New Technology Improves ATP Testingfor Food Safety Applications

Table of Contents
Introduction

2Principle of ATP Testing3

University of Wisconsin-Madison Study4

Why A3 Chemistry is Different

5Conclusion

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Introduction
Sanitation is critical to food safety. Ineffective cleaning
can affect the appearance and taste of food, harbormicroorganisms and promote the production ofbiofilms. ATPi hygiene monitoring tests are one of
the most commonly used methods to verify cleaning
effectiveness as they are simple, easy-to-use andprovide immediate results. ATP is a critical metaboliteand energy source for living organisms and ATP-based
hygiene assessments rely on the principle that surface
contaminants contain ATP in concentrations sufficientfor detection. The principle also takes advantage ofthe assumption that changes in ATP are proportional
to the degree of residual contamination remaining onfood processing surfaces or equipment. If cleaning isineffective, organic debris from food may remain on food processing surfaces or equipment and remaining food residuals
may promote microbial growth, shield bacteria from the action of sanitizing agents or allow allergen-containing residuals
to remain. ATP testing is designed to verify the effectiveness of such cleaning and most food processing facilities in every
processing category use ATP tests for sanitation verification and experts estimate that more than 50 million tests are
conducted every year worldwide.

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Principle of ATP Testing
The method of action of conventional ATP tests involves the reaction of ATP with firefly luciferin (Figure 1) - thebiochemical that allows a firefly to produce light. The amount of light produced is proportional to the amount of ATP in asample and measured in Relative Light Units (RLU) using a luminometer.
The assumption underlying the principle in the use of ATP tests is that the concentration of ATP residuals remains
consistent over time as long as the residuals remain in place. This may frequently not be the case.

Light
Figure 1. Principle of Conventional ATP tests

The problem with using ATP measurements as an indicator of sanitation is that the ATP molecule can be unstable andcan rapidly decompose into adenosine diphosphate (ADP) and adenosine monophosphate (AMP) (Figure 2). If the ATP infood residue or biofilm has degraded, conventional ATP tests that indicate ATP levels alone can fail to be a true sanitation
indicator and can show false negatives. This problem has been shown to be a particular problem in the production ofmeats, including beef, pork and chicken. As ATP degrades, the concentration of total adenylate (ATP+ADP+AMP) remains
relatively stable and a test that could detect the total concentration of adenylate (or “A3”) would provide higher sensitivity
due to an increase in the signal to be detected, would be less likely to produce false negative results, and would provide
for an overall more accurate verification of sanitation.

ATPADPAMP

Figure 2. Degradation of ATPKikkoman Biochemifa Company3

University of Wisconsin-Madison Study
Research recently published in the Journal of Food Protectionii has uncovered
very interesting data about changes in ATP concentrations in foods and bacteriathat confirm this question about the effectiveness of ATP tests. The results of thisstudy revealed that ATP concentrations can vary by several orders of magnitude
as the cells present in residual food or bacteria continue to metabolize ATP andproduce higher concentrations of its metabolic products – ADP and AMP.
In this study, the researchers from the University of Wisconsin-Madison first examined the contribution of residual ATP fromdifferent types of raw meat (beef, pork and chicken). As with other foods, reliable cleaning and sanitation after the processingof raw meat is important to hygiene and ATP tests are frequently used by meat processors to assess the effectiveness of theircleaning processes. What the data from the Wisconsin-Madison study showed was that the concentration of ATP degraded in
the raw meat samples over time, reducing the concentration of ATP present in the meat residuals and thus reducing the signal
available to a test that detects only ATP (Figure 3). This can reduce the effectiveness of a traditional ATP test.
The study also showed that one of the ATP degradation products - ADP - was shown to remain stable in concentration in
the raw beef, pork and chicken samples. The Wisconsin-Madison study provides quantitative data of the shift in concentrationfrom predominately ATP to a state where ADP and AMP become the predominant adenylate species (Figure 3).
Food products are not the only source of residual ATP in food processing; microorganisms will also contribute to ATP on
food processing surfaces. Bacteria that may be present may be growing or may exist in various states of nutrient deficiency
or injury changing the growth phase at which they exist. In this same study, it was also shown that the concentrations of thedifferent adenylates varied in that ATP was predominant at initial stages, but AMP became predominant at later time points(Figure 4). These results indicate that
detection of ATP + ADP + AMP will be

Figure 3. The change of ratios of adenylate homologs in raw poultry.

more effective than measuring ATP
alone as an indicator for the potential
presence of bacteria.iii
Under these conditions, a test that
detects only ATP will fail to detect theconcentration of either the ADP or

6h

T0

AMP present and consequently fail26h

Figure 4. The change of ratios of adenylate homologs after
incubation of Escherichia coli

to serve as an effective indicator of
the presence of residuals. This agrees
with another previously publishedstudyiv that suggested that a test that
detects ATP plus the degradation
products of ATP will be able todetect raw meat residue much more
effectively than a test that detects

T0ATP alone.
ATPKikkoman Biochemifa Company

26hADP

12 days

AMP4

Why A3 Chemistry is Different
A test that can detect all three adenylates will be more effective
than one that detects ATP alone.
Kikkoman’s new LuciPac A3 Sanitation System detects
ATP+ADP+AMP with one swab to give you the whole picture.
With the use of two additional enzymatic reactions (Figure 5),
AMP and ADP can be recycled back to ATP. This allows the test
to detect the total adenylate concentration and dramaticallyincreases the signal available to the test.

Figure 5. Principle of A3 Technology

Conclusion
When considering the use of ATP-based hygiene tests, foodprocessors and hygiene specialists should consider thatvariables such as time prior to cleaning, source of the residual
contamination, and physical conditions such as temperature
may alter the quantity of ATP available for an ATP test potentiallymaking them less effective for the intended purpose. The
patentedv Kikkoman A3 technology has been proven to detect
residues that others miss. And it is just as easy to use as aconventional ATP test. Just swab the way you always have, but
you’ll detect what you have been missing.

From left to right: Lumitester Smart,LuciPac A3 Surface, LuciPac A3 Water

Kikkoman LuciPac A3 Surface is AOAC-PTM certified.

i

Adenosine triphosphateii

Quantities of adenylate homologues (ATP+ADP+AMP) change over time in Prokaryotic and Eukaryotic cells. Smith N. W. et al., J. Food Prot. 2019, 82, 2088.

iii

The presence of ATP of other adenylates is not a confirmatory test for the presence of bacteria but is used as an indicator of the effectiveness of cleaning processes.

iv

Bakke M. et al., J. Food Prot. 2018, 81, 729.

v

Patent pending

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