【作者】 刘睿杰；

【导师】 王兴国；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2011， 博士

【摘要】 本论文研究了黄曲霉毒素B1(以下简称AFB1)在不同介质中降解行为、降解产物、路径及机理,并通过鼠伤寒沙门氏菌致突变实验和细胞毒理实验对降解产物的致突变性和毒性进行了研究,同时对紫外光降解AFB1后的花生油品质做安全性评估。首先在乙腈体系中研究不同初始浓度、照射光类型、照射强度和时间等因素对AFB1紫外光降解行为的影响。结果表明AFB1紫外光降解速度与初始浓度无关,符合一级动力学模型(R2≥0.9),受照射光类型、强度以及光照时间等因素影响。在不同波长照射光、强度以及照射时间下降解速率按照以下顺序递减v(UVA+UVC)>vUVC>vUVA,v800μw/cm2 > v400μw/cm2 >v200μw/cm2。运用UPLC-Q-TOF/MS技术鉴定出乙腈中AFB1三种紫外光降解产物为A(C14H10O6)、B(C17H14O6)和D(C16H12O5),利用光化学反应机理解释三种产物的生成机理:AFB1通过分子间氢提取反应生成B,接着在C(9c)-O(10)位发生光加成反应,在C(6a)-C(9a)处发生光消去反应,生成较稳定的A物质。D产物则由AFB1母核在C(11)-O(15)发生光消去反应生成。其次,研究不同降解体系中AFB1紫外光降解行为,发现在不同降解介质中AFB1的降解速率不同,花生油中最快,水中次之,乙腈中最慢,产物也不尽相同。同时解释此现象是由三种溶剂中自由基丰富程度所致。水和花生油两种体系中AFB1的降解规律得到验证与乙腈模型中一致,与AFB1初始浓度无关,符合一级动力学模型,受光照强度及时间影响。AFB1在水中生成三种紫外光降解产物为P1(C17H14O7)、P2(C16H14O6)和P3(C16H12O7),降解过程首先由AFB1C(8)-C(9)位发生水致光加成反应生成中间产物P1,P1在C(1)-O(14)位发生的光还原和C(4)-O(12)发生的光消去反应形成P2,同时P1在C(12)-O(13)位发生的光消去反应产生P3。三种降解产物结构与AFB1相似,只在部分基团发生变化。花生油中AFB1紫外光降解反应剧烈,推测AFB1分子吸收紫外光后,迅速发生光氧化反应,断裂为R-COOH、R-CO-R’等微量小分子物质。利用光化学反应原理对两种介质中可能的降解路径给予合理解释。应用Ames实验检测AFB1在水、花生油两种体系中不同紫外光降解产物的致突变性,实验结果表明经紫外照射去除两种体系中AFB1后,水中紫外光降解产物致突变性已大幅度降低,但没有彻底去除;而花生油中紫外光降解产物毒性完全消失。采用MTT及流式细胞术研究AFB1及其紫外光降解产物对HepG2细胞的影响。结果表明:作为阳性对照的AFB1对HepG2细胞有很强的细胞毒性,导致细胞活性降低78.6%。水中紫外光降解产物处理细胞的细胞活性降低8.5%左右,花生油中紫外光降解产物对HepG2细胞活性无任何影响。通过酶联免疫技术,研究了三类测试物对人类肿瘤相关性最高的抑癌基因p53表达量的影响,从而对AFB1及其紫外光降解产物的致癌性做初步评估。实验结果表明,AFB1使p53的表达显著降低,水中紫外光降解产物处理细胞的p53基因表达有微量减少,花生油中产物对p53基因的表达量无影响。致突变和细胞毒性实验结果与本论文前两章推测的降解产物结构鉴定结果相符,AFB1水中紫外光降解产物结构与AFB1结构相似,AFB1中毒性活性位点之一呋喃环仍然残存,因此,仍然保留部分毒性。相反,在花生油体系中的紫外光降解产物已失去与AFB1相似的结构,其化学性质发生巨大变化,因此相应的毒性和致癌性也随之消失。最后研究紫外照射技术应用于花生油时对花生油品质的影响。在可完全降解2μg/mL(远大于实际污染量)AFB1照射剂量范围内,研究紫外照射对花生油酸价、碘价、羰基值、过氧化值及维生素E及反式脂肪酸等重要因素的影响。结果表明紫外照射除使花生油过氧化值显著增加(p<0.01)外,对花生油其他品质并无影响。说明利用紫外照射技术去除花生油中黄曲霉毒素的方法不仅有效、安全,对花生油的品质及营养成分也无影响。更多还原

【Abstract】 The photodegradation behavior, products, pathway and mechanisms of AFB1 in different media were studied by the technique of UPLC-Q-TOF/MS in this article. The Ames test and cytotoxicity experiment were used here to evaluate the mutagenesis and toxicity of the photodegradation products of AFB1 in food, and an overall assessment of the safety of this detoxification method of Aflatoxins applied in peanut oil were carried out at last.Firstly, the photodegradation behavior at various initial concentration, wavelength, irradiation time and intensities were studied. The results indicate that the degradation ratio reduced at below order, v(UVA+UVC) > vUVC> vUVA , v800μw/cm2 > v400μw/cm2 >v200μw/cm2,vpeanut oil> vwater >vacetonitrile, and is not affected by the initial concentration, meanwhile AFB1 was proved to follow first-order reaction kinetics (R2≥0.9). At the same time, three photodegradation products A (C14H10O6), B (C17H14O6), D (C16H12O5) and the pathway of AFB1 in acetonitrile were identified using UPLC-Q-TOF/MS and elucidated by photochemistry mechanism. Hydrogen abstraction reaction of AFB1 lead to the generation of A, and then photoaddition at cite C(9c)-O(10) along with photoelimination reactions at cite C(6a)-C(9a) generated A product, at last the product D was generated by photoelimination at cite C(11)-O(15). Secondly, the photodegradation ratio and degradation products in various media were studied and discovered different in each media. The ratio in peanut oil is the fastest; in acetonitrile is the slowest, which was elucidated by the quantity of free electron in various medias.The degradation regular in water and peanut oil were verified the same to that in acetonitrile model, which follows the first-order reaction kinetics (R2≥0.9). Three photodegradation products P1 (C17H14O7), P2 (C16H14O6) and P3 (C16H12O7), which generated by photoaddition at cite C(8)-C(9) from AFB1 to P1, photoelimination at C(4)-O(12) and photoreduction reactions at O(1)-C(14) from P1 to P2 and photoelimination at C(12)-O(13) from P1 to P3, were identified and find that the main structures of them were similar to that of the AFB1. The possible photodegradation reaction in peanut oil was severe and the structure of AFB1 was destroyed thoroughly to minor molecular substances, such as R-COOH and R-CO-R’. The photodegradation pathway mechanism of AFB1 in two medias were elucidated seasonable by photochemistry principle.The Ames test was used to detect the mutagenesis of the photodegradation products in water and peanut oil. The results indicated that the reversion rate of the photodegradation in water (Pw) is higher than that of negative control (0ng AFB1), while there is no differences of the reversion rate between the photodegradation in oil (Po) and the negative control. The mutagenesis of the Pw decreased significantly but not be removed thoroughly, while that of Po is disappeared absolutely.MTT and FCM were employed to study the cytotoxicity of Pw, Po and AFB1 in HepG2 cell line. The results indicate that: the cell viability of AFB1-treated cell, used as positive control, decreas 78.6%. The cell viability of the Pw-treated cell reduce about 8.5%, while that of Po has no differences compared to the untreated cells, p>0.05. The expression of the antioncogene p53 protein in HepG2 cells treated by three tested compounds was detected by ELISA to evaluate the carcinogenicity of Po and Pw. The results showe that the expression of p53 protein significantly decrease in AFB1-treated cells; the concentration of p53 in Pw-treated cells slightly reduce and that in Po-treated cells has no difference compared with the untreated cells.The conclusion of the Ames test and the cytotoxicity are in agreement with the results of the identification of the photodegradation products in two kinds of media. The main structures of Pw are similar with the AFB1, and the furan ring, one of the toxic activity cites of AFB1 was not destroyed, remaining the corresponding toxicity. In contrast, the possible structures of Po are different from AFB1, following great change of the chemical nature, for that the corresponding toxicity and the carcinogenicity disappeared.At last, acidity value, iodine value, peroxide value, concentration of VE and total trans fatty acids as well as oil stability index were selected to study the effect of UV detoxification irradiation treatment on the quantity of peanut oil. The results show that there is no significant change of these parameters (p>0.05) except the significant increase of the peroxide value (p<0.01) after the selected dose (2ppm AFB1 can be destroyed absolutely) of UV irradiation treatment, which indicating that detoxification method used in peanut oil is not only effective, but also has no bad effect on the quantity of peanut oil.更多还原