Digestion of Proteins in Milk: Comparing different in vitro systems with in vivo data

Digestion is a highly organized sequence of biochemical, enzymatic and mechanical processes needed to release nutrients, such as peptides or free amino acids from proteins structuring complex food matrices. During the gastric phase, they are gradually denatured through the acid released into the stomach and hydrolysed by pepsin. At the intestinal phase of digestion, proteases, such as trypsin, chymotrypsin, elastase and others, are secreted to further degrade the proteins and peptides until they are absorbed through the intestinal epithelium. Static or dynamic in vitro digestion models are widely used to study this step-by-step degradation of proteins to peptides and free amino acids, raising the question on the differences between digestion models. Generation and destruction of specific peptides during in vitro digestion of skim milk powder, following either the static international consensus COST Infogest digestion protocol or the dynamic digestion model DIDGI were compared, and verified in vivo in pigs. The hydrolysis of the five most abundant milk proteins during digestion – their peptides and free amino acids – were investigated by gel electrophoresis, mass spectrometry and HPLC after the gastric and the intestinal -phase. Moreover, time-resolved in vitro digestion experiment, generation and degradation of specific peptides and release of free amino acids were followed in the two in vitro digestion models and compared with the digestion in pigs. The protein specific hydrolysis for each digestion was visualized and compared by generation of abundance-dependent peptide patterns. Highly abundant peptides observed after complete digestion indicate digestion-resistant protein sequences and represent interesting sources of bioactive peptides or allergenic epitopes. Partial-least-square data analysis (PLS-DA) comparing static and dynamic in vitro digestion with the in vivo pig data showed a high correlation of gastric and intestinal samples at the peptide level. The same analysis at the level of free amino acids showed a good correlation at the gastric phase. However, at the intestinal level, the three models formed distinct groups which could be due to absorption mechanisms in the in vivo situation