Ultrasound-assisted extraction of Litsea cubeba cake protein: Process optimization, structural characterization and functional evaluation

Metabolically dysregulated steatotic liver disease (MASLD) poses an increasingly significant threat to human health. In this study, ultrasound-assisted extraction (UAE) was employed to isolate proteins from Litsea cubeba ( L. cubeba ) seed cake, aiming to enhance both protein functionality and nutritional quality. Response surface methodology (RSM) was used to determine the optimal extraction parameters: an ultrasonic time of 31.84 min, a power of 343.93 W, and a temperature of 34.07 °C. Under these optimized conditions, the extraction yield of ultrasound-treated L. cubeba cake protein (ULCAP) increased from 58.87% to 62.20%. Mechanistically, ultrasonic cavitation effectively induces secondary structure remodeling and micro-morphological alterations in proteins through mechanical effects generated by cavitation, including intense shear forces and shockwaves, which result in the formation of physical pores. Fourier transform infrared spectroscopy (FTIR) revealed a significant decrease in the relative α-helix content in ULCAP, accompanied by an increased amide I/amide II ratio. Furthermore, scanning electron microscopy (SEM) confirmed a porous surface structure on the ULCAP. Consequently, the protein's solubility, foaming capacity and stability, emulsifying activity index (EAI), and emulsion stability index (ESI) were all significantly improved. Amino acid analysis further indicated that the UAE method enhanced the extractability of essential amino acids. In vivo animal experiments, combined with transcriptomic and metabolomics analysis, demonstrated that the ultrasound-optimized protein could reverse hepatic steatosis in mice, thereby ameliorating the progression of MASLD by modulating lipid metabolism pathways.