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Rheonix® – Beer Spoileralert™ Assay

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Rheonix® – Beer Spoileralert™ Assay Food and Drink Innovation Rheonix® Inc. Evaluation of Rheonix® Beer SpoilerAlert™ Assay www.campdenbri.co.uk 1 Summary In this study the Rheonix Beer SpoilerAlert™ Assay (PCR technology) using the Rheonix® Encompass Optimum™ Workstation was evaluated. The specificity of the assay was good with all the target organisms (P. claussenii, L. brevis, S. cerevisiae/pastorianus) being efficiently detected in beer samples. Additionally, beer-spoiler associated markers were detected at low concentrations, this being a very useful feature for brewers. However, although the assay is designed to identify Brettanomyces bruxellensis, detection of this organism in our tests was poor (NB one of the 2 strains, thought to be Brettanomyces bruxellensis, used in the study was subsequently identified as Saccharomyces cerevisiae var diastaticus). The system was found to be very sensitive with cell numbers down to ~ 103 cells/ml being detected. But a disadvantage, common for PCR based analyses, is the detection of dead non-culturable cell DNA. This resulted in the sterile beer sample showing some false positive results for yeast, a problem that may be circumvented by the manufacturer fine tuning the detection/reporting thresholds. Testing of a number of common brewery sample matrices showed that good results were obtained with bright beer and wort samples. However, when analysing yeast– containing samples (e.g. yeast slurry, fermentation sample) there was competition of the species reactions with those for yeast cells resulting in a suppression of the species signals. However, any spoiler-markers were consistently detected in all matrices. The system was very easy to use and required minimal sample handling and hands-on time. There is a guided easy-to-follow protocol on the user interface and results are obtained within 5 hours. Results are presented in a clearly laid out table with any positive beer spoiler results being highlighted in red. Technical training and on-line/phone support were excellent. Introduction Rheonix® has developed a fully automated and integrated molecular testing device (Encompass workstation) which provides a one-stop solution from sample application to result. The client approached Campden BRI to assess their Beer SpoilerAlert™ Assay (PCR technology) using the Rheonix® Encompass Optimum™ Workstation. The Beer SpoilerAlert™ assay targets a number of different genes specific for Pediococcus claussenii, Saccharomyces cerevisiae, Brettanomyces/Dekkera bruxellensis, Lactobacillus brevis, Pediococcus species, Lactic acid bacteria (LABS), LAB horA spoiler gene, LAB horC spoiler gene, P. claussenii bsrA gene, P. claussenii bsrB gene (the latter 4 are considered to confer beer-spoiling ability to organisms as they contribute to hop resistance). Samples are dispensed into sample tubes. In the workstation the samples are undergoing an automated process: DNA extraction, PCR, hybridisation for detection. The sample processing primarily occurs in the specially designed Rheonix CARD® cartridges which are loaded before an experiment is performed. Each cartridge can process 4 samples in parallel and a maximum of 6 cartridges can be used in one experiment, i.e. a maximum of 24 samples can be processed in one experiment. Positive results give a colour reaction on the cartridge. A camera detects the positive results as dark spots on a light background. Results are then displayed as presence/absence table in pdf format and highlighting the presence of beer spoilers as well as other organisms (from the above list) without spoiler genes. Methods The following micro-organisms were either retrieved from the Campden BRI microbiological collection or sourced externally: Pediococcus claussenii, Saccharomyces cerevisiae (4 strains), Saccharomyces pastorianus, Brettanomyces/Dekkera bruxellensis (2 strains, one of these was later identified as Saccharomyces cerevisiae var diastasticus), other Brettanomyces species, 2 contaminant yeast, Lactobacillus brevis (beer spoiler, 2 species), Lactobacillus brevis (non-beer spoiler, 2 species), Pediococcus sp. (P. damnosus or P. pentosaceus), two other lactic acid bacteria spp. (L. plantarum, L. paracasei), L. buchneri (containing the horA gene), L. paracollinoides (containing the horC gene), Bacillus (2 species). The species identities of the microbial strains, primarily isolates from 2 contaminated beverages, originally designated via DNA sequencing or biochemical analysis, is correct to the best of our knowledge, however no guarantee can be given and genetic changes due to sub- culturing cannot be ruled out. A commercial lager beer, believed to have undergone filtration and pasteurisation, was purchased. The beer was microbiologically analysed by 100 ml membrane filtration and was found to be free of yeast and bacteria. The Encompass Optimum™ Workstation was supplied by Rheonix® and a training session provided. The Beer SpoilerAlert™ reagent kits including the reagent packs as well as the cell scrapers and sample tubes (with caps and barcode adhesive labels) were all provided by Rheonix®. Specificity of assay Cell numbers in the microbial stock cultures were determined microscopically in counting chambers. The commercial beer was then spiked with individual micro-organisms at a concentration of approximately 5 x 104 cells/ml. Samples were dispensed into provided tubes, 1 ml sample per tube. Individual tubes were analysed once. For replicates multiple tubes of a sample were prepared. Sample tubes containing 1 ml solution were placed into the provided sample rack. The sample rack, Rheonix CARD® cartridges, and reagent kit were loaded onto the Encompass Optimum™ workstation following the instructions on the user interface (UI). Two runs of 24 samples each were analysed to evaluate the specificity of the Beer SpoilerAlert™ assay which included some replicates. The specificity tests performed on the Rheonix® equipment are listed in Table 1 below. In parallel, the spiked beer samples were analysed by traditional microbiology – serial dilution, plating 100 µl onto agar plates (either Raka Ray for anaerobic bacteria, WLN for aerobic bacteria or YM for yeast). Live/culturable cell numbers were determined by growth of colonies on the plates. Sensitivity of assay Cell numbers in the microbial stock cultures of P. claussenii, L. brevis (spoiler), B. bruxellensis and S. cerevisiae were determined microscopically in counting chambers. Based on these counts, the commercial beer was then spiked with the individual micro-organisms at a concentration of approximately 2.5 x 105 cells/ml. From the spiked beer samples 4 four-fold serial dilutions (in beer) were prepared down to approximately 1x103 cells/ml. All of the 20 samples (4 organisms at 5 concentrations) were analysed by dispensing 1 ml into 4 replicate sample tubes. The tubes were placed into the sample rack, loaded on the workstation along with Rheonix CARD® cartridges and reagents following the UI instructions. In parallel, the spiked beer samples were analysed by traditional microbiological techniques. The samples were serially diluted as required and 100 µl plated onto agar plates (either Raka Ray for anaerobic bacteria or YM for yeast). 3 CARD Lane Experiment 1 Experiment 2 1 Negative Lactic acid bacterium species 1 2 P. claussenii Lactic acid bacterium species 1 1 3 P. claussenii Bacillus species 1 4 P. claussenii Bacillus species 2 5 S. cerevisiae strain 1 Lactic acid bacterium possessing horA gene species 1 6 S. cerevisiae strain 2 Lactic acid bacterium possessing horA gene species 1 2 7 S. cerevisiae strain 3 Lactic acid bacterium possessing horA gene species 2 8 S. cerevisiae strain 4 Lactic acid bacterium possessing horA gene species 2 9 B. bruxellensis strain 1 P. claussenii 10 B. bruxellensis strain 1 Non-Brettanomyces wild yeast 3 11 B. bruxellensis strain 2* P. claussenii 12 B. bruxellensis strain 2* Negative 13 L. brevis non-spoiler strain 1 Lactic acid bacterium possessing horC gene species 1 14 L. brevis non-spoiler strain 1 Lactic acid bacterium possessing horC gene species 1 4 15 L. brevis non-spoiler strain 2 Lactic acid bacterium possessing horC gene species 2 16 L. brevis non-spoiler strain 2 Lactic acid bacterium possessing horC gene species 2 17 L. brevis spoiler strain 1 S. pastorianus 18 L. brevis spoiler strain 1 Non-Saccharomyces wild yeast 5 19 L. brevis spoiler strain 2 Negative 20 L. brevis spoiler strain 2 Brettanomyces species (not bruxellensis) 21 Pediococcus species 1 Lactic acid bacterium species 2 22 Pediococcus species 1 Lactic acid bacterium species 2 6 23 Pediococcus species 2 Lactic acid bacterium species 3 24 Pediococcus species 2 Lactic acid bacterium species 3 Table 1 Tests performed for specificity testing of the Beer SpoilerAlert™ assay. * Subsequently identified as S. cerevisiae var diastaticus Sample matrix compatibility of assay Bright beer – low cell concentration Cell numbers in the microbial stock cultures of P. claussenii, L. brevis (spoiler), L. brevis (non-spoiler), B. bruxellensis (strain 1) were determined microscopically in counting chambers. Based on these counts, the commercial bright beer was then spiked with the individual micro-organisms at a concentration of ~5-10 cells/100 ml. The spiked beers were filtered (100 ml, 0.45 µm membrane filter), the filters placed into 50 mm Petri dishes and covered with 2 ml sterile broth (MRS broth for bacteria, YM for yeast). The filters were enriched for 21 hrs at 25°C under the appropriate atmosphere. After enrichment, the filters were carefully scraped with cell scrapers
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