Date of Award


Degree Name

MS in Agriculture - Dairy Products Technology


Dairy Science


Dr. Phillip S. Tong


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 solubility.

In this experimental design, raw bovine milk was skimmed and pasteurized then enzyme-coagulated at natural pH to separate the whey. Liquid whey was then split into three aliquots and each received one of the following treatments: 1) mild heat/ freeze dry, 2) mild heat/spray dry and 3) high heat/spray dry. Heat treatment was applied to liquid whey prior to concentration. Heat treated whey was then concentrated and dried. Powders were reconstituted and analyzed for denaturation using 1) bicinchoninic acid assay for soluble protein and 2) fluorescence spectroscopy and for solubility using an insolubility index.

pH 4.6 solubility and fluorescence spectroscopy for quantifying denaturation correlated well to one another. Both found that the low heat treated samples were less denatured in structure than the sample which received the high heat treatment, regardless of drying method. However, the drying method of the protein powders was correlated to solubility rather than heat treatment. A correlation of denaturation measured in whey protein powders and solubility was apparent for the low heat, freeze dried sample and the high heat, spray dried sample.

Several conclusions were made in this research. 1) Centrifugal force causes less denaturation than membrane filtration and enzyme coagulation, thus unheated liquid whey obtained by centrifugal force can be used as a control in research on denaturation. 1) Fluorescence spectroscopy is a better method for quantifying denaturation in liquid and powdered whey compared to native PAGE and pH 4.6 solubility measured by BCA. 3) Functional solubility is dependent on denaturation and can be correlated to analytical methods of measuring denaturation.