- The same steps already mentioned in a last report are followed(10).
To purify the produced penicillin we had pursued different three protocol.
The first (P1) without using charcoal (just: filtration-ethyl acetate-centrifugation- sodium bicarbonate)
The second by using charcoal and V/V from the ethyl acetate
The third by using charcoal and V[14:2] from the ethyl acetate( for each 14 ml of the filtrate use 2 ml of ethyl acetate)
Then we have tested the filtrate when we had obtain a lot of contaminant.
For this reason the protocol was retried with the autoclaving of the liquid medium before adding the penicillium colony. Then the quantification will be tried.
2- Corrected protocol for Penicillin production :
2.1 Purification of the isolate:
We took a colony from the cultivated dish and we recultivate into another tryptone medium to obtain a pure strain that will be used below.
- We weigh 2 g of glucose powder, 0.8 g of lactose, 0.8g of peptone, 1g yeast extract, 0.15g of MgCl2, 0.15g of KCl, and 1g of KH2PO4, 0.2g CaCO3, 0.1g corn oil
- It is important to add penicillin precursor which called phenyl acetate at proportion 0.5%
- We add 100ml of distilled water
- We put the mixture in an erlenmeyer flasks
- We heat them with mixing for 15min by using a magnetic hot plate stirrer
- We autoclave the liquid medium, then let it cool down for about 30 min
- We inoculate it with the cultivated petri dish already prepared (about two or three colony)
- We incubate them at room temperature for 7 to 10 days with shaking
- Harvest liquid medium which contain penicillin by filtration
- Chill to 5-10oC (because penicillin is highly reactive and destroyed by alkali and enzyme)
- Acidify filtrate to PH 2.0-2.5 with H2SO4 (to convert penicillin to its anionic form)
- Extract penicillin from aqueous filtrate by adding ethyl acetate (at this very low ph as soon as possible)
- Discard aqueous fraction
- Allow the organic fraction to pass through charcoal to remove impurities and extract penicillin (this step is not important)
- Extract penicillin from ethyl acetate by adding 2% aqueous phosphate buffer ( here the PB can be replaced by distilled wter) at PH 7.5
- Acidify the aqueous fraction to PH 2-2.5 with mineral acid and re-extract penicillin into fresh ethyl acetate
- Add sodium bicarbonate to the solvent to christallise the antibiotic as sodium salt
- Recover crystal in filter centrifuge or by filtration.