2.4. Characterization of the antimicrobial activity
2.4.1. Determination of hydrogen peroxide
In order to evaluate the presence of hydrogen peroxide in the CFS, it was treated with 2 mg/ml of catalase (Sigma, USA) for 1 h at 37 °C. Then, the residual antimicrobial activity against Listeria innocua ATCC 33090 was determined by AWDA.
2.4.2. Enzyme sensibility
In order to evaluate the chemical nature of the inhibitory compound, the CFS sensitivity to hydrolytic enzymes (using trypsin (Type II-S), lysozyme (Grade III) and lipase (Type VII) at a final concentration of 1 mg/ml) was studied. All enzymes were purchased from Sigma Aldrich (USA) and the assays were carried out at the pH and temperature recommended by the supplier. The CFS was incubated in enzyme solutions for 2 h at 37 °C and the remaining activity was determined by AWDA against L. innocua ATCC 33090. Enzyme solutions alone and an aliquot of CFS were used as negative and positive controls, respectively.