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Postharvest Browning of Litchi Fruit by Water Loss and its Prevention by Controlled Atmosphere Storage at High Relative Humidity
Lebensm.-Wiss. u.-Technol., 32, 278}283 (1999)
Postharvest Browning of Litchi Fruit by Water Loss and its Prevention by Controlled Atmosphere Storage at High Relative Humidity Y. M. Jiang* and J. R. Fu
Y. M. Jiang: South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650 (P.R. China) J. R. Fu: School of Life Science, Zhongshan University, Guangzhou 50275 (P.R. China) (Received June 25, 1998; accepted March 18, 1999)
Postharvest browning of litchi fruit caused by water loss was investigated in relation to anthocyanin content, polyphenol oxidase (PPO) activity, pH value, and membrane permeability. Total anthocyanin concentration decreased with pericarp desiccation. Fruit stored at 90% relative humidity (RH) had the lowest loss of total anthocyanins over the storage period, while total anthocyanin content of fruit stored at 60 and 70% RHs signixcantly declined. Polyphenol oxidase activity with 4-methylcatechol as a substrate tended to increase with reduced storage RH, but no activity towards anthocyanins was detected. Furthermore, in the presence of 4-methylcatechol, the oxidation of anthocyanins by PPO was observed. The pH value was initially low, and then increased with pericarp desiccation, which was associated with increased PPO activity. Conversely, the integrity of membrane systems, which increased with reduced storage RH and increased storage time, showed that membrane structure became more vulnerable. These results indicate that oxidation of both phenolic and anthocyanins by PPO seems to awect the response of litchi fruit to water loss in terms of browning, and suggest that substrate-enzyme contact should be emphasized as this could promote enzymatic reaction leading to browning. Storage at 1 3C under controlled atmosphere (3}5% O2 and 3}5% CO2 ) at 90% RH gave good browning control and fruit quality maintenance.
1999 Academic Press Keywords: browning; controlled atmosphere; litchi; storage; water loss
Introduction Litchi (¸itchi chinensis Sonn.) is a tropical and subtropical fruit of high commercial value in the international market. However, litchi fruit rapidly lose their attractive red colour after harvest due to super"cial pericarp browning. Pericarp browning reduces litchi commercial value and has been considered the main postharvest problem (1}3). Postharvest browning of litchi was thought to be a rapid degradation of the red pigments by polyphenol oxidase, forming brown-colour byproducts (4, 5). Prasad and Jha (6), and Lee and Wicker (7) identi"ed the red pigments in litchi pericarp as anthocyanins. Anthocyanin degradation occurred concurrently with pericarp browning (8), while Polyphenol oxidase (PPO) activity increased (9, 10). Polyphenol oxidase can degrade anthocyanins in some plants (11, 12), but it has a low a$nity for pigments (13). Water loss results in a series of biochemical and physiological changes, including substrate-enzyme contact. Scott et al. (14), and Underhill and Simons (15) reported that postharvest browning of litchi fruit, due to skin
*To whom correspondence should be addressed.
0023-0438/99/0502780#06 $30 00/0 1999 Academic Press
desiccation, was highly correlated with weight loss. Chen et al. (16), and Paull and Chen (17) also reported that wrapping fruit helped reduce desiccation and delay colour change. Application of chitosan coating which minimized fruit desiccation signi"cantly delayed pericarp browning (18). However, to what extent reduced water loss rate can o!set browning, is unknown. The objectives of this study were to investigate the relationship between water loss in controlled relative humidity environments and fruit browning in relation to anthocyanin, polyphenol oxidase, pH, and membrane permeability, and then to control fruit browning during controlled atmosphere storage. Materials and Methods Litchi (¸itchi chinensis Sonn.) fruit cv. Huaizhi (a major cultivar in China), at the fully coloured, commercially matured stage, were obtained from a commercial orchard in Guangzhou. The fruit were dipped for 3 min in 0.1% TBZ (thiabendazole, Deco Chemicals, U.S.A.), and then air dried. Thiabendazole gives good control of postharvest diseases of litchi fruit (16). Fruit were sorted for freedom from visual defects and uniformity of shape,
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colour and size, and then randomly divided into two lots for storage at various relative humidities (RHs) and for controlled atmosphere storage at high RH. Storage at various RHs Fruit from the "rst lot were stored at various RHs at 20 3C and water loss, browning index, anthocyanin content, polyphenol oxidase activity, pH value, and relative leakage rate monitored through the experimental period.
Storage conditions Relative humidity was maintained in a temperature controlled room (24.5 m), set at 20 3C, with the following procedures. A constant pressure air supply was divided into two streams, one dried to about 35%, and the other humidi"ed to '95% RH. These streams were then recombined, via metering values, to give the desired RHs (60, 70, 80, or 90%) and #ow rate (1 L/min). The RHs can be maintained once adjusted throughout the course of experiments, with a #uctuation of only about 0.5%. The RH of the air leaving the storage container (2.12 m) was measured at a position midway between the top of the fruit samples and the exhaust port. An electrohygrometer sensor (model PCRC-55T, Phys-Chemical Research Corp, U.S.A.) was used to measure RH. The RH sensor was calibrated within an appropriate humidity range using saturated salt solutions. A rotometer was used to maintain #ow rate.
Measurements of water and weight losses Water loss from pericarp was calculated progressively, based on a comparison of the water contents in the pericarp at harvest and various storage times, using 18 fruit and expressed as a percentage (di!erence in the water content of the pericarp between at-harvest and storage time, divided by that at harvest). Water content was determined by drying the pericarp, while percent weight loss from the whole fruit, and aril plus seed was also recorded.
Browning assessment Browning was assessed by measuring the extent of the total browned area on each fruit pericarp from each of the RHs using 18 fruit on the following scale: 1"no browning (excellent quality); 2"slight browning; 3"(1/4 browning; 4"1/4}1/2 browning; 5"'1/2 browning (poor quality). The browning index was calculated using the formula & (browning scale;percentage of corresponding fruit within each class). Fruit evaluated at an index'3.0 were considered unacceptable for marketing.
Extraction and purixcation of litchi peel anthocyanins According to the method of Lee and Wicker (7), an ethanolic solution of litchi peel extract was prepared by homogenizing the pericarp of litchi fruit (25 g) with acidi-
"ed ethanol (15 mL of 1.5 mol/L HCl in 85 mL of 95% ethanol, per 100 mL), refrigerating overnight, "ltering through Whatman C 41, and diluting to 250 mL with acidi"ed ethanol. To remove the polymerized dark brown pigments from concentrated litchi skin, extract (1.0 mL) was run through a Sephadex G-25 (60;2.5 cm) column and eluted with 50% acetone/1% formic acid/water. The brownish-red colour pigments, eluted from the column, were discarded. The bright, red-coloured anthocyanin pigments were collected, combined, and concentrated to a small volume by rotary evaporation to remove the acetone. These partially puri"ed, aqueous anthocyanin pigments were absorbed onto a Water Sep-Pak C-18 cartridge, U.S.A., washed with 2 mL of water, eluted with 3 mL of acidi"ed methanol, and concentrated by #ushing with nitrogen gas to (0.5 mL. Anthocyanin assay Anthocyanin content was measured according to the method of Pirie and Mullins (19). Litchi fruit pericarp (2 g) from nine fruit was "nely sliced and extracted with 50 mL HCl-methanol (1 mL HCl in 99 mL methanol for 2 h. The extract was "ltered and diluted, and its absorbance measured at 530 nm. Anthocyanin content was expressed as absorbance at 530 nm/g dry weight (DW) based on the water content of the pericarp. Enzyme assay and protein determination Polyphenol oxidase (PPO) activity was assayed with 4methylcatechol as a substrate according to the method of Zauberman et al. (20). Two grammes of litchi peel from nine fruit was ground with 10 mL of 0.1 mol/L sodium phosphate bu!er (pH 6.8) and 0.2 g of polyvinylpyrrolidone (PVP, insoluble). After centrifugation at 19000 g for 20 min, the supernatant was collected as the crude enzyme extract. The assay of the enzyme activity was performed using 1.0 mL of 0.1 mol/sodium phosphate bu!er (pH 6.8), 0.5 mL of 100 mmol/L 4-methylcatechol, and 0.5 mL enzyme solution. The increase in absorbance at 410 nm at 25 3C was recorded automatically for 5 min (Beckman DU-7). One unit of enzyme activity was de"ned as an increase of 0.01 in absorbance per min. To measure anthocyanin oxidation catalyzed by PPO, 0.1 mL of the concentrated anthocyanins was diluted to 50 mL with 0.1 mol/L sodium phosphate bu!er (pH 6.8), and 0.5 mL of the dilution used as the PPO substrate instead of 4-methylcatechol. However, the percent decrease in absorbance at 510 nm at 25 3C, as compared to the initial anthocyanin solution, was recorded automatically for 30 min to monitor anthocyanin degradation by the method of Pi!eri and Cultrera (21). Protein content was determined according to the dyebinding method of Bradford (22) with bovine serum protein as the standard. pH measurement The pH value was measured according to the method of Underhill and Critchley (8). Pericarp tissues (6 g) from
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nine fruit was washed in distilled water to remove adhesive juice. The tissues were "nely sliced and homogenized in 50 mL distilled water for 1 min. The pH was measured while stirring the homogenate.
Measurement of relative leakage rate Membrane permeability, expressed by relative leakage rate, was determined according to the method of Autio and Bramlage (23). Discs were removed with a cork borer (10 mm in diameter) from the equatorial region of nine fruit pericarps. Thirty discs were rinsed twice and then incubated in 25 mL of distilled water at 2 3C, and shaken for 30 min. Electrolyte leakage was determined with a conductivity meter (model DDS-11A, Shanghai Scienti"c Instruments, China), and again after boiling another batch of discs for 15 min and cooling to 25 3C (total electrolytes). Relative leakage rate was expressed as percent of total electrolytes.
Controlled atmosphere storage at high RH The second batch of fruit was stored at low temperature under controlled atmosphere at high RH. A controlled atmosphere of 3}5% O and 3}5% CO at 13C was considered the optimum storage condition (16, 24). In this experiment, the atmosphere was applied as a continuous #ow (100 mL/min, about 90% RH) over fruit using gas controllers (model II-2, China Agricultural University). The container consisted of an impermeable plastic bag with a gas inlet and outlet at the opposite end of the bag. One end of the bag could be temporarily opened to allow fruit withdrawal during the trial. Fruit stored under air instead of controlled atmosphere conditions was used as a control. After 30 d of storage, the fruit were measured for weight loss, browning index, eating quality, and disease incidence. The weight loss of fruit and browning index of pericarp were determined according to the method described previously. Eating quality of pulp was assessed hedonically using a sixmember panel; 36 fruit were randomly selected and rated for quality (1"poor, 9"excellent). Eating quality rating was analysed by analysis of variance for a completely randomized design. Disease development was monitored by randomly collecting 18 fruit and recording the percent of fruit showing visible fungal growth or bacterial lesions on the peel surface. The contents of total soluble solids, titratable acidity and ascorbic acid were analysed at harvest and the end of the experiment. A 20 g sample of pulp from 12 fruit, was homogenised in a grinder and the supernatant after centrifugation at 20,000 g (Beckman J2-2, U.S.A.) for 15 min was collected to determine the contents of total soluble solids, titratable acidity and ascorbic acid. The total soluble solids were measured using a hand refractometer (J1}3A, Guangdong Scienti"c Instruments, China), while titratable acidity, expressed as percent citric acid, was determined by a 0.1 mol/L NaOH titration method, and ascorbic acid was analysed using the 2.6}dichlorophenolindophenol titration method (16).
Results Storage at various relative humidities E!ects of storage under various RH conditions at 20 3C on water loss, browning index, anthocyanin content, polyphenol oxidase activity, pH value, and relative leakage rate are shown in Fig. 1.
Water loss Water loss rate from the pericarp was signi"cantly reduced by increasing relative humidity during storage (Fig. 1A). The maximum water loss from the pericarp of the fruit after 3 d of storage was '50% at 60% RH, while it was only 19% at 90% RH. Fruit also lost weight progressively with storage time, but no marked weight loss in aril and seed part was observed (data not shown). These results indicate that selective dehydration, particularly in pericarp. occurred during storage, with little movement of water between aril and pericarp.
Browning Litchi pericarp was highly susceptible to desiccation, and skin browning increased with storage time (Fig. 1B). After 2 d of storage at 60 and 70% RHs, the initial red colour of the fruit had largely disappeared and the fruit were commercially unacceptable for marketing due to rapid browning in appearance caused by water loss, while fruit stored at 90% RH took a longer time (about 7 d) to develop this level of browning.
Anthocyanins As shown in Fig. 1C, total anthocyanin concentration decreased with storage time. A greater percent anthocyanin loss occurred during storage from 1 to 2 d than from 2 to 3 d, especially at lower RH storage. Fruit stored at 90% RH has the lowest loss of total anthocyanins over the storage period, while total anthocyanin content of fruit stored at 60 and 70% RHs signi"cantly reduced.
Polyphenol oxidase activity Polyphenol oxidase activity increased with reduced storage RH. After 2 d of storage at lower RHs (60 and 70%) fruit showed decreased enzymatic activity, but the PPO activity of fruit at 90% RH did not markedly change (Fig. 1D). In this study, no activity of PPO towards anthocyanins was detected, but in the presence of 4methylcatechol, PPO can oxidize anthocyanins and the enzyme activity was much higher than that with 4methylcatechol alone (data not shown).
pH The pH value was initially low, and tended to increase with pericarp desiccation (Fig. 1E) However, no marked change in pH was observed during storage at 80 and 90% RHs. This can re#ect a better maintenance of the
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Fig. 1 E!ects of various RHs in storage on water loss (A), browning index (B), anthocyanin content (C), PPO activity (D), pH value (E), and relative leakage rate (F). The water loss, browning index, anthocyanin content, PPO activity, pH value, and relative leakage rate were 0%, 1.24, 1.23 (530 nm/g DW), 1.05;10 unit/mg protein, 4.12, and 27.06% at harvest, respectively. The values of the water loss and browning index are the means for 18 fruit, while those of anthocyanin content, PPO activity, pH value, and relative leakage rate are based on extraction in triplicate derived from nine fruit. The vertical bars indicate standard errors. (䊉)"60% RH; (䊏)"70% RH; (䉱)"80% RH; and (䉲)"90% RH
Table 1 Weight loss, browning index, eating quality and disease incidence of litchi fruit after 30 d of storage at 13C under controlled atmosphere at high relative humidity
Air (control) 3}5% O #3}5% CO
Weight loss (%)
Browning index
Eating quality
Disease incidence (%)
4.8a 1.2b
2.4a 1.4b
6.3b 7.8a
5.5a 1.5b
Weight loss was expressed as percentage, while browning index was calculated using the formula &(browning scale;percentage of corresponding fruit within each class). Eating quality rating of pulp was assessed hedonically by a six-member panel and disease incidence monitored by recording the percentage of fruit showing visible fungal growth or bacterial lesions on the peel surface. Weight loss, browning index, eating quality, and disease incidence were 0%, 1.2, 8.1 and 0% at harvest, respectively The corresponding means within a column followed by the same letter are not signi"cantly di!erent at the 5% level
cellular environment of fruit stored under high RH conditions. Relative leakage rate The integrity of membrane systems can be expressed as relative leakage rate (23). The data illustrated in Fig. 1F, in which the relative leakage rate increased with reduced RHs and increased storage time, showed that membrane systems became more vulnerable to leakage. As a result, this may lead to enhanced substrate-enzyme contact.
Controlled atmosphere storage at high RH The weight loss, the decline in eating quality, and the disease incidence were generally accelerated with storage time. These were delayed by controlled atmosphere storage at high RH (Table 1). After 30 d of storage, the fruit under controlled atmospheres retained their bright colour, and had a higher content of ascorbic acid and lower loss in weight (Table 2), whereas the initial red colour of control fruit had largely disappeared.
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Table 2 Total soluble solids, total titratable acidity and ascorbic acid of litchi fruit after 30 d of storage at 13C under controlled atmosphere at high relative humidity
Air (control) 3}5% O #3}5% CO
Total soluble solids (%)
Total titratable acidity (%)
Ascorbic acid (mg/100 g pulp)
15.3ab 16.1a
0.58ab 0.63a
17.9b 24.5a
The contents of total soluble solids, total titratable acidity and ascorbic acid were 16.6%, 0.67% and 28.6 mg/100 g pulp at harvest, respectively The corresponding means within a column followed by the same letter are not signi"cantly di!erent at the 5% level
Discussion Water is the major constituent of all fruit, and, therefore, regulating its loss is an important requirement for maintaining postharvest quality. Litchi browning correlated well with desiccation, which is the major postharvest problem (15). At the lower humidities litchi fruit rapidly lost their attractive red colour and skin browning became commercially unacceptable after 2 d of storage (Fig. 1B), while fruit stored at 90% RH took a long time to develop the same level of browning. In this study, the "ndings that no marked weight loss from #esh over the storage period was observed suggests that water loss of litchi fruit was from peel rather than pulp. Thus, the browning control and quality maintenance of litchi fruit stored under controlled atmosphere at high RH were attributed to minimizing desiccation (Tables 1 and 2). Postharvest browning of litchi was thought to be due to anthocyanin degradation, but no evidence of browning was observed in the anthocyanin-containing mesocarp, and application of 0.1 mol/L HCl to already brown tissue signi"cantly improved pericarp redness, independently of anthocyanin synthesis (8, 20). As the colourless anthocyanin forms were more degradable by PPO, anthocyanin decolorization may involve its structural changes (13). Anthocyanin structure and, more importantly, colour are directly dependent on pH (25). Anthocyanin decolorisation due to changes in pH has also been reported in the visual colour of cherry and peach fruits (21, 26). We found that pH values of litchi pericarp homogenate changed from 4.12 to 4.82 after 3 d of storage at 60% RH, which was associated with increased PPO activity with a range of pH from 4.2 to 7.0 (27) Polyphenol oxidase is considered the enzyme responsible for browning of fruit and vegetables (28). Lin et al. (9) reported that PPO activity in litchi pericarp rapidly increased after harvest. Fruit stored at lower RHs showed higher activities of this enzyme (Fig. 1D). In the presence of 4-methylcatechol, the oxidation of anthocyanins by PPO was signi"cantly enhanced. Conversely, the rapid increase of relative leakage rate (Fig. 1F), which may result from the breakdown of membrane system, provided evidence that some phenolics could spill from vacuoles, being consistent with results from Liu et al. (29). As a result, these changes can lead to mixing of anthocyanins, phenolics and PPO, and in turn, accelerate the degradation of anthocyanins. Our results
indicate that oxidation of both phenolics and anthocyanins by PPO seems to a!ect the response of litchi fruit to water loss in terms of browning, and suggest that substrate-enzyme contact should be emphasized as this can promote enzymatic reactions leading to browning. Acknowledgment The "nancial support provided by International Foundation for Science (E/2265), Stockholm, Sweden, is greatly appreciated. References 1 HUANG, P. Y. AND SCOTT, K. J. Control of rotting and browning of litchi fruit after harvest at ambient temperature in China. ¹ropical Agriculture, 62, 2}4 (1985) 2 NIP, W. K. Handling and preservation of lychee (¸itchi chinensis Sonn.) with emphasis on colour retention. ¹ropical Science, 28, 5}11 (1988) 3 HOLCROFT, D. M. AND MITCHAM, E. J. Postharvest physiology and handling of litchi (Litchi chinensis Sonn.) Postharvest Biology & ¹echnology, 9, 265}281 (1996) 4 AKAMINE, E. K. Preventing the darkening of fresh lychees prepared for export. Hawaii Agricultural Experimental Station, University of Hawaii. Technical Programme Report, 127, 1}17 (1960) 5 JAISWAL, B. P., SAH, N. L. AND PRASAD, U. S. Regulation of colour break during litchi (¸itchi chinensis Sonn.) ripening. Indian Journal of Experimental Biology, 25, 66}72 (1987) 6 PRASAD, U. S. AND JHA, O. P. Changes in pigmentation patterns during litchi ripening: #avonoid production. Plant Biochemical Journal, 5, 44}49 (1978) 7 LEE, H. S. AND WICKER, L. Anthocyanin pigments in the skin of lychee fruit. Journal of Food Science, 56, 466}468, 483 (1991) 8 UNDERHILL, S. J. R. AND CRITCHLEY, C. Anthocyanin decolorisation and its role in litchi pericarp browning. Australian Journal of Experimental Agriculture, 34, 115}122 (1994) 9 LIN, Z. F., LI, S. S., ZHANG, D. L., LIU, S. X., LI, Y. B., LIN, G. Z. AND CHEN, M. D. The changes of pigments, phenolics contents and activities of polyphenol oxidase and phenylalanine ammonia-lyase in pericarp of postharvest litchi fruit. Acta. Botanica Sinica, 30, 40}45 (1988) (In Chinese with English abstract) 10 HUANG, S., HART, H., LEE, H. S. AND WICKER, L. Enzymic and colour changes during postharvest storage of lychee fruit. Journal of Food Science, 55, 1762}1763 (1990). 11 SISTRUNK, W. A. AND GASCOIGNE, H. L. Stability of colour in &concord' grape juice and expression of colour. Journal of Food Science, 48, 430}433, 440 (1983)
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12 MORRIS, J. R., SISTRUNK, W. A., JUNEK, J. AND SIMS, C. A. E!ect of fruit maturity, juice storage, and juice extraction temperature on quality of &concord' grape juice. Journal of the American Society for Horticulture Science, 111, 742}746 (1986) 13 MARKAKIS, P. Stability of anthocyanin in food In: MARKAKIS, P. (Ed.) Anthocyanin as Food Colour. New York: Academic Press, pp. 163}180 (1982) 14 SCOTT, K. J., BROWN, B. I., CHAPLIN, G. R. WILCOX, M. E. AND BAIN, J. M. The control rotting and browning of litchi fruit by hot benomyl and plastic "lm. Scientia Horticulture, 16, 253}262 (1982) 15 UNDERHILL, S. J. R. AND SIMONS, D. H. Lychee (¸itchi chinensis Sonn.) pericarp desiccation and the importance of postharvest micro-cracking. Scientia Horticulture, 54, 287}294 (1993) 16 CHEN, F., LI, Y. B. AND CHEN, M. D. Production of ethylene of litchi fruits during storage and its control. Acta Horticulturae Sinica, 13, 151}156 (1986). (In Chinese with English abstract) 17 PAULL, R. E. AND CHEN, N. J. E!ect of storage temperature and wrapping on quality characteristics of litchi fruit. Scientia Horticulturae, 33, 233}226 (1987) 18 ZHANG, D. L. AND QUANTICK, P. C. E!ects of chitosan coating on enzymatic browning and decay during postharvest storage of litchi (¸itchi chinensis Sonn.) fruit. Postharvest Biology and ¹echnology, 12, 195}202 (1997). 19 PIRIE, A. AND MULLINS, M. G. Changes in anthocyanin and phenolic content of grapevine leaf and fruit tissue treated with sucrose, nitrate and abscisic acid. Plant Physiology, 58, 468}472 (1976) 20 ZAUBERMAN, G., RONEN, R., AKERMAN, M., WEKSLER, A., ROT, I. AND FUCHS, Y. Postharvest retention of the red colour of litchi fruit pericarp. Scientia Horticulturae, 47, 89}97 (1991)
21 PIFFERI, P. G. AND CULTRERA, R. Enzymatic degradation of anthocyanins: the role of sweet cherry polyphenol oxidase. Journal of Food Science, 39, 786}791 (1974) 22 BRADFORD, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Analytical Biochemistry, 72, 248}254 (1976) 23 AUTIO, W. R. AND BRAMLAGE, W. J. Chilling sensitivity of tomato fruit in relation to ripening and senescence. Journal of the American Society for Horticultural Science, 111, 201}204 (1986) 24 KADER, A. A. Modi"ed and controlled atmosphere storage of tropical fruits. In: CHAMP, B. R., HIGHLEY, E. AND JOHNSON, G. I. (Eds), Postharvest Handling of Tropical Fruit. Chiang Mai, Thailand: ACIAR Proceedings, No. 50, 239}249 (1993) 25 NAKAYAMA, T. O. M. AND POWERS, J. J. Absorption spectra of anthocyanin in vivo. In: CHICHESTER, C. O. (Ed.), ¹he Chemistry of Plant Pigments. New York: Academic Press, pp. 193}199 (1982) 26 DENNY, E. G. COSTON, D. C. AND BALLARD, R. E. Peach skin discolouration. Journal of the American Society for Horticultural Science, 111, 549}553 (1986) 27 JIANG, Y. M., ZAUBERMAN, G. AND FUCHS, Y. Partial puri"cation and some properties of polyphenol oxidase extracted from litchi fruit pericarp. Postharvest Biology and ¹echnology, 10, 221}228 (1997) 28 MAYER, A. M. AND HAREL, E. Polyphenol oxidases in plants. Phytochemistry, 18, 193}215 (1979) 29 LIU, S. X., JIANG, Y. M., CHEN, F., ZHANG, D. L. AND LI, Y. B. The relationship between the browning in the pericarp of litchi (¸itchi chinensis Sonn.) fruit and polyphenol oxidase, peroxidase, phenolics and their compartmentation. Acta Botanica Austro Sinica, 7, 95}98 (1991) (In Chinese with English abstract)
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Postharvest Browning of Litchi Fruit by Water Loss and its Prevention by Controlled Atmosphere Storage at High Relative Humidity Y. M. Jiang* and J. R. Fu
Y. M. Jiang: South China Institute of Botany, Chinese Academy of Sciences, Guangzhou 510650 (P.R. China) J. R. Fu: School of Life Science, Zhongshan University, Guangzhou 50275 (P.R. China) (Received June 25, 1998; accepted March 18, 1999)
Postharvest browning of litchi fruit caused by water loss was investigated in relation to anthocyanin content, polyphenol oxidase (PPO) activity, pH value, and membrane permeability. Total anthocyanin concentration decreased with pericarp desiccation. Fruit stored at 90% relative humidity (RH) had the lowest loss of total anthocyanins over the storage period, while total anthocyanin content of fruit stored at 60 and 70% RHs signixcantly declined. Polyphenol oxidase activity with 4-methylcatechol as a substrate tended to increase with reduced storage RH, but no activity towards anthocyanins was detected. Furthermore, in the presence of 4-methylcatechol, the oxidation of anthocyanins by PPO was observed. The pH value was initially low, and then increased with pericarp desiccation, which was associated with increased PPO activity. Conversely, the integrity of membrane systems, which increased with reduced storage RH and increased storage time, showed that membrane structure became more vulnerable. These results indicate that oxidation of both phenolic and anthocyanins by PPO seems to awect the response of litchi fruit to water loss in terms of browning, and suggest that substrate-enzyme contact should be emphasized as this could promote enzymatic reaction leading to browning. Storage at 1 3C under controlled atmosphere (3}5% O2 and 3}5% CO2 ) at 90% RH gave good browning control and fruit quality maintenance.
1999 Academic Press Keywords: browning; controlled atmosphere; litchi; storage; water loss
Introduction Litchi (¸itchi chinensis Sonn.) is a tropical and subtropical fruit of high commercial value in the international market. However, litchi fruit rapidly lose their attractive red colour after harvest due to super"cial pericarp browning. Pericarp browning reduces litchi commercial value and has been considered the main postharvest problem (1}3). Postharvest browning of litchi was thought to be a rapid degradation of the red pigments by polyphenol oxidase, forming brown-colour byproducts (4, 5). Prasad and Jha (6), and Lee and Wicker (7) identi"ed the red pigments in litchi pericarp as anthocyanins. Anthocyanin degradation occurred concurrently with pericarp browning (8), while Polyphenol oxidase (PPO) activity increased (9, 10). Polyphenol oxidase can degrade anthocyanins in some plants (11, 12), but it has a low a$nity for pigments (13). Water loss results in a series of biochemical and physiological changes, including substrate-enzyme contact. Scott et al. (14), and Underhill and Simons (15) reported that postharvest browning of litchi fruit, due to skin
*To whom correspondence should be addressed.
0023-0438/99/0502780#06 $30 00/0 1999 Academic Press
desiccation, was highly correlated with weight loss. Chen et al. (16), and Paull and Chen (17) also reported that wrapping fruit helped reduce desiccation and delay colour change. Application of chitosan coating which minimized fruit desiccation signi"cantly delayed pericarp browning (18). However, to what extent reduced water loss rate can o!set browning, is unknown. The objectives of this study were to investigate the relationship between water loss in controlled relative humidity environments and fruit browning in relation to anthocyanin, polyphenol oxidase, pH, and membrane permeability, and then to control fruit browning during controlled atmosphere storage. Materials and Methods Litchi (¸itchi chinensis Sonn.) fruit cv. Huaizhi (a major cultivar in China), at the fully coloured, commercially matured stage, were obtained from a commercial orchard in Guangzhou. The fruit were dipped for 3 min in 0.1% TBZ (thiabendazole, Deco Chemicals, U.S.A.), and then air dried. Thiabendazole gives good control of postharvest diseases of litchi fruit (16). Fruit were sorted for freedom from visual defects and uniformity of shape,
Article No. fstl.1999.0546 All articles available online at http://www.idealibrary.com on
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colour and size, and then randomly divided into two lots for storage at various relative humidities (RHs) and for controlled atmosphere storage at high RH. Storage at various RHs Fruit from the "rst lot were stored at various RHs at 20 3C and water loss, browning index, anthocyanin content, polyphenol oxidase activity, pH value, and relative leakage rate monitored through the experimental period.
Storage conditions Relative humidity was maintained in a temperature controlled room (24.5 m), set at 20 3C, with the following procedures. A constant pressure air supply was divided into two streams, one dried to about 35%, and the other humidi"ed to '95% RH. These streams were then recombined, via metering values, to give the desired RHs (60, 70, 80, or 90%) and #ow rate (1 L/min). The RHs can be maintained once adjusted throughout the course of experiments, with a #uctuation of only about 0.5%. The RH of the air leaving the storage container (2.12 m) was measured at a position midway between the top of the fruit samples and the exhaust port. An electrohygrometer sensor (model PCRC-55T, Phys-Chemical Research Corp, U.S.A.) was used to measure RH. The RH sensor was calibrated within an appropriate humidity range using saturated salt solutions. A rotometer was used to maintain #ow rate.
Measurements of water and weight losses Water loss from pericarp was calculated progressively, based on a comparison of the water contents in the pericarp at harvest and various storage times, using 18 fruit and expressed as a percentage (di!erence in the water content of the pericarp between at-harvest and storage time, divided by that at harvest). Water content was determined by drying the pericarp, while percent weight loss from the whole fruit, and aril plus seed was also recorded.
Browning assessment Browning was assessed by measuring the extent of the total browned area on each fruit pericarp from each of the RHs using 18 fruit on the following scale: 1"no browning (excellent quality); 2"slight browning; 3"(1/4 browning; 4"1/4}1/2 browning; 5"'1/2 browning (poor quality). The browning index was calculated using the formula & (browning scale;percentage of corresponding fruit within each class). Fruit evaluated at an index'3.0 were considered unacceptable for marketing.
Extraction and purixcation of litchi peel anthocyanins According to the method of Lee and Wicker (7), an ethanolic solution of litchi peel extract was prepared by homogenizing the pericarp of litchi fruit (25 g) with acidi-
"ed ethanol (15 mL of 1.5 mol/L HCl in 85 mL of 95% ethanol, per 100 mL), refrigerating overnight, "ltering through Whatman C 41, and diluting to 250 mL with acidi"ed ethanol. To remove the polymerized dark brown pigments from concentrated litchi skin, extract (1.0 mL) was run through a Sephadex G-25 (60;2.5 cm) column and eluted with 50% acetone/1% formic acid/water. The brownish-red colour pigments, eluted from the column, were discarded. The bright, red-coloured anthocyanin pigments were collected, combined, and concentrated to a small volume by rotary evaporation to remove the acetone. These partially puri"ed, aqueous anthocyanin pigments were absorbed onto a Water Sep-Pak C-18 cartridge, U.S.A., washed with 2 mL of water, eluted with 3 mL of acidi"ed methanol, and concentrated by #ushing with nitrogen gas to (0.5 mL. Anthocyanin assay Anthocyanin content was measured according to the method of Pirie and Mullins (19). Litchi fruit pericarp (2 g) from nine fruit was "nely sliced and extracted with 50 mL HCl-methanol (1 mL HCl in 99 mL methanol for 2 h. The extract was "ltered and diluted, and its absorbance measured at 530 nm. Anthocyanin content was expressed as absorbance at 530 nm/g dry weight (DW) based on the water content of the pericarp. Enzyme assay and protein determination Polyphenol oxidase (PPO) activity was assayed with 4methylcatechol as a substrate according to the method of Zauberman et al. (20). Two grammes of litchi peel from nine fruit was ground with 10 mL of 0.1 mol/L sodium phosphate bu!er (pH 6.8) and 0.2 g of polyvinylpyrrolidone (PVP, insoluble). After centrifugation at 19000 g for 20 min, the supernatant was collected as the crude enzyme extract. The assay of the enzyme activity was performed using 1.0 mL of 0.1 mol/sodium phosphate bu!er (pH 6.8), 0.5 mL of 100 mmol/L 4-methylcatechol, and 0.5 mL enzyme solution. The increase in absorbance at 410 nm at 25 3C was recorded automatically for 5 min (Beckman DU-7). One unit of enzyme activity was de"ned as an increase of 0.01 in absorbance per min. To measure anthocyanin oxidation catalyzed by PPO, 0.1 mL of the concentrated anthocyanins was diluted to 50 mL with 0.1 mol/L sodium phosphate bu!er (pH 6.8), and 0.5 mL of the dilution used as the PPO substrate instead of 4-methylcatechol. However, the percent decrease in absorbance at 510 nm at 25 3C, as compared to the initial anthocyanin solution, was recorded automatically for 30 min to monitor anthocyanin degradation by the method of Pi!eri and Cultrera (21). Protein content was determined according to the dyebinding method of Bradford (22) with bovine serum protein as the standard. pH measurement The pH value was measured according to the method of Underhill and Critchley (8). Pericarp tissues (6 g) from
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nine fruit was washed in distilled water to remove adhesive juice. The tissues were "nely sliced and homogenized in 50 mL distilled water for 1 min. The pH was measured while stirring the homogenate.
Measurement of relative leakage rate Membrane permeability, expressed by relative leakage rate, was determined according to the method of Autio and Bramlage (23). Discs were removed with a cork borer (10 mm in diameter) from the equatorial region of nine fruit pericarps. Thirty discs were rinsed twice and then incubated in 25 mL of distilled water at 2 3C, and shaken for 30 min. Electrolyte leakage was determined with a conductivity meter (model DDS-11A, Shanghai Scienti"c Instruments, China), and again after boiling another batch of discs for 15 min and cooling to 25 3C (total electrolytes). Relative leakage rate was expressed as percent of total electrolytes.
Controlled atmosphere storage at high RH The second batch of fruit was stored at low temperature under controlled atmosphere at high RH. A controlled atmosphere of 3}5% O and 3}5% CO at 13C was considered the optimum storage condition (16, 24). In this experiment, the atmosphere was applied as a continuous #ow (100 mL/min, about 90% RH) over fruit using gas controllers (model II-2, China Agricultural University). The container consisted of an impermeable plastic bag with a gas inlet and outlet at the opposite end of the bag. One end of the bag could be temporarily opened to allow fruit withdrawal during the trial. Fruit stored under air instead of controlled atmosphere conditions was used as a control. After 30 d of storage, the fruit were measured for weight loss, browning index, eating quality, and disease incidence. The weight loss of fruit and browning index of pericarp were determined according to the method described previously. Eating quality of pulp was assessed hedonically using a sixmember panel; 36 fruit were randomly selected and rated for quality (1"poor, 9"excellent). Eating quality rating was analysed by analysis of variance for a completely randomized design. Disease development was monitored by randomly collecting 18 fruit and recording the percent of fruit showing visible fungal growth or bacterial lesions on the peel surface. The contents of total soluble solids, titratable acidity and ascorbic acid were analysed at harvest and the end of the experiment. A 20 g sample of pulp from 12 fruit, was homogenised in a grinder and the supernatant after centrifugation at 20,000 g (Beckman J2-2, U.S.A.) for 15 min was collected to determine the contents of total soluble solids, titratable acidity and ascorbic acid. The total soluble solids were measured using a hand refractometer (J1}3A, Guangdong Scienti"c Instruments, China), while titratable acidity, expressed as percent citric acid, was determined by a 0.1 mol/L NaOH titration method, and ascorbic acid was analysed using the 2.6}dichlorophenolindophenol titration method (16).
Results Storage at various relative humidities E!ects of storage under various RH conditions at 20 3C on water loss, browning index, anthocyanin content, polyphenol oxidase activity, pH value, and relative leakage rate are shown in Fig. 1.
Water loss Water loss rate from the pericarp was signi"cantly reduced by increasing relative humidity during storage (Fig. 1A). The maximum water loss from the pericarp of the fruit after 3 d of storage was '50% at 60% RH, while it was only 19% at 90% RH. Fruit also lost weight progressively with storage time, but no marked weight loss in aril and seed part was observed (data not shown). These results indicate that selective dehydration, particularly in pericarp. occurred during storage, with little movement of water between aril and pericarp.
Browning Litchi pericarp was highly susceptible to desiccation, and skin browning increased with storage time (Fig. 1B). After 2 d of storage at 60 and 70% RHs, the initial red colour of the fruit had largely disappeared and the fruit were commercially unacceptable for marketing due to rapid browning in appearance caused by water loss, while fruit stored at 90% RH took a longer time (about 7 d) to develop this level of browning.
Anthocyanins As shown in Fig. 1C, total anthocyanin concentration decreased with storage time. A greater percent anthocyanin loss occurred during storage from 1 to 2 d than from 2 to 3 d, especially at lower RH storage. Fruit stored at 90% RH has the lowest loss of total anthocyanins over the storage period, while total anthocyanin content of fruit stored at 60 and 70% RHs signi"cantly reduced.
Polyphenol oxidase activity Polyphenol oxidase activity increased with reduced storage RH. After 2 d of storage at lower RHs (60 and 70%) fruit showed decreased enzymatic activity, but the PPO activity of fruit at 90% RH did not markedly change (Fig. 1D). In this study, no activity of PPO towards anthocyanins was detected, but in the presence of 4methylcatechol, PPO can oxidize anthocyanins and the enzyme activity was much higher than that with 4methylcatechol alone (data not shown).
pH The pH value was initially low, and tended to increase with pericarp desiccation (Fig. 1E) However, no marked change in pH was observed during storage at 80 and 90% RHs. This can re#ect a better maintenance of the
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Fig. 1 E!ects of various RHs in storage on water loss (A), browning index (B), anthocyanin content (C), PPO activity (D), pH value (E), and relative leakage rate (F). The water loss, browning index, anthocyanin content, PPO activity, pH value, and relative leakage rate were 0%, 1.24, 1.23 (530 nm/g DW), 1.05;10 unit/mg protein, 4.12, and 27.06% at harvest, respectively. The values of the water loss and browning index are the means for 18 fruit, while those of anthocyanin content, PPO activity, pH value, and relative leakage rate are based on extraction in triplicate derived from nine fruit. The vertical bars indicate standard errors. (䊉)"60% RH; (䊏)"70% RH; (䉱)"80% RH; and (䉲)"90% RH
Table 1 Weight loss, browning index, eating quality and disease incidence of litchi fruit after 30 d of storage at 13C under controlled atmosphere at high relative humidity
Air (control) 3}5% O #3}5% CO
Weight loss (%)
Browning index
Eating quality
Disease incidence (%)
4.8a 1.2b
2.4a 1.4b
6.3b 7.8a
5.5a 1.5b
Weight loss was expressed as percentage, while browning index was calculated using the formula &(browning scale;percentage of corresponding fruit within each class). Eating quality rating of pulp was assessed hedonically by a six-member panel and disease incidence monitored by recording the percentage of fruit showing visible fungal growth or bacterial lesions on the peel surface. Weight loss, browning index, eating quality, and disease incidence were 0%, 1.2, 8.1 and 0% at harvest, respectively The corresponding means within a column followed by the same letter are not signi"cantly di!erent at the 5% level
cellular environment of fruit stored under high RH conditions. Relative leakage rate The integrity of membrane systems can be expressed as relative leakage rate (23). The data illustrated in Fig. 1F, in which the relative leakage rate increased with reduced RHs and increased storage time, showed that membrane systems became more vulnerable to leakage. As a result, this may lead to enhanced substrate-enzyme contact.
Controlled atmosphere storage at high RH The weight loss, the decline in eating quality, and the disease incidence were generally accelerated with storage time. These were delayed by controlled atmosphere storage at high RH (Table 1). After 30 d of storage, the fruit under controlled atmospheres retained their bright colour, and had a higher content of ascorbic acid and lower loss in weight (Table 2), whereas the initial red colour of control fruit had largely disappeared.
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Table 2 Total soluble solids, total titratable acidity and ascorbic acid of litchi fruit after 30 d of storage at 13C under controlled atmosphere at high relative humidity
Air (control) 3}5% O #3}5% CO
Total soluble solids (%)
Total titratable acidity (%)
Ascorbic acid (mg/100 g pulp)
15.3ab 16.1a
0.58ab 0.63a
17.9b 24.5a
The contents of total soluble solids, total titratable acidity and ascorbic acid were 16.6%, 0.67% and 28.6 mg/100 g pulp at harvest, respectively The corresponding means within a column followed by the same letter are not signi"cantly di!erent at the 5% level
Discussion Water is the major constituent of all fruit, and, therefore, regulating its loss is an important requirement for maintaining postharvest quality. Litchi browning correlated well with desiccation, which is the major postharvest problem (15). At the lower humidities litchi fruit rapidly lost their attractive red colour and skin browning became commercially unacceptable after 2 d of storage (Fig. 1B), while fruit stored at 90% RH took a long time to develop the same level of browning. In this study, the "ndings that no marked weight loss from #esh over the storage period was observed suggests that water loss of litchi fruit was from peel rather than pulp. Thus, the browning control and quality maintenance of litchi fruit stored under controlled atmosphere at high RH were attributed to minimizing desiccation (Tables 1 and 2). Postharvest browning of litchi was thought to be due to anthocyanin degradation, but no evidence of browning was observed in the anthocyanin-containing mesocarp, and application of 0.1 mol/L HCl to already brown tissue signi"cantly improved pericarp redness, independently of anthocyanin synthesis (8, 20). As the colourless anthocyanin forms were more degradable by PPO, anthocyanin decolorization may involve its structural changes (13). Anthocyanin structure and, more importantly, colour are directly dependent on pH (25). Anthocyanin decolorisation due to changes in pH has also been reported in the visual colour of cherry and peach fruits (21, 26). We found that pH values of litchi pericarp homogenate changed from 4.12 to 4.82 after 3 d of storage at 60% RH, which was associated with increased PPO activity with a range of pH from 4.2 to 7.0 (27) Polyphenol oxidase is considered the enzyme responsible for browning of fruit and vegetables (28). Lin et al. (9) reported that PPO activity in litchi pericarp rapidly increased after harvest. Fruit stored at lower RHs showed higher activities of this enzyme (Fig. 1D). In the presence of 4-methylcatechol, the oxidation of anthocyanins by PPO was signi"cantly enhanced. Conversely, the rapid increase of relative leakage rate (Fig. 1F), which may result from the breakdown of membrane system, provided evidence that some phenolics could spill from vacuoles, being consistent with results from Liu et al. (29). As a result, these changes can lead to mixing of anthocyanins, phenolics and PPO, and in turn, accelerate the degradation of anthocyanins. Our results
indicate that oxidation of both phenolics and anthocyanins by PPO seems to a!ect the response of litchi fruit to water loss in terms of browning, and suggest that substrate-enzyme contact should be emphasized as this can promote enzymatic reactions leading to browning. Acknowledgment The "nancial support provided by International Foundation for Science (E/2265), Stockholm, Sweden, is greatly appreciated. References 1 HUANG, P. Y. AND SCOTT, K. J. Control of rotting and browning of litchi fruit after harvest at ambient temperature in China. ¹ropical Agriculture, 62, 2}4 (1985) 2 NIP, W. K. Handling and preservation of lychee (¸itchi chinensis Sonn.) with emphasis on colour retention. ¹ropical Science, 28, 5}11 (1988) 3 HOLCROFT, D. M. AND MITCHAM, E. J. Postharvest physiology and handling of litchi (Litchi chinensis Sonn.) Postharvest Biology & ¹echnology, 9, 265}281 (1996) 4 AKAMINE, E. K. Preventing the darkening of fresh lychees prepared for export. Hawaii Agricultural Experimental Station, University of Hawaii. Technical Programme Report, 127, 1}17 (1960) 5 JAISWAL, B. P., SAH, N. L. AND PRASAD, U. S. Regulation of colour break during litchi (¸itchi chinensis Sonn.) ripening. Indian Journal of Experimental Biology, 25, 66}72 (1987) 6 PRASAD, U. S. AND JHA, O. P. Changes in pigmentation patterns during litchi ripening: #avonoid production. Plant Biochemical Journal, 5, 44}49 (1978) 7 LEE, H. S. AND WICKER, L. Anthocyanin pigments in the skin of lychee fruit. Journal of Food Science, 56, 466}468, 483 (1991) 8 UNDERHILL, S. J. R. AND CRITCHLEY, C. Anthocyanin decolorisation and its role in litchi pericarp browning. Australian Journal of Experimental Agriculture, 34, 115}122 (1994) 9 LIN, Z. F., LI, S. S., ZHANG, D. L., LIU, S. X., LI, Y. B., LIN, G. Z. AND CHEN, M. D. The changes of pigments, phenolics contents and activities of polyphenol oxidase and phenylalanine ammonia-lyase in pericarp of postharvest litchi fruit. Acta. Botanica Sinica, 30, 40}45 (1988) (In Chinese with English abstract) 10 HUANG, S., HART, H., LEE, H. S. AND WICKER, L. Enzymic and colour changes during postharvest storage of lychee fruit. Journal of Food Science, 55, 1762}1763 (1990). 11 SISTRUNK, W. A. AND GASCOIGNE, H. L. Stability of colour in &concord' grape juice and expression of colour. Journal of Food Science, 48, 430}433, 440 (1983)
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21 PIFFERI, P. G. AND CULTRERA, R. Enzymatic degradation of anthocyanins: the role of sweet cherry polyphenol oxidase. Journal of Food Science, 39, 786}791 (1974) 22 BRADFORD, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Analytical Biochemistry, 72, 248}254 (1976) 23 AUTIO, W. R. AND BRAMLAGE, W. J. Chilling sensitivity of tomato fruit in relation to ripening and senescence. Journal of the American Society for Horticultural Science, 111, 201}204 (1986) 24 KADER, A. A. Modi"ed and controlled atmosphere storage of tropical fruits. In: CHAMP, B. R., HIGHLEY, E. AND JOHNSON, G. I. (Eds), Postharvest Handling of Tropical Fruit. Chiang Mai, Thailand: ACIAR Proceedings, No. 50, 239}249 (1993) 25 NAKAYAMA, T. O. M. AND POWERS, J. J. Absorption spectra of anthocyanin in vivo. In: CHICHESTER, C. O. (Ed.), ¹he Chemistry of Plant Pigments. New York: Academic Press, pp. 193}199 (1982) 26 DENNY, E. G. COSTON, D. C. AND BALLARD, R. E. Peach skin discolouration. Journal of the American Society for Horticultural Science, 111, 549}553 (1986) 27 JIANG, Y. M., ZAUBERMAN, G. AND FUCHS, Y. Partial puri"cation and some properties of polyphenol oxidase extracted from litchi fruit pericarp. Postharvest Biology and ¹echnology, 10, 221}228 (1997) 28 MAYER, A. M. AND HAREL, E. Polyphenol oxidases in plants. Phytochemistry, 18, 193}215 (1979) 29 LIU, S. X., JIANG, Y. M., CHEN, F., ZHANG, D. L. AND LI, Y. B. The relationship between the browning in the pericarp of litchi (¸itchi chinensis Sonn.) fruit and polyphenol oxidase, peroxidase, phenolics and their compartmentation. Acta Botanica Austro Sinica, 7, 95}98 (1991) (In Chinese with English abstract)
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