Cryopreservation and chemotaxonomy in Saccharomyces meyen ex reess

[摘要] English: The ability to preserve microorganisms can be considered a major. biological achievement. Of special importance is the understanding of theprinciples of culture preservation with minimal occurrence ofcontamination, genetic and viability change. At present, cryopreservationis considered the most successful preservation method for yeasts yieldinghigh survival levels and good phenotypic stability. As a result, one of theaims of this study was the application and evaluation of a cryopreservationprotocol used in the maintenance of a Saccharomyces cerevisiae strainused by a major brewing company in South Africa. In order to ensure thatonly pure and stable yeasts with high viability are used after revival frommaintenance protocol, it is essential that appropriate, rapid andinexpensive quality control methods are implemented. Elaborate and timeconsuming tests are used today in the brewing industry and includeestimation of mutants and bacteria using Wallerstein Laboratory NutrientMedium (WLN), estimation of respiratory deficient (RDs) yeasts usingwort agar overlaid with Triphenyl- Tetrazolium-Chloride, detection of thewild 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 fermentingmicroorganisms using the Lactose-Peptone-Broth (LP) and detection of brewery bacteria using the Universal Liquid Medium (ULM). Accordingto the results, a decrease in the percentage RDs as well as Variants (i.e.other mutants of Saccharomyces cerevisiae) was evident in brewinginocula when maintained through cryopreservation. When yeasts fromslants (not cryopreserved) and yeasts subjected to cryopreservation were cultivated in wort contained in round bottom flasks, no significant changesin the percentage RDs, variants or maximum growth rate (J.lmax) could bedetected. A 4x3x3x2 nested experimental design was performed in order. to determine the sources of variation in yeast viability, stability andcontamination after preservation in liquid nitrogen for 136 days.Consequently, results on Variants and RDs suggest that the largest sourceof variation in the cryopreservation maintenance process was the errorarising from the analytical tests. Cryopreservation also influenced thevariation in the number of RDs obtained, though to a lesser extend. Nocontamination was found to occur during the cryopreservation protocol.From this study it is now possible to construct statistical quality controlcharts that can be used as an aid in the manufacture of brewing inoculamaintained through cryopreservation. Another aim of this study was theevaluation of chemotaxonomie characters such as sterols and polar lipidsin, as a first step, determining contamination of preserved yeasts withclosely related species. According to the results, total lipid content as wellas polar lipid fractions and associated fatty acid (FA) composition showedno obvious taxonomic value as the different Saccharomyces species couldnot be differentiated. Both linoleic acid (18:2) and linolenic acid (18:3) were detected in strains characterised by the absence of these FAs in totalFA composition, a situation believed to be due to the 'dilution' of theseFAs by the dominating NL fraction. Sterol content showed promise in thedemarcation of the Saccharomyces sensu strictu group.

[发布机构] University of the Free State