产赭曲霉毒素A黑曲霉的PCR法检测

【目的】快速检测产赭曲霉毒素A(OTA)的黑曲霉。【方法】根据黑曲霉(Aspergillus niger)CBS513.88中An15g07920基因编码聚酮合酶的酰基转移酶(AT)域设计引物,建立针对产OTA黑曲霉的聚合酶链式反应(PCR)检测方法。【结果】对72株曲霉属菌株(黑曲霉、炭黑曲霉、赭曲霉、佩特曲霉、寄生曲霉和塔宾曲霉)进行检测,发现产OTA的黑曲霉能够扩增出特异性条带,而产OTA的其它菌株不能扩增出条带;检测出3株假阳性的产OTA黑曲霉,实时定量PCR分析此3株菌中An15g07920的同源基因表达情况,发现在产毒条件下可正常表达,排除了因基因无法表达导致假阳性的可能。本方法的检测灵敏度为25 pg的DNA含量,在污染所试农产品孢子浓度大于4.0×10~4–4.0×10~5个/g时可有效检测出产毒菌株。【结论】本方法虽会产生4%的假阳性结果,但是仍可作为产毒黑曲霉有效的快速检测方法,并在农产品污染产毒黑曲霉时进行有效预警。     

赭曲霉毒素A的微生物脱毒研究进展

赭曲霉毒素A(Ochratoxin A,OTA)是一类聚酮化合物,是由曲霉属(Aspergillus spp.)和青霉属(Penicilliumspp.)等霉菌产生的次级代谢产物,具有强烈毒性。近年来,国内外已有大量关于OTA生物脱毒的研究,主要是OTA脱毒功能菌株的筛选,包括细菌、霉菌和酵母菌等。此外,对不同微生物脱毒机理的探索也是研究热点。对上述两方面的研究进展进行归纳和讨论。     

赭曲霉毒素A的产生、毒性机制与生物合成研究进展

赭曲霉毒素A(ochratoxin A, OTA)是曲霉属和青霉属等真菌的次生代谢物,广泛污染谷物、葡萄、坚果等农产品和饲料,造成严重的经济损失。此外,OTA的肝肾毒性和三致作用(致畸、致癌、致突变)已经得到越来越多的数据支撑,证实其对人类健康存在巨大威胁。OTA及其衍生物的理化性质已经有较为全面的研究,但是其合成过程及调控机理尚不明确。本文整理了赭曲霉毒素A的理化性质及产毒菌株,总结了OTA污染及致病情况的最新研究进展和各国的限量标准,最后分析了OTA的合成机制,讨论了未来OTA理论及应用层面的研究方向,为赭曲霉毒素A的风险评估提供数据支持,同时为OTA生物合成和调控机制提供理论参考。     

赭曲霉G蛋白偶联受体的鉴定及生物信息学分析

【目的】预测并分析赭曲霉(Aspergillus ochraceus)中存在的G蛋白偶联受体(G-protein- coupled receptors,GPCRs)的结构特征和理化性质,探究赭曲霉GPCR超家族蛋白的结构及所接收配体的聚类情况以及与其他同源蛋白的进化关系,为深入开展赭曲霉中GPCRs的定位、功能研究提供理论基础,也有望从G蛋白信号途径角度抑制赭曲霉毒素的产生,进一步控制粮食的真菌毒素污染。【方法】基于已经报道的曲霉属典型GPCRs序列,在赭曲霉全基因组中进行BLASTp比对以获取候选GPCRs蛋白。通过SMART及多种软件进行保守结构域,特别是跨膜结构的分析,进一步分析候选序列的理化性质、信号肽、二级结构及亚细胞定位等特征。最后,利用MEGA构建赭曲霉中GPCRs与同源蛋白的系统发育树进行遗传关系的比较。【结果】明确赭曲霉存在 15个具有典型7次跨膜结构的GPCRs蛋白,不存在信号肽及转运肽,均含有较高比例的α螺旋结构,15个蛋白质中有7个定位在细胞膜。赭曲霉中的GPCRs与黄曲霉等曲霉属中相应的同源序列具有较近的亲缘关系。【结论】本研究首次对赭曲霉的GPCR超蛋白家族进行了预测,分析其结构及理化性质,探讨了其与同源蛋白的聚类情况,为深入开展赭曲霉GPCRs的功能研究提供理论基础。     

一株产赭曲霉毒素A黑曲霉的筛选与鉴定

【目的】筛选出可产生赭曲霉毒素A (OTA)的霉菌菌株。【方法】利用CYA和YES培养基从实验室32个霉菌样品中筛选目的菌株。利用高效液相色谱-荧光检测法对OTA产生菌进行初筛,利用高效液相色谱-质谱联用对OTA初筛菌株进行复筛。通过菌落形态、菌丝及分生孢子形态、ITS DNA序列、β-Tubulin基因序列及Calmodulin基因序列分析等鉴定目的菌株。【结果】得到一株OTA产生菌株1062,该菌株能在25 °C条件下,在CYA、YES和CA培养基中很好生长。结合形态学、对培养基的要求以及上述3个基因序列的进化树分析,该菌株属于黑曲霉(Aspergillus niger)。【结论】菌株1062具有OTA产生能力,是一株黑曲霉。     

赭曲霉毒素A降解菌株的筛选及脱毒酰胺水解酶的基因克隆和表达

【背景】赭曲霉毒素A (ochratoxin A, OTA)是一种可以致癌的真菌毒素,其污染严重影响食品安全,危害人类健康。生物降解法去除OTA污染是近些年的研究热点,发掘高效的OTA降解脱毒酶资源具有重要的意义。【目的】筛选高效的OTA降解菌株并从中克隆降解基因,为生物脱毒方法的开发提供基因和酶资源。【方法】利用OTA为唯一碳源的筛选培养基从土壤中筛选纯化OTA降解菌株,通过16S rRNA基因序列分析确定其分类地位,利用高效液相色谱(high performance liquid chromatography, HPLC)分析其降解产物。通过同源序列比对的方法克隆降解基因并与载体pET-29a(+)相连,然后在大肠杆菌BL21(DE3)中表达。利用Ni²⁺亲和层析对表达产物进行纯化,研究其对OTA的降解活性和酶学特征。【结果】筛选到一株高效的OTA降解菌株,在12 h内能够完全降解1 μg/mL的OTA;初步鉴定该菌株属于Niastella,编号为JX-6;菌株JX-6通过酰胺键断裂途径降解OTA生成无毒的OTα;从菌株JX-6中鉴定了一个OTA酰胺水解酶,命名为NcOTase;NcOTase与已报道的OTA酰胺水解酶序列相似性较低,仅为31%–53%;纯化的NcOTase具有OTA水解活性,比酶活为60.3 U/mg,活性显著高于大部分已报道的OTA降解酶。【结论】NcOTase是一个高效的OTA降解脱毒酶,在去除食品和饲料中OTA污染方面具有很好的应用前景。     

赭曲霉毒素A和玉米赤霉烯酮-二联胶体金免疫层析试纸条的制备及应用

【背景】真菌毒素为真菌的有毒次级代谢产物,混合污染时毒性显著增强,可对人类和动物健康造成严重伤害。制备二联胶体金免疫层析试纸条,实现对常见真菌毒素混合污染的快速监测,具有重要意义。【目的】制备赭曲霉毒素A (Ochratoxin A,OTA)和玉米赤霉烯酮(Zeralenone,ZEN)金标单克隆抗体,基于免疫层析原理,采用竞争反应模式,建立二联胶体金免疫层析检测法用于污染样品中OTA和ZEN的同时快速检测。【方法】采用柠檬酸钠还原法制备胶体金颗粒,并标记获得两种真菌毒素金标单克隆抗体,通过优化相关条件,建立稳定的二联胶体金免疫层析检测方法,用于同时检测谷物和饲料样品中的OTA和ZEN。【结果】制备的OTA和ZEN二联胶体金试纸条对OTA和ZEN的检测限分别为0.625 ng/mL和1.25 ng/mL,且与谷物和饲料中其它真菌毒素(黄曲霉毒素B1、伏马毒素B1、桔青霉毒素、展青霉毒素和呕吐毒素)均无交叉反应,人工添加试验结果准确。对天然样本检测结果表明该方法与LC-MS/MS一致性良好。【结论】本研究制备的二联胶体金试纸条可用于实际样品中OTA和ZEN的同时快速筛查。     

黑曲霉中生物合成赭曲霉毒素A的非核糖体肽合成酶基因的鉴定

赭曲霉毒素A(ochratoxin A,OTA)是国际癌症研究机构认定的"2B"类致癌物。黑曲霉Aspergillus niger是美国食品药品监督局认可的食品安全菌。然而近年来陆续发现某些黑曲霉菌株能够产生OTA,这会对人类健康构成潜在威胁。阐明黑曲霉生物合成OTA的关键基因有助于理解OTA生物合成机制,这对OTA污染的防控具有重要意义。本研究克隆了产OTA黑曲霉中非核糖体肽合成酶(NRPS)编码基因(An15g07910),并对其进行了生物信息学分析,在此基础上采用同源重组的方法敲除了该基因,获得了一株性能稳定的敲除突变株Δnrps。与野生株相比,Δnrps突变株的表型在CYA培养基中并无明显改变,但在7d培养期间完全失去了合成赭曲霉毒素α(ochratoxinα,OTα)和OTA的能力,而赭曲霉毒素β(ochratoxinβ,OTβ)的合成不受影响。在野生株培养过程中,该nrps基因前4d表达量逐渐增大,并在第4天达到最高,随后基因表达量逐渐下降并趋于稳定,这与OTA的含量变化基本一致。结果表明该nrps基因(An15g07910)参与OTA的生物合成,其编码的NRPS可能负责催化苯丙氨酸部分和二氢异香豆素部分的交联。     

曲霉属三新记录种(英文)

报道了曲霉属的3个中国新记录种:Aspergillus dimorphicus,A.fumigatiaffinis以及A.westerdijkiae。比较了其与相近种的形态及分子序列β‐tubulin基因的差异,并采用高效液相色谱‐质谱联用技术对菌株产赭曲霉毒素A(OTA)的能力进行了测定。检测结果表明Aspergillus westerdijkiae CGMCC3.15268在大米培养基上能够产生OTA,单位菌丝的产毒量为17.26μg/kg;而A.fumigatiaffinis CGMCC3.15275以及A.dimorphicus CGMCC3.17045未检出OTA。     

一株产赭曲霉毒素A黑曲霉及其产毒条件

以甘肃河西走廊葡萄酒产区分离的曲霉属黑色组H1菌株为试验材料,依据形态特征和基于rDNA ITS、β-tubulin及calmodulin基因序列的系统发育分析,将其鉴定为黑曲霉Aspergillus niger。利用HPLC-FLD和UPLC-MS/MS检测分析确认H1菌株具有产生赭曲霉毒素A(OTA)的能力。进一步研究了培养条件对H1菌株产毒的影响。试验结果表明,YES培养基比CYA培养基更利于H1菌株的营养生长和产OTA;18–25℃,该菌生长较慢,但OTA产量高;18℃H1菌株在YES培养基上培养9d时OTA的产量达到最大值,为26.01μg/g YES;30–37℃该菌的生长速率显著提高,但OTA产量很低,甚至不产OTA。试验结果表明,温度和营养条件是影响H1菌株产生OTA能力的重要因素。     

Synthesis of hydroxy fatty acids from linoleic acid by human blood platelets

The metabolism of linoleic acid by washed human platelets was investigated. [1.14C] linoleic acid was converted to [1.14C] hydroxy octadecadienoic acids (HODEs) at about the same rate with which [1.14C] 12-HETE was produced from [1.14C] arachidonic acid. The total radioactivity in HODEs was distributed among two isomers: 13-HODE (85%) and 9-HODE (15%) as defined by CG-MS. The production of HODEs by intact washed platelets was inhibited by indomethacin (IC50:5 x 10(-7) M) which suggest that hydroxy fatty acids were produced by PGH-synthase. By contrast, the production of HODEs by platelet cytosolic fractions was not modified under indomethacin treatment but completely abolished by NDGA (10(-3) M) and inhibited by the platelet lipoxygenase inhibitors 15-HETE (2.10(-5) M) and baicalein (10(-5) M). Platelets thus contain two different active systems which may convert linoleic acid to hydroxy fatty acids. Since these compounds remained essentially associated with the platelets, their presence may significantly participate in the mechanisms of platelet activation.     

Steric analysis of 8-hydroxy- and 10-hydroxyoctadecadienoic acids and dihydroxyoctadecadienoic acids formed from 8R-hydroperoxyoctadecadienoic acid by hydroperoxide isomerases

8-Hydroxyoctadeca-9Z,12Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8E,12Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10S-HODE by Lentinula edodes, and 10R-HODE by Epichloe typhina. Racemic [8-(2)H]8-HODE and [10-(2)H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess-Martin periodinane followed by reduction to hydroxy fatty acids with NaB(2)H(4). The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with 8R-HODE and 10S-HODE as standards. 8R-HODE eluted after 8S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10S-HODE eluted before 10R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5S,8R-Dihydroxyoctadeca-9Z,12Z-dienoic acid (5S,8R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8R,11S-dihydroxyoctadeca-9Z,12Z-dienoic acid (8R,11S-DiHODE) by Agaricus bisporus. 8R-Hydroperoxylinoleic acid (8R-HPODE) can be transformed to 5S,8R-DiHODE and 8R,11-DiHODE by Aspergillus spp., and 8R,13-dihydroxy-9Z,11E-dienoic acid (8R,13-DiHODE) can also be detected. We prepared racemic [5,8-(2)H(2)]5,8- and [8,11-(2)H(2)]8,11-DiHODE by oxidation and reduction as above and 8R,13S- and 8R,13R-DiHODE by oxidation of 8R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC-MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8R-HODE (>95% R), 10R-HODE (>70% R), and 5S,8R- and 8R,11S-DiHODE with high stereoselectivity (>95%), whereas 8R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8R,11S-DiHODE.     

Influence of water activity, temperature and time on mycotoxins production on barley rootlets

AIMS: The objective of this study was to determine the ochratoxin (OT) and aflatoxin (AF) production by three strains of Aspergillus spp. under different water activities, temperature and incubation time on barley rootlets (BR). METHODS AND RESULTS: Aspergillus ochraceus and Aspergillus flavus were able to produce mycotoxins on BR. Aspergillus ochraceus produced ochratoxin A (OTA) at 0.80 water activity (a(w)), at 25 and 30 degrees C as optimal environmental conditions. The OTA production varies at different incubation days depending on a(w). Aflatoxin B(1) (AFB1) accumulation was obtained at 25 degrees C, at 0.80 and 0.95 a(w), after 14 and 21 incubation days respectively. Temperature was a critical factor influencing OTA and AFB(1) production. CONCLUSIONS: This study demonstrates that BR support OTA and AFB(1) production at relatively low water activity (0.80 a(w)) and high temperatures (25-30 degrees C). SIGNIFICANCE AND IMPACT OF THE STUDY: The study of ecophysiological parameters and their interactions would determine the prevailing environmental factors, which enhance the mycotoxin production on BR used as animal feed.     

Fungi and ochratoxin A detected in healthy grapes for wine production

AIMS: The mycoflora of healthy grapes (i.e. without visible symptoms of rot) for wine production in Portuguese wine-making regions was assessed and its potential for ochratoxin A (OTA) production evaluated. The OTA content of grapes was also determined. METHODS AND RESULTS: A total of 386 fungal strains were isolated by plating methods. The most frequent genera found in grapes were non-ochratoxigenic species: Cladosporium (28%), Penicillium (24%), Botrytis (13%) and Aspergillus (9%). Two OTA-producing strains were isolated, belonging to the species Aspergillus carbonarius and Aspergillus ochraceus. OTA was detected in three of four grape samples, up to 116 ng l(-1). CONCLUSIONS: OTA is being produced in healthy berries by Aspergillus species, namely A. carbonarius, at levels below the maximum recommended limit of 2,000 ng l(-1) in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The OTA concentration detected in healthy Portuguese grapes does not represent a risk to wine regarding the legal limit established.     

9- and 13-hydroxy-linoleic acid possess chemotactic activity for bovine and human polymorphonuclear leukocytes

The presence of polymorphonuclear leukocytes (PMNs) within the airways is a characteristic feature of a variety of lung diseases. Pulmonary alveolar macrophages (PAMs) and epithelial cells release many different factors which contribute to the recruitment of inflammatory cells into infected airways. PAMs and tracheal epithelial cells are able to produce linoleic acid metabolites (9-HODE and 13-HODE) besides arachidonic acid metabolites. The objective of the present study was to determine whether 9-HODE and 13-HODE possess chemotactic activity for isolated PMNs. It was found that 9-HODE and 13-HODE induced a chemotactic response of both human and bovine PMNs in vitro. The HODEs evoked chemotaxis with a linear dose response from 10(-10) to 10(-6) M to the same extent as the arachidonic acid metabolite 15-HETE. At 10(-8) M, 9-HODE and 13-HODE were approximately half as potent in inducing chemotaxis as compared to LTB4.     

Improved enantioselective analysis of polyunsaturated hydroxy fatty acids in psoriatic skin scales using high-performance liquid chromatography

Enantioselective analysis is used as a valuable tool for determining the biological origin of chiral derivatives of arachidonic, 11,14-eicosadienoic and linoleic acid in psoriatic skin scales and for clarifying their role in pathogenesis. This paper reports on a simple and rapid enantioselective determination (without any derivatization) of the fatty acid derivatives 13(R,S)-hydroxyoctadecadienoic acid [13(R,S)-HODE], 9(R,S)-hydroxyoctadecadienoic acid [9(R,S)-HODE] and 12(R,S)-hydroxyeicosatetraenoic acid [12(R,S)-HETE], using high-performance liquid chromatography (HPLC) with Chiralpak AD as the chiral selector and electrospray ionisation mass spectrometry (ESI-MS). The enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE in psoriatic skin scales of untreated patients (untreated during the last 4 weeks before sampling) was evaluated in comparison to psoriatic skin scales of patients underlying systemic treatment. The enantiomeric distribution of 12-HETE and 9-HODE showed no remarkable differences, whilst samples of patients under systemic treatment exhibited a lower predominance of 13(S)-HODE than samples of untreated patients. Furthermore, the effect of UVB phototherapy on the enantiomeric distribution of 12-HETE, 9-HODE and 13-HODE was studied and a semiquantitation of these compounds in psoriatic skin scales performed. The detected amounts of 9-HODE in samples of untreated patients were remarkably lower than those in samples of patients underlying systemic treatment. In the case of UVB phototherapy, no influence on the enantiomeric distribution could be observed.     

Triglyceride-rich lipoprotein lipolysis releases neutral and oxidized FFAs that induce endothelial cell inflammation

Triglyceride-rich lipoprotein (TGRL) lipolysis products provide a pro-inflammatory stimulus that can alter endothelial barrier function. To probe the mechanism of this lipolysis-induced event, we evaluated the pro-inflammatory potential of lipid classes derived from human postprandial TGRL by lipoprotein lipase (LpL). Incubation of TGRL with LpL for 30 min increased the saturated and unsaturated FFA content of the incubation solutions significantly. Furthermore, concentrations of the hydroxylated linoleates 9-hydroxy ocatadecadienoic acid (9-HODE) and 13-HODE were elevated by LpL lipolysis, more than other measured oxylipids. The FFA fractions elicited pro-inflammatory responses inducing TNFalpha and intracellular adhesion molecule expression and reactive oxygen species (ROS) production in human aortic endothelial cells (HAECs). The FFA-mediated increase in ROS was blocked by both the cytochrome P450 2C9 inhibitor sulfaphenazole and NADPH oxidase inhibitors. Compared with linoleate, 13-HODE was found to be a more potent inducer of ROS production in HAECs, an activity that was insensitive to both NADPH oxidase and cytochrome P450 inhibitors. Therefore, although the oxidative metabolism of FFA in endothelial cells can produce inflammatory responses, TGRL lipolysis can also release preformed mediators of oxidative stress (e.g., HODEs) that may influence endothelial cell function in vivo by stimulating intracellular ROS production.     

Large-Scale Transformation of Steroids by Fungal Spores

Spores of Aspergillus ochraceus and Septomyxa affinis were produced on a large scale by surface sporulation on moist wheat bran and barley. 11alpha-Hydroxylation of progesterone and Reichstein's compound S by spores of A. ochraceus and 1-dehydrogenation of compound S by spores of S. affinis were carried out in 5-liter fermentors. It was shown that, above a certain minimum, increase in aeration and agitation did not significantly affect steroid conversion. The industrial feasibility of the spore process was further demonstrated by 11alpha-hydroxylation of 6alpha-fluoro-16alpha,17alpha-dihydroxypregn-4-ene-3,20-dione in a modified 200-gal stainless-steel vessel with spores of A. ochraceus. Strict aseptic conditions are not necessary, either during harvesting of spores or during steroid transformation.     

Oxidative metabolism of linoleic acid by human leukocytes

Upon incubation with human leukocytes, [1-14C] linoleic acid is almost exclusively transformed into 13-hydroxy-9Z, 11E-octadecadienoic acid (13-HODE) if the linoleic acid concentration is lower than 50 microM. Identification of 13-HODE was done by GLC-MS at the level of its methyl ester, trimethylsilyl ether and by comparison with authentic 13-HODE in two different HPLC systems. Analysis of the products by chiral phase HPLC shows that 13(S)-hydroxy-9Z, 11E-octadecadienoic acid is by far the major metabolite formed by human leukocytes. Comparison of reactions performed with intact or lyzed cells suggests that the formation of 13(S)-HODE by human leukocytes occurs in two steps, a dioxygenation catalyzed by a 15-lipoxygenase and a reduction of intermediate 13-HPODE by a glutathione-dependent peroxidase.