CN103320365B - Fish-sourced aeromonas hydrophila disease antagonistic strain and application thereof - Google Patents
Fish-sourced aeromonas hydrophila disease antagonistic strain and application thereof Download PDFInfo
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- CN103320365B CN103320365B CN201310285415.3A CN201310285415A CN103320365B CN 103320365 B CN103320365 B CN 103320365B CN 201310285415 A CN201310285415 A CN 201310285415A CN 103320365 B CN103320365 B CN 103320365B
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Abstract
The invention relates to a fish-sourced aeromonas hydrophila disease antagonistic strain and an application of the antagonistic strain and belongs to the field of aquaculture technology. A fish-sourced aeromonas hydrophila disease antagonistic strain is screened out, cultivated and added into a feed or a medicine as probiotics. The antagonistic strain is a novel antagonistic strain which is high in biological activity stability and high in specificity, is safe for cultured fish and environment, can be used as the probiotics strain for preventing and treating aeromonas hydrophila caused explosive hemorrhagic disease in large freshwater fish farming, and can be used as the main strain to prepare a microbial preparation.
Description
Technical field
The present invention relates to the Antergic Germ against Aeromonas hydrophila strain of a kind of source of fish and application thereof, it is high to be specifically related to a kind of activity resistent ingredient stability, can be used as Antagonistic Fungi and the application thereof of Aeromonas hydrophila, belongs to technical field of aquaculture.
Background technology
Aeromonas hydrophila (
aeromonas hydrophila, A.hy) be subordinate to Aeromonas section (
aermonadaceae), Aeromonas (
aeromonas), this bacterium is present in water, soil and hydrocoles body.Aeromonas hydrophila causes fish acute hemorrhagic septicemia.This disease, also Outbreak disease, hemorrhagic disease, hemorrhagic ascites disease, Ascites Disease etc. are claimed, the main breed variety of fresh water can be infected, as the cultured freshwater fish such as grass carp, silver carp, flathead, carp, crucian carp, mandarin fish, black carp, tilapia, common eel, white crucian carp, yellow tail silver xenocypris, bream, goldfish, swamp eel, it is the popular the most a kind of hydrocoles conditioned pathogen of China.This bacterium is Grain-negative tyrothricin, and extreme single flagellum, without gemma, without pod membrane, is dispersed in arrangement, and all can grow at 0 ~ 41 DEG C, the optimum pH scope of growth is 5.5 ~ 9.0.In recent years at the popular Outbreak-infective disease of south China each province cultured freshwater fish, a lot of report is septicemia caused by this bacterium, Aeromonas hydrophila is one of the main pathogenic fungi of cultured freshwater fish Outbreak-infective disease.
The prevention and controls of this bacterium mainly comprises: microbiotic control, herbal control, vaccine prevention and control and probiotic bacterium prevention and control.1) microbiotic control.Microbiotic is used to the choice drug for the treatment of bacterial infectious disease, and microbiotic has significant the suppression or killing action to pathogenic bacteria.Research confirms that quinolones and some aminoglycoside medicaments have stronger fungistatic effect to Aeromonas hydrophila.But widely use along with antibiotic, in recent years different areas due to types of medicines different with usage, create a large amount of Resistant strain, Susceptibility to antibiotics is widely different.2) herbal control.Herbal medicine is without resistance, and noresidue, non-environmental-pollution, can not cause drug-induced disease and restraining and sterilizing bacteria successful, more and more cause the concern of people.Research generally shows that folk prescription uses the herbal medicine such as Turkey-galls, rheum officinale, garden burnet, peppery few and dark plum, myrobalan, pomegranate rind, cloves, garlic, the bark of ash, borneol, catechu, Root of Sixpetal Clematis to have and very strong presses down sterilizing ability.In actual production, the herbal medicine with bacteriostatic action is combined into compound, the disease suitably utilizing different herbal medicine mutually to act synergistically to prevent and treat Aeromonas hydrophila to cause, can play the bacteriostatic action of herbal medicine so better.Impact is there is in different Chinese herbal medicine extracting working methods on the bacteriostatic action of pathogenic bacteria.3) vaccine prevention and control.The vaccine that aquaculture uses mainly is divided into 2 classes: a class is attenuated vaccine, and another kind of is dead or the vaccine of deactivation.Attenuated vaccine contains the cause of disease alive of attenuation, energy inducing specific antibody, and similar to natural infection.The vaccine of deactivation is made up of the toxin of some compositions as bacteria lipopolysaccharide or deactivation of dead cause of disease or cause of disease, can the immunity system of stimulating animal.Although Evaluation of Aeromon As Hydrophila Vaccine prevention fish can be applied at present, and this disease occurs, cause immune protective effect difference or unstable because of reasons such as this bacterium serotype numerous immune response level are lower.4) probiotic bacterium prevention and control.Along with the appearance of drug resistance problems and the deterioration of environment make antibiotic use be subject to increasing test.Compared with controlling with medicine, probiotic bacterium (probiotic) is by means of the advantage of its non-toxic efficient, by improving the water body environment of cultivation system, at host internal secretion nutritive substance and digestive ferment, improve the disease resistance of host, improve its immunity system, reduce the sickness rate of disease, and then improve aquaculture production and economic benefit, just receive increasing concern.At present, aquatic products Disease management uses more probiotic bacterium is milk-acid bacteria, genus bacillus, yeast and the composite reactive probiotic bacterium that is made up of the multiple beneficial such as photosynthetic bacterium, milk-acid bacteria bacterial classification.
Summary of the invention
The object of the invention is to overcome above-mentioned weak point, a kind of novel Antagonistic Fungi is provided, its biological activity stability, specificity be high, to cultured fishes and environmental safety, can be used as in large Fish-water Fish Farming and prevent and treat the probiotic strain that Aeromonas hydrophila causes fulminant hemorrhagic disease.
According to technical scheme provided by the invention, the source of fish provided by the invention Antergic Germ against Aeromonas hydrophila strain is bacillus amyloliquefaciens (FFRC-S24), is separated from fish intestines, and this bacterium antagonistic ability is strong, and because being separated from fish body enteron aisle, security is high.The microbial preparation being effective active composition with bacillus amyloliquefaciens (FFRC-S24) can the infection of effective antagonism Aeromonas hydrophila.
Genus bacillus is generally and amphimicrobian aerobic addicted to temperature, can produce resistance statospore to tolerate various bad envrionment conditions, and in the fermentative production and shipping storage of product, other bacterium relative has stronger advantage.Genus bacillus obtains large-scale promotion application as the probiotic products on aquatic products in China, its production technique is very ripe with use flow process, and bacillus amyloliquefaciens can improve the transformation efficiency of phosphorus in animal feed industries as probiotic additive, therefore, the bacillus amyloliquefaciens FFRC-S24 of described antagonism Aeromonas hydrophila will have wide market outlook.
One strain source of fish Antergic Germ against Aeromonas hydrophila strain, is preserved in China typical culture collection center, and deposit number is CCTCC NO:M2013191, and preservation date is on May 9th, 2013, and Classification And Nomenclature is bacillus amyloliquefaciens FFRC-S24
(Bacillus amyloliquefaciens)fFRC-S24.
Get bacillus amyloliquefaciens FFRC-S24 and be inoculated in LB liquid nutrient medium, inoculum size is 20 μ L/L-10mL/L, and cultivates 18-48h in 25-30 DEG C, makes the concentration of bacterium reach 1 × 10
9– 3 × 10
10cFU/mL; The component of LB liquid nutrient medium is: yeast extract 4-6g/L, peptone 8-15g/L, sodium-chlor 8-12g/L.
The bacillus amyloliquefaciens FFRC-S24 being inoculated in LB liquid nutrient medium is stirred in fish feed, makes the probiotic concentration contained in feed be 1 × 10
10– 5 × 10
12cFU/KG.
To be inoculated in the bacillus amyloliquefaciens FFRC-S24 of LB liquid nutrient medium, bacterial concentration is 1 × 10
9-1 × 10
11cFU/mL, directly splash to large freshwater fish culturing pool mouth, usage quantity of splashing is 0. 05-0.5L/m
3.
Beneficial effect of the present invention: the present invention's screening is stronger from the source of fish antagonistic ability of Antergic Germ against Aeromonas hydrophila strain to Aeromonas hydrophila of bacillus amyloliquefaciens FFRC-S24, to aquatic animal safety, as the microbial preparation preventing and treating the large freshwater fish hemorrhagic disease caused by Aeromonas hydrophila, there is very wide application prospect.
Biological material specimens preservation: a strain source of fish Antergic Germ against Aeromonas hydrophila strain, be preserved in China typical culture collection center, address Wuhan, China, Wuhan University, deposit number is CCTCC NO:M2013191, preservation date is on May 9th, 2013, and Classification And Nomenclature is bacillus amyloliquefaciens FFRC-S24
(Bacillus amyloliquefaciens FFRC-S24).
Accompanying drawing explanation
Add (experimental group) in Fig. 1 embodiment 3 feed and do not add the quantity of bacillus amyloliquefaciens (FFRC-S24) at different time sections enteron aisle A.hy43.
Embodiment
The in-vitro screening of embodiment 1 source of fish Antergic Germ against Aeromonas hydrophila strain
In large fresh-water fishes enteron aisle, water surrounding and bed mud, utilizing LB solid medium separation of bacterial, being prepared into bacteria suspension by being separated the bacterium obtained, cut-off footpath is that the little scraps of paper of 6mm soak, for bacteria screening after drying.
Getting indicator (Aeromonas hydrophila) 400uL is spread evenly across on LB agar plate, be that the little scraps of paper of 6mm are immersed in bacillus amyloliquefaciens (FFRC-S24) bacterium liquid by diameter, after the flat board scribbling indicator dries naturally, get above the scraps of paper are attached to, then put into 25-30 DEG C of constant incubator to face up and cultivate after 1h, carry out inversions cultivation 48h.Result Explicit solutions bacillus amyloliquefaciens (FFRC-S24) antibacterial circle diameter to Aeromonas hydrophila reaches 7-13mm.
According to identical working method, result Explicit solutions bacillus amyloliquefaciens (FFRC-S24) all has antagonistic ability to the Aeromonas hydrophila strain (morbidity fish body, healthy fish body, water body, bed mud) coming from different sources.Bacillus amyloliquefaciens (FFRC-S24) is to other pathogenic bacterias common in aquaculture: Aeromonas veronii, Vibrio parahaemolyticus, Channel-catfish fish tarda, Streptococcus iniae, streptococcus agalactiae are without obvious antagonism.
Embodiment 2
Get bacillus amyloliquefaciens FFRC-S24 and be inoculated in LB liquid nutrient medium, inoculum size is 20 μ L/L, and cultivates 48h in 25 DEG C, makes the concentration of bacterium reach 1 × 10
9cFU/mL;
The component of LB liquid nutrient medium is: yeast extract 4g/L, peptone 8g/L, sodium-chlor 8g/L.
The bacillus amyloliquefaciens FFRC-S24 being inoculated in LB liquid nutrient medium is stirred in fish feed, makes the probiotic concentration contained in feed be 1 × 10
10cFU/KG.
Embodiment 3
Get bacillus amyloliquefaciens FFRC-S24 and be inoculated in LB liquid nutrient medium, inoculum size is 10mL/L, and cultivates 18h in 30 DEG C, makes the concentration of bacterium reach 2 × 10
10cFU/mL;
The component of LB liquid nutrient medium is: yeast extract 6g/L, peptone 10g/L, sodium-chlor 12g/L.
The bacillus amyloliquefaciens FFRC-S24 being inoculated in LB liquid nutrient medium is stirred in fish feed, makes the probiotic concentration contained in feed be 3 × 10
12cFU/KG.
Embodiment 4
Get bacillus amyloliquefaciens FFRC-S24 and be inoculated in LB liquid nutrient medium, inoculum size is 5mL/L, and cultivates 30h in 28 DEG C, makes the concentration of bacterium reach 3 × 10
10cFU/mL;
The component of LB liquid nutrient medium is: yeast extract 5g/L, peptone 10g/L, sodium-chlor 10g/L.
The bacillus amyloliquefaciens FFRC-S24 being inoculated in LB liquid nutrient medium is stirred in fish feed, makes the probiotic concentration contained in feed be 5 × 10
12cFU/KG.
Embodiment 5
Get bacillus amyloliquefaciens FFRC-S24 and be inoculated in LB liquid nutrient medium, inoculum size is 1mL/L, and cultivates 48h in 26 DEG C, makes the concentration of bacterium reach 2.5 × 10
10cFU/mL; The component of LB liquid nutrient medium is: yeast extract 4g/L, peptone 8g/L, sodium-chlor 12g/L.
To be inoculated in the bacillus amyloliquefaciens FFRC-S24 of LB liquid nutrient medium, bacterial concentration is 1 × 10
10cFU/mL, directly splash to large freshwater fish culturing pool mouth, usage quantity of splashing is 0. 05L/m
3.
Embodiment 6
Get bacillus amyloliquefaciens FFRC-S24 and be inoculated in LB liquid nutrient medium, inoculum size is 3mL/L, and cultivates 25h in 27 DEG C, makes the concentration of bacterium reach 1 × 10
10cFU/mL; The component of LB liquid nutrient medium is: yeast extract 6g/L, peptone 15g/L, sodium-chlor 8g/L.
To be inoculated in the bacillus amyloliquefaciens FFRC-S24 of LB liquid nutrient medium, bacterial concentration is 9 × 10
9cFU/mL, directly splash to large freshwater fish culturing pool mouth, usage quantity of splashing is 0.2L/m
3.
Embodiment 7
Get bacillus amyloliquefaciens FFRC-S24 and be inoculated in LB liquid nutrient medium, inoculum size is 8mL/L, and cultivates 31h in 29 DEG C, makes the concentration of bacterium reach 3 × 10
10cFU/mL; The component of LB liquid nutrient medium is: yeast extract 6g/L, peptone 15g/L, sodium-chlor 12g/L.
The bacillus amyloliquefaciens FFRC-S24 of LB liquid nutrient medium, bacterial concentration 1 × 10 will be inoculated in
11cFU/mL, directly splash to large freshwater fish culturing pool mouth, usage quantity of splashing is 0.4L/m
3.
Embodiment 3 bacillus amyloliquefaciens (FFRC-S24) is to enteropathogenic restraining effect
By A.hy43 shaking culture 18h in 30 DEG C of LB liquid nutrient mediums, put in aquarium, make the concentration of Aeromonas hydrophila in aquarium reach 10
7cFU/mL, soaks and gets three tail Megalobrama amblycephalas after one day and carry out enteron aisle sampling, be designated as 0h.Then by A.hy43
gFPthe Megalobrama amblycephala soaked is divided into two groups, puts into new aquarium, and one group is experimental group, and one group is control group.
Experimental group is thrown something and fed and is mixed with the feed of probiotic bacterium S24, and control group is thrown something and fed normal diet, gets experimental group and each 3 tail fishes of control group respectively at 12h, 24h, 36h, 48h, by spirit lamp, before, during and after aseptic technique collection Megalobrama amblycephala, each about 1cm of intestines, is placed in 1.5 mL centrifuge tubes after sterilizing, weighs.
Collection enteron aisle sterile saline is washed, adds 2mL sterile saline afterwards respectively and carry out homogenate.Homogenate sterile saline is diluted respectively 10 times, 100 times, 1000 times, get the diluted liquid of 3 gradients and organize each 100 μ L of stoste to be spread evenly across LB nutrient agar plate (containing kantlex 60 μ g/mL) containing kantlex, being placed in 30 ° of C biochemical cultivation cases and cultivating 18 h.At fluorescence microscopy Microscopic observation bacterium colony fluorescence, and count.
In result display experimental group enteron aisle, the quantity of A.hy43 is all less than control group.Between 0-12h, the quantity of Aeromonas hydrophila is in the decline stage, and after 12h, the amount of bacteria of each group all starts to increase.But the experimental group of the S24 that throws something and feeds gathers way, comparatively control group is slow.
Concrete data are as shown in table 1.
Add (experimental group) in table 1 feed and do not add the quantity of bacillus amyloliquefaciens (FFRC-S24) at different time sections enteron aisle A.hy43
Embodiment 4 protection ratio is tested
Select 30 urosomes to weigh the healthy Megalobrama amblycephala of 75 ± 0.5g, in aquarium, support 7d temporarily, start test.Test is divided into two groups, often organizes each 15 tails.Control group is thrown something and fed normal diet, and experimental group throws something and feeds 2.1 containing bacillus amyloliquefaciens feed.Throw something and feed after 2w, carry out immersion respectively attack poison with A.hy43, the A.hy43 concentration in each aquarium is 10
7cFU/ml, breeding observing 7d, add up the mortality ratio of each group, according to following formula Computation immunity protection ratio.Immune protective rate (RPS)=[1-(immune group mortality ratio/control group mortality ratio)] × 100%.
The mortality of each group is in table 2, and the protection ratio that we calculate S24 from table is 60%.
Table 2 protection ratio experimental result
。
Claims (1)
1. a strain source of fish Antergic Germ against Aeromonas hydrophila strain, is preserved in China typical culture collection center, and deposit number is CCTCC NO:M2013191, and preservation date is on May 9th, 2013, and Classification And Nomenclature is bacillus amyloliquefaciens FFRC-S24
(Bacillus amyloliquefaciens)fFRC-S24.
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| CN106318880B (en) * | 2015-06-15 | 2019-09-20 | 中国科学院微生物研究所 | One bacillus amyloliquefaciens, bacteriostatic agent prepared therefrom and purposes |
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| CN113663001A (en) * | 2021-08-24 | 2021-11-19 | 四川成康动物药业有限公司 | Essence composition containing rhubarb and gallnut and preparation method and device thereof |
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