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.

Graphic Abstract

中文翻译：

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铜结合肽的鉴定和乙酰丙酮乙酰丙酮水解产物功能特性的研究

在这项研究中，在日本醋菌的蛋白水解物中鉴定出两种铜结合肽。首先，考察了水解条件，包括酶的类型，pH，温度，酶：底物（E：S）的比例和水解时间对铜结合能力（CBC）的影响。其次，为了优化水解，通过E：S比和水解时间应用响应表面方法（RSM）来最大化蛋白水解产物的CBC。之后，评估蛋白水解产物的氨基酸组成和功能特性。同时，在进行CBC测试之前，使用超滤技术从蛋白水解物中回收了10–30 kDa，3–10 kDa，1–3 kDa和<1 kDa的四个肽级分。最后，用质谱仪/质谱仪（MS / MS）测定生物活性肽的分子量和氨基酸序列。结果表明，当使用Flavourzyme，pH 6，60°C，E：²⁺ / g蛋白质。蛋白水解产物，其中Leu为主要氨基酸，必不可少的氨基酸约占总氨基酸含量的40％。该蛋白水解物还具有很大的溶解性，热稳定性，起泡和乳化性能，油和水保持能力（分别为OHC和WHC）。1-3 kDa馏分的CBC最高，为2754.73±91.87μgCu ²⁺ / g蛋白，其中两个肽段（Asp-Tyr-Met-Leu-Pro-Thr-Asp-Lys-Tyr-Pro-His（1378.6 Da）和Gly-Tyr-Pro-Phe-Asp-Ala-Asp-Ser-Val-Asn-Phe-Pro-Val-His-Gly（1620.7 Da））被检测到。这是首次从小虾中回收铜亲和力肽，可将其用作增强铜的天然来源。

图形摘要