Apple Allergy: The cDNA Sequence of the Major Allergen of Apple,Determined by Performing PCR with a Primer Based on the N-Terminal Amino Acid Sequence,is Highly Homologous to the Sequence of the Major Birch Pollen Allergen

Considering the known N-terminal amino acid sequence of the major apple allergen, a polymerase chain reaction (PCR) primer was selected to amplify cDNA encoding this protein. A single PCR product was obtained, cloned into Escherichia coli and subsequently sequenced. The missing 5′-end of the apple cDNA sequence was obtained by a 5′-RACE method. The cDNA sequence showed 72% identity with the coding region of one of the known isoforms of Bet v 1, the major allergen of birch pollen. The deduced amino acid sequence resulted in a 158-residue protein with a calculated molecular mass of 17·5 kDa and 63% amino acid sequence identity to Bet v 1. In addition, further protein alignments showed a high degree of identity with allergens from other tree pollens and some ‘pathogenesis-related proteins’ from food plants. According to international regulations the allergen was termed Mal d 1 for this protein, it being the first major allergen discovered and characterised in fruits of apple (Malus domestica).     

Anti-inflammatory activity of American yam Dioscorea trifida L.f. in food allergy induced by ovalbumin in mice

Tropical ecosystems are particularly rich in edible plant species with different bioactive substances. Among the plants with promising benefits for health are species from the genus Dioscorea (Dioscoreaceae), especially those named yam. Recent studies have shown the beneficial effects of different species of Dioscorea, and its main constituent, diosgenine, in the treatment of food allergy. In this study we evaluated the potential of D. trifida, the only yam native from South America, in the treatment of ovalbumin (OVA) induced food allergy in Balb/c mice. HPLC/DAD analysis showed the presence of three very distinctive groups of natural products in extracts and fractions: (I) very polar substances, including allantoin, (II) phenolic substances as flavonoids and phenolic acids and (III) diosgenin and derivatives. Sensitive mice received casein feed with supplementation of crude extract (CE) and fractions. The supplementation with all products from D. trifida reduced IGE, intestinal oedema and mucus production, parameters observed in OVA allergic mice. The results showed the potential of this food to prevent or treat this disease and the necessity to be better explored.     

MALDI-based identification of stable hazelnut protein derived tryptic marker peptides

Food allergy is an important health problem especially in industrialised countries. Tree nuts, among which are hazelnuts (Corylus avellana), are typically causing serious and life-threatening symptoms in sensitive subjects. Hazelnut is used as a food ingredient in pastry, confectionary products, ice cream and meat products, therefore undeclared hazelnut can be often present as a cross-contaminant representing a threat for allergic consumers. Mass spectrometric techniques are used for the detection of food allergens in processed foods, but limited information regarding stable tryptic peptide markers for hazelnut is available. The aim of this study was to detect stable peptide markers from modified hazelnut protein through the Maillard reaction and oxidation in a buffered solution. Peptides ³⁹⁵Gly-Arg⁴⁰³ from Cor a 11 and ²⁰⁹Gln-Arg²¹⁷, ³⁵¹Ile-Arg³⁶³, ⁴⁶⁴Ala-Arg⁴⁷⁸ and ⁴⁰¹Val-Arg⁴¹⁷ from Cor a 9 hazelnut allergens proved to be the most stable and could be detected and confirmed with high scores in most of the modified samples. The identified peptides can be further used as analytical targets for the development of more robust quantitative methods for hazelnut detection in processed foods.     

多酚氧化酶交联β-酪蛋白的抗原性变化初步研究

利用多酚氧化酶催化牛乳β-酪蛋白交联,并通过ELISA方法检测交联前后β-酪蛋白抗原性的变化。SDS-PAGE结果显示,多酚氧化酶可以有效地催化β-酪蛋白交联。同时,间接竞争ELISA检测的β-酪蛋白交联后的半抑制浓度(IC50值)由2.63μg/mL变为4.07μg/mL,表明交联后β-酪蛋白抗原性明显降低。本研究工作为通过多酚氧化酶交联制备低致敏性乳制品提供了部分理论依据。     

Sensitive and specific detection of almond (Prunus dulcis) in commercial food products by real-time PCR

Almond has been widely used in all sorts of food products, mostly due to its pleasant flavor and health benefits. However almonds can become an important health problem since they are responsible for triggering adverse immune responses in allergic individuals, and since they are present in many processed foods they are considered as a potential hidden allergen. Consequently, it's important for food processors and regulatory agencies to be able to ensure accurate labeling of foods to protect the safety of the public and to avoid expensive recalls. We propose a simple and highly sensitive approach to detect almond in a wide range of processed foods. The method consists of a real-time PCR assay targeting the gene encoding for the ITS1 in almond, using a nuclease (TaqMan) probe labeled with FAM and BBQ. Sensitivity of real time PCR was determined by analysis of raw and heat treated almond-wheat flour mixtures with a range of detection of 0.1–100,000 mg/kg. The assay was successfully trialed on a total of 214 commercial foodstuffs allowing the detection of trace amounts of almond down to the level of 0.1 mg/kg, and is therefore proposed as a ready-to-use analytical tool to trace almond allergens in foods.     

Development of an epitope-specific analytical tool for the major peanut allergen Ara h 2 using a high-density multiple-antigenic peptide strategy

Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.     

Hazelnut Allergens: Molecular Characterization,Detection, and Clinical Relevance

In last few years, special attention has been given to food-induced allergies, in which hazelnut allergy is highlighted. Hazelnut is one of the most commonly consumed tree nuts, being largely used by the food industry in a variety of processed foods. It has been regarded as a food with potential health benefits, but also as a source of allergens capable of inducing mild to severe allergic reactions in sensitized individuals. Considering the great number of reports addressing hazelnut allergens, with an estimated increasing trend, this review intends to assemble all the relevant information available so far on the following main issues: prevalence of tree nut allergy, clinical threshold levels, molecular characterization of hazelnut allergens (Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 14, and Cor a TLP) and their clinical relevance, and methodologies for detection of hazelnut allergens in foods. A comprehensive overview of the current data about the molecular characterization of hazelnut allergens is presented, relating to biochemical classification and biological function with clinical importance. Recent advances in hazelnut allergen detection methodologies are summarized and compared, including all the novel protein-based and DNA-based approaches.     

Peanut varieties with reduced Ara h 1 content indicating no reduced allergenicity

Peanut allergy is a major cause of food‐induced severe anaphylactic reactions. To date, no medical care is available to prevent and treat peanut allergy and therefore hypoallergenic peanut varieties are of considerable health political and economic interest. Major allergens that induce IgE‐responses in peanut‐sensitive patients are Ara h 1, Ara h 2 and Ara h 3/4. In order to identify hypoallergenic peanuts, commercially locally available peanut varieties were screened for their allergen content. Ara h 1‐deficient peanuts from Southeast Asia were identified by SDS‐PAGE, immunoblotting, inhibition assays and ELISA. 2‐D PAGE analyses demonstrated the different compositions of the tested extracts and revealed a number of variations of the allergen patterns of peanuts from different varieties. Mediator release experiments of these peanut extracts demonstrated similar allergenicities as compared with standard peanut extract. These results indicate that the allergenicity of peanuts with reduced Ara h 1 content might be compensated by the other allergens, and thus do not necessarily cause a reduction of allergenicity.     

低过敏性花生制品开发

花生过敏严重影响健康.综述了花生主要过敏原及其性质、花生制品变应原性研究进展,以及低过敏性花生制品的开发途径.     

Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of cashew nut in foods

The presence of undeclared cashew can pose a health risk to cashew-allergic consumers. The food industry has the responsibility to declare the presence of cashews on packaged foods even when trace residues are or might be present. The objective of this study was to develop a rapid, sensitive, and specific enzyme-linked immunosorbent assay (ELISA) for the detection of cashew residues. Raw and roasted cashews were defatted and used separately to immunize sheep, goats, and rabbits. The cashew ELISA was developed using sheep and rabbit polyclonal anti-roasted cashew sera as capture and detector reagents, respectively, with visualization through an alkaline phosphatase-mediated substrate reaction. The cashew ELISA was shown to have a limit of quantification of 1 ppm (1 μg cashew/g). The ELISA was highly specific except that substantial cross-reactivity was noted with pistachio and a lesser degree of cross-reactivity was noted with hazelnut. The performance of the ELISA was assessed by manufacturing cookies, ice cream, and milk chocolate with added known amounts (0 to 1000 ppm) of cashew. The mean percent recoveries for ice cream, cookies, and milk chocolate were 118%± 2.9%, 84.3%± 4.0%, and 104%± 3.0%, respectively. In a limited retail survey, 4/5 retail samples with cashew declared on ingredient labels tested positive for cashew compared to 5/36 samples of foods with precautionary labels indicating the possible presence of one or more tree nuts and 0/18 samples without cashew declared on the label in any manner. The cashew ELISA can be used to detect undeclared cashew residue in foods and as a potential tool for the food industry to assess the effectiveness of allergen control strategies and to guarantee compliance with food labeling regulatory requirements.