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Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor the infrared absorption spectra of 79 mushroom samples from 29 Pleurotus ostreatus, P. eryngii and P. nebrodensis strains cultivated on wheat straw, grape marc and/or by-products of the olive industry. The spectroscopic analysis provided a chemical insight into the mushrooms examined, while qualitative and quantitative differences in regions related to proteins, phenolic compounds and polysaccharides were revealed among the species and s