Metagenome and metabolome integration reveals microbial signatures of sheep milk flavor

The flavor of ruminant milk largely determines consumer acceptance and dietary preferences for dairy products. Milk volatile organic compounds (VOCs) constitute and characterize the key sensory attributes of milk. However, the relationships between rumen microbial variation and inter-individual differences in these flavor compounds, as well as their potential functional links, remain insufficiently understood. In 447 dairy sheep, milk volatilomics detected 306 VOCs, and 29 compounds with key contributions to sheep milk flavor were further identified, including (E)-2-octenal, diallyl sulfur compounds, and p-cymene. Notably, for 20 of these key flavor compounds, a larger proportion of phenotypic variance was explained by the rumen microbiome than by host genetic factors. Rumen metagenomic analysis revealed significant differences in microbial community structure between the high and low VOC-profile groups and identified 133 differential species, among which Paludibacteraceae bacterium , Hallella mizrahii , and Sodaliphilus pleomorphus emerged as key microbial signatures distinguishing the two flavor groups. At the functional level, flavor-associated differences were mainly observed in the metagenomic functional potential related to sulfur metabolism, nitrogen metabolism, and unsaturated fatty acid biosynthesis. Reconstruction of 2,041 SGBs linked differentially abundant functional genes to their putative species-level carriers. In addition, the aldehyde flavor cluster was positively correlated with ruminal linoleic acid and negatively correlated with stearic acid. Compared with the low VOC-profile group, the high VOC-profile group exhibited significantly lower abundances of PUFA biohydrogenation related BBI and PAI type isomerases and reductases, together with a clear divergence in enzyme-type preference between groups. These results reveal a close relationship between rumen microbial PUFA biohydrogenation potential and variation in milk flavor-associated volatile compounds. This study demonstrates that variation in the rumen microbiome is closely associated with inter-individual variation in key sheep milk flavor compounds. By systematically characterizing the relationships between rumen microbial taxa, functional features, and sheep milk flavor compounds, this study provides new evidence for elucidating the potential mechanisms by which rumen microbes contribute to flavor formation in ruminant milk.