Alan R. Mackie

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.

The role of bile salts in digestion.

Bile salts (BS) are bio-surfactants present in the gastrointestinal tract (GIT) that play a crucial role in the digestion and absorption of nutrients. The importance of BS for controlled release and transport of lipid soluble nutrients and drugs has recently stimulated scientific interest in these physiological compounds. BS are so-called facial amphiphiles showing a molecular structure that is very distinct from classical surfactants. This peculiar molecular structure facilitates the formation of dynamic aggregates able to solubilise and transport lipid soluble compounds. The detergent nature of BS has been studied in the literature, mostly concentrating on the self-assembly behaviour of BS in solution but also in relation to protein denaturation and its effect on improving proteolysis. In contrast, the affinity of BS for hydrophobic phases has received less attention and studies dealing directly with the interfacial behaviour of BS are very limited in the literature. This is despite the fact that the interfacial activity of BS plays a vital role in fat digestion since it is closely involved with lypolisis. BS adsorb onto fat droplets and can remove other materials such as proteins, emulsifiers and lipolysis products from the lipid surface. The unusual surface behaviour of BS is directly related to their intriguing molecular structure and further knowledge could provide an improved understanding of lipid digestion. This review aims to combine the new insights gained into the surface properties of BS and their role in digestion. A better understanding of surface activity of BS would allow manipulation of physico-chemical and interfacial properties to modulate lipid digestion, improve bioavailability of lipid soluble nutrients and reduce absorption of saturated fats, cholesterol and trans fats.

The prevalence, cost and basis of food allergy across Europe

The development of effective management strategies to optimize the quality of life for allergic patients is currently hampered by a lack of good quality information. Estimates of how many individuals suffer from food allergy and the major foods involved vary widely and inadequacies of in vitro diagnostics make food challenges the only reliable means of diagnosis in many instances. The EuroPrevall project brings together a multidisciplinary partnership to address these issues. Cohorts spanning the main climatic regions of Europe are being developed in infants through a birth cohort, community surveys in school‐age children and adults and an outpatient clinic study. Confirmatory double‐blind placebo‐controlled food challenge diagnosis is being undertaken using foods as they are eaten with titrated doses to allow no‐effect and lowest‐observable effect levels for allergenic foods to be determined. The cohorts will also facilitate validation of novel in vitro diagnostics through the development of the EuroPrevall Serum Bank. Complementary studies in Ghana, western Siberia, India and China will allow us to gain insights into how different dietary patterns and exposure to microorganisms affect food allergies. New instruments to assess the socioeconomic impact of food allergy are being developed in the project and their application in the clinical cohorts will allow, for the first time, an assessment to be made of the burden this disease places on allergy sufferers and their communities.

Binding of lipopeptide to CD14 induces physical proximity of CD14, TLR2 and TLR1

Lipoproteins or lipopeptides (LP) are bacterial cell wall components detected by the innate immune system. For LP, it has been shown that TLR2 is the essential receptor in cellular activation. However, molecular mechanisms of LP recognition are not yet clear. We used a FLAG‐labeled derivative of the synthetic lipopeptide N‐palmitoyl‐S‐[2,3‐bis(palmitoyloxy)‐(2R,S)‐propyl]‐(R)‐cysteinyl‐seryl‐(lysyl)3‐lysine (Pam3CSK4) to study the roles of CD14, TLR2 and TLR1 in binding and signaling of LP and their molecular interactions in human cells. The activity of Pam3CSK4‐FLAG was TLR2 dependent, whereas the binding was enabled by CD14, as evaluated by flow cytometry and confocal microscopy. Using FRET and FRAP imaging techniques to study molecular associations, we could show that after Pam3CSK4‐FLAG binding, CD14 and Pam3CSK4‐FLAG associate with TLR2 and TLR1, and TLR2 is targeted to a low‐mobility complex. Thus, LP binding to CD14 is the first step in the LP recognition, inducing physical proximity of CD14 and LP with TLR2/TLR1 and formation of the TLR2 signaling complex.

Emulsification alters simulated gastrointestinal proteolysis of β-casein and β-lactoglobulin

We have studied the effect of the adsorption of milk proteins at the oil-water interface on their digestibility in simulated gastrointestinal environment. The investigations aimed to characterize how both the breakdown of the adsorbed proteins and the interactions with physiological surfactants, phosphatidylcholine (PC) and bile salts (BS), influence structural transformations of model, protein-stabilized food emulsions in the gastrointestinal track. Proteolysis of two contrasting proteins, β-casein (β-Cas) and β-lactoglobulin (β-Lg), was compared between the protein presented in solution or in emulsion, after adsorption at the oil-water interface. Digestion of β-Cas was faster when presented as an emulsion and led to the persistence of a 6 kD peptide not seen when the protein was presented in solution. Adsorption gave rise to a pepsin-susceptible form of β-Lg. Complex interactions were observed with PC introduced to the system in the vesicular form. Measurements of interfacial tension revealed that PC displaced the proteins from the oil droplets after only 30 s for β-Lg and 12 min for β-Cas, so that the gastric digestion largely took place in solution. Pepsinolysis of adsorbed β-Cas played a dominant role in emulsion destabilization. In contrast, collapse of β-Lg-stabilized emulsion under gastric conditions was mainly dependent on protein-PC interactions. β-Lg was significantly protected through simulated duodenal digestion as a result of a complex formed with the PC. In the absence of PC, the proteins were completely broken down after duodenal digestion, during which the duodenal surfactants, BS, displaced any remaining protein from the interface and governed the final structure of emulsion.

Impact of food processing on the structural and allergenic properties of food allergens

This article reviews recent studies that address one of the major unanswered questions in food allergy research: what attributes of food or food proteins contribute to or enhance food allergenicity?

Interfacial Characterization of β-Lactoglobulin Networks : Displacement by Bile Salts

The competitive displacement of a model protein (beta-lactoglobulin) by bile salts from air-water and oil-water interfaces is investigated in vitro under model duodenal digestion conditions. The aim is to understand this process so that interfaces can be designed to control lipid digestion thus improving the nutritional impact of foods. Duodenal digestion has been simulated using a simplified biological system and the protein displacement process monitored by interfacial measurements and atomic force microscopy (AFM). First, the properties of beta-lactoglobulin adsorbed layers at the air-water and the olive oil-water interfaces were analyzed by interfacial tension techniques under physiological conditions (pH 7, 0.15 M NaCl, 10 mM CaCl2, 37 degrees C). The protein film had a lower dilatational modulus (hence formed a weaker network) at the olive oil-water interface compared to the air-water interface. Addition of bile salt (BS) severely decreased the dilatational modulus of the adsorbed beta-lactoglobulin film at both the air-water and olive oil-water interfaces. The data suggest that the bile salts penetrate into, weaken, and break up the interfacial beta-lactoglobulin networks. AFM images of the displacement of spread beta-lactoglobulin at the air-water and the olive oil-water interfaces suggest that displacement occurs via an orogenic mechanism and that the bile salts can almost completely displace the intact protein network under duodenal conditions. Although the bile salts are ionic, the ionic strength is sufficiently high to screen the charge allowing surfactant domain nucleation and growth to occur resulting in displacement. The morphology of the protein networks during displacement is different from those found when conventional surfactants were used, suggesting that the molecular structure of the surfactant is important for the displacement process. The studies also suggest that the nature of the oil phase is important in controlling protein unfolding and interaction at the interface. This in turn affects the strength of the protein network and the ability to resist displacement by surfactants.

The membrane-permeabilizing effect of avenacin A-1 involves the reorganization of bilayer cholesterol.

Avenacin A-1 is a member of a group of naturally occurring compounds called saponins. It is found in oat plants, where it protects against fungal pathogens. A combined electrical and optical chamber was used to determine the interaction of avenacin A-1 with Montal-Mueller planar lipid bilayers. This system allowed simultaneous measurement of the effect of avenacin A-1 on the fluorescence and lateral diffusion of a fluorescent lipid probe and permeability of the planar lipid bilayer. As expected, cholesterol was required for avenacin A-1-induced bilayer permeabilization. The planar lipid bilayers were also challenged with monodeglucosyl, bis-deglucosyl, and aglycone derivatives of avenacin A-1. The results show that the permeabilizing activity of the native avenacin A-1 was completely abolished after one, two, or all three sugar residues are hydrolyzed (monodeglucosyl, bis-deglucosyl, and aglycone derivatives, respectively). Fluorescence recovery after photobleaching (FRAP) measurements on cholesterol-containing planar lipid bilayers revealed that avenacin A-1 caused a small but significant reduction in the lateral diffusion of the phospholipid probe N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Similarly, with the sterol probe (22-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-Chol), avenacin A-1, but not its derivatives, caused a more pronounced reduction in the lateral diffusion than that observed with the phospholipid probe. The data indicate that an intact sugar moiety of avenacin A-1 is required to reorganize membrane cholesterol into pores.

Thresholds for food allergens and their value to different stakeholders

Thresholds constitute a critical piece of information in assessing the risk from allergenic foods at both the individual and population levels. Knowledge of the minimum dose that can elicit a reaction is of great interest to all food allergy stakeholders. For allergic individuals and health professionals, individual threshold data can inform allergy management. Population thresholds can help both the food industry and regulatory authorities assess the public health risk and design appropriate food safety objectives to guide risk management. Considerable experience has been gained with the double‐blind placebo‐controlled food challenge (DBPCFC), but only recently has the technique been adapted to provide data on thresholds. Available data thus vary greatly in quality, with relatively few studies providing the best quality individual data, using the low‐dose DBPCFC. Such high quality individual data also form the foundation for population thresholds, but these also require, in addition to an adequate sample size, a good characterization of the tested population in relation to the whole allergic population. Determination of thresholds at both an individual level and at a population level is influenced by many factors. This review describes a low‐dose challenge protocol developed as part of the European Community‐funded Integrated Project Europrevall, and strongly recommends its wider use so that data are generated that can readily increase the power of existing studies.

Lateral diffusion in planar lipid bilayers: a fluorescence recovery after photobleaching investigation of its modulation by lipid composition, cholesterol, or alamethicin content and divalent cations.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.