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Type: Theses
Title: Enhanced wine-making efficiency through fool-proof malolactic fermentation: evolution of superior lactic acid bacteria
Author: Betteridge, Alice Livingston
Issue Date: 2015
School/Discipline: School of Agriculture, Food and Wine
Abstract: Malolactic fermentation (MLF), also known as the secondary fermentation in winemaking, involves the enzymatic decarboxylation of L-malic to L-lactic acid, by lactic acid bacteria, usually Oenococcus oeni. This conversion improves the organoleptic properties of wine and also decreases the risk of microbial spoilage. O. oeni is a notoriously fastidious microbe prone to slow growth, especially in the harsh physiochemical environment of wine: high ethanol, presence of sulfur dioxide, low pH and low temperature. Each of these factors influences the growth rate and metabolism, including MLF, of this organism. This study aimed to generate an improved strain of O. oeni with the ability to withstand the environmental pressures of wine, particularly high ethanol, using directed evolution (DE). Directed evolution is a non-recombinant method of generating improved strains. The process involves an organism mutating and potentially adapting to a high stress environment, in this case a high ethanol environment, over several hundred generations. This method has been used successfully to generate improved strains of other lactic acid bacteria and its efficacy as a method for the production of bacterial strains for the wine industry is detailed here. A continuous culture of O. oeni was established in MRS supplemented with 20 % (v/v) apple juice medium at 30 °C and 5 % (v/v) ethanol. Over the next 290 days and approximately 260 generations the ethanol concentration in the medium was gradually increased to 15 % (v/v) ethanol. A sample of this culture was screened for malic acid consumption (MLF) compared to the original parent. With proof of concept achieved, individual isolates from the DE culture were obtained in order to identify clones that demonstrated the ethanol tolerant phenotype to the greatest degree. An individual isolate, strain 90, was selected and its fermentation performance was characterised under a range of different ethanol (13, 15, 17 and 19 % (v/v)) concentrations and temperatures (15, 22 and 30 °C). This strain also retained viability for 48 hours in a medium supplemented with 22 % (v/v) ethanol, a condition that lead to an almost total loss of viability in the parent strain after only one hour. Finally the parent and two evolved strains (90 and 89) were sequenced using whole genome sequencing. 32 single nucleotide polymorphisms (SNPs) were discovered in the evolved strains compared to the parent. Twenty of these are non-synonymous mutations located in nineteen different genes; five of these are located in both strains. None of the mutations appear in known O. oeni ethanol stress response genes. GO analysis, BLAST and current literature were used to analyse these changes and propose possible reasons for the new phenotype. This study is the first known use of DE for O. oeni strain improvement and results have confirmed DE can be successfully used as a technique for developing new strains. Furthermore these findings form the basis of exciting new studies further exploring the genetic basis for tolerance to ethanol stress.
Advisor: Grbin, Paul Ramon
Jiranek, Vladimir
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2015
Keywords: wine-making
oenococcus oeni
directed evolution
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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