A standardised static in vitro digestion method suitable for food - an international consensus.

Simulated gastro-intestinal digestion is widely employed in many fields of food and nutritional sciences, as conducting human trials are often costly, resource intensive, and ethically disputable. As a consequence, in vitro alternatives that determine endpoints such as the bioaccessibility of nutrients and non-nutrients or the digestibility of macronutrients (e.g. lipids, proteins and carbohydrates) are used for screening and building new hypotheses. Various digestion models have been proposed, often impeding the possibility to compare results across research teams. For example, a large variety of enzymes from different sources such as of porcine, rabbit or human origin have been used, differing in their activity and characterization. Differences in pH, mineral type, ionic strength and digestion time, which alter enzyme activity and other phenomena, may also considerably alter results. Other parameters such as the presence of phospholipids, individual enzymes such as gastric lipase and digestive emulsifiers vs. their mixtures (e.g. pancreatin and bile salts), and the ratio of food bolus to digestive fluids, have also been discussed at length. In the present consensus paper, within the COST Infogest network, we propose a general standardised and practical static digestion method based on physiologically relevant conditions that can be applied for various endpoints, which may be amended to accommodate further specific requirements. A frameset of parameters including the oral, gastric and small intestinal digestion are outlined and their relevance discussed in relation to available in vivo data and enzymes. This consensus paper will give a detailed protocol and a line-by-line, guidance, recommendations and justifications but also limitation of the proposed model. This harmonised static, in vitro digestion method for food should aid the production of more comparable data in the future.

In Vitro Models for Studying Secondary Plant Metabolite Digestion and Bioaccessibility.

There is an increased interest in secondary plant metabolites, such as polyphenols and carotenoids, due to their proposed health benefits. Much attention has focused on their bioavailability, a prerequisite for further physiological functions. As human studies are time consuming, costly, and restricted by ethical concerns, in vitro models for investigating the effects of digestion on these compounds have been developed and employed to predict their release from the food matrix, bioaccessibility, and assess changes in their profiles prior to absorption. Most typically, models simulate digestion in the oral cavity, the stomach, the small intestine, and, occasionally, the large intestine. A plethora of models have been reported, the choice mostly driven by the type of phytochemical studied, whether the purpose is screening or studying under close physiological conditions, and the availability of the model systems. Unfortunately, the diversity of model conditions has hampered the ability to compare results across different studies. For example, there is substantial variability in the time of digestion, concentrations of salts, enzymes, and bile acids used, pH, the inclusion of various digestion stages; and whether chosen conditions are static (with fixed concentrations of enzymes, bile salts, digesta, and so on) or dynamic (varying concentrations of these constituents). This review presents an overview of models that have been employed to study the digestion of both lipophilic and hydrophilic phytochemicals, comparing digestive conditions in vitro and in vivo and, finally, suggests a set of parameters for static models that resemble physiological conditions.

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of baker's yeast.

During food processing such as baking, phytate is dephosphorylated to produce degradation products, such as myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates. Certain myo-inositol phosphates have been proposed to have positive effects on human health. The position of the phosphate groups on the myo-inositol ring is thereby of great significance for their physiological functions. Using a combination of high-performance ion chromatography analysis and kinetic studies the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme from baker's yeast (Saccharomyces cerevisiae) was established. The data demonstrate that the phytate-degrading enzyme from baker's yeast dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), D-Ins(1,2)P(2), to finally Ins(2)P (notation 3/4/5/6/1). Knowledge of the absolute stereochemical specificity of the baker's yeast phytase allows use of the enzyme to produce defined myo-inositol phosphates for kinetic and physiological studies.

Production process for high-quality pea-protein isolate with low content of oligosaccharides and phytate.

A process for pea-protein isolate production, resulting in low content of phytate and oligosaccharides, has been developed. Oligosaccharides were removed from the protein fraction through ultrafiltration. Ultrafiltration of 50- and 100-kD molecular-weight cutoffs (MWCOs) were tested, and both effectively separated the oligosaccharides from the protein. Phytate degradation was achieved by incubation of the pea-protein solution by addition of exogenous phytase enzyme. An almost complete degradation of inositol hexa-, penta-, tetra-, and triphosphates was reached using an incubation time of 1 h. The reduced content of oligosaccharides and inositol phosphates is likely to result in reduced flatulence and improved mineral bioavailability. These qualities of the pea-protein isolate make it a suitable protein source for infant formula production.

Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the stereospecificity of myo-inositol hexakisphosphate dephosphorylation by the phytate-degrading enzyme P2 of Escherichia coli was established. High-performance ion chromatography revealed that the phytate-degrading enzyme P2 of E. coli degrades myo-inositol hexakisphosphate by stepwise dephosphorylation via D/L-Ins(1,2,3,4,5)P(5), D/L-Ins(2,3,4,5)P(4), D/L-Ins(2,4,5)P(3) or D/L-Ins(1,2,4)P(3), D/L-Ins(1,2)P(2) or Ins(2, 5)P(2) or D/L-Ins(4,5)P(2) to finally Ins(2)P or Ins(5)P. Kinetic parameters for myo-inositol pentakisphosphate hydrolysis by E. coli and wheat phytase, respectively, showed that the myo-inositol pentakisphosphate intermediate produced either by the phytate-degrading enzyme of wheat or E. coli are not identical. The absolute configuration of the myo-inositol pentakisphosphate isomer produced by the E. coli enzyme was determined by taking into consideration that wheat phytase produces predominantly the D-Ins(1, 2,3,5,6)P(5) isomer (Lim, P.E., Tate, M.E., 1973. The phytases: II. Properties of phytase fraction F(1) and F(2) from wheat bran and the myo-inositol phosphates produced by fraction F(2). Biochim. Biophys. Acta 302, 326-328). The data demonstrate that the phytate-degrading enzyme P2 of E. coli dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,3,4,5)P(5), D-Ins(2,3,4,5)P(4), D-Ins(2,4,5)P(3), Ins(2,5)P(2) to finally Ins(2)P (notation 6/1/3/4/5).