CN109576174A - Bacillus subtilis Bacillus subtilis CS30 and its application - Google Patents
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Abstract
The invention belongs to functional microorganism screenings, applied technical field, and in particular to a kind of bacillus subtilis Bacillus subtilis CS30 and its application.Generate the bacillus subtilis Bacillus subtilis CS30 of lipopeptid, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 (Institute of Microorganism, Academia Sinica), the deposit date is on October 31st, 2018, deposit numbers: CGMCC NO.16666.Bacillus subtilis Bacillus subtilis CS30 is located away from the cold spring deposit of deep-sea in the present invention, this plant of bacterium has the characteristics that inhibit plant pathogenic fungi rice blast fungus and growth of tumour cell.
Description
Technical field
The invention belongs to field of biotechnology, a kind of bacillus subtilis Bacillus subtilis CS30 (ocean bud
Spore bacillus) application.
Background technique
Currently, being directed to the microbial agricultural disease of pathogenic, common control method is largely to use chemical pesticide.With
The excessive use of chemical pesticide, the bacterial strain of drug resistance pathogenic bacteria also constantly emerge in large numbers.In addition, the excessive of chemical pesticide uses
A series of problem is easily caused, the problem of environmental pollution as caused by pesticide residue and Food Security etc. have caused
The strong interest of people.Chemical pesticide not only has toxicity to target organism, can also constantly be delivered in difference with food chain
It is enriched in organism, and certain products also have the toxic effects such as certain carcinogenic, teratogenesis and mutagenesis to the mankind
[1].Biological pesticide as a kind of novel environment friendly agricultural, have control efficiency it is good, it is degradable, be not likely to produce drug resistance, toxicity
The advantages that small and the concern [2] for gradually causing people.
In recent years, it was concerned by the lipopeptid that microorganism generates as emerging biological pesticide.Lipopeptid is mainly by fat
Sour chain and amino acid composition, since it is with amphipathic structure, can be done directly on the lipid bilayer of cell membrane and lead to cell membrane
Rupture, so as to cause the death [3] of cell.Compared with traditional fungicide, lipopeptid is not easy to cause the appearance of drug-fast strain,
Degradable, small toxicity, and it is stronger to the tolerance of soda acid, temperature, it is the excellent selection for developing environmentally friendly new pesticide.Rouge
Peptide occupies sizable ratio [4] in the microbial bacterial agent for having obtained registration and business application.
Bacillus is the major microorganisms for generating lipopeptid, and generated lipopeptid is broadly divided into three classes according to structure:
3 surfactin class, iturin class and fengycin class major families, related derivatives reach more than 100 kinds.Additionally include
The antibacterial peptides such as mycosubtilin, lichenysin and pumilacidin.The antibiotic of lipopeptid class not only has the anti-of spectrum
Bacterium activity, to gram-positive bacteria, Gram-negative bacteria, fungi have a good inhibiting effect, also in antiviral, anti-inflammatory and
Anti-tumor aspect all has good inhibiting effect [5].Nevertheless, its bioactivity difference of different lipopeptids is very big.Due to big
Most lipopeptids are all to be synthesized by non-ribosomal system, therefore they are in the shape of fatty chain length, the composition of amino acid and ester ring
Biggish difference is had at position etc., to have different bioactivity.
The features such as marine environment is unique, with high salt, high pressure, low temperature and low illumination imparts marine microorganism and generates novel life
A possibility that active substances are more.The novel bioactive substance separated from marine microorganism is not only in antimycotic and bacterium
Aspect has stronger effect, it is anti-stick, the formation of antibiont film, anti-tumor aspect also have extraordinary potential application foreground
[6].Therefore, marine microorganism is the important source for screening novel biological agent.
Bibliography:
[1]Kavlock R J,Daston G P,Derosa C,et al.Research needs for the risk
assessment of health and environmental effects of endocrine disruptors:a
report of the U.S.EPA-sponsored workshop[J].Environmental Health
Perspectives,1996,104Suppl 4(Suppl 4):715.
[2]Cochrane SA,Vederas JC.2016.Lipopeptides from Bacillus and
Paenibacillus spp.:a gold mine of antibiotic candidates.Med Res Rev 36:4-31.
[3]Ines M,Dhouha G.2015.Lipopeptide surfactants:Production, recovery
and pore forming capacity.Peptides 71:100-112.
[4]Ongena M,Jacques P.Bacillus lipopeptides:versatile weapons for
plant disease biocontrol[J].Trends in Microbiology,2008, 16(3):115-125.
[5]Zhao HB,Shao DY,Jiang CM,Shi JL,Li Q,Huang QS,Rajoka MSR,Yang H,
Jin ML.2017.Biological activity of lipopeptides from Bacillus.Appl Microbiol
Biot 101:5951-5960.
[6]Gudina EJ,Teixeira JA,Rodrigues LR.2016.Biosurfactants produced by
marine microorganisms with therapeutic applications. Mar Drugs 14,38.
Summary of the invention
It is an object of that present invention to provide a kind of bacillus subtilis Bacillus subtilis CS30 (ocean gemma bars
Bacterium) and its application.
To achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of bacillus marinus, bacillus subtilis Bacillus subtilis CS30, the bacterial strain are preserved in China
Microbiological Culture Collection administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 (China
Institute of microbiology, the academy of sciences), the deposit date is on October 31st, 2018, deposit numbers: CGMCC NO.16666.
The bacillus subtilis is that Bacillus subtilis CS30 is located away from the cold spring deposit of deep-sea, described withered
Careless bacillus subtilis CS30 strain isolation is in deep-sea cold spring deposit, at 28 DEG C, LB solid culture
(peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, distilled water 1L, agar 20g, pH 7.0) is cultivated 2 days on base
Grow bacterium colony.
A kind of application of bacillus marinus, the bacillus subtilis Bacillus subtilis CS30 prevention and treatment by
Application in agricultural disease caused by the phytopathogens such as rice blast fungus.
A kind of application of bacillus marinus, the bacillus subtilis Bacillus subtilis CS30 prevention and
Treat the application in malignant tumour.
The bacillus subtilis Bacillus subtilis CS30 vegetative cell or whole cultures.
The bacillus subtilis Bacillus subtilis CS30 be the culture concentrate of bacterium, bacterial suspension,
Fermentation liquid or fermented supernatant fluid.
A kind of application of bacillus marinus, the bacillus subtilis Bacillus subtilis CS30 are preparing rouge
Application in peptide surfactin A1 or surfactin A3.
Further, above-mentioned bacillus subtilis Bacillus subtilis CS30 is fermented training in the medium
It supports, fermented supernatant fluid is collected by centrifugation after fermentation, supernatant is acidified, is freeze-dried, and methanol extracting and rotatory vacuum evaporate
Crude extract；Crude extract is further passed through to silica gel column chromatography and high performance liquid chromatography separation and purifying, active material is after purification
Obtaining is surfactin A1 and surfactin A3.
Lipopeptid surfactin A1 prepared by the bacillus subtilis Bacillus subtilis CS30 and
Surfactin A3 is inhibiting the application in plant pathogenic fungi；
Lipopeptid surfactin A3 prepared by the bacillus subtilis Bacillus subtilis CS30 is inhibiting
Application in growth of tumour cell.
A kind of preparation method of surfactin exists the bacillus subtilis Bacillus subtilis CS30
Fermented supernatant fluid is collected by centrifugation in fermented and cultured in fermentation medium after fermentation, supernatant is acidified, is freeze-dried, methanol extracting
And rotatory vacuum evaporates to obtain crude extract；Crude extract is further passed through to silica gel column chromatography and high performance liquid chromatography separation and pure
Change, active material is surfactin A1 and surfactin A3 to obtain the final product after purification.
Wherein, fermented and cultured are as follows: single colonie is inoculated in fluid nutrient medium (peptone 10g/L, yeast powder 5g/L, chlorine
Change sodium 10g/L, distilled water 1L, pH 7.0), then at 28 DEG C, 160 rpm shaker fermentation 48h.
Described isolate and purify is HPLC using 1260 series of Agilent, and mobile phase is methanol/water, using gradient elution, from
80% methanol to 100% methanol, elution time 45min are the elution of 100% methanol later, and chromatographic column is that C18 prepares column,
Flow velocity is 2mL/min, and the appearance time of Detection wavelength 230nm, surfactin and its derivative is 40-45min.Active group
Surfactin A1 is divided to be eluted out in 41.2min, surfactin A3 is eluted out in 42.7min
By the stronger component of activity in above-mentioned acquisition surfactin and its derivative by mass spectrum and mass spectrometric hyphenated technique into
Row structural analysis, show bacillus subtilis Bacillus subtilis CS30 generate lipopeptide compound in be
Surfactin and its derivative, wherein active stronger derivative is surfactin A1 and surfactin A3.
Advantage for present invention:
Bacillus subtilis Bacillus subtilis CS30 separates China deep-sea cold spring deposit in the present invention, belongs to
Marine microorganism, the lipopeptid which generates have the activity for inhibiting Pyricularia oryzae and tumour cell, pass through analysis, bacillus subtilis
Lipopeptid surfactin A1 in bacterium Bacillus subtilis CS30 has the work of strong inhibition plant pathogenic fungi
With surfactinA3 can not only inhibit the growth of plant pathogenic fungi, moreover it is possible to inhibit the growth of tumour cell.The bacillus
And its lipopeptid generated is in exploitation broad-spectrum antifungal class biological pesticide, improvement grain and food safety and anticancer drug and anticancer health-care
Product etc. have potential using value.
Detailed description of the invention:
Fig. 1 is that the bacillus subtilis Bacillus subtilis CS30 fermentation liquid that present invention implementation provides inhibits rice blast
Germ activity figure.
Fig. 2 is the active material for the bacillus subtilis Bacillus subtilis CS30 that present invention implementation provides
Separation and appearance time of surfactin A1 and the surfactin A3 in high performance liquid chromatograph device (HPLC).
Fig. 3 is the active material for the bacillus subtilis Bacillus subtilis CS30 that present invention implementation provides
The mass spectroscopy map of surfactin A1 and surfactin A3；Wherein, Fig. 3 A is Surfactin A1 mass spectrogram, Fig. 3 B
Surfactin A3 mass spectrogram.
Fig. 4 is the active material for the bacillus subtilis Bacillus subtilis CS30 that present invention implementation provides
The sequence chart of surfactin A1 and surfactin A3；Wherein, Fig. 4 A is Surfactin A1 sequence chart, and Fig. 4 B is
Surfactin A3 sequence chart.
Fig. 5 is that the bacillus subtilis Bacillus subtilis CS30 that present invention implementation provides is generated
The variation of the form and ultra microstructure of plant pathogenic fungi Pyricularia oryzae caused by surfactin A1 and surfactin A3.
Fig. 6 is that the bacillus subtilis Bacillus subtilis CS30 that present invention implementation provides is generated
The variation of the form and ultra microstructure of tumour cell caused by surfactin A3.
Specific embodiment
The invention will be further elaborated in following experimental example, however, the present invention is not limited thereto.
Bacillus subtilis Bacillus subtilis CS30 is located away from the cold spring deposit of deep-sea in the present invention, the strain
Bacterium has the characteristics that inhibit plant pathogenic fungi rice blast fungus and growth of tumour cell.Pass through analysis, bacillus subtilis
Lipopeptid surfactin A1 in bacterium Bacillus subtilis CS30 has the work of strong inhibition plant pathogenic fungi
With surfactin A3 can not only inhibit the growth of plant pathogenic fungi, moreover it is possible to inhibit the growth of tumour cell.Utilize the present invention
The bacillus subtilis Bacillus subtilis CS30 of acquisition and its lipopeptid of generation can be used for developing the micro- life of novel sea
Object preparation cannot be only used for the microbial agricultural diseases of plant pathogenics such as prevention and treatment rice blast fungus, also have one to malignant tumour
Fixed preventive and therapeutic action.
Embodiment 1:
Bacillus subtilis Bacillus subtilis CS30 is located away from the cold spring deposit of China deep-sea, marine bacteria
The 16S rDNA sequence of bacillus subtilis Bacillus subtilis CS30 is referring to SEQ ID NO.1.The bacterial strain is solid in LB
After body culture basal growth 2day, milky is presented in bacterium colony, and irregular shape is presented in colony edge, and many irregular pleats occurs in surface
Wrinkle.
16S rDNA (SEQ ID NO.1) sequence of the bacillus subtilis Bacillus subtilis CS30 are as follows:
CCTTCGGCGGCTGGCTCCTAAAGGTTACCTCACCGACTTCGGGTGTTAAA ACTCTCGTGGTGTGACG
GGCGGTGTGTACAAGGCCCGGGAACGTATTCAC CGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCAC
GCAGTCGA GTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAAC CTCGCGGTTTCGCTGC
CCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCC CAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCT
TCCTCCGGT TTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAG ATCAAGGGTTGCGCT
CGTTGCGGGACTTAACCCAACATCTCACGACACGA GCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGA
AGGGGACGTC CTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCG TTGCTTCGAATTAA
ACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAA TTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGC
GGAGTGCTTAA TGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTC ATCGTTTACGGCG
TGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCAC GCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTC
GCCTTCGCCACT GGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCAC TCTCCTCTTCTG
CACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTT GAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCC
TGCGAGCCCTTTA CGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGC TGGCACGTAGT
TAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGC CCTATTCGAACGGTACTTGTTCTTCCCTAACAACAG
AGCTTTACGATCCG AAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTG CGGAAGATTC
CCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAG TCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTA
CGCATCGTCGCCTTG GTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAA GTGGTAGCC
GAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACC ATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCC
CAGTCTTACAGGCAGG TTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTC
CCATCTGTCCGCTCGACTTGC
Above-mentioned production lipopeptid marine bacteria is bacillus subtilis Bacillus subtilis CS30, during which is preserved in
State's Microbiological Culture Collection administration committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 (in
Institute of microbiology, the academy of sciences, state), the deposit date is on October 31st, 2018, deposit numbers: CGMCC NO.16666.
Inhibition of the 2 bacillus subtilis Bacillus subtilis CS30 of embodiment to Pyricularia oryzae and Gibberella zeae bacterium
Effect.
Pyricularia oryzae is inoculated on potato glucose solid medium (PDA) and cultivates 2-5 days under the conditions of 28 DEG C, so
Afterwards by fermentation liquid obtained by the subtilis bacillus subtilis CS30 fermented and cultured through filter membrane mistake
On the small filter paper of sterilizing, filter paper is placed on after air-drying from Pyricularia oryzae fungus block 2cm drop after filter.It is cultivated under the conditions of 28 DEG C
3-5 days.After culture, observes and subtilis bacillus subtilis CS30 fermentation is being added dropwise
On the culture medium of liquid side, the growth of Pyricularia oryzae receives apparent inhibition, without bacillus subtilis is added dropwise
The side Growth of Magnaporthe Grisea of Bacillus subtilis CS30 fermentation liquid is good, is not suppressed (Fig. 1).Experimental result
Illustrate that marine bacteria bacillus subtilis Bacillus subtilis CS30 has apparent inhibiting effect to Pyricularia oryzae.
Fermentation, separation and the mirror of 3 the produced active material of bacillus subtilis Bacillus subtilis CS30 of embodiment
It is fixed.
(peptone 10g/L, yeast powder 5g/L, chlorination in 28 DEG C, 160rpm fluid nutrient medium by bacterial strain in embodiment 1
Sodium 10g/L, distilled water 1L, pH 7.0) fermented and cultured 48h, after fermentation, 8000rpm is centrifuged 15min, collects fermentation supernatant
PH is adjusted to 2.0 with hydrochloric acid and then stayed overnight at 4 DEG C, collected acid precipitation product, extracted, will be obtained with methanol by liquid, supernatant
Extract product carry out rotatory vacuum evaporation process, i.e., acquisition bacillus subtilis Bacillus subtilis CS30 active matter
The crude extract of matter.
Crude extract is further passed through to silica gel column chromatography and high performance liquid chromatography chromatography (HPLC, UV 210nm) has carried out point
From and purifying.It being separated by HPLC and obtains active material fraction, for HPLC using 1260 series of Agilent, mobile phase is methanol/water,
Using gradient elution, from 80% methanol to 100% methanol, elution time 45min is the elution of 100% methanol, chromatography later
Column is that C18 prepares column, flow velocity 2mL/min, Detection wavelength 230nm；According at 230nm wavelength detecting and it is antibacterial, suppression is swollen
Tumor activity testing result, active component surfactin A1 are eluted out in 41.2min, and surfactin A3 is in 42.7min quilt
Elute (Fig. 2).By the above-mentioned active material obtained from HPLC through mass spectroscopy, the molecular weight of surfactin A1 is
The molecular weight of 1022.71, surfactin A3 is 1036.72 (Fig. 3).It is concatenated mass spectroscopy and sequence analysis,
The sequence of surfactin A1 are as follows: β-OH fatty acid-acid-Pro-Leu/Ile-Leu/Ile-Val-Leu/Ile-
The sequence of Asp-Leu/Ile, surfactin A3 are as follows: β-OH fatty acid-Asp-Leu/Ile-Leu/Ile-Val-Glu-
Val-Leu/Ile (Fig. 4).
Embodiment 4: bacillus subtilis Bacillus subtilis CS30 active component is to Pyricularia oryzae form and surpasses
The influence of micro-structure.
Influence measurement of the active component to Pyricularia oryzae: Pyricularia oryzae is seeded in fresh PDA culture medium, 28 DEG C of trainings
After supporting 2-3 days, respectively by surfactin A1 and surfactin the A3 drop of 1ug after purification on the small filter paper of sterilizing, to
It is placed on after filter paper is dry from Pyricularia oryzae mycelia edge 2cm, continuation is cultivated at 28 DEG C.After culture 2-3 days, cut close to filter
1h is fixed with 2.5% glutaraldehyde in the mycelium of sheet edge, it is fixed after successively with various concentration (30%, 50%,
70%, 80%, 90% and alcohol 100%) sample is carried out dehydrating, each concentration handles 10min, by sample according to
Usual manner is prepared into scanning electron microscope example or transmission electron microscope sample respectively, is then accordingly observed.
The above-mentioned sample prepared is observed, observes figure (Fig. 5 A) and transmission electricity referring to scanning electron microscope
The micro- sem observation figure (Fig. 5 B) of son.It is from Fig. 5 A-a as can be seen that raw without the Pyricularia oryzae mycelia by surfactin processing
Long normal, organelle is complete, and cell wall and cell membrane growth are complete, by the processed rice blast of active component surfactin A1
Germ mycelia occurs significantly expanding (5A-b), also occurs by the mycelia of the surfactin A3 Pyricularia oryzae handled similar existing
As (5A-c).It is under transmission electron microscope as can be seen that raw without the Pyricularia oryzae mycelia by active component surfactin processing
Long normal, cellular content is abundant complete (5B-a).And pass through in the processed Pyricularia oryzae hyphal cell of surfactinA1
Tolerant seriously to leak, organelle obviously lacks, and so as to cause cell death (5B-b), and treated by surfactin A3
Also there is similar phenomenon (5B-c) in the mycelia of Pyricularia oryzae bacterium.
Embodiment 5: influence of the active component surfactin A3 to tumour cell form and ultra microstructure.
Influence measurement of the active component surfactin A3 to tumour cell: liver cancer cells Huh7.5 is transferred in 6 orifice plates
In, 37 DEG C are cultivated 24 hours, are then respectively adding the surfactin A3 of 150ug/ml and 300ug/ml, are continued to cultivate.Culture
After 24 hours, cell is collected, is washed 3 times with the PBS of 10mM, 1h is fixed with 2.5% glutaraldehyde, is successively used after fixed
Sample is carried out dehydrating by 30%, 50%, 70%, 80%, 90% and 100% alcohol of various concentration, each concentration processing
Sample is prepared into scanning electron microscope example or transmission electron microscope sample by 10 min respectively in a conventional manner, is then carried out corresponding
Observation.
Before tumour cell is without surfactin A3 processing, adherent growth, and surrounding has a large amount of filiform and surrounding ring
Border is connected (Fig. 6 A-a, d), and after surfactin A3 processing, the filiform being connected with ambient enviroment reduces (Fig. 6 A-b, e), companion
With increasing for concentration for the treatment of, filiform is completely disappeared, and tumour cell finally becomes spherical (Fig. 6 A-c, f).It is seen under transmission electron microscope
It examines and shows that the tumor cell film without surfactin A3 processing is complete, cytoplasm is evenly distributed (Fig. 6 B-a, d), and
After surfactin A3 processing, membranolysis, cytoplasm is revealed serious (Fig. 6 B-b, e), after higher concentration processing, cell
Cracking completely, is barely perceivable the presence (Fig. 6 B-c, f) of cell membrane.
Sequence table
<110>University Of Qingdao
The Institute of Oceanology of the Chinese Academy of Sciences
<120>bacillus marinus Bacillus subtilis CS30 and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1421
<212> DNA
<213>bacillus marinus (Bacillus subtilis CS30)
<400> 1
ccttcggcgg ctggctccta aaggttacct caccgacttc gggtgttaaa actctcgtgg 60
tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc tgatccgcga 120
ttactagcga ttccagcttc acgcagtcga gttgcagact gcgatccgaa ctgagaacag 180
atttgtggga ttggcttaac ctcgcggttt cgctgccctt tgttctgtcc attgtagcac 240
gtgtgtagcc caggtcataa ggggcatgat gatttgacgt catccccacc ttcctccggt 300
ttgtcaccgg cagtcacctt agagtgccca actgaatgct ggcaactaag atcaagggtt 360
gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 420
acctgtcact ctgcccccga aggggacgtc ctatctctag gattgtcaga ggatgtcaag 480
acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 540
cccccgtcaa ttcctttgag tttcagtctt gcgaccgtac tccccaggcg gagtgcttaa 600
tgcgttagct gcagcactaa ggggcggaaa ccccctaaca cttagcactc atcgtttacg 660
gcgtggacta ccagggtatc taatcctgtt cgctccccac gctttcgctc ctcagcgtca 720
gttacagacc agagagtcgc cttcgccact ggtgttcctc cacatctcta cgcatttcac 780
cgctacacgt ggaattccac tctcctcttc tgcactcaag ttccccagtt tccaatgacc 840
ctccccggtt gagccggggg ctttcacatc agacttaaga aaccgcctgc gagcccttta 900
cgcccaataa ttccggacaa cgcttgccac ctacgtatta ccgcggctgc tggcacgtag 960
ttagccgtgg ctttctggtt aggtaccgtc aaggtaccgc cctattcgaa cggtacttgt 1020
tcttccctaa caacagagct ttacgatccg aaaaccttca tcactcacgc ggcgttgctc 1080
cgtcagactt tcgtccattg cggaagattc cctactgctg cctcccgtag gagtctgggc 1140
cgtgtctcag tcccagtgtg gccgatcacc ctctcaggtc ggctacgcat cgtcgccttg 1200
gtgagccgtt acctcaccaa ctagctaatg cgccgcgggt ccatctgtaa gtggtagccg 1260
aagccacctt ttatgtttga accatgcggt tcaaacaacc atccggtatt agccccggtt 1320
tcccggagtt atcccagtct tacaggcagg ttacccacgt gttactcacc cgtccgccgc 1380
taacatcagg gagcaagctc ccatctgtcc gctcgacttg c 1421
Claims (9)
1. a kind of bacillus marinus, it is characterised in that: bacillus marinus is bacillus subtilis Bacillus subtilis
CS30, the bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: Chaoyang District, Beijing City
The institute 3 of North Star West Road 1 (Institute of Microorganism, Academia Sinica), the deposit date is on October 31st, 2018, deposit number:
CGMCC NO.16666。
2. a kind of application of bacillus marinus described in claim 1, it is characterised in that: the bacillus subtilis
Bacillus subtilis CS30 answering in the agricultural disease as caused by the phytopathogens such as rice blast fungus in prevention and treatment
With.
3. a kind of application of bacillus marinus described in claim 1, it is characterised in that: the bacillus subtilis
Bacillus subtilis CS30 is preventing and treating the application in malignant tumour.
4. by the application of bacillus marinus described in claim 1 described in claim 2 and 3, it is characterised in that: described withered
Careless bacillus subtilis CS30 is vegetative cell or whole cultures.
5. the application of bacillus marinus according to claim 4, it is characterised in that: the bacillus subtilis Bacillus
Subtilis CS30 is culture concentrate, bacterial suspension, fermentation liquid or the fermented supernatant fluid of bacterium.
6. a kind of application of bacillus marinus described in claim 1, it is characterised in that: the bacillus subtilis
Bacillus subtilis CS30 is preparing the application in lipopeptid surfactin A1 or surfactin A3.
7. the application of bacillus marinus according to claim 6, it is characterised in that: by above-mentioned bacillus subtilis
Bacillus subtilis CS30 fermented and cultured in the medium, is collected by centrifugation fermented supernatant fluid, supernatant is through acid after fermentation
Change, freeze-drying, methanol extracting and rotatory vacuum evaporate to obtain crude extract；Crude extract is further passed through into silica gel column chromatography and height
Effect liquid phase chromatogram separation and purifying, active material is surfactin A1 and surfactin A3 to obtain the final product after purification.
8. by the application of bacillus marinus described in claim 6 or 7, it is characterised in that: the bacillus subtilis
Lipopeptid surfactin A1 and surfactin A3 prepared by Bacillus subtilis CS30 is inhibiting pathogenic true
Application in bacterium；
Lipopeptid surfactin A3 prepared by the bacillus subtilis Bacillus subtilis CS30 is inhibiting tumour
Application in cell growth.
9. a kind of preparation method of surfactin, it is characterised in that: by bacillus subtilis described in claim 1
Bacillus subtilis CS30 fermented and cultured in the fermentation medium, is collected by centrifugation fermented supernatant fluid, supernatant after fermentation
Acidified, freeze-drying, methanol extracting and rotatory vacuum evaporate to obtain crude extract；Crude extract is further passed through into silica gel column chromatography
With high performance liquid chromatography separation and purifying, active material is surfactin A1 and surfactin A3 to obtain the final product after purification.
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| CN110295132A (en) * | 2019-08-01 | 2019-10-01 | 哈尔滨天齐人类第二基因组技术开发应用科技有限责任公司 | One plant has the active bacillus CCPM7645 of powerful anticancer and its application |
| CN110607265A (en) * | 2019-09-29 | 2019-12-24 | 中南民族大学 | Bacillus Subtilis CZ162 strain and application thereof |
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