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.     

Isolation and characterization of natural Ara h 6: evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat

Peanut allergy is a significant health problem because of its prevalence and the potential severity of the allergic reaction. The characterization of peanut allergens is crucial to the understanding of the mechanism of peanut allergy. Recently, we described cloning of the peanut allergen Ara h 6. The aim of this study was isolation and further characterization of nAra h 6. We purified nAra h 6 from crude peanut extract using gel filtration and anion exchange chromatography. The preparation was further characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with subsequent immunoblotting. Stability of nAra h 6 was studied by an in vitro digestibility assay as well as by resistance against thermal processing. Sequencing of nAra h 6 identified the N-terminal amino acid sequence as MRRERGRQGDSSS. Further results clearly demonstrated stability of nAra h 6 against pepsin digestion and heating. Immunoglobulin G (IgE) binding analysis and its biological activity shown by RBL 25/30-test of natural Ara h 6 supported the importance of this peanut allergen. Investigation of nAra h 6 revealed evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat.     

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.     

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.     

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.     

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。