Stabilization of alfalfa (Medicago sativa) protein following multiple postharvest treatments coupled with protease inactivation and optimization of protein extraction

Alfalfa is the highest-yielding, multiple-harvest, perennial forage crop, with multiple agricultural & environmental benefits such as nitrogen fixation and improving soil quality. Primarily used as animal feed, alfalfa is high in RuBisCo protein, which is a highly digestible, soluble, complete source of protein, making it an excellent novel protein source for human consumption. The biggest challenge, however, is the presence of proteases that hydrolyze the protein after harvest, thus limiting the success of protein extraction to produce an isolate. Hence, the objective of this research was to stabilize alfalfa protein following specific post-harvest techniques, inactivate proteases, and optimize protein isolation from alfalfa total herbage.

Alfalfa total herbage was subjected to different post-harvest treatments including flash freezing on farm, juicing, freeze drying, oven drying, steam blanching, and/or spray drying, followed by an acid-heat enzyme inactivation. Protein extraction conditions were optimized following alkaline solubilization with isoelectric precipitation. Protease activity, degree of hydrolysis (DH), and protein profile were evaluated to monitor and validate protein stability and impact of inactivation and extraction conditions on the integrity of the protein. Protein yield and purity were monitored.

The combination of flash freezing on farm, juicing, and freeze or spray drying contributed to lowest initial DH. The lowest protease activity and the minimal change in DH was observed after an acid-heat protease inactivation of 95°C at pH 3 for 7 min. Accordingly, protein isolation was successful with a protein yield and purity of 32% and 74%, respectively. Protein yield was successfully increased to 45% following double solubilization at pH 7, purification at pH 4.5, and dialysis.

This project contributed to significant advances towards enhancing the potential of  alfalfa protein as a novel plant protein for human consumption. Research will continue to unravel the functional and nutritional perspective of alfalfa protein for various high-value applications.