**2.6. Characterization of the inhibitory effect**

In order to determine the biological nature of the antimicrobial activity of bacteria, CFS (pH 6.0) of 24-h lactobacilli cultures of two selected isolates (*Lactobacillus casei* AP8 & *Lactobacillus plantarum* H5) incubated at 30°C, were tested for their sensitivity to the proteolytic enzymes. One ml of CFS was treated for 2 h with 1 mg ml-1 final concentration of the following enzymes: papain, trypsin, proteinase K, pronase E and α-amylase (Sigma, London). To clarify whether the antimicrobial activity detected derives from the production of hydrogen peroxide, 2600 IU/ml of catalase (Sigma, London) were added to 1 ml portions of extracellular extracts of LAB exhibiting antimicrobial activity and incubated for 24 h at ambient temperature. Chemicals were added to the CFS and the samples incubated for 5 h before being tested for antimicrobial activity. To determine the sensitivity of potential bacteriocin activities to the temperatures, samples of CFS were incubated under defined conditions. The effect of pH on bacteriocin activity was determined by adjusting the pH of the CFS (cell free supernatant (pH 6.5) of 24-h lactobacilli cultures incubated at 30°C) with diluted appropriate volumes of HCl and NaOH (Table 3). After incubating for 2 h, the pH of the samples was readjusted to 6.5 followed by sterilization (0.2 µm, Sigma, UK). In all cases, the remaining bacteriocin activity was assessed exemplarily by using strain *L. monocytogenes* ATCC 19115 as the indicator bacterium and by applying the agar disk diffusion plate bioassay. Untreated cell-free supernatants were used as controls and experiments were performed in duplicate.
