Abstract: Abstract: The use of peanut protein has attained an increasing attention, primarily attributed to its high nutrition value, steady supply, and low cost compared with other proteins with different sources. Peanut protein isolate (PPI) is the most refined peanut protein product, containing 90% protein on a moisture-free basis, and is used as a kind of important protein material in food industry. However, the poor protein solubility and functional properties of commercial PPI limit its application in food and non-food products. Because of small side reactions and safeness, proteolysis has been widely used to improve protein functionalities and to prepare bioactive peptides. However, peanut proteins are resistant to proteolysis due to their compact structures that protect many of the hydrolysis sites. Recently, several studies have reported that hydrothermal treatment could not only alter the spatial conformations of globular proteins, but also break up protein aggregates into smaller pieces, which may cause the exposure of previously buried hydrolysis sites. However, little work has been done so far to investigate this possibility. Therefore, this work aimed to investigate the influences of hydrothermal pretreatment on the proteolysis pattern and the structure of PPI. Hydrothermally cooked PPI (HPPI) was prepared using a CJ-200 autoclave, and protease Protamex was used for the preparation of PPI hydrolysates (PPIH) and HPPI hydrolysates (HPPIH). Response surface methodology (RSM) was used to optimize the processing conditions of hydrothermally cooking, and the optimal conditions were as follows: the pressure was 90 MPa, the temperature was 85 ℃, and the time was 20 min. The actual degree of hydrolysis (DH) of HPPIH obtained under this pretreatment condition was 16.3%±0.2%, which was not significantly different (P>0.05) from the predicted value (DH=16.2%). And the analysis of variance of the regression equation for the response surface quadratic model showed that the sequence of the importance for influential factors was pressure > temperature > time. The analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of PPI, HPPI, PPIH and HPPIH showed that hydrothermal pretreatment substantially improved the enzymatic accessibility of the major subunits of conarachin and arachin in PPI, which were initially resistant to Protamex hydrolysis. As a result, more peanut proteins in HPPI could be readily hydrolyzed and become soluble, causing a strong increase in protein recovery (PR) for HPPIH. Under the 90 MPa pressure, HPPIH showed a much higher PR of 73.4% than that of PPIH (PR=41.5%). In addition, it was somewhat surprising that the observations of SDS-PAGE profiles and DH measurement both showed that hydrothermal pretreatment enhanced the spontaneous hydrolysis of PPI. Analysis of the intrinsic emission fluorescence spectra of PPI and HPPI demonstrated that compared with the control PPI, HPPI showed a decrease in the intensity of the fluorescence peak at 323 nm, and a red-shift for the peak at 332 nm, which suggested that hydrothermal treatment caused the unfolding of tertiary structure for PPI. Analysis of Fourier transform infrared spectra (FTIR) of PPI and HPPI demonstrated that compared with the control PPI, HPPI showed a significant decrease in α-helix and β-turn, and an increase in β-sheet and random coil, which suggested that hydrothermal treatment could loosen the secondary structure of peanut protein. So it is inferred that the unfolded tertiary structure and the loosened secondary structure for HPPI after hydrothermal pretreatment may be the main causes for its improved enzymatic accessibility. In conclusion, this study shows that hydrothermal pretreatment is a highly effective technique to accelerate and enhance the proteolysis of PPI.