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IMPACT OF THERMAL AND HIGH PRESSURE PROCESSING ON THE ANTIGENICITY AND DETECTABILITY OF EGG AND MILK ALLERGENS
Title
IMPACT OF THERMAL AND HIGH PRESSURE PROCESSING ON THE ANTIGENICITY AND DETECTABILITY OF EGG AND MILK ALLERGENS
Creator
Yang, Shuopeng
Date
2013, 2013-12
Description
Cow’s milk allergy and egg allergy are the two most prevalent food allergies in the United States. Enzyme-linked immunosorbent assay (ELISA),...
Show moreCow’s milk allergy and egg allergy are the two most prevalent food allergies in the United States. Enzyme-linked immunosorbent assay (ELISA), based on antigen-antibody reactions, is the most popular method used by the food industry to detect allergen residues. Thermal treatments are the traditional food processing methods, while high pressure processing (HPP) has increasingly been applied as an alternative food processing technology. This research examined how thermal and high pressure processing may affect the antigenicity and detectability of milk and egg allergens. The first part of this study examined the performance of four ELISA test kits (Veratox for Total Milk Allergen, Biokits BLG Assay Kit, ELISA Systems Casein and BLG Residue Assays) for quantitation of milk residues in packaged foods that have been subjected to different degrees of processing. Whether standard reference materials (SRM) can be used to compare the relative performance of these test kits was also examined. Commercial food products including ice creams, cookies, fried fish sticks, and canned soups were chosen to represent foods that have been pasteurized, baked, fried, and autoclaved, respectively. Calcium caseinate, NIST non-fat dry milk (NFDM) SRM #1549 and USDA certified whey protein were used as reference standards. Veratox and ELISA Systems Casein kits, both of which use NFDM as the calibrator, showed similar readings for ice cream. However, the Casein kit tended to register a lower level of milk residues in other thermal processed products, especially for canned soup where the test kit showed a 50-fold lower level of milk residue than that reported by the Veratox kit. Using whey protein as a reference standard, the two BLG test kits showed a similar level of whey protein equivalent in ice cream, but for fish stick, cookie, and canned soup samples, the x ELISA Systems BLG kit registered values about 4-, 39-, and 17-fold lower than the values obtained by the Biokits BLG test kit, suggesting that the ELISA Systems kit has a greatly reduced sensitivity for foods that have received heat treatments. When using NIST NFDM as the reference standard, the Veratox and Biokits registered the same level of NFDM equivalent in ice cream, but the Biokits gave a higher level for the fish, cookies, and soup samples, indicating that this kit has a better sensitivity for heat processed food. Overall, different test kits registered different levels of milk even for the same commercial sample. The use of common reference materials provides a means for comparison, but the quantitation of milk residues is still complicated by the different heat sensitivity of each test kit. The second part of this study evaluated the impact of thermal processing on the solubility and antigenicity of egg allergens. Solutions (5 mg/ml sample in PBS) of Henningsen dehydrated whole egg, ovalbumin (OVA), and ovomucoid (OVO) were subjected to three different thermal processing conditions (heated in water at 60℃, 100℃, and autoclaved) for 10 minutes. The solubility of the unheated and heat-treated samples was determined by the BCA total protein assay. Changes in the antigenicity (IC50 value) of egg proteins after thermal treatments were determined by inhibition ELISA. Differences in the antigenicity determined as affected by the use of different target protein, coating antigen, and primary antibody were determined. At 60℃, the solubility and antigenicity of heat treated samples showed similar results as those of untreated sample. For sample treated under other conditions, different heat treatments affect the solubility of egg protein samples differently. Different heat processes, different antigen-antibody combinations, and different extraction methods could affect the IC50 values xi differently, which means the antigenicity changed differently due to different influence factors (e.g. IC50 values of OVO always increased after boiling or autoclaving treatment no matter which coating antigen and primary antibody chose, however OVA and whole egg powder showed uncertain changes). The last part of this study evaluated the impact of high pressure processing on the solubility, antigenicity, and the detectability of egg allergens. Solutions of dehydrated whole egg, OVA, OVO, and Egg Beater (ConAgra Foods) were subjected to high pressure treatments for 3 min under 4 conditions (400MPa 20℃, 400MPa 60℃, 600MPa 20℃, and 600MPa 60℃). The amount of protein in the high pressure treated samples was determined by BCA assay and the Veratox egg allergen test kits. Changes in protein antigenicity were determined by inhibition ELISA assay. The results showed that high pressure treatments under all 4 conditions did not affect the solubility and antigenicity of the egg proteins in the treated samples. HPP did not affect the detectability of these proteins by Veratox test either.
M.S. in Food Safety and Technology, December 2013
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