伏马毒素分离纯化及毒性试验研究

发布时间：2018-03-04 10:22

本文选题：串珠镰刀菌　切入点：伏马毒素　出处：《上海海洋大学》2015年硕士论文　论文类型：学位论文

【摘要】：为了研究伏马毒素毒性作用,本试验研究了串珠镰刀菌在不同培养基上产毒量随时间的变化及伏马毒素的纯度,优化得到大米培养基为最适产毒培养基。在大米培养基上接种串珠镰刀菌得到大量产毒培养基,通过MAX固相萃取小柱对提取液净化,然后经过制备液相色谱分两步制备得到FB1、FB2、FB3。给BALB/c小鼠注射制备得到的FB1,观察小鼠体重、采食量、脏器系数、血常规以及血清生化指标。旨在填补我国FB1毒理学基础研究的空白,为FB1的分离纯化和毒性作用机制的深入研究和畜禽生产中真菌毒素污染防控提供数据参考和理论依据。本试验主要研究内容和结论如下:(1)伏马毒素主要是由自然界的串珠镰刀菌(Fusarium moniliforme)产生的,本试验在4种不同培养基上接种串珠镰刀菌,PDA、PDB培养基26℃黑暗条件下培养15 d,每3 d采样一次;大米和玉米培养基培养5周,每周采样一次。通过液相色谱-串联质谱法(LC-MS/MS)检测四种培养基中FB1、FB2的含量,结果表明:PDA与PDB培养基中FB1、FB2在第9 d达到最大含量,但含量较低;玉米和大米培养基中FB1、FB2在第28 d达到最大,含量较高,大米培养基中FB1、FB2含量分别为817.08 mg/kg、329.01 mg/kg,玉米培养基中FB1、FB2含量分别为1143.40mg/kg、460.88 mg/kg。紫外全扫描法对四种培养基提取液进行全扫描,结果表明:PDA、PDB中杂质峰明显少于大米、玉米,而大米中杂质峰又明显少于玉米。综上,在不影响制备效率的前提下,选择含色素等杂质较少的大米作为培养基,不仅有利于伏马毒素的分离纯化,还能延长色谱柱的寿命。(2)以大米作为培养基,接种串珠镰刀菌得到大量产毒基质,以MAX固相净化小柱作为产毒基质提取液的前处理净化柱,通过Unitary C18分析色谱柱(4.6mm×250 mm,5μm)借助LC-MS/MS对流动相、梯度进行优化,使FB1、FB2、FB3与杂质峰之间达到满意的分离度,然后等比例放大到制备色谱柱上,利用制备液相色谱分两步分离纯化得到大量FB1、FB2、FB3。所得伏马毒素经LC-MS/MS法定性鉴别,结果表明:在正负离子模式下(ESI-和ESI+)进行全扫描,均得到单一、高峰度的色谱峰。对特征峰在正离子模式下进行母离子扫描,FB1、FB2、FB3化合物的主要分子离子峰为722.30[M+H]+、706.30[M+H]+、706.30[M+H]+;在ESI+模式下分子离子峰722.30的主要二级碎片为352.30[M+H-2TCA-H2O]+、334.30[M+H-2TCA-2H2O]+和704.35[M+H-H2O]+;分子离子峰706.30的主要二级碎片为354.30[M+H-2TCA]+和336.30[M+H-2TCA-H2O]+、318.30[M+H-2TCA-2H2O]+。通过UPLC-PDA全扫描和NMR对FB1、FB2、FB3制备品进一步定性分析,结果均表明:制备得到FB1、FB2、FB3的是高纯度伏马毒素。最后经过LC-MS/MS外标法对所得伏马毒素的纯度进行测定,证明其纯度分别为98.760±0.12%、99.009±0.08%、98.838±0.65%。24H能制备得到FB1、FB2、FB3的量分别为80mg、16mg、16mg,回收率约为60%。本方法操作简单,成本低,制备效率高,制备得到的FB1、FB2、FB3能够作为高纯度的伏马毒素供分析研究使用,为伏马毒素的深入研究和应用提供基础。(3)将80只BALB/c小鼠随机分成4组,每组20只,分别为对照组(灌胃灭菌生理盐水)、低剂量组(0.5 mg/kg.bw)、中剂量组(1.5 mg/kg.bw)和高剂量组(4.5 mg/kg.bw),喂饲实验室分离纯化的FB1,观察不同剂量的FB1对BALB/c小鼠体重、采食量、脏器系数、血常规以及血清生化指标,结果表明:随着FB1添加剂量的增加,小鼠采食量呈逐渐降低趋势,小鼠体增重也呈降低趋势,Sa/So的比值逐渐增大,但未见统计学差异;不同剂量FB1处理,小鼠心脏和肝脏指数未呈现变化,但FB1灌胃剂量超过1.5 mg/kg.bw可使小鼠的脾脏、肺和肾脏显著肿胀,4.5 mg/kg.bw FB1能对肝脏组织产生损伤;不同剂量FB1处理,中性粒细胞随FB1剂量的增加逐渐增高,其中1.5 mg/kg.bw组显著高于对照组(P?0.05),4.5mg/kg.bw显著高于其他三组(P?0.05或P?0.01);血红蛋白和血小板指标亦随FB1浓度的增加增高,其中1.5和4.5 mg/kg.bw组显著高于前两组(P?0.05或P?0.01),表明高剂量的FB1可导致血液粘稠、凝血不良;不同浓度FB1处理,血液生化指标白蛋白、碱性磷酸酶、谷丙转氨酶、葡萄糖、总蛋白和尿素氮未发生变化,但1.5 mg/kg.bw和4.5 mg/kg.bw组谷草转氨酶活性均显著高于对照组和0.5 mg/kg.bw组(P?0.05);4.5 mg/kg.bw组总胆红素浓度极显著高于前三组(P?0.01),肌酐浓度显著高于对照组和0.5 mg/kg.bw组(P?0.05)。
[Abstract]:In order to study the effect of fumonisin toxicity, this paper studied the purity of Fusarium moniliforme in different culture medium on toxin production changes with time and fumonisins, optimized the rice culture medium was the most suitable toxin producing medium. In the rice culture medium inoculated with Fusarium moniliforme was a toxin producing medium, by MAX solid phase extraction column to extract purification, followed by preparative liquid chromatography in two steps was prepared by FB1, FB2, FB3. to BALB/c mice with the prepared FB1, observing the mice body weight, feed intake, organ coefficient, blood routine and serum biochemical indexes. Designed to fill gaps in basic research of FB1 toxicology in China, to provide reference data and the theoretical basis for the prevention and control of mycotoxin contamination research and animal production mechanism for isolation and purification of FB1 and toxicity. In this experiment, the main research contents and conclusions are as follows: (1) fumonisin is mainly composed of self The natural world of Fusarium moniliforme (Fusarium moniliforme) produced by the test in 4 different medium inoculated with Fusarium moniliforme, PDA, PDB medium 26 Deg. C for cultivation under the condition of 15 d, every 3 D sampling time; rice and corn were cultured for 5 weeks, once a week. The sampling by tandem mass spectrometry liquid chromatography (LC-MS/MS) detection of four kinds of FB1 in the culture medium, the results showed that the content of FB2, PDA and PDB medium FB1, FB2 content reached the maximum at ninth D, but the content is relatively low; corn and rice medium, FB1, reached the maximum at twenty-eighth D FB2, higher content of the rice culture medium in FB1, the content of FB2 was 817.08 mg/kg, 329.01 mg/kg, maize culture medium FB1, FB2 contents were 1143.40mg/kg, 460.88 mg/kg. UV scanning method of four kinds of medium extract full scan, the results showed that PDA, PDB impurities significantly less in rice, corn, rice and impurities again Less than corn. In summary, in the premise of not affecting the preparation efficiency, containing pigments and other impurities less rice as a medium, not only conducive to the purification of fumonisins, but also long column life. (2) using rice as culture medium, inoculation of Fusarium moniliforme received a large number of toxigenic matrix, MAX as the production of solid purification cartridge pretreatment matrix poison extract purification column, through Unitary C18 analysis column (4.6mm * 250 mm, 5 m) with the LC-MS/MS mobile phase, gradient optimization, FB1, FB2, FB3 and impurity peak reached the satisfactory degree of separation, and then scale up to system preparation of chromatographic column, preparative liquid chromatography using two steps purified by a large number of FB1, FB2, FB3. of the fumonisins by LC-MS/MS qualitative identification, the results showed that: in the negative ion mode (ESI- and ESI+) were obtained by full scanning, single peak, the degree of color The characteristic peak peak. The parent ion scan in positive ion mode FB1, FB2, FB3 compound main molecular ion peak was 722.30[M+H]+, 706.30[M+H]+, 706.30[M+H]+; in the ESI+ mode of the 722.30 main molecular ion peak of two pieces of 352.30[M+H-2TCA-H2O]+, 334.30[M+ H-2TCA-2H2O]+ and 704.35[M+H-H2O]+; the 706.30 main molecular ion peak of two pieces for 354.30[M+H-2TCA]+ and 336.30[M+H-2TCA-H2O]+, 318.30[M+H-2TCA-2H2O]+. by UPLC-PDA full scan and NMR on FB1, FB2, FB3 analysis of preparation further qualitative results show that the prepared by FB1, FB2, FB3 of high purity of fumonisins. Finally through LC-MS/MS external standard method for the purity of fumonisins were determined to prove its purity respectively. 98.760 + 0.12%, 99.009 + 0.08%, 98.838 + 0.65%.24H can be prepared by FB1, FB2, FB3 for the amount of 80mg, 16mg, 16mg, the recovery rate of this method is about 60%. Simple operation, low cost, high preparation efficiency, prepared by FB1, FB2, FB3 can be used as fumonisins with high purity for analysis and study, provide the basis for further research and application of fumonisins. (3) 80 BALB/c mice were randomly divided into 4 groups, 20 rats in each group, the control group respectively (gavage of saline), low dose group (0.5 mg/kg.bw), middle dose group (1.5 mg/kg.bw) and high dose group (4.5 mg/kg.bw), fed with laboratory purified FB1, observation of different doses of FB1 on BALB/c mice body weight, feed intake, organ coefficient, the results showed that blood and serum biochemical indexes. With increasing dietary FB1 intake, mice decreased gradually, mice body weight also decreased, the ratio of Sa/So increased gradually, but there was no significant difference; different doses of FB1 treatment, the index of heart and liver were not changed, but the FB1 gavage dose more than 1 .5 mg/kg.bw in the mice spleen, lung and kidney were swelling, 4.5 mg/kg.bw FB1 can cause damage to the liver tissue; different doses of FB1 treatment, increased neutrophils with the dose of FB1 increased gradually, and the 1.5 mg/kg.bw group was significantly higher than control group (P? 0.05), 4.5mg/ kg.bw significantly higher than the other three groups (P? 0.05 or P? 0.01); hemoglobin and platelet index also increased with the increase of the concentration of FB1, of which 1.5 and 4.5 of mg/kg.bw group was significantly higher than that of the former two groups (P? 0.05 or P? 0.01), showed that high dose of FB1 can cause blood viscosity, blood coagulation defect; different concentrations of FB1, biochemical index of blood albumin, alkaline phosphatase, alanine aminotransferase, glucose, total protein and urea nitrogen did not change, but the 1.5 mg/kg.bw and 4.5 mg/kg.bw groups of aspartate aminotransferase activity were significantly higher than those in control group and 0.5 mg/kg.bw group (P? 0.05); the concentration of serum total bilirubin of 4.5 mg/kg.bw was significantly higher than before In the three group (P? 0.01), the creatinine concentration was significantly higher than that in the control group and the 0.5 mg/kg.bw group (P? 0.05).

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8;R114

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