CN105695334B - A kind of new trichoderma asperellum and application thereof - Google Patents
A kind of new trichoderma asperellum and application thereof Download PDFInfo
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- CN105695334B CN105695334B CN201410690333.1A CN201410690333A CN105695334B CN 105695334 B CN105695334 B CN 105695334B CN 201410690333 A CN201410690333 A CN 201410690333A CN 105695334 B CN105695334 B CN 105695334B
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- disinfectant
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention discloses a kind of new trichoderma asperellums and application thereof.Trichoderma asperellum of the present invention, it is the trichoderma asperellum LB-N2 for being CGMCC No.3.17461 by the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation.The metabolite of trichoderma asperellum of the present invention has the activity for inhibiting the bacteriums such as Escherichia coli, salmonella, staphylococcus aureus, can prepare anti-bacterial drug or disinfectant, potential applicability in clinical practice is good.
Description
Technical field
The present invention relates to a kind of new trichoderma asperellums and application thereof.
Background technique
Trichoderma (Trichoderma) fungi is attributed to Deuteromycotina, Hyphomycetes, hyphomycetales, glues spore mushroom.It is nature
Generally existing a kind of fungi, is common in plant residue and animal wastes in boundary, passes through from plant rhizosphere and seed, bulb surface
It can often be separated to.Trichoderma (Trichoderma) has extensive preventive and therapeutic effect to plant pathogenic fungi, and trichoderma is at least to corruption
29 kinds of important plant pathogenic fungis such as mould, sickle-like bacteria, rhizoctonia, sclerotinite, Sclerotium rolfsii have antagonism.Extensively at present
It is general a variety of by fungus-caused plant disease, especially Rhizoctonia solani, sickle-like bacteria, Sclerotium rolfsii, phytophthora for preventing and treating
Bacterium, pythium spp, chain lattice embrace damping-off of larch Seedlings caused by bacterium etc., broken disease, southern blight, phytophthora root rot etc. soil-borne diseases have preferably
Control efficiency.
Trichoderma asperellum (Trichoderma asperellum) can generate the objects such as chitinase, dextranase and protease
Matter, by the cell wall of degrading plant pathogen, and then inhibit plant pathogenic fungi growth (such as rhizoctonia Rhizoctonia,
Phytophthora Phytophthora and pythium spp Pythium etc.), it is applied to peanut sclerotium rolfsii, the silver leaf, cucumber of prevention and treatment fruit tree
The samping off of verticillium wilt and various crop.
Currently, having no that trichoderma asperellum and its metabolite have the active report for inhibiting bacterium.
Summary of the invention
The present invention provides a kind of new trichoderma asperellum, the preparation method of antibacterial metabolite and its it is used to prepare anti-thin
The purposes of bacterium drug or disinfectant.
Trichoderma asperellum of the present invention, it is by China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation
Deposit number is the trichoderma asperellum LB-N2 of CGMCC No.3.17461.
Aforementioned trichoderma asperellum LB-N2 (Trichoderma asperellum LB-N2) is preserved in Chinese microorganism strain guarantor
Hide administration committee's common micro-organisms center, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences are micro-
Biological study institute, preservation date on October 23rd, 2014, deposit number are as follows: CGMCC No.3.17461.
The present invention also provides the methods for the antibacterial metabolite for preparing aforementioned trichoderma asperellum, include the following steps:
(1) ferment: taking deposit number is the trichoderma asperellum LB-N2 of CGMCC No.3.17461, is inoculated in added with final concentration
For in the PD culture medium of 20g/L soluble starch and 8g/L peptone, shaking table is trained under conditions of 28 DEG C ± 1 DEG C, 180 revs/min
It supports, cultivates 7~10 days, filtering obtains fermented supernatant fluid and mycelium；
(2) it extracts:
The fermented supernatant fluid of step (1) is taken, is concentrated under reduced pressure, obtains concentrate, alcohol precipitation is extracted with ethyl acetate 3 times, merges second
Acetoacetic ester phase, the as acetic acid ethyl ester extract of fermented supernatant fluid；
The mycelium of step (1) is taken, is dried, is ground, the dehydrated alcohol ultrasonic extraction 60min of 50 times (v/w) is added, it is rear to use
Ethyl acetate extraction, obtains ethyl acetate phase, as mycelial acetic acid ethyl ester extract；
Merge the acetic acid ethyl ester extract and mycelial acetic acid ethyl ester extract for collecting fermented supernatant fluid, is concentrated under reduced pressure into
Medicinal extract to get.
In step (2), alcohol precipitating method is: the dehydrated alcohol of concentrate three times volume is added, stands 12h.
It the antibacterial metabolite that is prepared the present invention also provides preceding method and its is preparing anti-bacterial drug or is disappearing
Purposes in toxic agent.
The anti-bacterial drug or disinfectant are the drugs of anti-Escherichia coli, salmonella and/or staphylococcus aureus
Or disinfectant.
The present invention also provides a kind of antibacterials or disinfectant, it be using aforementioned antibacterial metabolite as active constituent,
In addition on drug or acceptable auxiliary material is prepared on disinfectant preparation.
The anti-bacterial drug or disinfectant are liquid preparation, gaseous formulation, solid pharmaceutical preparation or semisolid preparation.
The metabolite of new trichoderma asperellum provided by the invention, which has, inhibits Escherichia coli, salmonella, golden yellow Portugal
The activity of the bacteriums such as grape coccus can prepare anti-bacterial drug or disinfectant, and find the important microbe of new antibiotic
Resource, potential applicability in clinical practice are good.
The specific embodiment of form by the following examples is further described above content of the invention in detail bright.
But this should not be interpreted as to the scope of the above subject matter of the present invention is limited to the following embodiments.It is all to be based on above content institute of the present invention
The technology of realization all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is form of the Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski endogenetic fungus trichoderma asperellum LB-N2 of the present invention on PDA plate.
Fig. 2 is the form of trichoderma asperellum LB-N2 in the fermentation medium.
Fig. 3 is trichoderma asperellum LB-N2 spore shape feature.
Fig. 4 is the fungistatic effect figure of metabolite.
Specific embodiment
Experimental material and reagent:
The separation of the trichoderma asperellum of the present invention of embodiment 1 is identified
1, it separates
1.1 conventional endogenetic fungus partition methods
Healthy Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski blade middle section is cut into 2mm small pieces, small pieces are dipped in 75% alcohol 30S by tweezers, sterile
Water rinses 2~3 times, 2~3min of 3%NaClO, then immerses 75% alcohol 30S, and rinsed with sterile water 2~3 times, with last time nothing
Bacterium water compares, and aseptic nipper taking-up vanelets are smooth on PDA plate by its, and 6 small pieces are placed in every ware, are sealed with sealed membrane
The colonial morphology of statistics appearance is observed continuously in room temperature decentralization afterwards after setting 5 days and quantity is said if control group does not grow any bacterium
Bright surface sterilization is complete.
1.2 endogenetic fungus purifying agarics
3 point point of picking typical strain plant in dual anti-PDA plate (in 100%PDA plate contain ampicillin 40ppm,
Streptomycin sulphate 80ppm inhibits bacterial growth) on, 20 DEG C after culture 5 days, picking colony edge vitellarium mycelia tip is pure
Change, single colonie inclined-plane saves, and the fungi isolated and purified is numbered.
1.3 endogenetic fungus conservations
Picking Tip Splitting is inoculated in sterile inclined-plane PDA culture medium, and 5-7 days are cultivated in 28 DEG C of constant incubators to spore cloth
Full entire inclined-plane has no varied bacteria growing, 4 DEG C of envelope hiding of sterile paraffin oil is added, additional amount is to be higher by beveled top end about when upright test tube
1cm is advisable.
2, it identifies
2.1 identification method
The bacterial strain that preceding method is isolated is taken, is identified with the following method:
(1) colony morphology characteristic
Using microscope and visually observe colonial morphology on cell and solid plate.
(2) molecular biology identification
Using round pcr, measure the 18S rDNA/ITS sequence of aforementioned bacterial strain, using BLAST software by the sequence with
The DNA sequence dna included in GenBank is compared, and constructs chadogram with relevant bacteria species, identifies the kind of bacterial strain.
2.2 qualification result
(1) colony morphology characteristic:
As shown in Figures 1 to 3, growth rate is fast, can be paved within bacterium colony 3-5 days on PDA plate entire plate, and initial stage is white
Cotton-shaped mycelia, is scattered from center to edge, and center is small green spot, and mid-term generates light green color spore, and the later period is bottle green spore,
White hypha gradually decreases.Conidiophore is elongated, is bent, and conidium is spherical, subsphaeroidal, is formed within fermented and cultured 7-9 days straight
Diameter is the spherical mycelia of 6-7mm, and culture solution is transparent limpid.
(2) 18S rDNA/ITS sequence are as follows:
TGGGGGATCGGAGCTCACTCCCAACCCAATGTGAACGTTACCAAACTGTTGCCTCGGCGGGGTCACGCC
CCGGGTGCGTCGCAGCCCCGGAACCAGGCGCCCGCCGGAGGAACCAACCAAACTCTTTCTGTAGTCCCCTCGCGGAC
GTATTTCTTACAGCTCTGAGCAAAAATTCAAAATGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGA
TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTG
CGCCCGCCAGTATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCAACCCTCGAACCCCTCCGGGGGATCGGCGTTG
GGGATCGGGACCCCTCACACGGGTGCCGGCCCCGAAATACAGTGGCGGTCTCGCCGCAGCCTCTCCTGCGCAGTAGT
TTGCACAACTCGCACCGGGAGCGCGGCGCGTCCACGTCCGTAAAACACCCAACTTTCTGAAATGTTGACCTCGGATC
AGGTAGGAATACCCGCTGAACTTAAGCATATCAA
In conjunction with aforementioned colony morphology characteristic and 18S rDNA/ITS sequence, isolated bacterial strain is accredited as trichoderma asperellum
(Trichoderma asperellum) names as trichoderma asperellum LB-N2 (Trichoderma asperellum LB-N2), and
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, preservation date on October 23rd, 2014,
Deposit number are as follows: CGMCC No.3.17461.
Embodiment 2 prepares antibiosis metabolic product using strain fermentation of the present invention
1, experimental material
PDA solid medium (1L): 200 grams of potato, 20 grams of glucose, 15~20 grams of agar, remaining is water；Preparation side
Method: weighing 200g potato and be cut into small pieces, and adds boiling rotten (boiling 20~30 minutes, can be poked by glass bar), with eight layers
Agar is added in filtered through gauze, continues heating stirring and mixes, and after agar has dissolved, glucose is added, stirs evenly, slightly cold
But moisture is supplied after again to 1000 milliliters.
PD fluid nutrient medium (1L): 200 grams of potato, 20 grams of glucose, remaining is water；Preparation method: 200g horse is weighed
Bell potato is cut into small pieces, and adds boiling rotten (boiling 20~30 minutes, can be poked by glass bar), with eight layers of filtered through gauze, is added
Glucose stirs evenly, and supplies moisture again to 1000 milliliters after slightly cooling down.
Escherichia coli: Escherichia coli ATCC8739 bacterial strain；
Salmonella: salmonella ATCC14028 bacterial strain；
Staphylococcus aureus: 27217 bacterial strain of staphylococcus aureus gold Portugal ATCC.
2, preparation method
(1) the trichoderma asperellum LB-N2 strain that the separation of embodiment 1 saves the activation culture of strain: is moved into PDA solid plate
On, it is cultivated under the conditions of 28 DEG C ± 1 DEG C, until mycelia covers entire culture dish, it is that the punching of 4mm punch obtains bacteria cake with diameter,
For inoculation；
(2) fermented and cultured: fermentation culture is to be added in every 1000mL PD culture medium, soluble starch 20g, peptone
8g, every bottled fermentation culture 200mL of 500mL triangle；121 DEG C of sterilizing 20min, are cooled to 40 DEG C in aseptic condition after preparation
Lower 1 diameter of picking is 4mm bacteria cake, under the conditions of 28 DEG C ± 1 DEG C, 180 revs/min of shaking speed, fermented and cultured 7-10 days.
(3) fermentation material extracts: taking fermentation liquid, filters, be divided into mycelia and fermented supernatant fluid, fermented supernatant fluid 5L is concentrated under reduced pressure
To 1L, alcohol precipitation removes impurity (dehydrated alcohol of three times volume being added, stand 12h), filters removal precipitating, filtrate acetic acid second
Ester 1:1 is extracted 3 times, combined ethyl acetate phase；Mycelium is after baking oven drying, and taking-up is ground mycelia with mortar, with anhydrous second
Alcohol (w/v=1/50) ultrasonic extraction 60min, after ethyl acetate phase is extracted with ethyl acetate to obtain, finally merge collect fermentation supernatant
Liquid acetic acid ethyl ester extract and mycelia acetic acid ethyl ester extract, Rotary Evaporators are concentrated under reduced pressure into medicinal extract to get trichoderma asperellum LB-
N2 metabolite is saved backup with 4 DEG C of refrigerators of acetone solution.
3, antibacterial activity detects
The 3.1 trichoderma asperellum LB-N2 metabolite 0.1g for taking preceding method to prepare, and the acetone of 10mL volume is added, surpass
Sound wave shock sufficiently dissolves, and is finally configured to the sample solution that concentration is 10g/L；
Escherichia coli, salmonella, staphylococcus aureus after 3.2 activation are diluted to 10 with sterile water8Cfu/mL takes
0.1mL bacteria suspension is coated on LB plate.Oxford cup is put, 100ul sample is added, acetone is compareed, is repeated 3 times, 37 DEG C of trainings
Support 1~2d, observe inhibition zone size and whether there is or not.
4, antibacterial activity testing result
As a result as shown in the following table 1 and Fig. 4:
Inhibitory effect of the 1 trichoderma asperellum LB-N2 of table for examination pathogenetic bacteria
The trichoderma asperellum LB-N2 metabolite of preceding method of the present invention preparation is to large intestine bar it can be seen from table 1 and Fig. 4
Bacterium, salmonella, staphylococcus aureus all have good inhibiting effect, can be prepared into antibacterial drug or disinfection
Agent.
To sum up, the present invention separates one plant of new trichoderma asperellum from plant Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski stem, and metabolin is to big
Enterobacteria, salmonella, staphylococcus aureus have compared with high inhibition effect, research and develop new antibacterials for field of medicaments or disappear
Toxic agent provides new source.
Claims (7)
1. a kind of trichoderma asperellum (Trichoderma asperellum), it is characterised in that: it is protected by Chinese microorganism strain
The deposit number for hiding the preservation of administration committee's common micro-organisms center is the trichoderma asperellum LB-N2 of CGMCC No.3.17461.
2. the method for preparing the metabolite of trichoderma asperellum described in claim 1, characterized by the following steps:
(1) ferment: taking deposit number is the trichoderma asperellum LB-N2 of CGMCC No.3.17461, is inoculated in added with final concentration of
In the PD culture medium of 20g/L soluble starch and 8g/L peptone, the shaking table culture 7 under conditions of 28 DEG C ± 1 DEG C, 180 revs/min
~10 days, filtering obtained fermented supernatant fluid and mycelium；
(2) it extracts:
The fermented supernatant fluid of step (1) is taken, is concentrated under reduced pressure, obtains concentrate, alcohol precipitation is extracted with ethyl acetate 3 times, merges acetic acid second
Ester phase, the as acetic acid ethyl ester extract of fermented supernatant fluid；
Take the mycelium of step (1), dry, grind, be added 50 times (v/w) dehydrated alcohol ultrasonic extraction 60min, after use acetic acid
Ethyl ester extraction, obtains ethyl acetate phase, as mycelial acetic acid ethyl ester extract；
Merge the acetic acid ethyl ester extract and mycelial acetic acid ethyl ester extract for collecting fermented supernatant fluid, is concentrated under reduced pressure into leaching
Cream to get.
3. according to the method described in claim 2, it is characterized by: alcohol precipitating method is in step (2): being added three times of concentrate
The dehydrated alcohol of volume stands 12h.
4. the metabolite that Claims 2 or 3 the method is prepared.
5. metabolite described in claim 4 is preparing the purposes in anti-bacterial drug or disinfectant, it is characterised in that: described anti-
Bacterial drug or disinfectant are the drug or disinfectant of anti-Escherichia coli, salmonella and/or staphylococcus aureus.
6. a kind of antibacterials or disinfectant, it is characterised in that: it be using metabolite described in claim 4 as active constituent,
In addition on drug or acceptable auxiliary material is prepared on disinfectant preparation；
The microorganism that the antibacterials or disinfectant are directed to is Escherichia coli, salmonella and/or staphylococcus aureus.
7. antibacterials according to claim 6 or disinfectant, it is characterised in that: the anti-bacterial drug or disinfectant are
Liquid preparation, gaseous formulation, solid pharmaceutical preparation or semisolid preparation.
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| CN108407007A (en) * | 2018-01-30 | 2018-08-17 | 桐乡市林达木业有限公司 | A kind of deinsectization mildew-proof treatment method of fence board |
| CN109251098A (en) * | 2018-10-31 | 2019-01-22 | 周口师范学院 | A kind of quick returning to the field method of straw bacterial manure |
| CN109749943B (en) * | 2019-03-06 | 2022-02-11 | 重庆市农业科学院 | Trichoderma asperellum and application thereof |
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| CN102911878A (en) * | 2012-09-27 | 2013-02-06 | 浙江大学 | Trichoderma asperellum strain and application thereof |
| CN104211671A (en) * | 2013-05-30 | 2014-12-17 | 福州大学 | Alkyl pyranone compound and preparation process and application |
| CN104211780A (en) * | 2013-05-30 | 2014-12-17 | 福州大学 | Cyclic depsipeptides compound, preparation method and purpose thereof |
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| CN102911878A (en) * | 2012-09-27 | 2013-02-06 | 浙江大学 | Trichoderma asperellum strain and application thereof |
| CN104211671A (en) * | 2013-05-30 | 2014-12-17 | 福州大学 | Alkyl pyranone compound and preparation process and application |
| CN104211780A (en) * | 2013-05-30 | 2014-12-17 | 福州大学 | Cyclic depsipeptides compound, preparation method and purpose thereof |
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| Sesquiterpenes and Cyclopeptides from the Endophytic Fungus Trichoderma asperellum Samuels, Lieckf. & Nirenberg;Gang Ding et al;《Chemistry & Biodiversity》;20120615;第9卷(第6期);第1205页摘要，第1210页第4-6段，第1211页第1-2段 * |
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