【作者】 张晋博；

【导师】 杨晓泉；

【作者基本信息】 华南理工大学 ， 粮食、油脂及植物蛋白工程， 2013， 博士

【摘要】 多糖对蛋白质的修饰能够改善蛋白质的多种功能性质，可以作为功能配料应用于食品及医药领域，已经受到越来越多的关注。多糖与蛋白质发生相互作用的主要方式有共价接枝和静电相互作用。本文采用高温、短时干热法制备了大豆分离蛋白-麦芽糊精糖基化产物，研究了糖基化产物的乳化性质，为工业化生产蛋白质-多糖糖基化产物提供了一定的理论依据。利用Maillard反应和限制性酶解对大豆蛋白进行两亲性修饰，得到了具有两亲性结构的接枝共聚物，并且考察了其界面和乳化性质。采用反溶剂法制备了大豆蛋白-葡聚糖糖基化产物的纳米颗粒，研究了纳米颗粒的结构、界面和乳化性质。制备了大豆蛋白-葡聚糖糖基化产物凝胶，考察了凝胶的流变学和质构性质。利用蛋白质与多糖的静电相互作用，制备了大豆蛋白/甜菜果胶可溶性复合物，分析了复合物稳定水包油型乳液的机理。本文通过多糖对蛋白质的修饰作用来提高大豆蛋白的界面、乳化和凝胶性质，为其作为功能性配料应用于食品工业提供理论指导。主要研究结果如下：（1）采用高温（90、115和140℃）、短时（2h）干热Maillard反应制备了大豆分离蛋白（SPI）-麦芽糊精（MD）糖基化产物样品。利用蛋白质中游离氨基减少的数量和SDS-PAGE证实了糖基化产物的生成，并且发现提高热处理的温度可以增加糖基化产物的接枝度。与SPI、SPI-MD糖基化产物（90和115℃）稳定的水包油型乳液相比，SPI-MD糖基化产物（140℃）稳定的乳液对于pH的改变，离子强度和热处理，均表现出较高的稳定性。这是由于SPI-MD糖基化产物（140℃）具有较高的接枝度，较多的接枝的MD可以吸附到乳液液滴表面，为液滴间提供了较强的空间排斥作用，提高了乳液的稳定性。（2）采用胰蛋白酶对β-伴大豆球蛋白（7S）-葡聚糖糖基化产物进行水解，得到了水解度（DH）为2.2%和6.5%的糖基化产物水解物。与7S、7S-葡聚糖糖基化产物和DH为6.5%的糖基化产物水解物相比，DH为2.2%的7S-葡聚糖糖基化产物水解物在油-水界面具有较高的界面压和吸附量。这是由于限制性酶解可以使蛋白质分子展开，暴露出部分疏水基团，导致DH为2.2%的7S-葡聚糖糖基化产物水解物具有较高表面疏水性。与7S、7S-葡聚糖糖基化产物和DH为6.5%的糖基化产物水解物稳定的乳液相比，DH为2.2%的7S-葡聚糖糖基化产物水解物稳定的乳液对于环境因素（pH、离子强度和热处理）的改变，均表现出较高的稳定性，经过4周的储藏后，未出现明显的乳析现象。这是因为DH为2.2%的7S-葡聚糖糖基化产物水解物具有较高的吸附量，较多的接枝的葡聚糖增强了乳液液滴间的空间排斥作用，抑制了乳液的聚集和絮凝。（3）利用反溶剂法制备了7S、7S-葡聚糖糖基化产物和DH为2.2%的糖基化产物水解物的纳米颗粒。动态光散射、小角X-光散射和透射电镜结果显示，所有的纳米颗粒均为球形，并且DH为2.2%的7S-葡聚糖糖基化产物水解物纳米颗粒具有核壳结构。此外，与7S、7S-葡聚糖糖基化产物及其纳米颗粒相比，DH为2.2%的7S-葡聚糖糖基化产物水解物纳米颗粒在油-水界面具有较高的界面压和膨胀模量。这是由于DH为2.2%的7S-葡聚糖糖基化产物水解物纳米颗粒具有较高的表面疏水性。DH为2.2%的7S-葡聚糖糖基化产物水解物纳米颗粒稳定的水包油型乳液具有较高的储藏稳定性，经过30天的储藏后，乳液的微观结构未发生明显的变化。（4）研究了微生物转谷氨酰胺酶（MTGase）交联7S-葡聚糖糖基化产物的凝胶性。实验发现，与7S、干热7S和7S-葡聚糖混合物形成的凝胶相比，7S-葡聚糖糖基化产物具有较高的储存模量和损耗模量，并且具有较高的硬度、脆性、弹性和内聚性。7S-葡聚糖糖基化产物中的接枝的葡聚糖可以抑制MTGase交联过程中蛋白质分子间发生过度的相互作用，导致形成更加有序的和较强的凝胶网络结构。（5）利用MTGase和漆酶（La）交联7S/甜菜果胶（SBP）可溶性复合物，制备了7S-SBP-MTGase、7S-SBP-La和7S-SBP-MTGase-La三种交联产物。实验发现，与7S-SBP、7S-SBP-MTGase和7S-SBP-La稳定的水包油型乳液相比，7S-SBP-MTGase-La稳定的乳液对于添加盐离子和热处理，均表现出较高的稳定性，经过1个月的储藏后，未发生明显的乳析现象。这是由于7S/SBP相继经过MTGase和La的交联后，可以在乳液液滴表面形成具有层层沉积结构的界面膜，增强了液滴间的空间排斥作用，提高了乳液对盐离子和热处理的稳定性。更多还原

【Abstract】 The functional properties of proteins could be improved by modification ofpolysaccharides. As functional material, protein modified by polysaccharide have gained wideattention because of their potential application in the field of food and medicine.Protein-polysaccharide interactions mostly originated from conjugation and electrostaticinteractions. Soy protein isolate (SPI)-maltodextrin (MD) conjugates were prepared byhigh-temperature, short-time dry-heating Maillard reaction, and the emulsifying properties ofSPI-MD conjugates were investigated in this study, which will provide theoretic supports forindustrialized production of protein-polysaccharide conjugates. The amphiphilic graftcopolymers were synthesized using Maillard reaction and controlled enzymatic hydrolysis.The interfacial and emulsifying properties of the amphiphilic graft copolymers were analyzed.Nanoparticles were prepared from soy protein-dextran conjugates by desolvation method. Thestructure, interfacial and emulsifying properties of the nanoparticles were investigated. Thetexture and gelation properties of soy protein-dextran conjugates gels were investigated. Thesoluble complexes of soy protein/sugar beet pectin (SBP) were formed by electrostaticinteractions. The stability mechanism of emulsions stabilized by soy protein/SBP complexeswas also investigated. The main results are as follows:(1) SPI-MD conjugates were synthesized using Maillard reaction under high-temperature(90,115and140°C), short-time (2h) dry-heating conditions. The loss of free amino groups inproteins and SDS-PAGE profile confirmed that SPI-MD conjugates were formed and higherdry-heated temperatures could increase the glycosylation degree. The emulsifying propertiesof SPI and SPI-MD conjugates were evaluated in oil-in-water emulsions. The emulsionsstabilized with SPI-MD conjugates (synthesized at140°C) exhibited higher emulsifyingstability and excellent storage stability against pH, ionic strength and thermal treatmentcompared with SPI-MD conjugates (synthesized at90,115°C), and SPI stabilized emulsions.This might be due to a greater proportion of conjugated MD in SPI-MD conjugates(synthesized at140°C) because of the higher glycosylation degree, and more conjugated MDon the droplet surface could provide steric effect and enhance the stability of the droplets inthe emulsions. (2) A emulsifier was prepared by conjugating soy β-conglycinin and dextran underdry-heated Maillard reaction followed by trypsin hydrolysis with the DH at2.2%and6.5%.Hydrolysates of β-conglycinin-dextran conjugates DH2.2%had a much higher interfacialpressure and fraction of protein adsorption at the oil-water interface compared withβ-conglycinin, β-conglycinin-dextran conjugates and hydrolysates of β-conglycinin-dextranconjugates DH6.5%. This might be due to controlled enzymatic hydrolysis could induceprotein unfolding and hydrophobic regions exposed to exterior, which could lead to increasein surface hydrophobicity of hydrolysates of β-conglycinin-dextran conjugates DH2.2%.Hydrolysates of β-conglycinin-dextran conjugates DH2.2%were capable of forming a fineemulsion against the changes of pH, ionic strength and thermal treatment, which remainedstable during4weeks of storage compared with β-conglycinin, β-conglycinin-dextranconjugates and hydrolysates of β-conglycinin-dextran conjugates DH6.5%stabilizedemulsions. This might be due to hydrolysates of β-conglycinin-dextran conjugates DH2.2%had much higher fraction of protein adsorption, which could led to more conjugated dextranon the droplet surface and improve the steric repulsion between the droplets, thus dropletaggregation and flocculation were inhibited.(3) Nanoparticles were prepared from β-conglycinin, β-conglycinin-dextran conjugatesand hydrolysates of β-conglycinin-dextran conjugates DH2.2%by desolvation method. Allthe nanoparticles exhibited spherical structures, as evidenced by dynamic light scattering,small-angle X-ray scattering and transmission electron microscopy. In addition, thenanoparticles prepared from hydrolysates of β-conglycinin-dextran conjugates DH2.2%hadan obvious core-shell structure. Furthermore, the nanoparticles prepared from hydrolysates ofβ-conglycinin-dextran conjugates DH2.2%showed higher interfacial pressure anddilatational modulus during absorption at the oil-water interface compared with β-conglycininnanoparticles and β-conglycinin-dextran conjugates nanoparticles. Emulsions stabilized withnanoparticles prepared from hydrolysates of β-conglycinin-dextran conjugates DH2.2%hadhigher emulsion stability after30days of storage. There was no significant change in themicrostructure of the emulsions stabilized with hydrolysates of β-conglycinin-dextranconjugates DH2.2%nanoparticles after30day of storage.(4) The gelation properties of β-conglycinin-dextran conjugates induced by MTGase was investigated. The gels of β-conglycinin-dextran conjugates exhibited higher G’, G’’, hardness,fracturability, springiness and cohesiveness values compared to those of dry-heatedβ-conglycinin, β-conglycinin and β-conglycinin-dextran mixture. The conjugated dextran inβ-conglycinin-dextran conjugates could inhibit extensive protein-protein interactions whichmight result in the formation of more ordered and stronger gel network structures duringMTGase cross-linking process.(5) β-conglycinin/SBP-MTGase, β-conglycinin/SBP-La and β-conglycinin/SBP-MTGase-La were prepared by MTGase and/or La cross-linking the soluble complexesof β-conglycinin/SBP. β-conglycinin/SBP-MTGase-La stabilized emulsions had higherstability against the changes of ionic strength and thermal treatment compared withβ-conglycinin, β-conglycinin/SBP-MTGase and β-conglycinin/SBP-La stabilized emulsions.This might be because that the soluble complexes of β-conglycinin/SBP were cross-linked byMTGase and/or La, which could form a layer by layer deposition interfacial film, and increaseemulsions stability against ionic strength and thermal treatment due to the steric repulsionbetween the oil drops.更多还原