Food Hydrocolloids 24 (2010) 49–59
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j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d h y d
a
b
a
,
*
a
Laboratory of Food Chemistry and Biochemistry, Department of Food Science and Technology, School of Agriculture, Aristotle University, GR-541 24 Thessaloniki, Greece b
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 3 June 2009
Accepted 6 August 2009
Keywords:
Sodium lactate
3 -Polylysine
Beef
Antimicrobial
Thermal properties
Mechanical properties
Whey proteins
Active packaging a b s t r a c t
The effectiveness of antimicrobial films against beef’s spoilage flora during storage at 5 C and the impact of the antimicrobial agents on the mechanical and physical properties of the films were examined. Antimicrobial films were prepared by incorporating different levels of sodium lactate (NaL) and 3 -polylysine ( 3 -PL) into sorbitol-plasticized whey protein isolate (WPI) films. The moisture uptake behavior and the water vapor permeability (WVP) were affected only by the addition of NaL at all concentrations used since an increased water uptake and permeability were observed with the addition of NaL into the protein matrix.
An increase of the glass transition temperature (5–15 C) of the sorbitol region, as determined by Dynamic
Mechanical Thermal Analysis (DMTA), was caused by the addition of 3 -PL into the WPI specimens. Instead, incorporation of NaL into the protein matrix did not alter its thermo-mechanical behavior. The addition of
NaL at concentrations of 1.0% and 1.5% w/w in the film-forming solution resulted in a decline of maximum tensile strength ( s max
) and Young modulus (E). A decrease of E and elongation at break (%EB), was also observed with increasing s max
, accompanied with an increase in
3 -PL concentration, at moisture contents higher that 10% (w/w). The antimicrobial activity of the composite WPI films was tested on fresh beef cut portions. The maximum specific growth rate ( m max
) of total flora (total viable count, TVC) was significantly reduced with the use of antimicrobial films made from 0.75% w/w 3 -PL in film-forming solutions ( p < 0.05), while the growth of Lactic Acid Bacteria was completely inhibited. Significant inhibition of growth of the total flora and pseudomonads was also observed with the use of films made with protein solutions containing 2.0% w/w NaL. These results pointed to the effectiveness of the antimicrobial whey protein films to extend the shelf life of fresh beef.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
The increased interest in ‘‘ready to eat’’ and easy to consume products enhances the obligation for greater control on food quality and safety. Outbreaks of foodborne diseases brought the necessity for alternative methods in controlling microbial growth in food products (
Appendini & Hotchkiss, 2002 ). A new trend in food
preservation consists of the use of active packaging in order to enlarge the safety margin and reassure high quality products.
Antimicrobial packaging materials can effectively control the microbial contamination of solid or semi-solid food products by inhibiting the growth of spoilage or pathogenic microorganisms on the surface of the food. Incorporation of antimicrobial compounds into films results in decreased diffusion rates from the packaging
* Corresponding author. Tel./fax: þ 30 2310 991797.
E-mail address: biliader@agro.auth.gr
(C.G. Biliaderis).
0268-005X/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2009.08.003
material into the product, thus assisting the maintenance of high concentrations of the active ingredient where it is required (
Kristo,
Koutsoumanis, & Biliaderis, 2008 ). Antimicrobial packaging and its
applications in the food industry has been thoroughly reviewed
recently ( Cagri, Ustunol, & Ryser, 2004; Cha & Chinnan, 2004;
Coma, 2008; Gennadios, Hanna, & Kurth, 1997 ;
Ozdemir & Floros,
2008; Quintavalla & Vicini, 2002
).
Lactate salts such as sodium lactate (NaL) are widely used as flavor enhancers in meat and poultry products, contributing to increased cooking yields and water holding capacity (
Aymerich,
Jofre, Garriga, & Hugas, 2005; Lungu & Johnson, 2005; Shelef, 1994
).
Various salts of lactic or other organic acids have also demonstrated antimicrobial activity in laboratory media or food products
(
Barmpalia et al., 2005; Koutsoumanis et al., 2004; Mbandi & Shelef,
2001, 2002; Samelis et al., 2001
). Surprisingly, more pronounced antibacterial effects of lactates in meat than in broth have been
reported ( Shelef, 1994
). NaL has been characterized Generally
Recognised as Safe (GRAS) and is usually added at a level of 2–3%
based on the finished product weight ( Kristo et al., 2008
).

50
3
E-polylysine ( 3
-amino groups of
L
-lysine residues (
K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59
-PL) is a cationic homopolymer of 25–35 L -lysine units interlinked by a peptide bond between the carboxyl and
Hiraki, Hatakeyama, Morita, &
Izumi, 1998; Yoshida & Nagasawa, 2003 ). This compound is heat
stable even under acidic conditions and exhibits a wide antimicrobial activity against Gram( þ ) and Gram( ) bacteria, yeasts and
moulds ( Hiraki, 2000; Shih, Shen, & Van, 2006; Shima, Matsuoka,
Iwamoto, & Sakai, 1984; Yoshida & Nagasawa, 2003 ). Additionally, it
has been suggested that 3 -PL is able to suppress dietary fat adsorption from the small intestine by inhibiting pancreatic lipase activity (
Kido et al., 2003 ). The safety of
3 -PL as a food additive has been demonstrated by experiments in rats and for this reason it has been used in Japan for the preservation of fish sushi, cooked vegetables, noodles and other products (
Hiraki et al., 2003
). In
2004, this compound was recognised as safe (GRAS) by the U.S.
Food and Drug Administration for use as an antimicrobial agent in
cooked or sushi rice at levels up to 50 mg/kg of rice ( USFDA, 2004
).
In contrast to the large amount of information on the use of various antimicrobial films for controlling meat pathogens, little is known about their effect on the spoilage microflora of these products. In the present study fresh beef cuts were wrapped into
WPI films containing NaL or 3 -PL at two different levels. The effectiveness of these films against beef’s spoilage flora during storage at 5 C was studied. Additionally, the impact of the antimicrobial agents on the mechanical and physical properties of the films was studied since the overall performance of the films depends strongly on their physicochemical properties.
(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 (BET) or Guggenheim–
Anderson–DeBoer (GAB) sorption isotherm models.
The BET model is given by the equation: a w
ð 1 a w
Þ m
¼
1 m m
K
þ
K 1 m m
K a w where m m is the BET monolayer value, and
The constants m m and K
K is a constant.
were calculated from the linear regression of the experimental data for a w values up to 0.64.
The three-parameter GAB isotherm model is written as: m m m
¼
CKa w
ð 1 Ka w
Þ½ 1 þ ð C 1 Þ Ka w where m m is the GAB monolayer value, and K and C are constants.
All sorption measurements were performed at least in triplicate.
2.3. Water vapor permeability
2. Materials and methods
2.1. Film preparation
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,
Avena-Bustillos, and Krochta (1993)
. Film discs, previously equilibrated at 53% RH for 48 h, were sealed in cups containing distilled water and the cups were placed in an air-circulated oven at 25 C that was equilibrated at 53% RH using a saturated solution of MgCl
2
6H
2
O (Merck KgaA, Darmstadt, Germany). Film permeabilities were determined as described by
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.
Bipro, a whey protein isolate (Davisco Foods International, Le
Sueur, MN, USA), was dissolved in distilled water under continuous stirring to obtain film-forming solutions of 5% (w/w). The 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. This process is necessary to obtain a flexible film via covalent and non-covalent cross-linking that retains its integrity at 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
(Sigma, St. Louis, MO, USA) was added as a plasticizer at a constant concentration of 37.5% (sorbitol/(WPI þ sorbitol)). Such a concentration of sorbitol was necessary to overcome the brittleness of WPI films, which otherwise are very difficult to handle without breaking.
Appropriate amounts of the antimicrobials were added in the filmforming solution resulting in a final concentration of 1.0%, 1.5% and
2.0% (w/w) for NaL (50% solution, Merck KGaA, Germany) and 0.25%,
0.50% and 0.75% (w/w) for 3 -PL (Chisso Corp., Tokyo, Japan). The solutions were kept overnight at 4 C to remove air bubbles. Portions of 12.5 g solution were cast on Petri dishes ( 4 8.5 cm) and allowed to dry in an oven at 35 C for w
24 h. In order to prepare thick specimens for the dynamic mechanical thermal analysis (DMTA), Petri dishes ( 4 13.0 cm) were completely filled with the protein solution and allowed to dry in an oven at 35 C for w
72 h. Film thickness was determined using a manual micrometer at 5 random positions on the film to obtain an average value.
2.2. Moisture sorption isotherms
Moisture sorption isotherms were determined for all films according to
Biliaderis, Lazaridou, and Arvanitoyannis (1999)
. Film samples ( w
300 mg) were placed in previously weighed aluminum dishes and dried at 45 C in an air-circulated oven over silica gel
2.4. Dynamic mechanical thermal analysis
Thick WPI specimens (0.5
0.6
0.15 cm
3
) prepared for DMTA analysis were previously conditioned at various RH environments
(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 the DMTA measurement at 110 C for 2 h. The thermo-mechanical measurements were performed with a Mark III analyzer (Polymer Labs. Loughborough, UK) operated in the single cantilever bending mode (heating rate 2 C min
1 and a strain level equal to a maximum displacement of 16 m m). The DMTA thermal scans were performed at five frequencies, i.e. 1, 3, 5, 10 and 20 Hz, and the T g values were taken as the peaks in tan polymeric matrix.
d of the protein
2.5. Large deformation mechanical testing
Films were cut in dumbell form strips and stored at appropriate
RH environments (11%, 23%, 43%, 53% and 75%) for at least 10 days to obtain specimens with different moisture contents. Film thickness was evaluated 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 min
1
. Young’s

modulus (E), tensile strength ( s max
) and % elongation at break
(% EB) were calculated from the load-deformation curves of tensile
testing ( Lazaridou, Biliaderis, & Kontogiorgos, 2003 ). The data
represent an average of at least eight samples. The moisture content of samples, after storage, was determined by drying at
110 C for 2 h.
2.6. Meat sample preparation and storage
2.7. Microbiological analyses
K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59
Freshly cut beef was purchased from a local retail store. The meat was cut into small pieces (2.1
2.5
1 cm) and these were wrapped into cross-shaped antimicrobial films that covered the entire meat surface. Samples that were not covered with the films, or were covered with films that did not contain antimicrobial agents, served as controls. The meat samples were placed into a sterile plastic dish covered with plastic film and were 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 microbiological quality
(0, 2, 4, 6, 8, 10 and 12 days).
51 a
100
80
60
40
20
0
0
0 % NaL
1.00 % NaL
1.50 % NaL
2.00 % NaL
0.2
0.4
0.6
0.8
1 b
100
80
60
40
20
0
0
0 %
ε
-PL
0.25 %
ε
-PL
0.50 %
ε
-PL
0.75 %
ε
-PL
0.2
0.4
0.6
0.8
1
Fig. 1.
Effect of sodium lactate (NaL) (a) and 3 -polylysine ( 3 -PL), (b) concentration (w/w in the film-forming solution) on the moisture sorption isotherms of antimicrobial sorbitol-plasticized WPI films.
Throughout storage of the beef cuts samples were taken and analyzed as follows. Beef samples (5 g) with the surrounding films were aseptically removed from the plastic disk, added into 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 that were wrapped in antimicrobial films, the film was carefully removed and added in the ringer solution, together with the meat sample, to wash off the bacteria that could be attached on its surface. Decimal dilutions in quarter-strength Ringer solution were prepared and 1- or 0.1-mL sample aliquots 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 interval. 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 ( m max
) and the lag phase.
2.8. Statistical analysis
WVP and moisture sorption data were averages of five and three 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 the two
antimicrobial agents ( Fig. 1 ). The moisture sorption isotherms for
the films displayed sigmoidal relationships, indicating that the equilibrium moisture content increased slowly with an increase in a w up to 0.53 above which there was a steep rise in moisture content. The shape of the sorption isotherm is characteristic of materials rich in hydrophilic polymers and is frequently reported in literature (
Cho & Rhee, 2002; Dadalioglu & Evrendilek, 2004; Diab,
Biliaderis, Gerasopoulos, & Sfakiotakis, 2001; Kristo & Biliaderis,
2006; Kristo et al., 2008 ). This form of the curves was similar to
those observed elsewhere for films formed from WPI and plasticized with glycerol (
Coupland, Shaw, Monahan, O 0 Riordan, &
O
0
Sullivan, 2000
). As shown in
Fig. 1
(a and b) the equilibrium water content of WPI films containing 3 -polylysine did not change at any of the concentrations used, while it increased substantially with increasing levels of Na lactate. This may be attributed to the sodium ions present that create regions of high dielectric constant
(
Eisenberg & Navratil, 1973 ). However, higher moisture uptake was
observed for the films containing 1.5% NaL compared to the ones containing 2.0% of the antimicrobial agent. High levels of salt ions result in a screening effect of the proteins charge thus lowering electrostatic repulsion between the charged molecules. This may result in a more structured network with lower water holding capacity.
The GAP and the BET equations were fitted to the experimental sorption data and the calculated parameters are given in
Table 1 .
The three-parameter GAP model successfully described the water sorption data up to the a w of 0.95 confirming the applicability of
this equation to sorption data in the multilayer region ( Biliaderis et al., 1999
). On the other hand, the BET model was applicable only within the a w range 0–0.64. The range of monolayer moisture values ( m m
) was similar between the two models (5.76–14.08 for
GAP and 5.62–10.81 g H
2
O/100 g for BET). As expected, no differences were observed between the m m values of the control films and the ones containing different amounts of 3 -PL. On the other hand, incorporation of NaL into the sorbitol-plasticized WPI films resulted in higher m m values and a higher water holding capacity.

52 K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59
Table 1
Estimated parameters for water sorption data of WPI films containing antimicrobial agents (25 C) using the BET and GAB isotherm models.
Sample K r
2
K
0% antimicrobial
1.00% Na L
1.50% Na L
2.00% Na L
0.25% 3 -PL
0.50%
0.75%
3
3
-PL
-PL
BET ( a w
:0.11–0.64) m m
(g H
2
O/100 g)
6.14
8.91
10.81
10.07
5.78
5.62
6.10
5.86
2.38
2.40
2.65
11.39
12.98
5.38
0.94
0.96
0.97
0.94
0.98
0.98
0.99
GAB ( a w
:0.11–0.94) m m
(g H
2
O/100 g)
6.09
10.36
14.08
10.78
5.76
5.79
6.65
0.97
0.93
0.80
0.96
0.95
0.96
0.95
C
6.91
2.13
2.13
2.56
14.17
13.82
4.95
Greater moisture uptake of hydrocolloid films in the presence of increasing plasticizer concentration is frequently reported in the literature (
Cho & Rhee, 2002; Herna´ndez-Mun
Gavara, 2003; Kristo & Biliaderis, 2006 , 2008
).
3.2. Barrier properties
The WVP values of the films along with their thickness and the estimated RH values 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 antimicrobial free WPI films (8.6
h m
2 kPa) were similar to those reported by
0.6 g mm/
Anker, Stading, and
Hermansson (2000, 2001)
for sorbitol-plasticized WPI films tested under similar conditions. On the contrary, lower values (4.1 g mm/ h m
2 kPa) have been reported for glycerol plasticized WPI films
(
Wang et al., 2008 ). However, in the latter study lower amounts of
plasticizer were used. The amount of plasticizer used is crucial since it influences positively the moisture content (
Cho & Rhee, 2002;
2003; Kristo & Biliaderis, 2006
).
Mate and Krochta (1996)
reported that WVP increases with moisture content. The theory behind this fact is that the water molecules disrupt polymer chain interactions causing an increase in the interstices. This results in faster water vapor diffusion (
Anker, Stading, & Hermansson, 1998
). Even lower
WVP values (3.5 g mm/h m
2 kPa) for whey protein-lipid emulsion
films have been reported in the past ( McHugh & Krochta, 1994b
).
Nevertheless, in that study the tests were carried out under less severe experimental conditions and the results cannot be directly compared.
The experimental conditions for permeability measurements should always be reported in order to obtain comparable results since they play a vital role in the measured
barrier properties ( Greener & Fennema, 1989 ).
Incorporation of NaL in the WPI matrix (at any of the three levels) resulted in a significant increase of WVP. This may be partly
Table 2
Effect of the antimicrobial concentration on the water vapor permeability of sorbitol-plasticized WPI films.
Antimicrobial Antimicrobial concentration
(% film-forming solution containing 5% w/w WPI and 3% w/w sorbitol)
Thickness
( m m)
Sodium Lactate 0
1.00
1.50
2.00
179
190
177
180
3 -Polylysine 0
0.25
0.50
0.75
179
164
155
186
RH
(%)
WVP
(g s
1
10 10 m
1
Pa
1
)
76 23.9
1.3
a
74 30.3
1.7
b
72 35.3
8.8
c
72 36.8
8.2
c
76 23.9
1.3
b
73 27.6
3.7
bc
78 17.8
1.4
a
80 17.6
1.8
a
* Different letters within the same column and for the same antimicrobial agent indicate significant differences ( P < 0.05).
attributed to the higher moisture content of the films with increasing NaL concentration as shown in
Fig. 1b
. On the contrary, the WVP was lower for the films that contained medium and high levels of 3 -PL. It is important to point out that since 3 -PL is a cationic peptide its addition into the film-forming solution resulted in an increase of the pH. The pH values of the film-forming solutions containing 0%, 0.25% and 0.75% of the antimicrobial agent were 7.1,
8.6 and 8.8, respectively. It has been previously reported that WPI
films exhibit better barrier properties at higher pH values ( Anker et al., 1998; Cagri, Ustunol, & Ryser, 2001
). Whey protein films are formed by heat-mediated protein–protein interactions involving disulphide, hydrogen and hydrophobic bonds. Heating results in an
exposure of internal SH and hydrophobic groups ( Shimada &
Cheftel, 1998
) which induces intermolecular S–S formation upon drying. Moreover, the SH reactivity increases under alkaline pH
( Banerjee & Chen, 1995 ) which promotes a better protein network
structure and thereby a lower WVP.
3.3. Thermo-mechanical properties
The thermo-mechanical properties of sorbitol-plasticized WPI films in the glass transition zone were examined by DMTA.
Representative DMTA traces (log E 0 and tan d ) of WPI film specimens equilibrated at different RH environments, thus containing different moisture contents, are presented in
Fig. 2
. At all moisture contents two relaxation processes, manifested by respective peaks in tan d , were observed. The peak recorded at higher temperatures corresponds to the protein’s transition temperature ( a -relaxation) and it shifts to lower temperature as the water content increases.
Moreover, with increasing moisture content an increased breadth of this transition was observed, implying a broader distribution of relaxation times as it has been earlier reported for other amorphous
biopolymer matrices ( Lazaridou & Biliaderis, 2002
). Previous studies on the thermal properties of WPI films cannot provide comparable information since the experiments were either conducted at lower temperatures (
Anker, Stading, & Hermansson,
1999
), or whey protein concentrates of lower impurity were used
( Ghanbarzadeh & Oromiehi, 2008 ). Apart from the main relaxation,
a secondary one was observed at lower temperatures that corresponded to the transition temperature of sorbitol. Similar results have been previously reported for WPI films containing oregano oil
( Zinoviadou, Koutsoumanis, & Biliaderis, 2009 ), for sorbitol-plas-
ticized WPI films studied with differential scanning calorimetry
(DSC) (
Shaw, Monahan, O 0 Riordan, & O 0 Sullivan, 2002
) and fructose containing sodium caseinate films (
Kalichevsky, Blanshard, &
Tokarczuk, 1993
), suggesting the occurrence of immiscibility between polyols and the polymeric components at a microscopic level. However, other studies on sugar-polymer blends did not reveal a separate transition for the polyol when the plasticizer was
added up to 20% d.p. ( Lazaridou & Biliaderis, 2002
).
The effect of the antimicrobial agents on the transition temperature of the sorbitol-plasticized WPI specimens is presented in
Fig. 3
. Incorporation of NaL into the WPI matrix did not change r
2
0.88
0.98
0.99
0.97
0.92
0.94
0.97

11
10
9 a a
K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59 d b c
Symbol a b c d m.c (%)
15.9
8.2
5.6
3.8
0.4
b
0.3
a b c d
0.2
0.1
53
8
7
-70 -20 30 80
o
130
0
-70 -20 30 80
o
Fig. 2.
DMTA plots log E 0 (a) and tan d (b) for sorbitol-plasticized WPI films at different moisture contents.
130 significantly the two transition temperatures as shown in
Fig. 3a .
A small increase in the temperature that corresponded to the transition of sorbitol was noticed when 1.5% of the antimicrobial agent was added into the film formulation. Similar results have been reported for sorbitol-plasticized sodium caseinate films and it was attributed to the preferential binding of water by the Na
þ ions
( Kristo et al., 2008 ). On the other hand, the addition of
3 -PL, especially at the level of 0.75%, into the protein solution resulted in a significant increase of the low-T transition. For the samples that did not contain any antimicrobial agent the low-T transition varied from 33.3 to 0.7
C (depending on the moisture content of the specimens), while for the samples that contained 0.75% 3 -PL these values ranged between 22.8 and 13.0
C. Since 3 -PL consists of small molecules with an average molecular weight of w
4000 Da they can co-exist in the sorbitol-rich regions of the film matrix.
Such an organisation would result in an increase of the ‘average’
molecular weight of the sorbitol-rich domains ( Cherian, Gennadios,
Weller, & Chinachoti, 1995
) and this effect would be more pronounced as the concentration of 3 -PL increases. Similarly to the
110 a
0 % NaL
1.00 % NaL
1.50 % NaL
0 % NaL
1.00 % NaL
1.50 % NaL
80 samples that contained NaL the the presence of 3 a -transition was not affected by
-PL. Instead, the plasticizing effect of water dominated this transition as often reported in a large number of studies on the thermo-mechanical properties of biopolymer based
films ( Biliaderis et al., 1999; Gontard & Ring, 1996; Kalichevsky,
Jaroszkiewicz, Ablett, Blanshard, & Lillford, 1992; Kristo & Biliaderis,
2007; Slade & Levine, 1991
).
Fig. 4a
shows representative tan d curves from multi-frequency
DMTA scans of sorbitol-plasticized WPI samples at 3.7% moisture content. It is clearly demonstrated that the tan d peaks of both transitions shifted to higher temperatures with increasing frequency, as expected for all thermally activated processes. Based on the DMTA data Arrhenius plots of ln frequency versus 1/ T were conducted for both transitions of all samples in order to allow the calculation of the apparent activation energy ( Ea ). Typical Arrhenius plots for sorbitol-plasticized WPI films are presented in
Fig. 4b
. The calculated values of the Ea of the samples that contained different levels of NaL or 3 -PL and different moisture contents are presented in
Table 3
. The Ea values of WPI samples containing or not
110 b
80
0 % -PL
0.25 % -PL
0.50 % -PL
0.75 % -PL
0 % -PL
0.25 % -PL
0.50 % -PL
0.75 % -PL
50
20
50
20
-10
-10
-40
0 5 10 15 20
25
-40
0 5 10 15 20
25
Fig. 3.
Transition temperature of sorbitol-plasticized WPI films containing different concentrations of a) sodium lactate (NaL) and b) 3 -polylysine ( 3 -PL) (w/w in the film-forming solution) as a function of sample water content; open symbols correspond to the low-temp. transition (sorbitol-rich phase), whereas the filled symbols refer to the high-temp.
transition (polymer-rich phase).
T g was determined from the temperature position of the respective tan d peaks (3 Hz).

54
0.4
a 1Hz b 3Hz c 5Hz d 10Hz e 20Hz
K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59 diffusion processes are limited, it is of great importance to study the thermo-mechanical properties of the antimicrobial films in order to determine the parameters that may govern diffusion of the antimicrobial agents from the film matrix into the food product.
0.3
a b c d e
0.2
0.1
4
3
2
1
0
2.5
2.7
3.5
(1/T) x 10
3
3.6
3.7
0
-40 -20 0 20 40 60
80 100 120 140
Fig. 4.
Multi-frequency DMTA thermal scans for sorbitol-plasticized WPI samples
(m.c. 3.7% w/w) and Arrhenius plots of ln (frequency) versus reciprocal temperature
(inset).
antimicrobial agents varied within 172–302 kJ/mol, which is in agreement with previous studies that determined Ea of the a -relaxation to be in the range of 200–400 kJ/mol (
Champion, Le
Meste, & Simatos, 2000 ). The slightly higher
Ea values that were observed for the samples that contained NaL could be attributed to a denser protein packaging due to the Na þ ions. In general, the activation energy values of the high-temperature transition decreased with increasing moisture content as it has also been
reported in previous studies ( Kristo & Biliaderis, 2006; Lazaridou &
Biliaderis, 2002, 2003; Zhang & Han, 2006
). Since the glassy state is frequently described as a state of relatively high stability where
Table 3
Apparent activation energy ( Ea ) of the a-relaxations and low-T transitions estimated from the Arrhenius plots applied to multi-frequency DMTA data for samples containing different levels of the antimicrobial agents, at different moisture contents.
Antimicrobial agent
Antimicrobial concentration in film-forming solution (% w/w) m.c.
(% w/w) a -Relaxation
Ea (kJ/mol) r
2
Reference 0%
0%
0%
0%
3.8
5.6
8.2
16.0
264
228
203
172
Low-T relaxation
Ea (kJ/mol) r
2
0.95
a
223
0.98
208
0.98
180
0.97
169
0.94
a
0.99
0.99
0.99
NaL 1.00%
1.00%
1.00%
1.00%
1.50%
1.50%
3.8
6.6
9.1
21.2
5.3
11.5
302
241
214
184
292
247
0.94
275
0.99
224
0.97
189
0.98
138
0.96
272
0.97
217
0.97
0.98
0.98
0.99
0.96
0.97
3 -PL 0.25%
0.25%
0.25%
0.25%
0.50%
0.50%
0.50%
0.50%
4.1
5.3
7.6
16.7
4.0
5.3
7.5
14.9
222
215
199
180
272
263
258
261
0.98
0.95
0.91
0.94
0.98
0.98
0.99
0.99
202
195
182
163
189
164
147
132
0.75%
0.75%
0.75%
0.75%
4.0
5.1
7.5
15.9
255
228
207
189
0.98
233
0.95
191
0.94
180
0.99
164
0.99
0.95
0.99
0.98
a r
2 values refer to the respective Arrhenius plots of the ln f versus 1/ T data.
0.97
0.98
0.96
0.99
0.96
0.95
0.97
0.97
3.4. Tensile properties
The profiles of large deformation mechanical properties under tensile mode of sorbitol-plasticized WPI films, as affected by film moisture content and NaL or 3 -PL concentration, are represented in
Figs. 5 and 6
. Antimicrobial films were conditioned at five different
RH levels (11%, 23%, 43%, 53% and 75%) at 25 C. For the films containing NaL a gradual decrease in tensile strength with increasing moisture content was observed. Water is known as a very effective plasticizer and its action is reflected by lowering the tensile strength and the elastic modulus and by increasing the
extensibility of the film ( Chang, Cheah, & Seow, 2000; Kristo et al.,
2007, 2008; Lazaridou & Biliaderis, 2002
;
McHugh & Krochta,
1994a; Shaw et al., 2002; Slade & Levine, 1991 ). The addition of NaL
in concentrations of 1.0% and 1.5% in the film-forming solution resulted in a decline of s max and E. Similar results have been also observed in previous studies on the effect of NaL in pectin and
alginate and sodium caseinate films ( Kristo et al., 2008; Parris,
Coffin, Joubran, & Pessen, 1995 ). However, higher concentrations of
NaL showed an opposite trend; i.e. incorporation of 2.0% NaL in the film-forming solution resulted in a much higher E value and significantly lowered the film’s extensibility. The Na þ cations can interact with the negatively charged proteins, reduce the electrostatic repulsion between the molecules and in this way facilitate
a denser protein packing ( Ganzevles, Zinoviadou, van Vliet, Cohen
Stuart, & de Jongh, 2006 ). It is worthy to mention here that besides
NaL, the WPI films contained other plasticizers as well, such as sorbitol and water, at different concentrations which make such systems very complex from a structural and physicochemical point of view.
The effect of moisture content in the case of the control films and the ones containing 3 -PL was more complicated. The tensile strength increased with an increase in moisture content up to 10% for the control films and for the ones containing 3 -PL as it can be seen in
Fig. 6 . On further hydration, water appeared to act as
a typical plasticizer by reducing the measured s max
. On the other hand, the Young modulus showed a general decline with increasing moisture content. The effects of water on the mechanical properties of the films measured under low or higher deformation modes, such as tensile testing often imply an anti-plasticization effect.
Similar behavior has been reported in the past for cereal products
( Harris & Peleg, 1996 ), zein films ( Lai & Padua, 1998
) and tapioca
starch films ( Chang et al., 2000 ). The anti-plasticizing effect of
water at intermediate hydration levels has been thoroughly reviewed by
Pitia and Sacchetti (2008)
who concluded that the anti-plasticization effects of water are mostly observed in systems which at low moisture content are characterized by a T g higher than ambient temperature, permitting partial relaxation of the chain segments in the amorphous domains of the biopolymer matrix.
For the control films at a moisture level of 11% the measured s max was 12 MPa, the % EB 5.7% and the E 480 MPa. These results come in agreement with
Ozdemir and Floros, (2008) , who reported
comparable values for sorbitol-plasticized WPI films tested under similar conditions. Addition of 3 -PL into the WPI films at concentrations of 0.50% and 0.75% (w/w) in the film-forming solution resulted in significantly smaller tensile strength and improved extensibility compared to the control film. The 3 -polylysine preparation consists of relatively small molecules and their incorporation into the protein matrix may induce plasticization. The exact action of plasticizers is complicated and several theories such as the

2500
2000
1500
1000
500
K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59
25
20
0 % NaL
1.00 % NaL
1.50 % NaL
2.00 % NaL
20
0 % NaL
1.00 % NaL
1.50 % NaL
2.00 % NaL
15
15
10
10
5
5
0 % NaL
1.00 % NaL
1.50 % NaL
2.00 % NaL
55
0
0 10 20 30 40
Mo is tu re co nt en t (% w/ w)
0
0 10 20 30 40
Mo is tu re co nt en t (% w/ w)
0
0 10 20 30 40
Mo is tu re co nt en t (% w/ w)
Fig. 5.
Effect of sodium lactate (NaL) concentration (% w/w in the film-forming solution) and water content on tensile strength ( s max
), tensile modulus (E), and % elongation at break
(% EB), as determined from large deformation mechanical testing of the antimicrobial sorbitol-plasticized WPI films.
lubrication, the free volume and the gel theory have been previously proposed (
Suyatma, Tighzert, & Copinet, 2005 ). However,
a small addition of 3 -PL resulted in an increase of the s max and E, especially at low moisture contents. This behavior is described as anti-plasticization and has been previously reported for starch-
glycerol ( Lourdin, Bizot, & Colomna, 1997
), starch-sorbitol (
Gaudin,
Lourdin, Botlan, LLari, & Colonna, 1999
) and chitosan-propylene glycol films (
Suyatma et al., 2005
). According to these studies a possible explanation for this phenomenon is that a strong interaction may be occurring between the polymer and the small amount of the plasticizer that may decrease the free volume and thus restrict the molecular mobility of the polymer chains.
3.5. Growth of spoilage bacteria
The growth responses of the total flora, Pseudomonas spp. population and LAB on beef cuts and the effect of WPI containing the two antimicrobial agents are presented in
Fig. 7 . As it can be seen from
the respective graphs no significant differences were observed
( P < 0.05) between the bacteria growth of the control meat samples and those covered with antimicrobial free films, indicating that the presence of whey protein or sorbitol did not affect the growth of any of the bacteria studied. For these samples, the pseudomonads population was slightly lower at day 0 (2 log CFU/cm
2
), but reached the same value as the total flora by the end of the study. The LAB population was kept at lower levels by increasing from approximately 1–5 log CFU/cm
2
. As shown in
Fig. 7a
and b, pseudomonads dominated the spoilage flora, a result that was not surprising since under aerobic conditions, a few species of the genus Pseudomonas are generally recognised to dominate the meat system and to contribute to spoilage. This can be attributed to their ability to degrade glucose and amino acids even under refrigeration condi-
tions ( Ercolini, Ruso, Torrieri, Masi, & Villani, 2006 ). The same
authors have reported that mesophilic bacteria grew by
2000
1500
0 % -PL
0.25 % -PL
0.50 % -PL
0.75 % -PL
25
20
30
0 % -PL
0.25 % -PL
0.50 % -PL
0.75 % -PL
25
20
0 % -PL
0.25 % -PL
0.50 % -PL
0.75 % -PL
15
1000 15
10
10
500
5
5
0
0 10 20
Mo is tu re co nt en t (% w/ w)
0
0 10 20
Mo is tu re co nt en t (% w/ w)
0
0 10 20
Mo is ture co nt en t (% w/ w)
Fig. 6.
Effect of 3 -polylysine ( 3 -PL) concentration (% w/w in the film-forming solution) and water content on tensile strength ( s max
), tensile modulus (E), and % elongation at break
(% EB), as determined from large deformation mechanical testing of the antimicrobial sorbitol-plasticized WPI films.

56 K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59 a
10
8
6 b ab a ab ab a c bc b ab b a c a a b ab ab b b ab ab a a d cd bcd abc ab a b b a a ab a a
4
2 control
0 % antimicrobial
1.00 % NaL
2.00 % NaL
0.25 % -PL
0.75 % -PL
0
0 2 4 6
Time (days)
8 10 12 b
10
8 b b ab a ab a b b ab a ab a b b ab a a a
6 b a ab ab ab b b ab ab a a c bc abc a ab a
4
2 control
0 % antimicrobial
1.00 % NaL
2.00 % NaL
0.25 % -PL
0.75 % -PL
0
0 2 4 6
8 10 12 c
10
8
6
4
2 control
0 % antimicrobial
1.00 % NaL
2.00 % NaL
0.25 % -PL
0.75 % -PL d c bcd abc ab a c a b b ab b ab ab a e d cd c b a c c c c a b b b b b b a
0
0 2 4 6
8 10 12
Fig. 7.
Effect of antimicrobial agent concentration (% w/w in the film-forming solution) in the films on beef’s spoilage flora upon storage at 5 C: a) total viable count; b) pseudomonads; c) Lactic Acid Bacteria. Points represent average values ( n ¼ 4) and different letters for the data points at each sampling period indicate significant differences ( P < 0.05).
approximately 8 log CFU/gr in beef when stored at 5 C for 14 days.
Up to day 7 the pseudomonads dominated the bacterial flora, whereas the LAB grew to higher levels upon further storage.
The use of films containing the highest level of NaL (2.0% w/w in the film-forming solution) resulted in a significant reduction of the total flora and pseudomonads population during the entire storage period ( P < 0.05). However, low antimicrobial action was observed in the case of the LAB. Similar findings have been reported in previous studies where it was shown that acid decontamination may shift the meat flora from Gram( ) to Gram( þ ) bacteria
(
Koutsoumanis et al., 2004; Samelis, Sofos, Kendall, & Smith, 2002;
Van Netten, Huis in’t Veld, & Mossel, 1994 ). As shown in
Table 4 ,
wrapping of beef cuts with the films that contained a high concentration of NaL resulted in a significant reduction of total bacterial population growth rate ( P < 0.05). Although many explanations have been proposed for the antimicrobial effect of lactates there is no clear evidence concerning a specific mechanism.
Shelef and Yang (1991)
have attributed the antimicrobial activity of
NaL to the lactate anion, while it has also been suggested that high levels of lactate ions may shift the reduction of pyruvate to lactate closer to its thermodynamic equilibrium, resulting in an inhibition of a major anaerobic metabolism pathway essential for growth.
In a previous study, the antilisterial efficacy of chitosan-coated plastic films containing NaL was tested on ham steaks. The use of those films resulted in significant inhibition of Listeria monocytogenes counts after 10 days of storage at room temperature.
Similar results were observed when the steaks were stored at 4 C.
After 10 weeks of storage under refrigerated conditions the
L. monocytogenes population reached a value of 7 log CFU/cm
2 for the control samples, while the use of NaL containing films reduced the counts from 2.7 to 1.5 log CFU/cm
2 for the same storage period
( Ye, Neetoo, & Chen, 2008 ). Lower antimicrobial activity was

K.G. Zinoviadou et al. / Food Hydrocolloids 24 (2010) 49–59
Table 4
Effect of antimicrobial concentration on the growth rate and lag phase of the total viable count, pseudomonas and lactic acid bacteria population of beef tissue wrapped in sorbitol-plasticized WPI films during storage of at 5 C.
Total viable count
Treatment Lag phase (hours) Growth rate
Control 55.8
5.6
a
No antimicrobial 38.2
10.0
a
1.00% NaL
2.00% NaL
74.0
35.4
bc
61.2
17.9
ab
0.25%
0.75%
3
3
-PL
-PL
57.9
12.6
ab
98.9
29.8
c
0.049
0.008
b
0.042
0.010
ab
0.041
0.010
ab
0.025
0.004
a
0.035
0.008
ab
0.045
0.020
b
R
2 range
0.99
0.98
0.87–0.99
0.97–0.98
0.91–0.99
0.92–0.99
Pseudomonas Control 33.3
1.1
a
No antimicrobial 36.4
8.7
a
1.00% NaL
2.00% NaL
0.25%
0.75%
3
3
-PL
-PL
71.7
44.5
a
77.2
44.8
a
54.1
24.5
a
70.6
12.2
a
0.043
0.005
a
0.045
0.005
a
0.054
0.021
a
0.036
0.020
a
0.034
0.007
a
0.033
0.005
a
0.99
0.96–0.99
0.93–0.99
0.99
0.91–0.99
0.94
Lactic acid bacteria
Control 39.7
19.2
a
No antimicrobial 21.0
14.9
a
1.00% NaL No lag phase a
2.00% NaL
0.25%
0.75%
3
3
-PL
-PL
59.0
49.8
a
46.7
11.1
a
No growth b
0.028
0.007
a
0.027
0.010
a
0.020
0.005
ab
0.021
0.007
ab
0.014
0.006
b
No growth c
0.99
0.98
0.92–0.97
0.84–0.97
0.74–0.97
–
* Different letters within the same column and for the same bacterial type indicate significant differences ( P < 0.05).
observed when casein films containing NaL were tested on TSANaCl plates inoculated with L. monocytogenes . Only when the NaL concentration was increased to 40% (w/w on a dry basis) a significant reduction of the pathogen population was observed. However, although the bacterial counts were significantly reduced, the difference did not exceed 2 log CFU/cm
2
(
Kristo et al., 2008
). No inhibition of Escherichia coli , Bacilus subtilis and Aspergillus niger has been reported when plastic films incorporating NaL were used
( Vartiainen, Skytta, Enqvist, & Ahvenainen, 2003 ), while other
researchers found that NaL incorporation into a zein-ethanolglycerol coating improved its antilisterial activity when tested on
turkey frankfurters ( Lungu and Johnson, 2005
). As it has been already mentioned, more pronounced effects of lactates in real food systems than in broth media have been reported, and this may explain the contradictions observed among different studies
( Shelef, 1994
).
When the beef cuts were wrapped with the films made with
0.75% (w/w) 3 -PL solutions a significant reduction of the total flora and the pseudomonads population was observed at all sampling times. For example, the TVC population of those samples at day 8 was approximately 5.5 log CFU/cm
2
, while for the control films it was 8.5 log CFU/cm
2
. Similar results were observed for the pseudomonads population, while complete inhibition of the Gram ( þ )
LAB was observed when these films were applied and significant reductions were found for the films that contained lower concentrations of the antimicrobial agent. The proposed mechanism of the inhibitory effect of 3 -PL on bacterial growth has been attributed to its electrostatic adsorption to the cell surface due to its positive charge. This interaction leads to the stripping of the outer membrane and abnormal distribution of cytoplasm as it has been demonstrated by electron microscopy (
Shima et al., 1984
). The lack of external lipopolysaccharide wall surrounding the peptinoglycal cell wall results in a decreased sustainability of the Gram( þ ) bacteria to antimicrobial agents such as the 3 -PL, explaining the greater inhibitory effect that was observed against the LAB.
Limited studies have been conducted on the antimicrobial activity of 3 -PL on spoilage or pathogenic bacteria.
Shima et al.
(1984)
performed studies using broth media and determined that the minimum inhibitory concentration (MIC) for growth of various bacteria was below 100 m g/mL. These values were reported at pH below the isoelectric point of 3 -PL, because under alkaline
57 conditions its activity can be significantly reduced. The inhibition of growth of food pathogens in a culture broth media was observed even at an abusive storage temperature (24 C) and the measured
MIC were 0.02% for E. coli O157:H7 and L. monocytogenes and 0.04% for Salmonella sp. Additional experiments on L. monocytogenes conducted at 4 C indicated that the antimicrobial activity was
significantly enhanced at lower temperatures ( Geornaras & Sofos,
2005
). Similar results were reported when food extracts (10%) were used as substrates and the antimicrobial activity was more pronounced in the extracts that did not contain high protein levels
(
Geornaras, Yoon, Belk, Smith, & Sofos, 2007
). However, it is not possible to make comparisons between the findings of the present study and previous ones since there are no results about the inhibitory effect of 3 -PL in real food systems.
As it can be seen from the data of
Table 4
the use of WPI antimicrobial films containing a high concentration of 3 -PL also resulted in a significant increase of the lag phase for the TVC. The lag phase of total bacteria population was about 40 h for the samples that were covered with the antimicrobial free films, while the use of antimicrobial films increased the lag phase at least by a factor of 2.
A similar trend was observed for the pseudomonads as well. In the case of the LAB, even the use of the films that contained the lower concentration of the 3 -PL resulted in a significant decrease of the growth rate from 0.028 (d
1
) to 0.014 (d
1
). When modified or vacuum packaging is applied to fresh meat or meat products there is a shift from a Gram( ) aerobic flora towards Gram( þ ) bacteria, especially lactic acid bacteria (
Borch, Kant-Muermans, & Blixt,
1996; Brooks et al., 2008; Ercolini et al., 2006; Tsigarida, Skandamis,
& Nychas, 2000
). Consequently, the application of antimicrobial films containing 3 -PL in combination with modified or vacuum packaging seems rather promising to extend of the shelf life of such products.
4. Conclusions
The present study indicated that whey protein isolate films can sustain their structural integrity at the high a w of beef surface and serve as effective carriers of antimicrobial agents. The results of this study clearly demonstrated that addition of effect as evidenced by a reduction of the permeability. Incorporation of
Acknowledgments
3 antimicrobial active films containing 3
3 -PL into the whey protein isolate film matrix did not alter the WVP and the water sorption properties of the films, while it induced a plasticizing s max and an increase of elongation at break. On the other hand, addition of NaL assisted a higher moisture uptake that resulted in higher water vapor
-PL resulted in an increase of the transition temperature of sorbitol, while the thermo-mechanical properties of the polymer region were not affected. The use of the
-PL resulted in a significant inhibition of beef’s spoilage flora by reducing the m max of the bacteria, while NaL did not seem to suppress the growth of LAB.
This research was partly supported by the EU Framework VI program Food Quality and Safety (acronym: ProSafeBeef Food-CT-
2006-36241). The author K. Zinoviadou would like to thank the
State Scholarship Foundation (IKY) for awarding her a graduate fellowship.
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