CN105168265B - A kind of hypha extract of marine fungi umbrella branch trichoderma strain culture is preparing the application in antifungal drug - Google Patents
A kind of hypha extract of marine fungi umbrella branch trichoderma strain culture is preparing the application in antifungal drug Download PDFInfo
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Abstract
The invention discloses a kind of hypha extracts of marine fungi umbrella branch trichoderma strain culture to be prepared by the following method in the hypha extract for preparing marine fungi umbrella branch trichoderma strain culture described in the application wherein in antifungal drug: 1) picking deposit number is mould (Umbelopsis sp.) the QZ042 mycelium inoculation of marine fungi umbrella branch of CGMCC No.8101 in PDA solid medium ramp culture；2) the good fungi of inclined-plane culture is inoculated on PD fluid nutrient medium and cultivates day, obtain fermentation culture medium；3) fermentation culture medium is filtered, isolates mycelium and fermentation liquid；4) take mycelium to be extracted with mixed solvent, leaching liquor concentration to get.Applicant it is discovered by experiment that above-mentioned hypha extract to bulk bacteria, rod method is mould, the double spores of hair are mould and the fungies such as stem canker have apparent bacteriostasis.
Description
The application is the divisional application of " a kind of marine fungi umbrella branch trichoderma strain and its hypha extract and application ", former Shen
The applying date please are as follows: on May 15th, 2014, application No. is: 201410203253.9, a kind of denomination of invention are as follows: marine fungi umbrella
Branch trichoderma strain and its hypha extract and application.
Technical field
The present invention relates to marine microorganism strains, and in particular to a kind of marine fungi umbrella branch trichoderma strain and its mycelium extract
Object and application.
Background technique
Pathogenetic bacteria and fungi can cause the multiple diseases of animals and plants, not only influence crop yield and economic animal is raw
Long production causes great economic loss；And human health is influenced, threaten human life's safety.In order to inhibit and eliminate disease
Indigenous bacteria and fungi, people have been devoted to find new effective antibacterials from varying environment, more and more tighter to alleviate
The appearance of the endurance strain of weight.
In the research of antifungal drug, nineteen thirty-nine comes from Penicillium notatum (Penillicilium griseofulvum) thallus
Griseofulvin (Griseofulvin) become discovery first antifungal antibiotic, first antifungal drug two of nineteen fifty-five
Property mycin come out, develop the antifungal drugs such as Flucytosine and azole successively again later.But antifungal drug is still at present
Have the shortcomings that type lacks, selectivity is small, toxic side effect is big and drug resistance is strong, therefore researches and develops novel, efficient, low
Poison, the antifungal drug of wide spectrum are very necessary.
In the research of anti-bacterial drug, from the appearance of first antibacterials penicillin, tetracycline, chlorine finally is mould
The antibiotic such as element, kanamycins, rifampin, all in the mankind to having played more important effect on pathogenic bacteria, but with antibiosis
The rotten use of element.More and more Multidrug resistance pathogens start to occur, this is the another challenge of facing mankind, excavates new antibacterial
Drug and new antibacterial mechanisms are that the mankind solve the problems, such as one of this effective ways.
Under the more and more exhausted background of current terrestrial biological resources, it is one that novel drug is found from marine microorganism
The very promising approach of item.Wherein in January, 2010 by 2 months 2013, just finds 895 from marine bacteria, fungi, actinomyces
It is a it is new have antiviral, antibacterial, anti-inflammatory, antitumor and fouling resistance natural active matter (Zhao Chengying, Zhu Tonghan, Zhu Weiming,
The marine microorganism new natural product of 2010-2013, organic chemistry, 2012,32,1-41).In marine microorganism product development most
It is to be separated to cephalosporium sp from the sludge of ocean in 1945 for successful example, therefrom has found cephalosporin, develops later
At the cephalo antibiotics of series.And evidence suggests be considered as the important work from marine organisms such as sponges originally
Property substance, such as dolphin toxin, actinocongestin, numb shell ichtyhotoxisin actually and by microorganism generate, these all make to utilize sea
Foreign microorganism exploitation newtype drug becomes 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 its hypha extract and
Using.
Mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch of the present invention, the bacterial strain is in August 19 in 2013
It is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Beijing's southern exposure day
No. 3 institutes of microbiology of the Chinese Academy of Sciences of BeiJing, China of institute of area North Star West Road 1), deposit number is CGMCC No.8101.
Mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch of the present invention is to separate from the rotten wood of ocean, is pure
Change obtains.Its isolation and purification method are as follows: take ocean rotten wood sample, a certain amount of sterile physiological salt is added after being smashed to pieces with sterile glass rod
Water adds bead and takes after concussion 20~30min (carrying out under the conditions of usually at 20~30 DEG C) on shaking table, stratification
Layer liquid does 10 times of gradient dilutions, takes former times liquid, 10 times and 100 times of dilution figure cloth PDA solid plates, 26~30 DEG C of constant temperature trainings
It supports, after waiting bacterium colonies to grow, picking colony scribing line purifying is obtained.
The feature of mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch of the present invention is to produce spore, in seawater
Well-grown in PDA culture medium, bacterium colony initial stage are brown, and later period color gradually becomes shallower as, and are in take turns line shape to outgrowth, colony edge
Clear-cut, bacterium colony growth is slower, thin and flat, is close to culture medium, is not easy picking, has more chlamydospore, there is sporangiospore
And sporangium；Conidium is ball-type, and surface has apparent kick, and sphere diameter size is about 18~22um；Sporangium
For ball-type, sphere diameter size is about 54~60um.
The DNA for extracting the bacterial strain according to conventional methods utilizes the universal primer ITS1:5 '-in the area ITS of fungi
TCCGTAGGTGAACCTGCGG-3 ' and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' expands its area ITS, and gained ITS is surveyed
Sequence sequence is as follows:
CAGTGGGAAGTAAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCAAAAGATAATCTTTC
AACTCGAAAGATTTTTTCCTTTGTGCTGGCTTTGACCGTATGTAATTTTGGGACTTAAACATGGCAGCCTTTATGGT
TTGCCGGTCCCAAAAACAATATATCATCCTTATGAAAAACTTACTGAACAACTAAACAATGATTTTAATAATCTGTT
TAAAACAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAA
TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCA
GTATCATGAGCACTCTCACTCCTAACCTTTGTGGTTATGATGTGGAATTGGGATGCGCCGATTTTTACTAGTCGGCA
CTCCTAAAATGTAGCTCTTGGCTGTTTCCTACTACAGCAGTTTGGCCTAATAGTTTTGACTTTTGTCAAATCTTTGG
CTCCATTTGCTTCTGGAAGTCAGTCTTGATAATACAGAAAACTCATTCAAACTTGATCTAAATCAGGAGTTC。
The DNA for extracting the bacterial strain according to conventional methods, using universal primer 5 '-GTAGTCATATGCTTGTCTC-3 ' and
NS4:5 '-CTTCCGTCAATTCCTTTAAG-3 ' expands fungi 18s rRNA gene, the following institute of gained 18s rRNA sequencing sequence
Show:
ACTTCTCGTCGGAACCGACTGTTGCCAATCAGTTTCCAACAATCCAAAGGACTCACTAAGCCGTTCAATCGGTAGTA
GCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCGAGCTGATGACTCACGCTTACTAGGAATTCCTCGT
TGAAGAGCAATAATTGCAATGCTCTATCCCCAGCACGATG
AAGTTTCAAAAGATTACCCAGACCTTCCGGCCAAGGTTATAAACTCGTTGACTTCATCAGTGTAGCGCGCGTGCGGC
CCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCATCAATTAAACATTGATAGTCTCTCTAAGA
AGCCAAAAAGACACGACCAAAGTCATGCTGGCTATTTAGCAGAGTAAGGTCTCGTTCGTTATCGGAATTAACCAGAC
AAATCACTCCACGAACTAAGAACGGCCATGCACCACCACCCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCT
TACTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTGCCCTTC
CGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCGGAACCCAAAAACTTGGCTTTCGCTGAAATGCCG
AATGGGTCAATATAAAATATAACACCATCCGATCCTTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGA
TCGTCTTCGATCCCCTAACTTTCGTTCTTGATTAATGAAAACATCCTTGACAAATGCTTTCGCAGAAGTTAGTCTTC
AATAAATCCAAGAATTTCACCTCTGACAATTGAATACTAATGTCCCCAACTATCCCTATTCATCATTACTTTGGCTT
TAGAAACCAACGAAATAAGGCCAAAGTCCTATTTCATTATTCCATGCTAATATGTTCAGGCTTGAAAGCCTGCTTTA
AACACTCCAATTTTTTCAAAGTAAAAGTTCTGGTTCACCAGCCGCCACCGAAATGACGACTGGCTAACCCAGAAGGT
GGAGCCCCGCCCGTTGAGGTACCGATCAATGAAGACCGAACCCCACAGGCGAAGGCCAAAATTCAACTACGAGCTTT
TTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATT
GTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTACAAGACCCGTAAAGGCCCTGTATTGTTATTTATTGTC
ACTACCTCCCCGTGTCGGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTC
CCTCTCCGGAATCGAACCCTAATTCCCCGTTACCCGTTAAAAGCATGGTAGGCCACTAACCTACCATCGAAACTTGA
TAGGGCAGAAATTTGAATGCATCATCGCCGGCACAAGGCCATGCGATTCGATTAATTATTATGAATCACCATACAAG
CGGTTGCCCGCGTTGGCTTTTTATCTAATAAGTGCACCTCTTCCAGAAGTCGAGGTTATGTACGCATGTATTAGCTC
TAGAATTACCACGGTTATCCAAGTAGTAAGTGAATATCAAATAAATTATAACTGATTTAATGAGCCATTCGCAGTTT
CACTGTATAAATTTGTT。
In summary Morphological Identification and molecular biology identification are as a result, can determine that the bacterial strain is that Umbelopsis belongs to true
Bacterium, molecular biology identification is the result shows that its affiliation and Umbelopsis isabellina are nearest, but are reflected by form
It surely, can it can be found that its conidium and production spore device morphosis and size are all variant with Umbelopsis isabellina
It can be a new mutation of Umbelopsis isabellina, we fix tentatively the bacterial strain for Umbelopsis
sp.QZ042。
Second object of the present invention is to provide above-mentioned marine fungi umbrella branch mould (Umbelopsis sp.) QZ042 culture
The hypha extract of object, the extract are using mould (Umbelopsis sp.) QZ042 of above-mentioned marine fungi umbrella branch as fermentation
Bacterial strain, liquid fermentation obtain fermentation culture medium, separate mycelium and fermentation liquid, take mycelium with by ethyl acetate, methanol and second
Acid composition mixed solvent extracted, leaching liquor concentration to get.Wherein, ethyl acetate, the methanol of the composition mixed solvent
Volume ratio with acetic acid is preferably 70~80:15~20:4~8, further preferably 75~80:15~18:5~8；Described
Liquid fermentation is that QZ042 strain inoculated is into PD fluid nutrient medium by marine fungi umbrella branch mould (Umbelopsis sp.), in 25
Shaking table culture 5~7 days or stationary culture 15~30 days under the conditions of~32 DEG C, to obtain fermentation culture medium；In shaking table culture, turn
Speed is preferably 130~150r/min.
Third object of the present invention is to provide above-mentioned marine fungi umbrella branch mould (Umbelopsis sp.) QZ042 culture
The preparation method of the hypha extract of object, comprising the following steps:
1) seed culture: mould (Umbelopsis sp.) the QZ042 mycelium inoculation of picking marine fungi umbrella branch is trained in PDA solid
It supports the culture of base ramp 5~7 days, temperature is controlled at 26~30 DEG C；
2) fermented and cultured: the good fungi of inclined-plane culture is inoculated on PD fluid nutrient medium, is shaken under the conditions of 25~32 DEG C
Bed culture 5~7 days or stationary culture 15~30 days, obtains fermentation culture medium；
3) it filters: fermentation culture medium being filtered, mycelium and fermentation liquid are isolated；
4) it extracts: taking mycelium to be extracted with the mixed solvent being made of ethyl acetate, methanol and acetic acid, leaching liquor is dense
It contracts to get the hypha extract of mould (Umbelopsis sp.) the QZ042 culture of marine fungi umbrella branch is arrived.
In the step 2) of the above method, when shaking table culture, revolving speed is preferably 130~150r/min；In step 4), described group
Volume ratio at the ethyl acetate of mixed solvent, methanol and acetic acid is preferably 70~80:15~20:4~8, and the temperature of extraction is excellent
It is selected as 0~8 DEG C, the time of extraction is preferably 3~4 days.
Applicants experimentally found that mould (Umbelopsis sp.) the QZ042 training of marine fungi umbrella branch of the present invention
The hypha extract of object is supported in 50 μ g/ml, to Escherichia coli (E.Coli), Bacillus cercus (B.cereus), tobacco
Arthrobacterium (Arthrobacter nicotianae), klebsiella (Klebsiella peneumoniae), secondary haemolysis arc
The inhibition loop diameter d of the bacteriums such as bacterium (Vibrio parahaemolyticus) be respectively 12.3mm, 14.7mm, 17.2mm,
13.4mm and 22.5mm shows the hypha extract pair of mould (Umbelopsis sp.) the QZ042 culture of marine fungi umbrella branch
Bacterium has antibacterial activity, can be used to prepare anti-bacterial drug.
Therefore, fourth object of the present invention is to provide above-mentioned marine fungi umbrella branch mould (Umbelopsis sp.)
The hypha extract of QZ042 culture is preparing anti-Escherichia coli, Bacillus cercus, nicotianae, citric acid bar
Application in bacterium or vibrio parahaemolytious drug.
5th purpose of the invention be then to provide a kind of anti-Escherichia coli, Bacillus cercus, nicotianae, gram
The drug of thunder Bai Shi bacillus or vibrio parahaemolytious, the drug include that above-mentioned marine fungi umbrella branch is mould (Umbelopsis sp.)
The hypha extract of QZ042 culture is as active constituent.
Applicants experimentally found that mould (Umbelopsis sp.) the QZ042 training of marine fungi umbrella branch of the present invention
The hypha extract of object is supported in 100 μ g/ml, to bulk bacteria (Eurotium rubrum), the mould (Alternaria of rod method
Sp.), hair double spores mould (Lasiodiplodia pseudotheobromae), stem canker (Diaporthe
) etc. phaseolorum fungies have apparent bacteriostasis, show mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch
The hypha extract of culture has antifungal activity to fungi, can be used to prepare antifungal drug.
Therefore, of the invention the 6th to be designed to provide above-mentioned marine fungi umbrella branch mould (Umbelopsis sp.)
The hypha extract of QZ042 culture prepare that anti-bulk bacteria, rod method are mould, the double spores of hair are mould or stem canker drug in
Using.
7th purpose of the invention is then to provide that a kind of anti-bulk bacteria, rod method is mould, the double spores of hair are mould or stem canker is sick
The drug of bacterium, the drug include that the mycelium of mould (Umbelopsis sp.) the QZ042 culture of above-mentioned marine fungi umbrella branch extracts
Object is as active constituent.
In the application, the composition of the PD fluid nutrient medium are as follows: potato 200g, glucose 20g, 50% artificial seawater
1000mL；The composition of the PDA solid medium are as follows: potato 200g, glucose 20g, agar 20g, 50% artificial seawater
1000mL。
Compared with prior art, the present invention provides a kind of new marine fungi umbrella branch is mould (Umbelopsis sp.)
QZ042 bacterial strain, it is found by the applicant that the hypha extract of the strain culture has the antibacterium of wide spectrum, antifungal activity,
Active material and antimicrobial mechanism may be different from existing antibacterials, and there are the potentiality of new drug development.
Detailed description of the invention
Mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch according to the present invention was protected on August 19th, 2013
It is stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Chaoyang District, Beijing City north
No. 3 institutes of microbiology of the Chinese Academy of Sciences of BeiJing, China of institute of occasion West Road 1), deposit number is CGMCC No.8101.
Fig. 1 is mould shape of (Umbelopsis sp.) the QZ042 bacterial strain on culture medium of marine fungi umbrella branch of the present invention
State, wherein A indicate PDA culture medium on bacterium colony photo, B indicate under an optical microscope spore photo (amplification factor 10 ×
10), C indicates the chlamydospore under Electronic Speculum, and D indicates that the sporangium under Electronic Speculum, E indicate the conidiophore and mycelia under Electronic Speculum；
Fig. 2 is the ITS sequence chadogram of mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch of the present invention；
Fig. 3 is the 18s rRNA sequence evolution of mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch of the present invention
Tree.
Specific embodiment
Below by embodiment, the invention will be further described, but the invention is not limited to these embodiments.
Embodiment 1: the separation and identification of mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch
1. the separation of fungi
Ocean rotten wood sample is taken with sterile triangular flask, weighs 5g, 50ml sterile physiological is added after smashing to pieces using sterile glass rod
Salt water is added bead and shakes 20min on shaking table, upper liquid is taken to do 10 times of gradient dilutions after stratification, takes former times liquid, 10
Again with 100 times of dilution figure cloth PDA solid mediums, 26~30 DEG C of constant temperature incubations, after waiting bacterium colonies to grow, picking colony scribing line is pure
Change obtains.
2. culture and the morphological observation of fungi
Cultural characteristic observation: strain is inoculated in PDA culture medium, and 26 DEG C of constant temperature incubations obtain bacterium colony.Record nascent bacterium
Silk color, surface characteristics, has non-pigment generation at colony colour variation.
Film-making and microscopy: each bacterial strain of picking produces spore device and water seal piece is made in mycelia, and the dyeing of cotton indigo plant solution see under mirror
It examines, record mycelia feature and branch situation, spore and produces spore device structure, and photograph to record.
As a result: fungi well-grown in PDA culture medium, bacterium colony initial stage are brown, and later period color gradually becomes shallower as, in wheel line
For shape to outgrowth, colony edge is clear-cut, and bacterium colony growth is slower, thin and flat, is close to culture medium, is not easy picking, has more
Chlamydospore has sporangiospore and sporangium, and (a indicates the bacterium colony in PDA culture medium as shown in Figure 1 for the bacterium colony and spore of fungi
Photo, b indicate that spore photo (amplification factor 10 × 10) under an optical microscope, c indicate the chlamydospore under Electronic Speculum, d table
Show that the sporangium under Electronic Speculum, e indicate the conidiophore and mycelia under Electronic Speculum).Sporangiospore, sporangium, mycelia electron microscope
As seeing Fig. 1, the conidium of QZ042 is ball-type as can be seen from Figure 1, and surface has apparent kick, sphere diameter size
About 18~22um；Sporangium is ball-type, and sphere diameter size is about 54~60um.
3. the extraction of fungi total DNA
Isolated endogenetic fungus is inoculated into PD fluid nutrient medium, in 26 DEG C of shaking flask culture 4d.Bacterium is collected by filtration
Filament.Mycelium uses frozen-thawed 2 times, and " the fungal gene of USA Omega Bio-Tek company is used after the Glass rod grinding that sterilizes
Group DNA Mini Kit " extracts the genome of fungi.
4. the ITS Molecular Identification of fungi
With universal primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' (SEQ ID NO:1) and ITS4:5 '-
TCCTCCGCTTATTGATATGC-3 ' (SEQ ID NO:2) expand fungi rDNA intervening sequence (area containing ITS1, the area 5.8S,
The area ITS2) sequence.PCR reaction condition are as follows: 94 DEG C of denaturation 5min, then 94 DEG C, 30s → 55 DEG C, 40s → 72 DEG C, 1.0min into
35 cyclic amplifications of row, last 72 DEG C of extensions 10min.PCR product is directly served to the raw work biotechnology clothes in sea after purification
Be engaged in Co., Ltd, is sequenced with primer I TS1, and gained ITS sequencing sequence is as follows:
CAGTGGGAAGTAAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCAAAAGATAATCTTTC
AACTCGAAAGATTTTTTCCTTTGTGCTGGCTTTGACCGTATGTAATTTTGGGACTTAAACATGGCAGCCTTTATGGT
TTGCCGGTCCCAAAAACAATATATCATCCTTATGAAAAACTTACTGAACAACTAAACAATGATTTTAATAATCTGTT
TAAAACAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAA
TTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCA
GTATCATGAGCACTCTCACTCCTAACCTTTGTGGTTATGATGTGGAATTGGGATGCGCCGATTTTTACTAGTCGGCA
CTCCTAAAATGTAGCTCTTGGCTGTTTCCTACTACAGCAGTTTGGCCTAATAGTTTTGACTTTTGTCAAATCTTTGG
CTCCATTTGCTTCTGGAAGTCAGTCTTGATAATACAGAAAACTCATTCAAACTTGATCTAAATCAGGAGTTC(SEQ
ID NO:3).
Resulting ITS sequencing sequence carries out similarity analysis using Blastn in GenBank, from GenBank
In to find with the highest bacterial strain of its similarity be Umbelopsis isabellina (AJ876493.1), similitude is up to 97%.
By the Phylogenetic Analysis to bacterial strain, it is found that isolated bacterial strain Phylogenetic Relationships are very unique, system into
With Umbelopsis isabellina, Umbelopsis ramanniana etc. in the same branch, gained ITS sequence in change relationship
Column chadogram is as shown in Figure 2.
5. the 18s rRNA gene molecule of ocean rotten wood fungi is identified
With primer NS1:5 '-GTAGTCATATGCTTGTCTC-3 ' (SEQ ID NO:4) and NS4:5 '-
CTTCCGTCAATTCCTTTAAG-3 ' (SEQ ID NO:5) expands fungi 18s rRNA gene order.PCR reaction condition are as follows: 94
DEG C denaturation 5min, then 94 DEG C, 30s → 50 DEG C, 40s → 72 DEG C, 2min carry out 35 cyclic amplifications, it is last 72 DEG C extension
10min.PCR product is directly sent to Services Co., Ltd of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd after purification, is sequenced with primer NS1, institute
It is as follows to obtain 18s rRNA sequencing sequence:
ACTTCTCGTCGGAACCGACTGTTGCCAATCAGTTTCCAACAATCCAAAGGACTCACTAAGCCGTTCAATCGGTAGTA
GCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCGAGCTGATGACTCACGCTTACTAGGAATTCCTCGT
TGAAGAGCAATAATTGCAATGCTCTATCCCCAGCACGATGAAGTTTCAAAAGATTACCCAGACCTTCCGGCCAAGGT
TATAAACTCGTTGACTTCATCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCC
TCAAACTTCCATCAATTAAACATTGATAGTCTCTCTAAGAAGCCAAAAAGACACGACCAAAGTCATGCTGGCTATTT
AGCAGAGTAAGGTCTCGTTCGTTATCGGAATTAACCAGACAAATCACTCCACGAACTAAGAACGGCCATGCACCACC
ACCCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTACTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAG
TCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACT
CCCCCCGGAACCCAAAAACTTGGCTTTCGCTGAAATGCCGAATGGGTCAATATAAAATATAACACCATCCGATCCTT
AGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCTTCGATCCCCTAACTTTCGTTCTTGATTAATG
AAAACATCCTTGACAAATGCTTTCGCAGAAGTTAGTCTTCAATAAATCCAAGAATTTCACCTCTGACAATTGAATAC
TAATGTCCCCAACTATCCCTATTCATCATTACTTTGGCTTTAGAAACCAACGAAATAAGGCCAAAGTCCTATTTCAT
TATTCCATGCTAATATGTTCAGGCTTGAAAGCCTGCTTTAAACACTCCAATTTTTTCAAAGTAAAAGTTCTGGTTCA
CCAGCCGCCACCGAAATGACGACTGGCTAACCCAGAAGGTGGAGCCCCGCCCGTTGAGGTACCGATCAATGAAGACC
GAACCCCACAGGCGAAGGCCAAAATTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTG
GAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAA
TTACAAGACCCGTAAAGGCCCTGTATTGTTATTTATTGTCACTACCTCCCCGTGTCGGGATTGGGTAATTTGCGCGC
CTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCCCCGTTACCCGT
TAAAAGCATGGTAGGCCACTAACCTACCATCGAAACTTGATAGGGCAGAAATTTGAATGCATCATCGCCGGCACAAG
GCCATGCGATTCGATTAATTATTATGAATCACCATACAAGCGGTTGCCCGCGTTGGCTTTTTATCTAATAAGTGCAC
CTCTTCCAGAAGTCGAGGTTATGTACGCATGTATTAGCTCTAGAATTACCACGGTTATCCAAGTAGTAAGTGAATAT
CAAATAAATTATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTATAAATTTGT T (SEQ ID NO:6).
Resulting 18s rRNA sequencing sequence carries out similarity analysis using Blastn in GenBank, from nucleic acid sequence
Being found in database with the highest bacterial strain of its sequence similarity is Umbelopsis isabellina (AF157166), similitude
Up to 99%.
By the Phylogenetic Analysis to bacterial strain, find isolated bacterial strain on Phylogenetic with Umbelopsis
For isabellina etc. in the same branch, gained 18s rRNA sequence evolution tree is as shown in Figure 3.
Comprehensive strain morphology is identified with molecular biology identification as a result, can determine that the bacterial strain is that Umbelopsis belongs to true
Bacterium, molecular biology identification is the result shows that its affiliation and Umbelopsis isabellina are nearest, but are reflected by form
It surely, can it can be found that its conidium and production spore device morphosis and size are all variant with Umbelopsis isabellina
It can be a new mutation of Umbelopsis isabellina, we fix tentatively the bacterial strain for Umbelopsis
sp.QZ042。
Embodiment 2: the extraction of mould (Umbelopsis sp.) the QZ042 active material of marine fungi umbrella branch
1. seed culture
Culture medium: 180~220g of potato, 18~22g of glucose, agar 20g, 50% artificial seawater 1000mL.Picking
Hypha,hyphae, which is inoculated on culture medium, carries out inclined-plane culture, and 28 DEG C are cultivated 5~7 days.Wherein, artificial seawater (salinity 3.34%)
Be formulated as follows: every liter of water contains: sodium chloride nacl 26.726g, magnesium chloride Mg Cl22.26g, magnesium sulfate MgSO43.248g calcium chloride
CaCl21.153g sodium bicarbonate NaHCO30.198g, potassium chloride KCI 0.721g, sodium bromide NaBr 0.058g, boric acid
H3BO30.058g, sodium metasilicate Na2SiO30.0024g, sodium metasilicate Na2Si4O90.0015g, phosphoric acid H3PO40.002g, chlordeneization two
Aluminium Al2Cl60.013g, ammonia NH30.002g, lithium nitrate LiNO30.0013g。
2. fermented and cultured
Fermentation medium: 180~220g of potato, glucose 18~22g, 50% artificial seawater 1000mL.Artificial seawater
The formula of (salinity 3.34%) is same as above.The good fungi of inclined-plane culture is chosen into fermentation medium, under the conditions of 25~32 DEG C with
Revolving speed shaking table culture 5~7 days (or using stationary culture 15~30 days) of 140r/min.
3. 4 layers of filtered through gauze of above-mentioned fermentation liquid are collected mycelium and fermentation liquid respectively；
4. the preparation of the hypha extract of culture
The mycelium of collection is rinsed with water, fermentation liquid is removed, it is dry；Then with by ethyl acetate, methanol and acetic acid (body
Product is 0~8 DEG C, time 4d than the mixed solvent extraction for 80:15:5) composition, the temperature of extraction, and filtering, leaching liquor revolves
Turn evaporator RE-3000 55 DEG C with the revolving speed of 50r/min under rotation be concentrated by evaporation to small size, 70 DEG C of water-baths volatilize solvent,
Obtaining the hypha extract of mould (Umbelopsis sp.) the QZ042 culture of marine fungi umbrella branch, (hereinafter referred to as mycelium mentions
Take object), it is spare.
Embodiment 3: the hypha extract of the culture of mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch resists
Bacterium activity
1. the preparation of indicator bacteria
By E.Coli, B.cereus, Arthrobacter nicotianae, Klebsiella peneumoniae,
Vibrio parahaemolyticus is inoculated in respectively in beef extract-peptone fluid nutrient medium, with 150r/ on 37 DEG C of shaking tables
The revolving speed overnight incubation of min.Indicate fungi Eurotium rubrum, Alternaria sp., Lasiodiplodia
Pseudotheobromae, Diaporthe phaseolorum, Pestalotiopsis microspora are inoculated in PDA respectively
It cultivates on plate to the 1/2 of culture dish.These bacteriums and fungi are saved by this laboratory.Every plant of indicator bacteria connects 2 plates.
2. the measurement of antibacterial activity
Weigh hypha extract made from embodiment 2 respectively, by be added sterile 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, using plate filter paper AGP test
Method measures its antibacterial activity.The instruction bacteria suspension for moving into 500 μ L respectively in culture dish, pours into the beef extract melted to 45 DEG C
Peptone culture medium is uniformly mixed immediately.After to be solidified, the 5~6cm aseptic filter paper piece liquid to be leached for having prepare liquid will be inhaled and volatilized
After dry, it is affixed on plate.After 26 DEG C of 8~10h of culture, the size of bacteriostatic diameter is measured with crossing method, and is recorded.With sky
After white aseptic filter paper absorption leaching liquor volatilization is dry, as blank control.As a result as described in Table 1:
Table 1: hypha extract (the 50 μ g/ of the culture of mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch
Ml antibacterial activity)
Note 1: I: control solvent ethyl acetate: methanol: acetic acid (80:15:5)；II: hypha extract (50 μ g/ml).Note
2: inhibition zone (d) size: :+: (d) < 10.0mm；++:10.0mm<d<20.0mm；+++:d>20.0mm；: it is inactive.
3. the detection of antifungal activity
Weigh hypha extract made from embodiment 2 respectively, by be added sterile 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, using punch method detection prepare liquid
Antifungal activity.With the punch of diameter 5mm, punched in the colony edge for the instruction fungi for having cultivated 2-4d.Then it draws
The prepare liquid of 40uL after 26 DEG C of culture 2d, observes antibacterial result in hole.Leaching liquor is drawn as blank control.Experiment repeats
Three times.As a result as described in Table 2:
Table 2: hypha extract (the 100 μ g/ of the culture of mould (Umbelopsis sp.) QZ042 of marine fungi umbrella branch
Ml antifungal activity)
Note 1: I: control solvent ethyl acetate: methanol: acetic acid (70~80:15~20:4~8)；II: hypha extract
(100μg/ml)。
Note 2:+: there is antibacterial activity；: without antibacterial activity.
Claims (2)
1. anti-bulk bacteria, rod method are mould or stem canker is sick preparing for the hypha extract of marine fungi umbrella branch trichoderma strain culture
Application in bacterium drug；Wherein, the hypha extract of the marine fungi umbrella branch trichoderma strain culture by the following method into
Row preparation:
1) seed culture: picking deposit number be CGMCC No.8101 marine fungi umbrella branch it is mould (Umbelopsis sp.)
In PDA solid medium ramp culture 5~7 days, temperature was controlled at 26~30 DEG C QZ042 mycelium inoculation；
2) fermented and cultured: the good fungi of inclined-plane culture is inoculated on PD fluid nutrient medium, and shaking table is trained under the conditions of 25~32 DEG C
It supports 5~7 days or stationary culture 15~30 days, obtains fermentation culture medium；
3) it filters: fermentation culture medium being filtered, mycelium and fermentation liquid are isolated；
4) it extracts: mycelium being taken to be extracted with the mixed solvent being made of ethyl acetate, methanol and acetic acid, leaching liquor concentration,
To obtain the final product；Wherein the volume ratio of the ethyl acetate of composition mixed solvent, methanol and acetic acid is 70~80:15~20:4~8.
2. a kind of anti-bulk bacteria, rod method be mould or the drug of stem canker, it is characterised in that: the drug includes marine fungi umbrella
The hypha extract of branch trichoderma strain culture is as active constituent；Wherein, the marine fungi umbrella branch trichoderma strain culture
Hypha extract prepared by the following method:
1) seed culture: picking deposit number be CGMCC No.8101 marine fungi umbrella branch it is mould (Umbelopsis sp.)
In PDA solid medium ramp culture 5~7 days, temperature was controlled at 26~30 DEG C QZ042 mycelium inoculation；
2) fermented and cultured: the good fungi of inclined-plane culture is inoculated on PD fluid nutrient medium, and shaking table is trained under the conditions of 25~32 DEG C
It supports 5~7 days or stationary culture 15~30 days, obtains fermentation culture medium；
3) it filters: fermentation culture medium being filtered, mycelium and fermentation liquid are isolated；
4) it extracts: mycelium being taken to be extracted with the mixed solvent being made of ethyl acetate, methanol and acetic acid, leaching liquor concentration,
To obtain the final product；Wherein the volume ratio of the ethyl acetate of composition mixed solvent, methanol and acetic acid is 70~80:15~20:4~8.
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| CN201510559089.XA CN105168265B (en) | 2014-05-15 | 2014-05-15 | A kind of hypha extract of marine fungi umbrella branch trichoderma strain culture is preparing the application in antifungal drug |
| 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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| CN102181488A (en) * | 2011-03-14 | 2011-09-14 | 昆明理工大学 | Method for preparing active phenolic compounds through rosin-induced bioconversion |
| WO2012035209A1 (en) * | 2010-09-16 | 2012-03-22 | Metsäntutkimuslaitos | Fungal extracts and uses thereof |
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| WO2012035209A1 (en) * | 2010-09-16 | 2012-03-22 | Metsäntutkimuslaitos | Fungal extracts and uses thereof |
| CN102181488A (en) * | 2011-03-14 | 2011-09-14 | 昆明理工大学 | Method for preparing active phenolic compounds through rosin-induced bioconversion |
| CN103255061A (en) * | 2012-02-15 | 2013-08-21 | 中国医学科学院医药生物技术研究所 | Penicillium griseofulvum, antibacterial active compound generated thereby and application |
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| CN103952322B (en) | 2016-01-06 |
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| CN103952322A (en) | 2014-07-30 |
| CN105087397A (en) | 2015-11-25 |
| CN105087396A (en) | 2015-11-25 |
| CN105168265A (en) | 2015-12-23 |
| CN105076220B (en) | 2017-11-03 |
| CN105087397B (en) | 2018-02-02 |
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