基于计算机模拟与超声辅助酶法制备高生物利用度玉米蛋白的研究

发布时间：2018-06-16 02:46

本文选题：模拟酶切 + 超声波　；参考：《江苏大学》2015年博士论文

【摘要】：玉米蛋白是湿法生产玉米淀粉的副产物,约含70%的蛋白质,其独特的理化组成以及必需氨基酸的缺乏严重限制了其在食品工业中的应用。本文旨在通过酶法水解以提高玉米蛋白的生物利用度,通过玉米蛋白的超声波预处理提高其酶解效率和产物得率。本文开发了基于生物信息学平台的蛋白质酶切过程计算机模拟软件,利用该软件完成蛋白酶的自动筛选；根据筛酶结果,研究了不同酶解程度(水解度)下玉米蛋白生物利用度的变化规律及其分子机制；针对玉米蛋白存在因富含疏水性氨基酸而导致的溶解性低、结构致密这一分子结构缺陷,研究了双频功率超声波(聚能式、发散式)预处理对玉米蛋白酶解特性的影响；通过紫外光谱、荧光光谱、傅里叶变换红外光谱、圆二色谱、扫描电镜和原子力显微镜等多尺度分析方法和测试手段研究了双频功率超声波促进玉米蛋白酶解的作用机理；根据超声的作用机制和玉米蛋白的分子结构特征,采用米氏方程、一级动力学模型、阿伦尼乌斯方程以及过渡态理论研究了发散式双频扫频超声预处理对玉米蛋白酶解反应动、热力学参数的影响。本文得出以下主要研究结论：(1)开发出了可以满足蛋白质酶切基本过程模拟与结果分析的计算机软件,该软件能对蛋白质进行单(双)酶定向靶位酶切,能以指定的蛋白水解度为特征指标完成蛋白酶的自动筛选,并可以对酶切产物的分子量分布及其端点疏水性氨基酸的含量进行统计分析。(2)酶解反应显著提高了玉米蛋白的生物利用度,且提高幅度与蛋白水解度直接相关。体外模拟消化表明,水解度通过影响酶解液的多肽分子量分布和氨基酸组成来影响其生物利用度。酶解液中分子量大小为200-500 Da组分的含量几乎不受胃蛋白酶、胰蛋白酶的影响。随水解度的增大,游离疏水性氨基酸尤其Ala、Met、Ile、Val和Phe的含量增加,Thr和Leu含量减小；酶解液中的总氨基酸、疏水性氨基酸尤其是Leu、Pro和Ala含量随水解度的增大而增加。氨基酸消化率表明,酶解液中疏水性氨基酸Met、Ile、Pro、Ala的消化率显著下降,Thr的消化率显著上升,水解度对亲水性、疏水性氨基酸的平均消化率无显著性影响。(3)针对蛋白质的超声预处理,以水解度和蛋白转化率为指标,分别优化得出了聚能式和发散式两种功率超声波的最佳工作模式及其工作参数。与无超声的常规酶解相比,玉米蛋白经聚能顺序双频超声预处理后,其水解度和蛋白转化率分别提高了45.3%和59.3%；经发散双频扫频超声预处理后,其水解度和蛋白转化率分别提高了38.8%和54.7%。玉米蛋白经超声尤其是聚能顺序双频超声预处理后,其酶解液的分子量分布范围更窄,主要集中在200-1000 Da之间。(4)紫外光谱、荧光发射/激发光谱、表面疏水性结果表明,超声预处理能暴露出埋藏在分子内部的疏水性内核、使得蛋白质分子舒展,有利于酶解反应的进行。傅里叶变换红外光谱和圆二色谱分析结果表明,经超声预处理后,蛋白各二级结构(a-螺旋、β-折叠、β-转角和无规则卷曲)的相对含量发生了变化,且超声对玉米蛋白中的谷蛋白的作用强于对醇溶蛋白的作用。扫描电镜结果表明,尽管顺序双频处理后的醇溶蛋白出现了一定的聚集,但超声预处理能破坏蛋白的微观结构、减小颗粒的尺寸,增大酶与蛋白质的接触机会。亚显微结构及纳米机械力学特性分析表明,超声预处理能减小谷蛋白的颗粒高度和表面粗糙度,能减小醇溶蛋白的杨氏模量和刚度,增加蛋白的粘附力。发散式双频扫频超声适合于疏水性较强的蛋白预处理,而聚能式顺序双频超声适合于疏水性较弱的蛋白预处理。(5)动力学研究表明,玉米蛋白经双频扫频超声预处理后,其酶解反应的表观常数(KM值)降低了26.1%、表观崩解常数(kA值)增加了7.3%,说明超声处理后的玉米蛋白与酶的亲和力和结合频率得到提高；热力学分析表明,玉米蛋白经双频扫频超声预处理后,在293、303、313和323 K的反应温度下的酶解反应速率常数(k)分别提高了84.8%、41.9%、28.9%和18.8%,酶解反应的活化能(Ea)、活化焓(ΔH)、活化熵(△S)分别降低了23.0%、24.3%和25.3%,意味着酶解反应更容易进行。
[Abstract]:Corn protein is a by-product of corn starch production by wet method. It contains about 70% protein. Its unique physical and chemical composition and the lack of essential amino acids seriously restrict its application in the food industry. This paper aims to improve the bioavailability of corn protein by enzymatic hydrolysis and improve its enzymatic hydrolysis through ultrasonic pretreatment of corn protein. Efficiency and product yield. In this paper, a computer simulation software based on bioinformatics platform was developed. The software was used to screen the protease automatically. According to the results of the enzyme, the changes of the bioavailability of corn protein and its molecular mechanism under different degree of hydrolysis (degree of hydrolysis) were studied. There is a structural defect of low solubility and compact structure caused by rich hydrophobic amino acids. The effect of dual frequency power ultrasound (concentrated energy, divergent) pretreatment on the hydrolysis of corn protease is studied. Ultraviolet spectroscopy, fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular two chromatography, scanning electron microscopy and atomic force microscopy are used. The mechanism of dual frequency power ultrasound promoting the hydrolysis of corn protease is studied by multiscale analysis and testing. According to the mechanism of ultrasound and the molecular structure of corn protein, the Michaelis equation, the first order kinetic model, the Arrhenius equation and the over state theory are used to study the divergent dual frequency frequency sweep ultrasound. The effect of pretreatment on enzymatic hydrolysis of corn protease and the influence of thermodynamic parameters. This paper draws the following main conclusions: (1) a computer software can be developed to simulate the basic process simulation and result analysis of protein enzyme cutting. The software can cut the protein to target enzyme with a single (double) enzyme and can be characterized by the specified degree of protein hydrolysis. The index completes the automatic screening of protease, and can analyze the molecular weight distribution of the enzyme cut products and the content of the hydrophobic amino acids in the endpoint. (2) the enzymatic hydrolysis reaction significantly improves the bioavailability of the corn protein, and the increase in the amplitude is directly related to the degree of protein hydrolysis. The molecular weight distribution and amino acid composition of the polypeptide affect its bioavailability. The content of the molecular weight of 200-500 Da in the hydrolysate is almost unaffected by pepsin and trypsin. With the increase of the degree of hydrolysis, the content of free hydrophobic amino acids especially Ala, Met, Ile, Val and Phe is increased, the content of Thr and Leu is reduced; the content of Thr and Leu is reduced; the content of Thr and Leu is reduced; the content of Thr and Leu is reduced; The content of total amino acids, hydrophobic amino acids, especially Leu, Pro and Ala increased with the increase of the degree of hydrolysis. The digestibility of the amino acid digestibility in the hydrolysate showed that the digestibility of Met, Ile, Pro, Ala decreased significantly, the digestibility of Thr increased significantly, and the degree of hydrolysis had no significant influence on the average digestibility of hydrophobicity and hydrophobic amino acids. (3) needle The optimum working mode and its working parameters of two kinds of power ultrasound were optimized, including the degree of hydrolysis and the conversion rate of protein. Compared with the conventional enzymatic hydrolysis without ultrasonic, the hydrolysis degree and the protein conversion rate of corn protein were respectively compared with that of the two frequency ultrasonic pretreatment. After 45.3% and 59.3% higher, the hydrolysis degree and protein conversion rate increased by 38.8% and 54.7%. corn protein after ultrasonic pretreatment, and the molecular weight distribution of the hydrolysate was narrower, mainly between 200-1000 Da. (4) ultraviolet spectrum, fluorescence emission / excitation. The results of spectrum and surface hydrophobicity show that ultrasonic pretreatment can expose the hydrophobic core that is buried inside the molecule, which makes the protein molecules stretch and facilitate the enzymatic reaction. Fourier transform infrared spectroscopy and circular two chromatographic analysis show that after ultrasound pretreatment, the two structure of protein (a- spiral, beta fold, beta corner and) The relative content of irregular curling changed, and the effect of ultrasound on glutenin in corn protein was stronger than that of gliadin. The results of scanning electron microscopy showed that although a certain concentration of gliadin appeared after the sequence double frequency treatment, the ultrasonic pretreatment could destroy the microstructure of the protein, reduce the size of the particles, and increase the enzyme. The analysis of the microstructure and mechanical mechanical properties of the protein shows that the ultrasonic pretreatment can reduce the grain height and the surface roughness of the protein, reduce the young's modulus and stiffness of the gliadin, and increase the adhesion of the protein. The energetic sequence dual frequency ultrasound is suitable for weak hydrophobic protein pretreatment. (5) kinetic studies show that the apparent constant (KM value) of the enzymolysis reaction of corn protein decreased by 26.1% and the apparent disintegration constant (kA value) increased by 7.3%, indicating the affinity and binding frequency of the corn protein with the enzyme after ultrasonic treatment. The thermodynamic analysis showed that the enzyme hydrolysis rate constant (k) increased by 84.8%, 41.9%, 28.9% and 18.8% at the reaction temperature of 293303313 and 323 K, and the activation energy (Ea), activation enthalpy (delta H), and the activation entropy (delta S) decreased by 23%, 24.3% and 25.3% respectively. The enzymolysis reaction is more easily carried out.
【学位授予单位】：江苏大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：TS239;TQ936.2

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