Article pp.431-438 du Vol.27 n°6 (2007)

(1)

doi:10.3166/sda.27.431-438 SCIENCES DES ALIMENTS, 27(2007) 431-438

NOTE

Evolution of anthocyanins in pomegranate juice ( Punica granatum L.) of two cultivars “Mollar”

and “Assaria” during cold storage

G. Miguel¹*, C. Fontes¹, D. Martins¹, A. Neves¹, D. Antunes¹

SUMMARY

Pomegranate fruits of two cultivars (cv. “Mollar” and cv. “Assaria”) were harvested in an orchard of Algarve and stored at 5ºC, in the dark for several months. The evolution of anthocyanins was followed and compared in pomegranate juice during this storage period. In both cultivars, the 3,5- diglucosides and the 3-glucosides of cyanidin, delphinidin and pelargonidin were detected, nevertheless in different amounts. Generally, pomegranate juice of cv. “Assaria” had higher concentrations of anthocyanins than the one of cv. “Mollar”. The decrease of anthocyanin monoglucosides in cv.

“Mollar” juice during the storage period was significant, reaching a minimal value at the end, that is, after three months of storage. This was not so evident in cv. “Assaria”, in which the mono- and diglucosides of anthocyanins content was not significantly different. Delphinidin-3-glucoside was the most important anthocyanin in cv. “Assaria” being the sole anthocyanin, which amounts significantly differed in the juice of both cultivars (p<0.05).

Keywords

anthocyanins, pomegranate, Mollar, Assaria, storage.

1 – INTRODUCTION

Pomegranate (Punica granatum, Punicaceae) is a popular fruit of tropical and subtropical regions. Several cultivars can be detected in different regions of the planet, being some of them very promising in commercial terms (Roy &

Waskar, 1997). Pomegranate is a well-known orchard crop in Mediterranean countries, being some of their cultivars the object of many researches (Gil et al.,

1. Faculdade de Engenharia de Recursos Naturais – Universidade do Algarve – Campus de Gambelas – 8005-139 Faro – Portugal.

* Correspondence: Graça Miguel – e-mail: [email protected] SDA27_6.book Page 431 Lundi, 18. août 2008 4:28 16

(2)

432 Sci. Aliments 27(6), 2007 G. Miguel et al.

1995). In Portugal, two main cultivars can be found: “Mollar” and “Assaria”. The cultivar “Assaria” is found in Portugal, more precisely in Algarve, a southern province of this country. Its edible seeds are a favorite snack due to sweet taste and tenderness, and its fruits are mainly used for direct consumption (Miguel et al., 2004). Pomegranate juice is an important source of phenolic compounds, being the anthocyanins one of the most important, especially the 3-glucosides and 3,5-diglucosides of delphinidin, cyanidin and pelargonidin. However their relative amounts in fruit juice change according to the cultivar, the stage matu- ration of fruits, storage conditions among other factors (Gil et al., 1995, 1996;

Melgarejo et al., 2000; Miguel et al., 2004). The purpose of this work was to evaluate and compare the anthocyanin content in pomegranate seeds of “Mol- lar” and “Assaria” fruits, harvested in an orchard located in Tavira (Algarve, Southern Portugal) after storage for four months in the dark, at 5ºC.

2 – MATERIALS AND METHODS

2.1 Fruits

Sweet pomegranates (Punica granatum cv. “Mollar” and Punica granatum cv. “Assaria”) were harvested in an orchard in eastern Algarve (Portugal). Pome- granate fruits were harvested at the eating ripe stage (table 1). Fruits were transported, on the same day, to the laboratory at the University of Algarve.

After selection (diseased, bruised and injured fruit were rejected), healthy fruits of uniform size and appearance were randomly distributed into alveolated boxes and stored in the dark, at 5ºC and about 90-95% relative humidity.

At harvest and monthly, for 3-4 months, 10 fruits of each replication were removed and the concentration of anthocyanins was measured. For each sam- pling point, there were 4 replications.

Table 1

Pomegranate cv. “Mollar” and cv. “Assaria” ripening parameters at harvest.

Ripening parameters “Mollar” “Assaria”

Pressure Force ( N)

(1cm deformation) 164 ± 34 194 ± 21

Puncture Force (N)

(Conical plunger, 0.7cm depth) 28 ± 7 46 ± 7.2

Soluble solids content

(ºBrix) 14.6 ± 0.7 14.0 ± 0.3

Titratable acidity

(% citric acid) 0.78 ± 0.04 0.71 ± 0.04

Juice content

(ml/100 seeds) 78 ± 8 114 ± 21

Values are means of 4 replications ± standard deviation.

SDA27_6.book Page 432 Lundi, 18. août 2008 4:28 16

(3)

Evolution 0f anthocyanins in pomegranate juice (Punica granatum L.) of two cultivars “Mollar”… 433

2.2 Standard and reagents

Delphinidin-3,5-diglucoside, delphinidin-3-glucoside, cyanidin-3,5-diglucoside, cyanidin-3-glucoside, pelargonidin-3,5-diglucoside and pelargonidin-3- glucoside standards were purchased from Apin Chemicals Ltd, UK. Methanol (HPLC gradient grade) was purchased from Sigma-Aldrich Quimica, SA (Spain).

Formic acid was purchased from Riedel-de-Haën (Germany). The ultrapure water was purified with the MilliQ system, from Millipore, USA.

2.3 Anthocyanins quantification

Pomegranates were manually peeled and the seeds liquefied by hand. The tegmens were discarded. The juice sample (1 mL) was centrifuged (2 min at 1 000 rpm) and filtered through a 0.45 μm filter.

The identification of anthocyanins was performed by HPLC with a System Gold Programmable Detector Module 166-UV-Vis (Beckman Coulter, USA) using a LiChroCART 100 RP-18 column (25 cm x 0.4 cm i.d.; 5 μm particle size, Merck, Ger- many). The mobile phase was 5% formic acid (A) and methanol (B) in a linear gradient starting with 15% B to reach 35% B at 15 min, then isocratic until 20 min, at a flow rate of 1 mL/min. Chromatograms were recorded at the absorbance of 510 nm.

Injection volume was 20 μl using an injector with a 20 μL loop (Rheodyne, USA).

Anthocyanins were identified by comparison of their retention times with those of pure standards.

The anthocyanins were quantified individually based on standard curves of each anthocyanin type: delphinidin-3,5-diglucoside, delphinidin-3-glucoside, cyanidin- 3,5-diglucoside, cyanidin-3-glucoside, pelargonidin-3,5-diglucoside and pelargonidin-3-glucoside, at four concentrations (0.01, 0.02, 0.04 and 0.08 mg/mL). Total amount of anthocyanin in the mixture was the sum of the mean of each component.

3 – RESULTS

The total amounts of anthocyanins in both cultivars of pomegranate (“Mol- lar” and “Assaria”) are presented in figure 1. These values resulted from the sum of the anthocyanins (delphinidin-3,5-diglucoside, cyanidin-3,5-diglucoside, pelargonidin-3,5-diglucoside, delphinidin-3-glucoside, cyanidin-3-glucoside and pelargonidin-3-glucoside) quantified individually and based on standard curves of each anthocyanin. The juice of cv. “Assaria” had more anthocyanins than juice of cv. “Mollar”, as seen in figure 1. An exception was observed at the moment of harvesting in which higher levels of those pigments were observed in pomegranate cv. “Mollar” juice. While the amounts of total anthocyanins in cv. “Mollar” juice decreased over time attaining a minimal value at the end of the storage, in cv. “Assaria”, it was observed an increase of anthocyanin accu- mulation during the first storage month, from which their amounts declined.

This raise, after first month, was mainly due to the relative high amounts of monoglucosides (figure 2), delphinidin-3-glucoside and cyanidin-3-glucoside (figure 3).

(4)

Both cultivars possessed similar levels of anthocyanin diglucosides indepen- dent on the storage period. The same was not observed for anthocyanin monoglucosides, because pomegranate cv. “Assaria” showed significant higher levels of this group of compounds than in cv. “Mollar” (figure 2). Therefore for cv. “Mollar”, anthocyanin diglucosides dominated the pomegranate juice whe- reas in “Assaria” both mono- and diglucosides were in similar amounts.

0 50 100 150 200 250

0 1 2 3 4 5

Storage period (months)

Anthocyanin concentration (mg/l)

Assaria Mollar

Figure 1

Evolution of total anthocyanin concentration in juice of the pomegranate cv. “Mollar” and cv.

“Assaria”, during storage of the fruits, in the dark, at 5°C. Total amount of anthocyanins in the sample was calculated as the sum of the individual pigments.

Bars represent standard deviation of 4 replications.

0 20 40 60 80 100 120

0 1 2 3 4 5

Diglucosides Assaria Diglucosides Mollar Monoglucosides Assaria Monoglucosides Mollar

Figure 2

Evolution of mono- and diglucoside anthocyanins in juice of the pomegranate cv.

“Mollar” and cv. “Assaria”, during storage of the fruits, in the dark, at 5°C.

Total amounts of monoglucosides and diglucosides anthocyanins in the sample were calculated as the sum of the individual monoglucosides and diglucosides pigments,

respectively. Bars represent standard deviation of 4 replications.

(5)

One of the reasons for the great importance of anthocyanin monoglucosides in the juice of pomegranate cv. “Assaria” was the relative high amounts of delphinidin-3-glucoside that represents the most important anthocyanin present in that juice (figure 3) and being the sole anthocyanin which amounts significantly differed in juices of both varieties (p<0.05). Cyanidin-3,5-diglucoside was the second most important anthocyanin. In contrast, this pigment dominated in the juice of pomegranate cv. “Mollar” (figure 4).

Pomegranate cv. “Mollar” showed higher decay than pomegranate cv.

“Assaria” because after four months of storage there were no fruits of cv. “Mol- lar” in contrast to those registered for cv. “Assaria”. This decay was mainly due to Penicillium spp. as well as to the high husk-scald development.

0 10 20 30 40 50 60 70 80 90

0 1 2 3 4 5

Dp3,5 Cy3,5 Pg3,5 Dp3 Cy3 Pg3

Figure 3

Evolution of individual anthocyanin concentration in pomegranate cv.

“Assaria” juice, during storage of the fruits in the dark, at 5°C.

0 10 20 30 40 50 60 70 80 90

0 0,5 1 1,5 2 2,5 3 3,5

Dp3,5 Cy3,5 Pg3,5 Dp3 Cy3 Pg3

Figure 4

Evolution of individual anthocyanin concentration in pomegranate cv.

“Mollar” juice, during storage of the fruits in the dark, at 5°C.

(6)

4 – DISCUSSION

The anthocyanins identified in both “Assaria” and “Mollar” cultivars of pomegranate are the same of those as already reported by some authors: delphinidin-3,5-diglucoside, cyanidin-3,5-diglucoside, pelargonidin-3,5-diglucoside, delphinidin-3-glucoside, cyanidin-3-glucoside and pelargonidin-3-glucoside (Du et al., 1975).

The continued biosynthesis of phenolic compounds after harvest, related to the ripening process may partly explain the increase in the total amount of anthocyanins during the first month of storage in cv. “Assaria”. Some authors had also previously reported an increase of anthocyanins in pomegranates (Gil et al., 1995; Holcroft et al., 1998). They correlated such raise with the activity of the enzymes of the anthocyanin biosynthetic pathway: phenylalanine ammonia lyase (PAL) and UDP-glucose: flavonoid-3-O-glucosyltransferase (GT). Holcroft et al. (1998) reported that in juice of pomegranates stored in different atmos- pheres the increase in the total amount of anthocyanins was correlated with PAL activity but not with flavonoid-3-O-glucosyltransferase activity.

Generally the amounts of total anthocyanins decreased over time. The decay of total anthocyanins during storage period suggests a degradation of the anthocyanins. Some authors (Gil et al., 1996) working with pomegranate cv.

“Mollar” stored at different temperatures, concluded that the degradation of anthocyanins was significant during a storage at 8ºC. According to the same authors, at 1ºC the pigments remained quite stable. Martí et al. (2001) also reported a degradation of anthocyanins that was more significant for delphinidin-3-glucoside, followed by delphinidin-3,5-diglucoside, cyanidin-3- glucoside, pelargonidin-3-glucoside and cyanidin-3-glucoside when pomegranate juices were stored at 5ºC. The results obtained for cv. “Mollar” are in accordance with those obtained by some authors (Gil et al., 1996), because they suggested that the 3,5-diglucosides were more stable than 3-glucosides in all temperatures tested during the storage period assayed.

The different content of individual anthocyanins was already reported by other authors that also observed the concentrations of such pigments could change according to several factors: cultivar, clone, ripening of fruits and storage conditions (Gil et al., 1995, 1996; Artés et al., 2000a; Melgarejo et al., 2000). For example, the highest level of cyanidin-3,5-diglucoside detected in the present work for cv. “Mollar” (figure 4), mainly after the storage period, was already observed elsewhere (Artés et al., 2000b) when fruits of Spanish “Mollar de Elche” were submitted to a specific treatment before storing at 5ºC, or at the end of the ripening process for one clone of pomegranate cv. “Borde de Alba- tera” (Melgarejo et al., 2000).

The decay registered for pomegranate fruits of cv. “Mollar” at the end of storage may be due to an increase in polyphenoloxidase activity, as already reported by some authors (Artés et al., 2000a) for pomegranate cv. Spanish “Mollar de Elche”.

In the present work it was possible to stress the following remarks:

– Pomegranate fruits cv. “Mollar” stored at 5ºC decay easier than the fruits of cv. “Assaria”.

(7)

– Over time, cv. “Assaria” accumulated higher levels of anthocyanins than cv. “Mollar”.

– A permanent diminution of total anthocyanins was verified in cv. “Mollar”

during all storage period (3 months), not so evident in cv. “Assaria”.

– A great decrease of anthocyanin monoglucosides was registered over time in cv. “Mollar” in comparison to those of diglucosides, while in cv. “Assa- ria” the levels of anthocyanins of both di-and monoglucosides were not so different.

– The main pigment in cv. “Assaria” was delphinidin-3-glucoside.

It seems that the Portuguese cultivar “Assaria” performs better in terms of keeping anthocyanins through storage than cultivar “Mollar”.

5 – ACKNOWLEDGMENTS

This work was supported by the Centro de Desenvolvimento de Ciências e Técnicas de Produção Vegetal, Portugal (CDCTPV).

REFERENCES

ARTÉS F., VILLAESCUSA R., TUDELA J. A., 2000a. Modified atmosphere packaging of pomegranate. Journal of Food Science, 65, 1112-1116.

ARTÉS F., TUDELA J. A., VILLAESCUSA R., 2000b. Thermal postharvest treatments for improving pomegranate quality and shelf life. Postharvest Biology and Tech- nology, 18, 245-251.

DU C. T., WANG P. L., FRANCIS F. J., 1975.

Anthocyanins of pomegranate, Punica granatum. Journal of Food Science, 40, 417-418.

GIL M. I., ARTÉS F., TOMAS-BARBERAN F.

A., 1995. Changes in pomegranate juice pigmentation during ripening. Journal of Science and Food Agriculture, 68, 77-81.

GIL M. I., ARTÉS F., TOMÁS-BARBERÁN F.

A., 1996. Minimal processing and modified atmosphere packaging effects on pigmentation of pomegranate seeds. Journal of Food Science, 61, 161-164.

HOLCROFT D. M., GIL M. I., KADER A. A., 1998. Effect of carbon dioxide on anthocyanins, phenylalanine ammonia lyase and glucosyltransferase in the arils of stored pomegranates. Journal of the American Society of Horticultural Science, 123, 136-140.

MARTI N., PEREZ-VICENTE A., GARCIA- VIGUERA C., 2001. Influence of storage temperature and ascorbic acid addition on pomegranate juice. Journal of the Sci- ence of Food and Agriculture, 82, 217- 221.

MELGAREJO P., HERNANDÉZ F., MARTÍNEZ J., TOMÁS-BARBERÁN F. A., ARTÉS F., 2000. Evolution of pomegranate juice anthocyanins during the ripening of fruit of three clones: ME16, VA1 and BA1.

Options Méditerranéennes, 42, 123-127.

MIGUEL G., DANDLEN S., ANTUNES D., NEVES A., MARTINS D., 2004. The effect of two methods of pomegranate (Punica SDA27_6.book Page 437 Lundi, 18. août 2008 4:28 16

(8)

granatum L.) juice extraction on quality during storage at 4ºC. Journal of Biomedi- cine and Biotechnology, 5, 332-337.

ROY S. K., WASKAR D. P., 1997. Pomegran- ate. In Postharvest Physiology and Sto- rage of Tropical and Subtropical Fruits (Mitra, S. K. Ed.) pp. 365-374, CAB Inter- national.