本文選題：串珠鐮刀菌　切入點(diǎn)：伏馬毒素　出處：《上海海洋大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：為了研究伏馬毒素毒性作用,本試驗(yàn)研究了串珠鐮刀菌在不同培養(yǎng)基上產(chǎn)毒量隨時(shí)間的變化及伏馬毒素的純度,優(yōu)化得到大米培養(yǎng)基為最適產(chǎn)毒培養(yǎng)基。在大米培養(yǎng)基上接種串珠鐮刀菌得到大量產(chǎn)毒培養(yǎng)基,通過(guò)MAX固相萃取小柱對(duì)提取液凈化,然后經(jīng)過(guò)制備液相色譜分兩步制備得到FB1、FB2、FB3。給BALB/c小鼠注射制備得到的FB1,觀察小鼠體重、采食量、臟器系數(shù)、血常規(guī)以及血清生化指標(biāo)。旨在填補(bǔ)我國(guó)FB1毒理學(xué)基礎(chǔ)研究的空白,為FB1的分離純化和毒性作用機(jī)制的深入研究和畜禽生產(chǎn)中真菌毒素污染防控提供數(shù)據(jù)參考和理論依據(jù)。本試驗(yàn)主要研究?jī)?nèi)容和結(jié)論如下:(1)伏馬毒素主要是由自然界的串珠鐮刀菌(Fusarium moniliforme)產(chǎn)生的,本試驗(yàn)在4種不同培養(yǎng)基上接種串珠鐮刀菌,PDA、PDB培養(yǎng)基26℃黑暗條件下培養(yǎng)15 d,每3 d采樣一次;大米和玉米培養(yǎng)基培養(yǎng)5周,每周采樣一次。通過(guò)液相色譜-串聯(lián)質(zhì)譜法(LC-MS/MS)檢測(cè)四種培養(yǎng)基中FB1、FB2的含量,結(jié)果表明:PDA與PDB培養(yǎng)基中FB1、FB2在第9 d達(dá)到最大含量,但含量較低;玉米和大米培養(yǎng)基中FB1、FB2在第28 d達(dá)到最大,含量較高,大米培養(yǎng)基中FB1、FB2含量分別為817.08 mg/kg、329.01 mg/kg,玉米培養(yǎng)基中FB1、FB2含量分別為1143.40mg/kg、460.88 mg/kg。紫外全掃描法對(duì)四種培養(yǎng)基提取液進(jìn)行全掃描,結(jié)果表明:PDA、PDB中雜質(zhì)峰明顯少于大米、玉米,而大米中雜質(zhì)峰又明顯少于玉米。綜上,在不影響制備效率的前提下,選擇含色素等雜質(zhì)較少的大米作為培養(yǎng)基,不僅有利于伏馬毒素的分離純化,還能延長(zhǎng)色譜柱的壽命。(2)以大米作為培養(yǎng)基,接種串珠鐮刀菌得到大量產(chǎn)毒基質(zhì),以MAX固相凈化小柱作為產(chǎn)毒基質(zhì)提取液的前處理凈化柱,通過(guò)Unitary C18分析色譜柱(4.6mm×250 mm,5μm)借助LC-MS/MS對(duì)流動(dòng)相、梯度進(jìn)行優(yōu)化,使FB1、FB2、FB3與雜質(zhì)峰之間達(dá)到滿意的分離度,然后等比例放大到制備色譜柱上,利用制備液相色譜分兩步分離純化得到大量FB1、FB2、FB3。所得伏馬毒素經(jīng)LC-MS/MS法定性鑒別,結(jié)果表明:在正負(fù)離子模式下(ESI-和ESI+)進(jìn)行全掃描,均得到單一、高峰度的色譜峰。對(duì)特征峰在正離子模式下進(jìn)行母離子掃描,FB1、FB2、FB3化合物的主要分子離子峰為722.30[M+H]+、706.30[M+H]+、706.30[M+H]+;在ESI+模式下分子離子峰722.30的主要二級(jí)碎片為352.30[M+H-2TCA-H2O]+、334.30[M+H-2TCA-2H2O]+和704.35[M+H-H2O]+;分子離子峰706.30的主要二級(jí)碎片為354.30[M+H-2TCA]+和336.30[M+H-2TCA-H2O]+、318.30[M+H-2TCA-2H2O]+。通過(guò)UPLC-PDA全掃描和NMR對(duì)FB1、FB2、FB3制備品進(jìn)一步定性分析,結(jié)果均表明:制備得到FB1、FB2、FB3的是高純度伏馬毒素。最后經(jīng)過(guò)LC-MS/MS外標(biāo)法對(duì)所得伏馬毒素的純度進(jìn)行測(cè)定,證明其純度分別為98.760±0.12%、99.009±0.08%、98.838±0.65%。24H能制備得到FB1、FB2、FB3的量分別為80mg、16mg、16mg,回收率約為60%。本方法操作簡(jiǎn)單,成本低,制備效率高,制備得到的FB1、FB2、FB3能夠作為高純度的伏馬毒素供分析研究使用,為伏馬毒素的深入研究和應(yīng)用提供基礎(chǔ)。(3)將80只BALB/c小鼠隨機(jī)分成4組,每組20只,分別為對(duì)照組(灌胃滅菌生理鹽水)、低劑量組(0.5 mg/kg.bw)、中劑量組(1.5 mg/kg.bw)和高劑量組(4.5 mg/kg.bw),喂飼實(shí)驗(yàn)室分離純化的FB1,觀察不同劑量的FB1對(duì)BALB/c小鼠體重、采食量、臟器系數(shù)、血常規(guī)以及血清生化指標(biāo),結(jié)果表明:隨著FB1添加劑量的增加,小鼠采食量呈逐漸降低趨勢(shì),小鼠體增重也呈降低趨勢(shì),Sa/So的比值逐漸增大,但未見(jiàn)統(tǒng)計(jì)學(xué)差異;不同劑量FB1處理,小鼠心臟和肝臟指數(shù)未呈現(xiàn)變化,但FB1灌胃劑量超過(guò)1.5 mg/kg.bw可使小鼠的脾臟、肺和腎臟顯著腫脹,4.5 mg/kg.bw FB1能對(duì)肝臟組織產(chǎn)生損傷;不同劑量FB1處理,中性粒細(xì)胞隨FB1劑量的增加逐漸增高,其中1.5 mg/kg.bw組顯著高于對(duì)照組(P?0.05),4.5mg/kg.bw顯著高于其他三組(P?0.05或P?0.01);血紅蛋白和血小板指標(biāo)亦隨FB1濃度的增加增高,其中1.5和4.5 mg/kg.bw組顯著高于前兩組(P?0.05或P?0.01),表明高劑量的FB1可導(dǎo)致血液粘稠、凝血不良;不同濃度FB1處理,血液生化指標(biāo)白蛋白、堿性磷酸酶、谷丙轉(zhuǎn)氨酶、葡萄糖、總蛋白和尿素氮未發(fā)生變化,但1.5 mg/kg.bw和4.5 mg/kg.bw組谷草轉(zhuǎn)氨酶活性均顯著高于對(duì)照組和0.5 mg/kg.bw組(P?0.05);4.5 mg/kg.bw組總膽紅素濃度極顯著高于前三組(P?0.01),肌酐濃度顯著高于對(duì)照組和0.5 mg/kg.bw組(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).

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S859.8;R114

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本文編號(hào)：1565270