Hello. We are working on LAB bacteriocins to detect the antimicrobial activities against pathogenic bacteria. We grew LAB on MRS broth and obtained CFS, it showed inhibition zone , but what is the next step? We want to remove any other components that may show antimicrobial action to make sure it's caused by bacteriocins only. We want to add NaOH to adjust the PH, but what about adding the catalase? can someone suggest us any protocol ?
Hello
Catalase and hydrogen peroxide are only expressed under aerobic condition, so, you can excluded the inhibitory effect by incubation under anaerobic conditions using anaerobic jar Kit. Also, you can use catalase test.
In order to eliminate the possibility of interference with antagonism due to organic acids production you can use buffered culture media (at pH 7.2 with phosphate buffer 0.2 M)
For protocols, you can see : Ananou et al., 2020. Production, Recovery and Characterization of an Enterocin with Anti-Listerial Activity Produced by
Enterococcus hirae OS1. Appl Food Biotechnol 2020; 7(2): 103-114. http://dx.doi.org/10.22037/afb.v7i2.27582
Good luck!
Why don't you start purifying the activity from the supernatant. Doing any sort of size discrimination (gel filtration etc) should distinguish between peptide molecules which are larger and small molecules.
I agree with Mr Ananou, also most (if not all) LABs are catalase-negative bacteria. We usually check in a stepwise manner: 1) colony vs pathogen to screen about 100-200 putative LABs, 2) culture in MRS broth, centrifuge/filter and neutralised CFS then agar well diffusion or spot test against pathogen to eliminate organic acids. This can be followed by peptide purification and activity testing.
Ref: DOI: https://doi.org/10.14710/ik.ijms.20.1.33-37
Good luck!
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