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 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 , Saccharomyces cerevisiae, Brettanomyces/Dekkera bruxellensis, brevis, Pediococcus species, Lactic acid (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 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).

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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 (provided by Rheonix®) to ensure release of any cells that may be attached to the filters. The enriched broth containing the cells was then transferred into the 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.

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In parallel, 100 ml of the spiked beers were filtered and the filters incubated on RakaRay or YM.

Bright beer, yeast slurry, FV sample, wort

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, samples of commercial beer, yeast slurry, fermentation sample (FV) and standard wort were then spiked with the individual micro-organisms at a concentration of ~ 5 x 104 cells/ml. Sample aliquots of 1 ml volume were transferred to 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, 100 µl of the spiked beer, yeast slurry, FV sample and wort were plated onto RakaRay or YM agar plates and incubated. Colony numbers were then enumerated.

Data analysis The results obtained were analysed to determine the specificity of the assay, its sensitivity of detection, the compatibility with the different sample matrices tested and the reproducibility of data (replicates).

These results were also compared with those obtained by traditional microbiology.

Results

Specificity of assay

Figure 1A shows the results of the first specificity run (experiment 1) and Table 2 shows the spiked beer cell concentrations as determined by culturing on agar plates. The results output from the Encompass (see Figure 1A) shows which organisms have been detected and whether any hop resistance genes have been identified. The key for the multi-detection targets (PED, LABS, HORA, HORC) can be found in the Appendix 1. Additionally, warnings are given if any of the hop resistance genes have been detected (spoiler alert column) and any of the target organisms detected are highlighted as ‘potential problem’. Finally, the 2 internal control reactions (MM1 and MM2) are checked. If any of these have failed, the software alerts the user (see Control column) and the sample should be re-tested.

As can be seen in Table 2 the cell counts in the spiked beer samples were lower than the target concentration of 5 x 104 cells/ml, in some instances very low. This could be due to inaccuracies when determining cell counts in the microbial stock solutions by microscope in cell counting chambers. This would be especially true for small cells such as Pediococcus species. Other factors that could have lead to discrepancies between the microscopic and the plate counts may be the presence of dead cells (although all stocks were freshly prepared) and/or viable but non-culturable cells (VBNC). The microbial cultures were grown in broth (MRS, YM) so that when introducing the cells into the beer the environmental conditions change potentially leading to cell stress and as a result cells being compromised. Dead and VBNC cells are likely to be picked up by the PCR reaction used in the Rheonix system. The limit of detection (LOD) claimed by Rheonix is 1 x 104 cells/ml.

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Organism Plate count (cells/ml) Pediococcus claussenii 6.7 x 102 Saccharomyces cerevisiae, strain 1 3.4 x 104 Saccharomyces cerevisiae, strain 2 7.0 x 103 Saccharomyces cerevisiae, strain 3 2.9 x 104 Saccharomyces cerevisiae, strain 4 5.4 x 104 Brettanomyces bruxellensis, strain 1 1.3 x 102 Brettanomyces bruxellensis*, strain 2 5.0 x 104 Lactobacillus brevis, non-spoiler, strain 1 6.5 x 102 Lactobacillus brevis, non-spoiler, strain 2 7.6 x 102 Lactobacillus brevis, spoiler, strain 1 5.0 x 103 Lactobacillus brevis, spoiler, strain 2 5.8 x 103 3.1 x 103 Pediococcus pentosaceus 7.0 x 101 Table 2 Cell concentrations in the spiked beer samples (experiment 1) * Subsequently identified as S. cerevisiae var diastaticus

Pediococcus claussenii was detected in all 3 replicates giving positive results for PED, PC and LABS as well as for the hop-resistance genes bsrA and bsrB. So despite the plate count being below the reported LOD, the organism was correctly picked up and gave the expected results. Three of the four Saccharomyces cerevisiae strains were positively detected as SC. In the case of the fourth strain, the internal positive controls (MM1/MM2) were also negative indicating that the reactions in this sample did not perform correctly and therefore the results for this sample are not valid. A repeat testing of this yeast strain showed a positive identification for SC (data not shown). One of the duplicates of Brettanomyces bruxellensis strain 1 was rightly identified as BRETT. The second replicate of Brettanomyces bruxellensis strain 1 was not detected as BRETT. This sample was also negative for the related control (MM1) and for both replicates the MM2 reactions were negative indicating that this test should be repeated. However, a more in-depth inspection of the spot results (Figure 1B) indicates that the MM1 and MM2 reactions were all positive but are reported negative due to the signals being below the threshold level (thresholds are set to eliminate false negatives as a result of background noise). SC rather than BRETT was identified in the beer spiked with the second strain of B. bruxellensis. Subsequently this organism was identified as S. cerevisiae var. diastaticus, consistent with its SC detection. Both strains of non-beer spoiling Lactobacillus brevis were detected as LB and no hop-resistance genes were found. But for the beer spoiling L. brevis strains, which were also positive for LB, HORA was picked up and also HORC for the second strain. Some unexpected positive results were also obtained: SC in three of the L. brevis tests and PC/BSRA for one replicate of the L. brevis spoiler strain 1. The manufacturer has noted the presence of SC detection in various media types that are typically made with yeast extract. Detection of SC from media is most likely due to residual DNA in the yeast extracts. Alternatively, the base beer may contain a low level of yeast DNA (from live or dead cells) that is picked up in some of the samples. The ‘false positives’ could also be due to some cross-contamination, although all efforts were made for this not to happen. The beer samples spiked with Pediococcus damnosus and Pediococcus pentosaceus were both correctly identified as PED (even at the low plate count of 70 cells/ml for P. pentosaceus). However, the system was unable to evaluate one of the P. pentosaceus results reporting an error due to an image issue. Direct inspection of the spots (not displayed here) showed that this sample gave the same result as the other duplicate. It is believed that both Pediococcus species were originally isolated from spoiled beer, but no spoiler hop-resistance genes were detected. These genes are located on plasmids which can be lost when sub-culturing the organism. Therefore, it is possible that the hop-resistance of the 2 Pediococcus strains used here may have lost these genes. As for the L. brevis samples some positive SC results were obtained for some of the beers spiked with Pediococcus.

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Figure 1B Spot images for beer sample containing Brettanomyces bruxellensis strain 1. Far left: Replicate 1, middle: Replicate 2, right: key to spots. RS = reference spot (orients imaging software)

Figure 2 shows the results of the second specificity testing experiment and Table 3 the corresponding plate counts. Lactobacillus paracasei paracasei was not detected as a lactic acid bacterium nor did it show the presence of beer-spoiler genes which would have been expected as this was a spoiled beer isolate. Not all known strains of this species carry the LABS marker and also it is located on a plasmid which means that it may have been lost over when sub-culturing. Equally, the hor genes may have been lost over time. One of the replicates showed the presence of SC as did 3 further samples in this experiment (B. megaterium sample, one control unspiked beer sample, L. backi sample). As described above, these ‘false positives’ could be due to a low level of dead cell DNA in the beer. The two Bacillus species (subtilis, megaterium) did not show any positive reactions (apart from one positive SC), however the plate counts for these two samples were very low (possibly due to cells dying once spiked into the beer). The two samples spiked with the strains of L. brevis containing the horA gene were both detected for HORA, the second strain also being positive for HORC. Strain 1 showed a positive reaction for LB but strain 2 didn’t. However, upon examination of the spot image (not shown) a weak positive reaction for LB is detectable (below threshold). As in experiment 1, all expected Pediococcus claussenii markers were successfully identified in the beer samples containing this bacterium (note the very low plate count). Both wild yeast – non-Brettanomyces/H. anomala and non-Saccharomyces/R. mucilaginosa – were rightly not detected as BRETT or SC. The sample spiked with the horC containing Lactobacillus backi, showing a very low live and culturable cell concentration, was not detected as LABS as this species does not carry this marker (see Appendix) but was shown to contain the HORA and HORC markers. The other lactic acid bacterium sample (Lactobacillus paracollinoides believed to contain the horC gene) did however not show positive for HORC (either due to this particular strain not having the gene or due to the loss of the associated plasmid), but was positive for HORA. Testing with Saccharomyces pastorianus showed a positive reaction for SC; the SC detection is designed to pick up both Saccharomyces cerevisiae and pastorianus. Beer spiked with Brettanomyces anomala did not show any positive reaction, indeed the BRETT reaction is designed to be specific for Brettanomyces bruxellensis. The sample inoculated with Lactobacillus plantarum showed positive for LABS and HORA as well HORC. But additionally, an unexpected positive reaction was recorded for LB. This questions whether the bacterial L. plantarum inoculum may have been contaminated with L. brevis which is possible as colonies of different Lactobacillus species are difficult to discriminate. The last sample of this experiment was a beer spiked with Pediococcus damnosus. Both replicates were positive for PED but no hop resistance genes were detected (maybe due to the loss of the corresponding plasmid).

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Organism Plate count (cells/ml) Lactobacillus paracasei paracasei 4.7 x 103 Bacillus subtilis ss.subtilis 1.6 Bacillus megaterium 2.9 Lactobacillus brevis with horA, strain 1 4.4 x 103 Lactobacillus brevis with horA, strain 2 8.4 x 103 Pediococcus claussenii 1.2 Hansenula anomala 2.2 x 104 Lactobacillus backi with horC, strain 1 0.2 Lactobacillus paracollinoides with horC, strain 1 6.1 x 102 Saccharomyes pastorianus 6.1 x 104 Rhodotorula mucilaginosa 2.8 x 104 Brettanomyces anomala 3.2 x 104 Lactobacillus plantarum 2.9 x 103 Pediococcus damnosus 1.6 x 103 Table 3 Cell concentrations in the spiked beer samples (experiment 2)

Experiments 3 to 7 were conducted to establish the sensitivity of the Encompass workstation and SpoilerAlert kit. The first experiment in this series considered the limit of detection for Pediococcus claussenii in beer. A serial dilution was performed achieving 5 cell concentrations covering a range above and below the reported LOD. Every concentration was analysed in quadruplet. The results of this test are displayed in Figure 3 and Table 4 shows the corresponding plate counts. The growth on the agar plates was very poor with most of the samples, even at the highest cell concentration, not showing any growth. This organism had been sourced externally and this was the first experiment in which it was used. it is hypothesised that spiking the broth-based inoculum directly into the beer may have been too extreme an environmental change and an additional adaptation stage may have been advised. Nevertheless, detecting DNA from non-culturable cells, the Rheonix system correctly picked up the organism as PED, PC, LABS and BSRA at all concentrations. The BSRB gene marker was only missed on 2 of the 4 replicates at the very lowest cell concentration. Two of the 24 samples were positive for SC (at the lowest bacterial concentration), potentially due to dead yeast DNA in the base beer.

Organism Plate count (cells/ml) Pediococcus claussenii, target: 9.8 x 102 cells/ml <10 Pediococcus claussenii, target: 3.9 x 103 cells/ml <10 Pediococcus claussenii, target: 1.6 x 104 cells/ml <10 Pediococcus claussenii, target: 6.3 x 104 cells/ml 10 Pediococcus claussenii, target: 2.5 x 105 cells/ml <10 Table 4 Cell concentrations in the spiked beer samples (experiment 3)

The results for the sensitivity experiment of a beer-spoiling Lactobacillus brevis strain are shown in Figure 4 with the corresponding plate counts tabulated in Table 5. The plate counts are lower than expected, possibly due to the beer conditions compromising cell viability/culturability when spiking the cells into the beer sample. The HORA marker was consistently detected at all cell concentrations (this being the most important marker for deciding whether a beer will spoil). On the other hand, the LB marker was only picked up at cell concentrations  1.6 x 104 cells/ml and at the borderline concentration detection was partial (3 of the 4 replicates positive). A few unexpected/false positives

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(SC and LABS) were also seen in 3 of the 24 samples tested here, most likely due to dead cell DNA in these samples.

Organism Plate count (cells/ml) Lactobacillus brevis, target: 9.8 x 102 cells/ml 30 Lactobacillus brevis, target: 3.9 x 103 cells/ml 70 Lactobacillus brevis, target: 1.6 x 104 cells/ml 2.9 x 102 Lactobacillus brevis, target: 6.3 x 104 cells/ml 1.4 x 103 Lactobacillus brevis, target: 2.5 x 105 cells/ml 4.9 x 103 Table 5 Cell concentrations in the spiked beer samples (experiment 4)

Brettanomyces bruxellensis (strain 1) spiked beer was investigated in experiment 5 for which results are shown in Figure 5 and Table 6. BRETT was only detected in two of the replicates at the highest cell concentration. Four of the 24 samples had failed control reactions and for one sample an image quality error was reported. These 5 samples would need to be re-tested, however inspection of the spot images (not shown) showed all control reactions to be positive but probably below threshold or undetectable by the image analysis software. Four samples also showed a positive SC reaction as seen in the previous experiments and 1 sample reported a false positive LABS, all believed due to low level dead cell DNA.

Organism Plate count (cells/ml) Brettanomyes bruxellensis, target: 9.8 x 102 cells/ml 7.9 x 102 Brettanomyes bruxellensis, target: 3.9 x 103 cells/ml 3.5 x 103 Brettanomyes bruxellensis, target: 1.6 x 104 cells/ml 1.6 x 104 Brettanomyes bruxellensis, target: 6.3 x 104 cells/ml 5.0 x 104 Brettanomyes bruxellensis, target: 2.5 x 105 cells/ml 1.8 x 105 Table 6 Cell concentrations in the spiked beer samples (experiment 5)

The final experiment of the sensitivity tests investigated Saccharomyces cerevisiae spiked into beer. Results are displayed in Table 7 and Figure 6. SC was detected for all but 2 of the samples – one at the lowest cell concentration, the other at 6.3 x 104 cells/ml. However, the SC spot was clearly visible, but below threshold, on the spot image. Five of the 24 samples gave failed control reactions, however the spot image shows all controls to be positives (but below threshold).

Organism Plate count (cells/ml) Saccharomyces cerevisiae, target: 9.8 x 102 cells/ml 8.2 x 102 Saccharomyces cerevisiae, target: 3.9 x 103 cells/ml 3.0 x 103 Saccharomyces cerevisiae, target: 1.6 x 104 cells/ml 1.4 x 104 Saccharomyces cerevisiae, target: 6.3 x 104 cells/ml 4.7 x 104 Saccharomyces cerevisiae, target: 2.5 x 105 cells/ml 2.0 x 105 Table 7 Cell concentrations in the spiked beer samples (experiment 6)

The remaining runs were performed to determine how efficiently micro-organisms would be detected in various brewing relevant matrices (beer, yeast slurry, fermentation sample, wort). For experiment 7 bacteria and yeast were spiked into bright beer at low cell concentrations (target: 5-10 cells/100ml). The Rheonix system is not designed to pick up micro-organisms at such low concentrations directly. A

9 pre-enrichment step is required before carrying out the analysis. The samples were membrane filtered and the filters incubated submerged in liquid broth for 21hrs (18-24 hrs recommended). Thereafter the membranes were scraped with cell scrapers and the broth containing cells analysed. The plate counts in Table 8 are for the pre-enrichment samples. Most of the counts were somewhat higher than the target, but would still be considered below Rheonix’ LOD and as such requiring enrichment for detection. As can be seen in Figure 7, all 24 samples showed a positive SC reaction. This could be due to the yeast extract present in the enrichment media and/or yeast DNA in the base beer. Any DNA present in the samples may have been concentrated above LOD during membrane filtration. In the case of P. claussenii both the PC and BSRA markers gave positive results for all 5 replicates indicating that this spoiler can be detected reliably. Other P. claussenii associated markers (PED, LABS, BSRB) were detected in some but not all replicates. Unexpectedly, HA and HC were detected in one of the replicates each and HA was also detected in one of the unspiked control samples. It may be possible that during membrane filtration any plasmid DNA containing these genes in the sample could have been accumulated above LOD. Four out of five of the enriched samples containing the spoiler strain of L. brevis showed positive results for HA. The last replicate sample had a failed control reaction so this sample should be analysed again to be able to make a judgement. Only one of the replicates was positive for the LB marker. It may be that there was insufficient enrichment in these replicates to allow reliable detection of the LB marker. For the non-spoiler strain of L.brevis none of the replicates were identified correctly presumably due to the very low cell count (8 cfu/100ml) and insufficient enrichment in these samples. One replicate had a failed internal control and another a ‘false’ positive HC reaction. Finally, one of the five B. bruxellensis (strain 1) spiked replicates was picked up as BRETT.

Organism Plate count (cells/100ml) Pediococcus claussenii 39 Lactobacillus brevis, spoiler 62 Lactobacillus brevis, non-spoiler 8 Brettanomyces bruxellensis 147 Table 8 Cell concentrations in the spiked beer samples pre-enrichment (experiment 7)

In experiment 8 bright beer was spiked with the same micro-organisms but at a higher cell concentration (target: 5 x 104 cells/ml, actual cell concentrations are listed in Table 9). Pediococcus claussenii and both Lactobacillus brevis strains (spoiler and non-spoiler) were identified with all the correct markers and for all replicates (see Figure 8). However, Brettanomyces bruxellensis (strain 1) was not identified as BRETT. One of the unspiked beer samples showed positive for SC as seen for some samples in previous experiments.

Organism Plate count (cells/ml) Pediococcus claussenii, bright beer 6.5 x 103 Lactobacillus brevis, spoiler, bright beer 6.0 x 103 Lactobacillus brevis, non-spoiler, bright beer 9.1 x 102 Brettanomyces bruxellensis, bright beer 3.9 x 104 Table 9 Cell concentrations in the spiked beer samples (experiment 8)

The results for experiment 9, micro-organisms spiked individually into a yeast slurry, are shown in Figure 9 and the corresponding plate counts are tabulated in Table 10. Despite the yeast having been inoculated with Brettanomyces, the differential agar did not show any well established colonies (hence the zero count for this sample). As expected, for all samples the presence of SC was reported. Analysis of the beer sample spiked with Pediococcus claussenii showed positive results for PED, LABS, BSRA and BSRB in all 3 replicates, but only 2 of the 3 replicates were positive for PC. This could be due to competition for chemicals and the high yeast count, as the SC and the PC reaction use the same MasterMix (MM1). This argument is also valid for the missing LB signal for the samples 10 spiked with Lactobacillus brevis as the LB reaction also uses this Mastermix. On the other hand, the HA marker was successfully detected in all replicates in the presence of the spoiler strain. The Brettanomyces bruxellensis cells (strain 1) were not detected, again this could be due to competition with the yeast as the BRETT reaction also uses the same MM1 MasterMix although this organism was not detected in many of the brewing yeast free samples either. For one of the unspiked yeast slurry samples there was a failed control reaction (although visible on the spot image, below threshold).

Organism Plate count (cells/ml) Pediococcus claussenii, yeast slurry 3.4 x 103 Lactobacillus brevis, spoiler, yeast slurry 1.7 x 103 Lactobacillus brevis, non-spoiler, yeast slurry 1.0 x 101 Brettanomyces bruxellensis, yeast slurry 0 Table 10 Cell concentrations in the spiked yeast slurry samples (experiment 9)

Figure 10 and Table 11 show the results of experiment 10 in which fermentation samples were spiked with the 4 organisms. The outcome was similar to that of experiment 9 (inoculated yeast slurry) in that the species markers are not detected: PC, LB and BRETT most likely due to competition with the high number of yeast cells in the samples (use of the same MasterMix). But most of the spoiler genes - HA and BSRA/BSRB – were correctly identified (just one P.claussenii replicate was negative for BSRA). The PED and LABS markers were also all positive for the P. claussenii containing fermentation sample. Two of the 17 samples reported failed control reactions. The first unspiked fermentation sample was a true fail, whereas for the other B. bruxellensis spiked replicate the spot image shows positive for the control reaction (but below threshold).

Organism Plate count (cells/ml) Pediococcus claussenii, fermentation sample 5.6 x 103 Lactobacillus brevis, spoiler, fermentation sample 3.5 x 103 Lactobacillus brevis, non-spoiler, fermentation sample 7.0 x 102 Brettanomyces bruxellensis, fermentation sample 2.6 x 103 Table 11 Cell concentrations in the spiked fermentation samples (experiment 10)

The final experiment (no. 11) investigated whether the system is able to detect the micro-organisms when added to a wort sample. P. claussenii was reliably detected (PC, PED, LABS, BSRA, BSRB) as can be seen in Table 11 (cell concentrations are tabulated in Table 12). Both L. brevis strains (spoiler and non-spoiler) were also successfully detected, although one of the spoiler replicates was negative for LB (but just visible on the spot image, i.e. just below reporting threshold). Again, Brettanomyces bruxellensis (strain 1) was not detected by the system. One control reaction in this experiment failed, but on the spot image all control reactions were positive, i.e. the failed one was below reporting threshold.

Organism Plate count (cells/ml) Pediococcus claussenii, wort 1.5 x 103 Lactobacillus brevis, spoiler, wort 3.4 x 103 Lactobacillus brevis, non-spoiler, wort 1.0 x 103 Brettanomyces bruxellensis, wort 9.2 x 103 Table 12 Cell concentrations in the spiked wort samples (experiment 11)

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Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Unspiked beer - control - - - - Pass ------Pass Pediococcus claussenii - - - + Pass + + - - + + Pass BSRA, BSRB PED, LABS, PC Pediococcus claussenii - - - + Pass + + - - + + Pass BSRA, BSRB PED, LABS, PC Pediococcus claussenii - - - + Pass + + - - + + Pass BSRA, BSRB PED, LABS, PC Saccharomyces cerevisiae, strain 1 + - - - Pass ------Pass SC Saccharomyces cerevisiae, strain 2 + - - - Pass ------Pass SC Saccharomyces cerevisiae, strain 3 + - - - Pass ------Pass SC Saccharomyces cerevisiae, strain 4 - - - - Fail ------Fail Unknown MM1/2 fail Brettanomyces bruxellensis, strain 1 - + - - Pass ------Fail Unknown BRETT MM2 fail Brettanomyces bruxellensis, strain 1 - - - - Fail ------Fail Unknown MM1/2 fail Brettanomyces bruxellensis, strain 2 + - - - Pass ------Pass SC Brettanomyces bruxellensis, strain 2 + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler, strain 1 - - + - Pass ------Pass LB Lactobacillus brevis, non-spoiler, strain 1 - - + - Pass ------Pass LB Lactobacillus brevis, non-spoiler, strain 2 - - + - Pass ------Pass LB Lactobacillus brevis, non-spoiler, strain 2 + - + - Pass ------Pass SC, LB Lactobacillus brevis, spoiler, strain 1 + - + - Pass - - + - - - Pass HA SC, LB Lactobacillus brevis, spoiler, strain 1 - - + + Pass - - + - + - Pass HA, BSRA LB, PC Lactobacillus brevis, spoiler, strain 2 + - + - Pass - - + + - - Pass HA, HC SC, LB Lactobacillus brevis, spoiler, strain 2 - - + - Pass - - + + - - Pass HA, HC LB Pediococcus damnosus + - - - Pass + - - - - - Pass SC, PED Pediococcus damnosus - - - - Pass + - - - - - Pass PED Pediococcus pentosaceus + - - - Pass + - - - - - Pass SC, PED Pediococcus pentosaceus Err Err Err Err Err Err Err Err Err Err Err Err Image quality issue Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 1A Results of first set of specificity tests (experiment 1)

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Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Lactobacillus paracasei paracasei + - - - Pass ------Pass SC Lactobacillus paracasei paracasei - - - - Pass ------Pass Bacillus subtilis ss subtilis - - - - Pass ------Pass Bacillus megaterium + - - - Pass ------Pass SC Lactobacillus brevis with horA, strain 1 - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis with horA, strain 1 - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis with horA, strain 2 - - - - Pass - - + + - - Pass HA, HC Lactobacillus brevis with horA, strain 2 - - - - Pass - - + + - - Pass HA, HC Pediococcus claussenii - - + - Pass + + - - + + Pass BSRA, BSRB LB, PED, LABS Hansenula anomala - - - - Pass ------Pass Pediococcus claussenii - - + - Pass + + - - + + Pass BSRA, BSRB LB, PED, LABS Unspiked beer - control + - - - Pass ------Pass SC Lactobacillus backi with horC + - - - Pass - - + + - - Pass HA, HC SC Lactobacillus backi with horC - - - - Pass - - + + - - Pass HA, HC Lactobacillus paracollinoides with horC - - - - Pass - - + - - - Pass HA Lactobacillus paracollinoides with horC - - - - Pass - - + - - - Pass HA Saccharomyces pastorianus + - - - Pass ------Pass SC Rhodotorula mucilaginosa - - - - Pass ------Pass Unspiked beer - control - - - - Pass ------Pass Brettanomyces anomala - - - - Pass ------Pass Lactobacillus plantarum - - + - Pass - + + + - - Pass HA, HC LB, LABS Lactobacillus plantarum - - + - Pass - + + + - - Pass HA, HC LB, LABS Pediococcus damnosus - - - - Pass + - - - - - Pass PED Pediococcus damnosus - - - - Pass + - - - - - Pass PED Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 2 Results of second set of specificity tests (experiment 2)

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Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Pediococcus claussenii, 9.8 x 102 cells/ml - - - + Pass + + - - + - Pass BSRA PC, PED, LABS Pediococcus claussenii, 9.8 x 102 cells/ml + - - + Pass + + - - + + Pass BSRA, BSRB SC,PC, PED, LABS Pediococcus claussenii, 9.8 x 102 cells/ml - - - + Pass + + - - + - Pass BSRA PC, PED, LABS Pediococcus claussenii, 9.8 x 102 cells/ml + - - + Pass + + - - + + Pass BSRA, BSRB SC, PC, PED, LABS Unspiked beer - control - - - - Pass ------Pass Pediococcus claussenii, 3.9 x 103 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 3.9 x 103 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 3.9 x 103 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 3.9 x 103 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Unspiked beer - control - - - - Pass ------Pass Pediococcus claussenii, 1.6 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 1.6 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 1.6 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 1.6 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Unspiked beer - control - - - - Pass ------Pass Pediococcus claussenii, 6.3 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 6.3 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 6.3 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 6.3 x 104 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Unspiked beer - control - - - - Fail ------Fail Unknown MM1/2 fail Pediococcus claussenii, 2.5 x 105 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 2.5 x 105 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 2.5 x 105 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, 2.5 x 105 cells/ml - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 3 Results of first set of sensitivity tests – Pediococcus claussenii (experiment 3) 14

Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Lactobacillus brevis, 9.8 x 102 cells/ml - - - - Pass - - + - - - Pass HA Lactobacillus brevis, 9.8 x 102 cells/ml - - - - Pass - - + - - - Pass HA Lactobacillus brevis, 9.8 x 102 cells/ml - - - - Pass - - + - - - Pass HA Lactobacillus brevis, 9.8 x 102 cells/ml + - - - Pass - - + - - - Pass HA SC Unspiked beer - control - - - - Fail ------Fail Unknown MM1/2 fail Lactobacillus brevis, 3.9 x 103 cells/ml + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, 3.9 x 103 cells/ml - - - - Pass - - + - - - Pass HA Lactobacillus brevis, 3.9 x 103 cells/ml - - - - Pass - - + - - - Pass HA Lactobacillus brevis, 3.9 x 103 cells/ml - - - - Pass - - + - - - Pass HA Unspiked beer - control - - - - Pass ------Pass Lactobacillus brevis, 1.6 x 104 cells/ml - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, 1.6 x 104 cells/ml - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, 1.6 x 104 cells/ml - - - - Pass - - + - - - Pass HA Lactobacillus brevis, 1.6 x 104 cells/ml - - + - Pass - - + - - - Pass HA LB Unspiked beer - control - - - - Pass ------Pass Lactobacillus brevis, 6.3 x 104 cells/ml - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, 6.3 x 104 cells/ml - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, 6.3 x 104 cells/ml - - + - Pass - + + - - - Pass HA LB, LABS Lactobacillus brevis, 6.3 x 104 cells/ml - - + - Pass - - + - - - Pass HA LB Unspiked beer - control - - - - Pass ------Pass Lactobacillus brevis, 2.5 x 105 cells/ml - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, 2.5 x 105 cells/ml - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, 2.5 x 105 cells/ml - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, 2.5 x 105 cells/ml - - + - Pass - - + - - - Pass HA LB Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 4 Results of second set of sensitivity tests – Lactobacillus brevis (experiment 4) 15

Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Brettanomyces bruxellensis,9.8 x 102 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,9.8 x 102 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,9.8 x 102 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,9.8 x 102 cells/ml - - - - Pass ------Pass Unspiked beer - control - - - - Fail ------Fail Unknown MM1/MM2 fail Brettanomyces bruxellensis,3.9 x 103 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,3.9 x 103 cells/ml Err Err Err Err Err Err Err Err Err Err Err Err Image quality issue Brettanomyces bruxellensis,3.9 x 103 cells/ml + - - - Pass ------Pass SC Brettanomyces bruxellensis,3.9 x 103 cells/ml - - - - Pass ------Pass Unspiked beer - control - - - - Pass ------Pass Brettanomyces bruxellensis,1.6 x 104 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,1.6 x 104 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,1.6 x 104 cells/ml - - - - Fail ------Fail Unknown MM1/MM2 fail Brettanomyces bruxellensis,1.6 x 104 cells/ml - - - - Pass - + - - - - Pass LABS Unspiked beer - control - - - - Pass ------Pass Brettanomyces bruxellensis,6.3 x 104 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,6.3 x 104 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,6.3 x 104 cells/ml - - - - Fail ------Fail Unknown MM1/MM2 fail Brettanomyces bruxellensis,6.3 x 104 cells/ml - - - - Pass ------Pass Unspiked beer - control + - - - Pass ------Fail Unknown SC MM2 fail Brettanomyces bruxellensis,2.5 x 105 cells/ml + - - - Pass ------Pass SC Brettanomyces bruxellensis,2.5 x 105 cells/ml - - - - Pass ------Pass Brettanomyces bruxellensis,2.5 x 105 cells/ml - + - - Pass ------Pass BRETT Brettanomyces bruxellensis,2.5 x 105 cells/ml + + - - Pass ------Pass BRETT SC Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 5 Results of third set of sensitivity tests – Brettanomyces bruxellensis (experiment 5)

16

Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Saccharomyces cerevisiae, 9.8 x 102 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 9.8 x 102 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 9.8 x 102 cells/ml - - - - Pass ------Pass Saccharomyces cerevisiae ,9.8 x 102 cells/ml + - - - Pass ------Pass SC Unspiked beer - control - - - - Pass ------Pass Saccharomyces cerevisiae, 3.9 x 103 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 3.9 x 103 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 3.9 x 103 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 3.9 x 103 cells/ml + - - - Pass ------Fail Unknown SC MM2 fail Unspiked beer - control - - - - Fail ------Fail Unknown MM1/MM2 fail Saccharomyces cerevisiae, 1.6 x 104 cells/ml + - - - Pass ------Fail Unknown SC MM2 fail Saccharomyces cerevisiae, 1.6 x 104 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 1.6 x 104 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 1.6 x 104 cells/ml + - - - Pass ------Pass SC Unspiked beer - control - - - - Pass ------Pass Saccharomyces cerevisiae, 6.3 x 104 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 6.3 x 104 cells/ml - - - - Pass ------Pass Saccharomyces cerevisiae, 6.3 x 104 cells/ml + - - - Pass ------Fail Unknown SC MM2 fail Saccharomyces cerevisiae, 6.3 x 104 cells/ml + - - - Pass ------Pass SC Unspiked beer - control - - - - Pass ------Pass Saccharomyces cerevisiae, 2.5 x 105 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 2.5 x 105 cells/ml + - - - Pass ------Fail Unknown SC MM2 fail Saccharomyces cerevisiae, 2.5 x 105 cells/ml + - - - Pass ------Pass SC Saccharomyces cerevisiae, 2.5 x 105 cells/ml + - - - Pass ------Pass SC Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 6 Results of fourth set of sensitivity tests – Saccharomyces cerevisiae (experiment 6)

17

Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Pediococcus claussenii + - - + Pass + + - + + - Pass HC, BSRA SC, PC, PED, LABS Pediococcus claussenii + - - + Pass + - - - + - Pass BSRA SC, PC, PED Pediococcus claussenii + - - + Pass + - - - + - Pass BSRA SC, PC, PED Pediococcus claussenii + - - + Pass + - + - + + Pass HA, BSRA, BSRB SC, PC, PED Pediococcus claussenii + - - + Pass - + - - + + Pass BSRA, BSRB SC, PC, LABS Unspiked beer - control + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, spoiler + - + - Pass - - + - - - Pass HA SC, LB Lactobacillus brevis, spoiler + - - - Pass ------Fail Unknown SC MM2 fail Lactobacillus brevis, spoiler + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, spoiler + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, spoiler + - - - Pass - - + - - - Pass HA SC Unspiked beer - control + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler + - - - Pass ------Fail Unknown SC MM2 fail Lactobacillus brevis, non-spoiler + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, non-spoiler + - - - Pass ------Pass SC Unspiked beer - control + - - - Pass ------Pass SC Brettanomyces bruxellensis + + - - Pass ------Pass BRETT SC Brettanomyces bruxellensis + - - - Pass ------Pass SC Brettanomyces bruxellensis + - - - Pass ------Pass SC Brettanomyces bruxellensis + - - - Pass ------Pass SC Brettanomyces bruxellensis + - - - Pass ------Pass SC Unspiked beer - control + - - - Pass ------Pass SC Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 7 Results of first matrix compatibility test – Bright beer, low cell concentration/enrichment (experiment 7)

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Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Pediococcus claussenii, bright beer - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, bright beer - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Pediococcus claussenii, bright beer - - - + Pass + + - - + + Pass BSRA, BSRB PC, PED, LABS Unspiked beer - control + - - - Pass ------Pass SC Lactobacillus brevis, spoiler, bright beer - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, spoiler, bright beer - - + - Pass - - + - - - Pass HA LB Lactobacillus brevis, spoiler, bright beer - - + - Pass - - + - - - Pass HA LB Unspiked beer - control - - - - Pass ------Pass Lactobacillus brevis, non-spoiler, bright beer - - + - Pass ------Pass LB Lactobacillus brevis, non-spoiler, bright beer - - + - Pass ------Pass LB Lactobacillus brevis, non-spoiler, bright beer - - + - Pass ------Pass LB Unspiked beer - control - - - - Pass ------Pass Brettanomyces bruxellensis, bright beer - - - - Pass ------Pass Brettanomyces bruxellensis, bright beer - - - - Pass ------Pass Brettanomyces bruxellensis, bright beer - - - - Pass ------Pass Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 8 Results of second matrix compatibility test – Bright beer (experiment 8)

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Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Unspiked yeast slurry - control + - - - Pass ------Pass SC Pediococcus claussenii, yeast slurry + - - + Pass + + - - + + Pass BSRA, BSRB SC, PC, PED, LABS Pediococcus claussenii, yeast slurry + - - - Pass + + - - + + Pass BSRA, BSRB SC, PED, LABS Pediococcus claussenii, yeast slurry + - - + Pass + + - - + + Pass BSRA, BSRB SC, PC, PED, LABS Unspiked yeast slurry - control + - - - Pass ------Pass SC Lactobacillus brevis, spoiler, yeast slurry + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, spoiler, yeast slurry + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, spoiler, yeast slurry + - - - Pass - - + - - - Pass HA SC Unspiked yeast slurry - control + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler, yeast slurry + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler, yeast slurry + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler, yeast slurry + - - - Pass ------Pass SC Unspiked yeast slurry - control + - - - Pass ------Fail Unknown SC MM2 fail Brettanomyces bruxellensis, yeast slurry + - - - Pass ------Pass SC Brettanomyces bruxellensis, yeast slurry + - - - Pass ------Pass SC Brettanomyces bruxellensis, yeast slurry + - - - Pass ------Pass SC Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 9 Results of third matrix compatibility test – Yeast slurry (experiment 9)

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Presence/Absence Hop resistance Warnings Sample SC BRETT LB PC MM1 PED LABS HA HC BSRA BSRB MM2 Spoiler Alert Potential Problem Control Unspiked fermentation sample - control - - - - Fail ------Fail Unknown Unknown MM1/MM2 fail Pediococcus claussenii, fermentation sample + - - - Pass + + - - + + Pass BSRA, BSRB SC, PED, LABS Pediococcus claussenii, fermentation sample + - - - Pass + + - - - + Pass BSRB SC, PED, LABS Pediococcus claussenii, fermentation sample + - - - Pass + + - - + + Pass BSRA, BSRB SC, PED, LABS Unspiked fermentation sample - control + - - - Pass ------Pass SC Lactobacillus brevis, spoiler, fermentation sample + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, spoiler, fermentation sample + - - - Pass - - + - - - Pass HA SC Lactobacillus brevis, spoiler, fermentation sample + - - - Pass - - + - - - Pass HA SC Unspiked fermentation sample - control + - - - Pass ------Pass SC Lactobacillus brevis, non-spoiler, fermentation + - - - Pass ------Pass SC sample Lactobacillus brevis, non-spoiler, fermentation + - - - Pass ------Pass SC sample Lactobacillus brevis, non-spoiler, fermentation + - - - Pass ------Pass SC sample Unspiked fermentation sample - control + - - - Pass ------Pass SC Brettanomyces bruxellensis, fermentation sample + - - - Pass ------Fail Unknown SC MM2 fail Brettanomyces bruxellensis, fermentation sample + - - - Pass ------Pass SC Brettanomyces bruxellensis, fermentation sample + - - - Pass ------Pass SC Unspiked fermentation sample - control + - - - Pass ------Pass SC Key: SC=Saccharomyces cerevisiae, BRETT = Brettanomyces bruxellensis, LB = Lactobacillus brevis, PC = Pediococcus claussenii, PED = Pediococcus spp., LABS = Lactic acid bacteria plasmid marker, HA = hop resistance gene horA, HC = hop resistance gene horC, BSRA = hop resistance gene bsrA, BSRB = hop resistance gene bsrB, MM1 = Mastermix 1 control, MM2 = Mastermix 2 control Indicates incomplete information due to PCR failure, re-run sample

Figure 10 Results of fourth matrix compatibility test – Fermentation sample (experiment 10)

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Food and Drink Innovation

Conclusions

This study was conducted to evaluate the Beer SpoilerAlert™ Assay using the Rheonix® Encompass Optimum™ Workstation. The experiments were designed to assess the system’s specificity for particular micro-organisms, the sensitivity of the assay and its performance when presented with different sample formats.

For the specificity testing, the system was challenged with a number of yeast and bacteria, some of which the system is setup to detect and others, which were employed to check for cross-reactions. The microorganisms Pediococcus claussenii, Saccharomyces cerevisiae and Lactobacillus brevis were successfully identified as being present in the spiked beer samples. Spoiler and non-spoiler strains of L. brevis were correctly differentiated, with the former showing a positive signal for a hop-resistance gene target associated with beer spoilers. The two strains of Brettanomyes bruxellensis however were not reliably detected even at high cell concentrations (N.B. Subsequently one of the 2 strains was identified as S. cerevisiae var diastaticus). The Pediococcus species tested were all identified as belonging to this genus. Non-target organisms (such as Bacillus spp. and non-B. bruxellensis wild yeast) did not result in any false positives. Several Lactobacillus species were used in the study, which were believed to be beer spoilers. Indeed, most gave positive results for one or more of the target spoiler genes.

The second part of the study focused on assessing the sensitivity of the assay. The 4 species detected in the assay – Pediococcus claussenii, Lactobacillus brevis, Brettanomyces bruxellensis and Saccharomyces cerevisiae – were tested at cell concentrations as low as ~ 1 x 103 cells/ml. All, except B. bruxellensis, were detected at this lowest concentration. For L. brevis the spoiler gene detection was more sensitively picked up than the species marker (at the 2 lowest cell concentrations the spoiler gene gave a positive signal but not the L. brevis marker).

In a further experiment, a bright beer was inoculated with low cell counts that would traditionally be found by membrane filtration but would not be expected to be picked up directly by the Rheonix system. For such low cell count samples filtration followed by 18-24hr enrichment in broth is prescribed, after which the filters are scraped to release any adhered cells and the enriched broth is analysed. Pediococcus claussenii and one of its spoiler genes, BSRA, was reliably detected in all 5 replicates while HA, HC and BSRB spoiler genes were only detected in some of the replicates. On the contrary, for the L. brevis beer spoiler strain the spoiler gene was consistently positive but not the species marker. The non-spoiler L. brevis was not picked up (the enriched cell concentration may have been too low) and again the analysis did not perform well for B. bruxellensis.

Bright beer, yeast slurry, fermentation sample and wort were inoculated with the same 4 organisms to determine any sample matrix effect on the testing. For bright beer the analyses were successful for all bacteria but the yeast strain was not detected. The results for the yeast slurry and fermentation matrices were very similar in that P. claussenii was not detected as a species but all associated spoilage markers were. Similarly, the L. brevis spoiler gene was consistently detected but not the species. However, the non-spoiler L. brevis and B. bruxellensis were not identified in these brewing yeast containing samples. The wort sample on the other hand showed very similar results to the bright beer with all species (except B. bruxellensis) being detected including their spoiler genes.

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Food and Drink Innovation

In summary, the system was proven to be specific as it correctly detected the organisms it is designed to as well as picking up gene markers for spoiler organisms. The latter is a useful feature to establish whether any organisms present may cause beer spoilage or not (even if the organism may not be identified to species level). In fact, sensitivity for the spoilage genes appeared to be higher than for species genes. No cross-reactions with other non-target organisms were found. The assay is very sensitive, picking up very low DNA concentrations, but also picking up dead cell DNA (a common problem for PCR reactions). Quite a few of the samples of beer (spiked or unspiked) showed positive for Saccharomyces cerevisiae (most likely due to dead cell DNA rather than actual live contaminants as none were detected by traditional microbiology). Further fine adjustment of the detection/reporting threshold by the manufacturer may help in suppressing any such false positive results. Although the imaging software does work well there are instances where internal controls are reported as failed which would mean the sample needed a repeat analysis. In these cases evaluation of the spot images (available to the user) can help in understanding whether these are true fails or just an imaging/threshold issue. Reproducibility was very good with replicates giving the same results when above the LOD.

In general, the system is very easy to use with a user-friendly guided protocol. There is minimal sample handling involved (minimising any risk of cross-contamination and human error) with samples simply being dispensed into tubes which are then placed into the workstation. All other sample processing thereafter is automated and results are obtained within 5 hours, a great advantage compared to traditional microbiological analysis requiring up to 5-7 days incubation. Results are reported in a clear tabular format with positive spoiler results highlighted in red, making it easy to pick these out quickly. Training and technical support were excellent throughout the study with detailed responses being given rapidly.

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Food and Drink Innovation

Appendix 1

Key for multi-detection targets Pediococcus spp. LABS plasmid biomarker§ horA or horC§ bsrA or bsrB§ P. acidilactici Lactobacilii L. backii P. claussenii P. cellicola L. amylovorus L. brevis P. claussenii L. backii L. buchneri (horA only) P. damnosus L. delbrueckii L. jensenii (horA only) P. inopinatus L. helveticus L. lindneri P. parvulus L. johnsonii L. paracollinoides P. pentosaceus L. mucosae P. claussenii P. stilesii L. paracasei P. damnosus L. paracollinoides P. inopinatus (horA only) L. plantarum L. reuteri Pediococci P. claussenii P. damnosus Other organisms mesenteroides Oenococcus oeni

§ These markers are detectable in some strains of the indicated bacteria.

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