ARTICLE: FOOD PREPARATION & MANUFACTURING - RESEARCH ARTICLE

Detection of Bacterial Contamination Measured Using A Bioluminescent Assay

Research Conducted by Jane-marie Hawronskyj

Funded by Campden & Chorleywood Food Research Association and The University of Birmingham.

The aim of this research is to develop a novel method of detecting ATP with particular relevance to detection on food processing surfaces.
We are seeking an Industrial Collaborator for this work to develop the assay into a commercial system. For further enquiries, contact Dr C.Wharton or Dr R.Chittock.

Introduction
The future of food processing lies in the instigation of process controls such as HACCP and hygiene monitoring and for these measures to be effective results must be obtained as quickly as possible. ATP bioluminescence is currently used in the food industry to detect bacterial contamination or food soil within minutes on food processing surfaces. However the sensitivity of this system is about 1000-10000 bacteria per ml and there is currently a demand for this sensitivity to be increased. An ATP recycling system is capable of providing a large amplification of ATP therefore increasing the sensitivity of an ATP assay for contamination.

Methods
By using an amplification system consisting of luciferin, luciferase, phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), myokinase and pyruvate kinase it is possible to detect limits of 26pM. The amplification reaction is as follows:
Analysis of the kinetics of the amplification allow the concentration of ATP to be estimated by the time for bioluminescence to reach half its maximum amplitude.

Results and Discussion
The minimum concentration of ATP able to be detected by this method in a 'pure system' was 26pM corresponding to about 103 to 104 'average' bacteria (figure 1).
Figure 1: Concentration of ATP is determined using the time for the light output to reach half its maximum amplitude (t_). t_ is proportional to the log of the ATP concentration. Minimum amount of ATP detected was 26pM, t_ when no ATP was added was at 880 seconds.

As the ultimate sensitivity of this system is limited by low levels of ATP contamination present in the system, purification of the individual components of the amplification reaction, using HPLC, FPLC and dialysis techniques is being successfully carried out to further improve sensitivity.

The system has been tested with bacteria and food soil and been found to be equally sensitive. However, extractants used to release microbial ATP interfere with the recycling reaction, although the use of neutralisers minimises this quenching allowing sensitive measurement. Further research into suitable extractants and neutralisers is ongoing.

Significance
This assay has the potential for extreme sensitivity using relatively simple luminometers since the time for bioluminescence to reach half its maximum amplitude is proportional to the log of the ATP concentration. The recycling reaction can also be used to increase the sensitivity of a sophisticated luminometer and to increase the dynamic range over which ATP can be measured.
There is also potential for ATP bioluminescence to be used as a tool in the assessment of the hygienic design of food production equipment, using this novel amplification technique and direct measurements, for example through the use of a CCD camera.

References
Hawronskyj et al (1994). Low level bacterial contamination measured using a novel bioluminescent assay. Bioluminescence and Chemiluminescence: Fundamentals and Applied Aspects. p.411- 414.

Holah et al (1995). The use of ATP bioluminescence to monitor surface hygiene. The European Food and Drink Review. p.82 - 88.

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