2.2. Tyrosinase activity inhibition assay

The inhibition assays performed to determine the activities of mushroom tyrosinase were carried out by spectrophotometrically measuring the rate of melanin formation. Equal amount of tyrosinase was added to a phosphate buffer solution (PBS) saturated with tyrosine (2.5 mM), to which, solutions of EF-5 at different concentrations were sequentially added. After incubation for 24 h at 37 °C, the visible light absorbance was measured at 475 nm using a spectrophotometer (U-3010, HITACHI, Japan). All measurements were repeated thrice. The value of light absorbance of the corresponding group in the absence of tyrosinase was subtracted. The rate of enzyme inhibition was calculated using following formula:

where A, B and C are the values of light absorbance in the absence and presence of the peptide inhibitor, and tyrosinase dissolved in PBS, respectively.