| Summary: | The leguminous plant species, <i>Indigofera linnaei</i> and <i>Indigofera spicata</i> are distributed throughout the rangeland regions of Australia and the compound indospicine (L-2-amino-6-amidinohexanoic acid) found in these palatable forage plants acts as a hepatotoxin and can accumulate in the meat of ruminant livestock and wild camels. In this study, bovine rumen fluid was cultivated in an in vitro fermentation system provided with <i>Indigofera spicata</i> plant material and the ability of the resulting mixed microbial populations to degrade indospicine was determined using UPLC–MS/MS over a 14 day time period. The microbial populations of the fermentation system were determined using 16S rRNA gene amplicon sequencing and showed distinct, time-related changes occurring as the rumen-derived microbes adapted to the fermentation conditions and the nutritional substrates provided by the <i>Indigofera</i> plant material. Within eight days of commencement, indospicine was completely degraded by the microbes cultivated within the fermenter, forming the degradation products 2-aminopimelamic acid and 2-aminopimelic acid within a 24 h time period. The in vitro fermentation approach enabled the development of a specifically adapted, mixed microbial population which has the potential to be used as a rumen drench for reducing the toxic side-effects and toxin accumulation associated with ingestion of <i>Indigofera</i> plant material by grazing ruminant livestock.
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