CN104195087B - Liquefied Serratia A13 and fermentation culture method thereof and purposes - Google Patents
Liquefied Serratia A13 and fermentation culture method thereof and purposes Download PDFInfo
- Publication number
- CN104195087B CN104195087B CN201410454712.0A CN201410454712A CN104195087B CN 104195087 B CN104195087 B CN 104195087B CN 201410454712 A CN201410454712 A CN 201410454712A CN 104195087 B CN104195087 B CN 104195087B
- Authority
- CN
- China
- Prior art keywords
- culture medium
- serratia
- liquefied
- ypg
- liquefied serratia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention be a kind of liquefied Serratia (Serratia liquefaciens) A13, its preserving number is: CGMCC No.9490.The invention also discloses the fermentation culture method of liquefied Serratia A13: selection YPG or NYBD is culture medium, liquid amount is 125mL/500mL triangular flask, and culture medium initiates pH7.0, and cultivation temperature is 40 DEG C, and harvest time is 22-26h；The method of liquefied Serratia A13 or described of the present invention cultivate the bacterium solution obtained possess all grand Aeromonass of suppression (Aeromonas veronii) bacteriostatic application, may be used for the preparation of loach breeding medicine, or for adding in loach breeding feedstuff as antibacterial medicines.
Description
Technical field
The present invention relates to a kind of microorganism fungus kind, particularly a kind of liquefied Serratia A13；The invention still further relates to this bacterium
Fermentation culture method and purposes.
Background technology
Misgurni anguillicaudati (Misgurnus anguillicaudatus), with its excellent trophic component structure by the green grass or young crops of people
Look at, in recent years its aquaculture fast development.But bringing cuisines, and while bringing economic benefit to fisherman, its disease
Problem is outstanding day by day.The bacterial disease of Misgurni anguillicaudati is mainly by Aeromonas sobria, Vibrio vulnificus, Misgurni anguillicaudati Aeromonas and Fan Long gas
Zymomonas mobiliss etc. infect and cause, and this type of pathogenic bacteria is prevalent in the environment such as fresh water, sea water, mud and sewage, the most all has point
Cloth.Anti-Zhiduo County of Misgurni anguillicaudati disease uses the traditional method such as chemical synthetic drug and antibiotic at present, and this not only makes bacterial drug resistance
Increasing, cause the ecological disturbance of microorganism, produce superinfection, but also make antibiotic remain in vivo, human body is long-term
Absorption may result in chronic poisoning etc., and therefore, disease has had a strong impact on the sustainable development of culture fishery.
Antibacterial peptide, has that molecular weight is little, good water solubility, is not easy degeneration and can suppress many under the conditions of high temperature and alkalescence
Planting the features such as pathogen so that it is the substitute as antibiotic is increasingly subject to people's attention, it can be aquatic dynamic by stimulating
The immune system of thing body or the growth of suppression pathogenic bacterium improve aquatic animal immunity and premunition.Antibacterial peptide can be produced
Probiotic bacteria most from breeding water body, cultivated animals body surface or internal normal bacteria fauna, cultivated animals will not be caused
Harm, is considered as one of a kind of effective BIOLOGICAL CONTROL approach by people, therefore researchs and develops prevention, treat and contain Misgurni anguillicaudati
The microorganism formulation that bacterial disease occurs just seems particularly significant.
In the most multiple Misgurni anguillicaudati disease, the main pathogenic fungi be diagnosed as all grand Aeromonass (Aeromonas veronii), it can produce a series of virulence factor, such as gas lysin, enterotoxin and cell adhesion factor etc., participates in it right
The course of infection of aquatic animal, the mankind and poultry, by destroying host cell membrane, causing haemolysis etc. to cause host cell dead,
Cause disease.
Summary of the invention
The technical problem to be solved is for the deficiencies in the prior art, it is provided that a kind of new liquefied Serratia
A13。
Present invention also offers fermentation culture method and the purposes of aforementioned liquefied Serratieae A13.
The technical problem to be solved is to be realized by following technical scheme.The present invention is the husky thunder of liquefaction
Salmonella (Serratia liquefaciens) A13, it being characterized in, its preserving number is: CGMCC No.9490.
Liquefied Serratia of the present invention (Serratia liquefaciens) fermentation culture method of A13, it is special
Point is that its fermentation condition is: selection YPG or NYBD is culture medium, and liquid amount is 125 mL/ 500mL triangular flasks, culture medium
Initial pH 7.0, cultivation temperature is 40 DEG C, and harvest time is 22-26 h；Described culture medium YPG consists of: peptone 5 g/
L, glucose 5 g, yeast extract 5 g, distilled water 1 L；Described culture medium NYBD consists of: glucose 10 g, beef extract 8
G, yeast extract 5 g, distilled water 1 L.
The bacteria liquid has suppression that liquefied Serratia A13 of the present invention or fermentation culture method of the present invention obtain is all
Grand Aeromonas (Aeromonas veronii) purposes.
Liquefied Serratia involved in the present invention (Serratia liquefaciens) A13 is on August 8th, 2014
Being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number is CGMCC
No.9490；Depositary institution address: Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, phone: 010-
64807355。
Bacterial strain liquefied Serratia A13(hereinafter referred to as A13 to the present invention below) it is described in detail.
1.A13 the screening of bacterial strain
1.1 A13 bacterial strain screening and preservations
Ill Misgurni anguillicaudati is gathered, with aseptic water washing sick body surface and collect punching in loach breeding field, Donghai County, Lianyungang
Washing liquid.Gradient dilution collects liquid, and is coated with in LB culture medium, cultivates after 48 h for 30 DEG C, according to colonial morphology, including shape,
The features such as size, neat in edge degree and quality, selecting different single bacterium colonies and carry out line separation, repeatedly ruling repeatedly, until obtaining
Obtain pure culture.The present invention tests and is separated to 30 bacterial strains altogether, including A13.Take bacterium solution 500 μ L respectively and load 1.5 mL
In centrifuge tube, mix with the glycerol of final concentration 15% ,-40 DEG C of Refrigerator stores.
A13 antagonistic activity detects
The test strains separated point is received be coated with all grand Aeromonass (Aeromonas veronii) LB double
On layer flat board, cultivate 24 h, the size of record inhibition zone for 30 DEG C.Result shows, in 30 bacterial strains of separation, A13 is to all grand gas lists
Born of the same parents' bacterium bacteriostasis is the strongest, such as Fig. 1.
2.A13 the morphologic observation of bacterial strain
By A13 tri-ride on LB flat board, observe after 30 DEG C of constant temperature culture 24 h.Result shows, Antagonistic Fungi
A13 is creamy white, bacterium colony hemispherical, translucent, center dimpling, and smooth surface is tightly combined with culture medium, is difficult to provoke.Warp
Microexamination after Gram’s staining, A13 is gram negative bacteria, rod-short, and size is (1.0 ~ 1.2) μ m (0.5 ~ 0.8) μm,
Such as Fig. 2.
3.A13 bacterial strain Molecular Identification
3.1 A13 strain gene group DNAs extract and 16S rDNA amplification
A13 genomic DNA is extracted by bacterial genomes DNA general extraction methods, and general with bacterial 16 S rRNA gene
Primer 2 7F/1492R expands A13 16S rDNA fragment, result such as Fig. 3.
3.2 A13 bacterial strain 16s rDNA sequencings
Pcr amplification product directly serves Hai Shenggong biotechnology Services Co., Ltd, and sequencing result has been filed on DDBJ
Data base, its accession number is AB981192.
The determination of 3.3 A13 bacterial strain Phylogenetics
By the BLAST software in NCBI website, by known bacterial strain in the 16S rDNA sequence of strains A 13 and gene bank
16S rDNA sequence carries out tetraploid rice, and result shows, the 16S rDNA sequence of strains A 13 is husky with GenBank data base
The 16S rDNA sequence homology of the bacterial strains such as thunder Bordetella is the highest.20 are downloaded again from http://rdp.cme.msu.edu website
The type strain that individual tetraploid rice is high, builds A13 phylogenetic tree, result such as Fig. 4 with MEGA5.1.From fig. 4, it can be seen that
Strains A 13 and liquefied Serratia (Serratia liquefaciens ) sibship is nearest, therefore A13 is namedSerratia liquefaciens A13, is abbreviated asS. liquefaciens A13。
4.S. liquefaciens A13 fermentation condition optimization
4.1 A13 type of culture medium select
It is respectively configured 7 kinds culture medium (1) LB: peptone 10 g/L by formula as below, yeast extract 5 g/L, sodium chloride 10
G/L, distilled water 1 L, pH 7.15-20 g agar powder is added during solid.(2) KB: peptone 20 g/L, glycerol 10 mL,
K2HPO4 1.5 g/L, MgSO4 •7H2O 1.5 g/L, distilled water 1 L, pH 7.(3) CM: peptone 20 g/L, beef extract 3
G, distilled water 1 L, pH 7.(4) NA: peptone 20 g/L, beef extract 1 g, yeast extract 2 g, NaCl 5 g, distilled water 1
L, pH 7.(5) YPG: peptone 5 g/L, glucose 5 g, yeast extract 5 g, distilled water 1 L, pH 7.(6) NYBD: glucose
10 g, beef extract 8 g, yeast extract 5 g, distilled water 1 L, pH 7.(7) PPM: peptone 20 g/L, glucose 18 g,
KNO3 5 g, NaCl 1 g, distilled water 1 L, pH 7.
In 250 mL triangular flask, it is respectively charged into above 7 kinds of culture medium 100 mL, after sterilizing, accesses the A13 strain of activation,
At 160 rpm, at 30 DEG C, 24 h cultivated by shaking table.Take fermentation liquid respectively and carry out bacteriostatic activity detection, result such as Fig. 5.Permissible from Fig. 5
Find out, in YPG or NYBD culture medium top fermentationS. liquefaciens The A13 bacterium solution bacteriostatic activity to all grand Aeromonass
Relatively strong, antibacterial circle diameter respectively reaches 1.67 cm and 1.43 cm.Cost in view of YPG culture medium is relatively low, therefore after conduct
The cultivation formula of continuous experiment.
4.2 culture medium initiate the pH impact on A13 bacteriostatic activity
The LB fluid medium preparing 500 mL is sub-packed in the triangular flask of 5 bottle of 500 mL, with 1 mol/L hydrochloric acid and 1
Mol/L NaOH regulation medium pH is respectively 5.0,6.0,7.0,8.0,9.0；High temperature sterilize；Bacterium solution by A13 incubated overnight
It is inoculated in 5 bottle of 100 mL fluid medium with the inoculum concentration of 1 %, cultivates the bacterium solution after 24 h and be used for doing bacteriostatic experiment, pass through
Observe its fungistatic effect and measure inhibition zone size,
Result is shown in Fig. 6.From figure, culture medium initiates pH from 5-9, can cultivate and obtain highly active A13 fermentation liquid,
When wherein culture medium initiates pH 7, bacteriostatic activity is maximum, and antibacterial circle diameter reaches 1.67 cm, it is thus determined that pH 7 is optimum pH.
The impact on A13 bacteriostatic activity of 4.3 cultivation temperature
The YPG culture medium of preparation pH 7.0, after inoculation A13 respectively at 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C and 40 DEG C
Cultivate, after cultivating 24 h, measure bacteriostatic activity.Result shows, the A13 fermentation liquid fungistatic effect cultivated under the conditions of 40 DEG C is
Good, see that Fig. 7, antibacterial circle diameter reach 1.9 cm.As can be seen here, the suitableeest cultivation temperature of A13 generation active substance with 40 DEG C is
Preferably.
The impact on A13 bacteriostatic activity of 4.4 liquid amounts
Preparation YPG culture medium, fills 50 mL, 75 mL, 100 mL, 125 mL, 150 mL in 500 mL triangular flasks respectively,
PH value is 7.0, high temperature sterilize；The A13 bacterium solution of incubated overnight is inoculated in 5 bottles of YPG culture medium respectively with the inoculum concentration of 1 %,
40 DEG C, 160 rpm/min cultivate 24 h, and culture fluid is used for doing bacteriostatic experiment, result such as Fig. 8.Visible, sample-loading amount 50 mL ~
During 150 mL, all can obtain the fermentation liquid that bacteriostatic activity is stronger.But with 500mL triangle bottled culture medium 125 mL best results,
Antibacterial circle diameter reaches 1.67 cm.
Considering factors above, optimal conditions of fermentation is: select YPG liquid amount 125 mL/ 500mL triangular flask, cultivates
Base initiates pH 7.0, and cultivation temperature is 40 DEG C.
S. liquefaciens A13 strain growth curve
At optimum culture condition bottom fermentation A13, in 36 h, every 2 h sample once；Measure the taken sample of different time respectively
Absorbance value A600 under wavelength 600 nm, with growth time as abscissa, A600 is vertical coordinate, draws growth curve, knot
Fruit is such as Fig. 9.As can be seen from Figure 9,0 h ~ 2 h is lag phase, and 2 h ~ 8 h are logarithmic (log) phase, enters stable phase, be always maintained at after 8 h
Being slightly decreased after 30 h, decline phase performance is inconspicuous.
A13 growth curve combines and measures protein content, bacteriostatic activity in growth course, confirms when most preferably gathering in the crops of A13
Between be 22 26 h.
Owing to Misgurni anguillicaudati is when morbidity, it will usually induce corresponding microbe groups and breed rapidly to resist disease and spread.Institute
The Antagonistic Fungi that all grand Aeromonass can be suppressed to grow has been isolated from ill Misgurni anguillicaudati body surface, by optimizing fermentation bar with the present invention
Part, establishes fermentation technology flow process.The research of A13 of the present invention can be that the microorganism formulation of exploitation prevention Misgurni anguillicaudati bacterial disease carries
For theoretical foundation and Applied Materials.
Compared with prior art, the invention provides a kind of new liquefied Serratia (Serratia liquefaciens) A13, this bacterial strain or the bacteria liquid has obtained through the inventive method cultivation suppress all grand Aeromonass
(Aeromonas veronii) purposes, may be used for the preparation of loach breeding medicine, or for adding as antibacterial medicines
In loach breeding feedstuff.
Accompanying drawing explanation
Fig. 1 is that strains A 13 is to all grand Aeromonas bacteriostasis figures；
Fig. 2 is strains A 13 Gram’s staining result figure；
Fig. 3 is A13 bacterial strain STb gene and 16S rDNA amplified production figure；
Fig. 4 is the phylogenetic tree of strains A 13；
What Fig. 5 was different culture media on A13 strain fermentation culture fluid activity affects figure；
Fig. 6 is that medium pH affects figure, wherein to A13 bacterial strain bacteriostatic activity: ck-aseptic culture medium, numeral is pH value；
Fig. 7 is that culture medium temperature affects figure, wherein to A13 bacterial strain bacteriostatic activity: ck-aseptic culture medium, and numeral is temperature
Degree (DEG C)；
Fig. 8 is that culture medium liquid amount affects figure, wherein to A13 bacterial strain bacteriostatic activity: ck-aseptic culture medium, numeral is
Culture volume (500mL triangular flask)；
Fig. 9 is A13 strain growth curve chart under optimal conditions.
Detailed description of the invention
The concrete technical scheme of the present invention described further below, in order to those skilled in the art is further understood that
The present invention, and do not constitute the restriction to its right.
Embodiment 1, a kind of liquefied Serratia (Serratia liquefaciens) A13, its preserving number is: CGMCC
No.9490。
Embodiment 2, the fermentation culture method of a kind of liquefied Serratia A13 as described in Example 1, its fermentation condition
For: selection YPG or NYBD is culture medium, and liquid amount is 125 mL/ 500mL triangular flasks, and culture medium initiates pH 7.0, cultivates
Temperature is 40 DEG C, and harvest time is 22-26 h；Described culture medium YPG consists of: peptone 5 g/L, glucose 5 g, ferment
Female cream 5 g, distilled water 1 L；Described culture medium NYBD consists of: glucose 10 g, beef extract 8 g, yeast extract 5 g, steams
Distilled water 1 L.
Liquefied Serratia A13 described in embodiment 1 or the method described in embodiment 2 are cultivated the tool of the bacterium solution obtained and are used
Bacteriostasis, can be used to suppress all grand Aeromonass (Aeromonas veronii), may be used for the system of loach breeding medicine
Standby, or for adding in loach breeding feedstuff as antibacterial medicines.
Claims (3)
1. liquefied Serratia (Serratia liquefaciens) A13, it is characterised in that its preserving number is: CGMCC
No.9490。
2. the fermentation culture method of a liquefied Serratia A13 as claimed in claim 1, it is characterised in that its bar that ferments
Part is: selection YPG or NYBD is culture medium, and liquid amount is 125mL/500mL triangular flask, and culture medium initiates pH 7.0, cultivates
Temperature is 40 DEG C, and harvest time is 22-26h；Described culture medium YPG consists of: peptone 5g/L, glucose 5g, yeast extract
5g, distilled water 1L；Described culture medium NYBD consists of: glucose 10g, beef extract 8g, yeast extract 5g, distilled water 1L.
3. the bacterium solution that the liquefied Serratia A13 described in claim 1 or the method fermentation culture described in claim 2 obtain
Purposes in preparing antibacterial medicines, the bacterium pressed down is all grand Aeromonass (Aeromonas veronii).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410454712.0A CN104195087B (en) | 2014-09-09 | 2014-09-09 | Liquefied Serratia A13 and fermentation culture method thereof and purposes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410454712.0A CN104195087B (en) | 2014-09-09 | 2014-09-09 | Liquefied Serratia A13 and fermentation culture method thereof and purposes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104195087A CN104195087A (en) | 2014-12-10 |
| CN104195087B true CN104195087B (en) | 2016-09-14 |
Family
ID=52080457
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410454712.0A Active CN104195087B (en) | 2014-09-09 | 2014-09-09 | Liquefied Serratia A13 and fermentation culture method thereof and purposes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104195087B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255778A (en) * | 2015-11-12 | 2016-01-20 | 淮海工学院 | Serratia grimesii C13 and application thereof |
| CN105861384B (en) * | 2016-05-20 | 2019-06-11 | 南京林业大学 | A kind of efficient corrosion bacterium liquefied Serratia NLX-15 of silicate rock and its application |
| CN109548764B (en) * | 2018-12-06 | 2022-05-06 | 辰溪县凤凰山生态农业开发有限公司 | High-efficient loach excavates and treatment facility |
-
2014
- 2014-09-09 CN CN201410454712.0A patent/CN104195087B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104195087A (en) | 2014-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105238715B (en) | Bacillus DY26-004 and its prevention phytopathogenic fungi application | |
| CN104031960B (en) | Extracting method and application of extracellular antimicrobial lipopeptides of bacillus amyloliquefaciens | |
| CN107090419A (en) | Bacillus amyloliquefaciens and its application | |
| CN107267412A (en) | Methylotrophic bacillus and its application | |
| CN107974421A (en) | A kind of lactobacillus acidophilus and its screening technique and application, a kind of microbial inoculum | |
| CN108192829A (en) | A kind of gamboge streptomyces verticillus and its biological control agent and application | |
| CN104195087B (en) | Liquefied Serratia A13 and fermentation culture method thereof and purposes | |
| CN105420164B (en) | South Pole source bacillus N311 and its application on prevention and treatment phytopathogenic fungi | |
| CN105132339B (en) | One plant of broad-spectrum antifungal bacillus and its application in preventing wheat scab | |
| CN105219653B (en) | Produce Aspergillusclavatus (Aspergillus clavatus) Ac-32 bacterial strains and its application of Lovastatin | |
| CN112608860B (en) | Bacillus amyloliquefaciens YB130 and application thereof | |
| CN104726359B (en) | One bacillus pumilus BP and application thereof | |
| CN104845897A (en) | Bacillus subtilis, and breeding method and application thereof | |
| CN105132338B (en) | Bacillus DY26-020 and its application in preparing prevention phytopathogenic fungi fermented supernatant fluid | |
| CN105838658B (en) | A method of improving lactic acid bacteria biomass under high salt conditions | |
| CN104371957A (en) | Bacillus pumilus viable preparation and solid fermentation method and applications thereof | |
| CN103205383B (en) | Bacillus pumilus E14, culturing method thereof and application | |
| CN106544280B (en) | A kind of resistance meat duck yeast for animal feeds bacterial strain and application | |
| CN102424802B (en) | Bacillus pumilus, strain culture method, and application thereof | |
| CN103710269A (en) | Coniothyrium minitans strain JN-CM for preventing and controlling stalk break | |
| CN104974957B (en) | Bacillus（Bacillus sp.）ZY bacterial strains and its application | |
| CN106701867B (en) | Method for improving output of nikkomycin | |
| CN104450548A (en) | Paenibacillus polymyxa XD6 strain for preventing and controlling phytophthora capsici and biocontrol agent of paenibacillus polymyxa XD6 strain | |
| CN103589660A (en) | Endophytic bacterium capable of producing triptolide | |
| CN106754437A (en) | A kind of high activity meat duck tailored version yeast for animal feeds bacterium and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |