磁致伸缩生物传感器尺寸对沙门氏菌检测灵敏度的影响

为了研究不同尺寸传感器的检测灵敏度,该文以磁致伸缩膜片为物理传感器,多克隆抗体为生物识别元件,采用Langmuir-Blodgett(LB)技术将多克隆抗体固定在磁致伸缩膜片表面,制备了用于检测沙门氏菌的磁致伸缩生物传感器。检测时,将不同尺寸的生物传感器浸在含有沙门氏菌的被测溶液里,由于生物传感器对沙门氏菌的吸附使传感器质量增加,从而使传感器共振频率发生改变。采用尺寸为2 mm×0.4 mm×15μm,5 mm×1 mm×15μm和25 mm×5 mm×15μm的传感器检测浓度为102~109 CFU/m L的沙门氏菌溶液,并利用扫描电子显微镜观察传感器表面。研究结果表明,由传感器共振频率变化计算获得的沙门氏菌浓度与扫描电子显微镜观察统计分析获得的直观浓度吻合得很好,两者最大相对差值小于10%。传感器尺寸越小,检测灵敏度越高。尺寸为2 mm×0.4 mm×15μm,5 mm×1 mm×15μm和25 mm×5 mm×15μm的检测极限分别为5×103 CFU/m L,105 CFU/m L和107 CFU/m L。研究结果可为磁致伸缩生物传感器实际应用时,根据预测的细菌浓度,在保证灵敏度的前提下选择合适尺寸的传感器提供参考。     

检测食品沙门氏菌的生物传感器持久性研究

为了研究温度对生物传感器检测的持久性的影响,制备了用于检测食品中沙门氏菌的磁致伸缩生物传感器,以磁致伸缩膜片作为物理传感器,多克隆抗体作为生物识别元件,采用Langmuir-Blodgett(LB)技术将多克隆抗体固定在磁致伸缩膜片表面。当食品中沙门氏菌吸附在生物传感器上时,将引起其共振频率漂移。通过测试与分析磁致伸缩生物传感器共振频率漂移值,并利用扫描电子显微镜(scanning electron microscope)观察吸附了沙门氏菌的生物传感器表面,对生物传感器在25(室温)、45及65℃的持久性进行研究。结果表明:多克隆抗体磁致伸缩生物传感器与沙门氏菌的结合能力随着时间的延长逐渐降低;且温度越高,传感器的持久性越差;在25、45及65℃时,多克隆抗体磁致伸缩生物传感器的持久期分别为30、8和5 d,由此获得了多克隆抗体生物传感器在常用温度下的持久性。并结合阿伦尼乌斯方程,计算得到该生物传感器的激活能为13.024 kJ/mol。进一步证实了磁致伸缩生物传感器可用于定量检测实际溶液中沙门氏菌的浓度,表明生物传感器可应用于食品中细菌的实时快速定量检测。     

红树内生细菌AiL3菌株鉴定及其胞外抗菌活性物质特性

红树（Acanthus ilicifolius）内生细菌AiL3菌株通过形态学观察、生理生化特征测定及16S rDNA序列分析鉴定为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。菌株培养滤液经有机溶剂萃取和硫酸铵沉淀分析发现,硫酸铵沉淀物具有抑菌活性,说明AiL3菌株产生的抗菌活性物质可能是蛋白类物质。该蛋白粗提液对芒果炭疽病菌（Colletotrichum gloeosporioides）、黄瓜枯萎病菌（Fusarium oxysporum f.sp.cucumerinum）等8种植物病原菌均有较强的拮抗作用;对芒果炭疽病菌菌丝生长和分生孢子萌发的EC50分别为9.32和5.81μg/mL,当提取物浓度为20.33μg/mL时,可导致芒果炭疽病菌分子孢子的消解。     

六种重要瓜类种传细菌可视基因芯片筛查方法研究

种传细菌是瓜类作物及健康种子生产中关注的重要对象。针对细菌性白枯病菌(Pseudomonas viridiflava)、细菌性角斑病菌(P.syringae pv.lachrymans)、丁香假单胞菌丁香致病变种(P.syringae pv.syringae)、瓜类果斑病菌(Acidovorax citrulli)、细菌性叶斑病菌(Xanthomonas cucurbitae)、甜瓜黄单胞菌(X.melonis)6种重要瓜类种传细菌,利用多重PCR扩增及后续可视芯片检测技术,建立了可视基因芯片筛查方法。实验结果表明,以细菌纯培养液为模板,6种目标菌检测灵敏度平均可达N×103～N×104CFU/mL,并能从人工模拟带菌的西瓜(Citrullus vulgaris)、甜瓜(Euphorbia meloformis)、黄瓜(Cucumis sativus)的种子样品及自然感染瓜类果斑病菌的西瓜种子中检测到目标细菌。该研究为瓜类种传细菌的快速初筛及健康种子质量控制提供了新手段。     

堆肥作为微生物菌剂载体的研究

以自然风干的堆肥为载体,吸附3株功能芽胞细菌液体菌剂制成不同生物肥料,通过不同时间取样比较堆肥、有机无机肥和生物有机肥以及生物复混肥中功能芽胞细菌和普通微生物数量以及pH等指标变化,探讨堆肥作为载体生产生物肥料的可行性。研究结果表明,经过自然风干的堆肥与蛭石比较,吸附液体微生物菌剂后无论外观、手感还是功能芽胞细菌死亡率,差异均不大。含水量小于15%堆肥吸附液体菌剂比例为6%比较合适,吸附比例高时,生物肥料含水量和pH较高,影响保存效果。生物有机肥和生物复混肥保存6个月时,3株功能芽胞细菌总数分别为0.59×108 CFU.g-1和0.38×108 CFU.g-1,依然可达到农业行业标准要求。生物有机肥中功能芽胞细菌数量最高,生物复混肥集合了三大肥料优点,堆肥中普通微生物数量和多样性最高。完全腐熟的堆肥经过自然风干后可作为微生物菌剂载体。     

洋葱腐烂病菌荧光定量PCR检测体系的建立

唐菖蒲伯克霍尔德菌洋葱致病型(Burkholderia gladioli pv.alliicola),是重要的植物病原细菌检疫性有害生物.本研究的目的是建立特异、灵敏、快速的TaqMan探针荧光定量PCR方法用于检测洋葱腐烂病菌(B.gladioli pv.alliicola).利用洋葱腐烂病菌保守基因序列设计和筛选特异性引物和TaqMan探针,优化PCR反应体系和反应条件.荧光定量PCR菌液检测灵敏度达到1.02×102 CFU/mL,DNA检测灵敏度达到1.73×10-4 ng/μL,反应时间仅需1h左右,对14种伯克氏菌属Burkholderia)参比菌种的特异性检测结果显示,洋葱腐烂病菌具有明显的扩增曲线生长,表明建立的荧光定量PCR体系具有非常高的特异性.同时对洋葱(Allium cepa)种子进行了模拟带菌检测,结果表明,建立的洋葱腐烂病菌荧光定量PCR检测体系能够检出模拟样品中的洋葱腐烂病菌,验证了检测体系的实用性.本研究建立的洋葱腐烂病菌荧光定量PCR检测体系能够有效用于洋葱腐烂病菌的鉴定,为进出口口岸和基层检验检疫部门提供了一种简便、快速、准确的洋葱腐烂病菌分子检测手段.     

生物传感器在纤维素乙醇测定中的应用

为探讨生物传感器在纤维素乙醇测定中的应用,采用乙醇生物传感器分析仪对发酵液中的纤维素乙醇含量进行测定,分析其特点、规律。结果：乙醇生物传感分析仪线性关系良好,线性范围为0.2～20.0 mg/100 mL,回归方程为：Y=5.00225X+0.72273,R＝0.99976;对乙醇的检测快速而准确,检测时间为20 s,回收率为99.3%～100.4 %;与气相色谱方法、酒精计法相比较,差异不显著（P>0.05）;专一性强,且仪器重现性和稳定性较好,检测灵敏度高,检测下限为0.2mg/100mL。乙醇生物传感分析仪用于纤维素乙醇检测,具有检测精度高、速度快（20s）、稳定性好、使用成本低等优势,有望代替酒精计、气相色谱仪等,成为检测纤维素乙醇含量的新方法、新手段。     

电化学免疫传感器快速检测农产品中的毒死蜱

研究了一种无标记的电化学免疫传感器,用于农产品中的毒死蜱农药残留的快速检测。将毒死蜱人工抗原作为生物识别元件固定在金电极的表面,采用间接竞争法原理,样品中的被测组分与电极上的固定化包被抗原竞争性结合溶液中的抗体。抗体抗原结合反应通过电化学阻抗谱和石英晶体微天平进行表征。将该免疫传感器用于检测青菜、苹果等农产品中的毒死蜱农药残留。结果表明,此免疫传感器灵敏度好、准确度高;对毒死蜱农药的检测限为0.01μg/mL,回收率大于85%,检测时间小于1 h,变异系数小于5%,传感器经过再生处理后能重复使用,经济性较好。该研究可为实现快速检测农产品中农药残留传感器的商品化提供参考。     

细菌在两种土壤矿物表面吸附的热力学分析

运用表面热力学方法和扩展的DLVO理论,对两种典型土壤细菌恶臭假单胞菌(Pseudom onasputida)和枯草芽孢杆菌(Bacillus subtilis)在代表性土壤黏粒矿物高岭石和蒙脱石表面的吸附进行了分析,获得了黏粒矿物与细菌作用的疏水自由能变(ΔGH)和静电力自由能变(ΔGEL)及总自由能变(ΔG)。发现疏水自由能为负,显示疏水作用为引力,有利于细菌在黏粒矿物表面的吸附;而静电力自由能为正,表明细菌-矿物间存在静电斥力。疏水自由能显著大于静电力自由能,表明疏水作用在黏粒矿物对细菌吸附时的贡献大于静电力。两种细菌与两种矿物间的总吸附自由能为负值,意味着细菌在矿物表面的吸附是热力学自发过程。高岭石对细菌的吸附自由能大于蒙脱石对细菌的吸附自由能,表明细菌与高岭石间的亲和力较高,吸附更容易发生,这与化学吸附及滴定量热结果一致。表面热力学方法和XDLVO理论在预测细菌-矿物相互作用中有重要意义,但该方法未考虑多种非DLVO效应,如细胞表面多聚物、细菌鞭毛等在吸附反应中的作用,因此还存在一定的局限性,在揭示细菌-矿物相互作用的热力学机理方面还需与其他研究技术结合。     

细菌-矿物互作及其复合体在重金属修复中的应用

利用功能细菌辅助植物固定重金属是目前农田土壤污染修复中高效且环境友好的方式,其中细菌与矿物间相互作用广泛存在,包括细菌对矿物的溶解作用、矿物对细菌活性的影响以及细菌-矿物复合体的形成等,并贯穿整个修复过程。一方面,细菌与矿物互作会影响细菌的活性和表面特性,如带电性、表面官能团位点类型及浓度等,进而影响细菌对重金属的生物吸附行为以及辅助植物修复作用的发挥;另一方面,细菌-矿物结合形成的复合体较单一细菌、矿物组分对重金属的固定行为不同,在重金属修复过程中发挥不可忽视的作用。本文综合分析细菌与矿物的结合作用、细菌对矿物的溶解作用以及矿物对细菌活性的影响,阐述细菌-土壤矿物(矿物材料)复合体在重金属污染修复中的应用潜能,为复合体应用于重金属污染土壤环境提供理论依据。     

Use of fatty acid profiles to identify food-borne bacterial pathogens and aerobic endospore-forming bacilli

Capillary gas chromatography (GC) with flame ionization detection was used to determine the cellular fatty acid profiles of various food-borne microbial pathogens and to compare the fatty acid profiles of spores and vegetative cells of the same endospore-forming bacilli. Fifteen bacteria, representing eight genera (Staphylococcus, Listeria, Bacillus, Yersinia, Salmonella, Shigella, Escherichia, and Vibrio) and 11 species were used to compare the extracted fatty acid methyl esters (FAMEs). Endospore-forming bacilli were processed to obtain pure spores and whole cell FAMEs for GC analysis. A data set for each bacterial agent was prepared using fatty acid profiles from five replicates prepared on different days. The results showed that these fatty acid intensity profiles were unique for each of the 11 species and that they could be used as a fingerprint for the organisms. The cellular fatty acid profiles for Bacillus anthracis and Bacillus cereus show that there are two branched chain fatty acids, iso 17:1 omega10c and 17:1 anteiso, which are unique in these species. Iso 17:1 omega10c is present in B. cereus vegetative cells and spores but is not observed in B. anthracis. The 17:1 anteiso fatty acid is present in B. anthracis cells but not in B. cereus cells. Fatty acids 16:0 2OH and 17:0 iso 3OH are present in B. anthracis and B. cereus spores but not in the vegetative cells. In summary, analysis of FAMEs from bacteria and spores can provide a sensitive procedure for the identification of food-borne pathogens.     

Glycopeptide derived from hen egg ovomucin has the ability to bind enterohemorrhagic Escherichia coli O157:H7

Ovomucin glycopeptide (OGP) was prepared by size exclusion chromatography after Pronase digestion of hen egg ovomucin, and the binding of OGP to foodborne pathogens (Bacillus cereus,Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium, and Staphylococcus aureus) was investigaed. Binding assays with biotinylated bacteria as probes in microtiter plates showed that OGP bound to only E. coli O157:H7 among these foodborne pathogens. Periodate treatment markedly reduced the binding ability, indicating that E. coli O157:H7 bound to carbohydrate moieties of OGP. Lectin blot analysis with Maackia amurensis (MAA) and Sambucus nigra (SNA), which are specific for oligosaccharides containing sialic acid, revealed their binding sites in OGP were similar to the E. coli O157:H7 binding sites that were probed with biotinylated E. coli O157:H7 after Western blotting of OGP. Sialydase treatment of OGP abolished its ability to bind E. coli O157:H7, demonstrating that sialic acid played an important role in the binding. These results suggest that OGP has E. coli O157:H7-specific binding sites that consist of sialic acid. On the basis of these properties, OGP has the potential to be an ingredient with a protective effect against E. coli O157:H7 infection and to be a novel probe for the detection of E. coli O157:H7 in the food hygiene field.     

Biosensor measurements of polar phenolics for the assessment of the bitterness and pungency of virgin olive oil

Bitterness and pungency, sensory quality attributes of virgin olive oil, are related to the presence of phenolic compounds. Fast and reliable alternatives for the evaluation of sensory attributes and phenolic content are desirable, as sensory and traditional analytical methods are time-consuming and expensive. In this study, two amperometric enzyme-based biosensors (employing tyrosinase or peroxidase) for rapid measurement of polar phenolics of olive oil were tested. The biosensor was constructed using disposable screen-printed carbon electrodes with the enzyme as biorecognition element. The sensor was coupled with a simple extraction procedure and optimized for use in flow injection analysis. The performance of the biosensor was assessed by measuring a set of virgin olive oils and comparing the results with data obtained by the reference HPLC method and sensory scores. The correlations between the tyrosinase- and peroxidase-based biosensors and phenolic content in the samples were high (r = 0.82 and 0.87, respectively), which, together with a good repeatability (rsd = 6%), suggests that these biosensors may represent a promising tool in the analysis of the total content of phenolics in virgin olive oils. The correlation with sensory quality attributes of virgin olive oil was lower, which illustrates the complexity of sensory perception. The two biosensors possessed different specificities toward different groups of phenolics, affecting bitterness and pungency prediction. The peroxidase-based biosensor showed a significant correlation (r = 0.66) with pungency.     

Mapping pathways of possible phage-mediated genetic interchange among soil bacilli

Bacillus pumilus (Strain W43) has been shown to sustain the growth of an unusually large number of different phages. From 22 isolates 16 distinguishable phages have been obtained. Phage BPPX which is similar to the defective particle PBSX of B. subtilis is induced by four of the phages. When tested against a variety of Bacillus spp 12 of the non-defective phages had host ranges crossing at least one species line: Phage K13 infected 14 of 25 strains, distributed among six species some of which are considered to be taxonomically distant. It is suggested that the relatively restricted host ranges noted for most Bacillus phages may result from the use. as test organisms, of bacteria isolated from soils ecologically distinct from the source(s) of phage. A genetic “circuit diagram” constructed from the host range table, maps possible genetic connexions between various soil bacilli made possible by the phages. These data are set in the context of recent theories which postulate that viruses are agents of accelerated cell evolution.     

Kombucha fermentation and its antimicrobial activity

Kombucha was prepared in a tea broth (0.5% w/v) supplemented with sucrose (10% w/v) by using a commercially available starter culture. The pH decreased steadily from 5 to 2.5 during the fermentation while the weight of the "tea fungus" and the OD of the tea broth increased through 4 days of the fermentation and remained fairly constant thereafter. The counts of acetic acid-producing bacteria and yeasts in the broth increased up to 4 days of fermentation and decreased afterward. The antimicrobial activity of Kombucha was investigated against a number of pathogenic microorganisms. Staphylococcus aureus, Shigella sonnei, Escherichia coli, Aeromonas hydrophila, Yersinia enterolitica, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus epidermis, Campylobacter jejuni, Salmonella enteritidis, Salmonella typhimurium, Bacillus cereus, Helicobacterpylori, and Listeria monocytogenes were found to be sensitive to Kombucha. According to the literature on Kombucha, acetic acid is considered to be responsible for the inhibitory effect toward a number of microbes tested, and this is also valid in the present study. However, in this study, Kombucha proved to exert antimicrobial activities against E. coli, Sh. sonnei, Sal. typhimurium, Sal. enteritidis, and Cm. jejuni, even at neutral pH and after thermal denaturation. This finding suggests the presence of antimicrobial compounds other than acetic acid and large proteins in Kombucha.     

Morphology and general characteristics of phages specific to Lens culinaris rhizobia

Four lytic phages, namely LRP-1, LRP-4, LRP-13, and LRP-15, active against indigenous rhizobial strains of Lens culinaris were isolated and characterized for their individual morphology, host range, plaque characteristics, lytic behavior, and restriction endonuclease profiling of phage DNA. All phages had a typical polyhedral head and long non-contractile tail, representing the bacteriophage family close to Siphoviridae. Phages produced distinct types of plaques on their indicator bacterial strains. The host range of the phage isolates was restricted to Rhizobium leguminosarum biovars and no cross infectivity among susceptible strains was observed. A study on the lytic cycle of the phages under identical conditions exhibited distinct latent period and burst size. Inactivation pattern of phages with temperature and UV light was quite distinct. Phage LRP-1 showed higher thermal resistance, though greater sensitivity to UV light, as compared to other phages. Genome sizes of the phages were estimated to vary between 50–72 kbp. The 16S rRNA sequence analysis of the phage indicator rhizobial strains revealed 81% to 100% similarity with R. leguminosarum bv. viciae. The phages could thus prove to be considerably useful in typing and investigating into the genetic variability which might exist amongst the soil rhizobia nodulating Lens culinaris.     

Interactions between bacteriophages and bacteria in soil

The population biology of certain phages for Bacillus species in soil is described. In soil at 37°C the growth profile of B. circulans phage ST1 shows a lag phase, a phase of exponential growth and a plateau phase, much as occurs in monoculture except that the time scale is transposed into hours rather than minutes. Individual strains of replicating bacteria have been identified by a phage typing technique. At 37°C the number of different phage-sensitive strains falls sharply during the first 6 h but has risen again by 10h. After 10 h at 37°C, 76% of the bacterial types scored represent new strains, as judged by their susceptibility to the eleven phages used for typing. The data presented indicate that a sensitive equilibrium exists between phage and host bacterial cells in soil and that this may be altered very quickly under selective pressures.     

Frequency of phage-infected bacterial cells in the floodwater of a Japanese paddy field

In aquatic environments, viruses play an important role in the microbial food web through microbial mortality from viral lysis. Bacteriophages (phages) compose the majority of viral communities in the floodwater of paddy fields. The present study evaluated bacterial mortality from phage lysis in the floodwater of a Japanese paddy field based on the frequency of phage-infected bacterial cells by transmission electron microscopy. Floodwater was sampled five times during the rice cultivation period from two plots of NPK plus lime (no-compost plot) and NPK plus lime and compost (compost plot) under a long-term and ongoing fertilizer trial that began in 1925. The frequency of visibly infected cells in the compost plot was larger, ranging from 2.4 to 3.6% (average 3.0%), than that in the no-compost plot, which ranged from 1.6 to 2.9% (average 2.0%). The fractions of bacterial mortality from phage lysis in the floodwater samples were estimated to range from 12.8 to 27.3% (average 17.2%) and from 21.7 to 35.0% (average 27.9%) for the samples collected from the no-compost plot and the compost plot, respectively. This is the first study to estimate bacterial mortality from phage lysis in the paddy field ecosystem, and the frequency of phage lysis in floodwater was found to be within the frequency ranges observed in other aquatic environments.     

Identification of antiadhesive fraction(s) in nonimmunized egg yolk powder: in vitro study

The presence of antiadhesive component(s) in the hen egg yolk against foodborne pathogens was anticipated from results of a previous animal study conducted by the authors. The previous work showed egg yolk powder without specific antibodies is effective in controlling Salmonella enteritidis,Salmonella typhimurium, and Escherichia coli O157:H7 colonization in laying hens. Therefore, this study was necessary to locate the activity and identify the effective component(s). In vitro experiments were conducted using confluent Caco-2 cell monolayers. S. enteritidis, S. typhimurium, and E. coli O157:H7 were investigated against the various extracted granule and plasma fractions in three different assays: adhesion elimination, adhesion prevention, and antimicrobial. This study revealed original findings and identified the protective yolk fraction against the foodborne pathogens as the granule component, high-density lipoproteins (HDL). The protective activity conveyed by HDL was confirmed to remain intact despite peptic and tryptic enzymatic digestion and to have antiadhesive but not antimicrobial effect.     

Production and isolation of aflatoxin M1 for toxicological studies

One hundred mg aflatoxin M1 was produced and purified for toxicological studies. Aspergillus flavus NRRL 3251 was cultured on rice to produce aflatoxins B1, B2, M1, and M2, B1 and B2 were separated from M1 and M2 by a normal phase low pressure liquid chromatography (LC) column. M1 was then separated from M2 by a reverse phase low pressure LC column. Recoveries of aflatoxins from the LC columns were about 90%. The purified M1 was confirmed by ultraviolet-visible spectrometry, mass spectrometry, nuclear magnetic resonance spectrometry, optical rotation, and its mutagenicity to Salmonella typhimurium TA98.