IBD egg yolk antibody microspheres and preparation method thereof

Abstract

The present invention belongs to the technical field of yolk antibody preparation, and specifically relates to an IBD yolk antibody microsphere and a preparation method thereof. The method uses polygonatum polysaccharide, IBD yolk antibody and bupleurum essential oil as the inner phase of the microsphere, and polylactic-co-glycolic acid copolymer as the outer phase. The special structure of the microspheres prepared by the present invention is conducive to presenting yolk antibodies and polygonatum polysaccharide to immune-competent cells. Phagocytic cells engulf the microspheres and can destroy the shell structure of the microspheres, releasing yolk antibodies and polygonatum polysaccharide, neutralizing the IBD virus infected by the body, playing a preventive and protective role for chickens not infected with IBD, and quickly controlling the condition of chickens infected with IBD.

Description

Technical Field

[0001] The present invention belongs to the technical field of egg yolk antibody preparation, and particularly relates to IBD egg yolk antibody microspheres and a preparation method thereof. Background Art

[0002] Infectious bursal disease (IBD), also known as infectious bursal disease, is an acute, highly contagious disease caused by the infectious bursal virus (IBD). Due to its sudden onset, short course, high mortality rate, and immunosuppression in chickens, it remains one of the major infectious diseases in the poultry industry, causing significant economic losses. Vaccination is an effective measure to prevent IBD in chickens. By immunizing laying hens, the corresponding yolk antibodies are extracted from the yolks they produce for disease prevention and treatment, and are widely used in clinical practice. Yolk antibodies have many advantages: (1) Large-scale breeding, automatic collection and sorting of eggs are conventional technologies, and the cost of raising chickens is lower than that of mammals. (2) The damage caused by producing antibodies in hens is very small. Only eggs need to be collected, and no blood sampling is required, so the stress stimulation caused to the animals is also very small. (3) Compared with mammalian serum antibodies, egg yolk only contains IgY antibodies, so it is very easy to purify them by precipitation technology. (4) Once purified, IgY can be stored stably at 4°C for several years. (5) Chicken antibodies can also recognize antigenic epitopes that are different from mammalian antibodies, thus forming an antibody repertoire that is different from mammalian antibodies. (6) Compared with IgG, IgY does not activate the mammalian complement system or bind to mammalian Fc receptors in the gastrointestinal tract to cause an inflammatory response.

[0003] Although egg yolk antibodies have significant advantages, current IBD IBD IBD antibodies still have some drawbacks, such as the large injection volume and the potential for stress reactions. Research on IBD IBD IBD antibodies is ongoing. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide an IBD egg yolk antibody microsphere, which can effectively improve the immune efficacy of egg yolk antibodies and reduce the injection volume.

[0005] The technical solution adopted in the present invention is:

[0006] A method for preparing IBD egg yolk antibody microspheres comprises the following steps:

[0007] 1) Mix the Polygonatum sibiricum polysaccharide and IBD egg yolk antibody and stir evenly;

[0008] 2) adding the solution obtained in step 1) to bupleurum essential oil and homogenizing and emulsifying the mixture using a homogenizer to form the inner phase of the microspheres;

[0009] 3) dissolving poly(lactic acid-co-glycolic acid) (PLGA) in dichloromethane to obtain a solution as the external phase of the microspheres, wherein the concentration of the poly(lactic acid-co-glycolic acid) in the solution is 0.5-2%;

[0010] 4) mixing the solution from step 2) and the solution from step 3) in a ratio of 1:3-10 to obtain a mixed solution, adding a surfactant, PEG400, to the mixed solution for homogenization, and performing high shear emulsification at a rate of 10,000 r / min to increase the affinity between the inner and outer phases, so that the active ingredient of the inner phase is encapsulated in the PLGA outer phase;

[0011] 5) The mixture from step 4) was placed in a magnetic stirrer and continuously stirred at 250 r / min at room temperature to obtain microspheres, thereby completely evaporating the dichloromethane in the emulsion droplets. The microspheres were then centrifuged to obtain microspheres. The microspheres were then washed three times with distilled water to remove excess organic solvent to obtain IBD egg yolk antibody microspheres.

[0012] In the step 2), every 10 ml of bupleurum essential oil contains 3-7 g of polygonatum polysaccharide and 5 ml of IBD egg yolk antibody.

[0013] The particle size of the IBD egg yolk antibody microspheres prepared in step 5) is 25-1000 nm.

[0014] The bupleurum essential oil in the present invention is also called bupleurum volatile oil. During preparation, crushed bupleurum is distilled and extracted by steam distillation, and the distillate is collected to obtain the upper volatile oil.

[0015] The IBD egg yolk antibody used in the present invention was prepared by the following method:

[0016] a. Antigen preparation: The bursa of Fabricius and spleen with typical IBD pathological changes were collected, minced, and homogenized at high speed. The supernatant was collected by centrifugation, and 0.3% formaldehyde was added and inactivated in a 37°C incubator for 48 hours.

[0017] b. Antigen emulsification: ① Preparation of the oil phase: Take 94 parts of No. 10 white oil, 6 parts of Tween-80, and 2 parts of aluminum stearate. Mix the aluminum stearate with a small amount of white oil and heat to dissolve. Then, replenish the white oil and add Tween-80. Mix thoroughly to obtain the oil phase. ② Preparation of the aqueous phase: Separately, take 96 parts of the antigen prepared in step a and 4 parts of Tween-80 and mix thoroughly to obtain the aqueous phase. ③ Mix 3 parts of the oil phase and 1 part of the aqueous phase. Add the aqueous phase to a high-speed homogenizer and stir slowly. Slowly add the oil phase. After addition, stir at high speed and add thimerosal to a final concentration of 0.01% to prepare a milky white, viscous IBD oil-emulsion inactivated vaccine.

[0018] c. Chicken immunization: The test hens were vaccinated with the IBD oil-emulsion inactivated vaccine prepared in step b at 18 weeks of age. 1.5 ml was injected subcutaneously into the neck of each chicken. A booster immunization was performed every two weeks for three consecutive immunizations. The antibody titer was then measured every seven days until the titer reached 1:128. Eggs produced by the test chickens were collected.

[0019] d. Crude yolk antibody preparation: Take the yolk of the egg produced in step c, add 6 times the amount of distilled water to the yolk suspension to dilute it, adjust the pH value of the dilution to 5.0-5.2 with hydrochloric acid, centrifuge, take the supernatant, and concentrate the supernatant to obtain the yolk antibody solution;

[0020] e. Purification of egg yolk antibodies: The egg yolk antibody solution in step d is ultrafiltered and purified using an ultrafiltration membrane to obtain refined IBD egg yolk antibodies.

[0021] Compared with the prior art, the beneficial technical effects of the present invention are:

[0022] 1. Microspheres are a drug delivery system that encapsulates active pharmaceutical ingredients within a polymer to achieve a long-term, steady release. Microspheres offer the advantages of improved drug stability and sustained or controlled release. They also prevent drug inactivation in the body, concentrating the drug in the target area, improving efficacy and reducing toxic side effects. While the advantages of microspheres are clear, in actual research, not all drug ingredients can be formulated into microspheres, due to the specific characteristics of the drug. For example, the production of microspheres by combining IBD egg yolk antibodies with Polygonatum sibiricum polysaccharides presents numerous challenges. Because egg yolk antibodies (protein-like substances) and Polygonatum sibiricum polysaccharides are components of traditional Chinese medicine, direct encapsulation using conventional microsphere outer phases consistently fails to meet the required encapsulation efficiency, easily rendering the egg yolk antibodies inactive.

[0023] 2. During the preparation of the microspheres of the present invention, the selected Bupleurum essential oil can effectively encapsulate Polygonatum sibiricum polysaccharides and IBD egg yolk antibodies. Bupleurum essential oil can encapsulate IBD egg yolk antibodies and Polygonatum sibiricum polysaccharides within it, forming a preliminary encapsulation state. As the inner phase continues to encapsulate, the microspheres with the Bupleurum essential oil outer layer can be more ideally encapsulated by PLGA. This is because PLGA is a high-molecular organic compound that effectively encapsulates Bupleurum essential oil. Bupleurum essential oil also has antipyretic properties, which complements its immune-enhancing effects.

[0024] 3. The present invention combines IBD egg yolk antibodies with polygonatum polysaccharide as the main active ingredient in the microsphere inner phase. Polygonatum polysaccharide, due to its many hydroxyl groups in its structure, can provide a stabilizing effect by replacing the hydrogen bonds between protein and water, increasing the stability of egg yolk antibodies. At the same time, it enhances the immune efficacy of IBD egg yolk antibody microspheres and helps activate immune-active cells and cytokines to jointly increase the immune effect. Ultimately, when the microspheres of the present invention are used, a large injection volume of IBD egg yolk antibodies is not required to achieve the ideal prevention and treatment effect. Polygonatum polysaccharide can increase the viscosity of the water phase, improve the stability of colostrum (W / O), increase the resistance of the drug to the external water phase, and improve the encapsulation efficiency of the microspheres to a certain extent.

[0025] 4. The average particle size of the microspheres produced by the present invention is 25-1000 nm. The unique structure of these microspheres facilitates the presentation of egg yolk antibodies and Polygonatum sibiricum polysaccharides to immune-competent cells. Phagocytic cells engulf the microspheres and destroy the microsphere shell structure, releasing egg yolk antibodies and Polygonatum sibiricum polysaccharides, which neutralize the IBD virus infecting the body. This provides a preventive and protective effect for chickens not infected with IBD and rapidly controls the condition of chickens infected with IBD. At the same injection volume, the egg yolk antibody microspheres of the present invention are more effective than ordinary egg yolk antibodies. The egg yolk antibody microspheres of the present invention achieve a lower injection volume for equivalent immune effects, and as the injection volume is reduced, the stress response is also reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result diagram obtained by laser particle size analyzer. DETAILED DESCRIPTION

[0027] The specific embodiments of the present invention are described below with reference to the examples. However, the following examples are only used to illustrate the present invention in detail and are not intended to limit the scope of the present invention in any way.

[0028] Example 1:

[0029] A method for preparing IBD egg yolk antibody microspheres comprises the following steps:

[0030] 1) Mix 5g Polygonatum polysaccharide and 5ml IBD egg yolk antibody and stir evenly;

[0031] 2) Add the solution obtained in step 1) to 10 ml of bupleurum essential oil and homogenize and emulsify using a homogenizer to serve as the inner phase of the microspheres;

[0032] 3) Dissolve 1 g of poly(lactic-co-glycolic acid) (PLGA) in 75 ml of dichloromethane to obtain a solution as the external phase of the microspheres;

[0033] 4) The solution from step 2) was mixed with the solution from step 3) to obtain a mixed solution (internal and external phase volume ratio of 1:5). 9 ml of the surfactant PEG400 was added to the mixed solution, and high shear emulsification was performed at a rate of 10,000 r / min to increase the affinity between the internal and external phases, so that the active ingredients of the internal phase were encapsulated in the PLGA external phase.

[0034] 5) The mixture from step 4) was placed in a magnetic stirrer and continuously stirred at 250 r / min at room temperature to obtain microspheres, thereby completely evaporating the dichloromethane in the emulsion droplets. The microspheres were then centrifuged to obtain microspheres. The microspheres were then washed three times with distilled water to remove excess organic solvent to obtain IBD egg yolk antibody microspheres.

[0035] Example 2:

[0036] A method for preparing IBD egg yolk antibody microspheres comprises the following steps:

[0037] 1) Mix 7g Polygonatum polysaccharide and 5ml IBD egg yolk antibody and stir evenly;

[0038] 2) Add the solution obtained in step 1) to 10 ml of bupleurum essential oil and homogenize and emulsify using a homogenizer to serve as the inner phase of the microspheres;

[0039] 3) Dissolve 1 g of poly(lactic-co-glycolic acid) (PLGA) in 60 ml of dichloromethane to obtain a solution as the external phase of the microspheres;

[0040] 4) The solution from step 2) was mixed with the solution from step 3) to obtain a mixed solution (internal and external phase volume ratio of 1:4). 7 ml of the surfactant PEG400 was added to the mixed solution and homogenized. High shear emulsification was performed at a rate of 10,000 r / min to increase the affinity between the internal and external phases, so that the active ingredients of the internal phase were encapsulated in the PLGA external phase.

[0041] 5) The mixture from step 4) was placed in a magnetic stirrer and continuously stirred at 250 r / min at room temperature to obtain microspheres, thereby completely evaporating the dichloromethane in the emulsion droplets. The microspheres were then centrifuged to obtain microspheres. The microspheres were then washed three times with distilled water to remove excess organic solvent to obtain IBD egg yolk antibody microspheres.

[0042] Example 3:

[0043] A method for preparing IBD egg yolk antibody microspheres comprises the following steps:

[0044] 1) Mix 3g Polygonatum polysaccharide and 5ml IBD egg yolk antibody and stir evenly;

[0045] 2) Add the solution obtained in step 1) to 10 ml of bupleurum essential oil and homogenize and emulsify using a homogenizer to serve as the inner phase of the microspheres;

[0046] 3) Dissolve 1 g of poly(lactic-co-glycolic acid) (PLGA) in 120 ml of dichloromethane to obtain a solution as the external phase of the microspheres;

[0047] 4) The solution from step 2) was mixed with the solution from step 3) to obtain a mixed solution (internal and external phase volume ratio of 1:8). 13 ml of the surfactant PEG400 was added to the mixed solution and homogenized. High shear emulsification was performed at a rate of 10,000 r / min to increase the affinity between the internal and external phases, so that the active ingredients of the internal phase were encapsulated in the PLGA external phase.

[0048] 5) The mixture from step 4) was placed in a magnetic stirrer and continuously stirred at 250 r / min at room temperature to obtain microspheres, thereby completely evaporating the dichloromethane in the emulsion droplets. The microspheres were then centrifuged to obtain microspheres. The microspheres were then washed three times with distilled water to remove excess organic solvent to obtain IBD egg yolk antibody microspheres.

[0049] The properties of the IBD egg yolk antibody microspheres obtained in Examples 1-3 are shown in the following table:

[0050]

[0051] Example 4: Investigation of Polygonatum sibiricum Polysaccharide

[0052] The addition amounts of Polygonatum sibiricum polysaccharide in Example 1 were adjusted to 0.5 g, 1 g, and 10 g, respectively, and sucrose 0.5 g, 1 g, 3 g, and 5 g was used as a control. Other conditions remained unchanged. The results of the investigation are shown in the following table:

[0053]

[0054] The dosage of Polygonatum sibiricum polysaccharide and the choice of sucrose had little effect on the particle size of the microspheres, but had a significant impact on the encapsulation efficiency of the microspheres. The microspheres prepared with Polygonatum sibiricum polysaccharide had a significantly higher encapsulation efficiency than those prepared with sucrose. However, as the amount of Polygonatum sibiricum polysaccharide decreased, the encapsulation efficiency decreased. When the amount of Polygonatum sibiricum polysaccharide was increased to 10 parts, due to its high concentration, the Polygonatum sibiricum polysaccharide was not completely dissolved, and the presence of Polygonatum sibiricum polysaccharide particles in the microspheres resulted in larger microspheres, a sudden release of egg yolk antibodies, and a low encapsulation efficiency. As a traditional Chinese medicine extract, Polygonatum sibiricum polysaccharide has a more complex and diverse composition. When preparing IBD egg yolk antibody microspheres, it can effectively prevent the diffusion of active ingredients and improve the encapsulation efficiency.

[0055] Example 5: Investigation of Bupleurum essential oil

[0056] The bupleurum essential oil in Example 1 was adjusted to chitosan and houttuynia cordata essential oil (prepared using the same method as bupleurum essential oil), and the morphology, particle size, and encapsulation efficiency of the microspheres were investigated.

[0057]

[0058] When the present invention uses chitosan, a commonly used encapsulating material, the resulting microsphere morphology, particle size, and encapsulation efficiency all fail to reach ideal levels. Continuous research has revealed that this is because chitosan must be dissolved in an acidic solution during preparation, and the acidic substance causes the egg yolk antibody to denature and form clumps, resulting in poor morphology, sudden release, and low encapsulation efficiency. Using Houttuynia cordata essential oil as the internal phase carrier, while the microsphere morphology and particle size meet the requirements, the relatively thin Houttuynia cordata essential oil results in poor encapsulation, resulting in a microsphere encapsulation efficiency of only 68.6%. However, the patent's use of Bupleurum chinense essential oil makes it easier to form a W / O structure when preparing the microsphere internal phase, improving the egg yolk antibody encapsulation efficiency.

[0059] Example 6: Investigation of PLGA

[0060] The amount of polylactic acid-glycolic acid copolymer in Example 1 was adjusted to 0.1 g, 0.5 g, and 2 g, and the particle size and encapsulation efficiency of the microspheres were investigated.

[0061]

[0062] Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable functional polymer organic compound formed by the random polymerization of two monomers, lactic acid and glycolic acid. It has good biocompatibility, is non-toxic, and has excellent capsule- and film-forming properties, making it an ideal framework material for preparing microspheres. Results showed that when the amount of PLGA was too low, the microspheres tended to collapse during curing and did not form into spheres properly. When the amount of PLGA was too high, the microspheres formed had more irregular crystals and were difficult to form into spheres.

[0063] Example 7: Investigation of internal and external phases

[0064] Bupleurum essential oil wraps egg yolk antibodies and Polygonatum polysaccharide inside it, serving as the inner phase of the microspheres, while PLGA is the outer phase of the microspheres. During the preparation of the microspheres, the inner phase needs to be fully wrapped by the outer phase within its skeleton.

[0065] The ratio of the internal and external phases in Example 1 was adjusted to 1:1, 1:2, 1:3, 1:4, 1:8, and 1:10, and the appearance, particle size, and encapsulation efficiency of the microspheres were investigated.

[0066]

[0067] The experimental results show that as the external phase ratio continues to increase, the appearance of the microspheres continues to optimize. When the internal and external phase ratio is 1:5, the prepared microspheres are smooth, uniform in size, with an average particle size of 582.3nm and an encapsulation efficiency of 90.5%. When the external phase ratio continues to increase, the appearance and encapsulation efficiency of the microspheres are not much different from those at 1:5, but the particle size continues to increase. Therefore, the preferred internal and external phase ratio is 1:5.

[0068] Effect example 1:

[0069] 1) Microsphere morphology and particle size evaluation results

[0070] The appearance and particle size of the egg yolk antibody microspheres prepared in Example 1 were analyzed and evaluated using an optical microscope and a laser particle size analyzer, respectively.

[0071] The surface of the microspheres was smooth and the particles were uniform under the optical microscope. The results of the laser particle size analyzer showed that the D90 particle size of the prepared microspheres was 582.3nm. Figure 1 .

[0072] 2) Drug loading, encapsulation efficiency and yield of microspheres

[0073] Accurately weigh 1 g of the egg yolk antibody microspheres prepared in Example 1 and dissolve them in 5 ml of dichloromethane. Once completely dissolved, add 5 ml of 5% acetic acid and vortex to mix thoroughly. Then, centrifuge at 4000 rpm for 10 minutes to remove the carrier material. Extract three times with distilled water, combine the supernatants, and make the volume up to 10 ml. Determine the absorbance of Polygonatum sibiricum polysaccharide using UV-spectrophotometry, and calculate its content. Calculate the drug loading, encapsulation efficiency, and yield of the microspheres using the following formulas.

[0074] Drug loading = drug content in microspheres / microsphere mass × 100%

[0075] Encapsulation efficiency = actual drug loading / theoretical drug loading × 100%

[0076] Yield = total weight of microspheres after freeze-drying / actual feed amount × 100%

[0077] The present invention uses polygonatum polysaccharide as a content investigation index, and it is measured that the drug loading capacity of the prepared microspheres is more than 86%, the encapsulation efficiency is 90.5% on average, and the microsphere yield is 75%.

[0078] Effect Example 2: IBD Yolk Antibody Microsphere Safety Test

[0079] (1) Safety inspection test methods

[0080] Ten mice weighing 18-22g were subcutaneously injected with 0.5ml of this product. Ten 14-day-old SPF chicks were subcutaneously injected with 1ml of this product. The animals were observed for 14 consecutive days.

[0081] (2) Safety inspection test results

[0082] Safety test results showed that the experimental mice and chicks were all healthy, with normal mental state, behavioral activities and diet; respiration and body temperature were within normal range; there were no systemic reactions; and there were no other adverse reactions at the injection site.

[0083] Effect Example 3: IBD Yolk Antibody Microsphere Efficacy Test

[0084] (1) Effectiveness test method

[0085] Sixty four-week-old SPF chickens were randomly divided into four groups, 15 each. Group 1 served as a healthy control group, receiving no treatment and housed individually. Each chicken in Groups 2, 3, and 4 was inoculated with 0.1 ml (100 LD50) of IBDV SNJ93 virus through the eyes and nose. Twenty-four hours later, each chicken in Group 2 received a subcutaneous injection of 0.5 ml of the egg yolk antibody microspheres from Example 1, each chicken in Group 3 received a subcutaneous injection of 0.5 ml of a standard IBD egg yolk antibody (Tianjin Ruipu Biotechnology Co., Ltd.), and each chicken in Group 4 received 0.5 ml of normal saline. Morbidity and mortality in each group were observed until day 10.

[0086] (2) Efficacy test results

[0087] The results of the efficacy test showed that all the experimental chickens in Group 1 were alive and well. The experimental chickens in Group 4 began to become ill on the second day after the challenge, and all of them were ill on the third day, and some died. Five chickens had died on the fifth day after the challenge, and by the seventh day, only two experimental chickens in Group 4 were still alive. The experimental chickens in Group 2 began to become ill on the second day after the challenge, but after being injected with the yolk antibody microspheres of Example 1, their condition improved. On the third day after the challenge, except for one chicken that died, the overall condition of the flock began to improve, and by the sixth day, the flock had basically recovered. The experimental chickens in Group 3 began to become ill on the second day after the challenge. After being treated with ordinary yolk antibodies, their condition improved, but some chickens still died. As of the third day after the challenge, four chickens had died. The test results show that, under the same injection volume, compared with ordinary IBD egg yolk antibodies, the IBD egg yolk antibody microspheres prepared by the present invention can significantly improve the cure rate of bursal disease virus infection and reduce the mortality rate of diseased chickens.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and are not limiting. Other modifications or equivalent substitutions made to the technical solution of the present invention by ordinary technicians in this field should be included in the scope of the claims of the present invention as long as they do not depart from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing IBD egg yolk antibody microspheres, characterized in that: The following steps are involved: 1) Mix Polygonatum sibiricum polysaccharide and IBD egg yolk antibody; 2) adding the material obtained in step 1) to bupleurum essential oil, wherein each 10 ml of bupleurum essential oil contains 3-7 g of polygonatum polysaccharide and 5 ml of IBD egg yolk antibody, and mixing them uniformly to form the inner phase of the microspheres; when preparing the bupleurum essential oil, steam distillation is performed on the crushed bupleurum, and the distillate is collected to obtain the upper layer of volatile oil; 3) dissolving poly(lactic acid-co-glycolic acid) in dichloromethane to obtain a solution as the outer phase of the microspheres, wherein the mass volume concentration of the poly(lactic acid-co-glycolic acid) in the solution is 0.5-2%; 4) mixing the solution from step 2) and the solution from step 3) at a volume ratio of 1:3-10 to obtain a mixed solution, adding a surfactant PEG400 to the mixed solution and homogenizing the mixture so that the active ingredient of the inner phase is encapsulated in the poly(lactic acid-co-glycolic acid) outer phase; 5) The mixture of step 4) is stirred to obtain microspheres, which are then centrifuged and washed to obtain IBD egg yolk antibody microspheres.

2. The preparation method according to claim 1, wherein: The particle size of the IBD egg yolk antibody microspheres prepared in step 5) is 25-1000 nm.

3. IBD egg yolk antibody microspheres prepared by the preparation method according to claim 1.

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