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Changes during the ripening of the very late season Spanish peach cultivar Calanda
Scientia Horticulturae 105 (2005) 435–446 www.elsevier.com/locate/scihorti
Changes during the ripening of the very late season Spanish peach cultivar Calanda Feasibility of using CIELAB coordinates as maturity indices Ana Ferrer *, Sara Remo´n, Angel I. Negueruela, Rosa Oria Ciencia y Tecnologı´a de Alimentos, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Received 30 April 2004; received in revised form 26 January 2005; accepted 2 February 2005
Abstract Physiological maturity of peach at harvest greatly influences the post-harvest quality. Changes in colour during the last ripening stages of a very late and firm peach cultivar [Prunus persica (L.) Batsch cv. Calanda] have been studied to assess the feasibility of using reflectance colour references as non-destructive indices of maturity. The respiratory peak was simultaneous to the ethylene rise, and both induced important changes in the peaches. The physiological changes brought about a rise in polyphenoloxidase and peroxidase enzyme activity. The amount of carotenoids increased as maturity advanced and more markedly at the climacteric rise. Reflectance spectra and CIELAB coordinates reflected changes in skin and pulp colour and a* and hab evolved nearly in a near linear way. Values of the b* coordinate show changes as well. These findings agree with and extend previous results that reflectance changes can be used as reliable indices to establish the time of physiological maturity in Calanda peaches. # 2005 Elsevier B.V. All rights reserved. Keywords: Prunus persica; CIELAB; Pigments; Polyphenoloxidase; Peroxidase; Ethylene
* Corresponding author. Tel.: +34 976 76 15 84; fax: +34 976 76 15 90. E-mail address: [email protected] (A. Ferrer). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.002
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1. Introduction The physiological maturity of peach fruit at harvest greatly influences fruit quality (Shewfelt et al., 1987). Fruits harvested at an unripe stage are more prone to shrivelling, internal breakdown, and mechanical damage and are also of inferior quality when ripe. Over-mature fruits are likely to become soft and mealy and have insipid flavour soon after harvest (Kader and Mitchell, 1989), but present serious constraints for efficient handling and transportation. In peaches, advanced stages of ripeness have been characterized by marked changes in colour, firmness, acidity, and soluble solids content (Rood, 1957; Kader et al., 1982; Sims and Comin, 1963; Delwiche and Baumgardner, 1985; Heyes and Sealey, 1996). Various maturity indices, most of them including colour, have been used to monitor fruit development. There are conflicting reports in the literature on the correlation between colour measurements, pigment composition, physiological maturity and visual appreciation. Changes in colour in many fruits involve the loss of chlorophyll, the synthesis of new pigments such as carotenoids and/or anthocyanins, and the unmasking of other pigments that had previously been formed during the development of the fruit. Chemical analyses of pigment concentration are tedious and destructive; rapid, non-destructive measurements are preferable. Studies of the evolution of carotenoid pigments throughout the ripening period are difficult because many of them are present and the composition varies qualitatively and quantitatively and they are susceptible to isomerisation and oxidation. Sidwell et al. (1961) studied Elberta peaches and concluded that the chlorophyll content measured by light transmittance techniques might be an adequate indicator of ripeness degree. Other agents implicated in colour changes are the oxidative enzymes polyphenoloxidase (PPO) and peroxidase (POD), which promote browning reactions (Reyes and Luh, 1960) and discolouration in harvested peaches (Jen and Kahler, 1974). As variation in production area, season, and cultivar affect maturity indices and quality parameters (Kader and Mitchell, 1989), specific studies must be carried out on each species and variety. No previous studies are available about the indigenous Spanish variety Calanda although it is very much appreciated by European consumers and commands high prices in the fresh market due to its special characteristics: It matures very late, has a large size and high quality. They are clingstone, pale-yellowish, non-melting fleshed peaches which are individually wrapped in a paper bag during their development on the tree, avoiding insect attack, protecting them from pesticides, and reaching a characteristic uniform colour. They are expected to be uniformly cream or straw-coloured. Only slight blush is accepted, but green or orange-yellowish colours are refused. Its production, marketing and quality factors are strictly regulated under the protected designation of origin ‘‘Melocoto´ n de Calanda’’. It is of great interest to study the evolution of the principal colour parameters during the ripening of this cultivar, as a uniform development and maturity grade at harvest is mandatory. This study was undertaken to determine the relationship between colour measurements, physiological maturity and enzymatic browning potential and to determine if such changes could be useful indices of maturity for Calanda peaches.
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2. Materials and methods 2.1. Material studied Calanda peaches were hand-harvested in Teruel (Spain), from marked trees from the same orchard over three consecutive seasons on 10 different dates. Every harvest date, fruits were immediately transported to our laboratory, selected, sized and weighted, and their firmness, acidity and soluble solid content (SS) were determined. After a statistical Cluster analysis and using the SS/acidity index, firmness and fruit weight as indices, we have re-classified the peaches into 10 grades. 2.2. Analysis of soluble solids content, titrable acidity, and ripening index Soluble solids were determined in duplicate in juice extracted from the fruits by a refractometer at 20 8C and values were expressed in 8 Brix. Acidity was quantified in juice by potentiometric titration with 0.1 N NaOH to an end point of pH 8.1. The ripening index was obtained as the ratio of soluble solids to titrable acidity. 2.3. Fruit firmness determination Fruit firmness was measured with a texture analyzer TA-XT2 (Stable Micro Systems, Goaldming, England) by a penetration test, using a 6 mm diameter probe. 2.4. Physiological parameters The measurement of respiratory activity was carried out following the closed-system method at 20 8C. Peaches (450 50 g) were placed in hermetic glass containers (1000 mL) equipped with rubber sampling ports. Three replicates were prepared from each maturity stage. The initial atmosphere composition was that of air. Headspace was periodically sampled. Concentrations of O2 and CO2 were determined using a thermal conductivity detector. A Chrompack CP-Carboplot P7 column (inside diameter 0.53 mm, length 27.5 m) was used with helium as a carrier gas at a flow rate of 12.6 mL min1. The initial temperature of the oven was set at 40 8C and after 2.5 min it was increased at a rate of 45 8C min1 to a final temperature of 115 8C. The temperature of the injector block was 59 8C and the detector temperature was 120 8C. Concentration of C2H4 was determined using a Hewlett Packard 4890 chromatograph with a flame ionisation detector. A Porapack QS 80/100 mesh (6 feet, 1/800 ) was used with N2 as a carrier gas at a flow rate of 19 mL min1. The temperatures of the different components were as follow: injector 50 8C, oven 50 8C and detector 200 8C. Between each sample injection, jars were opened to re-equilibrate the normal air composition around the fruits. 2.5. Enzyme activity assays Polyphenoloxidase (E.C. 1.14.18.1; PPO) and peroxidase (E.C. 1.11.1.7; POD) were extracted as described by Cano et al. (1995). PPO was spectrophotometrically determined
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by monitoring the rate of dopachrome formation from DL-dopa at 475 nm and 20 8C (Lo´ pez et al., 1994). POD was determined by a method based on monitoring the decomposition of hydrogen peroxide by peroxidase with o-dianisidine as hydrogen donor by measuring the rate of colour development at 460 nm and 20 8C (Lo´ pez et al., 1994) and measured from the initial linear change in optical density. We define one absorbance unit (au) as an increase of 0.1 units of absorbance per minute. 2.6. Pigment analysis Carotene and xanthophyll content were determined from frozen pulp by measuring the absorbance of saponified samples with a UV–vis spectrophotometer using the AOAC (1990) guidelines. The samples were saponified with KOH (15% p,v) and 30% AA (p,v). This analysis was done in total darkness and in an inert atmosphere using ultrapure solvents. 2.7. Skin and pulp colour Colorimetric measurements were carried out on 10 peaches from every batch (both on the skin and in the pulp) with an Instrument System Spectroradiometer IS CAS 140 (Instrument System, Munich, Germany) using a TOP 100 probe with an AFNikor f:4, 200 mm lens; the instrument was controlled by the ISCOLOR software implemented on a PC. Reflectance spectra were measured during 8 s while the peach completed two rotations, in order to determine a global colour spectrum. Spectra were measured between 380 and 900 nm every 1 nm. From these spectra CIELAB coordinates L*, a*, b*, C* and hab were calculated with the standard observer CIE64 and the D65 illuminant. Coordinate a* is related with the red-green visual opposition and b* is related with the yellow-blue opposition. Coordinates L* (lightness), C* (chroma) and hab (hue) are related with the physiological attributes of visual response. 2.8. Statistical analysis Statistical analysis was carried out using the Statistical Package for the Social Science software version 11.5 (SPSS, 2002).
3. Results The main parameters of the groups, including SS/acidity, firmness and medium weight are shown in Table 1. Medium weight increases during the ripening of the peaches. Weight gain is more important during the first grades and less significant beyond grade 7. Maximum equatorial diameter values show the same evolution as the weight. The ripening index evolves in a nearly linear way, triplicating the initial value at the end of the study. Firmness decreases linearly from values of 14 kg cm2 to 4.5 kg cm2. It has been described previously that for optimal sensorial characteristics, peaches should show a ripening index of 15 (Robertson et al., 1992). Values above 15 are achieved
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Table 1 Main parameters of Calanda peach batches Ripeness degree
Fruit weight (g)
Transversal diameter (mm)
SSC/acidity (ripening index)
Firmness (kg cm2)
1 2 3 4 5 6 7 8 9 10
146 13 171 18 185 38 210 23 217 39 234 25 265 24 274 27 278 51 290 29
64.9 3.3 69.2 4.0 72.3 5.6 75.1 3.7 76.0 5.8 78.7 3.6 80.1 3.8 79.3 3.3 78.6 0.7 81.0 0.3
10.5 1.5 11.5 1.7 15.4 1.5 12.4 1.8 14.3 1.4 15.0 1.3 21.6 1.9 25.7 2.1 25.3 2.3 35.1 2.5
14.5 2.3 14.4 2.0 10.7 1.6 10.4 1.8 9.5 1.7 8.6 1.8 8.6 1.5 5.8 1.4 5.1 1.5 4.5 1.4
in Calanda peaches of grades 7–10. On the other hand, firmness values at maturity grades beyond 7 are in the limit of those accepted by the designation of origin. Beyond this point the fruits do not increase either weight nor size appreciably. According to those data appropriate harvest maturity for Calanda peaches correspond to maturity stage 7. In Calanda peaches, C2H4 biosynthesis remains at values between 1000 and 2000 nL kg1 h1 (Fig. 1) before the climacteric rise is observed. At that moment the production peaks to about 4000 nL kg1 h1 decreasing subsequently. This means that the climacteric rise in Calanda peaches happens when the fruit is considerably ripe. The respiratory peak coincides with the ethylene increase. The CO2 production is equal to 20 mL kg1 h1 at the beginning of the study, increases to 25 mL kg1 h1 showing the climacteric response and decreases afterwards to levels below the initial ones. The O2 consumption experiences a similar evolution as that of CO2. Similar results have been
Fig. 1. Respiratory activity (left axis) and ethylene production (right axis) during ripening. Data represent mean values.
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Fig. 2. Evolution on carotenoids content on the pulp.
observed in other late peach varieties (El-Agamy et al., 1981; Biggs et al., 1982). The data obtained suggest that respiratory activity and ethylene production are slightly lower in Calanda peaches than in other varieties. Brovelli et al. (1999) reported that the firmer varieties always produce lower ethylene rates than softer ones, and Calanda are nonmelting firm peaches. In this variety, total carotenoid content increases during the maturity process (Fig. 2), to about 0.5 mg (100 g fresh weight)1 at the end of the study. This value is similar to that found in the var. Redhaven studied by Gross (1979). Xanthophylls are the predominant pigments throughout the study, with ratios xanthophylls/carotenes varying between 3.5 and 1.4. Xanthophylls content show a significant increase between grades 4 and 5 and after that point, the increase is moderate. The amount of carotenes increased at the beginning of the study and again between grades 6 and 8. Spectral reflectance curves obtained for the different stages of maturity are plotted in Fig. 3. Considering that the maximum absorbance of chlorophyll can be set at 675 nm, two different groups can be established, maturity grades before or after 7. The lower values of reflectance are related to higher chlorophyll content. Corresponding to the decrease in chlorophyll content, the reflectance between 580 and 670 nm increases as a result of the ripening process. Changes between 450 and 530 nm are related to variations in carotenoid content and distribution. The observed asymmetry is associated to the transformations that take place between the carotenoids, due to hydroxylation and oxidation processes. With advancing maturity grade, the spectral reflectance curves flatten. All CIELAB parameters show similar evolution in the measurements made on the pulp or on the whole fruit, and only values of b* are significantly different in the measurements made on pulp or on skin, being higher for the pulp, because of the higher content of yellowish pigments and lower content of green pigments. At the end of the ripening, differences are minimal in b*. Evolution of the colour of the whole fruit is shown in the plane a*,b* (Fig. 4). Values of * a increase during the ripening period from negative values to positive ones, denoting the
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Fig. 3. Spectral reflectance curves during ripening. Each curve corresponds to one different degree of ripeness.
loss of green colour related to the disappearance of chlorophyll. Values of b* tend to increase through the ripening period, levelling off after the optimum time of harvest. The values of hab are quite similar on the pulp or on the skin of Calanda peaches (Fig. 5). Generally, hue is not used as a maturity index, in spite of being described as the coordinate
Fig. 4. Plane a*,b* during ripening.
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Fig. 5. Changes in coordinate hab during ripening and differences between measurements made in the whole fruit (wf) or in the pulp (p).
Fig. 6. Evolution of PPO activity (left) and POD activity (right) during peach ripening.
that best reflects the visual colour. However, our results show that the value of hab is a useful indicator, combined with other parameters, as it decreases linearly throughout ripeness, showing that peaches change from yellow-greenish to orange-yellowish. L* values (data not shown) represent the lightness of the peach. As reflection is greater on a yellowish area, Calanda peaches present higher values than those obtained for other more reddish varieties. PPO activity does not increase until grade 7 (Fig. 6, left) and increases 1.5 times after the ethylene and respiratory peaks. In contrast, POD activity (Fig. 6 right) shows two rise of activity; the first and smaller one at the beginning of the study and the second and more consistent one after the ethylene and respiratory maximum.
4. Discussion Previous studies (Pech et al., 1994) showed that in peach fruit the transition from the immature to the mature stage happens rather late, and this physiological consideration determines the crucial importance of the harvest date.
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Respiratory peak in Calanda peaches coincides with the ethylene increase. Amoro´ s et al. (1989), found the same results for Baby Gold, but in other species both peaks are not coincident (Saltveit, 1999). In Calanda peaches, ethylene seems to act as an integrator of all the maturity phenomena and is a useful predictor of fruit maturity, as observed in other yellow-fleshed peach varieties (Walsh et al., 1989). Luchsinger and Walsh (1998) showed good correlation between ethylene production and colour (coordinate a*) in Redhaven and Elegant Lady peaches. However, we obtained better correlation with the b* coordinate in Calanda peaches. The climacteric point in Calanda peaches promotes several changes in colour as a result of an enhancement of enzyme activity and an increased carotenoid synthesis. Tonutti et al. (1991) also reported that sequential events were observed in different fruit tissues from the climacteric stage. However, ethylene measurements are neither practical nor simple to carry out as a maturity index. PPO and POD activities show a final peak, after the climacteric rise, which could be associated with the beginning of the senescence phase. Flurkey and Jen (1978) described a similar evolution, finding that both enzymatic activities peaked at the end of the fruit development. It has been reported (Brennan and Frenkel, 1977) that peroxidase activity and levels of hydrogen peroxide increase during the ripening and senescence of fruits. POD activity has been suggested as a possible agent in the ripening and senescence of many fruits, such as Golden Delicious apples (Gorin and Heidema, 1976) and apricots (Va´ mosVigya´ zo´ et al., 1985). The involvement of both enzymes in browning processes is possible as it has been proposed that PPO could act as promoter of POD activity (Richard-Forget and Gaulliaard, 1997). In Calanda peach, ethylene accumulation may induce the synthesis of peroxidase isoenzymes and consequently provokes an increase in total peroxidase activity as observed in other species (Abeles et al., 1989). In general, ethylene is considered to promote de-greening in peaches. Increases in carotenoid content and in transmittance at 675 nm (chlorophyll decrease) in the first ripening stages suggest that this variety is sensitive to low ethylene concentrations and is capable to stimulate the autocatalytic pathway of ethylene production. However, both phenomena are enhanced at the climacteric stage. Peroxidase could play a role in chorophyll degradation, together with the chlorophyllase and the chlorophyll oxidase, and so is associated with the yellowing of Calanda peaches as observed in other vegetable products (Matile et al., 1987; Abeles et al., 1988). We must as well consider that this variety is kept inside a paper bag during the maturation processes, and pigment evolution will be conditioned by the light levels and temperature inside. Light, temperature and O2 concentration may have a profound effect in the colour development (Kays, 1991). The measurements of the transmittance spectra of the paper shows that it allows the light to pass at all the wavelengths at the same rate (data not shown). However, the light is attenuated, and this could be related to the absence of anthocyanin stretches, a feature of Calanda peaches. Indeed, Calanda peaches are uniformly coloured. That is the reason why global surface colour has been measured instead of the blush colour used by other authors (Shewfelt et al., 1987; Delwiche and Baumgardner, 1983; 1985). Colour measurements yield important data as the reflectance spectrum has been proposed as a maturity index, by itself or combined with other parameters. The increase in a* observed in Calanda peaches has previously been described for other varieties as
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Redhaven, Redglobe and Rı´oOsso (Delwiche and Baumgardner, 1985). The value of a* has been suggested as the primary coordinate of change near harvest (Delwiche and Baumgardner, 1983; 1985) and has been described as the better colour index of maturity (Delwiche and Baumgardner, 1983; Corey and Schlimme, 1988; Byrne et al., 1991). These authors associated this evolution with chlorophyll degradation and an increase in anthocyanin content. However, Calanda peaches do not contain anthocyanin pigments as they are yellowish coloured and the increase in a* is exclusively due to a loss of chlorophyll. Similar results were obtained in a study of the surface of watermelons (Wright and Kader, 1997). The hab value is not usually reported to be a good maturity index. However, our results show that it decreased linearly with time and that it correlated well with all the parameters related to maturity. We consider this value the best indicator of visual appreciation, as it decreases linearly and shows the most important variation. The trend observed in b* during the ripening of Calanda peaches has also been described for Redhaven peaches, another late variety (Delwiche and Baumgardner, 1985). The decrease observed at the climacteric point could be used in Calanda peaches as an indicator of the enhancement of browning enzymes and beginning of senescence phenomena. 5. Conclusions Ethylene production and respiratory activity showed a peak at the same point and triggered off several changes in pigments and enzyme activity. However, the fruits responded to low ethylene concentrations throughout ripening, showing continuous changes in colour. Modifications in CIELAB values were good indicators of the maturity stages in Calanda peaches, and they changed in a linear way during ripening (a* and hab). These changes correlated to a decrease in chlorophyll content and to an increase in the concentration of carotenoid pigments, and reflect the changes in the activity of phenolic enzymes. Changes in the b* coordinate can be used as well as a ripening indicator. We propose for Calanda peaches a combination of indices to estimate the harvest point, including the ripening index, the colour coordinates a* and hab and the firmness to avoid the inherent variation existing when working with fruit. Delaying the harvest does not lead to significant benefits because the climacteric rise implies such important changes in POD and PPO activities that the post-harvest life of the fruits would be markedly reduced. References Abeles, F.B., Dunn, L.J., Morgans, P., Callahan, A., Dinterman, R.E., Schmitt, J., 1988. Induction of 33-kD and 60-kD peroxidases during ethylene induced senescence of cucumber cotyledons. Plant Physiol. 87, 609–615. Abeles, F.B., Biles, C.L., Dunn, L.J., 1989. Hormonal-regulation and distribution of peroxidase isoenzymes in the cucurbitaceae. Plant Physiol. 91, 1609–1612. Amoro´ s, A., Serrano, M., Riquelme, F., Romojaro, F., 1989. Levels of ACC and physical and chemical parameters in peach development. J. Hort. Sci. 64, 673–677. Biggs, R., El-Agamy, S., Aly, M., 1982. Ethylene production by mature peach fruit. HortScience 17, 62. Brennan, T., Frenkel, C., 1977. Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol. 59, 411–416.
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Brovelli, E.A., Brecht, J.K., Sherman, W.B., Sims.F C.A., 1999. Non-melting flesh trait in peaches is not related to low ethylene production rates. HortScience 34, 313–315. Byrne, D.H., Nikolic, A.N., Burns, E.E., 1991. Variability in sugars, acids, firmness, and color characteristics of 12 peach genotypes. J. Am. Soc. Hort. Sci. 116, 1004–1006. Cano, M., De Ancos, B., Lobo, G., 1995. Peroxidase and polyphenoloxidase activities in papaya during postharvest ripening after freezing/thawing. J. Food Sci. 60, 815–820. Corey, K.A., Schlimme, D.V., 1988. Relationship of rind gloss and groups of colour to flesh quality of watermelon fruits during maturation. Sci. Hort. 34, 211–218. Delwiche, M.J., Baumgardner, R.A., 1983. Ground colour measurements of peach. J. Am. Soc. Hort. Sci. 108, 1012–1016. Delwiche, M.J., Baumgardner, R.A., 1985. Ground colour as a peach maturity index. J. Am. Soc. Hort. Sci. 110, 53–57. El-Agamy, S., Aly, M., Biggs, R., 1981. Ethylene as related to fruit ripening in peaches. Proc. Fla. State Hort. 94, 284–289. Flurkey, W.H., Jen, J.J., 1978. Peroxidase and polyphenol oxidase activities in developing peaches. J. Food Sci. 43, 1826–1831. Gorin, N., Heidema, F.T., 1976. Peroxidase activity in Golden Delicious apples as a possible parameter of senescence. J. Agric. Food Chem. 24, 200. Gross, J., 1979. Carotenoids changes in the mesocarp of Redhaven peach (Prunus persica) during ripening. Z. Pflanzenphysiol. 94, 461–468. Heyes, J.A., Sealey, D.F., 1996. Textural changes during nectarine development and ripening. Sci. Hort. 65, 49–58. Jen, J.J., Kahler, K.R., 1974. Characterization of polyphenol oxidase in peaches grown in the southeast. HortScience 9, 590. Kader, A.A., Heintz, C.M., Chordas, A., 1982. Management of fruit ripening. Postharvest Horticulture Series 9. Postharvest Outreach Program, Department of Pomology, UC-Davis. Kader, A.A., Mitchell, F.G., 1989. Peaches, Plums and Nectarines: Growing and Handling for Fresh Market, Publication 3331. University of California, California, pp. 1191–1196. Kays, S.J., 1991. Postharvest Physiology of Perishable Plant Products. Van Nortrand Reinhold, New York. Lo´ pez, P., Sala, F.J., De la Fuente, J.L., Condo´ n, S., Raso, J., Burgos, J., 1994. Inactivation of peroxidase, lipoxygenase and polyphenol oxidase by manothermosonication. J. Agric. Food Chem. 42, 252–256. Luchsinger, L.E., Walsh, C.S., 1998. Development of an objective and non-destructive harvest maturity index for peaches and nectarines. Acta Hort. 465, 679–687. Matile, P., Ginsburg, S., Schellenberg, M., Thomas, H., 1987. Catabolites of chlorophyll in senescent leaves. J. Plant Physiol. 129, 219–228. Pech, J.C., Balague, C., Latche, A., Bouzayen, M., 1994. Postharvest physiology of climacteric fruits: recent developments in the biosynthesis and action of ethylene. Sci. Alim. 14, 3–15. Reyes, P., Luh, B.S., 1960. Characteristics of browning enzymes in Fay Elberta freestone peaches. Food Technol. 14, 570. Richard-Forget, F., Gaulliaard, F.A., 1997. Oxidation of chlorogenic acid, catechins and 4-methylcatechol in model solutions by combinations of pear (Pyrus Communis cv. Williams) polyphenol oxidase and peroxidase: a possible involvement of peroxidase in enzymatic browning. J. Agric. Food Chem. 45, 2472–2476. Robertson, J.A., Meredith, F.I., Forbus, W.R., Lyon, B.G., 1992. Relationship of quality characteristics of peaches (cv. Loring) to maturity. J. Food. Sci. 57, 1401–1404. Rood, P., 1957. Development and evaluation of objective maturity indices for California freestone peaches. Proc. Am. Soc. Hort. Sci. 70, 104–112. Saltveit, M.E., 1999. Effect of ethylene on quality of fresh fruits and vegetables. Postharvest Biol. Technol. 15, 279–292. Shewfelt, R.L., Myers, S.C., Resurreccio´ n, A.V.A., 1987. Effects of physiological maturity at harvest on peach quality during low temperature storage. J. Food Qual. 10, 9–20. Sidwell, A.P., Birth, G.S., Ernest, J.V., Golumbic, C., 1961. The use of light transmittance techniques to estimate the chlorophyll content and stage of maturation of Elberta peaches. Food Technol. 2, 75–78. Sims, E.T., Comin, D., 1963. Evaluation of objective maturity indices for Halehaven peaches. Proc. Am. Soc. Hort. Sci. 82, 125–130.
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Changes during the ripening of the very late season Spanish peach cultivar Calanda Feasibility of using CIELAB coordinates as maturity indices Ana Ferrer *, Sara Remo´n, Angel I. Negueruela, Rosa Oria Ciencia y Tecnologı´a de Alimentos, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Received 30 April 2004; received in revised form 26 January 2005; accepted 2 February 2005
Abstract Physiological maturity of peach at harvest greatly influences the post-harvest quality. Changes in colour during the last ripening stages of a very late and firm peach cultivar [Prunus persica (L.) Batsch cv. Calanda] have been studied to assess the feasibility of using reflectance colour references as non-destructive indices of maturity. The respiratory peak was simultaneous to the ethylene rise, and both induced important changes in the peaches. The physiological changes brought about a rise in polyphenoloxidase and peroxidase enzyme activity. The amount of carotenoids increased as maturity advanced and more markedly at the climacteric rise. Reflectance spectra and CIELAB coordinates reflected changes in skin and pulp colour and a* and hab evolved nearly in a near linear way. Values of the b* coordinate show changes as well. These findings agree with and extend previous results that reflectance changes can be used as reliable indices to establish the time of physiological maturity in Calanda peaches. # 2005 Elsevier B.V. All rights reserved. Keywords: Prunus persica; CIELAB; Pigments; Polyphenoloxidase; Peroxidase; Ethylene
* Corresponding author. Tel.: +34 976 76 15 84; fax: +34 976 76 15 90. E-mail address: [email protected] (A. Ferrer). 0304-4238/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.02.002
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1. Introduction The physiological maturity of peach fruit at harvest greatly influences fruit quality (Shewfelt et al., 1987). Fruits harvested at an unripe stage are more prone to shrivelling, internal breakdown, and mechanical damage and are also of inferior quality when ripe. Over-mature fruits are likely to become soft and mealy and have insipid flavour soon after harvest (Kader and Mitchell, 1989), but present serious constraints for efficient handling and transportation. In peaches, advanced stages of ripeness have been characterized by marked changes in colour, firmness, acidity, and soluble solids content (Rood, 1957; Kader et al., 1982; Sims and Comin, 1963; Delwiche and Baumgardner, 1985; Heyes and Sealey, 1996). Various maturity indices, most of them including colour, have been used to monitor fruit development. There are conflicting reports in the literature on the correlation between colour measurements, pigment composition, physiological maturity and visual appreciation. Changes in colour in many fruits involve the loss of chlorophyll, the synthesis of new pigments such as carotenoids and/or anthocyanins, and the unmasking of other pigments that had previously been formed during the development of the fruit. Chemical analyses of pigment concentration are tedious and destructive; rapid, non-destructive measurements are preferable. Studies of the evolution of carotenoid pigments throughout the ripening period are difficult because many of them are present and the composition varies qualitatively and quantitatively and they are susceptible to isomerisation and oxidation. Sidwell et al. (1961) studied Elberta peaches and concluded that the chlorophyll content measured by light transmittance techniques might be an adequate indicator of ripeness degree. Other agents implicated in colour changes are the oxidative enzymes polyphenoloxidase (PPO) and peroxidase (POD), which promote browning reactions (Reyes and Luh, 1960) and discolouration in harvested peaches (Jen and Kahler, 1974). As variation in production area, season, and cultivar affect maturity indices and quality parameters (Kader and Mitchell, 1989), specific studies must be carried out on each species and variety. No previous studies are available about the indigenous Spanish variety Calanda although it is very much appreciated by European consumers and commands high prices in the fresh market due to its special characteristics: It matures very late, has a large size and high quality. They are clingstone, pale-yellowish, non-melting fleshed peaches which are individually wrapped in a paper bag during their development on the tree, avoiding insect attack, protecting them from pesticides, and reaching a characteristic uniform colour. They are expected to be uniformly cream or straw-coloured. Only slight blush is accepted, but green or orange-yellowish colours are refused. Its production, marketing and quality factors are strictly regulated under the protected designation of origin ‘‘Melocoto´ n de Calanda’’. It is of great interest to study the evolution of the principal colour parameters during the ripening of this cultivar, as a uniform development and maturity grade at harvest is mandatory. This study was undertaken to determine the relationship between colour measurements, physiological maturity and enzymatic browning potential and to determine if such changes could be useful indices of maturity for Calanda peaches.
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2. Materials and methods 2.1. Material studied Calanda peaches were hand-harvested in Teruel (Spain), from marked trees from the same orchard over three consecutive seasons on 10 different dates. Every harvest date, fruits were immediately transported to our laboratory, selected, sized and weighted, and their firmness, acidity and soluble solid content (SS) were determined. After a statistical Cluster analysis and using the SS/acidity index, firmness and fruit weight as indices, we have re-classified the peaches into 10 grades. 2.2. Analysis of soluble solids content, titrable acidity, and ripening index Soluble solids were determined in duplicate in juice extracted from the fruits by a refractometer at 20 8C and values were expressed in 8 Brix. Acidity was quantified in juice by potentiometric titration with 0.1 N NaOH to an end point of pH 8.1. The ripening index was obtained as the ratio of soluble solids to titrable acidity. 2.3. Fruit firmness determination Fruit firmness was measured with a texture analyzer TA-XT2 (Stable Micro Systems, Goaldming, England) by a penetration test, using a 6 mm diameter probe. 2.4. Physiological parameters The measurement of respiratory activity was carried out following the closed-system method at 20 8C. Peaches (450 50 g) were placed in hermetic glass containers (1000 mL) equipped with rubber sampling ports. Three replicates were prepared from each maturity stage. The initial atmosphere composition was that of air. Headspace was periodically sampled. Concentrations of O2 and CO2 were determined using a thermal conductivity detector. A Chrompack CP-Carboplot P7 column (inside diameter 0.53 mm, length 27.5 m) was used with helium as a carrier gas at a flow rate of 12.6 mL min1. The initial temperature of the oven was set at 40 8C and after 2.5 min it was increased at a rate of 45 8C min1 to a final temperature of 115 8C. The temperature of the injector block was 59 8C and the detector temperature was 120 8C. Concentration of C2H4 was determined using a Hewlett Packard 4890 chromatograph with a flame ionisation detector. A Porapack QS 80/100 mesh (6 feet, 1/800 ) was used with N2 as a carrier gas at a flow rate of 19 mL min1. The temperatures of the different components were as follow: injector 50 8C, oven 50 8C and detector 200 8C. Between each sample injection, jars were opened to re-equilibrate the normal air composition around the fruits. 2.5. Enzyme activity assays Polyphenoloxidase (E.C. 1.14.18.1; PPO) and peroxidase (E.C. 1.11.1.7; POD) were extracted as described by Cano et al. (1995). PPO was spectrophotometrically determined
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by monitoring the rate of dopachrome formation from DL-dopa at 475 nm and 20 8C (Lo´ pez et al., 1994). POD was determined by a method based on monitoring the decomposition of hydrogen peroxide by peroxidase with o-dianisidine as hydrogen donor by measuring the rate of colour development at 460 nm and 20 8C (Lo´ pez et al., 1994) and measured from the initial linear change in optical density. We define one absorbance unit (au) as an increase of 0.1 units of absorbance per minute. 2.6. Pigment analysis Carotene and xanthophyll content were determined from frozen pulp by measuring the absorbance of saponified samples with a UV–vis spectrophotometer using the AOAC (1990) guidelines. The samples were saponified with KOH (15% p,v) and 30% AA (p,v). This analysis was done in total darkness and in an inert atmosphere using ultrapure solvents. 2.7. Skin and pulp colour Colorimetric measurements were carried out on 10 peaches from every batch (both on the skin and in the pulp) with an Instrument System Spectroradiometer IS CAS 140 (Instrument System, Munich, Germany) using a TOP 100 probe with an AFNikor f:4, 200 mm lens; the instrument was controlled by the ISCOLOR software implemented on a PC. Reflectance spectra were measured during 8 s while the peach completed two rotations, in order to determine a global colour spectrum. Spectra were measured between 380 and 900 nm every 1 nm. From these spectra CIELAB coordinates L*, a*, b*, C* and hab were calculated with the standard observer CIE64 and the D65 illuminant. Coordinate a* is related with the red-green visual opposition and b* is related with the yellow-blue opposition. Coordinates L* (lightness), C* (chroma) and hab (hue) are related with the physiological attributes of visual response. 2.8. Statistical analysis Statistical analysis was carried out using the Statistical Package for the Social Science software version 11.5 (SPSS, 2002).
3. Results The main parameters of the groups, including SS/acidity, firmness and medium weight are shown in Table 1. Medium weight increases during the ripening of the peaches. Weight gain is more important during the first grades and less significant beyond grade 7. Maximum equatorial diameter values show the same evolution as the weight. The ripening index evolves in a nearly linear way, triplicating the initial value at the end of the study. Firmness decreases linearly from values of 14 kg cm2 to 4.5 kg cm2. It has been described previously that for optimal sensorial characteristics, peaches should show a ripening index of 15 (Robertson et al., 1992). Values above 15 are achieved
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Table 1 Main parameters of Calanda peach batches Ripeness degree
Fruit weight (g)
Transversal diameter (mm)
SSC/acidity (ripening index)
Firmness (kg cm2)
1 2 3 4 5 6 7 8 9 10
146 13 171 18 185 38 210 23 217 39 234 25 265 24 274 27 278 51 290 29
64.9 3.3 69.2 4.0 72.3 5.6 75.1 3.7 76.0 5.8 78.7 3.6 80.1 3.8 79.3 3.3 78.6 0.7 81.0 0.3
10.5 1.5 11.5 1.7 15.4 1.5 12.4 1.8 14.3 1.4 15.0 1.3 21.6 1.9 25.7 2.1 25.3 2.3 35.1 2.5
14.5 2.3 14.4 2.0 10.7 1.6 10.4 1.8 9.5 1.7 8.6 1.8 8.6 1.5 5.8 1.4 5.1 1.5 4.5 1.4
in Calanda peaches of grades 7–10. On the other hand, firmness values at maturity grades beyond 7 are in the limit of those accepted by the designation of origin. Beyond this point the fruits do not increase either weight nor size appreciably. According to those data appropriate harvest maturity for Calanda peaches correspond to maturity stage 7. In Calanda peaches, C2H4 biosynthesis remains at values between 1000 and 2000 nL kg1 h1 (Fig. 1) before the climacteric rise is observed. At that moment the production peaks to about 4000 nL kg1 h1 decreasing subsequently. This means that the climacteric rise in Calanda peaches happens when the fruit is considerably ripe. The respiratory peak coincides with the ethylene increase. The CO2 production is equal to 20 mL kg1 h1 at the beginning of the study, increases to 25 mL kg1 h1 showing the climacteric response and decreases afterwards to levels below the initial ones. The O2 consumption experiences a similar evolution as that of CO2. Similar results have been
Fig. 1. Respiratory activity (left axis) and ethylene production (right axis) during ripening. Data represent mean values.
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Fig. 2. Evolution on carotenoids content on the pulp.
observed in other late peach varieties (El-Agamy et al., 1981; Biggs et al., 1982). The data obtained suggest that respiratory activity and ethylene production are slightly lower in Calanda peaches than in other varieties. Brovelli et al. (1999) reported that the firmer varieties always produce lower ethylene rates than softer ones, and Calanda are nonmelting firm peaches. In this variety, total carotenoid content increases during the maturity process (Fig. 2), to about 0.5 mg (100 g fresh weight)1 at the end of the study. This value is similar to that found in the var. Redhaven studied by Gross (1979). Xanthophylls are the predominant pigments throughout the study, with ratios xanthophylls/carotenes varying between 3.5 and 1.4. Xanthophylls content show a significant increase between grades 4 and 5 and after that point, the increase is moderate. The amount of carotenes increased at the beginning of the study and again between grades 6 and 8. Spectral reflectance curves obtained for the different stages of maturity are plotted in Fig. 3. Considering that the maximum absorbance of chlorophyll can be set at 675 nm, two different groups can be established, maturity grades before or after 7. The lower values of reflectance are related to higher chlorophyll content. Corresponding to the decrease in chlorophyll content, the reflectance between 580 and 670 nm increases as a result of the ripening process. Changes between 450 and 530 nm are related to variations in carotenoid content and distribution. The observed asymmetry is associated to the transformations that take place between the carotenoids, due to hydroxylation and oxidation processes. With advancing maturity grade, the spectral reflectance curves flatten. All CIELAB parameters show similar evolution in the measurements made on the pulp or on the whole fruit, and only values of b* are significantly different in the measurements made on pulp or on skin, being higher for the pulp, because of the higher content of yellowish pigments and lower content of green pigments. At the end of the ripening, differences are minimal in b*. Evolution of the colour of the whole fruit is shown in the plane a*,b* (Fig. 4). Values of * a increase during the ripening period from negative values to positive ones, denoting the
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Fig. 3. Spectral reflectance curves during ripening. Each curve corresponds to one different degree of ripeness.
loss of green colour related to the disappearance of chlorophyll. Values of b* tend to increase through the ripening period, levelling off after the optimum time of harvest. The values of hab are quite similar on the pulp or on the skin of Calanda peaches (Fig. 5). Generally, hue is not used as a maturity index, in spite of being described as the coordinate
Fig. 4. Plane a*,b* during ripening.
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Fig. 5. Changes in coordinate hab during ripening and differences between measurements made in the whole fruit (wf) or in the pulp (p).
Fig. 6. Evolution of PPO activity (left) and POD activity (right) during peach ripening.
that best reflects the visual colour. However, our results show that the value of hab is a useful indicator, combined with other parameters, as it decreases linearly throughout ripeness, showing that peaches change from yellow-greenish to orange-yellowish. L* values (data not shown) represent the lightness of the peach. As reflection is greater on a yellowish area, Calanda peaches present higher values than those obtained for other more reddish varieties. PPO activity does not increase until grade 7 (Fig. 6, left) and increases 1.5 times after the ethylene and respiratory peaks. In contrast, POD activity (Fig. 6 right) shows two rise of activity; the first and smaller one at the beginning of the study and the second and more consistent one after the ethylene and respiratory maximum.
4. Discussion Previous studies (Pech et al., 1994) showed that in peach fruit the transition from the immature to the mature stage happens rather late, and this physiological consideration determines the crucial importance of the harvest date.
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Respiratory peak in Calanda peaches coincides with the ethylene increase. Amoro´ s et al. (1989), found the same results for Baby Gold, but in other species both peaks are not coincident (Saltveit, 1999). In Calanda peaches, ethylene seems to act as an integrator of all the maturity phenomena and is a useful predictor of fruit maturity, as observed in other yellow-fleshed peach varieties (Walsh et al., 1989). Luchsinger and Walsh (1998) showed good correlation between ethylene production and colour (coordinate a*) in Redhaven and Elegant Lady peaches. However, we obtained better correlation with the b* coordinate in Calanda peaches. The climacteric point in Calanda peaches promotes several changes in colour as a result of an enhancement of enzyme activity and an increased carotenoid synthesis. Tonutti et al. (1991) also reported that sequential events were observed in different fruit tissues from the climacteric stage. However, ethylene measurements are neither practical nor simple to carry out as a maturity index. PPO and POD activities show a final peak, after the climacteric rise, which could be associated with the beginning of the senescence phase. Flurkey and Jen (1978) described a similar evolution, finding that both enzymatic activities peaked at the end of the fruit development. It has been reported (Brennan and Frenkel, 1977) that peroxidase activity and levels of hydrogen peroxide increase during the ripening and senescence of fruits. POD activity has been suggested as a possible agent in the ripening and senescence of many fruits, such as Golden Delicious apples (Gorin and Heidema, 1976) and apricots (Va´ mosVigya´ zo´ et al., 1985). The involvement of both enzymes in browning processes is possible as it has been proposed that PPO could act as promoter of POD activity (Richard-Forget and Gaulliaard, 1997). In Calanda peach, ethylene accumulation may induce the synthesis of peroxidase isoenzymes and consequently provokes an increase in total peroxidase activity as observed in other species (Abeles et al., 1989). In general, ethylene is considered to promote de-greening in peaches. Increases in carotenoid content and in transmittance at 675 nm (chlorophyll decrease) in the first ripening stages suggest that this variety is sensitive to low ethylene concentrations and is capable to stimulate the autocatalytic pathway of ethylene production. However, both phenomena are enhanced at the climacteric stage. Peroxidase could play a role in chorophyll degradation, together with the chlorophyllase and the chlorophyll oxidase, and so is associated with the yellowing of Calanda peaches as observed in other vegetable products (Matile et al., 1987; Abeles et al., 1988). We must as well consider that this variety is kept inside a paper bag during the maturation processes, and pigment evolution will be conditioned by the light levels and temperature inside. Light, temperature and O2 concentration may have a profound effect in the colour development (Kays, 1991). The measurements of the transmittance spectra of the paper shows that it allows the light to pass at all the wavelengths at the same rate (data not shown). However, the light is attenuated, and this could be related to the absence of anthocyanin stretches, a feature of Calanda peaches. Indeed, Calanda peaches are uniformly coloured. That is the reason why global surface colour has been measured instead of the blush colour used by other authors (Shewfelt et al., 1987; Delwiche and Baumgardner, 1983; 1985). Colour measurements yield important data as the reflectance spectrum has been proposed as a maturity index, by itself or combined with other parameters. The increase in a* observed in Calanda peaches has previously been described for other varieties as
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Redhaven, Redglobe and Rı´oOsso (Delwiche and Baumgardner, 1985). The value of a* has been suggested as the primary coordinate of change near harvest (Delwiche and Baumgardner, 1983; 1985) and has been described as the better colour index of maturity (Delwiche and Baumgardner, 1983; Corey and Schlimme, 1988; Byrne et al., 1991). These authors associated this evolution with chlorophyll degradation and an increase in anthocyanin content. However, Calanda peaches do not contain anthocyanin pigments as they are yellowish coloured and the increase in a* is exclusively due to a loss of chlorophyll. Similar results were obtained in a study of the surface of watermelons (Wright and Kader, 1997). The hab value is not usually reported to be a good maturity index. However, our results show that it decreased linearly with time and that it correlated well with all the parameters related to maturity. We consider this value the best indicator of visual appreciation, as it decreases linearly and shows the most important variation. The trend observed in b* during the ripening of Calanda peaches has also been described for Redhaven peaches, another late variety (Delwiche and Baumgardner, 1985). The decrease observed at the climacteric point could be used in Calanda peaches as an indicator of the enhancement of browning enzymes and beginning of senescence phenomena. 5. Conclusions Ethylene production and respiratory activity showed a peak at the same point and triggered off several changes in pigments and enzyme activity. However, the fruits responded to low ethylene concentrations throughout ripening, showing continuous changes in colour. Modifications in CIELAB values were good indicators of the maturity stages in Calanda peaches, and they changed in a linear way during ripening (a* and hab). These changes correlated to a decrease in chlorophyll content and to an increase in the concentration of carotenoid pigments, and reflect the changes in the activity of phenolic enzymes. Changes in the b* coordinate can be used as well as a ripening indicator. We propose for Calanda peaches a combination of indices to estimate the harvest point, including the ripening index, the colour coordinates a* and hab and the firmness to avoid the inherent variation existing when working with fruit. Delaying the harvest does not lead to significant benefits because the climacteric rise implies such important changes in POD and PPO activities that the post-harvest life of the fruits would be markedly reduced. References Abeles, F.B., Dunn, L.J., Morgans, P., Callahan, A., Dinterman, R.E., Schmitt, J., 1988. Induction of 33-kD and 60-kD peroxidases during ethylene induced senescence of cucumber cotyledons. Plant Physiol. 87, 609–615. Abeles, F.B., Biles, C.L., Dunn, L.J., 1989. Hormonal-regulation and distribution of peroxidase isoenzymes in the cucurbitaceae. Plant Physiol. 91, 1609–1612. Amoro´ s, A., Serrano, M., Riquelme, F., Romojaro, F., 1989. Levels of ACC and physical and chemical parameters in peach development. J. Hort. Sci. 64, 673–677. Biggs, R., El-Agamy, S., Aly, M., 1982. Ethylene production by mature peach fruit. HortScience 17, 62. Brennan, T., Frenkel, C., 1977. Involvement of hydrogen peroxide in the regulation of senescence in pear. Plant Physiol. 59, 411–416.
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