Oxidation of whey protein isolate after thermal convection and microwave heating and freeze-drying: Correlation among physicochemical and NIR spectroscopy analyses

Abstract

This study investigated the oxidative susceptibility of whey protein isolate (WPI) dispersions treated by microwave or thermal convection before freeze-drying. WPI (20 mg protein/mL) in distilled water (DW) was heated at 63 ± 2 °C for 30 min by microwave (WPI-MW) or convection heating (WPI-CH) and freeze-dried. Untreated WPI (WPI-C), WPI solubilized in DW and freeze-dried (WPI-FD), and WPI solubilized in DW, heated at 98 ± 2 °C for 2 min and freeze-dried (WPI-B) were also evaluated. Structural changes (turbidity, ζ potential, SDS-PAGE, and near-infrared spectroscopy (NIR)) and protein oxidation (dityrosine, protein carbonylation, and SH groups) were investigated. WPI-FD showed alterations compared to WPI-C, mainly concerning carbonyl groups. Microwave heating increased carbonyl groups and dityrosine formation compared to conventional heating. NIR spectrum indicated changes related to the formation of carbonyl groups and PCA analysis allowed us to distinguish the samples according to carbonyl group content. The results suggest that NIR may contribute to monitoring oxidative changes in proteins resulting from processing.

Keywords: Carbonylated protein; Dityrosine; Protein oxidation.

Fig. 1

General flowchart of sample preparation and treatment conditions.

Fig. 2

Fluorescence spectra of dityrosine (a) and tyrosine fluorescence (b): WPI-C untreated (control); WPI-FD freeze-dried sample; WPI–CH heated at 63 ± 2 °C under magnetic stirring for 30 min; WPI-MW microwave heated at 63 ± 2 °C under magnetic stirring for 30 min; and WPI-B heated at 98 ± 2 °C under magnetic stirring for 2 min. All samples were lyophilized after thermal treatment and diluted in 20 mM sodium phosphate buffer (pH 7.0) containing 0.6 M KCl for analysis. Results expressed as fluorescence intensity/mg protein.

Fig. 3

SDS-PAGE electrophoresis profile under reducing conditions of WPI samples (columns 2–6) and standard molecular mass marker (column 1). WPI-C untreated (control); WPI-FD freeze-dried sample; WPI-B heated at 98 ± 2 °C under magnetic stirring for 2 min; WPI-MW microwave heated at 63 ± 2 °C under magnetic stirring for 30 min; and WPI–CH heated at 63 ± 2 °C under magnetic stirring for 30 min.

Fig. 4

NIR spectra after application of SNV (standard normal variation): WPI-C untreated (control); WPI-FD freeze-dried sample; WPI–CH heated at 63 ± 2 °C under magnetic stirring for 30 min; WPI-MW microwave heated at 63 ± 2 °C under magnetic stirring for 30 min; and WPI-B heated at 98 ± 2 °C under magnetic stirring for 2 min.

Fig. 5

Score graph (PC1 vs PC2) of pre-processed NIR spectra with SNV (a) and loadings graph of the first four main components (b) of samples: WPI-C untreated (control); WPI-FD freeze-dried sample; WPI–CH heated at 63 ± 2 °C under magnetic stirring for 30 min; WPI-MW microwave heated at 63 ± 2 °C under magnetic stirring for 30 min; and WPI-B heated at 98 ± 2 °C under magnetic stirring for 2 min.