Antimicrobial activity of soy edible lms incorporated with thyme and oregano

essential oils on fresh ground beef patties

Zehra Karagz Emirolu
a
, Gke Polat Yemi
b
, Betl Kodal Cokun
c
, Kezban Candoan
b,

a
Ministry of Agriculture and Rural Affairs, General Directorate of Protection and Control, Public Health Department, Bakanlklar, Ankara, Turkey
b
Department of Food Engineering, Faculty of Engineering, Ankara University, Dkap Campus, Ankara 06110, Turkey
c
Ministry of Agriculture and Rural Affairs, Department of Food Additives, Ankara Provincial Control Laboratory, Yenimahalle, Ankara, Turkey
a b s t r a c t a r t i c l e i n f o
Article history:
Received 16 November 2009
Received in revised form 12 March 2010
Accepted 11 April 2010
Keywords:
Soy edible lms
Oregano essential oil
Thyme essential oil
Antimicrobial packaging
Ground beef
Antibacterial activity
Antibacterial activity of soy protein edible lms (SPEF) incorporated with 1, 2, 3, 4 and 5% oregano (OR) or
thyme (TH) essential oils was evaluated against Escherichia coli, E. coli O157:H7, Staphylococcus aureus,
Pseudomonas aeruginosa and Lactobacillus plantarum by the inhibition zone test. Effects of SPEF containing 5%
OR and TH or a mixture of OR+TH (ORT) were also tested on fresh ground beef during refrigerated storage
(at 4 C). OR and TH incorporated SPEF exhibited similar antibacterial activity against all bacteria in
inhibition zone test. While E. coli, E. coli O157:H7 and S. aureus were signicantly inhibited by antimicrobial
lms, L. plantarum and P. aeruginosa appeared to be the more resistant bacteria. SPEF with OR, ORT, and TH
did not have signicant effects on total viable counts, lactic acid bacteria and Staphylococcus spp. when
applied on ground beef patties whereas reductions (pb0.05) in coliform and Pseudomonas spp. counts were
observed.
2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Meat and meat products being primary causes of food borne dis-
eases are also prone to spoilage during storage as with all proteinous
foods (Brown, 1982; Cardenas, Giannuzzi, & Zaritzkay, 2008). As in
many refrigerated foods, microbial growth is generally responsible for
the spoilage in meats and meat products together with biochemical
and enzymatic deteriorations (Devlieghere, Vermeiren, & Debevere,
2004). One of the traditional ways of controlling microbial growth in
these products, thus, improving safety and delaying spoilage is
application of antimicrobial dips or sprays on the surface of the
product (Kolsarici & Candogan 1995; Kerry, O'Grady, & Hogani, 2006).
However, in these applications, efciency of the antimicrobial
substances is restricted due to uncontrolled migration into the food
and partial inactivation of the active compounds because of the
interaction with food components (Quintavalla & Vincini, 2002). One
newapproachto overcome these limitations is the use of antimicrobial
packaging techniques as a promising type of active packaging. In
antimicrobial packaging, antimicrobial agents canbe incorporatedinto
the packaging material, coated on the surface of packaging lm or a
sachet containing antimicrobial compound can be added into the
package (Devlieghere et al., 2004).
Many studies have demonstrated that antimicrobial agents when
incorporated into the packaging lms could be effective for reducing
levels of foodborne organisms (Quintavalla & Vincini, 2002; Cutter,
2006). In order to meet consumer demands for more natural,
disposable, potentially biodegradable and recyclable food packaging
materials, research has focused on the incorporation of natural
antimicrobial compounds such as plant extracts and bacteriocins
into the biobased packaging materials instead of plastic lms
(Devlieghere et al., 2004; Lopez-Rubio, Almenar, Hernandez-Munoz,
Lagaron, Catala, & Gavara, 2004; Cutter, 2006). Within the biobased
packaging materials, edible lms and coatings have drawn attention
in recent years and a variety of edible lms and coatings has been
developed for fresh and further processed meats and poultry due to
their numerous advantages (Han, 2000; Cagri, Ustunol, & Ryser, 2004;
Cutter, 2006; Oussalah, Caillet, Salmieri, Saucier, & Lacroix, 2006;
Beverlya, Janes, Prinyawiwatkula, & No, 2008).
The ability of plant essential oils to protect foods against pathogenic
andspoilage microorganisms have beenreportedbyseveral researchers
(Lis-Balchin, Deans, & Eaglesham, 1997; Nelson, 1997; Hao, Brackett, &
Doyle, 1998; Burt & Reinders, 2003; Rojas-Gra, Avena-Bustillos, Olsen,
Friedman, Henika, & Martin-Belloso, 2007). In order to achieve effective
antimicrobial activity in direct food applications, high concentrations of
essential oils are generally needed, which might impact inappropriate
avors and odors in the product (Seydim & Sarikus 2006; Gutierez,
Meat Science 86 (2010) 283288
Corresponding author. Tel.: + 90 312 596 1797; fax: + 90 312 317 8711.
E-mail address: candogan@eng.ankara.edu.tr (K. Candoan).
0309-1740/$ see front matter 2010 The American Meat Science Association. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2010.04.016
Contents lists available at ScienceDirect
Meat Science
j our nal homepage: www. el sevi er. com/ l ocat e/ meat sci
Barry-Ryan, &Bourke, 2009). Therefore, recent researchshould focus on
incorporation of essential oils to edible lms as a supplementary
application in food packaging (Seydim & Sarikus 2006).
Among the most effective essential oils, thyme andoreganoessential
oils have been pointed out to posses better antimicrobial potential for
meat applications, which could be ascribed to the presence of phenolic
compounds, particularly thymol and carvacrol (Paster, Juven, & Shaaya,
1990; Hammer, Carson, & Riley, 1999; Burt, 2004; Kintzios, 2004;
Oussalah, Caillet, Salmieri, Saucier & Lacroix, 2004; Burt, Vlielander,
Haagsman, & Veldhuizen, 2005; Lpez, Snchez, Batlle & Nern, 2005;
Oussalah et al., 2006; Lpez, Snchez, Batlle & Nern, 2007; Solomakos,
Govaris, Koidis, & Botsoglou, 2008). Antimicrobial activity of thyme or
oregano essential oil incorporated edible lms have beenevaluated by a
number of researchers, however, limited data exist on the application of
antimicrobial edible lms incorporated with essential oils in real food
systems (Seydim & Sarikus 2006; Chi, Zivanovic, & Peneld 2006;
Oussalah et al., 2006; Du, Olsen, Avena-Bustillos, Mchugh, Levin, &
Friedman, 2008). Thus, the objectives of the present study were (1) to
evaluate the antibacterial activity of thyme (Thymus vulgaris L.) and
oregano (Oreganum heracleoticum L.) essential oils incorporated soy
protein based edible lms against selected meat associated bacteria and
(2) to determine effectiveness of these essential oil incorporated lms
on microbiological characteristics of fresh ground beef during refriger-
ated storage.
2. Materials and methods
2.1. Materials
Isolated soy protein with 90% protein content, and two types of
commercial essential oils fromOreganum heracleoticumL. and Thymus
vulgaris L. were kindly supplied by GURHAKFood and Chemicals Corp.,
(Istanbul, Turkey), and TIMTAS Agriculture and Medicine Products
Corp. (Mersin, Turkey) and ERDOMU Industries Corp. (Istanbul,
Turkey), respectively.
Bottom round beef and beef back fat were purchased from a local
butcher's shop (in different months for the two replications) on the day
of slaughter, transferred to the meat technology laboratory of the Food
Engineering Department at Ankara University in refrigerated contain-
ers, and stored at 4 C for 48 h for aging before patty preparation.
2.2. Soy protein based lm preparation
Isolated soy protein based edible lms were prepared by using the
methods described by Choi, Kim, Hanna, Weller, & Kerr (2003) and
Zivanovic, Chi, & Draughon (2005) with slight modications. Film
solutionwas preparedby slowly dissolving5%isolatedsoy protein(w/v)
in distilled water while stirring. Glycerol (3.5% wt/vol) was added as a
plasticiser and pH was adjusted to 10.0 with 0.1 N NaOH. The lm
solutions were heated to 90 C in a water bath for 30 min followed by
cooling to 40 C, and thenltering throughfour layers of cheese cloth by
a vacuumpump. Essential oils fromO. heracleoticumL. (OR) or T. vulgaris
L. (TH) were incorporated into the lm solution at various concentra-
tions of 0% (ISP as control), 1%, 2%, 3%, 4% and 5% (v/v) of edible lm
solution. The lmsolution (150,1 g) was cast onto sterile plastic petri
dishes (9 mmdiameter) followedby ovendrying at 30 Cfor 72 h. Films
were peeled and stored in a vacuum desicator for further use. Film
thicknesses were measured with a Digimatic Mitutoyo Micrometer
(Japan) to the nearest 0.001 mm. Measurements were taken at fteen
random locations of the lms. Average thicknesses for ISP, OR, and TH
lms were 0.1670.013, 0.2840.015, 0.2950.013 mm(standard
error), respectively. For the antibacterial activity test, prepared lms
were cut into disks of 17 mm diameter using a circular knife, and
weighed. Average weights of disks from ISP, OR, and TH lms were
0.0630.002, 0.0890.003, and 0.0870.003 g, respectively.
2.3. Experiment 1Antibacterial activity of the lms
For the antibacterial activity test, E. coli, S. aureus, E. coli O157:H7,
P. aeruginosa and L. plantarum cultures obtained from the culture
collections of the Food Engineering Department of Ankara University
were used. Overnight culture of L. plantarum was grown in MRS Broth
at 28 C. E. coli, S. aureus, E. coli O157:H7 and P. aeruginosa were grown
in Tryptic Soy Broth at 37 C.
Antibacterial activity testing was carried out using agar diffusion
method. Disks cut from the lms were placed on Nutrient agar (Merck,
Germany) plates, previously surface spreaded with the inoculum
containing indicator microorganisms in the range of 10
5
10
6
CFU/mL.
The plates were then incubated at 37 C for 24 h. The diameter of
inhibitory zone surrounding lm disks as well as contact area of edible
lms with agar surface were then measured in mm(Pranoto, Salokhe, &
Rakshit, 2005).
2.4. Experiment 2Application of lms on ground beef patties
Based on the results obtained fromthe antibacterial activity test and
considering possible diffusion of essential oils fromthe lm matrix into
the ground beef patties, the highest essential oil concentration tested
(5%) was used for preparation of the lms to evaluate antimicrobial
activity on fresh ground beef.
On the day of the patty preparation, visible connective tissue was
trimmed, and the meat and fat were cut into approximately 55 cm
pieces using a stainless steel knife, ground twice together using an
electric meat grinder (ARI, Istanbul, TURKEY) through a plate with
4 mm orices, and aseptically hand mixed for 1 min. This ground beef
contained 57.900.60% moisture, 23.910.91 fat, 16.840.17%
protein, and 1.150.04% ash [chemical composition was determined
using AOAC (1990) standard methods].
The ground beef mixture was divided into ve treatments.
Treatment groups consisted of 1) control samples (C), 2) samples
coated with isolated soy protein lms without addition of essential oil
(ISP), 3) samples coatedwith5%ORessential oil addedlms, 4) samples
coatedwith5%THessential oil addedlms, and(5) samples coatedwith
5% OR+THessential oil mixture with a ratio of 1:1 (ORT). Ground beef
from each treatment was weighed into 500.5 g portions and formed
into individual patties using sterile plastic petri plates (9 cmdiameter).
The lm materials prepared as described above were applied to the
upper and bottom surfaces of the patties except for control (C). Patties
with edible lms and C patties without application of edible lms were
then vacuum packaged in polyamide-ionomer polyethylene pouches
having anoxygenpermeability of 45 cm
3
/m
2
/24 h/690 mmHg(at 25 C
and 0% RH), heldat 41 Cfor 12 days, and sampled at Days 0, 1, 3, 6, 8,
10 and 12 for further analyses.
2.4.1. pH value of ground beef
The pH value was measured by homogenizing duplicate 10 g of
sample with 100 mL distilled water for 1 min using an Ultra Turrax
T25 (IKA Labortechnik, Staufen, Germany), and measuring the pHby a
Hanna pH meter, HI 221 (Romania). The pH measurements were
performed at Days 0, 1, 3, 6, 8, 10 and 12 of refrigerated storage.
2.4.2. Microbiological analysis of ground beef
At each sampling interval, duplicate packages from each treatment
were aseptically opened and a 10 g-portion from the center of the
patties was homogenized in sterile maximumrecovery diluent (Merck)
in a Seward stomacher for 1 min to make the initial dilution.
Appropriate serial dilutions (10
1
10
6
) were spread plated on Plate
Count Agar (PCA; Merck, Darmstadt, Germany) for total viable counts
(TVC), Man, Rogosa and Sharpe Agar (MRS; Merck) for lactic acid
bacterial (LAB) counts, Violet Red Bile Agar (VRB; Merck) for coliforms,
Baird Parker agar+egg yolk tellurite emulsion (BPA, Merck) for
Staphylococcus spp. counts, and Glutamat Starch Phenol Red agar
284 Z.K. Emirolu et al. / Meat Science 86 (2010) 283288
(GSP, Merck) for Pseudomonas spp. counts followedby incubationat 28
30 C for 48 h for TVC and Pseudomonas spp., at 28 C for 48 h for LAB, at
37 C for 48 h for coliforms and Staphylococcus spp. Bacterial counts
were expressed as log
10
colony forming units (CFU)/g sample.
2.5. Statistical analysis
Data from two replications for all response variables were analyzed
with the SAS general linear model (GLM) procedure. Separation of
means was accomplishedusingtheleast signicant difference(LSD) test
and PDIFF command at the 5% level of probability (SAS, 1996).
3. Results and discussion
3.1. Experiment 1Antibacterial activity of the lms
Inhibition zone diameters yielded by soy protein based edible lm
disks with various concentrations (0, 1, 2, 3, 4 and 5%) of oregano (OR)
and thyme (TH) essential oils against test organisms are shown in
Table 1. No inhibition zone against test organisms was observed for ISP
lms without the incorporationof essential oils. ORand THessential oils
showed inhibition against all test organisms even at minimum
concentration (1%) applied into the lm formulation. In general, both
OR and TH incorporated edible lms exhibited similar antimicrobial
activity with a few exceptions.
For S. aureus, increasing concentration of both OR and TH essential
oils in edible lms up to 3%resulted in signicantly higher antimicrobial
activity. Film disks containing 3% and 4% OR or TH essential oils had
similar inhibitory effect. On the other hand, the highest effective
concentration of OR tested against S. aureus was 5%. Inhibition zone
diameter in 5% TH incorporated lms was higher than at 3%
concentration while concentrations of 4% and 5% for TH essential
oil did not differ (pN0.05) in inhibiting S. aureus. Paster et al. (1990)
noted that direct application of essential oils of oregano (250 g/mL)
and thyme (350 g/mL) inhibited growth of S. aureus to some extent.
In another study, Sad (2003) determined the inhibitory effect of
four Turkish thyme and oregano hydrosols against four food
pathogens including S. aureus, and noted that S. aureus was the
most sensitive of the bacteria against the spice hydrosols and
increasing hydrosol concentrations generally showed an increase in
the growth inhibition levels and a reduction in the rate of growth. It
was also reported in the aforementioned study that thyme and
oregano hydrosols at 50 and 75 mL/100 mL concentrations were
completely inhibitive on S. aureus growth in broth culture. Similar
antimicrobial activities of essential oils from oregano and thyme
against S. aureus were also determined by Dadalioglu & Evrendilek
(2004); Donaldson, Warner, Cates & Young (2005); and Nedorostova,
Kloucek, Kokoska, Stolcova, & Pulkrabek (2009). Seydim & Sarikus
(2006) evaluated antimicrobial activity of whey protein isolate-based
edible lms incorporated with essential oils including oregano essential
oil and found that oregano essential oil added lms exhibited larger
inhibitory zone onS. aureus with2%of the minimumamount of oregano
essential oil level that showed inhibition and with the greatest zone of
inhibition 4% level against S. aureus.
TH essential oil in lm disks had strong antimicrobial activity
against E. coli and E. coli O157:H7. As concentration increased, TH
incorporated lms had greater (pb0.05) inhibitory effects against
those bacteria. E. coli was also signicantly inhibited by OR essential
oil, and OR concentration at 5% in the lmdisks was the most effective
concentration tested (pb0.05). For E. coli O157:H7, inhibitory effect
was observed with increasing essential oil concentration up to 4% OR.
No difference in antibacterial activity between 4% and 5% concentra-
tions of OR essential oil was determined against E. coli O157:H7
(pN0.05). This concentration dependent antimicrobial activity of OR
and TH essential oils against E. coli O157:H7 was also reported by
Sad (2003), Dadalioglu & Evrendilek (2004) and Burt (2004).
Oussallah et al. (2004) showed that 1% oregano essential oil addition
into the milk protein based edible lms inhibited E. coli O157:H7.
Zivanovic et al. (2005) determined antimicrobial activity of chitosan
lms enriched with four types of pure essential oils including oregano
essential oil. Within the four essential oils, oregano essential oil
showed the most intense antibacterial activity against E. coli O157:H7.
Oregano essential oil added whey protein based lms also inhibited E.
coli O157:H7 growth in zone inhibition assay (Seydim & Sarikus
2006). Solomakos et al. (2008) noted that 0.6% thyme essential oil had
an inhibitory effect against E. coli O157:H7 when applied directly in
the minced meat during refrigerated storage at 10 C. According to the
results of another study (Du et al., 2008) carvacrol containing tomato-
based edible lms inactivated the E. coli O157:H7, with the
inactivation related to carvacrol levels in the lms. Carvacrol and
thymol, active components of oregano and thyme are reported to
have inhibitory effects against E. coli O157:H7 by disintegration of the
outer membrane of microorganism and by release of outer mem-
brane-associated materials from the cells to the external medium
(Friedman, 2006; Rojas-Gra et al., 2007).
Among the bacteria tested, P. aeruginosa was the second resistant
bacteriumagainst ORand THessential oils after L. plantarum. While OR
concentration was a signicant factor (pb0.05) in inhibiting P.
aeruginosa, increases in concentration of TH did not have inhibitory
effect at concentrations higher than 2%. Gram-negative P. aeruginosa is
known to possess a high level of intrinsic resistance to most of the
antimicrobial agents due to a very restrictive outer membrane barrier
(Mann, Cox, &Markham, 2000). Resistance of P. aeruginosa to essential
oils was also reported by other researchers. Paster et al. (1990) stated
Table 1
Inhibition zone diameters yielded by soy protein based edible lm disks with various
concentrations (0, 1, 2, 3, 4 and 5%) of oregano (OR) and thyme (TH) essential oils
against test organisms.
Test organisms Concentrations
(%)
Inhibition zone diameters (mm)
OR incorporated
lm
TH incorporated
lm
Staphylococcus aureus 0 0 0
1 27.500.50
dB
30.000.00
dA
2 42.001.00
cA
38.000.00
cA
3 44.500.50
bA
46.001.00
bA
4 45.500.50
bA
48.001.00
abA
5 49.500.50
aA
49.500.50
aA
Escherichia coli 0 0 0
1 32.000.00
cB
36.500.50
eA
2 42.000.00
bA
41.000.00
dB
3 42.501.00
bA
43.000.00
cA
4 43.500.50
bB
47.000.00
bA
5 45.500.50
aB
49.000.00
aA
Escherichia coli O157:H7 0 0 0
1 35.500.50
dA
36.500.50
eA
2 41.001.00
cA
40.500.50
dA
3 44.500.50
bA
42.500.50
cA
4 49.500.71
aA
45.500.50
bB
5 50.500.50
aA
49.500.50
aA
Pseudomanas aeruginosa 0 0 0
1 27.001.00
dA
32.501.50
bA
2 34.500.50
cB
40.000.00
aA
3 37.001.00
bcA
40.000.00
aA
4 40.000.00
aA
41.001.00
aA
5 39.500.50
abA
42.001.00
aA
Lactobacillus plantarum 0 0 0
1 22.500.50
dA
20.500.50
dA
2 23.500.50
dB
29.500.50
cdA
3 30.000.00
cA
29.500.50
cdA
4 35.000.00
bA
32.500.50
bB
5 37.000.00
aA
36.500.50
aA
The meanstandard error.
AB: For a test organism, means within a row (between OR and TH incorporated lms)
not having a common superscript letter are different (pb0.05).
ad: For a test organism, means within a column (between concentrations) not having
a common superscript letter are different (pb0.05).
285 Z.K. Emirolu et al. / Meat Science 86 (2010) 283288
that P. aeruginosa was not affected by either oregano or thyme
essential oils at concentrations up to 500 g/mL. Cosentino et al.
(1999) assessed antimicrobial activity of Sardinian thymus essential
oils and their components against some spoilage and pathogenic
bacteria including P. aureginosa isolated from food products, and
reported that among the reference strains tested, P. aureginosa was the
least sensitive both to growth inhibition and lethal effect.
Inhibition zone results indicated that gram positive L. plantarum
was the most resistant bacterium to OR and TH essential oils among
the bacteria tested in the current study. However, inhibitory effects of
OR and TH incorporated lms against L. plantarum were observed
although these effects were not as much as against the other bacteria
tested. For both essential oils, concentration was a signicant factor in
this inhibition. Con, Ayar, & Gokalp (1998) determined antimicrobial
activities of extracts from six different spices including thyme against
eight bacterial strains of meat origin and noted that thyme extract
showed the highest antimicrobial activity against all test bacteria with
the lowest inhibitory effect against L. plantarum. This limited
antimicrobial activity of oregano essential oil when incorporated
into whey protein based edible lms against L. plantarum was also
reported by Seydim & Sarikus (2006).
3.2. Experiment 2Application of lms on ground beef patties
3.2.1. The pH value of ground beef
Ground beef had an initial pH of 5.87 before the patty preparation
and application of edible lms (Table 2). Isolated soy protein based
edible lm application onto the surface of the beef patties resulted in
increases in pH value of the samples, likely due to the high pH of the
lms (pH, 10.00, as determined in the lmsolution). This increase from
the initial pH was signicant after Day 1 for all of the lm treated
samples which had higher pH values as compared to the control
throughout the storage periods (pb0.05) except for ISP which had
similar pH value to C at Day 3 (pN0.05). The pH values of all samples
other than C generally decreased after Day 6 over time (pb0.05). This
decrease in pH value of the ground beef samples could be attributed to
the predominance of lactic acid bacteria, dominant microora in
vacuum packaged meats, which was in agreement with previous
studies (Sakala et al., 2002; Jones, 2004; Fik & Leszczyska-Fik, 2007).
3.2.2. Microbiological analysis of ground beef
Total viable counts (TVC) of the ground beef samples packaged
with or without edible lms during refrigerated storage are presented
in Fig. 1. Although slightly lower TVC (pN0.05) was determined in the
samples treated with essential oils containing lms at Day 3 and Day 6
as compared to C and ISP groups, no signicant differences (pN0.05)
in TVC was observed between the ground beef patty groups in general.
This might be due to the predominance of lactic acid bacteria (LAB)
counted within the TVC. LAB are known as predominant microora in
vacuum packaged fresh meats and are resistant to antimicrobial
treatments. Zinoviadou, Koutsoumanis, & Biliaderis (2009) reported
that 1.5% oregano essential oil incorporated whey protein isolated
edible lms resulted in 3.3 log reduction of TVC on fresh beef cuts as
compared with the control at day 8 of refrigerated storage. This
greater antimicrobial activity determined against TVC in aforemen-
tioned study as compared to ours might be due to the differences in
the meat material used. Ground beef patties have a complicated
structure and it would not be easy to effectively inhibit the growth of
bacteria on these products.
LAB counts showed increases over time in all groups (pb0.05)
(Fig. 2) and no signicant effect (pN0.05) of TH or OR incorporated
edible lms on LAB counts was observed over the storage periods.
Results from inhibition zone test supported these ndings in that, L.
Table 2
pH values of the ground beef packaged with antimicrobial edible lms during
refrigerated storage.*
Storage
days
C ISP OR TH ORT
0 5.870.02
A
5.870.02
B
5.870.02
C
5.870.02
B
5.870.02
B
1 5.830.01
bA
5.990.04
aA
6.090.04
aA
6.030.03
aA
5.990.01
aA
3 5.810.01
bA
5.810.03
bB
5.980.01
aB
5.970.01
aA
5.940.02
aAB
6 5.500.03
cB
5.720.02
bC
5.890.01
aC
5.880.02
aB
5.920.02
aB
8 5.440.05
cB
5.620.01
bD
5.750.03
aD
5.740.01
aC
5.740.04
aC
10 5.430.02
bB
5.530.03
aE
5.560.02
aE
5.620.05
aD
5.610.02
aD
12 5.470.01
dB
5.510.02
cdE
5.560.01
bcE
5.590.01
bD
5.690.01
aC
The meanstandard error.
AE: Means in a column, within a treatment group (between storage days), not having
a common superscript letter are different (pb0.05).
ad : Means in a row, within a storage period (between treatment groups) not having a
common superscript letter are different (pb0.05).
Fig. 1. LS-means for total viable counts (TVC) of ground beef packaged with
antimicrobial soy edible lms. C: control, ISP, OR, ORT and TH are samples packaged
with isolated soy protein lms without addition of essential oil, 5% OR essential oil
added lms, 5% OR+THessential oil mixture added lms, and 5% THessential oil added
lms, respectively. SEM within storage periods and within treatment groups are 0.098
and 0.083, respectively.
Fig. 2. LS-means for lactic acid bacterial counts (LAB) of ground beef packaged with
antimicrobial soy edible lms. C: control, ISP, OR, ORT and TH are samples packaged
with isolated soy protein lms without addition of essential oil, 5% OR essential oil
added lms, 5% OR+THessential oil mixture added lms, and 5% THessential oil added
lms, respectively. SEM within storage periods and within treatment groups are 0.168
and 0.142, respectively.
286 Z.K. Emirolu et al. / Meat Science 86 (2010) 283288
plantarum as a representative of LAB was found the most resistant
bacteriumamong the organisms tested to both THand OR essential oils
incorporated lms.
Initial Staphylococcus spp. counts for all patty groups (4.2 logCFU/g)
increased over time until the end of storage periods and showed
decreases after Day 10 reaching to 5.25.4 logCFU/g at Day 12 (Fig. 3).
Nosignicant difference inStaphylococcus spp. counts was found within
the patty groups (pN0.05) in general. Although OR and THessential oils
incorporated lmdisks had aninhibitory effect on S. aureus ininhibition
zone test (Table 1) the same inhibitory effect was not observed on the
ground beef patties against Staphylococcus spp. over 12-day refrigerated
storage (Fig. 3). This was probably due to the fact that there could be
different sub-strains of Staphylococcus strain in the ground beef used in
the present study, which all could have different specic resistance
mechanisms against antimicrobial treatments.
Counts of Pseudomonas spp., one of the common meat spoilage
bacteria at refrigerated temperatures, are given in Fig. 4. Initial
Pseudomonas spp. count of 6.36 logCFU/g was reduced to 5.62, 5.23,
and 5.09 logCFU/g for OR, TH, andORT groups, respectively, at the endof
the storage period. This decrease in Pseudomonas spp. count was
signicant (pb0.05) whenthe ground beef was coated with THand ORT
lms, which resulted in 1.13 and 1.27 log reductions, respectively, as
comparedtothe initial count. Similarly, Ousallahet al. (2004) reporteda
0.95 log reduction in Pseudomonas spp. counts in whole beef muscle
coated with milk protein based lms containing 1% oregano oil after
7 days during storage at 4 C.
Results of coliformcounts (Fig. 5) showedthat initial count increased
at Day 1 of the storage in the ground beef patties coated with essential
oil containing lms; however, after Day 3 through Days 10, 1.6, 1.9 and
2.0 log reductions (pb0.05) in coliform counts were observed with
incorporation of OR, ORT, and TH, respectively. Zivanovic et al. (2005)
found that chitosan lm application alone ensured a decrease of 13
logarithmic units in pathogen number while chitosan base edible lm
containing 1% and 2% of oregano essential oil ensured a decrease of 4
logarithmic units inthenumber E. coli O157:H7intheir study performed
on bologna slices at 10 C during 5-day storage. Chitosan, which is a
natural material, having antimicrobial activity, yielded more successful
results in inhibition of E. coli O157:H7, which is one of the important
food pathogens in the coliform group, in combination with thyme
essential oil synergistically. Del Nobile, Conte, Cannarsi, & Sinigaglia
(2009) reported that thymol, an active component from thyme, when
added at levels of 250, 500 and 75 mg per kg fresh minced beef patties
was effective on coliforms either under ordinary atmosphere packaging
conditions or in modied atmosphere packaging during refrigerated
storage at 4 C for 16 days. In another study, it was reported that, a
decrease of 23 logarithmic units in the most of the bacterial population
was ensured by applying 0.8% of essential thyme oil on the meat surface
(Tsigarida, Skandamis, & Nychas, 2000).
4. Conclusions
Greater antimicrobial activities of soy edible lms incorporated
with essential oils from OR and TH were demonstrated against S.
Fig. 3. LS-means for Staphylococcus spp. counts of ground beef packaged with
antimicrobial soy edible lms. C: control, ISP, OR, ORT and TH are samples packaged
with isolated soy protein lms without addition of essential oil, 5% OR essential oil
added lms, 5% OR+THessential oil mixture added lms, and 5% THessential oil added
lms, respectively. SEM within storage periods and within treatment groups are 0.235
and 0.189, respectively.
Fig. 4. LS-means for Pseudomonas spp. counts of ground beef packaged with
antimicrobial soy edible lms. C: control, ISP, OR, ORT and TH are samples packaged
with isolated soy protein lms without addition of essential oil, 5% OR essential oil
added lms, 5% OR+THessential oil mixture added lms, and 5% THessential oil added
lms, respectively. SEM within storage periods and within treatment groups are 0.116
and 0.098, respectively.
Fig. 5. LS-means for coliform bacterial counts of ground beef packaged with
antimicrobial soy edible lms. C: Control, ISP, OR, ORT and TH are samples packaged
with isolated soy protein lms without addition of essential oil, 5% OR essential oil
added lms, 5% OR+THessential oil mixture added lms, and 5% THessential oil added
lms, respectively. SEM within storage periods and within treatment groups are 0.223
and 0.189, respectively.
287 Z.K. Emirolu et al. / Meat Science 86 (2010) 283288
aureus, E. coli and E. coli O157:H7 as compared to P. aureginosa and L.
plantarum in growth media. The same inhibitory effect was not
observed when the antimicrobial lms were applied on ground beef
patties against Staphylococcus spp., total viable bacteria and lactic acid
bacteria. However, OR and TH essential oils incorporated lms
resulted in reductions in the counts of Pseudomonas spp. and coliform
bacteria of ground beef patties during refrigerated storage. This
limited antimicrobial activity of essential oils incorporated antimi-
crobial edible lms on ground beef patties could be due to the
complexity of ground beef matrix.
It has been recently shown that antimicrobial activity of OR and TH
essential oils was greater at acidic pH values and high concentrations
of protein with a moderate levels of simple sugars (Gutierrez et al.,
2009). Thus, future research in this area should focus on application of
antimicrobial edible lms on processed meat products, preferably, on
fermented sausages possessing acidic pH and high protein content.
Acknowledgements
This study was supported by the Research Foundation of Ankara
University (Project No: 2006-0745-046). Authors would like to
acknowledge Doctors A. K. Halkman, A. Bayrak, B. zen and F. Soyer
for their technical support.
References
AOAC. (1990). Ofcial methods of analysis of Association of Ofcial Chemists.Arlington,
VA: AOAC Inc. 15th Edi.
Beverlya, R. L., Janes, M. E., Prinyawiwatkula, W., & No, H. K. (2008). Edible chitosan
lms on ready-to-eat roast beef for the control of Listeria monocytogenes. Food
Microbiology, 25, 534537.
Brown, M. H. (1982). Introduction. In M. H. Brown (Ed.), Meat microbiology (pp. 112).
London: Applied Science Publishers Ltd.
Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications
in foodsA review. International Journal of Food Microbiology, 94, 223253.
Burt, S. A., & Reinders, R. D. (2003). Antibacterial activity of selected plant essential oils
against Escherichia coli O157:H7. Letters in Applied Microbiology, 36, 162167.
Burt, S., Vlielander, R., Haagsman, P. H., & Veldhuizen, J. A. E. (2005). Increase in activity
of essential oil components carvacrol and thymol against Escherichia coli O157:H7
by addition of food stabilizers. Journal of Food Protection, 68, 919926.
Ca'rdenas, F. C., Giannuzzi, L., & Zaritzkay, N. E. (2008). Mathematical modelling of
microbial growth in ground beef from Argentina. Effect of lactic acid addition,
temperature and packaging lm. Meat Science, 79, 509520.
Cagri, A., Ustunol, Z., & Ryser, E. (2004). Antimicrobial edible lms and coatings. Journal
of Food Protection, 67(4), 833848.
Chi, S., Zivanovic, S., & Peneld, M. P. (2006). Application of chitosan lms enriched
with oregano essential oil on bolognaActive compounds and sensory attributes.
Food Science and Technology International, 12(2), 111117.
Choi, S. G., Kim, K. M., Hanna, M. A., Weller, C. L., & Kerr, W. L. (2003). Molecular
dynamics of soy-protein isolate lms plasticized by water and glycerol. Journal of
Food Science, 68(8), 25162522.
Con, A. H., Ayar, A., & Gkalp, H. Y. (1998). Baz baharat uucu yalarnn eitli
bakterilere kar antimikrobiyal etkisi.Gda, 23(3), 171175 (in Turkish).
Cosentino, S., Tuberoso, C. I. G., Fadda, M. E., Pisano, B., Satta, M., Mascia, V., et al. (1999).
Antimicrobial activity and chemical composition of essential oils from Sardinia.
Igiene Moderna, 112(4), 14111421.
Cutter, N. C. (2006). Opportunities for bio-based packaging technologies to improve the
quality and safety of fresh and further processed muscle foods. Meat Science, 74(1),
131142.
Dadalioglu, I., & Evrendilek, G. (2004). Chemical compositions and antibacterial effects
of essential oils of Turkish oregano (Origanum minutiorum), bay laurel (Laurus
nobilis), Spanish lavender (Lavandula stoechas L.), and fennel (Foeniculum vulgare)
on common foodborne pathogens. Journal of Agriculture and Food Chemistry, 52,
82558260.
Del Nobile, M. A., Conte, A., Cannarsi, M., & Sinigaglia, M. (2009). Strategies for
prolonging the shelf life of minced beef patties. Journal of Food Safety, 29, 1425.
Devlieghere, F., Vermeiren, L., & Debevere, J. (2004). New preservation technologies:
Possibilities and limitations. International Dairy Journal, 14, 273285.
Donaldson, J. R., Warner, S. L., Cates, R. G., & Young, D. G. (2005). Assessment of
antimicrobial activity of fourteen essential oils when using dilution and diffusion
methods. Pharmaceutical Biology, 43(8), 687695.
Du, W. X., Olsen, C. W., Avena-Bustillos, R. J., Mchugh, T. H., Levin, C. E., & Friedman, M.
(2008). Antibacterial activity against E. coli O157:H7, physical properties, and
storage stability of novel carvacrol-containing edible tomato lms. Journal of Food
Science, 73(7), M378M383.
Fik, M., & Leszczyska-Fik, A. (2007). Microbiological and sensory changes in minced
beef treated with potassium lactate and sodium diacetate during refrigerated
storage. International Journal of Food Properties, 10(3), 589598.
Friedman, M. (2006). Antibiotic activities of plant compounds against non-resistant and
antibiotic-resistant foodborne human pathogens. In V. K. Juneja, J. P. Cherry, & M. H.
Tunick (Eds.), Advances in microbial food safetyACS Symposium Series. (pp. 167183)
Washington, DC: American Chemical Society.
Gutierrez, J., Barry-Ryan, C., & Bourke, P. (2009). Antimicrobial activity of plant essential
oils using food model media: Efcacy, synergistic potential and interactions with
food components. Food Microbiology, 26, 142150.
Hammer, K. A., Carson, C. F., & Riley, T. V. (1999). Antimicrobial activity of essential oils
and other plant extracts. Journal of Applied Microbiology, 86, 985990.
Han, J. (2000). Antimicrobial food packaging. Food Technology, 54, 5665.
Hao, Y. Y., Brackett, R. E., & Doyle, M. P. (1998). Efcacy of plant extracts in inhibiting
Aeromonas hydrophila and Listeria monocytogenes in refrigerated, cooked poultry.
Food Microbiology, 15, 367378.
Jones, R. J. (2004). Observations on the succession dynamics of lactic acid bacteria
populations in chill-stored vacuum-packaged beef. International Journal of Food
Microbiology, 90, 273282.
Kerry, J. P., O'Grady, M. N., & Hogani, S. A. (2006). Past, current and potential utilisation
of active and passive packaging systems for meat and muscle-based products: A
review. Meat Secience, 74, 113130.
Kintzios, K. E. (2004). Handbook of herbs and spices, Vol. 2,Greece: Agricultural
University of Athens.
Kolsarici, N., & Candogan, K. (1995). Effects of potassium sorbate and lactic acid on the
shelf life of vacuum-packed chicken meats. Poultry Science, 74(11), 18841894.
Lis-Balchin, M., Deans, S. G., & Eaglesham, E. (1997). Relationship between bioactivity
and chemical composition of commercial essential oils. Flavour Fragrance Journal,
13(2), 98104.
Lpez, P., Snchez, C., Batlle, R., & Nern, C. (2005). Solid- and vapor-phase antimicrobial
activities of six essential oils: Susceptibility of selected foodborne bacterial and
fungal strains. Journal of Agricultural and Food Chemistry, 53, 69396946.
Lpez, P., Snchez, C., Batlle, R., & Nern, C. (2007). Vapor-phase activities of cinnamon,
thyme, and oregano essential oils and key constituents against foodborne
microorganisms. Journal of Agricultural and Food Chemistry, 55, 43484356.
Lopez-Rubio, A., Almenar, E., Hernandez-Munoz, P., Lagaron, J. M., Catala, R., & Gavara,
R. (2004). Overview of active polymer-based packaging technologies for food
applications. Food Reviews International, 20, 357387.
Mann, C. M., Cox, S. D., & Markham, J. L. (2000). The outer membrane of Pseudomonas
aeruginosa NCTC6749 contributes to its tolerance to the essential oil of Melaleuca
alternifolia (tea tree oil). Letters in Applied Microbiology, 30, 294297.
Nedorostova, L., Kloucek, P., Kokoska, L., Stolcova, M., &Pulkrabek, J. (2009). Antimicrobial
properties of selected essential oils in vapour phase against foodborne bacteria. Food
Control, 20, 157160.
Nelson, R. R. (1997). In vitro activities of ve plants essential oils against methicillin-
resistant Staphylococcus aureus and vancomycin ressistant Enterococcus faecium.
Journal of Antimicrobial Chemotherapy, 40, 305306.
Oussalah, M., Caillet, S., Salmieri, S., Saucier, L., & Lacroix, M. (2004). Antimicrobial and
antioxidant effects of milk protein based lm containing essential oils for the
preservation of whole beef muscle. Journal of Agricultural and Food Chemistry, 52,
55985605.
Oussalah, M., Caillet, S., Salmieri, S., Saucier, L., & Lacroix, M. (2006). Antimicrobial
effects of alginate-based lmcontaining essential oils for the preservation of whole
beef muscle. Journal of Food Protection, 69(10), 23642369.
Paster, N., Juven, B. J., & Shaaya, E. (1990). Inhibitory effects oregano and thyme essential
oils on moulds and food born bacteria. Letters in Applied Microbiology, 11, 3337.
Pranoto, Y., Salokhe, V. M., & Rakshit, S. K. (2005). Physical and antibacterial properties
of alginate-based edible lm incorporated with garlic oil. Food Research
International, 38, 267272.
Quintavalla, S., & Vincini, L. (2002). Antimicrobial food packaging in meat industry.
Meat Science, 62, 373380.
Rojas-Gra, M., Avena-Bustillos, J. R., Olsen, C., Friedman, M., Henika, P. R., & Martin-
Belloso, O. (2007). Effects of plant essential oils and oil compounds on mechanical
barrier and antimicrobial properties of alginate-apple puree edible lms. Journal of
Food Engineering, 81, 634641.
Sad, O. (2003). Sensivity of four patogenic bacteria to Turkish thyme and oregano
hydrosols. LWTFood Science and Technology, 36, 467473.
Sakala, R. M., Hayashidani, H., Kato, Y., Hirata, T., Makino, Y., Fukushima, A., et al. (2002).
Change in the composition of the microora, on vacuum-packaged beef during
chiller storage. International Journal of Food Microbiology, 74(12), 8799.
SAS. (1996). SAS/STAT user's guide.Cary, NC: Statistical Analysis System Institute, Inc.
Release 6.12.
Seydim, A. C., & Sarikus, G. (2006). Antimicrobial activity of whey protein based edible
lms incorporated with oregano, rosemary and garlic essential oils. Food Research
International, 39, 639644.
Solomakos, N., Govaris, A., Koidis, P., & Botsoglou, N. (2008). The antimicrobial effect of
thyme essential oil, nisin and their combination against Escherichia coli O157:H7 in
minced beef during refrigerated storage. Meat Science, 80, 159166.
Tsigarida, E., Skandamis, P., & Nychas, G. -J. E. (2000). Behaviour of Listeria monocytogenes
and autochthonous ora on meat stored under aerobic, vacuum and modied
atmosphere packaging conditions with or without the presence of oregano essential
oil at 5 C. Journal of Applied Microbiology, 89, 901909.
Zivanovic, S., Chi, S., & Draughon, A. F. (2005). Antimicrobial activity of chitosan lms
enriched with essential oils. Journal of Food Science, 70(1), 4550.
Zinoviadou, K. G., Koutsoumanis, K. P., & Biliaderis, C. G. (2009). Physico-chemical
properties of whey protein isolate lms containing oregano oil and their
antimicrobial action against spoilage ora of fresh beef. Meat Science, 82, 338345.
288 Z.K. Emirolu et al. / Meat Science 86 (2010) 283288