Food Protection Trends - July/August 2022 - 280

temperature. A germicidal UV lamp (Lumalier, Memphis, TN)
was the emission source, and a research radiometer (ILT1700,
International Light Technologies, Peabody, MA) was used
to monitor the intensity (W/cm2
) of the UV irradiation.
Coupons with 4-day-old mature biofilms were placed 20 cm
from the emission source and treated for 0, 15, or 30 min.
Because biofilms developed on both sides of the coupons,
coupons were flipped halfway through the total exposure time.
Exposed coupons were then aseptically transferred to 10 ml
of phosphate buffered saline (PBS; VWR, Radnor, PA).
Combined treatment application
The combined application of chemical sanitizers and UV-C
light was evaluated to determine any increase in antimicrobial
activity. Surfaces with mature biofilms were exposed for 10 min
to chemical sanitizers and then for 0, 15, and 30 min to UV-C
light treatment. Because no differences among holding times
without UV-C light treatment were observed, this parameter
was not added to the experimental design. Treatment sequence
effect was also evaluated based on differences in antimicrobial
effectiveness when UV-C was applied before or after the
sanitizer step. After each treatment, coupons were aseptically
placed in 10 ml of D/E neutralizer broth. Control treatments
consisted of coupons with untreated biofilm in 10 ml of PBS or
D/E neutralizer broth.
Cells recovery and microbial counts
After exposure to single or combined treatments, coupons
were sonicated for 30 s at 45 kHz and then vortexed for 30
s, following ASTM standard E2871-19 (2). This process
was repeated three times to assure complete detachment of
biofilm cells. Serial dilutions were made in 0.1% peptone water
(Difco, BD) and spread plated in duplicate on TSA. Plates
were incubated at 37 ± 2°C for 24 h. Colonies were manually
counted, and results were recorded as log CFU per square
centimeter.
Statistical analysis
Treatments were randomized across coupons, and experiments
were run six times to reduce experimental error. Statistical
significance was defined at P < 0.05. An analysis of variance
and a multiple comparison of means test were used to evaluate
differences in the treatment results. Data were analyzed using the
GLM procedure of SAS 9.4 (SAS Institute, Cary, NC).
RESULTS
Effect of single treatment application on the reduction
of L. monocytogenes biofilms
The effects of UV-C light, lactic acid, peroxy acid, and
quaternary ammonium treatments alone on L. monocytogenes
biofilms are shown in Table 1. After 4 days at 30°C in TSBYE,
control biofilms reached a population of 6.04 ± 0.49 log CFU/
cm2
. When mature biofilms were exposed to UV-C light for 15
or 30 min, significant reductions (P < 0.05) of 1.73 ± 0.79 and
280 Food Protection Trends July/August
1.68 ± 0.97 log CFU/cm2
, respectively, were observed. However,
no significant differences were found between the 15- and 30min
exposures to UV light. After 10 min of exposure to 4% lactic
acid, a reduction of 3.06 ± 0.85 log CFU/cm2
was obtained,
exposure to 400 ppm of quaternary ammonium resulted in 2.61
± 0.91 log CFU/cm2
reduction, and exposure to 100 ppm of
peroxy acid achieved a 3.66 ± 0.90 log CFU/cm2
reduction. No
significant differences were observed between lactic acid and
quaternary ammonium treatments. Peroxy acid was the most
effective of the single treatment applications (P < 0.05). Overall,
all the chemical sanitizers investigated in this study significantly
reduced L. monocytogenes biofilms compared with the untreated
controls (P < 0.05).
Effect of combined treatment application on the
reduction of L. monocytogenes biofilms
Table 1 shows L. monocytogenes biofilm reductions obtained
after exposure to the combination treatments of chemical
sanitizers and UV-C light.
Lactic acid
Evaluation of the order of treatment application revealed a
significant effect (P < 0.05) for the combination of lactic acid
and UV-C light. When lactic acid was followed by 15 or 30 min
of UV-C light, biofilms were reduced by 5.11 ± 0.66 and 4.78
± 1.02 log CFU/cm2
, respectively, compared with the control.
Conversely, when lactic acid was preceded by 15 min of UV-C
light, no significant significance (P > 0.05) in biofilm reduction
(3.26 ± 0.62 log CFU/cm2
use of lactic acid alone (3.06 ± 0.85 log CFU/cm2
.
) was observed compared with the
). However,
a significant effect (P < 0.05) was observed when UV-C
exposure time was extended to 30 min: a reduction of 4.02
± 0.67 log CFU/cm2
Quaternary ammonium
Evaluation of the order of treatment application revealed
no significant effect (P > 0.05) for the combination of
quaternary ammonium and UV-C light. Nevertheless, an
enhanced log reduction was observed when the antimicrobial
treatments were used together compared with their use alone.
When quaternary ammonium was applied before UV-C light,
a significant difference was found (P < 0.05) between
the 15- and 30-min treatments, with 3.28 ± 1.32 and 4.02
± 1.19 log CFU/cm2
reductions, respectively. However, when
UV-C light was used first, no difference (P > 0.05) was found
between the 15- and 30-min treatments, with 3.45 ± 0.93 and
3.85 ± 0.84 log CFU/cm2
reductions, respectively.
Peroxy acid
Evaluation of the order of treatment application revealed a
significant effect (P < 0.05) for peroxy acid and UV-C light.
Greater reduction was observed when peroxy acid was applied
before the UV-C light. However, no significant difference
(P > 0.05) was observed between the reduction achieved by
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