Study on Enzymatic Preparation and in vitro Stability of ACE Inhibitory Peptides from Cottonseed Protein(棉籽蛋白ACE抑制肽的酶法制备及其体外稳定性研究)
摘要
Angiotensin-I-converting enzyme (ACE) inhibitory peptides play an important role in the prevention and treatment of hypertension to improve human health, because of their characteristics of safety, no side effects, and easy absorption. The objective was to prepare ACE inhibitory peptides from cottonseed protein using an appropriate protease (e.g., alcalase, protamex, flavourzyme, papain, and bromelain) under optimum conditions. The single-factor experiment was used to optimize hydrolysis conditions. The hydrolysate was then purified to identify sequences of bioactive peptides, and in vitro digestion and absorption stabilities of the hydrolysate was evaluated. It was found that the cottonseed protein hydrolyzed by protamex (1500U/g) at pH 8.0 and 55℃ for 5h resulted in the protein recovery rate of 39.8% and the highest ACE inhibitory activity (93.7%). The cotton protein hydrolysate was then separated into five fractions, in which fraction 4 with the highest inhibitory activity (IC<sub>50</sub>=220.1μg/mL). Three novel ACE inhibitory peptides were identified from fraction 4:VFNNNPQE, LLSQTPRY and VFPGCPET. LLSQTPRY exhibited the highest ACE inhibitory activity (IC<sub>50</sub>=105.2μmol/L). After in vitro gastrointestinal digestion and absorption by Caco-2 cell monolayer, the cotton protein hydrolysate retained ACE inhibitory activities of 53.8% and 20.5%, respectively. Therefore, the cottonseed protein hydrolysate prepared by protamex could be potentially used as a functional ingredient to develop antihypertensive food products.(食源性血管紧张素转化酶(angiotensin-I-converting enzyme, ACE)抑制肽因安全、无副作用、易吸收等优点,对防治高血压、提高居民健康水平具有重要作用。利用碱性蛋白酶、复合蛋白酶、风味蛋白酶、木瓜蛋白酶和菠萝蛋白酶水解棉籽蛋白制备ACE抑制肽,通过单因素实验优化水解条件,分离纯化水解物并鉴定其活性肽段组成,评价水解物的体外消化吸收稳定性。结果表明,棉籽蛋白经复合蛋白酶(1500U/g)在pH值为8.0和55℃下水解5h,蛋白回收率为39.8%,ACE抑制率可达93.7%。棉籽蛋白复合蛋白酶水解物经分离得到5个组分,其中组分4(F4)的ACE抑制活性最高(IC<sub>50</sub>=220.1μg/mL)。从该组分中发现3条新型ACE抑制肽:VFNNNPQE、LLSQTPRY和VFPGCPET,其中LLSQTPRY的活性最高(IC<sub>50</sub>=105.2μmol/L)。经人工胃肠液消化和Caco-2细胞单层膜吸收后,棉籽蛋白复合蛋白酶水