T-2毒素降解菌株的筛选、鉴定与降解机制分析

为获得高效降解T-2毒素的微生物菌株并探究其降解机制，本研究以T-2毒素为底物从小麦样品中筛选降解微生物并进行鉴定，探究该菌株对T-2毒素的降解特性，利用超高效液相色谱-飞行时间质谱仪分析其代谢产物与降解机制。结果表明，从50份小麦样品中筛选到2株T-2毒素高效降解菌株AFJ-2和AFJ-3，通过形态学观察和16S rDNA序列分析，初步鉴定为短小杆菌属（Curtobacterium sp.）菌株和芽孢杆菌属（Bacillus sp.）菌株。菌株AFJ-2和AFJ-3分别能在7 h和12 h内将5μg/mL T-2毒素完全降解，2株菌的胞内酶均对T-2毒素具有显著降解效果（P<0.05），不存在吸附作用。菌株AFJ-2可将T-2毒素转化为HT-2毒素和T-2三醇，菌株AFJ-3降解T-2毒素产生新茄镰孢菌醇（neosolaniol,NEO），基于降解位点推测，2株菌共同作用于T-2毒素可产生新的产物4-脱乙酰-NEO。研究结果丰富了生物降解T-2毒素的菌种资源库，为T-2毒素降解酶的分离纯化及功能基因的挖掘提供一定参考依据。     

双色实时荧光聚合酶链式反应法检测产玉米赤霉烯酮毒素的黄色镰孢菌

目的 建立双色实时荧光聚合酶链式反应(polymerase chain reaction,PCR)法同时检测黄色镰孢菌(Fusarium culmorum)及其所产玉米赤霉烯酮毒素合成基因的方法。方法 依据EF1α基因和PKS基因,优化建立双色实时荧光PCR反应体系和扩增条件;建立粮食霉变模型,检测霉变粮食中产毒真菌,验证方法的特异性、灵敏度和重复性。结果 应用本研究设计的引物和探针,仅产玉米赤霉烯酮毒素的黄色镰孢菌EF1α和PKS基因出现特异性扩增,其余真菌扩增结果均为阴性;检出限达3.5×10²CFU/mL,重复性良好;且采用此方法对粮食霉变模型中选取的样品进行检测,可检出产玉米赤霉烯酮的黄色镰孢菌。结论 该方法可准确鉴定霉变粮食中的产玉米赤霉烯酮毒素的黄色镰孢菌,实现在真菌毒素未产生前或者产生早期,对粮食受真菌及真菌毒素污染的情况进行早期监控。     

花生黄曲霉毒素污染拮抗菌B25-5的筛选、鉴定及控制效果

黄曲霉毒素是花生中重要的污染物,对人畜的健康具有极强的危害性。本研究筛选对黄曲霉毒素污染具有较强抑制作用的有益微生物,为花生中黄曲霉毒素污染治理提供支持。本研究从河北省滦南县、滦县、玉田、丰南等主产区县的花生中分离有益微生物,通过室内外实验,获得对产毒黄曲霉较强拮抗菌株B25-5。经分子生物学及理化性鉴定,该菌株为解淀粉芽孢杆菌。进一步开展了B25-5对黄曲霉孢子萌发的抑制作用、黄曲霉毒素的消减作用及黄曲霉毒素的分解作用等几个方面的研究,结果表明,拮抗菌可以显著的抑制产毒黄曲霉孢子的萌发、生长、菌丝的生长,降低黄曲霉毒素的产生及消除黄曲霉毒素。     

温度和pH对不同镰刀菌生长及产毒的影响

以从小麦、玉米、大米、大麦等作物中分离得到的12株不同种类的镰刀菌菌株为研究对象,探究不同温度和p H对其生长及产毒的影响。结果表明,镰刀菌在10~35℃和p H3~11范围内均能够生长,最适温度为20~30℃,最适p H为6~8。供试镰刀菌能够产生的毒素主要为A型单端孢霉烯族毒素、B型单端孢霉烯族毒素、伏马毒素和镰刀菌酸,产毒温度为5~40℃,但不同菌株的最适产毒温度存在差异;产毒过程受p H影响较大,过酸(p H3~5)及过碱(p H9~11)的条件下均未检测到任何毒素。总体上看,不同种类镰刀菌产毒类型存在差异,毒素产量受温度和p H影响比生长更大,且大多数菌株的生长与产毒最适条件并不一致。     

用LC/MS/MS分析麦芽和啤酒中的单端孢菌毒素(Trichothecene)

建立了一种简单、灵敏的用液相色谱电子喷射离子化联用质谱(LC/MS/MS-ESI)分析五种单端孢菌毒素(trichothecene)的方法.这五种单端孢菌毒素分别为:HT-2毒素(HT-2)、T-2毒素(T-2)、脱氧雪腐镰刀菌烯醇(DON)、瓜萎镰菌醇(NIV)和3-乙酰基脱氧雪腐镰刀菌烯醇(3-AcDON).分析了7种进口麦芽和17种国产啤酒(日本),仅从一种麦芽中检测出了5ng/g的HT-2和23ng/g的NIV,啤酒中仅检测出了0.5～1.4ng/g的DON.该方法可应用在食品质量检测方面.     

山西葡萄中链格孢菌分离鉴定与污染水平分析

本研究对山西葡萄主产区的巨峰葡萄（Kyoho grapes）和红提葡萄（Redglobe table grapes）上的主要病原真菌（链格孢菌）进行纯化分离、真菌形态学鉴定、分子生物学鉴定,并对链格孢菌产毒能力进行检测分析。研究表明,链格孢菌（Alternaria alternata）侵染山西葡萄具有一定的品种选择性。链格孢菌菌株产生的毒素主要以细交链孢菌酮酸（TEA）为主。本研究初步明确了山西葡萄（巨峰葡萄和红提葡萄）中链格孢菌的污染水平。     

重要产毒镰刀菌合成白僵菌素和恩镰孢菌素研究进展

白僵菌素(beauvericin,BEA)和恩镰孢菌素(enniatins,ENNs)是由镰刀菌属的多种真菌产生的六酯肽类真菌毒素,该类毒素对上皮细胞、免疫细胞、卵巢细胞等具有很强的毒性作用。本文主要针对能产生BEA和ENNs的重要产毒镰刀菌的形态学特征、分子遗传学特征以及影响BEA和ENNs产生的环境条件如温度、底物等因素进行概述。重点阐述了产毒镰刀菌在两类毒素合成酶的基因水平和氨基酸水平的差异及影响产毒等主要因素,为BEA和ENNs及其产毒镰刀菌的预防和控制提供理论依据。     

小麦中致病真菌的分离鉴定及熏蒸对其毒力的影响

小麦受镰刀菌的感染会导致赤霉病、真菌毒素污染和减产,镰刀菌对粮食安全有着严重威胁。通过对小麦中镰刀菌株进行分离鉴定,分离出的三种菌株N1、N2、N3分别属于禾谷镰刀菌属、亚洲镰刀菌属和高秆镰刀菌属。利用产毒基因检测和真菌毒素检测对三株菌株玉米赤霉烯酮、呕吐毒素和伏马毒素的产毒情况进行分析,结果一致显示N1和N3能产生玉米赤霉烯酮和呕吐毒素,均含有PSK和Tri5两种产毒基因,而N2不产生这三种毒素。进一步通过臭氧和二氧化氯进行气体熏蒸,探究气体熏蒸对分离出的三株菌株的影响,通过观察孢子形态、菌丝体长度、菌丝形态,结果表明二氧化氯熏蒸能有效抑制菌丝生长和孢子萌发,低浓度二氧化氯（300 mg/L）处理0.5 h,可明显抑制菌丝的生长。而臭氧只能抑制孢子萌发,对菌丝的生长没有明显的抑制作用。     

臭氧处理对交链孢菌生长及其毒素积累的抑制作用

交链孢菌（Alternaria spp.）易侵染农作物,引起农产品病害,而且能够代谢产生交链孢毒素,包括细交链孢酮酸（tenuazonic acid,TeA）、交链孢酚（alternariol,AOH）、交链孢酚单甲醚（alternariol monomethyl ether,AME）等,严重影响人体健康。因此,亟需高效、安全的方法用以防控交链孢菌及其毒素积累。本实验研究了臭氧处理对体外互隔交链孢（A. alternata）生长及其产毒能力的影响。结果表明,臭氧处理组的菌落直径显著低于对照组（P＜0.05）,且臭氧处理可显著抑制互隔交链孢产生TeA、AOH、AME这3 种交链孢毒素;利用扫描电子显微镜观察臭氧处理后互隔交链孢的微观形态,发现孢子和菌丝发生了凹陷、褶皱、断裂等异常现象;臭氧处理后的交链孢菌对番茄果实的致病力明显减弱,同时交链孢菌的产毒能力明显降低,20 mg/L臭氧处理条件下TeA、AOH、AME含量比对照组分别减少了36.1%、89.9%、93.2%。此外,臭氧对TeA、AOH、AME具有降解作用,降解率随着臭氧质量浓度的增加和处理时间的延长而显著提高,TeA经过20 mg/L臭氧处理30 min即可被完全降解,AOH及AME经20 mg/L臭氧处理120 min后降解率可达90%以上。综上,臭氧处理可以作为农产品及其制品中互隔交链孢及其毒素污染的防治手段。     

番茄交链孢病原菌鉴定及产毒分析

目的 通过对番茄病果中链格孢病原菌的分离鉴定及产毒分析,为番茄采收后病害防控提供理论依据。方法 采集福建省5个县市番茄病果,通过切片和组织分离法从番茄病害部位分离纯化病原菌,结合形态学特征和分子生物学对病原菌进行鉴定。根据柯赫氏法则确定病原菌的致病性,并利用超高效液相串联质谱法对分离得到的交链格孢菌产毒能力进行检测分析。结果 在发病番茄病害部位共分离得到的 3株互隔链格孢菌株均具产毒能力,但毒素种类和产毒能力各有不同,其中NH06菌株产毒量最高,可产生细交链格孢酮酸(tenuazonic acid,TeA )、交链孢酚 (alternariol, AOH)、交链孢酚单甲醚(alternariol monomethyl ether, AME)、交链孢烯(altenuene,ALT )、腾毒素 (tentoxin,TEN ) 5种毒素,而NP002和NH07仅检出产生4种毒素。结论 福建省番茄种植产区内存在交链格孢菌的污染,且产毒种类多,因此,在番茄种植产区内病果的处理应引起关注和重视。     

Detection of toxigenic Fusarium strains, producing T-2 toxin, in wheat grain mycoflora by microbiologic assay

Twenty-three Fusarium strains were isolated from wheat grain harvested in the Moscow region. The ability of the fungi cultures isolated for producing T-2 toxin was studied by the microbiological assay with the use of Saccharomyces lactis culture (BKMU-459) susceptible to T-2 toxin. The toxigenic properties were shown by 9 cultures. Six strains with unestablished species appurtenance grown on A. Capek's agar in Perti dishes were found to produce T-2 toxin in an amount of 2 to 50 micrograms/ml agar. Three strains grown on sterilized wheat grain and attributed to Fusarium sporotrichiella v. poae according to the morphological characteristics were discovered to produce T-2 toxin in an amount from 50-100 to 400-600 micrograms/g. Production of T-2 toxin by the strains isolated was confirmed by thin-layer chromatography. Experiments made on young rats have demonstrated that extracts from F. sporotrichiella v. poae strains producing T-2 toxin appeared highly toxic for the animals.     

Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium

This study was conducted to examine the ability of selected strains of Lactobacillus and Propionibacterium to remove common Fusarium toxins, trichothecenes, from liquid media. The trichothecenes studied were deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), fusarenon (FX), diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2). The Lactobacillus rhamnosus strain GG (LGG), Lactobacillus rhamnosus strain LC-705 (LC-705) and Propionibacterium freudenreichii ssp. shermanii JS (PJS) were incubated in PBS buffer containing 20 µg toxin ml -1 for 1h at 37°C, and after centrifugation the concentration of the toxins was measured in the supernatant fraction. Both viable and heat-killed forms of LGG and PJS were more efficient than LC-705 in removing the toxins from the liquid media. LGG and PJS removed four of the seven tested toxins (the removal varying from 18 to 93%) and LC-705 two toxins (10-64%). Of the toxins, 3-AcDON was not removed by any of the bacteria; HT-2 was removed by the non-viable LGG and also slightly by non-viable LC-705; DAS was removed by all three bacteria tested. Binding is postulated as the possible mechanism of the removal, since no difference was observed between the ability of viable and heat-killed bacteria in removing the trichothecenes, and no degradation products of the toxins were detected by gas chromatography (GC)-mass spectrometry (MS) analysis. It is concluded that significant differences exist in the ability of the bacteria to bind trichothecenes in vitro.     

采收期谷物中真菌毒素产毒菌的筛选鉴定

为筛选东北地区采收期小麦、玉米和水稻中的真菌毒素产毒菌,以酶联免疫吸附测定、超高效液相色谱-串联质谱联用技术和聚合酶链式反应为分析工具,通过真菌分离、液体培养、产毒菌初筛、产毒性定性分析和分离菌的基因序列比对,筛选出产玉米赤霉烯酮、呕吐毒素和伏马毒素的产毒菌6?株,经DNA分析鉴定,分别属于Fusarium asiaticum、F. poae、F. graminearum和F. fujikuroi 4?种真菌。本实验结果为我国针对性的防控粮食中产毒性真菌污染提供了基础性实验数据。     

Fate of trichothecene mycotoxins during the processing: milling and baking.

Toxic secondary metabolites produced by fungi representing Fusarium genus are common contaminants in cereals worldwide. To estimate the dietary intake of these trichothecene mycotoxins, information on their fate during cereal processing is needed. Up-to-date techniques such as high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC-MS/MS) was used for the analysis of seven trichothecenes (deoxynivalenol, nivalenol, HT-2 toxin, T-2 toxin, 15- and 3-acetyldeoxynivalenol, and fusarenon-X) in bread production chain (wheat grains, intermediate products collected during milling and baking process, breads). Regardless of whether the grains were naturally infected or artificially inoculated by Fusarium spp. in the field, the fractions obtained from the grain-cleaning procedure contained the highest mycotoxin levels. During milling the highest concentrations of deoxynivalenol were found in the bran, the lowest in the reduction flours. Baking at 210 degrees C for 14 min had no significant effect on deoxynivalenol levels. The rheological properties of dough measured by fermentograph, maturograph, oven rise recorder, and laboratory baking test were carried out, and based on the obtained results the influence of mycotoxin content on rheological behaviour was investigated.     

Fate of trichothecene mycotoxins during the processing: milling and baking

Toxic secondary metabolites produced by fungi representing Fusarium genus are common contaminants in cereals worldwide. To estimate the dietary intake of these trichothecene mycotoxins, information on their fate during cereal processing is needed. Up-to-date techniques such as high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC-MS/MS) was used for the analysis of seven trichothecenes (deoxynivalenol, nivalenol, HT-2 toxin, T-2 toxin, 15- and 3-acetyldeoxynivalenol, and fusarenon-X) in bread production chain (wheat grains, intermediate products collected during milling and baking process, breads). Regardless of whether the grains were naturally infected or artificially inoculated by Fusarium spp. in the field, the fractions obtained from the grain-cleaning procedure contained the highest mycotoxin levels. During milling the highest concentrations of deoxynivalenol were found in the bran, the lowest in the reduction flours. Baking at 210 degrees C for 14 min had no significant effect on deoxynivalenol levels. The rheological properties of dough measured by fermentograph, maturograph, oven rise recorder, and laboratory baking test were carried out, and based on the obtained results the influence of mycotoxin content on rheological behaviour was investigated.     

小豆内生真菌的筛选鉴定及其产玉米赤霉烯酮的可能性探究

玉米赤霉烯酮(zearalenone,ZEN)是目前全球范围污染谷物最广泛的霉菌毒素之一,对人体和动物健康都会产生极大威胁,目前从市售小豆检测到ZEN。【目的】筛选暴露ZEN的小豆内生真菌,结合经典方法和分子生物学方法鉴定所筛选内生真菌,并探索菌株潜在的ZEN产生风险。【方法】以暴露ZEN的小豆为样本,将分离纯化后的内生真菌进行经典形态观察;同时扩增真菌核糖体保守序列ITS1、ITS4,镰刀菌特异基因序列FU1、FU2确定小豆中镰刀菌种属,同时通过ZEN玉米赤霉烯酮产毒控制基因pks13进行PCR扩增,分析菌株ZEN合成的潜在风险。【结果】鉴别出3株镰刀菌,其中2株初步鉴定为砖红镰刀菌(Fusarium lateritium)、1株为花腐镰刀菌(Fusarium napiforme),未检测出ZEN合成关键基因;1株玛利节菱孢霉(Arthrinium marii),分子检测具有产ZEN玉米赤霉烯酮毒素的可能性。将以上4株菌分别回接至ZEN的产毒培养基,LC-MS/MS法未检测出ZEN毒素。【结论】暴露ZEN的小豆具有内生镰刀菌,存在可能产ZEN的内生节菱孢霉;回接培养基未检测到ZEN,但不排除小豆产生ZEN的风险。     

T-2毒素对凡纳滨对虾的经口急性毒性效应研究

通过T-2毒素微胶囊毒饵料一次性染毒凡纳滨对虾,采用模型拟合法测定T-2毒素对凡纳滨对虾经口LD50,并分析Ca2+-ATPase、多酚氧化酶(PPO)活力以及肌肉病理组织学变化,进而探明T-2毒素对凡纳滨对虾急性毒性效应。结果表明,T-2毒素对凡纳滨对虾的急性毒性经口一次性暴露的LD50为1.22 mg/(kg·bw),T-2毒素对PPO酶活性和Ca2+-ATPase活性的急性毒性效应ED50分别为0.05和3.22 mg/(kg·bw),比较风险评估指数RI可知,PPO活性可作为生物学效应标记描述T-2毒素对对虾的急性毒性效应,且比Ca2+-ATPase更加灵敏。T-2毒素急性暴露可导致肌纤维间隙面积比增大,可导致对虾肌肉品质劣化。采用LC-MS/MS定量检测染毒对虾肌肉中的T-2毒素,但是没有发现游离态T-2毒素残留,说明对虾中T-2毒素急性暴露不会引起物质蓄积,但却产生功能蓄积,可能是T-2毒素以隐蔽态形式存在,导致初始轻微损伤逐渐累加的结果。     

Production of trichothecene mycotoxins by Australian Fusarium species.

Australian isolates of Fusarium species were grown on potato dextrose agar. Trichothecenes produced by these species were extracted by ethyl acetate followed by methanol and a silica gel column was used to clean-up the extract. The extracted samples were derivatized by acetylation with trifluoroacetic anhydride and the derivatives analysed by gas chromatography/mass spectrometry (GC/MS). Multiple ion detection was used to trace ions characteristic of the trichothecenes expected to be present. Quantitation of those found was based on a known mass of pentabromophenol that was added as an internal standard. Eight species of Fusarium (nineteen strains) were surveyed, of which three species, F. acuminatum, F. equiseti and F. sporotrichioides, produced the trichothecenes scirpentriol, diacetoxyscirpenol, neosolaniol, HT-2 toxin, T-2 toxin, T-2 tetraol and deoxynivalenol. Wheat samples were inoculated with four different species of Fusarium, F. acuminatum, F. equiseti, F. graminearum and F. sporotrichioides, and in these samples diacetoxyscirpenol, neosolaniol, HT-2 toxin and T-2 toxin were found.     

降解亚硝酸盐乳酸菌的筛选及鉴定

以家制的大豆酱、酸菜、酱黄瓜、生拌黄瓜、腌制芥菜以及市售的辣白菜、乌江榨菜、五原榨菜、咸萝卜块为来源,从中分离出了18株乳酸菌菌株.经测定其在MRS液体培养基中降解亚硝酸盐的能力,最终从家制发酵的大豆酱中筛选出了1株优良的亚硝酸盐降解菌D j-1,其在1.0×107cfu/mL时,对0.125mg/mL亚硝酸盐22h内降解率为93.63％.经过形态特征观察、生理生化试验和16S rRNA基因序列相似性分析,鉴定该菌株为屎肠球菌.     

Transfer of Fusarium mycotoxins and 'masked' deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer

The fate of five Fusarium toxins--deoxynivalenol (DON), sum of 15- and 3-acetyl-deoxynivalenol (ADONs), HT-2 toxin (HT-2) representing the main trichothecenes and zearalenone (ZON) during the malting and brewing processes--was investigated. In addition to these 'free' mycotoxins, the occurrence of deoxynivalenol-3-glucoside (DON-3-Glc) was monitored for the first time in a beer production chain (currently, only DON and ZON are regulated). Two batches of barley, naturally infected and artificially inoculated with Fusarium spp. during the time of flowering, were used as a raw material for processing experiments. A highly sensitive procedure employing high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was validated for the analysis of 'free' Fusarium mycotoxins and DON-conjugate in all types of matrices. The method was also able to detect nivalenol (NIV), fusarenon-X (FUS-X) and T-2 toxin (T-2); nevertheless, none of these toxins was found in any of the samples. While steeping of barley grains (the first step in the malting process) apparently reduced Fusarium mycotoxin levels to below their quantification limits (5-10 microg kg(-1)), their successive accumulation occurred during germination. In malt, the content of monitored mycotoxins was higher compared with the original barley. The most significant increase was found for DON-3-Glc. During the brewing process, significant further increases in levels occurred. Concentrations of this 'masked' DON in final beers exceeded 'free' DON, while in malt grists this trichothecene was the most abundant, with the DON/DON-3-Glc ratio being approximately 5:1 in both sample series. When calculating mass balance, no significant changes were observed during brewing for ADONs. The content of DON and ZON slightly decreased by a maximum of 30%. Only traces of HT-2 were detected in some processing intermediates (wort after trub removal and green beer).