Evaluation of Irradiation and Heat Treatment on Antioxidant Properties of Fruit Peel Extracts and Its Potential Application During Preservation of Goat Fish Parupenaeus indicus

ORIGINAL PAPER

Evaluation of Irradiation and Heat Treatmenton Antioxidant Properties of Fruit Peel Extractsand Its Potential Application During Preservationof Goat Fish Parupenaeus indicus

Alagesan Paari & Hari Krishnam Naidu & Paulraj Kanmani & Ramraj Satishkumar &

Neelakandan Yuvaraj & Vellaiyan Pattukumar & Venkatesan Arul

Received: 14 August 2010 /Accepted: 2 March 2011 /Published online: 19 March 2011# Springer Science+Business Media, LLC 2011

Abstract The quenching capacities of Vitus vinifera seedextract, Citrus limon peel extract, Punica granatum peelextract, and Citrus sinensis peel extract were studied togetherwith their antioxidant activity in goat fish (Parupenaeusindicus). The functionality of the extracts was evaluatedusing β-carotene-linoleic acid model system, reducingpower assay, DPPH, hydroxyl, and nitrite radical scavengingassay. V. vinifera and P. granatum extract demonstrated bestradical scavenging potential in all multifunctional antioxi-dant assays. Radical scavenging activity measured byelectron paramagnetic resonance against a stable radical1,1,-diphenyl-2-picrylhydrazil revealed radical peaks oflower intensity in antioxidant-infused samples. Compoundspossessing antioxidant properties were identified frompurified fruit extracts by GC–MS analysis. Treatments withthese extracts increased the stability of irradiated goat fishagainst lipid oxidation. TBARS values for irradiated controlwas 4.26±0.10, whereas it was 2.44±0.14, 2.97±0.01, 2.93±0.03, 3.89±0.05, and 4±0.01 mg of MDS per kilogramfish for BHA, P. granatum peel, V. vinifera seed, C. sinensispeel, and C. limon peel extract-treated samples, respectively.This study also elucidated the relationship between heatingtemperature and irradiation dose on the antioxidant activityof extracts. Maximum antioxidant activity was observed at150 °C heated and 10-kGy irradiated extracts. These resultssuggest that fruit peels will be a potential material forextracting antioxidants.

Keywords Plant antioxidants . Lipid oxidation . Foodanalysis . Free radical

Introduction

Ever since the global necessity for fishery products isincreasing, there is a call for efficient preservative approaches.The serious outstanding problem with respect to distributionof seafood is their vulnerability to spoilage, primarily due tothe contamination of spoilage microorganisms (Niemira2010). Gamma irradiation has been employed for decontam-ination of animal foods, and there are ample publishedevidence showing the immense potential of this method forprolonging shelf life of fish and fishery products (Chwla etal. 2003). Though irradiation and frozen storage mayessentially stop the growth of pathogenic microorganisms,deterioration of quality brought about by chemical andphysical origins can happen (Ahn et al. 1998). The radiolyticproducts formed out of irradiation react with other musclecomponents and spoil the organoleptic and nutritionalattributes of fish species (Gardner 1979). Therefore, additionof either synthetic or natural radical scavengers to quenchthese unwanted radiolytic products is of high importance forthe successful preservation of food. Antioxidant combina-tions displayed the distinct rewarding reduction in inhibitingthe negative shades of irradiation in fish products and act asexcellent lipid stabilisers (Pazos et al. 2005). The interest inusing natural antioxidants as additives by the food industrycontinues to grow because they are presumed to be safesince they occur in foods and the question of safety ofsynthetic compounds can thus be avoided. Natural antiox-idants extracted from plants such as grapes (Yerlikaya and

A. Paari :H. K. Naidu : P. Kanmani :R. Satishkumar :N. Yuvaraj :V. Pattukumar :V. Arul (*)Department of Biotechnology, School of Life Sciences,Pondicherry University,Pondicherry 605014, Indiae-mail: [email protected]

Food Bioprocess Technol (2012) 5:1860–1870DOI 10.1007/s11947-011-0552-4

Gokoglu 2010), pomegranate (Gokoglu et al. 2009), andcitrus peel (Kang et al. 2006) have been used as alternativesto the synthetic antioxidants because of their potential effecton inhibition of lipid oxidation. Many of the abovementioned plant products contain antioxidant compounds inbound form with insoluble polymer (Niwa and Miyachi1986). Curiosity in the role of natural antioxidants in foodpreservation has promoted research in food science toresolve how their activity can be ameliorated via processingtechnologies (Avila-Sosa et al. 2010). Gamma irradiationrecommended for sterilisation of food has been reported tohave some enhancing effects on the antioxidant aspects ofplant extracts (Choi et al. 2006). This technology along withheat treatment was used to enhance the antioxidant proper-ties of selected fruit extracts. The aim of this work was toassess the radio-protecting capability of four Indian fruitextracts subjected to various doses of gamma irradiation andheat treatments and to demonstrate their practical utility in asea food system.

Materials and Methods

Sample Collection

Grape (Vitus vinifera), lemon (Citrus limon), pomegranate(Punica granatum), and orange (Citrus sinensis) wereobtained from a local market. In this study, heating and/orirradiation effects on the radical scavenging activity (RSA) offruit peels, under the experimental conditions of heating at 50,100, and 150 °C for 60 min and/or irradiating at 2.5, 5, and10 kGy, was determined. Fruit peels were heated in a heatfurnace and irradiated using cobalt 60 gamma chamber beforeextraction. Extracts from P. granatum was prepared using themethod described by Singh et al. (2002). Briefly, peels weremanually separated and shade-dried, and were extracted with100 ml of methanol at RT for 24 h. The extracts were filtered,and were re-extracted with same solvent and concentratedunder reduced pressure through rotator evaporator. Method ofLi et al. (2006) was followed for extraction from C. limon andC. sinensis. Briefly, frozen peels were dipped in liquidnitrogen and ground to fine powder. The powdered peelswere extracted using 100 ml of ethanol for 24 h for completeextraction. V. vinifera seeds were powdered and extracted in asoxhlet extractor with hexane for 6 h for the removal of fattymatter. The defatted powder was extracted in a soxhletextractor for 24 h with 100 ml methanol at 60–70 °C asdescribed by Jayaprakasha et al. (2001).

DPPH Radical Scavenging Activity

The capacity to scavenge the “stable” free radical DPPH wasmonitored according to themethod of Blois (1958). Methanolic

extracts from fruit peels (0.1 ml) were mixed with 0.9 ml ofmethanolic solution containing DPPH radicals (0.041 mM).The mixture was shaken vigorously and left to stand for60 min in the dark. The reduction of the DPPH radical wasdetermined by measuring the absorption at 517 nm. Theradical-scavenging activity (RSA) was calculated as apercentage of DPPH discoloration using the equation:

%RSA ¼ ADPPH � Asð ÞADPPH

� �� 100

Methanol was used as blank. AS is the absorbance of thesolution when the sample extract has been added, andADPPH is the absorbance of the DPPH solution.

Reducing Power

The reducing power of extracts was determined accordingto the method of Oyaizu (1986). Methanolic extract (1 ml)was mixed with 1 ml of 0.2 M phosphate buffer (pH 6.6)and 1 ml of potassium ferricyanide (10 mg/ml). Themixture was incubated at 50 °C for 20 min. After 1 ml oftrichloroacetic acid (100 mg/ml) was added, the mixturewas centrifuged at 13,400×g for 5 min. The upper layer(1.0 ml) was mixed with 1 ml of deionised water and 0.1 mlof ferric chloride (1.0 mg/ml), and the absorbance wasmeasured at 700 nm. Blank was prepared by adding everyother solution but without extract and ferric chloride(0.1%). Increased absorbance of the reaction mixtureindicates stronger reducing power.

β-Carotene Bleaching Assay

β-Carotene bleaching assay was done according to themethod of Matthaus (2002). A solution of β-carotene wasprepared by dissolving 1 mg of β-carotene in 1 ml ofchloroform. After the chloroform was removed at 40 °Cunder vacuum, 40 mg of linoleic acid, 100 ml of distilledwater, and Tween 80 emulsifier were added to the flaskwith vigorous shaking. Aliquots (5 ml) of this emulsionwere transferred into different test tubes containing 0.2 mlof extracts. The tubes were shaken and incubated at 50 °Cin a water bath. The zero time absorbance was measured at470 nm using a spectrophotometer. Absorbance readingswere then recorded at 30 min intervals until the controlsample had changed colour. A blank, devoid of β-carotene,was prepared. Antioxidant activity was calculated using thefollowing equation:

Antioxidant activity

¼ b � carotene content after 120 min of assay

Initial b � carotene content

� �� 100

Food Bioprocess Technol (2012) 5:1860–1870 1861

Nitrite Scavenging Activity

This assay was carried out as described by Saha et al.(2004) with some modifications. Methanolic extracts fromfruit peels (3 ml) were mixed with 2 ml of citric acid buffer(pH 3.0) and 0.1 ml of 200 μg/ml NaNO2. One hundredmillilitres of distilled water was added to the flask withvigorous shaking. After incubation for an hour at 37 °C,equal volume of Griess reagent (1% sulphanilamide, 0.1%N-(1-naphthyl)-ethyline diamine hydrochloride, 2.5%H3PO4) was added to the above mixture. The absorbancewas determined after 10 min at 538 nm. BHA was used asthe positive control. NaNO2 scavenging activity wascalculated using the following equation:

% Nitrite scavenging activity

¼ Astandard � Asample � Acontrol

� �Astandard

� 100

where AStandard is without NaNO2 and without extract, andAcontrol is without NaNO2.

Hydroxyl Radical Scavenging Activity

Hydroxyl radical scavenging activity was determined accord-ing to the method of Smirnoff and Cumbes (1989) with a fewmodifications. 0.3 ml of 5 mmol/l phenanthroline, 0.8 ml of0.75 mol/l phosphate buffer (pH 7.4), and 0.3 ml of7.5 mmol/l Feso4 were added to 0.5 ml of extract. To thismixture, 0.2 ml of 1% H202 was added and incubated at37 °C for 60 min. The absorbance of the resulting solutionwas measured spectrophotometrically at 532 nm, and theactivity was calculated using the following formula:

% Hydroxyl radical scavenging activity

¼ 1� Asample

Acontrol

� �� 100

Phosphate buffer was used as blank. Acontrol is theabsorbance of control without the tested samples, andAsample is the absorbance in the presence of testedsamples.

Electron Paramagnetic Resonance Spectroscopy

Electron paramagnetic resonance (EPR) investigations onRSA of extracts against a stable radical were measuredusing the method described by Nanjo et al. (1996). Amethanol solution of 60 μl each sample was added to 60 μlof DPPH (60 μmol l−1). After mixing vigorously, thesolutions were transferred into an aqueous cell quartz tubeand fitted into the cavity of the EPR spectrometer. The spinadduct was measured exactly 2 min later in an X-band EPR

spectrometer at room temperature using standard rectangularcavity operating at 9.4 GHz with a 100-kHz modulationfrequency. The microwave power and modulation amplitudewere 4 mW and 1 G, respectively.

Gas Chromatography–Mass Spectrometry Analysisof Natural Extracts

Dry powder of selected material showing maximumantioxidant properties (10-kGy irradiated sample) wassubjected to column chromatography employing silica gel(60–120 mesh size) and eluted stepwise using a lineargradient of chloroform:methanol. Fractions after analysisby TLC were pooled into their complementary groups. Theactive fraction, which showed maximum DPPH radicalscavenging activity in each extracts, was subjected for GC–MS analysis. Extracts were dissolved in ethanol (200 mg/ml) and centrifuged at 13,400×g for 5 min to precipitateundissolved materials. The supernatant was mixed with 4volumes of BSA [N,O-bis(trimethylsilyl)acetamide] andderivatised in a water bath (70 °C) for 15 min. Thecompounds in extracts were identified using a gas chro-matograph–mass spectrometer (GC6890/MS5973, Hewlett-Packard Co., Wilmington, DE). A split inlet (100:1) wasused to inject sample (5 μL) into an HP-5 column (30 m,0.32 mm i.d., 0.25 μm film; Hewlett-Packard Co.). Aramped oven temperature was used (100 °C for 2 min,increased to 270 °C at 10 °C/min, and held for 6 min). Theinlet temperature was 250 °C, and the carrier gas was He ata constant flow of 1.5 ml/min. The ionisation potential ofthe mass selective detector was 70 eV. Identification ofcompounds detected was achieved by comparing massspectral data of samples.

Evaluation of Antioxidant Potential in Goat Fishafter Irradiation Treatment

Antioxidant activity in the meat system was determinedusing the method of Moller et al. (1999) with modifica-tions. Parupenaeus indicus (goat fish) were separated intosections in sterile plastic bags and were treated withselected natural extracts as well as with BHA, a syntheticantioxidant. The percentage of extract that was used is 1%for all cases prior to irradiation. The irradiated sampleswere stored at 4 °C to examine the efficacy of naturalextracts in irradiated samples.

Peroxide Value

Peroxide value (PV) was determined using the methoddescribed by the American Oil Chemists Society (1990).Five grams of sample was dissolved in 30 ml glacial aceticacid–chloroform solution (3/2 v/v), and 1 ml KI solution

1862 Food Bioprocess Technol (2012) 5:1860–1870

(14 g/10 ml) was added. Distilled water was added after1 min, and the mixture was titrated with 0.01 N sodiumthiosulphate until the blue colour disappeared. PV wascalculated using the following formula:

PV mEq=Kgð Þ ¼ V � B� Nð ÞW

� �� 100

where V is the volume of sodium thiosulphate consumed, B isthe volume of normal sodium thiosulphate consumed duringa blank titration, W is the weight of the sample, and N is thenormality of sodium thiosulphate multiplied by a factor.

TBA Assay

Oxidation of lipids is assessed by the TBA assay which isbased on the reaction between thiobarbituric acid andmalondialdehyde as described by Ruberto and Baratta (1999).Briefly, 0.05 g of each sample was mixed with 1 ml ofdistilled water, 1.5 ml of 20% acidic acid, and 1.5 ml 0.8% ofTBA in 1.1% SDS and heated to 100 °C for 60 min in a waterbath. After cooling, 5 ml butan-1-ol was added. Samples werethen centrifuged at 10,000 rpm for 10 min. The absorbance ofthe upper layer was determined at 532 nm.

DPPH Assay

Measurements with the DPPH assay were taken using amethod used by Tepe et al. (2005). 0.05 g of the fishsamples and 5 ml of 0.004% DPPH in methanol weremixed. The samples were incubated at room temperature for30 min. Absorbance was measured at 517 nm, using methanolas a blank. Measurements were expressed as absorbance.Decreasing absorbance levels indicated increasing antioxidantactivity of the extracts.

Statistical Analysis

One-way analyses of variance were conducted to determinesignificant differences between treatment means. Differ-ences at p<0.05 were considered significant. Experimentswere replicated thrice on different occasions with differentfish samples. Data were expressed as mean ± standarddeviation. Analysis was performed using an SPSS package(SPSS 7 for Windows, SPSS Inc, USA).

Results and Discussion

Scavenging Activity of Natural Extracts

Hydroxyl radical is the most effective radical that react withlipids, proteins, and oxygen resulting in the production of

peroxy radical. Thus, removing hydroxyl radical is impor-tant for antioxidant defence in food systems (Aruoma1998). Effect of fruit extracts on hydroxyl radicals wasmoderate for C. sinensis (47.41±4.54%) and P. granatumpeel (57.6±4.05%) as compared to BHA, which showed68.26±2.95% inhibition (Fig. 1a). The antioxidant activityhas been reported to be concomitant with the reducingpower. The high reducing power (0.554 (C. limon)–0.874(V. vinifera seed) ASE/ml) indicated their potential aselectron donors to scavenge free radicals efficiently. Thereducing properties are generally associated with thepresence of reductones (Singh et al. 2002) which breakdown the free radical chain by donating a hydrogen atom.The polyphenols of the antioxidant extracts are believed toact in a similar fashion. Reducing powers obtained forextracts were in the following order: V. vinifera seed > P.granatum peel > C. sinensis peel > C. limon peel. Exposureto excess nitrite from the diet is implicated as a potentialetiological factor in the development of colorectal cancers(Kato and Puck 1971). Nitrite scavenging effect of naturallyoccurring fruit peel extract proved it to be a potential nitritescavenger. As shown in Fig. 1b, the scavenging capacities ofC. limon (53.5±5.15%) and C. sinensis (59±5.90%) towardsnitrite were relatively feeble, whereas the scavenging activitiesbecame stronger with V. vinifera seed (78.53±6.55%) and P.granatum peel (76.70±5.41%). Figure 1c shows the antiox-idant activity of the fruit peel and seed extracts as measuredby the bleaching of β-carotene. Antioxidant activities were 42±4.71%, 47.7±4.40%, 70.1±2.13%, and 67.4±1.73% for C.limon, C. sinensis, V. vinifera seed, and P. granatum extracts,respectively, but antioxidant activity of BHA reached 79.5±3.9%. The absorbance decreased rapidly in samples withoutantioxidant, whereas, in the presence of an antioxidant, theyretained their colour for a longer time indicating the antioxidantpotential of extracts. It is apparent that the components in the

Fig. 1 Antioxidant potential of fruit peel extracts: A Hydroxyl radicalscavenging activity, B nitrite scavenging activity, C β-carotene bleaching,D DPPH, E reducing power. Results are shown as means ± SD of threeindependent experiments


fruit extracts reduce the extent of β-carotene destruction byneutralising the linoleate-free radical formed in the system.

DPPH assay is a standard test in antioxidant studies andoffers a rapid technique for screening the RSA of specificextracts. The reduction capability of the DPPH radical isdetermined by the decrease in its absorbance at 517 nminduced by antioxidants (Bidchol et al. 2009). Protonradical scavenging potential was higher in V. vinifera seedextract (64.9±2.65%) followed by P. granatum peel (61.6±1.1%) and C. sinensis peel extract (39.7±1.0%). MinimalDPPH scavenging activity was observed in C. limon extract(23.3±3.41%), whereas positive control BHA exhibited anRSA of 78.61±3.0%. The better free radical scavenging byBHA is credited to their methoxy group, which increasesthe accessibility of the radical centre of DPPH to BHA(Gow-Chin and Pin-Der 1994).

Effect of Heating Conditions on the Antioxidant Activityof Natural Fruit Extracts

Effect of heat and irradiation treatment on the antioxidantcapacities of extracts were evaluated by two assays, DPPHradical scavenging activity (Fig. 2a) and beta-carotenebleaching assay (Fig. 2b). One can notice that the RSAsignificantly increased after the heat treatment at highertemperatures (150 and 100 °C) compared to lowertemperature (50 °C). Thermal treatment at 150 °C increasedthe RSA of fruit peel extracts from 30.19±3.46, 40.98±1.98,68.26±1.13, 69.02±1.38 to 38.96±1.33, 49.46±3.80,79.2±2.64, and 80.17±2.37 for C. limon peel extract, C.sinensis peel extract, V. vinifera seed extract, and P.granatum peel extract, correspondingly. We believe that theincrease is primarily due to the degradation and release of

bound antioxidant compounds from the matrix. Similar tothe results of DPPH assay, the scavenging potentialincreased in beta-carotene assay as the heating temperatureis increased. Maximum activity was observed in P. granatumpeel (84.53±1.12) and V. vinifera seed extract (86.3±4.20)when heated to 150 °C, suggesting the possibility of using itas a natural antioxidant for applications at higher temper-atures. The enhancing effect of simple heat treatment on theantioxidant activity of fruit peel extracts represented in thisstudy was consistent with that of previous reports (Lin et al.2008; Xu et al. 2007). However, this result contradicts thefindings of Bchir et al. (2010) who reported that an increasein temperature caused a decrease of the antioxidant activityin pomegranate.

Effect of Irradiation Conditions on the Antioxidant Activityof Extracts

Radical scavengers were assessed after exposing it to threedifferent doses 2.5, 5, and 10 kGy. The percentageantioxidant activity increased significantly upon irradiationtreatment from 69±4.09 and 69.51±1.59 for 2.5 kGyirradiated samples to 84.15±3.60 and 81.8±4.01 byincreasing the dose to 10 kGy for V. vinifera seed and P.granatum peel extracts, respectively, in DPPH assay.Similar to the results of DPPH assay, the scavenging potentialincreased in beta-carotene assay with higher doses. Theantioxidant potential elevated from 75.82±2.73 to 84.29±2.06 for P. granatum peel and from 72.33±1.90 to 81.5±3.23for V. vinifera seed extract with increasing dosage of gamma-irradiation from 2.5 to 10 kGy. Maximum activity wascontemplated at 150 °C and 10 kGy treated samples whichelucidates the affinity between the temperature and radiation

Fig. 2 a Effect of heat treatments (A 50 °C; B 100 °C; C 150 °C) andirradiation dose (D 2.5 kGy; E 5 kGy; F 10 kGy) on DPPH radicalscavenging activity. Results are shown as means ± SD of threeindependent experiments. b Effect of heat treatments (A 50 °C; B

100 °C; C 150 °C) and irradiation dose (D 2.5 kGy; E 5 kGy; F10 kGy) on antioxidant potential of fruit peel extracts in β-carotenemodel system. Results are shown as means ± SD of three independentexperiments


dose with the radical scavenging activity. Release of phenoliccompounds from unextractable insoluble polymers to anextractable form was considered a reasonable mechanism tobe responsible for the elevated antioxidant activity.Radiation-induced enhancement of antioxidant activity wasshown by Variyar et al. (2004) in soybean.

Electron Paramagnetic Resonance Studies on RadicalScavenging Activity

EPR spectrum of the DPPH radical is recognised by itsfive lines of relative intensities. It is important to note thatthe higher the antioxidant potential of fruit peel extracts,the smaller the signal of DPPH. The EPR signal intensityin the system with fruit peel and seed extracts is smaller(Fig. 3B–E) than that observed without the extracts(Fig. 3A). The decline in the signal after incubation withextract preparations may be due to the pairing of oddelectron of DPPH, reflecting the protective role of theseextracts in restricting the generated radicals. It is also veryapparent from the spectra that P. granatum peel andV. vinifera seed extract minimised the intensity of theradical peak to a greater extent in comparison to otherextracts. Similar decrease in the amount of EPR spectrawas observed after the addition of grape skin extracts byStavikova et al. (2008).

Gas Chromatography–Mass Spectrometry of PartiallyPurified Extracts

GC–MS analysis of active fraction of fruit peel extractsrevealed the presence of significant quantities of n-alkanes,namely tridicane, hexadecane, and pentadecane in the V.

Fig. 3 The typical EPR spectra of DPPH methanolic solution(60 μmol l−1) recorded in the presence of fruit extracts illustratingtheir antioxidant action (A control, B C. limon, C C. sinensis, D V.vinifera, F P. granatum). Spectra were recorded 2 min after theextracts addition

S. no RT Compound Area

1 6.34 Tridecane 1.02

2 8.0 hexadecane 8.06

3 8.0 Tetradecane 2.63

4 9.349 Heneicosane 2.49

Heptadecane

5 9.98 Phenol, 2,4-bis(1,1-dimethylethyl) 4.12

6 10.0 1-Iodo-2-methylundecane 1.1

7 10.17 Benzoic acid, 4-ethoxy-, ethyl ester 7.71

8 11.2 Hexadecane 3.87

9 12.9 Nonadecane, 2-methyl- 2.57

10 14.573 Tetradecanoic acid 3.31

11 14.9 Octadecane 2.44

12 23.47 Benzenamine, 3-methoxy-2,4,6-trimethyl 3.71

13 23.58 Benzenamine, 3-methoxy-2,4,6-trimethyl 1.70

14 27.0 4 H-1-Benzopyran-4-one 1.96

15 27.6 1,2-Benzenedicarboxylic acid 2.46

16 28.69 Octadecane, 1-chloro- 3.60

17 29.3 Stigmasta-5,22-dien-3-ol 7

18 31.1 7-Oxabicyclo[4.1.0]heptane, 1-methyl-4-(2-methyloxiranyl)- 10.82

19 31.6 2-Butyn-1-ol, 4-methoxy- 1.74

20 32.57 2-Ethoxycarbonyl-3-methyl-7-nitro 13.66

21 33.35 10,11-(3′-6′-Dihydrobenzo)[3.2]par acyclophane-4′-carboxylic acid 2.20

22 33.53 1-Heptatriacotanol 5.07

Table 1 Detected compoundsin gas chromatography from V.vinifera seed extracts


vinifera seed extracts (Table 1). Liolios et al. (2009) alsoreported the presence of those compounds in fruits of datepalm. Abundance of carboxylic acids like propanoic acidand butanoic acid was seen in C. limon samples. Hexade-coinic acid, an antioxidant from tea leaves (Jang et al.2010), was also found in C. limon extract (Table 2). Themain component Benzopyran 2 one in C. limon extract hasbeen reported for its biological activity in lemon peel oil bySu and Horvat (1987). Benzoic acid, tetradecane, dodecane,Phenol, 2, 4-bis (1, 1-dimethylethyl), tetradeconic acid, andnonadecane comprise the antioxidant properties for C.sinensis extract (Table 3). Presence of benzoic acid, 2-

Furancarboxaldehyde in orange juices, and their antioxidantnature has already been proven in many studies (Jerkovićand Marijanović 2010). Tetradecanoic acid as one amongthe fatty acid compositions has been studied for its radicalscavenging properties by Kırmızıgül et al. (2007). Benzene dicarboxylic acid, the major compound in P. granatum extractin our study (Table 4), was also detected in heat-treated citruspeels by Lee et al. (2003). Some compounds, which havebeen reported as major constituents in our study, were absentor expressed in low quantity from other authors reports. Thismay be due to different chemotype of the plant other thangeographical divergence and ecological condition.

Effect of Natural Extracts on TBARS and Peroxide ValueEvolution in Irradiated Samples

Addition of fruit peels extracts delayed the generation ofperoxides (Fig. 4) and TBARS values (Fig. 5). The intensesusceptibility of goat fish to suffer oxidation duringirradiation and storage is mainly due to the synchronouspresence of high amounts of unsaturated lipids. Theproblem associated with lipid oxidation during irradiationand storage is the fat content (5.78%) of the goat fish. Anincrease in the peroxide value is most useful as an index ofthe earlier stages of oxidation. The initial peroxide valuewas 1.2±0.11 and 1.7±0.30 in 2.5 and 5-kGy irradiatedgoat fish, respectively. By the end of storage time,significant differences (P>0.05) were observed betweenthe control (3.26±0.11) and each of V. vinifera, P.granatum, C. sinensis, and C. limon extract-treated sam-ples, which exhibited values of 2.06±0.11, 2.2±0.26, 3.33


1 8.6 Bicyclo [3.1.1]hept-2-ene, 2,6-dime thyl-6-(4-methyl-3-pentenyl)- 1.75


3 6.95 Benzocycloheptatriene 1.16

4 12.6 1 H-Pyrrole, 2-ethyl-4-methyl- 1.02

5 18.5 Hexadecanoic acid, ethyl ester 1.09

6 18.82 2 H-1-Benzopyran-2-one, 5,7-dimethoxy 12.82

7 23.07 Pimpinellin 2.92

7 H-Furo[3,2-g][1]benzopyran-7-one 4,9-dimethoxy-

8 29.07 Pyridine, 3-methyl-2,6-diphenyl 1.23

Dicyclooctanopyridazine

9 29.83 1-Naphthalenamine, N-phenyl- 1.02

10 31.8 Butanoic acid, 4.02

11 31.9 Propanoic acid 1.67

12 32.14 Beta-D-Fructopyranose, 3.35

13 32.36 4 H-1-Benzopyran-4-one 3.43

14 33.15 5-Hydroxymethyl tricyclazole 9.34

15 33.3 4 H-1-Benzopyran-4-one 7.09

16 33.875 Benzene, 1,2,4-trimethyl-5-(1-methylethyl)- 1.53

Table 2 Detected compoundsin gas chromatography from C.limon peel extracts

Table 3 Detected compounds in gas chromatography from C.sinensis peel extracts


1 6.1 2-Furancarboxaldehyde, 5-(hydroxymethyl) 2.36

2 6.34 Dodecane, Eicosane 2.08

3 6.95 Benzocycloheptatriene 1.16


5 9.3 1-Iodo-2-methylundecane 1.71

6 9.9 Phenol, 2,4-bis(1,1-dimethylethyl) 3.14



9 12.9 Nonadecane, 2-methyl- 2.28


11 14.9 Octadecane 1.44

12 20.74 Eicosane, 2-methyl- 1.64

13 28.07 Hentriacontane 1.22


±0.30, and 3.26±0.11, respectively (Fig. 4a). Analogoustrend was observed in 5-kGy irradiated samples, where theV. vinifera seed extract and P. granatum extract exhibitedbetter PV compared with the control and other counterparts(Fig. 4b). The decline of the PV at the end of storage mayoccur owing to decomposition of hydroperoxides intosecondary oxidation products. As peroxide value measure-ment are not reliable in assessing oxidation of highlyunsaturated fish samples because of their nature to formsecondary oxidation products, PV was used in conjunctionwith TBA and DPPH assay.

Initial TBA values were low in all treated samplescompared to the control as in the PV. A significant increasein TBARS was observed at the 12th day of storage, perhaps

due to the fact that TBARS are secondary products of lipidoxidation formed from decomposed lipid hydroperoxides.No difference in TBARS was noticed amid the dippingtreatments during initial days of storage. However, final dayvalue was 4.26±0.10, 4.086±0.01, 3.89±0.05, 2.97±0.01,2.93±0.03, and 2.44±0.14 for control, C. limon peel, C.sinensis peel, P. granatum peel, and V. vinifera seedextracts, respectively (Fig. 5a). It was prominent thatformation of TBARS in goat fish P. indicus increased whenthe irradiation dose was increased from 2.5 to 5 kGy. Finalday TBARS value for 5-kGy irradiated control sample was4.97±0.08, whereas it was 4.3±0.03, 4.26±0.06, 3.13±0.09, 3.769±0.01, and 3.04±0.09 for C. limon peel, C.sinensis peel, V. vinifera seed, P. granatum peels, and BHA

Table 4 Detected compounds in gas chromatography from P. granatum peel extracts


1 6.1 2-Furancarboxaldehyde, 5-(hydroxymethyl) 5.74


3 9.9 Phenol, 2, 4-bis (1, 1-dimethylethyl) 1.53



6 12.9 Heptacosane 1.00


8 26.1 2 H,8 H-Benzo[1,2-b:3,4-b′]dipyran-6-propanol, 5-methoxy-2,2,8,8-tetra methyl-, acetate 1.01

9 27.6 1,2-Benzenedicarboxylic acid 44.77

10 27.9 Tetratetracontane 1.86

11 31.4 4-[p-Chlorostyryl]-6-methoxy-8-[2, 5-dimethylpyrryl]quinoline 2.16

12 31.78 Disulphide, di-tert-dodecyl 1.55

13 32.3 4-Azafluorenone, phenylimine 11.6

14 32.4 1,3-Dioxo-2-[4-(3,4-xylyloxy)pheny l]-5-isoindolinecarboxylic acid 5.79

15 32.5 4 H-1-Benzopyran-4-one, 2-(3,4-dime thoxyphenyl)-5,6,7-trimethoxy 2.98

Fig. 4 a Formation of peroxides (mEq/kg) in 2.5-kGy irradiated goatfish without and with exogenous antioxidative treatment duringrefrigerated storage. Results are shown as means ± SD of threeindependent experiments. b Formation of peroxides (mEq/kg) in 5-

kGy irradiated goat fish without and with exogenous antioxidativetreatment during refrigerated storage. Results are shown as means ±SD of three independent experiments


respectively (Fig. 5b). Inhibition of lipid oxidation inirradiated samples could be due to either suppression offree radicals during initiation step or interruption of freeradicals chain reaction by acting as electron donours. BHAsupplied samples showed better TBARS value than thenatural extracts treated group. However, the difference wasnot significant for P. granatum and V. vinifera seed extracts.A similar result was observed by Kang et al. (2006) in fishhomogenate by the addition of citrus peel extract.

Demonstration of Antioxidant Activity in Irradiated FishSamples by DPPH Assay

Data generated using the DPPH assay in goat fish P. indicusirradiated at 2.5 and 5 kGy are presented in Fig. 6a, b,respectively. Throughout the study period, antioxidant

supplied samples exhibited lower DPPH absorbance valuesindicative of higher antioxidant activity than the controlsamples which are irradiated without antioxidant. Overall,fish samples infused with P. granatum and V. vinifera seedextract displayed higher antioxidant activity (i.e., lowerabsorbance) than did the C. limon, C. sinensis, and controlsamples. A final DPPH absorbance value of 0.270±0.003was seen for control, while it was 0.254±0.003, 0.232±0.002, 0.219±0.002, 0.200±0.003, and 0.193±0.004 for C.limon, C. sinensis, P. granatum, V. vinifera seed, and BHArespectively. Analogous reduction was observed in 5-kGyirradiated samples. A final DPPH value of 0.915±0.003was observed for control samples, while DPPH absorbancevalue for samples supplied with C. limon, P. granatum, V.vinifera seed, and C. sinensis extracts was 0.81±0.001,0.66±0.002, 0.60±0.004, and 0.72±0.001, respectively.

Fig. 5 a TBARS values (mg MDA kg−1 of fish) of 2.5-kGy irradiatedgoat fish added with natural fruit extracts during refrigerated storage.Results are shown as means ± SD of three independent experiments. b

TBARS values (mg MDA kg−1 of fish) of 5-kGy irradiated goat fishadded with natural fruit extracts during refrigerated storage. Resultsare shown as means ± SD of three independent experiments

Fig. 6 a Profiles of DPPH antioxidant activity level in 2.5-kGyirradiated goat fish infused with natural fruit extracts duringrefrigerated storage. Results are shown as means ± SD of threeindependent experiments. b Profiles of DPPH antioxidant activity

level in 5-kGy irradiated goat fish infused with natural fruit extractsduring refrigerated storage. Results are shown as means ± SD of threeindependent experiments


The purple-coloured DPPH (a stable free radical) is reducedto the yellow-coloured 1,1,-diphenyl-2-picrylhydrazine react-ing with the free radicals of the sample (Kirby and Schmith1997). The lower gathering of free radicals in antioxidantsupplied samples might be the reason for having lowerabsorbance (i.e., higher antioxidant reactivity). Overall, theantioxidant activity of all samples lessened with storagetime. Fasseas et al. (2007) demonstrated similar lower DPPHabsorbance in meat samples preserved with oregano andsage essential oils.

Conclusion

Oxidative damage caused by free radicals and other reactiveoxygen species is believed to be associated with thedevelopment of a range of diseases. This research hasdemonstrated that the extract from fruits, which is com-monly used as a natural food, is an excellent free radicalscavenger. Furthermore, the outcomes of the present studysuggest that antioxidant activity can be enhanced byexternal stimuli. Supply of food samples with naturalantioxidants prior to gamma irradiation treatment hadreduced the occurrence of lipid oxidation to a larger extentduring preservation. In the near future, it is possible that theapplication of synthetic antioxidants will decrease stillfurther and consumers will become more willing to acceptsafe natural antioxidants for preservation of sea foods.

Acknowledgements We duly acknowledge the financial supportreceived from the Department of Biotechnology, New Delhi, India.

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Documents

Evaluation of Irradiation and Heat Treatment on Antioxidant Properties of Fruit Peel Extracts and Its Potential Application During Preservation of Goat Fish Parupenaeus indicus