The protocol standardizes how researchers simulate the three main stages of digestion:

1. Oral phase (mouth)
   • Food is mixed with simulated saliva.
   • Enzymes like amylase begin breaking down carbohydrates.
   • Mechanical mixing mimics chewing.
2. Gastric phase (stomach)
   • The mixture is acidified (low pH
   • Pepsin and Gastric lipase are added to digest proteins.
   • Controlled time and agitation simulate stomach conditions (pH and gastric residence time).
3. Intestinal phase (small intestine)
   • pH is raised to physiological conditions.
   • Pancreatic enzymes (lipase, amylases, proteases, etc.) and bile salts are added.
   • This phase models where most nutrient release and absorption would occur.

Compared to other digestion models, INFOGEST 2.0:

• Refines enzyme activities and concentrations to better match human physiology
• Improves the electrolyte composition of digestive fluids
• Standardises timing, pH adjustments, and mixing conditions.
• Enhances reproducibility across labs globally
• It has been validated through inter-laboratory studies and shows improved alignment with in vivo digestion data.

• Enables direct comparison of results across studies through the use of standardized methodologies
• Evaluates bioaccessibility (how much of a nutrient becomes available for absorption)
• Useful for testing and developing functional foods, supplements, and novel ingredients
• Reduces reliance on animal and human trials in early research stages

• The INFOGEST 2.0 protocol is used to estimate nutrient bioaccessibility, i.e. the fraction of carbohydrates,  proteins, lipids, and micronutrients released during digestion and made available for absorption.
• It serves as a screening tool for protein digestibility and quality, often supporting work related to DIAAS estimation.
• Widely applied in food product development, especially for plant-based, functional, and novel foods.
• Enables the study of food matrix and structure effects,
• helping to explain how processing and formulation influence digestion and nutrient release.
• Used to evaluate delivery and stability of bioactive compounds (e.g., vitamins, probiotics, polyphenols).
• Reduces reliance on animal and human trials by enabling controlled, reproducible lab-based digestion studies.
• Can be coupled with colonic fermentation models to assess downstream effects on the gut microbiome.

• Assessment of protein digestibility: Evaluating the nutritional quality of novel and alternative proteins, including plant-based and hybrid formulations, using DIAAS-relevant approaches.
  • Perez-Moral N, et al (2022). In vitro protein bioaccessibility and human serum amino acid responses to white bread enriched with intact plant cells. Food Chemistry; http://dx.doi.org/10.1016/j.foodchem.2022.134538

• Glycaemic response prediction: Characterising carbohydrate digestion kinetics to estimate the glycaemic impact of food products.
  • Edwards et al (2019); A single-enzyme system for starch digestibility screening and its relevance to understanding and predicting the glycaemic index of food products. Food and Function, DOI: 10.1039/c9fo00603f
  • Butterworth et al. (2022); Enzyme kinetic approach for predictions of starch digestibility and glycaemic index; DOI: 10.1016/j.tifs.2021.11.015
  • Petropoulou K. et al (2020); A natural mutation in Pisum sativum L. (pea) alters starch assembly and improves glucose homeostasis in humans; Nature Food, 2020, http://dx.doi.org/10.1038/s43016-020-00159-8
  • Freitas et al (2025); Starch digestion: a comprehensive update on the underlying modulation mechanisms and its in vitro assessment methodologies; Trends in Food Science and Technology 159 ; http://dx.doi.org/10.1016/j.tifs.2025.104969

• Bioaccessibility studies: Investigating the release and stability of micronutrients and bioactive compounds during digestion.
  • Brodkorb et al. (2019) – INFOGEST static in vitro simulation of gastrointestinal food digestion, Nature Protocols 10.1038/s41596-018-0119-1
  • Eldridge et al (2026); Addressing Vitamin B12 deficiency through aeroponic fortification of a salad crop (Pisum sativum); Communications Biology http://dx.doi.org/10.1038/s42003-026-09764-y
  • Perez-Moral et al (2018); Comparative bio-accessibility, bioavailability and bioequivalence of quercetin, apigenin, glucoraphanin and carotenoids from freeze-dried vegetables incorporated into a baked snack versus minimally processed vegetables: Evidence from in vitro models and a human bioavailability study; Journal of Functional Foods ; http://dx.doi.org/10.1016/j.jff.2018.07.035

•  Functional ingredient evaluation: Assessing the survival and performance of probiotics and the delivery of prebiotic compounds through the gastrointestinal tract.
  • Bajka et al (2021); The impact of replacing wheat flour with cellular legume powder on starch bioaccessibility, glycaemic response and bread roll quality: A double-blind randomised controlled trial in healthy participants; Food Hydrocolloids; http://dx.doi.org/10.1016/j.foodhyd.2020.106565

• Processing impact analysis: Understanding how formulation, processing, and storage conditions influence digestion behaviour and nutrient availability.
  • Cañas et al (2020); Effect of cooking, cold storage, reheating and particle size on in vitro starch digestibility of pasta; Food and Function; http://dx.doi.org/10.1039/D0FO00849D

The Static Model is a simple in vitro system that uses small anaerobic culture vessels such as 96-well plates and serum bottles. It is a high-throughput approach that allows many independent control conditions to be run in parallel and it is widely used for screening interactions between the colonic microbiota and diets/foods, prebiotics, or probiotics. Typical experiment run for approximately 3-5 days.

Advantages 

1. Simple
2. Fast
3. Economical (cheap)
4. Relatively less work compared to other models.
5. high throughput well which is good for screening.
6. Anaerobic condition
7. Temperature controlled.  

1. Media ingredient depletion
2. Accumulation of the microbial by-products 
3. Small incubation volume if you need bigger samples for various analyses
4. Not pH-controlled systems.
5. No stirring

The Batch Colon Model is the simplest and most cost-effective way to replicate in vitro gut fermentation. Designed for short-term experiments, it is widely used to study interactions between the colonic microbiota and dietary components, including prebiotics and probiotics. Operating under anaerobic conditions, it provides a flexible platform for investigating microbial activity, inter-individual variability, and metabolite pathways over a period of five to seven days.

Specifications:

• Capacity: Up to 12 vessels; available in two sizes—100 mL vessels incubating 50–60 mL volumes, and 300 mL vessels incubating 100–150 mL volumes.
• Applications: Short-term microbial fermentation studies, microbiota–diet interactions, and pathway investigations.
• Control Features: pH control, stirring, anaerobic conditions, and precise temperature regulation.
• Advantages: Flexible design, frequent sampling at any time, supports larger sample volumes for multiple analyses.
• ​Considerations: Labour-intensive; nutrient depletion and metabolite accumulation may occur; limited to a maximum of 12 vessels.

The One-Stage Continuous Colon Model is designed for long-term experiments, maintaining a stable microbial ecosystem under physiologically relevant colon conditions. By continuously replenishing feed and removing waste products, the model supports sustained microbial activity, making it ideal for studying adaptation of the gut microbiota to dietary compounds and tracking microbial metabolism over time. Runs typically last three to five weeks, allowing detailed temporal analysis.

Specifications:

• Capacity: Up to 6 vessels; available in 100 mL (incubating 50–60 mL) or 300 mL (incubating 100–150 mL) sizes.
• Applications: Long-term microbial adaptation studies, metabolic profiling, and dietary compound assessment.
• Control Features: Automated pH regulation, stirring, anaerobic conditions, and temperature control.
• Advantages: Continuous feed replenishment sustains microbial ecosystems; allows frequent sampling at any time; supports larger volumes for multiple analytical endpoints.
• ​Considerations: Labour-intensive; requires extended operational time (3–4 weeks); limited to 6 vessels, which may restrict experimental scale.

The Three-Stage Continuous Colon Model is a sophisticated multivessel system that replicates the distinct environments of the ascending, transverse, and descending colon. Each vessel is maintained at a region-specific pH (5.5, 6.2, and 6.8), enabling detailed study of microbial activity and metabolism across different sections of the colon. Continuous feeding and waste removal sustain microbial ecosystems for up to five weeks, making this model ideal for spatial and temporal investigations of gut microbiota adaptation.

Specifications:

• Capacity: 3 vessels per setup, with the option to run up to 2 arms with 2 replicates (6 vessels total); available in 100 mL (50–60 mL incubated) or 300 mL (100–150 mL incubated) sizes.
• Applications: Region-specific studies of microbial activity, dietary compound metabolism, and microbiota adaptation.
• Control Features: Independent pH regulation per vessel, continuous feeding, stirring, anaerobic conditions, and precise temperature control.
• Advantages: Accurately simulates regional colon environments; allows frequent sampling; supports large volumes for comprehensive analysis.
• ​Considerations: Labour-intensive; long experimental runs (3–4 weeks); limited to 6 vessels, which may restrict the number of arms and replicates

The Micro-Matrix is a compact, high-throughput system designed for short-term in vitro gut studies. It features 24 independent bioreactors within a microtiter plate format, each with individual control of key parameters. This makes it ideal for rapid screening of dietary components, prebiotics, and probiotics under controlled anaerobic conditions. Typical runs last two to three days, providing fast and reproducible insights.

Specifications:

• Capacity: 24 wells per cassette, each with a 10 mL chamber incubating up to 6 mL
• Applications: High-throughput screening of compounds, microbial interactions, and fermentation profiles.
• Control Features: Independent pH regulation, stirring, anaerobic environment, and temperature control for each well.
• Advantages: Efficient, lower labour requirement, supports multiple conditions and replicates simultaneously.
• ​Considerations: Small incubation volumes may limit downstream analyses; best suited to start- and end-point sampling rather than interval time points.

Our models have been used in multiple peer-reviewed studies, including:
Clinical Trial: NCT02653001

Blogs featuring our work:

• How our Colon Model Facility helps us study gut microbiota - Quadram Institute
• Bringing Physiological Research to Parliament
• QIB Students Develop Their Entrepreneurial Skills

• Haarhuis et al. (2026). Food & Function. DOI: https://doi.org/10.1039/D5FO04781A
• Haarhuis et al. (2025). Molecular Nutrition & Food Research. DOI
• Shehata et al. (2023). Molecular Nutrition & Food Research.DOI
• Day-Walsh et al. (2021). European Journal of Nutrition.DOI
• ​Vardakou et al. (2007). International Journal of Biological Macromolecules. DOI