CN101275119A - Application of bdellovibrio to eliminating pathogen in freshwater product and culture water thereof - Google Patents
Application of bdellovibrio to eliminating pathogen in freshwater product and culture water thereof Download PDFInfo
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Abstract
The present invention provides an application of a bdellovibrio eliminating pathogenic bacteria in freshwater products and its aquaculture water, especially fit for eliminating pathogenic bacteria carried before freshwater products eating or processing, by transportation process or its aquaculture water. Bdellovibrio concentrated solution is added in aquaculture water, before eating, processing or in the course of transportation, the concentration achieves 10<3>pfu/ml. When the method is applied in freshwater products processing or in the course before processed, or in the course of transportation and in aquaculture water, the concentrations of bdellovibrio telotroch are 10<5>-10<13> pfu/ml, 10<4>-10<12> pfu/ml and 10 <3>-10<7> pfu/ml respectively. The clearance ratios of the bdellovibrio are all above 99%. The invention is fit for the whole process from freshwater products cultivation to the process before eating. The method not only improving the safety factor of consumer raw eating freshwater products, protecting health of consumer, but also playing an important role in green processing production of freshwater products.
Description
Technical field
The invention belongs to biological technical field, relate to bacterium and application thereof, specifically be meant the application of Bdellovibrio pathogenic bacterium in eliminating freshwater product and aquaculture water thereof, be specially adapted to eliminate the edible or processing of freshwater product before, the pathogenic bacterium of carrying in transportation and the aquaculture water thereof.
Background technology
Bdellovibrio (Bdellovibrio) is to separate a class of finding by Stolp and Petzhold from Kidney bean leaf blight aeruginosa atcc 11355 to finish the bacterial parasite that self grows and breed with attack and other bacterium of cracking.Bdellovibrio has the function of similar phage, the pathogenic bacterium that can cause fishery cultivating disease and human foods to be poisoned is had good cracking remove effect, but animals and plants are had no side effect.
Aeromonas hydrophila (Aeromonas hydrophila), pseudomonas putida (Pseudomonasputida), Wdwardsiella tarda (Edwardsiella tarda) and intestinal bacteria (Escherichia coli) all are the pathogenic bacterium that can cause that human foods is poisoned, and freshwater product is that its food source sexuality is dyed of paramount importance communication media.The pathogenic bacterium that freshwater product is entrained except causing the human foods poisoning, also can cause this disease during culturing of freshwater product.In order to control fresh water fishes and shrimps shellfish crab bacterial disease, culture producer and have to use microbiotic, so cause the residual of fishing medicine again.The residual of medicine exceeds standard, and brought great pressure for the outlet of China's freshwater product.
For the removing of pathogenic bacterium in the freshwater product, still at present methods that adopt physics or chemicals more, but the method for physics or chemicals all has its tangible drawback.Various antibiotic abuses have caused pathogenic bacterium to antibiotic resistance.The development new antibiotic is not only costly and the lead time is long.The method of physics then can only be applied to a certain link in " from the source to the dining table ", but not whole process.Yet, Bdellovibrio has the special bacterium of cracking pathogenic bacterium as a class, not only may be used on the whole process that freshwater product is produced, nor can cause bacterium resistance to occur, also do not have the too single-minded shortcoming of phage host, so Bdellovibrio has suitable advantage as the biological removers of class pathogenic bacterium.
If can be during freshwater product be being cultured, before transportation or edible, the processing; carry out the biology elimination work of pathogenic bacterium earlier; then can reduce or eliminate the probability of food poisoning on the one hand; reduce or eliminate the possibility of drug residue; the safety coefficient of improving the quality of products; protection human consumer's health, the processing that also can be freshwater product on the other hand provide the facility in high-quality raw material and the processing, for foreign exchange earning provides strong guarantee.
Summary of the invention
The objective of the invention is to overcome the drawback of prior art, find out the Bdellovibrio that is applicable to elimination freshwater product pathogenic bacterium by research and development, the application of a kind of Bdellovibrio pathogenic bacterium in eliminating freshwater product and aquaculture water thereof is provided, be specially adapted to eliminate the edible or processing of freshwater product before, the pathogenic bacterium of carrying in transportation and the aquaculture water thereof.
In order to achieve the above object, main technical schemes of the present invention is as follows:
The application of Bdellovibrio pathogenic bacterium in eliminating freshwater product and aquaculture water thereof, application method comprises following concrete steps and condition thereof:
Step 1: the separation and purification of Bdellovibrio
Fresh water or the bed mud of gathering carried out pre-treatment, the Aeromonas hydrophila of inoculation propagation Bdellovibrio, fall double-layer plate, plaque appears on the double-layer plate that contains the host bacterium, single spot that picking constantly enlarges, again by micro-biological process liquid multiplication culture, double-layer plate check, single spot of constantly enlarging of picking once more, and repeated several times, going down to posterity to single plaque, to form all consistent plaque of shape, size, transparency be the pure bacterial strain of a strain Bdellovibrio, by the big subtotal of single spot in the same time, separablely go out the pure bacterial strain of some strain Bdellovibrios; Described Bdellovibrio is preserved in Chinese typical culture collection center (CCTCC), and deposit number is BDF01 CCTCC M 208008, BDF02 CCTCC M 208009, BDF03 CCTCC M208010;
Step 2: the preparation of Bdellovibrio concentrated solution
Some strain Bdellovibrios are carried out fermentation culture respectively, and making concentration respectively is 10
11～10
13The Bdellovibrio concentrated solution of pfu/ml, stand-by;
Step 3: the application of Bdellovibrio in the pathogenic bacterium that the elimination freshwater product carries
In having polluted the freshwater product aquaculture water of pathogenic bacterium, edible before, add the Bdellovibrio concentrated solution of step 2 preparation in processing or the transportation, make the concentration of Bdellovibrio reach 10 at least
3Pfu/ml.
Described Bdellovibrio comprises Bdellovibrio telotroch and Bdellovibrio leech plastid.
Described application method adopts 2 strains or the above Bdellovibrio of 2 strains to mix and uses, and effect is better.
Described application method is specially adapted to eliminate pathogenic bacterium edible or that the preceding freshwater product of processing carries, and Bdellovibrio telotroch optimum concentration range is 10 during application
5～10
13Pfu/ml.
Described application method also is applicable to the pathogenic bacterium of eliminating in the freshwater product transportation, and Bdellovibrio telotroch optimum concentration range is 10 during application
4～10
12Pfu/ml.
Described application method also is applicable to the pathogenic bacterium of eliminating in the freshwater product aquaculture water, and Bdellovibrio telotroch optimum concentration range then is 10 during application
3～10
7Pfu/ml.
Bdellovibrio of the present invention eliminate freshwater product before edible, in the transportation and the effect of aquaculture water pathogenic bacterium as follows:
1, adopt in this law elimination freshwater product the pathogenic bacterium effect remarkable before the edible or processing at freshwater product
As shown in Figure 1, be example with tilapia, Macrobrachium rosenbergii, Corbicula fluminea, river crab, Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and intestinal bacteria clearance rate all can reach more than 99%.
2, the pathogenic bacterium security of adopting the present invention to eliminate in the freshwater product is good
Bdellovibrio is eliminated Aeromonas hydrophila in the freshwater product, pseudomonas putida, Wdwardsiella tarda and colibacillary method are biological methods, Bdellovibrio have can infect, the characteristic of cracking host bacteria, make it to be suitable as the biopurification factor of restraining or removing pathogenic bacterium in organism and the environment thereof, and it can be withered away automatically because of hungry behind the intact host bacteria of cracking, therefore humans and animals is had no side effect.Not only can improve the safety coefficient that the human consumer eats freshwater product raw, protection human consumer's health also provides safeguard for the green processing production of freshwater product.
3, apply the present invention in the freshwater product transportation, convenient, safe and effective
Freshwater product adopts Bdellovibrio to eliminate pathogenic bacterium in transportation and can prevent water quality deterioration, improve the freshwater product survival rate, avoids using the banning drugs of microbiotic and malachite green class simultaneously.
4, the present invention is fit to be applied to the breeding process of freshwater product
During culturing, promptly carry out the elimination work of pathogenic bacterium, be particularly conducive to and reduce or eliminate chemicals such as antibiotic use and residual, guarantee the high-quality of freshwater product, with tilapia, Macrobrachium rosenbergii, Corbicula fluminea, culture of Chinese mitten crab is example, the Bdellovibrio telotroch to the effect of the eliminations of pathogenic bacterium in fresh water fishes and shrimps shellfish crab aquaculture water test as shown in Figure 3, the quantity of pathogenic bacterium in the aquaculture water can be controlled in the level of 4cfu/ml.
5, the present invention provides a kind of new method for the entrained food-borne pathogens of elimination freshwater product and cultures in the edible preceding whole process applicable to freshwater product, reduce or eliminate chemicals such as residues of antibiotics effectively, guarantee the high-quality of freshwater product, fundamentally avoid the generation of freshwater product food poisoning, for foreign exchange earning provides strong guarantee.
Though 6, Bdellovibrio leech plastid also can be used for eliminating pathogenic bacterium Bdellovibrio leech plastid entrained in freshwater product and the aquaculture water thereof and eliminates the effect of pathogenic bacterium not as the Bdellovibrio telotroch, equally also can be used for eliminating pathogenic bacterium entrained in freshwater product and the aquaculture water thereof.
Description of drawings
Fig. 1 is tilapia, Macrobrachium rosenbergii, Corbicula fluminea, the river crab pathogenic bacterium concentration logarithmic value-time diagram when using three strain Bdellovibrio telotroches before edible and removing pathogenic bacterium;
Fig. 2 is the pathogenic bacterium concentration logarithmic value-time diagram when using three strain Bdellovibrio telotroches removing pathogenic bacterium in tilapia, Macrobrachium rosenbergii, Corbicula fluminea, the river crab transportation;
Fig. 3 is the pathogenic bacterium concentration logarithmic value-time diagram when using three strain Bdellovibrio telotroches removing pathogenic bacterium in tilapia, Macrobrachium rosenbergii, Corbicula fluminea, the culture of Chinese mitten crab water body;
Fig. 4 is that tilapia, Macrobrachium rosenbergii, Corbicula fluminea, river crab are used three strain Bdellovibrios respectively, two strain Bdellovibrios, the pathogenic bacterium concentration logarithmic value-time diagram when a strain Bdellovibrio is removed pathogenic bacterium before edible;
Fig. 5 is tilapia, Macrobrachium rosenbergii, Corbicula fluminea, the river crab pathogenic bacterium concentration logarithmic value-time diagram when using three strain Bdellovibrio leech plastids and three strain Bdellovibrio telotroches before edible and removing pathogenic bacterium;
Embodiment
The application of blended three strain Bdellovibrios pathogenic bacterium in eliminating edible tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab arbitrarily
Application method comprises following concrete steps and condition thereof:
Step 1: the separation and purification of Bdellovibrio
(1) the Bdellovibrio plaque is observed and is differentiated
Fresh water or the bed mud of gathering carried out pre-treatment, the inoculation Aeromonas hydrophila, the propagation Bdellovibrio, double-layer plate plaque occurs on the double-layer plate that contains the host bacterium, single spot that picking constantly enlarges, again by the check of micro-biological process liquid multiplication culture, double-layer plate, single spot of constantly enlarging of picking once more, and repeat repeatedly, extremely single plaque goes down to posterity and forms till all consistent plaque of shape, size, transparency, is the pure bacterial strain of Bdellovibrio.
(2) propagation of Aeromonas hydrophila is with concentrated
When occurring the transparent plaque of Bdellovibrio in the flat board, then breed Aeromonas hydrophila, centrifugal preparation concentrated solution.
(3) the single spot of Bdellovibrio phagocytosis selects and breeds
For the single spot of the phagocytosis that is defined as producing by Bdellovibrio, put into an amount of dilution nutrient broth (DNB) liquid nutrient medium after choosing, add the Aeromonas hydrophila concentrated solution of (2) simultaneously, constant temperature shaking table 250rpm, 28 ℃ of cultivation 24h.
(4) double-layer plate check
After the Bdellovibrio propagation, 5000rpm, 4 ℃ of centrifugal 20min remove the Aeromonas hydrophila thalline, and supernatant liquor obtains the Bdellovibrio bacterial sediment through 16000rpm, 4 ℃ of centrifugal 20min, and the DNB liquid nutrient medium is suspended sediment again, through the 1.2um filtering with microporous membrane, gained filtrate is diluted to 10 with this suspension
-1Pfu/ml, 10
-3Pfu/ml, 10
-5Pfu/ml, 10
-7Pfu/ml, 10
-9Pfu/ml, 10
-11Six extension rates of pfu/ml.From stoste and each dilution diluent, respectively get 0.1ml, mix with 0.3ml Aeromonas hydrophila concentrated solution respectively, mix with 50 ℃ of DNB upper strata substratum that contain 0.6% agar again, be poured in the DNB lower floor flat board and pave, get double-layer plate, in 28 ℃ of cultivations of constant incubator.
(5) purifying of Bdellovibrio
The flat board that plaque occurs uses the method for (3), (4), constantly single plaque, liquid multiplication culture, the double-layer plate check of expansion of picking once more, and repeat repeatedly, single plaque is passaged to and forms till all consistent plaque of shape, size, transparency, can think that Bdellovibrio is purified, be the pure bacterial strain of Bdellovibrio.Isolate three strain Bdellovibrios altogether, its bacterial strain number is respectively BDF01, BDF02, (described Bdellovibrio is being positioned at the Chinese typical culture collection center preservation of Wuhan City, Hubei Province Wuhan University on January 13rd, 2008 to BDF03, deposit number is respectively CCTCC NO:M 208008, CCTCC NO:M 208009, CCTCC NO:M 208010, the classification name is respectively Bdellovibrio BDF01, Bdellovibrio sp.BDF01 and Bdellovibrio BDF02, Bdellovibrio sp.BDF02 and Bdellovibrio BDF03, Bdellovibrio sp.BDF03);
Step 2: the preparation of Bdellovibrio telotroch concentrated solution
The bacterial strain that step 1 obtains number is respectively the Bdellovibrio of BDF01, BDF02, BDF03, and joins in the erlenmeyer flask that contains the DNB substratum after the Aeromonas hydrophila concentrated solution mixes, and constant temperature shaking table 250rpm, 28 ℃ cultivate 24h.Culture is got supernatant liquor through 6000rpm, 4 ℃ of centrifugal 20min, and centrifugal 20min under 16000rpm, the 4 ℃ of conditions abandons supernatant liquor again, adds an amount of DNB liquid nutrient medium Bdellovibrio throw out that suspends again, and making concentration is 10
11～10
13The Bdellovibrio telotroch mixed concentrated liquid of pfu/ml.
Step 3: Bdellovibrio is to Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and colibacillary elimination experiment in edible or the preceding tilapia of processing, Macrobrachium rosenbergii, Corbicula fluminea and the river crab
(1) Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and colibacillary preparation are cultivated with common nutrient broth medium shaking table, and 160rpm cultivates 12-16h for 28 ℃ and breeds Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and intestinal bacteria respectively.Nutrient solution is abandoned supernatant liquor through 5000rpm, 4 ℃ of centrifugal 20min, and the precipitation thalline suspends again with sterile distilled water, and adjusting separately respectively, concentration is 10
9Cfu/ml.
(2) Bdellovibrio is to Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and colibacillary elimination test
Test chamber is the aquarium of 25L, is divided into test and contrasts two big series.Test water is the breed fresh water of membrane filtration, and each aquarium injects the 16L filtered water.Test used tilapia and nearly weigh 140～1909, it is 8～10cm that the Macrobrachium rosenbergii body is about, and the Corbicula fluminea average body is long to be 40mm, and the river crab specification is 30～40g.Each aquarium is put each 10 of tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crabs.Through the sterilization of isocyanide dichloride uric acid sodium, water temperature is 26 ℃ before experiment.The continuous charge of experimental session water.3 aquariums are serial in contrast, only add four kinds of pathogenic bacterium, do not add Bdellovibrio.In the series of trials, 3 aquariums are one group, totally five groups, add a certain amount of Bdellovibrio telotroch concentrated solution respectively, and make the Bdellovibrio final concentration reach 10 respectively
5Pfu/ml, 10
7Pfu/ml, 10
9Pfu/ml, 10
11Pfu/ml and 10
13Pfu/ml.
During test, in corresponding aquarium, add the Aeromonas hydrophila that above-mentioned steps three (1) prepares, pseudomonas putida, Wdwardsiella tarda and intestinal bacteria make its final concentration reach 1 * 10 respectively
3Cfu/ml puts into tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab wherein then, adds the Bdellovibrio telotroch concentrated solution of different amounts again in the series of trials aquarium, makes them reach above-mentioned final concentration.Rimler-shotts (R-S substratum) selective medium is adopted in the detection of Aeromonas hydrophila, NMS (a kind of minimal medium) selective medium is adopted in the detection of pseudomonas putida, HE selectivity nutrient agar is adopted in the detection of Wdwardsiella tarda, and TBX (a kind of color developing culture medium) selectivity nutrient agar is adopted in colibacillary detection.
The three strain Bdellovibrios that bacterial strain number is respectively BDF01, BDF02, BDF03 mix, to the effect of the elimination tests of four kinds of pathogenic bacterium in the edible aquatic products (tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab) as shown in Figure 1.Among Fig. 1 ,-◆-be Aeromonas hydrophila concentration logarithmic value change curve ,-■-be pseudomonas putida concentration logarithmic value change curve ,-▲-be Wdwardsiella tarda concentration logarithmic value change curve ,-●-be e. coli concentration logarithmic value change curve.More than be minimum Bdellovibrio telotroch final concentration group (10 in the series of trials
5Pfu/ml) the concentration logarithmic value change curve of pathogenic bacterium in, because the elimination better effects if of other high density Bdellovibrio group, so expression no longer in the drawings.All data are the mean value of three parallel samples (i.e. three aquariums).
As can be known from Fig. 1, behind the interpolation Bdellovibrio telotroch, pathogenic bacterium concentration promptly begins to descend, to 4h, and only remaining 1～2cfu/ml.Compare starting point concentration and the final concentration of four kinds of pathogenic bacterium, three strain Bdellovibrios are respectively 99.21%, 99.49%, 99.75%, 99.87% to Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and colibacillary elimination factor as can be known.
Any three strain Bdellovibrio telotroch mixed solutions Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and colibacillary application application method in eliminating tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab transportation comprise following concrete steps and condition thereof:
Step 3 (2) Bdellovibrio telotroch is to Aeromonas hydrophila in tilapia, Macrobrachium rosenbergii, Corbicula fluminea and the river crab transportation, pseudomonas putida, Wdwardsiella tarda and colibacillary elimination test
The used tilapia of test chamber, test water and test, Macrobrachium rosenbergii, Corbicula fluminea, river crab specification are all with embodiment 1.Put 4 intact grids in each aquarium, in each grid, put into tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab respectively, immediately it is kept flat in the aquarium after sealing the grid lid, discharge unnecessary water in the case, make its water level just cover grid.Through the sterilization of isocyanide dichloride uric acid sodium, water temperature is 26 ℃ before experiment.The continuous charge of experimental session water.3 aquariums are serial in contrast, only add four kinds of pathogenic bacterium.In the series of trials, 3 aquariums are one group, totally five groups, add a certain amount of Bdellovibrio telotroch concentrated solution respectively, and make the Bdellovibrio final concentration reach 10 respectively
4Pfu/ml, 10
6Pfu/ml, 10
8Pfu/ml, 10
10Pfu/ml and 10
12Pfu/ml.
During test, in corresponding aquarium, add the Aeromonas hydrophila that above-mentioned steps three (1) prepares, pseudomonas putida, Wdwardsiella tarda and intestinal bacteria, and make its final concentration reach 1 * 10 respectively
3Cfu/ml.The concentration of four kinds of pathogenic bacterium detects all with embodiment 1.
Any three strain Bdellovibrio telotroches mix to the effect of the elimination test of four kinds of pathogenic bacterium in tilapia, Macrobrachium rosenbergii, Corbicula fluminea and the river crab transportation as shown in Figure 2.All same Fig. 1 of the used symbol of concentration logarithmic value change curve among Fig. 2.More than be minimum Bdellovibrio telotroch final concentration group (10 in the series of trials
4Pfu/ml) the concentration logarithmic value change curve of pathogenic bacterium in.
As can be known from Fig. 2, behind the interpolation Bdellovibrio telotroch, pathogenic bacterium concentration promptly begins to descend.The starting point concentration and the final concentration that compare four kinds of pathogenic bacterium, use three strain Bdellovibrio telotroches as can be known and remove Aeromonas hydrophila in the freshwater product transportation, pseudomonas putida, Wdwardsiella tarda and colibacillary elimination factor reach 99.05%, 99.37%, 99.68%, 99.84% respectively.
Mix three strain Bdellovibrio telotroches arbitrarily and in tilapia, Macrobrachium rosenbergii, Corbicula fluminea and culture of Chinese mitten crab process, eliminate Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and colibacillary application
Application method comprises following concrete steps and condition thereof:
The application in the breeding process in tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab of step 3 (2) Bdellovibrio telotroch
Before the test, build 12 temporarily and culture ponds (0.5m * 0.5m * 0.5m=125L).
To culture the pond is divided into test and contrasts two big series.Test water is not filtering breed fresh water, and 100L is injected in each pond.Test used tilapia, Macrobrachium rosenbergii, Corbicula fluminea, river crab specification all with embodiment 1.Each 20 of tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crabs are put in each pond.Through the sterilization of isocyanide dichloride uric acid sodium, the duration of test water temperature is 26 ℃ before experiment, and the every day of each bait throwing in sooner or later once.3 ponds are control series, only add this four kinds of pathogenic bacterium.In the series of trials, 3 ponds are one group, add a certain amount of Bdellovibrio telotroch concentrated solution, make it reach a predetermined Bdellovibrio final concentration.So have the Bdellovibrio telotroch final concentration of 3 different gradients, that is: 10
3Pfu/ml, 10
5Pfu/ml and 10
7Pfu/ml.
During test, in corresponding pond, add the Aeromonas hydrophila that above-mentioned steps three (1) prepares, pseudomonas putida, Wdwardsiella tarda and intestinal bacteria, and make its concentration reach 1 * 10 respectively
3Cfu/ml puts into tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab wherein then, adds the Bdellovibrio telotroch concentrated solution of different amounts again in the series of trials pond, makes them reach different final concentrations.Three strain Bdellovibrio telotroches to the effect of the eliminations of four kinds of pathogenic bacterium in aquaculture water test as shown in Figure 3.All same Fig. 1 of the used symbol of concentration logarithmic value change curve among Fig. 3.More than be minimum Bdellovibrio telotroch final concentration group (10 in the series of trials
3Pfu/ml).
As can be known from Fig. 3, starting point concentration and the final concentration of four strain pathogenic bacterium relatively, three strain Bdellovibrio telotroches are to Aeromonas hydrophila in the aquaculture water as can be known, pseudomonas putida, and Wdwardsiella tarda and colibacillary elimination factor are respectively 99.21%, 99.50%, 99.68%, 99.80%.
For comparing individual plant, many strains Bdellovibrio effect, use any blended three strain Bdellovibrios and two strain Bdellovibrios, strain Bdellovibrio telotroch Aeromonas hydrophila in eliminating edible tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab respectively, pseudomonas putida, Wdwardsiella tarda and colibacillary application
Application method comprises following concrete steps and condition thereof:
Step 3 (2) is used three strain Bdellovibrios, two strain Bdellovibrios, a strain Bdellovibrio telotroch respectively to Aeromonas hydrophila in edible tilapia, Macrobrachium rosenbergii, Corbicula fluminea and the river crab, pseudomonas putida, Wdwardsiella tarda and colibacillary elimination test
The used tilapia of test chamber, test water and test, Macrobrachium rosenbergii, Corbicula fluminea, river crab specification are all with embodiment 1.The continuous charge of experimental session water.3 aquariums are serial in contrast, only add four kinds of pathogenic bacterium.In the series of trials, 3 aquariums are one group, add the telotroch mixed concentrated liquid of three strain Bdellovibrios (BDF01, BDF02, BDF03) respectively, the telotroch mixed concentrated liquid of two strain Bdellovibrios, one strain Bdellovibrio telotroch concentrated solution makes it reach predetermined final concentration 1 * 10
5Pfu/ml.
During test, in corresponding aquarium, add the Aeromonas hydrophila that above-mentioned steps three (1) prepares, pseudomonas putida, Wdwardsiella tarda and intestinal bacteria make its final concentration reach 1 * 10 respectively
3Cfu/ml puts into tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab wherein then, adds above three kinds of Bdellovibrio telotroch concentrated solutions again in the series of trials aquarium respectively.
Use three strain Bdellovibrios, two strain Bdellovibrios, a strain Bdellovibrio telotroch as shown in Figure 4 to the effect of the elimination test of four kinds of pathogenic bacterium in edible tilapia, Macrobrachium rosenbergii, Corbicula fluminea and the river crab.Among Fig. 4,-◆-be the total concn logarithmic value change curve of four kinds of pathogenic bacterium when using a strain Bdellovibrio telotroch,-■-be the total concn logarithmic value change curve of four kinds of pathogenic bacterium when using two strain Bdellovibrio telotroches ,-▲-be the total concn logarithmic value change curve of four kinds of pathogenic bacterium when using three strain Bdellovibrio telotroches.All data are the mean value of three parallel samples (aquarium).
The result shows as shown in Figure 4, and behind the interpolation Bdellovibrio, four kinds of pathogenic bacterium concentration promptly descend in three test group.Use the concentration logarithmic value change curve of three strain Bdellovibrios, two strain Bdellovibrios, four kinds of pathogenic bacterium of strain Bdellovibrio elimination in the comparison test series, to use effect with best for three strain Bdellovibrio telotroches as can be known, and two strain Bdellovibrio telotroches are used effect with and also are better than single with a strain Bdellovibrio telotroch.Use with and refer to that any three strain Bdellovibrios, two strain Bdellovibrios mix.
Mix Aeromonas hydrophila, pseudomonas putida, Wdwardsiella tarda and the colibacillary application in eliminating edible tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab of three strain Bdellovibrio telotroches and three strain Bdellovibrio leech plastids arbitrarily
Application method comprises following concrete steps and condition thereof:
Picking list spot Bdellovibrio from the double-layer plate and joins in the erlenmeyer flask that contains the DNB substratum after the Aeromonas hydrophila concentrated solution mixes, and constant temperature shaking table 250rpm, 28 ℃ cultivate 24h.Culture is got supernatant liquor through 6000rpm, 4 ℃ of centrifugal 20min, more centrifugal 20min under 16000rpm, the 4 ℃ of conditions, abandon supernatant liquor, add an amount of DNB liquid nutrient medium Bdellovibrio throw out that suspends again, add the Aeromonas hydrophila concentrated solution again and mix and shake a bottle 30min, be Bdellovibrio leech plastid.It is 10 that Bdellovibrio is made concentration
11～10
13The Bdellovibrio leech plastid concentrated solution of pfu/ml.
Any three strain Bdellovibrio telotroches of step 3 (2) and any three strain Bdellovibrio leech plastids are to eating Aeromonas hydrophila in tilapia, Macrobrachium rosenbergii, Corbicula fluminea and the river crab, pseudomonas putida, Wdwardsiella tarda and colibacillary elimination test
The used tilapia of test chamber, test water and test, Macrobrachium rosenbergii, Corbicula fluminea, river crab specification are all with embodiment 1.The continuous charge of experimental session water.3 aquariums are serial in contrast, only add four kinds of pathogenic bacterium.In the series of trials, 3 aquariums are one group, add the mixed concentrated liquid of three strain Bdellovibrio telotroches, the mixed concentrated liquid of three strain Bdellovibrio leech plastids respectively in corresponding aquarium, make it reach predetermined Bdellovibrio final concentration 1 * 10
5Pfu/mi
During test, in corresponding aquarium, add the Aeromonas hydrophila that above-mentioned steps 3 (1) prepares, pseudomonas putida, Wdwardsiella tarda and intestinal bacteria make its final concentration reach 1 * 10 respectively
3Cfu/ml puts into tilapia, Macrobrachium rosenbergii, Corbicula fluminea and river crab wherein then, adds the Bdellovibrio concentrated solution of different amounts again in the series of trials aquarium, makes them reach different Bdellovibrio final concentrations.The concentration of four kinds of pathogenic bacterium detects all with embodiment 1.
Three strain Bdellovibrio telotroches and leech plastid thereof are to the effect of the elimination test of four kinds of pathogenic bacterium in edible tilapia, Macrobrachium rosenbergii, Corbicula fluminea and the river crab as shown in Figure 5.Among Fig. 5-◆-be the total concn logarithmic value change curve of four kinds of pathogenic bacterium when using three strain Bdellovibrio leech plastids ,-▲-be the total concn logarithmic value change curve of four kinds of pathogenic bacterium when using three strain Bdellovibrio telotroches.All data are the mean value of three parallel samples (aquarium).
Result shown in Figure 5 shows, after beginning to add Bdellovibrio, owing to have host bacterium Aeromonas hydrophila in its culturing process in the leech plastid concentrated solution, so a little higher than telotroch test group of starting point concentration logarithmic value of four kinds of pathogenic bacterium of leech plastid test group.The concentration logarithmic value curve of four strain pathogenic bacterium in Bdellovibrio telotroch test group and the Bdellovibrio leech plastid test group in the comparison test series, the Bdellovibrio telotroch is better than Bdellovibrio leech plastid to the removing effect of four kinds of pathogenic bacterium as can be known.
Claims (6)
1, the application of Bdellovibrio pathogenic bacterium in eliminating freshwater product and aquaculture water thereof, it is characterized in that: application method comprises following concrete steps and condition thereof:
Step 1: the separation and purification of Bdellovibrio
Fresh water or the bed mud of gathering carried out pre-treatment, the Aeromonas hydrophila of inoculation propagation Bdellovibrio, fall double-layer plate, plaque appears on the double-layer plate that contains the host bacterium, single spot that picking constantly enlarges, again by micro-biological process liquid multiplication culture, double-layer plate check, single spot of constantly enlarging of picking once more, and repeated several times, going down to posterity to single plaque, to form all consistent plaque of shape, size, transparency be the pure bacterial strain of a strain Bdellovibrio, by the big subtotal of single spot in the same time, separablely go out the pure bacterial strain of some strain Bdellovibrios; Described Bdellovibrio is preserved in Chinese typical culture collection center, and deposit number is BDF01 CCTCCM 208008, BDF02 CCTCC M 208009, BDF03 CCTCC M 208010;
Step 2: the preparation of Bdellovibrio concentrated solution
Some strain Bdellovibrios are carried out fermentation culture respectively, and making concentration respectively is 10
11～10
13The Bdellovibrio concentrated solution of pfu/ml, stand-by;
Step 3: the application of Bdellovibrio in the pathogenic bacterium that the elimination freshwater product carries
In having polluted the freshwater product aquaculture water of pathogenic bacterium, edible before, before the processing or add the Bdellovibrio concentrated solution of step 2 preparation in the transportation, make the concentration of Bdellovibrio reach 10 at least
3Pfu/ml.
2, the application of Bdellovibrio according to claim 1 pathogenic bacterium in eliminating freshwater product and aquaculture water thereof, it is characterized in that: described Bdellovibrio comprises Bdellovibrio telotroch and Bdellovibrio leech plastid.
3, the application of Bdellovibrio according to claim 1 and 2 pathogenic bacterium in eliminating freshwater product and aquaculture water thereof is characterized in that: described application method adopts 2 strains or the above Bdellovibrio of 2 strains to mix and uses.
4, the application of Bdellovibrio according to claim 1 and 2 pathogenic bacterium in eliminating freshwater product and aquaculture water thereof, it is characterized in that: described application method is applicable to eliminates pathogenic bacterium edible or that the preceding freshwater product of processing carries, and Bdellovibrio telotroch concentration range is 10 during application
5～10
13Pfu/ml.
5, the application of Bdellovibrio according to claim 1 and 2 pathogenic bacterium in eliminating freshwater product and aquaculture water thereof, it is characterized in that: described application method is applicable to the pathogenic bacterium of eliminating in the freshwater product transportation, and Bdellovibrio telotroch concentration range is 10 during application
4～10
12Pfu/ml.
6, the application of Bdellovibrio according to claim 1 and 2 pathogenic bacterium in eliminating freshwater product and aquaculture water thereof, it is characterized in that: described application method is applicable to the pathogenic bacterium of eliminating in the freshwater product aquaculture water, and Bdellovibrio telotroch concentration range then is 10 during application
3～10
7Pfu/ml.
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