【作者】 廖国周；

【导师】 周光宏；

【作者基本信息】 南京农业大学 ， 食品科学， 2008， 博士

【摘要】 饮食是诱发癌症的一个重要因素,而杂环胺是肉品在高温加工过程中通过美拉德反应与自由基机制而形成的一类致癌致突变化合物。杂环的形成受物理与化学因素的影响,如加工温度与时间,加工方式与设备,以及前体物等。目前肉品加工过程形成的杂环胺已引起研究者的广泛关注,并对杂环胺的各个方面展开研究,如在不同食品中的形成与出现,暴露的水平,生物利用效率,生物转移和致癌性,从而确定其对人类癌症形成的影响。然而中国传统肉制品的加工方法与国外的有较大差异,且国内尚未见相关的研究报道,因此本研究旨在探讨传统烧烤肉制品中杂环胺的形成量与形成机制,并进一步探讨天然抗氧化剂对烧烤肉制品中杂环胺形成的影响。具体研究内容和结果如下:1.传统肉制品中杂环胺检测方法的研究选取烤鸭、烤羊肉、烧鸡、卤牛肉和肉松5种传统肉制品为对象分析其杂环胺含量,样品净化前处理通过串联Extrelut-PRS-C₁₈柱进行固相萃取,经TSK-gelODS-80TM柱分离并以配备二极管阵列和荧光检测器的HPLC对杂环胺进行测定。结果表明,建立的方法中15种杂环胺的线性范围为0.01 ng·μL^-1-1.0 ng·μL^-1,检测限为0.02 ng·μL^-1-0.3 ng·μL^-1,回收率为61%-94%,相对标准偏差为2.4%-8.0%,该方法达到定量分析的要求。在传统肉制品中检测出6种杂环胺,且不同的肉制品中杂环胺的种类与含量不一致。9H-吡啶并[3,4-6]吲哚（Norharman）与1-甲基-9H-吡啶并[3,4-b]吲哚（Harman）均存在于所有肉制品中,含量分别为42.75 ng·g^-1和33.9 ng·g^-1。3-氨基-1-甲基-5H-吡啶并[4,3-b]吲哚（Trp-P-2）只存在于烤鸭与烤羊肉中,2-氨基-1-甲基-6-苯基-咪唑并[4,5-b]吡啶（PhIP）在烤鸭、烤羊肉和肉松中检出,而2-氨基-9H-吡啶并[2,3-b]吲哚（AaC）、2-氨基-3-甲基-9H-吡啶并[2,3-b]吲哚（MeAaC）只存在于肉松中,其含量均小于2 ng·g～（-1）。2.加工方式对鸭肉中杂环胺形成的影响将鸭肉分别进行蒸煮、微波、炭烤、烘烤、油炸与煎烤加工处理,加工鸭肉中的杂环胺分析前处理通过串联Extrelut-PRS-C₁₈柱进行固相萃取,经TSK-gel ODS-80TM柱分离并以配备二极管阵列和荧光检测器的HPLC对杂环胺进行测定。结果表明,加工鸭肉的烹调损失及杂环胺的含量与种类随着加工温度的升高与时间的延长而增加。煎烤鸭肉中形成的杂环胺种类与含量最大,其次为炭烤、油炸、烘烤、微波与蒸煮鸭肉。9H-吡啶并[3,4-b]吲哚（Norharman）与1-甲基-9H-吡啶并[3,4-b]吲哚（Harman）存在于所有加工鸭肉中,其含量在0.1 ng·g^-1-33 ng·g^-1之间。2-氨基-1-甲基-6-苯基-咪唑并[4,5-b]吡啶（PhIP）在煎烤与炭烤鸭肉中形成较多,含量在0.9 ng·g^-1-17.8 ng·g^-1之间。2-氨基-3甲基咪唑并[4,5-f]喹啉（IQ）只在煎烤与炭烤鸭肉中检出,含量在0.39 ng·g^-1-4.19 ng·g^-1之间。2-氨基-3,8-二甲基咪唑并[4,5-f]喹喔啉（MeIQx）在炭烤、烘烤、油炸与煎烤鸭肉中检出,含量高达4.49 ng·g^-1,其他杂环胺含量均小于10 ng·g^-1。加工鸭肉色泽随着加工温度升高而增加,但与杂环胺的形成量不存在相关性。3.原料肉中前体物含量对杂环胺形成的影响将牛、猪、羊、鸡、鸭和鹅肉分别在200℃下煎烤10 min,分析其原料肉中前体物的含量与加工肉制品中杂环胺的形成量,以探讨不同动物原料肉中杂环胺的形成量及原料肉中前体物的含量对杂环胺形成的影响。结果表明,6种原料肉中前体物含量差异较大,肌酸与葡萄糖的摩尔浓度比在0.89-9.84之间。加工肉制品中共检测出10种杂环胺,其中9H-吡啶并[3,4-b]吲哚（Norharman）与1-甲基-9H-吡啶并[3,4-b]吲哚（Harman）在加工肉品中含量最高,分别在10.90 ng·g^-1-24.16 ng·g^-1与4.64 ng·g^-1-14.04 ng·g^-1之间。而2-氨基-3,8-二甲基咪唑并[4,5-f]喹喔啉（MeIQx）在牛肉中形成量较高,达3.55 ng·g^-1,2-氨基-1-甲基-6-苯基-咪唑并[4,5-b]吡啶（PhIP）在禽肉中易于产生,其中鸡肉中含量最高,达15.26 ng·g^-1。PhIP与肌酸和葡萄糖的摩尔浓度比存在相关性（P<0.05）,随着葡萄糖浓度的增高,PhIP的形成量减少。4.模型体系中杂环胺形成的动力学研究以羊肉模型体系为对象对杂环胺形成的动力学进行研究,探讨加热温度、时间及前体物对杂环胺形成的影响。羊肉分别在四个不同温度（160、180、200、220℃）下加热四个时间段（5、10、15、20 min）,肉样中形成的杂环胺采用固相萃取并以配合二极管阵列和荧光检测器的HPLC进行分析。结果表明,羊肉中共检出IQ、MeIQx、4,8-DiMeIQx、PhIP、Norharman、Harman、Trp-P-2、Trp-P-1、AaC与MeAaC 10种杂环胺。随着加热温度与时间的增加,杂环胺的形成量增加,220℃下加热20 min,IQ、MeIQx、4,8-DiMeIQx、PhIP、Norharman、Harman、Trp-P-2、Trp-P-1、AaC与MeAaC的形成量分别为4.51 ng·g^-1、10.99 ng·g^-1、5.75 ng·g^-1、7.44 ng·g^-1、25.71 ng·g^-1、22.04 ng·g^-1、2.17 ng·g^-1、1.80 ng·g^-1、6.55 ng·g^-1、6.32 ng·g^-1。杂环胺前体物葡萄糖与肌酸的含量随着杂环胺形成量的增加而减少,呈负相关;而肌酸酐则相反,呈正相关。杂环胺形成的机制遵循一种反应物浓度过量的双分子反应机制,为准一级反应。5.天然抗氧化剂对烤羊肉中杂环胺形成的影响将0.05%VE,0.1%与0.5%葡萄籽提取物,0.1%与0.5槲皮素添加于羊肉表面,分别在200℃下煎烤10 min,利用固相萃取-配备二极管阵列检测器的HPLC分析烤羊肉中15种杂环胺的形成量,以探讨天然提取物对烤羊肉中杂环胺的形成量的影响。结果表明,烤羊肉中共检测出10种杂环胺,其中极性杂环胺4种,非极性杂环胺6种,总量达51.68 ng·g^-1。在极性杂环胺中PhIP的形成量较高,达10.50 ng·g^-1;非极性杂环胺中Norharman与Harman形成量较高,分别达19.66 ng·g^-1与12.23 ng·g^-1。烤羊肉中添加天然抗氧化剂只对极性杂环胺有抑制作用。其中添加0.05%VE、0.1%槲皮素与0.5%槲皮素对杂环胺抑制作用不明显,添加0.5%葡萄籽提取物可以显著抑制IQ,MeIQx,4,8-DiMeIQx与PhIP 4种极性杂环胺的生成（p<0.05）,其生成量分别减少36.65%,67.73%,35.63%与30.00%。天然抗氧化剂对烤羊肉中非极性杂环胺的抑制作用不明显。更多还原

【Abstract】 Diet is an important factor in the global variation of human cancer. Heterocyclic aromatic amines （HAAs） are mutagenic/carcinogenic compounds formed at low levels via the Maillard reaction and a free radical mechanism during cooking of animal tissue at high temperature. The formation and yield of HAAs are dependent on physical parameters, such as cooking temperature and time, cooking technique and equipment, heat and mass transport, and on chemical parameters, especially the precursors to HAAs. To date, researchers all over the world are busy studying various aspects of HAAs such as their formation, occurrence in different food products, bioavailability, bio-transformation, carcinogenicity, levels of exposure to consumers, in efforts to determine their significance in human cancer development. However, the processing methods and conditions of Chinese traditional food is different from Western, and to date little is known about HAAs content in the traditional meat products of China. The objectives of the present study were to investigate the formation of HAAs in Chinese traditional food, and to evaluate their inhibition through the addition of natural antioxidants to meat before cooking. The contents and results are as follows.1. Detection of heterocyclic aromatic amines in traditional meat products by HPLCHAAs content was investigated in five traditional meat products （including roasted duck, charbroiled mutton, dried pork floss, marinated beef, roasted chicken）. The clean-up procedure included tandem solid-phase extraction （SPE） using Extreltut-type columns filled with diatomaceous earth, propylsulphonic acid and chemically bounded phase-C₁₈. Separation was achieved using TSK-gel ODS-80TM column and identification and quantitative analysis of HAAs fraction was carried out using a HPLC system with DAD and FLD. Results showed that the linear ranges were 0.01 ng·μL^-1-1.0 ng·μL^-1 and the limit of detection were0.02 ng·μL^-1-0.3 ng·μL^-1 for 15 HAAs. Recoveries were 61%-94%, the relative standard deviation were 2.4%-8.0%. Six HAAs were detected in the traditional meat products, the various and content of HAAs differentiated among the meat products. 9H-pyrido[4,3-b]indole （Norharman） and 1-methyl-9H-pyrido[4,3-b]indole （Harman） were detected in all of the meat products, and the content of it were up to 42.75 ng·g^-1, 33.9 ng·g^-1 respectively. 3-amino-1-methyl-5H-pyrido[4,3-b]indole （Trp-P-2） was only detected in roasted duck and charbroiled mutton. 2-amino-1-methyl-6-phenylimidazo [4,5-f]pyridine （PhIP） was detected in roasted duck, charbroiled mutton and dried pork floss. However, 2-amino-9H-pyrido[2,3-b]indole （AaC） and 2-amino-3-methyl-9H-pyrido[2,3-b] indole （MeAaC） were only detected in dried pork floss. The content of it were less 2 ng·g^-1.2. Formation of heterocyclic aromatic amines during processing of duck meatThe effects of various processing methods, boiling, microwave cooking, charcoalgrilling, roasting, deep-frying and pan frying on the formation of heterocyclic aromatic amines （HAAs） in duck breast were studied. Results showed that both the varieties and contents of HAAs and the cooking loss of duck breast increasing along with increasing cooking temperature and time. Pan frying of duck breasts contained the highest amount of total HAAs, followed by charcoal grilling, deep-frying, roasting, microwave cooking and boiling. 9H-pyrido[4,3-b]indole （Norharman） and 1-methyl-9H-pyrido[4,3-b]indole （Harman） were detected in all of the cooked duck meat, and the content of it were from 0.1 ng·g^-1 up to 33 ng·g^-1. 2-amino-1-methyl-6-phenylirnidazo[4,5-f]pyridine （PhIP） was formed easily in cooked duck meat by pan frying and charcoal grilling, the content of it was from 0.9 ng·g^-1 up to 17.8 ng·g^-1. 2-amino-3-methylimidazo[4,5-f]quinoline （IQ） was identified in duck meat by charcoal grilling and pan frying, the content of it was from 0.39 ng·g^-1 up to 4.19 ng·g^-1. 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline （MeIQx） was detected in amounts below 4.49 ng·g^-1 in duck meat by charcoal grilling, roasting, deep-frying and pan frying. The other HAAs were detected in amounts below 10 ng·g^-1. Colour development increases with cooking temperature, but no correlation with HAAs content was observed.3. Effect of precursor composition in raw meat on the formation on heterocyclic aromatic amines in pan fried meatsThe meat pieces of following animals species, beef, pork, mutton, chicken, duck and goose were pan fried at 200℃for 10 min to study the occurrence of HAAs in fried meat of various animal species and the effect of precursor composition in raw meat on the formation of HAAs in pan fried meat. The composition of precursor （free amino acids, creatine and glucose） was examined in raw meat and the HAAs were detected in pan fried meat by HPLC with DAD and FLD detection. Results showed that great differences were found between the precursor content, both between the animal species and within any species. The molar ratio of total creatine/glucose was from 0.89 up to 9.84. Ten HAAs were detected in pan fried meat. 9H-pyrido[4,3-b]indole（Norharman） and 1-methyl-9H-pyrido [4,3-b]indole （Harman） were detected at high levels in cooked meat, the concentrations of the two compounds ranged from 10.90 ng·g^-1 up to 24.16 ng·g^-1 and from 4.64 ng·g^-1 to 14.04 ng·g^-1 respectively. 2-amino-3, 8-dimethyl-imidazo[4,5-f]quinoxaline（MeIQx） was detected in amounts up to 3.55 ng·g^-1 in beef. 2-amino-1-methyl-6-phenylimidazo-[4, 5-f]pyridine （PhIP） was formed easily in poultry, the content of PhIP in chicken was up to 15.26 ng·g^-1. A significant linear correlation only existed between PhIP and the molar ratio of total creatine/glucose（P<0.05）, the increased glucose contents reduced the concentrations of PhIP.4. Kinetics of heterocyclic aromatic amines formation in meat model systemMutton was used as a model system to examine the kinetics of the formation ofherterocyclic aromatic amines and elucidate the effects of time, temperature, and precursors on the formation of HAAs in cooked ground mutton. Freshly ground mutton was heated at four different temperatures （160, 180, 200, 220℃） for four different time intervals （5, 10, 15, 20 min）. HAAs were extracted and purified using solid-phase extraction and quantified using reverse-phase HPLC equipped with DAD and scanning fluorescence detectors. The results showed that 10 HAAs （IQ, MeIQx, 4, 8-DiMeIQx, PhIP, Norharman, Harman, Trp-P-2, Trp-P-1, AaC and MeAaC） were identified. The formation of HAAs increased with increasing time and temperature. The highest amount of HAAs formation of 4.51 ng·g^-1, 10.99 ng·g^-1, 5.75 ng·g^-1, 7.44 ng·g^-1, 25.71 ng·g^-1, 22.04 ng·g^-1, 2.17 ng·g^-1、1.80 ng·g^-1, 6.55 ng·g^-1、6.32 ng·g^-1, respectively were found after heating for 20 min at 220℃. Glucose and creatine decreased with increasing time and temperature, while creatinine increased with increasing time and temperature. The formation of HAAs and the degradation of precursors were inversely correlated. The formation of HAAs followed a bimolecular mechanism with one reactant concentration in large excess.5. Inhibition effect of natural extract on the formation of heterocyclic aromatic amines in cooked muttonThe effects of vitamin E, grape seed extract and quercetin on HAAs formation in fried ground mutton patties were studied. Antioxidants were added directly to the surface of the patties before frying. Patties were fried at 200℃for 10 min. HAAs were isolated by solid phase extraction and quantitated by HPLC with DAD and FLD detection. Results showed that 10 HAAs were detected in fried ground mutton patties, the total amount of HAAs was up to 51.68 ng·g^-1. 9H-pyrido[4,3-b]indole （Norharman）, 1-methyl-9H-pyrido [4,3-b]indole （Harman） and 2-amino-1-methyl-6- phenylimidazo-[4,5-f]pyridine （PhIP） were detected at high levels in fried ground mutton patties, the concentrations of the two compounds were 19.66 ng·g^-1,12.23 ng·g^-1 and 10.50 ng·g^-1 respectively. Compared to the control, the HAAs were not significantly decreased by 0.05% vitamin E, 0.1% quercetin and 0.5% quercetin, but it showed the decreased trend. IQ was effectively decreased by 0.5% grape seed extract in the treatment compared to the control. IQ concentrations in cooed patties were reduced by 10.32%. Grape seed extract at 0.5% levels significantly reduced the formation of IQ, MeIQx, 4,8-DiMeIQx and PhIP by 36.65%, 67.73%, 35.63%and 30.00% respectively （p<0.05）. Antioxidants were not effectively inhibited the formation of apolar HAAs in cooked mutton.更多还原