CN103251938B - Industrial production process of eimeria coccidiosis attenuated vaccine - Google Patents
Industrial production process of eimeria coccidiosis attenuated vaccine Download PDFInfo
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Abstract
The invention discloses an industrial production process of eimeria coccidiosis attenuated vaccine. The industrial production process of the eimeria coccidiosis attenuated vaccine comprises the following steps of: preparation of oocyst-containing chicken manure, high-efficiency mixing, relay screening, continuous flow centrifugal and crude separation, saturated salt water subdivision, centrifugal desalination, oocyst disinfection, sporogony, oocyst counting, oocyst proportioning, microencapsulation process and package. The industrial production process disclosed by the invention is applied to industrial production of eimeria coccidiosis attenuated polyvalent vaccine liquid and solid preparation, the vaccine production efficiency is greatly improved, the requirements on scale production can be met, and the popularization and the application of products are facilitated.
Description
Technical field
The present invention relates to the technology of separation from chicken infectious feces, purification Eimeria egg capsule egg capsule vaccine and preparation is lived, refer in particular to chicken Eimeria attenuated vaccine industrialized manufacturing technique.
Background technology
The preparation of the precocious attenuated vaccine of chicken Eimeria depends on and be separated egg capsule from infectious feces, and the isolation and purification of coccidian oocyst is a very complicated job.Existing production technology transplants the isolation technics in laboratory, and application saturated saline floatation is separated egg capsule.Its cardinal principle is: because the density (1.103 ~ 1.064) of coccidian oocyst is between water and the density of saturated brine, when the egg capsule solution containing impurity is placed on centrifuge in water, sink together with egg capsule is larger than water density with a small amount of material, due to buoyancy, the impurity less than water density floats, after centrifugal, abandon supernatant, eliminate the impurity less than water density; When the egg capsule solution containing impurity being placed on centrifugal in saturated brine (sucrose solution, saturated magnesium sulfate and saturated chromatosulfuric acid), due to buoyancy, float together with egg capsule is less than saturated brine density with a small amount of material, after centrifugal, abandon precipitation, eliminate the impurity larger than saturated brine density.Subsequently, the egg capsule sodium hypochlorite process of separation, removes the bur on antibacterial and egg capsule surface, preparation vaccine.
Saturated saline floatation is the most frequently used coccidian oocyst isolation technics of laboratory, and this technology is relatively simple, is easy to grasp, favorable repeatability, reliable results.But produce for industrialization and have major defect, main manifestations is: 1. screening efficiency is low, channelization can not be realized and connect and mechanization production; 2. centrifugal material amount is excessive, and centrifuge quantitative requirement is many, and centrifugation time is long.These reasons cause the direct factor that vaccine production capacity is little, industrialization level is low, and cause the cost increase of vaccine further, have impact on popularization and the use of vaccine.
Eimeria (Eimeria) coccidiosis (Coccidium) is the class obligatory parasitism protozoon parasitizing chicken intestinal, causes chicken coccidiosis (Coccidiosis).According to statistics, the Eimeria species that the whole world has been reported has 15 kinds, wherein, have 5 kinds to have significantly pathogenic, they are: Eimeria tenella (E.tenella), Eimeria Necatrix (E.necatrix), Eimeria maxima (E.maxima), Eimeria acervulina (E.acervulina) and Eimeria mitis (E.mitis).In these 5 kinds of coccidiosiss, have 3 kinds to the hazardness of chickling tool, they are: Eimeria tenella, Eimeria maxima and Eimeria acervulina.
Coccidiosis is a kind of ubiquitous infectious epidemic disease, in the world every generation having the place of chicken just to have Eimeria species and chicken coccidiosis, and the scale chicken house incident suitable place that is chicken coccidiosis, usually there is the outburst of this disease.Chicken coccidiosis a kind of infects rapidly, propagates fast and extensively, and the high lytic infection of pathogenicity rate, fatality rate is sick, and preventing and treating improperly in situation, infection rate and mortality rate are all up to more than 50%.Therefore, chicken coccidiosis causes serious economic loss to aviculture, and the economic loss that the whole world is caused because of this disease is every year up to tens billion of yuans, and the loss that China causes because of this disease is every year quite a few in 10,000,000,000 yuan.In poultry husbandry intensive today improved constantly, control chicken coccidiosis is indispensable link in the work of poultry husbandry control and prevention of disease.
From the eighties in last century, the prevention and control of chicken coccidiosis achieve major progress, mainly have benefited from the appearance of a large amount of coccidiostat, and obtain gratifying effect.But a large amount of coccidiostats mostly is chemical classes product (as Robenidine, nicarbazine, dinitolmide, diclazuril, nicarbazine and sulfa drugs) and antibiotics product (as monensin, Salinomycin, Lasalocid, that draws rhzomorph, Maduramicin, Hainanmycin).The life-time service of these products must cause the appearance of Drug resistance and drug residue phenomenon.And the use of various coccidiostat is the administration of companion's material.In fact the nineties in last century drug resistance phenomenon very general, the development pressure pcl of new drug increases in this case, and the prevention and control difficulty of poultry enterprise strengthens day by day.
Since entering 21 century, in global range, chemical drugs and antibiotic use are restricted, food safety system receives increasing concern, and developed country prohibits the use of chemical coccidiostas thing substantially, and the use of antibiotics coccidiostat is also subject to significant restrictions.Prohibite this series products in the U.S., Japan and the European Community to appear in feedstuff.In China due to the application without restriction of various coccidiostat, Drug resistance generally produces, and some products (as monensin) are worth without Clinical practice.The food safety policy of China comes into effect in recent years, the health perception of consumer self strengthens day by day simultaneously, the market day by day atrophy of coccidiostat, and, China also implements to forbid or limit the marketing policy using coccidiostat by the recent period, and the prevention and control situation of such chicken coccidiosis is suddenly nervous.
In order to effectively prevent and treat chicken coccidiosis, guarantee the sound development of poultry husbandry, in the urgent need to finding new prevention and controls, this wherein vaccination prevention be one of promising approach of tool.But traditional vaccine manufacturing technology is not suitable for industrialization and produces, and needs in a hurry to be suitable for large-scale production technical matters.Patent of invention " chicken Eimeria egg capsule the batch preparation " (patent No.: the lot splitting purification technique 2011100291271.6) disclosing coccidian oocyst, it is one of key technology of vaccine to chicken coccidiosis production, but whole techniques prepared by vaccine, particularly vaccine solid preparation can not be solved.
The production of vaccine to chicken coccidiosis depends on and be separated egg capsule from infection chicken manure, then egg capsule rational proportion is prepared vaccine.Currently available vaccines, existing vaccines production technology is summarized as follows: divide filtration → 200 mesh sieves to divide filtration → centrifugal rough segmentation → saturated brine segmentation → centrifugal desalination → egg capsule sterilization → Sporulated → egg capsule counting → egg capsule proportioning → subpackage containing egg capsule chicken manure → dilution mixture → 60 mesh sieve
This production technology is actually the common method of transplanting in laboratory, and its weak point is apparent: 1. inefficiency is filtered in screening, and screening process is divided into 2 operations, wastes time and energy; And non_mechanical screening can not produce enough " screening power ", and screening effect is poor.2. centrifugal treating equipment needed thereby quantity is too much or consuming time long, and as front analysis, conventional centrifugation technique is unsuitable for processing a large amount of material, even use vast capacity centrifugation apparatus also not reach the effect of continuous flow centrifugation.3. lack the microencapsulation process to egg capsule, can only liquid dosage form be prepared, limit use and the effect of vaccine.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of chicken Eimeria attenuated vaccine industrialized manufacturing technique, this production technology by high efficient mixed, relay screening, continuous flow centrifugation rough segmentation, saturated brine segments and the complete sets of Techniques such as egg capsule microencapsulation process forms, and makes that the preparation efficiency of vaccine significantly improves, preparation cost significantly reduces.This production technology is used for the production of the weak malicious polyvalent vaccine liquid of chicken Eimeria and solid preparation, it is on the basis of prior art, by improving egg capsule screening plant and combining with novel technique, not only define complete production of vaccine technique, and solve the production technology of solid preparation.
For making the production technology of vaccine to chicken coccidiosis meet industrialized requirement, improving production capacity, reducing product cost, must upgrade to prior art processes, and realize channelization connection and mechanization production.
The technical problem to be solved in the present invention is realized by following scheme: chicken Eimeria attenuated vaccine industrialized manufacturing technique is completed by following steps: containing egg capsule chicken manure → high efficient mixed → relay screening → continuous flow centrifugation crude separation → saturated brine segmentation → centrifugal desalination → egg capsule sterilization → Sporulated → egg capsule counting → egg capsule proportioning → microencapsulation process → subpackage.Concrete steps are:
(1) containing egg capsule chicken manure high efficient mixed, get infected chicken fresh excreta in mixer, add 3 ~ 4 times of weight clear water dilutions, the pepsin of above-mentioned weight 0.05 ~ 0.1% is added again after stirring and evenly mixing, continue mixing 1 ~ 2min, add the clear water of 5 ~ 7 times of weight after leaving standstill 5 ~ 6min again, continue stirring and evenly mixing and become liquid manure;
(2) relay screening, screening machine divides two-layer up and down, upper screen cloth 60 order, lower screen 200 order, open mixer discharging opening, divides machine inlet capable once to add described liquid manure 25 ~ 35 liters from upper sieve, liquid manure directly enters lower screen extension set and carries out the 2nd screening after screening is filtered, the filtrate of the 2nd screening enters fluid reservoir, changes screen cloth, then adds 25 ~ 35 liters of liquid manures, proceed screening, so until the screening of all liquid manures is complete;
(3) continuous flow centrifugation crude separation, fluid reservoir feeding device is connected with centrifuge feed arrangement, open centrifuge charging switch, centrifuge speed is set as 4000rpm, the complete material of described screening enters centrifuge by charging aperture, the effect of centrifugal force is subject in piece-rate system, egg capsule is retained by filter screen, supernatant enters filtering cabin through screen cloth and is discharged by liquid outlet, the complete material of described screening imports continuously in centrifugal process, egg capsule precipitation is stayed in centrifugal storehouse, and supernatant is then constantly discharged, and the supernatant collection of discharge is in container;
(4) saturated brine segmentation, after continuous centrifugal completes, takes out centrifugal storehouse, and in centrifugal storehouse, shift out centrifugal egg capsule precipitate weigh, the saturated brine adding 5 times of weights fully mixes, then to add in centrifuge tube with 3000rpm centrifugal 10 minutes;
(5) centrifugal desalination, last operation is centrifugal complete after taking-up centrifuge tube, draw centrifuge tube surface 2 ~ 3cm liquid, add in another same volume centrifugal tube, add pure water again to former centrifuge tube scale mixing, centrifuge tube loads centrifugal basket subrack, open centrifuge, setting speed is 3000rpm, centrifugal 15 minutes, takes out centrifuge tube after centrifugal stopping, abandoning supernatant adds pure water again to former centrifuge tube scale, carry out under arranging equally the 2nd time centrifugal, repeat above-mentioned centrifugally operated and carry out the 3rd centrifugal desalination, the precipitate after the 3rd centrifugal desalination is egg capsule;
(6) egg capsule scrubbing, sterilization, take out centrifuge tube abandoning supernatant, add 1% liquor natrii hypochloritis in precipitate egg capsule, mixing, acts on 5 minutes, water for injection is added again to centrifuge tube scale in centrifuge tube, centrifugal 15 minutes of 3000rpm, washes away postassium hypochlorite solution, repeated washing 1 ~ 2 time, add 2.5% potassium dichromate aqueous solution in egg capsule precipitation after scrubbing sterilization, egg capsule content is adjusted to 5 × 10
5/ ml;
(7) Sporulated, above-mentioned egg capsule is sub-packed in large plate, liquid level≤7mm, be placed on horizontal shaker object stage, start shaking table, under the prerequisite guaranteeing oxygen supply, egg capsule to 80% completes Sporulated to be stopped at once, and the whole Sporulated time needs 24 ~ 72 hours;
(8) egg capsule counting, counting Sporulated Oocysts number, calculates every ml egg capsule content, Sporulated Oocysts 2 × 5 × 10
5/ ml;
(9) egg capsule proportioning,
As being polyvalent vaccine, vaccine need be prepared in proportion.For trivalent Seedling, according to the form below is prepared.
Chicken Eimeria trivalent precocious Attenuate vaccine worm strain configuration proportion
Calculate the dosing amount of each worm strain by egg sac number, 1000 packaged loading amounts are 10ml/ bottle, and egg capsule liquid 1ml, adds 2.5% potassium dichromate 9ml; 500 packaged is 5ml/ bottle, and egg capsule liquid 0.5ml adds 2.5% potassium dichromate 4.5ml.Every ml vaccination 100 chickens.
(10) microencapsulation process, the 1st step: 3% sodium alginate adds in Agitation Tank, by 3% sodium alginate 10 by egg capsule mixing: egg capsule adds in sodium alginate by Sporulated Oocysts liquid 1, v/v ratio, and limit edged stirs;
2nd step: reaction encystation is after 15 ~ 20 minutes, open Agitation Tank leakage fluid dram, utilizing pressure (or potential difference) to make pot liquid evenly add another is equipped with in the Agitation Tank of 5% calcium chloride solution, stir pot liquid gently, make to add liquid reactions and form microcapsule, microcapsule diameter 2.0 ~ 3.0mm, is surrounded by about 60 ~ 72, egg capsule, average 66 in each capsule;
3rd step: cleaning sedimentation collects microcyst, with pure water cleaning, 30 DEG C of wind dryings;
4th step: counting calculates encapsulation with preparation and forms number, and using Semen Maydis powder as excipient, adjustment egg capsule content is 15 ~ 16/gram, and be example with 10 liters of Sporulated Oocysts liquid (10000 plumage parts, 1ml/ plumage), egg capsule adds up to 1 × 10
7individual, after above-mentioned preparation, form about 15 ~ 160,000 microcysts, can add in excipient in proportion, fully mix;
(11) subpackage, completes direct packaging after microencapsulation process.
Advantage of the present invention: by such scheme, this production technology substantially increases the efficiency that vaccine manufactures, and can meet the requirement of large-scale production, and significantly reduce production cost, be conducive to applying of product.Be mainly manifested in following several respects:
(1) high efficient mixer and screening machine relay screening is applied
Infectious chicken manure mixed processing and relay screening all employ the higher equipment of automaticity, and this increases work efficiency more than 2 times than traditional artificial screening.Mechanization screening is used not only to shorten sieving time, the more important thing is and improve screening efficiency, test shows, uses and is sieving through the egg capsule harvest yield raising more than 30% of filter than artificial screen cloth, this is because " screening power " that mechanical grading produces is much larger than manual method.
(2) application of continuous flow centrifugation technology and vast capacity centrifuge
In traditional coccidian oocyst isolation technics, centrifugalize is the core of overall technology, and this production technology be unable to do without centrifugalize equally.But traditional centrifugal method is subject to the restriction of technology and equipment, the demand that industrialization is produced can not be met.Produce for the industrialization that day process 300kg chicken manure is split run production capacity, mixed liquid manure amount can reach 3000 liters, after 60 orders and 200 mesh sieves divide, the liquid manure of 90% becomes filtrate (excrement slag < 10%), and whole filtrate reaches 2700 liters.The centrifuge of current largest domestic capacity is 6 × 2400ml, 1 time can centrifugal treating 11 liters of filtrates (by 76.4% loading amount), and process 2700 liters of filtrates, 1 desk centrifuge needs continuous operation 246 times; As 1 desk centrifuge work in 1 day 10 times, then need 25 super-magnum centrifuges, it is centrifugal that this does not also comprise follow-up a large amount of centrifugal purification, desalination and washing.Therefore, only have improvement centrifugal separation technology, be only the outlet of vaccine to chicken coccidiosis large-scale production.This production technology uses the continuous flow centrifuge that 1 flow velocity is 100L/h, and 27 little can process 2700 liters of materials immediately, if need complete the centrifugal of 2700 liters of materials within the one-shift operation time, then only need increase by 3 equipment.Relatively conventional centrifugal, the efficiency of continuous flow centrifugation improves 6 ~ 10 times, and single processing apparatus use amount reduces 20, and operator reduce by 40 people, significantly reduce production cost.Show through measuring and calculating, in 1 medium scale vaccine (producing 2 ~ 300,000,000 parts per year) factory, the production cost of subunit vaccine can reduce by more than 300%.In addition, this production technology saturated brine segmentation, desalination and de-sodium hypochlorite washing in all employ vast capacity (6 × 2400ml) centrifuge, this kind of centrifuge is the new architecture just appeared on the market in recent years, 1 time centrifugal processes 10 ~ 12 liters of materials, and work efficiency improves at least 3 times compared with conventional centrifuge.
(3) egg capsule microencapsulation process
The Clinical practice of vaccine to chicken coccidiosis mainly by with feedstuff, drink water mix after take in through mouth, but 2 problems can be run in actual use: one is when mixing with feedstuff, because the large property of forage volume is done, and the little property of amount of vaccine wets, uniform mixing cannot be accomplished, its result causes a part of chicken not take in enough egg capsules making immuning failure, and another part chicken then takes in too much egg capsule, occurs infecting even falling ill; Two is that when carrying out mixed drink, egg capsule falls to bottom due to action of gravity, the intake of egg capsule is had a greatly reduced quality, thus have impact on the immune quality of vaccine.Implement microencapsulation process to egg capsule and mainly contain following benefit: the solid dosage form 1. achieving vaccine, solid vaccine is when mixing with feedstuff, and the mobility of the two increases greatly, is easy to mix homogeneously; 2. make the volume of egg capsule increase, add by the probability initiatively taken in, as the color by manual change's microcyst, be more easily ingested; 3. microcyst produces protective effect to egg capsule, improves the quality of vaccine.
It is worthy of note, solid vaccine makes chicken while absorption egg capsule, has also taken in sodium alginate, the latter is the macromolecular compound that a kind of viscosity is very large, platymiscium polysaccharide, is also a kind of typical hydrophilic colloidal sol, is widely used in the industries such as food, printing and dyeing, medicine, papermaking.Be widely used as pharmaceutical preparation adjuvant at pharmaceutical field sodium alginate, there is good stability, dissolubility, viscosity and safety, and there is the ability of concentrated solution, formation gel and film forming.This product avirulence, LD
50value > 5000mg/kg, does not affect the quality and safety of vaccine.
Accompanying drawing explanation
Fig. 1 is the flow chart of this production technology.
Detailed description of the invention
Embodiment is completed by following steps: containing egg capsule chicken manure → high efficient mixed → relay screening → continuous flow centrifugation crude separation → saturated brine segmentation → centrifugal desalination → egg capsule sterilization → Sporulated → egg capsule counting → egg capsule proportioning → microencapsulation process → subpackage
(1) mixer high efficient mixed
1. condition
The higher mixer of automaticity is adopted to replace original container to mix, not only make mixed effect better, and, mixer has agitating function (speed of agitator≤60rpm), egg capsule sedimentation can be prevented, with malleation (+0.4P) or tool potential difference in blending tank, the liquid manure mixed can be made to be delivered to subsequent processing.The swept volume of mixer is no less than 500 liters.
2. operate
Get infected chicken fresh excreta 10kg in mixing all, add the dilution of 30Kg clear water, start agitator mixing, add pepsin 0.5g afterwards, continue mixing 1min, after leaving standstill 5min, add the clear water of 60Kg excrement amount again, continue stirring and evenly mixing.
(2) single-deck vibrating screen divides filter relay to sieve
1. condition
Vibrating motor: the vibration force that motor produces acts on compass screen surface by linking springs, makes screen cloth produce vibration force, increases the contact of material and screen cloth and prevent clogging;
Uneven weight: the converting rotary motion of motor is become level, inclination, the motion of vertical three-D, again this Motion Transmission to compass screen surface, material is produced on compass screen surface extend out gradually to open, whirlpool and to the multi-motion form in centralized, improve the running orbit of material to greatest extent, thus improve wire side utilization rate.By regulating the phase angle of upper and lower two ends weight, change the motion of material on screen cloth, to realize the clean cut separation to material.
Rotate: due to the rotation of motor, screen cloth rotates motion thereupon, rotating speed controls in 60 circles per minute, the existence of this centrifugal force can cause material to be piled up to the diffusion of wire side periphery, change the center of gravity angle of screen movement, make it to produce to the power in centralized, prevent solid accumulation.
Screening machine diameter 100cm (in screen cloth diameter), upper strata screen cloth 60 order, lower screen 200 order.
2. operate
Open mixer discharging opening, divide machine inlet capable once to add liquid manure 30 liters from upper sieve, liquid manure directly enters lower screen extension set and enters the 2nd screening after screening is filtered, and the filtrate of the 2nd screening enters fluid reservoir.
After 1st screening terminates, change screen cloth, then add 30 liters of liquid manures, carry out the 2nd screening, so circulation repeatedly, complete to all liquid manure screenings.
(3) continuous flow centrifugation crude separation
1. condition
Fluid reservoir capacity is not less than 500 liters, tool automatic stirring and reinforced function; Continuous flow centrifuge rotating speed is not less than 4000rpm, rotating speed control accuracy ± 20rpm, flow velocity >=50L/h.Running time controls many items chooses, 4 hours minimum continuous centrifugal time, automatic locking closure and internal lock system, unbalance protection, state autognostic, multiple circuit protection.
Tool programmable function and the display of digital rotating speed, controllable temperature.Meet pharmacy GMP requirement.
2. operate
The reinforced dress of fluid reservoir is connected with centrifuge feed arrangement, and open centrifuge charging switch, centrifuge speed is set as 4000rpm.Material enters centrifuge by charging aperture, is subject to the effect of centrifugal force in piece-rate system, retains egg capsule (comprising the impurity with egg capsule same volume) by filter screen, and liquid enters filtering cabin through screen cloth and discharged by liquid outlet.Like this, material imports continuously in centrifugal process, and egg capsule precipitation is stayed in centrifugal storehouse, and supernatant is then constantly discharged.The supernatant collection of discharging is in container, and can carry out continuous centrifugal again, egg capsule harvest yield can increase by 10%.
(4) saturated brine segmentation
1. condition
Saturated brine: add chemical grade sodium chloride (NaC1) in 90 DEG C of pure water, limit edged uses after cooling till being stirred to and occurring precipitation.
Vast capacity Centrifuge Techniques parameter:
Displacement volume >=2400ml × 6, horizontal rotor;
Maximum (top) speed >=4000rpm, LED numeral method and speed setting;
Controllable temperature, tool internal lock, unbalance protection and state self-checking function;
Meet pharmacy GMP specification, rotor easy cleaning, sterilization.
2. operate
After continuous centrifugal completes, take out centrifugal storehouse, shift out centrifugal sediment, be weighed as 3.82Kg in centrifugal storehouse, add 19.13Kg saturated brine and fully mix, equivalent adds centrifugal in centrifuge tube (3000 turns 10 minutes).
(5) centrifugal desalination
1. condition
Vast capacity Centrifuge Techniques parameter:
Displacement volume >=2400ml × 6, horizontal rotor;
Maximum (top) speed >=4000rpm, LED numeral method and speed setting;
Controllable temperature, tool internal lock, unbalance protection and state self-checking function;
Meet pharmacy GMP specification, rotor easy cleaning, sterilization.
2. operate
Last operation is centrifugal complete after taking-up centrifuge tube, careful draw centrifuge tube surface 2cm liquid, add in another centrifuge tube, then add pure water to original volume, mixing.Centrifuge tube loads centrifugal basket subrack, and open centrifuge, setting speed is 3000 turns, centrifugal 15 minutes.
Take out centrifuge tube after centrifugal stopping, abandoning supernatant adds pure water again to scale, carry out under arranging equally the 2nd time centrifugal.
Repeat above-mentioned centrifugally operated and carry out the 3rd centrifugal desalination.
Precipitate after 3rd centrifugal desalination, is egg capsule.
(6) egg capsule scrubbing, sterilization
1. condition
Original liquid concentration is 5.25% sodium hypochlorite, is mixed with 0.5% aqueous solution for subsequent use during use with distilled water by 1:9.
2. operate
Take out centrifuge tube abandoning supernatant, add 1% liquor natrii hypochloritis in precipitate (egg capsule), mixing, acts on 5 minutes.Add water for injection in centrifuge tube again to scale, 3000 leave the heart washes away postassium hypochlorite solution in 15 minutes, repeated washing 2 times.
Add 2.5% potassium dichromate aqueous solution in egg capsule precipitation after scrubbing sterilization, egg capsule content is adjusted to 5 × 10
5/ ml.
(7) Sporulated
1. condition
Sporulated place should meet ten thousand grades of purification conditions, Sporulated temperature 22 ~ 28 DEG C;
Horizontal shaker important technological parameters:
Rocking-turn mode: horizontal 360-degree rotates;
Speed: 30 ~ 300rpm;
Maximum load: >=10kg;
Stepless speed regulation, numerical monitor, meets pharmacy GMP standard.
2. operate
Above-mentioned egg capsule is sub-packed in large plate, liquid level≤7mm, is placed on horizontal shaker object stage, and start shaking table, under the prerequisite guaranteeing oxygen supply, the egg capsule to 80% completes Sporulated to be stopped at once, about 48 hours whole Sporulated time;
(8) egg capsule counting
Counting Sporulated Oocysts number, calculates every ml egg capsule content, Sporulated Oocysts 2 × 5 × 10
5/ ml;
(9) egg capsule proportioning
As being polyvalent vaccine, vaccine need be prepared in proportion.For trivalent Seedling, according to the form below is prepared.
Chicken Eimeria trivalent precocious Attenuate vaccine worm strain configuration proportion
Calculate the dosing amount of each worm strain by egg sac number, 1000 packaged loading amounts are 10ml, and egg capsule liquid 1ml, adds 2.5% potassium dichromate 9ml; 500 packaged is 5ml, and egg capsule liquid 0.5ml adds 2.5% potassium dichromate 4.5ml.Every ml vaccination 100 chickens.
(10) microencapsulation process
1. condition
3% sodium alginate and 5% calcium chloride solution;
Agitation Tank 1: tool agitating function, controllable temperature, capacity >=100 liter;
Agitation Tank 2: tool agitating function, controllable temperature, capacity is the twice of Agitation Tank 1.
2. operate
1st step: 3% sodium alginate adds in Agitation Tank by egg capsule mixing, and egg capsule adds in sodium alginate by (3% sodium alginate 10: Sporulated Oocysts liquid 1, v/v) in proportion, and limit edged stirs.
2nd step: reaction encystation is after 15 minutes, open Agitation Tank leakage fluid dram, utilizing pressure (or potential difference) to make pot liquid evenly add another is equipped with in the Agitation Tank of 5% calcium chloride solution, stir pot liquid gently, make to add liquid reactions and form microcapsule, microcapsule diameter 2.0 ~ 3.0mm, is surrounded by about 60 ~ 72, egg capsule, average 66 in each capsule.
3rd step: cleaning sedimentation collects microcyst, with pure water cleaning, 30 DEG C of winds are done.
4th step: counting calculates encapsulation with preparation and forms number, and using Semen Maydis powder as excipient, adjustment egg capsule content is 15 ~ 16/gram.Be example with 10 liters of Sporulated Oocysts liquid (10000 plumage parts, 1ml/ plumage), egg capsule adds up to 1 × 10
7individual, after above-mentioned preparation, form about 15 ~ 160,000 microcysts, can add in excipient in proportion, fully mix.
(11) subpackage
Complete direct packaging after microencapsulation process.
Claims (1)
1. chicken Eimeria attenuated vaccine industrialized manufacturing technique, it is characterized in that being completed by following steps: containing egg capsule chicken manure → mixing → relay screening → continuous flow centrifugation crude separation → saturated brine segmentation → centrifugal desalination → egg capsule sterilization → Sporulated → egg capsule counting → egg capsule proportioning → microencapsulation process → subpackage, concrete steps are:
(1) mix containing egg capsule chicken manure, get infected chicken fresh excreta in mixer, add 3 ~ 4 times of weight clear water dilutions, the pepsin of 0.05 ~ 0.1% of mixed solution gross weight is added again after stirring and evenly mixing, continue mixing 1 ~ 2min, add the clear water of 5 ~ 7 times of weight after leaving standstill 5 ~ 6min again, continue stirring and evenly mixing and become liquid manure;
(2) relay screening, screening machine divides two-layer up and down, upper screen cloth 60 order, lower screen 200 order, open mixer discharging opening, divides machine inlet capable once to add described liquid manure 25 ~ 35 liters from upper sieve, liquid manure directly enters lower screen extension set and carries out the 2nd screening after screening is filtered, the filtrate of the 2nd screening enters fluid reservoir, changes screen cloth, then adds 25 ~ 35 liters of liquid manures, proceed screening, so until the screening of all liquid manures is complete;
(3) continuous flow centrifugation crude separation, fluid reservoir feeding device is connected with centrifuge feed arrangement, open centrifuge charging switch, centrifuge speed is set as 4000rpm, the complete material of described screening enters centrifuge by charging aperture, the effect of centrifugal force is subject in piece-rate system, egg capsule is retained by filter screen, supernatant enters filtering cabin through screen cloth and is discharged by liquid outlet, the complete material of described screening imports continuously in centrifugal process, egg capsule precipitation is stayed in centrifugal storehouse, and supernatant is then constantly discharged, and the supernatant collection of discharge is in container;
(4) saturated brine segmentation, after continuous centrifugal completes, takes out centrifugal storehouse, and in centrifugal storehouse, shift out centrifugal egg capsule precipitate weigh, the saturated brine adding 5 times of weights fully mixes, then to add in centrifuge tube with 3000rpm centrifugal 10 minutes;
(5) centrifugal desalination, last operation is centrifugal complete after taking-up centrifuge tube, draw centrifuge tube surface 2 ~ 3cm liquid, add in another same volume centrifugal tube, add pure water again to former centrifuge tube scale mixing, centrifuge tube loads centrifugal basket subrack, open centrifuge, setting speed is 3000rpm, centrifugal 15 minutes, takes out centrifuge tube after centrifugal stopping, abandoning supernatant adds pure water again to former centrifuge tube scale, carry out under arranging equally the 2nd time centrifugal, repeat above-mentioned centrifugally operated and carry out the 3rd centrifugal desalination, the precipitate after the 3rd centrifugal desalination is egg capsule;
(6) egg capsule scrubbing, sterilization, take out centrifuge tube abandoning supernatant, add 1% liquor natrii hypochloritis in precipitate egg capsule, mixing, acts on 5 minutes, water for injection is added again to centrifuge tube scale in centrifuge tube, centrifugal 15 minutes of 3000rpm, washes away liquor natrii hypochloritis, repeated washing 1 ~ 2 time, add 2.5% potassium dichromate aqueous solution in egg capsule precipitation after scrubbing sterilization, egg capsule content is adjusted to 5 × 10
5/ ml;
(7) Sporulated, above-mentioned egg capsule is sub-packed in large plate, liquid level≤7mm, be placed on horizontal shaker object stage, start shaking table, under the prerequisite guaranteeing oxygen supply, egg capsule to 80% completes Sporulated to be stopped at once, and the whole Sporulated time needs 24 ~ 72 hours;
(8) egg capsule counting, counting Sporulated Oocysts number, calculates every ml egg capsule content, Sporulated Oocysts 2 × 5 × 10
5/ ml;
(9) egg capsule proportioning,
As being polyvalent vaccine, need prepare vaccine in proportion, for trivalent Seedling, according to the form below is prepared,
Chicken Eimeria trivalent precocious Attenuate vaccine worm strain configuration proportion
Calculate the dosing amount of each worm strain by egg sac number, 1000 packaged loading amounts are 10ml/
Bottle, egg capsule liquid 1ml, adds 2.5% potassium dichromate 9ml; 500 packaged is 5ml/ bottle, and egg capsule liquid 0.5ml adds 2.5% potassium dichromate 4.5ml;
(10) microencapsulation process, the 1st step: 3% sodium alginate adds in Agitation Tank, by 3% sodium alginate 10 by egg capsule mixing: egg capsule adds in sodium alginate by Sporulated Oocysts liquid 1, v/v ratio, and limit edged stirs;
2nd step: reaction encystation is after 15 ~ 20 minutes, open Agitation Tank leakage fluid dram, utilizing pressure or potential difference to make pot liquid evenly add another is equipped with in the Agitation Tank of 5% calcium chloride solution, stir pot liquid gently, make to add liquid reactions and form microcapsule, microcapsule diameter 2.0 ~ 3.0mm, is surrounded by 60 ~ 72, egg capsule in each capsule, average 66;
3rd step: cleaning sedimentation collects microcyst, with pure water cleaning, 30 DEG C of wind dryings;
4th step: counting calculates encapsulation with preparation and forms number, and using Semen Maydis powder as excipient, adjustment egg capsule content is 15 ~ 16/gram, and with 10 liters of Sporulated Oocysts liquid, 10000 plumage parts, 1ml/ plumage is example, and egg capsule adds up to 1 × 10
7individual, after above-mentioned preparation, form 15 ~ 160,000 microcysts, can add in excipient in proportion, fully mix;
(11) subpackage, completes direct packaging after microencapsulation process.
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| CN1951374A (en) * | 2006-11-07 | 2007-04-25 | 广西大学 | A chicken Coccidiosis pill form vaccine and preparation method thereof |
| CN101045159A (en) * | 2007-04-30 | 2007-10-03 | 吉林农业大学 | Method for preparing vaccine to chicken coccidiosis |
| CN102174408A (en) * | 2011-01-10 | 2011-09-07 | 内蒙古神元生物工程股份有限公司 | Method for batch preparation of chicken Eimeria coccidium oocysts |
| CN102796669A (en) * | 2012-08-07 | 2012-11-28 | 广西壮族自治区兽医研究所 | Method for extracting and purifying eimeria tenella merozoite |
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| JP2011521955A (en) * | 2008-05-29 | 2011-07-28 | インターベツト・インターナシヨナル・ベー・ベー | Coccidiosis vaccine |
| US20100150958A1 (en) * | 2008-12-15 | 2010-06-17 | Vectogen Pty Ltd. | Methods and Compositions for Use of a Coccidiosis Vaccine |
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| CN1951374A (en) * | 2006-11-07 | 2007-04-25 | 广西大学 | A chicken Coccidiosis pill form vaccine and preparation method thereof |
| CN101045159A (en) * | 2007-04-30 | 2007-10-03 | 吉林农业大学 | Method for preparing vaccine to chicken coccidiosis |
| CN102174408A (en) * | 2011-01-10 | 2011-09-07 | 内蒙古神元生物工程股份有限公司 | Method for batch preparation of chicken Eimeria coccidium oocysts |
| CN102796669A (en) * | 2012-08-07 | 2012-11-28 | 广西壮族自治区兽医研究所 | Method for extracting and purifying eimeria tenella merozoite |
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Denomination of invention: Industrial production process of eimeria coccidiosis attenuated vaccine Effective date of registration: 20180703 Granted publication date: 20150715 Pledgee: Zhang Yongping Pledgor: Inner Mongolia Shenyuan Biomedical Co.,Ltd. Registration number: 2018990000521 |
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