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 conditionsand/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