Thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on Listeria monocytogenes

ARTICLE IN PRESS

FOOD

HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 373–386 www.elsevier.com/locate/foodhyd

Thermal, mechanical and water vapor barrier properties of sodium caseinate films containing antimicrobials and their inhibitory action on Listeria monocytogenes Eleana Kristo, Konstantinos P. Koutsoumanis, Costas G. Biliaderis Department of Food Science and Technology, Laboratory of Food Chemistry and Biochemistry, School of Agriculture, Aristotle University, GR-541 24 Thessaloniki, Greece Received 22 August 2006; accepted 6 December 2006

Abstract Antimicrobial films were prepared by incorporating different concentrations of sodium lactate (Na lactate), potassium sorbate (K sorbate) and nisin into sorbitol-plasticized sodium caseinate (SC) films. The impact of antimicrobial compounds on the water sorption, water vapor barrier properties and thermo-mechanical behavior of SC films was investigated. Furthermore, their antimicrobial effectiveness against Listeria monocytogenes was studied as a function of antimicrobial concentration. The water content and water vapor permeability (WVP) of SC films increased with increasing Na lactate and K sorbate concentration, with Na lactate-containing SC films showing higher capacity to absorb moisture and greater WVP values than the films containing K sorbate. On the other hand, nisin addition did not cause significant changes in the water uptake and WVP of SC films. The incorporation of Na lactate and K sorbate affected in different way the glass transition temperature (Tg defined as tan d peak) of antimicrobial SC films, with Na lactate increasing and the K sorbate depressing the Tg of the system. The addition of increasing Na lactate and K sorbate concentration resulted in reduction of Young modulus (E) and maximum tensile strength (smax) and increasing of elongation at break (% EB) suggesting that both antimicrobials acted as plasticizers for the SC films. Nisin-containing SC films were the most effective in reducing growth of L. monocytogenes, followed by K sorbate-impregnated SC films, whereas films containing Na lactate were slightly effective in this respect and only at the higher concentration (40% w/w film dry basis). The results indicated that for effective applications of antimicrobial coatings in foods, sufficient knowledge is required not only on the independent properties of the coating film and the antimicrobial compound but also on their interactions. r 2007 Elsevier Ltd. All rights reserved. Keywords: Edible films; Sodium caseinate; Antimicrobial; Sodium lactate; Potassium sorbate; Nisin; Mechanical properties; Thermal properties; Water barrier; Listeria monocytogenes

1. Introduction The post-process contamination caused by product mishandling and faulty packaging is responsible for about two-thirds of all microbiologically related class I recalls in the USA, with most of these recalls originating from contamination of ready-to-eat and other meat products with Listeria monocytogenes (Cagri, Ustunol, & Ryser, 2004; Franklin, Cooksey, & Getty, 2004). To control undesirable microorganisms in foods during storage and Corresponding author. Tel.: +30 2310 991797; fax: +30 2310 471257.

E-mail address: [email protected] (C.G. Biliaderis). 0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2006.12.003

distribution, antimicrobial substances can be added into the product formulation, coated onto the surface of food or incorporated into food-packaging materials. Direct surface application of active substances by spraying or dipping is not highly effective because the active substances can react with food components, can evaporate or diffuse into the food showing reduced antimicrobial activity, resulting in the need for large antimicrobial concentrations to be applied (Han & Floros, 1998; Ouattara, Simard, Piette, Begin, & Holley, 2000; Quintavalla & Vicini, 2002; Vojdani & Torres, 1990). Instead, the incorporation of antimicrobial agents to packaging materials slows down their release and helps keeping high concentrations of the active

ARTICLE IN PRESS 374

E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

compounds on the product surface for extended periods of time. Numerous studies have demonstrated that antimicrobial compounds (e.g. organic acids and their salts or the bacteriocin nisin) are more effective in reducing the levels of foodborne pathogens when incorporated into an edible film or gel and applied to the product surface than when applied on the surface via a spray solution or directly added to the product (Cutter & Siragusa, 1996, 1997; Gill & Holley, 2000; Janes, Kooshesh, & Johnson, 2002; Padgett, Han, & Dawson, 1998; Sebti & Coma, 2002; Sebti, Carnet, Blanc, Saurel, & Coma, 2003; Torres, 1987; Vojdani & Torres, 1990). During the last decades, a number of antimicrobial compounds have been characterized as Generally Recognized As Safe (GRAS). Sodium lactate (Na lactate) is used in meat and poultry products as a humectant, flavoring and antimicrobial agent at a level of 2–3%, based on the finished product weight. The antimicrobial activity of Na lactate is attributed to lactate anion (Shelef & Yang, 1991). However, the specific action of lactates on the microbial cell is not completely elucidated since neither any intracellular pH reduction was sufficiently demonstrated nor any aw decrease caused by lactates was adequate to explain the antimicrobial effect (Barmpalia et al., 2005; Shelef, 1994; de Wit & Rombouts, 1990). Potassium sorbate (K sorbate) is among the most widely used food preservatives. It is used in dairy, meat, fish, bakery products etc. as yeast, mold and bacteria inhibitor. It is effective in most foods in the concentration range 0.05–0.3% by weight. Although its activity is greatest at low pH values, K sorbate is effective at pH values as high as 6.5 (Buazzi & Marth, 1991; Sofos & Busta, 1993). The retention of sorbic acid and sorbates by edible and plastic food-packaging films has been demonstrated with zein films (Torres, 1987), fatty acid-incorporated methylcellulose and hydroxypropyl methylcellulose (Rico-Pen˜a & Torres, 1991; Vojdani & Torres, 1990), wheat gluten-lipid films (Redl, Gontard, & Guilbert, 1996), and polyethylene and poly(ethylene-co-methacrylic acid) materials (Weng & Chen, 1997; Weng, Chen, & Chen, 1999). Nisin is a polypeptide produced by certain strains of the food grade lactic acid bacterium Lactococcus lactis subsp. lactis. In 1969, the Joint FAO/WHO Expert Committee on Food Additives approved nisin for use as an antimicrobial agent in foods. Since then, nisin is permitted for use in over 50 countries (Delves-Broughton, 2005). Nisin is very stable to thermal treatments and shows increased solubility and activity at acidic pH. However, its activity can be reduced by interaction with lipids (Sebti & Coma, 2002) or it can be inactivated by proteolytic enzymes, particularly in fresh meat products (Delves-Broughton, 2005). Nisin is effective against gram-positive bacteria such as L. monocytogenes as well as gram-negative bacteria when the cell wall is previously weakened by using chelating agents, such as EDTA. The mode of action of nisin includes pore formation in cell membranes through interaction with lipid II, a precursor of peptidoglucan biosynthesis (Bauer &

Dicks, 2005). The use of edible and plastic films to deliver nisin to a variety of microbiological media or food surfaces has been the aim of many studies. Nisin-incorporated polyethylene and polyethylene oxide films (Cutter, Willett, & Siragusa, 2001), methylcellulose/ hydroxypropyl methylcellulose films (Franklin et al., 2004), poly(vinyl chloride), linear low-density polyethylene, and nylon films (Natrajan & Sheldon, 2000a), calcium-alginate and agar films (Natrajan & Sheldon, 2000b), corn zein films (Hoffman, Han, & Dawson, 2001; Janes et al., 2002), whey protein, soy protein, egg albumin and wheat gluten films (Ko, Janes, Hettiarachchy, & Johnson, 2001) are examples of the enormous polymeric materials explored as potential antimicrobial packaging materials. In the present study, sodium caseinate (SC) films containing Na lactate, K sorbate or nisin as antimicrobials were applied on top of tryptose soy agar, containing 3% NaCl (TSANaCl) (which served as a model solid food system), that was inoculated with L. monocytogenes Scott A strain and the antimicrobial effectiveness of the composite films against this pathogen was evaluated during storage at 10 1C for 10 days. A comparison of the antimicrobial action was further made with systems carrying equivalent concentration of the antimicrobial, which was mixed with the agar medium to resemble the direct addition of the antimicrobial into a food product. Since the incorporation of antimicrobials might influence the physical and mechanical properties of the film the impact of the antimicrobials on thermo-mechanical, moisture sorption and water vapor barrier properties of the composite SC films were also studied. 2. Materials and methods 2.1. Film preparation SC (Wako Chemicals, Japan) was dissolved in distilled water under continuous stirring to obtain film-forming solutions of either 7.5% (w/w) for preparing thick specimens for dynamic mechanical thermal analysis (DMTA) or 4% (w/w) concentration for the rest of measurements. Sorbitol (St. Louis, MO, USA) as plasticizer was added to the polymer solution in the constant concentration of 25% (sorbitol/(SC+sorbitol)). Such a concentration of sorbitol was necessary to overcome brittleness of the SC films. The concentrations of antimicrobials used, expressed in different ways, are presented in Table 1. Nisin solution was prepared using nisaplin (Aplin and Barrett Ltd., Dorset, UK), a commercial source of nisin, that contains 2.5% nisin and the rest are mainly sodium chloride and milk proteins. According to the supplier, the activity of 1 g nisaplin is 106 IU. Nisin stock solution was prepared by dissolving 40 mg nisaplin per ml of solution in HCl 0.01 M and filtered using 0.45-mm membrane filters (Corning Incorporated, Germany). Appropriate amounts of Na lactate (50% solution, Merck KGaA, Germany), K sorbate (Cheminova, Denmark) and nisin stock solution were

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386 Table 1 Concentrations of antimicrobials, expressed on weight basis of film dry matter, on agar weight basis or film basis Antimicrobial agent Antimicrobial concentration AM AMþSCþS

a b c  100 g AM /100 g agar mg AM/film

Sodium lactate

10 20 40

0.37 0.83 2.17

73.8 165.3 434.8

Potassium sorbate

10 25

0.37 1.10

73.8 219.8

Nisin

0.075 0.0075

0.0025 0.00025

0.50 0.05

a

AM—antimicrobial, SC—sodium caseinate, S—sorbitol. The concentration in g AM/100 g agar represents the amount of AM directly added into the agar which is equivalent to the concentration of AM incorporated into the film in order to have the same total antimicrobial concentration supplied to the agar in both treatments. c The film has the diameter of the petri dish; i.e. the film completely covers the surface of TSANaCl agar plates. b

added into the film-forming solution. SC solutions were subsequently vacuum-degassed to remove air bubbles. Portions of 12.5 g solution were cast on Petri dishes (j 8.5 cm) and allowed to dry in an oven at 35 1C for 24 h. Film thickness was determined using a manual micrometer at 5 random positions on the film. Films with Na lactate and K sorbate were more flexible than the antimicrobialfree films or the films containing nisin. However, the addition of 40% Na lactate makes them very sticky and difficult to handle. 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 1C in an aircirculated 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) at 25 1C for 21 days, a time sufficient to reach constant weight and hence practical equilibrium. The water content of samples, after storage, was determined by drying at 110 1C for 2 h. Measurements were performed in triplicate. 2.3. Water vapor permeability Water vapor permeability (WVP) measurements of films were conducted at 25 1C using the ASTM (E96-63T) procedure modified for the vapor pressure at film 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 1C that

375

was equilibrated at 53% RH using 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 40.99. At least six replicates of each film type were tested for WVP.

2.4. Dynamic mechanical thermal analysis Thick SC specimens (0.5  0.7  0.15 cm3) containing either Na lactate or K sorbate prepared for DMTA analysis were previously conditioned at various RH environments (11%, 43%, 53%, 64% and 75%) over saturated salt solutions for at least 1 month. The water content of each film was evaluated using the respective sorption isotherm. Before performing the DMTA analysis, the films were coated with silicon grease to avoid water evaporation. DMTA measurements were performed with a Mark III analyzer (Polymer Labs. Loughborough, UK) operated in the single cantilever bending mode (heating rate 2 1C min–1 and a strain level equal to a maximum displacement of 16 mm). The Tg was determined as the peak in tan d at 3 Hz.

2.5. 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 min1. Young’s modulus (E), tensile strength (smax) and % elongation at break (% EB) were calculated from load-deformation curves of tensile measurements. Measurements represent an average of at least eight samples. The water content of samples, after storage, was determined by drying at 110 1C for 2 h.

2.6. Microbiological analysis 2.6.1. Bacterial strain L. monocytogenes Scott A was used throughout the study. Stock cultures were kept frozen (18 1C) in tryptic soy broth (TSB) (International Diagnostics, UK) supplemented with 20% glycerol, and were regenerated by transferring 50 ml into 10 ml of TSB and incubating at 30 1C for 24 h. Appropriately, diluted culture was then used for inoculation of the agar plates in order to obtain a target inoculum of 102 CFU/cm2.

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

376

2.7. Statistical analysis

2.6.2. Antimicrobial effectiveness of films Tryptose soy agar (International Diagnostics, UK) with 3% NaCl (Sigma-Aldrich GmbH, Germany) was used as a model solid food system (TSANaCl) of high pH (6.5) and high aw (aw at 22 1C was 0.98). Portions (20 g) of TSANaCl were poured into Petri dishes, allowed to solidify and stored under refrigeration until used. Aliquots (0.1 ml) of the properly diluted culture were inoculated on the surface of TSANaCl and different test films of the same diameter as the Petri dishes (containing or not antimicrobial substance) were placed on the inoculated surface. Inoculated uncoated TSANaCl were used as control. For the treatment with the direct addition of antimicrobials into the agar, amounts of antimicrobials equivalent to the levels incorporated into films, as shown in Table 1, were added into the molten TSANaCl medium and weighted portions of molten agar (20 g) were poured into circular Petri dishes (j 8.5 cm). The appropriate amounts of Na lactate and K sorbate were added during preparation of TSANaCl medium, whereas the nisin solution was filtersterilized and added into the molten agar before it was poured into Petri dishes. Plates were then covered with Parafilm (Pechiney, Chicago) to avoid dehydration and stored in controlled high-precision (70.2 1C) low-temperature incubators (model MIR 153; Sanyo Electric Co., OraGun, Gunma, Japan) at 10 1C for 10 days. L. monocytogenes counts on TSANaCl plates were examined immediately after inoculation and every day over the entire storage period. The agar was removed aseptically from Petri dishes and placed in a sterile BagPage (Interscience, France). A measure of 100 ml of ringer solution (International Diagnostics, UK) was added to each bag and stomached for 2 min (Stomacher Interscience, France). Samples were diluted appropriately with ringer solution, surface plated on PALCAM agar (Merck KgaA, Darmstadt, Germany) and the plates were incubated for 48 h at 30 1C before colonies were counted. All tests were run in duplicate and repeated twice.

a

g H2O/100g dry matter

120

WVP values and microbial numbers were averaged over the six and four replications, respectively. Data were analyzed by the general linear model (GLM) procedure of the Minitab Statistical Software, Release 13.1. Tukey’s multiple comparisons were used to determine any significant differences in mean log CFU/cm2 among treatments at a 95% confidence interval. 3. Results and discussion 3.1. Water sorption isotherms Water sorption isotherms were constructed for sorbitolplasticized SC films containing different concentrations of the antimicrobials Na lactate, K sorbate and nisin. The isotherms obtained were sigmoid in shape, showing a gradual initial increase in moisture content up to aw ¼ 0:53 and a rapid increment in film water content with further augmentation of aw (Fig. 1). Such an intense water uptake could be attributed mainly to the ability of low molecular weight constituents to adsorb more readily water than biopolymers at aw 40:53 (Biliaderis et al., 1999; Kapsalis, 1987; Lourdin, Coignard, Bizot, & Colonna, 1997). Such sigmoid water sorption isotherms are characteristic of materials rich in hydrophilic polymers and are frequently reported in the literature (Biliaderis et al., 1999; Cho & Rhee, 2002; Diab, Biliaderis, Gerasopoulos, & Sfakiotakis, 2001; Herna´ndez-Mun˜oz, Kanavouras, Perry, & Gavara, 2003; Herna´ndez-Mun˜oz, Lagaron, Lopez-Rubio, & Gavara, 2004). The water sorption isotherms of antimicrobial SC films were affected by the type and concentration of the antimicrobial incorporated. As shown in Fig. 1(a and b) the equilibrium water content of SC films containing Na lactate and K sorbate at a specified aw increased with increasing antimicrobial concentration. Na lactate-containing SC films showed higher water sorption capacity at a

c

b 0% NaL 10% NaL 20% NaL 40% NaL

100 80

0% KS 10% KS 25% KS

Nisin free Nisin (L) Nisin (H)

60 40 20 0 0.0

0.2

0.4

0.6 aw

0.8

0.0

0.2

0.4

0.6 aw

0.8

0.0

0.2

0.4

0.6

0.8

1.0

aw

Fig. 1. Effect of sodium lactate (NaL) (a), potassium sorbate (KS) (b) and nisin (c) concentration on the water sorption isotherms of antimicrobial, sorbitol-plasticized sodium caseinate films; Nisin (H) and Nisin (L) correspond to high (0.5 mg nisin/film) and low (0.05 mg nisin/film) concentrations of nisin, respectively.

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

given aw than films containing similar K sorbate concentrations, possibly due to the higher moisture affinity of Na lactate, which is a recognized humectant widely used in meat and poultry products to increase the water-holding capacity and cooking yields (Shelef, 1994). Sodium ions might be responsible for the considerably higher water uptake of Na lactate-containing films since they create regions of high dielectric constant, which are favorable for water (Eisenberg & Navratil, 1973). On the other hand, sorbates are larger and more hydrophobic molecules than lactates; lactates have a shorter carbon chain as well as a hydroxyl group in addition to the carboxylic group, and this explains their higher water absorption. The increasing moisture affinity of films with increasing levels of Na lactate and K sorbate is an indication that both antimicrobials may function as plasticizers for SC films. It is often reported in the literature the greater moisture uptake of hydrocolloid films with increasing plasticizer concentration (Cho & Rhee, 2002; Herna´ndezMun˜oz et al., 2003, 2004; Kristo & Biliaderis, 2006; Mahmoud & Savello, 1992). On the other hand, nisin addition did not affect the water content of SC films except for film impregnated with the high level of nisin, which showed slightly higher water uptake than the antimicrobial-free film, at aw 40:75. Nisin was in fact incorporated in very low concentrations relative to the dry matter of the films and is not expected to influence significantly the film structure.

3.2. Water vapor permeability The WVP values of the films along with their thicknesses and RH estimates at the film underside are given in Table 2. The RH values at film underside were lower than the assumed 100% RH, due to the mass transfer resistance of a stagnant air layer between the water surface and the film mounted in the cup, since the resistance to water transfer of

377

a highly hydrophilic film is relatively small (McHugh et al., 1993). The WVP value of the sorbitol-plasticized antimicrobialfree SC films (10  1010 g s1 m1 Pa1) was similar to the WVP values of glycerol-plasticized SC films (8.3  1010 g s1 m1 Pa1) reported by Schou et al. (2005), soy protein films (10.5–12  1010 g s1 m1 Pa1) reported by Kunte, Gennadios, Cuppet, Hanna, and Weller (1997) and soy protein isolate films (8–8.5  1010 g s1 m1 Pa1) reported by Cho and Rhee (2004), all tested under similar conditions of temperature and RH gradient. The addition of Na lactate and K sorbate significantly increased (Po0:05) film WVP, with Na lactate inducing greater increase than K sorbate at similar concentrations (Table 2). The ability of both Na lactate and K sorbate to absorb water particularly at high RH (with Na lactate having higher affinity for water than K sorbate) contributes to the augmentation of the diffusion constant of water vapor and hence the WVP (Chang, Cheah, & Seow, 2000; Kester & Fennema, 1989). Furthermore, lactates (owing to the presence of OH-group) could compete more strongly with water for active sites on the polymer network than do sorbates, thus assisting in water clustering (Kilburn, Claude, Schweizer, Alam, & Ubbink, 2005), which, in turn, increases the free volume and polymer permeability. In contrast, the addition of nisin did not cause significant changes (P40:05) in the WVP of SC films. Similarly, Grower, Cooksey, and Getty (2004) reported that nisin addition made no difference in the WVP of LDPE films coated with a nisin-incorporated cellulose-based coating. The above results imply a plasticizing action of Na lactate and K sorbate on the SC films and are in agreement with previous reports on biopolymer plasticization by polyols and other small molecules which showed an increase of WVP (Biliaderis et al., 1999; Herna´ndez-Mun˜oz et al., 2003, 2004; Kristo et al., 2007; Siew, Heilmann, Easteal, & Cooney, 1999; Sobral, Menegalli, Hubinger, & Roques, 2001).

Table 2 Effect of antimicrobial type and concentration on the water vapor permeability (WVP) of sorbitol-plasticized sodium caseinate films Antimicrobial

Antimicrobial concentration   AM AMþSCþS  100

Thickness (mm)

WVP (g s1 m1 Pa1)  1010

RH at film underside (%)

Sodium lactate

0 10 20 40

81 87 73 76

10.070.7a* 19.171.7b 28.172.7c 65.378.1d

79 72 67 60

Potassium sorbate

0 10 25

81 77 76

10.070.7a 15.770.9b 22.871.8c

79 75 71

Nisin

0 0.0075 0.075

81 69 73

10.070.7a 9.870.1a 11.071.0a

79 78 76

*

Different letters within the same column and for the same antimicrobial indicate significant differences (Po0:05).

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

378

Despite the increasing interest on the incorporation of antimicrobial compounds on edible films the literature is rather scarce on findings about their effect on hydrocolloid film structure and properties. In agreement with the present results, Pranoto, Rakshit, and Salokhe (2005) and Ozdemir (1999) reported an increase of WVP of chitosan and whey protein films, respectively, with increasing of K sorbate concentration. In contrast, Flores, Fama´, Rojas, Goyanes, and Gerschenson (2007) did not observe changes in the WVP of tapioca starch films by inclusion of sorbate. However, they used a lower level of sorbates in their films (4–5% film dry basis) compared to 10% and 25% in the present investigation. Moreover, Parris, Coffin, Joubran, and Pessen (1995) showed that the addition of 20% or 30% Na lactate in alginate or pectin films resulted in higher WVP values than when equivalent concentrations of glycerol or sorbitol were used as plasticizers; this may reflect molecular size and compatibility effects between the plasticizers and the polymer. 3.3. Dynamic mechanical thermal analysis The thermo-mechanical behavior of sorbitol-plasticized SC films, containing different levels of Na lactate or K sorbate was studied by DMTA. Representative DMTA traces (log E0 and tan d vs. temperature at 3 Hz) of antimicrobial SC film specimens impregnated with Na lactate and K sorbate are shown in Fig. 2a and b, respectively. Similar traces were obtained for the rest of samples equilibrated at different RH environments. Two relaxation processes defined by a maximum of tan d associated with a drop-in storage modulus (E0 ) were observed in these curves. The relaxation observed at higher temperatures corresponded to the glass transition of SC and is referred as the main relaxation. As suggested

9

b

m.c 11%

log E' (Pa)

log E' (Pa)

a

by Fig. 2a, the increase in Tg caused by Na lactate could be related to a chain rigidification mechanism due to electrostatic interactions involving the sodium cations (Eisenberg & Navratil, 1973; Gidley, Cooke, & WardSmith, 1993). Addition of sodium ions through incorporation of Na lactate in SC films, besides those already present in SC, may enhance polymer–polymer associations by inducing hydrophobic interactions through a salting out phenomenon or by neutralizing the negative charge of phosphate groups present in casein (Kalichevsky, Blanshard, & Tokarczuk, 1993; Timasheff & Arakawa, 1989). Similarly, Eisenberg and Navratil (1973) reported an increase of Tg of the styrene-sodium methacrylate copolymers with increasing sodium ion concentration. On the other hand, potassium ions added with K sorbate in the SC films, might act in a similar way as Na+ ions do; i.e. they could preferentially bind water, decreasing the amount of water available for solvation of caseinates and thus enhancing protein–protein interactions, but they do so to a lesser extent than Na+ (Carr, Munro, & Campanella, 2002). Furthermore, sorbate being more hydrophobic than lactate, could interact more readily with casein (considering its inherent hydrophobicity) and may act as a more efficient plasticizer than lactate. Incorporation of Na lactate in starch materials caused Tg depression at Na lactate concentrations up to 13.3% (w/w, starch dry basis), but the Tg remained unchanged at higher plasticizer levels (Lourdin et al., 1997). Besides the main relaxation, a secondary relaxation was observed at low temperatures (o10 1C) (Fig. 2a and b). The properties of the latter relaxation in SC films plasticized with different levels of sorbitol were described in a previous investigation (Kristo & Biliaderis, 2006), where it was shown that the temperature of this low temperature (low-T) relaxation corresponded to the Tg

8 7 0% NaL 10% NaL 20% Nal

6 0.6

m.c. 12%

9 8 7 0% KS 10% KS 25% KS

6

0.4

tan δ

tan δ

0.6

0.2

0.4 0.2

0.0

0.0 -25

0

25 50 75 Temperature (˚C)

100

125

-25

0

25 50 75 Temperature (˚C)

100

125

Fig. 2. DMTA plots (log E0 , top curves and tan d, bottom curves) for sorbitol-plasticized sodium caseinate films containing different concentrations of sodium lactate (NaL) (a) and potassium sorbate (KS) (b) with water content (m. c.) 11 and 12% (w/w), respectively (single cantilever bending mode, heating rate 2 1C/min, frequency 3 Hz).

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

region of sorbitol. However, this relaxation occurred at temperatures somewhat higher temperature than the Tg of sorbitol (at the respective water content), reflecting most likely a mechanical relaxation of a sorbitol-rich phase (Kristo & Biliaderis, 2006). A similar relaxation was observed in the region of the Tg of fructose-containing SC films (Kalichevsky et al., 1993). As shown in Fig. 2a and b, increasing of Na lactate and K sorbate concentration in SC films moved this relaxation towards higher temperatures, with the increase being more pronounced for Na lactate and only slightly distinguishable for K sorbate. Similar behavior was observed at all hydration levels studied (data not shown). The preferential binding of water by Na+ and K+ ions might have an impact in the increase of temperature of the low-T relaxation.

120

Tg (°C)

100

80

AMfree 10% NaL 20% NaL 10% KS 25% KS

60

40 3

6

9

12

15

18

21

Water content (% w/w)

Fig. 3. Glass transition temperature (Tg) of sorbitol-plasticized sodium caseinate (SC) films containing different concentrations of sodium lactate (NaL) and potassium sorbate (KS) as a function of sample water content; Tg was determined from the temperature position of the respective tan d peaks (3 Hz); AM—antimicrobial-free SC film.

3000

0% NaL 10% NaL 20% NaL 40% NaL

2500

3.4. Large deformation mechanical testing The evolution of large deformation mechanical properties of sorbitol-plasticized SC films as affected by film hydration level and Na lactate, K sorbate or nisin concentration is represented in Figs. 4–6. Antimicrobial films were conditioned at four RH levels (11%, 43%, 53% and 75%) at 25 1C. As shown in Figs. 4–6, the plasticizing action of water was quite obvious in all cases, independently of the type and antimicrobial content of the film. In general, E and smax decreased and of % EB increased as RH increased from 11% to 75%. Water is known to be a very effective plasticizer for most biopolymers (Slade & Levine, 1991) and its plasticizing action is reflected in lowering of the fracture strength, elastic modulus and increasing of flexibility of the film (Biliaderis et al., 1999; Chang et al., 2000; Chang, Abd Karim, & Seow, 2006; Diab et al., 2001; Kalichevsky et al., 1993; Kristo et al., 2007; Lazaridou & Biliaderis, 2002; Lazaridou, Biliaderis, & Kontogiorgos, 2003; Ollett, Parker, & Smith, 1991; van Soest, de Wit, J, & Vliegenthart, 1996). The rate of decrease of E and smax for all films was greater for film water contents between 5 and 10% (Fig. 4–6) indicating a moisture-induced brittle-ductile transition of the failure mode. Usually a steep drop in the Young’s modulus is reported to happen in the region around the glass transition (Ollett et al., 1991; van Soest et al., 1996). However, this probably is not the case for SC films

80 0% NaL 10% NaL 20% NaL 40% NaL

60

c

60 50 40

σ max (MPa)

2000 E (MPa)

b

Fig. 3 demonstrates the temperature dependence of the main relaxation (tan d peak at 3 Hz of the polymer-rich phase) on water content of SC films containing different concentrations of Na lactate and K sorbate. The Tg depression by water for all the films studied is indicative of the well-known plasticizing action of water (Slade & Levine, 1991). It seems that the pattern of Tg reduction by water for films with different type and concentration of the antimicrobial was not very different from that of antimicrobial-free sorbitol-plasticized SC films.

1500 1000

%EB

a

379

40

20 20

0

0 4

8

12

16

20

24

Water content (% w/w)

28

0% NaL 10% NaL 20% NaL 40% NaL

10

500 0

30

4

8 12 16 20 24 28 Water content (% w/w)

4

8 12 16 20 24 28 Water content (% w/w)

Fig. 4. Effect of sodium lactate (NaL) concentration and water content on tensile modulus (E), tensile strength (smax) and % elongation at break (% EB) as determined from large deformation tensile tests of antimicrobial, sorbitol-plasticized sodium caseinate films.

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

380 3000 2500

160

100

0% KS 10% KS 25% KS

0% KS 10% KS 25% KS

80

0% KS 10% KS 25% KS

140 120

1500 1000 500

100

60 %EB

σ max (MPa)

E (MPa)

2000

40

80 60 40

20

20

0 0 4

8

12 16 20 24 28

0 4

8

Water content (%w/w)

12 16 20 24 28

4

Water content (% w/w)

8

12 16 20 24 28

Water content (%w/w)

Fig. 5. Effect of potassium sorbate (KS) concentration and water content on tensile modulus (E), tensile strength (smax) and % elongation at break (% EB) as determined from large deformation tensile tests of antimicrobial, sorbitol-plasticized sodium caseinate films.

3000

80 Nisin free Nisin (L) Nisin (H)

2500

35 Nisin free Nisin (L) Nisin (H)

70 60

1500 1000

25

50 %EB

σ max (MPa)

E (MPa)

2000

Nisin free Nisin(L) Nisin (H)

30

40 30

20 15 10

20 500

5

10 0

0 4

8

12 16 20 24 28

Water content (% w/w)

0 4

8

12 16 20 24 28

Water content (% w/w)

4

8

12 16 20 24 28

Water content (%w/w)

Fig. 6. Effect of nisin concentration and water content on tensile modulus (E), tensile strength (smax) and % elongation at break (% EB) as determined from large deformation tensile tests of antimicrobial, sorbitol-plasticized sodium caseinate films; nisin (H) (0.5 mg nisin/film) and nisin (L) (0.05 mg nisin/ film) correspond to high and low concentrations of nisin, respectively.

investigated here. As shown in Fig. 3, at the indicated moisture region (5–10%), the films have greater Tg (determined from the tan d peak) than 80 1C, well above the ambient temperature (at which the tensile measurements were conducted). Overall, the SC films impregnated or not with the indicated amounts of Na lactate, K sorbate and nisin, seemed to exhibit the brittle-ductile transition at temperatures well below their Tg, suggesting that considerable changes in film fracture properties occur in the glassy state, a conclusion that is in agreement with previous investigations concerning SC (Kristo et al., 2007) and starch specimens (Nicholls, Appelqvist, Davies, Ingman, & Lillford, 1995). The addition of Na lactate into SC films resulted in the continuous decline of E and smax with increasing Na lactate concentration (Fig. 4), a behavior typical of small molecules causing biopolymer plasticization. Similarly, Parris et al. (1995), who screened Na lactate, sorbitol and glycerol as plasticizers for alginate and pectin films, indicated lowering of tensile strength and modulus and increase of EB with increasing Na lactate concentration. The more pronounced differences between Na lactate-free

SC films and films impregnated with 10%, 20% and 40% Na lactate occurred at low hydration levels. Thus, the addition of only 10% Na lactate brought about a drastic decrease in E (from 2443 MPa for antimicrobial-free sample to 1400 MPa for film with 10%Na lactate) and smax (from 63 MPa for antimicrobial-free sample to 38 MPa for film with 10%Na lactate) at 5% water content. However, SC films containing 20% Na lactate had only slightly lower E and smax values than films with 10% Na lactate at similar water contents, whereas the inclusion of 40% Na lactate induced large reductions in E and smax; these differences become narrower with increasing water content of the films. On the other hand, the % EB increased as a function of Na lactate concentration at low water content (up to 10%), but at higher hydration levels, films with 10% and 20% Na lactate exhibited greater % EB than the antimicrobial-free films or the films containing 40% Na lactate. The extensibility of samples with 40% Na lactate increased only slightly with water content and leveled off to 26%, a behavior that could be attributed to an excess of plasticizer molecules that do not interact with the protein matrix (Herna´ndez-Mun˜oz et al., 2003; Siew

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

3.5. Antimicrobial effectiveness of films The TSANaCl represented a model system of high pH (6.5) and high aw (aw at 22 1C was 0.98). SC film, being transparent and almost colorless, was nearly invisible and adhere well on the wet surface of agar (Avena-Bustillos & Krochta, 1993). Growth of L. monocytogenes (10 days storage at 10 1C) as affected by the antimicrobials Na lactate, K sorbate and nisin is presented in Figs. 7–9; growth curves of L. monocytogenes in control TSA plates and in TSA plates covered with the antimicrobial-free SC film are also shown in Figs. 7–9 for comparison. No significant differences (P40:05) were observed between growth of L. monocytogenes on control TSA plates and plates coated with the antimicrobial-free film during storage; L. monocytogenes populations increased from about 2 to 8 log CFU/cm2 by the end of the study for both samples. Similarly, Janes et al. (2002) found that L.

Control 0% NaL(F) 20% NaL (F) 40% NaL (F) 2.2%NaL (A)

8

log CFU/cm2

7 6 5

a

4 b ab ab a a

3 2

ac ac bc b

a

a a a b b

a a a

a a ab

a a a

b

b

a a a a

a

aa aa

b

c bc

b

b b

a ac ac

a aa a

b

1 0

1

2

3

4

5

6

7

8

9

10

Days of storage

Fig. 7. Effect of sorbitol-plasticized sodium caseinate films impregnated with different sodium lactate (NaL) levels on the growth and survival of Listeria monocytogenes on TSANaCl agar stored at 10 1C and comparison of this treatment with the direct addition into the agar medium of the equivalent antimicrobial amount. Means with different superscript letters are significantly different (Po0:05) for each sampling time; F—the corresponding Na lactate amount has been incorporated into the film; A— the corresponding concentration of Na lactate directly added into agar medium and being equivalent with the higher Na lactate concentration incorporated into the film (40%).

Control 0% KS (F) 10% KS (F) 25% KS (F) 1.1% KS (A)

8 7 log CFU/cm2

et al., 1999). It is noteworthy that, besides Na lactate exhibiting plasticization, the SC films contain other plasticizers as well, namely sorbitol (25%) and water at different concentrations, which make the system very complex. Thus, with addition of 40% Na lactate, particularly at high hydration levels, the total amount of plasticizers increases drastically and consequently, the cohesiveness and continuity of the film network might be lost. K sorbate also acted as plasticizer, since increasing amounts of K sorbate resulted in reduction of E and smax and increasing of % EB (Fig. 5), particularly for films with 25% K sorbate. At a water content of 6%, the SC films containing 10% K sorbate showed slightly higher E and smax values than the K sorbate-free film, suggesting an antiplasticization effect, which was not observed with the Na lactate. However, at higher water levels both E and smax of films containing 10% K sorbate became lower than the respective values of the antimicrobial-free films. The extensibility of SC films containing 25% K sorbate was extremely higher than that of antimicrobial-free and 10% K sorbate-containing films when the moisture content exceeded 10%. The results of the present study on the impact of K sorbate in the mechanical properties of SC films are in agreement with previous findings. Incorporation of K sorbate brought about a decrease in the tensile modulus and strength and an increase in EB of whey protein films (Ozdemir, 1999), tapioca starch films (Flores et al., 2007) and chitosan films (Pranoto et al., 2005). Since nisin was included in notably low concentrations, relative to the protein level, it is not expected that its addition into SC films will significantly alter their mechanical properties. However, at water contents higher than 8%, E and smax decreased and % EB increased with increasing nisin level (Fig. 6). Instead, Grower et al. (2004) reported that nisin addition made no difference in the mechanical properties of LDPE films coated with a nisincontaining cellulose-based coating.

381

6

a

5 4 3

a a ab b b

2 1 0

1

a a ab b b 2

a a ab b b

3

a b

b b

4

c ac

d

b

a

ad ab

ab

a a

a

bc

b

c

5

6

b

c bc

b b

b

7

8

a a b

a a ac

b bc c

b

9

10

Days of storage

Fig. 8. Effect of sorbitol-plasticized sodium caseinate films impregnated with different potassium sorbate (KS) levels on the growth and survival of Listeria monocytogenes on TSANaCl agar stored at 10 1C and comparison of this treatment with the direct addition into the agar medium of the equivalent antimicrobial amount. Means with different superscript letters are significantly different (Po0:05) for each sampling time; F—the corresponding K sorbate amount has been incorporated into the film; A— the corresponding concentration of K sorbate directly added into agar medium and being equivalent with the higher K sorbate concentration incorporated into the film (25%).

monocytogenes grew to more than 8 log CFU/g of ready-toeat chicken after 24 days of storage at 4 and 8 1C, regardless of the initial inoculum level (6.8 or 2.6 log CFU/g). Moreover, Samelis et al. (2001) and Samelis et al. (2005) reported that L. monocytogenes numbers increased from 2–3 log CFU/cm2 to more than 8 log CFU/cm2 during prolonged storage at 4 1C of vacuum packaged control bologna slices and inferred that pathogen growth was more pronounced on the surface of the processed meat products

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

382

Control Nisin free Nisin(L) Nisin (H) Nisin(A)

8

log CFU/cm2

7 6

a a

5 4

a a

3

a a

2

b

b

b

c c

c

c

1

2

3

a ad

a

d

b b

b

a a ac

b b

b

a

b

a a

ab a

a a b

b bc c

a

a

b

b d c

bc c

c

c

1 0

4 5 6 Days of storage

7

8

9

10

Fig. 9. Effect of sorbitol-plasticized sodium caseinate films impregnated with different nisin levels on the growth and survival of Listeria monocytogenes on TSANaCl agar stored at 10 1C and comparison of this treatment with the direct addition into the agar medium of the equivalent antimicrobial amount. Means with different superscript letters are significantly different (Po0:05) for each sampling time; F—the corresponding nisin amount has been incorporated into the film; A—the corresponding concentration of nisin directly added into agar medium and being equivalent with the higher nisin concentration incorporated into the film (0.5 mg nisin/film); nisin (H) and nisin (L) correspond to high (0.5 mg nisin/film) and low concentrations of nisin (0.05 mg nisin/film), respectively.

that show high pH and moisture content. The growth data indicated that the SC films were not effective by themselves to inhibit growth of L. monocytogenes. Janes et al. (2002) and Lungu and Johnson (2005) also showed that zein films per se had no effect on suppressing the growth of L. monocytogenes on coated chicken samples stored at 4 or 8 1C for 24 days or on turkey frankfurter pieces stored at 4 1C for 28 days, respectively. Moreover, coating the surface of ham or bologna with antimicrobial-free gelatin gel did not affect the L. monocytogenes growth (Gill & Holley, 2000). Pathogen growth in the presence of SC film containing 20% (w/w, db) Na lactate was similar to that observed for control as well as for the antimicrobial-free film-coated TSANaCl plates, since non-significant differences (P40:05) were noticed over the 10-day storage (Fig. 7). Films with 10% Na lactate showed similar behavior and are not shown in Fig. 7. Increasing the Na lactate concentration to 40% resulted in a significant reduction (Po0:05) in L. monocytogenes counts in comparison to control TSANaCl plates from the 3rd to the 9th day of storage except for the 8th day results, which were not significantly different (P40:05). However, although the pathogen count reduction was statistically significant, such difference was relatively small and did not exceed 2 log CFU/cm2. Overall, the growth results showed a limited efficacy of Na lactate on inhibition of L. monocytogenes under the conditions employed in this work (e.g. Na lactate concentrations, storage temperature, etc.). Koutsoumanis et al. (2004) reported that dipping of fresh beef into a lactic acid solution did not allow growth of L. monocytogenes during storage at 4 1C, but the growth was

not limited by the same treatment followed by storage at 10 1C. Similarly, Barmpalia et al. (2005) found lower growth rates and extended lag phase in L. monocytogenes growth when pork bologna samples containing 1.8% Na lactate were stored at 4 instead of 10 1C. In addition, combination of Na lactate with other antimicrobial agents, such as nisin, sodium diacetate, glucono-delta-lactone (Barmpalia et al., 2005; Lungu & Johnson, 2005; Ukuku & Fett, 2004) significantly increased its antimicrobial efficacy. Studies on active films and coatings containing Na lactate as antimicrobial agent reported variable results with respect to the degree of inhibition of the studied microorganism. Thus, Vartiainen, Skytta, Enqvist, and Ahvenainen (2003) reported no inhibition of Escherichia coli, Bacillus subtilis and Aspergillus niger from Na lactate incorporated into a plastic packaging material. Other researchers (Lungu & Johnson, 2005) found that Na lactate was more effective against L. monocytogenes, inoculated onto the surface of turkey frankfurters, when incorporated into a zein-ethanol-glycerol coating (count reduction from 3.05 log CFU/g at day 0 to 0.43 log CFU/g after 28 days of storage, 4 1C), compared to a zeinpropylene glycol coating (count reduction from 3.55 log CFU/g at day 0 to 3.06 log CFU/g after 28 days of storage, 4 1C). In the present study, the lack of any inhibitory activity of Na lactate added into the SC films at lower concentrations (10% and 20% film dry weight basis, corresponding to 0.37% and 0.83% agar weight basis, respectively) may be due to the fact that such low concentrations of antimicrobial are inadequate to suppress pathogen growth, particularly at an abusive temperature of 10 1C employed herein. A limited number of papers have dealt with films or coatings impregnated with Na lactate (Lungu & Johnson, 2005; Vartiainen et al., 2003). Instead, several studies reported on the antimicrobial effect of lactic acid or lactates, particularly on meat and meat products, after dipping of meat pieces into their solutions or incorporation of these antimicrobial agents into the product formulation (Barmpalia et al., 2005; Koutsoumanis et al., 2004; Lungu, & Johnson, 2005; Samelis et al., 2001; Shelef & Yang, 1991; Wederquist, Sofos, & Schmidt, 1994). The incorporation of antimicrobial agent into film would localize the antimicrobial effect at the food surface as opposed to mixing the active compound directly with food, which has a limited effectiveness owing to the low availability of the antimicrobial substance on the surface where the contamination is prevalent. Therefore, it is of interest to compare the effectiveness of Na lactate when it is embodied in the edible film structure with its effectiveness as part of the TSA medium, with the last case representing the incorporation of the antimicrobial in the product formulation. The highest concentration of Na lactate (40% w/w on dry matter), expressed as the concentration of total antimicrobial supplied to the product (here represented with the agar model system) is equivalent with the direct addition of 2.17 g Na lactate/100 g agar (Table 1), and this is in

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

accordance with the level of 2% Na lactate incorporation (based on the product weight) in meat product formulations (Shelef & Yang 1991; Wederquist et al., 1994). The direct addition of 2.17% Na lactate (agar weight basis) into the agar medium or the coating of TSA plates with SC film containing Na lactate at the equivalent concentration (40% w/w film dry basis) provided pathogen numbers that were not significantly different (P40:05) during the 10-day storage period, except for the ninth day when the treatment with the Na lactate-containing film showed lower pathogen levels (Fig. 7). These results indicated that the antimicrobial films did not enhance the effectiveness of Na lactate against L. monocytogenes at 10 1C when compared to the efficacy of Na lactate directly added into the TSA medium. The effect of SC films impregnated with 10% and 25% K sorbate (w/w, film dry basis, corresponding to 0.37% and 1.1% agar weight basis) on the growth of L. monocytogenes was also investigated. Films containing both K sorbate concentrations significantly reduced (Po0:05) the L. monocytogenes counts from the forth and first day of storage, respectively, in comparison to the control and antimicrobial-free SC film-coated TSA plates (Fig. 8). At the end of the 10-day storage at 10 1C L. monocytogenes reached populations of 5 and 3.95 log CFU/cm2 in TSA agar plates covered with films containing 10% and 25% K sorbate, respectively (i.e. 3.25 and 4.3 log cfu/cm2 lower than the control). Cagri, Ustunol, and Ryser (2002) also found the incorporation of 0.75 and 1% sorbic acid (w/v based on film forming solution) into whey protein films quite effective in inhibiting L. monocytogenes growth on bologna slices during storage at 4 1C for 21 days. Although the treatment with the film containing 25% K sorbate showed lower L. monocytogenes counts than that with 10% K sorbate, the differences were not statistically significant (P40:05), except for samples of 6 and 9 days of storage. These results might indicate that with a further increase of K sorbate concentration in the SC films (above 10%) there is no additional inhibitory effect on L. monocytogenes. Similarly, Carlin, Gontard, Reich, and Nguyen (2001) found no additional effect on L. monocytogenes growth in zein-coated sweet corn when the concentration of sorbic acid into a zein coating increased above 1 mg/g sweet corn (storage for 8 days at 10 1C). Pranoto et al. (2005) also reported a lack of further antimicrobial activity of K sorbate-containing chitosan films against L. monocytogenes when the concentration of the antimicrobial exceeded 100 mg/g chitosan. As was discussed for Na lactate, equivalent concentrations of K sorbate (the high concentration of K sorbate used in this study) were incorporated in SC films (which were coated on top of TSANaCl plates) as well as mixed with the TSANaCl medium and the effectiveness of both treatments on inhibition of L. monocytogenes was compared; i.e. incorporation of 25% (w/w film dry basis) K sorbate into SC film was equivalent to mixing of 1.1% K sorbate (w/w agar weight basis) with the agar medium (Table 1). Both treatments reduced pathogen numbers at

383

similar levels up to the 7th day of storage. However, the antimicrobial was more effective when incorporated into film than directly added into the agar during the last 3 days of storage (Po0:05). This finding may imply a higher effectiveness of antimicrobial films in long-term storage, which is one of the main goals in using antimicrobial films. While the direct addition of antimicrobials into food results in an immediate and short-term reduction of bacterial populations, antimicrobial films may inhibit bacterial growth during extended storage after packaging (Hoffman et al., 2001). The incorporation of nisin (0.05 and 0.5 mg nisin per film) in SC films also resulted in significantly lower (Po0:05) L. monocytogenes counts in comparison to the control and TSANaCl agar plates coated with antimicrobial-free film, regardless of the nisin concentration (Fig. 9). The reduction in pathogen growth increased with nisin concentration of the film; the differences in counts caused by the two levels of nisin were significant (Po0:05), except for the first and last day of storage. Thus, at the 9th day of storage, L. monocytogenes counts in TSANaCl plates covered with film containing 0.05 and 0.5 mg nisin per film were 5.22 and 3.67 log CFU/cm2, respectively, considerably lower than that of control samples at the same day (8.17 log CFU/cm2). These findings are in accordance with previous investigations on the inhibitory activity of nisin (Grower et al., 2004; Natrajan & Sheldon, 2000a; Padgett et al., 1998). Furthermore, nisin at the high concentration (0.5 mg per film) completely inhibited pathogen growth for the first 3 days of storage, since the L. monocytogenes numbers were below the detection limit (that was 1.24 log CFU/cm2); however, once the recovery of cells was assumed, the rate of growth seemed to be similar to that observed for cells grown in control and nisin-free film-coated TSANaCl plates. Similarly, Bell and De Lacy (1987) observed that addition of high nisin levels (400 and 500 IU/g) in cooked luncheon meat extended the lag phase of Bacillus licheniformis, but the rate of growth was similar to that observed in the nisin-free product. A number of recent studies have demonstrated the antimicrobial effectiveness of nisin as a component of edible films, particularly against pathogens like L. monocytogenes (Franklin et al., 2004; Hoffman et al., 2001; Ko et al., 2001; Lungu & Johnson, 2005). The efficacy of nisin-containing SC films coated on top of agar inoculated with L. monocytogenes was also compared with the antimicrobial action of incorporating equivalent amount of nisin in the agar. The incorporation of 0.5 mg nisin per film into SC film was equivalent to mixing of 2.5 mg nisin per 100 g agar. L. monocytogenes numbers of nisin-treated film-coated TSANaCl plates were significantly lower (Po0:05) than the pathogen levels obtained in nisin-containing agar medium throughout the storage period. Moreover, the growth of pathogen on the agar mixed with 2.5 mg nisin/100 g agar was similar to that observed in TSANaCl plates coated with film containing 10 times less nisin (0.05 mg nisin per film) for up to 8 days of

ARTICLE IN PRESS 384

E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

storage (Fig. 9). These results clearly indicate that SC films enhanced the antimicrobial activity of nisin against L. monocytogenes and contributed in a long-term efficacy of this antimicrobial. These findings are consistent with a reported greater retention and enhancement of antimicrobial activity of nisin (1000 IU/g) against L. monocytogenes when added to zein coatings compared to its direct application on the surface of ready-to-eat chicken (Janes et al., 2002). Also, 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. Gill and Holley (2000) indicated greater effectiveness of antimicrobials (25.5 g/L of lysozyme-nisin (1:3) and EDTA) when incorporated into a gelatin gel and applied on the surface of ham and bologna pieces, as opposed to a direct addition of an equivalent amount of total antimicrobials supplied to the product (e.g. 450 mg/kg directly mixed into the sausage batter). Overall, during the entire storage period, the nisincontaining SC films were more effective in reducing growth of L. monocytogenes, followed by K sorbate-impregnated SC films, whereas films containing Na lactate were only slightly effective and only at the higher concentration (40% w/w film dry bases) employed. Although the release of antimicrobial agents from the SC matrix on the TSA surface was not studied here, other studies (Ouattara et al., 2000) indicated that the release of acetic and propionic acids from chitosan films on the surface of bologna, ham and pastrami was initially fast when the gradient of agent concentration between the inside of the composite polymer matrix and the outside environment was high, and then decreased as the release of acids progressed; in this context, the antimicrobial agent will almost completely be released from the polymeric matrix within a few hours of film application, limiting the effectiveness of the film to extend the delivery of the active compound (Ouattara et al., 2000). Other factors might also affect the effectiveness of antimicrobials incorporated in the SC films. The desorption of Na lactate, K sorbate or nisin from the SC matrix could be different and related to the electrostatic interactions between SC and organic acid salts or nisin or with structural changes in the polymer induced by the presence of the antimicrobial agents, making the release of these compounds from the SC matrix a complex phenomenon (Ouattara et al., 2000). Thus, nisin and K sorbate might have been retained by the SC matrix at a higher degree than Na lactate, and, therefore, were more effective during storage. Ouattara et al. (2000) demonstrated that the acetic acid retention in chitosan films and its effectiveness is related to the characteristics of the meat surface, with bologna (containing less water than ham and pastrami) showing more acetic acid to remain in the chitosan film during storage. In the present study, nisin for example, being a bulkier molecule than the two salts of organic acids, may be released more slowly from the SC matrix and also slowly diffuse through the agar medium,

maintaining thus, an effective concentration on the agar surface. 4. Conclusions SC films could serve as carriers of antimicrobial compounds, enhancing the antimicrobial activity, particularly of nisin and to a lesser degree of K sorbate over prolonged storage. The incorporation of nisin in SC films substantially reduced the levels of L. monocytogenes on TSANaCl medium for 10 days at 10 1C, with the antimicrobial film being advantageous over the direct nisin addition into the agar. Furthermore, the results clearly demonstrated that the antimicrobial agents might substantially alter the thermo-mechanical and water vapor barrier properties of the composite film; e.g. Na lactate and K sorbate acted as plasticizers increasing the equilibrium water content, WVP and extensibility of SC films, but decreasing the Young modulus and tensile strength. Acknowledgments The author E. Kristo would like to thank the State Scholarship Foundation (IKY) for awarding her a graduate fellowship. This research was supported by EU Framework VI programme on Food Quality and Safety, ProSafeBeef ‘‘Food-CT-2006-36241’’. References Avena-Bustillos, R. J., & Krochta, J. M. (1993). Water vapor permeability of caseinate-based edible films as affected by pH, calcium crosslinking and lipid content. Journal of Food Science, 58, 904–907. Barmpalia, I. M., Koutsoumanis, K. P., Geornaras, I., Belk, K. E., Scanga, J. A., Kendall, P. A., et al. (2005). Effect of antimicrobials as ingredients of pork bologna for Listeria monocytogenes control during storage at 4 or 10 1C. Food Microbiology, 22, 205–211. Bauer, R., & Dicks, L. M. T. (2005). Mode of action of lipid II-targeting lantibiotics. International Journal of Food Microbiology, 101, 201–216. Bell, R. G., & De Lacy, K. M. (1987). The efficacy of nisin, sorbic acid and monolaurin as preservatives in pasteurized cured meat products. Food Microbiology, 4, 277–283. Biliaderis, C. G., Lazaridou, A., & Arvanitoyannis, I. (1999). Glass transition and physical properties of polyol-plasticized pullulan-starch blends at low moisture. Carbohydrate Polymers, 40, 29–47. Buazzi, M. M., & Marth, E. H. (1991). Mechanisms in the inhibition of Listeria monocytogenes by potassium sorbate. Food Microbiology, 8, 249–256. Cagri, A., Ustunol, Z., & Ryser, E. T. (2002). Inhibition of three pathogens on bologna and summer sausage slices using antimicrobial edible films. Journal of Food Science, 67, 2317–2324. Cagri, A., Ustunol, Z., & Ryser, E. T. (2004). Antimicrobial edible films and coatings. Journal of Food Protection, 67, 833–848. Carlin, E., Gontard, N., Reich, M., & Nguyen, C. (2001). Utilization of zein coating and sorbic acid to reduce Listeria monocytogenes growth on cooked sweet corn. Journal of Food Science, 66, 1385–1389. Carr, A. J., Munro, P. A., & Campanella, O. H. (2002). Effect of added monovalent or divalent cations on the rheology of sodium caseinate solutions. International Dairy Journal, 12, 487–492. Chang, Y. P., Abd Karim, A., & Seow, C. C. (2006). Interactive plasticizing–antiplasticizing effects of water and glycerol on the tensile properties of tapioca starch films. Food Hydrocolloids, 20, 1–8.

ARTICLE IN PRESS E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386 Chang, Y. P., Cheah, P. B., & Seow, C. C. (2000). Plasticizing–antiplasticizing effects of water on physical properties of tapioca starch films in the glassy state. Journal of Food Science, 65, 445–451. Cho, S. Y., & Rhee, C. (2002). Sorption characteristics of soy protein films and their relation to mechanical properties. Lebensmitte Wissenschaft und Technologie, 35, 151–157. Cho, S. Y., & Rhee, C. (2004). Mechanical properties and water vapor permeability of edible films made from fractionated soy proteins with ultrafiltration. Lebensmitte Wissenschaft und-Technologie, 37, 833–839. Cutter, C. N., & Siragusa, G. R. (1996). Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels. Letters in Applied Microbiology, 23, 9–12. Cutter, C. N., & Siragusa, G. R. (1997). Growth of Brochothrix thermosphacta in ground beef following treatments with nisin in calcium alginate gels. Food Microbiology, 14, 425–430. Cutter, C. N., Willett, J. L., & Siragusa, G. R. (2001). Improved antimicrobial activity of nisin-incorporated polymer films by formulation change and addition of food grade chelator. Letters in Applied Microbiology, 33, 325–328. de Wit, J. C., & Rombouts, F. M. (1990). Antimicrobial activity of sodium lactate. Food Microbiology, 7, 113–120. Delves-Broughton, J. (2005). Nisin as a food preservative. Food Australia, 57, 525–527. Diab, T., Biliaderis, C. G., Gerasopoulos, D., & Sfakiotakis, E. (2001). Physico-chemical properties and application of pullulan edible films and coatings in fruit preservation. Journal of Science of Food and Agriculture, 81, 988–1000. Eisenberg, A., & Navratil, M. (1973). Ion clustering and viscoelastic relaxation in styrene-based ionomers. II. Effect of ion concentration. Macromolecules, 6, 604–612. Flores, S., Fama´, L., Rojas, A. M., Goyanes, S., & Gerschenson, L. (2007). Physical properties of tapioca-starch edible films: Influence of filmmaking and potassium sorbate. Food Research International, 40, 257–265. Franklin, N. B., Cooksey, K. D., & Getty, K. J. K. (2004). Inhibition of Listeria monocytogenes on the surface of individually packaged hot dogs with a packaging film coating containing nisin. Journal of Food Protection, 67, 480–485. Gidley, M. J., Cooke, D., & Ward-Smith, S. (1993). Low moisture polysaccharide systems: Thermal and spectroscopic aspects. In J. M. V. Blanshard, & P. J. Lillford (Eds.), The glassy state in foods (pp. 303–316). Nottingham: University Press. Gill, A., & Holley, R. A. (2000). Surface application of lysozyme, nisin, and EDTA to inhibit spoilage and pathogenic bacteria on ham and bologna. Journal of Food Protection, 63, 1338–1346. Grower, J. L., Cooksey, K., & Getty, K. J. K. (2004). Development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes. Journal of Food Protection, 67, 475–479. Han, J. H., & Floros, J. D. (1998). Potassium sorbate diffusivity in American processed and Mozzarella cheeses. Journal of Food Science, 63, 435–437. Herna´ndez-Mun˜oz, P., Kanavouras, A., Perry, K. W. N., & Gavara, R. (2003). Development and characterization of biodegradable films made from wheat gluten protein fractions. Journal of Agriculture and Food Chemistry, 51, 7647–7654. Herna´ndez-Mun˜oz, P., Lagaron, J. M., Lopez-Rubio, A., & Gavara, R. (2004). Gliadins polymerized with cysteine: Effects on the physical and water barrier properties of derived films. Biomacromolecules, 5, 1503–1510. Hoffman, K. L., Han, I. Y., & Dawson, P. L. (2001). Antimicrobial effects of corn zein films impregnated with nisin, lauric acid, and EDTA. Journal of Food Protection, 64, 885–889. Janes, M. E., Kooshesh, S., & Johnson, M. G. (2002). Control of Listeria monocytogenes on the surface of refrigerated, ready-to eat chicken coated with edible zein film coatings containing nisin and/or calcium propionate. Journal of Food Science, 67, 2754–2757.

385

Kalichevsky, M. T., Blanshard, J. M. V., & Tokarczuk, P. F. (1993). Effect of water content and sugars on the glass transition of casein and sodium caseinate. International Journal of Food Science and Technology, 28, 139–151. Kapsalis, J. G. (1987). Influences of hysteresis and temperature on moisture sorption isotherms. In L. B. Rockland, & L. R. Beuchat (Eds.), Water activity: Theory and applications to food (pp. 173–215). New York: Marcel Dekker, Inc. Kester, J. J., & Fennema, O. (1989). An edible film of lipids and cellulose ethers: Barrier properties to moisture vapor transmission and structural evaluation. Journal of Food Science, 54, 1383–1389. Kilburn, D., Claude, J., Schweizer, T., Alam, A., & Ubbink, J. (2005). Carbohydrate polymers in amorphous states: An integrated thermodynamic and nanostructural investigation. Biomacromolecules, 6, 864–879. Ko, S., Janes, M. E., Hettiarachchy, N. S., & Johnson, M. G. (2001). Physical and chemical properties of edible films containing nisin and their action against Listeria monocytogenes. Journal of Food Science, 66, 1006–1011. Koutsoumanis, K. P., Ashton, L. V., Geornaras, I., Belk, K. E., Scanga, J. A., Kendall, P. A., et al. (2004). Effect of single or sequential hot water and lactic acid decontamination treatments on the survival and growth of Listeria monocytogenes and spoilage microflora during aerobic storage of fresh beef at 4, 10, and 25 1C. Journal of Food Protection, 67, 2703–2711. Kristo, E., & Biliaderis, C. G. (2006). Water sorption and thermomechanical properties of water/sorbitol-plasticized composite biopolymer films: Caseinate-pullulan bilayers and blends. Food Hydrocolloids, 20, 1057–1071. Kristo, E., Biliaderis, C. G., & Zampraka, A. (2007). Water vapor barrier and tensile properties of composite caseinate–pullulan films: Biopolymer composition effects and impact of beeswax lamination. Food Chemistry, 101, 753–764. Kunte, L. A., Gennadios, A., Cuppet, S. L., Hanna, M. A., & Weller, C. L. (1997). Cast films from soy protein isolates and fractions. Cereal Chemistry, 74, 115–118. Lazaridou, A., & Biliaderis, C. G. (2002). Thermophysical properties of chitosan, chitosan–starch and chitosan–pullulan films near the glass transition. Carbohydrate Polymers, 48, 179–190. Lazaridou, A., Biliaderis, C. G., & Kontogiorgos, V. (2003). Molecular weight effects on solution rheology of pullulan and mechanical properties of its films. Carbohydrate Polymers, 52, 151–166. Lourdin, D., Coignard, L., Bizot, H., & Colonna, P. (1997). Influence of equilibrium relative humidity and plasticizer concentration on the water content and glass transition of starch materials. Polymer, 38, 5401–5406. Lungu, B., & Johnson, M. G. (2005). Fate of Listeria monocytogenes inoculated onto the surface of model turkey frankfurter pieces treated with zein coatings containing nisin, sodium diacetate, and sodium lactate at 4 1C. Journal of Food Protection, 68, 855–859. Mahmoud, R., & Savello, P. A. (1992). Mechanical properties of and water vapor transferability through whey protein films. Journal of Dairy Science, 75, 942–946. McHugh, T. H., Avena-Bustillos, R., & Krochta, J. M. (1993). Hydrophilic edible films: Modified procedure for water vapor permeability and explanation of thickness effects. Journal of Food Science, 58, 899–903. Natrajan, N., & Sheldon, B. W. (2000a). Efficacy of nisin-coated polymer films to inactivate Salmonella typhimurium on fresh broiler skin. Journal of Food Protection, 63, 1189–1196. Natrajan, N., & Sheldon, B. W. (2000b). Inhibition of Salmonella on poultry skin using protein- and polysaccharide-based films containing a nisin formulation. Journal of Food Protection, 63, 1268–1272. Nicholls, R. J., Appelqvist, I. A. M., Davies, A. P., Ingman, S. J., & Lillford, P. J. (1995). Glass transitions and fracture behaviour of gluten and starches within the glassy state. Journal of Cereal Science, 21, 25–36.

ARTICLE IN PRESS 386

E. Kristo et al. / Food Hydrocolloids 22 (2008) 373–386

Ollett, A. L., Parker, R., & Smith, A. C. (1991). Deformation and fracture behaviour of wheat starch plasticized with glucose and water. Journal of Materials Science, 26, 1351–1356. Ouattara, B., Simard, R. E., Piette, G., Begin, A., & Holley, R. A. (2000). Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan. International Journal of Food Microbiology, 62, 139–148. Ozdemir, M. (1999). Antimicrobial releasing edible whey protein films and coatings. Ph.D. thesis. Purdue University. Padgett, T., Han, I. Y., & Dawson, P. L. (1998). Incorporation of foodgrade antimicrobial compounds into biodegradable packaging films. Journal of Food Protection, 61, 1330–1335. Parris, N., Coffin, D. R., Joubran, R. F., & Pessen, H. (1995). Composition factors affecting the water vapor permeability and tensile properties of hydrophilic films. Journal of Agriculture and Food Chemistry, 43, 1432–1435. Pranoto, Y., Rakshit, S. K., & Salokhe, V. M. (2005). Enhancing antimicrobial activity of chitosan films by incorporating garlic oil, potassium sorbate and nisin. Lebensmitte Wissenschaft und Technologie, 38, 859–865. Quintavalla, S., & Vicini, L. (2002). Antimicrobial food packaging in food industry. Meat Science, 62, 373–380. Redl, A., Gontard, N., & Guilbert, S. (1996). Determination of sorbic acid diffusivity in edible wheat gluten and lipid based films. Journal of Food Science, 61, 116–120. Rico-Pen˜a, D. C., & Torres, J. A. (1991). Sorbic acid and potassium sorbate permeability of an edible methylcellulose-palmitic acid film: Water activity and pH effects. Journal of Food Science, 56, 497–499. Samelis, J., Bedie, G. K., Sofos, J. N., Belk, K. E., Scanga, J. A., & Smith, G. C. (2005). Combinations of nisin with organic acids or salts to control Listeria monocytogenes on sliced pork bologna stored at 4 1C in vacuum packages. Lebensmitte Wissenschaft und Technologie, 38, 21–28. Samelis, J., Sofos, J. N., Kain, M. L., Scanga, J. A., Belk, K. E., & Smith, G. C. (2001). Organic acids and their salts as dipping solutions to control Listeria monocytogenes inoculated following processing of sliced pork bologna stored at 48 1C in vacuum packages. Journal of Food Protection, 64, 1722–1729. Schou, M., Longares, A., Montesinos-Herrero, C., Monahan, F. J., O’ Riordan, D., & O’Sullivan, M. (2005). Properties of edible sodium caseinate films and their applications as food wrapping. LebensmittelWissenschaft und-Technologie, 38, 605–610. Sebti, I., Carnet, A. R., Blanc, D., Saurel, R., & Coma, V. (2003). Controlled diffusion of an antimicrobial peptide from a biopolymer film. Transactions of IchemE, 81, 1099–1104. Sebti, I., & Coma, V. (2002). Active edible polysaccharide coating and interactions between solution coating compounds. Carbohydrate Polymers, 49, 139–144.

Shelef, L. A. (1994). Antimicrobial effects of lactates: A review. Journal of Food Protection, 57, 445–450. Shelef, L. A., & Yang, Q. (1991). Growth suppression of Listeria monocytogenes by lactates in broth, chicken and beef. Journal of Food Protection, 54, 283–287. Siew, D. C. W., Heilmann, C., Easteal, A. J., & Cooney, R. P. (1999). Solution and film properties of sodium caseinate/glycerol and sodium caseinate/polyethylene glycol edible coating systems. Journal of Agriculture and Food Chemistry, 47, 3432–3440. Slade, L., & Levine, H. (1991). Beyond water activity: Recent advantages based on an alternative approach to the assessment of food quality and safety. Critical Reviews in Food Science and Nutrition, 30, 115–360. Sobral, P. J. A., Menegalli, F. C., Hubinger, M. D., & Roques, M. A. (2001). Mechanical, water vapor barrier and thermal properties of gelatin based edible films. Food Hydrocolloids, 15, 423–432. Sofos, J. N., & Busta, F. F. (1993). Sorbic acid and sorbates. In P. M. Davidson, & A. L. Branen (Eds.), Antimicrobials in foods (pp. 49–94). New York: Marcel Dekker, Inc. Timasheff, S. N., & Arakawa, T. (1989). Stabilization of protein structure by solvents. In T. E. Creighton (Ed.), Protein structure a practical approach (pp. 331–345). Oxford: Oxford University Press. Torres, J. A. (1987). Microbial stabilization of intermediate moisture food surfaces. In L. B. Rockland, & L. R. Beuchat (Eds.), Water activity: Theory and applications to food (pp. 329–368). New York: Marcel Dekker, Inc. Ukuku, D. O., & Fett, W. F. (2004). Effect of nisin in combination with EDTA, sodium lactate, and potassium sorbate for reducing Salmonella on whole and fresh-cut cantaloupe. Journal of Food Protection, 67, 2143–2150. van Soest, J., J, G., de Wit, D., J, F., & Vliegenthart, G. (1996). Mechanical properties of thermoplastic waxy maize starch. Journal of Applied Polymer Science, 61, 1927–1937. Vartiainen, J., Skytta, E., Enqvist, J., & Ahvenainen, R. (2003). Properties of antimicrobial plastics containing traditional food preservatives. Packaging Technology and Science, 16, 223–229. Vojdani, F., & Torres, J. A. (1990). Potassium sorbate permeability of methylcellulose and hydroxypropyl methylcellulose coatings: Effect of fatty acids. Journal of Food Science, 55, 841–846. Wederquist, H. J., Sofos, J. N., & Schmidt, G. R. (1994). Listeria monocytogenes inhibition in refrigerated vacuum packaged turkey bologna by chemical additives. Journal of Food Science, 59 498–500, 516. Weng, Y. M., & Chen, M. J. (1997). Sorbic acid as antimycotic additive in polyethylene food packaging films. Lebensmitte Wissenschaft und Technologie, 30, 485–487. Weng, Y. M., Chen, M. J., & Chen, W. (1999). Antimicrobial food packaging materials from poly(ethylene-co-methacrylic acid). Lebensmitte Wissenschaft und Technologie, 32, 191–195.