CN104328077A - Avibacterium paragallinarum fermentation culture method - Google Patents
Avibacterium paragallinarum fermentation culture method Download PDFInfo
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Abstract
The invention discloses an avibacterium paragallinarum fermentation culture method, belonging to the field of avibacterium paragallinarum fermentation culture. The avibacterium paragallinarum fermentation culture method comprises the following steps: (1) preparation of primary seed solution; (2) preparation of secondary seed solution; (3) preparation of tertiary seed solution; and (4) fermentation culture of bacterial liquid. According to the avibacterium paragallinarum fermentation culture method, the fermentation culture conditions of the avibacterium paragallinarum are improved in the aspects of inoculation method, culture time and initial inoculation amount; a four-step inoculation method is used and the inoculation proportion is 2.5-10%; the culture manner is fermentation tank culture; and under the condition of not changing the total volume of each fermentation and not increasing the personnel and raw material cost, the avibacterium paragallinarum fermentation culture method has the effects that the fermentation end viable count of the avibacterium paragallinarum is up to 9.8*10<8>CFU/mL, the total count is up to 5.67 billion/mL, the yield of each batch is increased by 5 times, the seedling distribution requirements can be achieved without concentration and large-scale production and application can be carried out.
Description
Technical field
The present invention relates to the cultural method of secondary chicken fowl bacillus, particularly relate to the fermentation culture method of secondary chicken fowl bacillus, belong to the fermentation culture field of secondary chicken fowl bacillus.
Background technology
Infectious coryza of chicken (Infectious Coryza, IC) is a kind of Acute respiratory infectious disease being caused chicken by secondary chicken fowl bacillus (Avibacterium paragallinarum, Apg), is multiplely born in cold wet season.This disease can make Growing Chicken retarded growth and laying hen lay eggs obvious decline (laying rate can be caused to decline 10% ~ 40%), is one of important epidemic disease at the centralization-breeding factory that China is large-scale.At present in the world, many places have and to occur and popular this disease, China to be separated to first in 1987 this cause of disease (Feng Wenda. the Isolation and Identification [J] of Beijing infectious coryza of chicken pathogenic bacteria. microbiology is circulated a notice of, 1987,5:216 ~ 219.), also many strains secondary chicken fowl bacillus (Yang Guoliang is separated in Shandong Province of China, Beijing successively in recent years, the Isolation and ldentification [J] of the .A type para bacillus fowl blood philis (Shandong strain) such as Zheng Jie. Chinese animal and veterinary, 2013,40 (2): 189-192.).Along with the development of poultry husbandry, breeding environment constantly complicated, the occurrence frequency of infectious coryza of chicken is more and more higher, causes very large financial loss to poultry husbandry.
At present, medicine or vaccine immunity two kinds of methods are mainly taked in the control for infectious coryza of chicken.But after chicken medication, the original shape of medicine or its meta-bolites and relative substance may be accumulated, remain in the tissue of animal, organ or edible product, cause residual in animal food of veterinary drug, serious harm is caused to human body.So the infectious coryza of chicken vaccine of current high-quality is still the first-selection of this disease of prevention.But the cultural method of existing secondary chicken fowl bacillus exists the defect yielded poorly, concentration need be carried out to bacterium liquid and just can reach and join seedling requirement (" regulations " requires that work in-process total count is 5,000,000,000/mL).Therefore, be urgently optimized effectively to improve unit output to the high density fermentation critical process of secondary chicken fowl bacillus, be convenient to carry out large-scale industrial production.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of fermentation culture method of secondary chicken fowl bacillus (Avibacterium paragallinarum), the method significantly improves unit output, make fermentation termination total count reach 56.7 hundred million/mL, need not concentrate to reach and join seedling requirement.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
The invention discloses a kind of fermentation culture method of secondary chicken fowl bacillus (Avibacterium paragallinarum), comprise the following steps: the preparation of (1) primary seed solution; (2) preparation of secondary seed solution; The preparation of (3) three grades of seed liquor; (4) bacterium liquid fermentation culture.
Wherein, the preparation of step (1) described primary seed solution comprises: by secondary chicken fowl bacillus dilute, gets 4mL and is seeded to 160mL semisynthetic medium cultivation 11h; Preferably, described dilution is that the PBS being 7.2 by the secondary chicken fowl bacillus specie poison 5mL pH value of freeze-drying preservation dilutes.
The preparation of step (2) described secondary seed solution comprises: 160mL primary seed solution is seeded to 1600mL semisynthetic medium and cultivates 4h.
The preparation of described primary seed solution and secondary seed solution is 37 DEG C, and 200r/min shaking table is cultivated.
The preparation of the described three grades of seed liquor of step (3) comprises: 1600mL secondary seed solution is seeded to 30000mL semisynthetic medium and cultivates 4h; Described cultivation is that 37 DEG C of 200r/min magnetic agitation or shaking table are cultivated; Be preferably 200r/min magnetic agitation to cultivate.
The preparation of primary seed solution of the present invention, secondary seed solution and three grades of seed liquor, the terminal of described cultivation is to the OD of seed liquor
550value is 0.50 ± 0.05.
The fermentation culture method of secondary chicken fowl bacillus of the present invention, step (4) described bacterium liquid fermentation culture comprises: 30000mL tri-grades of seed liquor are seeded to 300L fermentor tank, and magnetic agitation is cultivated; Fermentation termination is bacterium liquid OD
550value is 0.55 ± 0.05.Substratum is semisynthetic medium; Culture temperature is 37 DEG C, and pH is 7.2, and mixing speed is 200r/min.
Utilize the fermentation culture method of secondary chicken fowl bacillus of the present invention to cultivate secondary chicken fowl bacillus, during fermentation ends, viable count reaches 9.8 × 10
8cFU/mL, total count reaches 56.7 hundred million/mL.
The fermentation culture method of secondary chicken fowl bacillus of the present invention is applicable to the secondary chicken fowl bacillus strain that any one can obtain.
The present invention improves from the fermentation culture of aspect to secondary chicken fowl bacillus C-Apg-8 (Page A type) such as vaccination ways, incubation time, initial inoculums.The present invention changes the two step inoculation methods that existing secondary chicken fowl bacillus produces: first order seed → secondary seed (7500mL) → 300L is four step inoculation method: 4mL → 160mL → 1600mL → 30000mL → 300L.
With regard to seed liquor inoculum size in bacterial fermentation processes, increase inoculum size and obviously can shorten the adaptive process of seed liquor to substratum, thus comparatively fast enter logarithmic phase, shorten lag phase.But increase inoculative proportion and also have certain limit, there are some researches show, time inoculum size is excessive, the nutritive ingredient that can consume excessively in substratum produces the meta-bolites that a large amount of anti-bacterias continues to increase, and is so all unfavorable for fermentation culture simultaneously.Meanwhile, larger inoculum size also means the increasing to the workload preparing seed, for the production work of reality, is also very disadvantageous.The present invention, through the inoculative proportion relatively and in conjunction with production practical situation selecting 2.5 ~ 10%, produces a desired effect.
In the production process of bacterial vaccine, the best incubation time of seed liquor is that the logarithmic growth end of term, (now the viable count of this bacterium was maximum value, and cell age is still in logarithmic phase), this is because in logarithmic phase the generation time of bacterial population constant, number of bacteria increases with geometricprogression amount; In logarithmic phase, the split speed of thalline is the fastest, between thalline individuality, cell composition component difference is less, O antigen property is relatively stable, consistent, therefore uses the seed liquor of logarithmic phase to inoculate seedling bacterium liquid and can make that the antigenic quality of seedling bacterium liquid is relatively stable, lag phase shortens greatly thus make whole culture cycle the shortest.Under culture condition of the present invention, the incubation time of primary seed solution should control within 11h.
Under certain condition (as substratum, temperature, pH value etc.), the generation time of each bacterium is constant (Li Jilun, Zhang Weixin etc. microbial physiology [M]. Beijing: press of Beijing Agricultural University, 1993, 422-426), therefore in the actual production process of inactivated vaccine of infectious coryza of chicken antigen, when needing incubation time or the bacterial concentration adjusting seed liquor, the generation time of the seed liquor recorded and required seed liquor incubation time or bacterial concentration just can be utilized to calculate optimum initial inoculum size as long as ensure that culture condition is constant, thus reach the object of production control technical process.
In actual production, because the repeatability of biological test is poor, wish that OD value when accurately finding seed liquor viable count the highest is difficult.The present invention passes through the corresponding data of a large amount of OD values and viable count, OD value scope (550nm) when finding logarithm end of term viable count to reach certain numerical value, namely for seed liquor be: 0.50 ± 0.05, for seedling bacterium liquid fermentation termination be: 0.55 ± 0.05 (because fermentor cultivation secondary chicken fowl bacillus needs ventilation, stir, mend alkali, therefore viable count increases to some extent, cause OD value slightly higher than seed liquor), so just can estimate viable count now, easy solves the problem measuring viable count length consuming time, the results terminal of secondary chicken fowl bacillus can be judged fast by this method, thus finally carry out Instructing manufacture.
The two step inoculation methods that the present invention produces by changing existing secondary chicken fowl bacillus: first order seed → secondary seed (7500mL) → 300L is four step inoculation method: 4mL → 160mL → 1600mL → 30000mL → 300L, inoculative proportion becomes 2.5 ~ 10% from 2.5% before, training method becomes fermentor cultivation from large bottle, do not changing single Batch fermentation cumulative volume simultaneously, when not increasing personnel and material cost, fermentation termination viable count is finally made to improve close to 13 times (9.8 × 10 before optimizing
8→ 7.7 × 10
7cFU/mL), total count improves close to 5 times (11.5 hundred million/mL → 56.7 hundred million/mL), and namely single batch of output increases by 500, and bacterium liquid need not concentrate to reach joins seedling antigen requirement (regulatory requirements work in-process total count is 5,000,000,000/mL).The secondary chicken fowl bacillus fermentation cultural method of optimizing application of the present invention shows in the good result of the secondary chicken fowl bacteroides antigen of veterinary biologics GMP workshop continuous seepage six batches, and the fermentation culture method of the present invention secondary chicken fowl bacillus completely can large-scale production and application.
In the production of vaccine of reality, the secondary chicken fowl bacillus emulsification seedling that the present invention will cultivate after process modification, carries out the Immunization protection test of vaccine, and it is attacked malicious protection ratio and reaches 100% qualified.
the term definition arrived involved in the present invention
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.
Term " inoculation " means object microorganism to be moved by aseptic technique requirement the process received in culture medium.
The term " vaccine " be used interchangeably or " vaccine composition " refer to such pharmaceutical composition, and it is included at least one immunogenic composition of induce immune response in animal.Vaccine or vaccine composition can watch for animals from the disease owing to infecting or possible death, and can comprise or not comprise one or more other components immunocompetent of enhanced activity component.Vaccine or vaccine composition can comprise in addition for vaccine or the typical component further of vaccine composition, comprise such as adjuvant or immunomodulator.The immunoactive component of vaccine can comprise using the complete live organism of its primitive form or in modified living vaccine as the organism through attenuation, through kill or deactivation vaccine in by the organism of appropriate method deactivation, or comprise the subunit vaccine of one or more immunogenic components of virus, or the genetic modification prepared by method known to those skilled in the art, sudden change or clone vaccine.Vaccine or vaccine composition can comprise one or exceed a kind of said components simultaneously.
Accompanying drawing explanation
Fig. 1 is seed liquor growth curve;
Fig. 2 is seed liquor logarithmic phase straight-line equation;
Fig. 3 is that the impact of different training method on secondary chicken fowl bacillus viable count is compared.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.It should be understood that described embodiment is only exemplary, any restriction is not formed to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments or replacement all fall into protection scope of the present invention.
1, experiment material
1.1 bacterial strain
Secondary chicken fowl bacillus strain C-Apg-8 (Page A type) is purchased from China Veterinery Drug Inspection Office.
1.2 key instrument
KC-06-57-300L type fermentor tank, Beijing Ke Nuode fluid device company limited produces; ZHWY-211C type constant temperature culture oscillator, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd.; 752 type ultraviolet-visible pectrophotometers, Shanghai Spectrum Apparatus Co., Ltd.; The super clean bench of DL-CJ-2ND type, Beijing Dong Lianhaer instrument manufacturing company limited; HF90 type CO2 incubator, Shanghai Lishen Scientific Equipment Co., Ltd.; Standard opacity tube, National Institute for Food and Drugs Control.
1.3 substratum
Semisynthetic medium, semi-synthetic nutrient agar with reference to Feng Wenda etc. method preparation (Feng Wenda. the isolation identification [J] of Beijing infectious coryza of chicken pathogenic bacteria. microbiology is circulated a notice of, 1987,5:216 ~ 219.); It is identical that the formula of semisynthetic medium and People's Republic of China (PRC) regulations version in 2000 specifies.
1.4 other
Chicken serum, purchased from the peaceful thing Engineering Co., Ltd in Zhengzhou one hundred; Cozymase, place of production Germang.The fermentation culture of embodiment 1 secondary chicken fowl bacillus
(1) preparation of primary seed solution
Secondary chicken fowl bacillus C-Apg-8 kind poison 5mL PBS (PH=7.2) dilution of freeze-drying being preserved, get 4mL and be seeded to 160mL semisynthetic medium 37 DEG C, 11h cultivated by 200r/min shaking table, cultivates the OD of terminal seed liquor
550value is 0.450, and viable count is the highest.
(2) preparation of secondary seed solution
160mL primary seed solution to be seeded in 1600mL semisynthetic medium 37 DEG C, 4h cultivated by 200r/min shaking table.Viable count is up to 1.3 × 10
9cFU/mL, now bacterium liquid at 550 nm OD value be 0.507.
The preparation of (3) three grades of seed liquor
1600mL secondary seed solution is seeded to 30000mL semisynthetic medium, and 37 DEG C of 200r/min magnetic agitation cultivate 4h; It is 9.0 × 10 that viable count reaches peak value
8cFU/ml, now bacterium liquid at 550 nm OD value be 0.467.
(4) bacterium liquid fermentation culture
30000mL tri-grades of seed liquor are seeded to 300L fermentor tank, and magnetic agitation is cultivated, and mixing speed is 200r/min; Fermentor cultivation temperature maintains about 37 DEG C by automatic temperature control system; Substratum is semisynthetic medium; By the NaOH control pH of auto-feeding 5 ~ 10% about 7.2.Fermentation termination bacterium liquid OD
550value is 0.501.
During fermentation ends, viable count is 7.6 × 10
8cFU/mL, total count is 56.7 hundred million/mL.
The fermentation culture of embodiment 2 secondary chicken fowl bacillus
(1) preparation of primary seed solution
Secondary chicken fowl bacillus C-Apg-8 kind poison 5mL PBS (PH=7.2) dilution of freeze-drying being preserved, get 4mL and be seeded to 160mL semisynthetic medium 37 DEG C, 11h cultivated by 200r/min shaking table, cultivates the OD of terminal seed liquor
550value is 0.550, and viable count is the highest.
(2) preparation of secondary seed solution
160mL primary seed solution to be seeded in 1600mL semisynthetic medium 37 DEG C, 4h cultivated by 200r/min shaking table.Viable count is up to 1.3 × 10
9cFU/mL, now bacterium liquid at 550 nm OD value be 0.507.
The preparation of (3) three grades of seed liquor
1600mL secondary seed solution is seeded to 30000mL semisynthetic medium, and 4h cultivated by 37 DEG C of 200r/min shaking tables; It is 1.4 × 10 that viable count reaches peak value
9cFU/ml, now bacterium liquid at 550 nm OD value be 0.523.
(4) bacterium liquid fermentation culture
30000mL tri-grades of seed liquor are seeded to 300L fermentor tank, and magnetic agitation is cultivated, and mixing speed is 200r/min; Fermentor cultivation temperature maintains about 37 DEG C by automatic temperature control system; Substratum is semisynthetic medium; By the NaOH control pH of auto-feeding 5 ~ 10% about 7.2.Fermentation termination bacterium liquid OD
550value is 0.60.
During fermentation ends, viable count is 3.3 × 10
9cFU/mL, total count is 57.4 hundred million/mL.
The fermentation culture of embodiment 3 secondary chicken fowl bacillus
(1) preparation of primary seed solution
Secondary chicken fowl bacillus C-Apg-8 kind poison 5mL PBS (PH=7.2) dilution of freeze-drying being preserved, get 4mL and be seeded to 160mL semisynthetic medium 37 DEG C, 11h cultivated by 200r/min shaking table, cultivates the OD of terminal seed liquor
550value is 0.502, and viable count is the highest.
(2) preparation of secondary seed solution
160mL primary seed solution to be seeded in 1600mL semisynthetic medium 37 DEG C, 4h cultivated by 200r/min shaking table.Viable count is up to 1.2 × 10
9cFU/mL, now bacterium liquid at 550 nm OD value be 0.507.
The preparation of (3) three grades of seed liquor
1600mL secondary seed solution is seeded to 30000mL semisynthetic medium, and 4h cultivated by 37 DEG C of 200r/min shaking tables; It is 1.45 × 10 that viable count reaches peak value
9cFU/ml, now bacterium liquid at 550 nm OD value be 0.512.
(4) bacterium liquid fermentation culture
30000mL tri-grades of seed liquor are seeded to 300L fermentor tank, and magnetic agitation is cultivated, and mixing speed is 200r/min; Fermentor cultivation temperature maintains about 37 DEG C by automatic temperature control system; Substratum is semisynthetic medium; By the NaOH control pH of auto-feeding 5 ~ 10% about 7.2.Fermentation termination bacterium liquid OD
550value is 0.578.
During fermentation ends, viable count is 8.5 × 10
8cFU/mL, total count is 57.21 hundred million/mL.
The fermentation process optimization experiment of experimental example 1 secondary chicken fowl bacillus
1, experimental technique
The preparation of 1.1 primary seed solution
C-Apg-8 kind poison 5mL PBS (PH=7.2) dilution that freeze-drying is preserved, get 4mL and inoculate 160mL semisynthetic medium 37 DEG C, 16h cultivated by 200r/min shaking table, period every 1h sampling and measuring viable count and OD value, draw growth curve, the feature the highest according to logarithmic growth end of term viable count determines incubation time and OD value scope.Repeat 3 times.
Detect OD value by the sample obtained during above-mentioned cultivation, detect with 752 type ultraviolet-visible pectrophotometers, represent with absorbancy, get zymocyte liquid 3mL, measure absorbancy at 550 nm, and detailed recorded data.
The preparation of 1.2 secondary seed solution
By determine viable count the highest time 160mL primary seed solution to be seeded in 1600mL semisynthetic medium 37 DEG C, 6h cultivated by 200r/min shaking table, period every 0.5h and 1h sampling and measuring viable count and OD value.Repeat 3 times, using the mean value of 3 repetitions as final result.
The preparation of 1.3 3 grades of seed liquor
According to test operability and inoculative proportion, 1600mL → 30000mL stage to be substituted with 160mL → 3000mL test, 37 DEG C of cultivations, and according to existing working condition, select magnetic agitation and shaking table to cultivate and synchronously carry out simultaneous test, rotating speed 200r/min.
1.4 fermentation culture
Inoculative proportion according to 10% is seeded to 300L fermentor tank, and all the other conditions are consistent with two step inoculation methods, carry out fermentation culture respectively, detects viable count and OD value, to compare.
1.5 viable count method
Adopt colony counting method.Get often kind of sample respectively and do 10 times of dilutions, with pipettor, PBS liquid good for sterilizing is added in tubule, often pipe 9mL, then getting 1mL after being shaken up by sample adds in the first pipe, afterwards by that analogy, selects three suitable extent of dilution, each extent of dilution inoculates 2 flat boards, inoculum size is 500 μ L, inoculates semi-synthetic nutrient agar and makes its natural diffuseness, in 37 DEG C of 5%CO
218h ~ 20h is cultivated under condition.CFU/mL is calculated according to the method in GB4789.2-94.
1.6 total bacteria count measuring method
Employing standard turbidimetry.By seedling bacterium liquid high speed centrifugation 30min, revolution is 4000r/min, supernatant discarded, gets the PBS that precipitation adds PH7.2 and makes suspension liquid, compare with biological products national standard " Chinese bacterial turbidity standard (C-Apg-8 bacterial strain: 2,800,000,000/mL) ", record total count.
The drafting of 1.7 secondary chicken fowl bacillus growth curves
Take incubation time as transverse axis, seed liquor viable count logarithm is the longitudinal axis, draws out seed liquor growth curve.
The calculating of 1.8 seed liquor generation times
Bacterium often divides a required time at logarithmic phase, is called generation time or doubling time, represents with g.According to statistical calculations, the mean value of bacterial population all cells generation time is certain, is referred to as the generation time of bacterial population.According to Zhang Hong etc. (Zhang Hong, Wang Wenquan etc. the research [J] of para bacillus fowl blood phili growth characteristics in production of vaccine. Chinese veterinary drug magazine .2003,37 (5): 15-17,20.) test method calculate generation time.
2, experimental result
The mensuration of 2.1 primary seed solution viable counts and OD value
The viable count measurement result of each time point of seed liquor and corresponding logarithmic value thereof, OD value are in table 1.
The each time point viable count of table 1 C-Apg-8 bacterial strain, logarithmic value and OD value
The drafting of 2.2 seed liquor growth curves
According to the data described point that table 1 records, take incubation time as transverse axis, seed liquor viable count logarithm is the longitudinal axis, and from 0h to 16h, each point mapping, draws out seed liquor growth curve, the results are shown in Figure 1.
As can be seen from Figure 1,0-2h after inoculation, this period is in growth lag phase, 2-11h (test 1), 2-12h (test 2,3) thalline enter logarithmic phase, 11-12h (test 1), 12-13h (test 2,3) enter stationary phase, and after this thalline enters decline phase.Known according to growth curve, under 37 DEG C of 200r/min shaking table culture condition, select the bacterium liquid of 11 hours as primary seed solution.
The data of 2.3 seed liquor g generation time calculate
With reference to (Zhang Hong such as Zhang Hong, Wang Wenquan etc. the research [J] of para bacillus fowl blood phili growth characteristics in production of vaccine. Chinese veterinary drug magazine .2003,37 (5): 15-17,20.) method, in growth curve of bacteria, logarithmic phase straight-line equation can be expressed as log
2n=Kt+b, wherein, N represents bacterium viable count at a time in logarithmic phase, and t represents the logarithmic phase a certain moment.
log
2N
1＝Kt
1+b log
2N
2＝Kt
2+b
K＝log
2(N
1/N
2)/t
1-t
2
g＝t
1-t
2/log
2(N
1/N
2)＝1/K
The function calculated line equation K value provided in computer software Excel is provided.That is: g generation time of bacterium equals the inverse of the slope K of bacteria log straight-line equation in vegetative period, the results are shown in Figure 2, table 2.
The logarithmic phase straight-line equation of table 2 C-Apg-8 bacterial strain and generation time
The preparation of 2.4 secondary seed solution
Result is as shown in table 3, and in 160mL → 1600mL stage, secondary chicken fowl bacillus is at 37 DEG C, and during 200r/min shaking table cultivation 4h, viable count is up to 1.3 × 10
9cFU/mL, now OD value is 0.507.
Each time point viable count of table 3 C-Apg-8 bacterial strain and OD value
Note: above data are the mean value of 3 test-results.
The preparation of 2.5 3 grades of seed liquor
As can be seen from Fig. 3 growth curve (mean values of 3 test-results), at about 4h, it is 1.4 × 10 that the viable count that shaking table and magnetic agitation are cultivated all reaches peak value
9cFU/ml, 9.0 × 10
8cFU/ml, and use SPSS 11.5 to carry out statistical analysis respectively to it, the two difference is not significantly (P=0.1365 > 0.05).According to the existing practical situation of production, adopt magnetic agitation to cultivate and substitute shaking table cultivation.
The determination of 2.6 OD values
In logarithmic phase, because the concentration of bacterial suspension and the quantity of rhinitis bacterium are directly proportional to optical density(OD) (OD value), the corresponding data of a large amount of OD values and viable count is passed through in production practice, successfully find the OD scope when logarithm end of term, viable count was the highest, be: 0.50 ± 0.05 be: 0.55 ± 0.05 for seed liquor for fermentation termination.
Contrast after 2.7 process modification with before improvement
Fermentor cultivation temperature maintains about 37 DEG C by automatic temperature control system; Mixing speed is 200r/min; By the NaOH control pH of auto-feeding 5 ~ 10% about 7.2.
Table 4 is that before and after process modification, six batch fermentations cultivate detected result, can obtain drawing a conclusion according to this result, the two step inoculation methods that C-Apg-8 (the Page A type) strain of secondary chicken fowl bacillus is produced by changing existing large bottle: first order seed → secondary seed (7500mL) → 300L is four step inoculation method: 4mL → 160mL → 1600mL → 30000mL → 300L, inoculative proportion becomes 2.5 ~ 10% from 2.5% before, do not changing single Batch fermentation cumulative volume simultaneously, when not increasing personnel and material cost, thalline viable count and the total count of cultivation are more satisfactory, be applied in 300L fermentor tank, achieve cell density OD value, viable count and total count are respectively 0.566, 9.8 × 10
8cFU/mL, 56.7 hundred million/mL, before and after process modification of comparing, viable count differs about 13 times (9.8 × 10
8→ 7.7 × 10
7cFU/mL), total count increases close to 5 times (11.5 hundred million/mL → 56.7 hundred million/mL), and secondary chicken fowl bacillus fermentation list finally can be made to criticize output increase about 5 times.
C-Apg-8 strain fermentation result before and after table 4 process modification
The protection test of experimental example 2 Immunization
The optimum result of the present invention's experimentally example 1; in the production of vaccine of reality by process modification after the secondary chicken fowl bacillus emulsification seedling of cultivating; and according to " People's Republic of China's veterinary biologics quality standard " calendar year 2001 version (The Ministry of Agriculture of the People's Republic of China, MOA; 2001) carry out the Immunization protection test of vaccine, it is attacked malicious protection ratio and reaches 100% qualified.
Claims (10)
1. a fermentation culture method for secondary chicken fowl bacillus (Avibacterium paragallinarum), is characterized in that, comprise the following steps: the preparation of (1) primary seed solution; (2) preparation of secondary seed solution; The preparation of (3) three grades of seed liquor; (4) bacterium liquid fermentation culture.
2. according to fermentation culture method according to claim 1, it is characterized in that, the preparation of step (1) described primary seed solution comprises: by secondary chicken fowl bacillus dilute, gets 4mL and is seeded to 160mL semisynthetic medium cultivation 11h; Preferably, described dilution is that the PBS being 7.2 by the secondary chicken fowl bacillus specie poison 5mL pH value of freeze-drying preservation dilutes.
3. according to fermentation culture method according to claim 1, it is characterized in that, the preparation of step (2) described secondary seed solution comprises: 160mL primary seed solution is seeded to 1600mL semisynthetic medium and cultivates 4h.
4. according to the fermentation culture method described in Claims 2 or 3, it is characterized in that: described cultivation is 37 DEG C, 200r/min shaking table is cultivated.
5. according to fermentation culture method according to claim 1, it is characterized in that, the preparation of the described three grades of seed liquor of step (3) comprises: 1600mL secondary seed solution is seeded to 30000mL semisynthetic medium and cultivates 4h.
6. according to fermentation culture method according to claim 5, it is characterized in that: described cultivation is 37 DEG C, 200r/min magnetic agitation or shaking table are cultivated; Be preferably 200r/min magnetic agitation to cultivate.
7. according to the fermentation culture method of claim 1,2,3 or 5 described in any one, it is characterized in that: the terminal of described cultivation is to the OD of seed liquor
550value is 0.50 ± 0.05.
8. according to fermentation culture method according to claim 1, it is characterized in that, step (4) described bacterium liquid fermentation culture comprises: 30000mL tri-grades of seed liquor are seeded to 300L fermentor tank, and magnetic agitation is cultivated; Fermentation termination is bacterium liquid OD
550value is 0.55 ± 0.05; Preferably, substratum is semisynthetic medium; Culture temperature is 37 DEG C, and pH value is 7.2, and mixing speed is 200r/min.
9. the secondary chicken fowl bacillus cultivated of the fermentation culture method of claim 1,2,3,5,6 or 8 described in any one.
10. described in claim 9, secondary chicken fowl bacillus is preparing the purposes in inactivated vaccine of infectious coryza of chicken.
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| CN (1) | CN104328077A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296407A (en) * | 2015-12-07 | 2016-02-03 | 山东华宏生物工程有限公司 | Method for culturing avibacterium paragallinarum bacterial solution |
| CN105296406A (en) * | 2015-12-07 | 2016-02-03 | 山东华宏生物工程有限公司 | Culture method and inactivation method of haemophilus parasuis |
| CN106267176A (en) * | 2015-05-12 | 2017-01-04 | 普莱柯生物工程股份有限公司 | Infectious coryza of chicken vaccine combination and its preparation method and application |
-
2014
- 2014-11-18 CN CN201410659185.7A patent/CN104328077A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| S.AKTER 等: "Isolation and identification of Avibacterium paragallinarum, the causal agent of infectious coryza (IC) from layer chickens in Bangladesh", 《J. BANGLADESH AGRIL. UNIV.》 * |
| 沈旭: "副鸡嗜血杆菌HB株的分离、鉴定及其高密度发酵的研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106267176A (en) * | 2015-05-12 | 2017-01-04 | 普莱柯生物工程股份有限公司 | Infectious coryza of chicken vaccine combination and its preparation method and application |
| CN106267176B (en) * | 2015-05-12 | 2020-03-10 | 普莱柯生物工程股份有限公司 | Infectious coryza vaccine composition, preparation method and application thereof |
| CN105296407A (en) * | 2015-12-07 | 2016-02-03 | 山东华宏生物工程有限公司 | Method for culturing avibacterium paragallinarum bacterial solution |
| CN105296406A (en) * | 2015-12-07 | 2016-02-03 | 山东华宏生物工程有限公司 | Culture method and inactivation method of haemophilus parasuis |
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Inventor after: Yang Guoliang Inventor after: Zhang Hong Inventor after: Chen Qiuping Inventor before: Zhang Hong Inventor before: Jia Guizhen Inventor before: Cao Ning Inventor before: Yang Guoliang Inventor before: Liang Shuang Inventor before: Ding Chunyu Inventor before: Chen Qiuping Inventor before: Zhang Lingyun Inventor before: Wang Yali Inventor before: Zhou Hane Inventor before: Li Jing |
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Application publication date: 20150204 |