Protein Fractionation of Green Leaves as an Underutilized Food Source—Protein Yield and the Effect of Process Parameters

Abstract

Green biomass has potential as a sustainable protein source for human consumption, due to its abundance and favorable properties of its main protein, RuBisCO. Here, protein fractionation outcomes of green leafy biomass from nine crops were evaluated using a standard protocol with three major steps: juicing, thermal precipitation, and acid precipitation. Successful protein fractionation, with a freeze-dried, resolubilized white protein isolate containing RuBisCO as the final fraction, was achieved for seven of the crops, although the amount and quality of the resulting fractions differed considerably between crops. Biomass structure was negatively correlated with successful fractionation of proteins from biomass to green juice. The proteins in carrot and cabbage leaves were strongly associated with particles in the green juice, resulting in unsuccessful fractionation. Differences in thermal stability were correlated with relatedness of the biomass types, e.g., Beta vulgaris varieties showed similar performance in thermal precipitation. The optimal pH values identified for acid precipitation of soluble leaf proteins were lower than the theoretical value for RuBisCO for all biomass types, but with clear differences between biomass types. These findings reveal the challenges in using one standard fractionation protocol for production of food proteins from all types of green biomass and indicate that a general fractionation procedure where parameters are easily adjusted based on biomass type should instead be developed.

Comparison of Wet Fractionation Methods for Processing Broccoli Agricultural Wastes and Evaluation of the Nutri-Chemical Values of Obtained Products

“…This could be attributed to the fact that the MW-LPC is largely composed of coagulated protein aggregates, which could be solubilized to a small extent. Typically, imperfectly coagulated copies of some abundantly expressed proteins such as Rubisco subunits are seen with weak appearance [ 1 ]. Even fewer and weaker bands could be detected in the BJs than in the LPCs due to the vacuum filtration, which retains the precipitated/coagulated proteins in solid phase.…”

“…The protein composition of green leaves is more heterogeneous than the stored protein in the seed endosperm or cotyledon. Along with it, the most abundant leaf protein is the RuBisCo enzyme (ribulose-1,5-bisphosphate carboxylase/Oxygenase enzyme), which can constitute 30–50% of the soluble cell protein in C3 plants [ 1 ]. It is located in the stroma of the chloroplast.…”

Is now the time for a Rubiscuit or Ruburger? Increased interest in Rubisco as a food protein

“…Alternatively, leaves can be frozen or dried before processing. While several small-scale studies freeze plant material for storage before processing ( Nynäs et al , 2021 ), and freezing does not appear to be detrimental to subsequent processing, large-scale freezing of plant material has a high energy cost ( Tamayo Tenorio et al , 2017 ). Similarly, drying the leaves also has a high energy cost, and extraction of protein from dried leaves is impaired by low yields and purity ( Hojilla-Evangelista et al , 2017 ).…”

“…1 ). Other methods of extraction include sugar cane rolls, hammer mills, or shredders, but the use of twin-screw presses has been the main method described for plant material such as sugar beet leaves, lucerne, or other crop plants ( Tamayo Tenerio et al ., 2017 ; Edwards et al , 1975 ; Nynäs et al , 2021 ). During the extraction process, different compounds are sometimes included to improve the yield of product, as described for the extraction of chlorophyll from spinach ( Özkan and Bilek, 2015 ).…”

“…Maintaining native-structure Rubisco throughout the extraction process is often crucial for the final product; in this case, the methods used must ensure Rubisco is not denatured. One simple process is to heat the juice solution to 50–55 °C for 20–30 min, which causes a majority of chlorophyll and associated proteins (‘green protein’) to aggregate ( Tamayo Tenorio et al , 2016 ; Nynäs et al , 2021 ). This temperature is below that necessary to denature Rubisco, and the precipitated compounds can be removed by centrifugation or decanting.…”