Ethylenediaminetetraacetate and lysozyme improves antimicrobial activities
of ovotransferrin against Escherichia coli O157:H7
K. Y. Ko,* A. F. Mendoncam,† H. Ismail,* and D. U. Ahn*1
*Department of Animal Science, and †Department of Food Science and Human Nutrition,
Iowa State University, Ames 50011
ABSTRACT The aim of this study was to evaluate the
effect of EDTA, lysozyme, or the combination of EDTA
and lysozyme on the antibacterial activity of ovotransferrin against Escherichia coli O157:H7. Ovotransferrin solutions (20 mg/mL) containing 100 mM NaHCO3
(OS) with added EDTA (2.0 or 2.5 mg/mL), lysozyme
(1.0, 1.5, or 2.0 mg/mL), or both were prepared. The
antibacterial activities of OS, OSE (OS + EDTA), or
OSL (OS + lysozyme) against E. coli O157:H7 in model systems were investigated by turbidity and viability
tests. In addition, OSE, OSL, or OSEL (OS + EDTA
+ lysozyme) was applied to irradiated pork chops and
commercial hams to determine whether the solutions
had antibacterial activity on meat products. The effect
of the initial cell population on the antibacterial activity of OSE, OSL, and OSEL was determined. Ethylenediaminetetraacetate at 2 mg/mL plus OS induced a
reduction of approximately 3 to 4 log in viable E. coli
O157:H7 cells in brain heart infusion broth media, and
1 mg/mL of lysozyme plus OS resulted in a reduction
of approximately 0.5 to 1.0 log during a 36-h incubation
at 35°C. However, neither OSE nor OSEL showed a
significant antibacterial effect on pork chops and hams
during storage at 10°C. The initial cell number in media
did not affect the antibacterial activity of OSE or OSEL
against E. coli O157:H7. This study demonstrates that
combinations of ovotransferrin, NaHCO3, and EDTA
have the potential to control E. coli O157:H7.
Key words: ovotransferrin, antibacterial activity, ethylenediaminetetraacetate, lysozyme,
Escherichia coli O157:H7
2009 Poultry Science 88:406–414
doi:10.3382/ps.2008-00218
INTRODUCTION
the availability of iron required for microbial growth,
was considered the major antibacterial mechanism of
ovotransferrin (Alderton et al., 1946; Fraenkel-Conrat
and Feeney, 1950; Arnold et al., 1980). However, subsequent studies have suggested that the antimicrobial
action of ovotransferrin could be intimately related to
direct interactions of ovotransferrin with the bacterial
surface, resulting in damage to the outer membranes of
microorganisms or destruction of their microbial function, such as the proton motive force (Arnold et al.,
1981; Ibrahim, 1997; Ibrahim et al., 2000; Aguilera et
al., 2003; Ahlborn and Sheldon, 2006). In addition, Ko
and Ahn (2008) recently developed a simple purification method for the large-scale production of ovotransferrin, and the application of ovotransferrin as a natural
antimicrobial agent in the food or meat industry seems
to be of interest.
Escherichia coli O157:H7 possesses specific cell-surface appendages such as fimbria or colonization factor
antigens that facilitate their adhesion to host tissuematrix components such as fibronectin, collagens, elastin, and mucin so that it can attach tightly to beef tissues (Naidu, 2002). In addition, illnesses caused by E.
coli O157:H7 are frequently linked to cattle and cattle
Natural antimicrobials that are environmentally
friendly, medically acceptable, highly effective, and
economical to manufacture have gained attention because of the consumer desire for natural food products
(Payne et al., 1994). Ovotransferrin, one of the main
iron-binding glycoproteins present in egg white, transports and scavenges Fe(III) in poultry eggs (Kurokawa
et al., 1995). It is a major contributor to the defense
of the egg against microbial infection and rotting.
Many studies have described the antibacterial properties of ovotransferrin against various microorganisms, including Escherichia coli (Schade and Caroline,
1944), Pseudomonas spp., Streptococcus mutans (Valenti et al., 1983), Staphylococcus aureus, Bacillus cereus
(Ibrahim, 1997), Salmonella Enteritidis (Baron et al.,
2000), and Candida (Valenti et al., 1985). Initially, the
iron-binding capability of ovotransferrin, which limits
©2009 Poultry Science Association Inc.
Received May 29, 2008.
Accepted October 1, 2008.
1
Corresponding author: duahn@iastate.edu
406
ANTIMICROBIAL ACTIVITIES OF OVOTRANSFERRIN
products (e.g., undercooked ground beef and raw milk;
Weeratna and Doyle, 1991; Bell et al., 1994).
Combinations of antimicrobials with different mechanisms can increase their antibacterial effectiveness.
Ethylenediaminetetraacetate has been used in several
food products as a chelating agent to prevent oxidative
and deteriorative reactions that are catalyzed by metal
ions (Hansen et al., 2001). In addition, EDTA is known
to enhance the effectiveness of antimicrobials and antibiotics, especially against gram-negative bacteria. For
instance, EDTA promotes the antimicrobial activity of
nisin, lysozyme, and monolaurin against gram-negative
microorganisms (Hughey and Johnson, 1987; Stevens
et al., 1991; Razavi-Rohani and Griffiths, 1994). Ethylenediaminetetraacetate destabilizes the outer membrane of gram-negative bacteria by chelating Ca2+ and
Mg2+ salts, which function as bridges between lipopolysaccharides (LPS) in the microbial outer membrane,
resulting in the release of LPS from gram-negative bacteria (Vaara, 1992).
Lysozyme catalyzes the hydrolysis of 1,4-glycosidic
linkages between N-acetylmuramic acid and N-acetylglucosamine in cell wall peptidoglycan. Because the
cell walls of gram-negative bacteria are protected by
an outer membrane, gram-negative microorganisms
are relatively resistant to the antimicrobial activity of
lysozyme; thus, the application of lysozyme in foods has
been limited (Gill and Holley, 2003). However, lysozyme
has gained considerable interest for use in food systems
because it is a natural enzyme produced by many animals and its activity targets a specific cellular structure
of microorganisms (Proctor and Cunningham, 1988).
Ovotransferrin alone has shown little antibacterial
activity against E. coli O157:H7 in previous studies.
According to Ko et al. (2008), an ovotransferrin solution with 100 mM NaHCO3 added (OS) was found to
be bacteriostatic against the pathogen, but not bactericidal. The aim of the present study was to promote the
antimicrobial activity of OS against E. coli O157:H7
by combining OS with EDTA, lysozyme, or both and
to identify the possibility of applying this combination
(i.e., EDTA or lysozyme, ovotransferrin, and 100 mM
NaHCO3) on commercial hams and irradiated pork
chops to control the growth of E. coli O157:H7.
MATERIALS AND METHODS
Ovotransferrin
The apo-ovotransferrin (iron-free) used in this study
was prepared by the method of Ko and Ahn (2008). After iron saturation of a 2×-diluted egg white solution,
holo-ovotransferrin (iron-saturated) was separated by
using ethanol. Iron was removed from the holo-ovotransferrin by using AG 1-X2 resin (chloride form, Bio-Rad
Laboratories Inc., Hercules, CA) and then freeze-dried.
The dried apo-ovotransferrin was dissolved in distilled
water, its pH was adjusted to 7.4, and 0.15 M NaCl
was added. The purity of the ovotransferrin used in this
407
study was approximately 80%, and the residual iron in
the prepared apo-ovotransferrin solution was less than
0.5 ppm. Before microbial study, the ovotransferrin solution was sterilized by filtering through a 0.22-µm syringe filter (Whatman Inc., Florbam Park, NJ).
Bacterial Strains
Five different strains of E. coli O157:H7 (ATCC
43890, C467, FRIK 125, ATCC 43895, and 93-062)
were used. Before inoculation, each strain was individually cultured in brain heart infusion (BHI) broth (Remel Inc., Lenexa, KS) for 24 h at 35°C and harvested to
activate the strain. An aliquot (5 mL) of each culture
solution was transferred to a sterilized centrifuge bottle
and centrifuged at 10,000 × g for 10 min at 4°C. The
pellet was collected, washed with saline, and then resuspended in 25 mL of saline. An inoculation cocktail
was prepared by mixing equal volumes of each cell suspension to have approximately the same population of
5 strains of E. coli O157:H7.
Turbidity Test
After adding 2 mL of ovotransferrin solution (40 mg/
mL) or OS to 2 mL of 2×-strength BHI broth, stock
solutions of EDTA or lysozyme (Sigma-Aldrich Inc.,
St. Louis, MO) were added to the media. In the broth
media, the final concentration of EDTA was 2.0 or 2.5
mg/mL, whereas the final concentration of lysozyme
was 1.0, 1.5, or 2.0 mg/mL. In addition, 40 µL of cell
suspension containing approximately 106 to 107 cells of
activated E. coli O157:H7 cocktail was inoculated into
the BHI broth to make the initial population of E. coli
O157:H7 approximately 104 to 105 cfu/mL. During a
36-h inoculation period at 35°C, samples were taken
at approximately 3- to 6-h intervals. The antimicrobial
capacities against E. coli O157:H7 of ovotransferrin solutions combined with EDTA or lysozyme were determined by measuring the turbidity with a spectrophotometer at 620 nm.
Viability Test
The antibacterial activity of ovotransferrin plus either EDTA or lysozyme was investigated by using a
viability test. First, 2 mL of ovotransferrin solution
(40 mg/mL) was added to 2 mL of 2×-concentrated
BHI broth media containing 100 mM NaHCO3 (OS). A
stock solution of either EDTA or lysozyme was added
to the OS solution so that the solutions contained OS
+ 2 mg/mL of EDTA (OSE2) or 1 mg/mL lysozyme
(OSL1). In addition, 40 µL of cell suspension containing approximately 106 to 107 cells of actively growing E.
coli O157:H7 was inoculated into the prepared solution
to make approximately 104 to 105 cfu/mL in the initial solution. Ethylenediaminetetraacetate or lysozyme
alone was prepared as a control group. After inoculation, the prepared culture solutions were incubated for
408
KO ET AL.
36 h at 35°C. 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 for 36
h at 35°C. The number of survivors on BHI agar plates
was counted as colony-forming units per milliliter of
sample.
Effect of Initial Number
on Antimicrobial Activity
This study was performed to determine whether the
population of cells would affect the antibacterial activity of ovotransferrin solutions combined with various solutions. The BHI broth media containing ovotransferrin
were prepared by using the same method as described
above. The OS plus 2 mg/mL of EDTA (OSE) or 1
mg/mL of lysozyme (OSEL) was added to prepared
BHI broth media. The BHI broth culture containing
EDTA (2 mg/mL) and lysozyme (1 mg/mL) without
added ovotransferrin was prepared to identify whether
the solution alone had antibacterial activity against E.
coli O157:H7. In addition, 40 µL of cell suspension containing 106, 107, or 108 cells of actively growing E. coli
O157:H7 was inoculated into 4 mL of BHI broth media
to make the number of E. coli O157:H7 104, 105, or 106
cfu/mL. After the prepared culture solutions were incubated for 24 h at 35°C, the viability of each treatment
was measured by using the same method as described
above.
Application of Ovotransferrin
on Pork Chops
Fresh, boneless pork loin chops from different animals were purchased from the Meat Laboratory at Iowa
State University and sliced into 1-cm-thick pieces (7 ×
5 cm). The sliced pork chops were vacuum-packaged
(−1 bar of vacuum with a 10-s dwell time) in low-oxygen-permeable bags (nylon-polyethylene, Koch, Kansas
City, MO; 9.3 mL of O2/m2 per 24 h at 0°C). After
vacuum-packaging, the sliced pork chops were irradiated at 5 kGy with a Linear Accelerator (Circe IIIR,
Thomason CSF Linac, Saint-Aubin, France) to kill any
bacteria that may have been present in the pork chops.
After irradiation, the pork chops were frozen, and were
completely defrosted before use for the microbial study.
Each package was opened aseptically with an alcoholsterilized scissors. A cocktail stock suspension (0.2 mL)
of 106 cfu/mL of E. coli O157:H7 was inoculated onto
the surface of each pork chop to make the initial number inoculated 104 cfu/cm2, and the pork chop samples
were then randomly divided into 5 groups. Inoculated
strains were spread manually for 30 s to distribute the
inocula evenly.
Lysozyme, EDTA, or their combination was added
to the OS solution. The treatments were as follows: 20
mg/mL of ovotransferrin containing 100 mM NaHCO3
(OSA) plus 2 mg/mL of EDTA (OSEA), OSA plus 2
mg/mL of EDTA and 1 mg/mL of lysozyme (OSELA),
30 mg/mL of ovotransferrin (OSB) plus 2 mg/mL of
EDTA (OSEB), and OSB plus 2 mg/mL of EDTA and
1 mg/mL of lysozyme (OSELB). One milliliter of each
ovotransferrin solution was distributed onto the same
surface of the pork chops and spread manually for approximately 30 to 60 s to distribute the solution homogeneously. For the control group, E. coli O157:H7
alone was inoculated onto the pork chops. After adding ovotransferrin to the pork chops, the samples were
stored in a 10°C incubator. Viable cells on the pork
chops were analyzed after 0, 2, 4, 8, and 12 d of storage; approximately 30 to 50 mL of sterile 0.1% peptone
water solution was added to each bag, followed by pummeling for 1 min in a Stomacher (model 80, Seward
Medical Ltd, London, UK) at normal speed. After serial dilution of the sample with 0.1% peptone water,
0.1 mL of diluted sample was spread homogeneously on
a MacConkey agar plate (Difco Laboratories, Detroit,
MI) in duplicate. The survivors were enumerated as
colony-forming units per gram by the same method as
presented above.
Application of Ovotransferrin on Ham
Commercial hams were purchased from 3 local retail
stores, and hams from each store were used as a replicate. Because hams provide several factors that restrict
the growth of some microorganisms, including low storage temperature, nitrite, salt, and reduced water activity (Gill and Holley 2003), they were not irradiated, unlike the pork samples. Hams were sliced to 0.2-cm-thick
pieces and vacuum-packaged (−1 bar of vacuum with a
10-s dwell time) in low-oxygen-permeable bags (nylonpolyethylene, 9.3 mL O2/m2 per 24 h at 0°C; Koch). Before inoculation, each package was opened aseptically
with an alcohol-sterilized scissors. A 0.2-mL quantity of
actively growing E. coli O157:H7 cocktail stock suspension (105 cfu/mL) was inoculated aseptically onto the
surface of the sliced ham to make 103 cfu/cm2. After inoculation, the ham samples were mixed manually for 30
s to distribute the inoculum evenly, and the packaged
samples were randomly divided into 5 groups. In addition, 1 mL of the 4 different ovotransferrin solutions,
prepared as in the pork chop study, was distributed
evenly on the surface of the hams. Samples inoculated
with E. coli O157:H7 alone without any solution added
were used as controls. All samples were repackaged under vacuum and stored at 10°C in an incubator. After incubation for 0, 3, 8, and 13 d, the numbers of
surviving bacteria were enumerated by using the same
method as in the pork chop study.
Statistical Analysis
All experiments were replicated 3 times, and data
were analyzed by JMP software (version 5.1.1, SAS
Institute., Cary, NC). The differences in mean values
ANTIMICROBIAL ACTIVITIES OF OVOTRANSFERRIN
409
Figure 1. Turbidity of brain heart infusion broth cultures inoculated with an Escherichia coli O157:H7 and ovotransferrin (20 mg/mL) solution
combined with NaHCO3, EDTA, or both during a 35°C incubation. C = control, only 104 cfu/mL of E. coli O157:H7; E2 = 2 mg/mL of EDTA;
E2.5 = 2.5 mg/mL of EDTA; OS = 20 mg/mL of ovotransferrin + 100 mM NaHCO3; OSE2 = OS + 2 mg/mL of EDTA; OSE2.5 = OS + 2.5
mg/mL of EDTA.
were compared by Tukey’s honestly significant differences, and mean values and SEM are reported (Kuehl,
2000). Statistical significance for all comparisons was
obtained at P < 0.05.
RESULTS AND DISCUSSION
Antibacterial Activity of EDTA Alone
or in Combination with Ovotransferrin
Escherichia coli O157:H7 in BHI broth multiplied
rapidly after 6 h of incubation and entered a stationary phase after 10 h of incubation at 35°C. Escherichia
coli O157:H7 in the media containing 2 or 2.5 mg/mL
of EDTA alone showed a lag time for 10 h and entered
a stationary phase after 18 h of incubation (Figure 1),
indicating that the E. coli O157:H7 strains were resistant to 2 or 2.5 mg/mL of EDTA. These results can be
attributed to the harvest time of the E. coli O157:H7
strains for inoculation in this study. Gill and Holley
(2003) reported that cells harvested from a log- or
stationary-phase culture are a poor model for assessing antimicrobial activities because the repairability of
bacteria at the log phase or the stationary phase is not
equivalent to that of cells at the lag adaptation phase.
Payne et al. (1994) reported that more than 1 mg/
mL of EDTA inhibited E. coli O157:H7 in ultra-high
temperature-processed milk. Generally, in gram-nega-
tive bacteria, the outer membrane lies outside the peptidoglycan, but it is protected by LPS molecules and
stabilized by divalent cations, which act as salt bridges
to neighboring LPS molecules and proteins (Marvin et
al., 1989). Ethylenediaminetetraacetate could destabilize the outer membrane of the microorganism and
increase its permeability by chelating divalent cations
(Vaara, 1992; Boland et al., 2004). Jarvis et al. (2001)
reported that 30 mM EDTA resulted in a complete loss
of viability of E. coli.
The OS, OSE2, and OSE2.5 (OS + 2.5 mg/mL of
EDTA) treatments had ≤0.1 optical density at 620 nm
(Figure 1), indicating that these treatments inhibited
the growth of E. coli O157:H7 during incubation at
35°C. However, all the turbidity values of OS, OSE2,
and OSE2.5 were ≤0.1, so the effect of EDTA on the
antibacterial activity of OS could not be distinguished
by the turbidity test (Figure 1). Therefore, the viability test was performed to determine the influence of
EDTA on the antibacterial activity of OS. The number
of survivors in 2 mg/mL of EDTA alone was similar to
that of the control. However, when 2 mg/mL of EDTA
was added to OS, the antibacterial activity of OS increased significantly, indicating that it was bactericidal
against E. coli O157:H7 (Figure 2). Yakandawala et
al., (2006) reported that a combination of ovotransferrin, protamine sulfate, and EDTA reduced biofilm
formation by catheter-associated bacteria, including
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KO ET AL.
Figure 2. Antibacterial activity of ovotransferrin (20 mg/mL) containing 100 mM NaHCO3 and lysozyme (1 mg/mL) or EDTA (2 mg/mL)
against the growth of Escherichia coli O157:H7 in brain heart infusion broth culture during a 36-h incubation at 35°C. C = control, only 104 cfu/
mL of E. coli O157:H7; L1 = 1 mg/mL of lysozyme; E2 = 2 mg/mL of EDTA; OS = 20 mg/mL of ovotransferrin + 100 Mm NaHCO3; OSL1 =
OS + 1 mg/mL of lysozyme; OSE2 = OS + 2 mg/mL of EDTA. a–cDifferent letters show that the means are significantly different (P < 0.05; n
= 3).
Klebsiella pneumoniae, Pseudomonas aeruginosa, and
Staphylococus epidermidis. In addition, Al-Nabulsi and
Holley (2007) found that EDTA enhanced the antibacterial activity of lactoferrin against E. coli O157:H7.
The improved antimicrobial activity by EDTA could
be explained by its divalent cation-chelating attributes
(Vaara, 1992; Boland et al., 2004).
Consequently, even though OS was bacteriostatic
against E. coli O157:H7, the OSE2 combination resulted in a reduction of E. coli O157:H7 of approximately 3
to 4 log. Therefore, OSE2 has potential as an antibacterial agent for controlling E. coli O157:H7.
Antibacterial Activity of Lysozyme Alone
or in Combination with Ovotransferrin
In the turbidity test, lysozyme alone at 1.0, 1.5,
and 2.0 mg/mL could not inhibit the growth of E. coli
O157:H7. The OS, OSL1, OSL1.5 (OS + 1 mg/mL
lysozyme), and OSL2 (OS + 2 mg/mL of lysozyme)
had <0.1 optical density at 620 nm (Figure 3). Even
though Park (1997) reported that approximately 0.1 to
0.4 g/L of lysozyme had antibacterial activity against
E. coli in a nutrient broth and vegetable juice, the current study suggests that E. coli O157:H7 had resistance
to lysozyme alone in the concentrations used. When either 1 or 2 mg/mL of lysozyme was combined with OS,
however, the combinations (OSL group) had antibacterial activity against E. coli O157:H7 (Figure 3).
A viability test was performed to determine whether
OSL was bactericidal or bacteriostatic against E. coli
O157:H7. As shown in Figure 2, there was no significant difference in antibacterial activity between 1 mg/
mL of lysozyme and the control. The population of viable E. coli O157:H7 cells after 36 h of incubation at
35°C (Figure 2) with the OS treatment was 4.5 log cfu/
mL, whereas that of OSL1 was 3.2 log cfu/mL (the
initial cell number was 4.0 log cfu/mL). The combination (OSL1) was bacteriostatic against E. coli O157:H7
(P < 0.05), indicating that the antimicrobial activity
of OSL1 could be ascribed to OS. Therefore, results
of this study suggest that 1 mg/mL of lysozyme did
not significantly affect the antimicrobial activity of OS
against E. coli O157:H7.
However, several reports show conflicting results regarding the effects lysozyme. Ellison and Giehl (1991)
claimed that a combination of 2 mg/mL of lactoferrin
and 0.5 mg/mL of lysozyme showed bactericidal capacity against E. coli. When lysozyme was combined
with bovine lactoferrin, it showed bactericidal activity
against E. coli even though the lactoferrin was bacteriostatic at most (Boesman-Finkelstein and Finkelstein,
1985; Yamauchi et al., 1993). Naidu et al. (2003) reported that some strains of E. coli with a low binding
capacity to lactoferrin recovered easily from the antimicrobial actions of lysozyme, whereas other strains of E.
coli with a high binding capacity failed to be repaired.
Considering these suggestions, the use of several strains,
as in this study, can be assumed to reduce the effect of
ANTIMICROBIAL ACTIVITIES OF OVOTRANSFERRIN
411
Figure 3. Turbidity of brain heart infusion broth cultures inoculated with an Escherichia coli O157:H7 and ovotransferrin (20 mg/mL) solution combined with NaHCO3, lysozyme, or both during a 35°C incubation. L1 = 1 mg/mL of lysozyme; L1.5 = 1.5 mg/mL of lysozyme; L2 = 2
mg/mL of lysozyme; OS = 20 mg/mL of ovotransferrin + 100 mM NaHCO3; OSL1 = OS + 1 mg/mL of lysozyme; OSL1.5 = OS + 1.5 mg/mL
of lysozyme; OSL2 = OS + 2 mg/mL of lysozyme; C = control, only 104 cfu/mL of E. coli O157:H7.
lysozyme on the antibacterial activity of ovotransferrin
by inducing the rapid repair of some strains.
Effect of Initial Cell Number
on Antimicrobial Activity
This study was performed to determine whether the
initial cell population would influence the antibacterial activity of OSE or OS combined with 2 mg/mL
of EDTA and l mg/mL of lysozyme (OSEL). After
24 h of incubation at 35°C, OS did not completely restrain the growth of E. coli O157:H7. In addition, the
antibacterial action of OS against E. coli O157:H7 was
more effective when initial cell numbers were 104 cfu/
mL than when they were 106 cfu/mL. However, the
antibacterial activities of OSE and OSEL, which were
bactericidal against E. coli O157:H7, were not affected
by the number of initial bacteria (Figure 4). In addition, combinations consisting of EDTA (2 mg/mL) and
lysozyme (1 mg/mL) could not inhibit the growth of E.
coli O157:H7 in BHI broth media when inoculated with
105 and 106 cfu/mL of cells initially. However, when
EDTA (2 mg/mL) and lysozyme (1 mg/mL) were combined with OS, the combination (OSEL), exhibited a
strong bactericidal activity against E. coli O157:H7 in
BHI broth, approximately 1 log cfu/mL. Many research-
ers have reported that the antibacterial activities of
lysozyme against gram-negative and gram-positive microorganisms are related to membrane disruption (Kalchayanand et al., 1992). Ethylenediaminetetraacetate
treatment increased the permeability of the outer membrane by releasing LPS, and it enhanced the antibacterial action of lysozyme. Branen and Davidson (2004)
claimed that when EDTA and lysozyme are combined,
bactericidal activity against E. coli increases. However,
this study revealed that 2 mg/mL of EDTA plus l mg/
mL of lysozyme did not inhibit the growth of E. coli
O157:H7. Gill and Holley (2003) reported that even
though lysozyme and EDTA enhanced antibacterial activity against gram-positive organisms, their combination did not show antimicrobial activity against some
gram-negative organisms. The susceptibility of E. coli
O157:H7 to EDTA plus lysozyme may be dependent on
the state of strains at inoculation. Arnold et al. (1981)
demonstrated that cultures at the early exponential
phase were more susceptible to the antimicrobial action
of lactoferrin than those at the early stationary phase,
which were more resistant. Based on this suggestion,
the current study used E. coli O157:H7 at the stationary phase, and the susceptibility of E. coli O157:H7 to
EDTA plus lysozyme was much lower than the susceptibilities obtained from other studies.
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KO ET AL.
Figure 4. Effect of initial cell population on the antibacterial activity of ovotransferrin (20 mg/mL) containing 100 mM NaHCO3 and EDTA
(2 mg/mL) or lysozyme (1 mg/mL) against Escherichia coli O157:H7 in brain heart infusion (BHI) broth culture during a 24-h incubation at 35°C.
104 = 104 cfu/mL of BHI broth; 105 = 105 cfu/mL of BHI broth; 106 = 106 cfu/mL of BHI broth; OS = 20 mg/mL of ovotransferrin + 100 mM
NaHCO3; OSE = OS + 2 mg/mL of EDTA; OSEL = OS + 2 mg/mL of EDTA + 1 mg/mL of lysozyme; EL = 2 mg/mL of EDTA + 1 mg/mL
of lysozyme. a–cDifferent letters shows that the means are significantly different (P < 0.05; n = 3).
Ovotransferrin Applications on Pork Chops
and Commercial Hams
Escherichia coli O157:H7 inoculated at 104 cfu/cm2
grew on the pork chops and increased to 107 cfu/mL
after 12 d of storage at 10°C. In addition, OSEA, OSELA, OSEB, and OSELB showed no antimicrobial activity (Table 1). Unlike on the pork chop samples, the
viable E. coli O157:H7 cells inoculated on commercial
hams decreased slowly during 13 d of storage at 10°C
(Table 2). Escherichia coli O157:H7 grew better on pork
chops, which contained more drip on defrosting than
commercial hams, whereas the viable E. coli O157:H7
cells inoculated on hams seemed to decrease slowly, un-
like those on pork chops. In addition, OSEA, OSELA,
OSEB, and OSELB treated on hams showed no antibacterial activity against E. coli O157:H7, and there
was no significant difference in the number of survivors
between the control and other treatments in both pork
chops and commercial hams (P < 0.05; Table 2).
The antibacterial activities of some antimicrobials
can be modulated by the nutrient or laboratory medium. Many divalent cations, such as Ca2+ and Mg2,
can block the biological activity of lactoferrin by forming tetramers (Bennett et al., 1981) or by inhibiting the
direct interaction of lactoferrin with LPS (Ellison and
Giehl, 1991; Yamauchi et al., 1993; Rossi et al., 2002).
In contrast to the in vitro tests, the lack of antibacterial
Table 1. Changes in the number of Escherichia coli O157:H7 survivors (number of viable cells, log10
cfu/mL) on e-beam-irradiated pork chops not treated or treated with ovotransferrin solutions contained 100 mM NaHCO3 plus either EDTA (2 mg/mL) or lysozyme (1 mg/mL) during storage at
10°C1
Storage period (d)
Treatment2
Control
OSEA
OSELA
OSEB
OSELB
1
0
4.1
4.1
4.2
4.2
4.0
±
±
±
±
±
2
0.1
0.1
0.1
0.0
0.2
4.2
4.5
4.0
4.3
4.2
±
±
±
±
±
4
0.1
0.2
0.2
0.6
0.1
5.0
4.7
5.0
5.4
5.9
±
±
±
±
±
8
0.1
0.6
0.8
0.6
0.4
5.2
6.0
5.9
6.2
5.9
±
±
±
±
±
12
0.1
1.5
0.7
0.3
1.2
7.3
7.2
7.2
6.9
7.0
±
±
±
±
±
0.1
0.7
1.3
1.0
1.3
All values within a column are significantly different (P < 0.05; n = 3).
Control = only E. coli O157:H7; OSA = ovotransferrin (20 mg/mL) + 100 mM NaHCO3; OSEA = OSA + 2
mg/mL of EDTA; OSELA = OSA + 2 mg/mL of EDTA + 1 mg/mL of lysozyme; OSB = ovotransferrin (30 mg/
mL) + 100 mM NaHCO3; OSEB = OSB + 2 mg/mL of EDTA; OSELB = OSB + 2 mg/mL of EDTA + 1 mg/
mL of lysozyme.
2
413
ANTIMICROBIAL ACTIVITIES OF OVOTRANSFERRIN
Table 2. Survivors of Escherichia coli O157:H7 (number of viable cells, log10 cfu/g) in commercial
hams not treated or treated with ovotransferrin solutions containing 100 mM NaHCO3 plus EDTA or
lysozyme during storage at 10°C1
Storage period (d)
2
Treatment
Control
OSEA
OSELA
OSEB
OSELB
0
3.7
3.5
3.5
3.4
3.3
±
±
±
±
±
3
0.0
0.0
0.1
0.1
0.2
3.8
3.5
3.5
3.5
3.5
±
±
±
±
±
8
0.2
0.1
0.3
0.1
0.3
3.3
3.6
3.2
3.7
3.1
±
±
±
±
±
13
0.4
0.1
0.3
0.3
0.5
2.8
2.9
3.2
2.7
2.7
±
±
±
±
±
0.1
0.2
0.3
0.2
0.2
1
All values with within a column are significantly different (P < 0.05; n = 3).
Control = only E. coli O157:H7; OSA = ovotransferrin (20 mg/mL) + 100 mM NaHCO3; OSEA = OSA + 2
mg/mL of EDTA; OSELA = OSA + 2 mg/mL of EDTA + 1 mg/mL of lysozyme; OSB = ovotransferrin (30 mg/
mL) + 100 mM NaHCO3; OSEB = OSB + 2 mg/mL of EDTA; OSELB = OSB + 2 mg/mL of EDTA + 1 mg/
mL of lysozyme.
2
activity of ovotransferrin in pork chops and commercial
hams may have been due mainly to divalent ions such
as Ca2+ and Mg2+, which were assumed to exist on
hams and pork chops.
Cutter and Siragusa (1995a,b) demonstrated that the
combination of nisin with chelators showed antibacterial activity against E. coli and Salmonella spp. in buffer, but this combination showed no antibacterial effectiveness against the same organisms on beef. Branen
and Davidson (2004) reported that the combination of
EDTA and nisin, which exhibited antimicrobial activity toward E. coli, showed no antimicrobial activity in
2% fat ultra-high temperature-processed milk. In addition, even though a combination consisting of lysozyme
and nisin enhanced bactericidal activity in de Man, Rogosa, Sharpe media, it did not have such an effect in
pork juice (Gill and Holley, 2003). According to these
reports, the results obtained from the application of
antimicrobials to food products were significantly different from those of the model systems. Generally, food
products provide a nutrient-rich environment, so microorganisms injured by certain antimicrobials are considered to recover more easily in these systems than under
conditions of cell starvation or in laboratory media.
In addition, the distribution or dilution effect of
ovotransferrin on pork chops and hams could explain
the low antibacterial activity of ovotransferrin in these
products. Ellison and Giehl (1991) claimed that the
bactericidal effect is affected by the concentration of
lactoferrin as well as by the degree of iron saturation
in the lactoferrin. Even though in the present study
approximately 20 to 30 mg/mL of ovotransferrin was
distributed on the surface of the pork chops and hams,
the concentration was likely diluted on the surface of
these products. In addition, because the ovotransferrin
was spread manually, a homogeneous distribution of
ovotransferrin on whole products might be impossible.
Moreover, the large amount of drip formed during the
defrosting process for pork chops might have prevented
the complete incorporation of ovotransferrin on the surface of the pork chops. Therefore, the antimicrobial action of ovotransferrin in pork chops and hams seemed to
be modulated because of the reasons described above.
In addition, it is necessary to overcome some of these
limitations and the limitations of the approaches used
in various studies before ovotransferrin can be applied
in meat or meat products.
Conclusions
Ethylenediaminetetraacetate at 2 mg/mL and
lysozyme at 1 mg/mL enhanced the antibacterial activity of OS against E. coli O157:H7 in BHI broth. Ethylenediaminetetraacetate at 2 mg/mL plus OS resulted
in a reduction of approximately 3 to 4 log, whereas 1
mg/mL of lysozyme plus OS resulted in a reduction of
approximately 0.5 to 1 log during incubation at 35°C.
In addition, the initial cell numbers in media did not affect the antibacterial activity of OSE or OSEL against
E. coli O157:H7. Moreover, OSE or OSEL showed no
antimicrobial activity on pork chops and commercial
hams. The antimicrobial activity of OSE or OSEL
against this strain was significantly different from the
results of the model system using BHI broth. Therefore, we suggest that it is necessary to perform further
studies on the application of ovotransferrin to provide
solutions to these possible limitations.
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