Pectinatus and Megasphaera RNA based novel detection method

Abstract

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. The morphological and physiological characterisation of the presumed Pectinatus and Megasphaera isolates along with NCBI nucleotide blast search assignments and maximum parsimony phylogenetic analysis showed good agreement on their identity. All the Pectinatus and Megasphaera isolates showed ability to spoil low alcohol beer, and the presence of these microorganisms in the brewery environments could be a potential threat to low alcohol and unpasteurised beer. Novel implementation of Hybridisation Protection Assay for detection of beer spoilage microorganisms, Pectinatus and Megasphaera was demonstrated. DNA probes specific for 16S ribosomal RNA 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 thymidine base with 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 detect 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.