Identifying Endogenous Proteins of Perennial Ryegrass (Lolium perenne) with Ex Vivo Antioxidant Activity

<b>Background</b>: 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 <i>ex vivo</i> could help mitigate these reactions for improved product quality. <b>Methods</b>: In this study, we investigated perennial ryegrass (<i>Lolium perenne</i> var. Abosan 1), 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 <i>in vitro</i> antioxidant properties and coupled this with quantitative bottom-up proteomics, GO-term analysis, and fraction-based enrichment. <b>Results</b>: Our findings revealed that several enzymes, such as superoxide dismutase and peroxiredoxin proteoforms, already known for their involvement in <i>in vivo</i> oxidative protection, are enriched in fractions displaying increased <i>in vitro</i> antioxidant activity, indicating retained activity <i>ex vivo</i>. Moreover, this study provides the most detailed characterization of the <i>L. perenne</i> proteome today and delivers new insights into protein-level partitioning during wet fractionation. <b>Conclusions</b>: Ultimately, this work contributes to a better understanding of the first steps of green biorefining and provides the basis for process optimization.