Sustainable locally harvest protein crops for food, screening of varieties for low enzymatic activity

In Denmark, the biorefinery of protein from green biomasses, such as clovers, grasses, and alfalfa, is running at commercial scale for production of monogastric feed [1]. No commercial breakthrough has been made for food grade protein to-date [2]. Aligned with the circular biorefinery principles [3], we aim to separate the white protein from the green protein but are facing problems with endogenous proteases and redox enzymes, which has been shown to actively degrade the proteins into peptides and amino acids [4,5] as well crosslinking the proteins [6]. The loss of intact protein due to protoelytic activty significantly reduces the protein yield to ≤ 10% [7], while crosslinkages due to redox enzyme activity decrease the protein digestibility [6]. Hence indigenous enzymes present a significant barrier for commercialization of food protein from green biomass. Despite this, investigation on harnessing the potential of the major protein from green biomass, RuBisCO, green biomass remains interesting due to its promising nutritional value [8,9]. Furthermore, alfalfa and clovers are perennial legume crops, i.e. they require less N-fertilization, have longer growth season than seasonal crops, and can be harvested multiple times a year, resulting in a higher protein yield [8]. Overcoming the challenges with endogenous enzymes will enable green biomass as a high quality, locally sourced dietary protein in Denmark. 

The present project will help the transformation of sustainable protein feed crops into food protein sources. The  aim is to identify good varieties for upcycling of feed crops to food crops. Screening of clovers and grasses to identify species with low proteolytic and redox enzyme activity along with mass spectrometry idenfication of the enzymes using a proteomic approach.
 

References

1. Olesen, J.E., et al., AgriFoodTure: Roadmap for sustainable transformation of the Danish Agri-Food system. 2021.
2. Bjerre, M. New project aims to revolutionise Sustainable Protein Production for Feed, Food and Aquaculture. 2023; Available from: https://food.au.dk/currently/news/nyhed/artikel/new-project-aims-to-revolutionise-sustainable-protein-production-for-feed-food-and-aquaculture
3. Tan, E.C. and P. Lamers, Circular bioeconomy concepts—a perspective. Frontiers in sustainability, 2021. 2: p. 701509.
4. Fremery, D.D.; Kohler, G.O.; Bickoff, E.M. Pro-Xan Process - Stability of Proteins and Carotenoid Pigments in Freshly Expressed Alfalfa Juice. J Agr Food Chem 1972, 20, 1155-1158.
5. Nissen, S.H.; Lübeck, M.; Møller, A.H.; Dalsgaard, T.K. Protein recovery and quality of alfalfa extracts obtained by acid precipitation and fermentation. Bioresource Technology Reports 2022, 19, 101190. https://doi.org/10.1016/j.biteb.2022.101190
6. Amer, B, Juul, L, Møller, AH, Møller, HS & Dalsgaard, TK, (2021). Improved solubility of proteins from white and red clover - inhibition of redox enzymes Int J Food Sci Tech, 56, 302–311 doi.org/10.1111/ijfs.14632
7. Tanambell, H.; Danielsen, M.; Devold, T.G.; Møller, A.H.; Dalsgaard, T.K. In vitro protein digestibility of RuBisCO from alfalfa obtained from different processing histories: Insights from free N-terminal and mass spectrometry study. Food Chemistry 2024, 434, 137301. https://doi.org/10.1016/j.foodchem.2023.137301
8. Manevski, K.; Lærke, P.E.; Jiao, X.R.; Santhome, S.; Jorgensen, U. Biomass productivity and radiation utilisation of innovative cropping systems for biorefinery. Agr Forest Meteorol 2017, 233, 250-264. https://doi.org/10.1016/j.agrformet.2016.11.245
9. Møller, AH, Hammershøj, M., Passos, N, Tanambell, H, Stødkilde, Lene, Ambye-Jensen, M, Danielsen, M, Jensen, SK, Dalsgaard, TK (2021). Biorefinery of green biomass – how to extract and evaluate high quality leaf protein for food? J. Agric. Food Chem. 69 (48), 14341 – 14357 https://doi.org/10.1021/acs.jafc.1c04289