牛血红蛋白肽的酶解工艺优化及其亚铁螯合物结构、稳定性研究
Optimization of Enzymatic Hydrolysis of Bovine Hemoglobin Peptide and Study on Structure and Stability of Ferrous Chelate
祝超智 1温耀涵 1许龙 1张秋会 1王兴辉 1赵改名 1韩广星2
作者信息
- 1. 河南农业大学食品科学技术学院,河南郑州 450002
- 2. 临沂综合实验站,山东临沂 273300
- 折叠
摘要
本研究以牛血为原料,提取牛血红蛋白进行酶解,筛选最适的酶解蛋白酶,采用单因素结合响应面试验探究最佳酶解工艺,通过扫描紫外光谱,傅里叶红外光谱,扫描电镜和全自动氨基酸分析仪等技术对制备的牛血红蛋白肽铁螯合物(Bovine Hemoglobin Peptide Iron Chelate,BHP-Fe)进行结构表征,并通过热重分析和体外模拟胃肠道消化探究其体外稳定性.结果表明:胃蛋白酶和碱性蛋白酶分步酶解为最适酶解方法.在胃蛋白酶初步酶解的基础上,得到碱性蛋白酶最佳酶解条件:料液比 1:3,酶解pH9.8,酶解温度 41℃,酶添加量 5900 U/g,酶解时间 2h.在此条件下,酶解得到牛血红蛋白肽(Bovine Hemoglobin Peptide,BHP)的 Fe2+螯合能力达72.11%,牛血红蛋白水解度达 35.07%.紫外光谱和傅里叶红外光谱结果显示,Fe2+与BHP肽链上的羧基氧和氨基氮发生螯合反应生成了不同于多肽的新物质;扫描电镜结果显示,多肽与Fe2+螯合后从表面光滑的碎片状变成表面粗糙的块状,两者存在明显差异;氨基酸含量变化显示天冬氨酸、谷氨酸、赖氨酸为多肽与金属离子的结合提供结合位点;热重分析结果表明,BHP与BHP-Fe在 300℃高温状态下依然能保持较好的稳定性;体外模拟消化稳定性分析结果表明,BHP-Fe相较于其它铁补充剂在胃肠道中能保持较高的稳定性.本实验制备的多肽源性补铁剂螯合力强、消化稳定性好、具有广阔应用前景,可为牛血副产物的高效利用和新型多肽源补铁剂的未来发展前景提供建设性参考.
Abstract
In this study,bovine blood was utilized as raw material,the extracted bovine hemoglobin was enzymolized,and then the most suitable enzymolysis protease was screened.Single factor combined response surface experiments were used to explore the optimal enzymolysis process.The structure of the prepared bovine hemoglobin peptide iron chelate(BHP-Fe)was characterized by technologies,such as scanning ultraviolet spectrum,Fourier infrared spectrum,scanning electron microscopy,and fully automated amino acid analyzer.Its stability in vitro was investigated through thermogravimetric analysis and simulated digestion of gastrointestinal tract in vitro.The results showed that the stepwise enzymatic hydrolysis of pepsin and alkaline protease was the most suitable enzymatic solution.Based on the preliminary enzymolysis of pepsin,optimal conditions for alkaline protease enzymolysis were obtained:A solid-liquid ratio of 1:3,an enzymolysis pH of 9.8,an enzymolysis temperature of 41℃,an enzyme dosage of 5900 U/g,and an enzymolysis time of 2 h.Under these conditions,the Fe2+ chelating capacity of bovine hemoglobin peptide(BHP)obtained through enzymatic hydrolysis was 72.11%,and the degree of bovine hemoglobin hydrolysis was 35.07%.The results of ultraviolet and Fourier infrared spectra showed that Fe2+ chelated with carboxyl oxygen and amino nitrogen on the BHP peptide chain,producing new substances different from polypeptides.Scanning electron microscopy revealed that the peptides changed from smooth fragments to rough patches after chelating Fe2+,and there were obvious differences between them.The change in amino acid content indicated that aspartate,glutamic acid,and lysine serve as binding sites for polypeptides and metal ions.Thermogravimetric analysis results demonstrated that BHP and BHP-Fe remain stable at high temperatures,maintaining good stability even at 300℃.The results of in vitro simulated digestive stability analysis showed that BHP-Fe could maintain higher stability in the gastrointestinal tract than other iron supplements.In conclusion,the polypeptide-derived iron supplement prepared in this experiment had strong chelating force,good digestive stability,and broad application prospects,which could provide constructive reference for the efficient utilization of bovine blood by-products and the future development prospect of new polypeptide-derived iron supplement.
关键词
牛血红蛋白肽/响应面优化/肽铁螯合物/结构表征/体外模拟消化Key words
bovine hemoglobin peptide/response surface optimization/peptide iron chelate/structural characterization/in vitrosimulated digestion引用本文复制引用
基金项目
现代农业产业技术体系建设项目(CARS-37)
国家自然科学基金(31701626)
出版年
2024
国家科技期刊平台
NSTL
万方数据