If i used (Amicon 10,000 MWCO) to concentrate the bacteriocin from MRS broth, the bacteriocin will be at the top part near to the filter or it will be in the bottom container? and what is the time, speed and temperature for centrifugation?
It will depend on the size of your bacteriocin. My guess is that most bacteriocins will NOT be concentrated in this manner as most of the ones from lactic acid bacteria (that have relevance as food pathogen inhibitors) are small molecules, around 2,000-3,500 mol. wgt, so unless they complex with some other molecules, you won't be concentrating them. But that is OK, because you will be 'cleaning up' the sample from most of the larger molecules that will be retained, but I would suspect the bacteriocins would elute through the filter and be in the filtrate. You can easily determine the amount retained/lost by doing activity titer before, and after Centricon ultrafiltration. If you have a vacuum dryer, one easy way would be to use Sep-Pak C18 cartridges hooked up to a syringe.....pass the bacteriocin-containing culture broth through that....everything will bind to the C18 matrix (eluate should look clear; cartridge will appear 'brown' by retained media proteins), and then using step-wise 10-ml rinses pushed through the cartridge by way of a syringe....wash with Isopropanol: 0% (water), 10%, 20%, 30%, 40%, 50%, 60%, 100%......dry these down with a vacuum concentrator if available and resuspend with water/buffer of your choosing. You will likely find that much of the 'brown' protein content elutes somewhere in the 10-20% fractions and hydrophobic bacteriocin molecules in the 30-50% fractions. A simple way to use hydrophobic nature of these molecules to your advantage in quasi-purification. Similarly, another way would be ammonium sulfate fractionation....stirring different media samples with different levels of ammonium sulfate saturation to selectively precipitate bacteriocins (0% ammonium sulfate, 20%, 40%, 60%), centrifuge, decant, resuspend pellet with water/buffer and check to see which percent cutoff may selectively precipitate your bacteriocin peptide. If you produce your bacteriocin in MRS broth, ammonium sulfate will cause the Tween 80 to flocculate at the surface upon centrifugation and you may want to collect that as I often found that is where the bacteriocin is found....in that surface flocculated film that can actually be scooped off the top (and resuspend that in water). Good luck....these procedures can be found in the literature.
if the bacteriocin (MW is less than or equal the filter ) is in a solution containing components higher than the filter, this will be good for the concentration. other wise it will not help.
the time and speed and temperature of ultrafiltration depend on the manufacturer instructions for the filter.
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