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。     

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。     

黄曲霉毒素B1降解菌的筛选及在玉米贮藏中的应用

【目的】本研究分离筛选出一株对黄曲霉既有抑制作用又能降解其毒素的拮抗细菌菌株,并将其应用于玉米中的黄曲霉污染防治研究。【方法】试验通过平板筛选法结合玉米活体筛选法对黄曲霉毒素B1 (AFB1)的拮抗细菌进行初筛,以AFB1的降解率和抑制率为指标进行复筛。【结果】分离到的菌株对黄曲霉菌的抑制率为79.20%,对1μg/mL的黄曲霉毒素B1的降解率为68.39%。贮藏期玉米含水量在15%–30%时,该菌株对黄曲霉污染的抑制率与玉米含水量成反比,即玉米含水量在15%时其抑制率达92.46%,玉米含水量在30%时其抑制率为19.41%。玉米含水量在28%时,菌株对黄曲霉污染玉米的防治效果达到36.39%。【结论】筛选出的拮抗菌株为枯草芽孢杆菌,该菌株不仅对黄曲霉菌有抑制作用,而且能减少AFB1对玉米的污染。     

黄曲霉分生孢子色素合成基因pks1的鉴定及其对生长发育和侵染性的影响

【目的】分生孢子色素是真菌细胞壁的重要成分,对真菌的生长发育极为重要,并有助于真菌抵御各种环境胁迫。本研究鉴定了黄曲霉分生孢子色素合成基因,并研究了分生孢子色素对黄曲霉生长发育及其对抗紫外照射和侵染能力的影响。【方法】通过已知真菌孢子色素合成基因蛋白序列同源比对确定了黄曲霉分生孢子色素合成基因及其所在的基因簇,利用同源重组策略对目标基因进行敲除,获得了该色素合成基因缺失的突变菌株,并研究该基因敲除后对表型、产孢、菌核形成、黄曲霉毒素产生、抗紫外照射和侵染性等影响。【结果】与野生型菌株相比,黄曲霉pks1基因缺失菌株的分生孢子颜色变为白色,生长速度、孢子产量、菌核形成和黄曲霉毒素B_1的产生均没有显著性变化,但该基因的缺失导致孢子对紫外线照射的抵御能力明显减弱,降低了黄曲霉对玉米和花生种子的侵染能力。【结论】pks1(AFLA_006170)基因是黄曲霉分生孢子色素合成的关键基因,影响黄曲霉分生孢子对紫外线照射等不利环境因子的抵抗能力和对粮食种子的侵染能力。     

微嗜酸寡养单胞菌中磷酸吡哆胺氧化酶基因pnpox生物学功能研究北大核心

【目的】研究微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila)CW117中磷酸吡哆胺氧化酶基因pnpox(phosphopyridoxamine oxidase,pnpox)在维生素B6(VB6)合成中的贡献及对黄曲霉毒素B1(aflatoxin B1,AFB1)的降解活性。【方法】采用基因插入突变方式,对菌株CW117中磷酸吡哆胺氧化酶基因pnpox进行突变,得到突变菌株。通过高效液相色谱法(high performance liquid chromatography,HPLC)检测突变株对AFB1的降解活性,以及突变株中吡哆醇和吡哆醛的合成情况,确定基因pnpox在寡养单胞菌体内VB6合成中的贡献和黄曲霉毒素降解代谢作用。【结果】成功构建了磷酸吡哆胺氧化酶基因突变子pnpox::pK19mobΩ2HMB,突变子吡哆醛的合成量较野生型菌株显著减少,吡哆醇合成量与野生型菌株无显著性差异;同时,突变子与野生型株CW117对AFB1的降解活性未发现显著性差异。【结论】菌株CW117中磷酸吡哆胺氧化酶在吡哆醛合成的过程中起着重要作用,该基因突变会导致VB6的严重缺乏,影响寡养单胞菌正常生长,但该基因对CW117降解黄曲霉毒素无显著性贡献。     

微嗜酸寡养单胞菌中磷酸吡哆胺氧化酶基因pnpox生物学功能研究

【目的】研究微嗜酸寡养单胞菌(Stenotrophomonas acidaminiphila)CW117中磷酸吡哆胺氧化酶基因pnpox(phosphopyridoxamine oxidase,pnpox)在维生素B6(VB6)合成中的贡献及对黄曲霉毒素B1(aflatoxin B1,AFB1)的降解活性。【方法】采用基因插入突变方式,对菌株CW117中磷酸吡哆胺氧化酶基因pnpox进行突变,得到突变菌株。通过高效液相色谱法(high performance liquid chromatography,HPLC)检测突变株对AFB1的降解活性,以及突变株中吡哆醇和吡哆醛的合成情况,确定基因pnpox在寡养单胞菌体内VB6合成中的贡献和黄曲霉毒素降解代谢作用。【结果】成功构建了磷酸吡哆胺氧化酶基因突变子pnpox::pK19mobΩ2HMB,突变子吡哆醛的合成量较野生型菌株显著减少,吡哆醇合成量与野生型菌株无显著性差异;同时,突变子与野生型株CW117对AFB1的降解活性未发现显著性差异。【结论】菌株CW117中磷酸吡哆胺氧化酶在吡哆醛合成的过程中起着重要作用,该基因突变会导致VB6的严重缺乏,影响寡养单胞菌正常生长,但该基因对CW117降解黄曲霉毒素无显著性贡献。     

黄曲霉素合成相关基因表达与环境因素的关系

简单介绍了黄曲霉毒素的发现、分布、危害和范围,详细叙述了黄曲霉毒素生物合成中相关基因的表达与调控,概述了黄曲霉毒素合成中的关键基因、酶和调控因子重要性,分析了影响黄曲霉毒素合成的环境因素。不仅在基础理论上对黄曲霉毒素合成机理进行了深入探讨,而且在应用研究上为减少粮食和食品受到重金属污染和黄曲霉毒素危害提供了新的思路。     

黄曲霉毒素生物合成的遗传调控研究进展

黄曲霉毒素是一类具有较强毒性和致癌力的次级代谢产物,在小麦、水稻、玉米和花生等多种粮食、油料、饲料和食品中检出率均比较高。因此,黄曲霉毒素不仅给人和动物的健康造成极其严重的威胁,而且也给食品和饲料等行业造成了巨大的经济损失。黄曲霉毒素主要由黄曲霉和寄生曲霉产生。自上个世纪60年代首次发现黄曲霉毒素以来,研究者在黄曲霉毒素合成途径、降解、合成机制和致病机理等方面做了大量研究。本文主要综述近年来国内外以黄曲霉为对象的黄曲霉毒素合成的遗传调控机制研究进展。从转录调控、蛋白翻译后修饰、信号转导途径、参与生长发育和形态建成的蛋白和其他酶等方面对黄曲霉毒素合成机制展开综述,为今后进一步深入系统研究黄曲霉毒素合成机制奠定基础,同时为制定防治黄曲霉及其毒素的策略提供理论基础。     

动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌的分离及安全性

【背景】芽孢杆菌是仅次于乳酸菌常用于微生态制剂中的菌种,然而部分芽孢杆菌微生态制剂规范不严,应用存在安全隐患。【目的】调查我国在售动物用芽孢杆菌微生态制剂中蜡样芽孢杆菌携带情况,揭示蜡样芽孢杆菌应用的潜在风险。【方法】对微生态制剂预处理,选择性筛选分离蜡样芽孢杆菌,通过全基因组测序测绘细菌毒素基因谱与耐药基因谱,细胞计数试剂盒-8法测定菌株对细胞的毒性,利用微量肉汤稀释法确定菌株耐药值。【结果】从50份微生态制剂产品中筛选分离得到23株蜡样芽孢杆菌群细菌,它们对氨苄西林、林可霉素和泰妙菌素3种抗生素均耐药,主要毒力基因nhe、hbl、cytK、ces的检出率分别为100%、30%、39%和4%,分离株均有溶血性且39%菌株产生热稳定毒素,不同菌株对非洲绿猴肾细胞呈现出不同程度的毒性。【结论】微生态制剂来源的蜡样芽孢杆菌毒性与耐药性严重,携带毒素基因与耐药基因广泛,多株菌株呈高细胞毒性且产生热稳定毒素。芽孢杆菌微生态制剂存在安全性问题,应加强对蜡样芽孢杆菌的质量安全监管力度,规范微生态制剂的市场秩序,杜绝安全隐患。     

黄曲霉群菌种产生黄曲霉毒素B1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。     

Description of a Novel Actinobacterium Microbacterium assamensis sp. nov., Isolated from Water Sample Collected from the River Brahmaputra, Assam, India

A Gram-positive, yellow pigmented actinobacterium, strain S2-48(T) was isolated from water sample collected from the river Brahmaputra, Assam, India and subjected to a polyphasic taxonomic study. Most of the physiological and biochemical properties, major fatty acids (C(15:0) Anteiso, iso C(16:0) and C(17:0) Anteiso), estimated DNA G+C content (70.2 mol%) and 16S rRNA gene sequence analysis showed that strain S2-48(T) belonged to the genus Microbacterium. Strain S2-48(T) exhibited highest 16S rRNA gene sequence similarity with Microbacterium testaceum (97.0%); however, the DNA-DNA relatedness value between strain S2-48(T) and M. testaceum was 9.1%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain S2-48(T) should be classified within the genus Microbacterium as a novel species, for which the name Microbacterium assamensis is proposed. The type strain is S2-48(T) (=MTCC 10486(T) = DSM 23998(T)).     

Microbe‐mediated control of Aspergillus flavus in stored rice grains with a focus on aflatoxin inhibition and biodegradation

Biological control of mycotoxigenic fungi using antagonistic microbes is a promising alternative to agricultural chemicals for postharvest storage. In this study, we evaluated rice‐derived bacterial strains to identify biocontrol agents to inhibit Aspergillus flavus in stored rice grains. Consequently, we obtained three potential biocontrol strains (Microbacterium testaceum KU313, Bacillus megaterium KU143 and Pseudomonas protegens AS15) from 26 tested strains that were prescreened from the 460 strains isolated from rice grains. The three selected strains proved to be effective biocontrol agents showing antifungal activity against A. flavus and good colonisation ability on rice grains, along with inhibition of the fungal growth and aflatoxin production. In particular, P. protegens AS15 greatly inhibited the aflatoxins produced by A. flavus on rice grains to 8.68 (percent aflatoxin reduction relative to control = 82.9%) and 18.05 (68.3 %) ng g^?1 dry weight of rice grains, compared with the 50.89 and 56.97 ng g^?1 dry weight of rice grains of the MgSO₄ control at 1 and 2 weeks after inoculation, respectively. In addition, strain AS15 had a significant ability to not only degrade aflatoxin B₁ (the most harmful aflatoxin), but also utilise the toxin for bacterial growth in a nutrient‐deficient medium. Therefore, the selected bacterial strains could be environmentally sound alternatives for the management of A. flavus and aflatoxin production by reducing the fungal damage to stored rice grains. This would also reduce the human and animal health hazards associated with the consumption of fungus‐contaminated rice grains. To our knowledge, this is the first report of the potential of the bacterial species M. testaceum and P. protegens as biocontrol agents for controlling aflatoxigenic A. flavus on stored rice grains.     

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B₁ complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B₁ remained bound. Nonviable bacteria retained the highest amount of aflatoxin B₁. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B₁ from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B₁ to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B₁. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B₁ released. Binding of aflatoxin B₁ appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.     

The influence of the irradiation regime upon mycotoxins production under experimental conditions

The paper handles the problem of the inactivation of the toxinogenic strain Aspergillus flavus following the application of gamma radiation to wheat. The amount of the applied dose and of the absorbed dose of ionizing radiation upon the inhibition of mycelium growth and toxin production were defined. The aflatoxin B1 was determined by extracting in chloroform and developed on Silufol R within the choroform; aceton system. The applied doses of gamma radiation (3-30 kGy) have show that the absorbed dose does not inhibit aflatoxin production. By combining the action of gamma radiation with humidity of the wheat (humidity 13-15%; 25% irradiation 6 kGy) an inactivation was reached. With the help of toxicologico-genetical tests (the Dominant Lethal Mutations Test, the Three Generations Test) the influence was traced of contaminated, irradiated substrates upon the health of experimental animals. It follows from the results obtained that in long-term feeding with contaminated wheat irradiated by gamma rays no positive mutagenic activity has been recorded. It allows to presume that wheat of humidity of 25% contaminated by a weakly toxigenic strain Aspergillus flavus irradiated by a dose of 6 kGy, and wheat of a humidity of 13-15%, contaminated by a strongly toxinogenic strain of Aspergillus flavus, irradiated by a dose of 6 kGy, are no genetic risk for white rats.     

Genome Sequence of Microbacterium yannicii, a Bacterium Isolated from a Cystic Fibrosis Patient

Microbacterium yannicii is a Gram-positive, aerobic, yellow-pigmented, rod-shaped, nonmotile, oxidase-negative, and catalase-positive bacterium isolated on Columbia colistin-nalidixic acid (CNA) agar with 5% sheep blood from the sputum of a cystic fibrosis patient. The present study reports the draft genome of a Microbacterium yannicii strain.     

Regulation of aflatoxin biosynthesis. 1 Comparative study of mycelial composition and glycolysis in aflatoxigen and nonaflatoxigenic strains.

A comparative biochemical study of an aflatoxigenic strain Aspergillus parasiticus NRRL 3240 and a nonaflatoxigenic strain A. flavus NRRL 3237 was carried out in order to have a better idea of regulation of aflatoxin biosynthesis. The results obtained revealed continuous primary metabolic activity (protein synthesis) in the nonaflatoxigenic strain while the aflatoxigenic stain showed inhibition of protein and nucleic acid synthesis. The aflatoxigenic strain showed higher levels of oxygen uptake, RNA, NAD, FMN and activities of glycolytic enzymes. Furthermore, it had lower of lipids and reduced activity of glucose-6-phosphate dehydrogenase, which is a source for NADPH. The differences observed have been discussed in relation to aflatoxin biosynthesis and its regulation.     

Regulation of aflatoxin production by Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation

To elucidate Ca(2+)-mediated regulation of aflatoxin production, the status of Ca(2+)/calmodulin-dependent protein phosphorylation and dephosphorylation was investigated employing toxigenic and non-toxigenic strains of Aspergillus parasiticus. Incubation of cytoplasmic extracts with [gamma-(32)P]ATP followed by SDS-PAGE and autoradiography revealed total absence of protein phosphorylation during periods corresponding to aflatoxin production in the toxigenic strain (NRRL 2999). In contrast, protein phosphorylation was unaffected in the non-toxigenic strain (SRRC 255). Aflatoxin production in the toxigenic strain was also accompanied by enhanced (26-fold) activity of calcineurin (calmodulin-dependent protein phosphatase 2B) concomitant with a lowered (6-fold) activity of calmodulin-dependent protein kinase. In addition, the in vitro activity of Ca(2+)/calmodulin-dependent protein kinase was susceptible to dose-dependent inhibition by aflatoxin. Since calcineurin remains active in the absence of phosphorylation by calmodulin-dependent protein kinase, it is suggested that calcineurin-mediated dephosphorylation of regulatory enzymes ensures continued production of aflatoxins.