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Development of an active wheat gluten film with L. curvatus
CRL705 bacteriocins, antimicrobial performance study
during aging


Journal: Food Additives and Contaminants
Manuscript ID: TFAC-2013-272.R1
Manuscript Type: Original Research Paper
Date Submitted by the Author: 22-Oct-2013
Complete List of Authors: Blanco Massani, Mariana; Instituto Nacional de Tecnología Industrial,
Botana, Adrián
Eisenberg, Patricia
Vignolo, Graciela
Methods/Techniques: Microbiology, Bioassay
Additives/Contaminants: Packaging - active, Packaging - food simulants
Food Types: Meat
Abstract:
Antimicrobial wheat gluten film was obtained at pilot scale by Lactobacillus
curvatus CRL705 bacteriocins inclusion in the film forming solution.
Bacteriocins minimum inhibitory concentration for the film activation was
2133 AU cm-3 (lactocin AL705) and 267 AU cm-3 (lactocin 705). Mechanical
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and barrier properties as well as film aging kinectics were not significantly
affected by bacteriocins addition. Antimicrobial film performance during
aging was assessed. Film activity against Listeria innocua 7 and
Lactobacillus plantarum CRL691 was observed over 50 days of aging. Even
when bacteriocins release from the film upon water contact was observed
for both bacteriocins at the beginging of aging period, and anti-Listeria
activity was delivered to the simulant up to the 15th day of aging, film
residual activity for both bacteriocins was observed over 50 days. The
results achieved confirm the potential of a gluten-film doped with L.
curvatus CRL705 bacteriocins as a bacteriocins carrier to avoid Listeria and
LAB growth thus enhancing quality and safety in foods.



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Development of an active wheat gluten film with L. curvatus CRL705
bacteriocins and a study of its antimicrobial performance during aging

Blanco Massani Marianaa*, Botana Adriána,b, Eisenberg Patriciaa,b and Vignolo
Gracielac

aINTI-Plásticos. Gral Paz 5445. Buenos Aires, Argentina
b3iA-UNSAM, Argentina
cCentro de Referencia para Lactobacilos (CERELA), CONICET, Tucumán, Argentina.

*Corresponding author: M. Blanco Massani, E-mail: blanco@inti.gob.ar
Telephone: (54 11) 4724 6200 Int: 6636
Fax: (54 11) 4753 5773

Abstract
Antimicrobial wheat gluten film was obtained at pilot scale by Lactobacillus curvatus
CRL705 bacteriocins inclusion in the film forming solution. Bacteriocins minimum
inhibitory concentration for the film activation was 2133 AU cm-3 (lactocin AL705) and
267 AU cm-3 (lactocin 705). Mechanical and barrier properties as well as film aging
kinectics were not significantly affected by bacteriocins addition. The antimicrobial film
performance during aging was assessed. Film activity against Listeria innocua 7 and
Lactobacillus plantarum CRL691 was observed over 50 days of aging. Even when
bacteriocins release from the film upon water contact was observed for both
bacteriocins at the beginging of aging period, and anti-Listeria activity was delivered to
the simulant up to the 15th day of aging, film residual activity for both bacteriocins was
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observed over 50 days. The results aconfirm the potential of a gluten-film doped with L.
curvatus CRL705 bacteriocins as a bacteriocins carrier to avoid Listeria and LAB
growth thus enhancing quality and safety in foods.

Keywords: active bio-based polymer, anti-Listeria, bacteriocins, antimicrobial film
performance during aging

Abbreviations
WG (Wheat gluten)
LAB (Lactic acid bacteria)
LLDPE (Linear low denstity polyethylene)
PHB (Polyhydroxybutyrate)
BCE (bacteriocins crude extract)
MIC (minimum inhibition concentration)

Introduction
Food packaging is designed not only to contain and protect food, but also to keep food
safe and secure, to retain food quality and freshness, and to increase its shelf-life (Imam
et al 2008). Determining factors in food consumption include price, income, culture,
and food safety. Food-related health crises in recent times have decreased consumer
confidence (Pllana et al 2012). Meat and meat food products are consumed extensively
throughout the world, and among the meat borne pathogens, L. monocytogenes has been
associated with cooked, ready-to-eat (RTE) meat and poultry products (Williams et al
2011; Hereu et al 2012; Thippareddi, 2012). To solve the problem comprising RTE food
contamination, antimicrobial additives have been included in polymers as a preservation
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strategy (Vermeiren et al 2002; De Jong et al 2005; Cooksey 2005; Blanco Massani et al
2008; Koontz et al 2010). Furthermore, as an eco-friendly hurdle technology, bio-based
polymers have been used keeping safe and extending shelf life of foods (Cagri et al
2004; Cha & Chinnan 2004; Juneja et al 2006; Coma 2008; Iturriaga et al 2012). In the
last few years, special attention was given to bacteriocin incorporation into these
materials for food applications. Many studies can be found on active packaging with
antimicrobial action by the inclusion of nisin and other bacteriocins such as enterocin,
pediocin and lacticin 3147 (Scannell et al 2000; Grower et al 2004; Luchansky & Call
2004; Lungu, & Johnson 2005; Guiga et al 2010 ; Ibarguren et al 2010). Regulatory
requirements for active packaging technologies in the United States are not very
different from the requirements for conventional antimicrobial additives. Packaging
materials that has no intented technical effect on the food should be notified to the FDA
at least 120 days before its introduction to the market and can be sold unless the FDA
objets to the notification. However, the material exerting antimicrobial effect on food
throuhg migration or controlled release would constitute a ‘‘direct additive” and would
be subject to much stricter FDA regulatory requirements (Cho et al, 2009; Restuccia et
al 2010). On the other hand, for Mercosur member states, plastic articles intended to
come in contact with foods should comply with the requirements of Mercosur
Regulation (GMC/RES 32/07), which is updated acoording to the European Union
Regulation (1935/2004/EC). Even when current Mercosur regulation on active
packaging materials is not available, general requirements stated in Regulation
1935/2004/EC for the safe use of active and intelligent packaging have been recently
integrated by Regulation 450/2009/EC (Restuccia et al 2010). Lactocin 705 and lactocin
AL705, are bacteriocins produced by Lactobacillus curvatus CRL705, with antagonist
effect against Lactic acid bacteria (LAB), Brochothix thermosphacta, and Listeria
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species, respectively (Castellano & Vignolo 2006). Previous studies have demonstrated
activity retention when lactocin 705 and AL705 were adsorbed on a synthetic LLDPE-
based film (Blanco Massani et al 2008). Either lactocin 705 or AL705 were found to be
stable in a range of pH from 3.0 to 6.0, and up to 70ºC (Palacios 2000; Castellano
2005). Among the renewable resources available for biodegradable polymers obtaining,
our laboratory has worked with PHB (Botana et al 2010), starch, corn zein, soy and WG
proteins. While for PHB, starch and corn zein, film processing temperatures are higher
than 70ºC (Zhang & Han 2006; Marcos et al 2007; Ghanbarzadeh & Oromiehi 2009;
Botana et al 2010), films from wheat gluten or soy proteins can be obtained at lower
temperatures (Irissin-Mangata et al 2001; Denavi et al 2009). Proteins are hetero-
polymers comprising amino acids generally classified by groups that could interact via
hydrogen bonds (non-ionized polar amino acids), ionic interactions (ionized polar
amino acids), non-polar interactions (non-polar amino acids) or covalent bonds
(disulfide or dityrosine bonds). This heterogeneous structure provides many reaction
sites for potential cross linking or chemical grafting (Guilbert & Cuq 2005). Proteins
must be denatured by heat, acid, base, and/or solvent in order to form the more extended
structures that are required for film formation. Once extended, protein chains can
associate through hydrogen, ionic, hydrophobic and covalent bonding (Bourtoom 2008).
Film and film-forming properties are strongly dependent on the pH of the dispersion,
and they are normally lower close to the isoelectric point. Thus, soy proteins isolated
films cannot be formed close to a pH of 4.5 (Wu & Bates 1972; Sian & Ishak, 1990)
and are mostly formed at alkaline conditions (Ou et al 2005; Wan et al 2005; Denavi et
al 2009). Many studies have focused on the production of gluten films either by casting
a film forming solution obtained in humid conditions, or by thermal processing
(Gennadios, Brandenburg et al 1993; Gennadios, Weller et al 1993; Hochstettera et al
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2006; Song et al 2008; Marcuzzo et al 2010; Zhang et al 2010). As wheat gluten films
can be formulated either at basic or acid conditions (isoelectric point around 7.5)
(Herald et al 1995; Olabarrieta, Cho et al 2006), this protein could be an addecuate
support for L. curvatus CRL705 bacteriocins, since either 705 or AL705 are able to
withstand film forming conditions (acid pH and temperatures lower than 70ºC). The use
of gluten films has been studied for food applications such as lipid barriers reducing fat
uptake in deep-fried products, postharvest shelf life extention for refrigerated
strawberries, reducing breakage of egg shell and egg microbial contamination, among
others (Albert & Mittal 2002; Xie et al 2002; Tanada-Palmu & Grosso 2005). Moreover,
gluten films containing bacteriocins and other antimicrobials has been proposed for
foods that can be potentially contaminated with Listeria monocytogenes, such as meat
foods, fruits and vegetables (Ko et al 2001; McCormick et al 2005; Ibarguren et al
2010). Nevertheless, gluten films are known to suffer from aging due to plasticizer lost
(glycerol), protein aggregation or oxidation, and as a consequence material ductility
lowers, while stiffness and strength raises, incrementing film brittleness (Morel et al
2000; Micard et al 2000; Olabarrieta, Cho et al 2006). The purpose of our study was to
obtain WG-film containing L. curvatus CRL705 bacteriocins and to assess the influence
of aging on its antimicrobial performance, for the potential use to avoid bacteria
proliferation on meat products.

Materials and Methods
Bacterial strains and growth conditions
Lactobacillus curvatus CRL705, lactocin 705 and lactocin AL705 producer, and
Lactobacillus plantarum CRL691, used as an indicator of lactocin 705, were isolated
from dry-fermented sausages (Vignolo et al 1993) and grown in MRS broth (Britania
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Laboratories, Buenos 20 Aires, Argentina) at 30º C. Listeria innocua 7, used as an
indicator of lactocin AL705, was obtained from the Unité de Recherches Laitières et
Génétique Appliquée, INRA (France) and grown in trypticase soy broth (TSB, Britania)
with 5mg cm-3 of added yeast extract (YE, Britania) at 30ºC. All strains were
maintained and stored at -20ºC in 0.15 g cm-3 of glycerol until use.

Bacteriocins preparation and antimicrobial assays
An active powder containing lactocin 705 and lactocin AL705 was obtained as earlier
reported (Blanco Massani et al 2008). A bacteriocins crude extract (BCE) was prepared
by the active powder water re-suspension.
Lactocin 705 and AL705 antimicrobial activity in aqueous solution expressed as AU
cm-3 was determined by the agar well diffusion method (Pongtharangkul & Demirci
2004).
Antimicrobial activity of the activated and control films (see below) was determined by
placing 1.0 cm diameter punched circles directly on the semisolid agar plates seeded
with the sensitive organisms. Film activity was evidenced as an inhibition zone of the
indicator organisms beneath and around the film and expressed as relative inhibition
area (Blanco Massani et al 2008).

Antimicrobial WG-film pilot scale preparation
Wheat gluten (77,9% of protein, 13,3% starch and 1,0% lipids w/w, on dry weight base)
was kindly supplied by Molinos Juan Semino S.A. (Carcarañá, Pcia de Santa Fe). To
obtain a WG-film at pilot scale, a film forming solution was prepared by stirring wheat
gluten, sodium sulfite (Merck, Germany), glycerol (Cicarelli, Argentina) and ethanol
96% (Merck, Germany) with a mechanical stirrer (Heidolph RZR 2041). After a
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homogeneous solution was attained, water and the BCE were added, and the pH was
adjusted to 5.0 with acetic acid (Sintorgan, Argentina). The film forming solution was
spread onto a continuous Teflon® tape and dried in a warm tunnel with forced air at
50ºC for 4 hours.
Concerning the investigation on antimicrobial minimum inhibitory concentration
(MIC), BCE different concentrations were added to the film forming solution (0.01; 0.1
y 1% v/v, BCE on formulation base). The obtained films were assayed for antimicrobial
activity as previously described and compared to a film without bacteriocins (control).

Film properties as a function of aging time
Active and control (without bacteriocins) films were conditioned for 2 days in an
environmental chamber at 50% relative humidity and 23 ºC. The films were aged in the
mentioned conditions for 50 days and different properties were determined as a fuction
of time.
Water content, mechanical and barrier properties.
Residual water content in the bio-based films (active and control) was determined by
drying each sample at 100ºC until constant weight. The water content of the films was
determined from the weight difference between films before and after drying.
Control and active films´ tensile strength and elongation at break were evaluated in
quintuplicates with an Instron universal testing machine (model 1125), according to
ASTM D638-10. Initial grip separation was set at 65 mm and cross-head speed was set
at 50 mm/min.
Films water vapor permeability (WVP) was gravimetrically determined as described in
ASTM E96/E 96M-12. The tested films were placed on the top of hermetically sealed
aluminum cups, containing anhydrous silica desiccant (0.00181 m2 exchange film area)
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and placed in a chamber (75% RH and 23ºC). At least three samples of each type of
film were tested. The WVP, determined by the increase in cup weight over time at the
mass transfer steady-state, was calculated from the following equation:
ptAwxWVP ∆∆∆= / (mol m-1 s-1 Pa-1)
Where w∆ is the weight gain of the permeation cell in the steady state (mol), x is the
film thickness (m), t∆ is the time of weight gain (s), A is the area of exposed film (m2),
and p∆ (Pa) is the water vapor pressure differential across the film.
The thickness of each sample was taken as the average of three measurements made at
random points in the film using a hand-held micrometer.
Lactocin 705 and lactocin AL705 residual antimicrobial activity on the films after water
and sunflower oil contact
Active and control wheat gluten films (2.5 cm2) were contacted with water and
sunflower oil (2 cm3) representing hydrophilic and hydrophobic food simulant media,
respectively. After 10 days of contact (5ºC), films were removed. Residual
antimicrobial activity of the films was determined by placing them directly on the
semisolid agar plates seeded with the sensitive organisms. Bacteriocins activity in water
and sunflower oil after the films were removed, was determined by the well agar
diffusion assay. Experiment was run in duplicates.

Statistical analysis
Experimental data were subjected to analysis of variance (ANOVA), and the Tukey test
was applied with a level of significance of 95%.
All statistical analyses were performed using Minitab Statistic Program, release 12.
Results are informed by mean ± Standar Deviation of replications.

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Results and discussion
Active film preparation. MIC determination
Different concentrations of de BCE (0.01%, 0.1 and 1%) were added to the wheat
gluten formulation. Films doped with BCE 0.1 % (267 AU cm-3 lactocin 705, 2133 AU
cm-3 AL705) and 1% (4267 AU cm-3 lactocin 705, AU cm-3 12800 AL705) exerted
antimicrobial activity against L. plantarum CRL691 and L. innocua 7; while for films
with BCE 0.01% added (67 AU cm-3 lactocin 705, 400 AU cm-3 AL705) antagonistic
effect was displayed only against L. innocua 7 (Figure 1). Thus 0.1% BCE was defined
as the MIC, since at this concentration the WG-films were active against both sensitive
microorganisms. Even when the activation method as well as the polymer matrix were
different from the present work, lactocin 705 and AL705 showed similar MIC for a
LLDPE-based film activation by contact (bacteriocins adsorption) with a solution from
L. curvatus CRL 705 (Blanco Massani et al 2008).

Film properties as a function of aging time
The functional properties of the wheat gluten film formulated with BCE (0.1%) and
stored for 50 days (23ºC, RH 50%) were periodically determined and compared to a
control film without bacteriocins. During the storage time, both the control and active
wheat gluten residual water content was around 10%. Tensile strength and percentage
elongation at break of the control and active film as a function of storage time are
shown in Figure 2a and 2b. At the beginning of the experiment, the mechanical
properties of the control and active films (Figure 2a and b) were significantly different
(P<0.05) (tensile strength 2.0 ± 0.1 MPa and 2.7 ± 0.3 MPa; elongation at break 251 ±
14% and 190 ± 13%, respectively for the control and active film); nevertheless, the
observed difference was negligible (Figure 2 a and b). Mechanical properties values
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obtained in our work are in the range of those already reported for WG-films
(Olabarrieta, Cho et al 2006, Irissin-Mangata et al 2001). After 5 days of aging, the
tensile strength as well as elongation at break of the active film reached the values of
those obtained for the control (3.5 ± 0.3 MPa and 3.4 ± 0.4 MPa; 155 ± 21.% and 163 ±
14%, respectively). Until the 20th day of storage, strength marginally increased, while
elongation decreased (3.8 ± 0.3 MPa and 3.8 ± 0.3 MPa; 137 ± 29 % and 146 ± 18 %,
respectively for the control and active film), (Figure 2 a and b). From the 20th day up to
the 50th, mechanical properties remained fairly constant reaching tensile strength values
of 3.3 ± 0.3 MPa, 3.6 ± 0.2 MPa, and elongation at break, 177 ± 29 % and 160 ± 22 %,
respectively for the control and active films (Figure 2 a and b). WG-films change in
mechanical properties as a function of time was already studied and explained by an
increase in thiol oxidation during aging, leading to the formation of protein polymers of
large molecular size (crosslinking) (Morel et al 2000; Micard et al 2000, Olabarrieta,
Cho et al 2006). Water vapor permeability was not significantly (P≥0.05) different
between the control and active film (8 10-12 ± 3 10-13, 8 10-12 ±3 10-13, respectively),
maintaining the magnitude order until the end of the storage period (8 10-12 ± 6 10-13, 8
10-12 ±8 10-13, respectively). Water vapor permeability values obtained in our work are
in the order of those found for wheat gluten films obtained at acid pH (Irissin-Mangata
et al 2001). When activating a packaging polymer to render antimicrobial properties,
some physico-mechanical properties as well as processability can be affected. General
properties of packaging materials include mechanical properties such as tensile strength,
elongation, burst strength, tearing resistance, stiffness, and physical properties such as
oxygen (and other gas) permeability, water vapor permeability, among others (Han
2000). From the comparison between active and control WG-films mechanical and
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barrier properties it could be observed that addition of bacteriocins had no effect on the
kinetics of WG film aging.
To fulfill the objective of protecting and extending food shelf life, active packaging
materials should maintain their activity properties over a time period, determined in
agreement with shelf life of the food they packed. To study the effect of aging in the
WG-film antimicrobial properties, activity of the bacteriocins in the wheat gluten film
with elapsed time (50 days at 23ºC) was assayed. A slight decrease in anti-Listeria
activity (latocin AL705) was observed by the end of the storage period (Figure 3); while
for lactocin 705, activity decrease was more pronounced. Nevertheless, antimicrobial
activity of the wheat gluten film was not completely lost during storage (Figure 3). The
polymer matrix is the internal network with free space in which the antimicrobials are
entrapped. The distribution and compactness of the polymer molecules, as well as
interactions between antimicrobials and polymer determine the movement of these
additives within the matrix (Balasubramanian 2012). Electrostatic interactions between
the cationic nisin and the anionic stearic fatty acid, added to a HPMC film was found to
decrease bacteriocin desorption from the film leading to a decrease in antimicrobial
activity (Sebti & Coma 2002). Nevertheless, when HPMC films were made at acid
conditions, nisin was released from the films since protonated species from nisin and
stearic acid were favored (Sebti et al 2002). Chemical crosslinking in a HPMC matrix
decreased antimicrobial activity of nisin in the polymer (Sebti et al 2003). In our work,
even when mechanical properties variations could suggest that WG-film suffered from
aging (crosslinking), no correlation between antimicrobial activity behavior (Figure 3)
and structural changes (Figure 2) was observed. Contrary, to our results on lactocin 705
and AL705 antimicrobial activity from the WG-film, are similar to that obtained for the
bacteriocins adsorbed on a PE-based film; in which case film activity was related to the
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bacteriocin stability over the time, rather than to an interaction with the film matrix, and
more stability of AL705 than 705 in the film was found (Blanco Massani et al 2012).
Antimicrobial stability was earlier reported for bacteriocins immobilized in different
bio-based matrices. Activity retention of cellulose based inserts showed an initial
decrease in the first week of storage but remained stable for the remaining 3 months of
the trial, while a cellulose based coating conserved its activity during 12 weeks both at
room temperature and under refrigeration (Scannell et al 2000, Neetoo et al 2007).
Other factor to be considered in active packaging food application is the migration of
the antimicrobial substances from the film, as well as its antimicrobial activity
performance on the presence of food simulants. The release kinetics of antimicrobial
agents has to be designed to maintain the concentration above the critical inhibitory
concentration with respect to the microorganisms’ growth kinetic. Foods have different
chemical and biological characteristics; they provide different environmental conditions
to microorganisms and included antimicrobial agents (Han 2000). The influence of
hydrophobic (sunflower oil) and hydrophilic (water) simulants media on antimicrobial
activity of the wheat gluten film; as well as the residual activity in the media after
contact with the film was periodically assayed. Results of these experiments are shown
in Figure 4 and Table 1. Wheat gluten films residual activity after water contact was
observed in each day of the experiment as shown in Figure 4 b and e (day 50 of
storage). Lactocin 705 activity in water after active WG-film contact was observed at
the beginning of the experiment, while for lactocin AL705, activity was also detected in
water contacted with films that were aged for 15 days (Table 1). Antimicrobial release
from polymers is influenced by several factors; (i) active component size, (ii)
antimicrobial and polymer matrix compatibility, (iii) polymer matrix characteristics, (iv)
processing method, (v) food composition, (vi) storage conditions. Controlled release of
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antimicrobials is feasible only if the antimicrobials are physically entrapped within the
polymer matrix rather than being chemically bound (Balasubramanian 2012). In our
work, even when lactocin 705 and AL705 were delivered from the active WG-film after
water contact, bacteriocins activity was still retained inside the film matrix (Figure 4 b
and e). Even when WG-films did not age as dramatically as other WG films obtained in
acid conditions (Olabarrieta, Cho et al 2006; Olabarrieta, Gällstedt et al 2006), film
cross-linking during the elapsed time (Micard et al 2000; Morel et al 2000; Irissin-
Mangata et al 2001) could lead to the observed bacteriocins entrapment within the WG-
matrix and loss in film bacteriocins release upon water contact after 15 days of film
storage. After active film contact with sunflower oil (Figure 4 c and f, Table 1), the film
exerted antimicrobial activity of lactocin AL705 only (Figure 4 f) and no activity in the
hydrophobic media was detected for each assayed storage time (Table 1). L. curvatus
CRL705 bacteriocins lack of activity in sunflower oil could be related to their
insolubility in that media, while inactivation of lactocin 705 in the WG-film after
contact is in coincidence with the results found for this bacteriocin adsorbed in a PE-
based film, which is related to its mode of action and interaction with hydrophobic
compounds (Castellano et al 2007; Blanco Massani et al 2012; Blanco Massani et al
2013). An active compound which is immobilized into polymeric materials could act
directly from the film without being released into the packaged foodstuff (Conte et al
2007). Protein packaging films can act as a reservoir and gradually release antimicrobial
agents to maintain a constant microbial inhibitory effect (Dawson et al 2003). In our
work, changes in properties observed after WG-film aging favored activity retention
inside the film without a significant change in antimicrobial activity, thereby allowing
the active agent to act at a food surface level. Furthermore, the efficacy of the
bacteriocin activity could be improved by control of migration of the bacteriocin into
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the packaged media, enabling its antimicrobial effect to be preserved beyond consumer
purchase (Sobrino-López & Martín-Belloso 2008). According to our results, it should
be expected that in the time scale of vacuum packaged cooked sausages (aproximately
30-40 days) (Korkeala & Björkroth, 1997), bacteriocins activity in the film and the
intimate contact provided by vacuum, give an efficient anti-microbial effect over these
products.
The positive impact on antimicrobial activity properties with gluten aging demonstrated
in this work, could help to understand the film performance applied in more complex
real cases (RTE meat food products), which is part of current studies.

Conclusions
A WG-film obtained at pilot scale demonstrated effectiveness as an antimicrobial
support for L. curvatus CRL705 bacteriocins. Mechanical properties as well as aging
kinectics were not significantly affected by bacteriocins addition. Promising
antimicrobial release properties were observed in contact with substances commonly
used as food simulants for cooked meat products (sunflower oil and water). Moreover,
anti-Listeria activity was retained in the film even after contact with simulants. Changes
in the properties observed after WG-film aging (50 days) favored activity retention
inside the film without a significant change in antimicrobial activity, this time period
being cosistent with the shelf life of some vacuum packaged cooked sausages. The
results suggest that a WG-film could be used as lactocin 705 and AL705 carrier to avoid
contamination in RTE meat products such as cooked sausages.



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Acknowledgments
This study was supported by funds from 3iA-UNSAM. Subsidio para proyectos de
investigación UNSAM-2006, Argentina. The authors would like to acknowledge to the
microbiological laboratory from INTI-Carnes.

References
Albert S, Mittal GS. 2002. Comparative evaluation of edible coatings to reduce fat
uptake in a deep-fried cereal product. Food Res Int. 35: 445–458.
Angellier-Coussy H, Gastaldi E, Gontard N, Guillard V. 2011. Influence of
processing temperature on the water vapour transport properties of wheat gluten based
agromaterials. Ind Crop Prod 33: 457–461.
ASTM E96 / E96M – 12.2012. Standard Test Methods for Water Vapor
Transmission of Materials. Philadelphia, PA: American Society for Testing and
Materials.
ASTM D 638-10. 2010. Standard test method for tensile properties of plastics.
Philadelphia, PA: American Society for Testing and Materials.
Balasubramanian A. 2012. Predicting target release profile of antimicrobials from
controlled release packaging. Available in:
http://mss3.libraries.rutgers.edu/dlr/showfed.php?pid=rutgers-lib:37363
Bastarrachea L, Dhawan S, Sablani SS. 2011. Engineering Properties of Polymeric-
Based Antimicrobial Films for Food Packaging. Food Eng Rev 3:79–93.
Marcos B, Aymerich T, Monfort JM, & Garriga M. 2007. Use of antimicrobial
biodegradable packaging to control Listeria monocytogenes during storage of cooked
ham. Int J Food Microbiol. 120: 152–158.
Page 16 of 30
http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk
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1
2
3
4
5
6
7
8
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13
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15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

For Peer Review Onl
y
Blanco Massani M, Fernandez M R, Ariosti A, Eisenberg P, Vignolo G. 2008.
Development and characterization of an active polyethylene film containing
Lactobacillus curvatus CRL705 bacteriocins. Food Addit Contam. 25: 1424-1430.
Blanco Massani M, Morando P, Vignolo G, Eisenberg P. 2012. Characterization of
a multilayer film activated with Lactobacillus curvatus CRL705 bacteriocins. J Sci
Food Agric. 92: 1318-1323.
Blanco Massani M, Vignolo G, Eisenberg P, Morando J. 2013. Adsorption of the
bacteriocins produced by Lactobacillus curvatus CRL705 on a multilayer- LLDPE film
for food-packaging applications, LWT - Food Sci Technol. 53:128-138.
Botana A., Mollo M, Eisenberg P, Torres Sanchez RM. 2010. Effect of modified
montmorillonite on biodegradable PHB nanocomposites. Appl Clay Sci. 47: 263–270.
Bourtoom T. (2008). Edible films and coatings: characteristics and properties. Int
Food Res J. 15: 237-248.
Castellano P, Vignolo G. 2006. Inhibition of Listeria innocua and Brochothrix
thermosphacta in vacuumpacked meat by addition of bacteriocinogenic Lactobacillus
curvatus CRL705 and its bacteriocins. Lett Appl Microbiol. 43: 194-199.
Castellano P, Vignolo G, Farías RN, Arrondo JL, Cheín R. 2007. Molecular view
by Fourier transform infrared spectroscopy of the relationship between lactocin 705 and
membranes: speculations on antimicrobial mechanism. Appl Environ Microbiol.
73:415-420.
Cagri A, Ustunol Z, Ryser ET. 2004. Antimicrobial edible films and coatings. J
Food Protect. 67: 833-848.
Castellano, P.H. (2005) [Tesis doctoral]. Bacteriocinas de bacterias lacticas como
biconservadores en carne envasada de atmosfera modificadas. Estrategias de aplicación.
Page 17 of 30
http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk
Food Additives and Contaminants
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

For Peer Review Onl
y
San Miguel de Tucumán: Universidad Nacional de Tucumán, Facultad de Bioquímica,
Química y Farmacia.
Cha DS, Chinnan MS. 2004. Biopolymer-based antimicrobial packaging: a review.
Crit Rev Food Sci Nutr. 44: 223-237.
Cho SY, Lee DS, Han JH. 2009. The Wiley encyclopedia of packaging technology.
In: Kit L.Yam, editors, Hoboken, NJ, USA: John Wiley & Sons, Inc. Antimicrobial
packaging, p. 50-59.
Coma V. 2008. Bioactive packaging technologies for extended shelf life of meat-
based products. Meat Sci. 78: 90-103.
Conte A, Buonocore GG, Sinigaglia M, Del Nobile MA. 2007. Development of
immobilized lysozyme based active film. J Food Eng. 78: 741–745.
Cooksey K. 2005. Effectiveness of antimicrobial food packaging materials. Food
Addit Contam. 22: 980–987.
Dawson PL, Hirt DE, Rieck JR, Acton JC, Sotthibandhu A. 2003. Nisin release
from films is affected by both protein type and film-forming method. Food Res Int. 36:
959–968.
De Jong AR, Boumans H, Slaghek T, Van Veen J, Rijk R, Van Zandvoort M. 2005.
Active and intelligent packaging for food: Is it the future? Food Addit Contam. 22: 975–
979.
Denavi G, Tapia-Blácido DR, Añón MC, Sobral PJA, Mauri AN, Menegalli FC.
2009. Effects of drying conditions on some physical properties of soy protein films. J
Food Eng. 90: 341–349.
Page 18 of 30
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Food Additives and Contaminants
1
2
3
4
5
6
7
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

For Peer Review Onl
y
Domenek S, Feuilloley P, Gratraud J, Morel M-H, Guilbert S. 2004.
Biodegradability of wheat gluten based bioplastics. Chemosphere. 54: 551–559.
EC, 2004, COMMISSION REGULATION 10/2011 of 27 October 2004 relating to
plastic materials and articles intended to come into contact with food. Off J the Eur
Commun. L338: 4-17.
Gennadios A, Brandenburg AH, Weller CL, Testin RF. 1993. Effect of pH on
properties of wheat gluten and soy protein isolate films. J Agric Food Chem. 41: 1835-
1939.
Gennadios A, Weller CL, Testin RF. 1993. Modification of physical and barrier
properties of edible wheat gluten- based films. Cereal Chem. 70: 426-429.
Ghanbarzadeh B, Oromiehi AR. 2009. Thermal and mechanical behavior of
laminated protein films. J Food Eng. 90: 517–524.
GMC/RES. 32/07 Lista positiva de aditivos para materiales plásticos destinados a
la elaboración de envases y equipamientos en contacto con alimentos. 1-72.
Grower JL, Cooksey K, Getty K. 2004. Release of nisin from methylcellulose-
hydroxypropyl metilcellulose film formed on Low-density Polyethylene film. J Food
Sci. 69: 107–111.
Guiga W, Swesi Y, Galland S, Peyrol E, Degraeve P, Sebti I. 2010. Innovative
multilayer antimicrobial films made with Nisaplin® or nisin and cellulosic ethers:
Physico-chemical characterization, bioactivity and nisin desorption kinetics. Innov Food
Sci Emerg Technol. 11: 352–360.
Guilbert S, Cuq B. 2005. Handbook of Biodegradable Polymers. In: Catia Bastioli,
editors. Shropshire, UK: Rapra Technology Limited. Chapter 11, Material Formed from
Proteins; p 339-384.
Page 19 of 30
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Food Additives and Contaminants
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2
3
4
5
6
7
8
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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29
30
31
32
33
34
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

For Peer Review Onl
y
Han JH. 2000. Antimicrobial food packaging. Food Technol. 54: 56–65.
Herald TJ, Gnanasambandam R, McGuire BH, Hachmeister. 1995. Degradable
Wheat Gluten Films: Preparation, Properties and Applications. J Food Sci. 60: 1147-
1150.
Hereu A, Dalgaard P, Garriga M, Aymerich T, Bover-Cid S. 2012. Modeling the
high pressure inactivation kinetics of Listeria monocytogenes on RTE cooked meat
products. Innov Food Sci Emerg Technol. 16: 305–315.
Hochstettera A, Taljaa RA, Heléna HJ, Hyvönena L, Jouppila K. 2006. Properties
of gluten-based sheet produced by twin-screw extruder. LWT - Food Sci Technol. 39:
893-901.
Ibarguren C, Vivas L, Bertuzzi M A, Apella MC, Audisio MC. 2010. Edible films
with anti-Listeria monocytogenes activity. Internat J Food Sci Technol. 45: 1443–1449.
Imam S., Glenn G, Chiou BS, Shey J, Narayan R, Orts W. 2008. Environmentally
compatible food packaging. In: E. Chiellini, editors. Boca Raton Boston, NY, USA:
CRC Press. Chapter 2, Types, production and assessment of biobased food packaging
materials; p 29-62.
Irissin-Mangata J, Bauduin G, Boutevin B, Gontard N. 2001. New plasticizers for
wheat gluten films. European Polym J. 37: 1533–1541.
Iturriaga L, Olabarrieta I, Martínez de Marañón I. 2012. Antimicrobial assays of
natural extracts and their inhibitory effect against Listeria innocua and fish spoilage
bacteria, after incorporation into biopolymer edible films. Int J Food Microbiol. 158:58–
64.
Page 20 of 30
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7
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

For Peer Review Onl
y
Juneja VK, Thippareddi H, Bari L, Inatsu Y, Kawamoto S, Friedman M. 2006.
Chitosan Protects Cooked Ground Beef and Turkey Against Clostridium perfringens
Spores During Chilling. J Food Sci. 71: 236-240.
Kraśniewska K, Małgorzata G. 2012. Substances with Antibacterial Activity in
Edible Films – A Review. Pol J Food Nutr Sci. 62: 199-206.
Ko S, Janes ME, Hettiarachchy NS, Johnson MG. 2001. Physical and Chemical
Properties of Edible Films Containing Nisin and Their Action Against Listeria
Monocytogenes. J Food Sci. 66:1006-1011.
Koontz JL, Moffitt RD, Marcy JE, O’Keefe SF, Duncan SE, Long TE. 2009.
Controlled release of α-tocopherol, quercetin, and their cyclodextrin inclusion
complexes from linear low-density polyethylene (LLDPE) films into a coconut oil
model food system. Food Additives and ContaminantsVol. 27, No. 11, November 2010,
1598–1607
Lee D S. 2008. Food Packaging and Shelf Life a Practical Guide. In: Gordon L.
Robertson, editor. Boca Raton, FL, USA: CRC Press. Chapter 4, Packaging and the
Microbial Shelf Life of Food; p. 55-80.
Luchansky JB, Call JE. 2004. Evaluation of nisin-coated cellulose casings for the
control of Listeria monocytogenes inoculated onto the surface of commercially prepared
frankfurters. J Food Protect. 67: 1017-1021.
Lungu B, Johnson GM. 2005. Potassium sorbate does not increase control of
Listeria monocytogenes when added to zein coatings with nisin on the surface of full fat
turkey frankfurter pieces in a model system at 4ºC. J Food Sci. 70: 95-99.
Page 21 of 30
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Food Additives and Contaminants
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16
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18
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23
24
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51
52
53
54
55
56
57
58
59
60

For Peer Review Onl
y
Marcuzzo E, Peressini D, Debeaufort F, Sensidoni A. 2010. Effect of ultrasound
treatment on properties of gluten-based film. Innov Food Sci Emerg Technol. 11: 451–
457.
McCormick KE, Han IY, Acton JC, Sheldon BW, Dawson PL. 2005. In-package
Pasteurization Combined with Biocideimpregnated Films to Inhibit Listeria
monocytogenes and Salmonella Typhimurium in Turkey Bologna. J Food Sci. 70: 52-57.
Micard V, Belamri R, Morel M-H, Guilbert S. 2000. Properties of Chemically and
Physically Treated Wheat Gluten Films. J Agric Food Chem. 48: 2948-2953.
Morel MH, Bonicel J, Micard V, Guilbert S. 2000. Protein insolubilization and
thiol oxidation in sulfite-treated wheat gluten films during aging at various temperatures
and relative humidities. J Agric Food Chem. 48: 186–192.
Neetoo H, Ye M, Chen H. 2007. Effectiveness and stability of plastic films coated
with nisin for inhibition of Listeria monocytogenes. J Food Protect. 70: 1267–1271.
Olabarrieta I, Cho SW, Gällstedt M, Sarasua JR, Johansson E, Hedenqvist MS.
2006. Aging properties of films of plasticized vital wheat gluten cast from acidic and
basic solutions. Biomacromoleules. 7: 1657-1664.
Olabarrieta I, Gällstedt M, Ispizua I, Sarasua J-R, Hedenqvist MS. 2006. Properties
of Aged Montmorillonite-Wheat Gluten Composite Films. J Agric Food Chem. 54:
1283-1288.
Ou S, Wang Y, Tang S, Huang C, Jackson, MG. 2005. Role of ferulic acid in
preparing edible films from soy protein isolate. J Food Eng. 70: 205–210.
Palacios, J.M. (2000) [Tesis doctoral]. San Miguel de Tucumán: Universidad
Nacional deTucumán, Facultad de Bioquímica, Químíca y Farmacia, p 44-45.
Page 22 of 30
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43
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46
47
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49
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For Peer Review Onl
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Pllana M, Gjergjizi H, Miftari I, Gjonbalaj M. 2012. The Food Safety and
Consumer Behavior. J Food Sci Eng. 2: 462-468.
Pongtharangkul T, Demirci A. 2004. Evaluation of agar diffusion bioassay for nisin
quantification. Appl Microbiol and Biotechnol. 65: 268–272.
Restuccia D, Spizzirri GU, Parisi OI, Cirillo G, Curcio M, Iemma F, Puoci F, Vinci
G, Picci N. 2010. New EU regulation aspects and global market of active and intelligent
packaging for food industry applications. Food Control. 21: 1425–1435
Robertson G. (2008). Environmentally compatible food packaging. In: E. Chiellini,
editor. Boca Raton, FL, USA: CRC Press. Chapter 1, State-of-the-art biobased food
packaging materials; p 3-28.
Scannell AGM, Hill C, Ross RP, Marx S, Hartmeier W, Arendt KE. 2000.
Development of bioactive food packaging materials using immobilised bacteriocins
lacticin 3147 and Nisaplin ®. Internat J Food Microbiol. 60: 241-249.
Sebti I, Coma V. 2002. Active edible polysaccharide coating and interactions
between solution coating compounds. Carbohydr Polym. 49: 139-144.
Sebti I, Ham-Pichavant F, Coma V. 2002. Edible Bioactive Fatty Acid-Cellulosic
Derivative Composites Used in Food-Packaging Applications. J Agric Food Chem. 50:
4290-4294.
Sebti I, Delves-Broughton J, Coma V. 2003. Physicochemical Properties and
Bioactivity of Nisin-Containing Cross-Linked Hydroxypropylmethylcellulose Films. J
Agric Food Chem. 51: 6468-6474.
Sian NK, Ishak S. 1990. Effect of pH on formation, proximate composition and
rehydration capacity of winged bean and soybean protein-lipid film. J Food Sci. 55:
261–262.
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50
51
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Sobrino-López A, Martín-Belloso O. 2008. Use of nisin and other bacteriocins for
preservation of dairy products. Int Dairy J. 18: 329–343.
Song Y-h, Zheng Q, Lai Z-z. 2008. Properties of thermo-molded
gluten/glycerol/silica composites. Chinese J Polym Sci. 26: 631−638.
Thippareddi, H. 2012. J Food Sci Eng. 2: 473.
Tanada-Palmu PS, Grosso CRF. 2005. Effect of edible wheat gluten-based films
and coatings on refrigerated strawberry (Fragaria ananassa) quality. Postharvest Biol
Tech. 36: 199–208
Vermeiren L, Devlieghere F, Debevere J. 2002. Efectiveness of some recent
antimicrobial packaging concepts. Food Addit Contam. 19: 163-171.
Vignolo G, Suriani F, Ruiz Holgado AP, Oliver G. 1993. Antibacterial activity of
Lactobacillus strains isolated from dry fermented sausages. J Appl Bacteriol. 75: 344-
349.
Wan VCh-H, Kim MS, Lee S-Y. 2005. Water vapor permeability and mechanical
properties of soy protein isolate edible films composed of different plasticizer
combinations. J Food Sci. 70: 387-391.
Williams SK, Roof S, Boyle EA, Burson D, Thippareddi H, Geornaras I, Sofos
JN,Wiedmann M, Nightingale K. 2011. Molecular ecology of Listeria monocytogenes
and other Listeria species in small and very small ready-to-eat meat processing plants. J
Food Protect. 74: 63-77.
Wu LC, Bates RP. 1972. Soy protein-lipid films. 2. Optimization of film formation.
J Food Sci. 37: 40-4.
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Xie L, Hettiarachchy NS, Ju ZY, Meullenet J, Wang H, Slavik MF, Janes ME.
2002. Edible Film Coating to Minimize Eggshell Breakage and Reduce Post-Wash
Bacterial Contamination Measured by Dye Penetration in Eggs, J Food Sci. 67: 280-
284.
Zhang X, Burgar I, Do MD, Lourbakos E. 2005. Intermolecular Interactions and
Phase Structures of Plasticized Wheat Proteins Materials. Biomacromolecules. 6: 1661-
1671.
Zhang Y, Han JH. 2006. Plasticization of pea starch films with monosaccharides
and polyols. J Food Sci. 71: 253-261.
Zhang X, Do MD, Kurniawan L, Qiao GG. 2010. Wheat gluten-based renewable
and biodegradable polymer materials with enhanced hydrophobicity by using
epoxidized soybean oil as a modifier. Carbohydrate Res. 345: 2174–2182.
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Figure legends
Figure 1 Lactocin 705 (a) and lactocin AL705 (b) antimicrobial activity on WG-films
doped with BCE (0.01, 0.1 and 1%). Control film had no antimicrobials in its
formulation.
Figure 2 Mechanical properties, of the active WG-film (▼) and a control without
bacteriocins (●) as a function of storage time. Lines illustrate trends.
Figure 3 Lactocin 705(●) and AL705 (○) antimicrobial activity on the WG-film as a
function of storage time. Lines illustrate trends.
Figure 4 Lactocin 705 and AL705 activity of the control (a), (d), and active WG-films
(b), (e) after water contact, and sunflower oil (c), (f) contact. C+ is the inhibition area
exerted by a spot of BCE.
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Lactocin 705 (a) and lactocin AL705 (b) antimicrobial activity on WG-films doped with BCE (0.01, 0.1 and
1%). Control film had no antimicrobials in its formulation.
118x54mm (96 x 96 DPI)


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Figure 2 Mechanical properties, of the active WG-film (▼) and a control without bacteriocins (●) as a
function of storage time. Lines illustrate trends.
46x21mm (600 x 600 DPI)


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Figure 3 Lactocin 705(●) and AL705 (○) antimicrobial activity on the WG-film as a function of storage time.
Lines illustrate trends.
43x37mm (600 x 600 DPI)


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Figure 4 Lactocin 705 and AL705 activity of the control (a), (d), and active WG-films (b), (e) after water
contact, and sunflower oil (c), (f) contact. C+ is the inhibition area exerted by a spot of BCE.
254x127mm (96 x 96 DPI)


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Table 1. Residual antimicrobial activity in water and sunflower oil after WG-film direct
contact.
Storage time
(days)
Activity in the media (AU cm-3)
Water Sunflower oil
0 19* 122** ND* ND**
15 ND 32 ND ND
30 ND ND ND ND
44 ND ND ND ND
50 ND ND ND ND
ND: not detected.
* Lactocin 705 activity
** Lactocin AL705 activity


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