【作者】 张伟；

【导师】 乔旭光； 赵善仓；

【作者基本信息】 山东农业大学 ， 食品加工与安全（专业学位）， 2019， 硕士

【摘要】 由禾谷镰刀菌复合种引起的小麦赤霉病是我国主要的麦类真菌病害之一。赤霉病的发生不仅会造成严重的产量损失,还会产生多种镰刀菌毒素污染,如单端孢霉烯族毒素、玉米赤霉烯酮和伏马毒素等,严重威胁人畜健康。近年来,随着全球气候变化和耕作方式的改变,我国小麦赤霉病频繁发生且伴随北扩西移的现象。山东是我国小麦种植第二大省,小麦食用消费量占比较大。因此,掌握山东省小麦镰刀菌毒素污染状况,明确山东省产毒镰刀菌种群类型及产毒化学型,对保障山东省小麦产品质量安全具有重要的现实意义。本研究在建立固相萃取-高效液相色谱串联质谱检测小麦中9种镰刀菌毒素方法的基础上,以2017和2018年山东省小麦主产区为重点取样地区,共采集了收获期小麦样品约598份,分析了影响我国小麦产品质量安全的DON、3ADON、15ADON、NIV、4ANIV、ZEN、FB1、FB2和FB3 9种镰刀菌毒素污染水平,并对样品中产毒镰刀菌种群及产毒化学型进行了分析,主要研究结果如下:1.建立了小麦镰刀菌毒素固相萃取-高效液相色谱串联质谱检测方法基于固相萃取的方式,比较4种不同的SAX、PSA、C18和NH2固相萃取柱的净化效果,优化了6种不同比例的乙腈-水提取液以及针对伏马毒素不同比例酸化甲醇的洗脱液,确定了乙腈-水(85∶15,v/v)提取液提取,NH2柱进行净化,乙腈-水(85∶15,v/v)一次淋洗,甲醇-甲酸(85∶15,v/v)二次洗脱的两步法测定,此外检测方法中选择较优的色谱及质谱方法进行测定,得出测定9种镰刀菌毒素的相关系数R2均大于0.99,回收率为80%~100.0%之间,相对标准偏差在5.8%~9.0%之间,均小于10%。DON、3ADON和15ADON检出限均为3.0μg/kg,NIV为2.0μg/kg,4ANIV为4.0μg/kg,ZEN为0.3μg/kg,FB1为3.0μg/kg,FB2为2.0μg/kg,FB3为2.0μg/kg。研究建立了一种同一前处理方式对多种镰刀菌毒素进行同时测定的色谱串联质谱方法进行数据采集分析的方法,避免同一批样品多次处理多次分析的弊端,节省仪器成本,提高工作效率。2.掌握了山东省小麦镰刀菌毒素污染的情况采用以上方法对2017和2018年山东省小麦主产区598批次小麦样品进行测定:结果得出禾谷镰刀菌毒素污染主要以DON、3A-DON、15A-DON为主,2017年整体毒素检出率为25.33%,2018年整体毒素检出率为48.51%,呈逐年上升趋势,总体污染率不高;鲁西南,鲁中南地区的污染率较其余地方严重,呈现区域化特点。泰农18,临麦2号,济麦22均是中抗赤霉病的品种,鲁原502为高感赤霉病品种,其他品种种类较杂,受气候等外部条件影响较大,基本为低感或中感赤霉病品种。3.明确了山东省小麦中产毒镰刀菌种群类型及产毒化学型经过对山东省小麦中禾谷镰刀菌的分离纯化,对纯化菌株进行DNA提取,并将分离出的镰刀菌菌株采用多重PCR方法对所分离的镰刀菌进行产毒化学型的鉴定,得出山东省内产毒镰刀菌主要以亚洲镰刀菌(F.asiaticum)禾谷镰刀菌(F.graminearum)为主,其中15ADON化学型的禾谷镰刀菌为禾谷镰刀菌复合种优势群体,3ADON化学型亚洲镰刀菌产毒能力强于15ADON化学型的禾谷镰刀菌和NIV化学型的禾谷镰刀菌。更多还原

【Abstract】 Wheat scab caused by Fusarium gramineae is one of the important wheat fungal diseases in China.The occurrence of scab will not only cause serious yield loss,but also produce a variety of fusarium toxin pollution,such as trichothecene,zearalenone(ZEN)and fumonisins(FBs),which seriously threaten human and animal health.In recent years,with the change of both global climate and farming practices,wheat scab in China has frequently occurred and accompanied by the phenomenon of northward expansion and westward movement.Shandong is the second largest province of wheat cultivation in China,and the consumption of wheat accounts for a large proportion.Therefore,it is of great practical significance to grasp the contamination status of Wheat Fusarium toxin in Shandong Province and clarify the population types and chemical types of Fusarium toxin-producing in Shandong Province for ensuring the quality and safety of wheat products in Shandong Province.In this study,a method of solid phase extraction,high performance liquid chromatography tandem mass spectrometry was developed to detect 9 kinds of fungus toxins in wheat.Based on the detection method,I focused on the major wheat producing areas in Shandong province in 2017 and 2018,and 540 wheat samples were collected in harvest season.The pollution level of the 9 kinds of fungus toxins,DON,3 A-DON,15 A-DON,NIV,4 A-NIV,ZEN,FB1,FB2 and FB3,which have influenced on wheat product quality and safety was analyzed.The strain types and toxic chemical types of those samples were also illuminated.1.A detection method of fusarium toxin in wheat using solid phase extraction-high performance liquid chromatography tandem mass spectrometry was established.Based on solid phase extraction,the purification effects of four different SAX,PSA,C18 and NH2 solid phase extraction columns were compared.Six different proportions of acetonitrile-water extracts and eluents for fumonisin acidified methanol in different proportions were optimized.It was found that acetonitrile-water(85:15,v/v)extracts were used to extract,NH2 column was purified,acetonitrile-water(85:15,v/v)was leached once,Methanol-methyl alcohol-methyl alcohol was used.Two-step method of acid(85:15,v/v)secondary elution was used.In addition,the better methods of chromatography and mass spectrometry were selected to determine the nine Fusarium toxins.The correlation coefficients R2 were all greater than 0.99.The recoveries were between 80%-100.0%.The relative standard deviations were between 5.8%-9.0%,all less than 10%.The detection limits of DON,3ADON and 15 ADON were 3.0 μg/kg,NIV 2.0 μg/kg,4ANIV 4.0 μg/kg,ZEN 0.3 μg/kg,FB1 3.0 μg/kg,FB2 2.0 μg/kg and FB3 2.0 μg/kg.A method of data acquisition and analysis for simultaneous determination of various Fusarium toxins by gas chromatography tandem mass spectrometry with the same pretreatment method was established to avoid the disadvantages of multiple analysis for the same batch of samples,save the cost of the instrument and improve the work efficiency.2.To grasp the contamination of Wheat Fusarium toxins in Shandong Province.The above methods were used to determine about 598 batches of wheat samples in the main wheat producing areas of Shandong Province in 2017 and 2018.The results showed that DON,3A-DON and 15A-DON were the main contaminants of Fusarium graminearum toxins.The overall detection rate of toxins was 25.33% in 2017 and 48.51% in 2018,showing an annual upward trend,and the overall contamination rate was not high.The locality is serious,showing the characteristics of regionalization.Tainong 18,Linmai 2 and Jimai 22 are all moderately resistant to scab.Luyuan 502 is highly susceptible to scab.Other varieties are relatively miscellaneous.They are mainly low or moderately susceptible to scab because of the influence of climate and other external conditions.3.Strain types and toxic chemical types of fusarium virulent in wheat in Shandong province was illuminated.After isolation and purification of Fusarium graminearum from wheat in Shandong Province,DNA was extracted from the purified strains,and the Fusarium strains were identified by multiplex PCR.It was concluded that the main Fusarium producing Fusarium in Shandong Province were F.asiaticum and F.graminearum,of which 15 ADON chemical types were the main one.Fusarium graminearum was the dominant population of Fusarium graminearum complex.The toxicity of 3ADON chemical type Fusarium graminearum was stronger than 15 ADON chemical types Fusarium graminearum and NIV chemical type Fusarium graminearum.更多还原