Food Control 26 (2012) 194e199

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Food Control
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Assessing manufacturers recommended concentrations of commercial sanitizers

on inactivation of Listeria monocytogenes
Cristina D. Cruz*, Graham C. Fletcher
The New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland 1025, New Zealand

a r t i c l e i n f o a b s t r a c t

Article history: Listeria monocytogenes is the causative agent of listeriosis in humans. Its wide distribution in the envi-
Received 1 November 2011 ronment is one of the main reasons it is a source of food poisoning. L. monocytogenes also forms biolms,
Received in revised form making cleaning and sanitation difcult. Microtiter plate assays have previously been used to assess
9 January 2012
biolm formation of L. monocytogenes and to evaluate the antimicrobial activity of disinfectants in
Accepted 17 January 2012
suspension. In this study, we have used a microplate assay to evaluate the minimum sanitizer concen-
trations required for a 5 log10 CFU/ml decrease of L. monocytogenes cells in suspension and in biolm.
Keywords:
Twenty-one sanitizers were tested against 20 strains of L. monocytogenes. The results showed that all
Listeria monocytogenes
Biolm
tested sanitizers achieved a 5-log10 reduction of viable cells in suspension at concentrations below the
Suspension manufacturers recommended concentrations, of a biguanide-based product. When tested against
Inactivation L. monocytogenes in biolm, only the peroxyacetic acid, chlorine dioxide and acidied sodium chorite-
Sanitizers based products gave a 5-log10 decrease, within or close to the manufacturers recommended concen-
trations. No relationship was observed between susceptibility to chemical sanitizers and other charac-
teristics of the isolates: pulsotypes, sources, biolm forming ability or persistence. This work suggests
that caution must be taken in selecting an appropriate sanitizer for use in food processing plants and an
effective cleansing programme is required to ensure thorough inactivation and removal of
L. monocytogenes in biolms.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Listeria monocytogenes is capable of attaching to inert surfaces
and subsequently forming biolms on food processing equipment
and environments (Autio et al., 1999; Norton et al., 2001; Rorvik,
Caugant, & Yndestad, 1995; Wong, 1998). Individual cells from the
growing biolm may be released during production, colonizing
new substrates or becoming a direct source of cross-contamination
to nal products.
In the food industry, the use of sanitizers and cleaners has been
incorporated into good manufacturing practices regimes to prevent
the accumulation of microbial cells and consequent biolm
formation (Hood & Zottola, 1995; Zottola & Sasahara, 1994).
However, various sanitizers extensively used by food processors
may not be very effective against some bacterial biolms (Bagge-
Ravn, Gardshodn, Gram, & Vogel, 2003; Lunden, Autio, Markkula,
* Corresponding author. Tel.: 64 9 926 3531; fax: 64 9 926 3570.
E-mail addresses: Cristina.Cruz@plantandfood.co.nz (C.D. Cruz), Graham.Fletcher@
plantandfood.co.nz (G.C. Fletcher).
Hellstrom, & Korkeala, 2003; Minei, Gomes, Ratti, DAngelis, & De
Martinis, 2008; Ryu & Beuchat, 2005; Surdeau, Laurent-Maquin,
Bouthors, & Gelle, 2006) and alternative removal strategies have
been studied. Because of innate differences in antimicrobial
susceptibilities, and the altered physiological state of some cells in
biolms, the effectiveness of industrial sanitizers may be affected. A
safe and effective sanitization process should ensure an acceptable
reduction in microbial levels without the presence of toxic
residuals.
Methods have been developed to evaluate the inactivation
efcacy of sanitizers against biolm bacteria using various
substrate materials such as stainless steel, rubber, and polystyrene
coupons (Folsom, Siragusa, & Frank, 2006; Parkar, Flint, & Brooks,
2004; Somers & Wong, 2004; Yang, Kendall, Medeiros, & Sofos,
2009). However, these methods are time-consuming and are
unlikely to produce biolms with consistent numbers of bacterial
cells for comparison. Moreover, these methods do not allow easy
testing of a variety of sanitizer products, a wide range of concen-
trations or different bacterial strains. A polyvinyl chloride (PVC)
microtiter plate assay has been used to evaluate the biolm
formation of L. monocytogenes and it was considered to be a rapid

0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2012.01.041
 C.D. Cruz, G.C. Fletcher / Food Control 26 (2012) 194e199 195

and simple method to screen for differences in biolm production
between strains (Djordjevic, Wiedmann, & McLandsborough, 2002;
Gamble & Muriana, 2007; Harvey, Keenan, & Gilmour, 2007; Moltz
& Martin, 2005). Microplate-based assays have also been used to
evaluate the antimicrobial activity of disinfectants in suspension
(Anonymous, 1988; Bloomeld, Arthur, Looney, Begun, & Patel,
1991).
The AOAC ofcial method proposed that, for a product to receive
the status of a sanitizer, it must meet the standard effectiveness of
a 99.999% reduction in number of viable microorganisms within
30 s (AOAC, 2005). Although the minimum inhibitory concentration
(MIC) is widely used to determine the efcacy of a sanitizer against
a pathogen, the determination of the bactericidal effect at the
manufacturers recommended concentration of a sanitizer is of
practical interest to the food industry (Heir, Sundheim, & Holck,
1995).
In this study, we developed a convenient microtiter plate assay
to assess the bactericidal effectiveness of different concentrations
of sanitizers that are commercially available in New Zealand against
L. monocytogenes cells both in suspension and in biolm. Results
were evaluated in comparison to the manufacturers recommended
concentrations. Relationship between susceptibility to the sani-
tizers and L. monocytogenes strains other characteristics was also
assessed.
2. Materials and methods
2.1. L. monocytogenes isolates
Twenty L. monocytogenes strains were selected from culture
collections to represent a range of biolm intensities, pulsotypes
and sources (Table 1). Frozen (85  C) stock cultures (Cruz &
Fletcher, 2011) were resuscitated in trypticase soy broth supple-
mented with 0.6% yeast extract (TSBYE, Difco) and held on
Columbia blood Agar (Difco) at 4  C until used in the experiments.
2.2. Sanitizers
Twenty-one sanitizers with six different active ingredients were
tested in this study based on their intended usage in food pro-
cessing environments, differences in chemical composition and
commercial availability in New Zealand (Table 2). All sanitizers
were kindly supplied by the manufacturers as concentrated liquids
and kept at 4e6  C upon receipt. Sanitizers were diluted in reverse
osmosis (RO) sterile water with 200 ppm added hardness (desig-
nated as 200 ppm hard water) containing MgCl2, CaCl2 and
NaHCO3 according to AOAC protocol (2005) immediately before
testing. Sanitizers were prepared in 2-fold dilutions in order to
obtain a range of six concentrations that included the maximum
concentration recommended by the manufacturer. Diluted sani-
tizers were used within 15 min of preparation. Sanitizers contain-
ing chlorine dioxide were prepared with a food grade acid provided
by the respective company.
2.3. Determination of the minimal effective concentration (MEC) of
sanitizers on L. monocytogenes in suspension
L. monocytogenes strains were grown overnight at 37  C in TBSYE
to achieve stationary phase with a nal concentration of ca. 2  107
colony forming units (CFU)/ml. Bacterial suspensions were centri-
fuged at 3200  g for 5 min and then cells were washed in sterile
200 ppm hard water, collected by centrifugation and resuspended
in a nal volume of 10 ml of sterile 200 ppm hard water.
An aliquot of the washed planktonic cell suspension (50 ml/well)
was added to the 96-well plate, followed by 50 ml/well of diluted
sanitizers. Plates were lightly tapped three times on the sides to
mix the suspensions and then left for 5 min (contact time) at 25  C.
After the contact time, 150 ml of a neutralizer solution containing 5%
egg yolk emulsion (Difco), 1% sodium thiosulphate (AnalaR, BDH
Chemicals Ltd, Poole, England) and 0.5% Tween-80 (Spectrum,
Gardena, CA) in TSBYE (AOAC, 2005) was applied to each well to
neutralize the antimicrobial effect of the sanitizers and was mixed

Table 1
Characteristics of Listeria monocytogenes isolates.

Culture collectiona Strain code Serotype Source Pulsotypec Biolm forming abilityb
PFR 15A04 1/2a or 3a Environment 3814 (p)d 0.41
15A07 1/2a or 3a Environment 6502 (p) 0.53
15B06 1/2a or 3a Environment 6502 (p) 0.52
15C02 1/2a or 3a Environment 5132 (p) 0.95
15D01 1/2a or 3a Environment 5132 (p) 0.94
15D10 1/2a or 3a Environment 5132 (p) 0.90
16A01 1/2a or 3a Seafood 3860 0.50
16G02 1/2a or 3a Environment 5132 (p) 0.56
16H02 1/2a or 3a Environment 0101 0.33
16H04 1/2a or 3a Environment 3814 (p) 0.39
16J10 1/2a or 3a Environment 5132 (p) 0.31
17A02 1/2a or 3a Environment 7002 0.87
18C09 1/2b or 3b or 7 Seafood 4048 0.64
18D05 1/2b or 3b or 7 Environment 3527 0.61
NZRCC LM01-07 1/2a or 3a Human 5132 (p) 1.21
LM00-75 1/2a or 3a Human 0203 0.97
CPH0413645 1/2a or 3a Food 0101 1.43
LM04-12 1/2a or 3a Human 3846 1.26
FSA 2655 1/2b or 3b or 7 Food 9006 1.03
ILSINA FSL J2-064 1/2b or 3b or 7 Animal 9937 1.05
a
PFR The New Zealand Institute for Plant & Food Research Limited (Cruz & Fletcher, 2011), NZRCC New Zealand Reference Culture Collection of Listeria strains,
FSA Food Science Australia (Hayman, Baxter, ORiordan, & Stewart, 2004), ILSINA International Life Sciences Institute North America (Fugett, Fortes, Nnoka, & Wiedmann,
2006).
b
As previously dened and used (Cruz & Fletcher, 2011).
c
BFA: biolm Forming Ability values are based on Crystal violet staining measured at OD595nm (low 0.0e0.7; intermediate 0.7e1.1 and high >1.1 biolm formers)
(Cruz & Fletcher, 2011).
d
(p): persistent pulsotypes recovered from premises over 6 months.
196 C.D. Cruz, G.C. Fletcher / Food Control 26 (2012) 194e199

Table 2
Commercial sanitizers tested in this study.

Manufacturer Commercial name Active ingredient (%) Recommended concentration (mg/ml)

Ecolab Sanova Acidied sodium chlorite 600e1000
XY-12 Sodium hypochlorite (10%) 100e200
Ecosafe Envirosan QHF Glutaraldehyde (GA, 10%)/Benzalkonium chloride (QAC, 5%) 1000 (GA)/500 (QAC)
Iodophor Multi Iodine 15e25
Tsunami Peroxyacetic acid (POAA) (15%) 120e250
Vortexx POAA/Peroxyoctanoic acid (4.5e5%) 80e150
Ster-bac Benzalkonium chloride (10%) 200e400
Jasol Hypostat 135 Sodium hypochlorite (13.5%) 200e800
Anthium Dioxcide Chlorine dioxide (2%) 200
Iodosan Iodine 25
Peroxysan 30 POAA (5%) 200e400
Powerquat Blue Benzalkonium chloride (15%) 200e400
Freedom Blend of QAC 150
Johnson Diversey Divosan Hypochlorite Sodium hypochlorite (13%) 100e200
JonClean 410 Chlorine dioxide (3.35%) 100e500
Divosan Iodophor Iodine 25e50
Divosan NR Benzalkonium chloride (10%) 200e1000
Orica Chemicals Enviroxyde Chlorine dioxide (2%) 200
Perform POAA (5%) 100e500
Bactoquat Benzalkonium chloride (10%) 200e800
Vantosan Poly biguanide hydrochloride (3%) 300

with light tapping. The control wells contained only 200 ppm hard
water and the bacterial suspension for each strain. The number of
viable cells in the suspension was enumerated by 10-fold serially
diluting in 0.85% saline solution and using the drop plate counting
method (5  25 ml) on TSAYE agar plates. The plates were incubated
at 35  C overnight. The most diluted suspension of the tested
sanitizers to show a viable bacterial reduction of 5-log10 CFU/ml
was considered as the minimal effective concentration (MEC).
2.4. Determination of minimal effective concentration (MEC) of
sanitizers on L. monocytogenes in biolm
The microtiter plate assay described by Djordjevic et al. (2002)
was used to form a 2-day mono-cultural biolm of L. mono-
cytogenes (Cruz & Fletcher, 2011). Sanitizers were 2-fold diluted in
200 ppm hard water, as previously described, but at 10-fold higher
concentrations. Diluted sanitizers (50 ml/well) were added to wells
containing the freshly grown and washed bacterial biolm. The
plate was lightly tapped three times on the side to facilitate even
distribution of the sanitizer solution. The biolms were incubated
with sanitizer for 5 min at 25  C. The control wells contained only
200 ppm hard water and the bacterial biolm for each strain. At the
end of incubation, 200 ml of neutralization solution was added to
each well to quench the antimicrobial activity of the sanitizer. The
bacterial cells in the biolm were scrapped from the plates using
a sterile cotton wool swab (Nanjing Foreign Economic & trade
Development CO., Ltd, China) in circular movements (alternatively
clockwise and anti-clockwise) 10 times to detach them from the
bottom of the wells. Detached bacterial cells were separated from
the swab by rotating the shaft of the swab between palms of hands
calculated from the combined measurements and analysed using
analysis of variance (ANOVA). Signicance differences (p < 0.05)
were analysed by Tukeys HSD test.
3. Results and discussion
The microplate assay method was found to be a convenient,
simple, and reproducible method that can be used to compare the
bactericidal efcacies of multiple sanitizers at multiple concentra-
tions and against multiple isolates whether in suspension or in
biolm.
The overall variation of the chemical concentrations to achieve
a 99.999% reduction (MEC) of L. monocytogenes cells in suspension
is shown in Table 3.
For Listeria cells in suspension, lower concentrations of most
commercial sanitizers were needed to achieve a 5-log10 decrease of
initial inoculum (MEC) than the manufacturers recommended
concentration (MRC). With the exception of the poly biguanide
hydrochloride-based sanitizer, equivalent kill rates were achieved
for all products at both half and the full recommended use
concentrations. POAA and QAC-based products had the lowest MEC
in comparison to their respective MRC. The MEC of acidied sodium
Table 3
Mean minimal effective concentrations (MEC) and manufacturers maximum rec-
ommended concentrations (MRC) of different sanitizers chemical group for Listeria
monocytogenes cells in suspension and in biolm.
Chemical group MECa (mg/ml) MRCb (mg/ml)
(or type of sanitizer)
Suspension Biolm

six times in either direction within the wells. The viable cells were Meanc SEM Meanc SEM
enumerated following the drop plate counting method. To allow Acidied sodium chlorite 121a 31.1 242a 31.2 1000
comparison, the results were calculated as cell mortality in Biguanide 241b 16.1 812b 105 300
Chlorine 28c 5.40 3,600c 208 200
comparison with their own control biolm for each independent
Chlorine dioxide 83a 5.90 111d 8.28 500
experiment/replicate. Iodine 15d 0.82 468e 34.9 50
Peroxyacetic acid 39c 5.29 172f 11.1 500
Quaternary ammonium 66e 5.03 2,023g 98.7 1000
2.5. Statistical analysis
compound
a
Duplicate replicates were performed in each of two indepen- Minimum Effective Concentration to achieve a 5-log10 CFU/ml in viable cells.
b
Manufacturers Recommended Concentration (maximum value stated for the
dent experiments for each sanitizerestrain combination (420 group based on the active ingredient concentration).
combinations). The viable cell count data were log-transformed to c
Means with the same letter in same column are not signicantly different
stabilise variance and the standard error of the mean was (p > 0.05).
 C.D. Cruz, G.C. Fletcher / Food Control 26 (2012) 194e199
chlorite (ASC) was similar to chlorine dioxide (p > 0.05) and that of
chlorine was similar to POAA (p > 0.05) formulations (Table 3).
When bacterial cells were cultivated as a simple 48-h biolm on
polyvinyl surfaces, the MEC of the commercial sanitizers increased
measurably compared with those in suspension, independent of
the active antimicrobial agent(s) present in the formulation
(p > 0.05). Only the ASC, peroxyacetic acid (POAA) and chlorine
dioxide products had average MEC values below or at the MRC, for
the inactivation of cells in the Listeria biolm (Table 3).
The antimicrobial activity of the halogen-based sanitizers was
most adversely affected by the biolm (chlorine MEC > 18 times the
highest MRC; iodine MEC > 10 times the highest MRC). The anti-
microbial activity of quaternary ammonium compounds (QAC) was
also markedly reduced against the Listeria biolm, with MEC values
at least 2 times higher than the MRC (Table 3). The differences
between measured average MEC values for Listeria cells in
suspension and the measured average MEC values for cells in the
biolm were even larger; chlorine had an MEC about 120 times
greater for Listeria in biolm and QAC and iodine compounds more
than 30 times (Table 3).
Clearly, the efcacy of the commercial sanitizers tested in this
study was markedly affected by whether the bacterial cells were in
suspension or in a biolm. Planktonic cells of L. monocytogenes
were found to be sensitive to most commercial sanitizers, when
evaluated at the MRC for an exposure time of 5 min. These results
were not unexpected, as previous reports have shown that Listeria
in suspension culture is not particularly difcult to destroy with
sanitizers (Wirtanen, Salo, Allison, Mattila-Sandholm, & Gilbert,
1998). However, four classes of sanitizers tested in this study could
not achieve a 5-log10 kill of L. monocytogenes cells present as
a simple biolm when tested at their MRC. Chlorine, iodine,
biguanide and most QAC-based products required higher concen-
trations than the manufacturers recommended concentrations to
be used as a biolm control agent. Previous studies have demon-
strated that bacteria in biolms, whether present as a single species
or multi-species biolm, show a greater level of resistance to
several physicochemical stresses such as antibacterial agents
(Berrang, Frank, & Meinersmann, 2008; Folsom et al., 2006; Pan,
Breidt, & Kathariou, 2006; van der Veen & Abee, 2011; Yang et al.,
2009). Studies have also shown that biolm resistance is related
to the age of the biolm (Belessi, Gounadaki, Psomas, & Skandamis,
2011; Chavant, Gaillard-Martinie, & Hebraud, 2004; Ibusquiza,
Herrera, & Cabo, 2011). In the present work this resistance was
clearly observed in a 48-h biolm.
ANOVA revealed no signicant difference (p > 0.05) between
the 20 L. monocytogenes strains regardless of the active chemical
present in the formulation of the sanitizer. No correlation was seen
when the isolates were grouped by pulsotypes, sources or biolm
forming ability (p > 0.05). In particular, the three pulsotypes that
persisted in mussel factories (strain codes 15A04, 15A07, 15B06,
15C02, 15D01, 15D10, 16G02, 16H04, 16J10; Cruz & Fletcher, 2011)
did not show any particular resistance to any of the sanitizers tested
in this study when compared with the other strains (p > 0.05).
These results indicate that this persistence was not due to resis-
tance to sanitizers. Other studies have found resistance to chlorine
(El-Kest & Marth, 1988; Folsom et al., 2006; Lunden et al., 2003) or
QAC-based products (Aase, Sundheim, Langsrud, & Rorvik, 2000;
Mereghetti, Quentin, Marquet-Van Der Mee, & Audurier, 2000; Pan
et al., 2006). Resistance to benzalkonium chloride, the major
component of commonly used industrial QAC-based sanitizers, has
been demonstrated for different bacteria, not only in biolms but
also in suspension cells (Heir et al., 2004; Romanova, Favrin, &
Grifths, 2002). This resistance appears not to be an independent
factor leading to persistence of certain strains in food processing
environments, but one of the contributing factors together with
197
biolm formation ability of the isolates (Aase et al., 2000;
Mereghetti et al., 2000; Pan et al., 2006). The test conditions chosen
in this study provided challenging conditions for QAC-based sani-
tizers with water hardness >100 ppm known to adversely affect
QAC performance (Mafu & Roy, 2001). According to manufacturers
information sheets, all tested QAC products contained benzalko-
nium chloride as the main active ingredient, with the exception of
Freedom, which was described as a blend of QACs.
Our results suggest that three classes of antimicrobial agents:
acidied sodium chlorite, POAA (including combination peroxy-
acetic and peroxyoctanoic mixtures) and chlorine dioxide, reliably
achieved a 5-log reduction against Listeria biolm cells within or
close to their MRC. However, it should be noted that the biolms
used in this study were simple mono-specic biolms formed
under static high nutrient conditions. Biolms that form in nature
are typically formed under more dynamic conditions of changing
(typically lower) nutrient loads and ows and typically consist of
a variety of bacterial species, which may make them more resistant
to sanitizers (Bremer, Monk, & Osborne, 2001; Mai & Conner, 2007;
van der Veen & Abee, 2011; van der Veen et al., 2010).
Berrang et al. (2008) have also shown the relative effectiveness
of POAA-based products on Listeria biolms within polyvinyl drain
pipes, in comparison to QAC- and chlorine-based products. The
efcacy of peroxyacetic-based products against biolms is
proposed to be a function of several factors: the ability of the small
molecule to penetrate the biolm extracellular polymeric
substance (EPS) matrix, the mode of action of the antimicrobial, and
its tolerance to moderate levels of organic matter (Burnett, Cords,
Finley, Magnuson, & Hilgren, 2005). Peroxide-based compounds
have also been reported to be effective for the removal of bacterial
biolms and are still widely used in the food industry (Fatemi &
Frank, 1999; Ibusquiza et al., 2011; Pan et al., 2006; Stopforth,
Samelis, Sofos, Kendall, & Smith, 2002).
Chlorine dioxide has received similar focus as POAA for control
of biolms because of its antimicrobial activity in the presence of
high levels of organic matter, either external to the biolm or part
of the EPS, and its strong oxidizing power (3.5-fold more than
chlorine). In addition it is often favoured over chlorine as it does not
react with nitrogen-containing compounds or ammonia to form
dangerous chloramines (Matthews, 2006).
In the present work, the intrinsic ability of each strain to form
biolms did not contribute to an increase in the sanitizers MEC.
Earnshaw and Lawrence (1998) and Folsom et al. (2006) also
showed no correlation among subtypes of L. monocytogenes cells,
biolm forming ability and sanitizer susceptibility. The ability of
L. monocytogenes strains to survive exposure to sanitizers may be
more associated with the amount of extracellular polysaccharide
produced by the biolm cells than the number of cells contained
within the biolm, or to other factors of its genetic subtype.
The effectiveness of individual sanitizers on L. monocytogenes
planktonic cells (in suspension) is shown in Fig. 1. All products had
an MEC signicantly below the MRC, independent of the brand.
However, some products with the same active ingredient supplied
by different companies showed signicant differences in MEC
(p < 0.05) with the iodine-based products being the only group that
did not differ among brands (p > 0.05) (Fig. 1).
When considering the performance of individual sanitizers
against Listeria in biolms, differences were also observed among
sanitizers depending on the brand and active agent, as illustrated in
Fig. 2. All chlorine dioxide-based products had similar performance
for Listeria cells in biolm, each performing well with an MEC
below the MRC. All POAA-based products also performed as good
biolm control agents but efciency varied with brand (p > 0.05).
This work has demonstrated that products in the same chemical
group show different efcacies against L. monocytogenes. It is
198 C.D. Cruz, G.C. Fletcher / Food Control 26 (2012) 194e199

1250
MEC
MRC
1000

Concentration (g/mL)
750

500

250
c c
b a a b
a,b a a a, b
b a a, a b
a a a a
0

Acidified Biguanide Chlorine Chlorine Dioxide Iodine Peroxyacetic Quaternary Ammonium Compound
Sodium acid
Chlorite

Sanitizers

Fig. 1. Average minimal effective concentrations (MEC) of 20 sanitizers required to achieve a 5-log10 reduction of Listeria monocytogenes in suspension after a 5-min contact time at
25  C. Manufacturers recommended concentrations (MRC) are displayed as the maximum values advised. Error bars depict standard error of the means (SEM). Equal letters mean
no signicant differences between sanitizers MEC of the same chemical group (p > 0.05).

important to note that the performance of a sanitizer will be the increased resistance of Listeria in biolms for some antimi-
affected by the type and stage of the microorganism to be inacti- crobial agents. Costs and practicability should be taken into
vated and by the food matrix present in the processing plant (Gram, account in the selection process of the best combination of
Bagge-Ravn, Ng, Gymoese, & Vogel, 2007). Conclusions outlined in commercial sanitizers for each food processor. Recommended
this work may thereby be limited to the conditions tested. concentrations of products should be based on harsh conditions
A common observation for all the tested sanitizers was that the and their suitability for specic applications should be also
manufacturers recommended use concentrations were signi- mentioned (i.e. effective against biolm). The biguanide-based
cantly higher than the tested MEC for effective kill of cells in sanitizer, all tested sanitizers would offer effective listericidal
suspension. It is acknowledged commercial practice for suppliers control, if applied at the recommended dose and after an effective
to endeavour to provide an adequate margin of safety for use of cleansing program, removing substrates for any possibility of
their products to cover many variances in actual use, including soil biolm formation. POAA, chlorine dioxide and ASC sanitizers may
loads, water conditions, application methods and human vari- also be effective in inactivating Listeria that are embedded in
ability. Despite this, it is clear that these allowances may not cover biolms.

6000
a MEC
MRC
5000
a
Concentration (g/mL)

4000

3000
a
a a
b
a,b
b
2000

c
c
1000
b
b a, b
a a a a a
a
0

Acidified Biguanide Chlorine Chlorine Dioxide Iodine Peroxyacetic acid Quaternary Ammonium
Sodium Compound
Chlorite
Sanitizers

Fig. 2. Average minimal effective concentration (MEC) of 20 sanitizers required to achieve a 5-log10 reduction of Listeria monocytogenes biolm after a 5-min contact time at 25  C.
Manufacturers recommended concentration (MRC) is displayed as the maximum value advised. Error bars depict standard error of the means (SEM). Equal letters mean no
signicant differences among sanitizers MEC of the same chemical group (p > 0.05).
 C.D. Cruz, G.C. Fletcher / Food Control 26 (2012) 194e199
Acknowledgements
This project was funded by the New Zealand Foundation for
Research, Science & Technology (Contract CAWX0703). The authors
are grateful to New Zealand seafood companies for supplying the
cultures and to the sanitizer manufacturers for providing the san-
itizer samples used in this study. We would also like to thank
Guangjin Lu, Annette White, and Fiona McKenzie for technical
assistance and David Lowry and Anthony Mutukumira for their
critical review of the manuscript.
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