Enzymatic Profiling of Bacterial Strains

Enzymatic activities were evaluated out by placing 5 µL of a TSB-1 culture of each strain at OD₆₀₀ ≈ 0.5 on the surface of TSA-1 plates supplemented with the appropriate substrate. All assays were repeated three times to ensure reproducibility of results. TSA-1 plates supplemented with 1% egg yolk were used to determine phospholipase activity [35 (link)]. TSA-1 plates with 1% Tween 20 were used to confirm lipolytic activity, as Tween 20 is easily hydrolyzed by esterase because it contains esters of lower chain fatty acids [36 (link)]. Both lipolytic activities (phospholipase and esterase) were evaluated after 24 h incubation at 25 °C. The gelatinase activity test was carried out on TSA-1 plates supplemented with 0.4% gelatin, and the results were recorded after 48 h incubation at 25 °C by covering the surface of the agar plate with a 12.5% (w/v) HgCl₂ solution. For the hemolysis test, 5 µL of an inoculum in TSB-1 at OD₆₀₀ ≈ 0.5 was placed on sheep blood agar plates (Oxoid, Hampshire, UK) and incubated at 25 °C for 48 h.