My actinobacteria grow in 2.5% soluble starch supplemented to the media. After I obtain the supernatant and store at 4 degree Celsius for a couple of days, a white mass which I believe to be starch precipitates in the cell free supernatant. What could be the possible reason? I could observe the same white precipitate during ammonium sulfate precipitation. After centrifugation the precipitate is large amounts of starch. How to get rid of this?
Are you sure the white precipitate is a starch? Do not you think the bacteria used the starch as their source of nutrient? Did you characterize the white precipitate in terms of its chemical composition? I am speculating that the white precipitate might be aggregates of extracellular proteins produced by bacteria. You can re-suspend the precipitate using appropriate buffer and run samples on SDS-PAGE for possible detection of proteins.
However, if the white precipitate is a starch, then the causes of its precipitation might be decreased its solubility at low temperature and/or change of pH of the solution up on storage at low temperature.
Ammonium sulphate is one of the most commonly used salt for precipitation of proteins and other biomolecules from solutions. Obviously, addition of ammonium sulphate to your supernatant results a precipitate depending on the concentration of the salt, incubation temperature, incubation time and concentration of proteins and other biomolecules in the solution.
Thank you. Yeah I believe that it's starch, I am attaching the picture of the precipitate before and I added iodine. Yeah the bacteria need starch for growth though.
Please note that starch is not readily soluble in water, it requires to be heated gently.
The same kind of problem I was facing during amylase production. Therefore, you are advised to reduce the concentration of starch in the production medium. The rheological property of starch is increased at higher concentrations and the medium will be viscous.
If you do not want to do this then you may use Whatman filter paper to retain the non-soluble starch.
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