CN105754875B - One plant of marine fungi Trichoderma harzianum DLEN2008005 and its application - Google Patents
One plant of marine fungi Trichoderma harzianum DLEN2008005 and its application Download PDFInfo
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Abstract
The invention discloses one plant of marine fungi Trichoderma harzianum (Trichoderma harzianum) DLEN2008005 and its application.The marine fungi Trichoderma harzianum DLEN2008005 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number on January 29th, 2016 are as follows: CGMCC No.12105；Preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.The bacterial strain and its culture have anti-oxidant and acetylcholine esterase inhibition activity, have a good application prospect in terms of preparing anti senile dementia drug, and in terms of preparing the drug of anti-oxidation medicine and/or acetylcholine esterase inhibition activity.
Description
Technical field
The invention belongs to microorganisms technical fields.More particularly, to one plant of marine fungi Trichoderma harzianum DLEN2008005
And its application.
Background technique
As disease incidence is or not the aggravation of world population ages and senile dementia (Alzheimer ' s Disease, AD)
Disconnected to increase, the new drug for finding anti-senile dementia is extremely urgent.
Currently, widely accepted senile dementia pathology theory is cholinergic nerve damage hypothesis.In the guidance of the theory
Under, acetylcholinesterase (Acetylcholinesterase, AChE) inhibitor is anti-senile dementia disease most widely used at present
Drug.It is many simultaneously the study found that the superoxide radical generated in aerobic cell metabolic process can damage brain tissue, promote
Into the aging and death of brain cell, and free radical can also damage cell chromosome, make No. 21 chromosome aberration, to occur
AD.And antioxidant can not only stabilizing cell membrane, Scavenging active oxygen, while can also inhibit and remove intracerebral amyloid beta
Deposition, thus to prevent and protect nerve cell from damage, to delay the development process of AD.
Because acetylcholinesterase inhibitor and antioxidant play the role for the treatment of or delaying AD, screening has suppression
The drug of AChE activity processed and anti-oxidant dual function is of great significance.
Summary of the invention
The technical problem to be solved by the present invention is to overcome the deficiencies of existing senile dementia therapeutic agent, and it is true to provide one plant of ocean
Bacterium Trichoderma harzianum (Trichoderma harzianum) DLEN2008005 and its application, the bacterial strain and its culture have fine
Anti-oxidant and acetylcholine esterase inhibition activity, had a good application prospect in terms of preparing anti senile dementia drug.
The object of the present invention is to provide one plant of marine fungi Trichoderma harzianum DLEN2008005.
Another object of the present invention is to provide the application of above-mentioned marine fungi Trichoderma harzianum DLEN2008005.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
One plant of marine fungi Trichoderma harzianum (Trichoderma harzianum) DLEN2008005, January 29 in 2016
Day is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (it is referred to as CGMCC), deposit number are as follows:
CGMCC No.12105；Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
The marine fungi Trichoderma harzianum (Trichoderma harzianum) DLEN2008005 be from DaLian, China sea
Separation obtains in the Intertidal Algae asparagus frond tissue in domain, has anti-oxidant and acetylcholine esterase inhibition effect, foundation
" Fungal identification handbook " (Wei Jingchao, 1979) is accredited as Trichoderma harzianum bacterial strain.
In addition, the ITS1-5.8S-ITS2 rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1.
The bacterial strain has the following properties: being inoculated on seawater potato sucrose culture medium (seawater PSA) plate, 28 DEG C of trainings
Support, bacterium colony starts as white cotton fiber shape, round, extend around, after from bacterium colony center to edge generate celadon spore, colony edge
Color is slightly shallow, and entire bacterium colony is in loose cotton-shaped；Mycelia is very thin colourless under microscope, and tool separates, multi-branched；Conidiophore is from bacterium
Born on the side shoot of silk, to raw or alternate, generally there is 2~3 branches, sporogenic stigma doleiform or taper estranged；It is mitogenetic
Spore is mostly spherical shape, and sporoderm has pustule, blue-green；26~30 DEG C of optimum growth temp range, optimum growh pH be 6.0~
7.0, using carbon sources such as glucose, sucrose, lactose；The bacterial strain is left to ferment in seawater potato sucrose fluid nutrient medium can
Generate anti-oxidant and acetylcholinesterase inhibitor active material.
The growth salt concentration range of the bacterial strain is 2.0%~5.0%.
In addition, above-mentioned marine fungi Trichoderma harzianum DLEN2008005 application, specifically in preparing anti senile dementia drug
Application, and the application in the drug for preparing anti-oxidation medicine and/or acetylcholine esterase inhibition activity, also in the present invention
Protection scope within.
A kind of culture of marine fungi Trichoderma harzianum DLEN2008005 includes thallus and thallus fermented and cultured product.
Further, the culture is the liquid by the way that marine fungi Trichoderma harzianum DLEN2008005 to be inoculated in sterilizing
In body culture medium, 28 DEG C of natural lighting obtain for culture 21~24 days；Wherein, the fluid nutrient medium the preparation method comprises the following steps: every
20.0g sucrose and 20.0g sea salt, natural ph are added in 500mL potato liquor；The potato liquor are as follows: take fresh horse
1cm is cut into the peeling of bell potato3Size is blocky, and every 200g potato ball adds 500 mL of distilled water to boil 20 min, the double-deck absorbent gauze
Filtering, and 500mL is settled to distilled water.
More preferably specifically, the preparation method of the culture is as follows:
(1) seed culture: strain is inoculated into sterile seed solid medium tablets from inclined-plane, culture dish is put down
Plate, which is placed in constant incubator at 28 DEG C, is inverted culture, and incubation time is 4~5 days；
(2) expand culture: 1L fluid nutrient medium being filled in 3L triangular flask, is gone out after being sealed with breathable sealing film in high pressure
Bacterium pot in 121 DEG C, 0.1 Mpa sterilize 20min；According to every 1000mL fluid nutrient medium inoculation above-mentioned steps (1) culture after cooling
Seed plate afterwards carries disease germs 40~80cm of culture block2Ratio inoculation, sealing, be placed in constant incubator and cultivate, natural light
According to incubation time is 21~24 days, obtains culture.
Wherein, used culture medium is as follows:
Seed solid medium are as follows: 20.0g sucrose, 20.0g sea salt and 15.0g fine jade are added in every 500mL potato liquor
Rouge, natural ph；
Fluid nutrient medium are as follows: 20.0g sucrose and 20.0g sea salt, natural ph are added in every 500mL potato liquor；
Wherein, potato liquor: fresh potato peeling is taken to be cut into 1cm3Size is blocky, and every 200g potato ball adds distillation
500 mL of water boils 20 min, the double-deck absorbent gauze filtering, and is settled to 500mL with distilled water.
The culture of above-mentioned marine fungi Trichoderma harzianum DLEN2008005 is preparing the application in anti senile dementia drug,
And the application in the drug for preparing anti-oxidation medicine and/or acetylcholine esterase inhibition activity, also all in guarantor of the invention
Within the scope of shield.
In addition, by a large amount of research and test, marine fungi bacterial strain Trichoderma harzianum of the invention (Trichoderma harzianum) DLEN2008005 can use conventional standing method culture, it can be containing being used for microculture in culture medium
Carbon source, nitrogen source and other nutrient sources be subject to illumination without stringent limitation and be suitable for the strain growth.
When preparing anti-oxidant and acetylcholine esterase inhibition activity product as purpose, fermented and cultured should be selected such that culture
Activity extract the highest condition of yield preferably.By culture obtained as above, just by some methods appropriate
Can extract active component therein, these methods are commonly used for the method for extracting metabolin, such as using activity extract with
Other impurity solubility, ions binding power, absorption affinity and in terms of difference extract, these methods can
To be used alone, can also suitably cooperate or Reusability.
Wherein, marine fungi bacterial strain Trichoderma harzianum is cultivated with following culture medium, cultural method and extracting method
It is best that DLEN2008005 generates activated product:
(1) culture medium:
Seed solid medium are as follows: sucrose 20.0g, potato liquor 500mL, sea salt 20.0g, agar 15.0g, natural pH
Value.
Fluid nutrient medium are as follows: sucrose 20.0g, potato liquor 500mL, sea salt 20.0g, natural ph.
Wherein, potato liquor: fresh potato peeling is taken to be cut into 1cm3Size is blocky, and every 200g potato ball adds distillation
500 mL of water boils 20 min, the double-deck absorbent gauze filtering, and is settled to 500mL with distilled water.
(2) culture and extracting method:
A. seed culture: strain is inoculated into sterile seed solid medium tablets from inclined-plane, culture dish is put down
Plate, which is placed in constant incubator at 28 DEG C, is inverted culture, and incubation time is 4~5 days；
B. expand culture: 1L fluid nutrient medium being filled in 3L triangular flask, is gone out after being sealed with breathable sealing film in high pressure
Bacterium pot in 121 DEG C, 0.1 Mpa sterilize 20min；After being cultivated after cooling according to every 1000mL fluid nutrient medium inoculation above-mentioned steps A
Seed plate carry disease germs 40~80cm of culture block2Ratio inoculation, sealing, be placed in constant incubator and cultivate, natural lighting,
Incubation time is 20~25 days, obtains fermentation liquid；
C. extract: the fermentation liquid obtained after culture is sterilized overnight with 500 mL ethyl acetate, is then ultrasonically treated
Suction filtered through kieselguhr is added in 30min, and filtrate is extracted 3 times with isometric ethyl acetate, obtains ethyl acetate extract；Filter cake is used
After 200mL methanol impregnates 12~24 hours, ultrasonic 30min filters, obtains methanolic extract；So it is repeated 2 times；
It uses Rotary Evaporators to be concentrated under reduced pressure in 45 DEG C respectively ethyl acetate extract and methanolic extract, finally merges and mention
After taking object and being centrifuged, continue to obtain a dry solid in 45 DEG C of evaporated under reduced pressure with Rotary Evaporators, is gross activity crude extract；
D. it purifies: above-mentioned gross activity crude extract being dissolved in a small amount of (preferably 5~20mL) methanol, is added to equal with its dry weight
100~200 mesh silica gel, mix and grind uniformly to dry, be splined on 200~300 mesh silica gel of 20~50 times of sample dry weights
On chromatographic column, with eluant, eluent, gradually gradient elution, eluant, eluent used are chloroform: acetone=25:1~0:1(v/v), using thin layer color
It composes bioactivity autography method and tracks active component.By the component sample of active component using liquid phase preparation method carry out into
The purifying of one step, obtains white powder component 1 and 2.
Wherein, liquid phase preparation uses normal phase column and reversed-phase column, detects UV absorption under 254 nm, the flowing that normal phase column uses
It is mutually chloroform-methanol, the mobile phase that reversed-phase column uses is methanol-water.Component 1 be prepared by reversed-phase liquid chromatography (condition:
Chromatographic column SinoChrom ODS-AP, partial size 15mm, having a size of the mm of 20.0 mm × 250, mobile phase is methanol: water=8:2, stream
Speed is 2 mL/min), the retention time at peak is in 4~9 min；Component 2 be prepared by positive liquid chromatogram (condition:
SepaFlash standard type quick separating silicagel column -12 g, 40-63 μm of partial size, having a size of the mm of 17 mm × 85, chloroform: first
Alcohol=5:1, flow velocity are 4 mL/min), the retention time at peak is in 10~15 min.
In addition, tlc analysis is shown: chromatography solvent is methylene chloride: when methanol=5:1, component 1 is in GF254 thin-layer silicon
R on offset platefValue is that tool bluish violet absorbs under 0.85,254nm ultraviolet lamp, and the colour developing of anisaldehyde sulfuric acid is brownish red；Component 2 exists
R on GF254 thin layer silica gel platefValue is weaker to absorb under 0.42,254 nm ultraviolet lamps, and the colour developing of anisaldehyde sulfuric acid is light gray.
HPLC analysis also is carried out to the two components, liquid phase model Agilent 1260, chromatographiccondition is as follows:
Chromatographic column is Luna 5u C18 (2) 100A, and having a size of the mm of 150 mm × 4.6,5 μm of partial size, system is methanol-water, 0~
15~100% methanol linear elution of 15min, 15.01~30min, 100% methanol isocratic elution, 1 ml/min of flow velocity.As a result table
Bright: the main chromatographic peak retention time for the anti-oxidant and acetylcholine esterase inhibition activity characteristic component that component 1 contains is 19.7
~21.4 min；Main chromatographic peak retention time 11.9 of the component 2 with acetylcholine esterase inhibition activity characteristic component~
12.6 min。
The present invention shows that component 1 has anti-oxidant and acetylcholine esterase inhibition by thin-layer chromatography bioactivity autography
Double activity, component 2 have acetylcholine esterase inhibition activity.Pass through the activity of the two components of enzyme mark colorimetric method for determining, group
Divide 1 there is anti-oxidant and acetylcholine esterase inhibition activity simultaneously, removes the EC of DPPH free radical50With inhibition acetylcholine
The IC of esterase50Respectively 1.76 mg/mL, 2.43 mg/mL, component 2 have acetylcholine esterase inhibition activity, IC50Value is
1.87 mg/mL。
Marine fungi Trichoderma harzianum DLEN2008005 fermentation liquid of the invention and its activity extract and therein have
Component is imitated, can be used for preparing the drug of anti-senile dementia.
The invention has the following advantages:
The isolated one plant of marine fungi Trichoderma harzianum of present invention screening (Trichoderma harzianum)
DLEN2008005 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms on January 29th, 2016
The heart, deposit number are as follows: CGMCC No.12105；Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology.The bacterial strain and its culture have anti-oxidant and acetylcholine esterase inhibition activity, are preparing anti-ageing year
In terms of anti-dementia agent, and with fine in terms of preparing the drug of anti-oxidation medicine and/or acetylcholine esterase inhibition activity
Application prospect.
Specific embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but embodiment the present invention is not done it is any type of
It limits.Unless stated otherwise, the present invention uses reagent, method and apparatus is the art conventional reagents, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are commercially available.
The separation and identification of 1 marine fungi Trichoderma harzianum DLEN2008005 of embodiment
1, separation method
(1) sample: the Intertidal Algae asparagus frond in DaLian, China sea area.
(2) separation method
By above-mentioned fresh seaweed sample with twice of seawater flushing after 20 min high pressure sterilizations at 121 DEG C, it is transferred into 200
Bead and antiseptic sea water 50 mL of the addition after 20 min high pressure sterilizations at 121 DEG C in mL conical flask, in 60 rpm revolving speeds
10 min of lower oscillation discard supernatant liquid, then are rinsed frond 2 times with antiseptic sea water.
It will be taken out after aqueous solution soaking 10 seconds of 70% ethyl alcohol of 50mL by the seaweed of above-mentioned processing, it is sterile with 50 mL
Seawater flushing removes surface residual ethyl alcohol, blots remaining seawater with sterile blotting paper, with the blade after flame calcination sterilizing by seaweed
Beveling is segment, and is transplanted on Solid media for plates at 28 DEG C and cultivates 3 days.
The initial bacterium colony for then showing celadon in white cotton fiber shape extension growth is cut into mycelia tip with sterile razor blade again, is shifted
Onto new Solid media for plates, continuation is cultivated 3 days at 28 DEG C, so in triplicate, obtains pure bacterial strain.
In addition, Solid media for plates used in separation process is seawater potato sucrose culture medium (seawater PSA),
Its ingredient are as follows: contain 500 mL potato liquors, 20g sucrose, 0.2g chloramphenicol, 20g coarse sea salt, 20 min at 121 DEG C in every liter
It can be used after high pressure sterilization.
Wherein, potato liquor: fresh potato peeling is taken to be cut into 1cm3Size is blocky, and every 200g potato ball adds distillation
500 mL of water boils 20 min, the double-deck absorbent gauze filtering, and is settled to 500mL with distilled water.
2, identification method:
(1) Morphological Identification: by obtained pure strain inoculated to seawater PSA plate, bacterium colony body is observed in 28 DEG C of cultures
Feature, quality and color including bacterium colony center and marginal portion；And enter sterile cover slips in colony edge oblique cutting, it is raw to mycelia
When length is extended on coverslip, the coverslip with mycelia is taken out with tweezers and is attached on glass slide, in optical microphotograph microscopic observation bacterium
Silk, conidiophore, conidial morphological feature.Morphology is carried out to it referring to overstaff " the Fungal identification handbook " of work of Wei Jing
Identification.
(2) one plant of bacterial strain for belonging to trichoderma is identified, molecular biology identification further is carried out to it:
After being cultivated 7 days in strain inoculated to seawater PSA plate 28 DEG C, mycelia is scraped with sterile razor blade, is transferred into nothing
In the mortar of bacterium, liquid nitrogen grinding is added, then uses the Plant Genome extracts kit of Beijing Tiangen biotech company
DP305 extracts DNA according to specification step, and gained DNA sample is dissolved in 50 μ L TE buffers.
Using the DNA sample as template, using fungi universal primer ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') and
ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ') is reacted by PCR expands its ribosomal gene internal transcribed spacer sequence
ITS1-5.8S-ITS2, PCR reaction condition are as follows: 1) originate 94 DEG C of 5 min of denaturation；2) 94 DEG C of 0.5 min of denaturation；3) 55 DEG C of annealing
0.5 min；4) 72 DEG C of 1 min of extension；5) 5 min of final 72 DEG C of extensions.Wherein, step 2 to step 4) recycles 30 times.
PCR reaction product is purified using agarose electrophoresis, purified product gene sequencer direct Sequencing, sequencing result with
Sequence in Genbank database carries out BLAST comparison and the bacterial strain is accredited as Trichoderma harzianum according to sequence similarity.
3, identify, the results show that screening obtains one plant of marine fungi Trichoderma harzianum, have anti-well by above-mentioned separation
Oxidation and acetylcholine esterase inhibition activity.
In the strain inoculated to seawater potato sucrose culture medium (seawater PSA) plate, 28 DEG C of cultures, bacterium colony starts to be white
It is cotton-shaped, it is round, extend around, after from bacterium colony center to edge generate celadon spore, colony edge color is slightly shallow, entire bacterium
It falls in loose cotton-shaped.
Under microscope, mycelia is very thin colourless, and tool separates, multi-branched.Conidiophore is born from the side shoot of mycelia, to life
Or alternate, generally have 2~3 branches, sporogenic stigma doleiform or taper estranged.Conidium is mostly spherical shape, sporoderm tool
Pustule, blue-green.26~30 DEG C of optimum growth temp range, optimum growh pH is 6.0~7.0, using glucose, sugarcane
The carbon sources such as sugar, lactose.
The bacterial strain is left to ferment in seawater potato sucrose fluid nutrient medium can produce anti-oxidant and inhibition acetylcholine
The substance of esterase active.
By the Strain Designation be marine fungi Trichoderma harzianum (Trichoderma harzianum) DLEN2008005, and
Being deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on January 29th, 2016, (it is referred to as
CGMCC), deposit number are as follows: CGMCC No.12105；Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese section
Institute of microbiology, institute.
2 marine fungi Trichoderma harzianum DLEN2008005 fermentation culture medium active testing of embodiment
1, Trichoderma harzianum DLEN2008005 fermentation liquid obtains
(1) seed culture: strain is inoculated into sterile seed solid medium tablets from inclined-plane, culture dish is put down
Plate, which is placed in constant incubator at 28 DEG C, is inverted culture, and incubation time is 4~5 days；
(2) expand culture: 1L fluid nutrient medium being filled in 3L triangular flask, is gone out after being sealed with breathable sealing film in high pressure
Bacterium pot in 121 DEG C, 0.1 Mpa sterilize 20min；According to every 1000mL fluid nutrient medium inoculation above-mentioned steps (1) culture after cooling
Seed plate afterwards carries disease germs 40~80cm of culture block2Ratio inoculation, sealing, be placed in constant incubator and cultivate, natural light
According to incubation time is 21~24 days, obtains fermentation culture medium.
Wherein, used culture medium:
Seed solid medium are as follows: sucrose 20.0g, potato liquor 500mL, sea salt 20.0g, agar 15.0g, natural pH
Value.
Fluid nutrient medium are as follows: sucrose 20.0g, potato liquor 500mL, sea salt 20.0g, natural ph.
Wherein, potato liquor: fresh potato peeling is taken to be cut into 1cm3Size is blocky, and every 200g potato ball adds distillation
500 mL of water boils 20 min, the double-deck absorbent gauze filtering, and is settled to 500mL with distilled water.
2, by test, the fermentation culture medium of marine fungi Trichoderma harzianum DLEN2008005 has anti-oxidant and inhibition second
Acetylcholinesterase activity.
The extraction of 3 marine fungi Trichoderma harzianum DLEN2008005 fermentation liquid active material of embodiment
1, culture medium
(1) seed solid medium are as follows: sucrose 20.0g, potato liquor 500mL, sea salt 20.0g, agar 15.0g, from
Right pH value.
(2) fluid nutrient medium are as follows: sucrose 20.0g, potato liquor 500mL, sea salt 20.0g, natural ph.
Wherein, potato liquor: fresh potato peeling is taken to be cut into 1cm3Size is blocky, and every 200g potato ball adds distillation
500 mL of water boils 20 min, the double-deck absorbent gauze filtering, and is settled to 500mL with distilled water.
2, extracting method
(1) seed culture: strain is inoculated into sterile seed solid medium tablets from inclined-plane, culture dish is put down
Plate, which is placed in constant incubator at 28 DEG C, is inverted culture, and incubation time is 4~5 days；
(2) expand culture: 1L fluid nutrient medium being filled in 3L triangular flask, is gone out after being sealed with breathable sealing film in high pressure
Bacterium pot in 121 DEG C, 0.1 Mpa sterilize 20min；According to every 1000mL fluid nutrient medium inoculation above-mentioned steps (1) culture after cooling
Seed plate afterwards carries disease germs 40~80cm of culture block2Ratio inoculation, sealing, be placed in constant incubator and cultivate, natural light
According to incubation time is 20~25 days, obtains fermentation liquid；
(3) extract: the fermentation liquid that step (2) obtains is sterilized overnight with 500mL ethyl acetate, is then ultrasonically treated 30min,
Suction filtered through kieselguhr is added, filtrate is extracted 3 times with isometric ethyl acetate, obtains ethyl acetate extract；Filter cake 200mL methanol
After impregnating 12~24 hours, ultrasonic 30min filters, obtains methanolic extract；So it is repeated 2 times；
It uses Rotary Evaporators to be concentrated under reduced pressure in 45 DEG C respectively ethyl acetate extract and methanolic extract, finally merges and mention
After taking object and being centrifuged, continue to obtain a dry solid in 45 DEG C of evaporated under reduced pressure with Rotary Evaporators, is gross activity crude extract；
(4) it purifies: above-mentioned gross activity crude extract being dissolved in a small amount of (such as 5~20mL) methanol, is added to and its dry weight
100~200 equal mesh silica gel mix and grind uniformly to drying, are splined on 200~300 mesh of 20~50 times of sample dry weights
On silica gel column chromatography, with eluant, eluent, gradually gradient elution, eluant, eluent used are chloroform: acetone=25:1~0:1(v/v), use is thin
Layer chromatography bioactivity autography method tracks active component；By the component sample of active component using liquid phase preparation method into
Row is further purified, and obtains white powder component 1 and 2.
Wherein, liquid phase preparation uses normal phase column and reversed-phase column, detects UV absorption under 254 nm, and normal phase column uses mobile phase
For chloroform-methanol, reversed-phase column is methanol-water using mobile phase.
Component 1 be prepared by reversed-phase liquid chromatography (condition: chromatographic column SinoChrom ODS-AP, 15 μm of partial size, ruler
Very little is the mm of 20.0 mm × 250, and mobile phase is methanol: water=8:2, and flow velocity is 2 mL/min), the retention time at peak is 4~9
min。
(condition: SepaFlash standard type quick separating silicagel column-is prepared by positive liquid chromatogram in component 2
12g, 40~63 μm of partial size, having a size of 17mm × 85mm, chloroform: methanol=5:1, flow velocity are 4 mL/min), the retention time at peak
In 10~15min.
3, tlc analysis is shown: chromatography solvent is methylene chloride: when methanol=5:1, component 1 is in GF254 thin layer silica gel plate
Upper RfValue is that tool bluish violet absorbs under 0.85,254nm ultraviolet lamp, and the colour developing of anisaldehyde sulfuric acid is brownish red；Component 2 is thin in GF254
R on layer silica gel platefValue is weaker to absorb under 0.42,254 nm ultraviolet lamps, and the colour developing of anisaldehyde sulfuric acid is light gray.
4, in addition, having carried out HPLC analysis, liquid phase model Agilent 1260, chromatographiccondition to the two components
As follows: chromatographic column is Luna 5u C18 (2) 100A, and having a size of the mm of 150 mm × 4.6,5 μm of partial size, system is methanol-water,
0~15min, 15~100% methanol linear elution, 15.01~30min, 100% methanol isocratic elution, 1 ml/min of flow velocity.
5, the result shows that: the main chromatography for the anti-oxidant and acetylcholine esterase inhibition activity characteristic component that component 1 contains
Peak retention time is 19.7~21.4 min；Component 2 has the main chromatographic peak of acetylcholine esterase inhibition activity characteristic component
11.9~12.6 min of retention time.
The determination of activity of 4 marine fungi Trichoderma harzianum DLEN2008005 fermentation liquid active material of embodiment
1, thin-layer chromatography bioactivity autography is tested:
(1) two blocks of identical GF254 silica gel plates are taken, with clean methanol by its preview it is clean after dry, use capillary respectively
Pipe takes 10 μ L components 1 and each point sample of component 2 on chromatoplate, is unfolded with methylene chloride: methanol=25:1.
Anti-acetylcholinesterase activity experiment is uniformly to spray 0.5U/mL AChE, dries, fixes enzyme solution, then 37
DEG C constant incubator in moisturizing be incubated for 20min, after incubation, DTNB is mixed with ATCh with 1:1, is sprayed on plank,
37 DEG C of incubation 10min again, observation experiment phenomenon.
Anti-oxidant experiment is the DPPH methanol solution that 1.28mmol/L is sprayed on lamellae, after 5~8min is placed in dark place, is seen
Examine experimental phenomena.
(2) experimental result
Thin-layer chromatography bioactivity autography shows that component 1 has anti-oxidant and acetylcholine esterase inhibition double activity,
Component 2 has acetylcholine esterase inhibition activity.
2, enzyme mark colorometric assay:
(1) acetylcholine esterase inhibition activity experimental method: sequentially added in 96 orifice plates 200 μ L, 100 μ L, 40 μ L,
20 μ L, 10 μ L mass concentrations are 5 mg/mL sample solutions, and the PBS of 10 0.1 M of μ L 10%DMSO, 40 μ L is sequentially added after drying
20 μ L are added in (pH 7.4), 10 μ L final concentration of 0.02 U/mL AChE, 20 μ L 5 mM DTNB, 37 DEG C of 10 min of incubation
30 μ L 1%SDS are added after 10 mM ATCh, then 37 DEG C of 10 min of incubation, detect OD value at 405 nm with microplate reader, value is
ODSample.Each sample sets a corresponding sample copy bottom simultaneously, ibid detects, is set as ODSample copy bottom.Negative control 10%DMSO
It instead of sample, ibid detects, is set as ODIt is negative；Negative background is to replace enzyme solution with BSA, the same OD of other reaction conditionsIt is negative, other
It ibid detects, is set as ODNegative background.All tests are repeated 2 times and calculate average and standard deviation, and positive control is Tacrine.If
AChE inhibiting rate is I (%), and following formula calculates[13]: I%=[(ODIt is negative-ODNegative background)-(ODSample-ODSample copy bottom)]/(ODIt is negative-
ODNegative background)×100%。
Figure is done with inhibiting rate-concentration, takes logarithm Trendline, calculates IC50。
(2) 200 μ L, 100 μ L, 50 μ L, 25 μ L, 12.5 μ L mass anti-oxidant experimental method: are sequentially added in 96 orifice plates
Concentration is 5 mg/mL sample solutions, and 100 μ L DMSO are added after drying, it is molten to add 100 μ L, 0.16 mmol/L DPPH methanol
Liquid, mixes, and control replaces DPPH solution with methanol；Blank group is that 100 μ L DMSO mixing, control are added in 100 μ L DPPH
Group replaces DPPH with 100 μ L methanol solutions.30 min are placed in room temperature dark place, and absorbance is measured at 540 nm, is denoted as A respectively1、
A2、A3、A4。
Clearance rate %=100- (A1-A2)×100/(A3-A4)
In formula, A1For the light absorption value of experimental group；A2For the light absorption value of experimental comparison group；A3For the light absorption value of blank group；A4For
The light absorption value of blank control group.
It is measured in parallel 2 times, calculates average value, and positive control (replacing the measurement of sample liquid with Vc) is made with Vc.
Figure is done with free radical scavenging activity-concentration, takes logarithm Trendline, calculates EC50。
(3) experimental result
By the activity of the two components of enzyme mark colorimetric method for determining, component 1 has anti-oxidant and anti-acetylcholine ester simultaneously
The activity of enzyme removes the EC of DPPH free radical50With the IC of anti-acetylcholinesterase50Respectively 1.76 mg/mL, 2.43 mg/
mL；Component 2 has anti-acetylcholinesterase activity, IC50Value is 1.87 mg/mL.
SEQUENCE LISTING
<110>Guangdong Ocean University
<120>one plants of marine fungi Trichoderma harzianum DLEN2008005 and its applications
<130>
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 612
<212> DNA
<213>the ITS1-5.8S-ITS2 rDNA sequence of Trichoderma harzianum DLEN2008005
<400> 1
ttttgatatg cttaagttca gcgggtattc ctacctgatc cgaggtcaac atttcagaag 60
ttgggtgttt aacggctgtg gacgcgccgc gctcccgatg cgagtgtgca aactactgcg 120
caggagaggc tgcggcgaga ccgccactgt atttcggaga cggccaccgc caaggcaggg 180
ccgatcccca acgccgaccc cccggagggg ttcgagggtt gaaatgacgc tcggacaggc 240
atgcccgcca gaatactggc gggcgcaatg tgcgttcaaa gattcgatga ttcactgaat 300
tctgcaattc acattactta tcgcatttcg ctgcgttctt catcgatgcc agaaccaaga 360
gatccgttgt tgaaagtttt gattcatttt cgaaacgcct acgagaggcg ccgagaaggc 420
tcagattata aaaaaaaccc gcgagggggt atacaataag agttttggtt ggtcctccgg 480
cgggcgcctt ggtccggggc tgcgacgcac ccggggcaga gatcccgccg aggcaacagt 540
ttggtaacgt tcacattggg tttgggagtt gtaaactcgg taatgatccc tccgcaggtc 600
acccctacag aa 612
Claims (8)
1. one plant of marine fungi Trichoderma harzianum (Trichoderma harzianum) DLEN2008005, which is characterized in that in
On January 29th, 2016 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number are as follows:
CGMCC No.12105；Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
2. according to claim 1 marine fungi Trichoderma harzianum (Trichoderma harzianum) DLEN2008005,
It is characterized in that, the ITS1-5.8S-ITS2 rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1.
3. according to claim 1 marine fungi Trichoderma harzianum (Trichoderma harzianum) DLEN2008005,
It is characterized in that, which has the following properties: being inoculated on seawater potato sucrose culture medium flat plate, 28 DEG C of cultures, bacterium colony is opened
Begin to be white cotton fiber shape, it is round, extend around, after from bacterium colony center to edge generate celadon spore, colony edge color is slightly shallow,
Entire bacterium colony is in loose cotton-shaped；Mycelia is very thin colourless under microscope, and tool separates, multi-branched；Side shoot of the conidiophore from mycelia
On bear, to raw or alternate, there is 2~3 branches, sporogenic stigma doleiform or taper estranged；Conidium is mostly spherical shape,
Sporoderm has pustule, blue-green；26~30 DEG C of the bacterial strain optimum growth temp range, optimum growh pH are 6.0~7.0, most preferably
Growing salt concentration range is 2.0%~5.0%, using glucose, sucrose, lactose；The bacterial strain is in seawater potato sucrose liquid
Standing for fermentation can produce anti-oxidant and acetylcholinesterase inhibitor active material in culture medium.
4. marine fungi Trichoderma harzianum DLEN2008005 described in claim 1 is preparing anti-oxidation medicine and/or is inhibiting acetyl gallbladder
Application in the drug of alkali esterase active.
5. the culture of marine fungi Trichoderma harzianum DLEN2008005 described in claim 1, which is characterized in that comprising thallus and
Thallus fermented and cultured product.
6. the culture of marine fungi Trichoderma harzianum DLEN2008005 according to claim 5, which is characterized in that the training
Feeding object is 28 DEG C of natural lighting by the way that marine fungi Trichoderma harzianum DLEN2008005 to be inoculated in the fluid nutrient medium of sterilizing
It obtains within culture 21~24 days；Wherein, the fluid nutrient medium the preparation method comprises the following steps: 20.0g is added in every 500mL potato liquor
Sucrose and 20.0g sea salt, natural ph；The potato liquor are as follows: fresh potato peeling is taken to be cut into 1cm3Size is blocky, often
200g potato ball adds 500 mL of distilled water to boil 20 min, the double-deck absorbent gauze filtering, and is settled to 500mL with distilled water.
7. the culture of marine fungi Trichoderma harzianum DLEN2008005 according to claim 6, which is characterized in that the training
The preparation method for supporting object is as follows:
(1) seed culture: strain is inoculated into sterile seed solid medium tablets from inclined-plane, culture dish plate is set
Culture is inverted at 28 DEG C in constant incubator, incubation time is 4~5 days；
(2) expand culture: 1L fluid nutrient medium being filled in 3L triangular flask, in high-pressure sterilizing pot after being sealed with breathable sealing film
In 121 DEG C, 0.1 Mpa sterilize 20min；After being cultivated after cooling according to every 1000mL fluid nutrient medium inoculation above-mentioned steps (1)
Seed plate carries disease germs 40~80cm of culture block2Ratio inoculation, sealing, be placed in constant incubator and cultivate, natural lighting, training
Supporting the time is 21~24 days, obtains culture；
Wherein, used culture medium is as follows:
Seed solid medium are as follows: 20.0g sucrose, 20.0g sea salt and 15.0g agar are added in every 500mL potato liquor, from
Right pH value；
Fluid nutrient medium are as follows: 20.0g sucrose and 20.0g sea salt, natural ph are added in every 500mL potato liquor；
Wherein, potato liquor: fresh potato peeling is taken to be cut into 1cm3Size is blocky, and every 200g potato ball adds distilled water 500
ML boils 20 min, the double-deck absorbent gauze filtering, and is settled to 500mL with distilled water.
8. the culture of marine fungi Trichoderma harzianum DLEN2008005 described in claim 5 is preparing anti-oxidation medicine and/or suppression
Application in the drug of acetylcholine esterase active processed.
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| biological effects of trichoderma harzianum peptaibols on mammalian cells;Joanna peltola et al.;《applied and environmental.microbiology》;20040831;第70卷(第8期);4996-5004 * |
| 乙酰胆碱酯酶抑制剂治疗老年痴呆症的研究进展;刘艳玲等;《山西医药杂志》;20080131;第37卷(第1期);64-66 * |
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