Study on screening potential allergenic proteins from infant milk powders based on human mast cell membrane chromatography and histamine release assays

Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.

Structure and allergenicity of a-lactalbumin:effects of ultrasonic prior to glycation and subsequent phosphorylation

Bovineα-lactalbumin(BLA)induced severe cow's milk allergy.In this study,a novel strategy combining ultrasonication,performed before glycation,and phosphorylation was proposed to reduce BLA allergenicity.Result showed that IgE-and IgG-binding capacities and the release rates of histamine and interleukin-6 from RBL-2 H3 were reduced.Moreover,intrinsic fluorescence intensity and surface hydrophobicity were decreased,whereas glycated sites(R10,N44,K79,K108,N102 and K114)and phosphorylated sites(Y36 and S112)of BLA were increased.Minimum allergenicity was detected during BLA treatment after ultrasonic prior to glycation and subsequent phosphorylation because of considerable increase in glycated and phosphorylated sites.Therefore,the decrease in allergenicity of BLA,the effect correlated well with the shielding effect of glycated sites combined with phosphorylated sites and the conformational changes.This study provides important theoretical foundations for improving and using the ultrasonic technology combined with protein modification in allergenic protein processing.

HPLC Analysis of α-lactalbumin and β-lactoglobulin in Bovine Milk with C_4 and C_(18) Column

Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were compared. Shiseido Proteonavi C4 column （250 mm×4.6 mm×5μm） and Shiseido CAPCELL PAK SG 300 C18 column （250 mm× 4.6 mm×5 μm） were used in the experiment. Phase A was composed of 0.1% （V/V） trifluoroacetic acid （TFA） in ultrapure water and Phase B （organic phase） was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL min-1. The detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist acid and more stable, and the method with C4 column had excellent sensitivities and good separation.

Fabrication and evaluation of a portable and reproducible quartz crystal microbalance immunochip for label-free detection ofβ-lactoglobulin allergen in milk products

In this study,a label-free,portable and reproducible immunochip based on quartz crystal microbalance(QCM)was developed for the qualitative detection ofβ-lactoglobulin(β-LG),an allergen,in milk products.Experimental parameters in the fabrication and regeneration procedure such as pH of the coupling microenvironment,amount of anti-β-LG antibody and regeneration reagent were optimized in detail.Under optimal conditions,the proposed QCM immunochip exhibited good recognition of β-LG,with a calibration curve of ΔF=12.877 C_(β-LG)^(0.4809)(R^(2)=0.9982)and limit of detection of 0.04μg/mL.Additionally,this portable QCM immunochip had good stability,high specificity,and no obvious cross-reaction to three other milk proteins(α-casein,α-lactalbumin,and lactoferrin).It could compete a qualitative measurement within5 min,and could be reused at least ten times.In the β-LG analysis of actual milk samples,the developed QCM immunochip yielded reliable and accurate results,which correlated strongly with those from the standard HPLC method(R^(2)=0.9969).Thus,the portable,stable,and reproducible QCM immunochip developed in this study allowed the rapid,cost-effectively and sensitively measure theβ-LG in milk products.

Determination of egg and milk allergen in food products by liquid chromatography-tandem mass spectrometry based on signature peptides and isotope-labeled internal standard

The aim of this work was to develop a liquid chromatography-tandem mass spectrometry method for the determination of milk allergen and egg allergen in food products.Signature peptides GGLEPINFQTAADQAR,VGINYWLAHK,VLVLDTDYK,FFVAPFPEVFGK,and NAVPITPTLNR were confirmed and synthesized as the quantitative peptide of ovalbumin,α-lactalbumin,β-lactoglobulin,α_(S1)-casein andα_(S2)-casein,the relative isotope-labeled internal standards were used in the quantitative analysis.Linear range was in the range of0.5-5000.0 nmol/L for egg and milk allergen in bread,cake,cookie,rice crust and wheat flour samples with free from egg and milk,the limits of detection of milk allergens and egg allergen were in the range between0.94 mg/100 g and 56.71 mg/100 g,limits of quantification of milk allergens and egg allergen were in the range between 2.36 mg/100 g and 141.78 mg/100 g.The recoveries ranged from 76.7%to 122.8%,the relative standard deviations were in the range of 1.60%-15.60%.The developed method has been successfully used for the detection of egg and milk allergen in various food samples.

Dietary polyphenols reduced the allergenicity ofβ-lactoglobulin via non-covalent interactions:a study on the structure-allergenicity relationship

Studies showed that complexation of polyphenols with milk allergens reduced their immunogenic potential.However,the relationship between structures of polyphenols and their hypoallergenic effects on milk allergens in association with physiological and conformational changes of the complexes remain unclear.In this study,polyphenols from eight botanical sources were extracted to prepare non-covalent complexes withβ-lactoglobulin(β-LG),a major allergen in milk.The dominant phenolic compounds bound toβ-LG with a diminished allergenicity were identified to investigate their respective role on the structural and allergenic properties ofβ-LG.Extracts from Vaccinium fruits and black soybeans were found to have great inhibitory effects on the IgE-and IgG-binding abilities ofβ-LG.Among the fourteen structure-related phenolic compounds,flavonoids and tannins with larger MWs and multi-hydroxyl substituents,notably rutin,EGCG,and ellagitannins were more potent to elicit changes on the conformational structures ofβ-LG to decrease the allergenicity of complexedβ-LG.Correlation analysis further demonstrated that a destabilized secondary structure and protein depolymerization caused by polyphenol-binding were closely related to the allergenicity property of formed complexes.This study provides insights into the understanding of structure-allergenicity relationship ofβ-LG-polyphenol interactions and would benefit the development of polyphenol-fortified matrices with hypoallergenic potential.

Efficacy of fermented milk and whey proteins in Helicobacter pylori eradication:A review

Helicobacter pylori (H. pylori) eradication is considered a necessary step in the management of peptic ulcer disease, chronic gastritis, gastric adenocarcinoma and mucosa associated lymphoid tissue lymphoma. Standard triple therapy eradication regimens are inconvenient and achieve unpredictable and often poor results. Eradication rates are decreasing over time with increase in antibiotic resistance. Fermented milk and several of its component whey proteins have emerged as candidates for complementary therapy. In this context the current review seeks to summarize the current evidence available on their role in H. pylori eradication. Pertinent narrative/systematic reviews, clinical trials and laboratory studies on individual components including fermented milk, yogurt, whey proteins, lactoferrin, α-lactalbumin (α-LA), glycomacropeptide and immunoglobulin were comprehensively searched and retrieved from Medline, Embase, Scopus, Cochrane Controlled Trials Register and abstracts/proceedings of conferences up to May 2013. A preponderance of the evidence available on fermented milk-based probiotic preparations and bovine lactoferrin suggests a beneficial effect in Helicobacter eradication. Evidence for α-LA and immunoglobulins is promising while that for glycomacropeptide is preliminary and requires substantiation. The magnitude of the potential benefit documented so far is small and the precise clinical settings are ill defined. This restricts the potential use of this group as a complementary therapy in a nutraceutical setting hinging on better patient acceptability/compliance. Further work is necessary to identify the optimal substrate, fermentation process, dose and the ideal clinical setting (prevention/treatment, first line therapy/recurrence, symptomatic/asymptomatic, gastritis/ulcer diseases etc.). The potential of this group in high antibiotic resistance or treatment failure settings presents interesting possibilities and deserves further exploration.

Gold nanoparticle-based lateral flow immunoassay for the rapid and on-site detection of wheat allergen in milk

Wheat gliadin is one of the most important causes of gluten sensitivity.This study aimed to develop a sandwich enzyme-linked immunosorbent assay(ELISA)and gold nanoparticle-based lateral flow(LFIA)strips to detect wheat allergen in food.Through cell screening,ten monoclonal antibodies(mAbs)against gliadin and after optimization were obtained,a pair of mAbs suitable for its detection(capture antibody:mAb 7;detection antibody:HRP-labeled mAb 6)were identified.Based on two of these antibodies,a sandwich ELISA with limit of detection(LOD)of 60 ng/mL in negative milk,and more importantly,negligible cross-reactivity to other allergens was developed.The average recoveries for gliadin in negative milk were 99.16%-100.07%using the sandwich ELISA.We also developed LFIA strips for the rapid detection of gliadin with visual limit of detection(vLOD)of 25 ng/mL and calculated LOD value of 6.56 ng/mL in negative milk.The test results of positive samples obtained from the LFIA strips were highly consistent with those of the sandwich ELISA.Thus,the developed LFIA strip is an effective and reliable tool for the rapid and on-site detection of wheat allergen in milk.

Cow milkα_(s1)-casein induces allergic responses in a mouse model of atopy

α_(s1)-Casein is a potential allergen to induce hypersensitivity in cow milk.We had identifiedα_(s1)-casein and its epitopes in previous studies.The present study aimed to evaluate the allergic mechanism ofα_(s1)-casein in a BALB/c mouse model.The levels of specific IgE,mast cell proteinase,histamine and cytokines in sensitized mice were determined,and the clinical and pathological observation were evaluated.Results showed that the levels of specific IgE,mast cell proteinase,histamine,IL-4,IL-5,IL-10 increased significantly with a dose-dependent trend.The local alveolar septum collapsed or thickened,and lymphatic foci were produced in the spleen and thymus,and the inflammatory cells infiltrated in small intestinal mucosa mesenchyme.In conclusion,the levels of specific IgE,mast cell proteinase,histamine,IL-4,IL-5,IL-10 and some inflammatory factors could possibly serve as allergic biomarkers ofα_(s1)-casein,however,additional studies on signal transduction and gene expression are necessary in future.

牛乳过敏及其降敏技术研究进展

牛乳蛋白是常见的食物过敏原,牛乳过敏已成为全球范围内常见的公共卫生问题。探索乳蛋白致敏机制,开发绿色、高效降敏技术已成为食品领域的重要研究课题。本文总结了牛乳过敏原的抗原表位,介绍了牛乳过敏的机制与症状,讨论了不同加工技术(如热加工、高压、超声、糖基化、偶联多酚、酶处理、发酵以及转基因)对牛乳蛋白降敏作用及潜在机制,并对牛乳降敏技术的发展做出展望。

猪笼液蛋白酶消减牛乳蛋白致敏表位的研究

目的:利用废弃猪笼液中的蛋白酶来消减牛乳主要致敏原蛋白的致敏性。方法:首先采集了米兰达猪笼草(Nepenthes×Miranda)的猪笼液,经过滤、5 kDa膜超滤、真空冷冻干燥和复溶获得酶活为126.276 U/mL的猪笼液蛋白酶液。然后用此酶对牛乳蛋白进行酶解,通过液相色谱-二级质谱法(LC-MS/MS)鉴定酶解后肽段产物,对比致敏原表位数据库以判断蛋白酶对表位的酶切情况。结果:牛乳主要致敏原蛋白中P1位的K、V、F、R、Y、I、S和L以及P1’位的D、A、V、I、L、F、E、R、Y、N、T、Q、S、W和P的肽键被有效酶解,酶切位点位于相对应的表位内,且位于蛋白表面和内部的表位均有被酶解。其中,来自α-LA的表位Eα-LA1中的K-V以及表位Eα-LA2中的K-V和K-A被酶解频率较高,来自β-LG的被酶解频率高的表位处的肽键为Eβ-LG1和Eβ-LG2中的L-D、V-Y和Y-V,来自αs1-CN的表位Eαs1-CN1中大部分的R-F和F-F被高频酶解,来自αs2-CN的表位Eαs2-CN1中的L-N、R-Y、K-F和K-T和表位Eαs2-CN2中的K-T、K-L和K-I被酶解频率较高,β-CN中被酶解频率较高的表位为Eβ-CN1和Eβ-CN2中的L-Q、S-W、D-V、L-T、T-D和E-N。结论:猪笼液蛋白酶水解对牛乳蛋白致敏原蛋白表位表现出不同程度的破坏作用,猪笼液有望成为新型蛋白酶开发的资源,实现农业废弃物再利用。

牛乳和鸡蛋中致敏原物质的检测

牛乳、鸡蛋及乳、蛋制品富含蛋白质、维生素和矿物质等,是人们饮食结构中的常见食物,在为人类提供丰富营养素的同时,也容易引起部分人群的过敏。针对目前过敏发生情况和过敏原检测技术的发展现状,该文简述了牛乳和鸡蛋中的主要过敏蛋白、酶等,并介绍了过敏原常见检测方法,比较各检测方法的优缺点,为牛乳和鸡蛋过敏原的检测提供理论依据和指导。

碱性及复合蛋白酶复配水解对脱脂牛乳致敏性的影响

牛乳中致敏蛋白在一定程度上限制了其在婴幼儿配方奶粉中的使用。以脱脂牛乳为原料,以水解度、脱脂牛乳IgG抑制率、过敏患者血清IgE抑制率为指标,研究碱性、复合蛋白酶复配处理对其致敏性的影响。结果表明,复配酶的酶活与底物比(E/S)为6000 U/g,碱性蛋白酶与复合蛋白酶质量比为3∶1,酶解时间60 min时,脱脂牛乳的水解度为9.21%,对脱脂牛乳IgG及过敏患者血清IgE抗体抑制率分别为86.94%与43.46%,显著高于单一蛋白酶(P<0.05)的处理效果。研究结果为以脱脂牛乳为婴幼儿奶粉基料的开发提供了重要参考。

酶水解降低牛奶蛋白致敏性研究进展

酶水解及其与加工技术的组合已经被广泛开发并用于降低牛奶蛋白的致敏性。文章综述了酶水解及其与其他加工技术(热处理、超声、高静水压、冷等离子体、糖基化和超滤)的组合在影响牛奶蛋白致敏性方面的最新进展和作用机制,并对比、分析和总结这些加工与酶水解技术组合优缺点。研究发现,酶水解与加工技术的组合在降低牛奶蛋白致敏性方面比单一处理更有效。主要归因于加工技术可以通过诱导蛋白质结构发生变化,使其暴露更多的酶切位点,从而提高酶对牛奶蛋白致敏表位的破坏。为低致敏性牛奶蛋白生产提供参考。

144例婴幼儿牛奶蛋白过敏的临床分析

目的分析婴幼儿牛奶蛋白过敏患儿临床特征,为早期诊断和有效治疗婴幼儿牛奶蛋白过敏提供科学依据。方法回顾性调查河南省儿童医院消化内科2020年11月1日-2022年5月1日住院的婴幼儿牛奶蛋白过敏144例病例,对婴儿婴幼儿牛奶蛋白过敏临床特征进行描述性分析,分析婴幼儿牛奶蛋白过敏患儿的临床特征。结果144例患儿中完全人工喂养者62例,纯母乳喂养64例,混合喂养18例。患儿以腹泻、便血为主要症状,母亲规避致敏高蛋白致敏食物或更换为氨基酸配方奶粉喂养后症状多明显缓解。结论牛奶蛋白过敏的患儿愈后良好,但很难诊断。本病患儿无特异性的临床表现,通过详细的病史及一般检查,当食物过敏被高度怀疑时,回避可疑过敏食物可能改善临床表现,如果诊断仍不明确或疗效较差,则需要进一步筛查食物过敏原,以确定明确致敏原,如仍不能明确诊断,需进一步行电子结肠镜检查及肠黏膜组织病理学检查协助诊断。治疗本病通过采用食物回避的方法,效果明显,早期诊断有利减少抗生素的滥用,减轻家庭经济负担,有利于对婴儿的早期营养提供指导,促进健康发育。

牛奶中过敏原组分的分离纯化及主要过敏原的鉴定

目的:分离纯化并鉴定牛奶中引起过敏反应的主要致敏原组分,并分析其与组胺释放的关系。方法:等电点沉淀、Sephadex G-50凝胶层析和DEAE-Sepharose离子交换层析等技术分离纯化牛奶过敏原组分。脱脂乳浸液免疫C57/BL6小鼠制备抗血清。Western-Blotting分析脱脂乳致敏原组分。间接ELISA检测抗血清中sIgE和sIgG;同时荧光法检测各致敏组分体外诱发免疫小鼠腹腔肥大细胞组胺释放率。结果:分离纯化出酪蛋白、β-乳球蛋白、α-乳白蛋白等致敏组分(纯度>90%)。免疫印迹表明酪蛋白是主要致敏原组分。抗血清sIgG水平差异显著,sIgE水平没有大的变化;组胺释放率与sIgG水平是一致的,而与sIgE水平没有明显相关性。酪蛋白抗血清sIgG效价最高为1∶25 600,组胺释放率高达(73.28±9.31)%。结论:牛奶中主要过敏原组分鉴定为酪蛋白,对牛奶过敏症的体外诊断治疗以及低/无过敏原配方奶的研制有一定的意义。

基于α-乳白蛋白基因序列的牛奶过敏原PCR检测

采用收集牛奶中脱落体细胞的方法提取牛奶DNA,针对牛奶过敏原蛋白α-乳白蛋白基因序列设计引物α-La-F/R,筛选适宜的实验条件,建立了检测牛奶过敏原的PCR方法。结果表明:PCR反应的最佳退火温度为56.5℃,引物浓度0.15μmol/L,循环数35,进一步实验表明该方法特异性良好,检测灵敏度可达到0.04ng DNA。将建立的PCR方法应用于10种食品的牛奶过敏原检测,实验结果与商品标示一致,表明该方法具有较高的准确性和可靠性,适用于实际商品中牛奶过敏原的检测。

牛奶大分子蛋白的水解及其抗原性变化

目的 :将婴儿乳粉以不同的食品级蛋白酶水解 ,降低牛奶的分子量 ,考察牛奶酶水解产物过敏原性的变化。方法 :采用胃蛋白酶、胰蛋白酶和自制胰酶制品对婴儿奶粉进行酶解 ,以快速蛋白质液相色谱测得酶解产物分子量分布和所占比例。通过实验动物主动、被动皮肤反应和口服致敏肠腔通透性变化观察不同酶水解后牛奶的过敏原性变化。结果 :快速蛋白质液相色谱结果表明蛋白酶水解牛奶后 ,牛奶蛋白分子量明显降低 ,尤以自制胰酶对乳粉的水解程度最高 (分子量 <10 0 0占 92 % ;10 0 0～10 0 0 0占 8% )。胃蛋白酶、胰蛋白酶和胰酶水解产物可以显著抑制由牛奶蛋白引起的小鼠耳速发型主动皮肤过敏 ,抑制率分别为 5 1%、83%和 86 %。小鼠同种及异种被动皮肤过敏反应实验中 ,与未水解奶粉组比较 ,同种皮肤过敏反应三种酶解产物炎性渗出降低率分别为 2 9%、70 %和 82 % ,异种皮肤过敏反应的降低率分别为 34%、70 %和 82 %。口服致敏小鼠肠腔通透性及分泌功能测定结果显示 ,胰酶水解产物致敏小鼠的肠腔伊文思蓝渗出率比未水解前降低了 2 7%。结论 :酶水解后牛奶大分子蛋白物质降解 ,其过敏原性有不同程度的降低 ,尤以胰酶水解产物变化最为显著。

酶法降低牛乳蛋白致敏性的研究进展

酶解牛乳是牛乳加工中的常用技术之一,并且作为低致敏牛乳制备的关键技术已成功应用于实际中。本文综述牛乳中主要过敏原及酶解机制,并详细介绍不同种类的单一酶解和复合酶解用于降低牛乳蛋白致敏性的研究,以期为制备和生产低致敏性乳制品提供一定依据和理论基础。

牛乳蛋白过敏原改性的研究

对牛乳中的主要过敏原酪蛋白、β-乳球蛋白(β-LG)及α-乳白蛋白(α-LA)进行了介绍。重点论述了热处理、蛋白水解、糖基化作用和乳酸发酵等技术对乳蛋白过敏改性的研究现状,提出了牛乳中过敏蛋白原改性的研究方向。