Synergistic effects of dual-frequency ultrasound on Stropharia rugosoannulata protein extraction: Mechanism unraveled through proteomics and physicochemical properties

Although dual-frequency ultrasound (DFU) shows promise in overcoming the limitations caused by localized intense cavitation in single-frequency ultrasound (SFU), its efficiency-enhancing mechanism in mushroom protein extraction, particularly the underlying molecular-level mechanisms, remains unclear. In this study, proteins were extracted from Stropharia rugosoannulata using SFU (23 kHz) and DFU (23/25, 23/28, 23/40 kHz). The synergistic effects and mechanisms of DFU were investigated through a combination of physicochemical characterization and proteomics. DFU significantly increased the protein content by over 50%, and the proteins exhibited superior thermal stability (denaturation temperature range: 134.87–136.10 °C) and a smaller particle size (240.6–443.7 nm). 727 proteins were found by proteomic analysis to be substantially more prevalent in the DFU-treated groups than in the SFU-treated groups. The proteins that were differentially expressed were mainly soluble functional proteins that were widely involved in processing genetic information, central carbon metabolism, and amino acid metabolism. This indicates that DFU achieves a synergistic enhancement effect by efficiently releasing the intracellular core functional proteome while concurrently altering protein structures to suppress molecular aggregation. This study deepens the understanding of the DFU mechanism at the molecular level and provides a theoretical foundation for its application in the sustainable production of high-quality alternative proteins. • DFU boosts protein yield from mushroom by >50% versus single-frequency ultrasound. • Extracted proteins show high thermal stability (up to 136 °C) and reduced aggregation. • Proteomics reveals DFU releases functional intracellular enzymes and ribosomal proteins. • The 23/28 kHz frequency combination is optimal for balanced yield and protein quality. • A mechanism model illustrates DFU's synergistic cell disruption and protein protection.