CN102258774A - Preparation method of Brucellosis live vaccine and product thereof - Google Patents
Preparation method of Brucellosis live vaccine and product thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a Brucellosis live vaccine and a product thereof. The method comprises primary strain preparation, secondary strain preparation and bacterial solution culture, wherein in the step of bacterial solution culture, the secondary strain is inoculated onto a liquid culture medium, and air is introduced in a manner of gradually increasing ventilation for culture; and after the bacteria strain is cultured for 30 hours, pure oxygen is introduced till the bacterial strain is cultured for 36 hours. In the method disclosed by the invention, pure oxygen is introduced at a time when it is most suitable for bacteria to prorogate in a large amount in the bacterial solution culture step, so the quantity of the cultured bacteria is increased greatly to 150 to 190 billion per milliliter. When the method is used, the bacterial solution is not required to be concentrated; and the method has the advantages of high yield, low production cost and the like.
Description
Technical field
The present invention relates to a kind of preparation method of animal vaccine, relate in particular to the preparation method of Brucella disease vaccine and, the invention belongs to the production field of Brucella disease vaccine by the vaccine that this method prepares.
Background technology
(full name Bu Lushi bacillosis (brucellosis) is the acute and chronic infectious disease of the infecting both domestic animals and human that caused by Brucella to brucellosis.Clinical characters is slow onset, long-term heating, hyperhidrosis, weakness, pantalgia and arthralgia, and acute stage symptom is many to disappear in 3～June.Brucella is divided into and is six kinds of cattle, sheep, pig, sarin Mus, sheep and dog Brucellas.First three kind that is mainly in China's discovery.
Brucella is tiny rod-short or club shape, does not produce the brood cell, the bacillus of Gram-negative.Brucella is to thermo-responsive, 70 ℃ 10 minutes can be dead; Sunlight direct projection death in 1 hour; In corrupt pathological material of disease, lose vigor rapidly; General disinfectant commonly used can both kill it very soon.In two weeks of weak point person incubation period, the elder can reach half a year.The cow miscarriage is the cardinal symptom of primary disease, and the miscarriage pilosity is born in conceived 5-7 month, output stillborn fetus or weak fetus.Cow post-abortion, intravaginal continued to discharge bronzing stench liquid often with placenta retention or endometritis, and in sustainable 2-3 week, orchitis or epididymitis often take place ill bull.The people also can infect this disease, shows as symptoms such as long-term heating, hyperhidrosis, arthralgia, neuralgia and liver, splenomegaly. the health of this disease grievous injury humans and animals, and OIE classifies it as category-B animal epidemic, and China classifies them as two class animal epidemics.
Brucellosis all constitutes a serious threat to animal husbandry and human health, and vaccine immunity is the major measure of prevention and control brucellosis.Because the entozoic characteristics of Brucella born of the same parents, several antibiotic that have only minority such as rifampicin to its treatment effectively, but these antibiotic side effect are very big, can not take for a long time, in case therefore fall ill and transfer to chronic then can't effect a radical cure, and can show effect repeatedly.Therefore, vaccination is the most effective means of prevention brucellosis.
Brucella live vaccine A19 strain is the very effective attenuated vaccine strain of anti-system brucellosis, and this vaccine is applicable to cattle and sheep, and every part immunizing dose of cattle is 60,000,000,000-80,000,000,000.The producting rule method of existing Brucella A19 strain live vaccine is as follows:
The first class inoculum preparation: behind the freeze-drying lactobacillus Kaifeng, with the peptone water dissolving, line is transplanted on Yi Dan Bai Shi agar plate or other appropriate medias, cultivates 2-3 day at 36-37 ℃.Choose qualified bacterium colony more than 10, transplant agar slant culture-medium, cultivated 48-72 hour for 36-37 ℃, as first order seed in Yi Dan Bai Shi.2-8 ℃ of preservation, the operating period should be no more than 1 month.
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate medias of Yi Dan Bai Shi agar, putting 36-37 ℃ cultivated 48-72 hour, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the suitable fluid medium of 10,000-20,000 ml, put 36-37 ℃ and cultivated 48-72 hour, check rearmounted 2-8 ℃ preservation purely, the operating period should be no more than 30 days.
Bacterium liquid is cultivated: add appropriate amount of defoamer by cultivating base unit weight, the sterilization back is by the 1%-2% inoculation second class inoculum of cultivating base unit weight, increase ventilation gradually at 36-37 ℃, cultivated 36 hours, can add 50% glucose solution in the incubation as required, each addition is the 1%-2% of culture medium total amount.
Carrying out Brucella live vaccine (A19 strain) the production viable count that obtains bacterium liquid of cultivating by existing rules is 800-1000 hundred million/ml, need carry out concentration to this bacterium liquid, and problems such as existence yields poorly, production cost height have much room for improvement.
Summary of the invention
Technical problem to be solved by this invention be overcome existingly in the production method of existing Brucella live vaccine yield poorly, problem such as production cost height, a kind of production method of new Brucella live vaccine is provided, this production method is cultivated the bacterium number that obtains bacterium liquid can reach 1500-1800 hundred million/ml, has advantages such as the output of existence height, production cost be low.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of preparation method of Brucella disease live-vaccine comprises first class inoculum preparation, second class inoculum preparation and the cultivation of bacterium liquid; Wherein, at first cultivate behind the fluid medium inoculation second class inoculum, begin to feed pure oxygen when being cultured to 30h and finish until cultivating according to the mode bubbling air that increases ventilation gradually at bacterium liquid cultivation stage.
The present invention further finds, the mode of different feeding pure oxygens has the difference of highly significant on the bacterium number of culture fluid, in order to improve the bacterium number of culture fluid to greatest extent, comparison of the present invention and the mode of having screened different feeding pure oxygens, the final discovery, feed pure oxygen in the following ways, it is the highest that the bacterium in the culture fluid is counted output, can reach 1700-1800 hundred million/ml: when being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 2m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 95%-98%, cultivate until 36h and finish.
The present invention finds that also at bacterium liquid cultivation stage, the mode of bubbling air also has certain influence for the bacterium number of culture fluid.The present invention finds by a large amount of experiment, bacterium liquid cultivation stage in such a way progressively bubbling air can effectively improve culture fluid bacterium number:
Behind the fluid medium inoculation second class inoculum at first according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h.
In addition, the present invention also finds, at bacterium liquid cultivation stage, the adding mode of 50% glucose solution also has certain influence for culture fluid bacterium number: by existing rules, the adding method of 50% glucose solution is to add 50% glucose solution in the incubation as required, and each addition is the 1%-2% of culture medium gross mass.The present invention found through experiments, if be the bacterial number that 2wt% can effectively improve the disposable adding of 50% glucose solution culture fluid by final content just when inoculation.
The inventive method selects the prolific period of this antibacterial of optimum to feed pure oxygen in bacterium liquid cultivation stage, cultivation bacterium number is greatly improved, the bacterium number can reach 1500-1900 hundred million/ml in the culture fluid, when using, need not this bacterium liquid is carried out concentration, have advantages such as output height, production cost are low.
Description of drawings
The process chart of Fig. 1 preparation method of the present invention.
The specific embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Embodiment 1
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 3 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 72 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 72 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 10,000 ml, put 37 ℃ and cultivated 72 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation; The composition of every 1000ml martin's bouillon culture medium is:
Steamed beef soup: 500ml
Gaster Sus domestica Digestive system: 500ml
Sodium chloride: 2.5g
PH6.4-6.7
Sterilize 116 ℃ 30-40 minute
Bacterium liquid is cultivated: the quality of pressing the martin's bouillon fluid medium adds 0.01% defoamer, sterilization back is that 2wt% is with the disposable adding of 50% glucose solution, by bubbling air behind the 2% inoculation second class inoculum of martin's bouillon fluid medium quality and increase the feeding amount of air gradually by final content; When being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 2m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 95%-98%, cultivate until 36h and finish.Through check, the bacterium number is 1,600 hundred million/ml in the culture fluid.
Embodiment 2
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 3 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 72 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 72 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 10,000 ml, put 37 ℃ and cultivated 72 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: the quality of press the martin's bouillon fluid medium adds 0.01% defoamer, the sterilization back by final content be 2wt% with the disposable adding of 50% glucose solution, press 2% of martin's bouillon fluid medium quality and inoculate behind the second class inoculum just according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h; When being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 2m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 95%-98%, cultivate until 36h and finish.Through check, the bacterium number is 1,850 hundred million/ml in the culture fluid.
Embodiment 3
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 3 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 72 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 72 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 20,000 ml, put 37 ℃ and cultivated 72 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: the quality of press the martin's bouillon fluid medium adds 0.01% defoamer, the sterilization back by final content be 2wt% with the disposable adding of 50% glucose solution, press 2% of martin's bouillon fluid medium quality and inoculate behind the second class inoculum just according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h; When being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 2m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 95%-98%, cultivate until 36h and finish.Through check, the bacterium number is 1,790 hundred million/ml in the culture fluid.
Embodiment 4
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 2 at 36 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 48 hours for 36 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 36 ℃ cultivated 48 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 10,000 ml, put 36 ℃ and cultivated 48 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: the quality of pressing the martin's bouillon fluid medium adds 0.01% defoamer, sterilization back by final content be 2wt% with the disposable adding of 50% glucose solution, the 1%-2% that press martin's bouillon fluid medium quality inoculates behind the second class inoculum just according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h; When being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, cultivates until 36h and finishes.Through check, the bacterium number is 1,500 hundred million/ml in the culture fluid.
Embodiment 5
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 2 at 36 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 60 hours for 36 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 36 ℃ cultivated 60 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 1.5 ten thousand ml, put 36 ℃ and cultivated 60 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: the quality of press the martin's bouillon fluid medium adds 0.01% defoamer, the sterilization back by final content be 2wt% with the disposable adding of 50% glucose solution, press 1% of martin's bouillon fluid medium quality and inoculate behind the second class inoculum just according to 8m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 12m
3/ h, the ventilation with air behind the cultivation 20h further increases to 16m
3/ h; When being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 2m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 95%-98%, cultivate until 36h and finish.Through check, the bacterium number is 1,720 hundred million/ml in the culture fluid.
Embodiment 6
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 3 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 48 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 48 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 20,000 ml, put 37 ℃ and cultivated 72 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: the quality of pressing the martin's bouillon fluid medium adds 0.01% defoamer, sterilization back by final content be 2wt% with the disposable adding of 50% glucose solution, the 1%-2% that press martin's bouillon fluid medium quality inoculates behind the second class inoculum just according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h; When being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 2m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 95%-98%, cultivate until 36h and finish.Through check, the bacterium number is 1,800 hundred million/ml in the culture fluid.
Embodiment 7
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 3 at 36 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 72 hours for 36 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 72 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 10,000 ml, put 37 ℃ and cultivated 72 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: the quality of pressing the martin's bouillon fluid medium adds 0.01% defoamer, sterilization back by final content be 2wt% with the disposable adding of 50% glucose solution, the 1%-2% that press martin's bouillon fluid medium quality inoculates behind the second class inoculum just according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h; When being cultured to 30h according to 6m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 1m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 94%, cultivate until 36h and finish.Through check, the bacterium number is 1,650 hundred million/ml in the culture fluid.
Embodiment 8
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 2 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 48 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 72 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 10,000 ml, put 37 ℃ and cultivated 72 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: the quality of pressing the martin's bouillon fluid medium adds 0.01% defoamer, sterilization back by final content be 2wt% with the disposable adding of 50% glucose solution, the 1%-2% that press martin's bouillon fluid medium quality inoculates behind the second class inoculum just according to 12m
3The feeding amount of/h feeds normal air, since 6h the ventilation of air is increased to 12m
3/ h, the ventilation with air behind the cultivation 12h further increases to 16m
3/ h; When being cultured to 30h according to 6m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 1m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 97% until 36h cultivation end.Through check, the bacterium number is 1,620 hundred million/ml in the culture fluid.
The comparative example 1
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 3 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 72 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 72 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 20,000 ml, put 37 ℃ and cultivated 72 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: add appropriate amount of defoamer by cultivating base unit weight, the sterilization back is by the 1%-2% inoculation second class inoculum of cultivating base unit weight, increase ventilation gradually at 36-37 ℃, cultivated 36 hours, can add 50% glucose solution in the incubation as required, each addition is the 1%-2% of culture medium total amount.Through check, the bacterium number is 82,000,000,000/ml in the culture fluid.
The comparative example 2
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 2 at 36 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 48 hours for 36 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 36 ℃ cultivated 48 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 10,000 ml, put 36 ℃ and cultivated 48 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: add appropriate amount of defoamer by cultivating base unit weight, the sterilization back is by the 1%-2% inoculation second class inoculum of cultivating base unit weight, and inoculation 36-37 ℃ of cultivation in back is according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h cultivates until 36h and finishes.Through check, the bacterium number is 98,000,000,000/ml in the culture fluid.
The comparative example 3
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 2 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 48 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 48 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 20,000 ml, put 37 ℃ and cultivated 48 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: add appropriate amount of defoamer by cultivating base unit weight, the sterilization back is by the 1%-2% inoculation second class inoculum of cultivating base unit weight, and inoculation 36-37 ℃ of cultivation in back is according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h cultivates until 36h and finishes.Through check, the bacterium number is 90,000,000,000/ml in the culture fluid.
The comparative example 4
The first class inoculum preparation: behind Brucella disease live-vaccine A19 strain freeze-drying lactobacillus (available from China Veterinery Drug Inspection Office) Kaifeng, with the peptone water dissolving, line is transplanted on TA flat board or other appropriate media, cultivates 2 at 37 ℃.Choose qualified bacterium colony more than 10, transplant, cultivated 60 hours for 37 ℃, as first order seed, 2-8 ℃ of preservation in the TA slant medium;
Second class inoculum preparation: get first class inoculum and be inoculated in flat or other appropriate media of TA, putting 37 ℃ cultivated 60 hours, after macroscopy is pure, each is flat, and to add peptone water an amount of, lawn is washed, be inoculated in the martin's bouillon fluid medium of 1.5 ten thousand ml, put 37 ℃ and cultivated 60 hours, obtain second class inoculum after checking purely, put 2-8 ℃ of preservation;
Bacterium liquid is cultivated: add appropriate amount of defoamer by cultivating base unit weight, the sterilization back is by the 1%-2% inoculation second class inoculum of cultivating base unit weight, and inoculation 36-37 ℃ of cultivation in back is according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h cultivates until 36h and finishes.Through check, the bacterium number is 85,000,000,000/ml in the culture fluid.
Claims (7)
1. the preparation method of a Brucella disease live-vaccine comprises first class inoculum preparation, second class inoculum preparation and the cultivation of bacterium liquid; It is characterized in that:, cultivate according to the mode bubbling air that increases ventilation gradually behind the fluid medium inoculation second class inoculum at bacterium liquid cultivation stage; Beginning to feed pure oxygen when being cultured to 30h finishes until cultivating.
2. according to the described preparation method of claim 1, it is characterized in that: when being cultured to 30h according to 8m
3The feeding amount of/h feeds pure oxygen, and later every 2h increases 2m with ventilation
3, make culture fluid dissolved oxygen amount DO value remain on 95%-98%, cultivate until 36h and finish.
3. according to the described preparation method of claim 1, it is characterized in that: behind the fluid medium inoculation second class inoculum at first according to 12m
3The feeding amount of/h feeds normal air, since 10h the ventilation of air is increased to 16m
3/ h, the ventilation with air behind the cultivation 20h further increases to 18m
3/ h.
4. according to the described preparation method of claim 1, it is characterized in that: by mass percentage, contain 2% glucose in the described fluid medium.
5. by the preparation-obtained Brucella live vaccine of any one preparation method of claim 1-4.
6. the described Brucella live vaccine of claim 5 is preparing the purposes of preventing in the brucellosis reagent.
7. the described Brucella live vaccine of claim 5 is preparing the purposes for the treatment of in the Brucella medicine.
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| CN103289986A (en) * | 2013-04-27 | 2013-09-11 | 华南农业大学 | Cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof |
| CN104436181A (en) * | 2013-09-16 | 2015-03-25 | 北京中科拜克生物技术有限公司 | Novel brucella vaccine and production method thereof |
| CN104560781A (en) * | 2014-12-01 | 2015-04-29 | 中牧实业股份有限公司 | Method for improving living bacterium rate of brucellosis live vaccine product |
| CN106511990A (en) * | 2016-11-04 | 2017-03-22 | 金宇保灵生物药品有限公司 | A concentration process and preparing process for a freeze-dried brucellosis live vaccine for veterinary use |
| CN107058193A (en) * | 2017-06-15 | 2017-08-18 | 金宇保灵生物药品有限公司 | A kind of fermentation process in high density of brucellosis live vaccine S2 thalline |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103289986A (en) * | 2013-04-27 | 2013-09-11 | 华南农业大学 | Cattle Brucella recombination strain S19-delta bp 26-BL and preparation method and application thereof |
| CN104436181A (en) * | 2013-09-16 | 2015-03-25 | 北京中科拜克生物技术有限公司 | Novel brucella vaccine and production method thereof |
| CN104560781A (en) * | 2014-12-01 | 2015-04-29 | 中牧实业股份有限公司 | Method for improving living bacterium rate of brucellosis live vaccine product |
| CN104560781B (en) * | 2014-12-01 | 2017-05-10 | 中牧实业股份有限公司 | Method for improving living bacterium rate of brucellosis live vaccine product |
| CN106511990A (en) * | 2016-11-04 | 2017-03-22 | 金宇保灵生物药品有限公司 | A concentration process and preparing process for a freeze-dried brucellosis live vaccine for veterinary use |
| CN107058193A (en) * | 2017-06-15 | 2017-08-18 | 金宇保灵生物药品有限公司 | A kind of fermentation process in high density of brucellosis live vaccine S2 thalline |
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