Current in vitro digestion systems for understanding food digestion in human upper gastrointestinal tract

Trends in Food Science & Technology 96 (2020) 114–126

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Current in vitro digestion systems for understanding food digestion in human upper gastrointestinal tract

T

Cheng Lia,b,c, Wenwen Yud, Peng Wue,∗, Xiao Dong Chene a

School of Medical Instrument and Food Engineering, University of Shanghai for Science and Technology, Shanghai, 200093, China Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China c Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou, 225009, China d The University of Queensland, Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation, Brisbane, QLD, 4072, Australia e Suzhou Key Laboratory of Green Chemical Engineering, School of Chemical and Environmental Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China b

ARTICLE INFO

ABSTRACT

Keywords: Upper gastrointestinal tract In vitro digestion system Stomach Dynamic model Morphology Anatomy

Background: Food digestion rate and location within the gastrointestinal (GI) tract are important for human health. Ideally, food digestion studies should be performed in vivo but this is not always technically, ethically and financially possible. Thus, various in vitro digestion systems have been developed, from static mono-compartmental to dynamic multi-compartmental models, to simulate food digestive behaviors within the GI tract. Scope and approach: In this review, food digestion process along the GI tract is briefly described. The current in vitro digestion systems with regards to the human GI physiology, and their advantages versus limitations in the understanding of various food digestion processes in the upper GI tract are critically discussed. There is an emphasis on the “near real” dynamic rat (DRSD) and human (DHSI) gastric-intestinal systems, which not only mimic the peristaltic movements and biochemical conditions found in vivo, but also incorporate the gastric morphology and anatomical structures. Key findings and conclusions: Although some in vitro digestion systems reported in literature can be statistically correlated with certain perspectives of food digestion processes in vivo, many physiological, anatomical and geometrical factors that play important roles in determining the digestion rate and extent have been overlooked. The DRSD and DHSI are advantageous in terms of being able to resemble the gastric morphology and anatomy in the rats and humans, respectively. It is of importance that the upper GI anatomy and morphology along with the related biochemical environments and peristaltic movements occurring in vivo should be considered in the development of more advanced and biologically relevant in vitro digestion systems.

1. Introduction Food digestion rate and location in the upper GI tract, including mouth, esophagus, stomach and duodenum, significantly affect postprandial metabolism and human health (Fig. 1) (Lehmann & Robin, 2007; Miao, Jiang, Cui, Zhang, & Jin, 2015; Raigond, Ezekiel, & Raigond, 2015; Zhang & Hamaker, 2009). For example, in terms of starch digestion, if the digestion were sufficiently slow that the residual starch enters the large intestine, short-chain fatty acids (SCFA) will be produced by the fermentation process (Fig. 1B). The major SCFA are acetate, propionate, and butyrate, among which butyrate is of particular importance as an energy source for the colonocytes and is

believed to protect against colorectal cancer (Conlon et al., 2012). When starch reaches the ileum or colon, glucagon-like peptide 1 (GLP1) and peptide YY (PYY) can be stimulated, which are able to reduce the gastric emptying rate and increase the satiety via the gut-brain feedback (Fig. 1B) (Lee, Bello-Pérez, Lin, Kim, & Hamaker, 2013). On the other hand, rapid starch digestion in the duodenum after food ingestion can result in a large load of glucose over a short period of time, and further substantial increase of glucose level (Fig. 1A) and insulin concentrations in human blood stream. This is of a risk factor of reducing the insulin sensitivity and developing type 2 diabetes mellitus. Thus, a slowly and sustainably digested starch, e.g. digested in the lower parts of small intestine and fermented in the large intestine, is preferred for

∗ Corresponding author. School of Chemical and Environmental Engineering, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China. E-mail address: [email protected] (P. Wu).

https://doi.org/10.1016/j.tifs.2019.12.015 Received 28 June 2019; Received in revised form 9 December 2019; Accepted 19 December 2019 Available online 23 December 2019 0924-2244/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. A scheme showing the effects of starch digestion rates and locations on postprandial glycemic response (A) and simulating the GLP1 and PYY hormones to decrease food intake and gastric emptying rate (B). RDS is rapidly digestible starch. SDS is slowly digestible starch. RS is resistant starch. GLP-1 is glucagon-like peptide 1 and PYY is peptide YY. Fig. 1 B is adapted from (Lee et al., 2013) with permission.

human health. Ideally, a reliable and accurate study of the food digestion should be performed in vivo on humans. However, the human clinical studies are challenging, because they are not always technically, financially and ethically feasible (Guerra et al., 2012; Hur, Lim, Decker, & McClements, 2011; M.; Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, Carrière et al., 2014; Yoo & Chen, 2006), and associated with poor repeatability due to individual variations. Besides, clinical human tests have to rely on some advanced and expensive instruments, such as nuclear magnetic resonance, scintiscanner and ultrasonic scanner. Intubation techniques including gastric barostat and intraluminal manometry could also be applied for assessing gastric motility and emptying, whereas the disadvantage is that the invasive nature could be poorly tolerated by patients and there are possible disturbances of the normal physiology and motility patterns (Kong & Singh, 2008). Therefore, there is an increasing interest to obtain reliable in vitro digestion systems that can closely mimic the human GI tract. The in vitro GI systems have been widely applied in the past decades for investigating the fates of foods during digestion within the upper GI tract and their physiological effect on human health. Compared to the human clinical trials, in vitro approaches are more efficient, straightforward, cheaper, and more importantly do not have ethical restrictions (Chen et al., 2011). However, designing and making of an idea in vitro digestion system that is able to fully reproduce what is going on in the real human GI tract is challenging. This is due to the inherent complexity of the human GI tract, which involves complicated GI motility, anatomical structures, numerous digestion-related enzymes and hormones that are crucial for food digestion whereas difficult to simulate completely in vitro. Further efforts and technological innovations are therefore needed to develop more advanced and biologically relevant in vitro GI models. This review presents a general description of food digestion process in humans, followed by a critical evaluation of the advances and limitations of current in vitro digestion systems already reported in literature, with an emphasis on the “near real” dynamic in vitro rat and human stomach-intestine systems. Based on the shortcomings of these in vitro systems, the future trends of making more advanced in vitro GI models are proposed. The aim of this review is to show the way forward for the development of more realistic in vitro GI models, which is practically meaningful to gain a mechanistic understanding of the digestion process and to produce more healthy food products with specific functionality.

processing causes the reduction of food particle size and the formation of food bolus (Hiiemae, 2004). Liquids are however almost ready to be swallowed and require minimal processing other than to equilibrate to body temperature and to be diluted by saliva (Engelen et al., 2003). The perception of final changes in bolus properties is assumed to indicate to the central nervous system (CNS) that the bolus is ready to be swallowed without pain, discomfort or risk of dysphagia. This has been conceptualized as the ‘swallowing threshold’ and described as a function of particle size (Peyron, Mishellany, & Woda, 2004), the maximum cohesive force, the degree of lubrication and the structure of food boluses (Prinz & Lucas, 1997). Saliva is a complex viscous aqueous medium produced by salivary glands, containing 99.5% of water, 0.3% of proteins/enzymes, and various electrolytes like sodium, potassium, calcium, magnesium, phosphate and bicarbonate (Salles et al., 2011). The enzymes are able to partially modify the food structures and compositions. Based on their catalytic properties, around 30 different enzymes have been found in human saliva (Salles et al., 2011). Other proteins found in the saliva are immunoglobulin A (IgA), lysozyme, lactoferrin, as well as mucosal glycoproteins (mucins) (Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, Carriere et al., 2014). In addition, the composition and flow rate of saliva are modulated by physical and chemical properties of ingested foods as well as psychological and physiological factors (Salles et al., 2011). Mastication is a complex oral motor behavior modulating jaw movements by CNS and many peripheral sensory inputs from epithelial mechanoreceptors, joints, and muscles. The mandible moves not only vertically, but also anteroposteriorly and laterally, and teeth are the grinding implement responsible for breaking food into fragments through mechanical cutting, crushing, grinding, compressing and shearing during mastication. Depending on mechanical characteristics of the foods, fragments normally reach a critical particle size (~0.8–3 mm) before bolus formation and this size tends to be rather similar among subjects (van der Bilt & Fontijn-Tekamp, 2004). 2.2. Gastric processing The resulted food bolus is carried by esophageal peristalsis into the stomach, which has four main motor functions: storage, mixing, grinding and emptying (Fig. 2). Human stomach is anatomically divided into four major sections (fundus, body, antrum, and pylorus) and the initially resting (fasted state) volume is ~25 mL, which can expand to 1.5–4 L to accommodate large volumes of food (Norton, Wallis, Spyropoulos, Lillford, & Norton, 2014). The proximal stomach including the fundus and body, mainly acts as the reservoir for undigested material, whereas the distal stomach (antrum) is the grinder, mixer and siever of solid foods. The antrum also acts as a pump for gastric emptying of solids by propelling actions, which allows small particles to pass through the pyloric sphincter into the duodenum (Kong & Singh,

2. Human gastrointestinal digestion 2.1. Oral processing Food digestion in the human GI tract is a continuous and dynamic process. It starts from mouth, with mastication and salivation being the two complementary oral mechanisms. For solid foods, the oral 115

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external factors can affect the gastric emptying rate, such as the composition (liquids or solids), and macronutrient content (fats, proteins, and carbohydrates) of the food bolus. Liquid and small food particles empty more quickly than larger ones. Larger-sized food solids are retained in the stomach for further processing and are only emptied through the pylorus when reduced to a particle size of < 2 mm (Norton et al., 2014). The overall emptying of solid food mainly presents an initial lag phase of 20–40 min upon the food ingestion followed by a linear pattern. The complete gastric emptying of solids usually takes place within 3–5 h mainly depending on food physiochemical properties (Norton et al., 2014). The low-nutrient liquids usually empty in a monoexponential pattern without the lag phase and the half-emptying time for non-nutrient liquids is approximately 20 min (Norton et al., 2014). In terms of meal viscosity, contradictory results have been reported in literature. Some studies showed that the viscosity delayed the gastric emptying rate (Camps, Mars, de Graaf, & Smeets, 2016), while in other studies, the gastric emptying rate was not affected by meal viscosity (van Nieuwenhoven, Kovacs, Brummer, Westerterp-Plantenga, & Brouns, 2001). The degree to which meal viscosity influences gastric emptying might be markedly reduced by rapid dilution by gastric juice or increased motor function. This can be illustrated by a study that when participants consumed meals that varied 1000-fold in viscosity, their gastric emptying rate only differed by a factor of 1.3 (Marciani et al., 2000). Besides, it has also been shown that increasing the energy density was a more effective way than increasing the viscosity in decreasing gastric emptying (Camps et al., 2016). In addition, there is a complex bidirectional relationship existing between gastric emptying and glycaemia. Gastric emptying accounts for ~35% of the variance in peak postprandial blood glucose levels in both healthy and diabetic individuals, while the rate of emptying is itself regulated by acute changes in glycaemia (Phillips, Deane, Jones, Rayner, & Horowitz, 2015). The entry of glucose into the small intestine is decreased in response to hyperglycaemia and accelerated to mitigate hypoglycaemia. When macronutrients reach the ileum or colon, hormone like GLP-1 and PYY can be stimulated, which are able to reduce the gastric emptying rate and increase the satiety via the gut-brain feedback (Lee, Bello-Pérez, Lin, Kim, & Hamaker, 2013). Many other factors such as subject gender and physical activity, have been shown to play a role in the gastric emptying (Bornhorst & Singh, 2014). Overall, comprehensive studies that examine food properties, antral motility, gastric chyme properties, and perhaps even hormonal responses after a meal are required to fully explain the complicated feedback and control of gastric emptying rate.

Fig. 2. Region specificity and functionality of the human gastrointestinal tract relevant to food digestion.

2008). Gastric motility significantly affects the rate of food breakdown in the stomach by changing the forces, pressures, and specific flow profiles encountered by food particles. It is characterized during the fasting state by a cyclic contractive pattern, while the contractions become continuous during the fed state, moving food from the top to the pylorus at an averaged propagation velocity of 2.5 mm/s and frequency of 2.6–3 waves/min. The peristaltic waves cause the chyme to be propelled back into the stomach's main body via retropulsion. Retropulsion is responsible for drastic mixing and emulsifying the food with gastric juices. The contraction forces are reported ranging from 0.2 N to 1.89 N, depending on the stomach's fasting or fed state (Norton et al., 2014). The ingested food bolus is then redistributed to the distal stomach, further ground into particles with a diameter of < 1–2 mm by irregular antral, tonic and phasic pyloric contractions. Significant variance can exist within these contractions, which has important roles in affecting the gastric emptying and digestion patterns of different foods. For example, elders and females perhaps have weaker antrum contractions thus resulting in a slower gastric emptying (Houghton et al., 1988). These small food particles together with the gastric juice form the chyme and enter the duodenum in a pulsatile fashion against pyloric resistance (Stevens, Jones, Rayner, & Horowitz, 2013). The stomach secretes an average of 2–3 L of gastric fluids (~pH 2) each day containing gastric acid, salts, and digestive enzymes (pepsin, lipase) (Bornhorst & Singh, 2014). There is a gel-like mucous layer throughout the gastric lumen to protect the inner cells and musculature of the stomach from its own acid secretions. Foveolar cells are located in the superficial mucosal compartment of the stomach (cardia) and secrete mucus as well as bicarbonate ions (Bornhorst & Singh, 2014). The gastric fundus and body contain gastric crypts filled with mucous neck cells, which also secretes mucus to protect the gastric epithelium, as well as parietal and chief cells. Parietal cells are triangular cells, containing H+-K+-ATPase, which is a proton pump to exchange K+ for H+, resulting in gastric acid (HCl) secretion. Chief cells secrete pepsinogen, an inactive precursor that is converted into proteolytic enzyme pepsin upon contact with acid. Chief cells also secrete gastric lipase, which is responsible for 10–30% of dietary triglyceride hydrolysis. The antrum has a smooth surface filled mainly with mucus-secreting cells. For more details about gastric secretions, refer to (Bornhorst & Singh, 2014). The rate of gastric emptying is a critical determinant of postprandial digestion rate and extent of macronutrients. Lower gastric emptying rate would result in slower delivery of food into the small intestine, where most of the starch is digested and absorbed. Many internal and

2.3. Small intestinal digestion The major digestion of the food is continued in the small intestine following gastric digestion. Small intestine has two main roles in digestion and absorption, that is, breakdown of macromolecules and absorption of water and nutrients (Fig. 2). It anatomically includes duodenum (0.25–0.3 m), jejunum (~1.22 m), and ileum (~1.52 m). Most digestion happens in the duodenum while the digested nutrients are absorbed at jejunum and ileum. Pancreatic enzymes (a complex mixture of proteases, amylases, and lipases) and other digestive enzymes produced by the inner wall of the small intestine act together in the breakdown of food constituents. Besides, pancreas secretes bicarbonate into the duodenum to maintain the pH of small intestine around 6–7, which is suitable for these enzymes to digest fats, carbohydrates and proteins. Carbohydrate is digested into monosaccharides (glucose, fructose, and galactose), protein into dipeptides, tripeptides, and amino acids, and fat into free fatty acids and 2-monoglycerides before the absorption (Campbell, Berry, & Liang, 2019). Bile from gallbladder also enter into the duodenum and plays a specific role in lipid digestion by emulsifying dietary fats into small droplets thus promoting pancreatic lipase activity. Most bile salts are actively reabsorbed in the ileum and reused in the bile through enterohepatic 116

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cycling. The complex topology of the inner lining of the small intestine gives it a huge absorptive surface area. Water and nutrients are absorbed by villus enterocytes via simple diffusion, facilitated diffusion, or active transport. For example, dipeptides and tripeptides are more easily absorbed by the enterocyte because they are transported via a transmembrane H+ gradient, while amino acids require active transport that is Na+ dependent (Campbell et al., 2019). This prevents buildup of digestion products in the lumen of the small intestine, which could inhibit enzyme activities. Chyme is transported through the intestines by segmentation and peristaltic muscle contractions, which are regulated through a combination of myogenic, neural, and hormonal factors (Campbell et al., 2019). Among these three factors, myogenic factors are the most important ones, while neural and hormonal factors act to modify myogenic-initiated motor patterns. The neural systems, including the autonomic nervous system and the enteric nervous system can control the small intestinal motility via direct innervation of smooth muscle, secretory, and endocrine cells (Campbell et al., 2019). Besides, many hormones like gastrin, cholecystokinin, secretin, and glucagon have been found able to affect the small intestinal motility (Campbell et al., 2019). Segmentation is characterized by a pattern of pressure waves traveling short distances that aid in the mixing of chyme and enhance its contact with the villous surfaces. Peristaltic pattern results in a large forward movement of the chyme at a rate of 2–25 cm/s by a muscular contraction (Bornhorst & Singh, 2014). Motor activity continues during the fasting state via the migratory motor complex (MMC), in order to propel undigested material in the small intestine into the colon for fermentation. In addition, small intestinal regions harbor distinct microbial populations that may play a role in human health and diseases (Booijink et al., 2010).

Despite the wide applications of static in vitro models for assessing the digestibility of various food components, they oversimplify the digestive physiology, failing to mimic the dynamic aspects of the digestive process especially the mechanical forces and fluid dynamics in a series of rigid beakers under continuous stirring. Therefore, the static models are mainly used for mechanistic studies and hypothesis building with specific applications for screening purposes. More sophisticated dynamic in vitro digestion models ranging from single to multi-compartmental systems have thus been developed in the past decades. Compared to the static models, the dynamic models allow simulation of the dynamic aspects of digestion, such as transport of digested meals, continuous secretions of digestive fluids, variable enzyme concentrations, and pH changes over times that occur in in vivo conditions (Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, Carrière et al., 2014; Shani-Levi et al., 2017). Some representative dynamic in vitro systems include the SHIME, TIM, DGM, HGS, GDS, IMGS, DRSD and DHSI. The origin, structures, mechanisms, limitations and applications of these models are critically reviewed here and their main characteristics are summarized in Table 1. 3.1. Simulator of the human intestinal microbial ecosystem (SHIME) The SHIME developed by Molly, Vande Woestyne, and Verstraete (1993) is the first multi-compartmental in vitro digestion system reported in literature which integrates the whole gastrointestinal tract (except mouth). It consists of six double-jacketed vessels maintained at 37 °C, simulating the stomach, duodenum/jejunum, ileum, caecum/ ascending colon, transverse colon and descending colon, respectively (De Boever, Bart, & Willy, 2000; Molly et al., 1993) (Fig. 4A and Table 1). The first two reactor vessels of the SHIME are based on the filland-draw principles to simulate food uptake and digestion in the stomach and duodenum/jejunum. The last four vessels are continuously stirred tank reactors where suspensions are mixed continuously with the aid of magnetic stirrers. There is no gas exchange between the different vessels and N2 is continuously passed into each reactor from the headspace of the culture system to ensure anaerobic conditions. The SHIME has been used for nutritional and pharmaceutical studies, general safety assessments and particularly for the interactions of food components with human resident microbiota (De Boever, Wouters, Vermeirssen, Boon, & Verstraete, 2001; De Boever et al., 2000; Molly et al., 1993; Patrick; Van de Wiele, Van den Abbeele, Ossieur, Possemiers, & Marzorati, 2015). Compared to in vivo studies, the SHIME provides more reproducible results and allows several parameters under control for mechanistic studies (Yoo & Chen, 2006). As an oversimplified model, however, it doesn't take into account of the absorption mechanisms of metabolites, the effects of colonization in the microbiota, immune and adhesion of micro-organisms to the reactor vessels and tubing (De Boever et al., 2000; Van de Wiele et al., 2015). Moreover, the magnetic stirring action in the rigid structure of the vessels is significantly different from the in vivo peristaltic forces resulting in completely distinct mechanistic mixing and digestion behaviors. The human fecal bacteria used in the different parts of the colon vessels are not the representative of the microbial community compositions existed in vivo (De Boever et al., 2000; Possemiers, Verthé, Uyttendaele, & Verstraete, 2004). Some conflicting results were thus reported (De Boever et al., 2000; Possemiers et al., 2004) and the extent of reliability of the SHIME remains to be answered. In addition, it was reported that at least 2 weeks were required to form a representative microbial ecosystem in the SHIME (Possemiers et al., 2004), which is of course not an efficient tool for digestion studies.

3. In vitro digestion models A number of in vitro digestion models ranging from single static systems to multi-compartmental dynamic systems have been developed particularly in the recent years and widely used as an alternative tool to study food digestion in vivo. Most of previous and recent attempts to understand the food digestion behavior have used static tools such as water bath shaking, air bath rotation, and a pH-stat. These static models simulate the stomach and/or intestine using a succession of stirred vessels recreating a single set of biochemical conditions (i.e. temperature, enzymes, pH and bile salts) in the different compartments of the GI tract. However, a major concern for the in vitro static protocols is the significant variation in the use of digestion parameters among different individual models. The parameters mainly include pH and duration of each digestion step, amount and type of digestive enzymes used, stirring/agitation speed, amount of food sample and etc. These variations impede the possibility to compare results across research-groups and to deduce general findings (Lorena Barros, Retamal, Torres, Zúñiga, & Troncoso, 2016; Guerra et al., 2012; Kong & Singh, 2008; Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, Carrière et al., 2014). For example, a popular static digestion model reported by Englyst, Kingman, and Cummings (1992) included the oral and gastric phases. Recently, an international consensus was reached within the COST INFOGEST network. A standardized static in vitro digestion protocol (Fig. 3) based on the current state of knowledge on in vivo digestion conditions was published (Brodkorb et al., 2019; Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, Carriere et al., 2014). The characteristics of this model are summarized in Table 1. As described in the INFOGEST consensus protocol, a mincer is applied for mimicking the mastication process. A polypropylene centrifuge tube anchored in a rotating wheel mixer at 37 °C is employed to simulate the rhythmic peristaltic contractions and mixing of the human upper GI tract. Salivary α-amylase, pepsin, and pancreatin solutions are used as the digestive enzymes in the oral, gastric, and intestinal phase, respectively. Sample aliquots can be taken at various incubating times for the resulting digested micronutrient test.

3.2. TNO's gastrointestinal model (TIM) TIM, developed at TNO Nutrition and Food Research (Zeist, The Netherlands), is a well-known mutli-compartmental artificial gastrointestinal system. TIM-1 (Fig. 4B and Table 1) consists of four 117

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Fig. 3. Overview and flow diagram of the static in vitro digestion procedure proposed by COST INFOGEST, including the oral, gastric, and intestinal phases. The figure is reproduced from (Brodkorb et al., 2019). SSF, SGF and SIF represent Simulated Salivary Fluid, Simulated Gastric Fluid and Simulated Intestinal Fluid, respectively. Enzyme activities are in units per mL of final digestion mixture at each corresponding digestion phase.

(Blanquet et al., 2004). Therefore, further quantitative validations of the TIM-1 are required especially for pharmaceutical studies. Furthermore, the absence of antral contraction suggests that the retropulsion found in vivo cannot be created in the TIM-1 and the shape of the glass jackets is also remarkably different from a real human stomach. These differences indicate that the TIM-1 system is unable to reproduce the in vivo fluid hydrodynamics and shear/grinding forces (Kong & Singh, 2008).

computer-controlled chambers simulating the conditions of stomach, duodenum, jejunum and ileum, which was thoroughly described by Mans Minekus et al. (1995). Later on a large intestine model was added for TIM-2 to study the microbial digestion of humans and monogastric animals (Blanquet, Meunier, Minekus, Marol-Bonnin, & Alric, 2003). Each compartment of the TIM-1 and TIM-2 is composed of glass jackets with flexible inner wall where water (37 °C) is pumped into the space between the inner and outer walls with variable water pressure, causing the inner wall to expand and contract to mimic the peristaltic movements. The system is kept anaerobic by flushing it with nitrogen. The pH of each compartment is computer-monitored and controlled using pH electrodes and altered by injection of water or acid solution into the stomach. Gastric, biliary, and pancreatic secretions are maintained by computer-controlled pumps. Water, small molecules such as digested products are removed from the jejunum and ileum of TIM-1 by pumping of dialysates through the hollow fiber membrane units (Blanquet et al., 2003; Mans Minekus et al., 1995). This is to simulate the nutrient absorption in small intestine. The TIM system provides full simulation of the GI tract which is advantageous compared to other in vitro digestion devices. It integrates key parameters of human digestion: temperature, kinetics of gastric and intestinal pH, transit time, peristaltic mixing and transport, sequential addition of digestive secretions, and passive absorption of water and small molecules through a dialysis system. TIM-1 is a commercially available system that so far offers the closest simulation of the dynamic behaviors occurring within the human gastrointestinal tract (Guerra et al., 2012; Kong & Singh, 2008). It has been successfully applied to study the gastro-intestinal behavior of a wide variety of feed, food and pharmaceutical products (Blanquet et al., 2003; Bussolo de Souza et al., 2014; Havenaar et al., 2013; Ribnicky et al., 2014; Van Loo-Bouwman et al., 2014). As an alternative to the expensive and time consuming human trials, the TIM-1 was successfully used to predict the glycemic response after intake of 21 carbohydrate-based food products with a good coefficient (r = 0.91) of the in vivo results (Bellmann et al., 2010). However, it has been reported in a pharmaceutical study that no quantitative link between in vivo and in vitro data could be found due to the following possible in vivo factors: metabolization by epithelial cells, first pass effect, larger distribution volume in vivo, and renal clearance

3.3. Dynamic gastric model (DGM) The DGM developed at the Institute of Food Research (Norwich, UK) is a computer-controlled single compartmental model designed to simulate the human gastric compartment of the fundus and antrum with a funnel-shaped or cone-shaped device (~800 mL) (Fig. 4C and Table 1). It has two functionally distinct zones, the fundus/main body (proximal stomach, the upper part) and the antrum (distal stomach, the lower part), differing markedly in the mechanical forces applied to the food bolus (Mercuri, Faulks, Craig, Barker, & Wickham, 2009; Mercuri, Lo Curto, Wickham, Craig, & Barker, 2008). Within the rigid fundus and flexible main body of the DGM, the food bolus is subjected to low physical force generated by gentle rhythmic peristaltic contractions from cyclical pressurization of the 37 °C water jacket surrounding the main body. The gastric acid and enzyme secretions are delivered dynamically by computer-controlled pumps depending on pH and meal volume changes and then distributed around outside of the DGM main body. The gastric contents moved into the DGM antrum are subjected to higher shear and grinding forces from the upward and downward movement of the barrel during processing, resulting in higher efficiency of mixing and particle size reduction. The DGM is able to mimic the gastric digestion and disintegration as well as emptying of the solid foods in real-time due to the incorporation of the strong mechanical processing at the antrum part. Hence, it has been used extensively for both food and pharmaceutical studies. In terms of starch digestion study, it has been used to evaluate gastric processing and duodenal digestion of starch in six cereal meals on the associated glycemic response (Ballance et al., 2013; Mandalari et al., 2013, 2014). Nevertheless, for the food materials such as liquid meal or 118

119

–

–

–

–

In vitro Mechanical Gastric System, IMGS

–

–

Simulator of the Human Intestinal Microbial Ecosystem, SHIME TNO's Gastrointestinal Model, TIM

–

–

–

Human Gastric Simulator, HGS

–

–

–

Dynamic Gastric Model, DGM

SSF, CaCl2, salivary amylase

Gastric Digestion Simulator, GDS

Mincer

–

–

–

–

–

–

37 °C

–

–

–

–

–

–

7

pH

–

+

2–3 h

–

+

+

2h

Emptying time

Temp.

Mastication

Salivation

Gastric phase

Oral phase

COST INFOGEST static model

In vitro digestion system

–

+

+

+

+

–

Dynamic gastric secretions

Motor-driven four pairs of pistons

Computer-controlled water pressure device

Computer-controlled water pressure device; Low physical force around the main body and strong mechanical force at the antrum part Rollers secured on belts that are driven by motor and pulleys to create a continuous contraction The amplitude and frequency of the contractions is controllable by adjusting the position of the rollers and the motor speed Magnetic stirring

Rotating mixer

Mixing

Table 1 Key characteristics of some representative in vitro digestion systems reported in literaturea.

37 °C; water

37 °C; water

37 °C; water

37 °C; a ribbon heater and a temperature sensor

37 °C; light bulbs and a mini-fan

37 °C; water

37 °C; incubator

Temp.

+

+

2

+

+

+

3

pH

–

+

+

–

–

–

–

Dynamic intestinal secretions

–

+

8h

–

–

–

2h

Transit time

Small intestinal phase

–

Computercontrolled water pressure device

Magnetic Stirring

–

–

–

Rotating mixer

Mixing

Removed by pumping of dialysates through the hollow fiber membrane units –

–

–

–

–

–

Intestinal absorption

–

37 °C; water

37 °C; water

–

–

–

37 °C

Temp

(Blanquet et al., 2003; Minekus, Marteau, Havenaar, & Huis in ’t Veld, 1995) Barros et al. (2016)

(De Boever et al., 2000; Molly et al., 1993

Kozu et al. (2014)

Kong and Singh (2010)

(Brodkorb et al., 2019; Minekus, Alminger, Alvito, Ballance, Bohn, Bourlieu, Carrière et al., 2014) Verhoeckx et al. (2015)

(continued on next page)

–

+

6.5–7.0

–

–

–

7

pH

Ref.

C. Li, et al.

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+

+

37 °C; heating lamps

37 °C; heating lamps and water bath

3.4. Human gastric simulator (HGS)

+

+

+

Rolling extrusion rig; contraction frequency/ amplitude adjustable

–

The HGS, developed by Kong and Singh (Kong & Singh, 2010) at the University of California, Davis, is specifically designed to quantitatively simulate the peristaltic activity of antrum contraction waves (ACWs) generated in the lower part of the human stomach (Fig. 4D and Table 1). The ACWs are known to play significant role in creating strong mechanical shear and grinding forces contributing to the disintegration and mixing of gastric contents during digestion (Schulze, 2006; Schwizer, Steingoetter, & Fox, 2006). The HGS mainly consists of a round cylindrical stomach chamber made of latex rubber, mechanical driving device, gastric secretion device, and an insulated plastic foam chamber for temperature control. The stomach chamber has a collective volume of 5.7 L with a thin polyester mesh bag (net pore size of 1.5 mm) placed inside, simulating a sieving effect of pylorus. The mechanical driving device consists of a series of rollers secured on belts that are driven by motor and pulleys to create a continuous contraction of the latex wall. The contraction force imposed by the rollers on the gastric contents can be controlled by adjusting the distance between the opposite rollers through changing the screw engagement depth inside the aluminum rod. The rate of gastric emptying is controlled by means of a peristaltic pump connected to the bottom of the gastric compartment through a plastic tube (internal diameter of 3.2 mm). A mini peristaltic pump is used to control the delivery rate of the simulated gastric juice into the compartment through five polyethylene tubes with an inner diameter of 0.86 mm. The entire HGS system is housed in an insulated plastic foam chamber. Two light bulbs and a mini-fan are installed to maintain the system operating at uniform and constant temperature of 37 °C. The most noteworthy characteristic of the HGS is the ability to produce a reasonable range of amplitude and frequency of mechanical forces (6738–8922 N/m2) as those presented in vivo (Kong & Singh, 2010; Marciani et al., 2001). The performance of the HGS was evaluated by digesting cooked rice and apple slices in the simulated stomach and analyzing the gastric digesta properties (pH profiles, particle size distribution and solids content). The results indicated that the HGS had higher efficiency of solid foods disintegration and nutrient release than the shaking bath due to the incorporation of the “antrum” mechanical force (Kong & Singh, 2010). Another research carried out by the same team studied the influence of cooked rice digestion in the HGS on the physical properties, including pH, particle size distribution, solid leaching and rheology of the gastric digesta and they concluded that the bran layer on brown rice had a profound effect in gastric digestion resulting in lower glycemic responses than white rice (Kong, Oztop, Singh, & McCarthy, 2011). Besides, HGS has also been used for the digestion study of apples, almond butter and protein emulsion gel (Guo, Ye, Lad, Dalgleish, & Singh, 2014). However, to the authors’ knowledge, no systematic comparison between the HGS and in vivo digestion has been conducted. Furthermore, a major drawback of the HGS is that the distribution of gastric contents in the HGS depends on gravity, resulting in the aggregation of a large amount of gastric digesta at the bottom of the inverse conical gastric chamber. This contradicts with the food distribution observed in the real “J-shaped” stomach, where ingested solid foods are stored initially in the proximal stomach and then move gradually into the distal stomach (Kong & Singh, 2008; Urbain et al., 1989). In addition, the HGS fails to mimic the real shape of the human stomach.

a

“+” and “-” represent that the digestion system is or not equipped with the function.

37 °C; heating lamps and water bath + + – – – Dynamic Human Stomach-Intestine System, DHSI

–

+ – – – – Dynamic Rat StomachDuodenum System, DRSD

Temp. Salivation In vitro digestion system

Table 1 (continued)

Electromechanical instrument composed of a series of motors, rollers and eccentric wheels to produce peristaltic contractions

– Rolling extrusion rig; contraction frequency/ amplitude adjustable + + + 37 °C; heating lamps Electromechanical compression-rolling extrusion rig

Transit time Dynamic intestinal secretions Emptying time Mastication

pH

Gastric phase Oral phase

Dynamic gastric secretions

Mixing

Temp.

pH

Small intestinal phase

Mixing

Intestinal absorption

Ref.

(Chen et al., 2013; Wu, Bhattarai, et al., 2017; Wu, Bhattarai, et al., 2017; Wu et al., 2014; Wu, Bhattarai, et al., 2017) (Chen et al., 2016; Wang et al., 2019) pH

the meal with few large particles that are rarely affected by the gastric physical forces, other simpler methods are sufficient (Culen, Rezacova, Jampilek, & Dohnal, 2013). The in vivo fluid dynamics and distribution of gastric contents cannot be replicated due to the vertical alignment of main body and antrum, which is completely different from that in vivo (Verhoeckx et al., 2015). In addition, visual observations are not possible during antral processing as the DGM antrum is not transparent.

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Fig. 4. Representative dynamic in vitro digestion systems reported in literature. (A) simulator of the human intestinal microbial ecosystem (SHIME); (B) TNO's gastrointestinal model-1 (TIM-1); (C) dynamic gastric model (DGM); (D) human gastric simulator (HGS); (E) gastric digestion simulator (GDS) and (F) in vitro mechanical gastric system (IMGS). Fig. 4A-F are adapted from (De Boever et al., 2000; Molly et al., 1993), (Blanquet et al., 2003; Minekus et al., 1995), (Mercuri et al., 2008, 2009), (Kong & Singh, 2010), (Kozu et al., 2014) and (Barros et al., 2016), respectively.

3.5. Gastric digestion simulator (GDS)

through the transparent parallel windows of the gastric vessel. The Tofu particles were disintegrated to a larger extent by showing a significantly higher proportion of smaller particles in the GDS than that in a flaskshaking water bath where there was the absence of peristaltic movements. Similarly, the cooked brown rice grains were observed to show slowly particle breakdown due to the presence of their fibrous bran layers compared to the white rice when subjected to gastric digestion in both the GDS and flask-shaking bath. This result is consistent with that reported by Kong et al. (2011). Despite the advantage and successful application for simulated gastric disintegration of solid foods, the GDS is unable to mimic the emptying of gastric digesta which could dramatically influence the gastric digestion and subsequent nutrient release (Bornhorst & Singh, 2014; Kong & Singh, 2008; Marciani et al., 2013). The GDS has not been validated against in vivo data on human gastric digestion although similar digestive behaviors of the cooked white and brown rice such as particle size distribution were found between the gastric digestion in the GDS and in pigs (Bornhorst, Chang, Rutherfurd, Moughan, & Singh, 2013). Similar to the DGM, TIM and HGS, the gastric vessel of the GDS doesn't account for the real shape of the human stomach. As described above, the SHIME, TIM, DGM, HGS and GDS focus on simulation of the biochemical environments and physical movements occurred in vivo. However, the morphological and anatomical features of the GI tract have rarely been considered among them. These features, however, have been reported to dramatically affect the rate and extent

The GDS, developed by Kozu and co-workers (Kozu et al., 2014) at Tsukuba University, mainly consists of a gastric vessel that simulates the antrum and a roller device for creating peristaltic motion (Fig. 4E and Table 1). The gastric vessel has a collective total volume of 550 mL and parallel transparent windows are equipped allowing the direct realtime observation of the disintegration of gastric contents in the presence of quantitatively simulated peristaltic contractions. The amplitude and frequency of the contractions is controllable by adjusting the position of the rollers and the motor speed. The rollers progress at several millimeters per second with a frequency of a few cycles/min, corresponding to human ACW parameters (Kozu et al., 2014). The large food particles can be disintegrated into smaller particulates due to the combined actions of the physical force induced by the simulated antrum peristaltic contractions and biochemical reactions (Kobayashi, Kozu, Wang, Isoda, & Ichikawa, 2017; Kozu et al., 2014). A temperature control system consisting of a ribbon heater and a temperature sensor is used to automatically keep the temperature inside and around the gastric vessel of 37 °C. The key advantage of the GDS is the combination of the controllable gastric peristalsis and direct observation of gastric digestion behaviors in real-time (Kobayashi et al., 2017). As such, it has been applied to investigate the gastric disintegration of solid or semisolid foods such as Tofu (Kozu et al., 2014) and cooked rice (Wang et al., 2015), the disintegration of which can be directly observed 121

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of digestion and absorption of ingested food materials (Chen et al., 2016; Chen, Wu, & Chen, 2013; DeSesso & Jacobson, 2001). For example, the wrinkled surface of inner small intestine possesses thousands of microvilli, which could increase the surface area of human small intestine for nutrients absorption by a factor of 20 (DeSesso & Jacobson, 2001). According to the in vivo study (Schulze, 2006), due to the characteristic “J” shape of human gastric morphology, heavier food particles than water are fell onto the greater curvature (proximal stomach), while lighter particles suspended in the water, filling up the pylorus canal. The suspension of water and the lighter particles (liquid phase) thus is emptied earlier than the heavier ones. However, this effect could not be achieved for in vitro gastric models such as the DGM, HGS, and GDS with inversely conical shape or the TIM with tubular geometry. In these in vitro systems, solid or semisolid food is stacked in the inverse conical cavity by gravity and emptied together with the aid of peristaltic waves on the gastric wall (Kong & Singh, 2010). This suggests that they could not reproduce similar gastric distributions and emptying patterns such as the lag phase of gastric digestion of heavier solid foods. The lag phase observed for solid foods may however be very important for evaluating the semisolid food digestion in human stomach, as many active ingredients from the solid food matrix such as probiotics is sensitive to the gastric acid and gastric enzymes. As a result, survival ratios of probiotics from the actual in vivo experiments may vary significantly from the values obtained with the in vitro gastric models. In recent years, several gastric human models with similar shape of the human or animal stomach were developed such as the IMGS, DRSD and DHSI. Their major features are also shown in Table 1.

3.7. Dynamic rat stomach-duodenum system (DRSD) Several generations of the DRSD (DRSD-I, DRSD-II and DRSD-III) aiming to simulate rat stomach and duodenum has been successively reported by Chen and co-workers (Chen, 2012; Chen & Yoo, 2006). The DRSD is a “near real” in vitro rat digestion model which was designed based on in vivo parameters (such as stomach dimensions, anatomical structures, flow rate of gastric fluids and enzyme activities) from living tests on rats. The key idea is not only to simulate the peristaltic contractions and biochemical conditions as those presented in the real rat stomach and duodenum, but also to follow the morphological and anatomical structures as close as possible (Chen, 2012; Chen & Yoo, 2006). As seen from the latest version of the in vitro rat digestion system (Fig. 5A and Table 1), DRSD-III, it is mainly composed of the soft-elastic rat stomach and duodenum model, the electromechanical device, the digestive fluid secretion and digesta emptying device, and the temperature controlled box (Chen, Jayemanne, & Chen, 2012; Chen et al., 2013). The soft rat stomach model (Fig. 5C) made of semi-transparent silicone material was fabricated with the aid of 3D-printing technology (Fig. 5B). It has similar morphology and geometrical dimensions with the real rat stomach, occupying a box of around 40 mm length × 30 mm width × 25 mm thickness with an approximately total volume of 9.0 mL (Fig. 5C). The consistent morphological inner structures are also obtained accordingly, with wrinkled inner surface at the glandular portion whereas smooth inner structure at the fore-stomach (DeSesso & Jacobson, 2001; Gärtner, 2002). The electromechanical device consists of a series of rollers and eccentric wheels acting in parallel to produce a powerful peristaltic rolling-extrusion movement and relatively weak vertical compression on the wall of the soft rat stomach model. The vertical compression imposed on the fore-stomach is designed to gradually drive the food materials from the fore-stomach to the glandular portion, whereas the rolling-extrusion is responsible for grinding the large food particles into smaller size and propelling the gastric digesta into the duodenum for further digestion. The rat duodenum device mainly consists of a silicone tube (~15 cm in length and 3 mm in inner diameter) and an electromechanical driving instrument to produce peristaltic contractions. The duodenum model is made up of a silicone tube with a length of ~15 cm and inner diameter of 3 mm. It is installed between the fixed pulley and eccentric wheel, which is intermittently rolling-extruded to simulate intestinal peristalsis. The segmented rolling extrusion functions to mix the duodenal digesta with pancreatic enzymes and bile juice (Lentle et al., 2012). The gastric secretions of the digestive enzymes and fluids into the rat stomach and duodenum models are accurately controlled by syringe pumps. Another syringe pump is applied to regulate the gastric emptying rate. The temperature inside the plexiglass box is maintained at 37 °C through the computer-controlled heating lamps. The DRSD-I has been validated by showing consistent digestion trends of the digestive and gastric emptying behaviors such as gastric pH, particle size distribution of casein powder suspensions (Chen et al., 2013) and raw rice particles (Wu, Chen, Wu, & Chen, 2014) with those obtained from the in vivo tests on rats. Zhang, Liao, Wu, Chen, and Chen (2018) illustrated that it was possible to achieve similar casein digestibility in the DRSD-II by optimizing the operating parameters to that found in vivo (Fig. 5D). As presented previously, the in vitro digestibility of the casein powder suspensions was significantly higher in the rat stomach model with wrinkled inner surface than that with smooth inner surface (Chen et al., 2013). Pleasingly, the peristaltic contractions exerted by the DRSD-II seem rather not vigorous, whereas these could produce comparable in vitro casein digestibility with a rigorously standard stirred tank reactor (Zhang et al., 2018). These results indicate the significant role of the gastric morphological and anatomical structures in enzymatic hydrolysis efficiency perhaps by producing an improved fluid mixing and gastric fluid distribution. By applying the DRSD which is relatively simple, less cost and particularly suitable for samples with limited amount, a number of food

3.6. In vitro mechanical gastric system (IMGS) The IMGS was reported in 2016 and developed by Barros and coworkers at Universidad Tecnológica Metropolitana, Chile (Barros, Retamal, Torres, Zuniga, & Troncoso, 2016). The device is mainly composed of a J-shaped human stomach model and a mechanical system (Fig. 4F and Table 1). The gastric model made of natural liquid latex was fabricated with the aid of a 3D-printed virtual prototype with realistic human morphology (“J” shape) and dimensions. The latex stomach model has an internal volume of 900 mL with a thickness of 1 mm. The mechanical system are coupled with four pairs of acrylic pistons, arranged on each side of the stomach model to reproduce an operational frequency of the peristaltic waves of three contractions per minute and mechanistic forces ranging between 0.20 and 1.89 N (Barros et al., 2016). The compressive force exerted by the mechanical system is monitored by a cylindrical Teflon probe attached to a texturometer. The pH changes during gastric digestion are directly measured using a pH-stat automatic titration unit. The human body temperature is mimicked by submerging the latex stomach model into a water bath at 37 °C and regulated by water recirculation with thermostatic control. A significant improvement of the IMGS in comparison with the SHIME, TIM, DGM, HGS and GDS is that it takes the stomach shape and dimensions into account. As stated earlier, this characteristic allows a more realistic simulation of the gastric distribution and emptying of the solid or semisolid foods. Moreover, the IMGS is able to produce similar peristaltic movements as found in the real human stomach, including propulsion, retropropulsion and grinding by controlling the moving frequency and distances of the four pairs of pistons (Barros et al., 2016). With the IMGS system, the O/W emulsion stability was shown to play a crucial role in controlling the final extent of lipolysis of protein-stabilized O/W emulsions (Barros et al., 2016). However, it is noted that IMGS cannot simulate the gastric emptying process, as the whole stomach model is a closed system. Furthermore, the continuous gastric secretions of enzymes and digestive fluids was not considered during the simulated digestion. As a new in vitro gastric system, it has not been directly validated against with the in vivo data and limited applications have been reported to date. 122

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Fig. 5. (A) The “near real” dynamic in vitro rat stomach-duodenum system-III (DRSD-III); (B) fabrication of the silicone rat stomach model with the aid of 3D-printing technology; (C) the soft-elastic silicone rat stomach model; (D) comparison of soluble peptides of the gastric digesta of the casein suspensions during gastric digestion in the in vivo, DRSD-I, DRSD-II and DRSD-III. Fig. 5A–D are reproduced from (Chen et al., 2013; Wu, Bhattarai, et al., 2017; Wu, Liao, Luo, Chen, & Chen, 2017; Zhang et al., 2018).

rates for variant foods can thus be determined by adjusting the auxiliary emptying device to reproduce the in vivo gastric emptying kinetics. The gastric sieving function of the pylorus is achieved through the pyloric squeezing device by which the opening size of the pylorus model can be changed with time. The temperature-controlled system consisting of water bath, heating lamp, thermal sensor and heat preservation box is used to maintain the surrounding temperature at 37 ± 1 °C. The DHIS-I was validated by showing consistent trends of gastric distribution and emptying of cooked buckwheat with that in vivo data reported in literature (Chen et al., 2016). As demonstrated by Wang et al. (2019), the DHIS-II was able to generate consistent gastric emptying profiles of both solid and liquid fractions in the mixed meal of beef stew and orange juice (300 g) with that reported in vivo (Moore, Christian, & Coleman, 1981) through optimizing the operating parameters like stomach tilting angle and squeezing frequency of the pylorus (Fig. 6C). The performance of the DHIS-II was further evaluated by showing good qualitative matches of the gastric pH, particle size distribution and emptying profiles of cooked rice with the in vivo data from literature. These results indicated that it was possible to achieve whatever gastric emptying situations in vivo by optimizing the operating parameters of the DHIS-II. However, the biggest disadvantage of the DHIS-II is the difficulty to find appropriate operating parameters to follow pre-set gastric emptying profiles. Moreover, the digestion rate and extent of real foods in the DHIS-II are generally lower than that in vivo due to the insufficient mixing between the substrates and digestive enzymes.

digestion-related studies have been performed (Zhao, Sun et al., 2019; Zhao, Wang, Zheng, Chen, & Guo, 2019). For examples, Wu, Deng, Wu, Dhital, and Chen (2017) investigated in vitro gastric digestion of cooked white and brown rice using the DRSD-I and illustrated the importance of the bran layer in the brown rice in decreasing the gastric emptying rate and starch digestibility. Wu, Bhattarai et al. (2017) used the DRSDII to illustrate the mechanisms of pectin in delaying the gastric emptying and hydrolysis rate of starch and protein in the starch-based diets either by increasing the digesta viscosity or encapsulating the starch granules in the pectin gel network. It was recently applied to monitor the release of bioaccessible sulforaphane content in the microwavepretreated cabbages during simulated gastric and duodenal digestion (Pongmalai et al., 2018). Despite the broad applications of the DRSD, it is noted that the rat digestion system should be prudently applied to study digestion in humans because of the considerable variations in GI morphology and anatomy between the rats and humans (DeSesso & Jacobson, 2001; Gärtner, 2002). 3.8. Dynamic human stomach-intestine system (DHIS) The initial version of the “near real” in vitro human stomach and small intestine system, DHIS-I, was also reported by (Chen et al., 2016). Later, an updated version named DHIS-II was shown by (Wang et al., 2019). The DHIS-II mainly consists of the J-shaped silicone human stomach model, the electromechanical instrument composed of a series of motors, rollers and eccentric wheels to produce peristaltic contractions, the auxiliary emptying device and temperature-controlled system (Fig. 6A) (Wang et al., 2019). The human stomach model (Fig. 6B) has similar gastric morphology (“J” shape), dimensions and inner wrinkles to the real human stomach, which is made with the aid of 3D-printing technology. By means of the auxiliary emptying device, the gastric emptying rate can be reasonably controlled by modifying the tilting angle of the whole stomach system in the range of −45° to +45° (“-” and “+” mean anticlockwise and clockwise rotation, respectively). Clockwise rotation can delay the emptying rate by impairing the gravity effect, while the gastric content is more readily to flow out resulting in an accelerated emptying rate by anticlockwise rotation. The emptying

4. Challenges and future prospects of the in vitro digestion systems In vitro digestion systems are increasingly popular and widely used for food nutritional or pharmaceutical studies in the recent years, due to their advantages of being more rapid, better reproducibility, lower cost, less labor intensive, and no ethical restrictions compared to in vivo tests on animals or humans. Although substantial advances have been made on improving the current in vitro digestion systems, there are still significant amount of work needed to be performed in the future. The followings are a summary of the limitations of the current in vitro 123

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Fig. 6. (A) The “near real” advanced dynamic in vitro human stomach-intestine system (DHIS-II); (B) wrinkled internal mold of the 3D-printed human stomach model; and (C) comparison of gastric retention ratio of the solid and liquid fractions in the mixture of beef stew and orange juice between in the DHIS-II and in vivo (reproduced from (Wang et al., 2019)). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

and 25% of the starch in spaghetti can already be hydrolyzed (Burton & Lightowler, 2008). As a consequence, an effective in vitro mouth device is of importance to couple with the gastric and intestinal models for full simulation of food digestion in vivo. 4) It is known that human digestive physiology (i.e. transit time, digestion and gastric emptying rate) gastrointestinal motility (i.e. peristaltic contraction frequency and force) and biochemical environment (such as pH, secretion rate of digestive juice and enzyme activity) are influenced by many biological factors including age, gender, posture, diseased states and etc. (Bourlieu et al., 2014; Houghton et al., 1988; Nguyen, Bhandari, Cichero, & Prakash, 2015). For example, gastric emptying is slower in elders and females perhaps due to the weaker antrum contractions in elders and women (Houghton et al., 1988). The availability of some digestive enzymes such as salivary amylase, pepsin and pancreatic α-amylase, their concentration, and gastric pH are different between infants and adults (Bourlieu et al., 2014). Therefore, in addition to mimicking normal adult human digestion systems like most of the current models, different populations due to their distinct differences in biological aspects should have their corresponding in vitro systems. 5) The necessity for most in vitro digestion systems to collect and store samples for further characterization is a major challenge. This sometimes involves the operation inconvenience and a problem for collecting representative samples. To avoid sampling, various advanced analytical techniques such as neutron or X-ray scattering, nuclear magnetic resonance, mass spectrometry and interfacial techniques are preferred to characterize physicochemical properties (such as gastric digesta pH and gastric emptying rate) directly within a digestion system (in situ) in a noninvasive way for continuous monitoring of digestion kinetics (Marze, 2017). 6) A careful interpretation of in vitro results is always required. It is difficult to ascertain which of the current models provides the most accurate values in terms of the human situation. Selection of the most appropriate model requires careful evaluation of the study objectives to assess the advantages and limitations afforded by each type of system, and a compromise between technical complexity and physiological relevance has often to be made.

digestion systems and a few recommendations for the future development of more advanced in vitro devices. 1) The morphology and anatomical structures of the GI tract are rarely incorporated for most in vitro digestion systems. As illustrated in this review paper, the gastric morphology and anatomical structures (e.g. wrinkles on the inner surface of stomach wall) have been shown to play significant role in gastric digestion and emptying rates. However, only a few digestion models including the IMGS and DHSI have considered the “J-shaped” morphology of the human stomach, while none of them has completely mimicked the morphological and anatomical aspects of human or animal GI tract. In order to make more realistic in vitro digestion systems, more anatomical details such as villi, wrinkles and dimensions of the GI tract along with the related peristaltic movements and biochemical conditions should be taken into account. 2) The in vitro and in vivo correlations (IVIVCs), which reliably associates in vitro and in vivo data, remains a high priority to validate in vitro systems. Despite the potential and broad applicability, none of the current in vitro digestion models reported in literature has been fully validated against in vivo data and recognized with widespread applications in scientific community. This is mostly perhaps due to the inherently complex anatomy, motility biochemical conditions and physiological functions of human gastrointestinal tract. Particularly, hormonal and nervous control, feedback mechanisms, mucosal cell activity, complexity of peristaltic movements, and involvement of the local immune system are extremely difficult and even impossible to reproduce in vitro (Guerra et al., 2012; Marze, 2017). However, these physiological factors are undoubtedly of significance in food digestion and may be investigated using Caco-2 cellular models in the future. 3) None of the current in vitro systems involve all the digestion steps from mouth to large intestine. For example, the consumption of a solid food inside mouth involves various oral operations, including first bite, chewing and mastication, transportation, bolus formation, swallowing and etc. (Chen, 2009). The oral step is rapid but plays an important role in the overall digestive process and particularly on gastric emptying rate of solid foods (Bornhorst & Singh, 2012; Chen, 2009; Marze, 2017). For starchy food, the simulation of oral digestion must be considered, as it has also been reported that even during 20–30 s of oral food processing, 50% of the starch in bread

In conclusion, the construction of a well-recognized in vitro digestion model with widespread applications is challenging while worthwhile, which is a process requiring a long time and technological 124

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innovations to gather experience by improving the current models to a higher level that is closer to the real situations in vivo.

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5. Conclusions Different in vitro digestion systems have been developed that can be applied for food digestion study, where indeed valuable scientific insights into the assessment of food digestion have been learnt from these in vitro models. It is recommended that careful evaluations of the study purposes are required for selection of the most appropriate digestion system as each type of system has its own advantages and limitations. Given the limitations of both human and animal experiments, a logistic way is that correlations are firstly established based on data from human cohort trials, while mechanistic studies are conducted by in vitro digestion systems to prove the causation. If combined with the appropriate functional assays, these studies have the potential to provide unique insight into the mechanisms by which the food is digested. Author contributions X. D Chen conceived the research topic. W Yu partially researched the literature and proposed valuable suggestions for the work. C Li and P Wu drafted and revised the paper scientifically, and P Wu prepared it in the final format for publication. All authors read and approved the final version of the manuscript. Declaration of competing interest The authors declare that there is no conflict of interest. Acknowledgements We are grateful for the kind help of Dr. Wei Zou from The University of Queensland for his in-depth discussion. This study was supported by the the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (19KJD550001), the Youth Fund of Jiangsu Natural Science Foundation (BK20190906), the National Key Research and Development Program of China (International S&T Cooperation Program, ISTCP, 2016YFE0101200), a grant from COFCO Nutrition and Health Research Institute (Beijing) and the National Natural Science Foundation of China-the Optimized Research on In vitro Soft-elastic Rat Stomach Biomimetic Digestive System (general program, No. 21676172). The first author would like to thank the funding support from Jiangsu Entrepreneurship and Innovation Phd Program, Yangzhou Lvyangjinfeng Talent Program, Jiangsu Yangzhou Key Research and Development Program (No. SSF2018000008), Yangzhou University Young Scientist Innovation Fund, Yangzhou University Open Research Fund, and Yangzhou University Talent Acquisition Startup Fund. References Ballance, S., Sahlstrøm, S., Lea, P., Nagy, N., Andersen, P., Dessev, T., et al. (2013). Evaluation of gastric processing and duodenal digestion of starch in six cereal meals on the associated glycaemic response using an adult fasted dynamic gastric model. European Journal of Nutrition, 52, 799–812. Barros, L., Retamal, C., Torres, H., Zúñiga, R. N., & Troncoso, E. (2016). Development of an in vitro mechanical gastric system (IMGS) with realistic peristalsis to assess lipid digestibility. Food Research International, 90, 216–225. Bellmann, S., Minekus, M., Zeijdner, E., Verwei, M., Sanders, P., Basten, W., et al. (2010). TIM-carbo: A rapid, cost-efficient and reliable in vitro method for glycaemic response after carbohydrate ingestion. In J. W. V. D. Kamp (Ed.). Dietary fibre new frontiers for food & health (pp. 467–473). Wageningen: Wageningen Academic Publishers. van der Bilt, A., & Fontijn-Tekamp, F. A. (2004). Comparison of single and multiple sieve methods for the determination of masticatory performance. Archives of Oral Biology, 49, 193–198. Blanquet, S., Meunier, J. P., Minekus, M., Marol-Bonnin, S., & Alric, M. (2003). Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: A model for biodetoxication in the human digestive environment. Applied and Environmental Microbiology, 69, 2884–2892. Blanquet, S., Zeijdner, E., Beyssac, E., Meunier, J.-P., Denis, S., Havenaar, R., et al.

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