CN104031844A - Cladosporium sp. strain, and extract and application thereof - Google Patents
Cladosporium sp. strain, and extract and application thereof Download PDFInfo
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Abstract
The invention discloses a Cladosporium sp. strain, and an extract and application thereof. The Cladosporium sp. FS86 is collected to China Center for Type Culture Collection (CCTCC) on March 27th, 2014, wherein the address is Wuhan University, Wuhan City, PRC, and the collection number is CCTCC NO: M2014108. The fermentation extract has obvious inhibitory effects on Candida albicans, Aspergillus niger, Aspergillus flavus, Trichoderma viride, Colletotrichum gloeosporioides, Cylindrocladium scoparium or Curvularia lunata. Therefore, the invention provides scientific references for developing new anti-pathogenic fungus drugs and exploring deep-sea-fungus-derived natural active substances.
Description
Technical field:
The invention belongs to biological and medical technical field, be specifically related to strain deep-sea fungi branch spore mould (Cladosporium sp.) FS86, and the fermented product extract of preparing from the liquid culture product of this bacterial strain, and the application of this extract in the disease-resistant fungal pathogens medicine of preparation.
Background technology:
Candida albicans (Candida albicans) is a kind of important opportunistic fungus; conventionally be present in normal people oral cavity; the upper respiratory tract; enteron aisle and vagina; generally in normal body, quantity is few; do not cause disease, when body's immunity or general phylactic power defensive power declines or the mutual restrictive function imbalance of normal microflora, this bacterium amount reproduction change growth forms (blastogenesis mycelia phase) and invade cell and cause disease.In recent years along with the application of heavy dose of microbiotic, hormone, immunosuppressor, and the carrying out of transplant operation, make deep fungal infection increasing and can threat to life.Candidiasis is mainly acute, the subacute or chronic infection causing, is modal mycosis, and day by day serious resistance phenomenon and limited medicine make candida albicans infection become problem in the urgent need to address clinically.
Plant pathogenic fungi is the main pathogenic microbes of Plant diseases, is the big factors that threatens plant life, all produces and brings massive losses to agroforestry every year.At present fungal diseases of plants is still taking the Agro-chemicals control of organic synthesis as main, but the use of a large amount of chemical pesticides has not only caused serious harm to human environment, and increased the resistance of pathogenic fungi.In the mankind more and more today of concern for the environment quality, strengthen the application of biological control in Plant diseases, be the inevitable choice of disease control.Therefore, from Microbial resources, finding new active substance replaces chemical pesticide and uses natural antibacterial compound cover crop to become the emphasis of current research.
Ocean account for earth surface long-pending 71%, contain abundant Microbial resources, wherein microorganism accounts for 90% of marine organisms quantity, controlling important biomolecule and the biochemical circulation of all maintenance terrestrial ecosystem balances, existing data shows that Marine Microorganisms storehouse is large more than Microbial resources storehouse, land, and also not yet fully excavated and utilize by the mankind at present, visible marine microorganism is the abundant medicine source Biological resources of the mankind.
In ocean, exist the special environment of many natural condition, especially deep-sea ocean environment, make the marine microorganism that moves in deep-sea in unique physics, chemistry and ecotope, under forever the toxic substance of thermograde, high salinity and the high density of dark, high hydraulic pressure, poor nutrition, drastic change surrounds, they have formed very special biological structure, metabolic mechanism system.Due to this extreme environment, make deep-sea thalassiomycetes can produce novel structure, active unique meta-bolites, there is huge using value, the exploitation that can be new drug and biological pesticide provides excellent germ plasm resource.Therefore, Deep-Sea Microorganisms active substance is the inevitable of research and development marine drug thing and effectively selecting, and is also the focus of current Deep-Sea Microorganisms development of resources.
Summary of the invention:
First object of the present invention is to provide strain deep-sea fungi branch spore mould (Cladosporium sp.) FS86, this bacterial strain is preserved in Chinese Typical Representative culture collection center (CCTCC) on March 27th, 2014, address: Wuhan University of Wuhan, China city, its deposit number is CCTCC NO:M2014108.
Deep-sea of the present invention fungi branch spore mould (Cladosporium sp.) FS86 be in the settling of (12 ° of 58.551 ' E, 17 ° of 0.646 ' N) 1598m depths from the South Sea, separate, purifying obtains.Its separation purification method is: ooze sample, be diluted to 10%, 27 DEG C of vibration 20min containing the sterilized water of 3% thick sea salt, is got to 0.2mL and is uniformly coated in isolation medium, and 26～28 DEG C of thermostat containers are cultivated, and after bacterium colony grows, picking colony line purifying obtains.
Described isolation medium obtains in the following manner: with poach 200g potato 20min, filtration is succeeded in one's scheme, then adds glucose 20g, KH
2pO
43g, MgSO
40.75g, B
110mg, thick sea salt 30g, agar 20g, water complements to 1L, pH nature.
Study by morphology, and according to " fungi identification handbook " (Wei Jingchao, 1979), this bacterial strain is tentatively accredited as a spore mould (Cladosporium sp.).
Its morphological specificity is as follows:
In isolation medium, cultivate after 3 days for 28 DEG C, bacterium colony is micro-exceeds substratum, shallow blackish green, has rill, edge tool white down shape mycelia; Cultivate after 5 days, colony colour is blackish green, and edge mycelia is canescence; Cultivate after 7 days, bacterium colony becomes olive brown to chocolate, has radial rill (Fig. 1); Mycelia has every, khaki or tawny, and conidiophore is upright or bending, 10-50 × 3 μ m, a top tool 1-4 spore trace; Conidium is khaki to dark olive, cylindrical, oval, fusiform or olive shape, and 2.0～9.0 × 2.5～4.5 μ m, 0～1 barrier film, most of without barrier film, 1～4 cell, spore trace and spore navel be (Fig. 2) obviously.
Extract according to conventional methods the DNA of this bacterial strain, and utilize the universal primer amplification QiITS district in fungi ITS district, the base sequence in QiITS district is as shown in SEQ ID NO.1.By carry out Blast retrieval in GenBank, the two sequences the highest with its similarity is a spore mould (Cladosporium sp.), and similarity reaches 99.2%.
Combining form is learned feature and ITS the sequencing results, and this bacterial strain is accredited as a spore mould (Cladosporium sp.), called after branch spore mould (Cladosporium sp.) FS86.
This bacterial strain is preserved in Chinese Typical Representative culture collection center (CCTCC), address on March 27th, 2014: Wuhan University of Wuhan, China city, its deposit number is CCTCC NO:M2014108.
Second object of the present invention is to provide a kind of deep-sea fungi branch spore mould (Cladosporium sp.) fermented product extract with disease-resistant fungal pathogens effect.
Deep-sea of the present invention fungi branch spore mould (Cladosporium sp.) fermented product extract is prepared by the following method, it is characterized in that, using the mould FS86 of above-mentioned branch spore as fermentation strain, liquid fermenting obtains fermenting culture, with ethyl acetate or dehydrated alcohol or the more than 95% aqueous ethanolic solution cold soaking of volume fraction, preferably use ethyl acetate cold soaking, cooling bath obtains after concentrated.
The described cold soaking time is preferably 24～48 hours.
Described liquid fermenting, preferably mould branch spore FS86 bacterium is inoculated in fermention medium, 120r/min, cultivate 7d for 26 DEG C, obtain fermenting culture, described every liter of fermention medium obtains in the following manner: with poach 200g potato 20min, filtration is succeeded in one's scheme, add glucose 10g, N.F,USP MANNITOL 20g, yeast extract paste 3g, peptone 5g, monosodium glutamate 5g, thick sea salt 3.0g, water complements to 1L again, pH nature.
The 3rd object of the present invention is to provide the application of deep-sea fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract in the disease-resistant fungal pathogens medicine of preparation.
The present invention found through experiments, deep-sea of the present invention fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract is under 250 μ g/ sheet filter paper concentration, to Candida albicans (Candida albicans), aspergillus niger (Aspergillus niger), flavus (Aspergillus flavus), viride (Trichoderma viride), colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides), the antibacterial circle diameter of the two spores mould (Cylindrocladium scoparium) of post branch and curvularia lunata (Curvularia lunata) is respectively 45.2mm, 41.0mm, 40.2mm, 30.8mm, 36.8mm, 35.2mm, 30.5mm.Select 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.2mg/mL, 0.1mg/mL, carries out minimum inhibitory concentration (MIC value) test under 0.05mg/mL7 concentration, determine that this fermented product extract is respectively 0.1mg/mL, 1mg/mL, 2mg/mL, 2mg/mL, 1mg/mL, 1mg/mL, 0.25mg/mL to Candida albicans, aspergillus niger, flavus, viride, colletotrichum gloeosporioides Penz, the two spore MIC values mould and curvularia lunata of post branch.As can be seen here, the mould FS86 fermented product extract of branch spore of the present invention has significant disease-resistant fungal pathogens activity, can be used to prepare disease-resistant fungal pathogens medicine.
The 4th object of the present invention is to provide a kind of disease-resistant fungal pathogens medicine, it is characterized in that, this disease-resistant fungal pathogens pharmaceutical pack contains the mould FS86 fermented product extract of branch spore as activeconstituents.
Described disease-resistant fungal pathogens medicine is preferably the medicine of the mould or curvularia lunata of the two spores of anti-candida albicans, aspergillus niger, flavus, viride, colletotrichum gloeosporioides Penz, post branch.
The mould FS86 fermented product extract of branch spore of the present invention has broad-spectrum disease resistance fungal pathogens activity, show that this fermented product extract has good application prospect, therefore the present invention for developing new disease-resistant fungal pathogens medicine, the natural active matter of excavating deep-sea originated from fungus provides scientific basis.
Branch spore mould (Cladosporium sp.) FS86 of the present invention is preserved in Chinese Typical Representative culture collection center (CCTCC) on March 27th, 2014, address: Wuhan University of Wuhan, China city, its deposit number is CCTCC NO:M2014108.
Brief description of the drawings:
Fig. 1 is the colony characteristics of mould (Cladosporium sp.) FS86 of a spore in isolation medium, and wherein A, B, C are respectively and cultivate 3 days, 5 days, 7 days.
Fig. 2 is the photo of mould (Cladosporium sp.) FS86 of a spore under opticmicroscope and electron microscope, and A is the light micrograph of conidium and conidiophore; B is conidial light micrograph; C-D is the stereoscan photograph of conidium and conidiophore; D-F is conidial stereoscan photograph.
Embodiment:
Come by specific embodiment below that the present invention is further described, but and unrestricted the present invention.
Embodiment 1: separation and the qualification of deep-sea fungi branch spore mould (Cladosporium sp.) FS86
Deep-sea fungi branch spore mould (Cladosporium sp.) FS86 be in the settling of (12 ° of 58.551 ' E, 17 ° of 0.646 ' N) 1598m depths from the South Sea, separate, purifying obtains.Its separation purification method is: ooze sample, be diluted to 10%, 27 DEG C of vibration 20min containing the sterilized water of 3% thick sea salt, is got to 0.2mL and is uniformly coated in isolation medium, and 26～28 DEG C of thermostat containers are cultivated, and after bacterium colony grows, picking colony line purifying obtains.Every liter of acquisition in the following manner of described isolation medium: with poach 200g potato 20min, filtration is succeeded in one's scheme, then adds glucose 20g, KH
2pO
43g, MgSO
40.75g, B110mg, thick sea salt 30g agar 20g, water complements to 1L, pH nature.
Study by morphology, and according to " fungi identification handbook " (Wei Jingchao, 1979), this bacterial strain is tentatively accredited as a spore mould (Cladosporium sp.).
Its morphological specificity is as follows:
In isolation medium, cultivate after 3 days for 28 DEG C, bacterium colony is micro-exceeds substratum, shallow blackish green, has rill, edge tool white down shape mycelia; Cultivate after 5 days, colony colour is blackish green, and edge mycelia is canescence; Cultivate after 7 days, bacterium colony becomes olive brown to chocolate, has radial rill (Fig. 1); Mycelia has every, khaki or tawny, and conidiophore is upright or bending, 10-50 × 3 μ m, a top tool 1-4 spore trace; Conidium is khaki to dark olive, cylindrical, oval, fusiform or olive shape, and 2.0～9.0 × 2.5～4.5 μ m, 0～1 barrier film, most of without barrier film, 1～4 cell, spore trace and spore navel be (Fig. 2) obviously.
Extract according to conventional methods the DNA of this bacterial strain, and utilize the universal primer amplification QiITS district in fungi ITS district, the base sequence in QiITS district is as shown in SEQ ID NO.1.By carry out Blast retrieval in GenBank, the two sequences the highest with its similarity is a spore mould (Cladosporium sp.), and similarity reaches 99.2%.
Combining form is learned feature and ITS the sequencing results, and this bacterial strain is accredited as a spore mould (Cladosporium sp.), called after branch spore mould (Cladosporium sp.) FS86.
This bacterial strain is preserved in Chinese Typical Representative culture collection center (CCTCC), address on March 27th, 2014: Wuhan University of Wuhan, China city, its deposit number is CCTCC NO:M2014108.
Embodiment 2: the preparation of deep-sea fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract
Mould branch spore FS86 bacterium is inoculated in fermention medium, and 120r/min, cultivates 7d for 26 DEG C, obtains the mould FS86 fermenting culture of branch spore.
By the fermenting culture of the mould FS86 of branch spore, with ethyl acetate cold soaking 24h, cooling bath must obtain the extract of brown paste again through concentrating under reduced pressure, be the mould FS86 fermented product extract of a spore.
Described every liter of fermention medium obtains in the following manner: with poach 200g potato 20min, filtration is succeeded in one's scheme, then adds glucose 10g, N.F,USP MANNITOL 20g, yeast extract paste 3g, peptone 5g, monosodium glutamate 5g, thick sea salt 3.0g, and water complements to 1L, pH: nature.
Embodiment 3: the disease-resistant fungal pathogens activity of deep-sea fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract
One, Determination of Antibacterial Activity:
Adopt filter paper method to measure the anti-microbial activity of the mould FS86 fermented product extract of branch spore or curvularia lunata mould to Candida albicans, aspergillus niger, flavus, viride, colletotrichum gloeosporioides Penz, the two spores of post branch.
The mould FS86 fermented product extract of the branch spore DMSO that embodiment 2 is obtained dissolves, and is diluted to 50mg/mL concentration.
The above-mentioned pathogenic fungi bacterium liquid that liquid fermenting is obtained is diluted to respectively bacterium number with physiological saline or spore count content is the concentration of 106/mL, draw concentration and be bacterium night of 106/mL or spore suspension 1mL in culture dish, pour the PDA substratum that is cooled to suitable temp into, mix, with aseptic nipper gripping thickness be 1.5mm, diameter is that the filter paper of 6mm is placed in containing on bacterium flat board, on filter paper, drip respectively the mould FS86 fermented product extract of branch spore diluent 5 μ L as sample sets simultaneously, replace this fermented product extract diluent as blank group using DMSO, flat board is placed in to 26 DEG C of thermostat containers and cultivates 72h, measure the size of antibacterial circle diameter with cross method of masurement, experiment in triplicate, evaluate anti-microbial activity with antibacterial circle diameter size.
Experimental result is as shown in table 1:
Table 1: the disease-resistant fungal pathogens effect of deep-sea fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract
Two, the mensuration of minimum inhibitory concentration:
Adopt filter paper method to measure that the mould FS86 fermented product extract of branch spore is mould to Candida albicans, aspergillus niger, flavus, viride, colletotrichum gloeosporioides Penz, the two spores of post branch, the minimum inhibitory concentration (MIC value) of curvularia lunata.
The mould FS86 fermented product extract of the branch spore DMSO of embodiment 2 is diluted to respectively to 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.2mg/mL, 0.1mg/mL, 0.05mg/mL concentration.Pathogenic fungi bacterium liquid is adjusted to respectively to bacterium number with physiological saline or spore count content is 10
6individual/mL, be that the filter paper that 1.5mm, diameter are 6mm is placed in containing on bacterium flat board with aseptic nipper gripping thickness, on filter paper, drip respectively this extract 5 μ L of above-mentioned concentration, replace this fermented product extract diluent as blank taking DMSO, being placed in 26 DEG C of thermostat containers cultivates after 72h, observe filter paper inhibition zone around, have the minimum concentration of the contained sample of filter paper of inhibition zone around taking filter paper as minimum inhibitory concentration (MIC value).Experiment in triplicate.Experimental result is as shown in table 2:
Table 2: the minimum inhibitory concentration (MIC value) of deep-sea fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract to pathogenic fungi
Note: "+" indicates there is inhibition zone, and "-" indicates without inhibition zone
Can be found out by table 1 and table 2, the mould FS86 fermented product extract of branch spore of the present invention, under 250 μ g/ sheet filter paper concentration, is respectively 45.2mm, 41.0mm, 40.2mm, 30.8mm, 36.8mm, 35.2mm, 30.5mm to Candida albicans, aspergillus niger, flavus, viride, colletotrichum gloeosporioides Penz, the two spore antibacterial circle diameters mould or curvularia lunata of post branch.Select 4mg/mL, 2mg/mL, 1mg/mL, 0.5mg/mL, 0.2mg/mL, 0.1mg/mL, carries out minimum inhibitory concentration (MIC value) test under 0.05mg/mL7 concentration, determine that this fermented product extract is respectively 0.1mg/mL, 1mg/mL, 2mg/mL, 2mg/mL, 1mg/mL, 1mg/mL, 0.25mg/mL to Candida albicans, aspergillus niger, flavus, viride, colletotrichum gloeosporioides Penz, the two spore MIC values mould or curvularia lunata of post branch.As can be seen here, the mould FS86 extract of branch spore of the present invention has significant disease-resistant fungal pathogens activity, can be used to prepare disease-resistant fungal pathogens medicine.
Described in comprehensive, to pathogenic fungi Candida albicans, aspergillus niger, flavus, viride, colletotrichum gloeosporioides Penz, the two spores of post branch, mould or lunata has remarkable inhibition to the mould FS86 extract of branch spore of the present invention.Therefore, realization of the present invention, for the natural active matter of developing new disease-resistant fungal pathogens medicine, excavation deep-sea originated from fungus provides scientific basis.
Claims (7)
1. strain deep-sea fungi branch spore mould (Cladosporium sp.) FS86, its deposit number is CCTCC NO:M2014108.
2. a deep-sea fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract, it is characterized in that, it is prepared by the following method: using deep-sea claimed in claim 1 fungi branch spore mould (Cladosporium sp.) FS86 as fermentation strain, liquid fermenting obtains fermenting culture, with ethyl acetate or dehydrated alcohol or the more than 95% aqueous ethanolic solution cold soaking of volume fraction, cooling bath obtains the mould FS86 fermented product extract of deep-sea fungi branch spore after concentrated; The described cold soaking time is 24～28 hours.
3. deep-sea according to claim 2 fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract, it is characterized in that, described liquid fermenting is that mould deep-sea fungi branch spore (Cladosporium sp.) FS86 bacterium is inoculated in fermention medium, 120r/min, cultivate 7d for 26 DEG C, obtain fermenting culture, every liter of acquisition in the following manner of described fermention medium: with poach 200g potato 20min, filtration is succeeded in one's scheme, add again glucose 10g, N.F,USP MANNITOL 20g, yeast extract paste 3g, peptone 5g, monosodium glutamate 5g, thick sea salt 3.0g, water complements to 1L, pH nature.
4. the application of deep-sea claimed in claim 2 fungi branch spore mould (Cladosporium sp.) FS86 fermented product extract in the disease-resistant fungal pathogens medicine of preparation.
5. application according to claim 4, it is characterized in that, described disease-resistant fungal pathogens medicine is the medicine of anti-candida albicans (Candida albicans), aspergillus niger (Aspergillus niger), flavus (Aspergillus flavus), viride (Trichoderma viride), colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides), the two spores mould (Cylindrocladium scoparium) of post branch or curvularia lunata (Curvularia lunata).
6. a disease-resistant fungal pathogens medicine, is characterized in that, this disease-resistant fungal pathogens pharmaceutical pack containing deep-sea fungi branch spore mould (Cladosporium sp.) the FS86 fermented product extract described in claim 2 as activeconstituents.
7. disease-resistant fungal pathogens medicine according to claim 6, it is characterized in that, described disease-resistant fungal pathogens medicine is the medicine of anti-candida albicans (Candida albicans), aspergillus niger (Aspergillus niger), flavus (Aspergillus flavus), viride (Trichoderma viride), colletotrichum gloeosporioides Penz (Colletotrichum gloeosporioides), the two spores mould (Cylindrocladium scoparium) of post branch or curvularia lunata (Curvularia lunata).
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| CN112336754A (en) * | 2020-11-25 | 2021-02-09 | 北京大学 | Application of cladosporium compound microbial inoculum in preparation of medicines for treating metabolic diseases and culture method |
| CN113930346A (en) * | 2021-10-25 | 2022-01-14 | 广西中医药大学 | Application of marine-derived aspergillus and fermentation product thereof in mango anthracnose resistance |
| CN114410477A (en) * | 2021-11-29 | 2022-04-29 | 深圳大学 | Inhibitor of inducible NO synthetase, production strain and preparation method thereof |
| CN115093974A (en) * | 2022-06-24 | 2022-09-23 | 自然资源部第三海洋研究所 | Application of deep sea Cladosporium and its fermented product in preparing antibacterial agent |
| CN116376708A (en) * | 2022-12-05 | 2023-07-04 | 青岛农业大学 | Cladosporium fungus and application thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112336754A (en) * | 2020-11-25 | 2021-02-09 | 北京大学 | Application of cladosporium compound microbial inoculum in preparation of medicines for treating metabolic diseases and culture method |
| CN113930346A (en) * | 2021-10-25 | 2022-01-14 | 广西中医药大学 | Application of marine-derived aspergillus and fermentation product thereof in mango anthracnose resistance |
| CN114410477A (en) * | 2021-11-29 | 2022-04-29 | 深圳大学 | Inhibitor of inducible NO synthetase, production strain and preparation method thereof |
| CN114410477B (en) * | 2021-11-29 | 2023-10-10 | 深圳大学 | Inhibitor of inducible NO synthetase, and production strain and preparation method thereof |
| CN115093974A (en) * | 2022-06-24 | 2022-09-23 | 自然资源部第三海洋研究所 | Application of deep sea Cladosporium and its fermented product in preparing antibacterial agent |
| CN116376708A (en) * | 2022-12-05 | 2023-07-04 | 青岛农业大学 | Cladosporium fungus and application thereof |
| CN116376708B (en) * | 2022-12-05 | 2024-06-07 | 青岛农业大学 | Cladosporium fungus and application thereof |
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| CN104031844B (en) | 2016-06-22 |
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