Study On The Correlation Between S-ovalbumin And Egg Freshness,and The Purification And Characteristics Of S-ovalbumin

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.