Plant small molecules steer lipase selectivity and free fatty acid profiles in enzyme-modified cheese revealed by machine learning–guided analysis

Mint and cumin extracts regulate lipase activity in enzyme-modified cheese. • Epicatechin and 7-hydroxycoumarin promote medium-chain FFAs, suppress long-chains. • Molecular simulations reveal selective inhibition of long-chain triglyceride binding. • Sensory analysis confirms enhanced aroma, reduced off-flavors in modified cheeses. • Plant-derived compounds offer a novel approach to optimizing cheese flavor. In enzyme-modified cheese (EMC), controlling the release of specific free fatty acids (FFAs) by lipases remains challenging. Eight plant extracts were screened, and Mentha haplocalyx and Cuminum cyminum showed pronounced regulatory effects on lipolysis. These extracts were further fractionated, purified, and identified. This study demonstrates that epicatechin and 7-hydroxycoumarin can selectively modulate lipase-mediated fatty acid release, promoting the accumulation of aroma-active medium-chain free fatty acids while suppressing long-chain fatty acid release during EMC production. The results revealed a concentration-dependent, bidirectional “pendulum effect,” where changing additive levels shifted lipase selectivity and apparent activity, enabling tunable FFA profiles. Sensory evaluation confirmed the chemical data, identifying one desirable-aroma EMC and two low off-flavor EMCs. Integrated AlphaFold3 modelling, molecular dynamics, docking, machine-learning analyses, and kinetic experiments firstly provided molecular-level insights into the mechanisms underlying the promotion of medium-chain fatty acid release and the suppression of long-chain fatty acid liberation during EMC production. Download: Download high-res image (183KB) Download: Download full-size image