20.1 Introduction Consumer satisfaction is related to fresh product quality. This quality is generally associated with visual appearance, colour being one of the most important aspects in the consumer’s purchase decision. The association of certain colours with the acceptance of fruits and vegetables begins early and is maintained through life. For instance, when the red colour of fruit is enhanced, the perceived sweetness level increases. Colour is normally used to determine acceptable limits for a given grade of product and to define colour tolerances for both harvest and trade. Combined with other characteristics it can be used to establish indices of maturity, enabling us to know whether a commodity can be harvested and to predict postharvest life of the product. For this reason colour requirements are more and more prevalent in retailer’s specifications. A knowledge of fruit and vegetable pigment composition allows us to evaluate the input of postharvest treatments on colour and quality. In fresh as well as in minimally processed products it is crucial to know the main factors affecting pigment stability as well as the main changes associated with processing. Analysing pigment composition of fruit and vegetables and their derivatives is important for optimising postharvest treatments during harvest, handling, storage and distribution. In fact, lowering O 2 and increasing CO 2 around fruit and vegetables by using controlled atmosphere (CA) or active or passive modified atmosphere packaging (MAP) techniques is commonly a good method for keeping colour stability. On the other hand, one of the main problems that reduces shelf-life of minimal processed fruit and vegetables is the enzymatic browning that occurs on the cut surface area. In this review, an update on the main tools for controlling colour changes is given. To prevent adverse 20 Active packaging and colour control: the case of fruit and vegetables F. Arte′s Calero, Technical University of Cartagena, Spain and P. A. Go′mez, National Institute for Agricultural Technology, Argentina changes, physical treatments, specially MAP to minimise enzymatic activity as well as combinations with antibrowning agents are considered. 20.2 Colour changes and stability in fruit and vegetables The colour of fruit and vegetables is a direct consequence of their natural pigment composition resulting mainly from three families of pigments, chlorophylls and carotenoids, located in the chloroplasts and chromoplasts respectively, and the water-soluble phenolic compounds anthocyanins, flavonols and proanthocyanins, located in the vacuole. Betalains (e.g. betacyanins and betaxanthins) are the fourth family of plant pigments and are responsible for the red and yellow colours that occur only rarely. Chlorophylls and their derivatives are responsible for green, blue green and olive brown colours, while carotenoids are responsible for red-yellow colours. Anthocyanins are responsible for orange, red, blue, and purple and black, and intermediate colours. It is very useful to know the composition of fruit and vegetable pigments in order to evaluate the possible incidence of postharvest treatments for keeping colour and quality and extending their shelf-life, as well as that of their derived products (Arte′s et al., 2002c; Kidmose et al., 2002; Lancaster et al., 1997). During ripening chloroplasts are gradually replaced by chromoplasts containing only carotenoids, although exceptionally in some fruits, such as avocado, chlorophyll is retained in the pulp of the ripe fruit. However, in most fruits carotenoids become unmasked when chlorophyll disappears upon ripening, and usually this is accompanied by a marked biosynthesis of carotenoids. In many fruits (apple, apricot, artichoke, asparagus, blackberry, blueberry, red-carrot, cherry, cranberry, eggplant, fig, grape, red-lettuce, nectarine, olive, red-onion, ‘Sanguine’ orange, peach, pear, plum, pomegranate, red-skinned potato, radish, raspberry, red and black-currant, purple sweet potato, strawberry, etc.) ripening is associated with an intense anthocyanins biosynthesis. Although all these colour and pigment composition changing processes occur at the same time, very different biochemical pathways are involved for each class of pigment (Arte′s et al., 2002c). External colour is also influenced by physical factors, such as the presence of waxes and geometry of the fruit surface. That is the reason why colorimeters work better for liquids than they do for whole fruits and vegetables. The key problem that prevents accurate colour measurement of them is that they have non-uniform surfaces. This has a pronounced effect on how light and colour are reflected and perceived. For example, the colour measurement of a bean depends on where on the curvature of the bean’s surface the measurement is made. If the angle of measuring is different from reading to reading, the quantitative colour reading will be different. If significant texture or granulation is present on the sample’s surface, some light coming from the equipment may be scattered at different angles and escape detection. To compensate for this problem, specific colorimeters have been constructed using a spherical geometry that diffusely Active packaging and colour control: the case of fruit and vegetables 417 illuminates samples, eliminating the directionality of the light (Marsili, 1996). Placing the head-reader of the apparatus on the skin of the fruit or vegetable is preferable to measuring at a distance. From 200 measurements on ten ‘Golden Delicious’ apples, measuring at a distance of 4mm from the fruit, produced higher standard deviations in colour parameters than placing the device on the apple (Madieta, 2002). 20.3 Colour measurement Internal and external colour can be both subjectively and objectively determined, in the latter case employing accurate devices. For determining pigment composition and defining colour quality indices in fruit and vegetables some methods are currently available, including the use of colour charts and chromatographic (HPLC, TLC) and spectrophotometric (UV-vis, colourimetry, etc.) analytical techniques. In the past few years, there has been a trend to use colorimetric rather than chemical analysis of pigment for describing colour changes and characterisation. Tristimulus colorimetric measurements are quicker and cheaper than conventional methods (Francis, 1969) and, overall, they are of a non-destructive nature. Colour is monitored in a three-dimensional colour space in terms of the chromatic colour coordinates L* (lightness), a* and b*, based on the CIELAB colour measurement system (Commission Internationale de l’Eclairage – International Commission on Illumination, CIE, 1986). In fact the CIE specified two colour spaces; one of these was intended for use with self-luminous colours and the other for use with surface colours. These notes are principally concerned with the latter known as CIE 1976 (L* a* b*) colour space or CIELAB (McGuire, 1992). The quantification of tristimulus data is based upon trigonometric functions. These coordinates, after a correct manipulation, provide an indication of several aspects of colour. Values of a*/b* ratio have been considered a good indicator of changes in ripening in tomatoes and citrus (Arias et al., 2000; Arte′s and Escriche, 1994; Arte′s et al., 2000b). A more accurate measurement of colour can be obtained indicating that angle, named hue angle (ho arctg b*/a*), which represents the basic tint of a colour, and chroma [(a* 2 b* 2 ) 1/2 ], an index analogous to colour saturation or intensity (McGuire, 1992). On average, the human eye perceives hue differences first, chroma or saturation differences second, and lightness/darkness last (Marsili, 1996). Hue index is adequate to predict colour when pigment degradation has taken place. It could be used to follow dilution, heat effects, browning, etc. The analysis of colour is used in those cases to determine the efficacy of postharvest treatments, including packaging, storage and distribution. However, there are conflicting reports in the literature on the correlation between colour measurements and pigment composition. For example, tristimulus colour measurements did not correlate well with changes in pigment composition of several apple cultivars (Lister, 1994). The -carotene pigment, an important 418 Novel food packaging techniques nutritional component as a precursor of vitamin A and the main carotenoid in green leafy vegetables and responsible for the orange colour in fruit and vegetables, was one of the first studied when trying to find a relationship between pigment content and colour (Francis, 1969). The applicability of using skin colour measurements to predict changes in pigment composition was investigated by analysing a wide range of fruit and vegetables. There were linear relationships between hue and anthocyanin concentration and between L* and log of chlorophyll concentration. However, there was not a unique linear combination of pigments that gave a unique point in the colour space and, at the same time, a given set of colour coordinates could be achieved by many combinations of pigments (Lancaster et al., 1997). Colour measurement in cranberry products is a good example of the interrelation between colour and pigment. The colour of cranberry juice is due to four anthocyanin pigments. There are other minor red pigments as well as six yellow flavonoid pigments but their contributions are less important. In fresh juice fruits, where pigments are homogeneously distributed, the relationship is stronger than for the whole fruit, where pigments are unevenly located in the cell layers below the epidermis (Francis, 1969). A recent study revealed that colour parameters were not good estimators of anthocyanin levels in raspberry, a highly perishable fruit with a storage life limited by decay and darkening of the typical red colour (Haffner et al., 2002). However, it was found that values of a*/b* ratio were well related with changes in lycopene (the predominant carotenoid) content in tomatoes (Arias et al., 2000). This agrees with cautions given in previous reports for interpreting changes in colour coordinates as simple changes in pigment composition (Lancaster et al., 1997). It could be concluded that there is a wide range in the degree of correlation between colour measurements and pigment composition. In order to find a high correlation each pigment would have to be carefully weighted for its contribution to the colour. For precise predictions, colour values should be checked against a chemical method to make sure that changes in colour are actually due to these pigments. 20.4 Process of colour change The structure of chlorophyll present in fruits and vegetables is affected during development, ripening, and senescence, and throughout postharvest treatments, with a consequent effect on external and internal colour. During fruit ripening and leaf senescence chlorophyll catabolism takes place. In fact chlorophyll degradation is a normal process of the ageing phenomenon in leafy vegetables and occurs to provide energy for the senescing leaves (O’Hare and Wong, 2000). In this way, because leaves are still alive after harvest and continue to respire using energy in the process, chlorophyll is metabolised to maintain life. Active packaging and colour control: the case of fruit and vegetables 419 Colour change is primarily related to a reduction in the amount of chlorophyll, which highlights other pigments such as carotenoids and anthocyanins. During fruit ripening the chlorophyll usually disappears due to chloroplast degeneration to gerontoplast. In leaves the chloroplasts commonly disintegrate but some of them remain, masking the yellow carotenoid colour. However, in ripe fruits chloroplasts degenerate into chromoplasts, con- comitantly with a massive biosynthesis of carotenoids (Matile et al., 1997; Ho¨rtensteiner, 1999). This change from chloroplast to chromoplast is particularly important in the case of fruits called carotenogenic (e.g. pepper, tomato, orange and persimmon), characterised by this extensive new synthesis of carotenoids, usually accompanied by a change in the carotenoid profile of the fruit (Arte′s et al., 2002c). In climacteric fruits, the maximum degradation of chlorophyll takes place during the climacteric rise, although generally slight quantities of chlorophyll are always present in the internal tissues. It has been found in apples and pears that degradation of chlorophyll could be mainly due to hydrolytic activity of chlorophyllase enzyme (EC 3.1.1.14) that transforms chlorophyll into phytol and porphyrin and the resultant chlorophyllide has no effect on colour changes. However, this effect was not found in tomatoes and disintegration of chloroplast membranes occurs before the loss of green colour (Pantastico, 1979). Pigment changes during tomato ripening imply a loss of chlorophyll and an accumulation of lycopene. If ripening proceeds under sub-optimal conditions for lycopene synthesis, -carotene accumulates resulting in yellow fruit (Shewfelt et al., 1988). As tomatoes turn from green to red, changes in the L*a*b* parameters during ripening are characterised by a decrease in hue and a concomitant increase in chroma. Non-climacteric fruits do not ripen off the tree and should be picked when fully ripe to ensure their best flavour. During ripening of non-climacteric fruits like citrus or sweet peppers, the process of natural colour break from green to the typically ripe orange/yellow/red is called degreening and takes place very gradually. During degreening of citrus the loss of chlorophyll accumulated into the chromoplasts of the epidermis (flavedo) and vesicles and the concomitant manifestation and new biosynthesis of carotenoids generally occur very slowly (Eaks, 1977). Shippers usually accelerate the degreening process of harvested citrus or sweet peppers (Fig. 20.1) both to advance the marketing period, when prices are higher, and to make fruits more attractive to consumers. The industrial technique commonly consists of applying low concentrations (5–50 ppm) of exogenous ethylene at 18–24oC and 90–95 % RH for two to four days (Arte′s et al., 2000b; Go′mez et al., 2002). Yellowing of green minimally processed products is not appealing to consumers and has a negative effect on sales of the product. It has been demonstrated that colour change from bright green to brown in fresh as well as in minimally fresh processed green vegetables is related to the presence of pheophytin, formed when chlorophyll loses its bound magnesium atom, which is substituted by hydrogen (Schwartz and von Elbe, 1983). More than 50% 420 Novel food packaging techniques conversion of the chlorophyll to pheophytin can occur before a change of colour from bright green to olive brown could be observed (Lau et al., 2000). The rate of chlorophyll degradation can be lowered by several means. However, a combination of them is more effective than one single method. The most important tool is chilling although there is a limit to temperature because some fruit and vegetables are susceptible to damage caused by low temperatures below their freezing point, suffering chilling injuries commonly accompanied by undesirable colour changes. Ethylene greatly accelerates chlorophyll degradation. Usually, leafy vegetables do not produce much ethylene, but can be affected from ethylene coming from other sources. The inclusion of ethylene scavengers within packages containing these vegetables could provide protection against ethylene action (O’ ′ Hare and Wong, 2000). Operations involved in fresh processing, like cutting, grating or peeling, stimulate ethylene biosynthesis that could cause physiological disorders, lowering the quality of the products. Some changes affecting colour include accumulation of phenolic compounds in carrots, red discoloration in chicory and endive, or russet spotting in lettuce (Arte′s, 2000b; Van de Velde and Hendrickx, 2001; Verlinden et al., 2001). It has been reported that ethylene produced during cutting of fresh processed spinach notably accelerates the loss of chlorophyll and damage is proportional to the ethylene level reached (Abe and Watada, 1991). Also celery sticks stored in atmospheres where ethylene is present showed a decrease in hue (Fig. 20.2) as colour changed from dark green to yellowish-green (Arte′s et al., 2002b). On the other Fig. 20.1 Changes in L* and a* parameters of bell peppers with 65% and 85% of initial red colour degreened with 50ppm c 2 H 4 during 2 days at 18oC followed by 3 days at 7oC in air (Go′mez et al., 2002). Active packaging and colour control: the case of fruit and vegetables 421 hand, antioxidants are related to chlorophyll retention in leafy products. Two antioxidants commonly present in fruits and vegetables are ascorbic acid and - carotene, which protect chlorophyll by inhibiting the reactions that degrade it, retarding yellowing (Schwartz and von Elbe, 1983). 20.4.1 Anthocyanin degradation Anthocyanins are very unstable pigments, particularly once removed from their natural environment and the protection provided by co-pigmentation, leading to unattractive yellowish and brownish pigments. This is particularly evident when minimal fresh processed products are prepared. When conditioning fruit and vegetables by techniques like peeling, cutting, slicing, etc., cell membranes are disrupted, allowing the mixing of phenolic substrates located in the vacuole and specific polyphenol oxidases enzymes (PPO; EC 1.14.18.1) associated to cell membranes, (mainly in the plastids). Washing the product immediately after cutting removes sugars and other substrates at the cut surfaces minimising reactions responsible for changes in colour and nutritional quality. It is well known that colour due to anthocyanins is particularly degraded by the enzymic hydrolysis in harvested products as recently reviewed (Arte′s et al., 2002c). Anthocyanins are oxidised in the vacuole of the plant cells in the presence of molecular O 2 and under appropriate conditions of pH, temperature and water activity, by the action of the enzyme tyrosinase (EC 1.10.3.1) or polyphenol oxidase Fig. 20.2 Colour changes (oHue) of celery sticks stored for 14 days in air and in controlled atmosphere (5% CO 2 + 5% O 2 ) free of ethylene at 0 and 5oC (Arte′s et al,. 2002b). 422 Novel food packaging techniques (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 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 CO 2 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 CO 2 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 (Mart?′ 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 O 2 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 CO 2 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 Peroxidases (POD; EC 1.11.1.7.) could also be involved in browning although to a lesser extent, due to low availability of H 2 O 2 within the plant cell (Arte′s et al., 1998; Sa′nchez-Ferrer et al., 1995). Lipoxygenase (EC 1.13.11) and lipase (EC 3.1.1.3) have been considered as the main causes for the breakdown of some vegetables like cucumbers. The reaction between lipoxygenase and lipids substrates generates hydroperoxides that are related to senescence and scald induction. Fatty acid radicals induced by peroxidase can react with cell components leading to further breakdown. Particularly, bleaching of -carotene and chlorophyll a occurs as a consequence of lipoxygenase catalysed reactions. Browning could easily be evaluated by colorimetric methods. For example, visual scores for browning of cut lettuce were well correlated with hue (values decreased as browning occurred) and a*, although correlation with b* was lower while it was not significant with L*, Hue angle values decreased as browning occurred (Peiser et al., 1998). 20.5 Colour stability and MAP To remain competitive in the fruit and vegetable market, suppliers must offer products with an optimal overall quality. Thus, the entire chain from producers and processors to retailers must be increasingly sensitive to consumer requirements, particularly as they relate to colour. In fact, perception of sweetness, sourness and flavour intensity was highly correlated to skin colour as has been reported for sweet cherries; full dark red cherries, measured by both visual and colourimetry, had higher consumer acceptance than full bright red (Crisosto et al., 2002). Atmospheres with reduced O 2 and/or elevated CO 2 concentrations are known to extend the storage life of fruit and vegetables. MAP can bring the lowering of respiratory activity and ethylene production, delay in ripening and softening, limiting weight losses and reduced incidence of physiological disorders and decay-causing pathogens (Ahvenainen, 1996; Arte′s, 2000b). As MAP slows the rate at which energy reserves are used it can be applied in combination with chilling storage for improving shelf-life of fruit and vegetables. At the same time, MAP affects biochemical reactions related to pigment synthesis and degradation (Arte′s, 1993 and 2000a), although responses to MAP depend on the kind of fruit or vegetable. In addition to this, the effect of respiratory gases on the metabolic behaviour of plant materials depends on temperature of application due to its influence on solubility of these gases. The effects of low O 2 and/or high CO 2 on colour changes in packaged fruit and vegetables will be examined using several examples recently reported. 20.5.1 Low oxygen effects It has been observed that the activity of tyrosinase, responsible for mushroom browning, is dependent on O 2 concentration. MAP induced higher L* values and 424 Novel food packaging techniques lowered the difference between ideal mushroom target and sample than those observed for mushrooms stored in conventional packages (non-MAP). The improved colour might also be due to lower microbial growth resulting from low O 2 (Roy et al., 1996). Strawberries stored under low O 2 2kPa showed a better colour, high anthocyanin concentration and organic acids content than those stored in air. Fruits became darker red and accumulated anthocyanin, although O 2 was not as effective at high CO 2 levels in reducing decay (Holcroft and Kader, 1999b). Freshly harvested white asparagus spears stored in air or in CA having increased O 2 concentrations (1 to 15kPa) showed a concomitant increment in anthocyanin content, resulting in an intense purple colour of the tips. Hue values were lower than those at harvest, with a decline in L* values. The lowest anthocyanin accumulation was observed at the tips of the spears stored under the lowest O 2 level (Siomos et al., 2000). Red discolouration of chicory after seven days of storage at 12oC was strongly reduced from 90% in air to 35% under 2kPa O 2 and 0kPa CO 2 (Verlinden et al., 2001). Fresh processed potato slices stored in MAP with low O 2 showed a better colour retention when the O 2 level was lowered from 3.5 to 1.4kPa, probably due to reduction of oxidase activity such as PPO, ascorbic acid oxidase (AAO, EC 1.10.3.3) and glycolic acid oxidase (GAO, EC 1.1.3.15). Slices in air showed a decrease in L* compared to MAP. It was advantageous to have almost no initial O 2 within packages by flushing N 2 . This active MAP was very important for the keeping quality of slices, taking into consideration that residual O 2 in the packages was enough to prevent anaerobiose (Gunes and Lee, 1997). To prevent browning of minimally processed potatoes, dipping in some chemical agents was essential because MAP alone did not avoid this disorder. Browning is closely related with O 2 and CO 2 levels in the package and O 2 must be decreased to an acceptable minimum level as soon as possible, it being advantageous to have almost no O 2 initially within packages (Gunes and Lee, 1997). The intensity of browning of ready-to-eat apples depends on the atmosphere composition. Apple cubes in MAP were efficiently preserved from browning and showed the lowest colour losses when initially displacing O 2 by injecting 100kPa N 2 and a film with low O 2 permeability was used. This atmosphere was the main factor affecting lightness, and L* changes occurred four times more slowly than when O 2 was about 2kPa or when medium O 2 permeability films were used (Soliva-Fortuny et al., 2001). It has been suggested that O 2 levels greater than 21kPa may influence the postharvest life of intact and fresh processed fruit and vegetables and PPO may be substrate-inhibited by high O 2 levels (Day, 1994). Superatmospheric O 2 could have an effect on respiratory activity and ethylene synthesis and action, although response depends on the commodity, ripening stage, O 2 level, length of storage and temperature. Levels of CO 2 and C 2 H 4 should also be considered. When focusing on pigment changes, Kader and Ben-Yehoshua (2000) reported Active packaging and colour control: the case of fruit and vegetables 425 that 40–50kPa O 2 accelerated ripening of tomatoes, with 60–100kPa stimulating synthesis of lycopene in rin varieties. 40–80kPa O 2 improved the colour of endocarp and juice of orange cultivars, increasing the deepest of orange colour. However, an undesirable change from yellow to orange has been observed in grapefruit. An increase in the red colour of flesh and juice of blood-orange ‘Sanguine’ cv has been related to anthocyanin synthesis. Superatmospheric O 2 was particularly effective in inhibiting browning of different fresh processed vegetables including mixed salads and chicory endive (Jacxsens et al., 2001, Allende et al., 2002). However, ‘Bartlett’ pear slices kept in 40, 60 or 80kPa O 2 exhibited similar severity of cut surface browning during storage at 10oC (Gorny et al., 2002). 20.5.2 High carbon dioxide effects Tolerance to high CO 2 is commonly reduced in fruit and vegetables. As an example, levels of 3–5kPa induced superficial scald and abnormal flavour in citrus during cold storage (Arte′s, 1995; Kader, 1990). For this reason, the use of CO 2 for keeping colour in plant materials must be particularly adapted for each species. Exposure of mango fruits to 50kPa CO 2 at 40–44oC for 160 minutes used as a quarantine treatment affected colour during cold storage. After 20 days at 10oC control fruits showed a higher decrease in hue than those heated under CO 2 enriched CA. Decrease in hue was a good estimator of the green to yellow colour turn. Yellowing increased in the absence of CO 2 and chroma (colour intensity) decreased (Ortega-Zaleta and Yahia, 2000). MAP stored peaches for 21 days at 2oC, with equilibrium CO 2 level about 20kPa, showed a residual effect of high CO 2 during the subsequent three days at 20oC. Colour development was slow and L* and chroma ground values were maintained as at harvest (Ferna′ndez-Trujillo et al., 1998). However, very high CO 2 levels (73kPa in MAP) destabilised cyanidin derivatives in the skin of ‘Starkimson’ apples (Remo′n et al., 2000). Despite the benefits of CO 2 enriched atmospheres in controlling postharvest decay, anthocyanin concentration is affected and more particularly in the internal tissues. During storage of strawberries in air plus 10 or 20kPa CO 2 , skin chroma increased with time and was not as affected by gas composition as flesh colour which turned pale. The hue of berries held in air was lower than that in CA and chroma under 10kPa CO 2 was slightly higher than under 20kPa CO 2 (Holcroft and Kader, 1999a). In contrast to the effect observed in strawberries, CO 2 did not affect anthocyanin in pomegranate fruit. Juice red colour increased in intensity during postharvest storage (8 weeks, 5oC) when 5kPa CO 2 was combined with 5kPa O 2 while air-stored fruits showed a slight pale red (low a* value) colour (Arte′s et al., 1996). Chroma of the skin was better maintained when fruits were stored in air plus 10kPa CO 2 while L* of the internal integuments decreased with time due to browning. Integuments were darker after six weeks at 10oC and 20kPa 426 Novel food packaging techniques CO 2 , as a consequence of CO 2 injury, while chroma and hue did not change (Holcroft et al., 1998). Inconsistent results have been found in sweet cherries. For red and purple cherries 11kPa of CO 2 inhibited anthocyanin synthesis and when initially packaged with a high CO 2 level maintained their anthocyanin content during storage (Remo′n et al., 2000). In order to avoid risk of high CO 2 on undesirable colour changes ‘Ambrune′s’ sweet cherries were treated for 26 days at 1–2oC with CO 2 shocks (air plus 20% CO 2 ) once a week for 24 hours every week followed by active aeration, or in continuous 20% CO 2 enriched air atmosphere. Air control and both CO 2 treatments were followed by three days in air at 13oC. At the end of shelf-life an increase of cyanidin derivatives and in total anthocyanins content was detected in fruit under both CO 2 treatments without differences between them. In all treatments L* increased while a*, hue and chroma decreased, indicating that no undesirable changes in colour were induced by high CO 2 . This CO 2 shocks technique could be useful as a commercial alternative to continuous CO 2 for controlling decay in sweet cherries, being easier and cheaper to apply than continuous CA storage, without adverse collateral effects (Arte′s et al., 2002d). The presence of anthocyanins in white asparagus spears, evident from purple colour development in their tips, is an undesirable change that affects quality. Anthocyanin accumulation was slow in MAP (1kPa O 2 and 5–7kPa CO 2 ) during six days at 2.5, 10 and 20oC, and tips were still white (a* did not change) at the end of storage. On the contrary, for spears stored in air, the anthocyanin content increased at all temperatures, resulting in an intense purple colour of the tips, the presence of light having little or no effect on any of these treatments (Siomos et al., 2000). Later studies revealed that the increase in the anthocyanin concentration was prevented when spears were stored under higher than 5kPa CO 2 in the dark or than 10kPa in the light. Hue showed no changes from harvest values. Brief exposure to 100kPa CO 2 before air storage was as effective as continuous storage under the above conditions in preventing development of red colour (Siomos et al., 2001). Since pH has a marked effect on anthocyanin stability and colour expression, changes in pH induced by CA could cause significant losses in colour. The effect of CO 2 enriched atmospheres on colour and anthocyanin concentration depends on fruit species. Those that have a high buffering capacity prevent pH changes and maintain pigmentation better. However, it is possible to correlate reduced pericarp pH with low PPO activity. A pH of 4.2 or less may almost abolish PPO activity (Jiang et al., 1997). It has been reported that high CO 2 can improve colour and chlorophyll retention during storage of broccoli. MAP with 11kPa O 2 plus 7.5kPa CO 2 throughout 96h at 5oC resulted in no differences in green colour retention of florets evaluated by hue, compared to the level at harvest (Barth and Zhuang, 1996). Experiments combining low temperature and CA revealed that increasing the CO 2 level to 2.6 or 4.7kPa (O 2 21kPa) maintained the appearance, as well as chlorophyll content of stored snow peas (Ontai et al., 1992). Active packaging and colour control: the case of fruit and vegetables 427 High CO 2 levels produced injury in lettuce by increasing PAL activity, but were effective for avoiding brown stain. Under air plus 15kPa CO 2 production of phenolic compounds was blocked and did not change during storage. If CO 2 was replaced by air, phenolics content increased, quickly at 20oC and slowly at 0oC. Previous reports indicated that in lettuce tissue, CO 2 prevented browning by both blocking the production of new phenolic compounds and inhibiting PPO (Siriphanich and Kader, 1985). For determining browning in ‘Baby’ and ‘Romaine’ lettuce midribs L* was a good parameter (a decrease indicates darkening), and an increase in a* was associated with reddish colours due to browning (Castan?er et al., 1999). Prepared diced yellow onion did not always develop enzymatic browning during storage. However, discoloration resulting from yellowing has been reported (Mencarelli et al., 1990, Blanchard et al., 1996). Keeping O 2 at 2kPa, while CO 2 increased from 0 to 15kPa, the b* values were lowered. CO 2 effect was also observed after cooking. While control diced onion showed an intensification of browning and blackening of the cut surfaces, browning of cooked onion pure′e (expressed as b*) was slowed down slightly by lowering O 2 , and more strongly by increasing CO 2 (Blanchard et al., 1996). Ready-to-eat apple is susceptible to browning and several methods have been tried to inhibit PPO. By using active MAP of 2.5kPa O 2 and 7kPa CO 2 a depletion of 62% of PPO activity was found. A first-order model fitted with enough accuracy was proposed for predicting colour changes. Browning was better described through decrease in L* and increase in total colour difference with respect to the initial values (Soliva-Fortuny et al., 2001). On the other hand, CO 2 injury in whole and fresh processed apple has been reported. Concentrations over 10kPa for ‘Golden Delicious’, ‘Renatta’ and ‘Imperatore’ cv produced flesh browning (Gorini, 1979). CA of air plus 10 or 20kPa CO 2 accelerated tissue browning and necrosis of fresh cut pear slices compared to those stored in air. Necrosis and severe cut surface browning first occurred in the flesh tissue closest to the core and spread radially (Gorny et al., 2002). A level of 39kPa CO 2 with O 2 close to 1kPa considerably affected anthocyanin concentration in apple tissue. The decline was attributed to non- enzymatic browning of anthocyanins with free amino acid released by damaged tissue under high CO 2 , indicating that CO 2 was a key factor in destabilising those anthocyanins (Lin et al., 1989). Sliced persimmon stored in CA containing 12kPa CO 2 resulted in colour retention. It maintained good visual quality when stored under air plus 12kPa CO 2 or 2kPa O 2 plus 12kPa CO 2 . However, air and 2kPa O 2 -stored fruit developed areas of black pigmentation on the cut surfaces at the limit of marketability (Wright and Kader, 1997). 428 Novel food packaging techniques 20.6 Combining low oxygen, high carbon dioxide and other gases The colour development of tomatoes is influenced by gas composition. Several authors have developed empirical models to calculate colour parameters for tomatoes stored in constant or varying gaseous environments. When fruits were exposed to 20 different gas atmospheres involving O 2 and CO 2 levels of 5– 20kPa and 0–20kPa respectively, it was found that changes in O 2 had a greater effect on colour development than changes in CO 2 . Models require modifications depending on initial maturity stage, temperature and cultivar. Delayed tomato ripening at 3–5kPa CO 2 could in part be attributed to their effect on inhibiting ethylene synthesis (Yang and Chinnan, 1987). For cherries, it was observed that skin colour exhibited a similar trend in four MAP (all of them with an equilibrium atmosphere of 1–3kPa O 2 and 9– 12kPaCO 2 ). The hue shifted from red to blue/red and then back to red. Thus, at the end of three weeks storage, the skin colour remained effectively unchanged. Since any change was temporary, it is difficult to explain. Variation in pH due to MAP can affect the colouration of anthocyanins producing qualitative differences in this pigment (Remo′n et al., 2000, Gil et al., 1996b). The anthocyanin level of raspberry fruit increased during storage in normal air while no changes were found after storage in CA of 10kPa O 2 plus 15 or 30kPa CO 2 . Air stored fruits were darker (lower L*), less intense in red (lower a*) and less yellow (lower b*) and more red-bluish (lower hue). CA with 10kPa O 2 plus 30kPa CO 2 resulted in darker berries but CA with 10kPa O 2 plus 15kPa CO 2 had no influence on any colour parameters (Haffner et al., 2002). Sweet pomegranate stored at 2 or 5oC for 12 weeks in MAP (15kPa O 2 and 3kPa CO 2 at the steady state) was compared with fruits packaged with perforated film. Air atmosphere maintained red skin-colour, with anthocyanin levels decreasing in all treatments. Chroma and hue values of the arils in air stored pomegranates suffered slight or no changes. However, MAP stored fruits showed a decrease in chroma (Arte′s et al., 2000a). Changes in skin and flesh colour of ‘Paraguayo’ peaches stored for three weeks at 0.5oC were analysed, and hue was the best index. Intermittent warming (IW) for one day at 20oC every six days of cold storage and MAP was applied. The high CO 2 (19.5kPa) and low O 2 (5.1kPa) generated by MAP alone or combined with IW, delayed colour development even during post storage ripening. Ground colour measured by hue angle was the best index for monitoring colour evaluation (Ferna′ndez-Trujillo and Arte′s, 1997). Shrinkable packaging has been used to create MAP with lower O 2 and higher CO 2 inside the products than in the surroundings. This inhibits ethylene production and action. In this way, the hue of apples stored unwrapped decreases, while the hue of shrink-wrapped apples increased indicating that the unpacked apples were more reddish and less yellowish (Lin et al., 1989). Low O 2 (0.25–0.5kPa), high CO 2 (air enriched with 5, 10 or 20kPa CO 2 ) or superatmospheric O 2 (40–80kPa) applied alone did not prevent cut surface Active packaging and colour control: the case of fruit and vegetables 429 browning of fresh cut ‘Bartlett’ pears slices. However, a post cut dipping in a solution of ascorbic acid, calcium lactate and cysteine (adjusted to pH 7) reduced browning and extended shelf-life of pear slices for up to eight days at 0oC (Gorny et al., 2002). Each vegetable responds differently to MAP. For example mizuna (an Asian vegetable of the Brassica family) kept its green colour with less than 2kPa O 2 , but not with CO 2 higher than 5kPa. Pak choy (another Asian leafy vegetable) responds well to either enhanced CO 2 (up to 15kPa) or reduced O 2 , while Chinese mustard responds well to a combination of reduced O 2 and enhanced O 2 (O’Hare and Wong, 2000). High CO 2 (29kPa) and low O 2 (1kPa) led to anaerobic respiration, causing a great degradation of chlorophyll in snow pea pods (Ontai et al., 1992). However, bagging pea pods with a polymeric film (5kPa O 2 and 5kPa CO 2 ) and stored 28 days at 5oC, led to maintenance of appearance and colour, as well as internal quality (chlorophyll, ascorbic acid and sugar contents). MAP reduced the breakdown of chlorophyll to pheophytin, and control pods had increased yellowing and were less intensely green (Pariasca et al., 2001). For keeping quality and avoiding browning of fresh-cut butter head lettuce, Varoquaux et al. (1996) found that levels of O 2 lower than 3kPa combined with CO 2 lower than 10kPa were optimal. For generating these atmospheres in MAP, plastic polymers with low permeability to O 2 and high to CO 2 must be applied. To ensure that MAP salad products have no brown edges (the major quality defect) in commercial production, less than 0.5kPa O 2 and high CO 2 (more than 7kPa) are used. However, these conditions may lead to fermentative metabolism with production of ethanol and acetaldehyde, and off-odours development (Cantwell and Suslow, 2002) A corn zein based polymer was evaluated to investigate its ability to perform as biodegradable MAP for avoiding yellowing of fresh broccoli. Atmospheres of 2–8kPa O 2 and 5–15kPa CO 2 maintained original colour and texture for six days at 5oC. This behaviour was attributed to the low respiration rate induced by high CO 2 that also inhibited the metabolic reactions leading to colour and texture loss (Rakotonirainy et al., 2001). Endives stored for five days at 7oC were highly susceptible to red discoloration and browning, both factors reducing their shelf-life, and minimal processing enhances quality degradation by increasing the respiration rate. A gas mixture of 10kPa CO 2 and 10kPa O 2 avoids colour changes and showed the best quality from consumers’ point of view (Van de Velde and Hendrickx, 2001). When several CA were applied to chicory in O 2 levels ranging from 2–21kPa and CO 2 from 0– 19kPa, the best composition to discourage red discoloration was provided by low O 2 combined with high CO 2 . The effect of O 2 was found to be more important than that of CO 2 . In addition to atmosphere composition effects, the higher the storage temperature (5, 12 and 20oC) or the storage duration (3, 7, 10 and 14 days) the more red discoloration developed (Verlinden et al., 2001). Minimally processed pomegranate seeds have a greatly reduced postharvest life compared to whole fruit, and MAP is an excellent method for extending 430 Novel food packaging techniques their shelf-life. The quality of ready-to-eat pomegranate seeds has been evaluated by using active (5kPa O 2 and 0kPa CO 2 ) and passive MAP at 0– 5oC. In general, a slight decrease in the juice anthocyanin content was found. L* increased under MAP, showing an opposite effect to unpacked seeds, and compared to air atmosphere, visual appearance was improved (Gil et al., 1996a; Villaescusa et al., 2001). However, MAP (11–13kPa O 2 and 9–12kPa CO 2 ) did not show any effect on delaying browning of fennel dice stored for 14 days at 0oC followed by four days in air at 15oC, very probably due to the relatively high O 2 levels reached. A decrease of hue and chroma for MAP and air treatments, without differences among them, was found. During storage in air or MAP yellowing occurred, probably due to the fact that enzymatic browning on fennel dice was only a surface reaction. After storage, colour of fennel juice changed without differences among control and MAP treatments although in contrast with results in dice, chroma increased (Arte′s et al., 2002a). Active (flushed with 4kPa O 2 plus 10kPa CO 2 ) MAP maintained the quality of fresh cut cantaloupe cubes for nine days at 5oC better than passive MAP. Among the benefits of MAP better colour retention was reported. A high lightness and bright orange colour on cube surfaces were observed. The initial L* hue and chroma values remained unchanged in MAP but decreased in the control (Bai et al., 2001). In treatments having higher than 15kPa O 2 and in treatments without CO 2, translucency (a typical cause of low quality in cantaloupes) occurred (O’Connor-Shaw et al., 1995). 20.6.1 Other gases It has been suggested that argon and other noble gases could be used for MAP applications. Apparently, although they are chemically inert, they are biochemically active. Their solubility in water is higher compared with N 2 and O 2 . Probably, they interfere with cell membrane fluidity and O 2 receptor sites of enzymes. It was observed that Ar suppress enzyme activity and controls adverse chemical reactions, reducing respiration rates and, consequently, having a direct effect on extending shelf-life (Day, 1994). However, there is a lack of conclusive evidence supporting the view that partial or total substitution of N 2 with Ar has beneficial effects in terms of quality for MAP systems. Comparative experiments using both Ar and N 2 on two enzymes, one involved in the respiration process (malic dehydrogenase EC 1.1.1.37) and the other involved in the oxidative browning of fruit (tyrosinase) confirmed that there was only a slight reduction in activity using Ar compared with N 2 (Zhang et al., 2000). The combination of CO at concentrations between 5–10kPa with O 2 levels lower than 5kPa has been shown to be able to retard browning and extending shelf-life in fresh-cut lettuce and other products (Cantwell, 1992). The use of exogenous NO by initial short-term fumigation could extend the postharvest life of fresh horticultural products through inhibiting ethylene production and action. Thus disadvantageous changes in colour of harvested fruits and vegetables could Active packaging and colour control: the case of fruit and vegetables 431 be delayed, although NO application was more effective in non-climacteric than in climacteric species (Leshem, 2000). 20.7 Future trends Increasingly, efforts to understand and control colour changes in fruit and vegetables must be focused to ensure that they satisfy consumer demand. Only then can emergent packaging techniques for avoiding undesirable colour changes be developed on a commercial scale. The use of physical treatments like active MAP (including the use of CO and superatmospheric O 2 ), or edible coatings that restrict O 2 entrance, and moderate thermal treatments must be developed. The combination of physical treatments with natural anti-browning agents like organic acids or derivatives of 4-hexilresorcinol should also be explored (Arte′s et al., 1998). Some examples follow. For cut apple and potato, the use of a cellulose-based edible coating, as carrier of antioxidants, acidulants and preservatives, prolonged storage life by one week when stored in overwrapped trays at 4oC. Ascorbic acid and some of its derivatives formulated as a coating delayed browning more effectively than when applied in an aqueous solution (Baldwin et al., 1996). Combination of several browning inhibitors (like potassium sorbate and isoascorbic acid) was more effective to reduce browning of fresh cut mangoes than those applied individually. Browning in air stored product was attributed to degradation of the tissue induced by dryness of the surface, and use of MAP to maintain high humidity was helpful (Gonza′lez Aguilar et al., 2000). 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