Cryopreservation and chemotaxonomy in Saccharomyces meyen ex reess

English: The ability to preserve microorganisms can be considered a major . biological achievement. Of special importance is the understanding of the principles of culture preservation with minimal occurrence of contamination, genetic and viability change. At present, cryopreservation is considered the most successful preservation method for yeasts yielding high survival levels and good phenotypic stability. As a result, one of the aims of this study was the application and evaluation of a cryopreservation protocol used in the maintenance of a Saccharomyces cerevisiae strain used by a major brewing company in South Africa. In order to ensure that only pure and stable yeasts with high viability are used after revival from maintenance protocol, it is essential that appropriate, rapid and inexpensive quality control methods are implemented. Elaborate and time consuming tests are used today in the brewing industry and include estimation of mutants and bacteria using Wallerstein Laboratory Nutrient Medium (WLN), estimation of respiratory deficient (RDs) yeasts using wort agar overlaid with Triphenyl- Tetrazolium-Chloride, detection of the wild yeasts using the Swartz-Differential Medium (SDM) protocol, estimation of the non-Saccharomyces species using Lysine-Medium (LYS), detection of the lactose assimilating and lactose fermenting microorganisms using the Lactose-Peptone-Broth (LP) and detection of brewery bacteria using the Universal Liquid Medium (ULM). According to the results, a decrease in the percentage RDs as well as Variants (i.e. other mutants of Saccharomyces cerevisiae) was evident in brewing inocula when maintained through cryopreservation. When yeasts from slants (not cryopreserved) and yeasts subjected to cryopreservation were cultivated in wort contained in round bottom flasks, no significant changes in the percentage RDs, variants or maximum growth rate (J.lmax) could be detected. A 4x3x3x2 nested experimental design was performed in order . to determine the sources of variation in yeast viability, stability and contamination after preservation in liquid nitrogen for 136 days. Consequently, results on Variants and RDs suggest that the largest source of variation in the cryopreservation maintenance process was the error arising from the analytical tests. Cryopreservation also influenced the variation in the number of RDs obtained, though to a lesser extend. No contamination was found to occur during the cryopreservation protocol. From this study it is now possible to construct statistical quality control charts that can be used as an aid in the manufacture of brewing inocula maintained through cryopreservation. Another aim of this study was the evaluation of chemotaxonomie characters such as sterols and polar lipids in, as a first step, determining contamination of preserved yeasts with closely related species. According to the results, total lipid content as well as polar lipid fractions and associated fatty acid (FA) composition showed no obvious taxonomic value as the different Saccharomyces species could not be differentiated. Both linoleic acid (18:2) and linolenic acid (18:3) were detected in strains characterised by the absence of these FAs in total FA composition, a situation believed to be due to the 'dilution' of these FAs by the dominating NL fraction. Sterol content showed promise in the demarcation of the Saccharomyces sensu strictu group.