Circular use of supercritical CO₂-extracted Peppermint (Mentha × piperita L.) residues in ruminant nutrition: An In Vitro fermentation model

The growing focus on circular economy strategies has encouraged the valorization of agro-industrial by-products as functional feed ingredients for ruminants. This study evaluated the effects of peppermint (Mentha × piperita L.) biomass and its post-distillation residues on in vitro rumen fermentation, volatile fatty acids (VFA), gas kinetics, methane production, and microbial composition. Four treatments were tested: control forage (Lolium multiflorum Lam., CTRL), raw peppermint (Mp), residue after supercritical CO₂ extraction (rMp), and the same residue supplemented with peppermint extract (erMp). Compared to CTRL, rMp promoted gas production per gram of dry matter, while Mp and rMp increased acetate and decreased butyrate, indicating a shift of hydrogen flow from methanogenesis to acetogenesis. Methane concentration (CH₄ % v/v) was significantly reduced in Mp, confirming inhibition of methanogenesis, whereas erMp showed delayed fermentation, higher methane yield, and reduced microbial diversity, likely due to excess essential oil compounds. Microbial analysis revealed treatment-specific shifts: Mp and rMp promoted acetogenic and lactate-utilizing taxa (e.g., Lachnospiraceae, Selenomonas ruminantium) and suppressed Prevotella. Overall, peppermint-based substrates significantly altered microbial community structure (PERMANOVA, UniFrac). These results highlight that peppermint biomass and its extraction residues can beneficially modulate rumen fermentation, enhance acetate production and lower methane, if inclusion levels are properly managed. Post-extraction residues retain functional bioactivity, offering a sustainable strategy for waste valorization in circular livestock systems.