New insights into the destabilization of fat globules in ultra-instantaneous UHT milk induced by added plasmin: Molecular mechanisms and the effect of membrane structure on plasmin action.

ABSTRACT

Residual plasmin activity in whole ultra-instantaneous UHT (UI-UHT) milk causes rapid fat rise during storage, seriously affecting consumers' purchase intentions. In this work, the molecular mechanisms underlying fat destabilization in whole UI-UHT milk by added plasmin were investigated based on the hydrolysis behavior of interfacial proteins. By using SDS-PAGE and peptidomic analysis, we found that the hydrolysis of interfacial proteins by plasmin led to a decrease in the amount and coverage of interfacial proteins and an increase in zeta-potential value, causing the flocculation and coalescence of fat globules. Moreover, the hydrolysis pattern varied in different categories of interfacial proteins by plasmin. In total, 125 peptides in all samples were identified. Plasmin tended to hydrolyze most major milk fat globule membrane (MFGM) proteins into protein fragments (>10 kDa) rather than peptides (<10 kDa). In contrast, peptides derived from caseins were more preferentially identified within a relatively short incubation time. It was the co-hydrolysis of caseins and some major MFGM proteins as anchors that destroyed the stability of MFGM. Furthermore, studies on the effect of trilayer membrane structure remaining at the interface on the hydrolysis rate of major MFGM proteins by plasmin revealed that ADPH and BTN were very sensitive to plasmin action, while PAS 7 was very resistant to plasmin action. Overall, membrane structure reduced the susceptibility of some major MFGM proteins to plasmin and provided protective effects. Therefore, this study provided important insights into the hydrolysis behavior of interfacial proteins in whole UI-UHT milk induced by plasmin.