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Development and characterization of an active polyethylene film containing
Lactobacillus curvatus CRL705 bacteriocins
M. Blanco Massani a; M. R. Fernandez b; A. Ariosti b; P. Eisenberg b; G. Vignolo a
a Centro de Referencia para Lactobacilos (CERELA), Argentina b INTI-Plásticos (Centro de Investigación y
Desarrollo Tecnológico para la Industria Plástica-INTI), Buenos Aires, Argentina
Online Publication Date: 01 November 2008
To cite this Article Blanco Massani, M., Fernandez, M. R., Ariosti, A., Eisenberg, P. and Vignolo, G.(2008)'Development and
characterization of an active polyethylene film containing Lactobacillus curvatus CRL705 bacteriocins',Food Additives &
Contaminants: Part A,25:11,1424 — 1430
To link to this Article: DOI: 10.1080/02652030802227219
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Food Additives and Contaminants
Vol. 25, No. 11, November 2008, 1424–1430
Development and characterization of an active polyethylene film containing Lactobacillus
curvatus CRL705 bacteriocins
M. Blanco Massania*, M.R. Fernandezb, A. Ariostib, P. Eisenbergb and G. Vignoloa
aCentro de Referencia para Lactobacilos (CERELA), CONICET, Chacabuco 145, T4000ILC
Tucuma´n, Argentina; bINTI–Pla´sticos (Centro de Investigacio´n y Desarrollo Tecnolo´gico para la
Industria Pla´stica–INTI), Av. General Paz 5445, B1650WAB San Martı´n, Buenos Aires, Argentina
(Received 26 February 2008; final version received 25 May 2008)
The development and characterization of a bacteriocin-containing polyethylene-based film is described,
incorporating lactocin 705 and lactocin AL705, produced by Lactobacillus curvatus CRL705, and nisin.
Three different procedures to obtain lactocin 705 and AL705 solution were evaluated, with the partially purified
aqueous bacteriocin solution showing the highest inhibitory activity against indicator strains (Lactobacillus
plantarum CRL691 and Listeria innocua 7). Pouch contact, soaking and a contact method were compared for
incorporating bacteriocins onto PE-based films. Contact between the PE film and bacteriocin solution was the
most effective, resulting in a more uniform distribution of bacteriocins on the film surface and using less active
solution. The minimal inhibitory concentration of bacteriocin solution was 267AUcm3 (lactocin 705) and
2133AUcm3 (lactocin AL705), while the minimal contact time was 1 h. When relative inhibition area for
antilisterial activity of the active films was compared, those treated with L. curvatus CRL705 bacteriocins
displayed higher inhibitory activity than nisin-treated films. Functional properties of active PE-films containing
lactocin 705 and AL705 showed no differences compared with non-active control films. Bacteriocin-active
PE-based films are shown to be highly effective in inhibiting growth of Listeria. The potential use of
commercially available packaging films as bacteriocins carriers may benefit active-packaging systems.
Keywords: food packaging; active packaging; bacteriocins; antilisterial activity
Abbreviations: LAB, lactic acid bacteria; YE, yeast extract; PE, polyethylene; PPABs, partially purified aqueous
bacteriocin solutions, containing L. curvatus CRL705 bacteriocins and obtained by method (i); PPIBs, partially
purified isopropanol bacteriocin solutions, containing L. curvatus CRL705 bacteriocins and obtained by method
(ii); SPEBs, solid-phase extraction bacteriocin solutions, containing L. curvatus CRL705 bacteriocins and
obtained by method (iii); MIC, minimal inhibitory concentration; MCT, minimal contact time; WVTR, water
vapour transmission rate.
Introduction
Active packaging has been introduced as an innovative
food-packaging concept in response to the continuous
changes in consumer demands and market trends.
Active packaging performs some important roles other
than providing an inert barrier between the product
and external environment (Church and Parsons 1995).
The incorporation of antimicrobial agents directly into
polymeric packaging allows the industry to combine
the preservative function of antimicrobials with the
protective function of packaging. Active packaging
technologies are designed to extend shelf-life, while
maintaining the nutritional quality and safety of
foods, and involve interactions between the food, the
packaging material and the internal gaseous
atmosphere (Labuza 1990).
In recent years, attention has focused on the
prevention of initial adhesion of microbial
contaminants by the application of an antimicrobial
substance to the surface rather than trying to remove
the undesirable bacteria once they have adhered.
Unfortunately, some of the traditional methods used
for food preservation (thermal processing, drying,
freezing, refrigeration, irradiation, modified atmo-
sphere packaging, and adding antimicrobial agents or
salts) cannot be applied to foods such as fresh meats and
ready-to-eat (RTE) products. Direct surface applica-
tion of antibacterial substances onto foods (sprays or
dip solutions) had limited benefits since the active
substances can become neutralized on contact or diffuse
rapidly from the surface to the bulk food (Quintavalla
and Vicini 2002). On the other hand, incorporation of
antimicrobial agents into food formulations may
result in partial inactivation of the active substances
by food constituents and is, therefore, expected to
have only a limited effect on surface microflora.
*Corresponding author. Email: marianablanco@cerela.org.ar
ISSN 0265–203X print/ISSN 1464–5122 online
2008 Taylor & Francis
DOI: 10.1080/02652030802227219
http://www.informaworld.com
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8

The literature provides evidence that antimicrobial
substances may be effective as indirect food
additives incorporated into food packaging materials,
for the reduction in surface contamination of processed
foods (Vermeiren et al. 2002; Cooksey 2005;
Joerger 2007).
Various types of active substances can be incorpo-
rated into the packaging material to improve its
functionality and give it new or extra functions.
A range of antimicrobials have been incorporated
into or coated onto different film types, such as
bacteriocins, acids and their salts and anhydrides, plant
extracts, enzymes, triclosan and antifungal agents.
Even though it is difficult to distinguish between the
technical function of an additive as solely a packaging
antimicrobial without any impact on the food itself,
the regulatory status of packaging in the European
Union permits the use of antimicrobial additives
(Directive 2002/72/EC and its amendments). Among
the bacteriocins used as antimicrobials, nisin produced
by Lactococcus lactis has GRAS status and has been
approved as a food additive by both the US FDA and
WHO. It is also the antimicrobial most commonly
incorporated into films, either as singly or combined
with other antimicrobials (Joerger 2007). Since the
target of these antimicrobial peptides are Gram
positive organisms, they have been applied to natural
as well as synthetic materials as an effective approach
to reduce Listeria monocytogenes in foods. For meat
and meat products in particular, different antilisterial
packaging materials and coatings have been evaluated
using nisin (Ming et al. 1997; Siragusa et al. 1999;
Scannell et al. 2000; Franklin et al. 2004; Grower
et al. 2004a; Guerra et al. 2005), pediocin (Nielsen
et al. 1990; Ming et al. 1997) and an antilisterial
bacteriocin produced by Lactobacillus curvatus 32Y
(Mauriello et al. 2004). Despite intensive research and
development and increasing commercialization of
active and intelligent packaging systems, no specific
method exist in national and international legislation
to determine their suitability in direct contact with
foods. The result is that the legislation applying to
traditional packaging materials has been applied
to active and intelligent packaging systems
(Robertson 2006).
Lactocin 705 and lactocin AL705, two bacteriocins
produced by Lactobacillus curvatus CRL705 of meat
origin, a two-component (class IIb) and an anti-
Listeria (class IIa) compound, respectively, have been
characterized (Castellano et al. 2003, 2004; Cuozzo
et al. 2003). Lactobacillus curvatus CRL705 was used
as a protective culture in fresh beef and shown to be
effective against Listeria innocua and Brochothrix
thermosphacta, as well as the indigenous lactic acid
bacteria (LAB) contaminants, with lactocin 705 and
AL705 being inhibitory under vacuum-packaging
conditions and low temperatures (Castellano et al.
2004; Castellano and Vignolo 2006). The overall
objective of this work was to develop and characterize
a PE-based packaging film containing the bacteriocins,
lactocin 705 and lactocin AL705, both produced by
L. curvatus CRL705, for its potential use in meats and
RTE products.
Materials and methods
Bacterial strains and growth conditions
Lactobacillus curvatus CRL705 (producer of lactocin
705 and lactocin AL705) and Lactobacillus plantarum
CRL691 (an indicator of lactocin 705), isolated from
dry-fermented sausages and in the CERELA culture
collection, were grown in MRS broth (Britania,
Buenos Aires, Argentina) at 30C. Listeria innocua 7
(an indicator of lactocin AL705) was obtained from the
Unite´ de Recherches Laitie`res et Ge´ne´tique Applique´e,
INRA (France) and was grown in trypticase soy broth
(TSB) (BBL, Cockeysville, MD, USA) with 5mg cm3
of added yeast extract (YE) at 30C. All strains
were maintained and stored at 20C in 0.15 g cm3
glycerol.
Bacteriocin preparation
Lactocin 705 and lactocin AL705 were obtained by
growing L. curvatus CRL705 in MRS broth at 30C for
18 h. The culture was centrifuged (2500 g for 15 min),
the supernatant precipitated using 0.44 g cm3 ammo-
nium sulphate, centrifuged (20,000 g for 20 min) and
freeze-dried. We evaluated various bacteriocin
solutions:
(i) Aqueous bacteriocin solution: the active
powder was re-suspended in water and used
as a partially purified aqueous bacteriocins
solution (PPABs);
(ii) Isopropanol extraction: NaCl was added to
the aqueous bacteriocin solution to saturation
and the solution extracted three times with 1/4
volume of isopropanol; the organic phases
were then pooled (PPIRs);
(iii) Solid-phase extraction: the modified method
of Berjeaud and Cenatiempo (2004) was used,
in which the aqueous bacteriocin solution was
applied to a SPE-C18 cartridge (Varian),
washed successively with 10 and 30% isopro-
panol þ20mM ammonium acetate and the
bacteriocins eluted using four bed volumes of
40% isopropanol þ20mM ammonium acetate
(SPEBs).
A stock nisin solution was prepared by dissolving
100mg of Nisaplin (Danisco Argentina SA, Buenos
Aires, Argentina) in 0.02N HCl to assure complete
solubilization. Finally, 1N NaOH was added until
Food Additives and Contaminants 1425
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pH 2.5, and distilled water to give a final volume of
10 cm3 and a nisin concentration of 10mg cm3.
A solution of 1mg cm3 was prepared from
10mg cm3 solution.
Bacteriocins activity quantification
Activity of the bacteriocin solutions, expressed in
arbitrary units (AU) per cm3, was determined by a
modification of the agar diffusion and critical dilution
assay described by Pongtharangkul et al. (2004).
Briefly, 15 ml of serial twofold dilutions of the
bacteriocin solution was added to 5mm diameter
wells cut in semisolid MRS and TSAþ 5mg cm3 YE
seeded with L. plantarum CRL691 and L. innocua 7,
respectively, as indicator organisms. The plates were
stored at 4C for 24 h to allow pre-diffusion, incubated
for 16–18 h at 30C and examined for zones of
inhibition. The titer was defined as the reciprocal of
the highest dilution yielding a visible zone of inhibition
on the indicator lawn. Each determination was
performed in duplicate.
Preparation of active film and activity determination
T6040B linear low-density polyethylene (PE)-based
film with a barrier layer of ethylene vinyl alcohol
copolymer (Cryovac; Sealed Air Co., Buenos Aires,
Argentina) was used as the packaging material. For the
adsorption of bacteriocins on the plastic film, three
methods were evaluated using PPABs (see above)
containing lactocin 705 (4267AUcm3) and lactocin
AL705 (8533AUcm3).
(i) Pouch-contact: pouches made of T6040B film
were prepared and thermo-sealed at 150C for
1 s and 2.5 Pa using a thermo-sealing machine
(TP-701S Heat Seal Tester, Tokyo, Japan).
The active solution (46.9 cm3) was placed
inside the pouch, clip-closed and shaken
gently for 24 h.
(ii) Soaking: T6040B films were soaked in 1.5 cm3
of PPABs for 24 h.
(iii) Contact: T6040B films were placed in con-
tacted with 4.6 cm3 of the PPABs active
solution during 24 h.
The area/volume ratio in each method was 4.26, 3.00
and 4.26, respectively. After each treatment, the
bacteriocin solution was removed, the plastic rinsed
with sterile distilled water and dried for 10 min at 50C.
Antimicrobial activity of impregnated and control
plastic films was determined by placing 1.0 cm
diameter punched circles directly on the semisolid
agar plates seeded with the sensitive organisms. Film
bioactivity was evidenced as an inhibition zone of the
indicator organisms beneath and around the packaging
material and was expressed as relative inhibition area
(inhibition zone around packaging film/film area).
The diameter of the inhibition zone around each film
was measured using a caliper. Samples were run in
triplicate.
MIC and MCT determination
MIC and MCT of PPABs necessary to reach the
adsorption equilibrium of the bacteriocins onto the
film surface were determined using three concentra-
tions of PPABs (0.1, 1 and 10mg cm3). The films were
placed in contact with the active solutions for 5, 20, 60,
480 and 1440 min. Nisin solutions (1 and 10mg cm3)
were also placed in contact with PE films for 1 h.
Triplicate samples were run simultaneously.
Functional properties of the active film
Tensile strength and elongation of treated and non-
treated films were compared using an Instron model
1125 according to ASTM D 638-07 with a grips
separation of 65mm and test speed 500mmmin1. The
water vapour transmission rate (WVTR) of the films
was evaluated accordingly ASTM E 96/E 96M-05. The
assayed PE film was used to seal an aluminum cup
containing anhydrous silica desiccant (0.048m2
exchange film area) and placed in a controlled
humidity and temperature (50% RH and 25C)
room. The WVTR (g H2Om2 day1) was determined
by the increase in cup weight over time at the mass
transfer steady-state. Overall migration into the aqu-
eous food simulant was carried out according to
European Prestandards ENV 1186–7 at 40C during
10 days. All tests were conducted in triplicate.
Results
The antimicrobial substances produced by L. curvatus
CRL705 were partially purified from the culture
supernatant and used as a source of bacteriocins for
the active PE-based film. When antimicrobial activity
of the bacteriocin solutions containing lactocin 705
and lactocin AL705 was compared, PPABs and PPIBs
showed higher titers than SPEBs (4267, 2133 and
533AUcm3 for lactocin 705 and 8533, 2133 and
1067AUcm3 for lactocin AL705, respectively).
In addition, PPABs exhibited a higher inhibitory
activity than PPIBs against L. plantarum CRL691
and L. innocua as indicator microorganisms.
Films containing L. curvatus CRL705 bacteriocins
were obtained by pouch-contact, soaking and contact.
Results showed that soaking was more effective than
the pouch-contact method as a larger inhibition zone
and more uniform distribution of bacteriocins on the
film surface was observed for both lactocin 705 or
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AL705 (Figure 1a and b). When relative inhibition
areas of the films obtained by soaking and contact
were compared (Figure 2), no significant difference was
observed (p4 0.05) but a lesser volume of active
solution was required with the contact method.
Three different PPAB concentrations and contact
times were assayed to determine the MIC of the
contact solution plus the MCT. A relative inhibition
area 41.0 was obtained after a 1 h contact when
PPABs at a concentration of 1 and 10mg cm3
(corresponding to 2133 and 12800AUcm3 of lactocin
AL705) was applied. On the other hand, no anti-
microbial activity was observed on films surface
treated with a 400AUcm3 solution (0.1mg cm3
PPABs) against L. innocua 7 (Figure 3). When the
active film was assayed against L. plantarum CRL691,
the MIC for lactocin 705 was 267AUcm3
(PPABs, 1mg cm3) after a 1 h contact; no activity
was observed using a 0.1mg cm3 PPABs solution
(67AUcm3) (Figure 4). Antimicrobial activities of PE
films treated with PPABs (1mg cm3) and nisin against
both L. plantarum CRL691 and L. innocua 7 were
compared. Nisin-treated films (1 and 10mg cm3)
displayed large inhibition zones when L. plantarum
CRL691 was used as indicator organism, but failed to
inhibit L. innocua 7 on the agar plates (Figure 5).
In addition, even when PE films treated with PPABs
(1mg cm3) exhibited a lower relative inhibition area
against L. plantarum CRL691 than nisin, they were
more effective in inhibiting L. innocua 7.
When functional properties of active and control
films were evaluated, the values obtained for
tensile strength (49 3 vs. 48 3MPa), elongation
(377 19 vs. 37213%) and WVTR (0.55 0.08
vs. 0.55 0.03 g day1m2), respectively, showed no
significant differences (p4 0.05). Moreover, when an
aqueous simulant was used, overall migration values
were lower than 1mgdm2 for both active and non-
active films.
Discussion
In this study, conditions for the development of active
PE-based films using the bacteriocins, lactocin 705 and
lactocin AL705, produced by Lactobacillus curvatus
CRL705 were evaluated. Results clearly demonstrated
a retention of antimicrobial activity when adsorbed
onto PE-based films. The various procedures used to
obtain bacteriocin solutions from L. curvatus CRL705
showed that PPABs and PPIBs displayed higher
antimicrobial activity than SPEBs, in agreement with
dilutions carried out with SPE-C18 cartridges.
Moreover, since PPABs was easier to obtain and its
Figure 1. Antimicrobial activity of PE-based films treated with PPABs using different methods against (a) L. plantarum CRL691
and (b) L. innocua 7.
Figure 2. Relative inhibition areas of antimicrobial
PE-based films developed by soaking and contact method
assayed against (g) L. plantarum CRL691 and ( )
L. innocua 7.
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activity was higher than PPIBs, it was selected as the
active solution.
Three methods of binding bacteriocins from
L. curvatus CRL705 onto PE-based films were
evaluated. Soaking and a contact procedure were
demonstrated to be more effective than pouch-contact.
Clean, homogeneous and confined inhibition areas
were observed, suggesting that the bacteriocins were
uniformly bound to the surface of the film and diffused
regularly into the agar. Soaking was also applied to
develop active PE-oriented polyamide (OPA) films
with an antilisterial bacteriocin from Lactobacillus
curvatus 32Y (Mauriello et al. 2004), in which non-
uniform bacteriocin diffusion from the film into the
agar was obtained. Since only attached bacteriocins
could exert antimicrobial activity, the washing step of
the active PE-based film after soaking may be
responsible for the uniform diffusion of lactocin 705
and lactocin AL705 into the agar. Several studies have
reported the efficacy of the contact method in
providing active films incorporating antimicrobial
substances. Nisaplin was incorporated into cello-
phane-based packaging by contact, which was effective
for the control of microbial growth in chopped meat
(Guerra et al. 2005). In addition, Daeschel et al. (1992)
reported the antimicrobial activity of nisin after
adsorption onto hydrophobic and hydrophilic silicon
surfaces using the contact method. Although the
contact and soaking methods yielded similar results
in our study, less bacteriocin solution was required
with the contact method; therefore, this method was
chosen for obtaining active PE-based films.
Bacteriocins other than nisin were also applied by
various methods to develop active plastic materials for
L. monocytogenes inhibition in agar media or food
systems, such as the bacteriocin from L. curvatus 32Y
and CWBI-B28 (Mauriello et al. 2004; Ercolini et al.
2006; Ghalfi et al. 2006), lacticin 3147 produced by
Lactococcus lactis subsp. lactis (Scannell et al. 2000)
and pediocin from Pediococcus acidilactici (Ming
et al. 1997).
The MIC of the antibacterial solution, defined as
the lowest concentration of bacteriocin yielding films
that result in complete inhibition of visible growth
beneath and around the active film, corresponded to
PPABs with a concentration of 1mg cm3
(2133AUcm3 for lactocin AL705 and 267AUcm3
for lactocin 705). An adsorption plateau was attained
after 1 h of contact with the active solution, indicating
saturation of both bacteriocins in the PE film after this
contact period. In other words, an antimicrobial
Figure 3. Effect of lactocin AL705 activity and contact time
on relative inhibition areas of PE-based films against
L. innocua 7. (f) PPABs, 0.1mg cm3 (400AUcm3),
(*) PPABs, 1mg cm3 (2133AUcm3) and (g) PPABs,
10mg cm3 (12800AUcm3).
Figure 4. Effect of lactocin 705 activity and contact time on
relative inhibition areas of PE-based films against
L. plantarum CRL691. (f) PPABs, 0.1mg cm3
(67AUcm3), (r) PPABs, 1mg cm3 (267AUcm3) and
(g) PPABs, 10mg cm3 (4267AUcm3).
Figure 5. Relative inhibition areas of PE-based films treated
with nisin (1 and 10mg cm3) and PPABs (1mg cm3)
against (g) L. plantarum CRL691 and ( ) L. innocua 7.
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PE-based film was developed using the PPABs
(1mg cm3) containing lactocin AL705 and lactocin
705 from L. curvatus CRL705 after 1 h of contact time.
Due to the fact that nisin is the only bacteriocin
approved for food applications, we used it in this study
for comparative purposes. In this study, the lack of
sensitivity of L. innocua to nisin-active films compared
to L. plantarum CRL691 (Figure 5) is in agreement
with Cutter and Siragusa (1994, 1996) and Cutter et al.
(2001), who found Brochothrix thermosphacta to be
more sensitive to nisin than L. innocua. Moreover,
using well-diffusion assays, these authors also found
that L. innocua was more resistant to nisin than
L. monocytogenes Scott A. On the other hand,
sensitivity evaluations of various Listeria strains to
lactocin AL705, enterocin CRL35 and nisin
indicated that the non-nisin antilisterial bacteriocins
were more inhibitory than nisin – their susceptibility
to bacteriocins being strain-dependent (Castellano
et al. 2001).
To establish the effect of bacteriocin solutions on
the functional properties of PE-based film, they were
compared with those of the developed active films.
When choosing a packaging film for any application, it
is important to select the film on the basis of product
requirements (Grower et al. 2004a). No differences
were found between the active and control films
when mechanical properties and WVTR were
evaluated. Grower et al. (2004a) reported variations
in tensile strength and moisture barrier properties
of a low density polyethylene film coated with a
cellulose-based solution incorporating nisin with
increasing nisin concentration. In a similar study, the
release of nisin from a coating was found to be
uncontrolled, with L. monocytogenes inhibition
being inconsistent (Grower et al. 2004b). These
variations in mechanical properties may be ascribed
to the coating methodology used for the
incorporation of antimicrobial substances onto the
film. For example, the performance of the
contact-obtained films in this study showed no
differences compared to non-treated films. Overall
migration limit is an estimate of the inertness of a
plastic material to prevent unacceptable changes
in the composition of foodstuffs (Czerniawski
and Pogorzelska, 1997). Our results showed overall
migration values below the required limit specified in
EU Directive 02/72/EC (2002).
In conclusion, the potential for incorporating
antimicrobial peptides from LAB onto PE-based
plastic materials has been demonstrated. The active
PE film obtained by ‘‘contact’’ with lactocin
705þ lactocin AL705, produced by L. curvatus
CRL705, proved to be more effective than nisin in
inhibiting the growth of Listeria, without changing the
functional properties of the film.
Acknowledgements
This study was supported by funds from Agencia Nacional
de Promocio´n Cientı´fica y Tecnolo´gica (ANPCyT),
PICT2003 9-13499 and CONICET, PIP 6229, Argentina.
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