利用改良的生物活性指引分群方法探索牛奶之胃腸道蛋白酶水解產物中血管收縮素轉化酶抑制肽

An Exploration of Angiotensin-I Converting Enzyme Inhibitory Peptide Derived from Gastrointestinal Protease Hydrolysate of Milk using Modified Bioassay-Guided Fractionation Approach

蘇吉亞(Sugiyati Ningrum)

指導教授 ： 徐睿良

國立屏東科技大學/農學院/生物科技系/碩士(2020年)

https://doi.org/10.6346/NPUST202000346

摘要

高血壓，與多種代謝途徑有關，例如腎素-血管緊張素-醛固酮系統（RAAS）。在該系統中，血管緊張素I轉換酶（ACE）將血管緊張素I轉換為有效的血管收縮劑血管緊張素II，從而增加血壓。臨床上已經使用了幾種合成的ACE抑制藥物，但合成藥物長期使用可能對人體健康產生副作用。長期以來，源自食用食品蛋白質的ACE抑制（ACEI）肽被視為更溫和，更安全的替代品。牛奶是哺乳動物常見的可食用蛋白質來源之一，可提供功能性化合物，包括生物活性肽。因此，胃腸道蛋白酶用於釋放生物活性肽。這項研究旨在探索負責ACE抑制活性的商業牛奶的ACE抑制肽。
本研究使用改良式生物活性指引的分群，從“安佳”牛奶蛋白水解物中，有效地篩選ACEI肽。親水作用液相層析（HILIC）(可視作適用於水相的正相層析法)，以固相萃取（SPE）短柱的形式，用於第一次分群，具有最佳ACEI活性（IC50 61.75±5.74 µg / mL）的HILIC分群為95％ACN + 0.1％FA洗脫液。此分液再用逆相（RP）SPE短柱繼續進行第二次生物活性指引的分群。最佳的RP-SPE分液為20％ACN + 0.1％FA之洗脫液，其ACEI活性為IC50 = 36.22±1.18 µg / mL。接著，將最佳的RP-SPE分液進行LC-MS / MS鑑定，鑑定可提供ACEI活性的活性胜肽HL-7。HL-7為從β-酪蛋白衍生的短鏈胜肽，其IC50值為16.87±0.3 µM。
第三純化步驟後，HL-7之IC50值顯著降低了8.92倍。同時，根據多重反應監測（MRM）實驗，從奶粉的胃腸道蛋白酶水解產物(1mg / ml)和RP-SPE分液(1mg / ml)中，可定量出其分別含有19.86±1.14 pg和14545±572.9 pg ACEI肽HL-7。此外，對HL-7的動力學研究表明，HL-7同時為競爭性及受質類型之抑製劑，此特性與分子對接的模擬結果一致，發現HL-7藉由3個氫鍵與ACE相互作用在三個關鍵殘基Ala354（S1），Gln281（S2'）和Lys511（S2'），同時在活性位點與Lys511（S2'）發生1個電荷相互作用。因此，我們認為HL-7可以作為替代性的天然ACE抑制劑，可用於控制血壓以及開發功能性食品

關鍵字

血管收縮素轉化酶 ； 活性胜肽 ； 牛奶 ； 改良式生物活性指引分群 ； 競爭性抑制劑

並列摘要

Hypertension is also known as high blood pressure that has associated with multi-metabolic pathways such as the renin-angiotensin-aldosterone system (RAAS). In this system, angiotensin I-converting enzyme (ACE) converts angiotensin I into a potent vasoconstrictor angiotensin II that increases blood pressure. There have been several synthetic ACE inhibitory drugs used clinically. However, synthetic drugs show side effects on human health after long-term use. ACE inhibitory (ACEI) peptides derived from edible food proteins have long been regarded as milder and safer alternatives. Milk is one of the common edible protein sources from mammalian, which provides functional compounds, including bioactive peptides. Therefore, gastrointestinal proteases were used for releasing bioactive peptides. The study aims to explore ACE-inhibitory peptides of commercial milk that responsible for ACE-inhibitory activity.
In this study, a modified bioassay-guided fractionation was performed to efficiently screen ACEI peptides from milk protein hydrolysate. The aqueous normal phase chromatography namely hydrophilic interaction liquid chromatography (HILIC) was used as a format of solid-phase extraction (SPE) short column for the first fractionation, then the HILIC-SPE fraction with the best ACEI activity (IC50 61.75±5.74 µg/mL) was obtained when eluted by 95%ACN+0.1%FA. The best HILIC-SPE fraction was continued to the second fractionation using reversed-phase (RP) SPE short column. The best RP-SPE fraction was eluted by 20%ACN+0.1%FA with an ACEI activity of IC50 36.22±1.18 µg/mL Afterward, the best RP-SPE fraction was subjected to LC-MS/MS to identify bioactive peptides that responsible for ACEI activity. HL-7 was successfully screened from the milk protein hydrolysate that derived by β-casein with an IC50 value of 16.87±0.3 µM.
The IC50 value of HL-7 was significantly decreased by 8.92 folds after the third purification step. According to a multiple reaction monitoring (MRM) experiment, 19.86±1.14 pg and 14545±572.9 pg of ACEI peptide HL-7 were obtained in the gastrointestinal protease hydrolysate and RP-SPE fraction derived from 1mg/ml of milk protein. Furthermore, the kinetic study of HL-7 showed characteristics of a competitive inhibitor and substrate-type inhibitor, as well as HL-7, was consistent with the simulation results of molecular docking, which found that HL-7 interacts with ACE via 3 hydrogen bonds at three key residues such as Ala354 (S1), Gln281(S2’), and Lys511 (S2’), and 1 charge interaction with Lys511 (S2’) in the active site. Hence, we believe HL-7 could be an alternative natural ACE inhibitor for controlling blood pressure as well as for the development of functional food.

並列關鍵字

Angiotensin-converting enzyme ； Bioactive peptide ； Milk ； Modified bioassay-guided fractionation ； HL-7 ； Competitive inhibitor

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