| Ovomucoid had been studied since early20th century, although up to now, the basic structure, the nature and characteristics are known, there has been no simple and effective way to reduce its allergenicity. In this study, ovomucoid was isolated and purified from the egg white, and its conformation and allergenicity was studied, major findings are as follows:In this paper, the crude ovomucoid was isolated by TCA-acetone method, then separated on DEAE-32. The purity of CHOM sample determinated by SDS-PAGE was about95.3%. About300mg ovomucoid was obtained from50mL egg white. Protein content of egg white was10%, CHOM accounted for11%of egg white proteins, so recovery of CHOM23.8%can be calculated. The activity of CHOM is up to7200BAEE unit/mgCHOM by measureing the trypsin inhibitory, indicating that CHOM did not denaturalize. Conformation of CHOM was characterized by FTIR, and secondary structure content of CHOM was calculated as follows, a-helix26.7%, β-sheet50%,β-turn10%, random coil13.3%, which agreed with the previous results and indicated that the conformation of CHOM did not change. Therefore, CHOM can be used in the subsequent allergy test.This study has successfully established a allergy model of Guinea Pig. The titer of antiserum from allergic Guinea Pig was4,000, measured by Ci-ELISA. So CHOM was the allergen of Guinea Pigs suffered with allergic reaction.The conformation and allergenicity changes of CHOM during heat treatment was studied. The CD spectra results showed that the content of a-helix, β-sheet, β-turn, random coil of untreated CHOM are respectively27.8%,50.2%,10%,12%, when treated by100℃for120min, its content are respectively6.3%,20.5%,20%,53.2%. the content of a-helix, β-sheet respectively decreased by77.3%,59.2%, while random coil increased by77.4%. Therefore, the content of a-helix, B-sheet gradually reduced, while B-turn and the random coil increased as increasing heating temperature and heating time. Simultaneously, surface hydrophobicity of CHOM decreased mearsured by ANS fluorescence probe spectroscopy. UV spectra results showed that the maximum absorbance at280rm of all samples did not shift obviously, but the maximum absorbance changed. With the temperature and time increasing, the maximum absorbance trended upward. During heat treatment, the allergenicinity changes of CHOM was deteminated by Ci-ELISA. Datas displayed that detected concentrations of CHOM treated by70℃,80℃,90℃,100℃for60min are respectively37.47μg/mL,20.52μg/mL,8.06μg/mL,6.24μg/mL, detected concentrations of CHOM treated by100℃for120min was only3.22μg/mL. The results showed that the intact CHOM combined with sIgG became less and less with the heating time and heating temperature increasing, which indicated that the allergenicinity of CHOM decreased. Thermal kinetic parameters D value obtained as follows, D70is2.13×102, D80is1.49×102, D90was1.41×102, and D100is1.15×102. D value decreased with increasing temperature. Within the range of70～90℃, the decrease of the immune activity is greater than at100℃. Z value obtained according to D value was12℃. So conclusions can be drawn, the immune activity of CHOM gradually decreased with increasing temperature and time.MR can affect the allergenicity of CHOM. MR products was prepared under different conditions and the relative fluorescence intensity and relative absorbance values characterized the MR process was measured. The results showed that, with the reaction time increasing, the relative fluorescence intensity and absorbance increased of the MR product. When3th day, the relative fluorescence intensity and absorbance were the largest. When4th day, it changed no longer significantly. Therefore,3d is the best reaction time. When the initial pH value of the reactants is less than7, the relative fluorescence intensity and absorbance values of the product is very low. When pH value of the reactants is higher than7, the rate of reaction significantly increased. When pH9, the value was the largest, when pH11, the value decreased. Therefore, initial pH9was the most suitable. When fructose:CHOM is1:1, the relative fluorescence intensity and the absorbance value is very low, indicating that MR level is very low. When1:2, the relative fluorescence intensity and absorbance values is the highest, when the proportion continues to increase, its value is declining, indicating the MR process gradually slowed down. Therefore,1:2 was selected. Ci-ELISA method was used to determine of the allergenicity changes of CHOM after MR. When2d, the concentration of intact CHOM is5μg/mL.When3d to4d, it only about1.5μg/mL. When initial pH<7, concentration of intact CHOM is about10p.g/mL, when pH>7, it became lower and lower, when pH9, only1.5μg/mL is detected. When the ratio of fructose and CHOM is1:1, the concentration of intact CHOM of the product is about10μg/mL, but when1:2, it is only about1.5μg/mL. The concentration of intact CHOM increased with the proportion larger. Therefore, the MR response may mask allergen epitopes of CHOM, resulting in reducing allergenactivity.The combination of SDS-PAGE and Ci-ELISA was used to study the allergenicity changes of CHOM digested in vitro simulated gastrointestinal tract. The results showed that the amount of intact CHOM combined with sIgG changed little after digesting in vitro simulated gastrointestinal tract. When CHOM was digested by pepsin for or after2min, the detected concentration of CHOM remained45.5μg/mL. Considering the result of SDS-PAGE, when digested for or after2min, high molecular weight band remained almost unchanged, while the low molecular weight bands became more and more fuzzy, which concluded that the allergenicity of CHOM digested by pepsin resuled from a larger molecular weight peptides. The products digested by pepsin for30min were digested by trypsin, the28kDa molecular weight bands disappeared, the24kDa molecular weight bands and17kDa obscured bands came out. It can be speculated that peptides from CHOM digested by trypsin was still sensitizated, and the peptides of larger molecular weight was likely to play a major role. |
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