Sensitization Potency of Sunflower Seed Protein in a Mouse Model: Identification of 2S-Albumins More Allergenic Than SFA-8.

SCOPE: Food allergy to sunflower seed (SFS) protein is not frequent and only non-specific lipid transfert protein (nsLTP) Hel a 3 is officially recognized as a food allergen. Out of the eleven seed storage 2S-albumins (SESA) detected in SFS, only SFA-8 allergenicity has been investigated so far. The study aimed then to evaluate SFS protein allergenicity and particularly, to compare the sensitization potency of SESA in a mouse model. METHODS AND RESULTS: The most abundant SESA and nsLTP were isolated from SFS through a combination of chromatographic methods. Purified proteins were then used to measure specific IgG1 and IgE responses in BALB/c mice orally sensitized to different SFS protein isolates. The study, thus, confirmed the allergenicity of SFA-8 and Hel a 3 but mice were also highly sensitized to other SESA such as SESA2-1 or SESA20-2. Furthermore, competitive inhibition of IgE-binding revealed that SFA-8 IgE-reactivity was due to cross-reactivity with other SESA. 11S-globulins were weakly immunogenic and were rapidly degraded in an in vitro model of gastroduodenal digestion. In contrast, Hel a 3, SESA2-1 and SFA-8 were more resistant to proteolysis and gastroduodenal digestion did not affect their IgE-reactivity. CONCLUSIONS: SESA2-1 or SESA20-2 were more potent allergens than SFA-8 in this mouse model. Allergenicity of SESA must be now confirmed in SFS-allergic patients.

Digestion and Transport across the Intestinal Epithelium Affects the Allergenicity of Ara h 1 and 3 but Not of Ara h 2 and 6.

SCOPE: No accepted and validated methods are currently available which can accurately predict protein allergenicity. In this study, the role of digestion and transport on protein allergenicity is investigated. METHODS AND RESULTS: Peanut allergens (Ara h 1, 2, 3, and 6) and a milk allergen (ß-lactoglobulin) are transported across pig intestinal epithelium using the InTESTine model and afterward basophil activation is measured to assess the (remaining) functional properties. Additionally, allergens are digested by pepsin prior to epithelial transport and their allergenicity is assessed in a human mast cell activation assay. Remarkably, transported Ara h 1 and 3 are not able to activate basophils, in contrast to Ara h 2 and 6. Digestion prior to transport results in a significant increase in mast cell activation of Ara h 1 and 3 dependent on the length of digestion time. Activation of mast cells by Ara h 2 and 6 is unaffected by digestion prior to transport. CONCLUSIONS: Digestion and transport influences the allergenicity of Ara h 1 and 3, but not of Ara h 2 and 6. The influence of digestion and transport on protein allergenicity may explain why current in vitro assays are not predictive for allergenicity.

Circulating Ara h 6 as a marker of peanut protein absorption in tolerant and allergic humans following ingestion of peanut-containing foods.

BACKGROUND: Bioaccessibility of food allergens may be a key determinant of allergic reactions. OBJECTIVE: To develop a protocol allowing the detection of the major peanut allergen, Ara h 6, in the bloodstream following ingestion of low amounts of peanut and to compare Ara h 6 bioaccessibility by food matrix. We further assessed for differences in absorption in healthy versus peanut-allergic volunteers. METHODS: A blood pretreatment combining acidic shock and thermal treatment was developed. This protocol was then applied to blood samples collected from human volunteers (n = 6, healthy controls; n = 14, peanut-allergic patients) at various time-points following ingestion of increasing levels of peanut incurred in different food matrices (cookies, peanut butter and chocolate dessert). Immunodetection was performed using an in-house immunoassay. RESULTS: An original pretreatment protocol was optimized, resulting in irreversible dissociation of human antibodies-Ara h 6 immune complex, thus rendering Ara h 6 accessible for its immunodetection. Ara h 6 was detected in samples from all volunteers following ingestion of 300-1000 mg peanut protein, although variations in the kinetics of passage were observed between individuals and matrices. Interestingly, in peanut-allergic subjects, Ara h 6 could be detected following ingestion of lower doses and at higher concentrations than in non-allergic volunteers. CONCLUSIONS AND CLINICAL RELEVANCE: The kinetics and intensity of Ara h 6 passage in bloodstream depend on both individual and food matrix. Peanut-allergic patients appear to demonstrate higher absorption rate, the clinical significance of which warrants further evaluation.

The Effect of the Food Matrix on the In Vitro Bio-Accessibility and IgE Reactivity of Peanut Allergens.

SCOPE: Factors such as food processing, the food matrix, and antacid medication may affect the bio-accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. METHODS AND RESULTS: Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio-accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS-PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut-allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro-duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of Mr < 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral-gastro-duodenal digestion irrespective of digestion conditions employed. CONCLUSION: Peanut allergen bio-accessibility is unaffected by the dessert or cookie matrices whilst high intra-gastric pH conditions render allergens more resistant to digestion.

Effect of pH regulation on the components and functional properties of proteins isolated from cold-pressed rapeseed meal through alkaline extraction and acid precipitation.

Cold-pressed rapeseed meal with high protein content (38.76% protein dry weight basis) was used to prepare rapeseed protein isolates (RPIs) by alkaline extraction (pH 8.0, 9.0, 10.0, 11.0, 12.0 and 13.0) and acid precipitation (pH 3.0, 3.5, 4.0, 4.5, 5.0 and 5.5). The protein with an intact structure and the highest yield (65.08%) was obtained at extraction pH 9.0 and precipitation pH 4.5, accompanied by the lowest D-amino acid content, the lightest colour and the lowest contents of glucosinolates (2.85 mmol/kg), phytic acid (1.05 mg/g) and sinapine (0.68 mg/g). Additionally, water/oil absorption, foaming and emulsifying capacities decreased with decreasing precipitation pH, while the solubility showed the reverse trend. During gastric simulation digestion, the α-polypeptide of cruciferin and napin in the RPIs showed digestive resistance. Overall, pH regulation might be an effective method to isolate high quality RPIs for use in the food processing industry.