CN105168265A - Application of mycelium extract of umbelopsis sp. strain culture in preparing antifungal drugs - Google Patents
Application of mycelium extract of umbelopsis sp. strain culture in preparing antifungal drugs Download PDFInfo
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Abstract
The invention discloses application of a mycelium extract of an umbelopsis sp. strain culture in preparing antifungal drugs, wherein the mycelium extract of the umbelopsis sp. strain culture is prepared by a method as follows: 1) picking hyphae of umbelopsis sp. QZ042 with preservation number as CGMCC No.8101, and inoculating the hyphae on a PDA sodium culture medium for slant culture; 2) inoculating and culturing fungi obtained from slant culture on a PD liquid culture medium for several days so as to obtain a fermented culture; 3) filtering the fermented culture, and separating mycelia and a fermentation broth; and 4) leaching the mycelia by virtue of a mixed solvent, and concentrating a leaching solution so as to obtain the mycelium extract. Upon experiments, the applicant discovers that the mycelium extract has a significant bacteriostatic action on eurotium rubrum, alternaria sp., lasiodiplodia pseudotheobromae, diaporthe phaseolorum and like fungi.
Description
The application is the divisional application of " a kind of marine fungi umbrella branch trichoderma strain and mycelium extract and application " thereof, the applying date of original application is: on May 15th, 2014, application number is: 201410203253.9, and denomination of invention is: a kind of marine fungi umbrella branch trichoderma strain and mycelium extract thereof and application.
Technical field
The present invention relates to Marine microorganism strain, be specifically related to a kind of marine fungi umbrella branch trichoderma strain and mycelium extract thereof and application.
Background technology
Pathogenetic bacteria and fungus can cause vegeto-animal multiple diseases, and it not only affects crop yield and economic animal growth is produced, and causes great economic loss; And affect health, threaten human life's safety.In order to suppress and eliminate pathogenetic bacteria and fungus, people are devoted to from varying environment, find new effective antibacterials always, to alleviate the appearance of more and more serious endurance strain.
In the research of antifungal drug, nineteen thirty-nine becomes first antifungal antibiotic of discovery from the griseofulvin (Griseofulvin) of penicillium (Penilliciliumgriseofulvum) thalline, nineteen fifty-five, first antifungal drug amphotericin came out, and developed the antifungal drug such as flucytosine and azole successively again later.But at present antifungal drug still exists the shortcoming that kind lacks, selectivity is little, toxic and side effects is large and drug resistance is strong, it is very necessary therefore to research and develop novel, efficient, low toxicity, wide spectrum antifungal drug.
In the research of anti-bacterial drug, from the appearance of first antibacterials penicillin, the antibiotic such as tetracycline finally, chloromycetin, kanamycin, rifampicin, have all played more important effect on mankind's enantiopathy indigenous bacteria, but along with antibiotic rotten use.Increasing Multidrug resistance pathogen starts to occur, this is the another challenge of facing mankind, excavates new antibacterials and new antibacterial mechanisms, is one of effective ways of addressing this problem of the mankind.
Under the background that current terrestrial biological resources are more and more exhausted, from Marine microorganism, find novel medicine is a very promising approach.Wherein in January, 2010 is in February, 2013, from marine bacteria, fungus, actinomycetes, just find 895 new natural active matter (Zhao Chengying with antiviral, antibacterial, antiinflammatory, antitumor and fouling resistance, Zhu Tonghan, Zhu Weiming, the Marine microorganism new natural product of 2010-2013, organic chemistry, 2012,32,1-41).In Marine microorganism product development, the most successfully example from the mud of ocean, is separated to cephalosporium sp in 1945, therefrom found cephalosporin, develops into the cephalo-type antibiotics of series later.And evidence suggests, originally be considered to derive from the halobiontic important active substances such as sponge, as dolphin toxin, actinocongestin, numb shell ichtyhotoxisin etc., be in fact also produced by microorganism, these all make to utilize Marine microorganism development of new medicine to become focus.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of marine fungi umbrella branch trichoderma strain and mycelium extract thereof and application.
Marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042, this bacterial strain is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 19th, 2013 and (is called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences of BeiJing, China), its deposit number is CGMCCNo.8101.
Marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042 is separation from the rotten wood in ocean, purification obtains.Its isolation and purification method is: get the rotten wooden sample in ocean, a certain amount of physiological saline solution is added after smashing to pieces with aseptic glass rod, add bead again on shaking table, shake 20 ~ 30min (usually carrying out under 20 ~ 30 DEG C of conditions), get upper liquid after stratification and do 10 times of gradient dilutions, get former times of liquid, 10 times and 100 times of diluent figure cloth PDA solid plates, 26 ~ 30 DEG C of constant temperature culture, after waiting bacterium colony to grow, picking colony line purification obtains.
The feature of marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042 is for producing spore, well-grown in sea water PDA culture medium, the bacterium colony initial stage is brown, and later stage color shoals gradually, in wheel stricture of vagina shape to outgrowth, colony edge clear-cut, colony growth is comparatively slow, thin and smooth, be close to culture medium, not easily picking, has more chlamydospore, has sporangiospore and Sporangium; Conidium is ball-type, and surface is with obvious kick, and sphere diameter size is about 18 ~ 22um; Sporangium is ball-type, and sphere diameter size is about 54 ~ 60um.
Extract the DNA of this bacterial strain according to conventional methods, utilize universal primer the ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' in the ITS district of fungus and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' to increase its ITS district, gained ITS sequencing sequence is as follows:
CAGTGGGAAGTAAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCAAAAGATAATCTTTCAACTCGAAAGATTTTTTCCTTTGTGCTGGCTTTGACCGTATGTAATTTTGGGACTTAAACATGGCAGCCTTTATGGTTTGCCGGTCCCAAAAACAATATATCATCCTTATGAAAAACTTACTGAACAACTAAACAATGATTTTAATAATCTGTTTAAAACAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCAGTATCATGAGCACTCTCACTCCTAACCTTTGTGGTTATGATGTGGAATTGGGATGCGCCGATTTTTACTAGTCGGCACTCCTAAAATGTAGCTCTTGGCTGTTTCCTACTACAGCAGTTTGGCCTAATAGTTTTGACTTTTGTCAAATCTTTGGCTCCATTTGCTTCTGGAAGTCAGTCTTGATAATACAGAAAACTCATTCAAACTTGATCTAAATCAGGAGTTC。
Extract the DNA of this bacterial strain according to conventional methods, utilize universal primer 5 '-GTAGTCATATGCTTGTCTC-3 ' and NS4:5 '-CTTCCGTCAATTCCTTTAAG-3 ' to increase fungus 18srRNA gene, gained 18srRNA sequencing sequence is as follows:
ACTTCTCGTCGGAACCGACTGTTGCCAATCAGTTTCCAACAATCCAAAGGACTCACTAAGCCGTTCAATCGGTAGTAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCGAGCTGATGACTCACGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCAGCACGATGAAGTTTCAAAAGATTACCCAGACCTTCCGGCCAAGGTTATAAACTCGTTGACTTCATCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCATCAATTAAACATTGATAGTCTCTCTAAGAAGCCAAAAAGACACGACCAAAGTCATGCTGGCTATTTAGCAGAGTAAGGTCTCGTTCGTTATCGGAATTAACCAGACAAATCACTCCACGAACTAAGAACGGCCATGCACCACCACCCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTACTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCGGAACCCAAAAACTTGGCTTTCGCTGAAATGCCGAATGGGTCAATATAAAATATAACACCATCCGATCCTTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCTTCGATCCCCTAACTTTCGTTCTTGATTAATGAAAACATCCTTGACAAATGCTTTCGCAGAAGTTAGTCTTCAATAAATCCAAGAATTTCACCTCTGACAATTGAATACTAATGTCCCCAACTATCCCTATTCATCATTACTTTGGCTTTAGAAACCAACGAAATAAGGCCAAAGTCCTATTTCATTATTCCATGCTAATATGTTCAGGCTTGAAAGCCTGCTTTAAACACTCCAATTTTTTCAAAGTAAAAGTTCTGGTTCACCAGCCGCCACCGAAATGACGACTGGCTAACCCAGAAGGTGGAGCCCCGCCCGTTGAGGTACCGATCAATGAAGACCGAACCCCACAGGCGAAGGCCAAAATTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTACAAGACCCGTAAAGGCCCTGTATTGTTATTTATTGTCACTACCTCCCCGTGTCGGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCCCCGTTACCCGTTAAAAGCATGGTAGGCCACTAACCTACCATCGAAACTTGATAGGGCAGAAATTTGAATGCATCATCGCCGGCACAAGGCCATGCGATTCGATTAATTATTATGAATCACCATACAAGCGGTTGCCCGCGTTGGCTTTTTATCTAATAAGTGCACCTCTTCCAGAAGTCGAGGTTATGTACGCATGTATTAGCTCTAGAATTACCACGGTTATCCAAGTAGTAAGTGAATATCAAATAAATTATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTATAAATTTGTT。
Comprehensive above-mentioned Morphological Identification and molecular biology identification result, can determine that this bacterial strain is that Umbelopsis belongs to fungus, molecular biology identification result show its sibship and Umbelopsisisabellina nearest, but can find that its conidium and product spore device morphosis and size are all variant with Umbelopsisisabellina by identification of morphology, may be the new mutation of of Umbelopsisisabellina, this bacterial strain be fixed tentatively as Umbelopsissp.QZ042 by we.
Second object of the present invention is to provide above-mentioned marine fungi umbrella branch mould (Umbelopsissp.) the QZ042 mycelium extract of culture, this extract is using above-mentioned marine fungi umbrella branch mould (Umbelopsissp.) QZ042 as fermentation strain, liquid fermentation obtains fermentation culture medium, separation of mycelial and fermentation liquid, get the mycelium mixed solvent be made up of ethyl acetate, methanol and acetic acid and carry out lixiviate, lixiviating solution concentrates, and to obtain final product.Wherein, the volume ratio of the ethyl acetate of described composition mixed solvent, methanol and acetic acid is preferably 70 ~ 80:15 ~ 20:4 ~ 8, more preferably 75 ~ 80:15 ~ 18:5 ~ 8; Described liquid fermentation is by mould for marine fungi umbrella branch (Umbelopsissp.) QZ042 inoculation in PD fluid medium, and under 25 ~ 32 DEG C of conditions, shaking table is cultivated 5 ~ 7 days or quiescent culture 15 ~ 30 days, to obtain fermentation culture medium; When shaking table is cultivated, rotating speed is preferably 130 ~ 150r/min.
3rd object of the present invention is the preparation method of the mycelium extract providing above-mentioned marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture, comprises the following steps:
1) seed culture: picking marine fungi umbrella branch mould (Umbelopsissp.) QZ042 mycelium inoculation is cultivated 5 ~ 7 days in PDA solid medium ramp, and temperature controls at 26 ~ 30 DEG C;
2) fermentation culture: by fungal inoculum good for slant culture on PD fluid medium, under 25 ~ 32 DEG C of conditions, shaking table is cultivated 5 ~ 7 days or quiescent culture 15 ~ 30 days, obtains fermentation culture medium;
3) filter: fermentation culture medium is filtered, isolates mycelium and fermentation liquid;
4) extract: get the mycelium mixed solvent be made up of ethyl acetate, methanol and acetic acid and carry out lixiviate, lixiviating solution concentrates, and namely obtains the mycelium extract of marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture.
The step 2 of said method) in, when shaking table is cultivated, rotating speed is preferably 130 ~ 150r/min; Step 4) in, the volume ratio of the ethyl acetate of described composition mixed solvent, methanol and acetic acid is preferably 70 ~ 80:15 ~ 20:4 ~ 8, and the temperature of lixiviate is preferably 0 ~ 8 DEG C, and the time of lixiviate is preferably 3 ~ 4 days.
Applicant found through experiments, the mycelium extract of marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042 culture is when 50 μ g/ml, to escherichia coli (E.Coli), Bacillus cercus (B.cereus), nicotianae (Arthrobacternicotianae), klebsiella (Klebsiellapeneumoniae), the suppression loop diameter d of the antibacterials such as vibrio parahaemolytious (Vibrioparahaemolyticus) is respectively 12.3mm, 14.7mm, 17.2mm, 13.4mm and 22.5mm, show that the mycelium extract of marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture has antibacterial activity to antibacterial, can be used for preparing anti-bacterial drug.
Therefore, the 4th object of the present invention is to provide the application of the mycelium extract of above-mentioned marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture in preparation Chinese People's Anti-Japanese Military and Political College enterobacteria, Bacillus cercus, nicotianae, klebsiella or vibrio parahaemolytious medicine.
5th object of the present invention is then to provide the medicine of a kind of Chinese People's Anti-Japanese Military and Political College enterobacteria, Bacillus cercus, nicotianae, klebsiella or vibrio parahaemolytious, this pharmaceutical pack containing the mycelium extract of above-mentioned marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture as active component.
Applicant found through experiments, the mycelium extract of marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042 culture is when 100 μ g/ml, to loose capsule bacterium (Eurotiumrubrum), rod method mould (Alternariasp.), the two spore mould (Lasiodiplodiapseudotheobromae) of hair, the funguses such as stem canker (Diaporthephaseolorum) have obvious bacteriostasis, show that the mycelium extract of marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture has antifungal activity to fungus, can be used for preparing antifungal drug.
Therefore, the 6th object of the present invention is to provide the mycelium extract of above-mentioned marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture the anti-loose capsule bacterium of preparation, rod method is mould, the two spore of hair is mould or application in stem canker medicine.
7th object of the present invention is then to provide a kind of anti-loose capsule bacterium, rod method is mould, the two spore of hair is mould or the medicine of stem canker, and this pharmaceutical pack contains the mycelium extract of above-mentioned marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture as active component.
In the application, consisting of of described PD fluid medium: Rhizoma Solani tuber osi 200g, glucose 20g, 50% artificial seawater 1000mL; Consisting of of described PDA solid medium: Rhizoma Solani tuber osi 200g, glucose 20g, agar 20g, 50% artificial seawater 1000mL.
Compared with prior art, the invention provides a kind of new marine fungi umbrella branch mould (Umbelopsissp.) QZ042 bacterial strain, applicant finds that the mycelium extract of this strain culture has antibacterium, the antifungal activity of wide spectrum, its active substance and antimicrobial mechanism may be different from existing antibacterials, there are the potentiality of new drug development.
Accompanying drawing explanation
Marine fungi umbrella branch mould (Umbelopsissp.) QZ042 involved in the present invention, be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 19th, 2013 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences of BeiJing, China), its deposit number is CGMCCNo.8101.
Fig. 1 is the form of marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042 bacterial strain in culture medium, wherein, A represents the bacterium colony photo in PDA culture medium, B represents spore photo (amplification 10 × 10) under an optical microscope, C represents the chlamydospore under Electronic Speculum, D represents the Sporangium under Electronic Speculum, and E represents conidiophore under Electronic Speculum and mycelia;
Fig. 2 is the ITS sequence cladogram of marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042;
Fig. 3 is the 18srRNA sequence evolution tree of marine fungi umbrella branch of the present invention mould (Umbelopsissp.) QZ042.
Detailed description of the invention
Below by embodiment, the invention will be further described, but the present invention is not limited to these embodiments.
Embodiment 1: the separation andpreconcentration of marine fungi umbrella branch mould (Umbelopsissp.) QZ042
1. the separation of fungus
The rotten wooden sample in ocean is got with aseptic triangular flask, take 5g, 50ml physiological saline solution is added after adopting aseptic glass rod to smash to pieces, add bead and shake 20min on shaking table, get upper liquid after stratification and do 10 times of gradient dilutions, get former times of liquid, 10 times and 100 times of diluent figure cloth PDA solid mediums, 26 ~ 30 DEG C of constant temperature culture, after bacterium colony grows, picking colony line purification obtains.
2. the cultivation of fungus and morphological observation
Cultural characteristic is observed: by strain inoculation in PDA culture medium, and 26 DEG C of constant temperature culture obtain bacterium colony.Record primary hyphae color, colony colour change, surface character, have non-pigment to produce.
Film-making and microscopy: each bacterial strain of picking produces spore device and mycelia makes water seal sheet, and cotton blue solution-dyed, carries out Microscopic observation, record mycelia feature and branch situation, spore and product spore device structure, and Taking Pictures recording.
Result: fungus is well-grown in PDA culture medium, the bacterium colony initial stage is brown, later stage color shoals gradually, in wheel stricture of vagina shape to outgrowth, colony edge clear-cut, colony growth is slower, thin and smooth, be close to culture medium, not easily picking, there is more chlamydospore, there are sporangiospore and Sporangium, (a represents the bacterium colony photo in PDA culture medium as shown in Figure 1 for the bacterium colony of fungus and spore, b represents spore photo (amplification 10 × 10) under an optical microscope, c represents the chlamydospore under Electronic Speculum, d represents the Sporangium under Electronic Speculum, e represents conidiophore under Electronic Speculum and mycelia).The sem image of sporangiospore, Sporangium, mycelia is shown in Fig. 1, and the conidium of QZ042 is ball-type as can be seen from Figure 1, and surface is with obvious kick, and sphere diameter size is about 18 ~ 22um; Sporangium is ball-type, and sphere diameter size is about 54 ~ 60um.
3. the extraction of fungus STb gene
Be inoculated in PD fluid medium, in 26 DEG C of shake-flask culture 4d by being separated the endogenetic fungus obtained.Collecting by filtration mycelium.Mycelium adopts frozen-thawed 2 times, extracts the genome of fungus after sterilizing Glass rod grinding with " the fungal genomic DNA Mini Kit " of USAOmegaBio-Tek company.
4. the ITS Molecular Identification of fungus
Universal primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' (SEQIDNO:1) and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' (SEQIDNO:2) is used to increase intervening sequence (containing ITS1 district, 5.8S district, the ITS2 district) sequence of fungus rDNA.PCR reaction condition is: 94 DEG C of degeneration 5min, then 94 DEG C, 30s → 55 DEG C, 40s → 72 DEG C, and 1.0min carries out 35 cyclic amplifications, and last 72 DEG C extend 10min.To directly serve Hai Shenggong biotechnology Services Co., Ltd after PCR primer purification, check order with primer I TS1, gained ITS sequencing sequence is as follows:
CAGTGGGAAGTAAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCAAAAGATAATCTTTCAACTCGAAAGATTTTTTCCTTTGTGCTGGCTTTGACCGTATGTAATTTTGGGACTTAAACATGGCAGCCTTTATGGTTTGCCGGTCCCAAAAACAATATATCATCCTTATGAAAAACTTACTGAACAACTAAACAATGATTTTAATAATCTGTTTAAAACAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCAGTATCATGAGCACTCTCACTCCTAACCTTTGTGGTTATGATGTGGAATTGGGATGCGCCGATTTTTACTAGTCGGCACTCCTAAAATGTAGCTCTTGGCTGTTTCCTACTACAGCAGTTTGGCCTAATAGTTTTGACTTTTGTCAAATCTTTGGCTCCATTTGCTTCTGGAAGTCAGTCTTGATAATACAGAAAACTCATTCAAACTTGATCTAAATCAGGAGTTC(SEQIDNO：3)。
The ITS sequencing sequence of gained adopts Blastn to carry out similarity analysis in GenBank, and from GenBank, find the bacterial strain the highest with its similarity to be Umbelopsisisabellina (AJ876493.1), similarity reaches 97%.
By the Phylogenetic Analysis to bacterial strain, find that the bacterial strain Phylogenetic Relationships be separated is very unique, on Phylogenetic with Umbelopsisisabellina, Umbelopsisramanniana etc. in same branch, gained ITS sequence cladogram is as shown in Figure 2.
5. the 18srRNA gene molecule qualification of the rotten wooden fungus in ocean
Primer NS1:5 '-GTAGTCATATGCTTGTCTC-3 ' (SEQIDNO:4) and NS4:5 '-CTTCCGTCAATTCCTTTAAG-3 ' (SEQIDNO:5) is used to increase fungus 18srRNA gene order.PCR reaction condition is: 94 DEG C of degeneration 5min, then 94 DEG C, 30s → 50 DEG C, 40s → 72 DEG C, and 2min carries out 35 cyclic amplifications, and last 72 DEG C extend 10min.To directly serve Hai Shenggong biotechnology Services Co., Ltd after PCR primer purification, check order with primer NS1, gained 18srRNA sequencing sequence is as follows:
ACTTCTCGTCGGAACCGACTGTTGCCAATCAGTTTCCAACAATCCAAAGGACTCACTAAGCCGTTCAATCGGTAGTAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCGAGCTGATGACTCACGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCAGCACGATGAAGTTTCAAAAGATTACCCAGACCTTCCGGCCAAGGTTATAAACTCGTTGACTTCATCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCATCAATTAAACATTGATAGTCTCTCTAAGAAGCCAAAAAGACACGACCAAAGTCATGCTGGCTATTTAGCAGAGTAAGGTCTCGTTCGTTATCGGAATTAACCAGACAAATCACTCCACGAACTAAGAACGGCCATGCACCACCACCCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTACTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCGGAACCCAAAAACTTGGCTTTCGCTGAAATGCCGAATGGGTCAATATAAAATATAACACCATCCGATCCTTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCTTCGATCCCCTAACTTTCGTTCTTGATTAATGAAAACATCCTTGACAAATGCTTTCGCAGAAGTTAGTCTTCAATAAATCCAAGAATTTCACCTCTGACAATTGAATACTAATGTCCCCAACTATCCCTATTCATCATTACTTTGGCTTTAGAAACCAACGAAATAAGGCCAAAGTCCTATTTCATTATTCCATGCTAATATGTTCAGGCTTGAAAGCCTGCTTTAAACACTCCAATTTTTTCAAAGTAAAAGTTCTGGTTCACCAGCCGCCACCGAAATGACGACTGGCTAACCCAGAAGGTGGAGCCCCGCCCGTTGAGGTACCGATCAATGAAGACCGAACCCCACAGGCGAAGGCCAAAATTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTACAAGACCCGTAAAGGCCCTGTATTGTTATTTATTGTCACTACCTCCCCGTGTCGGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCCCCGTTACCCGTTAAAAGCATGGTAGGCCACTAACCTACCATCGAAACTTGATAGGGCAGAAATTTGAATGCATCATCGCCGGCACAAGGCCATGCGATTCGATTAATTATTATGAATCACCATACAAGCGGTTGCCCGCGTTGGCTTTTTATCTAATAAGTGCACCTCTTCCAGAAGTCGAGGTTATGTACGCATGTATTAGCTCTAGAATTACCACGGTTATCCAAGTAGTAAGTGAATATCAAATAAATTATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTATAAATTTGTT(SEQIDNO：6)。
The 18srRNA sequencing sequence of gained adopts Blastn to carry out similarity analysis in GenBank, and from GenBank, find the bacterial strain the highest with its sequence similarity to be Umbelopsisisabellina (AF157166), similarity reaches 99%.
By the Phylogenetic Analysis to bacterial strain, find the bacterial strain that is separated on Phylogenetic with Umbelopsisisabellina etc. in same branch, gained 18srRNA sequence evolution tree is as shown in Figure 3.
Comprehensive strain morphology qualification and molecular biology identification result, can determine that this bacterial strain is that Umbelopsis belongs to fungus, molecular biology identification result show its sibship and Umbelopsisisabellina nearest, but can find that its conidium and product spore device morphosis and size are all variant with Umbelopsisisabellina by identification of morphology, may be the new mutation of of Umbelopsisisabellina, this bacterial strain be fixed tentatively as Umbelopsissp.QZ042 by we.
Embodiment 2: the extraction of marine fungi umbrella branch mould (Umbelopsissp.) QZ042 active substance
1. seed culture
Culture medium: Rhizoma Solani tuber osi 180 ~ 220g, glucose 18 ~ 22g, agar 20g, 50% artificial seawater 1000mL.Picking fungal mycelia is inoculated in culture medium and carries out slant culture, cultivates 5 ~ 7 days for 28 DEG C.Wherein, the formula of artificial seawater (salinity 3.34%) is as follows: every premium on currency contains: sodium chloride nacl 26.726g, magnesium chloride Mg Cl
22.26g, magnesium sulfate MgSO
43.248g, calcium chloride CaCl
21.153g, sodium bicarbonate NaHCO
30.198g, potassium chloride KCI0.721g, sodium bromide NaBr0.058g, boric acid H
3bO
30.058g, sodium silicate Na
2siO
30.0024g, sodium silicate Na
2si
4o
90.0015g, phosphoric acid H
3pO
40.002g, chlordeneization two aluminum Al
2cl
60.013g, ammonia NH
30.002g, lithium nitrate LiNO
30.0013g.
2. fermentation culture
Fermentation medium: Rhizoma Solani tuber osi 180 ~ 220g, glucose 18 ~ 22g, 50% artificial seawater 1000mL.The formula of artificial seawater (salinity 3.34%) is the same.The fungus that slant culture is good chooses fermentation medium, cultivates 5 ~ 7 days (or adopting quiescent culture 15 ~ 30 days) under 25 ~ 32 DEG C of conditions with the rotating speed shaking table of 140r/min.
3., by above-mentioned fermentation liquid 4 layers of filtered through gauze, collect mycelium and fermentation liquid respectively;
4. the preparation of the mycelium extract of culture
The mycelium use water collected is rinsed, removes fermentation liquid, dry; Then with the mixed solvent lixiviate be made up of ethyl acetate, methanol and acetic acid (volume ratio is 80:15:5), the temperature of lixiviate is 0 ~ 8 DEG C, time is 4d, filter, lixiviating solution rotary evaporator RE-3000 55 DEG C with the rotating speed of 50r/min under rotate evaporation and concentration to small size, 70 DEG C of water-baths volatilize solvent, obtain the mycelium extract (hereinafter referred to as mycelium extract) of marine fungi umbrella branch mould (Umbelopsissp.) QZ042 culture, for subsequent use.
Embodiment 3: the antibacterial activity of the mycelium extract of the culture of marine fungi umbrella branch mould (Umbelopsissp.) QZ042
1. the preparation of indicator bacteria
E.Coli, B.cereus, Arthrobacternicotianae, Klebsiellapeneumoniae, Vibrioparahaemolyticus are inoculated in respectively in beef extract-peptone fluid medium, with the rotating speed overnight incubation of 150r/min on 37 DEG C of shaking tables.Instruction fungus Eurotiumrubrum, Alternariasp., Lasiodiplodiapseudotheobromae, Diaporthephaseolorum, Pestalotiopsismicrospora are inoculated in respectively on PDA flat board and are cultured to 1/2 of culture dish.These antibacterials and fungus preserve by this laboratory.Every strain indicator bacteria connects 2 flat boards.
2. the mensuration of antibacterial activity
Take the mycelium extract that embodiment 2 is obtained respectively, by adding sterilized water dilute solution to 400 μ g/ml, 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, dull and stereotyped filter paper agar diffusion method is adopted to measure its antibacterial activity.In culture dish, move into the indicator bacteria suspension of 500 μ L respectively, pour the beef-protein medium being melted up to 45 DEG C into, immediately mix homogeneously.After to be solidified, after suction being had the volatilization of 5 ~ 6cm aseptic filter paper sheet of liquid to be measured liquid to be leached dry, be affixed on flat board.After cultivating 8 ~ 10h at 26 DEG C, measure the size of bacteriostatic diameter by decussation method, and record.After dry with the volatilization of blank aseptic filter paper absorption lixiviating solution, as blank.Result is as described in Table 1:
Table 1: the antibacterial activity of the mycelium extract (50 μ g/ml) of the culture of marine fungi umbrella branch mould (Umbelopsissp.) QZ042
Note 1: I: control solvent ethyl acetate: methanol: acetic acid (80:15:5); II: mycelium extract (50 μ g/ml).Note 2: inhibition zone (d) size: :+: (d) <10.0mm; ++: 10.0mm<d<20.0mm; +++: d>20.0mm;-: non-activity.
3. the detection of antifungal activity
Take the mycelium extract that embodiment 2 is obtained respectively, by adding sterilized water dilute solution to 400 μ g/ml, 200 μ g/ml, 100 μ g/ml, 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, punch method is adopted to detect the antifungal activity of liquid to be measured.With the card punch of diameter 5mm, punch at the colony edge of the instruction fungus having cultivated 2-4d.Then draw the liquid to be measured of 40uL in hole, after 26 DEG C of cultivation 2d, observe antibacterial result.Draw lixiviating solution as blank.Experiment in triplicate.Result is as described in Table 2:
Table 2: the antifungal activity of the mycelium extract (100 μ g/ml) of the culture of marine fungi umbrella branch mould (Umbelopsissp.) QZ042
Note 1: I: control solvent ethyl acetate: methanol: acetic acid (70 ~ 80:15 ~ 20:4 ~ 8); II: mycelium extract (100 μ g/ml).
Note 2:+: have antibacterial activity;-: without antibacterial activity.
Claims (2)
1. the mycelium extract of marine fungi umbrella branch trichoderma strain culture the anti-loose capsule bacterium of preparation, rod method is mould, the two spore of hair is mould or application in stem canker medicine; Wherein, the mycelium extract of described marine fungi umbrella branch trichoderma strain culture is prepared by the following method:
1) seed culture: picking deposit number is that marine fungi umbrella branch mould (Umbelopsissp.) the QZ042 mycelium inoculation of CGMCCNo.8101 is cultivated 5 ~ 7 days in PDA solid medium ramp, and temperature controls at 26 ~ 30 DEG C;
2) fermentation culture: by fungal inoculum good for slant culture on PD fluid medium, under 25 ~ 32 DEG C of conditions, shaking table is cultivated 5 ~ 7 days or quiescent culture 15 ~ 30 days, obtains fermentation culture medium;
3) filter: fermentation culture medium is filtered, isolates mycelium and fermentation liquid;
4) extract: get the mycelium mixed solvent be made up of ethyl acetate, methanol and acetic acid and carry out lixiviate, lixiviating solution concentrates, and to obtain final product; The volume ratio wherein forming the ethyl acetate of mixed solvent, methanol and acetic acid is 70 ~ 80:15 ~ 20:4 ~ 8.
2. resist loose capsule bacterium, rod method is mould, the two spore of hair is mould or a medicine for stem canker, it is characterized in that: this pharmaceutical pack contains the mycelium extract of marine fungi umbrella branch trichoderma strain culture as active component; Wherein, the mycelium extract of described marine fungi umbrella branch trichoderma strain culture is prepared by the following method:
1) seed culture: picking deposit number is that marine fungi umbrella branch mould (Umbelopsissp.) the QZ042 mycelium inoculation of CGMCCNo.8101 is cultivated 5 ~ 7 days in PDA solid medium ramp, and temperature controls at 26 ~ 30 DEG C;
2) fermentation culture: by fungal inoculum good for slant culture on PD fluid medium, under 25 ~ 32 DEG C of conditions, shaking table is cultivated 5 ~ 7 days or quiescent culture 15 ~ 30 days, obtains fermentation culture medium;
3) filter: fermentation culture medium is filtered, isolates mycelium and fermentation liquid;
4) extract: get the mycelium mixed solvent be made up of ethyl acetate, methanol and acetic acid and carry out lixiviate, lixiviating solution concentrates, and to obtain final product; The volume ratio wherein forming the ethyl acetate of mixed solvent, methanol and acetic acid is 70 ~ 80:15 ~ 20:4 ~ 8.
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| CN201410203253.9A CN103952322B (en) | 2014-05-15 | 2014-05-15 | A kind of thalassiomycetes umbrella branch trichoderma strain and mycelium extract thereof and application |
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| CN201510559167.6A Expired - Fee Related CN105087396B (en) | 2014-05-15 | 2014-05-15 | A kind of preparation method of the hypha extract of marine fungi umbrella branch trichoderma strain culture |
| CN201510559187.3A Expired - Fee Related CN105087397B (en) | 2014-05-15 | 2014-05-15 | A kind of application of hypha extract of marine fungi umbrella branch trichoderma strain culture in anti-bacterial drug is prepared |
| CN201510559190.5A Expired - Fee Related CN105076220B (en) | 2014-05-15 | 2014-05-15 | A kind of hypha extract of marine fungi umbrella branch trichoderma strain culture |
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| CN201510559167.6A Expired - Fee Related CN105087396B (en) | 2014-05-15 | 2014-05-15 | A kind of preparation method of the hypha extract of marine fungi umbrella branch trichoderma strain culture |
| CN201510559187.3A Expired - Fee Related CN105087397B (en) | 2014-05-15 | 2014-05-15 | A kind of application of hypha extract of marine fungi umbrella branch trichoderma strain culture in anti-bacterial drug is prepared |
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| CN105076220A (en) | 2015-11-25 |
| CN103952322B (en) | 2016-01-06 |
| CN105168265B (en) | 2019-02-15 |
| CN105087396B (en) | 2018-02-02 |
| CN103952322A (en) | 2014-07-30 |
| CN105087397A (en) | 2015-11-25 |
| CN105087396A (en) | 2015-11-25 |
| CN105076220B (en) | 2017-11-03 |
| CN105087397B (en) | 2018-02-02 |
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