Biopolymer-based films as carriers of antimicrobial agents

Procedia Food Science 1 (2011) 190 – 196

2211–601X © 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive Committee.doi:10.1016/j.profoo.2011.09.030

Available online at www.sciencedirect.com

Procedia – Food Science 00 (2011) 000–000

Procedia Food Science

www.elsevier.com/locate/procedia

11th International Congress on Engineering and Food (ICEF11)

Biopolymer-based films as carriers of antimicrobial agents

Kyriaki G. Zinoviadoua, Konstantinos P. Koutsoumanisa, Costas G. Biliaderisa a

*

aDepartment of Food Science and Technology, School of Agriculture Aristotle University of Thessaloniki, GR-54124, Thessaloniki, Greece

Abstract

Films were prepared by incorporating different levels of antibacterial agents such as oregano oil, sodium lactate (NaL) and ε-polylysine (ε-PL) into sorbitol-plasticized whey protein isolate (WPI) films. The moisture uptake behaviour and the water vapour permeability (WVP) of the films were only affected by NaL, as the water sorption and permeability increased with addition of NaL into the protein matrix. An increase of the glass transition temperature of the sorbitol regions, as assessed by Dynamic Mechanical Thermal Analysis (DMTA), was caused by the addition of ε-PL, while incorporation of the oregano oil caused plasticization of the film that was depicted by a decrease in the transition temperature of the polymer-rich regions. On the other hand, incorporation of NaL into the films did not significantly alter their thermo-mechanical properties. However, the addition of NaL or ε-PL in the film forming solution resulted in a decline of maximum tensile strength (σmax). Wrapping of beef cuts with the antimicrobial films resulted in a significant reduction of the bacterial population levels. The maximum specific growth rate (µmax) of total flora (Total Viable Count, TVC) and pseudomonads was significantly reduced (P<0.05), with the use of antimicrobial films containing relatively high levels of oregano oil (1.5 % w/w in the film forming solution) or ε-PL (0.75 % w/w in the film forming solution), while the growth of Lactic Acid Bacteria (LAB) was completely inhibited. © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of ICEF11 Executive Committee Members Keywords: Edible films; Beef; Antimicrobial; Thermal properties; Mechanical properties; Whey proteins; Active packaging

1. Introduction

Active packaging remains an on-going development in food preservation technologies aiming at controlling of food spoilage and the production of high quality products with improved sensorial attributes. Apart from acting as selective barriers for moisture exchange, gas and solute migration, edible films may operate as carriers of many functional ingredients such as antioxidants, antimicrobial agents,

a* Corresponding author: Tel. & Fax +30-2310-991797 E-mail: [email protected]

© 2011 Published by Elsevier B.V. Selection and/or peer-review under responsibility of 11th International Congress on Engineering and Food (ICEF 11) Executive Committee.

191Kyriaki G. Zinoviadou et al. / Procedia Food Science 1 (2011) 190 – 1962 Author name / Procedia – Food Science 00 (2011) 000–000

flavours and spices [1]. When applied, antimicrobial packaging materials can effectively control the microbial contamination of solid or semi-solid food products by inhibiting the growth of spoilage or pathogenic microorganisms on the surface of the food item. Incorporation of antimicrobial compounds into the films results in decreased diffusion rates from the packaging material into the product, compared to simple spraying of the antimicrobial agents on top of the food system, thus assisting the maintenance of relatively high concentrations of the active ingredient where it is required [2].

Essential oils (EOs) extracted from plants or spices are rich sources of biologically active compounds such as terpenoids and phenolic acids. It has been long recognised that a lot of the EOs have antimicrobial properties and oregano oil that contains large amounts of carvacrol is considered to be one of the most active plant extracts against pathogens [3]. Lactate salts such as sodium lactate (NaL) are widely used as flavor enhancers in meat and poultry products, contributing to increased cooking yields and water holding capacity of processed meats [4]. Additionally, various lactate salts have demonstrated antimicrobial activity in laboratory media or food products [5]. Ε-polylysine (ε-PL) is a cationic homopolymer of 25 to 35 L-lysine units interlinked by a peptide bond between the carboxyl and the ε-amino groups of the L-lysine residues. This compound is heat stable even under acidic conditions and exhibits a wide antimicrobial activity against Gram(+) and Gram(-) bacteria, yeasts and moulds [6].

In contrast to the large amount of information on the use of various antimicrobial films for controlling meat pathogens, little is known about their effect on spoilage flora of these products. In the present study, fresh beef cuts were wrapped into whey protein isolate (WPI) films containing different antimicrobial agents (oregano oil, NaL and ε-PL). The effectiveness of the films against the beef’s spoilage flora during storage at 5°C was investigated. Additionally, the impact of the antimicrobial agents on the mechanical and physical properties of the films was examined since the overall performance of the films depends strongly on their physicochemical properties.

2. Materials & Methods

Bipro, a whey protein isolate (Davisco Foods International), was dissolved to obtain film-forming solutions of 5% (w/w) concentration. Protein solutions were placed in a water bath at 90ºC for 30 min under stirring. Sorbitol (St. Louis, MO, USA) was added as a plasticizer in the constant concentration of 37.5% (sorbitol/(WPI+sorbitol)). Appropriate amounts of the antimicrobials were added in the film forming solution resulting in a final concentration of 1.0%, 1.5% and 2.0% (w/w) for NaL (50% solution, Merck KGaA, Germany), 0.25%, 0.5% and 0.75% (w/w) for ε-PL (Chisso Int, Japan) and 0.5%, 1.0% and 1.5% (w/w) for oregano oil (Origanum vulgare ssp. Hirtum, Ecopharm, Greece); the latter were homogenized at room temperature for 2 min at 13000 rpm and 2 min at 19000 rpm using an Ultra-Turrax homogenizer (T-25 basic, IKA, Werbe). Portions of 12.5 g solution were cast on Petri dishes (φ 8.5 cm) and allowed to dry in an oven at 35°C for ~24 h. Moisture sorption isotherms were conducted for all the samples according to [7] and WVP measurements were performed at 25°C using the ASTM (E96-63T) procedure modified for the vapor pressure at film underside [8]. The thermo-mechanical properties of the films were determined by DMTA performed with a Mark III analyzer (Polymer Labs. Loughborough, UK) operated in the single cantilever bending mode (heating rate 2°C min–1 and a strain level equal to a maximum displacement of 16 µm). The Tg of the samples was determined as the peak in tan δ at 3 Hz. The mechanical properties of the films were evaluated by large deformation mechanical testing using a TA-XT2i instrument (Stable Micro systems, Godalming, Surrey, UK) [9]. In order to determine the antimicrobial properties of the films beef cuts were wrapped with antimicrobial films and stored at 5°C. Throughout storage samples were analyzed and the populations of Total Viable Counts, pseudomonads and Lactic Acid Bacteria were measured. Microbial growth data were modeled as a function of time with the model of Baranyi using the in-house software Dmfit [10].

192 Kyriaki G. Zinoviadou et al. / Procedia Food Science 1 (2011) 190 – 196 Author name / Procedia – Food Science 00 (2011) 000–000 3

3. Results & Discussion

The moisture uptake behaviour and the WVP of the films were only affected when NaL was incorporated into the protein matrix. The WVP value of the antimicrobial-free films was 23.9 (g s-1 m-1 Pa-1), while for those containing NaL it levelled up to 36.8 (g s-1 m-1 Pa-1). The increased water sorption and the higher permeability observed for those films was ascribed to the sodium ions that create regions of high dielectric constant. Although, other studies on incorporation of fats or lipids into edible films have shown improvements in water vapor barrier properties [11] this was not observed in the present study, probably due to the lower concentrations of the oregano oil added in the films.

The results obtained by DMTA indicated the existence of two relaxation processes that were manifested by a peak in tanδ. The peak that was recorded at higher temperatures, corresponding to the protein’s transition temperature (α-relaxation), shifted to lower levels as the water content increased. A secondary relaxation was also observed at low temperatures (<0°C) that corresponded to the transition temperature of sorbitol and was highly affected by the water content as well. Similar results have been previously reported for sorbitol-plasticised WPI films studied with differential scanning calorimetry (DSC) [12] and fructose containing sodium caseinate films [13], suggesting the occurrence of immiscibility between polyols and the polymeric components at a microscopic level.

As shown in Fig. 1, incorporation of oregano oil, at two different levels in the whey protein films resulted in a decrease of the transition temperature, implying a plasticizing effect, while no effect was noted for the lower temperature transition (Fig. 2). On the other hand, the addition of ε-PL into the protein solution resulted in a significant increase of the low-T transition. This effect may be attributed to the increased “average” molecular weight of the sorbitol-rich region when the antimicrobial is incorporated. A small increase in the temperature that corresponded to the transition of sorbitol was noticed when 1.5% NaL was added into the film formulation, and it was attributed to the preferential binding of water by the Na+ ions.

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Fig. 1. Transition temperature of the polymer rich phase of sorbitol-plasticized WPI films containing different levels of: a) ε- polylysine (ε-PL); b) sodium lactate (NaL); and c) oregano oil


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Fig. 2. Transition temperature of the sorbitol rich phase of sorbitol-plasticized WPI films containing different levels of: a) ε- polylysine (ε-PL); b) sodium lactate (NaL); and c) oregano oil.

The results of large deformation mechanical testing of sorbitol-plasticized WPI films, as affected by

film moisture content and antimicrobial agent concentration, are presented in Fig. 3 a and b. For films containing NaL a gradual decrease in tensile strength at higher moisture contents was observed accompanied by an increase in elongation at break values (%EB). The effect of moisture content was more complicated for the rest of the treatments. The tensile strength increased with an increase in moisture content up to 10 % for the control films and for the ones containing oregano oil or ε-PL, implying an antiplasticization effect. On further hydration water appeared to act as a plasticizer by reducing the measured σmax. The anti-plasticizing effect of water at intermediate hydration levels has been thoroughly reviewed by Pitia [14] who concluded that the anti-plasticization effects of water are mostly observed in systems which at low moisture content are characterized by a Tg slightly higher than ambient temperature, permitting partial relaxation of the chain segments in the amorphous domains of the biopolymer matrix. The addition of oregano oil in the sorbitol-plasticized WPI films resulted in a decrease of σmax with increasing oil concentration, while addition up to 1.0 % in the film forming solution resulted in an increase in the elongation properties. Addition of ε-PL into the WPI films at concentrations of 0.50% and 0.75% (w/w) in the film forming solution resulted in significantly smaller tensile strength and improved extensibility compared to the control film.


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Fig. 3. Effect of antimicrobial agent concentration (w/w in the film forming solution) and water content on: (a) tensile strength (σmax); and (b) elongation at break (% EB).

The growth of the total flora (TVC), pseudomonads and lactic acid bacteria (LAB) on beef cuts and the effect of WPI containing antimicrobials are presented in Fig. 4 a-c. As it can be seen on the graphs no significant differences were observed (P<0.05) between the bacteria growth of the control meat samples and those covered with antimicrobial free films. The use of films containing any of the three antimicrobial agents resulted in a significant reduction of the TVC and pseudomonads population throughout the entire storage period. Additionally, the use of films that contained the EO or ε-PL resulted in a complete inhibition of LAB growth, as it can be seen in Fig. 4c. The maximum specific growth rate (µmax) of total flora (Total Viable Count, TVC) and pseudomonads was significantly reduced with the use of antimicrobial films containing EO or ε-PL (P<0.05). Since microbial loads higher than 107 CFU/ cm2 are usually associated with the occurrence of off-odors [15] it may be suggested that the use of WPI films containing oregano oil or ε-PL could double the shelf life of fresh beef cuts stored under refrigerated conditions.

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Fig. 4. Effect of antimicrobial agent (w/w in the film forming solution) in the films on beef’s spoilage flora upon storage at 5°C: a) Total Viable Count; b) Pseudomonas spp; c) Lactic Acid Bacteria. Points represent average values (n=4) and different letters for the data points at each sampling period indicate significant differences (P<0.05)

4. Conclusion

The results of this study clearly demonstrated that addition of ε-PL into the whey protein isolate matrix did not significantly alter the WVP and the water sorption properties of the films while it induced a plasticizing effect as evidenced by a reduction of the σmax and an increase of elongation at break. On the other hand, addition of NaL resulted in higher moisture uptake and higher water vapour permeability. Incorporation of ε-PL brought about an increase of the transition temperature of sorbitol, while the thermo-mechanical properties of the polymer regions were not affected. Incorporation of oregano oil resulted in a plasticizing effect, reducing the Tg of the films, as shown by DMTA analysis. The tensile properties of the films were altered to some extent by incorporation of the oregano oil, but such differences were diminished at higher moisture contents. The present study indicated that whey protein isolate films can sustain their structural integrity at the high αw environment of beef surface and additionally serve as effective carriers of antimicrobial agents. The use of the antimicrobial active films containing ε-PL or oregano oil resulted in a significant inhibition of beef’s spoilage flora by reducing the µmax of the bacteria, while NaL did not suppress the growth of LAB.

Acknowledgements

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

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Biopolymer-based films as carriers of antimicrobial agents