Check https://www.sciencedirect.com/science/article/pii/S0717345815000871

Lyna,

Some bacteriocins have a pH range to induce bioactivity.

While it is recommended to adjust your supernatant preparation to pH 7 before antimicrobial testing, the initial bacteriocin screening can be performed by an antagonism test on media supplemented with 0.1% CaCO3.

-Spot your producer on the surface of MRS agar supplemented with 0.1% CaCO3 and let it grow overnight.

-Kill the producer cells by exposure to chloroform vapours. followed by aeration to get rid of chloroform residues.

- Mix your target with a low melting point soft agar containing 0.1% CaCO3.

-Overlay the target over the killed producer.

-Incubate overnight and observe inhibition zones.

Once you confirm that antagonism assay works on CaCO3 supplemented media, high chance that the inhibition stems from bacteriocins.

On the other hand, you can extract the supernatant that you already have either by organic extraction (ethyl acetate/chloroform/methanol, butanol..etc.) or by ammonium sulphate precipitation.

Another thing that you can try is to treat your supernatant with proteinase K and test for inhibitory activity. Usually, bacteriocins are of protein or peptide nature and you should lose the activity upon proteases treatments.

You can not simply use MRS-HCl as a control because your supernatant contains lactic acid, not HCl and lactic acid is a bacterial metabolite that can only kill some bacteria, while HCl is cytotoxic in general.

Best,

-AB

Also check https://www.researchgate.net/figure/Effect-of-pH-and-temperature-on-the-antimicrobial-activity-of-the-supernatant-from-L_tbl1_263403611/amp

Look at the temperature and the concentration or molarity of the two solution.

Thank you all for your help. I will take your suggestions into account and hope it works next time.

Bests,

lyna