食品中交链孢毒素污染、生物利用及风险评估研究

发布时间：2018-09-01 09:55

【摘要】：目的：建立食品中交链孢菌酮酸(TeA)、交链孢酚(AOH)、交链孢酚单甲醚(AME)和腾毒素(TEN)的UPLC-MS/MS协同测定方法,并利用所建方法对国产小麦粉及其制品、番茄及其制品、柑橘及其制品中的4种交链孢毒素污染状况进行研究；体外建模,探讨食品中交链孢毒素经口的生物可及性和生物利用率；利用毒理学关注阈值(TTC)方法,对我国居民通过小麦及其制品暴露TeA、AOH、AME和TEN对其健康的影响风险进行评估。方法：(1)通过对质谱、色谱条件条件的优化,确定各种方法学参数,建立协同检测食品中TeA、AOH、TEN和AME的UPLC-MS/MS方法。(2)利用所建立的方法对采自我国河南、山东、安徽、吉林和北京5个省(市)的323份小麦粉及其制品、70份番茄及其制品和86份柑橘及其制品中的4种交链孢毒素污染水平进行测定。(3)体外条件下,模拟胃肠道生理消化过程,构建小麦粉制品中交链孢毒素在口腔、胃和小肠中生物可及性评价模型,并用真菌毒素吸附剂对模型进行验证；以人克隆结肠腺癌细胞(Caco-2)单层细胞系为工具,构建Caco-2细胞转运TeA、AOH和AME 3种毒素的肠转运体外模型并进行了验证,结合3种交链孢毒素的经口生物可及性,对3种交链孢毒素的生物利用率进行研究。(4)运用TTC方法对我国居民通过小麦粉及其制品暴露4种交链孢毒素对其健康的影响风险进行评估,结合研究获得的生物利用数据对其膳食内暴露量进行初步评估。结果：(1)建立的UPLC-MS/MS检测四种交链孢毒素方法的检出限在0.1-8 μg/kg之间,加标回收率在58.9-116.0%之间,等同或高于国际同类方法的回收率,相对标准偏差(RSD)在3.0-12.4%之间,RSD低于20%,可满足实际检测要求。(2)TeA为污染三类食品的主要交链孢毒素,在小麦粉中的检出率为99.4%,小麦粉中TeA污染水平范围为1.76-520 μg/kg(均值：88.4μg/kg)。小麦粉中AOH、 AME和TEN的检出率分别为6.1%、91.2%和97.2%,污染水平范围分别为16.0-98.7 μg/kg(均值：30.2 μg/kg)、0.320-61.8 μg/kg(均值：3.77 μg/kg)和2.25-129 μg/kg(均值：27.1 μg/kg)。小麦粉中TeA的污染水平存在地域差异,含量由高到低依次为：河南、安徽、北京、山东、吉林。小麦粉及其制品样品中四种毒素含量呈正相关,分别为：AOH与AME(r=0.904, P0.05)、TeA与AME(r=0.757, P0.01)、TeA与AOH+AME (r=0.860, P0.01)、TEN与TeA(r=0.747,P0.01。番茄酱和番茄汁中TeA的检出率均为100%,含量范围分别为10.2-1787 μg/kg均值：338μg/kg)和7.4-278 μg/kg(均值：87 μg/kg)。进口番茄酱样品中TeA含量显著高于国产样品(P0.05)。番茄酱中AOH和AME的含量范围分别为2.5-300μg/kg(均值：80 μg/kg)和0.32-38 μg/kg(均值：8.0 μg/kg); TEN的含量范围1.53-15.8 μg/kg(均值：4.9 μg/kg)。番茄汁中AME含量范围为0.20-5.8 μg/kg (均值：1.42μg/kg)。柑橘汁样品中TeA检出率为25.0%,污染水平范围为1.21-4.3μg/kg(均值：1.80 μg/kg),其他毒素均未检出。(3)建立了评价交链孢毒素生物可及性和生物利用率体外消化模型。馒头样品中TeA、AOH和AME的经口生物可及性分别为72.1±5.4%、7.0±0.8%和10.2±1.0%。成功构建了体外评价交链孢毒素肠转运的Caco-2细胞膜型。AOH、AME和TeA在不同浓度和转运时间的吸收率范围为1.5%和63.0%,TeA为易于吸收的毒素。(4)全人群和各年龄组人群通过小麦及其制品暴露AOH和AME量的均值和P97.5位数均超过TTC值,其中以2-6岁人群的暴露量最高,其次是7-17岁年龄组人群；全人群和各年龄组人群中高食物量消费者AOH的内暴露上限(UB)也超过TTC,应引起关注。全人群和各年龄组人群通过小麦及其制品暴露TeA和TEN的均值和P97.5位数均低于TTC值,对健康影响的风险较低。结论：建立的协同检测4种交链孢毒素的方法灵敏、特异,可满足不同基质中4种毒素分析需要；我国小麦及其制品、番茄制品和柑橘制品被交链孢毒素污染,以TeA污染最重；构建的生物可及性体外消化模型稳定、重复性好,完全可以满足对食品中各类交链孢毒素生物可及性的研究；所建3种毒素Caco-2单层细胞模型稳定,可用于3种毒素体外生物利用率研究中。我国居民高通过小麦及其制品中暴露AOH和AME对其健康影响的风险较高,应引起关注。在充分获取数据的基础上,有必要对污染水平和污染率较高的TeA做进一步的评估。
[Abstract]:OBJECTIVE: To establish a UPLC-MS/MS method for the simultaneous determination of alternosporin ketoacid (TeA), alternosporin (AOH), alternosporin monomethyl ether (AME) and tenotoxin (TEN) in food, and to study the contamination status of four kinds of alternosporin in wheat flour and its products, tomato and its products, citrus and its products. To investigate the oral bioavailability and bioavailability of Alternaria toxins in foods, and to assess the health risk of TeA, AOH, AME and TEN exposure to wheat and its products by toxicological concern threshold (TTC) method. A UPLC-MS/MS method for the simultaneous determination of TeA, AOH, TEN and AME in food was developed. (2) The contamination levels of 4 cross-linked sporotoxins in 323 wheat flour and its products, 70 tomatoes and their products and 86 citrus and their products from Henan, Shandong, Anhui, Jilin and Beijing provinces (cities) were determined in vitro. The bioavailability of Alternaria toxin in wheat flour products in oral, stomach and small intestine was evaluated by simulating gastrointestinal physiological digestion process, and the model was validated by mycotoxin adsorbent. Human colon adenocarcinoma cell (Caco-2) monolayer cell line was used to construct the intestine of Caco-2 cells transporting TeA, AOH and AME toxins. The in vitro transport model was validated, and the bioavailability of three Alternaria toxins was studied in combination with the bioavailability of three Alternaria toxins. (4) The risk of exposure to four Alternaria toxins from wheat flour and its products was assessed by TTC method, and the bioavailability of the three Alternaria toxins was also evaluated. Results: (1) The detection limits of the four methods were 0.1-8 ug/kg, and the recovery rates were 58.9-116.0%. The recoveries were equivalent to or higher than those of the international methods. The RSD ranged from 3.0-12.4%, and RSD was lower than 20%. (2) TeA was the main cross-linked spore toxin in three kinds of food. The detection rate of TeA in wheat flour was 99.4%. The contamination range of TeA in wheat flour was 1.76-520 ug/kg (mean: 88.4 ug/kg). The detection rates of AOH, AME and TEN in wheat flour were 6.1%, 91.2% and 97.2% respectively, and the contamination range was 16.0-98.7 ug/kg (mean: 30.2%). The contamination levels of TeA in wheat flour varied from high to low in order: Henan, Anhui, Beijing, Shandong, Jilin. The contents of four toxins in wheat flour and its products were positively correlated, respectively: AOH and AME (r = 0.904, P 0.05). TeA and AME (r = 0.757, P 0.01), TeA and AOH + AME (r = 0.860, P 0.01), TEN and TeA (r = 0.747, P 0.01). The detection rates of TeA in tomato sauce and tomato juice were 100%, and the contents ranged from 10.2 to 1787 ug/kg with an average value of 338 ug/kg and 7.4 to 278 ug/kg with an average value of 87 ug/kg. The TeA content in imported tomato sauce samples was significantly higher than that in domestic samples (P 0.05). The contents of AOH and AME in sauce ranged from 2.5 to 300 ug/kg (mean: 80 ug/kg) and 0.32 to 38 ug/kg (mean: 8.0 ug/kg), TEN ranged from 1.53 to 15.8 ug/kg (mean: 4.9 ug/kg), AME ranged from 0.20 to 5.8 ug/kg (mean: 1.42 ug/kg) in tomato juice, TeA detection rate in citrus juice was 25.0%, and pollution level ranged from 1.2%. 1-4.3 ug/kg (mean: 1.80 ug/kg) and no other toxins were detected. (3) An in vitro digestion model was established to evaluate the bioavailability and bioavailability of Alternaria toxin. The oral bioavailability of TeA, AOH and AME in steamed bread samples were 72.1 (+ 5.4%), 7.0 (+ 0.8%) and 10.2 (+ 1.0%) respectively. The absorption rates of AOH, AME and TeA were 1.5% and 63.0% at different concentrations and transport times, and TeA was easily absorbed toxin. (4) The mean and P97.5 digits of AOH and AME exposures to wheat and its products exceeded TTC values in the whole population and all age groups, with the highest exposure in the 2-6 year-old group, followed by the 7-17 year-old group. The upper limit of internal exposure (UB) of AOH in high-food-intake consumers was also higher than that of TTC in the whole population and all age groups. The mean and P97.5 digits of TEA and TEN exposure to wheat and its products were lower than that of TTC in the whole population and all age groups, and the risk of health effects was lower. The method is sensitive and specific, and can meet the needs of four toxins analysis in different substrates; wheat and its products, tomato products and citrus products are polluted by Alternaria toxin, TeA pollution is the most serious; the bioavailability in vitro digestion model is stable and reproducible, and it can completely meet the requirements of Alternaria toxin bioavailability in food. The single-layer cell model of three toxins Caco-2 is stable and can be used to study the bioavailability of the three toxins in vitro. TeA for further evaluation.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R155.5

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