Dana Tančinová

The in vitro effect of selected essential oils on the growth and mycotoxin production of Aspergillus species

abstract The aim of the present study was to assess the antifungal and anti-toxinogenic activity of 15 essential oils (EOs) against three fungi of the genus Aspergillus (A. parasiticus KMi-227-LR, A. parasiticus KMi-220-LR and A. flavus KMi-202-LR). The minimum inhibitory doses (MIDs) of the tested essential oils and their antifungal activity were determined using the micro-atmosphere method. The original commercial essential oil samples of Jasminum officinale L., Thymus vulgaris L., Syzygium aromaticum (L.) Merrill & Perry, Rosmarinus officinalis L., Ocimum basilicum L., Eucalyptus globulus Labill., Salvia officinalis L., Citrus limon (L.) Burm, Origanum vulgare L., Lavandula angustifolia Mill., Carum carvi L., Citrus sinensis (L.) Osbeck., Zingiber officinalis Rosc., Mentha piperita L. and Cinnamomum zeylanicum Nees. (C. verum J.S.Presl.) were produced in Slovakia (Calendula a.s., Nová Ľubovňa, Slovakia). All essential oils exhibited activity against all tested strains of fungi. After 14 days of incubation, A. flavus (KMi-202-LR) showed the highest susceptibility with a growth inhibition percentage (GIP) of 18.70% to C. limon and 5.92% to C. sinensis, while A. parasiticus (KMi-220-LR) exhibited a GIP of 20.56% to J. officinale. The minimum inhibitory doses (MIDs) of EOs with the most significant activity were recorded. The best antifungal activity, using the micro-atmosphere method was found in S. aromaticum with an MID of 62.5 μL L−1 air, T. vulgaris (MID of 62.5 μL L−1 air) and O. vulgare (MID of 31.5 μL L−1 air) against all tested strains. Mycotoxin production of the tested strains was evaluated by the thin layer chromatography (TLC) method. Mycotoxin production of AFB1 and AFG1 was inhibited following all treatments with C. carvi, R. officinale and S. officinale, Eucalyptus globulus L. and O. basilicum L. Essential oils exhibited a potential inhibition activity against toxic fungi, although, these affected only the production of AFB1.

Mycobiota of Slovak wine grapes with emphasis on Aspergillus and Penicillium species in the small carpathian area

The Slovak wine-growing region is divided into six viticulture areas. The largest in size and the most important over the centuries has been the Small Carpathian area (around 5800 ha of vineyards) spreads in the western of Slovakia. The objectives of this study were: to gain more knowledge about mycobiota on grapes originating from Slovakia, with a focus on genera Aspergillus and Penicillium and their ability to produce mycotoxins in in vitro conditions by thin layer chromatography method. From the twelve vineyards were collected 14 samples of wine grapes (white 6, blue 8) during harvesting 2011, 2012 and 2013. Fifty wine grapes per bunch (approximately 7-8 berries per plate) that showed no symptoms were randomly selected on Dichloran Rose Bengal Chloramphenicol agar medium. The plates were then incubated aerobically at 25 ±1 °C for 5 to 7 days in the dark. Of these samples were identified 22 genera. Ninety-three percent of samples were colonies by the genus Penicillium and 79% by the genus Aspergillus . During the survey, 251 isolates belonging to 14 Penicillium species ( P. aurantiogriseum, P. citrinum, P. coprophylum, P. crustosum, P. expansum, P. funiculosum, P. glabrum, P. griseofulvum, P. chrysogenum, P. oxalicum, P. polonicum, P. purpurogenum, P. roqueforti and P. thomii ) and 37 isolates belonging to 7 Aspergillus species ( A. clavatus, A. flavus, A. section Nigri, A. ostianus, A. parasiticus, A. versicolor and A. westerdijkiae ) were isolated and identified from exogenous contamination. The main occurring penicillium species of the samples were P. chrysogenum (36% Fr), followed P. crustosum (29% Fr), P. griseofulvum (21% Fr) and P. expansum (21% Fr). The main occurring aspergillus species of the samples were A. section Nigri (64%). Thirteen potentially toxigenic species were tested for their toxigenic ability. It was confirmed the production of various mycotoxins such as aflatoxin B 1 , G 1 , citrinin, griseofulvin, patulin, cyclopiazonic acid, penitrem A, roquefortin C and sterigmatocystin. Out of 124 strains, 84% produced at least one mycotoxin.

Growth of microorganisms in the pre-fermentation tanks in the production of ethanol

Our research was carried out to determine the plate count with a special observation Saccharomyces cerevisiae in the prefermenters cereal grains using the classical microscopic method. The cell counts were performed in the Bürker chamber. We followed changes in the plate count, number of Saccharomyces cerevisiae and changes during the yeast propagation in the mash. The mash would present only cultivated yeast Saccharomyces cerevisiae but may occur in a small number of other microorganism’s types. Samples were taken during the propagation process in distillery factories. During this period, 30 samples of corn mash were examined. Samples were collected from two tanks during the fifteen generations. The total number of Saccharomyces cerevisiae was reduced and we got a number of unwanted microbiota. The statistical evaluation demonstrated that the growth of unwanted microbiota is directly related to the increase in the propagation of generation in corn mash. The maximum number of yeast cells was found in the twelfth generation 3.052 x 10 mL in the propagation tank. The total number of microorganisms in this generation was 3.149 x 10 mL and yeasts represent 96.92% of the total microbiota. In the sample B, 95.62% were Saccharomyces cerevisiae during the fifteenth generation. Our results showed that the optimal exchange of the yeast is in 15th generation. Subsequently, repeat the whole process but now with new yeast. These results confirmed our understanding of the relationship between Saccharomyces cerevisiae and contamination during the ethanol fermentation.

Colonization of grapes berries by Alternaria sp. and their ability to produce mycotoxins.

Our research focused on identify the Alternaria species from grapes (surface sterilized berries and non-surface sterilized berries) of Slovak origin and characterize their toxinogenic potential in in vitro conditions. We analyzed 47 samples of grapes, harvested in years 2011, 2012 and 2013 from various wine-growing regions. For the isolation of species, the method of direct plating berries and surface-sterilized berries (using 1 % freshly pre-pared chlorine) on DRBC (Dichloran Rose Bengal Chloramphenicol agar) was used. For each analysis was used 50 berries. Only undamaged berries have been used for analysis. The cultivation was carried at 25 ±1°C, for 5 to 7 days in dark. After incubation, the colonies of Alternaria were transferred on PCA - potato-carrot agar and CYA - Czapek-yeast extract agar and cultured for 7 days at room temperature and natural light. A total 4 species-groups of the genus Alternaria were isolated from grapes berries: Alternaria alternata (1369 isolates), Alternaria arborescens (734 isolates), Alternaria infectoria (143 isolates), and Alternaria tenuissima (3579 isolates). According to European Union legislation mycotoxins produced by species genus Alternaria are not monitored in foods and food commodities. Mycotoxins such as alternariol and alternariol monomethylether are mutagenic and genotoxic in various in vitro systems. Selected strains were tested for production of altenuene, alternariol monomethylether and alternariol. In neither case of A. infectoria species-group isolates was confirmed the production of tested mycotoxins in in vitro conditions by TLC method. The ability to produce altenuene, alternariol monomethylether and alternariol in in vitro conditions was detected in isolates of Alternaria alternata , Alternaria arborescens and Alternaria tenuissima species-groups. Isolates of Alternaria alternata species-group (44 tested isolates) were able to produce altenuene (24 isolates), alternariol monomethyleter (42 isolates) and alternariol (43 isolates). Only one isolate did not produce any mycotoxins. Isolates of Alternaria arborescens species-group (38 tested isolates) were able to produce altenuene (24 isolates), alternariol monomethyleter (33 isolates) and alternariol (36 isolates). Only two isolates did not produce any mycotoxins. Isolates of Alternaria tenuissima species-group (87 tested isolates) were able to produce altenuene (42 isolates), alternariol monomethyleter (41 isolates) and alternariol (73 isolates). Thirteen isolates did not produce any mycotoxins.