CN106011019A - Production method of bdellovibrio bacteriovorus preparation - Google Patents

Production method of bdellovibrio bacteriovorus preparation Download PDF

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CN106011019A
CN106011019A CN201610513338.6A CN201610513338A CN106011019A CN 106011019 A CN106011019 A CN 106011019A CN 201610513338 A CN201610513338 A CN 201610513338A CN 106011019 A CN106011019 A CN 106011019A
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preparation
production method
host strain
suspension
turbid liquor
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薛恒平
薛彦青
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Nanjing Qian Sheng Source Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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    • A61K2035/11—Medicinal preparations comprising living procariotic cells
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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Abstract

The invention discloses a production method of a bdellovibrio bacteriovorus preparation. The production method comprises the following: (1) preparing host bacteria mixed suspension: taking rhodopseudomonas sphaeroides P4 or rhodopseudomonas palustris 101 in photosynthetic bacteria and E.Coli600 as mixed host bacteria for producing and culturing bdellovibrio bacteriovorus; (2) preparing a culture solution; (3) carrying out a fermentation process of the bdellovibrio bacteriovorus. The viable count in the prepared bdellovibrio bacteriovorus preparation is obviously improved and a lot of plaques can be produced, so that the use effect of the bdellovibrio bacteriovorus preparation is improved, and the cracking activity, stability and disease prevention and treatment effect of the bdellovibrio bacteriovorus are enhanced.

Description

A kind of production method of phage bdellovibro preparation
Technical field
The invention belongs to microbial technology field, be specifically related to the production method of a kind of phage bdellovibro preparation.
Background technology
Both at home and abroad in aquaculture and animal diseases control, it is currently mainly used the chemicals such as antibiotic, this The side effect of a little preparations, such as drug residue, the Drug resistance of pathogen, suppresses profitable strain etc., to human health And animal husbandry and fishery to produce the harmful effect that brings more and more obvious.The research and development of microbial ecological preparation in recent years Being paid close attention to by each side, the particularly research of bacteriophagic Bdellovibrio is favored by people.
Bacteriophagic Bdellovibrio since being found for 1963, because it is a kind of bacterial parasite, can crack escherichia coli, The animal pathogens such as Salmonella, Aeromonas hydrophila, vibrio, and itself is nontoxic to humans and animals, Environment etc. can also be improved, be increasingly subject to the concern of people.In research and development, people make with escherichia coli For Host Strains.Cultivate with the escherichia coli lived and produce bacteriophagic Bdellovibrio, limited by multiple technologies condition, be difficult to Carry out large-scale industrial production, simultaneously influence ecological environment.Thus it has been proposed that with high temperature (70～150 DEG C) Or escherichia coli are killed by chemicals (chloroform etc.), manufacture the Host Strains of inactivation, be used for producing phagocytosis trematodiasis arc Bacterium.It was verified that the phage bdellovibro preparation produced in this way, the bacteriophagic Bdellovibrio place to living can be reduced The cracking ability of main bacterium, even drops to without cracking ability, therefore, unstable to preventing and treating animal bacteria disease effects Fixed, the said preparation holding time is short simultaneously, makes formulation development application be restricted, is more made in aquaculture About.In patent ZL200610039346.8, applicant succeeds in developing with Rhodobacter sphaeroidcs etc. as host's Production method, the phage bdellovibro preparation produced nearly ten years, deeply welcome by user, but its phagocytosis trematodiasis Vibrio preparation still also exists some defects, and the plaque number such as produced is less, and bacterium number is not sufficiently stable, in order to Overcoming these not enough, single Host Strains is improved by we.
Summary of the invention
Goal of the invention: the problem existed for prior art, the present invention provides the life of a kind of phage bdellovibro preparation Product method, the present invention prepares viable count in phage bdellovibro preparation and significantly improves, and can produce substantial amounts of plaque, Further increasing the using effect of phage bdellovibro preparation, the industrialized production for bacteriophagic Bdellovibrio provides one New composite reactive Host Strains.
Technical scheme: to achieve these goals, the producer of a kind of phage bdellovibro preparation Method, comprises the steps:
1, the production method of a kind of phage bdellovibro preparation, it is characterised in that comprise the steps:
(1) preparation of host strain turbid liquor: cultured photosynthetic bacteria suspension is stood 24～96 hours and (trains Breeding method is shown in that China's microecology magazine is P.78.1990.3), remove supernatant, add and remove the bodies such as supernatant The mass fraction 0.2%～0.85%NaCl solution of accumulated amount, shakes up, then stands 24～72 hours, removes supernatant Liquid, adds mass fraction 0.2%～0.85%NaCl solution, be made into former photosynthetic bacteria suspension volume 1/2～ Host strain turbid liquor A of 2/3, by 5%～15% addition inactivation E.Coli600 of host strain turbid liquor A volume Bacteria suspension (strain is bought in Jiangsu Sanitation and Antiepidemic Station), two liquid mixing after prepare host strain turbid liquor B, Standby.
(2) preparation culture fluid: by the phosphate buffer dilute with water of pH6.5～8.0, be subsequently adding CaCl2 And MgCl2, make culture fluid.
(3) fermentation technology of bacteriophagic Bdellovibrio: adding host strain turbid liquor B in culture fluid, volume ratio is 100:(5～30), obtain suspension C, (strain includes Bd1 to add bacteriophagic Bdellovibrio liquid in suspension C Or Bd6, buy in Jiangsu Sanitation and Antiepidemic Station；Cultural method is shown in animal microecological progress p190,2000 Year, China Agricultyre University Press), volume ratio is 100:(2～3), at 28～30 DEG C of temperature, 150-180rpm Under the conditions of cultivate 72～120 hours, i.e. make phage bdellovibro preparation.
As preferably, step (1) described photosynthetic bacteria is Rhodobacter sphaeroidcs P4 or Rhodopseudomonas palustris 101 (strain is bought in biotechnology institute of Shanghai Communications University).Use photosynthetic bacteria as host, improve and bite The lytic activity of bacterium Bdellovibrio and stability, improve controlling disease effect.
As preferably, step (1) described photosynthetic bacteria suspension is the photosynthetic bacteria suspendible cultivating stable phase Liquid.
As preferably, the bacteria containing amount of step (1) described host strain turbid liquor A is 30～30,000,000,000/mL.
As preferably, the bacteria suspension of step (1) described inactivation E.Coli600 is the inactivation cultivating stable phase The bacteria suspension of E.Coli600.
As preferably, step (2) described CaCl2And MgCl2Content is 10～100mg/L.Culture fluid The CaCl that middle addition is a small amount of2And MgCl2Help lend some impetus to the growth of bacteriophagic Bdellovibrio below
As preferably, step (2) described CaCl2And MgCl2Content is 20mg/L.
As preferably, step (3) described bacteriophagic Bdellovibrio liquid bacteria containing amount is more than 105pfu/mL。
The present invention uses photosynthetic bacteria itself to be aquaculture and the probiotics improving water quality, and and bacteriophagic Bdellovibrio There is synergism, therefore the present invention takes the muddy ball in photosynthetic bacteria or the Rhodopseudomonas palustris can be as life Produce the new host bacterium cultivating bacteriophagic Bdellovibrio.
Beneficial effect: compared with prior art, present invention have the advantage that the present invention uses photosynthetic carefully Rhodobacter sphaeroidcs P4 in bacterium or Rhodopseudomonas palustris 101 are as the place of productive culture bacteriophagic Bdellovibrio Main bacterium, the bacteria suspension adding appropriate inactivation E.Coli600 prepares host strain turbid liquor；The present invention prepares In phage bdellovibro preparation, viable count significantly improves, and can produce substantial amounts of plaque, improve bacteriophagic Bdellovibrio The using effect of preparation, enhances the lytic activity of bacteriophagic Bdellovibrio and stability and controlling disease effect.
The phage bdellovibro preparation that the present invention prepares effect in terms of improving water quality is obvious, makes water PH value stable, Reduce COD, ammoniacal nitrogen and content of nitrite etc. in water body.Chinese Fishery academy's fresh water fishery Research center experiment shows, after using bacteriophagic Bdellovibrio, ammoniacal nitrogen is reduced to 0.22 (mg/L), COD by 0.35 4.2 (mg/L) are reduced to by 6.4.This preparation is to preventing and treating the white scour of chicken, and children's livestock and poultry diarrhea such as yellow and white dysentery of piglet also has Remarkable result.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described.
Embodiment 1
1, the preparation of host strain turbid liquor: the photosynthetic bacteria Rhodobacter sphaeroidcs P4 of stable phase will be cultivated Suspension, stands 24 hours, removes and accounts for complete soln volume 1/2 supernatant；Add and remove supernatant etc. The mass fraction 0.2%NaCl solution of volume, shakes up, then stands 24 hours, removes and accounts for complete soln body The supernatant of long-pending 1/2, adds mass fraction 0.2%NaCl solution, is made into former photosynthetic bacteria suspension volume Host strain turbid liquor A of 1/2, bacteria containing amount 3,000,000,000/ml, add the training of host strain turbid liquor A volume 5% Support the inactivation E.Coli600 bacteria suspension of stable phase, it is thus achieved that host strain turbid liquor B, standby.
2., preparation culture fluid: by phosphate buffer (PBS) 10 times of the water dilution of pH6.5, then add Enter 10mg/L CaCl2And MgCl2Make culture fluid.
3., the fermentation technology of bacteriophagic Bdellovibrio: adding host strain turbid liquor B in culture fluid, volume ratio is 100: 5, obtain suspension C, suspension C adds bacteriophagic Bdellovibrio liquid bacteria containing amount 5x105Pfu/ml, volume ratio is 100:2,28 DEG C of temperature, cultivates 120 hours under the conditions of 150rpm, i.e. makes phage bdellovibro preparation.
Embodiment 2
1, the preparation of host strain turbid liquor: the photosynthetic bacteria Rhodobacter sphaeroidcs P4 of stable phase will be cultivated Suspension, stands 60 hours, removes and accounts for complete soln volume 2/3 supernatant；Add and remove supernatant etc. The mass fraction 0.5%NaCl solution of volume, shakes up, then stands 48 hours, removes and accounts for complete soln body The supernatant of long-pending 2/3, adds mass fraction 0.5%NaCl solution, is made into former photosynthetic bacteria suspension volume Host strain turbid liquor A of 2/3, bacteria containing amount 17,000,000,000/ml, add host strain turbid liquor A volume 10% Cultivate the inactivation E.Coli600 bacteria suspension of stable phase, it is thus achieved that host strain turbid liquor B, standby.
2, preparation culture fluid: by phosphate buffer (PBS) 10 times of the water dilution of pH7.5, then add Enter 60mg/L CaCl2And MgCl2Make culture fluid.
3, the fermentation technology of bacteriophagic Bdellovibrio: adding host strain turbid liquor B in culture fluid, volume ratio is 100: 18, obtain suspension C, suspension C adds bacteriophagic Bdellovibrio liquid bacteria containing amount 7x105Pfu/ml, volume ratio For 100:3,30 DEG C of temperature, cultivate 96 hours under the conditions of 160rpm, i.e. make phage bdellovibro preparation.
Embodiment 3
1, the preparation of host strain turbid liquor: the photosynthetic bacteria Rhodopseudomonas palustris 101 of stable phase will be cultivated Suspension, stands 96 hours, removes and accounts for complete soln volume 1/2 supernatant；Add and remove supernatant etc. The mass fraction 0.85%NaCl solution of volume, shakes up, then stands 72 hours, removes and accounts for complete soln body The supernatant of long-pending 1/2, adds mass fraction 0.85%NaCl solution, is made into former photosynthetic bacteria suspension volume Host strain turbid liquor A of 1/2, bacteria containing amount 30,000,000,000/ml, add host strain turbid liquor A volume 15% Cultivate the inactivation E.Coli600 bacteria suspension of stable phase, it is thus achieved that host strain turbid liquor B, standby.
2, preparation culture fluid: by phosphate buffer (PBS) 10 times of the water dilution of pH8.0, then Add 100mg/L CaCl2And MgCl2Make culture fluid.
3, the fermentation technology of bacteriophagic Bdellovibrio: adding host strain turbid liquor B in culture fluid, volume ratio is 100: 30, obtain suspension C, suspension C adds bacteriophagic Bdellovibrio liquid bacteria containing amount 9x105Pfu/ml, volume ratio For 100:3,30 DEG C of temperature, cultivate 72 hours under the conditions of 180rpm, i.e. make phage bdellovibro preparation.
Embodiment 4
1, the preparation of host strain turbid liquor: the photosynthetic bacteria Rhodobacter sphaeroidcs P4 of stable phase will be cultivated Suspension, stands 48 hours, removes and accounts for complete soln volume 1/2 supernatant；Add and remove supernatant etc. The mass fraction 0.5%NaCl solution of volume, shakes up, then stands 48 hours, removes and accounts for complete soln body The supernatant of long-pending 1/2, adds mass fraction 0.5%NaCl solution, is made into former photosynthetic bacteria suspension volume Host strain turbid liquor A of 1/2, bacteria containing amount 30,000,000,000/ml, add host strain turbid liquor A volume 10% Cultivate the bacteria suspension of inactivation E.Coli600 of stable phase, it is thus achieved that host strain turbid liquor B, standby.
2, preparation culture fluid: by phosphate buffer (PBS) 10 times of the water dilution of pH7.0, then add Enter 20mg/L CaCl2And MgCl2Make culture fluid.
3, the fermentation technology of bacteriophagic Bdellovibrio: adding host strain turbid liquor B in culture fluid, volume ratio is 100: 18, obtain suspension C, suspension C adds bacteriophagic Bdellovibrio liquid bacteria containing amount 7x105Pfu/ml, volume ratio For 100:3,30 DEG C of temperature, cultivate 72 hours under the conditions of 180rpm, i.e. make phage bdellovibro preparation.
Test example 1
Phage bdellovibro preparation to the production of embodiments of the invention, successively 5 batches of extraction, detect respectively Produce plaque situation, the results are shown in Table 1.
Comparative example 1 Rhodobacter sphaeroidcs P4 is added without inactivateing E.Coli600 bacteria suspension as host, its He is identical with embodiment 3 method.
Comparative example 2 Rhodopseudomonas palustris 101 is added without inactivateing E.Coli600 bacteria suspension as host, its He is identical with embodiment 3 method.
The plaque situation that table 1 phage bdellovibro preparation produces
Rhodobacter sphaeroidcs P4 in photosynthetic bacteria or Rhodopseudomonas palustris 101 and E.Coli600, as The mixing Host Strains of productive culture bacteriophagic Bdellovibrio, cultivates viable count in the phage bdellovibro preparation made and substantially carries High.We, to cultivating preparation successively sampling Detection 5 times, are shown by table 1 result, and comparative example 1 is with single muddy ball Rhodopseudomonas P4 bacterium solution cultivation gained phage bdellovibro preparation averagely produces plaque number and is 3.7×105.4Pfu/ml, comparative example 2 cultivates gained bacteriophagic Bdellovibrio by single Rhodopseudomonas palustris 101 bacterium solution It is 4.5 × 10 that preparation averagely produces plaque number5, and embodiment 1, embodiment 2, embodiment 3, embodiment 4 Gained phage bdellovibro preparation averagely produces plaque number and is respectively 4.2 × 106、2.5×106.1、2.8×106.1, 3.5×106.4Pfu/ml, embodiment plaque number relatively comparative example significantly improves.
Test example 2
The phage bdellovibro preparation using the embodiment of the present invention to make, carries out prawn culturing test, and research preventing and treating is right Shrimp disease, each consumption 5ppm, it is spilled into pond, within 10～15 days, uses once, embodiment of the present invention prawn is put down All survival rate more than 90%, and blank is only 70%, and (blank is feeding and management routinely, no Use phage bdellovibro preparation), use biting of single Rhodobacter sphaeroidcs P4 or Rhodopseudomonas palustris 101 Bacterium bdellovibrio bacteriovorus preparation survival rate is less than 80%, it was demonstrated that the phage bdellovibro preparation that the present invention is produced is to preventing and treating fish and shrimp Disease, improving water quality all has remarkable result.

Claims (8)

1. the production method of a phage bdellovibro preparation, it is characterised in that comprise the steps:
(1) preparation of host strain turbid liquor: cultured photosynthetic bacteria suspension is stood 24～96 hours, Removing supernatant, adding the mass fraction with removing supernatant equal volume amounts is 0.2%～0.85%NaCl solution, Shaking up, stand 24～72 hours, remove supernatant, it is 0.2%～0.85%NaCl molten for adding mass fraction Liquid, is made into host strain turbid liquor A of former photosynthetic bacteria suspension volume 1/2～2/3, by host strain turbid liquor A The 5% of volume～15% bacteria suspension adding inactivation E.Coli600, prepares host strain turbid liquor B after two liquid mixings, Standby；
(2) preparation culture fluid: by the phosphate buffer dilute with water of pH6.5～8.0, be subsequently adding CaCl2 And MgCl2, make culture fluid；
(3) fermentation technology of bacteriophagic Bdellovibrio: adding host strain turbid liquor B in culture fluid, volume ratio is 100:(5～30), obtain suspension C, suspension C adds bacteriophagic Bdellovibrio liquid, volume ratio is 100: (2～3), 28～30 DEG C of temperature, cultivate 72～120 hours under the conditions of 150-180rpm, i.e. make phagocytosis Bdellovibrio bacteriovorus preparation.
The production method of phage bdellovibro preparation the most according to claim 1, it is characterised in that step (1) Described photosynthetic bacteria is Rhodobacter sphaeroidcs P4 or Rhodopseudomonas palustris 101.
The production method of phage bdellovibro preparation the most according to claim 1, it is characterised in that step (1) Described photosynthetic bacteria suspension is the photosynthetic bacteria suspension cultivating stable phase.
The production method of phage bdellovibro preparation the most according to claim 1, it is characterised in that step (1) The bacteria containing amount of described host strain turbid liquor A is 30～30,000,000,000/mL.
The production method of phage bdellovibro preparation the most according to claim 1, it is characterised in that step (1) The bacteria suspension of described inactivation E.Coli600 is the bacteria suspension of the inactivation E.Coli600 cultivating stable phase.
The production method of phage bdellovibro preparation the most according to claim 1, it is characterised in that step (2) Described CaCl2And MgCl2Content is 10～100mg/L.
The production method of phage bdellovibro preparation the most according to claim 5, it is characterised in that step (2) Described CaCl2And MgCl2Content is 20mg/L.
The production method of phage bdellovibro preparation the most according to claim 1, it is characterised in that step (3) Described bacteriophagic Bdellovibrio liquid bacteria containing amount is more than 105pfu/mL。
CN201610513338.6A 2016-07-01 2016-07-01 Production method of bdellovibrio bacteriovorus preparation Pending CN106011019A (en)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107988135A (en) * 2017-12-14 2018-05-04 华南理工大学 Application of the magnesium ion in promoting ocean bdellovibrio bdelloplast bacterial to be formed
CN110169982A (en) * 2019-06-06 2019-08-27 厦门惠盈动物科技有限公司 A kind of phage bdellovibro preparation and its application for preventing batrachia torticollis disease
CN110184222A (en) * 2019-06-06 2019-08-30 厦门惠盈动物科技有限公司 A kind of bacteriophagic Bdellovibrio freeze-dried powder preparation and its application
CN110179833A (en) * 2019-06-06 2019-08-30 厦门惠盈动物科技有限公司 A kind of phage bdellovibro preparation and its application for preventing grouper ulcer
CN110215466A (en) * 2019-06-06 2019-09-10 厦门惠盈动物科技有限公司 A kind of phage bdellovibro preparation and its application for preventing common eel bacteriosis

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107988135A (en) * 2017-12-14 2018-05-04 华南理工大学 Application of the magnesium ion in promoting ocean bdellovibrio bdelloplast bacterial to be formed
CN107988135B (en) * 2017-12-14 2020-06-19 华南理工大学 Application of magnesium ions in promoting formation of bdelloplast of marine bdellovibrio bacteriovorus
CN110169982A (en) * 2019-06-06 2019-08-27 厦门惠盈动物科技有限公司 A kind of phage bdellovibro preparation and its application for preventing batrachia torticollis disease
CN110184222A (en) * 2019-06-06 2019-08-30 厦门惠盈动物科技有限公司 A kind of bacteriophagic Bdellovibrio freeze-dried powder preparation and its application
CN110179833A (en) * 2019-06-06 2019-08-30 厦门惠盈动物科技有限公司 A kind of phage bdellovibro preparation and its application for preventing grouper ulcer
CN110215466A (en) * 2019-06-06 2019-09-10 厦门惠盈动物科技有限公司 A kind of phage bdellovibro preparation and its application for preventing common eel bacteriosis

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