High heterogeneity in the size distribution of the micellar fraction from in vitro digestions: sample preparation and reporting recommendations

Background: Understanding the size and surface charge (ζ-potential) of particles in the mixed micellar fraction produced by in vitro digestion is crucial to understand their cellular absorption and transport. The inconsistent presentation of micellar size data, often limited to average particle diameter, makes comparison of studies difficult. The present study aimed to assess different size data representations (mean particle diameter, relative intensity- or volume-weighted size distribution) to better understand physiological mixed micelle characteristics and to provide recommendations for size reporting and sample handling.

Results: Dietary compounds (RRR-α-tocopherol, retinyl-palmitate, β-carotene, curcumin and naringenin) underwent a simplified in vitro digestion, whereas foods (spinach and red cabbage) were subjected to both a simplified and the INFOGEST 2.0 digestions. Dynamic light scattering was used to measure size and surface charge of the mixed micelles. A significant percentage of particles above the 200 nm filter cut-off was observed, indicating aggregation and dynamic size changes in the mixed micellar fraction. Freezing of the mixed micelles notably enhanced the aggregation.

Conclusion: The determination of particle size in polydisperse mixed micellar fractions is challenging, and relying solely on average particle diameter can be misleading. Especially in more polydisperse samples, parameters such as polydispersity index and volume-weighted distribution should accompany average particle diameter data. To minimize the effect of freezing on particle size, we recommend filtering the digesta after storage (freezing), as this leads to similar size distribution compared to mixed micellar fraction measured directly after digestion. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.