HPLC determination of organic acids, sugars, phenolic compositions and antioxidant capacity of orange juice and orange wine made from a Turkish cv. Kozan

Microchemical Journal 91 (2009) 187–192

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Microchemical Journal

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HPLC determination of organic acids, sugars, phenolic compositions and antioxidantcapacity of orange juice and orange wine made from a Turkish cv. Kozan

Hasim Kelebek ⁎, Serkan Selli, Ahmet Canbas, Turgut CabarogluUniversity of Cukurova, Faculty of Agriculture, Department of Food Engineering, 01330-Adana, Turkey

⁎ Corresponding author. Tel.: +90 322 338 68 60; fax:E-mail address: [email protected] (H. Kelebek).

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Keywords:Orange juiceWinePhenolic compoundsOrganic acidsHPLCAntioxidant activity

olic compositions and antioxidant capacities of orange juice and orange wineobtained from the cv. Kozan of Turkey were determined. High-performance liquid chromatographic methodswere used to identify and quantify of these compounds. Three organic acids (citric, malic and ascorbic acids)and three sugars (sucrose, glucose and fructose) were determined. The major organic acid was found as citricacid. With regard to sugars, sucrose was present in the largest amounts for orange juice and wine. A total of13 phenolic compounds were identified and quantified in orange juice and wine, including hydroxybenzoicacids (2), hydroxycinnamic acids (5), and flavanones (6). Hesperidin, narirutin and ferulic acid were the mostabundant phenolic compounds in orange juice and wine. Antioxidant activities of orange juice and winewere measured using the DPPH• (2,2-diphenyl-1-picrylhydrazyl) assay, and the antioxidant capacity oforange juice was found to be higher than that of orange wine.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

The most widely grown citrus fruit in Turkey is orange, with anannual production of 1535806 tons in 2006 [1]. Oranges is grownthroughout the world in tropical and subtropical areas, where suitablesoils and climates are found. Among oranges, Kozan is a native orangevariety of Citrus sinensis and is produced on a large scale in the Kozanarea of the Adana province in southern Turkey. It is one of the mostimportant varieties used in orange juice and wine. Kozan orangesharvested in February and March have been said to be of the bestcommercial quality for processing, having a good balance of sweet tasteand a refreshing aroma. The fruit is medium size with a bright orangeflesh [2].

Wine is defined as an alcoholic beverage, which is produced byfermentation of fresh grapes or must. Grapes and apples are the cropsmostwidely grown for production of juices for winemaking. Citrus fruit,such as orange, mandarin, and grapefruit, are also used for wine [3–5]and wine cooler [6]. General composition and antioxidant properties offoodstuff were major factors in determining consumer acceptance andpreference. Organic acid, sugar and phenolic compounds are among themajor compounds of citrus fruits. Their nature and concentration largelyaffect taste characteristic and organoleptic quality. Organic acids are auseful index of authenticity in fruit product. The organic acidcomposition of fruits is alsoof interest because of its important influenceon the sensory properties of fruit juices. Themain organic acids of citrusfruits are citric and malic acids. In addition, traces of benzoic, oxalic and

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succinic acids have also been reported [7]. Sugars are the majorcomponents of citrus juice soluble solids and sweetness of orange juiceis intrinsic to its sugar composition. Sucrose is present in the largestamounts in orange juice. In recent years, more attention had been paidon phenolic compounds of citrus fruits, and some publications havesuggested theymight play an important role on the antioxidant capacityof citrus fruits [8–10]. Phenolic acids and flavanones are the two maingroups of phenolic compounds in orange juices. The most importantphenolic acid inorange juice is hydroxycinnamic acidand its derivatives:ferulic, p-coumaric, sinapic, caffeic and chlorogenic acids [9,11]. Peleget al. [12] investigated the distribution of bound and free caffeic, p-coumaric, ferulic, and sinapic acids in the Shamuti orange fruit andconfirmed ferulic acid predominance over the other hydroxycinnamicacids. Flavanones in oranges occur mainly as glycosides; glycosilationtakes place at position 7 either by rutinose or neohesperidose [13,14].Hesperidin and narirutin are known as the main flavanones in orangejuices [9,15,16]. Other flavanone glycosides identified in orange juicesinclude didymin, neohesperidin and naringin [16]. Orange flavonones,especially hesperidin, have shown a wide range of therapeuticalproperties such as anti-inflammatory, antihypertensive, diuretic,analgesic and hypolipidemic activities [14,17,18].

Recent studies have focused on the antioxidant capacity of citrusjuices [19,20]. Gorinstein et al. [8] observed that phenolic compounds inorange juices strongly correlate with the antioxidant capacity deter-mined in DPPH and FRAP assays. Arena et al. [21] studied the evaluationof antioxidant capacity of blood orange juice as influenced byconstituents, concentration process and storage. They reported thatblood orange juices have higher total antioxidant activity than blondjuices, and freshly-squeezed juices are higher thanprocessed.Miller and

188 H. Kelebek et al. / Microchemical Journal 91 (2009) 187–192

Rice-Evans [22] have underlined the significant contributory role ofpolyphenols (particularly hesperidin and narirutin) in the totalantioxidant activity of orange juice, even if vitamin C was the mostabundant antioxidant.

Although it is an important cultivar, organic acids, sugars, phenoliccompositions and antioxidant activity of Kozan orange juice and winehave not been investigated before. This research was undertaken todetermine the organic acids, sugars, phenolic composition andantioxidant capacity of the orange juice and wine obtained from thecv. Kozan.

2. Material and methods

2.1. Chemicals

Milli-Q water (Millipore, Bedford, MA) was used in all work. HPLC-grade acetonitrile and formic acid (Merck, Darmstadt, Germany) wereused after filtration through a 0.45-µm pore size membrane filter. 2,2-diphenyl-1-picryl hydrazyl (DPPH•) was obtained from Sigma–Aldrich(St. Louis, MO) and ascorbic acid was purchased from Merck(Darmstadt, Germany). Phenolic acids (gallic, protocatechuic, caffeic,chlorogenic, p-coumaric, ferulic, and sinapic acids) and flavanones(narirutin, naringin, hesperidin, neohesperidin, didymin) were pur-chased from Sigma-Aldrich (Steinheim, Germany).

2.2. Oranges

The mature oranges of Kozan variety (500 kg) were harvested in themiddle of March 2006 from three different orchards in the province ofKozan and transported to the Pilot Winery of the Department of FoodEngineering, Faculty of Agriculture, University of Cukurova. The orangejuice was obtained using the “Indelicato Super Automatic, Type A2104”extractor. For chemical and HPLC analysis, the freshly squeezed juiceswere centrifuged at 4000 rpm in a centrifuge (Eppendorf 3810 R,Hamburg, Germany) for 20 min, the supernatant were then filteredthrough 0.45-µm pore size membrane filters and were kept attemperature of −18 °C until analysis.

2.3. Wine making

Orange wine was produced as described previously [2,3]. Orangejuice obtained from the extractor was passed through the finisher toremove the seeds and pulp, and 50 mg L−1 SO2 was added. The orangejuiceswere then transferred into two stainless steel tanks (100 L) for thefermentation using spontaneous yeasts. Fermentationwasperformed induplicate at controlled temperatures (19±2 °C). During the alcoholicfermentation, 145 g L−1 sugar was also added to obtain a higher ethanollevel. After fermentation, thewinewas racked by adding 50mg L−1 SO2.

2.4. Standard chemical analysis

The total titratable acidity was assessed by titration with sodiumhydroxide (0.1 N) and expressed as citric acid. The pH value wasmeasured using a digital pHmeter (WTW Inolab pH-L1, Germany). Totalsoluble solids were measured as Brix using a refractometer (Carl Zeiss,Jena, Germany) [23]. Ash was determined by igniting aweighed samplein a muffle furnace at 550 °C to a constant weight, and extract wasmeasured by the method of AOAC [23].

2.5. Liquid chromatographic analysis of organic acids and sugars

A HPLC system (LC-10A HPLC Series, Shimadzu, Kyoto, Japan)equipped with a pump system, a refractive index detector (RID-10A)for sugar analysis, and a UV/Vis detector (SPD-20A) monitored at210 nm, for the analysis of organic acids. Sugars and organic acids weresimultaneously analyzed onto an Aminex HPX-87H column

(300×7.8 mm) (Bio-Rad) and kept at 55 °C. The analytical conditionsused were as follows: flow 0.3 mLmin−1, eluent 0.045 N H2SO4 with 6%acetonitrile (v/v) [24–26].

2.6. Liquid chromatographic analysis of phenolic compounds

Samples were filtered through a 0.45-µm pore size membrane filterbefore injection. An Agilent 1100 HPLC system (Agilent Technologies,Palo Alto CA-USA) operated by Windows NT based ChemStationsoftware was used. The HPLC equipment was used with a diode arraydetector (DAD). System consisted of a binary pump, degasser and autosampler. The columnusedwas a BeckmanUltrasphereODS (Roissy CDG,France): 4.6 mm×250 mm, 5 μm equipped with a precolumn4.6 mm×10 mm (same granulometry). The mobile phase consisted oftwo solvents: Solvent A, water/formic acid (95:5; v/v) and Solvent B,acetonitrile/solvent A (60:40; v/v). Phenolic compounds were elutedunder the following conditions: 1 mL min−1

flow rate and thetemperature was set at 25 °C, isocratic conditions from 0 to 10 minutewith 0% B, gradient conditions from 0% to 5% B in 30min, from5% to 15%B in 18 min, from 15% to 25% B in 14 min, from 25% to 50% B in 31 min,from 50% to 100% B in 3 min, followed by washing and reconditioningthe column. The ultra-violet-visible spectra (scanning from 200 nm to600nm)were recorded for all peaks. Triplicate analyseswere performedfor each sample. The identification of phenolic compounds wereobtained out by using authentic standards and by comparing theretention times and ultra-violet-visible spectra with those found in theliterature [10,16,18,26], while quantification was performed by externalcalibration with standards.

2.7. Antioxidant activity determination

The free radical scavenging activity of orange juice and wine weremeasured according to the DPPH• method reported by Brand-Williamset al. [27] with modifications [28]. Five different dilution of each orangejuice andwine (30/100, 20/100,15/100,10/100, 5/100)were prepared inethanol/water (v/v). An aliquot of 0.1 mL of diluted orange juice wasadded to 3.9 mL of DPPH• solution in methanol (6×10−5 M) andvortexed. The decrease in absorbance of DPPH• at 515 nmwasmeasuredat different time intervals by a Shimadzu UV-1700 spectrophotometer(Kyoto-Japan) until the reaction reached plateau (time at the steadystate). The DPPH• concentration (CDPPH as mg/ml) in the reactionmediumwas calculated from the following equation:

A515nm= 31:275CDPPH R2 = 0:9998� �

:

The percentage of remaining DPPH• (% DPPHREM) was calculated asfollows:

k DPPHREM = 100×CDPPH=CDPPH ; t = 0

where CDPPH, t=0 is the initial DPPH• concentration and CDPPH is theDPPH• concentration at the steady state. The percentage of remainingDPPH• at the steady state for the five dilution was plotted versus theratio mg DPPH• mL−1 orange juice and wine. The parameters EC50 andTEC50 were calculated graphically, and the AEwas determinedwith thefollowing equation:

AE = 1=EC50TEC50:

All tests were performed in triplicate.

3. Results and discussion

3.1. Chemical composition of the orange juice and wine

The chemical composition of theorange juice andwinewere given inTable 1. The juice yield was 40.0 L/100 kg (40.0%). The contents of total

Table 1General composition of Kozan orange juice and wine

Juice compositionJuice yield (%) 40.0±0.71Density (20 °C/20 °C) 1.052±0.01Total acidity (g L−1)a 9.11±0.01pH 3.35±0.01Brix 11.8±0.00Ash (g L−1) 3.7±0.08Extract (g L−1) 123±0.14

Wine compositionDensity (20 °C/20 °C) 1.021±0.01Ethanol (v/v %) 12.6±0.02Total aciditya (g L−1) 6.3±0.01pH 3.6±0.01Volatile acidityb (g L−1) 0.29±0.22Ash (g L−1) 2.8±0.24Extract (g L−1) 70.2±1.26Free SO2 (mg L−1) 8.2±0.08Bound SO2 (mg L−1) 73.3±0.12

aAs citric acid; bas acetic acid.

Table 2Sugar, organic acid, phenolic compositions of the orange juice and wine

Compounds Juice Wine

Sugars (g L−1)Sucrose 59.34±2.04 44.68±1.27Glucose 32.30±0.86 1.06±0.36Fructose 28.55±0.94 3.04±1.08Total 120.19±3.84 48.78±2.71

Organic acids (g L−1)Citric acid 12.66±0.16 6.03±0.08Ascorbic acid 0.49±0.01 0.23±0.01Malic acid 1.06±0.01 0.34±0.01Total 14.21±0.18 6.60±1.01

Hydroxybenzoic acids (mg L−1)Gallic acid 3.33±0.22 1.93±0.14Protocatechuic acid 0.96±0.06 0.45±0.03Total 4.28±0.29 2.38±0.18

Hydroxycinnamic acids (mg L−1)Caffeic acid 5.66±0.38 2.57±0.19Chlorogenic acid 8.49±0.57 4.66±0.35p-Coumaric acid 3.52±0.24 1.58±0.12Ferulic acid 24.06±1.62 9.91±0.74Sinapic acid 18.65±1.06 7.78±0.58Total 60.38±3.87 26.50±1.69

Flavanones (mg L−1)Narirutin 39.91±2.69 21.67±1.61Naringin 2.23±0.15 1.29±0.10Hesperidin 171.17±4.87 90.65±5.26Neohesperidin 0.95±0.06 0.55±0.04Didymin 6.07±0.41 3.52±0.26Apigenin 32.37±4.20 16.12±2.69Total 252.7±12.38 133.8±9.96

Results are the means of three repetitions.In Fig. 3.

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acidity, pH, ascorbic acid, ash and extract were in agreement with thosefound by Selli et al. [2,3]. Similarly Nagy and Smoot [29] reportedconcentrations of total acidity (%) from 0.77 to 1.11, and Brix (totalsoluble solids) from 10.2 to 12.6 in Valencia and Hamlin orange juices.The general composition of the wine was in accordance with previousstudies carried out on orange wine [2–4].

3.2. Sugar, organic acid, phenolic compositions of the orange juice andwine

Table 2 shows the sugar, organic acid and phenolic compositions ofthe orange juice and wine, which correspond to the three analyticalreplicates.

3.2.1. Sugar compositionSucrose, glucose and fructose were determined as sugar components

in orange juice andwine (Fig.1). Total amounts of sugar were 120.19 g L−1

and48.78g L−1 in juice andwine, respectively (Table2). Themainportionsof carbonhydrates in citrus fruits are the three simple sugars: sucrose,glucose, and fructose. Together, they represent about 80% of the totalsoluble solidsoforange juice, and the ratiosof sucrose:glucose:fructosearegenerally about 2:1:1 [24]. This ratio was similar for Kozan orange juicesample. Sucrose is present in the largest amounts for orange juice (59.34 gL−1) andwine (44.68g L−1), account for about%49.12of orange juice and%91.6 of wine's total sugar content (Table 2). Kozan orange juice containedboth reducing and nonreducing sugar in about equal amounts. The sugarcontent in Kozan orange juice was agreeable to the previous data; 23.7%glucose, 25.8% fructose, and 50.5% sucrose based on total sugars presentfrom 86 Florida Valencia orange juices [24]. The sugar profile and ratios ofspecific sugars have been suggested as an indicator for determining theauthenticity of juice samples [24,25].

3.2.2. Organic acid compositionOrganic acids most of soluble solids founds in citrus juices that are

not carbonhydrates. Three organic acids were separated and identifiedin orange juice and wines: citric, ascorbic and malic acids (Fig. 2). Themajor organic acid was found as citric acid in orange juice (12.66 g L−1)and wine (6.03 g L−1) (Table 2.). Reported results for citric acid level infresh, hand-squeezed Navelina juice from different region were 8.4–12.6 g L−1 [30]. Malic acid was second abundant organic acid in theorange juice (1.06 g L−1) andwines (0.34 g L−1). The lower concentrationof organic acids in the wine compared to the juice can be explained bylosses during fermentation. Citric and malic acids of sour orange juice

from Antalya were reported by Karadeniz as 48.8 and 2.2 g L−1,respectively [7].

Citrus juices, especially orange juice are rich sources of ascorbicacid, which is an important antioxidant. Concentration of ascorbic acidis a significant indicator of orange juice quality [21]. In our study,concentration of ascorbic acid in orange juice and wine were found by490.1 mg L−1 and 230.0 mg L−1, respectively (Table 2). It was foundthat the ascorbic acid content of the juices analyzed in the presentstudy was very similar to that of Navel orange juices produced inAustralia (498 mg L−1), although higher than those of Valencia orangejuices analyzed in the same study (406mg L−1) and others produced inthe Mediterranean area from different orange varieties, ranging from386.2 to 620.0 mg L−1 [31].

3.2.3. Phenolic composition of orange juice and wineA total of 13 phenolic compounds were identified and quantified in

orange juice and wine (Fig. 3), including hydroxybenzoic acids (2),hydroxycinnamic acids (5) and flavanones (6) compounds. The totalamount of phenolic compounds was 317.36 mg L−1 in orange juice and162.68 mg L−1 in wine. The total content of phenolics in several juicesobtained from five different Citrus sinensis (L.) varieties ranges from 361(Washington navel variety) to 1147 (Moro variety) mg of ferulic acidequivalents per liter [32]. According to Gil-Izquierdo et al. [15] hand-squeezed navel orange juice contains 839 mg of phenolics per liter.

Two hydroxybenzoic acids; gallic and protocatechuic acid, weredetected in orange juice and wine (Table 2). The major hydroxybenzoicacid is gallic acid (3, 4, 5-trihydroxybenzoic acid). Gallic acid is anaturally abundant plant phenolic compound. It is present in food ofplant origin, and since it was found to exhibit antioxidative properties, ithas attracted considerable interest [33].

The five hydroxycinnamic acids identified in the analysis werecaffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, and sinapic

Fig. 1. HPLC chromatogram of sugars in orange juice (Peaks: 1. Sucrose; 2. Glucose; 3. Fructose).

190 H. Kelebek et al. / Microchemical Journal 91 (2009) 187–192

acid. Ferulic acid was the most dominant hydroxycinnamic acids inorange juice (24.06mg L−1) andwine (9.91mg L−1), as it accounted forthe largest proportion of the total hydroxycinnamic acids contents

Fig. 2. HPLC chromatogram of organic acids in orange juic

(Table 2). Ferulic acid is a precursor for 4-vinyl guaiacol, thetransformation catalysed by the enzyme ferulic acid decarboxylase.Objectionable off-flavours resulting from 4-vinyl guaiacol have been

e (Peaks: 1.Citric acid; 2. Ascorbic acid; 3. Malic acid).

Fig. 3. Simultaneously recorded DAD chromatograms of the orange juice at 280 nm (for hydroxybenzoic acids and flavanones) and 320 nm (for hydroxycinnamic acids). (1. Gallic acid; 2.Protocatechuic acid; 3. Caffeic acid; 4. Chlorogenic acid; 5. p-Coumaric acid; 6. Ferulic acid; 7. Sinapic acid; 8. Narirutin; 9. Naringin; 10. Hesperidin; 11. Neohesperidin; 12 .Didymin; 13. Apigenin).

Table 3Free radical scavenging parameters of orange juice and wine

EC50 (ml mg−1 of DPPH) TEC50 (min) AE×10−3

Juice 0.31±0.2 105±2.0 30.72±2.0Wine 0.46±0.1 124±1.5 17.54±1.0

191H. Kelebek et al. / Microchemical Journal 91 (2009) 187–192

reported for improperly stored orange juice [12]. Sinapic acid(18.65 mg L−1) was the second most abundant hydroxycinnamicacid and followed by chlorogenic, caffeic, p-coumaric acids in orangejuice andwine. The study of Rapisarda et al. [32] found that ferulic acid(37.7 mg L−1) was the main phenolic acid in Valencia juices. Caffeicacid (2.1 mg L−1), sinapic acid (9.0 mg L−1), and p-coumaric acid(8.0 mg L−1) were also quantified. The levels of ferulic and p-coumaricacids in Kozan orange juice slightly lower than those reported byRapisarda et al. [32].

Flavanone is the major flavonoids in orange varieties. Sixflavanones; narirutin, naringin, hesperidin, neohesperidin, didyminand apigenin were identified in orange juice and wine. Table 2 showsthat, of the five flavanones, hesperidinwas the most abundant in bothsamples. Neohesperidin was the least abundant flavanone in thesamples. Hesperidin is tasteless and therefore does not contribute tothe taste of orange juice [13]. The level of hesperidin reported here forthe orange juice (171.17 mg L−1) is in good agreement with previouslyreported by Tomás-Barberán and Clifford [13], 104-637 mg L−1 andGorinstein et al. [8], 122–254 mg L−1. The literature reports thathesperidin level in the orange juice depends on an extraction method,

technological treatment and storage [13]. Narirutin was the secondmost abundant flavanone (39.91 mg L−1) in orange juice. Narirutinlevels in sweet orange juices have been reported at 26.3–54.2 [34] and30.0–80.4 mg L−1 in different varieties [35].

In the literature narirutin-to-hesperidin ratio has been proposedfor quality control of orange juices. The ratio obtained for Kozanorange juices was 0.233. According to earlier work on orange juices,the narirutin-to-hesperidin ratio ranged from 0.151 to 0.262 [14].Rouseff [36] reported that this ratio has to be b0.339 for authenticorange juices.

192 H. Kelebek et al. / Microchemical Journal 91 (2009) 187–192

3.3. Antioxidant activity of orange juice and wine

In our study, the antioxidant activities of orange juice and winewere evaluated using DPPH• free radical-scavenging assays. Thismethod is recommended by many authors [20,28] as easy andaccurate assays for measuring the antioxidant activity of orangejuices and other fruits. EC50 is inversely related to the antioxidantcapacity of a compound, as it expresses the amount of antioxidantneeded to decrease the radical concentration by 50%. The lower EC50

value the higher the antioxidant activity of a compound [20]. The EC50

value of orange juice (0.31 mg mL−1) was found lower than orangewine (0.46 mg mL−1) (Table 3). Orange juice has a higher antioxidantactivity contained higher concentration of phenolic compounds.TEC50 is the time need to reach the steady state to EC50 concentration.Time at steady state depends on the reactivity of antioxidants and theconcentrations used. The antiradical efficiency (AE) is a newparameter for the measurement the free radical scavenging ofsamples, and it combines the EC50 and TEC50 [28]. AE value of orangejuice (30.72×10−3) was found higher than wine (17.54×10−3). In theliterature, antioxidant activity of citrus juices depends on the type(position and number of hydroxyl in the molecule) and theconcentration of the phenolic compounds, as well on that of thetransition metal [18,19].

4. Conclusion

In this study, for the first time, organic acids, sugars, phenoliccontents, and antioxidant activity of the orange juice andwine obtainedfrom the cv. Kozan have been examined. The results indicated that totalantioxidant activity and phenolic contents of orange juice were higherthanorangewine. From the all phenolic compounds identified in orangejuice and wine, hesperidin, naruritin and ferulic acid were the mostabundant phenolic compounds. Quantitatively the major organic acidand sugar were found as citric acid and sucrose in the samples,respectively.

Acknowledgements

The authors would like to thank the University of Cukurova forfinancial support to this research project (Project no. ZF-2006-BAP-18).

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