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Effects of pH on the Morphology and Mechanical Property of Heat-induced Whey Protein Aggregates
Whey proteins denature and aggregate when they are heated in aqueous environments. It is known that the morphology of resulting aggregates varies, depending on pH. However, the structure-property relationship of heat-induced whey protein aggregates has not been fully understood. The objectives of this study were to study the effect of pH on the morphology of heat-induced whey protein aggregates, and to seek correlations between their morphologies and mechanical properties.
Whey protein was dissolved in de-ionized water, adjusted to pH 3-7, and heated at 80±0.2°C. Subsamples were taken at pre-specified time intervals and quenched in a 0°C water bath. The sample solutions were diluted to a protein concentration of 10-100 ppm, deposited on to freshly cleaved mica surfaces, air-dried, and imaged using atomic force microscopy (AFM) operated in peak-force tapping mode in air. Further mechanical tests were done with AFM force spectroscopy, where the whey protein aggregates were indented directly to obtain interaction forces. These force curves were analyzed where the Young’s modulus (E) of the samples can be fitted and calculated using the Hertzian model. The data was used to verify the mechanical and surface properties of the samples with different pH obtained with AFM imaging.
At pH 3, a relatively small fraction of protein aggregates revealed fibrillar morphologies, while the majority of aggregates appeared to be particulate. At other pH’s, only particulate aggregates were observed. All of these particulate aggregates were composed of smaller elementary particles, suggesting that the heat-induced aggregation was a two-step process regardless of pH, consisting of the formation of primary aggregates, followed by the secondary aggregation between primary aggregates. The Feret’s diameter, representing the diameter of the smallest circle that entirely covers an individual whey protein aggregate, became more dependent of the aggregate size with increasing pH, indicating that whey protein aggregates became more extended, coarse, or anisotropic with increasing pH. Furthermore, the surface roughness evaluated based on the cross-sectional height data decreased by a factor of 2 with increasing pH from 5.5 to 7. This suggested that the protein aggregates collapsed, meaning that the primary aggregates were denser and more tightly packed within the aggregate. From the force spectroscopy analysis, the samples prepared at pH 7 showed larger E values than the samples prepared at pH 5.5. This suggested that the samples at pH 7 were stiffer, which confirmed with the previous morphological results.
Keywords: Whey Protein, Heat-induced Aggregation, AFM