Removing peanut allergens by tannic acid

Tannic acid (TA) forms insoluble complexes with proteins. The aims here were to remove major peanut allergens as insoluble TA complexes and determine if they would dissociate and release the allergens at pH 2 and 8 (gut pH). Release of the allergens in the gut could lead to absorption and consequently an allergic reaction. TA (0.25, 0.5, 1, and 2 mg/ml) was added to a peanut butter extract (5 mg/ml; pH 7.2), stirred, and centrifuged. The precipitates were then suspended in buffer at pH 2, centrifuged, re-suspended at pH 8, and centrifuged. Supernatants from each step were analysed by SDS–PAGE, ELISA, and Western blots. The effect of NaCl (1 M) on complexes was also determined. Results showed that complexes formed at a TA concentration >0.5 mg/ml did not release major peanut allergens at pH 2 and 8, regardless of 1 M NaCl being present or not. IgE binding of the extracts was reduced substantially, especially at a TA concentration of 1–2 mg/ml. Animal or clinical studies are still needed before TA can find an application in the development of low-allergen peanut products/beverages or the removal of peanut allergens due to accidental ingestion.     

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.     

Towards a better understanding of the pectin structure-function relationship in broccoli during processing: Part I—macroscopic and molecular analyses

To investigate the structure-function relationship of pectin during (pre)processing, broccoli samples (Brassica oleracea L. cultivar italica) were subjected to one of the following pretreatments: (i) low-temperature blanching (LTB), (ii) LTB in combination with Ca²⁺ infusion, (iii) high-pressure pretreatment (HP), (iv) HP in combination with Ca²⁺ infusion, or (v) no pretreatment (control sample), whether or not in combination with a thermal treatment of 15 min at 90 °C. The macroscopic attributes of broccoli were linked to the chemical structure of broccoli pectin. By enhancing the cross-linking of pectic polymers, both LTB and HP reduced the texture loss that occurred during thermal processing of broccoli. During these pretreatments, homogalacturonan was de-esterified by pectin methylesterase, which led to changes in pectin solubility. When LTB or HP was combined with Ca²⁺ infusion, changes in the structure of pectin occurred, however not always reflected at the macroscopic level. The degree of esterification of pectin in Ca²⁺-soaked broccoli samples was lower compared to non-Ca²⁺-soaked samples and, in addition, a higher amount of ionically cross-linked pectin was retrieved.     

Development of a direct sampling method for verifying the cleanliness of equipment shared with peanut products

A direct sampling method was developed to verify the cleanliness of stainless steel equipment used to produce peanut products. The protocol consisted of sampling the product-contact area with two Critical Swab^® flat zapped-head foam swabs pre-moistened with 20 mM phosphate, pH 7.2 (PBS), mixing each swab in PBS for 1 h, and analyzing the extract for peanut residues. Quantitation of peanut residues was accomplished with a non-specific assay while SDS–PAGE was used to ensure the recovery of the allergen, Ara h 1. This sampling approach was able to recover 90–95% of the peanut proteins at sub-microgram levels and Ara h 1 at low nanogram levels directly from a stainless steel surface.     

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.     

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.     

花生过敏原Ara h 1蛋白的原核表达及致敏性分析

从花生中提取总RNA,用反转录聚合酶链式反应得到花生过敏原Ara h 1基因,构建pET-28a-Ara h 1表达载体,转入Rosetta（DE3）宿主表达菌中诱导产物表达,用镍离子亲和层析法纯化得到目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示目的蛋白分子质量约为75 kDa,与预计相符;经质谱鉴定为Ara h 1蛋白。用BALB/c小鼠模型评价重组Ara h 1蛋白的致敏性结果显示,重组Ara h 1蛋白致敏小鼠血清中特异性抗体、Th2型细胞因子、组胺含量升高,空肠和肺组织发生病变,表明重组Ara h 1蛋白可以导致小鼠发生Th2型过敏反应,且有与天然Ara h 1蛋白相似的致敏性。同时RBL细胞模型结果显示重组Ara h 1蛋白还可导致RBL细胞脱颗粒,释放β-己糖苷酶,进一步表明重组Ara h 1蛋白具有致敏性。     

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。     

A practical test system for sensitive, rapid screening and authentication of peanut allergens in imported and exported food products in Chinese Customs

Proper food allergen labeling protects consumers with serious food allergies, such as peanut (Arachis hypogaea) allergies. Currently, there is no widely accepted standard peanut allergen detection protocol for use by Customs agencies, which could avoid trade disputes caused by improper labeling. Herein we developed a peanut allergen screening and confirmation system for sensitive, rapid identification of peanut allergen ingredients which used gold immunochromatography assay strips followed by confirmatory western blotting. Gold immunochromatography assay strips were prepared with polyclonal antibodies against total peanut proteins for preliminary peanut allergen screening in foods labeled peanut-free. Western blotting with Ara h1-specific monoclonal antibodies was performed to confirm the results to exclude false positive results. Of 285 food samples tested, 164 were labeled as containing peanut allergens. The gold immunochromatography assay determined that 116 were negative, in accordance with their original labels. Five samples were positive, which was not consistent with their labels. These 5 positive samples were subjected to western blotting confirmatory tests. Only one was confirmed to be positive. We reported this result to the manufacturer and suggested they change the product label. This system, which includes sequential classification, screening, confirmation and reporting steps, was useful for monitoring peanut contamination in imported and exported foods by Chinese Customs.