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Varietal Blends as a Way of Optimizing and Preserving the Anthocyanin Content of Pomegranate (Punica granatum L.) Juices Pedro Mena,† Nuria Martí,§ and Cristina García-Viguera*,† †

Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, E-30100 Espinardo, Murcia, Spain IBMC-JBT Corp., FoodTech R&D Alliance, Universidad Miguel Hernández, E-03312 Orihuela, Alicante, Spain

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ABSTRACT: Anthocyanins are unstable compounds prone to degradation during storage of pomegranates juices, leading to disadvantageous color changes. Blending varietal pomegranate juices could be useful not only to preserve the genuine characteristics of fresh juices but also to study different factors affecting anthocyanin stability while maintaining to the utmost the matrix studied. The effects of critical factors such as anthocyanin concentration, pH, and endogenous ascorbic acid on pigment integrity were assessed through the study of the degradation kinetics of pomegranate phytochemicals in blended juices made from two distinct cultivars (‘Wonderful’ and ‘Mollar de Elche’). Pigment concentration and pH were the factors affecting anthocyanin stability, whereas ascorbic acid did not alter the degradation of anthocyanins. These results contributed to the definition of the so-called “cultivar effect” and to preserving to a great extent the anthocyanin load and color characteristics of fresh varietal juices, avoiding phytochemical degradation and browning development during storage. KEYWORDS: degradation kinetics, storage, color, vitamin C, phytochemical stability

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far.9−12 Therefore, the effects of critical factors such as anthocyanin concentration, pH, and endogenous ascorbic acid remain unknown. This fact represents a major gap to address research efforts properly toward the responsible factor(s) of pigment degradation and to tackle color degradation in pomegranate juice and other anthocyanin-rich products. A novel approach to protect anthocyanin content in pomegranate juices could be performed through varietal blends.10 The cultivar used for pomegranate juice elaboration affects anthocyanin stability and, thus, the sensorial properties of pomegranate juice.13,14 In this sense, beverages derived from the Spanish ‘Mollar de Elche’ variety change into nondesirable brown colors owing to a fast degradation of anthocyanins.15−17 On the contrary, other juices produced with more acid varieties (and less acceptable taste) protect better the anthocyanin content during storage.10 Consequently, appetizing pomegranate juices able to preserve to a great extent the anthocyanin load could be optimized by blending different varieties, as usually done for other fruit juices. In addition, varietal blends can be used as a suitable model to study different factors affecting anthocyanin stability while maintaining to the utmost the matrix studied. To fulfill this idea, different pomegranate juices made from two distinct cultivars (‘Mollar de Elche’ and ‘Wonderful’) were developed to study the effect of pH, pigment concentration, endogenous ascorbic acid, and storage temperature on anthocyanin stability. The relationship between anthocyanins and vitamin C and its influence on color changes were also stressed.

INTRODUCTION Pomegranate (Punica granatum L.) juice is a valuable source of (poly)phenolic compounds, such as anthocyanins and ellagitannins, with proven health-promoting features that have boosted worldwide marketing.1 Pomegranate fruits are usually earmarked for direct fresh consumption and are also processed by the food industry for the elaboration of juices, wines, and jams, among other products. However, the industrial transformation of the fruit is not exempt from some disadvantages such as color changes and the degradation of its phenolic composition. Hence, solving industrialization problems of pomegranate juice by preserving their phytochemical compounds and genuine characteristics is a major sticking point for the juice industry. Anthocyanins are a group of water-soluble natural pigments responsible for the attractive red-blue color of flowers and many fruits, including pomegranate. Pomegranate presents an anthocyanin profile characterized by six anthocyanins, cyanidin 3,5-di- and 3-O-glucoside, delphinidin 3,5-di- and 3-O-glucoside, pelargonidin 3,5-di- and 3-O-glucoside,2 although three new cyanidin derivatives have recently been described in pomegranate juice, pentoside, pentoside-hexoside, and rutinoside.3 Anthocyanins are rather unstable compounds and are prone to degradation during processing and storage, which results in color changes and declines of important marketable quality attributes.4 A number of factors influence anthocyanin stability, including pH, anthocyanin concentration and structure, temperature, light, oxygen, and enzymes as well as the presence of ascorbic acid, sugars, sulfur dioxide or sulfite salts, metal ions, and copigments.5−7 Although anthocyanin degradation has been extensively studied for pomegranate juice during processing and storage,8−10 not enough mechanistic information on the factors affecting pigment integrity has been provided, and only the effects of light, temperature, packaging material, and ascorbic acid addition have been considered so © XXXX American Chemical Society

Special Issue: International Workshop on Anthocyanins (IWA2013) Received: November 13, 2013 Revised: February 17, 2014 Accepted: March 10, 2014

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dx.doi.org/10.1021/jf405129q | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Color Measurement. Color measurement was determined as in Pérez-Vicente et al.11 Samples were measured in glass cells of 2 mm path length (CT-A22) at 520 nm using a Minolta CM-508i tristimulus color spectrophotometer (Osaka, Japan) coupled with a CM-A760 transmittance adaptor, illuminant D65, and 10° observer according to the CIELAB 76 convention. Data (CIE L*, CIE a*, CIE b*, chroma, and hue angle) were processed using the Minolta Software Chromacontrol S, PC-based colorimetric data system. Statistical Analyses and Kinetics of Phytochemical Degradation. Analysis of variance (ANOVA) and multiple-range tests (Scheffé’s test) were carried out using the IBM SPSS Statistics 19 software package (SPSS Inc., Chicago, IL, USA). Pearson correlation analyses were also performed. The degradation kinetics of most phytochemicals follows the firstorder eq 1 reaction21,22

MATERIALS AND METHODS

Chemicals and Reagents. Ascorbic acid, dehydroascorbic acid, and cetrimide (cetyltrimethylammonium bromide) were purchased from Sigma-Aldrich (Steinheim, Germany); formic acid, methanol, potassium dihydrogen phosphate, and sodium benzoate, all of analytical grade, were from Panreac Quı ́mica S.A. (Barcelona, Spain). 1,2-Phenylenediamine dihydrochloride (OPDA) was from Fluka Chemika (Neu-Ulm, Germany). Cyanidin 3-glucoside was from Polyphenols (Sandnes, Norway). Milli-Q water used was produced using an Elix3Millipore water purification system coupled to a Milli-Q module (model Adventage10) (Molsheim, France). Experimental Design. Pomegranate fruits from cv. Mollar de Elche (M) and cv. Wonderful (W), harvested in the Alicante region (southeastern Spain), were provided by “Cambayas Coop. V.” (Elche, Alicante, Spain). Pomegranates were cut in halves, and the juices of each cultivar were obtained by pressure with a laboratory pilot press (Zumonat C-40; Somatic AMD, Valencia, Spain).11,14 Pomegranate varietal juices were mixed in different proportions: 75% M + 25% W, 50% M + 50% W, and 25% M + 75% W, keeping also two control juices of 100% ‘Mollar de Elche’ (100% M) and 100% ‘Wonderful’ (100% W), respectively. Homogenized blended and pure juices were centrifuged (3 min at 2754g), and sodium benzoate (200 mg/L) was added to prevent spoilage.12 Juices were placed in screwcapped vials (10 mL) and stored in the dark at both 5 and 25 °C for 70 days. Triplicate solutions were prepared for each juice, and the mean values were reported in each case. Analyses were carried out at days 0, 6, 13, 20, 28, 42, 56, and 70. Quality Parameters of Juices. pH, titratable acidity (TA), and total soluble solids (TSS) were evaluated as quality indices.18 The pH values were measured using a pH-meter (GLP 21, Crison Ltd., Barcelona, Spain). The TA was determined by titrating 2 mL of juice (raised to a 20 mL final volume with Milli-Q water) with 0.1 N NaOH (pH 8.1). Results were expressed as grams of anhydrous citric acid per 100 mL. TSS contents were recorded in a hand refractometer (Atago N1, Atago, Tokyo, Japan) at 20 °C with values being expressed as °Brix. Measurements were repeated twice for each replicate. Ratio, also known as maturity index (MI), was calculated as the relationship between TSS and TA. Identification and Quantification of Anthocyanins. The anthocyanin profile of pomegranate juices was assessed by liquid chromatography coupled to mass spectrophotometry (HPLC-ESIMSn), according to the method of Gironés-Vilaplana.19 HPLC analyses for anthocyanin quantification were performed on a Merck-Hitachi liquid chromatograph (Tokyo, Japan), equipped with a diode array detector UV−vis L-7455, an autosampler L-7200, a pump L-7100, and an interface D-7000. Chromatograms were recorded and processed on a Merck-Hitachi D-7000 HSM PC-based chromatography data system. A 20 μL sample was analyzed on a Luna C18 column (25 cm × 0.46 cm, 5 μm particle size; Phenomenex, Macclesfield, UK) with a security guard C18-ODS (4.0 × 3.0 mm) cartridge system (Phenomenex), using a mobile phase of water/formic acid (95:5, v/v) (solvent A) and HPLC grade methanol (solvent B). Elution was performed at a flow rate of 1 mL/min. The linear gradient started with 1% B, remaining at isocratic conditions during 5 min, reaching 20% B at 20 min, 40% B at 30 min, 95% B at 35 min, and 1% B after 41 min. UV chromatograms were recorded at 520 nm. Anthocyanins were quantified by the absorbance of their corresponding peaks as cyanidin 3-glucoside. Prior to injection, samples were centrifuged during 5 min at 10480g (model EBA 21, Hettich Zentrifugen, Tuttlingen, Germany) at room temperature. The supernatant was filtered through a 0.45 μm nylon membrane (Simplepure; Membrane Solutions, Plano, TX, USA) before HPLC analysis. Extraction and Analysis of Vitamin C. Ascorbic acid (AA) and dehydroascorbic acid (DHAA) contents were determined by HPLCUV as described elsewhere and fully detailed in González-Molina et al.20 The vitamin C content was calculated by the addition of ascorbic acid and dehydroascorbic acid contents, and results were expressed as milligrams per 100 mL.

Ct = C0 exp(± kt )

(1)

where Ct and C0 are the concentration of bioactive compounds (anthocyanins and vitamin C) (mg/100 mL) at time t and t0, respectively, k is the rate constant, and t is the storage time (days). Moreover, the half-life value (t1/2) was calculated as t1/2 = ((ln 2)/k). D value (time required for 90% degradation of bioactive) was also calculated as D = (1/k).

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RESULTS AND DISCUSSION Quality Parameters of Juices. No significant differences (p > 0.05) in the quality parameters (pH, TSS, TA, and MI) assayed during storage were noted and, hence, results are presented as mean values for all of the experiments (Table 1). Table 1. Quality Parameters of Pure and Blended Varietal Pomegranate Juices through the Storage Period juice

pH

100% M 75% M + 25% W 50% M + 50% W 25% M + 75% W 100% W LSDe

d

3.58 a 3.11 b 2.92 c 2.81d 2.76 e 0.01***

TSSa

TAb

MIc

16.75 e 17.10 d 17.50 c 17.85 b 18.20 a 0.04***

0.27 e 0.64 d 1.09 c 1.39 b 1.78 a 0.01***

63.2 a 26.9 b 16.1 c 12.9 cd 10.3 d 0.86***

a

TSS, total soluble solids. bTA, titratable acidity (g citric acid/100 mL). cMI, maturity index (TSS/TA). Statistical treatment notes. d Means (n = 3) within a column followed by different letters are significantly different at P < 0.05 according to the Scheffé multiple range test. eLSD, least significant difference. Significant at (*) P < 0.05, (**) P < 0.01, and (***) P < 0.001.

The pH values in the juices decreased with the percentage of ‘Wonderful’ juice added, varying from ∼3.60 to ∼2.70 for 100% M and 100% W, respectively. TSS ranged between ∼16.7 for 100% M and ∼18.2 for 100% W, which cannot be considered an important difference despite its statistical significance. Otherwise, TA displayed severe differences among juices, oscillating between ∼0.30 and ∼1.80 g/100 mL for 100% M and 100% W, respectively. Acidity was augmented with increasing percentage of ‘Wonderful’ juice added in a dose− response effect, as expected. Consequently, the maturity index (MI), which is mainly influenced by TA for pomegranate juices,14 was depleted as the ‘Wonderful’ juice proportion was increased in the blended juices. The pomegranate cultivars chosen to develop the blended juices were widely distinct. ‘Wonderful’ is a worldwide variety rich in phytochemicals, with a deep red color, but too much acid. On the other hand, the Spanish ‘Mollar de Elche’ exhibits interesting organoleptical properties (high lightness and B



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Major individual anthocyanins for all juices were cyanidin 3glucoside, accounting for 31% of the total anthocyanin amount; delphinidin glycosides, both 3,5-diglucoside and 3-glucoside, 23% each one; and cyanidin 3,5-diglucoside, 20%. Pelargonidin glycosides accounted for only 3% of the total anthocyanins. The loss of anthocyanins during storage (Figure 3) fitted well to exponential curves for the two selected temperatures (Table 3). These results indicated that pigment degradation at isothermal conditions followed first-order reaction kinetics, as usually reported for red juices.4,10,21,22 The rate constant (k) increased at higher storage temperature, pointing out the notable influence of temperature on accelerated anthocyanin degradation.8,22 Half-life values (t1/2) ranged from 98 to 374 days at 5 °C and between 28 and 87 for 25 °C. D values pointing out the time required for the degradation of 90% of anthocyanins ranged between 141 and 539 days for juices stored at 5 °C and between 40 and 125 days at 25 °C. These differences of pigment shelf life between storage temperatures showed the role of the storage conditions in the preservation of the phytochemical composition of pomegranate juices. A point worth mentioning is that the shelf life values herein reported were considerably higher than those reported for thermally pasteurized pomegranate juices.8−10 This fact emphasizes the need for developing novel approaches avoiding thermal treatments and limiting microbial load at fresh juice to preserve as much as possible the anthocyanin content of commercial pomegranate juices. Kinetic parameters (Table 3) revealed a significant cultivar effect on the evolution of anthocyanins during storage at both 5 and 25 °C. The rate constant was clearly higher as the proportion of ;Mollar de Elche; juice was increased, the highest rate constants being recorded for 100% M. One hundred percent W juice registered rate constants 3.7- and 3.1-fold lower than those found for 100% M at 5 and 25 °C, respectively. The blended juices used in the present work could be regarded as suitable samples to shed light on the factors behind the so-called cultivar effect. They displayed intermediate k values proving that drops in anthocyanins were diminished in the mixtures proportionally to the percentage of ‘Wonderful’ juice added (Table 3). Then, the blend containing merely 25% of ‘Wonderful’ juice (75% M + 25% W) could not only increase the anthocyanin content of ‘Mollar de Elche’-based pomegranate juices but also preserve to a great extent the pigment composition through storage. In this sense, data herein obtained have shown that the higher pigment concentration, the lower degradation rate (Table 3). This phenomenon linking superior preservation to high anthocyanin concentrations could be attributed mainly to the formation of self-association complexes of copigmentation (the aggregation of different anthocyanin monomers in relatively concentrated matrixes).26 Similarly, other complexes, adducts between flavan-3-ols and anthocyanins, have been found in fresh pomegranate juices.27 Another explanation behind the cultivar effect observed and the higher preservation of anthocyanins in those juices containing a higher percentage of ‘Wonderful’ varietal juice could be related to the effect of pH. Actually, a higher stability of anthocyanins is linked to lower pH values.6 Differences of pH recorded for 50% M + 50% W, 25% M + 75% W, and 100% W (between 2.92 and 2.76, Table 1) were not so dramatic as the differences observed for anthocyanin shelf life in these same juices (Table 3). However, the stability of anthocyanins could be severely compromised at this restricted pH range

elevated sweetness/sourness balance), although it is prone to suffer browning during processing and storage.14 Both varietal juices might serve as complementary ones because the addition of ‘Wonderful’ to ‘Mollar de Elche’ juices could ameliorate the visual appearance of browning10 as well as ‘Mollar de Elche’ addition to ‘Wonderful’ juices could improve their sensory properties by smoothing their high acidity. Moreover, a point worth mentioning is that pomegranate juices mixed at different percentages of each variety presented intermediate values of pH (Table 1). This fact turned these mixtures into valuable samples to study the degradation of anthocyanins at different pH conditions within a similar complex matrix, avoiding the oversimplification of common model systems.23,24 Evolution of Anthocyanins in Blended Varietal Pomegranate Juices. The anthocyanin profile of pomegranate juices was characterized by delphinidin, cyanidin, and pelargonidin 3-glucosides and 3,5-diglucosides (Table 2; Table 2. Identification of Anthocyanins in Pomegranate Juice by HPLC-ESI-MSn (Positive Mode) ID 1 2 3 4 5 6

compound delphinidin 3,5diglucoside cyanidin 3,5-diglucoside pelargonidin 3,5diglucoside delphinidin 3-glucoside cyanidin 3-glucoside pelargonidin 3-glucoside

RT (min)

[M]+ (m/z)

MS2 ion fragments (m/z)a

MS3 ion fragments (m/z)

25.1

627

465,b 303

303

26.6 28.1

611 595

449, 287 433, 271

287 271

29.5 31.2 32.7

465 449 433

303 287 271

a

Fragment ions are listed in order of relative abundances. bMS2 ions in bold were those subjected to MS3 fragmentation.

Figure 1. Chemical structures of the anthocyanins found in pomegranate juices. Glc, glucose.

Figures 1 and 2), in accordance with previous papers.2,25 The amounts of these colored flavonoids were largely affected by the cultivar group, and total anthocyanin concentrations ranged from 23 to 171 mg/100 mL for 100% M and 100% W, respectively, in good agreement with those previously reported by Mena et al.14 Varietal blends exhibited intermediate values according the proportion of varietal juices used, as expected. C



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Figure 2. Chromatographic profile at 520 nm of anthocyanins registered in pomegranate juices. Peaks: (1) delphinidin 3,5-diglucoside; (2) cyanidin 3,5-diglucoside; (3) pelargonidin 3,5-diglucoside; (4) delphinidin 3-glucoside; (5) cyanidin 3-glucoside; (6) pelargonidin 3-glucoside.

Figure 3. Total anthocyanin content of pomegranate juices and their first-order degradation kinetics under different storage temperatures: (A) 5 °C; (B) 25 °C. Error bars are presented as standard error of the mean (SEM). For corresponding kinetic parameters cf. Table 3.

Table 3. Kinetic Parameters of Anthocyanin Degradation in Pomegranate Juices during Storage at Different Temperatures storage temp (°C)

juice

5

100% M 75% M + 25% W 50% M + 50% W 25% M + 75% W 100%

y y y y y

= = = = =

25

100% M 75% M + 25% W 50% M + 50% W 25% M + 75% W 100% W

y y y y y

= = = = =

R2

−k × 103 (day−1)

t1/2 (days)

D value (days)

23.8 exp(−0.00707x) 56.7 exp(−0.00588x) 89.4 exp(−0.00384x) 128.4 exp(−0.00186x) 173.4 exp(−0.00190x)

0.984 0.931 0.954 0.907 0.922

7.1 5.9 3.8 1.9 1.9

98.1 117.8 180.3 373.6 365.6

141.5 169.9 260.1 539.0 527.5


0.988 0.988 0.981 0.983 0.986

24.8 16.9 11.5 9.5 8.0

28.0 41.0 60.4 72.6 86.8

40.4 59.2 87.2 104.8 125.2

variation kinetics (concentration in mg/100 mL)

Concerning structure stability, it could be pointed out that diglucosides were more stable than monoglucosides in those samples stored at 5 °C (5 and 24% of loss, respectively), as it is usually described attending to anthocyanin stability.6,9,11,13,18 However, juices stored at 25 °C showed a different trend, and monoglucosides were revealed as the most stable anthocyanins (48% of loss for monoglucosides and 56% for diglucosides). On the whole, decreases in the anthocyanin amounts could be explained attending to several processes taking part during storage: (1) polymerization reactions involving the condensa-

considering how pH affects the equilibrium of anthocyanin structures (quinoidal base/flavylium cation) and pigment stability.6 On the basis of these results, both pigment concentration and pH should be regarded as factors affecting anthocyanin stability and contributing to the cultivar effect. Finally, the superior content in citric acid of ‘Wonderful’ juice compared to ‘Mollar de Elche’14 may also contribute to the definition of the cultivar effect observed on anthocyanin stability. D



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Figure 4. Vitamin C content of pomegranate juices and their first-order degradation kinetics under different storage temperatures: (A) 5 °C; (B) 25 °C. Error bars are presented as SEM. For corresponding kinetic parameters cf. Table 4.

Table 4. Kinetic Parameters of Vitamin C Degradation in Pomegranate Juices during Storage at Different Temperatures storage temp (°C)

juice

5

100% M 75% M + 25% W 50% M + 50% W 25% M + 75% W 100% W

y y y y y

= = = = =

25

100% M 75% M + 25% W 50% M + 50% W 25% M + 75% W 100% W

y y y y y

= = = = =

R2

−k × 103 (day−1)

t1/2 (day)

D value (day)


0.957 0.991 0.951 0.963 0.952

72.5 72.2 56.1 47.2 95.3

9.6 9.6 12.4 14.7 7.3

13.8 13.9 17.8 21.2 10.5

12.1 exp(−0.26761x) 11.8exp(−0.27190x) 9.5 exp(−0.25262x) 7.4 exp(−0.23705x) 7.5 exp(−0.28721x)

0.999 0.999 0.999 0.990 0.999

267.6 271.9 252.6 237.1 287.2

2.6 2.5 2.7 2.9 2.4

3.7 3.7 4.0 4.2 3.5

variation kinetics (concentration in mg/100 mL)

the presence of anthocyanins.12 However, vitamin C has been preserved in juices combining lemon and pomegranate juices at different rates, where the influence of pH seemed to be negligible and, hence, this hypothesis should be ruled out.18 On the other hand, whereas anthocyanins seem to play an essential role in vitamin C degradation,7 decreases in vitamin C content were observed among all samples regardless of the kind of juice (Figure 4) and, thus, regardless of the anthocyanin content in the sample. Consequently, it is possible that endogenous vitamin C of pomegranate juices decreases without attending any contribution of external factors and due exclusively to its natural oxidative deterioration. Color Parameters and the Role of Anthocyanins in Color Changes. Juice color varied significantly among varietal juices through storage (Figure 5). Lightness of pomegranate juices, represented as CIEL* values, was in accordance with a previous study.14 One hundred percent M showed the highest lightness, whereas 100% W displayed the lowest one. Significant differences were noted among varietal juices by the end of the storage period (p < 0.001) (Figure 5A,B). Whereas lightness of blended juices (75% M + 25% W, 50% M + 50% W, and 25% M + 75% W) and 100% W increased only slightly from day 42, a reduction was registered for 100% M for the first 42 days, prior to the final augment recorded (Figure 5A,B). General increases of lightness were in agreement with the trend followed by other unpasteurized pomegranate juices.6,9,11,13,18

tion of anthocyanins with byproducts from the degradation of monosaccharides and organic acids, yielding furfurals and aldehyde compounds; (2) the occurrence of enzymes degrading anthocyanins; and (3) direct oxidation of anthocyanin with oxygen.6,28,29 Vitamin C Degradation and Its Relationship with Anthocyanins. Although pomegranate juice is not distinguished by possessing high vitamin C concentrations, it may play an important role in browning processes and anthocyanin degradation.12 Vitamin C was detected only in its oxidized form (dehydroascorbic acid). Its initial concentrations were 12.0 and 7.8 mg/100 mL for 100% M and 100% W, respectively. Vitamin C decreased following a first-order reaction kinetic at both selected temperatures through storage (Figure 4; Table 4). The rate constant (k) increased at higher storage temperature, the rate constant at 25 °C being up to 4-fold the rate constant recorded for the samples stored at 5 °C. This fact had a remarkable effect on the shelf life of vitamin C. Actually, whereas the half-life value (t1/2) of juices stored at 5 °C ranged from 7 until 15 days, samples stored at 25 °C solely exhibited half-life values of 2.5 days. D values varied between 10 and 21 days for storage at 5 °C and were about 4 days at 25 °C. The degradation of vitamin C was not related to a certain cultivar pattern (Figure 4; Table 4). The fast reduction in vitamin C content of pomegranate juices with added ascorbic acid has been attributed to pH and E



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Figure 5. Evolution of color parameters in pure and blended varietal pomegranate juices through storage: CIEL* in samples stored at (A) 5 °C and (B) 25 °C; chroma at (C) 5 °C and (D) 25 °C (D); hue angle at (E) 5 °C and (F) 25 °C. Error bars are presented as SEM.

Initial chroma values of blended juices were not the sum of their parts, and a different trend occurred among juices attending to their composition (Figure 5C,D). One hundred percent W and blended juices decreased and, then, increased during storage. On the contrary, 100% M was augmented and then decreased. These results stressed the significance of cultivar effect on the evolution of color intensity just as its influence on mixtures of different varietal juices. Similarly, a notable varietal effect was observed for hue angle (Figure 5E,F), especially in those samples stored at room temperature. Hue values of 100% W juice were preserved during storage at both

temperatures, and they increased only slightly in blended juices. On the other hand, hue of 100% M juice was significantly increased during the first days, especially at 25 °C (Figure 5F), and then remained constant for the rest of the experiment. These color changes were consistent with the general increase of brownish hues reported for pomegranate juices.9,11,12,18 Changes oin CIEL* and chroma values could be attributed to anthocyanins as a strong significant correlation was found among them (r = −0.86, r = −0.89, respectively; p < 0.01). However, anthocyanin degradation cannot fully account for the variations in hue angle values (r = −0.66; p < 0.01), so other F



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granatum L.) peel, mesocarp, aril and differently produced juices by HPLC-DAD-ESI/MSn. Food Chem. 2011, 127, 807−821. (4) Maskan, M. Production of pomegranate (Punica granatum L.) juice concentrate by various heating methods: colour degradation and kinetics. J. Food Eng. 2006, 72, 218−224. (5) Timberlake, C. F. Anthocyanins − occurrence, extraction and chemistry. Food Chem. 1980, 5, 69−80. (6) Castañeda-Ovando, A.; Pacheco-Hernández, M. d. L.; PáezHernández, M. E.; Rodríguez, J. A.; Galán-Vidal, C. A. Chemical studies of anthocyanins: a review. Food Chem. 2009, 113, 859−871. (7) Jurd, L. Some advances in the chemistry of anthocyanin-type plant pigments. Chem. Plant Pigments 1972, 123−142. (8) Alighourchi, H.; Barzegar, M. Some physicochemical characteristics and degradation kinetic of anthocyanin of reconstituted pomegranate juice during storage. J. Food Eng. 2009, 90, 179−185. (9) Fischer, U. A.; Dettmann, J. S.; Carle, R.; Kammerer, D. R. Impact of processing and storage on the phenolic profiles and contents of pomegranate (Punica granatum L.) juices. Eur. Food Res. Technol. 2011, 233, 797−816. (10) Mena, P.; Martí, N.; Saura, D.; Valero, M.; García-Viguera, C. Combinatory effect of thermal treatment and blending on the quality of pomegranate juices. Food Bioprocess Technol. 2013, 6, 3186−3199. (11) Pérez-Vicente, A.; Serrano, P.; Abellán, P.; García-Viguera, C. Influence of packaging material on pomegranate juice colour and bioactive compounds, during storage. J. Sci. Food Agric. 2004, 84, 639− 644. (12) Martí, N.; Pérez-Vicente, A.; García-Viguera, C. Influence of storage temperature and ascorbic acid addition on pomegranate juice. J. Sci. Food Agric. 2002, 82, 217−221. (13) Alighourchi, H.; Barzegar, M.; Abbasi, S. Anthocyanins characterization of 15 Iranian pomegranate (Punica granatum L.) varieties and their variation after cold storage and pasteurization. Eur. Food Res. Technol. 2008, 227, 881−887. (14) Mena, P.; García-Viguera, C.; Navarro-Rico, J.; Moreno, D. A.; Bartual, J.; Saura, D.; Martí, N. Phytochemical characterisation for industrial use of pomegranate (Punica granatum L.) cultivars grown in Spain. J. Sci. Food Agric. 2011, 91, 1893−1906. (15) Mena, P.; Vegara, S.; Martí, N.; García-Viguera, C.; Saura, D.; Valero, M. Changes on indigenous microbiota, colour, bioactive compounds and antioxidant activity of pasteurised pomegranate juice. Food Chem. 2013, 141, 2122−2129. (16) Vegara, S.; Martí, N.; Mena, P.; Saura, D.; Valero, M. Effect of pasteurization process and storage on color and shelf-life of pomegranate juices. LWT−Food Sci. Technol. 2013, 54, 592−596. (17) Vegara, S.; Mena, P.; Martí, N.; Saura, D.; Valero, M. Approaches to understanding the contribution of anthocyanins to the antioxidant capacity of pasteurized pomegranate juices. Food Chem. 2013, 141, 1630−1636. (18) González-Molina, E.; Moreno, D. A.; García-Viguera, C. A new drink rich in healthy bioactives combining lemon and pomegranate juices. Food Chem. 2009, 115, 1364−1372. (19) Gironés-Vilaplana, A.; Valentão, P.; Moreno, D. A.; Ferreres, F.; García-Viguera, C.; Andrade, P. B. New beverages of lemon juice enriched with the exotic berries maqui, açai,́ and blackthorn: bioactive components and in vitro biological properties. J. Agric. Food Chem. 2012, 60, 6571−6580. (20) González-Molina, E.; Moreno, D. A.; García-Viguera, C. Genotype and harvest time influence the phytochemical quality of fino lemon juice (Citrus limon (L.) Burm. F.) for industrial use. J. Agric. Food Chem. 2008, 56, 1669−1675. (21) Garzón, G. A.; Wrolstad, R. E. Comparison of the stability of pelargonidin-based anthocyanins in strawberry juice and concentrate. J. Food Sci. 2002, 67, 1288−1299. (22) Kirca, A.; Ö zkan, M.; Cemeroǧlu, B. Storage stability of strawberry jam color enhanced with black carrot juice concentrate. J. Food Process. Preserv. 2007, 31, 531−545. (23) Hernández-Herrero, J. A.; Frutos, M. J. Degradation kinetics of pigment, colour and stability of the antioxidant capacity in juice model

compounds such as those formed as a consequence of browning processes (e.g., ascorbic acid degradation) or anthocyanin polymerization may contribute to hue variations.8,12 Color parameters as well as anthocyanin load in blended varietal pomegranate juices was mainly influenced by ‘Wonderful’ cultivar and not by ‘Mollar de Elche’. In fact, despite the significant color variations in 100% M, a high stability of color parameters was observed in the blended juices, even for that juice containing merely 25% of ‘Wonderful’ juice (75% M + 25%W). This blend may also exhibit a great organoleptic potential as it preserves the genuine sensorial characteristics of the cultivar ‘Mollar de Elche’ (data not shown).14 Consequently, blending different pomegranate juices could be regarded as an easy way to preserve juice color characteristics and pigment concentration of those varieties prone to color alterations such as ‘Mollar de Elche’. These protective effects on the anthocyanin composition and color attributes of some of these blends could remain after realistic pasteurization treatments.10 Therefore, the results herein presented may have direct implications for the pomegranate juice industry to solve browning problems during juice storage. Blending varietal pomegranate juices was useful not only to preserve the genuine characteristics of fresh juices but also to study different factors affecting anthocyanin stability while maintaining to the utmost the matrix studied. The role of anthocyanin concentration, pH, and the presence of ascorbic acid in pigment integrity was discussed, and it could contribute to the definition of cultivar effect in pomegranate juices. Finally, the present data could help to tackle anthocyanin degradation in other food matrices.

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AUTHOR INFORMATION

Corresponding Author

*(C.G.-V.) CEBAS-CSIC Phytochemistry Lab, Research Group on Quality, Safety and Bioactivity of Plant Foods Food Science and Technology Department, P.O. Box 164, E-30100 Espinardo, Murcia, Spain. Phone: +34 968 396304. Fax: +34 968 396213. E-mail: [email protected]. Funding

We thank Fundación Agroalimed for financial support of this work (Project “Producción de Zumo de Caqui e Industrialización de Granada”). We are grateful to the ConsoliderIngenio 2010 Fun-C-Food project (CSD2007-00063) and “Grupo de Excelencia de la Región de Murcia” (04486/ GERM/06). P.M. was funded by a grant of the FPU Fellowship Program from the Spanish Ministry of Education. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Raúl Domı ́nguez-Perles for technical support with the graphic design.

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REFERENCES

(1) Mena, P.; Gironés-Vilaplana, A.; Moreno, D. A.; García-Viguera, C. Pomegranate fruit for health promotion: myths and realities Funct. Plant Sci. Biotechnol. 2011, 5, 33−42. (2) Gil, M. I.; García-Viguera, C.; Artés, F.; Tomás-Barberán, F. A. Changes in pomegranate juice pigmentation during ripening. J. Sci. Food Agric. 1995, 68, 77−81. (3) Fischer, U. A.; Carle, R.; Kammerer, D. R. Identification and quantification of phenolic compounds from pomegranate (Punica G



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systems from six anthocyanin sources. Int. J. Food Sci. Technol. 2011, 46, 2550−2557. (24) Sadilova, E.; Stintzing, F. C.; Kammerer, D. R.; Carle, R. Matrix dependent impact of sugar and ascorbic acid addition on color and anthocyanin stability of black carrot, elderberry and strawberry single strength and from concentrate juices upon thermal treatment. Food Res. Int. 2009, 42, 1023−1033. (25) Mena, P.; Calani, L.; Dall’Asta, C.; Galaverna, G.; GarcíaViguera, C.; Bruni, R.; Crozier, A.; Del Rio, D. Rapid and comprehensive evaluation of (poly)phenolic compounds in pomegranate (Punica granatum L.) juice by UHPLC-MSn. Molecules 2012, 17, 14821−14840. (26) Boulton, R. The copigmentation of anthocyanins and its role in the color of red wine: a critical review. Am. J. Enol. Vitic. 2001, 52, 67− 87. (27) Sentandreu, E.; Navarro, J. L.; Sendra, J. M. LC-DAD-ESI/MSn determination of direct condensation flavanol-anthocyanin adducts in pressure extracted pomegranate (Punica granatum L.) juice. J. Agric. Food Chem. 2010, 58, 10560−10567. (28) Ochoa, M. R.; Kesseler, A. G.; Vullioud, M. B.; Lozano, J. E. Physical and chemical characteristics of raspberry pulp: storage effect on composition and color. LWT−Food Sci. Technol. 1999, 32, 149− 153. (29) Eiro, M. J.; Heinonen, M. Anthocyanin color behavior and stability during storage: effect of intermolecular copigmentation. J. Agric. Food Chem. 2002, 50, 7461−7466.

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