Real-time monitoring of casein gel microstructure during simulated gastric digestion monitored by small-angle neutron scattering

The evolving structure of protein-based foods during the digestion process is critical to the release of nutrients. However, traditional in vitro monitoring of the gel micro- and nano-structure during digestion involves analysing sample aliquots taken at different digestion time periods. This can pose issues for some gels, such as casein-based gels, as they are sensitive to sample manipulation and environmental changes. Herein, a newly developed flow setup was utilised to monitor (at the micro- and nano-length scales) the gel protein network of rennet-induced (RG) and transglutaminase-induced acid gels (TG) in situ and in real-time during simulated gastric digestion using ultra-small and small-angle neutron scattering (USANS and SANS). The proteolysis kinetics of the gels were investigated at two different pepsin enzyme concentrations (2000 and 8000 U mL-1) and in two different solvent environments (H2O and D2O). Results indicate that the flowing in situ system had a greater effect on the microstructural breakdown of TG relative to the acid-sensitive RG, compared to the traditional static method. This is the first in situ digestion study observing the structural changes of large protein gel particles with USANS or SANS in real-time. Our findings advance the understanding of the kinetics of casein gel disintegration under simulated conditions of gastric digestion relating to pepsin enzyme concentration and solvent environment, and critically, the utilisation of a new in situ and real-time setup for neutron studies.