Transglutaminase dosage governs proteolytic kinetics and cleavage patterns during in vitro digestion of pea proteins: a proteomics-driven molecular analysis

Protein structuring in foods strongly governs their digestion behaviour. To study that, pea protein was crosslinked using varying transglutaminase (TG) dosages (0-10 U TG/g protein, 40 °C for 1 h) and subjected to in vitro gastrointestinal digestion using the INFOGEST static protocol. Gel texture was analysed prior to digestion to link macroscopic properties with digestion outcomes. Digestive behaviour was followed kinetically by measuring trichloroacetic acid (TCA)-soluble protein as well as the TCA-soluble hydrolysed protein fraction. High performance size exclusion chromatography (HP-SEC) was used to unravel changes in peptide size distribution in the digestive supernatant. In general, increasing crosslinking intensity produced firmer gels and led to slower proteolysis during digestion, with most prominent impact in the gastric phase. Moreover, levels of bioaccessible protein (<1 kDa) were also impacted especially in the gastric phase due to restricted accessibility of pepsin to substrate sites. Proteomic analysis further revealed that more missed cleavages were present in peptides released from samples with extensive crosslinking.