Non-migratory bioactive polymers(NMBP)in food packaging &1 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. bailin 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 far as pac carin uses go, chitosan activity has been investigated 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" 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
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 104 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
82 Novel food packaging techniques 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 nvlon 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 tchkiss, 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*. 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 significantl 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 Saureus, he activity of the treated nylon increased with increasing temperature, no effect was observed a 4C or 15oC Protein(0. 1% Bovine Serum Albumin) completely inhibited the antimicrobial activity. Shearer and colleagues(2000) compared the ntimicrobial 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
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/cm2 . 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