CN101638625A - Method for culturing orange myxobacter JCH-04 and antibiosis metabolic product - Google Patents
Method for culturing orange myxobacter JCH-04 and antibiosis metabolic product Download PDFInfo
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Abstract
The invention discloses a method for culturing orange myxobacter JCH-04 and an antibiosis metabolic product. The orange myxobacter JCH-04 (Myxococcus fulvus JCH-04) has a preservation number of CGMCCNo. 2893. A ring of a strain with the preservation number of CGMCC No. 2893 is grafted in a shake flask containing seed culture medium, and seed liquor can be obtained after 5-day culture at a temperature of 30 DEG C and a rotational speed of 180 rpm. The seed liquor is grated in a triangular flask containing fermentation culture medium according to a volume ratio of 1:10, and fermentation liquorcan be obtained after 8-day culture at a temperature of 30 DEG C and a rotational speed of 180 rpm. The fermentation liquor then undergoes 10-minute centrifuging at a rotational speed of 4000 rpm so as to obtain resin thallus and filtrate. The resin thallus is analyzed by methanol for 24 h at a temperature of 30 DEG C and a rotational speed of 180 rpm and then sequentially undergoes rotary evaporation at 50 DEG C and condensation so as to obtain an antibiosis metabolic product A. The filtrate is sequentially extracted by n-hexane and ethylacetate which are equal in volume, biomolecules with amolecular weight within 1 KD are cut off by an ultrafiltration membrane, and an antibiosis metabolic product B is obtained by using a nanofiltration membrane. The method provides microorganism resources and reduces the repetition rate of new antibiotic selection.
Description
Technical field
The present invention relates to the cultural method of myxococcus and antimicrobial substance, be specifically related to the cultural method of orange myxobacter JCH-04 and antimicrobial metabolite, belong to biological technical field.
Background technology
Slime bacteria (Myxobacteira) is the prokaryotic organism that a class has complicated many cells behavior, be a class slidably, the aerobic gram negative bacillus of obligate, in cytodifferentiation, growth and organic evolution research, occupy critical role.The secondary metabolite of slime bacteria is similar to actinomycetes, comprise multiple compound structure type, structure type according to the slime bacteria secondary metabolite of having reported, the overwhelming majority only finds novel compound in slime bacteria, up to the present, have only 4 kinds of compounds in other biological, to find, the bacterial strain of slime bacteria can produce a kind of many derivatives of basic structure, for example, Sorangium cellulosum one strain bacterium can produce the different reactive derivative of kind more than 50 of soraphen.It also is different that different strains of the same race produces the antimicrobial substance kind, differs greatly, and the distribution of the generation bacterium of identical antimicrobial substance also can cross over kind, belong to even the boundary of section.Therefore, slime bacteria is important, as to have huge drug development potentiality medicine source mushroom group.
Come from last decade, especially the fritz finds that from sorangium cellulosum after the similar structures thing epothilone of anti-cancer medicine paclitaxel, slime bacteria can produce the microbe groups that enriches secondary metabolite as a class and come into one's own day by day.Biologically active substance synthetic aspect, the positive rate of slime bacteria is higher than actinomycetes, and can produce the active substance of many brand news, outer phlegmatic temperament of born of the same parents that slime bacteria is abundant and special zymoprotein etc. also have fabulous using value.According to the discovery substance classes rank of having reported, slime bacteria is after actinomycetes and genus bacillus, and before the false pseudomonas bacillus, but the positive rate of generation active substance is the highest.
In addition, the repetition probability of screening new antibiotic is more and more higher from microorganisms such as actinomycetes at present, and slime bacteria has special advantages in this respect.See that with regard to present circumstances 50% bacterial strain can produce biologically active substance in molten bacterial flora slime bacteria, and fibrinolysin group slime bacteria even up to 96%, this screening for new antimicrobial substance provides a good quasi-microorganism resource.And be subjected to world's favor gradually as a kind of microbe groups that produces abundant and useful secondary metabolites in recent years, but wherein some antibacterium, some can be antimycotic, some is anticancer, some thrombolysis, therefore, utilize the slime bacteria fermentation of separation and purification, it is significant to seek more effective antimicrobial substance.
Summary of the invention
The objective of the invention is to: the cultural method that orange myxobacter JCH-04 and antimicrobial metabolite are provided, obtain antimicrobial substance by the orange myxobacter JCH-04 fermentation culture, for the screening of antimicrobial substance provides a quasi-microorganism resource, reduce the repetition probability of screening new antibiotic.
Technical solution of the present invention is: this orange myxobacter JCH-04 (Myxococcus fulvus JCH-04), preservation date is on 01 22nd, 2009, in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is: CGMCC No.2893.
The cultural method of antimicrobial metabolite of the present invention may further comprise the steps:
(1) go bail for and hide the bacterial classification of the orange myxobacter JCH-04 be numbered CGMCC No.2893, get a ring bacterial classification inoculation in the shaking in the bottle of seed culture medium is housed, 30 ℃ of 180rpm cultivated 5 days, seed liquor; Wherein, the 1000ml seed culture medium is made up of 10g Zulkovsky starch, 15g analysis for soybean powder, 2.5g glucose, 0.5g yeast extract paste, 1g calcium chloride, 0.5g sal epsom and 970.5g water, and pH is 7.4;
(2) above-mentioned seed liquor is inoculated in volume ratio in the triangular flask that contains fermention medium at 1: 10,30 ℃ of 180rpm cultivated 8 days, got fermented liquid; Wherein, the 1000ml fermention medium is made up of 10g Zulkovsky starch, 15g analysis for soybean powder, 2.5g glucose, 0.5g yeast extract paste, 1g calcium chloride, 0.5g sal epsom, 5gSIPI-8 resin and 965.5g water, and pH is 7.4;
(3), get resin thalline and filtrate with the centrifugal 10min of above-mentioned fermented liquid 4000rpm;
(4) the resin thalline is used and 30 ℃ of 180rpm of the isopyknic methyl alcohol of fermented liquid resolve 24h, 50 ℃ of rotary evaporations, concentrate antimicrobial metabolite A;
(5) filtrate with interior biomolecules, gets antimicrobial metabolite B through nanofiltration membrane with ultra-filtration membrane molecular weight cut-off 1KD successively by isopyknic normal hexane and ethyl acetate extracting.
Advantage of the present invention is: 1, cultivate the antimicrobial substance that obtains with orange myxobacter JCH-04 bacterium, fungi are had broad-spectrum antibacterial activity, many strains pathogenic bacterium and spoilage organism there is significant anti-microbial activity, antibacterial product A has obvious restraining effect to multiple unwanted bacteria and mould, especially aquatic product cultivation pathogenic bacterium is shown the high-efficiency broad spectrum characteristic, sanitas, feeding additive aquatic animal and medical aspect higher actual application value and vast market prospect are arranged; 2, cultural method is simple and convenient, and it is low that the microbiotic that screening obtains repeats probability.
Description of drawings
Fig. 1 contains spirogram for the G+C of the DNA of orange myxobacter JCH-04 of the present invention.
Embodiment
Example: orange myxobacter JCH-04 according to following steps cultivate antimicrobial metabolite:
(1) go bail for and hide the bacterial classification of the orange myxobacter JCH-04 be numbered CGMCC No.2893, get a ring bacterial classification inoculation in the shaking in the bottle of seed culture medium is housed, 30 ℃ of 180rpm cultivated 5 days, seed liquor; Wherein, the 1000ml seed culture medium is made up of 10g Zulkovsky starch, 15g analysis for soybean powder, 2.5g glucose, 0.5g yeast extract paste, 1g calcium chloride, 0.5g sal epsom and 970.5g water, and pH is 7.4;
(2) above-mentioned seed liquor is inoculated in volume ratio in the triangular flask that contains fermention medium at 1: 10,30 ℃ of 180rpm cultivated 8 days, got fermented liquid; Wherein, the 1000ml fermention medium is made up of 10g Zulkovsky starch, 15g analysis for soybean powder, 2.5g glucose, 0.5g yeast extract paste, 1g calcium chloride, 0.5g sal epsom, 5gSIPI-8 resin and 965.5g water, and pH is 7.4;
(3), get resin thalline and filtrate with the centrifugal 10min of above-mentioned fermented liquid 4000rpm;
(4) the resin thalline is used and 30 ℃ of 180rpm of the isopyknic methyl alcohol of fermented liquid resolve 24h, 50 ℃ of rotary evaporations, concentrate antimicrobial metabolite A;
(5) filtrate with interior biomolecules, gets antimicrobial metabolite B through nanofiltration membrane with ultra-filtration membrane molecular weight cut-off 1KD successively by isopyknic normal hexane and ethyl acetate extrct oil-soluble impurities.
The morphological specificity of orange myxobacter JCH-04 of the present invention: it is crown that the bacterium colony of orange myxobacter JCH-04 is the reddish orange circle, has the bright expansion of looking for, and single colony diameter is less than 5mm, and the film like expansion has obvious radial ripple, the secretion yellow substance; Myxospore subglobular, diameter are between 0.45 μ m * 0.45 μ m～0.51 μ m * 0.47 μ m, according to " form of uncle's Jie Shi Bacteria Identification handbook (the 9th edition) is described, and this bacterial strain meets the myxococcus morphological specificity.
The biological characteristic of orange myxobacter JCH-04 of the present invention:
1) sporophore is observed
The orange myxobacter JCH-04 sporophore is bulbous protrusions, and is soft, and shrink the lower end, and have a tangible handle, and handle usually can not be lasting; Begin in succession, under the situation of humidity afterwards with regard to deliquescence; Be generally yellowish-orange; Become scarlet after having done to brown; Diameter is about 60～300 μ m.
2) dyeing is observed
(1) with gram staining method bacterial strain is observed, oily sem observation is used in the dyeing back under opticmicroscope, and all shown in red, bacterial strain is a Gram-negative bacteria.
(2) with the Schaeffer-Fulton Albert'stain Albert method of improvement the slime bacteria of strain culturing after one week carried out Victoria Green WPB dyeing, under oily mirror, do not observe green gemma, illustrate that this strain bacterium does not produce gemma.
3) flagellum situation
Orange myxobacter JCH-04 is inoculated in puncture method in the CY substratum that contains 0.3% agar, cultivated 3 days for 30 ℃, take out and observe, the bacterium colony shape that is in line, to around diffusion, all atrichias of 8 strain slime bacterias are not described; Wherein, CY substratum (g/L): casein protolysate peptone 3, yeast extract paste 1, calcium chloride 1, agar 15, deionized water is fixed molten to 1000mL, pH7.4.
4) whether aerobic judgement
Myxococcus 04 is made bacteria suspension, get an articulating and go into to be equipped with in the test tube of CY substratum, cultivated 3 days in 30 ℃ of thermostat containers, have only the liquid level place to form one deck mycoderm, thalli growth is seldom arranged under the liquid level, illustrate that bacterial strain belongs to aerobic bacteria.
5) physiological and biochemical test
(1) the dull and stereotyped test of CMC-Na: bacterial strain does not all have transparent circle to occur, and illustrates that these bacterial strains do not have cellulolytic ability.
(2) activity test of enzyme: press middle method tests of volume " microbiology experiment " such as Shen Ping, amylase, proteolytic enzyme, lipase, nuclease, urease, oxydase and hydrogen peroxide enzyme positive illustrate that bacterial strain can produce above-mentioned each enzyme.
(3) to antibiotic drug sensitive test: with its suitableeest coating concentration (OD
600Be 0.02-0.03) coat on the CY flat board, at the Oxford cup of the equidistant placement high-temperature sterilization of dull and stereotyped different positions, again different concns gradient microbiotic 200 μ l are added on the Oxford cup (Oxford cup diameter 5mm, microbiotic is a chloromycetin, Streptomycin sulphate, kantlex, concentration gradient is respectively 5,10,15,20 μ g/ml), two of every extent of dilution are parallel 30 ℃ of following cultivations 2-3 days, observe the formation of sporophore; The result shows: the Streptomycin sulphate of anti-15 micrograms of orange myxobacter JCH-04, and to the chloromycetin and the kantlex sensitivity of 15 micrograms.
6) the G+C assay of the DNA of orange myxobacter JCH-04:
(1) strain culturing and collection: the inoculation bacterial classification is in the triangular flask that the CY substratum is housed, the shaking table of putting 200r/min is cultivated behind the 24h centrifugal, wash cell with 0.15mol/LNaCl and 0.1mol/L EDTA (pH 8.0) damping fluid, finally be dissolved in the same damping fluid, concentration is 2～3g wet cell/25ml damping fluid.
(2) extraction and purification of DNA: in above-mentioned cell suspension, add N,O-Diacetylmuramidase 0.5～1mg/ml (final concentration), put 37 ℃ of water-bath 30min; Add mass concentration 20% sodium laurylsulfonate to 2%, 60 ℃ of water-bath 10min of final concentration; Add 5mol/LNaClO
4To final concentration 1mol/L, add equal-volume chloroform-primary isoamyl alcohol (24: 1v/v), the vibration 10min, the centrifugal 10min of 5000r/min; Absorption contains the supernatant water layer of DNA, adds 2 times of volume mass concentration 95% ethanol of supernatant water layer, rolls out the DNA silk with glass rod, is dissolved in 1SSC (0.015mol/LNaCl-0.015mol/L Trisodium Citrate); Adding final concentration is the RNA enzyme of 50 μ g/ml, puts 37 ℃ of water-bath 30min; Add equal-volume chloroform-primary isoamyl alcohol and shake 10min, the centrifugal 5min of 5000r/min draws water, adds 2 times of volume mass concentration 95% ethanol of water, rolls out the DNA silk with glass rod; Add 1ml 3mol/L NaAC-0.001mol/L EDTA pH7.0, add the primary isoamyl alcohol of 0.54 times of volume while stirring, water-soluble with ultraviolet spectrophotometer measure test pure, if A
260/ A
280Pure at 1.9 explanation DNA.
(3) Tm measures: the wavelength of ultraviolet spectrophotometer is fixed in 260nm, at first writes down 25 ℃ absorbancy, temperature rises to 50 ℃ then, and every 2 ℃ of stable for some time, record temperature and absorbancy finish until the sex change of absorbancy invariant representation.
(4) the Tm value is calculated: with the temperature is X-coordinate, is ordinate zou with relative absorbancy (institute's absorbancy of being surveyed is divided by the absorbancy under 25 ℃), the drafting thermal denaturation curve, and the corresponding temperature of thermal denaturation curve mid point is and melts chain temperature (Tm value).
(5) G+C mol% cubage: under the equal conditions, be reference with the Tm value of subtilis bacterial strain DNA, G+C mol% cubage can be used following formula: 1SSC among the DNA, G+C mol%=42.2+2.44 (Tm tests bacterium-Tm Bacillus subtilus); Big absorbancy during divided by 25 ℃ draws relative absorbancy at each temperature with dna solution absorbancy under the differing temps; Result such as Fig. 1.
The result shows that the Tm value of orange myxobacter JCH-04 is 94 ℃; With this understanding, Tm value with subtilis bacterial strain DNA is 83 ℃, G+C mol% cubage can be used following formula: 1SSC among the DNA, G+Cmol%=42.2+2.44 (Tm tests bacterium-Tm subtilis bacterial strain), reach a conclusion, G+C mol%=42.2+2.44 among the DNA of bacterial strain JCH-04 * (94-83)=69.0%; By " uncle's Jie Shi Bacteria Identification handbook (the 9th edition) and related data as can be known, the feature of this data fit slime bacteria.
7) 16srDNA sequence analysis method:
The extraction of DNA is by aforementioned 6) method; Conventional pcr amplification 16SrDNA, the PCR product checks order behind Shanghai Jie Sheng company purifying; Sequence is as follows:
aacgaacgctggcggcgtgcctaacacatgcaagtcgagcgcgaataggggcaaccctta
gtagagcggcgcacgggtgcgtaacacgtggataatctgcctggatgctcgggataacca
gtcgaaagattggctaataccggataagcccacggtttcttcggagactgagggaaaagg
tggcctctgtatacaagctatcacaaccagatgagtccgcggcccatcagctagttggcg
gggtaatggcccaccaaggcgacgacgggtagctggtctgagaggacgatcagccacact
ggaactgagacacggtccagactcctacgggaggcagcagtggggaattttgcgcaatgg
gcgaaagcctgacgcagcaacgccgcgtgtgtgatgaaggtctttggattgtaaagcact
ttcgaccgggaagaaaacccgtagcccaacacgctacggcttgacggtaccgggagaaga
agcaccggctaactctgtgccagcagccgcggtaatacagagggtgcaagcgttgttcgg
aattattgggcgtaaagcgcgtgtaggcggcgtgacaagtcgggtgtgaaagccctcagc
tcaactgaggaagtgcgcccgaaactgtcgtgcttgagtgccggagagggtggcggaatt
ccccaagtagaggtgaaattcgtagatatggggaggaacaccggtggcgaaggcggccac
ctggacggtaactgacgctgagacgcgaaagcgtggggagcaaacaggattagataccct
ggtagtccacgccgtaaacgatgagaactaggtgtcgtgggagttgacccccgcggtgcc
gtagctaacgcattaagttctccgcctgggaagtacggtcgcaagactaaaactcaaagg
aattgacgggggcccgcacaagcggtggagcatgtggtttaattcgacgcaacgcgcaga
accttacctggtcttgacatcctcggaatctctcagagatgagggagtgcccgcaaggga
accgagagacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagt
cccgcaacgagcgcaaccctcgcctttagttgccacgcaagtggatctctagagggactg
ccggtgttaaaccggaggaaggtggggatgacgtcaagtcctcatggcctttatgaccag
ggctacacacgtgctacaatggccggtacagagcgttgccaacccgcgagggggagctaa
tcgcataaaaccggtctcagttcagattggagtctgcaactcgactccatgaaggcggaa
tcgctagtaatcgcagatcagcacgctgcggtgaatacgttcccgggccttgtacacacc
gcccgtcacaccatgggagtcgattgctccagaagtcatctcaccaagaggtgcccaagg
agtggtcggtaactggggtgaag
The evaluation of orange myxobacter JCH-04 of the present invention:
The 16SrDNA nucleotides sequence of orange myxobacter JCH-04 is classified 1463bp as, will carry out maximum homology in the databases such as itself and GeneBank relatively, finds that the 16SrDNA sequence and the Myxococcus 16SrDNA sequence homology of orange myxobacter JCH-04 reaches 100%; Orange myxobacter JCH-04 and myxococcus fulvus 16SrDNA nucleotide sequence differ 16bp, and at NCBI net BLAST, sequence homology is 99%; Orange myxobacter JCH-04 bacterial strain of the present invention is more similar on physio-biochemical characteristics with Myxococcus fulvus, difference to some extent in amylase and oxidase activity and drug sensitive test only, this shows that the sibship of two bacterial strains in phylogenetic systematics is very approaching; Comprehensive analysis results from morphological specificity, cultural characteristic, physio-biochemical characteristics and 16SrDNA sequence, in conjunction with " principle that uncle's Jie Shi Bacteria Identification handbook (the 9th edition) is set forth, we think that orange myxobacter JCH-04 is that Myxococcus belongs to the next subspecies of member fulvus kind.
The antimicrobial spectrum of the fermentating metabolism product of orange myxobacter JCH-04 of the present invention is measured:
1) for the examination bacterial classification:
Intestinal bacteria (Escherichia coli), vibrio cholerae (Vibri cholera), inferior Vibrio parahemolyticus (Vibrio parahaemolyticus), Aeromonas hydrophila (Aeromonas hydrophila), Pseudomonas aeruginosa (Pseudomonsaeruginosa), Acinetobacter baumannii (Acinetobacter.baumannii), flavobacterium aquatile (Flavobacterium aquatile), bacillus cereus (Bacilluscereus), Sarcina lutea (Sarcina Lutea), subtilis (Bacillus subitilis), streptococcus aureus (Staphylococcus aureus), aspergillus oryzae (Aspergillus oryzae), Mucor (Mucorjavanicus), aspergillus niger (Aspergillus niger), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is provided by the court Microbiological Lab; Fusarium graminearum (Fusariumgraminearum), cotton-wilt fusarium (Fusarium vasinfectumACCC3.224), Pyricularia oryzae (Magnaporthe grisea) are provided by the academy of agricultural sciences, Huaian.
2) bacterium antibacterial tests: microbial culture adopts nutrient agar MH, wherein, adds the salt of quality 3% when cultivating time Vibrio parahemolyticus among the MH; Be diluted to different gradients with stroke-physiological saline solution, be coated with flat board and measure OD
600, determine the suitableeest coating concentration (OD of different bacterium
600Be 0.02-0.03, it is the 9cm planar surface that different indicator 0.2ml evenly are applied to diameter, after drying, Oxford cup at the equidistant placement high-temperature sterilization of dull and stereotyped different positions, again antibacterial product 200 μ l are added on Oxford cup (Oxford cup diameter 5mm), cultivated 16-24 hour record antibacterial circle diameter size down at 37 ℃.
3) fungi antibacterial tests: fungus culture adopts potato PDA substratum; With stroke-physiological saline solution spore is washed, make spore suspension, be diluted to the suitableeest coating concentration (being covered with plate with the indicator growth is advisable), draw 0.2ml and be coated with flat board, after drying,, again antibacterial product 200 μ l are added on the Oxford cup at the Oxford cup of the equidistant placement high-temperature sterilization of dull and stereotyped different positions, cultivated 48-72 hour record antibacterial circle diameter size for 30 ℃ down.
4) the fermentating metabolism product A of orange myxobacter JCH-04 and the antimicrobial spectrum of B, as table 1:
The fermentating metabolism product A of table 1 orange myxobacter JCH-04 and the antimicrobial spectrum of B
Annotate: antibacterial circle diameter (mm) is 3 repetition mean values, A: meta-bolites A antibacterial circle diameter, B: meta-bolites B antibacterial circle diameter
By table 1 fungistatic effect as seen, the antibacterial product A of orange myxobacter JCH-04 (Myxococcusfulvus JCH-04) is to 4 kinds of gram-positive microorganisms (wax sample bud pole bacterium, streptococcus aureus, Sarcina lutea, withered grass bud pole bacterium) and 7 kinds of Gram-negative bacteria (bacillus colis, vibrio cholerae, inferior Vibrio parahemolyticus, Aeromonas hydrophila, Acinetobacter baumannii, flavobacterium aquatile, Pseudomonas aeruginosa) and 7 kinds of fungi (cereuisiae fermentum, aspergillus oryzae, aspergillus niger, Mucor, fusarium graminearum, cotton-wilt fusarium, Pyricularia oryzae) all has significant fungistatic effect, especially to causing the pathogenic bacterium Aeromonas hydrophila of aquaculture direct economic loss, Acinetobacter baumannii, flavobacterium aquatile shows efficient resistance, therefore can develop becomes a kind of natural water product cultivating with fodder additives and sanitas or medicine, has significant application value; Orange myxobacter JCH-04 (Myxococcus fulvus JCH-04) though fermentating metabolism product B only 4 kinds of gram-positive microorganisms are had anti-microbial activity, its resistance is better than fermentating metabolism product A, also is a kind of anti-microbial activity product of waiting to develop.
Sequence table
＜110〉Huaiyingong College
＜120〉cultural method of orange myxobacter JCH-04 and antimicrobial metabolite
<160>1
<210>1
<211>1463
<212>DNA
<222>(1)…(1463)
<400>1
aacgaacgctggcggcgtgcctaacacatgcaagtcgagcgcgaataggggcaaccctta
gtagagcggcgcacgggtgcgtaacacgtggataatctgcctggatgctcgggataacca
gtcgaaagattggctaataccggataagcccacggtttcttcggagactgagggaaaagg
tggcctctgtatacaagctatcacaaccagatgagtccgcggcccatcagctagttggcg
gggtaatggcccaccaaggcgacgacgggtagctggtctgagaggacgatcagccacact
ggaactgagacacggtccagactcctacgggaggcagcagtggggaattttgcgcaatgg
gcgaaagcctgacgcagcaacgccgcgtgtgtgatgaaggtctttggattgtaaagcact
ttcgaccgggaagaaaacccgtagcccaacacgctacggcttgacggtaccgggagaaga
agcaccggctaactctgtgccagcagccgcggtaatacagagggtgcaagcgttgttcgg
aattattgggcgtaaagcgcgtgtaggcggcgtgacaagtcgggtgtgaaagccctcagc
tcaactgaggaagtgcgcccgaaactgtcgtgcttgagtgccggagagggtggcggaatt
ccccaagtagaggtgaaattcgtagatatggggaggaacaccggtggcgaaggcggccac
ctggacggtaactgacgctgagacgcgaaagcgtggggagcaaacaggattagataccct
ggtagtccacgccgtaaacgatgagaactaggtgtcgtgggagttgacccccgcggtgcc
gtagctaacgcattaagttctccgcctgggaagtacggtcgcaagactaaaactcaaagg
aattgacgggggcccgcacaagcggtggagcatgtggtttaattcgacgcaacgcgcaga
accttacctggtcttgacatcctcggaatctctcagagatgagggagtgcccgcaaggga
accgagagacaggtgctgcatggctgtcgtcagctcgtgtcgtgagatgttgggttaagt
cccgcaacgagcgcaaccctcgcctttagttgccacgcaagtggatctctagagggactg
ccggtgttaaaccggaggaaggtggggatgacgtcaagtcctcatggcctttatgaccag
ggctacacacgtgctacaatggccggtacagagcgttgccaacccgcgagggggagctaa
tcgcataaaaccggtctcagttcagattggagtctgcaactcgactccatgaaggcggaa
tcgctagtaatcgcagatcagcacgctgcggtgaatacgttcccgggccttgtacacacc
gcccgtcacaccatgggagtcgattgctccagaagtcatctcaccaagaggtgcccaagg
agtggtcggtaactggggtgaag
Claims (2)
1. orange myxobacter JCH-04, it is characterized in that: the deposit number of this orange myxobacter JCH-04 (Myxococcus fulvus JCH-04) is: CGMCCNo.2893.
2. the cultural method of the antimicrobial metabolite of orange myxobacter JCH-04 according to claim 1 is characterized in that: orange myxobacter JCH-04 according to following steps cultivate antimicrobial metabolite:
(1) get the bacterial classification that the orange myxobacter JCH-04 be numbered CGMCC No.2893 is hidden in an environmental protection, bacterial classification inoculation is in being equipped with the shaking in the bottle of seed culture medium, and 30 ℃ of 180rpm cultivated 5 days, seed liquor; Wherein, the 1000ml seed culture medium is made up of 10g Zulkovsky starch, 15g analysis for soybean powder, 2.5g glucose, 0.5g yeast extract paste, 1g calcium chloride, 0.5g sal epsom and 970.5g water, and pH is 7.4;
(2) above-mentioned seed liquor is inoculated in volume ratio in the triangular flask that contains fermention medium at 1: 10,30 ℃ of 180rpm cultivated 8 days, got fermented liquid; Wherein, the 1000ml fermention medium is made up of 10g Zulkovsky starch, 15g analysis for soybean powder, 2.5g glucose, 0.5g yeast extract paste, 1g calcium chloride, 0.5g sal epsom, 5gSIPI-8 resin and 965.5g water, and pH is 7.4;
(3), get resin thalline and filtrate with the centrifugal 10min of above-mentioned fermented liquid 4000rpm;
(4) the resin thalline is used and 30 ℃ of 180rpm of the isopyknic methyl alcohol of fermented liquid resolve 24h, 50 ℃ of rotary evaporations, concentrate antimicrobial metabolite A;
(5) filtrate with interior biomolecules, gets antimicrobial metabolite B through nanofiltration membrane with ultra-filtration membrane molecular weight cut-off 1KD successively by isopyknic normal hexane and ethyl acetate extracting.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102132788A (en) * | 2011-03-22 | 2011-07-27 | 淮阴工学院 | Method for preparing and applying myxococcus fulvus probiotics for aquaculture |
| CN103045581A (en) * | 2012-12-21 | 2013-04-17 | 淮阴工学院 | Method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite |
| CN104789496A (en) * | 2015-04-01 | 2015-07-22 | 广东省微生物研究所 | Application of five myxobacteria to predation of drug-resistance bacteria and preparation of drug-resistance bacteria suppression drugs |
| CN104962507A (en) * | 2015-08-04 | 2015-10-07 | 湖南师范大学 | Myxobacteria strain and antitumor activity metabolite thereof |
| CN105648001A (en) * | 2016-01-28 | 2016-06-08 | 淮阴工学院 | Method for extracting mycosubtilin through separation and purification of myxococcus fulvus JCH-04 secondary metabolite |
-
2009
- 2009-02-26 CN CN2009100246782A patent/CN101638625B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102132788A (en) * | 2011-03-22 | 2011-07-27 | 淮阴工学院 | Method for preparing and applying myxococcus fulvus probiotics for aquaculture |
| CN102132788B (en) * | 2011-03-22 | 2012-11-21 | 淮阴工学院 | Method for preparing and applying myxococcus fulvus probiotics for aquaculture |
| CN103045581A (en) * | 2012-12-21 | 2013-04-17 | 淮阴工学院 | Method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite |
| CN103045581B (en) * | 2012-12-21 | 2014-03-12 | 淮阴工学院 | Method for immobilizing myxococcus fulvus coated by chitosan loaded with attapulgite |
| CN104789496A (en) * | 2015-04-01 | 2015-07-22 | 广东省微生物研究所 | Application of five myxobacteria to predation of drug-resistance bacteria and preparation of drug-resistance bacteria suppression drugs |
| CN107574130A (en) * | 2015-04-01 | 2018-01-12 | 广东省微生物研究所（广东省微生物分析检测中心） | Coral coccus is in predation drug-fast bacteria and is preparing the application in suppressing drug-fast bacteria medicine |
| CN107574130B (en) * | 2015-04-01 | 2020-07-28 | 广东省微生物研究所（广东省微生物分析检测中心） | Application of coral coccus in predation of drug-resistant bacteria and preparation of drug for inhibiting drug-resistant bacteria |
| CN104962507A (en) * | 2015-08-04 | 2015-10-07 | 湖南师范大学 | Myxobacteria strain and antitumor activity metabolite thereof |
| CN104962507B (en) * | 2015-08-04 | 2018-02-13 | 湖南师范大学 | One plant of slime bacteria bacterial strain and its antitumor activity metabolite |
| CN105648001A (en) * | 2016-01-28 | 2016-06-08 | 淮阴工学院 | Method for extracting mycosubtilin through separation and purification of myxococcus fulvus JCH-04 secondary metabolite |
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