亚油酸对美拉德反应模型中晚期糖基化终产物的影响

发布时间：2018-08-02 09:32

【摘要】：晚期糖基化终产物(AGEs)是在体内产生的糖基化代谢产物,与一些与衰老相关的疾病相关。AGEs也会在食品加工期间产生,在蛋白、糖等营养丰富的成分之间累积,尤其是含脂食品。研究表明,食源性AGEs对人体健康存在隐患。由于真实食品的复杂性和异质性,目前的研究不是很详尽。本文采用接近真实食品贮藏的模拟体系,在加速贮藏的条件下,研究亚油酸(LA)的加入对模拟体系中味感、自由基、中间产物及终产物的影响,为探索AGEs的生成机理奠定基础。论文建立两个模拟体系,分别是氨基酸体系:(1)GLY:D-葡萄糖(D-Glc)+L-赖氨酸(L-Lys)+LA;(2)GL:D-Glc+L-Lys;蛋白质体系:(1)GBY:D-Glc+牛血清蛋白(BSA)+LA;(2)GB:D-Glc+BSA。氨基酸体系采用电子舌(E-tongue)、电子自旋共振光谱(ESR)、超高效液相色谱-串联质谱(UPLC-MS/MS)、比色法分析味感变化、AGEs以及自由基变化之间的关系,探究LA对氨基酸体系中味感、反应中间产物和自由基的影响。蛋白质体系采用聚丙烯酰胺凝胶电泳(SDS-PAGE)、红外色谱(FT-IR)、电导率、荧光色谱、圆二色谱(CD)等方法分析糖化BSA结构的变化,进而研究LA对蛋白质糖化及蛋白质二级变化的影响。主要研究结果如下:1.E-tongue分析说明LA的加入影响了苦味、涩味(代表性聚合物的味感)的变化,苦味和涩味含量增加体现聚合物含量增加。因此,LA是反应味感变化的关键因素,同时LA也是触发糖化反应的关键因素。2.ESR分析,在GL中存在高反应活性的自由基,并且这些基团主要是美拉德反应(MR)组分(D-Glc、L-Lys)产生的。GLY中的自由基主要由LA过氧化反应产生,而不是MR。因此,LA促进AGEs形成主要通过LA过氧化途径,而不是MR途径。3.UPLC-MS/MS和比色法分析具有代表性AGEs的变化,结果显示,在GL中AGEs主要通过Strecker降解反应形成。而在GLY中,.AGEs主要通过LA过氧化产生,并且生成量更大。4.SDS-PAGE分析得出,在糖基化反应过程中,天然BSA含量逐渐降低,蛋白聚合物和交联产物的含量逐渐增加。LA的加入使交联、聚合反应更加剧烈,促进更多AGEs生成。5.FT-IR的数据看出,GB、GBY中蛋白二级结构α-螺旋均低于天然BSA,而β-折叠与之相反。这说明LA的参与,更加明显地促进BSA二级结构的松散,导致蛋白质糖化、交联、聚合反应加剧。6.随着反应进行,GB和GBY的电导率在逐渐增加,但是GBY电导率低于GB,这是因为LA的加入会使蛋白质结构发生改变,生成大分子量的聚合产物,使电阻率增加。7.GB和GBY的样品,随反应进行荧光值呈现规律性的降低。GBY-Oh样品与GB-0h样品的荧光值差异显著(p0.01),这说明LA的加入,使得模型的荧光值降低。GBY样品吸收峰发生蓝移,这表征结构改变和新物质生成,说明模型中加入的LA是蛋白质结构变化的主要因素。8.CD主要是在远紫外光谱区(190-250 nm)对蛋白质二级结构的糖基化效果进行定量分析。结果表明,GB和GBY中BSA二级结构松散,GBY的变化程度更加明显。实验结果有别于天然BSA二级结构,说明LA对蛋白糖基化反应有促进作用,导致BSA二级结构的变化更加明显。通过以上试验可以说明,LA的加入对MR模型(GL、GB)影响较大,主要得出两方面结论:一方面,LA促进高反应活性自由基生成,从而促进脂质过氧化反应,进一步触发Strecker降解反应,生成了大量AGEs和风味物质,这些风味物质是GL味感变化的关键因素;另一方面,LA促进BSA二级结构的松散,更加明显的导致蛋白质糖化、交联、聚合反应加剧。
[Abstract]:Advanced glycosylation end product (AGEs) is a glycosylated metabolite produced in the body, and.AGEs, associated with aging related diseases, is also produced during food processing, accumulating between protein, sugar and other nutritious ingredients, especially fatty foods. Studies have shown that food derived AGEs is a potential hazard to human health. In this paper, the effects of the addition of linoleic acid (LA) on the taste, free radicals, intermediate products and final products in the simulation system are studied with the simulation system close to the real food storage and the effects of the addition of linoleic acid (LA) on the simulation system. This paper establishes two simulative bodies. The system, respectively, is an amino acid system: (1) GLY:D- glucose (D-Glc) +L- lysine (L-Lys) +LA; (2) GL:D-Glc+L-Lys; protein system: (1) GBY:D-Glc+ bovine serum protein (BSA) +LA; (2) GB:D-Glc+BSA. amino acid system using electronic tongue (E-tongue), electron spin resonance spectroscopy (ESR), ultra high performance liquid chromatography tandem mass spectrometry, colorimetric analysis The relationship between taste change, AGEs and free radical changes, and to explore the effect of LA on the taste, intermediate products and free radicals in the amino acid system. The protein system uses polyacrylamide gel electrophoresis (SDS-PAGE), infrared chromatography (FT-IR), conductivity, fluorescence chromatography, circular two chromatography (CD) and other methods to analyze the changes of the structure of saccharification BSA, and then study the changes of the structure of saccharifying BSA. The effect of LA on protein saccharification and protein two level changes. The main results are as follows: 1.E-tongue analysis shows that the addition of LA affects the change of bitterness, the taste of the astringency (the taste of the representative polymers), the increase of the content of bitterness and astringency. Therefore, the LA is the key factor of the change of the reaction taste, and the LA is also triggered. The key factor of saccharification reaction.2.ESR analysis shows that there is a highly reactive free radical in GL, and these groups are mainly produced by the mallard reaction (MR) component (D-Glc, L-Lys), and the free radicals produced in.GLY are mainly produced by LA peroxidation rather than MR., and LA promotes AGEs to form mainly through LA peroxidation, rather than MR pathway -MS/MS and colorimetric analysis were used to analyze the variation of representative AGEs. The results showed that AGEs was mainly formed by Strecker degradation in GL. In GLY,.AGEs was mainly produced by LA peroxidation, and the amount of production was more.4.SDS-PAGE analysis. In the glycosylation process, the content of Tian ran BSA gradually decreased, protein polymer and cross linked products were found. As the content increased gradually, the addition of.LA made the crosslinking, the polymerization reaction more intense, and the data to promote more AGEs to produce.5.FT-IR showed that the two grade structure of protein in GB and GBY was lower than that of natural BSA, and the beta folding was the opposite. This shows that the participation of LA is more obvious in promoting the loosening of the structure of BSA two, resulting in protein saccharification, crosslinking and polymerization. The electrical conductivity of GB and GBY increased gradually as the reaction progressively increased, but the conductivity of GBY was lower than that of GB, because the addition of LA would make the protein structure change and produce the polymerization products of large molecular weight, which made the resistivity of the samples of.7.GB and GBY increased, and the fluorescence values regularly reduced the.GBY-Oh samples and GB-0h samples with the reaction. The difference of fluorescence value is significant (P0.01), which indicates that the addition of LA makes the fluorescence value of the model reduce the blue shift of the.GBY sample absorption peak, which represents the structural change and the formation of new substances, indicating that the LA in the model is the main factor of the structural change of protein,.8.CD is mainly the sugar base of the two grade structure of the protein in the far ultraviolet spectrum region (190-250 nm). The results show that the BSA two structure in GB and GBY is loose and the degree of GBY change is more obvious. The experimental results are different from the natural BSA two structure, which indicates that LA has a promoting effect on the reaction of protein glycosylation, which leads to the more obvious changes in the structure of BSA two. Through the above experiments, the addition of LA to MR model (GL, GB) The main results are two conclusions: on the one hand, LA promotes the formation of highly reactive free radicals, thus promoting lipid peroxidation, further triggering the Strecker degradation reaction, and producing a large number of AGEs and flavor substances, which are the key factors for the change of GL taste, and on the other hand, LA promotes the loosening of the BSA structure of the BSA. Obviously, protein saccharification, cross-linking and polymerization are aggravated.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TS201.2

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