紫苏叶提取物对美拉德体系晚期糖基化终末产物生成的抑制作用研究

目的 探究紫苏叶提取物(Perilla frutescens leaf extract,PLE)对美拉德体系晚期糖基化终末产物(advanced glycation end-products, AGEs)的抑制作用。方法 通过构建葡萄糖-赖氨酸模型体系,利用液相色谱-串联质谱法探究其在加热过程中PLE对N^ε-羧甲基赖氨酸[N^ε-(1-carboxymethyl)-L-lysine,CML]、N^ε-羧乙基赖氨酸[N^ε-(1-carboxyethyl)-L-lysine, CEL]和吡咯素(pyrraline, Pyr) 3种AGEs的抑制作用,同时通过评价PLE的抗氧化性、测定PLE对α-二羰基化合物抑制率,以探讨PLE对AGEs的可能抑制途径。结果 PLE对AGEs的生成具有显著的抑制作用,其抑制率与PLE的添加浓度成正比,当添加量为2.0 mg/mL时,对CML、CEL和Pyr的抑制率分别为47.23%、29.59%和37.77%。PLE中的主要活性物质为迷迭香酸、芹菜素-7-O-二葡萄糖苷和野黄...     

亚油酸对食品加工中晚期糖基化终产物的影响

研究一种不饱和脂肪酸——亚油酸对糖基化反应体系主要中间产物及终产物的作用,探讨在食品加工中不饱和亚油酸对晚期糖基化产物的影响。建立亚油酸、D-葡萄糖和L-赖氨酸反应的微乳体系,采用紫外-可见吸光光度法检测中间产物乙二醛、丙二醛和果糖胺的生成量;荧光法检测戊糖素和荧光性晚期糖基化终产物(advanced glycation end-products,AGEs)的生成量;高效液相色谱法检测终产物羧甲基赖氨酸[Nε-(carboxymethyl)lysine,CML]的生成量,分别比较各生成产物的变化,探讨亚油酸对反应途径的影响。结果显示:从中间产物上看,亚油酸能有效参与羰胺的糖基化反应,提高中间产物乙二醛含量,增加丙二醛含量,减少果糖胺的生成;从终产物来看,亚油酸的存在对戊糖素的生成量影响不明显,但能明显抑制羧甲基赖氨酸的形成并抑制荧光性AGEs的累积。在糖基化反应体系中亚油酸的存在一方面能够氧化葡萄糖,使参与羰胺反应的葡萄糖减少;另一方面因氧化促进了乙二醛等活性醛的生成,从而加速乙二醛与赖氨酸之间的反应,减少了参与羰胺反应的赖氨酸含量;亚油酸参与反应使中间产物丙二醛含量增加,进而增加了羧甲基赖氨酸同源的羧乙基赖氨酸(CEL)的生成量,减少了非荧光性产物羧甲基赖氨酸和荧光性AGEs生成量。     

加工条件对果糖-赖氨酸体系中羧甲基赖氨酸和羧乙基赖氨酸形成的影响

晚期糖基化终产物(AGEs)是食品加工与贮藏过程中蛋白质与还原糖发生非酶褐变所生成的化合物,已证实与多种疾病相关。本文主要研究加工条件对晚期糖基化终产物羧甲基赖氨酸(CML)和羧乙基赖氨酸(CEL)形成的影响。采用果糖-赖氨酸模拟体系,使用同位素内标法进行LC-MS/MS分析,分析底物配比【果糖/赖氨酸,0.25∶(1~4∶1)】、反应液p H值(4.0~8.0)、加热温度(20~120℃)和加热时间(20~120 min)对CML及CEL形成的影响。CML和CEL的含量分别在果糖与赖氨酸比1∶1以及4∶1时达到最大值4.26μg/m L和5.39μg/m L;在p H 4.0~8.0范围,CML与CEL的含量都随p H值的增加而增加;在温度20~120℃范围,CML及CEL含量都随温度的升高而增加;100℃时,CML和CEL生成量均随加热时间的延长呈现先增加后不变的趋势,且在反应60min时,分别达到最大值4.94μg/m L和3.34μg/m L。适当的底物配比、碱性p H值、高温和反应时间都加剧CML和CEL的生成。     

大黄素对食品模型中晚期糖基化终产物的抑制作用

通过建立L-赖氨酸-葡萄糖(Lys-Glc)反应体系,以大黄素作为抑制剂,利用NBT还原法、紫外分光光度法、高效液相色谱法及荧光分光光度法分别研究大黄素对果糖胺、乙二醛(GO)、5-羟甲基糠醛(5-HMF)、类黑精、羧甲基赖氨酸(CML)及荧光性AGEs和戊糖素的抑制作用。结果表明:大黄素对氧化产物及由氧化产物进一步生成的中、末期产物GO、CML、类黑素有较好的抑制作用,对果糖胺、5-HMF有一定的抑制作用。大黄素对戊糖素、荧光性AGEs无明显抑制作用,延迟其产生。可见大黄素作为一种蒽醌类物质,对糖基化反应中氧化产物的抑制作用与其抗氧化性有关。     

添加糖与酱油对油炸鲟鱼晚期糖化终末产物形成的影响研究

本论文首次分析糖和酱油添加对油炸鲟鱼晚期糖化终末产物（AGEs）形成的影响。以鲟鱼肉为原料，添加绵白糖（0%、1%、2%、3%）、酱油（0%、1%、2%）并制备油炸鲟鱼饼（170 ℃/9 min），分析鲟鱼饼AGEs以及其前体物质糠氨酸、二羰基化合物的变化规律。结果表明，仅添加绵白糖时，油炸鲟鱼饼AGEs以及其前体物质糠氨酸、二羰基化合物含量随绵白糖添加水平增加而逐渐增加；仅添加酱油时，油炸鲟鱼饼糠氨酸、荧光AGEs、羧乙基赖氨酸（CEL）含量随着酱油添加水平增加而逐渐增加，而羧甲基赖氨酸（CML）、甲基乙二醛氢咪唑酮（MG-H1）含量随着酱油添加水平的增加而显著减少（P<0.05），添加酱油对AGEs抑制作用与酱油成分对二羰基化合物消耗有关。当绵白糖和酱油同时添加时，进一步促进糠氨酸、荧光AGEs和MG-H1的形成，两者对油炸鲟鱼饼AGEs形成具有显著交互作用（P<0.05）。因此，添加绵白糖会促进AGEs的形成，而酱油由于其复杂成分导致其添加对不同AGEs标志物形成影响各异。这些工作将为油炸水产品AGEs形成与控制提供理论依据与指导。     

反应条件对葡萄糖-甘氨酸模拟美拉德反应体系中α-二羰基化合物生成的影响

采用邻苯二胺(OPD)衍生和HPLC定性定量考察了反应条件(温度、p H、时间和底物浓度比)对葡萄糖-甘氨酸模拟美拉德反应体系中三种α-二羰基化合物3-葡萄糖醛酮(3-DG)、乙二醛(GO)和丙酮醛(MGO)生成的影响。结果表明:在80~140℃范围内温度的升高会促进体系中MGO和GO的生成,而3-DG在温度达到110℃时达到峰值,随后则开始下降。p H在5~9范围内对三种物质的影响趋势和温度类似,MGO和GO的生成随p H的升高而不断增加,而3-DG在p H为6时含量达到最高,随后急剧降低。3-DG和GO生成的速率随着时间的延长逐渐降低,而在2 h内MGO的生成速率则趋于恒定。葡萄糖的相对含量升高有利于3-DG的生成,而当甘氨酸的相对含量较高时则会促进MGO和GO的生成。本研究能够为美拉德反应中以α-二羰基化合物为中间产物的风味物质的生成机制研究提供一定的实验依据。     

基于蛋白沉淀法超高效液相色谱-串联质谱法检测面包中两种蛋白结合型晚期糖基化终末产物

目的 建立面包中两种典型晚期糖基化终末产物（AGEs）的超高效液相色谱-串联质谱检测方法。方法以结合型羧甲基赖氨酸（CML）和羧乙基赖氨酸（CEL）为检测对象，面包样品经还原孵育、蛋白沉淀、加同位素内标、酸水解、氮吹、九氟戊酸水溶液复溶后，多反应监测定性及定量分析。结果 方法的检出限CML为4.5 ng/g,CEL为0.5 ng/g；定量限CML为20 ng/g,CEL为2 ng/g;CML加标回收率为89.62%～95.65%,CEL的加标回收率为86.38%～97.17%；标准曲线线性范围为CML2.5～800 ng/mL和CEL0.25～80 ng/mL，决定系数均大于0.999。结论 该方法准确且灵敏，能够满足面包中AGEs的检测需求。     

山奈、白芷和香叶对猪肉加热过程中晚期糖化终末产物含量的影响

采用超高效液相色谱—串联质谱联用仪结合羧甲基赖氨酸(Nε-carboxymethyllysine,CML)和羧乙基赖氨酸(Nε-carboxyethyllysine,CEL)的同位素内标物作为AGEs的分析方法,研究了山奈、白芷和香叶3种食用香料的添加以及加热(100℃,5～120 min)对猪肉中结合态CML和CEL含量的影响。研究表明:加热对猪肉中AGEs的产生有促进作用,且AGEs的含量随着加热时间的延长而增加,经120min加热后的猪肉中CML和CEL的含量约为生肉的5～8倍,分别达到(35.60±2.20)～(40.68±5.66)mg/kg(CML)和(13.32±1.49)～(16.85±2.44)mg/kg(CEL)。在0～40min的加热时间过程中,加入或未加入香料的猪肉中CML[R~2=0.971～0.996;k=0.355～0.590mg/(kg·min)]和CEL[R~2=0.942～0.997;k=0.150～0.368mg/(kg·min)]的形成均符合零级反应动力学方程。香料的添加对于猪肉在加热过程中AGEs形成的影响因香料的种类、加热时间、肉的批次的不同而不同,总体来说,香料对猪肉加热过程中CML的形成影响较小,但是对CEL的形成有一定的促进作用,尤其是加热5 min处理的肉样,其CEL含量增加13%～61%。     

反应条件对糖-酸反应体系中3-脱氧葡萄糖醛酮及5-羟甲基糠醛形成的影响

为了分析糖-酸反应体系中3-脱氧葡萄糖醛酮（3-deoxyglucosone,3-DG）及5-羟甲基糠醛（5-hydroxymethylfurfural,5-HMF）的形成规律,本研究构建了果糖/蔗糖/葡萄糖-柠檬酸反应体系,探究糖种类、pH、温度、金属离子种类和含不同价态硫的化合物对3-DG及5-HMF形成的影响,并分析了二者的形成动力学。结果表明:三种糖对3-DG和5-HMF形成的影响不同,果糖和蔗糖反应体系中3-DG和5-HMF生成量远高于葡萄糖反应体系,pH降低和温度升高可促进3-DG和5-HMF形成;Ca2+、Mg2+、Al3+均对3-DG和5-HMF的形成有促进作用;K+能促进3-DG的形成,但对5-HMF的形成无显著影响（P>0.05）。Na₂S₂O₃和Na₂SO₃可以抑制3-DG及5-HMF的形成,而Na₂S₂O₅对3-DG的形成无影响,但能抑制5-HMF的形成（P<0.05）;Na₂SO₄可促进3-DG及5-HMF的形成;70~90 ℃时3-DG的形成符合零级动力学模型,而100 ℃时符合二级动力学模型;70~100 ℃时5-HMF的形成符合零级动力学模型。     

L-半胱氨酸盐酸盐对饼干中4种有害醛类和荧光晚期糖基化终产物的影响

将食品添加剂L-半胱氨酸盐酸盐添加到面团中制作饼干,研究了添加不同量(0.3,0.6,1.0,1.5 g/kg)的L-半胱氨酸盐酸盐对饼干外观、4种有害醛类和荧光晚期糖基化终产物(AGEs)含量的影响。结果表明:L-半胱氨酸盐酸盐能使饼干色泽变亮,pH小幅下降。L-半胱氨酸盐酸盐显著降低5-羟甲基糠醛(HMF)、3-脱氧葡萄糖醛酮(3-DG)、乙二醛(GO)、丙酮醛(MGO)和荧光AGEs含量且呈剂量效应。当L-半胱氨酸盐酸盐的添加量为0.3 g/kg时,饼干中HMF、3-DG、GO和MGO含量分别降低84.7%,13.7%,82.0%,72.1%,荧光AGEs降低8.9%。     

Identification and quantification of α-dicarbonyl compounds produced in different sugar-amino acid Maillard reaction model systems

A mixture of α-dicarbonyl compounds generated from Maillard reaction (MR) model systems, comprising of fructose with glycine (Fru-Gly) and lysine (Fru-Lys); glucose with glycine (Glu-Gly) and lysine (Glu-Lys); ribose with glycine (Rib-Gly) and lysine (Rib-Lys), were identified and quantified. α-Dicarbonyl compounds generated in the hexose models were predominantly glucosone and 3-deoxyglucosone (3-DG), with 3-deoxypentosone (3-DP) and pentosone being the major α-dicarbonyls produced in the pentose models. Ethyl acetate extraction of model MRPs resulted in poor recovery of 2,3-diaminonaphthalene benzoquinoxalines of glucosone, 3-DG, pentosone, 3-DP and methylglyoxal (MGO), respectively. The temporal profiles of pentosone, 3-DP, glyoxal (GO) and MGO were similar in the pentose models, with maximum concentration occurring within 5 min. These four α-dicarbonyl compounds were higher (P < 0.05) in Rib-Gly than in Rib-Lys MR systems. The quantity of α-dicarbonyl compounds was affected by the interaction among the type of sugar and amino acid and the reaction time. The type of sugar is the most important factor that affected both the quantity and quality of α-dicarbonyl compounds produced in MR mixtures.     

色氨酸抑制体外模型中晚期糖基化终末产物形成机理

食源性晚期糖基化终末产物(advanced glycation end products,AGEs)与多种慢性疾病密切相关,尤其是糖尿病和肾脏疾病.为减少AGEs对机体的健康风险,降低食品中AGEs含量,本研究从13种氨基酸中筛选出具有良好AGEs抑制效果的色氨酸(tryptophan,Trp),利用牛血清白蛋白(bo...     

冻融及加热过程鲢鱼鱼糜制品中晚期糖化终末产物的形成机制

将鲢鱼（Hypophthalmichthys molitrix）鱼糜在不同冻藏温度下进行冻融循环后加热，分析不同加热过程鱼糜制品中壬醛、1-辛烯-3-醇、内源性荧光物质、蛋白质亚基组成、赖氨酸、α-二羰基化合物（乙二醛（glyoxal,GO）和丙酮醛（methylglyoxal,MGO））及晚期糖化终末产物（advanced glycation end-products,AGEs）-羧甲基赖氨酸（Nε-carboxylmethyl-lysine,CML）和荧光AGEs的变化情况。结果显示，冻藏温度对鱼糜制品中各指标含量无显著影响（P>0.05）；随着冻融次数的增加，鱼糜制品中壬醛、1-辛烯-3-醇、内源性荧光物质、CML和荧光AGEs含量显著增加，赖氨酸含量增加，GO、MGO含量显著降低（P<0.05）；加热过程中，1-辛烯-3-醇、GO、MGO、CML和荧光AGEs含量显著增加，内源性荧光物质显著降低（P<0.05），赖氨酸含量先增加后降低，肌球蛋白重链逐渐降解成小分子蛋白。以上结果表明原料的冻融循环为鱼糜制品中AGEs的形成提供大量前体物质；热加工过程进一步促进了...     

Effect of combined UV-thermosonication and Ecklonia cava extract on advanced glycation end-products in soymilk

Thermal treatment is used to inactivate microbes in soymilk, but it increases the amount of advanced glycation end products (AGEs). Therefore, this study examined if ultraviolet light with thermo-sonication (UVTS) and Ecklonia cava extract (EX) could provide an alternative process to prevent AGEs formation in processed soymilk. A coiled tube UV reactor was used simultaneously with an ultrasonic generator for UVTS treatment, while an autoclave was employed for thermal treatment. UVTS treatment was examined at different temperatures and flow rates to achieve a 5-log reduction of pre-inoculated Escherichia coli and Salmonella typhimurium in soymilk. After confirming EX's anti-glycation effects against fructosamine, ɑ-dicarbonyl compounds, protein carbonyl content, and AGEs formation, it was added to soymilk before the UVTS. The ɑ-dicarbonyl compounds (glyoxal and methylglyoxal) and AGEs (N^ε-(1-carboxymethyl)-l -lysine [CML] and N^ε-(1-carboxyethyl)-l -lysine [CEL]) in soymilk increased after autoclaving (AC). Compared with AC, the UVTS with .05% EX decreased glyoxal, methylglyoxal, CML, and CEL by 78%, 82%, 32%, and 59%, respectively. These results indicated that UVTS with EX could be an alternative pasteurization process for soymilk that minimizes AGEs formation.     

Effects of oxidised linoleic acid on the formation of Nε‐carboxymethyl‐lysine and Nε‐carboxyethyl‐lysine in Maillard reaction system

This study investigated the effects of oxidised linoleic acid (18:2) on N^ε‐carboxymethyl‐lysine (CML) and N^ε‐carboxyethyl‐lysine (CEL) formation in Maillard reaction systems. Model systems of lysine/glucose (L/G), lysine/18:2 (L/18:2), lysine/18:2/glucose (L/18:2/G), myofibrillar protein/glucose (MFP/G), MFP/18:2 and MFP/18:2/G were maintained at 37 °C for 6 weeks. The results showed that CML/CEL contents in L/G (6.99 and 0.96 mmol mol^?1 lysine, respectively) were significantly higher than those in L/18:2/G (1.43 and 0.41 mmol mol^?1 lysine, respectively), and there is a small amount of CML/CEL generation in L/18:2. However, the CML/CEL levels in MFP/G (197.2 and 83.8 ng mg^?1 protein, respectively) were markedly lower than those in MFP/18:2/G (283.2 and 118.5 ng mg^?1 protein, respectively). 18:2 favours the formation of CML/CEL in MFP/18:2/G, not in L/18:2/G. All these findings indicated that the role of 18:2 on CML/CEL formation in Maillard reaction system was complex, and depended on CML/CEL formation rate and substrate types (lysine or lysine residue in protein).     

市售鱼加工食品中3 种晚期糖基化终末产物分析及与组分的相关性

分析市售烘烤类、油炸类、干制类、罐头类等鱼加工食品中羧甲基赖氨酸（Nε-carboxymethyllysine,CML）、羧乙基赖氨酸（Nε-carboxyethyllysine,CEL）、甲基乙二醛氢咪唑酮（Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine,MG-H1）3 种晚期糖基化终末产物（advanced glycation end products,AGEs）含量及与组分的相关性。使用ACQUITY UPLC BEH Amide色谱柱建立超高效液相色谱-串联质谱检测AGEs的方法,应用该方法分析65 种市售鱼加工食品AGEs含量。结果表明,鱼加工食品中MG-H1含量高于CEL和CML含量,MG-H1含量为CEL含量的2～11 倍、CML含量的2～20 倍;罐头类鱼加工食品中AGEs含量呈较高水平;油炸类鱼加工食品中乙二醛、甲基乙二醛（methylglyoxal,MGO）含量相对较高。通过主成分分析与相关性分析发现,鱼加工食品中3 种AGEs含量间呈显著正相关（r≥0.54,P＜0.001）;其AGEs含量与脂肪含量呈显著正相关,与蛋白含量呈显著负相关;烘烤类、干制类鱼加工食品中MG-H1含量与MGO含量呈正相关。     

Simultaneous Analysis of Nε‐(Carboxymethyl)Lysine and Nε‐(Carboxyethyl)Lysine in Foods by Ultra‐Performance Liquid Chromatography‐Mass Spectrometry with Derivatization by 9‐Fluorenylmethyl Chloroformate

Isotope dilution ultra‐performance liquid chromatography–electrospray tandem mass spectrometry with derivatization by 9‐fluorenylmethyl chloroformate was successfully applied to quantify N^ε‐(carboxymethyl)lysine (CML) and N^ε‐(carboxyethyl)lysine (CEL) in processed foods. We demonstrate that this analytical method is well validated for the determination of CML and CEL contents in processed foods. Relative standard deviations (RSD) indicate repeatability (RSD < 6% for CML and CEL) and reproducibility (RSD < 6% for CML and < 7% for CEL) in this method. Percent recovery is also good. We obtain recoveries of 102% to 112% for CML and 86% to 114% for CEL. CML levels detected in the samples vary from 2.29 to 480 mg/kg food, whereas CEL is detected in significantly lower concentrations ranging from 0.56 to 107 mg/kg food. These data could help consumers make better food choices by monitoring intake of advanced glycation end‐products, which may pose a risk to human health.     

Radio fluorination and positron emission tomography (PET) as a new approach to study the in vivo distribution and elimination of the advanced glycation endproducts Nϵ‐carboxymethyllysine (CML) and Nϵ‐carboxyethyllysine (CEL)

After synthesis of fluorine‐18 labelled analogues by [¹⁸F]fluorobenzoylation at the α‐amino group, biodistribution and elimination of individual advanced glycation endproducts, namely N^ε‐carboxymethyllysine and N^ε‐carboxyethyllysine, were studied in comparison to lysine in rats after intravenous injection using positron emission tomography (PET). The [¹⁸F]radiofluorinated amino acids were fast distributed via the blood, followed by a rapid excretion through the kidneys. Elimination kinetics were similar for both AGEs and lysine. For CML and CEL, but not for lysine, a temporary liver accumulation could be observed, which was not connected with any metabolisation or enterohepatic circulation. No further accumulation in any tissues was observable, indicating that increased tissue levels of CML or CEL, which have been described for certain disorders, are exclusively derived from endogenous origin and should not depend on a dietary intake. However, under uremic conditions, an impaired kidney function might result in a significant increase of the AGE‐load of blood and tissues. PET based on¹⁸F‐labelled AGEs proved to be a promising tool to elucidate the physiological fate of post‐translationally modified amino acids and to clarify the role of AGEs as possible “glycotoxins”.     

Selective fortification of lysinoalanine,fructoselysine and Nϵ‐carboxymethyllysine in casein model systems

In the present study, a promising strategy to study nutritional effects of selected chemical reaction products formed in heat treated protein containing foods is addressed. In due course, a selective fortification of different marker compounds for lysine damage in casein‐sugar mixtures was performed to provide model systems being applicable to investigate biological effects of the cross‐link lysinoalanine (LAL( the MRPs fructoselysine (FL) and N^ε‐carboxymethyllysine (CML) in a casein‐linked preparation. The three different model proteins, casein‐LAL, casein‐FL and casein‐CML were prepared by heating casein either in strong alkaline conditions at 105°C for 1 h, in the presence of glucose at 65°C for 68 h, or in the presence of glyoxylic acid at 37°C for 19 h. Finally, the degree of lysine modification achieved was 39%, 75% and 55% for the casein‐LAL, casein‐FL and casein‐CML, respectively. The calculation of lysine recovery and the respective analysis of each single modified casein (LAL‐, FL‐ and CML‐MP) for the selected fortified compound and each other compound vice versa proved that the individual procedure provides a specific fortification for LAL, FL and CML, respectively. The modified proteins are suitable as reference model proteins to be investigated for specific biological and toxicological effects of casein‐linked LAL, FL and CML.