高效液相色谱法定量测定玉米中腐马素毒素(Fumonisins)

本研究介绍了一种简单、快速分析测定玉米中腐马素毒素(fumonisins)残留的方法。样品用甲醇+水混合液提取,对提取液用小型强酸型阴离子固相萃取柱(SAX)纯化。纯化后的样品经化学衍生处理后,用反相高效液相色谱分离,并以荧光分光光度检测器定量测定。该方法对于添加已知量、添加水平在0.3—2mg·kg-1腐马素的玉米样的测定证实:其平均回收率在80%以上,而该方法对于腐马素三种同系物的最低检出限分别为0.1—0.05mg·kg-1。此外,本方法也对有限数目的大米、大麦、小米、高粱样中存在腐马素毒素污染的可能性进行了验证,结论是否定的。

Evaluation of maize inbred lines for resistance to pre-harvest aflatoxin and fumonisin contamination in the field

Two important mycotoxins, aflatoxin and fumonisin, are among the most potent naturally occurring carcinogens, contaminating maize(Zea mays) and affecting crop yield and quality.Resistance of maize to pre-harvest mycotoxin contamination, specifically aflatoxin produced by Aspergillus flavus and fumonisin produced by Fusarium verticillioides, is a goal in breeding programs that screen for these important traits with the aim of developing resistant commercial hybrids. We conducted two years of field evaluations on 87 inbred lines originating primarily in China and Mexico and not previously screened for resistance.The objectives of our study were to identify resistant germplasm for breeding purposes and to examine possible relationships between resistances to the two mycotoxins. Aflatoxin and fumonisin were present in samples harvested from all lines in both years.Concentrations of total aflatoxin ranged from 52.00 ± 20.00 to 1524.00 ± 396.00 μg kg^(-1),while those of fumonisin ranged from 0.60 ± 0.06 to 124.00 ± 19.50 mg kg^(-1). The inbred lines TUN15, TUN61, TUN37, CY2, and TUN49 showed the lowest aflatoxin accumulation and CN1, GT601, TUN09, TUN61, and MP717 the lowest fumonisin accumulation. TUN61 showed the lowest accumulation of both mycotoxins. This study confirmed previous observations that high levels of aflatoxin can coexist with fumonisin, with 55 maize lines showing a positive correlation coefficient between the concentrations of aflatoxin and fumonisin and 32 lines showing a negative correlation coefficient. These selected lines,particularly TUN61, may provide sources of resistance to mycotoxin contamination in breeding programs. However, the mechanism of resistance in this germplasm remains to be identified. Future research should also address factors that influence the fungus–plant interaction, such as herbivory and environmental stress.

Effects of Fumonisin B1 on Biomechanics and Cytoskeleton of Human Umbilical Vein Endothelial Cells

Objective Fumonisin B1(FB1)is an important mycotoxin in nature worldwide.The biomechanical properties of cells are closely related to their structure and function,and the cytoskeleton is the structural and functional basis of cells motility,and therefore,from a biomechanical point of view,the purpose of this study is to investigate the effects of FB1 on the biomechanical properties,migration capacity and cytoskeletal structure of human umbilical vein endothelial cells(HUVECs),which may lay an experimental foundation for further exploration of the toxicity mechanism of fumonisin.Methods HUVECs were cultured and treated with different concentrations of FB1.Then,CCK-8 kit was used to detect the effect of FB1 on the survival rate.The osmotic fragility of the cells was measured after treatment with different osmotic pressures for30 min.The cell membrane fluidity was measured by fluorescence polarization method.The cell electrophoretic mobility was measured by cell electrophoretic apparatus.The migration capacity of the cells was observed by scratch repair assay.The changes of reactive oxygen species and cytoskeletal structure were observed by confocal laser scanning microscopy.Finally,the mRNA and protein relative expression levels of cytoskeletal binding proteins were detected by real-time PCR,Western blotting and confocal laser scanning.Results The results of CCK-8 showed that FB1 could significantly inhibit the proliferation of HUVECs in a dose-and time-dependent manner.After treatment of HUVECs with FB1,the hypotonic resistance of the cell,cell surface charge,cell membrane fluidity and migration capacity were all weakened,while reactive oxygen species were significantly increased and the cytoskeletal structure was significantly reorganized.Furthermore,RTPCR results showed that the mRNA relative expression levels of cytoskeletal binding proteins,exception of actin,were down-regulated after treated with FB1.Besides,Western blotting and statistical analysis based on fluorescence intensity of laser confocal microscopy confirmed theses changes in protein level.Conclusions FB1 can significantly affect the biomechanical properties and motility of HUVECs,which may be directly correlated to the remodel of F-actin cytoskeleton,as well as the relative expression changes of cytoskeletal binding proteins.It is significant for further exploring the toxicity mechanism of fumonisin.

Ameliorated effects of Lactobacillus delbrueckii subsp. lactis DSM 20076 and Pediococcus acidilactici NNRL B-5627 on Fumonisin B1-induced Hepatotoxicity and Nephrotoxicity in rats

Oxidative stress has been implicated in a number of human regeneration and disease processes including atherosclerosis, pulmonary fibrosis, cancer, and different neurodegenerative diseases.The aim of this study was to evaluate the protective effects of Lactobacillus delbrueckii subsp. lactis DSM 20076 (LL-DSM) and Pediococcus acidilactici NNRL B-5627 (PA-NNRL) against the hepatic-and nephro-toxicity of fumonisin B1 (FB1) in FB1-treated rats for an experimental period of 4-weeks. Eighty mature male Sprague-Dawley rats were divided to 12 groups: 1 untreated group;3 groups fed by a FB1-contaminated diet (50, 100 and 200 mg FB1/kg diet,respectively);1 group fed orally by LL-DSM(1 ml/d);1 group fed orally by PA-NNRL (1 ml/d);3 groups co-administered by FB1-contaminated diet and LL-DSM (1 ml/d), and 3 groups coadministered by FB1-contaminated diet and PA-NNRL(1 ml/d). Malonaldehyde (MDA) nitric oxide, glutathione content, SOD activity, total antioxidant capacity (TAC), total oxidant status (TOS) and oxidative stress index (OSI) were determined. DPA assay was used to assess apoptosis in liver and kidney tissues.The animals fed with FB1-contaminated diet showed a significant increase in oxidative stress markers and DNA fragmentation accompanied with significant decrease in GSH content, SOD activity, and TAC in liver and kidney tissues, especially at highdosage of FB1 (T200). Probiotics antioxidant strains (LL-DSM and PA-NNRL) relatively succeeded to restore almost all parameters investigated as well as to reduce DNA fragmentation in liver and kidney tissues. As a conclusion, probiotics may induce its protective role via increasing the antioxidant capacity, inhibition of lipid peroxidation, scavenging of free radicals and decreasing DNA lesions in liver and kidney of experimental animals tested.

Effect of Planting Density, Irrigation Regimes, and Maize Hybrids with Varying Ear Size on Yield, and Aflatoxin and Fumonisin Contamination Levels

Corn (maize, Zea mays L.) hybrids expressing the flexibility trait in ear size (number of kernels per ear) are marketed for ability to give higher yields under adverse conditions. Altered kernel number is associated with altered number of silk, a major route for infection of kernels by aflatoxin-producing fungi such as Aspergillus flavus. The effect of plant density and irrigation level on yield and accumulation of aflatoxins and fumonisins in harvested grain was compared in a fixed-ear hybrid (Pioneer 33K81), a semi-flexible ear hybrid (Pioneer 3223) and a flexible ear hybrid (Golden Acres 8460) over a range of seeding densities (49,400, 61,750, 74,700, 86,450, and 98,800 seeds·ha–1) in non-irrigated, moderately-irrigated (6.4 cm soil water deficit) and well-irrigated plots (3.8 cm soil water deficit), during three years with variable rainfall. Irrigation increased yields in all hybrids, but in the absence of irrigation, yields were highest with the semi-flexible ear trait hybrid. In general, the hybrid with the flexible ear trait had lower optimal seeding densities than the other hybrids for each soil water regime. In general, kernel number was least affected by seeding density in the hybrid with fixed-ear trait compared to the semi- and flexible ear hybrids. The lowest levels of aflatoxin and of fumonisin contamination in harvested grain were associated with the flexible ear trait at all rainfall and irrigation levels, but there was no evidence that reducing stress by lowering seeding density reduced mycotoxin contamination. Inoculation with A. flavus resulted in much higher levels of aflatoxin and significantly higher levels of fumonisin contamination in grain of all hybrids under most conditions of rainfall and irrigation, suggesting that factors that promote A. flavus infection can affect production of both mycotoxins.

Growth Performance and Serum Lipids Profile of Clarias gariepinus Catfish Following Experimental Dietary Exposure to Fumonisin B1

Fumonisin B1 (FB1) is produced by fungus of the genus Fusarium (Fusarium verticiloides and F. proliferatum), and occurs predominantly in maize. The consumption of feed contaminated with FB1 has been reported to cause deleterious effects in some fish species. This study was designed to determine the effects of dietary FB1 on growth and lipids profile of Clarias gariepinus. 450 juvenile catfish were stocked into 5 groups of tanks consisting of 3 tanks per group and fed one of five diets amended with FB1 (0.0 mg;10.0 mg;20.0 mg;40.0 mg and 80.0 mg FB1/kg) for 56 days. At time point’s day 7, 14, 28 and 56, five fish were sampled from each tank weighted, length measured and bled for of lipids profile determinations. Results show that there was a significant reduction (P < 0.05), in the mean body length of the fish fed diets amended with various amounts of FB1 compared with those fed control diet;also, there was a significant reduction (P < 0.05) in the weight gain of fishes fed diets amended with FB1 compared with the control. The specific growth rate and the feed conversion ratio at 56 days shows fish fed 0.0 mg FB1/kg had the highest specific growth rate (0.39 ± 0.14%/day) and the lowest feed conversion ratio (0.59 ± 0.01) whereas, fish fed 80.0 mg FB1/kg had the least specific growth rate (0.07% ± 0.01%/day) and the highest feed conversion ratio (1.95 ± 0.11). Dietary FB1 caused significant increases (P < 0.05) in serum cholesterol, HDL-C;LDL-C;triglycerides and the sphinganine-sphingosine ratio. Dietary FB1 at an inclusion rate ≥ 20 mg FB1/kg of diet produced significant reduction in weight gain and hyperlipidemia marked by hypercholesterolemia, increased blood high-density lipid cholesterol, increased blood low-density lipid cholesterol, elevated blood triglycerides and elevated sphinganine-sphingosine ratio.

Aflatoxin and Fumonisin in Corn Production Chain in Bafia, Centre Cameroon： Impact of Processing Techniques

Food safety is to be a vital component of food security, with mycotoxin contamination, a major contributing factor. In linewith this, this study aimed at investigating the effect of maize maturity at harvest, and processing techniques on the aflatoxin andfumonisin levels in maize and maize products. Three maize maturity stages （80, 85, and 90 days after sowing）, two drying processes（sun and barn drying）, three storage periods （one, two and three months） and subsequent maize derivatives under these conditionswere sampled. These were analysed for total aflatoxins and total fumonisins using quantitative ELISA and samples with totalaflatoxins and total fumonisins exceeding regulated levels were analyzed by liquid chromatography-tandem mass spectrometry（LC-MS/MS） to determine the sub-types of toxins present. Results obtained showed that all analyzed samples were contaminatedwith total aflatoxins （range： 0.8 to 20 μg/kg） and total fumonisins （range： 10 to 5990 μg/kg）. Sun or barn drying for one weekfollowed by one month usual storage resulted in significant total fumonisins contamination, emphasizing the need of at least twoweeks of drying maize. It was also observed that processing techniques partly reduced the levels of toxins, mainly in maize productsthat have a sieving step.

Probable Effects of Dual Inoculation of Maize (<i>Zea mays</i>) Stem with <i>Fusarium verticillioides</i>and Certain <i>Trichoderma</i>Species on Fumonisin Content of Maize Seeds

Seeds from maize (Z. mays) plants whose stems received various treatment combinations of pathogen (F. verticillioides) and four antagonists (i.e. Trichoderma harzianum strain 2, T. hamatum, T. pseudokoningii strains 2 and 5) in the field were subjected to fumonisin analysis. Three pairing methods were employed for the inoculation of pathogen and the antagonists into stem of the maize plant, viz., “Pathogen inoculated before Antagonist”, “Antagonist inoculated before Pathogen”, and “Antagonist and Pathogen inoculated simultaneously”. Controls include “Inoculation of pathogen alone”, “Inoculation of antagonist alone”, and “Inoculation of sterile toothpicks”. Inoculation method used was the toothpick method. Seeds were harvested five weeks after inoculation and subjected to fumonisin analysis. Resulting data were subjected to ANOVA using the GLM procedure of SAS. There was a high significance among treatments i.e. there were varying levels of fumonisin occurrence among the treatments and varying Fusarium occurrences within the blocks. Seeds from treatments involving “Inoculating T. pseudokoningii strain 5 alone” and “Inoculating T. harzianum strain 2 alone” had the highest mean fumonisin content (P > 0.01) which were not significantly higher than in control. Seeds from treatments involving “Inoculating T. pseudokoningii strain 5 and pathogen simultaneously” and “Inoculating T. harzianum strain 2 before pathogen” were significantly low in fumonisin content compared to seeds from other treatments. Seeds which received “Inoculation of T. hamatum alone” were also significantly low (P > 0.01) in fumonisin content compared to others. It could thus be said that treatments involving Trichoderma species applied in the maize stem might have an effect on the fumonisin content and hence Fusarium occurrence in the seeds depending on the occurrence pattern of the Trichoderma within the maize stem.

伏马菌素B_(1)脱毒研究进展

伏马菌素B_(1)(FB_(1))是由镰刀菌产生的水溶性代谢产物,存在于镰刀菌污染了的谷物、油料作物、坚果、饲草和饲料中,是常见的真菌毒素之一。FB_(1)会对神经系统、呼吸系统、消化系统和生殖系统产生不同毒性作用,给农牧业造成不可估量的经济损失。如何对FB_(1)进行脱毒,以降低其毒性作用,是目前研究的热点。因此,本文对FB_(1)的物理脱毒、化学脱毒、生物脱毒及其应用进行综述,重点对常用于FB_(1)生物脱毒的菌株和酶及其脱毒效果、脱毒机制进行了深入探讨,以期为提高FB_(1)脱毒效率和开发新的FB_(1)脱毒方法提供参考和思路。

Free and Hidden Fumonisins in Hordei Fructus Germinatus and Their Transfer to the Decoction

Objective:This study aimed to determine the free and hidden fumonisins in Hordei Fructus Germinatus samples and to investigate the transfer rates of these mycotoxins from Hordei Fructus Germinatus to its decoction.Materials and Methods:The contamination levels of free and hidden fumonisins in a total of 60 Hordei Fructus Germinatus samples were analyzed using high-performance liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS).The decoction procedure was simulated with a highly contaminated Hordei Fructus Germinatus sample,and fumonisins in the resulting decoction preparation were determined by LC-MS/MS.Results:Among all the samples,8.3%were contaminated with free fumonisins(FB1 and/or FB2)and 13.3%were contaminated with total fumonisins(free+hidden,measured as hydrolyzed fumonisins,i.e.,HFB1 and/or HFB2).The concentrations of FB1 and HFB1 reached up to 83 and 95μg/kg,respectively,whereas FB2 and HFB2 were detected only in traces.The transfer rates of free and total fumonisins from Hordei Fructus Germinatus to the decoction were 71.8%and 83.3%for FB1 and FB2,respectively.In comparison,much lower transfer rates were found for total fumonisins,i.e.,38.2%and 24.7%for HFB1 and HFB2,respectively.Conclusion:The incidence and contamination levels of free and hidden fumonisins in Hordei Fructus Germinatus samples were generally low.Regarding decoction preparation,the transfer rates of free fumonisins into the decoction were high,whereas a large part of hidden fumonisins were retained in Hordei Fructus Germinatus rather than migrating into water.

基于氧化应激和线粒体功能障碍的伏马毒素B1诱导线虫神经毒性机制

为研究线粒体功能和氧化应激在伏马毒素B1(fumonisin B1,FB1)诱导神经毒性中的作用,以秀丽隐杆线虫为模型,对FB1处理后的线虫行为表型以及包括氧化应激和线粒体功能相关指标变化进行分析。结果表明,20~200μg/mL的FB1暴露24 h以剂量依赖方式诱导细胞色素氧化酶、超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)相关基因(cyp35A2、sod-1、sod-3、ctl-2和ctl-3)的异常表达。此外,100~200μg/mL的FB1处理降低了线虫线粒体密度,并显著降低了三磷酸腺苷(adenosine triphosphate,ATP)水平和线粒体膜电位(P<0.05)。200μg/mL的FB1处理显著抑制了线虫线粒体呼吸链复合物I和Ⅴ的表达,提高了线粒体分裂基因drp-1的表达水平。相关性分析显示,氧化应激和线粒体功能相关指标的表达与线虫行为学表型具有显著相关性。综上,FB1可能通过参与线虫氧化应激、线粒体呼吸链和线粒体动力学过程诱导神经毒性。

外源氨基酸对层出镰刀菌菌丝生长和伏马毒素合成的影响

由层出镰刀菌引起的水稻穗腐病,不仅造成水稻产量损失,而且因产生真菌毒素而导致稻米品质下降。本研究通过在生长培养基中添加外源氨基酸,评价其对层出镰刀菌菌丝生长和伏马毒素合成的影响。结果表明,不同氨基酸和不同添加量对菌丝生长均有影响。与未添加氨基酸的对照相比,在MM培养基上添加L-精氨酸、L-谷氨酸、L-脯氨酸和L-瓜氨酸,在PDA培养基上添加L-谷氨酸,均抑制菌丝生长。添加氨基酸显著提高了伏马毒素含量,且随着氨基酸浓度升高,在PDA中产毒量减少,在MM中则呈增加趋势。外源氨基酸明显改变了伏马毒素生物合成基因在转录水平上的表达,但基因表达模式与毒素含量并不一致。综合分析表明,外源氨基酸会影响层出镰刀菌菌丝生长和伏马毒素含量,且不同氨基酸组分与菌丝生长和伏马毒素合成的关联性不同。

利用时间分辨荧光免疫层析法同时定量检测玉米中的伏马毒素B_(1)、B_(2)和B_(3)

建立了一种可同时定量检测玉米中伏马毒素B_(1)、B_(2)和B_(3)的时间分辨荧光免疫层析方法。利用活化酯法制备时间分辨荧光微球和伏马毒素抗体的偶联物,将伏马毒素抗原包被到硝酸纤维素膜上作为T线,通过优化不同T线包被条数,最终建立伏马毒素竞争性免疫层析方法。并对所建立方法的灵敏度、特异性、准确度、精密度和与国标方法分析结果的相关性进行评价。包被两条T线建立的检测方法检出限为107.68~168.28μg/kg,定量限为283.46~444.63μg/kg;与伏马毒素B_(1)、伏马毒素B_(2)和伏马毒素B_(3)的交叉反应率分别为100%、85.59%和72.72%,与其它5种常见的真菌毒素均无明显交叉;加标回收率范围88.37%~117.42%,变异系数小于10%;该方法与国标方法GB 5009.240-2016中规定的免疫亲和柱净化-高效液相色谱法(IAC-HPLC)检测结果的符合度在92.17%~107.21%之间。建立的时间分辨荧光免疫层析检测方法能满足对玉米中伏马毒素B_(1)、B_(2)和B_(3)的现场快速定量检测。

我国饲料原料和配合饲料中T-2毒素、赭曲霉毒素A和伏马毒素的污染研究

本试验旨在研究我国饲料原料和配合饲料中T-2毒素、赭曲霉毒素A(OTA)和伏马毒素(FUM)的单独及联合污染情况。分别对从我国不同地区采集474、385和1905份饲料原料和配合饲料样品,进行T-2毒素、OTA和FUM含量检测。结果显示:饲料原料和配合饲料中T-2毒素、OTA和FUM总污染率分别为26.8%、61.8%和59.3%,其含量平均值分别为0~45.8μg/kg、0.3~11.1μg/kg和0~16.3 mg/kg。值得注意的是,分别有0.4%、0.3%和0.5%的饲料原料和配合饲料中T-2毒素、OTA和FUM含量超过了我国《饲料卫生标准》,并且,60%以上的饲料原料中霉菌毒素的联合污染率达30.3%~88.9%。由此可见,我国饲料原料和配合饲料中T-2毒素、OTA和FUM的污染较严重,生产中加强监测我国饲料原料和配合饲料中上述霉菌毒素的污染情况和实施相关防控措施尤为重要。

伏马毒素的污染现状及其生物脱毒研究进展

伏马毒素及其类似物是由镰刀菌属为主的真菌在其生长代谢过程中产生的一类溶于水、分子量小、具有毒害作用的次级代谢产物。伏马毒素广泛污染粮食作物及其相关制品,严重威胁人类和动物健康。传统的物理、化学脱毒方法不仅脱毒效率低,而且还会破坏谷物的营养成分,影响口感和风味。随着生物技术的发展,利用生物脱毒法对伏马毒素进行消减脱除是未来解决这一问题的关键。本文对伏马毒素的种类、污染现状、毒性机理及其降解菌或酶进行了归纳总结,并对生物脱毒研究中存在的问题进行了讨论,以期为深入研究伏马毒素的高效脱毒提供参考。

小鼠亚慢性伏马毒素B_(1)中毒对血液生化指标的影响及组织病理变化的观察

为探究小鼠伏马毒素B_(1)(FB_(1))亚慢性中毒对血液生化指标和组织病理学的影响,选用40只雄性昆明小鼠,通过饲料添加方式建立小鼠FB_(1)的亚慢性中毒模型,分别为对照组(标准基础饲料,C组)和伏马毒素组(5 mg/kg FB_(1)混合基础饲料,FB_(1)组),连续饲喂42 d。每天称重,于试验第21天和第42天取小鼠各器官称重,计算脏器比;测定血液生化指标;HE染色观察肝脏等12种脏器病理组织学变化。结果表明,与C组相比,21 d和42 d中,FB_(1)组肝脏等器官系数显著下降;FB_(1)组血清ALT等含量显著升高,UA等含量显著降低;血常规指标明显异常;病理组织学结果显示,FB_(1)组中肝脏等12种脏器存在一定病理损伤。提示5 mg/kg FB_(1)染毒能够影响小鼠的血液生化指标,且对小鼠各组织器官造成一定损伤。

Occurrence of mycotoxins in food,feed,and milk in two counties from different agro-ecological zones and with historical outbreak of aflatoxins and fumonisins poisonings in Kenya

Aflatoxins and fumonisins contaminate cereals during pre-and post-harvest periods.In this study,household or market maize,sorghum,millet,cow or goat milk,and animal feed samples collected from two counties(Makueni and Nandi)of Kenya and were analyzed for aflatoxins and fumonisins using competitive enzyme-linked immunosorbent assay and confirmation with high performance liquid chromatography.There was a significant difference(P<0.005)in the levels of aflatoxins between the home grown and market-sourced maize,sorghum,and millet samples.In Makueni,24.8%of home maize and 44.6%of the market maize samples exceeded the 10 ppb limit for aflatoxins.In all,93%and 90%of the maize samples were contaminated with fumonisins and 34%and 6%exceeded the 2 ppm limit in Makueni and Nandi,respectively;30%and 37%of homegrown sorghum and millet samples exceeded the 10 ppb limit for aflatoxin in Makueni and Nandi,respectively;and 89%and 81%of homegrown millet samples in Makueni and Nandi,respectively,were positive for fumonisins and 22%and 7%in Makueni and Nandi,respectively,exceeded the 2 ppm fumonisins limit.In total,52%and 87%of the milk samples in Nandi and Makueni,respectively,were contaminated with aflatoxin M1 and 8%of the samples from Makueni exceeded the 50 ppt limit.There is an urgent need to build capacity among the households on cheap,practical,and effective technologies that would reduce the proportions of food samples contaminated with aflatoxins and fumonisins.