Antioxidative And ACE-inhibitory Activities Of Zein Hydrolysate In Vitro Digests

Zein protein hydrolysate showed a strong antioxidant activity and ACE-inhibitory activity, but little is known about the in vivo antioxidant activity and ACE-inhibitory activity. The objective of this study was to assess the survivability of antioxidative activity and ACE-inhibitory activity of Alcalase-treated zein hydrolysate (ZH), and the relationship between antioxidant activity and ACE-inhibitory activity.Firstly, the antioxidative activity of zein hydrolysate treated by Alcalase (ZH) and the antioxidant potential of ZH during a two-stage (1 h pepsin→2 h pancreatin, 37 oC) in vitro digestion was studied. Free Amino acid composition, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+·) and 1,1-diphenyl-2-picrylhydrazyl (DPPH?) free radical scavenging activity, reducing power, and Cu2+ chelation ability were tested to determine the antioxidant efficacy of ZH. High-performance size exclusion chromatography (HPSEC) was run to evaluate the molecular weight of ZH digests. Results showed that ZH exhibited a strong ABTS+·free radical scavenging activity, reducing power, and Cu2+ chelation ability. The antioxidant activity of ZH (5 mg/mL) was exceeded (P < 0.05) that of 0.01 mg/mL of ascorbic acid and 0.1 mg/mL BHA. After in vitro digestion, the reducing power of ZH increased one-fold (P < 0.05), the ABTS+·free radical scavenging activity and Cu2+ chelation ability were fully recovered when compared with nondigested ZH. The ZH samples tested in the ABTS+·system, though at a lower concentration (1 mg/mL), had a Trolox equivalence of approximately 50 times that of ZH sample (8 mg/mL) tested in the DPPH? system, suggested that the ABTS assay was an appropriate method for the measurement of antioxidant activity of water-soluble proteins and peptides in an aqueous solution.The ACE-inhibitory activity of ZH and it's digests was determined by Cushman Ultraviolet spectrophotometer. Sephadex G-15 gel filtration was used to separate the final digest of ZH into fractions. ABTS+·free radical scavenging activity, reducing power, Cu2+ chelation ability and ACE-inhibitory activity were tested to determine the antioxidant and ACE-inhibitory efficacy of each fractions, the effect of Amino acid composition and the molecular weight profile was also considered. Results showed that ZH can inhibit ACE activity by 80.6±2.67% at 8 mg/mL, and also exhibited a good ACE-inhibitory activity after in vitro digestion. Four fractions were yielded after Sephadex G-15 gel filtration, of which peptides with MW120-568 Da in fraction 2 representing 70.07% of the total mass, the composition of basic amino acid, branched-chain amino acid, hydrophobic amino acid and the amino acid with antioxidant activity was higher in fraction 2, showed the strongest antioxidant activity and ACE-inhibitory activity.