OSMOTIC DEHYDRATION OF POMEGRANATE SEEDS (PUNICA ...

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OSMOTIC DEHYDRATION OF POMEGRANATE SEEDS (PUNICA GRANATUM L.): EFFECT OF FREEZING PRE-TREATMENT BRAHIM BCHIR1,2,3, SOUHAIL BESBES2, HAMADI ATTIA2 and CHRISTOPHE BLECKER1 1

Gembloux Agro-Bio Tech University of liège, Passage des Déportés 2, B- 5030 Gembloux, Belgium 2 Unité Analyses Alimentaires Ecole Nationale d’Ingénieurs de Sfax Route de Soukra, 3038 Sfax, Tunisia

Accepted for Publication January 8, 2010

ABSTRACT The osmotic dehydration of pomegranate seeds was compared using fresh and frozen seeds. The process was carried out at 50C in a 55°Brix solution of sucrose. Freezing pomegranate seeds before osmotic dehydration involved an increase of effective diffusivity and a reduction in dehydration time. The most significant changes of water loss (WL) (46 g/100 g of fresh seeds [FS]) and solids gain (SG) (7 g/100 g of FS) took place during the first 20 min for frozen seeds. After this period, seeds WL and SG ranged on average close to 43 and 8 g/100 g of FS, respectively. Osmotic dehydration was slower starting from fresh fruits but led to a higher rate of WL (62 g/100 g of FS) at the end of the process. Both scanning electron microscopy and texture analysis showed a destruction of cell structure and seed texture during the pretreatment (freezing). The same techniques also revealed a texture/structure modification induced by the osmotic dehydration process.

PRACTICAL APPLICATIONS In Tunisia, the research of addition value to pomegranate seeds is very limited and presents a traditional feature such as jam preparation or direct consummation of fruit during the crop season (between September and December). However, other perspectives of transformation and exploitation of 3

Corresponding author. TEL: +32(0)81/62.23.03; FAX: +32(0)81/60.17.67; EMAIL: b.bchir@ student.ulg.ac.be

Journal of Food Process Engineering 35 (2012) 335–354. All Rights Reserved. © 2011 Wiley Periodicals, Inc. DOI: 10.1111/j.1745-4530.2010.00591.x

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the pomegranate seeds should be undertaken to give an added value to this typical fruit. Osmotic dehydration can be an alternative process to increase the conservation time of fruit and to give a new osmodehydrated fruit that can be included in some food formulation such as ice cream, cereals, dairy, confectionery and bakery products.

INTRODUCTION Pomegranate (Punica granatum L.) is one of the most important fruits in Tunisia. Its total production in 2008 reached more than 70,000 tons. Pomegranate is composed by a nonedible part formed by 30% of skin (external part) and 13% of internal lamel and an edible part formed by seeds (50–70%). Pomegranate seeds are composed of 15% pips (woody part), which determines the hardness, and 85% pulp (the juicy part) depend on cultivar (Al-Maiman and Ahmad 2002).The edible part of the fruit contains considerable amounts of sugars, vitamins, organic acids, phenolic compounds and minerals (Espiard 2002). Pomegranate seeds need preservation methods to increase their shelf life because of insect attacks and microorganism growths. Methods we have noted include drying, freezing, pasteurization, osmotic dehydration, etc. (Raoultwack et al. 1991). Osmotic dehydration has received a considerable attention because of its low energy and cost compared with other dehydration methods. Osmotic dehydration can be an alternative process to increase the conservation time of fruits (Kowalska et al. 2008). Therefore, this method allows the comsumption of pomegranate seeds during the off season (other than September– December). Other benefits of osmotic dehydration include effective inhibition of polyphenoxidase and prevention of loss of volatile compounds, even under vacuum and reduction of heat damage to color and flavor during dehydration (Krokida et al. 2001). The other major application is to reduce the water activity of many food materials so that microbial growth will be inhibited (Bolin et al. 1983). Osmotic dehydration gives two major simultaneous counter-current mass transfer fluxes, namely water flow from the product to the surrounding solution and solute infusion into the product (Escriche et al. 2000; Lewicki and Porzecka-Pawlak 2005). There is a third flow of natural solutes such as sugars, organic acids, minerals and salts leaching from the food into the solution (Waliszewski et al. 1997), which is quantitatively negligible, but may have an important effect on the organoleptic and nutritional value of the product. Mass transfer was studied in several research papers in order to describe osmotic dehydration (Lazarides et al. 1995; Jiokap Nono et al. 2001). Nowadays, the industry uses osmotic dehydration for some previously cut fruit like

OSMOTIC DEHYDRATION OF P. GRANATUM

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apple, banana, mango and apricot, among others. However, this process has not been used for the conservation of whole pomegranate seeds by the industry. Moreover, to our knowledge, we are the first to have previously studied the osmotic dehydration of pomegranate seeds (Bchir et al. 2009). The cellular membrane exerts high resistances to transfer and slows down the rate of osmotic dehydration (Erle and Shubert 2001). Therefore, pretreatments such as freezing, high-pressure, high-intensity electric field pulses have been reported to enhance mass transfers (Tedjo et al. 2002) using mango fruits). The aim of this research was to investigate the kinetics of osmotic dehydration and to determine the influence of freezing on mass transfer during osmotic dehydration and to characterize textural and structural change in osmotically dehydrated pomegranate seeds.

MATERIAL AND METHODS Preparation of Pomegranate Seeds Fresh pomegranate fruits (P. granatum L.) of the El Gabsi variety were obtained from a local research center in Gabes, Tunisia. Pomegranate fruits were collected at the same ripening stage. The fruits (20 kg) were washed with cold tap water and then frozen at -50C. Some pomegranates (20 kg) were conserved at 4C until analysis. Seeds were recuperated immediately prior to the osmodehydration process. Osmodehydration Process Sucrose was dissolved in water in order to obtain 55°Brix solutions. About 10 g of seeds was soaked in the sugar solution and placed in bottles (Schott, Bad Schwartau, Germany) of 100 mL. The volume ratio between the fruit and the sugar solution was kept at 1:4. Osmotic dehydration process was conducted for 10–420 min in a shaking water bath (GFL instrument D 3006, Burgwedel, Germany; oscillation rate 160 rpm) at 50C (Bchir et al. 2009). Mass Transfer Kinetics Seeds were removed from the immersion solution at selected time intervals (0, 10, 20, 40, 60, 80, 120, 180, 240, 300, 360 and 420 min for frozen and fresh) and were quickly rinsed (with distilled water), and the excess of solution at the surface was removed with absorbent paper. Soluble solids were then measured as described below. The material was weighed before and after osmodehydration to calculate the percentage of weight reduction (WR). The

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moisture content was determined to calculate water loss (WL) and solids gain (SG), based on the following equations (Mavroudis et al. 1998):

WR g 100 g of fresh seeds =

SG g 100 g of fresh seeds =

(Wi − Wf ) Wi

( Wsf − Wsi ) Wi

⋅ 100

(1)

⋅ 100

(2)

WL g 100 g of fresh seeds = SG + WR

(3)

where Wi is the initial weight of the sample (g), Wf the final weight of the sample (g), Wsi the initial total solids content (g) and Wsf the final total solids content (g). Each value is the mean of three determinations. Mathematical modeling Diffusion coefficients were calculated using Fick’s second law equation, applied to a sphere, by modifying the Fourier number F0 = Deff t R 2 , using shape factor, because of the ellipsoids shape of pomegranate seeds. The following assumptions were taken into account in order to determine the effective diffusion coefficient: homogeneous body, the external resistance to mass transfer was negligible compared with internal resistance, the initial moisture content was uniform throughout the sample, and the diffusion coefficient was constant (Crank 1975). The solution for Fick’s equation law for diffusion out of a sphere is given by Eq. (4), using the following boundary conditions of internal resistance (Luikov 1968; Crank 1975): Uniform initial amount: t = 0, 0 < r < R, MC(t) = MC0 ∂MC (t ) Symmetry of concentration: t > 0, r = 0, =0 ∂r Equilibrium content at surface: t > 0, r = R, MC(t) = MCeq

WAouS =

MC (t ) − MCeq MC0 − MCeq

∞

= ∑ Bn exp [ − μ n2 F0 ]

(4)

n =1

where Bn = 6 μ n2 ; mn = nP; F0 = Deff, AorS t/R2; n = 1, 2, 3, . . . , Deff, AorS is the effective diffusivity of WL or SG (m2/s); n is the number of series terms, R is the equivalent radius of sphere (m), r is the distance in the radius direction (m)


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and t is the time (s). WA and WS are the dimensionless amount of WL and solids gain, respectively; MCeq is the equilibrium amount of moisture or solids content (g/g DM) calculated using Peleg’s equation. Peleg’s equation parameters were obtained using Eq. (5) (Peleg 1988). This two-parameter model was redefined by Palou et al. (1994) in terms of soluble solids and moisture content, and describes sorption curves that approach equilibrium asymptotically.

MC (t ) = MC0 ±

t k1 + k2 t

(5)

where MC (t ) is the amount of moisture or solids at the instant t (g/g dry matter (DM)), MC0 is the initial amount of moisture or solids (g/g DM), k1 and k2 are Peleg’s parameters and t is the time (s). The value of the amount moisture or solids content at the equilibrium was then calculated using Eq. (6) (Park et al. 2002).

1 t ⎞ ⎛ MCeq = lim ⎜ MC0 ± = MC0 ± ⎟ t →∞ ⎝ k1 + k2 t ⎠ k2

(6)

As stated earlier, in this work, pomegranate seeds were assumed to be ellipsoids, having three characteristic diameters (2rM1 - 2rM2 ⱕ 2RM). According to Alvarez et al. (1995), the diffusion coefficient (D′eff) must be corrected by the factor Y2 when the product shape can be assumed as an ellipsoid. The shape factor (Y) in Eq. (7) is defined as Ss/Sp and Ss is the surface area of a sphere of volume equal to that of fruit with surface area Sp, which is assumed to be an ellipsoid (Alvarez et al. 1995). The intrinsic diffusivity Deff is given by Y2 D′eff (Alvarez et al. 1995).

ψ=

Ss = Sp

4π Re2 ⎛ rM RM 2π r 2 + 2π ⎜ ⎝ 1 − ( rM RM )2

⎞ −1 2 ⎟ sin 1 − ( rM RM ) ⎠

(7)

Physico-chemical Analysis All analytical determinations were performed in triplicate. Values were expressed as the mean ⫾ standard deviation. Dry Matter and Moisture Contents. The dry matter was calculated using oven drying, according to AOAC method 934.01 (1990). Approximately 5 g of seeds was oven dried at 103C ⫾ 2C until constant weight. Moisture

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content was estimated by difference of mean values, 100% - % of dry matter (Chenlo et al. 2007). Protein Content. Total nitrogen was determined by the Kjeldahl method. Protein was calculated using the general factor (6.25) (AOAC 1990, method number: 920.152). Lipids Content. To determine total lipid content, about 5 g of seeds were mixed with chloridric acid. Fat was then extracted with a soxtherm automatic S 306 AK solvent extractor equipped with six Soxhlet posts (Gerhardt soxtherm, Switzerland) and command unit (Gerhardt Variostat, Switzerland) using petroleum ether 40–60C in each Soxhlet post. The result was expressed as the percentage of lipids in the dry matter. Ash Content. To determine ash content, about 5 g of seeds was incinerated in a muffle furnace (Gelman, Germany) at about 550C for 8 h. The total ash content was expressed in dry weight percentage (AOAC 1990, method number: 940.26). Carbohydrate Content. Carbohydrate content was estimated by difference of mean values, 100 - (Sum of percentages of moisture, ash, proteins and lipids) (Barminas et al. 1999). Total Soluble Solids and Water Activity (Aw). The soluble solids of seeds and osmotic solution were determined by measuring the °Brix at 20C using an ATAGO digital refractometer (DBX-55, Atago Co. Ltd, Tokyo, Japan). Aw was measured using an Aqualab (Lozanne, Switzerland) instrument at 20C. pH and Conductivity. pH measurements were performed using a Hanna instrument 8418 pH meter (Kallang, Singapore) at 20C. Conductivity was measured using a conductimeter LF 597-5 (Weilheim, Germany) instrument at 20C. Color. The CieLab coordinates (L*, a*, b*) were directly read with a spectrophotocolorimeter Mini Scan XE (Reston, VA, Germany) with a lamp (D 65). In this coordinate system, the L* value is a measure of lightness, ranging from 0 (black) to +100 (white), the a* value ranges from -100 (greenness) to +100 (redness) and the b* value ranges from -100 (blueness) to +100 (yellowness). Microscopic Observations. Microscopic observations were carried out according to Attia et al. (1993). Samples were deposed on supports and


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underwent three successive operations. First, the dehydration was carried out in an increasing ethanol gradient, going from 10–100% (v/v). Second, the drying was performed at critical point CO2 (75 bars, 40–42C) using a Bal-Tec apparatus (Bal-Tec CPD030, St. Francis, MN). Finally, the metallization with gold was carried out using a Bal-Tec apparatus (Bal-Tec MET 020). The observations were performed with a scanning electron microscope (SEM) Philips XL 30 (Philips, Nancy, France). Texture Analysis. Texture analysis were carried out using a texture profile analyzer (TA.XT2, Stable Micro Systems, Haslemere, U.K.), with a 75 mm compression probe. The operating conditions of the instrument were as follows: 1.5 mm/s pre-test speed, 0.5 mm/s-test speed, 10.0 mm/s post-test speed, 0.10 N trigger force and 85% sample deformation. The hardness and toughness of seeds were the means of 20 single seed measurements. The hardness (N) of the seed was taken as the force in compression that corresponded to the breakage of samples, while the toughness (N mm) is the energy required to crush the sample completely (Kingsly et al. 2006; Al-said et al. 2009). Statistical Analysis Statistical analyses were carried out using a statistical software program (SPSS for Windows version 11.0, SPSS Inc., Chicago, IL). The data was subjected to analysis of variance using the general linear model option (Duncan test) to determine significant differences between samples (P < 0.05). RESULTS AND DISCUSSION Chemical composition of pomegranate seeds before osmotic dehydration is shown in Table 1. Seeds are rich in carbohydrate (~85% DM), followed by TABLE 1. CHEMICAL CHARACTERISTIC OF POMEGRANATE SEEDS Seeds Dry matter (DM %) Protein g/100 g DM Lipid g/100 g DM Ash g/100 g DM Carbohydrate g/100 g DM pH aw °Brix

16.00 ⫾ 0.05 7.79 ⫾ 0.86 4.55 ⫾ 0.40 2.87 ⫾ 0.19 84.93 ⫾ 0.25 4.17 ⫾ 0.20 0.989 ⫾ 0.002 15.50 ⫾ 0.09

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protein (~8% DM), lipid (~5% DM) and ash (~3% DM). This composition is quite similar to pomegranate seeds cultivated in Egypt (El-nemr et al. 1990). Seeds also had a low pH (~4.17); this could be attributed to their high content in organic acids such as citric and malic acids, which are important for sensory properties and preservation (Poyrazoglu et al. 2002). Mass Transfer Kinetics The effect of dehydration time on WL, WR and SG was studied starting from both frozen and fresh pomegranate seeds at 50C, using sucrose solution. The WL increased in time during the osmotic process using fresh seeds (FS), but mostly before 120 min, reaching 47 g/100 g of FS (FS). After this period, only a slight increase was observed during the rest of the process reaching 62 g/100 g of FS after 420 min (Fig. 1). The WR and SG for FS followed also the same evolution reaching 55 and 7 g/100 g of FS at the end of the process, respectively. Several works reported similar dewatering and impregnation kinetics for osmotic dehydration of many fresh fruits (Khoyi and Hesari, 2007 and Falade et al. 2007). Using frozen seeds, the most significant changes took place during the first 20 min of dewatering, as shown in Fig. 1. During this time, WL in seeds

SG and WL (g/100g of fresh seeds)

70

60

50

40

30

20

10

0

0

50

100

150

200

250

300

350

400

450

Time (min)

FIG. 1. COMPARISON OF WL AND SG USING FRESH (¥ WL, 䊉 SG) AND FROZEN (DWL, 䊊SG) SEEDS DURING THE OSMOTIC DEHYDRATION PROCESS


343

was 46 g/100 g of FS, and after that, it varied slightly and ranged, on average, close to 43 g/100 g of FS. The same trend was also observed for WR. Under the same conditions, the SG of frozen seeds was also increased significantly during the first 20 min, reaching 7 g/100 g of FS, and tended to be stable at the end of the process. Similar trends for osmotic process of frozen pumpkin, apple and carrot were observed by Kowalska and Lenart (2001) and Kowalska et al. (2008). The increase of WL, as shown in Fig. 1, at the beginning of the process, is due to the large osmotic driving force between the dilute sap of seeds and the surrounding hypertonic medium. Then, after this period, the slower water transfer is mainly influenced by the reduction of the difference in concentration between the seeds and the osmotic solution, which could involve a slower driving force. Indeed, the reverse trend of °Brix observed in seeds and osmotic solution confirms these facts (Table 2). During the first 20 min, WL was the result of both the osmotic process and the defrosting of seeds. The trend observed in SG (Fig. 1) could be explained by the migration of sucrose to the seeds through the cell wall, and accumulating between the cell wall and the cellular membrane, due to the important gradient of sugar between the seeds and the osmotic solutions. Using FS, the dewatering was slow at the beginning of the process compared with frozen seeds. In fact, during 60 min, the percentage of WL in frozen seeds was 11 g/100 g higher than observed in FS. However, a higher rate of WL (62 g/100 g of FS) was obtained using FS after 420 min of the process. These results are in accordance with previous findings (Kowalska et al. 2008) comparing osmotic dehydration of fresh and frozen pumpkin and carrot. Moreover, Fig. 1 shows that SG in FS was lower than those of frozen seeds. In fact, after 60 min of the process, the SG of frozen seeds was 6 g/100 g higher than FS. Even after 420 min, the percentage of SG of FS was lower. So pretreatment before osmotic dehydration proved to increase SG, in comparison with samples without pretreatment. Our results were similar to those observed by Kowalska et al. (2008), showing that the use of freezing before osmotic dehydration increased the percentage of SG. Differences in WL and SG between fresh and frozen seeds can be explained by the damaged cell structure of frozen fruit due to freezing. In fact, Delgado and Rubiolo (2005) showed that during freezing, a part of the aqueous fraction freezes out and forms ice crystals that damage the integrity of the cellular compartments. Thus, the cellular membranes loose their osmotic status and their semi-permeability, favoring a large osmotic driving force between the dilute sap of the seeds and the surrounding osmotic solution (Torreggiani and Bertolo 2001; Kovacs and Meresz 2004). This fact was behind the higher WL and SG for frozen seeds, at the begging of the process. The difference in water lost from 180 min was due to the fast reduction of the

49.70 ⫾ 0.07c 41.60 ⫾ 0.14c 4.60 ⫾ 0.27ab 35.50 ⫾ 0.35c 42.70 ⫾ 0.08c 61.40 ⫾ 0.96cde 2.40 ⫾ 0.92b 6.50 ⫾ 0.26bc

52.30 ⫾ 0.14d 29.30 ⫾ 0.14b 4.71 ⫾ 0.02b 26.57 ⫾ 0.04b 34.88 ⫾ 1.05b 62.21 ⫾ 0.76e 2.09 ⫾ 0.02b 5.36 ⫾ 0.15b 54.90 ⫾ 0.25g 17.55 ⫾ 0.07ab 5.82 ⫾ 0.03d 3.13 ⫾ 0.04b 18.25 ⫾ 0.35ab 64.68 ⫾ 0.16cde -0.59 ⫾ 0.14a 3.54 ⫾ 0.01a

55.00 ⫾ 0.00e 15.50 ⫾ 0.09a 8.27 ⫾ 0.03c 0.90 ⫾ 0.01a 16.00 ⫾ 0.05a 65.76 ⫾ 0.01f -0.60 ⫾ 0.02a 3.60 ⫾ 0.01a

55.00 ⫾ 0.00g 15.50 ⫾ 0.09a

8.27 ⫾ 0.03e 0.90 ⫾ 0.01a 16.00 ⫾ 0.05a 65.76 ⫾ 0.01e -0.60 ⫾ 0.02a 3.60 ⫾ 0.01a

°Brix of solution °Brix of seeds pH of solution Conductivity of solution (ms/cm) Dry matter of seeds (%) CieLab-coordinate of solution L* a* b* Fresh seeds °Brix of solution °Brix of seeds

pH of solution Conductivity of solution (ms/cm) Dry matter of seeds (%) CieLab-coordinate of solution L* a* b*

5.62 ⫾ 0.02cd 3.61 ⫾ 0.23bc 20.79 ⫾ 0.10b 64.5 ⫾ 0.12de -0.59 ⫾ 0.01a 3.48 ⫾ 0.99ab

54.9 ⫾ 0.14g 19.45 ⫾ 0.07ab

20 min

10 min

0 min

Frozen seeds

54.75 ⫾ 0.07g 20.30 0.01b 5.15 ⫾ 0.64bc 4.10 ⫾ 0.17bc 23.53 ⫾ 1.01c 64.00 ⫾ 0.96cde -0.53 ⫾ 0.02a 4.68 ⫾ 0.01b

49.30 ⫾ 0.01bc 45.30 ⫾ 0.14d 4.50 ⫾ 0.20a 39.00 ⫾ 1.06d 46.80 ⫾ 0.65d 60.90 ⫾ 0.40cde 2.90 ⫾ 0.13bc 7.80 ⫾ 0.07cd

40 min

5.39 ⫾ 0.02cd 4.83 ⫾ 0.07cd 30.94 ⫾ 1.44d 64.10 ⫾ 0.69cd 0.16 ⫾ 0.02b 5.41 ⫾ 0.13b

54.55 ⫾ 0.21g 25.45 ⫾ 0.35b

49.10 ⫾ 0.21ab 46.40 ⫾ 0.14e 4.50 ⫾ 0.01ab 40.10 ⫾ 0.07de 47.80 ⫾ 0.83de 60.50 ⫾ 0.47bcde 3.30 ⫾ 0.46 bcd 8.10 ⫾ 0.17de

60 min

5.21 ⫾ 0.01c 6.00 ⫾ 0.31de 33.06 ⫾ 0.86d 64.00 ⫾ 0.51cd 0.35 ⫾ 0.02bc 5.22 ⫾ 0.52b

53.95 ⫾ 0.07f 26.95 ⫾ 0.07c

48.90 ⫾ 0.28ab 46.90 ⫾ 0.14e 4.40 ⫾ 0.02a 40.50 ⫾ 0.21def 48.30 ⫾ 0.41ef 59.80 ⫾ 0.75bcd 4.10 ⫾ 0.30bcde 9.60 ⫾ 1.98e

80 min

TABLE 2. EVOLUTION OF OSMOTIC DEHYDRATION PARAMETERS IN SUCROSE SOLUTION USING FROZEN AND FRESH SEEDS

344 B. BCHIR ET AL.

48.70 ⫾ 0.35a 49.30 ⫾ 0.35f 4.30 ⫾ 0.07a 42.10 ⫾ 2.19efg 49.30 ⫾ 0.17f 59.90 ⫾ 0.05bc 5.30 ⫾ 0.77 cde 9.30 ⫾ 0.22ed 52.05 ⫾ 0.07c 40.90 ⫾ 0.42e 4.60 ⫾ 0.09a 10.70 ⫾ 0.60g 51.73 ⫾ 1.41ef 63.60 ⫾ 0.12bc 1.00 ⫾ 0.33d 6.03 ⫾ 0.43b

48.60 ⫾ 0.21a 49.20 ⫾ 0.70f 4.40 ⫾ 0.01a 42.90 ⫾ 0.84fg 49.29 ⫾ 0.03f 59.90 ⫾ 0.58bcd 5.30 ⫾ 0.14bc 9.30 ⫾ 0.10de 52.75 ⫾ 0.35d 34.60 ⫾ 0.63d 4.60 ⫾ 0.11a 9.10 ⫾ 0.13f 50.52 ⫾ 0.47e 63.6 ⫾ 0.12cd 0.50 ⫾ 0.22bc 5.81 ⫾ 0.44b

49.00 ⫾ 0.21ab 49.10 ⫾ 0.07f 4.30 ⫾ 0.07a 41.20 ⫾ 0.28defg 49.50 ⫾ 0.76f 58.80 ⫾ 0.15b 4.60 ⫾ 0.24bcde 9.30 ⫾ 0.09de

53.30 ⫾ 0.14e 30.80 ⫾ 0.07cd 4.70 ⫾ 0.24ab 7.10 ⫾ 0.96e 49.62 ⫾ 0.24e 64.20 ⫾ 0.27c 0.80 ⫾ 0.14cd 5.50 ⫾ 0.19b

Means in line with different letters are significantly different (P < 0.05).

°Brix of solution °Brix of seeds pH of solution Conductivity of solution (ms/cm) Dry matter of seeds (%) CieLab-coordinate of solution L* a* b* Fresh seeds °Brix of solution °Brix of seeds pH of solution Conductivity of solution (ms/cm) Dry matter of seeds (%) CieLab-coordinate of solution L* a* b*

240 min

180 min

120 min

Frozen seeds

52.40 ⫾ 0.28cd 45.80 ⫾ 0.28f 4.60 ⫾ 0.08a 13.50 ⫾ 1.45h 53.33 ⫾ 0.55f 63.80 ⫾ 0.83bc 1.50 ⫾ 0.01e 6.38 ⫾ 0.63b

48.60 ⫾ 0.21a 49.20 ⫾ 0.07f 4.40 ⫾ 0.02a 42.90 ⫾ 1.13fg 49.50 ⫾ 0.16f 56.20 ⫾ 0.60a 5.90 ⫾ 0.34de 9.40 ⫾ 0.43ed

300 min

51.15 ⫾ 0.07b 48.4 ⫾ 0.56f 4.40 ⫾ 0.02a 15.20 ⫾ 0.46i 53.94 ⫾ 0.56f 62.50 ⫾ 0.18ab 1.80 ⫾ 0.44e 6.08 ⫾ 0.80b

48.60 ⫾ 0.14a 49.30 ⫾ 0.14f 4.40 ⫾ 0.01a 43.60 ⫾ 1.13g 49.60 ⫾ 0.17f 55.40 ⫾ 1.63a 6.30 ⫾ 0.17e 9.40 ⫾ 0.02ed

360 min

48.45 ⫾ 0.31a 48.40 ⫾ 0.57f 4.38 ⫾ 0.27a 15.50 ⫾ 0.99i 53.93 ⫾ 0.45f 61.33 ⫾ 1.43a 1.81 ⫾ 0.24e 6.12 ⫾ 0.47b

49.00 ⫾ 0.28ab 49.00 ⫾ 0.28f 4.39 ⫾ 0.14a 48.2 ⫾ 2.55h 49.60 ⫾ 0.01f 55.20 ⫾ 1.98a 6.38 ⫾ 0.79e 9.42 ⫾ 0.92ed

420 min

OSMOTIC DEHYDRATION OF P. GRANATUM 345

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TABLE 3. WATER AND SOLIDS EFFECTIVE DIFFUSIVITIES CALCULATED BY FICK’S MODEL AND VALUES OF PELEG’S EQUATION PARAMETERS Water loss 2 -1

Solids gain 2

Deffw (m s ) R (%) K1 Frozen 9.44 ¥ 10-12 Fresh 0.58 ¥ 10-12

99.92 98.96

K2

R (%) Deffs (m2s-1) R2(%) K1 2

28.05 0.23 99.99 670.00 0.21 99.66

4.81 ¥ 10-12 99.72 0.20 ¥ 10-12 98.12

K2

R2(%)

12.08 0.03 99.92 318.63 0.02 99.51

difference in concentration between seed and osmotic solution using frozen seeds (Table 2). Different studies on various fresh fruit dewatering (papaye apple, pumpkin, carrot and kiwi) mentioned that the most significant changes were observed between 30 and 100 min using sucrose solutions (60 and 62°Brix) at different temperatures (30 and 50C) (Saurel et al. 1994; Kowalska et al. 2008). All these authors reported that WL ranged between 39 and 50 g/100 g of FS. Results obtained in this study showed that WL in frozen seeds was in accordance with the literature; however, FS were slightly higher. This comparison should, however, take into account the experimental conditions and the differences in states of the various fruits. Indeed, given their small size, pomegranate seeds can be kept intact and do not need to be cut. On the contrary, in the literature, most fruits (apples, kiwis) used for the osmotic process have to be cut into small volumes because of their large sizes. Thus, cutting these fruit creates more external lesions, leading to a higher contact of cells with the osmotic solution in a shorter time. The longer time needed to dewater pomegranate FS could be explained by their external membrane ability to protect cells. The value of WL was higher than SG and depended on the advancement of the dewatering process. In addition, effective diffusivity of water was also higher than that of the solids (Table 3). According to Vial et al. (1991), the difference between WL an SG was essentially due to the diffusional differences between water and sugar as related to their different molar mass, as well as to the presence of “semi-permeable” vegetal membranes. Raoult-wack et al. (1989) described an antagonistic effect of water and solute transfer, signaling that this is probably due to the combination of sugar penetration by diffusion and sugar transportation by the water outflow as a function of the water flow rate. Evaluation of the Peleg and Fick Mathematical Models The Peleg equation’s parameters (K1 and K2) were determined for WL and SG (Table 3). This model showed a good fit to the experimental data, with


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correlation coefficients (R2) close to 0.99. Effective diffusivity values for water and solids were calculated using Fick’s model, which also presented a good fit to experimental data, showing an average correlation coefficients (R2) close to 0.99. Freezing pretreatment involved a strong increase of effective diffusivity of water and solids compared with untreated samples (Table 3). Physico-chemical Characteristics of the Osmodehydrated Fruit Preparation The effect of time on physico-chemical characteristics of the osmodehydrated seeds (frozen and fresh) and osmotic solution during osmotic process are shown in Table 2. Using frozen seeds, the most significant change occurred during the first 20 min of dewatering. In fact, statistical analysis shows a significant difference (P < 0.05) between 0, 10 and 20 min for all parameters. The FS, on the contrary, showed a progressive evolution. At the beginning of the process the °Brix of the solution decreased as the °Brix of seeds increased; after that, °Brix tended toward an equilibrium. This was a consequence of osmosis, inducing a balance of concentration between the seeds and the sucrose solution. The decrease of the osmotic solution pH and the increase of conductivity could be attributed to the diffusion of some organic acids from pomegranate seeds to the aqueous solution. Moreover, the measure of color parameters L*, a*, b* showed a slight variation of these parameters. This variation could be explained by a migration of pigment from pulp to solution and the nonenzymatic browning (Masmoudi et al. 2007). Compared with FS, the speed of the osmodehydration process using frozen seeds was higher. Indeed, using frozen seeds, the concentration equilibrium (°Brix) was reached earlier between the two compartments (seeds and osmotic solution) compared with FS. In fact, the °Brix and pH (solution) for frozen and FS reached 46 and 25°Brix, pH 4 and pH 5, respectively, after 60 min. Moreover, conductivity of frozen seeds solution was six times higher than the conductivity of the FS solution. These differences could be explained by the increase of the exchange through the seeds membranes due to the irreversible damage and a loss of selectivity of cells induced by freezing. These results showed the utility of freezing for a better transfer of solutes and water, respectively, into and out of the fruits. Microstructure and Texture Analysis To better understand the effect of freezing and osmodehydration treatment at the cell level and on texture characteristics, SEM and texture analysis were used. Figure 2a shows a fresh seed which was not submitted to treatments other than the preparation for SEM. The bright regions in the micrograph are mainly the cytoplasmic membrane and the cell walls; the darker regions are holes where

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FIG. 2. SCANNING ELECTRON MICROSCOPY PHOTOGRAPHS OF (A) FRESH; (B) FROZEN; AND OSMODEHYDRATED FRUITS PREPARED WITH (C) FRESH; AND (D) FROZEN SEEDS

cell contents were before. Frozen cells have a different appearance than fresh cells (Fig. 2b). In fact, tissues of FS showed isodiametric cells with a regular shape and well organized. On the contrary, frozen seed cells appeared torn and irregular in shape, due to the loss of turgor, with the presence of many empty regions (regions that were not occupied by cells). This difference is due to the freezing treatment. Indeed, empty regions indicated that ice nucleation and crystal growth damaged the cell wall. Numerous studies described that during freezing, there is a gradual breakdown in the organization of the protoplasmic structure and, in most cases, rupture of the plasmalemma with subsequent loss of turgor pressure in cells. In addition, some degradation and separation of cell walls were also noted (Jewell 1979; Torreggiani and Bertolo 2001). Decompartmentalization caused by ice crystals prevents the return of water to the intracellular medium during thawing, causing loss of turgidity. This has practical consequences in terms of the loss of the ability to act as a semi-permeable membrane or diffusion barrier, and also the modification of fruit texture (Kovacs and Meresz 2004). Texture analysis showed three zones characteristic of seed compression (Fig. 3). The first (a) corresponds to the pulp resistance (~13 N). The second


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FIG. 3. CHARACTERISTIC FORCE–DISTANCE CURVE FOR TEXTURE ANALYSIS USING FRESH SEEDS

(b) corresponds to the fracture of pip (~30 N) and the third zone (c) corresponds the crushing of seed characterized by the increased in force through a short distance. In fact, using the same analytical procedure, Al-said et al. (2009) showed that pulp and pip hardness varied between 9 and 14 N and 24–45 N, respectively, for pomegranate seeds cultivated in Oman. The peak force attained during the test is referred to as hardness and area under the curve as toughness. Statistical analysis shows a difference (P < 0.05) between fresh and frozen seeds (Table 4). Frozen-thawed seeds have the lower values of both hardness (47 ⫾ 2 N) and toughness (54 ⫾ 3 N mm) then FS (53 ⫾ 4 N and 74 ⫾ 3 N mm, respectively). This can be explained by cell membrane deterioration during freezing inducing the loss of binding capacity among cell walls, which is in accordance with the result observed by SEM. As a consequence, frozen seeds lose their firmness and reduce their thickness. Hence, the probe penetrates more in the seed and touches the pip at a smaller distance compared with FS. In fact, the distance before pip crushing using FS (5.45 ⫾ 0.73 mm) was higher than frozen seeds (4.34 ⫾ 0.51 mm). Figure 2c,d show that osmotic dehydration process changed the tissue structure compared with an untreated sample. In fact, cells appeared irregular

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TABLE 4. TEXTURAL PROPERTIES OF POMEGRANATE SEEDS Without treatment

Toughness (N mm) Hardness (N)

Osmotic dehydration treatment

Frozen seeds

Fresh seeds

Frozen seeds

Fresh seeds

54.55 ⫾ 3.96a 46.73 ⫾ 2.47a

74.22 ⫾ 3.45b 53.41 ⫾ 4.23a

67.21 ⫾ 5.55b 63.46 ⫾ 3.04b

89.59 ⫾ 5.85c 90.10 ⫾ 5.01c

Means in line with different letters are significantly different (P < 0.05).

in shape, slightly distorted but well organized. This fact was probably due to the solubilization of polysaccharides (cellulose, hemicellulose and pectin) that composes the cell walls, the WL and the pre-concentration of sucrose on the surface of the tissue (Raoult-wack et al. 1991; Torreggiani and Bertolo 2001; Delgado and Rubiolo 2005). Indeed, pectin is the major constituent of the middle lamella, and thus contributing to the cell adhesion and firmness (Nunes et al. 2008). Moreover, WL induces the plasmolysis of cells and SG gives consistency to the tissues. Nunes et al. (2008) showed that diffusion of sucrose into the fruit tissue, during osmotic dehydration process, and its interaction with the cell wall and middle lamella might result in the formation of a jam-like structure that gives consistency to the tissues. Likewise, Delgado and Rubiolo (2005) showed that osmotically dehydrated strawberry tissue with the sucrose concentration used did not greatly affect the tissue structure. Textural properties of seeds are closely linked to cellular structure (Torreggiani and Bertolo 2001; Sajeev et al. 2004). Indeed, the osmotic dehydration process induced an increase of textural parameters (toughness and hardness) compared with the untreated sample (Table 4). Statistical analysis shows a difference (P < 0.05) between hardness and toughness of seeds before and after osmotic process. In fact, hardness and toughness increased to 17 N and 13 N mm using frozen seeds and 36 N–15 N mm for FS. This could be a consequence of the exchange (WL and SG) between seeds and osmotic solution. Sajeev et al. (2004) showed that the increase in textural parameters can be attributed to the dehydration of cormels (Taro: Colocasia esculenta L.). As a consequence of this exchange, the products will more or less lose weight and will shrink eventually. Indeed, the peaks of pip crushing using frozen and FS were observed at 3.7 ⫾ 0.34 mm and 3.2 ⫾ 0.34 mm, respectively. Thus seeds reduce their thickness between an average close to 0.64 and 2.25 mm, after osmotic dehydration process, using frozen and FS, respectively. Fresh osmodehydrated seeds had higher texture parameters compared with frozen osmodehydrated seeds. Table 4 shows a statistical difference (P < 0.05) between hardness and toughness of osmodehydrated fresh and


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frozen seeds. These results are in accordance with previous findings (Van buggenhout et al. 2007) using fresh and frozen carrots. In fact, hardness and toughness of osmodehydrated fresh seed was 26 N and 22 N mm higher than those observed in osmodehydrated frozen seed, respectively. In addition, the thickness of osmodehydrated FS was 0.6 mm lower than osmodehydrated frozen seeds. These differences were due to higher WL using FS. In fact, Kingsly et al. (2006) found that the hardness and toughness of pomegranate seeds decreased when the moisture content increased.

CONCLUSION Freezing treatment prolongs the conservation of pomegranate seeds; however, it involves the destruction of the cell and seed textures. As a consequence, frozen seeds cannot be consumed directly. However, the osmotic dehydration process could add value to frozen pomegranate seeds. Indeed, freezing before osmotic dehydration provided 1.4 and 3.5 times more WL and SG, respectively, than an untreated sample at the beginning of the process. As a consequence, the process could be stopped after 20 min, implying a substantial gain of time and thermal energy. On the contrary for FS, it is better to continue the process up to 420 min. After the osmotic dehydration process, microstructure and texture analysis revealed a modification of the seed and cell structures. This could essentially be due to WL and SG. On the basis of seed texture, osmodehydrated frozen fruit may be considered more suitable for incorporating in many foods products, while osmodehydrated fresh fruit, due to their hard texture, could be air-dried to produce dried fruit. The finished product has an attractive color and presents good texture in the mouth, a pleasant sugar taste and a good aroma. Osmotic dehydration reduced water activity from 0.989 to an average of 0.900. At this Aw value, a complementary treatment, such as drying, freezing and pasteurization, should be necessary to ensure its good conservation.

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