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Animal Science Publications Animal Science

11-1-2011

Influence of nisin and selected meat additives on the

Influence of nisin and selected meat additives on the

antimicrobial effect of ovotransferrin against Listeria

antimicrobial effect of ovotransferrin against Listeria

monocytogenes

monocytogenes

S. H. Moon Konkuk University H.-D. Paik Konkuk University S. White

Iowa State University

A. Daraba

University Dunarea de Jos

Aubrey F. Mendonca

Iowa State University, amendon@iastate.edu

See next page for additional authors

Follow this and additional works at: https://lib.dr.iastate.edu/ans_pubs

Part of the Agriculture Commons, Animal Sciences Commons, and the Food Science Commons The complete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ ans_pubs/877. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

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Influence of nisin and selected meat additives on the antimicrobial effect of

Influence of nisin and selected meat additives on the antimicrobial effect of

ovotransferrin against Listeria monocytogenes

ovotransferrin against Listeria monocytogenes

Abstract

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.

Keywords Keywords

Listeria monocytogenes, ovotransferrin, nisin, meat additive, growth inhibition

Disciplines Disciplines

Agriculture | Animal Sciences | Food Science

Comments Comments

This article is published as Moon, S. H., H-D. Paik, S. White, A. Daraba, A. F. Mendonca, and D. U. Ahn. "Influence of nisin and selected meat additives on the antimicrobial effect of ovotransferrin against Listeria monocytogenes." Poultry science 90, no. 11 (2011): 2584-2591. doi:10.3382/ps.2010-01275.

Creative Commons License Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

Authors Authors

S. H. Moon, H.-D. Paik, S. White, A. Daraba, Aubrey F. Mendonca, and Dong U. Ahn

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INTRODUCTION

Trends toward the use of natural food preservatives are growing as consumers become more health con-scious and concerned about the long-term health effects of synthetic chemicals in foods. In response to such con-sumer demands, the food industry showed increased in-terests 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 pos-sible way of circumventing this problem is combined use of antimicrobials (Sofos et al., 1998).

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 scav-enges 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 bacterio-cin 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

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

deter-mine the effect of nisin and selected meat additives (salt, lactate, lactate–diacetate combination, and poly-phosphate) on the antimicrobial activities of ovotrans-ferrin (OTF) against the growth of Listeria

monocyto-genes. 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 us-ing frankfurters. Frankfurters were inoculated with L.

monocytogenes (~6.0 log10 cfu/frankfurter); treated

with OTF, meat additives, and nisin singly or in com-bination; 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 combina-tion of OTF (40 mg/mL) and nisin (1,000 IU) inhibited the growth of L. monocytogenes in BHI and in frank-furters 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

Received December 2, 2010. Accepted March 12, 2011.

1 Corresponding author: duahn@iastate.edu

© 2011 Poultry Science Association Inc.

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struction of transmembrane pH gradients, diffusion of the proton motive force, and inhibition of ATP synthe-sis (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 individu-als, 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 ex-tensively used in meat and poultry products as anti-microbial 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 Kant-Muermans, 2001). To our knowledge limited published reports exist on the effect of commonly used meat ad-ditives 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 experi-ments involving the Bioscreen C turbidometer (version 2.6; Oy Growth Curves AB Ltd., Helsinki, Finland), all OTF solutions were prepared in brain heart infu-sion (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.

mono-cytogenes Scott A strain was obtained from Irene

Wes-ley at the National Animal Disease Center (Agricul-tural Research Service, USDA, Ames, IA). All other strains were obtained as clinical isolates from the mul-tistate outbreak of 1998 and 1999 (CDCP, 2002). Each

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 se-quential 24-h transfers of individual stock cultures were performed in 10 mL of tryptic soy broth (Difco Labo-ratories, 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 com-bined to prepare a 5-strain mixture of L.

monocyto-genes. 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.

mono-cytogenes 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 hexameta-phosphate (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 lac-tate 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 ni-sin was investigated uni-sing 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 ni-sin. Cell suspension (100 µL) containing approximately 106 to 107 actively growing L. monocytogenes cells was

inoculated to the prepared solution to make approxi-mately 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 inocula-tion, 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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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.

mono-cytogenes 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 stor-age. Wash solution (20 mL) was added to each bag and the bag was vigorously shaken by hand to wash the sur-face 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 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 (▬).

Figure 2. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL)

com-bined 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 (●).

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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.

mono-cytogenes 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 frankfurt-ers 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 dilu-tions were surface plated, in duplicate, on MOX (Difco Laboratories). All inoculated agar plates were incubat-ed 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 incu-bation 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 incuba-tion time. All concentraincuba-tions 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 concentra-tions 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 mi-crobial 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 combina-tions] 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 concen-trations 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 inhibit-ing 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). Ovotransfer-rin (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). Ovo-transferrin (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 reduc-tion 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.

monocyto-genes in OTF (40 mg/mL) decreased to 3.18 log cfu/ Table 1. Number of viable cells (log cfu/mL of Listeria monocytogenes on brain heart infusion broth treated with or without ovo-transferrin

Sample1

Incubation time (h)

0 2 4 8 12 24

Control 5.33 ± 0.06Y,t 5.83 ± 0.15X,t 6.64 ± 0.12W,t 8.40 ± 0.21V,t 9.32 ± 0.12U,t 8.75 ± 0.04T,t

OTF (20 mg/mL) 5.36 ± 0.07V,t 3.30 ± 0.79W,u 3.67 ± 0.43W,u 6.89 ± 0.63U,u 8.67 ± 0.06T,tu 7.09 ± 0.33U,tu

OTF (40 mg/mL) 5.36 ± 0.07U,t 3.15 ± 0.63V,u 3.18 ± 0.31V,uv 5.51 ± 0.27U,v 7.73 ± 0.39T,u 6.31 ± 0.34TU,u

N (500 IU) 5.36 ± 0.04U,t 2.22 ± 0.27V,u 2.15 ± 0.45V,vw 2.96 ± 0.67V,w 3.36 ± 0.47V,v 7.06 ± 1.60T,tu

N (1,000 IU) 5.36 ± 0.04T,t 2.55 ± 0.23U,u 1.77 ± 0.22U,wx 1.83 ± 0.51U,xy 1.60 ± 0.90U,wx 4.03 ± 0.07U,vw

N (2,000 IU) 5.34 ± 0.05T,t 2.32 ± 0.06U,u 1.77 ± 1.03U,wx 0.00 ± 0.00V,z 0.87 ± 1.51UV,x 0.69 ± 1.19UV,w

OTF (20 mg/mL) + N (1,000 IU) 5.36 ± 0.07T,t 1.71 ± 1.49UV,u 0.90 ± 0.78V,xy 1.54 ± 0.10UV,y 1.37 ± 1.41UV,x 2.99 ± 0.74U,vw

OTF (40 mg/mL) + N (500 IU) 5.39 ± 0.00T,t 2.92 ± 0.01U,u 2.48 ± 0.68U,vw 2.56 ± 1.16U,x 3.07 ± 1.06U,vw 4.13 ± 0.63TU,v

OTF (40 mg/mL) + N (1,000 IU) 5.36 ± 0.04T,t 1.57 ± 0.51UV,u 0.49 ± 0.85UVW,y 0.23 ± 0.40VW,z 0.00 ± 0.00W,x 1.71 ± 1.48U,w T–YMeans between incubation times with different superscripts differ significantly (P < 0.05).

t–zMeans between treatments with different superscripts differ significantly (P < 0.05). 1OTF = ovotransferrin; N = nisin.

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mL but increased to 5.51 after 8 h and 6.31 log cfu/ mL after 24 h of incubation. It seems that the inhibi-tory 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, indicat-ing 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 signifi-cantly 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 bac-teriostatic effects on L. monocytogenes. Arnold et al. (1982) demonstrated that lactoferrin enhanced the ac-tivity 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.

monocy-togenes (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.

mono-cytogenes 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 ir-respective 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) + ni-sin (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 distribu-tion problems or diludistribu-tion effects of the antimicrobial agents, state of the microorganisms in inocula, or sta-bility 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 (□).

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BHI broth at 35°C. Salt at 1 or 2% (wt/vol) inhibited the growth of L. monocytogenes compared with con-trol. 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 cul-tures exposed to OTF alone or OTF + 1% salt. Addi-tion 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.

monocyto-genes inhibition in buffalo meat.

Figure 5 shows the effect of SL on the growth inhibi-tion 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 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 (●).

Figure 5. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL)

com-bined 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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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 hexameta-phosphate 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 val-ues (at 2 h) compared with control (at 6 h).

In conclusion, OTF delayed the growth of L.

mono-cytogenes 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 6. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL)

com-bined with 0.78% sodium lactate + 0.052% disodium acetate. CTRL = control treatment.

Figure 7. Turbidity of brain heart infusion broth cultures inoculated with Listeria monocytogenes and ovotransferrin (OTF; 40 mg/mL)

com-bined with phosphate (sodium hexametaphosphate). CTRL = control treatment.

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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 disap-pears 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 Univer-sity, Ames) for his technical assistance. This research was supported by the Technology Development Pro-gram for Agriculture and Forestry Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea, and WCU (World Class University) program (R31-10056) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology.

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