Edible nuts deliver polyphenols and their transformation products to the large intestine: An in vitro fermentation model combining targeted/untargeted metabolomics

The fate of polyphenols from edible tree nuts was investigated using a simulated in vitro intestinal fermentation system. The digested food matrix was fermented for 48 h and the changes in the phenolic profiles were evaluated by both untargeted UHPLC-QTOF and targeted UHPLC-Orbitrap mass spectrometry. The untargeted metabolomics approach allowed us to monitor the comprehensive changes in phenolic profiles from 0 up to 48 h of in vitro fermentation. Multivariate statistics (i.e., orthogonal projection to latent structures discriminant analysis) applied to this untargeted data allowed us to identify the most discriminating phenolic metabolites and to further understand the colonic transformation pathways involved. In particular, 13 putatively identified compounds derived from flavonoids, lignans and phenolic acids were found to have the highest discrimination potential. Six phenolic metabolites were then quantified by means of targeted metabolomics (using a UHPLC-Orbitrap). These metabolites included 3,4-dihydroxyphenylacetic acid, 4-hydroxybenzoic acid, hippuric acid, caffeic acid, protocatechuic acid and protocatechuic aldehyde. Using the targeted data, a clear matrix effect was observed over time, with an increase of some phenolic metabolites moving from 8 to 48 h of in vitro fermentation. Based on these data, catabolic pathways for colonic microbial degradation of flavonoids, hydroxycinnamic acids, tyrosols and lignans are proposed. Our findings show that edible tree nuts deliver polyphenols to the colon, where several microbial transformations occur that lead to smaller phenolic metabolites being observed. Furthermore, we found that the combined use of targeted and untargeted metabolomics can be particularly effective for investigating the fate of polyphenols in the large intestine.