CN104357354A - Production method of phage bdellovibrio microecological preparation - Google Patents

Production method of phage bdellovibrio microecological preparation Download PDF

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CN104357354A
CN104357354A CN201410610577.4A CN201410610577A CN104357354A CN 104357354 A CN104357354 A CN 104357354A CN 201410610577 A CN201410610577 A CN 201410610577A CN 104357354 A CN104357354 A CN 104357354A
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bdellovibrio
production
buffered saline
phosphate buffered
saline buffer
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CN201410610577.4A
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Chinese (zh)
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CN104357354B (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 the technical field of cultivation, and discloses a production method of a phage bdellovibrio microecological preparation. The production method comprises the following steps of (1) preparing host bacteria suspension; (2) preparing slow release particles; (3) preparing cultivation liquid; (4) preparing seeds; (5) preparing the bdellovibrio preparation. By the slow release technology of host bacteria, the defects that an existing phage bdellovibrio preparation is short in quality guarantee period and unstable in bacteria content are overcome, and the effects of preventing and treating aquatic livestock diseases and improving the water quality of the phage bdellovibrio preparation are enhanced.

Description

A kind of production method of bacteriophagic Bdellovibrio probiotics
Technical field
The invention belongs to cultural technique field, be specifically related to a kind of production method of bacteriophagic Bdellovibrio probiotics.
Background technology
Bacteriophagic Bdellovibrio is that a class is specially with the parasitics bacterium that predator bacteria is made a living, can vibrios, pseudomonas, Aeromonas etc. in the short period of time in cracking water body, can by pathogenic bacterium quantity limitation at lower level, effectively can control again the content of the COD of aquaculture water, sulfide and ammonia nitrogen simultaneously, and cultivation body intestinal absorption and immune level can be improved.Bdellovibrio active bacteria formulation not only can use as a kind of fodder additives, and can use as a kind of improver of water quality aquaculture water of directly splashing.Therefore, profit uses it as natural biological purification factor in cultivation water environment and " living antibiotics ", has vast potential for future development, has great importance to developing aquatic animal disease biological control new way.
At present, the bdellovibrio bacteriovorus preparation shelf time of some manufacturer production is shorter.Have tiring of research display Bdellovibrio preservation very unstable, room temperature preservation is after 36 days, and the concentration of Bdellovibrio is by original 10 8pfu/mL is reduced to 10pfu/mL.In addition, due to Bdellovibrio, it has strong endogenous respiration, easily dead at low temperatures, thus seems more difficult to the preservation of Bdellovibrio.Strengthening the research of the long-acting stable preservation method of Bdellovibrio, to realizing the popularization of Bdellovibrio in the application of aquatic animal biological disease-preventing, there is important theory significance.
Summary of the invention:
The object of this invention is to provide a kind of bacteriophagic Bdellovibrio probiotics and production method thereof, adopt the slow release method of Host Strains, solve the problems such as bacteriophagic Bdellovibrio quality guaranteed period in commercial process short, lytic activity reduction, improve the effect of phage bdellovibro preparation to disease control of aquatic animal and water quality improvement.
In order to realize foregoing invention object, the technical solution used in the present invention comprises the following steps:
1. the preparation of host bacteria suspension
Cultured intestinal bacteria are centrifugal and collect thalline with tubular-bowl centrifuge after autoclaving, and being diluted to concentration with phosphate buffered saline buffer is 10 11~ 10 13the host bacteria suspension of cfu/mL, saves backup at being placed in 4 DEG C.
2. the preparation of slow-releasing granules
By host bacteria suspension thin up 0.5 ~ 5 times, then add the gelifying agent of 1% ~ 15%, abundant mixed dissolution, in instillation linking agent, crosslinked 20min ~ 12h, cleans for subsequent use.
3. the preparation of nutrient solution
By the phosphate buffered saline buffer dilute with water 10 ~ 200 times of pH6.0 ~ 8.5, then add the CaCl of 10 ~ 100mg/L 2, and the MgCl of 10 ~ 100mg/L 2.
4. the preparation of seed
In phosphate buffered saline buffer, add host bacteria suspension 2% ~ 20% prepared by step 1, then access Bdellovibrio, 18 ~ 35 DEG C, 180rpm cultivates 18 ~ 60h.
5. the preparation of bdellovibrio bacteriovorus preparation
In phosphate buffered saline buffer, add host grain 5 ~ 20 prepared by step 2, then access the Bdellovibrio seed 1 ‰ ~ 10 ‰ of step 4 preparation, namely make 10 7~ 10 9the bdellovibrio bacteriovorus preparation of pfu/mL.
Host Strains described in step 1 is employing 115 DEG C of autoclaving 30min, then uses tubular-bowl centrifuge 10000rpm centrifugal, and being diluted to concentration with phosphate buffered saline buffer is 10 11~ 10 13the host bacteria suspension of cfu/mL;
The preparation of slow-releasing granules described in step 2 is by host bacteria suspension thin up 0.5 ~ 5 times, then adds the gelifying agent of 1% ~ 15%, abundant mixed dissolution, and in instillation linking agent, crosslinked 20min ~ 12h, cleans for subsequent use.
The gelifying agent that described in step 2, the preparation of slow-releasing granules uses can be 5% ~ 15% polyvinyl alcohol, adopts freeze-thaw method to carry out the solidification of particle; The gelifying agent that described in step 2, the preparation of slow-releasing granules uses also can be 1% ~ 6% alginates, and linking agent is 1% ~ 8%CaCl 2; The gelifying agent that described in step 2, the preparation of slow-releasing granules uses can also be 2% ~ 10% gelatin and 1% ~ 6% alginates mixed solution, and linking agent is 1% ~ 8%CaCl 2.
Phosphate buffer soln pH all described in step 3 is 6.0 ~ 8.5, and extension rate is 10 ~ 200 times, and NaCl concentration is 0 ~ 30 ‰, CaCl 2concentration is 10 ~ 100mg/L, MgCl 2concentration is 10 ~ 100mg/L.
Bdellovibrio seed described in step 4 is in phosphate buffered saline buffer, add host bacteria suspension 2% ~ 20%, then accesses Bdellovibrio, 18 ~ 35 DEG C, 180rpm cultivates 18 ~ 60h.
The preparation of bdellovibrio bacteriovorus preparation described in step 5 is the host grain 5 ~ 20 adding step 2 preparation in phosphate buffered saline buffer, then accesses the Bdellovibrio seed 1 ‰ ~ 10 ‰ of step 4 preparation, namely makes 10 7~ 10 9the bdellovibrio bacteriovorus preparation of pfu/mL.
The present invention has following advantage and effect relative to prior art:
(1) Host Strains adopted in production method of the present invention is through the gram negative bacterium of the environmental sound after deactivation.
(2) the present invention adopts Host Strains slow release method, not only substantially prolongs the quality guaranteed period 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 is produced, after three months room temperature storage, Bdellovibrio viable count still can reach 1.0 × 10 6more than pfu/mL.
(3) the Bdellovibrio slow-releasing granules that the present invention produces can keep the long-term stability of particle and do not dissolved while its effective constituent slow releasing of guarantee.
Embodiment: the present invention is described in further detail below in conjunction with embodiment, embodiments of the present invention are not limited thereto.
Embodiment 1: Host Strains is fixed with polyvinyl alcohol, detailed process is as follows:
(1) preparation of host bacteria suspension
By cultured intestinal bacteria through 115 DEG C of autoclaving 30min, then use tubular-bowl centrifuge 10000rpm centrifugal, and collect thalline, being diluted to concentration with phosphate buffered saline buffer is 10 12the host bacteria suspension of cfu/mL, saves backup at being placed in 4 DEG C;
(2) preparation of slow-releasing granules
By host bacteria suspension thin up 2 times, then add the polyvinyl alcohol of 10%, abundant mixed dissolution, the vegetables oil adding 1 times of volume fully stirs, and forms spherule, at-20 DEG C of sizing and solidifying 8h, cleans for subsequent use.
(3) preparation of nutrient solution
By the phosphate buffered saline buffer dilute with water 100 times of pH7.4, then add the CaCl of 40mg/L 2with the MgCl of 40mg/L 2.
(4) preparation of seed
In phosphate buffered saline buffer, add host bacteria suspension 10% prepared by step (1), then access Bdellovibrio, 28 ~ 30 DEG C, 180rpm cultivates 28h.
(5) preparation of bdellovibrio bacteriovorus preparation
In phosphate buffered saline buffer, add host grain 20 prepared by step (2), then access Bdellovibrio seed 10 ‰ prepared by step (4), namely make 10 8the 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 intestinal bacteria through 115 DEG C of autoclaving 30min, then use tubular-bowl centrifuge 10000rpm centrifugal, and collect thalline, being diluted to concentration with phosphate buffered saline buffer is 10 12the host bacteria suspension of cfu/mL, saves backup at being placed in 4 DEG C;
(2) preparation of slow-releasing granules
By host bacteria suspension thin up 1 times, add the sodium alginate of 2%, abundant mixed dissolution, then instill the CaCl of 3% 2in, crosslinked 30min, cleans for subsequent use.
(3) preparation of nutrient solution
By the phosphate buffered saline buffer dilute with water 20 times of pH7.2, then add the CaCl of 20mg/L 2with the MgCl of 20mg/L 2.
(4) preparation of seed
In phosphate buffered saline buffer, add host bacteria suspension 5% prepared by step (1), then access Bdellovibrio, 28 ~ 30 DEG C, 180rpm cultivates 32h.
(5) preparation of bdellovibrio bacteriovorus preparation
In phosphate buffered saline buffer, add host grain 15 prepared by step (2), then access Bdellovibrio seed 1 ‰ prepared by step (4), namely make 10 7the bdellovibrio bacteriovorus preparation of pfu/mL.
Embodiment 3: be fixed Host Strains with gelatin and sodium alginate, detailed process is as follows:
(1) preparation of host bacteria suspension
By cultured intestinal bacteria through 115 DEG C of autoclaving 30min, then use tubular-bowl centrifuge 10000rpm centrifugal, and collect thalline, being diluted to concentration with phosphate buffered saline buffer is 10 12the host bacteria suspension of cfu/mL, saves backup at being placed in 4 DEG C;
(2) preparation of slow-releasing granules
By host bacteria suspension thin up 1 times, and add the gelatin of 7% and the sodium alginate of 3%, abundant mixed dissolution, then instill the CaCl of 4% 2in, crosslinked 60min, cleans for subsequent use.
(3) preparation of nutrient solution
By the phosphate buffered saline buffer dilute with water 10 times of pH7.2, then add the CaCl of 60mg/L 2with the MgCl of 60mg/L 2.
(4) preparation of seed
In phosphate buffered saline buffer, add host bacteria suspension 15% prepared by step (1), then access Bdellovibrio, 28 ~ 30 DEG C, 180rpm cultivates 28h.
(5) preparation of bdellovibrio bacteriovorus preparation
In phosphate buffered saline buffer, add host grain 8 prepared by step (2), then access Bdellovibrio seed 10 ‰ prepared by step (4), namely make 10 8the bdellovibrio bacteriovorus preparation of pfu/mL.
Above-described embodiment 1 ~ 3 bdellovibrio bacteriovorus preparation is sub-packed in the aseptic bottle of 1L and seals after having prepared; Buy commercially available prod, as " commercially available prod contrast 1 "; With not adding the test group of slow-releasing granules as " positive control 1 "; Three groups of samples are room temperature preservation (commercially available prod with its date manufactured for starting point calculates its retention period) simultaneously, and timing adopts tap water agar double-layer agar technique to detect viable count, and detected result is in table 1:
Table 1 Bdellovibrio Detection of Stability result (pfu/mL)
Cycle Commercially available prod contrast 1 Positive control 1 Embodiment 1 Embodiment 2 Embodiment 3
30d 7.0×10 5 2.0×10 6 3.5×10 8 4.3×10 8 4.0×10 8
60d 3.0×10 4 3.5×10 5 7.2×10 7 8.6×10 7 1.5×10 7
90d 5.0×10 2 1.2×10 4 7.4×10 6 4.5×10 7 4.0×10 6
Above-mentioned test-results shows, adopts the bdellovibrio bacteriovorus preparation that method of the present invention prepares, and after three months room temperature storage, Bdellovibrio viable count still can reach 1.0 × 10 6more than pfu/mL, far away higher than " commercially available prod contrast 1 " and " positive control 1 ", the liquid Bdellovibrio that therefore the present invention produces has better stability.
Embodiment 4: the application example of Bdellovibrio in culture of Penaeus vannamei
1. test site and material: Example 3 bdellovibrio bacteriovorus preparation concentration is 10 8pfu/ml, selects Sha Bei culture of Penaeus vannamei field, Fanyu to be application test place.
2. Bdellovibrio test of products: by every 1m 3water body products applied 2 ~ 3mL, carries out detecting for continuous 8 days to the pond situation before and after test, record testing data.
3. Bdellovibrio supports bacteriosis experiment of preventive effects to Penaeus vannamei: by every 1m 3water body products applied 2 ~ 3mL, carries out detecting for continuous 6 days to the pond situation before and after test, record testing data.
4. the collection of water sample and index determining: use TCBS substratum to do 4 parallel simultaneous tests to each water sample and detect water sample vibrios numbers, and to pond pH, NO 2 --N, NH 3-N, DO, transparency, total alkalinity, amount of vibrio, prawn mortality, the situation of ingesting carries out observed and recorded; PH, NO 2 --N, NH 3the test kit that the mensuration of-N and DO adopts our company to produce detects, and total alkalinity adopts the reagent of institute of company configuration to detect (with reference to " water and waste water method for monitoring and analyzing ").Its result is as follows:
Table 2: liquid Bdellovibrio application test (20A)
Time pH NO 2 --N NH 3-N DO Transparent Total alkali Vibrios 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 /
Test-results shows: bdellovibrio bacteriovorus preparation can reduce vibrios sum in prawn culturing pond fast, and after bdellovibrio bacteriovorus preparation uses pond to use 6d, vibrios sum reduces an order of magnitude; Bdellovibrio bacteriovorus preparation has no significant effect pH, but suitably can reduce total alkalinity.
Table 3: Bdellovibrio is to the prophylactic tria (2A) of bacteriosis
Test-results shows: after using bdellovibrio bacteriovorus preparation of the present invention, vibrios sum in Chi Shui and prawn liver obviously declines, 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 (7)

1. a production method for bacteriophagic Bdellovibrio probiotics, is characterized in that comprising the following steps:
(1) preparation of host bacteria suspension
Cultured intestinal bacteria are centrifugal and collect thalline with tubular-bowl centrifuge after autoclaving, and being diluted to concentration with phosphate buffered saline buffer is 10 11~ 10 13the host bacteria suspension of cfu/mL, saves backup at being placed in 4 DEG C.
(2) preparation of slow-releasing granules
By host bacteria suspension thin up 0.5 ~ 5 times, then add the gelifying agent of 1% ~ 15%, abundant mixed dissolution, in instillation linking agent, crosslinked 20min ~ 12h, cleans for subsequent use.
(3) preparation of nutrient solution
By the phosphate buffered saline buffer dilute with water 10 ~ 200 times of pH6.0 ~ 8.5, then add the CaCl of 10 ~ 100mg/L 2, and the MgCl of 10 ~ 100mg/L 2.
(4) preparation of seed
In phosphate buffered saline buffer, add host bacteria suspension 2% ~ 20% prepared by step (1), then access Bdellovibrio, 18 ~ 35 DEG C, 180rpm cultivates 18 ~ 60h.
(5) preparation of bdellovibrio bacteriovorus preparation
In phosphate buffered saline buffer, add host grain 5 ~ 20 prepared by step (2), then access Bdellovibrio seed 1 ‰ ~ 10 ‰ prepared by step (4), namely make 10 7~ 10 9the bdellovibrio bacteriovorus preparation of pfu/mL.
2. the production method of a kind of bdellovibrio bacteriovorus ecological preparation according to claim 1, it is characterized in that: the described cultured intestinal bacteria of step (1) are through 115 DEG C of autoclaving 30min, then use tubular-bowl centrifuge 10000rpm centrifugal, being diluted to concentration with phosphate buffered saline buffer is 10 11~ 10 13the host bacteria suspension of cfu/mL.
3. the production method of a kind of bdellovibrio bacteriovorus ecological preparation according to claim 1, it is characterized in that: the preparation of step (2) described slow-releasing granules is by host bacteria suspension thin up 0.5 ~ 5 times, then the gelifying agent of 1% ~ 15% is added, abundant mixed dissolution, in instillation linking agent, crosslinked 20min ~ 12h, cleans for subsequent use.
4. the production method of a kind of bdellovibrio bacteriovorus ecological preparation according to claim 1, it is characterized in that: the gelifying agent that the preparation of step (2) described slow-releasing granules uses can be 5% ~ 15% polyvinyl alcohol, adopt freeze-thaw method to carry out the solidification of particle; The gelifying agent that the preparation of step (2) described slow-releasing granules uses also can be 1% ~ 6% alginates, and linking agent is 1% ~ 8%CaCl 2; The gelifying agent that the preparation of step (2) described slow-releasing granules uses can also be 2% ~ 10% gelatin and 1% ~ 6% alginates mixed solution, and linking agent is 1% ~ 8%CaCl 2.
5. the production method of a kind of bdellovibrio bacteriovorus ecological preparation according to claim 1, is characterized in that: the described all phosphate buffer soln pH of step (3) are 6.0 ~ 8.5, and extension rate is 10 ~ 200 times, and NaCl concentration is 0 ~ 30 ‰, CaCl 2concentration is 10 ~ 100mg/L, MgCl 2concentration is 10 ~ 100mg/L.
6. the production method of a kind of bdellovibrio bacteriovorus ecological preparation according to claim 1, it is characterized in that: the described Bdellovibrio seed of step (4) is in phosphate buffered saline buffer, add host bacteria suspension 2% ~ 20%, access Bdellovibrio again, 18 ~ 35 DEG C, 180rpm cultivates 18 ~ 60h.
7. the production method of a kind of bdellovibrio bacteriovorus ecological preparation according to claim 1, it is characterized in that: the preparation of step (5) described bdellovibrio bacteriovorus preparation is in phosphate buffered saline buffer, add host grain 5 ~ 20 prepared by step (2), access Bdellovibrio seed 1 ‰ ~ 10 ‰ prepared by step (4) again, namely make 10 7~ 10 9the 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 (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1857327A (en) * 2006-04-06 2006-11-08 薛恒平 Production process of bdellophage preparation
CN101948784A (en) * 2010-08-31 2011-01-19 华南理工大学 Bdellovibrio bacteriovorus preparation and fermentation method and application thereof
CN103719535A (en) * 2012-10-10 2014-04-16 上海海洋大学 Bdellovibrio bacteriovorus microcapsule and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1857327A (en) * 2006-04-06 2006-11-08 薛恒平 Production process of bdellophage preparation
CN101948784A (en) * 2010-08-31 2011-01-19 华南理工大学 Bdellovibrio bacteriovorus preparation and fermentation method and application thereof
CN103719535A (en) * 2012-10-10 2014-04-16 上海海洋大学 Bdellovibrio bacteriovorus microcapsule and preparation method thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
张小能等: "噬菌蛭弧菌颗粒剂制备条件的优化", 《上海海洋大学学报》 *

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