Active packaging and colour control: the case of fruit and vegetables F. Artes Calero, Technical University of Cartagena, Spain and P.A. Gomez, National Institute for Agricultural Technology, Argentina 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 consumers 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 cceptable 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 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 O2 and increasing CO 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
20.1 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 O2 and increasing CO2 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
Active packaging and colour control: the case of fruit and vegetables 417 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 espectively, 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(Artes 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 anthe 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(Artes 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 beans 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 diffuse
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
418 Novel food packaging techniques 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 preferable to measuring at a distance From 200 measurements on tenGolden 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 nternal 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 hey are of a non-destructive nature. Colour is monitored in a three-dimensiona colour space in terms of the chromatic colour coordinates L*(lightness), a* and b", based on the CieLAB colour measurement system(Commission nternationale de Ecla 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; Artes and Escriche, 1994; Artes et al., 2000b). A more accurate measurement of colour can be obtained indicating that angle, named hue angle(h= arct b*/a*), which presents the basic tint of a colour, and chroma [(a*+b*)"I, 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 nalysis 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 B-carotene pigment, an important
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
Active packaging and colour control: the case of fruit and vegetables 419 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 nthocyanin 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, 199 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 lally 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 afy 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
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
420 Novel food packaging 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 hloroplast 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 HOrtensteiner, 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(Artes et al., 2002c) In climacteric fruits, the maximum degradation of chlorophy ll 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, B-carotene accumulates resulting in yellow fruit(Shewfelt et al 1988). As tomatoes turn from green to red, changes in the L*a** 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(artes et al., 2000b; Gomez 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%
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, concomitantly 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
Active packaging and colour control: the case of fruit and vegetables 421 50 ppm C2H4-65% initial red colour 50 ppm C2H4-85% initial red colour After 2 days at 180 Air-65% initial red colou Air-85%o initial red colour After 3 days at 7C Fig. 20.1 Changes in L' and a'* parameters of bell peppers with 65% and 85% of initial red colour degreened with 50ppm c2H4 during 2 days at 18%C followed by 3 days at 7C in air( Gomez et al., 2002) 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(Artes, 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 anged from dark green to yellowish-green(Artes et al., 2002b). On the other
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 c2H4 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
422 Novel food packaging techniques 115 Air0°C AC5%C02+5%O2-0°C -AC5%CO2+5%02-5°C Initial 14 days Fig 20.2 Colou ges ( Hue)of celery sticks stored for 14 days in air and in ontrolled atmosphere (5%CO2+ 5%O2) free of ethy lene at 0 and 5C(Artes et al, hand, antioxidants are related to chlorophyll retention in leafy products. Two antioxidants commonly present in fruits and vegetables are ascorbic acid and B- 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 pecific 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 It is well known that colour due to anthocyanins is particularly degraded by the nzymic hydrolysis in harvested products as recently reviewed (artes et al., 2002c) Anthocyanins are oxidised in the vacuole of the plant cells in the presence of molecular O2 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
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 O2 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% CO2 + 5% O2) free of ethylene at 0 and 5ºC (Arte´s et al,. 2002b). 422 Novel food packaging techniques
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
424 Novel food packaging techniques Peroxidases (POD; EC 1 11. 1.7. )could also be involved in browning although to a lesser extent, due to low availability of H2O2 within the plant cell (Artes et al, 1998; Sanchez-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 an scald induction. Fatty acid radicals induced by peroxidase can react with cell components leading to further breakdown. Particularly, bleaching of B-carotene and chlorophyll a occurs as a consequence of lipoxygenase catalysed reactio 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 O2 and/or elevated CO2 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, Artes, 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(Artes, 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 O2 and/or high CO2 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 O2 concentration MAP induced higher L values and
Peroxidases (POD; EC 1.11.1.7.) could also be involved in browning although to a lesser extent, due to low availability of H2O2 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 O2 and/or elevated CO2 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 O2 and/or high CO2 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 O2 concentration. MAP induced higher L* values and 424 Novel food packaging techniques
Active packaging and colour control: the case of fruit and vegetables 425 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 O2(Roy et al, 1996) Strawberries stored under low O, 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 O2 was not as effective at high COz levels in reducing decay(Holcroft and Kader, 1999b Freshly harvested white asparagus spears stored in air or in CA having increased O2 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, level (Siomos et al., 2000) days of strongly reduced from 90% in air to 35% under 2kPa O2 and OkPa CO2 (Verlinden et al, 2001). Fresh processed potato slices stored in MAP with low O, showed a better colour retention when the o, 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. 15) Slices in air showed a decrease in L* compared to MAP. It was advantageous to have almost no initial O2 within packages by flushing N2. This active MAP was very important for the keeping quality of slices, taking into consideration that residual O2 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 O2 and COz levels in the package and Oz must be decreased to an acceptable minimum level as soon as possible, it being advantageous to have almost no O2 initially within packages( Gunes and Lee, The intensity of browning of ready-to-eat apples depends on the atmosphere omposition. Apple cubes in MAP were efficiently preserved from browning and showed the lowest colour losses when initially displacing O2 by injecting 100kPa N2 and a film with low O2 permeability was used. This atmosphere was the main factor affecting lightness, and l' changes occurred four times more slowly than when O2 was about 2kPa or when medium O2 permeability films were used( Soliva-Fortuny et al., 2001) It has been suggested that O2 levels greater than 2lkPa may influence the postharvest life of intact and fresh processed fruit and vegetables and PPO may be substrate-inhibited by high O2 levels(Day, 1994). Superatmospheric could have an effect on respiratory activity and ethylene synthesis and action, although response depends on the commodity, ripening stage, O2 level, length of storage and temperature. Levels of COz and C2 Ha should also be considered When focusing on pigment changes, Kader and Ben-Yehoshua(2000)reported
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 O2 (Roy et al., 1996). Strawberries stored under low O2 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 O2 was not as effective at high CO2 levels in reducing decay (Holcroft and Kader, 1999b). Freshly harvested white asparagus spears stored in air or in CA having increased O2 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 O2 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 O2 and 0kPa CO2 (Verlinden et al., 2001). Fresh processed potato slices stored in MAP with low O2 showed a better colour retention when the O2 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 O2 within packages by flushing N2. This active MAP was very important for the keeping quality of slices, taking into consideration that residual O2 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 O2 and CO2 levels in the package and O2 must be decreased to an acceptable minimum level as soon as possible, it being advantageous to have almost no O2 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 O2 by injecting 100kPa N2 and a film with low O2 permeability was used. This atmosphere was the main factor affecting lightness, and L* changes occurred four times more slowly than when O2 was about 2kPa or when medium O2 permeability films were used (Soliva-Fortuny et al., 2001). It has been suggested that O2 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 O2 levels (Day, 1994). Superatmospheric O2 could have an effect on respiratory activity and ethylene synthesis and action, although response depends on the commodity, ripening stage, O2 level, length of storage and temperature. Levels of CO2 and C2H4 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
