# In Vitro Simulation of Human Colonic Fermentation:
## 1 Introduction  
The role of diet in human health has been repeatedly addressed and confirmed. However, elucidating the relationship between nutrition and health requires an accurate understanding of the digestion process through which foods are transformed and interact with the organism to exert their eventual biological function [1]. Along digestion, chemical and enzymatic reactions, along with mechanical forces, transform food matrices and their main macronutrients (lipids, protein, and carbohydrates) into their conforming constituents, which are absorbed by the enterocytes in the small intestine [1]. Digestion is a very efficient process, but a small fractions of food constituents still escape the hydrolytic action of enzymes and subsequent absorption, thus remaining in the lumen and passing to the colonic stage, where the colonic microbiota can further interact with them, forming what is considered to be a relevant research topic nowadays [2, 3, 4]. Studying the digestion process in situ presents several vicissitudes due to the issue of access to the digestive system, the impossibility of continuous monitoring, and burden for the subjects as well as ethical restrictions [5]. Animal models have been extensively applied to assess the digestion process, but the fact that animals' microbiota differ from humans' microbiota is widely accepted [6]. For these reasons, alternative methodologies to study digestion have increased, including in vitro digestion models, which enable thesimulation of digestion in the laboratory [7; 8; 9; 10]. These models allow for the possibility of sampling at any point of the process, assessing digestion of any food or nutrient and establishing its effect on the microbiota profile, or determining the products generated by the microbiota metabolism [7; 11]. In vitro digestion studies address such diverse research questions as macronutrient digestibility, bioactive compounds bioaccessibility, the release of encapsulated substances, or the effect of gastrointestinal conditions on digestibility, among others. In relation to the microbiota, these models can be useful to assess the effect of prebiotics (nutritional compounds present in foods, such as fibres), probiotics (ingestion of alive microorganisms), or symbiotics (a combination of both) on microbiota populations or the role of these populations in the production of metabolites related to diet. Other relevant fields that could benefit from applying in vitro colonic fermentation relate to microbiota transmission from mothers to infants through breastmilk, the effect of antibiotics in the area of pharmacology, or in agronomics to determine if the presence of pesticides in some foods were to be evaluated in colonic microbiota. Depending on the goals, the digestion and fermentation variables must be adapted to represent the physiological conditions to simulate [5], such as transit time, pH, digestive secretions and composition [10], or microbiota profile [12]. Compared to the standard digestion and fermentation conditions, these amendments are crucial in the context of food digestion studies in chronic disease (pancreatic insufficiency, celiac disease, etc. ), special physiological states such as pregnancy, or life stages (infancy, adulthood, elderly). Great efforts are being made by the scientific community to standardize in vitro protocols for mimicking the luminal digestion of foods [13; 14]. Thanks to the adherence of many researchers to these protocols, which establish the basic characteristics that should be considered in the process (e.g., duration, pH of the stages, concentration of salts, and digestive enzymes), the comparison of results and the progress towards establishing general knowledge has been enabled. However, despite this progress, the in vitro simulation of the colonic stage still remains without a common consensus, probably because of the underpinning complexity to the reproduction of a microbial population and to all of the factors involved.