黑曲霉对黄曲霉生长、产毒及黄曲霉毒素B1的影响

目的研究黑曲霉对黄曲霉生长、产毒的抑制作用及对AFB1的降解作用。方法将黑曲霉分别与黄曲霉、AFB1共同培养,定期测定培养液pH、菌丝体干重、黄曲霉孢子数、AFB1含量。结果黑曲霉与黄曲霉混合培养时,黄曲霉孢子数、AFB1含量均比单独培养的低,2组之间差异有统计学意义(P<0.05),抑制率达到68.06%~91.52%;加入黑曲霉后,AFB1含量降低,实验组与对照组之间差异有统计学意义(P<0.05),降解率为46.19%。结论黑曲霉既能抑制黄曲霉生长、产毒,又能降解AFB1。     

饲喂不同浓度黄曲霉毒素B1饲料对异育银鲫成鱼的生长和毒素积累的影响

以含不同浓度黄曲霉毒素B1(AFB1)的配合饲料饲喂异育银鲫(Carassius auratus gibelio)成鱼56d,研究异育银鲫成鱼[(122.3±0.7)g]生长、生理反应、肝脏组织学变化、卵巢发育以及鱼体各组织中的AFB1的毒素积累状况。实验分为5个实验组,不同实验组饲料中AFB1含量分别为0、5、20、50、500μg/kg饲料(实测值分别为2.59、4.12、12.39、46.23、454.07μg/kg饲料),每个处理3个平行。在整个实验过程中各实验组均未表现出外部形态和行为异常,各组存活率均达到100%。各实验组异育银鲫成鱼终末体重、摄食率(FR)、特定生长率(SGR)和饲料效率(FE)均无显著差异。饲料AFB1水平对异育银鲫血清总胆固醇(TC)含量、血清谷丙转氨酶(GPT)、谷草转氨酶(GOT)和碱性磷酸酶(AKP)活性均无显著影响。各毒素组血清超氧化物岐化酶(SOD)活性与对照无显著差异。各毒素组肝脏和卵巢均未见明显的组织学病理变化。肌肉和性腺中的AFB1积累量低于FDA食品安全限定标准(5μg/kg)。肝胰脏中的AFB1积累和饲料中的AFB1水平呈对数关系。饲喂AFB1≥50μg/kg饲料使异育银鲫成鱼肝脏AFB1积累超过安全限量标准。结果表明,异育银鲫成鱼至少可耐受AFB1含量达500μg/kg饲料(实测值:454.07μg/kg饲料)56d。     

饲喂不同浓度黄曲霉毒素B1饲料对草鱼幼鱼生长和毒素积累的影响

以含不同浓度黄曲霉毒素B₁(AFB₁)(0、10、20、100、1000和5000 μg/kg饲料)的6种等氮等能(32.96%蛋白质, 14.55 kJ/g能量)配合饲料饲喂平均初始体质量为(2.90±0.16) g草鱼(Ctenopharyngodon idellus)幼鱼84d, 探讨AFB₁对草鱼幼鱼生长、肝胰脏和肾脏组织结构以及鱼体肌肉中的毒素积累的影响。实验分为6个实验组, 每组3个平行。结果表明, 在整个实验过程中各实验组幼鱼的行为均未表现出异常, 各组幼鱼的存活率、终末体重、摄食率、特定生长率、饲料效率、肝体比、脏体比均无显著差异。饲料AFB₁水平对草鱼幼鱼血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(AKP)、超氧化物岐化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性均无显著影响。各毒素组和对照组肝胰脏、肾脏组织学观察中未发现病理变化。摄食AFB₁≤1000 μg/kg的草鱼幼鱼肌肉中未检测出AFB₁残留, 仅在5000 μg/kg实验组中检测出肌肉中含有(1.21±0.18) μg/kg的AFB₁, 低于FDA食品安全限定标准。由此可见, 草鱼幼鱼至少可耐受AFB₁含量达5000 μg/kg饲料(实测值: 4979.2 μg/kg饲料) 84d。     

产黄曲霉毒素B1降解酶菌株的筛选鉴定及发酵条件优化

以香豆素为唯一碳源筛选到27株能高效降解黄曲霉毒素B1(AFB1)的微生物菌株.用高效液相色谱检测AFB1含量的方法进行AFB1降解酶活力测定.以不同菌株发酵上清液中AFB1降解酶活力高低为复筛条件,筛选到AFB1降解酶活力最高的一株菌并命名为HSD8.该菌株经形态学、生理生化及系统发育学方法鉴定为Sinomonas sp..筛选所得最优菌株的发酵上清液中酶活达443 U/mL,通过单因素试验对其产酶发酵条件进行优化,以提高酶活.优化所得最佳发酵条件为:装液量50 mL/250 mL,发酵周期48 h,初始pH 5.0,接种量8％,发酵温度37℃,摇床转速160r/min.在最佳发酵条件下,该菌株发酵上清液中酶活可达548 U/mL,比优化前提高23.7％.优选菌株HSD8在生物降解黄曲霉毒素B1方面具有应用潜力,值得进一步研究开发.     

饲喂不同浓度黄曲霉毒素B1饲料对花鳗鲡幼鱼生长、抗氧化能力和毒素积累的影响

以含不同浓度黄曲霉毒素B₁(AFB₁)(0、10、100和1000μg/kg饲料)的4种饲料饲喂平均初始体重为(6.41±0.10) g的花鳗鲡(Anguilla marmorata)幼鱼56d,探讨AFB₁对花鳗鲡幼鱼生长性能、抗氧化能力、肝脏组织结构及鱼体肌肉中的毒素积累的影响。结果表明,各实验组幼鱼均未表现出行为及体色的异常。1000μg/kg毒素组幼鱼的存活率、终末体重、摄食率、特定生长率和饲料效率显著低于对照组, 10μg/kg毒素组和100μg/kg毒素组与对照组无显著差异。10μg/kg毒素组幼鱼肝脏超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽S转移酶(GST)活性和丙二醛(MDA)含量与对照组无显著差异。饲料AFB₁含量≥100μg/kg显著影响花鳗鲡幼鱼肝脏的超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSHPx)和谷胱甘肽转移酶(GST)活性。对照组和10μg/kg毒素组幼鱼肝脏组织学观察未发现明显病理变化。1000μg/kg毒素...     

一株枯草芽孢杆菌降解黄曲霉毒素B1产物分析

为丰富生物降解黄曲霉毒素B1资源,研究菌株降解机制及产物毒性.通过液相色谱串联三重四级杆质谱仪(UPLC-MS/MS)及核磁共振氢谱(1H NMR)检测一株枯草芽孢杆菌WTX1降解黄曲霉毒素B1(aflatoxin B1,AFB1)产物,并将降解产物进行细胞毒理实验.菌株WTX1降解AFB1机制是破坏AFB1结构中的呋...     

黄曲霉毒素B1生物脱毒的研究进展

黄曲霉毒素是一组由黄曲霉、寄生曲霉等多种真茵产生的次级代谢产物,具有强烈的毒性,可以引起动物肝脏肿大、病变甚至癌变,对人和家畜的健康产生极大的威胁。本文简介了黄曲霉毒素B_1的分子结构、理化性质、污染现状,综述了黄曲霉毒素B_1生物脱毒方面及其应用的研究进展,重点讲述通过微生物降解黄曲霉毒素B_1的研究近况。     

黄曲霉毒素B1的免疫检测Ⅱ．抗体的产生及应用

将黄曲霉毒素B₁肟(AFB₁O)与牛血清白蛋白(BSA)的连接物,通过多点、多次免疫法注射免疫兔子。分析了抗体的产生进程、效价以及特异性。注射抗原后的第60天开始有较明显抗体产生,第120天达到高峰,维持15天左右后开始下降;抗体的ELISA效价高达1：30000;和黄曲霉毒素B₁(AFB₁)的结构类似物的竞争ELISA表明,抗体有很好的特异性。运用该抗体,以ELISA分析检测了几种农产品及饲料中污染AFB₁,的含量,并和薄层层析法的分析结构进行了比较,结果表明当AFB₁．的含量大于等于5ng／ml时。两者间有很好的相关性。     

羊源丁酸梭菌HDRyYB1发酵工艺的优化

【目的】目前,国内外鲜有关于羊源丁酸梭菌的报道。本课题选用羊源丁酸梭菌HDRy YB1为研究对象,对其发酵工艺进行优化,为该菌株作为饲料添加剂应用于畜牧业生产奠定基础。【方法】采用Plackett-Burman(PB)试验设计法和响应面法分析并优化显著影响HDRy YB1菌株发酵液中芽胞数的培养基成分。【结果】发酵培养基中的面粉浓度、鱼粉浓度和米粉浓度显著影响发酵液中的芽胞数,优化后的发酵培养基组分(质量体积比)为:面粉3.72%、鱼粉0.90%、米粉3.96%、酵母粉0.60%、Na Cl 0.19%、Mg SO4·7H2O 0.19%、KH2PO4 0.01%、Na HCO3 0.01%、Ca CO3 0.48%;培养参数为:37°C,初始p H为7.2-7.4,瓶装量100/250,接种量3%。在此条件下,HDRy YB1菌株发酵完全(18 h)的芽胞数为1.478×108 CFU/m L,是优化前的2.7倍。【结论】HDRy YB1菌株发酵培养基得到了优化,优化后的培养基可用于后期的扩大发酵试验,验证其在实践生产中的应用价值。     

食用菌对黄曲霉毒素B1的脱毒作用研究

目的 研究平菇、金针菇、黑木耳等七种食用菌对黄曲霉毒素B1（AFB1）的脱毒效果。方法 首先通过降解圈直径与菌落直径之比（Da/Dm）筛选出具有脱毒作用的株菌;然后通过AFB1残留量筛选出脱毒能力最强的菌株;最后将该菌株接种于含AFB1的玉米粉和大米中,考查其对粮食中AFB1的脱毒效果。结果 初筛发现平菇、黑木耳、金针菇对AFB1有较强脱毒作用,其Da/Dm分别为1.6±0.02、1.5±0.01、1.4±0.02;复筛发现黑木耳的脱毒能力最强,与AFB1共培养10 d后能清除88.16%的AFB1;进一步发现黑木耳对玉米粉和大米中AFB1有一定去除作用,清除率分别为62.4%和15.73%。结论 黑木耳对受AFB1污染的粮食作物有较好的脱毒作用,可用其控制食品与饲料中的AFB1。     

丁酸梭菌影响肠炎沙门氏菌对SPF小鼠致病性的作用

【目的】旨在探究丁酸梭菌影响肠炎沙门氏菌(CVCC3377)对SPF小鼠的致病性及对小鼠的保护作用。【方法】选用72只6周龄SPF级雌性C57BL/6小鼠,按照完全随机区组设计分为阴性对照(CON)、饲喂丁酸梭菌组(CB)、沙门氏菌感染组(SE)和丁酸梭菌保护组(CB+SE)4组,每组18只。CON组和SE组饲喂基础日粮和饮水,CB组和CB+SE组饲喂基础日粮和每天更换添加丁酸梭菌(1×10⁷CFU/mL)的饮水,连续饲喂一周;第8天进行肠炎沙门氏菌感染实验,分别对SE组和CB+SE组灌服0.2mL 1.1×10⁴CFU/只的肠炎沙门氏菌,同时对阴性对照组和丁酸梭菌组灌服0.2mL生理盐水。在感染后第6天处死小鼠并采集肝脏、脾脏和盲肠及内容物样品。采用H.E染色检测肝脏和脾脏组织的病理学变化,免疫组化检测脾脏组织中TLR4、MyD88蛋白的表达水平,荧光定量PCR检测盲肠组织中TNF-α和IL-6的表达水平,并通过高通量测序方法分析其对肠道菌群的影响。【结果】结果显示,丁酸梭菌能够缓解肠炎沙门氏菌感染引起的小鼠体重下降;H.E染色结果显示,丁...     

丁酸梭菌替代金霉素对肉鸡腿肌肉品质和抗氧化功能的影响

丁酸梭菌(Clostridium butyricum)是一种产丁酸等短链脂肪酸的有益菌,添加到饲料中可改善动物的生产性能、提高饲料利用率和机体的抗氧化功能.[目的]本文研究了丁酸梭菌替代金霉素添加到肉鸡日粮中,对肉鸡腿肌肉品质和抗氧化功能的影响.[方法]随机选取1日龄健康艾拔益加(AA)肉仔鸡360只,分为3组,每组8...     

24株鸡源丁酸梭菌的分离鉴定及耐药基因与毒力基因携带情况

[目的]旨在对鸡源丁酸梭菌进行分离鉴定与安全性评估.[方法]利用厌氧培养方法对源自汶上芦花鸡与SPF鸡粪便样品进行丁酸梭菌的分离与纯化,挑选可疑菌落进行微生物质谱鉴定,进一步通过16S rRNA基因测序进行鉴定,16S rRNA测序结果与NCBI核苷酸数据库中丁酸梭菌的16S rRNA序列进行同源性分析;同时,进行所有...     

A signal-amplified electrochemical immunosensor for aflatoxin B1 determination in rice

A sensitive and simple electrochemical immunosensor based on enzymatic silver deposition amplification was constructed for the detection of aflatoxin B₁ (AFB₁) in rice. The immunosensor was based on an indirect competitive format between free AFB₁ and aflatoxin B₁-bovine serum albumin (AFB₁-BSA) conjugate immobilized on the electrode surface for binding to a fixed amount of anti-AFB₁ antibody. Then the alkaline phosphatase (ALP)-labeled anti-mouse immunoglobulin G (IgG) secondary antibody was bound to the electrode surface through reaction with primary antibody. Finally, ALP catalyzed the substrate, ascorbic acid 2-phosphate, into ascorbic acid that reduced silver ions in solution to metal silver deposited onto the electrode surface. Linear sweep voltammetry was carried out to quantify the metal silver, which indirectly reflected the amount of the analyte. The experimental parameters, such as the dilution ratio of antibody and the concentration of AFB₁-BSA conjugate, have been evaluated and optimized. At the optimal conditions, the working range of the electrochemical immunosensor was from 0.1 to 10 ng/ml with a detection limit of 0.06 ng/ml. Good recoveries were obtained for the detection of spiked rice samples. So, the proposed method in this article could find a good use for screening AFB₁ in real samples.     

A comparative evaluation of aflatoxin B1 genotoxicity in fish models using the Comet assay

Aflatoxin B₁ (AFB₁) is classified as a Group I hepatocarcinogen in humans by the International Agency for Research on Cancer (IARC). The alkaline Comet assay is a simple and rapid method by which DNA damage can be demonstrated as a function of tail moment. The present work is the first to evaluate the genotoxicity of AFB₁ in fish using the Comet assay. Two different species of fish were selected as models due to previously established sensitivity to AFB₁: rainbow trout (sensitive) and channel catfish (resistant). Fish were i.p. injected with 0.5 mg AFB₁/1 ml DMSO/1 kg body weight. The Comet assay was performed after 4 and 24 h on whole blood, liver, and kidney cells of both species. Trout blood and kidney tissue tested displayed significant (p<0.05) and extensive DNA damage (shown by increased tail moment) after 4 h which then decreased by 24 h. In liver cells, damage progressively increased over time. Conversely, similarly treated catfish showed no elevation in DNA damage over controls at the same doses. These results suggest that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB₁ and for evaluating organ specific effects of these agents in different species.     

Processing of glutathionylcobalamin by a bovine B12 trafficking chaperone bCblC involved in intracellular B12 metabolism

Glutathionylcobalamin (GSCbl) is a biologically relevant vitamin B₁₂ derivative and contains glutathione as the upper axial ligand thought formation of a cobalt-sulfur bond. GSCbl has been shown to be an effective precursor of enzyme cofactors, however processing of the cobalamin in intracellular B₁₂ metabolism has not been fully elucidated. In this study, we discovered that bCblC, a bovine B₁₂ trafficking chaperone, catalyzes elimination of the glutathione ligand from GSCbl by using the reduced form of glutathione (GSH). Deglutathionylation products are base-off cob(II)alamin and glutathione disulfide, which are generated stoichiometrically to GSH. Although cob(I)alamin was not detected due to its instability, deglutathionylation is likely analogous to dealkylation of alkylcobalamins, which uses the thiolate of GSH for nucleophilic displacement. The catalytic turnover number for the deglutathionylation of GSCbl is ?1.62 ± 0.13 min⁻¹, which is, at least, an order of magnitude higher than that for elimination of upper axial ligands from other cobalamins. Considering the prevalence of GSH at millimolar concentrations in cells, our results explain the previous finding that GSCbl is more effective than other cobalamins for synthesis of enzyme cofactors.     

Naturally occurring chlorophyll derivatives inhibit aflatoxin B1-DNA adduct formation in hepatoma cells

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B₁ (AFB₁)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB₁-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB₁ totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB₁. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB₁-induced DNA damage in the Hepa-1 cell by direct trapping of AFB₁. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB₁ metabolites.     

拟南芥抗凋亡突变体fbr136的分离鉴定与分析

植物细胞程序性死亡(programmed cell death,PCD)在植物的生长发育进程以及防御生物与非生物胁迫的过程中具有重要的作用.Fumonisin B1(FB1)是一种真菌毒素,是鞘脂生物合成途径中关键酶神经酰胺合酶(ceramide synthase)的竞争性抑制剂.FB1在动植物细胞中均能够诱导PCD.为了探索植物PCD的机制,通过筛选拟南芥抗FB1的突变体,分离鉴定了11个,fumonisin B1 resistant (fbr)突变体.遗传分析表明,这些突变体分别是由9个相同或者不同的遗传座位突变造成的.对其中一个代表性的突变体fbr136进行了详细的表型分析和初步遗传定位.fbr136对其他PCD诱导剂,例如H2O2或paraquat也表现出一定的抗性或耐受性,而且在fbr136突变体中FB1不能正常诱导PR1基因的表达,说明fbr136突变体PCD的发生可能受到阻碍.硝基四唑(Nitroblue tetrazolium,NBY)染色表明,FBl处理fbr136突变体后产生和积累活性氧(reactive oxygenspecies)比野生型植物显著降低,暗示其抗凋亡表型可能与活性氧的产生有关.推测FBR136可能是FB1在诱导PCD过程中,从鞘脂含量变化到活性氧积累变化这一途径的一个重要的调控因子.fbr136被定位于染色体Ⅲ上,与以往鉴定的抗FB1突变基因的定位都不同,因此,FBR136可能是FB1诱导PCD信号途径中的一个新基因.     

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。