Bioinformatic screening and detection of allergen cross‐reactive IgE‐binding epitopes

Protein allergens can be related by cross‐reactivity. Allergens that share relevant sequence can cross‐react, those lacking sufficient similarity in their IgE antibody‐binding epitopes do not cross‐react. Cross‐reactivity is based on shared epitopes that is based on shared sequence and higher level structure (charge and shape). Epitopes are important in predicting cross‐reactivity potential and may provide the potential to establish criteria that identify homology among allergens. Selected allergen's IgE‐binding epitope sequences were used to determine how the FASTA algorithm could be used to identify a threshold of significance. A statistical measure (expectation value, E‐value) was used to identify a threshold specific to identifying cross‐reactivity potential. Peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. Alignments with allergens were noted for the ability to match the epitope's source allergen as well as any cross‐reactive or other homologous allergens. A FASTA expectation value range (1 × 10^?5–1 × 10^?6) was identified that could act as a threshold to help identify cross‐reactivity potential.     

A Cherry Seed‐Derived Spice,Mahleb, is Recognized by Anti‐Almond Antibodies Including Almond‐Allergic Patient IgE

There are a number of examples of immunologic cross‐reactivity elicited by pollens, fruits, seeds, and nuts of closely related plant species. Such cross‐reactivity is of particular concern for patients with food allergies. In this report, we investigated a spice (mahleb) that is prepared from the kernel of the St. Lucie cherry, Prunus mahaleb, for cross‐reactivity with almond (Prunus dulcis), using enzyme‐linked immunosorbent assay (ELISA) and Western blot. Almond and mahleb are members of the same genus. Cross‐reactivity between the mahleb and almond was demonstrated by reaction of cherry and almond kernel protein extracts with antibodies raised against almond proteins. Almond‐specific murine monoclonal IgG, rabbit polyclonal IgG, and almond‐allergic serum IgE each exhibited cross‐reactivity with cherry kernel protein. Because of the demonstrated cross‐reactivity between almond and mahleb, these findings should be of special concern to almond‐allergic patients and attending medical personnel.     

Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies

The major heat-stable shellfish allergen, tropomyosin, demonstrates immunological cross-reactivity, making specific differentiation of crustaceans and molluscs for food labelling very difficult. The aim of this study was to evaluate the application of allergen-specific monoclonal antibodies in differential detection of shellfish-derived tropomyosin in 11 crustacean and 7 mollusc species, and to study the impact of heating on its detection. Cross-reactive tropomyosin was detected in all crustacean species, with partial detection in molluscs: mussels, scallops and snails but none in oyster, octopus and squid. Furthermore, we have demonstrated that heating of shellfish has a profound effect on tropomyosin detection. This was evident by the enhanced recognition of multiple tropomyosin variants in the analysed shellfish species. Specific monoclonal antibodies, targetting the N-terminal region of tropomyosin, must therefore be developed to differentiate tropomyosins in crustaceans and molluscs. This can help in correct food labelling practices and thus protection of consumers.     

Tropomyosin Contains IgE‐Binding Epitopes Sensitive to Periodate but Not to Enzymatic Deglycosylation

Glycosylation has been reported to affect the epitopes of food allergens, however, there are few reports on its role in crab allergen. In the present study, the effect of glycosylation on the IgE‐binding activity of tropomyosin (TM), a major allergen in Scylla paramamosain, was investigated. The results showed that TM was a glycoprotein with a 0.2% carbohydrate moiety and contained O‐glycan. Moreover, enzymatic deglycosylation of TM by glycosidase had no effect on the IgE‐binding activity of TM. In contrast, treatment with periodate resulted in a significant reduction in its IgE‐binding activity.     

Differentiated Patterns of Legume Sensitisation in Peanut-Allergic Patients

Patients with peanut allergy are often serologically sensitised to other legumes. If primary peanut allergy was contracted, secondary allergies to other legume plants may develop. The cross-sensitisation implies an enhanced risk of food-related anaphylaxis for the patients even if its clinical relevance is under discussion. In the present study, 32 patients with anti-peanut immunoglobulin E-levels (IgE) of at least class 1 according to the ImmunoCap™ classification system were included. The patients had been referred to the Norwegian National Register and Reporting System for Severe Allergic Reactions to Food with histories of anaphylaxis by the consumption of legume-containing foods. The patient sera were analysed for IgE that reacted to peanut (Arachis hypogea), soybean (Glycine max), pea (Pisum sativum), lupin (Lupinus albus) and fenugreek (Trigonella foenum graecum). Immunoblot experiments were performed using extracts of in total eight different legume plants. IgE measurements and blot analyses revealed differentiated patterns of legume sensitisation. One third of the patients had anti-peanut IgE class 4 to 6. In contrast, just one patient had specific IgE higher than class 4 to respectively pea, soybean and fenugreek. However, ten patients had elevated anti-fenugreek IgE (≥class 3), significantly more than for the other legumes. The second highest levels were found for soybean, followed by pea and lupin. The immunoblots showed particularly strong IgE binding to proteins at 12, 22, 30, 36 and 50 kDa for all legumes, indicating that the major peanut allergens Ara h 1, Ara h 2 and Ara h 3 are strong candidates for causing cross-sensitivity.     

Identification of hemocyanin as a novel non-cross-reactive allergen from the giant freshwater shrimp Macrobrachium rosenbergii

Scope : Sensitization to giant freshwater shrimp Macrobrachium rosenbergii (Mr) was recently reported. However, the allergens have yet to be identified. This study aimed to identify and characterize a novel allergen of Mr shrimp. Methods and results: Extracted proteins were separated and purified by anion and in some experiments, size‐exclusion chromatography. Serum IgE from shrimp allergic donors identified a candidate protein, which was characterized by LC‐MS/MS. The specificity of IgE binding was tested using immunoblotting and inhibition ELISA. The IgE‐binding profiles from 12 of 13 Mr allergic subjects that were pre‐incubated with an extract of Penaeus monodon showed residual binding to ～60–80 kDa proteins. The 60–80 kDa IgE‐bound proteins were fractionated in the flow‐through of anion chromatography showing a high IgE reactivity. Peptides identified by LC‐MS/MS showed the proteins closely match subunits of hemocyanin (Hcs). Purified Hcs from hemolymph markedly inhibited binding of IgE from sera of Mr allergic subjects to solid‐phased Mr proteins in inhibition ELISA. Conclusion: Hcs were identified as heat‐stable, non‐cross‐reactive, high‐molecular‐weight (MW) allergens from Mr shrimp. Since circulatory organs are not always removed during food preparation, high concentrations of Hcs may be present along with shrimp meat, which contains the known cross‐reactive tropomyosin protein.     

Bos d 13, A Novel Heat-Stable Beef Allergen

Scope

Red meat, a staple food of Western diets, can also induce IgE-mediated allergic reactions. Yet, apart from the heat-labile protein serum albumin and the carbohydrate α-Gal, the molecules causing allergic reactions to red meat remain unknown.

Methods and results

IgE reactivity profiles of beef-sensitized individuals are analyzed by IgE-immunoblotting with protein extracts from raw and cooked beef. Two IgE-reactive proteins are identified by peptide mass fingerprinting as myosinlight chain 1 (MYL1) and myosin light chain 3 (MYL3) in cooked beef extract and are designated Bos d 13 isoallergens. MYL1 and MYL3 are produced recombinantly in Escherichia coli. ELISAs proved their IgE reactivity and circular dichroism analysis showed that they represent folded molecules with remarkable thermal stability. In vitro gastrointestinal digestion experiments showed the higher stability of rMYL1 as compared to rMYL3. Exposure of a monolayer of Caco–2 cells to rMYL1 indicated that the molecule is able to cross intestinal epithelial cells without disturbing the integrity of the tight junctions, suggesting the sensitizing capacity of MYL1.

Conclusion

MYLs are identified as novel heat-stable bovine meat allergens.     

Tropomyosin, the Major Oyster Crassostrea gigas Allergen and its IgE-binding Epitopes

The major allergen ( Cra g 1) isolated from the oyster Crassostrea gigas was digested with lysylendopeptidase. Twenty-one peptide fragments were isolated from the digest by reverse-phase HPLC and some of them were sequenced. Cra g 1 was identified as tropomyosin. In competitive ELISA inhibition analyses of lysylendopeptidase fragments, a peptide (K21) showed maximum inhibition of the binding of IgE antibodies in sensitive human serum to Cra g 1. On further tryptic hydrolysis of K21, two peptides exhibited high inhibitory ability equivalent to K21. These results suggested that Cra g 1 had an IgE-binding epitope in the segment 92–105 (IQLLEEDMERSEER) which was dissimilar to those of the major allergen (tropomyosin) from the shrimp Penaeus indicus.     

Characterization of lupin major allergens (Lupinus albus L.)

White lupin is considered to be a rich source of protein with a notable content of lysine and is being increasingly used in bakery, confectionery, snacks and pastry products due to its multifunctional properties, in addition to its potential hypocholesterolemic and hypoglycemic properties. However, lupin seed flour has been reported as a causative agent of allergic reactions, especially in patients with allergy to peanut since the risk of immunological cross‐reactivity between lupin and peanut is higher than with other legumes. Previously, we had identified two proteins as major lupin allergens (34.5 and 20 kDa) as determined by IgE immunoblotting using sera of 23 patients with lupin‐specific IgE. The aim of this study was to purify and characterize the two major lupin allergens. The results using in vitro IgE‐binding studies and MS analysis have shown that the 34.5 kDa allergen (Lup‐1) is a conglutin β (vicilin‐like protein) while the 20 kDa allergen (Lup‐2) corresponds to the conglutin α fraction (legumin‐like protein). The high level of amino acid sequence homology of Lup‐1 and Lup‐2 with the major allergens of some legumes explains the IgE cross‐reactivity and clinical cross‐reactivity of lupin and other legumes.     

Glycation of the Major Milk Allergen β‐Lactoglobulin Changes Its Allergenicity by Alterations in Cellular Uptake and Degradation

1 Scope

During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β‐lactoglobulin (BLG), in their interactions with cells crucially involved in allergy.

2 Methods and results

BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T‐cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco‐2 monolayer. Uptake of glycated BLG by bone marrow–derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor‐mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4⁺ T‐cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG‐specific IgE sensitized basophil cells.

3 Conclusions

This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy.     

Development and validation of a SYBR‐Green I Real‐Time PCR test to detect bivalves including Mytilus species in foods

The incidence of allergy to seafood, and in particular to molluscs is second only to that of nuts. To protect consumers, the regulators of food products insisted on identifying molluscs as allergens. The aim was to develop quantitative assay for the presence Mytilus species in processed food products. The chosen platform was real‐time PCR (qPCR) targeting either the gene encoding mitochondrial cytochrome C oxidase I or the nuclear gene encoding β‐actin. Recombinant plasmids containing each of target regions were used as a reference for quantification purposes. Limit of detection (LOD) and of quantification (LOQ) were determined. Spiked food samples containing 50–500 μg g^?1 of Mytilus chilensis were analysed both by qPCR and by ELISA. The former assay gave a positive outcome over this range, whereas the latter was sensitive down to a concentration of 125 μg g^?1.     

Preparation and optimisation of liposome‐in‐alginate beads containing oyster hydrolysate for sustained release

Oyster (Crassostrea gigas) hydrolysate shows antihypertensive effect in our previous study. Oral administration of oyster hydrolysate can loss bioactive peptides due to enzymatic degradation in vivo. To maximise its bioavailability, liposome‐in‐alginate (LA) beads were used to encapsulate the oyster hydrolysates to protect from degradation and obtain sustained release. The preparation conditions of the LA beads were optimised by response surface method using a model peptide of tyrosylalanine (YA). Their characterisation, swelling and release properties were investigated. The optimised conditions for the concentration of calcium chloride, sodium alginate and the amount of ethanol‐dissolved lecithin (EDL) were 0.5 m , 3% and 95.4 mg, respectively. The encapsulation efficiencies of YA and the oyster hydrolysate in the optimised condition were 74.9% and 84.3%, respectively. The release time of the oyster hydrolysate in the simulated gastrointestinal fluid was up to 16 h. The LA beads can be recommended to encapsulate oyster hydrolysate for bioavailability improvement.     

Gel‐enhancing effect and protein cross‐ linking ability of tilapia sarcoplasmic proteins

BACKGROUND: Tilapia (Oreochromis niloticus) sarcoplasmic proteins contain substantial transglutaminase (TGase) activity. The enzyme catalyzes the protein cross‐linking reaction, resulting in a more elastic gel. The objective was to investigate the gel‐enhancing effect of sarcoplasmic proteins from tilapia as related to TGase activity. RESULTS: Total TGase activity of sarcoplasmic proteins concentrate (SpC) increased about 3.6‐fold after ultrafiltration using 30 kDa membrane, but specific activity remained unchanged, indicating minimal TGase purification by ultrafiltration. Addition of 1 mg mL^?1 SpC containing 40 units TGase activity induced cross‐linking of tilapia actomyosin, and the extent of cross‐linking increased with added level of SpC. Myosin heavy chain (MHC) and troponin were preferably cross‐linked by tilapia SpC, while actin and tropomyosin were not affected. Higher retention of MHC was observed concomitantly with greater content of cross‐linked protein when SpC was added to lizardfish surimi. Lizardfish surimi with 10 g kg^?1 SpC added and pre‐incubated at 37 °C for 1 h exhibited 91.6% and 26.7% increase in breaking force and deformation, respectively, when compared to the control. CONCLUSIONS: Residual TGase activity in SpC played an important role in catalyzing the protein cross‐linking and enhancing actomyosin gelation. SpC could be a potential ingredient for improving textural properties of fish protein gel. Copyright © 2007 Society of Chemical Industry     

IDENTIFICATION OF GOAT MEAT USING HIGHLY SPECIES‐SPECIFIC POLYMERASE CHAIN REACTION

ABSTRACT

A highly species‐specific polymerase chain reaction (PCR) assay was developed for the authentic identification of goat. A product of 436 bp was amplified using newly designed primers against mitochondrial D‐loop region. The possibility of cross‐amplification was ruled out by considering as many as 25 other animal species. Suitability of the developed goat species‐specific PCR assay was confirmed for in raw, cooked (60, 80 and 100C for 30 min) and micro‐oven‐processed meat samples (n = 20 each). A sensitivity of 0.1% was established for detection of adulteration and limit of detection of goat DNA was 0.1 pg. This investigation presents a novel PCR assay with its newly designed primers that could be used for the authentic identification of goat species.

PRACTICAL APPLICATIONS

This work details about a novel diagnostic polymerase chain reaction, which could be used for authentic identification of goat species. This approach could be used for the confirmation of goat tissues in raw, cooked, as well as adulterated samples. The developed technique has also applications in the forensic analysis of wild animal‐related disputes, where this work could solve the problem of goat‐related issues.     

Determination of Iriflophenone 3‐C‐β‐d‐Glucoside From Aquilaria spp. by an Indirect Competitive Enzyme‐linked Immunosorbent Assay Using a Specific Polyclonal Antibody

Polyclonal antibody against iriflophenone 3‐C‐β‐d ‐glucoside (IP3G), a major compound from the leaves of Aquilaria spp., was produced for the development of an enzyme‐linked immunosorbent assay (ELISA). The results showed that the antibodies were specific for IP3G. The produced antibody has low cross reactivity with iriflophenone 3,5‐C‐β‐d ‐diglucopyranoside (13%), genkwanin 5‐O‐β‐primeveroside (3.55%) and no cross reactivity found in other compounds. The range of ELISA assay extends from 100 to 1560 ng/mL with coefficient of variation (CV) 1.19% to 2.07% for intra‐assay and 3.76% to 7.15% for inter‐assay precision levels. The recovery rates of IP3G in the leaves of Aquilaria spp. were in the range of 96.0% to 99.0% with CV 4.50% to 5.32%. A correlation between ELISA and high‐performance liquid chromatography methods was obtained when analysis of IP3G in the plant samples (R² = 0.9321). These results suggest that the developed ELISA method can be applied to determine IP3G content with high specificity, rapidity, and simplicity. The developed immunosorbent assay in this study provides a useful tool for the analysis of IP3G in plant samples and products.     

Effects of enzymatic hydrolysis on lentil allergenicity

Enzymatic hydrolysis and further processing are commonly used to produce hypoallergenic dietary products derived from different protein sources, such as cow's milk. Lentils and chickpeas seem to be an important cause of IgE‐mediated hypersensitivity in the Mediterranean area and India. Some studies have investigated the effects of enzymatic treatments on the in vitro immunological reactivity of members of the Leguminosae family, such as soybean, chickpea, lentil, and lupine. Nevertheless, there are only a few studies carried out to evaluate the effect on IgE reactivity of these food‐hydrolysis products with sera from patients with well‐documented allergy to these foods. In this study, lentil protein extract was hydrolyzed by sequential action of an endoprotease (Alcalase) and an exoprotease (Flavourzyme). Immunoreactivity to raw and hydrolyzed lentil extract was evaluated by means of IgE immunoblotting and ELISA using sera from five patients with clinical allergy to lentil. The results indicated that sequential hydrolysis of lentil results in an important proteolytic destruction of IgE‐binding epitopes shown by in vitro experiments. However, some allergenic proteins were still detected by sera from four out of five patients in the last step of sequential hydrolyzation.     

ANTIFUNGAL,AFLATOXIN INHIBITORY AND FREE RADICAL‐SCAVENGING ACTIVITIES OF SOME MEDICINAL PLANTS EXTRACTS

ABSTRACT

The antifungal, aflatoxin inhibitory and antioxidant activity of methanol–aqueous extract (2:1) of 62 medicinal plants was explored. Based on the antifungal results, the extracts of 25 plants showed more than 50% antifungal activity and were further investigated for their aflatoxin inhibition and antioxidant properties. Methanol–aqueous extracts of Phyllanthus emblica and Terminalia chebula fruits caused 100% inhibition of aflatoxin production by the toxigenic strain of Aspergillus flavus in semisynthetic medium at 1 mg/mL. In addition, P. emblica (IC₅₀ = 4.1 µg/mL) and T. chebula (IC₅₀ = 6.9 µg/mL) fruits extracts exhibited strong antioxidant activity during the 2,2‐diphenyl‐1‐picrylhydrazyl radical‐scavenging assay in comparison with butylated hydroxytoluene (IC₅₀ = 8.1 µg/mL) and butylated hydroxyanisole (IC₅₀ = 6 µg/mL).

PRACTICAL APPLICATIONS

Based on the results of the present study, methanol–aqueous extracts of Phyllanthus emblica and Terminalia chebula, being endowed with strong antifungal, aflatoxin inhibitory and antioxidant activity, may be recommended as plant‐based preservatives for the enhancement of shelf life of food items and their protection from the undesirable harmful effects of molds, aflatoxin and free radical‐mediated damages.     

Identification and origin of the character‐impact compounds of raw oyster Crassostrea gigas

The French consume large amounts of raw oysters. The study of the aroma of oyster Crassostrea gigas is of economic interest because it is a good method of checking the sensory quality. Aromas were extracted by vacuum steam distillation at 20 °C using whole oyster flesh. This extract presented similar sensory characteristics to raw oyster. The odour‐active compounds were characterised by gas chromatography coupled with olfactometry using a panel of 10 judges trained in seafood aroma recognition. Fifty‐nine volatile compounds were identified in oyster aroma extract. Among these, 25 were responsible for the overall odour of raw oyster. Four compounds identified in oysters were characterised by fresh and marine odour: 3‐(E)‐hexen‐1‐ol, decanal, 2‐undecanone and 3,6‐(E,Z)‐nonadien‐1‐ol. Some compounds were identified for the first time in oysters: 4‐(Z)‐heptenal (white boiled fish odour), which comes from n‐3 polyunsaturated fatty acid oxidation, and 3‐octanol (moss and sulphury odour), 2‐nonanol (cucumber odour) and octanoic acid, which arise from n‐6 polyunsaturated fatty acid oxidation. © 2002 Society of Chemical Industry     

Lupine,a source of new as well as hidden food allergens

The present review summarizes current knowledge about lupine allergy, potential sensitization routes, cross‐reactions between lupine and other legumes, and the respective IgE‐binding proteins. Since the 1990s, lupine flour is used as a substitute for or additive to other flours, mostly wheat flour, in several countries of the EU. In 1994, the first case of an immediate‐type allergy after ingestion of lupine flour‐containing pasta was reported. Since then, the number of published incidents following ingestion or inhalation of lupine flour is rising. So far, the Lupinus angustifolius β‐conglutin has been designated as the allergen Lup an 1 by the International Union of Immunological Societies Allergen Nomenclature Subcommittee. Initially, publications focussed on the fact that peanut‐allergic patients were at risk to develop anaphylaxis to lupine due to cross‐reactivity between peanut and lupine. At present, however, the ratio between cases of pre‐existing legume allergy (mostly peanut allergy) to de novo sensitization to lupine seed is nearly 1:1. Although in December 2006, lupine and products thereof were included in the EU foodstuff allergen list according to the Commission Directive 2006/142/EC amending Annex IIIA of Directive 2000/13/EC in order to prevent severe reactions caused by “hidden food allergens”, the majority of patients and medical personnel are still not aware of raw lupine seed as potentially dangerous food allergen.     

Determination of proximate composition,fatty acid content and amino acid profile of five lesser‐common sea organisms from the Mediterranean Sea

Proximate composition, fatty acid and amino acid analysis of an echinoderm (Paracentrotus lividus), a crustacean (Penaeus kerathurus), a tunicate (Microcosmus sulcatus), and two gastropod molluscs (Littorina littorea and Patella coerulea) were determined. All organisms except M. sulcatus, were found to be good protein sources. P. lividus contained on average 15.1 g/100 g, P. kerathurus 15.6 g/100 g, L. littorea 8.3 g/100 g and P. coerulea 9.2 g/100 g protein. The crustacean contained high percentages of n‐3 fatty acids (28.3 g/100 g fatty acids), the tunicate high percentages of saturated fatty acids (62.2 g/100 g fatty acids) while the gastropod molluscs and the echinoderm had a balanced content of all fatty acid families. The most abundant amino acid in P. kerathurus, M. sulcatus, L. littorea and P. coerulea was glutamic acid (11.13 ± 0.9, 1.05 ± 0.3, 5.39 ± 0.3, 5.55 ± 0.8 g/100 g freeze‐dried sample respectively), while glycine was the most abundant amino acid in P. lividus (10.34 ± 1.0 g/100g freeze‐dried sample).