Food Chemistry 129 (2011) 982990

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Food Chemistry
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Changes on phenolic and carotenoid composition of high intensity pulsed electric eld and thermally treated fruit juicesoymilk beverages during refrigerated storage
M. Morales-de la Pea, L. Salvia-Trujillo, M.A. Rojas-Gra, O. Martn-Belloso
Department of Food Technology, University of Lleida, Rovira Roure 191, 25198 Lleida, Spain

a r t i c l e

i n f o

a b s t r a c t
The effects of high intensity pulsed electric elds (HIPEF) (35 kV/cm with 4 ls bipolar pulses at 200 Hz for 800 or 1400 ls) or thermal (90 C, 60 s) treatments over phenolic and carotenoid compounds of a fruit juicesoymilk (FJSM) beverage stored at 4 C were evaluated and compared, having the untreated beverage as a reference. Coumaric acid, narirutin and hesperidin were the most abundant phenolic compounds in the FJSM beverage, while the main carotenoids were lutein, zeaxanthin and b-carotene. Immediately after HIPEF or heat processing, hesperidin content of the beverage showed a huge rise, resulting in a signicant increase on the total phenolic concentration. Regarding carotenoid concentration, HIPEF or thermal treatment lead to a signicant decrease; lutein, zeaxanthin and b-cryptoxanthin being the most affected compounds. In contrast, the content of some individual phenolics and carotenoids increased with time, while others tended to decrease or remained with no signicant changes with regards to their initial values. Total phenolic concentration seemed to be highly stable during storage; while, total carotenoid content gradually diminished, irrespectively of the treatment applied. Overall, the changes observed in HIPEF treated FJSM beverage were less than those in the heat processed one. Hence, HIPEF is a feasible technology to obtain FJSM beverages with extended shelf-life and a similar prole of antioxidant compounds to freshly made beverages. 2011 Elsevier Ltd. All rights reserved.

Article history: Received 21 January 2011 Received in revised form 28 March 2011 Accepted 11 May 2011 Available online 15 May 2011 Keywords: High intensity pulsed electric elds Fruit juicesoymilk beverages Phenolic compounds Carotenoid compounds

1. Introduction Phenolics and carotenoids are abundant compounds distributed in fruits and vegetables. They contribute signicantly to consumers health as well as the quality of foods (Klimczak, Maecka, Szlachta, & Gliszczynska-Swigo, 2007). On the one hand, phenols are multifunctional antioxidants and can act as oxidative enzymes inhibitors, metal chelators or free radical scavengers (Boskou, Gerothanassis, & Kefalas, 2006). They are also important because of their contribution to the sensory attributes of fruits (Macheix, Fleureliet, & Billot, 1990). On the other hand, carotenoids are considered potent antioxidants, due to their provitamin A activity and free radical scavengers (Bitton & Hornero-Mndez, 2001). In addition, they modulate the pathogenesis of cancers (Giovannucci, 1999) and coronary heart diseases (Kritchevsky, 1999). Hence, the daily consumption of fruits and vegetables rich in this kind of compounds is highly recommended (Willet, 1994). During the last decades, in order to meet consumers claims, the food industry has been developing an immense quantity of fruit
Corresponding author. Tel.: +34 973 702593; fax: +34 973 702596.
E-mail addresses: omartin@tecal.udl.cat, omartin@tecal.udl.es Belloso). (O. Martn-

products such as juices, drinks and mixed beverages. Among them, mixed beverages containing fruit juices with milk or soymilk have received considerable attention and their consumption in Europe has increased by 30% in the last 10 years (Zulueta, Esteve, & Frgola, 2007). In recent studies, Morales-de la Pea, Salvia-Trujillo, RojasGra, and Martn-Belloso (2010a), Morales-de la Pea, Salvia-Trujillo, Rojas-Gra, and Martn-Belloso (2010b) demonstrated that a FJSM beverage was a good source of vitamin C and isoavones, while having, at the same time, high antioxidant capacity. In addition, it could be considered that, being a mix of different fruit juices (orange, pineapple and kiwi) and soymilk, the beverage contains considerable amounts of phenolic and carotenoid compounds. Usually, this kind of products is preserved by thermal treatments, which are known to effectively inactivate microorganism and deleterious enzymes. However, the high temperature achieved during the processing adversely affects their organoleptical and nutritional properties (Wolbang, Fitos, & Treeby, 2008). Hence, in the last few decades, alternative technologies, for preserving foods, such as HIPEF, have been under continuous investigation by numerous research groups worldwide. Currently, the information available in open literature has demonstrated that the microbial and enzymatic inactivation levels achieved by HIPEF can be as high as those reached by heat pasteurization (Aguil-Aguayo,

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.05.058

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Soliva-Fortuny, & Martn-Belloso, 2010; Mosqueda-Melgar, Raybadui-Massilia, & Martn-Belloso, 2008). Moreover, it has been reported that HIPEF processing leads to high retention or enhancement of the concentration of some bioactive compounds in fruit and vegetable juices (Corts, Torregrosa, Esteve, & Frgola, 2006; Odriozola-Serrano, Soliva-Fortuny, Hernndez-Jover, & Martn-Belloso, 2009; Snchez-Moreno, Plaza, De Ancos, & Cano, 2003). Unfortunately, at present there is no available information focused on the phenolic and carotenoid composition of FJSM beverages and how HIPEF or thermal process affects them. Therefore, the main objectives of this study were: (i) identify the phenolic and carotenoid composition of a FJSM beverage and (ii) evaluate and compare the effects of HIPEF or thermal treatments over these compounds in the beverage during the storage at 4 C. 2. Materials and methods 2.1. FJSM beverage elaboration A blend of fruit juices was elaborated in the following proportions: orange (250 ml/l), kiwi (180 ml/l) and pineapple (70 ml/l) according to Morales-de la Pea et al. (2010a). Fruits were purchased at commercial maturity in a local supermarket. Each fruit was washed and disinfected before the juice extraction. They were ground and then mixed with commercial soymilk (YoSoy, Lleida, Spain) (425 ml/l), sugar (75 g/l) and citric acid up to pH 3.7. Finally, the beverage was ltered with cheese cloth by means of a vacuum pump to obtain a homogeneous liquid without any solid particles. 2.2. HIPEF equipment A continuous-ow bench scale system (OSU-4F, Ohio State University, Columbus, OH, USA), that held bipolar squared wave pulses was used to treat the FJSM beverage. The beverage was pumped through a system of eight collinear chambers serially connected. Each chamber had a treatment volume of 0.012 cm3 and a gap distance of 0.29 cm. The ow rate was adjusted to 60 ml/min and controlled by a variable speed pump (model 752210-25, Cole Palmer Instrument Company, Vermon Hills, IL, USA). According to previous studies, the beverage was subjected to a HIPEF treatment of bipolar square-wave pulses of 4 ls, at 35 kV/cm eld strength and a frequency of 200 Hz for two different treatment times: 800 or 1400 ls (Morales-de la Pea et al., 2010a). The energy density supplied to the product during HIPEF process for 800 and 1400 s was 4410 and 7717 kJ/l, respectively, and temperature was always kept below 32 C. 2.3. Thermal treatment The FJSM beverage was thermally processed (90 C, 60 s) in a tubular heat exchanger with 2.16 mm inner diameter and 1.10 m length (University of Lleida, Spain). A gear pump was used to maintain the beverage ow rate of 40.5 ml/min. After heating, the beverage was immediately cooled down to 5 1 C by a cooled-water bath. 2.4. Packaging and storage HIPEF and thermal uid handling system were disinfected rst with 4% NaOH and then with 10% chlorine and 20% ethanol solutions prior to processing. Polypropylene sterile bottles of 100 ml were used to store the untreated and treated FJSM beverages. Once lled, the receptacle was tightly closed and stored at 4 C in darkness. Considering shelf-like criteria the beverages were stored during 14 (untreated), 35 (HIPEF-800 s) and 56 (1400 s or heat

treated) days (Morales-de la Pea et al., 2010a). The samples were taken for analysis every 3 days during the rst 14 days, and from that period on, they were taken once per week. 2.5. Phenolic compounds Phenolic compounds were extracted and quantied by HPLC, following a procedure validated by Hertog, Hollman, and Venema (1992). 2.5.1. Extraction and hydrolysis Twenty milliliters of 62.5% aqueous methanol with 2 g/l of tert-butylhydroquinone and 5 ml of hydrochloric acid 6 M were carefully mixed with 0.5 g of freeze-dried FJSM beverage. After reuxing at 90 C for 2 h with regular swirling, the extract was cooled and subsequently made up to 50 ml with methanol and sonicated for 5 min. The extract was then passed through a 0.45 lm lter prior to injection. 2.5.2. Chromatography conditions An aliquot of 20 ll of the extracted samples was injected into the HPLC system, which was equipped with a 600 Controller, a 486 Absorbance Detector programmed to scan from 200 to 350 nm, a thermostatic column compartment, and a 717 Plus Auto Sampler with cooling system (Waters, Milford, MA). Phenolic compounds were separated using a reverse-phase C18 Spherisorb ODS2 (5 lm) stainless steel column (4.6 mm 250 mm) at room temperature with a ow rate of 1 ml/min. A gradient elution was employed with a solvent mixture of 2.5% HCOOH in water (solvent A) and 2.5% HCOOH in methanol (solvent B) as follows: linear gradient from 5% to 13% B, 015 min; linear gradient from 13% to 15% B, 1520 min; linear gradient from 15% to 30% B, 2832 min; isocratic elution 45% B, 3235 min; linear gradient 4590% B, 35 40 min; isocratic elution 90% B, 4045 min; linear gradient to reach the initial conditions after 5 min; post-time 10 min before the next injection. Individual phenols were identied by comparison of their UVvis spectral data and retention times with those of the reference standards (chlorogenic, caffeic, p-coumaric, ferulic and sinapic acids; hesperidin, rutin, narirutin, quercetin and apigenin). Quantication of phenolic compounds was carried out by integration of the peak areas. Data were compared to calibration curves of each phenolic compound and results were expressed as mg of phenolic compound in 100 ml of FJSM beverage. 2.6. Carotenoid compounds Carotenoids were extracted and quantied by HPLC, following a procedure validated by Corts, Esteve, Frgola, and Torregrosa (2004), with some modications. 2.6.1. Extraction and saponication Thirty milligrams of FJSM beverage were mixed with 50 mg magnesium hydroxide carbonate, 25 mg BHT and 35 ml ethanol:hexane solution (4:3 v/v) in an amber round-bottom ask under N2 atmosphere. After 45 min of continuous agitation, the sample was ltered in a low-pressure. The residue was washed with 35 ml of ethanolhexane solution (4:3 v/v) and ltered twice with 12.5 ml of ethanol and once with 12.5 ml of hexane (until it was colorless). All of the liquid ltrates were combined and washed twice with 50 ml of sodium chloride solution (10%) and then three times with 50 ml of water in an amber decanting funnel. The organic phase was evaporated at 40 C in a rotary evaporator. The residue obtained was saponied under N2 atmosphere (30 min) adding 10 ml of diethyl ether, 10 ml of methanolic KOH 0.5 M with 0.1% BHT (w/v). At the end of this time, 20 ml of diethyl ether were added. The solution obtained was washed again twice with 50 ml

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of sodium chloride solution (10%) and then three times with 50 ml of water until neutral pH was obtained. In order to ensure that the water was totally eliminated, 10 ml of absolute ethanol was added to the samples. The solution was evaporated at 45 C until dryness. The residue obtained was dissolved with 4 ml of diethyl ether and placed in an amber glass vial. The solvent was evaporated under N2 and the residue was stored at 18 C until it was time for the chromatographic analysis. Before injection into the HPLC system, the carotenoid extract was reconstituted with 1 ml of methanol:tert-butyl methyl ether solution (70:30, v/v). 2.6.2. Chromatography conditions An aliquot of 20 ll of the extracted samples were injected into the same HPLC system used for phenolic compounds determination. Carotenoids were separated using a reverse-phase C18 Spherisorb ODS2 (5 lm) stainless steel column (4.6 mm 250 mm). Gradient elution was needed for complete separation of the analytes (Table 1). The mobile phase consisted of four eluents: (A) methanol/ammonium acetate 0.1 M, (B) milli-Q water, (C) methyl tert-butyl ether and (D) methanol. The ow rate was xed at 1 ml/min and the total run time was 60 min. The column was set at 30 C, while sample vials (amber) on the auto sampler were preserved at 4 C. The carotenoids were identied by UVvis spectral data and their retention times (Corts et al., 2004; Mouly, Gaydou & Corsetti, 1999). Quantication of carotenoids was carried out by integration of the peak areas. Data were compared to calibration curves of each carotenoid compound and results were expressed as mg of carotenoid compound in 100 ml of FJSM beverage. 2.7. Statistical analysis Treatments were conducted in duplicate and two replicate analyses were carried out for each sample. Analysis of the variance (ANOVA) was performed to compare treatments. Least signicant difference (LSD) test was employed to determine differences between means immediately after processing and throughout storage. The condence interval was set at 0.95 for analysis and procedures. Results were analyzed using the Statgraphics Plus v.5.1 Windows package (Statistical Graphics Co., Rockville, Md). 3. Results and discussion 3.1. Phenolic and carotenoid prole The initial phenolic proles of the untreated and treated FJSM beverages are shown in Tables 2 and 3. Irrespective of the treat-

Table 1 Mobile phase gradient for determination of carotenoids by HPLC. Time (min) 0 10 12 17 22 29 30 40 45 50 55 60 Flow (ml/min) 1 1 1 1 1 1 1 1 1 1 1 1 A (%) 95 100 95 86 75 95 100 100 0 0 95 95 B (%) 5 0 0 0 0 0 0 0 0 0 5 5 C (%) 0 0 5 14 25 5 0 0 0 0 0 0 D (%) 0 0 0 0 0 0 0 0 100 100 0 0

A: methanol/ammonium acetate 0.1 M. B: milli-Q water. C: methyl tert-butyl ether (100%). D: methanol (100%).

ment applied, coumaric was the most abundant phenolic acid (3246%), followed by sinapic (35%), and, in lower concentrations, caffeic (1.42%), ferulic (0.821.7%) and chlorogenic (0.61.2%) acids (Table 2). On the other hand, narirutin was identied as one of the main avonoids present in the beverages before treatment, contributing to 19.527.5% of total avonoids (Table 3). Generally, phenolic content in fruits and vegetables is highly variable and is strongly inuenced by the maturity stage, growing areas, variety, lengths and condition of storage and processing techniques. To the best of the authors knowledge, information related to phenolic composition of FJSM beverages does not exist in open literature at present. Nonetheless, other studies have been carried out in orange juice or soymilk, separately. According to Kelebek, Canbas, and Selli (2008) and Klimczak et al. (2007) orange juice contains the ve hydroxycinnamic acids identied in the FJSM beverage; nevertheless, different to our ndings, ferulic acid predominated as the most abundant compound. In addition, hesperidin and narirutin are reported as the avonoids present in the highest amounts in orange juices (Gil-Izquierdo, Ferreres, & Toms-Barbern, 2001; Kelebek et al., 2008; Snchez-Moreno et al., 2003). Otherwise, soy-based products are good sources of p-hydroxybenzoic, coumaric, chlorogenic, caffeic, ferulic and coumaric acids, quercetin and hesperidin (Correa et al., 2010; Toda, Takahashi, Iwashina, & Hajika, 2010). Hence, the high quantities of coumaric acid, narirutin and hesperidin observed in the FJSM beverage may come from orange juice and soymilk, which were the major ingredients in the formulation. Table 4 shows the carotenoid compounds identied in the freshly prepared FJSM beverage, as well as in those that were HIPEF (800 and 1400 ls) and thermally treated. It was observed that, regardless of the treatment applied, lutein, zeaxanthin and b-carotene were present in higher concentrations reaching values between 0.066 to 0.108 mg/100 ml, 0.056 to 0.076 mg/100 ml and 0.012 to 0.021 mg/100 ml, respectively. It is well known that carotenoid distribution in citrus is extremely complex, with the largest number of carotenoids founded in any fruit (Melndez-Martnez, Vicario, & Heredia, 2003). Fruit juices, drinks and mixed beverages have widely different carotenoid content, which mainly depend on the ingredients of the samples under study (Mendiola et al., 2008; Zulueta et al., 2007). b-Carotene normally constitutes between 25% and 30% of total carotenoids in fruits (Bitton & Hornero-Mndez, 2001); however, unripe fruits usually show high contents of lutein (Gross, 1987). Corts et al. (2004) reported that 9-cisviolaxanthin + neoxanthin, anteraxanthin, b-carotene, lutein, -carotene, a-cryptoxanthin and b-cryptoxanthin were the major carotenoids in orange juices. Additionally, a signicant lutein concentration has been reported in different soy-based products (Correa et al., 2010; Toda et al., 2010). In this way, the higher amount of lutein quantied in the FJSM beverage, with regards to b-carotene, might be mostly inuenced by a high lutein content in the soymilk and the maturity stage of the oranges used to elaborate the FJSM beverage. Therefore, considering that orange juice (25%) and soymilk (42.5%) were the main ingredients of the FJSM beverage, they may have a signicant inuence on the phenolic and carotenoid composition of the samples. Hence, it can be established that phenolic and carotenoid proles of mixed beverages depend on the major ingredients contained on their formulation, as well as the variety and maturity stage of the fruits that are used to elaborate the beverages. Likewise, the individual phenolic or carotenoid compounds may interact when the different ingredients were mixed to formulate the FJSM beverage, leading to biochemical reactions that enhance or reduce the concentration of each compound in the nal product.

M. Morales-de la Pea et al. / Food Chemistry 129 (2011) 982990 Table 2 Effects of high intensity pulsed electric elds and heat pasteurization on phenolic acid content (mg/100 ml) of a fruit juicesoymilk beverage throughout storage at 4 C. Day 0 Treatment Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-1400 Thermal HIPEF-1400 Thermal HIPEF-1400 Thermal Caffeic 4.21 0.27a 5.17 0.27b 5.12 0.16b 4.70 0.01c 4.59 0.28ab 4.30 0.71ab 4.33 0.10a 4.57 0.09b 4.58 0.01a 4.03 0.12b 4.78 0.08c 5.02 0.03d 4.47 0.67ab 4.33 0.10a 4.30 0.18a 4.83 0.03b 4.74 0.21a 4.64 0.22ab 4.52 0.28ab 4.34 0.08b 3.80 0.05a 4.45 0.12b 4.35 0.06b 3.87 0.11a 4.32 0.18b 4.49 0.12b 4.64 0.22a 4.40 0.14a 4.53 0.34a 4.56 0.15a 4.30 0.25a 4.23 0.20a 4.35 0.12a 4.78 0.17a 4.09 0.05b Chlorogenic 2.27 0.10a 1.92 0.25ab 2.23 0.26ab 1.83 0.19b 1.99 0.01a 1.81 0.07b 1.92 0.31ab 1.78 0.06b 2.20 0.28ab 2.16 0.01a 2.28 0.25ab 1.94 0.15b 2.38 0.34a 1.84 0.09b 1.84 0.43ab 2.19 0.08a 2.22 0.31a 2.08 0.07a 2.09 0.18a 2.05 0.15a 1.60 0.12a 1.74 0.06a 2.16 0.08b 1.60 0.02a 1.75 0.10b 1.83 0.21b 1.81 0.21a 2.07 0.13a 1.85 0.16a 1.53 0.02a 1.90 0.09b 1.68 0.03a 1.77 0.17a 1.66 0.08a 1.54 0.10a Coumaric 89.96 0.59a 97.22 0.49b 100.38 0.99b 87.54 0.72c 93.62 3.26a 89.29 13.56ab 104.10 2.55b 94.61 6.87ab 90.38 7.42a 98.82 6.40a 96.63 0.51a 95.61 3.62a 90.33 3.25a 96.69 0.24b 97.57 4.25bc 100.21 0.42c 99.53 7.28a 94.22 1.84a 88.08 5.98a 89.53 4.30a 91.05 2.63a 79.15 8.82b 89.41 0.34a 84.15 4.44ab 86.50 1.11a 82.80 0.29b 100.38 0.83a 90.36 0.74b 87.55 5.73b 70.92 0.07a 75.93 1.84b 78.31 10.09a 87.63 7.17a 90.69 2.26a 94.29 0.34b Ferulic 1.99 0.04a 2.24 0.11bd 3.89 0.03c 2.57 0.42d 2.26 0.08a 2.19 0.97ac 4.16 0.26b 3.37 0.58bc 2.13 0.51a 3.17 0.06b 4.68 0.18c 3.12 0.07b 2.92 0.29ab 3.17 0.07a 3.46 0.88ab 2.77 0.04b 2.36 0.36a 3.30 0.75ab 3.35 0.18b 2.64 0.14a 3.25 0.26a 3.26 0.15a 2.17 0.26b 3.18 0.12a 3.82 0.42b 3.03 0.38a 4.05 0.80a 3.66 0.11a 3.46 0.22a 3.37 0.77a 4.03 1.10a 3.07 0.57a 4.13 1.04a 4.27 0.15a 3.32 0.46b Sinapic 9.84 0.23a 9.72 0.42ab 9.87 0.28a 8.56 0.94b 9.57 0.12a 8.97 1.61ab 9.98 0.21ab 9.70 0.70b 9.77 0.08a 10.47 0.24b 10.80 0.77b 9.15 0.13c 10.49 0.23a 10.15 0.07a 9.47 1.39a 11.00 0.79a 9.08 1.23a 8.88 1.04a 9.02 0.02a 8.77 0.76a 7.94 0.38ab 8.57 0.25a 8.07 0.18b 9.15 0.18a 7.68 0.01b 8.37 0.06c 9.55 0.13a 8.96 0.25b 7.79 0.26c 7.35 0.37a 7.58 0.07a 7.73 0.28a 8.82 0.45b 8.77 0.30a 9.20 0.01b TPA 108.27 0.01 116.27 0.24b 121.49 1.73c 105.20 1.56d

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112.03 2.94 106.57 16.21ab 124.49 2.30b 114.03 6.14 109.06 7.07 118.64 5.98ab 119.17 0.23b 114.85 3.43a 110.58 2.01a 116.21 0.19b 116.64 1.38b 121.00 0.28c 117.91 9.13a 113.26 1.51a 107.06 6.60a 107.33 5.43a 107.64 3.45a 97.17 0.23b 106.17 0.08a 101.95 4.63ab 104.07 0.99a 100.52 0.06b 120.43 1.31a 109.44 0.88b 105.18 6.28b 87.63 1.38a 93.74 2.85b 95.03 10.54a 106.71 6.53a 110.18 1.91a 112.44 0.87a

10

14

21

28

35

42 49 56

Control = Untreated FJSM beverage; HIPEF-800 = high intensity pulsed electric eld treatment at 35 kV/cm for 800 ls; bipolar 4 ls-pulses at 200 Hz; HIPEF-1400 = high intensity pulsed electric eld treatment at 35 kV/cm for 1400 ls; bipolar 4 ls-pulses at 200 Hz; thermal = thermal treatment (90 C, 60 s). TPA = total phenolic acids. A different letter in the same column corresponds to signicant differences amongst treatments (p < 0.05).

3.2. Effects of processing 3.2.1. Phenolic content The concentration of most of the individual phenolic compounds identied in the FJSM beverage was enhanced immediately after HIPEF (800 and 1400 s) or thermal treatments (Tables 2 and 3), except for chlorogenic, coumaric and sinapic acids, as well as rutin, which signicantly decreased in the heat treated beverage. Interestingly, hesperidin content showed a huge rise from 4% (untreated beverage) to 24% (treated beverages), irrespectively of the treatment applied (Table 3). As a result, the total phenolic concentration, obtained by the sum of the ve hydroxycinnamic acids and ve avonoids, was enhanced just after HIPEF or thermal treatments (Fig. 1 day 0). This increase was signicantly higher in HIPEF treated beverage than in that heat processed. At present, there are only a few works evaluating the effects of HIPEF or heat processes on the phenolic composition of individual fruit and vegetable juices, such as those from kiwi, orange and tomato (Dawes & Keene, 1999; Sentandreu, Navarro, & Sendra, 2007; Odriozola-Serrano et al., 2009). In general, results obtained within these works vary depending on the food matrix, the treatment applied and the process conditions. Sentandreu et al. (2007) reported that conventional pasteurization (90 C for 30 s) of orange juice has negligible effects on its phenolic content. Similarly, Odriozola-Ser-

rano et al. (2009) evaluated the variation in total phenolic compounds of tomato juice after HIPEF or thermal processes. Conversely to our ndings, these authors observed that there were no signicant differences in total phenolic content between untreated and HIPEF or thermally treated tomato juices. Nevertheless, our results are similar to those reported by Dawes and Keene (1999) who found that just after processing, an ultra pasteurized kiwi juice contained higher levels of phenolic acids in comparison to the fresh juice. Fruit antioxidants, such as phenolic compounds, are chemically diverse and found in different locations and forms in plant tissues and cells (Kalt, 2005). Individual phenolic concentration in fruit juices mainly depends on the technological treatment and storage conditions (Kelebek, Selli, Canbas, & Cabaroglu, 2009). The stress environment, such as UV radiation, air pollution and extreme temperatures, may provoke changes and reactions on the fruit phenolic content (Zobel, Lynch, & Jeffrey, 1997). Different reactions such as hydroxylation, methylation, isoprenylation, dimerization and/or glycosylation, which induce modications between the different phenolic compounds, can occur at various levels during processing (Rice-Evans, Miller, & Paganga, 1997). In addition, it is thought that the free phenolic acids are formed by partial degradation of the combined forms during extraction or processing of fruits (Fleuriet & Macheix, 1976) or by loosing the moieties between phenols and

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Table 3 Effects of high intensity pulsed electric elds and heat pasteurization on avonoid content (mg/100 ml) of a fruit juicesoymilk beverage throughout storage at 4 C. Day 0 Treatment Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-1400 Thermal HIPEF-1400 Thermal HIPEF-1400 Thermal Hesperidin 8.47 1.84a 61.07 1.26b 62.60 1.36b 58.45 0.14c 10.51 0.85a 61.40 0.21b 55.68 1.67c 44.42 16.22c 9.34 0.61 66.70 1.87b 65.50 0.64b 47.98 5.17c 11.31 4.39a 69.35 2.84b 58.41 3.70c 51.74 5.80c 11.61 1.57a 58.87 5.26b 50.19 2.34c 37.07 3.23d 54.00 4.72a 51.16 4.36a 43.45 1.15b 51.78 8.90a 54.40 1.38a 42.05 0.55b 51.14 10.20a 48.69 0.10a 47.30 9.49a 38.35 6.59a 46.16 4.67a 44.15 14.28a 39.44 2.62a 39.99 7.34a 37.92 0.01a Rutin 14.78 1.15a 14.99 0.03a 14.49 0.64a 8.42 1.48b 15.35 1.09a 12.38 0.45b 13.79 0.79a 10.18 0.51c 13.13 1.10a 12.74 0.68a 14.76 0.48b 10.08 0.04c 11.98 1.51a 11.17 2.05ac 16.87 0.58b 10.07 0.33c 12.85 3.65ab 12.83 1.31ab 14.83 0.96a 11.00 0.59b 14.34 1.18a 13.57 3.96a 14.93 0.19a 12.17 0.53a 14.16 0.53b 13.40 0.65b 14.42 0.18a 14.32 0.03a 13.63 0.21b 12.62 3.50a 10.18 0.60a 10.91 0.91a 10.37 0.92a 10.87 0.46a 9.55 1.66a Narirutin 40.91 5.17a 60.64 1.20b 54.37 0.09c 50.13 5.13c 56.63 3.08a 61.04 6.11ab 59.26 3.73a 66.90 1.95b 51.51 0.41a 53.32 0.71b 62.21 0.78c 64.21 0.96d 56.42 0.89a 54.43 1.30ab 53.13 0.47b 56.41 1.26a 52.53 1.08ab 52.33 0.22a 56.10 3.06b 61.86 1.50c 55.19 0.65a 57.29 0.66b 54.62 0.14a 61.99 3.57a 67.54 2.33b 60.46 4.21a 60.46 4.21a 50.15 0.90b 54.18 1.44c 58.64 4.96a 59.88 0.83a 57.40 1.25a 58.85 1.23a 62.40 0.65a 61.51 4.30a Quercetin 11.47 0.36a 14.10 0.18b 13.37 0.06c 13.37 0.21c 14.48 1.92a 13.31 1.19a 13.97 0.36a 14.15 0.41a 14.13 0.15a 15.33 0.77b 16.72 1.72b 13.81 0.24a 12.77 2.99a 14.23 0.46a 13.78 0.63a 13.73 0.13a 15.82 0.21a 14.40 0.91b 13.47 0.10c 14.17 0.05b 15.48 0.84a 14.57 2.06a 16.35 0.16a 14.10 0.27a 14.80 0.75ab 15.31 0.48b 13.70 1.04ab 13.63 0.10a 12.27 0.48b 15.70 0.18a 15.48 1.34a 15.93 2.13a 14.78 1.80a 15.56 0.34a 14.40 0.23b Apigenin 8.83 3.44ab 6.95 0.66ab 8.87 1.49a 6.07 0.11b 6.63 2.29abc 5.32 0.93a 6.31 0.10b 8.27 0.61c 6.68 1.02a 6.06 0.52a 9.12 2.56ab 8.46 0.46b 7.61 1.33a 7.50 1.08a 9.13 2.14a 8.66 0.09a 7.20 0.65a 6.02 0.66a 8.21 3.15a 6.33 1.01a 12.23 1.87a 8.44 1.89b 9.82 1.23ab 10.07 3.48a 6.67 0.25a 6.65 1.02a 13.14 0.94a 9.48 3.03a 7.72 0.62b 9.64 1.02a 10.88 3.02a 14.76 1.28a 9.66 3.78b 13.81 0.89a 13.27 1.21a TF 84.46 0.54a 157.76 0.38b 153.71 3.46c 136.44 6.79d 103.60 7.29a 153.45 7.03b 149.01 5.95b 143.91 13.56b 94.79 1.97a 154.14 4.04b 169.31 0.23c 144.54 5.82b 100.09 6.46a 156.68 7.74b 151.32 5.99b 150.94 2.02b 103.88 5.43a 144.65 8.13b 139.04 6.57b 124.67 0.79c 157.92 10.10a 142.94 0.23b 141.84 1.18b 142.73 13.33a 152.02 5.62a 149.94 1.03a 152.86 7.79a 136.27 0.77b 135.10 10.86ab 134.95 7.87a 142.58 1.41a 143.14 16.42a 133.10 4.43a 142.63 8.33a 133.66 2.87a

10

14

21

28

35

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Control = Untreated FJSM beverage; HIPEF-800 = high intensity pulsed electric eld treatment at 35 kV/cm for 800 ls; bipolar 4 ls-pulses at 200 Hz; HIPEF-1400 = high intensity pulsed electric eld treatment at 35 kV/cm for 1400 ls; bipolar 4 ls-pulses at 200 Hz; thermal = thermal treatment (90 C, 60 s). A different letter in the same column corresponds to signicant differences amongst treatments (p < 0.05). TF = total avonoids.

sugars (Dugo et al., 2005). Moreover, the presence of some enzymes, such as polyphenol oxidase (PPO) and phenylanine ammonialyase (PAL), can alter the phenolic composition of the fruits (Macheix et al., 1990; Spanos & Wrolstad, 1990). PAL is the key enzyme in phenolic biosynthesis, and induction of PAL activity generally corresponds to an increase in the accumulation of phenolic compounds (Macheix et al., 1990). Therefore, by taking into account the above mentioned statements, the changes observed on the phenolic content of the FJ SM beverage after HIPEF or thermal treatments could be due to some of the followed reasons: (i) biochemical reactions could have occurred during the HIPEF or heat processing, which led to the formation of new phenolic compounds; (ii) HIPEF or thermal processing might have caused signicant effects on cell membranes or in phenolic complexes with other compounds, releasing some free phenolic acids or avonoids; (iii) HIPEF and thermal process may inactivate PPO, preventing further loss of phenolic compounds; and (iv) HIPEF treatment might have induced favorable conditions to increase PAL activity, resulting in an enhancement of phenolic concentration in the beverage. 3.2.2. Carotenoid content Immediately after the HIPEF (800 or 1400 ls) or thermal treatments, a signicant loss in the content of lutein, zeaxanthin and b-

cryptoxanthin of the FJSM beverage was observed (Table 4). Lutein and zeaxanthin concentration showed the lowest concentration in thermally processed beverage, whereas the HIPEF treated for 800 or 1400 ls presented the highest losses of b-cryptoxanthin. Interestingly, the effects of HIPEF treatments on b-carotene content were different to those observed in other carotenoids, since it slightly rose just after processing. HIPEF and thermally treated FJSM beverages showed a signicant decrease on their initial total carotenoid content immediately after treatments (Fig. 2 day 0). However, the loss of total carotenoids was lower in both beverages treated by HIPEF for 800 ls (9.8%) and 1400 ls (18.4%) than in those that were heat treated (25.9%). Currently, studies related to the effects of HIPEF or thermal treatments on carotenoid composition of mixed beverages are scarce; but there are some works conducted in individual fruit and vegetable juices (Corts et al., 2006; Odriozola-Serrano et al., 2009; Torregrosa, Corts, Esteves, & Frgola, 2005). Different to our results, it was observed that HIPEF treated tomato juices showed a greater amount of total carotenoids regarding to the fresh juice (Odriozola-Serrano et al., 2009). However, Corts et al. (2006) stated that the effects of treatments over carotenoid composition mainly depended on processing conditions. They observed that immediately after HIPEF or heat processing, total carotenoid concentration of orange juice was signicantly diminished.

Table 4 Effects of high intensity pulsed electric elds and heat pasteurization on carotenoid content (mg/100 ml) of a fruit juicesoymilk beverage throughout storage at 4 C. Day 0 Treatment Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal Control HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-800 HIPEF-1400 Thermal HIPEF-1400 Thermal HIPEF-1400 Thermal HIPEF-1400 Thermal cis-Violaxanthin + anteraxanthin 0.012 0.001a 0.012 0.001a 0.011 0.002a 0.011 0.001a 0.013 0.003a 0.011 0.002a 0.010 0.002a 0.009 0.003a 0.013 0.003a 0.013 0.001a 0.012 0.001a 0.010 0.002a 0.015 0.001a 0.013 0.002a 0.015 0.001a 0.013 0.001a 0.013 0.001ab 0.011 0.002a 0.014 0.001b 0.012 0.002ab 0.013 0.003a 0.014 0.002a 0.012 0.001a 0.014 0.002a 0.015 0.002a 0.015 0.002a 0.014 0.003a 0.014 0.002a 0.016 0.004a 0.016 0.002a 0.017 0.001a 0.013 0.001a 0.015 0.001a 0.013 0.001a 0.012 0.001a cis-Anteraxanthin 0.023 0.001a 0.023 0.003a 0.022 0.006a 0.019 0.001a 0.022 0.001a 0.020 0.003a 0.023 0.003a 0.016 0.007a 0.021 0.001a 0.019 0.003a 0.024 0.003a 0.022 0.004a 0.023 0.001a 0.023 0.005a 0.026 0.002a 0.023 0.005a 0.021 0.003a 0.021 0.003a 0.024 0.001a 0.021 0.004a 0.022 0.007ab 0.027 0.002a 0.021 0.002b 0.023 0.003a 0.021 0.009a 0.023 0.003a 0.022 0.007a 0.022 0.003a 0.021 0.003a 0.027 0.003a 0.024 0.002a 0.021 0.003a 0.021 0.001a 0.019 0.003a 0.015 0.004a Anteraxanthin 0.015 0.002a 0.016 0.001a 0.015 0.003a 0.015 0.002a 0.014 0.002a 0.014 0.002a 0.013 0.001a 0.014 0.003a 0.013 0.001a 0.013 0.004a 0.012 0.001a 0.015 0.002a 0.013 0.001a 0.011 0.001b 0.013 0.002a 0.015 0.001a 0.011 0.003a 0.008 0.002b 0.011 0.003a 0.011 0.002a 0.010 0.003ab 0.012 0.001a 0.009 0.001b 0.011 0.002a 0.011 0.002a 0.012 0.001a 0.010 0.003a 0.009 0.003a 0.009 0.001a 0.013 0.003a 0.013 0.001a 0.011 0.003a 0.011 0.001a 0.010 0.001a 0.009 0.001a Lutein 0.108 0.006a 0.090 0.001b 0.080 0.001c 0.066 0.001d 0.085 0.013a 0.065 0.002a 0.062 0.006a 0.045 0.030a 0.076 0.021a 0.064 0.025a 0.064 0.001a 0.041 0.013a 0.082 0.002a 0.059 0.013b 0.069 0.004b 0.044 0.002c 0.071 0.013a 0.041 0.001b 0.055 0.001c 0.056 0.009c 0.045 0.015ab 0.059 0.004a 0.040 0.004b 0.050 0.002a 0.060 0.001b 0.054 0.001c 0.047 0.016ab 0.056 0.006a 0.044 0.001b 0.063 0.012a 0.052 0.001a 0.060 0.001a 0.049 0.001a 0.058 0.001a 0.037 0.008a Zeaxanthin 0.076 0.005a 0.072 0.001a 0.056 0.002b 0.056 0.014b 0.057 0.012a 0.051 0.001a 0.056 0.002a 0.053 0.001a 0.053 0.010a 0.047 0.019a 0.055 0.013a 0.054 0.001a 0.059 0.001a 0.043 0.007bc 0.059 0.002ab 0.044 0.001c 0.055 0.011a 0.036 0.001b 0.053 0.007ac 0.046 0.005c 0.040 0.013ab 0.050 0.005a 0.037 0.002b 0.048 0.001a 0.043 0.016a 0.048 0.001a 0.042 0.020a 0.040 0.002ab 0.038 0.001b 0.052 0.002a 0.041 0.006b 0.046 0.001a 0.048 0.005a 0.038 0.007a 0.040 0.008a

a-Cryptoxanthin
0.008 0.003a 0.008 0.003a 0.005 0.001b 0.006 0.001ab 0.006 0.001a 0.005 0.002a 0.005 0.001a 0.006 0.002a 0.006 0.001a 0.005 0.002a 0.005 0.002a 0.007 0.001a 0.006 0.003a 0.005 0.001b 0.005 0.003a 0.007 0.001b 0.005 0.003a 0.005 0.002a 0.005 0.001a 0.006 0.001a 0.005 0.002a 0.007 0.003a 0.005 0.001a 0.007 0.003a 0.007 0.002a 0.006 0.003a 0.006 0.002a 0.007 0.001a 0.005 0.002a 0.008 0.001a 0.008 0.001a 0.006 0.002a 0.007 0.001a 0.005 0.002a 0.006 0.001a

b-Cryptoxanthin 0.021 0.002a 0.012 0.002b 0.013 0.003b 0.016 0.005b 0.015 0.005a 0.011 0.005a 0.012 0.001a 0.016 0.002a 0.014 0.003a 0.012 0.005a 0.012 0.002a 0.016 0.003a 0.016 0.002ab 0.012 0.003a 0.013 0.001a 0.018 0.001b 0.014 0.002ab 0.011 0.002a 0.013 0.002ab 0.016 0.001b 0.012 0.005a 0.014 0.001a 0.015 0.001a 0.016 0.002a 0.013 0.006a 0.017 0.003a 0.014 0.006a 0.012 0.002a 0.015 0.004a 0.016 0.002a 0.019 0.002a 0.015 0.002a 0.017 0.001 0.012 0.003a 0.013 0.003a

a-Carotene
0.009 0.002a 0.007 0.001a 0.006 0.002a 0.009 0.003a 0.009 0.002a 0.006 0.003a 0.007 0.001a 0.008 0.001a 0.007 0.002a 0.006 0.002a 0.007 0.001a 0.009 0.002a 0.008 0.001ac 0.007 0.001b 0.008 0.001ab 0.010 0.001c 0.007 0.002a 0.007 0.001a 0.008 0.001a 0.009 0.001a 0.008 0.003a 0.010 0.001a 0.008 0.001a 0.010 0.001a 0.011 0.001a 0.010 0.001a 0.009 0.004a 0.011 0.001a 0.009 0.002a 0.013 0.001a 0.011 0.001a 0.009 0.002a 0.010 0.001a 0.008 0.002a 0.008 0.002a

b-Carotene 0.043 0.009ab 0.046 0.001b 0.050 0.003b 0.036 0.006a 0.033 0.009a 0.039 0.006a 0.041 0.003a 0.024 0.001b M. Morales-de la Pea et al. / Food Chemistry 129 (2011) 982990 0.022 0.001a 0.032 0.004a 0.032 0.003a 0.021 0.013a 0.020 0.001ab 0.028 0.001b 0.033 0.005a 0.017 0.001a 0.018 0.002a 0.027 0.003b 0.024 0.003c 0.018 0.002a 0.019 0.004a 0.017 0.005a 0.021 0.002a 0.022 0.002a 0.018 0.011a 0.025 0.012a 0.017 0.003a 0.02 0.003a 0.020 0.010a 0.025 0.003a 0.021 0.009a 0.024 0.003a 0.023 0.006a 0.017 0.003a 0.013 0.002a

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Control = Untreated FJSM beverage; HIPEF-800 = high intensity pulsed electric eld treatment at 35 kV/cm for 800 ls; bipolar 4 ls-pulses at 200 Hz; HIPEF-1400 = high intensity pulsed electric eld treatment at 35 kV/cm for 1400 ls; bipolar 4 ls-pulses at 200 Hz; thermal = thermal treatment (90 C, 60 s). A different letter in the same column corresponds to signicant differences amongst treatments (p < 0.05).

987

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350

TPC (mg/100mL)

300 250 200 150 100 50 0 0 10 20 30 40 50 60

Storage time (days)

Fig. 1. Total phenolic compounds (TPC) of untreated (), high intensity pulsed electric eld (35 kV/cm with 4 ls bipolar pulses at 200 Hz for 800 (j) or 1400 ls (N)) and thermal (d) (90 C, 60 s) treated fruit juicesoymilk beverages throughout storage at 4 C.

0.4

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 0 10 20 30 40 50 60

molecule, giving b-carotene as a product and increasing its concentration in treated sample. Similar to the behavior observed in phenolic composition, some biochemical reactions between carotenoids could take place during processing resulting in an augment or a loss of the individual compounds in the FJSM beverage. Taking into considerations the phenolic and carotenoid composition of the FJSM beverage, it could be considered as a good medium to include health related compounds in diet, irrespectively of the treatment applied. A regular portion of 200 ml of untreated, HIPEF or thermally treated FJSM beverage provide 211243 mg of phenolic acids and 169315 mg of avonoids, as well 0.072 0.1 (b-carotene), 0.0120.018 (a-carotene) and 0.02400.042 (b-cryptoxanthin) mg of vitamin A carotenoids. Thus high consumption of this beverage might be an approach to a healthier lifestyle. Nonetheless, considering the current consumers claim, beside nutritive characteristics, people are looking for high quality foods in terms of physicochemical and sensorial attributes. In this way, previous studies have proved that HIPEF treatment is able to better preserve physicochemical of the FJSM beverage than thermal treatments (Morales-de la Pea et al., 2010a) and sensorial quality of the beverage treated by HIPEF was not signicantly different to that of fresh samples, however after heat processing it was negatively affected (Salvia-Trujillo, Morales-de la Pea, ElezMartnez, & Martn-Belloso, 2009). Thus in order to meet current consumers requirements, HIPEF represents a feasible process to obtain FJSM beverages with a high content of antioxidant compounds and better fresh-like characteristics than thermal treatments. 3.3. Effects of storage 3.3.1. Phenolic content As seen in Fig. 1, storage time did not affect the total phenolic concentration of the FJSM beverage, irrespectively of the treatment applied. These results are in agreement with previous studies in which total phenolic content, determined by FolinCiocalteu technique, of untreated, HIPEF or thermally treated FJSM beverages remained stable along the storage at 4 C (Morales-de la Pea et al., 2010a). Likewise, Patthamakanokporn, Puwastein, Nitithamyong, and Sirichakal (2008) observed that the phenolic content of different selected fruits did not experience signicant changes over time. Nevertheless, other related works have indicated that generally, phenolic content may increase, decrease or remain stable in fruits during time depending on the storage conditions (Kalt, 2005). According to Macheix et al. (1990), the variation in fruit phenolic content results from the activity of the enzymes responsible for their biosynthesis or degradation. As PPO causes degradation of phenolic compounds, its activity might signicantly inuence the changes on the concentration of these compounds. It has been demonstrated that high inactivation levels of this enzyme can be achieved by HIPEF or thermal treatments and there is no reactivation throughout the storage (Aguil-Aguayo et al., 2010). Therefore, HIPEF and heat treatments applied to the FJSM beverage may allow high PPO inactivation and, as a result, the total phenolic concentration of the beverage remained stable throughout the entire storage period. The concentration of individual phenolic acids and avonoids underwent different effects over time in the untreated and treated FJSM beverages (Tables 2 and 3). Regarding phenolic acids (Table 2), it was observed that the content of chlorogenic and sinapic acids decreased as storage time increased, irrespectively of the treatment applied. Conversely, there was a slight increase in ferulic acid concentration during the storage; while the content of coumaric and caffeic acids remained with no signicant changes with respect to their initial values. On the other hand, Table 3 shows that,

TCC (mg /100 mL)

Storage time (days)

Fig. 2. Total carotenoid compounds (TCC) of untreated (), high intensity pulsed electric eld (35 kV/cm with 4 ls bipolar pulses at 200 Hz for 800 (j) or 1400 ls (N)) and thermal (d) (90 C, 60 s) treated fruit juicesoymilk beverages throughout storage at 4 C.

Interestingly, our results agree well with the effects observed by Torregrosa et al. (2005), who reported that a HIPEF treatment of 30 kV/cm caused a rise in the content of b-carotene of an orangecarrot juice and this increase was not statistically signicant. These authors pointed out that conversion amongst carotenoids take place during HIPEF or thermal processing. It is well known that carotenoids are highly unsaturated compounds with an extensive conjugated double-bonds system (Shi & Maguer, 2000). As a result, they are highly susceptible to oxidation, isomerization and other chemical changes during processing. Nonetheless, their extent of denaturation depends on their chemical structure (Bitton & Hornero-Mndez, 2001). According to Kidmose, Edelenbos, Nbk, and Christensen (2002), the major cause of carotenoid losses in vegetable products is the oxidation of the highly unsaturated carotenoid structure. In addition, it has been reported that the application of various industrial treatments can lead to the formation of cis isomers, which do not have the same bioactive activity as the trans isomers (Zulueta, Esteve, Frasquet, & Frgola, 2007). Zulueta et al. (2007) mentioned that lutein and zeaxanthin are highly susceptible to degradation during thermal treatments due to the presence of oxygen in their chemical structure. In this way, HIPEF or thermal processes applied in this study might have caused the stress conditions that led to the degradation of carotenoid content in the FJSM beverage. Nevertheless, HIPEF treatment was able to retain higher carotenoid concentration in the FJSM beverage than heat pasteurization process. In addition, the increase of b-carotene in HIPEF treated FJSM beverage coincide with the decrease in a-carotene content (Table 4). Thus, according to Bitton & Hornero-Mndez (2001), a-carotene may undergoes cyclization to form six member rings at one end of the

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with time, hesperidin content of untreated FJSM beverage showed a signicant rise. Nevertheless, it tended to decrease in HIPEF and thermally treated samples. Additionally, rutin and quercetin concentration remained with no signicant changes; narirutin and apigenin content was enhanced with no signicant differences between untreated and treated samples. Several studies have been carried out evaluating the effects of storage on the concentration of various phenolic compounds in different foods. The changes observed within these works vary widely, mainly due to the differences in the food matrix, treatment and storage conditions, as well as the specic phenolic compound under study. Odriozola-Serrano et al. (2009) found that chlorogenic acid content in tomato juices treated by HIPEF or heat signicantly decreased with time. On the other hand, Clifford (2000) reported that hydroxycinnamic acids, such as ferulic acid, may be released from conjugates by hydrolysis and subsequently decarboxylated by microorganisms. Moreover, Macheix et al. (1990) mentioned that methylation of hydroxycinnamic acids leads to the formation of ferulic acid and this reaction takes place by means of an o-methyltransferase. Del Caro, Piga, Vacca, and Agabbio (2004) evaluated possible changes of avonoids in minimally processed citrus fruits and juices during storage. Their results showed a signicant increase in hesperidin content of minimally processed fruits; nevertheless, some juices, such orange juice, showed a diminution of its avonoid content after a certain period of time. In this way, it could be said that the changes on the content of individual phenolic acids and avonoids throughout the time are highly inuenced by the treatment applied, the type of reaction that takes place within the food along the storage and the enzymatic activity which can induce the degradation or synthesis of each compound. 3.3.2. Carotenoid content The stability of carotenoid compounds during commercial shelf-life affects the quality of the product. Different reactions occurring during the storage such as isomerization and oxidation of carotenoids result in a loss of color, which is one of the most important attributes related to product quality affecting choice purchase (Baker & Gnter, 2004). Fig. 2 shows that total carotenoid content of untreated and treated FJSM beverages tended to decrease as the storage time increased. Even though there were no signicant differences between samples, HIPEF treated FJSM beverage showed slightly higher carotenoid concentration than the thermally treated beverage at the end of the storage. According to different authors, the losses of carotenoid content after a certain period of time usually depend on the maturity stage of the fruit and the storage and process conditions (SnchezMoreno et al., 2003). Corts et al. (2006) and Odriozola-Serrano et al. (2009) evaluated carotenoid stability of HIPEF and thermally treated tomato and orange juices under chilled storage, respectively. Similar to the effects observed in this work, they reported that the total carotenoid concentration of tomato and orange juices decreased during the time, regardless of the treatment applied. Moreover, thermally treated juices showed higher rate of degradation than those HIPEF treated. Although the pathways of carotenoids degradation have not been well established, Rodriguez-Amaya (1997) mentioned that the cause of instability and consequent degradation of these compounds is due to the susceptibility to oxidation and geometric isomerization of its polyene chain. Oxidation is the main cause of carotenoid loss, which is a spontaneous free-radical chain reaction in the presence of oxygen (Snchez-Moreno et al., 2003). This reaction is stimulated by light, metals, enzymes and peroxides (Rodrguez-Amaya, 1997; Snchez-Moreno et al., 2003). During autooxidation of carotenoids, alkylperoxyl radicals are formed and these radicals attack the double bounds resulting in formation

of epoxides (Odriozola-Serrano et al., 2009). Overall, the severity of this reaction depends on the carotenoid structure and environmental conditions (Ramakrishan & Francis, 1980). As seen in Table 4, the content of cis-violaxanthin + anteraxanthin, cis-anteraxanthin, a-carotene, a- and b-criptoxanthin remained with no signicant variations with time, whereas the concentration of anteraxanthin, lutein, zeaxanthin and b-carotene were signicantly depleted in untreated and treated beverages. These results are in agreement with the data reported by Wolbang et al. (2008), Snchez-Moreno et al. (2003) and Corts et al. (2006) who also observed that lutein, zeaxanthin and b-carotene showed the major losses among all carotenoids throughout storage in melon fruit and orange juice. According to Zulueta et al. (2007), owing to the presence of oxygen in the chemical structure, lutein and zeaxanthin are more susceptible to degradation with time. Moreover, Biacs and Wissgott (1997) reported that carotenoid losses are mainly due to the activity of enzymes, particularly in an oxygen environment. While lipoxygenase is the critical enzyme for the degradation of carotenoids (Li, Gong, Chen, & Chen, 2001), peroxidase is related to the oxidation of organic and inorganic matters and also participates in the degradation of carotenoids (Pang, Huang, Li, & Zhang, 2008). In a previous study, it was reported that the residual activity of lipoxygenase slowly increased after seven days of storage in HIPEF and thermally processed FJSM beverages (Morales-de la Pea et al., 2010a). Hence, the degradation of carotenoid concentration in the FJSM beverage could be due to the oxygen concentration in the bottles in which the samples were stored and/or to the presence of lipoxygenase in the FJSM beverages throughout the storage. However, further studies are needed to explain the mechanism of individual carotenoid reactions in fruit products as affected by storage time. 4. Conclusions Five phenolic acids, ve avonoids and nine carotenoids were identied in the FJSM beverage. Among the phenolic compounds, coumaric acid, narirutin and hesperidin were found in the highest concentration, whereas lutein, zeaxanthin and b-carotene were the most abundant carotenoids. Just after HIPEF or thermal treatments, the total phenolic concentration signicantly increased. Conversely, total carotenoid content showed a signicant degradation irrespective of the treatment applied. However, the HIPEF treated beverage for 800 and 1400 ls had a higher concentration of total phenolic and carotenoid compounds than the FJSM beverage processed by heat. Storage time did not affect the total phenolic content of the untreated and treated FJSM beverages. Total carotenoid content of all the evaluated samples tended to decrease as storage time increased. Individual phenolic and carotenoid compounds underwent different changes over the time, whereas some of them suffered signicant losses in their concentration, others remained without changes or were even enhanced. At the end of the storage, HIPEF treated FJSM beverage had a higher phenolic and carotenoid concentration than the heat treated beverage. Hence, the application of HIPEF processing could be a good alternative to thermal pasteurization in order to obtain FJSM beverages with a similar phenolic or carotenoid prole than the fresh beverages. Acknowledgements This study was supported by the Ministerio de Ciencia e Innovacin (Spain) throughout the Project AGL2006-12758-C02-02. Mariana Morales-de la Pea thanks the Consejo Nacional de Ciencia y Tecnologa (CONACYT) (Mxico) for the predoctoral grant. The ICREA Academia Award is also acknowledged by Olga MartnBelloso.

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