基于UPLC-QqQ-MS/MS同步检测热加工食品中典型晚期糖基化终末产物

建立超高效液相色谱-三重四极杆串联质谱法测定热加工食品中羧甲基赖氨酸（Nε-carboxymethyllysine,CML）和羧乙基赖氨酸（Nε-carboxyethyllysine,CEL）含量的同步检测方法。首先脱脂样品经硼氢化钠还原8 h,沉淀蛋白,酸水解后,经Oasis MCX固相萃取小柱净化和富集,以乙腈和含0.1%甲酸的1 mmol/L乙酸铵溶液为流动相梯度洗脱,采用ACQUITY UPLC HSS T3-C18色谱柱分离,多反应监测模式进行定性定量分析。CML和CEL在0.25～500 ng/mL范围内线性良好,相关系数高于0.999,方法检出限分别为8 ng/g和10 ng/g,定量限分别为36 ng/g和40 ng/g。平均回收率分别为96%～103%和94%～107%,相对标准偏差分别为1.48%～2.43%和1.23%～1.84%。利用该方法检测13 种市售热加工食品发现,婴儿肉松和婴儿饼干中CML和CEL含量显著高于烘焙食品和油炸食品（P＜0.05）。结果表明该方法快速、准确、高效,适用于热加工食品中CML和CEL的同步快速检测。     

UPLC-MS/MS分析食物煎炸过程中2种晚期糖基化终末产物的研究

建立了一种分析食用油煎炸食物中羧甲基赖氨酸(CML)和羧乙基赖氨酸(CEL)含量的超高效液相色谱-串联质谱法检测方法,并研究了煎炸温度、时间以及不同食用油对CML和CEL含量的影响。样品经硼氢化钠还原4 h后,采用HLB固相萃取小柱进行净化和富集,加标回收率达到86.0%～92.9%。CML和CEL在0.01～1.00μg/mL浓度范围内线性良好,相关系数R~2为0.999 4～0.999 5,检出限均为15.0μg/kg,定量限均为45.0μg/kg。在煎炸温度100～200℃,两者生成量随温度的升高而增加;在煎炸时间10～60 min,两者生成量随煎炸时间的延长呈现先增加后不变的趋势,在40 min时达到最大值;在比较不同食用油煎炸食物中发现,橄榄油煎炸的食物中CML和CEL含量最低。     

三文鱼中晚期糖基化终末产物含量测定及产地特征因子分析

建立超高效液相色谱-串联三重四极杆质谱同时检测三文鱼中羧甲基赖氨酸（N-carboxymethyllysine,CML）和羧乙基赖氨酸（N-carboxyethyllysine,CEL）的分析方法。色谱柱为ACQUITY UPLC HSS T3柱（2.1?mm×150?mm,1.8?μm）,流动相A为100%乙腈、B为0.1%甲酸,电喷雾离子源正离子模式扫描,多反应监测模式定性分析,同位素内标稀释法定量;样品经脱脂、还原、水解和Oasis?MCX固相萃取柱净化等步骤后上机分析;方法平均回收率为90%～95%,精密度相对标准偏差小于5%,CEL检出限为0.032?7?ng/g,定量限为0.142?2?ng/g。采用该法对来自挪威、智利和法罗群岛的21?份三文鱼样品进行含量测定,并对所获数据进行主成分分析。结果表明,即使是相同品种的三文鱼在不同国家和不同个体之间CML和CEL含量存在很大的生物差异性,并且发现CEL有望成为鉴别挪威三文鱼和智利、法罗群岛三文鱼的产地特征因子。     

绿茶中两种晚期糖基化终末产物同步检测研究

针对绿茶基质特性,采用HPLC-MS/MS技术构建绿茶中羧甲基赖氨酸(CML)和羧乙基赖氨酸(CEL)同步检测方法。结果表明,样品前处理时最优硼氢化钠还原时间为8 h,酸水解时间为24 h。方法在10~300 ng/m L浓度范围内线性关系良好,相关系数在0.999以上,检出限为1.0 ng/mg,定量限为5.0 ng/mg,加标回收率在76%~89%之间,日内精密度和日间精密度均在10%以内,表明该检测方法的准确性、重现性、稳定性较好,适于绿茶中CML和CEL的实际检测。     

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%。     

超高效液相色谱-稳定性同位素稀释质谱法测定预包装食品中不同阶段美拉德反应产物含量

建立基于稳定同位素内标稀释的超高效液相色谱-串联三重四极杆质谱同步检测预包装食品中羧甲基赖氨酸(N^ε-(carboxymethyl)-lysine,CML)、羧乙基赖氨酸(N^ε-(carboxyethyl)-lysine,CEL)、甲基乙二醛-羟基咪唑酮异构体(methyglyoxal-derived hydroimidazolones,MG-H1、MG-H2、MG-H3)、乙二醛-氢咪唑酮(glyoxal-derived hydroimidazolone,G-H1)、糠氨酸7种不同阶段美拉德反应产物(Maillard reaction products,MRPs)的含量。采用Kinetex C₁₈色谱柱进行分离，多反应监测模式进行定量分析。该方法的加标回收率为88.4%～111.3%，精密度相对标准偏差小于9%，检出限和定量限分别为2.1～14.2 ng/mL和7.4～41.2 ng/mL。采用该方法对134种常见预包装食品中的7种MRPs进行定量，并对得到的数据进行主成分分析和相关性分析。结果表明，CML、MG-H1...     

冻藏对原料肉加工前后羧甲基赖氨酸和羧乙基赖氨酸含量的影响

目的探究冻藏对原料肉加工前后羧甲基赖氨酸(N~ε-carboxymethyl-lysine,CML)和羧乙基赖氨酸(N~ε-carboxyethyllysine,CEL)含量的影响。方法以鸡胸肉为研究对象,使用反复冻融的方式模拟原料肉的冻藏过程,通过TBARs值与脂肪酸含量的变化评价原料肉脂肪氧化程度,同时考察原料肉与烤制后其中CML和CEL的含量。结果反复冻融会造成原料肉的汁液损失,游离氨基酸含量增加。在冻融前期(0～2 cycles)脂肪氧化程度较低, TBARs值为0.25 mg/kg,后期(4～6 cycles)程度加大,TBARs值为0.53 mg/kg。未冻融的新鲜原料肉中的CML及CEL含量均为最低,经1次冻融后CEL含量增加一倍达到(165.01±2.33)(μg/g干基),CML含量增加不显著(P0.05)。热加工后肉饼中CEL含量随原料肉冻融次数逐渐升高,于第6次冻融后达到最高值133.26±7.16(μg/g肉饼),与鲜肉对比含量增加15%,同时CML的含量增加44%。结论冻藏过程中,原料肉的反复冻融会导致脂肪氧化, CML和CEL的累积,同时会影响热加工后肉制品的品质。     

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。     

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 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 本研究通过构建葡萄糖-赖氨酸模型体系，利用高效液相色谱-串联质谱法探究其在加热过程中PLE对Nε-羧甲基赖氨酸（Nε-(1-Carboxymethyl)-L-lysine, CML）、Nε-羧乙基赖氨酸（Nε-(1-Carboxyethyl)-L-lysine, CEL）和吡咯素（Pyrraline, Pyr）三种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%。HPLC显示紫苏叶提取物中的主要活性物质为迷迭香酸、芹菜素-7-O-二葡萄糖苷和野黄芩苷等，推测其抑制途径可能是由于PLE的抗氧化性和清除二羰基化合物的共同作用。结论 PLE对AGEs有抑制作用，对食品安全和人体健康具有重要意义。     

单糖的类型对模拟体系中晚期糖基化终末产物生成的影响

为了研究单糖-赖氨酸模拟体系中单糖类型对晚期糖基化终末产物(AGEs)生成的影响。探讨了葡萄糖、半乳糖、果糖、山梨糖在120 ℃加热0~120 min期间pH、反应颜色、底物消耗、AGEs中间体包括3种二羰基化合物乙二醛(GO)、丙酮醛(MGO)和三脱氧葡萄糖醛酮(3-DG)以及果糖基赖氨酸(FL),以及最终三种AGEs包括羧甲基赖氨酸(CML)、羧乙基赖氨酸(CEL)、吡咯素(Pyr)的变化。结果显示随着反应进行,醛糖体系的pH下降程度以及颜色增加程度高于酮糖体系。反应结束时,葡萄糖-赖氨酸(Glu-Lys)、半乳糖-赖氨酸(Gla-Lys)、果糖-赖氨酸(Fru-Lys)和山梨糖-赖氨酸(Glu-Lys)体系中赖氨酸的损失分别为26.65%、28.95%、11.47%、14.90%;而所有体系中单糖在反应结束时的损失均达到了75.00%以上,要远高于赖氨酸。模拟体系中单糖会出现醛酮转换,但比例低于10%。所有体系中GO和3-DG的含量随着时间的延长而下降,这表明体系中GO和3-DG在反应前20 min内就已大量生成。α-二羰基化合物的生成量远高于FL的生成量。反应结束时,酮糖体系的CML和CEL含量要高于醛糖体系。所有体系中,三种AGEs生成量均为CEL > CML > Pyr。本文表明将来研究食品中AGEs的生成和抑制途径,需重点从中间体途径着手,尤其是α-二羰基化合物。     

Influence of Soluble Lysine Maillard Reaction Products on Escherichia coli Amino Acid Lysine Auxotroph Growth-Based Assay

ABSTRACT: Lysine Maillard reaction products (LMRP) were generated by autoclaving different concentrations and ratios glucose and lysine for 3 h. Effects of LMRP on the Escherichia coli lysine auxotroph (lys⁻) were examined in model systems. The slope and y-axis intercept of the standard curve in the absence of LMRP were significantly ( p < 0.05) greater than in the presence of LMRP. Our study shows that LMRP can decrease the growth extent of lys⁻. The E. coli available lysine assay may provide the food industry with a practical approach to rapid and accurate estimation of lysine for least cost formulation in foods.     

Effects of heat treatment on lysine in soya protein

Total lysine and available lysine were used For predicting the damage to lysine in soya protein heated with glucose and in alkali. The amounts of both types of lysine decreased during the treatments. The content of total lysine was much higher than that of available lysine. In soya protein heated with glucose, the available lysine content decreased by 78-85 % and that of total lysine by 17-40 %. In soya protein heated in alkali, available lysine decreased by 7-24% and total lysine by 7-13%. Since available lysine was more sensitive, changes in total lysine underestimated the nutritional damage.     

The rapid determination of chemically reactive lysine in the presence of carbohydrates by a modified trinitrobenzenesulphonic acid procedure

The trinitrobenzene sulphonic acid (TNBS) method has been shown by previous workers to give low results for chemically reactive lysine if carbohydrates are present in the sample. The major factor responsible has now been identified as loss of ?-trinitrophenyl (?-TNP) lysine during the hydrolysis stage, while adsorption of ?-TNP lysine by humins formed from carbohydrates during hydrolysis has been shown to be insignificant. The loss can be minimised by a reduction of the hydrolysis time without affecting the necessary degree of protein hydrolysis. A modified procedure is suggested which is shown to give very similar results to Carpenter's method (incorporating the Booth modification) for pure proteins and to the Silcock method in the presence of carbohydrates. ?-TNP lysine hydrolysed for 90 min or D,L-lysine taken through the trinitrophenylation procedure followed by hydrolysis for 120 min are the most suitable standards. The procedure can be completed in under 6 h and does not require the use of expensive equipment or hazardous chemicals.     

Nutritional characterization of high protein and high lysine barley lines

Six high protein and high lysine barley lines were derived from four crosses involving high protein andlor high lysine donors, ie Riso 1508 and SV 73608 (Hiproly×Mona⁵), with well adapted local strains V 5681 and V 4342. The trial was grown at the Wheat Research Institute, Faisalabad, Pakistan. The lines were chemically and nutritionally characterized and the results were compared with the parents. The biological value, net protein utilization and lysine contents were higher in all the derived lines than in their local parents, ie V 5681 and V 4342. The increased lysine levels in the lines B 82503 and B 82507 may be a consequence of the reduction of prolamin fractions with simultaneous increases in salt soluble fractions. In all the lines, lysine and nutritional quality increase were a reflection of high protein andlor high lysine parents. The line B 82503 had grain yield comparable to its local parent V 5681 but it was superior in lysine and some nutritional attributes to its high lysine donor parent Riso 1508. In some lines, lysine improvement was achieued without impairing grain yield.     

赖丙氨酸特性及检测

赖氨酸是食品中限制性氨基酸,在碱处理和加热时很易与蛋白质分子内其它氨基酸形成交联键,生成某些新氨基酸,如赖丙氨酸等。赖丙氨酸可由赖氨酸残基ε–氨基与脱氢丙氨酰残基缩合而成,后者可能通过胱氨酸残基碱降解、脱硫后生成,也可来自丝氨酸或磷酸丝氨酸残基分解。赖丙氨酸是一种金属螯合剂,因此,可使一些含有金属的酶,如羧肽酶A和羧肽酶B失去活性;且赖丙氨酸对小白鼠具有肾毒性。尽管至今为止,赖丙氨酸对人体毒性尚不明确,但在婴儿食品中,赖丙氨酸含量需严格控制;赖丙氨酸含量可作为蛋白质加工破坏程度和消化性能一个指标。常用的赖丙氨酸分析方法有气相色谱法、液相色谱法、氨基酸分析仪法和薄层层析法等。     

Blockage of available lysine at different stages of infant cereal production

Determination of available lysine was used to assess the heat damage produced during the processing of cereals for infant foods. The infant cereals analysed were: wheat, 7 and 8 cereals, rice, rice–corn, rice–corn–soy and oat. Lysine losses during the toasting of flours ranged between 14 and 29%. Roller‐drying treatment caused considerable heat damage, with a 53% decrease of available lysine in roller‐dried rice–corn–soy. However, oat flour was less susceptible, with a 5% reduction in available lysine during roller‐drying. © 1999 Society of Chemical Industry     

耐高浓度赖氨酸红曲霉突变菌株的选育

设计双层培养基选育耐高浓度赖氨酸红曲霉菌株，经紫外线长时间诱变获得一株新菌株102w，该菌株可在大米粉10％和赖氨酸1．6％的发酵液中正常生长，红曲色素产量达168．4U／mL，经多次传代试验，该菌株具有遗传稳定性。     

培养基某些生长因子对 ZL9601 菌株产赖氨酸的影响注

以谷氨酸棒杆菌诱变株 Ｚ Ｌ９６０１ 为赖氨酸生产菌,通过摇瓶发酵培养,分析了人工培养基中３ 种必需生长因子（ 生物素、蛋氨酸、苏氨酸） 对该菌产赖氨酸的影响,并通过正交试验设计确定了３ 种生长因子的最佳配比,结果表明,３ 种生长因子可明显增高赖氨酸的产量;在人工培养基中,３ 种生长因子的最佳配比为：生物素４００μｇ／ Ｌ、蛋氨酸２５０ｍｇ／ Ｌ、苏氨酸３００ｍｇ／ Ｌ。     

Digestible reactive lysine in selected milk-based products

Reactive lysine contents, true ileal reactive lysine digestibility, and true ileal digestible reactive lysine contents were determined in a wide range of processed milk products. A previously validated assay based on determining reactive lysine in both food and ileal digesta, after reaction of these materials with O-methylisourea, was applied. Semisynthetic diets containing milk products as the sole sources of protein and including chromic oxide as an indigestible marker were fed to growing rats. Digesta from the terminal ileum were collected posteuthanasia and, with samples of the diets, analyzed for reactive lysine (homoarginine) contents. True reactive lysine digestibility was determined after correcting for endogenous lysine loss at the terminal ileum of rats fed an enzyme hydrolyzed casein-based diet, followed by ultrafiltration (5000 Da) of the digesta. Digestible total lysine (determined using conventional methods) was also determined. The true ileal reactive lysine digestibility was high (>91%) in all the milk products tested, but was highest in the UHT milk (100%) and lowest in the infant formulas (91 to 93%). Total lysine digestibility (conventional measurement) significantly underestimated reactive lysine digestibility for all the products tested. The mean underestimation ranged from 1.3 to 7.1% units. The mean digestible total lysine content was significantly different from the available lysine content for most of the products examined. In some cases this difference was small (<3%), but for a number of the products (evaporated milk, whole milk protein, lactose hydrolyzed milk powder, and a sports formula) the difference was greater (6.5 to 14%). This would suggest firstly that total lysine and total lysine digestibility determined using conventional methods were inaccurate when applied to some milk-based foods, and secondly that some of the milk products have undergone lysine modification. In general, milk proteins are a highly digestible source of amino acids and lysine.     

Effect of elevated temperature storage on the digestible reactive lysine content of unhydrolyzed- and hydrolyzed-lactose milk-based products

The study aimed to evaluate the effects of storage at elevated temperatures on reactive Lys content and true ileal reactive Lys digestibility in a skim milk powder and a hydrolyzed-lactose skim milk powder. A validated bioassay based on guanidination of food and digesta samples was applied. Semisynthetic diets containing the milk powders as the sole sources of protein were formulated and fed to growing rats. Chromic oxide was included in each diet as an indigestible marker. Digesta were collected posteuthanasia and analyzed along with the diets for reactive Lys (homoarginine), and true ileal reactive Lys digestibility was calculated after correction for endogenous Lys loss. For the skim milk powder, there was no decrease in reactive Lys digestibility (Lys availability) when the powder was stored at 30 and 35°C for 18 mo. In contrast, when stored at 40°C for 12 mo, a small but statistically significant (6%) decrease was observed. For the hydrolyzed-lactose product, a 22% decrease in Lys availability was observed after storage at 35°C for 18 mo, and a 17% decrease was observed when stored at 40°C for only 6 mo. Digestible reactive (available) Lys content decreased by more than 20% for the skim milk powder stored at 30 and 35°C for 18 mo and 40% when stored at 40°C for 12 mo. Furthermore, available Lys decreased in the hydrolyzed-lactose skim milk powder by 41% when stored at 30°C for only 18 mo and 34 and 65% when stored at 35 and 40°C, respectively, for 6 mo. Elevated temperatures and prolonged storage periods negatively influenced the available Lys contents of both milk powders. The decrease in available Lys content and Lys availability was greater for the hydrolyzed-lactose skim milk powder compared with the normal skim milk powder, after storage at elevated temperatures.