1.1.4 Materials and methods

1.1.4.1 Microorganisms

The yeast Saccharomyces cerevisiae B-4 obtained from Institute of Agricultural and Food Biotechnology Warsaw, Poland, was used for assessment ultrasound exposition to ethanol production. The yeast cultures were cultivated on YPG slants (2% glucose, 2% peptone, 1% yeast extract) supplemented with 2% agar, at pH 5.0 and 30 °C for 24 h. The active cultures
for inoculation were prepared by growing the yeast in a 1 L baffled shake-flask containing sterile water and 100 mL YPG medium at 30 °C for 24 h on orbital shaker table at 200 rpm to a concentration of approximately 108 cells mL-1. The cultures in baffled shaken flasks of 100 mL were used to prepare the inocula. After 24 h of incubation at 30 °С, the precultures were centrifuged at 3800 rpm for 10 min and the cells were resuspended in sterile water to obtain 106 cells mL-1. Enzyme |3-D-galactosidase (optimum temperature 30 °С, optimum acidity pH 4.5-5.0, activity 8.7 AU mg-1 d. m. of the preparation), from Aspergillus oryzae manufactured by the SIGMA company (USA), was used for co-immobilization process. The amount of yeast and enzyme was 3% free cell inoculum and 4 cm3 enzyme solution. The yeast culture was co-immobilized in the 2% (w/v) sodium alginate by dropping yeast and enzyme into 150 cm3 0.09 mol L-1 solution of CaCl2 with 10% glucose. The cell beads were washed with sterile water and were stored as a fermentation medium in physiological solution at 8°C.