CN104357354B - A kind of production method of bacteriophagic Bdellovibrio microbial ecological agent - Google Patents

A kind of production method of bacteriophagic Bdellovibrio microbial ecological agent Download PDF

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CN104357354B
CN104357354B CN201410610577.4A CN201410610577A CN104357354B CN 104357354 B CN104357354 B CN 104357354B CN 201410610577 A CN201410610577 A CN 201410610577A CN 104357354 B CN104357354 B CN 104357354B
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bdellovibrio
slow
phosphate buffer
bacteria suspension
releasing granules
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CN201410610577.4A
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Chinese (zh)
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CN104357354A (en
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江文涛
马加军
储玉龙
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广州利洋水产科技股份有限公司
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/20Bacteria; Culture media therefor

Abstract

The invention belongs to cultural technique field, the invention discloses the production method of a kind of bacteriophagic Bdellovibrio microbial ecological agent, comprise the steps: 1) preparation of host bacteria suspension;2) preparation of slow-releasing granules;3) preparation of culture fluid;4) preparation of seed;5) preparation of bdellovibrio bacteriovorus preparation.The present invention adopts the slow release method of Host Strains, overcomes the current defect that the phage bdellovibro preparation shelf-life is short, bacterial content is unstable, improves the phage bdellovibro preparation effect to disease control of aquatic animal Yu water quality improvement.

Description

A kind of production method of bacteriophagic Bdellovibrio microbial ecological agent
Technical field
The invention belongs to cultural technique field, be specifically related to the production method of a kind of bacteriophagic Bdellovibrio microbial ecological agent.
Background technology
Bacteriophagic Bdellovibrio is the parasitics antibacterial that a class is made a living with predator bacteria specially, the vibrio in water body, pseudomonas, Aeromonas etc. can be cracked in the short period of time, pathogenic bacterium quantity can be limited in reduced levels, the content of the COD of breeding water body, sulfide and ammonia nitrogen can be efficiently controlled again simultaneously, and cultivation body intestinal absorption and immune level can be improved.Bdellovibrio active bacteria formulation acts not only as a kind of feed additive and uses, and can use as a kind of improver of water quality breeding water body of directly splashing.Therefore, use it as the natural biological purification factor in cultivation water environment and " living antibiotics ", there is vast potential for future development, developing aquatic animal disease Biological control new way is had great importance.
At present, the bdellovibrio bacteriovorus preparation holding time of some manufacturer production is shorter.The titer having research display Bdellovibrio preservation is very unstable, and after room temperature preservation 36 days, the concentration of Bdellovibrio is by original 108Pfu/mL is reduced to 10pfu/mL.Additionally, due to Bdellovibrio its have strong endogenous Repiration, easily dead at low temperatures, thus the preservation of Bdellovibrio is seemed relatively difficult.Strengthen the research of the long-acting stable preservation method of Bdellovibrio, to realizing Bdellovibrio popularization on aquatic animal biological disease-preventing is applied, there is important theory significance.
Summary of the invention:
It is an object of the invention to provide a kind of bacteriophagic Bdellovibrio microbial ecological agent and production method thereof, adopt the slow release method of Host Strains, solve the problems such as bacteriophagic Bdellovibrio shelf-life in commercial process short, lytic activity reduction, improve the phage bdellovibro preparation effect to disease control of aquatic animal Yu water quality improvement.
In order to realize foregoing invention purpose, the technical solution used in the present invention comprises the following steps:
1. the preparation of host bacteria suspension
Being centrifuged with tubular-bowl centrifuge after autoclaving by cultured escherichia coli and collect thalline, being diluted to concentration with phosphate buffer is 1011~1013The host bacteria suspension of cfu/mL, is placed at 4 DEG C and saves backup.
2. the preparation of slow-releasing granules
By host bacteria suspension dilute 0.5~5 times, it is subsequently adding the gel of 1%~15%, is sufficiently mixed dissolving, instill in cross-linking agent, cross-link 20min~12h, clean standby.
3. the preparation of culture fluid
By the phosphate buffer dilute with water 10~200 times of pH6.0~8.5, it is subsequently adding the CaCl of 10~100mg/L2, and the MgCl of 10~100mg/L2
4. the preparation of seed
Phosphate buffer adds the host bacteria suspension 2%~20% of step 1 preparation, then accesses Bdellovibrio, 18~35 DEG C, 180rpm cultivate 18~60h.
5. the preparation of bdellovibrio bacteriovorus preparation
Phosphate buffer adds the host grain 5~20 of step 2 preparation, then accesses Bdellovibrio seed 1 ‰~10 ‰ prepared by step 4, namely make 107~109The bdellovibrio bacteriovorus preparation of pfu/mL.
Host Strains described in step 1 is to adopt 115 DEG C of autoclaving 30min, is then centrifuged with tubular-bowl centrifuge 10000rpm, and being diluted to concentration with phosphate buffer is 1011~1013The host bacteria suspension of cfu/mL;
Being prepared by of slow-releasing granules described in step 2, by host bacteria suspension dilute 0.5~5 times, is subsequently adding the gel of 1%~15%, is sufficiently mixed dissolving, instills in cross-linking agent, cross-links 20min~12h, clean standby.
The gel that the preparation of slow-releasing granules described in step 2 uses can be 5%~15% polyvinyl alcohol, adopts freeze-thaw method to carry out the solidification of granule;The gel that the preparation of slow-releasing granules described in step 2 uses can also be 1%~6% alginate, and cross-linking agent is 1%~8%CaCl2;The gel that the preparation of slow-releasing granules described in step 2 uses can also is that 2%~10% gelatin and 1%~6% alginate mixed liquor, and cross-linking agent is 1%~8%CaCl2
All of phosphate buffered solution pH described in step 3 is 6.0~8.5, and extension rate is 10~200 times, and NaCl concentration is 0~30 ‰, CaCl2Concentration is 10~100mg/L, MgCl2Concentration is 10~100mg/L.
Bdellovibrio seed described in step 4 is addition host bacteria suspension 2%~20% in phosphate buffer, then accesses Bdellovibrio, 18~35 DEG C, 180rpm cultivate 18~60h.
Bdellovibrio bacteriovorus preparation described in step 5 be prepared by phosphate buffer add step 2 preparation host grain 5~20, then access step 4 preparation Bdellovibrio seed 1 ‰~10 ‰, namely make 107~109The bdellovibrio bacteriovorus preparation of pfu/mL.
The present invention has such advantages as relative to prior art and effect:
(1) Host Strains adopted in production method of the present invention is the gram negative bacteria of the environmental sound after inactivation.
(2) present invention adopts Host Strains slow release method, not only substantially prolongs the shelf-life of Bdellovibrio, improves the activity of product, enhances its result of use, also reduces the consumption of Host Strains simultaneously, reduces production cost.The liquid bdellovibrio bacteriovorus preparation that the method produces, after three months room temperature storage, Bdellovibrio viable count remains to reach 1.0 × 106More than pfu/mL.
(3) the Bdellovibrio slow-releasing granules that the present invention produces can keep the long-term stability of granule to be not dissolved while ensureing its effective ingredient slow releasing.
Detailed description of the invention: the present invention is described in further detail below in conjunction with embodiment, embodiments of the present invention are not limited to this.
Embodiment 1: Host Strains is fixed with polyvinyl alcohol, detailed process is as follows:
(1) preparation of host bacteria suspension
By cultured escherichia coli through 115 DEG C of autoclaving 30min, being then centrifuged with tubular-bowl centrifuge 10000rpm, and collect thalline, being diluted to concentration with phosphate buffer is 1012The host bacteria suspension of cfu/mL, is placed at 4 DEG C and saves backup;
(2) preparation of slow-releasing granules
By host bacteria suspension dilute 2 times, being subsequently adding the polyvinyl alcohol of 10%, be sufficiently mixed dissolving, the vegetable oil adding 1 times of volume is sufficiently stirred for, and forms spheroid, at-20 DEG C of sizing and solidifying 8h, cleans standby.(3) preparation of culture fluid
By the phosphate buffer dilute with water 100 times of pH7.4, it is subsequently adding the CaCl of 40mg/L2MgCl with 40mg/L2
(4) preparation of seed
Phosphate buffer adds step (1) host bacteria suspension 10% prepared, then accesses Bdellovibrio, 28~30 DEG C, 180rpm cultivate 28h.
(5) preparation of bdellovibrio bacteriovorus preparation
Phosphate buffer adds host grain 20 prepared by step (2), then accesses Bdellovibrio seed 10 ‰ prepared by step (4), namely make 108The bdellovibrio bacteriovorus preparation of pfu/mL.
Embodiment 2: Host Strains is fixed with sodium alginate, detailed process is as follows:
(1) preparation of host bacteria suspension
By cultured escherichia coli through 115 DEG C of autoclaving 30min, being then centrifuged with tubular-bowl centrifuge 10000rpm, and collect thalline, being diluted to concentration with phosphate buffer is 1012The host bacteria suspension of cfu/mL, is placed at 4 DEG C and saves backup;
(2) preparation of slow-releasing granules
By host bacteria suspension dilute 1 times, add the sodium alginate of 2%, be sufficiently mixed dissolving, then instill the CaCl of 3%2In, cross-link 30min, clean standby.
(3) preparation of culture fluid
By the phosphate buffer dilute with water 20 times of pH7.2, it is subsequently adding the CaCl of 20mg/L2MgCl with 20mg/L2
(4) preparation of seed
Phosphate buffer adds step (1) host bacteria suspension 5% prepared, then accesses Bdellovibrio, 28~30 DEG C, 180rpm cultivate 32h.
(5) preparation of bdellovibrio bacteriovorus preparation
Phosphate buffer adds host grain 15 prepared by step (2), then accesses Bdellovibrio seed 1 ‰ prepared by step (4), namely make 107The bdellovibrio bacteriovorus preparation of pfu/mL.
Embodiment 3: with gelatin and sodium alginate, Host Strains being fixed, detailed process is as follows:
(1) preparation of host bacteria suspension
By cultured escherichia coli through 115 DEG C of autoclaving 30min, being then centrifuged with tubular-bowl centrifuge 10000rpm, and collect thalline, being diluted to concentration with phosphate buffer is 1012The host bacteria suspension of cfu/mL, is placed at 4 DEG C and saves backup;
(2) preparation of slow-releasing granules
By host bacteria suspension dilute 1 times, and add the gelatin of 7% and the sodium alginate of 3%, be sufficiently mixed dissolving, then instill the CaCl of 4%2In, cross-link 60min, clean standby.
(3) preparation of culture fluid
By the phosphate buffer dilute with water 10 times of pH7.2, it is subsequently adding the CaCl of 60mg/L2MgCl with 60mg/L2
(4) preparation of seed
Phosphate buffer adds step (1) host bacteria suspension 15% prepared, then accesses Bdellovibrio, 28~30 DEG C, 180rpm cultivate 28h.
(5) preparation of bdellovibrio bacteriovorus preparation
Phosphate buffer adds host grain 8 prepared by step (2), then accesses Bdellovibrio seed 10 ‰ prepared by step (4), namely make 108The bdellovibrio bacteriovorus preparation of pfu/mL.
After prepared by above-described embodiment 1~3 bdellovibrio bacteriovorus preparation, it is sub-packed in the aseptic bottle of 1L and seals;Buy commercially available prod, as " commercially available prod comparison 1 ";With being not added with the test group of slow-releasing granules as " positive control 1 ";Three groups of sample room temperature preservation (commercially available prod calculates its retention cycle with its date of manufacture for starting point) simultaneously, timing adopts tap water agar double-layer agar technique detection viable count, and testing result is in Table 1:
Table 1 Bdellovibrio Detection of Stability result (pfu/mL)
Cycle Commercially available prod comparison 1 Positive control 1 Embodiment 1 Embodiment 2 Embodiment 3
30d 7.0×105 2.0×106 3.5×108 4.3×108 4.0×108
60d 3.0×104 3.5×105 7.2×107 8.6×107 1.5×107
90d 5.0×102 1.2×104 7.4×106 4.5×107 4.0×106
Above-mentioned result of the test shows, adopts the bdellovibrio bacteriovorus preparation that the method for the present invention prepares, and after three months room temperature storage, Bdellovibrio viable count remains to reach 1.0 × 106More than pfu/mL, is significantly larger than " commercially available prod comparison 1 " and " positive control 1 ", and the liquid Bdellovibrio that therefore present invention produces has better stability.
Embodiment 4: Bdellovibrio application example in culture of Penaeus vannamei
1. test site and material: Example 3 bdellovibrio bacteriovorus preparation concentration is 108Pfu/ml, selecting Sha Bei culture of Penaeus vannamei field, Fanyu is application test place.
2. Bdellovibrio product testing: by every 1m3Water body products applied 2~3mL, carries out detection in continuous 8 days, records test data the pond situation before and after test.
3. Penaeus vannamei is supported bacterial disease experiment of preventive effects by Bdellovibrio: by every 1m3Water body products applied 2~3mL, carries out detection in continuous 6 days, records test data the pond situation before and after test.
4. the collection of water sample and index determining: use TCBS culture medium that each water sample does 4 parallel contrast tests detection water sample vibrio numbers, and to pond pH, NO2 --N、NH3-N, DO, transparency, total alkalinity, amount of vibrio, prawn mortality, the situation of ingesting carries out observed and recorded;PH, NO2 --N、NH3-N and DO measures the test kit detection adopting our company to produce, and total alkalinity adopts the reagent detection (with reference to " water and effluent monitoring analyze method ") of company's institute configuration.Its result is as follows:
Table 2: liquid Bdellovibrio application test (20A)
Time pH NO2 --N NH3-N DO Transparent Total alkali Vibrio sum (cfu/ml)
0d 8.0 0.01 0.0 3.5 90+ 50 1200
1d 8.0 0.03 0.0 3.5 90+ 40 300
2d 8.0 0.01 0.0 3.5 90+ 30 300
3d 8.0 0.01 0.0 4.0 90+ 30 330
4d 8.0 0.03 0.0 5.0 90+ 20 320
5d 8.0 0.03 0.0 5.5 90+ 30 100
6d 8.0 0.06 0.0 5.0 90+ 30 210
7d 8.0 0.03 0.0 5.0 90+ 20 /
8d 8.0 0.01 0.0 5.5 90+ 20 /
Result of the test shows: bdellovibrio bacteriovorus preparation can quickly reduce vibrio sum in prawn culturing pond, and after bdellovibrio bacteriovorus preparation uses pond to use 6d, order of magnitude lower drops in vibrio sum;PH is had no significant effect by bdellovibrio bacteriovorus preparation, but can suitably reduce total alkalinity.
Table 3: the Bdellovibrio prophylactic tria (2A) to bacterial disease
Result of the test shows: after using bdellovibrio bacteriovorus preparation of the present invention, vibrio sum in Chi Shui and prawn liver is decreased obviously, pond prawn is without the sick death of outburst, and feeding volume is normal, and therefore bdellovibrio bacteriovorus preparation of the present invention prevents the effect of bacterial disease better in prawn culturing.

Claims (1)

1. the production method of a bacteriophagic Bdellovibrio microbial ecological agent, it is characterised in that comprise the following steps:
(1) preparation of host bacteria suspension
By cultured escherichia coli through 115 DEG C of autoclaving 30min, being then centrifuged with tubular-bowl centrifuge 10000rpm, being diluted to concentration with phosphate buffer is 1011~1013The host bacteria suspension of cfu/mL, is placed at 4 DEG C and saves backup;
(2) preparation of slow-releasing granules
By host bacteria suspension dilute 0.5~5 times, it is subsequently adding the gel of 1%~15%, is sufficiently mixed dissolving, instill in cross-linking agent, cross-link 20min~12h, clean standby;The gel that the preparation of described slow-releasing granules uses is 5%~15% polyvinyl alcohol, adopts freeze-thaw method to carry out the solidification of granule;Or the gel that the preparation of described slow-releasing granules uses is 1%~6% alginate, and cross-linking agent is 1%~8%CaCl2;Or the gel that the preparation of described slow-releasing granules uses is 2%~10% gelatin and 1%~6% alginate mixed liquor, and cross-linking agent is 1%~8%CaCl2
(3) preparation of culture fluid
By the phosphate buffer dilute with water 10~200 times of pH6.0~8.5, being subsequently adding concentration is 0~30 ‰ NaCl, and concentration is 10~100mg/LCaCl2, concentration is 10~100mg/LMgCl2
(4) preparation of seed
Phosphate buffer adds step (1) host bacteria suspension 2%~20% prepared, then accesses Bdellovibrio, 18~35 DEG C, 180rpm cultivate 18~60h;
(5) preparation of bdellovibrio bacteriovorus preparation
Phosphate buffer adds slow-releasing granules 5~20 prepared by step (2), then accesses Bdellovibrio seed prepared by 1 ‰~10 ‰ steps (4), namely make 107~109The bdellovibrio bacteriovorus preparation of pfu/mL.
CN201410610577.4A 2014-11-01 2014-11-01 A kind of production method of bacteriophagic Bdellovibrio microbial ecological agent CN104357354B (en)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN103719535A (en) * 2012-10-10 2014-04-16 上海海洋大学 Bdellovibrio bacteriovorus microcapsule and preparation method thereof

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CN100394926C (en) * 2006-04-06 2008-06-18 薛恒平 Production process of bdellophage preparation
CN103320341B (en) * 2010-08-31 2016-01-20 华南理工大学 A kind of bdellovibrio bacteriovorus preparation and fermentation process thereof and application

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CN103719535A (en) * 2012-10-10 2014-04-16 上海海洋大学 Bdellovibrio bacteriovorus microcapsule and preparation method thereof

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