CN103952322A - Umbelopsis sp. strain and mycelium extract thereof, as well as application - Google Patents
Umbelopsis sp. strain and mycelium extract thereof, as well as application Download PDFInfo
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- CN103952322A CN103952322A CN201410203253.9A CN201410203253A CN103952322A CN 103952322 A CN103952322 A CN 103952322A CN 201410203253 A CN201410203253 A CN 201410203253A CN 103952322 A CN103952322 A CN 103952322A
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Abstract
The invention discloses an Umbelopsis sp. strain and a mycelium extract thereof, as well as an application. The Umbelopsis sp. QZ042 was saved in China General Microbiological Culture Collection Center (CGMCC) on August 19, 2013 with the collection number of CGMCC No. 8101. The applicant finds that, the mycelium extract of an Umbelopsis sp. QZ042 culture disclosed by the invention has relatively good anti-bacterial activity against colon bacillus, bacillus cereus, arthrobacter nicotianae, klebsiella, bibrio parahemolyticus medicaments and other bacteria and has obvious antibacterial effect against eurotiales, alternaria, lasiodiplodia, stem canker fungi and other types of fungi through experiments and screening medicaments are provided for researching and developing new anti-bacterial and anti-fungal medicaments.
Description
Technical field
The present invention relates to marine microorganism bacterial classification, be specifically related to a kind of thalassiomycetes umbrella branch trichoderma strain and mycelium extract and application.
Background technology
Pathogenetic bacteria and fungi can cause vegeto-animal multiple diseases, and it not only affects crop yield and economic animal growth is produced, and causes great financial loss; And affect HUMAN HEALTH, threaten human life's safety.In order to suppress and to eliminate pathogenetic bacteria and fungi, people are devoted to find new effective antibacterials from varying environment 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 grisovin (Griseofulvin) of Penicillium notatum (Penillicilium griseofulvum) thalline, nineteen fifty-five, first antifungal drug amphotericin came out, and had developed successively again the antifungal drugs such as flucytosine and azole later.But at present antifungal drug still exists the shortcoming that kind lacks, selectivity is little, toxic side effect is large and resistance is strong, the antifungal drug of therefore researching and developing novel, efficient, low toxicity, wide spectrum is very necessary.
In the research of anti-bacterial drug, from the appearance of first antibacterials penicillin, the microbiotic such as tsiklomitsin finally, paraxin, kantlex, Rifampin have all been brought into play compared with vital role on mankind's enantiopathy indigenous bacteria, but along with antibiotic rotten use.Increasing Multidrug resistance pathogenic bacteria 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 more and more exhausted background of current Terrestrial biological resources, from marine microorganism, finding novel medicine is a very promising approach.Year February in January, 2010 to 2013 wherein, just from marine bacteria, fungi, actinomycetes, find 895 new natural active matter (Zhao Chengying with antiviral, antibacterial, anti-inflammatory, 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 is from the mud of ocean, to be separated to cephalosporium sp in 1945, has therefrom found cynnematin, develops into serial cephalosporin antibiotic later.And evidence suggests, originally be considered to derive from the halobiontic important active substances such as sponge, as dolphin toxin, congestin, numb shell ichtyhotoxisin etc., be in fact also by microorganisms, 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 thalassiomycetes umbrella branch trichoderma strain and mycelium extract and application.
Thalassiomycetes umbrella branch of the present invention mould (Umbelopsis sp.) 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 BeiJing, China institute of microbiology of the Chinese Academy of Sciences), its deposit number is CGMCC No:8101.
Thalassiomycetes umbrella branch of the present invention mould (Umbelopsis sp.) QZ042 is that separation from the rotten wood in ocean, purifying obtain.Its separation purification method is: get the rotten wooden sample in ocean, after smashing to pieces with aseptic glass rod, add a certain amount of stroke-physiological saline solution, add again granulated glass sphere on shaking table, to shake 20～30min (conventionally carrying out) under 20～30 ℃ of conditions, after stratification, get upper strata liquid 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 ℃ of constant temperature culture, after waiting bacterium colony to grow, picking colony line purifying obtains.
Thalassiomycetes umbrella branch of the present invention mould (Umbelopsis sp.) QZ042 is characterized as product spore, well-grown on seawater PDA substratum, the bacterium colony initial stage is brown, later stage color shoals gradually, be wheel line shape to outgrowth, colony edge clear-cut, colony growth is slower, thin and smooth, be close to substratum, be difficult for picking, have more chlamydospore, have sporangiospore and sporocyst; Conidium is ball-type, and surface is with obvious pimple, and sphere diameter size is about 18～22um; Sporocyst is ball-type, and sphere diameter size is about 54～60um.
Extract according to conventional methods the DNA of this bacterial strain, utilize universal primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' amplification QiITS district in fungi ITS district, gained ITS sequencing sequence is as follows:
CAGTGGGAAGTAAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCAAAAGATAATCTTTCAACTCGAAAGATTTTTTCCTTTGTGCTGGCTTTGACCGTATGTAATTTTGGGACTTAAACATGGCAGCCTTTATGGTTTGCCGGTCCCAAAAACAATATATCATCCTTATGAAAAACTTACTGAACAACTAAACAATGATTTTAATAATCTGTTTAAAACAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCAGTATCATGAGCACTCTCACTCCTAACCTTTGTGGTTATGATGTGGAATTGGGATGCGCCGATTTTTACTAGTCGGCACTCCTAAAATGTAGCTCTTGGCTGTTTCCTACTACAGCAGTTTGGCCTAATAGTTTTGACTTTTGTCAAATCTTTGGCTCCATTTGCTTCTGGAAGTCAGTCTTGATAATACAGAAAACTCATTCAAACTTGATCTAAATCAGGAGTTC。
Extract according to conventional methods the DNA of this bacterial strain, utilize universal primer 5 '-GTAGTCATATGCTTGTCTC-3 ' and NS4:5 '-CTTCCGTCAATTCCTTTAAG-3 ' amplification fungi 18s rRNA gene, gained 18s rRNA 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 fungi, molecular biology identification result show its sibship and Umbelopsis isabellina nearest, but can find that by identification of morphology its conidium and product spore device morphological structure and size are all variant with Umbelopsis isabellina, may be one of Umbelopsis isabellina new mutation, we fix tentatively the sp.QZ042 into Umbelopsis by this bacterial strain.
Second object of the present invention is to provide the mycelium extract of above-mentioned thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture, this extract is to using above-mentioned thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 as fermentation strain, liquid fermenting obtains fermenting culture, separation of mycelial and fermented liquid, getting mycelium uses the mixed solvent being comprised of ethyl acetate, methyl alcohol and acetic acid to carry out lixiviate, vat liquor is concentrated, obtains.Wherein, the volume ratio of the ethyl acetate of described composition mixed solvent, methyl alcohol and acetic acid is preferably 70～80:15～20:4～8, more preferably 75～80:15～18:5～8; Described liquid fermenting be by thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 inoculation in PD liquid nutrient medium, under 25～32 ℃ of conditions, shaking table is cultivated 5～7 days or standing cultivation 15～30 days, to obtain fermenting culture; When shaking table is cultivated, rotating speed is preferably 130～150r/min.
The 3rd object of the present invention is to provide the preparation method of the mycelium extract of above-mentioned thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture, comprises the following steps:
1) seed culture: picking thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 mycelium inoculation is cultivated 5～7 days in PDA solid medium ramp, and temperature is controlled at 26～30 ℃;
2) fermentation culture: the good fungi of slant culture is inoculated on PD liquid nutrient medium, and shaking table cultivation 5～7 days or standing cultivation are 15～30 days under 25～32 ℃ of conditions, obtain fermenting culture;
3) filter: fermenting culture is filtered, isolate mycelium and fermented liquid;
4) extract: get mycelium and use the mixed solvent being comprised of ethyl acetate, methyl alcohol and acetic acid to carry out lixiviate, vat liquor is concentrated, obtains the mycelium extract of thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture.
The step 2 of aforesaid 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, methyl alcohol and acetic acid is preferably 70～80:15～20:4～8, and the temperature of lixiviate is preferably 0～8 ℃, and the time of lixiviate is preferably 3～4 days.
Applicant found through experiments, the mycelium extract of thalassiomycetes umbrella branch of the present invention mould (Umbelopsis sp.) QZ042 culture is when 50 μ g/ml, to intestinal bacteria (E.Coli), bacillus cereus (B.cereus), tobacco Arthrobacter (Arthrobacter nicotianae), klebsiella (Klebsiella peneumoniae), the inhibition loop diameter d of the bacteriums such as Vibrio parahaemolyticus (Vibrio parahaemolyticus) is respectively 12.3mm, 14.7mm, 17.2mm, 13.4mm and 22.5mm, the mycelium extract that shows thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture has antibacterial activity to bacterium, can be used for preparing anti-bacterial drug.
Therefore the application of the mycelium extract that, the 4th object of the present invention is to provide above-mentioned thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture in preparation Chinese People's Anti-Japanese Military and Political College enterobacteria, bacillus cereus, tobacco Arthrobacter, klebsiella or Vibrio parahaemolyticus medicine.
The 5th object of the present invention is to provide the medicine of a kind of Chinese People's Anti-Japanese Military and Political College enterobacteria, bacillus cereus, tobacco Arthrobacter, klebsiella or Vibrio parahaemolyticus, and this pharmaceutical pack contains the mycelium extract of above-mentioned thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture as activeconstituents.
Applicant found through experiments, the mycelium extract of thalassiomycetes umbrella branch of the present invention mould (Umbelopsis sp.) QZ042 culture is when 100 μ g/ml, to loose capsule bacterium (Eurotium rubrum), chain lattice spore mould (Alternaria sp.), the two spores mould (Lasiodiplodia pseudotheobromae) of hair, the fungies such as stem canker (Diaporthe phaseolorum) have obvious bacteriostatic action, the mycelium extract that shows thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture has anti-mycotic activity to fungi, can be used for preparing antifungal drug.
Therefore the mycelium extract that, the 6th object of the present invention is to provide above-mentioned thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture the anti-loose capsule bacterium of preparation, chain lattice spore is mould, the two spores of hair are mould or stem canker medicine in application.
The 7th object of the present invention is to provide a kind of anti-loose capsule bacterium, chain lattice spore is mould, the two spores of hair are mould or the medicine of stem canker, and this pharmaceutical pack contains the mycelium extract of above-mentioned thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture as activeconstituents.
In the application, the consisting of of described PD liquid nutrient medium: potato 200g, glucose 20g, 50% artificial seawater 1000mL; Consisting of of described PDA solid medium: potato 200g, glucose 20g, agar 20g, 50% artificial seawater 1000mL.
Compared with prior art, the invention provides a kind of new thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 bacterial strain, applicant finds that the mycelium extract of this strain culture has the antibacterium of wide spectrum, anti-mycotic activity, its active substance and antibacterial mechanism may be different from existing antibacterials, have the potentiality of new drug development.
Accompanying drawing explanation
Thalassiomycetes umbrella branch involved in the present invention mould (Umbelopsis sp.) QZ042, on August 19th, 2013, be stored in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City BeiJing, China institute of microbiology of the Chinese Academy of Sciences), its deposit number is CGMCC No:8101.
Fig. 1 is the form of mould (Umbelopsis sp.) the QZ042 bacterial strain of thalassiomycetes umbrella branch of the present invention on substratum, wherein, A represents the bacterium colony photo on PDA substratum, B is illustrated in the spore photo (magnification 10 * 10) under opticmicroscope, C represents the chlamydospore under Electronic Speculum, D represents the sporocyst under Electronic Speculum, and E represents conidiophore and the mycelia under Electronic Speculum;
Fig. 2 is the ITS sequence evolutionary tree of thalassiomycetes umbrella branch of the present invention mould (Umbelopsis sp.) QZ042;
Fig. 3 is the 18s rRNA sequence evolutionary tree of thalassiomycetes umbrella branch of the present invention mould (Umbelopsis sp.) QZ042.
Embodiment
Below by embodiment, the invention will be further described, but the present invention is not limited to these embodiment.
Embodiment 1: the separated and evaluation of thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042
1. the separation of fungi
With aseptic triangular flask, get the rotten wooden sample in ocean, take 5g, after adopting aseptic glass rod to smash to pieces, add 50ml stroke-physiological saline solution, add granulated glass sphere on shaking table, to shake 20min, after stratification, get upper strata liquid 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 ℃ of constant temperature culture, after bacterium colony grows, picking colony line purifying obtains.
2. the cultivation of fungi and morphological observation
Cultural characteristic is observed: bacterial classification is inoculated on PDA substratum, and 26 ℃ of constant temperature culture obtain bacterium colony.Record primary hyphae color, colony colour variation, surface characteristic, have or not pigment formation.
Film-making and microscopy: each bacterial strain of picking produces spore device and mycelia is made water seal sheet, and cotton blue solution-dyed, carries out Microscopic observation, records mycelia feature and branch situation, spore and product spore device structure, and Taking Pictures recording.
Result: fungi is well-grown on PDA substratum, the bacterium colony initial stage is brown, later stage color shoals gradually, be wheel line shape to outgrowth, colony edge clear-cut, colony growth is slower, thin and smooth, be close to substratum, be difficult for picking, there is more chlamydospore, there are sporangiospore and sporocyst, (a represents the bacterium colony photo on PDA substratum as shown in Figure 1 for the bacterium colony of fungi and spore, b is illustrated in the spore photo (magnification 10 * 10) under opticmicroscope, c represents the chlamydospore under Electronic Speculum, d represents the sporocyst under Electronic Speculum, e represents conidiophore and the mycelia under Electronic Speculum).The sem image of sporangiospore, sporocyst, 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 pimple, and sphere diameter size is about 18～22um; Sporocyst is ball-type, and sphere diameter size is about 54～60um.
3. the extraction of the total DNA of fungi
The endogenetic fungus that separation is obtained is inoculated in PD liquid nutrient medium, in 26 ℃ of shake-flask culture 4d.Filter and collect mycelium.Mycelium adopts frozen-thawed 2 times, extracts the genome of fungi after sterilizing Glass rod grinding with " fungal genomic DNA extracts test kit in a small amount " of USA Omega Bio-Tek company.
4. the ITS Molecular Identification of fungi
Use intervening sequence (containing ITS1 district, 5.8S district, the ITS2 district) sequence of universal primer ITS1:5 '-TCCGTAGGTGAACCTGCGG-3 ' (SEQ ID NO:1) and ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ' (SEQ ID NO:2) amplification fungi rDNA.PCR reaction conditions is: 94 ℃ of sex change 5min, and then 94 ℃, 30s → 55 ℃, 40s → 72 ℃, 1.0min carries out 35 cyclic amplifications, and last 72 ℃ are extended 10min.To after PCR product purification, directly serve Hai Shenggong biotechnology Services Co., Ltd, with primer I TS1 order-checking, gained ITS sequencing sequence is as follows:
CAGTGGGAAGTAAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACCAAAAGATAATCTTTCAACTCGAAAGATTTTTTCCTTTGTGCTGGCTTTGACCGTATGTAATTTTGGGACTTAAACATGGCAGCCTTTATGGTTTGCCGGTCCCAAAAACAATATATCATCCTTATGAAAAACTTACTGAACAACTAAACAATGATTTTAATAATCTGTTTAAAACAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTCAGTATCATGAGCACTCTCACTCCTAACCTTTGTGGTTATGATGTGGAATTGGGATGCGCCGATTTTTACTAGTCGGCACTCCTAAAATGTAGCTCTTGGCTGTTTCCTACTACAGCAGTTTGGCCTAATAGTTTTGACTTTTGTCAAATCTTTGGCTCCATTTGCTTCTGGAAGTCAGTCTTGATAATACAGAAAACTCATTCAAACTTGATCTAAATCAGGAGTTC(SEQ?ID?NO：3)。
The ITS sequencing sequence of gained adopts Blastn to carry out similarity analysis in GenBank, and from GenBank, finding the bacterial strain the highest with its similarity is Umbelopsis isabellina (AJ876493.1), and similarity reaches 97%.
By the Phylogenetic Analysis to bacterial strain, find that separated bacterial strain Phylogenetic Relationships is very unique, on Phylogenetic with Umbelopsis isabellina, Umbelopsis ramanniana etc. in same branch, gained ITS sequence evolutionary tree is as shown in Figure 2.
5. the 18s rRNA gene molecule of the rotten wooden fungi in ocean is identified
Use primer NS1:5 '-GTAGTCATATGCTTGTCTC-3 ' (SEQ ID NO:4) and NS4:5 '-CTTCCGTCAATTCCTTTAAG-3 ' (SEQ ID NO:5) amplification fungi 18s rRNA gene order.PCR reaction conditions is: 94 ℃ of sex change 5min, and then 94 ℃, 30s → 50 ℃, 40s → 72 ℃, 2min carries out 35 cyclic amplifications, and last 72 ℃ are extended 10min.To after PCR product purification, directly serve Hai Shenggong biotechnology Services Co., Ltd, with primer NS1 order-checking, gained 18s rRNA sequencing sequence is as follows:
ACTTCTCGTCGGAACCGACTGTTGCCAATCAGTTTCCAACAATCCAAAGGACTCACTAAGCCGTTCAATCGGTAGTAGCGACGGGCGGTGTGTACAAAGGGCAGGGACGTAATCAACGCGAGCTGATGACTCACGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCAGCACGATGAAGTTTCAAAAGATTACCCAGACCTTCCGGCCAAGGTTATAAACTCGTTGACTTCATCAGTGTAGCGCGCGTGCGGCCCAGAACATCTAAGGGCATCACAGACCTGTTATTGCCTCAAACTTCCATCAATTAAACATTGATAGTCTCTCTAAGAAGCCAAAAAGACACGACCAAAGTCATGCTGGCTATTTAGCAGAGTAAGGTCTCGTTCGTTATCGGAATTAACCAGACAAATCACTCCACGAACTAAGAACGGCCATGCACCACCACCCATAGAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTACTATGTCTGGACCTGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGTGGTGCCCTTCCGTCAATTCCTTTAAGTTTCAGCCTTGCGACCATACTCCCCCCGGAACCCAAAAACTTGGCTTTCGCTGAAATGCCGAATGGGTCAATATAAAATATAACACCATCCGATCCTTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCTTCGATCCCCTAACTTTCGTTCTTGATTAATGAAAACATCCTTGACAAATGCTTTCGCAGAAGTTAGTCTTCAATAAATCCAAGAATTTCACCTCTGACAATTGAATACTAATGTCCCCAACTATCCCTATTCATCATTACTTTGGCTTTAGAAACCAACGAAATAAGGCCAAAGTCCTATTTCATTATTCCATGCTAATATGTTCAGGCTTGAAAGCCTGCTTTAAACACTCCAATTTTTTCAAAGTAAAAGTTCTGGTTCACCAGCCGCCACCGAAATGACGACTGGCTAACCCAGAAGGTGGAGCCCCGCCCGTTGAGGTACCGATCAATGAAGACCGAACCCCACAGGCGAAGGCCAAAATTCAACTACGAGCTTTTTAACTGCAACAACTTTAATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGCACCAGACTTGCCCTCCAATTGTTCCTCGTTAAGGGATTTAAATTGTACTCATTCCAATTACAAGACCCGTAAAGGCCCTGTATTGTTATTTATTGTCACTACCTCCCCGTGTCGGGATTGGGTAATTTGCGCGCCTGCTGCCTTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTAATTCCCCGTTACCCGTTAAAAGCATGGTAGGCCACTAACCTACCATCGAAACTTGATAGGGCAGAAATTTGAATGCATCATCGCCGGCACAAGGCCATGCGATTCGATTAATTATTATGAATCACCATACAAGCGGTTGCCCGCGTTGGCTTTTTATCTAATAAGTGCACCTCTTCCAGAAGTCGAGGTTATGTACGCATGTATTAGCTCTAGAATTACCACGGTTATCCAAGTAGTAAGTGAATATCAAATAAATTATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTATAAATTTGTT(SEQ?ID?NO：6)。
The 18s rRNA sequencing sequence of gained adopts Blastn to carry out similarity analysis in GenBank, and from GenBank, finding the bacterial strain the highest with its sequence similarity degree is Umbelopsis isabellina (AF157166), and similarity reaches 99%.
By the Phylogenetic Analysis to bacterial strain, find separated bacterial strain on Phylogenetic with Umbelopsis isabellina etc. in same branch, gained 18s rRNA sequence evolutionary tree is as shown in Figure 3.
Comprehensive strain morphology is learned and is identified and molecular biology identification result, can determine that this bacterial strain is that Umbelopsis belongs to fungi, molecular biology identification result show its sibship and Umbelopsis isabellina nearest, but can find that by identification of morphology its conidium and product spore device morphological structure and size are all variant with Umbelopsis isabellina, may be one of Umbelopsis isabellina new mutation, we fix tentatively the sp.QZ042 into Umbelopsis by this bacterial strain.
Embodiment 2: the extraction of thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 active substance
1. seed culture
Substratum: potato 180～220g, glucose 18～22g, agar 20g, 50% artificial seawater 1000mL.Picking hypha,hyphae is inoculated on substratum and carries out slant culture, cultivates 5～7 days for 28 ℃.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, Repone K KCI0.721g, Sodium Bromide NaBr0.058g, boric acid H
3bO
30.058g, water glass Na
2siO
30.0024g, water glass Na
2si
4o
90.0015g, phosphoric acid H
3pO
40.002g, chlordeneization two aluminium Al
2cl
60.013g,, ammonia NH
30.002g, lithium nitrate LiNO
30.0013g.
2. fermentation culture
Fermention medium: potato 180～220g, glucose 18～22g, 50% artificial seawater 1000mL.The formula of artificial seawater (salinity 3.34%) is the same.The fungi that slant culture is good is chosen to fermention medium, and the rotating speed shaking table with 140r/min under 25～32 ℃ of conditions is cultivated 5～7 days (or adopting standing cultivation 15～30 days).
3. by 4 layers of filtered through gauze for above-mentioned fermented liquid, collect respectively mycelium and fermented liquid;
4. the preparation of the mycelium extract of culture
The mycelium water of collecting is rinsed, remove fermented liquid, dry; Then use the mixed solvent lixiviate being formed by ethyl acetate, methyl alcohol and acetic acid (volume ratio is 80:15:5), the temperature of lixiviate is 0～8 ℃, time is 4d, filter, vat liquor rotates evaporation concentration to small volume with rotatory evaporator RE-3000 under 55 ℃ of rotating speeds with 50r/min, 70 ℃ of water-baths volatilize solvent, obtain the mycelium extract (hereinafter to be referred as mycelium extract) of thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture, standby.
Embodiment 3: the anti-microbial activity of the mycelium extract of the culture of thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042
1. the preparation of indicator
E.Coli, B.cereus, Arthrobacter nicotianae, Klebsiella peneumoniae, Vibrio parahaemolyticus are inoculated in respectively in beef extract-peptone liquid nutrient medium, on 37 ℃ of shaking tables with the rotating speed overnight incubation of 150r/min.Indication fungi Eurotium rubrum, Alternaria sp., Lasiodiplodia pseudotheobromae, Diaporthe phaseolorum, Pestalotiopsis microspora are inoculated in respectively and on PDA flat board, are cultured to 1/2 of culture dish.These bacteriums and fungi preserve by this laboratory.Every strain indicator connects 2 flat boards.
2. the mensuration of antibacterial activity
Take respectively the mycelium extract that embodiment 2 makes, by adding sterilized water diluting soln 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, adopt dull and stereotyped filter paper agar diffusion method to measure its anti-microbial activity.In culture dish, move into respectively the indicator suspension of 500 μ L, pour the beef-protein medium that is melted up to 45 ℃ into, mix immediately.After solidifying, suction is had to 5～6cm aseptic filter paper sheet of liquid to be measured after vat liquor volatilization is dry, be affixed on flat board.At 26 ℃, cultivate after 8～10h, by right-angled intersection method, measure the size of bacteriostatic diameter, and record.After dry with the volatilization of blank aseptic filter paper absorption vat liquor, 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 thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042
Note 1: I: control solvent ethyl acetate: methyl alcohol: 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 anti-mycotic activity
Take respectively the mycelium extract that embodiment 2 makes, by adding sterilized water diluting soln 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, adopt punch method to detect the anti-mycotic activity of liquid to be measured.With the punch tool of diameter 5mm, in the colony edge punching of having cultivated the indication fungi of 2-4d.Then draw the liquid to be measured of 40uL in hole, cultivate after 2d, observe antibacterial result for 26 ℃.Draw vat liquor as blank.Experiment in triplicate.Result is as described in Table 2:
Table 2: the anti-mycotic activity of the mycelium extract (100 μ g/ml) of the culture of thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042
Note 1: I: control solvent ethyl acetate: methyl alcohol: acetic acid (70～80:15～20:4～8); II: mycelium extract (100 μ g/ml).
Note 2:+: have anti-microbial activity;-: without anti-microbial activity.
Claims (10)
1. thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042, its deposit number is CGMCC No:8101.
2. the mycelium extract of a thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture, it is characterized in that: using thalassiomycetes umbrella branch claimed in claim 1 mould (Umbelopsis sp.) QZ042 as fermentation strain, liquid fermenting obtains fermenting culture, separation of mycelial and fermented liquid, getting mycelium uses the mixed solvent being comprised of ethyl acetate, methyl alcohol and acetic acid to carry out lixiviate, vat liquor is concentrated, obtains.
3. the mycelium extract of thalassiomycetes umbrella branch according to claim 2 mould (Umbelopsis sp.) QZ042 culture, it is characterized in that: described liquid fermenting be by thalassiomycetes umbrella branch claimed in claim 1 mould (Umbelopsis sp.) QZ042 inoculation in PD liquid nutrient medium, under 25～32 ℃ of conditions, shaking table cultivation 5～7 days or standing cultivation are 15～30 days, to obtain fermenting culture.
4. the mycelium extract of thalassiomycetes umbrella branch according to claim 2 mould (Umbelopsis sp.) QZ042 culture, is characterized in that: the volume ratio of the ethyl acetate of described composition mixed solvent, methyl alcohol and acetic acid is 70～80:15～20:4～8.
5. the preparation method of the mycelium extract of thalassiomycetes umbrella branch claimed in claim 2 mould (Umbelopsis sp.) QZ042 culture, comprises the following steps:
1) seed culture: picking thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 mycelium inoculation is cultivated 5～7 days in PDA solid medium ramp, and temperature is controlled at 26～30 ℃;
2) fermentation culture: the good fungi of slant culture is inoculated on PD liquid nutrient medium, and shaking table cultivation 5～7 days or standing cultivation are 15～30 days under 25～32 ℃ of conditions, obtain fermenting culture;
3) filter: fermenting culture is filtered, isolate mycelium and fermented liquid;
4) extract: get mycelium and use the mixed solvent being comprised of ethyl acetate, methyl alcohol and acetic acid to carry out lixiviate, vat liquor is concentrated, obtains the mycelium extract of thalassiomycetes umbrella branch mould (Umbelopsis sp.) QZ042 culture.
6. the preparation method of the mycelium extract of thalassiomycetes umbrella branch according to claim 5 mould (Umbelopsis sp.) QZ042 culture, it is characterized in that: step 4) in, the volume ratio of the ethyl acetate of described composition mixed solvent, methyl alcohol and acetic acid is 70～80:15～20:4～8.
7. the application of the mycelium extract of thalassiomycetes umbrella branch claimed in claim 2 mould (Umbelopsis sp.) QZ042 culture in preparation Chinese People's Anti-Japanese Military and Political College enterobacteria, bacillus cereus, tobacco Arthrobacter, klebsiella or Vibrio parahaemolyticus medicine.
The medicine of 8.Yi Zhong Chinese People's Anti-Japanese Military and Political College enterobacteria, bacillus cereus, tobacco Arthrobacter, klebsiella or Vibrio parahaemolyticus, is characterized in that: this pharmaceutical pack contains the mycelium extract of thalassiomycetes umbrella branch mould (Umbelopsis sp.) the QZ042 culture described in claim 2 as activeconstituents.
The mycelium extract of thalassiomycetes umbrella branch claimed in claim 2 mould (Umbelopsis sp.) QZ042 culture the anti-loose capsule bacterium of preparation, chain lattice spore is mould, the two spores of hair are mould or stem canker medicine in application.
Anti-loose capsule bacterium, chain lattice spore is mould, the two spores of hair are mould or a medicine for stem canker, it is characterized in that: this pharmaceutical pack containing the mycelium extract of thalassiomycetes umbrella branch mould (Umbelopsis sp.) the QZ042 culture described in claim 2 as activeconstituents.
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| CN201510559167.6A 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 |
| CN201410203253.9A CN103952322B (en) | 2014-05-15 | 2014-05-15 | A kind of thalassiomycetes umbrella branch trichoderma strain and mycelium extract thereof and application |
| CN201510559187.3A 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 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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| CN201510559089.XA Division 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 |
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Non-Patent Citations (3)
| Title |
|---|
| 何容等: "深黄伞形霉（Umbelopsis isabellina）华2-1产油发酵培养条件优化", 《应用与环境生物学报》 * |
| 吕书媛等: "长白山自然保护区北坡岳桦树根系内生真菌的种类及分布", 《菌物研究》 * |
| 陈士华等: "一株高产油脂微生物菌种的筛选与鉴定", 《河南工业大学学报（自然科学版）》 * |
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|---|---|---|---|---|
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| CN108949867B (en) * | 2018-08-01 | 2021-06-18 | 浙江海洋大学 | Method for preparing actitoxin by fermenting marine bacteria |
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