Food Bioscience 41 (2021) 101106

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Evaluation of freeze crystallization on pomegranate juice quality in

comparison with conventional thermal processing
Patricio Orellana-Palma a, *, María Guerra-Valle b, María Pía Gianelli b, Guillermo Petzold b
a
Department of Biotechnology, Universidad Tecnológica Metropolitana, Las Palmeras, 3360, P.O. Box, 7800003, Ñuñoa, Santiago, Chile
b
Department of Food Engineering, Universidad Del Bío-Bío, Av. Andrés Bello 720, Casilla 447, Chillán, Chile

A R T I C L E I N F O A B S T R A C T

Keywords: The objective of the present study was to evaluate the potential of freeze crystallization (FC) as concentration
Freeze crystallization technology for pomegranate juice. By comparing with conventional thermal treatment (evaporation), the effect
Evaporation on quality parameters (physicochemical properties, bioactive compounds, antioxidant activity, and aromatic
Pomegranate juice
profile) at three cycles was investigated. In the last cycle, the results showed that both FC and evaporation
Bioactive compounds
Antioxidant activity
technologies, concentrate similar total soluble solids (≈48 ◦ Brix). However, FC maintained a comparable color to
Aromatic profile the initial sample (red color), while the evaporated samples presented a browning color. FC efficiently retained
higher bioactive compound concentration than heat treatment (50 ◦ C), with values close to 87%, 71%, 69% and
67% for total polyphenol, anthocyanin, tannin, and flavonoid content, respectively. A similar behavior, with
highest values in FC, was observed in the antioxidant capacity, since FC resulted in an increase of 5.19, 5.12, 5.29
and 4.05-fold versus 3.11, 3.60, 3.87 and 1.97-fold in relation to the initial value, for 2,2-diphenyl-1-picrylhy­
drazyl (DPPH), 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) (ABTS), ferric reducing antioxidant
power (FRAP) and oxygen radical absorbance capacity (ORAC) assays, respectively. In aromatic profile, FC
allowed preservation of volatile compounds from the fresh juice. Instead, heat treatment only retained 68% of
the volatiles components present in the initial sample. Therefore, compared with evaporation, our results suggest
that FC is a potential alternative as non-thermal technology to preserve diverse and interesting components from
fresh juice.

1. Introduction
Pomegranates (Punica granatum L.) have been recognized worldwide
as a “super fruit” due to their abundant valuable effects on consumers,
because is a fruit with high level and variety of bioactive components.
Therefore, pomegranate consumption has been associated with relevant
health-beneficial properties against different diseases, as they exhibit
anti-inflammatory, anti-cardiovascular, anti-carcinogenic, anti-micro­
bial, anti-neurodegenerative, anti-diabetic and anti-mutagenic activities
(Melgarejo-Sánchez et al., 2021). Similarly, scientific investigations
have demonstrated positive properties for the prevention of allergic
signs, gastrointestinal problems and oral hygiene problems (Kandylis &
Kokkinomagoulos, 2020). The edible parts (arils) are consumed directly
or commonly used to elaborate diverse food products, such as fresh juice
jams, jellies, liqueurs, wine, and teas, among others non-food products
(Borges et al., 2019).
At present, traditional thermal treatments (evaporation,
pasteurization and/or sterilization) are the most used technology in the
food liquid industry to obtain a concentrate product with significant
improvement in quality and prolonged shelf life when compared to the
fresh juice. Unfortunately, the high-temperatures used in evaporation
processing have negative impacts on different quality properties (color
degradation, loss of nutritional and aroma characteristics, and alteration
in the final taste) due to the thermolabile and thermostable sensitivities
of each component in the original sample, and thus, these undesirable
effects leads to consumers’ dissatisfaction (Rehman et al., 2020).
Therefore, it is essential to find innovative non-thermal technologies to
preserve quality properties in the final concentrate from fresh juice
(Khan et al., 2018).
Previous studies on freeze crystallization (FC) have shown success in
maintaining different heat-labile components from fresh liquid foods
(Adorno et al., 2017; Orellana-Palma, Lazo-Mercado, et al., 2020), since
it is a green and emerging non-thermal preservation technology (Yoda
et al., 2021). Moreover, FC has more advantages than conventional

* Corresponding author.
E-mail address: p.orellanap@utem.cl (P. Orellana-Palma).

https://doi.org/10.1016/j.fbio.2021.101106
Received 26 March 2020; Received in revised form 30 March 2021; Accepted 25 April 2021
Available online 30 April 2021
2212-4292/© 2021 Published by Elsevier Ltd.
P. Orellana-Palma et al.
thermal or membrane technologies, since FC requires low energy costs
and presents high efficiency. Whereby, FC has become a technology with
great potential. Specifically, the FC process consists of freezing the liquid
sample below its freezing point, in order to achieve complete solidifi­
cation of the sample, and later, the unfrozen solution (cryoconcentrated
fraction) is separated from the ice fraction. The separation stage is
possible, since in the first step, as the temperature decrease the solutes
are rejected from the ice phase and accumulate at the solid-liquid
interphase, i.e., the cryoconcentrated fraction is between the ice crys­
tals (Orellana-Palma, Tobar-Bolaños, et al., 2020).
Among FC techniques, block freeze crystallization (BFC) has been
studied extensively due to their favorable characteristics, in operation,
equipment, and construction terms (Amran et al., 2016). In BFC, unlike
the other FC techniques, a liquid sample is totally freezing, then the
whole block is thawed and the concentrated phase is separated from the
ice phase by gravitational thawing (Aider & Ounis, 2012) or incorpo­
rated an assisted technique to increase some process parameters, among
them, efficiency, solute yield and percentage of concentrate (Petzold
et al., 2016, pp. 184–190). Particularly, centrifugation has been widely
used as assisted technique, since it takes advantage of the centrifugal
force, and thus, a high magnitude of driving force allows the occluded
solution extraction between the ice crystals (Casas-Forero et al., 2020).
Nevertheless, to the best of our knowledge, there is limited scientific
information that investigates the differences between FC with other
thermal or non-thermal technologies. Specifically, Balde and Aider
(2019) compared the impact of cryoconcentration by passive thawing,
vacuum evaporation and reverse osmosis on physicochemical and
rheological properties applied to skim milk, without emphasis on
nutritional or organoleptic properties. Meneses et al. (2021) studied the
effect of BFC on the solutes, catechin content, polyphenol content,
antioxidant activity and sensorial profile of green tea (Camellia sinensis)
extract, and later, the results post-BFC were compared with evaporation
test. Then, to date, BFC have not been compared with other concen­
tration thermal or non-thermal techniques to obtain a rich-solute final
concentrate with significant retention of values components (phenolic
compounds, antioxidant activity, volatile compounds, among others)
from fresh fruit juice with a broad interest by their multi-attribute health
effects (Puneeth & Chandra, 2020), e.g., pomegranate juice.
Thereby, the innovation and novelty on the use of BFC are based in
the significant information in terms of physicochemical characteristics,
nutritional and organoleptic properties that could be obtained in each
cryoconcentrated samples in comparison to other concentration tech­
niques, and in turn, this comparison could be essential information to
produce juices with important quality properties, and for future appli­
cations more focused on health and industrial scale.
Therefore, the aim of this study was to evaluate the effects of cen­
trifugal BFC (CBFC) and evaporation technique with regards to physi­
cochemical properties, bioactive compounds, antioxidant activity and
aromatic profile in fresh pomegranate juice.
2. Materials and methods
2.1. Chemicals and reagents
Sodium hydroxide (NaOH), sodium carbonate (Na2CO3), sodium
nitrite (NaNO2), aluminum chloride (AlCl3), hydrochloric acid (HCl),
potassium chloride (KCl), potassium persulfate (K2S2O8), sodium acetate
(CH3COONa), ferric chloride hexahydrate (FeCl3⋅6H2O), Folin-
Ciocalteu reagent, Folin-Denis reagent, gallic acid, cyanidin-3-
glucoside, (+)-catechin, tannic acid, fluorescein solution, DPPH (2,2-
dipheny-l-1-picrylhydrazyl), ABTS (2,2′ -Azino-bis(3-ethylbenzothiazo­
line-6-sulfonic acid)), TPTZ (2,4,6-tris(2-pyridyl)-S-triazine), AAPH
(2,2′ -azobis(2-methylpropionamide)-dihydrochloride) and Trolox
((±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Distilled
water was used throughout. All reagents were used in analytical grade.
2
Food Bioscience 41 (2021) 101106
2.2. Juice preparation
Fresh mature pomegranates (P. granatum cv. Wonderful) were pur­
chased in Chillán, Región del Ñuble, Chile, and the fruits were stored
under refrigeration condition (≈4 ◦ C) for a maximum of three days until
further analysis. The pomegranates were cleaned and washed, cutted in
half longitudinally, and the arils were removed manually. The arils were
crushed, and the juice was filtered through nylon cloth (0.8 mm mesh) to
discard the impurities and solid parts (peel and seeds) for avoiding the
presence of any residue that might interfere with the concentration
processes.
2.3. Concentration procedures
2.3.1. Block freeze crystallization (BFC) assisted by centrifugation
The CBFC procedure was carried out according to the method
described by Orellana-Palma et al. (2017) with some minor modifica­
tions. Pomegranate juice (45 mL) was put into plastic centrifugal tubes.
The tubes with juice were isolated with foam polystyrene (8 mm
thickness, K = 0.035 W/mK) in order for heat transfer to occur axially
(from bottom to top), and later, the samples were frozen in a static
freezer (-20 ◦ C, overnight).
The frozen samples were subjected to centrifugation (Hettich D-
7853, Tuttlingen, Germany) at 20 ◦ C for 15 min and 1600×g to force the
cryoconcentrated fraction (Cs) extraction from the frozen matrix (Cf).
The Cs at the first cycle was collected and used for the next cycle (i.e. the
cryoconcentrate juice was frozen under the same conditions and trans­
ferred to a refrigerated centrifuge), and so, the second Cs was used for
the third cycle. After each cycle, in the Cs, quality properties, i.e.,
physicochemical properties, bioactive compounds, antioxidant activity
and volatile compounds were determined.
2.3.2. Vacuum evaporation procedure
The fresh pomegranate juice was concentrated using a vacuum
evaporator (Rotavapor R-100, Büchi Labortechnik AG, Flawil,
Switzerland) at 50 ◦ C for 80 mbar, and the procedure was stopped when
the total soluble solids content (TSSC, ◦ Brix) of the evaporate juice was
similar to the CBFC sample obtained in each cycle. Similarly, quality
properties (physicochemical properties, bioactive compounds, antioxi­
dant activity and volatile compounds) at each evaporation cycle were
determined.
2.4. Physicochemical analysis
The TSSC was quantified using a digital refractometer (PAL-1, Atago
Co., Ltd., Tokyo, Japan). The pH was measured using a digital pH meter
(HI 2210, Hanna Instruments, Woonsocket, USA). The total titratable
acidity (TTA) and the density (ρ) were determined according to AOAC
standard procedures (AOAC, 1995), and the results were expressed as
grams (g) of citric acid (CA) per 100 mL of sample (g CA/100 mL), and as
grams (g) per milliliter (mL) of sample (g/mL), respectively. The color
properties were done in a spectrophotometer (CM-5, Konica Minolta,
Osaka, Japan) and the measurement was made using the CIELab system,
with illuminant D65 and 10◦ observer angle. The Hunter color values
were expressed as L* (lightness; 0 = black, 100 = white), a* (− a* =
greenness, +a* = redness) and b* (− b* = blueness, + b* = yellowness).
The CIELab values were used to calculate the total color difference (ΔE*)
between the concentrated samples and fresh pomegranate juice by the
equation (Eq. (1)). All physicochemical analysis were performed at
ambient temperature (≈22 ◦ C).
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE* = (ΔL* )2 + (Δa* )2 + (Δb* )2 (1)
where ΔL* = (L* – L0*), Δa* = (a* – a0*), Δb* = (b* – b0*). Subscript
0 indicates initial fruit pomegranate juice color.
P. Orellana-Palma et al.
2.5. Quantification of total bioactive compound (TBC)
Total polyphenol content (TPC) of fresh pomegranate juice and
concentrate samples was estimated colorimetrically by the
Folin–Ciocalteu method (Singleton & Rossi, 1965), with some modifi­
cations. First, 100 μL of sample was mixed with 500 μL of 10-fold diluted
Folin–Ciocalteu reagent and 1500 μL of 7.5% Na2CO3 solution. The
mixture was kept in dark at room temperature for 90 min (incubation),
and the absorbance was measured at 760 nm. The TPC was calculated
using gallic acid (GA) as standard curve and the results were expressed
as mg GA equivalents (GAE) per 100 g of dry matter (mg GAE/100 g d.
m.).
Total anthocyanin content (TAC) was estimated by pH differential
method as described by Çam et al. (2009), with modifications. 400 μL of
sample was added to 3600 μL of 0.025 M KCl (pH 1.0) and 3600 μL of
0.4 M CH3COONa (pH 4.5). The solutions were kept in dark at room
temperature for 30 min (incubation), and the absorbance was measured
at 520 (A520) and 700 (A700) nm. TAC was calculated using
cyanidin-3-glucoside (C3G) as standard curve and the results were
expressed as mg C3G equivalent per 100 g of dry matter (mg C3G/100 g
d.m.) by the following equation (Eq. (2)):
(mg)
A*MW*DF*1000
TAC = (2)
L ε*1
where A = [(A520 – A700)pH1.0] – [(A520 – A700)pH4.5], MW = Molecular
weight for cyanidin-3-glucoside (499.2 g/mol), DF = Dilution factor,
1000 = factor for conversion from (g/L) to (mg/L), ε = Molar extinction
coefficient for C3G (26900 L/mol*cm), l = Path length (1 cm).
Total tannin content (TTC) was calculated by the Folin–Denis
method (Li et al., 2015), with modifications. 100 μL of sample was
mixed with 1000 μL of distilled water, 500 μL of Folin–Denis reagent and
100 μL of 7.5% Na2CO3 solution. The mixture was kept in dark at room
temperature for 30 min (incubation), and the absorbance was measured
at 760 nm. TTC was calculated using tannic acid (TA) as standard curve
and the results were expressed as mg TA equivalent per 100 g of dry
matter (mg TAE/100 g d.m.).
Total flavonoid content (TFC) was determined by the aluminum
chloride colorimetric method following the procedure reported by
Dewanto et al. (2002), with minor modifications. 250 μL of sample was
mixed with 1000 μL of distilled water and 75 μL of 5% NaNO2 solution.
After 6 min, 150 μL of 10% AlCl3 solution, 500 μL of 1M NaOH and 2500
μL of distilled water were added to the solution. The solution was kept in
dark at room temperature for 40 min (incubation), and later, the
absorbance was measured at 510 nm. TFC was calculated using catechin
(C) as standard curve and the results were expressed as mg C equivalent
per 100 g of dry matter (mg CE/100 g d.m.).
All TBC were quantified on a UV/vis spectrophotometer (T70, Oasis
Scientific Inc., Greenville, USA).
2.6. Bioactive compound retention (BCR)
The BCR is the TBC percentage in the Cs respect to the fresh juice. The
BCR was calculated at each CBFC cycle using the following equation (Eq.
(3)) (Orellana-Palma et al., 2017).
( ) ( ) gradients at 4 ◦ C/min were applied, until reaching 130 ◦ C, 150 ◦ C, and
C0 BCf
BCR (%) = ∗ ∗100 (3)
Cf BC0
where C0 is the initial TSSC (◦ Brix), Cf is the TSSC value (◦ Brix) at each
cycle, BCf is the TBC at each CBFC cycle, and BC0 is the initial TBC.
2.7. Antioxidant properties
DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was assessed based on
Brand-Williams et al. (1995)’ method, with minor modifications. 150 μL
of sample was mixed with 2850 μL of DPPH methanolic solution. The
3
Food Bioscience 41 (2021) 101106
mixture was kept in dark at room temperature for 30 min (incubation).
Later, the absorbance was measured at 515 nm.
ABTS•+ (2,2ʹ-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid))
assay was estimated by the method established by Re et al. (1999), with
modifications. Briefly, the ABTS reagent was produced by reacting ABTS
stock solution (7 mM) with sodium acetate buffer (20 mM, pH 4.5).
Then, 7 mM ABTS solution was dissolved in potassium persulfate solu­
tion (2.45 mM) (1:1 ratio), and it was allowed to stand for 12 h and
stored for use. Thus, 50 μL of 10-fold diluted sample was added to 4000
μL of ABTS solution, and it was vigorously mixed for 5 min. The mixture
was kept in dark at room temperature for 30 min (incubation), and the
absorbance was measured at 734 nm.
Ferric reducing antioxidant power (FRAP) assay was performed ac­
cording to Benzie and Strain (1996) procedure, with some modifica­
tions. Briefly, FRAP reagent was prepared with 50 mL of sodium acetate
buffer (300 mM, pH 3.6), 5.0 mL of TPTZ (10 mM in hydrochloric acid
(40 mM)) and 5.0 mL of FeCl3⋅6H2O (20 mM) (10:1:1 ratio), respec­
tively, and then, the mixed solution was incubated at 37 ◦ C for 30 min.
150 μL of sample was mixed with 3000 μL of FRAP reagent. The solution
was kept in dark at room temperature for 10 min (incubation), and the
absorbance was measured at 595 nm.
DPPH, ABTS and FRAP assays were quantified on a spectropho­
tometer (T70 UV/VIS spectrophotometer, Oasis Scientific Inc., USA).
Oxygen-radical absorbance capacity (ORAC) method was deter­
mined using the method reported by Ou et al. (2001). Specifically, 30 μL
of sample and 20 μL of fluorescein solution (10 nM) were placed into
black 96-well microplates and incubated at 37 ◦ C for 30 min. Later, 50
μL of AAPH (600 mM) and 2900 μL of phosphate buffer (75 mM, pH 7.4)
were added to the solution. The absorbance was measured every 1 min
for 60 min at an excitation wavelength of 485 nm and an emission set of
520 nm using a multimode plate reader (Victor X3, PerkinElmer,
Hamburg, Germany).
For all assays, Trolox (T) was used as standard curve, and the results
were expressed as mM Trolox equivalents (TE) per 100 g of dry matter
(mM TE/100 g d.m.).
2.8. Quantification of volatile compounds
The volatiles compound identification was performed according to
Orellana-Palma, Zuñiga, et al. (2020). Briefly, 8 mL of sample was
placed in a 12 mL glass vial sealed with a PTFE-faced silicone septum
(Supelco, Bellefonte, PA, USA). Prior to analysis, samples were equili­
brated at 50 ◦ C for 60 min in a thermoblock (2050-ICE, Paris, France).
The volatile compounds were extracted from the vial headspace by solid
phase microextraction (SPME) technique, with a gas
chromatograph-flame ionization detector (GC-FID). An SPME holder
(Supelco, Bellefonte, PA, USA) containing a fiber carbox­
en/polydimethylsiloxane (85 μm, Car/PDMS) was used to adsorbed the
volatile compounds in the vial headspace, which was introduced in the
GC (PerkinElmer, Clarus 680, Shelton, CT, USA). The GC system was
equipped with a DB-624 (60 m × 0.25 mm × 1.8 μm) capillary column
(J&W Scientific, Folsom, CA, USA) to separate the compounds. In the
first 5 min, the oven was programmed to start at 50 ◦ C, and the tem­
perature was increased by 4 ◦ C/min until reaching 98 ◦ C. Later, three
230 ◦ C, respectively. Nitrogen was used as carrier gas and the flow rate
was 1.2 mL/min. The volatiles compounds were identified and verified
by their retention time, authentic standards and Kováts Index (KI).
2.9. Statistical analysis
The data were subjected to analysis of variance (ANOVA) and dif­
ferences between means were compared via least significant difference
(LSD) test (p ≤ 0.05) using Statgraphics Centurion software (Version
XVI, StatPoint Technologies Inc., Warrenton, VA, USA). All experiments
and analyses were performed in triplicate.
P. Orellana-Palma et al. Food Bioscience 41 (2021) 101106

Table 1
Physicochemical parameters of fresh pomegranate juice and concentrate samples.
Sample Cycle TSSC (◦ Brix) pH TTA (g CA/100 mL) Density (g/mL) L* a* b* ΔE*
a a a a a a a
Fresh juice 16 ± 1 3.3 ± 0.0 1.5 ± 0.0 1.1 ± 0.0 39 ± 0 14 ± 0 2.9 ± 0.1 –
CBFC 1 32 ± 2b 3.1 ± 0.0b 2.0 ± 0.0b 1.2 ± 0.0b 24 ± 0b 12 ± 0b 2.0 ± 0.0b 15 ± 0a
2 42 ± 2c 3.0 ± 0.0c 2.5 ± 0.1c 1.2 ± 0.0c 12 ± 0c 7.8 ± 0.3c 1.5 ± 0.0c 27 ± 0b
3 48 ± 2d 2.9 ± 0.0d 3.2 ± 0.1d 1.3 ± 0.0d 3.1 ± 0.1d 3.9 ± 0.1d 0.66 ± 0.01e 37 ± 0c
Evaporation 1 31 ± 1b 3.1 ± 0.0b 2.0 ± 0.0b 1.1 ± 0.0b 20 ± 0b 8.0 ± 0.1b 1.9 ± 0.0b 19 ± 0a
2 42 ± 2c 3.0 ± 0.0c 2.4 ± 0.1c 1.2 ± 0.0c 9.2 ± 0.0c 5.2 ± 0.1c 1.1 ± 0.0c 31 ± 0b
3 48 ± 1d 2.9 ± 0.0d 3.2 ± 0.1d 1.3 ± 0.0d 1.2 ± 0.0d 2.4 ± 0.1d 0.58 ± 0.14d 39 ± 0c
a,b,c,d
Different letters in the same column show significant differences at 5% between homogeneous groups in each variable to a least significant difference test (LSD).

3. Results and discussion
3.1. Evaluation of physicochemical parameters
Table 1 summarizes TSSC, pH, TTA, density and color of fresh
pomegranate juice and concentrates obtained by CBFC and evaporation
at three cycles. An important point, the TSSC values did not presented
significant statistical differences when compared between CBFC and
thermal processing at each cycle.
Firstly, the fresh pomegranate juice has an initial TSSC value close to
16 ◦ Brix, which is a value within the range established by Mena et al.
(2011) for cv. Wonderful (14.6–17.6 ◦ Brix), but slightly lower than those
indicated by Varela-Santos et al. (2012) (16.9 ◦ Brix). The differences or
similarities in the TSSC values of pomegranate juice can be connected to
the fact that the studies were carried out with fruits from different
harvest areas of the world, and these zones are characterized by various
climatic conditions and geographical characteristics. Additionally,
type/time of harvesting, ripening process, genetic and species variabil­
ities, growing season, and/or the interaction genotype-environment can
change the properties of the fruit, and in turn, in the final juice
(Topalović et al., 2020).
Thus, the TSSC increased significantly cycle to cycle with values of
approximately 32, 42 and 48 ◦ Brix, which is equivalent to a concen­
tration index (CI, ratio Cs/C0) of 2.0, 2.7 and 3.1 times, compared with
the initial TSSC value in the first, second, and third cycle, respectively.
In comparison with previous studies in our laboratory, the results (third
cycle) presented lower CI value than those achieved in orange juice (5.7)
(Orellana-Palma et al., 2019), apple juice (3.9) (Orellana-Palma,
Lazo-Mercado, et al., 2020), and pineapple juice (3.3) (Orellana-Palma,
Zuñiga, et al., 2020), but higher than those reached in blueberry juice
(2.5) (Petzold et al., 2015), and calafate juice (3.0) (Orellana-Palma,
Tobar-Bolaños, et al., 2020). Furthermore, the TSSC values were supe­
rior to those described by Moreno et al. (2015) and Ding et al. (2019),
who used block FC and suspension FC to cryoconcentrate coffee extract
and apple juice, respectively. These differences could be explained by
the freezing conditions and sized tubes capacity used in the BCC process.
Specifically, we used an axial freezing front propagation and a moderate
freezing rate temperature (-20 ◦ C), which allows an improve
counter-diffusion of solutes from the growing crystal surface. Moreover,
the centrifugal equipment has a sized tubes capacity of 50 mL-tube,
which favors the cryoconcentrate extraction from the frozen fraction in
the centrifugation process (Orellana-Palma et al., 2017). An important
point, Rehman et al. (2019) used osmotic distillation with membrane
contactor at a temperature of 25 ◦ C to concentrate pomegranate juice,
reaching a final solute concentration close to 52.0 ◦ Brix during 310 min
of operation, while our results (48 ◦ Brix) were reached during 45 min of
centrifugation (15 min by cycle). Thereby, CBFC presents a significant
advantage in the time used to obtain a slightly lower solute concentra­
tion than the treatment by osmotic distillation with membrane
contactor.
No significant statistical differences (p ≥ 0.05) were observed for the
pH, acidity and density values when comparing the same cycle between

Fig. 1. Visual appearance of fresh pomegranate juice and concentrate samples at each cycle.

4
P. Orellana-Palma et al.
Fig. 2. Effect of centrifugal block freeze crystalliazation (CBFC) and thermal treatment (evaporation) on total bioactive compound. (a) total polyphenol; (b)
anthocyanin; (c) tannin; and (d) flavonoid content applied to fresh pomegranate juice. Numbers represent the bioactive compound retention (BCR).
concentration techniques. The initial pH and TTA content values (fresh
juice) were comparable to those previously reported by Mphahlele et al.
(2016) in the same variety juice. Precisely, the pH decreased and TTA
increased gradually in relation to the fresh pomegranate juice. This
opposite performance has been recognized to the high solutes in the
sample at each cycle, which generates the organic acid content increase
(Khajehei et al., 2015). A similar behavior to TTA was observed in ρ,
since in the final cycle, ρ presented an increment of approximately 25%
compared to initial ρ value. This performance was equivalent with the
results informed by Orellana-Palma, Lazo-Mercado, et al. (2020) in
cryoconcentrate apple juice.
Moreover, in color property terms, the fresh pomegranate juice
values were lower (L* ≈39.0, a* ≈14.0, b* ≈3.0) than results obtained
with the same variety (Cano-Lamadrid et al., 2018). The difference in
the CIELab values could be related by various factors such as specific
fruit characteristics, agroclimatic conditions and/or method of fruit
pressing, among others (Beaulieu et al., 2015). Hence, the fruit juice was
significantly affected after applied CBFC or evaporation, since L*, a* and
b* values decreased at each cycle, i.e., the concentrates were darker than
the natural juice. This darkening of the samples could be accredited to
the increase in solutes, since as the cycles progressed; more concentrated
solute was separated from the ice fraction (CBFC) or water (evapora­
tion). The gradual decrease of CIELab values presented concordance
with previous results for cryoconcentrated strawberry juice (Adorno
et al., 2017), blueberry juice (Orellana-Palma et al., 2017) and apple
juice (Zielinski et al., 2019).
In particular, as shown in Fig. 1, the color in both CBFC and
5
Food Bioscience 41 (2021) 101106
evaporation samples changed from light red (fresh juice) to a dark color,
with the advance of the cycles. However, the evaporation samples pre­
sented a light brown (first cycle) to a dark brown color at the last cycle,
which indicates degradation during high temperature process.
The total color difference (ΔE*) was analyzed to assess the color
change, perceived under the human eye, between fresh juice and each
concentrated sample. According to the results, ΔE* values were over 15
CIELab units, and it reached ΔE* values of 37 and 39 for CBFC and
evaporation, in the final cycle, respectively. Thus, all the values indi­
cated that the human eye can find differences between fresh juice and
the concentrates based on the scale proposed by Krapfenbauer et al.
(2006) (ΔE*≥3.5 CIELab units allow denote visual differences by the
consumers between food products). The change in ΔE* values were
clearly depending on the TSSC in the sample, since a high ΔE* between
samples was obtained by increasing the TSSC (Casas-Forero et al., 2021).
In all cases, the ΔE* values acquired by evaporation were higher than
those obtained by CBFC, which indicates that CBFC can retain the
original attractive color of the fresh sample better than conventional
thermal processing (Orellana-Palma, Lazo-Mercado, et al., 2020).
3.2. Total bioactive compound (TBC) determinations
Fig. 2 shows the TBC of fresh juice and concentrate samples. The
fresh pomegranate juice presented TPC, TAC, TFC and TTC values close
to 422 mg GAE/100 g d.m., 192 mg C3G/100 g d.m., 72 mg TAE/100 g
d.m. and 51 mg CE/100 g d.m., respectively. The results are in agree­
ment with those reported by Sepúlveda et al. (2010), who study different
P. Orellana-Palma et al. Food Bioscience 41 (2021) 101106

Table 2
Antioxidant activity of fresh pomegranate juice and concentrates at each cycle.
Antioxidant activity DPPHa ABTSa FRAP** ORAC**

CBFC Evaporation CBFC Evaporation CBFC Evaporation CBFC Evaporation

Fruit juice 2.7 ± 0.4a 5.7 ± 0.2a 6.4 ± 0.3a 6.0 ± 0.4a
C1 6.7 ± 0.3b 4.2 ± 0.2b 15 ± 1b 11 ± 1b 19 ± 1b 13 ± 0b 11 ± 0b 6.6 ± 0.1b
C2 10 ± 1c 6.0 ± 0.4c 22 ± 0c 16 ± 0c 26 ± 1c 21 ± 0c 16 ± 1c 8.0 ± 0.4c
C3 14 ± 1d 8.3 ± 0.2d 29 ± 0d 21 ± 1d 34 ± 1d 25 ± 1d 24 ± 1d 12 ± 0d

Values with different superscript in a row are significantly different (p ≤ 0.05), according to LSD test or t-student test.
C1, C2 and C3 represents cycle 1, cycle 2 and cycle 3, respectively.
a
Free radical scavenging capacity; **Ferric reducing antioxidant power.

pomegranate cultivars present in Chile. The difference in TBC values
could be explained by several factors, such as harvesting year, ripening
stage, environmental/climate and storage conditions, final maturity, as
well as, the methodology in the sample preparation (fresh juice) (Gar­
cía-Pastor et al., 2020).
Compared with the fresh sample, TBC values significantly increased
(p ≤ 0.05) cycle to cycle, in both CBFC and evaporation concentration
techniques. However, the CBFC results were higher than the thermal
processing. The highest CBFC and evaporation values were achieved in
the last cycle, with an increase of 2.66, 2.16, 2.11 and 1.22-fold versus
2.12, 1.90, 1.85 and 1.25-fold in relation to the initial value, for TPC,
TAC, TTC and TFC, respectively. The TBC behavior on upward could be
influenced by the TSSC values at each cycle in the separation/extraction
process (see Table 1). A comparable tendency was observed in cry­
oconcentrated blueberry juice (Orellana-Palma et al., 2017), coffee
extract (Correa et al., 2018), Centella Asiatica extract (Amran et al.,
2020), broccoli extract (Azhar et al., 2020), and apple juice (Yoda et al.,
2021). Likewise, many studies have indicated that the high TBC values
in CBFC are due to the low processing temperatures to concentrate
solutes, and in turn, it allows an exceptional and high compound
retention (Petzold et al., 2016, pp. 184–190). Nevertheless, despite the
fact that the high temperature process allows to concentrate at a similar
level as CBFC, the temperature used (50 ◦ C) degraded bioactive com­
pounds, and this generates a low final retention compared to CBFC.
In each TBC, the retention was superior to 50%, and it was increased
along the cycles. Specifically, CBFC presented higher values than
evaporation with 73–87%, 60–71%, 59–69% and 56–67% from the first
to third cycle, for TPC, TAC, TTC and TFC, respectively. This phenom­
enal was in line with findings by Correa et al. (2018), Orellana-Palma,
Lazo-Mercado, et al. (2020), and Casas-Forero et al. (2020), in aqueous
coffee extract, apple juice, and blueberry juice, with final BCR values
close to 90%, 85%, and 71%–91%, respectively.
Hence, the TBC and retention values showed the positive effects of
subzero temperatures concentration technique to preserve susceptible
and thermolabile bioactive compounds in the concentrate samples,
which are highly degraded at high temperatures. Therefore, CBFC
technology can be visualized as a novel non-thermal technology to
concentrate, preserve and retain an endless number of phenolic com­
ponents such as polyphenols, anthocyanins and flavonoids, among
others, in the cryoconcentrated fraction (Gunathilake, 2020, pp. 39–69).

Table 3
Volatile compounds detected in fresh pomegranate juice, concentrates by CBFC and evaporation at the third cycle.
Peak Compound tRa KIb Fresh juice CBFC Evaporation
N◦ (min)
Experimental Reportedc Chemical Aread Areae Aread (μV∙s) Areae Aread Areae
group (μV∙s) (%) (%) (μV∙s) (%)

1 2-Methyl-propanal 5.02 594 594 Aldehydes 77 ± 19 0.57 61 ± 0 0.10 – 0.00

2 1-Propanol 6.27 614 614 Alcohols 76 ± 3 0.57 2765 ± 467 4.36 – 0.00
3 Butanal 6.66 620 624 Aldehydes 1336 ± 9 10.01 1357 ± 1 2.14 1217 ± 20 21.19
4 Ethyl acetate 7.36 631 633 Esters 100 ± 9 0.75 34629 ± 54.58 – 0.00
2159
5 2-Butanol 8.63 651 652 Alcohols 75 ± 1 0.56 94 ± 2 0.15 – 0.00
6 2-Methyl-3-buten- 8.89 655 654 Alcohols – 0.00 317 ± 15 0.50 1444 ± 71 25.13
2-ol
7 3-Methyl-2- 11.87 702 700 Ketones 7041 ± 217 52.77 10449 ± 16.47 1940 ± 33.74
butanone 1528 543
8 2-Pentanone 13.65 730 730 Ketones 196 ± 3 1.47 158 ± 23 0.25 – 0.00
9 Pentanal 14.10 737 737 Aldehydes 91 ± 8 0.69 265 ± 25 0.42 164 ± 65 2.85
10 3-Methyl-1-butanol 17.59 792 734 Alcohols 742 ± 86 5.56 65 ± 19 0.10 – 0.00
11 Octane 17.91 797 800 Alkanes – 0.00 76 ± 22 0.12 62 ± 2 1.08
12 Hexanal 20.77 842 842 Aldehydes 429 ± 88 3.22 66 ± 4 0.10 250 ± 70 4.35
13 Nonane 24.45 900 900 Alkanes 343 ± 5 2.57 3571 ± 470 5.63 – 0.00
14 3-Methylbutyl 24.90 907 906 Esters 2219 ± 65 16.63 8424 ± 1210 13.28 84 ± 4.5 1.46
acetate
15 Methyl hexanoate 27.70 951 950 Esters – 0.00 63 ± 4 0.10 246 ± 39 4.28
16 α-terpinene 33.10 1036 1037 Terpenes 99 ± 30 0.74 61 ± 2 0.10 74 ± 4 1.29
17 NI 35.26 1070 – – 254 ± 46 1.91 713 ± 190 1.12 98 ± 21 1.70
18 Linalool 40.28 1149 1148 Terpenoids 177 ± 30 1.32 232 ± 46 0.37 168 ± 83 2.93
19 1-Nonanol 43.96 1207 1207 Alcohols 87 ± 19 0.65 80 ± 22 0.13 – 0.00
Total 13343 ± 100 63447 ± 100 5746 ± 100
640 6209 921
a
Retention time.
b
Kováts index calculated for DB-624 capillary column.
c
Already reported in Di Cagno et al. (2017), Beaulieu and Obando-Ulloa (2017), and Mantzourani et al. (2020).
d
Total ion current (TIC) area of gas chromatography-flame ionization detector (GC-FID) ± standard deviation.
e
Percentage of the TIC area. NI: unidentified.

6
P. Orellana-Palma et al. Food Bioscience 41 (2021) 101106

3.3. Antioxidant activity (AA) determinations

The AA of the fresh pomegranate juice, CBFC and evaporation

samples are shown in Table 2. The AA values (mM TE in 100 g, on dry
matter) in the fresh juice were approximately 2.7, 5.7, 6.4 and 6.0 for
DPPH, ABTS, FRAP and ORAC, respectively. These values were lower
than those found by Sepúlveda et al. (2010) and Varela-Santos et al.
(2012) in pomegranate juice. The differences in AA values could be due
to climatic and agricultural conditions, since the studies were focused in
different Chilean grown pomegranate cultivars, i.e., the fruits were
harvested in various regions of Chile (North Region and Central Region),
which are highlighted by dry temperate climates, with temperature
varies from 10 ◦ C to 30 ◦ C throughout the year, while the Southern Chile
(Chillán) has constant rainy temperate climates in the year (Aceituno
et al., 2020, pp. 7–29). Furthermore, the variability of the AA values
depends on multiple factors such as fruit culture, maturity stage, storage
conditions, and extraction methods, and in turn, all these factors affect
the concentration of bioactive compounds, and thus, this concentration
has a direct relationship on the AA content in each fruit (Topalović et al.,
2020).
In both concentration techniques, as the cycles advanced, the vari­
ation of AA values between the cycles was statistically significant. This
upward behavior indicates a direct relationship with TSSC and TBC
values, since these increased as the cycles progressed, in agreement with
freeze crystallization reports applied to different liquid foods such as
strawberry juice (Adorno et al., 2017), yogurt enriched with cry­
oconcentrated strawberry (Jaster et al., 2018), apple juice (Zielinski
et al., 2019), orange juice (Orellana-Palma et al., 2019), and sapucaia
nut (Demoliner et al., 2020). Precisely, in the last cycle, each assay
presented a significant increase in relation to the initial value. However,
the highest AA values were observed in CBFC in comparison to evapo­
ration, with 13.9, 29.4, 34.1 and 24.3 (mM TE/100 g d.m.) versus 8.3,
20.1, 25.0 and 11.8 (mM TE/100 g d.m.), for DPPH, ABTS, FRAP and
ORAC, respectively. Hence, our results confirm the aforementioned in
physicochemical parameters and total bioactive compound sections,
which the temperature is the most influential factor to concentrate
different components, since low temperature allows a better AA con­
centration, and thus, the sensitive components preservation that
contribute to antioxidant capacity as total anthocyanin content, than
high temperatures, which finally leads to concentrate and preserve the
antioxidant components from the fresh juice (Gunathilake, 2020, pp.
39–69), and thus, the AA values support previous freeze crystallization
results in apple juice (Orellana-Palma, Lazo-Mercado, et al., 2020),
pineapple juice (Orellana-Palma, Zuñiga, et al., 2020), blueberry juice
(Orellana-Palma et al., 2021), and berry juices (Guerra-Valle et al.,
2021).

3.4. Volatile compounds determinations

Table 3 shows the volatile compounds of fresh pomegranate juice,

cryoconcentrate and evaporate sample at the third cycle. Firstly, a total
of sixteen volatile compounds were identified and quantified for the
fresh juice. Specifically, four alcohols, four aldehydes, two esters, two
ketones, one alkane, one terpene, one terpenoid and one unknown
compound were detected. Thus, 3-methyl-2-butanone, ethyl acetate and
3-methylbutyl acetate were the main compounds (high concentration)
in the fresh sample. These results are line with those informed by
Colantuono et al. (2018), who study the same pomegranate variety.
However, our results detected a higher number of volatile components
(16 vs 13), which may be due to the geographic differences that affect
agroclimatic conditions, as well as, type of harvest, juice preparation
and measurement conditions, among others, which could also fluctuate
the volatile content in the fruits.
As noted in the previous results, the volatile compounds were
concentrated as cycles advanced. The concentration variation for each
component in the cycles, i.e., the increase and decrease (or opposite
Fig. 3. GC-FID chromatograms from headspace volatile profile. (a) fresh
pomegranate juice; (b) cryoconcentrate; and (c) evaporate samples after the
third cycle.
behavior) from the first to the third cycle is due to a synergistic effect
between the volatile components in the juice (Mao et al., 2020) (see
supplemental material).
Notably, in the last cycle, CBFC as concentration technology allows
to detect three volatile compounds (2-Methyl-3-buten-2-ol: alcohol,
octane: alkane, and Methyl hexanoate: ester) that were not identified in
the fresh juice, which indicates a clear advantage compared to the use of
high temperatures, since, in the evaporation process, the volatiles were
significantly reduced, and thus, the third cycle indicated that only eight
volatile components (68%) were quantified, i.e., the temperature used
(50 ◦ C) degraded components from the original sample (Fig. 3).
The increase in volatile compounds by CBFC could be due to high

7
P. Orellana-Palma et al.
TSSC concentration, since several studies highlight that a high sugar
concentration improves the volatiles detection in the headspace (Pic­
cone et al., 2012), which underscores the FC importance, since it tech­
nology has repeatedly shown that significantly concentrates the initial
solutes of a liquid food (Petzold et al., 2016, pp. 184–190). The data
achieved for CBFC were in accordance with Gunathilake et al. (2014),
Moreno et al. (2015) and Miyawaki et al. (2016) for freeze crystalliza­
tion applied to coffee extract, pear juice, and apple juice, respectively.
As mentioned, the evaporation achieved a similar TSSC value at each
cycle than CBFC, but the temperature produce an irreversible negative
effect on physicochemical parameters, which can be corroborated in the
color visualization (Fig. 1), and thus, bioactive compounds and antiox­
idant capacity are affected by the components degradation, which is
finally reflected in a low number of volatile compounds detected by the
chromatograph.
4. Conclusions
In comparison with evaporation, which retained a low bioactive
compounds percentage and degraded various volatile components,
CBFC had significant effects on the quality parameters of fresh pome­
granate juice. Specifically, in the last cycle, the data demonstrated that
CBFC samples maintained the original juice color with a TSSC value
close to 48 ◦ Brix. Likewise, high TBC retention was reached, with 87%,
71%, 69% and 67% for TPC, TAC, TTC and TFC in relation to the initial
value, respectively. Moreover, the AA presented a similar behavior,
since CBFC increased up 5.2, 5.1, 5.3 and 4.1-fold with respect to the
initial AA value, for DPPH (2.7 mM TE/100 g d.m.), ABTS (5.7 mM TE/
100 g d.m.), FRAP (6.4 mM TE/100 g d.m.) and ORAC (6.0 mM TE/100
g d.m.) assays, respectively. In volatile compound terms, FC preserved
similar volatile compounds to the fresh juice. Instead, heat treatment
only retained 68% of the volatiles present in the initial sample. There­
fore, compared with evaporation, these results highlighted the CBFC
potential in retaining and improving the quality parameters of different
fresh juice and it could promote the preservation of physicochemical
properties, bioactive and volatile compounds, and antioxidant activity,
leading to the increased consumption of cryoconcentrated juice.
Author statement
Patricio Orellana-Palma: Conceptualization, Data curation, Formal
analysis, Funding acquisition, Investigation, Methodology, Project
administration, Software, Validation, Visualization, Writing - original
draft, Writing - review & editing. María Guerra-Valle: Conceptualiza­
tion, Data curation, Formal analysis, Investigation, Methodology, Soft­
ware, Validation, Writing - original draft. María Pía Gianelli:
Conceptualization, Data curation, Formal Analysis, Investigation,
Methodology, Software. Guillermo Petzold: Resources, Supervision,
Validation, Visualization, Writing - review & editing.
Declaration of competing interest
The authors confirm that they have no conflicts of interest with
respect to the work described in this manuscript.
Acknowledgments
Patricio Orellana-Palma is grateful for the financial support provided
by ANID-Chile through FONDECYT Postdoctoral Fellowship 2019
(Project Folio 3190420). The authors thank Mr. Marcelo Rodríguez
Vidal for his support during the gas chromatographic analysis at the
Laboratorio de Experimentación, Control y Certificación de la Calidad
de los Alimentos (LECYCA) at Universidad del Bío-Bío (Campus Fer­
nando May, Chillán).
8
Food Bioscience 41 (2021) 101106
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fbio.2021.101106.
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