Reduction of Allergenicity of Milk Protein by Conjugation with Acidic Oligosaccharides

为降低β-乳球蛋白（β-LG）的致敏性，改善其功能特性，采用Maillard反应制备了β-LG-海藻酸寡糖（β-LG-ALGO）偶联物，偶联物中β-LG与ALGO的摩尔比为1：6。结合物的等电点<4.6，低于β-LG的等电点，结合位点分别为60、77、100、138和141个赖氨酸。CD谱表明，β-LG与ALGO偶联后二级结构基本保持不变。荧光光谱分析表明，Trp周围的构象没有发生变化，结合物表面被糖链所覆盖。单克隆抗体结构分析表明，偶联物中15 Val-29 Ile和8Lys-19-Trp附近的构象发生了变化，而125 Thr-135 Lys附近的天然结构保持不变。通过与ALGO的结合，β-LG具有较高的热稳定性和乳化能力。免疫BALB/c、C57 BL/6和C3 H/He小鼠后，抗β-LG抗体反应明显降低。我们确定了β-LG和缀合物在这些小鼠中识别的B和T细胞表位，并发现β-LG- ALGO缀合物的线性表位谱与β-LG的线性表位谱相似，而对每个表位的免疫应答显著降低。认为ALGO对表位的掩蔽是缀合物中β-LG免疫原性降低的原因。

Bovine β-lactoglobulin-alginic acid oligosaccharide (β-LG-ALGO) conjugate was prepared by the Maillard reaction to reduce the allergenicity and improve the functional properties of β-LG. The molar ratio of β-LG to ALGO in the conjugates was 1 : 6. The isoelectric point of the conjugate was <4.6, which is lower than that of β-LG. Carbohydrate binding sites in β-LG were identified to be 60Lys, 77Lys, lOOLys, 138Lys and141Lys. CD spectra indicated that secondary structure of β-LG was almost maintained after conjugation with ALGO. Fluorescence studies suggested that the conformation around Trp had not changed in the conjugate and that the surface of the conjugate was covered with saccharide chain. Structural analyzes with monoclonal antibodies indicated that the conformation around 15Val29Ile and 8Lys-19-Trp in the conjugate had changed, while native structure was maintained around 125Thr-135Lys. By conjugation with ALGO, β-LG was endowed with high heat stability and improved emulsifying ability. The antiβ-LG antibody response was markedly reduced after immunization with the β-LG- ALGO conjugates in BALB/c, C57BL/6 and C3H/He mice. We determined the B and T cell epitopes of β-LG and the conjugate recognized in these mice and found that the linear epitope profiles of the β-LG- ALGO conjugate were similar to those of β-LG, while the immune response for each epitope was dramatically reduced. Masking of epitopes by ALGO was considered to be responsible for the decreased immunogenicity of the β-LG in the conjugate.