Influence of high hydrostatic pressure processing on physicochemical characteristics of a fermented pomegranate (Punica granatum L.) beverage

Innovative Food Science and Emerging Technologies 59 (2020) 102249

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Influence of high hydrostatic pressure processing on physicochemical characteristics of a fermented pomegranate (Punica granatum L.) beverage

T

Gabriela Rios-Corripioa, Jorge Welti-Chanesb, Verónica Rodríguez-Martínezb, ⁎ José Ángel Guerrero-Beltrána, a b

Departamento de Ingeniería Química y Alimentos, Universidad de las Américas Puebla, Ex hacienda Sta. Catarina Mártir, San Andrés Cholula, 72810, Puebla, Mexico Centro de Biotecnología FEMSA, Instituto Tecnológico de Monterrey, Av. Eugenio Garza Sada 2501, Monterrey, 64849, NL, Mexico

ARTICLE INFO

ABSTRACT

Keywords: High hydrostatic pressure (HHP) Punica granatum Pasteurization Fermented pomegranate beverage Antioxidant characteristics Microbial safety

The effect of high hydrostatic pressure at 500 MPa/10 min (HHP1), 550 MPa/10 min (HHP2) and 600 MPa/ 5 min (HHP3) on the microbiological, physicochemical, antioxidant and sensory characteristics of a fermented pomegranate (FP) beverage, stored for 42 days (4 ± 1 °C), was evaluated. The FP beverage was also pasteurized at 63 °C/30 min (VAT) and 72 °C/15 s (HTST). The high hydrostatic pressure (HHP) and VAT pasteurized beverages did not show microbial growth (< 10 CFU/mL) throughout 42 days of storage. The physicochemical characteristics were not affected (p > 0.05) by HHP or pasteurization. Color of the samples showed significant differences (p ≤ 0.05) in all HHP processed and pasteurized beverages. Antioxidant activity, total phenolic compounds, flavonoids and anthocyanins increased slightly after HHP processing. Antioxidants decreased throughout the storage in all treatments. Both HHP processed and pasteurized beverages were well accepted by average consumers when evaluated using a 9-points hedonic scale. Industrial relevance: The high hydrostatic pressure (HHP) improves the microbiological, antioxidant and sensorial stability of fermented pomegranate beverages during storage. The HHP is more common for processing fruit juices than for fermented beverages; therefore, it can be expanded to the fermented beverages industry, which could modify the today usual thermal processing methods and, or the addition of preservatives, that are not natural, for delivering high quality and healthier pomegranate fermented beverages to consumers.

1. Introduction It has been reported that fermented pomegranate (FP) beverages or pomegranate wines may have benefits to the humans health. It is known, from researches, that the fermentation process may increase the digestibility and bioavailability of bioactive components found in these beverages (Gumienna, Szwengiel, & Górna, 2016). The beneficial properties are ascribed to the antioxidants found in the beverages (Mousavi et al., 2013). In addition, a FP beverage is an important source of antioxidants such as anthocyanins, specifically, 3-O-glucoside and 3,5-O-diglucoside of cyanidin, delphinidin and pelargonidin (Ferrari, Maresca, & Ciccarone, 2010). Thermal treatments are the most used preservation methods for liquid foods to inactivate microorganisms and/or enzymes. However, when processing at high temperatures, irreversible losses of nutritious compounds may occur as well as unwanted changes in physicochemical and sensory properties (Sánchez-Moreno et al., 2005). A disadvantage of thermal processing is the slow conduction and convection of heat. The effectiveness of heat treatments can also be affected by the complexity of ⁎

the product and type and load of microorganisms in it. Another, negative effect of overheat processing is that sensory attributes of the food may change. The preservation of the sensory characteristics is of paramount importance in any food product (González & Barrett, 2010). In addition to the conventional thermal processing, there are other treatments or novel-technologies that do not use heat (non-thermal technologies) for processing foods. High hydrostatic pressure (HHP) has been applied in the food industry for processing foods such as vegetables, jams, jellies, sauces, purees, fruit juices, among others (Daher, Le Gourrierec, & Pérez-Lamela, 2017). Today, the HHP technology is used to deliver food products to create a new generation of value-added foods. This technology uses pressures in the range 200–800 MPa to inactivate microorganisms and/or enzymes (Hara, Nagahama, Ohbayashi, & Hayashi, 1990; Morild, 1981). It has been reported that the application of HHP to foods may maintain compounds such as antioxidants due to its short effect in covalent bonds (Cheftel, 1992). To increase the competitiveness in the food industry, several emerging technologies have been introduced in the winemaking process (Tao

Corresponding author. E-mail address: [email protected] (J.Á. Guerrero-Beltrán).

https://doi.org/10.1016/j.ifset.2019.102249 Received 26 February 2019; Received in revised form 12 September 2019; Accepted 24 October 2019 Available online 25 October 2019 1466-8564/ © 2019 Published by Elsevier Ltd.

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et al., 2012). In wine, the HHP processing, as a preservation method, has been tested at pressures in the range 200 to 500 MPa, demonstrating the inactivation of bacteria and yeasts; therefore, reducing the sulfite amounts used for wine preservation. The main changes observed in HHPtreated wines are the reactions of condensation and oxidation of phenolic compounds that lead to the formation of compounds with high degree of polymerization (Martínez-Monteagudo & Balasubramaniam, 2016; Nunes, Santos, Saraiva, Rocha, & Coimbra, 2017). It is a fact that it is necessary to minimize the degradation of functional molecules in processed foods, not only with the application of HHP, but also throughout their subsequent storage, to ensure optimal sensory, microbiological, physicochemical and antioxidant characteristics (Ferrari et al., 2010). It has been proven that anthocyanins in beverages are stable to the HHP processing (Cao et al., 2011; Zabetakis, Leclerc, & Kajda, 2000). There has also been reported an increased extractability of pigments from foods at very high pressures; therefore, the polyphenols content might increase. It has also been reported the release of anthocyanins, amino acids, and proteins with hydroxyl group after HHP treatment of fruit products (Kaşıkcı & Bağdatlioğlu, 2016). Enzymes such as polyphenoloxidase (affecting polyphenols), peroxidase (affecting anthocyanins), pectinmethylesterase (in citrus), lipoxygenase (affecting vegetables), and catalase have been involved in the degradation of chemical compounds in fresh and processed fruits and vegetable products (Torres et al., 2011). The use of HHP may lead to inactivate enzymes promoting (indirectly) an increase in phenols. However, the effect of HHP on some compounds in foods cannot be generalized because it may depends on the composition of the product, the working conditions, and the subsequent effect of the application of the treatment (Kaşıkcı & Bağdatlioğlu, 2016). After the application of HHP, the stability of the food during storage can be affected due to chemical (oxidation) and biochemical reactions, especially when the endogenous enzymes or microorganisms are incompletely inactivated (Hendrickx, Ludikhuyze, Van den Broeck, & Weemaes, 1998; Shigehisa, Ohmori, Saito, Taji, & Hayashi, 1991). HHP processing has been used for treating pomegranate juice; researchers observed different effects of this emerging technology. Ferrari et al. (2010) pointed out that the HHP improved the red color, the antioxidant capacity and total phenolic compounds of pomegranate juice. Chen et al. (2013) investigated the effect of HHP on the microbiological, physicochemical and antioxidant stability of non-clarified pomegranate juice using different pressures (300 and 400 MPa) and times (2, 5 10, 15, 20, 25 min). They mentioned an effective inactivation of microorganisms at 400 MPa/5 min; they also observed a great retention of color and an increase in antioxidants immediately after the application of HHP. Subasi and Alpas (2017) investigated the effect of HHP (200, 300, 400 MPa/ 10 min) in some quality properties of pomegranate juice, mentioning that the application of HHP at 400 MPa/10 min was enough to decrease the microbial load in about 4 logarithmic cycles in the pomegranate juice. Varela-Santos et al., (2012) evaluated the effect of HHP on the microbiological, physicochemical and antioxidant quality of pomegranate juice applying pressures of 350–550 MPa for 0.5, 1.5 and 2.5 min. Their results showed that pressures higher than 350 MPa, for 2.5 min, reduced the microbial load around 4 logarithmic cycles; the treatment was sufficient to maintain the microbial load below the limit of detection along the entire storage period. The treatment extend the microbiological shelf life of pomegranate juice stored at 4 °C for > 35 days. The aim of this study was to compare the effect of HHP and thermal processing on microbial, physicochemical, antioxidant and sensory characteristics of a fermented pomegranate beverage stored under refrigeration conditions.

were chosen free from physical and microbiological injuries; then, washed, and disinfected (1 min) with a 150 μL/L hypochlorite sodium solution. Arils were separated by hand from the fruit. The juice extraction from arils was performed using a Standard Turmix® juice extractor (México City, México). 2.2. Methods 2.2.1. Beverages The juice was adjusted in total soluble solids (TSSs) at 25 °Brix (% w/w) for the fermentation process. 10 L of fresh pomegranate juice was inoculated with five milliliters of inoculum of Saccharomyces cerevisiae [(2.60 ± 0.21) × 106 CFU/mL]. The fermentation of juice was performed at 25 ± 1 °C in an incubator until reaching a constant content of TSSs (Rios-Corripio & Guerrero-Beltrán, 2019). Once the fermented pomegranate (FP) beverage was obtained (20 days of fermentation), it was centrifuged at 6000 rpm for 5 min to eliminate insoluble solids. The fermentation process was performed in duplicate. FP beverage was packaged in polyethylene pouches of 13 cm2 (ULINE®, Wisconsin, USA), air evacuated, sealed, and then HHP processed or pasteurized. A nonprocessed sample was used as control. 2.2.2. Thermal treatment FP beverage was pasteurized at low (VAT) and high (HTST) temperatures. The VAT process consisted in heating the FP beverage in glass beaker at 63 ± 2 °C/30 min and the HTST pasteurization was carried out at 72 ± 2 °C/15 s. Afterward, beverages were cooled down rapidly in ice water. Both pasteurized samples were placed in sterile commercial glass bottles for immediate analysis to be stored at 4 °C. Physicochemical, microbiological and antioxidant characteristics were analyzed every 7 days. Sensory analysis was also performed at the end of the storage time. 2.2.3. HHP processing A 2 L AVURE HHP processing equipment (Technologies® Inc., Middletown, Ohio, USA) was used. Three batches of FP beverages were prepared to be treated at 500 MPa/10 min (HHP1), 550 MPa/10 min (HHP2), and 600 MPa/5 min (HHP3). After HHP processing, FP beverages were immediately analyzed or stored at 4 ± 1 °C. Physicochemical, microbiological and antioxidant characteristics were analyzed every 7 days. Sensory analysis was also performed only at the end of the storage. A FP beverage without pasteurization or HHP processing was used as control. 2.2.4. Microbial growth in FP beverages Psychrophilic (P), aerobic mesophilic bacteria (AMB) and molds plus yeasts (MY) were evaluated using the Plate Count Agar (PCA), PCA, and Dichloran Rose-Bengal Chloramphenicol agar (DIFCO, Sparks, MD, USA) methods, respectively. The inoculated Petri dishes were incubated at 4, 25 and 37 °C, respectively. Psychrophilic and yeast plus molds were counted after 96–120 h and mesophilic after 24–48 h of incubation. Microbial counts were reported as log10 of colony forming units per milliliter (CFU/mL). 2.2.5. Antioxidant capacity The evaluation of the free radical-scavenging effect on 1, 1-diphenyl-2-picrylhydrazyl (DPPH) was used to measure the antioxidant capacity (Brand-Williams, Cuvelier, & Berset, 1995). An aliquot of 10 μL of sample was mixed with 1990 μL of absolute ethanol and 2000 μL of DPPH (0.0039 g/100 mL of absolute ethanol). The mixture was totally homogenized and maintained in the dark for 45 min at room temperature (25 °C). Absorbances of the samples were measured at 517 nm using a JENWAY 6850 UV–Vis spectrophotometer (Stone, Staffordshire, UK). The antioxidant capacity was calculated using Eq. (1).

2. Materials and methods 2.1. Materials Pomegranate (Punica granatum L.) fruits, “Apaseo” variety, were purchased at a local market in Atlixco, Puebla, Mexico. Pomegranates

I= 2

Ac

As Ac

100

(1)

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G. Rios-Corripio, et al.

where I is the inhibition (%), As is the absorbance of the sample and Ac is the absorbance of the control. A standard curve was prepared at various concentrations of Trolox (6-hydroxy-2,5,7,8 tetramethylchrome-2 carboxylic acid 97%): 0–0.030 mg (R2 = 0.993). Results were calculated as mg of Trolox equivalents (TE)/100 mL of beverage using Eq. (2)

TE

mg I b = 100 mL m

DF

100

molar absorptivity coefficient (26,900 L/mol cm), F is the dilution factor, and 1 is the light pathway along the quartz cell (1 cm). 2.2.9. Total soluble solids (TSSs), pH and ethanol concentration TSSs were measured using an Atago refractometer (Atago Co. Ltd., Tokyo, Japan) according to the 932.14C AOAC (2000) method and reported as °Brix (% w/w). A Conductronic pH-meter (Conductronic S. A., Puebla, Mexico) was used for measuring pH at 20 ± 5 °C. The ethanol concentration was measured by specific gravity according to the 10.023 AOAC (2000) method.

(2)

where I is the inhibition (%) of the sample, b is the intercept (1.2497), m is the slope (3233.7 1/mg), and DF is the dilution factor of the sample.

2.2.10. Titratable (TA), volatile (VA) and fixed (FA) acidities For TA, VA, and FA, 5 mL of sample were placed in conical flasks with 25 mL of distilled water and titrated with 0.1 N NaOH solution according to the 942.15, 11.047 and 981.12 AOAC (2000) methods, respectively. TA and FA were calculated as grams of citric acid (CA)/ 100 mL and VA as grams of acetic acid (AA)/100 mL.

2.2.6. Total phenolic compounds Total phenolic compounds (TPCs) were analyzed by the Phenol Folin-Ciocalteu method (Singleton, Orthofer, & Lamuela-Raventos, 1999) with some modifications. An aliquot of 10 μL of sample (FP diluted 1:9 mL distilled water) was mixed with 3990 μL of distilled water, 250 μL of Folin-Ciocalteu reagent and 750 μL of Na2CO3 (20%). Samples were mixed and maintained at room temperature (25 ± 1 °C) for 2 h in the dark. The absorbance was measured at 765 nm using a JENWAY 6850 UV–Vis spectrophotometer (Stone, Staffordshire, UK). A standard curve was prepared with different concentrations of Gallic acid (GA): 0–0.064 mg (R2 = 0.990). TPCs content was calculated with Eq. (3). Results were reported as mg of GA/100 mL of beverage.

GA

mg = 100 mL

A

b m

DF

100

2.2.11. Color characteristics Ten milliliters of sample were placed in a small quartz cell (5 cm in height, 3.5 cm in length and 2 cm in width) for measuring color. The color parameters, in the transmittance mode, L*, a*, and b*, were measured using a tri-stimulus Chroma Meter CR-400 colorimeter (Konica Minolta Sensing Inc., Osaka, Japan) in the CIELab* scale. The total difference in color (ΔE*), Chroma and hue angle were calculated using Eqs. (7), (8), and (9), respectively.

(3)

E=

where A is the absorbance of the sample, b is the intercept (0.0014), m is the slope (22.395 1/mg), and DF is the dilution factor of the sample.

Chroma =

2.2.7. Total flavonoids Total flavonoids (TFs) were analyzed according to the Dewanto, Wu, Adom, and Liu (2002) method with some modifications. An aliquot of 10 μL of undiluted sample was mixed with 3265 μL of distilled water and 75 μL of 5% NaNO2, mixed and left in the dark. After 5 min, 150 μL of AlCl3-6H20 (10%) was added. After 6 min, 500 μL of NaOH (1 M) was added and mixed. Immediately, the absorbance was measured at 510 nm in a JENWAY 6850 UV–Vis spectrophotometer (Stone, Staffordshire, UK). A standard curve was prepared with different concentrations of quercetin (Q) in distilled water: 0–0.018 mg (R2 = 0.932). Total flavonoids were calculated with Eq. (4). Results were reported as mg of Q/100 mL of beverage.

Q

mg = 100 mL

A

b m

100

hue = tan

C3OG

mg 100 mL

=

Abs

MW

F 1

1000

Abs700nm )pH 4.5

1

2

+b

2

b a

bo 2)

(7) (8) (9)

2.2.13. Statistical analysis All experimental data were analyzed by ANOVA and Tukey tests using a Minitab v.17 Statistical Software (Minitab Inc., State College, PA, USA). A p ≤ .05 value was used to make a decision about significant differences within treatment means.

2.2.8. Total monomeric anthocyanins The pH differential method was used to measure the total monomeric anthocyanins (TMAs) content (Giusti & Wrolstad, 2001). An aliquot of 1 mL of sample was placed in an amber tube containing 3 mL of potassium chloride buffer pH 1.0. In another amber tube, 3 mL of sodium acetate buffer pH 4.5 and 1 mL of sample was also placed. Tubes were prepared in triplicate. Tubes were perfectly mixed and allowed to stand at room temperature (25 ± 1 °C) for 30 min. Absorbances were measured at 520 and 700 nm in a JENWAY 6850 UV–Vis spectrophotometer (Stone, Staffordshire, UK). Total monomeric anthocyanins (TMAs) were calculated with Eqs. (5) and (6). Results were reported as mg of cyaniding-3-O-glucoside (C3OG)/100 mL of juice.

(Abs520nm

a

ao 2) + (bt 2

2.2.12. Sensory analysis All beverages were sensory evaluated using a nine-point structured hedonic scale (Wichchukit & O'Mahony, 2015). The sensory analysis was performed at the end of storage (42 days) with 20 consumers: appearance, color, aroma, sweetness, flavor, and general acceptability.

(4)

Abs700nm )pH1.0

Lo 2 ) + (at 2

where Lo*, ao*, and bo* are the values of just processed FP beverages. Lt*, at*, and bt* are values of the FP beverages measured at different times.

where A is the absorbance of the sample, b is the intercept (0.0002), and m is the slope (2.930 1/mg).

Abs = (Abs520nm

(Lt 2

3. Results and discussion 3.1. Inactivation of microorganisms From day 0 until the end of storage (42 days), no growth of psychrophilic bacteria (< 10 CFU/ mL) was observed in all processed samples (HHP, VAT and HTST) and control. No growth of AMB or YM (< 10 CFU/ mL) were observed in all HHP and VAT processed FP beverages. Table 1 shows the AMB and YM counts in control and HTST pasteurized samples along the storage time. For HTST pasteurization, a reduction of 3 logarithmic cycles of AMB and YM was observed. Little growth of AMB (40 and 170 CFU/mL at 0 and 42 days, respectively) and YM (25 and 180 CFU/mL at 0 and 42 days, respectively) was observed through the storage time in FP HTST beverages. Therefore, the AMB and YM population increased only one log cycle from day 0 to day 42 of storage. Tonello, Largeteau, Demazeau, and Lonvaud-Funel (1996) applied HHP (300 MPa/6 min) to inactivate yeasts, lactic acid bacteria and acetic

(5) (6)

where MW is the molecular weight (449.2 g/mol) of C3OG, ε is the 3

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a better retention of antioxidants compared with the heat treatment. They observed that flavonoids and phenols remain stable at 600 MPa/4 min and greater antioxidant activity. Andrés, Villanueva, and Tenorio (2016) evaluated the effect 450 and 600 MPa/3 min in smoothies stored for 45 days; their results indicated higher antioxidant capacity in the food processed at 600 MPa. Patras, Brunton, Da Pieve, and Butler (2009) and Polydera, Stoforos, and Taoukis (2005) and Vega-Gálvez et al. (2014) found that the HHP may increase or maintain the antioxidant activity of strawberry, blackberry, tomato, carrot purees, orange juice and gooseberry pulp which may depend on the amount of pressure or treatment time. Fernández, Butz, Bognàr, and Tauscher (2001) and Indrawati, Van Loey, and Hendrickx (2004) showed that the antioxidant activity of orange and lemon juices did not decrease after HHP processing. They observed that thermal treatments might reduce the potential of free-radical scavenging activity, contrary to the HHP treatment, which does not affect the free-radical scavenging activity. In addition, the increase or decrease in antioxidant activity could be due to a combined effect of different compounds, which act synergistically or antagonistically. A series of factors that influence the antioxidant activity such as the oxidation system, the degree of glycosylation, the partition coefficient and the concentration of other antioxidant compounds that the fruit could have and that could be correlated with the antioxidant activity (Tsikrika, & Tsikrika & Rai, 2019; Hassimotto, Genovese, & Lajolo, 2005).

Table 1 Aerobic mesophilic bacteria (AMB), yeasts plus mold (YM) in HHP and pasteurization (VAT/HTST) pomegranate fermented beverages throughout storage. Time (days)

0 3 7 14 21 28 35 42

Control

HTST

AMB

YM

AMB

YM

1.0 × 104 1.4 × 104 1.9 × 104 2.0 × 104 4.6 × 104 5.6 × 104 7.1 × 104 8.6 × 104

1.0 × 104 1.1 × 104 1.5 × 104 1.9 × 104 4.1 × 104 5.2 × 104 6.4 × 104 8.3 × 104

4.0 × 101 4.1 × 101 4.8 × 101 6.3 × 101 8.8 × 101 9.1 × 101 9.9 × 101 1.7 × 102

2.5 × 101 2.6 × 101 4.0 × 101 5.5 × 101 5.8 × 101 6.0 × 101 1.0 × 101 1.8 × 102

acid bacteria in white, red and rosé wines; they reported a total inactivation of yeasts and bacteria in all wines. It has been observed complete inactivation of vegetative bacteria, yeasts and molds when pressures are above 200 MPa. In practice, pressures of up to 700 MPa and treatment times from seconds to minutes have been tested to inactivate microbial cells (Terefe, Buckow, & Versteeg, 2013). The bacterial spores, on the other hand, are highly resistant to pressure, showing a remarkable tolerance to pressures above 1000 MPa. However, by combining other intrinsic factors in foods, such as fermentation, it is possible to inactivate bacterial spores at pressures in the range 500–900 MPa (Terefe et al., 2013). Regarding yeasts and molds, they are less resistant to pressure than bacteria; they can be inactivated with pressures between 200 and 400 MPa. It has also been observed that Saccharomyces cerevisiae could be more resistant than Gram negative bacteria (Daher et al., 2017). Basak, Ramaswamy, and Piette (2002) demonstrated that S. cerevisiae was not inactivated under 400 MPa in orange juice. Shahbaz et al. (2015) reported a reduction of 5.8 logarithmic cycles of S. cerevisiae in apple juice treated with HHP at 500 MPa for 1 min.

3.3. Total phenolic compounds Fig. 2 shows the TPCs content throughout the storage time. The values of TPCs at day 0 and 3 for control and HHP1, HHP2, HHP3, VAT and HTST processed beverages were 389.71 ± 2.00 and 380.43 ± 1.02, 392.32 ± 1.00 and 389.65 ± 1.08, 395.02 ± 1.22 and 391.83 ± 1.06, 399.21 ± 2.04 and 395.85 ± 1.21, 324.37 ± 1.08 and 319.42 ± 1.20, 358.90 ± 1.04 and 346.11 ± 2.00 mg GA/100 mL, respectively. Significant differences (p ≤ .05) were observed for TPCs among pasteurized beverages (VAT and HTST) and control, HHP1, HHP2, and HHP3 pomegranate beverages. The HHP processed samples had higher TPCs content at day 0 than at day 42 of storage. The HHP3 beverage showed significant differences (p ≤ .05) in the content of TPCs compared to control, HHP1 and HHP2. HHP3 beverages was the one with the highest TPCs content just after pressurization and at the end of the storage: 399.21 and 383.40 mg of GA/100 mL, respectively. Nevertheless, the TPCs barely decreased in control and all processed samples throughout the storage. Varela-Santos et al. (2012) evaluated the effect of high hydrostatic pressure (350–550 MPa for 0.5, 1.5 and 2.5 min) on the microbiological, physicochemical and antioxidant quality of pomegranate juice. They reported that phenols did not decrease with the application of HHP. Phenols increased between 3.38 and 11.99% in treated samples at 350 and 550 MPa, respectively. From day 5 of storage, the phenolic compounds began to decrease in all HHP processed samples. Chen et al. (2013) also reported a significant increase (p ≤ .05) in the content of total phenols of pomegranate juice treated with HHP (300 and 400 MPa for 2.5, 5, 10, 15, 20 and 25 min). At day 0, their control had a total phenolic content of 120.48 ± 1.75 GAE mg/100 g and those treated with HHP had 124.63 ± 1.09 GAE mg/100 g. Alpas (2013) analyzed pomegranate juices treated with HHP in the range 200–400 MPa for 5–10 min; the author reported a significant decrease in total phenolic compounds. Ferrari, Maresca, and Ciccarone (2011) observed a significant reduction (30–60%) of total phenolic compounds in HHP processed pomegranate juice throughout the storage; they observed a stability until day 14; however, at the end of the storage, the phenolic compounds content remained barely stable. They suggested that these changes were probably due to some differences in the HHP processing of the pomegranate juice. In this study, the VAT pasteurized beverage was the one with the lowest content of phenolic compounds (324.37 mg of GA/100 mL at day 0); therefore, the reduction could be related to the temperature and time of processing.

3.2. Antioxidant capacity The antioxidant capacity (AC) results are shown in Fig. 1. A significant difference (p ≤ .05) was observed in the AC content within the VAT pasteurized and HHP2 and HHP3 processed beverages. Control, HHP1 and HTST beverages were not statistically different (p > .05) regarding AC. The antioxidant capacity values at day 0 and 3 for control, HHP1, HHP2 and HHP3, VAT and HTST were 317.76 ± 1.262 and 313.27 ± 1.109, 321.94 ± 1.076 and 320.01 ± 1.171, 323.55 ± 1.119 and 323.10 ± 1.101, 326.51 ± 1.199 and 324.88 ± 1.421, 296.48 ± 1.181 and 293.41 ± 1.102, 303.54 ± 1.233 and 300.73 ± 1.601 mg Trolox/100 mL of beverage, respectively. The beverage with the highest loss of AC was the VAT pasteurized pomegranate beverage: at day 0, it had 296.48 mg Trolox/100 mL and at day 42, it had 270.90 mg Trolox/100 mL. This could be due to the heat and the longer time used for pasteurization. The sample treated at 600 MPa for 5 min (HHP3) had the highest amount of AC. Queiroz et al. (2010) reported similar results to those found in this work and pointed out that the antioxidant activity of apple juice increased when treated at 250 MPa for 3 min. Plaza et al. (2006) found a greater reduction of antioxidant capacity in pasteurized (70 °C–30 s) orange juice than in samples processed at 400 MPa for 1 min and stored 40 days at 4 °C. Some studies have shown that pasteurization and HHP processing may exert different effects on the total phenolic compounds content and antioxidant activity in products under refrigeration (Zhao, Zhang, & Zhang, 2016). In this research, the HHP1 HHP2, and HHP3 beverages showed an increase of approximately 10% of AC in FP beverages, compared to the control. In Fig. 1, from day 7, a linear reduction (Table 2) in antioxidant capacity was observed for control and all treatments; the lower correlation coefficient (0.955) was observed for the HHP1 pomegranate beverage. The slope (m) indicates the reduction rate along the storage. Dede, Alpas, and Bayındırlı (2007) treated orange juice with HHP; they demonstrated 4

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Fig. 1. Effects of high hydrostatic pressure (HHP) processing and thermal pasteurization (VAT, HTST) on the antioxidant capacity of pomegranate beverages stored under refrigeration (4 ± 1 °C). Table 2 Antioxidant characteristics of control and processed pomegranate beverages. HHP/T (MPa/min)a

Characteristic LRP ACg TPCh TFi TMAj Ethk

a b c d e f g h i j k

mf b R2 mf b R2 mf b R2 mf b R2 mf b R2

b

c

Pasteurization

C

500/10

550/10

600/5

VATd

HTSTe

−0.715 304.61 0.971 −0.560 370.90 0.929 −0.401 97.57 0.998 −0.016 4.537 0.985 0.018 11.65 0.984

−0.898 315.46 0.955 −0.822 388.80 0.968 −0.304 100.96 0.937 −0.014 4.552 0.946 −0.004 11.64 0.771

−1.016 325.99 0.996 −0.345 388.66 0.950 −0.350 104.97 0.975 −0.021 4.935 0.952 −0.003 11.75 0.686

−1.064 330.57 0.985 −0.228 392.26 0.924 −0.316 107.49 0.791 −0.013 4.899 0.912 −0.004 11.74 0.771

−0.587 295.53 0.982 −0.692 313.17 0.983 −0.843 99.52 0.930 −0.016 4.396 0.987 −0.003 11.51 0.686

−0.654 302.19 0.978 −0.999 345.12 0.970 −0.845 101.74 0.958 −0.014 4.451 0.993 0.008 11.56 0.938

HHP/T: High hydrostatic pressure/time. LRP: Linear regression parameters. C: Control. VAT: Pasteurization at 63 °C/30 min. HTST: High Temperature-Shor Time pasteurization (72 °C/15 s). m: slope. AC: Antioxidant activity (mg Trolox/100 mL day). TPC: Total phenolic compounds (mg Gallic acid/100 mL day). TF: Total flavonoids (mg quercetin/100 mL day). TMA: Total monomeric anthocyanins (mg C3OG/100 mL day). Eth: Ethanol (mL/100 mL day).

The reduction rates of TPCs content is reported in Table 2. The highest reduction rates were observed in the HHP1 and HTST pomegranate beverages. The lowest reduction rates were observed in the HHP2 and HHP3 beverages. Nevertheless, little reduction of TPCs was observed in control and all processed pomegranate beverages along the storage.

HHP3, VAT and HTST processed FP beverages was 103.27 ± 1.11 and 101.40 ± 1.27, 108.67 ± 1.52 and 106.79 ± 1.95, 114.96 ± 1.01 and 112.43 ± 1.98, 121.54 ± 1.21 and 117.32 ± 1.00, 97.82 ± 1.61 and 94.56 ± 1.42, 99.01 ± 1.53 and 96.32 ± 1.02 mg Q/100 mL, respectively. The HHP3 processed FP beverage had the highest content of TFs at day 0 (121.54 mg of Q/100 mL) and day 42 (95.78 mg of Q/100 mL). Even though an increase in flavonoids was observed after the HHP processing, this increase did not show significant differences (p > .05). Briones-Labarca et al. (2017) studied the effect of HHP processing on the antioxidants and oenological quality characteristics of a young white

3.4. Total flavonoids Fig. 3 shows the TFs content in control and all processed FP beverages. The total flavonoids content at day 0 and 3 for control and HHP1, HHP2, 5

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Fig. 2. Effects of high hydrostatic pressure (HHP) and pasteurization (VAT, HTST) on total phenolic compounds of refrigerated (4 ± 1 °C) pomegranate beverages.

wine (Sauvignon blanc). They did not find significant differences (p > .05) in flavonoids content within all HHP processed wines in comparison with control. In this study, significant differences (p ≤ .05) were observed within the flavonoids content in VAT and HTST pasteurized beverages; they had the highest loss of flavonoids along the storage (Fig. 3, Table 4). The VAT beverage had 97.82 and 63.25 mg of Q/100 mL at day 0 and day 42, respectively. Approximately, 60% of flavonoids remained at day 42 of storage in comparison with the initial amount of control at day 0 (103.27 mg of Q/100 mL) (Fig. 3). On the other hand, similar reduction rates of TFs were observed (Table 2) throughout the storage in control, HHP1, HHP2, and HHP3 FP beverages.

4.33 ± 0.01, 4.41 ± 0.01 mg C3OG/100 mL, respectively. No significant differences were observed (p > .05) within control and pasteurized or HHP processed FP pomegranate beverages, similar results to other researches. Terefe, Matthies, Simons, and Versteeg (2009) reported an insignificant change in the content of anthocyanins in strawberries treated with HHP at 600 MPa for 10 min. They evaluated the effect of storage for three months and reported a reduction of anthocyanins, starting at day 27. There are some hypotheses about the mechanism of anthocyanins degradation in HHP processed fruit products. One hypothesis say that the anthocyanins degradation could be due to the remaining enzymes. A relationship between the remaining enzymes (β-glucosidase, peroxidase, and PPO) and the stability of anthocyanins has been observed in several fruit products (Ferrari et al., 2010). The enzymatic degradation of anthocyanins by β-glucosidase is due to the loss of the glycosidic group that leads to the formation of anthocyanidin; consequently, this may affect the color of the product (García-Palazon, Suthanthangjai, Kajda, & Zabetakis, 2004). However, it must be taken into account that pressure, temperature and time of processing as well as physicochemical properties such as TSSs, pH and acidity could have some effects on the enzymes responsible for the stability of anthocyanins in food products (García-Palazon et al., 2004). Corrales, Butz, and Tauscher (2008) pointed out that the HHP

3.5. Total monomeric anthocyanins Fig. 4 shows the effect of thermal and non-thermal treatments on the total monomeric anthocyanins content throughout the storage. It is known that anthocyanins are highly unstable to physical factors such as light, oxygen, pH, temperature, among others. The total anthocyanins content at day 0 for control, HHP1, HHP2, HHP3, VAT and HTST processed beverages was 4.68 ± 0.01, 4.76 ± 0.03, 4.79 ± 0.00, 4.81 ± 0.02, 4.35 ± 0.01, 4.44 ± 0.00 mg C3OG/100 mL and at day 3 was 4.58 ± 0.01, 4.74 ± 0.01, 4.76 ± 0.02, 4.77 ± 0.02,

Fig. 3. Effects of high hydrostatic pressure (HHP) and thermal treatments (VAT, HTST) on the total flavonoids content of pomegranate beverages during refrigeration (4 ± 1 °C). 6

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Control HHP1 HHP2 HHP3 VAT HTST

Total anthocyanins (mg C3OG/100 mL)

4.9

4.7

4.5

4.3

4.1

3.9

3.7

3.5 0

7

14

21

28

35

42

49

Time (days) Fig. 4. Effects of high hydrostatic pressure (HHP) and pasteurization (VAT, HTST) on total monomeric anthocyanins content in stored (4 ± 1 °C) pomegranate beverages.

3.7. Color characteristics

processing may affect the condensation reactions of anthocyanins in wines; therefore, they recommended using low pressures and short times for processing. In this work, the HHP processed FP beverages had high content of anthocyanins than untreated beverages. HHP3 for instance, had 4.81 and 4.28 mg of C3OG/100 mL at day 0 and day 42, respectively. In the VAT pasteurization FP beverage, the loss of anthocyanins was greater immediately upon pasteurization (4.35 mg of C3OG/100 mL). At the end of the storage, the VAT pasteurized FP beverage had 3.72 mg of C3OG/100 mL. Similar reduction rates (−0.013–0.021 mg C3OG/100 mL day) of TMAs were observed (Table 2) throughout the storage in control, HHP1, HHP2, HHP3, VAT and HTST processed FP beverages. The higher reduction rates of TMAs was observed in the HHP2 processed FP beverage (0.021 mg of C3OG/ 100 mL day).

Table 4 shows the color parameters of HHP processed, control and pasteurized FP beverages at days 0 and 42. Color is one of the most important physical properties of a food; it provides important information about the quality of a food. All FP beverages had an average L* value of 29.01 ± 0.37, indicating a “dark” luminosity; however, no significant differences were observed (p ≤ .05) within control and all processed beverages. The VAT pasteurized FP beverage was the one with the lowest luminosity (28.50). At day 42 of storage, the L* values barely decreased in all beverages. Tao et al. (2012) reported significant changes in L*, a*, b*, Chroma and hue color parameters of commercial red wine (Nero D'avola Syrah: 93% Nero D'avola, 7% Syrah) treated with HHP at 650 MPa for 5, 15, 60 and 120 min. Their L*, a*, b*, Chroma and hue color parameters values were in the range 77.63 ± 0.02–78.68 ± 0.06, 22.87 ± 0.06–22.08 ± 0.07, 8.70 ± 0.0–8.69 ± 0.03, 24.47 ± 0.05–23.73 ± 0.07 and 20.84 ± 0.04–21.48 ± 0.02°, respectively. The L* values barely increased as the pressure holding time increased; the HHP treated wines were a little bit more luminous than the untreated one. Contrary to reported in this study, where the luminosity parameter darkened. Therefore, the increase or decrease in the color parameters may be attributed to the type of fruit used in the fermented beverage, to the complexity of the food matrix, and its behavior when exposed to different pressures and times. The red color (a*) values, in the red-yellow segment of the color space (McLaren, 1986), were in the range 18.62–29.43 at days 0 and 42 for both control and processed FP beverages; however, at day 42 of the storage, the red color (a*) decreased slightly. High temperatures (pasteurization), combined with long times, may degrade color-related pigments such as anthocyanins. These changes are mostly due to the conversion of monomeric anthocyanins to more condensed compounds during storage. This condensation reaction, induced by high pressure and/or temperatures, involves covalent association of anthocyanins with other flavonols or organic acids, leading to the formation of new pyran rings by cycloaddition. Anthocyanins condensation may be responsible for the changes in red color during storage, forming other complexes (Marszałek, Woźniak, Kruszewski, & Skąpska, 2017). The HHP3 processed FP beverage showed the highest a* value, followed by HHP2 and HHP1. The beverages that changed more in the a* value were those pasteurized. Both VAT and HTST pasteurized FP beverages were statistically different (p ≤ .05), compared to the control, HHP1, HHP2 and HHP3 regarding the a* value. Concerning the b* color parameter, the highest values were for the HHP3 FP beverage. The

3.6. Physicochemical analysis 3.6.1. Total soluble solids and ethanol contents Figs. 5 and 6 show the TSSs and ethanol contents, respectively, of control and FP processed beverages. No significant changes (p ≤ .05) in the content of TSSs within processed and control beverages were observed. The storage time did not affect the TSSs content in beverages. For day 42, the beverages that decreased in TSSs were control (1.2°Bx) and HTST (0.7°Bx); therefore, a light increase in ethanol was observed (Fig. 6). Queiroz et al. (2010), Briones-Labarca et al. (2017) and Corrales et al. (2008) pointed out that no significant changes (p ≤ .05) were observed in the TSSs and pH of juices and wines treated with HHP. In this study, the samples that showed more changes in ethanol content were the control (increase in 0.7 mL/100 mL) and the HTST (increase in 0.3 mL/100 mL) pomegranate fermented beverages. 3.6.2. pH and acidity Table 3 shows the changes in pH and acidities (TA, FA and VA) within means of control and all processed beverages at days 0 and 42. No significant differences (p > .05) were observed in pH, TA and FA within means of all samples at day 0 or 42. The VA increased slightly in control and HTST pasteurized beverages. Corrales et al. (2008) pointed out that pH and acidities of 2004 red wines (Dornfelder variety, Niederkirchen, Germany) were barely affected by pressures below 600 MPa. Briones-Labarca et al. (2017) did not find significant differences (p > .05) in pH and acidities in white wines (Sauvignon blanc) treated with HHP at 300–500 MPa for 5, 10 and 15 min. 7

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12.6

Control HHP1 HHP2 HHP3 VAT HTST

Total soluble solids (% w/w)

12.4

12.2

12

11.8

11.6

11.4

11.2 0

7

14

21

28

35

42

49

Time (days) Fig. 5. Effects of high hydrostatic pressure (HHP) and thermal pasteurization (VAT, HTST) on total soluble solids in pomegranate beverages through refrigeration (4 ± 1 °C).

pasteurized beverages had the lower values (p ≤ .05) in comparison to the b* values of the HHP processed and control FP beverages. In general, the b* value decreased slightly from day 0 to day 42. About H°, no significant differences (p > .05) were observed within all beverages at day 0 or day 42 of storage. The H° or color angle for all FP beverages was in the range 23.6–25.0°, indicating a red color, found in the redyellow segment (0–90°) of the color space (McLaren, 1986). The hue value decreased slightly at the end of storage in control and pasteurized FP beverages. These changes were expected due to the values obtained for the a* parameter. The low reduction in H° was mainly due to the reduction of L* values. Significant differences (p ≤ .05) were observed within C values of control and processed FP beverages; however, no significant differences (p > .05) were observed within the three HHP processed FP beverages. The lower C values were for pasteurized beverages at days 0 and 42 of storage. Therefore, it could be said that anthocyanins were barely degraded due to the exposition to high temperature. The ΔE* values (total change in color) indicate the magnitude of color difference of a material (Pathare, Opara, & Al-Said, 2012; Patras, Brunton, Tiwari, & Butler, 2011). According to this value and the values of the a* and b* color parameters, the hue of all FP

Ethanol concentration (% v/v)

12.5

beverages barely changed. The higher change in color was observed in the pasteurized FP beverages (p ≤ .05) comparing with the HHP processed beverages. The ΔE* values were the result of the changes occurred in the L*, a* and b* color parameters. Puértolas, Saldaña, Álvarez, and Raso (2010) have pointed out that the color differences can be perceived by the naked eye if the value of ΔE* is > 3, value that was only observed, in this study, in the pasteurized beverages at days 0 and 42 of storage. Therefore, the HHP processing may maintain the anthocyanins that give color to the FP beverages. Keenan, Brunton, Gormley, and Butler (2011) reported ΔE* values lower than 3 in HHP processed (450 MPa-1.3, 5 min) strawberry, banana, orange and apple smoothies in comparison with thermally pasteurized smoothies. According to Keenan et al. (2011), despite of changes in chromatic characteristics in FP beverages treated with HHP, these changes could not be perceived by the naked eye. 3.8. Sensory analysis Table 5 shows the sensory evaluation data of FP beverages at the end of storage (42 days). Significant differences (p ≤ .05) were

Control HHP1 HHP2 HHP3 VAT HTST

12.3 12.1 11.9 11.7 11.5 11.3 0

7

14

21 28 Time (days)

35

42

49

Fig. 6. Effects of high hydrostatic pressure (HHP) and pasteurization on ethanol content in pomegranate beverages under low temperature (4 ± 1 °C). 8

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Table 3 pH and total, volatile and fixed acidities control and HHP processed and pasteurized FP beverages at days 0 and 42 of storage (4 ± 1 °C)a. HHP/T (MPa/min)b

Pasteurization

Characteristic

Control

Day 0 pH TA VA FA

3.09 0.69 0.06 0.62

± ± ± ±

0.01a 0.03a 0.01a 0.01a

3.08 0.68 0.05 0.63

± ± ± ±

0.02a 0.03a 0.01b 0.02a

3.09 0.69 0.05 0.64

± ± ± ±

0.02a 0.01a 0.01b 0.01a

3.10 0.69 0.06 0.63

± ± ± ±

0.01a 0.03a 0.02b 0.01a

3.10 0.68 0.05 0.63

± ± ± ±

0.02a 0.02a 0.01b 0.02a

3.10 0.69 0.06 0.63

± ± ± ±

0.01a 0.02a 0.02b 0.01a

Day 42 pH TA VA FA

3.10 0.74 0.11 0.63

± ± ± ±

0.02a 0.03a 0.02a 0.10a

3.07 0.69 0.06 0.63

± ± ± ±

0.02a 0.02a 0.01b 0.07a

3.09 0.68 0.06 0.62

± ± ± ±

0.01a 0.01a 0.01b 0.11a

3.09 0.69 0.05 0.64

± ± ± ±

0.01a 0.03a 0.02b 0.09a

3.10 0.69 0.06 0.63

± ± ± ±

0.01a 0.02a 0.01b 0.10a

3.10 0.71 0.08 0.63

± ± ± ±

0.03a 0.02a 0.01b 0.06a

a

b

500/10

550/10

600/5

VAT

HTST

Different letters within rows indicate significant differences (p ≤ .05). Pressure/time (MPa/min). TA (total acidity, citric acid, % w/v), VA (volatile acidity, acetic acid, % w/v), FA (fixed acidity, citric acid, % w/v).

Table 4 Color parameters of HHP processed and pasteurized fermented pomegranate beverages after 42 days of storage (4 ± 1 °C)a. HHP/T (MPa/min)

Pasteurization

Characteristic

Controlb

500/10

550/10

600/5

VAT

HTST

Day 0 L* a* b* H (°) C ΔE*

29.43 ± 0.02a1 23.87 ± 0.10a1 10.45 ± 0.04bc1 23.6c 26.06b NA

29.37 ± 0.03a 23.97 ± 0.07a 10.49 ± 0.06b 23.6bc 26.16ab 0.12b

29.12 ± 0.02a 23.99 ± 0. 11a 10.56 ± 0.04b 23. 8bc 26.21ab 0.35b

28.99 ± 0.01ab 24.51 ± 0.09a 11.23 ± 0.05a 24.6a 26.96a 1.10b

28.50 ± 0.02b 21.07 ± 0.10b 9.81 ± 0.06d 25.0ab 23.24c 3.02a

28.66 ± 0.03ab 21.23 ± 0.06b 9.90 ± 0.04 cd 25.0a 23.42c 2.80ª

Day 42 L* a* b* H (°) C ΔE*

28.03 ± 0.04a 21.86 ± 0.11a 9.12 ± 0.05bc 22.6c 23.69b 2.79b

28.01 ± 0.02a 22.05 ± 0.09a 10.10 ± 0.06b 24.6bc 24.25ab 2.33b

27.98 ± 0.01a 22.79 ± 0.08a 10.21 ± 0.05b 24.1bc 24.97ab 1.82b

27.76 ± 0.03ab 23.03 ± 0.10a 10.95 ± 0.03a 25.4a 25.50a 1.94b

27.49 ± 0.02b 18.62 ± 0.11b 8.11 ± 0.04d 23.5ab 20.31c 6.07a

27.65 ± 0.02ab 19.08 ± 0.10b 8.36 ± 0.04 cd 23.7a 20.83c 5.52a

a

b

Different letters within rows indicate significant differences (p ≤ .05). Data taken into account as a reference for the calculation of the net color change (ΔE*).

Table 5 Sensory evaluation data of fermented beverages treated with HHP and pasteurization (VAT and HTST) at the end of storage (42 days) at low temperature. HHP/T (MPa/min)a Characteristic

Controlb

Appearance Color Aroma Sweetness Flavor Acceptability

7.25 7.40 7.10 7.45 7.40 7.55

a

b

± ± ± ± ± ±

500/10 1.02ab 1.19ab 1.48a 1.23a 1.54a 1.39a

6.95 7.25 7.00 7.20 7.35 7.35

± ± ± ± ± ±

Pasteurization 550/10

1.05b 1.21ab 1.26a 1.54a 1.18a 0.87a

7.15 7.05 7.05 6.75 6.75 6.80

± ± ± ± ± ±

600/5 1.31b 1.43b 1.00a 1.92a 2.05a 1.70a

7.15 7.15 6.80 6.75 6.65 7.10

± ± ± ± ± ±

VAT 1.27b 1.31b 1.44a 1.48a 1.76a 1.41a

7.90 8.20 7.30 7.40 7.45 7.65

HTST ± ± ± ± ± ±

0.72ab 0.69a 1.56a 1.35a 1.43a 1.35a

8.15 8.25 7.25 7.00 7.35 7.40

± ± ± ± ± ±

0.74a 0.55a 1.41a 1.62a 1.76a 1.23a

Different letters within rows indicate significant differences (p ≤ .05). Data of control: untreated fermented beverage.

observed only within appearance and color of control and processed beverages. Sweetness, aroma, flavor and general acceptability did not have any statistical difference (p > .05). In most of the sensory attributes, a minimum average value of 7 (like much) was given by panelists to control and HHP processed and pasteurized FP beverages. Keenan et al. (2011) pointed out that no significant differences were observed about acceptability of HHP processed and pasteurized smoothies of fruits. Tao et al. (2012) reported differences in the appearance of red wines treated with HHP (650 MPa/45 min). It should be taken into account that in general there are “likes” and “dislikes” within any type of food and beverage and within qualities of

different types of foods and beverages. The results of the sensory evaluation are of paramount importance since samples treated with heat could lose color or be browning due to thermal caramelization (Tao et al., 2012). In this study, pomegranate FP beverages treated with heat were those with the highest loss of pigments and with the greatest color changes; nevertheless, they were still well accepted by panelists. 4. Conclusions The high hydrostatic pressure processed beverages, in the three HHP processing conditions (500 MPa/10 min, 550 MPa/10 min and 9

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600 MPa/5 min), were similarly microbiological stable as pasteurized beverages after 42 days of storage. HHP processing in FP beverages slightly increased the antioxidant compounds after pressurization; however, the storage time caused a slight decrease of them. The physicochemical parameters were not affected by thermal and HHP treatments or by the storage time. The color attributes of thermally processed beverages changed significantly, generating degradation of anthocyanins. The HHP processing did not affect the taste of FP beverages. All thermally and HHP processed FP beverages were well sensory accepted. The HHP processing could be an alternative for processing FP beverages, generating an innocuous and sensory acceptable beverage, containing adequate amounts of antioxidants. However, there is a need for making more studies about the effect of HHP processing over antioxidant compounds, sensory attributes and physicochemical characteristics of FP beverages and their shelf life.

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