Ueno Hiroshi M, Kato Teruhiko, Ohnishi Hidenori, Kawamoto Norio, Kato Zenichiro, Kaneko Hideo, Kondo Naomi, Nakano Taku
Research and Development Department, Bean Stalk Snow Co., Ltd., Kawagoe, Japan.
Department of Pediatrics, Graduate School of Medicine, Gifu University, Gifu, Japan.
Pediatr Allergy Immunol. 2016 Dec;27(8):818-824. doi: 10.1111/pai.12642. Epub 2016 Sep 16.
Optimally hydrolyzed β-Lactoglobulin (βLg) is a promising milk oral immunotherapy (OIT) candidate with respect to showing reduced B-cell reactivity but retaining the T-cell epitope. To demonstrate that an edible hypoallergenic βLg hydrolysate containing the T-cell epitope is suitable for OIT. We tested how chymotrypsin affected the retention of the T-cell epitope of βLg when preparing βLg hydrolysates using food-grade trypsin.
We investigated the effect of chymotrypsin activity on the formation of the T-cell epitope-containing peptide of βLg (βLg ) and prepared an edible βLg hydrolysate containing βLg using screened food-grade trypsins. B-cell reactivity was determined using immunoassays in which ELISA was performed with anti-βLg rabbit IgG and Western blotting was performed with a milk-specific IgE antiserum.
In βLg hydrolysis performed by varying the activity of trypsin and chymotrypsin, chymotrypsin activity inhibited the formation of βLg with an increase in hydrolysis time in a dose-dependent manner. βLg was generated by two of five food-grade trypsins used at a ratio of 1:50 (w/w, enzyme/substrate) for 20 h at 40°C. The edible βLg hydrolysate retained βLg and showed a reduction in molecular weight distribution and antigenicity against IgG and IgE.
Chymotrypsin activity inhibited the formation of βLg in the trypsin hydrolysate of βLg. This βLg trypsin hydrolysate is a novel candidate for peptide-based OIT in cow's milk allergy for safely inducing desensitization.
最佳水解β-乳球蛋白（βLg）是一种有前景的牛奶口服免疫疗法（OIT）候选物，因为它显示出降低的B细胞反应性，但保留了T细胞表位。为了证明含有T细胞表位的可食用低敏βLg水解产物适用于OIT。我们测试了在使用食品级胰蛋白酶制备βLg水解产物时，胰凝乳蛋白酶如何影响βLg的T细胞表位的保留。
我们研究了胰凝乳蛋白酶活性对βLg含T细胞表位肽（βLg ）形成的影响，并使用筛选出的食品级胰蛋白酶制备了含有βLg的可食用βLg水解产物。使用免疫测定法测定B细胞反应性，其中用抗βLg兔IgG进行ELISA，并用牛奶特异性IgE抗血清进行蛋白质印迹分析。
在通过改变胰蛋白酶和胰凝乳蛋白酶的活性进行的βLg水解中，胰凝乳蛋白酶活性以剂量依赖的方式随着水解时间的增加抑制βLg的形成。在40°C下以1:50（w/w，酶/底物）的比例使用五种食品级胰蛋白酶中的两种20小时产生了βLg。可食用的βLg水解产物保留了βLg，并显示出分子量分布和针对IgG和IgE的抗原性降低。
胰凝乳蛋白酶活性抑制了βLg胰蛋白酶水解产物中βLg的形成。这种βLg胰蛋白酶水解产物是用于牛奶过敏中基于肽的OIT以安全诱导脱敏的新型候选物。
