Food Chemistry 129 (2011) 16441651

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Food Chemistry
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Anthocyanin and colour changes during processing of pomegranate (Punica granatum L., cv. Hicaznar) juice from sacs and whole fruit
zge Turfan a, Meltem Trkylmaz b, Oktay Yemis c, Mehmet zkan b,
a

Municipality of Antalya, Water Control Laboratory, Antalya, Turkey Department of Food Engineering, Faculty of Engineering, Ankara University, Diskapi, Ankara 06110, Turkey c Department of Food Engineering, Faculty of Engineering, Pamukkale University, Denizli, Turkey
b

a r t i c l e

i n f o

a b s t r a c t
The effects of clarication and pasteurisation on anthocyanins (ACNs) and the colour of pomegranate juice (PJ) produced from sacs and whole fruits were investigated. Clarication caused a loss of 4% of ACNs in juice from sacs (JFS) and a loss of 19% in juice from whole fruit (JFWF). After pasteurisation, there was an 814% and 139% loss of ACNs from unclaried and claried JFS and JFWF samples, respectively. Polymeric colour was very high even in unclaried samples (2529%). Compared to JFS, higher polymeric colour was formed in JFWF. HPLC analyses of PJ revealed that cyanidin-3,5-diglucoside was the major ACN, followed by cyanidin-3-glucoside and delphinidin-3-glucoside. Cyanidin-3,5-diglucoside showed higher stability to clarication and pasteurisation than cyanidin-3-glucoside in both PJ samples. Cold clarication with only gelatin is recommended for PJ. To prevent excessive ACN loss and the formation of brown colouring, PJ should be subjected to minimal heating. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 15 February 2011 Received in revised form 6 April 2011 Accepted 15 June 2011 Available online 23 June 2011 Keywords: Pomegranate juice Punica granatum L. cv. Hicaznar Sacs Anthocyanin Polymeric colour Clarication Pasteurisation

1. Introduction Turkey is the fourth major producer of pomegranates in the world, following India, China and Syria. Production in Turkey accounts for approximately 5% of the total world production or approximately 2,000,000 metric tons per year (www.tagem.gov.tr). In recent years, there has been a steady increase in pomegranate production in Turkey due to a high demand for Turkish pomegranate juice (PJ) concentrates. In fact, production in metric tons was 80,000 in 2005, 91,000 in 2006 and 102,000 in 2007 (www.tarimmerkezi.com.tr). With new plantations, pomegranate production is expected to increase dramatically in the coming years. Turkey has a very rich genetic source of pomegranate cultivars. Over 30 registered varieties have been grown in addition to many local varieties. The pomegranate cultivars in highest demand by the Turkish fruit juice industry are Hicaznar and Silifke Asisi, which have a deep violet-red colour and a sweet and sour taste. These properties make the cultivars perfect for juice production. The high demand for PJ is the result of studies that have shown the health benets of biological compounds, specically, phytochemicals, in pomegranates. The primary phytochemicals in pomegranates are the polyphenols, including anthocyanin (ACN)
Corresponding author. Tel.: +90 312 596 1146; fax: +90 312 317 8711.
E-mail address: mozkan@ankara.edu.tr (M. zkan). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.06.024

pigments, avonol glycosides, procyanidins, phenolic acids and ellagic acid derivatives (Negi & Jayaprakasha, 2003). Polyphenols are found in all fruits and vegetables and play a major role in their colour, avour, texture as well as antioxidant (Hernandez, Melgarejo, Tomas-Barberan, & Artes, 1999) and antibacterial activities (Negi & Jayaprakasha, 2003). Due to their antioxidant properties, phenolic compounds including ACNs are thought to have preventive roles in a number of chronic diseases such as cardiovascular disease and cancers (Heinonen, Meyer, & Frankel, 1998). The primary antioxidative phenolics in PJ are punicalagins, followed by hydrolysable tannins, ACNs and ellagic acids (Gil, Tomas-Barberan, HessPierce, Holcroft, & Kader, 2000). An attractive red colour is the most important quality criteria for fruit juices containing anthocyanin, including PJ. ACNs are also responsible for the orange, red and blue colours of many fruits and vegetables. Unfortunately, ACNs are unstable and susceptible to degradation, leading to a brownish colour during processing and storage. The primary colour deterioration in fruit juices containing ACNs occurs as a result of the degradation of monomeric ACNs, polymerisation of ACNs and the subsequent formation of brown colour (Somers & Evans, 1986). These colour changes strongly affect consumer behaviour and result in a loss of marketability of processed pomegranate products. Various factors affect the stability of ACNs, including the temperature of processing and storage, the chemical nature of ACNs

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(acylation or glucosylation), pH, ascorbic acid, hydrogen peroxide, sugars, light and metals. Clarication and pasteurisation during the production of fruit juices also affect the stability of ACNs. Clarication is the most important step in the production of clear fruit juice. Clarication agents are used to achieve clarity and improve colour, avour and physical stability. After pressing, clarication agents such as bentonite, gelatin and kieselsol are added to cloudy juice to remove colloidally suspended particles that may be present in the form of proteins, polyphenols, pectins and gums. Removal of these particles is critical for improving the clarity and colour of fruit juices. Bentonite is added to fruit juices to remove proteins, while gelatin is used for the removal of high molecular weight (>500 Dalton) polyphenols that otherwise cause turbidity during storage. Kieselsol is added to fruit juices to remove excessive gelatin, which also causes turbidity during storage. The other important step in processing pomegranates is the heating of fruit juices at various stages. Heating right after pressing inhibits native polyphenol oxidase (PPO) enzymes that cause brown colour formation by oxidising polyphenols (Skrede, Wrolstad, & Durst, 2000). PPO also degrades monomeric ACNs indirectly by forming O-quinones from polyphenols during enzymatic browning that react with and degrade monomeric ACNs. Pasteurisation is the process of heating before packaging to inactivate pathogenic or spoilage microorganisms. Heat applied during juice processing causes the degradation of monomeric ACNs, polymerisation of ACNs and the formation of brown colour in red fruit products (Somers & Evans, 1986). To date, there have been no detailed studies that report changes in ACNs and the colour of PJ during processing. The primary purpose of this study was to determine the changes in ACN content and brown colour formation during clarication and pasteurisation of PJ from sacs and whole fruits. The secondary purpose was to identify the ACN prole of the major Turkish pomegranate cultivar Hicaznar, which is highly preferred by the Turkish fruit juice industry. 2. Materials and methods 2.1. Chemicals and reagents Cyanidin 3-O-glucoside and cyanidin 3,5-O-diglucoside standards were purchased from Sigma (St. Louis, MO); pelargonidin 3-O-glucoside and pelargonidin 3,5-O-diglucoside were purchased from Fluka (Seelze, Germany); and delphinidin-3-O-glucoside was purchased from Polyphenols Laboratories AS (Sandnes, Norway). All reagents used for liquid chromatography were HPLC grade and purchased from Merck (Darmstad, Germany). All other reagents were analytical grade and obtained from Merck. 2.2. Samples Pomegranates (Punica granatum L., cv. Hicaznar) were obtained from the Alata Horticultural Research Institute (Erdemli, Mersin) and processed immediately. A ow diagram of PJ processing is shown in Fig. 1. Before juice extraction, pomegranates were washed in cold tap water and drained. Damaged pomegranates were discarded. The top and bottom of the pomegranate husks were removed with a sharp stainless steel knife to prevent microbial contamination. After washing, 10 kg of pomegranates were selected and the outer skins were hand-peeled. The juicy sacs from fruit pericarp were separated by hand, placed in a muslin cloth and pressed with a laboratory hand press. The resulting juice was labelled juice from sacs (JFS). The rest of the pomegranates (50 kg) were cut into four pieces and processed into juice at the fruit juice pilot plant at

Ankara University. Pomegranate quarters were pressed on a rackand-cloth press (Bucher-Guyer, Niederweningen, Switzerland). The resulting juice was ltered through muslin cloth to remove particles and was labelled juice from whole fruits (JFWF). The resulting cloudy juices from sacs and whole fruits were divided into two groups. One group was claried using only gelatin at 5 C (cold clarication). The other group was not claried. Gelatin solutions at 0.5% and 1% (w/v) were used for the clarication of JFS and JFWF, respectively. lu, 1977), PJ Since pomegranates do not contain pectin (Cemerog samples were not depectinised. However, PJ, especially JFWF, contains a signicant amount of polyphenols and needs to be claried to remove some of the high molecular weight polyphenols that otherwise cause turbidity during storage. In fact, JFS and JFWF were shown to contain 2073 and 2773 mg of total phenolics expressed as gallic acid equivalents/l, respectively (Gzel, 2010). PJ obtained from whole fruits also contains high levels of tannins, which cause astringency (Gzel, 2010). Bentonite was not used during the clarication of PJ samples because pomegranates contain an insignicant amount of protein. Gelatin (A type, 80100 Bloom strength) was used at a concentration of 0.4 g/l for juice from sacs and 2 g/l for juice from whole fruits and was the only agent used for the clarication of PJ. There was no need to use kieselsol because cold clarication was applied and the PJ contained a high amount of polyphenols. After clarication, the turbidity of the juice from sacs and whole pomegranates was 2.23 and 1.75 NTU (Nephelometric turbidity unit), respectively. Claried and unclaried juices from sacs and whole fruits were again divided into two groups. One group was pasteurised, and one was not. The juice samples were transferred into 200 ml hermetically capped glass bottles, and pasteurisation was carried out in a water bath at 95 C for 10 min. After pasteurisation, the juice samples were immediately cooled to room temperature. 2.3. Compositional analysis The total soluble content (Brix) of PJ samples was determined by an automatic digital refractometer (Atago Rx-7000a, Tokyo, Japan). The brix was determined after the cloudy juice samples were ltered through a 0.45 lm PVDF (polyvinylidene uoride) lter (Millipore, Bedford, MA) to reduce turbidity. Brix measurements were carried out at 20 C. pH was measured potentiometrically with a pH metre (WTW Inolab Level 1, Weilheim, Germany). Titratable acidity was determined according to the method outlined by IFU (1968) and expressed as g anhydrous citric acid/100 ml or g sample. 2.4. Monomeric ACN analysis The total monomeric ACN content was determined using the pH differential method described by Giusti and Worlstad (2005). The pH of juice samples was brought to 1.0 with potassium chloride and 4.5 with sodium acetate buffers. The dilutions were then allowed to equilibrate for 15 min at room temperature (22 C). Prior to absorbance measurements, the solutions were ltered through a 0.45 lm PVDF lter to remove the haze. The absorbance of equilibrated solutions at 512 nm (kmax) for ACN content and 700 nm for haze correction was measured on a UVVIS double beam spectrophotometer (ThermoSpectronic Heliosa, Cambridge, England) with 1 cm path length disposable cuvettes (Brand Gmbh, Wertheim, Germany). All absorbance measurements were carried out at room temperature against distilled water as a blank. Pigment content was calculated as cyanidin-3-glucoside (Cy-3-glu) equivalents with a molecular weight of 449.2 and an extinction coefcient of 26 900 L cm1 mol1. The difference in absorbance

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. Turfan et al. / Food Chemistry 129 (2011) 16441651

Pomegranates

Washing

Seperation of husk and fruit pericarp by hand

Cutting into four pieces

Juicy sacs

Pressing

Pressing

Filtration

Filtration

Raw juice from sacs, JFS (control)a

Raw juice from whole fruit, JFWF (control)a

Pasteurisation (95 C, 10 min) Unclarified pasteurised juice (JFS)a

Clarification 4 C

Clarification 4 C

Pasteurisation (95 C, 10 min) Unclarified pasteurised juice (JFWF)a

Filtration Clarified juice (JFS)a

Filtration Clarified juice (JFWF)a

Pasteurisation (95 C, 10 min) Clarified pasteurised juice (JFS)a

Pasteurisation (95 C, 10 min) Clarified pasteurised juice (JFWF)a

Fig. 1. Pomegranate juice processing (aSamples taken for analysis).

values at pH 1.0 and 4.5 was directly proportional to ACN concentration. All ACN measurements were replicated two times. 2.5. Polymeric colour content Polymeric colour contents of PJ samples were determined using the bisulphite bleaching method described by Giusti and Worlstad (2005). The absorbance of bisulphite treated and non-treated solutions was measured at 420 nm for brown pigments, 512 nm (kmax) for monomeric ACNs and 700 nm for haze correction. Disposable cuvettes of 1 cm path length were used. All absorbance measurements were carried out at room temperature against distilled water as a blank. Polymeric colour measurements were replicated two times. 2.6. Colourimetric measurements The colour of PJ samples was measured with a light reectance spectrophotometer (Minolta CM-3600d, Osaka, Japan). Measurements were recorded in L (lightness), +a (redness) and +b (yellowness) CIE (Commission Internationale de IEclairage) colour coordinates. Chroma (C) and colour tone (hue angle, h) were cal-

culated from a and b colour coordinates using the following equations:

C a2 b 1=2 h arctan b =a :
During colour measurements, a specular component was included with C illuminant and a 10 observer angle. The juice samples were transferred to 1 cm path length quartz cuvettes with a 10 ml capacity. Measurements were replicated three times. 2.7. HPLC separation of ACNs 2.7.1. Extraction ACNs were extracted following the method described by Lee and Wrolstad (2004). Five milliliters of PJ was mixed with 10 ml acetone (100%) and homogenised at 9500 rpm for 2 min (Heidolph SilentCrusher M, Schwabach, Germany). The acetone extract was ltered on a Buchner funnel using Whatman No. 1 lter paper. ACNs on the homogenizer blade were washed rst with 10 ml 100% acetone and then with 10 ml 70% acetone. The resulting acetone extract was ltered through a Buchner funnel and combined


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with the previous acetone extract. The lter cake was also extracted with 70% acetone until the solution became colourless. The ltrates were combined and partitioned with chloroform (1:2 acetone:chloroform, v/v) in a separatory funnel and left overnight at 4 C for complete separation of organic and aqueous phases. The red coloured aqueous phase containing ACNs was collected and transferred to a rotary evaporator (Heidolph Laborota 4003, Schwabach, Germany) to remove residual acetone at 40 C. The aqueous extract was dissolved in puried water (containing 0.01% HCl, v/v), and the nal volume was brought to 10 ml with puried water. The resulting extract was ltered through a 0.45 lm PVDF lter directly into an amber coloured bottle. Two extracts were prepared from each sample. 2.7.2. ACN purication The ACNs were puried on a C-18 cartridge (Waters Co., Milford, MA) using a vacuum manifold system (Waters Co., Milford, MA). Prior to sample loading, the cartridge was activated with 5 ml ethyl acetate followed by 5 ml methanol (containing 0.01% HCl, v/v) and 2 ml aqueous 0.01% HCl (v/v). After loading 1 ml of ACN extract, the cartridge was washed with 2 ml aqueous 0.01% HCl to remove compounds not adsorbed by the column, such as sugars and organic acids. The cartridge was then dried under a stream of nitrogen for 10 min. Non-ACN phenolics were removed from the cartridge by rinsing with 5 ml ethyl acetate. Elution of ACNs was carried out by rinsing the cartridge with 2 ml methanol (containing 0.01% HCl, v/v). The methanolic extract containing ACNs was evaporated to dryness in a water bath (Memmert WB 14, Schwabach, Germany) at 35 0.1 C under a stream of nitrogen, and ACNs were dissolved in aqueous 0.01% HCl. The resulting extract was ltered through a 0.22 lm PVDF lter (Sartorious AG, Goettingen, Germany) directly into an amber coloured auto sampler vial, and the ltered extract was immediately injected into high performance liquid chromatography (HPLC). 2.7.3. Instrumentation and chromatography Separation and quantication of ACNs were performed using HPLC (Agilent 1200 series, Waldbronn, Germany) with a binary pump, a photo diode array (PDA) detector, a thermostatted autosampler, a degasser and a thermostatted column compartment. Chromatographic data were recorded and processed on Agilent 1200 series ChemStation rev.B.02.01 software. ACNs were separated on a C18 (5 lm) column (250 4.6 mm) (Phenomenex, Los Angeles, CA) with a C18 (5 lm) guard column (4 3 mm, 5 lm) (Phenomenex, Los Angeles, CA). The eluents used were (A) 100% acetonitrile and (B) O-phosphoric acid, acetic acid, acetonitrile and water (1:10:5:84; v/v/v/v) with a ow rate of 1 ml min1. Separation was performed with gradient elution using a modication of the elution prole described by Skrede et al. (2000). The linear gradient programme for the separation of pomegranate ACNs was as follows: from 0% to 12% A in 10 min, from 12% to 22% in 10 min, and holding at 22% A for 5 min. The sample injection volume was 50 ll, the column temperature was 25 C, and the detector was set at 520 nm. Identication of ACNs was carried out by comparing retention times and absorption spectra of unknown peaks with external reference standards. Quantication of ACNs was carried out using calibration curves of the following external reference standards: cyanidin 3-O-glucoside (R2 = 0.9995), cyanidin 3,5-O-diglucoside (R2 = 0.9834), delphinidin 3-O-glucoside (R2 = 0.9749), pelargonidin 3-O-glucoside (R2 = 0.9944) and pelargonidin 3,5-Odiglucoside (R2 = 0.8980). The calibration curves for each ACN standard contained seven data points. Quantication of total ACNs by HPLC was calculated based on cyanidin 3-O-glucoside. Five out of six ACNs in PJ samples were identied by comparing retention times and absorption spectras of unknown peaks with external reference standards. Since the commercial standard for

delphinidin-3,5-diglucoside was not available at the time of analysis, mass spectrometric detection was carried out for delphinidin3,5-diglucoside on the same HPLC system used for the identication of major ACNs in PJ. The mass/charge (m/z) ratio of 627 for delphinidin-3,5-diglucoside was used (Brito et al., 2007). The DAD detector was interfaced with a mass spectrometer (Agilent Series 1200 HPLC system) with an ESI source operating in positive ionisation mode. Nitrogen was used at a ow rate of 12 l min1 and a pressure of 35 psig both as a drying and a nebulising gas. The nebuliser temperature was set at 250 C, and a potential of 2000 V was used on the capillary. The mobile phase consisted of 100% acetonitrile (eluent A) and 1% formic acid (eluent B). The other chromatographic conditions were the same as described above for HPLC separation of major ACNs in PJ. 2.8. Determination of limits of detection and quantication The limit of detection (LOD) and limit of quantication (LOQ) for ACNs were determined based on the signal to noise (S/N) ratio. According to ICH guidelines for validation of analytical procedures, an acceptable S/N is 3:1 (or 2:1) for estimating the LOD and 10:1 for estimating the LOQ (Li, Chung-Wang, & Shih, 2002). 2.9. Statistical analyses Results of the ACN contents were analysed using the Minitab statistical software, version 14 (Minitab Inc., State College, PA.). Juice processing (JFS and JFWF), clarication and pasteurisation were considered as the main effects. Statistical differences amongst means were determined by Duncans multiple range test at a signicance level of 1%. 3. Results and discussion 3.1. Changes in compositional measurements during juice processing The pH, titratable acidity and soluble solid content (Brix) of the PJ samples were determined during processing (data not shown). There were small differences in the pH, titratable acidity and soluble solid contents of the PJ samples during processing, ranging from 3.35 to 3.40, 1.10 to 1.11 g/100 ml (as anhydrous citric acid) and 16.28 to 16.46, respectively. These results showed that clarication and pasteurisation did not affect the organic acid and sugar contents of the PJ samples. 3.2. Characterisation of PJ ACNs The total monomeric ACN content of juice from sacs (JFS) and juice from whole fruits (JFWF) were 382 mg and 291 mg ACN/L as determined by HPLC, respectively (Table 1). The ACN content and composition of pomegranates strongly depend on the cultivar and the stage of maturity. zkan, Trkylmaz, and Gzel (2009) determined the total ACN content of nine registered Turkish pomegranate varieties by HPLC and found large differences in ACN contents, ranging from 28 to 447 mg ACN/L juice. An HPLC chromatogram for the ACNs of PJ is shown in Fig. 2. Six different ACNs in PJ were detected and identied in HPLC elution order as delphinidin-3,5-diglucoside, cyanidin-3,5-diglucoside, delphinidin-3-glucoside, pelargonidin-3,5-diglucoside, cyanidin3-glucoside and pelargonidin-3-glucoside. The area of the peak for delphinidin-3,5-diglucoside was quantied based on cyanidin-3-glucoside, which is the most commonly found ACN in fruits and vegetables (Wrolstad et al., 2005). Quantications of other ACNs were carried out using calibration curves of the external reference standards. The same six ACNs have also been reported by

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Table 1 Total ACN content, polymeric colour and colour parameters of pomegranate juice samples from various processing stages. Pomegranate juice Total ACN content (mg/kg)b Spectrophotometric From sacs Control Claried Unclaried (P)a Claried (P) From whole fruit Control Claried Unclaried (P)a Claried (P)
a b

Polymeric colour (%) HPLC 382 355 363 331 291 227 278 254 25 30 27 33 29 31 29 33

Colour parameters L 33.04 38.92 21.63 38.53 26.06 36.54 24.21 34.98 a 60.32 63.52 47.52 62.35 55.08 63.06 52.57 61.70 b 54.91 59.71 36.56 56.12 44.46 59.02 41.15 55.68 C 81.57 87.18 59.96 83.89 70.79 86.37 66.76 83.11 h 42.31 43.23 37.56 41.99 38.91 43.10 38.05 42.07

364 349 335 299 365 296 316 269

P: Pasteurised juice. Expressed as cyanidin-3-glucoside.

mAU 700 600 500 400 300 200 100 0 3 4

1 3
5 6

4
7 8

6
9 10 11 min

Fig. 2. HPLC chromatograms of pomegranate juice ACNs. 1: delphinidin-3,5-diglucoside, 2: cyanidin-3,5-diglucoside, 3: delphinidin-3-glucoside, 4: pelargonidin-3,5diglucoside, 5: cyanidin-3-glucoside and 6: pelargonidin-3-glucoside.

Due, Wang, and Francis (1975), Hernandez et al. (1999), Gil et al. (2000) and Marti, Perz-Vicente, and Garcia-Viguera (2001). Amongst the six ACNs identied, cyanidin-3,5-diglucoside was the major ACN in PJ, with 56.4% of the total peak area, followed by cyanidin-3-glucoside (24.9%), delphinidin-3,5-diglucoside (9.1%), delphinidin-3-glucoside (4.3%), pelargonidin-3,5-diglucoside (3.4%) and pelargonidin-3-glucoside (1.8%). Harborne (1967) was the rst to report that the major ACN in PJ was delphinidin-3,5-diglucoside. However, Due et al. (1975) found that cyanidin-3-glucoside was the primary ACN in pomegranate sacs. Similarly, Marti et al. (2001) reported that juice obtained from the Mollar pomegranate (Spanish variety) contained cyanidin-3glucoside (40%) and cyanidin-3,5-diglucoside (38%) as the major ACNs. The primary ACN of the Wonderful pomegranate (American variety) is cyanidin-3-glucoside (42%), followed by delphinidin-3-glucoside (25%), cyanidin-3,5-diglucoside (17%) and delphinidin-3,5-diglucoside (14%) (Gil et al., 2000). Contrary to these studies, cyanidin-3,5-diglucoside was the major ACN found in PJ from the Turkish Hicaz variety. Maturity has a predominant effect on the ACN composition of pomegranates. In fact, Hernandez et al. (1999) found that delphinidin-3,5-diglucoside is the major ACN during the early stages of maturation. After maturation, cyanidin-3-glucoside and cyanidin3,5-diglucoside are the major ACN compounds. These ndings clearly show that the ACN composition of pomegranates depends on both the variety and maturity of the fruits. 3.3. Total ACN content obtained by spectrophotometer and HPLC The total ACN contents of the PJ samples were determined by both spectrophotometer (pH-differential method) and HPLC (Table 1). While the total ACN content of JFS obtained from the pH-dif-

ferential was 1.0 to 1.1 times lower than that obtained from HPLC, the total ACN content of JFWF obtained from the pH-differential was 1.1 to 1.3 times higher. In the literature, the total ACN content of juice samples determined by pH-differential was generally higher than those determined by HPLC. Lee, Durst, and Wrolstad (2002) found that the total ACN content of blueberry juices determined by the pH-differential method was 1.5 to 1.9 times higher than those obtained from HPLC. The difference may be partially due to spectral characteristics of ACNs inuenced by the different solvent systems utilised for HPLC and spectrophotometer (Lee, Rennaker, & Wrolstad, 2008). Additionally, the total ACNs were analysed by HPLC and spectrophotometer at different wavelengths of 520 nm and 512 nm, respectively. The sum of the peak area at a certain wavelength is used when quantifying ACNs by HPLC, which is generally close to the maximum wavelength of an individual ACN (Hundskopf, 1998). However, the different aglycons have different visible maximum wavelength values, ranging from 520 nm for pelargonidin to 546 nm for delphinidin, and their monoglucosides exhibit visible maximum wavelengths that are about 1015 nm lower than those from their respective aglycons (Hundskopf, 1998). Similarly, the maximum wavelength for an individual ACN is dependent upon the ACN chromophore. For example, the maximum wavelengths for cyanidin-3-glucoside and malvidin-3-glucoside in acidied (0.01% HCl) methanol are 523 nm and 534 nm, respectively (Durst & Wrolstad, 2005). The difference between the two methods may also be due to the presence of polymeric pigments in juice samples. Polymeric pigments may be retained in the HPLC column and therefore may not contribute to ACN measurements. In contrast, polymeric pigments are not separated prior to spectrophotometric measurements and may thus cause higher absorbance values (Lee et al.,

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2002). Juice samples obtained from whole pomegranates (JFWF) contained more polymeric pigments than juice obtained from only sacs (JFS) (Table 1). A high polymeric colour ratio is the result of the formation of a complex between ACNs and tannins and from ACN degradation (Wrolstad et al., 2005). In fact, Gzel (2010) reported that juice samples obtained by pressing whole pomegranates and sacs contained 127 and 60 mg/l of condensed tannins, respectively. JFWF samples in this study contained more condensed tannins, making the JFWF samples more likely to form polymeric colours with ACNs. This may in turn result in a higher total monomeric ACN content for JFWF samples. This study also revealed high correlations (r = 0.93 for JFS, r = 0.73 for JFWF) between the results for the ACN contents obtained from pH-differential and HPLC methods. A similar result was reported by Lee et al. (2008). 3.4. Changes in ACN composition during juice processing 3.4.1. General The total monomeric ACN content of unclaried juice, determined by the pH-differential method and expressed as cyanidin3-glucoside, was 364 mg ACN/L for JFS and 365 mg ACN/L for JFWF (Table 1). As expected, there was no difference between the total ACN contents of JFS and JFWF samples. As indicated previously, the total ACN content of pomegranates strongly depends on the cultivars. zkan et al. (2009) reported that the total monomeric ACN content of PJ obtained from nine registered cultivars ranged from 46 to 450 mg ACN/L. In their study, total monomeric ACN contents were also determined by the pH-differential method and expressed as cyanidin-3-glucoside. 3.4.2. Claried juice The total monomeric ACN content of unclaried juice samples from JFS and JFWF were almost the same (Table 1). However, after clarication, there was a 4% loss of ACNs for JFS and a 19% loss for JFWF samples. Previous reports have also shown 1418% decreases in ACN contents of claried red raspberry (Rommel, Heatherbell, & Wrolstad, 1990) and blackberry juices (Rommel, Wrolstad, & Heatherbell, 1992) compared to unclaried juice samples. The difference in total ACN contents of claried JFS and JFWF samples may be attributed to the use of different gelatin dosages during clarication. The results of ACN analyses clearly show that clarication signicantly affected the total ACN content of juice samples (P < 0.01), which agrees with previous reports. Vardin lu (2003) reported 5.66.2% losses in the total ACN and Fenerciog content of PJ when claried with different gelatin dosages. While gelatin is positively charged at the pH of PJ (ca. pH 3.5), phenolics are negatively charged. Therefore, during clarication, the interaction between positively charged gelatin and negatively charged phenolics leads to occulation. For gelatin to form ocks with phenolics, phenolics should have high molecular weight (>500 Daltons). As indicated previously, juice obtained from whole fruits contains higher molecular weight phenolics, such as tannins (both hydrolyzable and condensed), than the juice obtained from sacs (Gzel, 2010). Once ocks are formed between gelatin and phenolics, ACNs are also removed from PJ with the ock. Since JFWF contains higher molecular weight phenolics, a greater amount of gelatin was used for clarication. Therefore, more ACNs were lost during the clarication of JFWF samples compared to JFS samples. 3.4.3. Pasteurised juice There was an 8% loss in the total ACN content of JFS and a 13% loss in JFWF after pasteurisation. Lee et al. (2002) reported a substantial loss of ACNs (76%) in pasteurised blueberry juices. The increase in colour density is the result of the polymeric colour that forms from the degradation of ACNs. A plausible explanation for

the degradation of ACNs during the pasteurisation of PJ samples is a low-temperature, long-time treatment (LTLT). During the LTLT treatment, enzymes that degrade ACN, including polyphenol oxidase, may be activated. Rommel et al. (1992) found that high-temperature, short-time (HTST) heat-treated blackberry juices retained most ACNs. Similarly, Mikkelsen and Poll (2002) reported that much lower ACN losses occurred after HTST treatment of blackcurrant juice compared with LTLT treatment. 3.5. Changes in percentage of polymeric colour during juice processing The percentage of polymeric colour is a measure of the resistance of ACNs to bisulphite bleaching and shows the degree of ACN polymerisation during processing and storage of juices and concentrates. Changes in the polymeric colour of PJ samples were evaluated during various stages of processing (Table 1). Freshly squeezed fruit or vegetable juices usually contain less than 10% polymeric colour. However, even unclaried (control) PJ samples contained a much higher percentage of polymeric colour (29% in JFWF and 25% in JFS samples). Polymeric colour is dependent on processing and storage conditions and can be more than 30% higher when fruits or vegetables are subject to unfavourable storage conditions. In this study, the pomegranates were stored for a very short time after harvest (at 4 C for 2 days) prior to juice processing. In addition, all the juice samples, including unclaried juice samples, were immediately analysed for polymeric colour right after the depectinisation and clarication steps. Therefore, the high polymeric colour even in unclaried PJ samples may be due to the formation of polymeric ACN-tannin pigments before juice processing rather than inconvenient storage conditions. Potential mechanism for polymerisation involves condensation of ACNs with avan-3-ols such as catechins or polyavan-3-ols such as proanthocyanidins. As rich sources of ACNs and tannins (Gzel, 2010), high inherent polymeric colour values for pomegranates can be attributed to these substrates for condensation reactions. Unclaried JFWF (29%) had signicantly (P < 0.01) higher polymeric colour than unclaried JFS (25%) (Table 1). This may be attributed to the difference in phenolic compositions of JFS and JFWF. In JFWF, ACNs are mainly bound tannins extracted from the rinds during pressing. JFS samples contain tannins at much lower concentrations. Since the juicy sacs from the fruit pericarp were separated by hand and pressed without the rinds, JFS samples contained considerably less tannins compared to JFWF samples pressed with rinds. Gzel (2010) found that juice obtained from whole fruits had 970 mg of hydrolyzable tannins expressed as gallic acid equivalents/l, whereas juice obtained from only arils without the rinds had only 160 mg of hydrolyzable tannins expressed as gallic acid equivalents/l. These ndings clearly show that most of the tannins are located in the rinds of pomegranates. Polymeric colour increased in both JFS and JFWF samples after clarication (P < 0.01), which was also reported in blackberry juice by Rommel et al. (1990). Similarly, polymeric colour also increased in both juice samples after pasteurisation (P < 0.01). The increase in polymeric colour after pasteurisation may be attributed to the polymerisation and degradation of ACNs during heating (Gil et al., 2000). 3.6. ACN prole changes from processing 3.6.1. Claried juice Table 2 shows the change in individual anthocyanidin-glucosides of each sample from various processing stages. In general, clarication with gelatin caused losses in individual ACNs. Compared to the individual ACN contents of JFWF and JFS samples, there were more losses in JFWF samples than JFS samples. For example, there was a 4% loss in cyanidin-3,5-diglucoside in JFS

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Table 2 Total ACN content of pomegranate juice samples from various processing stages. Pomegranate Juice ACN content (mg/kg) Dp-3,5-diglua From sacs Control Claried Unclaried (P)b Claried (P) From whole fruit Control Claried Unclaried (P)b Claried (P)
a b c

Cy-3,5-diglu 58.7 56.6 49.5 47.7 44.8 36.4 34.7 35.2 (56.4%) (58.7%) (49.6%) (52.4%) (57.1%) (58.8%) (45.4%) (49.9%)

Dp-3-glu 4.5 2.8 7.2 5.5 2.6 1.4 7.0 4.8 (4.3%) (2.9%) (7.2%) (6.0%) (3.3%) (2.3%) (9.2%) (6.8%)

Pg-3,5-diglu 3.5 3.3 2.6 2.6 2.6 2.2 1.6 1.5 (3.4%) (3.4%) (2.6%) (2.9%) (3.3%) (3.6%) (2.1%) (2.1%)

Cy-3-glu 25.9 22.4 22.8 19.1 21.6 17.1 20.5 17.7 (24.9%) (23.2%) (22.8%) (21.0%) (27.6%) (27.6%) (26.8%) (25.1%)

Pg-3-glu 1.9 1.8 1.7 1.4 1.4 1.2 1.1 1.2 (1.8%) (1.9%) (1.7%) (1.5%) (1.8%) (1.9%) (1.4%) (1.7%)

9.5 (9.1%)c 9.5 (9.8%) 16.0 (16.0%) 14.7 (16.2%) 5.4 (6.9%) 3.6 (5.8%) 11.5 (15.0%) 10.1 (14.3%)

Delphinidin-3,5-diglucoside was expressed as cyanidin-3-glucoside. P: Pasteurised juice. Values in parenthesis are proportion of individual ACNs in total ACNs.

and a 19% loss in JFWF. Despite the decrease in individual ACNs, the ACN proles of both juice samples changed only slightly during the clarication step. The stability as a result of the clarication process was different for individual ACNs (Table 2). There was a 4% and 19% loss of cyanidin-3,5-diglucoside in JFS and JFWF samples, respectively, while the losses of cyanidin-3-glucoside were 14% and 19%, respectively. Delphinidin-3-glucoside was the least stable, with losses of 38% and 46% occurring right after clarication of JFS and JFWF samples, respectively. A similar nding was reported by Artes, Villaescusa, and Tudela (2000), who attributed the low stability of delphinidin-3-glucoside to the high reactivity of this pigment to enzymatic oxidation. 3.6.2. Pasteurised juice After pasteurisation, there was a 12% loss of cyanidin-3,5-diglucoside and a 16% loss of cyanidin-3-glucoside in the JFS samples. Except for the unclaried sample, there were no changes in cyanidin-3,5-diglucoside or cyanidin-3-glucoside after pasteurisation in JFWF samples, although the proportions and content of delphinidin-3,5-diglucoside increased after pasteurisation (Table 2). The delphinidin-3,5-diglucoside content in unclaried (control) JFS and JFWF samples was 9.1% and 6.9% of total ACNs, respectively. After pasteurisation, there was a 16% increase in delphinidin-3,5diglucoside in JFS and a 15% increase in JFWF. These increases were possibly due to the release of the pigment from the fruit cell wall by heat applied during LTLT treatment. Pelargonidin-3,5-diglucoside was the most unstable ACN during pasteurisation. 3.7. Changes in colour parameters (L, a, b, C and h)

A high correlation (r = 0.97) was found between the a and L values of PJ (gure not shown). The claried juice samples had higher h values than the unclaried juice samples. Higher h values indicate yellowing of the PJ samples. Generally, the increase in h values is the indication of ACN degradation as a result of heat treatment during juice processing. However, because there is no heat applied during clarication, the increase in h values of claried juice samples is believed to be due to the removal of ACNs by gelatin-phenolic ock rather than ACN degradation. 4. Conclusion This study revealed that cold clarication (using only gelatin) was sufcient for the production of clear PJ (1.75 NTU). There was no need to depectinize of PJ and, to use bentonite and kiselsol for clarication. However, due to the adverse effects of gelatin on ACNs, gelatin should be used with great care during clarication. Although results from total monomeric ACN analyses by HPLC and pH-differential methods revealed some differences, total ACN contents obtained from two methods were highly correlated (r = 0.93 for JFS, r = 0.73 for JFWF) with each other. This study was the rst study showing the cyanidin-3,5-diglucoside was the major ACN in pomegranates. High polymeric colour values for pomegranates juice is understandable because PJ contains high amount of tannins which react with ACNs to form brown coloured ACN-tannin complexes. Clarication and pasteurisation had a signicantly decreasing inuence on ACNs. Therefore, both processes should be carefully optimised to decrease ACN losses as well as the formation of polymeric colour. Acknowledgments

Lightness (L), redness (a), yellowness (b), chroma (C) and hue angle (h) were determined for JFS and JFWF samples during various processing stages (Table 1). All colour parameters increased in both juice samples after clarication. Rommel et al. (1990) also showed that L and b values increased in blackberry juice after clarication. This is likely because the particles susceptible to browning were removed by the gelatin used for clarication of PJ. This resulted in the increase in L values from 33.04 to 38.92 in JFS and from 26.06 to 36.54 in JFWF. This phenomenon was conrmed by Sapers (1992), who reported that the browning capacity of unclaried apple, grape and pear juices was associated with particulate fractions that could be removed by ltration with bentonite and diatomaceous earth. In contrast, pasteurisation resulted in substantial decreases in all colour parameters of PJ samples. This is in accordance with a previous study that attributed to these decreases to ACN degradation and sediment formation after pasteurisation (Rommel et al., 1990).

This study is part of Miss. Turfans masters of science thesis. The authors thank the Alata Horticultural Research Institute (Erdemli, Mersin) for providing the pomegranates. References
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