Application of abalone visceral glycoprotein nano-selenium in the preparation of aquatic feed additives

Abstract

The invention discloses the application of abalone visceral glycoprotein nano-selenium in the preparation of an aquatic feed additive for regulating the intestinal flora of tilapia. The abalone visceral glycoprotein nano-selenium of the invention can be used as an aquatic feed additive to improve the intestinal microbial diversity of tilapia, significantly increase the abundance of some beneficial bacteria such as Ceti in the intestinal tract of tilapia, significantly reduce the abundance of conditional pathogens such as Plesiomonas and Citrobacter and the abundance of potential pathogen Citrobacter freundii, and significantly increase the level of pyrrole-2-carboxylic acid in the intestinal tract of tilapia.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the application of abalone visceral glycoprotein nano-selenium in the preparation of aquatic feed additives. Background Art

[0002] Selenium (Se) is an essential trace element for organisms. It can not only improve the body's immunity, enhance reproductive function and promote growth, but also has anti-cancer, anti-oxidation, anti-stress, anti-aging, and participates in protecting cells from excessive H 2 O 2 Damage and antagonism of heavy metals such as cadmium and mercury. Selenium exists in the body mainly in two biologically active forms: selenoproteins and selenoamino acids. As many as 41 selenoproteins have been found in bony fish. The functions of some selenoproteins have been determined, such as glutathione peroxidase (GSH-Px), thioredoxin reductase (TrxRs) and selenoprotein P (SelP). SelP is the main protein responsible for selenium homeostasis and transport in the body, affecting sperm development. SelP has two different isoforms in fish, SelPa and SelPb; they have significant differences at the structural level and may play different roles in fish homeostasis.

[0003] Studies have shown that if selenium is deficient in an organism, it will inhibit the growth of the organism and reduce the antioxidant capacity. Severe selenium deficiency will damage the function of selenoproteins in the organism, thus affecting the health of the organism. Especially in growing animals, selenium deficiency can lead to some functional disorders, such as liver necrosis (rats), pancreatic atrophy (chickens), gizzard myopathy (turkey chicks) and "white muscle disease" (sheep). Selenium is usually present in large quantities in immune tissues such as the liver, spleen and lymph nodes. Selenium deficiency in the body may also impair cell-mediated immunity and B cell function, leading to a decline in the body's immune performance.

[0004] Traditional forms of selenium supplementation mainly include inorganic selenium and organic selenium. Inorganic selenium is mainly sodium selenite, and organic selenium is mainly selenomethionine. However, both inorganic and organic forms of selenium have disadvantages such as narrow safety range, high toxicity, and low biological activity, which limits their application in selenium supplementation. Nano-selenium is a new form of selenium supplementation discovered in recent years. Compared with traditional forms of selenium supplementation, nano-selenium (SeNPs) has the advantages of high biological activity, high bioavailability, and low toxicity. It is currently the selenium supplement with the lowest toxicity reported.

[0005] Nano-selenium is a selenium nanoparticle formed by using nanotechnology to attract red selenium on the amide plane of protein or the hydroxyl group of polysaccharide under redox system. It not only retains the biological activities of selenium such as growth promotion, immune regulation, and anti-oxidation, but also has the advantages of high biological efficacy and low toxicity. It is an efficient and low-toxic nutritional additive. At present, studies have shown that nano-selenium plays an important role in promoting animal growth and improving antioxidant capacity, but there are few studies on the regulatory effect of nano-selenium, especially abalone visceral glycoprotein nano-selenium, on the intestinal flora of aquatic animals. Summary of the invention

[0006] The purpose of the present invention is to provide a use of abalone visceral glycoprotein nano-selenium in the preparation of an aquatic feed additive for regulating tilapia intestinal flora.

[0007] In order to achieve the above object, the technical solution of the present invention is:

[0008] The invention aims to provide abalone visceral glycoprotein nano-selenium for preparing an aquatic feed additive for regulating tilapia intestinal flora.

[0009] Furthermore, the concentration of the abalone visceral glycoprotein nano-selenium is 3 mg / kg.

[0010] Furthermore, the regulation of tilapia intestinal flora and metabolism refers to:

[0011] (1) Improve the intestinal microbial diversity of tilapia;

[0012] (2) Significantly increased the abundance of beneficial bacteria Cetaceae in the intestine of tilapia;

[0013] (3) The abundance of Pseudomonas and Citrobacter in the intestine of tilapia was significantly reduced;

[0014] (4) Significantly reduced the abundance of Citrobacter freundii;

[0015] (5) Significantly increase the content of pyrrole-2-carboxylic acid related to intestinal amino acid metabolism in tilapia.

[0016] Furthermore, the preparation method of abalone visceral glycoprotein nano-selenium comprises the following steps:

[0017] S1. Mix 1 part of powdered abalone viscera (pre-ground) with 39 parts of ultrapure water;

[0018] S2. Add 1 part of papain at pH 9.5 and incubate the mixture at 50°C for 19 h to hydrolyze the abalone viscera.

[0019] S3. The hydrolyzate was heated in a 50°C water bath for 15 minutes to inactivate papain, and then centrifuged at 4°C, 4500 rpm for 20 minutes.

[0020] S4. Collect the supernatant and separate it with a 10 kDa ultrafiltration membrane, discard the permeate, collect the retentate and alcoholize it with ethanol (95% grade) at a volume ratio of 1:3, then centrifuge at 4°C, 12000 rpm for 20 minutes and collect the precipitate.

[0021] S5. Take 20 portions of ultrapure water to dissolve the precipitate, pass it through a DEAE Sepharose CL-6B ion exchange column, use 0.25 mol / L NaCl at a flow rate of 0.5 mL / min for gradient elution, and obtain abalone visceral glycoprotein mother solution with a purity of 96.38%.

[0022] S6. Take 20 parts of 1 mM sodium selenite, 100 parts of 5 mM Vc solution, 16 parts of abalone visceral glycoprotein mother solution, and 364 parts of sterilized ultrapure water, and react at 25°C and 600 rpm for 10 hours to finally obtain 500 parts of abalone visceral glycoprotein nano-selenium.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] Abalone visceral glycoprotein nano-selenium can be used to prepare aquatic feed additives to improve the intestinal microbial diversity of tilapia, significantly increase the abundance of some beneficial bacterial genera such as Cetaceae in the intestine of tilapia, significantly reduce the abundance of conditional pathogens such as Plesiomonas and Citrobacter and the abundance of potential pathogen Citrobacterfreundii, specifically enrich the probiotic Lactococcus, and upregulate the level of pyrrole-2-carboxylic acid in the intestine of tilapia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0026] Figure 1 Transmission electron microscopy, particle size distribution, Zeta potential and X-ray diffraction patterns of PSP-SeNPs.

[0027] Among them, a and b are transmission electron microscope images, c is the particle size distribution diagram, d is the Zeta potential diagram, and e is the X-ray diffraction (XRD) diagram.

[0028] Figure 2 This is the Alpha diversity map of tilapia intestinal community.

[0029] Figure 3 Beta diversity and NMDS diagram of tilapia intestinal flora.

[0030] Figure 4This is the bacterial composition at the phylum level in the intestine of tilapia after PSP-SeNPs treatment.

[0031] Figure 5 This is the KW rank sum test graph of the significant differences in bacterial phyla in the intestine of tilapia after PSP-SeNPs treatment.

[0032] a is Proteobacteria, b is Fusobacteria, c is Bacteroidetes, and d is Planctomycetes.

[0033] Figure 6 The intestinal community composition diagram of each group at the genus level and the KW rank sum test diagram of significantly different bacterial genera. Among them, a is the genus level bacterial community composition diagram, b is Plesiomonas, c is Ceti, d is Citrobacter, e is Fimbriiglobus, and f is Gemmata.

[0034] Figure 7 The intestinal community composition diagram of each group at the species level and the KW rank sum test diagram of significantly different bacterial species. Among them, a is the species level bacterial community composition diagram, b is Citrobacterfreundii, c is Gemmata sp_Br1-2, and d is Pantoea agglomerans. DETAILED DESCRIPTION

[0035] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials, reagents, instruments and equipment used in the following examples can be obtained from commercial sources.

[0038] Example 1 Preparation and characterization of abalone visceral glycoprotein nanoselenium (PSP-SeNPs)

[0039] S1. Preparation of abalone visceral glycoprotein (PSP) stock solution

[0040] 1. Mix 1 g of powdered abalone viscera (pre-ground) with 39 mL of ultrapure water;

[0041] 2. Add 1 g papain, pH 9.5, and incubate the mixture at 50 °C for 19 h to hydrolyze the abalone viscera.

[0042] 3. Heat the hydrolyzate in a 50°C water bath for 15 minutes to inactivate papain, and then centrifuge at 4500 rpm at 4°C for 20 minutes.

[0043] 4. Collect the supernatant and separate it with a 10 kDa ultrafiltration membrane, discard the permeate, collect the retentate and alcoholize it with ethanol (95% grade) at a volume ratio of 1:3, then centrifuge at 4°C, 12000 rpm for 20 minutes and collect the precipitate.

[0044] 5. Take 20 mL of ultrapure water to dissolve the precipitate, pass it through a DEAE Sepharose CL-6B ion exchange column, use 0.25 mol / L NaCl for gradient elution at a flow rate of 0.5 mL / min to obtain abalone visceral glycoprotein (PSP) mother liquor with a purity of 96.38%.

[0045] S2. Preparation of Abalone Visceral Glycoprotein Selenium Nanoparticles (PSP-SeNPs)

[0046] Take 20mL 1mM sodium selenite, 100mL 5mM Vc solution, 16mL PSP mother solution, and 364mL sterile ultrapure water, and react at 25°C and 600rpm for 10h. Finally, 500mL abalone visceral glycoprotein nanoselenium (PSP-SeNPs) was obtained.

[0047] Crystal structure of PSP-SeNPs: PSP-SeNPs were freeze-dried to powder using a vacuum freeze dryer and measured using an X-ray diffractometer in the 2θ range of 10-80° at a scanning rate of 3.0° / min. The X-ray source filtered copper Kα radiation (40 kV, 30 mA; ).

[0048] Morphology of PSP-SeNPs: The PSP-SeNPs solution was dropped onto a copper grid at 200 kV, and the morphology of PSP-SeNPs was observed by transmission electron microscopy (TEM).

[0049] Particle size distribution of PSP-SeNPs: At room temperature, pure water with a refractive index of 1.330, a viscosity of 0.8872 cP, and a dielectric constant of 78.3 was used as the dispersion medium, and a dynamic light scattering (DLS) was used to detect the particle size distribution of PSP-SeNPs.

[0050] Stability of PSP-SeNPs: At room temperature, the zeta potential of PSP-SeNPs was measured using a Malvern Zetasizer Nano-ZS90 and a folded capillary cuvette DTS1060 to evaluate the stability of PSP-SeNPs.

[0051] from Figure 1 a, b It can be seen that the prepared PSP-SeNPs particles are spherical and well dispersed. Figure 1c It can be seen that the particle size of the prepared PSP-SeNPs presents a unimodal distribution with an average diameter of 67.94 nm. Figure 1 d It can be seen that the Zeta potential of the PSP-SeNPs synthesized in this experiment is -36.1 mV, while the Zeta potential value of the SeNPs solution is 7.83 mV, indicating that the colloidal properties of PSP-SeNPs are stable. Figure 1 e shows that there is a characteristic diffraction peak of selenium at about 25°, which further confirms the presence of selenium. Example 2 Effect of abalone visceral glycoprotein nanoselenium (PSP-SeNPs) on the intestinal flora of tilapia Preparation of feed

[0052] Table 1 Feed formula

[0053]

[0054]

[0055] Note: Mineral premix provides: MgSO per kg of feed 4 7H 2 O 15mgNaCl 1.0mg, NaH 2 PO 4 ·2H 2 O 25mg, KH 2 PO 4 32mg, FeSO 4 ·H 2 O 2.5 mg, calcium lactate 3.5 mg, ZnSO 4 7H 2 O 0.353mg, MnSO 4 ·4H 2 O 0.162mg, CuSO 4 ·5H 2 O 0.031mg, CoCl 2 6H 2 O0.01 mg, KIO 3 0.003mg.

[0056] The basal diet contained about 31% crude protein and 4% crude fat. The basal feed formula is shown in Table 1. All dry raw materials were ground and passed through an 80-mesh sieve. The dry materials were thoroughly mixed with oil and boiled water (about 350 ml / kg dry raw materials) to form a uniform wet dough. The dough was made into wet pellet feed by a feed pelletizer with a 2.5 mm die (CD4 x 1TS, South China University of Technology, Guangzhou, China). The feed pellets were dried in a 54°C oven for 8-10 hours until the moisture content dropped to about 10%, and then packed into self-sealing bags, sealed, and stored in a -20°C refrigerator for later use.

[0057] Feeding and management

[0058] After tilapia was temporarily raised for two weeks, 540 active and healthy tilapia fry weighing 1.5±0.5g were selected and randomly divided into 6 groups. Each group had 90 fish, and 3 replicates were made, with 30 fish in each replicate. The control group (Group C) was fed with basic feed. The other groups added different doses and forms of selenium to the basic feed, namely Group M (PSP-SeNPs added at 0.3mg / kg, as selenium dose) and Group Y (sodium selenite added at 0.3mg / kg, as selenium dose).

[0059] Intestinal sample collection

[0060] Six anesthetized tilapia were taken from each tank (for high-throughput sequencing and metabolomics), and the weight and length of each fish were measured, and then the intestinal samples were collected by dissection. Before dissection, the body surface was wiped with 75% alcohol, and then a semicircle of the abdomen was cut open with sterile dissecting scissors to expose all the internal organs, and the entire intestine was taken out. The blood on the surface was wiped dry with absorbent paper, and the outer wall of the intestine was rinsed several times with 0.65% sterile saline; for the 6 fish used for 16S rDNA sequencing and metabolomics analysis, every 3 fish were combined and placed in a 1.5ml centrifuge tube (2 samples per tank, 6 replicates for each treatment), and the centrifuge tube was immediately placed in a liquid nitrogen tank for freezing, and then transferred to -80℃ for storage. For the 3 fish used for tissue sections, the intestinal midgut was cut and placed in 4% paraformaldehyde for sealing and storage.

[0061] Illunmina sequencing

[0062] After 7 weeks of breeding, the intestines of tilapia in groups C, M, and Y were sent to Guangzhou Kidio Biotechnology Co., Ltd. for high-throughput sequencing, with 4 replicates in each group, for a total of 12 samples.

[0063] Effects of PSP-SeNPs on Alpha Diversity of Intestinal Microorganisms in Tilapia Juveniles

[0064] Figure 2 The analysis results of the Alpha diversity index of each group and the significant differences between groups are shown. Compared with the control group, the Sobs, Chao, Ace, and PD indices of group M were significantly increased, indicating that 0.3 mg / kg PSP-SeNPs significantly increased the diversity of intestinal microorganisms in tilapia. Compared with group C, the Shannon and Simpson indices of group Y decreased significantly. In addition, compared with group Y, the Chao and Ace indices of group M increased significantly. It can be seen that the addition of 0.3 mg / kg of PSP-SeNPs significantly increased the microbial diversity, while the addition of 0.3 mg / kg of sodium selenite reduced the microbial diversity.

[0065] Figure 3It is an NMDS graph, which shows the similarities or differences in the community composition of each group of samples. It can be seen from the figure that the groups are separated from each other, especially the M group is significantly deviated from the other groups, indicating that the effect of the 0.3 mg / kg PSP-SeNPs added group on the intestinal flora of tilapia is more obvious than that of the other groups.

[0066] Effects of PSP-SeNPs on the composition and structure of intestinal microorganisms

[0067] At door level ( Figure 4 ), the control group (C) was mainly composed of Proteobacteria, Fusobacteria, and Firmicutes, with proportions of 80.33%, 15.03%, and 3.85%, respectively. After adding 0.3 mg / kg PSP-SeNPs (M group), Figure 5 As shown in the figure, the relative abundance of Fusobacteria increased significantly to 65.84% and became the most dominant phylum, and Planctomycetes became the second dominant phylum with an abundance of 12.86%. The Proteobacteria decreased significantly to 12.11%. In addition, the abundance of Bacteroidetes also increased significantly to 1.37%. The phyla in the intestine of tilapia in the group with 0.3 mg / kg sodium selenite were still mainly composed of Proteobacteria, Fusobacteria, and Firmicutes, but compared with Group C, Proteobacteria and Firmicutes decreased to 66.98% and 0.85%, and Fusobacteria increased to 30.02%.

[0068] Figure 6The following is a bar chart of the top 10 genera in abundance and a KW rank sum test chart of differential bacterial genera. The main bacterial genera in the intestine of tilapia are Citrobacter, Plesiomonas, Cetobacterium, Bacillus, and Aeromonas. The abundances of these genera in the control group (C group) are 38.78%, 29.72%, 15.03%, 3.32%, and 2.8%, respectively. Compared with the control group, the relative abundance of Cetobacterium, Fimbriiglobus, and Gemmata in the M group increased significantly, while the relative abundance of Citrobacter and Plesiomonas decreased significantly in the M group. The Plesiomonas in the Y group increased, and the Citrobacter decreased significantly.

[0069] At the species level, as shown in 7. Among the identified species with abundance > 0.1%, the main bacteria in the intestine of tilapia were Citrobacterfreundii (38.78%), Aeromonas hydrophila (2.02%), Pantoeaagglomerans (0.24%), and Gemmata sp_Br1-2 (0.19%). The KW rank sum test of the four groups found that compared with the C group, the Citrobacterfreundii in the M group decreased significantly, and the Gemmata sp_Br1-2 increased significantly. In addition, Aeromonas hydrophila decreased in the M group.

[0070] Intestinal flora is closely related to the body's intestinal barrier function, nutritional metabolism and immune response. At present, there are still few studies on the effects of nano-selenium on fish intestinal flora. In this experiment, the main bacterial phyla in the intestine of tilapia are Proteobacteria, Fusobacteria, and Firmicutes, which is consistent with the results of other tilapia intestinal microecological studies, confirming that these are the dominant bacterial phyla in the intestine of tilapia. Proteobacteria can increase intestinal permeability, but are often markers of intestinal disorders. Fusobacteria are anaerobic bacteria that produce butyrate, which can promote intestinal digestion and regulate immunity and anti-inflammatory properties. After adding 0.3 mg / kg of PSP-SeNPs, the abundance of Fusobacteria increased significantly, which was caused by the significant increase in the genus Cetobacterium (accounting for 100%) of this phylum. The abundance of this genus was 15.03% in the control group, while it increased to 65.85% in the M group. Cetobacterium can produce short-chain fatty acid acetate, thereby promoting the metabolism of proteins, carbohydrates and fats. This genus is also considered to be an important source of vitamin B12 (cobalamin) that can promote DNA synthesis, especially in tilapia. In addition, the genera Citrobacter and Plesiomonas of the Proteobacteria decreased significantly in the M group, and the genus Aeromonas of the same phylum also decreased in the M group.

[0071] The significant decrease in the genus Citrobacter in the M group of this experiment is because the abundance of Citrobacterfreundii of this genus has decreased significantly, from 38.78% to 0.43%. This bacterium is one of the important genera that cause global outbreaks of wild and farmed fish diseases. It can cause ulceration in the bodies of animals such as fish, shrimps and crabs, and especially brings huge losses to farmed fish disease outbreaks. At present, studies have found that Citrobacterfreundii infection in catfish, rainbow trout, tilapia, etc. brings typical hemorrhagic sepsis. Plesiomonas acts as an opportunistic pathogen in fish, especially when the water environment conditions of aquaculture are poor, it is easy to cause high mortality in farmed fish and reduced aquaculture productivity. Common bacteria in the genus Aeromonas are also conditional pathogens, among which Aeromonas hydrophila is a well-known pathogen of human-animal-aquatic organisms and the main pathogen causing fish motile Aeromonas septicemia. In the M group of this experiment, the abundance of this bacterium decreased from 2.02% to 0.08%. This suggests that nano-selenium has improved the intestinal bacteria of tilapia to a certain extent.

[0072] In addition, the abundance of Gemmata, Fimbriiglobus and Gemmata sp_Br1-2 was high in the M group. Studies have shown that Gemmata mainly exists in aquatic and soil environments. Species of this genus have good signal transduction capabilities and can adapt well to toxic and stressful habitats. Few studies have been reported on Fimbriiglobus, especially in aquatic animals. The genus Fimbriiglobus of the phylum Planctomycetes has been shown to be able to degrade chitin, especially the skeletons of arthropods living in mud, and therefore can enhance the stress resistance of tilapia to some chitin in the environment. The mechanism of the effect of this genus on tilapia needs to be studied. Screening and annotation of differential metabolites

[0073] Tables 2 and 3 show the names, expression patterns and metabolite categories of the extremely significant differential metabolites in the positive and negative ion modes. Compared with the control group, after adding PSP-SeNPs, one metabolite was significantly upregulated in the M group, which was pyrrole-2-carboxylic acid, which was related to organic acid metabolism, and no metabolites were downregulated in the M group. In the Y group, a total of 25 metabolites were extremely significantly downregulated, and one metabolite was significantly upregulated. Among the significantly downregulated metabolites, 15 were related to amino acid metabolism, 6 were related to nucleotide metabolism, 1 was related to vitamin metabolism, 2 were related to lipid metabolism, and 1 was related to signal molecule interaction. The significantly upregulated metabolite was isoleucine.

[0074] Although pyrrole-2-carboxylic acid is not an amino acid, it is involved in the metabolism of arginine and proline (as shown in Table 4). It is a common metabolite produced by bacteria, fungi, plants, animals and humans. It has special chemical and physical electronic properties and plays an important role in the metabolic regulation of many organisms. It is reported that pyrrole-containing compounds exhibit biological activities such as antibacterial, antifungal, anti-inflammatory or anti-tumor effects. In addition, in medical clinical research, the phenomenon of a significant reduction in pyrrole-2-carboxylic acid can also be used as an early prediction basis for cancer. The downregulated metabolites in group Y are related to the metabolism of amino acids, nucleotides, and lipids in tilapia. In short, 0.3 mg / kg of PSP-SeNPs does not cause disorders in the metabolism of amino acids in the intestine of tilapia, while the addition of 0.3 mg / kg of sodium selenite reduces the content of most amino acids in the intestine of tilapia.

[0075] Table 2 Differential metabolites in Cvs M group

[0076] Metabolite name Chinese name Ion mode Expression pattern Metabolite Class PathwayID Pyrrole-2-carboxylicacid Pyrrole-2-carboxylic acid POS Increase Pyrroles ko00330

[0077] Table 3 Differential metabolites in C vs Y groups

[0078]

[0079]

[0080] Table 4 Key differential metabolic pathways and their differential metabolites

[0081]

[0082] In summary, adding 0.3 mg / kg PSP-SeNPs (M group) to tilapia feed can improve the intestinal microbial diversity of tilapia, significantly increase the abundance of some beneficial bacterial genera such as Cetaceae in the intestine of tilapia, significantly reduce the abundance of conditional pathogens such as Plesiomonas and Citrobacter and the abundance of potential pathogen Citrobacter freundii, and significantly increase the level of pyrrole-2-carboxylic acid in the intestine of tilapia.

[0083] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. Application of abalone visceral glycoprotein nano-selenium in the preparation of aquatic feed additives for regulating tilapia intestinal flora, characterized in that: The regulation of the intestinal flora of tilapia refers to: (1) Improve the intestinal microbial diversity of tilapia; (2) increase the abundance of Cetiella in the intestine of tilapia; (3) reduce the abundance of Pseudomonas and Citrobacter in the intestine of tilapia; (4) Reducing the abundance of Citrobacter freundii or (5) Increase the content of pyrrole-2-carboxylic acid related to amino acid metabolism in the intestine of tilapia, The preparation method of abalone visceral glycoprotein nano-selenium comprises the following steps: S1. Mix 1 part of pre-ground powdered abalone viscera with 39 parts of ultrapure water; S2. 1 part of papain was added, pH was 9.5, and the mixture was incubated at 50°C for 19h to hydrolyze the abalone viscera; S3. The hydrolyzate was heated in a 50°C water bath for 15 minutes to inactivate papain, and then centrifuged at 4°C, 4500 rpm for 20 minutes; S4. The supernatant was collected and separated using a 10 kDa ultrafiltration membrane, the permeate was discarded, the retentate was collected and alcoholized with 95% ethanol at a volume ratio of 1:3, and then centrifuged at 4°C, 12000 rpm for 20 minutes to collect the precipitate; S5. Take 20 portions of ultrapure water to dissolve the precipitate, pass it through a DEAE Sepharose CL-6B ion exchange column, use 0.25 mol / L NaCl at a flow rate of 0.5 mL / min for gradient elution, and obtain abalone visceral glycoprotein mother solution with a purity of 96.38%; S6. Take 20 parts of 1 mM sodium selenite, 100 parts of 5 mM Vc solution, 16 parts of abalone visceral glycoprotein mother solution, and 364 parts of sterile ultrapure water, react at 25°C and 600 rpm for 10 hours, and finally obtain 500 parts of abalone visceral glycoprotein nano-selenium.

2. The use of abalone visceral glycoprotein nano-selenium as claimed in claim 1 in preparing an aquatic feed additive for regulating tilapia intestinal flora, characterized in that: The concentration of the abalone visceral glycoprotein nano-selenium is 3 mg / kg.

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