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.     

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.     

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...     

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.     

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.     

Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity

Scope : Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. Methods and results : Ara h 1 was purified from either raw (N‐Ara h 1) or roasted (R‐Ara h 1) peanuts. Boiling (100°C 15 min; H‐Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod‐like branched aggregates with reduced IgE‐binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G‐Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H‐ and G‐Ara h1 retained the T‐cell reactivity of N‐Ara h 1. R‐Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE‐binding capacity of the native protein. Conclusion : Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.     

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。     

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.     

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。     

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.     

Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases

BACKGROUND: The major peanut allergens are Ara h 1, Ara h 2 and Ara h 6. Proteolytic processing has been shown to be required for the maturation process of Ara h 6. The aim of this study was to examine whether Ara h 2 undergoes proteolytic processing and, if so, whether proteolytic processing influences its ability to bind human immunoglobulin E (IgE). RESULTS: Ara h 2 isolated from peanut extract under conditions of protease inhibition revealed a single additional peak for its two known isoforms (Ara h 2.01 and Ara h 2.02), corresponding to a C‐terminally truncated form lacking a dipeptide (RY). Ara h 2 isolated in the absence of protease inhibition, however, yielded two additional peaks, identified as C‐terminally truncated forms lacking either a dipeptide (RY) or a single tyrosine residue. The IgE‐binding capacity of the Ara h 2 truncated forms was not altered. CONCLUSION: Ara h 2 undergoes proteolytic processing by peanut proteases that involves C‐terminal removal of a dipeptide. Hence Ara h 2 isolated from peanut extract is a complex mixture of two isoforms expressed by different genes, Ara h 2.01 and Ara h 2.02, as well as truncated forms generated by the proteolytic processing of these isoforms. Copyright © 2010 Society of Chemical Industry     

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.     

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     

还原协同加热处理对花生过敏原Ara h2结构及抗原性的影响

花生过敏能引起荨麻疹、呕吐甚至休克等症状,通常是终生性过敏。本研究运用加热处理、半胱氨酸还原对Ara h2进行单独和复合处理,采用圆二色谱、紫外扫描光谱、间接竞争酶联免疫吸附法分别检测蛋白加工前后的结构与抗原性变化。结果显示,单一的加热和半胱氨酸还原处理过的蛋白结构与抗原性只发生部分变化。而复合处理中,在加热条件下,半胱氨酸还原蛋白的能力明显增强,还原后的蛋白二级结构与三级结构发生大幅变化,抗原性显著降低。研究结果表明,二硫键对于蛋白Ara h2结构稳定具有重要作用,二硫键被还原和加热过程破坏后,蛋白的抗原性和结构即发生重大变化。还原协同加热处理有望大幅改善2S球蛋白的结构和致敏性,可以作为蛋白脱敏加工的备选方法。     

High-pressure microfluidisation-induced changes in the antigenicity and conformation of allergen Ara h 2 purified from Chinese peanut

BACKGROUND: Peanut allergy is one of the most serious food allergies, and Ara h 2 is one of the most important peanut allergens as it is recognised by serum immunoglobulin E from more than 90% of peanut‐allergic individuals. Dynamic high‐pressure microfluidisation has been widely used in food processing as a new technology. The aim of this study was to investigate the effect of high‐pressure microfluidisation on the antigenicity and structure of Ara h 2. Extracted peanut allergen Ara h 2 was treated under a continuous pressure array of 60, 90, 120, 150 and 180 MPa. Immunoreactivity was measured by indirect enzyme‐linked immunosorbent assay with rabbit polyclonal antibodies. Secondary structure was analysed by circular dichroism. Surface hydrophobicity and sulfhydryl groups were assessed via fluorescence and UV absorption spectra respectively. RESULTS: High‐pressure microfluidisation treatment decreased the antigenicity of peanut allergen Ara h 2, changed its secondary structure and increased its UV absorption intensity and surface hydrophobicity. CONCLUSION: The change in conformation contributed to the decrease in antigenicity of Ara h 2, and the spatial conformation of peanut allergen Ara h 2 plays a critical role in its antigenicity. Copyright © 2011 Society of Chemical Industry     

Beneficial Influence of Short‐Term Germination on Decreasing Allergenicity of Peanut Proteins

Most allergenic storage proteins in peanuts are degraded during seed germination. By altering this natural physiological process, it might be possible to reduce peanut protein allergenicity. However, little is known about the change in allergenic proteins and their corresponding immunocreactivity, and the effects of major environmental conditions on their allergenicity during germination. In this study, the influence of different germination conditions (temperature and light) on the degradation of Ara h1 and allergenicity changes of peanut seeds was evaluated by ELISA and Western blotting. The results showed that the 40‐ and 65‐kDa proteins in peanut seeds degraded rapidly during the time course, beginning at 60 (at 25 °C) and 108 h (at 20 °C), and the corresponding immunocreactivity of Ara h1 decreased approximately one‐third after 5 to 7 d of germination. Compared with the cotyledons, the embryonic axes had a higher proportion of Ara h1, which was then degraded relatively faster during germination, resulting in a significant reduction in its allergenicity. Although a higher temperature improved the seed germination rate, it affected sprout quality (as did light); therefore, 25 °C and dark surroundings were suitable conditions under which peanut sprouts were processed; neither factor significantly affected the allergenicity of Ara h1. These results provided a theoretical basis for studies using biological methods to reduce peanut allergenicity.     

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.     

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。     

重组花生过敏原Ara h 2生物合成

为获得重组花生过敏原Ara h 2。通过RT-PCR 合成cDNA,并以此为模板进行PCR 扩增目的基因Ara h 2,扩增产物经纯化后克隆至pMD19-T Simple 载体中,构建重组质粒pMD19-T-Ara h 2。上述重组质粒经酶切纯化后定向克隆到pGEX-4T-1 表达载体中,构建原核表达载体pGEX-4T-1-Ara h 2,并转化表达宿主菌BL21-codonPlus(DE3)-RIPL 中,经IPTG 诱导表达。SDS-PAGE 电泳结果表明,该表达蛋白大小约为46kD,与理论值相符。通过Glutathione Sepharose 4B 凝胶亲和层析方法纯化融合蛋白GST-Ara h 2,获得融合蛋白纯度约为90%。Western blotting 分析表明,经纯化的融合蛋白能与抗Ara h 2 兔血清发生特异性反应,说明该蛋白具有良好的免疫原性。     

Legumin allergens from peanuts and soybeans: effects of denaturation and aggregation on allergenicity

Legumin proteins Ara h 3 from peanuts and glycinin from soybeans are increasingly described as important allergens. The stability of an allergen's IgE binding capacity towards heating and digestion is considered an important characteristic for food allergens. We investigated the effects of heating and digestion on the IgE binding of Ara h 3 and glycinin. Both proteins are relatively stable to denaturation, having denaturation temperatures ranging from 70 to 92 degrees C, depending on their quaternary structure and the ionic strength. Aggregates were formed upon heating, which were partly soluble for glycinin. Heating slightly decreased the pepsin digestion rate of both allergens. However, heating did not affect the IgE binding capacity of the hydrolyzates, as after only 10 min of hydrolysis no IgE binding could be detected any more in all samples. Peanut allergen Ara h 1, when digested under equal conditions, still showed IgE binding after 2 h of hydrolysis. Our results indicate that the IgE binding capacity of legumin allergens from peanuts and soybeans does not withstand peptic digestion. Consequently, these allergens are likely unable to sensitize via the gastro-intestinal tract and cause systemic food allergy symptoms. These proteins might thus be less important allergens than was previously assumed.