Size exclusion chromatography to evaluate in vitro proteolysis: A case study on the impact of microstructure in pulse powders

Cellular pulse ingredients are increasingly being studied but little knowledge on their proteolysis patterns upon digestion is available. This study investigated a size exclusion chromatography (SEC) approach to study in vitro protein digestion in chickpea and lentil powders, providing novel insights into proteolysis kinetics and the evolution of molecular weight distributions in the (solubilized) supernatant and (non-solubilized) pellet frac-tions. For the quantification of proteolysis, SEC-based analysis was compared to the commonly used OPA (o- phthaldialdehyde) approach and nitrogen solubilized upon digestion, leading to highly correlated proteolysis kinetics. Generally, all approaches confirmed that microstructure dictated proteolysis kinetics. However, SEC analysis delivered an additional level of molecular insight. For the first time, SEC revealed that while bio-accessible fractions reached a plateau in the small intestinal phase (around 45-60 min), proteolysis continued in the pellet, forming smaller but mostly insoluble peptides. SEC elutograms showed pulse-specific proteolysis patterns, unidentified using other current state-of-the-art methods.

Automated summary

This study used a size exclusion chromatography (SEC) approach to investigate in vitro protein digestion in chickpea and lentil powders, providing new insights into proteolysis kinetics and molecular weight distributions. SEC-based analysis was highly correlated with OPA approach and nitrogen solubility, confirming the role of microstructure in proteolysis kinetics. SEC analysis revealed that bio-accessible fractions reached a plateau in the small intestinal phase, while proteolysis continued in the pellet, forming smaller but mostly insoluble peptides. SEC elutograms showed pulse-specific proteolysis patterns not identified by other current state-of-the-art methods.