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Half-life periods for ACN degradation in BCJCs were 603, 137 and ... Keywords Anthocyanin, black carrots, degradation, kinetics, polymeric colour, storage.
International Journal of Food Science and Technology 2012, 47, 2273–2281

Original article Kinetics of anthocyanin degradation and polymeric colour formation in black carrot juice concentrates during storage Meltem Tu¨rkyılmaz1* & Mehmet O¨zkan2 1 Ministry of Food, Agriculture and Animal Husbandry, Golbasi District Directorate of Agriculture, Golbasi, 06830 Ankara, Turkey 2 Department of Food Engineering, Faculty of Engineering, Ankara University, Diskapi, Ankara 06110, Turkey (Received 22 November 2011; Accepted in revised form 24 April 2012)

The changes in anthocyanins (ACNs) and polymeric colour of black carrot juice concentrate (BCJC) samples were monitored during storage at )23, 5 and 20 C for 319 days and at 30 C for 53 days. While ACN degradation was fitted to a first-order reaction model, polymeric colour formation was fitted to a zero-order reaction model during the storage. Half-life periods for ACN degradation in BCJCs were 603, 137 and 29 days at 5, 20 and 30 C, respectively. The reaction rate constants for polymeric colour formation were 0.0207, 0.1435 and 0.5581% ⁄ days at 5, 20 and 30 C, respectively. HPLC-MS analyses of BCJC showed that cyanidin-3-galactoside-xyloside-glucoside-ferulic acid (56%) was the major ACN, followed by cyanidin3-galactoside-xyloside (19%) and cyanidin-3-galactoside-xyloside-glucoside-sinapic acid (10%). Cyanidin3-galactoside-xyloside-glucoside-ferulic acid was the most stable ACN in BCJC at storage temperatures. BCJCs should be kept at sub-freezing temperatures to minimise ACN degradation.

Summary

Keywords

Anthocyanin, black carrots, degradation, kinetics, polymeric colour, storage.

Introduction

Colour is an important indicator in the quality assessment of foods (Sadilova et al., 2006). The colour differences in fruit and vegetables during the seasons and the negative effects of processing and storage often make the use of colourants commercially necessary to maintain the colour preferred by the consumer. However, in recent decades, safety of food colourants has been a very contradictive matter and nearly all of the expostulation has been directed against the synthetic colourants. There is a remarkable interest in food colourants obtained from natural sources because of both legislative action and consumer concerns over the use of synthetic additives (Giusti & Wrolstad, 2003; Stintzing & Carle, 2004). However, the use of natural colourant in food has been limited by their relatively low stability to several processing (e.g. clarification, pasteurisation and drying), formulation and storage conditions. Moreover, they may contribute undesirable odour or flavour characteristics (Giusti & Wrolstad, 2003). Anthocyanins (ACNs) are well-known red natural colourants and allowed as natural food colourants in the USA under the category of fruit or vegetable juice *Correspondent: Fax: +(90) 312 484 7599; e-mail: [email protected]

colour (Giusti & Wrolstad, 2003). Extracts obtained from dried materials are allowed under this classification but only water can be used as an extracting solvent (Giusti & Wrolstad, 2003). As acylation of the ACNs enhances their stability by means of intramolecular and ⁄ or intermolecular copigmentation, and self-association reactions, sources of acylated ACNs may provide the desirable stability for food applications. Contrary to low colour stability of nonacylated ACN sources, numerous studies indicated that the acylated ACN sources such as red cabbage (Dyrby et al., 2001), radishes (Giusti & Wrolstad, 2003), red potatoes (Rodriguez-Saona et al., 1999) and black carrots (BCs) (Stintzing & Carle, 2004) had desirable colour stability as food colourants. Similarly, Sadilova et al. (2006) investigated thermal degradation of acylated and nonacylated ACNs from strawberry, elderberry and black carrot concentrates, and their findings also showed that black carrot ACNs were more stable than strawberry and elderberry pigments. When black carrot juice and concentrate added to foods as a colourant, they do not require declaration with an E-number on food labels (Kırca et al., 2007). Kırca & Cemerog˘lu (2003) coloured blond orange juice with black carrot juice concentrate (BCJC) as an alternative to blood orange juice and their results showed that colouring blond orange juice with BCJC

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resulted in a much more stable product against thermal degradation as compared with blood orange juice. Additionally, stability of BC ACNs was also studied in various fruit juices (grape juice, tangerine juice, apple juice, lemon juice, grapefruit juice and orange juice) nectars (peach nectar, apricot nectar and pineapple nectar) and strawberry marmalades by Kırca et al. (2007) who reported that the addition of BCJC to these fruit juices and nectars enhanced colour stability. The selection of food colourants usually is achieved by answering the following issues: regulartory issues, the target shade, the physical ⁄ chemical attributes of the food matrix and stability to processing and storage conditions (Giusti & Wrolstad, 2003). There is only one study found in literature for the storage stability of ACNs in BCJC (Kırca et al., 2007). However, in this study, the ACNs of the BCJC was not monitored by HPLC-MS, ACN contents of BCJCs were determined by only spectrophotometric method. Additionally, kinetics of polymeric colour formation during storage was not determined in this study. The objective of our study was as follows: (i) to determine the kinetics of ACNs degradation and polymeric colour formation in BCJC during storage at various temperatures and (ii) to characterise the ACNs of the BCJC by HPLC-MS techniques and monitor at the beginning and end of the storage.

Black carrots (205 kg)

Crushing

Pressing

Adjustment of pH (from pH 6.0 to 4.3) with citric acid (%50, w/v)

Raw juice (73.6 kg)

Depectinization (50°C 2h, 2.85 mL/L) Bentonite treatment (%10, w/v, 0.715 g/L)

Gelatin (%2, w/v, 7.5 mL/L), Kieselsol (%15, v/v, 2.6 mL/L) treatment*

Filtration

Storage at –23°C* (for 319 days) Storage at 5°C* (for 319 days)

Concentration (40°C)

Storage at 30°C* (for 53 days) Storage at 20°C* (for 319 days)

Figure 1 Flow sheet of BCJCs processing. *These samples were analyzed.

Materials and methods

Chemical and reagents

Standards of cyanidin 3-O-glucoside, used for quantifying the each ACN peak, and cyanidin 3,5-O-diglucoside, used for recovery studies, were purchased from Sigma (St. Louis, MO, USA). Pectinase enzyme and fining agents (bentonite, gelatin and kieselsol) were obtained from Do¨hler (Geisenheim, Germany) and Erblo¨sh (Geisenheim, Germany), respectively. 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. Fruits

Black carrots (BCs) (Daucus carota L. ssp. sativus var. atrorubens Alef.) were obtained from Ereg˘li, Konya and processed immediately. BCs were washed in cold tap water and drained. Damaged BCs were discarded. A flow diagram of BCJC processing is shown in Fig. 1. Before pressing, BCs were washed in cold tap water and drained. Damaged BCs were discarded. Processing

BCs were processed into juice in the fruit juice pilot plant at Ankara University. After washing, BCs were

International Journal of Food Science and Technology 2012

crushed and pressed on a-rack-and-cloth-press (BucherGuyer Ltd., Niederweningen, Switzerland). The clarification of BCJ included depectinisation and fining steps. To provide optimum pH for pectinase activity, the pH of the juice was adjusted from 6.0 to 4.3 with 50% (w ⁄ v) citric acid, and then, the juice was depectinised with Fructozym P at 50 C for 2 h. The depectinised juice was clarified using bentonite, gelatin (A type, 80–100 Bloom strength) and kieselsol. The bentonite solution at 10% (w ⁄ v) was used for the removal of proteins in BCJ. The bentonite was added at a concentration of 0.715 g L)1, and the juice was kept at 50 C for 2 h. Then, the 0.15 g L)1 gelatin (2%, w ⁄ v) and 0.39 mL L)1 kieselsol (15%, v ⁄ v) was added to the bentonite-treated juice. After clarification, the turbidity of the BCJ was 1.82 NTU. The clarified juices were concentrated to 68 Brix by a rotary low-pressure evaporator (Heidolph Laborota 4003; Schwabach, Germany) at 40 C under 20 mm-Hg pressure. Compositional analysis

The total soluble content (Brix) of BCJ samples was determined by an automatic digital refractometer (Atago Rx-7000a, Tokyo, Japan). Brix measurements were carried out at 20 C. pH was measured potentiometrically with a pH meter (WTW Inolab Level 1;

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¨ zkan Kinetics of anthocyanin degradation M. Tu¨rkyılmaz and M. O

Weilheim, Germany). Titratable acidity was determined according to the method outlined by IFU (1968) and expressed as ‘g anhydrous citric acid per 100 mL sample’. The turbidity was measured by turbidimeter as NTU (HACH Ratio ⁄ XR 43900; Loveland, CO, USA). Monomeric ACN analysis

Total monomeric ACN contents were determined using the pH differential method, as described by Giusti & Wrolstad (2005). The concentrate samples were diluted to their actual brix by weighing out 4-g concentrate in 25-mL volumetric flask and bringing the final volume with distilled water. The pH’s of diluted concentrate samples were 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 filtered through a 0.45-lm PVDF filter to remove the haze. The absorbance of equilibrated solutions at 530 nm (kmax) for ACN content and 700 nm for haze correction was measured on a UV-VIS double-beam spectrophotometer (ThermoSpectronic Helios–a, Cambridge, England) with 1-cm path length disposable cuvettes (Brand Gmbh, Wertheim, Germany). All absorbance measurements were carried out at room temperature and made against distilled water as a blank. Pigment content was calculated as cyanidin3-glucoside (Cy-3-glu) equivalents, with a molecular weight of 449.2 and extinction coefficient of 26,900 L (cm mol))1. The difference in absorbance values at pH 1.0 and 4.5 was directly proportional to ACN concentration. All ACN measurements were replicated two times. Polymeric colour content

Percent polymeric colour contents were determined using bisulphite bleaching method, as described by Giusti & Wrolstad (2005). All polymeric colour measurements were replicated two times. HPLC separation of ACNs Extraction

ACNs were extracted following the method described by Lee & Wrolstad (2004). The concentrate samples were diluted to their actual brix by weighing out 4-g concentrate in 25-mL volumetric flask and bringing the final volume with distilled water. The diluted concentrate sample (2 mL) was mixed with 10 mL of acetone (100%), and the acetonic extract was then filtered on a Buchner funnel using Whatman No 1 filter paper. The filter cake was also extracted with first 10 mL of 70% acetone and then 30 mL of 50% acetone until the

solution become colourless. The filtrates were then combined and partitioned with chloroform (1:2 acetone:chloroform, v ⁄ v) in a separatory funnel and left overnight at 4 C for the 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 the residual acetone at 40 C. The aqueous extract was dissolved in purified water (containing 0.01% HCl, v ⁄ v), and the final volume was brought to 25 mL with purified water. The resulting extract was filtered through a 0.45-lm PVDF (Millipore, Bedford, MA, USA) filter directly to an amber-coloured bottle. Two extracts were prepared from each sample. ACN purification

The ACNs were purified on a C-18 cartridge (200 mg, 1 mL) using a vacuum manifold system (Waters Co., Milford, MA, USA). Prior to sample load, the cartridge was activated with 5 mL of ethyl-acetate followed by 5 mL of methanol (containing 0.01% HCl, v ⁄ v) and 2 mL of aqueous 0.01% HCl (v ⁄ v). Upon loading 1 mL of ACN extract, the cartridge was washed with 2 mL of aqueous 0.01% HCL to remove compounds not adsorbed, such as sugars and acids. The cartridge was then dried under a stream of nitrogen for 10 min. The non-ACN phenolics were removed from the cartridge by rinsing with 5 mL of ethyl-acetate. Elution of ACNs was carried out by rinsing the cartridge with 2 mL of methanol (containing 0.01% HCl, v ⁄ v). The methanolic extract containing ACNs was then evaporated to dryness in a water bath (Memmert WB 14; Schwabach, Germany) at 35 ± 0.1 C under a stream of nitrogen. ACNs were then dissolved in aqueous 0.01% HCl. The resulting extract was filtered through a 0.2-lm PTFE (polytetrafluoroethylene) filter (Sartorious AG, Goettingen, Germany) directly to an amber-coloured auto sampler vial. The filtered extract was immediately injected to HPLC without further delay. Instrumentation and chromatography

ACNs were determined using an Agilent HPLC, series 1200 (Agilent, Waldbronn, Germany) equipped with ChemStation software, a binary pump, a photo diode array detector, a thermostatted auto sampler, a degasser and a thermostatted column compartment. ACNs were separated on a C18 (5 lm) column (250 · 4.6 mm) (Phenomenex Inc., Los Angeles, CA, USA) with a C18 (5 lm) guard column (4 · 3 mm, 5 lm) (Phenomenex Inc.). The mobile phase consisted of 100% acetonitrile (eluent A) and O-phosphoric acid ⁄ acetic acid ⁄ acetonitrile ⁄ water (1:10:5:84; v ⁄ v ⁄ v ⁄ v, eluent B). Separation was performed with gradient elution using a modification of the elution profile described by Skrede et al. (2000). The linear gradient programme for the separations of BC ACNs was as follows: from 99% to 88% B

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in 10 min, from 88% to 78% B in 10 min, holding at 78% B isocratic for 5 min and from 78% to 99% B in 5 min. Sample injection volume was 50 lL, and column temperature was set at 25 C. Monitoring was performed at 520 nm at a flow rate of 1 mL min)1. Quantification of total ACN was calculated based on cyanidin 3-O-glucoside. ACNs in BCJ samples were identified by HPLCDAD-MS and using mass ⁄ charge (m ⁄ z) ratios that were previously determined by Sadilova et al. (2006). The DAD detector was interfaced with a mass spectrometer (Agilent Series 1200 HPLC system) with an ESI source operating in the positive ionisation mode. Nitrogen was used at a flow rate of 12 L min)1 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 2,000 V was used on the capillary. The mobile phase consisted of 100% acetonitrile (eluent A) and formic acid (1%, eluent B). The other chromatographic conditions were the same as described previously for the HPLC separation of major ACNs in BCJC. Determination of limit of detection and quantification

The limit of detection (LOD) and limit of quantification (LOQ) for ACNs were determined based on signal to noise (S ⁄ N) ratio. According to ICH guideline 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 et al., 2002). Statistical analyses

Results from ACN content were analysed using the Minitab statistical software, version 14 (Minitab Inc., State College, PA, USA). Storage time at a storage temperature was considered as the main effect. Statistical differences among means were determined by the Duncan’s multiple range test at the 5% significance level.

Results and discussion

Changes in pH, titratable acidity and soluble solid content during storage

pH, titratable acidity and soluble solid content (Brix) of BCJC samples were determined during storage (data not shown). During storage, there were very small differences in pH, titratable acidity and soluble solid contents of BCJC samples ranging from 4.35 to 4.45, 3.50 to 3.83 g per 100 g (as anhydrous citric acid) and 67.1 to 68.0, respectively. Similar slight changes in the compositional measurements were also observed in red raspberry and blackberry juices during processing and storage (Rommel et al., 1990, 1992). Characterisation of ACNs and changes in ACNs of BCJC

To determine ACNs in BCJC as well as to monitor the changes in ACNs throughout the storage, HPLC-DADMS analyses were applied. The ACN peaks were identified by comparing their characteristic MS data with the published data for BC ACNs (Sadilova et al., 2007). The HPLC profile of the ACNs in the BCJC before storage is presented in Fig. 2. Five ACNs were identified in BCJC, exhibiting molecular ions M+ at m ⁄ z 743, 581, 949, 919 and 889, respectively. In a previous study, MS data of ACNs in BCs and their fragmentation patterns were presented by Sadilova et al. (2007). They reported that peak 1 (M+, m ⁄ z 743) had one MS2 fragment ions of m ⁄ z 287 (molecular weight, MW, of cyanidin). MS data indicated that this ACN contained one cyanidin, one pentose (MW–H2O, 132) and two hexoses (MW–H2O, 2 · 162 = 324). According to a former study carried out by Giusti et al. (1999), when these two hexoses appeared in one position (C-3), MS2 showed only a fragment ion of the aglycon. Thus, these sugars were likely linked with the same (C-3) position of cyanidin. Peak 2 had a molecular ion m ⁄ z 581 and a fragment ion m ⁄ z 287, which indicated that this ACN contained one

Figure 2 The HPLC profile of the ACNs in the BCJCs before storage. 1: Cyd-3-gal-xyl-glc, 2: Cyd-3-gal-xyl, 3: Cyd-3-gal-xyl-glc-sin, 4: Cyd-3-galxyl-glc-fer, 5: Cyd-3-gal-xyl-glc-coum.

International Journal of Food Science and Technology 2012

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¨ zkan Kinetics of anthocyanin degradation M. Tu¨rkyılmaz and M. O

cyanidin, one pentose (MW–H2O, 132) and one hexose (MW–H2O, 162). Peaks 3 (M+, m ⁄ z 949), 4 (M+, m ⁄ z 919) and 5 (M+, m ⁄ z 889) shared the same MS patterns (m ⁄ z 581 and 287). Loss of 368 (949–581=368) Da was ascribed to the cleavage of a hexose and one sinapoyl group (MW–H2O, 206). Similarly, for peak 4, loss of 338 (919–581=338) Da was ascribed to the cleavage of a hexose and one feruloyl group (MW–H2O, 176). And, for peak 5, the loss of 308 (889–581 = 308) Da was ascribed to the cleavage of a hexose and one coumaroyl group (MW–H2O, 146). If these groups had been linked with C-5 position of aglycone, more than two MS2 fragment ions would have been determined. In conclusion, the BCJC comprised two nonacylated ACNs and three mono-acylated ACNs with coumaric, ferulic and sinapic acids. The major compounds were cyanidin-3-galactoside-xyloside-glucoside-ferulic acid will be (cyd-3-gal-xyl-glc-fer, 56%), followed by cyanidin-3-galactoside-xyloside (cyd-3-gal-xyl, 19%), cyanidin-3-galactoside-xylosideglucoside-sinapic acid (cyd-3-gal-xyl-glc-sin, 10%), cyanidin-3-galactoside-xyloside-glucoside-coumaric acid (cyd-3-gal-xyl-glc-coum, 7%) and cyanidin-3-galactoside-xyloside-glucoside (cyd-3-gal-xyl-glc, 7%). The other studies also reported that five ACNs were determined, and cyd-3-gal-xyl-glc-fer was found as the major ACN of BCs (Kammerer et al., 2004; Sadilova et al., 2007). As BC ACNs especially provide an excellent bright strawberry-red shade at acidic pH’s (Kırca et al., 2007), black carrot juice and concentrate can be good choices for colouring fruit juices and nectars, soft drinks, conserves, jellies and confectionery (Downham & Collins, 2000). It is, therefore, also necessary that the BCJC be stored at a temperature that will minimise individual and total monomeric ACN degradation. The changes in total monomeric ACNs of BCJC samples stored for 319 days at )23, 5 and 20 C and for 53 days at 30 C are shown in Table 1. The higher storage temperatures caused the increases in monomeric ACN degradation. The highest degradation was observed in BCJC samples

Table 1 Changes in anthocyanin (ACN) contents and polymeric color of black BCJCs during storage ACN contenta (mg kg)1) Temperature Storage (°C) time (day) Spectrophotometric HPLC

)23 5 20 30 a

0 319 319 319 53

3747 3789 (0)b 2536 (32%) 745 (80%) 1065 (72%)

Expressed as cyanidin-3-O-glucoside. Values in parentheses are ACN losses (%).

b

4123 4008 (2%) 2206 (46%) 681 (83%) 1544 (63%)

Polymeric color (%) 18 19 25 36 49

stored at 30 C. There were no significant changes in ACN content of BCJC stored at )23 C. To prevent monomeric ACNs during storage, BCJC should preferably be stored under subfreezing temperatures. During the storage at 5, 20 and 30 C, cyd-3-gal-xylglc-coum had the highest stability (at pH 4.3), followed by cyd-3-gal-xyl-glc-fer and cyd-3-gal-xyl-glc-sin, respectively. However, the stability of acylated ACNs varies depending on different pH values. While ACNs acylated with the sinapoyl derivative exhibited the highest stability at pH 1 upon heating at 95 C, followed by the feruloylated and coumarolated cyanidin triglycosides (Sadilova et al., 2006), ACNs acylated with the feruloyl derivative exhibited the highest stability at pH 3.5 upon heating 95 C (Sadilova et al., 2007). Compared with acylated ACNs of BCJC, nonacylated ACNs (cyd-3-gal-xyl-glc and cyd-3-gal-xyl) showed the lowest stability to storage temperature and time, which agree with previous studies (Sadilova et al., 2006, 2007). For example, while the loss in cyd-3-gal-xyl-glc-coum content was 13% after storage at 30 C for 53 days, the loss in cyd-3-gal-xyl-glc-fer was up to 49% and nonacylated ACNs were not detected (Table 1) at the same storage conditions. Likewise, Sadilova et al. (2007) compared the half-life periods of acylated ACNs with nonacylates ACNs in purified pigment isolates at pH 3.5 upon heating at 95 C and found that cyd-3-gal-xyl-glcfer, cyd-3-gal-xyl-glc-coum and cyd-3-gal-xyl-glc-sin had 1.6, 1.4 and 1.3 times, respectively, higher half-life periods than that of cyd-3-gal-xyl-glc. The comparatively much higher stability of acylated ACNs with nonacylated ACNs was likely related to the natural synthesis of acylated organic acids and diversity of glycosidic linkages in relation to these acylated moieties (Rodriguez-Saona et al., 1999; Giusti & Wrolstad, 2003). The total ACN content of BCJC at 68Brix was 4,123 mg ACN per kg by HPLC at the beginning of the storage (Table 2). Much lower ACN content (1,146 mg kg)1) of BCJC at 64Brix was reported by Kırca et al. (2007). This difference was believed to be owing to different harvest time that causes great variations in the pigment contents within the same cultivar. In a previous study, the influence of harvest time on ACN concentration of BCs was evaluated and maximal monomeric ACN contents were found between mid-September and October (Kammerer et al., 2004). ACN contents of BCJC samples were also determined throughout the storage at )23, 5, 20 and 30 C. The ACN contents of BCJCs during storage were plotted as a function of time for different temperatures (figure not shown). The degradation of ACNs in BCJCs during storage followed the first-order reaction kinetics with respect to temperature because the curves in semi-log graph were linear and the high determination coefficients values (over 0.99) of each plot clearly showed a

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Table 2 Changes in individual ACN content of BCJCs during storage ACN content (mg kg)1)a Temperature (°C)

Storage time (day)

Cyd-3-gal-xyl-glca

Cyd-3-gal-xyla

Cyd-3-gal-xyl-glc-sina

Cyd-3-gal-xyl-glc-fera

Cyd-3-gal-xyl-glc-couma

–23 5 20 30

0 319 319 319 53

290 274 (5.5%) ND ND ND

789 775 (1.8%) ND ND ND

306 296 (3.3%) ND ND ND

2319 2254 (2.8%) 1831 (21%) 681 (71%) 1181 (49%)

419 409 (2.4%) 375 (11%) ND 363 (13%)

ND, not detected. a ACNs were expressed as cyanidin-3-glucoside.

very good fit of data. These results, which show that the rate of the degradation is directly proportional to the concentration of the pigments, agree with those of previous studies reporting first-order reaction kinetics for ACNs in strawberry (Garzon & Wrolstad, 2002), blood orange (Kırca & Cemerog˘lu, 2003), BC (Sadilova et al., 2006; Kırca et al., 2007) and blackberry (Wang & Xu, 2007). The first-order reaction rate constants (k) and halflives (t1 ⁄ 2), that is, the time needed for 50% degradation of ACNs, were calculated by the following equations: lnðCt =C0 Þ ¼ k  t; t1=2 ¼  ln 0:5=k; where C0 is the initial monomeric ACN content, and Ct is the monomeric ACN content after t min heating at a given temperature. The kinetic parameters of ACN degradation during storage are shown in Table 3, where the values of the first-order kinetic constants k and t1 ⁄ 2 were shown for each storage temperature. As the storage temperature

Table 3 Kinetic parameters for the ACN degradation and polymeric color formation of BCJCs during storage

Temperature (°C)

)k · 103 day)1

Q10 t1 ⁄ 2 Ea (day) 5–20 °C 20–30 °C (kJ mol)1)

ACN degradation 5 1.15 (0.9971)a 603 20 5.07 (0.9957) 137 30 23.03 (0.9976) 29 Polymeric color formation 5 2.07 (0.9605)a 20 14.35 (0.9995) 30 55.81 (0.9827) a

– – –

2.69

4.54

84

3.64

3.89

92

Numbers in parentheses are the determination coefficients (R2).

International Journal of Food Science and Technology 2012

increases, the corresponding k value also increases, which indicated that the greater the storage temperature, the greater the ACN degradation. The calculated half-life period (t1 ⁄ 2) at 5, 20 and 30 C were respectively, 603, 137 and 29 days in BCJC (at 68 Brix). However, Kırca et al. (2007) reported that the t1 ⁄ 2 values of ACNs of BCJCs at 64 Brix at 4, 20 and 37 C were 1,505, 252 and 28 days, respectively. As compared to t1 ⁄ 2 values, although t1 ⁄ 2 value (28 days) of ACNs at 37 C were almost the same as the t1 ⁄ 2 value (29 days) of ACNs at 30 C determined in our study, t1 ⁄ 2 values at 4 and 20 C were much higher than our t1 ⁄ 2 values. The differences between t1 ⁄ 2 values may be depend on different reasons. The ACN content of BCJC in the study of Kırca et al. (2007) was 3.3 times lower than that in our study. As known, for a first-order reaction, the rate of reaction is directly proportional to the reactant concentration which may be the reason why the reaction rates increase as the reactant concentrations increase. Other reasons may be different ACN compositions and the proportion of acylated ACNs in BCJCs. Previous studies showed that the t1 ⁄ 2 values of ACNs of sour cherry juice concentrate (at 71Brix) and blood orange juice concentrate (at 69Brix) at 20 C were 83 days (Is¸ık, 1993) and 0.74 days (Kırca & Cemerog˘lu, 2003), respectively. These observations clearly show that ACNs from BCJC have much higher stability than those from blood orange juice concentrate and sour cherry juice concentrate. Similarly, the half-life of ACN isolate from BC was two times higher than that of elderberry (Sadilova et al., 2006). The reason of the high ACN stability of BCs was attributed to their mono-acylated ACN contents. The aromatic (e.g. the derivatives of hydroxycinnamic acids, i.e. q-coumaric, ferulic, caffeic and sinapic acids) or aliphatic (e.g. malonic, acetic, malic, succinic, and oxalic acids) acyl group covalently bound to the ACNs were shown to stack on the planar, polarisable nuclei of the ACN, protecting the pyrylium nucleus from the nucleophilic attack of water at C-2 (Rodriguez-Saona et al., 1999). Therefore, acylation of ACNs considerably prolong their half-life, thus the stability, compared with that of nonacylated ACNs (Sadilova et al., 2007).

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Similarly, Pozo-Insfran et al. (2004) evaluated t1 ⁄ 2 values of ACN pigments in acai, hibiscus, BC and red cabbage, and they reported that t1 ⁄ 2 values of these fruit and vegetables at 37 C were as 13.3, 5.5, 20.3 and 47.9 days, respectively. The evaluation of t1 ⁄ 2 values of ACNs in these sources revealed that acylated ACN sources (BC and red cabbage) generally had higher stability compared with nonacylated sources (acai and hibiscus). Moreover, the t1 ⁄ 2 values revealed that ACNs in BCs showed lower stability than those in red cabbages during storage. The presence of diacylation in the red cabbage ACNs as compared to monoacylated ACNs in BCs might be responsible for its enhanced stability. Diacylated ACNs are stabilised by a sandwich-type stacking caused by hydrophobic interactions between the planar aromatic residues of the acyl groups and the positively charged pyrylium nucleus (Goto, 1987). This prevents the addition of nucleophiles, especially water, to the C-2 and C-4 positions of the ACN, diminishing the formation of the pseudobase (Pozo-Insfran et al., 2004). In the case of monoacylated ACNs, only one side of the pyrylium ring can be preserved towards the nucleophillic attack of water, and therefore, a weak intermolecular effect might occur (Brouillard, 1983). The mechanism of ACN degradation appears to be temperature dependent. The dependence of the degradation of ACNs in BCJCs over the temperature range of 5–30 C was determined by calculating the temperature quotient (Q10) and activation energy (Ea) values from the following equations: Q10 ¼ ðk2 =k1 Þ10=ðT2 T1 Þ k ¼ k0 eEa =RT ; Higher Q10 value (4.54) was obtained within the range of 20–30 C as compared to that (2.69) within the range of 5–20 C (Table 3). Our results are similar to previously published data (3.6 and 3.1 at the same temperature ranges, respectively) by Kırca et al. (2007). The high Q10 value indicates that the kinetic of ACN degradation was strongly affected by the temperature in this temperature range; therefore, low storage temperatures are needed to prevent the degradation of ACNs in BCJCs. Another way to express the influence of temperature on reaction rates is the use of Ea value. Activation energy for the degradation of ACNs in BCJC was 84 kJ mol)1 at 5–30 C (Table 3). The calculated Ea values for the ACN degradation in BCJC (at 68Brix) are similar to those reported by Kırca et al. (2007) for BCJC at 64Brix (86 kJ mol)1) at 4–37 C and by Kırca & Cemerog˘lu (2003) for blood orange juice concentrates at 69Brix (81 kJ mol)1) at the same temperature. However, lower Ea value for the ACN degradation in blackberry juice concentrate at 65Brix and 5–37 C

(65 kJ mol)1) were reported by Wang & Xu (2007). The higher Ea value implied that a smaller temperature change is needed to degrade of ACNs more rapidly. Kinetics of polymeric colour formation during storage

Percentage of polymeric colour is an index of degree of ACN polymerisation. Polymeric colour significantly increased (P < 0.05) throughout the storage of BCJC samples. Polymeric colour formation followed both zero- and first-order kinetics (figure not shown). Zeroorder kinetic model is chosen for the calculation of kinetic data of polymeric colour formation. Kinetic data for polymeric colour formation is presented in Table 3. At 30 C, BCJC samples stored showed a very fast rate of polymeric colour formation as compared with those of BCJC samples stored at 5 and 20 C. Rate constants (k) for polymeric colour formation in BCJC samples were 0.0207, 0.1435 and 0.5581% per day at 5, 20 and 30 C, respectively (Table 3). The k values for polymeric colour formation in all BCJC samples increased with increasing storage temperature. These findings agree well with previous reports (Rommel et al., 1990, 1992). Activation energy (Ea) for polymeric colour formation in BCJC was 92 kJ mol)1 at 5–30 C, (Table 3) and Q10 values obtained within the range of 5–20 C and 20– 30 C were 3.64 and 3.89, respectively. Our results are slightly higher than previously published Q10 values for polymeric colour formation in pomegranate juice concentrate samples (2.73–3.43 within the range of 5–20 C) by Turfan (2011). Higher Q10 value showed that polymeric colour formation in BCJC was more affected by changes in temperature than those in pomegranate juice concentrate samples. This result may be because of phenolic contents, including tannins. The BCJC obtained from clarified juice had 3.2-fold higher total phenolic contents (22,715 mg kg)1, Dereli, 2010) than pomegranate juice concentrate obtained from clarified juice (7,100 mg kg)1, Apaydın, 2008). As the ACNs are mainly bound to the phenolics, the polymerisation reactions of ACNs and other phenolic compounds (e.g. condensed tannin and phenolic acid etc.) may probably be more favourable in BCJC owing to higher phenolic content than that in pomegranate juice concentrate. As known, the polymeric pigments are resistant against bisulphite bleaching, while monomeric ACNs are readily bleached by bisulphite at pH of BCJC samples. A reason why polymeric colour formation increased as the storage temperature increased could be due to the various reactions, namely the polymerisation of ACNs, the degradation of ACNs and the formation of polymeric ACN-condensed tannin (polymers of flavan-3-ols) complexes and melanoidin pigments that result from the nonenzymatic browning reactions between reducing

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Table 4 Correlations between ‘ACN content and polymeric color’ with ‘turbidity and polymeric color’ during storage Storage temperatures (°C) Correlation between ACN and polymeric color Turbidity and polymeric color

5

20

30

y = )164.29x + 6766.6 (r = ) 0.967) y = 0.926x + 11.939 (r = 0.992)

y = – 89.248x + 5202.1 (r =) 0.956) y = 0.8183x + 15.582 (r = 0.930)

y = )90.4x + 5265.9 (r =) 0.970) y = 2.079x + 11.321 (r = 0.896)

sugars and amino acids (Giusti & Wrolstad, 2005). Of these reactions, one of the most probable reactions in BCJC samples during storage is the degradation of ACNs. As mentioned earlier, the rate of ACN degradation in BCJCs also increased at higher storage temperature. In parallel to ACN degradation, the rate of polymeric colour formation also increased by temperature elevations during storage. It was postulated that the formation of a chalcone is the first step in the degradation of ACNs (Adams, 1973) When temperature is increased, the formation of the unstable chalcone form is favoured, and the chalcone is further degraded to brown products (Jackman & Smith, 1996). During storage, there was a good negative correlation between the percentage of polymeric colour and ACN content (Table 4). Losses of monomeric ACNs in BCJC samples during storage were accompanied by the increases in polymeric colour values. The second most probable reason for the increase in polymeric colour during storage of BCJC samples is the reaction between ACNs and condensed tannins. In fact, the condensed tannin contents in the BCJC samples increased from 308 to 1,046, 1,330 and 1,557 mg catechin per kg after 8 months of storage at 5, 20 and 30C, respectively (Dereli, 2010). All BCJC samples stored at different temperatures (5, 20 and 30 C) showed increases in haze formation during storage. Good correlations were found between haze and polymeric colour formation (Table 4). Similarly, Rwabahizi & Wrolstad (1988) and Rommel et al. (1990) also reported a strong correlation between haze and polymeric colour formation for ACN-containing juices, such as strawberry and raspberry juices. Conclusion

Five ACNs were identified in BCJC, which comprised the cyanidin based two nonacylated ACNs and three mono-acylated ACNs with coumaric, ferulic and sinapic acids. ACNs of BCJC had higher stability than those of other sources (e.g. pomegranate, blueberryand elderberry). During the storage at 5, 20 and 30 C, cyd-3-galxyl-glc-coum had the highest stability (at pH 4.3), followed by cyd-3-gal-xyl-glc-fer and cyd-3-gal-xyl-glcsin, respectively. As the stability of acylated ACNs varies during storage depending on various factors (pH, Brix, ascorbic acid content, etc.), in the future studies,

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the effects of these factors on the colour stability of BCJC as food colourant should be investigated. Acknowledgments

This study is in part of Miss. Tu¨rkyılmaz’s preliminary studies of her PhD degree was funded by ‘Ankara University Scientific Research Projects Office’ Turkey (Grant # 07B4343002). Miss Tu¨rkyılmaz would like to thank The Scientific and Technological Research Council of Turkey-Graduate Research Scholarship Programme (TUBITAK-BIDEP) for their financial support during her PhD studies. The authors would like to thank Prof. Murat Kartal and Dr. Mahmoud Abu Asaker in the Faculty of Pharmacy at Ankara University for helping our MS analysis. The authors also thank the Yaka Group Co. (Eregˇli, Konya) for providing the BCs, and Do¨hler Co. (Istanbul) for providing pectolitic enzymes and clarifying agents. References Adams, J.B. (1973). Colour stability of red fruits. Food Manufacture, 48, 19–20, 41. Apaydın, E. (2008). Changes in antioxidant activity of pomegranate juice concentrate during production and storage. Master Thesis, Ankara: Ankara University. Brouillard, R. (1983). The in vivo expression of anthocyanin color in plants. Phytochemistry, 22, 1311–1323. Dereli, U. (2010). Changes in phenolic compounds of black carrot juice concentrate during production and storage and its relation with antioxidant activity. Master Thesis, Ankara: Ankara University. Downham, A. & Collins, P. (2000). Coloring our foods in the last and next millenium. International Journal of Food Science and Technology, 35, 5–22. Dyrby, M., Westergaard, N. & Stapelfeldt, H. (2001). Light and heat sensitivity of red cabbage extract in soft drink model systems. Food Chemistry, 72, 431–437. Garzon, G.A. & Wrolstad, R.E. (2002). Comparision of the stability of pelargonidin-based ACNs in strawberry juice and concentrate. Journal of Food Science, 67, 1288–1299. Giusti, M.M. & Wrolstad, R.E. (2003). Acylated anthocyanins from edible sources and their applications in food systems. Biochemical Engineering Journal, 14, 217–225. Giusti, M.M. & Wrolstad, R.E. (2005). Characterization and measurement of ACNs by UV-visible spectroscopy. Unit F1.2. In: Handbook of Food Analytical Chemistry (edited by R.E. Wrolstad, T.E. Acree, E.A. Decker, M.H. Penner, D.S. Reid, S.J. Schwartz, C.F. Shoemaker, D.M. Smith & P. Sporns). Pp. 19–31. New York: John Wiley & Sons.

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