Kim S B, Seo I S, Khan M A, Ki K S, Lee W S, Lee H J, Shin H S, Kim H S
Dairy Science Division, National Institute of Animal Science, Rural Development Administration, Cheonan, Chungnam 330-801, Republic of Korea.
J Dairy Sci. 2007 Sep;90(9):4033-42. doi: 10.3168/jds.2007-0228.
This study evaluated the influence of various enzymes on the hydrolysis of whey protein concentrate (WPC) to reduce its antigenic fractions and to quantify the peptides having iron-binding ability in its hydrolysates. Heated (for 10 min at 100 degrees C) WPC (2% protein solution) was incubated with 2% each of Alcalase, Flavourzyme, papain, and trypsin for 30, 60, 90, 120, 150, 180, and 240 min at 50 degrees C. The highest hydrolysis of WPC was observed after 240 min of incubation with Alcalase (12.4%), followed by Flavourzyme (12.0%), trypsin (10.4%), and papain (8.53%). The nonprotein nitrogen contents of WPC hydrolysate followed the hydrolytic pattern of whey. The major antigenic fractions (beta-lactoglobulin) in WPC were degraded within 60 min of its incubation with Alcalase, Flavourzyme, or papain. Chromatograms of enzymatic hydrolysates of heated WPC also indicated complete degradation of beta-lactoglobulin, alpha-lactalbumin, and BSA. The highest iron solubility was noticed in hydrolysates derived with Alcalase (95%), followed by those produced with trypsin (90%), papain (87%), and Flavourzyme (81%). Eluted fraction 1 (F-1) and fraction 2 (F-2) were the respective peaks for the 0.25 and 0.5 M NaCl chromatographic step gradient for analysis of hydrolysates. Iron-binding ability was noticeably higher in F-1 than in F-2 of all hydrolysates of WPC. The highest iron contents in F-1 were observed in WPC hydrolysates derived with Alcalase (0.2 mg/kg), followed by hydrolysates derived with Flavourzyme (0.14 mg/kg), trypsin (0.14 mg/kg), and papain (0.08 mg/kg). Iron concentrations in the F-2 fraction of all enzymatic hydrolysates of WPC were low and ranged from 0.03 to 0.05 mg/kg. Fraction 1 may describe a new class of iron chelates based on the reaction of FeSO4 x 7 H2O with a mixture of peptides obtained by the enzymatic hydrolysis of WPC. The chromatogram of Alcalase F-1 indicated numerous small peaks of shorter wavelengths, which probably indicated a variety of new peptides with greater ability to bind with iron. Alcalase F-1 had higher Ala (18.38%), Lys (17.97%), and Phe (16.58%) concentrations, whereas the presence of Pro, Gly, and Tyr was not detected. Alcalase was more effective than other enzymes at producing a hydrolysate for the separation of iron-binding peptides derived from WPC.
本研究评估了各种酶对乳清蛋白浓缩物（WPC）水解的影响，以降低其抗原成分，并对其水解产物中具有铁结合能力的肽进行定量。将加热（在100℃下加热10分钟）的WPC（2%蛋白质溶液）与2%的碱性蛋白酶、风味酶、木瓜蛋白酶和胰蛋白酶在50℃下分别孵育30、60、90、120、150、180和240分钟。与碱性蛋白酶孵育240分钟后观察到WPC的最高水解率（12.4%），其次是风味酶（12.0%）、胰蛋白酶（10.4%）和木瓜蛋白酶（8.53%）。WPC水解产物的非蛋白氮含量遵循乳清的水解模式。WPC中的主要抗原成分（β-乳球蛋白）在与碱性蛋白酶、风味酶或木瓜蛋白酶孵育60分钟内被降解。加热的WPC酶水解产物的色谱图也表明β-乳球蛋白、α-乳白蛋白和牛血清白蛋白完全降解。在碱性蛋白酶水解产物中观察到最高的铁溶解度（95%），其次是胰蛋白酶水解产物（90%）、木瓜蛋白酶水解产物（87%）和风味酶水解产物（81%）。洗脱级分1（F-1）和级分2（F-2）分别是用于水解产物分析的0.25和0.5M NaCl色谱阶梯梯度峰。WPC所有水解产物中，F-1的铁结合能力明显高于F-2。在碱性蛋白酶水解的WPC产物中观察到F-1的最高铁含量（0.2mg/kg），其次是风味酶水解产物（0.14mg/kg）、胰蛋白酶水解产物（0.14mg/kg）和木瓜蛋白酶水解产物（0.08mg/kg）。WPC所有酶水解产物的F-2级分中铁浓度较低，范围为0.03至0.05mg/kg。级分1可能描述了一类基于FeSO4·7H2O与WPC酶水解获得的肽混合物反应的新型铁螯合物。碱性蛋白酶F-1色谱图显示了许多较短波长的小峰，这可能表明有多种与铁结合能力更强的新肽。碱性蛋白酶F-1中丙氨酸（18.38%）、赖氨酸（17.97%）和苯丙氨酸（16.58%）浓度较高，而未检测到脯氨酸、甘氨酸和酪氨酸存在。在生产用于分离WPC衍生的铁结合肽的水解产物方面，碱性蛋白酶比其他酶更有效。
