CN101926829B - Bacillus subtillus degrading bacterial colony sensing signal and use of bacillus subtillus degrading bacterial colony as antiseptic - Google Patents
Bacillus subtillus degrading bacterial colony sensing signal and use of bacillus subtillus degrading bacterial colony as antiseptic Download PDFInfo
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- CN101926829B CN101926829B CN2010102504014A CN201010250401A CN101926829B CN 101926829 B CN101926829 B CN 101926829B CN 2010102504014 A CN2010102504014 A CN 2010102504014A CN 201010250401 A CN201010250401 A CN 201010250401A CN 101926829 B CN101926829 B CN 101926829B
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Abstract
The invention belongs to the field of hygiene and relates to a bacillus subtillus degrading bacterial colony sensing signal and use of bacillus subtillus degrading bacterial colony as an antiseptic, in particular to bacillus subtillus degrading gram-negative bacterial colony sensing signal and the formation of a bacterial biofilm of the bacillus subtillus degrading gram-negative bacterial colony. In the use of the degrading bacterial colony sensing signal, a proper concentration of the bacillus subtillus is 5*102 to 5*104cfu/ml. The bacillus subtillus is further used as antiseptic to inhibit the bacteria such as pseudomonas, agrobacterium, enterobacteriaceae and vibrionaceae. The antiseptic can be ointment for external use, liquid preparation for external use, oral tablet, oral liquid, disinfectant, clearing agents or water quality improving agent. The consistency of the bacillus subtillus serving as the antiseptic is 105 to 106 cfu/g. The use of the bacillus subtillus provided by the invention and the antiseptic inhibit the formation of biofilm of disease-causing bacteria and the growth of bacteria by using microbial quorum-sensing inhibition instead of preventing and sterilizing bacteria by compound which is adopted by most of the conventional antiseptic; therefore, the problem of drug resistance in the antimicrobial process is avoided.
Description
Technical field
The invention belongs to the hygiology field, relate to subtilis bacterium for degrading colony induction signaling and as the purposes of antiseptic-germicide.
Background technology
Subtilis (Bacillus stibillus) is the microorganism of strain separation and purification from breeding environment.Carry out the bacterial strain physiological and biochemical property according to uncle Jie Shi handbook and identify that subtilis is Jinyuan Garden shape on ordinary culture medium, and is opaque, surface drying has wrinkle Yi on every side, and the edge is uneven, the microscopically Gram-positive, and thalline is shaft-like.
Also have following biochemical characteristic: the acid of N.F,USP MANNITOL product+, the acid of pectinose product+, the glucose aerogenesis-, the formation indoles-, the V.P test+, the phenylalanine deamination-, V.p cultivates whole pH value 5.8, the acid of glucose product+, the starch hydrolysis+, the Citrate trianion utilization+, catalase+, nitrate reduction+, NaCl, KCl needs+, the 10%NaCl growth+, the 7%NaCl growth+, 5%NaCl growth+, the 2%NaCl growth+etc. characteristic (annotate: "+" represent positive, and "-" represents feminine gender).
Subtilis applied research at present relates to: 1. antibiotic and bacteriolysis; 2. enhance immunity power; 3. regulate the body intestinal microecology; 4. promoting digestion and body growth; 5. improve breeding environment.The pharmacological action of bibliographical information has following several respects, but does not all relate to its inhibition mechanism to the quorum sensing system.The relevant report of pharmacological research is as follows: antibiotic, antiviral, promoting digestion, raising disease resistance, improve multiple effects such as breeding environment.
At antibiosis, existing correlative study result has: separation and purification goes out a kind of proteolytic enzyme (29.0kD) in (1) subtilis T2 fermented liquid, its optimum temperuture is 65 ℃, optimal pH is 8.0, and this enzyme has obvious restraining effect to spore germination, the mycelial growth of cotton-wilt fusarium (Fusarium oxysporum f sp.vasinfectum).Show, subtilis have antifungic action (Xing Jieshuai, etc. Plant Pathology, 2008,38 (4): 377-381); (2) lipopeptide antibiotic that obtains after the extracting of fermentation of bacillus subtilis liquid has restraining effect to the various plants pathogenic bacteria; The volatility antibacterial substance of its generation can suppress the sprouting and the mycelial growth of ash arrhizus bacteria spore.Show that subtilis has the biological control effect, and (Chen Hua is etc. microbiology circular, 2008,35 (1): 1-4).These antibacterial research all are the material that extracts from subtilis have been done the research and the confirmation of antibacterial effect, and the antibacterial effect of bacillus subtilis strain are not made research.In addition bacillus subtilis strain be studies show that it has restraining effect in various degree to the isolating proteus bacterium of pouity dwelling place, streptococcus aureus, still, to intestinal bacteria, the little (Wang Cheng of enterococcus spp bacteria effect, Deng. Chinese agronomy newspaper, 2010,26 (3): 23-26).Because when existing as bacterial strain, its mechanism of action is not the inhibition of simple chemical substance to bacterium, but the interactional organic system between the bacterium of a complexity, therefore, the antifungal mechanism of bacillus subtilis strain, and the bacteria culture that can be suppressed, be still the problem an of the unknown at present.This has restricted the antibacterial application of subtilis to a certain extent.
Quorum sensing (Quorum sensing, QS) be a kind of bacterial cell communication mechanism, be that bacterium is called self-induction thing (autoinducer by synthetic, secretion, AI) signaling molecule, when AI concentration along with bacterium constantly grows, when population density reaches certain threshold value, starts the expression of specific gene.The quorum sensing system of gram negative bacterium is made of signaling molecule and receptor protein thereof.Signaling molecule is a class homoserine lactone molecule, the signaling molecule carbon chain lengths difference of different bacterium.It is higher that receptor protein belongs to LuxR family protein member similarity.When the density of bacterium acquired a certain degree, the signaling molecule concentration that exists around the bacterium rose thereupon.Signaling molecule concentration arrives certain thresholding, will combine with receptor protein.The conformation that combines the receptor protein of signaling molecule changes, and becomes activating transcription factor, starts the quorum sensing Expression of Related Genes such as release of biomembranous formation and virulence factor.Human or animals' such as Pseudomonas aeruginosa, secondary haemolysis, Vibrio harveyi pathogenic bacterium are regulated their group behavior by quorum sensing, regulate and control biomembranous formation, plasmid shifts and the expression of virulence factor etc.
And biomembranous formation can make the antibiotic effect of pathogenic bacterium tolerance greater concn, and medicine resistance ability can improve tens of times, even hundreds of times.Therefore,, suppress biomembranous synthetic, become the main R﹠D direction in present antibiotic field by inhibition/bacterium for degrading colony induction signaling molecule.Thereby suppressing biomembranous formation and other by the quorum sensing system that disturbs pathogenic bacterium is subjected to the physiological activity of this system regulation to reach antibiotic purpose to be considered to a kind of novel antibiotic strategy.What is more important, because the quorum sensing inhibitor does not directly suppress the growth of pathogenic bacterium, pathogenic bacterium can not produce resistance to it.The quorum sensing system inhibitor of therefore, exploitation safety has important use to be worth.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, the new application of subtilis be provided---the usefulness that reaches as antiseptic-germicide on the subtilis bacterium for degrading colony induction signaling is coated with.
The present invention finds first, and subtilis can the bacterium for degrading colony induction signaling, and suppresses bacterial biof iotalm and form.Can the degrade colony induction signaling of gram negative bacterium of subtilis, especially Rhodopseudomonas (Pseudomonas) (as Pseudomonas aeruginosa), the microbe groups of Agrobacterium (Agrobacterium), vibrionaceae (Vibrionaceae) (as vibrio alginolyticus) etc.The pathogenic bacterium of these Gram-negative monoids or harmful bacterium all have similar quorum sensing system, and the receptor protein of signaling molecule is LuxR or TraR, perhaps are structure and the regulatory mechanism albumen of two kinds of receptor-similars therewith.
In the application of bacterium for degrading colony induction signaling, the suitable concentration that subtilis exists is 5*10
2～5*10
4Cfu/ml.Find through overtesting: subtilis is at 5*10
3Can suppress the biomembranous formation of Pseudomonas aeruginosa during cfu/ml, at 5*10
4Suppress better effects if during cfu/ml; Subtilis is at 5*10
2Can suppress vibrio alginolyticus and the biomembranous formation of Vibrio anguillarum during cfu/ml; Subtilis can make quorum sensing receptor protein TraR concentration reduce, thereby can't effectively start quorum sensing Expression of Related Genes (as the expression of biomembranous formation and virulence factor), thereby reduced the pathogenic activity of pathogenic bacterium, and improved the sterilization effect of host immune system and other antiseptic-germicides.On the basis of these experiments, the investigator can determine effective therapeutic dose of subtilis by existing clinical experiment means.
Because this characteristic of subtilis, can be further with subtilis as antiseptic-germicide.Be applied to suppress the microbe groups of Rhodopseudomonas, Agrobacterium, enterobacteriaceae or vibrionaceae.Antiseptic-germicide can be external-applied ointment, liquid preparation for external use, oral tablets, oral liquid, sterilizing agent, sanitising agent or improver of water quality.As antiseptic-germicide, the concentration of subtilis is 10
5～10
6Cfu/g.Invention finds that because the characteristic of subtilis, subtilis can act on the flora in the environment, reaches effective restraining effect, and can not make these bacteriums produce resistance, is a kind of can the safe handling safely and the sterilant of useful effect.And make the antiseptic-germicide of this concentration, its activity can be kept preferably, and when dilution entered environment, subtilis was given full play to its bacterium for degrading colony induction signaling, go for the sterilization of multiple occasion, be particularly useful under water body environment, carrying out disinfection by adding antiseptic-germicide.
Because test shows the signaling molecule of subtilis degradable tool quorum sensing bacterium, disturb the quorum sensing system of bacterium, thereby make the bacterium can't effectively start quorum sensing Expression of Related Genes and regulation and control its physio-biochemical characteristics (as the expression of biomembranous formation and virulence factor), thereby reduced pathogenic activity, and improved host immune system.Therefore subtilis also can be used as antibacterials, and this pharmaceutical pack contains the subtilis and the pharmaceutically acceptable pharmaceutical carrier of effective bacterial content.The form of pharmaceutically acceptable solvate or hydrate provides.For example, can activate subtilis from flat board or inclined-plane, after its fermented liquid or the dilution, add to again in other carriers or the material.The formulation of medicine is the liquid or solid formulation that allows on the pharmaceutics, and the pharmaceutical dosage form for preparing with subtilis can be external-applied ointment etc.; Pharmaceutical dosage form with the subtilis preparation also can be liquid preparation for external use.
These medicines can be used for the inside and outside infected by microbes of body of the anti-bacterial animal of tool quorum sensing, perhaps to the inhibition of aquaculture water Related Bacteria, perhaps to the control of the relevant pathogenic bacteria of plant.Medicine with the subtilis preparation is used to prevent and treat bacterial various health problem, comprise other infectation of bacteria of enterobacterial infection, humans and animals, the pathogenic bacteria of control plant, can also regulate the body intestinal microecology, promoting digestion and body growth improve culture environment of aquatic products.
In order to verify effect of the present invention, signal degradation experiment and the vibrios microbial film experiment etc. of invention by tool quorum sensing bacterium verified that subtilis is a strain bacterium signal degradation bacteria, can be used as an effective quorum sensing inhibitor.By the bacterium for degrading colony induction signaling molecule, disturb the quorum sensing system of bacterium to suppress the activity of biomembranous formation and bacterium for degrading frizzled receptor molecule, thereby reach anti-microbial effect.
The quorum sensing inhibitor can pass through DOCK software, and the screening of utilization computer virtual obtains, and also can screen by making up indicator.Known some quorum sensing inhibitor can the bacterium for degrading quorum sensing signaling molecule, disturb the quorum sensing system of bacterium and suppress the formation of bacterial biof iotalm.Therefore, can identify whether this material has the quorum sensing inhibitor activity by the biomembranous formation situation of bacterial detection.
Compared with prior art, the present invention has following beneficial effect:
First Application of the present invention subtilis suppress bacterial population induced activity, utilize it to come the bacterium for degrading colony induction signaling, suppress the growth of bacterial biof iotalm.
2. application of subtilis provided by the present invention and antiseptic-germicide, by disturbing the pathogenic bacterium colony induction system to reach the effect that suppresses pathogenic bacteria, it is the strategy of a kind of new control pathogenic bacteria, rather than existing most antiseptic-germicides adopt utilize the direct antibiotic sterilization of compound, avoided the resistance problem in the antibiotic process thus.
3. the application of subtilis provided by the present invention when effectively utilizing its quorum sensing to suppress ability and antibacterial effect, can not cause negative impact to environment, and side effect is little.
4. antiseptic-germicide provided by the invention, safe in utilization, and its production cost is very cheap, is suitable for promoting the use of.
5. the present invention will help studying the biological prevention and control of tool quorum sensing bacterium, inquire into the new technology of anti-tool quorum sensing bacterium.
6. the present invention helps solving tool quorum sensing bacterium owing to a large amount of resistance problems of using microbiotic to produce from machine-processed aspect, and development non-antibiotic class is the main ecological prevention and control new technology of pathogenic bacterium.
Embodiment
Below further specify technical scheme of the present invention by specific embodiment.
Embodiment 1
The screening of subtilis, separation and purifying:
Dull and stereotyped 30 degree of 2216E were cultivated 2-3 days, and repeatedly line separates and purifying, the inoculation inclined-plane, and 4 degree are preserved standby.
Embodiment 2
The primary dcreening operation experiment of subtilis bacterium for degrading signaling molecule:
The primary dcreening operation experiment uses signaling molecule to be the OOHL molecule, and indicator is WCF47.
96 orifice plate primary dcreening operations:
1) from dull and stereotyped picking bacterium to be measured, in 96 well culture plate A, be cultured to growth logarithm late period with respective liquid substratum 200 μ l, set up negative control (LB+AHL) and positive control (Bt+AHL).
2) add 100 μ l MM liquid nutrient mediums in each hole of 96 well culture plate B, 0.2 μ l signaling molecule AHL (1mg/ml) gets bacterium 100 μ l to be measured with liquid-transfering gun to the hole of 96 well culture plate B correspondences from 96 well culture plate A, mixing.Cultivate 4hr altogether for 30 ℃.
3) in each hole of 96 well culture plate C, add 130 μ l MM liquid nutrient mediums, 20 μ l indicator WCF47 bacterium liquid, 1 μ l X-Gal.Get coculture 50 μ l with liquid-transfering gun to the hole of 96 well culture plate C correspondences from 96 well culture plate B, mixing.
4) 30 ℃ of incubated overnight.Can observations about 16hr.
Experimental result: after signaling molecule was degraded, the indication bacterium colony was white in color, otherwise was blue.According to color reaction, tentatively sift out subtilis microorganism strains with degraded signaling molecule AHL.
Embodiment 3
The multiple sieve experiment of subtilis bacterium for degrading signaling molecule:
Agar strip sieves again:
The MM culture medium flat plate that scribbles X-gal is cut into strip, surveys the mixture that adds bacterium to be measured and AHLs extract, add indicator WCF47 successively, observation experiment result behind 30 ℃ of cultivation 18h in another survey at slice one.LB or ddH
2The mixture of O and AHL is as negative control, with the positive contrast of mixture of active bacterium B.thuringiensis culture of AiiA and AHL.
Experimental result: according to color reaction, it is blue that negative control is, and positive control and subtilis are white in color, and shows that subtilis has the activity of degraded AHLs signaling molecule.
Embodiment 4
The biochemical identification experiment of subtilis
Carrying out the bacterial strain physiological and biochemical property according to uncle Jie Shi handbook identifies.The result shows that subtilis is Jinyuan Garden shape on ordinary culture medium, and is opaque, surface drying, and You Zhou Yi on every side, the edge is uneven, the microscopically Gram-positive, thalline is shaft-like.
Also have following biochemical characteristic: the acid of N.F,USP MANNITOL product+, gelatin hydrolysis+, lecithinase-, the acid of pectinose product+, the glucose aerogenesis-, tyrosine hydrolysis-, the formation indoles-, the V.P test+, the phenylalanine deamination-, V.p cultivates whole pH value 5.8, the acid of glucose product+, the propionic salt utilization-, the starch hydrolysis+, the acid of wood sugar product-, the Citrate trianion utilization+, catalase+, nitrate reduction+, junket Ruan hydrolysis+, NaCl, KCl needs+, the 10%NaCl growth+, the 7%NaCl growth+, the 5%NaCl growth+, 2%NaCl growth+(annotate: "+" represents positive, and "-" represents negative).
Experimental result according to bacterium colony characteristics, dyeing characteristic and biochemical indicator characteristic, determines that tentatively bacterial strain Zou 03 is a subtilis.
Embodiment 5
The Molecular Identification experiment of subtilis 16S rRNA:
With the bacteria total DNA is template, carry out the 16SrDNA sequence of pcr amplification bacterial strain Zou 03 with the Taq archaeal dna polymerase, primer is 27f:5 '-AGAGTTTGATCCTGGCTCAG-3 ' and 1492r:5 '-GGTTACCTTGTTACGACTT-3 ', the PCR reaction conditions: 94 ℃ of pre-sex change 5min; 94 ℃ of 1min, 45 ℃ of 1min, 72 ℃ of 1min, 30 circulations; 72 ℃ prolong extension 10min.The PCR product is connected Transformed E .coli DH5 with the pMD-18T carrier after glue reclaims column purification
Competent cell, the order-checking of picking positive transformant.The 16S rDNA sequence and the Genbank amplifying nucleic acid data that record are carried out the analysis of Blast sequence similarity, the 16S rDNA sequence of choosing the higher bacterium of homology adopts Clustalx 1.8 to carry out sequence alignment, utilization Phylip3.65 adds up and cluster analysis, Neighbor joining method constructing system is grown tree, by 1000 duplicate tests of Boostrap.
Experimental result: 1.0% agarose gel electrophoresis detects, and about the about 1.45kb of 16S rDNA sequence length of bacterium Zou 03, approaches total length, and is consistent with the expection size.Analyze by NCBI BLAST, the 16SrDNA sequence of sequence homology comparison result shows bacterium Zou 03 and the sequence fragment of Bacillus subtilis 16S rDNA (GQ861468.1) have 99% homology, determine that tentatively Y5 is the new genus bacillus of a strain.Choose homology 12 strain bacterium constructing system evolutionary trees more than 95%, bacterium Zou 03 is nearer with the close source relation of the Bacillus licheniformis hypotype Bacillus licheniformis (GenBank accession no.AB361368) that has reported as can be known.
Comprehensive traditional ne ar, physiological and biochemical property in conjunction with 16S rDNA sequence homology analysis and the structure bacterium decorum evolutionary tree of bacterium, can determine that bacterial strain Zou 03 is subtilis.
Embodiment 6
Subtilis suppresses vibrio alginolyticus and the biological film formed experiment of Vibrio anguillarum:
The vibrio alginolyticus and the Vibrio anguillarum bacterium liquid that in the 2ml plastic centrifuge tube, add 975 μ l incubated overnight respectively, the glycerine of 25 μ l 40%, 1 μ l (10
5Cfu/g) subtilis.With the processing that do not add subtilis as negative control.37 ℃ leave standstill cultivation 3 days.Carefully incline liquid in the centrifuge tube washes centrifuge tube for several times gently with the distilled water of sterilization, guarantees to rinse out free bacterium in the centrifuge tube.After the globule evaporation Deng the centrifuge tube inwall, add the crystal violet solution of 1ml 0.5%, room temperature left standstill 30 minutes.Viola crystallina is outwelled, wash centrifuge tube gently for several times with the distilled water of sterilizing, the Viola crystallina of guaranteeing not to be adsorbed on the microbial film is washed out.After treating the evaporation of the centrifuge tube inwall globule, add 1ml 95% ethanol, room temperature left standstill 15 minutes.At last ethanol in the centrifuge tube is poured in the cuvette, in spectrophotometer, surveyed the OD value with the 570nm wavelength.The OD value is low more, illustrates that the microbial film of vibrio alginolyticus and Vibrio anguillarum formation is few more.
Experimental result shows: subtilis is at this concentration (5*10
2Cfu/ml) can suppress vibrio alginolyticus and the biomembranous formation of Vibrio anguillarum down.But directly do not suppress the growth of vibrio alginolyticus and Vibrio anguillarum, show that restraining effect may be to reach by the quorum sensing system that suppresses bacterium.
Embodiment 7
Subtilis suppresses the biomembranous experiment of Pseudomonas aeruginosa:
The Pseudomonas aeruginosa bacterium liquid that in the 2ml plastic centrifuge tube, adds 975 μ l incubated overnight, the glycerine of 25 μ l 40%, 1 μ l 10
7The subtilis of cfu/g.With the processing that do not add subtilis as negative control.37 ℃ leave standstill cultivation 3 days.Carefully incline liquid in the centrifuge tube washes centrifuge tube for several times gently with the distilled water of sterilization, guarantees to rinse out free bacterium in the centrifuge tube.After the globule evaporation Deng the centrifuge tube inwall, add the crystal violet solution of 1ml 0.5%, room temperature left standstill 30 minutes.Viola crystallina is outwelled, wash centrifuge tube gently for several times with the distilled water of sterilizing, the Viola crystallina of guaranteeing not to be adsorbed on the microbial film is washed out.After treating the evaporation of the centrifuge tube inwall globule, add 1ml 95% ethanol, room temperature left standstill 15 minutes.At last ethanol in the centrifuge tube is poured in the cuvette, in spectrophotometer, surveyed the OD value with the 570nm wavelength.The OD value is low more, illustrates that the microbial film of Pseudomonas aeruginosa formation is few more.
Experimental result shows: subtilis is at this concentration (5*10
4Cfu/ml) can suppress the biomembranous formation of Pseudomonas aeruginosa down, but directly not suppress the growth of Pseudomonas aeruginosa, show that subtilis may disturb the quorum sensing system of bacterium and suppress biomembranous formation.
Embodiment 8
The experiment of subtilis degraded quorum sensing receptor protein TraR
TraR albumen is the receptor protein of root Agrobacterium quorum sensing system signal molecule, belong to the LuxR protein family together with the LasR albumen of Pseudomonas aeruginosa, the homology in their avtive spot zones is up to 70%, and therefore, the inhibitor that TraR albumen is worked often also can have restraining effect to LasR albumen.LuxR albumen becomes extremely unstable under the effect of quorum sensing inhibitor such as halogenation furanone, can be fallen by the rapid hydrolysis of the proteolytic enzyme of bacterium self in the short period of time (about 30 minutes), causes the proteic density loss of LuxR.Whether therefore, descend by the concentration after detection LuxR albumen and the inhibitor effect, whether inhibitor has the activity of inhibition as can be known, and suppresses active height.
Method with PCR obtains the proteic gene trar of TraR.This gene segment is inserted structure pETtrar plasmid among the expression vector PET-17b with the method for double digestion, again this plasmid is transformed in the e. coli bl21 with the calcium shifting method, obtain to efficiently express the proteic engineering bacteria of TraR at last.After this project bacterium was induced 4 hours with IPTG, centrifugal collection thalline was used the resuspended bacterium of sterilized water again, and what add that subtilis cultivates slightly puies forward antimicrobial fluid, cultivates 35 minutes for 37 ℃.Detect thalline TraR protein concentration with sds polyacrylamide gel electrophoresis at last.
Experimental result shows that subtilis can obviously reduce the concentration of quorum sensing receptor protein TraR in 35 minutes.After TraR concentration reduces, can't effectively start quorum sensing Expression of Related Genes (as the expression of biomembranous formation and virulence factor), thereby reduced pathogenic activity.This experiment has proved that from molecular level subtilis is an effective potential quorum sensing inhibitor.
Embodiment 9
With the subtilis is the external-applied ointment of effective constituent preparation
With reference to the pharmaceutics handbook, an amount of bacillus subtilis bacteria culture fluid is added appropriate amount of fluid paraffin or Yellow Vaselin, make external-applied ointment.
Embodiment 10
With the subtilis is the liquid preparation for external use of effective constituent preparation
With reference to the pharmaceutics handbook, an amount of bacillus subtilis bacteria culture fluid is dissolved in water, make liquid preparation for external use.
Embodiment 11
With the subtilis is the oral tablets of effective constituent preparation
With reference to the pharmaceutics handbook, an amount of bacillus subtilis bacteria culture fluid is added the moderate lubrication agent make softwood, granulate with 10 mesh sieves, drying, dry granular adds Magnesium Stearate, mixing, the tabletting machine compressing tablet becomes tablet, film coating, packing promptly gets coated tablet.
Embodiment 12
With the subtilis is the oral liquid of effective constituent preparation
With reference to the pharmaceutics handbook, an amount of bacillus subtilis bacteria culture fluid is dissolved in water, add correctives, add the water pondage, make oral liquid.
Claims (2)
1. subtilis prepares the purposes of the preparation of bacterium for degrading colony induction signaling, it is characterized in that described bacterium is the microbe groups of Rhodopseudomonas, Agrobacterium, enterobacteriaceae or vibrionaceae, the application concentration of described subtilis is 5*10
2～5*10
4Cfu/ml.
2. subtilis prepares the purposes of antiseptic-germicide, it is characterized in that described purposes is the microbe groups that is applied to Rhodopseudomonas, Agrobacterium, enterobacteriaceae or vibrionaceae, described Kang Junjishi external-applied ointment, liquid preparation for external use, oral tablets, oral liquid, sterilizing agent, sanitising agent or improver of water quality, the concentration of described subtilis are 10
5～10
6Cfu/g.
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| CN107937300A (en) * | 2017-11-08 | 2018-04-20 | 青岛农业大学 | A kind of bacillus subtilis and its application in aquaculture |
| CN107937300B (en) * | 2017-11-08 | 2020-09-01 | 青岛农业大学 | Bacillus subtilis and application thereof in aquaculture |
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