Traditional synthetic polymers made from petrochemicals that currently dominate the food packaging sector possess most of the specific properties required of useful packaging materials such as effective gas barrier properties (oxygen, water vapor, aroma, and light), transparency, ability to be sealed, chemical resistance, mechanical strength, as well as ease of processing. However, the long-term environmental sustainability of these polymers is now being questioned and interest is shifting to the search for biodegradable, biocompostable polymers, and composites as alternatives to conventional polymers (Mohanty and others 2005; Avella and others 2009; Ghosh and others 2011; Kuorwel and others 2011a; Pilla 2011). Commercial and academic interest in utilizing biodegradable polymers for food packaging applications has increased in recent years (Siracusa and others 2008; Ahmed and Varshney 2011; La Mantia and Morreale 2011). These materials also have the potential to reduce environmental impacts associated with the management of petroleum-based packaging wastes. There are 3 main categories of biodegradable polymers: (i) polysaccharides (starch and cellulose), proteins (gelatin and casein), and lipids that are derived directly from natural raw materials and renewable resources; (ii) poly(lactic acid) (PLA) that are chemically synthesized from bio-derived monomers; and (iii) poly(hydroxybutyrate) (PHB), poly(hydroxyvalerate), and poly(hydroxyalkanoates) that are made from microbiologically produced materials or genetically modified bacteria (Chandra and Rustgi 1998; Averous 2008).
The shelf life of most food products depends upon the biological, chemical, and physical interactions between the food, package, and the ambient environment (Robertson 2010). High levels of bacteria and microorganism in food products can potentially generate undesirable deteriorations in flavor, odor, color, sensory, and textural properties and may even become harmful to human health (Raouche and others 2011). The effort toward efficient food preservation continues to increase to fulfill consumers demand for natural, fresh, safer, and healthier products (Piližota 2012). Over the last few decades, the active packaging (AP) concept has been established and developed in order to improve the quality and extended the shelf life of food products (Miltz and others 1995; Rooney 1995; Appendini and Hotchkiss 2002). Some of the common AP concepts include gas scavengers, carbon dioxide emitters, moisture absorbing systems, and antioxidant and/or antimicrobial releasing and/or containing systems (Brody and others 2001; Suppakul and others 2003; López-Rubio and others 2006; Pérez-Pérez and others 2006).
To date, one of the extremely challenging AP technologies is the use of antimicrobial additives that can minimize the risk of food spoilage and contamination by suppressing the activities of targeted microorganisms (Labuza and Breene 1989; Lim and Mustapha 2003; Mauriello and others 2005; Joerger 2007; López-Mendoza and others 2007; Sung and others 2013; Guo and others 2014). A wide range of additives have been successfully incorporated directly into food products as well as into packaging materials, including organic acids and their salts, bacteriocins, enzymes, chelators such as ethylenediaminetetraacetic acid (EDTA), lactoferrin, and a range of plant extracts (Cooksey 2005; Sanchez-Garcia and others 2008; Jin and Niemira 2011; Barbiroli and others 2012; Jokar and others 2012; Torres and others 2014). This technology also combines antimicrobial additives with the packaging material that slowly and continuously release the additives over the required period of time and maintain a high concentration of the antimicrobial in the product (Quintavalla and Vicini 2002).
Different materials and methods can be used in order to develop antimicrobial packaging systems. For instance, petroleum-based and biodegradable polymers have been studied as potential candidates for the incorporation of antimicrobial substances in food packaging applications. These include polyolefins (polypropylene, low- and high-density polyethylene) and polyesters such as poly(ethylene terephthalate) (PET) and poly(caprolactone) (PCL) (Limjaroen and others 2003; Del Nobile and others 2009; Ramos and others 2012; Bastarrachea and others 2013). An antimicrobial activity between an antimicrobial package and food product can be achieved by either direct contact using a nonmigratory system or by indirect contact using a volatile antimicrobial releasing system (Guarda and others 2011). In addition, Sung and others (2013) have noted that even though there has been much research activity on the development of antimicrobial packaging systems that utilize various plastic materials and antimicrobial agents, only a few commercial antimicrobial packaging products are found in the market. They speculated that this might be due to strict safety and hygiene regulations, as well as limited consumer acceptance of high cost.
Synthesis and properties of PLA
The PLA polymers belong to the class of aliphatic polyesters that can be produced either by carbohydrate fermentation or by chemical synthesis of the lactic acid (LA) monomer (Averous 2008; Jamshidian and others 2010). The LA monomer can be obtained from renewable resources including starch-rich products such as corn, sugar beet, tapioca, and wheat (Auras and others 2004). The chemical structure of LA is such that it contains an asymmetric carbon atom creating 2 optically active configurations known as the L- and D-isomers. The ratio of L- to D-monomer units affects some of the key macromolecular properties such as the degree of crystallinity, melting temperature, and the ease of processing (Gupta and others 2007; Lim and others 2011). The synthesis of PLA is a relatively complex multistep process that starts from the production of LA, followed by the formation of the lactide monomer and finally, the polymerization process itself. Commercial PLA resins are usually produced from L-lactide with the resulting polymer, poly(L-lactic acid), being a semicrystalline material that has relatively high melting and glass transition temperatures (Albertsson and others 2011).
The beneficial properties of PLA include high mechanical strength, excellent thermoforming ability, biocompatibility, ability to be easily composted, and monomer renewability. In addition, the production of PLA has been reported to result in 15% to 60% lower carbon emissions and 25% to 55% lower energy consumption than petroleum-based polymers (Vink and others 2003; Dorgan and others 2006). This bio-based polymer can be processed by conventional plastic processing methods (which is of a commercial importance) to produce a range of products that are both recyclable and/or compostable. However, its inherent brittleness, low thermal resistance, and poor water-vapor barrier properties are disadvantages that currently limit its widespread use in packaging (Bhardwaj and Mohanty 2007).
A recent life cycle assessment (LCA) of PLA and PET for drinking water bottles comparing their impact on the environment was reported by Gironi and Piemonte (2011). In this study it was found that PLA has lower environmental impacts than PET in terms of fossil fuel resources and recycling capability. However, it was also noted that a reliance on PLA production can have adverse effects such as the clearing of forests for growing starch crops and the associated excessive water use. Such production may also indirectly affect human health and the environment because of the use of pesticides. Thus, it is important to note that LCA studies require a sufficient amount of data and information in order to avoid bias in the final findings.
Trends in PLA-based materials
A range of other polymers, additives, and fillers have been successfully combined with PLA in order to enhance material performance, reduce costs, and expand PLA into new applications (Martin and Avérous 2001). To date, the combination of biodegradable polymers and biofillers with PLA is receiving considerable academic and commercial attention mainly for the purpose of creating materials that potentially have lower environmental impacts than conventional synthetic polymers. The use of natural fibers as fillers or reinforcing agents (for example, wood, kenaf, flax, hemp, and ramie) and other biodegradable polymers (for example, starch, protein, PCL, and PHB) in PLA blends or composites is expected to considerably lower the price of the resulting products (due to the renewability and abundance of the raw materials) as well as to improve the properties that these materials offer without compromising the biodegradability (Akil and others 2011).
In addition, the use of nanoadditives, nanofibers, and nanocomposites, in particular, are some of the current approaches used in food packaging applications. Various types of nanofillers such as clay, silica, talc, montmorillonite, and nanofiber polymers have been utilized in order to improve the properties of PLA-based material composites (Sinha Ray and Okamoto 2003; Iwatake and others 2008; Torres-Giner and others 2008; Rhim and others 2009; Ray 2011; Abdul Khalil and others 2012; Tenn and others 2012). Packages containing nanomaterials are apparently more acceptable to the average consumer than nanotechnology-engineered food products (Siegrist and others 2007). However, some of the main drawbacks of PLA-based nanomaterials include their high cost, due to energy consumption in their production, as well as the possible migration of nanoparticles that might have a potential impact on human health (Abdul Khalil and others 2012; Rhim and others 2013).
PLA as a packaging material
With its classification being generally recognized as safe (GRAS), PLA has been approved for use in food packaging, including direct contact applications (Conn and others 1995). In addition, PLA is a good candidate for a variety of packaging applications due to its close similarity to commercial thermoplastics such as PET (Auras and others 2005). In the last decade, PLA has been developed for a wide range of primary packaging applications including oriented and flexible films, extruded and/or thermoformed packages suitable for common applications such as food and beverage containers, cups, overwrap, blister packages, as well as coated paper and board (Tullo 2000; Groot and others 2011). Recently, a Danish dairy company has used PLA, that was claimed to be biodegradable, for yoghurt cups that were traditionally made from high-impact polystyrene (Jessen 2007). Other commercial examples include the use of PLA for the production of lunch boxes and fresh food packaging (Mutsuga and others 2008), and containers for packaging of bottled water, bottled juices and yogurts (Ahmed and others 2009). Blends of PLA with starches, proteins, and other biopolymers have also been studied in order to develop fully renewable and degradable packaging materials (Raghavan and Emekalam 2001; Ke and Sun 2003; Suyatma 2004; Yew and others 2005; Bhatia and others 2007).
PLA antimicrobial packaging
The potential of PLA for use in antimicrobial packaging applications has been investigated in recent years by a number of researchers (Mustapha and others 2002; Rhim and others 2009; Chen and others 2012; Li and others 2012a; Jamshidian and others 2013; Fei and others 2014). There are also a number of patents worldwide on PLA-based materials containing antimicrobial agents (Auras and others 2010; Buonocore and others 2012; Chen and others 2012; Liu and others 2012). Several substances such as organic acids, bacteriocins (for example, nisin), plant extracts (for example, lemon extract), essential oils and extracts (for example, thymol), enzymes (for example, lysozyme), chelating agents (for example, EDTA), metals (for example, silver) have been incorporated into PLA to provide antimicrobial activity. In particular, PLA with the addition of natural antimicrobial agents such as nisin, lysozyme, and silver zeolite has shown inhibitory effects against selected microorganism such as Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Micrococcus lysodeikticus. Natural antimicrobial agents have also been incorporated into coatings on the surface of PLA and these were shown to be effective against spoilage and pathogenic microorganisms (Del Nobile and others 2009; Jin and others 2009; Liu and others 2009a; Rhim and others 2009). According to Jamshidian and others (2010), only a few studies have investigated the potential of PLA in general AP applications although there are a number of examples that use PLA in antimicrobial food packaging applications.
Current technologies enable effective antimicrobial packages to be prepared from PLA that has been blended with different compatible materials and plasticizers. The consumer preference for natural food products with few or no preservatives, with minimal microbial contamination while using sustainable packages has generated a growing interest in the use of PLA in antimicrobial packaging. An example of a commercial antimicrobial PLA packaging product is Antipack™ produced by Handary in Belgium, which is a film manufactured from a PLA-/starch-based material incorporated with an antifungal agent. This product is claimed to prevent the growth of yeast and mold during the shelf life period by gradually releasing chitosan-containing natamycin onto the surface of solid foods such as cheese, fruits, vegetables, meat, and poultry (Szafranska 2012).
Furthermore, it is believed that PLA can perform as a suitable carrier of antimicrobial agents without showing any indivertible impact on the compositing and potential biodegradation process. This is possible if the rate of dissipation of the antimicrobial agent or the controlled release during the shelf life of the system as a packaging material is fully understood, systematically performed, and accurately controlled (Balasubramanian and others 2009). Therefore, the study of antimicrobial agent migration in the system will be very important in the future to ensure the agent is dissipated before the packaging materials is disposed of in landfill. Ramos and others (2014) observed some improvement in the biodegradation of extruded PLA films containing thymol and silver nanoparticles as the antioxidant and antimicrobial agents, respectively, using a disintegration test in composting conditions.
Preparation of antimicrobial PLA-based materials
The choice of processing technique may significantly affect the properties of the resultant antimicrobial films, especially those made from biodegradable polymers (Rhim and others 2006). According to Han (2005), different factors affect the selection of the processing technique when preparing an antimicrobial packaging film. These include the type and properties of the polymer, the characteristics of the antimicrobial agent (such as polarity, compatibility, and thermal stability), storage temperature, type of packed foods and targeted microorganisms, and the residual antimicrobial activity after manufacturing.
It is also important to note that the selection of the processing method for the preparation of antimicrobial PLA-based materials depends on the type of system being developed. Basically, the antimicrobial system can be either a direct contact one using a nonmigratory antimicrobial agent or an indirect contact one using a volatile antimicrobial releasing system (Guarda and others 2011; Jin and Niemira 2011). In particular, the coating method is suitable for nonmigratory antimicrobial systems where a high concentration of antimicrobial agent is required on the surface of the film (Appendini and Hotchkiss 2002; Jin and others 2009). Solvent casting and extrusion are more suitable for migratory antimicrobial systems where the release of a volatile antimicrobial agent from the packaging material to the packaging headspace or the surface of the food product is required (Jin and Niemira 2011; Guo and others 2014).
In these systems, mathematical diffusion modeling can be used to predict the release profile of the antimicrobial agents. The initial and boundary conditions can be used to determine the distribution coefficient (Crank 1979). Moreover, a number of factors need to be considered in order to use the most appropriate diffusion model including film thickness, the ways in which the antimicrobial agent diffuses, and the volume of food simulant into which the migration occurs (Pérez-Pérez and others 2006). The various mass transfer models that can be used to analyze the migration of antimicrobial agents can also account for the influence of the molecular weight, ionic charge, and solubility of the agents (Han 2003).
The effectiveness of antimicrobial PLA-based material depends also on the selection of antimicrobial agents. Some volatile additives have low resistance to the conditions experienced during processing and fabrication that include high temperatures, shear, and pressure at the extrusion stage (Han 2003). The polarity and molecular weight of the antimicrobial agent are crucial material parameters that dictate the solubility or compatibility of the agent in the PLA matrix. For example, olive leaf extract (OLE) prepared using water as the solvent was found to be incompatible with PLA whereas OLE dissolved in chloroform and incorporated into a PLA film containing methylcellulose (MC) demonstrated a significant inhibition of S. aureus (Ayana and Turhan 2009).
Antimicrobial substances can be incorporated into PLA via wet or dry processing techniques, similar to those used for other biodegradable and synthetic polymers (Brody and others 2001). The wet processing technique consists of solvent casting/solvent evaporation using ambient or low temperature during mixing. According to Appendini and Hotchkiss (2002), biopolymers are good candidates for this type of film production when compared to polyolefins and other hydrophobic polymers. For the production of solvent cast films, PLA can be dissolved in different solvents such as chloroform (Rhim and others 2009), ethyl acetate (Mascheroni and others 2010), as well as methylene chloride (Jin and others 2010) and in many cases, both the PLA and the associated antimicrobial agents can be dissolved in the same solvent.
Solvent casting of additives onto a polymeric substrate involves solubilization, casting, and drying. This process is one of the more commonly used methods for laboratory-scale preparation of antimicrobial films from biopolymers (Kuorwel and others 2011a). The solvent casting method is also suitable for heat-sensitive antimicrobial agents such as bacteriocins (for example, nisin) and rather volatile compounds such as essential oils and their extracts (for example, thymol and carvacrol). Although some bacteriocins and peptides are relatively heat resistant, it is believed that their antimicrobial activity may be higher when minimal heating is applied during processing (Appendini and Hotchkiss 2002; Liu and others 2009a).
Dry or thermal processing techniques include the standard conventional processing methods used in the plastics industry such as extrusion, compression molding, blow molding, and injection molding. Extrusion is the most common method used for commercially processing polymeric packaging films (Raouche and others 2011). Compression molding of PLA can also be used to produce films that are relatively strong, thermally stable but brittle with a low thermal resistance compared to solvent-cast PLA films that are more ductile (Rhim and others 2006).
PLA is processed during extrusion at a temperature higher than 160 °C, which is crucial in order to ensure optimal melt viscosity as well as to complete the melting of the crystalline phase in the PLA matrix. This temperature is generally lower than the processing temperature of common thermoformed food packages made of PS, and PET. In addition, PLA can be processed using standard equipment with minimal equipment modifications (Jamshidian and others 2010; Lim and others 2011). It has been reported that PLA can be successfully extruded and/or compression molded with organic and inorganic antimicrobial agents and their combinations (Del Nobile and others 2009; Jin and others 2009; Busolo and others 2010; Prapruddivongs and Sombatsompop 2012). These processing techniques enable the antimicrobial agent to be evenly distributed in the amorphous regions of the polymeric material and can regulate its slow release from the film and maintain adequate concentrations against microorganisms (Suppakul 2004; Liu and others 2009a).
For antimicrobial PLA films, high temperatures and shear during extrusion processing can lead to a partial loss of the antimicrobial agent or its activity, especially if the antimicrobial agent has low thermal stability or high volatility (Han 2003; Del Nobile and others 2009). Therefore, the polymer and/or the antimicrobial agent may require modification prior to film processing in order to increase the compatibility between the 2 components (Kuorwel and others 2011a). Liu and others (2009a) have modified the steps in the processing method of co-extruded PLA membranes containing 5% (w/w) Nisaplin as the antimicrobial agent and lactide and/or glycerol triacetate (GTA) as the plasticizers. They reported that the maximum temperature at which Nisaplin retains its bioactivity is 120 °C but the temperature required to melt PLA is 160 °C. In order to address this problem, the processing temperature was reduced from 160 to 120 °C, by the addition of plasticizers, prior to blending with the antimicrobial agent. The incorporation of plasticizers lowered the temperature profile during manufacturing of the antimicrobial films. The resulting PLA/Nisaplin films showed no antimicrobial activity whereas, PLA/lactide and PLA/GTA blended membranes containing Nisaplin prevented the growth of L. monocytogenes in brain–heart infusion (BHI) broth.
It is clear that more research needs to be conducted to investigate the retention of natural antimicrobial agents when high temperatures are applied during processing. Microencapsulation is one important technique that can reduce the loss or the inactivation of antimicrobial agents by protecting volatile and heat-sensitive agents during thermal processing (Martins and others 2009; Guarda and others 2011; Joo and others 2012). The stability of microencapsulated antimicrobial agent during processing and storage is certainly a challenge and there are various possible negatives in such processes, including high cost as well as the complexity of the production process (Zuidam and Shimoni 2010).
Other studies have focused on coating methods where the antimicrobial substance is coated onto the surface of a primary polymeric film or substrate (Jin and Gurtler 2011; Jin and Niemira 2011; Li and others 2012a). The 2 common types of coating methods are spray gun coating and diffusion coating with each of these techniques involving the prior dissolution of the antimicrobial agent. Other coating techniques such as corona discharge, electro spinning, as well as sonification have been utilized to produce antimicrobial-coated PLA films and membranes (Li and others 2009; Rhim and others 2009; Theinsathid and others 2012). It was claimed that these methods do not significantly influence the loss of antimicrobial agent or result in an overall reduction in the mechanical properties of the films.
PLA-based materials containing antimicrobial agents can also be prepared in the form of active PLA nanofiber-based systems by using nanotechnology (Xu and others 2006; Torres-Giner and others 2008; Vega-Lugo and Lim 2009; Vargas-Villagran and others 2012). This recent and efficient technology features the use of electrical forces to produce ultrathin fibers known as polymer nanofibres. More than 200 types of both biodegradable and nonbiodegradable polymers have been designed and prepared by the electrospinning technique for specific applications and their properties have been characterized (Torres-Giner 2011). For instance, electrospun chitosan nanofiber mats made from a chitosan/PLA blend have been successfully prepared by Torres-Giner and others (2008). The main advantages of including the antimicrobial agent in the nanofiber in this way are that it can improve the material properties (for example, gas barrier and mechanical properties) and the release rate of the antimicrobial agent due to the high surface area and small pore size (micrometer to nanometer) (Kayaci and others 2013; Tanadi 2014). However, the disadvantages are that these materials are currently expensive to produce. Also, in some cases nanofiber mats might potentially disturb the release of the antimicrobial agent. Furthermore, these materials may not meet the requirements for FDA approval of biocides (Torres-Giner 2011).
Characteristics of antimicrobial PLA-based materials
Changes may occur in the mechanical and physical properties of many packaging materials after the incorporation of antimicrobial agents. When the antimicrobial agent is compatible with the packaging material, it can be incorporated with minimal physico–mechanical property deterioration. Conversely, an excess amount of an incompatible antimicrobial agent may reduce the physico–mechanical properties of the resulting composite material (Han 2003; Cooksey 2005). It is important to note that in the case of antimicrobial agents derived from natural polymers such as polysaccharides (for example, chitosan), the melt blending of 2 compatible polymers containing different glass transition temperatures may significantly alter the properties of the material. The engineering characteristics of various polymeric antimicrobial materials for food packaging have been reviewed by Bastarrachea and others (2011). They reported that a few changes occurred in the mechanical, thermal, and gas barrier properties of the polymer as well as in the surface morphology as a result of the addition of the antimicrobial agent. According to their literature review, only a few studies have measured the changes in PLA films loaded with antimicrobial agents.
In a recent study, Liu and others (2009a) extruded a thin membrane of PLA/pectin–Nisaplin microparticles with 1% (w/w) and 9% (w/w) concentrations of Nisaplin/pectin. They reported that the extruded PLA films impregnated with solvent-cast pectin–Nisaplin significantly reduced the tensile strength (by 49%), tensile modulus (by 41%), and toughness (by 51%) but no significant change in the flexibility occurred when compared to PLA/pectin membranes. They speculated that this phenomenon occurred due to the incompatibility between the inactive components in the Nisaplin (for example, 2.5% nisin, milk solids and salt) with the PLA. Nonetheless, the results seem contradictory because one would expect the flexibility to change along with the observed reduction in the tensile modulus and toughness. No explanation of this behavior was given.
In another study, Liu and others (2010) incorporated both 5% (w/w) loadings of Nisaplin and EDTA into plasticized PLA/GTA films and found a significant reduction in the elongation at break (from 108.5% to 62.5%) and in the impact strength (from 5.4 to 3.4 J/cm3) when compared to the plasticized only PLA. Although the PLA flexibility increased by adding GTA, the reduction in the mechanical properties of plasticized PLA incorporated with antimicrobial agents might be attributed to the “filler effect” (Liu and others 2010). The reported findings however, were unable to resolve the contradiction of the effect of the antimicrobial agent on the engineering properties. Generally, modification of the properties of neat PLA, or other biopolymers, by the addition of plasticizers is performed in order to increase the flexibility and to reduce the glass transition temperature (Tg) (Ljungberg and Wesslén 2002; Ozkoc and Kemaloglu 2009).
Recently, Prapruddivongs and Sombatsompop (2012) studied the effects of incorporating natural fibers such as wood flour on the mechanical properties of antimicrobial films. They reported that the addition of 1.5% (w/w) triclosan produced a slight effect on the mechanical properties of neat PLA. However, the inclusion of 10% (w/w) wood flour in the polymeric system containing 1.5% (w/w) triclosan was more pronounced than that of the inclusion of triclosan. The increase of wood flour loadings from zero to 10% (w/w) increased the stiffness of the active composites and, at the same time, reduced the flexibility and toughness. The latter observations are in agreement with those reported by Huda and others (2006) who also investigated the mechanical properties of PLA/wood composites and reported similar property changes. The incorporation of triclosan into the composite system did not cause any significant effect on the material properties and this might be due to the low percentage of antimicrobial agent in the formulation.
The type of fibers used and the uniformity of their dispersion within the matrix significantly affects the mechanical properties of polymer fiber composites. Key governing factors affecting the mechanical properties include the cellulose content of the fibers, their orientation, length, and diameter. According to Mukherjee and Kao (2011), the diameter of the fibers affects the length to diameter aspect ratio (L/D) and this may affect the mechanical performance of the composite. If the L/D ratio is too low, there will be insufficient stress transfer and the resulting reinforcement effect will be less significant. Conversely, if the L/D ratio is too high, the fibers may become entangled during mixing, and thus a lower stress transfer will be obtained (Nando and Gupta 1996). Bonilla and others (2013) investigated the effect of different particle sizes and loadings of chitosan powder (5% to 10%, w/w) incorporated into PLA on the physico–mechanical properties and antimicrobial activity of the resulting system. They observed that these films successfully inhibited the growth of total aerobic mesophilic and coliform microorganisms in minced pork meat as compared to the control, especially at a lower particle size of the chitosan powder. However, the incorporation of chitosan powder resulted in a less rigid and less stretchable film.
Generally, the mechanical properties of antimicrobial coated polymeric materials are not expected to be affected considerably by the antimicrobial coating. In these cases, the polymeric material is a supporting layer for the coating. However, some interaction between the polymer and coating system may occur at the interface between them, particularly when the antimicrobial agent concentration is high. Jin and others (2009) have developed an antimicrobial film consisting of PLA and pectin with nisin loaded into the film at 1% (w/w) concentration by a diffusion coating method. Although they reported that the coating of nisin did not affect the overall properties of the PLA/pectin film, significant changes in the mechanical properties were reported with the addition of approximately 20% (w/w) pectin and 6.7% (w/w) water into the PLA films.
Theinsathid and others (2012) reported some increase in the flexibility of PLA films (up to 14%) by incorporating 0.28% (w/w) of lauric arginate (LAE) via a coating method. However, it was reported that the change in elongation at break was not statistically significant. A corona discharge was used to modify the surfaces of the PLA films in the study to produce a more hydrophilic PLA as well as to increase the surface roughness. The surfaces of the modified films were expected to enhance the compatibility between the PLA and LAE. The authors reported that the slight increase in flexibility might be due to the weak secondary attraction forces between anionic charge on the surface of PLA and the cationic surfactant LAE. Similar findings were reported by Li and others (2012a) comparing uncoated films consisting of PLA and thermoplastic sugar beet pulp (TSBP) with PLA/TSBP films coated with Nisaplin in PLA/dichloromethane (DCM). They reported a slight increase in the percent of elongation at break (by 9%) for the coated films and a significant reduction in tensile modulus (by 64%) and tensile strength (by 36%). The reduction in the tensile properties was suggested to be due to the dissolution of PLA in DCM resulting in a decrease of the PLA's crystallinity as confirmed by dynamic mechanical analysis (DMA) and microscopy images.
There are only a few studies that have investigated the thermal properties of antimicrobial PLA packaging films. This might be due to the small or insignificant changes in the thermal profile by using thermogravimetric analysis (TGA) of the PLA films incorporated with antimicrobial agents as a result of the small sample size. The inclusion of low concentrations of antimicrobial agents at levels less than 10% (w/w) for example, will usually have only a very slight effect on the Tg, crystallinity temperature (Tc), melting temperature (Tm), or the percentage of crystallinity (Xc). In one example, Liu and others (2010) studied the thermal properties of PLA incorporated with 5% (w/w) nisin and EDTA that was plasticized by using 30% (w/w) GTA. A comparison between PLA films and the antimicrobial PLA films containing Nisaplin and EDTA showed that the antimicrobial agents had a smaller effect on the Tg than the plasticizer in the PLA film with 30% (w/w) GTA. From this study, it was suggested that the plasticizer content is more important than that of the antimicrobial agent in terms of the thermal profile of the materials. Furthermore, it was found that only a large amount of plasticizer (that is, 30%, w/w, GTA) incorporated into PLA lead to a significant decrease in Tc and Tm.
Prapruddivongs and Sombatsompop (2012) have reported that PLA/wood composites and antimicrobial PLA/wood composites (incorporated with triclosan) have lower Tg values than neat PLA. This is due to the reduction in the PLA content and the hydrophilic characteristic of wood as well as that of triclosan. In this study, the percent crystallinity of PLA/wood composites was reduced from 34.4% to 33.1% with the addition of 1.5% (w/w) of triclosan. A similar trend was observed for the percent crystallinity of antimicrobial PLA and neat PLA film with a reduction of 7.9% and 6.5%, respectively. However, no explanation for these phenomena was given. These findings are contradicted by the findings by Lui and others (2010) who reported that a PLA/Nisaplin–EDTA system exhibited a higher degree of crystallinity (61.1%) compared to neat PLA (57.9%). When an antimicrobial PLA film demonstrates a lower Tg value compared to a neat PLA film, it can be speculated that the antimicrobial agent is acting like a plastisizer and at the same time it will alter the thermogravimetric profile of the material. Lowering the Tg value of antimicrobial films due to the inclusion of antimicrobial agents may also reduce the required temperature profile during processing.
Most of the aforementioned studies have concentrated on the mechanical and thermal properties of antimicrobial PLA films, which are critical to developing effective film products. However, the retention of antimicrobial agents after thermal processing and the controlled release and migration of the antimicrobial agents from PLA films afterwards, have not been adequately and systematically addressed in the literature. These properties are of utmost importance in antimicrobial films in initiating and maintaining effective antimicrobial activity. Kumar and Münstedt (2005) investigated the release of silver ions from polyamides with a low percentage of crystallinity and found that these films inhibited the growth of microorganisms such as E. coli and S. aureus. They reported that the high crystallinity of polymeric materials results in a lower and slower release of antimicrobial agents to the surface of the film and hence affects its antimicrobial efficacy.
It is known that the hydrophilic nature of biopolymers such as polysaccharides lead to their low mechanical and water resistance properties. Thus like many other biopolymers, PLA may require some additional processing or modification in order to develop useful antimicrobial materials. This is mainly due to the intrinsic properties of unmodified PLA that include its high brittleness, poor water-vapor barrier, low crystallinity, slow biodegradation rate, hydrophobicity, and lack of reactive side-chain groups. Several types of modification have been developed to address these inadequacies and include chemical modification (for example, grafting, polymerization), addition of plasticizers (to reduce brittleness), blending with other biopolymers or biofibers, and the addition of compatibilizers to enhance its miscibility with otherwise incompatible polymers (Wu 2005; Yu and others 2006; Xu and others 2008; Signori and others 2009; Suryanegara and others 2009; Taib and others 2009; Van Den Oever and others 2010; Tudorachi and Lipsa 2011; Tawakkal and others 2012; Faludi and others 2013)
An important area that requires much further attention is the stability during the development of the antimicrobial packaging during production, distribution, and storage. In particular, the storage stability of the antimicrobial agents in the PLA materials is crucial and needs to be predicted from an end use standpoint. This is because antimicrobial packaging materials are often transported to and stored in warehouses under different temperature and humidity conditions that may lower the concentration and decrease the effectiveness of the antimicrobial agent (Suppakul and others 2011; Li and others 2012b). For instance, high storage temperatures might result in increasing the migration rate of the active agents and thus, reduce the antimicrobial activity of the active package system. To date, there is limited published scientific literature available on the loss and retention of antimicrobial agents in PLA-based materials during storage, especially at longer storage durations.
Antimicrobial activity of PLA films incorporated with antimicrobial agents
This section focuses on the capability of a PLA matrix to have incorporated within it various types of natural and synthetic antimicrobial agents via different methods of preparation. The purpose is to produce packaging systems that inhibit the growth of microorganisms. The antimicrobial activity of various types of natural and synthetic antimicrobial agents impregnated in PLA via melt blending, solvent casting, as well as their combinations is presented in Table 1. These antimicrobial PLA-based materials are purported to show inhibitory activity against the growth of different microorganisms such as E. coli O157:H7, S. aureus, Samonella, L. monocytogenes, S. typhimurium, Fusarium proliferatum, Botrytis cinerea, F. moniliforme, Aspergillus ochraceus, yeasts as well as molds. From Table 1, it is clear that most of the current studies are concerned with impregnated nonvolatile antimicrobial agents such as bacteriocin, enzymes, metals, as well as chelating agents.
Table 1. Applications of PLA as a primary and/or secondary material in antimicrobial film and coating systems designed for food packaging
|PLA/Microcrystalline cellulose (MCC)||Silver nanoparticles||5% (w/w)||E. coli||Agar media||SC||PLA/MCC/silver nanocomposites inhibited the growth of E. coli at 7.36 mm of zone of inhibition||Ali and Noori (2014)|
|PLA||Chitosan||5% and 10% (w/w)||Aerobic mesophilic and coliform microorganisms||Mince pork meat||E||PLA/chitosan showed an antimicrobial activity against microorganisms as compared to noncoated samples at 2 log CFU/mL||Bonilla and others (2013)|
|PLA||Silver-based nanoclay||1% to 10% (w/w)||Samonella spp.||Agar media Liquid media||SC||PLA containing 10% (w/w) silver-based nanoclay significantly inhibits the growth of Samonella spp. at 99.99% CFU reduction||Busolo and others (2010)|
|PLA||Lemon extract Thymol Lysozyme||3% to 7% (w/w) 7% to 15% (w/w) 3% to 10% (w/w)||M. lysodeikticus and Pseudomonas spp.||Agar media Liquid media||E||PLA retained slight antimicrobial activity of 10% lysozyme against M. lysodeikticus Lysozyme demonstrated higher thermal resistance||Del Nobile and others (2009)|
|PLA/MCC||Silver nanoparticles||1% (w/w)||S. aureus and E. coli||Liquid media Agar media||E||MCC reinforced the strength properties of PLA. PLA/MCC/silver nanocomposites demonstrated significant inhibition of E. coli than S. aureus||Fortunati and others (2012)|
|PLA/Zeolites||Silver (Ag+)||5% (w/w)||S. aureus and E. coli||Agar media Liquid media||SC/MM||Solvent casted PLA/silver zeolite reduce the colonies up to 0.8 and 1.02 log CFU/mL for S. aureus and E. coli, respectively, as compared to neat PLA films||Fernandez and others (2010)|
|PLA/Isolated soy protein (ISP)||Thymol Natamycin||2.5% to 25% (w/w) 0.33% to 0.52% (w/w)||Aspergillus sp., S. cerevisiae E. coli, and S. aureus||Agar media Tomato slice Cheese slice||SC||PLA/ISP film containing thymol inhibited the growth of E. coli and S. aureus but no zone of inhibition for mold or yeast was observed||González and Igarzabal (2013)|
|PLA||Lactic acid (LA) Sodium benzoate (SB) EDTA||2.5 mg/g for all||E. coli O157:H7 and S. stanley||Agar media||SC||Apple coated with PLA/SB+LA and PLA/SB+LA+EDTA inhibit E. coli O157:H7 and S. stanley at 4.7 log CFU/cm2 after 14 d of storage||Jin and Niemira (2011)|
|PLA||Allyl isothiocyanate (AIT) Nisin Zinc oxide||100 to 500 μL 250 mg/g 250 mg/g||Salmonella||Liquid egg albumen||SC||Glass jar coated with PLA/AIT/ nisin reduced Salmonella growth at <10 log CFU/mL after 21 d of storage||Jin and Gurtler (2011)|
|PLA||Nisin EDTA Sodium benzoate (SB) Potassium sorbate (PSorb)||250 mg/ g 250 mg/ g 47 mg/g 45 mg/g||E. coli O157:H7 molds and yeasts||Strawberry puree||SC||PLA/SB+PSorb films inhibit greater E. coli O157:H7 and microflora compared to direct addition of SB+PSorb to food||Jin and others (2010)|
|PLA||Nisin||80 to 500 mg/g||L. monocytogenes||Skim milk Liquid egg white||SC||Glass jar coated with PLA/250 mg nisin inactivated cell of L. monocytogenes in liquid egg white and skim milk at storage temperature of 4 and 10 °C||Jin (2010)|
|PLA/Pectin||Nisin||1% (w/v)||L. monocytogenes||Liquid egg White Orange juice BHI liquid media||E+C||PLA/pectin coated with nisin reduces more cells of L. monocytogenes up to 4.5 log CFU/mL in liquid egg white after 48 h at 24 °C. Pectin facilitated the access and adsorption of nisin||Jin and others (2009)|
|PLA||Nisin||0.25 g/g||L. monocytogenes, E. coli O157:H7, and S. enteritidis||Liquid egg white Orange juice Liquid media Agar media||SC||PLA/nisin films inhibit L. monocytogenes in BHI and liquid egg white up to approximately 50% reduction compared to control (4.5 log CFU/mL); PLA/nisin is less ineffective against E. coli O157:H7 in culture medium agar||Jin and Zhang (2008)|
|PLLA||Trans-2-hexenal trapped in β-cyclodextrins||(70:30) β-CDs:Trans-2-hexenal||A. solani, A. niger, B. cinerea, and C. acutatum Penicillium sp.||Agar media Liquid media||E||PLA/β-CD-trans-2-hexenal pellets completely inhibit the growth of A. solani when compared to the PLA/β-CD-trans-2-hexenal sheet||Joo and others (2012)|
|PLA||Triclosan/cyclodextrin inclusion complex (TR/CD-IC)||5% (w/w)||E. coli and S. aureus||Agar media||ES||PLA containing TR/CD-IC nanofibers showed slight improvement antibacterial activity against S. aureus and E. coli compared to PLA nanofibers containing only triclosan with 2.8 to 3.0 cm zone of inhibition, respectively||Kayaci and others (2013)|
|PLLA||Silver nanoparticles||5% (w/w)||E. coli and S. aureus||Agar diffusion Liquid media||SC||PLLA/nanosilver exhibit strong antimicrobial properties at 5 mm zone of inhibition||Li and others (2009)|
|PLA/Sugar beet pulp (SBP)||Nisaplin AIT||0.51 mg/cm2 20.4 μL/cm2||L. monocytogenes and Samonella||Liquid media||E+C||PLA/SBP coated with Nisaplin and AIT inhibit cells of L. monocytogenes up to 3.91 and 4.77 log CFU/mL, respectively, in BHI broth after 48 h at 24 °C||Li and others (2012a)|
|PLA/Plasticizer Glyceroltriacetate (GTA)||Nisaplin EDTA||5% (w/w) 5% (w/w)||E. coli O157:H7||Agar media Liquid media||E||Plasticized PLA impregnated with EDTA/Nisaplin inhibits E. coli O157:H7 up to 60% reduction (3 log CFU/mL) after 24 h compared to control||Liu and others (2010)|
|PLA/Plasticizers Lactic acid Lactide Glycerol triacetate||Nisaplin||5% (w/w)||L. monocytogenes||Agar media Liquid media||E||Films inhibit L. monocytogenes cell population; inclusion of plasticizers into PLA reduces tensile strength||Liu and others (2009a)|
|PLA/Pectin||Nisin Nisaplin®||1% (w/w)||L. monocytogenes||Agar media Liquid media||SC+E||PLA/pectin+Nisaplin films inhibit the growth of L. monocytogenes; pectin protects the biological activity of Nisaplin||Liu and others (2009b)|
|PLA/Pectin||Nisin||1% (w/v)||L. plantarum||Agar media Liquid media||SC+E||PLA/pectin pretreatment Nisin showed antimicrobial activity against L. plantarum at 1.5 log CFU/mL after 24 h||Liu and others (2007)|
|PLA/Calcium bentonite (CB)/PEG||Propolis||13% (w/w)||NA||Water Ethanol||SC||Modified PLA incorporated with propolis could provide a possible delivery system for food||Mascheroni and others (2010)|
|PLA/Wood flour||Triclosan||0.5% to1.5% (w/w)||E. coli||Liquid media||E||1.5% of triclosan in PLA/wood composite inhibits the growth of E. coli at 40% reduction after 1 h of contact time||Prapruddivongs and Sombatsompop (2012)|
|PLA/Plasticizer||Allyl isothiocyanate (AITC)||8% (w/w)||B. cinerea||Agar media||E||PLA containing AITC inhibits B. cinerea||Raouche and others (2011)|
|Polyethylene glycol (PEG)||AITC Carvacrol||8% (w/w)|| ||Liquid media|| ||PLA containing encapsulated AITC and carvacrol inhibits more B. cinerea|| |
|PLLA||Nanoclays||2.5 to 15 pph (parts clay per 100 parts PLLA)||S. aureus, L. monocytogene, E. coli O157:H7, and S. typhimurium||Agar media Liquid media||SC||Only PLA composite films compounded with nanoclay Cloisite 30B showed bacteriostatic activity against L. monocytogenes||Rhim and others (2009)|
|PLA/Plasticizer Polyethylene glycol (PEG)||Chitosan||70% to 90% (w/w)||F. proliferatum, F. moniliforme, and A. ochraceus||Agar media Liquid media||SC||Composites show positive inhibition against fungi. High flexibility with low water-vapor barrier properties of composites||Sébastien and others (2006)|
|PLA||Silver nanoparticles||8% to 32% (w/w)||E. coli, S. aureus, and V. parahaemolyticus||Agar media||SC||PLA/8% (w/w) silver nanocomposites inhibit the growth of E. coli, S. aureus, and V. parahaemolyticus at 1.43, 4, and 4 mm of zone of inhibition||Shameli and others (2010)|
|PLA||Lauric arginate (LAE)||0% to 2.6% (w/w)||L. monocytogenes and S. typhimurium||Agar media Cooked sliced ham||C||Population of L. monocytogenes and S. typhimurium decreased by log 4.80 and 5.13 CFU/mL||Theinsathid and others (2012)|
|PLA||Lactic acid (LA) Sodium lactate (SL)||5% to 15% (w/w) 10% to 30% (w/w)||L. monocytogenes, E. coli O157:H7, and S. typhimurium||Agar media Liquid media||E||Lactic acid more efficient in inhibiting L. monocytogenes; incorporation of LA increase rigidity and brittleness of the film||Theinsathid and others (2011)|
|PLA/Sawdust particle (SP)||Pediocin||0.2% (w/v)||L. monocytogenes||Agar media Raw slice meat||C||PLA/SP containing pediocin precondition by dry heat treatment inhibited L. monocytogenes in raw slice meat at 2 log cycle at 4 ˚C in 14 d of storage||Woraprayote and others (2013)|
Del Nobile and others (2009) have studied the effect of lemon extract, thymol, and lysozyme incorporated directly into PLA via a melt extrusion process. They reported that among the antimicrobial agents under investigation, lysozyme demonstrated the highest microbial inhibition against M. lysodeikticus when compared to the volatile antimicrobial agents (lemon extract and thymol). This was attributed to the good thermal resistance of lysozyme. Jin and Zhang (2008) have prepared PLA/nisin films and reported that these successfully inhibited the growth of L. monocytogenes in BHI broth and liquid egg white up to approximately 50% reduction when compared to the control. They reported that the PLA/nisin film is less effective against E. coli O157:H7 in culture medium agar than it is against L. monocytogenes.
Table 1 also shows that some researchers have investigated the incorporation of antimicrobial agents derived from metals, including silver ions, into PLA (Li and others 2009; Busolo and others 2010; Fernandez and others 2010). Fernandez and others (2010) studied the effect of different processing techniques that were used to impregnate silver ions into PLA/zeolite films on the antimicrobial activity of the resulting films against S. aureus and E. coli. In their findings, the solvent-cast PLA/silver zeolite reduced the colonies of S. aureus and E. coli up to 0.8 and 1.02 log CFU/mL respectively, which were greater than the reductions observed in the melt blended and compression molded samples.
Although in most studies PLA was incorporated with nonvolatile and volatile antimicrobial agents, some have used compounds that would typically be added directly into foods. Jin and others (2010) explored the effect of food additives such as potassium sorbate and sodium benzoate (SB) incorporated into PLA. These antimicrobial PLA films were prepared via the solvent casting method in order to investigate the inhibitory effect against E. coli O157:H7 and microflora in strawberry puree. The films showed greater inhibitory effects against E. coli when compared with the direct addition of the agents into the real food. It was suggested that the direct addition of antimicrobial agents into a targeted food might lead to a loss of activity due to the instant reaction with other components in the food such as lipids or proteins. Conversely, a more continuous antimicrobial effect could occur in the PLA films due to the slow release of the antimicrobial agents from the film and thus enable high enough concentrations against microorganisms to be sustained with time.
Encapsulation of antimicrobial agents is an emerging technique to introduce antimicrobial activity into polymeric substrates. This is particularly useful when volatile agents are used and thermal processes are required. Joo and others (2012) encapsulated trans-2-hexenal (which is a volatile antimicrobial agent) with beta-cyclodextrin (β-CD) and impregnated the complex into PLA via extrusion processing. They reported that during 7 d of storage at 23 °C the PLA/β-CD/trans-2-hexenal pellets completely inhibited the growth of A. solani in potato dextrose agar media when compared to the PLA/β-CD-trans-2-hexenal sheets. Encapsulation of volatile antimicrobial agents into β-CD has also been investigated by others using different types of polymeric materials (Raouche and others 2011; Ramos and others 2012).
Other materials such as fillers and plasticizers can be used to facilitate and increase the activity of antimicrobial agents in PLA. Some fillers such as natural fibers (for example, wood flour) and other polysaccharides (for example, pectin) can be used for this purpose. Prapruddivongs and Sombatsompop (2012) studied the incorporation of wood flour particles as an antibacterial promoter for triclosan-based wood flour/PLA composites. In these systems a higher loading of wood flour particles resulted in the migration of more triclosan onto the composite surface and a subsequent inhibition of the growth of nonpathogenic E. coli ATCC 25922 in a growing liquid media. They reported that by using the plate agar count technique, 1.5% (w/w) of triclosan in 10% (w/w) wood flour/PLA composite reduced the viable cell count of E. coli by 40% after 1 h of contact time in nutrient broth, which was greater than a PLA/triclosan film without wood flour. They speculated that since the wood flour is hydrophilic, the PLA also becomes more hydrophilic and thus, more water molecules can be adsorbed on the composite surface thereby facilitating the migration of the antimicrobial agent to the surface of the film. Liu and others (2009a) reported that a neat PLA film did not exhibit any antimicrobial activity with a small concentration of Nisiplin. Nevertheless, they found that co-extrusion of PLA and pectin–Nisaplin composites demonstrated a higher antimicrobial activity compared to neat PLA and suggested that the pectin protects the biological activity of the composite films and thus, significantly inhibits the growth of the test species L. monocytogenes. Other systems effective against L. monocytogenes include PLA/nanoclay composites using Cloisite 30B (Rhim and others 2009), although the use of nanoclays is yet to be fully established. Liu and others (2012) have patented their active materials combination containing PLA with bacteriocin (for example, nisin, generally in the form of Nisaplin®) and plasticizer (for example, LA, lactide, triacetin, GTA), and as an option at least one pore forming agent. Recently, Kayaci and others (2013) have prepared antimicrobial nanofibres or nanowebs using an electrospinning method that comprises a PLA and triclosan/cyclodextrin inclusion complex (TR/CD-IC). The triclosan/cyclodextrin complex consisted of both beta (β) and gamma (γ) complex. They reported that the zones of inhibition of E. coli and S. aureus were wider for the PLA containing TR/CD-IC nanowebs, suggesting that both PLA/TR/β-CD-IC and PLA/TR/γ-CD-IC nanowebs have better antibacterial properties than the PLA containing triclosan.
It is therefore clear that PLA has the potential to be used as a primary polymeric material in antimicrobial packaging applications since both volatile and nonvolatile additives demonstrate reasonably good compatibility with this polymer. Nevertheless, future and systematic work needs to be performed in order to confirm the potential of such antimicrobial systems through studies with food stimulants as well as with real food products.
Antimicrobial activity of PLA films coated with antimicrobial agents
Due to the heat sensitivity of antimicrobial agents, they are often either coated onto the packaging material after it is formed or added to cast films. For example, natural polyphenolics do not tolerate the high temperatures that are typically encountered during thermal processing of polymeric materials (Appendini and Hotchkiss 2002). Furthermore, antimicrobial agents are often coated onto the surface of packaging materials in order to provide a high concentration of the agent on the surface of the food product where microbial contamination is more significant (Jin 2010; Kuorwel and others 2011b). Only a few reports were found in the literature on the activity of antimicrobial agents coated onto PLA films or PLA incorporated with antimicrobial agents that have been then solution coated onto other substrates. These substrates can be glass jars, plastic films, or coated directly onto food products.
Table 1 depicts the antimicrobial activity of different types of antimicrobial agents incorporated in PLA via coating, solvent casting as well as their combinations with primary and/or secondary materials. Jin and others (2009) studied the effectiveness of PLA/pectin films coated with nisin against L. monocytogenes. They reported that PLA/pectin and PLA films coated with nisin (1%, w/v) were significantly different in terms of their antimicrobial activity based on the agar diffusion test. In this system, nisin can be easily coated on PLA/pectin due to the rough surface of the composite as compared to the relatively smooth surface of neat PLA films. The presence of pectin also facilitates the access and adsorption of nisin. The nisin-coated PLA/pectin system was found to reduce the count of L. monocytogenes up to 3.7 and 4.5 log CFU/mL in orange juice and liquid egg white, respectively.
In another study, Theinsathid and others (2012) prepared PLA films coated with LAE at various coating loadings from zero to 2.6% (w/w). An average 4 mm zone of inhibition was observed for all antimicrobial films starting from 0.07% to 0.28% (w/w) of LAE loading. The populations of L. monocytogenes and S. typhimurium were decreased with a higher loading of LAE (2.6%, w/w) by log 2 to 3 CFU/mL compared to the uncoated PLA films, as expected.
Table 1 also lists systems in which direct contact of the antimicrobial agent with the food product as well as the packaging material occurs. For example, Jin and Niemira (2011) prepared PLA coating systems using SB, LA, and EDTA. They coated apples with solvent-cast PLA/(SB+LA) and PLA/(SB+LA+EDTA) consisting of 2.5 mg of each antimicrobial substance per gram of PLA. They reported that PLA/(SB+LA) films inhibited E. coli O157:H7 and Salmonella stanley at 4.7 log CFU/mL after 14 d of storage. Jin (2010) coated glass jars with solvent-cast PLA and 250 mg of nisin and reported that in liquid egg white and in skim milk L. monocytogenes were inactivated by this system at storage temperatures of 4 and 10 °C. It is important to note that an antimicrobial film is more effective against microbial growth when the food and the packaging material are in direct contact but this is not always practical (Brody and others 2001; Appendini and Hotchkiss 2002).
Generally, if the concentration of an antimicrobial agent is at or above the minimum inhibitory concentration (MIC) on the food surface, the system will maintain effective antimicrobial activity (Suppakul 2004). According to Rardniyom (2009), a good selection of an antimicrobial agent together with the combination of different types of tested foods or food simulants is an important factor to be considered when designing an antimicrobial package. Since PLA is a relatively new polymer, it requires further investigation, particularly with regard to the antimicrobial activity of PLA systems that are intended for targeted food packaging materials.