De Angelis Maria, Rizzello Carlo G, Scala Enrico, De Simone Claudio, Farris Giovanni A, Turrini Francesco, Gobbetti Marco
Department of Plant Protection and Applied Microbiology, University of Bari, 70126 Bari, Italy
J Food Prot. 2007 Jan;70(1):135-44. doi: 10.4315/0362-028x-70.1.135.
This study was aimed at showing the capacity of probiotic VSL#3 to hydrolyze wheat flour allergens. Hydrolysis was investigated either by the use of baker's yeast bread treated with digestive enzymes and VSL#3, an experimental design that mimicked the activity of probiotics during gut colonization, or by the use of VSL#3 as a starter for dough fermentation, an experimental design that mimicked the predigestion of wheat flour proteins during food processing. Albumins, globulins, and gliadins extracted from wheat flour and chemically acidified and started dough and total proteins extracted from breads were analyzed by immunoblotting with pooled sera from patients with an allergy to wheat. Hydrolysis of wheat flour proteins was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Mass spectrometry matrix-assisted laser desorption and ionization-time of flight was used to identify some immunoglobulin E (IgE)-binding proteins. As shown by immunoblotting with sera from allergic patients, several IgE-binding proteins persisted after treatment of baker's yeast bread by pepsin and pancreatin. The signal of all these IgE-binding proteins disappeared after further treatment by VSL#3. As shown by SDS-PAGE and related immunoblotting and 2DE analyses, when VSL#3 was used as a starter for bread making, it caused a marked degradation of wheat proteins, including some IgE-binding proteins such as the putative transcription factor APFI and wheat alpha-amylase inhibitors. Indeed, the IgE-binding profile of the bread manufactured by VSL#3 was largely different from that of baker's yeast bread. The IgE-binding proteins that persisted in the bread made with VSL#3 were completely degraded by pepsin and pancreatin.
本研究旨在展示益生菌VSL#3水解小麦粉过敏原的能力。通过使用经消化酶和VSL#3处理的面包酵母面包(一种模拟益生菌在肠道定植过程中活性的实验设计)或使用VSL#3作为面团发酵的发酵剂(一种模拟食品加工过程中小麦粉蛋白质预消化的实验设计)来研究水解情况。用对小麦过敏患者的混合血清通过免疫印迹法分析从小麦粉中提取并经化学酸化的清蛋白、球蛋白和醇溶蛋白以及从面团和面包中提取的总蛋白。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和二维电泳(2DE)测定小麦粉蛋白质的水解情况。使用基质辅助激光解吸电离飞行时间质谱法鉴定一些免疫球蛋白E(IgE)结合蛋白。正如用过敏患者血清进行免疫印迹所显示的,用胃蛋白酶和胰蛋白酶处理面包酵母面包后,几种IgE结合蛋白仍然存在。在用VSL#3进一步处理后,所有这些IgE结合蛋白的信号消失。正如SDS-PAGE及相关免疫印迹和2DE分析所示,当VSL#3用作面包制作的发酵剂时,它会导致小麦蛋白显著降解,包括一些IgE结合蛋白,如推定的转录因子APFI和小麦α-淀粉酶抑制剂。事实上,用VSL#3制作的面包的IgE结合谱与面包酵母面包的IgE结合谱有很大不同。在用VSL#3制作的面包中持续存在的IgE结合蛋白被胃蛋白酶和胰蛋白酶完全降解。