Vicilin and the basic subunit of legumin are putative chickpea allergens

IgE-mediated reactions to food allergens constitute a major health problem in industrialized countries. Chickpea is consumed in Mediterranean countries, and reportedly associated with IgE-mediated hypersensitivity reactions. However, the nature of allergic reactions to chickpea has not been characterized.     

A food matrix reduces digestion and absorption of food allergens in vivo

Scope: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross‐reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens. Methods and results: Mal d 1 lost its IgE‐reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE‐binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein‐rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo‐2 monolayers. Finally, IgE‐reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding. Conclusions: A protein‐rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.     

Validation and comparison of a sandwich ELISA,two competitive ELISAs and a real-time PCR method for the detection of lupine in food

Methods applied in food allergen analysis should be specific, sensitive and applicable to both raw and highly processed foods. The performance of the most commonly used methods, ELISA and real-time PCR, may, however, be influenced by food processing steps, e.g., heat treatment. The present study compares the applicability of four in-house developed methods, one sandwich ELISA, two competitive ELISAs and a real-time PCR method, for the detection of lupine in four different food matrices, comprising bread, biscuits, rice patties and noodles. In order to investigate the influence of food processing on the detectability, not only the heat treated model foods but also the corresponding doughs were analysed. The sandwich ELISA proved to be the most sensitive method. The LOD was found to be 10 ppm lupine, independent from the food matrix and independent if the dough or the heat treated food was analysed. In addition, the methods were applied to the analysis of commercial foodstuffs differing in their labelling.     

Commercial soybean lecithins: a source of hidden allergens?

 Soybeans are known to be allergenic for adults as well as for infants. Processed products derived from soybeans are used in a wide spectrum of foods, drugs and other industrial products. In particular, soybean lecithins are used as stabilizers and emulsifiers and may not be suspected as possible source of allergens. To test this hypothesis, six commercial soy lecithins were investigated for residual allergenicity and compared with extracts from raw and heat-treated soybeans. They were characterized, the protein content was determined by enzyme-linked immunosorbent assay (ELISA) and allergens were analyzed with specific IgE from patients' sera using the enzyme allergosorbent test (EAST), EAST inhibition and protein blotting followed by immunodetection. For further characterization a polyclonal antiserum directed against soybean extract and a monoclonal antibody (mAb 025) directed against the acidic subunit of the soybean storage protein glycinin were used. The EAST studies revealed that three of six sera from patients with allergy to soybeans contained IgE to four soy lecithins (Topcithin 50, Topcithin 300, Emulfluid FD 12, Epikuron 100 P), the same lecithins which were found to contain residual proteins. Two lecithins with a protein content of less than 20 ppb did not bind IgE. EAST inhibition showed that the allergens from soy lecithin were immunologically more closely related to allergens from heat-treated soybeans than to those from raw soybeans. Protein blotting and immunodetection of the protein extract from the lecithins resulted in various allergen bands between 14 kDa and 94 kDa. A heat-stable allergen of 39 kDa was recognized by the monoclonal antibody and thus identified as a subunit of glycinin. The results obtained were confirmed by a mediator release assay based on a rat basophil leukemia cell line. Lecithins that contained residual proteins caused a specific mediator release, suggesting that these products may induce allergic symptoms. Our results show that soybean lecithins are capable of introducing hidden allergens to processed foods and that the IgE binding potential corresponds to the total protein determined by ELISA. Furthermore, it appears to be possible that by monitoring the protein content soy lecithins can be applied which may be safe for the allergic consumer. Received: 22 January 1998     

A simple procedure of lupin seed protein fractionation for selective food applications

A simple procedure aimed at fractionating the main proteins of lupin seeds is proposed. The protocol consisted of alkali extraction of all proteins and isoelectric precipitation of two classical storage globulins, namely conglutins and , and one albumin, conglutin . The supernatant of this separation contained conglutin , the other main lupin protein. Conglutin could easily be purified further by selective Zn²⁺ precipitation, leading to a more than eightfold enrichment with respect to the amount in the flour. Resuspension of the isoelectric precipitate in saline water/ethanol solution allowed us to isolate conglutin , a 2S sulphur-rich lupin protein, at a very high purity level. Conglutins and could not be separated further by simple procedures. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis analysis confirmed the identities of the isolated protein fractions. The distribution of each separated protein type is close to that of the corresponding proteins in the seed. The results are discussed in view of the applicability of the method and the potential use of each protein type as a food ingredient.     

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.     

Agro-industrial potential of exotic fruit byproducts as a source of food additives

Exotic fruit consumption and processing is increasing worldwide due to the improvement in preservation techniques, transportation, marketing systems and consumer awareness of health benefits. The entire body of tropical exotic fruits is rich in bioactive compounds, such as phenolic constituents, carotenoids, vitamins and dietary fiber. However, the fruit processing industry deals with the large percentage of byproducts, such as peels, seeds and unused flesh, generated in the different steps of the processing chains. In most cases, the wasted byproducts can present similar or even higher contents of bioactive compounds than the final produce does. The aim of this review is to promote the production and processing of exotic fruits highlighting the possibility of the integral exploitation of byproducts rich in bioactive compounds. Amongst the possible uses for these compounds that can be found in the food industry are as antioxidants (avoiding browning and lipid oxidation and as functional food ingredients), antimicrobials, flavoring, colorants and texturizer additives. Finally, the importance of extraction techniques of bioactive compounds designated as food additives is also included.     

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.     

食品过敏原生物传感检测技术研究进展

食品过敏原已成为全球性的食品安全隐患。应对食品过敏反应最直接、有效的方法就是避免食用和接触各种含有致敏成分的食品。因此,快速和灵敏地检测食品中过敏原对预防和控制食品过敏反应是十分重要的。食品过敏原检测主要可以分为基于核酸和蛋白质的两大类检测技术。与基于聚合酶链式反应技术、液相色谱-串联质谱法的传统检测手段相比,生物传感技术检测平台因具有操作简单、高通量、灵敏度高、现场便携等优点,在食品过敏原快速检测领域备受关注。本文综述了近年来食品过敏原的生物传感检测技术研究进展,包括电化学传感器、光学传感器和其他新型传感器及其在食品过敏原检测中的应用,并展望了生物传感技术在食品过敏原分析中面临的挑战和发展趋势。     

矿物质对食物过敏原结构和稳定性的影响

蛋白质是引发食物过敏的一类重要物质,食物过敏原与矿物质的结合会影响其结构和功能的改变。本文阐述了食物中矿物质与过敏原结合的影响因素和矿物质对过敏原结构与稳定性的影响,以及不同条件下金属离子对几种过敏原的影响。通过分析其结构和致敏性的关联变化,为一种既能丰富营养素又能降低过敏原致敏性的方法提供理论基础,以用于指导低致敏食物的研发。      

Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.     

质谱技术在食物过敏原检测中的研究进展

食物过敏是当前食品安全领域比较突出的问题,相应的过敏原检测方法研究也越来越受重视。相比于传统的免疫学检测方法,质谱技术在检测加工后以及复杂基质中的食物过敏原中,具有高通量和高灵敏性等优点,被广泛应用于食物过敏原的检测。本文主要从定性和定量2方面介绍了质谱技术在食品过敏原检测中的研究进展。相关研究对牛乳、鸡蛋、小麦和榛子(坚果类)等主要食物过敏原均有涉及。在定性研究中,以肽质量指纹图谱法和肽碎片离子鉴定法为主,鉴定食物中的过敏原蛋白。在定量研究中,通过标记/无标记技术,也能够实现对微量的目标蛋白进行相对/绝对定量。质谱技术应用于食物过敏原检测中,将有助于提升过敏原检测能力,降低食物过敏安全风险。     

Milk allergens, their characteristics and their detection in food: A review

Cow's milk allergy (CMA) is one of the most common food allergies in childhood. This allergy is normally outgrown in the first year of life, however 15% of allergic children remain allergic. Many studies have been carried out to define and characterise the allergens involved in CMA and described two major allergens: casein (αs1-CN) and β-lactoglobulin. In addition to this, many other milk proteins are antigenic and capable of inducing immune responses. Milk from sheep or goats differs from cow's milk (CM) in terms of composition and allergenic properties. Food processing such as heating affects the stability, structure and intermolecular interactions of CM proteins, thereby changing the allergenic capacity. Chemical and proteolytic treatments of milk to obtain milk hydrolysates have been developed to reduce allergic reactions. Prevention of CMA largely relies on avoidance of all food products containing cow's milk. To achieve this, interest has focused on the development of various technologies for detecting and measuring the presence of milk allergens in food products by immunoassays or proteomic approaches. This review describes the technologies implemented for the analysis of milk allergens (allergenicity, biochemistry) as well as their potential detection in food matrices.     

食品中过敏原及其检测方法的研究进展

近年来食物过敏作为食品安全的热点问题在全球引起了广泛关注。目前食物过敏仍无有效的根治方法,严格避免摄入致敏食品仍是过敏性疾病防治的最有效方式。因此,食品中过敏原的识别鉴定、性质分析及检测方法的研究至关重要。本文针对八大类过敏食物,从分子结构、免疫学特性等方面介绍了常见食物过敏原的研究进展;归纳了现阶段用于食品中过敏原分析的常规检测方法,以及在此基础上发展而来的新兴检测丰富,并比较分析了不同检测技术方法的优缺点。相比于植物源性过敏原,动物源性过敏原的相关研究有待深入,尤其是蛋白家族的确定及空间结构解析等是未来动物源性过敏原研究的重要方向;目前食品中常见过敏原的常规检测方法及新兴检测方法依然存在检测效率和准确性不高、成本昂贵等局限性,建立高效、准确、低成本的过敏原检测方法仍是该研究领域发展的重要趋势。     

Influence of food processing on the immunochemical stability of celery allergens

Celery roots were processed by microwave heating, cooking, drying, γ-irradiation, ultra high pressure treatment and high voltage impulse treatment. The immunochemical stabilities of the three known allergenic structures of celery were tested with sera from patients who were sensitised to celery. In addition, rabbit antisera were used to detect the allergens profilin and Api g 1 on celery immunoblots. The specificity and reactivity of IgE from the patients' sera were investigated by immunoblotting, by an enzyme allergosorbent test (EAST) and by dose-related IgE inhibition experiments. The results of all three methods agreed closely and indicated high antigenic and allergenic activity in native celery which was reduced by thermal processing. The heat-stability of the known celery allergens decreased in the following order: carbohydrate epitopes> profilin>Api g 1. In contrast, the allergenicity was only mildly reduced by non-thermal processing. The results obtained with human IgE were confirmed by an in vitro mediator-release assay that is based on rat basophil leukemia cells (RBL cells) which were passively sensitised with celery-specific murine IgE. With sera from mice that had been immunised with native celery, the native sample and non-thermal celery preparations elicited the strongest mediator release, whereas a weak response was obtained with samples from heat-processed celery. These results agreed closely with the data obtained in allergic patients whose IgE antibodies were directed against the major protein allergen Api g 1. Our results may be helpful in risk assessment and in selecting food preparations which can be consumed without symptoms by a subgroup of celery-allergic patients with a known sensitisation pattern. ©1997 SCI     

时间分辨免疫荧光法测定食物中花生过敏原成分

目的:建立双抗体夹心时间分辨荧光免疫法[Time-resolved Fluoroimmunoassay(TRFIA)]测定食物中花生过敏原蛋白成分,为进出口食品中花生过敏原成分检测和由花生导致的食物过敏性疾病的预防提供技术基础。方法:提取花生蛋白,免疫小鼠制备抗花生总蛋白的多克隆抗体,用该抗体包被酶标板,并用生物素标记该多抗作为捕捉抗体,Eu3+标记的链酶亲和素和以β-二酮体为主的增强液等试剂,建立双抗夹心TRFIA方法,检测该方法的灵敏度,同时用于13种食品中花生过敏原蛋白成分检测。结果:初步地研制出双抗体夹心TRFIA法检测食品中花生过敏原蛋白成分,具有特异性,和其最低检出限为0.1ng/mL,标准曲线在0.1～160ng/mL范围内线性良好;11种食品检测结果与食物过敏原标签标注内容相符,而2种标示不详的食品检测结果均呈现阳性。     

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。     

Optical thin-film biochips for multiplex detection of eight allergens in food

An innovative method for the rapid detection of food allergens is developed and validated. Here we reported the development of silicon-based optical thin-film biochip technology that simultaneously permits visible detection of eight food allergens including celery, almond, oat, sesame, mustard, lupine, walnut and hazelnut on the basis of two tetraplex PCR systems. The biochip detection time was about 30 min after PCR amplification. Briefly, the optical thin-film biochip detects the presence of PCR fragment targets by enzymatically converting the formation of nucleic acid hybrids to molecular thin films. The mass contributed by the thin film alters the interference pattern of light on the biochip surface, resulting in a visible color change on the chip surface. Therefore, this assay permits sensitive, specific and high-throughput detection of allergens in food samples.