Repositorio Institucional

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 (for review)

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



*Manuscript with Line Numbers Click here to view linked References

1 Active polymers containing Lactobacillus curvatus CRL705 bacteriocins: 2 effectiveness assessment in Wieners 3 4 Blanco Massani M.1, Molina V. 1, Sanchez M1, Renaud V1,2, Eisenberg P1,2 and 5 Vignolo G3. 6 7 1INTI Gral Paz 5445. Buenos Aires, Argentina; 23iA-UNSAM, Argentina; 3Centro de 8 Referencia para Lactobacilos (CERELA), CONICET, Tucumán, Argentina. 9 10 11 12 13 14 15 16 17 18 *Corresponding author: M. Blanco Massani, E-mail: blanco@inti.gob.ar 19 Telephone: (54 11) 4724 6200 Int: 6636 20 Fax: (54 11) 4753 5773 21 22 23 24 25

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

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1. Introduction



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Although food biotechnology has recently made important progresses, food



55 industry and particularly meat industry is still under scrutiny by consumers due to



56 sanitary episodes generated by meat and meat based products (Bremer et al., 2005;



57 CDC, 2007). Modern life conditions related to or as consequence of globalization,



58 contribute to the major incidence of food diseases outbreaks. The major challenges for



59 food safety are the emergent pathogens, among which L. monocytogenes is included



60 (Vignolo et al., 2008; Vignolo et al., 2012). During food chain distribution, food needs



61 to be protected from physical, chemical and microbiological spoilage. The shelf life of



62 food is controlled by (i) the product characteristics including formulation and



63 processing parameters (intrinsic factors), (ii) the package properties and (iii) the



64 environment to which the products are exposed during distribution and storage



65 (extrinsic factors). Among intrinsic factors, pH, water activity, enzymes,



66 microorganisms, and concentration of reactive compounds are included. Many of these



67 factors can be controlled by selection of raw materials and ingredients, as well as the



68 choice of processing parameters. However, extrinsic factors namely temperature,



69 relative humidity, light, total and partial pressure of different gases as well as



70 mechanical stresses including consumer handling may affect the rates of deteriorative



71 reactions occurring during the shelf life of food. The properties of package can exert a



72 significant effect on many of the extrinsic factors and thus indirectly on the rates of



73 deteriorative reactions (Robertson, 2006). Interaction of the packaged food with its



74 packaging and the external environment may also change intrinsic food factors, package



75 headspace acting as a buffer between food and packaging material. Due to these



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76 interactions, moisture content (i.e., water activity), dissolved O2 and CO2 contents, and



77 preservative concentration can be modified to affect the microbiota and its growth rate



78 (Lee, 2010).



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Muscle tissue from healthy animals is free of bacterial or viral pathogens. As



80 with spoilage organisms, pathogens are deposited on meat surfaces during processing



81 and handling of meat carcass. Potential sources of pathogen contamination comprise



82 animal-associated pathogens transferred to meat from the hide, skin, or feathers and the



83 intestinal tract of the animal during carcass processing; human-associated pathogens



84 transferred from personnel during handling of product, processing equipment and tools,



85 which if inadequately cleaned and sanitized may act not only as vehicles for pathogen



86 but also as sources of contamination (Gill and Gill, 2010). Most perishable foods are



87 vulnerable to microbial spoilage even under chilled conditions. Their shelf life is thus,



88 for the most part, terminated when they become unacceptable due to the growth of



89 undesirable microorganisms (Lee, 2010). Within a certain range of environmental



90 conditions, often only one member from the total microflora is responsible for spoilage



91 (specific spoilage organisms—SSO); for cooked meat products, lactic acid bacteria



92 were found as the prevalent spoilage microorganisms (Mataragas et al., 2006;



93 Audenaert, et al., 2010; Chenoll, et al., 2007). For shelf life studies, after determining



94 the SSO and the conditions under which this group of microorganisms is responsible for



95 food spoilage, the next step is to determine the number of SSO responsible for food



96 deterioration producing lack of acceptability (Dalgaard, 1995; Koutsoumanis and



97 Nychas, 2000). The acceptable limit of microbial growth that determines the shelf life 98 differs with food type and storage conditions. SSO counts of 105–108 bacteria per g1 or 99 cm2 are commonly used as a convenient upper limit of quality and are reached mostly



100 during microorganism growth exponential phase (Lee, 2010). Combined intrinsic



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101 factors are used to preserve food safety and ensure organoleptic quality, i.e. suitable 102 food shelf life can be obtained by incorporating low levels of additives, mild 103 dehydration and heat processes (Leistner and Gould, 2002). Several technologies can be 104 combined in order to improve food safety and extend shelf life of foods (Rybka105 Rodgers, 2001). During the last years, a number of biopreservation technologies has 106 been developed by the inclusion of antimicrobial extracts, lysozyme, bacteria and/or 107 bacteriocins among others, into polymer matrices (Marcos et al., 2008; Gialamas et al., 108 2010; Ramos et al., 2012; López de Dicastillo et al., 2013; Arcan and Yemenicioğlu, 109 2013). Nevertheless for bacteriocins biopreservation hurdles, it was found that 110 antimicrobial effect could be affected by food components (Zapico et al., 1999; Aasen 111 et al., 2003; Bhatti et al., 2004). Lactocin 705 and lactocin AL705, are bacteriocins 112 produced by Lactobacillus curvatus CRL705. Lactocin 705 has antagonist effect against 113 Lactic acid bacteria (LAB) and Brochothix thermosphacta, while AL705 is active 114 against Listeria species (Castellano and Vignolo, 2006). Both bacteriocins retained its 115 antimicrobial activity when included in polymer matrices such as LDPE (Blanco 116 Massani et al., 2008, 2012) and gluten (Blanco Massani et al., in press article). In the 117 present study, active LDPE and gluten films obtained by L. curvatus CRL705 118 bacteriocins incorporation were evaluated for antimicrobial effectiveness in 119 contaminated Wieners. 120 121 2. Materials and Methods 122 2.1. Bacterial strains and growth conditions 123 Lactobacillus curvatus CRL705 (producer of the bacteriocins lactocin 705 and lactocin 124 AL705) and Lactobacillus plantarum CRL691 (which is sensitive to the activity of 125 lactocin 705) from CERELA culture collection, were grown in MRS broth (Britania,

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126 Argentina) for 16 h at 30ºC. Listeria innocua 7 (sensitive to the activity of lactocin 127 AL705) obtained from the Unité de Recherches Laitières et Génétique Appliquée, 128 INRA (France) was grown in trypticase soy broth (Britania) with 5 mg/cm3 of yeast 129 extract added (Britania, Argentina) for 16 h at 30 ºC. All strains were maintained and 130 stored at −20 ºC in 0.15 g/cm3 of glycerol until use. 131 132 2.2. Wieners elaboration 133 Wieners were manufactured in a meat processing pilot plant, according to standard 134 procedure (C.A.A.). Beef and pork meat cuts were minced (Themis 32 mincer) and 135 processed together with fat in a vertical cutter (Robot Coupe). Ice (0.11 g/cm3), sodium 136 phosphate (3 mg/cm3), sodium erithorbate (0.5 mg/cm3), sodium chloride (0.017 g/cm3) 137 and sodium nitrite (0. 15 mg/cm3) were added and mixed to obtain a homogeneous mass. 138 Finally, starch and water (0.11 g/cm3) were added to form an emulsion which was filled 139 (Hidraulic filler, RISCO IV 20) into artificial casings (2 cm diameter). Wieners (5 cm, 14 140 g) were cooked in an oven (Lavaflux) at 80°C for 15 min, cooled in an ice bath to a core 141 temperature below 40°C and refrigerated at 3°C until manual peeled. After that re142 pasteurization of vacuum-packed wieners was performed (10 min, 80°C). 143 144 2.3. Active solution preparation and quantification 145 A powder containing lactocin 705 and lactocin AL705 from L. curvatus CRL705 was 146 obtained by ammonium sulfate precipitation as earlier reported (Blanco Massani et al., 147 2008). For activity determination the active powder was resuspended in water and the 148 agar well diffusion assay against L. plantarum CRL691 (lactocin 705 sensitive 149 organism) and L. innocua 7 (lactocin AL705 sensitive organism) was performed 150 (Blanco Massani et al., 2012). Antimicrobial activity was expressed as AU/cm3.

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

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

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

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

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251 h-1 for active and control, respectively). This result might suggest that gluten film 252 components could have been used by L. plantarum CRL691 as nutrients source. 253 On the other hand, a bacteriostatic effect against L. innocua 7 was observed in both 254 synthetic and gluten activated packages until the fourth week of storage, then exhibiting 255 a slight decrease in Listeria counts (P<0.05, Fig. 1b and 2b) with death rates of -0.0003 256 h-1 for synthetic and -0.0002 h-1 for gluten containing active packages. At the end of 257 storage at 5ºC (45 days), L. innocua 7 counts were 2.5-log cycles lower (1.7 x 104 and 258 1.5 x 104, respectively for the active synthetic and gluten containing packets) than each 259 respective control (7.4 106 for synthetic and 2.2 106, gluten containing packages, Fig. 1b 260 and 2b). These results are in agreement with those reported using various packaging 261 materials (PE, PE/PA, LDPE, celullosic inserts) containing bacteriocins (lacticin 3147, 262 nisin, enterocin 416K1, bacteriocin produced by L. curvatus 32Y) assayed in different 263 food systems such as sliced cheese and ham, pork steak, ground beef, frankfurters and 264 fresh cheeses (Scannell et al., 2000; Mauriello et al., 2004; Iseppi et al., 2008). In 265 cooked meat products, post-processing contamination represents a major safety concern; 266 product handling, processing surfaces, equipments and tools are often involved in this 267 type of contamination (Korkeala and Björkroth, 1997). Listeria inhibition in the wiener 268 samples depends on two opposite phenomena: the growth rate, which is principally 269 related to food characteristics and storage temperature, and the killing rate of the 270 antibacterial compounds (bacteriocins) as well as its diffusion rate out of the coating 271 (Iseppi et al., 2008). It is essential that preservatives applied have low diffusivity in their 272 host film to remain at the surface of the food, since diffusion into the food core results 273 in a preservative concentration reduction at the surface (Scannell et al., 2000). Anti274 Listeria activity was observed in ham wrapped with enterocins alginate films due to a 275 balanced ratio between the release rate of bacteriocins and the growth rate of L.

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276 monocytogenes (Marcos et al., 2007). On the contrary, results from Iseppi et al. (2008) 277 showed a decrease in anti-Listeria activity as a function of time when inoculated 278 frankfurters samples were packed with an enterocin-doped LDPE film, suggesting that 279 the diffusion out of the coating was fast for the bacteriocin contained within the first 280 layers of the coating, while enterocin release from deeper layers was slower than 281 Listeria growth rate. In our study, L. innocua 7 inhibition in both active packages would 282 indicate that the release rate of bacteriocin is higher than bacterium growth rate, anti283 listerial lactocin AL705 reaching a concentration equal or greater than the MIC 284 throughout the experiment (Blanco Massani et al., 2008). Some bacteriocins have 285 shown the same effect (bactericidal or bacteriolytic) over the target cells either in 286 culture media or in foods systems (Sabia et al., 2004; Ercolini et al., 2006; Iseppi et. al., 287 2008). Nevertheless, even when lactocin 705 and AL705 bactericidal effect on L. 288 plantarum CRL691 and Listeria 7 in laboratory media was demonstrated (Vignolo et 289 al., 1996; Cuozzo et al., 2003; Castellano et al., 2004), a bacteriostatic effect was 290 observed in meat and meat products (Vignolo et al., 1996; Castellano and Vignolo, 291 2006). Inactivation of peptide antimicrobial compounds by endogen meat enzymes or 292 fat particles may be responsible for the decreased antimicrobial activity in food systems 293 (Castellano et al., 2008). Moreover, even when bactericidal effect of lactocin 705 294 extract (256 AU/cm3) against L. plantarum CRL691 was earlier reported (Cuozzo et al. 295 2003), in our work active films with 267 AU/cm3 lactocin 705 added were not able to 296 inhibit L. plantarum CRL691 in inoculated wienners. Lactocin 705 inactivation by 297 contact with fatty substances during and after its adsorption on the synthetic film was 298 previously reported (Blanco Massani et al., 2012, 2013). Here, the presence of fat (20299 30%) in wieners could have negatively affected lactocin 705 antimicrobial activity, 300 decreasing its inhibitory ability against L. plantarum CRL691. This fact shows the

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301 impact of the food matrix composition on the effectiveness of post-process



302 technologies, highlighting the importance of validation procedures for each particular



303 application (Gálvez et al., 2007).



304



Residual antimicrobial activity in activated packaging at the end of storage and



305 in wieners exudates was evaluated. Results showed a lack of lactocin 705 and AL705



306 activity on synthetic multilayer films and wieners exudate after 45 days of storage at



307 5°C, (Fig. 3). On the contrary, although gluten pads and wieners exudate did not exhibit



308 residual activity for lactocin 705, a residual antilisterial activity due to lactocin AL705



309 was observed during 15 days at 5°C (Fig. 4 b). This result would indicate that this



310 bacteriocin is present in higher concentration in the gluten pads than in the synthetic



311 multilayer films. As was recently reported by Blanco Massani et al. (2013), only the



312 adsorbed lactocin AL705 was shown to exert antimicrobial activity, after synthetic films 313 activation and its saturation concentration (200 AU/cm3) was lower than the bacteriocin 314 present in the activation solution (2133 AU/cm3), whereas in the gluten pads, inner 315 lactocin AL705 concentration was that of the added (2133 AU/cm3), this resulting in a



316 higher gluten residual activity of lactocin AL705 after wieners contact. On the other



317 hand, reduced antimicrobial activity of bacteriocins was earlier reported when recovered



318 from complex matrixes such as food systems (Raju et al., 2003; Aasen et al., 2003).



319 Lack of lactocin 705 activity in films and wieners exudates found in our work is in line



320 with interferences produced by fat as earlier discussed.



321



Changes of wieners pH in packages inoculated whit L. plantarum CRL691



322 showed a decrease from 6.3 to 5.9 (day 19th ), a final value in the range of 5.7-5.8 being



323 reached towards the end of the experiment (Fig. 5a and b). In wiener packages



324 inoculated with L. innocua 7 and those uninoculated, pH values stayed around 6.3



325 throughout the experiment. The pH decrease in the presence of L. plantarum CRL691 is



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326 in agreement with its high acidogenic ability as was reported by Fadda et al. (2010).



327 When visual inspection of wiener packages during storage at 5°C was carried out, the



328 appearance of small bubbles from day 19 onwards was registered either in inoculated or



329 uninoculated samples (data not shown). Gas production in meat products is a



330 consequence of heterofermentative metabolism of the naturally present meat borne



331 Lactobacillus and Leuconostocs species (Korkeala, & Björkroth, 1997; Mataragas et al.,



332 2006; Chenoll et al., 2007). Even when L. plantarum CRL691 is a facultative



333 heterofermenter strain, gas production may not be ascribed to its metabolism. Since



334 vacuum packaging thermo-sealing of wieners was performed under non-sterile



335 conditions, contamination with gas-producer organisms could have been occurred.



336



337 4. Conclusions



338



The use of natural substances as biologically derived antimicrobials appears as



339 an important requirement in the active food packaging methodology for the microbial



340 control. Here, assayed as wieners packages, high anti-listerial efficacy for synthetic and



341 gluten containing packaging activated with lactocin AL705, from L. curvatus CRL705,



342 was obtained. However, no inhibition of L. plantarum CRL691 by lactocin 705 was



343 exerted due to the high fat content of wieners. These results show the importance of



344 particular food characteristics in the design of active packaging.



345



346



Acknowledgments



347



This study was supported by grants from 3iA-UNSAM 2006, Argentina. The



348 authors would like to acknowledge the assistance of INTI-Carnes laboratories and



349 Professor Claudia Melian.



350



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496



Figure Legends



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



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