Metabolomic Profiling and Functional Characterization of Biochar from Vine Pruning Residues for Applications in Animal Feed

Biochar has garnered interest as a feed ingredient in livestock nutrition, due to its functional properties, circularity, and effect on environmental impact reduction, and potential to reduce environmental impact, offering significant benefits aligned with sustainable agro-zootechnical practices. The in vivo effects of biochar are closely tied to its physical characteristics, which can vary depending on the biomass used as feedstock and the production process. This variability can result in heterogeneity among different biochar used in animal nutrition, leading to inconsistent outcomes reported in the literature. The aim of this study was to perform a metabolomic and functional characterization of an aqueous biochar extract from vine pruning waste, in order to predict its potential in vivo effects as a functional feed ingredient. Metabolomic analysis of the biochar extracts was performed using Quadrupole Time-of-Flight (QTOF) High-Performance Liquid Chromatography Tandem Mass Spectrometry (HPLC MS/MS). Antimicrobial activity against E. coli F18+ and E. coli F4+ was evaluated using standard growth inhibition assays, while quorum sensing of E. coli exposed to biochar extracts was evaluated by Realtime-PCR. Addition-ally, prebiotic activity was assessed by exposing selected Lactobacillus strains to the biochar extract with growth patterns monitored to determine species-specific responses. The metabolomic profile revealed several distinct molecular classes, including multiple peaks corresponding to phenolic compounds. The extract demonstrated hsignificant growth inhibitory activity against both E. coli pathotypes, reducing growth by 29% and 16% for F4+ and F18% respectively (p<0,001). The rela-tive expression of genes involved in quorum sensing (MotA, FliA for biofilm formation, and FtsE, HflX for cell division) suggested that the observed inhibitory effects were likely due to interfer-ence with flagellar synthesis and motility as well as a reduced cell division. The biochar extract also demonstrated species-specific prebiotic potential. In conclusion, biochar derived from vine pruning waste represents a valuable feed ingredient due to its functional properties that may contribute to reduced antibiotic use in livestock production.