Distribution, contributing factors, and dietary enrichment of equol in bovine milk

Equol, a bioactive metabolite of Isoflavones, is associated with diverse health benefits, yet its enrichment in bovine milk has not been fully clarified. In this study, we established a liquid chromatography-mass spectrometry method for detecting equol in milk, featured by an acid hydrolysis pretreatment. Equol was found predominantly in the milk fat fraction. A random forest regression model was developed to predict milk equol levels based on its significant correlations with various lactation parameters. Dietary supplementation with red clover Isoflavones markedly increased equol concentration in milk by up to 15.6 fold. The milk thermol processing and microbial fermentation showed negligible effects on equol level, indicating its strong stability. Collectively, these findings elucidate the distribution patterns and key determinants of equol in milk. Importantly, a strategy of red clover Isoflavones supplementation was developed to produce higher equol-enriched milk as a novel functional food for human health.