Identification of ice-binding proteins from Raphanus sativus and application in frozen dough

Cryopreservation is a widely employed method for processing and preserving food. However, conventional antifreeze agents often hard to mitigate the mechanical damage caused by ice recrystallization during freeze-thaw cycles. In this study, two ice-binding proteins (IBPs), COR15B and COR47, were identified from Raphanus sativus using bioinformatics and molecular biology techniques. Both IBPs exhibited significant ice recrystallization inhibition (IRI) and ice crystal morphology modification activity. A novel, high-yield Bacillus subtilis expression system was developed for the heterologous production of these IBPs, achieving approximately 50 μg/mL through response surface optimization. These proteins, even when used at thousandths of the ratio, retained their IRI activity. Notably, the heterologously expressed IBPs significantly reduced freeze-induced damage in flour-based products and improved yeast survival and fermentative capacity during repeated freeze-thaw cycles. These results highlight the considerable potential of radish-derived IBPs as cryoprotectants for enhancing food storage stability.