Lipolytic activity was assessed through an agar well diffusion assay. More accurately, the medium consisted of 0.5% peptone (LAB M), 0.3% meat extract (LAB M), 0.5% lecithin (Serva, Heidelberg, Germany), 1% tributyrin (Merck, Darmstadt, Germany), and 1.5% agar, with the addition of 2.5 mM CaCl2 (Applichem) and 5 mM MgSO4 (Applichem), according to Carrazco-Palafox et al. [29 (link)]. Then, wells were aseptically punched. Overnight yeast and bacterial cultures were centrifuged (12,000× g; 15 min; 4 °C) to obtain CFS. Twenty-five (25) μL of each CFS were added in each well. Incubation took place at 30 °C for 10 days. The presence of a clarification halo around each well was indicative of lipolytic activity.
Strains that exhibited lipolytic activity were subjected to further study. More accurately, overnight culture was centrifuged (12,000× g; 15 min; 4 °C), washed twice with sterile saline, and used to inoculate flasks containing the above medium without agar addition. Incubation took place under shaking (200 rpm) at 30 °C for 21 days. The pH value and total titratable acidity of the samples were determined at days 3, 6, 9, 12, 15, 18 and 21. Uninoculated flasks served as controls. Lipolytic activity was expressed in AU/mL; one arbitrary unit was defined as the amount of enzyme that catalyzed the release of 1 μmol of fatty acids.
Strains that exhibited lipolytic activity were subjected to further study. More accurately, overnight culture was centrifuged (12,000× g; 15 min; 4 °C), washed twice with sterile saline, and used to inoculate flasks containing the above medium without agar addition. Incubation took place under shaking (200 rpm) at 30 °C for 21 days. The pH value and total titratable acidity of the samples were determined at days 3, 6, 9, 12, 15, 18 and 21. Uninoculated flasks served as controls. Lipolytic activity was expressed in AU/mL; one arbitrary unit was defined as the amount of enzyme that catalyzed the release of 1 μmol of fatty acids.
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