Enzymatic hydrolysis: a method in alleviating legume allergenicity.

Legumes are involved in IgE mediated food allergy in many countries. Avoidance of allergenic food is the only way to avoid symptomatic reaction. The present study investigated the effect of enzymatic hydrolysis on the allergenicity of three legumes - kidney bean (Phaseolus vulgaris), black gram (Vigna mungo) and peanut (Arachis hypogaea). Soluble protein extracts of the study legumes were sequentially treated by Alcalase(®) and Flavourzyme(®). Allergenicity of hydrolysates was then determined by ELISA, immunoblot, stripped basophil histamine release and skin prick test (SPT). Hydrolysis resulted in the loss of all IgE binding fractions determined by immunoblot in the three legumes. Specific IgE binding in ELISA was reduced by 62.2 ± 7.7%, 87.1 ± 9.6% and 91.8 ± 7.2% in the hydrolysates of kidney bean, black gram and peanut, respectively (p < 0.01). The release of histamine was decreased significantly when sensitized basophils were challenged with hydrolysates as compared to raw extracts. Significant reduction in the biopotency of hydrolysates was also observed in SPT where only 1/10 kidney bean-sensitive individuals, 2/6 black gram-sensitive individuals and 1/7 peanut-sensitive individuals were found positive to their respective hydrolysates. In conclusion, enzymatic hydrolysis is effective in attenuating allergenicity of legume proteins and may be employed for preparing hypoallergenic food extracts.

Purification and immunobiochemical characterization of a 31 kDa cross-reactive allergen from Phaseolus vulgaris (kidney bean).

BACKGROUND: Legumes are a rich source of proteins but are also potential elicitors of IgE-mediated food allergy. This study aimed to isolate and characterize a major allergen of Phaseolus vulgaris (kidney bean) and determine its allergenicity. METHODOLOGY: Kidney bean allergen was purified using Q Sepharose column (anion exchanger) and eluates with high intensity were pooled to purify protein using Superdex 75 (gel filtration) and C18 column (RP-HPLC). Patients with history of kidney bean allergy were skin prick tested (SPT) with crude kidney bean extract and the purified protein. Specific IgE was estimated in sera by enzyme-linked immunosorbent assay (ELISA). Characterization of purified protein and its cross-reactivity was investigated by immunobiochemical methods. Identification of purified protein was carried out by tandem mass spectrometry. PRINCIPAL FINDINGS: Purified protein appeared as a single band at 31 kDa on SDS-PAGE and showed IgE binding to 88% patients' sera by ELISA and immunoblotting. SPT with purified protein identified 78% hypersensitive patients of kidney bean. Significant release of histamine from sensitized basophils was observed after challenge with purified protein. PAS staining suggested it to be a glycoprotein, but no change in IgE binding was observed after periodate oxidation. The 31 kDa protein remained stable for 60 min on incubation with pepsin. The purified protein had high allergenic potential since it required only 102 ng of self protein for 50% IgE inhibition. Mass spectrometric analysis identified it as Phytohemagglutinin. It also showed hemagglutination with human RBCs. Cross-reactivity was observed with peanut and black gram with IC50 of 185 and 228 ng respectively. CONCLUSION/SIGNIFICANCE: A 31 kDa major allergen of kidney bean was purified and identified as phytohemagglutinin with cross-reactivity to peanut and black gram.

First case report of anaphylaxis caused by Rajgira seed flour (Amaranthus paniculatus) from India: a clinico-immunologic evaluation.

The prevalence of food allergy is reported to be 3-4% in adults and about 6% in children. However food allergy across different countries accounts for 35-50 % all cases of anaphylaxis to foods. In the present study, we have reported a case of anaphylaxis to Amaranth grain (Amaranthus paniculatus) commonly known as Rajgira (Ramdana) in India. A 60 year old female suffered anaphylaxis after consuming Rajgira seed flour generally consumed during fasting. Food allergy to Amaranth seeds is not reported so far. The patient reported to hospital with complaints of itching in mouth, choking throat, redness and swelling of face and burning abdomen within 5 min of consuming Rajgira flour. Clinical and immunological investigations revealed SPT and oral challenge positivity beside high allergen specific IgE in the serum of the patient. Three IgE binding protein fractions were detected in roasted Rajgira seed flour extract which could be considered to be allergenically important for triggering anaphylaxis.

Effect of thermal processing and γ-irradiation on allergenicity of legume proteins.

Legumes are implicated in IgE mediated food allergy in different countries. The present study aimed to investigate the effect of different processing methods on allergenicity of legume proteins. The extracts were processed by boiling, γ-irradiation or by combination of both. The changes in soluble protein content, specific IgE binding and allergenic potential of legume proteins were assessed. Thermal processing resulted in a 3- to 4-fold reduction in soluble protein. Specific IgE binding was reduced 74±6.5%, 83±11.6% and 62±7.2% in the soluble protein of kidney bean, black gram and peanut, respectively, after boiling (p<0.01) whereas there was 34±5.2%, 74±15.6% and 44±11.1% IgE binding reduction in the insoluble protein fraction of respective legumes. Boiling followed by γ-irradiation reduced IgE binding significantly (p<0.05). Biopotency of soluble protein of kidney bean, black gram and peanut was reduced 7-, 3- and 26-folds (p<0.001), respectively, and that of insoluble protein decreased 6-, 4- and 8-folds (p<0.001), respectively, after boiling. Combination treatment was effective in reducing the potency of both soluble and insoluble protein significantly as compared to boiling alone (p<0.001). However, γ-irradiation alone did not bring any change in allergenicity. In conclusion, boiling followed by γ-irradiation is effective in attenuating allergenicity of legume proteins.

Isolation and characterization of a 28 kDa major allergen from blackgram (Phaseolus mungo).

Legumes are the major elicitors of IgE-mediated food allergy in many countries of the world. Purified major allergens are prerequisite for component resolved diagnosis of allergy. The present study was aimed to isolate and characterize a major allergenic protein from blackgram (Phaseolus mungo). Respiratory allergy patients with history of blackgram allergy were skin prick tested (SPT) and sera were collected from SPT positive patients. The blackgram extract was fractionated using a combination of anion exchange and hydrophobic interaction chromatography. The purified protein was characterized by indirect ELISA, immunoblot, ELISA inhibition, SPTs, stripped basophil histamine release, lymphoproliferation assay and digestibility assay. The purified protein separated at 28 kDa on 12% gel and showed IgE binding with 81% of blackgram hypersensitive patients' sera on immunoblot indicating it to be a major allergen. Periodic Acid Schiff's and meta-periodate treatment staining detected it to be a glycoprotein. The 28 kDa protein recognized 7/9 (77.8%) of blackgram positive patients by SPT, where as all 9 patients showed significant histamine release on stimulation with protein as compared to controls. The 28 kDa protein remained stable up to 15 min on incubation with SGF. Bands of 14-16 kDa appeared after 15 min of pepsin digestion that remained stable up to 60 min of incubation. However, purified protein degraded within 5 min after incubation with SIF. The N-terminus-12 residues sequence of 28 kDa protein was GRREDDYDNLQL. A stretch of residues 'DDYDNLQL' showed homology with Rho-specific inhibitor of transcription termination (E=0.42, Identity=87%) and NBS-LRR type disease resistant protein from peanut (Arachis hypogaea) (E=2, Identity=77%). In conclusion, the purified 28 kDa protein is a potent major allergen that may have implication in diagnosis of blackgram allergy.

Kidney bean: a major sensitizer among legumes in asthma and rhinitis patients from India.

BACKGROUND: The prevalence of IgE mediated food allergies has increased over the last two decades. Food allergy has been reported to be fatal in highly sensitive individuals. Legumes are important food allergens but their prevalence may vary among different populations. The present study identifies sensitization to common legumes among Indian population, characterizes allergens of kidney bean and establishes its cross reactivity with other legumes. METHODOLOGY: Patients (n = 355) with history of legume allergy were skin prick tested (SPT) with 10 legumes. Specific IgE (sIgE) and total IgE were estimated in sera by enzyme-linked immunosorbent assay. Characterization of kidney bean allergens and their cross reactivity was investigated by immunobiochemical methods. Identification of major allergens of kidney bean was carried out by mass spectrometry. PRINCIPAL FINDINGS: Kidney bean exhibited sensitization in 78 (22.0%) patients followed by chickpea 65 (18.0%) and peanut 53 (15%). SPT positive patients depicted significantly elevated sIgE levels against different legumes (r = 0.85, p<0.0001). Sera from 30 kidney bean sensitive individuals exhibited basophil histamine release (16-54%) which significantly correlated with their SPT (r = 0.83, p<0.0001) and sIgE (r = 0.99, p<0.0001). Kidney bean showed eight major allergens of 58, 50, 45, 42, 40, 37, 34 and 18 kDa on immunoblot and required 67.3±2.51 ng of homologous protein for 50% IgE inhibition. Inhibition assays revealed extensive cross reactivity among kidney bean, peanut, black gram and pigeon pea. nLC-MS/MS analysis identified four allergens of kidney bean showing significant matches with known proteins namely lectin (phytohemagglutinin), phaseolin, alpha-amylase inhibitor precursor and group 3 late embryogenesis abundant protein. CONCLUSION/SIGNIFICANCE: Among legumes, kidney bean followed by chick pea and peanut are the major allergic triggers in asthma and rhinitis patients in India. Kidney bean showed eight major allergens and cross reacted with other legumes. A combination of SPT, sIgE and histamine release assay is helpful in allergy diagnosis.