【作者】 付全意；

【导师】 李琳；

【作者基本信息】 华南理工大学 ， 制糖工程， 2012， 博士

【摘要】 食品加工过程美拉德反应在改变食品组分的结构和功能的同时也产生了部分不可忽视的有害物质。在众多美拉德反应产物中，以羧甲基赖氨酸（CML）为代表的晚期糖基化终末产物（AGEs）已经证实与糖尿病等疾病都存在着千丝万缕的关系，CML潜在的危害性已越来越受到食品科技界的关注。国际食品科技领域关于CML的研究主要集中在CML生理医学方面，对CML的分析方法和毒性等方面的研究取得了一些进展，然而，针对食源性CML在食品加工过程中形成机理的研究则寥寥无几。本文将研究食品模拟体系糖化反应过程中有害物质CML的形成和演化的量化方法，在此基础上，确定食品加工过程中CML控制、抑制和消除策略。首先，本文建立了快速和精确检测食品模拟体系中CML的HPLC－MS检测方法。采用HPLC－MS检测CML的重现性好，精密度高，CML的检测定量限（LOQ）可以达到50ng/mL。采用此方法用于定量检测本文食品加热模拟体系中的CML。然后，研究微波和传统加热方式下的食品模拟体系糖化反应过程中CML生成的宏观变化规律。研究发现，在美拉德反应中还原糖更有利于生成CML，不同的糖生成的CML总量的顺序为：乳糖>葡萄糖>蔗糖。而且，微波加热相对于传统加热能够生成更多的CML。其次，在了解CML生成规律的基础上，本文探讨了形成CML的中间产物乙二醛和果糖基赖氨酸的生成规律和相互之间的联系。研究发现，CML中间产物果糖基赖氨酸能够生成乙二醛，说明果糖基赖氨酸不仅是形成CML的中间产物，同时也是生成CML中间产物乙二醛的前体物质。同时，基于CML的中间产物乙二醛形成于美拉德反应的所有阶段，本文提出了新的观点，即CML不仅产生于糖化反应的终末阶段，而且产生于糖化反应的开始阶段，在理论上可以将CML称为糖化过程的产物。本文采用碳标记示踪技术（CAMOLA）研究CML中间产物参与复杂的化学反应，通过合成13C标记CML，重点研究了CML形成的复杂路径。不同于传统观点认为CML分子上的羧甲基仅来自于葡萄糖残基的C－1和C－2位基团，本文研究发现CML中羧甲基的来源有两个途径：（1）果糖基赖氨酸分子中的葡萄糖残基的C－1和C－2位基团参与了CML分子上的羧甲基的形成；（2）葡萄糖氧化裂解的过程中，C－1和C－2位基团，C－3和C－4位基团，C－5和C－6位基团分别参与了乙二醛的形成，来自于葡萄糖残基不同基团的乙二醛与赖氨酸反应生成了CML，在此生成CML的路径中，CML中羧甲基分别来葡萄糖残基的C－1和C－2位基团，C－3和C－4位基团，C－5和C－6位基团。在此基础上，本文提出了CML形成的新途径，拓展了研究CML在食品加工过程中形成的新领域。研究还发现在葡萄糖和氨基酸的微波加热模拟体系中，CML中间产物果糖基赖氨酸的生成量和生成速率明显高于乙二醛，而且通过中间产物果糖基赖氨酸生成CML的总量远远大于通过乙二醛。结合13C标记的CML和CML中间产物的变化，本文认为无论是从形成CML的中间产物果糖基赖氨酸和乙二醛的生成量，还是从通过此两种路径形成的CML的总量角度研究，在葡萄糖和氨基酸加热体系中果糖基赖氨酸是生成CML的主要中间产物，乙二醛是次要中间产物。因此，不同于传统观点认为通过中间产物乙二醛生成CML的路径是主要路径，本文认为通过中间产物果糖基赖氨酸生成CML的路径是主要路径。另外，本文首次采用了多级动力学模型考察CML的形成动力学规律。研究了反应底物葡萄糖和赖氨酸，中间产物果糖基赖氨酸和乙二醛，反应终产物CML和类黑精等的反应速率、形成速率和降解速率。通过多级动力学模型较为全面阐述CML的形成机理。中间产物果糖基赖氨酸产生的反应速率常数大于生成乙二醛的反应速率，生成果糖基赖氨酸的活化能Ea低于生成乙二醛的活化能，说明食品加工过程中更容易生成中间产物果糖基赖氨酸，而且果糖基赖氨酸生成CML的转化率大于高于乙二醛，进一步证实了果糖基赖氨酸是形成CML的主要中间产物。最后，本文研究三种维生素（维生素B1，维生素C，维生素E）和两种黄酮类物质（芦丁和槲皮素）以及还原剂硼氢化钠对葡萄糖和赖氨酸模拟体系中CML生成的抑制效果。维生素C和维生素E对CML生成的没有抑制效果。维生素B1的氨基与氨基酸或蛋白质中的赖氨酸残基存在竞争的关系，竞争还原糖的羰基，抑制CML的生成。硼氢化钠对羰基化合物具有很强的还原能力，终止其氧化裂解生成CML。芦丁和槲皮素特有的酚羟基苯环结构具有强抗氧化能力，能够抑制CML的生成。通过结合CML生成的多级动力学模型研究发现，黄酮物质芦丁抑制CML的生成主要发生在糖化反应第二阶段，即中间产物的生成和转化为CML的阶段。更多还原

【Abstract】 Maillard reaction not only changes the structure and function of food components, butalso generated a lot of harmful substances in food processing. Among those complex Maillardreaction products, Nε－(Carboxymethyl)lysine（CML）as the representative of advancedglycation end products (AGEs) has affinity relationship to diabetes and other diseases, CMLas a potentially harmful sector has attracted more and more attention. Correctly, theinternational food science and technology research focused on the physiology in medicine,analysis and toxicity of CML, however, the mechanism of formation of CML in foodprocessing was still unclear. The aim of this study was to evaluate the performance of formingCML during glycation reaction in food model systems, and then discuss the strategies oncontrolling, inhibiting and eliminating CML in food processingFirstly, a rapid and accurate HPLC-MS detection method was used to detect CML infood model system. The HPLC-MS detection method had good reproducibility and highaccuracy, the limit of quantification (LOQ) of CML detection was achieved50ng/mL. Thedetection method was used for the determination of CML formed in food model system in thepaper.Then, the performance of forming CML during glycation reaction in food model systemswas also evaluated. The study showed that reducing sugars in the Maillard reaction was moreconducive to form CML.The order of reactivity for the formation of CML was lactose>glucose> sucrose. Microwave heating treatment in food model systems produced more CMLthan traditional heating treatment.Secondly, we discussed the formation of CML intermediate glyoxal and fructoselysine,and made a relationship between the two intermediates. Fructoselysine had the capacity toproduce glyoxal, the result showed fructoselysine not only was the intermediate of CML, butalso was the precursors of CML intermediate glyoxal. Because CML intermediate glyoxal wasformed at all stages of glycation in the Maillard reaction, so CML may be called as a productof the glycation process in theory.In this study, carbohydrate modul labeling technique （CAMOLA）was used to explainthe formation of the CML intermediate and the complex chemical reactions pathway offorming CML. The traditional view was the carboxymethyl of CML was only formed from C－1and C－2of glucose residues, however, we found the source of carboxymethyl of CML had two ways:（1）C－1and C－2of glucose residues in fructoselysine were involved in theformation of carboxymethyl of CML;（2）C－1and C－2of glucose residues, C－3and C－4of glucose residues, C－5and C－6of glucose residues were involved in the formationof glyoxal during glucose oxidative cleavage, and then glyoxal and lysine formed CML, sothe carboxymethyl of CML was from C－1and C－2of glucose residues, C－3and C－4ofglucose residues, C－5and C－6of glucose residues. The result expanded the theory offormation of CML in food processing.During the glucose-amino acid model system, the formation of CML fromfructoselysine was great higher than from glyoxal.Combined with the concentration of13Clabeled CML and CML intermediate formation, fructoselysine was the main intermediate, andglyoxal was the minor intermediate of CML，the formation of fructoselysine was the mainpathway to form CML, the paper overturned the traditional view that carbonyl compoundsglyoxal is the main intermediate of CML.The traditional view indexed that the pathway offorming CML was mainly through the intermediate products glyoxal, we found forming CMLthrough the intermediate product of fructose lysine was the main path.Furthermore, a multi－stage kinetics model was also discussed for formation of CML.Glucose and lysine, intermediates fructoselysine and glyoxal, the reaction end product CMLand melanoidins，the parameters of the rate constants and activation energies were estimatedin the kinetics model. The reaction rate constant of fructoselysine was great higher thanglyoxal, and the activation energy Ea of fructoselysine was lower than glyoxal, the resultshowed the formation of fructoselysine was easier than glyoxal, and fructoselysine wasfavorer to form CML.We found fructoselysine was a major intermediate of CML.Lastly, we also examined the inhibition effect of CML by some inhibitors in glucose－lysine model systems, the inhibitors were three vitamins (vitamin B1, vitamin C and vitaminE), two flavonoids (rutin and quercetin) and reducing agent sodium borohydride sodium.Ascorbic acid and tocopherol did not affect inhibition of CML. There was a competition ofcarbonyl reducing sugar competition between the vitamin B1and amino acids or amino lysineresidues of protein, inhibiting the generation of CML. Sodium borohydride reduction has astrong ability to reduce carbonyl compounds, not only to reduce dicarbonyl compoundsglyoxal, but also to reduce the main intermediate fructoselysine to terminate the oxidation ofCML forming. Rutin and quercetin inhibited the formation of CML via vicinyl dihydroxylgroups, which has a strong antioxidant capacity. We also discussed the pathway of inhibiting CML by rutin in a multi－stage kinetics model. Rutin inhibited the formation of CML mainlyoccurred in the second phase of the glycosylation reaction, the formation of the intermediateproduct and transformed into the stage of CML.更多还原