A Comparison of Analytical Methods for Quantifying Denatured Whey Proteins and Their Correlation to Solubility

Protein structure affects the bioactivity and functionality of whey protein ingredients in food systems. Bioactivity of whey proteins and their derivatives are highly dependent upon primary, secondary and tertiary structure. The degree of denaturation of whey proteins is an important factor for determining how whey protein ingredients will perform in a food system. Several analytical methods have been developed to quantify protein denaturation of whey proteins. The goal of this project was to use a variety of analytical methods to quantify whey protein denaturation and to evaluate the correlation of denaturation to the functionality of whey protein powders. The objective of the first series of experiments was to compare three different analytical methods to measure denaturation of whey proteins in liquid whey obtained by various methods of separation and with varying degrees of heat treatment. A split plot experimental design was used. Raw bovine milk was skimmed and liquid whey was separated from the skim milk at natural pH. Three separation methods: 1) centrifugation, 2) membrane filtration and 3) enzyme coagulation, made up the first split plot. Each sub-plot of liquid whey was then divided into three split plots to receive heat treatment. Heat treatments were no heat, 76°C for fifteen seconds and 85°C for three minutes. Each of the resulting nine treatment combinations was analyzed by 1) polyacrylamide gel electrophoresis, 2) bicinchoninic acid-soluble protein assay and 3) fluorescence spectroscopy to determine the amount of denatured protein in the liquid whey. Fluorescence spectroscopy was found to be the most sensitive and reliable method for detecting differences in structure due to denaturation, while native polyacrylamide gel electrophoresis was found to be the least sensitive method. The sample which received the centrifugal treatment of isolation with no heat was found to be the most undenatured in structure while the sample which received the enzyme treatment of isolation with high heat was found to be the most denatured in structure. The objective of the second series of experiments was to evaluate the effect of denaturation on whey protein solubility in dried whey protein powders. Solubility is one of the most important functional properties to consider when selecting a whey protein ingredient, especially for beverage systems. Processing parameters are often manipulated in efforts to improve solubility. The protein structures of whey are considered to have an effect on solubility. Specifically, the degree of denaturation of whey proteins is thought to play a role in