CN102205122B - Method for preparing piglet diarrhea-resisting egg yolk antibody microcapsules by spray drying method - Google Patents
Method for preparing piglet diarrhea-resisting egg yolk antibody microcapsules by spray drying method Download PDFInfo
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Abstract
The invention discloses a method for preparing piglet diarrhea-resisting egg yolk antibody microcapsules by a spray drying method, which mainly comprises the following steps of: extracting an egg yolk antibody from egg yolk of eggs laid by specific immune healthy hens, and purifying; mixing the egg yolk antibody serving as a core material, gelatin and sodium alginate which serve as a wall material and Tween 80 serving as an emulsifier according to a certain ratio and emulsifying to obtain encystation emulsifying liquid; and finally, performing spray drying on the obtained encystation emulsifying liquid to obtain a microcapsule finished product. In the microcapsule egg yolk antibody prepared by the method, more than 85 percent of activity is kept, the envelop rate is 77.5 percent, the 2-hour release rate of artificial gastric juice is 8.5 percent, the 4-hour release rate of artificial intestinal juice is 81.1 percent, and the egg yolk antibody can be protected against the destruction of gastric acid and pepsase, so that the egg yolk antibody can reach intestinal tracts and can be released to exert effects. The egg yolk antibody microcapsules are a high-efficiency and environment-friendly biological preparation which can replace antibiotics and is used for preventing and treating piglet diarrhea.
Description
Technical field
The present invention relates to field of medicaments, be specifically related to a kind of method of utilizing spray drying method to prepare anti-piglet diarrhea egg yolk antibody microcapsules.
Background of invention
Piglet diarrhea is one of principal element that causes the dead and growth retardation of piglet, serious harm the development of China's pig industry.The cause of disease that causes piglet diarrhea is a lot, mainly contains to produce enterotoxin colon bacillus (ETEC), transmissible gastro-enteritis virus (TGEV), Porcine epidemic diarrhea virus (PEDV), rotavirus etc.The intestinal tract disease that special yolk antibody treatment and prevention are caused by antibacterial and virus, have safe noresidue, do not develop immunity to drugs, cost is low and take easily advantage, the effect in treatment and prevention of diarrhea in piglets has received the concern of Chinese scholars as the alternative antibiotic high-performance bio preparation of a class.But the special yolk antibody that traditional method prepares is subject to gastric acid and pepsic destruction easily, affects its effect.
At present, there is not yet about utilizing spray drying method to prepare the bibliographical information of research and the application of anti-piglet diarrhea egg yolk antibody microcapsules.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method of utilizing spray drying method to prepare anti-piglet diarrhea egg yolk antibody microcapsules; the microcapsule that the method makes can protect yolk antibody not to be subjected to gastric acid and pepsic destruction, makes yolk antibody can arrive intestinal and discharges the performance effect.
The method that the present invention utilizes spray drying method to prepare anti-piglet diarrhea egg yolk antibody microcapsules is to operate by following processing step:
1), with containing three kinds of serotypes of K88, K99 and 987P, and malicious Seedling immune health chicken groups each three times a little less than the escherichia coli inactivated vaccine of bacterium number at least 30 hundred million CFU/mL of each bacterium and transmissible gastroenteritis of swine, porcine epizootic diarrhea and the rotavirus three, each immune interval 20 days, two kinds of immune intervals 10 days, last immunity was gathered in the crops height and is exempted from ovum after 10 days;
2), soak height with 0.1% bromo geramine and exempt from ovum, sterilization 30min carries out the eggshell surface sterilization with 75% ethanol again, then gathers yolk liquid, and adopts the water-solubility protein in the water dilution method extraction yolk;
3) adopt, again the foreign protein in the saturated ammonium sulfate method removal water-solubility protein, obtain highly purified yolk antibody, and yolk antibody concentration is transferred to 10mg/mL;
4) being that 2～6% gelatin solution, mass percentage concentration are 0.5～1% sodium alginate soln and step 3 with mass percentage concentration) yolk antibody that obtains is to mix at 50: 50: 1 by volume, add again final concentration and be 0.5～1% Tween 80, under the condition of 50～70 ℃ of temperature, mixing speed 1000～2000rpm and mixing time 15～60min, carry out emulsifying and make the encystation emulsion;
5), with step 4) the encystation emulsion that obtains adopts spray drying method to make the microcapsule finished product, spray-dired condition is: 160～180 ℃ of inlet temperature, charging rate 5～15ml/min, drying time 60～80 ℃ of 5～15s and outlet temperatures.
The inventive method adopts spray drying method to combine with the microcapsule encapsulation technique; make the microcapsule that makes to protect yolk antibody not to be subjected to gastric acid and pepsic destruction; and make yolk antibody can arrive intestinal and discharge the performance effect, be a kind of high-efficiency environment friendly biological preparation of alternative antibiotic control piglet diarrhea.
The specific embodiment
The below is further described the inventive method:
Embodiment 1:
Prepare anti-piglet diarrhea egg yolk antibody microcapsules by following processing step:
(1). use the escherichia coli inactivated vaccine of bacterium number average more than 3,000,000,000 CFU/mL that contains K88, K99 and three kinds of serotypes of 987P and each bacterium by only immune 200 healthy chickens of 1mL/, totally three times, every minor tick 20 days.At 10 days then malicious Seedlings a little less than immune swine transmissible gastroenteritis, porcine epizootic diarrhea and the rotavirus three behind each immune escherichia coli inactivated vaccine, it is only immune press 0.5mL/, and totally three times, every minor tick 20 days.Last immunity was gathered in the crops height and is exempted from the about 250kg of ovum after 10 days;
(2). be the ovum of 0.1% bromo geramine soaking step (1) results with mass percentage concentration, sterilization 30min, be that 75% ethanol carries out the eggshell surface sterilization with mass percentage concentration again, open eggshell under the aseptic condition, egg white and yolk are separated, again yolk is placed sterilization to roll on the filter paper, sop up and puncture vitellinae membrana behind the residual egg white and collect yolk liquid.With aquesterilisa yolk liquid is done 10 times of dilutions again, stir evenly, and transfer PH to 5.5, hold over night is the centrifugal 30min of 10000rpm/min at 4 ℃, rotating speed again, collects supernatant;
(3). in the supernatant that step (2) is collected, add isopyknic PBS (0.01mol/L, PH6.8) dilute, add again saturated ammonium sulfate solution, make its volume account for 1/2 of cumulative volume, under 4 ℃, leave standstill 1h again, be the centrifugal 20min of 5000rpm/min at 4 ℃, rotating speed, abandon supernatant.Using PBS (0.01mol/L, the PH6.8) dissolution precipitation of supernatant volume in 2 times of steps (2) again, add saturated ammonium sulfate solution again, make its volume account for 1/3 of cumulative volume, is the centrifugal 20min of 5000rpm/min at 4 ℃, rotating speed again, abandons supernatant.Use PBS (0.01mol/L, PH6.8) dissolution precipitation, obtaining concentration is that 10mg/mL, purity are 88% the about 500ml of yolk antibody again.
(4). be 3% gelatin solution 25000ml with mass percentage concentration with mass percentage concentration be after 0.5% sodium alginate soln 25000ml mixes, add again the yolk antibody 500ml that step (3) obtains, add again final concentration and be 0.5% Tween 80, under the condition of temperature 60 C, mixing speed 1500rpm and mixing time 30min, carry out emulsifying and make the encystation emulsion;
(5). the encystation emulsion that step (4) is obtained carries out spray drying and makes the about 2.5kg of egg yolk antibody microcapsules under the condition of 170 ℃ of inlet temperature, charging rate 10ml/min, drying time 60 ℃ of 10s and outlet temperatures, pack by the 0.5kg/ bag with compound paper bag, namely obtain anti-piglet diarrhea egg yolk antibody microcapsules finished product.
Embodiment 2:
The operating procedure of present embodiment (1)-(4) are identical with embodiment 1, in (5) step, the encystation emulsion that step (4) is obtained carries out spray drying and obtains the about 2.5kg of egg yolk antibody microcapsules under the condition of 160 ℃ of inlet temperature, charging rate 5ml/min, drying time 80 ℃ of 7s and outlet temperatures, pack by the 0.5kg/ bag with compound paper bag, namely obtain anti-piglet diarrhea egg yolk antibody microcapsules finished product.
Embodiment 3:
The operating procedure of present embodiment (1)-(3) are identical with embodiment 1;
Operating procedure (4), be 4% gelatin solution 25000ml with mass percentage concentration with mass percentage concentration be after 0.75% sodium alginate soln 25000ml mixes, add the yolk antibody 500ml that step (3) obtains, add again final concentration and be 1% Tween 80, under the condition of 65 ℃ of temperature, mixing speed 2000rpm and mixing time 45min, carry out emulsifying and make the encystation emulsion;
Operating procedure (5) is identical with embodiment 1.
Embodiment 4:
The operating procedure of present embodiment (1)-(3) are identical with embodiment 1;
Operating procedure (4), be 6% gelatin solution 25000ml with mass percentage concentration with mass percentage concentration be after 1% sodium alginate soln 25000ml mixes, add the yolk antibody 500ml that step (3) obtains, add again final concentration and be 0.75% Tween 80, under the condition of temperature 50 C, mixing speed 1000rpm and mixing time 60min, carry out emulsifying and make the encystation emulsion;
Operating procedure (5) is identical with embodiment 1.
Embodiment 5:
Operating procedure (1) is pressed only 200 healthy chickens of immunity of 0.5ml/, totally three times, every minor tick 20 days with transmissible gastroenteritis of swine, porcine epizootic diarrhea and rotavirus three weak malicious Seedlings.Behind malicious Seedling a little less than each immune swine transmissible gastroenteritis, porcine epizootic diarrhea and the rotavirus three 10 days then immunity contain the escherichia coli inactivated vaccine of bacterium number average more than 3,000,000,000 CFU/mL of K88, K99 and three kinds of serotypes of 987P and each bacterium, press only immunity of 1ml/, totally three times, every minor tick 20 days.Last immunity was gathered in the crops height and is exempted from the about 250kg of ovum after 10 days;
Operating procedure (2)-(4) are identical with embodiment 1;
Operating procedure (5), the encystation emulsion that step (4) is obtained carries out spray drying and obtains the about 2.5kg of egg yolk antibody microcapsules under the condition of 180 ℃ of inlet temperature, charging rate 13ml/min, drying time 70 ℃ of 15s and outlet temperatures, pack by the 0.5kg/ bag with compound paper bag, namely obtain anti-piglet diarrhea egg yolk antibody microcapsules finished product.
Below be the performance measurement example of product of the present invention:
1. get product microcapsule 2g of the present invention in the 10ml centrifuge tube, add the broken capsule solution 5ml of 0.05mol/L sodium citrate, placing 37 ℃ constant-temperature table is that 12h is hatched in the 200rpm jolting in rotating speed, is the centrifugal 10min of 5000rpm in rotating speed, collects supernatant.Colibacillary antibody titer is 2 in the employing MA method mensuration supernatant
8, and former colibacillary antibody titer is 2
8Adopt indirect ELISA method to measure the antibody horizontal of transmissible gastroenteritis of swine, porcine epizootic diarrhea and rotavirus in the supernatant, the OD value is respectively 1.725,1.832 and 1.767, and former OD value is respectively 2.018,2.125 and 2.037, and result of the test shows: colibacillary antibody activity remains unchanged in the microcapsule preparation process; The antibody activity of transmissible gastroenteritis of swine, porcine epizootic diarrhea and rotavirus remains on respectively 85.5%, 86.2% and 86.7%.As a whole, the yolk antibody activity keeping is more than 85%.
2. after dissolving microcapsule as stated above, the concentration that adopts Folin-phenol method to measure yolk antibody in the solution is 0.62mg/mL, the actual mass of yolk antibody is 0.62mg/mL * 5ml=3.1mg, and the microcapsule of preparation 2500g has added the 5g (yolk antibody of 10mg/mL * 500ml=5000mg) altogether, therefore the Theoretical Mass of yolk antibody is 4mg in the 2g microcapsule, calculates the envelop rate of microcapsule by following formula.
In envelop rate (%)=microcapsule in yolk antibody actual mass/microcapsule yolk antibody Theoretical Mass * 100% apply mechanically formula and calculate: envelop rate (%)=3.1mg/4mg * 100%=77.5%
Result of the test shows: microencapsulated rate is 77.5%.
3. get product microcapsule 2g of the present invention in the 10ml centrifuge tube, add simulated gastric fluid 5ml, placing 37 ℃ constant-temperature table is that 2h is hatched in the 200rpm jolting in rotating speed, be the centrifugal 5min of 1000rpm in rotating speed, get supernatant ice bath cessation reaction, yolk antibody concentration is 0.053mg/mL in the employing Folin-phenol method mensuration supernatant, and the burst size of yolk antibody is 0.053mg/mL * 5ml=0.265mg, calculates the release rate of microcapsule by following formula.
Yolk antibody actual mass * 100% in the burst size/microcapsule of release rate (%)=yolk antibody
Applying mechanically formula calculates: release rate (%)=0.265mg/3.1mg * 100%=8.5%
Result of the test shows: yolk antibody is 8.5% at simulated gastric fluid 2h release rate in the microcapsule.
4. get product microcapsule 2g of the present invention in the 10ml centrifuge tube, add simulated intestinal fluid 5ml, placing 37 ℃ constant-temperature table is that 4h is hatched in the 200rpm jolting in rotating speed, be the centrifugal 5min of 1000rpm in rotating speed, get supernatant ice bath cessation reaction, yolk antibody concentration is 0.503mg/mL in the employing Folin-phenol method mensuration supernatant, and the burst size of yolk antibody is 0.503mg/mL * 5ml=2.515mg, calculates the release rate of microcapsule by following formula.
Yolk antibody actual mass * 100% in the burst size/microcapsule of release rate (%)=yolk antibody
Applying mechanically formula calculates: release rate (%)=2.515mg/3.1mg * 100%=81.1%
Result of the test shows: yolk antibody is 81.1% at simulated intestinal fluid 4h release rate in the microcapsule.
Claims (1)
1. a method of utilizing spray drying method to prepare anti-piglet diarrhea egg yolk antibody microcapsules is characterized in that, it comprises following processing step:
1), with containing three kinds of serotypes of K88, K99 and 987P, and malicious Seedling immune health chicken groups each three times a little less than the escherichia coli inactivated vaccine of bacterium number at least 30 hundred million CFU/mL of each bacterium and transmissible gastroenteritis of swine, porcine epizootic diarrhea and the rotavirus three, each immune interval 20 days, two kinds of immune intervals 10 days, last immunity was gathered in the crops height and is exempted from ovum after 10 days;
2), soak height with 0.1% bromo geramine and exempt from ovum, sterilization 30min carries out the eggshell surface sterilization with 75% ethanol again, then gathers yolk liquid, and adopts the water-solubility protein in the water dilution method extraction yolk;
3) adopt, again the foreign protein in the saturated ammonium sulfate method removal water-solubility protein, obtain highly purified yolk antibody, and yolk antibody concentration is transferred to 10mg/mL;
4) be that 2～6% gelatin solution, mass percentage concentration are that the yolk antibody that 0.5～1% sodium alginate soln and step 3) obtain is that 50 ︰, 50 ︰ 1 mix by volume with mass percentage concentration,, add again final concentration and be 0.5～1% Tween 80, under the condition of 50～70 ℃ of temperature, mixing speed 1000～2000rpm and mixing time 15～60min, carry out emulsifying and make the encystation emulsion;
5), encystation emulsion that step 4) is obtained adopts spray drying method to make the microcapsule finished product, spray-dired condition is: 160～180 ℃ of inlet temperature, charging rate 5～15ml/min, drying time 60～80 ℃ of 5～15s and outlet temperatures.
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| CN107970446B (en) * | 2017-10-31 | 2020-11-03 | 广西壮族自治区兽医研究所 | Piglet diarrhea-resistant high-immunity whole egg powder microcapsule and preparation method and application thereof |
| CN113171333B (en) * | 2021-04-25 | 2024-02-13 | 大连大学 | Multi-effect repairing mask liquid containing nano embedded egg yolk antibody and preparation method thereof |
| CN113929775A (en) * | 2021-10-26 | 2022-01-14 | 国药集团动物保健股份有限公司 | Yolk antibody for preventing and/or treating porcine diarrhea, preparation and preparation method thereof |
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