Perilla seed oil reshapes the rumen microbiome and increases fermentation end-products in vitro

Abstract Aims The effect of Perilla frutescens seed oil (PSO) on an in vitro rumen microbial ecosystem was evaluated by integrating fermentation measurements, microbiome profiling, metagenomics, and untargeted metabolomics. Methods and Results Rumen inoculum was incubated for 24 h with a control TMR substrate (CK), TMR supplemented with 23.7 mg of Perilla seeds per bottle (PS), or TMR supplemented with 8.5 μL of Perilla seed oil per bottle (PSO), with the PS and PSO treatments providing equivalent amounts of seed oil. Fermentation kinetics and volatile fatty acids were measured, and microbial and metabolic responses were characterized using 16S rRNA gene sequencing, metagenomics, KEGG and CAZy annotation, untargeted metabolomics and MetOrigin2 source tracing. PSO increased maximum gas production and total volatile fatty acid concentrations while maintaining pH within the physiological range. Community diversity was unchanged, but PSO altered microbial composition, including increases in Firmicutes, Verrucomicrobia, Vagococcus, Clostridium and Lactobacillus and decreases in Shigella sonnei and Methanosarcina sp. Ant1. PSO also altered microbial functional profiles and increased several lipid- and vitamin-associated metabolites, including linoleic acid, 13-HODE, 9-oxoODE, pantothenic acid and thiamine, while reducing lactate. Conclusions PSO changed rumen microbial community structure and functional potential in parallel with increased fermentation end-products and extensive metabolic shifts. These in vitro findings identify microbial and metabolic responses that warrant validation in vivo.