Innovative Food Science and Emerging Technologies 10 (2009) 203–207
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Innovative Food Science and Emerging Technologies j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i f s e t
Antimicrobial plastic film: Physico-chemical characterization and nisin desorption modeling W. Guiga ⁎, S. Galland, E. Peyrol, P. Degraeve, A. Carnet-Pantiez, I. Sebti 1 Université Lyon 1, Laboratoire de Recherche en Génie Industriel Alimentaire EA 3733, IUT A, Département Génie Biologique, Technopole Alimentec, rue Henri de Boissieu, 01060 Bourg-en-Bresse cedex 9, France
a r t i c l e
i n f o
Article history: Received 5 March 2008 Accepted 21 September 2008 Keywords: Nisaplin Plastic film Desorption
a b s t r a c t Antimicrobial active films represent an innovative concept in food packaging, developed to answer to consumer's expectation for better microbiological safety. In this study, the growth of pathogenic microorganisms on the surface of food is proposed to be controlled by coating, on the surface of polyethylene/ polyamide/polyethylene film (PE/PA/PE), a film-forming solution containing Nisaplin, a commercial form of bacteriocin produced by Lactococcus lactis subsp. lactis: nisin. The bioactivity of these multi-layer films coated with Nisaplin loaded HydroxyPropylMethylCellulose film is based on the release of this antimicrobial molecule towards a food simulant. Nisin mass transfer was studied and modeled, for different operating conditions, generally encountered in food products. pH didn't seem to interfere with nisin release kinetics, while the variation of NaCl concentration between 0.8% and 3.2% decreased the desorption coefficient (kd) by 18% and the temperature increase from 10 °C to 28 °C resulted in an increase of kd from 1.78 × 10− 2 m s− 1 to 2.10 × 10− 2 m s− 1. Coating of PE/PA/PE film with this antimicrobial layer induced little mechanical properties modifications without compromising industrial applications. Water barrier capacity was not altered. Industrial relevance: This paper concerns active packaging, considered as a new approach to preserve food shelf life. Active packaging is a real gain for plastic and Food industrials. Coating was used to obtain antimicrobial packaging. The impact of coating on film characteristics is investigated. Also, antimicrobial agent desorption is determined during storage conditions. © 2008 Elsevier Ltd. All rights reserved.
1. Introduction To prevent microbial contamination of food products, antimicrobial agents can be either incorporated in foods during their preparation, or applied on their surface (Kim et al., 2002). Both of these operations present a limited efficiency as they result in a rapid loss of antimicrobial activity. Antimicrobial active packaging — consisting in the incorporation of the active molecules in the packaging films — present an innovative option. In fact, they allow a better efficiency in food protection as they offer a better stability of the antimicrobial agent, and ensure the control of its release towards food. The commonly used antimicrobial molecules in food are organic acids (Cagri et al., 2001), enzymes (Padgett et al., 1998), essential oils (Tunc et al., 2007) or bacteriocins (Jacquet et al., 1998). Among the latter, only nisin (amphiphilic cationic peptide produced by Lactococcus lactis subsp. lactis) is allowed for use as food additive in the European Union (E 234). When used as antimicrobial agent in food packaging,
the efficiency of nisin depends on several parameters: pH, temperature, salt and fat concentrations (Jung et al., 1992; Dean & Zottola, 1996). These parameters play a major role on nisin solubility, bioactivity, stability, desorption rate from films and diffusion into food matrices. Some previous studies investigated nisin diffusion into food matrices (Sebti et al., 2004; Ripoche et al., 2006), but very few were interested in the desorption phenomenon of the antimicrobial agent from biopolymer films (Buonocore et al., 2003). The purpose of this study was to investigate the desorption phenomenon of nisin from a polyethylene/polyamide/polyethylene PE/PA/PE film coated with HydroxyPropylMethylCellulose (HPMC) film-forming solution containing Nisaplin. Nisaplin is a commercial form of nisin at 2.5% of purity. At the same time, this study proposed to verify the impact of Nisaplin incorporation in films on the mechanical and water barrier properties of films. 2. Materials and methods 2.1. Nisaplin solutions preparation
⁎ Corresponding author. Tel.: + 33 1 58 80 86 16; fax: + 33 1 40 27 20 66. E-mail address:
[email protected] (W. Guiga). 1 Deceased. 1466-8564/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2008.09.006
Nisaplin powder was from DANISCO (Denmark; 2.5% purity, 77.5% NaCl, 20% non-fat dry milk compounds). Nisaplin solutions
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Nomenclature A aw c Ca C1a C2a Cb ⁎ Csol
Area of mass exchange activity of water constant related to the heat of sorption water concentration in the air in contact with gel water concentration at the surface of agar gel water concentration at the film surface water concentration in the air equilibrium concentration of nisin between the solution and the HPMC film Cto,sol initial nisin concentration in the solution at t = 0 %EB percentage elongation at break g time gravity k constant related to the heat of sorption. Kc Effective water transfer coefficient kd desorption coefficient KEQ′ and KEQ equilibrium partition constant RH relative humidity rpm rotation per minute S film surface area TWDR Total Water Desorption Rate Vsol: volume of the desorption solution x water content (g water/100 g dry film) xm weight of water (g) in a complete monolayer per 100 g of polymer Y Young modulus (Pa)
were obtained by suspending Nisaplin powder in HCl 0.01 N (pH 2) at a concentration of 48 mg ml− 1 (corresponding to a calculated nisin concentration of 800 µg mL− 1) for desorption experiments and 10 mg ml− 1 (corresponding to a calculated nisin concentration of 166.7 µg mL− 1) for the characterization of Nisaplin-loaded films. Nisaplin suspensions had to be centrifuged at 4000 g (8500 rpm, rotor radius equal to 5 cm) for 15 min at 4 °C and the supernatant recovered. Centrifugation was necessary to take off insoluble fractions in acid solution. Nisaplin solutions were stored at 4 °C until their use. 2.2. Preparation of antimicrobial active films The film forming solution was 6% and 3% (w/w) of HydroxyPropylMethylCellulose (HPMC) for desorption experiments and film characterization respectively. HPMC (culminal 50, Aqualon France, le Pecq) is prepared in 1/3 (w/w) ethanol (96%; Chimie-Plus Laboratoires, Denice, France) and 2/3 (w/w) HCl 0.01 M with a requested final nisin concentration of 800 µg mL− 1 for desorption experiments and 166,7 µg mL− 1 for film characterization respectively. After 1–2 h of homogenization at room temperature, the film forming solution was used to coat the polyethylene/polyamide/ polyethylene (PE/PA/PE) on one side of the film using an automatic K control coater (Erichsen®, Rueil-Malmaison, France) equipped with a spiral film applicator (120 µm-wet thickness). Eight layers are deposited successively with a drying step at 60 °C during 10 min after each deposit. The final thickness was measured with a Käfer micrometer (Erichsen, Rueil-Malmaison, France). It was of 10 ± 1 µm for standard PE/PA/PE films and 60 ± 1 µm and 110 ± 8 µm for multilayer PE/PA/PE films coated with Nisaplin loaded HPMC film used for characterization and for film desorption experiments respectively. Before each experiment, the antimicrobial activity of films was verified using microbial agar diffusion method on Kocuria rhizophila ATCC 9341 (formerly Micrococcus luteus). All multilayer
PE/PA/PE films coated with Nisaplin loaded HPMC film showed clear zones of inhibition of about 10 mm surrounding the diameter of the circular film. 2.3. Evaluation of physico-chemical properties of plastic films 2.3.1. Sorption isotherms Sorption isotherms were established for PE/PA/PE films, multilayer PE/PA/PE films coated with nisin-loaded HPMC film and multilayer PE/ PA/PE films coated with Nisaplin-loaded HPMC film. Films were cut into 0.5 × 0.8 cm pieces. 0.12 g of each cut sample were put into aluminum dishes and stored in sealed glass jars containing saturated salt solutions corresponding to constant water activity values (aw) at 23 °C. aw values used were: 0.10, 0.22, 0.33, 0.44, 0.55, 0.68, 0.76 and 0.85. Equilibrium moisture content of samples was reached after a maximum of two months storage (Coupland et al., 2000; Sebti et al., 2007). It was then determined by drying cut samples at 103 °C during 2 h. The experimental sorption isotherms were then plot and fitted by the Guggenheim–Anderson–DeBoer (GAB) model (Eq. (1)), commonly used to describe water sorption in food products.
x=
xm × c × k × aw ð1−k × aw Þ × ð1−k × aw + c × k × aw Þ
ð1Þ
Where x is the weight (g) of sorbed water per 100 g of polymer, aw is the water activity, xm is the weight of water (g) in a complete monolayer per 100 g of polymer, c and k are constants related to the heat of sorption. 2.3.2. Total Water Desorption Rate (TWDR) measurements were determined using a modified method developed by Desobry and Hardy (1993) as described in the Fig. 1. Open agar (3% w/w) and agar coated with films were put in an environment with controlled humidity and temperature (50 ± 5% RH and 23 ± 1 °C) during 3 days. The slope of the linear part of the desorption curve (mass loss versus time) corresponds to TWDR, expressed as kgwater m− 2 s− 1. The effective water transfer coefficient Kc was then determined using the following equation: TWDRagar = Kc ðC1a −Cb Þ
ð2Þ
The water concentration of the air in contact with the agar gel (C1a) was calculated with Mollier diagram from air characteristics of open agar surface (100% RH and 23 °C) and Cb by surrounded air characteristics (50% RH and 23 °C). The water concentration of the air in contact with the film surface (C2a) was then determined using the following equation: TWDRfilm = Kc ðC2a −Cb Þ
ð3Þ
2.3.3. Mechanical properties A TA.XT2 Texture Analyzer (Texture Technology Corp.) was used to measure Young's modulus (Y (Pa)) and elongation at break (EB (%)) on 20 mm × 45 mm samples previously stored for 7 days at 23 °C and 50% RH. Films were uniaxially stretched at a constant velocity of 0.1 mm s− 1. The computer-recorded force–deformation curves were then used to determine Y and EB. Y reflects the film stiffness and is calculated from the slope of the initial linear region of the force–deformation curve, as follows: Y=
Curve slope Film section
The film section was: width ⁎ thickness.
ð4Þ
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Fig. 1. Models of surface water evaporation and of water diffusion in antimicrobial film: a) from open agar gel and b) from agar gel coated with multilayer PE/PA/PE film coated with Nisaplin loaded HPMC film.
The elongation at break is the maximum change in film length before breaking, expressed as a percentage.
3. Results and discussions 3.1. Sensitivity of film to water
2.4. Study of the nisin desorption 2.4.1. Method and experimental design The film side having been coated with Nisaplin loaded HPMC film was placed at the surface of a desorption solution (NaCl solution, volume of 125 mL) and the former was stirred during 4 h. A volume of 800 µL was periodically taken from this solution to quantify the desorbed nisin according to the BCA method (QBCA Sigma®). The impact of three parameters on the desorption kinetics was studied, using a full factorial experimental design (Table 1): the pH of the desorption solution (between 3.8 and 6.3), its NaCl concentration (between 0.8% and 3.2%) and temperature (between 10 °C and 28 °C). 2.4.1.1. Estimation of desorption coefficients. The desorption of nisin incorporated into multi-layer PE/PA/PE films coated with Nisaplin loaded HPMC film placed in the top of the desorption solution was mass balance studied in the desorption solution according to the following equation: Vsol
dCsol ⁎ = − A kd Csol −Csol dt
ð5Þ
Then; ⁎ ⁎ Csol = Csol e−ðkd⁎A=V Þ⁎t + Cto; sol −Csol
ð6Þ
The Eq. (5) could be solved with the following boundary conditions: t=0
Cto; sol = 0
8tN0
CPE=PA=PE = K VEQ ⁎CHPMC and Csol = KEQ ⁎CHPMC
The equilibrium partition constant KEQ′ between PE/PA/PE and HPMC can be neglected (= 0) because nisin goes preferentially to the desorption solution. Also, the equilibrium partition constant KEQ between desorption solution and HPMC is equal to 1, because HPMC film is water soluble very quickly. kd and Csol⁎ were estimated using Excel solver. 2.5. Statistical analyses Data were analyzed using the software Statgraphics plus version 1, Copyright© Manugistics, 1994–1998. Means and standard deviations were calculated. A t-test was used to compare the means of two samples. Experiments were replicated at least three times.
3.1.1. Sorption isotherms The sensitivity of films to water is an essential parameter, as it makes it possible to predict water and vapor transfer from/to food across the packaging. Therefore, sorption isotherms of standard PE/PA/ PE films, PE/PA/PE film coated with HPMC film and PE/PA/PE film coated with Nisaplin loaded HPMC film were established at 23 °C. The sorption curves are presented in Fig. 2 and calculated GAB parameters in Table 2. Fig. 2 shows that the behavior of these three films was different when they were in contact with moistened air. The moisture content of PE/PA/PE standard film didn't significantly increase when it was subjected to increasing aw values. This result was expected, since PE/PA/PE polymers are hydrophobic and cannot be hydrated. At the opposite, PE/PA/PE film coated with HPMC film and PE/PA/PE film coated with Nisaplin loaded HPMC film presented a significant rehydration when subjected to increasing aw values. This result can be explained by the hydrophilic nature of HPMC, which made it possible to hydrate the surface of coated PE/PA/PE films. However, Fig. 2 also shows that PE/PA/PE film coated with Nisaplin loaded HPMC film had a significantly higher rehydration potential than PE/PA/PE film coated with HPMC film without Nisaplin. This can only have resulted from the Nisaplin preparation used for film activation. In fact, Nisaplin® preparation contains the antimicrobial agent, nisin, which has a hydrophilic side, allowing an additional rehydration of films. Then, this preparation also contains 12% of non-fat milk proteins and 6% of carbohydrates that probably contributed to improve the hydrophilic character of the films. Finally, this preparation essentially contains sodium chloride (77.5% w/w of the Nisaplin® preparation), known to significantly modify the water sorption characteristics of foods (Comaposada et al., 2000) and biopolymer gels (Rougier et al., 2007). Knowing that the coating preparation contained 14.3% (dry basis) of Nisaplin®, this explains the difference in the water sorption curves between PE/PA/PE films coated with HPMC solution and PE/PA/
Table 1 Experimental design points Points
pH
Temperature (°C)
NaCl (%)
1 2 3 4 5 6 7 8
3.8 3.8 6.3 6.3 3.8 3.8 6.3 6.3
10 °C 28 °C 10 °C 28 °C 10 °C 28 °C 10 °C 28 °C
0.8 0.8 0.8 0.8 3.2 3.2 3.2 3.2
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Fig. 2. Sorption isotherms of standard PE/PA/PE film, PE/PA/PE film coated with HPMC film and PE/PA/PE film coated with Nisaplin loaded HPMC film. Experimental data were motifs and calculated data were lines (model).
PE films coated with Nisaplin®-containing-HPMC film forming solution. 3.1.2. Total Water Desorption Rate (TWDR) To check the water barrier function of packaging, TWDR from the film put onto a wet food material was measured. The results presented in Table 3 show that there was no significant difference between antimicrobial and standard films. In these experiments, the coated side of the film was in contact with the agar gel. The PE/PA/PE uncoated side was in contact with air, playing its barrier role. Consequently, even though film activation modified its water sorption characteristics, it didn't alter its barrier properties, since the external side of the PE/PA/PE film remained uncoated, thus hydrophobic, insensitive to external water activity changes and insoluble in water. 3.2. Film mechanical resistance The first function of a packaging is to physically protect the integrity of food. Its mechanical properties must not be altered by any treatment it undergoes. To check the preservation of PE/PA/PE-film mechanical properties after coating with Nisaplin-containing-filmforming solution, antimicrobial and standard films were analyzed for Young modulus (Y) and Elongation at Break (%EB). The comparative results are presented in Table 3. Percentages of elongation at break (EB) and the Young moduli were significantly different between standard PE/PA/PE films and PE/PA/PE films coated with Nisaplin loaded HPMC film. In fact, %EB for antimicrobial films was significantly lower than that for standard PE/PA/PE films (P b 0.05). The adsorption of antimicrobial-film-forming solution onto PE/PA/PE films also resulted in an increase of Y modulus. Coating of PE/PA/PE films with a Nisaplin/HPMC film-forming solution made the films plastic. This result was not so surprising. Antimicrobial film thickness was 6 to 10 times higher that that of initial PE/PA/PE films, which explained antimicrobial film stiffness. These observations were in accordance with the results presented by Kim et al. (2002) who observed variations in tensile strength, Y and %EB when Nisaplin antimicrobial solution was used for film coating. HPMC polymer used for coating
was probably not the whole cause in film stiffness. In fact, NaCl, milk proteins and carbohydrates present in the Nisaplin preparation can have interacted with HPMC and modified its mechanical behavior. Furthermore, HPMC but also proteins and salt (present in Nisaplin®) have high affinity to water, known to be a very effective plasticizer for most biopolymers, that induces lowering of elastic modulus (Lazaridou & Biliaderis, 2002; Lazaridou et al., 2003; Chang et al., 2006; Kristo et al., 2007). In effect, films were stored at 23 °C and 50% relative humidity during 7 days before mechanical analyses. As already shown in Fig. 2, PE/PA/PE films did not significantly rehydrate when stored under highly moistened air, while activated films did. 3.3. Nisin desorption The release of nisin from the antimicrobial films takes place in two steps: 1/ diffusion of nisin towards the film surface; 2/ transfer of nisin across the interface film/desorption solution. Nisin diffusion in the polymer (step 1) is regarded as diffusion — after film rehydration, swelling and nisin re-solubilization — in a porous solid matrix, where pores are connected with each other and filled with the solvent (NaCl desorption solution in the present study). Nisin desorption from antimicrobial films was studied using NaCl based desorption solutions at different concentrations, pH and temperatures. The desorption coefficients were then calculated for each operating condition and also reported after a statistical analysis (Table 4). Depending on the molecular weight and the affinity of the migrant agent with the film polymers or with the desorption solution, desorption coefficients could significantly vary (Sebti et al., 2004). For example, Schwartzberg and Chao (1982) reported that diffusivities decreased when molecular weight increased, which is in accordance with the Stokes–Einstein equation (Loncin, 1980) that shows the reverse proportionality between transfer coefficients and the effective radius of the diffusing molecule.
Table 3 Standard PE/PA/PE film and antimicrobial film characterization Plastic films
Table 2 Parameter values of the GAB model fitted to various films in the water activity range of 0.10–0.85 c
k
xm
PE/PA/PE film coated with HydroxyPropylMethylCellulose film 0.002 0.874 17.946 forming solution containing Nisaplin PE/PA/PE film coated with HydroxyPropylMethylCellulose film- 0.003 0.672 17.435 forming solution PE/PA/PE 0.028 0.036 18.334
Activated filmsa
Trends Analysis of variance
4.2 × 104 ± 0.7 × 104 ↗ Mechanical Y (Pa) 1.1 × 104 ± 0.5 × 104 271 ± 29 130 ± 10 ↘ properties EB (%) TWDR 1.11×10−3 ±0.01×10−3 1.19 × 10−3 ± 0.01 × 10− 3 ↔ (kg s− 1 m− 2)
⁎ ⁎ NS
Y: Young modulus. EB: Elongation at break. TWDR: Total Water Desorption Rate. ⁎: P b 5%, NS: not significant (P N 5%). a Activated film is PE/PA/PE film coated with HydroxyPropylMethylCellulose film forming solution containing Nisaplin.
W. Guiga et al. / Innovative Food Science and Emerging Technologies 10 (2009) 203–207
References
Table 4 Desorption coefficients obtained from the analysis of the experimental design Points
kd (× 10− 2) m s− 1
1 2 3 4 5 6 7 8
1.92 ± 0.13 2.23 ± 0.18 2.03 ± 0.16 2.24 ± 0.03 1.79 ± 0.19 1.62 ± 0.03 1.51 ± 0.23 1.98 ± 0.07
kd m s− 1 (× 10− 2) Mean ± Std deviation
Temperature (°C)
NaCl (%)
10
28
0.8
2.01 0.17
⁎⁎ 2.10 0.13
⁎ 1.78 0.16
207
pH 3.2
3.8
6.3
1.73 0.14
NS 1.89 0.17
1.92 0.21
NS: non-significant; ⁎: PPb 5%, b 0.1%. 5%, ⁎⁎:ce:italic>/ce:italic> ⁎⁎: P b 0.1%.
Despite the optimal solubility and bioactivity of nisin at low pH values (Sebti, 2002), results show that desorption coefficients obtained at pH 3.8 and pH 6.3 were not significantly different. Consequently, within the studied pH range, this parameter didn't alter desorption kinetics. Desorption would thus rather be controlled by mass transfer phenomena. This is confirmed by the results concerning NaCl concentration and temperature impacts on desorption. In fact, Table 4 shows that when NaCl concentration increased, the nisin desorption coefficient decreased. This result is in accordance with already published studies (Kim et al., 2002), showing that increasing concentrations of NaCl, sucrose or citric acid in the desorption solution resulted in a decrease of the desorption rate. In fact, the presence of these molecules significantly modifies the ionic strength of the desorption solution, which alters the mass transfer phenomena, in accordance with Fick's law. This result is particularly important, knowing that Nisaplin® preparations contain high amounts of salts (77.5%). Results presented in the Table 4 also show that when temperature increased, the desorption coefficient of nisin also increased, in accordance with Arrhenius law. 4. Conclusion and perspectives This work demonstrated the efficiency of the antimicrobial activation of PE/PA/PE films with Nisaplin-containing-film-forming solution, using HPMC as biopolymer coater. Additionally, film activation modified very slightly the mechanical resistance but not the permeability to water vapor. The kinetic study and modeling of bacteriocin desorption demonstrated that nisin release from the activated films depends on the ionic strength of the desorption solution, its temperature but not its pH. Further investigations would try to improve the control of nisin release kinetics from antimicrobial films, by micro-encapsulation methods or multilayer films using a barrier layer to delay the transfer of antimicrobial agent transfer. Acknowledgements Authors gratefully acknowledge the Rhône-Alpes Region (France) for the financial support of the project, and Mr. Emmanuel BRARD for his technical assistance and his contribution to this study.
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