CN114891066A - 一种磷酸肽及其肽钙螯合物的制备方法和应用 - Google Patents
一种磷酸肽及其肽钙螯合物的制备方法和应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,具体涉及到了一种磷酸肽及其肽钙螯合物的制备和应用。所述磷酸肽由5个氨基酸组成,序列为EDD‑pSer‑pSer(谷氨酸‑天冬氨酸‑天冬氨酸‑磷酸化丝氨酸‑磷酸化丝氨酸)。磷酸肽EDD‑pSer‑pSer制备的肽钙螯合物具有较高的钙螯合率,达到93.60±0.22%,即钙螯合能力为468mg/g。所述磷酸肽EDD‑pSer‑pSer与钙离子的螯合位点为氨基、羧基、磷酸基团以及肽键,结合方式以静电相互作用为主。本发明肽钙螯合物,其生物利用度显著高于CaCO3、D‑葡萄糖酸钙和乳酸钙。本发明拓展了磷酸肽在食品、医药和保健品等行业的应用,并为新型肽钙产品开发提供思路。
Description
技术领域
本发明属于生物技术领域,尤其是一种磷酸肽及其肽钙螯合物的制备方法和应用。
背景技术
钙作为膳食营养素对人体健康至关重要,能够调控人体一系列的生物功能。在健康人的身体中,钙含量约占人体重量的2%。一方面,一些组织和器官中的无机成分以钙为主,直接参与人体的构建;另一方面,钙参与调节人体的各种系统运转和细胞的新陈代谢,如凝血、神经传导、心脏健康等功能。人体中缺乏足够的钙会导致多种疾病,如骨质疏松、手足抽搐症、脆性骨折等。由于膳食摄入和生物利用度不足,全球估计有35亿人面临钙缺乏症风险。膳食中充足的钙可预防婴幼儿佝偻病的发生。而在青少年到30岁达到骨量峰值这个时期尤为重要,这能够减少成年后因骨骼逐渐脱矿发生骨质疏松的概率。而对于这些不能通过食物来源获得充足钙供应的人,可能需要在饮食之外口服钙补充剂,以达到推荐的每日摄入量。因此,发展钙补充剂逐渐成为了研究热点。
目前,钙补充剂主要有四代,第一代是无机钙,包括碳酸钙、氯化钙等。无机钙含钙量高,成本低,易于获得,但是其溶解度小,生物利用度低,而且摄入过量会对肠胃存在一些副作用。第二代钙补充剂主要是有机钙,包括葡萄糖酸钙、柠檬酸钙、乳酸钙等。有机钙能够很好的溶于水中,不需要消耗胃酸溶解,有利于肠胃吸收,但其含钙量低,补钙效果不理想。第三代是氨基酸螯合钙,通过与氨基酸螯合,使钙离子在肠道能够保持一定的浓度,因此它们也可以在一定程度上提高矿物质的生物利用度。但是由于其经济效益低,并未在市场上大范围推广。
与氨基酸相比,小肽在人体吸收中转运速度快、消耗能量少,同时作为载体不易饱和,称为第四代钙补充剂。此外,小肽还可以利用转运蛋白或非载体依赖机制完整的穿过肠壁。而作为补钙剂,人工合成的肽-矿物螯合物已经被发现比无机盐具有更高的生物利用度。因此,肽钙螯合物成了当前研究的热点。已经发现了多种来源的钙结合肽。但以肽钙螯合物作为钙补充剂的种类有限,产品单一。
发明内容
本发明目的在于克服现有技术的不足之处,提供一种具有高钙结合能力的磷酸肽及其肽钙螯合物的制备方法和应用。
本发明解决其技术问题所采用的技术方案是:
一种磷酸肽,由5个氨基酸组成,序列为EDD-pSer-pSer(谷氨酸-天冬氨酸-天冬氨酸-磷酸化丝氨酸-磷酸化丝氨酸)。
本发明还提供上述磷酸肽在食品、医药和保健品等行业的应用。
本发明还提供上述磷酸肽可应用于制备肽钙螯合物。
本发明还提供了一种肽钙螯合物的制备方法,将上述的磷酸肽和氯化钙进行反应,反应条件为:pH 5~7,肽钙质量比≥0.5:1,螯合温度为20~50℃,螯合时间≥20min。
本发明还提供了肽钙螯合物中所述磷酸肽与钙离子的螯合位点为氨基、羧基、磷酸基团以及肽键,结合方式以静电相互作用为主。
本发明还提供了上述的肽钙螯合物可应用于钙补充剂。
本发明取得的优点和积极效果为:
1、本发明提供的磷酸肽EDD-pSer-pSer(ES)制备成肽钙螯合物(ES-Ca),具有较高的钙螯合率,达到93.60±0.22%,即钙螯合能力为468mg/g。
2、本发明提供的磷酸肽与钙离子的螯合位点为氨基、羧基、磷酸基团以及肽键。
3、本发明提供的肽钙螯合物,其生物利用度显著高于CaCO3、D-葡萄糖酸钙和乳酸钙。
附图说明
图1为本发明中ES和ES-Ca的X射线衍射图谱;
图2为本发明中ES(A)和ES-Ca(B)的扫描电镜图(放大倍数为1000倍);
图3为本发明中ES和ES-Ca的傅里叶红外光谱图;
图4为本发明中ES和ES-Ca的核磁共振氢谱图;
图5为本发明中ES和ES-Ca的核磁共振磷谱图;
图6为本发明中ES和钙相互作用的ITC以及其结合参数图;
图7为本发明中消化时间对不同类型钙制剂在模拟肠液中溶解度的影响图;
图8为本发明中不同类型钙制剂的可透析钙百分比。
具体实施方式
下面详细叙述本发明的实施例,需要说明的是,本实施例是叙述性的,不是限定性的,不能以此限定本发明的保护范围。
本发明中所使用的原料,如无特殊说明,均为常规的市售产品;本发明中所使用的方法,如无特殊说明,均为本领域的常规方法。
实施例1,肽钙螯合物的制备
将磷酸肽ES(1mg/mL)与5mM的CaCl2溶液以不同肽钙比混合加入到0.02M不同pH的Tris-HCl缓冲液中。在不同温度的摇床中进行螯合反应,然后加入9倍混合溶液体积的无水乙醇,以10000g离心25min。所得沉淀用无水乙醇洗涤3次,待沉淀中的无水乙醇完全挥发后,用超纯水复溶并冻干得到肽钙螯合物(ES-Ca)。ES-Ca具有较高的钙螯合率,达到93.60±0.22%,即钙螯合能力为468mg/g。
实施例2,肽钙螯合物的表征
(1)X射线衍射分析
如图1所示,在ES的XRD图谱中,出现了一个宽而分散的衍射峰,表明ES是无规则的非晶态结构。而在ES和ES-Ca的XRD图谱之间出现明显的对比。在ES-Ca的XRD图谱中,原有的宽峰消失,出现了一些尖锐的衍射峰,例如在10.72°,15.28°等处出现的峰,表明ES-Ca形成了新的有序的晶体结构。
(2)扫描电子显微镜分析
利用扫描电镜观察ES和ES-Ca的微观形态。如图2所示,ES为表面光滑的球型结构,且分布较为松散;而肽钙螯合后,多肽的结构被破坏,形成了致密的纳米小球颗粒,粒径变小,分布密集。
(3)傅里叶红外光谱分析
红外光谱吸收峰的变化可以揭示多肽上金属离子的螯合位点。如图3所示,ES和ES-Ca的红外吸收曲线有显著差异。肽钙螯合后,ES在3404.63cm-1处的吸收峰转移至3385.81cm-1,这表明ES的氨基参与了Ca2+的螯合。肽钙螯合后2095.65cm-1处的峰消失,说明磷酸基团与钙螯合后O=P-O-H中的H被取代。肽螯合Ca2+后,酰胺I带的特征峰从1651.78cm-1移动到了1655.08cm-1,酰胺II带的特征峰从1577.53cm-1移动到了1560.59cm-1处,这表明酰胺中的C=O和N-H基团可能参与了与钙的螯合。羧酸基团中C=O的对称伸缩振动位于1420.21cm-1,肽与钙螯合后转移至1409.24cm-1,表明羧基可能与钙离子螯合,形成-COOCa。1096.11cm-1和993.51cm-1在肽螯合钙后分别移至1080.32cm-1和930.69cm-1,表明磷酸基团和肽键都有可能与钙离子螯合。以上这些结果表明了ES中的氨基、羧基、肽键和磷酸基团都参与了与钙离子的螯合。
(4)核磁共振氢谱分析
图4显示,位于D2O吸收峰右侧区域(4.0-4.76ppm)的信号峰信号强度显著下降,并且峰位置向高场移动,这表明在肽钙螯合过程中,多肽中的强电子吸收基团羧基-COO-与Ca2 +螯合。在2.56ppm和2.20ppm处的三重峰分别对应于Glu中-CH2-COO-的两个β-H自旋耦合开裂,肽钙螯合后,两个峰的信号强度显著降低并位移到2.39ppm和2.10ppm的高场区域。此现象是因为羧基参与螯合反应。2.90ppm和2.70ppm附近的峰可能对应于Asp的-CH2-COO-和Ser的-CH2-O-中H的自旋耦合开裂,两个峰值也发生了明显的下降并移动到了高场方向。这是由于Asp上的羧基和Ser上的磷酸集团也参与了螯合反应。这些结果表明Asp和Glu中的羧基以及Ser上的磷酸基团都参与了螯合反应。
(5)核磁共振磷谱分析
如图5所示,化学位移δ=-0.08ppm处的双重峰归因于磷酸化丝氨酸上的磷酸基团。肽钙螯合后,磷原子附近电子云的密度大小改变,-0.08ppm处的双峰化学位移向低场移动至0.03ppm处,同时峰强度显著降低。这个结果证明了ES中的磷酸基团是钙离子的结合位点。
(6)等温滴定量热法(ITC)分析
如图6,在CaCl2滴定ES的过程中出现了明显的放热峰,单位点模型可以较好地拟合反应等温线。ΔG<0,表明肽钙螯合反应是自发的放热反应。ΔH<0和ΔS>0,说明肽和钙结合过程主要以静电相互作用力为主。
实施例3,肽钙螯合物的生物利用度的测定
体外模拟消化是膳食营养研究中应用最广泛的模型,它是评估矿物质生物利用度的一种有效且可重复的方法。高溶解度和透析性与更好的生物利用度有关。
如图7所示,经过胃液消化后,监测了在肠道消化过程中四种钙制剂的溶解度变化。所有钙制剂的溶解度随着消化时间的延长而逐渐降低。消化2h后,CaCO3、D-葡萄糖酸钙、乳酸钙和ES-Ca的溶解度分别下降到40.49±1.35%、41.09±0.82%、31.18±1.11%和51.18±0.77%。由于钙离子在肠道的碱性条件下容易沉淀形成氢氧化钙等不溶性沉淀,CaCO3、D-葡萄糖酸钙和乳酸钙经过胃液消化后形成的游离钙离子在肠液中被沉淀,溶解度下降相对较高。相反,ES-Ca在肠道中一直保持相对较高的溶解度,其溶解度主要在胃的消化过程中丧失。与CaCO3、D-葡萄糖酸钙和乳酸钙相比,ES-Ca在体外消化模型中的溶解性更好。这些结果表明在人体消化过程中,ES-Ca能够在胃肠道复杂的化学环境中储存更多的钙离子进行下一步吸收,具有着相比于其他钙补充剂更加优异的持钙能力。
如图8所示,ES-Ca的可透析钙的百分比(56.22±0.69%)显著高于CaCO3、D-葡萄糖酸钙和乳酸钙。此外,ES-Ca的可透析钙百分比也高于已报道的卵黄高磷蛋白磷酸肽-钙螯合物DEEENDQVK-Ca(47.11±0.99%)。上述结果表明了ES-Ca在肠道吸收过程中能够完整通过的肠壁,并且其透过率相比于其他类型的钙制剂以及同类DK-Ca都更高。这为人体更好的吸收钙离子提供了可能。体外模拟胃肠道消化模型实验证明ES-Ca具有更好的溶解度和透过率,能够预测出ES-Ca作为钙补充剂,在人体消化吸收过程中具有优良的生物利用度。
尽管为说明目的公开了本发明的实施例,但是本领域的技术人员可以理解:在不脱离本发明及所附权利要求的精神和范围内,各种替换、变化和修改都是可能的,因此,本发明的范围不局限于实施例所公开的内容。
Claims (5)
1.一种磷酸肽,其特征在于:所述磷酸肽由5个氨基酸组成,序列为EDD-pSer-pSer(谷氨酸-天冬氨酸-天冬氨酸-磷酸化丝氨酸-磷酸化丝氨酸)。
2.如权利要求1所述的磷酸肽在制备肽钙螯合物方面中的应用。
3.一种利用如权利要求1所述的磷酸肽制备肽钙螯合物的方法,其特征在于:所述方法将所述的磷酸肽和氯化钙进行反应,反应条件为:pH 5~7,肽钙质量比≥0.5:1,螯合温度为20~50℃,螯合时间≥20min。
4.根据权利要求3所述的制备肽钙螯合物的方法,其特征在于:所述磷酸肽与钙离子的螯合位点为氨基、羧基、磷酸基团以及肽键,结合方式以静电相互作用为主。
5.如权利要求3或4所述的方法制得的肽钙螯合物在钙补充剂方面中的应用。
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