Browsing by Subject "Saccharomyces cerevisiae"

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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.
Development of a genetically defined diploid yeast strain for the application in spirit production
(2005) Schehl, Beatus; Heinisch, Jürgen
Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. In this work two haploid laboratory strains with suitable auxotrophic markers were used for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Different fruit mashes were fermented, subjected to distillation and analysed for fermentation parameters including growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols, uretahne and glycerol. All spirits produced were tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques adjusting yeast physiology to special production schemes.
Development of an on-line process monitoring for yeast cultivations via 2D-fluorescence spectroscopy
(2019) Assawajaruwan, Supasuda; Hitzmann, Bernd
An optimum process is required in the field of food, pharmaceutical and biotechnological industry with the ultimate goal of achieving high productivity and high-quality products. In order to achieve this goal, there are many different parameters to be realized and controlled, e.g., physical, chemical and biological aspects of microbial bioprocesses. Microbial cultivations are a very complex process, therefore, reliable and efficient tools are required to receive as much real-time information for an on-line monitoring as possible, so that the processes can be controlled in time. The primary objective of this research was to apply a two-dimensional (2D) fluorescence spectroscopy to monitor glucose, ethanol and biomass concentrations of yeast cultivations. The measurement of one spectrum has 120 fluorescence intensity variables of excitation and emission wavelength combinations (WLCs) without consideration of the scattered light. To investigate which WLCs carry important and relevant information regarding the analyte concentrations, the three wavelength selection methods were implemented: a method based on loadings, variable importance in projection (VIP) and ant colony optimization. The five selected WLCs from each method for a particular analyte were evaluated by multiple linear regression (MLR) models. The selected WLCs, which showed the best predictive performance of the MLR models, were relevant to the analyte concentrations. Regarding the results of the MLR models, the most significant WLCs contained seven different excitation and emission wavelengths. They can be combined to have 38 WLCs for one spectrum based on the principle of fluorescence. They were in the area of NADH, tryptophan, pyridoxine, riboflavin and FAD/FMN. The 38 WLCs were used to predict the glucose, ethanol and biomass concentrations via partial least squares (PLS) regression. The best prediction from the PLS models with 38 WLCs had the percentage of root mean square error of prediction (pRMSEP) in the range of 3.1-6.3 %, which was not significantly different from the PLS models with the 120 variables. Therefore, the specific fluorescence sensor for yeast cultivations could be built with less filters, which would make it a low-cost device. The following plan of the research goal was to investigate the attribute of fluorophores inside cells in real time using a 2D fluorescence spectrometer. The considered intracellular fluorophores, such as NADH, tryptophan, pyridoxine, riboflavin and FAD/FMN were observed during the yeast cultivations under three different conditions: batch, fed-batch with the glucose pulse during a glucose growth phase (GP) and fed-batch with the glucose pulse during an ethanol growth phase (EP) after a diauxic shift. With the help of principal component analysis, the different states of the yeast cultivations, particularly the glucose pulse during EP, can be recognized and identified from the on-line fluorescence spectra. On the other hand, the change of the fluorescence spectra in the fed-batch process with the glucose pulse during GP was not recognizable. Remarkably, the intensities of the fluorophores due to the glucose pulse during EP did not change in the same direction. The fluorescence intensities of NADH and riboflavin increased, but the intensity of tryptophan, pyridoxine and FAD/FMN decreased. The conversion between tryptophan and NADH intensities was quantified as a proportional factor. It was calculated from the ratio of the area of NADH and tryptophan fluorescence intensity after the glucose addition until depletion. The proportional factor was independent on various glucose concentrations with the coefficient of determination, R2 = 0.999. The correlative intensity changes of these fluorophores demonstrate a metabolic switch from ethanol to glucose growth phase. Based on the previous experiments, a closed-loop control has been implemented for yeast cultivations. 2D fluorescence spectroscopy was applied for an on-line monitoring and control of yeast cultivations to attain pure oxidative metabolism. A glucose concentration is an important factor in a fed-batch process of Saccharomyces cerevisiae. Therefore, it has to be controlled under a critical concentration to avoid overflow metabolism and to gain high productivity of biomass. The characteristic of the NADH intensity can effectively identify the metabolic switch between oxidative and oxidoreductive states. Consequently, the feed rates were regulated using the NADH intensity as a metabolic signal. With this closed-loop control of the glucose concentration, a biomass yield was obtained at 0.5 gbiomass/gglucose. Additionally, ethanol production could be avoided during the controlled feeding phase. The fluorescence sensor with the signal of the NADH intensity has potential to control a glucose concentration under the critical value in real time. The experiments carried out show that 2D fluorescence spectroscopy has great potential in on-line monitoring and process control of the yeast cultivations. Consequently, it is promising to build up a compact and economical fluorescence sensor with the specific wavelengths using light-emitting diodes and photodiodes. The sensor would be a cost-effective and miniaturized device for routine analysis, which could be advantageous to real-time bioprocess monitoring.