Saltiness Enhancement of Soy Peptides by Modulating Amiloride-Insensitive Salt-Responsive Cells and Interacting with Cell Membranes

Saltiness-enhancing peptides hold great potential for salt reduction in the food industry. This study investigated the saltiness-enhancing mechanism of soy peptides E (EDEGEQPRPF), DG (DEGEQPRPFP), and 9AA (DEGEQPRPF), focusing on their interactions with amiloride-insensitive taste cells and cell membranes. Sensory evaluation showed that adding E and DG (0.4 mg/mL) to 50 mM NaCl increased perceived saltiness to 61.4 and 54.78 mM NaCl, while 9AA had no effect. Calcium imaging of taste organoids highlighted the role of Cl^- in the amiloride-insensitive pathway. Peptide E enhanced the response of amiloride-insensitive salt-responsive cells by 35.19%, while DG and 9AA did not. Single-cell RNA sequencing revealed no functional ENaC heterotrimer and high Tmc4 expression in all types of taste cells, while Trpv1 was found in only one circumvallate papilla (CV) taste cell. E and DG form more stable bonds with TMC4 via hydrogen bonds and water bridges compared to 9AA, as evidenced by molecular dynamics simulations. Negatively charged peptide E, with an α-helical-like structure, adsorbed onto liposomes more than DG and 9AA due to its N-terminal Glu, suggesting E may indirectly modulate taste receptor function by altering membrane potential. These findings provide insights into the structure-function relationship of saltiness-enhancing peptides.