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Pectinatus and Megasphaera: RNA based novel Detection method

ABSTRACT: The study primarily aimed at investigation of occurrence of Gram negative, anaerobic beer spoilage microorganisms belonging to genera; Pectinatus and Megasphaera in the major UK breweries. The study also aimed at optimisation of multiplex PCR and use of optimised methodology for detection of beer spoilage Pectinatus, Megasphaera, Lactobacillus and Pediococcus species. The putative isolates of Pectinatus and Megasphaera were premeditated to be characterised initially using routine microbial methods and ultimately intended to be identified using partial 16S ribosomal gene sequencing. The main aim of this study was to develop a Luminometer based, chemiluminescence method for rapid detection of beer spoilage microorganisms belonging to genera; Pectinatus and Megasphaera. Acridinium ester labelled DNA probes were used for genus and species specific detection of these microorganisms. The project also focussed on evaluation of specificity and sensitivity of the developed acridinium ester labelled DNA probes. Finally the study aimed at development of routine protocols for different brewery environment samples and evaluated compatibility of routinely used beer enrichment media with the developed HPA assay.

PROVIDER: PRJEB4209 | ENA |

REPOSITORIES: ENA

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Project description:The yeast Dekkera bruxellensis is as ethanol tolerant as Saccharomyces cerevisiae and may be found in bottled wine. It causes the spoilage of wine, beer, cider and soft drinks. In wines, the metabolic products responsible for spoilage by Dekkera bruxellensis are mainly volatile phenols. These chemical compounds are responsible for the taints described as M-bM-^@M-^XM-bM-^@M-^XmedicinalM-bM-^@M-^YM-bM-^@M-^Y in white wines (due to vinyl phenols) and as M-bM-^@M-^XM-bM-^@M-^XleatherM-bM-^@M-^YM-bM-^@M-^Y, M-bM-^@M-^XM-bM-^@M-^Xhorse sweatM-bM-^@M-^YM-bM-^@M-^Y and M-bM-^@M-^XM-bM-^@M-^XstableM-bM-^@M-^YM-bM-^@M-^Y in red wines (due to ethyl phenols mainly 4-ethylphenol). Apart from the negative aroma nuances imparted by these yeasts, positive aromas such as M-bM-^@M-^XsmokyM-bM-^@M-^Y, M-bM-^@M-^XspicyM-bM-^@M-^Y and M-bM-^@M-^XtoffeeM-bM-^@M-^Y are also cited. Our goal was to identify the impact that the wine spoilage yeast Dekkera bruxellensis has on fermenting S. cerevisiae cells, especially on its gene expression level. To this end we co-inoculated both yeast species at the start of fermentation in a synthetic wine must, using S. cerevisiae-only fermentations without Dekkera bruxellensis as a control. All fermentations were employed in special membrane reactors (50 KDa pore size cut-off) physically separating Dekkera bruxellensis from wine yeast S. cerevisiae. Biomass separation with this membrane was done to abolish the possibility of hybridizing also D. bruxellensis probes on Agilent V2 (8x15K format) G4813 DNA microarrays designed just for S. cerevisiae ORF targets. The 50 KDa pore membrane separating both yeasts allowed the exchange of ethanol, metabolites and sugars during the fermentation. Fermentations were carried out in synthetic wine must in duplicate for both the control S. cerevisiae (strain Lalvin EC1118) and mixed fermentation. Sampling of yeast S. cerevisiae for RNA extractions were performed at 22 h of fermentation, during the exponential growth phase of S. cerevisiae, at 92 h and 144 h of fermentation, during its early and late stationary growth phase and at 187 h of fermentation, during its phase of growth decline.

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Pectinatus and Megasphaera: RNA based novel Detection method

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