5 Non-migratory bioactive polymers (NMBP)in food packaging M. D. Steven and J. H. Hotchkiss. Cornell University, USA 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 ponent does not migrate out of the polymer system into the surrounding
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
72 Novel food packaging techniques Food Food Fig. 5.1 Simplified visual comparison of (a) non-migratory and (b)migratory bioactive packaging. Adapted from Han(2000) medium(see Fig. 5. 1). Typically this is achieved through covalent attachment of he 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 in limitations, in application or utility, and frequently with an increase in 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 gulatory 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 Technical benefits nical 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 chardon and Hyslop, 1985). Conjugation to polymer supports has been shown to enhance dramatically the stability of these molecules. Topchieva and
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
Non-migratory bioactive polymers(NMBP)in food packaging 73 0 80 Fig 5.2 Activity of PEG-conjugated chymotrypsin and native chymotrypsin held at 45C. Activity is expressed in percent relative to the initial activity of each enzyme preparation. Adapted from Topchieva et al.(1995) 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 200C for 30 minutes and when autoclaved at 121C 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 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
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
74 Novel food packaging techniques Innoculum level PS-PEG-Peptide 5505.56.06.57.0 ph Fig. 5.3 Antimicrobial activity of a small synthetic peptide against E coli 0157: H7 in 0. IM 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) 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
(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 ChengAn, 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
Non-migratory bioactive polymers(NMBP)in food packaging 75 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 Kruif 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 Kruif et al, 2002, de Kruif 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, al though 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 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 ckaging 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
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