This study examined the effect of high-intensity ultrasound-assisted alkaline extraction (HUAE) on the extraction yield and the physicochemical and functional properties of soy protein (SP) using the two-pot multivariate method for the first time. Plackett-Burman Design (PBD) coupled with Response Surface Methodology (RSM) was systematically utilized to select and subsequently optimize the HUAE parameters. Based on PBD results, the significant extraction factors were liquid to solid ratio (LSR), temperature, ultrasonic amplitude, and extraction time. The optimum conditions for the maximal extraction yield and minimal energy consumption were 50:1 mL/g LSR, 50 °C, 48 % ultrasonic amplitude, and 10 min extraction time. At optimum conditions, the extraction yield (35.28 %) was significantly improved compared to traditional extraction (26.39 %). Besides, HUAE resulted in modification of the protein secondary and tertiary structures due to the unfolding of protein molecules and the exposure of hydrophobic groups or regions as shown by FTIR spectroscopy, free sulfhydryl analysis, and scanning electron microscopy. These structural changes led to decreased solubility and emulsifying activity but improved emulsion stabilization and antioxidant properties. With future development, HUAE could potentially produce soy protein for targeted applications, broadening its utilization and meeting the need for more sustainable alternative processing.
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