Abstract
High-quality silage enhances palatability and feed intake; however, the effects of co-fermentation with flavouring agents and lactic acid bacteria (LAB) on its flavour quality, core microbiota, and taste-active amino acids remain unclear. This study investigated the effects of fermentation using Lactiplantibacillus plantarum (LP) alone or in combination with phenethyl acetate (LPP) on the flavour profile of alfalfa silage and its subsequent influence on feed intake. Both LP and LPP significantly improved fermentation quality versus control (CK), with markedly higher feed intake-LP > CK and LPP > LP. Key flavour compounds, including dimethyl trisulfide, 4-ethylphenol and β-damascenone, were significantly increased in the LP alone group. Contrarily, essential taste-related amino acids including aspartic acid, alanine, proline, histidine, isoleucine, and valine were decreased, except for arginine. These metabolic shifts collectively contributed to enhanced feed intake. The addition of LPP increased phenylethyl alcohol, benzyl alcohol and hexanal, and decreased arginine, contributing to enhanced palatability. Aryl alcohol dehydrogenase, proline aminopeptidase, histidine dehydrogenase, and branched-chain amino acid transaminase from LP played a crucial role in the formation of phenylethyl alcohol, proline, histidine and isoleucine during fermentation. These results provide insights into how LAB and flavouring agents jointly regulate flavour development in high-quality alfalfa silage.