Effect of Conventional and Ultrasound-Assisted Extraction Conditions on the Physicochemical Properties, Phytochemical Content, Antioxidant Activity and Functional Properties of Alfalfa Protein Concentrates

Alfalfa (<i>Medicago sativa</i> L.) is an underutilized source of phytonutrients and easily digestible protein, containing all essential amino acids, highlighting its potential for food applications. This study aimed to produce alfalfa protein concentrates (APC) from frozen aerial parts and evaluate how conventional extraction and ultrasound-assisted extraction (UAE) affect the extraction yield, physicochemical properties, functional attributes, color parameters, phytochemical composition and antioxidant activity. The influence of extraction pH and the type of acid used for isoelectric precipitation was also evaluated. Paired <i>t</i>-tests (<i>p</i> ≤ 0.05) showed that UAE (37 kHz, 25 °C, 15 min) increased the extraction yield by 20.5-39.7%, the protein content in APC by 2.5-12.1% and the in vitro protein digestibility by 5.6-11.03%, depending on the extraction conditions. Ultrasound treatment decreased the levels of chlorophyll and carotenoids, modified the color parameters and increased the total polyphenols and flavonoids content. Improvements in the textural, foaming and emulsifying properties of APC were also observed. UAE also reduced the scavenging capacity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals. However, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•⁺) scavenging activity significantly increased in aqueous APC extracts, reaching 3118.8 mg TE/100 g DW. Overall, UAE proved effective in improving the yield and functionality of APC, supporting its application in the development of alfalfa-based protein ingredients.