Multi-omics profiling reveals microbial regulation of a key aromatic ester phenethyl acetate formation in fermented alfalfa and its impact on sheep feed preference

This study utilized headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS) to identify the key volatile flavor compounds in fermented alfalfa. The contribution of core microbiota to forming these key flavor compounds was investigated using a combination of absolute quantification of 16S rRNA gene copy number and metagenomic technology. Additionally, the critical roles of core fermentation microorganisms were quantitatively detected and validated through liquid chromatography mass spectrometry (LC-MS). Results revealed that <i>Lactiplantibacillus plantarum</i> B90 treated group achieved superior fermentation quality, with esters and aldehydes being the dominant volatile flavor compounds. Phenethyl acetate was the only aromatic ester that was significantly up-regulated after fermentation. The aryl alcohol dehydrogenase from <i>L. plantarum</i> facilitated the conversion of phenylacetaldehyde into phenylethyl alcohol, which serves as the precursor for phenethyl acetate. Furthermore, fermented alfalfa sprayed with phenethyl acetate was associated with increased feed intake in sheep. These findings propose new insights for microbial modulation of fermented flavor in fermented forage to enhance sheep feed intake.