生物膜干涉法检测花生蛋白与花生过敏患者血清IgE的结合能力

探索基于生物膜干涉技术对花生蛋白与花生过敏患者血清中免疫球蛋白E（immunoglobulin E,IgE）的结合能力进行检测的方法。利用链霉亲和素（streptavidin,SA）标记的传感器、生物素化的羊抗人IgE抗体、花生过敏患者血清池以及花生蛋白建立了一种测定花生蛋白与花生过敏患者血清IgE结合能力的新方法,优化检测条件为抗体1∶100稀释后线下固化20?min,血清1∶10稀释后过夜结合,完成传感器修饰。在线洗基线后用质量浓度为1?mg/mL的花生蛋白与传感器结合3?600?s,解离120?s。利用该法对不同热加工后花生蛋白与患者血清IgE的结合能力进行评估,并与常用的酶联免疫吸附实验（enzyme linked immunosorbent assay,ELISA）检测进行比较。结果表明,本方法可以直接评估过敏原蛋白与血清IgE的结合能力,与ELISA结果相关系数达到0.91。热加工中,油炸处理提高了花生蛋白的IgE结合能力,水煮和烘烤降低了花生蛋白的IgE结合能力,且去壳热加工比带壳热加工花生的蛋白的IgE结合能力更强。     

脂质过氧化物对花生过敏原致敏性的影响

目的 阐明加工过程中脂质过氧化物对花生过敏蛋白Ara h 1结构和过敏原性的影响。方法 通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳技术,圆二色谱法和内源荧光光谱法研究不同脂质过氧化物[2,2-偶氮二(2-甲基丙基咪)二盐酸盐(2,2’-azobis(2-methylpropanimidamide)dihydrochloride,AAPH)和丙烯醛]对花生过敏蛋白Ara h 1的结构影响,进一步采用免疫印迹技术、酶联免疫法,模拟胃液消化和细胞模型分析其过敏原性的变化。结果 脂质过氧化物修饰后的花生过敏蛋白Ara h 1二级结构变得更无序,内源荧光强度降低;花生过敏蛋白Arah1的免疫球蛋白E结合能力、消化稳定性和释放活性介质能力均降低。结论 在花生加工过程中脂质过氧化物能够改变花生过敏蛋白的结构,降低其过敏原性。     

不同处理方式对花生蛋白致敏性的影响

花生蛋白是主要食物过敏原之一,会引起严重的甚至致命的食物过敏。不同的加工处理方式都可以显著改变花生过敏原的分子结构,从而影响它们的致敏性。结合已有的花生致敏的研究成果和最新的研究进展,分析比较花生传统热处理如烘烤处理、水煮处理、油炸处理和新型非热处理如超高压微射流处理、辐射处理、酶法处理对花生致敏性的影响,为加工生产低致敏性或无致敏性的花生制品提供理论依据。     

酶解作用对牛乳β-lg抗原性影响的研究

牛乳β-乳球蛋白（β-lg）是胎儿出生后遇到的第一个外来过敏原,并且它是导致牛乳蛋白过敏的主要过敏原。胰蛋白酶对β-lg的水解作用可以导致其空间三维结构的变化,但是该酶诱导β-lg蛋白结构变化对其致敏性影响的意义还不清楚。为了探明水解作用对β-lg致敏性或者抗原性的影响,利用小鼠动物模型从体外（脾淋巴细胞增殖）和体内（IgE水平和组胺水平）两个方面研究了水解作用对β-lg致敏性的影响。体外细胞增殖实验结果表明,和β-lg水解物相比,β-lg能够显著刺激脾淋巴细胞增殖（P=0.01）;ELISA方法检测小鼠血清和小肠液IgE（免疫球蛋白E）抗体水平结果表明β-lg组小鼠血清IgE水平要高于β-lg水解物刺激组小鼠（P=0.03）,而小肠液中IgE水平和血清IgE水平变化趋势一致,但是整体水平要显著高于血清（P=0.002）。血浆组胺实验表明β-lg免疫组小鼠血浆中组胺水平明显高于β-lg水解物免疫组（P=0.001）。      

单酶水解豆芽蛋白及其产物的潜在致敏性

目的优化豆芽蛋白酶水解的条件,并探讨其致敏性的变化。方法利用Alcalase 2.4L碱性蛋白酶水解豆芽蛋白,以水解度为评价指标,根据单因素实验优化豆芽蛋白的酶水解条件,并通过IgG、IgE的结合实验评估酶解产物潜在致敏性的变化。结果酶水解豆芽蛋白的优化工艺条件:底物浓度为8%、酶与底物比(E/S)为1:20(m:m)、酶解时间为4 h。豆芽蛋白酶水解产物的抗原性低于大豆蛋白酶解产物的抗原性,但豆芽蛋白水解产物的IgE结合能力高于大豆蛋白酶解产物的IgE结合能力。结论大豆经过发芽处理后再用Alcalase2.4L轻度水解能有效降低大豆蛋白的潜在致敏性。     

高压结合酶法消减南美白对虾虾仁致敏性

以南美白对虾虾仁为原料,研究高压结合酶法对虾仁过敏原致敏性的消减作用。将南美白对虾去头去尾去壳去肠线后,采用超高压法和高压结合酶法消减其致敏性,用间接酶联免疫吸附法检测致敏性消减效果,确定消减条件。结果表明：采用超高压法处理,在压力100 MPa、温度25 ℃、保压时间15 min的条件下,虾仁过敏原的致敏性降低了67.09%,且与处理过程中蛋白溶出量有关;采用高压结合酶法处理,在盐水质量浓度1 g/100 mL、酶与虾仁质量比1∶130、压力450 MPa、温度40 ℃、保压时间55 min的条件下,虾仁过敏原的致敏性降低了86.58%。由此可见,高压处理对虾仁过敏原的致敏性有消减作用,高压结合酶法的消减效果更好。     

模拟消化方法在食物过敏原潜在致敏性评价中的应用进展

食物过敏已成为全球范围内日益严重的食品安全问题。对食物过敏原的潜在致敏性进行评价有助于更好地了解食物过敏原自身特性及其对过敏人群免疫系统的影响。模拟消化实验是食物过敏原潜在致敏性评价的有力手段。食物过敏原蛋白的消化稳定性为其潜在致敏性评价结果提供了重要参考依据,但并非所有食物过敏原均具有较强的消化稳定性。食物过敏原的潜在致敏性一方面取决于过敏原蛋白在通过消化道时的消化稳定性,另一方面取决于消化产物中具有免疫刺激能力的过敏原表位的丰度。不同模拟消化方法的应用对食物过敏原的消化结果及潜在致敏性评价结果的真实性和可比性具有重要影响。本文系统地介绍了体内及体外模拟消化方法的优缺点及在食物过敏原潜在致敏性评价中的应用研究进展,并综述了食物过敏原消化稳定性与其潜在致敏性评价结果的关系。以期对食物过敏原致敏性评价体系的进一步完善和发展有所帮助。     

芝麻过敏原及其致敏性消减技术研究进展

芝麻是新的“8大类”过敏食物之一,芝麻过敏反应由于其潜在危害性及其日益上升的发病率而越来越引起全世界的普遍重视,因此研究芝麻致敏性消减技术在保障食品安全方面具有重要意义。概述了芝麻主要过敏原(Ses i 1～Ses i 7)的结构和免疫特性,及其致敏性消减技术原理和研究进展;已报道的芝麻过敏原有7种,属于2S白蛋白、7S类豌豆球蛋白、油质蛋白和11S球蛋白,其中11S球蛋白和2S白蛋白是主要的过敏原蛋白。利用热加工技术消减芝麻致敏性,主要通过煮沸、微波、烘焙等工艺引起过敏原的解聚、变性进而破坏致敏表位;高压、辐照、发酵、酶解等非热加工技术通过氢键、疏水键等化学键的变化、多肽链的断裂引起蛋白结构变化,从而掩盖或直接降解致敏表位。另外,结合其他食品过敏原,对芝麻过敏原的潜在消减技术,包括脉冲电场、冷等离子体、超声波、脉冲光、糖基化改性和复合加工技术等进行了阐述分析。未来需要进一步研究芝麻不同过敏原的致敏表位、不同加工工艺对过敏原结构及致敏性影响、开展血清学、细胞和动物模型等致敏性评价等,以明确芝麻致敏性消减的主要机制,以期为生产低敏或脱敏芝麻产品奠定理论依据与科学指导。     

常见食物过敏原结构稳定因素与致敏性的关系研究进展

食物过敏原所引发的食品安全问题是目前亟待解决的卫生学问题,而分析过敏原蛋白结构稳定性与致敏性的关系,对于了解食品过敏的机理以及探索消减过敏原致敏性的方法具有十分重要的意义。本文选取几种主要食物过敏原蛋白,探讨维持过敏原蛋白结构稳定性的因素（二硫键、糖基、疏水作用以及氢键）,以及结构稳定与致敏性的关系,为如何消减过敏原致敏性提供一定的理论指导。     

碱性氨基酸对β-乳球蛋白结构和致敏性的影响

碱性氨基酸是一种自身带正电荷的小分子，可与蛋白质上的负电残基发生静电吸引，也可与蛋白侧链上某些基团构成空间位阻，进而导致蛋白质结构的改变。β-乳球蛋白(β-lactoglobulin, β-Lg)可引发过敏反应，通过不同碱性氨基酸对β-Lg进行处理，研究其结构和致敏性的变化规律。以L-精氨酸(L-Arg)、L-赖氨酸(L-Lys)和L-组氨酸(L-His)单独或混合处理的β-Lg为研究对象，通过电泳、圆二色谱、光谱等技术分析其结构的变化；采用酶联免疫吸附法、细胞实验等方法评价其抗氧化活性、免疫球蛋白E(immune globulin E,IgE)/免疫球蛋白G(immune globulin G,IgG)结合能力和KU812细胞释放组胺和白介素-6的能力。结果表明，L-Arg、L-Lys和L-His处理β-Lg后，改变了β-Lg的二级结构、紫外吸收强度、内源荧光强度和表面疏水性，同时增加其ABTS阳离子自由基清除能力、降低IgE/IgG结合能力以及KU812细胞中组胺和白细胞介素-6的分泌能力。因此，L-Arg、L-Lys和L-His破坏β-Lg的构象过敏表位，降低其致敏性，顺序依次为...     

Ferulic acid enhances IgE binding to peanut allergens in Western blots

Ferulic acid, a phenolic compound, is known to complex with proteins and peanut allergens. Preliminary studies indicated that ferulic acid could also complex with and inhibit IgE antibodies to peanut allergens in ELISA. However, results from Western blots were quite different. The objective of this study was to report the unexpected finding of IgE binding being enhanced rather than reduced by ferulic acid in Western blot. Ferulic acid, at various concentrations (0–10 mg/ml), was mixed with a pooled plasma (containing IgE antibodies) from peanut-allergic individuals before incubation with a peanut allergen-bound membrane and colorimetric detection of IgE. Results showed that an enhancement of allergen bands or IgE binding, compared to the control, was observed at a ferulic concentration of 10 mg/ml. Compounds with a similar structure, such as caffeic acid and chlorogenic acid, at the same concentration, did not have an enhancing effect on IgE binding. Tests with ferulic acid alone or soy proteins indicated that the enhanced IgE binding was due to the IgE–ferulic complexes and not ferulic acid itself. It was concluded that ferulic acid (10 mg/ml), in combination with IgE, enhanced IgE binding to peanut allergens in Western blots. The finding indicated that ferulic acid can help reduce the time for colour development of protein bands in Western blots.     

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.     

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.     

A food matrix reduces digestion and absorption of food allergens in vivo

Scope: Food allergy is caused by primary (class 1) food allergens, e.g. Bos d 5 (cow's milk) and Cor a 8 (hazelnut) or secondary (class 2) food allergens, e.g. Mal d 1 (apple). The latter cannot sensitize susceptible individuals but can cause allergy due to immunological cross‐reactivity with homologous respiratory allergens. Here, we studied the effects of food matrix on gastrointestinal proteolysis, epithelial transport and in vivo absorption of class 1 and class 2 food allergens. Methods and results: Mal d 1 lost its IgE‐reactivity immediately after simulated gastric digestion whereas Bos d 5 and Cor a 8 did not. Only Cor a 8 maintained IgE‐binding capacity after simulated intestinal proteolysis. The presence of hazelnut and peanut extracts, which served as protein‐rich model food matrices, delayed gastrointestinal degradation and reduced epithelial transport rates of all allergens through CaCo‐2 monolayers. Finally, IgE‐reactive allergens were assessed at different time points in sera from rats fed with all three allergens with or without hazelnut extract. The levels of all allergens peaked 2 h after animals were fed without matrix and increased over 8 h after feeding. Conclusions: A protein‐rich food matrix delays gastrointestinal digestion and epithelial transport of food allergens and thereby may affect their sensitizing capacity and clinical symptoms.     

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     

Mitigation of peanut allergenic reactivity by combined processing: Pressured heating and enzymatic hydrolysis

Among food allergens, peanut is one of the most critical. This study evaluates peanut allergenic features after the combination of heat, pressure, and enzymatic digestion under sonication, by immunodetection using serum IgE of sensitized patients and mass-spectroscopy. In the studied population, there was a predominance of patients with sensitization to Ara h 9 (nsLTP) followed by sensitization to seed storage proteins (Sprot, Ara h 1, 2, 3, and 6). The Sprot sensitized patients showed higher reactivity. The enzyme E5 was efficient for inducing protein fragmentation and allergenic reactivity reduction when it was used combined with pressured heating treatments such as autoclave and Controlled Instantaneous Depressurization (DIC). Only a few Ara h 1 and Ara h 3 peptides were identified after enzymatic digestion of DIC peanut samples. The combination of pressured heating treatments and enzymatic hydrolysis was the most efficient method to strongly mitigate or even eliminate the allergenic potential of peanut. Our findings set a possibility for a group of patients in which their allergy could be treated with a processed less-allergenic peanut and consequently less risky, more easy and quicker desensitization treatment.Industrial relevanceThe findings identify innovative thermal, pressure and enzymatic processing conditions highly effective to mitigate or even abolish the allergenic potency of peanut, which may be relevant for consumers, clinicians, regulatory agencies and the food industry. The applications of processed peanut with reduced IgE binding potency for tolerance induction might be a convenient strategy.     

Digestibility of peanut and hazelnut allergens investigated by a simple in vitro procedure

 Stability under digestion is thought to be an important prerequisite determining the allergenicity of food proteins. To test this hypothesis, two important allergenic plant-derived foods were selected for this investigation. Protein extracts from roasted peanuts and unprocessed (native) hazelnuts were digested by a static, two-step in vitro procedure with commercial enzyme tablets containing peptic and pancreatic enzymes, respectively. The food extracts were subjected to gastric digestion for 2 h followed by a 45-min treatment under duodenal conditions. Undigested control samples and the two digests were investigated by analytical sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), by SDS-PAGE immunoblotting, by an enzyme allergosorbent test (EAST) with human IgE, and by a rat basophil leukaemia (RBL) cell mediator release assay that depends on specific IgE raised in mice. Peanut proteins appeared to be more stable under digestion than hazelnut proteins, as shown by electrophoresis. These results were also underlined by immunblot data. The gastric digest from peanut contained various protein fragments that were detected by antibodies from a peanut-specific rabbit antiserum and by IgE from patients allergic to peanuts. These immunoblot reactivities decreased strongly after subsequent pancreatic digestion. In the gastric digest from hazelnuts, a rabbit antiserum with a broad reactivity against native hazelnut proteins exclusively recognized small protein fragments of <15 kDa. This serum showed no binding to blots of the pancreatic digest. Sera from hazelnut-allergic patients presented IgE reactivities to an 18-kDa major allergen with homology to major tree-pollen allergens, to a minor allergen of 12 kDa, and to multiple bands  1 30 kDa in native hazelnut extract. No binding was observed with these sera on blot strips of the two digests prepared from hazelnut extract. Under the native conditions of EAST, both digests from peanuts strongly reacted with human IgE. Their IgE binding capacity persisted at a level of about 50% when compared to undigested peanut. In the case of hazelnuts the IgE reactivity of the untreated samples was reduced to <10% by both gastric and combined gastric/duodenal digestion for a serum pool prepared from four patients and sera from three additional participants. By contrast, a constantly high immunoreactivity of the hazelnut digests was detected with serum from one patient. The results of the EAST were confirmed by dose-related mediator release experiments performed with RBL cells passively sensitized with allergen-specific murine IgE. As a whole, our results indicated that the EAST and the RBL cell assay are superior to immunoblotting for the immunologic testing of digests. In accordance with clinical observations, the allergenicity of peanut proteins was very persistent during digestion, whereas the native birch-pollen-related hazelnut allergens appeared to be relatively labile under identical conditions. Received: 6 July 1998     

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。     

Characterization of lupin major allergens (Lupinus albus L.)

White lupin is considered to be a rich source of protein with a notable content of lysine and is being increasingly used in bakery, confectionery, snacks and pastry products due to its multifunctional properties, in addition to its potential hypocholesterolemic and hypoglycemic properties. However, lupin seed flour has been reported as a causative agent of allergic reactions, especially in patients with allergy to peanut since the risk of immunological cross‐reactivity between lupin and peanut is higher than with other legumes. Previously, we had identified two proteins as major lupin allergens (34.5 and 20 kDa) as determined by IgE immunoblotting using sera of 23 patients with lupin‐specific IgE. The aim of this study was to purify and characterize the two major lupin allergens. The results using in vitro IgE‐binding studies and MS analysis have shown that the 34.5 kDa allergen (Lup‐1) is a conglutin β (vicilin‐like protein) while the 20 kDa allergen (Lup‐2) corresponds to the conglutin α fraction (legumin‐like protein). The high level of amino acid sequence homology of Lup‐1 and Lup‐2 with the major allergens of some legumes explains the IgE cross‐reactivity and clinical cross‐reactivity of lupin and other legumes.