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.     

Quantification of lupine (Lupinus angustifolius) in wheat flour using real-time PCR and an internal standard material

In the European Union, labelling of the 14 food allergens listed in Annex IIIa of Directive 2000/13/EC is mandatory. The implementation of upper limits for these allergens is under discussion. Therefore, quantitative analytical methods will be needed to verify compliance with regulatory requirements and to provide an improved basis for the legal assessment of allergen labelling. In this study, the lupine flour content in wheat flours was determined using real-time PCR and statice seeds as internal standard material. The method proved to be applicable to the quantification of lupine contents from 1 to 10?mg/kg, which is in the range relevant for allergic consumers.     

Sandwich Enzyme‐Linked Immunosorbent Assay (ELISA) for the Detection of Lupine Residues in Foods

ABSTRACT: Lupine has been increasingly used in food applications due to its high nutritional value and excellent functional properties. However, lupine provokes allergic reactions in susceptible individuals. The presence of undeclared lupine residues in foods can pose a serious health risk to lupine‐allergic individuals. Therefore, the objective of this research was to develop a sandwich‐type ELISA for the detection of lupine residues in foods. Lupine flour derived from Lupinus albus was used to immunize 3 rabbits and a sheep. Pooled lupine‐specific antibodies were partially purified from the sera by ammonium sulfate precipitation. A sandwich lupine ELISA with a limit of quantification (LOQ) of 1 ppm was developed by utilizing the rabbit antisera as the capture reagent and the sheep antiserum as the detector reagent. The binding of the antigen‐antibody complex was visualized by the addition of commercial rabbit antisheep IgG antibody labeled with alkaline phosphatase with subsequent addition of p‐nitrophenyl phosphate substrate to produce a colored product for quantification. Minor cross‐reactivity was observed with soy (Glycine max) and black bean (Castanospermum australe). The performance of the lupine ELISA was evaluated in reference food standards (beef frankfurter and apple cinnamon muffin) and laboratory‐prepared cooked frankfurters and corn muffins. The mean percent recovery for lupine spiked‐frankfurters and corn muffins were 108.4%± 8.8% and 103.1%± 11.5%, respectively. The sandwich‐type lupine ELISA developed in this study provides food manufacturers and regulatory agencies with an effective analytical tool to detect and quantify lupine residues in processed foods.     

花生过敏及其主要致敏原Ara h1的研究进展

花生是一种具有致敏作用的重要食品,能够引起严重的过敏反应。花生的致敏性研究是食物安全研究领域的一个重要课题。本文主要论述了近年来花生致敏现状及花生主要致敏原Ara h1研究进展,包括花生致敏特点、脱敏方法等方面的内容。对降低花生引起的过敏反应风险具有一定意义,同时为对花生过敏者的临床脱敏治疗提供理论依据。     

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.     

Peanut Allergy,Peanut Allergens,and Methods for the Detection of Peanut Contamination in Food Products

ABSTRACT: Attention to peanut allergy has been rising rapidly for the last 5 y, because it accounts for the majority of severe food‐related anaphylaxis, it tends to appear early in life, and it usually is not resolved. Low milligram amounts of peanut allergens can induce severe allergic reactions in highly sensitized individuals, and no cure is available for peanut allergy. This review presents updated information on peanut allergy, peanut allergens (Ara h1 to h8), and available methods for detecting peanuts in foods. These methods are based on the detection of either peanut proteins or a specific DNA fragment of peanut allergens. A summary of published methods for detecting peanut in foods is given with a comparison of assay formats, target analyte, and assay sensitivity. Moreover, a summary of the current availability of commercial peanut allergen kits is presented with information about assay format, target analyte, sensitivity, testing time, company/kit name, and AOAC validation.     

Impact of dietary factors and food processing on food allergy

Allergic reactions to food can significantly reduce the quality of life and even result in life‐threatening complications. In addition, the prevalence of food allergy has increased in the last decades in industrialized countries and the mechanisms underlying (increased) sensitization are still not fully understood. It is believed that the development and maintenance of oral tolerance to food antigens is a process actively mediated by the immune system and that this reaction is essential to inhibit sensitization. Ongoing research indicates that different dietary factors also may contribute to immune homeostasis and oral tolerance to food and that food processing modulates allergenicity. One of the major questions in food allergy research is therefore which impact nutrition and food processing may have on allergenicity of food and perhaps on sensitization. We summarize in this review the different dietary factors that are believed to contribute to induction of oral tolerance and discuss the underlying mechanisms. In addition, the functional consequences of allergen modification will be emphasized in the second part as severity of allergic reactions and perhaps sensitization to food is influenced by structural modifications of food allergens.     

Survey of peanut levels in selected Irish food products bearing peanut allergen advisory labels

Peanut allergy affects up to 2% of consumers and is responsible for the majority of fatalities caused by food-induced anaphylaxis. Peanut-containing products must be clearly labelled. Manufacturers are not legally required to label peanut if its inclusion resulted from unintentional cross contact with foods manufactured in the same facility. However, the use of allergen advisory statements alerting consumers of the potential presence of peanut allergen has increased in recent years. In previous studies, the vast majority of foods with precautionary allergen statements did not contain detectable levels of peanut, but no data are available on Irish food products. Thirty-eight food products bearing peanut/nut allergen-related statements were purchased from multiple locations in the Republic of Ireland and analysed for the presence of peanut. Peanut was detected in at least one lot in 5.3% (2 of 38) of the products tested. The doses of peanut detected ranged from 0.14 mg to 0.52 mg per suggested serving size (0.035–0.13 mg peanut protein). No detectable levels of peanut were found in the products that indicated peanut/nuts as a minor ingredient. Quantitative risk assessment, based on the known distribution of individual threshold doses for peanut, indicates that only a very small percentage of the peanut-allergic population would be likely to experience an allergic reaction to those products while the majority of products with advisory labels appear safe for the peanut-allergic population. Food manufacturers should be encouraged to analyse products manufactured in shared facilities and even on shared equipment with peanuts for peanut residues to determine whether sufficient risk exists to warrant the use of advisory labelling. Although it appears that the majority of food products bearing advisory nut statements are in fact free of peanut contamination, advice to peanut allergy sufferers to avoid said foods should continue in Ireland and therefore in the wider European Union.     

Food allergy,a summary of eight cases in the UK criminal and civil courts: effective last resort for vulnerable consumers?

Food allergy has a forensic context. The authors describe eight cases in the UK courts involving fatalities, personal injury or criminal non‐compliance with food law from mainly ‘grey’ literature sources. The potentially severe consequences for people with food allergy of contraventions of labelling law have led to enforcement action up to criminal prosecution for what might otherwise be regarded as ‘trivial’ non‐compliance. The authors suggest there should be central collation of such cases. Non‐compliances should be followed up in a more rapid and robust manner. Evidence of fraud in the catering supply chain supports recent calls for zero tolerance of food fraud. Businesses must guard against gaps in allergen management, for which there are readily available sources of training and guidance, but also against fraudulent substitution in the supply chain, about which training and guidance should be developed. New allergen labelling legislation and case law appear to place responsibility on food businesses even for the forensically problematic area of allergen cross‐contamination. The courts can be an effective last resort for vulnerable consumers; however, there is evidence of knowledge and skill gaps in both the investigation and prosecution of potentially serious incidents of food allergen mismanagement and mislabelling. Thorough investigation of food allergy deaths is required with a tenacious and skilled approach, including early realisation that samples of the food and/or stomach contents from a post mortem examination should be retained and analysed. The supply chain must be rigorously examined to find out where adulteration or contamination with the fatal allergen occurred. © 2014 Society of Chemical Industry     

DNA 提取方法对实时荧光定量 PCR 检测花生过敏原 Ara h 2 基因的比较研究

目前,食物过敏是一个世界性的公共卫生问题,其中花生过敏最为严重。基于DNA的分子生物学检测方法目前已广泛应用于花生过敏原检测,样品DNA的提取质量会显著影响检测的灵敏度及准确率。本研究比较了5种DNA提取方法,包括十六烷基三甲基溴化铵（cetyl trimethyl ammonium bromide,CTAB）法、柱式法及3种基于磁珠纯化技术的DNA快速提取法,对生花生、煮花生、炸花生、烤花生、花生酥和花生酱6种不同加工方式的花生基质样品进行DNA提取,考察了不同方法获得的DNA浓度、纯度指标,并采用实时荧光定量PCR（quantitative real-time PCR, qPCR）对花生过敏原Ara h 2基因进行了检测分析。结果表明,月桂酰肌氨酸钠（Sodium Lauroyl Sarcosine）磁珠法（简称SLS磁珠法）的适用性广、提取率高,对于6种花生基质提取的DNA均能高效检出花生过敏原Ara h 2基因;对于花生含量为0.05%～1.00%的小麦粉二元混合物,SLS磁珠法的DNA提取率总体优于CTAB法,并且能有效提取出与花生共用一条生产线的燕麦片中污染的花生DNA,证实SLS磁珠法提取的实际样品DNA能够满足花生过敏原检测目的。本研究为花生及其制品DNA提取方法提供了参考,特别是磁珠类方法,高效快速,提取质量能够保障后续基于DNA的花生过敏原分子生物学方法检测结果的准确性。     

食品过敏原标签要求及生产过程控制初探

食品过敏已成为世界关注的重大公共卫生学和食品安全问题。食品过敏原是引起食品过敏的直接诱因,具有耐加工和交叉反应的特性。国际食品法典委员会(Codex Alimentarius Commission,CAC)规定了必须在食品标签中标注的8种可能引起过敏反应的食品及其配料,包括含有麸质的谷类、甲壳动物及其制品、鸡蛋及其制品、鱼类及其制品、花生、大豆及其制品、奶和奶制品(包括乳糖)、坚果及其制品、浓度不低于10 mg/kg的亚硫酸盐。欧盟、美国、日本、澳大利亚和新西兰等已根据国际食品法典委员会对过敏原的要求出台强制性标识食品过敏原的法规,这在一定程度上对我国出口食品企业造成了影响。本文通过对比CAC以及欧盟、中国、美国、日本、澳大利亚和新西兰等地有关过敏原标签的要求,提醒出口食品加工企业减少因标签不合格造成的经济损失,并为其在生产加工过程中过敏原的控制提出建议。     

Food matrix standards for the quantification of allergenic food ingredients using real-time PCR

For the quantification of food allergens by real-time polymerase chain reaction (PCR), food matrix standards with defined levels of spiked allergenic food ingredients can be used. The production and homogeneity testing of selected materials as sausages, cookies and sauce hollandaise powder is described. Except for egg and milk, all relevant allergenic ingredients were spiked to each material. Allergens were spiked and quantified as food ingredients, for example, peanut or lupine flour, at levels of 5–400 mg/kg. Material with sufficient homogeneity could be produced even at low levels of 5–10 mg of the allergenic ingredient per kilogram. The effect of the food matrix on allergen quantification was checked. The bias caused by this effect was in an acceptable range for the tested materials. The materials produced within this study were used as samples and for calibration in inter-laboratory validation studies for the quantification of allergenic food ingredients by real-time PCR. The results of this study are a contribution how to produce such reference materials for allergen analysis in the near future. Before threshold or action values of allergens in food are set, the availability of reference materials is essential.     

Effect of Heat Processing on IgE Reactivity and Cross‐Reactivity of Tropomyosin and Other Allergens of Asia‐Pacific Mollusc Species: Identification of Novel Sydney Rock Oyster Tropomyosin Sac g 1

1 Scope

Shellfish allergy is an increasing global health priority, frequently affecting adults. Molluscs are an important shellfish group causing food allergy but knowledge of their allergens and cross‐reactivity is limited. Optimal diagnosis of mollusc allergy enabling accurate advice on food avoidance is difficult. Allergens of four frequently ingested Asia‐Pacific molluscs are characterized: Sydney rock oyster (Saccostrea glomerata), blue mussel (Mytilus edulis), saucer scallop (Amusium balloti), and southern calamari (Sepioteuthis australis), examining cross‐reactivity between species and with blue swimmer crab tropomyosin, Por p 1.

2 Methods and results

IgE ELISA showed that cooking increased IgE reactivity of mollusc extracts and basophil activation confirmed biologically relevant IgE reactivity. Immunoblotting demonstrated strong IgE reactivity of several proteins including one corresponding to heat‐stable tropomyosin in all species (37–40 kDa). IgE‐reactive Sydney rock oyster proteins were identified by mass spectrometry, and the novel major oyster tropomyosin allergen was cloned, sequenced, and designated Sac g 1 by the IUIS. Oyster extracts showed highest IgE cross‐reactivity with other molluscs, while mussel cross‐reactivity was weakest. Inhibition immunoblotting demonstrated high cross‐reactivity between tropomyosins of mollusc and crustacean species.

3 Conclusion

These findings inform novel approaches for reliable diagnosis and improved management of mollusc allergy.     

High-pressure microfluidisation-induced changes in the antigenicity and conformation of allergen Ara h 2 purified from Chinese peanut

BACKGROUND: Peanut allergy is one of the most serious food allergies, and Ara h 2 is one of the most important peanut allergens as it is recognised by serum immunoglobulin E from more than 90% of peanut‐allergic individuals. Dynamic high‐pressure microfluidisation has been widely used in food processing as a new technology. The aim of this study was to investigate the effect of high‐pressure microfluidisation on the antigenicity and structure of Ara h 2. Extracted peanut allergen Ara h 2 was treated under a continuous pressure array of 60, 90, 120, 150 and 180 MPa. Immunoreactivity was measured by indirect enzyme‐linked immunosorbent assay with rabbit polyclonal antibodies. Secondary structure was analysed by circular dichroism. Surface hydrophobicity and sulfhydryl groups were assessed via fluorescence and UV absorption spectra respectively. RESULTS: High‐pressure microfluidisation treatment decreased the antigenicity of peanut allergen Ara h 2, changed its secondary structure and increased its UV absorption intensity and surface hydrophobicity. CONCLUSION: The change in conformation contributed to the decrease in antigenicity of Ara h 2, and the spatial conformation of peanut allergen Ara h 2 plays a critical role in its antigenicity. Copyright © 2011 Society of Chemical Industry     

热加工花生的蛋白分离及其过敏原纯化方法研究进展

花生不仅本身是一种营养丰富的食品,而且作为原料或配料广泛应用于食品加工中。然而花生及其制品是FAO/WHO认定的八大类食物过敏原之一,可导致严重的过敏反应,通常伴随终身,甚至危及生命。不同地区的人们食用花生的加工方式不同,其花生过敏的患病率也有所不同,热加工是花生的主要加工方式,因此各类热加工导致的花生致敏性变化成为研究热点。过敏原蛋白的分离作为花生热加工研究中的重要步骤,也变得十分重要。本文主要对常见的3种热加工花生(水煮、油炸和烘烤)中的花生蛋白分离及其过敏原纯化的方法研究进行综述。现有的花生热加工研究中蛋白分离技术主要是通过溶剂浸提;而过敏原纯化技术主要是借助层析法,根据各组分在物理化学性质上的差异进行纯化;此外还可以根据最终研究目的的不同采用其他的辅助方法达到分离纯化效果。通过对现有分离纯化方法进行了解和比较,可为热加工花生过敏原蛋白的分离纯化甚至进一步的分析检测提供理论参考和指导。     

花生致敏原检测技术的研究进展

目前食物致敏原检测已经成为食品安全领域面临的重要话题。花生是一种主要的食物致敏原, 开展食 品中花生致敏原检测技术研究, 对预防过敏反应, 快速诊断病情, 保障食品安全具有重要意义。本文综述了近 十年来花生致敏原检测技术的研究进展, 介绍了花生的致敏机制, 分析了不同检测方法的优缺点, 举例说明了 这些方法的实际应用, 讨论了该领域面临的挑战, 旨在为花生的致敏原检测、脱敏技术等研究提供有价值的参 考意见。     

Peanut Allergy: Characteristics and Approaches for Mitigation

Peanut allergy has garnered significant attention because of the high sensitization rate, increase in allergy, and severity of the reaction. Sufficiently reliable therapies and efficient mitigating techniques to combat peanut allergy are still lacking. Current management relies on avoiding peanuts and nuts and seeds with homologous proteins, although adverse events mostly occur with accidental ingestion. There is a need for hypoallergenic peanut products to protect sensitized individuals and perhaps serve as immunotherapeutic products. Alongside traditional practices of thermal and chemical treatment, novel processing approaches such as high‐pressure processing, pulsed ultraviolet light, high‐intensity ultrasound, irradiation, and pulsed electric field have been performed toward reducing the immunoreactivity of peanut. Covalent and noncovalent chemical modifications to proteins also have the tendency to alter peanut allergenicity. Enzymatic hydrolysis seems to be the most advantageous technique in diminishing the allergenic potential of peanut. Furthermore, the combined processing approach (hurdle technologies) such as enzymatic hydrolysis followed by, or in conjunction with, roasting, high pressure and heat, ultrasound with enzymatic treatment, or germination have shown a significant reduction of peanut immunoreactivity and may emerge as useful techniques in reducing the allergenicity of peanut and other foods. This study represents our current knowledge about the alterations in allergenic properties of peanut via different processing mechanisms as well as evaluating its future potential, geographical based data on increasing sensitization, clinical relevance, eliciting dose, and current management of peanut allergy. Furthermore, the molecular characteristics and clinical relevance of peanut allergens have been discussed.     

Molecular characterization of Api g 2, a novel allergenic member of the lipid-transfer protein 1 family from celery stalks

Scope: Celery represents a relevant cross‐reactive food allergen source in the adult population. As the currently known allergens are not typical elicitors of severe symptoms, we aimed to identify and characterize a non‐specific lipid transfer protein (nsLTP). Methods and results: MS and cDNA cloning were applied to obtain the full‐length sequence of a novel allergenic nsLTP from celery stalks. The purified natural molecule consisted of a single isoallergen designated as Api g 2.0101, which was recombinantly produced in Escherichia coli Rosetta‐gami. The natural and recombinant molecules displayed equivalent physicochemical and immunological properties. Circular dichroism revealed a typical α‐helical fold and high thermal stability. Moreover, Api g 2 was highly resistant to simulated gastrointestinal digestion. As assessed by ELISA, thermal denaturation did not affect the IgE binding of Api g 2. Natural and recombinant Api g 2 showed similar allergenic activity in mediator release assays. Api g 2‐specific IgE antibodies cross‐reacted with peach and mugwort pollen nsLTPs. Conclusion: Based on our results, it can be anticipated that inclusion of recombinant Api g 2 in the current panel of allergens for molecule‐based diagnosis will facilitate the evaluation of the clinical relevance of nsLTP sensitization in celery allergy and help clinicians in the management of food allergic patients.     

Towards the development of peanut–wheat flour composite dough: Influence of reduced‐fat peanut flour on bread quality

The effect of partial substitution of wheat flour with reduced‐fat peanut flour at different levels (10, 20, 30, 40, and 50%) on physical parameters, proximate composition, sensory profile, and shelf stability of bread were investigated. Loaf volume, specific volume, and crumb density were significantly (p ≤ .05) reduced with increasing level of substitution with the peanut flour. Peanut flour had significant (p ≤ .05) improvement on the protein content and reduction in carbohydrate content of loaves. Consumers preferred the taste aroma and color of the peanut–wheat flour composite loaves at ≥20% peanut flour inclusion. Freshly baked composite peanut–wheat bread loaves with 10% level of peanut substitution had higher overall acceptability than 100% wheat flour formulation but less microbial stability during storage. Reduced fat‐peanut has potential application for improving the nutritional quality and shelf stability of wheat flour bread.

Practical applications

The demand for convenient alternative to conventional foods is on the increase with the dynamics of the world's social values, lifestyles, and demographic trends. Having peanut incorporated into dough (as one food system) will offer convenience to consumer and therefore add value to bread variety on market shelves. Assessing the influence of the peanut flour on bread quality provides first‐hand information that can facilitate optimization of the baking process toward commercial production of peanut–wheat flour bread.     

False Positive Detection of Peanut Residue in Liquid Caramel Coloring Using Commercial ELISA Kits

Initial food industry testing in our laboratory using enzyme‐linked immunosorbent assay (ELISA) methods indicated that the darkest caramel color (class IV) unexpectedly contained traces of peanut protein, a potential undeclared allergen issue. Caramel production centers on the heating of sugars, often glucose, under controlled heat and chemical processing conditions with other ingredients including ammonia, sulfite, and/or alkali salts. These ingredients should not contain any traces of peanut residue. We sought to determine the reliability of commercially available peanut allergen ELISA methods for detection of apparent peanut residue in caramel coloring. Caramel color samples of classes I, II, III, and IV were obtained from 2 commercial suppliers and tested using 6 commercially available quantitative and qualitative peanut ELISA kits. Five lots of class IV caramel color were spiked with a known concentration of peanut protein from light roasted peanut flour to assess recovery of peanut residue using a spike and recovery protocol with either 15 ppm or 100 ppm peanut protein on a kit‐specific basis. A false positive detection of peanut protein was found in class IV caramel colors with a range of 1.2 to 17.6 parts per million recovered in both spiked and unspiked liquid caramel color samples. ELISA kit spike/recovery results indicate that false negative results might also be obtained if peanut contamination were ever to actually exist in class IV caramel color. Manufacturers of peanut‐free products often test all ingredients for peanut allergen residues using commercial ELISA kits. ELISA methods are not reliable for the detection of peanut in class IV caramel ingredients and their use is not recommended with this matrix.