Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
Department of Food Science, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Apr 1;1236:124061. doi: 10.1016/j.jchromb.2024.124061. Epub 2024 Feb 28.
Typically, bioactive peptides were uncovered from complex hydrolysates using sequential bioassay-guided fractionation. To increase the efficiency of bioactive peptide screening, a simple and convenient tandem bioassay-guided fractionation based on solid-phase extraction (SPE) was conducted to screen the angiotensin-I-converting enzyme (ACE) inhibitory peptides from the hydrolysate of Inca nut cake protein (INCP). The so-called SCX-RP SPE system was constructed by assembling SCX (strong cation exchange) and RP (reversed phase) SPE cartridges. Using this tandem SCX-RP SPE, the INCP digested with combined gastrointestinal protease (INCP GP) was fractionated into 30 fractions. The fraction F11 exhibited the highest ACE inhibitory activity among 30 fractions. The ACE IC of fraction F11 was calculated to be 6.6 ± 0.5 µg/mL. The ACEI activity of fraction F11 was stronger than the INCP GP hydrolysate (ACE IC of 12.7 ± 0.4 µg/mL). The tandem SCX-RP SPE fractionation reduced the number of ACE inhibitory (ACEI) peptide candidates from 127 peptides in the INCP GP hydrolysate to only ten peptides in fraction F11. Subsequently, WALPTQSW (WW-8) and WLPTKSW (WW-7) from fraction F11 were synthesized, and their ACE IC was determined to be 4.7 ± 0.1 and 7.9 ± 0.1 µM, respectively. The dipeptidyl peptidase-4 (DPP4) inhibitory and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities of WALPTQSW (WW-8) were also explored to give IC values of 131.7 ± 5.2 and 191.8 ± 7.0 µM, respectively. The molecular docking and inhibition mechanism studies indicated that WW-8 inhibited ACE and DPP4 as competitive and non-competitive inhibitors, respectively. The pre-incubation experiment of WW-8 toward ACE and DPP4 demonstrated that WW-8 was a true-inhibitor type. Additionally, the amount of WW-8 was quantified to be 5.8 ± 0.2 and 35 ± 0.4 µg per milligram hydrolysate and fraction F11, respectively. This study demonstrated tandem bioassay-guided SCX-RP SPE fractionation efficiently screened ACEI peptide derived from INCP GP hydrolysate, adding more value to Inca nut cake (a leftover of the oil industry) as a bioactive peptide precursor.
通常，生物活性肽是使用顺序生物测定指导的分级分离从复杂的水解物中发现的。为了提高生物活性肽筛选的效率，采用了一种简单方便的基于固相萃取（SPE）的串联生物测定指导分级分离方法，从印加坚果蛋糕蛋白（INCP）水解物中筛选血管紧张素转化酶（ACE）抑制肽。所谓的 SCX-RP SPE 系统是通过组装 SCX（强阳离子交换）和 RP（反相）SPE 小柱构建的。使用这种串联 SCX-RP SPE，用组合胃肠蛋白酶（INCP GP）消化 INCP 被分成 30 个馏分。在 30 个馏分中，馏分 F11 表现出最高的 ACE 抑制活性。馏分 F11 的 ACE IC 计算为 6.6 ± 0.5 µg/mL。馏分 F11 的 ACEI 活性强于 INCP GP 水解物（ACE IC 为 12.7 ± 0.4 µg/mL）。串联 SCX-RP SPE 分级分离将 INCP GP 水解物中的 127 种 ACE 抑制肽候选物数量减少到仅馏分 F11 中的 10 种肽。随后，从馏分 F11 中合成了 WALPTQSW（WW-8）和 WLPTKSW（WW-7），并确定它们的 ACE IC 分别为 4.7 ± 0.1 和 7.9 ± 0.1 µM。还探索了 WALPTQSW（WW-8）的二肽基肽酶-4（DPP4）抑制和 2,2-二苯基-1-苦基肼（DPPH）清除活性，给出的 IC 值分别为 131.7 ± 5.2 和 191.8 ± 7.0 µM。分子对接和抑制机制研究表明，WW-8 分别作为竞争性和非竞争性抑制剂抑制 ACE 和 DPP4。WW-8 对 ACE 和 DPP4 的预孵育实验表明，WW-8 是一种真正的抑制剂类型。此外，WW-8 的量被定量为每毫克水解物和馏分 F11 分别为 5.8 ± 0.2 和 35 ± 0.4 µg。本研究表明，串联生物测定指导的 SCX-RP SPE 分级分离有效地筛选了 INCP GP 水解物中衍生的 ACEI 肽，为印加坚果蛋糕（油脂工业的剩余物）作为生物活性肽前体增加了更多价值。
