Active packaging and colour control: the case of fruit and vegetables 423 (PPO). But anthocyanins are not direct substrates for PPO, which catalyses the hydroxylation of monophenols to o-diphenols(cresolase activity EC 1. 14. 18. 1)and the oxidation of o-diphenols to o-quinones(catecholase activity EC 1.10.3.1) Catecholases were considered as the main PPO enzymes responsible for browning in fruit and vegetables. These o-quinones are very reactive molecules that rapidly condense by combining with amino or sulfhidril groups of proteins and with reducing sugars, producing different brown, black or red polymers of high molecular weight and unknown structure known as melanins (artes et al, 1998 ) In contrast to the ethylene effect on chlorophyll, anthocyanin synthesis and ethylene production seem to be correlated. In fact, red cherries stored in air reached high ethylene levels and the highest anthocyanin content by the end of cold storage(Remon et al., 2000 The increase in pH and decrease in titratable acidity induced by high CO during CA storage of fruit and vegetables have a strong effect in anthocyanin expression and stability. The red flavylium cation(AH+) remains stable only in acidic conditions. Changes in anthocyanin stability can result from nucleophilic attacks by water molecules on the anthocyanin molecule to form a colourless pseudobase, hemiacetal, or carbinol. The flavylium form can be restored by acidification. The colourless carbinol can form chalcone(a yellow pigment) by the opening of the ring structure. As pH increases above 4, a blue quinonoidal base is formed. Increase in pH above 7 can result in the loss of a proton from the hydroxyl group to form a second quinonoidal base(holcroft and Kader, 1999b) In addition, these authors reported that phenylalanine ammonia lyase(PAL, EC 4.3.1.5)and flavonoid glucosyltransferase(GT, EC 2. 4.1.28), two key enzymes in the synthetic pathway of anthocyanins in strawberry, were adversely affected by high CO2 levels during cold storage On the other hand, it has been demonstrated that the degrading effect of vitamin C on anthocyanin stability leads to undesirable colour changes in model olution and in natural pomegranate juice systems(Marti et al., 2001). Exposure to light and heat also induced these degrading reactions. However, glucosylation provides protection against photodegradation and the formation of molecular copigmentation complexes and ion-pairs lowered the degradation thocyanins(Brouillard et al, 1997) 20.4.2 Browning Browning is the result of a chain of reactions that very often occurs in fruit and vegetables. The first step of that process takes place in the vacuole and it is the deamination of the amino acid phenylalanine by PAL. The product of that reaction is the cinnamic acid which is hydroxy lated into various phenolic compounds. When O2 is present, the PPO located in the cytoplasm(plastids) oxidises the compounds to o-quinones, which polymerise into brown compounds (Siriphanich and Kader, 1985). The relationship between PPO and browning was reported when it was found that CO2 competitively inhibited PPO activity in mushrooms retaining their colour, although at high concentrations increasing browning(Murr and Morris, 1974)(PPO). But anthocyanins are not direct substrates for PPO, which catalyses the hydroxylation of monophenols to o-diphenols (cresolase activity EC 1.14.18.1) and the oxidation of o-diphenols to o-quinones (catecholase activity EC 1.10.3.1). Catecholases were considered asthe main PPO enzymes responsible for browning in fruit and vegetables. These o-quinones are very reactive molecules that rapidly condense by combining with amino or sulfhidril groups of proteins and with reducing sugars, producing different brown, black or red polymers of high molecular weight and unknown structure known as melanines (Arte´s et al., 1998). In contrast to the ethylene effect on chlorophyll, anthocyanin synthesis and ethylene production seem to be correlated. In fact, red cherries stored in air reached high ethylene levels and the highest anthocyanin content by the end of cold storage (Remo´n et al., 2000). The increase in pH and decrease in titratable acidity induced by high CO2 during CA storage of fruit and vegetables have a strong effect in anthocyanin expression and stability. The red flavylium cation (AH+) remains stable only in acidic conditions. Changes in anthocyanin stability can result from nucleophilic attacks by water molecules on the anthocyanin molecule to form a colourless pseudobase, hemiacetal, or carbinol. The flavylium form can be restored by acidification. The colourless carbinol can form chalcone (a yellow pigment) by the opening of the ring structure. As pH increases above 4, a blue quinonoidal base is formed. Increase in pH above 7 can result in the loss of a proton from the hydroxyl group to form a second quinonoidal base (Holcroft and Kader, 1999b). In addition, these authors reported that phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and flavonoid glucosyltransferase (GT, EC 2.4.1.28), two key enzymes in the synthetic pathway of anthocyanins in strawberry, were adversely affected by high CO2 levels during cold storage. On the other hand, it has been demonstrated that the degrading effect of vitamin C on anthocyanin stability leads to undesirable colour changes in model solution and in natural pomegranate juice systems (Martı´ et al., 2001). Exposure to light and heat also induced these degrading reactions. However, glucosylation provides protection against photodegradation and the formation of intermolecular copigmentation complexes and ion-pairs lowered the degradation of anthocyanins (Brouillard et al., 1997). 20.4.2 Browning Browning is the result of a chain of reactions that very often occurs in fruit and vegetables. The first step of that process takes place in the vacuole and it is the deamination of the amino acid phenylalanine by PAL. The product of that reaction is the cinnamic acid which is hydroxylated into various phenolic compounds. When O2 is present, the PPO located in the cytoplasm (plastids) oxidises the compounds to o-quinones, which polymerise into brown compounds (Siriphanich and Kader, 1985). The relationship between PPO and browning was reported when it was found that CO2 competitively inhibited PPO activity in mushrooms retaining their colour, although at high concentrations increasing browning (Murr and Morris, 1974). Active packaging and colour control: the case of fruit and vegetables 423