Título: | Improving ham shelf life with a polyhydroxybutyrate/polycaprolactone biodegradable film activated with nisin |
Fuente: | Food Packaging and Shelf Life, vol. 11 |
Autor/es: | Correa, Juan Pablo; Blanco Massani, Mariana R.; Molina, Vanesa; Sanchez, Mariana; Eisenberg, Patricia; Kainz, Cecilia |
Materias: | Jamón; Vida de anaquel; Conservación de alimentos; Películas comestibles; Películas orgánicas; Nisina; Nanocompuestos; Envases |
Editor/Edición: | ; 2017 |
Licencia: | https://creativecommons.org/licenses/by-nc-nd/4.0/ |
Afiliaciones: | Correa, Juan Pablo. Universidad Nacional de San Martín. Instituto de Investigación e Ingeniería Ambiental; Argentina Blanco Massani, Mariana R. Instituto Nacional de Tecnología Industrial. INTI-Plásticos; Argentina Molina, Vanesa. Instituto Nacional de Tecnología Industrial. INTI-Plásticos; Argentina Sanchez, Mariana. Instituto Nacional de Tecnología Industrial. INTI-Plásticos; Argentina Eisenberg, Patricia. Instituto Nacional de Tecnología Industrial. INTI-Plásticos; Argentina Kainz, Cecilia. Instituto Nacional de Tecnología Industrial. INTI-Plásticos; Argentina |
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Improving ham shelf life with a polyhydroxybutyrate/polycaprolactone biodegradable film activated with nisin Juan Pablo Correaa, b, , , Vanesa Molinac, Mariana Sanchezc, Cecilia Kainzc, Patricia Eisenbergb, c,Mariana Blanco Massania, c a National Council of Scientific and Technical Research-CONICET, Av. Rivadavia 1917 (C1033AAJ), Buenos Aires, Argentina b National University of San Martín – UNSAM 3iA. Campus Miguelete, Av. 25 de Mayo y Francia (CP 1650), San Martín, Buenos Aires, Argentina c National Institute of Industrial Technology-INTI, Av. Gral. Paz 5445, San Martín (B1650WAB), Buenos Aires, Argentina Food Packaging and Shelf Life Volume 11, March 2017, Pages 31–39 https://doi.org/10.1016/j.fpsl.2016.11.004 Received 11 June 2016, Revised 15 November 2016, Accepted 21 November 2016, Available online 1 December 2016 © <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/ 1 Improving ham shelf life with a Polyhydroxybutyrate/Polycaprolactone 2 biodegradable film activated with nisin 3 Juan Pablo Correa a,b*, Vanesa Molina c, Mariana Sanchez c, Cecilia Kainz c, Patricia 4 Eisenberg b,c and Mariana Blanco Massani a,c 5 6 a National Council of Scientific and Technical Research-CONICET, Av. Rivadavia 1917 7 (C1033AAJ), Buenos Aires, Argentina. 8 b National University of San Martín – UNSAM 3iA. Campus Miguelete, Av. 25 de Mayo y 9 Francia (CP 1650), San Martín, Buenos Aires, Argentina. 10 c National Institute of Industrial Technology-INTI, Av. Gral. Paz 5445, San Martín 11 (B1650WAB), Buenos Aires, Argentina. 12 * Corresponding author. Tel. / fax: +54 11 4753 5773. 13 E-mail address: jcorrea@inti.gob.ar (Juan Pablo Correa). 1 14 Abstract 15 Environmental polution and food shelf life extension are issues of global concern. In this 16 work, biodegradable Polyhydroxybutyrate/Polycaprolactone (PHB/PCL) films and organo17 clays (Cloisite® 30B and 10A) based nanocomposites were prepared. Tensile and thermal 18 properties, water vapor barrier and activation with nisin were studied. Organo-clays 19 addition promoted a reinforcement effect of the polymer blend, increasing barrier 20 properties and degradation temperature. The optimal parameters for nisin adsorption to 21 PHB/PCL film were 4000 IU cm-3, 40°C and 10 min. Organo-clays exerted antimicrobial 22 activity against Lactobacillus plantarum CRL691; nevertheless, their inclusion into the 23 polymer blend did not lead to antimicrobial films. Nisin adsorption to PHB/PCL film was 24 not afected by clays presence. PHB/PCL nisin activated film was effective against L. 25 plantarum CRL691 (used as processed meat spoilage bacterium model) inoculated on 26 sliced ham, thus extending its shelf life. PHB/PCL blend and its nanocomposites activated 27 with nisin showed potential for their application in processed meat packaging. 28 Keywords: Biodegradable nanocomposite films; active packaging; nisin; ham; shelf life 29 extension. 2 31 1. Introduction 32 Spoilage bacteria lead to economic losses in meat processing industry (Giaouris et al., 33 2014). To overcome this problem, and according to an increased negative perception 34 towards chemical agents, natural antimicrobial agents have been extensively screened and 35 tested for their effectiveness in meat and processed meat foods (Woraprayote et al., 2016). 36 Active packaging containing bacteriocins showed the ability to inhibit unwanted 37 microorganisms in various meat products (Blanco Massani et al., 2014; Marcos, Aymerich, 38 Garriga, & Arnau, 2013; Woraprayote et al., 2013). In contrast to plant extracts and other 39 natural antimicrobial preservatives, bacteriocins present colorless, odorless, and tasteless 40 characteristics. These characteristics make bacteriocins good candidates for their 41 application in active packaging, since their use does not interfere with sensory quality of 42 food products (Elsser-Gravesen & Elsser-Gravesen, 2013; Woraprayote et al., 2016). 43 Regulatory requirements for active packaging technologies in the United States are not 44 very different from the requirements for conventional antimicrobial additives. The material 45 exerting antimicrobial effect on food through migration or controlled release would 46 constitute a ‘‘direct additive” and would be subject to FDA regulatory requirements 47 (Restuccia et al 2010). For the European Union, regulation 450/2009/EC set legal basis for 48 the correct use, safety and marketing of active packaging. The use of the active substance 49 must accomplish a technological need and active substances migrating from the packaging 50 shall comply with the conventional rules laid down in the EU Directives (Commission 51 Regulation 1333/2008/EC, Reg 1129/2011 and Reg 1130/2011) (Restuccia et al 2010). 52 Among hundreds of bacteriocins, nisin is the only commercial bacteriocin approved for 53 food applications (Elsser-Gravesen & Elsser-Gravesen, 2013; Reg 1129/2011). Its 3 54 mechanism of action involves initial interaction with the membrane and further lipid II 55 binding. In this way, a stabilized poration complex is formed in the target site and at the 56 same time, sequestration of lipid II causes cell wall biosynthesis inhibition. This dual mode 57 of action, in which inhibition of peptidoglycan synthesis and pore formation are most 58 efficiently combined, makes nisin a very potent antimicrobial agent and impedes the 59 emergence of resistant strains (Islam, Nagao, Zendo, & Sonomoto, 2012). 60 Bacteriocins adsorption on polymer surfaces offers a way for setting up antibacterial 61 systems. Other strategy is adding antimicrobial agents into polymers. In this regard, 62 commercially montmorillonites modified with quaternary alkyl ammonium salts Cloisite® 63 10A and 30B showed potent antimicrobial activity; thus, active packaging obtained with 64 these and other modified clays was reported (Nigmatullin, Gao, & Konovalova, 2008; 65 Rhim, Hong, & Ha, 2009; Tornuk, Hancer, Sagdic, & Yetim, 2015). 66 Polyhydroxybutyrate (PHB) is a renewable thermoplastic material exhibiting intermediate 67 oxygen and water permeability compared to petroleum based polymers. Also, PHB is more 68 attractive than other biopolyesters like polylactic acid (PLA) in terms of barrier properties 69 (Corre, Bruzaud, Audic, & Grohens, 2012). Nevertheless, PHB has limited food packaging 70 applications due to its cost, narrow processability window, stiffness and brittleness 71 properties. Blending PHB with other polymers offers a way to obtain materials with 72 enhanced properties. Polycaprolactone (PCL) is a good candidate for this purpose due to 73 its biodegradable nature, its flexibility, toughness and thermal stability (Lovera et al., 74 2007). Aditionally, in order to improve PHB mechanical, gas barrier, and thermal 75 properties, nanofillers such as organo-clays could be incorporated at low filler content (less 76 than 5% by weight) and nanoscale distribution (Bordes, Pollet, Bourbigot, & Avérous, 4 77 2008; Botana, Mollo, Eisenberg, & Torres Sanchez, 2010). However, the incorporation of 78 nanofillers has raised concerns among several researchers, regarding the effects of particles 79 migration from the nanocomposites to the packaged food. The European regulation on 80 plastic materials and articles intended to come into contact with food (Commision 81 Regulation 10/2011) is rather specific with regard to nanoparticles and states that risk 82 assessment of materials in nanoform has to be performed on a case-by-case basis, until 83 more information is known in relation to this new technology. According to Souza & 84 Fernando (2016), nanoparticles could have potential to migrate to the packaged foodstuff, 85 but migration assays and risk assessment are still not conclusive. This scarce information 86 regarding migration is related to the lack of suitable and validated test methods for the 87 identification, characterization and detection of nanoparticles in complex matrices as food 88 (EFSA, 2011). Even when in vitro toxicological research on neat clays is of high interest 89 nowadays, studies dealing with nanocomposites containing clays are still scarcer. 90 Maisanaba et al. (2015) reported that different clays have their own cytotoxic profile with 91 dependence on the experimental conditions (type of clay, modifier, cell line, 92 concentrations used, etc). Despite neat clay Cloisite 30B showed high toxicity effects 93 (Maisanaba et al., 2015), particularly for a Organoclay/Poly(butylene adipate-co94 terephthalate) nanocomposite, no citotoxicity was observed with 10% of clay incorporation 95 (Fukushima, Wu, Bocchini, Rasyida & Yang, 2012). Similar results have been revealed for 96 in vitro and in vivo toxicity of organo-clays/PLA nanocomposites (Maisanaba et al. 2014a; 97 Maisanaba et al. 2014b). These authors also concluding that organo-clays/polymer based 98 nanocomposites need to be studied in greater detail, regarding safety issues, in order to 99 support their conclusions. 5 100 In this work, a PHB/PCL blend, with a high (50%) content of renewable polymer and 101 organo-clays (Cloisite® 30B and 10A) PHB/PCL based nanocomposites were activated 102 with nisin in order to develop antimicrobial films with enhanced material properties. The 103 effects of the type of organo-clay (Cloisite® 30B and 10A) on the intercalation of 104 polymeric chains into clay galleries, and on the final mechanical properties of the 105 biodegradable nanocomposites were studied. Nisin adsorption at different temperatures 106 was investigated for the PHB/PCL matrix looking for the best conditions to obtain an 107 antimicrobial biodegradable film. Nisin adsorption on PHB/PCL matrix with and without 108 clays was studied and compared to find out possible synergistic antimicrobial effects. 109 Finally, the ability of nisin activated PHB/PCL films to inhibit Lactobacillus plantarum 110 CRL691 (used as processed meat spoilage bacterium model) was explored for cooked ham 111 during 4-week period. 112 2. Materials and methods 113 2.1 Materials 114 Polyhydroxybutyrate (PHB) homopolymer commercial grade “Biocycle 1000” was 115 obtained from PHB Industrial S.A., Brazil, in the form of powder with a weight average 116 molecular weight of approximately 600,000 g/mol. Polycaprolactone (PCL) commercial 117 grade “FB-100” was obtained from Perstorp, United Kingdom, in the form of pellets with a 118 weight average molecular weight of 100,000 g/mol. Organo-clays Cloisite® 30B and 10A 119 (Clo 30B and Clo 10A) were obtained from Southern Clay Products, USA. 120 2.2 Bacterial strains and growth conditions 6 121 Lactobacillus plantarum CRL691 was grown in (Man, Rogosa and Sharpe) MRS broth 122 (Britania, Argentina) at 30ºC. The strain, kindly transferred by CERELA-CONICET 123 (Argentina), was maintained and stored at -20 ºC in 0.15 g cm-3 of glycerol until use. 124 2.3. Nanocomposite films preparation 125 Materials were dried during 8 h under vacuum at 80 °C (PHB and organo-clays) and 40 °C 126 (PCL). For this study, PHB/PCL in a 50/50% (w/w) proportion was used as the blend with 127 the highest content of renewable polymer without detrimental consequences on mechanical 128 properties and processabilty, due to the stiffness and high flowability of PHB. The 50/50 129 PHB/PCL blend and organo-clay based nanocomposites with a Clo10A and Clo30B fixed 130 content at 5% (w/w) were prepared by melt intercalation in an internal mixing chamber 131 Brabender Plasti-corder (30 cm3) at 165 °C and 50 rpm rotor speed for 5 min. 132 Nanocomposites were labeled as PHB/PCL/Clo10A and PHB/PCL/Clo30B. Films were 133 obtained by compression molding at 175 °C using an 8 min molding cycle. 134 2.4. Polymer and nanocomposites characterization 135 2.4.1. Oxidation onset temperature (OOT) 136 OOT was performed by dynamic scanning in a differential scanning calorimeter (DSC) 137 Mettler 822e/500/1473 using the method described in ASTM E 2009 (American Society 138 for Testing and Materials, 2014a). Measurements were carried out under an oxygen 139 atmosphere and analyzed from 30 °C to 350 °C at a heating rate of 10°C/min. Results of 140 two determinations were averaged and informed. 141 2.4.2. X-Ray diffraction (XRD) 142 Organo-clays structure and their PHB/PCL composites were evaluated with XRD 143 measurements. XRD patterns were taken with a Phillps PW 1730/10 X-ray diffractometer, 7 144 operated at 40 kV and 30 mA, equipped with Cu Ka radiation at a wavelength of 0.1546 145 nm. Diffraction data was collected over a 2θ range of 1°–10°, with a step width of 0.02° 146 and a counting time of 2.0 s/step. Silicate layer basal spacing (d001) was calculated using 147 the Bragg’s equation (1) 148 λ=2d sin θ (1) 149 Where λ is the wavelength of the X-ray radiation used (0.1546 nm), d is the spacing 150 between diffractional lattice planes and θ is the measured diffraction angle. 151 2.4.3. Tensile properties 152 Tensile testing was performed with an INSTRON universal testing machine model 5569 153 according to ASTM D 882-12 method (American Society for Testing and Materials, 2012). 154 The tests were carried out at 23 °C, with a constant rate of 5 mm/min, an initial grip 155 separation of 50 mm and ribbon-shape samples (10 mm width) according to ASTM D 882156 12 method. 157 2.4.4. Water Vapor Permeability (WVP) 158 Films WVP was measured using the cups method described in ASTM E 96/E96M -14 159 (American Society for Testing and Materials, 2014b). For this test, a cylindrical vessel 160 filled with desiccant (10 g of silica) was sealed with the investigated film and stored in a 161 chamber with controlled temperature and relative humidity (23°C and 50%, respectively). 162 Water mass uptake was monitored as a function of time and the Water Vapor Transmission 163 Rate (WVTR) was calculated from the slope of the mass uptake profile versus time at the 164 steady state. Then, WVP (g·s-1·m-1·Pa-1) was calculated using equation (2) 165 WVP = WVTR·d/ΔP (2) 8 166 Where WVTR is the water vapor transmission rate (g·s-1·m-2), d is the film thickness (m) 167 and ∆P is the water vapor partial pressure difference (Pa). Experiment was run in four 168 replicates. 169 2.5. Activation with nisin 170 2.5.1. Antimicrobial solutions 171 A nisin stock solution (8000 IU cm-3) was prepared by dissolving 800 mg of commercial 172 nisin powder (Maxinis®, AMG, Buenos Aires, Argentina) in 100 cm 3 of distilled water. 173 Thereafter different nisin concentration solutions (4000, 2000, 500, 200, 100 and 50 IU 174 cm-3) were prepared from the stock solution. 175 2.5.2. Adsorption kinetic and equilibrium. 176 500 IU·cm-3 of nisin was previously definded as the minimum inhibitory concentration 177 necessary to obtain a PHB/PCL film active against L. plantarum CRL691 (data not 178 shown). To optimize nisin adsorption times and temperatures the PHB/PCL film (0.95 179 cm2) was contacted with 0.260 cm3 of 500 IU cm-3 nisin solution during different times (1, 180 5, 10, 15, 30, 60 and 120 min) at 20, 30 and 40 °C. 181 Nisin adsorption isotherms were obtained by contacting the PHB/PCL film with different 182 peptide concentration solutions (4000, 2000, 500, 200, 100 and 50 IU cm-3 of nisin) during 183 30, 15 and 10 min, respectively at 20, 30 and 40 °C. 184 Bacteriocin adsorption capacity on PHB/PCL; PHB/PCL/Clo10A and PHB/PCL/Clo30B 185 was studied by contacting nisin (4000 IU cm-3) during 10 min at 40 °C. 186 After activation treatments, activity on the films and in nisin solutions were evaluated in 187 semisolid agar as described in section 2.5.3. In all cases, triplicate samples were run in two 188 independent experiments. 9 189 2.5.3. Activity quantification 190 Nisin activity in solution (titer) was determined by the agar well diffusion assay 191 (Pongtharangkul & Demirci, 2004). Serial two-fold dilutions of the bacteriocin solutions 192 (15 μl) were added to 5 mm diameter wells cut in semisolid MRS agar plates seeded with 193 L. plantarum CRL691 (107 CFU·cm-3). After incubation (18 h at 30 ºC) nisin titer, 194 expressed in arbitrary units (AU cm-3), was defined as the reciprocal of the highest dilution 195 yielding a visible zone of inhibition on the sensitive strain. All determinations were 196 performed in triplicate. 197 To quantify nisin on film surfaces the zone of inhibition assay was used. This method is 198 based on the fact that the area of each inhibition zone directly corresponds to the quantity 199 of bacteriocin retained by samples (Massani et al., 2013; Bower et al., 2002; Blanco 200 Massani et al., 2012; Green, Fulghum, & Nordhaus, 2011). For this evaluation, nisin 201 treated film circles (0.95 cm2) were placed face down on semisolid agar plates seeded with 202 L. plantarum CRL691 (107 CFU·cm-3) and analized for inhibition zones. Clo30B, Clo10A 203 and their PHB/PCL nanocomposites inhibition zones were also studied. ImageJ 1.47t 204 (Wayne Rasband, National Institutes of Health, USA) was used to measure antimicrobial 205 activity as relative inhibition areas (RIA=inhibition zone area/film area). 206 2.6. Sliced cooked ham active packaging 207 2.6.1. Cooked ham elaboration 208 Cooked ham was manufactured in a meat processing pilot plant according to Argentinean 209 regulations (“C.A.A.,” 1893). A raw pork leg was trimmed off and minced (Themis 32 210 mincer). Extension of 60% was obtained by mixing the pulp with brine (water, 211 carrageenan, sucrose, maltodextrin, sodium chloride, erythorbate, phosphate, nitrate and 10 212 nitrite). Massage during 40 min was performed (KitchenAid® Stand Mixer) to allow brine 213 homogeneous distribution. Pulp was cooked by a cook-in process in a 80 °C water bath to 214 a core temperature of 72 °C. After cooling (3 °C), the ham was sliced, vacuum-packed 215 (90%-Erlich Best Vacuum), post pasteurized (10 min, 80 °C) and stored (5-6 °C). 216 2.6.2. Ham inoculation and active packaging 217 L. plantarum CRL691 was resuspended in steryle saline solution (NaCl 8.5 mg cm-3) to 218 reach 105 CFU·cm-3 (0.15 Abs530). Ham slices (250 g) were inoculated by immersion (30 s) 219 in 25 cm3 of the L. plantarum CRL691 suspension, reaching a final concentration of 103 220 CFU per gram of ham. After drying, three PHB/PCL nisin activated films (36 cm2 each) 221 were used to separate five inoculated slices (25 g) which were included inside a packaging 222 made with untreated Cryovac films (96 cm2). Following the same procedure PHB/PCL 223 control (without nisin) films were used to obtain control ham packages. Packages were 224 thermo-sealed under vacuum (90%-Erlich Best Vacuum) and stored (5 °C, 4 weeks). This 225 experiment was run in two independet experiments by duplicates. 226 2.6.3. Microbiological determinations 227 After inoculation and at 7, 14, 21 and 28 days of storage (5 °C) microbiological 228 evaluations were performed in 10 g obtained by transversely cutting the sliced hams. Each 229 sample was minced with 90 cm3 of sterile saline solution for 1 min. Homogenate dilutions 230 were obtained with sterile saline solution and duplicate counts of L. plantarum CRL691 231 were obtained after incubation (anaerobic conditions, 35 °C, 48h) in MRS. Results were 232 expressed as log CFU g-1. Lag time λ was estimated using the MicroFit v 1.0 software 233 (Institute of Food Research, United Kingdom). pH was measured in all packages. 234 2.7. Statistical analysis 11 235 Experimental data was subjected to analysis of variance (ANOVA). Tukey test was applied 236 at 0.05 of significance level. All statistical analyses were performed using Minitab Statistic 237 Program, release 12 (Pennsylvania, USA). 238 3. Results and discussion 239 3.1. Polymer and nanocomposites characterization 240 Table 1 presents OOT values, mechanical and barrier properties of PHB/PCL blend and 241 PHB/PCL-based nanocomposites. The addition of organo-clays improved nanocomposites 242 degradation temperature compared to the blend without clays (18 °C for Clo10A and 24 °C 243 for Clo30B). It is known that layered silicates improve thermal stability of the polymer 244 matrix, acting as a heat barrier, raising the overall thermal stability of the system, and 245 enhancing oxygen and oxidation products barrier during thermal decomposition 246 (Alexandre & Dubois, 2000). From an industrial point of view, this is an interesting result 247 since the improvement of the OOT values for the nanocomposites indicates an increase in 248 the processing temperature upper limit for these materials without a significant level of 249 degradation, which implies a wider processing window. 250 Clo30B and Clo10A X-ray diffraction (XRD) patterns (Fig. 1) revealed diffraction peaks at 251 2θ= 4.94 and 2θ= 4.53 respectively, corresponding to 17.9 Å (Clo30B) and 19.5 Å 252 (Clo10A) layer distance. Clay´s peaks were shifted to lower angle for nanocomposites, 253 corresponding to higher interlayer distances for PHB/PCL/Clo30B (37.1 Å) and 254 PHB/PCL/Clo10A (36.9 Å). The higher basal spacings of clays in the nanocomposites as 255 compared to the pristine organo-clays are due to the intercalation of polymer chains inside 256 the clay layers (Alexandre & Dubois, 2000). The extent of this intercalation calculated as 257 an increase in d-spacing (∆d001) was higher for PHB/PCL/Clo30B than for 12 258 PHB/PCL/Clo10A (19.2 and 17.5 Å, respectively). This result could be related to the 259 strong hydrogen bonding and interactions between the carboxyl groups of PHB and PCL in 260 the blend and the hydroxyl group in the gallery of Clo30B. Similar results were reported 261 for the preparation of other biodegradable polyester/organo-clay nanocomposites (Bordes 262 et al., 2008; Botana et al., 2010; Ray & Bousmina, 2005). The peaks around 2θ= 4.54 263 (PHB/PCL/Clo30B) and 4.38 (PHB/PCL/Clo10A) may be originated by agglomerated 264 organo-clay particles caused by collapse of interlayer space of clays due to the thermal 265 degradation of the alkyl ammonium ions during nanocomposites processing and/or the d002 266 reflection (Botana et al., 2010; Najafi, Heuzey, & Carreau, 2012). 267 According to Alexandre & Dubois, (2000) and Carli, Crespo, & Mauler, (2011), clays 268 intercalation process increases tensile modulus. Results of mechanical properties (Table 1) 269 showed that clays addition promoted a reinforcement effect given by a significant increase 270 in the tensile modulus (around 27% for Clo10A and 50% for Clo30B), while tensile 271 strength was not affected in comparison with PHB/PCL neat blend. However this 272 enhancement in tensile properties was at the expense of the deformation at break with both 273 types of clays. This behavior is in agreement with published results on 274 PHA/montmorillonite nanocomposites, and can be attributed to either degradation of the 275 clay organomodifier that may alter physical and mechanical properties of the polymer 276 matrix, or embrittlement due to the presence of some non intercalated clay stacks (Bordes 277 et al., 2008; Carli et al., 2011). 278 WVP is an important property because it indicates the amount of water vapour that 279 permeates per unit of area and time through the packaging material. WVP values of the 280 PHB/PCL and PHB/PCL-based nanocomposites (Table 1) showed that organically 13 281 modified clays significantly increased barrier properties, since a decrease in 282 nanocomposites WVP when comparing to that of neat PHB/PCL 50/50 blend was 283 observed (59% for films compounded with Clo10A and 52% for films with Clo30B). 284 Reduction of the WVP values observed in the nanocomposites can be attributed to long 285 and tortuous paths created by clay platelets distributed in the polymer matrix, slowing 286 down the progress of water molecules through the matrix (Alexandre & Dubois, 2000). 287 This result is important since lower WVP values indicate better moisture protection to the 288 packaged food and, consequently, a potential reduction of food deterioration due to 289 excessive gain of water during storage (Siracusa, 2012). Also, WVP values of PHB/PCL290 based nanocomposites found in our work (Table 1) are similar to the informed for neat 291 PLA commercial extruded films (1.8 x 10-11 g·s-1 ·m-1·Pa-1) (Corre et al., 2012). 292 The nanocomposites obtained in this study presented improved thermal stability, 293 mechanical, and barrier properties, compared to the neat PHB/PCL blend. The feasibility 294 of improving these properties after organo-clays addition to the blend reveals true potential 295 to expand the range of biodegradable materials currently used for various packaging 296 applications. 297 3.2. Films activation 298 3.2.1. Adsorption kinetic and equilibrium 299 For the development of active polymers for potential food applications, the study of 300 bacteriocins adsorption on a determined surface yields to the knowledge of activation 301 parameters (optimal time/temperature, shape of adsorption curves and saturation point), 302 which can be obtained from the analyses of kinetics an equilibrium of adsorption (Massani, 303 Vignolo, Eisenberg, & Morando, 2013). When assaying relative inhibition areas, the size 14 304 of the inhibition zone is related to the diffusion constant for the antimicrobial agent 305 evaluated in the media as well as to the total amount of agent that is available to diffuse 306 (Green, Fulghum, & Nordhaus, 2011). That is to say, for the same experimental conditions 307 (adsorbed antimicrobial tested, culture media, sensitive strain and inoculum size, 308 incubation temperature for culture growth, etc.) and for bacteriocins that completely elute 309 from the surface, the area of each inhibition zone directly corresponds to the quantity of 310 bacteriocin adsorbed by samples (Bower et al., 2002; Green et al., 2011; Massani, Vignolo, 311 Eisenberg, & Morando, 2013). In order to find the kinetic of adsorption at different 312 temperatures, a 500 IU cm-3 nisin solution was contacted with the PHB/PCL film during 313 different times (1 to 120 min) and the correspondings RIA were analyzed. Adsorption 314 plateau was defined as the time, at a fixed temperature, from which RIA were not 315 significantly different (p≥ 0.05) (Fig. 2). It was observed that activation temperature 316 affected both, the maximum of adsorption (plateau) and the time needed to reach the 317 plateau. Thus, at lower activation temperature higher times were required to reach the 318 adsorption plateau, being respectively 30, 15 and 10 minutes, the necessary times for 319 adsorption equilibrium attainment at 20, 30 and 40 °C (Fig. 2). In contrast, Guerra et al., 320 (2005a) studying nisin adsorption in rubber, PET and stainless steel at 8, 25, 40 and 60 °C 321 reported that temperature had no influence on adsorption time (12 h). In our study 322 adsorption acceleration by temperature was observed, obtaining the lowest time for 323 adsorption equilibrium (10 min) at 40 °C this probably indicating that temperature increase 324 favoured dehydration of nisin in aqueous solution, promoting bacteriocin adsorption to the 325 surface as already explained by Norde (1996) and previously found for other bacteriocins 326 (Massani et al., 2013). Nevertheless, the highest RIA at the plateu attainment was observed 15 327 at 30°C, then films nisin activity following the order 20 °C>40 °C (Fig. 2). Temperature 328 rise could be leading to a decrease in bacteriocin active concentration available to be 329 adsorbed on the film, as earlier reported (Massani et al., 2013). The results on adsorption 330 kinetics in our system (nisin-PHB/PCL film) led to adsorption times (10 min at 40°C; 15 331 min at 30°C and 30 min at 20°C) lower than those reported for activation with nisin of 332 other surfaces such as cellophane, PET, and stainless steel (12 h at 8°, 25°, 40° and 60°C) 333 (Guerra et al., 2005a; Guerra, Macias, Agrasar, & Castro, 2005b). 334 For the purpose of modeling nisin adsorption to the PHB/PCL film at different 335 temperatures and constructing adsorption isotherms, the bacteriocin was contacted with the 336 film in different concentrations at 20, 30 and 40 °C. Results of nisin RIA as a function of 337 the bacteriocin titers after adsorption were fitted as shown in Figure 3. For all the assayed 338 temperatures, it was observed that nisin RIA on the activated film increased with the 339 concentration of nisin contact solution up to a point (nisin contact solution 4000 IU cm-3, 340 34133 AU·cm-3 after adsorption) from which RIA were not significantly different from 341 each other (p≥0.05). Thus, a steep initial slope of bacteriocin adsorption on PHB/PCL 342 followed by an adsorption plateau at higher concentrations was observed (Fig. 3). This 343 behavior could be subjected to an empirical treatment according to Langmuir equation 344 which implies monolayer adsorption (Guerra et al., 2005a). From this, a PHB/PCL film 345 saturation point begining with 4000 IU cm-3 nisin contact solution was defined. From 346 equilibrium determinations (Fig 3) the same adsorption plateau at different temperatures 347 was obtained, suggesting that 40 °C could be the best activation temperature, due to the 348 reduction of contact time in comparison to 20 and 30 °C. As a consequence, 40 °C, 10 min 16 349 and a 4000 IU cm-3 nisin contact solution were defined as the optimum parameters for 350 PHB/PCL matrix activation. 351 3.2.2. Materials antimicrobial activity and activation comparison 352 Clo10A, Clo30B, PHB/PCL and their nanocomposites were assayed against L. plantarum 353 CRL691 to study their antimicrobial activity. It was observed that even when Clo10A and 354 30B were active against the microorganism, antimicrobial activity was not observed for 355 either film without nisin treatment (Table 2). Clay/polymer nanocomposites biocidal 356 activity is a function of antimicrobial interchanged cation, and the composites 357 nanostructure. It appears that at least two mechanisms are responsible for the antimicrobial 358 activity of the organo-clays, i.e., activity of surfactants released from the clay and the 359 action of the solid surface (Nigmatullin et al., 2008). Partial degradation of alkyl 360 ammonium ions during nanocomposites processing as previously discussed (section 3.1; 361 Botana et al., 2010; Carli et al., 2011) could be a possible explanation for antimicrobial 362 activity lack in PHB/PCL nanocomposites. On the other hand, intimate contact between a 363 bacterial cell and organo-clay antimicrobial surface may be crucial to affect the cell 364 (Nigmatullin et al., 2008). In order to describe interlayer organo-clays structure, it is 365 believed that the cationic head group of the alkyl ammonium molecule preferentially 366 resides at the layer surface, leaving the organic tail radiating away from the surface 367 (Alexandre & Dubois, 2000). Botana et al., (2010) reported that when PHB crystallizes in 368 presence of clay mineral particles, crystals could grow on the particle surfaces. Moreover, 369 PHB/PCL nanocomposites thermal studies (DSC) suggested that clays may be acting as 370 crystallization agents (Correa et al., unpublished results). Then, interchanged cations from 371 clays filled into PHB/PCL matrix might be entrapped into the PHB crystals, being not 17 372 available to migrate in a concentration above the minimum inhibitory concentration needed 373 to inhibit the sensitive microorganism. Tornuk et al. (2015) reported antimicrobial activity 374 of LLDPE nanocomposites (based on MMT and halloysite) grafted with bioactive 375 compounds when using in processed meat packaging applications. In our work, since 376 organoclays addition did not impart antimicrobial activity to the PHB/PCL based film, 377 nisin adsorption was suggested in order to reach an active material capable of inhibiting 378 lactic acid bacteria (LAB) during cooked ham storage. 379 PHB/PCL organo-clay films were treated with nisin using the optimized parameters (40 380 °C, 10 min, 4000 IU·cm-3 nisin contact solution), assayed against L. plantarum CRL691 381 and compared to nisin activated PHB/PCL (Table 2). All surfaces exerted uniform 382 inhibition areas and RIA presented no significant differences (p≥0.05) (Table 2). As 383 aboved mentioned, the relative inhibition area displayed by a bacteriocin treated surface 384 has already been associated to the amount of bacteriocin adsorbed; i.e., the smallest 385 inhibition zone corresponded to the smallest bacteriocin adsorbed mass (Massani, Vignolo, 386 Eisenberg, & Morando, 2013; Bower et al., 2002). Based on this assumption, the lack of 387 significant differences between the RIA found after activating PHB/PCL and organo-clay 388 films with nisin (Table 2) could be indicating that nisin activation was not influenced by 389 organo-clays addition to the PHB/PCL film. Karam et al. (2013) performing XPS and 390 time-of-flight secondary ion mass spectrometry measurements on nisin adsorbed to LDPE 391 and the polymer grafted with acrylic acid confirmed that the peptide formed a multilayer 392 coverage, and films antimicrobial activity was related to nisin distribution on the surfaces. 393 In our work, nisin antimicrobial activity on a neat PHB/PCL and PHB/PCL nanocomposite 18 394 films was uniform (Table 2) and peptide adsorption seemed to occur by monolayer 395 coverage (Fig. 3). 396 As discussed in section 3.1, nanocomposites presented better properties when compared 397 with neat PHB/PCL blend. However, either the barrier or the mechanical properties 398 observed in our work were not sufficient to satisfy all the requirements imposed on the 399 materials used for meat products packaging (Sukhareva, Yakovlev, Legonkova, & Zaikov, 400 2008; Kerry, Kerry & Ledwar, 2002). For this reason, to asses the efectiveness in a 401 processed meat product, the materials obtained in this work were proposed as ham 402 separating films. Also, considering that either PHB/PCL film or organo-clays added 403 PHB/PCL films showed the same performance as nisin carriers, and until having more 404 insights into the safety issues concerning the use of nanocomposites in direct contact with 405 foods (European Commission, 2011; EFSA, 2011; Maisanaba et al, 2015; Souza & 406 Fernando, 2016), neat PHB/PCL films were used as nisin support for experiments 407 performed to asses effectiveness in food. 408 3.3. PHB/PCL film as active material for ham storage 409 During ham production, the aim of thermal processing is to reduce the number of bacteria 410 present to a reliable level which ensures the safety and stability of the product. 411 Nevertheless, cross-contamination occurs throughout subsequent product handling, slicing 412 and packaging (Dušková, Kameník, Lačanin, Šedo, & Zdráhal, 2016). LAB were informed 413 as a microbial population capable of reaching numbers corresponding to the spoilage 414 threshold (around 6 to 7 log CFU g-1) in cooked hams (Dušková et al., 2016; Pothakos, 415 Samapundo, & Devlieghere, 2012). Figure 4a shows the growth of L. plantarum CRL691 416 obtained for inoculated active (PHB/PCL nisin activated separating films) and control 19 417 (PHB/PCL separating films) ham packages. In control packages, typical LAB grown was 418 observed with an initial count of 3.48±0.13 log CFU g-1 units, raising to 6.60±0.20 log 419 CFU g-1 at the end of the experiment (28th day) (Fig 4a). Meanwhile for active packages, 420 inhibition of the LAB was observed up to the 21st day (3.16±0.56 log CFU g-1), then 421 reaching 3.99±0.20 log CFU g-1 (28th day) (Fig 4a). These results are in agreement with 422 Kalschne et al., (2014) who examined the possibility of controlling LAB development on 423 vacuum-packed sliced cooked ham by the addition of nisin. The mentioned authors found a 424 decrease of LAB growth by 2 or 3 log cycles upon antimicrobial addition. Data shown in 425 Fig 4a clearly indicates a bacteriostatic inhibition effect over L. plantarum CRL691 426 inoculated on ham, with 3.4 log cycles reduction at the 21st day, and 2.6 log cycles 427 reduction at the end of the experiment (28th day). Lag phase extention is essential in order 428 to avoid unwanted microorganisms growth. Lag phase, calculated with MicroFit software, 429 was extended from 7.03 to 22.39 days due to nisin effect in the active packages, avoiding 430 LAB counts to reach more than 6 log units (Fig 4a), thus prolonging ham shelf life up to 28 431 days (Dušková et al., 2016; Kalschne, Geitenes, Veit, Sarmento, & Colla, 2014; Pothakos 432 et al., 2012). 433 pH changes in active and control packages during storage time are shown in Figure 4b. A 434 slight decrease in control packages at the end of the storage was observed (6.36 to 6.10), 435 while pH levels remained unaffected for active packages (Fig 4b). pH decrease in control 436 packages could be explained by the inoculated LAB growth. Our results of pH invariability 437 around 6.3 due to LAB inhibition in packages containing nisin are in coincidence with data 438 reported by Kalschne et al. (2014). 439 4. Conclusions 20 440 PHB/PCL biodegradable polymer blend and organo-clay based nanocomposites were 441 studied and characterized as nisin carriers for their application in food packaging. 442 Cloisite® 30B showed better intercalation and interaction with the PHB/PCL matrix than 443 Cloisite® 10A. The nanocomposites obtained in this study presented better thermal 444 stability, mechanical and barrier properties compared to the neat PHB/PCL blend. The 445 feasibility of improving these properties offers the possibility to expand the range of 446 biodegradable materials that could be used for different packaging applications. The 447 optimal parameters for PHB/PCL film activation with nisin were 4000 IU·cm-3 contact 448 solution, 40 °C and 10 min. Organo-clay fillers did not exert synergistic antimicrobial 449 effects when combined with nisin upon films activation. PHB/PCL nisin activated film was 450 effective in inhibiting L. plantarum CRL691 inoculated on ham. This work opens the 451 possibility to consider nisin activated PHB/PCL polymer blend and/or PHB/PCL/organo452 clay based nanocomposites as active materials for potential food packaging applications. 453 The feasibility of obtaining an entirely biodegradable antimicrobial packaging with nisin, 454 PHB/PCL and nanocomposites involving organoclays is further to be studied. So far, we 455 demonstrated the workability of these blends upon nisin activation, and nisin-PHB/PCL 456 effectiveness as shelf life extender for vacuum-packed sliced cooked ham. 457 Acknowledgments 458 We acknowledge research grants and financial support from CONICET and ANPCyT 459 [PICT 2011-2690]. Acknowledgements are also extended to the assistance of Jorge Maggi 460 and Rosa Torres (CETMIC-CONICET) in XRD analyses, Mariela Giberti and Nora 461 Schicchi for substantial contribution during the statistical analysis of this work. 462 References 21 463 Alexandre, M., & Dubois, P. (2000). 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XRD patterns of pristine Cloisite 10A (Clo 10A), Cloisite 30B (Clo 30B) 634 organo-clays and their Polyhydroxybutirate/Polycaprolactone/Clay based nanocomposites 635 (PHB/PCL/Clo 10A and PHB/PCL/Clo 30B). d(001) basal spacings were included in Å. 636 Figure 2. Nisin adsorption on PHB/PCL at 20°C (), 30°C () and 40°C () during 637 different contact times. Continuous lines mark tendencies. Error bars indicate standard 638 deviations from two independent experiments by triplicates. 639 Figure 3. Nisin adsorption isotherms on PHB/PCL at 20°C (), 30°C () and 40°C (). 640 The curves drawn through the data follow Langmuir equation. Error bars indicate standard 641 deviations from two independent experiments by triplicates. 642 Figure 4. L. plantarum CRL961 growth (a) and pH changes (b) during 28 days at 5 ºC in 643 active () and control () ham packages. Lines between points mark tendencies. Error 644 bars indicate standard deviations from two independent experiments by duplicates. 645 30 646 Abbreviations and acronyms 647 PHB, Polyhydroxybutyrate/ 648 PCL, Polycaprolactone 649 IU, International units 650 AU, Arbitrary units 651 Clo 30B, Cloisite® 30B 652 Clo 10A, Cloisite®10A 653 OOT, Oxidation onset temperature 654 DSC, Differential scanning calorimeter 655 XRD, X-Ray diffraction 656 WVP, Water Vapor Permeability 657 RIA, relative inhibition areas 658 PLA, Polylactic acid 659 MMT, Montmorillonite 660 LAB, lactic acid bacteria 661 31 1 Table 1. Indicated material properties average for neat PHB/PCL 50/50 blend and PHB/PCL/clay 2 nanocomposite films. Property PHB/PCL PHB/PCL/ Clo 10A PHB/PCL/ Clo 30B OOT (°C)† 224.1±0.2 a 242.4±2.1 b 248.2±0.9 c Tensile Modulus (MPa)‡ 817 ± 45 a 1035 ± 81 b 1226 ± 46 c Tensile Strength (MPa) ‡ 6.29± 1.42 a 7.49± 0.89 a 7.06 ± 1.96 a Elongation at break (%) ‡ 3.03± 1.71 a 0.91 ± 0.16 b 0.72 ± 0.19 b WVP (x10-11 g·s-1·m-1·Pa-1)† 2.62± 0.15 a 1.05± 0.06 b 1.26± 0.07 c 3 a-c Different letters in the same line indicate significative differences (p <0.05). 4 † Mean of four determinations ± standard deviation. 5 ‡ Mean of five replications ± standard deviation. 6 PHB/PCL, Polyhydroxybutyrate/ Polycaprolactone 7 Clo10A, Cloisite® 10A 8 Clo30B, Cloisite® 30B 9 OOT, Oxidation onset temperature 10 WVP, Water Vapor Permeability 1 Table 2. Antimicrobial activity of organically modified montmorillonites, nisin treated and non2 treated films. Sample Relative inhibition area against L. plantarum CRL691 Inhibition behavior on semisolid agar Clo 10A 1.5±0.3 a Clo 30B 2.2±0.2 b PHB/PCL ND † PHB/PCL/Clo 10A PHB/PCL/Clo 30B ND † ND † PHB/PCL + nisin 5.5±0.5 c PHB/PCL/Clo 10A + nisin 5.9±0.7 c PHB/PCL/Clo 30B + nisin 5.9±0.9 c 3 a-c Different letters within the table indicate significative differences (p<0,05) from two 4 independent experiments by triplicates. 5 † ND, not detected 6 Clo10A, Cloisite® 10A 7 Clo30B, Cloisite® 30B 8 PHB/PCL, Polyhydroxybutyrate/ Polycaprolactone ! "#Ver+/- |