Abstract
Modern food chemistry and nutrition research stresses digestion and bioavailability. In vitro digestive models are needed to reconstruct the human gastrointestinal (GI) system and study food-derived chemical release, transformation, and absorption. This review examines major in vitro digestion platforms, including static and dynamic systems, organ-on-chip devices, and 3D-bioprinted gut models. Integrating these models with absorption simulators such as Caco-2 cells and advanced analytical instruments (e.g., HPLC, LC-MS/MS, FTIR, NMR, and OMICS technologies) enables comprehensive profiling of digestion products. These models assess bioaccessibility and bioavailability of polyphenols, omega-3 fatty acids, and encapsulated nutraceuticals. Computer modeling and artificial intelligence (AI) increase prediction for high-throughput functional food performance screening. Though promising, current models lack homogeneity and microbial representation often does not fully replicate in vivo gut microbiota complexity. In vitro digestive models assist food scientists enhance products. Priorities include harmonizing INFOGEST protocols, improving physiological relevance, and customizing nutrition and clinical validation. These developments may strengthen the utility of in vitro models in precision nutrition and industrial applications.
Highlight
- Comparison of model accuracy in mimicking GI conditions.
- OMICs tools boost understanding of digestion mechanisms.
- Analytical methods track nutrient release and changes.
- Advances in gut-on-chip and 3D models for relevance.
Introduction
Food digestion is a series of chemical reactions that involve the physical breaking of food, the enzymatic breakdown and the change of the molecules to smaller ones in the digestive tract. Knowledge about the release of nutrients and bioactive compounds from food under physiological conditions is a prerequisite for the evaluation of their nutritional and health-promoting properties. This knowledge has become the center of attention in modern food chemistry, which connects the gap between food composition and the way it is metabolized by the human body (Capuano & Janssen, 2021). Typically, the understanding of digestion and nutrient absorption has been based on the results of animal and human in vivo studies. Nevertheless, the development of in vitro digestion models has substantially advanced research on food functionality, nutrient release, and bioavailability, thus providing more ethical, cost-effective, and standardized alternatives (Bohn et al., 2018). However, they also entail several drawbacks. The controversies over the use of humans and animals for experiments, variability among subjects, elevated expenses, protracted periods of research, and challenges in managing the parameters of the control group are some of the constraints impeding their practicability (Abuhassira-Cohen & Livney, 2022; Mota et al., 2023). Moreover, in vivo techniques are often limited in their ability to provide mechanistic insights on the biochemical changes of food components in the gastrointestinal (GI) tract. In vitro digestion models simulate sequential digestive events and are widely used to investigate nutrient release, transformation, and predicted absorption.
They enable nutrient breakdown monitoring, identifying digestion intermediates, conducting studies on bioaccessibility (the portion of a compound released from the food matrix and available for absorption), and predicting bioavailability when used along with cellular absorption models (Ji et al., 2022; Minekus et al., 2014). By design, researchers achieve precise control of pH, enzyme concentrations, digestion duration, and mechanical mixing, making these models highly suitable for reproducible and mechanistic studies in food chemistry and nutrition. The invention of the INFOGEST static in vitro digestion protocol, developed as an international consensus method by the INFOGEST network (COST Action FA1005), has been a major milestone and is now the most widely used harmonized protocol for simulating gastrointestinal digestion in vitro (Brodkorb et al., 2019). Despite the wide adoption of the INFOGEST protocol, several practical challenges remain that limit its cross-laboratory reproducibility. One major source of variability arises from differences in enzyme sources and activities, especially for pepsin, pancreatin, and bile extracts, which may vary between suppliers or batches, leading to inconsistent proteolysis or lipolysis rates across laboratories (Bohn et al., 2018; Brodkorb et al., 2019). In addition, pH control during gastric and intestinal phases often introduces discrepancies, as slight variations in titration procedures, electrode calibration, or buffering capacity of the food matrix can alter enzyme activity and digestion kinetics (Verkempinck et al., 2022). Another critical factor is operator-dependent variations, such as mixing intensity, timing, temperature equilibration, and sample handling, which can significantly influence micelle formation, protein hydrolysis patterns, and bioaccessibility outcomes (Tan et al., 2022). Recent studies have therefore emphasized the need for harmonized reporting, enzyme activity normalization, and inter-laboratory ring trials to improve reproducibility and ensure that INFOGEST-based results can be reliably compared across institutions (Freitas et al., 2025). Addressing these practical issues is critical for strengthening the global utility of the INFOGEST protocol in food chemistry and nutrition research. In addition to enabling direct comparison across studies, such standardization also facilitates collaborative research between universities and industry.
Moreover, the consortium of the INFOGEST has also established protocols for infants, the elderly, and people with impaired digestion, which shows the flexibility of the model for different physiological conditions further (Egger et al., 2016; Zhou et al., 2023). In vitro digestion is a major research area that could be effectively combined with analytical and omics technologies. The employment of metabolomics, proteomics, lipidomics, and transcriptomics alongside digestion models enables a system-level understanding of the changes occurring during digestion. For instance, mass spectrometry and nuclear magnetic resonance (NMR) are methods that can quickly pinpoint digestion intermediates and metabolites (Madalena et al., 2022; Smeets et al., 2021). These data indicate the changes in nutrient release and transformation that are the result of the composition, structure, and processing of food, which is an area of research that is rapidly expanding due to the development of functional foods and personalized nutrition programs.
Moreover, combining in vitro digestion with cellular absorption models such as Caco-2 monolayers or intestinal organoids provides an initial indication of the intestinal transport of nutrients and bioactive compounds. This integration helps bridge bioaccessibility and bioavailability and provides a more detailed understanding of nutrient function (Antal et al., 2024; Rodrigues et al., 2022). These hybrid structures are still under development; however, they show potential as alternative platforms to reduce reliance on animal models. In addition, they may provide more humane, cost-effective, and scalable approaches for evaluating drug and food absorption. In vitro digestion models play an important role in nutrient research and are increasingly applied in food product development, fortification strategies, bioavailability enhancement, nutraceutical design, and food contaminant risk assessment (Lesmes, 2023; Marze, 2017). Researchers, for instance, have used a simulated digestion technique to measure the influence of encapsulation on curcumin release, the impact of processing on protein digestibility, and the effect of lipid formulation on omega-3 bioaccessibility. The use of digestive models is growing to anticipate the interactions between food and gut microbiota, especially through the visualization of colonic fermentation (Shah et al., 2016; Young et al., 2020). One problem with the current models is that they frequently oversimplify the gastrointestinal complexity and lack some characteristics, for example, mucus barriers, immune interactions, peristaltic motion, or the fluctuating presence of gut microbiota.
Besides, while standardized models such as INFOGEST serve as good benchmarks, the differences in enzyme activity, pH profiles, and digestion duration from one laboratory to another can still lead to variations in results. Further development of dynamic, physiologically relevant digestion systems such as TIM-1, SHIME, and Tiny-TIM remains necessary, as these platforms provide real-time control of pH, flow rates, and secretions to better simulate human digestion (Dupont et al., 2019; Singh, 2024). On the other hand, these sophisticated models have raised the complexity and the costs, which can make them less accessible for the usual use. Against this background, this review summarizes the current state of in vitro digestion models in food chemistry. We also discuss future directions in in vitro digestion research, including integration with OMICS, dynamic simulation, and cell-based absorption models. By outlining key improvements, applications, and limitations, this review provides a framework for understanding how in vitro digestion models may further advance food science and human nutrition.
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Farhang Hameed Awlqadr, Mohammed N. Saeed, Othman Abdulrahman Mohammed, Syamand Ahmed Qadir, Aryan Mahmood Faraj, Seyed Mohammad Najibi Hosseini, Muhammad Tayyab Arshad, Muhammed Adem Abdullahi, Tablo H. Salih, In vitro digestion models in food chemistry: advancements, challenges, and applications in nutrient bioaccessibility and bioavailability, Food Chemistry: X, Volume 36, 2026, 103951, ISSN 2590-1575, https://doi.org/10.1016/j.fochx.2026.103951.
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