Identifying Endogenous Proteins of Perennial Ryegrass (<i>Lolium perenne</i>) with <i>Ex Vivo </i>Antioxidant Activity

During the initial steps of green biorefining aimed at protein recovery, endogenous proteins and enzymes, along with e.g. phytochemical constituents, are decompartmentalized into a green juice. This creates a highly dynamic environment prone to a plethora of reactions including oxidative protein modification and deterioration. Obtaining a fundamental understanding of the enzymes capable of exerting antioxidant activity ex vivo could help mitigate these reactions for improved product quality. In this study, we investigated perennial ryegrass (Lolium perenne), one of the most widely used turf and forage grasses, as a model system. Using size exclusion chromatography, we fractionated the green juice to investigate in vitro antioxidant properties and coupled this with quantitative bottom-up proteomics, GO-term analysis, and fraction-based enrichment. Our findings revealed that several enzymes, already known for their involvement in in vivo oxidative protection, are enriched in fractions displaying increased in vitro antioxidant activity, indicating retained activity ex vivo. Moreover, this study provides the most detailed characterization of the L. perenne proteome today and delivers new insights into protein-level partitioning during wet fractionation. Ultimately, this work contributes to better understanding the first steps of green biorefining and provides the basis for process optimization.