Stabilization and extraction of alfalfa (Medicago sativa) protein following multiple postharvest processing techniques coupled with protease inactivation.

There is an ever-increasing demand for novel plant proteins that are non-allergenic, nutritionally complete, adequately functional, and can be sustainably sourced. RuBisCo is a protein that fulfills these requirements and can be sourced from alfalfa (Medicago sativa). Therefore, this study investigated several techniques to adequately extract alfalfa protein. Protein extraction from commercial sun-dried alfalfa was largely unsuccessful (≤ 5 % protein yield) due to endogenous proteolytic activity. Flash freezing of freshly harvested alfalfa followed by juicing and freeze drying (FF/J/FD) effectively preserved protein integrity with 12 % degree of hydrolysis (%DH) compared to 20 % DH in the oven dried (OD) sample. However, the FF/J/FD sample had significantly higher protease activity than the OD sample (26 vs. 11 Units/g protein). A combined acid (pH 3.0) and thermal (95 °C) treatment reduced protease activity to a negligible level in both FF/J/FD and FF/J/spray dried (FF/J/SD) samples. Protein extraction utilizing FF/J/FD sample subjected to acid/thermal protease inactivation was relatively successful with 32 % protein yield and 74 % protein purity. Protein polymerization due to thermal treatment contributed to reduced protein yield. This work provided not only foundational basis to determine the best post-harvest management practices to control proteolysis in alfalfa, but also essential information that will guide future attempts at protein extraction from green biomass to produce valuable protein ingredients for various food applications.