Food Science and Technology Department


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 Steven L. Taylor and Joseph L. Baumert. Lincoln, Nebraska: December, 2013

Copyright (c) 2013 Katherine O. Ivens


Analytical methods, such as enzyme-linked immunosorbent assays (ELISA), are used to detect and quantify residues from allergenic sources in food products. However, ELISAs have not been validated for use in foods that have been exposed to proteolysis. This thesis explores the specificities, sensitivities, and capabilities of commercially-available milk ELISA kits for detecting milk residues in cheeses that have undergone varying degrees of proteolysis.

The specificity, accuracy, and consistency of twelve commercially-available milk ELISA kits for individual milk proteins and commonly used milk-derived ingredients, including α-,β-, and κ-casein, β-lactoglobulin, α-lactalbumin, non-fat dry milk, sodium caseinate, and whey protein concentrate were evaluated. ELISA kits exhibited targeted specificities to milk proteins as opposed to broad-spectrum detection. Kits were able to detect milk residues in all derivative ingredients, although the kits were not quantitatively accurate.

Further, ELISA kits were used to investigate the effects of proteolysis in Cheddar cheese during aging. As cheese ripened and proteolysis continued, fewer milk residues were detected in samples using commercial milk ELISA kits. In a survey of retail cheeses produced with different degrees of proteolysis, the lowest concentration of milk residues were detected in Blue cheese, while Mozzarella cheese contained the highest milk residues. Emmentaler, Brie, and Limburger cheeses were also evaluated. Five samples of enzyme-modified cheese (EMC) were assessed for their milk residue content; again, not all ELISA kits were able to detect milk residues in samples that had been subjected to extensive proteolysis.

The recommendation of specific ELISA kits for detecting milk residues is highly product-specific. Current commercially-available milk ELISA kits are capable of detecting milk residues in a variety of cheeses, including some that have been exposed to extensive proteolysis. However, the quantitative accuracy of commercial milk ELISA kits is jeopardized when proteolysis has occurred. Some ELISAs may have further application to monitor proteolysis and indicate cheese maturity during ripening because of their specificities.

Advisers: Stephen L. Taylor and Joseph L. Baumert