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Saccharides protect biostructures against adverse environmental conditions mainly by preventing large scale motions leading to unfolding. The efficiency of this molecular mechanism, which is higher in trehalose with respect to other sugars, strongly depends on hydration and sugar/protein ratio. Here we report an Infrared Spectroscopy study on dry amorphous matrices of the disaccharides trehalose, maltose, sucrose and lactose, and the trisaccharide raffinose. Samples with and without embedded protein (Myoglobin) are investigated at different sugar/protein ratios, and compared. To inspect matrix properties we analyse the Water Association Band (WAB), and carefully decompose it into sub-bands, since their relative population has been shown to effectively probe water structure and dynamics in different matrices. In this work the analysis is extended to investigate the structure of protein-sugar-water samples, for the first time. Results show that several classes of water molecules can be identified in the protein and sugar environment and that their relative population is dependent on the type of sugar and, most important, on the sugar/protein ratio. This gives relevant information on how the molecular interplay between residual waters, sugar and protein molecules affect the biopreserving properties of saccharides matrices.

“Water Association” Band in Saccharide Amorphous Matrices: Role of Residual Water on Bioprotection