*2.2. Crude Enzyme Production and Cheese Whey Hydrolysis*

Crude enzyme production was determined during solid state fermentations (SSF) on wheat bran (WB) and further optimized under various parameters. More specifically, 5 g WB (dry basis) were weighed and sterilized into 250 mL Erlenmeyer flasks. To enhance secretion of fungal β-galactosidase, the medium was supplemented with 10 mg MgSO<sup>4</sup> in each SSF culture [13,29]. Suspensions of approximately 2 <sup>×</sup> <sup>10</sup><sup>6</sup> spores mL−<sup>1</sup> were prepared by collecting spores of 5 days old fungal pre-cultures as described above. Inoculated WB flasks were incubated at 28 ◦C under static conditions and enzyme activity was determined at regular time intervals until 120 h of incubation. In terms of enzyme production optimization, different initial moisture content of the substrate of 60, 65, 70 and 75% (*w*/*w* on a dry basis) was also examined. The varying moisture content was fixed by addition of deproteinized whey (pH 4.5) in order to stimulate enzyme production.

At the end of the fermentation process, the WB solids were mixed thoroughly with deproteinized whey (1:10 *w*/*v*) at 120 rpm for 1 h at room temperature [30]. Crude enzyme extracts were filtered through sterile gauze and centrifuged further at 4000 rpm for 20 min. The effect of temperature in the hydrolytic activity of the enzymes was evaluated at 40–70 ◦C for 60 h. Lactose hydrolysis assay was further optimized employing varying initial enzyme activities of 7.5, 11 and 15 U/mL and hydrolysis experiments were carried out at 500 mL final volume in a water bath for 60 h under agitation. Initial enzyme activities used in hydrolysis experiments were achieved by selecting the appropriate amount of crude enzymes (~150 U/g), which were produced under optimal SSF conditions. Samples for sugars and free amino nitrogen (FAN) determination were collected at regular time intervals and heated (100 ◦C) to inactivate enzymatic reaction. Subsequently, the pH value of hydrolysates was adjusted to 6.0, and they were sterilized to be used as nutrient supplements for BC production. All the experiments were performed in duplicates.
