【作者】 刘凤茹；

【导师】 陈正行；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2014， 博士

【摘要】 矿物元素钙对人体健康非常重要，膳食中缺乏钙元素会导致多种疾病。金属螯合肽在改善矿物元素缺乏上具有潜在应用价值，目前越来越多的具有促进和提高矿物元素生物利用度的金属螯合肽被发现和鉴定。食物源螯合肽因其营养、安全已经被用于钙强化相关的功能食品和保健品的开发。本研究采用蛋白酶水解麦胚蛋白释放出具有钙离子螯合活性的麦胚肽，可为相关食物源螯合肽的研究和开发提供实验依据。为了获得麦胚蛋白的最佳提取方案，论文采用含有2个独立变量（溶解pH值和沉淀pH值）的中心组合设计模型和双变量相关分析同时考察了碱溶和酸沉pH值对麦胚蛋白得率和功能性质的影响及其相关性。结果表明，碱溶pH值和酸沉pH值对麦胚蛋白得率影响均显著（p<0.05），且碱溶pH值与得率（r=0.785，p=0.000）、吸油性（r=0.752，p=0.000）、乳化性（r=0.697，p=0.001）均呈正线性相关；酸沉pH值与吸水性（r=-0.863，p=0.000）呈负相关；麦胚蛋白各功能性质中仅吸油性与乳化性（r=0.753，p=0.000）、吸水性与起泡性（r=0.503，p=0.034）正线性相关；得率只与吸油性（r=0.820，p=0.000）、乳化性（r=0.683，p=0.002）呈正线性相关，其余不显著；当S/P=9.5/4.0（S=碱溶pH值，P=酸沉pH值）时，麦胚蛋白具有最高得率值36.64%，同时泡沫稳定性表现出最大值91.67%，但最大吸水性（3.99g g-1）出现在S/P=10.2/3.3，吸油性、乳化性和起泡性（分别为2.98g g-1，0.207，150%）的最大值在S/P=9.5/3.0，乳化稳定性（76.09%）的最大值则在S/P=10.5/4.0。这使得加工者可以在不显著影响产量的前提下通过适当调节pH值来获得实际所需功能特性的分离蛋白。利用金属盐控制蛋白质聚集体的形成来提高溶液的溶解性对于蛋白质在食品中的应用是非常重要的。实验考察了麦胚球蛋白（WGG）在酸化、热处理和Ca2+诱导条件下的聚集-解聚集、溶解性、表面特性和蛋白微结构性质的变化。得到如下结论：麦胚球蛋白溶液在pH5.0（即等电点，pI）和95℃热处理时，具有最大浊度（A400=1.3）和最小溶解性（20.3%）；加入50mmol/L CaCl2至WGG的分散液中，溶解度呈现先增加和后下降的趋势：相对于不加钙的WGG溶液，pH<pI（pH5.0~1.0）时溶解度从38.9%提高到76.3%，Ca2+诱导解聚集；而对于pH>pI （pH6.0~9.0）的溶解度从20.3%降低到12.2%，Ca2+促进聚集；不同热处理（55~95℃，pH2.0）条件下，浊度随加热温度的升高而显著增加（p<0.05）；加入50mmol/L CaCl2后，溶解度显著提高（p<0.05）；在pH（pH1.0~5.0）和酸热（pH2.0，55~95℃）处理条件下的解聚集指数（代表聚合物解聚集程度）均是一个恒定值y=0.5；Ca2+浓度（100mmol/L）对溶解度影响显著（p<0.05），由95℃时对照样品（不加Ca2+）的26.9%提高到66.8%；麦胚球蛋白聚集-解聚集过程中的疏水性（H0）、δ-电位和蛋白微观形态（SEM）变化结果表明，WGG由粗糙的不规则大团块降解为均匀分散的微粒，加入二价阳离子Ca2+可以通过增强静电斥力促进蛋白聚集体的解离。以麦胚蛋白为原料，利用蛋白酶限制性酶解技术和金属离子螯合技术制备麦胚肽-钙络合物，并采用现代分析技术如紫外-可见（UV-Vis）光谱法和傅立叶变换红外（FTIR）光谱来表征该络合物的结构特性。由碱性蛋白酶（Alcalase2.4L）制备的水解度为21.5%的麦胚蛋白酶解液具有最高的钙结合能力和络合物得率，分别为18.0mg (g蛋白)-1、32.5%；经超滤膜截留后的组分P2（3K<Mw<5K）的钙离子结合能力显著高于其他分馏组分（p<0.05），其钙含量提高到67.5mg (g蛋白)-1；高钙螯合肽中的氨基酸残基Glu、Arg、Asp和Gly含量与疏水性氨基酸比例（44%）最高，表明酸性氨基酸的羧基与疏水性氨基酸含量对结合钙能力起到了重要作用；分子量<2000Da的肽段具有高钙亲和特性；紫外-可见光谱（UV-vis）和红外光谱图（FTIR）表征麦胚肽-钙络合物发现，Ca2+与麦胚肽中的酰胺键发生了作用，形成了基态络合物；Ca2+主要与羧基的氧原子和氨基氮原子相互作用键合到麦胚肽上。因此，麦胚蛋白是生产钙螯合肽的很有潜力的蛋白资源，可作为生物活性成分应用于健康食品中。通过体外模拟胃肠道消化实验比较了麦胚肽-钙络合物与无机钙和葡萄糖酸钙体外消化后钙的生物利用度（包括释放量、可溶性以及透析度）。结果表明，麦胚肽-钙络合物有着更高的生物利用度：胃中消化2h后，钙的释放率达90%以上；胃肠中消化8h后的溶解度和透析率分别维持在80%和43%的高水平；麦胚肽-钙络合物依赖于胃酸的酸化能缓慢释放Ca2+，相对于CaCO3和葡萄糖酸钙的快速释放能减少对胃的刺激；在植酸、草酸和膳食纤维等抑制剂的影响下，麦胚肽-钙络合物仍具有较高的生物利用度：植酸存在时，胃肠道消化2h后其溶解性和透析率分别降低至77%和35%；添加草酸后，分别降低到75%和24%；膳食纤维影响较大，分别降低到45%和21%；但生物利用度均高于CaCO3和葡萄糖酸钙，是理想的钙补充剂；麦胚肽-钙络合物在模拟胃肠道消化过程中具有生物稳定性，是吸收和输送钙元素的良好载体。更多还原

【Abstract】 Mineral calcium is an important element for human health. Deficiency of dietary calciumcan lead to numerous diseases. Mineral chelating peptides have shown potential application inthe management of mineral deficiencies. An increasing number of chelating peptides with theability to facilitate and enhance the bioavailability of minerals are being discovered andidentified. Food-derived chelating peptides have been used for development of functionalfoods and health products fortified with calcium due to their safety and multi-functionality. Inthis work, wheat germ protein is going to be hydrolyzed by proteinase to release the calciumchelating peptides, which might provide an experimental evidence for the related research anddevelopment of food-derived chelating peptides.A central composite design with two independent variables (solubilisation pH andprecipitation pH) and bivariate correlations were selected for the correlation analysis of theprotein separation conditions and the functional properties. The results showed that wheatgerm protein yield was sensitive to both solubilisation pH and precipitation pH (p<0.05). Theyield (r=0.785, p=0.000), fat absorption (FA, r=0.752, p=0.000) and emulsification (EA,r=0.697, p=0.697) of isolates showed a high positive linear correlation with solubilisation pH;whereas water absorption (r=0.863, p=0.000) showed a negative correlation to precipitationpH; For all the functional properties of wheat germ protein, only fat absorption toemulsification (r=0.753, p=0.753), water absorption to foaming capacity (r=0.503, p=0.503)were linear correlation; The protein yield showed high positive correlation with FA (r=0.820,p=0.000) and EA (r=0.683, p=0.683) but did not have significant correlations with otherproperties. In addition, when wheat germprotein showed the highest yield (36.64%) in9.5/4.0(solubilisation pH and precipitation pH), only foaming stability showed maximum (91.67%)and other functional properties were not necessarily the best, such as the maximum of waterabsorption (3.99g g-1) was at the extraction condition of10.2/3.3, while fat absorption,emulsifying activity, foaming capacity were at9.5/3.0(2.98g g-1,0.207,150%, respectively),and the maximum value of emulsifying stability (76.09%) was at10.5/4.0. This allowsprocessers to adjust pH values according to the protein isolate property requirements withoutaffecting the yields greatly.Metal salt was used to control the formation of protein aggregates to improve thesolubility, it is very important for the application of the protein in food. Effects of CaCl2ondisaggregation of wheat germ globulin (WGG) solution heat-treated ranging from55°C to95°C at pH2.0were investigated by turbidity, solubility, H0(surface hydrophobicity),-potential and microstructure. Maximum turbidity (A400=1.3) and minimum solubility (20.3%)occurred at the isoelectric point (pI5.0) and95°C. The turbidity of WGG at pH ranging from1.0to5.0(below pI) in the presence of CaCl2was rapidly decreased, while significantincrease in pH ranging from6.0to9.0(above pI). The addition of CaCl2to dispersions ofWGG produced an increase and followed a decrease in solubility: for pH<pI (pH5.0~1.0) thesolubility began to rise from38.9%to76.3%, compared with their controls without calcium(p<0.05), andat pH>pI (pH6.0~9.0) the decrease was20.3%to12.2%(p<0.05). A strong increase in the turbidity during heating was observed at low pH (2.0) for proteins with nocalcium ion. In the presence of50mmol/L CaCl2, solubility of thermally treatednative WGGsignificantly increased. The disaggregation extent of the aggregations, reported as thedisaggregation index, was observed as a constant (y=0.5) between pH-induced (from pH1.0to the pI) treatment or heat-induced (from55to95°C) treatment for WGG. At Ca100mmol/L a significant increase in the solubility of WGG was detected (66.8%at95°C)compared with its control without calcium (26.9%); The chages of hydrophobic (H0), zetapotential and the protein microstructure (SEM) of WGG during theaggregation-disaggregation process were investigated. The results suggested that theelectrostatic repulsion between the particles issufficiently strong to disintegrate proteinaggregates, leading to a degradation of large lumps and ahomogeneous distribution of WGGparticles.To produce and evaluate wheat germ protein-calcium complex from enzymatichydrolysis of wheat germ protein, the restrictive enzymolysis technology, metal chelatetechnology and analytical techniques such asultraviolet-visible (UV-vis) spectroscopy andFourier transforminfrared (FTIR) spectroscopy had been accepted. It is demonstrated that theamount of Ca bound depended greatly on the type of enzyme, degree of hydrolysis (DH),amino acid composition, and molecular mass distribution of different hydrolysates. Wheatgerm protein hydrolysate prepared by Alcalase2.4L at DH of21.5%had the maximum levelof Ca bound and the highest yield of complex (18.0mg (g protein)-1,32.5%), and wassubsequently fractionated through ultrafiltration membranes with molecular weight cutoffs.Calcium binding capacity of portions P2(3K<Mw<5K) was significantly higher (p<0.05)than other portions, its calcium content increased to67.5mg (g protein)-1; Peptide fragmentsexhibiting high calcium-binding capacity had molecular mass <2000Da. The calcium-bindingpeptides mainly consisted of Glu, Arg, Asp, and Gly, and the level of Ca bound was related tothe hydrophobic amino acid content in WGPHs. UV-visible and Fourier transform infraredspectrademonstrate that amino nitrogen atoms and oxygen atoms on the carboxylgroup wereinvolved in complexation. Therefore, wheat germ protein is a promising proteinsource for theproduction of calcium binding peptides and could be utilized as a bioactive ingredient fornutraceutical food production.In vitro gastrointestinal digestion experimentwas used to evaluate the wheat germpeptides on calcium bioavailability, comparing inorganic calcium and calcium gluconate. Thecalcium bioavailability included the release quantity, solubility and dialyzability. The resultsshowed that wheat germ peptide-calcium complex had a higher bioavailability: The releaserate of peptide-calcium complex was more than90%after2h of the stomach digestion; In thegastrointestinal digestion after8h the solubility and dialyzability remained at ahigh level with80%and43%, respectively; Wheat germ peptide-calcium complex was more slowly torelease of Ca2+under the gastricacid than CaCO3and calcium gluconate, which could reducethe stimulation of the stomach; Under the influence of inhibitors such as phytic acid, oxalicacid and dietary fiber, wheat germ peptide-calcium complex still had a high bioavailability;At the present of phytic acid, its solubility and dialyzability reduced to77%and35%respectively during gastrointestinal digestion for2h; After adding oxalic acid, reduced to75% and24%, respectively; Dietary fiber, reduced to45%and21%respectively; However, thebioavailability of wheat germ peptide-calcium complexwere higher than CaCO3and calciumgluconate. It means that wheat germ peptide-calcium complex is the ideal calciumsupplements; Wheat germ peptide-calcium complex had remarkable biological stabilityduring simulated gastrointestinal digestion, thus the calcium chelating peptides from wheatgerm protein is a good indicator for enhancement of calcium absorption.更多还原