Uri Lesmes

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.

Effects of resistant starch type III polymorphs on human colon microbiota and short chain fatty acids in human gut models

This study probed the possible effects of type III resistant starch (RS) crystalline polymorphism on RS fermentability by human gut microbiota and the short chain fatty acids production in vitro. Human fecal pH-controlled batch cultures showed RS induces an ecological shift in the colonic microbiota with polymorph B inducing Bifidobacterium spp. and polymorph A inducing Atopobium spp. Interestingly, polymorph B also induced higher butyrate production to levels of 0.79 mM. In addition, human gut simulation demonstrated that polymorph B promotes the growth of bifidobacteria in the proximal part of the colon and double their relative proportion in the microbiota in the distal colon. These findings suggest that RS polymorph B may promote large bowel health. While the findings are limited by study constraints, they do raise the possibility of using different thermal processing to delineate differences in the prebiotic capabilities of RS, especially its butryrogenicity in the human colon.

Impact of Interfacial Composition on Physical Stability and In Vitro Lipase Digestibility of Triacylglycerol Oil Droplets Coated with Lactoferrin and/or Caseinate

The physicochemical and functional properties of oil-in-water emulsions can be controlled by engineering the interfacial layer coating the oil droplets. This study examined the impact of interfacial deposition of lactoferrin (LF) and/or caseinate (Cas) onto oil droplets stabilized by the opposite protein on emulsion stability and lipase digestibility. Zeta potential measurements show both proteins can be deposited on droplet surfaces coated with the opposite protein. Secondary emulsion formulations containing droplets coated with mixed caseinate-lactoferrin layers (Cas-LF and LF-Cas emulsions) had enhanced stability to flocculation in 3 < pH < 7 and from 0 to 80 mM calcium chloride. The majority of emulsions studied were rapidly digested by lipase in an in vitro digestion model, demonstrating the improved secondary formulations are not expected to alter lipid bioavailability. This work provides information valuable in the design of emulsion formulations for applications in the food, pharmaceutical, and personal care industries.

Fabrication and morphological characterization of Biopolymer particles formed by electrostatic complexation of heat treated Lactoferrin and Anionic Polysaccharides

Biopolymer particles fabricated from proteins and/or polysaccharides can be used to encapsulate functional components or to modify various functional properties of materials. In this study, sub-micrometer biopolymer particles were fabricated by electrostatic complexation of heat-denatured protein (lactoferrin, LF) particles with anionic polysaccharides (alginate, carrageenan, or pectin). The aim of the study was to exploit macromolecular electrostatic interactions to form sub-micrometer sized particles and study their stability and morphological characteristics. Initially, protein particles were formed by heat treatment (91 degrees C, 20 min) of a lactoferrin solution (0.2% LF, pH 7), which led to a suspension of protein particles with mean diameter of 200-400 nm and isoelectric point of pI approximately 8.5. Biopolymer particles were then formed by mixing the protein particles with anionic polysaccharides at pH 8 and then lowering the pH to promote electrostatic deposition of polysaccharides onto the protein particle surfaces. The influence of pH (2-11) and ionic strength (0-200 mM NaCl) on the properties and stability of the complexes was studied using turbidity, dynamic light scattering, and electrophoresis measurements. Relatively stable particles could be formed from pH 5 to 8, but appreciable aggregation occurred at lower pH which was attributed to charge neutralization and bridging effects. LF-pectin complexes were relatively stable to salt addition, but LF-carrageenan and LF-alginate complexes exhibited aggregation at higher salt concentrations. These results have important implications for the application of lactoferrin-polysaccharide complexes as functional components in commercial products, such as pharmaceuticals, personal care products, and foods.

Impact of Electrostatic Deposition of Anionic Polysaccharides on the Stability of Oil Droplets Coated by Lactoferrin

The purpose of this study was to investigate the effects of three anionic polysaccharides (low methoxyl pectin (LMP), high methoxyl pectin (HMP) and alginate) on the physicochemical properties and stability of lactoferrin (LF)-coated lipid droplets. LMP, HMP and alginate were shown to adsorb to the surfaces of LF-coated droplets at neutral pH, which was primarily attributed to electrostatic attraction between anionic groups on the polysaccharide molecules and cationic patches on the protein surfaces. In the absence of polysaccharide, the LF-coated droplets were highly unstable to aggregation when heated above about 60 degrees C at pH 7, presumably because thermal denaturation of the adsorbed proteins increased droplet attraction. The addition of either LMP or HMP prior to heating greatly improved the thermal stability of the emulsions, with no aggregation being observed from 30 to 90 degrees C. On the other hand, the presence of anionic polysaccharides had little effect on emulsion stability or even promoted emulsion instability when 0 to 200 mM NaCl or CaCl2 was added. This study shows that the stability of LF-coated lipid droplets can be improved by careful selection of an appropriate type and amount of anionic polysaccharide to incorporate.

Correlation between in vitro and in vivo data on food digestion. What can we predict with static in vitro digestion models

ABSTRACT During the last decade, there has been a growing interest in understanding foods digestive fate in order to strengthen the possible effects of food on human health. Ideally, food digestion should be studied in vivo on humans but this is not always ethically and financially possible. Therefore, simple in vitro digestion models mimicking the gastrointestinal tract have been proposed as alternatives to in vivo experiments. Thus, it is no surprise that these models are increasingly used by the scientific community, although their various limitations to fully mirror the complexity of the digestive tract. Therefore, the objective of this article was to call upon the collective experiences of scientists involved in Infogest (an international network on food digestion) to review and reflect on the applications of in vitro digestion models, the parameters assessed in such studies and the physiological relevance of the data generated when compared to in vivo data. The authors provide a comprehensive review in vitro and in vivo digestion studies investigating the digestion of macronutrients (i.e., proteins, lipids, and carbohydrates) as well as studies of the bioaccessibility and bioavailability of micronutrients and phytochemicals. The main conclusion is that evidences show that despite the simplicity of in vitro models they are often very useful in predicting outcomes of the digestion in vivo. However, this has relies on the complexity of in vitro models and their tuning toward answering specific questions related to human digestion physiology, which leaves a vast room for future studies and improvements.

Digestive fate of dietary carrageenan: Evidence of interference with digestive proteolysis and disruption of gut epithelial function

Scope: The objective of this study was to interrogate two mechanisms by which commercial Carrageenans (E407) (CGN) may adversely affect human health: (i) Through modification of gastric proteolysis and (ii) Through affecting gut epithelial structure and function. Methods and results: Three commercial CGN samples with distinct zeta‐potentials (stable at the pH range of 3–7 and varied with physiological levels of CaCl2) were mixed with milk, soy or egg protein isolates, then subjected to a semi‐dynamic in vitro digestion model and analyzed by SDS‐PAGE. This revealed varying levels of interference with gastric digestive proteolysis and a significant decrease in pepsin activity. Further, a Caco‐2 cell model was used to explore various effects of physiologically digested CGN (pdCGN) on various epithelial cell functions and characteristics. Samples of pdCGN (0.005–0.5 mg/mL) affected the epithelial barrier function, including redistribution of the tight‐junction protein Zonula Occludens (Zo)‐1, changes in cellular F‐actin architecture and increased monolayer permeability to the transfer of macromolecules. Moreover, pdCGN induced elevation in the levels of the pro‐inflammatory IL‐8 receptor CXCR1. Conclusion: This work raises the possibility that CGN may reduce protein and peptide bioaccessibility, disrupt normal epithelial function, promote intestinal inflammation, and consequently compromise consumer health.

Prebiotics: Modulators of the Human Gut Microflora

Prebiotics are cost-effective and efficient tools to promote the growth and/or activity of certain bacteria in the indigenous flora of the human gastrointestinal tract to beneficially affect host health and well-being. Human studies have established inulin, fructo-oligosaccharides, galacto-oligosaccharides and lactulose as prebiotics. Their mechanism/s of action have been elucidated in part, mainly through various in vivo and in vitro methods outlined herein. Prebiotic efficacy has been demonstrated in healthy adults, pregnant and lactating women, elderly, infants, and various disease states including colon cancer. Prebiotics maintain a vivid field of research now using state of the art instruments, in vitro gastrointestinal models, advanced computerization tools and comprehensive approaches, e.g., metabolomics and metagenomics. It is not unlikely that future innovations and discoveries will expand and establish the use of prebiotics as modulators of the human gut microbiota and ultimately help support modern life.