*2.6. Preparation of Hazelnut Protein Hydrolysates*

Hazelnut protein was isolated as Tatar et al. reported [21]. Briefly, DHF dissolved in distilled water was adjusted to a pH of 8.0 with 1% NaOH solution, and stirred for 30 min. Then, the supernatant was collected by centrifugation (9391× *g*, 20 min) and adjusted to a pH of 4.5 by 1% HCl solution for precipitation. Next, the precipitates were collected by centrifugation (3000× *g*, 20 min) and freeze-dried for powder harvest after being neutralized to pH 7.0 by a 0.2% NaOH solution.

Hazelnut protein hydrolysates were prepared according to the method described by Liu et al. with some modification [22]. Briefly, 1 g freeze-dried protein, after being mixed with 50 mL distilled water, was denatured at 90 ◦C for 15 min. Selected proteases were added to mixtures at room temperature. The enzyme amount, reaction time, optimal pH, and reaction temperature were depended on different proteases. For alkaline and neutral proteinases, these parameters are 10,000 U/g protein, 120 min, pH 8.5, 54 ◦C [22], and 17,000 U/g protein, 120 min, pH 7.0, 44 ◦C [23], respectively. Obtained hazelnut hydrolysates, after being heated at 100 ◦C for 10 min to inactivate enzymes, were readjusted to neutral pHs (1% HCl solution for hydrolysates of alkaline protease). Next, the hydrolysates, after being centrifuged at 3910× *g* for 15 min to remove a little sediment, were freezedried for peptide preparation. The lyophilized peptide powders were stored at −20 ◦C prior to use. The purity of hazelnut hydrolysates was measured by Folin-phenol protein quantitative assay [22].
