Nanoparticles for preventing and treating pullorum disease as well as preparation method and application of nanoparticles

Abstract

The invention is applicable to the technical field of biological medicines, and provides a nanoparticle for preventing and treating pullorum disease as well as a preparation method and application of the nanoparticle. The preparation method comprises the following steps: adding zein and rutin into ethanol, uniformly stirring to obtain a zein-rutin nano-particle dispersion liquid, dropwise adding the zein-rutin nano-particle dispersion liquid into a soybean polysaccharide (ssps) solution, uniformly stirring to obtain an ssps-zein-rutin dispersion liquid, and freeze-drying to obtain the nano-particles. The nanoparticles can be used for preventing and treating pullorum disease, can relieve clinical symptoms and dissection symptoms of chicks with pullorum disease, and can improve the weight of the chicks with pullorum disease. Encapsulation materials are easy to obtain and food-borne, raw materials are easy to obtain, the cost is low, the effect is good, processing steps are simple, and potential application value is achieved in the aspect of preventing and treating the pullorum disease. Meanwhile, rutin is packaged through the nano-carrier, the rutin utilization rate is increased, the prevention and treatment effect is better, and a new means is provided for achieving the prevention and treatment strategy of reducing resistance and replacing resistance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a nanoparticle for preventing and treating pullorum disease and a preparation method and application thereof. BACKGROUND

[0002] Pullorum disease (PD) is an acute and septic infectious disease of poultry caused by salmonella pullorum (S. Pullorum) infection, which mainly prevails in chickens and turkeys. Its transmission modes include horizontal transmission and vertical transmission: horizontal transmission can be transmitted through chickens eating feed, drinking water contaminated by S. Pullorum or directly contacting with sick chickens; vertical transmission is that hens carry bacteria through egg production to pass to the offspring chicks. Chicken pullorum not only has various transmission modes, but also has susceptibility to chickens of all ages, among which chicks are more susceptible to S. Pullorum, and after infection, they often lead to poor growth and development, and reduce their potential production performance. With the increasing demand for poultry products in China, the scale of chicken farming is expanding, and chicken pullorum is showing a high incidence trend, which causes economic losses to poultry farming and restricts the healthy and sustainable development of poultry farming in China.

[0003] At present, antibiotics are often used to prevent and treat chicken pullorum. In order to achieve the national strategy of reducing antibiotics, it is urgent to find a replacement for antibiotics. Rutin, as a flavonoid compound extracted from plants, is a secondary metabolite widely existing in plants in nature, and has various biological activities and pharmacological effects. Rutin has been widely used in clinical practice as a traditional Chinese herbal medicine due to its strong antioxidant properties and low toxicity and side effects, and is considered for the treatment of chicken pullorum. However, rutin has defects such as low solubility and poor permeability, which leads to poor absorption and low bioavailability after oral administration, limiting its application effect.

[0004] With the development of nanomaterials and nanomedicine, the application of nanomaterials in drug delivery provides a new idea for enhancing the therapeutic effect of drugs. The encapsulation of drugs by nanomaterials can greatly improve the bioavailability of drugs. Among them, corn alcohol-soluble protein nanoparticles have many advantages such as natural source, easy preparation under mild conditions, avoidance of using synthetic and toxic reagents, high possibility of large-scale industrial production, biodegradability and good biocompatibility, and the protein molecules have strong affinity to bioactive substances, so they have high encapsulation efficiency and loading capacity. However, corn alcohol-soluble protein nanoparticles have the problem of low solubility, which easily leads to aggregation or precipitation, limiting their application in colloidal systems. Soluble soybean polysaccharide, as a highly water-soluble, low-viscosity, heat-stable and negatively charged polysaccharide, can be used to stabilize corn alcohol-soluble protein nanoparticles.

[0005] In summary, the existing prevention and control means have obvious deficiencies, based on this, exploring the nano-particle drug for preventing and treating chicken white diarrhea has important guiding significance for the research of chicken white diarrhea prevention and treatment and the development of nano-particle drug, and the present application proposes a kind of nano-particle for preventing and treating chicken white diarrhea and its preparation method and application. SUMMARY

[0006] The present application aims to provide a kind of nano-particle for preventing and treating chicken white diarrhea and its preparation method and application, to solve the problems proposed in the above background art.

[0007] The object of the present application is realized by the following technical solutions: A kind of nano-particle for preventing and treating chicken white diarrhea, which is composed of corn alcohol-soluble protein, soybean polysaccharide and rutin, wherein the mass ratio of rutin to corn alcohol-soluble protein is 1:10-1:40, and the mass ratio of corn alcohol-soluble protein to soybean polysaccharide is 1:2-2:1.

[0008] A preparation method of the nano-particle described above, comprising the following steps: Step 1: corn alcohol-soluble protein is added to a 75% ethanol solution and stirred to disperse uniformly, then rutin is added and stirred to obtain a zein-rutin nano-particle dispersion; Step 2: the zein-rutin nano-particle dispersion is added dropwise to a soybean polysaccharide solution and stirred to prepare an ssps-zein-rutin nano-particle dispersion, and the nano-particle is obtained after freeze-drying.

[0009] Further, in steps 1 and 2, the stirring speed is 800 rpm, and the stirring time is 30 min.

[0010] A composition comprising the nano-particle described above and a pharmaceutically or feed acceptable carrier.

[0011] The application of the nano-particle or composition described above in the preparation of a product for preventing and treating chicken white diarrhea.

[0012] Further, the application includes preparing a product for alleviating the clinical symptoms of chicken white diarrhea chicks.

[0013] Further, the application includes preparing a product for alleviating the necropsy symptoms of chicken white diarrhea chicks.

[0014] Further, the application includes preparing a product for increasing the body weight of chicken white diarrhea chicks.

[0015] According to the application described above, the product is a drug, a feed additive or a feed.

[0016] Compared with the prior art, the present application has the following beneficial effects: The nanoparticles prepared by encapsulating rutin using zein and soybean polysaccharide as nanoparticle carriers can be used to prevent and treat pullorum, reduce the clinical symptoms and autopsy symptoms of pullorum-infected chicks, and increase the weight of pullorum-infected chicks. The encapsulation material used in the nanoparticles is easy to obtain and has food-borne properties, and is practical when feeding chicks. The raw materials are easy to obtain, low in cost, and effective, and the processing steps are simple, and the nanoparticles have potential application value in preventing and treating pullorum. At the same time, the nanocarrier encapsulation of rutin solves the problem that rutin is not easily absorbed in the body and has poor bioavailability. Compared with pure rutin and rutin nanoparticles without soybean polysaccharides, the nanoparticles have a better prevention and treatment effect, providing a new means for achieving a prevention and treatment strategy of reducing and replacing antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The average weight change of chicks in each group from 2 days to 17 days of age.

[0018] Figure 2 is the average clinical symptom score of each group of chicks; A is the clinical symptom score at 12 days of age; B is the clinical symptom score at 17 days of age ( ** Indicates that there is a significant difference compared with the model group. ## There was a significant difference compared with the Example 1 group, P < 0.01).

[0019] Figure 3 is the average autopsy symptom score of each group of chicks; A is the autopsy symptom score at 12 days of age; B is the autopsy symptom score at 17 days of age ( ** Indicates that there is a significant difference compared with the model group. ## There was a significant difference compared with the Example 1 group, P < 0.01). DETAILED DESCRIPTION

[0020] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0021] The specific implementation of the present invention is described in detail below with reference to specific embodiments. In the present invention, the reagents used without indicating the manufacturer are all conventional products that can be purchased from the market. Example

[0022] (1) Zein was added to a 75% ethanol solution and stirred to disperse evenly (the mass fraction of zein was 2%). Rutin was then added to the solution (rutin:zein = 1:20) and stirred at 800 rpm for 30 min to obtain a zein-rutin nanoparticle dispersion.

[0023] (2) The zein-rutin nanoparticle dispersion liquid was added dropwise into a soybean polysaccharide (ssps) solution (zein: ssps = 2:1), stirred at 800 rpm for 30 min, to prepare an ssps-zein-rutin nanoparticle dispersion liquid, and the rutin nanoparticles were obtained after the dispersion liquid was frozen at -80°C for 24 h.

[0024] Example 2: Compared with Example 1, in this example, rutin: zein = 1:10, and the rest of the operations are the same as those in Example 1.

[0025] Example 3: Compared with Example 1, in this example, rutin: zein = 1:40, and the rest of the operations are the same as those in Example 1.

[0026] Example 4: Compared with Example 1, in this example, zein: ssps = 1:1, and the rest of the operations are the same as those in Example 1.

[0027] Example 5: Compared with Example 1, in this example, zein: ssps = 1:2, and the rest of the operations are the same as those in Example 1.

[0028] Comparative Example 1: Compared with Example 1, this comparative example is pure rutin without zein and ssps.

[0029] The preparation procedure is as follows: 10 mg of rutin is dissolved in 20 ml of distilled water.

[0030] Comparative Example 2: Compared with Example 1, this comparative example is rutin nanoparticles without ssps.

[0031] The preparation procedure is as follows: zein is added into a 75% ethanol solution and stirred to disperse uniformly (mass fraction of 2%), and then rutin is added into the solution (rutin: zein = 1:20), stirred at 800 rpm for 30 min, to obtain a zein-rutin nanoparticle dispersion liquid, and the rutin nanoparticles are obtained after the dispersion liquid is frozen at -80°C for 24 h.

[0032] Performance test: chicken white diarrhea model establishment and efficacy test; Male 1-day-old Hyline Brown chicks were adaptively fed for one day and then randomly divided into 9 groups, 20 chicks in each group. The grouping conditions are as follows: Blank group: intragastrically administered with normal saline; Model group: intragastrically administered with normal saline; Example 1~5 groups (hereinafter referred to as example groups): 30 mg of nanoparticles prepared in Examples 1~5 were dissolved in 1 ml of distilled water, respectively, and the intragastric dose was 1 mL per chick per day; Comparative Example 1 group: administrated with rutin prepared in Comparative Example 1, with a dose of 1 mL / animal / day; Comparative Example Group 2: 30 mg of the nanoparticles prepared in Comparative Example Group 2 were dissolved in 1 mL of distilled water, and the oral dose was 1 mL / animal / day.

[0033] Each group was given the drug once a day for 5 consecutive days. At 7 days old, the chicks except the blank group were challenged with bacteria to establish the pullorum model: Salmonella pullorum was inoculated into sterile nutrient broth for enrichment and prepared into 10 9 cfu / mL concentration of bacterial solution, and all chickens except the blank group were gavaged with 0.2 mL of bacterial solution to establish the pullorum disease model.

[0034] The weight, clinical symptom score, autopsy symptom score and other data of each group of chicks were recorded and analyzed.

[0035] (1) Weight changes; Record the average weight changes of chicks from 2 days to 17 days of age in each group. Figure 1 As shown in the figure, before infection with Salmonella pullorum, the weight of the chicks in each group was similar. After infection with Salmonella pullorum, the average weight gain of the model group and the control group was slow, while the weight of the chicks in the example group grew faster, with a growth rate close to that of the blank group. This indicates that the example preparation has a significant effect on preventing and treating the effects of pullorum on chick growth.

[0036] (2) Clinical symptom score; Clinical symptom scores were performed on chicks in each group 5 days after infection (12 days old) and 10 days after infection (17 days old). The scoring criteria are shown in Table 1. The average clinical symptom scores of chicks in each group were calculated. Figure 2 As shown in Figures A and B. It can be seen that at 12 and 17 days of age, the scores of the Example group were significantly lower than those of the Model group and the Comparative Example group, while the Comparative Example group score was not significantly lower than that of the Model group. This indicates that the Example group preparation has a good preventive and therapeutic effect on the clinical symptoms of pullorum in chicks.

[0037] Table 1 Clinical symptom scoring table

[0038] (3) autopsy symptom score; 5 days after infection (12 days old) and 10 days after infection (17 days old), 5 chicks were sampled from each group for autopsy symptom scoring. The scoring criteria are shown in Table 2. The average autopsy symptom score of each group of chicks was calculated. Figure 3 As shown in Figures A and B. It can be seen that at 12 and 17 days of age, the scores of the Example group were significantly lower than those of the Model group and the Comparative Example group, while the Comparative Example group score was not significantly lower than that of the Model group. This indicates that the Example group preparation has a good effect on preventing and treating symptoms caused by pullorum in chicks.

[0039] Table 2 Autopsy symptom score table

[0040] The above experimental results prove that the nanoparticles prepared by encapsulating rutin monomers with zein and soybean polysaccharide as nanoparticle carriers have good prevention and treatment effect on chicken white diarrhea. The effect is better than that of rutin monomers on chicken white diarrhea, and better than that of nanoparticles prepared by encapsulating rutin monomers with zein as nanoparticle carrier on chicken white diarrhea. A new strategy with effectiveness and safety is provided for the prevention and treatment of chicken white diarrhea, which is expected to promote the development of green prevention and control technology of poultry diseases.

[0041] The above is only the preferred embodiment of the present application, it should be pointed out that for those skilled in the art, without departing from the concept of the present application, can make a number of deformation and improvement, these should be considered as the protection scope of the present application, these will not affect the effect and practicality of the patent of the present application.

Claims

1. A nanoparticle for preventing and treating pullorum, characterized in that: The invention is composed of zein, soybean polysaccharide and rutin, wherein the mass ratio of rutin to zein is 1:10-1:40, and the mass ratio of zein to soybean polysaccharide is 1:2-2:

1.

2. A method for preparing nanoparticles according to claim 1, characterized in that: The following steps are involved: Step 1: Add zein to a 75% ethanol solution and stir to disperse it evenly, then add rutin and stir to obtain a zein-rutin nanoparticle dispersion; Step 2: adding the zein-rutin nanoparticle dispersion dropwise into the soybean polysaccharide solution and stirring to prepare the ssps-zein-rutin nanoparticle dispersion, and then freeze-drying to obtain the nanoparticles.

3. The preparation method according to claim 2, characterized in that In step 1 and step 2, the stirring speed is 800 rpm and the stirring time is 30 min.

4. A composition, characterized in that The invention comprises the nanoparticles according to claim 1 and a pharmaceutically or feed-acceptable carrier.

5. Use of the nanoparticles according to claim 1 or the composition according to claim 4 in preparing a product for preventing and treating pullorum.

6. The use according to claim 5, characterized in that The application includes preparing a product for alleviating clinical symptoms of pullorum in chickens.

7. The use according to claim 5, characterized in that The application includes preparing a product that alleviates the post-mortem symptoms of pullorum in chicks.

8. The use according to claim 5, characterized in that The application includes preparing a product for increasing the body weight of pullorum chickens.

9. The use according to any one of claims 6 to 8, characterized in that The product is a medicine, a feed additive or a feed.

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