Real-time-PCR-System zum Nachweis von Bacillus cereus (emetischer Typ) in Lebensmitteln

Bacillus cereus is a Gram-positive, facultative anaerobic, spore-performing bacterium. Some B. cereus strains have the ability to produce two different types of toxins: (a) diarrhoeic toxin: the disease is similar to a C. perfringens toxin-infection; it is caused by a heat-labile protein, (b) emetic toxin: the disease is similar to Staphylococcus-aureus intoxikation; it is caused by a heat-stable protein. Statements about the frequency of B. cereusfood-poisonings are difficult because a reporting system for this disease is missing in Germany and there exists no valid methodology to diagnose this disease with conventional microbiological or biochemical methods. Nevertheless B. cereus, beside S. aureus, seems to be the most important bacterium to cause food-associated intoxications. The detection of emetic toxin directly in food is difficult, time-consuming and expensive (HPLC-MS) and so a real-time-PCR assay including an Intern Amplification Control (IAC) for the detection of emetic B. cereus was developed and tested with different food matrices and routine samples. The assay is a rapid and reliable detection system for the routine laboratory to confirm in combination with microbiological methods (plating, colony-counting or enrichment) the suspicion of a food-associated B. cereus intoxication. This is a great progress from the epidemiological and legal point of view, particularly as a first diagnosis can be given after 30 hours. In addition it will be possible to make a risk assessment for different food products in order to protect the consumers’ health, because investigations can be performed more easily and frequently.

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Zusammenfassung: Bacillus cereus z?hlt zu den gram-positiven, fakultativ anaeroben, sporenbildenden Bakterien. Einige Bacillus cereus-St?mme sind in der Lage, zwei unterschiedliche Arten von Toxinen zu bilden: (a) diarrhoeisches Toxin: Die Erkrankung ?hnelt einer Toxin-Infektion durch C. perfringens, ausgel?st durch ein hitzelabiles Protein, (b) emetisches Toxin: Die Erkrankung ?hnelt einer Staphylokokken-Intoxikation, ausgel?st durch ein niedermolekulares hitzestabiles Protein. Aussagen über die H?ufigkeit von Bacillus cereus-bedingten Krankheitsausbrüchen sind schwierig, da keine generelle Meldepflicht besteht und die Diagnosestellung mittels konventioneller mikrobiologischer Methoden nicht m?glich ist. Man geht aber davon aus, dass Bacillus cereus neben Staphylococcus aureus zu den h?ufigsten Lebensmittel-Intoxikationserregern z?hlt. Da sich der direkte Toxinnachweis aus dem Lebensmittel für das emetische Toxin nach wie vor als schwierig und teuer erweist (HPLC-MS), wurde ein real-time-PCR-System einschlie?lich einer Internen Amplifikationskontrolle (IAC) zum Nachweis von Bacillus cereus, emetischer Typ etabliert, an unterschiedlichen Lebensmittelmatrices und anschlie?end an Probenmaterial aus dem t?glichen Probenaufkommen getestet. Das real-time-PCR-System hat sich als eine schnelle und zuverl?ssige Nachweismethode für die Routinediagnostik erwiesen, mit der sich in Kombination mit mikrobiologischen Methoden (Kontaktplattenverfahren, Keimzahlbestimmung, Anreicherungsverfahren) der Verdacht auf eine lebensmittelbedingte Bacillus cereus-Intoxikation (emetisches Toxin) best?tigen l?sst. Dies stellt aus epidemiologischer und lebensmittelrechtlicher Sicht einen gro?en Fortschritt dar, zumal eine erste Verdachtsdiagnose bereits nach 30 Stunden gestellt werden kann. Ebenso ist es m?glich, durch weitergehende Untersuchungen eine Risikoabsch?tzung für unterschiedliche Produktgruppen im Sinne des gesundheitlichen Verbraucherschutzes zu treffen.

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Eingegangen: 19. Januar 2007     

Production of triacylglycerol and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by the toluene-degrading bacterium Rhodococcus aetherivorans IAR1

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) produced by various bacteria has been intensively investigated as a promising biodegradable plastic, but required a supply of an expensive precursor as a secondary carbon source for its production. In a previous study, we identified a new bacterial strain, Rhodococcus aetherivorans IAR1, which synthesizes PHBV from toluene without the supply of a precursor. Toluene is the volatile organic compound most abundantly emitted to the environment. In the present paper, we show that R. aetherivorans IAR1 produces triacylglycerols (TAGs) simultaneously with PHBV. Both PHBV and TAGs were synthesized before the nitrogen source is completely exhausted. The cellular content of PHBV reached 10% of cell dry weight (CDW) and its synthesis ceased even during intermittent supply of toluene. However, accumulation of TAGs continued during cultivation and their cellular content reached 24% of CDW at the end of cultivation. Cerulenin inhibited TAG production and increased PHBV cellular content up to 30% of CDW. The mole fraction of 3-hydroxyvalerate (3HV) in PHBV produced from toluene increased from 60% to 80% during its accumulation. Fatty acid compositions of TAGs produced from acetate and toluene were different. At the end of cultivation, the mole fraction of C17:0, one of odd-carbon number fatty acids, was 5% on toluene or 10% on acetate while the mole fraction of 3HV in PHBV from toluene was as high as that in PHBV from acetate, suggesting that a C5 intermediate of toluene degradation might directly become a precursor of 3HV whereas propionyl-CoA is required for the incorporation of C17:0 into TAGs.     

HPLC-ICPMS联用测定废水中的三价铬与六价铬

建立了同时测定废水中三价铬和六价铬的液相色谱仪(HPLC)和电感耦合等离子体质谱仪(ICPMS)联用的分析方法.使用Hypersil GOLD TM(150伊4.6 mm,5滋m)色谱柱,流动相为5 mmoL/L(pH=7)EDTA,流速1.2 mL/min时三价铬与EDTA配位生成稳定的Cr(芋)原EDTA和六价铬可以在4 min内完成两种价态铬含量的分析.该方法由于样品制备和色谱条件的最优化,可以分析高基体样品,比如废水.当进样量为100滋L时,该方法对三价铬和六价铬的检测限分别为43.4 ng/L和82.8 ng/L.该方法灵敏度高,分析干扰少,高效准确.     

Fermentative production of (R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.     

Characterisation of 2-Cys peroxiredoxin isozyme (Prx1) from Taiwanofungus camphorata (Niu-chang-chih): Expression and enzyme properties

Peroxiredoxins (Prxs) are a family of antioxidant peroxidases. The functions of Prxs comprises of cell protection against oxidative stress and regulation of cell proliferation. A putative 2-Cys Prx isozyme (Prx1) cDNA was cloned from Taiwanofungus camphorata (commonly known as Niu-chang-chih in Taiwan). The deduced amino acid sequence is conserved amongst the reported Prxs. A 3-D homology structure was created for this Prx1. To characterise the T. camphorata Prx1, the coding region was subcloned into a pAVD10 and transformed into Escherichia coli. The recombinant 6His-tagged Prx1 was expressed and purified by Ni²⁺-nitrilotriacetic acid sepharose. The purified enzyme showed two forms using a 15% SDS–PAGE. The enzyme retained 60% activity at 60 °C for 2.5 min. The enzyme was stable under a broad pH range from 5 to 11. The enzyme showed 57% activity after 40 min of incubation at 37 °C with trypsin. The ability of the enzyme to protect intact supercoiled plasmid DNA from ^·OH induced nicking was demonstrated.     

Degradation of 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) by brown-rot fungi

Twelve species of brown-rot fungi (BRF) have been investigated for their ability to degrade 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT). In potato dextrose broth (PDB) medium, Gloeophyllum trabeum, Fomitopsis pinicola and Daedalea dickinsii showed a high ability to degrade DDT. 1,1-Dichloro-2,2-bis (4-chlorophenyl) ethane (DDD), 1,1-dichloro-2,2-bis (4-chlorophenyl) ethylene (DDE) and 4,4-dichlorobenzophenone (DBP) were detected as metabolic products of DDT degradation by G. trabeum in PDB medium. The DDT degradation pathway in G. trabeum is proposed, which differs from the proposed pathways in bacteria and other fungi, particularly in the transformation of DDE to DDD. On the other hand, DBP was not detected as a metabolic product of DDT degradation in FeSO(4)-deficient cultures of G. trabeum, whereas DDE and DDD were detected, indicating the involvement of an iron-dependent reaction. Only DBP was detected from DDT, DDE and DDD degradation via a chemical Fenton reaction under conditions similar to those in G. trabeum cultures. These compounds may be directly transformed to DBP via a Fenton reaction.     

Improvement of poly(3-hydroxybutyrate) [P(3HB)] production in Corynebacterium glutamicum by codon optimization, point mutation and gene dosage of P(3HB) biosynthetic genes

In our previous study, a system for producing poly(3-hydroxybutyrate) [P(3HB)] was established by introducing a polyhydroxyalkanoate (PHA) biosynthetic gene operon (phaCAB Re) derived from Ralstonia eutropha into Corynebacterium glutamicum. In this study, two experimental strategies have been applied to improve P(3HB) production in recombinant C. glutamicum. One is a codon optimization of the N-terminal-coding region of the PHA synthase (PhaC Re) gene focusing on the codon usage preference for the translation system of C. glutamicum. The other is the replacement of wild-type phaC Re with a modified gene encoding a mutation of Gly4Asp (G4D), which enhanced the production of PhaC Re and P(3HB) in Escherichia coli. The introduction of these engineered PHA synthase genes into C. glutamicum enhanced the production of PhaC(Re) and P(3HB). Interestingly, we found that these gene modifications also caused increases in the concentration of the translation products of the genes encoding monomer-supplying enzymes, beta-ketothiolase (PhaA Re) and acetoacetyl-CoA reductase (PhaB Re). This finding prompted us to carry out a gene dosage of phaAB Re for a double plasmid system, and the highest production (52.5 wt%) of P(3HB) was finally achieved by combining the gene dosage of phaAB Re with codon optimization. The molecular weight of P(3HB) was also increased by approximately 2-fold, as was P(3HB) content. Microscopic observation revealed that the volume of the cells accumulating P(3HB) was increased by more than 4-fold compared with the non-P(3HB)-accumulating cells without filamentous morphologenesis observed in E. coli.     

传统陈窖豆豉粑和霉菌型豆豉挥发性风味化合物研究

采用顶空固相微萃取（HS—SPME）和气质联用（GC—MS）方法，共鉴定出贵州传统陈窖豆豉粑和霉菌型豆豉四个样品中的挥发性化合物102种，其中DCB-1、DCB-2、DCB-3和LGM-DC分别为65、48、50和47种，这些化合物包括醇（16）、酸（13）、酯（18）、醛（5）、酮（11）、酚（2）、吡嗪（11）、呋喃（9）、吡喃酮（2）、含硫化合物（4）、酰胺类化合物（9）以及其它化合物（2）等12类。已报道的对大豆发酵调味品风味贡献较大的41种化合物至少在三个样品中同时检出。就Maillard反应特征产物之一的吡嗪类化合物而占，其含量仅次于酸，可能对产品主体风味的形成起着重要作用。     

普通肉食螨对粗脚粉螨的捕食功能研究

普通肉食螨广泛分布于粮库及仓储场所,是理想的天敌资源,对害螨及储粮害虫的卵和低龄幼虫有很好的防治潜能。为明确普通肉食螨对害螨的防控能力,以粗脚粉螨为对象,研究普通肉食螨原若螨、后若螨、雌成螨三种螨态对粗脚粉螨卵、幼螨、若螨和成螨的捕食功能反应。结果表明,普通肉食螨三种螨态对粗脚粉螨各螨态的功能反应均属于HollingⅡ型,普通肉食螨三种螨态中,雌成螨对粗脚粉螨卵、幼螨、若螨和成螨的攻击能力最强,攻击系数分别是0.913、1.030、1.017和0.989;普通肉食螨喜食粗脚粉螨幼螨,最大捕食效能为42.436头/d;在捕食能力方面,除粗脚粉螨卵以外,普通肉食螨对粗脚粉螨的捕食能力大小均为:雌成螨后若螨原若螨,普通肉食螨对粗脚粉螨具有很好的防治潜能。本研究为普通肉食螨防治储粮害螨奠定了基础参数,为进一步应用普通肉食螨控制害虫害螨提供参考。     

Determination of ethylenethiourea (ETU) and propylenethiourea (PTU) in foods by high performance liquid chromatography-atmospheric pressure chemical ionisation-medium-resolution mass spectrometry

A robust and sensitive method for the determination of ethylenethiourea (ETU) and iso-propylenethiourea (i-PTU) in foods is reported. ETU and i-PTU were extracted by blending with dichloromethane (DCM) in the presence of sodium sulphate, sodium carbonate, thiourea and ascorbic acid. ²H₄-ETU and n-PTU were used as internal standards. After filtration the DCM was removed by rotary evaporation and the extract re-dissolved in water before analysis by reversed-phase liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry using a double focusing mass spectrometer at a resolution of 5000. Mean recoveries of ETU and i-PTU from fruit-based, cereal-based and meat-based infant foods, potato chips and tinned potatos at 0.01 mg kg^-1 and from pizza and yoghurt at 0.02-0.1 mg kg^-1 were 95% and 97% respectively. Precision, including both repeatability and internal reproducibility, was in the range of 3.1-13.1%.     

Effects of gibberellic acid (GA3) on strawberry PAL (phenylalanine ammonia-lyase) and TAL (tyrosine ammonia-lyase) enzyme activities

Gibberellic acid (GA₃) treatments (30 and 60 μg litre⁻¹) were applied to young plants (Fragaria ananassa cv Chandler). Fruits were harvested at various developmental stages (14, 21, 28 and 35 days from fruit set). Weight and size, phenolic compounds (total polyphenols and anthocyanins) and enzyme activities, phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) were determined. Our aim was to obtain detailed information about PAL and TAL activities related to the strawberry colour during development and ripening processes and to determine the effects of exogenous treatments of GA₃ on PAL and TAL activities. Exogenous treatments of GA₃ improve weight, size and colour of strawberry fruits, and affect PAL and TAL activities. We found that the anthocyanin content and PAL activity are enhanced by the exogenous treatment of GA₃ in the range of 30 μg litre⁻¹. However, with the higher GA₃ treatment, only the anthocyanin content is affected in that way. These findings suggest that gibberellic acid effect on PAL, TAL and ultimately anthocyanin enhancement is dosage related and saturation of the response occurs at 30 μg litre⁻¹. © 1998 SCI.     

Cloning of inulin fructotransferase (DFA III-producing) gene from Arthrobacter globiformis C11-1

A gene encoding an inulin fructotransferase (DFA III-producing) [EC 2.4.1.93] from Arthrobacter globiformis C11-1 was cloned and the nucleotide sequence was determined. The cloned fragment contained a 1353 bp open reading frame. The initiation codon was estimated to be an unusual codon, GTG. The gene encoded a signal peptide (40 amino acid residues) for secretion. The molecular mass of the native enzyme was calculated as 43,400 Da from the sequencing data. The deduced amino acid sequence of the enzyme had 74.0 % homology with that of inulin fructotransferase (DFA III-producing) from Arthrobacter sp. H65-7. It also had 45.1% homology with that of inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter globiformis S14-3. The enzyme produced in the culture supernatant of an Escherichia coli clone was purified to the electrophoretically homogeneous stage. The N-terminal amino acid sequence of the cloned enzyme secreted in the broth was the same as that of the native enzyme from A. globiformis C11-1. Therefore, on this enzyme, it is estimated that the cleavage sites by the signal peptidase for secretion of A. globiformis C11-1 and E. coli JM109 are the same.     

Visualization of temporal differences between dominant perceptions in temporal dominance of sensations (TDS) and temporal check-all-that-apply (TCATA) perceptions using dominance-highlighted TCATA (dTCATA) curves

The temporal dominance of sensations (TDS) method measures dynamic changes of panelists’ attention to the sensory attributes of products. The temporal check-all-that-apply (TCATA) method measures all sensory attributes perceived at each moment of an evaluation. However, unlike in TDS, significant levels cannot be calculated in TCATA. This study proposes the use of dominance-highlighted TCATA (dTCATA) curves, which are highlighted TCATA curves that show significant time periods for the TDS data of different panels. Twelve R&D panelists evaluated five commercial corn soups using the TCATA method. Then, 125 consumer panelists evaluated the same products using the TDS method. The dTCATA curves showed TCATA curves for all attributes for each product evaluated by the R&D panel highlighted with the dominance rates identified by the consumer panel in the TDS evaluation. For example, for product 1, some attributes (sweet, viscosity) showed relatively high citation proportions in the TCATA evaluations of the R&D panel and significant dominance rates in the TDS evaluations of the consumer panel. In contrast, consommé flavor showed relatively low citation proportions in TCATA but significant dominance rates in TDS. By merging TDS and TCATA data, we could compare consumers’ dominant sensations with the evaluations of R&D panelists. This comparison could provide useful insights to product developers. In some cases, we observed attributes with significant dominance rates that were under-identified by the R&D panel in TCATA. This could suggest that most of the R&D panel may not have perceived these attributes; therefore, during product development, these attributes should be carefully considered.     

Rapid determination of polybrominated diphenyl ethers (PBDEs) in fish using selective pressurized liquid extraction (SPLE) combined with automated online gel permeation chromatography-gas chromatography mass spectrometry (GPC-GC/MS)

A rapid method for the determination of seven predominant polybrominated diphenyl ethers (PBDEs) in fish was developed. The analysis could be completed in 1 h. The PBDEs in the sample were extracted with fat by selective pressurized liquid extraction (SPLE) using acid alumina as a fat retainer. After concentration, the extract was directly injected into online gel permeation chromatography with gas chromatography-mass spectrometry (GPC-GC/MS) in negative chemical ionization (NCI) mode. Method detection limits of seven PBDEs were between 0.002 and 0.005 ng g^?1 fat. Quantitative recoveries ranging from 81.9% to 110.3% were obtained by analysis of spiked fish. The relative standard deviations were less than 15% (n = 6). The method was applied to a certified reference material of fish; all results were in the reference ranges. It was shown to be a rapid and reliable alternative in the routine analysis for PBDEs.     

Influence of the electron acceptor on nitrite reductase gene (nir) diversity in an activated sludge community

Analyses of the nitrite reductase gene diversities (nirK and nirS) in an activated sludge community fed with both nitrite and glucose were conducted. Results suggest that the topology of nirK and nirS gene fragment-based phylogenetic trees is influenced more by the available electron acceptor than by the carbon source. A denitrification reactor was operated for 53 days and a clone library constructed when the denitrifying communities in the SBR were supplied with both nitrite and glucose. Half of the nirK and nearly all the nirS gene fragments formed a cluster that was separate from a cluster containing nirK and nirS sequences derived from other communities in nitrate-fed reactors. On the other hand, nirK and nirS fragments obtained with glucose as the carbon source were similar to those detected in communities fed with other carbon sources.     

食品中Cr(Ⅲ)和Cr(Ⅵ)分离及ICP-MS法测定研究

本文报道了食品中Cr(Ⅲ)和Cr(Ⅵ)分离并用ICP-MS仪进行测定方法．食品经微波消化后，用离子交换树脂进行交换，以^45Sc为内标元素，采用内标法进行测定。该方法加标回收率是98．6％-99．6％，相对标准偏差RSD是2．1％～3．5％，检测限是0．006-0．01ng／ml，可用于食品以及饮用水、废水中Cr(Ⅲ)和Cr(Ⅵ)的测定。     

Determination of 2,6-diisopropylnaphthalene (DIPN) and n-dibutylphthalate (DBP) in food and paper packaging materials from US marketplaces

A gas chromatography-ion-trap tandem mass spectrometry procedure was developed for the determination of 2,6-diisopropylnaphthalene (DIPN) and n-dibutylphthalate (DBP) in domestic and imported paper packages and food sold in US marketplaces. The procedure involved ultrasonic extraction with dichloromethane, followed by analysis with the gas chromatography-ion-trap tandem mass spectrometry. Calibration curves for DIPN and DBP were achieved with concentrations ranging from 0.01 to 10 µg ml^?1 and the corresponding r ² values were 0.9976 and 0.9956, respectively. In most of the fortified samples the recoveries were higher than 80% with a relative standard deviation (RSD) <10%. Using this procedure, it was found that less than 20% of the tested domestic packages and more than 60% of the tested imported food packages contained both DIPN and DBP. The concentrations of DIPN and DBP ranged from 0.09 to 20 mg kg^?1 and 0.14 to 55 mg kg^?1, respectively, with most of the DINP and DBP levels lower than 20 mg kg^?1. DIPN was not detected (<0.01 mg kg^?1) in 41 food samples and DBP was only detected in two domestic and four imported food samples with concentrations ranging from <0.01 to 0.81 mg kg^?1.     

皮革中多环芳烃(PAHs)测定方法

介绍了皮革中多环芳烃的测定方法,样品用正己烷经加速溶剂萃取、凝胶渗透色谱净化、自动定量浓缩后,用气相色谱质谱联用仪测定。方法检测低限为0.20μg/mL,实验室内相对标准偏差小于5%,回收率除萘以外均大于80%。     

荧光增白剂在高得率浆漂白及含高得率浆高级纸中的应用

近年来,高得率浆(HYP)在高级纸中的应用逐渐增加,对高得率浆的视觉品质要求也相应提高,如何高效合理地提高浆张的白度以及白度稳定性成为广大造纸工作者关注的热点。传统的HYP过氧化氢漂白成本较高,污染负荷比较严重,而且以损失高得率浆的松厚度和光散射系数等特性为代价来取得较高的白度。本文主要围绕高得率浆的发展及应用,论述了近些年高得率浆的漂白工艺的研究进展。此外,还探讨了高得率浆的过氧化氢/OBA漂白工艺与传统高得率浆过氧化氢漂白工艺的优缺点,以及荧光增白剂在含高得率浆高级纸中的应用研究。     

多酚氧化酶及超氧化物歧化酶与花生霉变的关系

探讨多酚氧化酶(PPO)及超氧化物歧化酶(SOD)活力变化与花生霉变进程的关系,为食品质量检测及食品的储藏安全提供理论依据。分别采用邻苯二酚显色法和邻苯三酚自氧化法测定PPO及SOD的酶活力。结果表明,PPO及SOD酶活均随着花生霉变进程变化而变化,呈现出特定的规律性。安全可食阶段的花生PPO酶活最高,而SOD酶活则最低。当花生微量长霉时,PPO酶活减少率为38.42%,而SOD酶活增加率则超过500%。这些数据可以作为花生是否霉变的评判依据。