Escuela de Alimentos, Facultad de Recursos Naturales, Pontificia Universidad Catolica de Valparaiso, Valparaiso, Chile.
J Agric Food Chem. 2010 May 12;58(9):5300-7. doi: 10.1021/jf903580t.
Proteases in fish muscle often cause undesired softening of intact meat pieces during refrigerated storage or slow cooking. Several food-grade proteinaceous inhibitors can overcome this softening if properly delivered to the intracellular sites where proteases are located. Fluorescence recovery after photobleaching (FRAP) and laser scanning confocal microscopy (LSCM) were used to measure the translational diffusion of fluorescein isothiocyanate (FITC)-labeled protease inhibitors into intact muscle fibers of halibut. Diffusion coefficients (D) of alpha-2-macroglobulin (720 kDa), soybean trypsin inhibitor (21 kDa), and cystatin (12 kDa) were measured in both muscle fibers and dilute aqueous solutions. On the time scale of the observation (35 min), cystatin and soybean trypsin inhibitor diffused through the cell membrane (sarcolemma) and sarcoplasm, but at a considerably slower rate (>10-fold difference) than in dilute aqueous solution. alpha-2-Macroglobulin did not diffuse into muscle cells within the time frame of the experiment, but did completely penetrate the cell during overnight exposure. The present study thus shows a clear dependence of D on protein inhibitor size when moving within intact skeletal muscle fibers. Low molecular weight protease inhibitors such as cystatin can be effectively diffused into intact fish muscle cells to minimize proteolytic activity and meat softening.
鱼类肌肉中的蛋白酶在冷藏储存或缓慢烹饪过程中经常导致完整肉块产生不理想的软化。如果将几种食品级的蛋白质抑制剂适当递送到位于蛋白酶所在的细胞内位置，则可以克服这种软化。荧光恢复后光漂白（FRAP）和激光扫描共聚焦显微镜（LSCM）用于测量荧光素异硫氰酸酯（FITC）标记的蛋白酶抑制剂在比目鱼完整肌肉纤维中的平动扩散。在肌肉纤维和稀水溶液中测量了α-2-巨球蛋白（720 kDa），大豆胰蛋白酶抑制剂（21 kDa）和胱抑素（12 kDa）的扩散系数（D）。在观察的时间范围内（35 分钟），胱抑素和大豆胰蛋白酶抑制剂通过细胞膜（肌膜）和肌浆扩散，但扩散速度明显较慢（差异超过 10 倍）比在稀水溶液中。α-2-巨球蛋白在实验的时间范围内不会扩散到肌肉细胞中，但在过夜暴露期间完全穿透了细胞。因此，本研究清楚地表明，当在完整的骨骼肌纤维内移动时，D 与蛋白质抑制剂的大小有明显的依赖性。低分子量蛋白酶抑制剂，如胱抑素，可以有效地扩散到完整的鱼肌肉细胞中，以最小化蛋白水解活性和肉质软化。
