Identification and isolation of a novel antifreeze peptide from crayfish shells

In this study, a novel antifreeze peptide was isolated from crayfish shells and its antifreeze mechanism and activity were investigated. Firstly, the crayfish shells peptides (CSPs) were prepared by Enzymatic Hydrolysis Technology (alkaline protease and trypsin) and the CSPs could significantly enhance the survival rate of Saccharomyces cerevisiae from 12.5% to 88.5% after 24h freeze-thaw cycle. Then, a total 1004 peptides were identified from CSPs by liquid chromatography/tandem mass spectrometry (LC-MS/MS), among those peptides, two peptide sequences YWDHPIRDGFAPH (AFP1) and GPPGKPGIPDIVDW (AFP1) were selected as potential antifreeze polypeptides (AFPs) according the physicochemical properties: instability index, hydrophilicity, and confidence level. Furthermore, the antifreeze activity and mechanism of AFP1 and AFP2 were studied through molecular dynamics simulation, revealing that the AFP1 and AFP2 could adsorb to ice surface with 7 and 14 hydrogen bonds, respectively. Moreover, AFP2 has a more stable binding capacity and could interact with 33 water molecules. At last, the results of differential scanning calorimetry (DSC) and cold-stage polarization microscope indicated that the thermal hysteresis (TH) values of AFP2 were 1.62 - 2.09 degrees C and could inhibit ice recrystallization. Therefore, this study provides a theoretical basis for developing safe peptide-based cryoprotectants which will improve the quality of frozen foods.