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...     

己糖修饰的糖基化卵清蛋白产物及其致敏性分析

研究了不同己糖修饰的糖基化卵清蛋白的接枝度、表面疏水性、紫外光谱、荧光光谱及致敏性变化。结果表明:半乳糖修饰的卵清蛋白糖基化程度最高,果糖修饰的最低;糖基化反应使卵清蛋白表面疏水性降低;紫外光谱和荧光光谱构象表征表明,糖基化过程中的热处理以及分子基团的改变破坏了卵清蛋白原有构象,使芳香族氨基酸外露,且半乳糖对卵清蛋白构象的破坏程度最高;糖基化反应使卵清蛋白致敏性降低,但果糖修饰的卵清蛋白的IgG/IgE结合力下降轻微,远低于半乳糖和葡萄糖的。     

低致敏食品制备技术及其工业化应用研究进展

食物过敏是联合国粮农组织和世界卫生组织认定的全球性食品安全问题之一。在食品加工多元化的背景下,食物过敏患者要完全避免过敏原十分困难,研发低致敏食品对食物过敏患者的安全膳食至关重要。总结了低致敏食品制备技术的加工技术原理;以蒸煮、微波和烘烤为主的热加工技术通过加热诱导蛋白质变性的方式破坏致敏性构象性表位;高压、脉冲电场、脉冲光、低温等离子体、辐照和超声等非热加工技术可以通过过敏原蛋白结构修饰、多肽链断裂、新化学键的产生等方式直接破坏致敏性表位;酶水解、酶交联、糖基化、微生物发酵等其他加工方法则通过改变蛋白质构象或将蛋白质与糖类物质结合,破坏或隐藏过敏原致敏性表位。另外,对工业化低致敏蛋白配料的加工方法和生产现状进行了阐述分析。基于酶法水解的部分水解乳蛋白和深度水解乳蛋白已经可以工业化生产,其他消减食物致敏性的方法以及其他低致敏蛋白配料值得进一步研究。希望可以为工业化生产低致敏食品提供参考。     

超声协同糖基化对蛋清粉致敏性及结构的影响

以市售蛋清粉为研究对象,通过电泳、紫外光谱、荧光光谱和酶联免疫吸附等方法研究超声波协同糖基化修饰对其蛋白结构和致敏性的影响。结果表明,超声波协同糖基化修饰能显著降低蛋清粉的致敏性,致敏性随超声强度的增加呈先增加后降低的趋势,且在15 min、600 W的条件下有最大程度降低。这和其结构变化密切相关,经复合处理后,蛋清蛋白分子量显著增加,同时三级结构发生显著变化,使表面疏水性增强、内源荧光和自由氨基含量降低,导致其致敏性降低。超声波协同糖基化修饰是一种良好的降低蛋清粉致敏性的方法。     

糖基化鳕鱼小清蛋白消化过程中的致敏性

采用间接竞争ELISA、高效液相色谱等技术研究糖基化鳕鱼小清蛋白(PV)在体外模拟消化过程中致敏性及结构的变化规律。结果显示:糖基化PV经消化后其IgE/IgG结合能力显著降低,并于胃肠消化2h时达到最低,3h时略有增加;胃蛋白酶酶解产生的分子量500Da的肽段在胰蛋白酶的作用下会重新聚集;消化产物都出现新的发射峰并产生红移,发射峰的荧光强度也呈先降低后增加趋势。说明鳕鱼PV致敏性的变化与其结构的改变密切相关,糖基化改性结合模拟人体胃肠道消化可有效降低蛋白的致敏性。     

多重技术联合应用缓解牛乳蛋白致敏性的研究进展

本文论述了牛乳中主要的致敏蛋白：酪蛋白、 α-乳白蛋白与β-乳球蛋白的致敏机理,同时总结了物理技术联合生物技术（高压联合酶解,微波联合酶解,超声联合酶解,加热联合酶解,超声联合发酵）、物理技术联合化学技术（高压联合糖基化,超声波联合糖基化）、化学技术联合生物技术（酶解联合糖基化）对降低牛乳致敏性的作用,可为今后低致敏性乳及乳制品的开发提供理论依据和研究思路。     

超声波辅助糖基化对β-乳球蛋白消化过程中致敏性的影响

采用间接竞争ELISA、扫描电镜和动态光散射等技术,研究超声波结合乳糖糖基化修饰对β-乳球蛋白(β-Lg)在体外模拟消化过程中致敏性和结构变化的影响。结果表明:被修饰的β-Lg在消化过程中致敏性显著降低,胃肠消化的水解度也降低;被修饰的的β-Lg经胃消化形成更大的颗粒结构,在肠消化中发生聚集;消化后其荧光强度先升高后降低,最大发射波长出现红移。综上,超声波结合糖基化改性可有效降低β-Lg在消化过程中的致敏性,其致敏性变化与糖基化修饰对其结构的改变有关。     

人嗜碱性粒细胞肿瘤细胞系KU812F检测牛乳及其替代品致敏性方法的建立

比较牛乳及其替代品释放组胺的能力,以建立体外细胞检测牛乳及替代品疑似过敏原组分致敏性的检测方法。以嗜碱性粒细胞KU812F为研究对象,通过牛乳过敏患者血清的孵育,使细胞处于致敏状态,再以牛乳及其替代品过敏原作为激发物,使细胞脱颗粒释放组胺。结果表明：利用嗜碱性粒细胞KU812F检测牛乳及替代制品组胺释放量的方法具有较高的可靠性及重现性,细胞释放组胺的量与过敏原的剂量相关,在一定的剂量范围内,组胺的释放率与过敏原的剂量呈正比,组胺释放率达到最大之后,组胺释放率与剂量呈负相关。     

高盐刺激嗜碱性粒细胞产生促炎因子及其分子机制

本研究以食物过敏重要的效应细胞嗜碱性粒细胞（KU812）为对象，探究高盐条件对嗜碱性粒细胞脱颗粒以及相关细胞因子表达的影响，并初步探讨其分子机制。首先利用酶联免疫吸附试验（enzyme linked immunosorbent assay,ELISA）和实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction,qPCR）分析高盐条件下KU812细胞脱颗粒释放生物活性介质和促炎细胞因子的变化；并用q PCR分析血清和糖皮质激素调节蛋白激酶1(serum and glucocorticoid-regulated kinase 1,SGK1)抑制剂和P38抑制剂对高盐条件下KU812细胞产生白细胞介素（interleukin,IL）-4的影响。结果表明，高盐条件并不能促进KU812细胞脱颗粒释放生物活性介质组胺和β-氨基己糖苷酶，但能促进其产生促炎因子IL-6和肿瘤坏死因子（tumor necrosis factor,TNF）-α；高盐条件也可以促进KU812细胞产生IL-4，同时伴随着SGK1基因...     

4种婴幼儿奶粉的消化模式及致敏性评估

目的全面评估4种市售婴幼儿奶粉消化前后致敏性变化。方法通过间接竞争酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELLSA),建立体外KU812细胞模型检测KU812细胞脱颗粒释放的生物活性介质:β-HEX、组胺、IL-6以及胞内Ca~(2+)浓度变化。结果通过间接竞争ELISA法比较4种奶粉肠消化60 min产物IC50值可以得出抗原性的强弱关系为:FN≈AQ。但总体4种奶粉的消化产物致敏性差异并不明显。细胞实验中, A和F奶粉消化后所测4种细胞因子含量均下降,说明消化能降低A和F奶粉的致敏性;而N和Q奶粉消化后所测4种细胞因子含量变化规律不明显。结论 2种实验方法的结果为开发低致敏婴幼儿乳品配方提供了基本理论数据。     

Inhibitory effects of blueberry root methanolic extract on degranulation in KU812F cells

The effect of blueberry (Vaccinium angustifolium) root methanolic extract (BRM) on the A23187 plus phorbol myristate acetate (PMA)-induced degranulation in human basophilic KU812F cells was investigated. The total phenolic content (TPC) was 170±1.9 mg gallic acid equivalents (GAE)/g BRM. BRM reduced levels of histamine and β-hexosaminidase released from stimulated KU812F cells. Intracellular calcium concentration ([Ca²⁺]i) were determined by spectrofluorometric analysis using Fura 2-AM was found to be reduced by BRM with dose-dependent manner. Moreover, Western blot analysis revealed that protein kinase C (PKC) translocation was inhibited by BRM with dose-dependent manner. These results indicated that BRM inhibited histamine and β-hexosaminidase through the suppression of Ca²⁺ influx and PKC translocation. Therefore, we suggest that BRM is potent inhibitor of degranulation in mast cells and basophils, and may be useful in preventing the allergic reactions.     

Glycation and phosphorylation of α-lactalbumin by dry heating: Effect on protein structure and physiological functions

α-Lactalbumin (α-LA) was glycated with maltopentaose (MP) through the Maillard reaction (MP-α-LA) and subsequently phosphorylated by dry heating in the presence of pyrophosphate to investigate its structure and physiological functions. Glycation occurred effectively, and the sugar content of α-LA increased by approximately 22.3% through the Maillard reaction. The phosphorylation of MP-α-LA was enhanced with an increase in the dry-heating time from 1 to 5 d, and the phosphorous content of MP-α-LA increased by approximately 1.01% by dry heating at pH 4.0 and 85°C for 5 d in the presence of pyrophosphate. The electrophoretic mobility of α-LA increased with an increase in the phosphorylation level. The circular dichroism spectra showed that the change in the secondary structure of the α-LA molecule by glycation and subsequent phosphorylation was slight. However, the Trp fluorescence intensity was increased by phosphorylation after glycation. In addition, the differential scanning calorimetry thermograms of α-LA showed that the denaturation temperature of MP-α-LA was decreased by phosphorylation. These results indicated that molten (partially unfolded) conformations of α-LA were formed by dry heating in the presence of pyrophosphate after glycation. The anti-α-LA antibody response was significantly reduced by glycation and subsequent phosphorylation. The suppressive effect of α-LA on the production of proinflammatory cytokines such as IL-6 and tumor necrosis factor-α from THP-1 cells after stimulation with lipopolysaccharide was significantly enhanced by glycation with MP and was further enhanced by phosphorylation after glycation. The Ca phosphate-solubilizing ability of α-LA was enhanced by phosphorylation. The apoptotic activity of α-LA was reduced by glycation and subsequent phosphorylation. These results suggest that phosphorylation by dry heating in the presence of pyrophosphate after glycation with MP through the Maillard reaction is a useful method for improvement of the physiological functions of α-LA.     

Improvement of functional properties of bovine serum albumin through phosphorylation by dry-heating in the presence of pyrophosphate

ABSTRACT:  Bovine serum albumin (BSA) was phosphorylated by 2 methods. One is dry-heating in the presence of pyrophosphate, and the other is conjugation with maltopentaose through the Maillard reaction and subsequent dry-heating in the presence of pyrophosphate. The phosphorus content of BSA was increased to approximately 0.45% by dry-heating at pH 4.0 and 85 °C for 5 d in the presence of pyrophosphate, and approximately 0.91% by glycation and subsequent phosphorylation. The circular dichroism spectra showed that the change of secondary structure in the BSA molecule by phosphorylation was mild. However, tryptophan fluorescence intensity of BSA decreased by phosphorylation. The differential scanning calorimetry thermograms of BSA showed a disappearing of the 1st peak and a lowering of the 2nd peak denaturation temperature by phosphorylation. These results indicated molten (partially unfolded) conformations of BSA formed by both phosphorylation methods. The functional properties of BSA such as heat stability and calcium phosphate solubilizing ability were improved by phosphorylation alone and further by phosphorylation after glycation. Transparent gels of BSA with relatively high water-holding capacity were obtained by phosphorylation alone, and the immunogenicity of BSA was reduced significantly by glycation and phosphorylation, respectively.     

磷酸化对鲢小清蛋白抗原性降低的作用

通过建立鱼类主要过敏原小清蛋白（parvalbumin,PV）磷酸化反应的条件,研究磷酸化对PV的抗原性及结构影响。将PV和葡萄糖-6-磷酸二钠盐（D-glucose-6-phosphate disodium salt hydrate,G6P-Na2）混合,在不同质量比、反应时间以及反应温度条件下进行干法磷酸化反应。采用Tricine-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Dot-blotting检验其聚合类型以及抗原性变化。通过扫描电镜对聚合物的微观结构进行分析,利用圆二色谱仪和ANS探针法分析磷酸化产物的二级结构和疏水性变化。结果显示,在PV与G6P-Na2质量比为1∶4、反应温度80?℃、反应时间80?min条件下生成的磷酸化产物的抗原性最低。磷酸化反应后,产物呈现聚集状态,其二级结构变化较为明显,疏水性明显提高。磷酸化反应对蛋白结构的改变可能是影响PV抗原性降低的主要原因。     

Glycation of bovine β-Lactoglobulin: effect on the protein structure

Summary Since temperature and water activity are among the most important parameters that affect the Maillard reaction, the glycation sites in pure, native bovine β-lactoglobulin were determined after a mild heat treatment (60 °C) in an aqueous solution and after a treatment under a restricted water environment (50 °C, 65% relative humidity). In both systems, the results obtained underlined the structural heterogeneity of β-lactoglobulin (β-LG) glycoforms with respect to the number of lactose residues linked per protein molecule and to the binding sites involved. Subsequently, the effect of the glycation conditions on both the association behaviour and the conformational changes of the glycated β-LG were characterised by proteolytic susceptibility, binding of the fluorescent probe 8-anilino-1-naphtalene-sulfonic acid, SDS-PAGE and size exclusion chromatography. The results showed that dry-way glycation did not significantly alter the native-like behaviour of the protein while the treatment in solution led to important structural changes. These changes resulted in a specific denatured β-LG monomer, which covalently associated via the free thiol group. The homodimers thus formed and the expanded monomers underwent subsequent aggregation to form high molecular weight species, via non–covalent interactions. The use of monoclonal antibodies with defined epitopes, raised against native β-LG, confirmed that the protein conformation was much more modified when glycation was performed in a solution while the structural changes induced during dry-way treatment were limited to the AB loop region of the protein.     

Conformation affects the potential allergenicity of ovalbumin after heating and glycation

Ovalbumin (OVA), one of the major allergens in hen egg white, and has widespread use in experimental models of allergy. The aim of this research was to assess the effect of glycation and heat treatment on the potential allergenicity of OVA prepared from hen egg white. Secondary and tertiary structures of OVA were also characterised to show the relationship between potential allergenicity and the conformation of OVA after heating and glycation. Glycation significantly reduced the potential allergenicity of OVA tested with egg allergy patients’ sera, which was caused by conformation changes. An increased IgG reactivity was measured using rabbit anti-OVA and was supposed to be caused by protein unfolding which exposed hidden epitopes. Heating reduced the potential allergenicity of OVA at the expense of increased IgG reactivity. It is suggested that conformational changes of OVA induced by glycation and controlled heating significantly reduced its potential allergenicity.     

Stability and bioaccessibility of curcumin emulsions stabilized by casein hydrolysates after maleic anhydride acylation and pullulan glycation

The effects of maleic anhydride (MA) acylation and pullulan glycation on casein hydrolysates (CH) and the physicochemical stability of modified or unmodified CH-stabilized emulsions were explored. Compared with casein, the solubility of CH was improved, and CH₁ (hydrolysis degree 4%) exhibited the optimal emulsifying properties. After the acylation of MA, degrees of acylation (DA) increased with increasing addition of MA. Fourier-transform infrared spectroscopy revealed that a covalent bond was formed between MA and CH₁. The results of pullulan glycation indicated that the degree of glycation decreased with increasing DA. Acylation combined with glycation effectively reduced the surface hydrophobicity of CH. Results of analysis of physicochemical stability and gastrointestinal fate of curcumin in emulsions revealed that CH modified by MA acylation and pullulan glycation played a positive role in enhancing the stability and bioaccessibility of curcumin loaded in emulsions.     

高压脉冲电场结合糖基化对β-乳球蛋白过敏原性与功能性质的影响

采用高压脉冲电场(pulsed electric field,PEF)结合糖基化处理β-乳球蛋白(β-lactoglobulin,β-Lg),在β-Lg与半乳糖质量比1:2、反应温度60℃、反应时间3 h、p H 7.4和不同PEF强度反应条件下,研究改性前后β-Lg的过敏原性和功能性质的变化。结果表明,经过PEF结合糖基化处理后,β-Lg自由氨基含量显著降低,β-Lg过敏原性有一定程度下降,15 kV/cm PEF处理90μs后进行糖基化,过敏原性降低程度最大;在此条件下,β-Lg的溶解性、乳化性、乳化稳定性及抗氧化活性显著提高(P0.05)。这为制备低致敏性且功能性质好的β-Lg提供了一种新的方法。