Hello, i'm working on lactobacillus isolates from different dairy products and food samples , and focusing on the effects of bacteriocins against food-borne pathogens. How can i distinguish the action of bacteriocin from other antimicrobial compounds like organic acids and hydrogen peroxide ? can someone give the exact steps ? thank you
Dear Rawan hassan Alsh,
Treat your cell-free supernatant with different proteolitic enzime and, after enzime inactivaction, tested residual antimicrobial activity. The bioactivity due to proteinaceous compounds such as bacteriocins will reduced or completely loss.
Regards
Mariana Emilia Cozzolino thank you Mariana , but i don't want to inhibit the activity of bacteriocins, i need to compare the bacteriocins produced by different lactobacillus isolates. How to reduce or eliminate the effects of other antimicrobial compounds ?
Dear Rawan Alshahrani,
just , first test the potential Antimicrobial producing LAB,
then, attempt to rule out inhibition activity due to hydrogen peroxides( using catalase) and organic acids ( adjusting the PH). Evaluate antimicrobial activity of LAB with and without proteinase enzyme to test the portentous nature of compounds.
Further more, you can use different solvent extraction protocols
From a past experience, antimicrobial peptides and proteins should precipitate with 70-75% saturation of ammonium sulphate.
Ammonium sulphate precipitation of the cell-free supernatant will produce for sure brown pellets that contain most of the bacteriocins (if any).
You can then dissolve your pellets with PBS or water then desalt on Sephadex or any other method (Vivaspin, dialysis, c18 cartridges), then test for inhibitory activity.
This is the ultimate proof that you have proteinaceous antimicrobial substances that are worth further mass spectrometry analysis.
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