从麦胚清蛋白分离制备高活性抗氧化肽

doi:10.3864/j.issn.0578-1752.2016.12.013

Abstract

Abstract: 【Objective】 The aim of this study is to develop an effective preparation process for obtaining highly active antioxidant peptide derived from wheat germ albumin using two-step dual-enzymatic hydrolysis. 【Method】 A two-step dual-enzymatic hydrolysis and separation were employed to screen the antioxidant peptide (AOP) from wheat germ albumin. The in vitro AOP’s antioxidant activity was evaluated by DPPH radical scavenging activity and Fe²⁺ chelating ability assays. The hydrolysate derived from tryptic hydrolysis of wheat germ albumin was fractionated by ultrafiltration according to molecular weight; then the fractions with high antioxidant capacity were further isolated by gel filtration chromatography (Sephadex G-75). After that, the fraction with higher antioxidant activity was further hydrolyzed by an alcalase, and the hydrolysate was subjected to purification by Sephadex G-15 gel filtration chromatography and reversed-phase HPLC (RP-HPLC), respectively. The highest antioxidant fraction was achieved and the structure was identified by ESI-TOF MS/MS. 【Result】 Hydrolysate derived from tryptic hydrolysis of wheat germ albumin was fractionated by ultrafiltration according to molecular weight. Three fractions, P_a, P_b and P_c were obtained from the fraction with high antioxidant capacity. The fraction P_b showed the strongest antioxidant activity evaluated by DPPH radical scavenging activity and Fe²⁺ chelating ability assays and its extraction yield was 23.1%. The fraction P_b which had the highest antioxidant activity was further hydrolyzed by an alcalase, and the hydrolysate was subjected to gel filtration chromatography (Sephadex G-15) and two more groups, P_d and P_e were obtained. The extraction yield of fraction P_d which had higher antioxidant capacity was 52.1%. After further purification by RP-HPLC, five fractions, P₁, P₂, P₃, P₄ and P₅ were separated from the fraction P_d, and the fraction P₃ exhibited the highest antioxidant activity with the in vitro EC₅₀ values of DPPH radical scavenging activity at 1 mg/ml and Fe²⁺ chelating ability at 0.6 mg/ml, respectively. The extraction yield of the fraction P₃ was 7.3% in RP-HPLC step. Finally, employing the electrospray ionization-time-of-flight mass spectrometer (ESI-TOF MS/MS), the structure of the fraction P₃ was identified. Its amino acids sequence, molecular weight and purity were AREGETVVPG, 1013.51Da and 85%, respectively. Consistently, the antioxidant capacity of the synthetic peptide with the same sequences ( purity ≥95%) showed the similar activity to the fraction P₃ (P>0.05). 【Conclusion】 The preparation process of a two-step dual-enzymatic hydrolysis in combination with ultrafiltration, gel filtration chromatography, and RP-HPLC was effective in separation, screen and purification of a highly active antioxidant peptide.

Key words: wheat germ, albumin, a two-step dual-enzymatic hydrolysis, separation and purification, antioxidant peptide (AOP)

CHEN Si-yuan, LIU Yong-xiang, CAO Xiao-zhou, ZHANG Yi-jing, CHEN Hai-juan, SHEN Xin-chun. The Preparation Process for Isolation of a Highly Active Antioxidant Peptide Derived from Wheat Germ Albumin[J].Scientia Agricultura Sinica, 2016, 49(12): 2379-2388.

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