CN101974456A - Marine streptomyces strain and application thereof - Google Patents
Marine streptomyces strain and application thereof Download PDFInfo
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- CN101974456A CN101974456A CN2010102958290A CN201010295829A CN101974456A CN 101974456 A CN101974456 A CN 101974456A CN 2010102958290 A CN2010102958290 A CN 2010102958290A CN 201010295829 A CN201010295829 A CN 201010295829A CN 101974456 A CN101974456 A CN 101974456A
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Abstract
The invention relates to a marine streptomyces strain and application thereof. A marine streptomyces sp.HH-1 with the colony induction inhibiting activity is finally obtained by separating an endosymbiosis actinomycetes strain from a marine mollusk body and selecting by a pseudomonas aeruginosa screening model QAIS2 and a purple coli mode strain CV026. The invention, for the first time, proves that the extractive prepared by fermenting the streptomyces strain has double inhibiting functions to the pseudomonas aeruginosa and a purple coli colony induction system and does not restrict the growth of the screening model QAIS2 and the purple coli mode strain CV026 within the effective concentration range. The extractive can remarkably lower the output of the purple coli mycetin and weaken the drug resistance of disease germ. The invention has the advantages that the preparation materials can be easily obtained, the fermentation and cultivation condition of the strain is simple, the technical process for preparing the compound is controllable, the yield is high, and the like. The effective ingredients which are used for inhibiting bacterial colony induction and separated from the marine streptomyces sp.HH-1 ferment extractive can be used for researching and developing novel antibacterial medicines in the medical field.
Description
Technical field:
The invention belongs to the marine microorganism field, a kind of antibacterium quorum sensing activity extract and application thereof that belongs to bacterial strain (Streptomyces sp.HH-1) preparation by marine streptomyces of specific design.
Background technology:
(Quorum Sensing is a bacterium QS) carries out egosyntonic a kind of group behavior according to self change in cell density to quorum sensing.Bacterium utilizes signaling molecule, or be called the self-induction thing (autoinducers, AI) as alternative " language " mutually, bacterium constantly is secreted into signaling molecule outside the born of the same parents in reproductive process, thereby and detect the variation of its its population density of concentration perception; When its density reaches certain threshold value, will start some expression of gene.The quorum sensing system extensively is present in Gram-negative bacteria and the gram-positive microorganism, controlling a series of bacterium behavior and phenotypes such as comprising biofilm load formation, toxin generation, Disease-causing gene expression, exocellular polysaccharide generation, the pathogenic bacteria pathogenic course is being played conclusive effect.Lot of documents shows that its pathogenecity of bacterial strain that has lacked the quorum sensing system reduces greatly, therefore, by disturbing the bacterial population induction system to control pathogenic bacteria pathogenic is a very effective strategy, and the quorum sensing system with bacterium is the antibacterials of target sieving, different fully with the mechanism of action of those traditional antibiotic medicines, only suppress the pathogenic behavior of bacterial population induction regulation and control, do not influence the growth of bacterium, can not produce bacterial drug resistance in theory.
At present, people utilize different screening models, found furanone (J EMBO, 2003,22 (15): 3803-3815.), 4-NPO (J Bacteriol, 2005,187 (5): 1799-1814), solenopsinA (Infect Dis, 2008,198 (8): 1198-1201) wait compound to have bacterial population induction restraining effect.QSIS2 screening model (J Bacteriol, 2005,187 (5): be to utilize to be subjected to Pseudomonas aeruginosa (Pseudomonas the aeruginosa) (Nature of quorum sensing system 1799-1814), 2000,406:959-964) promotor of the downstream gene lasB of Tiao Jieing and sucrose lethal gene SacB are that the reporter gene structure forms; Under the situation that has the sucrose substrate to exist, signaling molecule 3-oxygen dodecanoyl homoserine lactone (3-oxo-C12-HSL) and the combination of lasR receptor protein, start the expression of lasB promotor downstream lethal gene SacB, if have quorum sensing supressor such as positive control C30 (furanone), lethal effect then can be remedied; Chromobacterium (Chromobacterium violaceum) is a kind of gram negative bacterium, when the increase of self bacterial cell density reaches certain threshold concentration, the signaling molecule caproyl homoserine lactone (C6-HSL) that discharges in environment can combine with transcription regulatory protein CviR in the born of the same parents, starts quorum sensing Expression of Related Genes such as mycetin; So a kind of easy, quorum sensing supressor filtering mode bacterial strain (Microbiology, 1997,143 (12): 3703-3711) intuitively of chromobacterium Chang Zuowei.
Marine microorganism is the huge treasure-house of biologically active substance.Special ocean environment is given marine microorganism with new activity, high salinity, high pressure, low temperature and the special complicated marine eco-environments such as illumination may make marine microorganism have bigger diversity on species, genomic constitution and ecological functions, produce the special outcome that is different from the source, land.The present invention separates the endosymbiosis actinomycetes in the sea mollusk body, utilize QSIS2 screening model and the painted method of TTC viable bacteria to obtain a strain and have the active streptomycete HH-1 of bacterial population induction inhibition, and its fermentation broth extract can suppress the output of the mycetin of chromobacterium quorum sensing system regulation.
Summary of the invention:
The objective of the invention is to overcome the shortcoming that exists in the prior art, a kind of extract by ocean Streptomyces sp.HH-1 fermentative preparation is provided, this extract has the activity that suppresses Pseudomonas aeruginosa and chromobacterium quorum sensing system, and can reduce the generation of the toxicity of pathogenic bacteria factor, weaken the resistance of pathogenic bacterium.
The preparation method of activity extract of the present invention mainly contains: ocean Streptomyces sp.HH-1 separates two steps of preparation with extract.Get the ocean Streptomyces sp.HH-1 after the activation, be inoculated into and carry out fermentation culture in the fermention medium, use equal-volume ethyl acetate extraction 3 times after the fermentation ends, extraction liquid is filtered, vacuum and low temperature is concentrated into dried, obtain brown medicinal extract, take by weighing an amount of brown medicinal extract and be used for biological activity determination after with dissolve with methanol.
The extract of Streptomyces sp.HH-1 preparation in ocean involved in the present invention has the effect that suppresses Pseudomonas aeruginosa and chromobacterium infection; This extract does not suppress the growth of chromobacterium CV026 in the 0-200mg/mL concentration range; But can significantly reduce the generation of chromobacterium mycetin, and the quorum sensing restraining effect of this extract is concentration dependent; This extract does not exert an influence to the growth of screening model QSIS2 in the scope of 0-500mg/mL.
The present invention compared with prior art, it is simple to have an ocean Streptomyces sp.HH-1 strain fermentation culture condition, its technological process of producing extract is simple and easy to control, the output height, the extract of preparation suppresses advantages such as the bacterial population influential action is obvious.
Description of drawings:
Fig. 1 is the screening active ingredients figure of activity extract of the present invention.
Fig. 2 is that extract of the present invention is to screening model QSIS2 growth curve test pattern.
Fig. 3 is that extract of the present invention is to chromobacterium CV026 growth curve test pattern.
Fig. 4 is the mensuration figure of the extract of different concns of the present invention to chromobacterium mycetin output.
Embodiment:
Also the present invention is described further in conjunction with the accompanying drawings below by example.
Embodiment 1: the separation and Culture of marine actinomycete
Sample collecting: gather wild sea mollusk marine rainbow, clam, oyster in January, 2009 from the different areas, Jiaozhou Bay, deposit in the aseptic bottle.
Actinomycetes separate and substratum is preserved on the inclined-plane: Soluble starch 20g; KNO
31g; K
2HPO
40.5g; MgSO
40.5g; FeSO
47H
2O 0.01g; Agar 20g; Sea salts 33.3g; Distilled water 1000mL; PH 7.2～7.4, and 121 ℃, 1.01 * 10
520min is standby in the Pa sterilization, and it is the cycloheximide of 50mg/L and the nalidixic acid of 25mg/L that isolation medium faces with before adding final concentration.
Actinomycete fermentation substratum: Glucose 10g; Soluble starch 10g; Yeast extract 10g; Peptone10g; K
2HPO
40.5g; NaCl 0.5g; MgSO
40.5g; Beefextract 0.3g; CaCO
32g; Sea salts 33.3g; Distilledwater 1000mL; PH 7.2～7.4, and 121 ℃, 1.01 * 10
520min is standby in the Pa sterilization.
The separation method of marine actinomycete: with aseptic sea water immersion sample, 75% alcohol surface sterilization, remove shell and get software, use 75% alcohol surface sterilization once more, aseptic seawater flushing 3 times, pulverize and grind, gradient dilution is applied on the isolation medium flat board, cultivates 6～10d at 28 ℃, bacterium colony is chosen the line separation and purification, observe and arrange heavy.
Zymotechnique: after the dull and stereotyped activation of marine actinomycete, an amount of inoculated by hypha block of picking is in the triangular flask of interior dress 150mL aforesaid liquid substratum, and 28 ℃ of temperature, rotating speed 140rpm shake and cultivated 10 days.After the fermentation ends, with fermented product mycelium and the ultrasonication of supernatant mixed solution, add the equal-volume ethyl acetate, stirred overnight at room temperature, 6000rpm centrifuging and taking supernatant liquor adds equal-volume ethyl acetate re-extract 2 times again, be concentrated into total extraction liquid vacuum and low temperature dried, obtain brown medicinal extract, become the 100mg/mL liquid storage with dissolve with methanol after the brown medicinal extract extract drying, with standby behind the 0.22 μ M membrane filtration in 4 ℃ of preservations.
Embodiment 2: ocean Streptomyces sp.HH-1 extract active testing
One. the extract quorum sensing suppresses active testing
The used substratum of screening model is the LB substratum: Tryptone 10g; Yeast extract 5g; NaCl 10g; Distilledwater 1000mL; Solid LB substratum adds 15g agar; 7.0,121 ℃ of pH, 1.01 * 10
520min is standby in the Pa sterilization.Pseudomonas aeruginosa quorum sensing supressor (QSI) screening model QSIS2 is by (JBacteriol such as Thomas Bovbjerg Rasmussen; 2005; 187 (5): 1799-1814) make up; this screening model is to utilize Pseudomonas aeruginosa to be subjected to the promotor of the startup subdomain of the pathogenic related gene lasB that the QS system regulates as reporter gene; with Polylevulosan transferase gene sacB (lethal gene) is that reporter gene makes up; when in substratum, adding sucrose; because sacB expression of gene; the growth of thalline can be suppressed; and when QSI exists; the sacB expression of gene is suppressed; protect the growth of thalline, thereby can filter out the inhibition of quorum sensing system effectively with the variation of thalli growth.Mycetin is the (Microbiology that is subjected to the rigorous regulation and control of quorum sensing in chromobacterium, 1997,143 (12): 3703-3711), chromobacterium CV026 is the signaling molecule deletion mycopremna, after adding signaling molecule C6-HSL, can start the generation of the mycetin of quorum sensing regulation and control, if the existence of quorum sensing supressor is arranged, then can suppress the generation of mycetin, on agar plate, show muddy but opaque circle.
The solid LB substratum that screening model QSIS2 plate screening method: 13.5mL melts is cooled to 40 ℃, the sucrose that adds 1.5mL 60%, the signaling molecule 3-oxygen dodecanoyl homoserine lactone 3-oxo-C12-HSL of the QSIS2 bacterium liquid of 150 μ L incubated overnight, 6 μ L, 500 μ mol/L, the viable bacteria staining agent TCC TTC of 75 μ L 5% (w/v) and the gentamicin of 24 μ L 50mg/mL fall dull and stereotyped behind the mixing.After treating that flat board solidifies, punch with punch tool, the extract that adds 3 μ L different concns in each hole respectively, 37 ℃ of incubators are cultivated then, bacterium is enclosed growing state around observing well, if having quorum sensing, extract suppresses active, there then should have bacterial growth to iris out around the well to be existing, the big more activity that is of bacterium loop diameter is strong more, detected result is as shown in the figure shown in the 1a: compare with positive control C30 (6.35 μ g), 0.45mg, 0.3mg, 0.15mg extract present positive findings, and become concentration dependent.
The solid LB substratum that chromobacterium plate screening method: 15mL melts is cooled to 40 ℃, add the chromobacterium CV026 bacterium liquid of 150 μ L incubated overnight, the signaling molecule hexanoyl homoserine lactone C6-HSL of 15 μ L, 500 μ mol/L and the kantlex of 15 μ L 20mg/mL, fall dull and stereotyped behind the mixing.After treating that flat board solidifies, punch with punch tool, add 3 μ L extracts in each hole, cultivate in 30 ℃ of incubators,, then can suppress the generation of mycetin if this extract is the quorum sensing supressor, on agar plate, show muddy but opaque circle, The selection result is shown in Fig. 1 b: compare with positive control C30 (6.35 μ g), 0.45mg, 0.3mg, 0.15mg extract present positive findings, and become concentration dependent.
Two. the different concns extract is to the QSIS2 growth effect
Picking screening model bacterial strain QSIS2 is in fresh LB substratum, and 37 ℃, 140rpm are cultured to logarithmic phase; The bacterium liquid that will be cultured to logarithmic phase with fresh LB is diluted to OD
600≈ 0.05,6 groups of packing, and 3 every group are parallel; Every group adds final concentration extract final concentration 0mg/mL respectively, 50mg/mL, 100mg/mL, 200mg/mL, 300mg/mL, 400mg/mL, 37 ℃, the cultivation of 140rpm shaking table; OD was respectively managed in survey respectively every 1-2 hour
600Until the logarithm later stage; With incubation time Time (h) is X-coordinate, absorbancy with the 600nm place is an ordinate zou, draw the growth curve of bacterial strain QSIS2 under the Streptomyces sp.HH-1 extract effect of ocean, from growth curve (Fig. 2) as can be seen, this extract does not suppress the growth of screening model QSIS2 in the 0-400mg/mL concentration range.
Three. the extract of different concns is to the influence of chromobacterium CV026 growth
Picking chromobacterium CV026 is in fresh LB substratum, and 30 ℃, 140rpm are cultured to logarithmic phase; The bacterium liquid that will be cultured to logarithmic phase with fresh LB is diluted to OD
600≈ 0.05,5 groups of packing, and 3 every group are parallel; Every group adds extract respectively to final concentration 0mg/mL, 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 37 ℃, the cultivation of 140rpm shaking table; OD was respectively managed in survey respectively every 1-2 hour
600Until the logarithm later stage; With incubation time Time (h) is X-coordinate, optical absorbance with the 600nm place is an ordinate zou, draw the growth curve (Fig. 3) of chromobacterium CV026 under this extract effect, this extract does not exert an influence to the growth of chromobacterium CV026 in the 0-200mg/mL concentration range.
Four. the extract of different concns is tested chromobacterium mycetin output
Picking chromobacterium CV026 is in fresh LB substratum, and 30 ℃, 140rpm are cultured to logarithmic phase; The bacterium liquid that will be cultured to logarithmic phase with fresh LB is diluted to OD
600Be 0.05,5 groups of packing, 3 every group are parallel; Adding signaling molecule (C6HSL) to final concentration in every group is 500n μ mol/L, and the final concentration of extract is respectively 0mg/mL, 50mg/mL, 100mg/mL, 150mg/mL, 200mg/mL, 30 ℃, 140rpm shaking culture 12h.Mycetin is quantitative: draw the above-mentioned cultured bacterium liquid of 1mL in 1.5mL Eppendorf pipe, the centrifugal 10min of 14000rpm fully precipitates mycetin and thalline.Remove supernatant liquor, add 1mL DMSO and manage in Eppendorf, vortex, fully vibration makes mycetin be dissolved in DMSO.The centrifugal again 10min of 14000rpm, precipitation thalline and chip.Draw supernatant and measure OD
585The photoabsorption at place, concentration is 50mg/mL, 100mg/mL, 150mg/mL, this extract of 200mg/mL reaches 25%, 47%, 65%, 82% (Fig. 4) to chromobacterium mycetin reduction amount respectively.
Claims (3)
1. a strain derives from from the sea mollusk body is intravital and has bacterial population induction and suppress active ocean Streptomycessp.HH-1, presents positive findings by the extract of this strain fermentation preparation through Pseudomonas aeruginosa screening model QSIS2 and chromobacterium type strain CV026 screening.
2. according to claim 1: this extract does not exert an influence to the growth of Pseudomonas aeruginosa screening model QSIS2 in the scope of 0-400mg/mL, and extract does not suppress the growth of chromobacterium CV026 in the 0-200mg/mL concentration range; But can significantly reduce the generation of chromobacterium mycetin, and the quorum sensing restraining effect of this extract is concentration dependent, along with concentration increases gradually, its restraining effect strengthens gradually, and this extract can be used for preventing and treating the research on the chromobacterium disease infection aspect.
3. according to claim 2: can be used for the research and development of field of medicaments novel antibacterial medicine by isolated inhibition bacterial population induced effective constituent in the Streptomyces sp.HH-1 fermented product extract of this ocean.
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| CN2010102958290A CN101974456A (en) | 2010-09-29 | 2010-09-29 | Marine streptomyces strain and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220271A (en) * | 2011-05-27 | 2011-10-19 | 中国科学院微生物研究所 | Marine Streptomyces strain and use thereof |
| CN103695358A (en) * | 2013-12-24 | 2014-04-02 | 扬州大学 | Oceanic streptomyces parvulus and application thereof on aspect of quorum sensing inhibition |
| CN104928220A (en) * | 2015-07-08 | 2015-09-23 | 中国石油大学(华东) | Rheinheimera aquimaris strain from ocean sludge and application thereof |
-
2010
- 2010-09-29 CN CN2010102958290A patent/CN101974456A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220271A (en) * | 2011-05-27 | 2011-10-19 | 中国科学院微生物研究所 | Marine Streptomyces strain and use thereof |
| CN102220271B (en) * | 2011-05-27 | 2012-12-19 | 中国科学院微生物研究所 | Marine Streptomyces strain and use thereof |
| CN103695358A (en) * | 2013-12-24 | 2014-04-02 | 扬州大学 | Oceanic streptomyces parvulus and application thereof on aspect of quorum sensing inhibition |
| CN103695358B (en) * | 2013-12-24 | 2015-08-26 | 扬州大学 | The one small streptomycete in strain ocean and the application in quorum sensing suppression thereof |
| CN104928220A (en) * | 2015-07-08 | 2015-09-23 | 中国石油大学(华东) | Rheinheimera aquimaris strain from ocean sludge and application thereof |
| CN104928220B (en) * | 2015-07-08 | 2018-07-06 | 中国石油大学(华东) | The water Rhein sea of one plant of ocean sludge source is write from memory Salmonella bacterial strain and its application |
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Application publication date: 20110216 |