黄曲霉群菌种产生黄曲霉毒素B_1的调查研究

本文对来自我国20个省、市、自治区的不同基物上分离的和中国科学院微生物研究所菌种保藏室以及其他单位提供的黄曲霉群菌种,经随机选取82株进行了黄曲霉毒素B_1的测定,证明在测试的9个已知分类群中产生黄曲霉毒素B_1的菌种只限于寄生曲霉和黄曲霉,另外4株种名未定者也能产生此种毒素。在黄曲霉中产毒菌株约占30％(28.3％),其在GAN(葡萄糖硝酸铵)和大米培养基中的黄曲霉毒素B_1的最高产量分别为133,333.3和160,000.0ppb。总的来说,大体上可以反映在我国一般基物上黄曲霉产毒菌株存在的现状。在实验过程中,还对黄曲霉群菌种在GAN和大米培养基中黄曲霉毒素B_1的产量和产毒菌株数作了比较。发现在大米培养基中黄曲霉毒素B_1的产量高于GAN,而且测试的黄曲霉产毒菌株在这两种培养基中均各有不能产毒的菌株,因此,在测定产毒菌株时,若仅采用其中一种产毒培养基,往往会有漏掉产毒菌株的可能性。     

传统发酵豆瓣中产毒黄曲霉高效拮抗菌的筛选

从自然发酵的豆瓣中筛选出对产毒黄曲霉菌的生长及其毒素合成均有抑制作用的细菌, 在蚕豆天然培养基(BAM)上利用菌落对峙实验初筛和滤纸片复筛得到1株有较高抑制产毒黄曲霉活性的菌株L4。对L4进行形态学、生理生化特征及16S rRNA序列同源性分析, 鉴定此菌株为枯草芽孢杆菌(Bacillus subtilis)。在抑制黄曲霉生长和黄曲霉毒素B1 (AFB1)合成的研究中表明, 在L4与黄曲霉菌共同培养15 d后, 黄曲霉菌丝产量和黄曲霉毒素B1 产量均比黄曲霉单独培养时显著降低(P < 0.01), AFB1合成受到明显抑制, 抑制率达93.7%。当黄曲霉孢子液与L4发酵上清液1: 1 (V/V)混合后接种在玉米粒上时, 黄曲霉在玉米上的生长和孢子萌发均得到完全抑制。     

黄曲霉和米曲霉的多相鉴定方法

【背景】黄曲霉(Aspergillus flavus)和米曲霉(Aspergillus oryzae)形态特征相近,基因组高度相似,较难区分。【目的】旨在总结一套准确鉴别二者的分类方法。【方法】利用22株标准菌株对传统形态学、产毒培养基、酶联免疫毒素检测、系统发育分析、产毒基因检测等5种鉴别方法分别进行验证。【结果】各鉴定方法的结果存在异同,单一的鉴定方法容易出现假阴性或假阳性结果。【结论】利用单一方法区分黄曲霉和米曲霉具有潜在风险,多相鉴定方法可以准确鉴别二者。     

黄曲霉毒素生物防控菌的筛选及鉴定

筛选黄曲霉毒素生物防控菌,为黄曲霉毒素的生物防控提供支持。以花生原产地土壤为材料,采用牛津杯法筛选所需菌株。对筛选出的拮抗菌株进行抑制产毒曲霉菌株的生长、产孢、降解黄曲霉毒素实验。筛选出2株黄曲霉毒素生防细菌,编号21-1-2、17-3,经鉴定,拮抗菌21-1-2为枯草芽胞杆菌,拮抗菌17-3为地衣芽胞杆菌。分别对拮抗菌对曲霉孢子萌发的抑制、抑制黄曲霉的生长和菌丝延长以及减少黄曲霉毒素的产生、对黄曲霉毒素的分解作用等几个方面进行研究,结果表明,拮抗菌可以明显抑制产毒曲霉孢子的萌发、生长、菌丝的延长,减少黄曲霉毒素的产生以及分解黄曲霉毒素。     

曲霉属黄绿组的一个产毒新种

本文报道曲霉属黄绿组(通常称黄曲霉群)的一个产毒新种肇庆曲霉(Aspergillus zhaoqingensis sp.nov.)。该菌分离广东肇庆土壤,在形态上近于米曲霉(A.oryzae),但作为该组的关键分类特征分生孢子纹饰很不相同:本种的分生孢子明显的粗疏粗糙至具不规则的脊状突起而米曲霉则为光滑或稍粗糙。本种能产生黄曲霉毒素B_1而米曲霉则不产生。本菌亦不同于组内其它菌种。文中对与黄曲霉密切相关诸种也作了简短的讨论。     

黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生相关联

为探讨黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生的关系,收集黄曲霉菌、米曲霉菌和寄生曲霉菌若干株。在有利于黄曲霉毒素产生的条件下培养后,提取各菌株的总RNA,RT-PCR法检测aflR基因的mRNA表达水平;并应用ELISA法检测各菌株产生黄曲霉毒素B1的情况。提取各菌株的基因组DNA,PCR扩增aflR基因启动子序列并测序。应用基因分析软件将不产毒素的黄曲霉菌与产毒黄曲霉菌的aflR基因启动子序列进行比较,找出不产毒菌株aflR基因启动子序列的变异位点。ELISA法和RT-PCR法结果表明,产毒的黄曲霉菌菌株均有明显的aflR基因转录,而在2株不产毒的黄曲霉菌菌株中,一株aflR基因无转录,另一株仅有较低水平的转录。序列比较结果表明,不产毒黄曲霉菌菌株的aflR基因启动子序列存在如下共同变异位点:-90、-236、-253、-262、-282位。米曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均为阴性,并且其aflR基因启动子序列中存在与上述不产毒黄曲霉菌菌株相同的变异位点。寄生曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均呈阳性,并且其aflR基因启动子序列的上述5个位点与产毒黄曲霉菌完全一致。在不产毒素的黄曲霉菌aflR基因启动子序列中发现了5个共同变异位点,实验结果提示这些变异位点可能与黄曲霉毒素的产生有关。     

黄曲霉毒素对费氏弧菌发光的影响

[目的]探讨黄曲霉毒素对一种发光细菌——费氏弧菌发光的抑制效应.[方法]黄曲霉毒素或产黄曲霉毒素的菌株培养液对费氏弧菌进行处理后,利用多功能酶标仪检测费氏弧菌的发光强度,研究黄曲霉毒素对费氏弧菌发光的影响.[结果]黄曲霉毒素浓度的对数值与费氏弧菌发光的抑制率呈线性关系,依据所得的回归方程可以快速准确地检测不同微生物产毒素的情况:6株不同来源的黄曲霉菌株均能够产毒素,以黄曲霉毒素含量表示的毒素量在14.94 - 46.45mg/L之间,1株米曲霉不产毒素.[结论]费氏弧菌发光强度的改变可以较准确地反映微生物产毒素的能力,尤其是微生物产黄曲霉毒素的能力,为在工农业生产中快速检测黄曲霉毒素提供了新的线索,有望发展成为一种检测黄曲霉毒素的新技术.     

黄曲霉产毒菌株的快速筛选实验

黄曲霉产毒菌株的常规鉴定方法多采用待检菌株经产毒培养后用薄层层析法测定其产毒能力。这种方法需要较完善的实验条件,操作也较为繁琐。1975年以来,为鉴定从谷物、食品和饲料中分离出的大批黄曲霉菌株的产毒性能,我们曾参考了     

黄曲霉毒素B1脱毒菌株LAB-10的分离、鉴定及降解能力分析

【目的】从乳酸菌群含量丰富的动物肠道中筛选具有黄曲霉毒素B1(AFB1)脱毒应用前景的乳酸菌种。【方法】通过设计脱毒乳酸菌种特异性富集分离培养基,从肉鸡肠道粪便中筛选具有AFB1脱毒能力的微生物菌种,对脱毒菌种进行脱毒机理初步分析,并通过形态学、生理生化和系统发育学方法,鉴定脱毒菌种的系统分类学地位。【结果】脱毒菌株LAB-10经脱毒机理及降解能力测试分析,初步确定其AFB1的脱毒机理为生物降解作用,菌株LAB-10对14μg/L浓度AFB1在48 h的降解率为63.4%。形态学、生理生化及系统发育学研究结果表明,菌株LAB-10系统分类学地位为乳酸杆菌属的发酵乳杆菌。【结论】发酵乳杆菌LAB-10属于益生菌群,生物安全性高,该菌株在黄曲霉毒素降解测试培养基中显示出显著的AFB1降解能力,具有一定的应用潜力。     

可污染食品及饲料的产黄曲霉毒素真菌的多重PCR检测

根据黄曲霉毒素生化途径中的关键调控基因aflR、omt-1和ver-1的序列以及真菌共有的5.8S rDNA的ITS序列分别设计ApaF/ApaR、OmtF/OmtR、VerF/VerR及ITS1/ITS4四对引物,研究建立产黄曲霉毒素真菌及其潜在饲料或食品污染的多重PCR快速灵敏检测体系。PCR扩增的4个DNA片段中,1032bp、797bp和600bp与基因库中对应基因或DNA序列的同源性达99%以上,仅452bp片段与对应基因ver-1的同源性为98%。通过优化主要影响因子,建立了快速检测产黄曲霉毒素真菌的单管多重PCR反应体系,并用于6种曲霉和1种青霉DNA样品的检测。结果显示,上述4个片段均平行地清晰出现在2株黄曲霉Aspergillus flavus和1株寄生曲霉A.parasiticus的DNA样品中,而其余菌种只检测到ITS片段,说明检测特异性很好。灵敏性分析表明,多重PCR检测的保守灵敏度为1ng/μL样品DNA,所有目标片段的条带均很清晰;即使DNA浓度降至0.1ng/μL,除aflR之外的所有条带也可分辨。     

Fungicidal and anti-aflatoxigenic effects of the essential oil of Cymbopogon citratus (DC.) Stapf. (lemongrass) against Aspergillus flavus Link. isolated from stored rice

AIMS: To develop a natural fungicide against aflatoxigenic fungi, to protect stored rice, using the essential oil of lemongrass. METHODS AND RESULTS: Aspergillus flavus Link. was isolated from stored rice and identified as an aflatoxigenic strain. Lemongrass oil was tested against A. flavus and the test oil was fungistatic and fungicidal against the test pathogen at 0.6 and 1.0 mg ml(-1), respectively. Aflatoxin production was completely inhibited at 0.1 mg ml(-1). The results obtained from the thin layer chromatographic bioassay and gas chromatography indicated citral a and b as the fungicidal constituents in lemongrass oil. During the fumigant toxicity assay of lemongrass oil, the sporulation and the mycelial growth of the test pathogen were inhibited at the concentrations of 2.80 and 3.46 mg ml(-1), respectively. CONCLUSION: Lemongrass oil could be used to manage aflatoxin formation and fungal growth of A. flavus in stored rice. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, fungicides are not used to control fungal pests or mycotoxin production on stored rice. Rice treated with the essential oil of lemongrass could be used to manage fungal pests as well as the insect pests in stored rice. The essential oil is chemically safe and acceptable to consumers, as synthetic chemical fungicides can cause adverse health effects to consumers.     

Different media and methodologies for the detection of aflatoxin production by Aspergillus flavus strains isolated from trout feed

We have studied the aflatoxin producing capacity of 41 Aspergillus flavus strains isolated from the mycoflora present of natural media (wheat, rice and mixed feed) synthetic medium (Aflatoxin Producing Ability Medium) and semisynthetic media (Coconut Agar Medium and Glucose Yeast Extract Agar) were compared. Aflatoxins were analysed on days 4 and 8 post-inoculation under an incubation temperature of 28 °C. A total of 30 strains (75.7%) were producers on natural media as detected by Thin Layer Chromatography: 23 strains on wheat, 27 on rice and 12 on mixed feed. The results by qualitative flourescence tests on synthetic and semisynthetic media were: 3 strains positive on Coconut Agar Medium (CAM) 1 on Glucose Yeast Extract Agar (GY + Agar) and none on Aflatoxin Producing Ability Medium (APA).     

产氨基酰化酶米曲霉高产菌株的选育

采用亚硝基瓜抑菌圈法对米曲霉菌株3042,AF92011,AF93018,AF93020,AF93022,AF93332进行诱变处理,从诱变菌株中筛选到几个高产氨基酰化酶菌株(3042-5,AF93020-2,AF93020-6和AF93022-5),对DL-蛋氨酸的拆分活力分别比出发菌株提高34.1%、157.6%、152.4%和145.4%,其中米曲霉菌株3042的诱变菌株3042-5最高酶活力达203.1U/ml     

Stimulation by Hyphopichia burtonii and Bacillus amyloliquefaciens of aflatoxin production by Aspergillus flavus in irradiated maize and rice grains

Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens. Aflatoxin production by A. flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees C). Both H. burtonii and B. amyloliquefaciens markedly stimulated growth and aflatoxin production by A. flavus on cracked maize, especially at 25 degrees C and 0.95 and 0.98 aw. No aflatoxin was detected in pure cultures of A. flavus on cracked rice after 12 days of incubation at 25 degrees C, but some was produced by mixed cultures at 16 degrees C and 0.98 aw. The morphological interactions among A. flavus, H. burtonii, and B. amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions.     

Stimulation by Hyphopichia burtonii and Bacillus amyloliquefaciens of aflatoxin production by Aspergillus flavus in irradiated maize and rice grains.

Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens. Aflatoxin production by A. flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees C). Both H. burtonii and B. amyloliquefaciens markedly stimulated growth and aflatoxin production by A. flavus on cracked maize, especially at 25 degrees C and 0.95 and 0.98 aw. No aflatoxin was detected in pure cultures of A. flavus on cracked rice after 12 days of incubation at 25 degrees C, but some was produced by mixed cultures at 16 degrees C and 0.98 aw. The morphological interactions among A. flavus, H. burtonii, and B. amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions.     

Cyclopiazonic acid production by submerged cultures of Penicillium and Aspergillus strains

Abstract 62 isolates of Penicillium and Aspergillus were screened for cyclopiazonic acid (CPA) production by surface and submerged culture on different media. The production of this mycotoxin was restricted to Penicillium camembertii group II (and its domesticated form P. camembertii ), P. griseofulvum , and Aspergillus flavus (and its domesticated form A. oryzae ). The best yield of CPA was obtained by a strain of P. griseofulvum , but several strains of P. camembertii group II were also good producers. Propionic acid (500 and 1000 mg/l medium) did not enhance the production of CPA. The best yields of CPA were obtained in submerged culture, but in some cases growth and CPA production only occured in surface culture. A simplified procedure for isolation of CPA is described.     

Soil fungi of some low-altitude desert cotton fields and ability of their extracts to inhibit Aspergillus flavus

Soil is presumed to be a major source of inoculum for Aspergillus flavus which contaminates cottonseed and produces the potent carcinogen, aflatoxin. Little is known about the mycoflora of the low desert soils of cotton fields where aflatoxin is a chronic problem. In this study, soils from cotton fields in southwestern Arizona and southeastern California were assayed for filamentous fungi. Forty-two taxa, predominantly in the genera Aspergillus, Penicillium and Fusarium, were isolated. To determine whether or not compounds produced by these fungi could be potential inhibitors of A. flavus, extracts of strains of each taxon were tested for their ability to inhibit growth of A. flavus. Twelve taxa produced compounds inhibitory to A. flavus, including several strains of Fusarium solani, Penicillium vinaceum and Aspergillus auricomus. This revised version was published online in June 2006 with corrections to the Cover Date.