【作者】 廖兰；

【导师】 赵谋明；

【作者基本信息】 华南理工大学 ， 食品科学， 2012， 博士

【摘要】 小麦面筋蛋白是小麦淀粉生产的副产品,因水溶性差,限制了其在食品工业中的应用。盐酸脱酰胺是其改性方法中较为有效的方法之一,但存在着水解程度难控制、氨基酸异构化、氯丙醇残留等难以解决的技术问题。本文采用三种有机弱酸在湿热条件下脱酰胺改性小麦面筋蛋白,寻找对小麦面筋蛋白水解度低和明显改善小麦面筋蛋白功能特性的改性条件,研究湿热条件下,有机酸脱酰胺改性小麦面筋蛋白的结构功能变化的内在规律和作用机制,并应用超声技术进一步改善改性小麦面筋蛋白的功能特性,深入探讨湿热酸脱酰胺对小麦面筋蛋白酶解敏感性和酶解产物呈味特征的影响,以及在酸奶和鱼油缓释体系的应用。选用含不同羧基的醋酸、琥珀酸、柠檬酸,以盐酸作为参照,在121 oC脱酰胺改性小麦面筋蛋白。改性蛋白脱酰胺程度、蛋白回收率、水解程度变化表明较低浓度的有机酸,能够显著提高小麦面筋蛋白溶解性,增大其蛋白回收率,且其样品水解度均小于1%。研究得到最佳的湿热酸处理条件是湿热处理10min,酸浓度分别为:醋酸0.082 mol/L,琥珀酸0.042 mol/L,柠檬酸0.024 mol/L。SDS-PAGE、拉曼光谱、傅里叶红外光谱、氨基酸分析和功能特性分析表明:有机酸脱酰胺有效地伸展了小麦面筋蛋白的二级、三级结构,显著改善小麦面筋蛋白的功能特性,保持并提高了小麦面筋蛋白营养特性。改性蛋白的溶解性在pH3-10,特别在pH6.0时,比对照显著提高(p <0.05),等电点pI向碱性方向偏移。通过研究改性过程中小麦面筋蛋白的静电斥力、分子表面电荷、分子内作用力、分子量分布、蛋白二级结构、热特性和分子形貌的变化趋势,系统地分析了小麦面筋蛋白湿热脱酰胺改性过程中分子聚集行为的变化规律,结果表明:改性过程中,酸脱酰胺产生的分子静电斥力和热效应产生的聚集力相互博弈,麦谷蛋白与醇溶蛋白经历了聚集、解离同时进行的过程。蛋白不断增大静电斥力可能是导致蛋白聚集体粒径、构象突变的主要原因。热处理6min左右是两种作用力的平衡点。疏水键和氢键是改性过程中前6 min湿热最主要的聚集力,6min后共价键作用(二硫键)是分子聚集体的主要作用。通过粒度、电泳、傅里叶红外光谱、分子作用力变化分析等手段研究了小麦面筋蛋白透析前后分子结构和作用力的变化,发现透析后改性蛋白溶解性降低的原因是透析过程中,蛋白分子表面电荷减小,引发蛋白分子间的氢键作用,减小了蛋白分子和水的作用。合理的超声处理进一步增加了改性小麦面筋蛋白分子的柔韧性,暴露了包埋的疏水性基团,并使蛋白功能特性得到恢复。本研究得出超声促溶的最优工艺条件是:超声功率100 W,超声时间10 min,超声温度44 oC。在此条件下,小麦面筋蛋白氮溶指数达到77.28%。利用胰酶,研究了有机酸和盐酸脱酰胺改性处理对小麦面筋蛋白酶解特性的影响,分析和比较了酶解产物肽分子量分布、游离氨基酸及风味的变化,研究表明有机酸脱酰胺改性提高了小麦面筋蛋白酶解敏感性,提升了小麦面筋蛋白酶解液的风味及营养价值,其酶解产物风味变化与底物脱酰胺程度有密切的关系。研究了改性面筋蛋白在酸奶和鱼油释控体系的应用。改性小麦面筋蛋白可替代部分奶粉进行酸奶发酵,缩短了酸奶发酵时间。当替代量为3 %时,缩短8%的发酵时间,质构参数和感官评价结果良好。在鱼油缓释体系中,鱼油-小麦面筋蛋白微粒包埋率较高(为81.8%),超微结构观察显示鱼油小麦面筋蛋白微粒呈球形,囊壁较光滑致密,无裂缝和明显褶皱。包埋后鱼油具有良好的释控特性和贮存稳定性,延缓了鱼油在人体胃液中的释放,近50%鱼油进人小肠,其主要作用机理是通过氢键和疏水作用力,使鱼油填充于蛋白分子网络结构的空隙。更多还原

【Abstract】 Wheat gluten is a byproduct of the wheat starch industry. The water-solubility of it is strongly limited so that its applications are very narrow in the food-processing. Hydrochloric acid was one of the most effective acid catalyst used frequently for protein deamidation. The disadvantages of deamidation on proteins with it, such as the uncontrollable hydrolysis degree, the production of potentially carcinogenic compounds, and the isomerization of certain amino acids in proteins, have yet to be solved. The aim of the present study was to modify wheat gluten by carboxylic acid deamidation upon hydrothermal treatment. The degree of hydrolysis, functional and conformational properties of deamidated wheat gluten (DWG) were evaluated. Optimal conditions and some intrinsic mechanisms of deamidation on wheat gluten with organic acids upon hydrothermal treatment were studied. Protective effect of DWG after dialysis subjected to ultrasound treatment was also investigated. Moreover, susceptibility of wheat gluten to enzymatic hydrolysis following deamidation and sensory characteristics of the resultant hydrolysates, in together with the applications of DWG on yoghourt and drug controlled delivery system were evaluated.Three carboxylic acids (acetic acid, succinic acid and citric acid) were used to deamidate wheat gluten with reference samples investigated of wheat gluten treated with HCl. Deamidation conditions were optimized. Changes in deamidation degree, hydrolysis degree, nitrogen soluble index, the foaming and emulsification properties, the tertiary and secondary conformation and nutritional property of wheat gluten deamidated with carboxylic acids were identified. Carboxylic acids were found to effectively deamidate the amides in wheat gluten proteins into carboxyl groups, which resulted in a significant increase of the nitrogen soluble index of wheat gluten. Deamidation of wheat gluten by carboxylic acids was found to be more efficient than that with HCl and significantly improved functional properties of wheat gluten. Wheat gluten deamidated with carboxylic acid exhibited improved flexible protein molecules, favorable changes in the tertiary and secondary structures and better nutritional characteristics. The solubility of wheat gluten after carboxylic acid deamidation increased in the isoelectric region of untreated wheat gluten (around pH 6.0). The isoelectricpoint of these proteins was shifted to a basic pH and existed in a broad pH regions. Changes of aggregation behavior of wheat gluten during carboxylic acid deamidation upon hydrothermal treatment were investigated to test the influences of deamidation on the aggregation extent of wheat gluten. Hydrothermal treatment induced that the size of soluble wheat gluten aggregate progressively increased due to the cross-linking of gliadins and slowly cleaved glutenins. But significant changes in molecular weight distribution, solubility under six denaturing agents’treatment and Zeta potential of wheat gluten aggregates were observed at 6 min heating time and distinct shift of intra-/inter-molecular interactions of wheat gluten aggregates occurred before and after 6 min heating treatment respectively. Moreover, as heating time increased, the island-like aggregates decreased markedly and the striped aggregates increased notably. To explain the aggregation behavior in this case, we postulated that the extent of aggregation of wheat gluten depended on the balance between intra-/inter-molecular electrostatic repulsion, the non-covalent and disulfide bonds formation in the system. Hence, a scheme was drawn, which appeared to be the mechanism responsible for the aggregation of wheat gluten through thermal cross-linking and opening up of the network structure of wheat gluten aggregates by deamidation.The reasons for the decreasing of solubility of DWG during dialysis were investigated. Results from DLS, SDS-PAGE, FTIR, morphology and molecular forces change of DWG during dialysis showed the increasing of hydrogen bonds due to the decreasing of protein surface potential increased the interaction of proteins. The effect of sonication factors, including sonication power percentage, process time and temperature, on the soluble nitrogen index (NSI) of succinic acid deamidated wheat gluten (SDWG) after dialysis was investigated. On base of the results of univariate factor analysis upon sonication, response surface analysis was used to optimize the process condition according to the response of NSI of SDWG. The optimal condition of sonication power percentage, treating time and sonication temperature obtained was 100 W, 10 min and 44℃, respectively, where the NSI of sample treated reached 77.28%. Changes of functional properties of the protein in SDWG upon sonication were determined. The percentages of enhancement in the foaming capacity and stability of sample after sonication were 11% and 20%, respectively. And a similar trend of significant growth in emulsifying properties of samples was also observed.Investigation on applications of DWG on yoghourt and controlled delivery system showed that the ferment time of yoghourt decreased after the addition of DWG, and it was with a good textile and sensory evaluation at the DWG addition percentage of 3% for total casein amount. For the application in the field of fish oil (FO) controlled delivery system, the most efficient condition was obtained at 50.4 mg/ml for SDWG and 3:3 (w/w) for FO/SDWG ratio, with an EE of 81.8%. In this condition, confocal microscopy showed FO well encapsulated in SDWG microspheres. SEM showed sunken pores and fractures inside microspheres after FO was extracted, confirming the presence of FO in microspheres. FTIR and electrophoresis showed during microspheres formation dramatically elevated SWDG aggregation resulted in intermolecular-crosslinking and enhanced interactions (hydrogen bonds and hydrophobic interactions) between SDWG and FO. In the evaluations of in vitro experiments in simulated gastric fluid and oxidation stability during storage, results indicated that SDWG matrix protected it from both oxygen and gastric fluid, resulting in improved storage stability and release property.更多还原