Selective quantification of free fatty acids reveals matrix-dependent differences in lipid bioaccessibility

Current in vitro approaches to lipid bioaccessibility typically quantify total fatty acid methyl esters (FAMEs) in the bioaccessible (micellar) fraction after digestion, assuming complete hydrolysis of esterified lipids. However, this operational definition does not discriminate between released and hydrolyzed lipids and may systematically overestimate the fraction immediately available for absorption. This study proposes an analytical approach that operationally differentiates between lipid release and free fatty acid formation. Following INFOGEST digestion, free fatty acids (FFAs) were isolated from the bioaccessible fraction by solid-phase extraction and quantified by gas chromatography. Four ready-to-eat foods differing in matrix structure (walnuts, canned mackerel, cream cheese, biscuits) were used as test matrices. Bioaccessibility, operationally defined as FFAs relative to total fatty acids in undigested food, ranged from 67.6% to 9.9%. In three of four matrices, a substantial proportion of solubilized lipids were not recovered as FFAs, indicating that conventional FAME-based quantification would likely overestimate bioaccessibility. The fatty acid profile of the bioaccessible fraction differed from the original composition, suggesting matrix-dependent differences in fatty acid release patterns. These findings highlight limitations of the prevailing analytical practice for lipid bioaccessibility assessment and provide a more compositionally rigorous framework, with implications for digestion studies, and food composition data interpretation.