ena-STUDY-ASHTAVINAYAK PARADH-25-06-2013-23:07:56:216-15
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.
The occurrence of beer spoilage bacteria belonging to the genera Pectinatus and Megasphaera in ten major UK breweries was investigated. The sampling points were selected from fermentation areas, beer conditioning areas and beer bottling and canning sites. Multiplex PCR methodology was used for detection of three Pectinatus and three Megasphaera species using species specific primers. The presence of six Lactobacillus and three beer spoilage Pediococcus species were also examined. Overall, 117 samples were analysed from ten breweries; six samples were positive for the presence of Pectinatus species and three samples were positive for the presence of Megasphaera species, while 34  samples were positive for the presence of Lactobacillus species and 23 samples were positive for Pediococcus species.  Lactobacillus and Pediococcus species appeared to be the major potential spoilage microorganisms. Although none of the actual beer samples were found to be positive for Pectinatus and Megasphaera species, their occurrence in aerobic brewery environments indicates sanitation problems and revealed the presence of highly established biofilms in some breweries.  DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of genera Pectinatus and Megasphaera using a Hybridization Protection Assay. All the probes were modified during synthesis by inserting an amino linker arm at the 5' end during synthesis and also internally modified by replacing amine modified thymidine base; synthesised probes were labelled with (AE) and purified using reverse phase HPLC. The designed internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032 pmol. All the designed probes showed high specificity towards target RNA and could detection bacterial contamination within the range of ca. 5x102 – 1x103 CFU using HPA assay. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media.
The occurrence of beer spoilage bacteria belonging to the genera Pectinatus and Megasphaera in ten major UK breweries was investigated. The sampling points were selected from fermentation areas, beer conditioning areas and beer bottling and canning sites. Multiplex PCR methodology was used for detection of three Pectinatus and three Megasphaera species using species specific primers. The presence of six Lactobacillus and three beer spoilage Pediococcus species were also examined. Overall, 117 samples were analysed from ten breweries; six samples were positive for the presence of Pectinatus species and three samples were positive for the presence of Megasphaera species, while 34  samples were positive for the presence of Lactobacillus species and 23 samples were positive for Pediococcus species.  Lactobacillus and Pediococcus species appeared to be the major potential spoilage microorganisms. Although none of the actual beer samples were found to be positive for Pectinatus and Megasphaera species, their occurrence in aerobic brewery environments indicates sanitation problems and revealed the presence of highly established biofilms in some breweries.  DNA probes specific for rRNA of selected target species were utilised for the detection of beer spoilage bacteria of genera Pectinatus and Megasphaera using a Hybridization Protection Assay. All the probes were modified during synthesis by inserting an amino linker arm at the 5' end during synthesis and also internally modified by replacing amine modified thymidine base; synthesised probes were labelled with (AE) and purified using reverse phase HPLC. The designed internally AE labelled probes were able to detect target RNA within the range of 0.016-0.0032 pmol. All the designed probes showed high specificity towards target RNA and could detection bacterial contamination within the range of ca. 5x102 – 1x103 CFU using HPA assay. The developed assay was also compatible with MRS, NBB and SMMP beer enrichment media.