Influence of nisin and selected meat additives on the antimicrobial effect
of ovotransferrin against Listeria monocytogenes
S. H. Moon,* H.-D. Paik,* S. White,† A. Daraba,‡ A. F. Mendonca,† and D. U. Ahn§#1
*Division of Animal Life Science, Konkuk University, Seoul 143-701, South Korea; †Department of Food Science
and Human Nutrition, Iowa State University, Ames 50010; ‡Faculty of Food Science and Engineering,
University Dunarea de Jos, Galati, 800008 Romania; §Department of Animal Science, Iowa State University,
Ames 50011; and #Department of Agricultural Biotechnology, Major in Biomodulation, WCU,
Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, Korea
ABSTRACT The objective of this study was to determine the effect of nisin and selected meat additives
(salt, lactate, lactate–diacetate combination, and polyphosphate) on the antimicrobial activities of ovotransferrin (OTF) against the growth of Listeria monocytogenes. A Bioscreen C turbidometer (Oy Growth Curves
AB Ltd., Helsinki, Finland) was used to evaluate the
effect of various concentrations of nisin and individual
meat additives on the antilisterial activity of OTF in
brain heart infusion (BHI) broth. The concentrations
of OTF, meat additives, nisin, and their combinations
that proved most inhibitory to L. monocytogenes were
selected and their antilisterial effects were tested using frankfurters. Frankfurters were inoculated with L.
monocytogenes (~6.0 log10 cfu/frankfurter); treated
with OTF, meat additives, and nisin singly or in combination; and held under vacuum at 4, 10, or 25°C. At
40 mg/mL, OTF strongly suppressed (3.46 log at 4 h
and 2.59 log at 12 h) the growth of L. monocytogenes
in BHI broth compared with the control. A combination of OTF (40 mg/mL) and nisin (1,000 IU) inhibited
the growth of L. monocytogenes in BHI and in frankfurters held at 25°C below the detection limit (1 cfu/
mL) at 12 h. However, the antimicrobial effect of OTF
(40 mg/mL) alone was not observed in frankfurters at
all temperatures used in this study. Nisin (1,000 IU),
OTF (40 mg/mL), and nisin (1,000 IU) combination
completely inhibited the growth of L. monocytogenes in
frankfurters at all temperatures during 3 d. Salt at 0.5
and 1%, lactate at 0.78 and 1.56%, and lactate (1.56%)
+ diacetate (0.01%) did not alter the inhibitory effect
of OTF against the pathogen in BHI, but salt at 2% or
polyphosphate at 0.05% negated the growth inhibitory
effect of OTF against L. monocytogenes. This study
demonstrated that combination of OTF and nisin was
effective in controlling L. monocytogenes.
Key words: Listeria monocytogenes, ovotransferrin, nisin, meat additive, growth inhibition
2011 Poultry Science 90:2584–2591
doi:10.3382/ps.2010-01275
INTRODUCTION
Trends toward the use of natural food preservatives
are growing as consumers become more health conscious and concerned about the long-term health effects
of synthetic chemicals in foods. In response to such consumer demands, the food industry showed increased interests in the use of antimicrobial preservatives that are
viewed as natural. Many antimicrobials from natural
sources are limited in their range of activity and exhibit
effectiveness at relatively high concentrations. One possible way of circumventing this problem is combined
use of antimicrobials (Sofos et al., 1998).
©2011 Poultry Science Association Inc.
Received December 2, 2010.
Accepted March 12, 2011.
1 Corresponding author: duahn@iastate.edu
Ovotransferrin (OTF) and nisin are both natural
antimicrobials. Ovotransferrin is the main component
in the antimicrobial defense system of hens’ egg. It is
an iron-binding glycoprotein that transports and scavenges Fe(III) in poultry eggs (Kurokawa et al., 1995).
The iron-binding action of OTF is considered to be its
main antimicrobial mechanisms (Arnold et al., 1981),
but the direct interactions of OTF with the bacterial
surface also seem to play an important role (Valenti et
al., 1983, 1985; Ibrahim, 1997; Ibrahim et al., 2000).
Nisin is a bacteriocin produced by Lactococcus lactis
spp. that is lethal to gram-positive bacteria (Ming and
Daeschel, 1993, 1995; Delves-Broughton et al., 1996;
Apostolidis et al., 2008). To date it is the only bacteriocin approved for use in a wide variety of food products
(Delves-Broughton, 1990). The cellular target for the
antibacterial action of nisin is cytoplasmic membrane
where bacteriocin produces pores, which results in de-
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GROWTH INHIBITION OF LISTERIA MONOCYTOGENES BY OVOTRANSFERRIN AND NISIN
struction of transmembrane pH gradients, diffusion of
the proton motive force, and inhibition of ATP synthesis (Okereke and Montville, 1992).
Listeria monocytogenes is an enteric pathogen that
has been implicated in several outbreaks traced to
contaminated cheese and other dairy products and to
various types of meat, including turkey meats (CDCP,
2002; Gandhi and Chikindas, 2007). Among the known
foodborne enteric pathogens, L. monocytogenes has the
second highest mortality rate (20%) and the highest
hospitalization rate (90%; Mead et al., 1990). Because
of its relatively high fatality rate and the uncertainty
of infectious dose for immune-compromised individuals, US regulatory agencies established a zero tolerance
for L. monocytogenes in cooked and ready-to-eat meat
products (USDA, 2003).
Meat additives such as salt, sodium lactate (SL),
sodium diacetate (SDA), and polyphosphate are extensively used in meat and poultry products as antimicrobial agents (Apostolidis et al., 2008). However,
published studies on the antimicrobial activities of
these substances on L. monocytogenes in cooked cured
meat products are limited (Stekelenburg and KantMuermans, 2001). To our knowledge limited published
reports exist on the effect of commonly used meat additives on the antibacterial effectiveness of OTF. The
objective of this study was to determine the effects of
nisin or selected meat additives on the ability of hen
egg OTF to control the growth of L. monocytogenes in
laboratory broth and frankfurters.
MATERIALS AND METHODS
OTF
Ovotransferrin was prepared from egg white using
the method by Ko and Ahn (2008). For the experiments involving the Bioscreen C turbidometer (version
2.6; Oy Growth Curves AB Ltd., Helsinki, Finland),
all OTF solutions were prepared in brain heart infusion (BHI) broth (Fisher Scientific, Pittsburgh, PA)
and filter sterilized using Millipore filters (0.22 µm
pore size; Millipore, Billerica, MA). Vacuum-packaged
frankfurters (beef–pork) were purchased from a local
grocery store and kept at 4°C until used. Nisaplin, a
commercial nisin product (2.5% nisin, 106 IU/g), was
provided by Danisco USA Inc. (Thomson, IL).
Bacterial Strains and Culture Conditions
A 5-strain mixture of L. monocytogenes, including
strains H7962 serotype 4b, H7762 serotype 4b, H7969
serotype 4b, H7764 serotype 1/2 a, and Scott A NADC
2045 serotype 4b, was used in this study. The L. monocytogenes Scott A strain was obtained from Irene Wesley at the National Animal Disease Center (Agricultural Research Service, USDA, Ames, IA). All other
strains were obtained as clinical isolates from the multistate outbreak of 1998 and 1999 (CDCP, 2002). Each
2585
organism was maintained as a frozen (−70°C) stock
culture in BHI broth (Danisco USA Inc.) supplemented
with 10% glycerol until used. For each experiment, 2 sequential 24-h transfers of individual stock cultures were
performed in 10 mL of tryptic soy broth (Difco Laboratories, Detroit, MI) supplemented with 0.6% yeast
extract (Difco Laboratories) and incubated at 35°C for
20 h.
Preparation of Inoculum
Equal amounts of each working culture were combined to prepare a 5-strain mixture of L. monocytogenes. Cells were harvested by centrifugation (10,000
× g, 10 min, 4°C) in a Sorvall Super T21 centrifuge
(DuPont Instruments, Wilmington, DE) and washed
once in sterile 0.85% (wt/vol) NaCl (saline; (Danisco
USA Inc.). The pelleted cells were suspended in fresh
saline and the resulting cell suspension was used as the
inoculum.
Sample Preparation and Inoculation
The growth-inhibitory effect of OTF on L. monocytogenes was determined by measuring the turbidity
of BHI broth culture (35°C) over a 24-h period using
a Bioscreen C turbidometer (Oy Growth Curves AB
Ltd.). The final concentration of OTF was adjusted
to 10, 20, and 40 mg/mL in BHI broth. The stock
solutions of NaCl, SL, SL + SDA, sodium hexametaphosphate (SHMP), or nisin were added to the media
containing 40 mg/mL of OTF. The final concentration
of NaCl was adjusted to 1 and 2% and potassium lactate was adjusted to 0.78 and 1.56%. The SL + SDA
combination solution was adjusted to 0.78% + 0.052%.
Sodium hexametaphosphate was used at 0.05% (final
concentration). Nisin concentration was adjusted to
500 or 1,000 IU (final).
The antibacterial activity of OTF as affected by nisin was investigated using a viability test. First, 5 mL
of OTF solution (80 mg/mL) was added to 5 mL of
BHI broth media containing 1,000 or 2,000 IU of nisin. Cell suspension (100 μL) containing approximately
106 to 107 actively growing L. monocytogenes cells was
inoculated to the prepared solution to make approximately 105 cfu/mL in the initial solution. The OTF
solutions (20 and 40 mg/mL), nisin (500 IU), and nisin
(1,000 IU) were prepared as controls. After inoculation, the prepared culture solutions were incubated at
35°C for 24 h. The number of viable cells was analyzed
by spread plating each culture solution (0.1 mL) after
diluting (1:10) with 0.1% sterile peptone water (Remel
Inc., Lenexa, KS). The samples were incubated at 35°C
for 48 h. The number of survivors on modified Oxford
medium (MOX) agar plates (Danisco USA Inc.) was
counted as colony-forming units per milliliter of sample.
Frankfurter samples (1 frankfurter/vacuum bag)
were inoculated with 0.1 mL of the 5-strain mixture to
give a final cell concentration of approximately 104 to
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Moon et al.
Figure 1. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 10–40 mg/mL).
Control (CTRL; no treatment; ♦); 40 mg/mL of OTF (▲); 20 mg/mL of OTF (○); and 10 mg/mL of OTF (▬).
105 cfu/sample. Each bag was manually massaged for
30 s to evenly spread the inoculum over the surface of
the frankfurters. Each prepared OTF solution (1 mL)
was distributed evenly on the surface of a frankfurter.
Control group was prepared by inoculating L. monocytogenes to frankfurters without adding any solution.
The final concentration of solutions was 40 mg/mL of
OTF solution, 1,000 IU of nisin solution, and 40 mg/
mL of OTF + 1,000 IU of nisin combination solution.
The bags were vacuum sealed using a Multivac A
300/51 vacuum packaging machine (Multivac, GmbH
& Co., Wolfertschwenden, Germany) and stored at
4, 10, and 25°C. Viable L. monocytogenes cells on the
frankfurter were analyzed after 0, 1, 2, and 3 d of storage. Wash solution (20 mL) was added to each bag and
the bag was vigorously shaken by hand to wash the surface of the frankfurter and release organisms into the
wash solution. Serial dilutions of the wash solution were
prepared in 0.1% peptone water (9 mL) and 0.1-mL
aliquots of appropriate dilutions were surface plated,
in duplicate, on MOX agar plates (Danisco USA Inc.).
All inoculated agar plates were incubated aerobically
at 35°C and typical L. monocytogenes colonies were
counted after 48 h.
Figure 2. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL) combined with nisin (N). Control (CTRL; no treatment; ♦); 40 mg/mL of OTF (▲); 500 IU of N (□); 1,000 IU of N (■); 1,000 IU of N + 40 mg/mL
of OTF (○); and 1,000 IU of N + 40 mg/mL of OTF (●).
2587
GROWTH INHIBITION OF LISTERIA MONOCYTOGENES BY OVOTRANSFERRIN AND NISIN
Table 1. Number of viable cells (log cfu/mL of Listeria monocytogenes on brain heart infusion broth treated with or without ovotransferrin
Incubation time (h)
Sample1
0
2
Control
OTF (20 mg/mL)
OTF (40 mg/mL)
N (500 IU)
N (1,000 IU)
N (2,000 IU)
OTF (20 mg/mL) + N (1,000 IU)
OTF (40 mg/mL) + N (500 IU)
OTF (40 mg/mL) + N (1,000 IU)
0.06Y,t
0.07V,t
0.07U,t
0.04U,t
0.04T,t
0.05T,t
0.07T,t
0.00T,t
0.04T,t
0.15X,t
0.79W,u
0.63V,u
0.27V,u
0.23U,u
0.06U,u
1.49UV,u
0.01U,u
0.51UV,u
5.33
5.36
5.36
5.36
5.36
5.34
5.36
5.39
5.36
±
±
±
±
±
±
±
±
±
5.83
3.30
3.15
2.22
2.55
2.32
1.71
2.92
1.57
±
±
±
±
±
±
±
±
±
4
6.64
3.67
3.18
2.15
1.77
1.77
0.90
2.48
0.49
±
±
±
±
±
±
±
±
±
0.12W,t
0.43W,u
0.31V,uv
0.45V,vw
0.22U,wx
1.03U,wx
0.78V,xy
0.68U,vw
0.85UVW,y
8.40
6.89
5.51
2.96
1.83
0.00
1.54
2.56
0.23
±
±
±
±
±
±
±
±
±
8
12
0.21V,t
0.63U,u
0.27U,v
0.67V,w
0.51U,xy
0.00V,z
0.10UV,y
1.16U,x
0.40VW,z
0.12U,t
0.06T,tu
0.39T,u
0.47V,v
0.90U,wx
1.51UV,x
1.41UV,x
1.06U,vw
0.00W,x
9.32
8.67
7.73
3.36
1.60
0.87
1.37
3.07
0.00
±
±
±
±
±
±
±
±
±
24
8.75
7.09
6.31
7.06
4.03
0.69
2.99
4.13
1.71
±
±
±
±
±
±
±
±
±
0.04T,t
0.33U,tu
0.34TU,u
1.60T,tu
0.07U,vw
1.19UV,w
0.74U,vw
0.63TU,v
1.48U,w
T–YMeans
between incubation times with different superscripts differ significantly (P < 0.05).
between treatments with different superscripts differ significantly (P < 0.05).
1OTF = ovotransferrin; N = nisin.
t–zMeans
Microbiological Analyses
At 0, 2, 4, 8, 12, and 24 h of incubation at 35°C,
each inoculated BHI broth was serially diluted (1:10) in
sterile peptone (0.1%) and surface plated on MOX agar
(Difco Laboratories). The numbers of viable L. monocytogenes in BHI broth with or without added OTF,
nisin, or OTF–nisin combinations were counted at 48 h.
For evaluating numbers of viable L. monocytogenes
in frankfurters, packages of inoculated frankfurters were aseptically opened using sterile scissors, and
sterile 0.1% peptone water (20 mL) was added to bag
containing 1 frankfurter. Each mixture was vigorously
shaken by hand to release organisms on the surface of
the frankfurter into the wash solution. Serial dilutions
of the wash solution were prepared with 0.1% peptone
water (9 mL), and 0.1 mL-aliquots of appropriate dilutions were surface plated, in duplicate, on MOX (Difco
Laboratories). All inoculated agar plates were incubated aerobically at 35°C and typical L. monocytogenes
colonies were counted at 48 h.
Statistical Analysis
Analysis of variance was performed with the GLM
procedure of SAS (version 9.1.1; SAS Institute, 1995).
Differences were considered statistically significant at P
< 0.05 unless otherwise stated.
RESULTS AND DISCUSSION
Figure 1 shows the optical density (OD) of BHI
broth inoculated with L. monocytogenes during incubation time at 35°C. A faster rate of increase in OD
and higher OD values were observed in control culture
compared with those with added OTF during incubation time. All concentrations of OTF (10, 20, and 40
mg/mL) suppressed the growth of L. monocytogenes,
with 20 and 40 mg/mL exhibiting a stronger inhibitory
effect than 10 mg/mL. None of the OTF concentrations tested were bacteriostatic. Preliminary efforts to
evaluate higher OTF concentrations to determine the
maximum inhibitory concentration were futile because
of high viscosity of the OTF solution, which made it
difficult to filter, and partial denaturation of OTF and
loss of antimicrobial activity. Therefore, for subsequent
experiments, OTF at 40 mg/mL was used alone or in
combination with nisin or selected meat additives. The
OD measurements used in this study were a reliable,
precise, and convenient method for collecting the microbial growth data. Using the Bioscreen, data points
were gathered continuously and used to prepare the
growth curve fittings.
Figure 2 indicated that all treatments [OTF (40 mg/
mL), nisin (500 or 1,000 IU), and OTF–nisin combinations] suppressed growth of L. monocytogenes during
the incubation period at 35°C. Ovotransferrin at 40
mg/mL was slightly more effective than 500 IU of nisin
in inhibiting the growth of L. monocytogenes. Growth
inhibition of L. monocytogenes increased as the concentrations of nisin increased. Nisin at 1,000 IU completely
inhibited the growth of the pathogen for about 7 h,
after which its growth increased rapidly and reached
to the OD values of 500 IU of nisin treatment. The
OTF–nisin combinations were very effective in inhibiting the growth of L. monocytogenes, and both OTF +
nisin (500 IU) and OTF + nisin (1,000 IU) treatments
completely inhibited the growth of the pathogen for 18
and 24 h, respectively.
The viability tests indicated that L. monocytogenes
grew rapidly in control and reached >9.0 log cfu/mL
after 12 h of incubation at 35°C (Table 1). Ovotransferrin (40 mg/mL), 500 IU of nisin, 1,000 IU of nisin, or
OTF–nisin combinations exhibited a microcidal effect
against L. monocytogenes within 2 h; log reduction in
viable counts ranged from 2.18 (OTF, 40 mg/mL) to
3.76 (40 mg/mL of OTF + 1,000 IU of nisin). Ovotransferrin (40 mg/mL), 500 IU of nisin, 1,000 IU of
nisin, or OTF–nisin combinations exhibited a microbial
effect against L. monocytogenes within 2 h; log reduction in viable counts ranged from 2.18 (OTF, 40 mg/
mL) to 3.76 (40 mg/mL of OTF + 1,000 IU of nisin).
After 4 h of incubation, the number of L. monocytogenes in OTF (40 mg/mL) decreased to 3.18 log cfu/
2588
Moon et al.
mL but increased to 5.51 after 8 h and 6.31 log cfu/
mL after 24 h of incubation. It seems that the inhibitory effect of OTF (40 mg/mL) alone was not strong
enough to control the growth of L. monocytogenes after
4 h at 35°C.
After 12 h of incubation, the viable counts of the
pathogen increased to 7.73, 3.36, and 1.60 log cfu/
mL with OTF, 500 IU of nisin, and 1,000 IU of nisin
treatments, respectively. However, OTF + 1,000 IU of
nisin produced a steady decline in viable counts with
numbers of survivors below the detection limit (1 cfu/
mL) at 12 h, but increased to 1.71 cfu/mL, indicating that their antimicrobial activities were short-lived.
Compared with control and all other treatments tested,
40 mg/mL of OTF + 1,000 IU of nisin had a significantly lower number of L. monocytogenes survivors at
24 h (P < 0.05). At 24 h, viable counts were 8.75, 6.31,
4.03 4.13, and 1.71 log cfu/mL for control, OTF, 500
IU of nisin, 1,000 IU of nisin, and OTF + 1,000 IU
of nisin treatment, respectively. Samilis et al. (2005)
reported that nisin enhanced the antilisterial activity
of chemicals that could be applied as postprocessing
antimicrobial solutions in meat products because of the
immediate bactericidal and strong but short-term bacteriostatic effects on L. monocytogenes. Arnold et al.
(1982) demonstrated that lactoferrin enhanced the activity of nisin against L. monocytogenes. However, the
levels of lactoferrin and nisin used did not inhibit any
of the gram-negative strains. Lactoferrin binds iron and
slows the growth of L. monocytogenes to facilitate nisin
action. Lactoferrin may also act directly on L. monocytogenes (Arnold et al., 1977, 1982).
The influence of OTF (40 mg/mL) or nisin (1,000
IU) alone or in combination on the viability of L. monocytogenes in frankfurters at 4, 10, and 25°C for 72 h is
shown in Figure 3. Except at 25°C, OTF alone produced
no reduction in viable counts of L. monocytogenes at all
temperatures tested. At 25°C, L. monocytogenes counts
increased by 3 log cfu/g after 72 h. Nisin (1,000 IU)
and OTF (40 mg/mL) + 1,000 IU of nisin both reduced
the initial numbers of L. monocytogenes within 2 h irrespective of temperature. However, differences in the
number of survivors in frankfurters exposed to those
treatments were not significant (P > 0.05). Hampikyan
and Ugur (2007) studied the effect of nisin in Turkish
fermented sausages and found that the addition of nisin
(50 μg/g) reduced L. monocytogenes counts by 2.5 log
cfu/g. However, Mangalassary et al. (2008) found no
significant differences in L. monocytogenes population
when 1,000 IU of nisin was used in ready-to-eat turkey
bologna. Nisin (1,000 IU) and OTF (40 mg/mL) + nisin (1,000 IU) showed a clear inhibition effect against
L. monocytogenes in frankfurters during storage, but
OTF and nisin did not show any synergistic effects.
Several studies also reported that combinations of nisin
with antimicrobial agents showed strong antibacterial
activities against L. monocytogenes in a model system
but did not show antibacterial effect in meat products
(Stiles, 1996; Gill and Holley, 2000, 2003; Arqués et al.,
2004). It is difficult to explain why they do not show
antibacterial activities in meat products, but distribution problems or dilution effects of the antimicrobial
agents, state of the microorganisms in inocula, or stability of the antimicrobial agents on the surface of meat
products could be part of the reason (Ko and Ahn,
2008).
Figure 4 shows the effect of NaCl (salt) on the growth
inhibition effect of OTF against L. monocytogenes in
Figure 3. Number of viable cells of Listeria monocytogenes on
frankfurters treated with or without ovotransferrin (OTF). Control
(CTRL; no treatment; ♦); 40 mg/mL of OTF (▲); 1,000 IU of nisin
(N; ■); and 1,000 IU of N + 40 mg/mL of OTF (□).
GROWTH INHIBITION OF LISTERIA MONOCYTOGENES BY OVOTRANSFERRIN AND NISIN
2589
Figure 4. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL)
combined with salt. Control (CTRL; no treatment; ♦); 40 mg/mL of OTF (▲); 1% NaCl (□); 2% NaCl (■); 1% NaCl + 40 mg/mL of OTF (○);
and 2% NaCl + 40 mg/mL of OTF (●).
BHI broth at 35°C. Salt at 1 or 2% (wt/vol) inhibited
the growth of L. monocytogenes compared with control. Ovotransferrin activity was initially enhanced in
the presence of 2% salt; however, at 16 to 24 h, OD
increased to values higher than those observed in cultures exposed to OTF alone or OTF + 1% salt. Addition of common salt to the medium has been reported
to increase the listericidal action of other antimicrobial
agents (Pawar et al., 2000. Pawar et al. (2000) reported
that NaCl caused little improvement in L. monocytogenes inhibition in buffalo meat.
Figure 5 shows the effect of SL on the growth inhibition of OTF against L. monocytogenes in BHI broth
(35°C). Sodium lactate at 0.78 or 1.56% (wt/vol) was
slightly inhibitory to the growth of L. monocytogenes,
but no difference in OD values was observed in broth
with OTF alone or in combination with SL at any of
the 2 concentrations tested. Similar results were ob-
Figure 5. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL) combined with sodium lactate (SL). Control (CTRL; no treatment; ♦); 40 mg/mL of OTF (▲); 0.78% SL (□); 1.56% SL (■); 0.78% SL + 40 mg/
mL of OTF (○); and 1.56% SL + 40 mg/mL of OTF (●).
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Moon et al.
Figure 6. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL) combined with 0.78% sodium lactate + 0.052% disodium acetate. CTRL = control treatment.
tained when the antimicrobial activity of OTF was
evaluated in the presence of a combination of 1.56%
SL and 0.01% SDA. No differences in OD values were
observed in broth with OTF alone or in combination
with 1.56% SL + 0.01% SDA (Figure 6).
Growth inhibition of OTF against L. monocytogenes
in BHI broth during incubation at 35°C as affected by
0.05% SHMP is shown in Figure 7. Sodium hexametaphosphate alone was stimulatory to the growth of L.
monocytogenes, with OD values reaching higher than
those of control. Ovotransferrin (40 mg/mL) in the
presence of SHMP exhibited a drastic reduction in its
antimicrobial effect. Sodium hexametaphosphate and
OTF + SHMP produced an earlier increase in OD values (at 2 h) compared with control (at 6 h).
In conclusion, OTF delayed the growth of L. monocytogenes in BHI broth for 24 h at 35°C. Ovotransferrin
at 40 mg/mL exhibited bacteriostatic activity whereas
OTF at 20 mg/mL did not show significant effect on
antibacterial activity against L. monocytogenes in BHI
broth. Nisin at 1,000 IU exhibited bactericidal effect
against L. monocytogenes in BHI broth. Nisin at 1,000
IU + 40 mg/mL of OTF resulted in approximately 6
to 7 log reduction during 12 h of incubation at 35°C.
However, OTF (40 mg/mL) or OTF (40 mg/mL) +
1,000 IU of nisin showed no antimicrobial activity on
Figure 7. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL) combined with phosphate (sodium hexametaphosphate). CTRL = control treatment.
GROWTH INHIBITION OF LISTERIA MONOCYTOGENES BY OVOTRANSFERRIN AND NISIN
frankfurters. To use these natural antimicrobial agents
in meat products, therefore, it is very important to find
why the antimicrobial effect of OTF and nisin disappears in meat products. Meat additives (salt, SL, SL +
SDA, SHMP) had no effect on the antilisterial effect of
OTF in BHI broth.
ACKNOWLEDGMENTS
The authors thank William Colonna (Department of
Food Science and Human Nutrition, Iowa State University, Ames) for his technical assistance. This research
was supported by the Technology Development Program for Agriculture and Forestry Ministry for Food,
Agriculture, Forestry and Fisheries, Republic of Korea,
and WCU (World Class University) program (R3110056) through the National Research Foundation of
Korea funded by the Ministry of Education, Science
and Technology.
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