Food Protection Trends - July/August 2022 - 282

CFU/cm2
was obtained after treatment with 400 ppm of
QAC. These differences could be attributed to differences in
biofilm age and sanitizer concentration. Chavant et al. (11)
observed a decrease in biofilm cell mortality from 98% after
6 h to 45% after 7 days of biofilm maturation when samples
were exposed to 20 ppm of QACs. In another study, solutions
of hydrogen peroxide and sodium hypochlorite were more
effective than QACs for controlling Staphylococcus aureus
and Pseudomonas aeruginosa biofilms (22). However, the
use of QACs is one of the most common strategies in the
food industry (16). Very few studies have been conducted
to investigate the combination of QACs with other control
strategies against biofilms (8, 9). Berrang et al. (8) found an
enhanced bactericidal effect when quaternary ammonium
(400 ppm) was combined with ultrasonication, with a 3.5-log
reduction of L. monocytogenes biofilms. Similar log reductions
were observed in our study when QACs were combined with
UV-C light (3.28 to 4.02 log CFU/cm2
).
Effective control of biofilms has been achieved by use of
compounds with strong oxidizing properties, such as peroxyacetic
acid and peroxyoctanoic acid (3, 29). Peroxyacetic acid
at 80 and 160 ppm against L. monocytogenes biofilms on polystyrene
achieved reductions of 3.29 and 4.34 log CFU per well,
respectively (21). Fatemi and Frank (14) compared hydrogen
peroxide, peroctanoic acid, and peracetic acid sanitizers. Peracetic
acid and peroctanoic acid (80 ppm) inactivated L. monocytogenes
biofilms on stainless steel, with 3.2 and 3.9 log CFU/
cm2
reductions after 5 min of exposure. Hydrogen peroxide
and peroxyacetic acid are strong oxidizing agents, whereas peroxyoctanoic
acid is a surface-active component retaining active
antimicrobial agents (3). In our study, peroxy acid followed by
UV-C light resulted in a high level of biofilm reduction (4.4 to
4.7 log CFU/cm2
), indicating enhancement of antimicrobial
activity when treatments were combined. Conversely, Berrang
et al. (8) found no enhancement in biofilm reduction with use
of peroxyacetic acid followed by ultrasonication. The use of
UV light at 0.3 and 0.6 J/cm2
against biofilms of L. monocytogenes
on stainless steel achieved reductions of 0.26 and 0.42 log
CFU/cm2
was combined with sodium hypochlorite (200 ppm), with
reductions of 3.1 and 3.8 log CFU/cm2
. A synergistic effect was observed when UV light
(19).
When intervention strategies are combined (e.g., as in this
study), bacteria sublethally injured by the first treatment (e.g.,
peroxy acid) do not have time to recover before the immediate
application of the second treatment (e.g., UV light). The bacteria
sublethally injured by the first treatment activate defense
REFERENCES
1. Agle, M. E. 2007. Biofilms in the food
industry, p. 3-17. In H. P. Blaschek, H. Wang,
and M. E. Agle (ed.), Biofilms in the food
environment. First Edition. Wiley-Blackwell,
Hoboken, NJ.
2. ASTM International. 2019. E2871-19.
Standard test method for determining
disinfectant efficacy against biofilm grown
in the CDC Biofilm Reactor using the
single tube method. In Book of standards,
vol. 11.08. ASTM International, West
Conshohocken, PA.
3. Azanza, M. P. V. 2004. Hydrogen
peroxide, peroxyacetic acid, octanoic
acid, peroxyoctanoic acid, and
1-hydroxyethylidene-1,1-diphosphonic acid
(Hedp) as components of antimicrobial
washing solution. 63rd Joint FAO/WHO
Expert Committee on Food Additives
meeting. Food and Agriculture Organization
of the United Nations, Rome.
282 Food Protection Trends July/August
mechanisms to address the damage caused by that particular
stress. However, with the immediate application of the second
treatment, the bacterial defense mechanisms are overwhelmed
and not promptly activated, leading to a much greater
population reduction than achieved by the application of
a single intervention (37). The response mechanisms of
L. monocytogenes to these interventions differ; chemical
sanitizers cause oxidative or acid stress conditions, activating
bacterial cell wall repair genes, whereas UV light creates
abnormal chemical bonds in the DNA, triggering the photo
reactivation repair system (28, 30, 34, 39). When bacterial
cells encounter several stresses simultaneously, the defense
mechanisms are overwhelmed, resulting in a much greater
population reduction than achieved with the application of a
single intervention (37). UV irradiation damages nucleic acids,
and chemical sanitizers weaken the cell wall and membranes.
This multiple damage mechanism was also observed by
Koivunen and Heinonen-Tanski (20); microbial repair
mechanisms are overloaded and unable to repair the injuries,
resulting in death of the bacterial cells. We hypothesized that
when the chemical sanitizer is followed by UV light, the sanitizer
disrupts the membrane protecting the biofilms, allowing
deeper penetration of the UV radiation. In our experiments,
this combination resulted in the highest reduction in biofilms;
however, more research is needed to determine whether the
interventions were additive or synergistic by evaluating the effect
of the second intervention on injured versus uninjured cells.
In the present study, we evaluated the effect of three
chemical sanitizers (lactic acid, quaternary ammonium, and
peroxy acid) alone or in combination with UV-C light to
control L. monocytogenes biofilms on stainless steel. When
lactic acid treatment was followed by UV-C light, the greatest
log reduction in biofilm was observed. In contrast, quaternary
ammonium followed by UV-C light had the least effect on the
biofilm. Further research is needed to benchmark the proposed
treatments in food processing environments. Nevertheless,
the results obtained in this investigation support the use of
combined treatments as effective strategies to control biofilms
of L. monocytogenes on stainless steel.
ACKNOWLEDGMENTS
The authors thank the Kansas Department of Agriculture
and the U.S. Department of Agriculture, National Institute
of Food and Agriculture Hatch Multistate Research Fund
(project 1014385) for funding support. We especially thank
Indu Seetharaman for her statistical expertise and guidance.
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