酶交联及乙醇去溶剂化法制备乳清蛋白—锌纳米微粒及其特性研究
发布时间：2018-11-22 16:58
【摘要】：本研究内容隶属于国家“十二五”科技支撑计划项目《乳清蛋白应用技术与开发(2013BAD18B07)》。本研究以乳清蛋白为原料,采用转谷氨酰胺酶酶交联作用,在中性条件下与七水硫酸锌结合,再通过乙醇去溶剂化法进一步优化制备乳清蛋白-锌纳米微粒。通过对制备条件的优化,得到装载率较高、稳定性及各方面性质良好的乳清蛋白-锌纳米微粒。随后,本文对制备的乳清蛋白-锌纳米微粒的二级结构、热变性、外观形态学特性、体外消化特性、细胞毒性以及贮藏稳定性等进行了研究。具体研究结论如下:(1)在酶交联法制备乳清蛋白-锌纳米分散液的技术研究中,本研究分别考察了乳清蛋白浓度、交联时间、加酶量、锌加入量对制备的乳清蛋白-锌纳米分散液的粒径、Zeta电位、装载率、单位蛋白载锌量及浊度等因素的影响。结果显示:当乳清蛋白浓度为5.0%、交联时间为4 h,加酶量为5.0 U/g、锌加入量为5.0 m M时,制备的乳清蛋白-锌纳米微粒的效果较好,其粒径大小为100.76±2.17nm,Zeta电位为-29.11±1.96 m V,装载率可达到54.56±3.19%,单位蛋白载锌量为8.28±0.48μg/mg。(2)在乙醇去溶剂化法优化乳清蛋白-锌纳米微粒的制备技术研究中,本研究分别考察了蛋白与乙醇加入比、锌加入量及加酶量对制备的乳清蛋白-锌纳米微粒的粒径、装载率及单位蛋白载锌量的影响。结果显示:当底物与乙醇加入体积比为1:4,锌加入量为5.0 m M,加酶量为5.0 U/g时,制备的乳清蛋白-锌纳米微粒的效果较好,其粒径大小为150.83±5.15 nm,装载率可达到99.22±3.31%,单位蛋白载锌量为10.43±0.27μg/mg。(3)在对酶交联及乙醇去溶剂化法制备的乳清蛋白-锌纳米微粒的特性进行研究中,结果显示:酶交联及乙醇去溶剂化处理后乳清蛋白的二级结构发生了改变。形成的乳清蛋白-锌纳米微粒在扫描电镜下呈现圆球状,粒度大小为100 nm左右,分布较为均匀。乳清蛋白经上述方法制备形成纳米微粒后,其热变性有所提高。乳清蛋白-锌纳米微粒在模拟胃液中锌的释放率较低,而在肠液中较高。细胞毒性试验结果表明,当乳清蛋白-锌纳米微粒浓度为0.1875 mg/m L时,对细胞作用24 h,乳清蛋白-锌纳米微粒对细胞无潜在毒性作用。贮藏期试验表明,制备的乳清蛋白-锌纳米微粒在25℃条件下,14 d内的稳定性良好。本研究表明通过转谷氨酰胺酶的交联及乙醇去溶剂化相结合的方法能够制备一种锌负载率较高、稳定性良好的乳清蛋白-锌纳米微粒,这有可能为解决锌吸收率低的问题提供了一种新的思路。
[Abstract]:The content of this study belongs to the National Science and Technology support Project of the Twelfth Five-Year Plan, whey protein Application Technology and Development (2013BAD18B07). In this study, whey protein was used as raw material, transglutaminase crosslinking was used to combine with zinc sulfate heptahydrate under neutral conditions, and then further optimized the preparation of whey protein-zinc nanoparticles by ethanol desolvation. By optimizing the preparation conditions, high loading rate, good stability and various properties of whey protein-zinc nanoparticles were obtained. Subsequently, the secondary structure, thermal denaturation, appearance morphology, in vitro digestion, cytotoxicity and storage stability of the prepared whey protein-zinc nanoparticles were studied. The specific conclusions are as follows: (1) in the preparation of whey protein-zinc nano-dispersion by enzyme crosslinking, the concentration of whey protein, the crosslinking time and the amount of enzyme were investigated. The effects of zinc addition on the particle size, Zeta potential, loading rate, zinc load per unit protein and turbidity of the prepared whey protein-zinc nanoparticles were investigated. The results showed that when the concentration of whey protein was 5.0%, the crosslinking time was 4 h, the amount of enzyme added was 5.0 U / g, and the amount of zinc was 5.0 mm, the effect of the prepared whey protein-zinc nanoparticles was better. The particle size is 100.76 卤2.17nmGV, the Zeta potential is -29.11 卤1.96mV, and the loading rate is 54.56 卤3.19mV, and the Zeta potential is -29.11 卤1.96mV. Zinc content per unit protein was 8.28 卤0.48 渭 g / mg 路(2). (2) in the optimization of preparation of whey protein-zinc nanoparticles by ethanol desolvation, the ratio of protein to ethanol was studied. The effects of zinc addition and enzyme addition on the particle size, loading rate and zinc content per unit protein of the prepared whey protein-zinc nanoparticles. The results show that when the volume ratio of substrate to ethanol is 1: 4, the amount of zinc added is 5.0 mm and the amount of enzyme is 5.0 U / g, the effect of the prepared whey protein-zinc nanoparticles is better, and the particle size is 150.83 卤5.15 nm,. The loading rate was 99.22 卤3.31 and the zinc load per unit protein was 10.43 卤0.27 渭 g / mg 路(3) in the study of the properties of whey protein-zinc nanoparticles prepared by enzymatic crosslinking and ethanol desolvation. The results showed that the secondary structure of whey protein changed after enzymatic crosslinking and ethanol desolvation. The resulting whey protein-zinc nanoparticles were spherical under scanning electron microscope with a particle size of about 100 nm and a uniform distribution. The thermal denaturation of whey protein was improved after the preparation of nano-particles by the above method. The release rate of zinc in simulated gastric juice was lower than that in intestinal fluid. The results of cytotoxicity test showed that when the concentration of whey protein-zinc nanoparticles was 0.1875 mg/m / L, the leukoprotein-zinc nanoparticles had no potential toxicity to cells at 24 h. The storage test showed that the stability of the prepared whey protein-zinc nanoparticles was good at 25 鈩，
本文编号：2349955
[Abstract]:The content of this study belongs to the National Science and Technology support Project of the Twelfth Five-Year Plan, whey protein Application Technology and Development (2013BAD18B07). In this study, whey protein was used as raw material, transglutaminase crosslinking was used to combine with zinc sulfate heptahydrate under neutral conditions, and then further optimized the preparation of whey protein-zinc nanoparticles by ethanol desolvation. By optimizing the preparation conditions, high loading rate, good stability and various properties of whey protein-zinc nanoparticles were obtained. Subsequently, the secondary structure, thermal denaturation, appearance morphology, in vitro digestion, cytotoxicity and storage stability of the prepared whey protein-zinc nanoparticles were studied. The specific conclusions are as follows: (1) in the preparation of whey protein-zinc nano-dispersion by enzyme crosslinking, the concentration of whey protein, the crosslinking time and the amount of enzyme were investigated. The effects of zinc addition on the particle size, Zeta potential, loading rate, zinc load per unit protein and turbidity of the prepared whey protein-zinc nanoparticles were investigated. The results showed that when the concentration of whey protein was 5.0%, the crosslinking time was 4 h, the amount of enzyme added was 5.0 U / g, and the amount of zinc was 5.0 mm, the effect of the prepared whey protein-zinc nanoparticles was better. The particle size is 100.76 卤2.17nmGV, the Zeta potential is -29.11 卤1.96mV, and the loading rate is 54.56 卤3.19mV, and the Zeta potential is -29.11 卤1.96mV. Zinc content per unit protein was 8.28 卤0.48 渭 g / mg 路(2). (2) in the optimization of preparation of whey protein-zinc nanoparticles by ethanol desolvation, the ratio of protein to ethanol was studied. The effects of zinc addition and enzyme addition on the particle size, loading rate and zinc content per unit protein of the prepared whey protein-zinc nanoparticles. The results show that when the volume ratio of substrate to ethanol is 1: 4, the amount of zinc added is 5.0 mm and the amount of enzyme is 5.0 U / g, the effect of the prepared whey protein-zinc nanoparticles is better, and the particle size is 150.83 卤5.15 nm,. The loading rate was 99.22 卤3.31 and the zinc load per unit protein was 10.43 卤0.27 渭 g / mg 路(3) in the study of the properties of whey protein-zinc nanoparticles prepared by enzymatic crosslinking and ethanol desolvation. The results showed that the secondary structure of whey protein changed after enzymatic crosslinking and ethanol desolvation. The resulting whey protein-zinc nanoparticles were spherical under scanning electron microscope with a particle size of about 100 nm and a uniform distribution. The thermal denaturation of whey protein was improved after the preparation of nano-particles by the above method. The release rate of zinc in simulated gastric juice was lower than that in intestinal fluid. The results of cytotoxicity test showed that when the concentration of whey protein-zinc nanoparticles was 0.1875 mg/m / L, the leukoprotein-zinc nanoparticles had no potential toxicity to cells at 24 h. The storage test showed that the stability of the prepared whey protein-zinc nanoparticles was good at 25 鈩，
本文编号：2349955
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