CN105695335A - New aspergillus flavus and use thereof - Google Patents

Abstract

The present invention discloses new aspergillus flavus and use thereof. The aspergillus flavus is aspergillus flavus LB-Y4 preserved in China General Microbiological Culture Collection Center, and the accession number is CGMCC No. 3.15413. Metabolites of the aspergillus flavus have activity for inhibition of staphylococcus aureus and other bacteria, can be used for preparing anti-bacterial drugs or disinfectants, and has good prospects for clinical application.

Description

A kind of new Aspergillus flavus and application thereof

Technical field

The present invention relates to a kind of new Aspergillus flavus and application thereof。

Background technology

Aspergillus flavus (Aspergillusflavus), Fungi Imperfecti, a kind of common saprophytic fungus。It is more common on the Organic substance of mouldy grain, grain goods and other mould corruption。Colony growth is very fast, and short texture, surface celadon, the back side is colourless or slightly brownish。Thalline has the branch mycelia of many complexity to constitute。Vegetative hyphae has separation；A part for aerial hyphae forms long and coarse conidiophore, and top produces flask shape or subsphaeroidal top capsule, and surface produces many stigmas (being generally bilayer), on stigma generate the shaggy spherical conidium of string。Conidiophore, top capsule, stigma and conidium synthesis spore head, can be used for producing amylase, protease and phosphodiesterase etc., be also the common strain in brewery industry。

At present, have no Aspergillus flavus and metabolite has the active report suppressing antibacterial。

Summary of the invention

The invention provides a kind of new Aspergillus flavus, the preparation method of its antibacterial metabolite and for preparing the purposes of anti-bacterial drug or disinfectant。

Aspergillus flavus of the present invention, it is characterised in that: it is by the Aspergillus flavus LB-Y4 that preserving number is CGMCCNo.3.15413 of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation。

Aforementioned Aspergillus flavus LB-Y4 (AspergillusflavusLB-Y4) is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, preservation date on October 23rd, 2014, deposit number is: CGMCCNo.3.15413。

The method that present invention also offers the antibacterial metabolite preparing aforementioned Aspergillus flavus, comprises the steps:

(1) fermentation: take the Aspergillus flavus LB-Y4 that preserving number is CGMCCNo.3.15413, it is inoculated in the PD culture medium being added with final concentration of 20g/L soluble starch and 8g/L peptone, when 28 DEG C ± 1 DEG C, 180 revs/min, shaking table is cultivated, cultivate 7～10 days, filter, obtain fermented supernatant fluid and mycelium；

(2) extract:

Take the fermented supernatant fluid of step (1), concentrating under reduced pressure, obtain concentrated solution, precipitate with ethanol, be extracted with ethyl acetate 3 times, combined ethyl acetate phase, be the acetic acid ethyl ester extract of fermented supernatant fluid；

Take the mycelium of step (1), dry, grind, add 50 times (v/w) dehydrated alcohol supersound extraction 60min, after be extracted with ethyl acetate, obtain ethyl acetate phase, be mycelial acetic acid ethyl ester extract；

Merge the acetic acid ethyl ester extract and mycelial acetic acid ethyl ester extract of collecting fermented supernatant fluid, be evaporated to extractum, to obtain final product。

In step (2), alcohol precipitating method is: add the dehydrated alcohol that concentrated solution triploid is long-pending, stands 12h。

Present invention also offers antibacterial metabolite that preceding method prepares and the purposes in preparing anti-bacterial drug or disinfectant thereof。

Described anti-bacterial drug or disinfectant are medicine or the disinfectant of anti-Staphylococcus aureus。

Present invention also offers a kind of antibacterials or disinfectant, it is with aforementioned antibacterial metabolite for active component, adds on medicine or the preparation that in disinfectant, acceptable adjuvant is prepared from。

Described anti-bacterial drug or disinfectant are liquid preparation, gaseous formulation, solid preparation or semi-solid preparation。

The metabolite of new Aspergillus flavus provided by the invention has the activity suppressing the antibacterials such as staphylococcus aureus, it is possible to prepare anti-bacterial drug or disinfectant, is also the important microbe resource finding new antibiotic, and potential applicability in clinical practice is good。

The detailed description of the invention of form by the following examples, further describes in detail bright to the foregoing of the present invention。But this should not being interpreted as, the scope of the above-mentioned theme of the present invention is only limitted to below example。All technology realized based on foregoing of the present invention belong to the scope of the present invention。

Accompanying drawing explanation

Fig. 1 is the Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski endogenetic fungus Aspergillus flavus LB-Y4 of the present invention form on PDA plate。

Fig. 2 is the Aspergillus flavus LB-Y4 form in fermentation medium kind。

Fig. 3 is Aspergillus flavus LB-Y4 spore shape feature。

Fig. 4 is the fungistatic effect figure of metabolite。

Detailed description of the invention

Experiment material and reagent:

The isolation identification of embodiment 1 Aspergillus flavus of the present invention

1, separate

1.1 conventional endogenetic fungus partition methods

Health Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski blade mid portion is cut into 2mm small pieces, small pieces are dipped in 75% ethanol 30S by tweezers, rinsed with sterile water 2～3 times, 3%NaClO2～3min, immerse 75% ethanol 30S again, rinsed with sterile water 2～3 times, compare with last sterilized water, aseptic nipper takes out vanelets, and it is smooth on PDA plate, 6 small pieces are placed in every ware, after after sealing with sealed membrane, ambient temperatare is put 5 days, Continuous Observation adds up the colonial morphology and quantity that occur, if matched group does not grow any bacterium, then illustrates that surface sterilization is complete。

1.2 endogenetic fungus purifying agarics

3 points of picking typical strain are planted in dual anti-PDA plate (containing ampicillin 40ppm in 100%PDA flat board, streptomycin sulfate 80ppm carrys out bacteria growing inhibiting) on, cultivate after 5 days for 20 DEG C, mycelia tip, vitellarium, picking colony edge purification, single bacterium colony inclined-plane preserves, and will separate the fungus numbering of purification。

1.3 endogenetic fungus conservations

Picking Tip Splitting is inoculated in aseptic inclined-plane PDA culture medium, cultivates and within 5-7 days, treat that spore is covered with whole inclined-plane and there is no varied bacteria growing in 28 DEG C of constant incubators, adds aseptic paraffin oil 4 DEG C envelope and hides, exceeds beveled top end and be about 1cm and be advisable when addition is with upright test tube。

2, identify

Take preceding method and separate the bacterial strain obtained, adopt and identify with the following method:

(1) colony morphology characteristic

Utilize the colonial morphology on microscope and perusal cell and solid plate。

(2) molecular biology identification

Utilize round pcr, measure the 18SrDNA/ITS sequence of aforementioned bacterial strain, utilize BLAST software the DNA sequence included in this sequence and GenBank to be compared, and build cladogram with relevant bacteria species, identify the kind of bacterial strain。

2.2 qualification results

(1) colonial morphology

As shown in Figures 1 to 3, colony growth is rapid, cultivates 7 days diameter 35-40mm for 25 DEG C；Compact colonies velvet shape or cotton-shaped, smooth or radial irregular rill；Conidium color is yellow green to grass green；Generally without transudate；Bacterium colony reverse side is colourless to filbert。It is spherical at the beginning of conidial head, afterwards in Radiation；Conidiophore is conigenous substrate mostly；Capsule is subsphaeroidal or flask shape on top, within fermentation culture 7-9 days, forms the brown mycelium pellet that diameter is 1-2mm, and culture fluid is transparent limpid。

(2) 18SrDNA/ITS sequence is:

ACGAGGGGTACGGTTCTAGCGAGCCCAACCTCCCACCCGTGTTTACTGTACCTTAGTTGCTTCGGCGGGCCCGCCATTCATGGCCGCCGGGGGCTCTCAGCCCCGGGCCCGCGCCCGCCGGAGACACCACGAACTCTGTCTGATCTAGTGAAGTCTGAGTTGATTGTATCGCAATCAGTTAAAACTTTCAACAATGGATCTCTTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAACTAGTGTGAATTGCAGAATTCCGTGAATCATCGAGTCTTTGAACGCACATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCATCAAGCACGGCTTGTGTGTTGGGTCGTCGTCCCCTCTCCGGGGGGGACGGGCCCCAAAGGCAGCGGCGGCACCGCGTCCGATCCTCGAGCGTATGGGGCTTTGTCACCCGCTCTGTAGGCCCGGCCGGCGCTTGCCGAACGCAAATCAATCTTTTCCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAAA

In conjunction with aforementioned colony morphology characteristic and 18SrDNA/ITS sequence, it is Aspergillus flavus (Aspergillusflavus) by separating the identification of strains obtained, name as Aspergillus flavus LB-Y4 (AspergillusflavusLB-Y4), and it is preserved in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preservation date on October 23rd, 2014, deposit number is: CGMCCNo.3.15413。

Embodiment 2 adopts strain fermentation of the present invention to prepare antibiosis metabolic product

1, experiment material

PDA solid medium (1L): Rhizoma Solani tuber osi 200 grams, glucose 20 grams, 15～20 grams of agar, all the other are water；Preparation method: weigh 200g Rhizoma Solani tuber osi and be cut into small pieces, add water and well-done (boil 20～30 minutes, can be poked by Glass rod), by eight layers of filtered through gauze, add agar, continue heated and stirred mixing, after agar has dissolved, add glucose, stir, somewhat supply moisture again to 1000 milliliters after cooling,。

PD fluid medium (1L): Rhizoma Solani tuber osi 200 grams, glucose 20 grams, all the other are water；Preparation method: weigh 200g Rhizoma Solani tuber osi and be cut into small pieces, add water well-done (boiling 20～30 minutes, can be poked by Glass rod), by eight layers of filtered through gauze, adds glucose, stirs, somewhat supplies moisture again to 1000 milliliters after cooling,。

Staphylococcus aureus: staphylococcus aureus gold Portugal ATCC27217 bacterial strain。

2, preparation method

(1) activation culture of strain: the Aspergillus flavus LB-Y4 strain that embodiment 1 separates preservation moves on PDA solid plate, cultivates under 28 DEG C ± 1 DEG C condition, covers whole culture dish to mycelia, is that the punching of 4mm card punch obtains bacterium cake with diameter, for inoculation；

(2) fermentation culture: fermentation culture is add in every 1000mLPD culture medium, soluble starch 20g, peptone 8g, the bottled fermentation culture 200mL of every 500mL triangle；Prepare rear 121 DEG C of sterilizing 20min, be cooled to 40 DEG C aseptically picking 1 diameter be 4mm bacterium cake, under 28 DEG C ± 1 DEG C condition, shaking speed 180 revs/min, fermentation culture 7-10 days。

(3) fermented product extracts: take fermentation liquid, sucking filtration, it is divided into mycelia and fermented supernatant fluid, fermented supernatant fluid 5L is evaporated to 1L, precipitate with ethanol removes impurity (namely with the dehydrated alcohol extraction that triploid is long-pending, stand overnight), and sucking filtration removes precipitation, filtrate extracts 3 times with ethyl acetate 1:1, combined ethyl acetate phase；Mycelium is after oven for drying, mycelia is ground by taking-up mortar, with dehydrated alcohol (w/v=1/50) supersound extraction 60min, after be extracted with ethyl acetate to obtain ethyl acetate phase, finally merge and collect fermented supernatant fluid acetic acid ethyl ester extract and mycelia acetic acid ethyl ester extract, Rotary Evaporators is evaporated to extractum, obtains Aspergillus flavus LB-Y4 metabolite, standby with 4 DEG C of Refrigerator stores of acetone solution。

3, antibacterial activity detection

3.1 take Aspergillus flavus LB-Y4 metabolite 0.1g prepared by preceding method, and add the acetone of 10mL volume, and ultrasonic wave concussion fully dissolves, and is finally configured to the sample solution that concentration is 10g/L；

Staphylococcus aureus after 3.2 activation is diluted to 10 with sterilized water⁸Cfu/mL,

Take 0.1mL bacteria suspension to coat on LB flat board。Putting Oxford cup, add 100ul sample, acetone compares, and repeats 3 times, cultivates 1～2d for 37 DEG C, the size of observation inhibition zone and with or without。

4, antibacterial activity testing result

Shown in result such as table 1 below and Fig. 4:

Table 1 Aspergillus flavus LBY4 is for the inhibition of examination pathogenetic bacteria

By table 1 and Fig. 4 it can be seen that staphylococcus aureus is had good inhibitory action by Aspergillus flavus LBY4 metabolite prepared by preceding method of the present invention, it is possible to preparation becomes antibacterial medicine or disinfectant。

To sum up, the present invention separates, from plant Ligularia virgaurea (Maxim.) Mattf. Ex Rehd. Etkobuski stem, the Aspergillus flavus that a strain is new, and staphylococcus aureus is had relatively high inhibition effect by its metabolite, researches and develops new antibacterials for field of medicaments or disinfectant provides new source。

Claims (8)

1. an Aspergillus flavus, it is characterised in that: it is by the Aspergillus flavus LB-Y4 that preserving number is CGMCCNo.3.15413 of China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation。

2. the method for the antibacterial metabolite of Aspergillus flavus described in preparation claim 1, it is characterised in that: comprise the steps:

(1) fermentation: take the Aspergillus flavus LB-Y4 that preserving number is CGMCCNo.3.15413, it is inoculated in the PD culture medium being added with final concentration of 20g/L soluble starch and 8g/L peptone, when 28 DEG C ± 1 DEG C, 180 revs/min, shaking table is cultivated, cultivate 7～10 days, filter, obtain fermented supernatant fluid and mycelium；

(2) extract:

Take the fermented supernatant fluid of step (1), concentrating under reduced pressure, obtain concentrated solution, precipitate with ethanol, be extracted with ethyl acetate 3 times, combined ethyl acetate phase, be the acetic acid ethyl ester extract of fermented supernatant fluid；

Take the mycelium of step (1), dry, grind, add 50 times (v/w) dehydrated alcohol supersound extraction 60min, after be extracted with ethyl acetate, obtain ethyl acetate phase, be mycelial acetic acid ethyl ester extract；

Merge the acetic acid ethyl ester extract and mycelial acetic acid ethyl ester extract of collecting fermented supernatant fluid, be evaporated to extractum, to obtain final product。

3. method according to claim 2, it is characterised in that: in step (2), alcohol precipitating method is: add the dehydrated alcohol that concentrated solution triploid is long-pending, stands 12h。

4. the antibacterial metabolite that method described in Claims 2 or 3 prepares。

5. antibacterial metabolite purposes in preparing anti-bacterial drug or disinfectant described in claim 4。

6. purposes according to claim 5, it is characterised in that: described anti-bacterial drug or disinfectant are medicine or the disinfectant of anti-Staphylococcus aureus。

7. antibacterials or disinfectant, it is characterised in that: it is with metabolite antibacterial described in claim 4 for active component, adds on medicine or the preparation that in disinfectant, acceptable adjuvant is prepared from。

8. purposes according to claim 7, it is characterised in that: described anti-bacterial drug or disinfectant are liquid preparation, solid preparation or semi-solid preparation。