Evaluation of quality changes of blueberry juice during refrigerated storage after high-pressure and pulsed electric fields processing

Innovative Food Science and Emerging Technologies 14 (2012) 18–24

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Evaluation of quality changes of blueberry juice during refrigerated storage after high-pressure and pulsed electric fields processing F.J. Barba a, H. Jäger b, N. Meneses b, M.J. Esteve a, A. Frígola a,⁎, D. Knorr b a b

Nutrition and Food Chemistry, University of Valencia, Avda. Vicent Andrés Estellés, s/n. 46100 Burjassot, Spain Department of Food Biotechnology and Food Process Engineering, Berlin University of Technology, Koenigin-Luise-Str. 22, D-14195 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 20 September 2011 Accepted 8 December 2011 Editor Proof Receive Date 13 January 2012 Keywords: HP PEF Physicochemical properties Bioactive compounds Color Blueberry juice

a b s t r a c t A better knowledge of the effect of refrigerated storage on the nutritional and physicochemical characteristics of foods processed by emerging technologies with regard to unprocessed juices is necessary. Thus, blueberry juice was processed by high pressure (HP) (600 MPa/42 °C/5 min) and pulsed electric fields (PEF) (36 kV/cm, 100 μs). The stability of physicochemical parameters, antioxidant compounds (ascorbic acid, total phenolics, total anthocyanins) and antioxidant capacity was studied just after treatment and during 56 days at refrigerated storage at 4 °C. Just after treatment, all treated blueberry juices showed a decrease lower than 5% in ascorbic acid content compared with the untreated one. At the end of refrigerated storage, unprocessed and PEF juices showed similar ascorbic acid losses (50%) in relation to untreated juice, although HP juices maintained better the ascorbic acid content during storage time (31% losses). All juices exhibited fluctuations in total phenolic values with a marked decrease after 7 days in refrigerated storage, however prolonged storage of the juices at 4 °C, up to 56 days resulted in another in the total phenolic content for all juices in comparison with day 7. HP preserved antioxidant activity (21% losses) more than unprocessed (30%) and PEF (48%) juices after 56 days at 4 °C. Color changes (a*, b*, L, Chroma, hº and ΔE) were slightly noticeable after refrigerated storage for all juices. Industrial relevance: Non-thermal technologies allow the acquisition of drinks that keep their characteristics similar to the fresh product. They must join second conservation treatment such as refrigerated storage. A better knowledge of the effect of refrigerated storage on the nutritional and physicochemical characteristics of foods processed by emerging technologies with regard to unprocessed juices is necessary. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Juices obtained from blueberries are increasingly promoted and consumed due to their reported nutritional and health benefits (Nindo, Tang, Powers, & Singh, 2005; Rossi et al., 2003). They are a rich source of vitamins C and E, minerals among other beneficial substances that are essential components for normal growth and development. Additionally, berry juices contain phenolic substances like flavonols, tannins, and anthocyanins with high antioxidant capacity (Atala, Vásquez, Speisky, Lissi, & López-Alarcón, 2009; Netzel et al., 2002). Vitamin retention studies to assess the effects of food processing on the nutritional value of foods are of great importance to food technologists and consumers. Researchers have used ascorbic acid as a quality indicator in fruits and vegetables because it is a sensitive bioactive compound providing an indication of the loss of other vitamins and therefore acting as a valid criterion for other organoleptic or

⁎ Corresponding author. Tel.: + 34 963544955; fax: + 34 963544954. E-mail address: [email protected] (A. Frígola). 1466-8564/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ifset.2011.12.004

nutritional components (Giannakourou & Taoukis, 2003). The concentration of ascorbic acid decreases during storage depending on the storage conditions such as temperature, oxygen content and light (Blasco, Esteve, Frígola, & Rodrigo, 2004; Zerdin, Rooney, & Vermuë, 2003). Likewise, a diet rich in phenolic compounds correlates with reduced risk of coronary heart diseases (Amiot, Fleuriet, Cheynier, & Nicolas, 1997). Anthocyanins are the most important phenolic compounds present in berry fruits that are of special significance because of their contribution to total antioxidant capacity of berry fruits. Anthocyanins also are responsible of taste and flavor of the fresh fruit and for the brilliant red color and its different hues in many fruits and berries. Attractive color is one of the main sensory characteristics of fruit and berry products, and this important quality parameter strongly affects consumer behavior and can be correlated with both sensorial and nutritional quality attributes (Rein & Heinonen, 2004). Thermal treatments are the most used methods to extend the shelf-life of liquid foods by the inactivation of microorganisms and enzymes; however, heat causes irreversible losses of nutritional compounds, undesirable changes in physicochemical properties, and alteration of their antioxidant properties (Plaza et al., 2006; Wang & Xu,

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2007). To facilitate the preservation of unstable nutrients many juice processors have investigated alternatives to thermal pasteurization. Non-thermal technologies have been reported to be a good option for obtaining food products with a fresh-like appearance while preserving their nutritional content (Odriozola-Serrano, Soliva-Fortuny, & Martin-Belloso, 2009; Zabetakis, Leclerc, & Kajda, 2000). Therefore, the evaluation of the potential use of non-thermal technologies, such as HP or PEF, is important because they inactivate microorganisms and enzymes to a certain extent and can avoid the negative effects of heat pasteurization (Buckow & Heinz, 2008; Toepfl, Mathys, Heinz, & Knorr, 2006). The aim of this work is to study whether the non-thermal technologies (HP and PEF) can be used to obtain a high quality blueberry juice and increase its shelf-life while maintaining its physicochemical and nutritional characteristics. 2. Material and methods 2.1. Samples Blueberries (Vaccinium myrtillus, harvested in Poland) were purchased at Berlin's wholesale market (Berlin, Germany). The fruits were washed, drained, chopped and pressed. Then, the obtained blueberry juice was centrifuged at 4000×g for 15 min. 2.2. Chemicals Trolox® (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), ABTS (2,2-azinobis(3-ethylbenzothiazoline 6-sulphonate)), potassium hydroxide, sodium and disodium phosphate, acetonitrile (special grade), magnesium hydroxide carbonate and tetrabutyl ammonium hydrogen sulfate were obtained from Fluka (Steinheim, Germany). L(+)-ascorbic acid, ethanol, methanol metaphosphoric acid and sodium chloride (special grade) were obtained from Merck (Darmstadt, Germany). 2.3. Microbiological assay The number of surviving cells, N, was determined after a proper dilution of the treated samples in distilled water, by plate count method. The count of microbial colonies, grown on PCA (Plate Count Agar, Oxoid) at 32 °C for 72 h, was expressed in cfu/ml (colony forming units per ml of sample). The survival fraction, S = N/ N0 and the level of inactivation, log (S) were evaluated for each test. 2.4. HP system The samples, 10 ml inserted in PE-LD bottles were placed in the HP unit (U 4000 Unipress, Poland). The pressurization liquid was a mixture of distilled water and 1, 2-propanediol (50:50 v/v). The pressure level, pressurization time, and temperature were controlled automatically. The samples were pressurized at 600 MPa during 5 min at a maximum temperature of 42 °C (initial temperature 25 °C) (the come-up and come-down pressure times for this treatment was 45 and 6 s, respectively). All the treatments were applied in triplicate, with three bottles per replicate. Immediately after pressurization the samples were transferred to an ice/water bath and then stored under refrigeration (4±1 °C) until needed for analysis. For HP treatment, literature reports 1–5 min at 350–500 MPa to achieve 5-log reduction of different foodborne pathogens in acidic fruit juices (Donsì, Ferrari, Di Matteo, & Bruno, 1998; Alpas, Kalchayanaud, Bozoglu, & Ray, 2000). The selection of the treatment was based on the results obtained by other authors, the retention of bioactive compounds and microbial inactivation in berry products (Patras, Brunton, Da Pieve, & Butler, 2009; Buckow, Kastell, Terefe, & Versteeg, 2010; Barba et al., in press).

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2.5. PEF treatment system Continuous treatment was performed using a 7 kW modulator (ScandiNova Systems AB, Uppsala, Sweden) providing rectangular pulses, pulse width 3 μs (setpoint 50% of maximum voltage), rise time 0.44 μs; falling time 0.27 μs, in the range of 3–8 μs with a maximum voltage of 50 kV and a repetition rate of 400 Hz. The co-linear type treatment chamber (Berlin University of Technology) was fed with a flow of 5 l/h using a peristaltic pump 323 Du (Watson Marlow, Wilmington USA). The treatment chamber consisted of one central high voltage electrode and two outer grounded electrodes (all stainless steel, inner diameter 6 mm) separated to a distance of 4 mm by two polyoxymethylene insulators with an inner diameter of 4 mm. This geometry provides two treatment zones of a total enclosed volume of 0.22 ml exposed to the electric field, resulting in a total residence time of the medium in the electrical field of 0.15 s at a flow rate of 5 l/h. Adjustment of inlet temperature was conducted by stainless steel cooling coils (Berlin University of Technology) immersed in a VWR 1160S circulating water bath (VWR, Darmstadt, Germany). A Takaoka fiber optic thermometer FT1110 (Chiyoda Corporation, Tokyo, Japan) served as temperature control during PEF treatment. Outlet temperature after treatment did not exceed 60 °C. Cooling to 10 °C was realized within 7 s using a cooling coil with an inner diameter of 2 mm submersed in a water bath (VWR, Darmstadt, Germany) at 5 °C. Treatment time was 100 μs and the electric field was set at 36 kV/cm. Samples were collected and packaged aseptically in 10 ml PET bottles after PEF treatment and stored in refrigeration (4 ± 1 °C). The experiments were performed in triplicate. For pulsed electric field processing, conditions recommended to inactivate Escherichia coli and other pathogens are 25–35 kV/cm during 20–300 μs (Wouters, Dutreux, Smelt, & Lelieveld, 1999; Alvarez & Heinz, 2007). However, for this technology, actual design of the equipment, especially treatment chamber, strongly influences the effectiveness of the treatment and the temperature reached during the PEF treatment itself (Mastwijk et al., 2007). PEF conditions were used that were in line with the literature data and that shown in previous studies in safe and stable acidic fruit juices (Torregrosa, Esteve, Frígola, & Cortés, 2006; Matser, Schuten, Mastwijk, & Lommen, 2007; Odriozola-Serrano et al., 2009; Timmermans et al., 2011). 2.6. Ascorbic acid Ascorbic acid was determined according to Rückemann (1980). All reagents were of analytical grade. The eluent contained 2.5 g of tetrabutyl ammonium hydrogen sulfate in 945 ml distilled water and 55 ml methanol. A standard ascorbic acid solution of 50 mg ascorbic acid in 6% metaphosphoric acid was prepared with appropriate dilutions. 400 μl of blueberry juice were diluted with 1600 μl of 6% metaphosphoric acid. The samples prepared in this way were filtered through filters with a pore size of 0.22 μm and injected in the chromatograph (HPLC column, pump, variable wavelength monitor, Knauer GmbH, Berlin, Germany). Elution is obtained at a flow rate of 1 ml/min and eluate absorbance is measured at 251 nm. Retention was expressed as mg/100 g. 2.7. Total phenolic compounds Total phenolic content was measured using the Folin–Ciocalteu method (Singleton & Rossi, 1965). Results were expressed as milligrams of gallic acid equivalents per gram of fresh weight. 2.8. Total monomeric, polymeric anthocyanins, monomeric index Total anthocyanins, monomeric and polymeric anthocyanins, and monomeric index were determined using a modified method of

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Mazza, Fukumoto, Delaquis, Girard, and Ewert (1999). A 10-fold diluted blueberry juice of 100 μl was mixed with 1700 μl of distilled water and 200 μl of 5% (v/v) HCl. The sample was hold at room temperature for 20 min before measuring the absorbance at 520 nm in a 10 mm cuvette. This reading corresponds to the total anthocyanins content after considering the relevant dilution (A ta). Separately, 100 μl of juice aliquot, 800 μl of 5% (w/v) SO2, 1700 μl of distilled water and 200 μl of 5% (v/v) HCl were mixed, and absorbance was measured at 520 nm (A SO2) after 20 min at room temperature. From this reading, polymeric anthocyanins were obtained and monomeric anthocyanins were calculated from the difference between total anthocyanin (A ta) and polymeric anthocyanins (A SO2) content as follows:   ta SO SO monomeric index ¼ A −A 2 =A 2 Calculations of total anthocyanins were based on malvidin-3glucosid (molar absorptivity 28,000 for blueberry). All spectrophotometric analyses were performed using a UV–visible spectrophotometer Lambda 20 (Perkin-Elmer, Überlingen, Germany). 2.9. Antioxidant capacity The ABTS method was adapted from Re et al. (1999). This method is based on the capacity of antioxidants to quench the radical cation 2,2-azino-bis(3-ethylbenzothiazoline 6-sulphonate) (ABTS), which has a characteristic long-wavelength absorption spectrum showing a maximal peak at 734 nm. The ABTS radical cation is formed by the interaction of ABTS (7 mM) with K2S2O8 (2.45 mM). 2.10. Physicochemical parameters pH and ºBrix (total soluble solid content (g/100 g)) were measured in accordance to IFU methods (2001). Conductivity was measured at 20 °C using the Conductivity Meter WTW LF 323 (WTW, Germany). Color measurements were done in a Hunter Lab Labscan spectrophotometer (CR200, Minolta, Japan) and the Hunter color parameters L*: lightness (0=black, 100=white), a* (−a*=greenness, +a*=redness) and b* (− b* = blueness, + b* = yellowness) were used. Tests for each sample were conducted in triplicate and the values were averaged. These values were then used to calculate hue degree (h 0 = arctangent [b*/ a*]), chroma [C = (a* 2 + b* 2) 1/2], which is the intensity or color saturation, and ΔE, total differences of color, [ΔE = ((ΔL*) 2 + (Δa*) 2 + (Δb*) 2) 1/2] Calvo (2004). 2.11. Statistical analysis Significant differences between the results were calculated by analyses of variance (ANOVA) and the possible interactions between the parameters. An LSD test was applied to indicate the samples between which there were differences. A multiple regression analysis was performed for each parameter to study the influence of pressure and treatment time. Also the correlations between a pair of variables were studied. All statistical analyses were performed using Statgraphics Plus 5.0 (Statistical Graphics Corporation, Inc., Rockville, MD, USA). 3. Results and discussion In order to compare the shelf life of unprocessed, HP-treated (600 MPa/5 min) and PEF treated blueberry juice (36 kV/cm, 100 μs), the microbial inactivation was studied as a first prerequisite to obtain shelf stable juice. The microbiological assays performed on the HP and PEF treated samples during refrigerated storage at 4 °C during the period of the study showed that the microbial load after

the all treatment cycles is always less than 10 cfu/ml. This result highlights the efficacy of HP and PEF on microbial inactivation and confirms the experimental data reported in literature for acidic fruit juices (Alpas et al., 2000; Elez-Martínez & Martín-Belloso, 2007). The degradation kinetics of ascorbic acid was analyzed during refrigerated storage at 4 °C. In Table 1 are shown the results for ascorbic acid retention obtained for each experimental condition. The initial concentration of ascorbic acid in blueberry juice was 16.3 mg/100 g. The results were in the range of those reported by Prior et al. (1998) for blueberry juice. Immediately after HP and PEF treatment ascorbic acid retention was 95% and 97% respectively. Several authors have reported that ascorbic acid was minimally affected by HP at mild temperatures (Barba, Esteve, & Frígola, 2010; Bull et al., 2004). On the other hand, the results obtained in this study after PEF treatment also were in accordance to those found by other authors in orange–carrot juice (Torregrosa et al., 2006) and tomato juice (Odriozola-Serrano, Aguiló-Aguayo, Soliva-Fortuny, Gimeno-Anó, & Martín-Belloso, 2008), they found 90% vitamin C retention when they applied an electric field of 35 kV/cm during 130 μs and 35 kV/cm during 1000 μs, respectively. The differences in vitamin C retention between PEFtreated juices may be due to the lower pH of blueberry juice (3.00) in comparison to orange–carrot juice and tomato juice, as more acidic conditions are known to stabilize vitamin C (Tannenbaum, Archer, & Young, 1985). Fig. 1 shows the ascorbic acid concentration obtained during refrigerated storage of the different types of blueberry juice studied. Plotting concentration of ascorbic acid against the refrigerated storage time of the juice gives the ascorbic acid degradation rate. 1.4th order reaction model is the best for explaining ascorbic acid degradation. In the first week, ascorbic acid retention of untreated, HP and PEF treated blueberry juice was around 80% compared with day 1. However, after day 7, it was observed higher vitamin retention in HP blueberry juice. At the end of the storage (Day 56), lower ascorbic acid loss rates

Table 1 Ascorbic acid, total phenolics, total anthocyanin (TACN) and TEAC in unprocessed blueberry juice and treated by PEF and HP and after 56 days of refrigerated (4 °C) storage. Storage time

Ascorbic acid (mg/ Total phenolics TACN 100 g) (mg/g) (mg/g)

TEAC (μmol/g)

Day 0 Untreated 16.3±0.0a 36 kV/cm 15.8±0.4a 600 MPa/5 min 15.5±0.3a

3.30±0.02a 3.32±0.02a 3.35±0.03a

2.52±0.07a 2.64±0.07b 2.75±0.05c

32.6±1.4abc 33.5±1.1c 34.2±0.9c

Day 7 Untreated 13.1±0.3b 36 kV/cm 13.3±0.4b 600 MPa/5 min 13.6±0.4b

2.83±0.05g 2.84±0.03fg 2.94±0.04def

2.56±0.06ab 2.40±0.04d 2.50±0.08a

24.9±0.3h 30.3±0.7e 33.0±0.5ac

Day 14 Untreated 10.9±0.8de 36 kV/cm 10.6±0.7ef 600 MPa/5 min 13.6±0.3b

3.01±0.08bcde 2.99±0.07bcde 2.97±0.12bcde

2.56±0.06ab 32.1±0.7ab 2.36±0.03de 30.5±1.0ef 2.55±0.08ab 33.7±0.8c

Day 21 Untreated 10.6±0.6ef 36 kV/cm 10.4±0.5ef 600 MPa/5 min 12.9±0.7bc

3.07±0.03bc 2.93±0.03defg 3.09±0.01b

2.56±0.02ab 2.24±0.11ef 2.57±0.09ab

32.2±0.5abcd 29.4±0.3f 32.8±0.4abc

Day 28 Untreated 9.7±0.3f 36 kV/cm 10.2±0.5ef 600 MPa/5 min 11.9±0.3de

3.02±0.07bcde 2.94±0.01cdefg 3.01±0.08bcde

2.56±0.02ab 2.28±0.05ef 2.60±0.13ab

31.4±1.7bde 31.5±0.3abde 30.4±0.6def

Day 56 Untreated 8.1±0.5g 36 kV/cm 8.2±0.2g 600 MPa/5 min 11.2±1.1de

2.98±0.22bde 2.89±0.09efg 3.04±0.02bcd

2.56±0.02ab 2.23±0.11f 2.81±0.03c

22.6±0.2i 17.3±1.3j 26.9±1.4g

a–i Different letters indicate significant statistical differences in function of the applied treatment.

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were found in the case of HP blueberry juice (73%) compared with the respective values of unprocessed (52%) and PEF treated samples (50%) at 4 °C storage. Esteve, Farré‚, and Frígola (1996), when they studied the stability of ascorbic acid in fresh orange juice and commercial orange juices stored at 4 and 10 °C, found that at 4 °C the retention of ascorbic acid was 90% after 7 days of storage. Polydera, Stoforos, and Taoukis (2005) obtained similar results for HP orange juice storage at 5 °C during one month. A possible explanation for the lower ascorbic acid degradation rates during storage of high pressure treated blueberry juice could be a loss of availability of metal ions (e.g. iron, copper), catalyzing the ascorbic acid degradation, due to their hydration or the formation of complexes with chelating agents, reported to be favored by high pressure (Cheah & Ledward, 1995). The destruction of peroxides by high pressure application may also be a possible reason for the retardation of ascorbic acid degradation after HP treatment of blueberry juice. Moreover, the different grade of enzymatic inactivation achieved with each treatment could be involved in the ascorbic acid degradation. Nagy (1980) reported two main reasons of vitamin C reduction which are: oxidative reactions by enzymes such as cytochrome oxidase, ascorbic acid oxidase and peroxidase found in fruits, aerobic and nonenzymatic anaerobic reactions. HP was reported to affect the hydrophobic and electrostatic bonds of proteins, therefore affecting their secondary, tertiary and quaternary structures. Such conformational changes can enhance enzyme activity by uncovering active sites and consequently facilitate catalytic conversion. The total phenolic (TP) content, total anthocyanin, and total antioxidant activity (expressed as TEAC value) in blueberry juice unprocessed and processed by HP and PEF are shown in Table 1. The concentration of TP compounds in unprocessed blueberry juices was 3.30 ± 0.02 mg/g. These values were in the range of those previously reported by Prior et al. (1998) and Koca and Karadeniz (2009) for blueberries. The application of HP or PEF treatment did not cause significant differences in TP content. Results obtained in this study were in accordance with those found by Barba et al. (in press) in blueberry juice treated by HP and Odriozola-Serrano et al. (2008) for tomato juice treated by PEF. During 56-day storage at 4 °C fluctuations in the TP content were observed for all juices (unprocessed and processed), as shown in Table 1. In the first 7 days, the TP content of all blueberry juices decreased significantly (p b 0.05). Prolonged storage of the juices at 4 °C, up to 56 days resulted in another increase in the TP content for all juices in comparison with day 7. Piljac-Žegarac, Valek, Martinez, and Belščak (2009) observed similar behavior in total phenolics content in dark fruit juices after storage at 4 °C. They observed a decrease in total phenolic content after 13 days storage at 4 °C and after an increase in TP 29 days storage 4 °C. Similarly, in their study of polyphenolic content and antioxidant activity of orange juices during storage,

110

Untreated

PEF

HPP

100 90

y =-6,592ln(x) + 101,21 r² = 0,9212

80

C/C0

70

y =-12,01ln(x) + 102,07 r² = 0,9612

60 50

y =-12,5ln(x) + 101,35 r² = 0,9882

40 30 20 10 0 0

7

14

21

28

35

42

49

56

63

Time (days) Fig. 1. Ascorbic acid remaining concentration in untreated, HP and PEF treated samples stored in refrigeration at 4 °C. The lines interpolating the experimental data points show the fit of a 1.4th-order reaction model.

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Klimczak, Małecka, Szlachta, and Gliszczyńska-Świgło (2007) first observed a decrease in the total phenol content after 4 months storage, followed by a significant increase at the end of storage (6 months). It is possible that during juice storage, some reducing compounds are formed that react with the unspecific Folin–Ciocalteu reagent and significantly enhance the phenolic content (Escarpa & González, 2001). Pérez-Vicente, Serrano, Abellán, and García-Viguera (2004) reported insignificant changes in TP compounds of pomegranate juices stored during 160 days at 18 °C. The maintenance of TP compounds during storage might be due to the inactivation of the enzymes responsible for its degradation such as polyphenoloxidase, peroxidase and βglucosidase. These enzymes are the main enzyme implicated in reactions that are associated with loss of quality in berry juices. In addition, these enzymes are involved in the oxidative degradation of phenolic compounds (Amiot et al., 1997; Tiwari, O'Donnell, & Cullen, 2009). It has been demonstrated that both HP and PEF treatments could inhibit these enzymes in different fruit juices (AguilóAguayo, Sobrino-López, Soliva-Fortuny, & Martín-Belloso, 2008; Cano, Hernández, & De Ancos, 1997; Garcia-Palazon, Suthanthangjai, Kajda, & Zabetakis, 2004). The concentration of anthocyanins in fresh blueberry juice was 2.52 ± 0.07 mg/g. It was observed that a statistically significant increase of total anthocyanin happened immediately after HP treatment (109% retention) and PEF treatment (105% retention). During storage, a degradation in total anthocyanins content in PEF treated samples was observed after 7 days of storage at 4 °C (91% retention). Tiwari et al. (2009) reported that partial inactivation of enzymes such as β-glucosidase, polyphenoloxidases and peroxidase can contribute to the degradation of anthocyanins during storage. Recently, Odriozola-Serrano et al. (2009) reported anthocyanin retention of >83% in strawberry juice processed with high intensity pulsed electric fields and Aguiló-Aguayo et al. (2008) reported a enhanced activity of 113% for β-glucosidase in strawberry juice during PEF treatment (50 Hz, pulse width 1-μs at 35 kV/cm for 1000 μs). This can explain total anthocyanin degradation during storage in samples treated by PEF. On the other hand, HP samples preserved anthocyanin content after 56 days storage (102% retention). The stability of anthocyanins can be due to a possible complete inactivation of polyphenoloxidase as reported by Garcia-Palazon et al. (2004) for strawberry and red raspberry treated by HP. Just after treatment, processes did not modify significantly the antioxidant activity as can be observed in Table 1. After 2 days storage (data not shown) a statistically significant (p b 0.05) increase in TEAC values was observed in all analyzed samples. According to Pinelo, Manzocco, Núňez, and Nicoli (2004) the increase in the antioxidant activity may be explained by the strong tendency of polyphenols to undergo polymerization reactions, whereby the resulting oligomers possess larger areas available for charge delocalization. When the degree of polymerization exceeds a critical value, the increased molecular complexity and steric hindrance reduce the availability of hydroxyl groups in reaction with the radicals, which causes a resultant decrease in the antiradical capacity. This may explain the observed decrease in antioxidant capacity of our blueberry juices, which followed after the initial transient increase. At the end of the 56-day storage period, the studied juices exhibited a significant decrease in TEAC (p b 0.05), which varied from 69% (unprocessed) to 52% (PEF treated) and 79% (HP), showing HP the highest antioxidant activity. Piljac-Žegarac et al. (2009) observed an increase in antioxidant activity in the first days followed of a significant loss TEAC values in dark fruit juices under refrigerated conditions during 29 days. Likewise, Kevers et al. (2007) when they studied the antioxidant activity of spinach, broccoli and leek they observed also a decreased more than 50% after 30 days in refrigerated storage. Plaza et al. (2006) observed better retention of antioxidant activity of HP orange juices after storage during 40 days at 4 °C than for PEF treated juices in the same period.

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To relate antioxidant activity to the total phenolics, total anthocyanins and ascorbic acid, it was studied whether there were correlations between a pair of variables. Thus, a statistically significant correlation between ascorbic acid (p= 0.0001) and TP (p= 0.0068) with TEAC values was observed. These results suggested the participation of ascorbic acid and phenolics in the antioxidant capacity as observed Amakura, Umino, Tsuji, and Tonogai (2000) measuring antioxidant capacity with DPPH method and Prior et al. (1998) using ORAC method also observed the same correlation between TP and antioxidant capacity in blueberries. The effects of HP and PEF treatments on instrumental color parameters b*(blueness and yellowness), a* (greenness and redness) and L* (lightness or darkness) of blueberry juice are shown in Table 2. No visual differences in juices were perceived immediately after HP or PEF treatments. Color parameters were compared in untreated, pressurized and PEF juices during refrigerated storage. The applied treatments did not significantly affect color parameters in comparison with untreated juice, whereas storage time had a significant influence on L*, a* and b*. During storage, b* value of unprocessed blueberry juice moved significantly towards the negative direction (pb 0.05). These results were in accordance to those founded by Zhang et al. (2008) when they studied the stability and color characteristics of cyaniding-3-glucoside solution during storage. However, b* value of HP and PEF treated juices moved significantly towards the positive direction (pb 0.05). Daoudi et al. (2002) obtained similar results for white grape juice treated by high pressure and storage at 4 °C during 60 days. On the other hand the a* values (0.25±0.04) gradually changed towards a more positive direction: (0.34 ± 0.04–1.11 ± 0.12) for the HP juice and (0.24 ± 0.02– 1.33 ± 0.03) for the PEF processed blueberry juice. The increase in a* values in the blueberry juice is similar to the results found by Patras, Brunton, Da Pieve, Butler, and Downey (2009) for high pressure processed tomato and carrot purées. L* values decreased in all the samples analyzed during storage. Similar results were reported by Daoudi et al. (2002) for grape juice treated by high pressure at 400 MPa/10 min.

They observed a decrease in L* values after storage at 4 °C during 30 and 60 days respectively. Lee and Coates (1999) found a slight decrease in L* value when red grapefruit was thermally processed and attributed it to partial precipitation of unstable particles in the juice as described by Genovese, Elustondo, and Lozano (1997) in cloudy apple juice after heat treatment. Likewise, h0 value significantly decreased (pb 0.05) after storage in all samples. It was observed a more intense decreasing in HP blueberry juice (87.20 ± 0.31–80.72± 0.99) and PEF (87.09± 0.21–78.73± 0.25) than in unprocessed samples (87.90 ± 0.36–81.96 ± 0.36), indicating the color changed towards more lilac. Similar results were found by Zhang et al. (2008). In blueberry juice, the total color change (ΔE) in all the processed samples immediately after treatment or during storage was significantly different (p b 0.05) from the unprocessed samples. The total color difference (ΔE*) indicates the magnitude of the color difference. Depending on the value of ΔE, the color difference between the treated and untreated samples can be estimated such as not noticeable (0–0.5), slightly noticeable (0.5–1.5), noticeable (1.5–3.0), well visible (3.0–6.0) and great (6.0–12.0) (Cserhalmi, Sass-Kiss, Tóth-Markus, & Lechner, 2006). As it is shown in Table 2, it is quite clear that the application of HP or PEF had a small effect on color changes. In addition, a significant negative correlation was observed between ΔE* and ascorbic acid (p=0.0016). Patras, Brunton, Da Pieve, and Butler (2009) observed similar results in non-thermally-processed samples of strawberry purées.

4. Conclusion HP and PEF retained similarly physicochemical properties and bioactive compounds to those of untreated blueberry juice. During refrigerated storage at 4 °C, HP juice showed higher ascorbic acid retention, phenolic content and antioxidant capacity than untreated and PEF juices. HP can be a potentially useful unit operation in

Table 2 Blueberry juice color evolution during 56 days of refrigerated (4 °C) storage of untreated, PEF and HP treated samples. Color parameters

a*

b*

L*

h0

ΔE

Day 0 Untreated PEF HP

0.25 ± 0.04ab 0.31 ± 0.07bcd 0.34 ± 0.04bcd

− 6.83 ± 0.02ab − 6.81 ± 0.04ab − 6.89 ± 0.01bc

31.54 ± 0.04a 31.44 ± 0.04a 31.52 ± 0.01a

87.90 ± 0.36ab 87.09 ± 0.21a 87.20 ± 0.31bcd

0a 0.11 ± 0.03e 0.11 ± 0.03e

Day 7 Untreated PEF HP

0.29 ± 0.13abc 0.42 ± 0.07de 0.37 ± 0.06bcde

− 6.82 ± 0.03ab − 6.76 ± 0.02ae − 6.81 ± 0.03a

31.35 ± 0.03b 31.24 ± 0.02cd 31.25 ± 0.03c

87.59 ± 1.06abc 86.59 ± 0.72de 86.89 ± 0.46cd

0.22 ± 0.02b 0.35 ± 0.04d 0.31 ± 0.03bc

Day 14 Untreated PEF HP

0.37 ± 0.01bcde 0.35 ± 0.01bcd 0.25 ± 0.04ab

− 6.98 ± 0.05d − 6.79 ± 0.02a − 6.82 ± 0.05ab

31.30 ± 0.02bc 31.23 ± 0.01cd 31.15 ± 0.03e

86.91 ± 0. 03cd 86.97 ± 0.17bcd 87.87 ± 0.35ab

0.31 ± 0.01bc 0.33 ± 0.01d 0.39 ± 0.03d

Day 21 Untreated PEF HP

0.38 ± 0.01cde 1.14 ± 0.17g 1.28 ± 0.09hi

− 6.98 ± 0.02d − 6.59 ± 0.13f − 6.79 ± 0.05a

31.25 ± 0.04c 31.16 ± 0.03de 30.71 ± 0.09fg

86.91 ± 0.10cd 79.29 ± 0.14hi 79.36 ± 0.63hi

0.35 ± 0.04d 1.00 ± 0.11f 1.32 ± 0.11gh

Day 28 Untreated PEF HP

0.48 ± 0.05e 1.19 ± 0.07gh 1.15 ± 0.04g

− 6.93 ± 0.05cd − 6.50 ± 0.03g − 6.75 ± 0.04ae

31.27 ± 0.05c 30.76 ± 0.03g 30.68 ± 0.05h

85.76 ± 0.27e 80.18 ± 1.40gh 80.31 ± 0.24g

0.38 ± 0.04d 1.26 ± 0.05g 1.25 ± 0.04g

Day 56 Untreated PEF HP

0.99 ± 0.06f 1.33 ± 0.03i 1.11 ± 0.12fg

− 7.01 ± 0.06d − 6.68 ± 0.01ef − 6.76 ± 0.00ae

30.97 ± 0.11f 30.62 ± 0.09h 30.48 ± 0.03i

81.96 ± 0.36f 78.73 ± 0.25j 80.72 ± 0.99g

0.95 ± 0.12f 1.43 ± 0.04h 1.36 ± 0.05gh

a–i

Different letters indicate significant statistical differences in function of the applied treatment.

F.J. Barba et al. / Innovative Food Science and Emerging Technologies 14 (2012) 18–24

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