms is a problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In view of this, the present invention provides a biological nano-selenium feed additive, its preparation method and application, to solve the problems of low utilization rate of organic selenium and its detrimental effect on the health of organisms in existing methods.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A bio-nano selenium feed additive, comprising the following components in parts by weight: 5-15 parts bio-nano selenium, 20-60 parts hydroxypropyl-β-cyclodextrin, 10-30 parts L-methionine, 30-80 parts betaine, 300-600 parts microcrystalline cellulose, 20-50 parts sodium butyrate; The bio-nano selenium was prepared using Bacillus subtilis strain 1A747.

[0008] Preferably, the method for preparing the bio-nano selenium includes the following steps: Bacillus subtilis strain 1A747 was transferred to LB liquid culture medium containing sodium selenite for cultivation. After cultivation, bio-nano selenium was extracted.

[0009] Preferably, the sodium selenite content in the LB liquid culture medium containing sodium selenite is 5-8 mmol / L.

[0010] Preferably, the culture temperature is constant at 35~40℃, the time is 45~50h, and the culture method is constant temperature shaking culture.

[0011] Preferably, the extraction step involves centrifuging the culture medium after cultivation, collecting the precipitate, incubating the precipitate with lysozyme, and then sequentially performing ultrasonic disruption and washing after incubation to obtain bio-nano selenium.

[0012] Preferably, the particle size of the bio-nano selenium is 150~600nm.

[0013] Preferably, the degree of substitution of the hydroxypropyl-β-cyclodextrin is 4-6, and the purity is ≥98%. The microcrystalline cellulose has a particle size D50 of 50~100μm and a crystallinity of 35~50%.

[0014] Another object of the present invention is to provide a method for preparing a bio-nano selenium feed additive, comprising the following steps: 1) Mix bio-nano selenium with water to obtain a bio-nano selenium dispersion, add L-methionine to obtain an L-methionine-bio-nano selenium complex dispersion; 2) Hydroxypropyl-β-cyclodextrin was added in batches to the L-methionine-bionano selenium complex dispersion, then betaine and sodium butyrate were added and mixed, and finally microcrystalline cellulose was added and mixed, and dried to obtain bionano selenium feed additive.

[0015] Preferably, the hydroxypropyl-β-cyclodextrin is added in batches 3 to 6 times.

[0016] Another object of the present invention is to provide an application of a bio-nano selenium feed additive in the formulation of feed, wherein the amount of the bio-nano selenium feed additive added to the feed is guaranteed to have a selenium content of 0.1~0.4 mg / kg; The feed is fish feed.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: 1. L-methionine forms a stable complex with the bio-corona on the surface of bio-nano selenium, which can be actively taken up by amino acid transport proteins of intestinal epithelial cells, significantly increasing the total selenium deposition in muscle compared to direct addition of nano-selenium. Hydroxypropyl-β-cyclodextrin can partially encapsulate bio-nano selenium, forming a molecular capsule that remains stable in the acidic gastric environment. Upon entering the mid-to-late intestinal tract, the cyclodextrin structure loosens, resulting in a targeted and sustained release. The sodium butyrate added in this invention serves as an energy source for the hindgut and slightly lowers the local pH, further regulating the release rate. The synergistic effect of hydroxypropyl-β-cyclodextrin and sodium butyrate extends the selenium release window to 2.5–3 hours, matching the intestinal transit time of the organism (fish) and synergistically promoting selenium absorption.

[0018] 2. The addition of hydroxypropyl-β-cyclodextrin and microcrystalline cellulose can also improve the dispersibility and storage stability of bio-nano selenium. The molecular capsules formed by hydroxypropyl-β-cyclodextrin will be further adsorbed on the surface of microcrystalline cellulose, so that bio-nano selenium will not agglomerate in feed mixtures and after contact with water for at least 4 hours.

[0019] 3. The bio-nano selenium feed additive of this invention can specifically increase the content of selenomethionine, selenocysteine, and methylselenocysteine ​​in the muscle of organisms (fish). The L-methionine added in this invention serves both as a coordination carrier for nano-selenium and as a direct precursor of selenomethionine; betaine, as a methyl donor, provides raw materials for the methylation modification of selenoamino acids. The combined use of these three substances forms a complete metabolic chain of "raw material-modification-delivery," enabling the efficient conversion of selenium into selenomethionine, selenocysteine, and methylselenocysteine.

[0020] 4. The components in this invention are widely available, inexpensive, and processed under mild conditions, without the need for organic solvents. This aligns with the development direction of green additives, has good industrialization prospects, and is suitable for large-scale production. Detailed Implementation

[0021] This invention provides a bio-nano selenium feed additive, comprising the following components in parts by weight: 5-15 parts bio-nano selenium, 20-60 parts hydroxypropyl-β-cyclodextrin, 10-30 parts L-methionine, 30-80 parts betaine, 300-600 parts microcrystalline cellulose, and 20-50 parts sodium butyrate; under the above mass ratio, the specific addition amount of bio-nano selenium can be 6 parts, 8 parts, 10 parts, 12 parts, or 14 parts; the specific addition amount of hydroxypropyl-β-cyclodextrin can be 25 parts, 30 parts, 35 parts, 40 parts, 45 parts, 50 parts, or 55 parts; The specific addition amounts of L-methionine can be 12, 15, 18, 20, 22, 25, or 28 parts; the specific addition amounts of betaine can be 35, 40, 45, 50, 55, 60, 65, 70, or 75 parts; the specific addition amounts of microcrystalline cellulose can be 320, 350, 380, 400, 420, 450, 480, 500, 520, 550, or 580 parts; and the specific addition amounts of sodium butyrate can be 25, 30, 35, 40, or 45 parts.

[0022] In this invention, the bio-nano selenium is prepared using Bacillus subtilis strain 1A747.

[0023] In this invention, the method for preparing the bio-nano selenium includes the following steps: In this invention, Bacillus subtilis strain 1A747 was transferred to LB liquid culture medium containing sodium selenite for cultivation. After cultivation, bio-nano selenium was extracted.

[0024] In this invention, the sodium selenite content in the LB liquid culture medium containing sodium selenite is 5~8 mmol / L, specifically 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, or 7.5 mmol / L.

[0025] In this invention, the culture temperature is constant at 35~40℃, specifically 36℃, 37℃, 38℃, or 39℃; the time is 45~50h, specifically 46h, 47h, 48h, or 49h; and the culture method is constant temperature shaker culture.

[0026] In this invention, the extraction step involves centrifuging the culture medium after cultivation, collecting the precipitate, incubating the precipitate with lysozyme, and then sequentially performing ultrasonic disruption and washing after incubation to obtain bio-nano selenium.

[0027] In this invention, the particle size of the bio-nano selenium is 150~600nm, specifically 180nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, and 550nm.

[0028] In this invention, the bio-selenium nanoparticles prepared by Bacillus subtilis strain 1A747 possess unique biological specificity due to their surface chemical composition (polysaccharide-peptide bio-corona), charge distribution, particle size, and coordination mode with L-methionine. Compared to chemically prepared selenium nanoparticles, the selenium nanoparticles obtained in this invention are not "naked selenium"; their surface adsorbs a layer of bacterial residue, which facilitates the binding of L-methionine. Chemically prepared selenium nanoparticles have a single coordination site and disordered spatial distribution, making effective binding difficult. The particle size of the bio-selenium nanoparticles obtained in this invention precisely balances the integrity of the bio-corona and intestinal uptake efficiency, and matches the inclusion size of hydroxypropyl-β-cyclodextrin. Too small a particle size leads to a thin bio-corona layer, easy detachment, excessively rapid intracellular release, and a short release window; while too large a particle size results in low uptake efficiency, poor complex stability, delayed release, and insufficient absorption.

[0029] In this invention, the degree of substitution of the hydroxypropyl-β-cyclodextrin is 4 to 6, specifically 4.2, 4.4, 4.5, 4.6, 4.8, 5, 5.2, 5.4, 5.5, 5.6, and 5.8; the purity is ≥98%, specifically 98%, 98.2%, 98.5%, 98.8%, 99%, 99.2%, 99.5%, and 99.8%.

[0030] In this invention, the particle size D50 of the microcrystalline cellulose is 50~100μm, specifically 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm; and the crystallinity is 35~50%, specifically 38%, 40%, 42%, 45%, 48%.

[0031] This invention also provides a method for preparing a bio-nano selenium feed additive, comprising the following steps: 1) Mix bio-nano selenium with water to obtain a bio-nano selenium dispersion, add L-methionine to obtain an L-methionine-bio-nano selenium complex dispersion; 2) Hydroxypropyl-β-cyclodextrin was added in batches to the L-methionine-bionano selenium complex dispersion, then betaine and sodium butyrate were added and mixed, and finally microcrystalline cellulose was added and mixed, and dried to obtain bionano selenium feed additive.

[0032] In this invention, the mass-to-volume ratio of the bio-nano selenium to water in step 1) is 5~15g:1.5~4.5L, preferably 10g:3L.

[0033] In this invention, the preferred temperature for mixing with L-methionine is 20~45℃, specifically 25℃, 30℃, 35℃, or 40℃; the preferred time is 1.5~3h, specifically 1.8h, 2h, 2.2h, 2.5h, or 2.8h; and the pH value is ≤7, specifically 6, 6.2, 6.5, or 6.8.

[0034] In this invention, the hydroxypropyl-β-cyclodextrin is added in batches 3 to 6 times, specifically 3, 4, 5, or 6 times, and the amount added in different batches is preferably the same.

[0035] The present invention also provides an application of a bio-nano selenium feed additive in the formulation of feed, wherein the amount of the bio-nano selenium feed additive added to the feed is guaranteed to have a selenium content of 0.1~0.4 mg / kg, specifically 0.15 mg / kg, 0.2 mg / kg, 0.25 mg / kg, 0.3 mg / kg, or 0.35 mg / kg.

[0036] In this invention, the feed is fish feed.

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] Nano-selenium synthesis: Bacillus subtilis strain 1A747 was transferred to LB liquid culture medium containing 8 mM sodium selenite. The conical flask containing the culture medium was placed in a 37°C shaker and cultured for 48 h until the bacterial culture medium turned bright red. The culture medium was then centrifuged at 12500 g for 10 min at 4°C, and the precipitate containing nano-selenium was collected. The precipitate was washed three times with PBS, and lysozyme was added and incubated at 37°C for 2 h. The sample was then sonicated at 1000 W for 30 min, with a 5-second interval between sonication cycles, and the sample was placed on ice during the sonication process. The disrupted sample was then rinsed with 1.5 M sodium selenite solution at pH 8.3 containing 1% sodium selenite. The precipitate was washed three times with Tris-HCl from SDS. The precipitate was then resuspended in 24 mL of deionized water, and 1 mL of n-octanol was added and mixed thoroughly. The mixture was centrifuged at 2000 g for 10 min at 4 °C, and then placed in a refrigerator at 4 °C for 24 h. After the solution separated into layers, the lower aqueous phase was removed and washed with chloroform, 100% ethanol, 70% ethanol and deionized water, respectively. Finally, the precipitate was collected by centrifugation at 16000 g for 5 min, yielding bio-nano selenium with a particle size of 150~600 nm.

[0040] For the above-mentioned bio-selenium nanoparticle sieving, 10 parts of the obtained bio-selenium nanoparticles with a particle size of 300-400 nm were fully dispersed in deionized water (mass-volume ratio of 10 g: 3 L), and ultrasonically assisted dispersion was performed at 200 W for 10 min to obtain a bio-selenium nanoparticle dispersion. Then, 15 parts of L-methionine were added, the pH of the system was adjusted to 6.5, the temperature was 35℃, and the mixture was stirred at 300 rpm for 3 h to obtain an L-methionine-bio-selenium nanoparticle complex (its particle size increased slightly compared to the initial particle size, approximately 18%). Then, 40 parts of hydroxypropyl-β-cyclodextrin (Wacker Cavasol W7) were added to the above dispersion. HP, DS4.5~5.5, purity ≥98%), the above hydroxypropyl-β-cyclodextrin was divided into 4 equal parts and added in batches. After each batch was added, the mixture was stirred for 5 minutes before adding the next batch. After all the batches were added, the mixture was stirred for 1 hour to mix evenly. Then, 40 parts of betaine and 30 parts of sodium butyrate were added and stirred for 10 minutes to dissolve. Finally, 400 parts of microcrystalline cellulose (Shandong Guangda MCC 95, D50=75μm, crystallinity 35~42%, divided into 4 equal parts and added in 4 batches) were added and mixed thoroughly. After the microcrystalline cellulose was completely added, the system was in the form of wet granules. After spray drying, the bio-nano selenium feed additive was obtained.

[0041] Example 2

[0042] The bio-selenium nanoparticles prepared in Example 1 were sieved, and 12 parts of bio-selenium nanoparticles with a particle size of 200-350 nm were fully dispersed in deionized water (mass-volume ratio of 10 g: 3 L). The dispersion was ultrasonically assisted at 200 W for 10 min to obtain a bio-selenium nanoparticle dispersion. Then, 20 parts of L-methionine were added, the pH of the system was adjusted to 6.5, the temperature was set to 35°C, and the mixture was stirred at 300 rpm for 3 h to obtain an L-methionine-bio-selenium nanoparticle complex. Then, 30 parts of L-methionine were added to the above dispersion. One part of hydroxypropyl-β-cyclodextrin (same as in Example 1) was added in three equal parts, with each part added in batches. After each batch was added, the mixture was stirred for 5 minutes before adding the next batch. After all the batches were added, the mixture was stirred for another hour until it was homogeneous. Then, 60 parts of betaine and 40 parts of sodium butyrate were added and stirred for 10 minutes to dissolve them. Finally, 500 parts of microcrystalline cellulose were added and mixed thoroughly (same as in Example 1, divided into five equal parts and added in five batches). After the microcrystalline cellulose was completely added, the mixture was spray-dried to obtain the bio-nano selenium feed additive.

[0043] Example 3

[0044] The bio-selenium nanoparticles prepared in Example 1 were sieved, and 15 parts of bio-selenium nanoparticles with a particle size of 200-350 nm were fully dispersed in deionized water (mass-volume ratio of 10 g: 3 L). The dispersion was ultrasonically assisted at 200 W for 10 min to obtain a bio-selenium nanoparticle dispersion. Then, 28 parts of L-methionine were added, the pH of the system was adjusted to 6.5, the temperature was set to 40 °C, and the mixture was stirred at 300 rpm for 3 h to obtain an L-methionine-bio-selenium nanoparticle complex. Finally, 48 parts of L-methionine were added to the above dispersion. 6 equal parts of hydroxypropyl-β-cyclodextrin (same as in Example 1) were added in batches, with each batch stirred for 5 minutes before adding the next batch. After all the batches were added, stirring was continued for 1 hour to mix them evenly. Then, 60 parts of betaine and 45 parts of sodium butyrate were added and stirred for 10 minutes to dissolve them. Finally, 500 parts of microcrystalline cellulose were added and mixed thoroughly (same as in Example 1, divided into 5 equal parts and added in 5 batches). After the microcrystalline cellulose was completely added, the bio-nano selenium feed additive was obtained by spray drying.

[0045] Example 4

[0046] The bio-selenium nanoparticles prepared in Example 1 were sieved, and 8 parts of bio-selenium nanoparticles with a particle size of 400-550 nm were fully dispersed in deionized water (mass-volume ratio of 10 g: 3 L). The dispersion was ultrasonically assisted at 200 W for 10 min to obtain a bio-selenium nanoparticle dispersion. Then, 12 parts of L-methionine were added, the pH of the system was adjusted to 6.5, the temperature was set to 40 °C, and the mixture was stirred at 300 rpm for 2.5 h to obtain an L-methionine-bio-selenium nanoparticle complex. Then, 2 parts of L-methionine were added to the above dispersion. Five parts of hydroxypropyl-β-cyclodextrin (same as in Example 1) were added in batches, with each batch stirred for 5 minutes before adding the next batch. After all the batches were added, the mixture was stirred for 1 hour until homogeneous. Then, 30 parts of betaine and 20 parts of sodium butyrate were added and stirred for 10 minutes to dissolve them. Finally, 350 parts of microcrystalline cellulose were added and thoroughly mixed (same as in Example 1, divided into five equal parts and added in five batches). After the microcrystalline cellulose was completely added, the mixture was spray-dried to obtain the bio-nano selenium feed additive.

[0047] Comparative Example 1

[0048] The 300-400 nm bio-nano selenium obtained in Example 1 was directly used as a bio-nano selenium feed additive.

[0049] Comparative Example 2

[0050] The only difference between this comparative example and the previous example is that L-methionine is not added.

[0051] Comparative Example 3

[0052] The only difference between this comparative example and the embodiment is that hydroxypropyl-β-cyclodextrin is not added.

[0053] Experimental Example 1

[0054] Experimental animals: Grass carp (250.79±1.57g) were randomly assigned to 24 experimental net cages, with 20 fish per cage; each treatment group had 3 replicate cages, and each cage had a water volume of approximately 400 liters. During the 60-day rearing period, the experimental ponds were continuously supplied with flowing water. Each cage was equipped with a microporous aeration device to ensure that the dissolved oxygen concentration was maintained at a minimum of 7 mg / L. During the rearing period, the water temperature fluctuated between 20 and 31℃, the average pH was 8.2, and the average dissolved oxygen was 7.8 mg / L.

[0055] Experimental Protocol: A basic feed was prepared, with the basic feed serving as the blank group. Control groups 1-3 were supplemented with bio-nano selenium feed additives from Comparative Examples 1-3, and experimental groups 1-4 were supplemented with bio-nano selenium feed additives from Examples 1-4 of this invention. The selenium content in the feed remained at 0.3 mg / kg in both control groups 1-3 and experimental groups 1-4 after the addition of the bio-nano selenium feed additives. Comparative Examples 1-3 corresponded to control groups 1-3, and Examples 1-4 corresponded to experimental groups 1-4. For the first two weeks, all fish were acclimatized in net cages with the basic feed, fed three times daily (08:30, 12:30, 16:30). Formal feeding: The experimental feed was fed three times daily until apparent satiety, and uneaten feed was collected. After 60 days of rearing, samples were collected according to the following steps: Euthanasia: Fish were euthanized using 0.5 g / L MS-222 (Sigma), and the final weight of fish in each cage was recorded. Muscle samples: Muscle from the left dorsal fin to the head above the lateral line was collected, flash-frozen in liquid nitrogen and stored at -80°C for total selenium and selenium speciation analysis.

[0056] Basic feed formulation:

[0057] The components in the basic feed formula are all commercially available components and are not intended to limit the invention.

[0058] Analysis of total selenium and selenium speciation in muscle

[0059] Total selenium content: Weigh 0.2g of dried muscle, add 8mL of HNO3, and let stand overnight at room temperature. Microwave digestion: 120℃ for 5min → 150℃ for 10min → 190℃ for 20min. Evaporate to approximately 1mL by electric heating, transfer to a 25mL volumetric flask, rinse the digestion tube three times with ultrapure water and make up to volume, and detect by ICP-MS (Agilent 7900). Calculate the total selenium content (wet weight, mg / kg freshweigh). The results are shown in Table 1.

[0060] Selenium speciation analysis: Muscle was freeze-dried at -60℃ and then ground into powder using liquid nitrogen. 0.2g of the powder was added to 20mL of 100mM Tris-HCl buffer (pH 7.4) and 0.2g of trypsin. After sonication for 1 hour, the mixture was shaken at 37℃ for 1 hour. Then, 0.2g of type XIV proteinase was added and shaken at 40℃ for 8 hours. Finally, 0.2g of proteinase K was added and shaken at 50℃ for 14 hours. The mixture was centrifuged at 10000g for 10 minutes. The supernatant was filtered through a 0.22μm filter membrane, processed in a 10kDa ultrafiltration centrifuge tube, and rotary evaporated at 43℃ to 2mL. ICP-MS was used to detect selenium speciation, and the dry weight (μg / kg dry weight) of selenocysteine-SeCys2, methylselenocysteine-MeSeCys, and selenomethionine-SeMet was calculated. The results are shown in Table 1.

[0061] Table 1. Results of total selenium and selenium speciation analysis in muscle.

[0062] Table 1 shows that, under the same conditions of 0.3 mg / kg selenium supplementation in the feed, the total selenium content in the muscle of grass carp in experimental groups 1-4 of this invention was significantly higher than that in control group 1. This indicates that the present invention significantly improves the absorption and utilization rate of selenium in grass carp through the synergistic effect of sustained-release protection by hydroxypropyl-β-cyclodextrin and active transport via L-methionine coordination. Comparison with control group 2 shows a significant increase in selenomethionine content in the muscle of grass carp in the experimental groups of this invention. This indicates that by providing the framework with exogenous L-methionine and the methyl donor with betaine, the conversion of selenium to high-value selenomethionine is directed, achieving efficient utilization and targeted conversion of selenium. Compared with control group 3, the sustained-release effect of hydroxypropyl-β-cyclodextrin is also very important.

[0063] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A bio-nano selenium feed additive, characterized in that, The components include the following parts by mass: 5-15 parts bio-nano selenium, 20-60 parts hydroxypropyl-β-cyclodextrin, 10-30 parts L-methionine, 30-80 parts betaine, 300-600 parts microcrystalline cellulose, 20-50 parts sodium butyrate; The bio-nano selenium was prepared using Bacillus subtilis strain 1A747.

2. The bio-nano selenium feed additive according to claim 1, characterized in that, The preparation method of the bio-nano selenium includes the following steps: Bacillus subtilis strain 1A747 was transferred to LB liquid culture medium containing sodium selenite for cultivation. After cultivation, bio-nano selenium was extracted.

3. The bio-nano selenium feed additive according to claim 2, characterized in that, The sodium selenite content in the LB liquid culture medium containing sodium selenite is 5~8 mmol / L.

4. The bio-nano selenium feed additive according to claim 3, characterized in that, The culture temperature is constant at 35~40℃ for 45~50 hours, and the culture method is constant temperature shaking culture.

5. The bio-nano selenium feed additive according to claim 4, characterized in that, The extraction steps involve centrifuging the culture medium after cultivation, collecting the precipitate, incubating the precipitate with lysozyme, and then sequentially performing ultrasonic disruption and washing after incubation to obtain bio-nano selenium.

6. A bio-nano selenium feed additive according to any one of claims 1 to 5, characterized in that, The particle size of the bio-nano selenium is 150~600nm.

7. The bio-nano selenium feed additive according to claim 6, characterized in that, The degree of substitution of the hydroxypropyl-β-cyclodextrin is 4-6, and the purity is ≥98%. The microcrystalline cellulose has a particle size D50 of 50~100μm and a crystallinity of 35~50%.

8. A method for preparing a bio-nano selenium feed additive according to any one of claims 1 to 7, characterized in that, Includes the following steps: 1) Mix bio-nano selenium with water to obtain a bio-nano selenium dispersion, add L-methionine to obtain an L-methionine-bio-nano selenium complex dispersion; 2) Hydroxypropyl-β-cyclodextrin was added in batches to the L-methionine-bionano selenium complex dispersion, then betaine and sodium butyrate were added and mixed, and finally microcrystalline cellulose was added and mixed, and dried to obtain bionano selenium feed additive.

9. The method for preparing the bio-nano selenium feed additive according to claim 8, characterized in that, The hydroxypropyl-β-cyclodextrin is added in batches 3 to 6 times.

10. The application of the bio-nano selenium feed additive prepared by the preparation method according to claim 8 or 9 in the formulation of feed, characterized in that, The amount of the bio-nano selenium feed additive added to the feed is guaranteed to have a selenium content of 0.1~0.4 mg / kg; The feed is fish feed.

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