CN101497867B - Isolated culture method and use of Streptomycete from sea - Google Patents
Isolated culture method and use of Streptomycete from sea Download PDFInfo
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- CN101497867B CN101497867B CN2009100251634A CN200910025163A CN101497867B CN 101497867 B CN101497867 B CN 101497867B CN 2009100251634 A CN2009100251634 A CN 2009100251634A CN 200910025163 A CN200910025163 A CN 200910025163A CN 101497867 B CN101497867 B CN 101497867B
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Abstract
The invention relates to a separation culture method of BM-2 (Streptomyces sp.BM-2) from sea, which is characterized in that the method comprises the following steps of accumulation culture, thalli separation and purification, strain screening, strain identification, and the like. According to the test results of morphology, physiology and biochemistry, and the analysis of the homology of 16SrDNA, separated and cultured stains are BM-2(Streptomyces sp. BM-2). The BM-2(Streptomyces sp. BM-2) has the function of inhibiting various plant pathogenic fungi, colon bacillus, bacillus subtilis and aeromonas hydrophila.
Description
Technical field
The present invention relates to a kind of culture of strains method, the isolation cultivation method of particularly a kind of streptomycete BM-2 from the ocean; The invention still further relates to the purposes of streptomycete BM-2.
Background technology
Many actinomycetic secondary metabolites have the purposes of medicine and plant protection aspect; antibacterium, antimycotic and antitumor drug have been widely used as; in the tens thousand of kinds of microbe-derived biologically active substances of now having found, have 70% to be approximately by actinomycetes institute synthetic.Separate the most actinomycetes that obtain at present and all derive from soil, it is antibiotic more than 2/3rds that the microbiotic that the Lu Sheng actinomycetes produce accounts for natural origin, yet along with the day by day raising of pathogenic micro-organism to antibiotics resistance, it is extremely urgent to seek the microbiotic with novel mechanism of action.Over nearly 20 years, the quantity of separating the lead compound that obtains from the Lu Sheng actinomycetes falls sharply, so people have invested more wide habitat-ocean to sight.The living environment of marine actinomycete is very special, as high salinity, high pressure, low nutrition, low temperature and with different biologies between relation etc.In the extreme environment of these so-called life, marine actinomycete has been developed and unique metabolic way, and this guarantees that not only it survives in extreme environment, the potentiality that produce antibiotic compounds also are provided.Therefore, ocean environment will become the important new source of actinomycetes and actinomycetes meta-bolites.
Though marine actinomycete is not the chief component in the marine microorganism ecosystem, increasing result of study shows that its meta-bolites has uniqueness.Teach the latest find of having reported them June calendar year 2001 in the 10th the international ocean natural product symposial that Japan holds from the W.Fenical of internationally famous marine natural product research institution University of California: they find and have successfully cultivated one first and belong to marine actinomycete, called after Marinospora., this bacterium can only grow having under the condition of salt, has unique 16S rRNA sequence.Carried out antibiotic and antitumor activity screening test to this bacterium of 100 strains, the result has 80% fermented liquid extract to demonstrate significantly antibiotic and anti-tumor activity.A strain fermented liquid is wherein carried out chemical research, and therefrom separation and purification goes out the compound of a series of novel structures, called after Salinosporamides, and these compounds have very strong antitumour activity.
Many actinomycetic secondary metabolites have the purposes of medicine and plant protection aspect, have been widely used as antibacterium, antimycotic and antitumor drug.According to incompletely statistics, at present caused people's attention about the research of marine actinomycete active substance, from ocean environment, separate the marine actinomycete that has obtained multiple generation new active substance, and to the fermentation condition of the bacterial strain that obtains, the isolation identification of biological activity and active substance is studied, obtained gratifying achievement, but existing research mainly concentrates in the exploitation of medicine, and marine actinomycete is also at the early-stage to the research of the antimicrobial substance of plant pathogenic fungi, and seeking the marine actinomycete with new antibacterial mechanisms from ocean environment, to be applied to the biological control of Plant diseases significant for developing the high-efficiency environment friendly novel agrochemical.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, and a kind of isolation cultivation method that the various plants pathogenic fungi is had the streptomycete BM-2 (Streptomyces sp.BM-2) of strong anti-microbial effect is provided.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is the isolation cultivation method of a kind of streptomycete BM-2 from the ocean (Streptomyces sp.BM-2), is characterized in that its step is as follows:
(1) enrichment culture: gather ooze from marine site, Lianyun Harbour, the triangular flask vibration of ooze 10g being put into the aseptic seawater that fills 50mL shakes up, and get 5mL suspension and join in the 50mL enrichment culture liquid, 190r/min, shaking table was cultivated 5 days under 25 ℃ of conditions;
(2) separation and purification of thalline: get 5 days nutrient solution 1.0mL of enrichment culture with liquid-transfering gun and join in the test tube that fills the aseptic seawater of 9.0mL, be prepared into 10
-1Sample diluting liquid; Adopt the method serial dilution of gradient dilution then, be prepared into 10 respectively
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Sample diluting liquid; Get 10 respectively then
-5, 10
-6With 10
-7Sample diluting liquid 0.2ml be put on No. 1 culture medium flat plate of seawater Gao Shi, after spreading rod coating evenly, place 25 ℃ incubator to cultivate, grow bacterium colony; Distinguish the bacterium colony on No. 1 culture medium flat plate of picking seawater Gao Shi again, method with three rides is inoculated on No. 1 culture medium flat plate of seawater Gao Shi, place 25 ℃ of incubators to cultivate, rule repeatedly until obtaining pure bacterial strain list bacterium colony, the bacterial strain of several purifying is transferred to respectively in No. 1 substratum test tube slant of seawater Gao Shi preserves;
(3) screening of bacterial strain: adopt dull and stereotyped face-off culture method, for being tried bacterium, measure the anti-fungal activity of plant pathogenic of the different strains that separation and purification obtains respectively for the plant pathogenic fungi fusarium graminearum of examination and cotton-wilt fusarium; During operation the plant pathogenic fungi of the inoculation culture 5d of PDA culture medium flat plate central authorities, the different strains that around plant pathogenic fungi, obtains apart from the symmetrical streak inoculation separation and purification in culture dish wall 15mm place, be inverted constant temperature culture for 25 ℃, making plant pathogenic fungi colony growth situation is measured antibacterial bandwidth behind the cultivation 5d; Each bacterial strain is a processing, and every processing repeats 3 times; Choose a strain to the antibacterial bandwidth mean value of aforementioned 2 kind of plant pathogenic fungies bacterial strain, be designated as the BM-2 bacterial strain greater than 15mm;
(4) evaluation of bacterial strain: the BM-2 bacterial strain was cultivated 5 days down in 25 ℃, and observed and recorded bacterium colony and cellular form are carried out physiological and biochemical test simultaneously, and carry out 16S rDNA sequencing analysis; Think tentatively that according to morphology and physiological and biochemical test result bacterial strain BM-2 belongs to streptomyces; The homology analysis of 16SrDNA shows that the BM-2 bacterial strain approaches Streptomyces sp. most, and the 16S rDNA sequence similarity of the two is 99%, and the BM-2 bacterial strain is streptomycete (Streptomyces sp.).
In the isolation cultivation method technical scheme of above-described streptomycete BM-2 (Streptomyces sp.BM-2) from the ocean, the optimization of fermentation conditions of described streptomycete BM-2 bacterial strain is: optimum medium is: starch 14g, lactose 16g, analysis for soybean powder 16g, dipotassium hydrogen phosphate 3g, lime carbonate 4g, Repone K 0.4g, sodium-chlor 30g, water 1000ml, pH are 7.5; Fermentation condition is: inoculum size 7%, bottling amount 250ml triangular flask 80ml/ bottle, shaking speed 190r/min, 28 ℃ of optimum temperutures, incubation time 5d.
Among the present invention related biomaterial " streptomycete BM-2 (Streptomyces sp.BM-2) " bacterial strain on February 4th, 2009 Genebank open (accession number is: FJ595715, is network address: (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi? db=nuccore﹠amp; Id=222145980).This bacterial strain is public material, rise in 20 years on this patent application date, the public if desired, Huaihai Institute of Technology oceanography institute laboratory can externally provide.
Streptomycete BM-2 of the present invention (Streptomyces sp.BM-2) possesses the purposes that suppresses the various plants pathogenic fungi.
It below is the correlation test of the streptomycete BM-2 that the contriver did (Streptomyces sp.BM-2) bacterial strain (hereinafter to be referred as the BM-2 bacterial strain).
1, separation screening test.
1.1 substratum
(1) enrichment medium: yeast extract paste 5g, extractum carnis 5g, peptone 10g, NaCl 5g, Chen Haishui 1000mL, pH 7.5-8.0.
(2) No. 1 substratum of seawater Gao Shi: Zulkovsky starch 20g, KNO
31g, sodium-chlor 0.5g, dipotassium hydrogen phosphate 0.5g, MgSO
47H
2O 0.5g, FeSO
4H
2O 0.01g, agar 20g, seawater 1000mL, pH7.2～7.4.
(3) PDA substratum: potato 200g, glucose 20g, agar 20g, water 1000mL, pH nature.
1.2 for the examination pathogenic bacteria
Southern corn leaf blight (Bipolaria maydis), corn circle pinta bacterium (Helminthosporiumcarbonum), fusarium graminearum (Fusarium graminearum), cotton-wilt fusarium (Fusariumoxysporum f.sp.vasinfectum), wheat is avenged rotten reaping hook germ (Fusarium nivale), spot defoliation bacterium (Alternaria alternata), root rotof flax bacterium (Bipolaris sorokiniana), alternaria tenuis germ (Alternaria tenuis), totally 9 kinds of tomato early blight bacteriums (Alternaria solani), provided by institute of the Chinese Academy of Agricultural Sciences Institute of Plant Protection, the Huaihai Institute of Technology marine microbiology laboratory is preserved.
1.3 separation method
(1) enrichment culture: gather ooze from marine site, Lianyun Harbour, the triangular flask vibration of ooze 10g being put into the aseptic seawater that fills 50mL shakes up, and get 5mL suspension and join in the 50mL enrichment culture liquid, 190r/min, shaking table was cultivated 5 days under 25 ℃ of conditions;
(2) separation and purification of thalline: get 5 days nutrient solution 1.0mL of enrichment culture with liquid-transfering gun and join in the test tube that fills the aseptic seawater of 9.0mL, be prepared into 10
-1Sample diluting liquid; Adopt the method serial dilution of gradient dilution then, be prepared into 10 respectively
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Sample diluting liquid; Get 10 respectively then
-5, 10
-6With 10
-7Sample diluting liquid 0.2ml be put on No. 1 culture medium flat plate of seawater Gao Shi, after spreading rod coating evenly, place 25 ℃ incubator to cultivate, grow bacterium colony; Distinguish the bacterium colony on No. 1 culture medium flat plate of picking seawater Gao Shi again, method with three rides is inoculated on No. 1 culture medium flat plate of seawater Gao Shi, place 25 ℃ of incubators to cultivate, rule repeatedly until obtaining pure bacterial strain list bacterium colony, the bacterial strain of several purifying is transferred to respectively in No. 1 substratum test tube slant of seawater Gao Shi preserves;
(3) screening of bacterial strain: adopt dull and stereotyped face-off culture method, for being tried bacterium, measure the anti-fungal activity of plant pathogenic of the different strains that separation and purification obtains respectively for the above-mentioned 9 kind of plant pathogenic fungies (comprising fusarium graminearum and cotton-wilt fusarium) of examination; During operation the plant pathogenic fungi of the inoculation culture 5d of PDA culture medium flat plate central authorities, the different strains that around plant pathogenic fungi, obtains apart from the symmetrical streak inoculation separation and purification in culture dish wall 15mm place, be inverted constant temperature culture for 25 ℃, making plant pathogenic fungi colony growth situation is measured antibacterial bandwidth behind the cultivation 5d; Each bacterial strain is a processing, and every processing repeats 3 times; Choose a strain to the antibacterial bandwidth mean value of aforementioned fusarium graminearum and cotton-wilt fusarium bacterial strain, be designated as the BM-2 bacterial strain greater than 15mm.
The concrete outcome of shaker test is as follows:
There are 7 strains that above-mentioned 9 kind of plant pathogenic fungies are had stronger restraining effect in the several strains that above-mentioned steps (2) separation and purification obtains, see Table 1.Wherein the bacteriostatic action of BM-2 bacterial strain is the strongest, it has stronger restraining effect to the various plants pathogenic fungi, restraining effect to cotton-wilt fusarium and fusarium graminearum is the strongest, antibacterial bandwidth has reached 18.0mm and 16.5mm respectively, and the antibacterial bandwidth of tomato early blight bacterium, southern corn leaf blight, corn being justified the pinta bacterium has all reached more than the 10mm.
Table 17 bacterial strains are to the restraining effect (mm) of 9 kinds of pathogenic fungies
2, the qualification test of BM-2 bacterial strain.
2.1 substratum
Related No. 1 substratum of Gao Shi in the qualification test, the PDA substratum, czapek agar medium, the inorganic salt starch culture-medium, the rolled oats nutrient agar, glucose asparagine nutrient agar, Wa Shi nutrient agar substratum, gelatine culture, starch culture-medium, the beef extract-peptone liquid nutrient medium, peptone water medium, the glucose peptone water substratum, lead acetate medium, urea medium, the grease substratum, the sugar alcohol fermention medium, litmus milk enrichment and phenylalanine substratum are conventional substratum, but the seawater with respective amount is replaced water when preparation.
2.2 morphological specificity and cultural characteristic are observed
2.2.1 morphological specificity is observed
Adopt skewer sheet method.Streak inoculation BM-2 bacterial strain on Gao Shi I nutrient agar flat board, the cover glass of sterilization is gone into (skewer is on the inoculation line) in the agar with about 45 skewer, the skewer sheet is inverted cultivation down for dull and stereotyped 28 ℃, purpose according to the observation, when cultivating 3d, 5d, 7d, carefully extract cover glass respectively, wipe back side culture with tweezers, to there be one of bacterium to face up and be placed on the slide glass, directly the morphological specificity of BM-2 bacterial strain on the microscopy cover glass.
Get skewer sheet and printingout through observation by light microscope, it is vigorous that the result shows that the substrate mycelium of this bacterium and aerial hyphae all grow, and branch is many; Fibrillae of spores is that linear and ripple are curved, and is no verticillate.The spore chain that forms after the fibrillae of spores maturation becomes beading.
2.2.2 cultural characteristic is observed
Main observation spore, aerial mycelium, the color of substrate mycelium and having or not of soluble pigment.The BM-2 bacterial strain is seeded in respectively on Gao Shi I nutrient agar, potato culture, czapek agar medium, inorganic salt starch culture-medium, rolled oats nutrient agar, glucose asparagine nutrient agar, the Wa Shi nutrient agar substratum, cultivate 7～15d for 28 ℃, whether the color of observed and recorded bacterial strain spore, aerial hyphae, substrate mycelium on various substratum and soluble pigment feature such as produce.
Results strain BM-2 is well-grown on nutrient agars such as Gao Shi I number, Wa Shi gravy, glucose asparagine, inorganic salt starch, and growing way is relatively poor on potato culture.The colour-change of aerial hyphae is little on different substratum, and the substrate mycelium colour-change is bigger, and no soluble pigment produces.The results are shown in Table 2.
The cultural characteristic of table 2 BM-2 bacterial strain
" ++ ++ ": well-grown; " +++": growth is general; "+": it is poor to grow.
2.3 physiological and biochemical test
Physiological and biochemical test comprises the Multitests such as oxidative fermentation test of gelatin liquification test, starch hydrolysis experiment, Mierocrystalline cellulose decomposition run, fat hydrolysis test, urease test, litmus milk test, methyl red test, V-P test, indole test, hydrogen sulfide production test, citrate test, catalase test, sugar alcohol fermentation test, amino acid decarboxylase enzyme test, phenylalanine deaminase test and glucose.Physiological and biochemical test the results are shown in Table 3.
The physio-biochemical characteristics of table 3 BM-2 bacterial strain
"+": the positive, "-": feminine gender
Sugar alcohol fermentation test result comprehensively sees Table 4.The explanation bacterium to be measured that product acid is positive in the sugar alcohol fermentation test can be decomposed this sugar or alcohol produces acid thereby make the indicator purpurum bromocresolis show yellow, and the explanation bacterium to be measured that the acid of sugar alcohol fermentation test product is negative can not be decomposed this sugar alcohol and be produced acid thereby do not make indicator manifest yellow; Sugar alcohol fermentation test aerogenesis is positive, and explanation bacterium to be measured can be decomposed this sugar or alcohol produces gas.
Table 4 BM-2 bacterial strain sugar alcohol fermentation test result
Observe and the physiological and biochemical test result according to form, cultural characteristic, consult that " uncle Jie Shi Bacteria Identification handbook the 9th edition and " streptomycete identification handbook ", the BM-2 bacterial strain meets the feature of Streptomycetaceae streptomyces.
2.4 BM-2 bacterial strain 16S rDNA Gene Sequence Analysis
2.4.1 the BM-2 strain liquid is cultivated
The BM-2 bacterial strain washes spore and to make spore suspension after slant activation with sterilized water, concentration is about 10
6Individual/mL, be inoculated in No. 1 substratum of liquid seawater Gao Shi (250mL triangular flask liquid amount 30mL), 28 ℃ of 180rpm shaking tables are cultivated 3d.
2.4.2 the extraction of DNA
Adopt the CTAB cracking process.
2.4.3 pcr amplification
The amplimer sequence is:
fD1：16SF：5′-AGAGTTTGATCATGGCTCAG-3′
rD1：16SR：5′-ACGGTTACCTTACCTTGTTACGACTT-3′
Synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Increase by table 5 reaction system and condition:
Table 5 pcr amplification system (25 μ l system)
Amplification condition is:
95 ℃ of pre-sex change 5min;
94 ℃ of sex change 1min, 65 ℃ of annealing 1min, 72 ℃ are extended 3min, 3 circulations;
94 ℃ of sex change 1min, 63 ℃ of annealing 1min, 72 ℃ are extended 3min, 3 circulations;
94 ℃ of sex change 1min, 61 ℃ of annealing 1min, 72 ℃ are extended 3min, 3 circulations;
94 ℃ of sex change 1min, 59 ℃ of annealing 1min, 72 ℃ are extended 3min, 3 circulations;
94 ℃ of sex change 1min, 57 ℃ of annealing 1min, 72 ℃ are extended 3min, 3 circulations;
94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 3min, 3 circulations;
94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 3min, 20 circulations;
72 ℃ are extended 10min once more; 4 ℃ of permanent preservations.
2.4.4 the detection of pcr amplification product and sequencing thereof
Adopt 1.0% agarose gel electrophoresis to detect the PCR product.The point sample amount is 5 μ l.The PCR product that amplifies is served sea living worker's biotechnology Services Co., Ltd to check order.Openly (accession number was: FJ595715) at Genebank on February 4th, 2009 to record sequence.In GenBank institute's calling sequence is carried out BLAST and analyze, the 16S rDNA gene order of BM-2 bacterial strain and Streptomyces sp. (FJ001761) homology reach 99%.
Combining form is learned and is observed and the Physiology and biochemistry measurement result, thinks that the BM-2 bacterial strain belongs to streptomycete (Streptomyces sp.)
3, BM-2 bacterial strain bacteriostatic test.
3.1 there be not the restraining effect of fermented liquid to the pathogenic fungi mycelial growth
(1) aseptic preparation of fermentation liquid.With the BM-2 inoculation on No. 1 medium slant of seawater Gao Shi, cultivate 4d, with the aseptic seawater flushing hypothallus of 5mL, be inoculated in the 250mL triangular flask that No. 1 substratum of 50mL liquid seawater Gao Shi is housed 25 ℃, 190r/min shaking culture 5d, nutrient solution is in 4 ℃, and the centrifugal 10min of 5000r/min removes thalline, supernatant liquor filters with the biofilter of diameter 0.22 μ m, obtains not having fermented liquid.
(2) no fermented liquid is measured the restraining effect of 9 kind of plant pathogenic fungi mycelial growths.PDA culture medium flat plate central authorities inoculation diameter be 5mm tried the plant pathogenic fungi lawn, apart from 15mm place, ware edge, symmetry is beaten the hole that diameter is 5mm, drip the no fermented liquid of 200 μ lBM-2 bacterial strains in three holes, another hole in contrast, No. 1 substratum of liquid seawater Gao Shi of interior equivalent is behind 25 ℃ of constant temperature culture 5d, observation is tried the colony growth situation of plant pathogenic fungi, measures antibacterial bandwidth.Repeat 3 times.
The scope of restraining fungi of the fermented liquid of BM-2 bacterial strain is wide, anti-microbial effect is strong, all are all had stronger restraining effect for the examination pathogenic fungi, and wherein the antibacterial bandwidth to fusarium graminearum and cotton-wilt fusarium has reached 25.1mm and 20mm respectively, has shown good prospects for application.
Table 6 BM-2 bacterial strain does not have the restraining effect of fermented liquid to pathogenic fungi
3.2 there be not the influence of fermented liquid to alternaria tenuis bacterium conidia germination
(1) preparation of alternaria tenuis bacterium conidial suspension.The alternaria tenuis bacterium of cultivating 7d is washed spore with sterilized water, and with 4 layers of filtered through gauze removal mycelia, filtrate is the centrifugal 5min of 3500r/min at room temperature, is precipitated as spore, and being mixed with concentration with the no fermented liquid of BM-2 bacterial strain is 10
6The spore suspension of individual spore/mL is contrast with No. 1 substratum of liquid seawater Gao Shi.
(2) mensuration of spore germination rate.Get the 0.1ml spore suspension and be coated on the aseptic slide, place in the culture dish that is covered with moistening filter paper, 25 ℃ of following dark constant temperature culture are respectively at 1,2,3,4,5,6h observes the spore germination situation, calculates spore germination rate, measures germ tube length.Spore germination rate as the 6h contrast is lower than 90%, then continues regularly to observe, and observes the destruction of no fermented liquid to pathogenic fungi spore and germ tube simultaneously.
Spore germination rate (%)=spore germination number/investigation spore sum
The fermented liquid of BM-2 bacterial strain has certain restraining effect to conidial sprouting of alternaria tenuis bacterium, and the spore germination rate after the processing reduces, and the germ tube elongation slowly.The fermented liquid of BM-2 bacterial strain is strong to the restraining effect of spore germination, spore germination rate after the processing obviously reduces, the 6h spore germination rate only is 30%, the germ tube growth is slow, 6h germ tube length is 140 μ m, and the germination rate height of contrast, the 6h spore germination rate reaches 96%, the germ tube fast growth, 6h germ tube length has reached 280 μ m.
Microscopic examination is the result show: the protoplasma of handling spore concentrates, the cell walls attenuation, and the germ tube growth is slower.Along with the prolongation in treatment time, the germ tube of handling spore begins deformity, distortion occurs, tubbiness, and the spore bodily form is unusual, even beading expands.And the germ tube of control group is elongated, and branch is many, seldom has vesicle to occur.
3.3 there be not the bacteriostatic action of fermented liquid to 5 kinds of bacteriums
(1) 5 kind of bacterium: intestinal bacteria (Escherichia coli), subtilis (Bacillussubtilis), streptococcus aureus (Staphylococcus aureus), Aeromonas hydrophila (Aeromonashydrophila), Yersinia (Yersinia sp.).Intestinal bacteria (Escherichia coli), subtilis (Bacillus subtilis), streptococcus aureus (Staphylococcus aureus) are cultivated with beef-protein medium.Aeromonas hydrophila (Aeromonas hydrophila), Yersinia (Yersinia sp.) are used the 2216E culture medium culturing.
(2) substratum:
Beef-protein medium: extractum carnis 0.3g, peptone 1.0g, sodium-chlor 0.5g, agar 20g, water 1000mL.
The 2216E substratum: peptone 5g, yeast extract paste 1g, agar 20g, Chen Haishui 1000mL, pH 8.0.
(3) the BM-2 bacterial strain fermentation liquor is to the bacteriostatic action measuring method of 5 kinds of bacteriums.
Adopt the punching diffusion process.5 kinds of bacteriums to cultivating 24h on the bacteria culture medium inclined-plane wash with 0.85% stroke-physiological saline solution, and being prepared into concentration is 5 * 10
6Individual/the mL bacteria suspension, get the 0.2mL bacteria suspension and drop in plate culture medium surface central authorities, evenly with the flat coating of aseptic spreading rod.With the punching around the bacterium flat board that contains at 1.5cm place, distance culture dish edge of the punch tool of diameter 5mm, the fermented liquid 50 μ L of BM-2 bacterial strain are injected in every hole respectively, are contrast with No. 1 liquid nutrient medium of aseptic seawater Gao Shi of equivalent.Constant temperature in 28 ℃ of incubators is observed behind the cultivation 24h and is measured antibacterial circle diameter, the results are shown in Table 7.
Table 7 BM-2 bacterial strain fermentation liquor is to the bacteriostatic action measurement result of bacterium
As can be seen from Table 7, the BM-2 bacterial strain fermentation liquor has very strong restraining effect to intestinal bacteria, subtilis, Aeromonas hydrophila, but to the restraining effect of streptococcus aureus and Yersinia a little less than.
4, BM-2 bacterial strain antibacterial substance produces condition optimizing
4.1 substratum produces the influence of antibacterial substance to the BM-2 bacterial strain.
Prepare 6 kinds of different liquid nutrient mediums (see Table 8, prepare), divide to install in the triangular flask of 250ml, each triangular flask 50ml that packs into Chen Haishui.Inoculum size is 7%, is under the condition of 190r/min at 28 ℃, rotating speed, cultivates 5d, and the fungistatic effect of different substratum fermented liquids is measured in 3 repetitions of every kind of substratum, determines optimum medium according to fungistatic effect.
Table 8 culture medium prescription
Found that, the bacteriostatic activity that substratum is formed the BM-2 bacterial strain fermentation liquor has considerable influence, the bacteriostatic action of No. 3 substratum fermented liquids is the strongest, the antibacterial bandwidth maximum of fermented liquid, be 9.0mm, next is 1, No. 6 substratum, thinks that No. 3 substratum are for producing the optimum medium of antimicrobial substance for BM-2 bacterial strain in the substratum of examination.
4.2 the BM-2 bacterial strain produces the antibacterial substance Optimizing Conditions of Fermentation.
Optimum medium prescription according to the generation antimicrobial substance that filters out carries out fermentation condition optimization.
4.2.1 incubation time produces the influence of antibacterial substance to the BM-2 bacterial strain.
The preparation optimum medium divides to install in the triangular flask of 250ml, every bottled 50ml that goes into.Inoculum size is 7%, puts into the shaking culture case, under 28 ℃, 180r/min condition, cultivate 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d respectively, each incubation time do 3 parallel, measure the fermented liquid fungistatic effect of different incubation times, determine the suitableeest incubation time according to fungistatic effect.
Found that, different incubation times are bigger to the bacteriostatic action influence of BM-2 bacterial strain fermentation liquor, lower at preceding 3d fermented liquid bacteriostatic activity, prolongation in time, bacteriostatic activity strengthens, when incubation time is 5d, and the antibacterial bandwidth maximum of fermented liquid, reach 7.0mm, determine that therefore it is 5d that the BM-2 bacterial strain produces the antibacterial substance Best Times.
4.2.2 culture temperature produces the influence of antibacterial substance to the BM-2 bacterial strain.
The preparation optimum medium divides to install in the triangular flask of 250ml, each triangular flask 50ml that packs into.Inoculum size 7%, rotating speed are 180r/min, respectively at 15 ℃, 20 ℃, 25 ℃, 28 ℃, 30 ℃, 35 ℃, cultivate 5d under 37 ℃ of conditions.Each temperature do 3 parallel, measure the fungistatic effect of fermented liquid.
Found that when the BM-2 bacterial strain was cultivated between 15 ℃ ~ 37 ℃, its fermented liquid all had bacteriostatic action, when culture temperature was 28 ℃, the antibacterial bandwidth maximum of fermented liquid reached 13mm, so BM-2 bacterial strain generation antibacterial substance optimum temps is 28 ℃.
4.2.3 pH produces the influence of antibacterial substance to the BM-2 bacterial strain.
The preparation optimum medium, adjust pH is respectively: 5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0, each pH value do 3 parallel.Inoculum size is to cultivate 5d under 7%, 28 ℃, 180r/min condition, measures the fungistatic effect of different pH value fermented liquids.
Measure that the antibacterial bandwidth result of BM-2 bacterial strain fermentation liquor shows under the different pH: pH is 7.5 o'clock antibacterial bandwidth maximums, can reach 13.0mm, is the best pH that produces antimicrobial substance.
4.2.4 inoculum size is produced the influence of antibacterial substance to the BM-2 bacterial strain.
The preparation optimum medium, inoculum size is respectively: 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, each inoculum size repeats 3 times.Put into 28 ℃, the shaking culture case of 180r/min and cultivate 5d, measure the fungistatic effect of different vaccination amount fermented liquid.The inoculum size experimental result shows: BM-2 is that 7% o'clock fungistatic effect is best in inoculum size.
4.2.5 the bottling amount produces the influence of antibacterial substance to the BM-2 bacterial strain.
The preparation optimum medium, liquid amount is respectively in the 250ml triangular flask: 30,40,50,60,70,80,90,100ml, each bottling amount do 3 parallel, inoculum size is 7%, 28 ℃, shaking culture 5d under the 180r/min condition measures the fungistatic effect of fermented liquid respectively.Measure the antibacterial bandwidth of BM-2 bacterial strain fermentation liquor under the different bottling amounts, the result shows: bottling amount antibacterial bandwidth when 30ml ~ 80ml constantly increases, antibacterial band was the wideest when the bottling amount was 80ml, after this increase liquid amount, fungistatic effect weakens gradually, so the bottling amount is best bottling amount during for 80ml.
4.2.6 salinity produces the influence of antibacterial substance to the BM-2 bacterial strain.
In optimum medium, add NaCl, respectively preparation contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, the 10%NaCl substratum, inoculum size is 7%, 28 ℃, 180r/min shaking culture 5d, measures the fungistatic effect of different N aCl content substratum fermented liquid.Found that, be 1%～3% o'clock at Nacl content, and along with the raising of salt concn, bacteriostatic action strengthens gradually, and concentration surpasses 3% o'clock bacteriostatic action and descends, and Nacl content is that 3% o'clock fungistatic effect is best.
4.2.7 rotating speed produces the influence of antibacterial substance to the BM-2 bacterial strain.
The preparation optimum medium, inoculum size is 7%, and shaking speed is adjusted to 160,170,180,190,200,210 respectively, 220r/min, each rotating speed do 3 parallel, cultivate 5d for 28 ℃, measure the fungistatic effect of different rotating speeds condition bottom fermentation liquid.
The antibacterial bandwidth result who measures different shaking speed bottom fermentation liquid shows: when the BM-2 bacterial strain was 190r/min at rotating speed, the antibacterial bandwidth maximum of its fermented liquid illustrated that fungistatic effect is best.When shaking speed was higher or lower than 190r/mi, bacteriostatic action reduced gradually.Therefore think that the best shaking speed of this bacterial strain is 190r/min.
Above-mentioned test is optimized BM-2 strain fermentation condition, think that the optimum medium of BM-2 bacterial strain generation antibacterial substance is: starch 14g, lactose 16g, analysis for soybean powder 16g, dipotassium hydrogen phosphate 3g, lime carbonate 4 g, Repone K 0.4g, sodium-chlor 30g, water 1000ml, pH are 7.5; Fermentation condition is: inoculum size is 7%, and the bottling amount is 80ml (a 250ml triangular flask), and shaking speed is 190r/min, and optimum temperuture is 28 ℃, and incubation time is 5d.
Embodiment
Below further describe concrete technical scheme of the present invention,, and do not constitute restriction its right so that those skilled in the art understands the present invention further.
Embodiment 1.The isolation cultivation method of a kind of streptomycete BM-2 (Streptomyces sp.BM-2) from the ocean, its step is as follows:
(1) enrichment culture: gather ooze from marine site, Lianyun Harbour, the triangular flask vibration of ooze 10g being put into the aseptic seawater that fills 50mL shakes up, and get 5mL suspension and join in the 50mL enrichment culture liquid, 190r/min, shaking table was cultivated 5 days under 25 ℃ of conditions;
(2) separation and purification of thalline: get 5 days nutrient solution 1.0mL of enrichment culture with liquid-transfering gun and join in the test tube that fills the aseptic seawater of 9.0mL, be prepared into 10
-1Sample diluting liquid; Adopt the method serial dilution of gradient dilution then, be prepared into 10 respectively
-2, 10
-3, 10
-4, 10
-5, 10
-6, 10
-7Sample diluting liquid; Get 10 respectively then
-5, 10
-6With 10
-7Sample diluting liquid 0.2ml be put on No. 1 culture medium flat plate of seawater Gao Shi, after spreading rod coating evenly, place 25 ℃ incubator to cultivate, grow bacterium colony; Distinguish the bacterium colony on No. 1 culture medium flat plate of picking seawater Gao Shi again, method with three rides is inoculated on No. 1 culture medium flat plate of seawater Gao Shi, place 25 ℃ of incubators to cultivate, rule repeatedly until obtaining pure bacterial strain list bacterium colony, the bacterial strain of several purifying is transferred to respectively in No. 1 substratum test tube slant of seawater Gao Shi preserves;
(3) screening of bacterial strain: adopt dull and stereotyped face-off culture method, for being tried bacterium, measure the anti-fungal activity of plant pathogenic of the different strains that separation and purification obtains respectively for the plant pathogenic fungi fusarium graminearum of examination and cotton-wilt fusarium; During operation the plant pathogenic fungi of the inoculation culture 5d of PDA culture medium flat plate central authorities, the different strains that around plant pathogenic fungi, obtains apart from the symmetrical streak inoculation separation and purification in culture dish wall 15mm place, be inverted constant temperature culture for 25 ℃, making plant pathogenic fungi colony growth situation is measured antibacterial bandwidth behind the cultivation 5d; Each bacterial strain is a processing, and every processing repeats 3 times; Choose a strain to the antibacterial bandwidth mean value of aforementioned 2 kind of plant pathogenic fungies bacterial strain, be designated as the BM-2 bacterial strain greater than 15mm;
(4) evaluation of bacterial strain: the BM-2 bacterial strain was cultivated 5 days down in 25 ℃, and observed and recorded bacterium colony and cellular form are carried out physiological and biochemical test simultaneously, and carry out 16S rDNA sequencing analysis; Think tentatively that according to morphology and physiological and biochemical test result bacterial strain BM-2 belongs to streptomyces; The homology analysis of 16SrDNA shows that the BM-2 bacterial strain approaches Streptomyces sp. most, and the 16S rDNA sequence similarity of the two is 99%, and the BM-2 bacterial strain is streptomycete (Streptomyces sp.).
The streptomycete BM-2 of present embodiment cultural method gained (Streptomyces sp.BM-2) has the effect that suppresses 9 kind of plant pathogenic fungi mycelial growths, and the conidial sprouting of alternaria tenuis bacterium is wherein had restraining effect; Also possess the restraining effect very strong to intestinal bacteria, subtilis and Aeromonas hydrophila.
Embodiment 2.In the isolation cultivation method of embodiment 1 described streptomycete BM-2 (Streptomyces sp.BM-2) from the ocean, the streptomycete BM-2 bacterial strain of gained? fermentation condition is: optimum medium is: starch 14g, lactose 16g, analysis for soybean powder 16g, dipotassium hydrogen phosphate 3g, lime carbonate 4g, Repone K 0.4g, sodium-chlor 30g, water 1000ml, pH are 7.5; Fermentation condition is: inoculum size 7%, bottling amount 250ml triangular flask 80ml/ bottle, shaking speed 190r/min, 28 ℃ of optimum temperutures, incubation time 5d.
Claims (1)
1. the purposes of the inhibition plant pathogenic fungi of streptomycete BM-2 (Streptomyces sp.BM-2), and the purposes that suppresses intestinal bacteria, subtilis and Aeromonas hydrophila; Described plant pathogenic fungi is that southern corn leaf blight, corn circle pinta bacterium, fusarium graminearum, cotton-wilt fusarium, wheat are avenged rotten reaping hook germ, spot defoliation bacterium, root rotof flax bacterium, alternaria tenuis germ or tomato early blight bacterium.
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| CN101919835B (en) * | 2010-07-16 | 2012-02-08 | 暨南大学 | Application of 2-acetylamino gentisic acid to preparing insulin sensitizer |
| CN102835423B (en) * | 2012-08-01 | 2014-04-16 | 淮海工学院 | Streptomyces mediolani ZW-1 bacterial strain, and bacteriostatic application of fermentation broth thereof |
| CN106434475B (en) * | 2016-11-01 | 2019-05-28 | 滕州市悟通香料有限责任公司 | One streptomycete category polysaccharide degradation bacteria and its cultural method and application |
| CN106978375A (en) * | 2017-05-12 | 2017-07-25 | 中国科学院海洋研究所 | A kind of streptomycete NHF165 bacterial strains and its application |
| CN114292785B (en) * | 2021-12-29 | 2023-07-28 | 暨南大学 | Marine streptomycete and application thereof in preventing and controlling aeromonas hydrophila from infecting aquatic animals |
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| CN108048380A (en) * | 2018-02-11 | 2018-05-18 | 海南大学 | One plant of Deccan streptomycete QY-3 and its application |
| CN108048380B (en) * | 2018-02-11 | 2021-04-02 | 海南大学 | Streptomyces debaryensis QY-3 and application thereof |
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