Active polymers containing Lactobacillus curvatus CRL705
bacteriocins: Effectiveness assessment in Wieners

M. Blanco Massania, , ,V. Molinaa, M. Sancheza, V. Renauda, b, P. Eisenberga, b, G. Vignoloc
a INTI Gral Paz 5445. Buenos Aires, Argentina
b 3iA-UNSAM, Argentina
c Centro de Referencia para Lactobacilos (CERELA), CONICET, Tucumán, Argentina

International Journal of Food Microbiology
Volume 178, 16 May 2014, Pages 7–12

Received 30 October 2013, Revised 29 January 2014, Accepted 15 February 2014, Available online 22
February 2014
https://doi.org/10.1016/j.ijfoodmicro.2014.02.013


© <2017>. This manuscript version is made available under the CC-BY-NC-ND
4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/

Highlights

Films used as L. curvatus CRL705 bacteriocins carriers showed antimicrobial activity
Anti-Listeria activity was observed in active inoculated wieners packets (45 days)
Inoculated Lactobacillus was slightly inhibited during two weeks of wieners storage
PH decrease and gas formation were observed in Lactobacillus inoculated packets
Wieners fat content reduced packaging effectiveness against lactic acid bacteria

*Highlights (for review)

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Active polymers containing Lactobacillus curvatus CRL705 bacteriocins: 1
effectiveness assessment in Wieners 2
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Blanco Massani M.1, Molina V. 1, Sanchez M1, Renaud V1,2, Eisenberg P1,2 and 4
Vignolo G3. 5
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1INTI Gral Paz 5445. Buenos Aires, Argentina; 23iA-UNSAM, Argentina; 3Centro de 7
Referencia para Lactobacilos (CERELA), CONICET, Tucumán, Argentina. 8
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*Corresponding author: M. Blanco Massani, E-mail: blanco@inti.gob.ar 18
Telephone: (54 11) 4724 6200 Int: 6636 19
Fax: (54 11) 4753 5773 20
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*Manuscript with Line Numbers
Click here to view linked References

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Abstract 26
Bacteriocins from lactic acid bacteria have potential as natural food preservatives. In 27
this study two active (synthetic and gluten) films were obtained by the incorporation of 28
lactocin 705 and lactocin AL705, bacteriocins produced by Lactobacillus curvatus 29
CRL705 with antimicrobial activity against spoilage lactic acid bacteria and Listeria. 30
Antimicrobial films effectiveness was determined in wieners inoculated with 31
Lactobacillus plantarum CRL691 and Listeria innocua 7 (104 CFU/g) stored at 5ºC 32
during 45 days. Active and control (absence of bacteriocins) packages were prepared 33
and bacterial counts in selective media were carried out. Visual inspection and pH 34
measurement of wieners were also performed. Typical growth of both inoculated 35
microorganisms was observed in control packages which reached 106-107 CFU/g at the 36
end of storage period. In the active packages, L. innocua 7 was effectively inhibited (2.5 37
log cycles reduction at day 45), while L. plantarum CRL691 was only slightly inhibited 38
(0.5 log cycles) up to the second week of storage, then counts around 106-107 CFU/g 39
were reached. Changes in pH values from 6.3 to 5.8 were produced and gas formation 40
was observed in active and control packages. The low inhibitory effectiveness against 41
lactic acid bacteria is in correlation with the low activity observed for lactocin 705 in 42
the presence of fat; wieners fat content (20-30%) may adversely affect antimicrobial 43
activity. This study supports the feasibility of using polymers activated with L. curvatus 44
CRL705 bacteriocins to control Listeria on the surface of wieners and highlights the 45
importance of evaluating antimicrobial packaging systems for each particular food 46
application. 47
48
Keywords: antimicrobial food packaging; bacteriocins; anti-Listeria; wieners; lactic 49
acid bacteria. 50

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51
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1. Introduction 53
Although food biotechnology has recently made important progresses, food 54
industry and particularly meat industry is still under scrutiny by consumers due to 55
sanitary episodes generated by meat and meat based products (Bremer et al., 2005; 56
CDC, 2007). Modern life conditions related to or as consequence of globalization, 57
contribute to the major incidence of food diseases outbreaks. The major challenges for 58
food safety are the emergent pathogens, among which L. monocytogenes is included 59
(Vignolo et al., 2008; Vignolo et al., 2012). During food chain distribution, food needs 60
to be protected from physical, chemical and microbiological spoilage. The shelf life of 61
food is controlled by (i) the product characteristics including formulation and 62
processing parameters (intrinsic factors), (ii) the package properties and (iii) the 63
environment to which the products are exposed during distribution and storage 64
(extrinsic factors). Among intrinsic factors, pH, water activity, enzymes, 65
microorganisms, and concentration of reactive compounds are included. Many of these 66
factors can be controlled by selection of raw materials and ingredients, as well as the 67
choice of processing parameters. However, extrinsic factors namely temperature, 68
relative humidity, light, total and partial pressure of different gases as well as 69
mechanical stresses including consumer handling may affect the rates of deteriorative 70
reactions occurring during the shelf life of food. The properties of package can exert a 71
significant effect on many of the extrinsic factors and thus indirectly on the rates of 72
deteriorative reactions (Robertson, 2006). Interaction of the packaged food with its 73
packaging and the external environment may also change intrinsic food factors, package 74
headspace acting as a buffer between food and packaging material. Due to these 75

4

interactions, moisture content (i.e., water activity), dissolved O2 and CO2 contents, and 76
preservative concentration can be modified to affect the microbiota and its growth rate 77
(Lee, 2010). 78
Muscle tissue from healthy animals is free of bacterial or viral pathogens. As 79
with spoilage organisms, pathogens are deposited on meat surfaces during processing 80
and handling of meat carcass. Potential sources of pathogen contamination comprise 81
animal-associated pathogens transferred to meat from the hide, skin, or feathers and the 82
intestinal tract of the animal during carcass processing; human-associated pathogens 83
transferred from personnel during handling of product, processing equipment and tools, 84
which if inadequately cleaned and sanitized may act not only as vehicles for pathogen 85
but also as sources of contamination (Gill and Gill, 2010). Most perishable foods are 86
vulnerable to microbial spoilage even under chilled conditions. Their shelf life is thus, 87
for the most part, terminated when they become unacceptable due to the growth of 88
undesirable microorganisms (Lee, 2010). Within a certain range of environmental 89
conditions, often only one member from the total microflora is responsible for spoilage 90
(specific spoilage organisms—SSO); for cooked meat products, lactic acid bacteria 91
were found as the prevalent spoilage microorganisms (Mataragas et al., 2006; 92
Audenaert, et al., 2010; Chenoll, et al., 2007). For shelf life studies, after determining 93
the SSO and the conditions under which this group of microorganisms is responsible for 94
food spoilage, the next step is to determine the number of SSO responsible for food 95
deterioration producing lack of acceptability (Dalgaard, 1995; Koutsoumanis and 96
Nychas, 2000). The acceptable limit of microbial growth that determines the shelf life 97
differs with food type and storage conditions. SSO counts of 105–108 bacteria per g1 or 98
cm2 are commonly used as a convenient upper limit of quality and are reached mostly 99
during microorganism growth exponential phase (Lee, 2010). Combined intrinsic 100

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factors are used to preserve food safety and ensure organoleptic quality, i.e. suitable 101
food shelf life can be obtained by incorporating low levels of additives, mild 102
dehydration and heat processes (Leistner and Gould, 2002). Several technologies can be 103
combined in order to improve food safety and extend shelf life of foods (Rybka-104
Rodgers, 2001). During the last years, a number of biopreservation technologies has 105
been developed by the inclusion of antimicrobial extracts, lysozyme, bacteria and/or 106
bacteriocins among others, into polymer matrices (Marcos et al., 2008; Gialamas et al., 107
2010; Ramos et al., 2012; López de Dicastillo et al., 2013; Arcan and Yemenicioğlu, 108
2013). Nevertheless for bacteriocins biopreservation hurdles, it was found that 109
antimicrobial effect could be affected by food components (Zapico et al., 1999; Aasen 110
et al., 2003; Bhatti et al., 2004). Lactocin 705 and lactocin AL705, are bacteriocins 111
produced by Lactobacillus curvatus CRL705. Lactocin 705 has antagonist effect against 112
Lactic acid bacteria (LAB) and Brochothix thermosphacta , while AL705 is active 113
against Listeria species (Castellano and Vignolo, 2006). Both bacteriocins retained its 114
antimicrobial activity when included in polymer matrices such as LDPE (Blanco 115
Massani et al., 2008, 2012) and gluten (Blanco Massani et al., in press article). In the 116
present study, active LDPE and gluten films obtained by L. curvatus CRL705 117
bacteriocins incorporation were evaluated for antimicrobial effectiveness in 118
contaminated Wieners. 119
120
2. Materials and Methods 121
2.1. Bacterial strains and growth conditions 122
Lactobacillus curvatus CRL705 (producer of the bacteriocins lactocin 705 and lactocin 123
AL705) and Lactobacillus plantarum CRL691 (which is sensitive to the activity of 124
lactocin 705) from CERELA culture collection, were grown in MRS broth (Britania, 125

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Argentina) for 16 h at 30ºC. Listeria innocua 7 (sensitive to the activity of lactocin 126
AL705) obtained from the Unité de Recherches Laitières et Génétique Appliquée, 127
INRA (France) was grown in trypticase soy broth (Britania) with 5 mg/cm3 of yeast 128
extract added (Britania, Argentina) for 16 h at 30 ºC. All strains were maintained and 129
stored at −20 ºC in 0.15 g/cm3 of glycerol until use. 130
131
2.2. Wieners elaboration 132
Wieners were manufactured in a meat processing pilot plant, according to standard 133
procedure (C.A.A.). Beef and pork meat cuts were minced (Themis 32 mincer) and 134
processed together with fat in a vertical cutter (Robot Coupe). Ice (0.11 g/cm3), sodium 135
phosphate (3 mg/cm3), sodium erithorbate (0.5 mg/cm3), sodium chloride (0.017 g/cm3) 136
and sodium nitrite (0. 15 mg/cm3) were added and mixed to obtain a homogeneous mass. 137
Finally, starch and water (0.11 g/cm3) were added to form an emulsion which was filled 138
(Hidraulic filler, RISCO IV 20) into artificial casings (2 cm diameter). Wieners (5 cm, 14 139
g) were cooked in an oven (Lavaflux) at 80°C for 15 min, cooled in an ice bath to a core 140
temperature below 40°C and refrigerated at 3°C until manual peeled. After that re-141
pasteurization of vacuum-packed wieners was performed (10 min, 80°C). 142
143
2.3. Active solution preparation and quantification 144
A powder containing lactocin 705 and lactocin AL705 from L. curvatus CRL705 was 145
obtained by ammonium sulfate precipitation as earlier reported (Blanco Massani et al., 146
2008). For activity determination the active powder was resuspended in water and the 147
agar well diffusion assay against L. plantarum CRL691 (lactocin 705 sensitive 148
organism) and L. innocua 7 (lactocin AL705 sensitive organism) was performed 149
(Blanco Massani et al., 2012). Antimicrobial activity was expressed as AU/cm3. 150

7

151
2.4. Active films preparation and antimicrobial activity determination 152
Two types of active films were prepared. Synthetic: Multilayer films kindly provided by 153
Cryovac (Sealed Air, Argentina) and commercially used as bottom and top of wiener 154
packages, were contacted (1 h, 30°C) with the active solution containing L. curvatus 155
CRL705 bacteriocins (1 mg/cm3, 267 AU/cm3 and 2133 AU/cm3 for lactocin 705 and 156
lactocin AL705, respectively) (Blanco Massani et al., 2012). Agro-protein polymer: Wheat 157
gluten (0.779 g of protein, 0.133 g starch and 0.01 g lipids, per gram of gluten on dry 158
weight base) kindly supplied by Molinos Juan Semino S.A. (Carcarañá, Santa Fe) was 159
stirred with sodium sulfite (Merck, Germany), glycerol (Cicarelli, Argentina) and ethanol 160
96% (Merck, Germany) using a mechanical stirrer (Heidolph RZR 2041). After a 161
homogeneous solution was attained, water and the L. curvatus CRL705 bacteriocins (1 162
mg/cm3, 267 AU/cm3 and 2133 AU/cm3 for lactocin 705 and lactocin AL705, respectively) 163
solution were added, and the pH was adjusted to 5.0 with acetic acid (Sintorgan, 164
Argentina). The film forming solution was spread onto a continuous Teflon® tape and 165
dried in a warm tunnel with forced air at 50ºC for 4 h (Blanco Massani et al., in press 166
article). Negative controls consisted on either synthetic or wheat gluten films in which 167
active bacteriocins solution was replaced by water. Films were sterilized by UV exposition 168
during 10 min and aseptically stored until use. Antimicrobial activity of the activated and 169
control (without bacteriocins) films was determined by directly placing on the semisolid 170
agar plates seeded with the sensitive organisms (MRS agar plates seeded with L. plantarum 171
CRL691 for lactocin 705 and trypticase soy agar + yeast extract seeded with L. innocua 7 172
for lactocin AL705 activity determinations). Antimicrobial activity was evidenced as an 173
inhibition zone of the indicator organisms beneath and around the films. 174
175

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2.5. Active packaging preparation and wieners inoculation 176
For synthetic packaging, each pair of bacteriocins treated Cryovac films (bottom and 177
top of wiener package) (96 cm2) was thermo-sealed in a sterile cabinet (Biosafety 178
cabinet Labcono, purifier class II), whereas active gluten film was included as a pad (48 179
cm2) inside packaging made with untreated Cryovac films (96 cm2). Control packaging 180
(without bacteriocins) were also prepared. All sets were aseptically stored at 5ºC until 181
use. 182
Wieners were separately inoculated under sterile conditions by immersion (30 s) in a 183
solution containing L. innocua 7 (104 CFU/g) and L. plantarum CRL691 (104 CFU/g). 184
After drying, three wieners (42 g) were placed into each active and control packaging 185
previously prepared. In parallel, control (without bacteriocins) uninoculated wieners 186
packages were included. All packages were thermo-sealed under vacuum (90%) (Erlich 187
Best Vacuum) and stored at 5ºC for 45 days. 188
189
2.6. Microbiological determinations 190
Immediately after inoculation and at 4, 13, 19, 29, 34 and 45 days of storage at 5°C, two 191
synthetic packages with each inoculated microorganism were aseptically opened and 192
microbiological evaluation was performed in 10 g obtained by transversely cutting each 193
wiener. The sample was minced with 90 cm3 of sterile saline solution (NaCl 8.5 194
mg/cm3) in a Stomacher (Seward Laboratory Blender, Stomacher 400) for 2 min. 195
Appropriate dilutions from the homogenate were prepared with sterile saline solution 196
and counts of L. innocua 7 and L. plantarum CRL691 were performed in MOX with 197
sodium moxalactame and MRS in anaerobic conditions, respectively. For the gluten 198
containing packages the same experiment was performed immediately after inoculation 199
and at 4, 19, 34 and 45 days of storage at 5°C. For non-inoculated packages total 200

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aerobic counts were performed at time 0 and at the end of storage (45th day, 5ºC) on 201
Plate Count Agar (Difco). For all the samples duplicate plates were incubated for 48 h at 202
35ºC. Results were expressed as log CFU/g. A DMFit manual Version 2.0. Program 203
(Baranyi and Roberts, 1994) was used to model inoculated microorganisms growth. 204
205
2.7. Residual antimicrobial activity, visual inspection and pH determination 206
Residual antimicrobial activity of the wieners contacted films and the supernatant liquid 207
from wiener packages (exudate) were determined in semisolid agar against the sensitive 208
microorganisms. Positive bacteriocin activity was evidenced as a zone of inhibition on 209
the indicator organism lawn. Homogenate pH measurements (Hanna Instruments 210
microprocessor pHmeter, HI1332B) and visual inspection of the packages were also 211
performed. 212
213
2.8. Statistical analysis 214
Three independent experiments were performed in duplicate. Data points are 215
represented by the mean, with the standard error indicated by error bars. All data were 216
subjected to analysis of variance (ANOVA), and the Tukey test was applied at the 0.05 217
level of significance. Statistical analyses were performed using Minitab Statistic 218
Program, release 12 (Pennsylvania, USA). 219
220
221
3. Results and Discussion 222
All control packages (without bacteriocins) either synthetic or those containing gluten 223
pads (Figs. 1 and 2, respectively), showed the typical growth of both L. plantarum 224
CRL691 and L. innocua 7 inoculated which reached maximum level of 107 CFU/g at 225

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day 45 of storage at 5ºC. Total aerobic counts in non-inoculated wieners at time 0 were 226
below the detection limit (30 CFU/g) either for synthetic or gluten containing control 227
packagesTotal aerobic counts in non-inoculated wieners at time 0 were below the 228
detection limit (30 CFU/g) either for synthetic or gluten containing control packages, 229
reaching values of 1 x 102 (synthetic packages) and 4 x 102 CFU/g (gluten containing 230
packages) at day 45 of storage. Food shelf life is defined as the time during which all of 231
the primary characteristics make the food acceptable for consumption. Thus, the shelf 232
life refers to the time period that food stays on both the retailer´s and consumer´s shelf 233
before it becomes unacceptable (Robertson, 2006). Counts of LAB have often been used 234
as a quality criteria for shelf life determination of chill stored cooked meats and fresh 235
vegetables packaged under vacuum, low O2, or high CO2 modified atmospheres (Lee, 236
2010). Vacuum-packaging and meat moisture inside the bags enable excellent contact 237
between the meat surface and bacteriocins (Ming et al., 1997). In this study, the 238
presence of lactocin 705 incorporated in synthetic packages produced a slight decrease 239
in L. plantarum CRL691 counts in wieners over two weeks of storage at 5ºC (0.5-log 240
CFU/g cycles lower than the control, Fig. 1a), and a slight delay in the microorganism 241
growth (μmax= 0.008 and μmax= 0.007 h
1, respectively for the control and active 242
packages). Nevertheless, from the 19th day to the end of storage (45 days), the same L. 243
plantarum CRL691 counts (P≥0.05) were observed for the control and active packets 244
(around 7.3±0.5 log CFU/g). When the growth of L. plantarun CRL691 was evaluated 245
in the packages containing the active gluten pad, even though different growth patterns 246
were observed, a lack of inhibition at the end of storage was also found (Fig. 2a). A 247
mildly extended lag phase was observed in the presence of lactocin 705 (193 h and 85 h 248
for active and control packaging, respectively), specific growth rates for gluten active 249
packages being higher than those for synthetic packages (μmax= 0.017 and μmax= 0.012 250

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h 1 for active and control, respectively). This result might suggest that gluten film 251
components could have been used by L. plantarum CRL691 as nutrients source. 252
On the other hand, a bacteriostatic effect against L. innocua 7 was observed in both 253
synthetic and gluten activated packages until the fourth week of storage, then exhibiting 254
a slight decrease in Listeria counts (P<0.05, Fig. 1b and 2b) with death rates of 0.0003 255
h 1 for synthetic and 0.0002 h 1 for gluten containing active packages. At the end of 256
storage at 5ºC (45 days), L. innocua 7 counts were 2.5-log cycles lower (1.7 x 104 and 257
1.5 x 104, respectively for the active synthetic and gluten containing packets) than each 258
respective control (7.4 106 for synthetic and 2.2 106, gluten containing packages, Fig. 1b 259
and 2b). These results are in agreement with those reported using various packaging 260
materials (PE, PE/PA, LDPE, celullosic inserts) containing bacteriocins (lacticin 3147, 261
nisin, enterocin 416K1, bacteriocin produced by L. curvatus 32Y) assayed in different 262
food systems such as sliced cheese and ham, pork steak, ground beef, frankfurters and 263
fresh cheeses (Scannell et al., 2000; Mauriello et al., 2004; Iseppi et al., 2008). In 264
cooked meat products, post-processing contamination represents a major safety concern; 265
product handling, processing surfaces, equipments and tools are often involved in this 266
type of contamination (Korkeala and Björkroth, 1997). Listeria inhibition in the wiener 267
samples depends on two opposite phenomena: the growth rate, which is principally 268
related to food characteristics and storage temperature, and the killing rate of the 269
antibacterial compounds (bacteriocins) as well as its diffusion rate out of the coating 270
(Iseppi et al., 2008). It is essential that preservatives applied have low diffusivity in their 271
host film to remain at the surface of the food, since diffusion into the food core results 272
in a preservative concentration reduction at the surface (Scannell et al., 2000). Anti-273
Listeria activity was observed in ham wrapped with enterocins alginate films due to a 274
balanced ratio between the release rate of bacteriocins and the growth rate of L. 275

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monocytogenes (Marcos et al., 2007). On the contrary, results from Iseppi et al. (2008) 276
showed a decrease in anti-Listeria activity as a function of time when inoculated 277
frankfurters samples were packed with an enterocin-doped LDPE film, suggesting that 278
the diffusion out of the coating was fast for the bacteriocin contained within the first 279
layers of the coating, while enterocin release from deeper layers was slower than 280
Listeria growth rate. In our study, L. innocua 7 inhibition in both active packages would 281
indicate that the release rate of bacteriocin is higher than bacterium growth rate, anti-282
listerial lactocin AL705 reaching a concentration equal or greater than the MIC 283
throughout the experiment (Blanco Massani et al., 2008). Some bacteriocins have 284
shown the same effect (bactericidal or bacteriolytic) over the target cells either in 285
culture media or in foods systems (Sabia et al., 2004; Ercolini et al., 2006; Iseppi et. al., 286
2008). Nevertheless, even when lactocin 705 and AL705 bactericidal effect on L. 287
plantarum CRL691 and Listeria 7 in laboratory media was demonstrated (Vignolo et 288
al., 1996; Cuozzo et al., 2003; Castellano et al., 2004), a bacteriostatic effect was 289
observed in meat and meat products (Vignolo et al., 1996; Castellano and Vignolo, 290
2006). Inactivation of peptide antimicrobial compounds by endogen meat enzymes or 291
fat particles may be responsible for the decreased antimicrobial activity in food systems 292
(Castellano et al., 2008). Moreover, even when bactericidal effect of lactocin 705 293
extract (256 AU/cm3) against L. plantarum CRL691 was earlier reported (Cuozzo et al. 294
2003), in our work active films with 267 AU/cm3 lactocin 705 added were not able to 295
inhibit L. plantarum CRL691 in inoculated wienners. Lactocin 705 inactivation by 296
contact with fatty substances during and after its adsorption on the synthetic film was 297
previously reported (Blanco Massani et al., 2012, 2013). Here, the presence of fat (20-298
30%) in wieners could have negatively affected lactocin 705 antimicrobial activity, 299
decreasing its inhibitory ability against L. plantarum CRL691. This fact shows the 300

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impact of the food matrix composition on the effectiveness of post-process 301
technologies, highlighting the importance of validation procedures for each particular 302
application (Gálvez et al., 2007). 303
Residual antimicrobial activity in activated packaging at the end of storage and 304
in wieners exudates was evaluated. Results showed a lack of lactocin 705 and AL705 305
activity on synthetic multilayer films and wieners exudate after 45 days of storage at 306
5°C, (Fig. 3). On the contrary, although gluten pads and wieners exudate did not exhibit 307
residual activity for lactocin 705, a residual antilisterial activity due to lactocin AL705 308
was observed during 15 days at 5°C (Fig. 4 b). This result would indicate that this 309
bacteriocin is present in higher concentration in the gluten pads than in the synthetic 310
multilayer films. As was recently reported by Blanco Massani et al. (2013), only the 311
adsorbed lactocin AL705 was shown to exert antimicrobial activity, after synthetic films 312
activation and its saturation concentration (200 AU/cm3) was lower than the bacteriocin 313
present in the activation solution (2133 AU/cm3), whereas in the gluten pads, inner 314
lactocin AL705 concentration was that of the added (2133 AU/cm3), this resulting in a 315
higher gluten residual activity of lactocin AL705 after wieners contact. On the other 316
hand, reduced antimicrobial activity of bacteriocins was earlier reported when recovered 317
from complex matrixes such as food systems (Raju et al., 2003; Aasen et al., 2003). 318
Lack of lactocin 705 activity in films and wieners exudates found in our work is in line 319
with interferences produced by fat as earlier discussed. 320
Changes of wieners pH in packages inoculated whit L. plantarum CRL691 321
showed a decrease from 6.3 to 5.9 (day 19th ), a final value in the range of 5.7-5.8 being 322
reached towards the end of the experiment (Fig. 5a and b). In wiener packages 323
inoculated with L. innocua 7 and those uninoculated, pH values stayed around 6.3 324
throughout the experiment. The pH decrease in the presence of L. plantarum CRL691 is 325

14

in agreement with its high acidogenic ability as was reported by Fadda et al. (2010). 326
When visual inspection of wiener packages during storage at 5°C was carried out, the 327
appearance of small bubbles from day 19 onwards was registered either in inoculated or 328
uninoculated samples (data not shown). Gas production in meat products is a 329
consequence of heterofermentative metabolism of the naturally present meat borne 330
Lactobacillus and Leuconostocs species (Korkeala, & Björkroth, 1997; Mataragas et al., 331
2006; Chenoll et al., 2007). Even when L. plan tarum CRL691 is a facultative 332
heterofermenter strain, gas production may not be ascribed to its metabolism. Since 333
vacuum packaging thermo-sealing of wieners was performed under non-sterile 334
conditions, contamination with gas-producer organisms could have been occurred. 335
336
4. Conclusions 337
The use of natural substances as biologically derived antimicrobials appears as 338
an important requirement in the active food packaging methodology for the microbial 339
control. Here, assayed as wieners packages, high anti-listerial efficacy for synthetic and 340
gluten containing packaging activated with lactocin AL705, from L. curvatus CRL705, 341
was obtained. However, no inhibition of L. plantarum CRL691 by lactocin 705 was 342
exerted due to the high fat content of wieners. These results show the importance of 343
particular food characteristics in the design of active packaging. 344
345
Acknowledgments 346
This study was supported by grants from 3iA-UNSAM 2006, Argentina. The 347
authors would like to acknowledge the assistance of INTI-Carnes laboratories and 348
Professor Claudia Melian. 349
350

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351
352
References 353
Aasen, I.M., Markussen, S., Møretrø, T., Katla, T., Axelsson, L., Naterstad, K. 2003. 354
Interactions of the bacteriocins sakacin P and nisin with food constituents. 355
International Journal of Food Microbiology 87, 35–43. 356
Arcan, I., Yemenicioğlu, A. 2013. Development of flexible zein–wax composite and 357
zein–fatty acid blend films for controlled release of lysozyme. Food Research 358
International 51, 208–216. 359
Audenaert, K., D’Haene, K., Messens, K., Ruyssen, T., Vandamme, P., Huys, G. 2010. 360
Diversity of lactic acid bacteria from modified atmosphere packaged sliced 361
cooked meat products at sell-by date assessed by PCR-denaturing gradient gel 362
electrophoresis. Food Microbiology 27, 12-18. 363
Baranyi, J., Roberts, T.A. 1994. A dynamic approach to predicting bacterial growth in 364
food. International Journal of Food Microbiology 23, 277-294. 365
Bhatti, M., Veeramachaneni A., Shelef, L.A. 2004. Factors affecting the antilisterial 366
effects of nisin in milk. International Journal of Food Microbiology 97, 215–219. 367
Blanco Massani, M., Fernandez, M. R., Ariosti, A., Eisenberg, P., Vignolo, G. 2008. 368
Development and characterization of an active polyethylene film containing 369
Lactobacillus curvatus CRL705 bacteriocins. Food Additives and Contaminants 370
25, 1424-1430. 371
Blanco Massani, M., Morando, P., Vignolo, G., Eisenberg, P. 2012. Characterization of 372
a multilayer film activated with Lactobacillus curvatus CRL705 bacteriocins. 373
Journal of the Science of Food and Agriculture 92, 1318-1323. 374

16

Blanco Massani, M., Vignolo, G., Eisenberg, P., Morando, P. 2013. Adsorption of the 375
bacteriocins produced by Lactobacillus curvatus CRL705 on a multilayer-LLDPE 376
film for food-packaging applications. LWT - Food Science and Technology 53, 377
128-138. 378
Blanco Massani, M., Botana, A., Eisenberg, P., Vignolo, G. Development of an active 379
wheat gluten film with L. curvatus CRL705 bacteriocins, antimicrobial 380
performance study during aging. Food Additives & Contaminants: Part A. In 381
press article DOI:10.1080/19440049.2013.859398. 382
Bremer, V., Bocter, N., Rehmet, S., Klein, G., Breuer, T., Ammon, A. 2005. 383
Consumption, knowledge, and handling of raw meat: A representative 384
crosssectional survey in Germany, March 2001. Journal of Food Protection 68, 385
785-789. 386
Castellano, P., Holzapfel,W., Vignolo, G. 2004. The control of Listeria innocua and 387
Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain 388
Lactobacillus casei CRL705. Food Microbiology 21, 291-298. 389
Castellano, P., Vignolo, G. 2006. Inhibition of Listeria innocua and Brochothrix 390
th ermosphacta in vacuum-packed meat by addition of bacteriocinogenic 391
Lactobacillus curvatus CRL705 and its bacteriocins. Letters in Applied 392
Microbiology 43, 194-199. 393
Castellano, P., Belfiore, C., Fadda, S., Vignolo, G. 2008. A review of bacteriocinogenic 394
lactic acid bacteria used as bioprotective cultures in fresh meat produced in 395
Argentina. Meat Science 79, 483-499. 396

17

CDC, Centers for Disease Control and Prevention. 2007. Preliminary Food Net data on 397
the incidence of infection with pathogens transmitted commonly through food-10 398
States, 2006. Morbidity and Mortality Weekly Report 56, 336-339. 399
Chenoll, E., Macián, M.C., Elizaquível, P., Aznar, R. 2007. Lactic acid bacteria 400
associated with vacuum-packed cooked meat product spoilage: population 401
analysis by rDNA-based methods. Journal of Applied Microbiology 102, 498–402
508. 403
Código Alimentario Argentino (C.A.A.). Capítulo VI: Alimentos cárneos y afines, p 44. 404
Dalgaard, P. 1995. Modelling of microbial activity and prediction of shelf life for 405
packed fresh fish. International Journal of Food Microbiology 26, 305-317. 406
Cuozzo, S.A., Castellano, P., Sesma, F., Vignolo, G., Raya, R.R. 2003. Differential 407
roles of the two-component peptides of lactocin 705 in antimicrobial activity 408
Current Microbiology. 46, 180 – 183. 409
Fadda, S., Vildoza, M.J., Vignolo, G. 2010. The acidogenic metabolism of 410
Lactobacillus plantarum CRL681 improves sarcoplasmic protein hydrolysis 411
during meat fermentation. Journal of Muscle Foods 21, 545–556. 412
Gálvez, A., Abriouel, H., Lucas López, R., Omar N. B. 2007. Bacteriocin-based 413
strategies for food biopreservation. International Journal of Food Microbiology 414
120, 51-70. 415
Gialamas, H., Zinoviadou, K.G., Biliaderis, C.G., Koutsoumanis, K.P. 2010. 416
Development of a novel bioactive packaging based on the incorporation of 417
Lactobacillus sakei into sodium-caseinate films for controlling Listeria 418
monocytogenes in foods. Food Research International 43, 2402–2408. 419

18

Gill, A. O., Gill, C. O. 2010. Packaging and the Shelf Life of Fresh Red and Poultry 420
Meats. In Gordon L. Robertson (Ed) Food Packaging and Shelf Life A Practical 421
Guide. Taylor & Francis Group, Boca Raton, FL. pp. 258-272. 422
Ercolini, D., La Storia, A., Villani, F., Mauriello, G. 2006. Effect of a bacteriocin-423
activated polythene film on Listeria monocytogenes as evaluated by viable 424
staining and epifluorescence microscopy. Journal of Applied Microbiology 100, 425
765–772. 426
Iseppi, R., Pilati, F., Marini, M., Toselli, M., Niederhäusern, S., Guerrieri, E., Messi, P., 427
Sabia, C., Manicardi, G., Anacarso, I., Bondi, M. 2008. Anti-listerial activity of a 428
polymeric film coated with hybrid coatings doped with enterocin 416K1 for use 429
as bioactive food packaging. International Journal of Food Microbiology 123, 430
281-287. 431
Korkeala, H.J., Björkroth, K.J. 1997. Microbiological spoilage and contamination of 432
vacuum-packaged cooked sausages. Journal of Food Protection 60, 724–731. 433
Koutsoumanis, K., Nychas, G.-J.E. 2000. Application of a systematic experimental 434
procedure to develop a microbial model for rapid fish shelf life predictions. 435
International Journal of Food Microbiology 60, 171-184. 436
Lee, D.S. 2010. Packaging and the microbial shelf life of food. In Robertson, G.L. (Ed), 437
Food Packaging and Shelf Life, A Practical Guide. Taylor & Francis Group, Boca 438
Raton, FL, pp 55-80. 439
Leistner, L., Gould, G.W. 2002. Hurdle technologies: combination treatments for food 440
stability, safety and quality. In Leistner, L., & Gould, G.W. (Ed), Food 441
Engineering Series. Kluwer Academic/Plenum Publishers, New York, pp 1-15 442

19

López de Dicastillo, C., Castro-López, M. M., López-Vilariño, J. M., González-443
Rodríguez, M. V. 2013. Immobilization of green tea extract on polypropylene 444
films to control the antioxidant activity in food packaging. Food Research 445
International 53, 522–528. 446
Marcos, B., Aymerich, T., Monfort, J. M., Garriga, M. 2007. Use of antimicrobial 447
biodegradable packaging to control Listeria monocytogenes during storage of 448
cooked ham. International Journal of Food Microbiology 120, 152–158. 449
Marcos, B., Aymerich, T., Monfort, J. M., Garriga, M. 2008. High-pressure processing 450
and antimicrobial biodegradable packaging to control Listeria monocytogenes 451
during storage of cooked ham. Food Microbiology 25, 177–182. 452
Mataragas, M., Drosinos, E.H., Vaidanis, A., Metaxopoulos, I. 2006. Development of a 453
predictive model for spoilage of cooked cured meat products and its validation 454
under constant and dynamic temperature storage conditions. Journal of Food 455
Science 71, 157-167. 456
Mauriello, G., Ercolini, D., La Storia, A., Casaburi, A., Villani, F. 2004. Development 457
of polythene films for food packaging activated with an antilisterial bacteriocin 458
from Lactobacillus curvatus 32Y. Journal of Applied Microbiology 97, 314-322. 459
Ming, X., Weber, G.H., Ayres, J.W., Sandine, W.E. 1997. Bacteriocins applied to food 460
packaging materials to inhibit Listeria monocytogenes on meats. Journal of Food 461
Science 62, 413–415. 462
Raju C. V., Shamasundar, B. A., Udupa, K. S. 2003. The use of nisin as a preservative 463
in fish sausage stored at ambient (28 ± 2ºC) and refrigerated (6 ± 2ºC) 464
temperatures. International Journal of Food Science and Technology 38, 171–185. 465

20

Ramos, O. L., Silva, S. I., Soares, J. C., Fernandes, J.C., Poças, M. F., Pintado, M. E., 466
Malcata, F.X. 2012. Features and performance of edible films, obtained from 467
whey protein isolate formulated with antimicrobial compounds. Food Research 468
International 45, 351–361. 469
Robertson, G.L. 2006. Food packaging principles and practice. Second ed. Taylor & 470
Francis, Boca Raton, FL. 471
Rybka-Rodgers, S. 2001. Improvement of food safety design of cook-chill foods. Food 472
Research International 34, 449-455. 473
Sabia, C., Messi, P., de Niederhaüsern, S., Manicardi, G., Bondi, M. 2004. Study of two 474
bacteriocins produced by Enterococcus casseliflavus and Ent. faecalis . Letters in 475
Applied Microbiology 38, 99–105. 476
Scannell, A.G., Hill, C., Ross, R.P., Marx, S., Hartmeier, W., Arendt, E.K. 2000. 477
Development of bioactive food packaging materials using immobilized 478
bacteriocins lacticin 3147 and Nisaplin. International Journal of Food 479
Microbiology 60, 241–249. 480
Vignolo, G., Cuozzo, S., de Kairuz, M.N., Holgado, A.P. de R., Oliver, G. 1996. 481
Listeria monocytogenes inhibition in meat slurry by in situ produced lactocin 705. 482
Microbiolie Aliments and Nutrition 14, 363-367. 483
Vignolo, G., Fadda, S., de Kairuz, M.N., Holgado, A.P. de R., Oliver, G. 1996. Control 484
of Listeria monocytogenes in ground beef by ‘Lactocin 705’, a bacteriocin 485
produced by Lactobacillus casei CRL 705. International Journal of Food 486
Microbiology 29, 397-402. 487
Vignolo, S., Fadda, S., Castellano, P. 2008. Bioprotective cultures. In Toldrá, F. (Ed), 488
Meat Biotechnology.Springer, New York, pp. 399-424. 489

21

Vignolo, G., Saavedra, L., Sesma, F., Raya, R. 2012. Food bioprotection: lactic acid 490
bacteria as natural preservatives. In: Bhat, R., Alias, A.K. & Paliyath, G. (Eds), 491
Progress in Food Preservation. Wiley-Blackwell, Oxford, UK, pp. 453-483 492
Zapico, P., de Paz, M., Medina, M., Nuñez, M. 1999. The effect of homogenization of 493
whole milk, skim milk and milk fat on nisin activity against Listeria innocua . 494
International Journal of Food Microbiology 46, 151–157. 495

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Figure Legends 496
Figure 1. L. plantarum (a) and L. innocua 7 (b) growth during 45 days at 5 ºC in the 497
active (▼) and control (●) synthetic packages. Lines between points mark tendencies. 498
499
Figure 2. L. plantarum (a) and L, innocua 7 (b) growth during 45 days at 5 ºC in the 500
active (▼) and control (●) gluten containing packages. Lines between points mark 501
tendencies. 502
503
Figure 3. Residual antimicrobial activity of lactocin 705 (a) and AL705 (b) in the active 504
synthetic films before (1) and after (2) contact with wieners (15 days at 5 ºC). Wells in 505
the plates correspond to residual analysis in wieners exudates. 506
507
Figure 4. Residual antimicrobial activity of lactocin 705 (a) and AL705 (b) in the active 508
gluten (A) and control (C) films after contact with wieners (15 days at 5 ºC) 509
510
Figure 5. Changes of pH during storage (45 days at 5ºC) in active (▼) and control (●) 511
synthetic (a) and gluten (b) wiener packages inoculated with L. plantarum CRL691 . 512

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