Food Protection Trends - November/December 2020 - 403

control programs, hygiene monitoring to provide rapid and
timely results is required so corrective actions can be taken
immediately. Visual assessment alone is not accurate and
reliable even though it is economical (5, 6). Traditional
bacterial cultures are often used for sanitation evaluation.
However, culture incubation takes a few days, precluding
immediate feedback and timely action. Protein swab tests
and lateral flow immunoassays are quick and simple methods
for identification of food debris and allergens. The swabbased protein test can be used to detect food residues and a
broad range of allergens, but these tests are qualitative and
nonspecific and may not be effective with low levels of residual
protein contamination (7). Lateral flow immunoassays can
be used to detect allergenic foods qualitatively and selectively
with high sensitivity and thus are suitable for the control
of allergens but are not appropriate for monitoring organic
debris that does not contain the target proteins (6). Hygiene
monitoring tests based on the presence of ATP have also
been widely used because of their speed and simplicity.
ATP is the energy molecule found in animals, plants, and
microorganisms. Although ATP tests are nonspecific, they can
reveal the presence of food debris and bacteria, indicating a
risk of bacterial proliferation and cross-contact with allergens.
ATP measurements is based on the firefly luciferase reaction in
which light is produced from luciferin and ATP. The amount
of light produced is proportional to the amount of ATP in a
sample and can be quantified by measuring the light with a
luminometer and is expressed as relative light units (RLU).
Benchmark values are set to determine baseline cleanliness in
a facility, and these benchmark values are important guidelines
for the evaluation of cleanliness. Cleanliness is designated as
"pass" when the measured RLU fall below the benchmark
value and as "fail" when the RLU are above the benchmark.
When the test indicates a failing result, the recommendation
is to reclean the test site, revise the cleaning procedures, or
replace the failed equipment.
However, conventional ATP test systems can be inefficient
because ATP is degraded to ADP and AMP by heat, acids, or
alkalis, and enzymes (3). Raw meat and fish contain mainly
ADP, regardless of species (3), and ADP is the predominant
adenylate in bovine, porcine, and poultry carcasses (14). In
contrast, raw whole eggs, shellfish (shrimp, oysters, scallops,
and abalone), and processed meats and seafood (sausage,
bacon, beef jerky, canned fish, and dried fish) contain large
amounts of AMP (3). AMP is also found abundantly in many
plant seeds and nuts, which are widely known as allergens
(3). Hence, conventional ATP tests that indicate only ATP
concentrations might fail to detect other food residues and
would fail to identify inadequately cleaned surfaces.
A novel ATP-ADP-AMP (A3) test has been developed
based on a luciferin-luciferase assay with advanced enzyme
chemistry (3, 4, 12). In the luciferase reaction, ATP is
degraded into AMP, which then inhibits luciferase, resulting in
a decrease in light production. To detect AMP simultaneously

and maintain light production, ATP is regenerated from AMP
with the pyruvate orthophosphate dikinase reaction (3).
ADP is then converted to ATP by a pyruvate kinase reaction.
A device to simultaneously detect ATP, ADP, and AMP has
been successfully developed. The limit of detection for each
adenylate is ca. 2.5 fmol (2). This A3 test has been validated for
the detection of ATP, ADP, and AMP on stainless steel surfaces
and has been approved by the Association of Official Analytical
Collaboration with a performance tested method certification
(2). This A3 test is expected to provide for a more sensitive and
reliable indicator of cleanliness than the conventional ATP test.
The protocol of an ATP test consists of sample collection by
swabbing, extraction of the swab material, a bioluminescence
reaction, and an instrument-based detection of light. In
our previous study comparing conventional ATP tests and
the A3 test, diluted food samples were pipetted onto each
swab (3, 12). Further comparisons that include swabbing
efficacy are also important because this essential component
of the sampling phase is also affected by the variability of
the performance of the swabbing devices and moistening
agents. In the present study, swabbing assays of food residues
on stainless steel coupons were carried out to compare the
sensitivity of the A3 test and conventional ATP tests.

FIGURE 1. The measurement of swabbing pressure on the scale pan
of the analytical balance using the A3 test swab. When the swab shaft
slightly bent, the balance showed approximately 70-100 g.

MATERIALS AND METHODS
A3 and ATP tests
The A3 test was performed with a commercially available
test kit (LuciPac A3 Surface/Lumitester PD-30, Kikkoman
Biochemifa, Tokyo, Japan) according to the manufacturer's
directions. The three ATP tests used in this study were also
commercially available and were performed in accordance
with manufacturers' instructions. The measurements in all tests
were recorded in RLU.

November/December Food Protection Trends

403

Food Protection Trends - November/December 2020

Table of Contents for the Digital Edition of Food Protection Trends - November/December 2020