Microstructural Analysis of Whey/Soy Protein Isolate Mixed Gels Using Confocal Raman Microscopy

This work explores the potential of confocal Raman microscopy to investigate the microstructure of mixed protein gel systems. Heat-set protein gels were prepared using whey protein isolate (WPI), soy protein isolate (SPI), and mixtures thereof, with a total of five different whey-to-soy protein ratios (100, 75, 50, 25, and 0%). These were analysed using confocal Raman microscopy, and different data analysis approaches were used to maximize the amount of structural and compositional information extracted from the spectral datasets generated, including both univariate and multivariate analysis methods. Small spectral differences were found between pure WPI and SPI gels, mainly attributed to conformational differences (amide bands), but SPI exhibited considerably greater auto-fluorescence than WPI. The univariate analysis method allowed for a rapid microstructural analysis, successfully mapping the distribution of protein and water in the gels. The greater fluorescence of the capsule-like structures found in the mixed gels, compared to other regions rich in proteins, suggested that these may be enriched in soy proteins. Further analysis, using a multivariate approach, allowed us to distinguish proteins with different levels of hydration within the gels and to detect non-proteinaceous compounds. Raman microscopy proved to be particularly useful to detect the presence of residual lipids in protein gels.