【作者】 章宇；

【导师】 张英； 任一平；

【作者基本信息】 浙江大学 ， 食品科学， 2008， 博士

【摘要】 近年来,食品中丙烯酰胺的发现、分析检测、形成机理和抑制途径引起了世界相关领域科学家的广泛关注。研究热加工过程中丙烯酰胺的形成机理和影响因素,寻找有效的抑制途径,对于食品中丙烯酰胺危害的防护和食品安全性研究具有重要意义。通过添加植物化学素的方法抑制食品中丙烯酰胺的形成,探讨其对美拉德反应相关前体物质、中间产物和终产物的影响,是目前研究的热点和难点。本论文系统地研究了生物黄酮抑制食品中丙烯酰胺形成的机理及其构效关系。论文首先建立了丙烯酰胺的气相色谱(GC)和液相色谱-串联质谱(LC-MS/MS)检测方法；通过对常规热加工食品、婴幼儿食品、复杂基质样品的检测,痕量分析以及食品化学分析测试水平计划(FAPAS)认证,对LC-MS/MS方法的可行性进行评价；以中式传统食品为对象,对两种方法进行比较,确定后者作为本文用于丙烯酰胺检测的定量分析方法。其次,以富含生物黄酮的竹叶抗氧化物(AOB)和绿茶提取物(EGT)为研究对象,分别采用0.1%和0.01%(w／w)的AOB浸渍处理,炸薯片和炸薯条的丙烯酰胺形成抑制率可达74.1%和76.1%；当AOB添加量为0.5%(w／w)时,炸鸡翅的丙烯酰胺形成抑制率可达59.0%；当AOB和EGT的添加量分别为0.1%和0.01%(w／w)时,油条的丙烯酰胺形成抑制率可达82.9%和72.5%。结合最大允许使用剂量和感官评定的结果最终选择了两者在上述食品中的最佳添加剂量水平。然后,在确认生物黄酮对食品中丙烯酰胺抑制作用的基础上,以响应面分析(RSM)和正交实验分析(OAM)为试验设计方法,以超高效液相色谱-串联质谱(UPLC-MS/MS)法为检测手段,系统研究了天冬酰胺-葡萄糖／果糖／蔗糖模式体系分别在低湿和微波加热条件下产生丙烯酰胺的规律。其中,天冬酰胺-葡萄糖模式体系产生丙烯酰胺的优化条件为：两种前体物质以等摩尔比例在180℃的低湿体系中加热15 min或在微波体系中加热5 min。选择天冬酰胺-葡萄糖模式体系,研究AOB和EGT对丙烯酰胺形成／消除动力学过程的影响。当两者在低湿和微波体系中的添加剂量水平分别为2×10-4 mg／g和10-6mg／mL时,对丙烯酰胺的抑制率达到最大值。分别采用Logistic-指数动力学模型和形成／消除一级动力学模型描述低湿和微波体系中AOB和EGT对丙烯酰胺动力学过程的影响,结果表明,AOB和EGT对两种体系中丙烯酰胺的形成动力学过程具有显著的抑制作用,而对消除动力学过程无显著影响。建立土豆微波模式体系,研究24种生物黄酮试样对丙烯酰胺的抑制作用及其构效关系。量效关系研究表明,当生物黄酮的添加剂量水平为10-9mol/L时,对丙烯酰胺的抑制率达到最大。DPPH.ABTS和FRAP抗氧化评价体系的测试结果表明,生物黄酮对丙烯酰胺的抑制率与反应体系抗氧化性的变化值(△TEAC)之间存在显著的相关性。采用定量结构-活性关系(QSAR)方法评价生物黄酮抑制丙烯酰胺的构效关系。结果表明,黄酮芳环羟基的数目和位置对其抑制作用具有重要影响；在芳环羟基数目相等的情况下,黄酮对丙烯酰胺的抑制率明显优于异黄酮；对于具有相同苷元的黄酮糖苷而言,碳苷对丙烯酰胺的抑制率优于氧苷；此外,生物黄酮的拓扑结构对其抑制丙烯酰胺的活性也有重要影响。最后,建立了同步检测天冬酰胺、葡萄糖、果糖和丙烯酰胺的同位素稀释UPLC-MS/MS检测方法。根据高抑制率和代表性原则,选取异荭草苷(AOB的特征性碳苷黄酮)、表没食子酸儿茶素没食子酸酯(EGT的特征性黄烷醇酯)、木犀草素-7-O-葡萄糖苷、木犀草素、染料木素和槲皮素为生物黄酮的典型试样,在其抑制丙烯酰胺的最适剂量水平(10-9 mol/L)上建立土豆微波模式体系的机理动力学模型,绘制天冬酰胺、葡萄糖、果糖、丙烯酰胺和类黑素的动力学曲线,计算相应动力学参数的变化,分析其作用位点。结果表明,生物黄酮可显著地抑制天冬酰胺与果糖反应生成以Schiff碱为代表的中间产物的过程、葡萄糖通过异构化作用向果糖转化的过程以及中间产物向丙烯酰胺转化的过程,但无法抑制天冬酰胺与葡萄糖反应的过程和丙烯酰胺生成后的消除和转化过程。此外,生物黄酮对抑制美拉德反应终产物类黑素的形成有一定的影响,但这种作用并不显著。更多还原

【Abstract】 Recently, the findings, analytical methods, formation mechanism and reduction pathways of acrylamide in foods have attracted wide attention all over the world. The formation mechanism, impact factors and effective reduction methods of acrylamide during heat processing play important roles in the defense of acrylamide hazard and food safety. Studies on the reduction effect of plant-derived components on the formation of acrylamide and its precursors and intermediates through Maillard reaction in foods become a research hotspot. This dissertation systematically studied the mechanism and structure-activity relationship of bio-flavonoids in reducing acrylamide formation in foods.Two analytical methods of gas chromatography (GC) and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantification of acrylamide were investigated. The LC-MS/MS method was further validated via the analysis in routine heat-treated foods, baby foods and complex food matrixes, trace analysis, and food analysis performance assessment scheme (FAPAS) proficiency test. The above two methods were compared with each other and applied for the analysis of acrylamide in Chinese traditional foods. Finally, the LC-MS/MS method was selected as the standard method for quantitative analysis of acrylamide.Two bio-flavonoid rich extracts of antioxidant of bamboo leaves (AOB) and extract of green tea (EGT) were first used to examine the effect on acrylamide reduction. The result showed that 74.1% and 76.1% of acrylamide were reduced in potato crisps and French fries when they were treated by 0.1% and 0.01%(w/w) of AOB solutions before frying, respectively. Similarly,59.0% of acrylamide was reduced in fried chicken wings when 0.5%(w/w) of AOB was used. Furthermore,82.9% and 72.5% of acrylamide were reduced in Chinese fried bread sticks when 0.1%(w/w) of AOB and 0.01%(w/w) of EGT were added in the flour, respectively. The optimal addition levels of AOB and EGT were finally obtained after taking the maximum permissible addition levels and sensory quality into consideration. Based on the confirmation of the reduction effect of bio-flavonoids on acrylamide in foods, the formation of acrylamide was studied in a model system of asparagine-glucose/fructose/ sucrose. The experimental design was performed by the response surface methodology (RSM) and orthogonal array methodology (OAM) while the acrylamide content was analyzed by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Results showed that acrylamide was optimally generated in model systems when the equimolar asparagine and glucose were heated 15 min in the low-moisture system at 180℃or 5 min in the microwave system at 180℃.The acrylamide content could be decreased either by inhibiting its formation or promoting its elimination. The effect of AOB and EGT on the formation and elimination kinetics of acrylamide was then investigated in the asparagine-glucose model system. The maximum reduction effect of acrylamide was observed in low-moisture and microwave systems when the addition levels of both extracts were 2×10-4 mg/g and 10-6 mg/mL, respectively. Meanwhile, the effect of both extracts on the kinetics of acrylamide in low-moisture and microwave systems was described via the Logistic-exponential and first-order formation/elimination kinetic models, respectively. Results indicated that addition of AOB and EGT could significantly reduce the formation kinetics of acrylamide while no effect on its elimination kinetics.The reduction effect and structure-activity relationship of totally 24 bio-flavonoid samples on the formation of acrylamide were then studied in a potato model system using the microwave heating method. Results of dose-response study showed a maximum reduction effect when the addition levels of bio-flavonoids were 10-9 mol/L. Results of antioxidant evaluation systems using DPPH, ABTS and FRAP methods revealed a significant positive correlation between the acrylamide reduction effect of bio-flavonoids and the antioxidant levels (ATEAC) in reaction systems. Meanwhile, the structure-activity relationship study on the reduction effect of bio-flavonoids was performed via the quantitative structure-activity relationship (QSAR) method, and it was demonstrated that the number and position of aromatic hydroxyl functional groups play an important role in the reduction effect of flavonoids. Furthermore, the reduction effect of flavones is more effective than isoflavones with the same number of aromatic hydroxyls. The reduction effect of flavone C-glycosides is better than flavone O-glycosides for the flavone glycosides with the same structure of aglycone. The topological structures of bio-flavonoids are also related to their reduction effects.To simultaneously quantify the contents of asparagine, glucose, fructose and acrylamide in final reaction products, an improved UPLC-MS/MS method was evaluated. Six bio-flavonoids including homoorientin (the characteristic flavone C-glycoside of AOB), (-)-epigallocatechin gallate (the characteristic flavanol ester of EGT), luteolin-7-O-glucoside, luteolin, genistein and quercetin were selected for the reduction mechanistic study according to their reduction abilities and universality. The mechanistic kinetic models were established in the potato microwave model system based on the optimal addition levels (10-9 mol/L) of 6 selected bio-flavonoids. Then, the kinetic profiles of asparagine, glucose, fructose, acrylamide and melanoidins were obtained. The reduction positions of bio-flavonoids were finally analyzed after estimating the change of kinetic parameters. Results indicated that bio-flavonoids can significantly reduce the formation of intermediates such as Schiff base in the reaction between asparagine and fructose, the transform action from glucose to fructose via isomerization, and the formation of acrylamide from intermediates. However, they cannot reduce the reaction between asparagine and glucose and no effect on the elimination of acrylamide. Furthermore, bio-flavonoids have reduction effect on the formation of melanoidins, which is one of the final products of the Maillard reaction. However, such effect seems not significant.更多还原