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The aim of this study was to characterise the individual human gastric and duodenal juices to be used in in vitro model digestion and to examine the storage stability of the enzymes. Gastroduodenal juices were aspirated, and individual variations in enzymatic activities as well as total volumes, pH, bile acids, protein and bilirubin concentrations were recorded. Individual pepsin activity in the gastric juice varied by a factor of 10, while individual total proteolytic activity in the duodenal juice varied by a factor of 5. The duodenal amylase activity varied from 0 to 52.6 U/ml, and the bile acid concentration varied from 0.9 to 4.5 mM. Pooled gastric and duodenal juices from 18 volunteers were characterised according to pepsin activity (26.7 U/ml), total proteolytic activity (14.8 U/ml), lipase activity (951.0 U/ml), amylase activity (26.8 U/ml) and bile acids (4.5 mM). Stability of the main enzymes in two frozen batches of either gastric or duodenal juice was studied for 6 months. Pepsin activity decreased rapidly and adjusting the pH of gastric juice to 4 did not protect the pepsin from degradation. Lipase activity remained stable for 4 months, however decreased rapidly thereafter even after the addition of protease inhibitors. Glycerol only marginally stabilised the survival of the enzymatic activities. These results of compositional variations in the individual gastrointestinal juices and the effect of storage conditions on enzyme activities are useful for the design of in vitro models enabling human digestive juices to simulate physiological digestion.

a b s t r a c tBackground: In vitro digestion models show great promise in facilitating the rationale design of foods. This paper provides a look into the current state of the art and outlines possible future paths for developments of digestion models recreating the diverse physiological conditions of specific groups of the human population. Scope and approach: Based on a collective effort of experts, this paper outlines considerations and parameters needed for development of new in vitro digestion models, e.g. gastric pH, enzymatic activities, gastric emptying rate and more. These and other parameters are detrimental to the adequate development of in vitro models that enable deeper insight into matters of food luminal breakdown as well as nutrient and nutraceutical bioaccessibility. Subsequently, we present an overview of some new and emerging in vitro digestion models mirroring the gastro-intestinal conditions of infants, the elderly and patients of cystic fibrosis or gastric bypass surgery. Key findings and conclusions: This paper calls for synchronization, harmonization and validation of potential developments in in vitro digestion models that would greatly facilitate manufacturing of foods tailored or even personalized, to a certain extent, to various strata of the human population.

Lactoferrin (LF) is a protein present in milk and other body fluids that plays important biological roles. As part of a diet, LF must survive gastrointestinal conditions or create bioactive fragments to exert its effects. The degradation of LF and formation of bioactive peptides is highly dependent on individual variation in intraluminal composition. The present study was designed to compare the degradation and peptide formation of bovine LF (bLF) following in vitro digestion under different simulated intraluminal conditions. Human gastrointestinal (GI) juices were used in a 2-step model digestion to mimic degradation in the stomach and duodenum. To account for variation in the buffering capacity of different lactoferrin-containing foods, gastric pH was adjusted either slowly or rapidly to 2.5 or 4.0. The results were compared with in vivo digestion of bLF performed in 2 volunteers. High concentration of GI juices and fast pH reduction to 2.5 resulted in complete degradation in the gastric step. More LF resisted gastric digestion when pH was slowly reduced to 2.5 or 4.0. Several peptides were identified; however, few matched with previously reported peptides from studies using nonhuman enzymes. Surprisingly, no bovine lactoferricin, f(17-41), was identified during in vitro or in vivo digestion under the intraluminal conditions used. The diversity of enzymes in human GI juices seems to affect the hydrolysis of bLF, generating different peptide fragments compared with those obtained when using only one or a few proteases of animal origin. Multiple sequence analysis of the identified peptides indicated a motif consisting of proline and neighboring hydrophobic residues that could restrict proteolytic processing. Further structure analysis showed that almost all proteolytic cutting sites were located on the surface and mainly on the nonglycosylated half of lactoferrin. Digestion of bLF by human enzymes may generate different peptides from those found when lactoferrin is digested by nonhuman enzymes. The degradation of LF in the GI tract should be taken into consideration when health effects are proposed, because LF has now been approved by the European Food Safety Authority as a dietary supplement in food products.

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