CN106318880B - One bacillus amyloliquefaciens, bacteriostatic agent prepared therefrom and purposes - Google Patents
One bacillus amyloliquefaciens, bacteriostatic agent prepared therefrom and purposes Download PDFInfo
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Abstract
The present invention relates to a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4, are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.10149.Bacillus amyloliquefaciens V4 bacterial strain and/or microbial inoculum provided by the invention can produce the bacteriocin of antagonism vibrio pathogen and pathogenicity Aeromonas at a temperature of the common nursery of breeding water body and cultivation; inhibit the growth and breeding of Vibrio vulnificus, Vibrio natriegen, Vibrio harveyi, Black Sea vibrios, Aeromonas hydrophila and aeromonas salmonicida; it can play the role of efficiently controlling the generation and sprawling of the infectious disease of aquatic animal as caused by vibrio pathogen and pathogenicity Aeromonas; the death rate and feed coefficient for reducing aquatic livestock, promote the weight gain of aquatic livestock.
Description
Technical field
The invention belongs to microorganism fields, more particularly, to a bacillus amyloliquefaciens, bacteriostatic agent prepared therefrom
And purposes.
Background technique
China is world aquaculture big country, year cultured output account for the 71% of Gross World Product up to more than 5,000 ten thousand tons.But
The lost units as caused by aquatic products disease accounts for 15% or more every year, and economic loss is up to 150,000,000,000 yuan.Known aquaculture disease
Evil has more than 100 kinds, and cause of disease type has more than 300 kinds, wherein bacterial pathogen type accounts for 54.9%.And kinds of pathogenic vibrio, kill salmon gas
Monad is most important pathogen in infectious disease of aquatic animal.The disease as caused by vibrios, popular area is wide, disease incidence
Height causes significant damage to aquaculture.Vibrio anguillarum (Vibrio anguillarum), Vibrio harveyi (V.harveyi), wound
Fish diseases can be caused by hurting a variety of vibrios such as vibrios (V.vulnificus), Vibrio salmonicida (V.salmonicida), certain
Type also results in people or the morbidity of other animals.In addition, aeromonas salmonicida (Aeromonas salmonicida), thermophilic aqueous vapor
Monad (Aeromonas hydrophila) is also the common pathogen of aquiculture animal.Aeromonas salmonicida
Furunculosis occurs for (Aeromonas salmonicida) main infection salmon fishes.Aeromonas hydrophila (Aeromonas
Hydrophila), infection leads to fulminant hemorrhage in production.
Broad-spectrum antibiotic is mainly used in aquatic animal disease prevention and treatment at present, according to expert investigation, the annual production of China is anti-
Raw about 210,000 tons of raw material of element accounts for antibiotic gross annual output amount wherein there is 9.7 ten thousand tons of antibiotic for animal husbandry and fishery aquaculture
46.1%.Although the generation and sprawling of cultivated animals disease can be efficiently controlled using antibiotic, deposit in actual production
Abuse of antibiotics in blindness use and drug abuse phenomenon, aquaculture has various negative effects.First, antibiotic
Blindness using beneficial microorganism normal growth and breeding in severe jamming breeding environment and aquatic livestock enteron aisle, destroy micro-
The ecological balance.Second, in aquaculture, long-term a large amount of, the nonstandard use of antibiotic causes aquatic livestock to pathogenic microorganism
Neurological susceptibility, more seriously cause the drug resistance of pathogen to enhance, a large amount of drug-resistant bacterias, even superbacteria occur.Third,
The use of antibiotic also results in it and remains, is enriched in aquatic animal body, will finally endanger the health of the mankind.Although aquatic products
The residual quantity of middle antibiotic is very low, but very serious to the potential hazard of human health, and has a far reaching influence.4th, antibiotic
Medicament residue also results in aquatic product quality reduction, becomes the limiting factor of aquatic products sales volume and export trade amount.
Probiotics are one of existing antibiotic and the most effective substitute products of synthetic drug species feed addictive, are had
The characteristics of manufacturing cost is low, mature technology, thus it should be able to it is superior with its, be conducive to human health and ecological ring
The properties of product of border protection, gradually substitute antibiotics.In breeding production the probiotics of practical application include: viable bacteria body, it is dead
The parts such as thallus, drive member, metabolite and active growth helping matter.Thus, it is found that new can be applied to water
The bacterial strain for producing the probiotics of cultivation is of great significance.
Summary of the invention
The present invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4, the bacterial strain,
It can inhibit the growth and breeding of vibrio pathogen and pathogenicity Aeromonas, moved to reduce the aquatic products as caused by these pathogens
The generation and sprawling of object communicable disease, reduce the death rate of aquatic livestock.
One aspect of the present invention provides a bacillus amyloliquefaciens (Bacillus amyloliquefaciens)
V4, the bacterial strain are deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 10th, 2014,
Deposit number is CGMCC No.10149.Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The bacillus amyloliquefaciens, are isolated from ocean circulating water cultivation environment, which has characteristics that
Colonial morphology of the bacterial strain on 2216E culture medium are as follows: white, translucent, smooth wet bacterium colony slightly swells, round
Neat bacterium colony；Gram's staining thallus purple is gram-positive bacteria, rod-shaped；Alkaline phosphatase experiment is positive；Naphthols-AS-BI
Phosphohydrolase experiment is negative；Alpha-glucosidase experiment is positive；Carbohydrate experiment: mannitol is positive；L-arabinose
It 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；Sorbierite is positive；Methyl-α D- glucopyranoside is positive；N-Acetyl-D-glucosamine is positive；Amarogentin is positive；
ARBULIN is positive；Aesculin ironic citrate is positive；Salicin is positive；D- cellobiose is positive；D-Maltose is positive；D- lactose
It is positive；D- melibiose is positive；D- sucrose is positive；D- trehalose is positive；D- raffinose is positive；Starch is positive；Glycogen is positive；Xylose
Alcohol is positive；D- gentiobiose is positive；The Toulon D- sugar is positive；K-IAO is positive.
Another aspect of the present invention, provide above-described bacillus amyloliquefaciens V4 inhibit vibrio pathogen and
The purposes of the growth of pathogenicity Aeromonas and/or breeding.
In the above-mentioned technical solutions, the vibrio pathogen includes Vibrio vulnificus, Vibrio natriegen, Vibrio harveyi and Black Sea
Vibrios, the pathogenicity Aeromonas includes Aeromonas hydrophila and aeromonas salmonicida.
The present invention also provides a kind of bacteriostatic agent, active constituent is above-described bacillus amyloliquefaciens
(Bacillus amyloliquefaciens)V4。
The preparation method of above-mentioned bacteriostatic agent, includes the following steps:
By above-described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4 access LB liquid training
It supports and is cultivated in base, condition of culture is 28~32 DEG C of temperature, 180~210rpm of hunting speed, incubation time 1~3 day, the training of acquisition
Nutrient solution, that is, the bacteriostatic agent.
Preferably, cultivation temperature is 30 DEG C, hunting speed 200rpm, incubation time 2 days.
In the above-mentioned technical solutions, the LB culture medium includes following ingredient: 8~12 mass parts of peptone, and yeast extracts
4~6 mass parts of object and/or yeast extract, 8~12 mass parts of sodium chloride, 970~980 mass parts of distilled water, NaOH tune pH 7~
7.5。
Preferably, 10 mass parts of peptone, 5 mass parts of yeast extract and/or yeast extract, 10 mass parts of sodium chloride,
975 mass parts of distilled water, with NaOH tune pH 7.2.
Another aspect of the invention provides a kind of prevention and/or killing aquatic products vibrio pathogen and aquatic products pathogenicity gas
The method of monad accesses above-described bacteriostatic agent into water body.
In the above-mentioned technical solutions, the vibrio pathogen includes Vibrio vulnificus, Vibrio natriegen, Vibrio harveyi and Black Sea
Vibrios, the pathogenicity Aeromonas includes Aeromonas hydrophila and aeromonas salmonicida.
It is still another aspect of the present invention to provide a kind of reduction aquatic livestock death rate and feed coefficient, improve rate of body weight gain
Method accesses above-described bacteriostatic agent into aquaculture of aquatic animal water body by 0.1%~0.3% volume ratio.
Bacillus amyloliquefaciens V4 bacterial strain and/or microbial inoculum provided by the invention are in the common nursery of breeding water body and cultivation temperature
The lower bacteriocin that can produce antagonism vibrio pathogen and pathogenicity Aeromonas of degree, inhibits Vibrio vulnificus, Vibrio natriegen, Ha Wei
The growth and breeding of family name vibrios, Black Sea vibrios, Aeromonas hydrophila and aeromonas salmonicida, can play and efficiently control by disease
The effect of generation and the sprawling of infectious disease of aquatic animal caused by originality vibrios and pathogenicity Aeromonas, reduces aquatic products
The death rate of animal promotes the weight gain of aquatic livestock.
Detailed description of the invention
The inhibition that Fig. 1 bacillus amyloliquefaciens V4 microbial inoculum supernatant grows Vibrio vulnificus (Vibrio vulnificus)；
The inhibition that Fig. 2 bacillus amyloliquefaciens V4 microbial inoculum supernatant grows Vibrio natriegen (Vibrio natriegens)；
Fig. 3 bacillus amyloliquefaciens V4 microbial inoculum supernatant grows Aeromonas hydrophila (Aeromonas hydrophila)
Inhibition；
Fig. 4 bacillus amyloliquefaciens V4 microbial inoculum supernatant is raw to aeromonas salmonicida (Aeromonas salmonicida)
Long inhibition；
The suppression that Fig. 5 bacillus amyloliquefaciens V4 microbial inoculum supernatant grows Vibrio harveyi (Vibrio harveyi PH4)
System；
The inhibition that Fig. 6 bacillus amyloliquefaciens V4 microbial inoculum supernatant grows Black Sea vibrios (Vibrio ponticus).
Specific embodiment
Below in conjunction with drawings and examples, a specific embodiment of the invention is described in more details, so as to energy
The advantages of enough more fully understanding the solution of the present invention and its various aspects.However, specific embodiments described below and reality
It applies example to be for illustrative purposes only, rather than limiting the invention.
The species identification of 1 bacterial strain of embodiment
Biological characteristics: colonial morphology of the bacterial strain on 2216E culture medium are as follows: white, translucent, smooth wet bacterium colony,
It slightly swells, round neat bacterium colony；Gram's staining thallus purple is gram-positive bacteria, rod-shaped；Alkaline phosphatase experiment sun
Property；The experiment of naphthols-AS-BI phosphohydrolase is negative；Alpha-glucosidase experiment is positive；Carbohydrate experiment: mannitol sun
Property；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；Sorbierite is positive；Methyl-α D- glucopyranoside is positive；N-Acetyl-D-glucosamine is positive；
Amarogentin is positive；ARBULIN is positive；Aesculin ironic citrate is positive；Salicin is positive；D- cellobiose is positive；D- malt
It is sugared positive；D- Lactose-positive；D- melibiose is positive；D- sucrose is positive；D- trehalose is positive；D- raffinose is positive；Starch is positive；
Glycogen is positive；Xylitol is positive；D- gentiobiose is positive；The Toulon D- sugar is positive；K-IAO is positive.
Molecular biology identification: the acquisition process of 16S rRNA gene and the comparison result with existing strain in sequence table
It is cultivated in bacterial strain V4 access LB liquid medium, condition of culture is 28~32 DEG C of temperature, hunting speed 180~
210rpm incubation time 24 hours, takes 1ml liquid medium in 1.5ml centrifuge tube, and 8000r/min is centrifuged 5min, abandons supernatant
500 μ l ddH are added in liquid2Cell is resuspended in O, and cell suspension 8000r/min is centrifuged 5min, abandons supernatant, 100 μ l are added
ddH2Cell is resuspended in O, then centrifuge tube is put into boiling water and boils 10min, makes cell cracking, takes after 8000r/min centrifugation 5min
Clear 2 μ l expands the 16S of the bacterial strain using 27F, 1492R as upstream and downstream primer as pcr template in 50 μ l reaction systems
RRNA gene.Shown in the 16S rRNA sequence SEQ ID NO:1 for obtaining V4 bacterial strain after gene sequencing.
It is searched in the databases such as the GenBank of NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) close
The 16S rRNA gene order of bacterial strain carries out multisequencing contraposition arrangement with MEGA software, using Neighbor-Joining method structure
Phylogenetic tree is built, it is as follows.
2 bacillus amyloliquefaciens V4 microbial inoculum supernatant of embodiment is raw to Vibrio vulnificus CZ-A2 (Vibrio vulnificus)
Long inhibition
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum supernatant:
By in the LB culture medium of bacillus amyloliquefaciens V4 strain inoculated to 100mL, 30 DEG C of cultures obtain microbial inoculum for 24 hours, will
Microbial inoculum 8000r/min is centrifuged 10 minutes, obtains microbial inoculum supernatant, which is obtained by filtration nothing with disposable syringe filter
Fermented liquid.
LB medium component: 10 mass parts of peptone, 5 mass parts of yeast extract, 10 mass parts of sodium chloride, distilled water 975
Mass parts, pH 7.2.
2. the inhibiting effect that microbial inoculum supernatant grows Vibrio vulnificus (Vibrio vulnificus) CZ-A2:
Vibrio vulnificus (Vibrio vulnificus) CZ-A2 is prepared into bacteria suspension, it is flat that 100 μ l bacteria suspensions are coated with LB solid
The aseptic filter paper piece of diameter 6mm is placed in planar surface by plate, and the bacillus amyloliquefaciens V4 without fermented liquid of 10 μ l is added dropwise in nothing
On bacterium filter paper, then culture dish is placed in 30 DEG C of incubators and is cultivated 24 hours.Visible bacillus amyloliquefaciens V4 is observed to wound
Hurt the inhibition zone of vibrios generation, inhibition zone area is 220.71 ± 6.61mm2(attached drawing 1).
Embodiment 3, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to Vibrio natriegen (Vibrio natriegens FS-1)
The inhibition 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 inhibiting effect that microbial inoculum supernatant grows Vibrio natriegen (Vibrio natriegens FS-1):
Vibrio natriegen (Vibrio natriegens) is prepared into bacteria suspension, 100 μ l bacteria suspensions are coated with LB solid plate, will
The aseptic filter paper piece of diameter 6mm is placed in planar surface, and the bacillus amyloliquefaciens V4 without fermented liquid of 10 μ l is added dropwise in sterile filter
On the scraps of paper, then culture dish is placed in 30 DEG C of incubators and is cultivated 24 hours.Visible bacillus amyloliquefaciens V4 is observed to needing sodium arc
The inhibition zone that bacterium generates, inhibition zone area are 50.24 ± 3.6mm2(attached drawing 2).
Embodiment 4, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to Aeromonas hydrophila (Aeromonas
Hydrophila) the inhibition 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 inhibiting effect that microbial inoculum supernatant grows Aeromonas hydrophila (Aeromonas hydrophila):
Aeromonas hydrophila (Aeromonas hydrophila) is prepared into bacteria suspension, 100 μ l bacteria suspensions are coated with LB solid
The aseptic filter paper piece of diameter 6mm is placed in planar surface by plate, be added dropwise the bacillus amyloliquefaciens V4 without fermented liquid of 10 μ l in
Aseptic filter paper on piece, then culture dish is placed in 30 DEG C of incubators and is cultivated 24 hours.Observe V4 pairs of visible bacillus amyloliquefaciens
The raw inhibition zone of Aeromonas hydrophila, inhibition zone area are 176.625 ± 5.18mm2(attached drawing 3).
Embodiment 5, bacillus amyloliquefaciens V4 microbial inoculum supernatant are to aeromonas salmonicida (Aeromonas
Salmonicida) the inhibition 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 inhibiting effect that microbial inoculum supernatant grows aeromonas salmonicida (Aeromonas salmonicida):
Aeromonas salmonicida (Aeromonas salmonicida) is prepared into bacteria suspension, 100 μ l bacteria suspensions are coated with LB solid
The aseptic filter paper piece of diameter 6mm is placed in planar surface by plate, be added dropwise the bacillus amyloliquefaciens V4 without fermented liquid of 10 μ l in
Aseptic filter paper on piece, then culture dish is placed in 30 DEG C of incubators and is cultivated 24 hours.Observe V4 pairs of visible bacillus amyloliquefaciens
The inhibition zone that aeromonas salmonicida generates, inhibition zone area are 240.41 ± 4.57mm2(attached drawing 4).
Embodiment 6, bacillus amyloliquefaciens V4 microbial inoculum supernatant are raw to Vibrio harveyi (Vibrio harveyi PH4)
Long inhibition
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 inhibiting effect that microbial inoculum supernatant grows Vibrio harveyi (Vibrio harveyi PH4):
Vibrio harveyi (Vibrio harveyi PH4) is prepared into bacteria suspension, 100 μ l bacteria suspensions are coated with LB solid plate,
The aseptic filter paper piece of diameter 6mm is placed in planar surface, the bacillus amyloliquefaciens V4 without fermented liquid of 10 μ l is added dropwise in sterile
On filter paper, then culture dish is placed in 30 DEG C of incubators and is cultivated 24 hours.Visible bacillus amyloliquefaciens V4 is observed to tie up Kazakhstan
The inhibition zone that family name vibrios generates, inhibition zone area are 47.78 ± 4.62mm2(attached drawing 5).
The suppression that embodiment 7, bacillus amyloliquefaciens V4 microbial inoculum supernatant grow Black Sea vibrios (Vibrio ponticus)
System
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 inhibiting effect that microbial inoculum supernatant grows Black Sea vibrios (Vibrio ponticus B8):
Black Sea vibrios (Vibrio ponticus B8) is prepared into bacteria suspension, 100 μ l bacteria suspensions are coated with LB solid plate, will
The aseptic filter paper piece of diameter 6mm is placed in planar surface, and the bacillus amyloliquefaciens V4 without fermented liquid of 10 μ l is added dropwise in sterile filter
On the scraps of paper, then culture dish is placed in 30 DEG C of incubators and is cultivated 24 hours.Visible bacillus amyloliquefaciens V4 is observed to Black Sea arc
The inhibition zone that bacterium generates, inhibition zone area are 63.59 ± 2.9mm2(attached drawing 6).
The influence of embodiment 8, addition different proportion bacillus amyloliquefaciens V4 microbial inoculum to rainbow trout growth and survival
1. the preparation of bacillus amyloliquefaciens V4 microbial inoculum
By in the LB liquid medium of ingredient described in bacillus amyloliquefaciens V4 strain inoculated to embodiment 2,30 DEG C are trained
It supports for 24 hours, gained culture solution is bacillus amyloliquefaciens V4 microbial inoculum.
2. adding different proportion bacillus amyloliquefaciens V4 preparation in Donaldson rainbow trout aquaculture pond
0.1%~0.3% ratio adds bacillus amyloliquefaciens V4 preparation into Donaldson rainbow trout aquaculture pond by volume, if
Control group is set, original body mass, latter stage weight, rate of body weight gain, feed coefficient, specific growth rate and the death rate of Donaldson rainbow trout are monitored.
After cultivation 45 days, compared with the control group, addition group can promote the weight gain of Donaldson rainbow trout, and promoting ratio is 17.08~71.22%.
Feed coefficient can be reduced simultaneously, highest rate is up to 19.49%.Meanwhile compared with the control group, addition group can reduce Donaldson rainbow trout
The death rate, ratio are 27.25~81.86% (being shown in Table 1).
Table 1
Claims (6)
1. a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) V4 is preserved in Chinese microorganism strain guarantor
Administration committee's common micro-organisms center is hidden, deposit number is CGMCC NO.10149.
2. bacillus amyloliquefaciens V4 described in claim 1 inhibits vibrio pathogen and the growth of pathogenicity Aeromonas in preparation
And/or the purposes of breeding drug.
3. purposes as claimed in claim 2, which is characterized in that the vibrio pathogen includes Vibrio vulnificus, Vibrio natriegen, Kazakhstan
Vickers vibrios and Black Sea vibrios, the pathogenicity Aeromonas includes aeromonas salmonicida.
4. a kind of bacteriostatic agent, which is characterized in that its active constituent is bacillus amyloliquefaciens described in claim 1
(Bacillus amyloliquefaciens)V4。
5. the preparation method of bacteriostatic agent as claimed in claim 4, which comprises the steps of:
By bacillus amyloliquefaciens described in claim 1 (Bacillus amyloliquefaciens) V4 access LB liquid training
It supports and is cultivated in base, condition of culture is 28~32 DEG C of temperature, 180~210rpm of hunting speed, incubation time 1~3 day, the training of acquisition
Nutrient solution, that is, the bacteriostatic agent.
6. preparation method as claimed in claim 5, which is characterized in that the LB culture medium includes following ingredient: peptone 8~
12 mass parts, 4~6 mass parts of yeast extract and/or yeast extract, 8~12 mass parts of sodium chloride, 970~980 matter of distilled water
Measure part, NaOH tune pH 7~7.5.
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| CN107937301B (en) * | 2017-11-08 | 2020-04-07 | 青岛农业大学 | Bacillus amyloliquefaciens and application thereof in aquaculture |
| US20210084905A1 (en) * | 2018-02-19 | 2021-03-25 | President And Fellows Of Harvard College | Methods and compositions for aquaculture |
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| CN111635878B (en) * | 2020-07-24 | 2022-05-10 | 宁波大学 | Bacillus amyloliquefaciens and application thereof in pomfret culture |
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| CN113005054B (en) * | 2021-02-04 | 2022-07-12 | 江苏海洋大学 | Bacillus amyloliquefaciens SS-ZC-26 and preparation method and application thereof |
| CN114716030B (en) * | 2022-04-29 | 2022-11-22 | 哈尔滨工业大学（深圳） | Spherical hydrogel and preparation method and application thereof |
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