【摘要】 酪蛋白磷酸肽（casein phosphopeptide,CPP）是从牛乳蛋白水解而来的生物活性磷酸化肽类，具有优越的螯合能力，本实验利用CPP作为载体，制备酪蛋白磷酸肽-硒螯合物（casein phosphopeptide selenium chelate,CPP-Se）作为一种新型硒补充剂。首先采用光谱学和热力学对其结构进行表征，利用分子对接技术模拟可能结合位点以及模型，并建立脂多糖（lipopolysaccharide,LPS）诱导RAW264.7巨噬细胞炎症模型对其免疫和抗炎活性进行分析。结果显示，在肽硒质量比为2∶1、螯合温度35.48℃、螯合pH 7.0的工艺条件下，CPP-Se的硒含量达到5578.66μg/g；紫外和傅里叶变换红外光谱结果表明CPP主要通过羰基、羧基、磷酸基团螯合Se；圆二色光谱显示螯合物中CPP肽链的β-折叠比例提高；热重分析结果表明CPP-Se比CPP的稳定性更强；等温滴定量热法联合分子对接技术分析表明肽硒之间的螯合位点主要是谷氨酸和丝氨酸中的氢键，距离为2.6?，结合能为-7.53 kJ/mol，主要以疏水相互作用作为驱动力；RAW264.7巨噬细胞实验结果显示，同等硒质量浓度下，CPP-Se的安全使用范围相较亚硒酸钠扩大了2倍，且CPP-Se使RAW264.7巨噬细胞的吞噬能力提高了25%，显著抑制了LPS诱导RAW264.7巨噬细胞中一氧化氮、肿瘤坏死因子-α和白细胞介素-6的分泌与释放。综上所述，酪蛋白磷酸肽-硒螯合物可作为一种具有安全性的新型硒补充剂，本研究可为CPP的广泛应用提供新的思路。更多还原

【Abstract】 Casein phosphopeptide(CPP) is a bioactive phosphorylated peptide derived from the hydrolysis of cow’s milk casein,which has superior chelating properties.In this study,CPP was used as a carrier to prepare selenium-chelating CPP(CPP-Se) for use as a novel selenium supplement.The structure of CPP-Se was characterized by spectroscopy and thermodynamics.Molecular docking was used to simulate the binding interaction between Se and CPP and the immunoregulatory and anti-inflammatory activities of CPP-Se were studied in lipopolysaccharide(LPS)-stimulated RAW264.7 macrophage cells.The results showed that the Se content of CPP-Se prepared under peptide/selenium mass ratio of 2:1,35.48 ℃ and pH 7.0 was 5 578.66 μg/g.Ultraviolet(UV) and Fourier transform infrared spectroscopy(FTIR)demonstrated that CPP chelated Se through carbonyl,carboxyl,and phosphate groups.Circular dichroism(CD) spectroscopy indicated that the proportion of β-sheet was higher in CPP-Se than in CPP.Thermogravimetric(TG) analysis indicated that CPP-Se was more stable than CPP.Furthermore,isothermal titration calorimetry(ITC) combined with molecular docking revealed that the chelating site between peptide and selenium was hydrogen bonds in glutamic acid and serine,with a distance of 2.6 ? and a binding energy of-7.53 kJ/mol,mainly driven by hydrophobic interactions.For the same selenium concentration,the safe range of CPP-Se for RAW264.7 macrophages was two times larger than that of sodium selenite,and CPP-Se increased the phagocytic capacity of macrophages by 25%.The inhibitory effect of CPP-Se on the secretion and release of NO,tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in LPS-stimulated RAW264.7 macrophages was more pronounced than that of sodium selenite.In summary,CPP-Se can be used as a new safe selenium supplement.This study provides new ideas for the widespread application of CPP.更多还原