Microbiology

The effects of different factors (temperature, light, enzymic activation) on the ability of selected mutagens and carcinogens to induce hereditary bleaching of Euglena gracilis were investigated. In the resting medium, the elevation of incubation temperature from 25°C to 37°C increased significantly the effect of all compounds tested on the frequency of bleached mutants of E. gracilis. The effect of light is not so unambiguous. While nitrosoguanidine (NG) exhibited practically the same bleaching activity both in the light and dark, the mutagenic effect of sodium azide (SA), nitrovin (NV), nitrosoethylurea (NEU), and benzo(a)pyrene (BP) was decreased in light. On the other hand, the light increased the bleaching activity of 5-nitro-2-furylacrylic acid (NFAA) significantly. The activation mixture S9 increased bleaching effect of NFAA and BP, whereas other mutagens were partially (NG and SA) or completely (NV and NEU) inactivated.

A method based upon polymerase chain reaction (PCR) for the detection of Phaseolus vulgaris beans in chestnut purØe was developed. The method involves DNA isolation by the GeneSpin kit and PCR with primers oriented to a sequence of a nuclear gene encoding PvLEA-18, a late embryogenesis-abundant protein of beans. The PCR method was shown to be specific for Ph. vulgaris, producing a 100 bp fragment with six Ph. vulgaris cultivars, and negative results with four non-Ph. vulgaris Phaseolus samples and five non-Phaseolus legumes. When evaluated with model mixtures of Ph. vulgaris beans and the chestnut purØe, a detection limit of 1% was determined.

Aims:  The aim of this study was to develop a 5′-nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes.Methods and Results:  Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 104 cfu ml−1 after 35 cycles and 102 cfu ml−1 after 45 cycles were achieved by PCR in both real-time and end-point fluorescence measurement modes. Linear calibration lines were obtained in the range from 102 to 109 cfu ml−1 for three L. monocytogenes strains in real-time PCR with 45 cycles.Conclusions:  The developed 5′-nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes.Significance and Impact of the Study:  In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.

Aim:  A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum.Methods and Results:  The method is a nested PCR targeting a specific region of rDNA of C. parvum, which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre.Conclusions:  The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format.Significance and Impact of the Study:  The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.

A polymerase chain reaction-bascd method for the detection of gluten-containing cereals in flours and "gluten-free" bakery products was optimized and its intriLlaboratory valid{tion was carried out. Thc optimized method involved DNA isolatioD by chaotropic solid-phase extraction and PCR with primcrs of Dahindcu ct al. [Dahinden L, von Biiren M.. Liithy J., 200]. A quantitative competitive PCR system to detect contamin{tion of wheal. barley and ryc in gluten-free food for coeliac patients. Europgan Food Research and Technology 212, 328-2331. Using purified DNA. intrinsic detection limit of42 t 12pg was determined, which corresponds to 10o genomc copies. B; the analysis of a pancl of 26 European wheat cultivars and flours from six non-gluten-containing plants. which are commonly used for the pfoductior of gluten-free bakery products, inclusivity of 100'% and exclusivity of 100% wcre determined. By the anal],sis of model samples of soya flour and cakes. dctcction limit of 0.1%, (w/w) of line wheat flour was determined, which is suitablc for thc analysis of "glulcD-llcc" food products, as it is approximately equivalent to the limit of lOmg per l00g for gluten stated b)' Codex Alinlentarius. The mcthod was successfully applied to four samplcs of flours and l3 brands of biscuits designated "gluten-free". oul of \\'hich nvo flours and onc brand of biscuits were found positive for glutencontaining cereals. The method proved to bc suitable lbr routinc use. it was relatively straightforward and could be completed in one working day. O 2004 Elsevier Ltd. All rights leservcd.

A method based upon polymerase chain reaction (PCR) for the detection of celery (Apium graveolens) in food was developed. The method involves DNA isolation by chaotropic or non-chaotropic solid-phase extraction and PCR with primers oriented to the sequence of the nuclear gene encoding mannitol dehydrogenase. The PCR method was shown to be specific for celery, producing a 279 bp fragment with four celery varieties and negative results with other species commonly present together with celery in food products (16 samples). The detection limit of PCR was 490–1530 pg DNA, which corresponds to 102 genome copies. When evaluated with model samples of celery in meat pâtés, a detection limit of 0.1% (w/w) was determined. When used to analyse food products from the market (dried vegetable seasonings, dehydrated bouillons), all four products declared to contain celery were correctly identified as positive and all three products in which celery was not declared were identified as negative.

A method based upon polymerase chain reaction (PCR) for the detection of Phaseolus vulgaris beans in chestnut purée was developed. The method involves DNA isolation by the GeneSpin kit and PCR with primers oriented to a sequence of a nuclear gene encoding PvLEA-18, a late embryogenesis-abundant protein of beans. The PCR method was shown to be specific for Ph. vulgaris, producing a 100 bp fragment with six Ph. vulgaris cultivars, and negative results with four non-Ph. vulgaris Phaseolus samples and five non-Phaseolus legumes. When evaluated with model mixtures of Ph. vulgaris beans and the chestnut purée, a detection limit of 1% was determined.

The potential mutagenic activity of three carbamate derivatives with local anesthetic activity was investigated. Genotoxic activity was observed after application of Carbisocaine on Euglena gracilis, whereas no activity was detected by Carbisocaine, Heptacaine and Pentacaine on Salmonella typhimurium, Escherichia coli and Drosophila melanogaster.

The potential mutagenic activity of hexachlorobenzene (HCB) and pentachloroanisole (PCA) was investigated. No genotoxicity after application on Salmonella typhimurium (Ames test), Escherichia coli, and human peripheral blood lymphocytes in vitro was observed.

A simple, rapid and reproducible procedure for the identification and determination of extracellular saccharase from culture medium of watermelon cell suspension cultures is described. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activity. Intracellular activity was estimated from the cell suspension. Watermelon cell suspension was permeabilized by Tween 80 and immobilized by glutaraldehyde. The highest saccharase activity was at pH 4.6 at a temperature of 50 1C. The hydrolysis of substrate was linear 5 h after reaching 60% conversion. The cells had high saccharase activity and good stability, and in long-term storage they showed convenient physico-mechanical properties.

Watermelon cells after permeabilization in Tween 80 were immobilized by glutaraldehyde without any carrier. Cells immobilized by cross-linking demonstrated significantly lower aminopeptidase activities than untreated cells. Pectate and alginate hydrogels were used successfully for immobilization of watermelon cells which retained activities of some aminopeptidases. A simple and rapid procedure for determination of extracellular aminopeptidases was developed using a synthetic substrate. p-Nitroanilides of amino acids were used as substrates for the determination of the extracellular and intracellular enzymatic activities. The culture medium (without cells) was used for the identification and determination of extracellular enzyme activities, whereas intracellular activities were estimated from the cell suspension.