Identification of Copper-Binding Peptides and Investigation of Functional Properties of Acetes japonicus Proteolysate

Abstract

In this research, two copper-binding peptides were identified in the proteolysate of Acetes japonicus. Firstly, the examination of the effects of hydrolysis conditions including enzyme type, pH, temperature, enzyme:substrate (E:S) ratio and hydrolysis time on the copper-binding capacity (CBC) was carried out. Secondly, to optimize the hydrolysis, response surface methodology (RSM) was applied via E:S ratio and hydrolysis time for maximizing the CBC of the proteolysate. After that, the amino acid composition and functional properties of the proteolysate was evaluated. Simultaneously, before being tested for CBC, four peptide fractions of 10–30 kDa, 3–10 kDa, 1–3 kDa, and < 1 kDa were recovered from the proteolysate using ultrafiltration. Finally, mass spectrometer / mass spectrometer (MS/MS) was used to determine molecular weight and amino acid sequence of bioactive peptide. The result showed that when using Flavourzyme, pH 6, 60 °C, E:S ratio of 40.33 U/g protein and hydrolysis time of 4.15 h, CBC reached its peak at 2699.72 µgCu2+/g protein. Proteolysate, of which Leu was the major amino acid, the indispensible amino acids comprised of approximately 40% total amino acid content. The proteolysate also exerted great solubility, heat stability, foaming and emulsifying property, oil and water holding capacity (OHC and WHC, respectively). The 1–3 kDa fraction exhibited the highest CBC of 2754.73 ± 91.87 μg Cu2+/g protein, from which two peptides (Asp-Tyr-Met-Leu-Pro-Thr-Asp-Lys-Tyr-Pro-His (1378.6 Da) and Gly-Tyr-Pro-Phe-Asp-Ala-Asp-Ser-Val-Asn-Phe-Pro-Val-His-Gly (1620.7 Da)) were detected. This is the first time that the copper affinity peptides were recovered from the small shrimp that could be utilized as a natural source for copper enhancement.