2.2. Preparation of Tree Peony Seed Protein Hydrolysate (TPSPH) and Five Fractions

Freeze-dried TPSP was dispersed in deionized water to obtain a concentration of 20 mg/mL and hydrolyzed at pH 8.0, 60 °C, and 4 h with Alcalase activity of 120 U/g (enzyme unit/substrate weight). After hydrolysis, the enzyme was then deactivated in boiling water for 10 min, followed by centrifugation at 8000× g at 4 °C for 20 min. The resulting supernatant was lyophilized and kept at −20 °C for further experiments.

Ultrafiltration of TPSPH was performed using an ultrafiltration stirred cell Amicon 8400 (Millipore Corp., Billerica, MA, USA). TPSPH was sequentially passed through ultrafiltration membranes with MW cut-off of 30, 10, 5, and 3 kDa, respectively. Five fractions with molecular weight of >30 kDa, 10–30 kDa, 5–10 kDa, 3–5 kDa, and <3 kDa were collected, lyophilized, and stored at −20 °C until required for further analysis. The concentration of peptides was determined by using the modified Lowry method with bovine serum albumin as the standard [15]. Peptide concentrations of TPSPH and five fractions were modified for further analysis based on the calculating peptide contents from the prepared standard curve.