A new Ultrasound-Ultrafiltration-Assisted Alkaline Isoelectric Precipitation (UUAAIP) technique was employed for producing alfalfa protein isolate from alfalfa leaves. A five-factor-three-level Box-Behnken Design (BBD) was used to optimize the UUAAIP process for maximizing both extraction yield and protein content simultaneously. The optimum conditions were as follows: solvent/solid material ratio of 43.3 mL/g, pH of 10.1, extraction temperature of 42.5 degrees C, extraction time of 102 min, and flow rate of 9.7 L/h. Under these conditions, the maximum predicted extraction yield and protein content were 14.5 g/100 g and 91.1 g/100 g, respectively. UUAAIP technique improved the average molecular weight and color of the isolate. Moreover, it was efficient in removing the saponins and phenolic compounds as anti-nutritional factors compared to heat-coagulation extraction (HCE) and alkaline isoelectric precipitation extraction (AIPE). The protein isolate extracted by UUAAIP had greater solubility, water-holding and oil-binding capacities but lower emulsifying and foaming properties than those extracted by HCE and AIPE.
