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1.
Peanut allergy is a public health issue. The culprits are the peanut allergens. Reducing the allergenic properties of these allergens or proteins will be beneficial to allergic individuals. In this study, the objective was to determine if peroxidase (POD), which catalyzes protein cross-linking, reduces the allergenic properties of peanut allergens. In the experiments, protein extracts from raw and roasted defatted peanut meals at pH 8 were incubated with and without POD in the presence of hydrogen peroxide at 37 degrees C for 60 min. The POD-treated and untreated samples were then analyzed by SDS-PAGE, western blots, and competitive inhibition ELISA. IgE binding or allergenicity was determined in blots and ELISA. Results showed that POD treatment had no effect on raw peanuts with respect to protein cross-linking. However, a significant decrease was seen in the levels of the major allergens, Ara h 1 and Ara h 2, in roasted peanuts after POD treatment. Also, polymers were formed. Despite this, a reduction in IgE binding was observed. It was concluded that POD induced the cross-linking of mainly Ara h 1 and Ara h 2 from roasted peanuts and that, due to POD treatment, IgE binding was reduced. The finding indicates that POD can help reduce the allergenic properties of roasted peanut allergens.  相似文献   

2.
Phytic acid would form soluble and insoluble complexes with proteins. Our objective was to determine if phytic acid forms insoluble complexes with major peanut allergens, and if such reaction results in a peanut extract with a lower level of soluble allergens and allergenic property. Extracts from raw and roasted peanuts were treated with and without phytic acid at various pH values and then analyzed by SDS-PAGE and a competitive inhibition ELISA (ciELISA). The ciELISA measured IgE binding using a pooled serum from peanut-allergic individuals. Results showed that phytic acid formed complexes with the major peanut allergens (Ara h 1 and Ara h 2), which were insoluble in acidic and neutral conditions. Succinylation of the allergens inhibited complex formation, indicating that lysine residues were involved. A 6-fold reduction in IgE binding or allergenic potency of the extract was observed after treatment with phytic acid. It was concluded that phytic acid formed insoluble complexes with the major peanut allergens, and resulted in a peanut extract with reduced allergenic potency. Application of phytic acid to a peanut butter slurry presented a similar result, indicating that phytic acid may find use in the development of hypoallergenic peanut-based products.  相似文献   

3.
Influence of thermal processing on the allergenicity of peanut proteins   总被引：3，自引：0，他引：3  
Peanuts are one of the most common and severe food allergens. Nevertheless, the occurrence of peanut allergy varies between countries and depends on both the exposure and the way peanuts are consumed. Processing is known to influence the allergenicity of peanut proteins. The aim of this study was to assess the effect of thermal processing on the IgE-binding capacity of whole peanut protein extracts and of the major peanut allergens Ara h 1 and Ara h 2. Whole proteins, Ara h 1, and Ara h 2 were extracted and purified from raw, roasted and boiled peanuts using selective precipitation and multiple chromatographic steps, and were then characterized by electrophoresis and mass spectrometry. The immunoreactivity of whole peanut extracts and purified proteins was analyzed by the enzyme allergosorbent test (EAST) and EAST inhibition using the sera of 37 peanut-allergic patients. The composition of the whole protein extracts was modified after heat processing, especially after boiling. The electrophoretic pattern showed protein bands of low molecular weight that were less marked in boiled than in raw and roasted peanuts. The same low-molecular-weight proteins were found in the cooking water of peanuts. Whole peanut protein extracts obtained after the different processes were all recognized by the IgE of the 37 patients. The IgE-binding capacity of the whole peanut protein extracts prepared from boiled peanuts was 2-fold lower than that of the extracts prepared from raw and roasted peanuts. No significant difference was observed between protein extracts from raw and roasted peanuts. It is noteworthy that the proteins present in the cooking water were also recognized by the IgE of peanut-allergic patients. IgE immunoreactivity of purified Ara h 1 and Ara h 2 prepared from roasted peanuts was higher than that of their counterparts prepared from raw and boiled peanuts. The IgE-binding capacity of purified Ara h 1 and Ara h 2 was altered by heat treatment and in particular was increased by roasting. However, no significant difference in IgE immunoreactivity was observed between whole protein extracts from raw and roasted peanuts. The decrease in allergenicity of boiled peanuts results mainly from a transfer of low-molecular-weight allergens into the water during cooking.  相似文献   

4.
Although many sequences and linear IgE epitopes of allergenic proteins have been identified and archived in databases, structural and physicochemical discriminators that define their specific properties are lacking. Current bioinformatics tools for predicting the potential allergenicity of a novel protein use methods that were not designed to compare peptides. Novel tools to determine the quantitative sequence and three-dimensional (3D) relationships between IgE epitopes of major allergens from peanut and other foods have been implemented in the Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/). These peptide comparison tools are based on five-dimensional physicochemical property (PCP) vectors. Sequences from SDAP proteins similar in their physicochemical properties to known epitopes of Ara h 1 and Ara h 2 were identified by calculating property distance (PD) values. A 3D model of Ara h 1 was generated to visualize the 3D structure and surface exposure of the epitope regions and peptides with a low PD value to them. Many sequences similar to the known epitopes were identified in related nut allergens, and others were within the sequences of Ara h 1 and Ara h 2. Some of the sequences with low PD values correspond to other known epitopes. Regions with low PD values to one another in Ara h 1 had similar predicted structure, on opposite sides of the internal dimer axis. The PD scale detected epitope pairs that are similar in structure and/or reactivity with patient IgE. The high immunogenicity and IgE reactivity of peanut allergen proteins might be due to the proteins' arrays of similar antigenic regions on opposite sides of a single protein structure.  相似文献   

5.
Detection of peptides from the peanut allergen Ara h 1 by liquid chromatography-mass spectrometry (LC-MS) was used to identify and estimate total peanut protein levels in dark chocolate. A comparison of enzymatic digestion subsequent to and following extraction of Ara h 1 from the food matrix revealed better limits of detection (LOD) for the pre-extraction digestion (20 ppm) than for the postextraction digestion (50 ppm). Evaluation of LC-MS instruments and scan modes showed the LOD could be further reduced to 10 ppm via a triple-quadrupole and multiple-reaction monitoring. Improvements in extraction techniques combined with an increase in the amount of chocolate extracted (1 g) improved the LOD to 2 ppm of peanut protein. This method provides an unambiguous means of confirming the presence of the peanut protein in foods using peptide markers from a major allergen, Ara h 1, and can easily be modified to detect other food allergens.  相似文献   

6.
Enzymatic digestion of total protein along with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to confirm the presence of a major peanut allergen in food. Several peptides obtained from the enzymatic digestion of the most abundant peanut allergen, Ara h 1, were identified as specific peptide biomarkers for peanut protein. Using ice cream as a model food matrix, a method was developed for the detection of the allergen peptide biomarkers. A key component of the method was the use of molecular mass cutoff filters to enrich the Ara h 1 in the protein extracts. By applying the method to ice cream samples containing various levels of peanut protein, levels as low as 10 mg/kg of Ara h 1 could routinely be detected. This method provides an unambiguous means of confirming the presence of the peanut allergen, Ara h 1, in foods and can easily be modified to detect other food allergens.  相似文献   

7.
Trypsin inhibitors are pathogenesis-related (PR) proteins, which play an important role in the plant defense mechanism against insects and pathogens. Peanut trypsin inhibitors are low molecular mass seed storage proteins. Like peanut allergens, they are stable to acid and heat, resistant to digestion, and can have a negative impact on human health. In peanut, five Bowman-Birk trypsin inhibitors (BBTI) have been isolated and amino acid sequences published. However, to date, no peanut BBTI sequence is available at both the cDNA and the genomic levels. The objectives of this investigation were (i) to synthesize degenerate oligonucleotides based on conserved regions of published amino acid sequences of BBTI, BII, and BIII; (ii) to isolate, sequence, and analyze at least one positive peanut trypsin inhibitor cDNA clone using the synthesized (32)P-labeled oligonucleotides as probes; and (iii) to determine its trypsin inhibitory activity. Thirty-two degenerate oligonucleotides DNA primers of 24 nucleotides each were synthesized based on the published amino acid sequences of peanut BBTI, and two were selected as probes to screen a peanut Lambda gt 11 cDNA library. Three putative positive clones were isolated, purified, and subcloned, and one was sequenced. Sequence analysis revealed a partial cDNA clone of 643 bp with a start codon. This clone shares 93 and 96% nucleotide sequence homology with peanut allergens Ara h 3 and Ara h 4 cDNA clones, respectively. A trypsin inhibitor assay revealed that peanut allergen Ara h 3 has a trypsin inhibitory activity of 11 238 TIA/mg protein. We concluded that peanut allergen Ara h 3 may also function as a trypsin inhibitor.  相似文献   

8.
Numerous food allergens of plant origin belong to the 2S albumin family, including peanut Ara h 2. In addition to Ara h 2, several other conglutins related to 2S albumins are present in peanut seeds. We evaluated the allergenicity of different peanut conglutins as compared with Ara h 2. Several conglutins were isolated from the kernel, i.e. Ara h 2, a new isoform of Ara h 6 and its derived product, which is likely to be naturally formed during seed processing. Enzyme allergosorbent tests performed on sera of peanut allergic patients showed that more than 94% of 47 analyzed patients had positive IgE responses to Ara h 6 isoform and to its degradation product. Skin prick tests with the new isoform of Ara h 6 led to a positive response in seven out of the eight tested patients. Both enzyme allergosorbent tests and skin prick tests showed that the reactivity of Ara h 6 was similar to, or even higher than, that of Ara h 2, suggesting that the present isoform of Ara h 6 is as allergenic as Ara h 2. In addition the IgE response to the plant processed (i.e., hydrolyzed) Ara h 6 new isoform is equivalent to the IgE response to the native isoform. The IgE immunoreactivity is mostly abrogated by chemical reduction and denaturation of Ara h 6 isoforms, which underlined the importance of tertiary structure in Ara h 6 immunoreactivity. These results, and particularly the high correlation between anti-Ara h 2 and anti-Ara h 6 IgE responses, emphasise the major role of 2S albumins in peanut allergenicity.  相似文献   

9.
The allergenicity of seed storage proteins, the major components of edible legume seeds, may cause serious reactions in both children and adult population. Updated methodologies for evaluation of the activity of these proteins are needed. In this paper we used two-dimensional (2D) electrophoretic techniques to investigate the immuno-cross-reactivities of anti Ara h 3 basic subunit IgG to the seed proteomes of three legume species, namely, peanut, soybean, and lupin. The seed proteins, extracted with two different procedures, were separated by 2D electrophoresis, and the electrophoretic maps were analyzed by Western blot. In peanut proteome the antibodies strongly reacted with the 23 kDa polypeptides, corresponding to Ara h 3 basic isoforms, the antigen they were raised to, and three unidentified acidic polypeptides near 45 kDa. Remarkable cross-reactivities with lupin and soybean Ara h 3 homologous polypeptides and nonrelated proteins, namely, lupin conglutin gamma and soybean Bg7S, were detected. Therefore, these proteins may be regarded as new putative allergens. The present findings show the potentiality of 2D electrophoresis in the identification of food allergens and open the way to the traceability of the new cross-reacting proteins in the food chain.  相似文献   

10.
Ara h 1 was purified from raw peanuts (Arachis hypogaea L.) in the presence or absence of protease inhibitors. N-Terminal amino acid sequences were determined after western blotting. Both purification procedures proved to be very consistent and resulted in identical chromatographic and electrophoretic behavior of Ara h 1 and in the isolation of identical proteins of approximately 64 kDa with RS/H_PPGERTRG as the N-terminal amino acid sequence. Consequently, purified Ara h 1 appears to be truncated at the N-terminal side. The observations strongly suggest that Ara h 1 occurs physiologically as a protein of which the first 84 and 78 amino acids, respectively, are cleaved off in planta upon maturation of the protein. On the basis of epitope mapping, the cleaved-off N-terminal peptide contains three allergenic epitopes, of which two are major. These truncated epitopes will go undetected in assays when purified Ara h 1 from peanuts is used as reference material. Patients' sera, however, contain IgE-type antibodies against the epitopes that are contained in the cleaved-off peptide, implying that the peptide, or part of it, is still present in peanuts that are consumed. Possible consequences of this exposure to these three epitopes are discussed. On the basis of literature data the cleaved-off peptide is hypothesized to have antifungal activity.  相似文献   

11.
An indirect competitive ELISA was developed allowing the detection of hidden peanut protein residues down to 2 ppm (micorgrams per gram) in various foods. The high-titer, peanut-specific polyclonal antiserum used recognized potentially allergenic proteins in both native and roasted peanuts. In the absence of a food matrix, extractable protein from roasted peanuts was detected at 104 +/- 13%. From various food items, peanut protein at > or =13 ppm was recovered between 84 and 126%, and at 2 ppm of peanut protein recovery was 143 +/- 6%. Intra- and interassay precision was <15%. In 5 of 17 commercial food products without declaration of peanut components, between 2 and 18 ppm of peanut protein was detected. This is the first assay based on commercially available reactants that allows the reliable determination of trace amounts of hidden peanut allergens in a variety of complex food matrices.  相似文献   

12.
Mildly extracted peanut allergen Ara h 1 was previously reported to occur as an oligomeric complex. In this paper we describe how the protein in this oligomeric complex interacts noncovalently with phenolic compounds of the proanthocyanidin type. These interactions are being disrupted during anion exchange chromatography, resulting in the dissociation of the oligomeric Ara h 1 complex into protein trimers. By use of the known three-dimensional structure of beta-conglycinin, a soy protein homologous to Ara h 1, proline-rich regions were observed in silico on both faces of its trimeric structure, which are conserved in Ara h 1. These proline-rich regions could explain the binding of proanthocyanidins to Ara h 1 and the formation of multiple Ara h 1 trimer complexes. This was supported by the observation that the addition of peanut proanthocyanidins to trimeric Ara h 1 and to beta-conglycinin resulted in the formation of soluble oligomeric protein complexes. The structurally related legumin proteins do not contain such proline-rich regions on both sides of the protein, and proanthocyanidins were shown to have a lower affinity for legumin proteins from peanuts and soybeans (peanut allergen Ara h 3 and soy glycinin, respectively). Ara h 1 present as the oligomeric complex is assumed to be the representative form of the allergen in which it is consumed by humans.  相似文献   

13.
This study was aimed at the determination of the pepsin-susceptible and pepsin-resistant epitopes in native and heat-treated Ara h 1, a major allergen from peanuts. Both the oligomeric structure and the trimeric structure of the allergen were investigated. Under the in vitro conditions applied, oligomeric Ara h 1, either unheated or preheated, was hydrolyzed by pepsin at a lower rate than trimeric Ara h 1. Peptides with relatively high molecular masses were shown to be able to bind IgE, whereas peptides with lower molecular masses (<2 kDa) did not. In these latter fractions, fragments of 15 previously published epitopes of mature Ara h 1 were identified. As a result, these epitopes are not likely responsible for the induction of systemic food allergic reactions to peanuts. Using sequential chymotrypsin digestion, the pepsin-resistant IgE-binding peptides were deduced to contain the previously identified intact epitopes EDWRRPSHQQ (amino acids 50-59) and PRKIRPEG (amino acids 60-67). The presence of four additional earlier published intact epitopes (covering amino acids 6-13, 14-21, 24-31, and 40-47) on the pepsin-resistant peptides could be neither deduced nor ruled out. The two deduced and four possible pepsin-resistant epitopes are all situated in the N-terminal part of Ara h 1, which does not show homology with other vicilin proteins. Consequently, this unique N-terminal part of Ara h 1 is proposed to be responsible for the allergen's ability to induce systemic allergic reactions.  相似文献   

14.
A modified version of the Conditt and Baumgardner gas chromatographic/mass spectroscopic (GC/MS) method for determination of daminozide in peanut butter and raw peanuts is described. Daminozide in the food product is hydrolyzed to unsymmetrical dimethylhydrazine (UDMH) by sodium hydroxide digestion. The generated UDMH is distilled from the food matrix and captured by reaction with salicylaldehyde in a condensation trap. Resulting high pH distillates generated by peanuts and peanut products are adjusted back to a pH of 5-6 through addition of glacial acetic acid. After thermal incubation and extraction into methylene chloride, salicylaldehyde dimethylhydrazone is separated from interferences by capillary GC and quantitated by MS using the selective ion monitoring (SIM) mode. Quantitation of daminozide is based on the ratio of the salicylaldehyde dimethylhydrazone molecular ion (m/z 164) to the molecular ion (m/z 153) of the internal standard, 4-nitroanisole. Confirmation of daminozide identity is determined by relative intensity of the m/z 164 ion to the m/z 120 (C7H4ON) ion. Improved m/z 164 ion intensity and reduction of neighboring interferences due to acetic acid treatment permitted a daminozide detection limit of 0.005 ppm in a 50 g sample and an associated 0.02 ppm limit of quantitation. This modification is specific for high protein samples that generate high pH distillates such as peanuts and peanut products and is not specifically intended for analysis of low protein samples.  相似文献   

15.
Allergenicity of Maillard reaction products from peanut proteins   总被引：2，自引：0，他引：2  
It is known that peanut allergy is caused by peanut proteins. However, little is known about the impact of roasting on the allergenicity of peanuts. During roasting, proteins react with sugars to form Maillard reaction products, which could affect allergenicity. To determine if the Maillard reaction could convert a nonallergenic peanut protein into a potentially allergenic product, nonallergenic lectin was reacted with glucose or fructose at 50 degrees C for 28 days. Browning products from heat-treated peanuts were also examined. The products were analyzed in immunoblot and competitive assays, using a pooled serum (i.e., IgE antibodies) from patients with peanut anaphylaxis. Results showed that the products were recognized by IgE and had an inhibitory effect on IgE binding to a peanut allergen. Thus, the findings suggest that these Maillard reaction products are potentially allergenic and indicate the need to verify whether the Maillard reaction products formed in peanuts during roasting increase their allergenicity.  相似文献   

16.
The binding of peanut protein allergens to activated charcoal (AC), used medically for gastric decontamination following the ingestion of toxic substances, was investigated for potential clinical application. Crude peanut extract (CPE) or purified peanut protein allergens Ara h 1 and 2 were co-incubated with AC under a variety of conditions followed by centrifugation to remove the AC and adsorbed protein. The resulting supernatant solution was analyzed for unadsorbed protein by gel electrophoresis and quantitative protein assay. The extent of protein adsorption by a known amount of AC was determined. Protein binding to AC was rapid and irreversible. The extent of adsorption was unaffected by pH, but was optimal near physiological salt concentrations. Denatured proteins, or those of larger molecular weight, required more AC than smaller or native proteins. The extent of protein binding increased with temperature, supporting the concept that protein molecules diffuse into vacant pores of appropriate size on the charcoal surface.  相似文献   

17.
The processes of peanut maturation, curing, and roasting are known to have an important role in peanut flavors. One of these processes (i.e., roasting) has been found to have an effect on allergenicity. To determine if the other processes (i.e., maturation and curing) affect allergenicity, mature and immature roasted peanuts and peanuts cured at different temperatures (35-77 degrees C) were, respectively, tested for IgE binding and advanced glycation end adducts (AGEs). Peanuts with and without stress proteins, which are associated with peanut maturation and curing, were also tested. Results showed that mature roasted peanuts exhibited a higher IgE binding and AGEs level than immature roasted peanuts. Curing temperatures between 35 and 60 degrees C gave no difference in the profiles. However, a higher curing temperature (i.e., 77 degrees C) exhibited a profile of higher levels of AGEs and IgE binding. These levels were higher in peanuts with stress proteins than without stress proteins. Roasting increased stress protein level and IgE binding. From these results, the processes of maturation and curing, in conjunction with roasting, may be associated with allergenicity, suggesting that these processes may lead to changes in the allergenic properties of peanuts.  相似文献   

18.
Ara h 1, a major peanut allergen, is known as a stable trimeric protein. Nevertheless, upon purification of native Ara h 1 from peanuts using only size exclusion chromatography, the allergen appeared to exist in an oligomeric structure, rather than as a trimeric structure. The oligomeric structure was independent of the salt concentration applied. Subjecting the allergen to anion exchange chromatography induced the allergen to dissociate into trimers. Ammonium sulfate precipitation did not bring about any structural changes, whereas exposing the allergen to hydrophobic interaction chromatography caused it to partly dissociate into trimers, with increasing amounts of trimers at higher ionic strengths. The (partial) dissociation into trimers led to a change in the tertiary structure of the monomeric subunits of the allergen, with the monomers in Ara h 1 oligomers having a more compact tertiary structure compared with the monomers in Ara h 1 trimers. As structural characteristics are important for a protein's allergenicity, this finding may imply a different allergenicity for Ara h 1 than previously described.  相似文献   

19.
罗春萍  胡纯秋 《核农学报》2019,33(7):1349-1355
为探讨辐照处理对花生Ara h 2蛋白结构与致敏活性的影响,采用不同剂量60Co-γ辐照处理分离纯化所得到的花生过敏原Ara h 2蛋白,结合紫外扫描光谱、圆二色谱(CD)和聚丙烯酰胺凝胶电泳(SDS-PAGE)评估辐照处理后Ara h 2蛋白的结构变化,并用免疫印迹法和间接酶联免疫吸附法检测辐照处理后Ara h 2的抗原性变化。结果表明,60Co-γ辐照处理可以显著改变花生Ara h 2蛋白的构象,使其降解、发生交联。随着辐照剂量的增大,Ara h 2蛋白与抗体的结合能力呈逐渐下降趋势,且与蛋白紫外吸光度的增强和α-螺旋含量的降低呈现良好的相关性。当辐照剂量为10 kGy时,可基本破坏 Ara h 2 蛋白的结构和免疫活性。60Co-γ辐照处理可以有效降低花生过敏原 Ara h 2 蛋白的致敏性,这为花生脱敏技术的研究提供了新思路。  相似文献   

20.
A gas chromatographic/mass spectrometric (GC/MS) method for determining daminozide in high protein products has been developed. Daminozide is hydrolyzed in the presence of a strong base to form unsymmetrical dimethylhydrazine (UDMH) which is then distilled from the food matrix. A stable derivative is formed by reacting UDMH with salicyladehyde to form salicyaldehyde dimethylhydrazone. This derivative is separated and quantitated by GC/MS using selected ion monitoring (SIM) of key ions in the fragmentation pattern: m/z 164 (molecular ion of hydrazone) and m/z 120 (C7H6ON). An internal standard, 4-nitroanisole, is monitored at m/z 153 (molecular ion) and m/z 123 (C6H5O2N). The limit of detection is 0.01 ppm daminozide in a 50 g sample; however, because of variation at low levels, the limit of quantitation is 0.1 ppm. Recoveries are 90% or greater from peanuts and peanut butter spiked at the 0.1-2 ppm level. Reproducibility of the method depends on the food matrix and is 26% RSD in the worst case. Data are compared for the GC/MS method and the official EPA colorimetric procedure. Results showed a high bias in the colorimetric method, especially when roasted peanut products were analyzed.  相似文献   

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