Food Chemistry 145 (2014) 312–318

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Food Chemistry

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Chemical and sensory quality of fresh pomegranate fruits exposedto gamma radiation as quarantine treatment

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.08.052

⇑ Corresponding author. Tel.: +82 53 950 5775; fax: +82 53 950 6772.E-mail address: jhkwon@knu.ac.kr (J.-H. Kwon).

Hafiz Muhammad Shahbaz a, Jae-Jun Ahn a, Kashif Akram a,b, Hyo-Young Kim a, Eun-Joo Park a,Joong-Ho Kwon a,⇑aSchool of Food Science & Biotechnology, Kyungpook National University, Daegu 702-701, Republic of KoreabInstitute of Food Science and Nutrition, University of Sargodha, Sargodha 40100, Pakistan

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 May 2013Received in revised form 9 August 2013Accepted 14 August 2013Available online 26 August 2013

Keywords:PomegranatesGamma-irradiationQuarantine disinfestation treatmentChemical propertiesAntioxidative capacitySensory profile

The U.S. Department of Agriculture in February 2012 approved the import of fresh pomegranates sub-jected to irradiation as a quarantine procedure with a minimum absorbed dose of 0.4 kGy against differ-ent pests. This study evaluated the application of different gamma-irradiation doses (0.4, 1, and 2 kGy) infresh pomegranate fruits and their effect on the chemical and sensory characteristics. The total solublesolids, titratable acidity, and pH values remained unaffected up to 1 kGy treatment. Irradiation causeda significant decrease in the total anthocyanins and phenolic content. A strong positive correlation wasobserved among the antioxidant activities, total phenolics and anthocyanin contents. In general, a stron-ger preference was shown by sensory panelists for the juice from irradiated fruits. This study providesresearch-based information about the application of irradiation as a quarantine disinfestation treatmentto enhance the marketing and consumer acceptance of pomegranates.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction consistency in quality. The Korea Food and Drug Administration

Pomegranates (Punica granatum L.) are one of the importantcommercial fruits extensively cultivated in many tropical and sub-tropical regions of the world (Tehranifar, Zarei, Nemati, Esfandiy-ari, & Vazifeshenas, 2010). The fruit has wide consumerpreference as the consumption of fresh arils or juice because ofits exceptional and unique sensory and nutritional properties(Varela-Santos et al., 2012). The arils contain a considerableamount of polyphenols, polysaccharides, sugars, acids, vitamins,and important minerals (Al-Maiman & Ahmad, 2002). A growingnumber of scientific studies have highlighted the potential humanhealth related benefits of pomegranate juice such as antiathero-genic, antioxidant, antihypertensive, etc. (Mena et al., 2011;Rajasekar, Akoh, Martino, & MacLean, 2012; Tehranifar et al.,2010; Varela-Santos et al., 2012; Zaouay, Mena, Garcia-Viguera, &Mars, 2012).

The fresh consumption of pomegranates has increased in Koreabut local production, that covers an area of about 161.4 ha, is notenough to fulfil market demand (Shahbaz, Akram, Ahn, & Kwon,2013). In the past few years, the United States (U.S.) has capturedthe major market share (97%) of all the imported pomegranates inthe Korean market. The fresh pomegranates imported from the U.S.are preferred by consumers because of their uniformity and

(KFDA) has been authorised to conduct inspections of pomegranatefruits upon arrival at ports in Korea. According to the KFDA estab-lished standards and phytosanitary import requirements, the im-ported fruits must contain proper labelling indicating sufficientinformation. In addition, pomegranates should not undergo anydisinfestation treatment such as fumigation, etc. by importers toqualify for ‘‘organic certification’’ as Korean consumers perceivelow-chemical products as healthy products (U.S. Agricultural TradeOffice, 2010).

Pomegranate fruits have a high risk of infestation with suckinginsects and mite pests during growth which deteriorates theirquality and constrain the international trade (Ananda, Kotikal, &Balikai, 2009). According to pest risk assessment prepared by theAnimal and Plant Health Inspection Service (APHIS) of the U.S.Department of Agriculture (USDA, 2012), 25 quarantine pestsincluding two mites (Tenuipalpus granati and Tenuipalpus punicae)could follow the pomegranate fruit pathway. The APHIS in Febru-ary 2012 authorised the import of fresh pomegranates into theU.S. mainland from India to a minimum irradiation dose of0.4 kGy as quarantine disinfection treatment. The recommendedirradiation dose, along with standard postharvest processes, willhelp to effectively neutralise the concerned insect pests and miti-gate the risks of their dissemination (USDA, 2012). The most feasi-ble application of irradiation technology in agricultural products,including fruits, is probably quarantine disinfestation without sig-nificantly affecting the chemical or sensory attributes (Fields &

H.M. Shahbaz et al. / Food Chemistry 145 (2014) 312–318 313

White, 2002). However, research is required to determine theappropriate irradiation doses and their subsequent effects on dif-ferent quality attributes in fruits. In general, the FDA restricts themaximum irradiation dose level to 1 kGy for disinfestation and de-layed maturation in fresh fruits (Boylston, Reitmeier, Moy, Mosher,& Taladriz, 2002).

The aim of this study was to investigate the effect of gamma-irradiation, as a quarantine disinfestation treatment, on the chem-ical and sensory qualities of fresh pomegranate fruits. In addition,total polyphenols and antioxidant activities were assessed usingtwo different radical scavenging assays. The results were used totest for correlations between different quality-related parameters.

2. Material and methods

2.1. Pomegranate fruits, irradiation, and juice extraction

This study was done using pomegranate fruits grown in Califor-nia (California cultivar; U.S. origin) and freshly imported to Korea(December 2012). Seventy commercially available fresh pome-granates, packed in cardboard boxes, were purchased from a localmarket in Daegu, South Korea. The fruits were divided into fourequal portions and labelled with the specific radiation dose.Approximately, 16 fruits were sampled for each irradiation treat-ment and kept overnight at 5 �C in the laboratory. The packed fruitsamples were then irradiated at the Korea Atomic Energy ResearchInstitute (Jeongeup, Korea) with doses of 0, 0.4, 1, and 2 kGy usinga Cobalt-60 gamma-ray source (AECL, IR-79, MDS Nordion Interna-tional Co. Ltd., Ottawa, Ontario, Canada).

The irradiation process was accomplished at room temperaturewith a dose rate of 1.5 kGy/hr. The absorbed doses (±5.6%) werecalibrated by alanine dosimeters with a 5 mm diameter (BrukerInstruments, Rheinstetten, Germany) in which the free-radical sig-nals were determined with a Bruker EMS 104 EPR analyzer (BrukerInstruments, Rheinstetten, Germany) (Shahbaz et al., 2013). Afterirradiation processing, the pomegranate fruits were taken to thelaboratory and manually cut with a sterile sharp blade to separatethe fleshy arils. Juice was extracted from the isolated arils using asolid fruit juice extractor (Juice Extractor, Model Le Duo, Magimix,France). The extracted juice was pour into labelled sterile glass bot-tles and immediately analysed or stored at 4 �C. The experimentaljuice samples were filtered with Whatman qualitative filter paper,Grade 4, before the chemical analyses. The pure juice samples werediluted at different proportions with distilled water for differentanalyses until the absorbance was within the linear range of thespectrophotometer (Optizen 2120UV, Mecasys Co. Ltd., Daejeon,Korea). All the analyses were independently repeated three timesto ensure accuracy. All the chemicals used were of analytical gradeand purchased from Sigma–Aldrich.

2.2. Chemical analyses

The titratable acidity (TA) of the juice was measured by titratingit against 0.1 N NaOH to the end point of pH 8.1, monitored with apH meter. The results were expressed as percentage of citric acid.The pH measurements were performed using a digital pH meter(Orion 3 star, Thermo Electron Co., Waltham, MA, USA) at 21 �C.The total soluble solids (TSS) in the juice were determined with adigital refractometer (Master-M, ATAGO, Brix 0–32%, Tokyo, Japan)at 20 �C. The instrument was calibrated with distilled water beforethe analysis. TSS values were expressed as �Brix (Rajasekar et al.,2012).

The total anthocyanins content in juice samples was deter-mined with the pH differential method using two buffer systems:potassium chloride buffer, pH 1.0 (0.025 M) and sodium acetate

buffer, pH 4.5 (0.4 M) according to Giusti and Wrolstad (2001).Briefly, 1 mL of diluted juice was mixed with 4 mL of correspond-ing buffers. Absorbance was measured at two wavelengths,510 nm and 700 nm, after 15 min of incubation in a spectropho-tometer against distilled water as a blank. The total anthocyaninscontent was calculated by applying the standard formula. The val-ues, three replications per sample, were expressed as mg cyanidin-3-glucoside per 100 mL of juice.

The juice colour measurements were done in a colorimeter(CM-3600d, Konica Minolta, Osaka, Japan) using the Hunter Labscale (‘L⁄’: Lightness; ‘a⁄’: redness; ‘b⁄’: yellowness) previously de-scribed by Rajasekar et al. (2012). The instrument was calibratedagainst a white reference plate provided with the chromameter be-tween different readings. The quartz cell was filled with filteredjuice and colour data were recorded with the Minolta SoftwareChroma control data system. The average values of 3 measure-ments were reported.

The amount of total sugars in the fruit juice was determinedusing a modified version of the phenol–sulphuric acid assay re-cently described by Nielson (2010). Accurately 1 mL of the dilutedjuice sample was mixed with 1 mL of 5% phenol solution and 5 mLof 96% sulphuric acid (rapidly added) in each tube. The tubes werevortexed and allowed to stand at room temperature for 20 min.The concentrated sulphuric acid converts all non-reducing sugarsto reducing sugars, so the method determines the concentrationof the total sugars present in the sample. A blank was preparedby substituting distilled water for the juice sample. The absorptionof the characteristic yellow-orange colour produced as a result ofthe interaction between the sugars and the phenol was measuredat 490 nm using a spectrophotometer. The typical colour of thisreaction is stable for several hours. The concentration of the totalsugars present in each sample was calculated by referring to astandard sucrose curve.

The content of the reducing sugars was measured with the Nel-son–Somogyi method (Somogyi, 1952) with minor modifications.The method is widely used for the quantitative determination ofreducing sugars in biological materials. Four types of required solu-tions were prepared according to standard procedures with highaccuracy. Arsenomolybdate reagent was incubated at 37 �C for24 h prior to use. The diluted juice sample (0.5 mL) was mixed withthe different solutions as previously described. The absorbance ofthe blue colour was read at 520 nm with a spectrophotometer.The amount of reducing sugars present in the fruit juice samplewas calculated from a standard curve graph drawn using a glucosesolution as the standard. The average results for triplicate determi-nations were expressed as g/100 mL of juice.

2.3. Total phenolics content and antioxidant capacities

The concentration of the total phenolics was measured by theFolin–Ciocalteu reagent method recently described by Rajasekaret al. (2012). To each 50 lL of diluted juice, 0.5 mL Folin–Ciocalteureagent and 1.5 mL of 7.5% sodium carbonate were added. Thesamples were allowed to stand at room temperature for 30 minincubated under dark conditions. The wavelength of spectropho-tometer was fixed at 765 nm for the absorbance reading. Resultswere expressed as mg of Gallic acid/100 mL of juice using a gallicacid (0–0.1 mg/mL) standard curve.

Several assays were done to estimate the antioxidant activity infresh fruits and their products. Most of the natural antioxidants aremultifunctional; therefore, for a more reliable evaluation, it isimportant to perform different antioxidant activity assessmentsto give proper consideration to the various mechanisms of antiox-idant action. In this study, DPPH (2,2-diphenyl-1-picrylhydrazyl)and ABTS (2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid)methods were used to measure the antioxidant activity of

314 H.M. Shahbaz et al. / Food Chemistry 145 (2014) 312–318

pomegranate fruit juice samples. The free radical-scavengingactivity of the juices was assessed against the stable 1,1-diphe-nyl-2-picryl hydrazyl radical (DPPH) according to the method ofMishra, Ojha, and Chaudhury (2012a) with slight modifications.The DPPH and Trolox solutions were freshly prepared in ethanoland adjusted to a 1.000 ± 0.002 absorbance at 517 nm. Each ofthe 1 mL juice samples was shaken vigorously with 5 mL of freshlyprepared ethanolic solution of DPPH (12 mg DPPH/100 mL etha-nol). The scavenging activity on the DPPH radical of the resultingsolution was measured (525 nm) after 30 s of reaction in the spec-trophotometer against an ethanol blank. The DPPH scavengingability of the extract was calculated from the equation (Mishraet al., 2012a).

For the ABTS assay, the procedure described by Re et al. (1999)was followed. Stock solution of ABTS�+ was prepared by reacting a7 mM aqueous solution of ABTS with 2.4 mM potassium persul-phate solution and allowed to stand for 16 h at room temperaturein dark conditions. The resulting solution was diluted in ethanol toan absorbance of 0.700 ± 0.002 units at 734 nm to obtain ABTS�+

working solution. Each of the 200 lL juice samples was blendedwith 4 mL of ABTS�+ working solution for a total reaction time of5 min. The absorbance was taken at a wavelength of 734 nmagainst ethanol as a standard using the spectrophotometer. Trolox(0.04–1.25 mg/mL) was used as a standard for the calibration curve(Re et al., 1999).

2.4. Sensory evaluation

Sensory evaluation was performed by randomly choosing agroup consisting of 30 students belonging to the School of FoodScience & Biotechnology, Kyungpook National University. The agedistribution of the panelists was between 22 and 30 years includ-ing both men and women. The students had basic knowledge onthe concepts and applications of sensory analysis. Appropriateinformation about the nature of the present evaluation was alsoprovided. Irradiated and control juice samples were placed ontop of white paper plates in a randomised order and identified with3-digit codes. Approximately 30 mL of each juice sample (8 �C) waspresented to the judges with 5 min intervals between samples.Deionised water and unsalted crackers were provided for palaterinsing in-between the samples. Panelists rated the intensity ofthe sensory attributes including color, flavor, aroma, sweetness,sourness, overall taste, and overall acceptances on a five-pointscale (5 = most like; 4 = good; 3 = fair; 2 = dislike a little; 1 = mostdislike). The scores from 2.5–5 were considered acceptable (Kim& Yook, 2009).

2.5. Statistical analysis

Statistical analyses were performed with the Statistical AnalysisSystem (SAS) (Version 9.2; SAS Inst. Inc., Cary, N.C., U.S.A) usingone-way analysis of variance ANOVA. Duncan’s multiple range(DMR) test was applied to calculate the significant difference be-tween different irradiation treatments. Results were expressed asthe average ± standard deviation. Data were also analysed to deter-mine whether there was any correlation between different fruitquality attributes using Pearson correlation.

3. Results and discussion

3.1. Effect of irradiation on total anthocyanins content

Anthocyanins are one of the major groups of pigments account-able for contributing different colors in fruits, including pomegran-

ate juice, and possible health benefits such as dietary antioxidants.During chemical determination analysis, monomeric anthocyaninpigments undergo a reversible structural transformation as a func-tion of pH. The pH differential method calculates the total mono-meric anthocyanin content based on the structural change of theanthocyanin chromophore between pH 1.0 and pH 4.5 (Tehranifaret al., 2010).

From the analysis, a slight gradual decrease in the anthocyanincontent of juice from the gamma-irradiated pomegranate fruit wasidentified (Table 1). The total anthocyanins content decreased asthe dose increment increased from 0.4 kGy to 2 kGy. However,the differences were only statistically significant for the 2 kGytreatment compared to the control samples. Comparable to theseresults, Alighourchi, Barzegar, and Abbasi (2008) directly exposedextracted pomegranate juice to ionizing radiation (0–10 kGy) andobserved a significant reduction in the total and individual antho-cyanins content at all applied doses, particularly at higher doses(3.5–10 kGy). However, from their study results, irradiation treat-ment of juice with more than 2 kGy was not recommended dueto the detrimental effects on the total anthocyanins content. Thedecrease in the anthocyanins content is mainly due to the degrada-tion of individual anthocyanins. The stability of diglycosides antho-cyanins to irradiation was higher than monoglycosides at lowerdoses (0.5 and 2 kGy) of gamma irradiation (Alighourchi et al.,2008). Furthermore, the relative stability of an individual anthocy-anin depends on its matrix, structural features, and the processingconditions (Alighourchi et al., 2008; Torskangerpoll & Andersen,2005).

In contrast, Ayed, Yu, and Lacroix (1999) reported an increase inthe anthocyanins content of grape pomace for increasing irradia-tion doses with an optimum dose at 6 kGy. The stability of antho-cyanins against irradiation is related to the juice composition. Theincrease in the anthocyanins content of grape pomace can beattributed to the extraction of bound pigments through the degra-dation of the cell wall. However, most of the available literature onthe applications of irradiation treatment is restricted to solid foodsand there is limited research available on fruit juices (Alighourchiet al., 2008).

3.2. Effect of irradiation on titratable acidity, pH, and total solublesolids

Comparison of the effects of irradiation (0, 0.4, 1 and 2 kGy) onthe different physicochemical parameters of the pomegranate juicesamples are presented in Table 1. The titratable acidity in the juicesamples remained unaffected at 0.4 kGy but a significant decreasewas observed at 1 kGy and 2 kGy treatments. The main organicacid accountable for the titratable acidity in pomegranate fruit iscitric acid. Similarly, the pH was unchanged up to a 1 kGy dose le-vel but a higher dose produced a significant increase in the value.The total soluble solids concentration of the pomegranates was notaffected at all by the applied irradiation doses.

Conflicting results have been reported about the irradiation ef-fect on pH, TA and TSS parameter in different fruit juices. The pres-ent results are in close agreement to the findings of Fan, Niemera,Mattheis, Zhuang, and Olson (2005) who did not observe any effectof irradiation at 0.5 and 1.0 kGy on the TA and pH values of slicedapples, which were initially treated with 7% calcium ascorbate.Similarly, Miller and McDonal (1996) reported no differences inthe pH values of blueberries when irradiated with gamma-rays(0.5–1.0 kGy). In contrast, Moreno, Castell-Perez, Gomes, Da Silva,and Moreira (2007) found that irradiation up to 3.2 kGy did not af-fect the pH in blueberry fruits. Yu et al. (1995) found no differencesin the pH values of electron beam irradiated strawberry fruits up to2.0 kGy.

Table 2Hunter’s colour values of juice from gamma-irradiated pomegranate fruit (n = 3).

Colour parameter Irradiation dose (kGy)

0 0.4 1 2

L⁄ Lightness 58.05 ± 0.10d 57.40 ± 0.06c 57.08 ± 0.04b 56.47 ± 0.04a

a⁄ Redness 28.43 ± 0.07d 28.80 ± 0.02c 29.81 ± 0.02b 30.81 ± 0.05a

b⁄ Yellowness 18.74 ± 0.05d 18.79 ± 0.08c 19.39 ± 0.04b 20.08 ± 0.03a

DE 0.00 ± 0.00d 0.51 ± 0.09c 1.74 ± 0.14b 3.09 ± 0.05a

Values with the same superscript letters (a–d) in a row are not significantly dif-ferent at p < 0.05.

Table 1Total anthocyanins content (TAC), titratable acidity (TA), pH, reducing sugars (RS), total sugars (TS) and total soluble solids (TSS) of juice from gamma-irradiated pomegranatefruit (n = 3).

Dose (kGy) Parameters

TAC (mg/100 mL) TA (% citric acid) pH RS (g/100 mL) TS (g/100 mL) TSS (oBrix)

0 30.45 ± 0.86a 0.24 ± 0.012a 3.45 ± 0.006b 16.37 ± 240.6b 18.82 ± 0.076a 16.3 ± 0.058a

0.4 29.61 ± 0.19ab 0.24 ± 0.015a 3.45 ± 0.006b 17.04 ± 277.3a 18.82 ± 0.076a 16.3 ± 0.058a

1 28.83 ± 0.26b 0.23 ± 0.044b 3.46 ± 0.006b 16.68 ± 243.9ab 18.81 ± 0.076a 16.3 ± 0.058a

2 28.33 ± 0.39c 0.22 ± 0.00c 3.54 ± 0.012a 16.47 ± 450.6b 18.82 ± 0.076a 16.3 ± 0.058a

Values with the same superscript letters (a–c) in a column are not significantly different at p < 0.05.

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3.3. Effect of irradiation on sugars content

Fruit sweetness is an important aspect of fruit quality. Glucoseand fructose are the most predominant sugars present in all fruitsincluding the pomegranate fruit. Sugars and acids in fruits signifi-cantly influence the flavor, appearance, chemical and sensory char-acteristics (Al-Maiman & Ahmad, 2002).

Most of the available literature describes the titration basedLane–Eynon method for the determination of sugars in fruit juices(Tehranifar et al., 2010). However, titration methods have severaldisadvantages such as the final results largely depend on precisereaction times, temperature and reagent concentration. In addi-tion, the method is susceptible to interference from other typesof molecules that act as reducing agents. In the present investiga-tion, spectroscopic procedures based on a calorimetric techniqueincluding the phenol–sulphuric acid assay and Somogyi–Nelsonmethod were used to quantify the amount of total sugars andreducing sugars, respectively.

Table 1 shows the changes in the sugar content of the fruit sam-ples upon irradiation. The total sugars content did not change inthe pomegranate fruit at all the applied irradiation doses. However,compared to the control, slight variations were observed amongdifferent dose levels for the reducing sugars content. The amountof reducing sugars varied differently at all applied irradiationdoses. Similar findings have been reported by Mitchell, McLauch-lan, Isaacs, Williams, and Nottingham (1992) in which the gammairradiation had no effect on the sucrose and fructose content ofcustard apples at 75 and 300 Gy but a significant increase was ob-served in the glucose levels. In the same experiment, no effect wasobserved in the fructose and glucose content in lemons at 75 Gybut an increase was recorded for the sucrose content. Our findingsare also in agreement with El-Samahy, Youssef, Askar, and Swailam(2000) in which no effect from the gamma radiation (0.5–1.5 kGy)was observed on the total sugars content of mangoes but thereducing sugars were slightly increased. Research studies haveshown that there is no substantial effect from irradiation onmacronutrients such as proteins and carbohydrates in plant mate-rials even up to a dose of 10 kGy (Crawford & Ruff, 1996).

3.4. Effect of irradiation on fruit juice color

Anthocyanin pigments are almost exclusively responsible for thedifferent colors in fruits. The attractive colour (ruby red) of pome-granate fruit juice has been an important quality attribute whichinfluences the consumer behaviour (Zaouay et al., 2012). It may besignificant to investigate the change in colour because irradiationcan result in the destruction of pigments in fruit and fruit juices.

Juice colour indices showed significant statistical differencesamong the control and irradiated fruit samples. The results (Ta-ble 2) show that the control fruit juice had a darker colour thanthat of the irradiated fruit samples with significant differences inthe luminosity dimension scale (Lightness; L⁄ = 0 denotes blackand L⁄ = 100 indicates diffuse white). The redness (a⁄) and yellow-ness (b⁄) indices increased directly with the irradiation dose and

represented significant differences among the samples at differentdose levels. Altogether, the colour difference values (DE) were sig-nificantly different among the various irradiation doses. The pres-ent results are consistent with Boylston et al. (2002) who reportedthat the colour of 0.75 kGy irradiated rambutan and orange fruitstended to be more intense than that of the control fruits visuallyevaluated by sensory judges. Opposite to these findings, a studydone by Mitchell et al. (1992) on mangoes showed a reduction inthe a⁄ values after gamma-rays treatment at 75 and 300 Gy. Thechange in colour can be ascribed to a decrease in the polyphenoloxidase activity by irradiation (Mishra et al., 2012b). Subsequently,the irradiation application produced an important improvement inthe sensory parameters which was also favored by the sensoryjudges for the 0.4 kGy and 1 kGy samples.

3.5. Effect of irradiation on total phenolics content

Phenolic compounds are important because they contribute tothe nutritional and sensory quality of fruits. Phenolic compoundshave been widely studied in many fruits and their activity is believedto be mainly because of the irredox properties, which play a signifi-cant role in adsorbing and neutralizing free radicals (Zaouay et al.,2012). Some of these compounds, particularly the flavonoids, pro-vide health benefits due to their antioxidant capacity. Pomegranatefruit juice contains a greater amount of phenolic compounds com-pared to other fruit juices (Tehranifar et al., 2010). Folin–Ciocalteuis a simple and widely used method to estimate total phenols basedon the mechanism of the transfer of electrons from phenolic com-pounds to the Folin–Ciocalteu reagent in an alkaline medium.

The effect of irradiation on the total phenolic compounds ofjuice samples was significant. Total phenols showed a linear trendof decrease with the gradual increase in irradiation dose. However,the irradiation effect was more prominent at higher doses of 1 kGyand 2 KGy (Fig. 1 A). In addition, a linear relationship was observedbetween the total phenolics content and free radical scavengingactivities of pomegranate juice.

Different results have been published for the irradiation effecton phenolic compounds in foods. The decrease in phenolic con-tents of pomegranate juice upon irradiation in present study corre-lates well with the previous findings of Song et al. (2006) on kalejuice in which a significant decrease was found in the total phenolsof fruit juice immediately after irradiation (0, 3, and 5 kGy). Ahn

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Fig. 1. Total phenolics content (A) and antioxidant capacity tested by DPPH (B),ABTS (C) assays for gamma-irradiated pomegranate fruit (n = 3). Values with thesame letter for each dose are not significantly different at p < 0.05.

316 H.M. Shahbaz et al. / Food Chemistry 145 (2014) 312–318

et al. (2005) also reported a significant reduction in the phenoliccontents of Chinese cabbage at 1 kGy or above. This phenomenonmay occur due to the immediate oxidation of phenolic compoundsthus playing an antioxidant role by reducing the free radicals andthe reactive oxygen species produced by irradiation.

In contrast, Song et al. (2006) found an increase in the total phe-nols in irradiated (0–5 kGy) carrot juice immediately after irradia-tion. El-Samahy et al. (2000) reported that the concentration of thetotal phenolic compounds was higher in irradiated mangoes (0.5–1.5 kGy) compared to the control. One possible reason for thisincreasing trend can be ascribed to the ability of b-carotene in thesefruits to scavenge radicals (Song et al., 2006).

3.6. Effect of irradiation on antioxidant activities with DPPH and ABTSassays

The antioxidant activity index of pomegranate juice wasobtained in terms of its radical scavenging potential by using two(DPPH and ABTS) in vitro assays. These assays have been exten-sively used for the quantification of antioxidant potential of pheno-lic samples from different fruits and vegetables. The DPPH assayrelies on the reduction of DPPH which is a stable free radical witha characteristic absorption and accepts an electron or hydrogenradical to become a stable diamagnetic molecule. The deep purplecolour of a freshly prepared DPPH solution fades in the presence ofan antioxidant and is converted to a colourless product resulting ina decrease in absorbance at 517 nm. The characteristic decrease inabsorbance is mainly due to the scavenging of the radical by hydro-gen donation (Mishra et al., 2012a). Similarly, the ABTS radical cat-ion is reactive towards most antioxidants particularly thephenolics. The blue ABTS radical cation is converted to a colorlessform during its interaction with antioxidants leading to a decreasein absorbance at 734 nm (Re et al., 1999).

As shown in Fig. 1 B, the DPPH values were significantly lowerin irradiated samples, especially at higher doses. The differencewas statistically significant for antioxidant activity between the1 kGy and 2 kGy treated samples. The minimum irradiated samples(0.4 kGy) represented a slight decrease in DPPH activity comparedto the control. However, the effect of irradiation on DPPH activitywas highly significant at the maximum irradiation dose (2 kGy).The ABTS assay profile also showed a corresponding trend to thetotal phenolic compounds upon irradiation. ABTS radical scaveng-ing ability was slightly lowered after irradiation without any sig-nificant differences. However, there were no differences for theABTS values between the 1 kGy and 2 kGy irradiated fruit juices.The main antioxidant compounds in pomegranate juice are hydro-lyzable tannins, but anthocyanins and ellagic acid derivatives alsocontribute to the total antioxidant capacity of the juice (Gil, Tomá-s-Barberán, Hess-Pierce, Holcroft, & Kader, 2000).

The results are consistent with the studies by Song et al. (2006)reporting a significant decrease in the DPPH radical-scavengingcapacity of kale juice upon irradiation (1–5 kGy gamma-rays) atday 1. Kim and Yook (2009) found that irradiation doses of 1 and2 kGy did not significantly affect the antioxidant potential in kiwi-fruits throughout a storage period of 3 weeks. In contrast, the DPPHscavenging ability has dissimilarity to the works of Song et al.(2006) who evaluated the effect of gamma-irradiation on carrotjuice during a storage period of 3 days at 10 �C. The antioxidantactivity of irradiated carrot juice was higher than that of the con-trol juice but decreased over the storage time.

3.7. Sensory evaluation

Sensory investigations are an important part of food controlbecause they help to measure and numerically define the qualitylevel perceived by judges or consumers. The information aboutthe sensory quality attributes plays a significant role in consumersatisfaction and hence influences further consumption (Escribano,Sanchez, & Lazaro, 2010). High doses of irradiation can induce anoff-odour called ‘‘irradiation odor’’ in fruit juices. Volatile sulphurcompounds, such as hydrogen sulphide, methanethiol, methyl sul-phide, dimethyl disulphide, and dimethyl trisulphide, play a signif-icant role in the development of the off-odor. It is observed thatirradiation exerts its effect by hydrolysis of water in foods wherewater is a dominant component. Irradiation of water generatesthree primary free radicals: hydroxyl, hydrogen atoms, andhydrated electrons. Use of specific scavengers in a model systemrevealed that hydroxyl radicals are involved in the formation ofvolatile sulphur compounds (Fan, Lee, & Ahn, 2011). In addition,

Table 3Correlation coefficients (r) of total anthocyanins content (TAC), titratable acidity (TA), pH, reducing sugars (RS), total sugars (TS), total soluble solids (TSS), total phenolics content(TP), DPPH and ABTS assays for antioxidant activity from juice of gamma-irradiated pomegranate fruit.

TAC TA pH RS TS TSS TP DPPH ABTS

TAC 1.0000 – – – – – – – –TA 0.6434 1.0000 – – – – – – –pH �0.6749 �0.9842 1.0000 – – – – – –RS 0.6038 0.9952 �0.9684 1.0000 – – – – –TS �0.8284 �0.8835 0.8889 �0.8592 1.0000 – – – –TSS �0.0658 0.0085 �0.0143 �0.0040 0.0000 1.0000 – – –TP 0.8572 0.7375 �0.7729 0.7104 �0.9304 �0.0178 1.0000 – –DPPH 0.8788 0.7671 �0.7957 0.7545 �0.8938 �0.0274 0.8882 1.0000 –ABTS 0.7686 0.5659 �0.6590 0.5093 �0.7496 0.2005 0.7700 0.7506 1.0000

Table 4Sensory evaluation of juice from gamma-irradiated pomegranate fruit (n = 3).

Parameter Irradiation dose (kGy)

0 0.4 1 2

Color 3.70 ± 1.02ab 4.22 ± 0.90a 3.78 ± 1.00ab 3.26 ± 0.92b

Flavor 3.52 ± 0.90bc 4.09 ± 0.85a 3.87 ± 0.92ab 3.26 ± 0.62c

Aroma 3.30 ± 0.76a 3.43 ± 0.59a 3.48 ± 0.67a 3.35 ± 0.57a

Sweetness 4.13 ± 0.69a 4.09 ± 0.51a 3.96 ± 0.47a 4.00 ± 0.43a

Sourness 3.13 ± 0.76a 3.26 ± 1.01a 3.17 ± 1.03a 3.04 ± 0.93a

Overall taste 3.83 ± 1.07a 3.98 ± 0.68a 3.89 ± 1.02a 3.09 ± 0.79b

Overallacceptance

3.43 ± 0.66b 3.98 ± 0.61a 3.83 ± 0.58a 3.13 ± 0.76b

Values with the same superscript letters (a–c) in a row are not significantly dif-ferent at p < 0.05.5 = Most like; 4 = good; 3 = fair; 2 = dislike a little; 1 = most dislike.

H.M. Shahbaz et al. / Food Chemistry 145 (2014) 312–318 317

irradiation may induce undesirable chemical changes, such as theaccumulation of volatile sulphur compounds including malondial-dehyde, formaldehyde, and tetrahydrofuran which ultimatelyleads to off-flavour in fruit juices (Fan, Niemira, & Thayer, 2004).

Juice from the untreated fruits as well as from the irradiatedfruits were offered to the panelists to rate their preference. Table 4shows the average and standard deviation values of the sensoryattribute ratings for pomegranate fruit juice. In general, the juicefrom low dose (0.4 kGy and 1 kGy) treated fruits were liked moreamong the panelists compared to the juice from the control andhigh dose (2 kGy) treated fruits. The irradiated fruit juice samplesshowed greater variance in the hedonic value at different dose lev-els. The maximum average score was attained with the low dose(0.4 kGy) treated fruit juice in respect to all sensory parameters.Moreover, none of the samples were in the unacceptable hedonicrange (<2.5).

Irradiation induced a significant impact on the colour of all thejuice samples similar to Hunter’ colour results. Noticeable changeswere observed and the colour of the juice from irradiated fruits(0.4 kGy and 1 kGy) was more liked by the judges. Similar to ourresults, Boylston et al. (2002) found that the colour of 0.75 kGyirradiated rambutans and oranges were more intense than the con-trol and preferred by panelists on the visual scale. Preferences foroverall taste were understood to increase with irradiation dose.The total soluble solids and titratable acidity of the fruit juice didnot differ significantly from irradiation (Table 1) correspondingto all the juice samples also equally scored for their sweetnessand sourness values. The sweetness was the ‘‘most liked’’ attributeby the panelists for all types of juices. This aspect also indicatedthat sweet varieties of pomegranate fruit are liked among consum-ers for fresh consumption as juice. The flavour and aroma wereamong the minimally affected sensory aspects as irradiation in-duced no off-flavors. Yu et al. (1995) also reported that sensorypanelists perceived no difference in the flavour of strawberriesirradiated at 2 kGy. Irradiation enhanced the overall acceptabilityof pomegranate fruit juice up to a 1 kGy dose.

In comparison to other studies, Kim and Yook (2009) found thatirradiation positively contributed to improve the sensory quality inkiwifruits. Panelists showed a strong preference for irradiatedkiwifruits fruits (1, 2 and 3 kGy) in sweetness, overall taste andoverall acceptability. El-Samahy et al. (2000) also reported thatmangoes irradiated up 1.0 kGy were more acceptable to the sen-sory panelists for their organoleptic properties.

Corresponding with the findings of McDonald et al. (2012)where consumers rated the overall acceptability of irradiated pea-ches (up to 0.90 kGy) higher than that of the untreated peaches.Mitchell et al. (1992) revealed that the sensory acceptability of or-ange juice prepared from irradiated orange (0.6 kGy) fruit reducedsignificantly. Furthermore, Moreno, Castell-Perez, Gomes, Da Silva,and Moreira (2007) stated that blueberries exposed to 3.2 kGy e-beam irradiation were scored unacceptable by sensory panelists.

The sensory evaluation provides information about the attri-butes of a product from consumers’ perspectives which determinesits acceptance. It is significant to develop a relationship betweenthe physical and chemical composition of a product and its sensoryattributes, as well as between sensory perceptions and acceptabil-ity for consumers (Escribano et al., 2010).

3.8. Correlation analysis

Correlation analysis of the data indicated (Table 3) that there isa strong positive correlation between the antioxidant activity,determined by the DPPH and ABTS assays, and total phenolic(r = 0.8882 and r = 0.7700) and anthocyanins contents (r = 0.7671and r = 0.5659). However, the correlation was higher for the DPPHradial scavenging assay compared to the ABTS antioxidant assay.These compounds are primarily responsible for the antioxidantactivity of pomegranate fruit juice. Similarly, total anthocyaninscontent showed a significant positive correlation with the totalphenolics content (r = 0.7375). A similar correlation was found byZaouay et al. (2012) between the total phenolics content and theantioxidant capacity for 15 Spanish pomegranate cultivars. Thefindings also corroborates with the results of Mena et al. (2011)who reported a linear correlation between total anthocyanins con-tent and antioxidant activity assays. This is an indication that thetotal anthocyanin compounds significantly contribute to the anti-oxidant activity in pomegranate fruit juice. Besides, there was asignificant negative correlation between pH and titratable acidity.

4. Conclusions

Variability in the chemical composition and sensory profiles ofthe pomegranate fruit juice was observed at different dose levels(0.4, 1 and 2 kGy). Irradiation improved the sensory profile and astronger preference was shown by the panelists for juice fromthe 0.4 kGy and 1 kGy treated fruits. The chemical, sensory andnutrient qualities of the juice from the fruits were most affectedby the higher 2 kGy treatment. Total phenolic compounds and

318 H.M. Shahbaz et al. / Food Chemistry 145 (2014) 312–318

antioxidant assays were not significantly affected up to the lower0.4 kGy dose. Furthermore, there is a strong positive correlationbetween the antioxidant activity, total phenolics and anthocya-nins. On the basis of results, irradiation up to 1 kGy can be adoptedas a quarantine disinfestation treatment against disinfestation ofpests in pomegranate fruits. These research-oriented scientificfacts about irradiated pomegranate can help to boost the interna-tional marketing and consumer acceptability of the fruit.

Acknowledgements

This research was supported by Export Promotion TechnologyDevelopment Program, Ministry of Agriculture, Food and Rural Af-fairs, South Korea.

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