Bioprocessing of Red Clover (Trifolium pratense) Releases an Isoflavone Compound that Targets Membrane Fluidity in Parasitic Nematodes.

Biorefining of grassland biomass (e.g., red clover) has attracted attention as a source of high-value protein that can serve as an alternative, more sustainable feed for livestock. In addition, extracts from red clover pulp (the byproduct of this process) have shown potent antiparasitic activity, suggesting an added valorization of the biorefining process through development of a dietary supplement that can potentially be used to control parasite infection. Here, we demonstrate that the biorefinery process disrupts plant cell walls, leading to an accumulation of the isoflavone biochanin A in the pulp fraction. Analytical HPLC coupled with bioassays confirmed biochanin A as the principal antiparasitic compound. Transcriptomic analyses with the parasitic nematode Ascaris suum indicated that biochanin A exerts its antiparasitic effect by interfering with membrane fluidity, while other compounds within the pulp extract may suppress its expulsion, thereby enhancing the effect of biochanin A. Finally, we developed an autohydrolysis-ethanol extraction method to achieve even greater recovery of the active compounds from the pulp. Together, these data indicate that biorefining of red clover biomass yields a side-stream with enhanced concentrations of active compounds that exert nematocidal effects against economically important parasites.