CN106318880A - Bacillus amyloliquefaciens and its bacterial depressant and use - Google Patents
Bacillus amyloliquefaciens and its bacterial depressant and use Download PDFInfo
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Abstract
The invention relates to Bacillus amyloliquefaciens V4. The Bacillus amyloliquefaciens V4 is preserved in the China general microbiological culture collection center and has a preservation number of CGMCC NO. 10149. The Bacillus amyloliquefaciens V4 strain and/or microbial agent can produce bacteriocin for antagonizing vibrio pathogens and pathogenic aeromonas at seedling growing and culture temperatures of common culture water, inhibit vibrio vulnificus, vibrio natriegen, vibrio harveyi, black sea vibrio, aeromonas hydrophila and aeromonas salmonicida growth and breeding, effectively control aquatic animal infectious disease generation and propagating caused by vibrio pathogens and pathogenic aeromonas, reduce aquatic animal mortality and a forage coefficient and promote aquatic animal weight gain.
Description
Technical field
The invention belongs to microorganism field, more particularly, to a bacillus amyloliquefaciens, by it
The antibacterial of preparation and purposes.
Background technology
China is world aquaculture big country, year cultured output reach more than 5,000 ten thousand tons, account for Gross World Product
71%.But the lost units caused by Aquatic product disease every year accounts for more than 15%, economic loss is up to
150000000000 yuan.Known aquiculture disease has kind more than 100, cause of disease kind to have a kind more than 300, wherein,
Bacterial pathogen kind accounts for 54.9%.And kinds of pathogenic vibrio, aeromonas salmonicida are aquatic animal infectiousness
Topmost pathogen in disease.By the microbial disease of arc, popular area is wide, and sickness rate is high, gives
Aquaculture causes significant damage.Vibrio anguillarum (Vibrio anguillarum), Vibro harveyi (V.
Harveyi), a variety of vibrios such as Vibrio vulnificus (V.vulnificus), Vibrio salmonicida (V.salmonicida) can
To cause fish diseases, some kind also results in people or the morbidity of other animals.It addition, kill salmon gas list
Born of the same parents bacterium (Aeromonas salmonicida), Aeromonas hydrophila (Aeromonas hydrophila) are also
The pathogen that aquatic animal is common.Aeromonas salmonicida (Aeromonas salmonicida) is main
Infect salmon fishes generation furunculosis.Aeromonas hydrophila (Aeromonas hydrophila), is producing
Middle infection causes fulminant hemorrhagic disease.
Broad ectrum antibiotic is mainly used at present, according to expert investigation, China in aquatic animal disease is prevented and treated
Annual production antibiotic raw material about 210,000 tons, wherein has 9.7 ten thousand tons of antibiotic to support for animal husbandry and fishery
Grow industry, account for the 46.1% of antibiotic gross annual output amount.Although it is dynamic to use antibiotic can efficiently control cultivation
The generation of thing disease and spreading, but exist in actual production and blindly use and drug dependence phenomenon, water
The abuse of antibiotics produced in cultivation has many negative effects.First, the blindness of antibiotic uses tight
Heavily disturb beneficial microorganism normal growth and breeding in breeding environment and aquatic animal intestinal, destroy
Microecological balance.Second, in aquaculture, antibiotic use the most a large amount of, nonstandard causes water
Produce the animal susceptibility to pathogenic microorganism, more seriously cause the drug resistance of pathogen to strengthen, go out
Now a large amount of drug-resistant bacteria, even superbacterias.3rd, the use of antibiotic also results in it aquatic
In animal body, residual, enrichment, endanger the health of the mankind the most at last.Although antibiotic is residual in aquatic products
Allowance is the lowest, but very serious to the potential hazard of health, and has a far reaching influence.4th, anti-
Raw element drug residue also results in aquatic product quality to be reduced, and becomes aquatic products sales volume and export trade amount
Limiting factor.
Microbial ecological agent is existing antibiotic and the maximally effective substitute products of synthetic drug species feed additive
One of, there is the feature of low cost of manufacture, mature technology, therefore it should be able to be superior with it
, be conducive to human health and the properties of product of ecological environmental protection, progressively substitute antibiotics.Cultivation
In production, the microbial ecological agent of actual application includes: viable bacteria body, dead thalline, drive member, metabolism are produced
The parts such as thing and active growth helping matter.Thus, it is found that new can be applicable to aquaculture
The bacterial strain of microbial ecological agent significant.
Summary of the invention
The invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4,
This bacterial strain, can suppress vibrio pathogen and the growth of pathogenicity Aeromonas and breeding, thus reduce by
The generation of the microbial infectious disease of aquatic animal of these cause of diseases and spreading, reduces aquatic animal
Mortality rate.
One aspect of the present invention, it is provided that a bacillus amyloliquefaciens (Bacillus
Amyloliquefaciens) V4, this bacterial strain is deposited in China Microbiological on 10th in December in 2014
Culture presevation administration committee common micro-organisms center, deposit number is CGMCC No.10149.Protect
Tibetan unit address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Described bacillus amyloliquefaciens, is isolatable from ocean circulating water cultivation environment, this bacterial strain have with
Lower characteristic:
Bacterial strain colonial morphology in 2216E culture medium is: white, translucent, smooth moistening bacterium colony,
Slightly swell, circular neat bacterium colony；Gram’s staining thalline purple, for gram positive bacteria, shaft-like；
The alkali phosphatase experiment positive；Naphthols-AS-BI phosphohydrolase experiment feminine gender；Alpha-glucosidase is real
Test the positive；Carbohydrate is tested: mannitol is positive；L-arabinose is positive；D-ribose is positive；
D-xylose is positive；D-Glucose is positive；D-Fructose is positive；D-MANNOSE is positive；Inositol is positive；Sweet
Dew sugar alcohol is positive；Sorbitol is positive；Methyl-α D-pyranglucoside is positive；N-Acetyl-D-glucosamine
Positive；Amygdaloside is positive；ARBULIN is positive；Esculin ferric citrate is positive；Salicin is positive；
D-cellobiose is positive；D-Maltose is positive；D-Lactose-positive；D-6-(.alpha.-D-galactosido)-D-glucose. is positive；D-sucrose sun
Property；D-trehalose is positive；D-cottonseed sugar is positive；Starch is positive；Glycogen is positive；Xylitol is positive；
D-gentiobiose is positive；D-Toulon sugar is positive；Potassium gluconate is positive.
Another aspect of the present invention, it is provided that above-described bacillus amyloliquefaciens V4 is in suppression disease
Originality vibrio and pathogenicity Aeromonas grow and/or the purposes of breeding.
In technique scheme, described vibrio pathogen includes Vibrio vulnificus, Vibrio natriegen, Ha Wei
Family name vibrio and Black Sea vibrio, described pathogenicity Aeromonas includes Aeromonas hydrophila and kills salmon gas unit cell
Bacterium.
Present invention also offers a kind of antibacterial, its active component is above-described solution starch spore bar
Bacterium (Bacillus amyloliquefaciens) V4.
The preparation method of above-mentioned antibacterial, comprises the steps:
Above-described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4 is accessed LB
Cultivating in fluid medium, condition of culture is temperature 28～32 DEG C, hunting speed 180～210rpm,
Incubation time 1～3 days, it is thus achieved that the most described antibacterial of culture fluid.
Preferably, cultivation temperature is 30 DEG C, hunting speed 200rpm, incubation time 2 days.
In technique scheme, described LB culture medium includes following composition: peptone 8～12 mass
Part, yeast extract and/or yeast extract 4～6 mass parts, sodium chloride 8～12 mass parts, distilled water
970～980 mass parts, NaOH adjusts pH 7～7.5.
Preferably, peptone 10 mass parts, yeast extract and/or yeast extract 5 mass parts, chlorination
Sodium 10 mass parts, distilled water 975 mass parts, adjust pH 7.2 with NaOH.
Another aspect of the invention, it is provided that one is prevented and/or kills Aquatic product vibrio pathogen and Aquatic product
The method of pathogenicity Aeromonas, accesses above-described antibacterial in water body.
In technique scheme, described vibrio pathogen includes Vibrio vulnificus, Vibrio natriegen, Ha Wei
Family name vibrio and Black Sea vibrio, described pathogenicity Aeromonas includes Aeromonas hydrophila and kills salmon gas unit cell
Bacterium.
It is still another aspect of the present invention to provide a kind of reduction aquatic animal mortality rate and feed coefficient, carry
The method of high rate of body weight gain, accesses above institute by 0.1%～0.3% volume ratio in aquaculture of aquatic animal water body
The antibacterial stated.
Bacillus amyloliquefaciens V4 bacterial strain and/or microbial inoculum that the present invention provides are educated breeding water body is common
Antagonism vibrio pathogen and the bacteriocin of pathogenicity Aeromonas can be produced at a temperature of Seedling and cultivation, press down
Vibrio vulnificus processed, Vibrio natriegen, Vibro harveyi, Black Sea vibrio, Aeromonas hydrophila and kill salmon gas
The growth of Zymomonas mobilis and breeding, it is possible to play and efficiently control by vibrio pathogen and pathogenicity gas unit cell
The generation of microbial infectious disease of aquatic animal and the effect spread, reduce the dead of aquatic animal
Die rate, promote the weightening finish of aquatic animal.
Accompanying drawing explanation
Vibrio vulnificus (Vibrio vulnificus) is grown by Fig. 1 bacillus amyloliquefaciens V4 microbial inoculum supernatant
Suppression；
Vibrio natriegen (Vibrio natriegens) is grown by Fig. 2 bacillus amyloliquefaciens V4 microbial inoculum supernatant
Suppression；
Fig. 3 bacillus amyloliquefaciens V4 microbial inoculum supernatant is to Aeromonas hydrophila (Aeromonas
Hydrophila) suppression grown；
Fig. 4 bacillus amyloliquefaciens V4 microbial inoculum supernatant is to aeromonas salmonicida (Aeromonas
Salmonicida) suppression grown；
Fig. 5 bacillus amyloliquefaciens V4 microbial inoculum supernatant is to Vibro harveyi (Vibrio harveyi PH4)
The suppression of growth；
Black Sea vibrio (Vibrio ponticus) is grown by Fig. 6 bacillus amyloliquefaciens V4 microbial inoculum supernatant
Suppression.
Detailed description of the invention
Below in conjunction with drawings and Examples, the detailed description of the invention of the present invention is carried out more detailed theory
Bright, so as to the advantage being more fully understood that the solution of the present invention and its various aspects.But, with
The detailed description of the invention of lower description and embodiment are only descriptive purpose rather than limitation of the present invention.
The species identification of embodiment 1 bacterial strain
Biological characteristics: bacterial strain colonial morphology in 2216E culture medium is: white, translucent,
Smooth moistening bacterium colony, slightly swells, circular neat bacterium colony；Gram’s staining thalline purple, for gram
Positive bacteria, shaft-like；The alkali phosphatase experiment positive；Naphthols-AS-BI phosphohydrolase experiment feminine gender；α-
The glucosidase experiment positive；Carbohydrate is tested: mannitol is positive；L-arabinose is positive；
D-ribose is positive；D-xylose is positive；D-Glucose is positive；D-Fructose is positive；D-MANNOSE is positive；
Inositol is positive；Mannitol is positive；Sorbitol is positive；Methyl-α D-pyranglucoside is positive；N-
Acetylglucosamine is positive；Amygdaloside is positive；ARBULIN is positive；Esculin ferric citrate is positive；
Salicin is positive；D-cellobiose is positive；D-Maltose is positive；D-Lactose-positive；D-6-(.alpha.-D-galactosido)-D-glucose. sun
Property；D-sucrose is positive；D-trehalose is positive；D-cottonseed sugar is positive；Starch is positive；Glycogen is positive；
Xylitol is positive；D-gentiobiose is positive；D-Toulon sugar is positive；Potassium gluconate is positive.
Molecular biology identification: in sequence table 16S rRNA gene acquisition process and with existing strain
Comparison result
Bacterial strain V4 accesses in LB fluid medium and cultivates, and condition of culture is temperature 28～32 DEG C, vibration
Speed 180～210rpm, incubation time 24 hours, take 1ml liquid medium in 1.5ml centrifuge tube
In, 8000r/min is centrifuged 5min, abandons supernatant, adds 500 μ l ddH2O, re-suspended cell, will be thin
Born of the same parents suspension 8000r/min is centrifuged 5min, abandons supernatant, adds 100 μ l ddH2O re-suspended cell, then will
Centrifuge tube is put into and is boiled 10min in boiling water, makes cell crack, and 8000r/min takes supernatant 2 μ l after being centrifuged 5min
As pcr template, expand in 50 μ l reaction systems using 27F, 1492R as upstream and downstream primer
Increase the 16S rRNA gene of this bacterial strain.The 16S rRNA sequence of V4 bacterial strain is obtained after gene sequencing
Shown in SEQ ID NO:1.
The data bases such as the GenBank at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/)
The 16S rRNA gene order of the close bacterial strain of middle search, carries out multisequencing para-position row with MEGA software
Row, use Neighbor-Joining method phylogenetic tree construction, as follows.
Embodiment 2 bacillus amyloliquefaciens V4 microbial inoculum supernatant is to Vibrio vulnificus CZ-A2 (Vibrio
Vulnificus) suppression grown
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum supernatant:
By in bacillus amyloliquefaciens V4 inoculation to the LB culture medium of 100mL, 30 DEG C of cultivations
24h obtains microbial inoculum, is centrifuged by microbial inoculum 8000r/min 10 minutes, obtains microbial inoculum supernatant, by this
Clear liquid is filtrated to get without fermented liquid by disposable syringe filter.
LB medium component: peptone 10 mass parts, yeast extract 5 mass parts, sodium chloride 10 matter
Amount part, distilled water 975 mass parts, pH 7.2.
2. the inhibitory action that Vibrio vulnificus (Vibrio vulnificus) CZ-A2 is grown by microbial inoculum supernatant:
Vibrio vulnificus (Vibrio vulnificus) CZ-A2 is prepared bacteria suspension, 100 μ l bacteria suspension coatings
LB solid plate, is placed in planar surface by the aseptic filter paper sheet of diameter 6mm, drips the solution of 10 μ l
Culture dish on aseptic filter paper sheet, then is placed in 30 DEG C of cultivations without fermented liquid by bacillus amyloliquefaciens V4
Case is cultivated 24 hours.Observe the inhibition zone that Vibrio vulnificus is produced by visible bacillus amyloliquefaciens V4,
Inhibition zone area is 220.71 ± 6.61mm2(accompanying drawing 1).
Embodiment 3, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to Vibrio natriegen (Vibrio
Natriegens FS-1) suppression that grows
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum supernatant:
Bacillus amyloliquefaciens V4 microbial inoculum supernatant is made by step 1 in embodiment 2.
2. the inhibitory action that Vibrio natriegen (Vibrio natriegens FS-1) is grown by microbial inoculum supernatant:
Vibrio natriegen (Vibrio natriegens) is prepared bacteria suspension, 100 μ l bacteria suspension coating LB solids
Flat board, is placed in planar surface by the aseptic filter paper sheet of diameter 6mm, drips the solution starch spore of 10 μ l
Culture dish on aseptic filter paper sheet, then is placed in 30 DEG C of incubators cultivation without fermented liquid by bacillus V4
24 hours.Observe the inhibition zone that Vibrio natriegen is produced by visible bacillus amyloliquefaciens V4, inhibition zone face
Amassing is 50.24 ± 3.6mm2(accompanying drawing 2).
Embodiment 4, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to Aeromonas hydrophila (Aeromonas
Hydrophila) suppression grown
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum supernatant:
Bacillus amyloliquefaciens V4 microbial inoculum supernatant is made by step 1 in embodiment 2.
2. the suppression that Aeromonas hydrophila (Aeromonas hydrophila) is grown by microbial inoculum supernatant is made
With:
Prepared by Aeromonas hydrophila (Aeromonas hydrophila) bacteria suspension, and 100 μ l bacteria suspensions are coated with
Cloth LB solid plate, is placed in planar surface by the aseptic filter paper sheet of diameter 6mm, drips 10 μ l's
Culture dish on aseptic filter paper sheet, then is placed in 30 DEG C of trainings without fermented liquid by bacillus amyloliquefaciens V4
Support in case and cultivate 24 hours.Observe visible bacillus amyloliquefaciens V4 to Aeromonas hydrophila life
Inhibition zone, inhibition zone area is 176.625 ± 5.18mm2(accompanying drawing 3).
Embodiment 5, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to aeromonas salmonicida (Aeromonas
Salmonicida) suppression grown
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum supernatant:
Bacillus amyloliquefaciens V4 microbial inoculum supernatant is made by step 1 in embodiment 2.
2. the suppression that aeromonas salmonicida (Aeromonas salmonicida) is grown by microbial inoculum supernatant is made
With:
Prepared by aeromonas salmonicida (Aeromonas salmonicida) bacteria suspension, and 100 μ l bacteria suspensions are coated with
Cloth LB solid plate, is placed in planar surface by the aseptic filter paper sheet of diameter 6mm, drips 10 μ l's
Culture dish on aseptic filter paper sheet, then is placed in 30 DEG C of trainings without fermented liquid by bacillus amyloliquefaciens V4
Support in case and cultivate 24 hours.Observe what aeromonas salmonicida was produced by visible bacillus amyloliquefaciens V4
Inhibition zone, inhibition zone area is 240.41 ± 4.57mm2(accompanying drawing 4).
Embodiment 6, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to Vibro harveyi (Vibrio harveyi
PH4) suppression grown
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum supernatant:
Bacillus amyloliquefaciens V4 microbial inoculum supernatant is made by step 1 in embodiment 2.
2. the inhibitory action that Vibro harveyi (Vibrio harveyi PH4) is grown by microbial inoculum supernatant:
Vibro harveyi (Vibrio harveyi PH4) is prepared bacteria suspension, 100 μ l bacteria suspension coating LB
Solid plate, is placed in planar surface by the aseptic filter paper sheet of diameter 6mm, drips the solution starch of 10 μ l
Culture dish on aseptic filter paper sheet, then is placed in 30 DEG C of incubators by bacillus cereus V4 without fermented liquid
Cultivate 24 hours.Observe the inhibition zone that Vibro harveyi is produced by visible bacillus amyloliquefaciens V4, press down
Bacterium circle area is 47.78 ± 4.62mm2(accompanying drawing 5).
Embodiment 7, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to Black Sea vibrio (Vibrio ponticus)
The suppression of growth
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum supernatant:
Bacillus amyloliquefaciens V4 microbial inoculum supernatant is made by step 1 in embodiment 2.
2. the inhibitory action that Black Sea vibrio (Vibrio ponticus B8) is grown by microbial inoculum supernatant:
Prepared by Black Sea vibrio (Vibrio ponticus B8) bacteria suspension, and 100 μ l bacteria suspension coating LB are solid
Body flat board, is placed in planar surface by the aseptic filter paper sheet of diameter 6mm, drips the solution starch bud of 10 μ l
Culture dish on aseptic filter paper sheet, then is placed in 30 DEG C of incubators training without fermented liquid by spore bacillus V4
Support 24 hours.Observe the inhibition zone that Black Sea vibrio is produced by visible bacillus amyloliquefaciens V4, antibacterial
Circle area is 63.59 ± 2.9mm2(accompanying drawing 6).
Embodiment 8, add different proportion bacillus amyloliquefaciens V4 microbial inoculum to rainbow trout growth and survival
Impact
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum
The liquid LB of composition described in bacillus amyloliquefaciens V4 inoculation to embodiment 2 is cultivated
In base, cultivating 24h for 30 DEG C, gained culture fluid is bacillus amyloliquefaciens V4 microbial inoculum.
2. in Donaldson rainbow trout culturing pool, add different proportion bacillus amyloliquefaciens V4 preparation
0.1%～0.3% ratio interpolation bacillus amyloliquefaciens V4 preparation is supported to Donaldson rainbow trout by volume
Grow in pond, matched group is set, monitor the original body mass of Donaldson rainbow trout, body weight in latter stage, rate of body weight gain, raise
Material coefficient, specific growth rate and mortality rate.After cultivating 45 days, compared with matched group, interpolation group can promote
Entering the weightening finish of Donaldson rainbow trout, promotion ratio is 17.08～71.22%.Feed coefficient can be reduced simultaneously,
Reach 19.49% at high proportion.Meanwhile, compared with matched group, interpolation group can reduce the mortality rate of Donaldson rainbow trout,
Ratio is 27.25～81.86% (being shown in Table 1).
Table 1
Claims (9)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4, in being preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number is CGMCC
NO.10149。
2. the bacillus amyloliquefaciens V4 described in claim 1 is in suppression vibrio pathogen and pathogenicity
Aeromonas growth and/or the purposes of breeding.
3. purposes as claimed in claim 2, it is characterised in that described vibrio pathogen includes wound
Vibrio, Vibrio natriegen, Vibro harveyi and Black Sea vibrio, described pathogenicity Aeromonas includes addicted to water
Aeromonas and aeromonas salmonicida.
4. an antibacterial, it is characterised in that its active component is the solution starch described in claim 1
Bacillus cereus (Bacillus amyloliquefaciens) V4.
5. the preparation method of the antibacterial described in claim 4, it is characterised in that comprise the steps:
By bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4 described in claim 1
Accessing in LB fluid medium and cultivate, condition of culture is temperature 28～32 DEG C, hunting speed 180～
210rpm, incubation time 1～3 days, it is thus achieved that the most described antibacterial of culture fluid.
6. preparation method as claimed in claim 5, it is characterised in that described LB culture medium includes
Following composition: peptone 8～12 mass parts, yeast extract and/or yeast extract 4～6 mass parts,
Sodium chloride 8～12 mass parts, distilled water 970～980 mass parts, NaOH adjusts pH 7～7.5.
7. preventing and/or kill vibrio pathogen and a method for pathogenicity Aeromonas, its feature exists
In, in water body, access the antibacterial described in claim 4 as 0.1%～0.3% volume ratio.
8. method as claimed in claim 7, it is characterised in that described vibrio pathogen includes wound
Vibrio, Vibrio natriegen, Vibro harveyi and Black Sea vibrio, described pathogenicity Aeromonas includes addicted to water
Aeromonas and aeromonas salmonicida.
9. reduce aquatic animal mortality rate and feed coefficient, a method for raising rate of body weight gain, its feature
It is, accesses described in claim 4 in aquaculture of aquatic animal water body as 0.1%～0.3% volume ratio
Antibacterial.
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