Food Science and Technology Department


First Advisor

Philip E. Johnson

Second Advisor

Michael G. Zeece

Date of this Version



A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Food Science and Technology, Under the Supervision of Professors Philip E. Johnson and Michael G. Zeece, Lincoln, Nebraska: December, 2016

Copyright 2016 Lee Palmer


Increasing global population increasingly limited by resources has spurred interest in novel food sources. Insects may be an alternative food source in the near future, but consideration of insects as a food requires scrutiny due to risk of allergens. Currently, the insect Dactylopius coccus, known as cochineal, is used to produce carmine, a natural red pigment used in food, which has caused allergic reactions. This study investigated allergens of cochineal focusing on purification from the pigment. Mass spectrometry identified a previously described major allergen of cochineal and a tropomyosin, although further work is required.

Tropomyosin is a major cross-reactive allergen across invertebrates including insects and shellfish and has multiple isoforms per species of varying function, sequence, and expression. Extractions of diverse insects must be sufficiently representative to be comparable. This study used a mass spectrometry compatible buffer and a zwitterionic-chaotropic buffer with sequential extractions. Both buffers were found to be sufficiently representative via rabbit anti-shrimp tropomyosin IgG. These extractions were used for further immunoblotting with shrimp-allergic sera and sera from subjects with self-reported shellfish allergy or sensitization to shellfish. Tropomyosins were cloned from several samples and their sequences investigated for epitopes and semi-quantitative massspectrometry. A pattern of low reactivity was found for several samples not corroborated by quantitative data. Further cloning is necessary to align these data sets.

Resistance to digestion is a common test for potential allergenicity as epitopes may persist after digestion. Use of pepsin is standard, although this may not be as representative as a direct assay of the source food. Simulated gastric pepsinolysis was performed with defatted Acheta domesticus cricket powder and immunoblotted against shrimp-allergic sera and rabbit anti-tropomyosin IgG. Patterns of reactivity were similar against non-reduced samples with relatively lower reactivity with allergic sera against reduced samples. The allergic sera was predominantly cross-reactive with tropomyosin with lesser reactivity against reduced forms of cricket tropomyosins.

It was found that insect based foods pose potential risk to shellfish allergic patients due to homologous proteins including tropomyosin.

Advisors: Philip E. Johnson and Michael G. Zeece