In this study, the relationship between protein hydrolysis and oil release was studied by fluorescence microscopy and Fourier Transform Infrared (FTIR) spectroscopy, respectively. Five commercial proteases (Flavourzyme 1000L, Neutrase 1.5MG, Protamex, Alcalase 2.4L, and Thermolysin) were evaluated for oil extraction from rapeseed, and Alcalase 2.4L was found to be the best enzyme for highest free oil yield (81.81%). When use this enzyme, the results showed that parts of proteins restricted the release of oil, and the oil released almost completely was achieved after hydrolysis for 3.0 h. To further reveal the relationship between oil release and protein hydrolysis, the effects of hydrolysis time on the degree of hydrolysis of protein, and free oil yield were studied. The results showed that the release of oil was only related to the hydrolysis of parts of proteins, and when the degree of hydrolysis of protein was more than 17.05%, the effect of protein hydrolysis on oil release was greatly reduced. The intrinsic relationship between the structural changes of proteins and the release of oil in the hydrolysis process was analyzed by comparing protein secondary structure of different hydrolysis time, and the results showed that the transition of protein structure from order to disorder was an important factor for oil release.
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