Extraction with ethyl acetate is good. However the yield in extraction depends on many factors such as how long you are extracting. You can try the following. Add twice the volume of the solvent to the culture broth; Keep it intermittently shaken in separation flask about 4 h. After separate the extract alone that means the solvent fraction and store it separately. Do the same procedure with the spent culture broth again for atleast twice. You then pool all the three fractions of ethyl acetate extracts and dry them through evaporation preferably in a rotary evaporation. You may get more extract. Better use a good quantity of culture broth. Atleast 500 ml broth is needed initially. All the best.

I am also facing the same problem..can anyone suggest any remedy?

First can u tell me what are all the solvent u tried based on the polarity.

R u tried with rotary evaporator.

Hi Smriti

I would suggest to add 10% Methanol to your centrifugate and then add remaining 90% voulume of Ethyl acetate (in 2 lots). Then separate EA layer and evaporate to get desired residue. e.g. for 1 L centrifugate if you want to do extraction in 1 L solvent. First add 100ml Methanol. stir it. then add 450ml EA-take out EA layer. Again add 450ml EA - separte EA layer and pool with earlier EA layer-concentrate both. Please try out. If any difficulty please free to ask. This is assuming that you want to do only EA extraction.

Check the level of the used evaporation temperature. 50 to 55 C used to work well

hi better u have to use starch caesin broth medium or soyabean meal medium for 15 days at 28 C. u will more amount. all the best

hello smriti. u can try other solvents also like chloroform, butanol, acetonitrile etc. but if u wanna use ethyl acetate as it is easy to evaporate, change the pH of the broth and try extraction at different pH.

Hi better u ill try to increase the incubation time up to 15 days at 250rpm of broth culture and you will try in starch casein broth and soya bean meal broth. You will get get good result all the best

You can try to use another solvent with different polarity or you preferably to make successive extraction beginning with the less polar solvent then the high and the higher polar. Generally the production of the maximum amount of metabolites or ingredients of an organism requires to do the optimization processes; to know the optimum growth medium, the optimum pH, the optimum incubation temperature and period, the optimum rpm and so on. you can also try to extract from the solid medium (agar medium) instead of the broth or the liquid medium, I had a trial in this regard when I despaired to extract the active ingredients from actinomycetes liquid culture I used the solid culture and had extracted the active metabolites using successive extraction with different solvents to determine the best solvent.

adjust different pH of extract before and find some suitable pH for extraction of metabolite from broth. if it does not work then check n hexane, chloroform, for extraction. if again results are not obtained then try HP Di-anion 20 resin fro extraction of metabolites. also check activated charcoal for extraction.

have u got the sufficient growth in your production medium? If the production is high surely u can get more amount of extract, 1st u should standardize the medium, then u can go for extraction, i think starch casein nitrate medium may be suppress the growth. then one more question u r incubation period is only 6 days is it enough? u check it out everything and monitor carefully all the best

Secondary metabolites are produced in small quantities, we need more than 20 L of production medium to obtain detecteble amount of secondary metabolites , try to cultivate producer bacterium in optimum production conditions under large scale production

Ok what is extracellular compound u want to extract?

Follow the standard extraction method of this compound from the literature review?

After that we can give u the best answer.

Be clear or define what u want to extract. Regards

Try on Starch casein agar as (Composition: Starch -10gm, Casein hydrolysate-0.3gm, Potassium nitrate-2gm, Sodium chloride-2gm, Potassium hydrogen phosphate-2gm, Magnesium sulphate-0.05gm, Ferrous sulphate-0.02gm, Calcium carbonate 0.01gm, Disstilled water-1000ml, pH-7.3±0.2) production medium and incubate for 8 to 10 days at 30 C without shaking.Filter the broth, extract with equal amounts of ethyl acetate, evaporate it and then check the weight of extract.

I suggest you to:

- Try the extraction of secondary metabolites from the solid medium used instead of the liquid medium. protocol you can find it in this article: Identification and antifungal activity of Streptomyces sp. S72 isolated from Lake Oubeira sediments in North-East of Algeria

- extending the incubation time (14 days),

- use other media recommended for the production of secondary metabolites (ISP1 ISP2 PELG, ...).

- Try to optimize the following factors: pH, temperature, salinity....

Hi. Is there any particular bioactivity that you are testing the culture filtrate extracts against? Did you get any activity if tested already? Extract yield may be low especially if you do small scale fermentation of actinobacteria. But you should test activity before anything. One more thing, have you grown your isolates in different production media - try a complex media which normally is better for production of high yield of bioactive metabolites (extracellular and intracellular). Another thing is the fermentation time - have you conducted a time profile study to see when is the optimum harvesting period to obtain maximum amount of secondary metabolites and bioactivity? If you are just doing a screening study on many actinobacteria isolates, then a period of 7-10 days should be ok to start with. Once you get the hit candidates that show activity in extract, immediately conduct time-profile studies to check optimum harvest time to increase yield. But make sure you use a range of production media (many literatures to refer). All the best !

I try NB, ISP2 and Starch casein nitrate broth for fermentation. I want to check antimicrobial activity of actinomycetes isolates against human pathogens.

Thanks to all of you. I follow all the steps mentioned in the refrence papers of actinomycetes, but dont know where is the problem. how to extract the compounds from agar plate.

extraction from agar plates will yield u very low quantity that barely meet ur needs to test for antimicrobial activity. Many have above suggested refinements. First try to fix ur own mind which to follow. All the best

Some actinomycetes do not produce active compounds in liquid medium, but show excellent activity in solid medium. In such case solid fermentation is the only option. Alternately you can grow the actinomycete in agar plates. Minimum of 15 days is essential for better results. After 15 days cut the agar medium in to small bits along with the actinobacterial growth and transfer one plate growth to 50 ml each of Ethyl acetate, chloroform, and n-butanol in conical flasks cover the mouth and put in shaker for two days filter the solvent fraction into a clean petriplate, dry and use for bioaactivity test. Hope you will get better results. If there is result then grow the actinobacteria in plates and proceed. good luck

you must be used large quantity i.e. about 5-10 L of broth in order to get sufficient quantity of crude.

1- it is not clear that do you obtain sufficient biomass or no? if no, please consider the situation of the growth.

2- Please use complex medium (e.g. ISP agar) rather than chemically define medium. It help to enhance the concentration of bioactive compounds.

3- Please increase the rpm from 120 to 220. Actinomycetes are aerobic, therefore increasing shaking help to more growth.

4- is the problem seen for the active strain of unknown activity strain?

5- I think your problem is due to culture not extraction. In screening program, there is no need to test various solvent to extract the fermentation broth, ethyl acetate is enough. However, carefully consider the growth condition.

6- to distinguish the problem (fermentation or extrtion), simply, check the activity of fermentation broth (before extraction) by bioassay on agar medium.

Good Luck.

Try activated charcoal and resin hp dianion

Dear Smriti

You can follow Dr. Javad Hamedi advices. His experiences on secondary metabolites production by actinomycetes are considerable.

Go for more than 10 or 20 liters of culture broth and from that you may get 1 or 2 g of crude extract after solvent extraction and you use Soya meal medium for production 14 days incubation at 150 rpm

First of all, please studied the culture characteristics, also used for suitable media composition incubate etc., after that, u have go through mass cultivation in mass scales, finally the culture supernate was obtained by centrifuge process at 10,0000 ram for 20 mint. after that, the culture supernante was mixed with mid polar solvent as ethyl acetate for vigor shaking and separating the the solvent base, than he solvent base was concentrated finally u get the crude extracts

Best of Luck

@ Santhiyagu Prakash, Can you please provide the reference for the protocol, Sir?

Rakesh Kumar Yadav please seen my publication list sir

Use minimal medium. Starch soluble as a carbon source and ammonium sulphate as a nitrogen source. PH 6or 6.5.shaking speed 250 rpm.