Influenza granules for poultry and preparation method thereof

Abstract

The invention provides flu granules for poultry and a preparation method of the flu granules, and belongs to the technical field of poultry medicines. The preparation method comprises the following steps: carboxylating a multi-walled carbon nanotube, compounding the carboxylated multi-walled carbon nanotube with compound selenium / germanium polysaccharide obtained by fermenting flammulina velutipes, shiitake mushrooms, grifola frondosa and nostoc sphaeroids kutz to prepare a polysaccharide compound carbon nanotube, coupling the obtained compound selenium / germanium protein with citric acid and cecropin, complexing with bismuth salt, and compounding with the polysaccharide compound carbon nanotube to prepare the influenza granules for poultry. The prepared influenza particles for poultry are high in bioavailability, good in safety and low in cytotoxicity, can effectively inhibit virus replication, reduce virus load, relieve diseases caused by viruses and enhance the immune function of the body, and can assist antigens to stimulate the body to generate immune response and improve the expression level of antibodies when being used together with immunogens or vaccines, so that the influenza particles can be used for preventing and treating the diseases. Wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of poultry medicines, and in particular to influenza particles for poultry and a preparation method thereof. Background Art

[0002] Avian influenza is the abbreviation of avian influenza. The characteristics of avian influenza in chickens are: ① The clinical symptoms are complex, and clinically it is often mixed with infectious bronchitis, infectious laryngotracheitis, Escherichia coli, etc.; ② The morbidity and mortality rates are related to the strain, gender of the flock, environment and complications, and secondary infection. For example, the invasion of avian influenza virus into the mucosa is conducive to the secondary Escherichia coli disease, which enhances the effect of avian influenza virus hemagglutinin, leading to increased morbidity and mortality in the flock; ③ There are many serotypes of avian influenza virus, and the cross-protection between types is weak. The virulence of different strains of the same serotype varies greatly; ④ The avian influenza virus mutates frequently, and pathogenic mutations, antigenic drift and transformation are prone to occur.

[0003] Current status of the harm and prevention of avian influenza in chickens: ① For more than 100 years since avian influenza was discovered, humans have not mastered specific prevention and treatment methods. They can only prevent its spread by disinfection, isolation, and mass slaughter of poultry and livestock, which has caused harm to the poultry industry. ② After poultry is infected with avian influenza, the body's immune defense system is first destroyed, resulting in the loss of the body's resistance and self-repair ability; ③ Western antiviral drugs have a partial antagonistic effect on virus particles after clinical use, but they also have toxic side effects on poultry's body cells. In veterinary clinical practice, the effect of Western antiviral drugs on the prevention and treatment of avian influenza in poultry is not ideal. ④ The use of Western antiviral drugs in the process of poultry breeding will lead to food safety and other problems. my country explicitly does not allow the use of Western antiviral drugs for the prevention and treatment of avian influenza in livestock and poultry. Therefore, there are currently no antiviral veterinary drugs for the prevention and treatment of avian influenza in poultry. ⑤ In the research and exploration of clinical prevention and treatment of avian influenza in poultry, it was found that some Chinese veterinary medicines such as astragalus, honeysuckle, isatis root, and momordica charantia have good clinical effects in preventing and treating avian influenza in poultry. In addition, the study also found that Chinese veterinary medicines such as astragalus not only have strong clinical antiviral capabilities, but also have the functions of enhancing the body's immunity and disease resistance, and improving the body's own repair capabilities.

[0004] Traditional Chinese veterinary medicines are processed in a relatively simple and rough manner. They are usually directly mixed into poultry feed or drinking water in the form of simple coarse or fine powder or traditionally boiled. The effective ingredients of Chinese veterinary medicines cannot be fully and effectively absorbed and utilized, resulting in less than ideal clinical application results. Therefore, the improvement of drugs for the prevention and treatment of avian influenza is an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to provide an influenza particle for poultry and a preparation method thereof, which has high bioavailability, good safety, low cytotoxicity, can effectively inhibit virus replication, reduce viral load, alleviate symptoms caused by viruses, enhance the body's immune function, and can be used together with an immunogen or a vaccine to assist antigens in stimulating the body to produce an immune response and improve the level of antibody expression, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way: The present invention provides a method for preparing influenza particles for poultry, comprising the following steps: S1. Pretreatment: Mixing multi-walled carbon nanotubes, concentrated sulfuric acid and concentrated hydrochloric acid, heating to reflux reaction, cooling to room temperature, washing, and then heating and extracting with tetrahydrofuran, and drying to obtain carboxyl-modified carbon nanotubes; S2. Fermentation: Wash Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Mylodon puerariae separately, dry, mix and crush, add to water, sterilize, inoculate with germanium-enriched yeast and selenium-enriched yeast seed liquid, ferment and culture, then add snail enzyme, heat and enzymolysis, filter, add ethanol to the filtrate, precipitate, filter, obtain solid and filtrate, wash the solid, dry, and obtain composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for graded precipitation, filter, wash, and dry, and obtain composite selenium / germanium protein; S3. Coupling: adding citric acid to water, adding NHS and EDC, stirring to activate, adding cecropin and composite selenium / germanium protein, stirring to react, adding bismuth salt, stirring, dialyzing, and freeze-drying the non-permeated solution to obtain bismuth citrate protein; S4. Preparation of polysaccharide composite carbon nanotubes: adding carboxyl modified carbon nanotubes to dimethyl sulfoxide, adding composite selenium / germanium polysaccharide and concentrated sulfuric acid, heating and stirring to react, centrifuging, washing, and drying to obtain polysaccharide composite carbon nanotubes; S5. Preparation of influenza particles for poultry: Add polysaccharide composite carbon nanotubes into water, add NHS and EDC, stir to activate, add bismuth citrate protein, stir to react, spray dry, and obtain influenza particles for poultry.

[0007] As a further improvement of the present invention, the mass ratio of the multi-walled carbon nanotubes, concentrated sulfuric acid and concentrated hydrochloric acid in step S1 is 1-2:200-250:35-50, the heating reflux reaction time is 7-10 hours, the heating extraction temperature is 75-85°C, the time is 20-24 hours, and the washing is performed to a pH value of 5-6.

[0008] As a further improvement of the present invention, the mass ratio of Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Mylostomia pueraria in step S2 is 3-5:2-4:1-3:7-10, the inoculation amounts of the germanium-enriched yeast and selenium-enriched yeast seed solutions are 2-3 v / v% and 1-2 v / v%, respectively, and the bacterial content of the seed solution is 10 8 -10 9 cfu / mL, the fermentation culture conditions are 25-30°C, 150-250r / min, fermentation culture 24-36h, the addition amount of the snail enzyme is 0.5-1wt% of the total mass of the system, the temperature of the heating enzymolysis is 35-38°C, and the time is 1-3h.

[0009] As a further improvement of the present invention, the mass ratio of citric acid, NHS, EDC, cecropin, composite selenium / germanium protein and bismuth salt in step S3 is 12-15:3-4:3-4:2-3:3-5:0.2-0.5, the bismuth salt is bismuth citrate, the stirring activation time is 20-30 min, and the stirring reaction time is 12-15 h.

[0010] As a further improvement of the present invention, in step S4, the mass ratio of the carboxyl modified carbon nanotubes, the composite selenium / germanium polysaccharide and the concentrated sulfuric acid is 10:3-4:0.5-1, and the temperature of the heating and stirring reaction is 130-140° C. for 5-7 hours.

[0011] As a further improvement of the present invention, in step S5, the mass ratio of the polysaccharide composite carbon nanotubes, NHS, EDC and bismuth citrate protein is 10-12:3-4:2-3:5-7, the stirring activation time is 10-20 min, and the stirring reaction time is 7-10 h.

[0012] As a further improvement of the present invention, the present invention specifically comprises the following steps: S1. Pretreatment: 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 were mixed uniformly, heated to reflux for 7-10 hours, cooled to room temperature, washed to a pH value of 5-6, and then heated to 75-85 ° C with tetrahydrofuran, extracted for 20-24 hours, and dried to obtain carboxyl modified carbon nanotubes; S2 fermentation: 3-5 parts by weight of Flammulina velutipes, 2-4 parts by weight of shiitake mushrooms, 1-3 parts by weight of Grifola frondosa, 7-10 parts by weight of Gexian rice algae were washed, dried, mixed and crushed, added to water, sterilized, inoculated with a bacterial content of 10 8 -10 9cfu / mL of germanium-rich yeast and selenium-rich yeast seed liquid, inoculation amounts of 2-3v / v% and 1-2v / v%, respectively, at 25-30°C, 150-250r / min, fermentation culture for 24-36h, then adding snail enzyme, the amount of snail enzyme added is 0.5-1wt% of the total mass of the system, heating to 35-38°C, enzymolysis for 1-3h, filtering, adding ethanol to the filtrate, precipitating, filtering, obtaining a solid and a filtrate, washing the solid, drying, and obtaining a composite selenium / germanium polysaccharide; recovering ethanol from the filtrate, adding ammonium sulfate for graded precipitation, filtering, washing, and drying, and obtaining a composite selenium / germanium protein; S3 coupling: 12-15 parts by weight of citric acid was added to water, 3-4 parts by weight of NHS and 3-4 parts by weight of EDC were added, and the mixture was stirred for activation for 20-30 min, 2-3 parts by weight of cecropin and 3-5 parts by weight of composite selenium / germanium protein were added, and the reaction was stirred for 12-15 h, 0.2-0.5 parts by weight of bismuth salt was added, stirred, dialyzed, and the non-permeated liquid was freeze-dried to obtain bismuth citrate protein; S4. Preparation of polysaccharide composite carbon nanotubes: 10 parts by weight of carboxyl modified carbon nanotubes were added to dimethyl sulfoxide, 3-4 parts by weight of composite selenium / germanium polysaccharide and 0.5-1 parts by weight of concentrated sulfuric acid were added, heated to 130-140°C, stirred for reaction for 5-7h, centrifuged, washed, and dried to obtain polysaccharide composite carbon nanotubes; S5. Preparation of influenza particles for poultry: 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 minutes, add 5-7 parts by weight of bismuth citrate protein, stir and react for 7-10 hours, spray dry, and obtain influenza particles for poultry.

[0013] The present invention further protects influenza particles for poultry prepared by the above preparation method.

[0014] The present invention further protects the use of the influenza granules for poultry in preparing veterinary drugs for treating or assisting in treating avian influenza.

[0015] The present invention has the following beneficial effects: The present invention prepares an immunopotentiator that can enhance the body's immune function and has the advantages of enhancing the body's specific and nonspecific immune responses, accelerating the induction of immune responses, improving the body's anti-infection ability, and correcting immune deficiencies. When used together with an immunogen or a vaccine, it can assist the antigen in stimulating the body to produce an immune response and improve the antibody expression level.

[0016] The present invention uses carbon nanotubes as carriers. On the one hand, they can complex a large number of polysaccharides and proteins. The nanomaterials have unique physical, chemical and biological properties, have high antigen carrying capacity characteristics, and are non-immunogenic themselves. On the other hand, carbon nanotubes can improve the animal body's immune response to inactivated viruses, genetically engineered recombinant proteins, polypeptides, and small molecule compounds, enhance the body's specific antibody expression, and play a good role in assisting antiviral.

[0017] The invention uses enoki mushroom, shiitake mushroom, maitake mushroom and kudzu vine as raw materials, which are rich in 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, has good anti-influenza virus, anti-HIV virus (HIV) and anti-type I herpes simplex virus (HSV-1) effects, and has a good inhibitory effect on avian influenza virus. Enoki mushroom polysaccharide and shiitake mushroom polysaccharide are safe, efficient and environmentally friendly new immunomodulators, which can inhibit or reduce harmful microorganisms in the intestine, regulate the balance of intestinal microecology, inhibit the proliferation of pathogenic bacteria such as Pasteurella multocida, Escherichia coli, Salmonella in vitro, and are also used as immunopotentiators. The synergistic effect with a variety of animal vaccines can significantly improve the immune effect. Kudzu vine vine is also a freshwater algae containing active polysaccharides and phycobiliproteins, and has good anti-inflammatory, antibacterial and antiviral effects.

[0018] After mixed fermentation with germanium-rich yeast and selenium-rich yeast, composite selenium / germanium polysaccharides and composite selenium / germanium proteins are obtained. Organic selenium can improve the humoral and cellular immunity of livestock and poultry, and enhance the body's resistance to infectious diseases. Selenium deficiency will increase the pathological nature of viral infection, and 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 selenoprotein modules encoded by viruses. Organic germanium has a significant immunomodulatory effect. It can stimulate T lymphocytes to produce lymphokines, such as inducing interferon γ-IFN, auxiliary factor IL-2, etc., activate macrophages to become cytotoxic macrophages, and activate the activity of natural killer cells (NK), thereby improving disease resistance. Both can significantly improve the growth performance of poultry, improve their immunity, reduce the impact of avian influenza on poultry, have good antiviral effects, and have a synergistic effect.

[0019] The composite selenium / germanium protein is coupled with cecropin and citrate, and bismuth is complexed. Cecropin, as a small molecule polypeptide, is an important component of insect innate immunity. Its special mode of action makes cecropin have many functions such as antibacterial and antiviral, enhancing the body's immunity, and tissue repair. The formed citrate protein bismuth has low cytotoxicity and high safety. It can effectively inhibit viral replication, reduce viral load, and alleviate virus-induced diseases. Through a unique metal replacement mechanism, it irreversibly deprives the virus helicase of the key zinc ion, and then forms a bismuth-bound helicase, thereby making it lose its biological activity and playing an antiviral role.

[0020] The influenza particles for poultry prepared by the present invention have high bioavailability, good safety, low cytotoxicity, can effectively inhibit virus replication, reduce virus load, alleviate symptoms caused by viruses, enhance the body's immune function, and can be used together with immunogens or vaccines to assist antigens in stimulating the body to produce immune responses and improve antibody expression levels, thus having broad application prospects. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Germanium-enriched yeast, 20 billion cfu / g, selenium-enriched yeast, 20 billion cfu / g.

[0023] Preparation of bacterial seed solution: Inoculate the bacterial strain into Gao's medium, activate and culture for 18-24 hours at 27℃ and 100r / min to obtain a culture medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution.

[0024] Snail enzyme, wall breaking rate 90%.

[0025] NHS, N-hydroxysuccinimide, EDC, 1-ethyl-(3-dimethylaminopropyl)carbodiimide.

[0026] Multi-walled carbon nanotubes, industrial products, diameter 10-20nm, length 10-30μm.

[0027] Example 1 The present invention provides a method for preparing influenza particles for poultry, which specifically comprises the following steps: S1. Pretreatment: 1 g of multi-walled carbon nanotubes, 200 g of concentrated sulfuric acid, and 35 g of concentrated hydrochloric acid were mixed for 15 min, heated under reflux for 7 h, cooled to room temperature, washed to a pH of 5, heated to 75 ° C with tetrahydrofuran, extracted for 20 h, and dried to obtain carboxyl-modified carbon nanotubes; S2. Fermentation: 3g of Flammulina velutipes, 2g of Lentinula edodes, 1g of Grifola frondosa, and 7g of Mytilus foetida were washed, dried, mixed and crushed, added to 200mL of water, sterilized, inoculated with germanium-rich yeast and selenium-rich yeast seed liquid, the inoculation amount was 2v / v% and 1v / v%, respectively, at 25°C, 150r / min, fermented and cultured for 24h, then added with snail enzyme, the amount of snail enzyme added was 0.5wt% of the total mass of the system, heated to 35°C, enzymolysis for 1h, filtered, ethanol was added to the filtrate, precipitated, filtered, solid and filtrate were obtained, the solid was washed, dried, and composite selenium / germanium polysaccharide was obtained; ethanol was recovered from the filtrate, ammonium sulfate was added for graded precipitation, filtered, washed, and dried to obtain composite selenium / germanium protein; S3. Coupling: 12 g of citric acid was added to 300 mL of water, 3 g of NHS and 3 g of EDC were added, and the mixture was stirred for 20 min. 2 g of cecropin and 3 g of composite selenium / germanium protein were added, and the reaction was stirred for 12 h. 0.2 g of bismuth citrate was added, and the mixture was stirred for 1 h. The mixture was dialyzed with a 5 kDa dialysis bag for 36 h, and the non-permeated solution was freeze-dried to obtain bismuth citrate protein. S4. Preparation of polysaccharide composite carbon nanotubes: 10 g of carboxyl modified carbon nanotubes were added to 200 mL of dimethyl sulfoxide, 3 g of composite selenium / germanium polysaccharide and 0.5 g of concentrated sulfuric acid were added, heated to 130 ° C, stirred for 5 h, centrifuged, washed, and dried to obtain polysaccharide composite carbon nanotubes; S5. Preparation of influenza particles for poultry: 10 g of polysaccharide composite carbon nanotubes was added to 200 mL of water, 3 g of NHS and 2 g of EDC were added, and the mixture was stirred for activation for 10 min. 5 g of protein bismuth citrate was added, and the mixture was stirred for reaction for 7 h. The mixture was spray-dried to obtain influenza particles for poultry.

[0028] Example 2 The present invention provides a method for preparing influenza particles for poultry, which specifically comprises the following steps: S1. Pretreatment: 2 g of multi-walled carbon nanotubes, 250 g of concentrated sulfuric acid, and 50 g of concentrated hydrochloric acid were mixed for 15 min, heated to reflux for 10 h, cooled to room temperature, washed to a pH of 6, heated to 85 ° C with tetrahydrofuran, extracted for 24 h, and dried to obtain carboxyl-modified carbon nanotubes; S2. Fermentation: Wash 5g of Flammulina velutipes, 4g of Lentinula edodes, 3g of Grifola frondosa, and 10g of Mytilus foetida, dry them, mix and crush them, add them to 200mL of water, sterilize them, inoculate them with seed liquid of germanium-rich yeast and selenium-rich yeast, the inoculation amounts are 3v / v% and 2v / v%, respectively, at 30°C, 250r / min, ferment and culture for 36h, then add snail enzyme, the amount of snail enzyme added is 1wt% of the total mass of the system, heat to 38°C, enzymolysis for 3h, filter, add ethanol to the filtrate, precipitate, filter, obtain solid and filtrate, wash the solid, dry, and obtain composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for graded precipitation, filter, wash, and dry to obtain composite selenium / germanium protein; S3. Coupling: 15 g of citric acid was added to 300 mL of water, 4 g of NHS and 4 g of EDC were added, and the mixture was stirred for activation for 30 min, 3 g of cecropin and 5 g of composite selenium / germanium protein were added, and the reaction was stirred for 15 h, 0.5 g of bismuth citrate was added, and the mixture was stirred for 1 h. The mixture was dialyzed with a 5 kDa dialysis bag for 36 h, and the non-permeated solution was freeze-dried to obtain bismuth citrate protein; S4. Preparation of polysaccharide composite carbon nanotubes: 10 g of carboxyl modified carbon nanotubes were added to 200 mL of dimethyl sulfoxide, 4 g of composite selenium / germanium polysaccharide and 1 g of concentrated sulfuric acid were added, heated to 140 ° C, stirred for 7 h, centrifuged, washed, and dried to obtain polysaccharide composite carbon nanotubes; S5. Preparation of influenza particles for poultry: Add 12 g of 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, spray dry, and obtain influenza particles for poultry.

[0029] Example 3 The present invention provides a method for preparing influenza particles for poultry, which specifically comprises the following steps: S1. Pretreatment: 1.5 g of multi-walled carbon nanotubes, 220 g of concentrated sulfuric acid, and 44 g of concentrated hydrochloric acid were mixed for 15 min, heated to reflux for 8 h, cooled to room temperature, washed to a pH value of 5.5, and then heated to 80 ° C with tetrahydrofuran, extracted for 22 h, and dried to obtain carboxyl-modified carbon nanotubes; S2. Fermentation: 4g of Flammulina velutipes, 3g of Lentinula edodes, 2g of Grifola frondosa, and 8g of Mytilus foetida were washed, dried, mixed and crushed, added to 200mL of water, sterilized, inoculated with germanium-rich yeast and selenium-rich yeast seed liquid, the inoculation amount was 2.5v / v% and 1.5v / v%, respectively, at 27°C, 200r / min, fermented and cultured for 30h, then added with snail enzyme, the amount of snail enzyme added was 0.7wt% of the total mass of the system, heated to 37°C, enzymolysis for 2h, filtered, ethanol was added to the filtrate, precipitated, filtered, solid and filtrate were obtained, the solid was washed, dried, and composite selenium / germanium polysaccharide was obtained; ethanol was recovered from the filtrate, ammonium sulfate was added for graded precipitation, filtered, washed, and dried to obtain composite selenium / germanium protein; S3. Coupling: 13 g of citric acid was added to 300 mL of water, 3.5 g of NHS and 3.5 g of EDC were added, and the mixture was stirred for 25 min. 2.5 g of cecropin and 4 g of composite selenium / germanium protein were added, and the reaction was stirred for 13 h. 0.35 g of bismuth citrate was added, and the mixture was stirred for 1 h. The mixture was dialyzed with a 5 kDa dialysis bag for 36 h. The non-permeated solution was freeze-dried to obtain bismuth citrate protein. S4. Preparation of polysaccharide composite carbon nanotubes: 10 g of carboxyl modified carbon nanotubes were added to 200 mL of dimethyl sulfoxide, 3.5 g of composite selenium / germanium polysaccharide and 0.7 g of concentrated sulfuric acid were added, heated to 135 ° C, stirred for 6 h, centrifuged, washed, and dried to obtain polysaccharide composite carbon nanotubes; S5. Preparation of influenza particles for poultry: 11 g of polysaccharide composite carbon nanotubes was added to 200 mL of water, 3.5 g of NHS and 2.5 g of EDC were added, and the mixture was stirred for activation for 15 min. 6 g of protein bismuth citrate was added, and the mixture was stirred for reaction for 8 h. The mixture was spray-dried to obtain influenza particles for poultry.

[0030] Comparative Example 1 Compared with Example 3, the difference is that the germanium-enriched yeast seed liquid is not inoculated in step S2.

[0031] The details are as follows: S2. Fermentation: 4g of Flammulina velutipes, 3g of Lentinus edodes, 2g of Grifola frondosa, and 8g of Mytilus foetida were washed, dried, mixed and crushed, added into 200mL of water, sterilized, inoculated with selenium-enriched yeast seed solution at an inoculation amount of 4v / v%, and fermented at 27°C, 200r / min for 30h, then added with snail enzyme in an amount of 0.7wt% of the total mass of the system, heated to 37°C, enzymolysis for 2h, filtered, added ethanol to the filtrate, precipitated, filtered, obtained a solid and a filtrate, washed the solid, dried, and obtained a composite selenium / germanium polysaccharide; recovered ethanol from the filtrate, added ammonium sulfate for graded precipitation, filtered, washed, and dried, and obtained a composite selenium / germanium protein.

[0032] Comparative Example 2 Compared with Example 3, the difference is that no selenium-enriched yeast seed liquid is inoculated in step S2.

[0033] The details are as follows: S2. Fermentation: 4g of Flammulina velutipes, 3g of Lentinula edodes, 2g of Grifola frondosa, and 8g of Mytilus foetida were washed, dried, mixed and crushed, added into 200mL of water, sterilized, inoculated with germanium-rich yeast seed solution at an inoculation amount of 4v / v%, and fermented at 27°C, 200r / min for 30h, then added with snail enzyme in an amount of 0.7wt% of the total mass of the system, heated to 37°C, enzymolysis for 2h, filtered, added ethanol to the filtrate, precipitated, filtered, obtained a solid and a filtrate, washed the solid, dried, and obtained a composite selenium / germanium polysaccharide; recovered ethanol from the filtrate, added ammonium sulfate for graded precipitation, filtered, washed, and dried, and obtained a composite selenium / germanium protein.

[0034] Comparative Example 3 Compared with Example 3, the difference is that the seed liquid of germanium-enriched yeast and selenium-enriched yeast species is not inoculated in step S2.

[0035] The details are as follows: S2. Fermentation: 4g of Flammulina velutipes, 3g of Lentinus edodes, 2g of Grifola frondosa, and 8g of Myrtillus foetida were washed, dried, mixed and crushed, added into 200mL of water, and snail enzyme was added in an amount of 0.7wt% of the total mass of the system. The mixture was heated to 37°C, and enzymolysis was performed for 2h. The mixture was filtered, and ethanol was added to the filtrate for precipitation. The solid was filtered to obtain a solid and a filtrate. The solid was washed and dried to obtain a composite selenium / germanium polysaccharide. Ethanol was recovered from the filtrate, and ammonium sulfate was added for graded precipitation. The mixture was filtered, washed, and dried to obtain a composite selenium / germanium protein.

[0036] Comparative Example 4 Compared with Example 3, the difference is that bismuth citrate is not added in step S3.

[0037] The details are as follows: 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, freeze-dry the non-permeated liquid to obtain citrate protein.

[0038] Comparative Example 5 Compared with Example 3, the difference is that no cecropin is added in step S3.

[0039] The details are as follows: 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, freeze-dry the non-permeated liquid to obtain bismuth citrate protein.

[0040] Comparative Example 6 Compared with Example 3, the difference is that step S4 is not performed.

[0041] The details are as follows: S1. Pretreatment: 1.5 g of multi-walled carbon nanotubes, 220 g of concentrated sulfuric acid, and 44 g of concentrated hydrochloric acid were mixed for 15 min, heated to reflux for 8 h, cooled to room temperature, washed to a pH value of 5.5, and then heated to 80 ° C with tetrahydrofuran, extracted for 22 h, and dried to obtain carboxyl-modified carbon nanotubes; S2. Fermentation: Wash 4g of Flammulina velutipes, 3g of Lentinula edodes, 2g of Grifola frondosa, and 8g of Mytilus foetida, dry them, mix and crush them, add them to 200mL of water, sterilize them, inoculate them with germanium-enriched yeast and selenium-enriched yeast seed liquid, the inoculation amounts are 2.5v / v% and 1.5v / v%, respectively, at 27°C, 200r / min, ferment and culture for 30h, then add snail enzyme, the amount of snail enzyme added is 0.7wt% of the total mass of the system, heat to 37°C, enzymolysis for 2h, filter, add ammonium sulfate to the filtrate for graded precipitation, filter, wash, and dry to obtain a composite selenium / germanium protein; S3. Coupling: 13 g of citric acid was added to 300 mL of water, 3.5 g of NHS and 3.5 g of EDC were added, and the mixture was stirred for 25 min. 2.5 g of cecropin and 4 g of composite selenium / germanium protein were added, and the reaction was stirred for 13 h. 0.35 g of bismuth citrate was added, and the mixture was stirred for 1 h. The mixture was dialyzed with a 5 kDa dialysis bag for 36 h. The non-permeated solution was freeze-dried to obtain bismuth citrate protein. S4. Preparation of influenza particles for poultry: 11 g of carboxyl-modified carbon nanotubes were added to 200 mL of water, 3.5 g of NHS and 2.5 g of EDC were added, and the mixture was stirred for activation for 15 min. 6 g of bismuth citrate protein was added, and the mixture was stirred for reaction for 8 h. The mixture was spray-dried to obtain influenza particles for poultry.

[0042] Comparative Example 7 Compared with Embodiment 3, the difference is that step S5 is not performed.

[0043] The details are as follows: S1. Pretreatment: 1.5 g of multi-walled carbon nanotubes, 220 g of concentrated sulfuric acid, and 44 g of concentrated hydrochloric acid were mixed for 15 min, heated to reflux for 8 h, cooled to room temperature, washed to a pH value of 5.5, and then heated to 80 ° C with tetrahydrofuran, extracted for 22 h, and dried to obtain carboxyl-modified carbon nanotubes; S2. Fermentation: 4g of Flammulina velutipes, 3g of Lentinula edodes, 2g of Grifola frondosa, and 8g of Mytilus foetida were washed, dried, mixed and crushed, added to 200mL of water, sterilized, inoculated with germanium-enriched yeast and selenium-enriched yeast seed liquid, the inoculation amounts were 2.5v / v% and 1.5v / v%, respectively, at 27°C, 200r / min, fermented and cultured for 30h, and then snail enzyme was added, the amount of snail enzyme added was 0.7wt% of the total mass of the system, heated to 37°C, enzymolysis for 2h, filtered, ethanol was added to the filtrate, precipitated, filtered, solid and filtrate were obtained, the solid was washed and dried, and composite selenium / germanium polysaccharide was obtained; S3. Preparation of polysaccharide composite carbon nanotubes: 10 g of carboxyl modified carbon nanotubes were added to 200 mL of dimethyl sulfoxide, 3.5 g of composite selenium / germanium polysaccharide and 0.7 g of concentrated sulfuric acid, heated to 135°C, stirred for reaction for 6 h, and spray dried to obtain influenza particles for poultry.

[0044] Test Example 1 100TCID 50 AIVNJ02 strain (AIV, avian influenza virus) was mixed with 0.5 mg / L poultry influenza particles water suspension prepared in Example 1-3 or Comparative Example 1-7 and the above concentration of hand-free disinfectant in equal volumes, and 200 μL of the mixture was added to MDCK monolayer cells (dog kidney passage cells) with a confluence of 80%. At the same time, a negative control group of cells (no virus and water suspension) and a virus control group (100 TCID50 virus was added, and no water suspension was added) were set up, and each group of cells was repeated in 6 wells. The cells of each group were placed at 37°C and 5% CO2 for 2h, the culture medium was discarded, and PBS was washed twice, and 200 μL of fresh cell culture medium was replaced, and cultured for 72h. Three duplicate wells of each group of cells were added with 10 μL CCK-8 solution, and cultured for 4h at 37°C and 5% CO2. The OD of each well was detected. 450nm The inhibition rate of each drug on the virus was calculated using the following formula.

[0045] Inhibition rate of virus (%) = (mean OD of drug group 450nm Value-average OD of negative control group 450nm value) / (average OD of cell control group 450nm Value-average OD of negative control group 450nm value)×100%; the results are shown in Table 1.

[0046] Table 1

[0047] It can be seen from Table 1 that the influenza particles for poultry prepared in Examples 1-3 of the present invention have a good virus inhibition rate.

[0048] Test Example 2 14-day-old Ross 308 white-feather broilers were randomly divided into 12 groups: control group, H9N2 infection group, Example 1-3 group, and Comparative Example 1-7 group, with 20 broilers in each group. The broilers in the control group were nasally dripped with 0.2 mL of sterile saline, and the broilers in the other groups were nasally dripped with H9N2 virus allantoic fluid (about 10 5 EID 50 H9N2 virus) 0.2mL nasal drops, Example 1-3 group and Comparative Example 1-7 group broilers were treated in the same way as the H9N2 infection group, and then used Example 1-3 or Comparative Example 1-7 poultry influenza particles (100mg / kg) + basic feed, and the other groups were fed with normal drinking water + basic feed. In this experiment, the animals were infected only once, and the day of infection was taken as the first day of the formal experiment, and the formal test period was 21 days. During this period, the clinical manifestations and deaths of the chickens were regularly observed and recorded. Online flat farming was adopted, and independent waterers and feed buckets were provided, and food and water were freely available.

[0049] The mortality rate of broiler chickens in each group was recorded 21 days after intervention.

[0050] On the 21st day after intervention, three chickens were randomly selected from each group for autopsy. The trachea and lungs were removed and placed in 4% formaldehyde and 2.5% glutaraldehyde solution for tissue fixation. Part of the lung was taken to detect the number of copies of H9N2 subtype AIV. 100 mg of the collected lung tissue was added to 1 mL TRIzol solution and ground and homogenized. The total RNA extraction method of animal tissue was followed. The H9N2 virus content in chicken lung tissue was determined. The results are shown in Table 2.

[0051] Table 2

[0052] It can be seen from Table 2 that the influenza particles for poultry prepared in Examples 1-3 of the present invention have a good effect of resisting avian influenza virus.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing influenza particles for poultry, characterized in that: The following steps are involved: S1. Pretreatment: Mixing multi-walled carbon nanotubes, concentrated sulfuric acid and concentrated hydrochloric acid, heating to reflux reaction, cooling to room temperature, washing, and then heating and extracting with tetrahydrofuran, and drying to obtain carboxyl-modified carbon nanotubes; S2. Fermentation: Wash Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Mylodon puerariae separately, dry, mix and crush, add to water, sterilize, inoculate with germanium-enriched yeast and selenium-enriched yeast seed liquid, ferment and culture, then add snail enzyme, heat and enzymolysis, filter, add ethanol to the filtrate, precipitate, filter, obtain solid and filtrate, wash the solid, dry, and obtain composite selenium / germanium polysaccharide; recover ethanol from the filtrate, add ammonium sulfate for graded precipitation, filter, wash, and dry, and obtain composite selenium / germanium protein; S3. Coupling: adding citric acid to water, adding NHS and EDC, stirring to activate, adding cecropin and composite selenium / germanium protein, stirring to react, adding bismuth salt, stirring, dialyzing, and freeze-drying the non-permeated solution to obtain bismuth citrate protein; S4. Preparation of polysaccharide composite carbon nanotubes: adding carboxyl modified carbon nanotubes to dimethyl sulfoxide, adding composite selenium / germanium polysaccharide and concentrated sulfuric acid, heating and stirring to react, centrifuging, washing, and drying to obtain polysaccharide composite carbon nanotubes; S5. Preparation of influenza particles for poultry: Add polysaccharide composite carbon nanotubes into water, add NHS and EDC, stir to activate, add bismuth citrate protein, stir to react, spray dry, and obtain influenza particles for poultry.

2. The preparation method according to claim 1, characterized in that: 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 heating reflux reaction time is 7-10 hours, the heating extraction temperature is 75-85°C, the time is 20-24 hours, and the washing is performed to a pH value of 5-6.

3. The preparation method according to claim 1, characterized in that: The mass ratio of Flammulina velutipes, Lentinula edodes, Grifola frondosa, and Mylostomia pueraria in step S2 is 3-5:2-4:1-3:7-10, the inoculation amount of the germanium-enriched yeast and selenium-enriched yeast seed liquid is 2-3 v / v% and 1-2 v / v%, respectively, and the bacterial content of the seed liquid is 10 8 -10 9 cfu / mL, the fermentation culture conditions are 25-30°C, 150-250r / min, fermentation culture 24-36h, the addition amount of the snail enzyme is 0.5-1wt% of the total mass of the system, the temperature of the heating enzymolysis is 35-38°C, and the time is 1-3h.

4. The preparation method according to claim 1, characterized in that: 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 stirring activation time is 20-30min, and the stirring reaction time is 12-15h.

5. The preparation method according to claim 1, characterized in that: In step S4, the mass ratio of the carboxyl modified carbon nanotubes, the composite selenium / germanium polysaccharide and the concentrated sulfuric acid is 10:3-4:0.5-1, and the temperature of the heating and stirring reaction is 130-140° C. and the time is 5-7 hours.

6. The preparation method according to claim 1, characterized in that: In step S5, the mass ratio of the polysaccharide composite carbon nanotubes, NHS, EDC and bismuth citrate protein is 10-12:3-4:2-3:5-7, the stirring activation time is 10-20min, and the stirring reaction time is 7-10h.

7. The preparation method according to claim 1, characterized in that: The specific steps include: S1. Pretreatment: 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 were mixed uniformly, heated to reflux for 7-10 hours, cooled to room temperature, washed to a pH value of 5-6, and then heated to 75-85 ° C with tetrahydrofuran, extracted for 20-24 hours, and dried to obtain carboxyl modified carbon nanotubes; S2 fermentation: 3-5 parts by weight of Flammulina velutipes, 2-4 parts by weight of shiitake mushrooms, 1-3 parts by weight of Grifola frondosa, 7-10 parts by weight of Gexian rice algae were washed, dried, mixed and crushed, added to water, sterilized, inoculated with a bacterial content of 10 8 -10 9 cfu / mL of germanium-rich yeast and selenium-rich yeast seed liquid, inoculation amounts of 2-3v / v% and 1-2v / v%, respectively, at 25-30°C, 150-250r / min, fermentation culture for 24-36h, then adding snail enzyme, the amount of snail enzyme added is 0.5-1wt% of the total mass of the system, heating to 35-38°C, enzymolysis for 1-3h, filtering, adding ethanol to the filtrate, precipitating, filtering, obtaining a solid and a filtrate, washing the solid, drying, and obtaining a composite selenium / germanium polysaccharide; recovering ethanol from the filtrate, adding ammonium sulfate for graded precipitation, filtering, washing, and drying, and obtaining a composite selenium / germanium protein; S3 coupling: 12-15 parts by weight of citric acid was added to water, 3-4 parts by weight of NHS and 3-4 parts by weight of EDC were added, and the mixture was stirred for activation for 20-30 min, 2-3 parts by weight of cecropin and 3-5 parts by weight of composite selenium / germanium protein were added, and the reaction was stirred for 12-15 h, 0.2-0.5 parts by weight of bismuth salt was added, stirred, dialyzed, and the non-permeated liquid was freeze-dried to obtain bismuth citrate protein; S4. Preparation of polysaccharide composite carbon nanotubes: 10 parts by weight of carboxyl modified carbon nanotubes were added to dimethyl sulfoxide, 3-4 parts by weight of composite selenium / germanium polysaccharide and 0.5-1 parts by weight of concentrated sulfuric acid were added, heated to 130-140°C, stirred for reaction for 5-7h, centrifuged, washed, and dried to obtain polysaccharide composite carbon nanotubes; S5. Preparation of influenza particles for poultry: 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 minutes, add 5-7 parts by weight of bismuth citrate protein, stir and react for 7-10 hours, spray dry, and obtain influenza particles for poultry.

8. Influenza particles for poultry prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the influenza granules for poultry as claimed in claim 8 in the preparation of veterinary drugs for treating or assisting in treating avian influenza.

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