CN102776143A - Fermentation production process of special bdellovibrio for mariculture - Google Patents
Fermentation production process of special bdellovibrio for mariculture Download PDFInfo
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- CN102776143A CN102776143A CN2012102623493A CN201210262349A CN102776143A CN 102776143 A CN102776143 A CN 102776143A CN 2012102623493 A CN2012102623493 A CN 2012102623493A CN 201210262349 A CN201210262349 A CN 201210262349A CN 102776143 A CN102776143 A CN 102776143A
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Abstract
The invention discloses a fermentation production process of special bdellovibrio for mariculture, which has few steps, is easy to operate, and beneficial to industrial production. The prepared Bdellovibrio bacteriovorus product has the advantages of high thallus content of 106-108 thallus per ml, long storage time of the product of more than one year, and easiness in use. The fermentation production process of the special bdellovibrio for the mariculture is particularly suitable for a fermentation tank of 1-5 tons.
Description
Technical field
The present invention relates to the fermentation manufacturing technique of a kind of Bdellovibrio, relate in particular to the fermentation manufacturing technique of the special-purpose Bdellovibrio of a kind of sea farming.
Background technology
Bacteriophagic Bdellovibrio is as one type of parasitics bacterium of making a living with predator bacteria specially; Biological characteristics with unique cracking bacterium; Can control the content of COD, sulfide and the ammonia nitrogen etc. of aquaculture water simultaneously again effectively with the pathogenic bacterium quantity limitation at lower level.Therefore, utilize it, developing aquatic animal disease biological control new way is had great importance as the natural biological purification factor in the aquaculture water environment and " living antibiotics ".At present, the manufacturer on the market replaces the special-purpose Bdellovibrio product of seawater with fresh water Bdellovibrio product, such Bdellovibrio product, and it is too short that Bdellovibrio contains quantity not sufficient, shelf time, is unfavorable for producing and using.
Summary of the invention
The present invention is directed to the deficiency of prior art, provide a kind of sea farming special-purpose Bdellovibrio fermentation manufacturing technique, to reach the Bdellovibrio product of producing, Bdellovibrio content is high, the shelf time is permanent.The special-purpose Bdellovibrio fermentation manufacturing technique of sea farming of the present invention is applicable to 1 ~ 5 ton of fermentor tank.
In order to realize the foregoing invention purpose, the technical scheme that the present invention adopted is:
The fermentation manufacturing technique of the special-purpose Bdellovibrio of a kind of sea farming; Comprise that the host bacterium is cultivated and bacteriophagic Bdellovibrio is cultivated; Wherein: said host bacterium is cultivated; May further comprise the steps: (1) one-level shake-flask seed is cultivated---and the host bacterium after the activated processing is inoculated in the one-level that is loaded with substratum shakes in the bottle, under the condition of 33 ± 1 ℃ of temperature, 180 ~ 220 rev/mins of rotating speeds, cultivate 18 ~ 24h, promptly make one-level and shake bottle host's bacterial classification; (2) the secondary shake-flask seed is cultivated---and shake at the secondary that is loaded with substratum and insert the cultured one-level of step (1) in the bottle and shake bottle host's bacterial classification; Under the condition of 33 ± 1 ℃ of temperature, 180 ~ 220 rev/mins of rotating speeds, cultivate 18 ~ 24h, promptly make the secondary shake-flask seed; (3) seed tank culture---the cultured secondary shake-flask seed of step (2) inserted according to 1% ~ 3% volume ratio be loaded with in the seeding tank of substratum, under the condition of 80 ~ 120 rev/mins of 37 ± 1 ℃ of jar temperature, tank pressure 0.045 ~ 0.55 MPa, mixing speed, cultivate 18 ~ 24h and promptly make host bacterium seed liquor; (4) fermentor cultivation---in being loaded with the fermentor tank of substratum, insert the cultured host bacterium of step (3) seed liquor, under the condition of 80 ~ 100 rev/mins of 35 ± 1 ℃ of jar temperature, tank pressure 0.05 MPa, mixing speed, cultivate 16 ~ 24h and get final product;
In the host bacterium culturing process, before shaking bottle, secondary to one-level and shaking the culture medium inoculated of bottle, seeding tank and fermentor tank, the one-level that is loaded with corresponding substratum is shaken bottle, secondary shakes bottle, seeding tank and fermentor tank and all passes through sterilising treatment;
Said bacteriophagic Bdellovibrio is cultivated; May further comprise the steps: 1) bacteriophagic Bdellovibrio after the activated processing is inoculated in and has cultivated one-level and shake the one-level of bottle host's bacterial classification and shake in the bottle; 33 ± 1 ℃ of temperature, shake under the condition of 180 ~ 220 rev/mins of bottle rotating speeds and cultivate 18 ~ 24h, promptly make one-level and shake a bottle bacteriophagic Bdellovibrio; 2) shake in the bottle at the secondary of having cultivated the secondary shake-flask seed, insert the cultured one-level of step 1) and shake a bottle bacteriophagic Bdellovibrio, 33 ± 1 ℃ of temperature, shake under the condition of 180 ~ 220 rev/mins of bottle rotating speeds and cultivate 18 ~ 24h, promptly make secondary and shake a bottle bacteriophagic Bdellovibrio; 3) in the seeding tank of having cultivated host bacterium seed liquor; Volume ratio access step 2 by 1% ~ 5%) cultured secondary shakes a bottle bacteriophagic Bdellovibrio; Under the condition of 80 ~ 100 rev/mins of 33 ± 1 ℃ of temperature, tank pressure 0.05 MPa, mixing speed, cultivate 16 ~ 24h, promptly make the bacteriophagic Bdellovibrio seed liquor; 4) in the fermentor tank of having cultivated the host bacterium; Volume ratio by 3% ~ 5% inserts the cultured bacteriophagic Bdellovibrio seed liquor of step 3); Under the condition of 80 ~ 100 rev/mins of 33 ± 1 ℃ of jar temperature, tank pressure 0.05 MPa, mixing speed, cultivate 72 ~ 96h, promptly make Bdellovibrio bacterium liquid;
The culture medium prescription that one-level is shaken bottle is: sodium-chlor 0.4 ~ 0.6 g/l; Yeast powder 0.2 ~ 0.4 g/l; Peptone or Tryptones 0.4 ~ 0.6g/l; The pH of this substratum is 6.5 ~ 6.8;
The culture medium prescription that secondary shakes bottle is: sodium-chlor 0.4 ~ 0.6 g/l; Yeast powder 0.2 ~ 0.4 g/l; Peptone 0.4 ~ 0.6g/l; The pH of this substratum is 6.5 ~ 6.8;
The culture medium prescription of seeding tank is: sodium-chlor 0.18 ~ 0.22 g/l; Yeast powder 0.08 ~ 0.12 g/l; Peptone or Tryptones 0.18 ~ 0.22 g/l; Skimmer 0.009 ~ 0.011 g/l; The pH of this substratum is 7.0 ~ 7.2;
The culture medium prescription of fermentor tank is: sodium-chlor 0.18 ~ 0.22 g/l; Yeast powder 0.08 ~ 0.12 g/l; Peptone or Tryptones 0.18 ~ 0.22 g/l; Skimmer 0.018 ~ 0.022 g/l; The pH of this substratum is 6.5 ~ 6.8.
In this technology, Aeromonas hydrophila or pseudomonas that said host bacterium is intestinal bacteria, non-virulent.
In this technology, one-level is shaken the pH regulator reagent that substratum adopted that bottle, secondary shake bottle, seeding tank and fermentor tank and is NaOH solution.
In this technology, the skimmer that is adopted in seeding tank and the fermentor cultivation based formulas is water-soluble silicon oil.
In this technology, one-level is shaken medium sterilization condition that bottle, secondary shake bottle, seeding tank and fermentor tank under 0.1MPa, 121 ℃ condition, lasting 20min.
In this technology, host bacterium and bacteriophagic Bdellovibrio that the access one-level is shaken in the bottle are bacteria suspension; The preparation method of said host bacterium bacteria suspension is: adopt ordinary method to produce inclined-plane host bacterium earlier, get 1 of inclined-plane host bacterium then, add 8 ~ 12ml sterilized water, promptly can be made into host bacterium bacteria suspension; The preparation method of said bacteriophagic Bdellovibrio bacteria suspension is: adopt ordinary method to produce the inclined-plane bacteriophagic Bdellovibrio earlier, get one of inclined-plane bacteriophagic Bdellovibrio, add 8 ~ 12ml sterilized water, promptly can be made into the bacteriophagic Bdellovibrio bacteria suspension.In addition, in this technology, can also adopt transfering loop to carry out one-level and shake host bacterium and bacteriophagic Bdellovibrio inoculation operation in the bottle, and need not to adopt bacteria suspension.
In this technology, the substratum loading amount that one-level is shaken bottle is 80 ~ 110ml/250ml culturing bottle, and host bacterium bacterial suspension inoculation amount is that 0.4 ~ 0.6ml/ bottle, bacteriophagic Bdellovibrio bacterial suspension inoculation amount are the 1-3ml/ bottle in the bottle and one-level is shaken; The substratum loading amount that secondary shakes bottle is 180 ~ 220ml/500ml culturing bottle, and secondary shakes one-level in the bottle and shakes the inoculum size that bottle host's bacterial classification, one-level shake bottle bacteriophagic Bdellovibrio and be 1 ~ 3ml/ bottle.
When the fermentor cultivation of this technology finished, it is clear that fermented liquid becomes, and sampling directly detects, and the absorbance under 560nm (OD) is below 0.8, if adopt tryptone to replace peptone in the culture medium prescription, then the absorbance under the 560nm will be below 0.5; The Bdellovibrio counting is 10
6Individual/as more than the ml, especially to concentrate on 10
7~ 10
8Individual/ml.
Therefore, the fermentation manufacturing technique of the special-purpose Bdellovibrio of sea farming provided by the invention, step is few, and is simple to operate, is beneficial to suitability for industrialized production.Prepared bacteriophagic Bdellovibrio product, thalline content is high, can reach 10
6~ 10
8Individual/ml, the shelf time of product is long, more than 1 year, is easy to use.The fermentation manufacturing technique of the special-purpose Bdellovibrio of sea farming of the present invention is specially adapted to the production of 1 ~ 5 ton of fermentor tank.
Embodiment
Below will combine each embodiment that technical scheme of the present invention at length is described.But protection scope of the present invention can not be thought and only is confined to following specific embodiment.For said those skilled in the art, under the basic premise that does not break away from the present invention's design, can also make some simple deductions or be equal to replacement, these are equal to the row that alternative still will be regarded as protection scope of the present invention.
Host bacterium of the present invention can be as bacteriophagic Bdellovibrio host's bacterium for the Aeromonas hydrophila of intestinal bacteria, non-virulent, pseudomonas or other; Related bacteriophagic Bdellovibrio is the China Committee for Culture Collection of Microorganisms common micro-organisms center that is preserved in (being called for short CGMCC), deposit number is CGMCC No.1045's
Bdellovibrio sp.S-23.
Embodiment 1
Selecting intestinal bacteria is the host bacterium of bacteriophagic Bdellovibrio.
(1) colibacillary cultivation
Preparation inclined-plane solid medium 150ml, filling a prescription is: peptone, 0.5g/l; Carnis Bovis seu Bubali cream, 3g/l; Sodium-chlor, 0.5g/l; Agar, 1.8g/l; Regulate the pH to 7.0 of solid medium with NaOH solution; 0.1MPa, 121 ℃, sterilization 20min, 10 test tubes of packing, cooled and solidified; The single bacterium colony of intestinal bacteria on the picking flat board, the inoculation inclined-plane, 37 ± 1 ℃ of temperature controls are cultivated 24h, promptly prepare the inclined-plane intestinal bacteria;
By prescription be: sodium-chlor 0.5 g/l, yeast powder 0.3 g/l, peptone 0.5g/l prepare one-level shake-flask seed substratum 2L, regulate the pH to 6.5 of substratum; Packing in the 250ml culturing bottle, every bottle of 100ml substratum, totally 20 bottles; Get 1 of inclined-plane intestinal bacteria, add the 10ml sterilized water, process bacteria suspension, go into the seed bottle by the 0.5ml/ bottle graft, totally 20 bottles; 33 ± 1 ℃ of temperature controls shake 200 rev/mins of bottle rotating speeds, cultivate 24h, promptly make one-level and shake a bottle intestinal bacteria seed;
By prescription be: sodium-chlor 0.5 g/l, yeast powder 0.3 g/l, peptone 0.5g/l prepare secondary shake-flask seed substratum 2L, regulate the pH to 6.5 of substratum; Packing in the 500ml culturing bottle, every bottle of 200ml substratum, totally 10 bottles; Get one-level and shake 1 bottle in bottle intestinal bacteria seed, go into secondary shake-flask seed bottle, totally 10 bottles by the 3ml/ bottle graft; 33 ± 1 ℃ of temperature controls shake 200 rev/mins of bottle rotating speeds, cultivate 24h, promptly make secondary and shake a bottle intestinal bacteria seed;
By prescription be: sodium-chlor 0. 2 g/l; Yeast powder 0.1 g/l; Peptone 0.2 g/l; Water-soluble silicon oil 0.01 g/l; With pH to 7.0 ~ 7.2 of sodium hydroxide adjusting substratum, preparation 150L seed tank culture base, at 0.1MPa, 20min sterilizes under 121 ℃ the condition; In the seed tank culture base, insert cultured secondary and shake a bottle intestinal bacteria 1.5L, 37 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.045 ~ 0.55 MPa, 100 rev/mins of mixing speed are cultivated 24h, promptly make the intestinal bacteria seed liquor;
By prescription be: sodium-chlor 0.2 g/l; Yeast powder 0.1 g/l; Peptone 0.2 g/l; Water-soluble silicon oil 0.02 g/l; Regulate pH to 6.5 ~ 6.8 of substratum with sodium hydroxide; Preparation fermentation tank culture medium 700L, at 0.1MPa, 20min sterilizes under 121 ℃ the condition; In fermentor tank, insert intestinal bacteria seed liquor 21L, 35 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 24h, get final product;
(2) bacteriophagic Bdellovibrio is cultivated
Preparation inclined-plane solid medium 150ml, filling a prescription is: peptone 0.5g/l; Carnis Bovis seu Bubali cream 3g/l; Sodium-chlor 0.5g/l; Agar 1.8g/l; Regulate the pH to 7.0 of solid medium with NaOH solution; Under 0.1MPa, 121 ℃, sterilization 20min, 10 test tubes of packing, cooled and solidified; Bacteriophagic Bdellovibrio list bacterium colony on the picking flat board, the inoculation inclined-plane, 37 ± 1 ℃ of temperature controls are cultivated 24h, promptly prepare the inclined-plane bacteriophagic Bdellovibrio;
Get 1 of inclined-plane bacteriophagic Bdellovibrio, add the 10ml sterilized water, process bacteria suspension, go into cultured one-level by the 1ml/ bottle graft and shake a bottle intestinal bacteria seed bottle, 33 ± 1 ℃ of temperature controls, shake-flask culture 24h promptly makes one-level and shakes a bottle bacteriophagic Bdellovibrio; Get cultured one-level and shake a bottle bacteriophagic Bdellovibrio, go into cultured secondary by the 1ml/ bottle graft and shake a bottle intestinal bacteria seed bottle, 33 ± 1 ℃ of temperature controls, shake-flask culture 24h promptly makes secondary and shakes a bottle bacteriophagic Bdellovibrio;
In cultured intestinal bacteria seeding tank, insert the 1.5L secondary and shake a bottle bacteriophagic Bdellovibrio, 33 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 24h, promptly make the bacteriophagic Bdellovibrio seed liquor.
In cultured Escherichia coli fermentation jar, move into bacteriophagic Bdellovibrio seed liquor 21L, 33 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 96h.
In Bdellovibrio is cultivated, when upper level Bdellovibrio content surpasses 10
6The time, can be inoculated in the next stage fermentor tank (or big triangular flask), the volume ratio of each inoculum size is preferably 1% ~ 5%; The volume ratio of the inoculum size in laboratory culture stage more preferably 1% ~ 3%; And the volume ratio of big jar inoculum size of last production usefulness is preferably 3% ~ 5%.
Sampling detects, and draws in every 1ml bacterium liquid and contains 10
8With last Bdellovibrio, the OD value that sampling directly detects under the 560nm is 0.556; Product is through preserving, and the time is more than 1 year.
Embodiment 2
Selecting the Aeromonas hydrophila of non-virulent is the host bacterium of bacteriophagic Bdellovibrio.
(1) cultivation of the Aeromonas hydrophila of non-virulent
Preparation inclined-plane solid medium 150ml, filling a prescription is: peptone, 0.4g/l; Carnis Bovis seu Bubali cream, 3.5g/l; Sodium-chlor, 0.6g/l; Agar, 1.7g/l; Regulate the pH to 7.1 of solid medium with NaOH solution; 0.1MPa, 121 ℃, sterilization 20min, 10 test tubes of packing, cooled and solidified; The single bacterium colony of the Aeromonas hydrophila of the non-virulent on the picking flat board, the inoculation inclined-plane, 37 ± 1 ℃ of temperature controls are cultivated 30h, the Aeromonas hydrophila of promptly preparing the inclined-plane non-virulent;
By prescription be: sodium-chlor 0.4 g/l, yeast powder 0.4 g/l, peptone 0.4g/l prepare one-level shake-flask seed substratum 0.8L, regulate the pH to 6.8 of substratum; Packing in the 250ml culturing bottle, every bottle of 80ml substratum, totally 10 bottles; Get 1 of the Aeromonas hydrophila of inclined-plane non-virulent, add the 12ml sterilized water, process bacteria suspension, go into the seed bottle by the 0.6ml/ bottle graft, totally 10 bottles; 33 ± 1 ℃ of temperature controls shake 220 rev/mins of bottle rotating speeds, cultivate 20h, promptly make the Aeromonas hydrophila seed that one-level is shaken bottle non-virulent;
By prescription be: sodium-chlor 0.4 g/l, yeast powder 0.4 g/l, peptone 0.4g/l prepare secondary shake-flask seed substratum 1.8L, regulate the pH to 6.8 of substratum; Packing in the 500ml culturing bottle, every bottle of 180ml substratum, totally 10 bottles; Get 1 bottle in the Aeromonas hydrophila seed that one-level shakes bottle non-virulent, go into secondary shake-flask seed bottle by the 1ml/ bottle graft, totally 10 bottles; 33 ± 1 ℃ of temperature controls shake 220 rev/mins of bottle rotating speeds, cultivate 20h, promptly make the Aeromonas hydrophila seed that secondary shakes bottle non-virulent;
By prescription be: sodium-chlor 0. 22 g/l; Yeast powder 0.08 g/l; Peptone, 0.22 g/l; Water-soluble silicon oil 0.011 g/l; With pH to 7.0 ~ 7.2 of sodium hydroxide adjusting substratum, preparation 100L seed tank culture base, at 0.1MPa, 20min sterilizes under 121 ℃ the condition; In the seed tank culture base, insert cultured secondary shake-flask seed 1.5L, 37 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.045 ~ 0.55 MPa, 120 rev/mins of mixing speed are cultivated 18h, promptly make the Aeromonas hydrophila seed liquor of non-virulent;
By prescription be: sodium-chlor 0.22 g/l; Yeast powder 0.12 g/l; Peptone, 0.18 g/l; Water-soluble silicon oil, 0.022 g/l; Regulate pH to 6.5 ~ 6.8 of substratum with sodium hydroxide; Preparation fermentation tank culture medium 700L, at 0.1MPa, 20min sterilizes under 121 ℃ the condition; In fermentor tank, insert the Aeromonas hydrophila seed liquor 28L of non-virulent, 35 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 20h, get final product;
(2) bacteriophagic Bdellovibrio is cultivated
Preparation inclined-plane solid medium 150ml, filling a prescription is: peptone, 0.5g/l; Carnis Bovis seu Bubali cream, 3g/l; Sodium-chlor, 0.5g/l; Agar, 1.8g/l; Regulate the pH to 7.0 of solid medium with NaOH solution; 0.1MPa, 121 ℃, sterilization 20min, 10 test tubes of packing, cooled and solidified; Bacteriophagic Bdellovibrio list bacterium colony on the picking flat board, the inoculation inclined-plane, 37 ± 1 ℃ of temperature controls are cultivated 24h, promptly prepare the inclined-plane bacteriophagic Bdellovibrio;
Get 1 of inclined-plane bacteriophagic Bdellovibrio,, go into the Aeromonas hydrophila seed bottle that cultured one-level is shaken bottle non-virulent by 2 ring/bottle grafts with the transfering loop inoculation, 33 ± 1 ℃ of temperature controls, shake-flask culture 18h promptly makes one-level and shakes a bottle bacteriophagic Bdellovibrio; Get cultured one-level and shake a bottle bacteriophagic Bdellovibrio, go into the Aeromonas hydrophila seed bottle that cultured secondary shakes bottle non-virulent by the 2ml/ bottle graft, 33 ± 1 ℃ of temperature controls, shake-flask culture 20h promptly makes secondary and shakes a bottle bacteriophagic Bdellovibrio;
In the seeding tank of the Aeromonas hydrophila of cultured non-virulent, insert the 4.5L secondary and shake a bottle bacteriophagic Bdellovibrio, 33 ± 1 ℃ of controlling tank temperature; Blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed; Cultivate 24h, promptly make the bacteriophagic Bdellovibrio seed liquor.
In the Aeromonas hydrophila fermentor tank of cultured non-virulent, move into bacteriophagic Bdellovibrio seed liquor 30L, 33 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 72h.
Sampling detects, and draws in every 1ml bacterium liquid and contains 10
7With last Bdellovibrio, the OD value that sampling directly detects under the 560nm is 0914; Product is through preserving, and the time is more than 1 year.
Embodiment 3
Selecting pseudomonas is the host bacterium of bacteriophagic Bdellovibrio.
(1) cultivation of pseudomonas
Preparation inclined-plane solid medium 150ml, filling a prescription is: peptone, 0.6g/l; Carnis Bovis seu Bubali cream, 2.5g/l; Sodium-chlor, 0.4g/l; Agar, 1.7g/l; Regulate the pH to 7.2 of solid medium with NaOH solution; 0.1MPa, 121 ℃, sterilization 20min, 10 test tubes of packing, cooled and solidified; The single bacterium colony of pseudomonas on the picking flat board, the inoculation inclined-plane, 37 ± 1 ℃ of temperature controls are cultivated 36h, promptly prepare the inclined-plane pseudomonas;
By prescription be: sodium-chlor 0.6 g/l, yeast powder 0.2 g/l, Tryptones 0.6g/l prepare one-level shake-flask seed substratum 1.1L, regulate the pH to 6.6 of substratum; Packing in the 250ml culturing bottle, every bottle of 110ml substratum, totally 10 bottles; Get 1 of inclined-plane pseudomonas, go into the seed bottle, totally 10 bottles with the every bottle graft of transfering loop picking 1 ~ 2 ring; 33 ± 1 ℃ of temperature controls shake 180 rev/mins of bottle rotating speeds, cultivate 18h, promptly make one-level and shake a bottle pseudomonas seed;
By prescription be: sodium-chlor 0.6 g/l, yeast powder 0.2 g/l, Tryptones 0.6g/l prepare one-level shake-flask seed substratum 2.2L, regulate the pH to 6.6 of substratum; Packing in the 500ml culturing bottle, every bottle of 220ml substratum, totally 10 bottles; Get one-level and shake 1 bottle in bottle pseudomonas seed, go into secondary shake-flask seed bottle, totally 10 bottles by the 2ml/ bottle graft; 33 ± 1 ℃ of temperature controls shake 180 rev/mins of bottle rotating speeds, cultivate 18h, promptly make secondary and shake a bottle pseudomonas seed;
By prescription be: sodium-chlor 0. 18 g/l; Yeast powder 0.12 g/l; Tryptones, 0.18 g/l; Water-soluble silicon oil 0.009 g/l; With pH to 7.0 ~ 7.2 of sodium hydroxide adjusting substratum, preparation 170L seed tank culture base, at 0.1MPa, 20min sterilizes under 121 ℃ the condition; In the seed tank culture base, insert cultured secondary shake-flask seed 2.1L, 37 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.045 ~ 0.55 MPa, 80 rev/mins of mixing speed are cultivated 20h, promptly make the pseudomonas seed liquor;
By prescription be: sodium-chlor 0.18 g/l; Yeast powder 0.08 g/l; Peptone, 0.22 g/l; Water-soluble silicon oil, 0.018 g/l; Regulate pH to 6.5 ~ 6.8 of substratum with sodium hydroxide; Preparation fermentation tank culture medium 700L, at 0.1MPa, 20min sterilizes under 121 ℃ the condition; In fermentor tank, insert pseudomonas seed liquor 35L, 35 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 16h, get final product;
(2) bacteriophagic Bdellovibrio is cultivated
Preparation inclined-plane solid medium 150ml, filling a prescription is: peptone, 0.5g/l; Carnis Bovis seu Bubali cream, 3g/l; Sodium-chlor, 0.5g/l; Agar, 1.8g/l; Regulate the pH to 7.0 of solid medium with NaOH solution; 0.1MPa, 121 ℃, sterilization 20min, 10 test tubes of packing, cooled and solidified; Bacteriophagic Bdellovibrio list bacterium colony on the picking flat board, the inoculation inclined-plane, 37 ± 1 ℃ of temperature controls are cultivated 24h, promptly prepare the inclined-plane bacteriophagic Bdellovibrio;
Get 1 of inclined-plane bacteriophagic Bdellovibrio,, go into cultured one-level by 2 ring/bottle grafts and shake a bottle pseudomonas seed bottle with the transfering loop inoculation, 33 ± 1 ℃ of temperature controls, shake-flask culture 20h promptly makes one-level and shakes a bottle bacteriophagic Bdellovibrio; Get cultured one-level and shake a bottle bacteriophagic Bdellovibrio, go into cultured secondary by the 2ml/ bottle graft and shake a bottle pseudomonas seed bottle, 33 ± 1 ℃ of temperature controls, shake-flask culture 18h promptly makes secondary and shakes a bottle bacteriophagic Bdellovibrio;
In the seeding tank of cultured pseudomonas, insert the 3L secondary and shake a bottle bacteriophagic Bdellovibrio, 33 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 24h, promptly make the bacteriophagic Bdellovibrio seed liquor.
In cultured pseudomonas fermentor tank, move into bacteriophagic Bdellovibrio seed liquor 30L, 33 ± 1 ℃ of controlling tank temperature, blowing air, control tank pressure 0.05 MPa, 80 ~ 100 rev/mins of mixing speed are cultivated 84h.
Sampling detects, and draws in every ml bacterium liquid and contains 10
6With last Bdellovibrio; The OD value that sampling directly detects under the 560nm is 0.732; Product is through preserving, and the time is more than 1 year.
Embodiment 4
The cultivation of the Aeromonas hydrophila of non-virulent and the cultural method of bacteriophagic Bdellovibrio increase to 1 ton to the fermentation volume of fermentor tank with embodiment 2, through detecting, contain 10 in every ml bacterium liquid
6With last Bdellovibrio; OD value under the 560nm is 0.687; Product is through preserving, and the time was in 1 year.
Embodiment 5
The cultivation of pseudomonas and the cultural method of bacteriophagic Bdellovibrio increase to 5 tons to the fermentation volume of fermentor tank with embodiment 3, through detecting, contain 10 in every ml bacterium liquid
6With last Bdellovibrio; OD value under the 560nm is 0.780; Product is through preserving, and the time is 1 year.
Embodiment 6
The cultural method of colibacillary cultivation and bacteriophagic Bdellovibrio increases to 3 tons to the fermentation volume of fermentor tank with embodiment 1, through detecting, contains 10 in every ml bacterium liquid
6With last Bdellovibrio; OD value under the 560nm is 0.562; Product is through preserving, and the time is 1 year.
Claims (7)
1. the fermentation manufacturing technique of the special-purpose Bdellovibrio of sea farming is characterized in that, comprises that the host bacterium is cultivated and bacteriophagic Bdellovibrio is cultivated, wherein:
Said host bacterium is cultivated; May further comprise the steps: (1) one-level shake-flask seed is cultivated---and the host bacterium after the activated processing is inoculated in the one-level that is loaded with substratum shakes in the bottle; Under the condition of 33 ± 1 ℃ of temperature, 180 ~ 220 rev/mins of rotating speeds, cultivate 18 ~ 24h, promptly make one-level and shake bottle host's bacterial classification; (2) the secondary shake-flask seed is cultivated---and shake at the secondary that is loaded with substratum and insert the cultured one-level of step (1) in the bottle and shake bottle host's bacterial classification; Under the condition of 33 ± 1 ℃ of temperature, 180 ~ 220 rev/mins of rotating speeds, cultivate 18 ~ 24h, promptly make the secondary shake-flask seed; (3) seed tank culture---the cultured secondary shake-flask seed of step (2) inserted according to 1% ~ 3% volume ratio be loaded with in the seeding tank of substratum, under the condition of 80 ~ 120 rev/mins of 37 ± 1 ℃ of jar temperature, tank pressure 0.045 ~ 0.55 MPa, mixing speed, cultivate 18 ~ 24h and promptly make host bacterium seed liquor; (4) fermentor cultivation---in being loaded with the fermentor tank of substratum, insert the cultured host bacterium of step (3) seed liquor, under the condition of 80 ~ 100 rev/mins of 35 ± 1 ℃ of jar temperature, tank pressure 0.05 MPa, mixing speed, cultivate 16 ~ 24h and get final product;
In the host bacterium culturing process, before shaking bottle, secondary to one-level and shaking the culture medium inoculated of bottle, seeding tank and fermentor tank, the one-level that is loaded with corresponding substratum is shaken bottle, secondary shakes bottle, seeding tank and fermentor tank and all passes through sterilising treatment;
Said bacteriophagic Bdellovibrio is cultivated; May further comprise the steps: 1) bacteriophagic Bdellovibrio after the activated processing is inoculated in and has cultivated one-level and shake the one-level of bottle host's bacterial classification and shake in the bottle; 33 ± 1 ℃ of temperature, shake under the condition of 180 ~ 220 rev/mins of bottle rotating speeds and cultivate 18 ~ 24h, promptly make one-level and shake a bottle bacteriophagic Bdellovibrio; 2) shake in the bottle at the secondary of having cultivated the secondary shake-flask seed, insert the cultured one-level of step 1) and shake a bottle bacteriophagic Bdellovibrio, 33 ± 1 ℃ of temperature, shake under the condition of 180 ~ 220 rev/mins of bottle rotating speeds and cultivate 18 ~ 24h, promptly make secondary and shake a bottle bacteriophagic Bdellovibrio; 3) in the seeding tank of having cultivated host bacterium seed liquor; Volume ratio access step 2 by 1% ~ 5%) cultured secondary shakes a bottle bacteriophagic Bdellovibrio; Under the condition of 80 ~ 100 rev/mins of 33 ± 1 ℃ of temperature, tank pressure 0.05 MPa, mixing speed, cultivate 16 ~ 24h, promptly make the bacteriophagic Bdellovibrio seed liquor; 4) in the fermentor tank of having cultivated the host bacterium; Volume ratio by 3% ~ 5% inserts the cultured bacteriophagic Bdellovibrio seed liquor of step 3); Under the condition of 80 ~ 100 rev/mins of 33 ± 1 ℃ of jar temperature, tank pressure 0.05 MPa, mixing speed, cultivate 72 ~ 96h, promptly make Bdellovibrio bacterium liquid;
The culture medium prescription that one-level is shaken bottle is: sodium-chlor 0.4 ~ 0.6 g/l; Yeast powder 0.2 ~ 0.4 g/l; Peptone or Tryptones 0.4 ~ 0.6g/l; The pH of this substratum is 6.5 ~ 6.8;
The culture medium prescription that secondary shakes bottle is: sodium-chlor 0.4 ~ 0.6 g/l; Yeast powder 0.2 ~ 0.4 g/l; Peptone 0.4 ~ 0.6g/l; The pH of this substratum is 6.5 ~ 6.8;
The culture medium prescription of seeding tank is: sodium-chlor 0.18 ~ 0.22 g/l; Yeast powder 0.08 ~ 0.12 g/l; Peptone or Tryptones 0.18 ~ 0.22 g/l; Skimmer 0.009 ~ 0.011 g/l; The pH of this substratum is 7.0 ~ 7.2;
The culture medium prescription of fermentor tank is: sodium-chlor 0.18 ~ 0.22 g/l; Yeast powder 0.08 ~ 0.12 g/l; Peptone or Tryptones 0.18 ~ 0.22 g/l; Skimmer 0.018 ~ 0.022 g/l; The pH of this substratum is 6.5 ~ 6.8.
2. according to the fermentation manufacturing technique of the special-purpose Bdellovibrio of the said sea farming of claim 1, it is characterized in that Aeromonas hydrophila or pseudomonas that said host bacterium is intestinal bacteria, non-virulent.
3. according to the fermentation manufacturing technique of the special-purpose Bdellovibrio of the said sea farming of claim 1, it is characterized in that one-level is shaken the pH regulator reagent that substratum adopted that bottle, secondary shake bottle, seeding tank and fermentor tank and is NaOH solution.
4. according to the fermentation manufacturing technique of the special-purpose Bdellovibrio of the said sea farming of claim 1, it is characterized in that the skimmer that is adopted in seeding tank and the fermentor cultivation based formulas is water-soluble silicon oil.
5. according to the fermentation manufacturing technique of the special-purpose Bdellovibrio of the said sea farming of claim 1, it is characterized in that one-level is shaken medium sterilization condition that bottle, secondary shake bottle, seeding tank and fermentor tank under 0.1MPa, 121 ℃ condition, lasting 20min.
6. according to the fermentation manufacturing technique of the special-purpose Bdellovibrio of the said sea farming of claim 1, it is characterized in that host bacterium and bacteriophagic Bdellovibrio that the access one-level is shaken in the bottle are bacteria suspension; The preparation method of said host bacterium bacteria suspension is: adopt ordinary method to produce inclined-plane host bacterium earlier, get 1 of inclined-plane host bacterium then, add 8 ~ 12ml sterilized water, promptly can be made into host bacterium bacteria suspension; The preparation method of said bacteriophagic Bdellovibrio bacteria suspension is: adopt ordinary method to produce the inclined-plane bacteriophagic Bdellovibrio earlier, get one of inclined-plane bacteriophagic Bdellovibrio, add 8 ~ 12ml sterilized water, promptly can be made into the bacteriophagic Bdellovibrio bacteria suspension.
7. according to the fermentation manufacturing technique of the special-purpose Bdellovibrio of the said sea farming of claim 6; It is characterized in that; The substratum loading amount that one-level is shaken bottle is 80 ~ 110ml/250ml culturing bottle, and host bacterium bacterial suspension inoculation amount is that 0.4 ~ 0.6ml/ bottle, bacteriophagic Bdellovibrio bacterial suspension inoculation amount are the 1-3ml/ bottle in the bottle and one-level is shaken; The substratum loading amount that secondary shakes bottle is 180 ~ 220ml/500ml culturing bottle, and secondary shakes one-level in the bottle and shakes the inoculum size that bottle host's bacterial classification, one-level shake bottle bacteriophagic Bdellovibrio and be 1 ~ 3ml/ bottle.
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| CN110241057A (en) * | 2019-07-23 | 2019-09-17 | 福建九为生物技术有限公司 | A kind of direct putting type Bdellovibrio leavening and preparation method thereof and application method |
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