76 Novel food packaging techniques negligible, negating food additive classification of that substance for that pecific 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 proces 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 ttached 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 fresl products, enhancing the acceptability and shelf-life of minimally processed foods
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
Non-migratory bioactive polymers(NMBP)in food packaging 77 5.2. 4 The food processor's perspective From the perspective of the food processor, NMBP would have several Ivantages. 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 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 ackaging 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 ckaging. With viscous liquids, the high viscosity makes it unlikely that ther 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
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
78 Novel food packaging techniques nd/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 53.2 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 bour 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 mmobilised peptide concentrations of 4umol/ml in growth media. In the immobilisation of res(1998)found that the enzy 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 zyme 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
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 4mol/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
Non-migratory bioactive polymers(NMBP)in food packaging 79 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 readil 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 nherent bioactivity(Oh et al, 2001; Ozdemir and Sadikoglu, 1998; Shearer ef al., 2000; Vigo, 1999, Vigo and Leonas, 1999)and new ones are currently being
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
80 Novel food packaging developed (Tew et al., 2002). Most examples of inherently bioactive polymer evolve 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-B-(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 MgCl2 in the media and resulted in increased uptake of a hydrophobic probe (1-M 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", reduced activity hitosan 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 yeast
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 MgCl2 in the media and resulted in increased uptake of a hydrophobic probe (1-Nphenylnaphthylamine) 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 Mg2+, 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 grampositive bacteria, fungi and yeasts. 80 Novel food packaging techniques