Investigation of bacteriocins from lactic acid bacteria and their impact in winemaking
Thesis (MSc (Wine Biotechnology))--University of Stellenbosch, 2007.
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria and are active against other bacteria, either in the same species (narrow spectrum) or across genera (broad spectrum). The application of bacteriocins during the vinification process might help to prevent the production of undesired compounds by inhibiting the indigenous bacterial microflora and allowing malolactic fermentation to be conducted by a selected bacterial strain. Furthermore, the use of bacteriocins might allow reducing the total sulphur dioxide amount in wine. The purpose of this study was the selection of lactic acid bacteria (LAB) belonging to the genera Oenococcus, Lactobacillus and Pediococcus with the ability to produce bacteriocins, with respective biological activity against undesired indigenous wine LAB and the capability to complete malolactic fermentation. The first objective of this study was the screening of LAB isolated from South African red wines for the production of bacteriocins. Only 27 strains out of 330 wine isolates, belonging to the species Lb. plantarum, Lb. paracasei, Lb. hilgardii and O. oeni, showed activity towards various wine-related and non wine-related indicator strains with the colony-overlay method. It is the first time that bacteriocin activity is reported in O. oeni. The second objective was the detection and identification of known structural bacteriocin genes of Lb. plantarum wine strains. Furthermore, the web server BAGEL was used to in silico analyse putative bacteriocin-encoding genes in the genome of O. oeni and primers were designed to amplify four possible bacteriocin-encoding genes. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Moreover, PCR analysis rendered positive results with all four chosen putative bacteriocin-encoding genes in the eight tested O. oeni strains with antimicrobial activity. The latter genes of O. oeni were heterologously expressed in different Escherichia coli host strains, but no antimicrobial activity could be detected. The third objective of this study was the transformation and expression of the heterologous bacteriocin genes nisin A and pediocin PA-1 in two selected Lb. plantarum strains. To enhance their antimicrobial activity a plasmid containing the nisin A gene was successfully cloned into the two strains. Indeed, an enhanced antimicrobial activity could be detected, but the transformed plasmid was not stable. The fourth objective in this project was the evaluation of bacteriocin production in liquid media. A co-culture experiment with a plantaricin producing Lb. plantarum strain and an Enterococcus faecalis strain as indicator was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 hours. The last objective was the evaluation of the impacts of phenolic compounds on the activity of nisin and pediocin. The short term influence of two phenolic acids, two flavan-3-ols, grape tannins and oak tannins on the activity of nisin and pediocin PA-1 was investigated. No influence on the activity was detected. Furthermore, synergistic effects on bacterial growth inhibition were observed. This study confirms the potential use of either bacteriocin additives or bacteriocin-producing LAB in order to control the bacterial microflora during the vinification process.