Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK.
Regul Toxicol Pharmacol. 2009 Dec;55(3):372-81. doi: 10.1016/j.yrtph.2009.08.010. Epub 2009 Aug 31.
Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.
最初，在一个实验室中使用“高蛋白酶检测法”和“低蛋白酶检测法”比较了两种牛奶过敏原β-酪蛋白和β-乳球蛋白（β-Lg）的抗消化能力。低蛋白酶检测法代表了一种替代的标准化方案，模拟了胃肠道中的条件。对于高蛋白酶检测法，两种蛋白质都与胃蛋白酶或胰蛋白酶一起孵育，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和反相高效液相色谱法监测消化情况。低蛋白酶检测法涉及胃十二指肠消化，存在或不存在磷脂酰胆碱（PC）。β-酪蛋白和β-Lg 在高蛋白酶检测法中都容易被胃蛋白酶和胰蛋白酶水解。相比之下，β-酪蛋白在低蛋白酶检测法中的消化动力学较慢，β-Lg 对胃蛋白酶有抗性。在十二指肠消化过程中，β-Lg 逐渐降解，添加 PC 会减缓消化速度。随后，在 12 个独立实验室中通过凝胶的视觉评估和密度计分析评估了低蛋白酶检测法的重现性：采样和电泳方法的使用影响了实验室间和实验室内的可变性。低蛋白酶检测法具有重现性。未来的研究将使用更广泛的蛋白质组来扩展这些发现。
