![Effect of a 7-day simulated home-refrigerated storage at 4 C on total microbial count [aerobic bacterial (A) and yeast and mold (B) count], total soluble solid (C), pH (D), and cloud value (E) of cold-pressed juices. Data are presented as mean AE standard deviation (SD) of three independent biological replicates. An asterisk (*) on the right top of the time-point indicates its significant difference compared to the fresh juice (control) (Student's t-test, p < 0.05).](https://www.researchgate.net/publication/333856911/figure/fig4/AS:779160226127872@1562777682162/Effect-of-a-7-day-simulated-home-refrigerated-storage-at-4-C-on-total-microbial-count_Q320.jpg)
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Effect of cold-pressed and normal centrifugal juicing on quality attributes of
fresh juices: do cold-pressed juices harbor a superior nutritional quality and
antioxidant capacity?
Gholamreza Khaksar
a
, Kitipong Assatarakul
b
, Supaart Sirikantaramas
a
,
c
,
*
a
Molecular Crop Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok, 10330, Thailand
b
Special Task Force of Activating Research (STAR) in Novel Technology for Food Packaging and Control of Shelf Life, Department of Food Technology, Faculty of Science,
Chulalongkorn University, Bangkok, 10330, Thailand
c
Molecular Sensory Science Center, Chulalongkorn University, Bangkok, 10330, Thailand
ARTICLE INFO
Keywords:
Food science
Antioxidant activity
Bioactive compound content
Cold-pressed juicing
Centrifugal juicing
Home-refrigerated storage
Physicochemical properties
ABSTRACT
Cold-pressed juices are claimed to contain higher levels of antioxidants and bioactive compounds compared to
normally centrifuged ones. Herein, we evaluated the antioxidant capacity and the bioactive compound contents of
some freshly prepared fruit juices, extracted by a cold-pressed juicer and compared them to those prepared by a
normal centrifugal juicer. We observed no significant differences between cold-pressed and normal centrifugal
juices in terms of the contents of bioactive compounds (ascorbic acid, total phenolic, and total carotenoid) and
antioxidant capacity (ferric ion reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical scavenging activity). Storage at room temperature (~28 C) adversely affected the ascorbic acid, total
phenolics, total carotenoids, FRAP and DPPH values of the cold-pressed juices within 48 h. However, under
simulated home-refrigerated storage conditions, the antioxidant capacity, contents of bioactive compounds and
physicochemical properties of the cold-pressed juices remained unchanged till day 5 post-storage. However, at
day 6, most of the parameters exhibited a decreasing trend and reached their lowest values at day 7. Principal
component analysis confirmed significant changes in the quality of juices at day 7 of storage related to the first
two principal components (ascorbic acid and FRAP). Our results strongly question the claim regarding the su-
perior quality of cold-pressed juices. Moreover, our findings provided compelling evidence regarding the possible
adverse effects of long storage under home-refrigerated conditions on the quality of cold-pressed juices.
1. Introduction
There is a large body of evidence suggesting an association between a
diet rich in fruits and vegetables and fewer risk factors related to major
chronic diseases in humans (Slavin and Lloyd, 2012), including cardio-
vascular disease (CVD) (Bazzano et al., 2002), several common cancers
(Van’t Veer et al., 2000), and age-related degeneration (De
Mello-Andrade and Fasolo, 2014). Health promotion guidelines and di-
etary recommendations strongly suggest a regular consumption of pure
(100%) fruit juice (PFJ) (Williams, 1995, see review Caswell, 2009). A
review of evidence published by Ruxton et al. (2006) recommended that
PFJs (without any added components, e.g. sweeteners) retain the major
health-promoting compounds of whole fruit; and therefore, can
contribute to improved health conditions, justifying the increasing con-
sumption of PFJs in recent years. Health-benefiting properties of fruit
juices are ascribed mostly to their bioactive compounds, such as vitamin
C, phenolic compounds, carotenoids, and tocopherols (Gardner et al.,
2000;Sun et al., 2002).
Lifestyle changes and increased awareness among health-conscious
consumers have driven the beverage industry to develop and introduce
functional drinks with added nutritional values and health-promoting
benefits (Siroet al., 2008). Moreover, the fast-growing demand for
freshly squeezed unpasteurized fruit and vegetable juices reflects
changes in consumer preferences for beverages without processing
(Raybaudi-Massilia et al., 2009). Consequently, some beverage manu-
facturers have introduced freshly squeezed, unpasteurized, cold-pressed
juices and have claimed that they are healthier and could be stored for
more days than the normal centrifugal juices. This claim is based on the
following justification. Regular fruit juices are extracted by centrifugal
juicers that utilize a fast-spinning metal blade juxtaposed against a mesh
* Corresponding author.
E-mail address: supaart.s@chula.ac.th (S. Sirikantaramas).
Contents lists available at ScienceDirect
Heliyon
journal homepage: www.heliyon.com
https://doi.org/10.1016/j.heliyon.2019.e01917
Received 2 May 2019; Received in revised form 30 May 2019; Accepted 4 June 2019
2405-8440/©2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Heliyon 5 (2019) e01917
filter. This process separates the juice extract from fruit flesh by cen-
trifugal force. When the metal blade spins at a very high speed, it gen-
erates heat, which can negatively affect the bioactive compound contents
of juice. On the contrary, cold-pressed juices are made by cold-pressed
extractors that first crush and then press the fruit to extract its juice at
a very low speed. This extraction process generates almost no heat and
preserves the nutritional quality of the juice. Notably, the market prices
of cold-pressed juices are much higher than those of normal centrifuged
ones. However, there is no solid scientific basis for this claim. Previous
studies have documented the effects of juice extraction methods on the
quality of fruit juices. Miguel et al. (2004) investigated the effects of two
different juice extraction methods (centrifugation by a Phillips Electric
juice centrifuge vs. squeezing by a Phillips Electric lemon squeezer) on
the quality of pomegranate juice. Interestingly, it was found that the two
extraction methods did not have any effect on the quality parameters
measured, including the composition of sugars and amino acids, color,
pH, and anthocyanin content. In a study by Rajasekar et al. (2012),
blending was reported to be a more effective approach to preserve the
nutritional quality (antioxidant capacity, total phenolic, and total
monomeric anthocyanin contents) of pomegranate juice compared to
that of a mechanical press juice extraction. Pyo et al. (2014) found that
Korean kernel fruit juices prepared by blending harbored a significantly
stronger antioxidant capacity and higher total phenolic content than
those obtained by juicing. However, the ascorbic acid contents of juices
made by juicing were significantly higher than those by blending. Kim
et al. (2017) compared the nutritional value of grape juice prepared by
three different household juicers: low-speed masticating juicer, a
high-speed centrifugal juicer, and a blender. It was found that the juice
extracted by the low-speed masticating juicer had a higher nutritional
quality than that of the other two types.
Another important factor associated with the quality of juices is the
effect of storage conditions, mainly temperature and time-period on the
nutritional value of juices. During storage, the degradation of some
bioactive compounds, such as vitamin C, total phenolic content (TPC),
and total carotenoids could occur, which is a critical factor regarding the
quality of juices. Generally, beverage manufacturers set a short period,
known as expiration date or shelf life, during which the physicochemical
properties of juices remain conserved. However, during this period, the
stability of bioactive properties, such as antioxidant capacity and the
means by which they are affected by storage conditions remain unclear.
To address this issue, a few studies have documented the effect of storage
conditions on antioxidant capacity and bioactive compounds of some
fruit juices (Del Caro et al., 2004;Piljac-Zegarac et al., 2009;Bhardwaj
and Nandal, 2014;Mgaya-Kilima et al., 2014;La Cava and Sgroppo,
2015;Touati et al., 2016). However, to the best of our knowledge, no
such information is available regarding the effect of storage conditions on
the nutritional quality of freshly prepared unpasteurized cold-pressed
juices. Moreover, there is no published study comparing the nutritional
values of cold-pressed juices to normal centrifuged ones. Pineapple
(Ananas comosus), guava (Psidium guajava L.), white-fleshed pitaya
(Hylocereus undatus), known as white dragon fruit, and red-fleshed pitaya
(Hylocereus costaricensis), known as red dragon fruit are among the highly
nutritive and widely popular fruits commonly found in fresh fruit mar-
kets of Southeast Asia, including Thailand. These fruits have also gained a
fast-growing popularity in international markets. Moreover, carrot
(Daucus carota subsp. sativus) is a nutritious root vegetable with a
worldwide consumption. Herein, we selected these fruit and vegetable
species for the preparation of juices and aimed to evaluate and compare
the antioxidant capacity of freshly prepared unpasteurized juices
extracted by a cold-pressed juicer, normal centrifugal juicer, and blender.
Moreover, we determined the effect of the storage temperature on the
quality of cold-pressed juices. Finally, we investigated the impact of
simulated home refrigeration-storage conditions of consumers on the
physicochemical properties and nutritional quality of cold-pressed juices.
The information obtained from this study will increase consumer
awareness regarding the content of bioactive compounds and antioxidant
capacity of cold-pressed juices and the effect of refrigerated storage on
the quality of this type of fruit beverage.
2. Materials and methods
2.1. Fruit juice preparation
Fresh fruits, including pineapple, guava, carrot, red dragon fruit, and
white dragon fruit of similar size and appearance for each type and free of
any external defects were purchased from a local market in Bangkok,
Thailand. The fruits were fully rinsed with tap water, wiped dry, peeled
(except for guava), and cut into small pieces. Equal portions (10 g of fruit
pulp from each fruit sample) were prepared prior to juice extraction.
Three different juice extraction methods, including cold-pressed juicing,
normal centrifugal juicing, and blending were used for each fruit type
using a LIVIVE vertical cold-pressed juicer (Foshan Geuwa Electric
Appliance Co. Ltd, China), Philips HR1866 centrifugal juicer (Philips,
Netherlands), and Otto blender (Otto Kingglass Co. Ltd, Thailand),
respectively (Fig. 1). The extracted juices were then filled in sterile 50-mL
conical centrifuge tubes and centrifuged at 12000 rpm for 15 min at 4 C.
The supernatants were used for further analyses.
To investigate the effect of storage conditions (temperature and time-
period) on the quality of cold-pressed juices, extraction was performed as
mentioned above; the extracted fruit juices were stored in sterile 50-mL
centrifuge tubes and divided into three groups. The first group was
immediately centrifuged, and the supernatant was used for analysis
(fresh samples). The second group was stored under dark conditions at
RT (~28 C) and sampled at 24 and 48 h. The third group was stored at 4
C (simulated home-refrigerated storage) and sampled every day for a 7-
day storage period. The juices were centrifuged immediately after the
time-periods mentioned above and the supernatants were used for
further analyses.
2.2. Chemicals
All reagents and internal standards used in this study were obtained
from Sigma-Aldrich Inc. St. Louis, MO, USA.
2.3. Ascorbic acid content
Ascorbic acid content was determined following the method
described by Klein and Perry (1982) with slight modifications. Briefly, a
100-
μ
L aliquot of each juice supernatant was mixed with 50
μ
Lof3%
metaphosphoric acid, and the mixture was incubated for 10 min at RT.
The mixture was then added to 150
μ
L of 0.8 mM 2,6-dichloroindophenol
(DCIP) and absorbance was measured within 30 s at 515 nm against a
blank (DCIP mixed with acid). Content of ascorbic acid was calculated
according to a standard curve derived from authentic L-ascorbic acid
(1–50 mg/100 mL). The results were expressed as mg ascorbic acid per
100 g fresh fruit.
2.4. Total phenolic content (TPC)
TPC was measured by the Folin-Ciocalteu's method following a pre-
viously described procedure (Swain and Hillis, 1959). A 150
μ
L aliquot of
each supernatant, 2400
μ
L of ultrapure water, and 150
μ
L of 0.5 N
Folin–Ciocalteu reagent were mixed well using a Vortex mixer and were
allowed to react for 30 min at RT (~28 C). Later, 300
μ
Lof1NNa
2
CO
3
solution was added to the mixture and mixed well. The mixture was then
incubated at RT (~28 C) for 2 h under dark conditions and the absor-
bance measured at 725 nm using a microplate reader. The TPCs of juices
were expressed as mg gallic acid equivalent (GAE) per 100 g fresh fruit
based on a standard curve obtained from authentic gallic acid (0.2–4
mg/100 mL).
G. Khaksar et al. Heliyon 5 (2019) e01917
2
2.5. Total carotenoid content
Total carotenoid content was assessed following the spectrophoto-
metric method (A
470
) described by Talcott and Howard (1999) and
expressed as β-carotene equivalent per 100 g fresh fruit using a standard
curve of authentic β-carotene (0.1–2 mg/100 mL).
2.6. Antioxidant capacity
Ferric ion reducing antioxidant power (FRAP) assay was performed
according to the method described by Benzie and Strain (1996) with
some modifications. The FRAP working solution was freshly prepared by
mixing 300 mM acetate buffer (3.1 g C
2
H
3
NaO
2
⋅3H
2
O and 16 mL
C
2
H
4
O
2
; pH 3.6), 10 mM 2, 4, 6-tripyridyl-s-triazine (TPTZ) in 40 mM
HCl, and 20 mM FeCl
3
⋅6H
2
O in a 10:1:1 ratio, respectively, and warmed
up at 37 C before use. A 40-
μ
L aliquot of the supernatant was mixed with
160
μ
L of the FRAP working solution in a 96-well plate and incubated at
RT for 30 min under dark conditions. FRAP values (A
593
) were reported
as
μ
mol trolox equivalent (TE) per 100 g fresh fruit based on a standard
curve of authentic Trolox (50–1700
μ
M).
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity
was performed according to a method described previously (Brand-Wil-
liams et al., 1995) with some modifications. The DPPH working solution
was obtained by mixing 10 mL of the stock solution (24 mg of DPPH
dissolved in 100 mL methanol) with 45 mL methanol. Next, the DPPH
solution (950
μ
L) was mixed with 50
μ
L of each supernatant and incu-
bated for 1 h under dark conditions. The A
515
was read, and the DPPH
values of the juices were calculated using the following equation:
DPPH radical scavenging activity ð%Þ¼AðA1A2Þ
A100;
where Absorbance ðAÞis the A
515
of DPPH solution without supernatant,
A1 is the A
515
of DPPH solution with supernatant, and A2 is the A
515
of
the supernatant.
2.7. Total microbial count
Each fruit juice was serially diluted (10
2
10
8
) using autoclaved
0.1 % peptone water. Subsequently, a 1-mL aliquot of each diluted
sample was placed on a Luria-Bertani (LB) agar plate and incubated at 37
C for 2 d to obtain a total aerobic bacterial count. For the total yeast and
mold count, a 1-mL aliquot was placed on a potato dextrose agar plate
and incubated at 30 C for 3–5 d. The results were expressed as log
colony-forming units (CFU) per mL of juice.
2.8. Physicochemical analysis
Total soluble solid (TSS) was measured using a digital refractometer
(HANNA HI96801, USA) and the results were reported in standard Brix
units. The pH of juices was determined with a digital benchtop pH meter
(METTLER TOLEDO, USA). Color analysis of the juices was performed
using a colorimeter (CR-410 chroma meter, Minolta, Japan). Three color
parameters, including L* (lightness), a* (redness/greenness), and b*
(yellowness/blueness) were measured. The color difference (ΔE) was
calculated compared to that of freshly prepared juice as a control using
the following equation:
ΔE¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ðΔL*Þ2þðΔa*Þ2þðΔb*Þ2
q
To measure the cloud value representing the degree of turbidity or
darkening of juices, A
660
was read using a spectrophotometer (Eppendorf
BioSpectrometer
®
, Eppendorf, USA) against a blank (distilled water)
(Aadil et al., 2013).
2.9. Statistical analysis
The data obtained in this study were subjected to statistical analysis
using SPSS software, version 20 (SPSS Inc., IBM). The data are presented
as means standard deviation (SD) of three independent replicates.
Statistical comparisons of the means were done using a one-way ANOVA,
followed by Duncan's multiple range test or Student's t-test at the 0.05
confidence level.
3. Results
3.1. Bioactive compound contents and antioxidant capacity of cold-pressed
juices compared to normal centrifugal ones
Three different types of juice extractors were used in this study: cold-
pressed juicer, normal centrifugal juicer, and blender. Interestingly, as
indicated in Fig. 2, no significant differences were observed among the
different types of extraction methods in terms of the contents of bioactive
compounds (ascorbic acid, total phenolic, and total carotenoid contents)
and antioxidant capacity (FRAP and DPPH values) of the juices. Our
observation, herein, questioned the claim that cold-pressed juice contains
higher antioxidants and bioactive compounds compared to that of the
normally extracted juice. Cold-pressed juices are claimed to have a longer
shelf life compared to normal centrifugal ones. Therefore, to evaluate this
claim, the effect of storage conditions (temperature and time-period) on
the quality parameters of cold-pressed juices was investigated.
3.2. Effect of storage conditions (temperature and time-period) on the
quality attributes of cold-pressed juices
Among health-conscious consumers, there is an ever-existing concern
regarding the effect of storage conditions on nutritional qualities of jui-
ces. Therefore, we aimed to study this phenomenon by comparing the
content of bioactive compounds and antioxidant capacity of five types of
cold-pressed juices kept at either RT (~28 C) or 4 C (stored under
simulated home-refrigerated conditions) over a period of 48 h. Interest-
ingly, storage at 4 C and RT during a 24-h period did not have any
negative impact on the content of bioactive compounds and antioxidant
capacity of all types of juices. The ascorbic acid, TPC, total carotenoids,
Fig. 1. Three types of juice extractor, including normal centrifugal juicer, cold-pressed juicer and blender were used in this study. A representative picture of each
extractor is presented.
G. Khaksar et al. Heliyon 5 (2019) e01917
3
FRAP and DPPH values of refrigerated juices and those kept at RT,
measured at different time-points, did not vary significantly compared to
those of the fresh ones, except for the red dragon juice kept at RT (Fig. 3).
Notably, the antioxidant capacity (FRAP and DPPH values) of the red
dragon juice stored at RT demonstrated a significant increase at 24 h
compared to that of the fresh one. However, this phenomenon was not
Fig. 2. Content of bioactive compounds (ascorbic acid (A), total phenolic (B) and carotenoid (C) contents) and antioxidant capacity (Ferric ion reducing antioxidant
power (FRAP) (D) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity (E)) of juices extracted by cold-pressed juicer, normal centrifugal juicer, and
blender. Data are presented as mean standard deviation (SD) of three independent replicates. For each measured parameter, comparisons are shown for different
extraction methods within each fruit juice; no significant differences were observed according to Duncan's multiple-range test (p<0.05). (GAE: gallic acid equivalent,
TE: Trolox equivalent, E: equivalent).
Fig. 3. Effect of storage at room temperature (~28 C) and 4 C on ascorbic acid, total phenolic, and carotenoid contents and antioxidant capacity (FRAP and DPPH)
of cold-pressed juices. Bars represent the mean standard deviation (SD) of three independent replicates. For each measured value, comparisons are shown between
the fresh juice and different time-points during storage of each fruit juice; an asterisk (*) above the bars indicates a significant difference at that time-point compared
to the fresh juice (control) (Student's t-test, p<0.05). (GAE: gallic acid equivalent, TE: Trolox equivalent, E: equivalent).
G. Khaksar et al. Heliyon 5 (2019) e01917
4
observed for the red dragon juice stored at 4 C. Additionally, storage for
2 days (48 h) had different effects on the quality of juices depending on
the temperature. Storage for 48 h at 4 C did not have any negative effect
on the contents of bioactive compounds and antioxidant capacity of
juices. Ascorbic acid, TPC, total carotenoids, FRAP and DPPH values of
the juices measured at 48 h did not vary significantly when compared to
the fresh ones. However, storage at RT for a 48-h period significantly
reduced the bioactive compound contents and antioxidant capacity of
juices; all the measured values at 48 h declined significantly compared to
those of the fresh juices (Fig. 3). Taken together, our results suggest that
storing juices at 4 C for 48 h can protect the bioactive compounds and
antioxidant capacity of juices, whereas keeping at RT can significantly
decrease these factors.
Surprisingly, antioxidant capacity of the red dragon juice stored at RT
increased significantly at 24 h compared to that of the fresh one. This
phenomenon could have occurred as a result of enzymatic activities
during storage at RT. Furthermore, we did not observe any increase in the
FRAP and DPPH values of the red dragon juice stored at 4 C; this
observation strengthened the possibility of an enzymatic reaction that
could have occurred at a much slower rate at 4 C. To further examine
this possibility, we investigated whether adding pure methanol to the
juice can denature proteins and inhibit the enzymatic reaction that is
involved in increasing the antioxidant capacity. Interestingly, for the red
dragon juice without methanol (control; juice þwater, 1:1), both FRAP
and DPPH values peaked at 24 h of storage life. However, for the juice
mixed with methanol (juice þabsolute methanol, 1:1), we did not
observe any increase in these values at 24 h of storage (Fig. 4). This result
provided convincing evidence that the increase in the antioxidant ca-
pacity of the red dragon juice could have occurred as a result of enzy-
matic reaction(s).
3.3. Effects of a 7-day simulated home-refrigerated storage on the quality
of the cold-pressed juices
It was observed that storing the juices refrigerated (4 C) over a 48-h
period did not cause any detrimental effect on the quality of cold-pressed
juices. This observation prompted us to further examine the effect of a
longer refrigerated storage period (a 7-day storage period) on physico-
chemical properties, content of bioactive compound, and antioxidant
capacity of juices.
3.3.1. Effect on total microbial count and physicochemical properties [total
soluble solid (TSS), pH, cloud value, and color]
Preserving juices for a 7-day period at 4 C did not affect the total
microbial count of all the juices. The total aerobic bacteria and yeast/
mold counts of juices did not vary significantly during the storage period
compared to those of the freshly squeezed ones. Moreover, the TSS and
pH values of the juices measured during the 7-day storage period did not
demonstrate significant changes compared to those of the freshly pre-
pared ones, except for pineapple juice, which showed a significant
decrease in the TSS value at day 7 of storage (Fig. 5). Furthermore, cloud
value showed significant changes during the storage period for some
juices. However, the cloud values of carrot and red dragon juice
remained constant during the storage period and showed no significant
differences compared to those of the fresh ones. In contrast, the cloud
value of white dragon juice declined significantly at day 3 and remained
constant during the remaining storage days. Moreover, for the pineapple
juice, the cloud value showed a significant decrease at day 6 of storage;
however, the guava juice was the only type of juice with an increased
cloud value during storage in the refrigerator (Fig. 5)a 1.16-fold in-
crease at day 7 of storage.
With respect to lightness (L
*
), a significant difference was recorded
only for the white dragon juice which showed a slight decrease in
lightness at day 7 compared to that of the fresh juice (Table 1). For the
redness/greenness (a
*
), significant changes were observed for the pine-
apple and carrot juices, whereas guava, white and red dragon fruits did
not show significant changes in the a
*
value during the storage. Notably,
the yellowness/blueness (b
*
) values of all juices did not demonstrate any
significant changes during the storage period when compared to those of
the freshly prepared ones (Table 1).
3.3.2. Effect on bioactive compounds (vitamin C, total phenolic, and total
carotenoid contents)
A significant decrease in ascorbic acid and total phenolic contents was
observed at day 6 of storage for all juices except for the red dragon juice,
which showed a slight decrease in vitamin C content at day 7 of storage
but did not show a significant change in TPC during the storage period
(Fig. 6). Total carotenoids of carrot, guava, and red dragon juices were
decreased significantly at day 6 of storage, whereas for pineapple and
white dragon, the total carotenoids did not change significantly during
the storage period (Fig. 6).
3.3.3. Effect on antioxidant capacity
FRAP and DPPH values of pineapple, guava, carrot, and white dragon
juices declined significantly at day 6 of storage. For the red dragon juice,
the FRAP value showed a similar pattern to that of other juices, while
significant changes in DPPH were not observed during the refrigerated
storage (Fig. 6).
Fig. 4. Effect of methanol on the antioxidant capacity of red dragon juice stored at room temperature for 24 h. Pure methanol was mixed with freshly prepared red
dragon juice at a ratio of 1:1 and stored at room temperature. Ferric ion reducing antioxidant power (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging activity were measured and compared to the control (red dragon juice without methanol; juice þwater, 1:1). Bars represent the mean standard deviation
(SD) of three independent replicates. An asterisk (*) above the bars indicates a significant difference at that time-ponit between red dragon juice with methanol and
control (Student's t-test, p<0.05).
G. Khaksar et al. Heliyon 5 (2019) e01917
5
4. Discussion
Dietary guidelines include frequent consumption of pure fruit juices,
the health-promoting properties of which are attributed to their rich
content of bioactive compounds, including vitamin C and phenolic
compounds. Over the past few years, there is an ever-growing demand
for the consumption of freshly prepared unpasteurized fruit juices with
no or minimal processing, due to its freshness, higher vitamin content,
and high nutritional value. Traditional thermal treatments, including
pasteurization adversely affect the nutritional values and sensory char-
acteristics of foods and beverages (Wolbang et al., 2008;Rawson et al.,
2011;Pilavtepe-Celik, 2013), and sometimes fail to produce a
Fig. 5. Effect of a 7-day simulated home-refrigerated storage at 4 C on total microbial count [aerobic bacterial (A) and yeast and mold (B) count], total soluble solid
(C), pH (D), and cloud value (E) of cold-pressed juices. Data are presented as mean standard deviation (SD) of three independent biological replicates. An asterisk (*)
on the right top of the time-point indicates its significant difference compared to the fresh juice (control) (Student's t-test, p<0.05).
Table 1
Effect of storage under simulated home-refrigerated conditions at 4 C on color analysis of cold-pressed juices.
Fresh Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
Lightness (L*)
Pineapple 34.02 0.56
ab
34.49 0.87
ab
35.22 0.59
a
33.98 0.83
ab
34.49 0.76
ab
33.50 1.01
b
35.29 0.78
a
35.45 0.73
a
Guava 43.61 0.13
b
44.01 0.6
ab
44.38 0.23
a
44.39 0.43
a
44.46 0.43
a
43.72 0.4
b
43.81 0.32
b
43.65 0.4
b
Carrot 35.09 0.54
a
33.54 1.08
b
33.83 0.87
b
33.08 0.65
bc
33.12 0.43
bc
32.49 0.83
c
33.35 0.64
b
35.78 0.43
a
White dragon fruit 33.56 0.21
a
29.63 0.67
c
30.47 1.32
c
31.87 0.78
b
32.15 0.54
b
31.99 0.32
b
31.97 0.42
b
31.90 0.43
b
Red dragon fruit 22.37 0.89
a
21.87 1.07
a
21.98 0.96
a
22.99 1.87
a
22.67 0.95
a
23.08 2.06
a
22.31 0.92
a
22.79 0.64
a
Redness/greenness (a*)
Pineapple 3.75 0.09
a
3.58 0.08
ab
3.18 0.08
b
3.44 0.09
ab
2.97 0.09
c
3.2 0.1
b
2.91 0.11
c
2.82 0.13
c
Guava 5.59 0.11
ab
5.43 0.2
b
5.45 0.09
b
5.57 0.08
ab
5.77 0.08
a
5.36 0.09
b
5.45 0.08
b
5.41 0.09
b
Carrot 3.74 0.11
b
3.35 0.2
b
3.59 0.09
b
3.51 0.08
b
3.61 0.098
b
3.74 0.09
b
3.68 0.1
b
4.97 0.21
a
White dragon fruit 0.93 0.1
ab
1.03 0.09
a
0.66 0.08
bc
0.76 0.11
bc
0.69 0.089
bc
0.7 0.09
bc
0.67 0.11
bc
0.73 0.13
bc
Red dragon fruit 4.92 0.32
a
4.84 0.08
a
5.11 0.53
a
4.88 0.31
a
4.99 0.46
a
4.67 0.11
a
4.78 0.23
a
5.08 0.07
a
Yellowness/blueness (b*)
Pineapple 6.49 0.1
a
6.50 0.11
a
5.77 0.23
b
5.60 0.21
b
4.56 0.23
c
4.58 0.22
c
2.63 0.1
d
2.75 0.22
d
Guava 3.23 0.09
a
3.1 0.08
a
3.09 0.09
a
3.2 0.088
a
3.23 0.08
a
2.4 0.16
b
2.57 0.11
b
2.54 0.09
b
Carrot 15.87 0.43
bc
15.71 0.34
bc
16.03 0.45
b
15.16 0.44
c
15.69 0.39
bc
15.50 0.23
bc
15.58 0.32
bc
16.91 0.49
a
White dragon fruit 0.97 0.1
a
1.01 0.089
a
1.16 0.09
a
0.22 0.088
b
0.2 0.03
b
0.09 0.007
c
0.08 0.007
c
0.07 0.004
c
Red dragon fruit 0.32 0.07
ab
0.27 0.03
b
0.31 0.08
ab
0.32 0.05
ab
0.28 0.03
b
0.33 0.04
a
0.34 0.09
a
0.33 0.05
a
Color difference (ΔE)
Pineapple 1.46 0.11
c
1.58 0.22
c
1.17 0.32
c
2.16 0.27
b
2.16 0.31
b
2.42 0.3
b
3.2 0.15
a
Guava 0.59 0.11
a
0.81 0.13
a
0.78 0.22
a
0.87 0.24
a
0.92 0.31
a
0.75 0.22
a
0.73 0.35
a
Carrot 1.62 0.32
ab
1.29 0.22
b
2.15 0.21
ab
2.00 0.13
ab
2.63 0.32
a
1.79 0.45
ab
1.78 0.76
ab
White dragon fruit 3.93 0.67
a
3.11 0.45
a
1.92 0.33
b
1.66 0.43
b
1.89 0.19
b
1.89 0.42
b
1.96 0.52
b
Red dragon fruit 0.36 0.04
a
0.41 0.09
a
0.35 0.08
a
0.34 0.07
a
0.35 0.06
a
0.32 0.07
a
0.33 0.06
a
Data are presented as the mean standard deviation (SD) of three independent replicates.
For each measured parameter, values in the same row followed by different superscripted letters are significantly different according to Duncan's multiple-range test (p
<0.05).
G. Khaksar et al. Heliyon 5 (2019) e01917
6
microbiologically stable product (Mohamed and Eissa, 2012).
Cold-pressed juices have been introduced as a new generation of fruit
beverages and are claimed by beverage manufacturers to have higher
contents of bioactive compounds compared to those of regular (normal)
centrifugal juices (The wonderful benefits of cold Pressed Juice, 2018;
The real benefits of cold-pressed juice, 2018). However, our findings in
the present study strongly question this claim. Five types of juices,
which are commonly found in the Thai markets, were prepared using
centrifugal (regular) and cold-pressed juicers. Numerous research
studies have estimated FRAP and DPPH values as indicators of antiox-
idant capacity and vitamin C, and total phenolic and carotenoid con-
tents as indicators of beneficial value of fruit beverages to health
(Piljac-Zegarac et al., 2009;Rajasekar et al., 2012;Mgaya-Kilima et al.,
2014;Pyo et al., 2014;Touati et al., 2016;Abountiolas and Nascimento
Nunes, 2018). When we measured these values of normal and
cold-pressed juices, we consistently found that the antioxidant capacity
and content of bioactive compounds of cold-pressed juices were not
significantly different from those of regular centrifugal juices (Fig. 2).
These results provide convincing evidence that the above-mentioned
claim by some beverage manufacturers is misleading. Notably, our
study is not the first scientific report, which questions the claim of these
beverage producers. Abountiolas and Nascimento Nunes (2018) re-
ported that the labels on some fruit beverages claiming high antioxidant
and/or phenolic contents were not consistent with the measured values;
thus, these claims were considered misleading. An important factor to
be considered about our results is the condition for preparing the juices.
We used a short time (30 seconds) for preparing each juice using the
centrifugal juicer. However, if a longer time is used for juice extraction
using the centrifugal juicer, the quality of the extracted juice might
differ due to the greater amount of heat produced by the juicer. In other
words, juicing conditions, including the juicing time and the juicer
specifications (e.g., rotating speed) might affect the quality of the
extracted juices. This phenomenon could explain the observation by
Kim et al. (2017), who found a higher nutritional quality of grape juice
extracted by a low-speed masticating juicer compared to the juice
extracted by a high-speed centrifugal one.
Interestingly, among the different types of juices, the guava juice
harbored the highest levels of ascorbic acid, TPC, FRAP, and DPPH.
Thaipong et al. (2006) also reported that ascorbic acid, total phenolic and
total carotenoid contents of guava extract were significantly higher than
those of other fruit crops.
Storage conditions (storage temperature and time-period) could
affect the content of bioactive compounds and antioxidant capacity of
fruit juices. Previous studies have addressed this phenomenon and have
found that storing juices under refrigerated conditions (4 C) could
strongly protect the nutritional quality and antioxidant capacity of juices
compared to storing them at RT (Bhardwaj and Nandal, 2014;Mgaya--
Kilima et al., 2014;Touati et al., 2016). Consistently, in our study,
storage at RT adversely affected the quality of cold-pressed juices within
48 h (Fig. 3), possibly due to the degradation of bioactive compounds,
including ascorbic acid, total phenolics and carotenoids, and/or reactions
with other compounds. However, for the juices stored at 4 C, no changes
were observed for all the measured values at 48 h. Our results clearly
confirmed that similar to regular juices (Bhardwaj and Nandal, 2014;
Mgaya-Kilima et al., 2014;Touati et al., 2016) the quality of cold-pressed
juices is negatively affected by storage at RT. Interestingly, the antioxi-
dant capacity (FRAP and DPPH) of red dragon juice stored at RT was
significantly increased at 24 h of storage, whereas we did not observe any
increase for the one stored at 4 C. This increase in antioxidant capacity
of red dragon juice could possibly be due to enzymatic reactions that
were much slower at 4 C than at RT. The intense red color of red dragon
juice is attributed to its rich level of betacyanin, a nitrogenous red-violet
pigment (see review Polturak and Aharoni, 2018). A possible enzymatic
reaction by β-glucosidase, which converts betacyanin to its aglycone
form, betanidin (Gandía-Herrero et al., 2004), might explain the increase
in antioxidant content of the red dragon juice. The high antioxidant ca-
pacity of some aglycones has been previously reported (K€
ahk€
onen and
Heinonen, 2003).
Fig. 6. Effect of a 7-day simulated home refrigerated storage at 4 C on ascorbic acid (A), total phenolic (B) and carotenoid (C) contents and antioxidant capacity
(FRAP (D) and DPPH (E)) of cold-pressed juices. Bars represent mean standard deviation (SD) of three independent replicates. For each measured value, com-
parisons are shown between the fresh juice and different time-points during storage of each fruit juice. An asterisk (*) above the bars indicates a significant difference
at that time-point compared to the fresh juice (control) (Duncan's multiple-range test, p<0.05) (GAE: gallic acid equivalent, TE: Trolox equivalent, E: equivalent).
G. Khaksar et al. Heliyon 5 (2019) e01917
7
Storing fruit juices in a refrigerator at home and consuming as desired
is a regular practice. However, as reported by Piljac-Zegarac et al. (2009),
this type of storage could affect the TPC and antioxidant capacity of
juices. Moreover, a slight decrease in ascorbic acid and total carotenoid
content of some fruit beverages kept in a refrigerator for a period of 8
d was reported by Castro-L
opez et al. (2016). Due to the possible adverse
effect of storage conditions on the safety and quality of fresh unpas-
teurized fruit juices, gaining a better understanding of this phenomenon
is of paramount importance. There is no definite guideline regarding the
exact shelf life of freshly prepared unpasteurized juices kept in
home-refrigerated conditions. However, most juice manufacturers sug-
gest a shelf life of 3–5d(Understanding shelf life of cold-pressed juice,
2016). Therefore, we investigated the effect of storage under home-
refrigerated conditions on the quality of cold-pressed juices for a period
of 7 d [we designed our experiment to include sampling of the juices
shortly before (1–2 d) and after (6–7 d) the commonly suggested shelf
life]. No changes were observed for the total microbial (bacterial and
yeast) count during the 7-d storage at 4 C for all the juices, indicating
stability of the juices during the storage period. Moreover, physico-
chemical properties (TSS and pH) of the juices measured in this study
remained constant for all the juices, excluding pineapple juice, which
exhibited a decreased value of TSS at day 7 (Fig. 5).
Cloud value (clarity) is ascribed to particles in the juice, such as
cellulose, hemicellulose, proteins, lipids, and some other minor particles
(Baker and Cameron, 1999). For fruit juices, stability of the cloud value is
a visual quality factor associated with the flavor and color and is inter-
connected to consumer acceptance (Beveridge, 2002). The cloud value of
carrot and red dragon did not exhibit any changes during the storage,
which clearly confirmed the stability of these juices during the storage at
4C. However, a significant decrease in the cloud value of white dragon
and pineapple was observed (Fig. 5), which indicated less turbidity of
these juices compared to that of the fresh ones. This phenomenon could
have occurred as a result of fewer suspended particles, possibly due to
reactions among the particles. Nevertheless, guava juice was the only
type of juice that showed a significant increase of its cloud value at day 7.
This increase could be due to the breakdown of larger molecules resulting
in higher numbers of suspended particles at day 7 of storage than those in
the fresh juice. Color stability is an important quality characteristic of
juices during storage. We observed the greatest value of color difference
(ΔE) for pineapple (3.2) than for other juices, which indicated a medium
color difference obvious to an untrained eye (2 <ΔE<3.5). This obvious
change in the color of pineapple juice was mainly attributed to the sig-
nificant decrease in the þb
*
value (yellowness), which might be due to
the degradation of pigments during the storage period. The change in the
color of pineapple juice, which coincided, with a significant decrease in
the clarity and TSS values clearly confirmed the impact of the storage on
the quality of pineapple juice. For the carrot and white dragon juices, a
very small color difference, obvious only to a trained eye was observed (1
<ΔE<2). Meanwhile, the guava juice also demonstrated a much smaller
color difference (invisible difference) (0 <ΔE<1); however, the smallest
difference in color was observed for the red dragon juice (0.33) (Table 1).
Taken together, because of the measured parameters, the red dragon
juice exhibited the greatest storage stability with minimum changes
during storage in a refrigerator compared to that of the other types of
juices.
Antioxidant capacity is widely used as a tool to evaluate the quality of
fruit beverages after processing and during storage. Accordingly, in our
study, we measured FRAP, DPPH, ascorbic acid, total phenolic and
Fig. 7. Principal component analysis (PCA) of nutritional (ascorbic acid, total phenolic and carotenoid contents), antioxidant (FRAP and DPPH), and physicochemical
(pH, total soluble solid, clarity, and color) properties of fruit juices during a 7-day simulated home-refrigerated storage at 4 C. Each spot represents one independent
biological replicate. PCA was performed using a web-based tool MetaboAnalyst 4.0.
G. Khaksar et al. Heliyon 5 (2019) e01917
8
carotenoid contents of cold-pressed juices during the 7-day storage at 4
C. Most of the measured values started to show similar declining trends
at day 6 of storage and reached their lowest values at day 7 (Fig. 6).
However, this trend was quite different for the red dragon juice, in which
the TPC and DPPH remained unchanged during the storage period and
ascorbic acid showed a slight decrease only at day 7 of storage (Fig. 6).
Our findings indicated that different storage conditions could have
different effects on the quality of juices depending on the type of the fruit,
possibly due to different compositions of bioactive compounds in
different fruit types. This phenomenon has previously been reported by
some studies (Castro-L
opez et al., 2016;Abountiolas and Nascimento
Nunes, 2018). In terms of the effect of storage on the quality of juices and
in accordance with the measured values, principal component analysis
(PCA) of pineapple, guava, carrot, and white dragon revealed a similar
pattern and clearly indicated significant changes in the quality of juices at
day 6 and especially at day 7 of storage. PCA of each fruit juice classified
the samples into eight groups (fresh, day 1, day 2, day 3, day 4, day 5, day
6, and day 7). Groups of fresh to day 5 were close together, which
indicated that there were no significant changes in the quality of juices
until day 5 post-storage. However, samples at day 6 and especially at day
7 were clearly separated, which pointed to the significant changes in the
quality of juices at days 6 and 7 of storage (Fig. 7). For the red dragon
juice, PCA revealed a different trend compared to that for the other jui-
ces. All groups (fresh to day 7) were located in close proximity to each
other, indicating minimal changes in the quality of red dragon juice
during the storage period (Fig. 7). Taken together, PCA strongly sug-
gested that the storage conditions affected fruit juices differently, based
on the fruit type –the red dragon juice was the most stable type during
the storage period.
During juice extraction, cells from the fruit pulp are disrupted. This
phenomenon could bring enzymes previously localized at different sub-
cellular compartments (e.g., cell-wall-localized β-glucosidase) and sub-
strates in close proximity to each other and subsequently stimulate
enzymatic reactions. These enzymatic reactions could be responsible for
the possible changes in the quality attributes of fruit juices during stor-
age. Further studies, investigating these biochemical reactions at mo-
lecular levels by using state-of-the-art mass spectrometers coupled with
cutting-edge metabolomic technologies, could provide new insights
into the effects of storage on the nutritional quality of fruit juices.
Maintaining the quality of fruit juices during storage is of utmost
importance and a better understanding in this area should lead to im-
provements to obtain optimal storage conditions. Sensory analysis is
widely used in food science and technology to assess the quality of a
product with respect to mouth feel, odor, taste, color, and creaminess. In
this regard, studying the sensory characteristics of freshly squeezed un-
pasteurized juices during home-refrigerated storage is of great interest
for obtaining optimal storage conditions that would satisfy the general
consumer acceptance. Hence, the parameters measured in this study
would provide in-depth knowledge regarding the effect of storage con-
ditions on the quality of cold-pressed juices. However, to expand our
knowledge, metabolomics and sensory analyses could be a subject for
future investigation.
In summary, we compared the content of bioactive compounds and
antioxidant capacity of cold-pressed juices to those of the regular
(normal) ones and found no significant differences between the two. Our
results thus provide compelling evidence that the claim regarding the
higher nutritional quality of cold-pressed juices could be misleading and
should be questioned. Moreover, the physicochemical properties, anti-
oxidant capacity and content of bioactive compounds of cold-pressed
juices remained unchanged until day 5 of storage under home-
refrigerated conditions. However, at day 6, most of the measured
values started to decline and reached their lowest levels at day 7 of
storage. This observation clearly confirmed that storage of juices in re-
frigerators could negatively affect the quality of cold-pressed juices, thus
questioning the claim regarding the longer shelf life of cold-pressed jui-
ces kept in home refrigerators.
Declarations
Author contribution statement
Gholamreza Khaksar: Performed the experiments; Analyzed and
interpreted the data; Wrote the paper.
Kitipong Assatarakul: Contributed reagents, materials, analysis tools
or data.
Supaart Sirikantaramas: Conceived and designed the experiments;
Analyzed and interpreted the data; Wrote the paper.
Funding statement
This research was financially supported by Chulalongkorn research
funding (GRU 6203023003-1) to S.S., and Ratchadapisek Somphot Fund
for Postdoctoral Fellowship, Chulalongkorn University to G.K.
Competing interest statement
The authors declare no conflict of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
We thank Ms. Krongkan Thongmat for her assistance in physico-
chemical analysis (color measurement).
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