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ATP tests may be Affected byFood Processing and Ingredients

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ATP tests
ATP hygiene monitoring tests are one of the most commonly used methods to verify cleaning effectiveness. This is because they
are simple, easy-to-use and provide immediate results. The principle of the test applies the assumption that changes in ATP areproportional to the degree of residual contamination remaining on food processing surfaces or equipment. If cleaning has beenineffective, ATP-containing organic debris from food will be detected on food processing surfaces or equipment.
However, the capabilities of conventional ATP tests are more frequently being questioned and their limitations are becoming
more recognized.

How ATP tests operate
Adenosine triphosphate (ATP) is a molecule found in many food materials including plant and animal tissues as well as within

microorganisms such as yeasts, molds, and bacteria. In food processing, these same sources – foods, plant and animal tissues,and microorganisms – constitute most residual contamination on food processing surfaces. If high levels of ATP are found, theassumption is that residual contamination remains, as would be the case after inadequate cleaning. The amount of ATP
measured strongly correlates with the level of contamination present. In practice, when surfaces are cleaned to the point thatassessments of ATP concentrations are below a defined threshold, the surface is declared sufficiently clean and the subsequent
processing steps can proceed.11

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Importance of monitoring offood residues for food safety
Residual contamination on food production surfaces is recognized as not only an indicator of insufficient cleaning but also a
processing hazard that may promote unsanitary, unsafe conditions for the following reasons:(1) Raw agricultural food commodities, such as fresh meat, fish, dairy, fruit, and vegetables are known sources of both spoilage
and pathogenic bacteria.
(2) Food residues can be sources of nutrients that promote or enable the growth of microorganisms including the proliferationof difficult to clean biofilms that serve as a reservoir of those same microorganisms.
(3) Food residues and other soils can deplete the antimicrobial activity of disinfectants in several ways including the reduction of
the germicidal effect of the disinfectant through chemical reactions as well as the protection or insulation of microorganisms
from contact with the disinfectant. These reasons and others have established that cleaning verification is a prominent part of
all robust food safety plans. To meet the need to assess these soils in a timeframe suitable for processing, ATP tests are frequently
used to monitor and verify cleaning and sanitation practices in food production and food service operations.

Limitations of conventional ATP tests
Recent research, however, has illustrated some of the limitations of conventional ATP tests. These limitations can lead to thegeneration of false-negative assessments. In short, because of the unique, unstable behavior of ATP, a conventional ATP test issusceptible to indicating that a surface is clean, when significant soils remain. These false-negative assessments can lead aquality manager in a manufacturing facility to conclude that processing surfaces and equipment are clean, when in fact residual
food debris or contamination remains. ATP tests are designed to detect only the ATP molecule. Recent work has demonstrated
that ATP readily degrades (hydrolyzes) to the chemical homologues of ADP (adenosine diphosphate) and/or AMP (adenosinemonophosphate). When this degradation happens, there is significant reduction in ATP even though the soil remains on theequipment. As a result, the test – or more accurately the assessment – becomes much less sensitive as shown in various studies.

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Recent research from the University ofWisconsin – Madison about ATP degradation
Research from the University of Wisconsin – Madison demonstrated
that the degradation of ATP within raw meat in typical processing
environments can be dramatic even exhaustive in spite of theremaining presence of food soil.2 This university study showed thatATP degrades quickly into related compounds, ADP then AMP, whichbecome the predominant adenylates, neither of which are detectible
by standard ATP-based assays.
A particular study examined the practical implications of ATP degradation by comparing the performance of a conventional ATP
assay against an assay that detects all three adenylates (Kikkoman A3)3 in two licensed, inspected food processing facilities - onea Grade A dairy and the other a meat processing plant.4 A total of 960 data points for each type of assay were collected inparallel over the course of several months and seasons for the purposes of hygiene assessment. The study showed that theKikkoman A3 assay detected contamination events as frequently as the existing technology, but, more importantly, atsignificantly higher rates where ATP degradation was likely to occur.
Furthermore, additional research recently published in the Journal of Food Protection5 demonstrated that ADP generallycomprised about 90% (as a mole fraction) of total adenylates across all processing treatments with the exception the final cook
step where AMP became predominant (Figure 1). Retail processed meat samples were further evaluated and they followed
general trends of most having minimal ATP concentrations with ADP predominant in uncooked samples and AMP predominant
in cooked samples (Figure 2).
As a result, the University of Wisconsin-Madison researchers recommend that facilities carefully consider these degradation
phenomena when selecting a technology for purposes of hygiene assessment and verification. Kikkoman A3 can be areasonable approach to avoid the effects of the ATP degradation phenomena, especially in meat processing facilities or wheresensitivity to plant hygiene is especially critical.

Figure 1: The Change of ratios of adenylates at select manufacturing
steps during smoked sausage production in UW-Madison
Meat Science and Muscle Biology Laboratory■ ATPAfter grinding & stuffing

Figure 2: Ratio of adenylate content of selected
retail processed meat products

eiusagSam■ AMPokedem

Pa
stra

Hausag■ ADPSmSumm

er

Sa

lian

Dr
yur
stIta

izoatw

orhes
Fresh

BrChBacon■ ATPFr3■ AMP

After final cook with heating

Ratio of adenylates (%)

After trimmings■ ADPKikkoman Biochemifa Company01-04_清掃向け技術資料.indd 3
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Kikkoman A3 TestIn 2017, Kikkoman researchers developed the technology used in thisUniversity of Wisconsin-Madison study to remedy this shortcoming of current
ATP test technologies. Known as “Kikkoman A3” the technology accounts forthis demonstrated loss of ATP by simultaneously accounting for andmeasuring the presence of all three homologues, ATP, ADP, and AMP. As aresult, Kikkoman A3 has shown markedly improved sensitivity to ATPdepletion.

Swab and get Better results in 10 seconds.
• Tests are collected in the same way as conventional ATP tests – a simple
surface swab
• The A3 advantage is able to reverse ATP depletion• This advantage results in increased confidence in soil detection

Conclusion
Cleaning and sanitation verification practices throughout the food manufacturing environment are critical and form the basis ofevery food safety plan. Rapid, sensitive verification assays are an important step in such programs and are the reason ATP tests
were first developed and continue to be used. Recent studies, however, demonstrate the implications of the depletion of ATPand the increased risk of false negative assessments when using from ATP-only assays. All food processors, especially those whoprocess meats where ATP depletion is prominent, should carefully consider the selection of the appropriate food hygieneverification technology where accurate residual soil detection is critical to process success.6,7

1 Mildenhall, K. B, and S. A. Rankin 2020. Implications of Adenylate Metabolism in Hygiene Assessment: A Review. J. Food Prot. 83: 1619–1631. https://doi.org/10.4315/JFP-20-087
2 Smith, N. W., J. J. Sindelar, S. A. Rankin 2019. Quantities of adenylate homologues (ATP+ADP+AMP) change over time in Prokaryotic and Eukaryotic cells. J. Food Prot. 82: 2088–2093. https://doi.org/10.4315/0362-028X.JFP-19-223
3 Total adenylate is ATP+ADP+AMP, known as A3
4 Whyte, C., T. F. Ma, J. Sindelar, and S. A. Rankin 2021. Rapid hygiene assay sensitive to cumulative adenylate homologues exhibits equal or higher frequencies of soil contamination
detection than assay limited to ATP detection. J. Food Prot. 84: 1937–1944. https://doi.org/10.4315/JFP-21-028
5 Smith, N. W., J. J. Sindelar, S. A. Rankin 2024. AMP, ADP, and ATP concentrations differentially affected by meat processing, manufacturing, and non-meat ingredients. J. Food Prot.
87: 100287. https://doi.org/10.1016/j.jfp.2024.100287
6 Bakke, M., and S. Suzuki. 2018. Development of a novel hygiene monitoring system based on the detection of total adenylate (ATP+ADP+AMP). J. Food Prot. 81:729-737. https://doi.org/10.4315/0362-028X.JFP-17-432org/10.4315/0362-028X.JFP-19-223
7 Bakke, M. 2022. A comprehensive analysis of ATP tests: practical use and recent progress in the total adenylate test for the effective monitoring of hygiene. J. Food Prot. 85:10791095. https://doi.org/10.4315/JFP-21-384

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Kikkoman Biochemifa Company2-1-1 Nishi-Shinbashi, Minato-ku, Tokyo105-0003 JAPANbiochemifa@mail.kikkoman.co.jp
https://biochemifa.kikkoman.com/e/kit©2024 Kikkoman Corp.(1702W240601)

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