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Bruising injury of persimmon (Diospyros kaki cv. Fuyu) fruits
Scientia Horticulturae 103 (2005) 179–185
Bruising injury of persimmon (Diospyros kaki cv. Fuyu) fruits Hee Jae Lee a,∗ , Tae-Choon Kim b , Su Jin Kim a , Seung Je Park c a b
School of Plant Science, Seoul National University, Seoul 151-742, Republic of Korea Department of Horticulture, Wonkwang University, Iksan 570-749, Republic of Korea c Department of Agricultural Machinery Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea Accepted 16 April 2004
Abstract This study was performed to monitor the deterioration of bruised persimmon (Diospyros kaki cv. Fuyu) fruits. Freshly harvested fruits were bruised by dropping them from a height of 50 cm onto a steel board and then stored at 0 or 20 ◦ C in temperature controlled chambers for up to 4 weeks. Immediately after the bruising, no visible injury on the fruits was evident, but the fruits deteriorated rapidly during storage. The skin tissues of the fruits stored at 20 ◦ C became more reddish with the duration of the storage, but no such changes were found with the fruits stored at 0 ◦ C. The increase in redness of the skin tissues appeared to be associated with storage temperature, but not with the bruising. The skin tissues also became darker when stored at 20 ◦ C than at 0 ◦ C and this tendency was more obvious with the bruised fruits. Flesh firmness decreased rapidly during storage except for the non-bruised fruits stored at 0 ◦ C. Even the non-bruised fruits rapidly lost their flesh firmness at 20 ◦ C. No significant changes in lipid peroxidation, as expressed by malondialdehyde production, were found between the bruised and the non-bruised fruits during the storage either at 0 ◦ C or at 20 ◦ C. This implies that the fruit deterioration caused by bruising is not due to the consequences of lipid peroxidation. Polyphenol oxidase activity increased more rapidly in the bruised fruits than in the non-bruised fruits during storage. The bruising had more effect on increasing polyphenol oxidase activity than did the storage temperature. Although the increase in polyphenol oxidase activity appeared to be associated with the visual deterioration of the bruised fruits, it did not exactly correspond to the physical deterioration. These results indicate that polyphenol oxidase is not the only factor influencing the deterioration associated with bruising. Cell wall hydrolases are currently being assayed to determine if they also contribute the deterioration following bruising. © 2004 Elsevier B.V. All rights reserved. Keywords: Bruising; Diospyros kaki; Lipid peroxidation; Mechanical injury; Polyphenol oxidase
∗
Corresponding author. Tel.: +82-2-880-4566; fax: +82-2-873-2056. E-mail address: [email protected] (H.J. Lee). 0304-4238/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2004.04.016
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1. Introduction With the increasing use of mechanical equipments for the harvesting and packing of fruits, the mechanical injury on fruits has become a very important problem (Brusewitz and Bartsch, 1989; Marshall and Brook, 1999). Mechanical injury, such as bruises, cuts, and abrasions, causes marked deterioration in fruit quality (Quintana and Paull, 1993; Shewfelt, 1993). Bruising can occur when the fruit is carelessly dropped onto hard surfaces or subjected to harsh impacts during harvesting and packing. The bruising is initiated by the breakage of cell membranes, allowing cytoplasmic enzymes to act on sequestered substrates (Rouet-Mayer et al., 1990; Shewfelt, 1993). The resultant browning is caused by the enzyme action on phenolic substrates. Polyphenol oxidase (PPO) is well known enzyme responsible for tissue browning in mechanically injured fruits (Van Lelyveld and Bower, 1984; Sciancalepore, 1985; Nicolas et al., 1994). PPO catalyzes the oxidation of phenolic compounds to o-quinones which subsequently polymerize to form dark-colored pigments (Rouet-Mayer et al., 1990). During harvesting, grading, and transport of persimmon fruits, the fruits often deteriorate due to bruising and subsequent storage. However, no comprehensive study has been published on bruising injury of persimmon fruits. In the present study, the deterioration of bruised persimmon (Diospyros kaki cv. Fuyu) fruits were monitored with respect to fruit discoloration, firmness decrease, and lipid peroxidation during storage. PPO activity was also monitored to determine if it is associated with the deterioration of the bruised fruits.
2. Materials and methods Unblemished persimmon fruits of ‘Fuyu’ cultivar were purchased from an orchard in Jinyoung county, Korea at their commercial harvest. The fruits were dropped from height of 50 cm onto a steel board and caught after one bounce. The impact area was marked with black indelible ink. Control fruits were not dropped. The fruits were then placed in cupped plastic trays and stored at 0 or 20 ◦ C in temperature controlled chambers for up to 4 weeks. One hour prior to sampling, the fruits to be examined were removed from the chambers and equilibrated to room temperature. The discoloration of the skin and the flesh was determined as changes in a∗ and L∗ values by using a chromameter (model CR-300, Minolta Co., Ltd., Japan) as suggested by Sapers and Douglas (1987). The a∗ value is negative for bluish-green and positive for red-purple. The L∗ value represents the lightness of colors from 0 to 100, being small for dark colors and large for bright colors (McGuire, 1992). The firmness of fruit skin and flesh was measured using a Rheometer (model TA.XT2, Stable Micro Systems) with an 8 mm tip. For extracting PPO, fruit skin and flesh samples were separately homogenized with a polytron twice for 10 s at maximum speed using a fresh weight to volume ratio of 1:4. The homogenization buffer consisted of 50 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES, pH 7.6), 2 mM ethylenediaminetetraacetic acid, 1 mM MgCl2 , 1 mM MnCl2 , 330 mM sucrose, and 5 mM dithiothreitol (McConchie et al., 1994). The homogenate was filtered through one layer of Miracloth (Calbiochem, La Jolla, CA, USA) and centrifuged at 6000 × g for 5 min. The pellet was resuspended in a solubilization buffer consisting of
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50 mM HEPES (pH 7.6), 2% (w/v) sodium dodecyl sulfate, 3% (w/v) polyvinylpyrrolidone, and 50 mM CaCl2 , and the suspension was then incubated on ice in the dark for 30 min with periodic mixing (Valero et al., 1988). The suspension was centrifuged at 25,000 × g for 20 min and the resulting supernatant was used as a crude enzyme extract. PPO activity was assayed by monitoring absorbance change at 420 nm in 100 mM sodium citrate buffer (pH 5.0) and 50 mM catechol with the crude enzyme extract (Coseteng and Lee, 1987). The PPO activity was expressed as A420 h−1 mg−1 protein, because the products have unknown extinction coefficients. Protein was determined by the method of Bradford (1976) with bovine serum albumin as a standard. Lipid peroxidation was estimated by the level of malondialdehyde (MDA) production using a slight modification of the thiobarbituric acid (TBA) method as previously described (Du and Bramlage, 1992). The separate samples of fruit skin and flesh were completely homogenized in a solution of 0.5% TBA in 20% trichloroacetic acid with a polytron at maximum speed using a fresh weight to volume ratio of 1:10 and 1:5 for fruit skin and flesh, respectively. The homogenate was centrifuged at 20,000 × g for 15 min and the supernatant collected. The supernatants were heated in a boiling water bath for 25 min and allowed to cool in an ice bath. Following centrifugation at 20,000 × g for 15 min, the resulting supernatants were used for spectrophotometric determination of MDA. Absorbance at 532 nm for each sample was recorded and corrected for non-specific turbidity at 600 nm and for soluble sugars at 440 nm (Du and Bramlage, 1992). MDA concentration was calculated using a molar extinction coefficient of 156 mM−1 cm−1 (Buege and Aust, 1978).
3. Results and discussion Freshly harvested fruits were bruised by dropping them from a height of 50 cm onto a steel board and then stored at 0 or 20 ◦ C in temperature controlled chambers for up to 4 weeks. Immediately after the bruising, no visible injury of split or puncture on the fruits was evident, but the fruits deteriorated rapidly during storage. The first bruising symptom, color change of fruit tissues, could be observed after 2 or 3 days of storage at 20 ◦ C, and the injury became more apparent with increasing the storage duration. When the non-bruised and the bruised fruits were stored at 20 ◦ C, their skin and flesh became more reddish with increased duration of storage (Fig. 1A and B). The increase in redness was more apparent in the flesh than in the skin. However, when the fruits were stored at 0 ◦ C, no such changes were found neither in the skin nor in the flesh (Fig. 1A and B). Since the redness change was not affected by the bruising at any storage temperatures, the redness increase appeared to be associated with storage temperature, not with bruising. Both skin and flesh of the fruits stored at 20 ◦ C also became darker with increasing the storage duration (Fig. 1C and D). A higher magnitude of the decrease in lightness was observed in the flesh than in the skin and the bruising accelerated the decrease. The lightness of the skin decreased almost linearly with increasing the storage duration (Fig. 1C), whereas that of the flesh decreased rapidly during the first week of the storage and then remained relatively constant thereafter (Fig. 1D). The lightness decrease was found to be more associated with the bruising than with storage temperature. For example, the flesh tissues of the bruised fruits stored at 0 ◦ C became darker more rapidly than those of the
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Fig. 1. Changes of chromaticity in skin and flesh tissues of non-bruised and bruised persimmon fruits stored at 0 or 20 ◦ C. (A) a∗ in skin tissue; (B) a∗ in flesh tissue; (C) L∗ in skin tissue; (D) L∗ in flesh tissue. Values are the mean ± S.E. of five replicates. In some cases error bars are obscured by the symbol.
non-bruised fruits stored at 20 ◦ C (Fig. 1D). However, the lightness of the skin and flesh did not change in the non-bruised fruits stored at 0 ◦ C (Fig. 1C and D). Skin and flesh firmness decreased rapidly during storage except for the non-bruised fruits stored at 0 ◦ C (Fig. 2). The decrease in firmness was more obvious in the flesh than in the skin and it was dependent on both storage temperature and bruising. The fruits stored at 20 ◦ C lost their skin and flesh firmness more rapidly than those at 0 ◦ C and the bruising accelerated the decrease in firmness. In skin tissues, the bruising had more effect on decreasing the firmness that the storage temperature (Fig. 2A). In flesh tissues, however, higher magnitude of firmness decrease was observed in the non-bruised fruits stored at 20 ◦ C than in the bruised fruits stored at 0 ◦ C (Fig. 2B). The firmness decrease of the bruised fruits did not appear to correspond to the lightness decrease. The flesh tissues of the bruised fruits lost their lightness rapidly during the first week of storage at 20 ◦ C (Fig. 1D), but their firmness gradually decreased up to 3 weeks (Fig. 2B). The flesh deterioration of the bruised fruits appeared to be associated with the increased activity of PPO. With increasing the storage duration, PPO activity of the flesh increased more rapidly in the bruised fruits than in the non-bruised fruits (Fig. 3). The PPO activity also increased even in the non-bruised fruits stored at 0 ◦ C. During the first week of storage, however, the PPO activities did not change except for the flesh of the bruised fruits stored at 20 ◦ C. The increase in PPO activity was higher in the fruits stored at 20 ◦ C than in those at 0 ◦ C. Even in the non-bruised fruits, PPO activity significantly increased when the
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Fig. 2. Changes of firmness in skin (A) and flesh tissues (B) of non-bruised and bruised persimmon fruits stored at 0 or 20 ◦ C. Values are the mean ± S.E. of five replicates.
Fig. 3. Changes of PPO activity in flesh tissues of non-bruised and bruised persimmon fruits stored at 0 or 20 ◦ C. Values are the mean ± S.E. of five replicates.
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fruits were stored at 20 ◦ C. The bruising had more effect on increasing PPO activity than storage temperature. However, PPO activities in the skin of the non-bruised and the bruised fruits were found to be negligible and changed insignificantly at any storage duration and temperatures (data not shown). PPO activity has been well known to have the enzymatic browning potential in fresh fruits (Lee et al., 1990; Gradziel and Wang, 1993) and to be responsible for tissue browning in bruised fruits (Van Lelyveld and Bower, 1984; Sciancalepore, 1985; Nicolas et al., 1994). The tissue browning following bruising is primarily due to phenolic oxidation resulting from the mixing of phenolics and PPO upon the collapse of cellular compartmentalization (Rouet-Mayer et al., 1990; Shewfelt, 1993). In our study, mechanical impacts resulting from the dropping may cause unseen physical damage below the surface of the persimmon fruits. These impacts may cause damage to the cellular membranes. Senescence also results in decompartmentalization of PPO and its substrates. Thus, the browning of olives (Sciancalepore and Longone, 1984) and other fruits (Vamos-Vigyazo and Nadudvari-Markus, 1983) is dependent on PPO activity, although the functional significance of PPO in senescence is not obvious. Considering that the tissue browning is both qualitatively and quantitatively substrate dependent (Coseteng and Lee, 1987), strict correlation between PPO activity and tissue browning potential may not exist. In the present study, the PPO activity change did not exactly correspond to the lightness and the firmness decrease. These results indicate that PPO is not the only factor influencing the deterioration associated with bruising. No significant changes in lipid peroxidation, as expressed by MDA production, were found between the bruised and the non-bruised fruits during storage either at 0 or 20 ◦ C (data not shown). Thus, the possibility that lipid peroxidation is associated with the fruit deterioration can be excluded. By contrast, increased lipid peroxidation has been observed during the development of chilling injury in many harvested fruits and vegetables and of superficial scald in apples (Shewfelt and del Rosario, 2000). We are currently assaying cell wall hydrolases to determine if they also contribute the deterioration following bruising.
Acknowledgements This work was supported by Wonkwang University in 2001 and Agricultural Plant Stress Research Center (grant no. R11-2001-9203003) funded by Korea Science and Engineering Foundation.
References Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein–dye binding. Anal. Biochem. 72, 248–254. Brusewitz, G.H., Bartsch, J.A., 1989. Impact parameters related to postharvest bruising of apples. Trans. Am. Soc. Agric. Eng. 32, 953–957. Buege, T.A., Aust, S.D., 1978. Membrane lipid peroxidation. Meth. Enzymol. 52, 302–310. Coseteng, M.Y., Lee, C.Y., 1987. Changes in apple polyphenoloxidase and polyphenol concentrations in relation to degree of browning. J. Food Sci. 52, 985–989.
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Du, Z., Bramlage, W.J., 1992. Modified thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. J. Agric. Food Chem. 40, 1566–1570. Gradziel, T.M., Wang, D., 1993. Evaluation of brown rot resistance and its relation to enzymatic browning in clingstone peach germplasm. J. Am. Soc. Hort. Sci. 118, 675–679. Lee, C.Y., Kagan, V., Jaworski, A.W., Brown, S.K., 1990. Enzymatic browning in relation to phenolic compounds and polyphenoloxidase activity among various peach cultivars. J. Agric. Food Chem. 38, 99–101. Marshall, D.E., Brook, R.C., 1999. Reducing bell pepper bruising during postharvest handling. HortTechnology 9, 254–258. McConchie, R., Lang, N.S., Lax, A.R., Lang, G.A., 1994. Reexamining polyphenol oxidase, peroxidase, and leaf-blackening activity in Protea. J. Am. Soc. Hort. Sci. 119, 1248–1254. McGuire, R.G., 1992. Reporting of objective color measurements. HortScience 27, 1254–1255. Nicolas, J.J., Richard-Forget, F.C., Goupy, P.M., Amiot, M.J., Aubert, S.Y., 1994. Enzymatic browning reactions in apple and apple products. CRC Crit. Rev. Food Sci. Nutr. 34, 109–157. Quintana, M.E.G., Paull, R.E., 1993. Mechanical injury during postharvest handling of ‘Solo’ papaya fruit. J. Am. Soc. Hort. Sci. 118, 618–622. Rouet-Mayer, M., Ralambosoa, J., Philippon, J., 1990. Roles of o-quinones and their polymers in the enzymic browning of apples. Phytochemistry 29, 435–440. Sapers, G.M., Douglas Jr., F.W., 1987. Measurement of enzymatic browning at cut surfaces and in juice of raw apple and pear fruits. J. Food Sci. 52, 1258–1262. Sciancalepore, V., 1985. Enzymatic browning in five olive varieties. J. Food Sci. 50, 1194–1195. Sciancalepore, V., Longone, V., 1984. Polyphenol oxidase activity and browning in green olives. J. Agric. Food Chem. 32, 320–321. Shewfelt, R.L., 1993. Stress physiology: a cellular approach to quality. In: Shewfelt, R.L., Prussia, S.E. (Eds.), Postharvest Handling: A Systems Approach. Academic Press, San Diego, CA, USA, pp. 257–276. Shewfelt, R.L., del Rosario, B.A., 2000. The role of lipid peroxidation in storage disorders of fresh fruits and vegetables. HortScience 35, 575–579. Valero, E., Varón, R., Garc´ıa-Carmona, F., 1988. Characterization of polyphenol oxidase from Airen grapes. J. Food Sci. 53, 1482–1484. Vamos-Vigyazo, L., Nadudvari-Markus, V., 1983. Inactivation of polyphenol oxidase and depletion of o-dihydroxy phenol content during the enzymatic browning reaction of fruit tissues. Acta Aliment. 12, 1–9. Van Lelyveld, L.J., Bower, J.P., 1984. Enzyme reactions leading to avocado fruit mesocarp discoloration. J. Hort. Sci. 59, 257–263.
Bruising injury of persimmon (Diospyros kaki cv. Fuyu) fruits Hee Jae Lee a,∗ , Tae-Choon Kim b , Su Jin Kim a , Seung Je Park c a b
School of Plant Science, Seoul National University, Seoul 151-742, Republic of Korea Department of Horticulture, Wonkwang University, Iksan 570-749, Republic of Korea c Department of Agricultural Machinery Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea Accepted 16 April 2004
Abstract This study was performed to monitor the deterioration of bruised persimmon (Diospyros kaki cv. Fuyu) fruits. Freshly harvested fruits were bruised by dropping them from a height of 50 cm onto a steel board and then stored at 0 or 20 ◦ C in temperature controlled chambers for up to 4 weeks. Immediately after the bruising, no visible injury on the fruits was evident, but the fruits deteriorated rapidly during storage. The skin tissues of the fruits stored at 20 ◦ C became more reddish with the duration of the storage, but no such changes were found with the fruits stored at 0 ◦ C. The increase in redness of the skin tissues appeared to be associated with storage temperature, but not with the bruising. The skin tissues also became darker when stored at 20 ◦ C than at 0 ◦ C and this tendency was more obvious with the bruised fruits. Flesh firmness decreased rapidly during storage except for the non-bruised fruits stored at 0 ◦ C. Even the non-bruised fruits rapidly lost their flesh firmness at 20 ◦ C. No significant changes in lipid peroxidation, as expressed by malondialdehyde production, were found between the bruised and the non-bruised fruits during the storage either at 0 ◦ C or at 20 ◦ C. This implies that the fruit deterioration caused by bruising is not due to the consequences of lipid peroxidation. Polyphenol oxidase activity increased more rapidly in the bruised fruits than in the non-bruised fruits during storage. The bruising had more effect on increasing polyphenol oxidase activity than did the storage temperature. Although the increase in polyphenol oxidase activity appeared to be associated with the visual deterioration of the bruised fruits, it did not exactly correspond to the physical deterioration. These results indicate that polyphenol oxidase is not the only factor influencing the deterioration associated with bruising. Cell wall hydrolases are currently being assayed to determine if they also contribute the deterioration following bruising. © 2004 Elsevier B.V. All rights reserved. Keywords: Bruising; Diospyros kaki; Lipid peroxidation; Mechanical injury; Polyphenol oxidase
∗
Corresponding author. Tel.: +82-2-880-4566; fax: +82-2-873-2056. E-mail address: [email protected] (H.J. Lee). 0304-4238/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2004.04.016
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H.J. Lee et al. / Scientia Horticulturae 103 (2005) 179–185
1. Introduction With the increasing use of mechanical equipments for the harvesting and packing of fruits, the mechanical injury on fruits has become a very important problem (Brusewitz and Bartsch, 1989; Marshall and Brook, 1999). Mechanical injury, such as bruises, cuts, and abrasions, causes marked deterioration in fruit quality (Quintana and Paull, 1993; Shewfelt, 1993). Bruising can occur when the fruit is carelessly dropped onto hard surfaces or subjected to harsh impacts during harvesting and packing. The bruising is initiated by the breakage of cell membranes, allowing cytoplasmic enzymes to act on sequestered substrates (Rouet-Mayer et al., 1990; Shewfelt, 1993). The resultant browning is caused by the enzyme action on phenolic substrates. Polyphenol oxidase (PPO) is well known enzyme responsible for tissue browning in mechanically injured fruits (Van Lelyveld and Bower, 1984; Sciancalepore, 1985; Nicolas et al., 1994). PPO catalyzes the oxidation of phenolic compounds to o-quinones which subsequently polymerize to form dark-colored pigments (Rouet-Mayer et al., 1990). During harvesting, grading, and transport of persimmon fruits, the fruits often deteriorate due to bruising and subsequent storage. However, no comprehensive study has been published on bruising injury of persimmon fruits. In the present study, the deterioration of bruised persimmon (Diospyros kaki cv. Fuyu) fruits were monitored with respect to fruit discoloration, firmness decrease, and lipid peroxidation during storage. PPO activity was also monitored to determine if it is associated with the deterioration of the bruised fruits.
2. Materials and methods Unblemished persimmon fruits of ‘Fuyu’ cultivar were purchased from an orchard in Jinyoung county, Korea at their commercial harvest. The fruits were dropped from height of 50 cm onto a steel board and caught after one bounce. The impact area was marked with black indelible ink. Control fruits were not dropped. The fruits were then placed in cupped plastic trays and stored at 0 or 20 ◦ C in temperature controlled chambers for up to 4 weeks. One hour prior to sampling, the fruits to be examined were removed from the chambers and equilibrated to room temperature. The discoloration of the skin and the flesh was determined as changes in a∗ and L∗ values by using a chromameter (model CR-300, Minolta Co., Ltd., Japan) as suggested by Sapers and Douglas (1987). The a∗ value is negative for bluish-green and positive for red-purple. The L∗ value represents the lightness of colors from 0 to 100, being small for dark colors and large for bright colors (McGuire, 1992). The firmness of fruit skin and flesh was measured using a Rheometer (model TA.XT2, Stable Micro Systems) with an 8 mm tip. For extracting PPO, fruit skin and flesh samples were separately homogenized with a polytron twice for 10 s at maximum speed using a fresh weight to volume ratio of 1:4. The homogenization buffer consisted of 50 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES, pH 7.6), 2 mM ethylenediaminetetraacetic acid, 1 mM MgCl2 , 1 mM MnCl2 , 330 mM sucrose, and 5 mM dithiothreitol (McConchie et al., 1994). The homogenate was filtered through one layer of Miracloth (Calbiochem, La Jolla, CA, USA) and centrifuged at 6000 × g for 5 min. The pellet was resuspended in a solubilization buffer consisting of
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50 mM HEPES (pH 7.6), 2% (w/v) sodium dodecyl sulfate, 3% (w/v) polyvinylpyrrolidone, and 50 mM CaCl2 , and the suspension was then incubated on ice in the dark for 30 min with periodic mixing (Valero et al., 1988). The suspension was centrifuged at 25,000 × g for 20 min and the resulting supernatant was used as a crude enzyme extract. PPO activity was assayed by monitoring absorbance change at 420 nm in 100 mM sodium citrate buffer (pH 5.0) and 50 mM catechol with the crude enzyme extract (Coseteng and Lee, 1987). The PPO activity was expressed as A420 h−1 mg−1 protein, because the products have unknown extinction coefficients. Protein was determined by the method of Bradford (1976) with bovine serum albumin as a standard. Lipid peroxidation was estimated by the level of malondialdehyde (MDA) production using a slight modification of the thiobarbituric acid (TBA) method as previously described (Du and Bramlage, 1992). The separate samples of fruit skin and flesh were completely homogenized in a solution of 0.5% TBA in 20% trichloroacetic acid with a polytron at maximum speed using a fresh weight to volume ratio of 1:10 and 1:5 for fruit skin and flesh, respectively. The homogenate was centrifuged at 20,000 × g for 15 min and the supernatant collected. The supernatants were heated in a boiling water bath for 25 min and allowed to cool in an ice bath. Following centrifugation at 20,000 × g for 15 min, the resulting supernatants were used for spectrophotometric determination of MDA. Absorbance at 532 nm for each sample was recorded and corrected for non-specific turbidity at 600 nm and for soluble sugars at 440 nm (Du and Bramlage, 1992). MDA concentration was calculated using a molar extinction coefficient of 156 mM−1 cm−1 (Buege and Aust, 1978).
3. Results and discussion Freshly harvested fruits were bruised by dropping them from a height of 50 cm onto a steel board and then stored at 0 or 20 ◦ C in temperature controlled chambers for up to 4 weeks. Immediately after the bruising, no visible injury of split or puncture on the fruits was evident, but the fruits deteriorated rapidly during storage. The first bruising symptom, color change of fruit tissues, could be observed after 2 or 3 days of storage at 20 ◦ C, and the injury became more apparent with increasing the storage duration. When the non-bruised and the bruised fruits were stored at 20 ◦ C, their skin and flesh became more reddish with increased duration of storage (Fig. 1A and B). The increase in redness was more apparent in the flesh than in the skin. However, when the fruits were stored at 0 ◦ C, no such changes were found neither in the skin nor in the flesh (Fig. 1A and B). Since the redness change was not affected by the bruising at any storage temperatures, the redness increase appeared to be associated with storage temperature, not with bruising. Both skin and flesh of the fruits stored at 20 ◦ C also became darker with increasing the storage duration (Fig. 1C and D). A higher magnitude of the decrease in lightness was observed in the flesh than in the skin and the bruising accelerated the decrease. The lightness of the skin decreased almost linearly with increasing the storage duration (Fig. 1C), whereas that of the flesh decreased rapidly during the first week of the storage and then remained relatively constant thereafter (Fig. 1D). The lightness decrease was found to be more associated with the bruising than with storage temperature. For example, the flesh tissues of the bruised fruits stored at 0 ◦ C became darker more rapidly than those of the
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Fig. 1. Changes of chromaticity in skin and flesh tissues of non-bruised and bruised persimmon fruits stored at 0 or 20 ◦ C. (A) a∗ in skin tissue; (B) a∗ in flesh tissue; (C) L∗ in skin tissue; (D) L∗ in flesh tissue. Values are the mean ± S.E. of five replicates. In some cases error bars are obscured by the symbol.
non-bruised fruits stored at 20 ◦ C (Fig. 1D). However, the lightness of the skin and flesh did not change in the non-bruised fruits stored at 0 ◦ C (Fig. 1C and D). Skin and flesh firmness decreased rapidly during storage except for the non-bruised fruits stored at 0 ◦ C (Fig. 2). The decrease in firmness was more obvious in the flesh than in the skin and it was dependent on both storage temperature and bruising. The fruits stored at 20 ◦ C lost their skin and flesh firmness more rapidly than those at 0 ◦ C and the bruising accelerated the decrease in firmness. In skin tissues, the bruising had more effect on decreasing the firmness that the storage temperature (Fig. 2A). In flesh tissues, however, higher magnitude of firmness decrease was observed in the non-bruised fruits stored at 20 ◦ C than in the bruised fruits stored at 0 ◦ C (Fig. 2B). The firmness decrease of the bruised fruits did not appear to correspond to the lightness decrease. The flesh tissues of the bruised fruits lost their lightness rapidly during the first week of storage at 20 ◦ C (Fig. 1D), but their firmness gradually decreased up to 3 weeks (Fig. 2B). The flesh deterioration of the bruised fruits appeared to be associated with the increased activity of PPO. With increasing the storage duration, PPO activity of the flesh increased more rapidly in the bruised fruits than in the non-bruised fruits (Fig. 3). The PPO activity also increased even in the non-bruised fruits stored at 0 ◦ C. During the first week of storage, however, the PPO activities did not change except for the flesh of the bruised fruits stored at 20 ◦ C. The increase in PPO activity was higher in the fruits stored at 20 ◦ C than in those at 0 ◦ C. Even in the non-bruised fruits, PPO activity significantly increased when the
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Fig. 2. Changes of firmness in skin (A) and flesh tissues (B) of non-bruised and bruised persimmon fruits stored at 0 or 20 ◦ C. Values are the mean ± S.E. of five replicates.
Fig. 3. Changes of PPO activity in flesh tissues of non-bruised and bruised persimmon fruits stored at 0 or 20 ◦ C. Values are the mean ± S.E. of five replicates.
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fruits were stored at 20 ◦ C. The bruising had more effect on increasing PPO activity than storage temperature. However, PPO activities in the skin of the non-bruised and the bruised fruits were found to be negligible and changed insignificantly at any storage duration and temperatures (data not shown). PPO activity has been well known to have the enzymatic browning potential in fresh fruits (Lee et al., 1990; Gradziel and Wang, 1993) and to be responsible for tissue browning in bruised fruits (Van Lelyveld and Bower, 1984; Sciancalepore, 1985; Nicolas et al., 1994). The tissue browning following bruising is primarily due to phenolic oxidation resulting from the mixing of phenolics and PPO upon the collapse of cellular compartmentalization (Rouet-Mayer et al., 1990; Shewfelt, 1993). In our study, mechanical impacts resulting from the dropping may cause unseen physical damage below the surface of the persimmon fruits. These impacts may cause damage to the cellular membranes. Senescence also results in decompartmentalization of PPO and its substrates. Thus, the browning of olives (Sciancalepore and Longone, 1984) and other fruits (Vamos-Vigyazo and Nadudvari-Markus, 1983) is dependent on PPO activity, although the functional significance of PPO in senescence is not obvious. Considering that the tissue browning is both qualitatively and quantitatively substrate dependent (Coseteng and Lee, 1987), strict correlation between PPO activity and tissue browning potential may not exist. In the present study, the PPO activity change did not exactly correspond to the lightness and the firmness decrease. These results indicate that PPO is not the only factor influencing the deterioration associated with bruising. No significant changes in lipid peroxidation, as expressed by MDA production, were found between the bruised and the non-bruised fruits during storage either at 0 or 20 ◦ C (data not shown). Thus, the possibility that lipid peroxidation is associated with the fruit deterioration can be excluded. By contrast, increased lipid peroxidation has been observed during the development of chilling injury in many harvested fruits and vegetables and of superficial scald in apples (Shewfelt and del Rosario, 2000). We are currently assaying cell wall hydrolases to determine if they also contribute the deterioration following bruising.
Acknowledgements This work was supported by Wonkwang University in 2001 and Agricultural Plant Stress Research Center (grant no. R11-2001-9203003) funded by Korea Science and Engineering Foundation.
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