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.     

Reduction of major peanut allergens Ara h 1 and Ara h 2, in roasted peanuts by ultrasound assisted enzymatic treatment

This study investigated the effects of ultrasound, enzyme concentration and enzyme treatment time on soluble protein and major allergenic proteins (Ara h 1 and Ara h 2) of roasted peanut kernels. A 3-factor, five-level orthogonal experimental design was implemented with various ultrasonication times, concentrations of trypsin or α-chymotrypsin and treatment times. The total soluble proteins were determined by the Bicinchoninic acid (BCA) method, Ara h 1 and Ara h 2 were evaluated by SDS–PAGE and sandwich ELISA. The IgE-binding of peanut extracts was analysed by a competitive inhibition ELISA. Results indicate that ultrasound treatment, followed by protease digestion of peanuts, significantly increased the solubility of peanut protein and decreased the concentrations of Ara h 1 and Ara h 2. The sequential treatment of peanuts by ultrasonication–trypsin–alpha chymotrypsin, resulted in maximum reductions of Ara h 1/Ara h 2, and lowest IgE-binding. This study provides an approach to significantly reduce allergenic proteins in peanut product.     

Enzymatic treatment of peanut kernels to reduce allergen levels

This study investigated the use of enzymatic treatment to reduce peanut allergens in peanut kernels as affected by processing conditions. Two major peanut allergens, Ara h 1 and Ara h 2, were used as indicators of process effectiveness. Enzymatic treatment effectively reduced Ara h 1 and Ara h 2 in roasted peanut kernels by up to 100% under optimal conditions. For instance, treatment of roasted peanut kernels with α-chymotrypsin and trypsin for 1–3 h significantly increased the solubility of peanut protein while reducing Ara h 1 and Ara h 2 in peanut kernel extracts by 100% and 98%, respectively, based on ELISA readings. Ara h 1 and Ara h 2 levels in peanut protein extracts were inversely correlated with protein solubility in roasted peanut. Blanching of kernels enhanced the effectiveness of enzyme treatment in roasted peanuts but not in raw peanuts. The optimal concentration of enzyme was determined by response surface to be in the range of 0.1–0.2%. No consistent results were obtained for raw peanut kernels since Ara h 1 and Ara h 2 increased in peanut protein extracts under some treatment conditions and decreased in others.     

中国花生致敏蛋白的识别

我国对花生过敏方面的研究很少。本实验利用中国常用花生品种识别鉴定了中国主要的致敏蛋白,比较了国内外花生品种致敏蛋白相对含量的差异,期望找到中国花生过敏发病率较低的原因,为临床食物过敏患者的治疗和低过敏花生品种的培育提供理论依据。研究结果表明:Ara h1和三条Ara h3多肽是中国主要的致敏蛋白,并发现了Ara h1的亚基,分子量为58kD的多肽。Ara h1和Ara h3的相对含量各品种之间差异显著,并且低于国外花生品种。因此中国花生主要致敏蛋白相对含量低可能是导致中国花生过敏发病率较低的主要原因。     

IDENTIFICATION AND QUANTIFICATION OF MAJOR PEANUT ALLERGENS (IN CHINA) WITH IGE-BINDING PROPERTY

Peanut allergens have not been studied in China. This study aimed to investigate (1) whether there are differences in the relative amounts of major peanut allergens between the Chinese peanut varieties and the American, and (2) the effect of cooking methods on peanut allergenicity. The allergenic property of raw peanuts and peanut preparations was assessed by immunoblotting and enzyme-linked immunosorbent assay. The relative contents of the major peanut allergens were quantified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis densitometry. For results, Ara h 1 and Ara h 3 were major peanut allergens in China. The amounts of Ara h 1 and Ara h 3 in peanut varieties differ significantly and were both lower than the American varieties. The immunoglobulin E (IgE)-binding ability of different processed peanuts to IgE was not significantly different. Therefore, peanut varieties may induce different amounts of allergens. The relative lower contents of Ara h 1 and Ara h 3 may lead to the lower prevalence of peanut allergy.

PRACTICAL APPLICATIONS

Because of its nutritional and rheological properties, peanut is used in a wide range of different foods, and peanut allergy represents an important health problem. It is essential to identify the compounds of peanut allergens and to study their characteristics in order to explore approaches for the therapies and to breed the nonallergenic peanut seed.     

Mitigation of Major Peanut Allergens by Pulsed Ultraviolet Light

Peanut allergy represents one of the most severe IgE-mediated reactions with food, but to date, the only effective way to prevent peanut allergy is total avoidance. If allergens could be mitigated during food processing before a product reaches the consumer, this would substantially lessen the food allergy problem. The efficacy of pulsed ultraviolet light (PUV), a novel food processing technology, on reducing peanut allergens, was examined. This study revealed for the first time that PUV was also capable of deactivating Ara h 2, the most potent allergenic protein of peanut. Protein extracts from raw and roasted peanuts were treated for 2, 4, and 6?min and peanut butter slurry was treated for 1, 2, and 3?min in a Xenon Steripulse XL 3000? PUV system. The distance from the central axis of the lamp was varied at 10.8, 14.6, and 18.2?cm. The SDS?CPAGE showed a reduction in the protein band intensity for Ara h 1, Ara h 2, and Ara h 3 at the energy levels ranging from 111.6 to 223.2?J/cm². Reduction of the protein band intensity for peanut allergens increased with treatment time but decreased with increased distance from the PUV lamp. The ELISA for peanut extracts and peanut butter slurry showed a reduction in IgE binding of up to 12.9- and 6.7-folds, respectively, compared to control.     

Allergenicity of roasted peanuts treated with a non-human digestive protease

Peanut allergy is a severe and lifelong type of food allergy triggered by allergenic proteins and peptides in peanuts. This study investigated the effects of ultrasound-assisted alcalase treatment on the concentrations of major allergenic proteins (Ara h 1 and Ara h 2) in roasted peanut kernels and the allergenicity of treated peanut extracts. Peanut kernels were sonicated for 1 h in buffer solution, incubated with different amount of alcalase for various time, then vacuum dried. The variations of Ara h 1 and Ara h 2 contents in soluble and insoluble portions of peanuts treatments were evaluated by sandwich ELISA and SDS-PAGE, respectively. The in vitro IgE-binding capacity of treated peanut extracts was determined by a competitive inhibition ELISA using pooled plasma of 10 peanut allergic patients. Samples with lower in vitro IgE-binding were used for human skin prick tests (SPTs) in peanut allergic individuals. Results indicate that alcalase digestion of sonicated peanuts significantly increased protein solubility while decreasing Ara h 1 and Ara h 2 concentrations in both soluble and insoluble portions of peanuts relative to untreated peanuts. The maximum reductions of Ara h 1 and Ara h 2 levels were obtained following 3 hour digestion with alcalase at concentrations of 4.54 and 6.05 U/100 g. Samples obtained under these conditions showed the lowest in vitro IgE-binding and caused the least allergic response in human SPTs. The current study suggests that the allergenic potential of peanuts could be reduced by postharvest processing such as ultrasound-assisted enzymatic treatment of peanuts kernels.     

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。     

Reducing the allergenic capacity of peanut extracts and liquid peanut butter by phenolic compounds

Phenolic compounds are known to form soluble and insoluble complexes with proteins. The objective of this study was to determine if phenolics such as caffeic, chlorogenic and ferulic acids form insoluble and irreversible complexes with major peanut allergens, and if such complexation reduces immunoglobulin E (IgE) binding. After adding each of the phenolics to peanut extracts and liquid peanut butter, the soluble materials were analysed by SDS–PAGE and inhibition ELISA. Results showed that addition of the phenolics precipitated most of the major peanut allergens, Ara h 1 and Ara h 2, and that complexation was irreversible. IgE binding was reduced approximately 10- to 16-fold. We concluded that reducing IgE binding by phenolics is feasible. The research, if proven by clinical studies, could lead to the development of less allergenic liquid peanut-based products.     

Polyphenol oxidase/caffeic acid may reduce the allergenic properties of peanut allergens

Polyphenol oxidase (PPO) catalyzes the oxidation of tyrosine residues of proteins and, therefore, their cross‐linking. Previously we demonstrated that cross‐links produced by peroxidase (POD), which also catalyzes tyrosine oxidation, led to a reduction in the allergenic properties of peanut allergens. 11 We postulated in this study that PPO can also reduce the allergenic properties by cross‐linking the allergens. Because caffeic acid, a phenolic compound, can cross‐link proteins, its effect on peanut allergens was also examined. In the experiments, peanut extracts were treated with and without PPO, PPO/caffeic (pH 8, 37 °C for 1 h) and caffeic acid (pH 10.5, overnight), respectively. The samples were then analyzed for cross‐links and IgE binding by SDS‐PAGE, Western blots, and competitive inhibition ELISA. Results showed that, in all cases, cross‐links and a decrease of the levels of two peanut major allergens, Ara h 1 and Ara h 2, were observed. Of the three treatments, PPO/caffeic was the most effective in reducing IgE binding or the allergenic properties of peanut allergens. The availability of tyrosine residues was also demonstrated in a POD‐treated system, using a monoclonal antibody against dityrosine. We concluded that PPO/caffeic acid reduced the allergenic properties of Ara h 1 and Ara h 2 by cross‐linking and decreasing the levels of allergens. Copyright © 2005 Society of Chemical Industry     

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。     

Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: A comparative in vitro study and partial characterization of digestion‐resistant peptides

Scope : There are differences in stability to pepsin between the major allergens in peanut; however, data are from different reports using different digestion models. This study provides a comprehensive comparison of the digestibility of the major peanut allergens. Methods and results : Peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were incubated with pepsin to mimic the effect of gastric digestion. Samples were analyzed using SDS‐PAGE. To further investigate resistance to digestion, Ara h 2 was additionally subjected to digestion with trypsin and residual peptides were characterized. Ara h 1 and Ara h 3 were rapidly hydrolyzed by pepsin. On the contrary, Ara h 2 and Ara h 6 were resistant to pepsin digestion, even at very high concentrations of pepsin. In fact, limited proteolysis could only be demonstrated by SDS‐PAGE performed under reducing conditions, indicating an important role for the disulfide bridges in maintaining the quaternary structure of Ara h 2 and Ara h 6. Trypsin digestion of Ara h 2 similarly resulted in large residual peptides and these were identified. Conclusion : Ara h 2 and Ara h 6 are considerably more stable towards digestion than Ara h 1 and Ara h 3.     

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。     

A potential practical approach to reduce Ara h 6 allergenicity by gamma irradiation

Peanut allergen Ara h 6 was isolated and irradiated at 1, 3, 5, or 10 kGy, and a whole peanut protein extract (WPPE) was also treated by irradiation. Alteration in structure of Ara h 6 was characterised by circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and SDS–PAGE, and antigenicity was evaluated by immunoblotting and indirect ELISA with anti-Ara h 6 polyclonal antibody. Irradiation induced significant changes in the secondary and tertiary structures of Ara h 6, and the antigenicity of both purified Ara h 6 and WPPE were reduced upon increasing the irradiation doses. Moreover, a good correlation between the loss in α-helix and IgG binding to Ara h 6 was observed. This indicated that irradiation might be an efficient approach to reduce or eliminate peanut allergenicity.     

高温高压处理对花生致敏蛋白Ara h1及其免疫反应性的影响

通过免疫学方法研究了高温高压处理后花生主要致敏蛋白Ara h1溶解性和免疫反应性的变化。结果表明:高温高压处理可使Ara h1及花生蛋白粗提物均发生显著变性和分解,以135 ℃处理最为显著;但Ara h1在121和135 ℃处理20 min后均表现出良好的热稳定性,可溶性蛋白含量不再随处理时间延长而发生明显减小;135 ℃处理20 min或更长时间后样品中15 kDa左右或更小分子量的蛋白片段成为分解物的主要组成部分。高温高压处理可使Ara h1的免疫反应性显著下降,且在蛋白粗提物的混合体系中更为显著:135 ℃处理20和60 min后,Ara h1的IgG结合能力分别仅为对照的1/3和1/6;而在混合体系中,相同条件下Ara h1的IgG结合能力分别仅为对照的2/9和1/9。     

花生品种对水媒法提取分离蛋白质与脂质及内源性蛋白酶活性的影响

为筛选适合水媒法加工的花生品种,降低花生蛋白的致敏性,收集了5个花生品种,首先比较了其脂质和蛋白质含量,然后采用水媒法提取分离蛋白质与脂质,考察了花生品种对脂质和蛋白质的提取率及脂质和蛋白质的分离效果的影响,最后比较了不同品种花生的内源性蛋白酶(内肽酶和外肽酶)活性。结果表明：脂质(46.95%～51.35%)和蛋白质(24.66%～29.68%)含量在花生品种间存在差异;花生品种对于蛋白质提取率(91.86%～97.77%)的影响显著,而对于脂质提取率(98%左右)的影响不大;不同品种花生浆中蛋白质和脂质的离心分离效果不同,蛋白质在蛋白液中的分布比例为85.32%～97.34%,脂质在乳状液中的分布比例为68.75%～81.24%,白沙308的分离效果最好;内源性蛋白酶活性(pH 3～5)在花生品种间存在差异,但是各品种的内肽酶均在pH 3时表现出最强活性,可有效水解花生致敏蛋白Ara h 1;各品种花生的外肽酶活性均随pH的增加而增加,在pH 5时,四粒红最高;对于内肽酶和外肽酶的协同活性,花育在pH 3时最强,而其他4个品种在pH 5时最强。综上,白沙308可作为花生水媒法加工的...     

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。     

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.     

质谱法分析烘焙对花生过敏原Ara h 1潜在致敏性的影响

为研究烘焙对花生过敏原Ara h 1潜在致敏性的影响,采用高离液序列盐溶液从鲜花生和烘焙花生中提取总蛋白,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析烘焙前后蛋白条带变化情况,并对其中的花生主要过敏蛋白Ara h 1条带进行质谱和Swiss-Model模型分析。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示,烘焙花生蛋白出现了大分子聚合物条带以及较多弥散状蛋白条带,说明烘焙过程中蛋白质会发生聚集,同时也可能发生降解。对鲜花生Ara h 1条带的质谱分析结果显示,检测出70 条肽段,覆盖率达到79.2%;而烘焙后其中40 条肽段未能检出,但新增1 条肽段,且覆盖率降至43.9%。全部71 个肽段涉及到Ara h 1的18 个过敏原线性表位,酶解后鲜花生中检出16 个过敏原线性表位被破坏,烘焙花生中仅发现12 个被破坏。结论：烘焙加工会破坏蛋白质高级结构,掩盖了部分酶切位点,减少了酶解对过敏原线性表位的破坏,这可能是导致烘焙加工后Ara h 1致敏性强于未加工样品的原因。     

Peanut allergen (Ara h 1) detection in foods containing chocolate

Inadvertent exposure to peanut in foods poses health risks for peanut-allergic individuals that can be reduced by improving detection systems for allergen contaminants in food products and manufacturing processes. Detection of peanut in chocolate has been especially difficult. We report the optimization of conditions for measuring a major peanut allergen, Ara h 1, in chocolate with the use of a two-site monoclonal antibody sandwich enzyme-linked immunosorbent assay. Ara h 1 was extracted from peanut in the presence or absence of chocolate with phosphate buffer, salt, and three dried milks (goat, soy, or nonfat) (0 to 25% wt/vol) for 15 min at 60 degrees C or for 2.5 h at room temperature. The best conditions for Ara h 1 extraction in the presence of chocolate were 5% nonfat dry milk for 2.5 h at room temperature. Spiking experiments of chocolate with peanut confirmed improvement of the extraction: Ara h 1 was detected in extractions of 0.16 to 0.33% peanut in chocolate. Interestingly, the best conditions for Ara h 1 extraction were different for peanut alone than with chocolate, regarding time, temperature, and percentage of nonfat dry milk in the extraction buffer. In chocolate with peanut foods, the total Ara h 1 values were 10-fold higher than when products were extracted with phosphate buffer alone and could be up to 400-fold higher for individual foods. The dramatic improvement of Ara h 1 extraction should allow specific allergen monitoring in chocolate-containing food products and assessment of Ara h 1 exposure.