Development of a novel bioactive packaging based ...

Food Research International 43 (2010) 2402–2408

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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s

Development of a novel bioactive packaging based on the incorporation of Lactobacillus sakei into sodium-caseinate films for controlling Listeria monocytogenes in foods Haralampos Gialamas b, Kyriaki G. Zinoviadou a, Costas G. Biliaderis a, Konstantinos P. Koutsoumanis b,⁎ a b

Laboratory of Food Chemistry and Biochemistry, Department of Food Science and Technology, School of Agriculture, Aristotle University, GR-541 24, Thessaloniki, Greece Laboratory of Food Microbiology and Hygiene, Department of Food Science and Technology, School of Agriculture, Aristotle University, GR-541 24, Thessaloniki, Greece

a r t i c l e

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Article history: Received 21 July 2010 Accepted 14 September 2010 Keywords: Active packaging Edible film Lactobacillus sakei Antilisterial activity Mechanical properties Beef biopreservation Sodium caseinate

a b s t r a c t A novel packaging technology was developed based on the incorporation of Lactobacillus sakei cells into sodiumcaseinate (SC) edible films. Incorporation was based either on direct addition of the cells in the film forming solution used for casting or by surface spraying of the culture on the preformed film, resulting in a population density of 106 cfu/cm2. Addition of sorbitol in the film matrix increased the viability of the cells, greater than 90%, upon storage under both refrigeration and ambient temperature conditions for 30 days. Incorporation of the viable protective culture did not affect the mechanical properties and the physico-chemical properties of the film. Application of the films to both laboratory medium (agar) and a food model system (fresh beef) inoculated with Listeria monocytogenes resulted in a rapid growth of L. sakei immobilized in the film following contact with the wet medium or the food surface and a significant inhibition of the pathogen growth compared to the control samples under both constant and dynamic storage temperature protocols. The present study indicated that biopolymerbased antimicrobial films containing cells of a protective culture can be used as an effective packaging technology for improving food safety. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Over the past years different changes have occurred in consumer demands for quality products, eating habits and the retail food market. The increased interest in “ready to eat” and easy to consume products enhances the obligation for greater control on food quality and safety. Furthermore, consumers prefer foods that are minimally processed, contain fewer preservatives and additives, but maintain an unimpaired sensorial quality. In this sense, preservation of foods by application of more natural technologies can be an advantageous approach in order to solve many of the food spoilage-safety related issues. Listeria monocytogenes is a foodborne pathogenic bacterium that causes listeriosis, a severe disease, especially to elderly, pregnant women and newborns (Alves, Martinez, Lavrador & De Martinis, 2006). Control of this pathogen is difficult due to its ability to grow even at refrigerated temperatures and its tolerance to low pH values and certain preservatives, such as sodium chloride, which are inhibitory to other food pathogens (Hugas, Pages, Garriga & Monfort, 1998). Additionally, it can survive attached to the surfaces of processing equipment, via biofilm formation, and thereby become a potential contamination source (Castellano, Belfiore, Fadda & Vignolo,

⁎ Corresponding author. Tel./fax: + 30 2310 991647. E-mail address: [email protected] (K.P. Koutsoumanis). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.09.020

2008). Contamination of food products with L. monocytogenes may occur in different stages of the production chain even during slicing or packaging of the product. Control of L. monocytogenes in foods is usually based on the use of chemical antimicrobials (Barmpalia et al., 2005). Over the last years, however, the food industry, in an effort to meet consumer demands for naturally preserved products, has focused on the research for alternative, chemical-free preservation methods. Biopreservation refers to the extended shelf life and enhanced safety of food products using their natural or controlled microflora and for that reason it has gained increasing attention. Lactic Acid Bacteria (LAB) are the main “tool” of biopreservation due to their ability to produce metabolic products with strong antimicrobial effect against pathogenic bacteria. Inhibition of pathogenic micro-organisms by LAB may be due to the effect of one or synergism between several mechanisms, such as competition for nutrients, lowering of pH, production of lactic acid, acetic acid, hydrogen peroxide, gas composition of atmosphere or production of antimicrobial substances such as bacteriocins (Skytta, Hereijers & Mattila-Sandholm, 1991; Vandenbergh, 1993; Drosinos & Board, 1994; Cleveland, Montville, Nes & Chikindas, 2001; Jones, Zagorec, Brightwell & Tagg, 2009). Up to now edible or biodegradable films have been used as carriers of many functional ingredients. Such ingredients may include antioxidants, antimicrobial agents, flavors, spices and colorants which improve the functionality of the packaging materials by adding novel

H. Gialamas et al. / Food Research International 43 (2010) 2402–2408

or extra functions (Salmieri & Lacroix, 2006). A lot of research has been conducted on the effectiveness and the applications of antimicrobial packaging in the food industry and this subject has been thoroughly reviewed (Cagri, Ustunol & Ryser, 2004; Cha & Chinnan, 2004; Coma, 2008; Quintavalla & Vicini, 2002). In contrast to the large amount of information on the antimicrobial activity of edible films containing antimicrobials, to our knowledge no information is available about the antimicrobial activity of bacterial cells immobilized in biopolymer films. Research on the possibility of using such polymeric materials as carriers of viable bacteria cells that exhibit antimicrobial activity against selected pathogens would lead to an alternative natural preservation method, easily applicable and of low cost. Therefore, the objectives of the present study were (a) to develop a novel edible bioprotective packaging film based on the incorporation of L. sakei cells into a sodium-caseinate film matrix; (b) to evaluate the impact of the bacterial cells present on the physicochemical properties of the films; (c) to examine the viability of the immobilized L. sakei in the protein films on storage under both refrigeration and ambient temperature conditions; and (d) to determine the antimicrobial effectiveness of the film against L. monocytogenes growth in laboratory media and an actual food model system. 2. Materials and methods 2.1. Bioprotective film preparation Sodium caseinate, SC (Wako Chemicals, Japan), was dissolved in distilled water under continuous stirring to obtain film-forming solutions of 4% (w/w) protein concentration. Sorbitol (St. Louis, MO, USA) was added as plasticizer to the polymer solution at a constant solids concentration of 30% (sorbitol/(SC + sorbitol)). Such a concentration of sorbitol was necessary to overcome the brittleness of the SC films. SC solutions were subsequently vacuum-degassed to remove air bubbles. Portions of 12.5 g solution were casted on Petri dishes (8.5 cm) and allowed to dry in an oven at 40 °C for 24 h. Film thickness was determined using a manual micrometer at 5 random positions on the film. A L. sakei strain (LQC 1089) isolated from meat (kindly provided by Prof. E. Drosinos, Laboratory of Food Quality Control and Hygiene, Agricultural University of Athens) was used throughout this study for the preparation of the bioactive films. The selection of the strain was based on its higher antimicrobial activity against L. monocytogenes observed in preliminary experiments compared to 10 other LAB strains tested (data not shown). The stock culture was kept frozen (−30 °C) in MRS/glycerol broth (70:30 v/v) and was regenerated by transferring aseptically 0.01 ml into 10 ml of MRS broth and incubating at 30 °C for 24 h. Cells were harvested by centrifugation at 6000 rpm for 20 min and washed twice with sterile Ringer's solution. L. sakei was incorporated either by adding the bacterial cells preparation into the film forming solution prior to casting (0.1 mL per 12.5 g of the film forming solution) or by spraying 0.1 mL of the bacterial cells preparation on top of a preformed SC film. 2.2. Water sorption isotherms Water sorption isotherms were determined for all films according to Biliaderis, Lazaridou and Arvanitoyannis (1999). Film samples (~300 mg) were placed in previously weighed aluminum dishes and dried at 45 °C in an air-circulated oven over silica gel (Sigma-Aldrich GmbH, Germany) until constant weight. The samples were subsequently kept in desiccators over saturated salt solutions of known relative humidity (RH), ranging from 11 to 84% at 25 °C for at least 21 days, a time sufficient to reach constant weight and hence practical equilibrium. The water content of samples, after storage at the specified relative humidity environment, was determined by drying at 110 °C for 2 h. Measurements were performed in triplicate.

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2.3. Water vapor permeability Water vapor permeability (WVP) measurements of the films were conducted at 25 °C using the ASTM (E96-63 T) procedure modified for the vapor pressure at the film's underside according to McHugh, Avena-Bustillos, and Krochta (1993). Film disks, previously equilibrated at 53% RH for 48 h, were sealed to cups containing distilled water and the cups were placed in an air-circulated oven at 25 °C that was equilibrated at 53% RH using a saturated solution of Mg(NO3)2 6H2O (Merck KgaA, Darmstadt, Germany). Film permeability was determined as described by Kristo, Biliaderis, and Zampraka (2007). The steady-state water vapor flow was reached within 2 h for all films. Slopes were calculated by linear regression and correlation coefficients for all reported data were 0.99. At least six replicates of each film type were tested for WVP. 2.4. Mechanical properties Films were cut in dumbbell form strips and stored at appropriate RH environments (11%, 43%, 53% and 75%) for 10 days to obtain films with different moisture contents. Film thickness was measured at three different points with a hand-held micrometer and an average value was obtained. Samples were analyzed with a TA-XT2i instrument (Stable Micro systems, Godalming, Surrey, UK) in the tensile mode operated at ambient temperature and a crosshead speed of 60 mm/min. Young's modulus (E), tensile strength (σmax) and % elongation at break (% EB) were calculated from load-deformation curves of tensile testing. Measurements represent an average of at least eight samples. The water content of samples, after storage, was determined by drying at 110 °C for 2 h. 2.5. Viability of L. sakei during storage of the film The viability of L. sakei was tested on films, with or without incorporation of sorbitol (at 30% on a dry basis), and stored at 4 °C and 25 °C, simulating refrigeration and ambient conditions of distribution and storage. The films were stored in Petri dishes in high-precision (±0.2 °C) low-temperature incubators (Sanyo MIR 153, Sanyo MIR 253, Sanyo Electric, Ora-Gun, Japan) in which the precise temperature during storage was recorded with the aid of electronic temperature recorders (Cox Tracer, Cox Technologies, Belmont, NC, USA). At appropriate time intervals the films were removed aseptically from the Petri dishes and placed in a sterile BagPage (Interscience, France) and 100 ml of ringer solution (International Diagnostics, UK) was added to each bag and stomached for 2 min (Stomacher Interscience, France). Decimal dilutions in quarter-strength Ringer solution were prepared and 1- or 0.1-mL sample of appropriate dilutions were spread on MRSA (1.10660, Merck) and incubated at 30 °C for 96 h. All tests were run in duplicate and repeated twice. 2.6. Antimicrobial activity of the film against L. monocytogenes The antimicrobial activity of the developed film was tested against L. monocytogenes Scott A inoculated in both laboratory media (Tryptose soy agar, International Diagnostics, UK) and fresh beef. Stock cultures were kept immobilized on beads (−30 °C), and were regenerated by transferring a bead aseptically into 10 ml of TSB and incubating at 30 °C for 24 h. Cells were harvested by centrifugation at 6000 rpm for 20 min and washed twice with sterile Ringer's solution. Appropriately diluted culture was then used for surface inoculation of TSA and fresh beef cuts (4x5x1cm) in order to obtain a target inoculum of 102 CFU/cm2. The developed films, that contained 30% w/w sorbitol, were cut at the same size dimensions as the samples, and were placed on the inoculated surface of the product; the top surface of the preformed antimicrobial films were brought in contact with the product surface. Samples that did not carry films served as controls. All the samples were kept into sterile

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Petri dishes, sealed with parafilm to avoid dehydration, and stored at 4 °C in controlled high-precision low-temperature incubators (model MIR 153; Sanyo Electric Co., Ora-Gun, Gunma, Japan). Additional experiments were performed for beef cuts that were stored under dynamic temperature conditions varying between 5 and 15 οC. The precise temperature history of samples during storage was recorded with the aid of electronic temperature recorders (Cox Tracer, Cox Technologies, Belmont, NC, USA). L. monocytogenes and L. sakei counts on the samples were examined immediately after inoculation and every 2 or 3 days over the entire storage period. The agar or the beef samples were removed aseptically from the Petri dishes and placed in a sterile BagPage (Interscience, France), 100 ml of ringer solution (International Diagnostics, UK) was added to each bag and stomached for 2 min (Stomacher Interscience, France). Decimal dilutions in quarter-strength Ringer solution were prepared and 1- or 0.1-mL sample of appropriate dilutions were poured or spread to the following media: PALCAM agar (Merck KgaA, Darmstadt, Germany) for L. monocytogenes incubated for 48 h at 30 °C and MRSA (1.10660, Merck) for L. sakei, overlaid with the same medium and incubated at 30 °C for 96 h. All tests were run in duplicate and repeated twice. 2.7. Statistical analysis All data were analyzed by the general linear model (GLM) procedure of the SPSS software, Release 13.0. Comparisons were made using the Duncan's multiple range test to determine any significant differences among the treatments at a 95% confidence interval. 3. Results and discussion 3.1. Water sorption isotherms Water sorption isotherms were constructed for sorbitol-plasticized SC films acting as carriers of the bacterial cells. A shown in Fig. 1 the isotherms obtained were sigmoid in shape and it was observed that the moisture content of the film increased slowly with increased humidity until αw ~ 0.64, after which small increases in the relative humidity lead to large weight gains. This type of water sorption isotherms is typical of materials rich in hydrophilic polymers and has been frequently reported in previous studies for biopolymer matrices (Biliaderis et al., 1999; Cho & Rhee, 2002; Diab, Biliaderis, Gerasopoulos & Sfakiotakis, 2001; Zinoviadou, Koutsoumanis & Biliaderis, 2009; Zinoviadou, Koutsoumanis & Biliaderis, 2010). The films used in the present study consisted of 30% sorbitol on a dry basis and reached a higher moisture content under storage at high RH when compared to films that did not contain sorbitol (Kristo & Biliaderis, 2006). The increasing moisture affinity of films with increasing plasticizer concentration is consistent with the findings of Coupland, Shaw, Monahan, O'Riordan, and O'Sullivan (2000) for

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glycerol-plasticized whey protein films, Cho and Rhee (2002) for sorbitol and/or glycerol-plasticized soy protein films, and Kristo et al (2006) for sorbitol-plasticized pullulan and SC films. The water sorption behavior of the SC films was not affected by the presence of the protective culture in neither of the two methods employed for incorporation of the bacterial cells. This can be attributed to the fact that the mass of the added biomass is minor compared to the dry matter of the films and in that sense is not expected to influence the water sorption behavior of the films. 3.2. Water vapor permeability The WVP values of the films along with their thicknesses and the estimated RH values at the film underside are presented in Table 1. The calculated RH values were lower than the expected 100% RH due to the water transfer resistance of a stagnant air layer between the film and the water surface in the cup, since the resistance to vapor transfer of hydrophilic materials is small (McHugh et al., 1993). The WVP of the control films was 16.2 ± 1 g·mm/h·m2·kPa and was slightly higher than the value that has been previously reported for sorbitol-plasticized SC films studied under similar conditions (Kristo, Koutsoumanis & Biliaderis, 2008). However, in the latter study less plasticizer was incorporated in the film matrix. The amount of plasticizer used is crucial since it influences positively the moisture content (Cho et al., 2002; Hernandez-Izquierdo & Krochta, 2008; Hernández-Muñoz, Kanavouras, Perry & Gavara, 2003). Increased plasticizer content decreases intermolecular forces between polymer coils resulting in an enhancement of material flexibility and a decrease in the barrier properties due to the augmentation of the free volume (Audic & Chaufer, 2005). Even lower values WVP have been reported for SC films that did not contain sorbitol and were studied under more severe conditions. However, all these findings are not directly comparable since the conditions under which permeability studies are performed play an important role in the measured barrier properties (Greener & Fennema, 1989). As it can be seen at Table 1 addition of the bioactive culture, either by incorporation or by spraying, did not alter significantly the barrier properties of the films. Once again the relatively small mass of the bacterial cells, compared to the total mass of the polymeric matrix, can explain why the WVP is not affected by the presence of the cells. Similarly, it has been reported in previous studies that addition of antimicrobial agents, such as nisin, at low concentrations did not cause significant changes in the WVP of SC films (Kristo et al., 2008) or LPDE films coated with a nisin-incorporated cellulose-based coating (Grower, Cooksey & Getty, 2004). 3.3. Tensile properties The profiles of large deformation mechanical properties under tensile mode of sorbitol-plasticized SC films, as affected by film moisture content and the presence of the bacterial cells, are represented in Fig. 2. Antimicrobial films were conditioned at four different RH levels (11%, 43%, 53% and 75%) at 25 °C. As it can be seen in Fig. 2 for all the films a gradual decrease in tensile strength with increasing moisture content at levels above 5% was observed. Water is known as a very effective plasticizer and its action is reflected in lowering fracture strength (σmax) and elastic modulus (E), and increasing of film flexibility (% EB) (Chang, Table 1 Effect of bacterial cells addition (incorporation or spraying) on water vapor permeability (WVP) of sorbitol-plasticized sodium-caseinate films.

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3.4. Viability of L. sakei during storage of the films The viability of L. sakei added to SC films was tested during storage at 4 °C and 25 °C. These two temperatures were selected in order to reproduce the usual distribution and storage conditions of the films. Changes in immobilized cell populations of L. sakei in the sorbitol-free film stored at 4 °C and 25 °C are shown in Fig. 3a. As it can be seen, when the bacterial cells were sprayed on the surface of preformed film, a decrease of the population was observed on storage at room temperature. The higher viability observed at 4 °C could be attributed to the reduced bacterial metabolism occurring at low temperatures. On the other hand, direct incorporation of the bacterial cells into the film forming solution resulted in greater viability since no reduction of the population was observed under both storage temperatures. It has been previously reported that stress adaptation of microbial cells enables them to survive better when they are subsequently exposed to the same or other type of stresses (Foley et al., 2005). In the case of direct cell incorporation, the bacteria have suffered a heat stress during the drying

step of film preparation. This may explain the fact that those cells exhibited higher viability at room temperature compared to the cells that were sprayed on top of the pre-formed films. In order to improve the viability of L. sakei in the film during storage at ambient temperature, sorbitol (30% on a dry basis) was added to the film solution. Sorbitol or other similar compounds have been reported to act as protective agents for microbial cells during drying or storage at low aw conditions (Leslie, Israeli, Lighthart, Crowe & Crowe, 1995;

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Cheah & Seow, 2000; Debeaufort & Voilley, 1997; Kristo & Biliaderis, 2007; Lazaridou & Biliaderis, 2002; McHugh & Krochta, 1994; Shaw, Monahan, O'Riordan & O'Sullivan, 2002). However, at low moisture contents (~5–7%), water may exert an antiplasticizing effect and in this case this was reflected by increased values of maximum tensile strength (σmax). The anti-plasticizing effect of water has been thoroughly reviewed by Pitia and Sacchetti (2008) who concluded that antiplasticization is mostly observed in systems of low moisture content that are characterized by a glass transition temperature (Tg) higher than ambient temperature. Similar behavior has been reported in the past for cereal products (Harris & Peleg, 1996), zein films (Lai & Padua, 1998), tapioca starch films (Chang et al., 2000) and whey protein isolate films (Zinoviadou et al., 2009; Zinoviadou et al., 2010). As far as the control films, at a moisture level of 6%, the measured σmax was 32 MPa, the EB 9% and the E 950 MPa. These results come in agreement with Kristo et al. (2008), who reported comparable values for sorbitol-plasticized WPI films tested under similar conditions. The tensile strength of sorbitol-plasticized SC films studied by Fabra, Talens and Chiralt (2008) was lower but this can be attributed to the fact that in that case the film specimens were conditioned in a higher RH environment. Overall, the incorporation or addition by spraying of the viable cells to the films did not affect significantly the tensile properties and this can be attributed to the relatively insignificant mass of the added cells.

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Linders, de Jong, Meerdink & vant Riet, 1997; Linders, Wolkers, Hoekstra & vant Riet, 1997). Indeed, as it can be seen in Fig. 3b, the use of the polyol increased the viability of the bacterial cells during storage of the films at 25 °C over a period of 30 days. Similarly, Carvalho et al. (2004) have reported that the addition of sorbitol during freeze-drying of Lactobacillus plantarum and Lactobacillus rhamnosus cultures resulted in higher survival during freeze drying and increased viability during storage. Similar findings have been reported by Siaterlis, Deepika and Charalampopoulos (2009), who studied the effect of growth medium and cryoprotectants on the growth and survival of different LAB during freeze drying. Several mechanisms have been proposed in order to explain this positive effect of added polyols on the survival of bacterial cells. It has been shown that sugars are able to lower the transition temperature (Tm) of dry membranes by replacing structural water molecules and/or preventing unfolding and aggregation of proteins (Leslie et al., 1995; Yoo & Lee, 1993). Within the membrane structure, according to the water replacement hypothesis, the protective effect is attributed to the interaction between sugars and membrane phospholipids (lipid head-groups). This assists the maintenance of membrane fluidity, which in the absence of polyols tends to decrease upon drying (Santivarangkna, Kulozik, Kienberger & Foerst, 2009). Last but not least, sorbitol has been reported to prevent lipid oxidation due to its antioxidant properties (Linders, Wolkers, et al., 1997). 3.5. Antimicrobial activity of the films against L. monocytogenes The antimicrobial activity of the developed film against L. monocytogenes was tested in both laboratory media (TSA) and a food model system (fresh beef). The effect of the films on the growth of L. monocytogenes on TSA stored at 4 °C is presented in Fig. 4. As it is shown in Fig. 4b, L. sakei grew rapidly immediately after the contact

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of the film with the surface of the medium and reached a level of 107 cfu/cm2 after 4 days of storage. This result was independent of the technique used for incorporation of the bacterial cells into the protein matrix; i.e. either by direct incorporation in the film forming solution or by spraying on the pre-formed SC film. The high levels L. sakei in samples with the films resulted in a significant reduction of L. monocytogenes compared to the control. Indeed, after 12 days of storage in the case of films with the incorporated culture there was a reduction by 3 log of the pathogen population, while in the case of the sprayed films the reduction was 3.6 log cycles compared to the control. It needs to be noted that the above inhibition can be attributed exclusively to the presence of L. sakei in the films since the SC films themselves do not cause any inhibition of the L. monocytogenes (Kristo et al., 2008). The growth of L. monocytogenes on fresh beef slices with or without the application of SC films with L. sakei is presented in Fig. 5. In samples without film (control) the initial level of LAB, naturally present on beef, was 3.1 logs cfu/cm2 and increased to a maximum level of 5.8–6.0 logs cfu/cm2 after 6–9 days of storage (Fig. 5b). In beef slices brought in contact with the films the total initial LAB level was 6 logs cfu/cm2 due to the presence of L. sakei in the films which increased to 7 logs cfu/cm2 during storage. The latter difference in LAB levels resulted in a significant inhibition of L. monocytogenes growth. As it is shown in Fig. 4a the level of the pathogen in samples with films was reduced by 2 log cycles compared to the control at the end of the storage period. The antilisterial effect of the developed films was also evaluated on beef stored under dynamic temperature conditions. Previous studies have shown that substantial temperature fluctuations may occur during distribution and storage of foods (Koutsoumanis, Pavlis, Nychas & Xanthiakos, 2010) which can affect significantly their safety status. The results of the present work showed that the application of SC films with L. sakei on beef protected the product by lowering the growth of L. monocytogenes during storage under an abusive temperature profile (Fig. 6). Temperature fluctuations from 4 to 14 °C resulted in a significant growth of the pathogen in control samples from 4.0 to 6.4 logs cfu/cm2, while for samples with the films the total growth of L. monocytogenes was by 1.5–2.0 log cycles lower at the end of storage period. The antilisterial effect of L. sakei has been reported in the past and was attributed to several mechanisms (Skytta et al., 1991; Vandenbergh, 1993; Drosinos et al., 1994; Cleveland et al., 2001; Jones et al., 2009). Bredholt, Nesbakken and Holck (2001) have reported that a non-bacteriocin producing strain of L. sakei was able to inhibit the growth of L. monocytogenes inoculated on cooked ham and stored at 4 °C and 8 °C. A plausible mechanism responsible for such an effect is product acidification through lactic acid production by LAB, while another hypothesis is directed toward competition for nutrients (Vermeiren, Devlieghere, Vandekinderen & Debevere, 2006). Similarly, Alves et al. (2006) have reported that the growth of two strains of L. monocytogenes was significantly suppressed in sliced cooked vacuum-packaged ham when the samples were co-inoculated with either bacteriocin producing or non-producing L. sakei strains. Significant inhibition of L. monocytogenes growth has also been observed for L. sakei strains producing bacteriocins such as sakacin P (Aasen, Moretro, Katla, Axelsson & Storro, 2000; Katla, Moretro, Aasen, Holck, Axelsson & Naterstad, 2001; Katla et al., 2002), sakasin A (Schillinger, Kaya & Lucke, 1991), lactosin S and sakasin K (Castellano et al., 2008). Several researchers have studied the application of LAB metabolic products in foods through edible films and showed that incorporation of bacteriocins, such as nisin, into films results in higher inhibitory activity compared to direct addition of the antimicrobial agent to the model food system. Sebti and Coma (2002) found greater inhibition of Listeria innocua inoculated on the surface of tryptose agar medium by a nisin-impregnated hydroxypropyl methylcellulose coating than by a free-nisin solution. Similarly, Kristo et al. (2008) have reported higher


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4 Fig. 5. Growth of Listeria monocytogenes (a) and Lactic Acid Bacteria (b) on fresh beef at 4 °C with or without sorbitol-plasticized sodium-caseinate films carrying the Lactobacillus sakei culture.

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Time (days) antilisterial activity of nisin when it was incorporated in SC films than when the bacteriocin was directly added into the TSA medium. This was attributed to the decreased diffusion rates from the packaging material into the product, which assists the relatively maintenance of high concentrations of the active ingredient where it is required (product surface). In the present study we show that the alternative approach of using viable bacterial cells, incorporated into biopolymerbased films, also constitutes an effective means in controlling pathogens and improving food safety. This technique is advantageous compared to the addition of the bacterial metabolic products into the films since it could reduce significantly the cost. 4. Conclusions The present study indicated that sodium-caseinate films can act as effective carriers of L. sakei bacterial cells in order to be used as antimicrobial agents (competitive culture). The results of this study clearly demonstrated that addition of the bacterial cells into the sodium-caseinate film matrix did not alter any of the physicochemical properties of the films. Addition of polyols such as sorbitol into the protein films does increase the viability of L. casei into the SC matrix, even when stored at room temperature. The use of the antimicrobial active films against L. monocytogenes, inoculated on laboratory media and food model systems and stored under constant and dynamic temperatures conditions, resulted in a significant inhibition of the pathogen, compared to the control samples. The above results indicate that biopolymer-based films carrying a L. sakei strain can be used as an effective, alternative packaging technology for improving food safety.

Fig. 6. Growth of Listeria monocytogenes (a) and Lactic Acid Bacteria (b) on fresh beef under dynamic temperature conditions with or without sorbitol-plasticized sodiumcaseinate films carrying the Lactobacillus sakei culture.

Acknowledgments This research was supported by the EU Framework VI program Food Quality and Safety (acronym: ProSafeBeef Food-CT-2006-36241).

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