March 2011 Instrument Assessment Report. Hygiena International Ltd. Campden BRI. Assessment of a novel ATP monitoring device.

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1 March 2 Instrument Assessment Report Campden BRI food and drink innovation Hygiena International Ltd Assessment of a novel ATP monitoring device Executive summary The Hygiena EnSURE luminometer was very simple and easy to use requiring very little instruction. Its hand-held sturdy format allows it to be used in a brewery situation where hygiene assessment may be required. The calibration of the equipment did not show any drift over a month period and the provided standards were also stable over this time. Measurements demonstrated good repeatability. In comparison with a competitor luminometer, the Hygiena unit appeared approximately times more sensitive at the low ATP levels of fmol and lower. This was also demonstrated by the difference in limit of detection which was.2 fmol ATP for the EnSURE luminometer and 2.72 fmol ATP for the competitor system. However, at very high ATP concentrations the unit showed signal saturation which is not considered an issue in the context of hygiene monitoring as such samples would be considered fails anyway. The EnSURE and SuperSnap swab system satisfactorily detected beer residue of most products down to at least : dilution. However, on both systems, one beer showed very low readings and so did the two wines and the alcopop tested indicating that this technology is not suitable for residue detection of these particular drinks. In our experiments yeast cells could be detected down to concentrations of cells/ml and there was a linear correlation between ATP bioluminescence and cell numbers up to 4 cells/ml, therefore this technique can give an indication of yeast cell numbers in solution. Background The objective of this project was to evaluate Hygiena s new EnSURE ATP detection system for use in the alcoholic beverage industry as part of a hygiene monitoring programme, specifically as a post cleaning verification test for product residue. In the alcoholic beverage industry the contamination of the process plant and drink

2 with foreign micro-organisms must be prevented, as the potential cost of lost batches and customer dissatisfaction due to poor hygiene is high. To ensure the microbiological integrity of the process, appropriate tests must be carried out. Although the traditional plate based microbiological method validates the microbiological integrity of the process, several days are usually required before a result is obtained, which is too slow for rapid countermeasures. Adenosine triphosphate (ATP) bioluminescence technology is being used for the rapid assessment of hygiene. ATP is an energy compound in living cells. Methods based on ATP-bioluminescence measure ATP using an enzyme which catalyses an ATP-specific bioluminescence reaction in which ATP is converted to produce light, quantified as Relative Light Units (RLU). The intensity of the emitted light is proportional to the concentration of ATP. Hygiena has developed a sensitive ATP detection system, which comprises of the EnSURE instrument, SuperSnap surface swabs and AquaSnap water testing dippers. Hygiena states that SuperSnap is the most sensitive ATP surface test in its portfolio of all-in-one devices. Whilst other ATP testing devices use freeze-dried enzymes, SuperSnap uses a liquid-stable enzyme, which is said to detect very small amounts of ATP and organic residue, giving consistent true results at low RLUs. The project compared the Hygiena + SuperSnap/AquaSnap detection system with another luminometer + surface/water sampler system from a leading global competitor company. Materials and methods Detection of ATP standards An ATP calibration curve was constructed to demonstrate the sensitivity and limit of detection of the systems and the ratio of RLUs to ATP concentration. The ATP standards were provided by Hygiena at.,.,, and femtomoles. Ten repeat measurements were taken at each ATP standard concentration for each instrument. The sample size was µl pipetted onto the swabs and the ATP readings recorded in the two luminometers. The stability of the instrument calibration was tested by using a stable positive and negative control provided by Hygiena, and measuring these standards after one week and one month. The stability of the 2 controls was ensured by freezing them over the month. Detection of ATP in alcoholic beverages The level of ATP bioluminescence was determined for products that represent the alcoholic beverages market (Table ). The beverages were serially diluted in sterile ATP free water (supplied by Hygiena) down to -4 dilution, except for the flavoured alcoholic beverage which was diluted down to - dilution. The sample size was µl sample pipetted onto the swabs and the ATP reading recorded in the two luminometers. The tests were repeated ten times for each system and each beverage at each dilution. Alcoholic beverage type Details Ale A Ale B Ale Ale C Ale D Ale E Lager F Lager G Lager Lager H Lager I Lager J Cider Red wine White wine Flavoured alcoholic drink Cider Red wine White wine Alcopop Table : Alcoholic beverages used in the study.

3 Detection of low levels of yeast contamination in diluents An isolate of Saccharomyces was grown aerobically at 25 C in Yeast and Mould (YM) broth for four days. The total yeast count was determined. The isolate was then serially diluted in quarter-strength Ringer's solution down to cells/ml. A µl sample of each dilution was collected using an AquaSnap dipper or the competitor dipper. These tests were repeated ten times for each system at each dilution. A negative control of YM broth diluted in Ringer's solution was also included. A fresh aliquot was used for each sampling to prevent carry-over of extractant from the dipper to the stock solutions. Results Equipment evaluation At the start of the equipment evaluation project a short training course on the EnSURE system was given by Hygiena. The trainers were friendly and helpful. All questions regarding the use of the system were answered. An instruction booklet was supplied and technical support was promptly provided through a local representative, either in person, over the phone or by . Instructions were simple and easy to understand. The system is very similar to the competitor system. It requires only minimal setting up making it almost immediately ready for use. It was easy to use, even suitable for an unskilled operator and required little training. The function allowing the user to scroll back through the results, which displays the number, time and date of results, was very useful and was used to double check that the manual recording of the readings was correct. The positive and negative control provided by Hygiena gave confidence to the user that the machine was correctly calibrated. Physically, the machine proved to be well built and strong withstanding the constant use it was subjected to over the course of the experimental days of this project. Its hand-held format allows the device to be conveniently used anywhere as it is easily transported. This is important since the brewery setting calls for a robust portable piece of kit. The EnSURE machine is battery powered and the battery did not require replacing during the length of the study whilst the competitor machine had to be charged on occasions. However, it is to be noted that both machines were in constant use over the course of the equipment evaluation, which is unlikely to reflect the way these machines would be used in a hygiene monitoring programme in a drinks manufacturing situation. A slight malfunction with the EnSURE system occurred on occasions when placing a SuperSnap swab in the machine, rather than measuring, it would ask the operator to remove the swab, close the lid and then replace the swab. The operator would then press the OK button to get the RLU reading. However, at this point the machine would restart. Detection of ATP standards The stability of the calibration of the EnSURE machine was proven by using a stable positive and negative control. The readings for the controls remained very similar throughout the 28 day period (Table 2). Days Control 7 28 Positive Negative Table 2: EnSURE system s positive and negative controls over 28 days. Hygiena claims that its new ATP detection system has good linear correlation of ATP concentration to bioluminescence even at low ATP concentrations. This was tested by constructing an ATP calibration curve to demonstrate the sensitivity and limit of detection of the system and the ratio of RLUs to ATP concentration. Table 3 shows the results. A good linear correlation is obtained between RLU and the ATP concentration (fmols) (see Figures and 2). The average ratio of RLUs to ATP concentration (slope) for the EnSURE + SuperSnap system was found to be fmol ATP 5 RLU.

4 Sample Mean for each ATP standard fmol. fmol fmol fmol fmol fmol Table 3: ATP bioluminescence of ATP standards Figure : Regression fit of ATP bioluminescence vs ATP standard concentration (fmols) for the EnSURE system. Figure 2: Logarithmic plot of ATP bioluminescence vs ATP standard concentration.

5 As all ten readings for the blank sample were zero, to be able to determine the LOD, the smallest difference in readings ( RLU) was used. Together with the slope of the calibration curve, the limit of detection (LOD) was calculated as: Smallest difference in readings LOD = = =.2 fmol ATP Slope of curve 4.78 Ten blank readings on the competitor s luminometer were also determined giving values between and 2 RLU. The limit of detection (LOD) for this system was calculated as: Mean blank = 5.3 RLU Standard deviation of blank = 2.63 RLU Slope of calibration curve = 8.4 RLU/fmol LOD = = = 2.72 fmol ATP Mean blank + (3 x SD) 23.9 Slope of curve 8.4 This shows that at low ATP concentrations the EnSURE system is approximately times more sensitive than the competitor system. Figure 3: The bioluminescence values for ATP standards measured by the EnSURE and competitor system on day, 7 and 28. Analysis of variance (ANOVA) was applied to determine any significant changes to the ATP bioluminescence of the five standards over time. Figure 3 shows the regression plots of ATP bioluminescence for each ATP concentration + luminometer combination. For a few ATP concentration + luminometer combinations there was statistically significant evidence of changes in RLU results over the 28 days (see: fmol + EnSURE; fmol + ; fmol + EnSURE). However, considering the changes are very small and not associated with a particular standard or device, the judgment is made that the ATP standards are stable over 28 days. Figure 4 shows the calibration curves for the EnSURE and competitor systems measured on the three different days. As one can see the EnSURE system was more sensitive compared to the competitor system at distinguishing between levels of ATP of. and fmols, showing better linearity at these low ATP concentrations whereas the competitor device shows a tailing off. As observed previously for the positive standard, the five ATP standards and the calibration of both devices appeared to be stable over the 28 days.

6 Mean of log(rlu) EnSURE... ATP standards (fmols). Figure 4: ATP calibration curves for the EnSURE and competitor systems on day, 7 and 28. Detection of ATP in alcoholic beverages Figure 5 shows the average ATP bioluminescence readings for the 4 diluted alcoholic drinks obtained with the EnSURE luminometer. The products appear to fall into 3 groups: those showing the highest readings (Ale A, Ale E, Ale B, Lager G, Ale C and Lager I); those showing somewhat lower values (Ale D, Lager J, Lager H and Cider) and finally those drinks with the lowest ATP values (Lager F, Red and White Wine). The ATP levels of the alcopop were practically undetectable with the EnSURE system. The reason for the differences in products is likely to do with the raw materials used, as the cellular ATP of the plant-matter will contribute to the ATP in the drinks. The flavoured alcoholic drink is a synthetic drink, essentially sugar added to an alcohol solution, whilst beer, cider and wine are derived from agricultural raw materials. The malt, apples and grapes from which beer, cider and wine are made from respectively would also have a microbial load additionally imparting ATP to these alcoholic beverages. Also some products may contain ingredients that exhibit an inhibiting/quenching effect on the bioluminescence reaction. The graphs in Appendix illustrate the ATP bioluminescence of the alcoholic beverages that were tested with each system at each dilution. From these graphs very few outlying individual data points can be identified (one outlier in the : dilution of white wine data and one outlier in the : flavoured alcoholic drink data, both measured using the competitor s system). The competitor luminometer read higher bioluminescense values for all samples. Repeatability of ATP measurements of the neat drinks seemed somewhat better for the EnSURE system than for the competitor machine (wider spread in repeat measurements) Days 7 28 Figure 5: Average ATP bioluminescence readings for the diluted alcoholic beverages. Measurements taken with the EnSURE system.

7 Generally, linearity between ATP bioluminescence and product concentration was achieved on both systems with linear correlation coefficients mostly lying between.92 and.97. The readings would be expected to decrease by one log order for each dilution and the bioluminescence measurements showed this for most of the drinks (down to dilution : for the beverages with the highest readings and down to : dilution for beverages with intermediate readings). However, the drinks showing the lowest values did not follow this pattern. There was no indication of signal saturation at the higher concentrations. However, a levelling off at lower concentrations was noticeable and this was more pronounced for the competitor s system for which ATP bioluminescence levelled off at about 5 RLU for both of the lowest product concentrations tested. The EnSURE system, on the other hand, did show better linearity at these low concentrations indicating that the sensitivity of the EnSURE system at these low ATP concentrations is better than for the competitor system. Detection of low levels of yeast contamination in diluents Following aerobic growth of Saccharomyces at 25 C in YM broth for four days, the total yeast count was calculated and found to be x7 cells/ml. The cell suspension was serially diluted down to cells/ml. The same dilution series was prepared for the YM broth containing no cells to serve as a control. As one can see from Figure 6 the results for the ATP bioluminescence of the neat (x7 cells/ ml) yeast solution measured on the competitor s system was very high compared to the same samples measured on the EnSURE machine. This is because the EnSURE machine gives a maximum reading of four digits, whilst the competitor machine can give readings of seven digits. The EnSURE machine gave its maximum reading of 9999 for all the neat (x7 cells/ml) yeast solution aliquots. The bioluminescence signal was saturated and showed a plateau effect at the three highest cell concentrations (5 to 7 cells/ml). This could be interpreted as a lack in accuracy for the Hygiena system at these high ATP concentrations. However, when put into the context of a hygiene monitoring programme, the brewing plant which underwent a post cleaning verification test for product residue would fail if the results reached these high levels anyway. Log Dilution Neat : : : : : : Neat : : : : : : Machine EnSURE Figure 6: Logarithmic plot of the ATP bioluminescence of different concentrations of yeast solution measured by the EnSURE and competitor system. The measurements of the corresponding diluted broth samples gave readings between 5 and 58 RLU for the Hygiena system and between 36 and 228 RLU for the competitor s system. The broth contains yeast and malt extract which is the likely cause for the ATP detected. The YM readings were subtracted from the yeast readings and the resulting data is presented in Figure 7. At the yeast cell concentration of x 4 cells/ml the datapoint for the competitor s system was negative (this appears to be an outlier). At the lowest cell concentration ( cells/ml) the values for both systems were

8 negative (the. ml used for the analysis may not have contained any yeast cells). The signal saturation at the highest concentrations for the EnSURE is still noticeable. In our experiment, the lowest detectable yeast cell concentration for both systems was cells/ml which corresponds to the detection of yeast cells in the analysed samples (. ml sample volumes). Linear regressions were determined for the Hygiena as well as the competitor product. For the EnSURE system only the data points below saturation (up to cell concentration of x4 cells/ml) were employed, whereas for the competitor system all data were used. EnSure: ATP (RLU) = 2E+6 x dilution factor (r 2 =.99) : ATP (RLU) = 9.5E5 x dilution factor (r 2 =.99) As all ten readings for the EnSURE blank sample (without any yeast cells) were zero, to be able to determine the LOD, the smallest difference in readings ( RLU) was used. Together with the slope of the calibration curve, the limit of detection (LOD) was calculated as: LOD = = = 4 cells/ml Smallest difference in readings Slope of curve.248 Figure 7: Logarithmic plot of the ATP bioluminescence of the yeast dilution series with the bioluminescence of the corresponding YM broth concentration subtracted. Discussion Hygiena s EnSURE system was found to be easy to use and the short training provided would allow even unskilled persons to operate the equipment without a problem. The small hand-held size allows the device to be employed at the manufacturing site and its robust build means that it will not suffer damage easily. The provided positive and negative controls were seen to be stable over 28 days. The Detection of ATP standards experiments demonstrated the sensitivity and repeatability of the EnSURE unit + SuperSnap swab to detect ATP over a range of concentrations ( to, fmol). The EnSURE machine did not drift following calibration and this was proven through testing the standards over a one month period. Only minor inconsistent instabilities over time were detected for some of the ATP standards, most showed good stability over the 28 days. The average ratio of RLUs to ATP concentration for the EnSURE system was found to be 5 RLU fmol ATP. Both systems would on occasion give outlier results. The Hygiena system proved to be sensitive at low ATP levels, more so than the competitor system which showed a tailing off at the lowest concentrations. The LOD was determined as.2 fmol ATP showing an approximately times higher sensitivity compared to the competitor s system (LOD = 2.72 fmol ATP). A range of alcoholic beverages (lager, ale, wine, cider, alcopop) were serially diluted and tested on both systems. The products fell into 3 groups with either high, medium or low ATP bioluminescence measurements. The expected one log reduction in readings per dilution was seen for the 2 groups exhibiting the higher light values. Comparison between results of the two systems showed that the

9 competitor s luminometer gave higher readings than the EnSURE device. And the sensitivity of the Hygiena equipment was higher than for the competitor s at the highest dilutions (lowest ATP levels). The repeatability of measurements seemed slightly better with the EnSURE system. A brewing yeast culture was serially diluted to give cell concentrations ranging between 7 and cells/ml. As a control a matching dilution series with the yeast growth medium only was also tested. A general increase in ATP bioluminescence with rising cell number was detected and this rise followed a linear relationship at cell concentrations up to 4 cells/ml. The slope of the resulting the linear curves on both systems did not strictly follow the expected one log bioluminescence decrease for each dilution step, even with the values obtained for the growth medium subtracted. It is conceivable that some compounds, possibly from the medium, interfere with the reaction. Also the yeast cells may not contribute all equally to the free ATP content in the suspending liquid possibly due to the ATP extraction not being sufficient and/or variation in cell size and ATP content. At the highest cell concentrations > 5 cells/ml the bioluminescence of the Hygiena system was not as sensitive as the competitor s. A signal saturation plateau was noticeable. This may be because the EnSURE luminometer gives a maximum reading of The LOD for the EnSURE system was determined as 3 cells/ml in our experiment cells/ml were reliably detected. This is a low yeast cell concentration determined very rapidly (5 secs) as compared to the standard microbiological methods. In summary, the EnSURE luminometer is an easy to use device which is very portable and sturdy. Its calibration is stable over 28 days and the provided ATP standards were also stable over this period. The limit of detection was very low below fmol ATP. In comparison with the competitor device the Hygiena luminometer appeared more sensitive at low ATP levels and showed slightly better repeatability. However, at the higher bioluminescence levels it showed signal saturation (EnSURE + AquaSnap) which was not seen with the competitor device. This is not a cause for concern as at these ATP levels of residue the hygiene test would be considered a fail anyway. The unit performed well with most of the alcoholic beverages tested and was able to detect beer and cider residues at a : dilution. ATP detection was not a suitable method for the detection of wine and alcopop as the bioluminescence values were very low even at the high product concentrations. Interestingly, one of the beers also showed very low readings; therefore it would be recommended to test a beverage s ATP bioluminescence output before using this technique for residue detection. There was a linear correlation between yeast cell concentration (up to x4 cells/ml) and bioluminescence output and yeast cells were reliably detected at cells/ml and above. The system can therefore be used for detection of yeast cell residue e.g. in CIP rinse waters. Appendix Ales: Ale C y = x R² =.9277 y = 72.x R² = Ale C y = 396.9x R² =.9274 y = 738.3x R² = ATP bioluminescence of dilution series of Ale C shown in linear and logarithmic plots. Linear

10 Ale A y = 3693x R² =.9484 y = x R² = ATP bioluminescence of dilution series of Ale A shown in linear and logarithmic plots. Linear Ale E y = 348.9x R² =.9326 y = 233.7x R² = Ale A y = 3752x R² =.9474 y = x R² = Ale E y = 356x R² =.9322 y = 244.x R² = ATP bioluminescence of dilution series of Ale E shown in linear and logarithmic plots. Linear Ale D y = 23.8x R² =.9484 y = 7.2x R² =.9683 Ale D y = x R² =.934 y = 7.53x R² = ATP bioluminescence of dilution series of Ale D shown in linear and logarithmic plots. Linear

11 ATP bioluminescence of dilution series of Ale B shown in linear and logarithmic plots. Linear Lagers: Ale B y = 332.7x R² =.9333 y = 23.5x R² = Lager I y = x R² =.738 y = 72.x R² = Ale B y = 3349.x R² =.9328 y = 264.4x R² = Lager I y = x R² =.7372 y = 284.6x R² = ATP bioluminescence of dilution series of Lager I shown in linear and logarithmic plots. Linear Lager G y = 55.9x R² =.9686 y = 475.2x R² =.973 y = 337.4x R² =.934 Lager G y = 372x R² = ATP bioluminescence of dilution series of Lager G shown in linear and logarithmic plots. Linear

12 Lager J y = 475.2x R² =.973 y = x R² = ATP bioluminescence of dilution series of Lager J shown in linear and logarithmic plots. Linear Lager H y = 48.98x R² =.99 y = x R² =.939 Lager J y = x R² =.886 y = x R² = Lager H y = 5.4x R² =.937 y = 37.8x R² = ATP bioluminescence of dilution series of Lager H shown in linear and logarithmic plots. Linear 7 Lager F Lager F y = x R² =.6974 y = x R² =.965 y = 43.86x R² = -.73 y = x R² = ATP bioluminescence of dilution series of Lager F shown in linear and logarithmic plots. Linear

13 Wine: ATP bioluminescence of dilution series of Red Wine shown in linear and logarithmic plots. Linear ATP bioluminescence of dilution series of White Wine shown in linear and logarithmic plots. Linear Other alcoholic beverages: ATP bioluminescence of dilution series of Cider shown in linear and logarithmic plots. Linear

14 ATP bioluminescence of dilution series of Alcopop shown in linear plot. Linear regressions have been fitted. Campden BRI, Centenary Hall, Coopers Hill Road, Nutfield, Surrey RH 4HY United Kingdom Tel: +44 () Web: