Polyphenol fate and superficial scald in apple

Postharvest

Biology.and Techn~0,:jy

Postharvest Biologyand Technology4 (1994) 213-224

ELSEVIER

Polyphenol fate and superficial scald in apple M.V. Piretti a, G. G a l l e r a n i b'*, G.C. P r a t e l l a b a Dipartirnento di Biochimica, Universitgzdi Bologna, Via Zanolini 3, 40126 Bologna, Italy b CRIOF,, Universitgl di Bologna, Via Filippo Re 8, 40126 Bologna, Italy

(Accepted 2 November 1993)

Abstract

Cv. Granny Smith apples were treated in attempts to influence the incidence of superficial scald by (a) storing at 20°C for 10 days, (b) dipping in 2000 ppm diphenylamine for 20 s, or (c) storing in 1% 02 and 2% CO2. The fruits, together with a control sample, were subsequently kept at 0°C, and samples were removed for analysis at 44, 109 and 205 days after the start of storage. Polyphenols in the skin were extracted from the apple samples and separated by HPLC using an internal standard. There was a general decline in the polyphenol fractions (epicatechin, quercetin glycosides, procyanidins and unknown polyphenols) in control fruit, which showed increasing scald after about 50 days storage. Of the treatments, (a) was most effective at preventing scald, followed by (b) and then (c). No evidence was found to involve flavonols, condensation between flavonoid glycosides and gallic acid or polymerisation of flavan-3,4-diols in the disorder of scald. The oxidative coupling of o-dihydroxyphenols in damaged tissue remains as the most likely explanation of the browning. Key words: Apples; Superficial scald; Polyphenols

1. Introduction

O f marked economic importance, superficial scald (SS) in apple is a physiological alteration that appears during cold storage. The latest studies suggest its onset as being induced by the oxidation of farnesene, a terpenoid compound, with the formation of conjugated triene hydroperoxides (CTH) that determine the resulting cell damage (Huelin and Coggiola, 1968, 1970; Pratella et al., 1986, 1989; Meir and Bramlage, 1988; Budini et al., 1989). Meigh and Filmer (1969) showed there is no * Corresponding author. Present address: Istituto Agrario S. Michele all'Adige, Via E. Mach 1, 38010 S. Michele all' Adige (TN), Italy. Fax: 461 650-956. 0925-5214/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved. SSDI 0925-5214(93)E0065-L

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direct link between farnesene concentration and the severity of SS. Yet this apparent incongruence may be due to the fact that the terpene's oxidation to CTH is reduced by the presence of natural antioxidants in the fruit. Anet (1974) maintains that there are various liposoluble antioxidants in the cuticle of the apple and that some are involved in the process determining the onset of SS. However, the thin-layer chromatography data this same author reports, are significant only in qualitative terms and limited to the cuticle alone, whereas it is the surface layer of the skin that is the site of histological damage (Bain and Mercer, 1963). While the quantitative relation between liposoluble antioxidants and SS has since been more accurately defined (Meir and Bramlage, 1988; Gallerani et al., 1990), little notable progress has been reported in identifying the chemical compounds involved. The assumption that the antioxidants implicated in the process leading to the onset of SS are lipid substances, rests mainly upon two premises. The first is that farnesene is mostly found in the fruit's cuticle, i.e. in a fatty environment, thus making it logical to suppose that CTH formation occurs therein (Huelin and Coggiola, 1968). The second is that diphenylamine (DPA), a liposoluble compound having an antioxidant/antiscald action, is mostly distributed in this part of the fruit, thereby evincing the apolarity of the CTH-formation environment. What has not yet been determined is the pattern of the cell-damaging process that leads to scald - - a process involving cell layers underneath the cuticle that have different chemical and physical traits from the latter. It is logical to assume the involvement of polyphenols, readily oxidizable compounds which are found in substantial amounts in the skin of apples. For, even though they are not liposoluble, they could induce browning (Duvenage and de Swardt, 1973) and at the same time play a protective role because of their antioxidant effectiveness. Of the natural antioxidants involved in the onset of SS, Anet (1974) identified a molecule having UV spectral characteristics similar to those of p-hydroxybenzoic acid. Duvenage and de Swardt (1973), investigating the interrelation of total polyphenol content and SS onset in the susceptible Granny Smith and the resistant White Winter Permain cultivars, reported that leucoanthocyanidines increased as fruit maturation progressed, suggesting that this increase could lead to lower SS susceptibility. They also found that as cold storage increased, so did high-molecular-weight flavanols, a finding attributed to the polymerization of oxidized flavanols and of leucoanthocyanidines. Kang and Seung (1987) showed that during storage total polyphenols decline in all parts of the fruit, whether in the SSsusceptible Mutsu or in the resistant Fuji cultivar, except in the skin, where the level remains practically constant. This decrease was matched, however, by an increase in polyphenoloxidase activity in the SS-susceptible cultivar, although these latter data were obtained employing the methods of Folin-Denis and of Folin-Ciocalteu which are known to be of limited reliability. The present study investigated the role of fruit-skin polyphenols leading to the onset of SS in apple.

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2. Materials and methods

Sampling Cv. Granny Smith apples from Ferrara Province were harvested at a 2.5 degree of maturity (preclimateric) as measured on the CRIOF starch-degradation scale. They were graded by size, sorted for external defects and then randomized so that each sample was representative of the orchard of origin. Four treatment samples were set up, each consisting of 700 fruits: one was dipped for 20 s in DPA at 2000 ppm, one stored at 20°C for 10 days until yellowing (over-ripening) prior to cold storage, one was stored in low oxygen (LO, 02 1%, COz 2% ), and the last left untreated as control; all the fruits were stored at 0°C and 95% R H for 205 days. It is a known fact that the first three treatments reduce fruit susceptibility to SS. Sample analyses were performed at about 50-day intervals except for the last lot, which was assayed when the incidence of SS was about 100%.

Polyphenol extraction Fourteen fruits per treatment were divided into two groups, weighed and peeled, any residual flesh attached to the skin being carefully scraped off. The peels were minced, immersed at once in 500 ml methanol and heated for 10 min at 30°C to induce softening. Polyphenols were extracted at room temperature under stirring for 24 h; thereafter the liquid phase was filtered and methanol distilled. The residue thus obtained was dissolved in 200 ml distilled water and extracted five times with 100 ml ethyl acetate. The water residue was discarded and the organic phase dried on anhydrous sodium sulphate, filtered and vacuum-dried. This residue was put in chloroform, and the polyphenols, which are insoluble in this solvent, were recovered by vacuum-filtering with a Whatman GF/D filter and weighed. The resulting material was treated with 400/zl anhydrous pyridine and 1600/zl anhydrous acetic anhydride and left overnight at room temperature; the peracetylated polyphenols were then precipitated by being poured into water and ice and recovered by filtration.

Polyphenol analysis A Varian 5060 equipped with a 10 /zl loop and coupled to a Varian UV 100 variable wavelength detector was used for high-performance liquid chromatography (HPLC). Retention times and quantitative parameters were determined with a Spectra-Physics 4270 data system set at 2 Ab U/V; the column was a Spherisorb S-5 Nitrile 5/zm, 250 × 4.6 mm i.d. (Phase Separation, Queensferry, UK), equipped with a Partisil-10 PAC pre-column, 100 x 4.6 mm i.d. (Whatman, Clifton, NJ, USA); separations were performed after Piretti and Doghieri (1990) in HPLC-grade n-hexane (A) and ethyl acetate (B) (Merck-Bracco, Milano, Italia). The elution program was as follows. Time 0: A - B (70 : 30); flow 1 ml/min. Time 35 min: A - B (50 : 50); flow 0.5 ml/min. Time 60 min: A - B (0 : 100); flow 1 ml/min. Time 100 min: A - B (0 : 100); flow 1 ml/min. Injection volume was 10/zl of sample and the detector was set at 278 nm. Each polyphenol extract was fractionated by thin-layer chromatography (TLC) after Piretti and Doghieri (1990).

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Glycoside hydrolysis The aglycones of the apple-skin glucoside polyphenols were determined by hydrolysing 20 mg of extract with ethanol-2M hydrochloridric acid after Harborne (1965) and extracted following ethanol evaporation from the reaction mixture via ethyl acetate as above; the resulting material was acetylated and analysed by HPLC (Piretti and Doghieri, 1990). Quantitative determination Peracetyl-(-)-epigaUocatechin-3-gallate (PEG) (0.5 mg/ml), extracted in the free state from green leaf tea after Piretti and Doghieri (1990) was added as internal standard to 20 mg extracted polyphenols dissolved in 10 ml ethyl acetate; a 10 /xl aliquot was then analysed by HPLC as above. The quantitative values for the separated components were referred to the internal standard and expressed per 100 g skin (mg of PEG/100 g skin): I P × (Wpp/2 x 10-5)1 W~s x 100 where P = (ACsp/ACstd). 5 x 10-3 (mg of polyphenol (as PEG) per 10/zl sample); ACsp = area count for the individual separated polyphenol; ACstd = area count for the PEG internal standard; Wpp = weight of the total peracetylated polyphenols extracted from the skin sample (g); Wss = weight of the skin sample (g).

Determination of polyphenol concentration in browned tissue A selection of about 40 fruits evincing extensive browning at SS onset (very pronounced and visible after one week at 200C) was sorted into two sample groups, one of fully browned and the other of completely healthy skin, for polyphenol determination as described above. Polyphenol variation during onset of browning at 20°C Variations in polyphenols were monitored during the first week of fruit storage at 20°C (subsequent to 0°C storage), given that most of the browning appears at this time. Fruits showing an increasing extent of browning (1 = healthy tissue; 2 = slightly brown; 3 -- almost fully brown) were sampled and analysed as reported above. CTH determination CTHs were extracted from two samplings, each of 10 fruits: each fruit was dipped in n-hexane (spectrophotometric reagent, Carlo Erba, Milano, Italy) for 2 min (Huelin and Coggiola, 1968). The extract was brought to volume and analysed by a Hewlett Packard 8452 UV-Vis spectrophotometer. The differences in absorption from 290 to 281 nm (OD 290-281) were recorded to determine CTH concentration (Anet, 1974); the resulting data were expressed as optical density cm -2 x 104 ( O D c m -2 x 104) (Gallerani and Pratella, 1991).

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Variation of SS incidence Two samples of 50 fruits per treatment were kept at 20°C for one week. Only fruits having a brown area greater than 1 cm2 were considered damaged; data are expressed as percentage of affected apples. 3. Results

SS appeared in untreated control after about 100 days cold storage, registering maximum intensity after about 200 days (Fig. 1). The SS incidence in DPA-treated, LO-stored and pre-ripened fruits was less than 20% of stored apples. The CTH pattern was consistent with that of SS incidence: it showed higher levels in nontreated control than in the fruits subjected to SS-inhibiting treatments (Fig. 2). This finding would appear to confirm a direct link between CTH concentration and SS development. HPLC analysis of polyphenol constituents showed at least 13 peracetyl derivatives: peracetyl-(-)-epicatechin, peracetyl quercetin glucosides and peracetyl procyanidin B2 were identified on the basis of HPLC retention time (RT-HPLC) and Rf in TLC, which proved identical to those of corresponding authentic standard (Fig. 3). As reported by Piretti and Doghieri (1990), most of the polyphenol constituents isolated from the skin were glycosides of quercetin, the latter being the only flavone found by hydrolysis of the extracted material. Detected in addition to the polyphenol constituents found in all analysed samples were the peracetylated triterpenoid compounds ursolic and oleanolic acids and fl-sitosteryl glucoside (Brieskorn and 10090-

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M. V. Piretti et al. / Postharvest Biology and Technology 4 (1994) 213-224

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Klinger, 1963), which were identified by TLC via comparison to known standards, as reported in Piretti et al. (1974). Although several compounds remained unidentified, their RT-HPLC as well as their conversion in anthocyanidins following hot HC1 treatment of the extract suggest they may be oligomerous procyanidins. The variations in four types of polyphenols of the skin during apple storage is shown in Fig. 4. The concentration of epicatechin remained practically steady from harvest to about day 100 of storage in all samples except the DPA-treated fruits, which registered a slight drop in the first 50 days and a subsequent rise to values comparable with the other samples. From day 100 to the end of storage the epicatechin content showed a progressive decline, albeit less marked in the DPAtreated sample. A declining trend in all samples was recorded for the quercetin glycosides, a decrease that was far more pronounced after the first 50 days' storage.

M.V Piretti et al./ Postharvest Biology and Technology 4 (1994) 213-224

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221

A diminution of procyanadin B2 was also found over the last 100 days of storage for all samples. Except for their rather marked decrease in untreated control, the presumed flavanoid oligomers showed no particular alterations over the last 100 days of storage. By contrast, the decrease of all polyphenols was evident in the control fruits throughout the period of 20°C-storage following cold storage (Fig. 5). The increasing severity of SS in the 5-6 days of fruit storage at room temperature is shown by the degree of browning (1, 2, 3 scale) (see Section 2). The comparison of affected and unaffected tissues clearly shows that scald is accompanied by the disappearance of all the previously detected types of polyphenols (Fig. 6). 4. Discussion

It can first be established that the most important polyphenols in apple fruits, especially in the skin, appear to be quercetin glycosides, together with minor amounts of (-)-epicatechin, procyanidine B2 and, possibly, flavan-3-ol oligomers. Yet the most noteworthy, and new, finding was the sudden disappearance of all polyphenols whatsoever from the areas of affected skin, whereas measurable amounts of both flavan- and flavon-oid polyphenols could still be extracted from the unaffected areas. This means that only ursolic and oleanolic acids and fl-sitosteryl glucoside could be extracted from the brown skin. The most likely explanation for the disappearance of polyphenols from the SSaffected peel would be their polymerisation into complex tannins, as reported by Duvenage and de Swardt (1973). However, it seems quite improbable that such complex tannins could result from the direct coupling of flavonol glycosides, large amounts of which are found in apple skin, since only some kinds of biflavonoids have heretofore been found in nature (Geiger and Quinn, 1988) and no evidence has been reported about flavonoid coupling to polyflavonoids. It seems just as improbable that, since apples do not contain gallic acid (Haslam, 1989a), glycoside flavonoids might have been converted into the correspondingly well-known galloyl macromolecular complex esters (Haslam, 1989c). Another possibility, at once both fascinating and disconcerting, might account for disappearing of flavonols. The ascertained farnesene oxidation might be linked to the reduction of the quercetin moiety of flavonol-glycosides to the corresponding flavan-3,4-diols, followed by the ready polymerisation of these latter to proantocyanadins - - a process that would accord with the generally accepted pattern reported by Haslam (1989b) (Fig. 7). Yet, whatever the case may be, there is no doubt that any attempt to provide a response to these issues must take into account the nature of any complex tannins. Note in this regard that the procedure employed in the present study does not allow the extraction of highly polymerised polyphenols. In effect, even if the water-methanol mixture is able to extract these substances from apple skin, the ethyl acetate can extract from the corresponding water solutions only those oligomer polyphenols having up to three to four coupled units (Lea and Timberlake, 1974). Nor would such conditions enable us to ascertain whether the disappearance of the polyphenols from the affected skin is due to the sudden

M.V. Piretti et al. /Postharvest Biology and Technology 4 (1994) 213-224

222

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formation of complex polymers or to other mechanisms. Continued investigation will be necessary to determine the nature and the chemical composition of any complex tannins produced in SS-affected skin.

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A second crucial point to consider is the browning of the affected skin area, which was most pronounced upon removal of the apples from cold storage and might be unrelated to the disappearance of the flavonoid glycoside. Our contention is that it is linked to the oxidation of o°diphenol moieties to o-benzoquinones, followed by the formation of brown coupled polymers (Weinges and MOiler, 1972). This pathway can be triggered by polyphenoloxidase (PPO), notable amounts of which have been found in apple skin (Walker and Hulme, 1966), and can involve both o-dihydroxy-flavanoids and -flavonoids, even if several authors (Roberts and Wood, 1951; Baruah and Swain, 1959; Roberts, 1960) report that quercetin derivatives, especially their glycosides, undergo PPO oxidation with some difficulty. However, since very low amounts of o-dihydroxyphenols can produce an intense brown colour by oxidation, the basic flavanoid content of apple skin may be responsible for the browning, without involving the o-dihydroxyphenols produced by the flavonoidfarnesene redox process tentatively suggested above.

Acknowledgements Research supported by National Research Council, Special Project RAISA, Subproject 4, Paper No. 916. References Anet, E.EL.J., 1974. Superficial scald, a functional disorder of stored apples, XI. Apple antioxidants. J. Sci. Food Agric., 25: 299-304. Bain, J.M. and Mercer, EV., 1963. The submicroscopic cytology of superficial scald,a physiological disease of apples. Aust. J. Biol. Sci., 16: 442-449. Baruah, P. and Swain, T., 1959. Action of potato phenolase on flavonoid compounds. J. Sci. Food Agric., 10: 125-130. Brieskorn, C.H. and Klinger, H., 1963. Chemical composition of apple peel, II. Composition of apple peel triterpenes. Z. Lebensmittelunters. Forsch., 120: 269. Budini, R.A., Pratella, G.C. and Gallerani, G., 1989. The role of peroxides in the pathogenesis of apple scald: a biochemical index for its prediction. Adv. Hortic. Sci., 3: 106-108. Duvenage, A.J. and de Swardt, G.H., 1973. Superficial scald on apples: the effect of maturity and diphenylamine on the flavonoid content in the skin of two cultivars. Z. Pflanzenphysiol., 70: 222234. Gallerani, G. and Pratella, G.C., 1991. Upgrading peroxide detection to prevent superficial scald of apples by HPLC. Adv. Hortic. Sci., 5: 139-143. Gallerani, G., Pratella, G.C. and Budini, R.A., 1990. The distribution and role of natural antioxidant substances in apple fruit affected by superficial scald. Adv. Hortic. Sci., 4: 144-146. Geiger, H. and Quinn, C., 1988. Biflavonoids. In: J.B. Harborne (Editor), The Flavonoids. Chapman and Hall, London, pp. 99-122. Harborne, J.B., 1965. Plant polyphenols, XIV. Characterization of flavonoid glycosides by acidic and enzymic hydrolyses. Phytochemistry, 4: 107-120. Haslam, E., 1989a. Proanthocyanidins. In: Plant Polyphenols - - Vegetable Tannins Revisited. Cambridge University Press, Cambridge, pp. 1-13. Haslam, E., 1989b. Proanthocyanidins. In: Plant Polyphenols - - Vegetable Tannins Revisited. Cambridge University Press, Cambridge, pp. 14-89. Haslam, E., 1989c. Gallic acid metabolism. In: Plant Polyphenols - - Vegetable Tannins Revisited. Cambridge University Press, Cambridge, pp. 90-153.

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Huelin, EE. and Coggiola, I.M., 1968. Superficial scald, a functional disorder of stored apples, IV. Effect of variety, maturity, oiled wraps and diphenylamine on the concentration of alpha-farnesene in the fruit. J. Sci. Food Agric., 19: 298-301. Huelin, EE. and Coggiola, I.M., 1970. Superficial scald, a functional disorder of stored apples, VII. Effect of applied alpha-farnesene, temperature and diphenylamine on scald and the concentration and oxidation of alpha-farnesene in the fruit. J. Sci. Food Agric., 21: 584-589. Kang, S.D. and Seung, K., 1987. Nature and control of apple scald during cold storage. J. Kor. Soc. Hortic. Sci., 28(4): 343-345. Lea, A.G.H. and Timberlake, E, 1974. The phenolics of ciders, 1. Procyanidines. J. Sci. Food Agric., 25: 1537-1545. Meigh, D.E and Filmer, A.A.E., 1969. Natural skin coating of the apple and its influence on scald in storage, III. Alpha-farnesene. J. Sci. Food Agric., 20: 139-143. Meir, S. and Bramlage, W.J., 1988. Antioxidant activity in Cortland apple peel and susceptibility to superficial scald after storage. J. Amer. Soc. Hortic. Sci., 113(3): 412-418. Piretti, M.V. and Doghieri, P., 1990. Separation of peracetylated flavanoid and flavonoid polyphenols by normal-phase high performance liquid chromatography on a cyano-siliea and their determination. J. Chromatogr., 514: 334-342. Piretti, M.V., Ghedini, M. and Weinges, K., 1974. VIII- Sull' imbrunimento dei vini bianchi. Nota IV. Isolamento di acido oleanolico e di fl-sitosteril glucoside dalle vinacce di uva albana. Ann. Chim., 64: 415-419. Pratella, G.C., Budini, R.A. and Gallerani, G., 1986. Influenza di un antiriscaldo suUa patogenesi del riscaldo comune delle mele. Atti Giornate Fitopatol., 2: 89-96. Pratella, G.C., Gallerani, G.C. and Budini, R.A., 1989. The etiology of apple common scald. In: Proceedings of the International Conference on Technical Innovations in Freezing and Refrigeration of Fruits and Vegetables, University of California, Davis Campus, July 1989. University of California, Davis, Calif., pp. 62-66. Roberts, E.A,H., 1960. Effect of glycosilation on the enzymic oxidation and translocation of flavonoids. Nature, 185:536-37 Roberts, E.A.H. and Wood, E.J., 1951. Oxidation of anthoxanthins by tea oxidases. Nature, 167:608 Walker, J.R.L. and Hulme, A.C., 1966. Study in the enzymic browning of apples, III. Purification of apple phenolase. Phytochemistry, 5: 259-262. Weinges, K. and MOiler, O., 1972. Ober die enzimatische oxydative Kupplung der nat0rlichen Polyhydroxyftavane. Chem. Z., 96: 612-618.