This study aimed to prepare and screen salty peptides and elucidate the factors triggering their saltiness properties and saltiness-enhancing effects. Electronic tongue results indicated that samples treated with Alcalase (AJ) exhibited the highest saltiness intensity (5.05 ± 0.11) and greater saltiness-enhancing compared to those treated with Flavourzyme (AF), Neutrase (AZ), and Papain (AM). Peptides identified by LC-MS/MS were docked to the TMC4 receptor, resulting in 23 peptides with good docked results. The key amino acid residues (Thr431, Arg433, Phe440, Phe432, and Ser273) were discovered. The salty peptides WPGFK (633.75 Da), YFDWPGFK (1059.17 Da), and YFDWPGFKT (1160.27 Da) were selected for salt reduction and molecular characterization studies. Electronic tongue results showed that the 0.01 % WPGFK solution matched the saltiness of the 0.24 % NaCl solution and demonstrated superior saltiness-enhancing compared to YFDWPGFK and YFDWPGFKT. Primary sequence analysis of three salty peptides suggested that the WPG amino acid segment might play a crucial role in generating saltiness. Circular dichroism (CD) analysis of the secondary structure indicated that increased random coil and decreased β-turn contents resulted in higher saltiness perception and increased random coil content led to greater saltiness-enhancing ability.
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