Hu Guanhua, Wang Debao, Sun Lina, Su Rina, Corazzin Mirco, Sun Xueying, Dou Lu, Zhang Min, Zhao Lihua, Su Lin, Jin Ye
College of Food Science and Engineering, Inner Mongolia Agricultural University, Hohhot 010018, China.
Agricultural and Animal Products Processing Research Institute, Inner Mongolia Academy of Agriculture and Animal Husbandry Academy, Hohhot 010018, China.
Foods. 2022 Sep 1;11(17):2655. doi: 10.3390/foods11172655.
To isolate a novel peptide with calcium-binding capacity, sheep bone protein was hydrolyzed sequentially using a dual-enzyme system (alcalase treatment following neutrase treatment) and investigated for its characteristics, separation, purification, and structure. The sheep bone protein hydrolysate (SBPH) was enriched in key amino acids such as Gly, Arg, Pro, Leu, Lys, Glu, Val, and Asp. The fluorescence spectra, circular dichroism spectra, and Fourier-transform infrared spectroscopy results showed that adding calcium ions decreased the α-helix and β-sheet content but significantly increased the random and β-turn content (p < 0.05). Carboxyl oxygen and amino nitrogen atoms of SBPH may participate in peptide−calcium binding. Scanning electron microscopy and energy dispersive spectrometry results showed that SBPH had strong calcium-chelating ability and that the peptide−calcium complex (SBPH−Ca) combined with calcium to form a spherical cluster structure. SBPH was separated and purified gradually by ultrafiltration, gel filtration chromatography, and reversed-phase high-performance liquid chromatography. Liquid chromatography-electrospray ionization/mass spectrometry identified the amino acid sequences as GPSGLPGERG (925.46 Da) and GAPGKDGVRG (912.48 Da), with calcium-binding capacities of 89.76 ± 0.19% and 88.26 ± 0.25%, respectively. The results of this study provide a scientific basis for the preparation of a new type of calcium supplement and high-value utilization of sheep bone.
为了分离出一种具有钙结合能力的新型肽，采用双酶系统（中性蛋白酶处理后再用碱性蛋白酶处理）对羊骨蛋白进行顺序水解，并对其特性、分离、纯化和结构进行研究。羊骨蛋白水解物（SBPH）富含甘氨酸、精氨酸、脯氨酸、亮氨酸、赖氨酸、谷氨酸、缬氨酸和天冬氨酸等关键氨基酸。荧光光谱、圆二色光谱和傅里叶变换红外光谱结果表明，添加钙离子会降低α-螺旋和β-折叠含量，但会显著增加无规卷曲和β-转角含量（p<0.05）。SBPH的羧基氧原子和氨基氮原子可能参与肽与钙的结合。扫描电子显微镜和能谱分析结果表明，SBPH具有很强的钙螯合能力，肽-钙复合物（SBPH-Ca）与钙结合形成球形簇结构。通过超滤、凝胶过滤色谱和反相高效液相色谱对SBPH进行逐步分离和纯化。液相色谱-电喷雾电离/质谱鉴定出氨基酸序列为GPSGLPGERG（925.46 Da）和GAPGKDGVRG（912.48 Da），其钙结合能力分别为89.76±0.19%和88.26±0.25%。本研究结果为新型钙补充剂的制备和羊骨的高值利用提供了科学依据。
