《食品化学》课程教学资源（文献资料）生物黄酮抑制食品中丙烯酰胺形成的机理及其构效关系研究（浙江大学博士学位论文）

浙江大学生物系统工程与食品科学学院 博士学位论文 生物黄酮抑制食品中丙烯酰胺形成的机理及其构效关系研究 姓名：章宇 申请学位级别：博士 专业：食品科学 指导教师：张英；任一平 20080501

浙江大学生物系统工程与食品科学学院 博士学位论文 生物黄酮抑制食品中丙烯酰胺形成的机理及其构效关系研究 姓名：章宇 申请学位级别：博士 专业：食品科学 指导教师：张英;任一平 20080501

浙江大学博士学位论文 病要 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交全性研究具有重要 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，是日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内烯酰胺形成的机理及其构效关系。 论文首先建立了内烯酰胺的气相色谱(GC)和液相色谱串联质谱(LC-MSMS) 检测方法：通过对常规热加丁食品、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-MSMS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%(ww)的AOB浸渍处理，炸薯片和炸薯条的内烯酰 胺形成抑制率可达74.1%和76.1%：当A0B添加量为0.5%(ww)时，炸鸡翅的丙 烯酰胺形成抑制率可达59.0%：当A0B和EGT的添加量分别为0.1%和0.01%(ww) 时，油条的内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巾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(OM)为试验设计方法，以超高效液相色谱-米联质谱 (UPLC-MSMS)法为检测手段，系统研究了天冬酰胺葡萄糖/果糖/蔗脚模式体系分 别在低湿和微波加热条件下产生内烯酰胺的规律。其中，天冬酰胺葡萄模式体系 产生内烯酰肢的优化条件为：两种前体物质以等摩尔比例在180℃的低湿体系巾加 热15min或在微波体系加热5min。 选择天冬酰胺葡萄糖模式体系，研究AOB和EGT对内烯酰胺形成/消除动力学

浙江大学博士学位论文 摘要 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)和液相色谱一串联质谱(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肭S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O．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O．5％(w／w)时，炸鸡翅的丙 烯酰胺形成抑制率可达59．O％；当AOB和EGT的添加量分别为O．1％和O．01％(w／w) 时，油条的内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巾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牛丙烯酰胺的规律。其中，天冬酰胺．筒萄糖模式体系 产牛内烯酰胺的优化条件为：两种前体物质以等摩尔比例在180 oC的低湿体系巾加 热15 min或在微波体系。Il力几热5 min。 选择天冬酰胺．葡萄糖模式体系，研究AOB和EGT对丙烯酰胺形成片肖除动力学 i

新江大学博上学位论文 病要 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2x10mgg和10 mgmL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/消除级动力学模型描述低湿和微波体系中AOB和EGT对丙烯酰肢动力学过 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10moL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 物黄酮对丙烯酰肢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，黄酮芳环羟基的数门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，黄酮对内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、果糖和丙烯酰肢的同位素稀释UPLC MSMS检测方法。根据高抑制率和代衣性原则，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-7-0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”moL)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，牛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 及中间产物向内烯酰胺转化的过程，何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，但这种作川并不显著。 关键词：生物黄酮：丙烯酰胺抑制：土豆微波模式体系：抗氧化活性：构效关系： 量效关系；作用位点：抑制动力学

浙江大学博士学位论文 摘要 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。6 mg／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母mol／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F1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 同昔元的黄酮糖苷而育，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)、木犀草素一7．D-葡 萄糖苷、木犀草素、染料木素和槲皮素为牛物黄酮的典型试样，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，牛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 及中问产物向内烯酰胺转化的过程，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，但这种作丌j并不显著。 关键词：牛物黄酮：丙烯酰胺抑制；土豆微波模式体系；抗氧化活性；构效关系； 量效关系；作用位点：抑制动力学

新江大学博士学位论文 Abstract 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 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 .1%and1%(w/w)of AOB solutions before frying. respectively.Similarly,590%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)ofAOB and 0.01%(w/w)of EGT were added in the flour,respectively.The optimal addition levels of AOB and EGTwere finally obtained after taking the maximum permissible addition levels and sensory quality into consideration

浙江大学博士学位论文 Abstract 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 fAOB)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 flies when they were treated by 0．1％and 0．0 1％(w／w)of AOB solutions before frying， respectively．Simi larly,59．0％of acrylam ide was reduced in flied 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 0．0 1％(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．

新江人学博士学位论文 Abstract 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 in the microwave system at 180C. The acrylamide content could be decreased either by inhibiting its formation or promoting its elimination.The effect of AOB and EGTon 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 2x10mg/g and 10mg/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 coud significantly reduce the formation kinetics of acrylamide while on 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 maximumduction effect when the addition levels of bio-flavonoids were1mol/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 iv

浙江人学博士学位论文 Abstraet Based on the confirmation of the reduction effect of bio—flavonoids on acrylamide i n foods， the formation ofacrylamide 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 1 5 min in the lOW—moisture system at 1 80 oC or 5 min in the microwave system at 1 80 oC． 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 2x 1 04 mg／g and 1 0七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 el imination 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 e虢ct when the addition levels of bio．flavonoids were l 0一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