二硫键分子构象对菜籽Napin蛋白消化性能影响及其消化产物吸收机制研究

After rapeseed processing, the remained cake meal resources are abundant, and the amino acid balance of rapeseed protein in the remained cake is superior to soy protein. The protein titer can be comparable to casein, and the value of high value processing and utilization is promising. Our previous study found that Rapeseed Napin protein (RNP) has excellent solubility and emulsification properties, but its digestive performance is poor and the absorption mechanism of digested products is not yet clear. Therefore, the RNP obtained in the previous project was used as raw material, and the conformation of disulfide bonds was changed by enzymatic reduction method. The advanced structure and physicochemical properties of RNP before and after the reaction were characterized by modern instrument analysis techniques; the disposition changes of disulfide bonds were mainly revealed. Elucidate the role and regulation of this change in promoting RNP digestion performance, and build its chromatographic fingerprinting; study the absorption mechanism of RNP digested intestinal epithelial cells based on the Caco-2 cell model. Using the in silico molecular docking simulation method, the interaction between RNP and hydrolase, enzyme and transporter during digestion and absorption was explored, and the conformational change, active site and signaling pathway of disulfide bond were identified. The RNP digestion and absorption mechanism is enriched from the molecular conformation of the characteristic bond sites, providing new options for the high value utilization of rapeseed protein.

油菜籽经加工后剩余饼粕资源丰富，饼粕中菜籽蛋白氨基酸平衡优于大豆蛋白，蛋白效价可以媲美酪蛋白，高值化加工利用前景广阔。我们前期研究发现菜籽Napin蛋白（RNP）溶解度与乳化性能优良，但其消化性能较差且消化产物的吸收机制尚不明确。因此，本项目以前期制备得到的RNP为原料，通过酶促还原法改变二硫键分子构象，采用现代仪器分析技术表征反应前后RNP高级结构与理化特性；重点揭示二硫键分子构象变化情况，阐明该变化对促进RNP消化性能提升的作用和规律，并构建其基于色谱学的指纹图谱；基于Caco-2细胞模型研究RNP消化产物小肠上皮细胞吸收机制。运用in silico分子对接模拟方法，探究RNP在消化吸收过程中与水解酶、生物酶、转运蛋白的相互作用关系，明确其二硫键分子构象变化、活性中心位点及信号通路。从特征键位分子构象角度丰富了RNP消化吸收机理，为菜籽蛋白的高值化利用提供新选择。

菜籽饼粕中含有40%左右的蛋白质，氨基酸平衡优于大豆蛋白，蛋白效价可以媲美酪蛋白。菜籽Napin蛋白（RNP）具有优良的溶解特性和乳化性能，但其消化性能较差且消化产物的吸收机制尚不明确。本项目采用多糖复合法优化RNP提取工艺，以RNP为研究对象，考察超声协同混合处理法对RNP中二硫键分子的改变作用，对反应前后RNP高级结构和理化特性进行了表征，揭示了二硫键构象变化与消化性的相关性；采用多种商业酶水解RNP，利用Caco-2细胞模型研究RNP消化衍生肽在小肠上皮细胞中的吸收机制；进一步采用分子对接手段模拟探究RNP消化衍生肽在消化吸收过程中与酶相互作用机制。本研究发现ι型卡拉胶与水溶性菜籽蛋白复合效果最佳，产物RNP含量丰富。超声协同蛋白混合处理可有效改善RNP消化性，处理后蛋白内源荧光强度和蛋白表面疏水性明显下降，部分二硫键分子被破坏，β-折叠含量降低（7.86%），无规卷曲含量增加（18.45%）；构建了消化率与蛋白质结构间的构效关系，发现RNP消化性改善与二硫键构象变化有关，主要表现为t-g-t模式的增加（14.77%）和g-g-g模式的减少（0.85）。RNP4种水解产物中RNPH-1的DPP-IV酶抑制活性最强，其洗脱馏分G2-R2的DPP-IV酶抑制活性最高，利用UPLC-Triple-TOF-MS/MS从该组分中鉴定出10条DPP-IV酶抑制肽，其中PAGPF、IPQVS、ELHQEPL和Q（-17.03）KTMPGP固相合成产物活性最高。IPQVS和ELHQEEPL均以剂量依赖型方式抑制Caco-2细胞单层中的DPP-IV酶活性，不影响DPP-IV酶mRNA的表达。IPQVS主要通过胞吞，ELHQEEPL主要通过细胞旁路的形式的跨Caco-2细胞单层转运。分子对接结果表明在Caco-2细胞单层模拟小肠细胞吸收实验中DPP-IV酶和寡肽IPQVS和ELHQEEPL降解产物的结合弱于母肽，结合的位点也发生了显著减少，极大地限制了母肽抑制DPP-IV酶抑制活性。综上，本项目系统分析和探究了二硫键分子构象对RNP消化性能影响及其消化产物吸收机制，从分子构象角度丰富了RNP消化吸收机理，为菜籽蛋白的高值化利用提供新选择。