黄鲫蛋白肽湿热杀菌条件下晚期糖基化终末产物（AGEs）的形成机理及其肠道吸收分子机制

It has been confirmed that many diseases, such as diabetes, are related with advanced glycation end products (AGEs). Food source AGEs intake is undoubtedly considered as the major way to accumulate in human body as endogenous AGEs. To date, the research work on the formation mechanism and kinetic absorption of peptide foods source AGEs are relatively scarce. In our previous study, we found that moist heat sterilization is an effective approach for half-fin anchovy peptides to enhance their biological activities. However, endogenous AGEs are also formed mainly by Maillard reaction during the heat processing of foods. The project is attempting to reveal the formation mechanism of AGEs and to investigate the in vitro mechanism of intestinal absorption using half-fin anchovy peptides as a sample of aquatic peptide foods through moist heat sterilization technology. The main works in this study are composed of: (1) characterization of the marker peptide of half-fin anchovy peptides during Maillard reaction, and establishment the reaction system between the marker peptide and glucose; (2) constructing multiple reaction formulas for kinetics study and revealing the formation mechanism of AGEs from half-fin anchovy peptides via heat sterilization using carboxymethyl lysine (CML) as the marker; (3) elucidating the mechanism of absorption in vitro of half-fin anchovy peptides AGEs after heated sterilization treatments in terms of digestibility and absorption pathway in intestine. The research results can establish the formation mechanism of AGEs from peptide products after moist heat sterilization. Moreover, the results will provide the technical guidelines for AGEs of aquatic protein peptides on impacting the mechanism of intestinal absorption in vitro.

内源性晚期糖基化终末产物（AGEs）已被证实与糖尿病等多种疾病发生有关，食源性AGEs的摄入无疑是人体内源性AGEs累积的主要途径，至今有关肽类AGEs的形成机制及动态吸收研究还相对薄弱。课题组前期研究发现黄鲫蛋白肽经湿热杀菌可显著增强其生物活性，但是加热过程中美拉德反应也是食源性AGEs形成主要途径。本课题以黄鲫蛋白肽湿热杀菌产物为研究对象，探究AGEs的形成机理，紧密围绕肠吸收研究热点，探讨黄鲫蛋白肽AGEs的肠吸收机制。拟开展主要研究有：分析黄鲫蛋白肽的美拉德反应标志肽特性，构建标志肽—葡萄糖反应体系，建立多元动力学反应方程揭示AGEs形成标志物—羧甲基赖氨酸（CML）的形成机理，阐明黄鲫蛋白肽AGEs的消化性、小肠吸收性。课题研究结果将为肽类AGEs的形成机理研究奠定理论基础，也为肽类AGEs的肠吸收机制研究提供技术依据。

食源性AGEs的摄入无疑是人体内源性AGEs累积的主要途径，至今有关肽类AGEs的形成机制及吸收研究还相对薄弱。本课题以黄鲫蛋白肽（HAHp）湿热杀菌产物为研究对象，主要研究内容有：(1) 分析黄鲫蛋白肽的美拉德反应标志肽特性；(2) 构建标志肽—葡萄糖反应体系，建立多元动力学反应方程揭示AGEs形成标志物—羧甲基赖氨酸（CML）的形成机理；(3) 阐明黄鲫蛋白肽AGEs的消化性、小肠吸收性。结果表明：HAHp经Sephadex G 25凝胶分离得到的F3组分，不仅自身抑菌作用最强，而且该组分经过美拉德反应后抑菌率也极显著地提高。经HPLC分析确定F3组分为分子量接近的肽类混合物，采用液质联用技术从F3中鉴定出82条小分子肽 （448.2322-1600.7803 Da），且大多数肽含有酪氨酸、苯丙氨酸和色氨酸残基。化学合成3条肽HLRVGW（H-1）、KWTRPMY（H-2）和KLWHHTF（H-3）作为HAHp代表肽，研究发现美拉德反应能有效提高代表肽对大肠杆菌的抑菌作用，选择自身及美拉德反应后抑菌效果最强的肽H-3为标志肽，进一步分析80℃、100℃和120℃下加热20~160 min时，标志肽-葡萄糖美拉德反应初级产物果糖胺和终端产物的形成特性。结果表明：蛋白浓度和葡萄糖含量对果糖胺生成符合二元方程，而终端产物（420 nm吸光值）生成量与反应体系中葡萄糖含量有关。体外模拟胃液消化可提高HAHp-葡萄糖美拉德反应产物对大肠杆菌的抑菌效果，其中蛋白质浓度、肽浓度随着胃液消化时间的延长而降低，但是游离氨基酸总量则随着消化时间的延长而显著地增加。此外，果糖胺含量在模拟胃液消化中增加显著。相比之下，模拟肠液消化会显著降低果糖胺的含量，但是CML含量保持稳定。采用小鼠体外肠攀模型确定HAHp美拉德肽在肠不同部位均有吸收，其中回肠对美拉德产物中的肽和CML吸收量最高。此外，HAHp美拉德肽的吸收会一定程度上降低肠黏膜的氧化程度。现阶段结果为肽类AGEs的形成机理研究奠定理论基础，也为肽类AGEs的肠吸收机制研究提供技术支持。