Regulating effects of hydroxytyrosol acyl esters with different acyl chain lengths and isomers on gut microbiota: insights from in vitro gastrointestinal digestion, fecal fermentation and Lactobacillus fermentation

BACKGROUND Phenolic compounds are increasingly recognized for their diverse health benefits, particularly their potent antioxidant capacities. To enhance its lipophilicity, stability and bioavailability, hydroxytyrosol has been successfully esterified with various alkyl chains lengths and different isomers (branched-chain and straight-chain), forming hydroxytyrosol-fatty acid acyl esters (Hty-Es). These synthesized Hty-Es, have garnered significant interest and are widely explored in fields such as food preservation because of their superior antioxidant properties. However, little is known about the digestion, fermentation characteristics and the effects on the intestinal microbiota in vitro . RESULTS This study evaluated the influence of hydroxytyrosol (HTy) esterified with fatty acids of varying chain lengths (C2:0, C3:0, C4:0 n , C4:0 i , C6:0, C8:0, C12:0, C14:0 and C16:0) on polyphenol sustained-release properties and gut microbiota modulation during simulated in vitro digestion, fecal fermentation and Lactobacillus fermentation. The findings demonstrated partial hydrolysis of HTy-Es in both simulated gastric fluid and intestinal fluid to slowly release a small amount of polyphenols, suggesting their potential to reach the colon and cecum. Analysis of fermented fecal samples revealed that HTy-Es supplementation modulated microbiota composition, elevating the diversity of Actinobacteriota, Firmicutes and Bacteroidetes , whereas it decreased the diversity of Desulfobacterota, Actinobacteriota, Chloroflexi and Verrucomicrobiota . At the genus level, the growth of detrimental genera ( Escherichia shigella ) was successfully suppressed. Notably, HTy-Es with distinct acyl chain lengths and isomer configurations exhibited divergent effects on polyphenol bioaccessibility and gut microbiome diversity, underscoring their structure-dependent functional potential for health applications. CONCLUSION Conclusively, the slow release of fatty acids and HTy clearly demonstrated that HTy-Es would be an effective approach for enhancing the beneficial health effects of fatty acids and HTy. © 2025 Society of Chemical Industry.