Meat Science 82 (2009) 338–345
Contents lists available at ScienceDirect
Meat Science
journal homepage: www.elsevier.com/locate/meatsci
Physico-chemical properties of whey protein isolate films containing oregano
oil and their antimicrobial action against spoilage flora of fresh beef
Kyriaki G. Zinoviadou a, Konstantinos P. Koutsoumanis b, Costas G. Biliaderis a,*
a
b
Laboratory of Food Chemistry and Biochemistry, Department of Food Science and Technology, School of Agriculture, Aristotle University, P.O. Box 235, GR-541 24 Thessaloniki, Greece
Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, School of Agriculture, Aristotle University, GR-541 24 Thessaloniki, Greece
a r t i c l e
i n f o
Article history:
Received 2 December 2008
Received in revised form 27 January 2009
Accepted 3 February 2009
Keywords:
Whey protein films
Oregano oil
Mechanical properties
Glass transition
Beef
Antimicrobial activity
a b s t r a c t
Antimicrobial films were prepared by incorporating different levels of oregano oil (0.5%, 1.0%, and 1.5% w/
w in the film forming solution) into sorbitol-plasticized whey protein isolate (WPI) films. The moisture
uptake behavior and the water vapor permeability (WVP) were not affected by the addition of oregano
oil at any of the concentrations used. A reduction of the glass transition temperature (10–20 °C), as
determined by dynamic mechanical thermal analysis (DMTA), was caused by addition of oil into the protein matrix. A decrease of Young modulus (E) and maximum tensile strength (rmax) accompanied with an
increase in elongation at break (%EB) was observed with increasing oil concentration up to a level of 1.0%
(w/w). Wrapping of beef cuts with the antimicrobial films resulted in smaller changes in total color difference (DT) and saturation difference (Dchroma) during refrigeration (5 °C, 12 days). The maximum specific growth rate (lmax) of total flora (total viable count, TVC) and pseudomonads were significantly
reduced (P < 0.05) by a factor of two with the use of antimicrobial films (1.5% w/w oil in the film forming
solution), while the growth of lactic acid bacteria was completely inhibited. These results pointed to the
effectiveness of oregano oil containing whey protein films to increase the shelf life of fresh beef.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Whey proteins are a by-product of the cheese-making industry
and have generally been disposed of as animal feed or used in infant formulas and sports food. Nowadays, great efforts are being
made to find new uses for whey proteins, e.g. production of edible
films (Anker, Stading, & Hermansson, 1998). Edible or biodegradable films constitute a convenient means to prolong the shelf life
of foods and increase their quality without contributing to environmental pollution. Apart from acting as selective barriers for moisture, gas and solute migration, these films may operate as carriers
of many functional ingredients. Such ingredients may include antioxidants, antimicrobial agents, flavors, spices and colorants which
improve the functionality of the packaging materials by adding novel or extra functions (Salmieri & Lacroix, 2006). Antimicrobial
packaging and its applications in the food industry has been thoroughly reviewed (Cagri, Ustunol, & Ryser, 2004; Cha & Chinnan,
2004; Coma, 2008; Gennadios, Hanna, & Kurth, 1997; Ozdemir &
Floros, 2004; Quintavalla & Vicini, 2002). Incorporation of antimicrobial compounds into films results in decreased diffusion rates
from the packaging material into the product, thus assisting the
maintenance of high concentrations of the active ingredient where
they are required (Kristo, Koutsoumanis, & Biliaderis, 2008).
* Corresponding author. Tel./fax: +30 2310 991797.
E-mail address: biliader@agro.auth.gr (C.G. Biliaderis).
0309-1740/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2009.02.004
Essential oils (EOs) extracted from plants or spices are rich
sources of biologically active compounds such as terpenoids and
phenolic acids. It has been long recognised that some of the EOs
have antimicrobial properties (Burt, 2004; Nychas, 1995; Shelef,
1983). Within a great variety of EOs, oregano oil that contains large
amounts of carvacrol is considered to be one of the most active
plant extracts against pathogens (López, Sánchez, Batlle, & Nerín,
2005, 2007). The hydroxyl group present in the structure of phenolic compounds confers antimicrobial activity and its relative position is very crucial for the effectiveness of these natural
components; this can explain the superior antimicrobial activity
of carvacrol compared to other plant phenolics. Although most of
the EOs are classified as Generally Recognized as Safe (GRAS) their
use as food preservatives is often limited due to flavouring considerations since effective antimicrobial doses may exceed organoleptically acceptable levels. However, incorporation of oregano oil in
edible films seems rather appealing since, due to the decreased diffusion rate of the active compounds smaller amounts will be
required to accomplish the desired antimicrobial effect.
In the present study, fresh beef cuts were wrapped in WPI films
containing oregano oil at three different levels. The effectiveness of
the films against the beef’s spoilage flora during storage at 5 °C was
investigated. Additionally, the impact of the oregano oil on the
mechanical and physical properties of the films was examined
since the functional behavior of the films is a combination of both
their antimicrobial and physico-chemical properties.
339
K.G. Zinoviadou et al. / Meat Science 82 (2009) 338–345
2. Materials and methods
2.1. Film preparation
Bipro, a whey protein isolate (WPI) (Davisco Foods International), was dissolved in distilled water under continuous stirring
to obtain film forming solutions of either 8% (w/w) for preparing
thick specimens for dynamic mechanical thermal analysis (DMTA)
or 5% (w/w) concentration for the rest of the measurements. Protein solutions were placed in a water bath at 90 °C for 30 min while
being stirred continuously. Heating the protein is essential for the
formation of intermolecular disulfide bonds to assist the establishment of a cross-linked polymeric network structure. This process is
necessary to obtain a flexible film that retains its structural integrity in high moisture environments (Le Tien et al., 2000; Vachon
et al., 2000). Solutions were then rapidly cooled in an ice water
bath, to avoid further denaturation and sorbitol (St. Louis, MO,
USA) was added as a plasticizer at the constant concentration of
37.5% (sorbitol/(WPI + sorbitol)). Such a concentration of sorbitol
was necessary to overcome the brittleness of WPI films, which
otherwise are very difficult to handle without breaking. Oregano
oil (Origanum vulgare sp. Hirtum, Ecopharm, Greece) at 0.5%, 1.0%
and 1.5% (w/w) ratios was added to the film forming solutions.
Equivalent amounts were also added for preparation of the DMTA
samples in order to obtain the same oil concentration on a dry basis. The solutions were homogenized at room temperature for
2 min at 13,000 rpm and 2 min at 19,000 rpm using an Ultra-Turrax (T-25 basic, IKA, Werbe). The solutions were then kept overnight at 4 °C to remove air bubbles. Portions of 12.5 g solution
were cast on Petri dishes (u 8.5 cm) and allowed to dry in an oven
at 35 °C for 24 h. Film thickness was determined using a manual
micrometer at five random positions on the film to obtain an average value.
2.1.1. Moisture sorption isotherms
Moisture sorption isotherms were determined for all films
according to Biliaderis, Lazaridou, and Arvanitoyannis (1999). Film
samples (300 mg) were placed in previously weighed aluminum
dishes and dried at 45 °C in an air-circulated oven over silica gel
(Sigma–Aldrich GmbH, Germany) until constant weight. The samples were subsequently kept in desiccators over saturated salt
solutions of known relative humidity (RH) at 25 °C for 21 days, a
time sufficient to reach constant weight and hence practical equilibrium. The moisture content of samples, after storage, was determined by drying at 110 °C for 2 h. The obtained data were fitted to
the Brunauer–Emmett–Teller (Durango et al., 2006) or the Guggenheim–Anderson–DeBoer (GAB) sorption isotherm models.
The BET model is given by the equation:
aw
1
K 1
aw
¼
þ
mm K
ð1 aw Þm mm K
where mm is the BET monolayer value, and K is a constant.
The constants mm and K were calculated from the linear regression of the experimental data for aw values up to 0.64.
The three-parameter GAB isotherm model is written as:
m
CKaw
¼
mm ð1 Kaw Þ½1 þ ðC 1ÞKaw where mm is the GAB monolayer value, and K and C are constants.
Measurements were performed at least in triplicate.
Avena-Bustillos, and Krochta (1993). Film discs (15.20 cm2), previously equilibrated at 53% RH for 48 h, were sealed into cups
containing distilled water and the cups were placed in an air-circulated oven at 25 °C equilibrated at 53% RH using a saturated solution
of MgCl2 6H2O (Merck KgaA, Darmstadt, Germany). Film permeability was essentially determined according to Kristo, Biliaderis,
and Zampraka (2007). The steady-state water vapor flow was
reached within 1 h for all films. Slopes were calculated by linear
regression and correlation coefficients for all reported data were
>0.99. At least five replicates of each film type were tested for WVP.
2.1.3. Dynamic mechanical thermal analysis
Thick WPI specimens (0.5 0.6 0.15 cm3) prepared for DMTA
analysis were conditioned at various RH’s (33%, 43%, 53% and 75%)
over saturated salt solutions for at least one month. The moisture
content of each film was evaluated by drying the sample after measurement at 110 °C for 2 h. The DMTA measurements were performed with a Mark III analyzer (Polymer Labs, Loughborough,
UK) operated in the single cantilever bending mode (heating rate
2 °C min1 and a strain level equal to a maximum periodical displacement of 16 lm). The Tg of the samples was determined as
the peak in tan d at 3 Hz.
2.1.4. Large deformation mechanical testing
Films were cut in dumbbell form strips and stored at appropriate
RH’s (11%, 23%, 43%, 53% and 75%) for at least 10 days to obtain
films with different moisture contents. Film thickness was measured at three different points with a hand-held micrometer and
an average value was obtained. Samples were analyzed with a
TA-XT2i instrument (Stable Micro systems, Godalming, Surrey,
UK) in the tensile mode operated at ambient temperature and a
crosshead speed of 60 mm min1. Young’s modulus (E), tensile
strength (rmax) and % elongation at break (%EB) were calculated
from the load–deformation curves of tensile testing (Lazaridou, Biliaderis, & Kontogiorgos, 2003). The data represent averages of measurements of at least eight samples. The moisture content of the
samples, after storage, was determined by drying at 110 °C for 2 h.
2.2. Meat sample preparation and storage
Freshly cut beef was purchased from a local retail store. The
meat was divided in small pieces (2.1 2.5 1 cm) and these were
wrapped in cross-shaped antimicrobial films that covered the entire meat surface. Samples that were not covered with the films
served as controls. The meat samples were placed into a sterile
plastic dish covered with plastic film and stored in high precision
(±0.2 °C) low-temperature incubators (model MIR 153; Sanyo Electric Co., Ora-Gun, Gumma, Japan) at 5 °C; all samples were evaluated periodically for color and microbiological quality (0, 2, 4, 6,
8, 10 and 12 days).
2.2.1. Colorimetric measurements
The changes in color of the beef pieces wrapped in the antimicrobial films were evaluated by measuring the L*, a* and b* parameters using a portable colorimeter (Chroma Meter, model CR-400;
Minolta, Osaka, Japan). The measured color parameters were used
to calculate changes in total color (DT) and saturation difference
(Dchroma) (Boakye & Mittal, 1996), according to the following
equations:
2
2.1.2. Water vapor permeability
Water vapor permeability (WVP) measurements of films were
conducted at 25 °C using the ASTM (E96-63T) procedure modified
for the vapor pressure at film underside according to McHugh,
DE ¼ ½ðL0 L Þ2 þ ða0 a Þ2 þ ðb0 b Þ2 1=2
2
1=2
Dchroma ¼ ða2
ða2 þ b Þ1=2
0 þ b0 Þ
The colorimeter was calibrated using a white standard plate. For
each treatment four samples were measured and on each beef
piece four readings were made.
K.G. Zinoviadou et al. / Meat Science 82 (2009) 338–345
2.2.2. Microbiological analyses
Throughout storage of the beef cuts samples were taken and
analyzed as follows. Beef samples (5 g) were aseptically removed
from the plastic disk, added to 45 mL of sterile quarter-strength
Ringer solution (LabM 100Z, Lancashire, UK) and homogenized in
a stomacher (Stomacher Interscience, France) for 2 min at room
temperature. In the case of the samples wrapped with antimicrobial films, the film was carefully removed and added in the ringer
solution to wash off the bacteria that could be attached to its surface. Decimal dilutions in quarter-strength Ringer solution were
prepared and 1 or 0.1 mL samples of appropriate dilutions were
poured or spread to the following media: plate count agar (PCA;
1.05463, Merck) for total viable count (TVC), incubated at 25 °C
for 72 h; MRS (1.10660, Merck) for lactic acid bacteria, overlaid
with the same medium and incubated at 30 °C for 96 h; cetrimide–fucidin–cephaloridine agar (CFC; with selective supplement
X108, LabM, Lancashire, UK) for Pseudomonas spp., incubated at
25 °C for 72 h. The storage experiments for the beef cuts were performed twice and duplicate samples for each treatment were analyzed for their microflora at each time. The microbial growth data
of the different spoilage bacteria of beef were modeled as a function of time with the model of Baranyi and Roberts (1994) using
the in-house software Dmfit, which allows the calculation of the
maximum specific growth rate (lmax) and the lag phase.
2.3. Statistical analysis
WVP, moisture sorption and color data were averages of five,
three and four replications, respectively. For the microbial analyses, the reported results are means of four measurements. All data
were analyzed by the general linear model (GLM) procedure of the
SPSS software, Release 13.0. Comparisons were made using the
Duncan’s multiple range test to determine any significant differences among the treatments at a 95% confidence interval.
3. Results and discussion
3.1. Moisture sorption isotherms
Water sorption isotherms were constructed for sorbitol-plasticized WPI films containing different concentrations of oregano
oil. The moisture content of the film increased slowly with increased humidity until aw 0.64, after which small increases in
humidity led to large weight gains. Such sigmoidal water sorption
isotherms are characteristic of materials rich in hydrophilic polymers (Biliaderis et al., 1999; Cho & Rhee, 2002; Diab, Biliaderis,
Gerasopoulos, & Sfakiotakis, 2001). The form of the curves was
similar to those observed elsewhere for films formed from WPI
and plasticized with glycerol (Coupland, Shaw, Monahan, O’Riordan, & O’Sullivan, 2000). Oregano oil addition at all three levels
did not markedly affect the water content of WPI films. Wang
and Padua (2004) reported a decrease in moisture adsorption of
zein films incorporating oleic acid. However, in that study the lipid
concentration was much higher (41% w/w) than in the present
work (Fig. 1).
The GAP and BET equations were fitted to the experimental
sorption data and the calculated parameters are shown in Table
1. The three-parameter GAP model is the most applicable since it
takes into account the properties of the adsorbed water in the multilayer region and can describe successfully the water sorption data
up to the aw of 0.95 (Kristo & Biliaderis, 2006). The range of monolayer moisture values (mm) was similar between the two models
(5.16–6.09 for GAP and 4.97–6.14 g H2O/100 g for BET). A small decrease in the monolayer value was observed as the oregano oil concentration increases. These findings are in agreement with the data
60
g H 2 0/100g dry matter
340
0 % oil
0.5 % oil
1.0 % oil
1.5 % oil
50
40
30
20
10
0
0
0.2
0.4
0.6
0.8
1
Water activity
Fig. 1. Effect of oregano oil concentration (w/w in the film forming solution) on the
moisture sorption isotherms of antimicrobial sorbitol-plasticized WPI films.
Table 1
Estimated parameters for water sorption data of oregano oil containing WPI films
(25 °C) using the BET and GAB isotherm models.
Sample
BET
(aw: 0.11–0.64)
K
R2
mm
(g H2O/100 g)
0% oil
0.5% oila
1.0% oila
1.5% oila
a
6.14
5.75
5.16
4.97
GAB
(aw: 0.11–0.94)
K
C
R2
0.97
0.98
0.98
0.98
6.91
7.63
8.02
17.2
0.88
0.90
0.96
0.93
mm
(g H2O/100 g)
5.86
6.56
7.26
19.7
0.94
0.96
0.98
0.98
6.09
5.75
5.16
5.20
Percent concentration (w/w) of oregano oil in film forming solution.
reported by Wang and Padua (2004), who also showed lower mm
values for zein films that, contained oleic acid.
3.2. Barrier properties
The WVP values of the films along with their thicknesses and
the estimated RH at the film underside are presented in Table 2.
The calculated RH values were lower than the expected 100% RH
due to the water transfer resistance of a stagnant air layer between
the film and the water surface in the cup (McHugh et al., 1993).
The WVP value of the oregano oil free WPI films (8.6 ±
0.6 g mm/h m2 kPa) were similar to those reported by Anker
et al. (1998), Anker, Stading, and Hermansson (2000) for sorbitolplasticized WPI films tested under similar conditions. On the other
hand, Wang et al. (2008) reported lower values (4.1 g mm/
h m2 kPa) for glycerol-plasticized WPI films under similar conditions. However, in the latter study less plasticizer was incorporated
in the film matrix. The amount of a compatible plasticizer in a
polymeric matrix is of great importance since as it increases the
interchain attractive forces become weaker and the energy of activation for diffusion (Ed) is reduced (Anker et al., 1998). Even lower
WVP values (3.5 g mm/h m2 kPa) have been reported for sorbitolplasticized WPI (1:1) films (McHugh & Krochta, 1994a). Neverthe-
Table 2
Effect of oregano oil concentration on the water vapor permeability (WVP) of sorbitolplasticized whey protein isolate films.
Oregano oil concentration
(% w/w film forming solution)
Thickness (lm)
RH
WVP*
(g mm/h m2 kPa)
0% oil
0.5% oil
1.0% oil
1.5% oil
179.2
177.2
168.7
187.3
75.6
76.6
73.1
75.8
8.6 ± 0.5a
8.5 ± 1.1a
11.0 ± 0.6a
9.1 ± 1.7a
*
Different letters within the same column indicate significant differences
(P < 0.05).
341
K.G. Zinoviadou et al. / Meat Science 82 (2009) 338–345
less, in the latter case, the tests were carried out under less severe
conditions and the results cannot be directly compared; for that
reason, all WVP values should be accompanied by information on
the testing conditions (Greener & Fennema, 1989).
In the present study, oregano oil incorporation in the WPI matrix did not affect significantly the WVP at any of the three levels
employed. Previous studies on the WVP of alginate-apple puree
edible films (Rojas-Graü et al., 2007) and apple puree films
(Rojas-Graü et al., 2006) containing EOs also showed no significant
differences for any of the EOs incorporated in the films, although
the concentrations used were lower than in the present work. In
contrast, other studies on incorporation of fats or lipids into edible
films have shown improvements in water vapor barrier properties
(Fabra, Talens, & Chiralt, 2008; Pérez-Gago & Krochta, 1999, 2000;
Shellhammer & Krochta, 1997); in these studies, however, higher
concentrations of the lipid components were added to the films.
Moreover, it has been demonstrated that the type of lipid used also
plays an important role on the final WVP of the polymeric film
(Shellhammer & Krochta, 1997).
3.3. Thermo-mechanical properties
The thermo-mechanical behavior of sorbitol-plasticized WPI
films containing different levels of oregano oil was studied by
DMTA. Representative DMTA traces (log E0 and tan d) of antimicrobial films conditioned at RH 33% are shown in Fig. 2. Similar traces
were observed for the other samples equilibrated at higher water
activities. The intense peak of tan d observed at higher temperatures corresponds to the glass–rubber transition of WPI. Incorporation of oregano oil, at two different levels in the protein matrix,
resulted in a decrease of the transition temperature, implying a
plasticizing action. This effect was even more apparent for the
11.00
log E' (MPa)
0 % oil
10.50
0.5 % oil
10.00
1.0 % oil
9.50
9.00
8.50
8.00
films that contained the medium level of the essential oil. Previous
studies investigating the thermal properties of WPI films cannot
provide comparable information since the experiments were either
conducted at lower temperatures (Anker, Stading and Hermansson,
1999), or a whey protein concentrate of lower impurity was used
(Ghanbarzadeh & Oromiehi, 2008).
Apart from the main a-relaxation, a secondary relaxation was
observed at low-temperatures (<0 °C) that corresponded to the
transition temperature of sorbitol, as has been demonstrated for
polyol-plasticized sodium caseinate and pullulan films (Kristo &
Biliaderis, 2006, 2007). Similar results have been found for WPI
plasticized with sorbitol containing 22% water, using DSC (Shaw,
Monahan, O’Riordan, & O’Sullivan, 2002). The addition of oregano
oil had no effect on this relaxation since it is a highly hydrophobic,
showing no preferential binding for water.
The dependence of both transition temperatures on water content is illustrated in Fig. 3. As the water content of the specimens
increased, both the low-temperature and the main relaxation
shifted to lower temperatures due to the well-known plasticizing
effect of water. As can be seen from Fig. 3, the transition temperature reduction by water for the samples containing different
amounts of oil did not differ from that of the antimicrobial-free
WPI films.
3.4. Tensile properties
In Fig. 4 the effect of moisture content and oregano oil concentration on the large deformation (tensile) properties of sorbitolplasticized films is presented. The most apparent feature of the
plots is an increase in stiffness for all the samples as the moisture
content increases from 4% to 8%. The anti-plasticizing effect of
water at intermediate hydration levels has been thoroughly reviewed by Pitia and Sacchetti (2008) who concluded that anti-plasticization is mostly observed in systems of low moisture content
that are characterized by a Tg higher than ambient temperature.
According to Harris and Peleg (1996) glassy biopolymers are brittle
and fragile at extremely low moisture contents. Nevertheless, at
moderate aw levels the partially plasticized matrix becomes more
cohesive and the structure is stiffer. Additionally, Fontanet, Davidou, Dacremont, and Le Meste (1997) proposed that better reorganization may occur upon hydration due to increased molecular
mobility and this may result in hardening. The typical plasticizing
action of water is observed at much higher moisture contents. This
effect is shown by the decreasing the stress at break, the elastic
modulus and by the increasing elongation properties of the film
(Chang, Cheah, & Seow, 2000; Kristo et al., 2007, 2008; Lazaridou
Transition Temp. ( oC )
0.4
tan
0.3
0.2
0.1
0
-60
120
100
80
60
40
20
0
-20
-40
0 % oil
0.5 % oil
1.0 % oil
0 % oil
0.5 % oil
1.0 % oil
0
-10
40
90
140
Temperature (o C)
Fig. 2. DMTA plots (log E0 , tan d) for sorbitol-plasticized WPI films containing
different concentrations of oregano oil (w/w in the film forming solution) with
moisture content 4% (w/w); single cantilever bending mode, heating rate 2 °C/min,
frequency 3 Hz.
5
10
15
20
Moisture content (% )
Fig. 3. Transition temperature of sorbitol-plasticized WPI films containing different
concentrations of oregano oil (w/w in the film forming solution) as a function of
sample water content; open symbols correspond to the low-temperature transition
(sorbitol-rich phase), whereas the filled symbols refer to the high-temperature
transition (polymer-rich phase). Tg was determined from the temperature position
of the respective tan d peaks (3 Hz).
342
K.G. Zinoviadou et al. / Meat Science 82 (2009) 338–345
2000
15
0 % oil
0.5 % oil
1.0 % oil
1.5 % oil
15
% EB
1000
0 % oil
0.5 % oil
1.0 % oil
1.5 % oil
20
max (MPa)
1500
E (MPa)
20
25
0 % oil
0.5 % oil
1.0 % oil
1.5 % oil
10
10
500
5
5
0
0
0
10
20
Moisture content (% w/w)
0
0
10
20
0
10
20
Moisture content (% w/w)
Moisture content (% w/w)
Fig. 4. Effect of oregano oil concentration (w/w in the film forming solution) and water content on tensile strength (rmax), tensile modulus (E), and % elongation at break
(%EB), as determined from large deformation mechanical testing of the antimicrobial sorbitol-plasticized WPI films.
3.5. Color variation
As can be seen in Fig. 5 an increase in Dchroma was observed over
time and this was more evident for the control samples. The smaller changes in chroma measured for the meat cuts wrapped with
the WPI films containing oregano oil reflect better color retention
due to stabilization of oxymyoglobin. The color variation of the
beef samples wrapped with films for different oregano oil concentration is well described by DT which provides a measure of the total color change since it takes into account all three color
parameters: lightness ‘L’, red-green ‘a’ and yellow-blue ‘b’. The
DT (Fig. 5b) exhibited a sharp increase during the first 2 days of
a
control
0 % oil
0.5 % oil
1.0 % oil
1.5 % oil
20
15
chroma
& Biliaderis, 2002; McHugh & Krochta, 1994b; Shaw, Monahan,
O’Riordan, & O’Sullivan, 2002).
For the control films, at a moisture level of 11.0%, the measured
rmax was 12 MPa, the % EB 5.7% and the E 480 MPa. These results
agree with Ozdemir and Floros (2008), who reported comparable
values for sorbitol-plasticized WPI films under similar conditions.
The addition of oregano oil in the sorbitol-plasticized WPI films resulted in a decrease of E and rmax with increasing oil concentration.
On the other hand, addition of oregano oil at a concentration up to
1.0% in the film forming solution resulted in an increase in elongation properties. The effect of lipid or EO addition in films has been
studied and in all the cases significant decreases in tensile strength
and elastic modulus have been reported (Fang, Tung, Britt, Yada, &
Dalgleish, 2002; Pérez-Gago & Krochta, 2000; Rojas-Graü et al.,
2006, 2007; Yang & Paulson, 2000; Zivanovic, Chi, & Draughon,
2005). Lipid addition induces the development of a heterogeneous
film structure, featuring discontinuities. The latter may affect the
stretching ability of the film based on the characteristics of the lipids added. Since oregano oil is liquid at room temperature, it will
be present in the film in the form of oil droplets that can easily
be deformed, enhancing the film’s extensibility (Fabra et al.,
2008). However, even at the highest oil content of 1.5% there was
no further plasticization and improvement of film extensibility,
but simply weakening of the film structure (lowering of the tensile
strength). Similar results have been reported on the plasticizing effect of soybean oil in whey protein films (Fang et al., 2002). It is
also worth noting that the differences among the samples became
less apparent at higher moisture contents, indicating again the
importance of water as a plasticizer in the WPI matrix.
10
5
0
-5
0
b
2
4
6
Time (days)
8
10
12
6
Time (days)
8
10
12
control
0 % oil
0.5 % oil
1.0 % oil
1.5 % oil
25
20
15
10
5
0
0
2
4
Fig. 5. Effect of oregano oil concentration (w/w in the film forming solution) on the
color properties (DT, part a; Dchroma, part b) of beef cuts wrapped in sorbitolplasticized WPI films on storage at 5 °C; points represent mean values (n = 4) and
half bars are standard deviations.
storage and then a gradual increase during the remaining storage
period. A sharp increase of DT during ageing of beef meat has been
reported previously (Boakye & Mittal, 1996). This result was obvious for all samples, with the greatest variability being noted for the
control sample (not wrapped with WPI films). The meat surface
discoloration largely depends on the oxidation rate of red oxymyoglobin to metmyoglobin giving meat an unattractive brown color
(Nerín et al., 2006). Oregano oil contains a large amount of terpenes and phenolic compounds that exhibit antioxidant activity
and reduce the changes in Dchroma and DT (Sánchez-Escalante,
Djenane, Torrescano, Beltrán, & Roncalés, 2003). Improved color
properties have been also reported for ostrich meat preserved with
rosemary, an extract also rich in phenolic compounds (Seydim, Gu-
343
K.G. Zinoviadou et al. / Meat Science 82 (2009) 338–345
zel-Seydim, Acton, & Dawson, 2006), and for low sulphite beef patties mixed with green tea extract (Bañón, Díaz, Rodríguez, Garrido,
& Price, 2007).
3.6. Growth of spoilage bacteria
The growth of the TVC, Pseudomonas spp. population and LAB on
beef cuts and the effect of WPI containing two concentrations of
oregano oil are presented in Fig. 6 (Goni et al., 2007). As can be
seen no significant differences were observed (P > 0.05) between
bacterial growth on the control meat samples and those covered
with antimicrobial-free films, indicating that the presence of whey
protein alone did not affect the growth of any of the bacteria studied. Even though it has been previously suggested that Escherichia
coli O157:H7 and Pseudomonas spp. can use milk protein based
films as a substrate for growth (Oussalah, Caillet, Salmieri, Saucier,
& Lacroix, 2004). Under the high aw conditions of the meat surface
the contacting films are highly hydrated and probably do not exhibit high O2 barrier properties and for this reason no differences in
the microflora profile were observed using the antimicrobial-free
film; i.e. the TVC population increased from 3 to 9 log CFU/cm2
by the end of the study for both control and antimicrobial-free film
treated samples. For the same samples, the pseudomonads popula-
a
10
b
b
b
log CFU/cm
2
8
b
b
b
6
b
4
b
b
b
b
b
a
a
control
0 % oil
0.5 % oil
1.5 % oil
a
b
2
b
a
b
a
b
a
b
a
0
0
2
4
6
8
10
12
Time (days)
b
10
b
b
8
log CFU/cm
2
b
b
b
b b
4
b
2
a
b
a
control
0 % oil
0.5 % oil
1.5 % oil
a
b
a
a
b
b
a
a
b
6
b
b
b
b
0
0
2
4
6
8
10
tion was slightly lower at day 0 (2 log CFU/cm2) but reached the
same value as the TVC by the end of the study. The LAB population
was kept at lower levels increasing from approximately 1–5 log CFU/cm2. By comparing Figs. 6a and b, it can be seen that pseudomonads dominated the spoilage flora, not surprising since spoilage of
fresh beef stored under aerobic conditions is mainly due the
growth and metabolic activity of pseudomonads (Koutsoumanis
et al., 2004). Ercolini, Ruso, Torrieri, Masi, and Villani (2006) reported that mesophilic bacteria grew by approximately 7 log CFU/gr in beef after 14 days of storage under refrigerated
conditions. Up to day 7 the dominant bacteria flora was pseudomonads, whereas upon further storage LAB grew to higher levels.
The use of films containing the highest level of oregano oil (1.5%
w/w in the film forming solution) resulted in a significant reduction of the TVC and pseudomonad population during the entire
storage period. The TVC population of the samples wrapped in
the films with the high oil content at day 8 was 5.1 log CFU/cm2,
while for the control it was 8.4 log CFU/cm2. Since microbial loads
( rchigher than 107 CFU/cm2 are usually associated with off-odors E
olini et al., 2006), it may be suggested that the use of WPI films
containing 1.5% w/w oregano oil could double the shelf life of fresh
beef stored under refrigerated conditions. The antimicrobial effect
was even more obvious against the Gram (+) LAB since complete
inhibition was noted when the 1.5% EO films were used, and significant reductions with films that contained lower levels of the
essential oil. The lower antimicrobial activity against pseudomonads can be attributed to the fact that Gram () are in general
more resistant due to the external lipopolysaccharide wall surrounding the peptidoglycan cell wall. However, the hydrophobic
constituents of the EOs are capable of gaining access to the periplasm of Gram (-) bacteria through the proteins of the outer membrane, as demonstrated by Confocal Scanning Laser Microscopy
(Lambert, Skandamis, Coote, & Nychas, 2001). The increase in
membrane permeability provokes a release of the cell constituents,
a decrease in ATP production in the cells and a decrease of the
intracellular pH (Oussalah, Caillet, Salmieri, Saucier, & Lacroix,
2006).
As can be seen from Table 3 the use of WPI antimicrobial films
resulted in a significant decrease in the maximum specific growth
rate (lmax) of TVC and pseudomonads. The lmax of total bacteria
population was 0.049 ± 0.008 (d1) for the control samples, while
the use of antimicrobial films decreased the rate by a factor of 2.
The results are expressed as log CFU/cm2 since the antimicrobial
films are active on the meat surface. The antimicrobial concentration of the films that contain the highest level of the oregano oil is
0.32 g/100 cm2. In a previous study on beef stored at 5 °C, the effect
of oregano oil addition (0.8% v/w or 0.3 g/100 cm2) against Salmo-
12
Time (days)
c
6
c
2
log CFU/cm
b
c
5
control
0 % oil
0.5 % oil
1.5 % oil
4
3
2
c
b
1
aa
b
c
c
c
Treatment
b
b
c
Table 3
Effect of oregano oil concentration on the lmax and lag phase of the total viable count,
pseudomonads and lactic acid bacteria population of beef wrapped in sorbitolplasticized WPI films during storage at 5 °C.
Total viable count
b
b
b
b
a
a
a
a
a
a
Pseudomonads
0
0
2
4
6
8
10
12
Time (days)
Fig. 6. Effect of oregano oil concentration (w/w in the film forming solution) in the
films on beef’s spoilage flora on storage at 5 °C: (a) Total viable count; (b)
pseudomonads; (c) lactic acid bacteria. Points represent average values (n = 4) and
different letters for the data points at each sampling period indicate significant
differences (P < 0.05).
Lactic acid bacteria
Control
0% oil
0.5% oil
1.5% oil
Control
0% oil
0.5% oil
1.5% oil
Control
0% oil
0.5% oil
1.5% oil
Lag phase (h)
a
55.8 ± 5.6
38.2 ± 10.0a
50.4 ± 8.0a
65.5 ± 30.3a
33.3 ± 1.1a
36.4 ± 8.7a
39.9 ± 7.3a
66.7 ± 35.9a
39.7 ± 19.2a
21.0 ± 14.9a
44.1 ± 28.9a
No growthb
R2
lmax (h1)
a
0.049 ± 0.008
0.042 ± 0.010a,b
0.038 ± 0.002b
0.023 ± 0.004c
0.043 ± 0.005a,b
0.045 ± 0.005a
0.038 ± 0.002b
0.026 ± 0.004c
0.028 ± 0.007a
0.027 ± 0.010a
0.021 ± 0.013a
No growthb
0.99
0.98
0.92–0.98
0.81–0.98
0.99
0.96–0.99
0.93–0.99
0.93–0.99
0.99
0.98
0.83–0.99
–
Different letters within the same column and for the same bacterial type indicate
significant differences (P < 0.05).
344
K.G. Zinoviadou et al. / Meat Science 82 (2009) 338–345
nella typhimurium, pseudomonads and LAB was evaluated (Skandamis, Tsigarida, & Nychas, 2002). Although similar concentrations
of the antimicrobial agent were used in both studies, less inhibitory activity was noted in the case of direct application of the antimicrobial instead of its incorporation into a film matrix.
Pseudomonas grew with a lmax of 0.033 (d1) and no lag phase
was observed. Moreover, the growth of LAB was observed at a rate
similar to that recorded for the untreated samples. As previously
demonstrated for foodborne pathogens, antimicrobial compounds
might be more effective in reducing the level of bacteria when
incorporated in a biopolymer film applied on the product surface
than when the antimicrobial is directly applied to the surface via
spraying or dipping (Kristo et al., 2008).
Alginate and milk protein films containing 1.0% oregano oil
were effective against foodborne pathogens inoculated on beef
(Oussalah et al.,2004, 2006). Alginate-apple puree films containing
low concentration of oregano oil (0.1% v/w) have been tested
against E. coli on agar and a large inhibitory zone was found (Rojas-Graü et al., 2007), demonstrating high antimicrobial activity.
In general, it can be said that a higher concentration of EO is required to achieve the same antimicrobial effect in food as
in vitro; in this context, it has been suggested that the greater availability of nutrients in food, compared to laboratory media, may enable bacteria to repair damaged cells (Gutierrez, Barry-Ryan, &
Bourke, 2008). Food composition can also affect the migration
mechanism of the antimicrobial agent into the food structure. For
example, the active compounds of the EOs are highly hydrophobic
substances and thus their diffusion into the product could be affected by the presence of fat. Studies on the use of antimicrobial
films on ham (15% fat) and bologna (25% fat) found that the availability of EOs in alginate based films was lower in the case of bologna, pointing to the significance of the affinity between the
antimicrobial agents and the product matrix (Oussalah, Caillet,
Salmieri, Saucier, & Lacroix, 2007).
4. Conclusions
This study indicated that WPI films can sustain their structural
integrity at the high aw of the beef surface and serve as effective
carriers of oregano oil. The use of the antimicrobial active films resulted in a significant inhibition of spoilage flora by reducing the
lmax of the bacteria. Application of WPI films containing 1.5% w/
w oil in the film forming solution was effective in increasing the
beef’s shelf life by a factor of 2, while minimising changes in color.
Furthermore, the results clearly demonstrated that the antimicrobial agent did not markedly alter the WVP and water sorption
properties of the film. Incorporation of oregano oil resulted in a
plasticizing effect, reducing the Tg of the films, as shown by the
DMTA analysis. The tensile properties of the films were also altered
by incorporation of the oregano oil, but such differences were
diminished at higher moisture contents.
Acknowledgments
This research was supported by the EU Framework VI program
Food Quality and Safety (acronym: ProSafeBeef Food-CT-200636241). The author K. Zinoviadou would like to thank the State
Scholarship Foundation (IKY) for awarding her a graduate
fellowship.
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