储粮中黄曲霉毒素B_1快速检测技术的研究

运用高效液相色谱方法检测储粮中黄曲霉毒素B1的含量,检测样品中黄曲霉毒素B1的含量为0.004 3 mg/kg,样品的RSD为0.033,回收率为97.41%,提取液是乙腈∶水=84∶16,流动相是甲醇∶乙腈∶水混合溶液=15∶13∶72,线性回归方程是y=188.97x+4.96相关系数是R2=0.999 8,检测器激发波长=460 nm,衍生法利用光化学衍生池,进化方法利用免疫亲和柱。高效液相色谱法测定黄曲霉毒素B1是一种操作简单、准确度好、灵敏度高的检测方法。     

免疫学法检测黄曲霉毒素的研究进展

黄曲霉毒素是黄曲霉菌和寄生曲霉菌的次生代谢产物,是世界卫生组织公认的致癌物。目前,对黄曲霉毒素的检测方法有薄层层析法、高效液相色谱法和酶联免疫吸附法等,其中免疫化学方法具有特异性强、灵敏度高和快速简便等优点,在黄曲霉毒素的检测中应用广泛。建立快速、简便和无毒的检测体系将成为今后检测AFT的研究热点。     

响应面法优化重组AhPR-1蛋白抑制黄曲霉菌侵染花生的条件

基于大肠杆菌重组花生Ah PR-1蛋白具有抑菌的作用功能,利用响应面法优化蛋白抑制黄曲霉菌在花生上生长的条件因素。以表观评价法测评的黄曲霉生长程度和HPLC法检测的毒素合成的含量为评价标准,考察Ah PR-1蛋白质量浓度、作用温度和水分活度对蛋白抑制黄曲霉菌侵染花生及黄曲霉毒素产生的影响。结果显示,在花生染黄曲霉菌浓度为4×106CFU/m L的条件下,随着蛋白质量浓度升高,抑菌效果明显增强,蛋白质量浓度为0.32 ng/μL和0.40 ng/μL,效果最好且差别不是很明显;在温度28℃,水分活度0.3~0.4的条件下,AhPR-1蛋白的抑制效果最显著。在一定的环境条件下,Ah PR-1蛋白能够一定程度上抑制黄曲霉菌在花生表面的繁殖生长和毒素的合成。     

紫苏醛-海藻酸钠复合涂膜抗花生黄曲霉菌研究

花生极易在储运过程中因黄曲霉菌的快速生长繁殖受到黄曲霉毒素的污染,为了更好地防控花生受到黄曲霉毒素的污染,本文以海藻酸钠和紫苏醛为原料,甘油为增塑剂,以抑制黄曲霉菌生长活性为主要评价指标,研究紫苏醛-海藻酸钠复合涂膜最佳配方及复合涂膜花生的储藏条件。结果表明,海藻酸钠浓度6 mg/m L,活性物紫苏醛0.015 mg/m L,甘油浓度6 mg/m L时制备的复合膜抗黄曲霉生长活性能力最佳,抑菌直径达到(7.23±0.11)cm。紫苏醛添加方式对涂膜花生黄曲霉菌抑制效果影响很大,最佳添加方式为熏蒸,涂膜花生贮藏温度应在25℃以下,贮藏环境相对湿度为60%以下。     

黄曲霉毒素高快准检测技术难题破解

据中国农业科学院最新消息,该院油料作物所研究员李培武带领农业部生物毒素检测重点实验室科研团队,成功破解了黄曲霉毒素高灵敏快速准确定量检测的技术难题,研制出黄曲霉毒素系列检测仪器和配套产品,如牛奶等单个样品从取样到结果打印最快9分钟即可完成,用时相当于国外同类产品的一半,检测技术达国际领先水平,打破了发达国家在该领域的垄断。黄曲霉毒素是迄今发现的毒性和致癌性最强的真菌毒素。其中,黄曲霉毒素B1的毒性是氰化钾的10倍,是砒霜的68倍,致癌力是标准致癌物二甲基硝胺的75倍。此前,国际通行的黄曲霉毒素检测方法为高效液相色谱法或高效液相色谱质谱联用法,不仅需大型仪器,而且相关设备价格昂贵(每台几十万元甚至几百万元)。     

不同贮藏条件下花生中黄曲霉毒素变化趋势

目的探寻不同贮藏条件下花生中黄曲霉毒素含量的变化趋势。方法以远杂9102和豫花15品种的花生和花生仁为研究对象,采用免疫亲和层析净化高效液相色谱法测定其在不同储藏的条件下黄曲霉毒素的含量。结果在整个贮藏过程中,远杂9102和豫花15品种的花生和含水量低于10%的花生仁的黄曲霉毒素含量为0,而含水量高于10%的花生仁的黄曲霉毒素含量随着贮藏时间的延长而升高。在接菌量相同的条件下,同一品种的花生仁黄曲霉毒素的含量随含水量增加而增高;在含水量相同的条件下,同一品种花生仁中黄曲霉毒素的含量随接菌量的增加而增高。在相同的贮藏条件下,远杂9102花生仁中黄曲霉毒素含量极显著高于豫花15。结论在贮藏过程中,花生中黄曲霉毒素的含量与花生是否带壳、含水量、初始带菌量和品种之间具有相关性。     

海洋巨大芽孢杆菌在培养基和花生中对黄曲霉产毒的抑制

目的 研究海洋巨大芽孢杆菌在PDA、MM培养基和花生中对黄曲霉产毒的抑制。方法 采用酶联免疫吸附法和高压液相色谱荧光检测法测定培养基和花生中黄曲霉毒素B1含量。结果 PDA、MM培养基培养的黄曲霉通过酶联免疫吸附法检测后, 实验组黄曲霉毒素B1含量分别在5.5158.1 pg/mL和2.210.3 pg/mL, 对照组毒素B1含量分别在2407.22986.3 pg/mL和4.42755.6 pg/mL。通过高压液相色谱检测28 ℃和37 ℃培养的被黄曲霉侵染的花生, 实验组黄曲霉毒素B1含量均未检出, 对照组28 ℃下, 黄曲霉毒素B1含量为(8.588?0.322)μg/kg, 37 ℃下黄曲霉毒素未检出。结论 不同培养基中, 海洋巨大芽孢杆菌对黄曲霉产黄曲霉毒素均有抑制作用。黄曲霉在28 ℃比37 ℃更容易在花生上产毒, 海洋巨大芽孢杆菌对花生上黄曲霉产毒素有显著抑制作用。     

免疫亲和柱净化-UPLC-MS/MS测定花生中黄曲霉毒素B_1

为提高花生中黄曲霉毒素B_1的检测效率和准确性,试验通过对提取溶剂、色谱条件和质谱条件的优化,建立了免疫亲和柱-超高效液相色谱串联质谱(UPLC-MS/MS)快速测定花生中黄曲霉毒素B_1的方法,并对方法进行了评估。结果表明,用甲醇-水(60∶40,V/V)对样品进行提取,采用黄曲霉毒素免疫亲和柱萃取、净化,以0.1%甲酸水溶液、0.1%甲酸乙腈溶液作为超高效液相色谱的流动相,在电喷雾离子源(ESI)正离子模式下采用多反应监测(MRM)对花生中黄曲霉毒素B_1进行定性、定量检测,在5.5 min内完成一个样品的分析。结果表明,黄曲霉毒素B_1在0.1~56.0 ng/m L的线性定量范围,相关系数高达0.999 42,检出限低至0.02μg/kg,定量限精确至0.1μg/kg,低、中、高浓度回收率为82%~92%,相对标准偏差5%。该方法具有前处理简单、净化效果好和准确度高的优点,适用于花生等复杂基质样品的黄曲霉毒素B_1定性和定量检测。     

多功能净化柱高效液相色谱法检测稻米中的黄曲霉毒素B1的研究

建立多功能净化柱净化高效液相色谱法检测稻米中的黄曲霉毒素B1的方法。稻米样品经V（乙腈）∶V（水）=84∶16提取,多功能净化柱净化,三氟乙酸衍生化,高效液相色谱法荧光检测器检测。黄曲霉毒素B1浓度在0.010~100 μg/L范围内呈良好的线性关系,回归方程为y=60.431x+0.163 8,相关系数R2=0.999 9。该方法的检出限为0.1 μg/kg,加标回收率为82.25%~98.52%,标准偏差（SD）为2.23%~3.62%,比普通的0.45 μm微孔滤膜净化的要高。该法操作简便易行、选择性高、检测效果好,适合大批量粮油作物中黄曲霉毒素的检测,有利于推广应用。     

间接竞争ELISA检测试剂盒测定粮油食品中的 黄曲霉毒素B1

目的建立一套适用于食品中黄曲霉毒素B1的快速、简便、准确的检测方法,同时,进一步验证本公司研制的黄曲霉毒素ELISA检测试剂盒的检测效果。方法用酶联免疫法和高效液相色谱法分别对市售的食用油、花生、谷物样品中AFB1的污染程度进行检测分析。结果用ELISA法和HPLC法测定食用油样品的回收率分别为87.1%~92.7%和85.8%~100.8%;花生样品的回收率分别为76.0%~92.8%和76.3%~92.9%;谷物样品的回收率分别为82.6%~92.7%和82.7%~106.4%,花生加标样品6次平行测定的RSD分别为1.29%~2.46%和5.15%~8.70%,11份阳性样品测定结果表明2种方法具有很好的一致性(r2=0.995)。结论 ELISA法和HPLC法均有很好的线性关系,且测定结果相近。本公司研制的黄曲霉毒素ELISA试剂盒可以快速地检测食品中黄曲霉毒素B1,分析周期短,分析效率高。     

Resistance to aflatoxin accumulation in kernels of maize inbreds selected for ear rot resistance in West and Central Africa

Thirty-six inbred lines selected in West and Central Africa for moderate to high resistance to maize ear rot under conditions of severe natural infection were screened for resistance to aflatoxin contamination using the previously established kernel screening assay. Results showed that more than half the inbreds accumulated aflatoxins at levels as low as or lower than the resistant U.S. lines GT-MAS:gk or MI82. In 10 selected aflatoxin-resistant or aflatoxin-susceptible inbreds, Aspergillus flavus growth, which was quantified using an A. flavus transformant containing a GUS-beta-tubulin reporter gene construct, was, in general, positively related to aflatoxin accumulation. However, one aflatoxin-resistant inbred supported a relatively high level of fungal infection, whereas two susceptibles supported relatively low fungal infection. When kernels of the 10 tested lines were profiled for proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, significant variations from protein profiles of U.S. lines were observed. Confirmation of resistance in promising African lines in field trials may significantly broaden the resistant germplasm base available for managing aflatoxin contamination through breeding approaches. Biochemical resistance markers different from those being identified and characterized in U.S. genotypes, such as ones inhibitory to aflatoxin biosynthesis rather than to fungal infection, may also be identified in African lines. These discoveries could significantly enhance the host resistance strategy of pyramiding different traits into agronomically useful maize germplasm to control aflatoxin contamination.     

Restriction fragment length polymorphism assessment of the heterogeneous nature of maize population GT-MAS:gk and field evaluation of resistance to aflatoxin production by Aspergillus flavus.

Aflatoxin, produced by Aspergillus flavus, is one of the most toxic and carcinogenic substances known and contaminates many agricultural commodities such as corn, peanuts, cottonseed, and tree nuts. The challenge to breeders/plant pathologists is to identify lines that have resistance to aflatoxin production. Maize population GT-MAS:gk has been identified and released as a germplasm with resistance to aflatoxin contamination. In the present study, we assessed genetic divergence in the GT-MAS:gk population using restriction fragment length polymorphism (RFLP) DNA markers to survey 11 selfed inbred lines and conducted field evaluations for the dissimilarities in aflatoxin production among these inbred lines in comparison with a sister population, GT-MAS:pw.nf. The 11 selfed inbred lines were assayed for DNA polymorphism using 113 RFLP markers in 10 linkage groups covering 1,518.2 centimorgans (cM; unit of gene or chromosome size). Considerable variation among the inbreds was detected with RFLP markers, of which 42 probe-enzyme combinations gave 102 polymorphic bands. Cluster analysis based on genetic similarities revealed associations and variations among the tested lines. Three polymorphic groups were distinguished by cluster analysis. Two years of field evaluation data showed that aflatoxin concentrations among the lines were significantly different in both years (P < 0.001). Maturity data were also different. Thus, this study demonstrates that the maize population GT-MAS:gk is heterogeneous and that individuals may be different in resistance to A. flavus infection and aflatoxin production. Therefore, the most resistant lines should be inbred to increase homogeneity, and resistance should be confirmed through progeny testing.     

花生重组近交系黄曲霉产毒抗性的变异与相关分析

以不同遗传背景的亲本远杂9102与中花5号杂交构建重组近交系（RIL）群体,进行人工接种和黄曲霉毒素含量测定。结果表明,RIL群体对黄曲霉的产毒抗性存在较大变异,产毒抗性为受亲本加性基因控制的数量性状,而且存在超亲优势,因此利用微效加性基因的累加和互补提高产毒抗性的潜力较大。相关性分析表明,RIL群体中黄曲霉产毒抗性与百果重、含油量、青枯病抗性之间相关性不显著,不存在紧密连锁关系。初步筛选到大果、高油、兼抗青枯病与黄曲霉产毒的优异材料,可以作为进一步育种研究的核心亲本。     

玉米抗黄曲霉毒素污染的研究进展

黄曲霉毒素污染是影响玉米食用安全的重要因素。筛选培育玉米抗性品种,从源头控制黄曲霉的侵染,是解决玉米田间及储存期黄曲霉污染的有效方法。对国内外玉米黄曲霉抗原种质的筛选鉴定、分子标记辅助选育及部分抗性机理等方面的研究进行了概述,并就目前存在的一些问题,探讨了我国玉米抗黄曲霉的研究方向。     

Resistance to aflatoxin contamination in corn as influenced by relative humidity and kernel germination.

Kernels of corn population GT-MAS:gk, resistant to aflatoxin B1 production by Aspergillus flavus, and susceptible Pioneer hybrid 3154 were tested for aflatoxin when incubated under different relative humidities (RH). High aflatoxin levels were not detected in either genotype at RH < 91%. Resistance in GT-MAS:gk was consistent across all RH levels (91 to 100%) at which significant aflatoxin accumulation was detected. Aflatoxin levels in GT-MAS:gk averaged about 98% less than those in susceptible Pioneer 3154, which suggests that storage of this or other genotypes with similar resistance mechanisms may be possible under moisture conditions less exacting than are required with susceptible hybrids. Results for fungus growth and sporulation ratings on kernel surfaces were similar to those for aflatoxin levels. When kernels of both genotypes were preincubated 3 days at 100% RH prior to inoculation with A. flavus, germination percentages increased to very high levels compared to those of kernels that were not preincubated. In preincubated kernels aflatoxin levels remained consistently low in GT-MAS:gk but decreased markedly (61%) in Pioneer 3154. When eight susceptible hybrids were evaluated for aflatoxin accumulation in preincubated kernels, seven of these supported significantly lower toxin levels than kernels not subjected to preincubation. Average reduction across hybrids was 83%, and reductions within hybrids ranged from 68 to 96%. Preincubated kernels of one susceptible hybrid (Deltapine G-4666) supported aflatoxin levels comparable to those in resistant GT-MAS: gk. Data suggest that an inhibitor of aflatoxin biosynthesis may be induced during kernel germination. Possible mechanisms for embryo effects on resistance to aflatoxin accumulation are discussed.     

加热对减少花生中黄曲霉毒素水平的作用

将花生在各种不同的加热温度和加热时间下进行实验,观察黄曲霉毒素在不同温度及不同加热时间下的减少水平。研究结果表明,加热可以减少花生中黄曲霉毒素的水平,并随着加热温度和加热时间的增加,毒素减少量也增加;黄曲霉毒素减少速率与样品最初被污染的水平有关;AFTG1的热稳定性比AFTB1差。     

西藏高原粮油作物曲霉菌污染及菌株产毒力研究

为探明西藏高原粮油作物曲霉菌污染状况及黄曲霉菌产毒能力,连续5年对西藏青稞、小麦、花生3种作物中曲霉菌污染情况进行分析,并对其分离到的黄曲霉菌株开展产毒力研究,结果表明,204份样品中,共分离出15种曲霉菌,曲霉菌污染率呈花生>青稞>小麦。青稞、小麦中曲霉属优势种均为黑曲霉(Aspergillus niger),真菌毒素主要为杂色曲霉毒素和赭曲霉毒素;花生优势种为黄曲霉(A.flavus);仅受黄曲霉毒素污染。来源于不同作物的黄曲霉菌,其产毒类型也有差异,麦类作物产毒菌株以产黄曲霉毒素B₁(AFB₁)、黄曲霉毒素B₂(AFB₂)为主;花生产毒菌株以产AFB₁、AFB₂、AFG₁、AFG₂为主。     

Assessment of Groundnut Varietal Tolerant to Aflatoxin Contamination in Indonesia

Aflatoxin is secondary metabolite produced by Aspergillus flavus and A. paraciticus that grow on the seed coat (testa) of groundnut. This toxin is a serious food safety issue throughout the world. The availability of resistant genotype to A. flavus infection and/or aflatoxin contamination urgently needed. The experiment found one genotype had aflatoxin contamination under the safe level (≤ 10 ppb), with <15% of seed number infected by A. flavus. Recently, the biggest peanut industry, where the main production is roasted-peanut (in shell) produced from fresh pods, grows and develops that variety.     

Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.     

Effect of different postharvest drying temperatures on Aspergillus flavus survival and aflatoxin content in five maize hybrids

After harvest, maize is dried artificially to halt fungal growth and mycotoxin production while in postharvest storage. The process often limits harvest capacity and has been a frequent cause of seed injury. Higher drying temperatures could lead to shorter drying periods and faster turnover; however, there is often a deterioration of the physical grain quality, including increased breakage susceptibility and loss of viability. The goals of this study were to determine the effect of different postharvest drying temperatures on Aspergillus filavus and Fusarium verticillioides survival and aflatoxin content in maize and to determine the viability of the seed. Five corn hybrids varying in resistance to A. flavus were side needle-inoculated with A. flavus, harvested at physiological maturity, and dried at temperatures ranging from 40 to 70 degrees C. Kernels were evaluated for aflatoxin, stress cracks, germination, and kernel infection by A. flavus and a natural infestation of F. verticillioides. Drying temperature had no effects on aflatoxin concentration given the heat stability of the toxin. With increased temperatures from 40 to 70 degrees C, germination decreased significantly, from 96 to 27%, and stress cracks increased significantly (1.4 up to 18.7). At temperatures above 60 degrees C, F. verticillioides kernel infection was significantly reduced to less than 18%. At 70 degrees C, there was a significant reduction in A. flavus kernel infection, from 11 to 3%. This information is useful in determining a range of temperatures that can be used for drying seed when fungal infection, stress cracks, and seed viability are of interest.