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Effects of ingredient composition on optical and mechanical properties of pullulan film for food-packaging applications
LWT - Food Science and Technology 44 (2011) 2296e2301
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Effects of ingredient composition on optical and mechanical properties of pullulan film for food-packaging applications V. Trinetta*,1, C.N. Cutter, J.D. Floros Department of Food Science, Penn State University, 202 Food Science Building, University Park, PA 16802, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2010 Received in revised form 8 July 2011 Accepted 11 July 2011
In the present research, the effects of pullulan (Pul), glycerin (Gly), xanthan gum (Xa) and locust bean (Lb) concentrations on pullulan film properties were investigated using a Central Composite Rotatable Design. Optimal ingredient combination was determined and antimicrobial activity of films combined with sakacin A was confirmed against Listeria monocytogenes. Using predictive models, contour plots and the characteristics of commercial LDPE films as constraints, the following combination within the optimal region was selected: Pul 100 g/l, Gly 10 g/l, Xa 1 g/l and Lb 1 g/l. Statistical analysis demonstrated that Pul and Gly significantly influenced the properties of pullulan films. Strong interactions were observed between Pul-Gly and Xa-Gly. When sakacin A was added to the film mixture, no significant influence on films optical properties was reported, while an increase in flexibility was observed. Results obtained indicate the potential application of pullulan film developed in this study as an effective antimicrobial biopolymer in food-packaging systems. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Pullulan Mechanical and optical characteristics Central Composite Rotatable Design (CCRD) Antimicrobial packaging
1. Introduction Antimicrobial packaging has attracted great interest in recent years. This kind of packaging is defined as the incorporation of antimicrobial substances in materials (Quintavalla & Vicini, 2002) in order to enhance the safety and quality of food products by controlling and/or reducing the growth of food borne and spoilage microrganisms (Cooksey, 2005). Among antimicrobial agents, bacteriocins are an example of naturally-occurring compounds that possess the potential to be used in bio-preservation. The controlled release of bacteriocins from packaging films can inhibit and/or inactivate the growth of target microorganisms over time, thus offering a more natural strategy for food preservation. In addition to the desire for safer and better quality products, food packaging has been influenced by other notable changes. Consumers are demanding bio-based, disposable, potentially biodegradable and recyclable materials (Cutter, 2006). Biopolymers therefore represent an innovative opportunity for the food industry: these films could be effective as antimicrobials carriers and help to reduce the use of plastics. In order to satisfy consumers requests, several researchers have investigated mechanical and optical properties of biopolymers and * Corresponding author. Tel.: þ1 765 494 1212; fax: þ1 765 494 7953. E-mail address: [email protected] (V. Trinetta). 1 Present address: Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, IN 47907, USA. 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.07.015
provided useful information about how their characteristics are affected by composition and storage conditions. For example, moisture barrier properties of protein films could be improved by inclusion of hydrophobic additives (Kim, Marx, Weller & Milford, 2003), while the mechanical and barrier properties of edible chitosan films were negatively affected by increasing plasticizer concentrations (Butler, Vergano, Testin, Bunn & Wiles, 1996). Whey protein films produced without plasticizer were very brittle and the addition of glycerol was required for flexible films (McHugh & Krochta, 1994). These results demonstrate the growing interest in biopolymer materials and their application in food packaging. In this study, the biopolymer pullulan was selected: pullulan is a microbially-derived polysaccharide, excellent film-former, that has been used as edible film with various flavors, herb extracts and spices (Kim, Ko & Park, 2002). The films produced are colorless, tasteless, odorless, transparent, flexible, highly impermeable to oil, heat-sealable, and with good oxygen barrier properties (Gounga, Xu, Wang & Yang, 2008). Investigation of its blends is a promising area of material science, as it may lead to new materials with improved functional properties and biodegradability at a relatively low cost (Gounga, Xu, Wang & Yang, 2008). Therefore, the objectives of this research were to: (a) investigate concentration effects of pullulan film components on mechanical and optical properties; (b) determine an optimal combination of pullulan film ingredients and offer a bio-alternative to plastic packaging materials; and (c) evaluate the influence of sakacin A addition, a bacteriocin produced by Lactobacillus sakei DSMZ 6333, on film properties.
V. Trinetta et al. / LWT - Food Science and Technology 44 (2011) 2296e2301
2. Materials and methods
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USA); xanthan gum (Xa) was purchased from TCI America (Portland OR, USA), and locust bean (Lb) from CP Kelco (Lille Skensved, Denmark).
2.1. Experimental design A Central Composite Rotatable Design (Cochran & Cox, 1957) with four variables was used to determine the constituent amount effects and the optimum combination for the development of pullulan films with good mechanical and optical properties for food-packaging use. Pullulan (Pul), glycerin (Gly), xanthan gum (Xa) and locust bean (Lb) were chosen as the input factors, each at five levels. The independent variables were coded as 2; 1; 0; 1; 2. The actual values of the independent variables were: 20, 70, 120, 170 and 220 g/l for Pul; 5, 20 35, 50 and 65 g/l for Gly; 1, 2, 3, 4 and 5 g/l for both Xa and Lb (Table 1). The minimum and maximum levels of each component were based on our preliminary experiments and according to the manufacturer instructions (Hayashibara, Okayama, Japan). Modde Software (Umtri, 1997) was used to construct the design and to determine the experimental points. The six replicates (runs 25e30) at the center of the design were included in order to reduce confounding (11%) (Baskarana et al., 2007). The complete design consisted of 30 runs and all experiments were performed in triplicate and in a randomized order. Water was considered as a bulk agent and was not included in the design. For each film combination, thickness (T), transparency (T600), ultimate tensile strength (UTS), elastic modulus (EM), elongation (EL) and puncture force (PUN) were measured.
2.3. Film formation Films were made following the instructions provided by the manufacturer with some modifications. Briefly, different aqueous solutions of Pul (20e220 g/l) were prepared by stirring the powder in distilled water (100 ml final volume) for 10 min; afterward various concentrations of Gly (5e65 g/l), to plasticize the films, and Xa and Lb (1e5 g/l), to stabilize the matrix solution, were added in sequence. When all the components were completely dissolved (w20 min), the solution was autoclaved at 121 C for 15 min. Films were cast by pipetting 10 ml of solution into sterile plastic plates with inner diameters of 10 cm and allowed to dry for 24 h at 25 C and 40% relative humidity (RH). 2.4. Film thickness measurement Thickness was measured with a micrometer (Max-Cal Inc, Japan) at five random locations on the film (Kyoungju & Song, 2007). The average films thickness (T) for three replicates of each film formulation is reported in Table 1. 2.5. Transparency
2.2. Materials Pullulan (Pul) was supplied by Hayashibara Company (Okayama, Japan). Glycerin (Gly) was obtained from VWR Company (Batavia IL,
A Minolta spectrophotometer type CM 3500 d (Konica Minolta Sensing, Inc, USA), was used to determine the transparency of films (Lee, Son & Hong, 2008). The percent of transmittance
Table 1 Experimental design and data. Responsesb
Factors (g/l) Sample number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17a 18 19 20 21 22 23 24 25 26 27 28 29 30
Pullulan
Glycerin
Xanthan gum
Locust Bean
T
T600
UTS
EM
EL
PUN
70 170 70 170 70 170 70 170 70 170 70 170 70 170 70 170 20 220 120 120 120 120 120 120 120 120 120 120 120 120
20 20 50 50 20 20 50 50 20 20 50 50 20 20 50 50 35 35 5 65 35 35 35 35 35 35 35 35 35 35
2 2 2 2 4 4 4 4 2 2 2 2 4 4 4 4 3 3 3 3 1 5 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 4 4 4 4 4 4 4 4 3 3 3 3 3 3 1 5 3 3 3 3 3 3
0.102 0.011 0.061 0.005 0.083 0.005 0.098 0.026 0.051 0.001 0.083 0.013 0.110 0.018 0.072 0.010 0.064 0.005 0.081 0.008 0.084 0.003 0.064 0.005 0.047 0.003 0.109 0.005 0.067 0.003 0.061 0.010 0.033 0.006 0.141 0.022 0.059 0.009 0.148 0.016 0.097 0.006 0.087 0.003 0.063 0.001 0.066 0.015 0.094 0.005 0.093 0.002 0.094 0.002 0.094 0.001 0.094 0.005 0.094 0.003
8.860 1.072 14.77 1.145 10.65 0.782 9.620 0.660 17.61 2.698 10.93 1.768 8.260 1.306 12.58 1.735 14.01 1.119 11.15 1.138 10.51 0.466 14.01 1.137 19.24 1.418 8.26 0.330 13.68 1.113 15.01 2.660 26.74 4.452 6.44 0.933 15.51 2.320 6.090 0.731 9.350 0.601 10.39 0.389 14.45 2.375 14.18 1.356 9.530 0.518 9.570 0.190 9.550 0.194 9.540 0.505 9.530 0.403 9.540 0.198
22.63 1.659 73.88 4.982 0.930 0.625 68.83 7.748 54.43 3.631 46.12 3.137 0.610 0.045 27.88 2.230 14.90 1.305 35.82 2.607 0.950 0.074 36.75 2.514 16.43 1.232 33.85 6.478 1.810 0.414 31.82 2.214 0 37.59 2.565 74.01 4.967 1.460 1.113 43.40 3.163 26.67 1.789 28.93 1.943 25.56 1.743 32.25 2.155 31.98 2.152 30.77 2.051 33.11 2.213 33.19 2.229 32.27 0176
1294 86.88 3312 231.0 25.74 1.724 344.16 23.10 2322 156.9 2520 184.5 16.49 2.435 2028 154.9 1048 81.03 2403 163.8 26.09 1.916 1907 147.9 1232 135.4 1272 108.8 54.64 19.0 2259 156.7 0 175.1 12.02 413.4 31.10 29.90 1.43 1999 142.5 133.5 24.68 1343 96.77 1808 130.4 1557.3 139.8 1572 140.1 1492 144.1 1599 112.5 1576 110.2 1711 13.10
112.0 19.69 116.0 31.75 248.7 7.57 140.0 13.85 130.0 16.37 110.7 10.08 250.0 20.34 82.00 13.11 102.0 12.05 84.67 12.74 247.7 15.27 118.0 7.21 94.67 3.57 106.0 6.92 203.7 41.35 75.00 10.81 0 116.7 15.53 158.0 14.59 248.7 23.09 114.7 17.09 86.33 1.61 124.7 18.01 93.00 4.18 120.0 12.16 117.3 12.85 120.5 19.92 121.3 18.03 118.3 13.21 118.0 17.99
29.34 1.38 35.24 2.49 27.55 1.93 42.92 4.30 23.69 3.07 32.95 2.30 12.93 1.64 6.849 0.44 4.756 1.02 25.27 1.66 7.609 1.29 7.335 1.63 9.967 0.97 10.29 0.83 2.578 0.30 25.06 7.14 0 17.44 1.43 11.08 0.73 21.52 1.34 10.96 0.87 30.18 3.78 40.88 1.43 22.45 1.98 22.63 1.40 22.54 1.42 22.39 1.69 22.54 1.87 21.81 1.55 22.40 2.52
Data are presented as mean values of three replicates standard deviation. a Films with composition: pullulan 20 (g/l), glycerin 35 (g/l), xathan gum 3 (g/l) and locust bean 3 (g/l) were very weak; thus mechanical properties measurements were not possible. Run 17 was excluded from all statistical analyses. b T: Thickness (mm); T600: Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N).
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(%Tr) at 600 nm was measured, and the transparency at 600 nm (T600) was obtained from the following equation (Han & Floros, 1997):
T600
ðlog%TrÞ ¼ TðmmÞ
2.6. Mechanical properties A Texture Analyzer TA.XT2i (Texture Technologies Corporation, USA) was used to determine ultimate tensile strength (UTS), elastic modulus (EM) and elongation at the break (EL). The tests were performed according to the Standard Test Methods, ASTM D882-02 (ASTM, 2002). Film samples were taken at a width of 1 cm and a length of over 5 cm (Han & Floros, 1997). Initial grip separation and crosshead speed were set at 2 cm and 25 mm/s, respectively. UTS (MPa) is defined as the maximum tensile strength that a film can sustain and was calculated as the ratio of maximum load and original minimum cross sectional area of specimen. EM (MPa) is instead the ratio of stress to strain over the linear part of stressstrain curve and it is a measure of film stiffness (Ozdemir and Floros, 2008). EL is the ratio between the extended length at the moment of rupture and the initial length. The texture analyzer was also used to determine the puncture resistance (PUN) of films. The specimen was placed in a circular holder and a pre-load of 0.1 N was applied to the film at a rate of 0.05 mm/min prior to the test. A hemispherical probe was used with a penetration rate of 10 mm/min. The area under the loaddeformation curve up to the point of puncture (load at puncture, N) was calculated as the force at break and expresses films toughness (Lange, Mokdad & Wyser, 2002). 2.7. Films combined with sakacin A The antimicrobial activity of pullulan films combined with sakacin A has been already demonstrated against several epidemic clones of Listeria monocytogenes (Trinetta, Floros & Cutter, 2010). As a verification test, sakacin A was finally added to the selected pullulan film composition within the optimal region and the influence of bacteriocin addition on the mechanical and optical properties of films was evaluated. Film antimicrobial activity was qualitatively verified against PSU J1-123 and PSU R2-674, two selected epidemic clones of L. monocytogenes, using a plate overlay assay (Trinetta, Floros & Cutter, 2010). Statistical analysis Experiments were performed in triplicate and means with standard deviation were presented. Data were analyzed by Multiple Linear Regressions (MLR), employing the MODDE Statistical Software and the RSGEG procedure implemented by SAS Software (SAS 9.1, Inst. Inc., Cary, N.C., U.S.A.) (SAS, 2008). The fit of the models was investigated through the coefficients of determination and the analysis of variance (ANOVA). Main effects and interactions were evaluated for each response independently, and contour plots were created. Predictive models, contour plots and the characteristics of commercial low density polyethylene (LDPE) films were used as constraints to graphically identify the optimum region (Ozdemir & Floros, 2008). Significant differences and comparison among means for predicted and experimental values at the selected optimal composition were determined, with data being significantly different at P < 0.05.
3. Results and discussion 3.1. Statistical analysis A Central Composite Design was used, experimental data of input factors and observed responses are shown in Table 1. Analysis of variance is given in Table 2: UTS, EM and EL models clearly showed that both linear and quadratic effects were significant (P 0.01), while PUN model indicated that only the linear effects were important (P 0.05). T and T600 models were not statistically significant (P 0.1); they were consequently considered approximations and used only for trend analysis (Kim, Marx, Weller & Milford, 2003). Estimated regression coefficients of the second order polynomial equations for the six responses are reported in Table 3. Following statistical analysis and considering significant factors and interactions, predictive models and contour plots were generated. Each response was analyzed independently and ingredient influence and effects on pullulan film properties are presented below.
3.2. Thickness and Transparency Film thickness affects most of the functional properties of a packaging system. Cuq, Gontard, Cuq & Guilbert (1996) attributed an increase of thickness in cellulose-based coatings to a lower rate of active molecule released. Other researchers demonstrated that an increase in film thickness caused an increase in film opacity (Mali, Grossmann & Garcya, 2004), but thicker films had better mechanical properties (Cuq, Gontard, Cuq & Guilbert, 1996). Therefore, thickness seems to influence not only optical, but also mechanical characteristics of packaging films, as we observed in our experiments. Table 3 indicates that Gly was the main factor influencing the T of pullulan film (P 0.1). By increasing the concentration of Gly, T values increased consequently. The relation was linear and positive: low T values were observed at low Gly concentrations, while higher values were obtained when the amounts of Gly increased. Gly was also the main factor influencing T600 (P 0.1). Transparency of films and coatings is of importance when packaging is used as a direct application on food surfaces, since consumers prefer thin and transparent films (Ozdemir & Floros, 2008). The effect of Gly addition on transparency is indicated in Table 3: the increased of Gly caused a decrease of T600. It was also reported that Gly addition produced whitish films, while films with low Gly concentration were more transparent. As expected, the most transparent film was the thinnest (run 13, Table 1), while the most opaque was the thickest film (run 20, Table 1). High concentrations of Gly probably increased the density of pullulan network in the solutions and consequently film thickness and transparency (Muller, Yamashite & Laurindo, 2008)
Table 2 Analysis of variance (ANOVA) of all six responses. Responsesc
R2 Linear Quadratic Cross product
T
T600
UTS
EM
EL
PUN
0.60 0.47 0.42 0.40
0.61 0.45 0.46 0.43
0.96 1.10a 0.11a 0.31a
0.85 33.61a 17.78a 13.40b
0.97 40703a 16381a 2087a
0.63 122294b 44420 33432
a, b The letters a and b show the level of significance: a ¼ 1%, b ¼ 5%. Numbers without superscripts are not significant. c T: Thickness (mm); T600; Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N).
V. Trinetta et al. / LWT - Food Science and Technology 44 (2011) 2296e2301 Table 3 Estimated regression coefficients of second order polynomial equations between factors and responses. Responsesd
Intercept
bPule bGlye bXae bLbe bPul*Pul bGly*Pul bGly*Gly bXa*Pul bXa*Gly bXa*Xa bLb*Pul bLb*Gly bLb*Xa bLb*Lb
T
T600
UTS
EM
EL
PUN
3.95b 0.04 0.45c 0.42 6.85 7.0E4 0.01 0.13 0.15 0.04 3.53 0.12 0.45 0.60 12.30b
3.90b 0.04 0.47c 0.35 7.20c 7.0E4 0.01c 0.01 0.16 0.02 3.56 0.13 0.44 0.31 12.89b
0.40b 0.05b 0.23a 0.51 0.12 2.4E3a 0.02a 0.02a 0.04 0.05 0.83 0.03 0.21c 1.83 1.41
7.78 0.49 1.27 5.85 12.87 0.03a 0.11a 0.23a 7.0E4 0.99 14.12 0.05 2.03 7.54 13.48
8.22 3.31 30.34b 678.48a 157.07 0.52a 4.28a 8.69a 6.62 70.97a 614.27b 3.46 2.08 181.27 405.94
586.01 41.98 44.67 39.55 378.31b 1.39 0.37 6.97 19.41 84.53 502.65 23.26 96.95 3846 22.71
a, b, c . The letters a, b and c show the level of significance: a ¼ 1%, b ¼ 5%, c ¼ 10%. Numbers without superscripts are not significant. d T: Thickness (mm); T600: Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N). e Pul: pullulan; Gly : glycerin; Xa: xanthan gum; Lb: locust bean.
Lb seems to be also a significant factor for transparency: the addition of this ingredient negatively affected the optical properties of pullulan films, as indicated in Table 3. 3.3. Mechanical properties UTS, EM and EL are material characteristics used to describe how film mechanical properties are related to the chemical structure (McHugh & Krochta, 1994). UTS represents the force required to break the material of a given area; EM is the fundamental measure of film stiffness, and EL is the maximum tensile strain that a film can sustain. In general, packaging films are required to be mechanically strong, but not brittle: these properties are reflected in high values of UTS, EM and a substantial percent of EL at break, when subjected to a tensile elongation test (Nagarsenker & Hegde, 1999). As indicated in Table 3, Pul and Gly are the primary factors influencing UTS and their interaction is also important. UTS values increased, raising Pul concentrations and conversely the response decreased when Gly was incorporated into the film solution. Gly-Pul interaction was also important for EM (P 0.01). Table 3 indicates also that Gly and Xa were the primary factors affecting films EL. Gly had a positive impact on EL values, while increasing Xa concentrations, lower values were observed. The interactions between PulGly and Xa-Gly were also important for this response. By increasing Pul concentrations, EL increased greater when Gly was set at low values; similar effects were observed by increasing Gly in film formulations and setting Xa at low levels. Another important mechanical property of packaging materials is the puncture resistance (Lange, Mokdad & Wyser, 2002). In many applications, packaging can be subjected to damage by penetration, leading to reduced barrier properties and loss of package integrity. For this reason, it is important to describe material behaviors such as “brittle” or “ductile”, when evaluating film puncture resistance. This property is typically expressed as load puncture or energy: the area under the loadedeformation curve (Lange, Mokdad & Wyser, 2002). Table 3 demonstrates that Lb was the main factor affecting the film puncture resistance, and its influence is negative: when high Lb amounts were included in films formulation, response values decreased.
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3.4. Influence of factors Pul was a significant factor in the film mechanical properties. Its presence negatively affected UTS and EL, while EM increased with increasing Pul concentrations (Table 3). Generally, pullulan films are colorless, tasteless, odorless, transparent, flexible, water-soluble and heat-sealable; these properties make this polysaccharide an ideal material for edible films and coatings, as well as a biodegradable and water-soluble packaging material. Pullulan is an exocellular homopolysaccharide produced by Aureobasidium pullulan; it is a linear mix of a-D-glucan consisting mainly of maltotriose repeating units interconnected by a-(1/6) linkages. The regular alternation of a-(1/4) and a-(1/6) bonds results in distinctive structural flexibility and enhances water-solubility (Wu, Jin, Tong & Chen, 2009). It is significant to note that when Pul concentration was set at its lowest value (20 g/l), no mechanical measurements were possible (run 17, Table 1), indicating the importance of this ingredient in making films with good mechanical properties. Therefore, pullulan was considered necessary for film formation. Also Gly proved to be an important factor for all responses, except for PUN (Table 3). Its presence negatively influenced T600 and EM, but positively affected T, UTS and EL. These behaviors are due to the plasticizing effects of Gly. A plasticizer is defined as “a substantially nonvolatile, high boiling, no separating substance”, that when added to another material, changes its physical and mechanical properties (McHugh & Krochta, 1994). Polyols, such as glycerin, plasticize effectively due to their ability to interact with biopolymer chains, reducing internal hydrogen bonding and increasing intermolecular space (Park, Wellwe, Vergano & Testin, 1993). These effects usually cause a decrease in stress rupture and an increase in flexibility, as we clearly observed in our analysis. These observations should be considered when locating the optimum region in order to obtain mechanically resistant pullulan film, but also acceptable by consumers. The ingredient Xa was significant only for elasticity. Xanthan gum is a microbial polysaccharide used often in food industry as an additive. It has very high gelling ability even at low concentrations, and it is often used in combination with other gums, such as locust bean, a vegetable galactomannan, to enhance film texture characteristics. As we observed in our analysis, the addition of Lb onto pullulan film solutions and its combination with Xa, may cause an interaction within the polysaccharide chains, facilitating their movement in the matrix network and consequently increase PUN values of films under stress (Gontard, Guilbert & Cuq, 1993). Even if Lb negatively affects puncture resistance, the combination of xanthan gum and locust bean gum was suggested by the film manufacturer (Harashibara), in order to obtain increased viscosity and facilitate film formation. Therefore, both ingredients were included in films formulation. 3.5. Locating the optimum region Pullulan films and coatings developed in this study were investigated as an alternative bio-material for direct packaging system with mechanical and optical characteristics similar to low density polyethylene films (LDPE). Therefore, our goal was to locate the optimum region with the following characteristics: high mechanical properties to maintain film integrity during shipping, handling and storage, and transparent and thin films to make packaging acceptable by consumers (Ozdermir & Floros, 2008). In order to obtain these characteristics, the following constraints from LDPE films were selected (MatWeb, 2008): T < 0.8 mm, T600 > 10 log %Tr/mm, UTS > 5 MPa, EM > 200 MPa, EL > 100% and PUN > 12 N. These constraints were graphically imposed in each
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contour plot obtained by RSM analysis. Graphs were generated by plotting each response to Pul and Gly factors, while Xa and Lb were maintained constant and set at the lowest level (1 g/l), since their influence was negative for almost all the measured responses. Constrains and regions that satisfy the conditions selected were identified. The six contour plots were then overlapped and the area that fulfilled all constraints was considered as the optimum region. The shaded area in Fig. 1 represents the optimal region of the proportions of Pul and Gly required to produce mechanically sound and optically preferred pullulan films. The area ranges were (g/l): Pul 60e135, Gly 5e28, while Xa and Lb levels were set at 1 g/l. Within this optimal region, a specific composition was selected (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l, Lb 1 g/l), in order to validate effects and interactions observed during the models analysis.
Table 4 Predicted and experimental values of responses at the selected optimal composition (pullulan 100 g/l, glycerin 10 g/l, xathan gum 1 g/l and locust bean 1 g/l) within the optimal region of pullulan films with and without sakacin A.
3.6. Optimum verification and sakacin A addition influence on pullulan films
Also in this study, the composition of selected pullulan films (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l, Lb 1 g/l) infused with sakacin A showed clear antimicrobial activity against the two reference epidemic clones, PSU J1-123 and PSU R2-674 of L. monocytogenes (data not shown), confirming the applicability of the pullulan film infused with sakacin A, as a means of delivering a natural antimicrobial compound directly onto a food surface. The optical and mechanical properties of optimal films composition after bacteriocin addition are reported in Table 4. The addition of sakacin A did not significantly influence T or T600, as compared to the bacteriocin-free films (P > 0.05). Conversely, a significant decrease in UTS and an increase in EL were observed (Table 4). The incorporation of sakacin A into film-forming solutions probably caused rearrangements and interactions within the pullulan network, resulting in an increase of film flexibility, as we reported. Additional studies are furthermore required in order to optimize sakacin A controlled release from pullulan films.
In an independent experiment, three replications of the selected ingredients combination within the optimal region were made, and their T, T600, UTS, EM, EL and PUN were experimentally determined. Predicted values by RSM models and experimental values obtained for the responses are reported in Table 4. Results clearly suggest that predicted and experimental values were not statistically different, at 5% significant level. The models developed in this study were useful to describe relations and interactions among factors and responses. Finally, in order to assess if the selected combination could represent an effective packaging system to suppress microbial spoilage and extend produce shelf-life (Nguyen, Gidley & Dykes, 2008; Ye, Neetoo & Chen, 2008), the effect of sakacin A addition on antimicrobial, mechanical and optical film properties was investigated. In a previous study the effectiveness of sakacin-A-containing pullulan films against epidemic clones of L. monocytogenes was evaluated on experimentally inoculate turkey deli meat slices (Trinetta, Floros & Cutter, 2010). The results showed the potential used of pullulan films as an antimicrobial coating: Listeria population on treated turkey breast was reduced 3 log cfu/g, as compared to untreated samples, after 3 weeks at refrigerated temperature conditions (Trinetta, Floros & Cutter, 2010).
Fig. 1. Optimum region obtained by overlapping contour plots of all six responses generated at 1 g/l xanthan gum and locust bean. Shaded area represents the optimum region with T < 0.8 mm, T600 > 10 log% Tr/mm, UTS> 5 MPa, EM> 200 MPa, EL> 100% and PUN> 12 N. The spot (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l, Lb 1 g/l) indicates the selected combination, within the optimal region, for the validation step.
Responsesc
Predicted
T T600 UTS EM EL PUN
0.062 14.32 124.9 3262 133.9 40.43
Experimental without sakacin
0.02a 2.97a 24a 982a 33.9a 3.68a
0.054 16.46 112 3304 146 40.58
0.013a 0.56a 8.25a 627a 4.86ab 8.78a
with sakacin 0.052 17.18 79.84 3122 167 43.80
0.026a 0.41a 11.11b 722a 1.30b 7.40a
Data are presented as mean values of three replicates standard deviation. Values with different letter in same row are significantly different (p < 0.05). c T: Thickness (mm); T600: Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N).
4. Conclusions In this study a Central Composite Rotatable Design, followed by a graphical optimization method was developed to understand the effects of the four selected factors: pullulan, glycerin, xanthan gum and locust bean, on mechanical and optical properties of pullulan films. Within the optimal region (Pul 60e135 g/l, Gly 5e28 g/l,while Xa and Lb at 1 g/l) a specific composition (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l and Lb 1 g/l), was selected to verify the models and obtain a mechanically strong and optically transparent film. Pullulan, glycerin, and their interaction significantly influenced the mechanical properties of the film: plasticizer addition caused a decrease in stress rupture and an increase in flexibility. Moreover, the interaction between xanthan gum and glycerin was important for pullulan film elasticity and the addition of xanthan gum and locust bean negatively affected the mechanical film characteristics. Conversely, the addition of sakacin A did not significantly influence optical film properties, an increased in elasticity was observed and the antimicrobial activity was confirmed. In conclusion, sakacin-A-containing pullulan films developed in this study represent a biopolymer alternative to plastic packaging materials. These films demonstrated good mechanical and optical properties, as well as antimicrobial effects. The results also suggest that pullulan films may be used to improve the microbial stability and safety of food products, given the inhibitory activity demonstrated against epidemic clones of L. monocytogenes. References ASTM. (2002). Designation D 882-02: standard test method for tensile properties of thin plastic sheeting. Annual book of ASTM Standards. Philadelphia, Pennsylvania: American Society for Testing and Materials. 167-176.
V. Trinetta et al. / LWT - Food Science and Technology 44 (2011) 2296e2301 Baskarana, R., Devia, U. S., Nayakb, C. A., Kudachikara, V. B., Prakash, M. N. K., Prakash, M., et al. (2007). Effect of low-dose g-irradiation on the shelf life and quality characteristics of minimally processed potato cubes under modified atmosphere packaging. Radiation Physics and Chemistry, 76, 1042e1049. Butler, B. L., Vergano, P. J., Testin, R. F., Bunn, J. M., & Wiles, J. L. (1996). Mechanical and barrier properties of edible chitosan films as affected by composition and storage. Journal of Food Science, 61, 953e956. Cochran, W. G., & Cox, G. M. (1957). Experimental designs (2nd ed.). New York: Wiley. Cooksey, K. (2005). Effectiveness of antimicrobial food packaging materials. Food Additives and Contaminants, 22, 980e987. Cuq, B., Gontard, N., Cuq, J. L., & Guilbert, S. (1996). Functional properties of myofibrillar protein-based biopackaging as affected by film thickness. Journal of Food Science, 61, 580e584. Cutter, C. N. (2006). Opportunities for bio-based packaging technologies to improve the quality and safety and further processed muscle foods. Meat Science, 74, 131e142. Gontard, N., Guilbert, S., & Cuq, J. L. (1993). Water and glycerol as plasticizer affect mechanical and water vapor barrier properties of an edible wheat gluten film. Journal of Food Science, 58, 206e211. Gounga, M. E., Xu, S. Y., Wang, Z., & Yang, W. G. (2008). Effect of whey protein isolate-pullulan edible coating on the quality and shelf life of freshly roasted and freeze-dried Chinese chestnut. Journal of Food Science, 73, 155e161. Han, J. H., & Floros, J. D. (1997). Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity. Journal of Plastic Film and Sheeting, 13, 287e298. Kim, K. M., Ko, C. J., & Park, H. J. (2002). Mechanical properties, water vapour permeabilities and solubilities of highly carboxymethylated starch-based edible films. Journal of Food Science, 67, 218e222. Kim, K. M., Marx, D. B., Weller, C. L., & Milford, A. H. (2003). Influence of sorghum wax, glycerin and sorbitol on physical properties of soy protein isolates films. American Journal of Oil Chemists’ Society, 80, 71e76. Kyoungju, K. U., & Song, K. B. (2007). Physical properties of nisin-incorporated gelatin and corn zein film and antimicrobial activity against Listeria monocytogenes. Journal of Microbiology and Biotechnology, 17, 520e523. Lange, J., Mokdad, H., & Wyser, Y. (2002). Understanding puncture resistance and perforation behaviour of packaging laminates. Journal of Plastic Film and Sheeting, 18, 231e244.
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Lee, J. W., Son, S. M., & Hong, S. I. (2008). Characterization of protein-coated polypropylene films as a novel composite structure for active food packaging application. Journal of Food Engineering, 86, 484e493. Mali, S., Grossmann, M. E., & Garcya, M. A. (2004). Barrier, mechanical and optical properties of plasticized yam starch films. Carbohydrate Polymers, 56, 129e135. MatWeb. (2008). MatWeb’s searchable database of material properties [Internet]. Available from http://www.matweb.com/search/DataSheet.aspx?MatGUID¼9ff 98d958a714b2a8a00990a929d6f14. McHugh, T. H., & Krochta, J. M. (1994). Sorbitol- vs glycerol- plasticized whey protein edible films: integrated oxygen permeability and tensile property evaluation. Journal of Agriculture and Food Chemistry, 42, 841e845. Muller, C. M. O., Yamashite, F., & Laurindo, J. B. (2008). Evaluatioin of the effects of glycerol and sorbitol concentration and water activity on the water barrier properties of cassava starch films throught a solubility approach. Carbohydrate Polymers, 72, 82e87. Nagarsenker, M. S., & Hegde, D. D. (1999). Optimization of the mechanical properties and water-vapor transmission properties of free films of Hidroxypropylmethylcellulose. Drug Development and Industrial Pharmacy, 25, 95e98. Nguyen, V. T., Gidley, M. J., & Dykes, G. A. (2008). Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meat. Food Microbiology, 25, 471e478. Ozdemir, M., & Floros, J. D. (2008). Optimization of edible whey protein films containing preservatives for mechanical and optical properties. Journal of Food Engineering, 84, 116e123. Park, H. J., Weller, C. L., Vergano, P. J., & Testin, R. F. (1993). Permeability and mechanical properties of cellulose-based edible films. Journal of Food Science, 58, 1361e1364. Quintavalla, S., & Vicini, L. (2002). Antimicrobial food packaging in meat industry. Meat Science, 62, 373e380. SAS/STATÒ. (2008). User’s Guide, Version 8. Cary, NC, USA: SAS Institute Inc. Trinetta, V., Floros, J. D., & Cutter, C. N. (2010). Sakacina A-containing pullulan film: an active packaging system to control epidemic clones of Listeria monocytogenes in ready to-eat foods. Journal of Food Safety, 30, 260e265. Umtri, A. B. (1997). Modde software, version 4.0. Umea, Sweden: Umetri, AB. Wu, S., Jin, Z., Tong, Q., & Chen, H. (2009). Sweet potato: a novel substrate for pullulan production by Aureobasidium pullulans. Carbohydrate Polymers, 76, 645e649. Ye, M., Neetoo, H., & Chen, H. (2008). Control of Listeria monocytogenes into ham steaks by antimicrobials incorporated into chitosan-coated plastic films. Food Microbiology, 25, 260e265.
Contents lists available at ScienceDirect
LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Effects of ingredient composition on optical and mechanical properties of pullulan film for food-packaging applications V. Trinetta*,1, C.N. Cutter, J.D. Floros Department of Food Science, Penn State University, 202 Food Science Building, University Park, PA 16802, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 November 2010 Received in revised form 8 July 2011 Accepted 11 July 2011
In the present research, the effects of pullulan (Pul), glycerin (Gly), xanthan gum (Xa) and locust bean (Lb) concentrations on pullulan film properties were investigated using a Central Composite Rotatable Design. Optimal ingredient combination was determined and antimicrobial activity of films combined with sakacin A was confirmed against Listeria monocytogenes. Using predictive models, contour plots and the characteristics of commercial LDPE films as constraints, the following combination within the optimal region was selected: Pul 100 g/l, Gly 10 g/l, Xa 1 g/l and Lb 1 g/l. Statistical analysis demonstrated that Pul and Gly significantly influenced the properties of pullulan films. Strong interactions were observed between Pul-Gly and Xa-Gly. When sakacin A was added to the film mixture, no significant influence on films optical properties was reported, while an increase in flexibility was observed. Results obtained indicate the potential application of pullulan film developed in this study as an effective antimicrobial biopolymer in food-packaging systems. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Pullulan Mechanical and optical characteristics Central Composite Rotatable Design (CCRD) Antimicrobial packaging
1. Introduction Antimicrobial packaging has attracted great interest in recent years. This kind of packaging is defined as the incorporation of antimicrobial substances in materials (Quintavalla & Vicini, 2002) in order to enhance the safety and quality of food products by controlling and/or reducing the growth of food borne and spoilage microrganisms (Cooksey, 2005). Among antimicrobial agents, bacteriocins are an example of naturally-occurring compounds that possess the potential to be used in bio-preservation. The controlled release of bacteriocins from packaging films can inhibit and/or inactivate the growth of target microorganisms over time, thus offering a more natural strategy for food preservation. In addition to the desire for safer and better quality products, food packaging has been influenced by other notable changes. Consumers are demanding bio-based, disposable, potentially biodegradable and recyclable materials (Cutter, 2006). Biopolymers therefore represent an innovative opportunity for the food industry: these films could be effective as antimicrobials carriers and help to reduce the use of plastics. In order to satisfy consumers requests, several researchers have investigated mechanical and optical properties of biopolymers and * Corresponding author. Tel.: þ1 765 494 1212; fax: þ1 765 494 7953. E-mail address: [email protected] (V. Trinetta). 1 Present address: Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, IN 47907, USA. 0023-6438/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2011.07.015
provided useful information about how their characteristics are affected by composition and storage conditions. For example, moisture barrier properties of protein films could be improved by inclusion of hydrophobic additives (Kim, Marx, Weller & Milford, 2003), while the mechanical and barrier properties of edible chitosan films were negatively affected by increasing plasticizer concentrations (Butler, Vergano, Testin, Bunn & Wiles, 1996). Whey protein films produced without plasticizer were very brittle and the addition of glycerol was required for flexible films (McHugh & Krochta, 1994). These results demonstrate the growing interest in biopolymer materials and their application in food packaging. In this study, the biopolymer pullulan was selected: pullulan is a microbially-derived polysaccharide, excellent film-former, that has been used as edible film with various flavors, herb extracts and spices (Kim, Ko & Park, 2002). The films produced are colorless, tasteless, odorless, transparent, flexible, highly impermeable to oil, heat-sealable, and with good oxygen barrier properties (Gounga, Xu, Wang & Yang, 2008). Investigation of its blends is a promising area of material science, as it may lead to new materials with improved functional properties and biodegradability at a relatively low cost (Gounga, Xu, Wang & Yang, 2008). Therefore, the objectives of this research were to: (a) investigate concentration effects of pullulan film components on mechanical and optical properties; (b) determine an optimal combination of pullulan film ingredients and offer a bio-alternative to plastic packaging materials; and (c) evaluate the influence of sakacin A addition, a bacteriocin produced by Lactobacillus sakei DSMZ 6333, on film properties.
V. Trinetta et al. / LWT - Food Science and Technology 44 (2011) 2296e2301
2. Materials and methods
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USA); xanthan gum (Xa) was purchased from TCI America (Portland OR, USA), and locust bean (Lb) from CP Kelco (Lille Skensved, Denmark).
2.1. Experimental design A Central Composite Rotatable Design (Cochran & Cox, 1957) with four variables was used to determine the constituent amount effects and the optimum combination for the development of pullulan films with good mechanical and optical properties for food-packaging use. Pullulan (Pul), glycerin (Gly), xanthan gum (Xa) and locust bean (Lb) were chosen as the input factors, each at five levels. The independent variables were coded as 2; 1; 0; 1; 2. The actual values of the independent variables were: 20, 70, 120, 170 and 220 g/l for Pul; 5, 20 35, 50 and 65 g/l for Gly; 1, 2, 3, 4 and 5 g/l for both Xa and Lb (Table 1). The minimum and maximum levels of each component were based on our preliminary experiments and according to the manufacturer instructions (Hayashibara, Okayama, Japan). Modde Software (Umtri, 1997) was used to construct the design and to determine the experimental points. The six replicates (runs 25e30) at the center of the design were included in order to reduce confounding (11%) (Baskarana et al., 2007). The complete design consisted of 30 runs and all experiments were performed in triplicate and in a randomized order. Water was considered as a bulk agent and was not included in the design. For each film combination, thickness (T), transparency (T600), ultimate tensile strength (UTS), elastic modulus (EM), elongation (EL) and puncture force (PUN) were measured.
2.3. Film formation Films were made following the instructions provided by the manufacturer with some modifications. Briefly, different aqueous solutions of Pul (20e220 g/l) were prepared by stirring the powder in distilled water (100 ml final volume) for 10 min; afterward various concentrations of Gly (5e65 g/l), to plasticize the films, and Xa and Lb (1e5 g/l), to stabilize the matrix solution, were added in sequence. When all the components were completely dissolved (w20 min), the solution was autoclaved at 121 C for 15 min. Films were cast by pipetting 10 ml of solution into sterile plastic plates with inner diameters of 10 cm and allowed to dry for 24 h at 25 C and 40% relative humidity (RH). 2.4. Film thickness measurement Thickness was measured with a micrometer (Max-Cal Inc, Japan) at five random locations on the film (Kyoungju & Song, 2007). The average films thickness (T) for three replicates of each film formulation is reported in Table 1. 2.5. Transparency
2.2. Materials Pullulan (Pul) was supplied by Hayashibara Company (Okayama, Japan). Glycerin (Gly) was obtained from VWR Company (Batavia IL,
A Minolta spectrophotometer type CM 3500 d (Konica Minolta Sensing, Inc, USA), was used to determine the transparency of films (Lee, Son & Hong, 2008). The percent of transmittance
Table 1 Experimental design and data. Responsesb
Factors (g/l) Sample number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17a 18 19 20 21 22 23 24 25 26 27 28 29 30
Pullulan
Glycerin
Xanthan gum
Locust Bean
T
T600
UTS
EM
EL
PUN
70 170 70 170 70 170 70 170 70 170 70 170 70 170 70 170 20 220 120 120 120 120 120 120 120 120 120 120 120 120
20 20 50 50 20 20 50 50 20 20 50 50 20 20 50 50 35 35 5 65 35 35 35 35 35 35 35 35 35 35
2 2 2 2 4 4 4 4 2 2 2 2 4 4 4 4 3 3 3 3 1 5 3 3 3 3 3 3 3 3
2 2 2 2 2 2 2 2 4 4 4 4 4 4 4 4 3 3 3 3 3 3 1 5 3 3 3 3 3 3
0.102 0.011 0.061 0.005 0.083 0.005 0.098 0.026 0.051 0.001 0.083 0.013 0.110 0.018 0.072 0.010 0.064 0.005 0.081 0.008 0.084 0.003 0.064 0.005 0.047 0.003 0.109 0.005 0.067 0.003 0.061 0.010 0.033 0.006 0.141 0.022 0.059 0.009 0.148 0.016 0.097 0.006 0.087 0.003 0.063 0.001 0.066 0.015 0.094 0.005 0.093 0.002 0.094 0.002 0.094 0.001 0.094 0.005 0.094 0.003
8.860 1.072 14.77 1.145 10.65 0.782 9.620 0.660 17.61 2.698 10.93 1.768 8.260 1.306 12.58 1.735 14.01 1.119 11.15 1.138 10.51 0.466 14.01 1.137 19.24 1.418 8.26 0.330 13.68 1.113 15.01 2.660 26.74 4.452 6.44 0.933 15.51 2.320 6.090 0.731 9.350 0.601 10.39 0.389 14.45 2.375 14.18 1.356 9.530 0.518 9.570 0.190 9.550 0.194 9.540 0.505 9.530 0.403 9.540 0.198
22.63 1.659 73.88 4.982 0.930 0.625 68.83 7.748 54.43 3.631 46.12 3.137 0.610 0.045 27.88 2.230 14.90 1.305 35.82 2.607 0.950 0.074 36.75 2.514 16.43 1.232 33.85 6.478 1.810 0.414 31.82 2.214 0 37.59 2.565 74.01 4.967 1.460 1.113 43.40 3.163 26.67 1.789 28.93 1.943 25.56 1.743 32.25 2.155 31.98 2.152 30.77 2.051 33.11 2.213 33.19 2.229 32.27 0176
1294 86.88 3312 231.0 25.74 1.724 344.16 23.10 2322 156.9 2520 184.5 16.49 2.435 2028 154.9 1048 81.03 2403 163.8 26.09 1.916 1907 147.9 1232 135.4 1272 108.8 54.64 19.0 2259 156.7 0 175.1 12.02 413.4 31.10 29.90 1.43 1999 142.5 133.5 24.68 1343 96.77 1808 130.4 1557.3 139.8 1572 140.1 1492 144.1 1599 112.5 1576 110.2 1711 13.10
112.0 19.69 116.0 31.75 248.7 7.57 140.0 13.85 130.0 16.37 110.7 10.08 250.0 20.34 82.00 13.11 102.0 12.05 84.67 12.74 247.7 15.27 118.0 7.21 94.67 3.57 106.0 6.92 203.7 41.35 75.00 10.81 0 116.7 15.53 158.0 14.59 248.7 23.09 114.7 17.09 86.33 1.61 124.7 18.01 93.00 4.18 120.0 12.16 117.3 12.85 120.5 19.92 121.3 18.03 118.3 13.21 118.0 17.99
29.34 1.38 35.24 2.49 27.55 1.93 42.92 4.30 23.69 3.07 32.95 2.30 12.93 1.64 6.849 0.44 4.756 1.02 25.27 1.66 7.609 1.29 7.335 1.63 9.967 0.97 10.29 0.83 2.578 0.30 25.06 7.14 0 17.44 1.43 11.08 0.73 21.52 1.34 10.96 0.87 30.18 3.78 40.88 1.43 22.45 1.98 22.63 1.40 22.54 1.42 22.39 1.69 22.54 1.87 21.81 1.55 22.40 2.52
Data are presented as mean values of three replicates standard deviation. a Films with composition: pullulan 20 (g/l), glycerin 35 (g/l), xathan gum 3 (g/l) and locust bean 3 (g/l) were very weak; thus mechanical properties measurements were not possible. Run 17 was excluded from all statistical analyses. b T: Thickness (mm); T600: Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N).
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(%Tr) at 600 nm was measured, and the transparency at 600 nm (T600) was obtained from the following equation (Han & Floros, 1997):
T600
ðlog%TrÞ ¼ TðmmÞ
2.6. Mechanical properties A Texture Analyzer TA.XT2i (Texture Technologies Corporation, USA) was used to determine ultimate tensile strength (UTS), elastic modulus (EM) and elongation at the break (EL). The tests were performed according to the Standard Test Methods, ASTM D882-02 (ASTM, 2002). Film samples were taken at a width of 1 cm and a length of over 5 cm (Han & Floros, 1997). Initial grip separation and crosshead speed were set at 2 cm and 25 mm/s, respectively. UTS (MPa) is defined as the maximum tensile strength that a film can sustain and was calculated as the ratio of maximum load and original minimum cross sectional area of specimen. EM (MPa) is instead the ratio of stress to strain over the linear part of stressstrain curve and it is a measure of film stiffness (Ozdemir and Floros, 2008). EL is the ratio between the extended length at the moment of rupture and the initial length. The texture analyzer was also used to determine the puncture resistance (PUN) of films. The specimen was placed in a circular holder and a pre-load of 0.1 N was applied to the film at a rate of 0.05 mm/min prior to the test. A hemispherical probe was used with a penetration rate of 10 mm/min. The area under the loaddeformation curve up to the point of puncture (load at puncture, N) was calculated as the force at break and expresses films toughness (Lange, Mokdad & Wyser, 2002). 2.7. Films combined with sakacin A The antimicrobial activity of pullulan films combined with sakacin A has been already demonstrated against several epidemic clones of Listeria monocytogenes (Trinetta, Floros & Cutter, 2010). As a verification test, sakacin A was finally added to the selected pullulan film composition within the optimal region and the influence of bacteriocin addition on the mechanical and optical properties of films was evaluated. Film antimicrobial activity was qualitatively verified against PSU J1-123 and PSU R2-674, two selected epidemic clones of L. monocytogenes, using a plate overlay assay (Trinetta, Floros & Cutter, 2010). Statistical analysis Experiments were performed in triplicate and means with standard deviation were presented. Data were analyzed by Multiple Linear Regressions (MLR), employing the MODDE Statistical Software and the RSGEG procedure implemented by SAS Software (SAS 9.1, Inst. Inc., Cary, N.C., U.S.A.) (SAS, 2008). The fit of the models was investigated through the coefficients of determination and the analysis of variance (ANOVA). Main effects and interactions were evaluated for each response independently, and contour plots were created. Predictive models, contour plots and the characteristics of commercial low density polyethylene (LDPE) films were used as constraints to graphically identify the optimum region (Ozdemir & Floros, 2008). Significant differences and comparison among means for predicted and experimental values at the selected optimal composition were determined, with data being significantly different at P < 0.05.
3. Results and discussion 3.1. Statistical analysis A Central Composite Design was used, experimental data of input factors and observed responses are shown in Table 1. Analysis of variance is given in Table 2: UTS, EM and EL models clearly showed that both linear and quadratic effects were significant (P 0.01), while PUN model indicated that only the linear effects were important (P 0.05). T and T600 models were not statistically significant (P 0.1); they were consequently considered approximations and used only for trend analysis (Kim, Marx, Weller & Milford, 2003). Estimated regression coefficients of the second order polynomial equations for the six responses are reported in Table 3. Following statistical analysis and considering significant factors and interactions, predictive models and contour plots were generated. Each response was analyzed independently and ingredient influence and effects on pullulan film properties are presented below.
3.2. Thickness and Transparency Film thickness affects most of the functional properties of a packaging system. Cuq, Gontard, Cuq & Guilbert (1996) attributed an increase of thickness in cellulose-based coatings to a lower rate of active molecule released. Other researchers demonstrated that an increase in film thickness caused an increase in film opacity (Mali, Grossmann & Garcya, 2004), but thicker films had better mechanical properties (Cuq, Gontard, Cuq & Guilbert, 1996). Therefore, thickness seems to influence not only optical, but also mechanical characteristics of packaging films, as we observed in our experiments. Table 3 indicates that Gly was the main factor influencing the T of pullulan film (P 0.1). By increasing the concentration of Gly, T values increased consequently. The relation was linear and positive: low T values were observed at low Gly concentrations, while higher values were obtained when the amounts of Gly increased. Gly was also the main factor influencing T600 (P 0.1). Transparency of films and coatings is of importance when packaging is used as a direct application on food surfaces, since consumers prefer thin and transparent films (Ozdemir & Floros, 2008). The effect of Gly addition on transparency is indicated in Table 3: the increased of Gly caused a decrease of T600. It was also reported that Gly addition produced whitish films, while films with low Gly concentration were more transparent. As expected, the most transparent film was the thinnest (run 13, Table 1), while the most opaque was the thickest film (run 20, Table 1). High concentrations of Gly probably increased the density of pullulan network in the solutions and consequently film thickness and transparency (Muller, Yamashite & Laurindo, 2008)
Table 2 Analysis of variance (ANOVA) of all six responses. Responsesc
R2 Linear Quadratic Cross product
T
T600
UTS
EM
EL
PUN
0.60 0.47 0.42 0.40
0.61 0.45 0.46 0.43
0.96 1.10a 0.11a 0.31a
0.85 33.61a 17.78a 13.40b
0.97 40703a 16381a 2087a
0.63 122294b 44420 33432
a, b The letters a and b show the level of significance: a ¼ 1%, b ¼ 5%. Numbers without superscripts are not significant. c T: Thickness (mm); T600; Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N).
V. Trinetta et al. / LWT - Food Science and Technology 44 (2011) 2296e2301 Table 3 Estimated regression coefficients of second order polynomial equations between factors and responses. Responsesd
Intercept
bPule bGlye bXae bLbe bPul*Pul bGly*Pul bGly*Gly bXa*Pul bXa*Gly bXa*Xa bLb*Pul bLb*Gly bLb*Xa bLb*Lb
T
T600
UTS
EM
EL
PUN
3.95b 0.04 0.45c 0.42 6.85 7.0E4 0.01 0.13 0.15 0.04 3.53 0.12 0.45 0.60 12.30b
3.90b 0.04 0.47c 0.35 7.20c 7.0E4 0.01c 0.01 0.16 0.02 3.56 0.13 0.44 0.31 12.89b
0.40b 0.05b 0.23a 0.51 0.12 2.4E3a 0.02a 0.02a 0.04 0.05 0.83 0.03 0.21c 1.83 1.41
7.78 0.49 1.27 5.85 12.87 0.03a 0.11a 0.23a 7.0E4 0.99 14.12 0.05 2.03 7.54 13.48
8.22 3.31 30.34b 678.48a 157.07 0.52a 4.28a 8.69a 6.62 70.97a 614.27b 3.46 2.08 181.27 405.94
586.01 41.98 44.67 39.55 378.31b 1.39 0.37 6.97 19.41 84.53 502.65 23.26 96.95 3846 22.71
a, b, c . The letters a, b and c show the level of significance: a ¼ 1%, b ¼ 5%, c ¼ 10%. Numbers without superscripts are not significant. d T: Thickness (mm); T600: Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N). e Pul: pullulan; Gly : glycerin; Xa: xanthan gum; Lb: locust bean.
Lb seems to be also a significant factor for transparency: the addition of this ingredient negatively affected the optical properties of pullulan films, as indicated in Table 3. 3.3. Mechanical properties UTS, EM and EL are material characteristics used to describe how film mechanical properties are related to the chemical structure (McHugh & Krochta, 1994). UTS represents the force required to break the material of a given area; EM is the fundamental measure of film stiffness, and EL is the maximum tensile strain that a film can sustain. In general, packaging films are required to be mechanically strong, but not brittle: these properties are reflected in high values of UTS, EM and a substantial percent of EL at break, when subjected to a tensile elongation test (Nagarsenker & Hegde, 1999). As indicated in Table 3, Pul and Gly are the primary factors influencing UTS and their interaction is also important. UTS values increased, raising Pul concentrations and conversely the response decreased when Gly was incorporated into the film solution. Gly-Pul interaction was also important for EM (P 0.01). Table 3 indicates also that Gly and Xa were the primary factors affecting films EL. Gly had a positive impact on EL values, while increasing Xa concentrations, lower values were observed. The interactions between PulGly and Xa-Gly were also important for this response. By increasing Pul concentrations, EL increased greater when Gly was set at low values; similar effects were observed by increasing Gly in film formulations and setting Xa at low levels. Another important mechanical property of packaging materials is the puncture resistance (Lange, Mokdad & Wyser, 2002). In many applications, packaging can be subjected to damage by penetration, leading to reduced barrier properties and loss of package integrity. For this reason, it is important to describe material behaviors such as “brittle” or “ductile”, when evaluating film puncture resistance. This property is typically expressed as load puncture or energy: the area under the loadedeformation curve (Lange, Mokdad & Wyser, 2002). Table 3 demonstrates that Lb was the main factor affecting the film puncture resistance, and its influence is negative: when high Lb amounts were included in films formulation, response values decreased.
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3.4. Influence of factors Pul was a significant factor in the film mechanical properties. Its presence negatively affected UTS and EL, while EM increased with increasing Pul concentrations (Table 3). Generally, pullulan films are colorless, tasteless, odorless, transparent, flexible, water-soluble and heat-sealable; these properties make this polysaccharide an ideal material for edible films and coatings, as well as a biodegradable and water-soluble packaging material. Pullulan is an exocellular homopolysaccharide produced by Aureobasidium pullulan; it is a linear mix of a-D-glucan consisting mainly of maltotriose repeating units interconnected by a-(1/6) linkages. The regular alternation of a-(1/4) and a-(1/6) bonds results in distinctive structural flexibility and enhances water-solubility (Wu, Jin, Tong & Chen, 2009). It is significant to note that when Pul concentration was set at its lowest value (20 g/l), no mechanical measurements were possible (run 17, Table 1), indicating the importance of this ingredient in making films with good mechanical properties. Therefore, pullulan was considered necessary for film formation. Also Gly proved to be an important factor for all responses, except for PUN (Table 3). Its presence negatively influenced T600 and EM, but positively affected T, UTS and EL. These behaviors are due to the plasticizing effects of Gly. A plasticizer is defined as “a substantially nonvolatile, high boiling, no separating substance”, that when added to another material, changes its physical and mechanical properties (McHugh & Krochta, 1994). Polyols, such as glycerin, plasticize effectively due to their ability to interact with biopolymer chains, reducing internal hydrogen bonding and increasing intermolecular space (Park, Wellwe, Vergano & Testin, 1993). These effects usually cause a decrease in stress rupture and an increase in flexibility, as we clearly observed in our analysis. These observations should be considered when locating the optimum region in order to obtain mechanically resistant pullulan film, but also acceptable by consumers. The ingredient Xa was significant only for elasticity. Xanthan gum is a microbial polysaccharide used often in food industry as an additive. It has very high gelling ability even at low concentrations, and it is often used in combination with other gums, such as locust bean, a vegetable galactomannan, to enhance film texture characteristics. As we observed in our analysis, the addition of Lb onto pullulan film solutions and its combination with Xa, may cause an interaction within the polysaccharide chains, facilitating their movement in the matrix network and consequently increase PUN values of films under stress (Gontard, Guilbert & Cuq, 1993). Even if Lb negatively affects puncture resistance, the combination of xanthan gum and locust bean gum was suggested by the film manufacturer (Harashibara), in order to obtain increased viscosity and facilitate film formation. Therefore, both ingredients were included in films formulation. 3.5. Locating the optimum region Pullulan films and coatings developed in this study were investigated as an alternative bio-material for direct packaging system with mechanical and optical characteristics similar to low density polyethylene films (LDPE). Therefore, our goal was to locate the optimum region with the following characteristics: high mechanical properties to maintain film integrity during shipping, handling and storage, and transparent and thin films to make packaging acceptable by consumers (Ozdermir & Floros, 2008). In order to obtain these characteristics, the following constraints from LDPE films were selected (MatWeb, 2008): T < 0.8 mm, T600 > 10 log %Tr/mm, UTS > 5 MPa, EM > 200 MPa, EL > 100% and PUN > 12 N. These constraints were graphically imposed in each
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contour plot obtained by RSM analysis. Graphs were generated by plotting each response to Pul and Gly factors, while Xa and Lb were maintained constant and set at the lowest level (1 g/l), since their influence was negative for almost all the measured responses. Constrains and regions that satisfy the conditions selected were identified. The six contour plots were then overlapped and the area that fulfilled all constraints was considered as the optimum region. The shaded area in Fig. 1 represents the optimal region of the proportions of Pul and Gly required to produce mechanically sound and optically preferred pullulan films. The area ranges were (g/l): Pul 60e135, Gly 5e28, while Xa and Lb levels were set at 1 g/l. Within this optimal region, a specific composition was selected (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l, Lb 1 g/l), in order to validate effects and interactions observed during the models analysis.
Table 4 Predicted and experimental values of responses at the selected optimal composition (pullulan 100 g/l, glycerin 10 g/l, xathan gum 1 g/l and locust bean 1 g/l) within the optimal region of pullulan films with and without sakacin A.
3.6. Optimum verification and sakacin A addition influence on pullulan films
Also in this study, the composition of selected pullulan films (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l, Lb 1 g/l) infused with sakacin A showed clear antimicrobial activity against the two reference epidemic clones, PSU J1-123 and PSU R2-674 of L. monocytogenes (data not shown), confirming the applicability of the pullulan film infused with sakacin A, as a means of delivering a natural antimicrobial compound directly onto a food surface. The optical and mechanical properties of optimal films composition after bacteriocin addition are reported in Table 4. The addition of sakacin A did not significantly influence T or T600, as compared to the bacteriocin-free films (P > 0.05). Conversely, a significant decrease in UTS and an increase in EL were observed (Table 4). The incorporation of sakacin A into film-forming solutions probably caused rearrangements and interactions within the pullulan network, resulting in an increase of film flexibility, as we reported. Additional studies are furthermore required in order to optimize sakacin A controlled release from pullulan films.
In an independent experiment, three replications of the selected ingredients combination within the optimal region were made, and their T, T600, UTS, EM, EL and PUN were experimentally determined. Predicted values by RSM models and experimental values obtained for the responses are reported in Table 4. Results clearly suggest that predicted and experimental values were not statistically different, at 5% significant level. The models developed in this study were useful to describe relations and interactions among factors and responses. Finally, in order to assess if the selected combination could represent an effective packaging system to suppress microbial spoilage and extend produce shelf-life (Nguyen, Gidley & Dykes, 2008; Ye, Neetoo & Chen, 2008), the effect of sakacin A addition on antimicrobial, mechanical and optical film properties was investigated. In a previous study the effectiveness of sakacin-A-containing pullulan films against epidemic clones of L. monocytogenes was evaluated on experimentally inoculate turkey deli meat slices (Trinetta, Floros & Cutter, 2010). The results showed the potential used of pullulan films as an antimicrobial coating: Listeria population on treated turkey breast was reduced 3 log cfu/g, as compared to untreated samples, after 3 weeks at refrigerated temperature conditions (Trinetta, Floros & Cutter, 2010).
Fig. 1. Optimum region obtained by overlapping contour plots of all six responses generated at 1 g/l xanthan gum and locust bean. Shaded area represents the optimum region with T < 0.8 mm, T600 > 10 log% Tr/mm, UTS> 5 MPa, EM> 200 MPa, EL> 100% and PUN> 12 N. The spot (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l, Lb 1 g/l) indicates the selected combination, within the optimal region, for the validation step.
Responsesc
Predicted
T T600 UTS EM EL PUN
0.062 14.32 124.9 3262 133.9 40.43
Experimental without sakacin
0.02a 2.97a 24a 982a 33.9a 3.68a
0.054 16.46 112 3304 146 40.58
0.013a 0.56a 8.25a 627a 4.86ab 8.78a
with sakacin 0.052 17.18 79.84 3122 167 43.80
0.026a 0.41a 11.11b 722a 1.30b 7.40a
Data are presented as mean values of three replicates standard deviation. Values with different letter in same row are significantly different (p < 0.05). c T: Thickness (mm); T600: Transparency (log %Tr/mm); UTS: Ultimate tensile strenght (MPa); EM: Elastic Modulus (MPa); EL: Elongation (%); PUN: Puncture force (N).
4. Conclusions In this study a Central Composite Rotatable Design, followed by a graphical optimization method was developed to understand the effects of the four selected factors: pullulan, glycerin, xanthan gum and locust bean, on mechanical and optical properties of pullulan films. Within the optimal region (Pul 60e135 g/l, Gly 5e28 g/l,while Xa and Lb at 1 g/l) a specific composition (Pul 100 g/l, Gly 10 g/l, Xa 1 g/l and Lb 1 g/l), was selected to verify the models and obtain a mechanically strong and optically transparent film. Pullulan, glycerin, and their interaction significantly influenced the mechanical properties of the film: plasticizer addition caused a decrease in stress rupture and an increase in flexibility. Moreover, the interaction between xanthan gum and glycerin was important for pullulan film elasticity and the addition of xanthan gum and locust bean negatively affected the mechanical film characteristics. Conversely, the addition of sakacin A did not significantly influence optical film properties, an increased in elasticity was observed and the antimicrobial activity was confirmed. In conclusion, sakacin-A-containing pullulan films developed in this study represent a biopolymer alternative to plastic packaging materials. These films demonstrated good mechanical and optical properties, as well as antimicrobial effects. The results also suggest that pullulan films may be used to improve the microbial stability and safety of food products, given the inhibitory activity demonstrated against epidemic clones of L. monocytogenes. References ASTM. (2002). Designation D 882-02: standard test method for tensile properties of thin plastic sheeting. Annual book of ASTM Standards. Philadelphia, Pennsylvania: American Society for Testing and Materials. 167-176.
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