Submitted to: Food Research International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/28/2024
Publication Date: 1/2/2025
Citation: Smovzhenko, A., Heuschele, D.J., Ismail, P.B. 2025. Stabilization and extraction of alfalfa (Medicago sativa) protein following multiple postharvest processing techniques coupled with protease inactivation. Food Research International. 201. Article 115588. http://doi.org/10.1016/j.foodres.2024.115588.
DOI: http://doi.org/10.1016/j.foodres.2024.115588

Interpretive Summary: 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 if sourced from alfalfa (Medicago sativa). Humans need to digest either intact or denatured proteins to promote efficient absorption into the blood stream. Unfortunately protein within alfalfa leaves begins to breakdown into individual amino acids once the plant has been harvested. This protein breakdown changes the physical and chemical properties of any food product using alfalfa leaf protein as an additive and will inhibit absorption of amino acids into the bloodstream. In this study, we investigated multiple methods of post-harvest treatment to determine methodology that would maintain protein structure after harvesting. Sun-dried alfalfa hay contained the least amount of intact protein, while alfalfa immediately flash frozen with liquid nitrogen, juiced and then freeze dried effectively preserved all of the protein intact. This preservation only lasted as long as no water was added to the protein powder. Most food processing methods require the addition of water at some stage. Therefore, we conducted further testing on methods to maintain the intact proteins during the protein purification steps of food processing. Heat treating the freeze dried powdered material before adding water resulted in the recovery of 74% pure intact protein compared to water (5%) and other methods tested by other lab groups (20 - 35%). This discovery is key to making the use of alfalfa leaf protein in human foods economical for processors.

Technical Abstract: 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 if 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 (less than 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 harvest 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.