CN100497589C - Fusarium - Google Patents
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- CN100497589C CN100497589C CNB2006101638176A CN200610163817A CN100497589C CN 100497589 C CN100497589 C CN 100497589C CN B2006101638176 A CNB2006101638176 A CN B2006101638176A CN 200610163817 A CN200610163817 A CN 200610163817A CN 100497589 C CN100497589 C CN 100497589C
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Abstract
The present invention relates to microbial technology, and is especially one kind of microbe strain with broad spectrum and high activity bacteriostatic function. The microbe strain is named as Fusarium oxysporum YM311977 in the preservation number of CCTCC No. 206112. Fusarium oxysporum YM311977 separated from poisonous plant Datura stramonium endogenetic fungi has its microbiological position determined. It is found that Fusarium oxysporum YM311977 possesses obvious bacteriostatic function on 17 kinds of pathogenic microbes, including 11 kinds of gram positive bacteria, 4 kinds of gram negative bacteria and 2 kinds of dermatosis pathogenic fungi. The present invention utilizes plant endogenetic fungi resource to obtain natural antibiotic medicine.
Description
Technical field
The present invention relates to microbial technology field, particularly relate to a kind of microorganism strains with broad spectrum and high activity bacteria resistance function.
Background technology
Thorn apple (Datura arborea L.) plant is famous poisonous plants, its leaf flower and seed contain Scopolamine (hyoscine, scopolamine, hyoscine), tropine (hyoscyamine), coromegine (atropine), atropyltropeine (apoatropine), noratropine (noratropine), thorn apple element (daturine), saldanine (meteloidine), tigloidine alkaloids such as (tigloidine).Wherein, the clinical cardiovascular system diseases (as coronary heart disease, stenocardia, heart failure, coronary artery dilator, increase coronary flow, improve the myocardial anoxia state and reduce myocardial consumption of oxygen) that is used for the treatment of of Scopolamine; Multiple pharmacologically active and multiple clinical medicine purposes such as septic shock (remove arteriovenous spasm, microcirculation improvement) and nervous system disorders (excited breathing, circulation maincenter, maincenter is had sedative effect).
Summary of the invention
The object of the present invention is to provide a kind of separation in Chinese yunnan province Kunming thorn apple (Daturaarborea L.) plant living body, to have the microorganism strains-fusarium of broad spectrum and high activity bacteria resistance function.
Fusarium called after fusarium Fusarium oxysporum YM311977 of the present invention now is deposited in specified depositary institution of State Intellectual Property Office, and deposit number is CCTCC NO:206112.
Fusarium of the present invention is to separate to obtain from Chinese Yunnan Province Kunming thorn apple (Datura arborea L.) plant living body.Now be deposited in Patent Office of State Intellectual Property Office and specify depositary institution's preservation, depositary institution's title: Chinese typical culture collection center, depositary institution address: China, Wuhan University, postcode: 430072.Preservation date is on October 31st, 2006, and deposit number is 206112.
This microorganism belongs to endogenetic fungus, through morphologic observation, cultural characteristic and molecular biology 18SrDNA gene sequencing, confirm that its taxonomy status belongs to the fusarium of imperfect fungi (Fungi Imperfecti) Moniliales (Moniliales) knurl Cuo spore section (Tuberculariaceae) Fusarium (Fusarium) (Fusarium oxysporum), strain number YM 311977 (Fig. 1,2,3).
Fusarium YM311977 is on the PDA substratum, and colony growth is very fast, cultivates 3 days colony diameter 31mm, white mycelium for 28 ℃; The bacterium colony middle part is little prominent, flat cotton-shaped, and growth is tight, neat in edge, back side purple.Colony diameter 24mm on malt extract medium, white mycelium, the fine and soft shape of the flat wadding of bacterium colony, growth is tight, neat in edge, the back side is light brown.And colony diameter 25mm on the czapek's solution, white mycelium, the fine and soft shape of the flat wadding of bacterium colony, neat in edge, back side white.Its representative configuration feature is that conidiophore is formed by the podocyte growth, obviously distinguishes with typical strain of the same race.
With 18SrDNA gene order homology is the phylogenetic tree that fundamental construction forms the relevant fungi of 16 strains that comprises bacterial strain YM311977, fusarium YM311977 base sequence similarity is 100% as can be known, with this genus bacterial classification Fusarium oxysporum indifference.
The culture medium culturing feature (see figure 1) of bacterial strain YM311977:
1. 28 ℃ of PDA substratum were cultivated 3 days, and colony diameter 31mm spread whole culture dish in 7 days.White mycelium; The bacterium colony middle part is little prominent, flat cotton-shaped, and growth is tight, neat in edge, back side purple.
2. 28 ℃ of malt extract mediums were cultivated 3 days, colony diameter 24mm, and 7 days 55mm spread whole culture dish in 11 days.White mycelium, the fine and soft shape of the flat wadding of bacterium colony, growth is tight, neat in edge, the back side is light brown.
3. 28 ℃ of czapek's solutions were cultivated 3 days, colony diameter 25mm, and 7 days 60mm spread whole culture dish in 11 days.White mycelium, the fine and soft shape of the flat wadding of bacterium colony, neat in edge, back side white.
The morphological specificity of YM311977 bacterial strain (seeing Fig. 2,3):
1. asexual generation white mycelium have every, conidiophore is arranged, macroconidium forms in pionnotes, the back becomes scattered about between mycelia more, and spore just is fusiform or avette, and is smooth, diameter 5.5-9.5 * 27.5-38 micron becomes sickleshaped after long-time the placement.
2. sexual generation is not found.
The molecular biological characteristics of YM311977 bacterial strain:
Adopt the molecular biology round pcr, the dna sequencing analysis, YM311977 endogenetic fungus 18SrDNA genome is by 1684 based compositions.With the 18SrDNA sequence is the phylogenetic tree of fundamental construction, and the evolutionary distance of bacterial strain YM311977 and Fusarium oxysporum is (Fig. 4) recently.
YM311977 strain fermentation cultural characteristic:
1. bacterium culture medium: (get fresh potato 200 grams, peeling is cut into small pieces solid PDA substratum, adds 1 liter in water and boils 30 minutes, filters and abandons potato, and filter adds water and complement to 1 liter night, adds glucose 20 grams, the PH nature.Add agar 15 grams in the above-mentioned potato glucose substratum, be solid PDA substratum).
2. fermention medium: (composition is the liquid czapek's solution: NaNO3 3g, K2HPO4 1g, KCL 0.5g, MgSO4.7H2O0.5g, FeSO4 0.01g, sucrose 30g, distilled water 1000mL, PH nature.Add agar 15 grams in the aforesaid liquid czapek's solution, be the solid czapek's solution).
3. culture temperature: 30 ℃ ± 2 ℃.
4. fermentation time: 5-7 days.
The fermentation culture feature: cultivated the 1st day, bacterial classification has odd white hypha point; Cultivated the 2nd day, a small amount of beige mycelium pellet is arranged; Cultivated the 3rd day, the fermented liquid mycelium increases, and mycelium pellet is beige, and mycelia is not in conjunction with tight in the mycelium pellet, and a small amount of mycelia is adherent, be purple, and it is muddy that fermented liquid shows slightly; Cultivated the mycelium pellet color burn brown that shoals, fermented liquid muddiness the 5th day; Cultivated the 7th day, and be full of mycelium in the fermented liquid, the muddy thickness of fermented liquid.
The extraction of fusarium of the present invention (YM311977) antibacterial substance, its processing step is: (28 ℃ ± 2 ℃ of bacterial classifications → activation (solid PDA substratum) → first order seed (solid czapek's solution) → cultivation, 3-4 days) → (shaking table rpm200 rev/min of inoculation (inoculating needle one ring) → secondary seed (liquid czapek's solution) → cultivation, 28 ℃ ± 5 ℃, 4-5 days) → inoculation (inoculum size 2-5%) → fermentation (50 liters of fermentor tanks, 28 ℃ ± 5 ℃, 12-15 days) → fermented product → extraction → tunning.In this step, fermented substrate is the Cha Shi substratum.This technology is suitable for the cultivation of other filamentous fungus quasi-microorganism of alkaloid one class.
In the above-mentioned zymotechnique, the fermentation condition of numbering YM311977 bacterial classification is as follows:
(1) fermentation mode: liquid fermenting.
(2) seed culture: seed culture is potato glucose liquid nutrient medium (liquid PDA).
Potato glucose substratum (PDA) preparation: get fresh potato 200 grams, peeling is cut into small pieces, and adds 1 liter in water and boils 30 minutes, filters and abandons potato, and filter adds water and complement to 1 liter night, adds glucose 20 grams, the PH nature.
Add agar 15 grams in the above-mentioned potato glucose substratum, be solid PDA substratum.
(3) fermentation culture: fermention medium is a liquid Cha Shi substratum.
The preparation of liquid czapek's solution: take by weighing NaNO3 3g respectively, K2HPO4 1g, KCL 0.5g, MgSO4.7H2O0.5g, FeSO4 0.01g mixes the back and adds sucrose 30g, distilled water 1000mL, abundant stirring and dissolving, PH nature.
(4) incubation time: strain activation and culture 48-72 hour, seed shaking table incubation time 72-96 hour, fermented incubation time 5-7 days.
(5) culture temperature: 28 ℃ ± 2 ℃ of seed culture temperature, fermentation culture temperature are 30 ℃ ± 2 ℃.
(6) fermentation finishes, and filtering fermentation liquor is removed mycelium, collects fermented liquid.Adopt domestic macroporous resin (model AB-8) that fermented liquid is adsorbed, after distilled water flushing is driven away impurity, adopt the ethanolic soln wash-out of 60-70% (v/v), elutriant obtains the tunning extract after the ethanol evaporation solution concentration.The tunning extract adds sterilized water and makes antibiotic tunning goods.
After 48 hours, extracting solution is concentrated into dried YM311977 endogenetic fungus tunning through certain volume 95% ethanol lixiviate, adds sterilized water and makes the tunning sample.In the 50ug/ml-5mg/ml dosage range, tunning has significant inhibitory effect to 11 kinds of Gram-positive pathogenetic bacterias, 4 kinds of Gram-negative pathogenetic bacterias, 2 kinds of skin pathomycetes respectively.
The tunning of bacterial strain YM311977 has the growth of inhibition effect to following 17 kinds of pathogenetic bacterias respectively.Wherein, gram positive bacterium is 11 kinds: bacillus cereus (Bacillus cereus); Subtilis (Bacillus subtilis); Streptococcus pneumoniae (Streptococcus pneumoniae); First type Streptococcus hemolyticus (Streptcoccus hemolyticus); Beta hemolysis suis (Ttreptococcushemolyticus); Sarcina lutea (Sarcina lutea); Staphylococcus albus (Staphylococcusalbus); Streptococcus aureus (Staphy-lococcus aureus); Mycobacterium tuberculosis (human-like) (Mycobacterium tuberculosis); M. smegmatics (Mycobacterium smegmatis); Staphylococcus epidermidis (Staphylococcus aureus).4 kinds of gram negative bacteriums: intestinal bacteria (Escherichia coli); Proteus vulgaris (Prate vulgaris); Salmonella typhi (Salmonella typhi) and sonne bacillus (Shigella sonnei).2 kinds of skin pathomycetes, that is: microsporon gypseum (Microsporum gypseum) and star gypsum sample hair moss bacterium (Trichophuton gypseum) (seeing Table 1).
Table 1
* bacteriostatic action symbol implication: "-" expression does not have bacteriostatic activity; "+" expression has bacteriostatic activity; " ++ " expression bacteriostatic activity is obvious; " +++" represent that bacteriostatic activity is remarkable.
The tunning of bacterial strain YM311977 has the growth of inhibition effect to bacillus cereus (Bacilluscereus), microsporon gypseum (Microsporum gypseum) under 50ug/ml concentration.Under 200ug/ml concentration, respectively subtilis (Bacillus subtilis), first type Streptococcus hemolyticus (Streptcoccus hemolyticus), M. smegmatics (Mycobacterium smegmatis), star gypsum sample hair moss bacterium (Trichophuton gypseum) are played the growth-inhibiting effect; Under 1mg/ml concentration, can suppress streptococcus pneumoniae (Streptococcus pneumoniae), beta hemolysis suis (Ttreptococcus hemolyticus), Sarcina lutea (Sarcina lutea), the growth of Staphylococcus albus (Staphylococcus albus) and mycobacterium tuberculosis (Mycobacterium tuberculosis): under 5mg/ml concentration, can suppress streptococcus aureus (Staphylococcus aureus), staphylococcus epidermidis (Staphylococcu saureus), intestinal bacteria (Escherichia coli), proteus vulgaris (Prate vulgaris), growth (Fig. 5 of salmonella typhi (Salmonella typhi) and bacillus ceylonensis A (Shigella sonnei), 6).
The tunning essential substance of bacterial strain YM311977 is a 5-butyl picolinate compound, and its content is up to 380mg-450mg/L.Secondly, the tunning of bacterial strain Ym311977 also contains 5-butyl picolinate ethyl ester, Xie Ansuan and N.F,USP MANNITOL compound, and these compounds are known compound, the present invention discloses a kind of new acquisition approach.
The meta-bolites (alkaloid compound) of endogenetic fungus-fusarium moniliforme of the present invention (Fusarium oxysporum) all has stronger restraining effect to multiple common Gram-positive, negative pathogenetic bacteria and the growth of minority skin pathogenic microorganism respectively.Simultaneously, this bacterial strain can obtain its main metabolites 5-butyl picolinate ethyl ester and 5-butyl picolinate compound by artificial pure culture, and 5-butyl picolinate compound is inhibited to human tumor cell's (as neck squamous cell (HNSCC), melanochrome, mammary gland, skin, thymus gland etc.).In addition, this compound is the potent inhibitor of dopamine, the effect that can improve maincenter Dopamine level and bring high blood pressure down.In addition, this compound can also suppress the effect of plant-root growth.
The present invention separates poisonous plants thorn apple (Datura arborea L.) endogenetic fungus first, in the microbiology status of the fusarium YM311977 that clearly has the broad spectrum antibiotic activity effect, find this bacterium to 11 kinds of gram positive bacteriums, 4 kinds of gram negative bacteriums, 2 kinds of skin pathomycetes, totally 17 kinds of pathogenic micro-organisms have comparatively obvious suppression growth effect.The present invention seeks to utilize the plant endogenesis epiphyte resource, in the hope of obtaining the natural medicinal ingredients of anti-human diseases new antibiotic class.
Description of drawings
Fig. 1 is the bacterium colony picture of fusarium YM311977 of the present invention.
Fig. 2 is a fusarium YM311977 strain morphology microgram of the present invention (400X).
Fig. 3 is a fusarium YM311977 conidium microgram of the present invention (400X).
Fig. 4 is the phylogeny tree graph of fusarium YM311977 homology of the present invention.
Fig. 5 is the restraining effect figure of fusarium YM311977 meta-bolites of the present invention to gram positive bacterium.
Fig. 6 is the restraining effect figure of fusarium YM311977 meta-bolites of the present invention to the skin pathomycete.
Embodiment
Embodiment:
The fermentation culture of fusarium YM311977 bacterial strain:
Fusarium (Fusarium oxysporum) YM311977 bacterial strain is the natural microbial medicine source that fermentation obtains anti-human diseases new antibiotic class medicinal ingredients.
Get numbering YM311977 bacterial classification, under aseptic condition (sterilisable chamber or Bechtop),, transfer in sterilized solid PDA substratum test tube (in 18 * 180mm) with a little bacterial classification of inoculating needle picking, put interior 28 ℃ ± 1 ℃ activation culture of incubator 72-96 hours, standby.
Take out 72-96 hours standby bacterial classification of activation, under aseptic condition, in sterilized first order seed liquid PDA substratum, (adopt 500 milliliters of glass triangle bottles with the bacterial classification that it is good that the same manner inserts activation, pack into 100 milliliters of the liquid PDA substratum that prepare, sterilized 30 minutes for 15 pounds), cultivated 48-72 hours at 28 ℃ ± 1 ℃ following shaking table, be the YM311977 first order seed.
(121 ℃ (1.01 * 105P) sterilizations are after 30 minutes, and are to be cooled to 30 ℃ for BIOENGINEERING, pack into 25 liters of the liquid czapek's solutions that prepare of model-R1) to adopt 50 liters of automatic fermenters.Under aseptic condition, get the YM311977 seed liquor, under aseptic condition, be inoculated in the fermentor tank that 25 liters of liquid czapek's solutions are housed, inoculum size is by 2-5% (v/v), 28 ℃ ± 2 ℃ of controlled temperature, stirred (200rpm) aerated culture 5-7 days, during fermentation, notice that the fermentor tank parameter changes, and inspection has or not the microbiological contamination phenomenon.
Fermentation finishes, filtering fermentating liquid, remove mycelium, collect fermented liquid, adopt macroporous adsorbent resin (model AB-8) absorption tunning activeconstituents, after distilled water flushing is driven away impurity, adopt the ethanolic soln wash-out of 60-70% (v/v), elutriant obtains the tunning extract after the ethanol evaporation solution concentration.The tunning extract is through silica gel (Silica G) column chromatography, and separation, purifying can obtain tunning antibacterial substance alkaloid one compounds-5-butyl picolinate ethyl ester (C
12H
17NO
2) and 5-butyl picolinate (C
10H
13NO
2) and Xie Ansuan and N.F,USP MANNITOL.Wherein, every liter of fermented liquid content of 5-butyl picolinate compound is 380-450mg/L.
The 18SrDNA genome of YM311977 of the present invention is made up of 1681 bases (bp), and its series is as follows:
1 gtctagtata?agcaattata?ccgcgaaact?gcgaatggct?cattatataa?gttatcgttt
61 atttgatagt?accttactac?ttggataacc?gtggtaattc?tagagctaat?acatgctaaa
121 aatcccgact?tcggaaggga?tgtatttatt?agattaaaaa?ccaatgccct?tcggggctca
181 ctggtgattc?atgataactc?ctcgaatcgc?atggccttgt?gccggcgatg?gttcattcaa
241 atttcttccc?tatcaacttt?cgatgtttgg?gtattggcca?aacatggttg?caacgggtaa
301 cggagggtta?gggctcgacc?ccggagaagg?agcctgagaa?acggctacta?catccaagga
361 aggcagcagg?cgcgcaaatt?acccaatccc?gacacgggga?ggtagtgaca?ataaatactg
421 atacagggct?cttttgggtc?ttgtaattgg?aatgagtaca?atttaaatcc?cttaacgagg
481 aacaattgga?gggcaagtct?ggtgccagca?gccgcggtaa?ttccagctcc?aatagcgtat
541 attaaagttg?ttgtggttaa?aaagctcgta?gttgaacctt?gggcctggct?ggccggtccg
601 cctcaccgcg?tgtactggtc?cggccgggcc?tttccctctg?tggaacccca?tgcccttcac
661 tgggtgtggc?ggggaaacag?gacttttact?gtgaaaaaat?tagagtgctc?caggcaggcc
721 tatgctcgaa?tacattagca?tggaataata?gaataggacg?tgtggttcta?ttttgttggt
781 ttctaggacc?gccgtaatga?ttaataggga?cagtcggggg?catcagtatt?caattgtcag
841 aggtgaaatt?cttggattta?ttgaagacta?actactgcga?aagcatttgc?caaggatgtt
901 ttcattaatc?aggaacgaaa?gttaggggat?cgaagacgat?cagataccgt?cgtagtctta
961 accataaact?atgccgacta?gggatcggac?ggtgttattt?tttgacccgt?tcggcacctt
1021?acgagaaatc?aaagtgcttg?ggctccaggg?ggagtatggt?cgcaaggctg?aaacttaaag
1081?aaattgacgg?aagggcacca?ccaggggtgg?agcctgcggc?ttaatttgac?tcaacacggg
1141?gaaactcacc?aggtccagac?acaatgagga?ttgacagatt?gagagctctt?tcttgatttt
1201?gtgggtggtg?gtgcatggcc?gttcttagtt?ggtggagtga?tttgtctgct?taattgcgat
1261?aacgaacgag?accttaacct?gctaaatagc?ccgtattgct?ttggcagtac?gctggcttct
1321?tagagggact?atcggctcaa?gccgatggaa?gtttgaggca?ataacaggtc?tgtgatgccc
1381?ttagatgttc?tgggccgcac?gcgcgctaca?ctgacggagc?cagcgagtac?ttccttgtcc
1441?gaaaggtccg?ggtaatcttg?ttaaactccg?tcgtgctggg?gatagagcat?tgcaattatt
1501?gctcttcaac?gaggaatccc?tagtaagcgc?aagtcatcag?cttgcgttga?ttacgtccct
1561?gccctttgta?cacaccgccc?gtcgctacta?ccgattgaat?ggctcagtga?ggcgtccgga
1621?ctggcccaga?gaggtgggca?actaccactc?agggccggaa?agctctccaa?actcggtcat
1681?taga。
Claims (1)
1, a kind of fusarium is characterized in that called after fusarium Fusarium oxysporumYM311977, now is deposited in specified depositary institution of State Intellectual Property Office, and deposit number is CCTCC NO:206112.
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| CN105803015A (en) * | 2016-05-28 | 2016-07-27 | 云南大学 | Method for converting dicentrine into (4S,6aR)-4-hydroxyl dicentrine through Clonostachys sp. Fermentation |
| CN110699264B (en) * | 2019-11-08 | 2021-03-05 | 山东省花生研究所 | Rapid culture method of pathogenic fungi of peanut rot |
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Non-Patent Citations (4)
| Title |
|---|
| 云南药用植物真菌病害研究（I）. 张宏等.云南农业大学学报，第13卷第1期. 1998 |
| 云南药用植物真菌病害研究（I）. 张宏等.云南农业大学学报，第13卷第1期. 1998 * |
| 利用非致病性尖镰孢霉诱导番茄抗枯萎病研究. 郑是琳等.山东农业大学学报，第23卷第4期. 1992 |
| 利用非致病性尖镰孢霉诱导番茄抗枯萎病研究. 郑是琳等.山东农业大学学报，第23卷第4期. 1992 * |
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