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 yeastdeveloped (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-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 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 gram￾positive bacteria, fungi and yeasts. 80 Novel food packaging techniques