Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.
Food Chem. 2013 Dec 1;141(3):2944-51. doi: 10.1016/j.foodchem.2013.05.050. Epub 2013 May 24.
Three bioactive peptides, angiotensin-converting enzyme-inhibitory peptides (ACEIPs), casein phosphopeptides (CPPs) and antimicrobial peptides (AMPs), were simultaneously prepared from casein by pancreatic hydrolysis via an enzymatic membrane reactor (EMR) and combinational chromatography. The reaction was performed at 37 °C and pH 8.0 for 3h followed by ultrafiltration. ACEIPs were purified by size exclusion chromatography (SEC) from permeate fractions with molecular weight (MW) below 1 kDa. Concurrently, strong cation exchange high-performance liquid chromatography (SCE-HPLC) was used to isolate CPPs and AMPs from retentate fractions ranging from 1 kDa and 5 kDa. Following reverse-phase high performance liquid chromatography-electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS) analysis, potential ACEIPs and a total of 34 CPPs were identified (18 corresponded to αs1-casein, 2 to αs2-casein, 10 to β-casein and 4 to κ-casein). Additionally, the ACEIPs in fraction B from SEC had the highest ACE-inhibiting-activity (73.5% in 1.0mg/ml), while total concentrations of AMPs were directly proportional to overall antibacterial activity. Uniquely, fraction 5 from SCE exhibited the highest activity against Staphylococcus aureus (97.49% in 1.0 mg/ml).
三种生物活性肽，血管紧张素转换酶抑制肽（ACEIPs）、酪蛋白磷酸肽（CPPs）和抗菌肽（AMPs），通过酶膜反应器（EMR）和组合色谱法从胰蛋白酶水解的酪蛋白中同时制备。反应在 37°C 和 pH8.0 下进行 3 小时，然后进行超滤。ACEIPs 通过尺寸排阻色谱（SEC）从分子量（MW）低于 1 kDa 的透过液部分中进行纯化。同时，强阳离子交换高效液相色谱（SCE-HPLC）用于从 1 kDa 和 5 kDa 之间的保留液部分中分离 CPPs 和 AMPs。经反相高效液相色谱-电喷雾串联质谱（RP-HPLC-ESI-MS/MS）分析，鉴定出潜在的 ACEIPs 和 34 种 CPPs（18 种对应于αs1-酪蛋白，2 种对应于αs2-酪蛋白，10 种对应于β-酪蛋白，4 种对应于κ-酪蛋白）。此外，SEC 中 B 级分的 ACEIPs 具有最高的 ACE 抑制活性（在 1.0mg/ml 时为 73.5%），而 AMPs 的总浓度与整体抗菌活性成正比。独特的是，SCE 的 5 级分对金黄色葡萄球菌的活性最高（在 1.0mg/ml 时为 97.49%）。
