食品包装技术（英文版）：36756_05

5.1 Introduction Non-migratory bioactive polymers (NMBP) are a class of polymers that possess biological activity without the active components migrating from the polymer to the substrate. This concept has existed for some time (Bachler et al., 1970; Brody and Budny, 1995; Katchalski-Katzir, 1993) and has been applied primarily to immobilised enzyme processing (Katchalski-Katzir, 1993; Mosbach, 1980). It is only now becoming of interest in packaging applications (Appendini and Hotchkiss, 1997; Soares, 1998). Bioactive materials are based on molecules that elicit a response from living systems. The goal is to use bioactive materials for which the response is desirable from the standpoint of the package or the product, for example inhibition of microbial growth or flavour improvement. Enzymes are classic examples of bioactive substances, as are many peptides, proteins, and other organic compounds. The definition, from the perspective of packaging, is based on function: the way the substance interacts with living systems. Purely physical processes, for example adsorption or diffusion, are excluded from this definition. Bioactive polymers can be formed by attachment of bioactive molecules to synthetic polymers, as in the case of enzyme immobilisation (Appendini and Hotchkiss, 1997; Soares, 1998), or may result from an inherent bioactive effect of the polymer structure, as with chitosan (Collins-Thompson and Cheng-An, 2000; Tanabe et al., 2002). They have potential applications in the packaging of food and other biological materials, in food processing equipment, on biomedical devices (Sodhi et al., 2001; Sun and Sun, 2002) and in textiles (Edwards and Vigo, 2001; Sun and Sun, 2002). Non-migratory polymers are defined to be those for which the bioactive component does not migrate out of the polymer system into the surrounding 5 Non-migratory bioactive polymers (NMBP) in food packaging M. D. Steven and J. H. Hotchkiss, Cornell University, USA medium (see Fig. 5.1). Typically this is achieved through covalent attachment of the active component to the polymer backbone, inherently bioactive polymer backbones, or entrapment of the active component within the polymer matrix. The first two of these will be discussed in this chapter. 5.2 Advantages of NMBP In order for any new technology to be considered, it needs to have advantages over existing technologies. Typically, however, these advantages come with certain limitations, in application or utility, and frequently with an increase in cost. Benefits and limitations will apply differently to the different types of NMBP. The benefits of NP can be divided into four main areas: technical benefits, regulatory advantages, marketing aspects and the food processor’s perspective. Note that this list is not exhaustive; particular applications will involve some or all of these plus other considerations specific to that application. 5.2.1 Technical benefits Technical benefits of NMBP include improved stability of the bioactive substance, and concentration of the bioactive effect at a specific locus. Improved stability is a consideration for covalently immobilised bioactive substances; biological molecules, e.g. enzymes, are typically very sensitive to environmental conditions. They are readily denatured by some solvents, by high, and in some cases low, temperatures; by high pressures, high shear or ionising radiation; by certain levels of pH and in the presence of high concentrations of electrolytes (Richardson and Hyslop, 1985). Conjugation to polymer supports has been shown to enhance dramatically the stability of these molecules. Topchieva and Fig. 5.1 Simplified visual comparison of (a) non-migratory and (b) migratory bioactive packaging. Adapted from Han (2000). 72 Novel food packaging techniques colleagues (1995) demonstrated improved thermal stability of chymotrypsin when conjugated to poly(ethylene glycol) (PEG) (see Fig. 5.2). Appendini and Hotchkiss (2001) similarly demonstrated the thermal stability of a small antimicrobial peptide when covalently attached to a PEG-grafted poly(styrene) (PS) support. The immobilised peptide remained active when dry-heated to 200oC for 30 minutes and when autoclaved at 121oC for 15 minutes. Polymers are often processed at temperatures that would denature native proteins; thermally stable protein-polymer conjugates will be resistant to high processing temperatures and suitable for polymer extrusion and other high temperature polymer and food processing applications. Appendini (1999) also demonstrated the improved activity of the conjugated peptide over a range of pH (see Fig. 5.3). Note that although there is some loss of activity caused by attaching the peptide to the surface (this will be discussed in section 5.3 below), the residual activity is retained over a broader pH range than for the native peptide. Other authors have also reported improved stability of polymer-conjugated enzymes to pH and temperature (Gaertner and Puigserver, 1992; Yang et al., 1996; Yang et al., 1995a; Yang et al., 1995b; Zaks and Klibanov, 1984). The extended range of pH stability will provide activity in a broader range of food products than would be the case for the native compound. The stability of proteins to inimical media, such as organic solvents, supercritical fluids and gases, is often improved by polymer conjugation and applications have developed to exploit this in non-aqueous enzymology Fig. 5.2 Activity of PEG-conjugated chymotrypsin and native chymotrypsin held at 45oC. Activity is expressed in percent relative to the initial activity of each enzyme preparation. Adapted from Topchieva et al. (1995). Non-migratory bioactive polymers (NMBP) in food packaging 73 (Mabrouk, 1997; Panza et al., 1997; Veronese, 2001; Yang et al., 1995a; Yang et al., 1995b; Zaks and Klibanov, 1984). This enhanced stability to organic solvents is useful in allowing a broader range of solvents and chemicals to be used in casting, cleaning/sterilising or treating polymer films prior to package filling without damaging the functional characteristics of immobilised bioactive constituents. The long-term stability of immobilised peptides and proteins is generally enhanced compared to the native compounds (Katchalski-Katzir, 1993; Panza et al., 1997). The improved stability will help ensure the activity of bioactive packaging is retained for the shelf-life of the packaged food product. Long-term stability is also important in ensuring adequate shelf-life of the NMBP packages before filling; packaging materials are often warehoused for extended periods prior to use; any modifications need to remain active after storage. The second technical benefit is concentration of the activity at a specific locus within the package and/or the food. This allows the activity to be concentrated where it will be most effective. For many minimally processed food products, such as fresh meat and fresh-cut fruit and vegetables, the majority of contaminating bacteria are located on the surface of the product (Collins-Thompson and Cheng- An, 2000; Hotchkiss, 1995). Concentrating antimicrobials on the surface of the product, as occurs with antimicrobial packaging, allows minimal amounts of the Fig. 5.3 Antimicrobial activity of a small synthetic peptide against E.coli 0157:H7 in 0.1M citrate buffer from pH 3.5 to 7.0. Activity is shown for the native peptide ( ) and peptide attached to a PS surface through a PEG spacer ( ). Equivalent peptide concentrations were used in each determination. Adapted from Appendini (1999). 74 Novel food packaging techniques active compounds to be used to maximum effect. Similarly, sampling the headspace of a product for substances indicative of microbial growth using an enzymatic spoilage indicator (de Kruijf et al., 2002), could be accomplished by locating the indicator in the package headspace in a position where it will be most visible to a consumer. This minimises use of expensive materials, e.g. enzymes, and possible undesirable interactions with the food. 5.2.2 Regulatory advantages Regulations relating to active food packaging are still evolving. As new technologies develop, regulations generally must be modified to encompass them. A detailed discussion of European Union regulations relating to food packaging, with specific discussions of the implications for active and intelligent packaging systems, is presented by de Kruif and Rijk in Chapter 22 of this text (de Kruijf and Rijk, 2003). It is important in interpreting this work from a NMBP standpoint to recall that NMBP do not result in migration of the active components into the food. As noted by various authors (de Kruijf et al., 2002; de Kruijf and Rijk, 2003; Meroni, 2000; Vermeiren et al., 2002; Vermeiren et al., 1999), there are no specific EU regulations for active or intelligent packaging; rather these packaging systems are subject to the same regulations as traditional packaging. These regulations require that all components used to manufacture food contact materials be on ‘positive lists’; active and intelligent agents are not typically included on these lists. Further, the regulations set down migration limits for both overall migration and migration of specific components. For NMBP the migration requirements should not be problematic, although a lack of migration will need to be established as detailed in the appropriate regulations. The compounds used to manufacture NMBP, however, will need to be included on the relevant positive lists. The key Directive (regulation) of concern is 89/109/EEC. De Kruijf and Rijk (2003) indicate that a new Directive, to replace 89/109/EEC, will soon be published and will allow the use of active and intelligent food contact materials. For more information, consult Chapter 22. In the United States, regulations relating to food contact materials can be found in the Code of Federal Regulations (CFR) Title 21 Parts 170 through 190 (Anon., 2002). The regulations revolve around determining if compounds in packaging materials are food additives. Food additives are defined as substances ‘the intended use of which results or may reasonably be expected to result, directly or indirectly, either in their becoming a direct component of food or otherwise affecting the characteristics of food’. Further, ‘If there is no migration of a packaging component from the package to the food, it does not become a component of the food and thus is not a food additive’ unless it is used ‘to give a different flavour, texture of other characteristic in the food’, in which case it ‘may’ be a food additive (21 CFR §170.3 (e) (1)). The regulations also establish guidelines for determining limits below which migration can be considered Non-migratory bioactive polymers (NMBP) in food packaging 75 negligible, negating food additive classification of that substance for that specific application. These US regulations can be interpreted that any substance for which it can be shown that there is negligible migration into a food product is not classified as a food additive (21 CFR §170.39). This would imply that NMBP needs to meet the regulations required of items for food contact use, but do not need to meet the more stringent food additive regulations, provided lack of migration is proven. However, additive classification may also depend on the intended function of the component. If it was intended that a packaging component be active in the food product, as with an immobilised antimicrobial on a packaging film intended to extend the shelf-life of packaged food, then the component might be classified as a Direct Food Additive and be required to comply with food additive regulations (Brackett, 2002). There is, therefore, some ambiguity as to the status of NMBP materials. If the active components of NMBP are not classified as food additives, then it will be a significant advantage, allowing the use of substances in food packaging that are not currently permitted as food additives, provided, of course, that negligible migration is proven. The process of obtaining food contact approval has been recently reviewed (Heckman and Ziffer, 2001). The above discussion on US regulations mainly focuses on the issues of attached or entrapped bioactive compounds where the active agent is added to the polymer backbone. For inherently bioactive polymers, which will be discussed in more depth shortly, a different situation may exist. A structural component of the packaging film may not be classified as a direct food additive, for example UV irradiated nylon (Shearer et al., 2000) with antibacterial properties, even if it has a direct effect in the food. This will probably not apply if an edible film is considered, as is often the case in applications involving chitosan (Coma et al., 2002) and other biopolymers. In these cases, food additive regulations will most likely apply. 5.2.3 Marketing aspects In recent years, consumers have become more aware and concerned about the composition and safety of their food. There have been increasing demands for safe, but minimally processed and preservative-free products (Appendini and Hotchkiss, 2002; Collins-Thompson and Cheng-An, 2000; Vermeiren et al., 1999). This is against a background of recent food-borne microbial disease outbreaks (Appendini and Hotchkiss, 2002; Mead et al., 1999). NMBP may have a key role to play in this area. Incorporating non-migratory antimicrobials in packaging materials may significantly reduce microbial contamination, while providing minimally processed, preservative-free food products. Similarly, immobilised enzyme packaging (Soares, 1998) may provide in-package processing opportunities which would not otherwise be possible for ‘fresh’ products, enhancing the acceptability and shelf-life of minimally processed foods. 76 Novel food packaging techniques 5.2.4 The food processor’s perspective From the perspective of the food processor, NMBP would have several advantages. A general benefit would be in achieving a more stable product with a longer shelf-life, but beyond that certain NMBP technologies and applications may offer specific benefits. As an example, consider the production of lactose-free milk. The demand for this product is not high, although there is a place for it in the market. It sells at a high price due to the high cost of production and the low sales volume. Processing requires significant plant down time for cleaning or dedicated production facilities and the high capacity of modern plants means that the minimum production volume may be greater than the demand, leading to product wastage and requiring the use of expensive UHT technology to extend shelf-life. Using lactase-active packaging, however, regular milk could be packed off a normal production run to obtain a lactose-reduced or lactose-free product after a short period of storage. A migratory enzyme, or the direct addition of lactase to milk, cannot be used in this application, due to the strict requirements of the pasteurised milk ordinance (Anon., 1999). Similarly for other products, some of the processing may be accomplished in package, instead of in the processing plant, reducing processing costs and increasing flexibility for the food processor. 5.3 Current limitations As noted above, any new technology must have benefits over current technologies in order to be successful, but these benefits also typically come with limitations. The most pertinent limitations of NMBP are: a limited locus of activity, specific requirements on the mechanism of activity of the active agent, reduced activity, availability of appropriate technology and an increase in packaging cost. 5.3.1 Limited locus of activity A significant limitation of NMBP is the need for the reaction constituents to be transported to the package-product interface. This limits the function to areas in intimate contact with the packaging material for solid and viscous liquid foods. For low-viscosity foods this is less of a problem, as agitation during distribution mixes the product and will bring the required constituents in contact with the packaging. With viscous liquids, the high viscosity makes it unlikely that there will be sufficient mixing during distribution to bring all the target constituents into contact with the packaging material. Additionally, the high viscosity will limit diffusive mixing. For solid products, diffusive migration of the target constituents will also be limited and unlikely to be an effective mechanism of ensuring adequate action of the NMBP. Even for applications where the surface of a product is the target, the need for intimate contact with the packaging material may prevent application of the active agent within crevices and folds of the packaged item. This can, however, be alleviated through package design Non-migratory bioactive polymers (NMBP) in food packaging 77 and/or vacuum packaging. Migratory bioactive packaging technologies are often similarly limited in their diffusion and mixing requirements, as the active agent may need to diffuse through the food to achieve the desired effect. 5.3.2 Mechanisms of action In order for a bioactive agent to be active when covalently anchored to a packaging material, the conformation of the active component in the immobilised state (compared to the free solution form), the location of the covalent link to the polymer, and the mechanism by which the agent interacts with the environment to achieve the desired function must all be considered. If, for example, an antimicrobial ingredient needs to enter the microbial cell to be effective, then it is unlikely to be active in a tethered state, whereas an antimicrobial agent that is active at the microbial surface may maintain activity when tethered. If attachment causes conformational changes in the bioactive compound, or an active site is altered, then activity will be disrupted. Consider also the attachment of an enzyme that requires a co-enzyme for activity. If this coenzyme is not present in the food or otherwise attached along with the primary enzyme, then the primary enzyme will be inactive. Understanding the mechanism of the active agent is a key requirement in creating NMBP. 5.3.3 Reduced activity One concern in immobilising bioactive compounds is the potential for loss of activity. In many cases, activity is reduced compared to the native compound (Katchalski-Katzir, 1993), and in some cases it is lost completely. With appropriate coupling methodology, however, activity can be retained, albeit normally at a lower level than for the free compound. The activity of a bound bioactive compound can vary drastically compared to the free soluble form. Appendini (1999) compared the activity of a small antimicrobial peptide when immobilised to PEG grafted poly(styrene) (PS) beads and found that it was 200– 7000 times less active than the free soluble peptide. It still possessed significant antimicrobial activity, however, and was effective against E. coli 0157:H7 at immobilised peptide concentrations of 4 mol/ml in growth media. In the immobilisation of naringinase, Soares (1998) found that the enzyme retained 23% of its free activity when immobilised. Soares also found that at a pH less than 3.1, the immobilised naringinase possessed higher activity than free naringinase. This often occurs with immobilised enzymes – their increased stability leads to higher activity compared to the free enzyme when conditions depart significantly from the optimum. Mosbach (1980) also suggested that for sequential enzyme pathways, the activity of the immobilised enzymes could be higher than that of the enzymes in free solution if the enzymes were immobilised in close proximity. In other words, although the activity of immobilised enzymes is typically reduced under optimum conditions, they still normally retain sufficient activity to be useful and may show improved activity under extreme conditions. 78 Novel food packaging techniques 5.3.4 Technology availability The commercial availability of technology required to produce NMBP could limit applications. Technologies for functionalising the surface of polymer films are readily available, but newer technologies, which provide controlled surface functionalisation, are still in development; this is especially true with respect to their application in high throughput continuous processes, as required for production of packaging materials. Surface functionalisation is discussed in more detail in section 5.5. Beyond basic surface functionalisation, further modification of film surfaces is not typically practised commercially. Production of most NMBP will require further processing, probably involving wet chemical treatments for immobilisation of active agents; adaptation of existing technologies will be required to implement these treatments. If NMBP becomes widespread, the technology will become more readily available and this will cease to be a limitation. 5.3.5 Cost The final limitation of NMBP is the likely increase in cost. NMBP will require further steps and additional materials in film manufacturing/converting processes, increasing production costs. Intensive modifications, such as the attachment of proteins, will incur significant cost increases due to the additional processing steps required, the chemicals used in processing, and from the cost of the agent itself. The peptides, proteins and enzymes involved can be quite expensive, although increased demand will undoubtedly result in cost reductions of these components in the long term. Additionally, the need to recover research and development expenses and new equipment requirements will also increase the film cost. Over time, new equipment will become cheaper and more readily available, increased material availability should lead to lower material costs and the overall cost of the films will decrease. This is typical of the cycle involved in introducing new technologies. 5.4 Inherently bioactive synthetic polymers: types and applications As previously mentioned, there are two main types of NMBP – inherently bioactive polymers and polymers with covalently immobilised bioactive agents. For inherently bioactive polymers, the structural polymer itself is bioactive. For example, polymers containing free amines have been shown to be antimicrobial (Shearer et al., 2000). Included in this definition are structural polymers with modified backbones. These polymers differ from those with immobilised bioactive compounds in that no previously synthesised bioactive compound is attached to the polymer chain. Several materials have been found to have inherent bioactivity (Oh et al., 2001; Ozdemir and Sadikoglu, 1998; Shearer et al., 2000; Vigo, 1999; Vigo and Leonas, 1999) and new ones are currently being Non-migratory bioactive polymers (NMBP) in food packaging 79 developed (Tew et al., 2002). Most examples of inherently bioactive polymers involve antimicrobial activity. 5.4.1 Chitosan Chitosan is the probably the most studied inherently bioactive NMBP to date (Coma et al., 2002; Oh et al., 2001; Tanabe et al., 2002). It possesses broad spectrum antimicrobial activity in simple media and is available commercially as an antifungal coating for shelf-life extension of fresh fruit (Appendini and Hotchkiss, 2002; Padgett et al., 1998). Chitosan is the deacetylated form of chitin (poly- -(1!4)-N-acetyl-D-glucosamine), a common natural biopolymer extracted from the shells of crustaceans. Production of chitosan from chitin involves demineralisation, deproteinisation, and deacetylation (Oh et al., 2001). The properties of chitosan films, including antimicrobial efficacy, mechanical and barrier properties, are significantly affected by the degree of deacetylation (Oh et al., 2001; Paulk et al., 2002). Recent research suggests that chitosan disrupts the outer membrane of bacteria (Helander et al., 2001; Tsai and Su, 1999); there were earlier suggestions that the activity was solely due to bacterial adsorption (Appendini and Hotchkiss, 2002), but the weight of evidence now suggests it possesses true antimicrobial activity. Given that chitosan is a large polymeric macromolecule, activity is unlikely to require penetration of the polymer to the intracellular area (Helander et al., 2001). Helander and colleagues (2001) comment that the key feature of the antimicrobial effect of chitosan is probably the positive charge that exists on the amino group at C-2 below pH 6.3. The positive charge on this group creates a polycationic structure, which may interact with the predominantly negatively charged components of the gram-negative outer membrane. They investigated the membrane interactions of chitosan with E. coli, P. aeruginosa and S. typhimurium in microbiological media and determined that the activity was affected by pH, being significant at pH 5.3, but non-existent at pH 7.2; was dependent on the absence of MgCl 2 in the media and resulted in increased uptake of a hydrophobic probe (1-N- phenylnaphthylamine) from the media, indicating increased membrane permeability. Activity was thought to result from chitosan binding to the outer membrane, and was reduced for mutant S. typhimurium strains with cationic outer membranes. Chitosan was found to significantly sensitise the outer membrane to the action of other compounds, for example bile acids and dyes. Tsai and Su (1999) similarly investigated the mechanism of activity of chitosan against E. coli and found that higher temperatures and an acidic pH increased chitosan activity, and that divalent cations, such as Mg 2+ , reduced activity. Chitosan caused leakage of glucose and lactate dehydrogenase from bacterial cells. They also concluded that the activity involves interaction between polycationic chitosan and anions on the bacterial surface, resulting in changes in membrane permeability. A similar mode of action can be assumed against gram- positive bacteria, fungi and yeasts. 80 Novel food packaging techniques The activity of chitosan has been tested against a broad range of microorganisms by researchers in many different fields, including dentistry and pharmaceuticals (Ikinci et al., 2002), textiles (Takai et al., 2002) and food packaging (Oh et al., 2001; Paulk et al., 2002; Tanabe et al., 2002). In microbial growth broths, chitosan has been found effective against gram-positive and gram-negative bacteria, along with some moulds and yeasts (Oh et al., 2001; Tsai and Su, 1999; Tsai et al., 2002). The minimum inhibitory concentration varies with organism and increases as the chitosan degree of deacetylation decreases. Chitosan activity has also been tested in mayonnaise against Z. bailii and L. fructivorans (Oh et al., 2001). The addition of chitosan to the mayonnaise formulation increased the bacterial inhibition compared to the control mayonnaise, although bacteria numbers also decreased in the control. Higher concentrations of chitosan were required for a significant inhibitory effect in mayonnaise than in growth broth. Tsai and colleagues (2000) investigated the antimicrobial efficacy of chitosan in milk, although strict milk composition regulations and the significant solubility of chitosan in milk mean that this application is unlikely to be commercially viable. At this stage, it is important to note that most research on chitosan activity has been conducted in solution, not with chitosan films, so extrapolation to packaging applications is difficult. Additionally, the high solubility of chitosan makes its use in liquid packaging applications unlikely, as it would dissolve into the food over time, violating the non-migratory principle. As far as packaging uses go, chitosan activity has been investigated as an edible antimicrobial film for fish fillets (Tsai et al., 2002). The indigenous microflora of fish was inhibited by films formed from 0.5% and 1.0% chitosan solutions. After ten days of storage, mesophilic and psychrotrophic bacteria were reduced compared to control samples. For both types of organism, counts were reduced by approximately 1 log. Additionally, volatile basic nitrogen evolution was decreased and pH increase was suppressed compared to the controls. Coliforms were inhibited throughout a 14-day storage trial, while Aeromonas and Vibrio species showed negligible initial inhibition but slower growth and decreased numbers during the second half of storage. Pseudomonas spp. were initially inhibited on the dipped fillets, but increased after five days to the same levels as the control fillets. Overall, chitosan appeared to be an effective antibacterial coating; it may be suitable for use as an antimicrobial edible film for processed fish products. In another edible film application, the antimicrobial effect of edible 98% deacetylated chitosan films was investigated against Listeria spp. on agar media and cheese. Significant anti-listerial activity was found in an agar plate assay: reductions of 5–8 log cycles were observed. The effect was also significant on the chitosan coated cheese samples. No viable cells were detected three and five days after dipping the cheese samples first in an inoculating solution of 10 4 cells/ ml and then in a chitosan film-forming solution. These results are promising for the application of chitosan edible films to help control pathogenic contamination on the surfaces of solid food products. Similarly, the antifungal properties of Non-migratory bioactive polymers (NMBP) in food packaging 81 chitosan may make it suitable for use as an edible film for low moisture applications where mould spoilage is a concern, e.g. bakery applications. 5.4.2 UV irradiated nylon A recent development is surface modification of polymers leading to antimicrobial activity, for example treatment of nylon with an excimer laser at UV frequencies (193 nm) (Ozdemir and Sadikoglu, 1998; Shearer et al., 2000). This has been described as a physical modification (Appendini and Hotchkiss, 2002), although the actual change which leads to the induced antimicrobial activity is a chemical change: amides on the nylon surface are converted to amines, which remain bound to the polymer chains, as observed with X-ray photoemission spectroscopy (XPS). Antimicrobial nylon-6,6 is prepared by irradiating with an UV excimer laser at 193 nm for a total exposure of 1-3 J/cm 2 . This results in conversion of approximately 10% of the surface amides and some etching of the film surface (see Fig. 5.4). The antimicrobial effect is strongly dependent on the wavelength of the laser used, with films treated at 193 nm showing a 5 log reduction in K. pneumoniae in one hour, while film treated at 248 nm had no antimicrobial effect (Ozdemir and Sadikoglu, 1998). XPS analysis of the surface of film treated at 193 nm indicated that surface amide groups were converted to amines, while film treated at 248 nm showed no such change. The mechanism of antimicrobial activity is presumably similar to that of chitosan, poly-L-lysine and other cationic polymers, involving interaction with negatively charged microbial membranes leading to membrane disruption and leakage of cellular constituents. The activity of UV irradiated nylon has been tested against various bacteria (Shearer et al., 2000). In comparisons with untreated nylon, the treated nylon resulted in slight reductions in viable cell numbers for E. coli and S. aureus. Some bacterial reduction was also observed for the untreated nylon, presumably due to bacterial adsorption. The treated and untreated nylons were not significantly different for reduction of E. faecalis and P. fluorescens. At least three hours of exposure were required for a significant reduction in cell counts and, for S.aureus, the activity of the treated nylon increased with increasing temperature; no effect was observed a 4oC or 15oC. Protein (0.1% Bovine Serum Albumin) completely inhibited the antimicrobial activity. Shearer and colleagues (2000) compared the antimicrobial effect of treated film to that of secondary amines (n-butyl butyl amine) in solution and found that, to obtain a significant effect, a ten fold higher concentration of soluble secondary amine was required compared to the calculated number of amines formed on the surface of the film. It is not mentioned if the increased surface roughness was factored into this calculation; increased surface roughness results in a significant increase in the absolute surface area of the film, with a consequent increase in the number of active sites. The results of the antimicrobial assays of UV irradiated nylons are not definitive. The data does not clearly show that the bacteria are killed as opposed to adsorbed on the surface of the film; the increased surface area resulting from 82 Novel food packaging techniques etching of the film by the laser treatment would result in increased bacterial adsorption compared to the native film. Bacterial adsorption is the most likely cause of the decreased bacterial populations observed for the untreated nylon films, and in most cases the differences observed between the treated and untreated films were small, typically of the order of one log. More investigation is needed to determine whether cells are adsorbed or inactivated. 5.4.3 Others In addition to the polymers mentioned above, many others have been investigated for potential bioactivity. An interesting recent development is the possibility of Fig. 5.4 Atomic force microscopy surface profiles of (a) untreated and (b) UV irradiated nylon film (from Shearer et al. (2000), reprinted by permission of Wiley-Liss, Inc., a subsidiary of John Wiley and Sons, Inc.) Non-migratory bioactive polymers (NMBP) in food packaging 83 designing biomimetic antimicrobial polymers. Tew and colleagues (2002) have designed a series of amphiphilic acrylic polymers that possess very similar structures to the magainin class of amphiphilic antimicrobial peptides. These peptides kill microbes by disrupting cell membranes and are active against a broad range of microorganisms. Similarly, the new synthetic biomimetic polymers were found to disrupt synthetic membranes and to be active against several bacteria: E. coli, K. pneumoniae, S. typhimurium, P. aeruginosa and E. faecium. These synthetic polymers might be suitable as independent polymer layers in packaging materials, or as a grafted layer on a structural-polymer backbone. Poly(ethylene glycol) (PEG) has also been reported to possess inherent antimicrobial activity (Jinkins and Leonas, 1994; Vigo, 1999; Vigo and Bruno, 1993; Vigo and Leonas, 1999). Unpublished results from our laboratory have been mixed as to the antimicrobial effectiveness of PEG: it does possess antimicrobial activity, but only at high concentrations (80mg/ml in liquid media). The water soluble nature of this polymer suggests it would be best applied as tethered graft chains on a structural polymer backbone, for example poly(ethylene) (PE), rather than as an independent polymer layer. PEG is also known to reduce the adhesion of proteins and cells to materials (Sofia and Merrill, 1998; Zalipsky and Harris, 1997). Poly-l-lysine and poly(lactic acid) have both been reported to possess limited antimicrobial activity (Appendini and Hotchkiss, 2002; Ariyapitipun et al., 2000; Mustapha et al., 2002). Poly-l-lysine is a cationic polymer that promotes cell adhesion and is thought to be active by a mechanism similar to that of chitosan; interaction with the negative charges on the cell membrane causing membrane disruption and leakage of cellular constituents (Appendini and Hotchkiss, 2002). Low molecular weight poly(lactic acid) has been found to be active against several organisms, although the mechanism of antimicrobial activity is not known (Ariyapitipun et al., 2000; Mustapha et al., 2002). It has been suggested that it releases lactic acid and that the activity derives from this. New antimicrobial compounds are constantly being discovered. Some are compounds that are well known, but for which the activity was not recognised, whereas others are novel compounds. Regardless, the possible applications for inherently antimicrobial polymers are significant and research in this area will continue. One important note: many of the abovementioned compounds are water soluble and initial trials are normally conducted on the soluble form of the polymer. Activity of the soluble form does not necessarily carry over to activity of the polymer in a film, either as an independent layer or as a graft layer on another backbone polymer. It is important to test the film activity as well as the solution activity if there is to be any application of these new materials. 5.5 Polymers with immobilised bioactive compounds The second major type of NMBP is a polymer backbone to which an active agent is covalently attached. The active agent may be a peptide, protein or 84 Novel food packaging techniques enzyme; it can be synthesised on the surface, or it can be synthesised or extracted separately and then covalently linked to the polymer. To date there has been more research conducted in this area than in that of inherently bioactive polymers, and a number of examples have been commercialised. Often, enzymes that are adsorbed on polymers are termed immobilised, however, these compounds are not truly immobilised as they readily migrate out of the polymer in suitable non-reactive solvents (solvents that do not result in significant disruption of the covalent linkages of the system; concentrated acids and bases, for example, will cleave some covalent linkages). The term immobilised, as used here, implies covalent attachment of bioactive molecules to the polymer backbone. In some cases large compounds can also be immobilised by entrapment in the polymer matrix such that they cannot migrate out of the polymer under ambient conditions, but this state is harder to preserve at elevated polymer processing temperatures. This review does not consider entrapment immobilisation. 5.5.1 Developing NMBP by immobilisation Some key considerations in developing NMBP by immobilising bioactive compounds are the nature of the polymer backbone and whether immobilisation is needed in the bulk polymer or just on the polymer surface. The nature of the polymer backbone is a significant consideration in designing attachment schemes. If the polymer is essentially inert, such as PE, then reactive functional groups need to be created on the polymer backbone to provide sites for attachment. This step is termed functionalisation of the polymer – the development of functional groups on the polymer backbone – and should be tailored to develop the maximum number of target groups for the desired coupling (immobilisation) chemistry while minimising polymer degradation and side reactions. For polymers which already possess suitable functional groups in the polymer backbone, for example poly(acrylic acid), the coupling chemistry needs to be chosen to target the available groups. Most of the functionalisation and coupling chemistries we will describe are surface centric, but bulk functionalisation can also be achieved with these technologies by treating the polymer as a fine powder and then heat processing, e.g. extruding, moulding or pressing. This will result in the bioactive component being distributed throughout the bulk of the polymer. Polymers can also be bulk modified by dissolution in appropriate solvents, followed by solution modification and then removal of the solvent. However, these solvents may also denature the bioactive compounds of interest, e.g. proteins; proteins can sometimes be protected from denaturation by conjugation with, for example, PEG oligomers prior to dissolution in a solvent. Bulk modification in solution is definitely an option for water-soluble biopolymers such as chitosan, zein and poly(lactic acid). For bulk covalent immobilisation, the active agent will not be able to migrate from the bulk of the polymer to the surface where it will be active, so the Non-migratory bioactive polymers (NMBP) in food packaging 85 applications of non-migratory bulk immobilisation in food packaging are limited. Covalent immobilisation in the bulk may be of interest for constructing food-processing equipment where surface wear is an issue. Many of the technologies used for surface coupling are also applicable to coupling reagents in solution. For surface modification of polymers, it is a good idea to have the polymer in as close to the final container form as possible to prevent surface rearrangement burying the active groups in the bulk polymer during heat processing/forming. Surface modification of a polymer film is the simplest situation. For more complex shapes and processes, such as blow moulding, it may be best to perform the covalent attachment with powdered polymer prior to forming the container. Extruding the active polymer as a thin layer in the appropriate location, e.g. an inner layer in a blow-moulding parison, will minimise wastage of the active agent. Similarly, covalently modified polymer powder could be used in co- extrusion of polymer films to provide an inner layer, but given the typically high cost of the active agent, it may be more economical to attach it only to the surface of the container. For both films and powders, the basic processes of surface immobilisation are the same and the active agent will be attached to the surface of either the individual powder grains or the film. Some organic solvents swell polymers without dissolving them, allowing reagents to penetrate the polymer matrix; polar polymers often swell in water. Polymer swelling allows increased modification of the polymer surface, but may bury some of the active agent in the polymer bulk if the food to be contained does not similarly swell the polymer. Tailoring the solvents used for the reactions can control swelling. Polymer functionalisation For inert polymers, such as PE, the polymer backbone requires functionalisation prior to attaching or generating the bioactive agent of interest. The polymer processing literature contains many examples of this. Some of the simplest methods for laboratory use involve wet chemical treatments of the polymers to oxidise the surface, for example concentrated chromic acid in sulphuric acid, potassium permanganate in concentrated sulphuric acid (Eriksson et al., 1984; Larsson et al., 1979) and potassium hypochlorite in concentrated sulphuric acid (Eriksson et al., 1984). Although these methods are relatively simple and do not require overly complex equipment, the hazardous nature of the reagents makes them undesirable in commercial applications. A recent development ameliorates this problem by utilising a microwave catalysed reaction between solid potassium permanganate and powdered polyolefins (Mallakpour et al., 2001a; Mallakpour et al., 2001b). This process reduces some of the problems inherent with wet chemical methods, but still produces waste water containing high concentrations of KMnO 4 . Wet chemical oxidations introduce various carbonyl groups, predominantly carboxylic acids, aldehydes and ketones, on polymer surfaces. The reaction can be optimised to produce the maximum concentration of the desired carbonyl 86 Novel food packaging techniques function (Eriksson et al., 1984; Holmes-Farley et al., 1985; Rasmussen et al., 1977). Side reactions include incorporation of sulphate groups and surface etching/ablation. Sulphate groups can be removed by nitric acid treatment post- oxidation and surface etching can be controlled by optimising the reaction conditions. For more information, a recent review of polymer surface modification using wet chemical procedures is recommended (Garbassi et al., 1994), as is a second review on the chemical modification of poly(ethylene) surfaces (Bergbreiter, 1994). Wet chemical modifications have numerous safety and environmental concerns that limit their commercial application. More common in commercial applications are physical surface treatments such as flame treatment and corona discharge. Corona discharge involves applying a high voltage (10–40 kV) at a high frequency (1–4 kHz) between a discharge electrode and an earthed roller carrying the film (Robertson, 1993). This oxidises the surface of the film, introducing a range of oxygen and nitrogen functional groups to the polymer backbone. Careful control is required to prevent excessive etching. Flame treatment also produces an oxidised film surface and introduces a range of oxygen and nitrogen functions, but is more difficult to control than corona treatment. Both treatments require specialised equipment, but this equipment is common in polymer processing and converting operations. The disadvantage of both these methods is that it is very difficult to control the exact chemical nature of the functional groups created on the surface of the film, increasing the difficulty of further coupling. It might be possible to control the chemical groups formed in corona discharge treatment by varying the gas composition of the treatment atmosphere, although the typical installation does not have such capabilities. Controlling the treatment atmosphere has been a successful strategy to control the chemical groups created in plasma surface treatment of polymers (Groning et al., 2001; Klapperich et al., 2001; Schroder et al., 2001; Terlingen et al., 1995). The disadvantage of classic plasma processing is that it requires a high vacuum to generate a stable plasma. As such, plasma processing is a batch process, which is not suited to high throughput polymer converting operations. A new development in plasma processing is the APNEP system, an atmospheric pressure plasma treatment system developed by EA Technology Ltd in the UK (Shenton and Stevens, 1999). This has been tested with a range of common polymers and various atmospheric compositions (Shenton et al., 2001; Shenton and Stevens, 1999; Shenton and Stevens, 2001; Shenton et al., 2002). Controlled surface functionalisation should be possible similar to that obtainable through vacuum plasmas. One disadvantage of the APNEP system compared to vacuum plasmas is that the plasma is at a very high temperature and care is needed to prevent thermal degradation of polymers during treatment. This is achieved by placing the films in the downstream afterglow region of the plasma rather than in the plasma itself – the distance from the plasma source is an important variable for this system. The APNEP system was also found to cause greater polymer etching than vacuum plasmas. Non-migratory bioactive polymers (NMBP) in food packaging 87 Plasma treatment technologies, or possibly controlled atmosphere corona discharge treatments, are likely to be the most useful techniques for controlled surface functionalisation of a broad range of polymers for coupling with bioactive compounds. Reviews of physical methods for modification/ functionalisation of polymer surfaces are available (Lane and Hourston, 1993; Ozdemir et al., 1999a; Ozdemir et al., 1999b), although these do not include the novel APNEP technology. One review by Ozdemir and colleagues (1999a) is particularly apt in that it approaches surface functionalisation from the food packaging standpoint, albeit with different intended applications. Polymeric spacers One of the difficulties in attaching bioactive agents to polymeric systems is the necessity of maintaining the conformation and structure of the attached compound. The activity of most bioactive compounds is closely related to their structure and is normally lost if this structure is disrupted. The hydrophobic nature of many common polymers will disrupt the structure of a hydrophilic bioactive compound if they are coupled directly. To prevent this, it may be necessary to use a hydrophilic spacer molecule between the bioactive compound and the hydrophobic polymer backbone. A spacer also helps reduce steric hindrances to the activity of the attached compound (Weetall, 1993). Many different oligomers can be used as spacers, although the one most commonly used is PEG. The main considerations in selecting a spacer are that it does not disrupt the structure of the bioactive compound, it is approved for food contact use and suitable chemistry exists for coupling it to both the polymer backbone and the bioactive compound. PEG is safe, well characterised (Zalipsky and Harris, 1997) and approved for food use (Anon., 2002). It has been used extensively for conjugation with peptides and proteins (Zalipsky and Harris, 1997) and a large range of derivatives are available for conjugation with different functional groups (Anon., 2001b). Methods are also well established for grafting it to polymer backbones, typically by attaching preformed PEG chains of defined molecular weight using various coupling chemistries (Bae et al., 1999; Emoto et al., 1998; Kang et al., 2001; Malmsten et al., 1998; Sofia and Merrill, 1998). PEG is water soluble, allowing coupling in aqueous media and increasing the probability that liquid food products will swell the polymer surface, so increasing the interactions between the attached active agent and the target constituents in the food. Coupling chemistries There are many coupling chemistries available for covalently linking bioactive compounds to polymers and many different types of linkage can be formed. Amide bonds are formed between an amino group (on either the bioactive agent or the polymer) and a carboxylic acid group. Other common linkages are esters and thioesters, formed by interactions between carboxylic acids and alcohols or thiols, respectively. All these groups are common constituents of peptides, proteins and enzymes. The coupling chemistries which have been explored for 88 Novel food packaging techniques polymer conjugation are generally the same as those used for peptide synthesis; texts on peptide synthesis are good sources of information on coupling techniques (Bodanszky, 1993a; Bodanszky, 1993b; Bodanszky and Bodanszky, 1994). The carbodiimide method is a well-established and relatively simple coupling technique (see Fig. 5.5). It can be used in organic solvents or aqueous systems, depending on the carbodiimide chosen. 1,3-Dicyclohexyl carbodiimide (DCC) is typically the carbodiimide of choice in organic solvents (Bodanszky and Bodanszky, 1994), whereas 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, often referred to as WSC or EDC, is the most commonly used carbodiimide for aqueous coupling (Bae et al., 1999; Carraway and Koshland, 1972; Hinder et al., 2002; Kang et al., 2001; Nakajima and Ikada, 1995; Plummer and Bohn, 2002; Valuev et al., 1998). The ideal situation is for carboxylic acid groups to be present on the film and amino functions on the bioactive compound. The carboxyl functions are activated by the carbodiimide; if amines are present within the structure of an activated compound, as with a peptide, then the activated carboxyl groups couple with them spontaneously, leading to inter- and intra-molecular cross-linking. The film is immersed in a buffered solution of carbodiimide to activate the carboxyl functions, removed and gently rinsed, then immersed in a buffered solution containing the bioactive compound to be attached. The exact conditions, e.g. time, temperature and pH, require fine tuning for each individual coupling system. A second common method for coupling bioactive agents to polymers is glutaraldehyde coupling. Glutaraldehyde has long been recognised as an efficient cross-linking agent for use with proteins and other biological molecules (Bigi et al., 2001; Kikuchi et al., 2002; Weissman, 1979) and has been used to immobilise bioactive materials onto polymeric backbones (Appendini and Hotchkiss, 1997; Molday et al., 1975; Soares, 1998). It has also been used clinically for cross-linking biological molecules in the construction of bioprosthetic implants (Bigi et al., 2001). Glutaraldehyde (OHC-CH 2 -CH 2 - CH 2 -CHO) is a bifunctional short-chain aldehyde that reacts with amines. It requires amines on both the support and the bioactive molecule, and forms three- dimensional cross-linked aggregates. A third possibility for coupling reagents are succinimidyl succinate (SS) active esters and their derivatives. These are commercially available (Anon., 2001b) and have been extensively employed for PEG conjugation to peptides and proteins. Succinimide esters react with free amine groups to form stable amide linkages. The most commonly used derivative is the succinimidyl propionic acid ester of PEG (PEG-SPA), which has been used to conjugate PEG with insulin (Caliceti and Veronese, 1999) and human growth hormone antagonist (hGHA) (Olson et al., 1997). The reaction conditions can be modified to suit the system of interest. A succinimidyl ester, N-hydroxy succinimide (NHS), can also be used in an extension of carbodiimide coupling (Hinder et al., 2002; Plummer and Bohn, 2002). Carbodiimide activated carboxylic acids are relatively unstable and may Non-migratory bioactive polymers (NMBP) in food packaging 89 not be suitable for two-step coupling processes, especially if there is a significant delay between the activation and the coupling steps. Instead, the activation can be performed in the presence of NHS, creating a more stable, but still reactive, NHS ester of the carboxylic acid. The NHS-activated polymer film is then immersed in a solution of the amine-containing compound to be coupled and coupling proceeds with the formation of amide linkages. NHS active esters of PEG can also be obtained commercially (Anon., 2001b). 5.6 Applications of polymers with immobilised bioactive compounds Although there are many possible applications of immobilised bioactives in food packaging, only a few have been explored to date. These can be broken into three main applications: in-package processing, antimicrobial packaging/shelf- life extension and intelligent packaging. 5.6.1 In-package processing In-package processing is the most novel form of NMBP packaging. It involves immobilising an enzyme on the surface of the packaging material so as to perform in the package what would otherwise be a processing step prior to packaging. An example is the hydrolysis of naringin, one of the bitter compounds in citrus juices, with immobilised naringinase (a mixture of - rhamnosidase and -glucosidase) (Soares and Hotchkiss, 1998; Soares, 1998). This packaging material was able to reduce significantly the bitter naringin content of grapefruit juice during storage, providing what was perceived as a sweeter product as storage progressed. Another potential application exists for immobilised lactase ( -galactosidase) packaging to produce lactose-reduced or Fig. 5.5 Simplified mechanism of attaching peptides to surface carboxyl functions using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (WSC). R1: ethyl, R2: 3- dimethylaminopropyl, R3: molecule to be attached. Based on the mechanism proposed by Nakajima and Ikada (1995). 90 Novel food packaging techniques lactose-free milk products at a reduced cost compared to current methods. Lactose-free milk products are required for consumers who are lactose- intolerant; these people are unable to consume regular milk products. Only the number of enzymes with potential processing applications limits the range of potential applications. Other examples include the use of hydrolases to inactivate enzymes which limit shelf-life (many of these are currently inactivated with thermal techniques which also cause loss of product ‘freshness’), glucose isomerase to convert glucose to fructose, so increasing the sweetness of products, and cholesterol reductase to reduce the cholesterol content of products. The concepts of lactase and cholesterol reductase immobilised packaging were explored by Pharmacal Biotechnologies in the early 1990s (Brody and Budny, 1995), although no known commercialisation has arisen to date. The basic principle of in-package processing with immobilised enzymes is shown using the example of cholesterol reductase in Figure 5.6. The interior surface of the packaging material contains immobilised cholesterol reductase in contact with the food. The enzyme substrate, in this case cholesterol, contacts the immobilised enzyme due to natural convective currents or diffusion within the product or mixing caused by product agitation during handling and transportation. The substrate is acted on by the enzyme, producing the desired Fig. 5.6 The principle of in-package processing with NMBP, using the example of cholesterol reduction of milk with covalently immobilised cholesterol reductase enzyme. Adapted from Brody and Budny (1995). Non-migratory bioactive polymers (NMBP) in food packaging 91 products, or as in this case, removing the undesirable component. Over storage, the composition of the packaged food changes to that desired by the food processor for optimum consumer acceptability, nutritional content or shelf-life. 5.6.2 Antimicrobial packaging/shelf-life extension Shelf-life extension and prevention of food-borne disease are the two goals of antimicrobial packaging. Considering the later goal, it is clearly important to maintain pathogen levels in food below the level that will cause illness, be it by toxin production or food-borne infection. To this end, it is important to prevent pathogen growth and to inactivate any that may be present in the food. Considering shelf-life extension, destruction and complete growth inhibition is not necessarily important. In order to extend the shelf-life of a product where microbial growth is the limiting factor, it is only necessary to reduce the growth rate or extend the lag phase of the organisms, i.e. to retard microbial growth. It is not necessary totally to inactivate the microbes. Even where microbial growth is not the limiting factor for shelf-life, immobilised enzymes may have a role in extending shelf-life by targeting shelf-life limiting reactions, e.g. oxidation, by removing catalysts or reactants. The primary concern of all food processors should be the safety of the food they produce. A recent review estimated that there were 76 million illnesses, 325,000 hospitalisations and 5000 deaths per year due to food-related illness in the United States (Mead et al., 1999). The role of antimicrobial packaging in preventing food-borne illness has two major aspects; sterilisation or sanitation of packaging materials and prevention of pathogen growth in packaged foods. Although less dire than food poisoning, food spoilage due to microbial action is also a problem, resulting in the loss of large quantities of food. Preventing microbial spoilage may allow food, which would otherwise spoil and be wasted, to be transported to places where it is needed. Several reviews of antimicrobial food packaging have recently been published (Appendini and Hotchkiss, 2002; Collins-Thompson and Cheng-An, 2000; Han, 2000; Vermeiren et al., 2002). Both synthetic and natural compounds have been investigated for attachment to polymers to create non-migratory antimicrobial packaging. Appendini and Hotchkiss (Appendini, 1996; Appendini and Hotchkiss, 1997) investigated the attachment of lysozyme, derived from hen egg white, to poly(vinyl alcohol), nylon and cellulose triacetate (CTA). Although lysozyme was successfully immobilised on all materials, its activity on nylon and poly(vinyl alcohol) was insufficient for commercial use when tested against M. lysodeikticus. Greater activity was retained on CTA films, with significant bacterial retardation observed in trypticase soy broth exposed to the modified CTA. There was still, however, a significant reduction in activity compared to free lysozyme. Activity of the lysozyme film also decreased with repeated usage, indicating that the lysozyme was either inactivated over time/use or migrated out of the film. Another natural antimicrobial system that could be used for antimicrobial packaging is the lactoperoxidase system of milk. The immobilisation of lactase 92 Novel food packaging techniques and glucose oxidase enzymes on nylon pellets has been investigated with the goal of producing hydrogen peroxide to activate the lactoperoxidase system naturally present in milk (Garcia-Garibay et al., 1995). The system investigated was designed for use in a bioreactor, rather than in packaging. The advantage of this system is that milk regulations in many countries prevent the addition of preservatives to milk; activating a natural antimicrobial system present in the milk provides antimicrobial effect without contravening regulations. Lactase and glucose oxidase were immobilised onto nylon pellets using glutaraldehyde coupling with a poly(ethyleneimine) spacer. The system resulted in reductions of 0.5–2 log cycles in the natural microflora of raw milk. Milk samples were exposed to the enzymes for only three minutes and microbial counts were taken 24 hours after exposure (storage at 8oC). Modifying this system for use in a package, with prolonged exposure of the milk to the hydroperoxide, would probably result in greater bacterial inhibition, but might also result in oxidation of milk components leading to undesirable sensory characteristics. A small synthetic antimicrobial peptide has been investigated for non- migratory antimicrobial packaging applications (Appendini, 1999; Appendini and Hotchkiss, 2001; Haynie et al., 1995; Haynie, 1998). The peptide was attached to a PEG grafted PS bead support during peptide synthesis, but this support was not amenable to further processing. A high activity was observed for the immobilised peptide, however, with significant antimicrobial activity against a broad range of microorganisms, gram positive and gram negative bacteria, yeasts and moulds. There is some doubt as to whether the peptide remained bound to the support: some evidence suggested that the PEG spacer was hydrolysing, releasing PEG-peptide conjugates into solution. The conditions that caused the hydrolysis were not investigated. The degree of activity was found to be dependent on the test media; positive activity was found in buffer, trypticase soy broth, apple juice and meat exudate. Further investigation of this system continues, with research focusing on producing a non-migratory antimicrobial film using this peptide. Other enzymes with indirect antimicrobial activity have also been immobilised onto polymers suitable for food packaging. Glucose oxidase was immobilised in conjunction with catalase for use as an oxygen scavenger (de Kruijf et al., 2002; Labuza and Breene, 1989). The glucose oxidase oxidises glucose to produce glucono-delta-lactone and hydrogen peroxide. Hydrogen peroxide could lead to potential undesirable oxidations of food components, so is degraded to water and oxygen by catalase. The net reduction in oxygen content is half a mole per mole of reactions. A review of the mechanism and kinetics of glucose oxidase can be found in the literature (Labuza and Breene, 1989). Alcohol dehydrogenase has also been immobilised to polymer films and can also be used as an oxygen scavenger (Labuza and Breene, 1989). Reduced oxygen concentration inhibits the growth of aerobic microbes, especially yeasts and moulds, however care must be taken that anaerobic, low acid, high moisture conditions do not result, as these may favour the growth of pathogenic Clostridium botulinum. Non-migratory bioactive polymers (NMBP) in food packaging 93 Immobilising suitable enzymes on packaging films could also control carbon dioxide concentration. Non-enzymatic carbon dioxide emitters and absorbers have been developed and commercialised (Labuza and Breene, 1989), although there has been no commercialisation of enzymatic carbon dioxide-reducing systems to date. Other enzymes may be used to produce ethanol, which has well known antimicrobial activity. Commercial ethanol emitters (Labuza and Breene, 1989) are not typically enzyme based, however, normally using encapsulated slow-release ethanol instead. The ethanol vapours released have been found to inhibit microbial growth. In addition to their indirect antimicrobial effect, many enzymes that modify the gaseous atmosphere of packaged products can also extend product shelf-life by inhibiting non-microbial degradative mechanisms. The main application of this is in reduced-oxygen packaging to inhibit the oxidation of food components and prevent resulting negative sensory and nutritional effects. Carbon dioxide control can also be important in extending shelf-life, as in the case of packaged coffee. Enzyme systems may be used to prevent taints and off-flavours by metabolising compounds of concern. Care needs to be taken, however, that the taints inhibited are not indicative of microbial spoilage. Off odours and flavours can be key indicators to consumers that food is spoiled and unfit for consumption; removing these indicators may result in consumption of spoiled, possibly pathogenic, foods. In all cases where indirect enzyme action is used to control microbial growth, and in fact for all immobilised enzyme reactions in food packaging, the by- products of the reaction must be carefully considered. As noted above, glucose oxidase produces hydrogen peroxide, which could cause potentially detrimental oxidations of food components. Other enzymes also produce by-products that may have detrimental effects on the sensory characteristics or shelf-life of the packaged food. Complete understanding of the catalysed reactions is required to ensure undesirable by-products are minimised. 5.6.3 Intelligent packaging Intelligent packaging, defined as packaging systems that monitor the condition of packaged food and communicate information on food quality during transport and storage (de Kruijf et al., 2002), has recently received a lot of attention and is dealt with in detail in other chapters of this text. The following discussion is a brief overview of potential NMBP applications in intelligent packaging. Immobilised enzymes and antibodies are common components of intelligent packaging systems, so NMBPs have many potential applications in this area. A range of different indicators involving immobilised bioactive compounds has been developed, including time-temperature integrators (de Kruijf et al., 2002; Labuza and Breene, 1989), spoilage indicators (Anon, 2001a) and indicators of chemical or other contamination (Woodaman, 2001). Time-temperature integrators (TTIs) based on enzyme catalysed reactions are available commercially (de Kruijf et al., 2002; Labuza and Breene, 1989). Although the 94 Novel food packaging techniques commercial versions do not include NMBP, this is an area in which NMBP could be effective. For microbial spoilage, enzymatic TTIs indicators may be particularly suited since microbial growth depends on enzyme-catalysed reactions. For indicators of microbial or toxicant contamination, there are two main methods by which immobilised bioactive compounds can be used: (i) enzyme catalysed reactions requiring microbial metabolites or contaminant chemicals as substrates, or (ii) immobilised antibodies specific to bacterial metabolites and toxins, or contaminating chemicals. Both the enzymes in (i) and the antibodies in (ii) could be used to develop NMBP which indicates contamination. A final paradigm for the use of immobilised bioactive compounds in intelligent packaging is as detection units on biosensors. The incorporation of biosensor systems in packaging films is an area for future research. Biosensors may allow remote monitoring of package conditions or point-of-sale testing of product condition by interfacing with appropriate electronic devices. These may be useful for both TTIs or for the detection of contaminating bacteria or toxicants. Various systems are in development. 5.7 Future trends Some clear trends and opportunities exist in the field of non-migratory bioactive polymers for food packaging. The use of immobilised enzymes in food packaging is well established and will become more so, leading to in-package enzymatic processing as an extension of the food processing factory. Antimicrobial packaging will become another weapon in the war against foodborne disease and spoilage indicators will help prevent consumption of food that does spoil, leading to fewer food-poisoning incidents. The technology will increasingly involve the combination of biotechnology and materials science leading to a broader range of materials with a bioactive function. Additionally, the understanding of the processes causing food deterioration will increase, as will knowledge of the structure and function of bioactive polymers, enzymes and antibodies, leading to still more opportunities for NMBP in food packaging. 5.8 References ANON. (1999) Grade ‘A’ pasteurized milk ordinance, 1999 revision, Website: http://vm.cfsan.fda.gov/~ear/p-nci.html: US Department of Health and Human Services: Public Health Service and Food and Drug Administration. ANON. (2001a) Plastic wrap detects spoiled food. J. Sci. Ind. Res. 60, 171. ANON. (2001b) Shearwater Corporation Catalog 2001: Polyethylene glycol and derivatives for biomedical applications. Huntsville, AL: Shearwater Corporation. Non-migratory bioactive polymers (NMBP) in food packaging 95 ANON. (2002) Code of Federal Regulations, Title 21, Parts 170-199. http:// www.access.gpo.gov/nara/cfr/waisidx_02/21cfrv3_02.html. Published: 1 Apr. 2002. APPENDINI, P. (1996) Immobilization of Lysozyme on synthetic polymers for application to food packages. Masters Thesis. Ithaca, NY: Cornell University. APPENDINI, P. (1999) Synthetic antimicrobial peptide. Potential application in foods and food packaging materials. PhD Thesis. Ithaca, NY: Cornell University. APPENDINI, P. and HOTCHKISS, J.H. (1997) Immobilisation of lysozyme on food contact polymers as potential anti-microbial films . Packag. Technol. Sci. 10, 271–9. APPENDINI, P. and HOTCHKISS, J.H. (2001) Surface modification of poly(styrene) by attachment of an antimicrobial peptide. J. Appl. Polym. Sci. 81, 609– 16. APPENDINI, P. and HOTCHKISS, J.H. (2002) Review of antimicrobial food packaging. Innovative Food Science and Emerging Technologies 3, 113–26. ARIYAPITIPUN, T., MUSTAPHA, A. and CLARKE, A.D. (2000) Survival of Listeria monocytogenes Scott a on vacuum-packaged raw beef treated with polylactic acid, lactic acid, and nisin. J. Food Prot. 63, 131–6. BACHLER, M.J., STRANDBE, G.W. AND SMILEY, K.L. (1970) Starch conversion by immobilized glucoamylase. Biotechnol. Bioeng. 12, 85. BAE, J.S., SEO, E.J. and KANG, I.K. (1999) Synthesis and characterization of heparinized polyurethanes using plasma glow discharge. Biomaterials 20, 529–37. BERGBREITER, D.E. (1994) Polyethylene surface-chemistry. Prog. Polym. Sci. 19, 529–60. BIGI, A., COJAZZI, G., PANZAVOLTA, S., RUBINI, K. and ROVERI, N. (2001) Mechanical and thermal properties of gelatin films at different degrees of glutaraldehyde crosslinking. Biomaterials 22, 763–68. BODANSZKY, M. (1993a) Peptide chemistry: a practical textbook. Berlin: Springer-Verlag. BODANSZKY, M. (1993b) Principles of peptide synthesis. Berlin: Springer-Verlag. BODANSZKY, M. and BODANSZKY, A. (1994) The practice of peptide synthesis. Berlin: Springer-Verlag. BRACKETT, R.E. (2002) Regulatory perspective on antimicrobials in packaging . A presentation at IFT 2002 Annual Meeting Session #24: Packaging tackles food safety: a look at antimicrobials, 16 June 2002, Anaheim, CA. BRODY, A.L. and BUDNY, J.A. (1995) Enzymes as active packaging agents. Rooney, M.L., (Ed.) Active food packaging pp.174–92. Glasgow: Blackie Academic & Professional. CALICETI, P. and VERONESE, F.M. (1999) Improvement of the physicochemical and biopharmaceutical properties of insulin by poly(ethylene glycol) conjugation. STP Pharma Sci. 9, 107–13. 96 Novel food packaging techniques CARRAWAY, K.L. and KOSHLAND, D.E. (1972) Carbodiimide modification of proteins. Methods Enzymol. 28, 616–23. COLLINS-THOMPSON, D. and CHENG-AN, H. (2000) Chilled storage of foods - packaging with antimicrobial properties. In: Robinson, R.K., Batt, C.A. and Patel, P.D. (Eds.) Encyclopedia of Food Microbiology, pp. 416–20. San Diego, CA: Academic Press. COMA, V., MARTIAL-GROS, A., GARREAU, S., COPINET, A., SALIN, F. and DESCHAMPS, A. (2002) Edible antimicrobial films based on chitosan matrix. J. Food Sci. 67, 1162–9. DE KRUIJF, N., VAN BEEST, M., RIJK, R., SIPILAINEN-MALM, T., LOSADA, P.P. and DE MEULENAER, B. (2002) Active and intelligent packaging: applications and regulatory aspects. Food Addit. Contam. 19, 144–62. DE KRUIJF, N. and RIJK, R. (2003) Legislative issues relating to active and intelligent packaging. In: Ahvenainen, R. (Ed.) Novel Food Packaging Techniques, Cambridge, UK: Woodhead Publishing Ltd. EDWARDS, J.V. and VIGO, T.L. (2001) Bioactive fibers and polymers. Washington, DC: American Chemical Society. EMOTO, K., VAN ALSTINE, J.M. and HARRIS, J.M. (1998) Stability of poly(ethylene glycol) graft coatings. Langmuir 14, 2722–9. ERIKSSON, J.C., GOLANDER, C.G., BASZKIN, A. and TERMINASSIANSARAGA, L. (1984) Characterization of KMnO 4 /H 2 SO 4 oxidized polyethylene surfaces by means of ESCA and Ca-45(2+) adsorption. J. Colloid Interface Sci. 100, 381–92. GAERTNER, H.F. and PUIGSERVER, A.J. (1992) Increased activity and stability of poly(ethylene glycol)-modified Trypsin. Enzyme Microb. Technol. 14, 150–5. GARBASSI, F., MORRA, M. and OCCHIELLO, E. (EDS.) (1994) Polymer surfaces: from physics to technology, pp. 243–73. West Sussex, England: John Wiley and Sons. GARCIA-GARIBAY, M., LUNA-SALAZAR, A. and CASAS, L.T. (1995) Antimicrobial effect of the lactoperoxidase system in milk activated by immobilized enzymes. Food Biotechnol. 9, 157–66. GRONING, P., COEN, M.C. and SCHLAPBACH, L. (2001) Polymers and cold plasmas. Chimia 55, 171–7. HAN, J.H. (2000) Antimicrobial food packaging. Food Technol 54, 56–65. HAYNIE, S.L., CRUM, G.A. and DOELE, B.A. (1995) Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin. Antimicrob. Agents Chemother. 39, 301–7. HAYNIE, S.L. (1998) Antimicrobial composition of a polymer and a peptide forming amphiphilic helices of the magainin-type. USA Patent. 5,847,047. HECKMAN, J.H. and ZIFFER, D.W. (2001) Fathoming food packaging regulation revisited. Food Drug Law J. 56, 179–95. HELANDER, I.M., NURMIAHO-LASSILA, E.L., AHVENAINEN, R., RHOADES, J. and ROLLER, S. (2001) Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. Int. J. Food Microbio. 71, 235–44. Non-migratory bioactive polymers (NMBP) in food packaging 97 HINDER, S.J., CONNELL, S.D., DAVIES, M.C., ROBERTS, C.J., TENDLER, S.J.B. and WILLIAMS, P.M. (2002) Compositional mapping of self-assembled monolayers derivatized within microfluidic networks. Langmuir 18, 3151–8. HOLMES-FARLEY, S.R., REAMEY, R.H., MCCARTHY, T.J., DEUTCH, J. and WHITESIDES, G.M. (1985) Acid-base behavior of carboxylic-acid groups covalently attached at the surface of polyethylene - the usefulness of contact-angle in following the ionization of surface functionality. Langmuir 1, 725–40. HOTCHKISS, J.H. (1995) Safety considerations in active packaging. Rooney, M.L., (Ed.) Active food packaging pp. 238–54. Glasgow: Blackie Academic & Professional. IKINCI, G., SENEL, S., AKINCIBAY, H., KAS, S., ERCIS, S., WILSON, C.G. and HINCAL, A.A. (2002) Effect of chitosan on a periodontal pathogen Porphyromonas gingivalis. Int. J. Pharm. 235, 121–7. JINKINS, R.S. and LEONAS, K.K. (1994) Influence of a polyethylene-glycol treatment on surface, liquid barrier and antibacterial properties. Text. Chem. Color. 26, 25–9. KANG, I.K., SEO, E.J., HUH, M.W. and KIM, K.H. (2001) Interaction of blood components with heparin-immobilized polyurethanes prepared by plasma glow discharge. J. Biomat. Sci.-Polym. E. 12, 1091–8. KATCHALSKI-KATZIR, E. (1993) Immobilized enzymes – learning from past successes and failures. TIBTECH 11, 471–8. KIKUCHI, M., TAGUCHI, T., MATSUMOTO, H.N., TAKAKUDA, K. and TANAKA, J. (2002) Cross-linkage of hydroxyapatite/collagen nano-composite with 3 different reagents. Key Eng. Mater. 218, 449–52. KLAPPERICH, C., PRUITT, L. and KOMVOPOULOS, K. (2001) Chemical and biological characteristics of low-temperature plasma treated ultra-high molecular weight polyethylene for biomedical applications. J. Mater. Sci.-Mater. M. 12, 549–56. LABUZA, T.P. and BREENE, W.M. (1989) Applications of active packaging for improvement of shelf-life and nutritional quality of fresh and extended shelf life foods. J. Food Process. Pres. 13, 1–70. LANE, J.M. and HOURSTON, D.J. (1993) Surface treatments of polyolefins. Prog. Org. Coat. 21, 269–84. LARSSON, R., ERIKSSON, J.C. and OLSSON, P. (1979) Effects of surface localized sulfate groups on the clotting time in plasma. Thromb. Res. 14, 941–52. MABROUK, P.A. (1997) The use of poly(ethylene glycol)-enzymes in nonaqueous enzymology. Harris, J.M. and Zalipsky, S., (Eds.) Poly(Ethylene Glycol): Chemistry and Biological Applications pp. 118–33. ACS Symposium Series #680. Washington, DC: American Chemical Society. MALLAKPOUR, S.E., HAJIPOUR, A.R., MAHDAVIAN, A.R., ZADHOUSH, A. and ALI- HOSSEINI, F. (2001a) Microwave assisted oxidation of polyethylene under solid-state conditions with potassium permanganate. Eur. Polym. J. 37, 1199–206. MALLAKPOUR, S.E., HAJIPOUR, A.R., ZADHOUSH, A. and MAHDAVIAN, A.R. (2001b) 98 Novel food packaging techniques Efficient and novel method for surface oxidation of polypropylene in the solid phase using microwave irradiation. J. Appl. Polym. Sci. 79, 1317–23. MALMSTEN, M., EMOTO, K. and VAN ALSTINE, J.M. (1998) Effect of chain density on inhibition of protein adsorption by poly(ethylene glycol) based coatings. J. Colloid Interface Sci. 202, 507–17. MEAD, P.S., SLUTSKER, L., DIETZ, V., MCCAIG, L.F., BRESEE, J.S., SHAPIRO, C., GRIFFIN, P.M. and TAUXE, R.V. (1999) Food-related illness and death in the United States. Emerg. Infect. Dis. 5, 607–25. MERONI, A. (2000) Active packaging as an opportunity to create package design that reflects the communicational, functional and logistical requirements of food products. Packag. Technol. Sci. 13, 243–8. MOLDAY, R.S., DREYER, W.J., REMBAUM, A. and YEN, S.P.S. (1975) New immunolatex spheres - visual markers of antigens on lymphocytes for scanning electron microscopy. J. Cell Biol. 64, 75–88. MOSBACH, K. (1980) Immobilized enzymes. Trends Biochem. Sci. 5, 1–3. MUSTAPHA, A., ARIYAPITIPUN, T. and CLARKE, A.D. (2002) Survival of Escherichia coli O157:H7 on vacuum-packaged raw beef treated with polylactic acid, lactic acid, and nisin. J. Food Sci. 67, 262–7. NAKAJIMA, N. and IKADA, Y. (1995) Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media. Bioconjugate Chem. 6, 123–30. OH, H.I., KIM, Y.J., CHANG, E.J. and KIM, J.Y. (2001) Antimicrobial characteristics of chitosans against food spoilage microorganisms in liquid media and mayonnaise. Biosci., Biotechnol., Biochem. 65, 2378–83. OLSON, K., GEHANT, R., MUKKU, V., O’CONNELL, K., TOMLINSON, B., TOTPAL, K. and WINKLER, M. (1997) Preparation and characterization of poly(ethylene glycol)ylated human growth hormone antagonist. Harris, J.M. and Zalipsky, S., (Eds.) Poly(ethylene glycol): Chemistry and Biological Applications pp. 170–81. ACS Symposium Series #680. Washinton, DC: American Chemical Society. OZDEMIR, M. and SADIKOGLU, H. (1998) A new and emerging technology: laser- induced surface modification of polymers. Trends Food Sci. Tech. 9, 159– 67. OZDEMIR, M., YURTERI, C.U. and SADIKOGLU, H. (1999a) Physical polymer surface modification methods and applications in food packaging polymers. Crit. Rev. Food Sci. 39, 457–77. OZDEMIR, M., YURTERI, C.U. and SADIKOGLU, H. (1999b) Surface treatment of food packaging polymers by plasma. Food Technol 53, 54–8. PADGETT, T., HAN, I.Y. and DAWSON, P.L. (1998) Incorporation of food-grade antimicrobial compounds into biodegradable packaging films. J. Food Prot. 61, 1330–5. PANZA, J.L., LEJEUNE, K.E., VENKATASUBRAMANIAN, S. and RUSSELL, A.J. (1997) Incorporation of poly(ethylene glycol) proteins into polymers. Harris, J.M. and Zalipsky, S., (Eds.) Poly(Ethylene Glycol): Chemistry and Biological Applications, pp.134–44. ACS Symposium Series #680. Washington, DC: Non-migratory bioactive polymers (NMBP) in food packaging 99 American Chemical Society. PAULK, M., COOKSEY, D., AND WILES, J. (2002) Characterization of Chitosan packaging films with differing levels of % deacetylation. A poster presented at 2002 IFT Annual Meeting 19 June 2002, Anaheim, CA. PLUMMER, S.T. and BOHN, P.W. (2002) Spatial dispersion in electrochemically generated surface composition gradients visualized with covalently bound fluorescent nanospheres. Langmuir 18, 4142–9. RASMUSSEN, J.R., STEDRONSKY, E.R. and WHITESIDES, G.M. (1977) Introduction, modification, and characterization of functional-groups on surface of low- density polyethylene film. J. Am. Chem. Soc. 99, 4736–45. RICHARDSON, T. and HYSLOP, D.B. (1985) Enzymes. Fennema, O.R., (Ed.) Food Chemistry 2nd edn, pp. 371–476. New York: Marcel Dekker. ROBERTSON, G.L. (1993) Food Packaging: Principles and Practise. New York: Marcel Dekker. SCHRODER, K., MEYER-PLATH, A., KELLER, D., BESCH, W., BABUCKE, G. and OHL, A. (2001) Plasma-induced surface functionalization of polymeric biomaterials in ammonia plasma. Contrib. Plasm. Phys. 41, 562–72. SHEARER, A.E.H., PAIK, J.S., HOOVER, D.G., HAYNIE, S.L. and KELLEY, M.J. (2000) Potential of an antibacterial ultraviolet-irradiated nylon film. Biotechnol. Bioeng. 67, 141–6. SHENTON, M.J., LOVELL-HOARE, M.C. and STEVENS, G.C. (2001) Adhesion enhancement of polymer surfaces by atmospheric plasma treatment. J. Phys. D: Appl. Phys. 34, 2754–60. SHENTON, M.J. and STEVENS, G.C. (1999) Investigating the effect of the thermal component of atmospheric plasmas on commodity polymers. Thermochim. Acta 332, 151–60. SHENTON, M.J. and STEVENS, G.C. (2001) Surface modification of polymer surfaces: atmospheric plasma versus vacuum plasma treatments. J. Phys. D: Appl. Phys. 34, 2761–8. SHENTON, M.J., STEVENS, G.C., WRIGHT, N.P. and DUAN, X. (2002) chemical-surface modification of polymers using atmospheric pressure non-equilibrium plasmas and comparisons with vacuum plasmas. J. Polym. Sci., Part A: Polym. Chem. 40, 95–109. SOARES, N.D.F.F. (1998) Bitterness reduction in citrus juice through naringase immobilized into polymer film. PhD Thesis, Cornell University, Ithaca, NY. SOARES, N.D.F.F. and HOTCHKISS, J.H. (1998) Naringinase immobilization in packaging films for reducing naringin concentration in grapefruit juice. J. Food Sci. 63, 61–5. SODHI, R.N.S., SAHI, V.P. and MITTELMAN, M.W. (2001) Application of electron spectroscopy and surface modification techniques in the development of anti-microbial coatings for medical devices. J. Electron Spectrosc. Relat. Phenom. 121, 249–64. SOFIA, S.J. and MERRILL, E.W. (1998) Grafting of PEO to polymer surfaces using electron beam irradiation. J. Biomed. Mater. Res. 40, 153–63. 100 Novel food packaging techniques SUN, Y. and SUN, G. (2002) Durable and regenerable antimicrobial textile materials prepared by a continuous grafting process. J. Appl. Polym. Sci. 84, 1592–9. TAKAI, K., OHTSUKA, T., SENDA, Y., NAKAO, M., YAMAMOTO, K., MATSUOKA, J. and HIRAI, Y. (2002) Antibacterial properties of antimicrobial-finished textile products. Microbiol. Immunol. 46, 75–81. TANABE, T., OKITSU, N., TACHIBANA, A. and YAMAUCHI, K. (2002) Preparation and characterization of keratin-chitosan composite film. Biomaterials 23, 817–25. TERLINGEN, J.G.A., GERRITSEN, H.F.C., HOFFMAN, A.S. and JEN, F.J. (1995) Introduction of functional-groups on polyethylene surfaces by a carbon dioxide plasma treatment. J. Appl. Polym. Sci. 57, 969–82. TEW, G.N., LIU, D.H., CHEN, B., DOERKSEN, R.J., KAPLAN, J., CARROLL, P.J., KLEIN, M.L. and DEGRADO, W.F. ( 2002) De novo design of biomimetic antimicrobial polymers. Proc. Natl. Acad. Sci. U.S.A. 99, 5110–14. TOPCHIEVA, I.N., EFREMOVA, N.V., KHVOROV, N.V. and MAGRETOVA, N. (1995) Synthesis and physicochemical properties of protein conjugates with water-soluble poly(alkylene oxides). Bioconjugate Chemistry 6, 380–8. TSAI, G.J. and SU, W.H. (1999) Antibacterial activity of shrimp chitosan against Escherichia coli. J. Food Prot. 62, 239–43. TSAI, G.J., SU, W.H., CHEN, H.C. and PAN, C.L. (2002) Antimicrobial activity of shrimp chitin and chitosan from different treatments and applications of fish preservation. Fisheries Sci. 68, 170–7. TSAI, G.J., WU, Z.Y. and SU, W.H. (2000) Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. J. Food Prot. 63, 747– 52. VALUEV, I.L., CHUPOV, V.V. and VALUEV, L.I. (1998) Chemical modification of polymers with physiologically active species using water-soluble carbodiimides. Biomaterials 19, 41–3. VERMEIREN, L., DEVLIEGHERE, F., VAN BEEST, M., DE KRUIJF, N. and DEBEVERE, J. (1999) Developments in the active packaging of foods. Trends Food Sci. Tech. 10, 77–86. VERMEIREN, L., DEVLIEGHERE, F. and DEBEVERE, J. (2002) Effectiveness of some recent antimicrobial packaging concepts. Food Addit. Contam. 19, 163– 71. VERONESE, F.M. (2001) Peptide and protein PEGylation: a review of problems and solutions. Biomaterials 22, 405–17. VIGO, T.L. (1999) Antimicrobial polymers and fibers: retrospective and prospective. Abstr. Pap. Am. Chem. 218, 31–CELL. VIGO, T.L. and BRUNO, J.S. (1993) Antimicrobial activity of fabrics containing cross-linked polyols. Abstr. Pap. Am. Chem. 206, 50–CELL. VIGO, T.L. and LEONAS, K.K. (1999) Antimicrobial activity of fabrics containing crosslinked polyethylene glycols. Text. Chem. Color. Am. D. 1, 42–6. WEETALL, H.H. (1993) Preparations of immobilized proteins covalently coupled Non-migratory bioactive polymers (NMBP) in food packaging 101 through silane agents to inorganic support. Appl. Biochem. Biotechnol. 41, 157–88. WEISSMAN, R.C. (1979) A microscopical and mechanical study of the gelation of collagen crosslinked with glutaraldehyde. PhD Thesis, Cornell University. WOODAMAN, J.G., INVENTOR. ASSIGNED TO CALIFORNIA SOUTH PACIFIC INVESTORS, P.C. (2001) Detection of contaminants in food. USA US 6,270,724 B1. YANG, Z., WILLIAMS, D. and RUSSELL, A.J. (1995a) Synthesis of protein-containing polymers in organic solvents. Biotechnol. Bioeng. 45, 10–17. YANG, Z., MESIANO, A.J., VENKATASUBRAMANIAN, S., GROSS, S.H., HARRIS, M.J. and RUSSELL, A.J. (1995b) Activity and stability of enzymes incorporated into acrylic polymers. J. Am. Chem. Soc. 117, 4843–50. YANG, Z., DOMACH, M., AUGER, R., YANG, F.X. and RUSSELL, A.J. (1996) Polyethylene glycol-induced stabilization of subtilisin. Enzyme Microb. Technol. 18, 82–9. ZAKS, A. and KLIBANOV, A.M. (1984) Enzymatic catalysis in organic media at 100-degrees-C. Science 224, 1249–51. ZALIPSKY, S. and HARRIS, J.M. (1997) Introduction to chemistry and biological applications of poly(ethylene glycol). Harris, J.M. and Zalipsky, S., (Eds.) Poly(ethylene glycol): Chemistry and Biological Applications. ACS Symposium Series #680. Washington, DC: American Chemical Society. 102 Novel food packaging techniques