A novel extraction method for peanut allergenic proteins in chocolate and their detection by a liposome-based lateral flow assay

In this study, conditions for extracting the major peanut allergen (Ara h1) from chocolate were optimized, and the extracted samples were analyzed by a lateral flow assay (LFA) using liposomal nanovesicles. The optimal conditions using peanut-spiked chocolate were found to be extraction with a mixture of phosphate buffered saline and hexane for 30 min at 35 °C. After centrifugation, the buffer portion was treated with insoluble poly(vinylpolypyrrolidone) to remove phenolic compounds, and then analyzed by the LFA. The entire analysis, including sample preparation and LFA, could be easily completed within 2 h, and the detection limit was 158 g of peanuts/g of chocolate.     

A two-site monoclonal antibody immunochromatography assay for rapid detection of peanut allergen Ara h1 in Chinese imported and exported foods

Food allergen labeling has not yet been implemented in China. Therefore, a gold immunochromatography assay (GICA) was developed using two monoclonal antibodies (mAb) against the peanut allergen Ara h1. The GICA was specific for standard peanut samples with a sensitivity of 10 ng/ml. Peanut protein traces extracted from 124 food products imported and exported by China Customs were easily and rapidly detected by GICA. 68 food samples originally labeled as containing peanuts were positive for Ara h1 and 54 food samples labeled as not containing peanuts were negative for Ara h1, indicating that the labels from the manufacturers were accurate. However, 2 food samples labeled as not containing peanuts tested positive for Ara h1. The present GICA provides a fast, simple, semi-quantitative method for the determination of peanut allergens in foods. This detection system can be used to ensure the safety of food imported and exported by China Customs.     

Enzymatic treatment of peanut butter to reduce the concentration of major peanut allergens

This study investigated the effects of enzymatic treatment of peanut butter on two‐major peanut allergens (Ara h 1 and Ara h 2). Home‐made and commercial peanut butter samples were treated with alpha‐chymotrypsin, trypsin or the combination of these enzymes and incubated at room temperature for 24 h or at 37 °C for 3 h. Treated peanut butter samples were sampled weekly for evaluation of total soluble proteins and extractable Ara h 1/Ara h 2. Data show that 1:1 alpha‐chymotrypsin: trypsin at 0.04% of enzyme‐to‐peanut butter ratio resulted in near complete reduction of extractable Ara h 1 and Ara h 2 respectively. Treatment of peanut butter with a combination of trypsin and alpha‐chymotrypsin resulted in a decrease in IgE‐binding, suggesting that enzymatic treatment has the potential to reduce the allergenicity. However, clinical tests are needed to confirm any reduction in allergenic potential. The amount of water used to disperse enzyme did not have significant effect on allergen reduction but affected the consistency and colour of treated products, especially when the amount of water added was above 5% of peanut butter weight.     

Investigation on sequential extraction of peanut allergens for subsequent analysis by ELISA and 2D gel electrophoresis

A two-step sequential extraction method of peanut proteins was proposed with the aim to investigate the protein composition and allergen content of peanut samples. The extraction procedure reported is fully compatible with subsequent analysis by enzyme-linked immunosorbent assays (ELISA) as well as 2D gel electrophoresis (2D PAGE). This sequential extraction method was used to study three different peanut varieties and three different types of food processing. Peanuts were analysed for total protein content and the extraction efficiency of raw and processed peanuts was determined. The total protein content of the three peanut varieties was found to be comparable, but their extraction efficiency varies. The peanut extracts were characterised by employing three different ELISA test kits specific to either the allergens Ara h 1 or Ara h 2, or to soluble peanut proteins. The content of both Ara h 1 and Ara h 2 differed in the raw peanut extracts of the three varieties. However, thermal processing resulted in much larger changes in detectability. Blanching significantly increases the detectability of Ara h 2, whereas Ara h 1 detection remains almost unchanged. After roasting a clear decrease of detectability was observed for both Ara h 1 and Ara h 2, although the effect is more severe for Ara h 1. 2D PAGE was employed to compare the protein profiles and abundances of peanut extracts. Statistically relevant differences were observed for the two different protein fractions obtained by using the described method, showing the relevance of this two-step sequential extraction method.     

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。     

花生过敏原在加工中的变化

简述了花生中主要的过敏原Arah1、Arah2、Arah3和Arah4的一般特征及其所存在的与IgE结合的表位特征,详细介绍了焙烤、风干、水煮和煎炸等加热方法及酶解、研磨、发芽和压榨等加工工艺对花生过敏原的影响,为开发无过敏或低过敏性花生制品提供了一定的科学依据。     

阴离子交换层析法分离纯化花生主要过敏原Ara h1的研究

Ara h1蛋白是花生的主要过敏原蛋白。本研究从天然的花生提取花生蛋白质,通过硫酸铵分级沉淀及阴离子交换层析法纯化花生主要过敏原Ara h1。通过聚丙烯酰胺凝胶电泳分析蛋白纯度,结合免疫印迹实验对Ara h1免疫活性进行鉴定。结果表明：采用阴离子交换层析法纯化出的Ara h1纯度达到90%以上。得率为23.1%。免疫印迹实验表明,此方法纯化出来的Ara h1仍具有免疫原性,能跟花生过敏病人血清特异结合。     

花生过敏原及其检测方法研究进展

花生过敏可导致某些人群严重的食品安全问题。过敏患者只能通过避免食用含有花生过敏原成分的食物来避免过敏。但是,食品在生产加工、储存、运输、销售过程中有可能被过敏原污染。因此,确定各类加工食品中是否含有花生过敏原成分,对于预防食用者发生花生过敏反应具有重要意义。花生中已确定的过敏原蛋白有13种(Ara h 1~Ara h 13)。本文综述了花生中过敏原蛋白的结构信息,当前流行的提取方法,以及各种定性定量的检测方法,总结了各种方法的优缺点。同时对建立一种具有特异性强、灵敏度高、定量准确的花生致敏蛋白检测方法的发展趋势进行了展望。     

脂质过氧化物对花生过敏原致敏性的影响

目的 阐明加工过程中脂质过氧化物对花生过敏蛋白Ara h 1结构和过敏原性的影响。方法 通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳技术,圆二色谱法和内源荧光光谱法研究不同脂质过氧化物[2,2-偶氮二(2-甲基丙基咪)二盐酸盐(2,2’-azobis(2-methylpropanimidamide)dihydrochloride,AAPH)和丙烯醛]对花生过敏蛋白Ara h 1的结构影响,进一步采用免疫印迹技术、酶联免疫法,模拟胃液消化和细胞模型分析其过敏原性的变化。结果 脂质过氧化物修饰后的花生过敏蛋白Ara h 1二级结构变得更无序,内源荧光强度降低;花生过敏蛋白Arah1的免疫球蛋白E结合能力、消化稳定性和释放活性介质能力均降低。结论 在花生加工过程中脂质过氧化物能够改变花生过敏蛋白的结构,降低其过敏原性。     

花生过敏患者血清中抗Ara h1的IgG与IgE分离纯化研究

Ara h1是花生的一种主要致敏原蛋白,能够使花生过敏患者产生特异的IgG与IgE,从而导致花生过敏症状。Ara h1与其特异IgG和IgE的结合是导致花生致敏的一个重要原因。本研究利用Protein A亲和层析柱与Ara h1作为配体的亲和层析柱纯化抗Ara h1的IgG与IgE。高纯度的抗Ara h1特异IgG与IgE对探讨研究其与Ara h1之间相互作用具有重要意义。     

花生过敏原Ara h 6的分离纯化及鉴定

为高效分离纯化花生过敏原Ara h 6,通过脱脂、蛋白浸提、阴离子交换层析分离得到目的蛋白,并用十二烷基磺酸钠- 聚丙烯酰胺凝胶电泳(SDS-PAGE)、基质辅助激光解吸/ 电离飞行时间质谱(MALDI-TOF/MS)及免疫印迹技术(Western blotting)对其进行鉴定。结果表明,该蛋白为花生过敏原Ara h 6,其分子质量约为15kD,纯度大于95%,得率为22.5%。该方法简单、高效,可为花生过敏的进一步研究提供实验材料。     

花生主要过敏原Ara h1的纯化

采用硫酸铵沉淀及凝胶过滤层析方法,纯化花生主要过敏原蛋白Ara h1,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)结合免疫印迹实验(Western-Blotting)进行鉴定。结果表明：可纯化出纯度90%以上的Ara h1过敏原蛋白。     

Survival, growth, and thermal resistance of Listeria monocytogenes in products containing peanut and chocolate

Outbreaks of listeriosis associated with the consumption of ready-to-eat foods have raised interest in determining growth, survival, and inactivation characteristics of Listeria monocytogenes in a wide range of products. A study was undertaken to determine the thermal tolerance of L. monocytogenes in a peanut-based beverage (3.1% fat), whole-fat (3.5%) milk, wholefat (4.0%) and reduced-fat (1.0%) chocolate milk, a chocolate-peanut spread (39% fat), and peanut butter (53% fat). The D60 degrees C value (decimal reduction time at 60 degrees C) in peanut beverage (3.2 min) was not significantly different (P > 0.05) than the D60 degrees C value in whole-fat milk (3.3 min) or whole-fat chocolate milk (4.5 min) but significantly lower (P < or = 0.05) than the D60 degrees C value in reduced-fat chocolate milk (5.9 min). The pathogen was significantly more resistant to heat when enmeshed in chocolate-peanut spread (water activity [aw] of 0.46; D60 degrees C = 37.5 min) and peanut butter (aw of 0.32; D60 degrees C = 26.0 min) than in liquid products. At 10 degrees C, the pathogen grew most rapidly in whole-fat chocolate milk and slowest in peanut beverage. At 22 degrees C, populations increased significantly within 12 and 16 h in whole-fat milk and reduced-fat chocolate milk, respectively, and within 8 h in whole-fat chocolate milk and peanut beverage. Initial populations (3.37 to 4.42 log CFU/g) of L. monocytogenes in chocolate-peanut spread and peanut butter adjusted to an aw of 0.33 and 0.65 declined, but the pathogen was not eliminated during a 24-week period at 20 degrees C. Survival was enhanced at reduced aw. Results indicate that a pasteurization process similar to that used for full-fat milk would be adequate to ensure the destruction of L. monocytogenes in peanut beverage. The pathogen survives for at least 24 weeks in chocolate-peanut spread and peanut butter at an aw range that encompasses that found in these products.     

Development of three real-time PCR assays to detect peanut allergen residue in processed food products

Hypersensitivity to peanut is a public health problem, since the ingestion of even low amounts of peanut can trigger severe allergic reactions. Allergic consumers rely on the information provided on the label of foodstuffs to identify products that might endanger their health. In order to protect the allergic consumer methods are required for the detection of allergenic ingredients. For this purpose we have developed three real-time polymerase chain reaction (PCR) assays, based on TaqMan chemistry, that are capable of detecting peanut specific DNA sequences in food products. The peanut specific sequence targeted for detection is located within the gene family of the allergen Ara h 3. The occurrence of multiple Ara h 3 sequences in the peanut genome increases the chance to achieve a good sensitivity. DNA extraction is also known to affect detection by PCR, therefore the efficiency of several different DNA extraction methods was compared. The methods reported here are capable of detecting 2.5 pg peanut DNA (less than one copy of peanut genome equivalent) and all three assays were successfully applied to detect peanut traces in a model food product where they could detect 10 mg kg⁻¹ peanut.     

超高效液相色谱-电喷雾质谱法检测花生 过敏原Ara h 2

目的建立烘培食品中花生过敏原Ara h 2的液相质谱联用定量检测方法。方法选择花生蛋白中的致敏蛋白Ara h 2作为目标蛋白,筛选出该致敏蛋白的特异肽,人工合成特异肽标准品和特异肽内标,从而建立直接检测花生致敏蛋白Ara h 2的准确定量方法。同时还对全国不同地区的20种花生中致敏蛋白Ara h 2的含量进行检测分析,初步统计得出致敏蛋白Ara h 2和花生蛋白的换算系数,并以Ara h 2作为生物标记物检测10种烘培食品中花生蛋白的残留量。结果花生样品中致敏蛋白Ara h 2的定量限为4.45μg/g,回收率在106.0%~107.8%之间。烘培食品中,定量限可达到6.23μg/g,回收率在107.0%~113.2%之间。结论本方法特异性强、灵敏度高、定量准确,具有良好的应用前景。     

Two-Site Antibody Immunoanalytical Detection of Food Allergens by Surface Plasmon Resonance

Bovine protein β-lactoglobulin (βLG) and peanut protein Ara h1 are considered good targets for detecting milk and peanut allergen, respectively. We used surface plasmon resonance (SPR) to detect βLG and Ara h1 by immobilizing the affinity-purified monoclonal antibodies on the biosensor chip. Proteins that bound to the antibody surface were detected by a shift in resonance angle. Adding polyclonal antibodies in the sandwich assay enhanced the sensitivity. βLG and Ara h1 were detected at 5.54 and 0.77 ng/mL, respectively, by SPR, and the results demonstrated good linear relation with relatively low concentrations of protein. The limit of detection with SPR was comparable to that with sandwich enzyme-linked immunosorbent assay (S-ELISA) with the same polyclonal and monoclonal antibodies. Use of the SPR biosensor is a simple, fast, and reliable way to detect βLG and Ara h1.     

加工方式和蛋白提取方法对花生蛋白致敏性的影响

加工方式影响花生蛋白的致敏性。本实验系统的研究了中国传统花生制品(水煮和油炸花生)和美国常食用的烘烤花生致敏性的差异,并比较了不同蛋白提取缓冲溶液对花生致敏蛋白提取效率的影响。结果表明:不同花生制品在不同的缓冲溶液中蛋白提取效率不同,水煮和油炸并没有较烘烤方式降低致敏蛋白的数量,而降低花生蛋白的致敏性。因此加工方式并不是导致中国花生过敏发病率低的主要原因。     

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.     

离子交换层析法分离花生过敏原Ara h2的研究

为了制备出花生中重要过敏原Arah2,以生花生为材料,采用脱脂、离心、膜透析、离子交换层析等方法,纯化花生过敏原Arah2。结果显示,采用阴离子交换层析方法,制取的Arah2蛋白纯度达90%,得率为21.9%,该方法为过敏原Arah2的分离研究提供了可行的实验参数。     

Effects of different extraction buffers on peanut protein detectability and lateral flow device (LFD) performance

The accidental uptake of peanuts can cause severe health reactions in allergic individuals. Reliable determination of traces of peanuts in food products is required to support correct labelling and therefore minimise consumers' risk. The immunoanalytical detectability of potentially allergenic peanut proteins is dependent on previous heat treatment, the extraction capacity of the applied buffer and the specificity of the antibody. In this study a lateral flow device (LFD) for the detection of peanut protein was developed and the capacity of 30 different buffers to extract proteins from mildly and strongly roasted peanut samples as well as their influence on the test strip performance were investigated. Most of the tested buffers showed good extraction capacity for putative Ara h 1 from mildly roasted peanuts. Protein extraction from dark-roasted samples required denaturing additives, which were proven to be incompatible with LFD performance. High-pH buffers increased the protein yield but inhibited signal generation on the test strip. Overall, the best results were achieved using neutral phosphate buffers but equal detectability of differently altered proteins due to food processing cannot be assured yet for immunoanalytical methods.