Cryopreservation and chemotaxonomy in Saccharomyces meyen ex reess
| dc.contributor.advisor | Kock, J. L. F. | |
| dc.contributor.author | Morakile, Gontse | |
| dc.date.accessioned | 2017-10-18T08:51:06Z | |
| dc.date.available | 2017-10-18T08:51:06Z | |
| dc.date.issued | 1998-11 | |
| dc.description.abstract | 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. | en_ZA |
| dc.description.abstract | Afrikaans: Die vermoë om mikroorganismes te bewaar kan as 'n belangrike . biologiese prestasie beskou word. Vanspesiale belang is die begrip van die beginsels van kultuurbewaring met minimale kontaminasie, genetiese en lewensvatbaarheidsveranderinge. Kriopreservering word huidiglik as die mees suksesvolle bewaringsmetode vir giste beskou en lewer hoë oorlewingsvlakke en goeie fenotipiese stabiliteit. Een van die doelwitte van hierdie studie was gevolglik die toepassing en evaluering van 'n kriopreserveringsprotokol vir die onderhoud van 'n Saccharomyces cerevisiae starn wat deur 'n groot Suid-Afrikaanse brouery gebruik word. Om te verseker dat slegs rein en stabiele kulture met 'n hoë lewensvatbaarheid gebruik word na opwekking vanuit die bewaringsprotokol, is die implementering van geskikte, vinnige en goedkoop kwaliteitsbeheermetodes noodsaaklik. Ingewikkelde en tydrowende toetse word tans in die brou-industrie gebruik insluitende bepaling van mutante en bakterieë met Wallerstein Laboratorium Nutrient Medium (WLN), bepaling van respiratories gebrekkige giste (RGGs) met wort agar bedek met Trifeniel-Tetrazolium-Chloried, bepaling van wilde giste met die Swartz-Differensiële Medium (SDM) protokol, bepaling van nie-Saccharomyces species met Lisien-Medium (LM), bepaling van laktose assimilerende en laktose fermenterende mikroorganismes met Laktose-Peptoon-Sop (LP) en bepaling van brouerybakterieë met Universele Vloeibare Medium (UVM). Volgens die resultate is 'n afname in die persentasie RGGs en variante (d.i. ander mutante van Saccharomyces cerevisiae) sigbaar ill brou-inokula wat deur kriopreservering onderhou is. In 'n vergelykende studie waar Giste vanaf skuinstes (nie gekriopreserveer nie) en giste wat wel gekriopreserveer is, in rondeboom:flesse met wort gekweek is, kon geen betekenisvolle verskille in die persentasies RGGs, variante of maksimum groeisnelheid . (Ilmaks) waargeneem word nie. Om die oorsprong van die variasie in lewensvatbaarheid, stabiliteit en kontaminasie van giste na preservering in vloeibare stikstof vir 136 dae te bepaal, is 'n 4 x 3 x 3 x 2 geneste eksperimentele ontwerp uitgevoer. Die resultate aangaande variante en RGGs dui gevolglik daarop dat die grootste bron van variasie in die kriopreserveringsproses die fout afkomstig vanaf die analitiese toetse was. Kriopreservering het die variasie in die hoeveelheid RGGs verkry tot 'n geringe mate beïnvloed. Geen kontaminasie het gedurende die kriopreserveringsprotokol voorgekom nie. Uit hierdie studie is dit nou moontlik om statistiese kwaliteitsbeheerkaarte op te stel wat as hulpmiddels in die vervaardiging en kriopreservering van brou-inokula kan dien. 'n Ander doelwit van hierdie studie is die evaluasie van chemotaksonomiese eienskappe soos sterole en polêre lipiede in die bepaling van kontaminasie van bewaarde giste met naby verwante spesies. Die resultate toon aan dat die totale lipiedinhoud en die polêre lipiedfraksies en geassosieerde vetsure (Vse) oënskynlik geen taksonomiese waarde het nie aangesien die verskillende Saccharomyces spesies nie onderskei kon word nie. Sterolinhoud behoort geëvalueer te word as 'n vinnige identifikasiehulpmiddel. Beide linoleïnesuur (18:2) en linoleensuur (18:3) is waargeneem in stamme gekenmerk deur die afwesigheid van hierdie Vse in die totale vetsuursamestelling. Dit kan toegeskryf word aan die verdunning van hierdie Vse deur die dominerende neutrale lipiedfraksie. Sterolinhoud blyk belowend te wees in die afbakening van die Saccharomyces sensu strictu groep. | af |
| dc.identifier.uri | http://hdl.handle.net/11660/7297 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Cultures (Biology) -- Cryopreservation | en_ZA |
| dc.subject | Plant cells and tissues -- Cryopreservation | en_ZA |
| dc.subject | Dissertation (M.Sc. (Microbiology and Biochemistry))--University of the Free State, 1998 | en_ZA |
| dc.title | Cryopreservation and chemotaxonomy in Saccharomyces meyen ex reess | en_ZA |
| dc.type | Dissertation | en_ZA |