An influenza granule for poultry and a preparation method thereof

Abstract

The present invention provides an influenza granule for poultry and a preparation method thereof, belonging to the technical field of poultry medicines. After carboxylating multi-walled carbon nanotubes, they are compounded with the composite selenium / germanium polysaccharide obtained by fermenting Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Nostoc commune Vaucher, to prepare a polysaccharide-compounded carbon nanotube. Additionally, the obtained composite selenium / germanium protein is coupled with citric acid and cecropin, complexed with bismuth salts, and then compounded with the polysaccharide-compounded carbon nanotube to prepare the influenza granule for poultry. The influenza granule for poultry prepared by the present invention has high bioavailability, good safety, low cytotoxicity, can effectively inhibit virus replication, reduce the viral load, relieve the symptoms caused by the virus, enhance the immune function of the body, and when used together with an immunogen or a vaccine, can assist the antigen to stimulate the body to produce an immune response and improve the antibody expression level, having broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry drugs, and particularly relates to an influenza granule for poultry and a preparation method thereof. Background Art

[0002] Avian influenza is the abbreviation of avian epidemic influenza. The morbidity characteristics of avian influenza in chickens are as follows: ① The clinical symptoms are complex and often co-infected with infectious bronchitis, infectious laryngotracheitis, colibacillosis, etc. clinically; ② The morbidity and mortality rates are related to the virus strain, chicken flock gender, environment and complications, secondary infections. For example, when the avian influenza virus invades the mucosa, it is conducive to the secondary infection of colibacillosis, strengthening the hemagglutinin effect of the avian influenza virus and resulting in an increase in the morbidity and mortality rates of the chicken flock; ③ There are many serotypes of avian influenza virus, and the cross-protection between different serotypes is weak, and the virulence differences of different strains of the same serotype are very large; ④ The avian influenza virus has a high mutation frequency and is prone to pathogenic mutations, antigen drift and antigen shift.

[0003] The current harm and prevention and treatment status of avian influenza in chickens are as follows: ① Since avian influenza was discovered more than 100 years ago, humans have not mastered specific prevention and treatment methods and can only prevent its spread by means of disinfection, isolation, and mass slaughter of poultry and livestock, which has caused harm to the chicken farming industry. ② Since poultry infected with avian influenza first destroys the body's immune defense system, resulting in the loss of the body's resistance and self-repair ability; ③ After western medicine antiviral drugs are used clinically, they have a partial antagonistic effect on virus particles, but they also have toxic and side effects on the body cells of poultry. The prevention and treatment effect of western medicine antiviral drugs on avian influenza in poultry in veterinary clinics is not ideal. ④ After using western medicine antiviral drugs in the process of raising poultry, problems such as food safety will occur. China clearly does not allow the use of western medicine antiviral drugs for the prevention and treatment of avian influenza in livestock and poultry. Therefore, there is currently no antiviral veterinary drug for the prevention and treatment of avian influenza in poultry. ⑤ In the clinical prevention and treatment research and exploration of avian influenza in poultry by humans, it is found that some traditional Chinese veterinary medicine varieties such as astragalus membranaceus, honeysuckle, isatis root, momordica cochinchinensis, etc. have good clinical effects on the prevention and treatment of avian influenza in poultry. Moreover, the research also finds that traditional Chinese veterinary medicines such as astragalus membranaceus not only have strong clinical antiviral ability, but also have the effects of enhancing the body's immunity and disease resistance and improving the body's self-repair ability.

[0004] Due to the relatively simple processing technology and relatively rough processing of traditional Chinese veterinary medicine, most of them are in the form of simple coarse powder, fine powder or directly mixed into poultry feed or drinking water after traditional decoction. The active ingredients of traditional Chinese veterinary medicine cannot be fully and effectively absorbed and utilized, resulting in less than ideal clinical application effects. Therefore, the improvement of drugs for the prevention and treatment of avian influenza is an urgent problem to be solved at present. Summary of the Invention

[0005] The object of the present invention is to provide an influenza granule for poultry and its preparation method, which has high bioavailability, good safety, low cytotoxicity, can effectively inhibit virus replication, reduce virus load, relieve the symptoms caused by the virus, enhance the immune function of the body, and when used in combination with an immunogen or vaccine, can assist the antigen to stimulate the body to produce an immune response, improve the antibody expression level, and has broad application prospects.

[0006] The technical solution of the present invention is realized as follows:

[0007] The present invention provides a preparation method of an influenza granule for poultry, comprising the following steps:

[0008] S1. Pretreatment: Mix multi-walled carbon nanotubes, concentrated sulfuric acid, and concentrated hydrochloric acid evenly, heat under reflux for reaction, cool to room temperature, wash, then heat and extract with tetrahydrofuran, and dry to obtain carboxylated modified carbon nanotubes;

[0009] S2. Fermentation: Wash Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Nostoc commune Vaucher respectively, dry, mix and pulverize, add to water, sterilize, inoculate with a seed solution of germanium-rich yeast and selenium-rich yeast strains, ferment and culture, then add snailase, heat and enzymolyze, filter, add ethanol to the filtrate, precipitate, filter to obtain a solid and a filtrate, wash the solid, dry to obtain a composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain a composite selenium / germanium protein;

[0010] S3. Coupling: Add citric acid to water, add NHS and EDC, stir and activate, add cecropin and composite selenium / germanium protein, stir and react, add bismuth salt, stir, dialyze, and freeze-dry the non-permeated liquid to obtain bismuth citrate protein;

[0011] S4. Preparation of polysaccharide composite carbon nanotubes: Add carboxylated modified carbon nanotubes to dimethyl sulfoxide, add composite selenium / germanium polysaccharide and concentrated sulfuric acid, heat and stir for reaction, centrifuge, wash, and dry to obtain polysaccharide composite carbon nanotubes;

[0012] S5. Preparation of influenza granule for poultry: Add polysaccharide composite carbon nanotubes to water, add NHS and EDC, stir and activate, add bismuth citrate protein, stir and react, and spray-dry to obtain an influenza granule for poultry.

[0013] As a further improvement of the present invention, in step S1, the mass ratio of the multi-walled carbon nanotubes, concentrated sulfuric acid, and concentrated hydrochloric acid is 1-2: 200-250: 35-50, the time of the heat under reflux reaction is 7-10 h, the temperature of the heat extraction is 75-85 °C, the time is 20-24 h, and the washing is carried out until the pH value is 5-6.

[0014] As a further improvement of the present invention, in step S2, the mass ratio of Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Nostoc sphaeroides is 3-5:2-4:1-3:7-10, the inoculation amounts of the Ge-enriched yeast and Se-enriched yeast strain seed liquors are 2-3 v / v% and 1-2 v / v% respectively, the bacteria content of the strain seed liquor is 10 8 -10 9 cfu / mL, the conditions for fermentation culture are 25-30 °C, 150-250 r / min, fermentation culture for 24-36 h, the addition amount of snailase is 0.5-1 wt% of the total mass of the system, and the temperature for heating enzymolysis is 35-38 °C, and the time is 1-3 h.

[0015] As a further improvement of the present invention, in step S3, the mass ratio of citric acid, NHS, EDC, cecropin, composite selenium / germanium protein, and bismuth salt is 12-15:3-4:3-4:2-3:3-5:0.2-0.5, the bismuth salt is bismuth citrate, the time for stirring activation is 20-30 min, and the time for stirring reaction is 12-15 h.

[0016] As a further improvement of the present invention, in step S4, the mass ratio of carboxylated modified carbon nanotubes, composite selenium / germanium polysaccharide, and concentrated sulfuric acid is 10:3-4:0.5-1, the temperature for heating and stirring reaction is 130-140 °C, and the time is 5-7 h.

[0017] As a further improvement of the present invention, in step S5, the mass ratio of polysaccharide composite carbon nanotubes, NHS, EDC, and bismuth citrate protein is 10-12:3-4:2-3:5-7, the time for stirring activation is 10-20 min, and the time for stirring reaction is 7-10 h.

[0018] As a further improvement of the present invention, it specifically includes the following steps:

[0019] S1. Pretreatment: Mix 1-2 parts by weight of multi-walled carbon nanotubes, 200-250 parts by weight of concentrated sulfuric acid, and 35-50 parts by weight of concentrated hydrochloric acid evenly, heat and reflux for 7-10 h, cool to room temperature, wash to a pH value of 5-6, and then heat to 75-85 °C with tetrahydrofuran and extract for 20-24 h, and dry to obtain carboxylated modified carbon nanotubes;

[0020] S2. Fermentation: Wash 3-5 parts by weight of Flammulina velutipes, 2-4 parts by weight of Lentinula edodes, 1-3 parts by weight of Grifola frondosa, and 7-10 parts by weight of Nostoc sphaeroides respectively, dry, mix and pulverize, add to water, sterilize, and inoculate with a bacteria content of 10 8 -10 9Ge-enriched yeast and Se-enriched yeast strain seed solutions with a concentration of cfu / mL, with inoculation amounts of 2-3 v / v% and 1-2 v / v% respectively, at 25-30 °C, 150-250 r / min, fermented and cultured for 24-36 h, then added with snailase, the addition amount of the snailase being 0.5-1 wt% of the total mass of the system, heated to 35-38 °C, enzymolyzed for 1-3 h, filtered, ethanol was added to the filtrate, precipitated, filtered, to obtain a solid and a filtrate, the solid was washed and dried to prepare a composite Se / Ge polysaccharide; the filtrate was recycled with ethanol, added with ammonium sulfate for fractional precipitation, filtered, washed and dried to obtain a composite Se / Ge protein;

[0021] S3. Coupling: Add 12-15 parts by weight of citric acid to water, add 3-4 parts by weight of NHS and 3-4 parts by weight of EDC, stir and activate for 20-30 min, add 2-3 parts by weight of cecropin and 3-5 parts by weight of the composite Se / Ge protein, stir and react for 12-15 h, add 0.2-0.5 parts by weight of bismuth salt, stir, dialyze, and the non-permeated liquid was freeze-dried to prepare bismuth citrate protein;

[0022] S4. Preparation of polysaccharide composite carbon nanotubes: Add 10 parts by weight of carboxylated modified carbon nanotubes to dimethyl sulfoxide, add 3-4 parts by weight of the composite Se / Ge polysaccharide and 0.5-1 part by weight of concentrated sulfuric acid, heat to 130-140 °C, stir and react for 5-7 h, centrifuge, wash and dry to prepare polysaccharide composite carbon nanotubes;

[0023] S5. Preparation of avian influenza granules: Add 10-12 parts by weight of polysaccharide composite carbon nanotubes to water, add 3-4 parts by weight of NHS and 2-3 parts by weight of EDC, stir and activate for 10-20 min, add 5-7 parts by weight of bismuth citrate protein, stir and react for 7-10 h, and spray dry to prepare avian influenza granules for poultry.

[0024] The present invention further protects an avian influenza granule prepared by the above preparation method.

[0025] The present invention further protects an application of the above avian influenza granule in the preparation of veterinary drugs for treating or assisting in treating avian influenza.

[0026] The present invention has the following beneficial effects:

[0027] The present invention prepares an immune enhancer, which can enhance the immune function of the body, has the advantages of enhancing the specific and non-specific immune responses of the body, accelerating the induction of immune responses, improving the body's anti-infection ability, correcting immune deficiencies, etc. When used in combination with an immunogen or vaccine, it can assist the antigen to stimulate the body to produce an immune response and improve the antibody expression level.

[0028] The present invention uses carbon nanotubes as a carrier. On the one hand, it can complex a large amount of polysaccharides and proteins. The nanomaterials with unique physical, chemical, and biological properties have the characteristic of high antigen loading capacity and no immunogenicity themselves. On the other hand, carbon nanotubes can enhance the immune response of the animal body to inactivated viruses, genetically engineered recombinant proteins, polypeptides, and small molecule compounds, enhance the expression of specific antibodies in the body, and play a good role in assisting antiviral.

[0029] The present invention uses Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Nostoc commune as raw materials, which themselves contain rich polysaccharides and proteins. Grifola frondosa polysaccharide is a kind of fungal polysaccharide rich in β-1-3-glycosidic bonds and β-1-2-glycosidic bonds, and has good effects against influenza virus, human immunodeficiency virus (HIV), and herpes simplex virus type I (HSV-1), and has a good inhibitory effect on avian influenza virus. Flammulina velutipes polysaccharide and Lentinula edodes polysaccharide are a kind of safe, efficient, and environmentally friendly new immune regulator, which can inhibit or reduce harmful microorganisms in the intestine, regulate the intestinal microecological balance, inhibit the proliferation of pathogenic bacteria such as Pasteurella multocida, Escherichia coli, and Salmonella in vitro, and are also used as immune enhancers. Acting together with a variety of animal vaccines can significantly improve the immune effect. Nostoc commune is also a freshwater alga containing active polysaccharides and phycobiliproteins, and has good anti-inflammatory, antibacterial, and antiviral effects.

[0030] After mixed fermentation with germanium-enriched yeast and selenium-enriched yeast, composite selenium / germanium polysaccharide and composite selenium / germanium protein are obtained. Organic selenium can improve the humoral and cellular immune functions of livestock and poultry, enhance the body's resistance to infectious diseases. Selenium deficiency will increase the pathogenicity of virus infection, while supplementing an appropriate amount of selenium can improve the body's resistance to viruses. Selenium may also affect the replication and pathogenicity of viruses by affecting the selenium protein module encoded by the virus. Organic germanium has an obvious immunomodulatory effect, and can stimulate T lymphocytes to produce lymphokines, such as inducing interferon γ-IFN, cofactor IL-2, etc., activate macrophages into cytotoxic macrophages, and activate the activity of natural killer cells (NK), thereby improving the disease resistance. The two can significantly improve the growth performance of poultry, enhance their immunity, reduce the impact of avian influenza on poultry, have a good antiviral effect, and have a synergistic effect.

[0031] Couple composite selenium / germanium protein with cecropin and citric acid, and complex with bismuth. As a small molecule polypeptide, cecropin is an important component of insect innate immunity. Its special mode of action endows cecropin with many functions such as antibacterial, antiviral, enhancing body immunity, and tissue repair. The formed bismuth citrate protein has low cytotoxicity, high safety, can effectively inhibit virus replication, reduce virus load, and relieve the symptoms caused by the virus. Through a unique metal replacement mechanism, it irreversibly deprives the key zinc ions in the virus helicase, and then forms bismuth-binding helicase, thereby making it lose biological activity and playing an antiviral role.

[0032] The influenza granules for poultry prepared by the present invention have high bioavailability, good safety, low cytotoxicity, can effectively inhibit virus replication, reduce virus load, relieve the symptoms caused by the virus, enhance the body's immune function. When used together with an immunogen or a vaccine, it can assist the antigen to stimulate the body to produce an immune response, improve the antibody expression level, and has broad application prospects. Detailed implementation mode

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] Germanium-rich yeast, 20 billion cfu / g, selenium-rich yeast, 20 billion cfu / g.

[0035] Preparation of the strain seed solution: Inoculate the strain into Gause's medium, culture at 27°C and 100 r / min for 18 - 24 h to obtain a strain seed solution with a bacterial content of 10 8 -10 9 cfu / mL.

[0036] Snail enzyme, with a cell wall breaking rate of 90%.

[0037] NHS, N-hydroxysuccinimide, EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0038] Multi-walled carbon nanotubes, industrial grade, with a tube diameter of 10 - 20 nm and a length of 10 - 30 μm.

[0039] Example 1

[0040] The embodiment of the present invention provides a preparation method of influenza granules for poultry, which specifically includes the following steps:

[0041] S1. Pretreatment: Mix 1 g of multi-walled carbon nanotubes, 200 g of concentrated sulfuric acid, and 35 g of concentrated hydrochloric acid for 15 min, heat under reflux for 7 h, cool to room temperature, wash until the pH value is 5, then heat to 75 °C with tetrahydrofuran, extract for 20 h, and dry to obtain carboxylated modified carbon nanotubes;

[0042] S2. Fermentation: Wash 3 g of Flammulina velutipes, 2 g of Lentinula edodes, 1 g of Grifola frondosa, and 7 g of Nostoc commune Vaucher respectively, dry, mix and pulverize, add to 200 mL of water, sterilize, inoculate with a seed solution of Ge-rich yeast and Se-rich yeast strains, with inoculation amounts of 2 v / v% and 1 v / v% respectively, ferment and culture at 25 °C and 150 r / min for 24 h, then add snailase, with the addition amount of snailase being 0.5 wt% of the total system mass, heat to 35 °C, enzymolyze for 1 h, filter, add ethanol to the filtrate, precipitate, filter to obtain a solid and a filtrate, wash the solid and dry to obtain composite Se / Ge polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain composite Se / Ge protein;

[0043] S3. Coupling: Add 12 g of citric acid to 300 mL of water, add 3 g of NHS and 3 g of EDC, stir and activate for 20 min, add 2 g of cecropin and 3 g of composite Se / Ge protein, stir and react for 12 h, add 0.2 g of bismuth citrate, stir for 1 h, dialyze with a 5 kDa dialysis bag for 36 h, freeze-dry the non-permeated liquid to obtain bismuth citrate protein;

[0044] S4. Preparation of polysaccharide composite carbon nanotubes: Add 10 g of carboxylated modified carbon nanotubes to 200 mL of dimethyl sulfoxide, add 3 g of composite Se / Ge polysaccharide and 0.5 g of concentrated sulfuric acid, heat to 130 °C, stir and react for 5 h, centrifuge, wash, and dry to obtain polysaccharide composite carbon nanotubes;

[0045] S5. Preparation of avian influenza granules: Add 10 g of polysaccharide composite carbon nanotubes to 200 mL of water, add 3 g of NHS and 2 g of EDC, stir and activate for 10 min, add 5 g of bismuth citrate protein, stir and react for 7 h, and spray dry to obtain avian influenza granules.

[0046] Example 2

[0047] The embodiment of the present invention provides a method for preparing avian influenza granules, specifically including the following steps:

[0048] S1. Pretreatment: Mix 2 g of multi-walled carbon nanotubes, 250 g of concentrated sulfuric acid, and 50 g of concentrated hydrochloric acid for 15 min, heat under reflux for 10 h, cool to room temperature, wash until the pH value is 6, then heat to 85 °C with tetrahydrofuran, extract for 24 h, and dry to obtain carboxylated modified carbon nanotubes;

[0049] S2. Fermentation: Wash 5 g of Flammulina velutipes, 4 g of Lentinula edodes, 3 g of Grifola frondosa, and 10 g of Nostoc commune Vaucher respectively, dry them, mix and pulverize them, add them to 200 mL of water, sterilize, inoculate with the seed solutions of germanium-enriched yeast and selenium-enriched yeast strains, with the inoculation amounts being 3 v / v% and 2 v / v% respectively, ferment and culture at 30 °C and 250 r / min for 36 h, then add snailase, the addition amount of the snailase is 1 wt% of the total mass of the system, heat to 38 °C, enzymatically hydrolyze for 3 h, filter, add ethanol to the filtrate, precipitate, filter, obtain a solid and a filtrate, wash the solid, dry it, and prepare a composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain a composite selenium / germanium protein;

[0050] S3. Coupling: Add 15 g of citric acid to 300 mL of water, add 4 g of NHS and 4 g of EDC, stir and activate for 30 min, add 3 g of cecropin and 5 g of the composite selenium / germanium protein, stir and react for 15 h, add 0.5 g of bismuth citrate, stir for 1 h, dialyze with a 5 kDa dialysis bag for 36 h, freeze-dry the undialyzed solution to obtain bismuth citrate protein;

[0051] S4. Preparation of polysaccharide composite carbon nanotubes: Add 10 g of carboxylated modified carbon nanotubes to 200 mL of dimethyl sulfoxide, add 4 g of the composite selenium / germanium polysaccharide and 1 g of concentrated sulfuric acid, heat to 140 °C, stir and react for 7 h, centrifuge, wash, and dry to obtain polysaccharide composite carbon nanotubes;

[0052] S5. Preparation of avian influenza granules: Add 12 g of the polysaccharide composite carbon nanotubes to 200 mL of water, add 4 g of NHS and 3 g of EDC, stir and activate for 20 min, add 7 g of bismuth citrate protein, stir and react for 10 h, and spray-dry to obtain avian influenza granules.

[0053] Example 3

[0054] The embodiment of the present invention provides a preparation method of avian influenza granules, which specifically includes the following steps:

[0055] S1. Pretreatment: Mix 1.5 g of multi-walled carbon nanotubes, 220 g of concentrated sulfuric acid, and 44 g of concentrated hydrochloric acid for 15 min, heat and reflux for 8 h, cool to room temperature, wash until the pH value is 5.5, then heat to 80 °C with tetrahydrofuran and extract for 22 h, dry to obtain carboxylated modified carbon nanotubes;

[0056] S2. Fermentation: Wash 4 g of Flammulina velutipes, 3 g of Lentinula edodes, 2 g of Grifola frondosa, and 8 g of Nostoc sphaeroides Kütz respectively, dry them, mix and pulverize them, add them to 200 mL of water, sterilize, inoculate with the seed solutions of germanium-rich yeast and selenium-rich yeast strains, and the inoculation amounts are 2.5 v / v% and 1.5 v / v% respectively. Ferment and culture at 27 °C and 200 r / min for 30 h, then add snailase, and the addition amount of the snailase is 0.7 wt% of the total mass of the system. Heat to 37 °C and enzymolyze for 2 h. Filter, add ethanol to the filtrate, precipitate, filter, obtain solids and filtrate, wash the solids, dry them, and prepare composite selenium / germanium polysaccharide; recycle ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain composite selenium / germanium protein;

[0057] S3. Coupling: Add 13 g of citric acid to 300 mL of water, add 3.5 g of NHS and 3.5 g of EDC, stir and activate for 25 min, add 2.5 g of cecropin and 4 g of composite selenium / germanium protein, stir and react for 13 h, add 0.35 g of bismuth citrate, stir for 1 h, dialyze with a 5 kDa dialysis bag for 36 h, and freeze-dry the non-permeated liquid to obtain protein bismuth citrate;

[0058] S4. Preparation of polysaccharide composite carbon nanotubes: Add 10 g of carboxylated modified carbon nanotubes to 200 mL of dimethyl sulfoxide, add 3.5 g of composite selenium / germanium polysaccharide and 0.7 g of concentrated sulfuric acid, heat to 135 °C, stir and react for 6 h, centrifuge, wash, and dry to obtain polysaccharide composite carbon nanotubes;

[0059] S5. Preparation of avian influenza granules: Add 11 g of polysaccharide composite carbon nanotubes to 200 mL of water, add 3.5 g of NHS and 2.5 g of EDC, stir and activate for 15 min, add 6 g of protein bismuth citrate, stir and react for 8 h, and spray-dry to obtain avian influenza granules.

[0060] Comparative Example 1

[0061] Compared with Example 3, the difference is that the seed solution of germanium-rich yeast strain is not inoculated in step S2.

[0062] Specifically as follows:

[0063] S2. Fermentation: Wash 4 g of Flammulina velutipes, 3 g of Lentinula edodes, 2 g of Grifola frondosa, and 8 g of Nostoc flagelliforme respectively, dry them, mix and pulverize them, add them to 200 mL of water, sterilize, inoculate with a seed solution of selenium-enriched yeast strain, the inoculation amount is 4 v / v%, at 27 °C, 200 r / min, ferment and culture for 30 h, then add snail enzyme, the addition amount of the snail enzyme is 0.7 wt% of the total mass of the system, heat to 37 °C, enzymatically hydrolyze for 2 h, filter, add ethanol to the filtrate, precipitate, filter, obtain solids and filtrate, wash the solids, dry, and prepare composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain composite selenium / germanium protein.

[0064] Comparative Example 2

[0065] Compared with Example 3, the difference is that the seed solution of selenium-enriched yeast strain was not inoculated in step S2.

[0066] Specifically as follows:

[0067] S2. Fermentation: Wash 4 g of Flammulina velutipes, 3 g of Lentinula edodes, 2 g of Grifola frondosa, and 8 g of Nostoc flagelliforme respectively, dry them, mix and pulverize them, add them to 200 mL of water, sterilize, inoculate with a seed solution of germanium-enriched yeast strain, the inoculation amount is 4 v / v%, at 27 °C, 200 r / min, ferment and culture for 30 h, then add snail enzyme, the addition amount of the snail enzyme is 0.7 wt% of the total mass of the system, heat to 37 °C, enzymatically hydrolyze for 2 h, filter, add ethanol to the filtrate, precipitate, filter, obtain solids and filtrate, wash the solids, dry, and prepare composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain composite selenium / germanium protein.

[0068] Comparative Example 3

[0069] Compared with Example 3, the difference is that neither the germanium-enriched yeast nor the seed solution of selenium-enriched yeast strain was inoculated in step S2.

[0070] Specifically as follows:

[0071] S2. Fermentation: Wash 4 g of Flammulina velutipes, 3 g of Lentinula edodes, 2 g of Grifola frondosa, and 8 g of Nostoc flagelliforme respectively, dry them, mix and pulverize them, add them to 200 mL of water, add snail enzyme, the addition amount of the snail enzyme is 0.7 wt% of the total mass of the system, heat to 37 °C, enzymatically hydrolyze for 2 h, filter, add ethanol to the filtrate, precipitate, filter, obtain solids and filtrate, wash the solids, dry, and prepare composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain composite selenium / germanium protein.

[0072] Comparative Example 4

[0073] Compared with Example 3, the difference is that bismuth citrate was not added in step S3.

[0074] Specifically as follows:

[0075] S3. Coupling: Add 13 g of citric acid to 300 mL of water, add 3.5 g of NHS and 3.5 g of EDC, stir and activate for 25 min, add 2.5 g of cecropin and 4 g of composite selenium / germanium protein, stir and react for 13 h, dialyze with a 5 kDa dialysis bag for 36 h, and lyophilize the non-permeate solution to obtain citric acid protein.

[0076] Comparative Example 5

[0077] Compared with Example 3, the difference is that cecropin was not added in step S3.

[0078] Specifically as follows:

[0079] S3. Coupling: Add 13 g of citric acid to 300 mL of water, add 3.5 g of NHS and 3.5 g of EDC, stir and activate for 25 min, add 6.5 g of composite selenium / germanium protein, stir and react for 13 h, add 0.35 g of bismuth citrate, stir for 1 h, dialyze with a 5 kDa dialysis bag for 36 h, and lyophilize the non-permeate solution to obtain bismuth citrate protein.

[0080] Comparative Example 6

[0081] Compared with Example 3, the difference is that step S4 was not carried out.

[0082] Specifically as follows:

[0083] S1. Pretreatment: Mix 1.5 g of multi-walled carbon nanotubes, 220 g of concentrated sulfuric acid, and 44 g of concentrated hydrochloric acid for 15 min, heat under reflux for 8 h, cool to room temperature, wash to a pH of 5.5, then heat to 80 °C with tetrahydrofuran and extract for 22 h, and dry to obtain carboxylated modified carbon nanotubes;

[0084] S2. Fermentation: Wash 4 g of Flammulina velutipes, 3 g of Lentinula edodes, 2 g of Grifola frondosa, and 8 g of Nostoc commune Vaucher respectively, dry, mix and pulverize, add to 200 mL of water, sterilize, inoculate with the seed solutions of Ge-rich yeast and Se-rich yeast strains, with inoculation amounts of 2.5 v / v% and 1.5 v / v% respectively, at 27 °C, 200 r / min, ferment and culture for 30 h, then add snail enzyme, and the addition amount of the snail enzyme is 0.7 wt% of the total mass of the system, heat to 37 °C, enzymolyze for 2 h, filter, add ammonium sulfate for fractional precipitation to the filtrate, filter, wash, and dry to obtain composite selenium / germanium protein;

[0085] S3. Coupling: Add 13 g of citric acid to 300 mL of water, add 3.5 g of NHS and 3.5 g of EDC, stir and activate for 25 min, add 2.5 g of cecropin and 4 g of composite selenium / germanium protein, stir and react for 13 h, add 0.35 g of bismuth citrate, stir for 1 h, dialyze with a 5 kDa dialysis bag for 36 h, freeze-dry the non-permeated liquid to obtain protein bismuth citrate;

[0086] S4. Preparation of avian influenza granules: Add 11 g of carboxylated modified carbon nanotubes to 200 mL of water, add 3.5 g of NHS and 2.5 g of EDC, stir and activate for 15 min, add 6 g of protein bismuth citrate, stir and react for 8 h, and spray dry to obtain avian influenza granules.

[0087] Comparative Example 7

[0088] Compared with Example 3, the difference is that step S5 is not carried out.

[0089] Specifically as follows:

[0090] S1. Pretreatment: Mix 1.5 g of multi-walled carbon nanotubes, 220 g of concentrated sulfuric acid, and 44 g of concentrated hydrochloric acid for 15 min, heat under reflux for 8 h, cool to room temperature, wash to a pH value of 5.5, then heat to 80 °C with tetrahydrofuran and extract for 22 h, and dry to obtain carboxylated modified carbon nanotubes;

[0091] S2. Fermentation: Wash 4 g of Flammulina velutipes, 3 g of Lentinula edodes, 2 g of Grifola frondosa, and 8 g of Nostoc commune Vaucher respectively, dry, mix and pulverize, add to 200 mL of water, sterilize, inoculate with the seed solutions of Ge-enriched yeast and Se-enriched yeast strains, with inoculation amounts of 2.5 v / v% and 1.5 v / v% respectively, at 27 °C, 200 r / min, ferment and culture for 30 h, then add snail enzyme, the addition amount of the snail enzyme is 0.7 wt% of the total mass of the system, heat to 37 °C, enzymolyze for 2 h, filter, add ethanol to the filtrate, precipitate, filter to obtain a solid and a filtrate, wash the solid and dry to obtain composite selenium / germanium polysaccharide;

[0092] S3. Preparation of polysaccharide composite carbon nanotubes: Add 10 g of carboxylated modified carbon nanotubes to 200 mL of dimethyl sulfoxide, add 3.5 g of composite selenium / germanium polysaccharide and 0.7 g of concentrated sulfuric acid, heat to 135 °C, stir and react for 6 h, and spray dry to obtain avian influenza granules.

[0093] Test Example 1

[0094] Add 100 TCID 50The AIVNJ02 strain (AIV, avian influenza virus) was respectively mixed in equal volume with the aqueous suspension of influenza granules for poultry at a concentration of 0.5 mg / L prepared in Examples 1-3 or Comparative Examples 1-7 and the hand-free disinfectant at the above concentration. 200 μL of the mixed solution was taken and added to MDCK monolayer cells (dog kidney passage cells) with a confluence of 80%. At the same time, negative control group cells (without virus and aqueous suspension) and virus control group (adding 100 TCID50 virus, without aqueous suspension) were set up, and each group of cells was repeated in 6 wells. The cells in each group were placed at 37 °C and 5% CO2 for 2 h, the culture medium was discarded, washed twice with PBS, replaced with 200 μL of fresh cell culture medium, and cultured for 72 h. 10 μL of CCK-8 solution was added to 3 duplicate wells of each group of cells, and cultured for 4 h under the conditions of 37 °C and 5% CO2, and the OD 450nm value of each well was detected. The inhibition rate of each drug on the virus was calculated using the following formula.

[0095] Inhibition rate on virus (%) = (average OD value of drug group 450nm - average OD value of negative control group 450nm value) / (average OD value of cell control group 450nm value - average OD value of negative control group 450nm value) × 100%; The results are shown in Table 1.

[0096] Table 1

[0097]

[0098] As can be seen from Table 1, the influenza granules for poultry prepared in Examples 1-3 of the present invention have a good virus inhibition rate.

[0099] Test Example 2

[0100] Fourteen-day-old Ross 308 white broilers were randomly divided into 12 groups, namely control group, H9N2 infection group, Examples 1-3 groups, and Comparative Examples 1-7 groups, with 20 broilers in each group. The broilers in the control group were instilled with 0.2 mL of sterile normal saline through the nasal cavity, and the broilers in other groups were instilled with 0.2 mL of allantoic fluid of H9N2 virus (about 10 5 EID 50 H9N2 virus) through the nasal cavity. After the broilers in Examples 1-3 groups and Comparative Examples 1-7 groups were treated in the same way as the H9N2 infection group, they were fed with the influenza granules for poultry in Examples 1-3 or Comparative Examples 1-7 (100 mg / kg) + basal diet, and the remaining groups were fed with normal drinking water + basal diet. The animals in this experiment were only infected once, and the day of infection was taken as the 1st d of the formal experiment, and the formal experiment period was 21 d. During this period, the clinical manifestations and death conditions of the chicken flock were regularly observed and recorded. The chickens were raised on the net floor, provided with independent waterers and feed buckets, and allowed to eat and drink freely.

[0101] Record the mortality rates of broiler chickens in each group 21 days after the intervention.

[0102] On the 21st day after the intervention, 3 chickens were randomly selected from each group for autopsy. The trachea and lungs were taken and fixed in 4% formaldehyde and 2.5% glutaraldehyde solutions for tissue fixation. Part of the lungs was taken to detect the copy number of H9N2 subtype AIV. 100 mg of the collected lung tissue was added to 1 mL of TRIzol solution and ground into a homogenate, and the operation was carried out according to the method for extracting total RNA from animal tissues. The content of H9N2 virus in chicken lung tissue was measured. The results are shown in Table 2.

[0103] Table 2

[0104]

[0105] As can be seen from Table 2, the influenza granules for poultry prepared in Examples 1-3 of the present invention have a good effect of resisting avian influenza virus.

[0106] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of influenza granules for poultry, characterized in that Specifically, it includes the following steps: S1. Pretreatment: Mix 1-2 parts by weight of multi-walled carbon nanotubes, 200-250 parts by weight of concentrated sulfuric acid, and 35-50 parts by weight of concentrated hydrochloric acid evenly, heat under reflux for 7-10 h, cool to room temperature, wash until the pH value is 5-6, then heat to 75-85 °C with tetrahydrofuran, extract for 20-24 h, and dry to obtain carboxylated modified carbon nanotubes; S2. Fermentation: Wash 3 - 5 parts by weight of Flammulina velutipes, 2 - 4 parts by weight of Lentinula edodes, 1 - 3 parts by weight of Grifola frondosa, and 7 - 10 parts by weight of Nostoc commune Vaucher respectively, dry them, mix and pulverize, add them to water, sterilize, inoculate with a seed solution of germanium - rich yeast and selenium - rich yeast strains with a bacterial content of 10 8 -10 9 cfu / mL. The inoculation amounts are 2 - 3 v / v% and 1 - 2 v / v% respectively. Ferment and culture at 25 - 30 °C and 150 - 250 r / min for 24 - 36 h, then add snailase. The addition amount of the snailase is 0.5 - 1 wt% of the total mass of the system. Heat to 35 - 38 °C and enzymolyze for 1 - 3 h. Filter, add ethanol to the filtrate, precipitate, filter to obtain a solid and a filtrate. Wash the solid, dry it to obtain a composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for fractional precipitation, filter, wash, and dry to obtain a composite selenium / germanium protein; S3. Coupling: Add 12-15 parts by weight of citric acid to water, add 3-4 parts by weight of NHS and 3-4 parts by weight of EDC, stir and activate for 20-30 min, add 2-3 parts by weight of cecropin and 3-5 parts by weight of composite selenium / germanium protein, stir and react for 12-15 h, add 0.2-0.5 parts by weight of bismuth salt, stir, dialyze, and lyophilize the non-permeated liquid to obtain bismuth citrate protein; S4. Preparation of polysaccharide composite carbon nanotubes: Add 10 parts by weight of carboxylated modified carbon nanotubes to dimethyl sulfoxide, add 3-4 parts by weight of composite selenium / germanium polysaccharide and 0.5-1 part by weight of concentrated sulfuric acid, heat to 130-140 °C, stir and react for 5-7 h, centrifuge, wash, and dry to obtain polysaccharide composite carbon nanotubes; S5. Preparation of avian influenza particles: Add 10-12 parts by weight of polysaccharide composite carbon nanotubes to water, add 3-4 parts by weight of NHS and 2-3 parts by weight of EDC, stir and activate for 10-20 min, add 5-7 parts by weight of bismuth citrate protein, stir and react for 7-10 h, and spray dry to obtain avian influenza particles for poultry.

2. An avian influenza particle for poultry prepared by the preparation method according to claim 1.

3. Use of the avian influenza particle for poultry according to claim 2 in the preparation of veterinary drugs for treating or assisting in treating avian influenza.

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