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Lactose is a common component found in many foods and dairy products. In this study, the vibrational signatures in the crystalline structure of α-, β-, and α-lactose monohydrate were calculated based on quantum chemistry calculation (QCC), whilst the vibrational spectra in freeze-dried lactose equilibrated at various aw and pre-humidified amorphous lactose (0.33 aw) stored from 25 to 95 °C were determined by using Raman and FT-IR spectroscopies. The vibrational signatures of crystalline lactose were affected by the presence of water acc