Actually I am extracting Polyhydroxy butyrate from bacillus by using MSM media but I am discarding the supernatant of that media because my concern is to the pellet only,so I am expecting the supernatant could be used for extracting of biosurfactant.for that I need protocol.
1) Inoculate in 50 ml R2B broth containing 1ml of petrol.
2) Incubate at 25°C for 7 days with shaking conditions. After incubation, centrifuge at 5000 rpm, 4°C for 30 minutes to remove the bacterial cells.
3) To the supernatant add 1M H2SO4 to adjust the pH to 2.
4) Add equal volumes of chloroform: methanol was in the ratio of 2:1. Shake well to
ensure proper mixing and leave overnight for evaporation. White coloured precipitate seen at the interface between the two liquids is the biosurfactant.
5) Take out the biosurfactant formed with the help of micropipette and keep
in eppendorf tubes. Add 1ml distilled water and vortex well to mix.
6) Centrifuge at 7000rpm, 4°C for 30 minutes. Discard the supernatant and dry the pellet for 24 h.
The dry pellet thus obtained is the crude extract of the biosurfactant.
Hi Swati
So you need protocol for extraction of biosurfactants from Bacillus using MSM?
Let me know
Ahmad Zulfiqar Thanks for your replay.
It will be better if I will get the protocol
Alcoholic extraction with sulphated precipitation in cold could transfer biosurfactant in to supernatant.
Before going for extraction, see the microbe has produced biosurfactant in the medium. Do emulsification and surface tension measurements for the culture broth. This will give you an idea, is it worth extraction.
Use the following protocols for biosurfactant extration:
1. Acidify the broth to pH 2.0 and extract with ethyl acetate (3:1, v/v, EA:Broth)
2. Use Chloroform methanol at 2:1 ratio, if needed acidify the broth to pH 2.0 prior to extraction.
In both step you have to evaporate the solvents in a rotatory evaporator under reduced pressure. If the strain produced biosurfactant, the crude product you get after evaporation is the biosurfactant.
If you need detailed information view my publication.
1) Inoculate in 50 ml R2B broth containing 1ml of petrol.
2) Incubate at 25°C for 7 days with shaking conditions. After incubation, centrifuge at 5000 rpm, 4°C for 30 minutes to remove the bacterial cells.
3) To the supernatant add 1M H2SO4 to adjust the pH to 2.
4) Add equal volumes of chloroform: methanol was in the ratio of 2:1. Shake well to
ensure proper mixing and leave overnight for evaporation. White coloured precipitate seen at the interface between the two liquids is the biosurfactant.
5) Take out the biosurfactant formed with the help of micropipette and keep
in eppendorf tubes. Add 1ml distilled water and vortex well to mix.
6) Centrifuge at 7000rpm, 4°C for 30 minutes. Discard the supernatant and dry the pellet for 24 h.
The dry pellet thus obtained is the crude extract of the biosurfactant.
Article Bacillus amyloliquefaciens ssp. plantarum F11 isolated from ...
All above said protocols are labourious
Just take 1:2 ratio of broth and ethylacetate
Shake it vigerously
Separate top layet
Allow top layer to evaporate
Add 50 ul to dried one
Spot on TLC and confirm the rhamnolipid
Very simple procedue, dont follow lenghty protocols and waste your time
People dont think innovatively and stick to old procedure and end up their lifed
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction