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Extended research has been carried out to clarify the ecological role of plant secondary metabolites (SMs). Although their primary ecological function is self-defence, bioactive compounds have long been used in alternative medicine or in biological control of pests. One single plant may contain a wide variety of bioactive compounds, making analytics rather costly. The total bactericide capacity can be quantified by either microbiological or ecotoxicological methods. Here, the principle and possible applications of a specific bacterial bioluminescence inhibition based ecotoxicological assay are reviewed.

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Poll. 1981 16 301 315 Nealson, K. H., Hastings, J. W. (1979) Bacterial bioluminescence: its

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A bioluminescent derivative of Bacillus subtilis containing a plasmid encoding a luxAB fusion under control of a vegetative promoter and gives bioluminescence upon addition of an exogenous long-chain aldehyde has been used as test organism. Its spore populations have been produced and their heat- and radiation survival curves established. Heat-sensitization effect of pre-irradiation of spores was proven not only by colony counting but also with differential scanning calorimetry. Under a linearly programmed temperature increase, the heat destruction of spores surviving 2.5 kGy gamma irradiation resulted in at a few centigrade lower temperature than that of untreated spores. Heat denaturation endotherms in the DSC-thermogram of irradiated spores were shifted to lower temperatures as well. Comparative turbidimetric, luminometric and phase-contrast microscopic studies of untreated, heat-treated and irradiated spore populations showed that the kinetics of germination and the light emission during germination of radiation-inactivated spores were the same as those of untreated spores, revealing that the pre-formed luciferase enzyme packaged into the spores during sporulation remained intact after an irradiation dose causing 90% decrease in number of colony forming spores. Therefore, in contrast to heat-treated spores, the initial bioluminescence reading upon germination of irradiated spores does not reflect the viable count of their population.

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A recombinant Bacillus subtilis strain containing a plasmid encoding a luxAB fusion, which gave bioluminescence upon addition of an exogenous long-chain aldehyde as substrate for the endogenous luciferase enzyme, was used as test organism. Its populations were treated with 300 MPa for 20 min, or 600 MPa for 20 min at around room temperature, and this treatment is foreseen as a quality-friendly, non-thermal pasteurisation of foods. Besides the estimation of viable cell counts, the extent of pressure-induced germination and post-process development were investigated by phase-contrast microscopy, turbidimetry and luminometry. Increased heat sensitivity of pressurized spore populations was observed both by viable cell counting during a linearly programmed elevation of temperature and a simultaneous differential scanning calorimetry. This was related to pressure-induced germination of spores, although a small fraction remained ungerminated. The luciferase pool built into the spores during their formation seemed to have withstood pressurization. Spore germination was accompanied by the emergence of bioluminescence which also indicated sensitively the characteristic changes of metabolic activity running parallel with the development of untreated cell populations and that of the survivors of the hydrostatic pressure treatments when the cells were incubated in a nutrient broth.

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Suspensions of a bioluminescent (luxAB) transformant of Listeria monocytogenes in pH 7.0 phosphate buffer were pressurised and the effect of the pressure treatment was monitored by plate counting. When the bacteria were suspended in NaCl- and nisin-free buffer the number of colony forming units (CFU) decreased by 3 and 6 log cycles after 300 MPA for 10 and 30 min, respectively. Supplementing the plating medium with 5% NaCl did not influence the colony forming capacity of non-pressurised cells, however, CFU of residual populations after respective treatments of 300 MPa for 10 and 30 min were reduced by a further 2 and 3.5 log cycles in case of salt containing plates. Nisin-addition to the plating medium caused less than one log unit decrease in the CFU of the non-pressurised population. However, the CFU of 10 min-pressurised sample was 4 log cycles less in the nisin-containing plates than in the nisin-free ones, whereas no colonies were formed in the nisin-containing plates even when 1 ml was inoculated from the originally 1010 CFU/ml population after 300 MPa for 30 min. The luciferase activities (bioluminescence intensities) decreased concomitant with the reduction of the viable cell counts, however, they were approx. 0.6-0.8 log units less in the presence of 5% NaCl in the pressurised suspension than those expected from the previously established linear correlation between the logarithmic light outputs and the logarithmic viable cell counts.

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Rapid analysis by coupling HPTLC with bioluminescence and mass spectrometry enables very fast response to bioactive substances in unknown samples. In this study marine sponges were screened for new bioactive compounds. After chromatographic separation of twelve methanolic marine sponge extracts the HPTLC plates were coated with bioluminescent bacteria ( Vibrio fischeri ) by a simple dipping procedure. If separated compounds were bioactive they inhibited or enhanced the bacterial luminescence and could be identified as dark zones on the luminescent background. This micro-biological detection revealed new compounds compared with physical (absorbance or fluorescence measurement) or chemical (microchemical derivatization) detection techniques. Effect-directed analysis turned out to be superior to target analysis in the search for natural products with a distinct effect. For identification of unknown bioactive zones the HPTLC system was coupled to a high-resolution mass spectrometer to obtain the exact masses of the unknowns. Thus, a Vibrio fischeri -bioactive zone was identified as avarone, a bioactive metabolite so far only known to be synthesized by the sponge Dysidea avara . This methodology proved very effective not only for detection but also for identification of unknown bioactive metabolites in sponges.

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We present a video-densitometric quantification method in combination with diode-array quantification for the methyl-, ethyl-, propyl-, and butylparaben in cosmetics. These parabens were separated on cyanopropyl bonded plates using water-acetonitrile-dioxane-ethanol-NH3 (25%) (8:2:1:1:0.05, v/v) as mobile phase. The quantification is based on UV-measurements at 255 nm and a bioeffectively-linked analysis using Vibrio fischeri bacteria. Within 5 min, a Tidas S 700 diode-array scanner (J&M, Aalen, Germany) scans 8 tracks and thus measures in total 5600 spectra in the wavelengths range from 190 to 1000 nm. The quantification range for all these parabens is from 20 to 400 ng per band, measured at 255 nm. In the V. fischeri assay a CCD-camera registers the white light of the light-emitting bacteria within 10 min. All parabens effectively suppress the bacterial light emission which can be used for quantifying within a linear range from 100 to 400 ng. Measurements were carried out using a 16-bit MicroChemi chemiluminescence system (biostep GmbH, Jahnsdorf, Germany), using a CCD camera with 4.19 megapixels. The range of linearity is achieved because the extended Kubelka-Munk expression was used for data transformation. The separation method is inexpensive, fast, and reliable.

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Acta Biologica Hungarica
Authors:
G. Paulovits
,
Nóra Kováts
,
A. Ács
,
Á. Ferincz
,
Anikó Kovács
,
B. Kakasi
,
Sz. Nagy
, and
Gy. Kiss

., Heinlaan, M., Kurvet, I., Kahru, A. (2008) High throughput kinetic Vibrio fischeri bioluminescence inhibition assay for study of toxic effects of nanoparticles. Toxicol. In Vitro 22 , 1402–1417. Kahru A

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JPC - Journal of Planar Chromatography - Modern TLC
Authors:
Wolfgang Schulz
,
Wolfram Seitz
,
Stefan Weiss
,
Walter Weber
,
Martin Böhm
, and
Dirk Flottmann

Combination of HPTLC with the bioluminescence test using Vibrio fischeri enables effective screening for bioactive substances in unknown samples. This paper describes several applications in environmental testing using bioluminescence inhibition in HPTLC. The samples were extracted by solid-phase extraction or applied directly to the TLC plate. After chromatography of the samples the plates were immersed in a suspension of bioluminescence bacteria. The bioactive substances were identified as dark or light zones in comparison with the background of the plate. By the use of this method several types of sports field granule were compared for their toxicity toward Vibrio fischeri . It was also possible to compare the bioactivity of expressway waste water or landfill leachate by use of the method. To evaluate the digital images taken by a CCD camera, inhibition chromatograms over the developed sample tracks were created.

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Direct bioautography performed with luminescence gene-tagged bacteria enables almost real-time detection of antimicrobial compounds in plant extracts. This method for the detection of chamomile ( Matricaria recutita ) components with antibacterial effect against Bacillus subtilis soil bacteria was more sensitive than commonly used bioautographic visualization by staining with a tetrazolium salt. Some compounds had a strong inhibiting effect only via the bioluminescence measurement. Extraction of antibacterial components of chamomile flowers was most effective with 50% ethanol; slightly lower efficiency was achieved with acetone and methanol, and hexane was least effective. The results were confirmed by using luminescent Pseudomonas syringae pv. maculicola plant pathogen bacteria.

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