Aix Marseille Université, CNRS, UMR7281 Bioénergétique et Ingénierie des Protéines, Marseille, France.
Food Funct. 2018 Nov 14;9(11):5975-5988. doi: 10.1039/c8fo01450g.
Rabbit gastric extract (RGE) is a source of gastric enzymes for in vitro digestion studies. While its gastric lipase activity has been characterized and compared to other lipases, its pepsin activity has not been studied. We measured pepsin activity in RGE using both hemoglobin and azocoll as substrates, and identified the protein separated by SDS-PAGE as a type II-4 mature pepsin of 328 amino acid residues using Edman sequencing, LC-MS/MS analysis and intact mass measurement. As a proof-of-concept that RGE was suitable for in vitro digestion of both proteins and lipids, it was used for studying the proteolysis of β-casein under conditions mimicking the early stages of intragastric digestion. β-Casein was displayed either in solution or at the surface of a β-casein-stabilized rapeseed oil emulsion to investigate the impact of lipids and lipolysis on proteolysis. Proteolysis of β-casein was quantified based on the kinetics of β-casein disappearance, the identification of various peptides generated upon digestion and their variation with time. The results obtained with RGE were highly similar to those obtained with equivalent amounts of porcine pepsin used as a reference standard. Digestion of β-casein was slower when it was displayed at the oil-water interface and some degradation peptides were transiently observed at higher levels and for a longer time than with β-casein in solution, or accumulated upon digestion. N-terminal sequencing of the main isolated peptides revealed a sequential action of pepsin starting from the hydrophobic C-terminal end of β-casein, which was impaired by the interaction of β-casein with lipids.
兔胃提取物（RGE）是体外消化研究中胃酶的来源。虽然其胃脂肪酶活性已被表征并与其他脂肪酶进行了比较，但它的胃蛋白酶活性尚未得到研究。我们使用血红蛋白和偶氮胶原作为底物测量了 RGE 中的胃蛋白酶活性，并通过 Edman 测序、LC-MS/MS 分析和完整质量测量，将 SDS-PAGE 分离的蛋白质鉴定为 328 个氨基酸残基的 II-4 成熟胃蛋白酶。作为 RGE 适合蛋白质和脂质体外消化的概念验证，它被用于研究在模拟胃内消化早期条件下β-酪蛋白的蛋白水解。β-酪蛋白以溶液或β-酪蛋白稳定的菜籽油乳液表面的形式存在，以研究脂质和脂肪水解对蛋白水解的影响。基于β-酪蛋白消失的动力学、消化产生的各种肽的鉴定及其随时间的变化，定量了β-酪蛋白的蛋白水解。用 RGE 获得的结果与用作参考标准的等量猪胃蛋白酶获得的结果高度相似。当β-酪蛋白在油水界面上显示时，其消化速度较慢，并且一些降解肽在更高水平和更长时间内短暂观察到，而不是在溶液中，或者在消化时积累。主要分离肽的 N 端测序揭示了胃蛋白酶从β-酪蛋白的疏水性 C 端开始的顺序作用，该作用被β-酪蛋白与脂质的相互作用所破坏。
