«Development of a genetically defined diploid yeast strain for the application in spirit production Dissertation zur Erlangung des Grades eines ...»
Institut für Lebensmitteltechnologie
Fachgebiet: Gärungstechnologie Prof. Dr. J. J. Heinisch
Development of a genetically defined
diploid yeast strain for the application in
zur Erlangung des Grades eines Doktors
der Fakultät Naturwissenschaften
der Universität Hohenheim
aus Landau in der Pfalz
Die vorliegende Arbeit wurde am 28.10.2003 von der Fakultät Naturwissenschaften der Universität Hohenheim als „Dissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften“ angenommen.
Tag der mündlichen Prüfung: 21.10.2005 Dekan: Prof. Dr. H. Breer Berichterstatter, 1. Prüfer: Prof. Dr. J. Heinisch Mitberichterstatter, 2. Prüfer: PD Dr. T. Senn
3. Prüfer: Prof. Dr. V. Kottke „Dosis facit venenum“ Paracelsus, 1493 - 1541
SCOPE AND OUTLINE OF THE Ph.D. THESISScope The diversity and the composition of the yeast micropopulation during fruit fermentations contributes significantly to the sensory characteristics of the spirits. The growth of each yeast species is characterized by specific metabolic activities, which determine concentrations of flavour compounds in the final product (Walker, 1998). However, it should be pointed out that, within each yeast species, significant strain variability has been recorded (Younis and Steward, 1998). The wide use of starter cultures of Saccharomyces cerevisiae, mainly applied to reduce the risk of spoilage and unpredictable changes of flavour, ensures a balanced quality. On the other hand it may also cause a loss of characteristic aroma and flavour determinants.
Therefore it could be of great benefit to select and combine certain characteristics of different yeast strains. These could be adjusted according to need not only in spirit production, but also in wine and beer making, to optimize and ensure a reproducible quality. Currently there is a large number of different yeast strains for spirit and wine production on the market.
These have been isolated, selected and cultivated from spontaneous fermentations, are readily available and are all claimed to have perfect fermentation skills. In general, little genetic research has been devoted to yeast strains used in fermentation and baking industries. If any, this has concentrated on the winery busyness (Pretorius, 2000). Since financial resources are very scarce for scientific investigations in spirit productions, little attention has been paid to biological improvements.
Accordingly, the yeast strains commonly employed for alcohol production are genetically largely undefined and highly heterogeneous (Benitez et al., 1996). Thus, little is known about their chromosomal constitution and aneuploidy is frequently observed (Bidenne et al., 1992, Cardinali and Martini, 1994, Vezinhet, 1981). 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 (Pretorius, 2000). The application of laboratory yeast strains for industrial purposes offers the potential of a genetic and physiological design, since the complete genome
sequence of S. cerevisiae is available (Goffeau et al., 1996; Zagulski et al., 1998). Recently, laboratory strains have been developed with improved metabolic features (van Dijken et al., 2000). The efficiency of fermentation could further be improved e. g. by a better sugar utilization, an increased ethanol tolerance, resistance to zymocins and heavy metals, reduced formation of foam, induced flocculance at the end of fermentation, the production of extracellular (or liberated) enzymes or the reduced formation of undesired metabolites. For example, ethyl carbamate (EC) which is mainly found in fermented foods and beverages, has been listed as a carcinogenic agent. Especially in stone fruit brandies EC can additionally origin from the fruit itself. EC forms in fermented food by the reaction of urea and ethanol (Ough et al., 1988a, Pretorius, 2000). It has been assumed that yeast contributes substantially to EC formation since urea is formed during arginine degradation (Ough et al., 1988b, Kitamoto et al., 1991).
Regarding the performance in alcoholic fermentation, it has been claimed that laboratory strains show worse ethanol production kinetics. Furthermore, it is generally believed, that such strains lead to the appearance of undesired aromatic compounds in fermented fruit.
Based on the prospect of strain improvement in this work, a genetically well defined prototrophic diploid laboratory yeast strain should be constructed and tested for its fermentative and sensory performances in spirit production. Such a strain offers the potential for further genetic modification by classical breeding and modern molecular genetic techniques, to adjust yeast physiology to special production schemes.
Outline Chapter II provides an introduction to (i) the fundamentals of the distillation process, (ii) yeast metabolism with regard to the degradation of carbohydrates and nitrogen compounds as well as the formation of secondary fermentation products and flavours, and (iii) the relevance of ethyl carbamate in spirits with a special focus on its origins.
Chapter III describes the construction of a laboratory yeast strain and its suitability for fermentation of fruit mashes in spirit production. The fermentation skills of the laboratory strain are compared to industrial yeast strains. Finally, the influence of the different yeast strains employed on the sensory quality of the spirits has been determined. An outline for future applied research is given, involving genetic possibilities for improvements in spirit
production. This chapter has been published in: Schehl, B., C. Müller, T. Senn, and J. J.
Heinisch: A laboratory strain suitable for spirit production. Yeast 21:1375-89, 2004.
Chapter IV comprises experiments evaluating the influence of the stone content on the quality and flavour of plum and cherry spirits combined with analytical assessments of the spirits using the laboratory strain and some industrial yeast strains. This chapter has been published in: Schehl B., T. Senn, and J. J. Heinisch. Effect of the stone content on the quality of plum and cherry spirits produced from mash fermentations with commercial and laboratory yeast strains. J. Agric. Food Chem. 53:8230-38, 2005.
Chapter V describes the characteristics of spirit production using the established laboratory strain HHD1 compared with its genetically modified mutant HHD1delCAR1 in laboratory scale experiments. Furthermore the dependence of the EC content on the yeast strain employed has been investigated. Finally, the data are related to the technological procedure used for spirit production. This chapter has been submitted for publication: Schehl, B., D.
Lachenmeier, T. Senn, and J. J. Heinisch: Reduction of ethyl carbamate in stone fruit spirits by manipulation of the fermenting yeast strain. Appl. Environ. Microbiol., submitted.
In chapter VI a statistical analysis of a database with regard to ethyl carbamate in stone fruit spirits in the Southern part of Germany over the last 15 years is reported. A discussion on acceptable methods of spirit production based on “state of the art technology” is supported by these data. This chapter has been published in: Lachenmeier, D. W., B. Schehl, T.
Kuballa, W. Frank, and T. Senn: Retrospective trends and current status of ethyl carbamate in German stone-fruit spirits. Food Additives and Contaminants 22:397-405, 2005.
CONTRIBUTIONS OF CO-AUTHORS
The co-authors Prof. Dr. Jürgen J. Heinisch and Priv. Doz. Dr. Thomas Senn (Chapters 3 to 5) contributed by their supervision, financial support and many fruitful suggestions to the publications and the studies which have been carried out at the University of Hohenheim, Institute of Food Technology, Section Fermentation Technology. Some of the genetic work was done during a short-term stay at the Department of Biology at the University of Osnabrück, Germany.
Dr. Dirk W. Lachenmeier also contributed by intensive discussions and carried out the ethyl carbamate analyses at the Chemisches und Veterinäruntersuchungsamt (CVUA), Karlsruhe, Germany.
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