Heat denaturation of Brazil nut allergen Ber e 1 in relation to food processing

Ber e 1, a major allergen from Brazil nuts, is very stable to in vitro peptic digestion. As heat-induced denaturation may affect protein digestibility, the denaturation behaviour of Ber e 1 was investigated. The denaturation temperature of Ber e 1 varies from approximately 80–110 °C, depending on the pH. Upon heating above its denaturation temperature at pH 7.0, the protein partly forms insoluble aggregates and partly dissociates into its polypeptides, whereas heating at pH 5.0 does neither induce aggregation, nor dissociation of the protein. The denaturation temperature of approximately 110 °C at pH values corresponding to the general pH values of foods (pH 5–7) is very high and is expected to be even higher in Brazil nuts themselves. As a result, it is unlikely that heat processing causes the denaturation of all Ber e 1 present in food products. Consequently, the allergen is assumed to be consumed (mainly) in its native form, having a high stability towards pepsin digestion.     

Germin-like protein Cit s 1 and profilin Cit s 2 are major allergens in orange (Citrus sinensis) fruits

Oranges are clinically relevant allergenic foods. To date, orange allergens have not been characterized in detail. The study is aimed at analyzing the sensitization profile in orange-sensitized subjects with and without clinical allergy, and to identify orange allergens. Fifty-six sensitized subjects with self-reported reactions to orange were grouped into reactors (anaphylaxis or multiple episodes of immediate reactions and/or positive challenge tests) and non-reactors (negative open food challenge tests). Allergens were characterized by IgE immunoblotting, N-terminal sequencing, IgE-inhibition assays, and mediator release assays were performed to determine the allergenic potency of orange profilin. Of 56 subjects, 23 were classified as orange allergic showing mainly an oral allergy syndrome. Of 23 subjects classified as orange allergic, 22 were sensitized to profilin, Cit s 2. In patients with mono-sensitization to profilin in vitro histamine releases up to 75% from basophils were induced using orange extract and purified plant profilins. Of the allergic patients 78% were sensitized to germin-like protein, Cit s 1. Both allergens showed retained IgE reactivity in heat-processed orange juice. Interestingly, subjects with and without clinical allergy showed a comparable sensitization profile. Profilin and germin-like proteins are major orange allergens. The potential clinical relevance of orange profilin was indicated by its strong capacity to release histamine from basophils. However, a predominant sensitization to both allergens in subjects without symptoms also indicates a high frequency of clinically insignificant sensitization.     

巴西坚果过敏原Ber e 1的分离纯化及表征鉴定

以巴西坚果仁为原料,通过粉碎、脱脂、浸提、阴离子交换层析纯化过敏原蛋白Ber e 1;利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、液相色谱-串联质谱联用、Western blot等方法对其进行鉴定,并通过圆二光谱仪和紫外分光光度计表征其二、三级结构。结果表明：纯化获得的巴西坚果过敏原Ber e 1,单轮制备量可达20 mg以上,且纯度大于95%,其蛋白质高级结构未被破坏,能够被巴西坚果过敏患者的血清准确识别。该纯化技术路线简单、对设备要求低且高效,为巴西坚果过敏原Ber e 1的相关研究奠定了一定的基础。     

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.     

Expanded bed adsorption as a fast technique for the large-scale purification of the complete isoform pool of Ber e 1, the major allergen from Brazil nuts

A new, fast, large-scale purification method for Ber e 1, the major allergen from Brazil nuts, using expanded bed adsorption (EBA) chromatography, is presented. Using EBA, crude extracts can be applied to a fluidized column, which allows the unhindered passage of particulate impurities, thereby avoiding time-consuming centrifugation or filtration steps. With this new purification method, 2.8 g of Ber e 1 was obtained from 85 g defatted Brazil nut meal, essentially within 1 day. Various structural as well as immunochemical characteristics of the purified protein were determined, and compared to those of Ber e 1 purified using conventional chromatographic techniques. The complete pool of Ber e 1 isoforms was collected using EBA. The most abundant isoforms were observed to have pI around 8 and heterogeneity was observed in both the large and the small subunit of the heterodimeric protein. Ber e 1 has a highly ordered secondary structure. No apparent differences in immune reactivity were observed between EBA purified Ber e 1 and conventionally purified Ber e 1, using IgE-binding experiments. Thus, using EBA, Ber e 1 can be purified fast and on gram-scale, while having purity equal to that of conventionally purified Ber e 1.     

Development of a real-time PCR method for the simultaneous detection of soya and lupin mitochondrial DNA as markers for the presence of allergens in processed food

Lupin and soya are members of the Leguminosae family which are recognised as some of the richest source of vegetable proteins. Lupin- and soya-containing products are available on the EU market and could cause severe adverse reactions in allergic individuals, even if consumed at low concentrations. In this context the development of methods for reliable detection of these allergens in food products is a useful tool for the surveillance of established legislation on food labelling within the EU. This work described the development of a duplex real-time PCR method allowing the simultaneous detection of traces of lupin and soya in processed food based on a specific TaqMan® probe designed on a mitochondrial tRNA-MET gene. A set of primers and probes was designed for the amplification of a 168 and 175 bp fragment of lupin and soya mitochondrial DNA, respectively. The performance of the method was established using lupin and soya flours and cookies baked from lupin- and soya-containing dough (different concentrations and baking times). The PCR platform yielded consistent and repeatable results. The specificity of the system was tested with DNA from 28 plant species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg per kg range. Both lupin and soya at a level of 2.5 mg per kg food matrix could be detected in cookies baked at 180 °C for 10 min. The method was successfully applied to bakery (e.g. bread) and vegetarian (e.g. non-meat sausages) food products that contain or may contain soya and/or lupin as ingredient or contaminant (according to the declaration on the product label).     

Ring trial validation of single and multiplex real-time PCR methods for the detection and quantification of the allergenic food ingredients sesame,almond, lupine and Brazil nut

The inter-laboratory (=ring-trial) validation of 4 food allergen quantification methods using real-time PCR is described. Three single real-time PCR methods for the specific detection and quantification of sesame, almond and Brazil nut were used. Additionally, a multiplex real-time PCR method combining the detection of sesame, almond, Brazil nut and lupine was tested in parallel. Matrix based calibrants (rice cookies) spiked (=incurred) with defined amounts of sesame, almond, lupine and Brazil nut were applied for quantitative evaluation. Cookies based upon wheat and rice flour as well as sauce hollandaise powder each incurred with these allergenic ingredients in the range of 10–123 milligram per kilogram were used as ring-trial samples. The lowest spike level of 10 mg/kg could reproducibly be detected by 6 of 7 PCR systems. In quantitative evaluation of the results, reproducibility standard deviations of approximately 50 % and below were obtained. In addition, the effect of the food matrix on allergen quantification was examined. The range of “recoveries” over all matrices and methods was from 43 to 109 %.     

Rapid method for the determination of meat in food products

A method for the determination of meat in food products is described based on the determination of protein-bound 3-methylhistidine. The method involved high pressure liquid chromatography and reduced the elution time of previous methods from 7 h to 10 min. The accuracy of the method was increased by using electronic integration. The method was used to determine the meat content of nine meat pies and three hamburger patties.     

检测食品中大肠杆菌O157的多重荧光PCR方法的建立

针对大肠杆菌O157的志贺毒素编码基因stx1、stx2和O157血清型的标志基因wzy的保守序列,设计特异性的引物和探针,反复优化反应体系和条件,建立了一种新的多重荧光PCR检验方法.结果显示,该方法灵敏度高,stx1、stx2的扩增效率分别为96.4%和94.6%,实际样品检测时stx1、six2的最低检出限分别为4.2×102cfu/mL和4.2×103cfu/mL.该方法特异性强,扩增结果与各参考菌株基因型一致,能良好的区分出O157菌株和非O157犁大肠杆菌.该方法能用于食品样品的检测和流行病学分离株的快速鉴定.     

Real-time PCR multiplex method for the quantification of Roundup Ready soybean in raw material and processed food

This paper describes a quantitative real-time multiplex PCR method optimised for the ABI PRISM^® 7700 Sequence Detection System (SDS) and TaqMan^® chemistry for Roundup Ready^® Soybean (RRS) in raw material and processed food. This method has the advantage of performing the amplification of the target taxon lectin gene and the genetically modified (GM) target sequence in the same test tube. The quantification is based on a calibration curve obtained with the DNA extracted from Certified Reference Material standards (CRM IRMM-410: R) in the range 0.1–5% RRS. The method was validated in-house by using CRMs and the applicability was verified on three mixtures of soybean flour at 1, 5, 10% of RRS respectively and on prepared baked products (biscuits and plum cakes). The statistical parameters of the method were found to be satisfactory both for soybean flour and baked products. In the process the absolute LOD and LOQ was 7.1 and 35.5 copy number respectively; the relative LOD and LOQ was 0.03 and 0.01% of RRS respectively.     

An enzymatic method for the determination of fructans in foods and food products

 We report a new and non-equipment demanding method of measuring the content of fructans as well as the contents of free glucose, free fructose and sucrose in foods and food products enzymatically. This method comprises hydrolysis of fructans into d-glucose and d-fructose enzymatically and measurement of the released sugars enzymatically. Sucrose is hydrolysed by α-glucosidase instead of β-fructosidase, which is normally used. In addition, sucrose is measured in the form of d-fructose instead of the typical d-glucose form, and the fructanase used to hydrolyse the fructans has fewer side effects than the fructanase reported as normally used. The method is tested on ten standard substances and five fructan products, and nine foods and food products are also analysed. The enzymatic measurement of the released sugars is confirmed by measurements done by high performance anion exchange chromatography with pulsed amperemetric detection. Received: 1 March 1999 / Revised version: 23 April 1999     

Nuclear DNA barcodes for cod identification in mildly-treated and processed food products

Gadoids are a group of fish with historical importance in the fishing industry. The high demand for cod is one of the reasons why cod products are often mislabelled, and numerous observations have been made on the replacement of Atlantic cod (Gadus morhua) by cheaper species or its illegal capture in contravention of fish quotas. Fish species identification is traditionally based on morphological features, but this may be difficult in case of heat-treated or processed products, or where the species look similar, as in the Gadoid group. DNA-based approaches (using either nuclear or mitochondrial DNA) are most commonly used in this case, due to their high specificity and to the high resilience of the target molecules to food processing techniques. In this article, we identified, using an automated screening approach, novel barcode regions and their associated primers in the nuclear genome, to be used for the efficient identification of Gadoids. The barcode regions were tested on official and commercial samples, raw or mildly treated products, like frozen, or salted, as well as pre-cooked complex mixtures and processed samples, using next-generation sequencing (NGS) technique. The method proposed could complement existing fish identification strategies in establishing an efficient framework to detect and prevent frauds along the food chain.     

Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products

The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg^?1 for sesame and 1.4 mg kg^?1 for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.     

食物过敏原及检测技术的研究进展

食物过敏是当前食品安全领域较为突出的问题,综述食品过敏原、过敏的免疫学发生机制及检测技术等方面的研究现状及发展展望,涉及致敏的食物种类及相应过敏原,过敏的免疫学分子发生机理,并对食物过敏原现存的检测技术做了相关的介绍。     

食品中亚硫酸盐的毒性和检测方法综述

亚硫酸盐作为一类食品添加剂,具有漂白、防腐、抗氧化、抑制细菌生长等作用,被广泛应用于食品加工中。然而摄入过量的亚硫酸盐会对人体造成全身性的危害。如何控制食品中亚硫酸盐含量成为食品安全面临的一大难题。为了深刻地认识亚硫酸盐对人体的危害,解决食品中二氧化硫残留量超标问题,本论文对食品中的亚硫酸盐进行综述,介绍了食品中亚硫酸盐的存在形式、作用、限量标准及现状,重点阐述了食品中亚硫酸盐对人体的毒害作用,列举了目前国内外食品中亚硫酸盐检测方法,为食品中亚硫酸盐控制提供一定的参考与借鉴。     

Development of sensitive and specific multiplex PCR method for the detection of microcystin producing cyanobacteria in spirulina food supplements

Food Science and Biotechnology - Spirulina has emerged as the next-generation dietary supplement owing to its health benefits. Despite the advantages, there have been reports of contamination by...     

A multiplex PCR method for the identification of commercially important salmon and trout species (Oncorhynchus and Salmo) in North America

Abstract: The purpose of this study was to develop a species-specific multiplex polymerase chain reaction (PCR) method that allows for the detection of salmon species substitution on the commercial market. Species-specific primers and TaqMan® probes were developed based on a comprehensive collection of mitochondrial 5′ cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) “barcode” sequences. Primers and probes were combined into multiplex assays and tested for specificity against 112 reference samples representing 25 species. Sensitivity and linearity tests were conducted using 10-fold serial dilutions of target DNA (single-species samples) and DNA admixtures containing the target species at levels of 10%, 1.0%, and 0.1% mixed with a secondary species. The specificity tests showed positive signals for the target DNA in both real-time and conventional PCR systems. Nonspecific amplification in both systems was minimal; however, false positives were detected at low levels (1.2% to 8.3%) in conventional PCR. Detection levels were similar for admixtures and single-species samples based on a 30 PCR cycle cut-off, with limits of 0.25 to 2.5 ng (1% to 10%) in conventional PCR and 0.05 to 5.0 ng (0.1% to 10%) in real-time PCR. A small-scale test with food samples showed promising results, with species identification possible even in heavily processed food items. Overall, this study presents a rapid, specific, and sensitive method for salmon species identification that can be applied to mixed-species and heavily processed samples in either conventional or real-time PCR formats. Practical Application: This study provides a newly developed method for salmon and trout species identification that will assist both industry and regulatory agencies in the detection and prevention of species substitution. This multiplex PCR method allows for rapid, high-throughput species identification even in heavily processed and mixed-species samples. An inter-laboratory study is currently being carried out to assess the ability of this method to identify species in a variety of commercial salmon and trout products.     

Two tetraplex real-time PCR for the detection and quantification of DNA from eight allergens in food

According to the EU and Swiss legislation, food has to be labelled for allergens to enable allergic consumers to avoid such food and its products. To provide efficient and reliable methods, two novel quantitative multiplex real-time polymerase chain reaction systems were developed and validated. They simultaneously determine DNA of peanut, hazelnut, celery, soy, egg, milk, almond and sesame, respectively. The tests exhibit good specificity and sensitivity in the range of 0.01%. Due to low DNA amounts, lower sensitivities for egg and milk were obtained. First comparisons of ELISA results with PCR results suggest a qualitative accordance, but a low correlation of quantitative results.     

Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food

A very sensitive and new real-time multiplex PCR method for the quantification of genetically modified (GM) maize crops in food materials was developed and validated for an ABI Prism 7700 Sequence Detection System. In the assay described, fluorescence-labelled TaqMan probes were chosen to detect the amplified DNA fragments during PCR. In this multiplex approach, maize-specific DNA (zein) and 35S-CaMV promoter-specific DNA fragments are amplified in the same tube. The method was tested for the detection and quantification of the four maize events that are approved in Europe and contain the 35S-CaMV promoter: Bt11, Bt176, Mon810 and T25 maize. Quantification was based on a standard curve prepared from certified maize flour reference material prepared by the Institute for Reference Materials and Measurements. Quantification within the range of the standard curve (0.05-1% GM maize) and up to 100% was possible. Repeatability of the method for each GM maize event was determined; coefficients of variations ranged from 28-40%. In addition, three internal Nestlé laboratories successfully applied this method and comparable results were obtained.