CN101781627A - Preparation method and application of sea bdellovibrio bacteriovorus ecological preparation - Google Patents

Abstract

The invention provides a preparation method and application of a sea bdellovibrio bacteriovorus ecological preparation, and relates to a sea microbial ecological preparation and a preparation method of a bdellovibrio bacteriovorus preparation for mariculture. The preparation method comprises the following steps of: preparing a host bacteria suspension; separating and purifying bdellovibrio bacteriovorus; and propagating and culturing bdellovibrio bacteriovorus. The preparation method is characterized in that after pseudomonas stutzeri is connected with an incline and is activated, adding physiological saline in a test tube to prepare the bacterial suspension; then propagating and culturing with nutrient bouillon; adding a flocculating agent in the obtained bacterial suspension; standing and precipitating after uniformly shaking; adding the physiological saline to obtain 1012 cfu/mL of host bacteria suspension; separating and purifying the bdellovibrio bacteriovorus of collected sea and bottom sediment samples with a double agar plate method; pouring culture media containing the host bacteria suspension and the collected samples into sea agar in the lower layer for culture after uniformly mixing; and adding the host bacteria and the bdellovibrio bacteriovorus obtained by separation into sterilized natural sea, and culturing until the concentration of the bdellovibrio bacteriovorus is 108 to 109 pfu/mL for future use.

Description

The preparation of sea bdellovibrio bacteriovorus ecological preparation and application

Technical field

The invention belongs to biological technical field, relate to a kind of preparation of seawater probiotics, be used for the control of sea farming process purification of water quality and bacteriosis.

Background technology

Since the eighties in 20th century, China's mariculture industry development is rapid, and not only breed variety is abundant, and equal tool higher economic value.But some are unfavorable for that the phenomenon of aquaculture sustainable and healthy development also comes one after another, and as the development disorder of single variety, blindly improve cultivation density etc. and all cause water quality deterioration and culture breaking out of disease.The frequent generation of breed variety bacteriosises such as prawn, large yellow croaker since 1993 has caused enormous economic loss for China's mariculture industry, and the intensification mode of production of breed variety has simultaneously more been aggravated the epidemic rate of disease to a certain extent.Remove the pathogenic bacterium that occur in the breeding process at present and adopt pharmacotherapy, but antibiotic blindness is used, abuse, has caused a series of problems such as resistance, drug residue.

Bdellovibrio (Bdellovibrio) is that a class of finding in 1962 is finished the bacterial parasite that self grows, breeds with attack and other bacterium of cracking.It extensively is present in natural water, soil, and in the ight soil of human and animal, the Gram-negative bacteria of the most of sections of energy cracking, genus especially all has good lytic activity to the aquatic animal common pathogen.Bdellovibrio has important scavenging(action) to the bacterium of the salmonella in the sewage.In the river under tap water, lake water and the simulating natural condition of sterilization, Bdellovibrio reaches more than 90% elimination efficiencies such as intestinal bacteria, Salmonellas, dysentery bacterium, vibrio cholerae, even 100%.So Bdellovibrio is being considered to good prospects for application aspect the biological control of purification of water quality and bacteriosis.Though at present existing both at home and abroad about the Bdellovibrio formulation products, their basic sources are in fresh water, and are not suitable for the sea farming process, and the aspect of tiring also remains further to be improved.

Seawater Bdellovibrio preparation can be improved the micro-ecological environment of seawater-culture pond, suppress the breeding of pathogenic bacterium such as vibrios, the water surrounding ecological balance damage that reduces or avoid chemostefilant to cause, and the abuse of antibiotics resistance, the drug residue problem that cause, improve the survival rate of growing seedlings, use safer.It has wide development space as a kind of " living antibiotics ".

Summary of the invention

The object of the present invention is to provide a kind of preparation and application method that is suitable for the Bdellovibrio preparation of used in mariculture.

Technical scheme of the present invention is made up of following steps: the preparation host bacteria suspension; The separation of Bdellovibrio, purifying; The Bdellovibrio multiplication culture, after it is characterized in that the non-virulent Pseudomonas stutzeri connect slant activation, the physiological saline that adds about 2ml in test tube makes bacteria suspension, use nutrient broth multiplication culture 18-24h then, in gained bacterium liquid, add flocculation agent, leave standstill 2-5h after shaking up, collecting precipitation adds physiological saline and promptly obtains 10 ¹²The host bacteria suspension of cfu/mL; Adopt double-deck agar plate method that the seawater of gathering is carried out separating of Bdellovibrio and purifying with the bed mud sample, to contain to pour on the previously prepared lower floor seawater agar behind the substratum mixing of host bacteria suspension and collected specimens and cultivate 6～7 days, plaque appears at the double-layer plate that contains the host bacterium, size and form, sharpness and pigment according to plaque are classified to separating the plaque that obtains, adopt single spot to go down to posterity, more than single spot triplicate that picking constantly enlarges, till the plaque that forms shape, size, the equal unanimity of transparency, promptly get the pure bacterial strain of Bdellovibrio; With the host bacterium with separate the Bdellovibrio that obtains join the sterilization natural sea-water in, seal with sterile gauze, shaking table is cultivated a week, to Bdellovibrio concentration be 10 ⁸～10 ⁹Pfu/mL, standby.The Bdellovibrio concentrated solution is mixed with milk sucrose stablizer, and blending ratio is 1: 5, places under-40 ℃ of conditions pre-freeze 3 hours, carries out lyophilize 18～24h then under 2～7mTorr vacuum degree condition, promptly obtains the Bdellovibrio freeze-dried preparation.Adding Bdellovibrio preparation application concentration range in marine culture water is 10～10 ⁴Pfu/mL.

The present invention has the strong characteristics of the ability of growing again under different habitats, guaranteed the lytic activity of Bdellovibrio, and the most aquatic products pathogenic bacterium of cracking have preferably been improved the aquatic products disease-controlling effect.The host bacterium of being adopted---Pseudomonas stutzeri is a non-pathogenic bacteria, has avoided environmental pollution, uses safer.Adding Bdellovibrio preparation application concentration range in marine culture water is 10～10 ⁴Pfu/mL not only can purify water, and also can play the effect of disease control in breeding process.Bacterial growth was suppressed after aquaculture water added Bdellovibrio, and total plate count and vibrios number average have reduced by 1～2 order of magnitude.Add Bdellovibrio in the Young Crab seedling raising process, not only water quality is purified, and each distortion ratio and surviving rate of the young also obviously improves phase.

Embodiment

Embodiment one:

The preparation of the separation of seawater Bdellovibrio, purifying and concentrated solution thereof:

1, the preparation of host bacteria suspension

Pseudomonas stutzeri (Microbe Inst., Chinese Academy of Sciences provides) is connect slant activation, cultivates 18～24h, and the physiological saline that adds about 2ml in test tube makes bacteria suspension, receives in the nutrient broth medium again, and 30 ℃ down with shaking table liquid culture 18～24h.The chitosan oligomer of adding 1% stirs with 150r/min speed as flocculation agent in gained bacterium liquid, adopts multi-layered polyester high efficiency filter bag filtration then, collecting precipitation.Add physiological saline at last and promptly obtain 10 ¹²The host bacteria suspension of cfu/mL can be preserved 10d under 4 ℃.

2, the separation of Bdellovibrio, purifying

Adopt double-deck agar plate method to the seawater of gathering, separation and the purifying that the bed mud sample carries out Bdellovibrio, method is to get 45 ℃ of insulations behind the medium sterilization of 5mL upper strata, add the host bacteria suspension of 200 μ L and the sample liquid of 500 μ L, be poured into behind the mixing on the previously prepared lower floor seawater agar, solidify back 28 ℃ and be inverted cultivation, observe 6～7d continuously.Size and form, sharpness and pigment according to plaque are classified to separating the plaque that obtains, adopt single spot to go down to posterity, plaque dug out from flat board insert the test tube that the 1/10YP leach liquor is housed temperature and bathe 8h, then with the leach liquor cultivation of falling the double-layer plate again, at least triplicate, till the plaque of size, form, transparency unanimity occurring on the flat board, the pure bacterial strain Bdh5221 of the Bdellovibrio that obtains sample is placed 4 ℃ of preservations.

3, Bdellovibrio multiplication culture

The liquid propagation of separating obtained Bdellovibrio is to add 0.2mL Pseudomonas stutzeri bacteria suspension and 5mL Bdellovibrio leach liquor mixing in the Erlenmeyer flask of the sterilization natural sea-water that 95mL is housed, seal with eight layers of sterile gauze, shaking table is cultivated under 28 ℃, pH 7.4～7.6,110r/min rotating speed, observe a week continuously, to concentration be 10 ⁸～10 ⁹The Bdellovibrio concentrated solution of pfu/mL is stand-by.The Bdellovibrio counting adopts plaque count method on the double-deck agar plate.

Culture medium prescription related in the implementation process is as follows:

Host's substratum: it is nutrient broth medium that the host cultivates with substratum, i.e. extractum carnis 3g, peptone 10g, sodium-chlor 15g, distilled water or natural sea-water 1000ml (salinity is about 30 ‰), 121 ℃ of sterilization 30min.

Nutrient agar: extractum carnis powder 3g, peptone 10g, sodium-chlor 5g, agar powder 14g, distilled water or natural sea-water 1000ml (salinity is about 30), 121 ℃ of sterilization 30min.

Double-deck substratum: Bdellovibrio screening, separation and purifying are double-deck agar with substratum, and lower floor is the seawater agar of 1.0% agar, and the upper strata is with the natural sea-water agar of 0.5% agar, 121 ℃ of 30min that sterilize.

Leach liquor: i.e. 1/10YP leach liquor, be yeast extract paste 0.03g, peptone 0.006g, natural old seawater 1000ml, 121 ℃ of sterilization 30min are used for Bdellovibrio is leached from double-layer plate.

Embodiment two:

The preservation of seawater Bdellovibrio:

Bdellovibrio concentrated solution and milk sucrose stablizer is even by 1: 5 mixed, obtain treating freeze dried mixed solution.Milk sucrose stablizer is formed: 5% sucrose+10% skim-milk+85% deionized water.Stablizer need be handled through autoclaving, and the qualified back of sterility test is standby.

With above-mentioned mixed solution packing, first pre-freeze is 3 hours under-40 ℃ of conditions, under-80 ℃, 2～7mTorr vacuum degree condition it is carried out lyophilize 18～24h with Freeze Drying Equipment then, promptly obtains the Bdellovibrio freeze-dried preparation.

Embodiment three:

The application of Bdellovibrio in the purifying sea water culturing pool:

Be the application of research Bdellovibrio in the purifying sea water culturing pool, the present invention handles the Penaeus vannamei culturing pool with Bdellovibrio Bdh5221.Test chamber is the aquarium of 125mL, and 25 ℃ of water temperatures, test water are untreated shrimp culture pond water, throws in Penaeus vannamei 10 tails in each aquarium, and the interpolation concentration of Bdellovibrio is respectively 1.5 * 10 ¹Pfu/mL (test group one), 1.5 * 10 ²Pfu/mL (test group two), 1.5 * 10 ³Pfu/mL (test group three), and the control group that does not add Bdellovibrio Bdh5221 is set, adopt the 24h aeration, one week of cultured continuously.The total bacterium of experimental session record bacterium, vibrios number and Bdellovibrio number, and observe prawn frequently and survive situation, the results are shown in Table 1.

Table 1. Bdellovibrio is handled the detected result of microbe population in the shrimp culture pond water of back

Can find out from table 1, the total plate count of each test group is compared with control group all with the vibrios number and is significantly descended, and differ nearly 1～2 order of magnitude, the quantity of Bdellovibrio rises also comparatively obvious, even minimum Bdellovibrio inoculum density also can reach proliferate effect preferably, illustrate that this Bdellovibrio has the stronger ability of growing again.Consider from the Financial cost of validity and pond application, select to add 1.5 * 10 ¹The strain of pfu/mL seawater Bdellovibrio gets final product.With regard to the prawn surviving rate, there is not difference with control group.The above results has not only illustrated the security of this seawater bacterial strain Bdh5221, has confirmed that also it is feasible that Bdellovibrio is applied to the purifying sea water culturing pool.

Embodiment four:

The application of Bdellovibrio in the Young Crab seed cultivation:

At first having carried out the safety testing of Bdellovibrio Bdh5221 to Young Crab Magna zoea larva, is 2.0 * 10 adding Bdellovibrio concentration ¹, 2.0 * 10 ², 2.0 * 10 ³, 2.0 * 10 ⁴Behind the pfu/mL, surviving rate and the distortion ratio of observing Young Crab each period are more or less the same with control group, obviously decline occurs, even the part test group is higher, show that seawater Bdellovibrio Bdh5221 can further be applied to the Young Crab seed cultivation.

The cultivation experiment container is 0.5m ³Black glass molten steel groove, each tank is placed two gas charging stones; The stocking rate of the mud crab Magna shape I phase young is 3.5 ten thousand in the tank; Water for larval nursing is through 5 * 10 ^-6After clorox is disinfected 12h, remove the sand filter seawater of chlorine residue with Sulfothiorine, salinity 30 ‰ is cultivated 27.5～30.5 ℃ of water temperatures, illuminance 1000～10000lux.If test group and control group, test group add Bdellovibrio Bdh5221 before the young is put into back and each juvenile crab metamorphosis, make the Bdellovibrio quantity of cultivating in the water reach 2.5 * 10 ¹Pfu/mL, my conventional seedbed system method of control group is added microbiotic before the young is put into back and each juvenile crab metamorphosis (Enrofloxacin, florfenicol etc., adding concentration is 1.5 * 10 ^-6).Duration of test detects index and comprises total plate count, amount of vibrio, Bdellovibrio quantity, pH, NO ₂-N, NO ₃-N, NH ₄-N, COD etc.And use the volumetry sampling technique abnormal night on the same day juvenile crab, calculate juvenile crab metamorphosis, surviving rate.Its result is as follows:

Table 2. Bdellovibrio is applied to microbe population and water-quality guideline detected result between the Young Crab nursery stage

As can be seen from Table 2, the vibrios number that adds Bdellovibrio Bdh5221 test group enters V at mud crab Magna zoea larva and maintains a level after the stage substantially and no longer rise, reduced by 1～2 order of magnitude than control group, the growth and decline of total plate count have shown the variation tendency same with amount of vibrio.Water quality detection result in addition shows, the NO of test group and control group ₂-N, NO ₃-N, NH ₄-N content was more or less the same at the initial stage of growing seedlings, and all was in lower level, but as time passes, test group three's content obviously rises slow than control group, especially NH ₄-N detected value is than the low 0.11～0.34mg/L of control group.As seen seawater Bdellovibrio Bdh5221 not only has restraining effect to vibrios number in the aquaculture water, and the water quality of whole water body is made moderate progress.

Each phase of the table 3. Young Crab young survives, the comparison of distortion ratio

Table 3 result can find out that there is evident difference in test group at survival, distortion ratio and the control group of each phase of the young, all is higher than control group.The adding of seawater Bdellovibrio Bdh5221, make the water body micro-ecological environment adjusted, keep balance, improved 6.6%, 6.3% than control group respectively to survival rate and the distortion ratio of megalopa larva.

Claims (6)

1. the preparation of sea bdellovibrio bacteriovorus ecological preparation and application is made up of following steps: the preparation host bacteria suspension; The separation of Bdellovibrio, purifying; The Bdellovibrio multiplication culture, after it is characterized in that the non-virulent Pseudomonas stutzeri connect slant activation, the physiological saline that adds about 2ml in test tube makes bacteria suspension, use nutrient broth multiplication culture 18-24h then, in gained bacterium liquid, add flocculation agent, leave standstill 2-5h after shaking up, collecting precipitation adds physiological saline and promptly obtains 10 ¹²The host bacteria suspension of cfu/mL; Adopt double-deck agar plate method that the seawater of gathering is carried out separating of Bdellovibrio and purifying with the bed mud sample, to contain to pour on the previously prepared lower floor seawater agar behind the substratum mixing of host bacteria suspension and collected specimens and cultivate 6～7 days, plaque appears at the double-layer plate that contains the host bacterium, size and form, sharpness and pigment according to plaque are classified to separating the plaque that obtains, adopt single spot to go down to posterity, more than single spot triplicate that picking constantly enlarges, till the plaque that forms shape, size, the equal unanimity of transparency, gained is the pure bacterial strain of Bdellovibrio; With the host bacterium with separate the Bdellovibrio that obtains join the sterilization natural sea-water in, seal with sterile gauze, shaking table is cultivated a week, to Bdellovibrio concentration be 10 ⁸～10 ⁹Pfu/mL.

2. the preparation of sea bdellovibrio bacteriovorus ecological preparation according to claim 1 and application is characterized in that flocculation agent is a chitosan oligomer, and consumption is 1%.

3. the preparation of sea bdellovibrio bacteriovorus ecological preparation according to claim 1 and application, it is characterized in that separating the double-deck agar lower floor that adopts with purifying is the seawater agar of 1.0% agar, the upper strata is with the natural sea-water agar of 0.5% agar, salinity about 30 ‰, 121 ℃ of sterilization 30min.

4. the preparation of sea bdellovibrio bacteriovorus ecological preparation according to claim 1 and application is characterized in that Bdellovibrio concentrated solution and milk sucrose stablizer evenly by 1: 5 mixed, obtains treating freeze dried mixed solution; Milk sucrose stablizer is formed: 5% sucrose+10% skim-milk+85% deionized water.

5. the preparation of sea bdellovibrio bacteriovorus ecological preparation according to claim 4 and application, it is characterized in that the Bdellovibrio concentrated solution was placed under-40 ℃ of conditions pre-freeze 3 hours with treating freeze dried mixed solution after milk sucrose stablizer mixes, under-80 ℃, 2～7mTorr vacuum degree condition, carry out lyophilize 18～24h at Freeze Drying Equipment then, promptly obtain the Bdellovibrio freeze-dried preparation.

6. according to the preparation and the application of claim 1 or 2 or 3 or 4 or 5 described sea bdellovibrio bacteriovorus ecological preparations, it is characterized in that the application concentration range of adding Bdellovibrio in marine culture water is 10～10 ⁴Pfu/mL.