E. N. Clare Mills

Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically relevant in vitro gastrointestinal digestion

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 °C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, ≈ 25% of the allergen remained intact, ≈ 50% corresponded to a large fragment of Mr 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the ‘large fragment’ remained, even after 2 h of digestion, with a mass of ≈ 5000 Da. The ‘large fragment’ comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37–M47) and to another peptide (P42–P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T‐cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B‐ and T‐cell epitopes.

Structural, Biological, and Evolutionary Relationships of Plant Food Allergens Sensitizing via the Gastrointestinal Tract

The recently completed genome sequence of the model plant species Arabidopsis has been estimated to encode over 25,000 proteins, which, on the basis of their function, can be classified into structural and metabolic (the vast majority of plant proteins), protective proteins, which defend a plant against invasion by pathogens or feeding by pests, and storage proteins, which proved a nutrient store to support germination in seeds. It is now clear that almost all plant food allergens are either protective or storage proteins. It is also becoming evident that those proteins that trigger the development of an allergic response through the gastrointestinal tract belong primarily to two large protein superfamilies: (1) The cereal prolamin superfamily, comprising three major groups of plant food allergens, the 2S albumins, lipid transfer proteins, and cereal α -amylase/trypsin inhibitors, which have related structures, and are stable to thermal processing and proteolysis. They include major allergens from Brazil nut, peanuts, fruits, such as peaches, and cereals, such as rice and wheat; (2) The cupin superfamily, comprising the major globulin storage proteins from a number of plant species. The globulins have been found to be allergens in plant foods, such as peanuts, soya bean, and walnut; (3) The cyteine protease C1 family, comprising the papain-like proteases from microbes, plants, and animals. This family contains two notable allergens that sensitize via the GI tract, namely actinidin from kiwi fruit and the soybean allergen, Gly m Bd 30k/P34. This study describes the properties, structures, and evolutionary relationships of these protein families, the allergens that belong to them, and discusses them in relation to the role protein structure may play in determining protein allergenicity.

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?

Food allergen protein families and their structural characteristics and application in component-resolved diagnosis: new data from the EuroPrevall project

AbstractA large number of food allergens able to induce allergic symptoms in predisposed individuals, including severe, even life-threatening reactions, have been identified and characterized. However, proteins able to cause such IgE-mediated reactions can be assigned to only a limited number of protein families. Detailed knowledge about the characteristics of food allergens, their 3D structures, biological activity and stability, will help to improve diagnosis of food allergy, avoid unnecessary exclusion diets and assess the risk of cross-reactive allergies to other food sources. This review is dedicated to summarizing current knowledge about the most important food allergen protein families and to presenting data from the EuroPrevall allergen library, a proof-of-concept collection of highly purified, characterized and authenticated food allergens from animal and plant food sources to facilitate improved diagnosis of food allergies. Relevant food allergen sources

Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.

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.

The impact of processing on allergenicity of food.

Purpose of reviewProcessing procedures and food structure may modulate the allergenic properties of foods. However, our lack of knowledge on this topic makes it difficult to both predict and minimize the impact of processing on allergenicity of foods and provide allergic patients with appropriate advice over what is safe to eat. Recent findingsNew data on the major birch pollen allergen, Bet v 1, show it is thermostable, whereas their complex interactions with lipids either enhancing or reducing its stability. Studies of cereal allergies have shown allergenic disulphide-bonded prolamin superfamily members (lipid transfer proteins, α-amylase inhibitors) are resistant to cooking although species differences in maize and wheat lipid transfer proteins have been identified. Novel methods are being sought to mitigate the allergenicity of foods using enzymes like transglutaminase and treatments with phytochemicals such as phytate. SummaryFurther research is needed to explain the subtle differences in the susceptibility of processing on the allergenic potential of Bet v 1 homologues in apple and celeriac and lipid transfer proteins from different cereals. The efficacy of new processing strategies in reducing food allergenicity needs to be demonstrated in allergic individuals. Studies are still lacking on the effect of the food matrix on allergenicity, and the impact of processing on sensitization potential.

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.

High pressure, thermal and pulsed electric-field-induced structural changes in selected food allergens.

SCOPE The effects of high-pressure/temperature treatment and pulsed electric field treatment on native peanut Ara h 2, 6 and apple Mal d 3 and Mal d 1b prepared by heterologous expression were examined. METHODS AND RESULTS Changes in secondary structure and aggregation state of the treated proteins were characterized by circular dichroism spectroscopy and gel-filtration chromatography. Pulsed electric field treatment did not induce any significant changes in the structure of any of the allergens. High-pressure/temperature at 20 °C did not change the structure of the Ara h 2, 6 or Mal d 3 and resulted in only minor changes in structure of Mal d 1b. Ara h 2, 6 was stable to HPP at 80 °C, whereas changes in circular dichroism spectra were observed for both apple allergens. However, these changes were attributable to aggregation and adiabatic heating during HPP. An ELISA assay of temperature treated Mal d 3 showed the antibody reactivity correlated well with the loss of structure. CONCLUSION In conclusion, novel-processing techniques had little effect on purified allergen structure. Further studies will demonstrate if these stability properties are retained in foodmatrices.