Dear Olga,

You start the extraction with ethyl acetate and then you extract with methanol.

I have copid some important paragraph from a recent review (attached) which will be helpful to select your isolation method:

The majority of isolation procedures still utilize simple

extraction procedures with organic solvents of different

polarity, water and their mixtures.1,71,72 The methods include

maceration, percolation, Soxhlet extraction, ultrasound-assisted

extraction and turbo-extraction. Maceration is carried out

at room temperature by soaking the material with the solvent

with eventual stirring. It has the advantage of moderate

extraction conditions but suffers from high solvent

consumption, long extraction times and low extraction yields.

Extraction yield is improved by percolation, i.e. packing the

pre-soaked plant material in a container which allows the

constantly controlled removal of the extract via a valve at the

bottom and adding fresh solvent from the top. Soxhlet

extraction is a popular method for extraction due to its

reduced solvent consumption; however, thermo-labile

compounds might be degraded during the extraction process.

For liquid samples extraction by organic solvents or heterogeneous

solvent mixtures can be done, either simply in a

separating funnel or similar to a Soxhlet apparatus in a

perforator. On a smaller scale, extraction of the liquid sample

absorbed on a porous matrix (like diatomaceous earth) packed

in a column with non-miscible solvents is an option (e.g.

Extrelut columns).

Hoping this will be helpful,

Rafik

Dear Olga,

You start the extraction with ethyl acetate and then you extract with methanol.

I have copid some important paragraph from a recent review (attached) which will be helpful to select your isolation method:

The majority of isolation procedures still utilize simple

extraction procedures with organic solvents of different

polarity, water and their mixtures.1,71,72 The methods include

maceration, percolation, Soxhlet extraction, ultrasound-assisted

extraction and turbo-extraction. Maceration is carried out

at room temperature by soaking the material with the solvent

with eventual stirring. It has the advantage of moderate

extraction conditions but suffers from high solvent

consumption, long extraction times and low extraction yields.

Extraction yield is improved by percolation, i.e. packing the

pre-soaked plant material in a container which allows the

constantly controlled removal of the extract via a valve at the

bottom and adding fresh solvent from the top. Soxhlet

extraction is a popular method for extraction due to its

reduced solvent consumption; however, thermo-labile

compounds might be degraded during the extraction process.

For liquid samples extraction by organic solvents or heterogeneous

solvent mixtures can be done, either simply in a

separating funnel or similar to a Soxhlet apparatus in a

perforator. On a smaller scale, extraction of the liquid sample

absorbed on a porous matrix (like diatomaceous earth) packed

in a column with non-miscible solvents is an option (e.g.

Extrelut columns).

Hoping this will be helpful,

Rafik

Thank you, this is extremely helpful. Have a great day

If you have grown the fungus in submerged culture, then you first separate the mycelium from the broth by filtration or centrifugation. The mycelium is treated with methanol to extract the metabolites. If you wish you can add ethyl acetate after the methanol extract is removed. 

The supernatent can be tested in wells or on disks. You can extract the supernatent with ethyl acetate,  but if the active compound is not extractable with ethyl acetate you can use an adsorbent resin (XAD 4 or 7) or ion exchange resin to 'extract' the active compound.

The suggestions mentioned above may be right but keep in mind that it all depends upon the characteristics of the intended metabolites in the living tissues . For the aqueous systems in the fungi , the Aqueous methanol will be best solution to bring the metabolites in the extract . Now extract with Ethyl acetate solvent ,evaporate this extract to very small volume, few ml , perform the TLC test on a square type ( 4 X 4 inches size ) to check first on a one dimensional side , the behaviour of the extract contents , the same plate can be eluted on the second dimension . this will show us the no / amount of the impurities being eluted in the first dimension. The solvent system we just have to find out through the trial basis & use that which gives maximum no. of compounds on the TLC plate. The Column chromatography solvent system has to be fairly stronger due to thickness of the silica bed or cellulose column bed . Collect the column fractions & do their UV , try to separate them as per their behaviour. Separate the one U R interested most , evaporate them for further study & chemical characterizations .

Thanks for your help. It is greatly appreciated

In practice the ethyl acetate extract is more effective than metanol extract in inhibition of microorganism growth, most of all. 

Dear Zahra , Olgas first aim is to have the proper & complete extraction of   the Metabolites from the Fungus Cultures . Once She has the extract , then only she can do the Antibiotic Activity assays .  The answers given above will resolve her objective for the purpose. Dr Abid Aziz Ajaz

 Thank you