食品源蛋白肽-钙的螯合作用模式及促钙吸收机制

Calcium is an essential element that plays an important role in body physiological function, and calcium deficiency is harmful to human health. Recently, the reality of lower bioavailability, which restricted the effectiveness of calcium uptake, has become a bottleneck problem in calcium supplement products. The utilization of calcium absorption could be significantly promoted by organic peptide-Ca chelate that was prepared by food-based peptides chelating with calcium element, and the key of revealing the nature of biological function is to clarify the interaction model of peptides chelated with calcium ions and mechanism of calcium uptake promotion. The project aims to research the interaction model of peptides chelated with calcium ions and mechanism of calcium uptake promotion, based on previous work that has been fabricated the peptide-calcium chelate and proved its elementary bioavailability. The specific calcium binding peptides are to be purified and characterized by HPLC, MS, CD and NMR, and the unique structure will be identified. The peptide-calcium chelating sites, thermodynamics/kinetics and the mechanism of chelating will be determined by ITC / MST methods, molecular dynamics, atomic force microscopy and NMR. The qRT-PCR, Western Blotting techniques and iTRAQ proteomics technique will be used for analyzing the effects of peptide-calcium chelate on cell metabolism, confirming the calcium ion uptake pathway and elucidating the mechanism of calcium uptake promotion. This study will provide the theoretical foundation for the development and application of food-based peptide-Ca chelate.

钙是人体必不可少且具重要生理功能的元素，缺钙危害人体健康。补钙产品层出不穷，但生物利用率不高的现实成为制约人体有效补钙的瓶颈问题。利用食品源蛋白肽对钙的螯合作用制备肽-钙有机螯合物能显著提高钙吸收利用率，而如何阐明肽-钙的螯合作用模式及促钙吸收机制是揭示其功效本质的关键。本项目拟在已实现可控制备肽-钙螯合物并初步证明其高生物利用率的基础上，利用HPLC、质谱、全序列测定、圆二色谱和核磁共振等手段分离鉴定钙螯合特征肽、确定其结构特征和螯合钙离子结构域；利用ITC/MST量热法、分子动力学法、原子力显微镜和扫描电镜等技术解析肽-钙之间螯合作用位点、热/动力学过程及螯合作用机理；利用iTRAQ蛋白质组学技术，结合qRT-PCR和Western Blotting技术，明确肽-钙螯合物对细胞代谢的影响作用，解析钙离子吸收通路，阐明促钙吸收机制。本研究将为食品源肽-钙螯合物的开发与应用奠定理论基础。

钙营养缺乏是全球性的健康问题，而生物利用率不高是制约有效补钙的瓶颈。食源性肽-钙螯合物能显著提高钙离子吸收利用率，近年来已成为开发补钙制剂的一个研究热点。本项目以食源性加工副产物如鱼鳞等为原料，酶解制备具有高效钙螯合力的鱼源胶原蛋白钙螯合肽。以钙螯合肽及功能性低聚糖类益生元为原料，优化制备糖基化钙螯合肽以提高钙螯合力。肽与钙发生螯合反应后，钙由高度结晶状态转变为无定形状态。红外光谱分析表明，肽上的氨基氮原子和羧基氧原子是主要的螯合位点。相比游离无机钙离子，螯合物在碱性pH范围内不会发生沉降，提示其可在人体肠道碱性pH环境保持稳定。同步热分析表明，螯合物相比多肽具有更稳定的结构及热稳定性。利用Caco-2细胞模型考察钙螯合肽的促钙吸收作用，结果表明，钙螯合肽可以促进细胞对钙离子的吸收作用，并可以显著减轻膳食抑制剂对钙离子的吸收抑制作用。钙螯合肽可通过提高TRPV6通路关键因子表达水平，发挥促钙跨膜转运作用。