高载钙胶原蛋白的化学修饰、胶囊化制备及其吸收机制

Calcium deficiency is a global nutritional problem. The novel method of calcium preparation is that using protein/polypeptide as calcium carrier which can help control release and absorption. We first prepared human-like collagen (HLC) as ligand to prepare edible HLC-Ca complex. To develop safe and high-efficiency calcium preparation, however, it still had some problem such as low loading, easily degradable and unclear absorption pathways. So in this project, we first chemically modified the HLC (mHLC) to increase the Ca2+ binding sites to get high-loading calcium complex. The preparation technics of mHLC-Ca microcapsule was established to reduce degradation against proteases. Also, the slow release kinetics of mHLC and Ca2+ in the microcapsule under simulated conditions of gastrointestinal tract was studied using dynamics and thermodynamics methods. The safety and activity of mHLC-Ca complex and its microcapsule was studied in cell and animal experiments. At last, the genes related to Ca2+ transport in gastrointestinal tract were determined and the absorption pathway of HLC/mHLC-Ca complex was elucidated to develop a novel calcium preparation.

缺钙是全球公认的营养学难题，以蛋白/多肽为载体的复合钙制剂对Ca2+的释放和吸收具有缓释作用，代表了未来钙制剂研发的新方向。本课题组率先以无毒、水溶性好、可食的类人胶原蛋白(HLC）为载体制备了HLC-Ca复合物，但要将其开发成安全、高效的补钙制剂，尚需解决载钙量低、易被蛋白酶降解及其Ca2+吸收途径不清楚等问题。为此，本项目首先拟对HLC进行化学修饰以增加Ca2+的结合位点，得到具有高载钙量的mHLC-Ca复合物；同时，建立mHLC-Ca微胶囊制备工艺以避免蛋白酶对其降解，从动力学和热力学角度探索模拟胃肠道体系中mHLC-Ca微胶囊中mHLC及Ca2+的缓释动力学行为，并在离体细胞和在体动物水平上综合评价mHLC-Ca复合物及其微胶囊的安全性和补钙活性；最后利用分子生物学手段监测胃肠道中与Ca2+转运相关基因的调控表达，结合营养学实验阐释HLC/mHLC-Ca复合物中Ca2+的吸收途径。

以蛋白质/多肽为载体制备新型复合钙，被认为是解决钙营养缺乏症的有效途径之一。选择钙离子结合能力强的载体蛋白是关键，本项目将水溶性好、生物相容性好、无毒、可食的类人胶原蛋白（HLC）进行化学修饰、微囊化处理，成功制备了高载钙量的胶原钙复合物，主要成果如下：.1. 制备了三种高载钙类人胶原蛋白复合物（mHLC-Ca）：PHLC-Ca、As-HLC-Ca和SH-HLC-Ca；利用光谱学方法对制备的三种mHLC-Ca复合物进行了结构表征，明确了修饰后HLC与钙离子的相互作用机制，对其作用位点、结合常数及热力学参数进行系统表征，并对复合物的稳定性剂溶解性进行了研究；利用细胞模型（Caco-2细胞和BHK21细胞）对mHLC-Ca复合物的细胞毒性进行了测定。利用维甲酸诱导的骨质疏松小鼠模型研究发现，相比市场上常见的葡萄糖酸钙制剂，PHLC-Ca、Ac-HLC-Ca和SH-HLC-Ca复合物显著改善了小鼠骨密度和骨骼钙质的含量，有效缓解了骨质疏松。.2. 以海藻酸钠(ALG)和壳聚糖(CS)为壁材，采用高压脉冲电场制备了PHLC-Ca的微胶囊(CS/ALG-(PHLC-Ca)；通过改变ALG和PHLC-Ca的质量比对微胶囊载药量和包封率进行了优化，最终确定ALG与PHLC-Ca的最佳质量比为：1:1/2；采用扫描电镜、粒径分析仪对所制备的微胶囊的形态和粒径分布进行了研究；通过热力学分析、红外光谱证明了PHLC-Ca是通过静电吸附被成功地包埋在微胶囊中；通过壳聚糖的分子量、浓度及反应时间与微胶囊成型规律的研究，确定了CS/ALG-(PHLC-Ca)微胶囊外层壳膜的厚度、在模拟连续胃肠液中的溶胀率以及PHLC-Ca的释放规律。最终确定壳聚糖分子量为55-100KDa、壳聚糖浓度1%、反应时间20min所制备的微胶囊对PHLC-Ca有很好的缓控释效果。.3. 利用MTT比色法证实Empty CS/ALG微胶囊和CS/ALG-(PHLC-Ca)微胶囊对细胞安全无毒；利用骨质疏松动物模型，微囊化处理的PHLC-Ca微胶囊可避免胃液对其活性以及钙稳定性的影响，延长了PHLC-Ca的吸收时间，进而提高了钙离子的生物利用率。