Browsing by Subject "Fluoreszenz-in-situ-Hybridisierung"

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    Detektion von Schadhefen in Wein mittels mit Flusszytometrie analysierter in situ Hybridisierung (Flow-FISH)
    (2021) Willberger, Ilka Nadine; Scharfenberger-Schmeer, Maren
    In oenological practice, mostly unpasteurised grape musts are used. This leads to an increased introduction of non-saccharomyces, which can have a lasting effect on the fermentation process. Disturbances in the fermentation process are usually only detected in practice on the basis of abnormalities in selected parameters such as sugar content or temperature or the occurrence of off-flavours. The fermenting yeast population may already be so affected at this point that intervention in the fermentation process can no longer prevent the occurrence of off-flavours in the end product or incomplete fermentation. With the help of flow cytometry, an efficient method using FISH (Fluorescence In Situ Hybridisation) was developed to detect and quantify the common representatives of the fermentation population such as Sacchoromyces cerevisiae and the harmful yeast population such as Hanseniaspora uvarum, Dekkera bruxellensis and Pichia anomala in the course of fermentation. Rapid detection enables countermeasures to be taken in good time before a harmful yeast population can have too great of an influence on the course of fermentation and the metabolites formed. Flow-FISH was established with pure cultures from strain collections in defined medium (YPD) and pasteurised white grape must. Samples are extracted and fixated directly from fermentation mixtures. For hybridisation, 18S- and 26S-rRNA probes with FITC-labelling are used. For the evaluation of the flowcytometric data, the Overton-subtraction method is used in this work. This allows a more accurate assessment of the hybridised cell population than the usual setting of a marker. For this purpose, an effective negative control with complementary sequence to the universal eukaryote probe (Euk516) is introduced. Subsequently, the method already known from the literature was optimised with regard to hybridisation conditions and cell fixation and thus adapted to the requirements of a quantitative flow cytometric analysis. With fixation in formaldehyde or in ethanol, fixation methods were developed that fulfil the requirements of both rapid and reliable fixation in the laboratory and rapid fixation in the cellar, if transport to the laboratory is not possible in a timely manner.Helper probes were designed to increase the fluorescence intensity. They are unlabelled and bind in the direct proximity of the specific probe. In all yeast species investigated, S. cerevisiae, H. uvarum, D. bruxellensis and P. anomala, the fluorescence intensity can be considerably increased by using the helper probes. In the case of D. bruxellensis and P. anomala, detection is only possible with the use of the helper probes. The helper probes allow the Flow-FISH assay to be used in a broader growth range of the yeast culture. Without helper probes, quantitative detection is limited to the middle logarithmic growth phase. With helper probes, hybridised cells can be reliably detected starting in the early logarithmic growth phase up until the stationary phase. This covers the critical phase of fermentative activity so that increasing contamination can be detected in the fermentation.The specificity of the probes is given. In part, there are slightly increased fluorescence intensities compared to the negative control, especially with the D. bruxellensis probe combination and non-specific yeasts, which can probably be attributed to increased binding due to the composition of this probe combination.The Flow-FISH assay is also reliable in mixtures of different yeast species and up to a cell count of 10³ cells / ml in the initial fermentation. This detection limit is also achieved by other methods in molecular biology for yeast detection. In contrast to most of these methods, Flow-FISH can also quantify the number of yeasts present. Additionally the use of the flow cytometer offers a simple variant to determine the total cell count of all yeasts in the fermentation. The detection limit of Flow-FISH allows detection before the damage threshold values of the yeasts examined are reached. The Flow-FISH method presented in this dissertation can also be applied to other yeast strains, some of which also originate from wild isolates. A transferability to native fermentations from oenological practice is given. It was possible to examine both spontaneous fermentations and inoculated fermentations in practice fermentations in steel tanks for their yeast population composition and to follow their development in the course of fermentation. Due to the use of flow cytometry and the helper probes and negative control used in this dissertation, the optimised Flow-FISH assay offers a stable basis for the continued development of a test system for use in oenological practice.