【作者】 鲍蕾；

【导师】 包振民；

【作者基本信息】 中国海洋大学 ， 遗传学， 2009， 博士

【摘要】 黄曲霉毒素是由黄曲霉菌和寄生曲霉菌产生的次生代谢产物,是国际肿瘤研究机构(IARC)界定的IA类强致癌物质,广泛地污染粮食、谷类、油料、调味品等农作物。由于生物控制和实验室检测是黄曲霉毒素污染控制体系的关键环节,本文以中国出口敏感农产品为研究对象,对黄曲霉毒素的生物积累及其检测技术质量控制标准体系进行了研究。一、黄曲霉毒素产生的因素及生物控制技术研究本文以黄曲霉和寄生曲霉标准菌株以及从花生、土壤样品中分离出来的野生菌株为研究对象,选择在不同的培养条件下(接种量、无机盐、温度、pH值、氧气等),菌株液体发酵液的产毒情况,通过正交实验的方法确定了产毒菌株最适生长条件和产毒最低条件。研究发现,通过控制改变储藏环境条件来控制产毒菌的产毒是一个高效易行的方法。较低的温度和良好的通风,即一个凉爽干燥的储藏环境,加上弱碱性的条件可以很好的降低毒素的产量。本研究采用RT-PCR方法,对黄曲霉毒素生物合成过程中的相关基因的表达情况进行了研究,在基因表达水平上探索了黄曲霉毒素产生的规律。研究发现aflO、aflD、aflP三个基因的表达与菌株是否产毒直接相关,与HPLC法检测黄曲霉毒素的结果一致。RT-PCR技术可用来鉴定菌株是否产毒。在上述实验的基础上,从我国的土壤和花生样品中筛选出不产毒的黄曲霉菌株,通过紫外诱变筛选出生长力强的不产毒黄曲霉菌株A1025作为拮抗菌株,该菌株经过5代传代培养后仍不产生毒素,具有较好的稳定性;当拮抗菌的孢子浓度为10%时,发酵液中已检测不到黄曲霉毒素;将拮抗菌以不同的抑制浓度接种在花生等农产品中,发现该拮抗菌株能够有效降低黄曲霉毒素的生成,对照组黄曲霉毒素的含量为89.5 ppb,接种10%拮抗菌孢子实验组黄曲霉毒素含量为1.26 ppb,当接种量达到15%时,已检测不到黄曲霉毒素。研究表明,A1025对黄曲霉毒素的产生具有有效的抑制作用,在花生等农作物的模拟实验中能够有效地抑制黄曲霉毒素的产生。二、黄曲霉毒素检测技术的质量控制标准体系的研究本研究针对世界贸易技术壁垒的严峻挑战,从方法验证、质量控制、不确定度评定、国际协作实验研究和能力评估体系研究等几个方面,对实验室黄曲霉毒素的分析检测质量控制体系进行了研究,以满足国际限量规定及我国出口农产品的贸易技术需求。1、免疫亲和柱净化高效液相色谱法检测花生中的黄曲霉毒素方法验证、质量控制和不确定度评定研究本研究建立了使用免疫亲和柱净化结合柱后衍生化、高效液相色谱法(IAC/PCD/HPLC)测定花生中的黄曲霉毒素,并对检测方法进行了验证、对实验室的质量控制进行了研究、对方法的不确定度进行了评定。经研究,本方法的检出限为0.1μg/kg(S/N≥3),检测限为0.3μg/kg(S/N≥9),四种黄曲霉毒素均显示线性良好,方法在黄曲霉毒素B1、G1含量为0-8μg/kg的范围内线性良好,相关系数分别为0.99982和0.99976。方法在黄曲霉毒素B2、G2含量为0-2.4μg/kg的范围内线性良好,相关系数分别为0.99934和0.99983。采用阳性质量控制样品进行重复性测试,测得黄曲霉毒素B1和总量的重复性相对标准偏差(RSDr)分别为6.10%和6.08%。采用FAPAS质控样品进行再现性测试,测得黄曲霉毒素B1和总量的再现性相对标准偏差(RSDR)分别为6.10%和6.08%。采用实验室参加国际FAPAS能力验证的Z评估值进行准确性测试,经统计,黄曲霉毒素B1和黄曲霉毒素总量结果Z≤2。采用阴性控制样品添加测试准确性,回收率结果为: B1为77.3%-94.5%。黄曲霉毒素总量为79.9-92.9%。实验室的质量控制采用添加样品测定回收率,得到黄曲霉毒素B1和总量的回收率分别是77.5%-86.3%和70.4%-81.7%。采用花生阳性质控样品进行测试,绘制质量控制图,将阳性质控样品与待测样同批测定,依据质控样品的检测结果在质量控制图的位置,判断检测结果的准确性。本文研究了来自样品制备均质性、标准品纯度、准确性和精密度四个方面的相对不确定度,并计算了合成相对不确定度ru和扩展相对不确定度rU。样品均质性产生的相对不确定度ru1是0.033(B1)和0.036(总量);标准品纯度产生的相对不确定度ru2是0.0132(B1)和0.0647(总量);准确性测试产生的相对不确定度ru3是0.064(B1)和0.049(总量);精密度测试产生的相对不确定度ru4是0.070(B1)和0.048(总量)。依据以上四个独立的相对不确定度分量,计算黄曲霉毒素B1和总量的合成相对不确定度(ru)为0.10,黄曲霉毒素B1和总量的扩展相对不确定度(rU)为0.20。取95%的可信区间,得到黄曲霉毒素B1和总量的扩展不确定度(U)为U=0.20×黄曲霉毒素B1或总量的检测值。测试结果的报告表述为:B1(总量)±0.20×B1(总量)。2、人参和生姜中黄曲霉毒素B1、B2、G1、G2和棕曲霉毒素A的国际间实验室IAC/PCD/HPLC法协作同检研究AOAC(美国官定分析化学家协会)协作研究是申请AOAC国际标准的关键步骤。本协作研究由来自世界7个国家的13个实验室共同参加,研究者依照AOAC提供的检测方法和测试程序,对人参和生姜中黄曲霉毒素B1、B2、G1、G2和棕曲霉毒素A的IAC/PCD/HPLC方法进行了验证,并对30个盲样,包括空白样品、黄曲霉毒素添加样品(0.25-16.0μg/kg)、棕曲霉毒素A添加样品(0.25-8.0μg/kg)以及含有毒素的天然污染样品进行了测定。测试样品经甲醇-0.5%的碳酸氢钠提取,提取液经离心、磷酸盐缓冲液稀释,过滤后,经过内含黄曲霉毒素和棕曲霉毒素A的单克隆抗体的免疫亲和柱进行净化,加入色谱极甲醇,将亲和柱中的毒素进行洗脱,洗脱液注入高效液相色谱仪进行分离、荧光检测器定量检测。结果显示,在0～4.0ng/mL的黄曲霉毒素B1、0～1.0ng/mL的黄曲霉毒素B2、0～2.0ng/ mL的黄曲霉毒素G1、0～1.0ng/mL的黄曲霉毒素G2范围内,黄曲霉毒素毒素含量与峰面积间线性良好。在0～4.0ng/mL的棕曲霉毒素A范围内,棕曲霉毒素A含量与峰面积间线性良好。阴性控制样品添加回收率试验显示本方法检测人参和生姜中黄曲霉毒素的回收率为80～90%,检测人参和生姜中棕曲霉毒素A的回收率为85～95%。实验室内相对标准偏差(RSDr)为:黄曲霉毒素2.6-8.3%,棕曲霉毒素2.5-10.7%。13家实验室间相对标准偏差(RSDR)为黄曲霉毒素5.7-28.6%,棕曲霉毒素A 5.5-10.7%。HorRat值≤2。研究结果显示,该方法简便快速,准确可靠,灵敏度高,重现性好,能够满足人参和生姜中黄曲霉毒素和棕曲霉毒素A检测的需要。协作研究已通过AOAC的审核,成为AOAC国际标准(Official Method 2008.02)。3、花生黄曲霉毒素实验室检测能力的评估技术体系研究为评价我国实验室黄曲霉毒素的检测能力,在国内外总计95家实验室进行了黄曲霉毒素检测能力评估研究。本研究依据了国际能力验证评估的统一规则(IUPAC Harmonised Technical Report 2006)、国际标准化组织制订的能力验证评估的导则(ISO 13528: 2005(E),对能力验证评估研究进行了实施和评价。测试样品采用天然黄曲霉毒素污染的花生粉,采用S s≤0.3σp检验法对待测样品进行了均匀性测试,采用x ? y≤0.3σp法,对样品进行了稳定性测试。采用Z比分数评定各参加实验室的测试结果。各参加实验室提交结果的Robust均数作为测试样品黄曲霉毒素含量的指定值x a。依据Horwitz方程的修正公式σp =0.22c/mr计算能力验证评估的标准偏差σp。在92个参加实验室中,49家实验室报送黄曲霉毒素总量的结果,46家实验室结果“满意”,3家实验室结果“可疑”; 80家实验室报送黄曲霉毒素B1的结果,73家结果“满意”,4家结果“可疑”,3家结果“不满意”。参加实验室采用的方法有液相色谱法、酶联免疫法、荧光光度计法和液质联用法。更多还原

【Abstract】 Aflatoxins are widely distributed mycotoxins produced mainly by Aspergillus species. It can contaminate many kinds of crops products, including peanuts, corn, cottonseed, brazil nuts, pistachios, spices, figs, and copra etc. The International Agency for Research on Cancer (IARC) has classified aflatoxin B1 as a Group 1 human carcinogen. The objective of this project was to study biological control and quality assurance in aflatoxin analysis, which are two important elements of strategies to manage aflatoxins based on those foods with aflatoxin problems.Aflatoxin production was evaluated under different fermentation conditions including the temperature, pH and rotational speed. A.flavus, A.parasiticus standard strain and strains isolated from soil and peanut samples were used in the experiment. The optimal and worst conditions for aflatoxin production were tested by orthogonal experiment. This experiment simulated the temperature, pH and ventilation condition of grain storage. Aflatoxin production could be reduced by modifying storage conditions. The ventilation, temperature and pH of storage are key elements affecting aflatoxin production. Study showed that low temperature, well ventilation ,dry and alkaline store condition were good practice to avoid aflatoxin contamination.Using RT-PCR technique, Several structural genes in aflatoxin biosynthesis pathway were studied. Three genes including aflO, aflD and aflP, whose expression were found to be closely related to aflatoxin productivity, which could be used to distinguish aflatoxigenic strains from nontoxigenic strains. The results from RT-PCR were consistent with those from HPLC determination for aflatoxins.A nontoxigenic and competitive strains named A1025 was isolated by UV irradiation in this study. It could not produce aflatoxins and kept stability during 5 generation’s study. Experiments were conducted to determine effect of A1025 to reduce aflatoxin production. When the spore concentration of A1025 was 10%, aflatoxins could not be detected in the fermentation broth. It was found that A1025 can effectively reduce aflatoxin production when it was inoculated in peanut and other agricultural products at different concentrations. When the inoculation concentration of A1025 is 10%, the aflatoxin in peanut is 1.26ppb, while the control group is 89.5ppb. When the inoculation concentration of A1025 reached to 15%, the aflatoxin could not be detected. The study showed that A1025 has effective inhibition for aflatoxin production in simulation experiment for peanut and other agricultural products.An HPLC method for the determination of aflatoxin B1 and total aflatoxins in peanut was validated, the routine qulity control measures were conducted and the measurement uncertainty were estimated. LOD was estimated to be 0.1μg/kg with signal to noise ratio at 3 to 1. LOQ was 0.3μg/kg(S/N=10). The correlation coefficient for B1 and G1 are 0.99982 and 0.99976 with concentrations ranging from 0 to 8μg/kg. And B2、G2 are 0.99934 and 0.99983 with concentrations from 0 to 2.4μg/kg. Positive quality control samples were analyzed to get the RSDr is 6.10% for aflatoxin B1 and 6.08% for Total aflatoxins. Using FAPAS surplus samples to get the RSDR is 7.0% for B1 and 4.8% for Total aflatoxins. Trueness were evaluated from FAPAS interlaboratory proficiency test. Z≤2 for both B1 and total aflatoxins. Blank samples were spiked and analyzed in routine quality control. The recovery for B1 and Total aflatoxins are 77.5%-86.3% and 70.4%-81.7%. An in-house positive quality control samples were prepared from a naturally contaminated whole peanut sample.The positive quality control samples were tested for homogeneity and quality control limits were estabilished.The approach in this method includes four main areas of the method which can affect the overall uncertainty: sample homogeneity, standard purity, accuracy and precision. The relative uncertainty which were calculated for each area were finally transferred to the Combined Relative Uncertainty and Expand Relative Uncertainty. Relative uncertainty from sample homogeneity (ru1) were 0.033 for aflatoxin B1 and 0.036 for total aflatoxins. Relative uncertainty from standard purity (ru2) were 0.0132 for B1 and 0.0647 for total aflatoxins. Relative uncertainty from accuracy (ru3) were 0.064 for B1 and 0.049 for total aflatoxins. Relative uncertainty from precision (ru4) were 0.070 for B1 and 0.048 for total aflatoxins. Based on above calculation, the combined relative uncertainty(ru) were 0.10 for both aflatoxin B1 and total aflatoxins. The expanded relative uncertainty(rU) is caculated using a coverage factor of 2 which give a level of confidence of 95%. The expanded uncertainty(U) were estimated as 0.20 multiply reported value of B1 or Total. The report for aflatoxin analysis is finally described as: B1( Total)±0.20×B1(total).A collaborative study was conducted to evaluate the method to determine aflatoxin B1、B2、G1、G2 and Ochratoxin A in ginseng and ginger. Coordinated by Dr. Mary Trucksess, Thirteen laboratories from 7 countries participated in this study. According to AOAC guidelines, participants validated the method and analyzed 30 blind samples including blank samples, aflatoxins spiked samples (with concentration range from 0.25 to 16.0μg/kg ), ochratoxin A spiked samples (with concentration range from 0.25 to 8.0μg/kg) and naturally contaminated samples. The test portion was extracted with methanol-0.5% aqueous sodium hydrogen carbonate solution(700+300,v/v). The sample extract was filtered, diluted with phosphate buffer and applied to immunoaffinity column containing antibodies specific for aflatoxins and ochratoxin A. The column was washed with water and the aflatoxins and ochratoxin A were eluted with methanol. Aflatoxins were quantified by reverse-phase liquid chromatography (HPLC) with electrochemical post column derivatization. The detection was achieved by fluorescence. In-house validation showed that the correlation coefficient for B1 and Ochratoxin A were 0.9999 and 0.99998 with concentrations ranging from 0 to 4.0ng/ml. B2 and G2 were 0.9997 and 0.9996 with concentrations 0 to 1.0 ng/ml. G1 was 0.9999 with concentration from 0 to 2.0 ng/ml. Recoveries by using spiked samples were 80% to 90% for total aflatoxins and 85% to 95% for Ochratoxin A. Collaborative study showed that RSDR was 5.7-28.6% for total aflatoxins and 5.5-10.7% for ochratoxin A. HorRat value was less than 2. The method showed acceptable accuracy and could satisfy analytical requirements to determine total aflatoxins and ochratoxin A simultaneously.The assessment was conducted according to ISO 13528: 2005(E) and the International Harmonized Protocol for Proficiency Testing. 95 laboratories participated in the study. The analytic samples for this testing scheme were prepared from naturally contaminated peanut butter. The Ss≤0.3σptest was used to evaluate the homogeneity of the test samples; Sample stability was confirmed with x ? y≤0.3σp. The performance of each laboratory was designated by a z-score that was calculated using robust statistics. The robust mean of the participants’results in this study was nearly coincident with the median. A modified Horwitz equation was used to determine the standard deviation. Laboratories whose performance ratings were questionable or unsatisfactory were re-evaluated in a second interlaboratory comparison. Of the 49 laboratories that reported results for total aflatoxins, 46 performed satisfactorily. 3 performed questionable. Of the 80 laboratories that reported results for aflatoxin B1, 73 performed satisfactorily. 4 performed questionable. 3 performed unsatisfactory; The analytic results were obtained by four methods: high performance liquid chromatography, enzyme-linked immunosorbent assay (ELISA), fluorometry and liquid chromatography with tandem mass spectrometry (LC/MS/MS).更多还原