【作者】 陈海英；

【导师】 徐学明；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2013， 博士

【摘要】 鸡蛋制品中活性功能成分的精深加工是提高蛋产品附加值,扩大其工业化应用范围的重要途径。蛋黄中卵黄高磷蛋白(Pv)是以带负电荷的磷酸酰丝氨酸为核心的细长链的形式存在的含有大量磷的蛋白。疏水末端赋予了Pv双极性,使其具备了作为天然生物表面活性剂的应用潜能。由于Pv在食品复杂体系中应用时必然与其他组分相互作用而影响其乳化行为,同时,针对Pv自身组成结构缺陷,包括缺乏分枝结构不能形成强有效空间位阻效应,疏水末端短小而限制其有效吸附在油水界面等,本课题分别从内外因素两个角度对Pv乳化特性进行改良,并对Pv的结构组成及理化特性进行表征,旨在对Pv乳化作用机理进行阐述。以鸡蛋蛋黄为原料,分别采用盐溶法和PEG法实现了Pv零有机溶剂的分离提取。盐溶法工艺的Pv得率为91.05%,氮/磷比(N/P)为3.45,极性氨基酸(AA)占总AA含量的70.7%,疏水性AA占19%左右。同时,Pv的二级结构比较松散,不规则展开占91%。Pv具有比酪蛋白酸钠(Sc)更高的乳化特性,乳化活性指数(EAI)为19.80,乳化稳定指数(ESI)为67.60,但是起泡特性远不如大豆分离蛋白,起泡活性(FC)为12.42%。PEG法工艺的Pv得率为49.28%,N/P为2.89,极性AA占总AA含量的80.2%,疏水性AA占13.5%左右。PEG6000的使用屏蔽了Pv的疏水特性,还引起了部分的二级结构的变化,旷螺旋和β-结构(包括p-折叠和p-转角)分别为17%和26%,不规则展开只占35.8%。因此,Pv的EAI只有11.37,ESI为49.89,FC也只有8.33。常见食品添加剂,包括甘油、果胶和海藻糖对Pv的乳化行为具有协同增效作用。结果表明,外源添加0.5%甘油显著地将Pv的EAI从19.8增加到20.9,ESI从67.6增加到75.1。0.1%果胶和0.5%海藻糖也增强了Pv的乳化稳定性。圆二色谱(CD)图谱显示,Pv呈现出典型的松散的不规则展开型二级结构,所有添加剂只稍微改变了Pv的二级结构。内源性荧光光谱也表明,甘油和海藻糖增加了Pv的疏水性,果胶则相反。实验中所有的乳化液都呈现处剪切变稀的流动特性,其流动行为都符合幂律模型。频率扫描表明,乳化液呈现出粘性液体的流动特性。从增加Pv空间位阻效应以提高Pv乳化稳定性的角度出发,Pv与葡聚糖(Dex)混合体系在高温液态体系下美拉德反应初级阶段实现了Pv的糖基化反应。具体反应条件为,Pv:Dex比为1：4,Dex分子量为40kDa,热处理温度为100。C反应时间为6h,Pv的自由氨基含量下降到77.4%。SDS-PAGE凝胶电泳上新条带Ⅱ及SE-HPLC洗脱图谱上主要峰出峰时间从9.16min迁移到8.87min,次要峰的出峰时间从6.56min迁移到6.13min共同证实了Pv-Dex交联物的生成。同时,Pv糖基化反应后,不规则展开结构下降到72.1%,α-螺旋增加到20.8%。Pv与Dex共价交联后在pH5.0～8.0之间的溶解度保持恒定,但显著地将Pv在pH4.0处的溶解度从53.0%提升到79.3%。Dex的共价结合还降低了Pv的疏水特性,对Pv表面电荷有轻微的屏蔽效应,但是,显著增加了Pv在酸性环境下的界面层厚度。从浊度法、粒径分布以及共聚焦激光显微镜三个角度来考察了糖基化对不同pH环境下Pv乳化特性的影响,所有结果都证实,Dex与Pv交联是提高Pv乳化稳定性及对极端pH环境抗性的有效手段。从增加Pv疏水末端以改善Pv乳化特性的角度出发,将脂肪酸(FA)酯化活化后,再与Pv酰化反应制备了不同碳链长度的Pv-FA系列衍生物。SDS-PAGE凝胶电泳图表明Pv-FA衍生物的分子量稍高,条带更宽。月桂酸(C12),肉豆蔻酸(C14),棕榈酸(C16)与Pv酰化后最大紫外吸收波长发生不同程度的偏移,最大吸收强度依次为Pv-C14>Pv-C16>Pv-C12,反应程度分别为16.45%,30.56%,27.02%。Pv与C12、C14、C16共价结合后Pv溶解度分别下降到51.00%、56.77%、22.14%,不规则展开型二级结构也从35.8%分别增加到38.3%、69.3%、45.6%。C14和C16在Pv末端的结合还增强了Pv的表面疏水性。Pv-C14在中性环境下EAI最高,为16.26,同时,ESI也高达74.41,FC高达33.33%。对极端pH环境的抵抗能力也最强。C12的引入也显著提高了Pv的EAI到14.54,ESI到62.23,FC到20.83%,对极端pH环境具有一定的抵抗能力。C16与Pv结合显著增强了Pv的FC到28.57%。研究表明,Pv疏水末端FA的引入是改善Pv加工特性的另一种有效手段。更多还原

【Abstract】 Deep processing of functional active ingredients was an effective way toimprove the value-added and expand the industrial application of egg products. Pv,from hen egg yolk, was a protein with high content of phosphours. The core in theform of an elongated chain contained negatively charged phosphatidylserine. Phosvtinacted as a bipolar molecule with the hydrophic amino acid in the C-terminal. Itshowed an application potential as the natural biosurfactant in food, medical, andcosmetic industry. Generally, the components in food system would have an interationwith Pv and then influenceits emulsifying behavior, and there was barely literature onthis subjuct. At the same time, the little hydrophobic terminal in Pv would limit itsabsorption behavior in the interfacial layer of oil-in-water. The lack of branchedstructure in Pv would aslo limit the forming of an effective steric-hinerance instabilizing the oil-in-water emulsion. In our research, the improvement technologyfrom both the external and internal points of view was tried to enhance theemulsifying property of Pv. The physicochemical properties were also characterizedto better understand the mechanism of emulsifying actions.Two Pv-isolated technology, salty and PEG, were applicated to extract Pv fromhen egg yolk with no organic solvent used. The yield and N/P ratio of Pv by saltymethod were91.05%and3.45respectively. The proportions of polar and hydrophobicAA residues were70.7%and19%respectively. At the same time, the secondarystructure of Pv was loose and the unfolding structure accounded for91%. Theemulsifying activity index (EAI) and emulsion stability index (ESI) of Pv were19.80and67.60resepctively, higher than that of sodium caseinate (Sc). However, thefoaming capacity (FC) was12.42%, lower than that of soybean protein isolated (SPI).The yield and N/P ratio of Pv by PEG method was49.28%and2.89respectively. Theproportions of polar and hydrophobic AA residues were80.2%and13.5%respectively. Part of hydrophic properties of Pv were screened and the secondarystructure was changed by PEG6000. The proportion of α–helix and β–structure(including β–folding and β–turn) were17%and26%respectively, and the unfoldingforms occupied of35.8%. The EAI and ESI of Pv isolated by PEG method were11.37and49.89respectively, lower than that by salty method. The FC was only8.33, alsolower than that of SPI.The normal food additives, such as glycerol, pectin and trehalose showed asynergistic interaction with Pv on the emulsifying behavior. The presence of0.5% glycerol significantly increased EAI of Pv from19.8to20.9and ESI from67.6to75.1. The addition of0.1%pectin and0.5%trehalose also resulted in a betteremulsion stability of Pv. The pattern of CD spectra indicated a typical unfoldingsecondary structure, and all the tested additives displayed slight effect on theunfolding structure of Pv. Furthermore, fuorescence spectra pattern demonstrated thatglycerol and trehalose increased the hydrophobicity of Pv, while pectin showed anopposite effect. All emulsions exhibited a shear-thinning behavior and the fowbehavior was ftted to the power law model. Frequency sweeps of all tested emulsionsillustrated that they behaved like a viscous liquid.To enhance the steric hindrance effect of Pv and in turn improve the emulsionstability, the Pv-Dex conjugates were prepared in an aqueous solution at the initialstage of Maillard reaction. The optimum conjugation condition chosen was per Pvglycosylated with4-fold dextran (Mw40kDa) at100℃for6h. The content of freeamino groups in the glycosylated Pv decreased to77.4%. The new bandII inSDS-PAGE gel and the shift of main peak in SE-HPLC from9.16min to8.87minand the minor peak from6.56min to6.13min both confirmed the formation ofcovalent conjugates. CD spectra demonstrated that the unfolding structure decreasedto72.1%, and α–helix increased to20.8%after glycosylation reaction. No significantdifference occurred to the solubility of Pv after conjugation with Dex when pH rangedfrom5.0to8.0, but a remarkable increase from53.0%to79.3%appeared at pH4.0.The hydrophobicity was decreased and the surface charges were slightly screenedafterDex attaching to Pv, while the interfacial thickness of Pv was prominentlyincreasedat the acidic condition. The emulsifying properties characterized byturbidimetric method, droplet-size distributions and confocal laser scanningmicroscopy illustrated that glycosylation of Pv would be an effective method toimprove the emulsion stability against unfavorable pH environment.To link a hydrophobic tail to Pv and then improve the emulsifying property andfoaming property, FA was activated through esterification and then attached to Pvthrough acylation. The slight higher andwider bands in SDS-PAGE gel indicated thatdifferent carbon chain length of FA had been convalent binded to Pv. Differentdegrees of shift of maximum UV absorbance wavelength were caused by lauric acid(C12), myristic acid (C14), palmitic acid (C16) grafting with Pv, and the absorbanceswere in the following order: Pv-C14＞Pv-C16＞Pv-C12. The reaction extent of C12,C14and C16with Pv were16.45%，30.56%，27.02%respectively and the solubilityof relevant products decreased to51.00%56.77%22.14%respectively. The unfolding structure was also increased from35.8%to38.3%69.3%45.6%respectively. Pv-C14displayed the highest EAI of16.26, ESI of74.41, and FC of33.33%in the neutral condition, and showed the strongest resistance to theunfavorable pH environment. Pv-C12also increased the EAI to14.54, ESI to62.23,and FC to20.83%, and resulted in a certain resistance to acidic conditions. Pv-C16enhanced FC to28.57%. All the resultes demonstrated that the covalent binding of FAto Pv was an effective way to improve the processing properties of Pv.更多还原