Kathy Messens

Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 degrees C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.

HETEROGENEOUS PHOTOBLEACHING IN CONFOCAL MICROSCOPY CAUSED BY DIFFERENCES IN REFRACTIVE INDEX AND EXCITATION MODE

The photobleaching of fluorescence emission during confocal laser scanning was studied on well-defined, stained objects [microspheres of polystyrene or fluorescent gels of fluorescein isothiocyanate (FITC)-labeled dextran] and on biological samples. X,Y laser scanning with confocal microscopy induces fundamental differences in exposure rate and time in different z-planes orthogonal to the optical axis. A heterogeneous bleaching rate was observed at different focal levels in the polystyrene spheres and in the gels. This phenomenon can be caused by refractive index differences or is correlated with a photobleaching rate, which is dependent not only on the excitation light intensity but also on the photon flux (total intensity per unit of time). Heterogeneous excitation induced by refractive index differences results in photobleaching differences but will not necessarily cause heterogeneous emission intensity. Altered emission originating from altered excitation will be annihilated if the emitted light returns to the image plane along the same inverse path, compensating for the proportional increase or decrease in excitation intensity with an increased or decreased emission intensity. High numerical aperture or increased scanning speed increases the photobleaching rate. This leads to the conclusion that photobleaching in confocal scanning laser microscopy is dependent on photon energy flux density (joule/m2s).

Detection of Genetically Modified Soy in Doughs and Cookies

ABSTRACT In many countries, including the European Union member states, Switzerland, Australia, New Zealand, and Japan, legislation has been set up for labeling of genetically modified organisms (GMOs) in food and feed products. To comply with these regulations, reliable detection methods are necessary. If the detection is based on DNA, a GMO analysis may contain several steps where qualitative and quantitative species-specific, GMO screening, GMO construct, and GMO line-specific polymerase chain reactions (PCRs) are used. A limit of detection (LOD) thereby defines to what extent a target molecule may be detected in a sample. In this study, cookies were made with variable levels of a soy sample containing 2 wt% Roundup Ready soy. For all PCRs described, detection limits based on dilution series and practical LODs were determined. The practical LODs are used to determine to what extent a GMO ingredient may be detected in a real food product. Results reveal that, due to the baking process, the overall DNA f...

Comparative Evaluation of Six Extraction Methods for DNA Quantification and PCR Detection in Cocoa and Cocoa-Derived Products

Six methodologies for extracting DNA in cocoa and cocoa-derived products (cocoa leaves, cocoa beans, cocoa powder, cocoa butter, cocoa mass, and dark chocolate) are described. These DNA extraction methods included four commercial kits DNeasy Plant kit, DNeasy Plant kit with polyvinyl polypyrrolidone (PVPP), QIAamp DNA Stool kit, Wizard Genomic DNA kit, and two CTAB based methods, CTAB-Proteinase K procedure and CTAB-SDS (sodium dodecyl sulfate) procedure. The DNA yield, purity, and quality for six different cocoa matrices are discussed. The yield and purity were determined through spectrophotometry. Three different conventional PCR reactions were used to assess the quality of the DNA extracted. The Wizard Genomic DNA kit and the CTAB-Proteinase K method were found to be the best for DNA quality and PCR detection in cocoa leaves. The CTAB-Proteinase K procedure is highly recommended for cocoa beans, while the Wizard Genomic DNA kit revealed the best PCR performance when applied to cocoa powder. The cocoa butter matrix gave the lowest DNA yield, purity, and amplification results for all the examined extraction methods. The QIAamp DNA Stool kit and the CTAB-SDS method showed the best result for PCR detection of cocoa mass. The Wizard Genomic DNA kit and the CTAB-SDS method showed the best PCR performance for dark chocolate. In general, the two CTAB based methods, namely the CTAB-Proteinase K and the CTAB-SDS were suitable for the DNA extraction of all types of cocoa-derived products with the exeption of cocoa butter. This evaluation can be especially useful for the detection of DNA in cocoa-derived samples.

Molecular characterization of Vietnamese cocoa genotypes (Theobroma cacao L.) using microsatellite markers

Vietnam has the appropriate climate, soil, and humidity for cocoa cultivation and is growing as a cocoa-producing country. To supply the international cocoa market, trees have been planted in Southern Vietnam. Cocoa quality depends on various factors, such as the genotype/cultivar, environment, and post-harvest processing. Until now, little research has been done on the genetic background of Vietnamese cocoa. Therefore, this study focused on the genetic relationships of 75 cocoa cultivars, sampled in Vietnam. Fourteen microsatellite markers were used to assay the genetic diversity and to genotype and differentiate the accessions. Descriptive statistics showed that most of the used microsatellite markers were sufficient to be used in further analysis, of which mTcCIR 15, 33, and 37 were the most polymorphic. The principal coordinate analysis (PCoA) and Bayesian clustering approach divided the samples in two groups, which were linked to the Trinitario and Forastero varieties. The Vietnamese Can Tho (CT) cultivars showed little variation, while the Thu Duc (TD) cultivars showed a continuous variation between the different reference cultivars. This indicates that the CT cultivars were Trinitario and that the TD cultivars were hybrids of Forastero or Forastero and Trinitario. The molecular characterization is an important step towards developing a strong genetic basis for the Vietnamese cocoa industry. It can be used to conserve valuable genetic material and to select promising cocoa cultivars which are disease resistant, high yielding, and fine flavored. In this way, the high-quality Vietnamese cocoa production can be improved and maintained.

DTREEv2, a computer-based support system for the risk assessment of genetically modified plants

Risk assessment of genetically modified organisms (GMOs) remains a contentious area and a major factor influencing the adoption of agricultural biotech. Methodologically, in many countries, risk assessment is conducted by expert committees with little or no recourse to databases and expert systems that can facilitate the risk assessment process. In this paper we describe DTREEv2, a computer-based decision support system for the identification of hazards related to the introduction of GM-crops into the environment. DTREEv2 structures hazard identification and evaluation by means of an Event-Tree type of analysis. The system produces an output flagging identified hazards and potential risks. It is intended to be used for the preparation and evaluation of biosafety dossiers and, as such, its usefulness extends to researchers, risk assessors and regulators in government and industry.

Discrimination of Cocoa Liquors Based on Their Odor Fingerprint: a Fast GC Electronic Nose Suitability Study

With the rising interest by consumers for high-quality cocoa products from a clear geographical origin, a rapid analytical method for quality control, authenticity and traceability assessment is of paramount importance. However, the complex mixture of volatiles present in cocoa liquor, the main ingredient for the chocolate production, complicates reaching this purpose. Hence, an analytical fingerprint approach using advanced electronic nose (E-nose) technology may offer a suitable technique. This study aimed to verify the suitability of an E-nose based on ultra-fast gas chromatography (GC) for the rapid discrimination between cocoa liquors from different origins. Fourteen cocoa liquors, produced of cocoa beans from ten different geographical origins, were analyzed. The obtained odor fingerprints were investigated using principal component analysis (PCA) which successfully discriminated most cocoa liquors, within one continent, according to their geographical origin. Besides, discriminant factor analysis (DFA) showed the possibility to differentiate between bulk and fine cocoa. Further tentative identification of predominant volatile compounds allowed the detection of compounds within a wide range of chemical classes occurring in cocoa products, such as acids, alcohols, aldehydes, ketones, esters, pyrazines, pyrones, and pyrroles. Most odorant compounds were previously described in literature as key volatiles in cocoa flavor, notable examples are acetic acid, 2-heptanol, 2/3-methylbutanal, acetophenone, isoamyl acetate, tetramethylpyrazine, maltol, and 2-acetyl-1-pyrroline. This study proves for the first time the usefulness of the GC E-nose for effective and rapid aroma profiling and discrimination between single origin cocoa liquors, which can be easily applied in the cocoa industry.

The suprachromosomal organization of DNA as a morphogenetic element in plant development

The suprachromosomal organization of DNA in cell nuclei is examined by the use of (whole mount) fluorescence in situ hybridization (FISH), fluorescence immunocytochemistry (FICC) and confocal scanning laser microscopy (CSLM). The distribution of ribosomal DNA (5.8S, 18S, 25S and 5S rDNA) and a chromosome specific highly repetitive sequence (a 500bp repeated sequence) were examined in the chromatin of diploid (2n) and tetraploid (4n) nuclei of different tissues of Arabidopsis thaliana (Bauwens et at. (1991), Chromosoma, 101, 41-48). Qualitative and quantitative analysis of the images recorded with a Bio-Rad MRC-600 confocal scanning laser microscope atler FISH with the 45S rDNA probe on diploid nuclei showed two larger and two smaller sites. Some of the nuclei exhibited more than the four expected spots, possibly due to polyploidy or differential amplification of the rDNA. Many of the interphase nuclei had a reduced number of hybridization spots, which could be the result of a (non random) chromosomal association. Some research was also done on 5S rDNA, where the diploid nuclei showed mostly two rDNA-spots and the tetraploid nuclei mostly four labeled sites. All this previous work was done on squashpreparations of four (2n) or five days (4n) old seedlings. Whole mount in situ hybridization (Bauwens et al. (1994), Plant Journal, 6, 123-131) was used to examine whether the suprachromosomal organization was tissue specific or not. Preliminary tests indicate that there exists such a correlation e.g. The hybridization of the 500bp repeated sequence is quite specific. In the root these spots seem to be oriented according to a line running parallel to the newly formed cell wall and therefore are oriented in a tissue specific manner. Another part of the research exits in optimizing protocols for the (whole mount) immunolocalization of tubulines in Arabidopsis thaliana. Our goal is to combine the immunolocalization of tubulines with (whole mount) in situ hybridization and to determine in this way the relation between the suprachromosomal organization and cell division. LOCALIZATION OF mRNAs AND PROTEINS OF P14 AND P20 IN RAT PITUITARY