CN103349782B

CN103349782B - The preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation

Info

Publication number: CN103349782B
Application number: CN201310238769.2A
Authority: CN
Inventors: 霍乃蕊; 张新荣; 巩红霞; 李宝霞; 李芳�; 王珺; 任志远; 韩克光; 郑明学; 古少鹏
Original assignee: Shanxi Agricultural University
Current assignee: Shanxi Agricultural University
Priority date: 2013-06-17
Filing date: 2013-06-17
Publication date: 2016-05-04
Anticipated expiration: 2033-06-17
Also published as: CN103349782A

Abstract

The present invention relates to a kind for the treatment of and preventing coccidiosis of chicken preparation, be specially a kind of preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation. Solve the problem of chicken coccidiasis prevention and treatment. The preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation, step is: preparation, separation and the purification of high immunity yolk antibody (IgY), then make microcapsules, the reaction condition that described microcapsules are prepared microcapsules is: chitosan concentration 0.05-0.8%(w/v), CaCl₂Concentration 0.5-1.5%(w/v), sodium alginate concentration 1.0-3.0%(w/v), IgY and sodium alginate mass ratio be 1-4:8. IgY microcapsules prepared by technical solution of the present invention are a kind of prophylactic agent, especially a kind of medicine. Microcapsules prepared by the present invention provide good example for antibiotic alternative medicine, and determined technique also can be used to prepare IgY or the IgG microcapsules of other pathogenic microorganisms, have promotional value, and are conducive to the sound development of green and organic poultry husbandry.

Description

The preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation

Technical field

The present invention relates to a kind for the treatment of and preventing coccidiosis of chicken preparation, be specially a kind of preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation.

Background technology

Chicken coccidiasis (coccidiosis) is to parasitize by one or more of pushing up in the Eimeria of answering device door Eimeria a kind of parasitic protozoa disease that on chicken intestinal mucosa, intracutaneous causes. Coccidiosis is that one generally occurs, and endangers very serious disease. The Eimeria parasitizing in chicken body it is reported 9 kinds more than, wherein draw attention as pathogen have Eimeria tenella (E.tenella), heap type Eimeria (E.acervulina), Eimeria maxima (E.maxima), Eimeria Necatrix (E.necatrix), E.brunetti (E.brunetti) five kinds. Eimeria tenella is the one the most common in chicken coccidia, pathogenicity is the strongest, mainly encroaches on chicken caecum and near zone thereof. 15～30 Japanese instar chickling incidences of disease are the highest, can reach 50%～70%, and the death rate is 20%～30%, when serious up to more than 80%; The Chicks ' Growth of the resistance to mistake of falling ill is slack-off, and Adult Chicken is seldom morbidity, and minority is carrier, but larger on Egg Production of Laying Hens impact. Often be only according to Allen report the economic loss that global-worm illness causes all over the world and just reach 8,000,000,000 dollars. Along with the continuous appearance of the growing of intensive poultry husbandry and the strain of drug resistance worm, the harm of this disease will be on the rise, and therefore, the disease of how preventing effecting a permanent cure is one of important topic of current poultry husbandry.

Present stage chicken coccidiasis remain taking chemicals as main means of prevention, but medical treatment is faced with the puzzlement of three day by day serious large problems: the appearance speed of drug-resistant worm plant has exceeded the development speed of new drug, and the generation of drug-resistant worm plant also can cause the unmanageable vicious circle of multiple coccidia mixed infection; Medicament residue serious harm human health and affect poultry product export; Chemicals is prevented and treated global-worm illness immunity is produced and suppressed. And existing anticoccidial drug is mainly in the early stage or mid-term (vegetative propagation phase) of coccidium infection, but there is the generative propagation phase of coccidia after the clinical symptoms such as bloody stool and injury-resistance is very micro-or without effect to infecting. Therefore, once chicken occurs clinical symptoms after infecting coccidia, anticoccidial drug used just produces little effect at present, thereby makes the death rate of chicken coccidiasis high, brings huge economic loss to poultry husbandry. Moreover use these medicine blood concentrations often to occur " peak valley " phenomenon, and strong toxicity, Animal stress is large, use inconvenience; In view of the above-mentioned defect of chemical prevention, global-worm illness immunoprophylaxis and treatment means have been proposed, hyper-immune serum and high-immunity yolk can carry out specific binding with the coccidia in each stage, its specificity is removed or killed, or its pathogenicity is weakened, but because of serum limited amount, cost is high and be prone to allergic reaction etc. and be difficult for being widely used.

Therefore research direction of the present invention selected wide material sources, cost low, study without the high immunity yolk antibody (IgY) of allergic reaction and nutritious effect, taking immunization therapy and immune defense as means, control the generation of chicken coccidiasis and be used for the treatment of.E.tenellaSpecial yolk antibody (IgY) can be combined in vitroE.tenellaOn the tegumental membrane antigen of zygoblast, have and reduce the pathogenicity of zygoblast and dissolve zygoblast effect; Experiment is also foundE.tenellaZygoblast and special yolk antibody reduce gradually with its quantity of prolongation of action time in vitro, effect 24h zygoblast can reduce 47.6%, and with the variation of action time, zygoblast becomes Pear-Shaped, fusiformis, pyriform and little shaft-like etc. from original banana-shaped or long spindle, and fracture, soft edge are unclear. Our result of study also shows: IgY starts to come across in caecum after the oral 3h of chicken, 6h peaks, and what antibody titer was 7.75, IgY tire has reduced by 3 titres compared with stoste, after 6h, the concentration of IgY declines gradually, in the time of oral rear 10h, is reduced to 3.00. In view of above-mentioned result of study, so be necessary to research and develop a kind of oral slow-releasing preparation, while making Yolk antibody targeting site, reduce the loss of antibody titer, and can make IgY with enough concentration at target site (chicken caecum and near zone thereof) long-acting, this is also one of main purpose of this research. Said preparation can reduce the loss of IgY titre, improves tire 1 ~ 2 titre of non-slow release IgY in enteron aisle, thereby reduces the use amount of Yolk antibody and strengthen the performance of drug effect.

Can improve medicine pharmacokinetic characteristic in vivo by slow release method, improve bioavilability, the duration of prolong drug valid density, reduce administration number of times, what heighten the effect of a treatment, alleviate fowl group stress. Sustained release agent is a kind of modern delivery system, can transmit more enduringly medicine, can continue release to reach the preparation of long-acting in the long period, thereby can reduce administration number of times and dosage after medication. It is the most frequently used although oral and a kind of administering mode more conveniently, but medicine discharges the impact that is often subject to intestines and stomach rate of evacuation in actual applications, oral bioadhesive microspheres is compared with general sustained release preparation, expand and GI contact area, and owing to having used bioadhesive material, microballoon adheres to gastrointestinal tract mucous upper for a long time, has extended drug release time, is conducive to release and the absorption of medicine. If time and fund allow, this research also will be attempted a kind of bioadhesive material of screening, makeE.tenellaSpecific IgY sustained release agent can be attached in chicken caecum and near zone continuous action.

In sum, chickenE.tenellaOnly in enteron aisle existence, but specific IgY as targeted drug only the enteron aisle concentration worm of could killing of remaining valid. But when the strong acidic environment of IgY process stomach, its activity is easily destroyed. For this reason, utilize specificity anti-E.tenellaYolk antibody (IgY), research can be specifically forE.tenellaThe polypide of development in different stages, but the target long-acting biological Prevention Technique of Eimeria tenella in chicken body, killed and medicine seems very urgent and important.

Summary of the invention

The present invention, in order to solve the problem of chicken coccidiasis prevention and treatment, provides a kind of preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation.

Technical scheme of the present invention is, a kind of preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation, step is: preparation, separation and the purification of high immunity yolk antibody (IgY), then make microcapsules, the reaction condition that described microcapsules are prepared microcapsules is: chitosan concentration 0.05-0.8%(w/v), CaCl₂Concentration 0.5-1.5%(w/v), sodium alginate concentration 1.0-3.0%(w/v), IgY and sodium alginate mass ratio be 1-4:8.

Concrete grammar is preparation, separation and the purification of (1) high immunity yolk antibody (IgY), and step is: by attacking poison to healthy chickE.tenella, utilize the chicken amplification of going down to posterity, obtain antigen; Then give healthy laying hen oral antigen, collect high-immunity egg; High-immunity egg is separated to yolk and obtain yolk stoste; Precooling sterile purified water 1:6-12 dilution by volume for yolk stoste, with 0.1mol/LHCL tune pH to 5.1, fully stirs rear 4 DEG C of standing 8h, centrifugal, collects supernatant and is yolk water-soluble component; Adopt sad-cold ethanol extraction process or ammonium sulfate secondary precipitation to separate, extract and obtain IgY IgY.

(2) prepare microcapsules: prepare microcapsules: the preparation of encystation liquid: take shitosan, add glacial acetic acid stirring and dissolving, add deionized water and be mixed with shitosan (w/v) solution, add CaCl₂, dissolve vacuum outgas; In the sodium alginate soln that IgY is dissolved in, above-mentioned solution is dripped in encystation liquid, reaction gained micro-capsule washs through deionized water, and vacuum freeze drying, obtains microcapsules.

In the present invention, be to protect the active material in it as the main feature of oral slow-releasing preparation microcapsules, the corrosion of opposing gastric juice and intestinal juice. While making Yolk antibody targeting site, reduce the loss of antibody titer, and can make IgY with enough concentration at target site (chicken caecum and near zone thereof) long-acting, so the technology of preparing of microcapsules is exactly one of key character of key of the present invention.

Research in the body of 1. microcapsules protections of the present invention and slow release effect specifically, the oral equivalent amount of active IgY(40ug that contains of SPF chicken that non-ball worm infects) not encapsulated Yolk antibody and dispensing than being the encapsulated Yolk antibody of 2:8, after 1h, catch and kill the IgY content detecting in caecum, result shows: the two all occurs after oral 3h in caecum, now the content in the every 10g cecal content of not encapsulated Yolk antibody is only 1.91 μ g, and encapsulated Yolk antibody detected level is 4.63ug/10g, when 5h, not encapsulated Yolk antibody concentration and encapsulated Yolk antibody concentration all reach highest level, the former is 3.161.91 μ g, the latter is 8.38 μ g, then decline gradually. when 10h, can't detect not encapsulated Yolk antibody, and now, the concentration of encapsulated Yolk antibody is still 4.37ug/10g. the capsule of preparation has not only played protection effect to Yolk antibody as can be seen here, and has played the slow release effect of medicine, can continue at caecum position specific effect.

2. couple E.Tenella infects the protection effect research of chicken; 96h starts oral IgY result and shows malicious from attacking time and attacking poison: along with the increase of oral capsule Yolk antibody amount; the death rate that infects E.tenella chicken all declines, and rate of body weight gain and ACI increase. Every day oral 1 encapsulated Yolk antibody, the death rate has reduced by 53.34% compared with attacking malicious control group, rate of body weight gain and ACI have improved respectively 49.19% and 72.40. But not encapsulated Yolk antibody group is under Isodose, and the death rate has only declined 14.8%, rate of body weight gain and ACI have only improved respectively 18.6% and 32.1%. Illustrate that IgY meets with coccidia zygoblast and merozoite in caecum, can be partly dissolved zygoblast and merozoite, weaken the virulence of coccidia zygoblast and merozoite, reduce the invasiveness of polypide, thereby reduced the pathogenicity of polypide.

In sum: the present invention has determined the microcapsule preparation process taking sodium alginate and shitosan as the applicable intestinal canal administration of wall material. Can improve medicine pharmacokinetic characteristic in vivo by slow release method, improve bioavilability, the duration of prolong drug valid density, reduce administration number of times, what heighten the effect of a treatment, alleviate fowl group stress.

Generation and the treatment succeeded in developing chicken coccidiasis of this sustained release agent have important clinical meaning and economic implications. Said preparation is a kind of efficient, safe novel form, both can be used as the prophylactic agent of chicken coccidiasis, also can be used as the global-worm illness medicine of the chicken that occurs clinical symptoms. Compared with other coccidiostats, can make the M & M of chicken coccidiasis obviously reduce, every plumage chicken is estimated to increase income 0.3 ~ 0.6 yuan.

Compared with not encapsulated IgY, vivo and vitro result of the test all shows that IgY capsule prepared by this technique not only has protection effect, has more slow-release function, can make Yolk antibody with higher concentration long-time continuous action in chicken caecum.

In challenge test, compared with attacking malicious control group, the capsule group chicken death rate of feeding has reduced by 53.34%, and rate of body weight gain and ACI have improved respectively 49.19% and 72.40. Therefore this capsule has not only reduced because of the dead direct economic loss of bringing of chicken, has also reduced the use of anticoccidial chemicals simultaneously, has saved cost; The raising of rate of body weight gain and ACI has increased cultivation efficiency, brings direct economic benefit. Owing to can reducing the use of anticoccidial chemicals, therefore reduce chicken veterinary drug residue, improve chicken meat quality, can increase export. Avoid chemicals to use specificity not strong simultaneously, chicken has been produced to the defect of immunosupress, reduction laying rate, there is indirect economy meaning.

This is a kind of novel veterinary drug production technology of highly effective and safe in a word, and first Application slow release method is developed anti-E.tenella coccidia Yolk antibody biological targeting sustained release preparation. Said preparation has overcome many defects of anticoccidial drug, has particularly overcome the generation of drug resistance worm strain, has advantages of that anticoccidial effect is strong, nuisanceless, the IgY enteron aisle valid density duration is long. This medicine can, specifically for the polypide of E.tenella development in different stages, have advantages of that anticoccidial chemicals is incomparable. The present invention has all done deep research to the performance of the embedding rate in capsule preparation process, medicine carrying amount, dispensing ratio and finished capsule product, can prepare IgY capsule or the IgG capsule of other antigens or enteropahtogenic microganism by this technique.

The economic results in society of technology of the present invention and popularizing application prospect, at present, China has become the first in the world poultry big country, and the number of animals raised is more than 10,000,000,000, and our province chicken standing crop is 1.2 hundred million left and right, and has the trend of development. But due to many reasons, the continuous generation of poultry disease has seriously affected the development of China's aviculture, wherein chicken coccidiasis is one of bottleneck of restriction aviculture development. At present average every plumage consumes anticoccidial expenses for medicine by 0.2 yuan～0.3 yuan calculating, and the annual expense for anticoccidial drug is just up to 20～3,000,000,000 yuans. Existing coccidiostat still has certain effect as the prophylactic agent of chicken coccidiasis, but as the medicine of chicken coccidiasis, it has little effect, and particularly arrives the sexual reproductive phase of chicken coccidiasis, and almost without any effect, the death rate is up to more than 80%. Medicine of the present invention can replace existing coccidiosis medicine, this biological targeting slow releasing pharmaceutical can act on coccidia pathogenetic early stage or mid-term, the chicken that occurs clinical symptoms is also had to certain curative effect, reduce the death rate of chicken body, improve its production performance, both can be used as the prophylactic agent of chicken coccidiasis, also can be used as the medicine of the chicken coccidiasis that occurs clinical symptoms. Compared with other coccidiostats, the M & M of chicken coccidiasis significantly can be reduced, make chicken production performance keep stable, can improve poultry husbandry benefit approximately 10%, increase poultry product export volume, reduce harm human body being caused because medicine is residual.

IgY microcapsules prepared by technical solution of the present invention are a kind of prophylactic agent, especially a kind of medicine. Microcapsules prepared by the present invention provide good example for antibiotic alternative medicine, and determined technique also can be used to prepare IgY or the IgG microcapsules of other pathogenic microorganisms, have promotional value, and are conducive to the sound development of green and organic poultry husbandry.

Brief description of the drawings

The comparison diagram of tri-kinds of degreasing method effect of settling of Fig. 1

The SDS-PAGE figure in each stage of Fig. 2 separation and purification IgY

Note: 1 and 2 is all standard I gY; 3.(NH4) 2SO4 secondary precipitation component; 4. sad-cold ethanol group 5.Marker

Fig. 3 IgY heat endurance

The absolute acid stability of Fig. 4 IgY

Fig. 5 indirect ELISA is surveyed the calibration curve of IgY content

In Fig. 6 varying environment, chicken IgY is with curve over time

The blank microballoon swelling curve of Fig. 7

Fig. 8 microcapsules are at the different manifestations state of simulation digestive environments

The medicine carrying amount of Fig. 9 chitosan concentration on BSA micro-capsule and the impact of envelop rate

The BSA release profiles of the prepared micro-capsule of the different chitosan concentrations of Figure 10

Figure 11 CaCl₂The impact of concentration on envelop rate and medicine carrying amount

Figure 12 Different Ca Cl₂The release profiles of concentration

The impact of Figure 13 sodium alginate concentration on envelop rate and medicine carrying amount

The release profiles of the different sodium alginate concentrations of Figure 14

The medicine carrying amount of Figure 15 sodium alginate concentration on BSA micro-capsule and the impact of envelop rate

The different dispensing comparison of Figure 16 micro-capsule release profiles.

Detailed description of the invention

The preparation technology of embodiment 1, a kind of Eimeria Tenella Yolk antibody target slow-release preparation, step is: preparation, separation and the purification of (1) high immunity yolk antibody (IgY), concrete steps are: by attacking poison to healthy chickE.tenella, utilize the chicken amplification of going down to posterity, obtain antigen; Then give healthy laying hen oral antigen, every 8d immunity once, immunity 3 times altogether, high-immunity egg is collected in last immunity; High-immunity egg is separated to yolk and obtain yolk stoste; Precooling sterile purified water 1:6 dilution by volume for yolk stoste, with 0.1mol/LHCL tune pH to 4.8, fully stirs rear 4 DEG C of standing 6h, and the centrifugal 15min of 10000r/min, collects supernatant and be yolk water-soluble component (WFS); Adopt sad-cold ethanol extraction process or ammonium sulfate secondary precipitation to separate, extract and obtain IgY IgY;

(2) prepare microcapsules: the reaction condition of preparing microcapsules is: chitosan concentration 0.1%(w/v), CaCl₂Concentration 0.5%(w/v), sodium alginate concentration 2.0%(w/v), IgY and sodium alginate mass ratio be 1 ︰ 8; The preparation of encystation liquid: take shitosan, add 10% the glacial acetic acid stirring and dissolving of 50mL, add deionized water and be diluted to 1000mL, be mixed with 0.1% shitosan (w/v), add CaCl₂, final concentration is 0.5%, dissolves vacuum outgas; IgY is dissolved in 2%(w/v) sodium alginate soln in, above-mentioned solution is dripped in encystation liquid, reaction 30min after, gained micro-capsule washs through deionized water, vacuum freeze drying obtains microcapsules.

Embodiment 2

A preparation technology for Eimeria Tenella Yolk antibody target slow-release preparation, step is: preparation, separation and the purification of (1) high immunity yolk antibody (IgY), concrete steps are: by attacking poison to healthy chickE.tenella, utilize the chicken amplification of going down to posterity, obtain antigen; Then give healthy laying hen oral antigen, every 10d immunity once, immunity 3 times altogether, high immune egg is collected in last immunity; High-immunity egg is separated to yolk and obtain yolk stoste; Precooling sterile purified water 1:9 dilution by volume for yolk stoste, with 0.1mol/LHCL tune pH to 5.1, fully stirs rear 4 DEG C of standing 8h, and the centrifugal 20min of 10000r/min, collects supernatant and be yolk water-soluble component (WFS); Adopt sad-cold ethanol extraction process or ammonium sulfate secondary precipitation to separate, extract and obtain IgY IgY;

(2) prepare microcapsules: the reaction condition of preparing microcapsules is: chitosan concentration 0.05%(w/v), CaCl₂Concentration 1.5%(w/v), sodium alginate concentration 3.0%(w/v), IgY and sodium alginate mass ratio be 4 ︰ 8; The preparation of encystation liquid: take shitosan, add 10% the glacial acetic acid stirring and dissolving of 50mL, add deionized water and be diluted to 1000mL, be mixed with 0.05% shitosan (w/v), add CaCl₂, final concentration is 1.0%, dissolves vacuum outgas; IgY is dissolved in 3%(w/v) sodium alginate soln in, above-mentioned solution is dripped in encystation liquid, reaction 30min after, gained micro-capsule washs through deionized water, vacuum freeze drying obtains microcapsules.

Embodiment 3

A preparation technology for Eimeria Tenella Yolk antibody target slow-release preparation, step is: preparation, separation and the purification of (1) high immunity yolk antibody (IgY), concrete steps are: by attacking poison to healthy chickE.tenella, utilize the chicken amplification of going down to posterity, obtain antigen; Then give healthy laying hen oral antigen, every 12d immunity once, immunity 3 times altogether, high-immunity egg is collected in last immunity; High-immunity egg is separated to yolk and obtain yolk stoste; Precooling sterile purified water 1:10 dilution by volume for yolk stoste, with 0.1mol/LHCL tune pH to 5.5, fully stirs rear 4 DEG C of standing 10h, and the centrifugal 25min of 10000r/min, collects supernatant and be yolk water-soluble component (WFS); Adopt sad-cold ethanol extraction process or ammonium sulfate secondary precipitation to separate, extract and obtain IgY IgY; (2) prepare microcapsules: the reaction condition of preparing microcapsules is: chitosan concentration 0.8%(w/v), CaCl₂Concentration 0.5%(w/v), sodium alginate concentration 1.5%(w/v), IgY and sodium alginate mass ratio be 3 ︰ 8; The preparation of encystation liquid: take shitosan, add 10% the glacial acetic acid stirring and dissolving of 50mL, add deionized water and be diluted to 1000mL, be mixed with 0.4% shitosan (w/v), add CaCl₂, final concentration is 1.5%, dissolves vacuum outgas; IgY is dissolved in 1.5%(w/v) sodium alginate soln in, above-mentioned solution is dripped in encystation liquid, reaction 30min after, gained micro-capsule washs through deionized water, vacuum freeze drying obtains microcapsules.

Embodiment 4

A preparation technology for Eimeria Tenella Yolk antibody target slow-release preparation, step is: preparation, separation and the purification of (1) high immunity yolk antibody (IgY), concrete steps are: by attacking poison to healthy chickE.tenella, utilize the chicken amplification of going down to posterity, obtain antigen; Then give healthy laying hen oral antigen, every 10d immunity once, immunity 3 times altogether, high-immunity egg is collected in last immunity; High-immunity egg is separated to yolk and obtain yolk stoste; Precooling sterile purified water 1:12 dilution by volume for yolk stoste, with 0.1mol/LHCL tune pH to 5.1, fully stirs rear 4 DEG C of standing 8h, and the centrifugal 30min of 10000r/min, collects supernatant and be yolk water-soluble component (WFS); Adopt sad-cold ethanol extraction process or ammonium sulfate secondary precipitation to separate, extract and obtain IgY IgY;

(2) prepare microcapsules: the reaction condition of preparing microcapsules is: chitosan concentration 0.3%(w/v), CaCl₂Concentration 0.8%(w/v), sodium alginate concentration 2.0%(w/v), IgY and sodium alginate mass ratio be 1 ︰ 8; The preparation of encystation liquid: take 2g shitosan, add 10% the glacial acetic acid stirring and dissolving of 50mL, add deionized water and be diluted to 1000mL, be mixed with 0.3% shitosan (w/v), add CaCl₂, final concentration is 0.8%, dissolves vacuum outgas; IgY is dissolved in 2%(w/v) sodium alginate soln in, above-mentioned solution is dripped in encystation liquid, reaction 30min after, gained micro-capsule washs through deionized water, vacuum freeze drying obtains microcapsules.

Experimental example 1: following for obtaining the deterministic process of IgY condition:

Determining of water dilution method optimum condition

The optimal pH of the 1 yolk aqueous solution is determined

Under the different pH values such as 4.6,4.8,5.0,5.1,5.2,5.5,6.0,7.0, dilute yolk stoste, watch by naked eyes, to the comparison of the lower supernatant turbidity of different pH values, and measure under different pH protein content and fat content in supernatant, experimental result is in table 1, table 2, and result shows, after yolk stoste is processed by pH5.1, supernatant is comparatively limpid, illustrates that most of lipid transfers precipitation to from yolk stoste; In supernatant, fat content is minimum, and its value is 1.42mg/mL; Protein content is the highest, and its value is 4.02mg/mL, and therefore pH5.1 is the optimum pH of extracting the yolk aqueous solution.

Table 1 dilutes the impact of yolk liquid pH on IgY extraction effect

Note :+be less precipitation; ++ be a small amount of precipitation; +++ more precipitation;

+ be clarification; ++ clarification; +++ comparatively muddy; ++++muddiness.

Protein content and determination of fat result in supernatant under table 2 condition of different pH

PH value	Protein content (mg/mL)	Fat content (mg/mL)
			4.6	3.2	5.3
4.8	3.31	4.26
			5.0	3.6	2.01
5.1	4.02	1.42
			5.2	3.89	1.63
5.5	3.39	1.89
			6.0	3.12	3.18
7.0	3.09	3.75

The optimum diluting multiple of 2 yolk liquid

Under 1:4,1:6,1:8,1:9, the different extension rates of 1:10,1:12, dilute yolk stoste, measure protein content and fat content in supernatant by Bradford method and chloroform-methanol method, measurement result is in table 3. Result shows: in the time that extension rate reaches 1:12, in supernatant, fat content is minimum, but its total protein content of comparing is also lower, and extension rate is while reaching 1:9, in supernatant, total protein content is the highest, and fat content and minimum are more or less the same, place spend the night after supernatant limpider, so determine optimum diluting multiple be 1:9; But consider centrifugally operated in follow-up test, extension rate large volume also increases, and brings larger workload to separation and purification, be extension rate so adopt 1:6 in follow-up test.

Protein content and fat content under the different extension rates of table 3 in supernatant

The best time of repose of 3 yolk water-soluble components

With the precooling distilled water dilution yolk aqueous solution of 8 times of volumes, and adjust pH to 5.1, under 4 DEG C of conditions, place respectively 2h, 4h, 5h, 6h, 8h, 10h, 15h and 20h, after with the naked eye observing placement 8h, the clarity of supernatant is better, along with the prolongation of standing time, the clarity of supernatant does not have significant change.

The effect of settling of 4 three kinds of degreasing methods

As can be seen from Figure 1, steam water at the degreasing effect of front 5h sodium citrate buffer solution and acetic acid-sodium acetate buffer apparently higher than cold acid, but along with the effect of three kinds of methods of prolongation of time remains basically stable. Consider the impact of salt pair IgY content, this experiment adopts cold acid to steam the water (> 6h that spends the night) method degreasing.

The comparison of the different method for concentration of 5IgY

Get two pieces of high-immunity eggs, collect the about 40mL of yolk stoste; Then add 5 times of distilled water to yolk volume and stir and evenly mix in yolk stoste, equal-volume is divided into two parts, and every part is 120mL; Use respectively sad-cold ethanol and ammonium sulfate secondary precipitation to IgY separation and Extraction. Yolk antibody separates, extraction the results are shown in Table 4.

Table 4 Yolk antibody separates, extracts result

Testing sample	Protein content (mg)	IgY content (mg)	IgY recovery rate (purity)
				WFS	546.895	119	21.83 %
Ammonium sulfate precipitation gained precipitation	434.96	110.8	25.47 %
				Sodium sulphate secondary precipitation,	170.776	89.8	52.58 %
Sad extracting supernatant	417.94	100.8	24.12 %
				Sad-cold ethanol precipitation	165.402	75.2	45.46 %
Sad-cold ethanol freeze drying	159.31	59.8	37.54 %

Two kinds of methods are few to the recovery rate weak effect of IgY as can be seen from the table, and ammonium sulfate secondary precipitation recovery rate is higher, but ammonium sulfate secondary precipitation need be centrifugal under low temperature hypervelocity, and extracting method is only applicable to experiment, is not suitable for factory's large-scale production. Sad-cold ethanol precipitation method, though recovery rate is low, extraction conditions is easy, therefore, adopts sad-cold ethanol precipitation method for further develop this experiment later.

The separation of 6IgY and purifying

As shown in Figure 2; sad-cold Ethanol Method and (NH4) contain the foreign protein such as alpha active albumen, 'beta ' activity albumen after 2SO4 secondary precipitation; in electrophoretic band, contain the band consistent with standard I gY heavy chain and light chain; want further to go out foreigh protein removing; must be by gel filtration; but consider that this experiment supplies raw materials for the preparation of later microcapsules, foreign protein likely protects IgY to enter caecum by hydrochloric acid in gastric juice, so do not need IgY to purify.

IgY Detection of Stability

1.IgY thermal stability determination

Adopt after different temperatures processes IgY, detect remaining specific IgY and antigen-binding activity with indirect elisa method. As can be seen from Figure 3, below 70 DEG C time, IgY has good heat endurance.

The absolute acid stability of 7.2IgY is measured

Measure IgY immunocompetent variation under condition of different pH with indirect ELISA. Within the scope of pH4-7, the vigor of IgY is substantially unaffected as shown in Figure 4; But in the time that pH value drops to 4, active beginning sharply declines, and therefore IgY is unstable in the low acid environment of hydrochloric acid in gastric juice, when oral application, need to carry out technical protection to it.

The mensuration of 8.IgY to hydrochloric acid in gastric juice and pepsic stability

From Fig. 5,6, low acid and the Protease Environment of stomach are very large to the activity influence of IgY, make its basic loss of activity in half an hour. Be necessary to take technological means to be protected it, microcapsules technology is a kind of technique that can inquire into.

The present invention studies as one of material being wrapped by using BSA, to obtain best microcapsules preparation condition. The microcapsules of preparation with this understanding, envelop rate is more than 70%, and medicine carrying amount is more than 20%, and in gastric acid environment 2h, accumulative total discharges and is less than 10%, and in intestinal environment, 4h accumulative total release rate is greater than 80%.

The mensuration of BSA properties of microcapsules and each factor affect it

Reagent and material

Sodium alginate (purity > 98%), shitosan (viscosity 50-800mpa), BSA, Coomassie brilliant blue G-250, CaCl₂Glacial acetic acid phosphoric acid, pepsin (1 ︰ 3000)

The preparation of microcapsule solution

1 encystation liquid

Take 2g shitosan, add 10% the glacial acetic acid stirring and dissolving of 50mL, add deionized water and be diluted to 1000mL, be mixed with 0.2% shitosan (w/v), add CaCl₂, dissolve vacuum outgas.

2 broken capsule liquid (0.2MNaHCO₃，0.06MNa₃C₄H₅O₇·2H₂O）

Take 16.802gNaHCO₃And 17.647gNa₃C₄H₅O₇·2H₂O is dissolved in the ultra-pure water of 1000mL surely, adjusts pH to 9.6,121 DEG C of sterilizing 20min, room temperature preservation.

3 SGFs

Take NaCl1.755g, pepsin 3.2g, concentrated hydrochloric acid 7mL, regulates pH to 1.2, is settled to lL.

4 simulated intestinal fluids

Take KH₂PO₄6.8g, trypsase 10g, adds 950ml deionized water to make it to dissolve, and with 4MNaOH adjusting pH to 6.8, is settled to 1L.

5 hydrochloric acid in gastric juice liquid

Concentrated hydrochloric acid 7mL is dissolved in the physiological saline of 1L surely, regulates pH to 1.2.

The making of blank microballoon

(1) take certain sodium alginate, add distilled water stirring and dissolving, be mixed with the solution that concentration is 2% (w/v).

(2) by 2%(w/v) sodium alginate soln, 4 DEG C of degassed 1min of 500r/min. Utilize constant flow pump that above-mentioned solution is dripped to encystation liquid from 8# syringe needle apart from liquid level 10cm with the speed of 3mL/min, after reaction 30min, gained micro-capsule washs through deionized water, and vacuum is 30～50pa, and heating-up temperature is 15 DEG C of vacuum freeze dryings.

The mensuration of blank microballoon swelling ratio

Take a certain amount of above-mentioned ready-made freeze-drying microcapsules, in manual simulation's hydrochloric acid in gastric juice liquid, after 2h, remove hydrochloric acid in gastric juice liquid, add simulated intestinal fluid, every 1h, take out micro-capsule, suck surperficial liquid with filter paper, claim its weight.

.... formula 1

The water content (quality of the quality-dry micro-capsule of swelling rear micro-capsule) that in formula 1, Ws is micro-capsule, the quality that Wd is dry micro-capsule.

The preparation of BSA microcapsules

Take a certain amount of BSA and be dissolved in 2%(w/v) sodium alginate soln in, naturally dissolve, 4 DEG C of degassed 1min of 500r/min. All the other processes are with above-mentioned blank microballoon preparation process. The gross mass of the dosage that records BSA and last freeze-drying microballoon, and the dosage of unit of account quality freeze-drying BSA microballoon while preparing, note is a.

The mensuration of BSA properties of microcapsules and each factor affect it

The mensuration of 1 medicine carrying amount and envelop rate

Take BSA microcapsule lyophilized powder 0.010g in triangular flask, add brokenly capsule liquid 5mL, put 37 DEG C, 150r/min shaking table vibration 2h, is broken to pasty state, 4 DEG C of centrifugal 15min of 4500r/min, with Bradford method survey protein content, taking standard BSA as standard protein, try to achieve sample BSA concentration by calibration curve, calculate medicine carrying amount and the envelop rate of BSA micro-capsule according to formula 2 and formula 3.

... ... ... .... formula 2

... ... ... ... ... formula 3

The mensuration of 2BSA microcapsules release in vitro performance

The holdup time of medicine in people's stomach is about 0.5 ~ 4.5h, is about 2～6h in the holdup time of small intestine, accordingly, because the microballoon of this test preparation will be applied to chicken, is 2h therefore choose the release time of micro-capsule in gastric acid environment, is 4h in intestinal environment. Get BSA microcapsule lyophilized powder 10mg, in 25mL tool plug conical flask, add hydrochloric acid in gastric juice liquid 10mL, jolting 2h on 37 DEG C of 150r/min shaking tables. Then micro-capsule is filtered, be transferred in 10mL simulation intestinal environment, timing sampling (simultaneously supplementing equivalent equality of temperature medium), measures BSA concentration in supernatant.

Calculate the preparation of BSA according to following formula, draw release profiles^[29]

×100%

... ... ... .... formula 4

In formula:

Cn: n time point institute sample thief concentration;

V: dissolution medium cumulative volume;

Vi: the sample volume of i time point;

Ci: i time point institute sample thief concentration. (Vo and Co are zero);

W:BSA micro-capsule weight;

BSAloading%: micro-capsule medicine carrying amount.

The impact of 3 chitosan concentrations on properties of microcapsules

For the impact of research chitosan concentration on micro-capsule performance, keep each liquor capacity constant, CaCl₂Concentration is 1.0%(w/v), sodium alginate concentration is 2%(w/v), BSA is 2 ︰ 8 with the ratio of sodium alginate, adopts different chitosan concentrations: 0%; 0.05%; 0.10%; 0.20%; 0.40%; 0.80%, prepare microcapsules, and measure its envelop rate and medicine carrying amount. Initial more than 0.8% chitosan concentration is also used, but because the Viscosity of Chitosan of high concentration is too high, the most of adhesion of prepared microcapsules, so the chitosan concentration scope of groping is 0～0.8%.

4CaCl₂The impact of concentration on properties of microcapsules

For research CaCl₂The impact of concentration on micro-capsule performance, keeps each liquor capacity constant, and chitosan concentration is 0.1%(w/v), sodium alginate concentration is 2%(w/v), BSA is 2 ︰ 8 with the ratio of sodium alginate, adopts different CaCl₂Concentration: 0.05%; 0.10%; 0.50%; 1.0%; 1.5%, prepare microcapsules, and measure its performance.

5 sodium alginate concentrations are to micro-capsule performance impact

For the impact of research sodium alginate variable concentrations on BSA properties of microcapsules, keep each liquor capacity constant, chitosan concentration is 0.1%(w/v), CaCl₂Concentration is 0.5%(w/v), BSA is 2 ︰ 8 with the ratio of sodium alginate, adopts different sodium alginate concentrations (w/v): 1.0%; 1.5%; 2.0%; 2.5% and 3.0%.

The impact of 6BSA dosage comparison micro-capsule performance

For research BSA dispensing impact on micro-capsule performance than (mass ratio of BSA and sodium alginate), keep each liquor capacity constant, chitosan concentration 0.1% (w/v), CaCl₂Concentration 0.5% (w/v), sodium alginate concentration 2.0% (w/v), the BSA that this test adopts offers medicine than being respectively 1 ︰ 8,2 ︰ 8,4 ︰ 8,6 ︰ 8 and 8 ︰ 8.

Result: blank microballoon swelling curve

The freeze-drying microcapsules quality that this test takes is 0.1012g(Wd), the difference of wet micro-capsule quality and dry micro-capsule quality is water content, the ratio of water content and dry micro-capsule is swelling ratio SR, sees formula 1.

The microspheres quality of table 5 different time and water content

Taking the time as abscissa, the swelling curve that swelling ratio does for ordinate as shown in Figure 7. Blank microballoon is within 0～2h time period, and in gastric acid environment, the swelling ratio of microcapsules is 1.79, and swelling ratio reaches 1.69 in the time of 1h, while can be understood as 1h dry microballoon water suction saturated, after this in 1h, water absorption rate becomes substantially. In the time being transferred in simulation intestinal environment, progressively rising of the swelling ratio of microcapsules, 4h swelling ratio reaches 21.18 times, and when 8h, swelling ratio reaches 33.58, but the mechanical strength of microcapsules dies down, and has and discloses broken sensation. The microballoon that preparation is described has pH sensitiveness, substantially not swelling in sour environment, slowly swelling in neutral slight alkali environment, meets the requirement of intestinal canal administration microcapsules.

Microcapsules release in vitro morphology observation

As can be seen from Figure 8, the variation of pattern when chitosan-sodium alginate microcapsules discharges in vitro, microcapsules freeze-dried powder is the granule of the slightly fold of white, its temperature in hydrochloric acid in gastric juice liquid is bathed after 2h, microcapsules almost do not change, when microcapsules temperature in gastric acid environment is bathed after 2h, proceeded in intestinal environment after 1h, microcapsules obviously expand, and keep complete spherical, the phenomenon of not breaking, in intestinal environment, temperature is bathed after 4h, microcapsules mechanical strength obviously reduces, and occurs breakage, the remains of visible microcapsules.

Microcapsules are in the release of simulated intestinal fluid, be one from expanding, corroding to the progressive formation breaking. Breaking of chitosan-sodium alginate microcapsules determined by residing environment pH. Sour environment, due to the existence of microcapsules intermediate ion key, microcapsules do not discharge obvious expansion. Once be exposed in neutral slight alkali environment but microcapsules leave sour environment, COOC-on sodium alginate strand on chitin-sodium alginate composite membrane can be replaced gradually by OH-, what is more important shitosan can be lost positive charge, composite membrane is depolymerization gradually, sodium alginate calcium gel beads in capsule is come out, cause the release of the capsule heart.

The impact of chitosan concentration on micro-capsule performance

The impact of 1 chitosan concentration on microencapsulated rate and medicine carrying amount

Taking chitosan concentration as abscissa, envelop rate and medicine carrying amount are ordinate, and drawing is as Fig. 9.

Result shows: the envelop rate that does not add the micro-capsule of the sodium alginate of shitosan is 23.01%, and adds the shitosan (0.05% of minimum flow; W/v) envelop rate obviously improves and reaches 43.19%. Along with the raising of shitosan solubility, envelop rate improves constantly. Shitosan solubility is 0.1%(w/v) time, the maximum while approaching 0.8%, differs only 4% left and right, the two statistical analysis, and difference is not remarkable.

As shown in Figure 9, medicine carrying amount increases along with the raising of chitosan concentration, but very similar to the variation tendency of envelop rate, is 0.1%(w/v in concentration) time, close to maximum, increasing and tend towards stability subsequently, statistical analysis difference is not remarkable.

The impact that 2 chitosan concentrations discharge microcapsule medicament

The BSA microcapsules of preparing with the shitosan of variable concentrations, its release rate profile as shown in figure 10,0～2h,, in gastric acid environment, in all samples, the release rate of BSA is all less than 10%, but 1h before proceeding to after intestinal environment, microcapsules have a sudden outburst process. 0%, the micro-capsule that prepared by 0.05%, 0.10%, 0.20%, 0.40%, 0.8% shitosan, in the time of 3h, discharge respectively 75.46%, 72.12%, 59.79%, 57.88%, 52.78%, 52.45% BSA, the raising of chitosan concentration has been described, the slow release effect of controlled pharmacy. Under the condition of shitosan (< 0.1%) of not adding shitosan or low concentration, in intestinal environment, in 2h, BSA almost all discharges. The shitosan of concentration 0.10% can delay the release of BSA, and has obvious difference with the release that concentration is 0.00% and 0.05%, and in intestinal environment 4h, accumulative total discharges approximately 83%. When chitosan concentration exceedes 0.1%, the release of microcapsules is without obvious difference.

With the rising of chitosan concentration, the release controllability of microcapsules strengthens gradually; Exceed this scope (> 0.10%; W/v) time, the release control substantially constant of microcapsules.

In sum, chitosan concentration is 0.01%(w/v) time, envelop rate and medicine carrying amount all reach maximum; Release aspect in vitro, meets release request. Therefore, finally the concentration of definite shitosan is 0.1%(w/v).

CaCl₂The impact of concentration on micro-capsule performance

1CaCl₂The medicine carrying amount of variable concentrations on BSA micro-capsule and the impact of envelop rate

Figure 11 has provided CaCl₂The impact of concentration on envelop rate and medicine carrying amount, the two has identical variation tendency, along with CaCl₂The rising of concentration, microencapsulated rate and medicine carrying amount all decline gradually, work as CaCl₂Concentration is 0.05%(w/v) time, medicine carrying amount and the envelop rate of micro-capsule are the highest, are respectively 80.44% and 22.41%; Along with CaCl₂The rising of concentration, envelop rate and medicine carrying scale reveal downward trend, but fall is very little, works as CaCl₂Concentration is 1.5%(w/v) time, envelop rate and medicine carrying amount only respectively 66.61% and 15.09%, the difference with 0.05% time is only 13.83% and 7.32%. While investigation for microcapsules mechanical performance, find CaCl₂When concentration is 0.05% and 0.1%, range estimation just can find out that capsule volume and water content are all very large, frangible in operating process. CaCl₂Three more than 1.0% concentration of concentration, volume and the mechanical strength of capsule are basic identical, range estimation no significant difference. In operating process, all there is good mechanical strength, but CaCl₂When concentration is 0.5%, medicine carrying amount and the envelop rate corresponding data higher than 1.0% and 1.5% time, statistical analysis significant difference. Therefore envelop rate and this experimental result of medicine carrying amount show to prepare the best CaCl of microcapsules₂Concentration is 0.5%.

2CaCl₂The impact of concentration on micro-capsule release

Figure 12 provides Different Ca Cl₂The influence curve of chitosan-sodium alginate microcapsules prepared by the concentration rate of release to BSA. From figure, can draw, each change in concentration trend is basic identical, and at front 3h, all curves are substantially overlapping, and the slowly-releasing process after 3h is also basic identical, without significant change. All curves are in gastric acid environment at front 2h() rate of release of BSA is all less than 10%. Proceeding to after intestinal environment, microcapsules are dashed forward and are released at front 1h, discharge percentage and are respectively 69.04%, 69.11%, 70.12%, 70.36% and 71.89%. After prominent releasing, rate of release is very slow; when 6h experiment finishes; total percentage that discharges is just respectively 89.23%, 89.87%, 89.55%, 88.32%, 90.32%; each concentration (being in gastric acid environment) accumulative total release rate in 2h is less than 10%; in intestinal environment 4h, accumulative total release rate is about 80% left and right, meets release request.

Envelop rate and medicine carrying component analysis result show, 0.5% CaCl₂Concentration is optimal concentration, and the research of tablets in vitro effect shows CaCl₂Concentration has no significant effect the release in vitro of BSA, considers, and determines optimum CaCl₂Concentration is 0.5%(w/v).

The impact of sodium alginate concentration on micro-capsule performance

The medicine carrying amount of 1 sodium alginate variable concentrations on BSA micro-capsule and the impact of envelop rate

Figure 13 shows, the impact of sodium alginate concentration on envelop rate and medicine carrying amount, and along with the rising of sodium alginate concentration, envelop rate raises gradually. Sodium alginate concentration is 2.0%(w/v) time, envelop rate reaches maximum 74.07%, but sodium alginate concentration is lower than 2.0%(w/v) time, envelop rate is 65.03% and 55.6%, differing and be respectively 11.04% and 17.47% with maximum, is significant difference by statistical analysis. This is because sodium alginate concentration is low, sodium alginate-chitosan polyelectrolyte is lepthymenia, also have the heterogeneous property degree that sodium alginate external source gel process produces to reduce, cause the slack and undisciplined of gel plasma membrane in heterogeneous gel network, the permeability of micro-capsule increases, impel the capsule heart to be diffused into water from microcapsules, reduce envelop rate. When sodium alginate concentration exceedes 2.0%(w/v) time, envelop rate declines very soon, decline respectively 24.55% and 28.74%, statistical analysis and concentration are 2.0%(w/v) time, significant difference, this is that system viscosity is large because sodium alginate concentration is too high, the microcapsules agglomerate that easily bonds forming, causes envelop rate bad. Medicine carrying amount is identical with envelop rate trend, be 2.0%(w/v at sodium alginate concentration) time, medicine carrying amount is up to 16.44%, be 1.0%, 1.5%, 2.5% and 3.0% to differ respectively 3.10%, 2.45%, 2.84% and 4.77% with concentration, statistical analysis significant difference, therefore the envelop rate of this experimental result and medicine carrying scale are bright, and the best sodium alginate concentration of preparing microcapsules is 2.0%(w/v).

The impact of 2 sodium alginate concentrations on micro-capsule release

As can be seen from Figure 14, under the different level of sodium alginate concentration, the BSA releasing trend of microcapsules is identical. In 2h (in gastric acid environment), the BSA release rate in microcapsules used is all less than 10%, proceeding in intestinal environment, front 1h, BSA microcapsules start prominent releasing, and prominent releasing is respectively 70.36%, 67.34%, 63.42%, 55.02% and 53.66%, after prominent releasing, have and continue slowly to discharge. When 6h, experiment finishes, total release ratio is 90.13%, 87.24%, 80.59%, 75.78% and 70.98%, each concentration (being in gastric acid environment) accumulative total release rate in 2h is less than 10%, in intestinal environment 4h, the release of BSA and sodium alginate concentration have obvious relation, and BSA rate of release raises and reduces with the concentration of sodium alginate. When sodium alginate concentration is 1.0%(w/v) time, BSA accumulative total in intestinal environment 4h discharges approximately 82%, sodium alginate concentration is 3.0%(w/v) time, BSA adds up in 4h to discharge approximately 64% in intestinal environment, discharges minimum, differ is 18%, it is larger that difference differs, and sodium alginate concentration is 3.0%(w/v) time, do not meet release request, consider, the best sodium alginate concentration of this experimental selection is 2.0%(w/v).

In sum, sodium alginate concentration is 2.0%(w/v) time, envelop rate and medicine carrying amount have all reached maximum; In simulation extracorporeal releasing experiment, when sodium alginate concentration is 2.0%(w/v) time, BSA accumulative total release rate in gastric acid environment 2h is 7.75%, in intestinal environment 4h, accumulative total is released to 72.84%, meets the release request of intestinal canal administration. Therefore, determine that sodium alginate optimal concentration is 2.0%(w/v).

The impact of dosage comparison micro-capsule performance

The medicine carrying amount of 1 dosage comparison BSA micro-capsule and the impact of envelop rate

Figure 15 shows, the envelop rate of micro-capsule reduces than the increase of (BSA and sodium alginate mass ratio) with dispensing, offers medicine when being 1 ︰ 8, and envelop rate is 92.25%; Further increase and offer medicine while comparing 2 ︰ 8,4 ︰ 8,6 ︰ 8 and 8 ︰ 8, envelop rate drops to 74.60%, 70.4%, 66.22% and 62.25% on the contrary, and differing is 17.65%, 21.85%, 26.03% and 30.00%, the mutual significant difference of statistical analysis; The medicine carrying amount of microcapsules increases with the increase of dispensing ratio, and dispensing is when being 1 ︰ 8, and medicine carrying amount is 13.96%, when dispensing is when increasing to 8 ︰ 8, medicine carrying amount reaches maximum 55.27%, and differing is 41.31%, and between each dispensing ratio, the mutual significant difference of statistical analysis.

2BSA dispensing is than the different impacts on micro-capsule release

As shown in Figure 16, dispensing is very larger than the BSA release impact of the different chitosan-sodium alginate microcapsules on preparation. In gastric acid environment 2h, dispensing is all less than 10% than the accumulative total that is 1 ︰ 8 and 2 ︰ 8, discharges and is more than 60% than the accumulative total that is 4 ︰ 8,6 ︰ 8 and 8 ︰ 8 but offer medicine, and causes in intestinal environment, discharging percentage few, does not meet the requirement of intestinal canal administration.

In sum, dispensing, than at low level (≤2:8), can obtain higher envelop rate; Release aspect in vitro, in gastric acid environment 2h, release rate is less than 10%, in intestinal environment 4h, add up to discharge percentage and be respectively 81.01% and 77.96%, meet release request, in addition, dispensing is than being 1 ︰ 8, seal cost though can obtain higher envelop rate but increase, consider, determine that optimum dispensing ratio is 2 ︰ 8.

Conclusion, above-mentioned experiment discussion reaction condition (chitosan concentration, CaCl₂Concentration, sodium alginate concentration and BSA and sodium alginate mass ratio) impact on BSA chitosan-sodium alginate microcapsules performance, chitosan concentration 0.1%(w/v), CaCl determined that the optimum reaction condition of preparing microcapsules is:₂Concentration 0.5%(w/v), sodium alginate concentration 2.0%(w/v), BSA and sodium alginate mass ratio be 2 ︰ 8, the BSA microcapsules of preparation with this understanding, envelop rate is more than 70%, medicine carrying amount is more than 20%, in gastric acid environment 2h, accumulative total discharges and is less than 10%, and in intestinal environment, 4h accumulative total release rate is greater than 80%.

This conclusion draws based on BSA, and the stomach environment of facing due to the microcapsules of different content thing is consistent, so content uses IgY, and the other materials such as IgG, preparation process is consistent, and its effect is all on all four. Show same character so be prepared into anti-E.tenellaIgY microcapsules according to above-mentioned process conditions.

Claims

1. the preparation technology of an Eimeria Tenella Yolk antibody target slow-release preparation, it is characterized in that: preparation, separation and the purification of high immunity yolk antibody (IgY), chitosan concentration 0.1%(w/v), CaCl then make microcapsules, the described reaction condition of preparing microcapsules is:₂Concentration 0.5%(w/v), sodium alginate concentration 2.0%(w/v), IgY and sodium alginate mass ratio be 2:8;

Preparation, separation and the purification of high immunity yolk antibody (IgY), concrete steps are: by attacking poison to healthy chickE.tenella, utilize the chicken amplification of going down to posterity, obtain antigen; Then give healthy laying hen oral antigen, every 10d immunity once, immunity 3 times altogether, high-immunity egg is collected in last immunity; Collect high-immunity egg; High-immunity egg is separated to yolk and obtain yolk stoste; Precooling sterile purified water 1:6-12 dilution by volume for yolk stoste, with 0.1mol/LHCL tune pH to 5.1, fully stirs rear 4 DEG C of standing 8h, centrifugal, collects supernatant and is yolk water-soluble component; Adopt sad-cold ethanol extraction process to separate, extract and obtain IgY IgY;

Prepare microcapsules: the preparation of encystation liquid: take shitosan, add glacial acetic acid stirring and dissolving, then add deionized water to be mixed with chitosan solution, add CaCl₂, dissolve vacuum outgas; In the sodium alginate soln that IgY is dissolved in, above-mentioned solution is dripped in encystation liquid, reaction gained micro-capsule washs through deionized water, and vacuum freeze drying, obtains microcapsules.