大豆蛋白凝胶结构与非网络蛋白扩散行为或网络蛋白性质关系的研究

发布时间：2018-12-15 05:42

【摘要】：凝胶性是大豆蛋白最突出的功能性质之一,为食品体系贡献弹性、硬度、持水性、风味成分保留等功能。目前,凝胶型大豆蛋白在肉制品、千叶豆腐、鱼糜制品等产品中应用广泛。认识凝胶结构、了解凝胶结构和性质的关系,对凝胶型大豆蛋白产品的开发和应用均有理论和实际指导意义。本论文从确定大豆蛋白凝胶中网络蛋白和非网络蛋白的组成入手,通过研究非网络成分的扩散行为以及凝胶的质构、流变和微观结构特性,对凝胶网络结构进行了表征;同时通过测定蛋白质的组成、网络蛋白的浓度和网络蛋白之间相互作用力的变化,揭示了凝胶网络结构变化的原因。首先,采用扩散法对凝胶中非网络蛋白和网络蛋白进行了分离,通过不同电泳技术对两类蛋白质的组成及存在形式进行了分析。以18%(w/v)蛋白质溶液在0.1 nol/L盐离子浓度和95℃条件加热30 min形成的大豆蛋白凝胶中的非网络蛋白为代表,其主要组成是11S的A、B肽链和胰凝乳蛋白酶抑制剂(BBI),此外含有少量的7S-α’、α亚基。其中B肽链以AB和A5B3的形式存在;A肽链除了这两种形式外,还包含单体、二聚体、三聚体和聚合体等形式;BBI以单体形式存在。针对网络蛋白组成的研究,讨论中结合分析了三种蛋白凝胶中的网络蛋白。其中,7S-α、α’亚基通过形成聚集体和多聚体两种形式参与到凝胶网络结构中;7S-β亚基是以聚集体的形式参与到凝胶网络结构中。11S-A3、A肽链主要以二硫键连接的聚合体的形式参与到凝胶网络结构中,而11SB肽链可以以单体、二聚体、聚合体、聚集体等形式参与到凝胶网络中。在此基础上,继续研究了非网络蛋白的扩散行为和凝胶结构变化的关系;并通过分析非网络蛋白和网络蛋白的组成及含量的变化,揭示了凝胶网络结构变化的原因。排阻色谱分析表明非网络蛋白由三部分组成,相对分子质量分别为253.9、44.8和9.7 kDa;三种成分在起始阶段的扩散符合Fick第二定律。制备凝胶时的蛋白质浓度增加或加热时间延长可引起非网络蛋白扩散系数的降低。扩散系数与相对分子质量之间符合幂次函数关系,其中特征指数因子α值随着蛋白质浓度增加、加热时间延长而变大,说明凝胶网络结构趋于致密。电泳和蛋白质浓度分析结果表明,11S蛋白变性程度和网络蛋白含量的增加是导致凝胶网络结构致密的原因。进一步通过研究外源性分子探针的扩散考察了7S/11S比率和盐浓度对凝胶网络结构的影响;并采用扫描电子显微镜对扩散实验结果进行了验证。在同一条件下制备的蛋白质凝胶中,探针的相对分子质量越大扩散系数越小;在不同的蛋白质凝胶中,探针相对分子质量越大,其扩散对凝胶网络结构的变化越敏感。凝胶中探针的扩散系数随着盐离子浓度或总蛋白中11S比率的增加而增大;扫描电子显微镜数据结果显示,随着盐离子浓度或11S比率增加,凝胶网络结构孔隙变大、蛋白质聚集体分布不均匀,导致探针在网络结构中容易扩散。通过测定凝胶在SDS溶液或SDS和DTT混合溶液中溶解速率,对凝胶网络蛋白中分子间作用力的类型和大小进行了表征,并与凝胶破裂力的测定结果进行了比较。结果表明,疏水、氢键相互作用和二硫键均参与了大豆蛋白凝胶网络结构的形成;作用力强弱的变化与凝胶破裂力大小的变化呈正相关。随着总蛋白中11S比率增加或加热温度升高,凝胶中疏水、氢键相互作用和二硫键均加强,凝胶破裂力的值逐渐增大;随着加热时间的延长,网络蛋白分子间相互作用和凝胶破裂力均呈现先增大后减小的变化趋势。此外,对比发现加热温度对三个作用力的影响最大,加热温度变化对凝胶破裂力影响更为显著。最后研究了大豆蛋白凝胶的弹性模量与非网络蛋白、网络蛋白含量以及蛋白质聚集体大小的关系。通过扩散法去除凝胶中的非网络蛋白,对去除前后凝胶弹性模量进行了测定,发现非网络蛋白对凝胶弹性模量没有贡献。随着加热温度升高,11S蛋白变性程度增加,网络蛋白占总蛋白的比率增加,进而导致凝胶弹性模量呈指数性增加。盐离子浓度和7S/11S比率对凝胶弹性模量的影响不完全取决于网络蛋白占总蛋白比率,此时凝胶弹性模量与蛋白质聚集体的平均粒径呈正相关。
[Abstract]:Gelatin is one of the most outstanding functional properties of the soybean protein, which is the function of the food system's contribution to elasticity, hardness, water-holding property and flavor component retention. At present, the gel-type soybean protein is widely used in the products such as meat products, bean curd, minced fish products and the like. To understand the gel structure, to understand the relationship between the structure and properties of the gel, the development and application of the gel-type soybean protein product are both theoretical and practical. Based on the determination of the composition of the network protein and the non-network protein in the soybean protein gel, the gel network structure was characterized by studying the diffusion behavior of the non-network component and the properties of the texture, the rheological and the microstructure of the gel, and the composition of the protein was also determined. The change of the interaction force between the concentration of the network protein and the network protein reveals the cause of the change of the gel network structure. First, the non-network protein and the network protein in the gel were separated by the diffusion method, and the composition and the existing form of the two kinds of proteins were analyzed by different electrophoretic techniques. The non-network protein in the soy protein gel, which was formed by heating for 30 min at a concentration of 0.1 mol/ L and at 95.degree. C. with an 18% (w/ v) protein solution, was represented by a peptide chain of 11S and a chymotrypsin inhibitor (BBI), in addition to a small amount of 7S-1. ', sub-subunit. in which the B-chain is present in the form of AB and A5B3; in addition to these two forms, the peptide chain also contains the form of monomers, dimers, trimers, and polymers; and the BBI is present in the form of a monomer. In order to study the network protein composition, the network protein in three kinds of protein gel was analyzed in the paper. Of which, 7S-1, 1' the subunits are involved in the gel network structure by forming aggregates and multimers; the 7s-subunits are in the form of aggregates that are involved in the gel network structure, The 11SB peptide chain may be incorporated into the gel network in the form of monomers, dimers, polymers, aggregates, and the like. On this basis, the relationship between the non-network protein's diffusion behavior and the change of the gel structure was studied, and the reason of the change of the gel network structure was revealed by analyzing the composition and the content of the non-network protein and the network protein. The analysis of exclusion chromatography showed that the non-network protein was composed of three parts, and the relative molecular weight was 253.9, 42.8 and 9.7kDa, respectively. The diffusion of the three components in the initial stage was in accordance with the Fick's second law. The increase in protein concentration or prolonged heating time during the preparation of the gel may result in a decrease in the non-network protein diffusion coefficient. The relationship between the diffusion coefficient and the relative molecular mass is a power-order function, wherein the value of the characteristic index factor is increased with the increase of the concentration of the protein, the heating time is prolonged, and the gel network structure tends to be compact. The results of the analysis of electrophoresis and protein concentration show that the degree of denaturation of 11S protein and the increase of the content of the network protein are the cause of the dense structure of the gel network. The effect of the ratio of 7S/ 11S and the salt concentration on the structure of the gel was further investigated by the study of the diffusion of the exogenous molecular probe, and the results of the diffusion experiment were verified by the scanning electron microscope. In the protein gel prepared under the same condition, the greater the relative molecular mass of the probe and the smaller the diffusion coefficient of the relative molecular mass of the probe, the greater the relative molecular mass of the probe in different protein gels, the more sensitive the diffusion of the probe to the gel network structure. the diffusion coefficient of the probe in the gel is increased with the increase of the concentration of the salt ions or the 11S ratio in the total protein; the scanning electron microscope data show that as the concentration of the salt or the 11S ratio increases, the pore of the gel network structure becomes large, the distribution of the protein aggregates is not uniform, resulting in an easy diffusion of the probe in the network structure. The type and size of the intermolecular force in the gel network protein were characterized by measuring the dissolution rate of the gel in the SDS solution or SDS and the DTT mixed solution, and compared with the determination result of the gel breaking force. The results showed that both the hydrophobic, hydrogen bond interaction and the disulfide bond were involved in the formation of the network structure of the soybean protein gel; the change of the strength of the acting force was positively related to the change of the gel breaking force. As the 11S ratio in the total protein is increased or the heating temperature is increased, the hydrophobic, hydrogen bond interaction and the disulfide bond in the gel are enhanced, the value of the gel breaking force is gradually increased, and as the heating time is prolonged, The interaction between the network protein molecules and the gel breaking force exhibit a tendency to decrease after the first increase. In addition, the effect of the heating temperature on the three forces was found to be the largest, and the change of the heating temperature was more significant to the gel breaking force. The relationship between the elastic modulus of the soybean protein gel and the non-network protein, the content of the network protein and the size of the protein aggregates was studied. The non-network protein in the gel was removed by the diffusion method, and the elastic modulus of the gel before and after removal was measured, and the non-network protein was found to have no contribution to the elastic modulus of the gel. As the temperature of the heating increases, the degree of denaturation of the 11S protein increases, and the ratio of the network protein to the total protein is increased, resulting in an exponential increase in the elastic modulus of the gel. The effect of the salt ion concentration and the ratio of 7S/ 11S on the elastic modulus of the gel is not entirely dependent on the total protein ratio of the network protein, at which point the elastic modulus of the gel is positively related to the average particle size of the protein aggregate.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TS201.21

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