Assessing iron, zinc and calcium bioaccessibility using the INFOGEST method: Challenges and opportunities

Iron, zinc and calcium are essential minerals required for key physiological functions, including oxygen transport, immune function, enzymatic activity and bone health. Despite their importance, deficiencies remain widespread globally, driven in part by the poor bioavailability of these minerals from many dietary sources, particularly plant-based foods. As dietary patterns increasingly shift towards plant-based and novel ingredients, robust methods for assessing mineral bioavailability are urgently needed to support food composition data, dietary modelling and product development. While in vivo approaches remain the reference standard, their cost, complexity and ethical constraints limit their routine application. Consequently, in vitro digestion models have become indispensable tools for screening and for mechanistic investigation of mineral bioaccessibility. The INFOGEST static in vitro digestion method is now the most widely adopted harmonised protocol, providing standardised digestion conditions, found to agree with in vivo data for protein digestion. However, its application to mineral bioaccessibility presents distinct challenges that remain unresolved. These include substantial interference from reagent-derived background minerals, strong matrix-dependent mineral-ligand interactions, and a lack of consensus on how the bioaccessible fraction should be defined and separated following digestion. This review critically examines the use of the INFOGEST method for assessing Fe, Zn and Ca bioaccessibility, highlighting key methodological limitations and recent advances. Future progress will require targeted optimisation of digestion conditions, standardised definitions of bioaccessibility, validation of separation techniques, and direct comparison with in vivo data. Addressing these challenges is essential to ensure that in vitro models generate physiologically meaningful, comparable data capable of informing nutrition research and policy.