【作者】 黄群；

【导师】 马美湖；

【作者基本信息】 华中农业大学 ， 食品科学， 2012， 博士

【摘要】 卵白蛋白是鸡蛋中最丰富的蛋白质组分,占蛋清蛋白总量的54%,为43.0kDa的单体、球状磷酸糖蛋白,自然卵白蛋白(N-卵白蛋白)在鸡蛋贮存或孵化期间不可逆地转化为热稳定形式(S-卵白蛋白)。S-卵白蛋白为N-卵白蛋白的构象异构体,S-构型转化进程中分子特性与理化性质发生相应变化。本课题研究S-卵白蛋白与鸡蛋鲜度的相关性,考察贮藏条件对S-卵白蛋白转化的影响；采用两步超滤技术、聚乙二醇沉淀和阴离子交换层析法分离纯化卵白蛋白；研究S-构型转化对卵白蛋白分子及乳化特性、微观结构的影响；考察白藜芦醇、核黄素、姜黄素等活性小分子与S-卵白蛋白的相互作用,研究超高压处理对S-卵白蛋白结构与功能特性的影响。探讨以S-卵白蛋白为指标、通过等价蛋龄评价商业鸡蛋鲜度的可行性。分别测定25℃和37℃条件下85枚鲜蛋的S-卵白蛋白含量、蛋黄指数、蛋清pH值和哈夫单位等指标,并进行相关性分析。结果表明,S-卵白蛋白与贮存时间及鸡蛋鲜度评价常用指标之间具有高度相关性。指数回归模型分析建立了等价蛋龄预测模型,将S-卵白蛋白含量转换为等价蛋龄(25℃),可评价不同条件下贮存鸡蛋的鲜度。研究证实以S-卵白蛋白为指标评价商业鸡蛋鲜度是可行的。为了探讨贮存条件对鸡蛋清S-卵白蛋白转化的影响,考察了贮存温度、湿度、二氧化碳浓度及涂膜处理等4个影响因素。结果表明,S-卵白蛋白转化与贮存温度呈正相关,高温能加速转化；S-卵白蛋白含量受湿度影响不显著,高湿仅能有限抑制S-卵白蛋白转化；低浓度CO2(2.5%)抑制S-卵白蛋白转化效果不明显,而高浓度CO2(≥5.0%)抑制效果显著；涂膜处理能显著抑制S-卵白蛋白转化,尤其是油溶性涂膜剂、矿物油涂膜处理抑制效果极显著。基于超滤机理分析了超滤过程中初始料液体积分数、pH值和跨膜压力等参数对膜通量、透过液蛋白质质量浓度的影响。在考察膜通量变化确定超滤时间的基础上,通过超滤试验获得最佳工艺条件：初始料液体积分数6.0%,pH值2.5,跨膜压力0.12Mpa。超滤产物纯度、回收率较高,卵白蛋白、溶菌酶的纯度分别为85.72%、87.21%,回收率分别为51.36%、62.58%。试验结果表明,两步超滤法可用于鸡蛋清卵白蛋白与溶菌酶的规模化、高效分离。采用聚乙二醇(PEG)使其它蛋清蛋白质沉淀,然后用阴离子交换层析进一步纯化上清液,获得高纯度卵白蛋白产物。结果表明,15%PEG-8000沉淀可有效地将卵白蛋白与其它蛋清蛋白分离,上清液经Q-Sepharose FF阴离子交换层析纯化,卵白蛋白产物纯度高达94.84%,回收率达71.45%。该法操作简单、周期短,纯度和回收率均理想,可用于工业化大规模生产。采用DEAE Sepharose C1-6B离子交换纯化N-卵白蛋白、碱性条件下热诱导制备S-卵白蛋白,对S-卵白蛋白分子及乳化特性进行研究,探讨二者之间内在联系。研究S-卵白蛋白转化进程中游离氨基含量、Zeta电位、疏水性、乳化液滴平均粒径(d32)与乳化活性的变化情况。在此基础上对以上因子进行相关性分析,并对S-卵白蛋白的乳化液进行显微镜观察。RP-HPLC显示N-卵白蛋白纯度达98%,差示扫描量热(DSC)分析显示诱导制备了S-卵白蛋白,在S-卵白蛋白转化进程中存在过渡产物(Ⅰ-卵白蛋白)。S-构型转化使游离氨基含量增加54.69%、Zeta电位降低7.1mV、乳化液滴平均粒径(d32)减少2.07μm,疏水性提高76.88%,乳化活性提高38.92%,而乳化稳定性减少51.07%。显微镜观察结果表明,S-卵白蛋白易于形成较小的乳化液滴。S-卵白蛋白的转化能有限改善卵白蛋白的乳化活性,却降低乳化稳定性,卵白蛋白的S-构型转化是蛋清蛋白品质劣化的原因之一。采用圆二色谱、X射线衍射、ANS荧光探针和紫外光谱研究了S-构型转化对卵白蛋白微观结构的影响。结果显示,不同诱导时间处理的卵白蛋白二级结构的α-螺旋,β-折叠,β-转角和无规卷曲之间相互转化,α-螺旋略有减少,β-折叠相应增加,分子有序性提高。S-构型转化后卵白蛋白晶体结构增加,72h处理后结晶区最大,说明蛋白结构的有序性提高,与CD分析结果一致。卵白蛋白的表面疏水性随诱导时间的延长而增强,72h处理后达到最大值。S-构型转化使卵白蛋白分子表面具有紫外吸收的芳香氨基酸残基包埋于分子内部,导致最大紫外吸光值随诱导时间的延长而下降。研究表明,卵白蛋白的微观结构变化与S-构型转化有关。采用荧光猝灭光谱、同步荧光光谱和紫外光谱研究白藜芦醇、核黄素、姜黄素等3种活性小分子与S-卵白蛋白的相互作用机制。根据Stern-Volmer方程研究了结合作用机理,由热力学参数的计算判断了主要作用力类型,并依据能量转移原理求得了结合距离和能量转移效率。研究表明,3种小分子对S-卵白蛋白内源性荧光产生强烈的猝灭作用,猝灭机理主要为静态猝灭和非辐射能量转移；3种小分子与S-卵白蛋白间结合位点数n≈1、结合距离均小于7nm；作用力均以氢键和范德华力为主,且为吉布斯自由能降低的自发过程；同步荧光、紫外光谱研究表明3种小分子能够使S-卵白蛋白的构象发生变化。采用激光拉曼光谱、荧光光谱分析超高压(UHP)处理对S-卵白蛋白构象的影响,并考察其对溶解性、乳化性和起泡性等功能特性的影响。结果表明,经UHP处理后S-卵白蛋白的分子量分布没有明显变化。随着压力的增加,无规卷曲含量逐渐增加,二硫键构象变化使得蛋白稳定性降低,酪氨酸残基包埋于侧链中,紧密折叠结构得以舒展。经100Mpa处理后S-卵白蛋白的荧光强度略有增强,此后急剧下降,无峰位位移现象,说明UHP引起蛋白分子构象变化。S-卵白蛋白的溶解性、乳化能力及起泡能力等功能特性均随压力增加而明显改善,以300Mpa处理较为理想,此后压力增加至500Mpa时反而下降。更多还原

【Abstract】 Ovalbumin, a monomer globular phosphate glycoprotein protein with a molecular mass of43.0kDa, which constitutes about54%of the total proteins in a newly deposited egg, is the most abundant protein component in shell egg. During storage or hatching period, native ovalbumin (N-ovalbumin) gradually converts into S-ovalbumin, which is an irreversible extreme heat-stable form compared with N-ovalbumin. S-ovalbumin is a conformational isomer of native ovalbumin rather than a chemically modified derivative, and certain functional properties of S-ovalbumin are different from that of N-ovalbumin, owing to a series of changes in the molecular and microscopic structure during the process of S-configuration transformation. In the present project, the correlation between S-ovalbumin and egg freshness, and the effect of storage condition on the S-ovalbumin formation in chicken egg white were investigated firstly. Secondly, ovalbumin was separated and purified using two-stage ultrafiltration technique or polyethylene glycol precipitation and anion-exchange chromatography. And then, the effect of S-configuration transformation on molecular characteristics, emulsifying properties and microstructure of ovalbumin was also researched. Finally, the interaction between active small molecules and S-ovalbumin was studied, such as resveratrol, riboflavin and curcumin, and the effect of ultra high pressure (UHP) on the strcture and functional characteristic of S-ovalbumin was also investigated.The aim of the first chapter was to study S-ovalbumin as a reference index for the freshness of commercial shell egg in terms of equivalent egg age. S-ovalbumin content, yolk index, albumen pH and haugh units were determined at the storage temperature of25℃and37℃respectively, using85fresh laid eggs. Correlation analysis showed a high correlation coefficient of S-ovalbumin content to storage time, as well as to the three frequently used freshness indexs (haugh unit, yolk index and albumen pH). Furthermore, by exponential equation analysis, the prediction model of equivalent egg age was established, which could be used to predict the freshness of commercial shell egg stored in any condition by converting S-ovalbumin content into equivalent egg age at25℃. This study confirmed the possibility of using S-ovalbumin as a reference index to express commercial shell egg freshness with equivalent egg age. In order to evaluate the effect of storage condition on the S-ovalbumin formation in chicken egg white, four influential factors, such as storage temperature, humidity, CO2concentration and film coating, were investigated in the second chapter. The results showed that S-ovalbumin formation positively correlated with the storage temperature, and high temperature could accelerate this formation. S-ovalbumin content was not significantly affected by humidity, and high humidity had only a limited inhibition of the S-ovalbumin formation. Low-concentration CO2(2.5%) did not inhibit evidently the formation of S-ovalbumin, but high-concentration CO2(≥5.0%) had a significant inhibitory effect. Film coating could significantly inhibit the S-ovalbumin formation, especially the oil soluble and mineral oil coating processing had a prominent inhibitory effect.Based on the mechanism of ultrafiltration, main factors that affecting the permeate flux and total proteins concentration in permeating liquor, such as initial feed concentration, pH value and trans-membrane pressure (TMP) were studied. After ascertaining the ultrafiltration time by examining the change of permeate flux, the optimum ultrafiltration conditions were supposed to be follows:initial feed concentration,1:1; pH value,2.5; TMP,0.12Mpa. In these optimum conditions, purity and yield of ovalbumin was85.72%and51.36%respectively, and that of lysozyme was87.21%and62.58%respectively, implying that separation efficiency was satisfied. The experimental results showed that it is feasible for two-stage ultrafiltration technique to separate ovalbumin and lysozyme from chicken egg white in bulk and high efficiently.High purity ovalbumin was obtained by using polyethylene glycol as a precipitate to separate ovalbumin from other proteins of chicken egg white, and then adopting anion exchange chromatography to further purify the supernatant, and the purity of objective product was determined with reversed-phase high-performance liquid chromatography. It was showed in the results that polyethylene glycol precipitation could efficiently separate ovalbumin from other proteins when the PEG-8000concentration is15%(M/V). After purifying the supernatant with Q-Sepharose Fast Flow anion exchange chromatography, the purity of ovalbumin product reached up to94.84%, and recovery of yields was71.45%. This method is simple, high-efficiency, low cost and environmentally friendly, and easy for scale-up preparation.N-ovalbumin was purified by ion-exchange with DEAE Sepharose C1-6B, S-ovalbumin prepared by thermal induction in alkalinity condition, and molecular characteristics and emulsifying properties of S-ovalbumin were studied to investigate the relationship between them. Content of free amino group, zeta potential, hydrophobicity, emulsifying activity and average droplet size of emulsion(d32) during S-ovalbumin information process were investigated, and correlation analysis of referred factors and microscopic observation of emulsion of S-ovalbumin were also conducted. The purity of ovalbumin reached up to98%determined with RP-HPLC, and S-ovalbumin was prepared testified with differential scanning calorimetry (DSC) which showed that there was transition-production (I-ovalbumin) during S-ovalbumin information process. S-configuration transformation resulted in increase of54.69%of free amino group,76.88%of hydrophobicity and38.92%of emulsifying activity, and decrease of7.1mV of zeta potential,2.07μm of average droplet size of emulsion(d32) and51.07%of emulsifying stability. Microscopic observation indicated that S-ovalbumin was prone to form small droplet of emulsion. Emulsify activity was improved to a limited extent, but emulsifying stability worsened by the formation of S-ovalbumin. Therefore, it’s concluded that S-configuration transformation of ovalbumin is one of the reasons to quality deterioration of chicken egg albumen.The effect of S-configuration transformation on the micro structure of ovalbumin was studied by CD spectra, XRD spectra, ANS fluorescence probe emission spectra and UV absorption spectra. CD spectra was used to examine the changes in the secondary structure of the ovalbumin during S-ovalbumin information process. When the induced time prolonged, the mutual transformation between a-helix,β-sheet,β-turn and the random coil was observed, and the orderliness of the secondary structure was increased with a-helix decreasing slightly andβ-sheet increasing correspondingly. XRD spectra analysis showed that the crystal structure content of the ovalbumin increased with prolonging the induced time and the largest data was observed at72h, indicating that the orderliness of the secondary structure was increased, similar to CD spectra analysis. The ANS fluorescence probe emission spectra analysis demonstrated that S-configuration transformation induced an increase in surface hydrophobicity with prolonging the induced time, and the largest data was also observed at72h. In addition, UV absorption spectra analysis indicated that S-configuration transformation resulted in a decrease in the UV-absorption maximum value with prolonging the induced time, indicating that the aromatic amino acid was buried in the molecular interior. The results indicated that the changes in the microstructure of ovalbumin were relevant to S-configuration transformation. The interaction between active small molecules and S-ovalbumin was studied by using fluorescence quenching spectra, synchronous fluorescence spectra and ultraviolet spectra. The active small molecules used in this work were resveratrol, riboflavin and curcumin. Results showed that the quenching of intrinsic fluorescence of S-ovalbumin was produced by the formation of a complex. The value obtained for the binding constant, according to the Stern-Volmer equation, deduced the existence of static quenching mechanism. According to the Forster theory of non-radiation energy transfer, the distance between active small molecules and S-ovalbumin was calculated to be less than7nm. The values obtained for the thermodynamic parameter AH and AS suggested the participation of vad der Waals force and hydrogen bonds in the binding of active small molecules to S-ovalbumin, and the negative value of AG revealed this process is spontaneous. Finally, the conformational change of S-ovalbumin in the presence of active small molecules was confirmed based on the information obtained from the evaluation of synchronous fluorescence spectra and UV-Vis absorption spectra.The effect of Ultra High Pressure (UHP) on conformation of S-ovalbumin was studied with raman spectra and fluorescence spectral analysis, and the effect on some functional properties of S-ovalbumin, such as solubility, emulsibility and foamability, were also investigated. It was showed no significant change in the molecular weight distribution of S-ovalbumin after UHP under various pressure treatments. Along with the increase of pressure, the content of random coil was increasing gradually, the stability decreasing owing to the conformation change of disulfide bonds, and tyrosine was buried in the side chain, accordingly the compactly plate sheet structure was to become loose. The fluorescence intensity showed a slight increase after UHP under100Mpa, then a sharp decline with the increase of pressure, no shift of the fluorescence emission peak happening, so it was pointed out that UHP induced conformational change. The solubility, emulsibility and foamability properties had been improved significantly with the increase of pressure, and achived the best effect at300Mpa, however, there was a drop down when pressure was added to500Mpa.更多还原