Preparation of phosphopeptides from phosvitin using the high-temperature and mild pressure pretreatment and enzyme combinations

Phosvitin is an excellent source for phosphopeptide production, but it is highly resistant to enzymatic hydrolysis. The high-temperature and mild pressure (HTMP) pretreatment hydrolyzed phosvitin at random sites and helped the subsequent enzyme hydrolysis of the peptides produced. With the HTMP pretreatment alone, 154 peptides were produced while the use of trypsin, Protex 6L, and Multifect 14L in combination with pretreatment produced 252, 280, and 164 peptides, respectively. The use of two enzyme combinations (Trypsin + Protex 6L and trypsin + Multifect 14L) helped the hydrolysis further. The number of phosphopeptides produced increased when the modifications within the same amino acid sequences were considered. This study indicated that HTMP pretreatment was a breakthrough method to improve the enzymatic hydrolysis of phosvitin that enabled an easy production of phosvitin phosphopeptides (PPPs) for their subsequent functional characterizations.