大豆过敏原的检测方法研究进展

大豆是八大食物过敏原之一,如何快速、准确、低成本地检测大豆过敏原已成为科研、食品工业以及医药卫生等行业关注的焦点。大豆中的主要过敏原是大豆球蛋白、β-conglycinin、Gly m Bd 60K、Gly m Bd 30K和Gly mBd 28K。其中,大豆过敏原的主要检测方法有电泳法、免疫学方法、PCR法、色谱法、质谱法以及生物芯片技术。本文通过对大豆中主要过敏原的检测方法进行综述,旨在为不同食物中大豆过敏原的检测提供方向,以保障大豆过敏患者安全。     

大豆主要过敏原及其脱敏方法的研究进展

大豆是引起食物过敏最常见的过敏原食物之一,如何降低大豆的致敏性,保证大豆食品的安全,已成为食品安全领域的一项重要课题。大豆中的过敏原蛋白很多,大致可分为种子储藏蛋白、结构蛋白和防御相关蛋白。其中7S球蛋白组分中的Gly m Bd 30 K和Gly m Bd 28 K,β-伴大豆球蛋白中的Gly m Bd 60K是3种主要的过敏原。近几年来,以热加工法、酶处理法、超高压法和基因工程法等为代表的大豆脱敏技术研究取得了很多新进展。其中,热加工法与酶处理法已经在食品工业中广泛应用。超高压法作为一种新的大豆脱敏技术,由于它对大豆的营养价值和风味影响较小,越来越受到食品工业的关注,具有潜在的应用前景。基因工程法则是食品脱敏的新技术,它可以消除食物原料过敏原性,但基因食品的安全性仍饱受争议,能否实际应用于脱敏仍待进一步研究。     

基于生物技术控制大豆过敏原的研究进展

大豆富含优质的蛋白质,具有很高的营养价值;同时,大豆也是公认的八大主要过敏食物之一。大豆中的主要过敏原包括大豆球蛋白、β-伴大豆球蛋白以及7S球蛋白组分中两个低丰度蛋白Gly m Bd 28K和Gly m Bd 30K等。迄今为止,生物育种、酶法改性和微生物发酵等生物技术是控制大豆过敏原致敏性的重要手段,同时这些方法还能改善大豆及其制品的功能特性。其中,生物育种是一种从根源上去除大豆过敏原蛋白的方法;酶法改性操作简便、条件温和,具有非常广泛的研究前景;而微生物发酵则是一种较为成熟的生产脱敏大豆蛋白制品的方法。     

处理方法对大豆致敏蛋白的影响

Gly m Bd 30K、Gly m Bd28K和β-伴大豆球蛋白的α亚基(MW68K)是大豆中的主要致敏蛋白，本文对经过不同处理的大豆进行SDS-PAGE电泳，结果显示，结合盐析和pH值处理可以有效去除Gly m Bd 30K和Glym Bd28K，但要同时去除三种致敏蛋白还需进一步研究。     

牛乳αS1-酪蛋白与大豆蛋白交叉过敏原的识别鉴定

为识别牛乳αS1-酪蛋白和大豆蛋白的交叉过敏原,鉴定其致敏特性,揭示交叉过敏机理,首先以牛乳过敏患者血清、αS1-酪蛋白小鼠单克隆抗体为探针,通过免疫印迹方法识别大豆蛋白交叉过敏原,然后通过生物信息学工具比对了交叉过敏原与αS1-酪蛋白的氨基酸序列相似性,进而通过体外消化实验和加热实验探究交叉过敏原的稳定性。结果表明：牛乳αS1-酪蛋白和大豆蛋白的交叉过敏原为β-伴大豆球蛋白α亚基（Gly m Bd 60K）,十二烷基硫酸钠-聚丙烯酰氨凝胶电泳结果表明Gly m Bd 60K在2 min消化完全,并且加热对过敏原的交叉反应性没有影响。     

大豆过敏原蛋白及致敏性消减技术的研究进展

大豆是我国重要的粮食作物之一,其蛋白质含量高达35%~40%（m/m）。与此同时,大豆蛋白是人们日常生活中最常见的一类食物过敏原,大豆过敏已经成为了急需解决的公共安全问题。β-伴大豆球蛋白（β-Conglycinin,7S）、大豆球蛋白（Glycinin,11S）、Gly m Bd 28K和Gly m Bd 30K（P34）被认为是大豆过敏原中引发机体发生过敏反应的主要成分。迄今为止,国内外对于大豆过敏尚无根治办法,唯一的预防策略是严格避免摄入来防止过敏反应的发生。但研究指出,通过特殊的加工方法或技术手段可以降低大豆过敏原的致敏性,其中以热加工法、超高压法、酶处理法和基因工程法等方法为代表的消减技术得到广泛关注。因此,该文综述了大豆过敏原的类型,常用的过敏蛋白致敏性消减技术及各项技术的优缺点,以期为低敏性大豆食品的开发提供参考。     

大豆主要过敏原Gly m Bd 28K的分离纯化及多抗血清的制备

该文从脱脂大豆粉中分离纯化28K(Gly m Bd 28K)蛋白,并制备多抗血清。方法用碱溶酸提法提取28K蛋白,盐析法制得28K粗蛋白,DEAE–Sepharose CL–6B层析纯化28K蛋白,SDS–聚丙烯酰胺凝胶电泳检测蛋白的纯度,制备兔抗血清,用间接ELISA法检测效价。该实验纯化出28K蛋白,制备了抗28K的多抗血清,效价为1∶51200。为研究大豆主要过敏原蛋白28K的单克隆抗体制备及28K的Ig G结合表位的确定奠定了基础。     

致敏大豆蛋白P34及其清除方法的研究进展

大豆作为"八大"过敏食品之一,其过敏患者数量约占食物过敏总人数的25%。如何降低大豆产品的致敏性,保证消费者的安全,已经成为食品安全领域一个热议的话题。大豆中含有多种致敏因子,如Gly-m Bd 30K蛋白、Gly-m Bd 28K蛋白和Gly-m Bd 28K蛋白等。其中Gly-m Bd 30K蛋白作为致敏性最强的过敏原,能被65%的大豆过敏患者血清识别,在几种过敏蛋白中占着较突出的地位。本文综述了Gly-m Bd 30K蛋白的抗原表位、去除方法的研究现状。其中,酶法处理、超高压法、挤压膨化法等降低大豆致敏性的研究已取得了一定的成果,这为进一步提高大豆及其制品的品质质量,保证其在食品工业和畜牧业方面的安全利用提供了科学参考。     

β-伴大豆球蛋白多克隆抗体的制备及纯化前后免疫学特性鉴定

以β-伴大豆球蛋白免疫新西兰兔,制备抗β-伴大豆球蛋白多克隆抗体。利用正辛酸-硫酸铵沉淀法纯化多抗血清,并分析纯化前后多抗血清蛋白含量、纯度、效价、敏感性及特异性。结果表明,经聚丙烯酰胺凝胶电泳分析纯化后血清纯度得到提高;间接ELISA法检测多抗血清效价及敏感性无明显变化;与大豆Gly m Bd 28K蛋白和花生蛋白的交叉反应率分别由25.28%、1.16%降至为0.79%、0.2%。制得的抗β-伴大豆球蛋白多克隆抗体特异性高。为β-伴大豆球蛋白致敏蛋白的检测,和大豆β-伴大豆球蛋白致敏亚分子结构的定位奠定了基础。     

热处理对大豆油体表面的油体蛋白和外源性蛋白影响

研究了热处理不同p H生豆浆和吸胀大豆种子对大豆油体上油体蛋白和外源性蛋白的影响,并通过SDS-聚丙烯酰胺凝胶电泳表征油体蛋白和外源性蛋白的变化。研究表明:热处理生豆浆和吸胀的大豆种子时,随着温度和时间的增加,都可以抑制内切蛋白酶P34的活性,有利于油体蛋白保留在油体上;而且热处理对吸胀的大豆种子效果更显著。热处理不同p H的生豆浆时,随着热处理强度和生豆浆p H的增加,外源性蛋白(主要是大豆球蛋白、伴大豆球蛋白和过敏性蛋白Bd 30K)会从油体上逐渐解离下来。在80℃、p H8.5下处理生豆浆5~30min时,油体纯度高达97%,且含有的过敏性蛋白Bd 30K低于1%。     

Reliable identification of grapevine rootstock varieties using RAPD PCR on woody samples

Since grapevine ( Vitis spp .) rootstock material is being traded increasingly as disbudded woody material a lack of distinctive morphological features on such material necessitates an alternative and reliable means of identification. Methods described here were developed for rapid and efficient extraction of DNA from woody samples rich in phenolic compounds and polysaccharides, and for subsequent identification of varieties by RAPD PCR. Using these methods, and with the application of only one selected RAPD primer, we were able to differentiate sixteen rootstock varieties, including the seven varieties most commonly used in Germany. Problems commonly encountered with reproducibility of RAPD patterns were avoided by choosing primers with a dinucleotide sequence and a high G/C content that allowed a rather high annealing temperature of 45°C. Methods described here should also be useful for other horticultural crops, especially those with woody tissues rich in phenolic compounds and polysaccharides.     

An internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics

An internet website (http://cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-ofcharge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.     

Aroma characterization of various apricot varieties using headspace–solid phase microextraction combined with gas chromatography–mass spectrometry and gas chromatography–olfactometry

The characterization of the aromatic profile of several apricot cultivars with molecular tracers in order to obtain objective data concerning the aromatic quality of this fruit was undertaken using headspace–solid phase microextraction (HS–SPME). Six apricot cultivars were selected according to their organoleptic characteristics: Iranien, Orangered, Goldrich, Hargrand, Rouge du Roussillon and A4025. The aromatic intensity of these varieties measured by HS–SPME–Olfactometry were defined and classified according to the presence and the intensity of grassy, fruity and apricot like notes. In the six varieties, 23 common volatile compounds were identified by HS–SPME–GC–MS. Finally, 10 compounds, ethyl acetate, hexyl acetate, limonene, β-cyclocitral, γ-decalactone, 6-methyl-5-hepten-2-one, linalool, β-ionone, menthone and (E)-hexen-2-al were recognized by HS–SPME–GC–O as responsible of the aromatic notes involved in apricot aroma and considered as molecular tracers of apricot aromatic quality which could be utilized to discriminate apricot varieties.     

Tests of potential functional barriers for laminated multilayer food packages. Part I: Low molecular weight permeants

The advent of the functional barrier concept in food packaging has brought with it a requirement for fast tests of permeation through potential barrier materials. In such tests it would be convenient for both foodstuffs and materials below the functional barrier (sub-barrier materials) to be represented by standard simulants. By means of inverse gas chromatography, liquid paraffin spiked with appropriate permeants was considered as a potential simulant of sub-barrier materials based on polypropylene (PP) or similar polyolefins. Experiments were performed to characterize the kinetics of the permeation of low molecular weight model permeants (octene, toluene and isopropanol) from liquid paraffin, through a surrogate potential functional barrier (25 μm-thick oriented PP) into the food simulants olive oil and 3% (w/v) acetic acid. These permeation results were interpreted in terms of three permeation kinetic models regarding the solubility of a particular model permeant in the post-barrier medium (i.e. the food simulant). The results obtained justify the development and evaluation of liquid sub-barrier simulants that would allow flexible yet rigorous testing of new laminated multilayer packaging materials.     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Occurrence of bisphenol-F-diglycidyl ether (BFDGE) in fish canned in oil

The levels of bisphenol-F-diglycidyl ether (BFDGE) were quantified as part of a European survey on the migration of residues of epoxy resins into oil from canned fish. The contents of BFDGE in cans, lids and fish collected from all 15 Member States of the European Union and Switzerland were analysed in 382 samples. Cans and lids were separately extracted with acetonitrile. The extraction from fish was carried out with hexane followed by re-extraction with acetonitrile. The analysis was performed by reverse phase HPL C with fluorescence detection. BFDGE could be detected in 12% of the fish, 24% of the cans and 18% of the lids. Only 3% of the fish contained BFDGE in concentrations considerably above 1mg/kg. In addition to the presented data, a comparison was made with the levels of BADGE (bisphenol-A-diglycidyl ether)analysed in the same products in the context of a previous study.     

Analysis of benzo[a]pyrene in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples

A simple, rapid and inexpensive method has been developed for the determination of benzo[a     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.     

Contribution of European research to risk analysis

The European Commission's, Quality of Life Research Programme, Key Action 1—Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches; harmonization of risk assessment principles, methodologies, and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries; to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women; evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.