Neil M. Rigby

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?

A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy

Individual pectin polymers and complexes, isolated from the pericarp of unripe tomato (Lycopersicon esculentum var. Rutgers), were subjected to a mild acid hydrolysis and visualised and characterised by atomic force microscopy (AFM). The AFM images confirm earlier studies showing that individual pectic polysaccharides often possess long branches. The AFM data have been used to construct size and molecular weight distributions for the single molecules and complexes, from which the calculated number-average and weight-average molecular weights can then be compared directly with the published literature data on the rheology of bulk samples. Loss of the neutral sugars arabinose, galactose and rhamnose from the pectin samples does not significantly alter either the size or the branching density of the individual polymers, but is reflected in a breakdown of the complexes. Significant loss of galacturonic acid at long hydrolysis times was found to be accompanied by changes in the size and branching of the single polymers and further breakdown of the complexes. The results suggest that rhamnose, arabinose and galactose are not the major components of the individual polymers but are, instead, confined to the complexes. The polysaccharides represent a previously unrecognised branched homogalacturonan with a minimum mean size some three times larger than that previously reported. The complexes consist of homogalacturonans (HGs) held together by rhamnogalacturonan I (RG-I) regions. Comparison of the rate of depolymerisation of the homogalacturonans and complexes with the published data on changes in the intrinsic viscosity of bulk pectin samples, subjected to similar acid hydrolysis, suggests that the different rates of depolymerisation of RG-I and HG contribute separately to the observed changes in intrinsic viscosity during acid hydrolysis. Thus data obtained using a single molecule microscopy technique provides new insights into the behaviour in the bulk.

Effect of Heating and Glycation on the Allergenicity of 2S Albumins (Ara h 2/6) from Peanut

Background Peanut allergy is one of the most common and severe food allergies, and processing is known to influence the allergenicity of peanut proteins. We aimed to establish the effect of heating and glycation on the IgE-binding properties and biological activity of 2S albumins (Ara h 2/6) from peanut. Methodology/Principal Findings Native Ara h 2/6 was purified from raw peanuts and heated in solution (15 min, 110°C) in the presence or absence of glucose. Ara h 2 and 6 were also purified from roasted peanut. Using PBMC and sera from peanut-allergic patients, the cellular proliferative potency and IgE reactivity (reverse EAST inhibition) and functionality (basophil degranulation capacity) of allergens were assessed. Heating Ara h 2/6 at 110°C resulted in extensive denaturation, hydrolysis and aggregation of the protein, whilst Ara h 2 and 6 isolated from roasted peanut retained its native conformation. Allergen stimulation of PBMC induced proliferation and Th2 cytokine secretion which was unaffected by thermal processing. Conversely, IgE reactivity and functionality of Ara h 2/6 was decreased by heating. Whilst heating-glycation further reduced the IgE binding capacity of the proteins, it moderated their loss of histamine releasing capacity. Ara h 2 and 6 purified from roasted peanut demonstrated the same IgE reactivity as unheated, native Ara h 2/6. Conclusions/Significance Although no effect of processing on T-cell reactivity was observed, heat induced denaturation reduced the IgE reactivity and subsequent functionality of Ara h 2/6. Conversely, Ara h 2 and 6 purified from roasted peanut retained the structure and IgE reactivity/functionality of the native protein which may explain the allergenic potency of this protein. Through detailed molecular study and allergenicity assessment approaches, this work then gives new insights into the effect of thermal processing on structure/allergenicity of peanut proteins.

Gastro‐duodenal digestion products of the major peanut allergen Ara h 1 retain an allergenic potential

Background The process of gastro‐duodenal digestion may play a role in determining the allergenic properties of food proteins. The sensitizing and allergenic potential of digestion products of highly degraded allergens, such as the major peanut allergen Ara h 1, is currently under debate. We evaluated the effect of in vitro gastro‐duodenal digestion of Ara h 1 on T cell reactivity and basophil histamine release.

Physiological phosphatidylcholine protects bovine β-lactoglobulin from simulated gastrointestinal proteolysis

We have investigated the effect of phosphatidylcholine (PC) on the resistance of bovine beta-lactoglobulin (beta-Lg) to simulated in vitro gastrointestinal proteolysis. Whilst addition of PC did not affect the resistance of beta-Lg to gastric pepsinolysis, it protected the protein from subsequent degradation under duodenal conditions. The effect was dependent on the ratio of PC to beta-Lg, 16% of the protein remaining intact in the presence of an equimolar ratio of PC/protein, which increased to 62% when a 60-fold molar excess of PC was included. PC also altered the pattern of digestion products observed by SDS-PAGE. Thermal denaturation of beta-Lg abolished this effect showing that it was dependent on the native folded structure of the protein. Since neither of the beta-Lg ligands retinol or palmitate exerted a protective effect, it is unlikely that PC is mediating its effect by occupying the central calyx. An alternative explanation may be that the lipids bind to a secondary fatty acid binding site in beta-Lg, thus blocking the action of proteases for steric reasons. These data indicate how biomolecular interactions between proteins and lipids may alter patterns of proteolysis and need to be taken into consideration in any in vitro model of digestion.

Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity

SCOPE Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. METHODS AND RESULTS Ara h 1 was purified from either raw (N-Ara h 1) or roasted (R-Ara h 1) peanuts. Boiling (100°C 15 min; H-Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod-like branched aggregates with reduced IgE-binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G-Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H- and G-Ara h1 retained the T-cell reactivity of N-Ara h 1. R-Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE-binding capacity of the native protein. CONCLUSION Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.

Calcium gelation of pectic polysaccharides isolated from unripe tomato fruit

Abstract Cell-wall material was prepared from unripe tomato fruit, and a pectic polysaccharide extracted with cyclohexanediaminetetraacetic acid. The structure of the purified pectic polysaccharide was examined by sugar and methylation analysis, and was typical of a rhamnogalacturonan from the primary cell wall. The physicochemical properties of the isolated polysaccharide were characterised by viscometry and size-exclusion chromatography. The polysaccharide was polydisperse, but of large molecular size as indicated by an intrinsic viscosity of 810 ml. g −1 . At concentrations above ∼ 0.2–0.6% w/w, coil entanglement was observed as an increase in the dependency of viscosity on concentration. For these concentrated solutions, clear elastic gels were formed on addition of calcium ions. At concentrations in the range 0.6–2.8% w/w the shear modulus of the gel showed a c 1.9 dependence on concentration. The modulus of the gel increased linearly with absolute temperature in a rubberlike way, enabling an estimate of cross-link density to be made.

Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays

Background Peanuts are often consumed after roasting, a process that alters the three‐dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity.

Fast: Towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.