Study of pomegranate ripening by dielectric spectroscopy

Postharvest Biology and Technology 86 (2013) 346–353

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Study of pomegranate ripening by dielectric spectroscopy M. Castro-Giráldez ∗ , P.J. Fito, M.D. Ortolá, N. Balaguer Instituto Universitario de Ingeniería de Alimentos para el Desarrollo, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain

a r t i c l e

i n f o

Article history: Received 1 March 2012 Accepted 13 July 2013 Keywords: Dielectric spectroscopy Pomegranate Polyphenols Maturity index

a b s t r a c t Pomegranate (Punica granatum L.) is one of the fruits most recently studied for its many health benefits and its high antioxidant capacity and total phenolic content. Currently, the industry uses destructive methods to ensure the quality standards demanded by consumers. In this context, dielectric spectroscopy is presented as an interesting technique to monitor, on-line, fruit quality standards and ripening changes. The aim of this study is to analyze the effect of the major components of pomegranate and its structure on the dielectric spectrum between 500 MHz and 20 GHz. Some physical, chemical and dielectric measurements were carried out in the arils, spongy white tissues and peel. A maturity index was defined based on dielectric properties of fruit at two different frequencies, 2.4 and 1.2 GHz. The results demonstrated the utility of this index for pomegranate. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Pomegranate (Punica granatum L.) is an ancient fruit that has been recently studied for its numerous health benefits such as the reduction of blood pressure, the anti-atherosclerotic effect and the reduction of LDL oxidation (Aviram et al., 2000, 2004, 2008; Aviram and Dornfeld, 2001; Kaplan et al., 2001); moreover, this fruit presents some components with anticarcinogenic action (Kim et al., 2002; Lansky et al., 2005; Malik et al., 2005; Malik and Mukhtar, 2006; Shishodia et al., 2006) and that inhibit tumour initiation and development (Adams et al., 2006; Khan et al., 2007). Some studies demonstrated that pomegranate fruit also confer an anti-inflammatory activity (Lansky and Newman, 2007; Larrosa et al., 2010; Lee et al., 2010; Shukla et al., 2008), antidiabetic properties (Bagri et al., 2009; Katz et al., 2007; Li et al., 2008; Parmar and Kar, 2007) and antimicrobial properties (Al-Zoreky, 2009; Choi et al., 2009; Gould et al., 2009; McCarrel et al., 2008; Reddy et al., 2007). These beneficial effects of pomegranate have been directly related to its high antioxidant capacity and total phenolic content (Aviram et al., 2000; Gil et al., 2000; Seeram et al., 2005; Basu and Penugonda, 2009; Viuda-Martos et al., 2010). The edible part of pomegranate fruit is the pulp (arils), which surrounds the seeds. The fruit also contains membranous walls and spongy white tissues that are called locula septa. Both locula septa and the pulp form the whole pericarp. Moreover, the fruit is surrounded by a thin peel (Kader, 2006). Some studies have shown that the functional components of pomegranate fruit are located

∗ Corresponding author. Tel.: +34 96 387 98 32; fax: +34 96 387 73 69. E-mail address: [email protected] (M. Castro-Giráldez). 0925-5214/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2013.07.024

in the different parts of the fruit, not only in the arils (Negi et al., 2003). Besides the polyphenols and the antioxidant components, the edible part of pomegranate is also rich in sugars, mainly glucose and fructose, vitamins, polysaccharides, minerals and organic acids, fundamentally, citric acid (Melgarejo et al., 2000). Pomegranate is a non-climacteric fruit and therefore a minimum maturity state is necessary at harvest (Elyatem and Kader, 1984). Kader (1999) defined the maturity state of fruits using the relationship between sugar content and acidity in liquid phase (maturity index), distinguishing between fruit that are not able to continue their ripening process once removed from the plant, and fruit that can continue the process once collected. Pomegranate fruit belong to the first group and have a low respiration rate and a non-climacteric respiration pattern, which has not received much study. Objective techniques for determining the maturation state after harvesting are needed in order to decide the best uses and storage time of this kind of fruit. There exist numerous instrumental techniques to carry out these determinations but these techniques require samples from fruit internal tissues and, therefore, are destructive tests. On the other hand, recent studies developed non-destructive techniques to predict soluble solids content in honeydew melons (Nelson et al., 2006) and watermelons (Nelson et al., 2007) with promising results. Moreover, a maturity index based on dielectric properties in the microwave frequency range was developed for predicting maturity of climacteric fruits (CastroGiráldez et al., 2010a,b). The aim of this paper was to study the changes in terms of respiration rate, chemical composition and dielectric properties of pomegranates during storage at 5 ◦ C for a period of 79 d, trying to evaluate the maturity index of the fruit by a non-destructive technique.

M. Castro-Giráldez et al. / Postharvest Biology and Technology 86 (2013) 346–353

2. Materials and methods 2.1. Raw material Pomegranates (cv Mollar Valenciana) were harvested at the end of September from a plantation located in Valencia, Spain. The fruit were selected for homogeneity in size. Pieces with superficial defects were refused. All the fruit were at the commercial maturity state and were stored at 5 ◦ C. 2.2. Experimental procedure Prior to the studies of pomegranate fruit, different standard solutions simulating the liquid phase of pomegranate fruit were prepared. The chemical composition of the standard solutions was based on bibliographic sources (Al-Maiman and Ahmad, 2002). The dielectric properties of these solutions were measured at 5 ◦ C in order to describe the different dispersions that occur in the microwaves range. The additive used for preparing standard solutions was: citric acid 1-hydrate (E-330, F.C.C., Panreac Quimica S.A.U., Barcelona, Spain). Standard solutions were prepared with Milli® -Q water. Citric acid concentrations of 0, 1, 1.5, 2, and 2.5 g L−1 were assayed in solutions of 0 and 15% mass fraction of sugar. The sugar composition was obtained by mixing glucose and fructose in the equal proportions simulating the composition of the pomegranate fruit (Melgarejo et al., 2000). The measurements of pomegranate fruit were made at 2, 9, 23, 37, 51, 65 and 79 d after the fruit harvest date. At each measurement date, three pomegranates were used to measure the respiration rate before the destructive measurements. After the respiration measurements were made, the pomegranates were divided in two halves. One of them was used for dielectric spectra measurements and afterwards for polyphenol content determination. The other half was used to measure water activity, sugar mass fraction in liquid phase (weight of sugar by weight of liquid phase), titratable acidity, and pH. In parallel, at each measurement date, other pomegranate fruit were analyzed as respiration control fruit (these fruit were kept intact till the end of the experiments). 2.3. Dielectric properties measurement The system used to measure dielectric properties consists of an Agilent 85070E open-ended coaxial probe connected to an Agilent E8362B vector network analyzer. The dielectric properties were measured by contacting the coaxial probe with the pomegranate surface. The dielectric properties of the standard solutions were measured by inserting the coaxial probe into the liquid. The dielectric properties of pomegranate fruit were measured in the arils, in the spongy white tissues of the locula septa, and in the peel. The mean values of five replicates are reported in this article. All determinations were made at 5 ◦ C from 500 MHz to 20 GHz. 2.4. Physical–chemical analysis Water activity was determined by using a dew point hygrometer, Aqualab® series 3 TE (Decagon Devices, Inc., Washington, USA). Water activity was measured in the arils and also in the spongy white tissues. Sugar content was determined by a refractometer (ABBE, ATAGO Model 3-T, Japan). Titratable acidity (expressed as citric acid; g L−1 ) was determined according to the AOAC (1984) method 22.008 (AOAC, 1984).

347

Analytical determinations described above were obtained by triplicate. The total phenolic content (TPC) was determined using the Folin–Ciocalteu’s reagent (Chang et al., 2006). Pomegranate arils (0.5 g) were extracted with 5 mL methanol for 1 h, then the methanolic extract (ME) was diluted 1:1 (L L−1 ) with distilled water. 125 ␮L of the diluted extract was mixed with 0.5 mL of distilled water in a test tube followed by addition of 125 ␮L of Folin–Ciocalteu reagent (FCR) and allowed to stand for 6 min. Then, 1.25 mL of 7% sodium carbonate mass fraction in water solution and 1 mL of distilled water were added. Each sample was allowed to stand for 90 min at 25 ◦ C. Absorption at 760 nm was measured with a UV–vis spectrophotometer (JASCO V-630) and compared to a gallic acid calibration curve. The results were expressed as gallic acid equivalents (GAE) by sample mass (mg kg−1 ). Each assay was carried out in triplicate. Optical measurements were made by an Apochromatic Stereomicroscope Leica MZ (Leica Microsystems Ltd., Heerbrugg, Switzerland). 2.5. Low temperature scanning electron microscopy (cryo-SEM) A Cryostage CT-1500C unit (Oxford Instruments, Witney, UK), coupled to a Jeol JSM-5410 scanning electron microscope (Jeol, Tokyo, Japan), was used. The sample was immersed in slush N2 (−210 ◦ C) and then quickly transferred to the Cryostage at 1 kPa, where sample fracture took place. Sublimation (etching) was carried out at −95 ◦ C; the final point was determined by direct observation in the microscope, working at 5 kV. Then, once again in the Cryostage unit, the sample was coated with gold in vacuum (200 Pa), applied for 3 min, with an ionization current of 2 mA. The observation in the scanning electron microscope was carried out at 15 kV, at a working distance of 15 mm and a temperature ≤−130 ◦ C. 2.6. Respiration analysis A closed or static system was chosen to measure the respiration rate. Fruit were placed in 2 L hermetic glass containers provided with a rubber septum and were stored at 5 ◦ C in a temperaturecontrolled chamber (J.P. Selecta S.A., Hot-Cold M, Barcelona, Spain). The volume of air from the headspace was withdrawn at different times with a needle connected to a gas analyzer. A head-space-gas analyzer (PBI Dansensor A/S, CheckMate 9900, Ringsted, Denmark) was used to determine the oxygen and carbon dioxide contents (L L−1 ). Gas sampling was carried out every 60 min for 8 h. The relative humidity was analyzed before and after the respiration analysis by a hygrometer (ϕ). The respiration rate, expressed as oxygen consumption rate and carbon dioxide production rate, was calculated by Eq. (1): RRi (␮gi kg−1 s−1 ) = ± F

dxi VHS · MWair · MFruit dt

(1)

In this equation, the subindex ‘i’ represents oxygen or carbon dioxide, the symbol ‘+’ is used to indicate the carbon dioxide production and the symbol ‘−’ for oxygen consumption. The mass fraction by time represents the slope of the fitting data of ‘i’ fraction measured by time; the head space volume (VHS ) was calculated from the volume of the glass and the volume of fruit obtained from its mass and density; MFruit represents the mass of the sample and MWair represents the molecular weight of air estimated with Eq. (2): MWair = xO2 · MWO2 + xCO2 · MWCO2 +



+



1 − xO2 + xCO2 +

ps5

◦C

P

·ϕ



ps5

◦C

·ϕ

P

· MWN2 –Ar

· MWH2 O (2)

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Fig. 1. Overview of the macro and microstructure of pomegranate fruit.

where the molecular weight for oxygen, carbon dioxide and water are: 32, 44 and 18, respectively, and MWN2 –Ar represents the molecular weight of nitrogen and argon in air in normal conditions (28.15 g mol−1 ); ps represents the saturation vapour pressure at 5 ◦ C, ϕ is the relative humidity, and P is the absolute pressure. 2.7. Statistical analysis Statistical analysis was carried out with the Statgraphic® Centurion XVI, version 16.1.11 (Statpoint Technologies, Inc., USA). ANOVA analysis and linear regression were used to find significant interaction between dielectric factor, days from harvest and other important parameters of fruit ripening. 3. Results and discussion In order to relate the dielectric properties of pomegranate fruit with the physiological changes, the different levels of tissue complexity were studied. Pomegranate fruit is formed by the grains (aril and seed), the spongy white tissues and the peel (Fig. 1). The arils are the fleshy covering that surround the seeds and represent the edible part of the fruit. The arils are formed by tubular cells which

are joined to the seed by a transition tissue of spherical cells. Tubular cells contain a high quantity of liquid, serving as the reservoir tissue of the fruit. These features are also identified in Fig. 1. It is important to highlight the high porosity (air space) of the spongy white tissues when compared with the rest of tissues. Pomegranate fruit were characterized by soluble solids content (SSC) and titratable acidity (TA). Titratable acidity ranged from 3.6 and 2.9 g L−1 (Fig. 2), results that agree with those obtained by other authors (Poyrazo˘glu et al., 2002). It is also noted that the acidity value decreased with the time of storage, stabilizing around of 3 g L−1 after 51 d. The reduction of acidity showed significant differences (p < 0.05) between the first month of storage and the last one. Acidity values vary widely with the cultivar, requiring a content of less than 1% to have an optimum index of commercial maturity (Kader, 2006). In this study, all fruit were harvested with optimum acidity. On the other hand, SSC was determined for each fruit, with values between 14.5 and 16.5% of sugar mass fraction in the liquid phase. Non-significant differences (p < 0.05) were found during the storage time. Based on the parameters already presented, the maturity index was determined as the ratio between SSC and TA. This parameter indicates the optimal time to collect the fruit and ranged between

M. Castro-Giráldez et al. / Postharvest Biology and Technology 86 (2013) 346–353 3,9

349

1,8

Acidity (g L-1)

RQ 1,6

3,7

1,4

3,5 1,2

3,3

1

3,1

0,8 0,6

2,9

0,4

2,7

0,2 0

2,5 0

10

20

30

40

50

60

70

80

90

0

10

20

30

40

50

60

70

80

t (d) Fig. 2. Evolution of titratable acidity (g L−1 ) during storage time of pomegranates.

Fig. 4. Respiration quote of pomegranate fruit during storage time at 5 ◦ C, where pomegranate control (♦) and pomegranate samples ().

39 and 54, indicating that all the fruit were in the commercial period (Melgarejo and Martínez, 1992). The maturity index is commonly used as indicator of the optimum harvest time, but it is not a good indicator of storage time. For this purpose, it is better to use the titratable acidity or the respiration rate (Al-Maiman and Ahmad, 2002). The metabolic processes of fruit ripening are described by the biochemical transformations and by the respiratory pathways that maintain the cell energy system. Thus, the respiration rate was studied in order to determine the effect of storage time on the physiological activity of fruit. In Fig. 3a, the respiration rate of pomegranate fruit, in terms of carbon dioxide generation, is shown for the studied storage period. It is possible to observe the very significant (p < 0.01) increase of respiration rate with storage time (at 5 ◦ C), which is the typical behaviour of pomegranate fruit at low temperature (Elyatem and Kader, 1984). The respiration rate, in terms of oxygen consumption, follows the same tendency as carbon dioxide respiration rate (Fig. 3b). It is important to highlight that the analyzed samples followed the same tendency as the control fruit. The respiratory quotient (RQ) was also calculated and is presented in Fig. 4. It is the molar ratio of carbon dioxide produced versus oxygen consumed (Artes et al., 1996). The RQ takes values around 1, indicating that the fermentation threshold has not reached. Looking at these results, the ripening state of the fruit can be determined by analyzing the respiration rate during the storage period.

3

a)

RRCO2 (µg kg-1 s-1)

90

t (d)

During storage, surface dehydration and evapotranspiration phenomena were analyzed by measuring water activity for different tissue components (Fig. 5). In the figure, it can be seen that water activity is reduced drastically with storage time in the spongy white tissues, but the reduction is less marked in the arils. Total polyphenolic content (TPC) is one of the most important quality parameter in pomegranate due to its high antioxidant capacity. This content changes during fruit maturation on the tree (Shwartz et al., 2009), therefore, it seemed appropriate to follow it during storage. The TPC was determined in pomegranate arils and its change during storage is shown in Fig. 6. It is important to note that the TPC suffers a dramatic exponential decrease during the first month of storage, reaching a stable value around 180 mg kg−1 . Some standard solutions were prepared in order to segregate the effect of the different chemical species of pomegranate arils in the microwave spectrum. Citric acid was added to standard solutions of water and sugars with 15% sugars mass fraction in the liquid, which is the average sugar content of pomegranate. The solutions were measured in order to analyze the effect of the organic acids on the loss factor spectra at microwave frequencies, because citric acid content is related with fruit ripening, as noted previously. The citric acid molecule has four conformation states depending on the number of hydrogen lost in the molecule, and the polarity of the molecule depends on this quantity, being maximum in the un-ionized molecule (C6 H8 O7 ). At any pH in a solution of citric acid, 40% of the mono-ionized species have their 4,5

b)

RRO2 (µg kg-1 s-1)

4

2,5 3,5 3

2

2,5 1,5 2 1,5

1

1 0,5 0,5 0

0 0

10

20

30

40

50

60

70

80

90 t (d)

0

10

20

30

40

50

60

70

80

90 t (d)

Fig. 3. (a) Carbon dioxide respiration rate (␮L kg−1 s−1 ) of pomegranate fruit during storage time at 5 ◦ C, where pomegranate control () and pomegranate samples (). (b) Oxygen respiration rate (␮L kg−1 s−1 ) of pomegranate fruit during storage time at 5 ◦ C, where pomegranate control () and pomegranate samples ().

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0,99

0,983

a)

aw

b)

aw

0,982 0,985

0,981 0,98

0,98 0,975

0,979 0,97

0,978 0,965

0,977 0,96

0,976

0,955

0,975

0,95

0,974 0

10

20

30

40

50

60

70

80

90

0

10

20

30

40

50

t (d)

60

70

80

90

t (d)

Fig. 5. Water activity evolution with storage time, where (a) the spongy white tissues and (b) arils.

600

TPC (mg kg-1 ) 500 400

300 200

100 0

0

10

20

30

40

50

60

70

80

90

t (d) Fig. 6. Evolution of total phenolic content (TPC) of pomegranate arils with storage time.

terminal carboxylic acid groups ionized (Martin, 1961). Moreover, the mono-ionized molecule is the most stable conformation between pK1 of 3.15 and pK2 of 4.76 (Loewenstein and Roberts, 1960). The pH of pomegranate and those of standard solutions were between 3.95 and 4.3. Therefore, the citric acid molecule behaves as a dipole with a dielectric constant value and a relaxation frequency lower than of the water molecule. Fig. 7b shows the spectra of the citric acid standard solutions, where it is possible to observe differences at the low frequencies of the spectra. Fig. 7a shows the relation of loss factor at different frequencies with regard to the acidity, showing good correlations in the range of 1.2–2.4 GHz. This can be due to the fact that the citric acid molecule presents a dipole orientation effect at lower frequencies than the water molecule; thus, it is possible to develop a dielectric factor (df) to predict the acidity of the liquid media by using these frequencies (Eq. (3)). df =

ε2.4 GHz ε1.2 GHz

(3)

Fig. 8 shows the relationship between the acidity and the dielectric factor of the standard solutions, with a highly significant linear correlation. It is important to highlight that the good correlation between both variables can indicate that the loss factor signal between 1.2 and 2.4 GHz could be related with the relaxation of the citric acid molecules. Dielectric properties were measured in the different tissues of pomegranate: peel, spongy white tissues and arils. The loss factor spectra of the different tissues of pomegranate at 9 d from harvest are shown in Fig. 9 as an example. In the figure, it is possible to

note that the spongy white tissue presents the highest ionic effect, which could be due to the fact that this part of the fruit is rich in ionic compounds with high mobility (Al-Maiman and Ahmad, 2002). On the contrary, the arils present higher dipolar effect due to their higher water activity and lower concentration of ions. The peel, which is the dried part of pomegranate, presents the lowest loss factor spectrum and the low mobility of compounds reduces the signal in the whole frequency spectrum. Pomegranate degradation throughout the ripening process can be followed by chemical analysis of the acidity (citric acid content), as was shown in Fig. 2. It has been demonstrated that the dielectric factor defined in Eq. (3), is controlled by the acidity of the liquid phase when the major organic acid is citric. Fig. 10 shows the relationship between the acidity of the pomegranate throughout the ripening process and the dielectric factor of the arils, which is the part of the tissue chosen by the industry to predict the maturity. Fig. 10 demonstrates the significant fit between both variables, physiologic and dielectric. As was explained above, the ripening process of pomegranate fruit can also be described by the respiration rate. The respiration rate monitored by dielectric techniques could be an excellent tool throughout storage. Fig. 11 shows the significant correlation between the respiration rate, expressed as carbon dioxide generation, and the dielectric factor of pomegranate arils. Therefore, if the ripening process (expressed as variation of citric acid content) can be predicted by the dielectric factor, then this factor could also predict the time after harvest. Fig. 12 shows the evolution of the dielectric factor from the day of harvest with good fitting and a very significant relation.

M. Castro-Giráldez et al. / Postharvest Biology and Technology 86 (2013) 346–353

R² = 0,8416

ε''

a)

8 GHz

35

b)

351

8 GHz

ε''

35

12 GHz

12 GHz R² = 0,4017

15 GHz

15 GHz R² = 0,0115

30

30

20 GHz

20 GHz

R² = 0,123 25

25 R² = 0,982 2.4 GHz

2,4 GHz

20

20

R² = 0,9692

1.4 GHz

15

1,4 GHz 15

1,2 GHz

1.2 GHz

Ac

R² = 0,987

10

10

0.5 GHz

0,5 GHz

R² = 0,9577 Ac (g

f (GHz)

L-1)

5

5 0

0,5

1

1,5

2

2,5

0,1

1

10

100

Fig. 7. (a) Relation of loss factor at different frequencies with regard to the acidity of standard solutions; (b) loss factor spectra of standard solutions of water and 15% of sugars mass fraction and different acidities: (–) 0 g L−1 , (–) 1 g L−1 , (– – –) 1.5 g L−1 , (- - -) 2 g L−1 , (– · –) 2.5 g L−1 .

2,5

35 ε''

f

Ac (g L-1 ) Ac (g L -1 ) = -20,614 df + 35,079 R² = 0,9898

2

30 aril

25 spongy white ssue

1,5 20

15

1

peel

10

0,5 5 dipolar effect

ionic effect

df 0

f (GHz)

1,54

1,59

1,64

1,69

1,74

Fig. 8. Relationship between the acidity of the standard solutions and the dielectric factor (Eq. (3)).

0 0,1

1

10

100

Fig. 9. Loss factor spectra of the different tissues of pomegranate at 9 d from harvest.

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4. Conclusions

3,9

Ac (g L-1 )

It was possible to follow the evolution of pomegranate ripening after harvest by monitoring the citric acid content and the respiration rate. Thus, the development of a sensor system to predict these variables could be useful to follow the ripening on-line in a rapid and non-destructive way. A dielectric factor (df) based on loss factor at 1.2 and 2.4 GHz for evaluating the citric acid content of standard solutions was defined. This factor was applied in pomegranate fruit, demonstrating its utility for determining the days from harvest, as well as the fruit’s physiological activity.

3,7

Ac = -2,8456 df + 7,2531 R² = 0,9719

3,5

3,3

3,1

References 2,9

2,7

df

2,5 1,25

1,3

1,35

1,4

1,45

1,5

1,55

Fig. 10. Relationship between the acidity and the dielectric factor of pomegranate arils.

Fig. 11. Relationship between the respiration rate, expressed as rate of carbon dioxide evolution, and the dielectric factor of pomegranate arils. 90

t (d) t = 440,43·df - 572,98 R² = 0,9921

80

70

60

50

40

30

20

10

0 1,25

df 1,3

1,35

1,4

1,45

1,5

Fig. 12. Relationship between the days from harvest and the dielectric factor of pomegranate arils.

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