*4.3. Assay of Porcine Pancreatic* α*-Amylase Inhibitory Activity*

The porcine α-amylase (PPA) inhibitory activity of the peptide fractions obtained by RP-HPLC was tested by the following procedure. Experimental samples (10 μL) were added to 80 μL of 50 mM sodium phosphate, 100 mM sodium chloride buffer (pH 7.0), and PPA (A4268) (1 μg/mL) (Sigma Aldrich, St. Louis, MO, USA), and incubated for 10 min at RT. The substrate solution, 2-chloro-4-nitrophenyl α-D-maltotrioside (CNPG3) (Sigma Aldrich, St. Louis, MO, USA), was added to the reaction mixture (1 mM) and incubated for 10 min at RT. The optical absorption was measured on an xMark microplate spectrophotometer (BioRad, Hercules, CA, USA) at 405 nm. Acarbose (1mg/mL) (Sigma Aldrich, St. Louis, MO, USA) was used as a positive control.

#### *4.4. Circular Dichroism Spectroscopy*

Circular dichroism (CD) spectra were recorded on Chirascan-plus CD spectropolarimeter (Applied Photophysics, Leatherhead, UK) in quartz cells with an optical path length of 0.1 cm for the peptide region spectrum. The cuvette with the solution of the native and recombinant peptides (50 μg/mL) in a 0.01 M sodium phosphate buffer was incubated at 25 ◦C for 20–25 min before recording the CD spectrum. The content of secondary structure elements of peptides was calculated by the Provenzer–Glockner method [20], using advanced Provencher calculation programs from the CDPro software package (Leatherhead, UK) [57].
