Spore inhibition-based enzyme substrate assay for monitoring of aflatoxin M1 in milk

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M₁ (AFM₁) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM₁ sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM₁ in milk. However, if there was no color change, presence of AFM₁ at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM₁ detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM₁ in milk.     

Early murine immune responses from endotracheal exposures to biotechnology-related Bacillus strains

An immunology-based in vivo screening regime was used to assess the potential pathogenicity of biotechnology-related microbes. Strains of Bacillus cereus (Bc), Bacillus subtilis (Bs), Bacillus thuringiensis (Bt), and Bt commercial products (CPs) were tested. Balb/c mice were endotracheally instilled with purified spores, diluted CP, or vegetative cells (VC) (live or dead). Exposed mice were evaluated for changes in behavioral and physical symptoms, bacterial clearance, pulmonary granulocytes, and pulmonary and circulatory pyrogenic cytokines (interleukins (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α), as well as acute phase biomarkers (fibrinogen and serum amyloid A). Except for some differences in clearance rates, no marked effects were observed in mice exposed to any spore at 10⁶ or 10⁷ colony forming units (cfu). In contrast, live Bc or Bt VCs (10⁵ or 10⁶?cfu) produced shock-like symptoms (lethargy, hunched appearance, ruffled fur, and respiratory distress), and 11–200-fold elevations in pyrogenic cytokines at 2-h post-exposure. In the study, 4-h effects included increased lethargy, ocular discharge, and 1.5–4-fold rise in circulatory acute phase markers, but no indications of recovery. Bs VC did not produce any changes in symptoms or biomarkers. After 2 or 4?h of exposure to dead VC, increases of only plasma IL-1β and TNF-α (4.6- and 12.4-fold, respectively) were observed. These findings demonstrate that purified spores produced no marked effects in mice compared to that of metabolically active bacteria. This early screening regime was successful in distinguishing the pathogenicity of the different Bacillus species, and might be useful for assessing the relative hazard potential of other biotechnology-related candidate strains.     

沙田柚根围AM真菌的生境适宜性和季节变化性

以山地长寿沙田柚成年果树为对象,研究不同生境和季节对丛枝菌根(Arbuscular mycorrhiza,AM)真菌侵染以及根围土壤中AM真菌孢子密度的影响.试验结果表明,不同生境下,菌根侵染率和AM真菌孢子密度都是以梯田最高,坡地次之,洼地最低;在坡地和洼地生境下,生草区均高于清耕区.两块试验地AM真菌的菌丝侵染率均为夏季最高(16.8%±1.9%和16.0%±1.8%),秋季次之,冬季最低;丛枝和泡囊的形成也是夏/秋季较高,春/冬季较低;而根围土壤中AM真菌孢子密度则是秋季最高[(159±19)个/100 g和(167±17)个/100 g],夏季次之,冬季最低.总之,对于长寿沙田柚成年果树,AM真菌的菌根侵染率在夏季梯田最高,根围土壤中的AM真菌孢子密度在秋季梯田最高;在坡地和洼地生境中,生草处理均可显著提高菌根侵染率和AM真菌孢子密度.图4参27     

大亚湾沉积物中浮游植物休眠体萌发研究

为了解孢囊在浮游植物种群动态及赤潮发生中的作用,于2005年4月~2006年6月采集大亚湾养殖海域沉积物样品,经孔径为125,20μm的网筛过滤,在25℃下进行萌发,观察培养20,40d的浮游植物休眠体的萌发状况.结果表明,共萌发出浮游植物27属37种.裸甲藻(Gymnodinium corii Schiller)为优势甲藻类,其占甲藻细胞数量平均百分比为73.1%,最高百分比可达99.7%.定鞭藻中的金色藻(Chrysochromulima spp.)和蓝藻中的拟鱼腥藻(Anabaenopsis spp.)也萌发出较高数量.不同时间培养、萌发的浮游植物种类组成有所差异,同时也随沉积物捕捉时间的不同而出现一定的季节差异.休眠体的萌发可在一定程度上反映水体浮游植物组成,但在萌发中大量出现的一些微型和微微型浮游植物可能会由于体型过小而在浮游植物监测中被忽略.     

乌蕨孢子繁殖技术的研究

研究了乌蕨(Stenoloma chusanum(L.)Ching)孢子无菌繁殖和常规繁殖方法。结果表明,乌蕨孢子在1/4MS培养基培养时的萌发率最高,达85.3%;在1/2MS培养基中的配子体成苗率最高,达85.9%;1/2MS培养基是培养优质孢子体苗的最佳培养基;河沙与草炭土体积比为1∶1的混合基质是播种孢子的理想基质,孢子萌发率和配子体成苗率分别达到82.6%和73.4%;无菌繁殖和常规繁殖所得的幼孢子体苗移栽成活率均达90%以上。     

Inhibition of the antioxidant activity of catalase and superoxide dismutase from Fusarium verticillioides exposed to a Jacquinia macrocarpa antifungal fraction

The aim of this study was to investigate the in vitro effect of an antifungal fraction obtained from Jacquinia macrocarpa plant (JmAF) in the generation of reactive oxygen species (ROS) and the activity of the catalase (CAT) and superoxide dismutase (SOD) enzymes from Fusarium verticillioides, as well as their influence in the viability of the fungus spores. The compounds present in the JmAF were determined by gas chromatography/quadrupole time-of-flight mass spectrometry (GC/QTOF-MS). The effect of the exposition to JmAF on the generation of ROS, as well as in the CAT and SOD activities in F. verticillioides, was determined. The main compounds detected were γ-sitosterol, stephamiersine, betulinol and oleic acid. JmAF showed very high ability in inhibiting the spore viability of F. verticillioides, and their capacity to cause oxidative stress by induction of ROS production. JmAF induced the highest ROS concentration and also inhibited CAT and SOD activities. The results obtained in this study indicate that JmAF is worthy of being considered for the fight against phytopathogenic fungi.     

北京和保定地区大气细颗粒物中可培养细菌的种群特征

在北京城区和河北省保定郊区(望都),于2015年1月10~17日雾-霾时期,使用大气颗粒物采样器收集大气细颗粒物(PM2.5),比较了两地雾-霾时期PM2.5中可培养细菌的种群结构特征异同;使用场发射扫描电镜(FESEM)观察PM2.5颗粒形态,利用后向轨迹模型NOAA/ARL HYSPLIT-4分析北京城区和保定郊区气团输送来源,采用离子色谱仪、流动分析仪和ICP-MS仪器监测PM2.5中主要水溶性离子和重金属元素含量.结果表明,北京城区和保定郊区分离得到的PM2.5可培养细菌为厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和α-变形菌门(α-Proteobacteria)三大菌门,其中厚壁菌门(Firmicutes)为优势菌门;分离纯化鉴定出17种细菌,革兰氏阳性菌占90%以上;优势菌属为产芽孢的芽孢杆菌属(Bacillus),分别占北京城区(RCEES)和保定郊区(WD)PM2.5样品中可培养细菌百分比的68.15%和75%.北京城区和保定郊区PM2.5中可培养细菌种群结构特征可能受到PM2.5理化性质和气团传输的影响.     

黄土高原典型林木根际土壤微生物群落结构与功能特征及其环境指示意义

通过研究黄土高原典型林木根际土壤微生物群落结构,为正确评价该生态恢复区土壤环境恢复状况提供理论依据.利用湿筛倾析法、涂平板法和BIOLOG法研究了陕北黄土高原4种典型林木丛枝菌根真菌(arbuscular mycorrhizal fungi,AMF)孢子密度与土壤微生物群落结构多样性.结果表明,沙棘AMF孢子密度为刺槐的2.24倍,顺序为沙棘>狼牙刺>柠条>刺槐,不同林木土壤细菌和放线菌数量达极显著差异(p<0.01),真菌数量差异不显著(p>0.05).同一林木土壤细菌对BIOLOG-ECO板中碳源的利用优势种类和利用程度呈现明显的差异性;主成分分析表明对主成分1和主成分2贡献大的碳源为14种和8种,柠条和沙棘土壤典型变量值的变异(离散)较小,而狼牙刺和刺槐土壤典型变量值的变异较大.相关性分析表明,AMF孢子密度与细菌数量、氨基酸类、多胺类和芳香化合物类碳源呈极显著正相关(p<0.01),与真菌数量、放线菌数量和细菌总代谢活性呈显著正相关(p<0.05),但是与羧酸类、糖类和多聚物类碳源相关性不显著(p>0.05).AMF孢子密度可以作为评价陕北黄土高原林木土壤细菌生理代谢类群及其多样性的环境生物学指标.     

短短芽胞杆菌及其芽胞对芘的降解

考察短短芽胞杆菌(Brevibacillus brevis)菌体及芽胞对四环多环芳烃芘的降解性能.结果表明,菌体5 d内对1 mg·L-1芘的降解率可达53%.菌体和胞内酶降解芘过程中检测到1-羟基芘、9-羟基菲、α-萘酚和β-萘酚这4种单羟基降解产物,在完整菌体降解体系中,产物呈现先积累后下降的趋势,而在胞内粗酶液降解体系中,大分子代谢产物表现为一直积累的趋势,说明B.brevis完整菌体在降解初期并不具备一些中间产物的降解酶,但随着时间推移,可被诱导产生相关酶对生成的新产物进行进一步降解.芽胞悬液在添加芘的无机盐培养基中5 d内萌发生成的营养细胞可达到1.5×109个·L-1,对1 mg·L-1芘的降解率达到15%.     

Volumetric assessment of airborne fungi in two sections of a rural indoor dairy cattle shed

There is increasing concern about the exposure to fungal aerosols in occupational environments and associated respiratory allergic diseases and asthma. A large number of people work in cattle sheds around the world, pulmonary function impairments and higher frequency of respiratory symptoms have been reported in dairy farmers; however, it appears that adequate information on the fungal aerosols from the cattle sheds are largely lacking. Volumetric assessment of airborne culturable and nonculturable fungal spores was performed in two sections of a large rural indoor cattle shed of West Bengal, India for 2 consecutive years. An Andersen Two Stage Viable Sampler was used for sampling culturable fungi and a Burkard Personal Slide Sampler was used to collect the total airborne fungal spores including both the culturable and nonculturable types. A total of 31 spore types and 35 types of viable colony-forming units were recorded. Average concentration range of total fungal spores was 233-2985/m(3) and concentration of viable colony-forming units ranged between 165 and 2225 CFU/m(3). Burkard Sampler showed higher frequencies of Aspergilli/Penicilli, Cladosporium, Alternaria, and smut spores. Andersen Sampler showed the prevalence of Aspergillus niger, Aspergillus flavus and Cladosporium cladosporioides colonies. Some recorded fungi were earlier reported as allergenic, toxic, and pathogenic for occupational workers as well as cattle population. Higher concentration levels of airborne total and culturable fungal spores were recorded during the months of November through February (winter) and June through September (late summer and rainy season).