Extended boiling of peanut progressively reduces IgE allergenicity while retaining T cell reactivity.
BACKGROUND: Current peanut oral immunotherapy is hampered by frequent adverse events. It has been shown that boiling can reduce peanut allergenicity. Hypoallergenic peanut products have the potential to reduce treatment-related reactions during desensitisation. OBJECTIVE: To show that extended boiling (for up to 12 hours) can progressively reduce peanut allergenicity while retaining T-cell reactivity. METHODS: Raw peanuts were boiled for half, 1, 2, 4 and 12 hours in deionized water. After dehydration, boiled and raw peanuts were ground, defatted and soluble proteins extracted in PBS and cooking water (leachate) retained. SDS-PAGE, Western blot, inhibition ELISA, mass spectrometry and skin prick test were used to characterize changes to peanut allergens and human IgE-reactivity. T cell responses to raw and boiled peanut extracts were determined by proliferation of CD4+/CD25+/CD134+ T cells in peanut-allergic and non-allergic individuals. RESULTS: Extended boiling progressively reduced peanut allergenicity through a combination of leaching of allergens into cooking water, fragmentation of allergens, and denaturation of conformational epitopes. Two hour boiling led to an 8-fold reduction in IgE binding capacity of boiled peanuts as determined by inhibition ELISA, while 12 hour boiling led to a 19-fold reduction. Mass spectrometry revealed an increasing number of unique allergen peptides with longer boiling times. Raw, 2 hour and 12 hour boiled peanut extracts were equivalent in their ability to stimulate T cell activation and proliferation. CONCLUSION AND CLINICAL RELEVANCE: Progressive reduction of peanut allergenicity with extended boiling does not affect T cell reactivity. Boiled peanuts may be a candidate for oral immunotherapy.
Quantitative proteomic profiling of peanut allergens in food ingredients used for oral food challenges.
Profiling allergens in complex food ingredients used in oral food challenges and immunotherapy is crucial for regulatory acceptance. Mass spectrometry based analysis employing data independent acquisition coupled with ion mobility mass spec-trometry-mass spectrometry (DIA-IM-MS) was used to investigate the allergen composition of raw peanuts and roasted peanut flour ingredients used in challenge meals. This comprehensive qualitative and quantitative analysis using label-free approaches identified and quantified 123 unique protein accessions. Semi-quantitative analysis indicated that allergens Ara h 1 and Ara h 3 were the most abundant proteins and present in approximately equal amounts, and were extracted in reduced amounts from roasted peanut flours. The clinically significant allergens Ara h 2 and 6 were less abundant but relative quantification was unaffected by roasting. Ara h 5 was undetectable in any peanut sample, whilst the Bet v 1 homologue Ara h 8 and the lipid transfer protein aller-gen, Ara h 9, were detected in low abundance. The oleosin allergens, Ara h 10 and 11 were moderately abundant in the raw peanuts but were 100-fold less abundant in the defatted roasted peanut flour than the major allergens Ara h 1, 3, 2 and 6. Certain isoforms of the major allergens dominated the profile. The relative quantitation of the major peanut allergens showed little variation between different batches of roasted peanut flour. These data will support future development of targeted approaches for absolute quantifica-tion of peanut allergens which can be applied to both food ingredients used in clinical studies, extracts used for skin testing and to identify trace levels of allergens in foods.
The effect of thermal processing on the behaviour of peanut allergen peptide targets used in multiple reaction monitoring mass spectrometry experiments.
Mass spectrometry-based methods offer an alternative means of determining allergens in foods. Whilst targeted methods are likely to offer the most robust approach for detection and quantification, little is known about how food processing may affect the behaviour of peptide targets. A systematic study has been undertaken to investigate the effects of thermal processing (boiling, roasting, frying) on the behaviour of a suite of peanut peptide targets representing the major clinically-relevant allergens. Initially the effect of thermal processing on protein extractability was investigated and a mass spectrometry-compatible buffer identified comprising 50 mM Tris-HCl, pH 8.8 containing 50 mM dithiothreitol and 0.04% (w/v) acid labile detergent which was able to extract 45-100% of protein from raw, boiled, roasted and fried peanuts using sonication at 60 °C. Eight peptide targets were identified including two peptides from each cupin allergen, Ara h1 and Ara h3 and four peptides from the prolamin superfamily allergens Ara h2, 6 and 7. AQUA peptide standards were synthesised and used to undertake multiple-reaction monitoring experiments, giving assay sensitivities of 0.1-30 amoles of peptide on-column (3 : 1 signal : noise), calculated limits of quantification between 96-1343 amoles of peptide on-column and a linear dynamic range of 4-5 orders of magnitude. Absolute quantification of individual peanut allergens in thermally processed samples showed that peptide targets in the cupin allergens were more prone to processing-induced effects than those from Ara h2, 6 and 7. Targets flanked by arginine residues showed greater thermostability. Identification of processing-stable targets, coupled with more efficient extraction procedures and a wide dynamic range, shows that targeted mass spectrometry methods have great potential as an additional method for quantifying peanut allergens in complex food matrices.
Identification and characterization of a new pecan [Carya illinoinensis (Wangenh.)
The 7S vicilin and 11S legumin seed storage globulins belong to the cupin protein superfamily and are major food allergens in many foods from the "big eight" food allergen groups. Here, for the first time, pecan vicilin was found to be a food allergen. Western blot experiments revealed that 30% of 27 sera used in this study and 24% of the sera from 25 patients with double-blind, placebo controlled clinical pecan allergy contained IgE antibodies specific to pecan vicilin. This allergen consists a low-complexity region at its N-terminal and a structured domain at the C-terminal that contains two cupin motifs and forms homotrimers. The crystal structure of recombinant pecan vicilin was determined. The refined structure gave R/Rfree values of 0.218/0.262 for all data to 2.65 Å. There were two trimeric biological units in the crystallographic asymmetric unit. Pecan vicilin is also a copper protein. These data may facilitate the understanding of the nutritional value and the allergenicity relevance of the copper binding property of seed storage proteins in tree nuts.
Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4+ T-cell epitopes with other tree nuts.
BACKGROUND: Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T-cell epitopes and T-cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. OBJECTIVES: In this study, we characterized cashew specific T-cell responses in cashew allergic subjects and examined cross-reactivity of these cashew specific cells toward other tree nut allergens. METHODS: CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T-cells was determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. RESULTS: CD4+ T-cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T-cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T-cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. CONCLUSIONS: Phylogenetically diverse tree nut allergens can activate cashew reactive T-cells and elicit a TH 2 type response at an epitope specific level.
A Comparison of Flavor Differences between Pecan Cultivars in Raw and Roasted Forms.
The objective of this research was to explore sensory differences among 8 different pecan cultivars ("Pawnee," "Witte," "Kanza," "Major," "Lakota," "Giles," "Maramec," "Chetopa") in raw and roasted forms. The cultivars were collected from 2 growing seasons (2013 and 2014) and evaluated separately. Trained panelists evaluated each cultivar from each season in raw and roasted forms, measuring intensities of 20 flavor attributes using descriptive analysis. The intensities of 10 of the 20 flavor attributes were higher for the roasted pecans across all cultivars. These included pecan ID, overall nutty, nutty-woody, nutty-grainlike, nutty-buttery, brown, caramelized, roasted, overall sweet, and sweet. The cultivars exhibited significant differences from one another for 8 attributes: pecan ID, nutty-buttery, caramelized, acrid, woody, oily, astringent, and bitter. Each of the cultivars displayed unique flavor profiles with some demonstrating extremes of certain attributes, for example the high astringency of "Lakota" or the buttery characteristics of "Pawnee." These results may help pecan growers and pecan product manufacturers understand flavor differences between different varieties of pecans, both in raw and roasted states, and the changes that occur during this process.
Modeling the survival kinetics of Salmonella in tree nuts for use in risk assessment.
Salmonella has been shown to survive in tree nuts over long periods of time. This survival capacity and its variability are key elements for risk assessment of Salmonella in tree nuts. The aim of this study was to develop a mathematical model to predict survival of Salmonella in tree nuts at ambient storage temperatures that considers variability and uncertainty separately and can easily be incorporated into a risk assessment model. Data on Salmonella survival on raw almonds, pecans, pistachios and walnuts were collected from the peer reviewed literature. The Weibull model was chosen as the baseline model and various fixed effect and mixed effect models were fit to the data. The best model identified through statistical analysis testing was then used to develop a hierarchical Bayesian model. Salmonella in tree nuts showed slow declines at temperatures ranging from 21°C to 24°C. A high degree of variability in survival was observed across tree nut studies reported in the literature. Statistical analysis results indicated that the best applicable model was a mixed effect model that included a fixed and random variation of δ per tree nut (which is the time it takes for the first log10 reduction) and a fixed variation of ρ per tree nut (parameter which defines the shape of the curve). Higher estimated survival rates (δ) were obtained for Salmonella on pistachios, followed in decreasing order by pecans, almonds and walnuts. The posterior distributions obtained from Bayesian inference were used to estimate the variability in the log10 decrease levels in survival for each tree nut, and the uncertainty of these estimates. These modeled uncertainty and variability distributions of the estimates can be used to obtain a complete exposure assessment of Salmonella in tree nuts when including time-temperature parameters for storage and consumption data. The statistical approach presented in this study may be applied to any studies that aim to develop predictive models to be implemented in a probabilistic exposure assessment or a quantitative microbial risk assessment.
The extraction of different proteins in selenium enriched peanuts and their antioxidant properties.
In this study, the selenium enriched peanuts and the different solubility proteins extracted from them were investigated. The dried defatted selenium enriched peanuts (SeP) powder (0.3147 μg/g) had a 2.5-fold higher mean total selenium concentration than general peanuts (GP) power (0.1233 μg/g). The SeP had higher concentration of selenium, manganese and zinc than that of GP, but less calcium. The rate of extraction of protein was 23.39% for peanuts and alkali soluble protein was the main component of protein in SeP, which accounted for 92.82% of total soluble protein and combined selenium was 77.33% of total selenium protein. In different forms of proteins from SeP, the WSePr due to higher concentration of selenium had higher DPPH free-radical scavenging activity, higher reducing activity and longer induction time than other proteins.
In Vivo Evaluation of Anti Diabetic, Hypolipidemic, Antioxidative Activities of Saudi Date Seed Extract on Streptozotocin Induced Diabetic Rats.
INTRODUCTION: Phoenix dactylifera (date palm) is major fruit of gulf region. In folk medicine; dates have been traditionally use. The date seed is used as hypoglycaemic, expectorant, tonic, aphrodisiac, antidiarrheic and mouth hygiene. AIM: This study intended to evaluate the anti-diabetic, hypolipidaemic and antioxidative activities of date seed extract in diabetes-induced rats. MATERIALS AND METHODS: Total of seven groups of rats, consisting of control rats and streptozotocin induced diabetic rats treated with aqueous seed extract in concentration of 100g/L in dosage of 10ml/day/rat. To evaluate the anti-diabetic property, glucose and weight was analysed weekly and at the end of eight week all rats were sacrificed. To evaluate the hypolipidaemic and antioxidative activities, serum cholesterol, triglyceride, malondialdehyde, superoxide dismutase, 8-hydroxy-2'-deoxyguanosine were estimated. Liver enzymes and kidney function tests were performed. Moreover to verify the glycaemic effect; glycated haemoglobin and serum insulin was performed. RESULTS: Aqueous seed extract in concentration of 100 gm/L in dosage of 10ml/day/rat brings a significant reduction of blood glucose levels in diabetic rats in comparison of control rats. There were significant differences in the investigated clinical chemistry and oxidative stress parameters between control and diabetic rats with both seed extract of Ajwa and Sukkari dates. CONCLUSION: Present study verifies the antidiabetic property, of aqueous seed extracts of two different varieties of dates namely Ajwa and Sukkari of Kingdom of Saudi on streptozotocin induced Diabetic rats. Prolong treatments with the extract restores the function of liver and kidney and balance the oxidative stress condition in diabetic treated rats.
Ethnic differences in prevalence of cashew nut, pistachio nut and almond allergy.
Allergy to peanuts and tree nuts is one of the most serious food allergies affecting children in English-speaking westernised countries like the USA, UK and Australia. The prevalence of peanut allergy (PA) in these regions is as high as 1-3% and is possibly increasing whereas it appears to be uncommon in developing communities in Africa and Asia (1-3). In both these high and low prevalence regions however differences of peanut sensitisation among various ethnic groups in the same geographical areas have been reported, with higher prevalences in immigrant ethnic minorities in the developed communities (4,5).