Purpose: This study was aimed to analyze the damage on S. cerevisiae in apple juice provoked by a US+PL treatment through the study of the physiological characteristics by FC and the ultrastructure by TEM.
Methods: Commercial (pH 3.5; 12.5°Brix) and natural squeezed apple juices were inoculated (pH: 3.4; 11.8°Brix) with S. cerevisiae KE162 (~1 x 106 CFU/ml). Ultrasonic treatments (20 kHz, 600 w; 95.2 µm; 20, 30 or 44°C; 10 and 30 min) were applied. US treated or not juice samples were exposed to PL (Xenon lamp; 3 pulses/s; 60 s; 71.6 J/cm2; Tinitial: 2; 30; 44°C) reaching different final temperatures due to the heat build-up during PL treatment (Tfinal: 13; 42; 56°C ). After treatments, cells were labelled with fluorescein diacetate (FDA) and propidium iodide (PI) for monitoring esterase activity and membrane integrity, respectively. After conventional fixation, cells were examined by TEM using a JEOL transmission electronic microscope.
Results: Yeast log reduction was up to 1.8-2.8 for single US, 2.0-3.8 for single PL, and 5.8-6.4 for US+PL in natural squeezed or commercial apple juice, respectively. When single US was applied, FC revealed that population gradually migrated from PI--FDA+ to PI+-FDA- quadrants, indicating rupture of membranes and progressive loss of esterase activity(EA) (10 min-US: 2.9-11.6 % EA; 30 min-US: EA non detected). When US+PL treatments were applied, EA was not detected and membrane permeability significantly increased, depending on juice type and temperature. In TEM, sonicated cells showed puncturing of walls with leakage of content and damage at subcellular level. When PL was applied, inner content did not leach out of the cells but appeared coagulated and the lumen looked coarse.
Significance: FC and TEM analysis helped to better understand inactivation mechanism by US+PL combined treatment.