Eur Food Res Technol (2006) 222: 321–329
DOI 10.1007/s00217-005-0073-3
ORIGINAL PAPER
Pedro Elez-Martı́nez · Robert C. Soliva-Fortuny ·
Olga Martı́n-Belloso
Comparative study on shelf life of orange juice processed by high
intensity pulsed electric fields or heat treatment
Received: 9 March 2005 / Revised: 6 June 2005 / Accepted: 7 June 2005 / Published online: 23 September 2005


C Springer-Verlag 2005
Abstract The effects of high intensity pulsed electric
fields (HIPEF) processing (35 kV/cm for 1,000 µs;
bipolar 4-µs pulses at 200 Hz) on the microbial shelf
life and quality-related parameters of orange juice were
investigated during storage at 4 and 22 ◦ C and compared
to traditional heat pasteurization (90 ◦ C for 1 min) and an
unprocessed juice. HIPEF treatment ensured the microbi-
ological stability of orange juice stored for 56 days under
refrigeration but spoilage by naturally occurring microor-
ganisms was detected within 30 days of storage at 22 ◦ C.
Pectin methyl esterase (PME) of HIPEF-treated orange
juice was inactivated by 81.6% whereas heat pasteurization
achieved a 100% inactivation. Peroxidase (POD) was
destroyed more efficiently with HIPEF processing (100%)
than with the thermal treatment (96%). HIPEF-treated or-
ange juice retained better color than heat-pasteurized juice
throughout storage but no differences (p<0.05) were found
between treatments in pH, acidity and ◦ Brix. Vitamin C re-
tention was outstandingly higher in orange juice processed
by HIPEF fitting recommended daily intake standards
throughout 56 days storage at 4 ◦ C, whereas heat-processed
juice exhibited a poor vitamin C retention beyond 14 days
storage (25.2–42.8%). The antioxidant capacity of both
treated and untreated orange juice decreased slightly dur-
ing storage. Heat treatments resulted in lower free-radical
scavenging values but no differences (p<0.05) were
found between HIPEF-processed and unprocessed orange
juice.
Keywords High intensity pulsed electric fields .
Orange juice . Shelf life . Microbiological stability .
Quality parameters
P. Elez-Martı́nez · R. C. Soliva-Fortuny ·
O. Martı́n-Belloso (
)
Department of Food Technology, UTPV-CeRTA, Universitat de
Lleida Av.,
Alcalde Rovira Roure, 191,
25198 Lleida, Spain
e-mail: omartin@tecal.udl.es
Tel.: +34-973-702593
Fax: +34-973-702596
Introduction
Orange juice (OJ) is the most popular fruit beverage of most
European countries [1]. Commercial orange juice has been
traditionally heat-processed to destroy spoiling microor-
ganisms and inactivate enzymes that curb the product qual-
ity during storage [2]. Heat processing often induces unde-
sirable changes in the color, flavor and nutritional value of
orange juice [3].
The demand for lightly processed foods that preserve
their fresh-like characteristics without compromising their
safety has increased rapidly in the last few years [4]. High-
intensity pulsed electric fields (HIPEF) is an emergent non-
thermal technology with promising results in the field of
fluid foods [5]. Therefore, HIPEF is potentially an alter-
native of choice for citrus juice processing, especially if
combined with other sub-lethal treatments such as refriger-
ation temperature, as an approach to the hurdle technology
[6].
Microbial inactivation of OJ with HIPEF has been shown
to reach values as high as those achieved with heat-
pasteurization [7–9]. The extent of microbial destruction in
HIPEF-treated OJ has been reported to be affected by sev-
eral factors such as electric field strength, treatment time,
pulse width, frequency, and polarity, among others [8–10].
Information about the effect of HIPEF on enzymes that
are detrimental for OJ quality is still incomplete. Most
studies refer to the effect of treatment parameters on en-
zyme inactivation but little is known about changes during
commercial shelf life. Pectin methyl esterase (PME) causes
cloud loss and gelation of commercial juices [11]. It has
been demonstrated that HIPEF treatments can inactivate
orange juice PME with different efficiency depending on
the assayed conditions [4, 12–15]. On the other hand, per-
oxidase (POD) is involved in the oxidation of a wide range
of natural compounds, thus being responsible for loss of
flavor quality in OJ [16]. HIPEF processing may achieve
high rates of OJ POD inactivation [17]. As far as we know,
studies on POD inactivation affecting shelf life of HIPEF-
processed OJ have not yet been published.
322
OJ is rich in vitamin C, a bioactive compound contribut-
ing to the antioxidant potential of OJ [18–20]. When com-
pared to heat processing, HIPEF caused higher retention of
vitamin C in OJ based-drinks [21, 22]. Its content in HIPEF-
treated OJ has been shown to be influenced by processing
and storage temperatures [23, 24]. Little is known about
the effects of HIPEF processing on the retention of other
bioactive compounds of OJ. Sánchez-Moreno et al. [22]
evaluated the impact of a HIPEF treatment on the flavanone,
carotenoid, vitamin C and free-radical scavenging capacity
of OJ. Nonetheless, no information is currently available
about the evolution of the antioxidant properties during the
commercial shelf life of OJ processed with HIPEF.
The objective of this work was to compare the effects
of HIPEF processing and heat pasteurization on the micro-
biological stability and several quality-related parameters
of OJ stored at 4 and 22 ◦ C, including PME activity, POD
activity, color, pH, acidity, ◦ Brix, vitamin C content and
antioxidant capacity.
Materials and methods
Sample preparation
OJ samples were obtained from fresh-squeezed Navelina
oranges (origin Valencia, Spain). Electrical conductivity
(Testo 240 conductivimeter; Testo GmBh & Co, Lenzkirch,
Germany) was determined in order to analyze the treated
product from the point of view of electrical properties.
HIPEF treatment
A bench scale system (OSU-4F, Ohio State University,
OH, USA) was used to treat the OJ samples. The juice
was pumped at a flow rate of 1 ml/s through a system of
eight collinear chambers connected in series. Each cham-
ber had a treatment volume of 0.012 cm3 that was de-
limited by two stainless steel electrodes separated by a
gap of 0.29 cm. The flow was controlled with a variable
speed pump (model 75210-25, Cole Palmer, Vernon Hills,
IL, USA). The treated OJ was passed through a cooling
coil connected between each pair of chambers and sub-
merged in an ice-water shaking bath, so that monitored
temperatures of the samples never exceeded 40 ◦ C. HIPEF
treatment consisted of bipolar square-wave pulses of 4-µs,
with a frequency of 200 Hz, and 35 kV/cm peak electric
field strength. The mean total treatment time (pulse width
× number of pulses) was calculated as 1,000 µs, thus re-
sulting in an energy input of 5390 MJ/m3 . The treatment
conditions were selected based on previous research where
pasteurization and high enzymatic inactivation levels were
achieved in orange juice processed by HIPEF [8, 9, 15, 17].
Thermal treatment
In order to compare the effectiveness of the HIPEF treat-
ment to a conventional thermal treatment, OJ samples were
heat pasteurized (90 ◦ C, 1 min). These conditions were se-
lected based on literature. Typical heat treatments of orange
juice vary from 95 ◦ C to 90 ◦ C for 15 s to 1 min [25]. OJ
was processed in a tubular stainless steel heat exchange coil
immersed in a hot water shaking bath using a gear pump
to maintain the desired flow rate (Universitat de Lleida,
Lleida, Spain). Once processed, the juice was immediately
cooled in a heat exchange coil immersed in an ice-water
bath.
Sample packaging and storage
HIPEF fluid handling system was disinfected first with 4%
NaOH and then with 10% chlorine and 20% ethanol solu-
tions prior to processing. Moreover, the first 200 ml treated
liquid were discarded to ensure stationary treatment con-
ditions. On the other hand, 200 ml-polypropelene bottles,
previously subjected to a sterilization process (121 ◦ C for
30 min) to ensure sterility, were used to store OJ. The juice
was bottled directly from the treatment system, leaving the
minimum amount of headspace volume. Once filled, the re-
ceptacle was tightly closed and stored under refrigeration
(4 ◦ C) or at ambient temperature (22 ◦ C) in darkness up to
analysis.
Microbiological stability
Serial dilutions of 10 ml OJ samples were prepared with
1% sterile peptone water. Microbial inactivation was stud-
ied by total aerobic plate counts using plate count agar
(PCA), and yeast and mould counts using chloramphenicol
glucose agar (CGA). Peptone and agar media were pur-
chased from Biokar Diagnostics (Beauvais, France). Each
dilution was plated in triplicate and incubated at 30 ◦ C
for 3 days in PCA plates, and 25 ◦ C for 5 days in CGA
plates.
Enzyme activity analysis
Pectin methyl esterase (PME)
PME activity was measured using the method described
by Kimball [11]. Pectin, sodium chloride and NaOH
were purchased from Acros Organics (New Jersey,
USA), Rectapur (Fontenay, France) and Panreac Quimica
(Barcelona, Spain), respectively.
A 10 ml aliquot of OJ was mixed with 40 ml of 1% pectin-
salt substrate. Reagents had been previously tempered at
30 ◦ C and were incubated at this same temperature. The
solution was adjusted to pH 7.0 with 2.0 N NaOH, and
then to pH 7.7 with 0.05 N NaOH. Once pH was stabilized,
0.10 ml of 0.05 N NaOH were added. The time required
for the solution’s pH to return to 7.7 was recorded.
PME activity expressed in pectin esterase units (PEU)
was calculated by the following equation:
[NaOH] · VNaOH
PEU =
Vjuice · t 

where [NaOH] is the NaOH concentration (0.05 N), VNaOH
is the volume of NaOH used (0.10 ml), Vjuice is the volume
of juice used (10 ml), and t is the time (in minutes) needed
for the pH to return to 7.7 after the addition of NaOH.
Percentage of residual PME activity was calculated in
relation to the activity of the untreated sample.
Peroxidase (POD)
POD activity in OJ was measured following a modification
of the method described by Cano et al. [26]. Chemicals
were purchased from Scharlau Chemie, SA (Barcelona,
Spain).
The enzyme extract was obtained by homogenizing 10 ml
of OJ with 20 ml of 200 mol/m3 sodium phosphate buffer
(pH = 6.5). The homogenate was centrifuged (24000×g,
15 min) at 4 ◦ C (Centrifuge AVANTITM J-25, Beckman
Instruments Inc., Fullerton, CA, USA) and the supernatant
was filtered through a Whatman No. 1 paper. The resulting
liquid constituted the enzymatic extract and was used with-
out delay to determine POD activity by spectrophotome-
try. To this purpose 2.7 ml 50 mol/m3 sodium phosphate
buffer (pH = 6.5), 0.2 ml p-phenylenediamine (10 g/kg)
as H-donor, 0.1 ml hydrogen peroxide (15 g/kg) as oxidant
and 0.1 ml of enzymatic extract were put in contact in a
1 cm path cuvette. The oxidation of p-phenylenediamine
was measured at 485 nm and 25 ◦ C using a CECIL CE
2,021 spectrophotometer (Cecil Instruments Ltd., Cam-
bridge, UK). POD activity was calculated from the initial
rate of the reaction which was computed from the linear
portion of the plotted curve. One unit of POD activity was
defined as a change in absorbance at 485 nm per minute
and milliliter of enzymatic extract.
Percentage of residual POD activity was calculated in
relation to the activity of the untreated sample.
Color measurement
All measurements were carried out at room temperature.
The color of the juice was measured with a Macbeth Color-
Eye 3,000 colorimeter (Macbeth-Kollmorgen Inst Corp,
Newburgh, NY, USA). Equipment was set up for illuminant
D75 and 10◦ observer angle. CIE L∗ (lightness), CIE a∗
(green to red chromaticity) and CIE b∗ (blue to yellow
chromaticity) were determined.
Determination of pH, titratable acidity and soluble
solids content
The pH was measured using a pH-meter CRISON 2001
(Crison Instruments S.A., Barcelona, Spain). Total acid-
ity was determined by dilution of 20 ml OJ sample into
100 ml distilled water and potentiometric titration with
0.1 N NaOH up to pH 8.1. A temperature-compensated re-
fractometer Atago RX-1000 (Atago Company Ltd., Tokyo,
323
Japan) was used to determine the soluble solids content of
the juice.
Vitamin C analysis
Vitamin C content in OJ was analysed by HPLC. The ex-
traction procedure was based on a method used by Soliva-
Fortuny et al. [27]. A sample of 25 ml OJ was mixed
with 10 ml of a solution containing 10% (w/v) metaphos-
phoric acid + 0.5% 2,3-dimercapto-1-propanol (BAL, a
thiol reducing reagent). The homogenate was centrifuged
at 15300×g for 15 min at 4 ◦ C (Centrifuge AvantiTM
J-25, Beckman Instruments Inc., Fullerton, CA, USA). The
supernatant was vacuum-filtered through Whatman No. 1
paper and then diluted to 50 ml with deionized water. The
samples were filtered with a Millipore 0.45-µm membrane.
An aliquot of 20 µl was injected into the HPLC system us-
ing a NH2 -Spherisorb S5 Column (250×4.6 mm, 5 µm).
The eluent was acetonitrile:5 mM potassium dihydrogen
phosphate buffer adjusted to pH 3.5 (40:60). The flow was
isocratic at a rate of 1 ml/min at room temperature. De-
tection was performed with a 486 Absorbance Detector
(Waters, Milford, MA) at 254 nm. Results were expressed
as vitamin C retention related to the untreated sample.
Antioxidant capacity measurement
The antioxidant capacity of OJ was studied through
the evaluation of free radical-scavenging effect on 1,1-
diphenyl-2-picrylhydrazyl (DPPH· ) radical. This determi-
nation was based on the method proposed by De Ancos et al.
[28]. Samples of fresh OJ were centrifuged at 6000×g for
15 min at 4 ◦ C (Centrifuge Medigifer; Select, Barcelona,
Spain) and aliquots of 0.010 ml of the supernatant were
mixed with 3.9 ml of methanolic DPPH· (0.025 g/l) and
0.090 ml of distilled water. The homogenate was shaken
vigorously and kept in a dark place for 30 min. Absorption
at 515 nm was measured with a spectrophotometer (CE-
CIL CE 2021; Cecil Instruments Ltd., Cambridge, UK).
Analysis grade methanol was used as a blank. Results were
expressed as percentage of inhibition of the radical DPPH· ,
which can be related to the decrease in absorbance with re-
spect to the control value (DPPH· initial absorption value).
Statistical analysis
Triplicate samples were packaged for analytical determina-
tion and triplicate measurements were performed for each
sample. Analysis of variance (ANOVA) was performed
to compare treatment mean values using the least sig-
nificant difference (LSD) test (p<0.05) using Statgraph-
ics Plus v 5.1 for Windows package (Statistical Graphics
Co., Rockville, MD, USA). Differences among treatments
throughout the time were analyzed using covariance anal-
ysis (ANCOVA).
324

Untreated OJ was rapidly spoiled by microorganisms

reaching 6.7 and 6.6 log CFU/ml for both total aerobic
plate counts and yeast and mold counts after 3 days at
22 ◦ C. Untreated samples stored under refrigeration (4 ◦ C)
kept microbial loads below 6 log CFU/ml during 35 days.
On the other hand, thermal pasteurization led to greater mi-
croorganism inactivation and maintained microbial counts
at less than 1 CFU/ml throughout 56 days at both 4 and
22 ◦ C. As well, the OJ samples processed with HIPEF and
stored at 4 ◦ C were shelf stable for at least 56 days, whereas
storage temperatures of 22 ◦ C could not ensure stability dur-
ing this period and counts higher than 6 log CFU/ml were
reached after 42 days. Min et al. [24] achieved microbial
stability of refrigerated OJ treated with a pulse strength of
40 kV/cm for 97 µs during 112 days. Beyond this period,
the growth of microorganisms was attributed to the survival
of mold and yeast ascospores, that are resistant to HIPEF
treatments.

Enzyme activities

Pectin methyl esterase (PME)

Inactivation of PME is desired to preserve citrus juice

cloud stability. Residual relative PME activity of raw and
processed OJ during storage at 4 and 22 ◦ C is shown in
Fig. 2. PME is a thermolabile enzyme and it is therefore
Fig. 1 Effects of HIPEF treatment and heat pasteurization (TT) on being currently inactivated by heat [25]. Our results are
the microbial counts (total aerobic bacteria (A) and yeasts and molds
(B)) of orange juice throughout storage at 4 and 22 ◦ C. No significant consistent with this fact, so that a 100% inactivation of the
differences (p<0.01) were found between TT at 4 and 22 ◦ C initial PME activity (14.6×10−4 PEU) was reached when
processing OJ at 90 ◦ C for 1 min. On the other hand, HIPEF
treatment inactivated 81.6% activity in the fresh-squeezed
juice without exceeding 40 ◦ C. Because temperature was
Results and discussion not high enough to inactivate PME, enzyme inactivation
was probably due entirely to the HIPEF treatment itself.
Microbiological stability The mechanism of inactivation has not been yet well
described but would be associated to protein unfolding and
The total aerobic and the yeast and mold plate counts of
HIPEF-processed and thermally-pasteurized OJ compared
to untreated juice are shown in Fig. 1. Microbial loads
of the fresh-squeezed juice were 3.5 and 2.9 log CFU/ml
for total aerobics and yeast and mold counts, respectively.
Populations of yeasts are more likely to be predominant
in OJ because of its low pH and high content in sugars
and organic acids [29]. In contrast, either HIPEF-treated
(35 kV/cm for 1,000 µs with 4-µs bipolar pulses at
200 Hz) or heat-treated (90 ◦ C for 1 min) juices had initial
microbial counts below 1 log CFU/ml. Microbial inacti-
vation achieved with the HIPEF treatment was similar to
that reported by Yeom et al. [23] for OJ preserved with a
treatment of 35 kV/cm for 59 µs. In their study, they report
an increase of the temperature of the OJ to 60.1 ◦ C during
processing, which could participate in the destruction of
some microorganisms, especially bacteria. In our research, Fig. 2 Effects of HIPEF treatment and heat pasteurization (TT) on
treatment temperatures did not exceed 40 ◦ C, thus ensuring the residual pectin methylesterase (PME) activity of orange juice
that all microorganisms were destroyed due to electropo- throughout storage at 4 and 22 ◦ C. PME activity of heat pasteur-
ration, and this would justify the prolonged treatment time ized orange juice was under the limit of detection. No significant
needed to reach a similar microbial inactivation. differences (p<0.01) were found between TT at 4 and 22 ◦ C

denaturation, breakdown of covalent bonds and oxidation-
reduction reactions, that are induced by intense electric
fields. [30]. These modifications would affect the active site
of the enzyme and would therefore difficult the assembling
of the substrate [31]. Yeom et al. [13, 14] reported inactiva-
tion of 83.2% and 90% in OJ treated at 35 kV/cm for 184 µs
with 2.2-µs pulses at 700 Hz and at 35 kV/cm for 59 µs with
1.4-µs pulses at 600 Hz with maximum treatment tem-
peratures of 61.9 and 60.1 ◦ C, respectively. A treatment
of 35 kV/cm for 1500 µs with 4-µs bipolar pulses at
200 Hz without exceeding 37.5 ◦ C caused 80% reduction
of PME activity [15]. In addition, it was shown that
PME inactivation is greatly dependent on the treatment
conditions. Van Loey et al. [12] reported reductions of
less than 10% of orange peel PME activity in orange juice
processed at 35 kV/cm for 1,000 µs.
Residual PME activity of HIPEF-treated OJ remained
significantly constant around 20% during 56 days storage
either at 4 or 22 ◦ C. These results agree with those of
Yeom et al. [14], who found that decreased PME activity
of treated OJ was not recovered during storage at 4 and
22 ◦ C for 112 days, concluding that PEF treatments cause
irreversible inactivation of PME. Heat pasteurization was
also effective in reaching irreversible activity depletions
irrespective of the storage temperature, which did not show
to have any influence on PME activity throughout storage
in any of the tested treatments or conditions. PME activity
in nontreated OJ followed an exponential decay reaching
values of 37.2–40.3% activity of the unprocessed fresh
juice at 56 day.
Peroxidase (POD)
Both the HIPEF and the heat treatment had a great impact
on the POD activity of OJ. POD activity of fresh-squeezed
OJ was 2.42
absorbance/(min·ml) (100%). The HIPEF
treatment, consisting of 35 kV/cm for 1000 µs with bipolar
pulses of 4 µs at 200 Hz, inactivated the 100% of the initial
activity (Fig. 3). Under these conditions POD inactivation
was the same than that reached in previous studies with a
treatment of 4-µs bipolar pulses at 35 kV/cm for 1500 µs
at 200 Hz without exceeding 35.0 ◦ C [17]. These results
contrast with the slighter inactivation reported in other food
matrixes such as milk [12, 32] or soybean [33, 34] and can
be attributed to different treatments and enzyme source.
Moreover, due to its thermostable nature [35], OJ POD
activity was less sensible to thermal treatments and was
reduced to a 4% of the initial value. The process of inacti-
vation is likely to be induced through denaturation of the
enzyme, by modifying its conformational state, and thus
preventing the substrate from fitting the active site of the
enzyme. However, in comparison to thermal treatments,
HIPEF probably produced greater changes in the enzyme
structure, starting with a strong polarization of the protein
molecules and ending with the formation of hydrophobic
interactions or covalent bonds and subsequent formation
of aggregates [36, 37]. Another hypothesis for enzyme in-
325
Fig. 3 Effects of HIPEF treatment and heat pasteurization (TT) on
the residual peroxidase (POD) activity of orange juice throughout
storage at 4 and 22 ◦ C. POD activity of HIPEF treated orange juice
was under the limit of detection. No significant differences (p<0.01)
were found between 4 and 22 ◦ C for HIPEF and TT, repectively
activation would be electrochemical reactions caused by
electrode corrosion. No visible consequences of the degra-
dation of the stainless steel electrodes was perceptible and
no information is available by the moment regarding the
effect of these phenomena on enzymes. Morren et al. [38]
concluded that the application of short bipolar pulses, used
in this study, may avoid most occurrence of electrode cor-
rosion in PEF treatment chambers. However, the effect of
treatment parameters and electrode materials on these pro-
cesses needs to be further investigated.
Residual POD activity of OJ subjected to HIPEF and ther-
mal pasteurization throughout storage at 4 and 22 ◦ C is dis-
played in Fig. 3. Non-treated juice maintained significant
residual POD activity values during storage, decreasing
gradually and reaching 18.7–21.3% of the initial activity at
56 days storage. The enzyme inactivation throughout stor-
age was not significantly (p<0.05) dependent on the storage
temperature. Regarding thermal and HIPEF processing,
both technologies yielded low stable enzyme activity
during 56 days, thus indicating that the changes induced in
the enzyme structure were irreversible for both treatments.
Therefore, HIPEF processing can be a good alternative
to heat processing in order to reduce oxidation of natural
compounds and to avoid undesirable POD-mediated
reactions in OJ that may account for losses in flavor quality
[16].
Color
Formation of dark compounds leading to browning, to-
gether with flavor changes and nutrient loss, has been tra-
ditionally linked to thermal over-processing of OJ. The
effects of HIPEF processing and thermal pasteurization
on the color of OJ and changes during storage at 4 and
22 ◦ C for 56 days are shown in Fig. 4. Differences in the
CIELab parameters between HIPEF and heat treatments
did not appear to be significant (p>0.05). L∗ a∗ b∗ values at
0 day storage were 43.4, 7.4, 30.3 and 44.1, 6.2, 31.3 for
326
Fig. 4 Effects of HIPEF treatment and heat pasteurization (TT) on
the color of orange juice throughout storage under refrigeration or at
ambient temperature
HIPEF-processed and thermally-processed juice, respec-
tively. Untreated OJ exhibited lower L∗ and b∗ values than
treated juice. Generally, HIPEF treatments have been re-
ported as a method for better preserving the color of OJ in
comparison to heat processing [23, 24].
The color of OJ processed with HIPEF remained constant
throughout storage at 4 and 22 ◦ C and was statistically
brighter than heat-pasteurized or untreated juice. By the
contrary, heat-treated juice underwent a dramatic change
in a∗ and b∗ from the first days of storage at ambient
temperature. L∗ values decreased especially during the
last 20 days storage at 22 ◦ C but no parallelism could be
pointed out at 4 ◦ C. The existence of an initial lag period,
in which colorless compounds are probably formed, would
explain this trend. According to these results, Yeom et al.
[23] reported a similar trend for browning of processed
OJ after prolonged storage. In their study, statistically
significant differences were found between HIPEF-treated
and thermally-treated OJ during storage at 4 ◦ C but similar
color was observed between samples stored at 22 ◦ C.
pH, titratable acidity and soluble solids content
Untreated juice had an electric conductivity of
0.439±0.012 S/m. The effects of HIPEF and heat treat-
ments on the pH, acidity and soluble solids content of
OJ are exhibited in Tables 1–3, respectively. The kind
of processing had little effect on the physical properties
of the juice at 0 day storage. Acidity and pH values of
thermally-processed and HIPEF-processed OJ were main-
tained around 3.5 and 0.7–0.8 g citric acid/100 ml orange
juice for 56 days storage. Nevertheless, a significant in-
crease in both parameters of untreated juice stored at 22 ◦ C
was detected from day 28 to day 56. These changes may be
related to the spoilage of the juice by microorganisms that
would contribute to an increase in acidity. No significant
changes were found in the soluble solids content of the
treated juices. However, mean values for HIPEF-treated
and heat-treated OJ throughout storage were 11.8 and
11.4 ◦ Brix and estimated differences between them were
found to be significant (p<0.05). A dramatic decrease in
soluble solids was observed between day 7 and day 14 in
nonprocessed samples. Consumption of sugars by develop-
ing microbial populations in the juice is the most probable
explanation for these changes. Eventually, storage temper-
ature did not have a significant influence on the evolution
of ◦ Brix. Nevertheless, covariance analysis reported sig-
nificantly higher acidity values for juices stored at 22 ◦ C
(0.77 g citric acid/100 ml juice) compared to samples stored
at 4 ◦ C (0.71 g citric acid/100 ml juice).
Vitamin C
Fresh-squeezed unprocessed juice had a vitamin C con-
tent of 52.1 mg/100 ml. Recommended daily intake (RDI)
of vitamin C is currently revised but should be never be-
low 60 mg, as established by the U.S. Food and Drug
Administration [39]. According to this, a 250 ml serving
size should contain 24 mg/100 ml in order to contribute
to the 100% of the RDI. Therefore, a retention of more
than 46% of the initial vitamin C content found in the fresh
juice qualified to provide a least the 100% of the RDI.
HIPEF-processing resulted in higher ascorbic acid content
in OJ (91.2%) than thermal pasteurization (82.8%). Dif-
ferent studies have proved the effectiveness of HIPEF in
achieving higher vitamin C retention in comparison with
heat treatments. Min et al. [24] did not found differences be-
tween fresh and HIPEF-processed OJ treated at 40 kV/cm
for 97 µs at 2,000 Hz with bipolar pulses of 2.6 µs and
a maximum temperature of 40 ◦ C. Consistently, Sánchez-
Moreno et al. [22] reported vitamin C retention of 93%
using a treatment of 35 kV/cm for 1,000 µs with bipo-
 327

Table 1 Effects of HIPEF Storage HIPEF 4 ◦ C HIPEF 22 ◦ C TT 4 ◦ C TT 22 ◦ C Control 4 ◦ C Control 22 ◦ C

treatment or thermal
pasteurization on pH of orange time (day)
juice throughout storage at 0 3.63±0.01b 3.63±0.01b 3.61±0.01ab 3.61±0.01ab 3.60±0.01a 3.60±0.01a
4 and 22 ◦ C
3 3.66±0.01b 3.54±0.04a 3.71±0.01c 3.57±0.01a 3.74±0.02c 3.65±0.01b
7 3.61±0.01c 3.55±0.01a 3.58±0.01b 3.55±0.00a 3.63±0.01d 3.54±0.01a
14 3.66±0.01bc 3.60±0.01a 3.61±0.00a 3.60±0.00a 3.65±0.01b 3.67±0.00c
HIPEF: HIPEF-processed juice;
TT: heat-processed juice; 21 3.65±0.00b 3.60±0.01a 3.61±0.01a 3.60±0.01a 3.65±0.01b 3.66±0.01b
Control: unprocessed juice 28 3.56±0.01a 3.64±0.01d 3.62±0.00c 3.59±0.01b 3.68±0.00e 3.71±0.00f
Values are expressed as mean ± 35 3.59±0.03a 3.61±0.06a 3.60±0.06a 3.60±0.04a 3.65±0.03ab 3.74±0.04b
standard deviation 42 3.57±0.01a 3.71±0.01c 3.64±0.01b 3.65±0.01b 3.70±0.00c 3.75±0.01d
Different letters in the same row 49 3.53±0.00a 3.65±0.04c 3.65±0.01c 3.60±0.01b 3.59±0.01b 3.81±0.00d
indicate significant differences 56 3.58±0.04a 3.63±0.04a 3.64±0.03a 3.61±0.04a 3.62±0.04a 3.82±0.06b
(p<0.05)

Table 2 Total acidity values (g Storage HIPEF 4 ◦ C HIPEF 22 ◦ C TT 4 ◦ C TT 22 ◦ C Control 4 ◦ C Control 22 ◦ C

citric acid/100 ml orange juice)
of HIPEF-treated or time (day)
thermally-processed 0 0.72±0.01a 0.72±0.01a 0.70±0.01a 0.70±0.01a 0.76±0.01b 0.76±0.01b
fresh-squeezed orange juice
throughout storage at 4 and 3 0.68±0.01bc 0.68±0.01bc 0.61±0.01a 0.65±0.01b 0.61±0.01a 0.68±0.01c
22 ◦ C 7 0.67±0.01b 0.67±0.01b 0.60±0.01a 0.74±0.01c 0.68±0.01b 0.67±0.01b
14 0.65±0.01a 0.86±0.01c 0.65±0.01a 0.86±0.02c 0.71±0.01b 0.71±0.01b
HIPEF: HIPEF-processed juice;
TT: heat-processed juice; 21 0.71±0.01a 0.88±0.01b 0.70±0.01a 0.86±0.01b 0.70±0.01a 0.72±0.01a
Control: unprocessed juice 28 0.76±0.01c 0.77±0.01c 0.68±0.01a 0.78±0.02c 0.72±0.01b 0.77±0.01c
Values are expressed as mean ± 35 0.79±0.01c 0.80±0.01c 0.69±0.01a 0.80±0.01c 0.74±0.01b 0.76±0.01b
standard deviation 42 0.82±0.01d 0.80±0.01cd 0.71±0.01a 0.79±0.01bc 0.78±0.02bc 0.76±0.01b
Different letters in the same row 49 0.76±0.01bc 0.78±0.01cd 0.71±0.01a 0.79±0.01d 0.76±0.01b 0.86±0.01e
indicate significant differences 56 0.81±0.01c 0.82±0.01c 0.71±0.01a 0.78±0.01b 0.77±0.01b 0.90±0.01d
(p<0.05)

Table 3 Soluble solids (◦ Brix) Storage HIPEF 4 ◦ C HIPEF 22 ◦ C TT 4 ◦ C TT 22 ◦ C Control 4 ◦ C Control 22 ◦ C

content of HIPEF-treated or
thermally-processed time (day)
fresh-squeezed orange juice 0 11.65±0.07b 11.35±0.07a 11.85±0.07c 11.85±0.07c 11.65±0.07b 11.35±0.07a
throughout storage at 4 and
22 ◦ C 3 11.65±0.24b 11.35±0.24ab 11.42±0.02ab 11.73±0.07ab 11.53±0.07b 10.93±0.32a
7 11.59±0.01b 11.41±0.33ab 11.42±0.02ab 11.79±0.16b 11.47±0.15ab 11.05±0.32a
14 11.65±0.24c 11.41±0.33c 11.97±0.07c 11.97±0.07c 7.51±0.30b 5.92±0.47a
HIPEF: HIPEF-processed juice;
TT: heat-processed juice; 21 11.29±0.10c 11.35±0.24c 11.79±0.02d 11.97±0.10d 7.57±0.05b 5.55±0.05a
Control: unprocessed juice 28 11.23±0.02c 11.47±0.24c 11.91±0.16d 11.91±0.19d 7.87±0.13b 5.74±0.04a
Values are expressed as mean ± 35 11.31±0.07d 11.13±0.07c 11.85±0.07e 11.75±0.07e 7.63±0.07b 5.42±0.07a
standard deviation 42 11.35±0.02c 11.71±0.25cd 12.09±0.07d 12.03±0.01d 7.99±0.04b 6.04±0.30a
Different letters in the same row 49 11.29±0.07c 11.11±0.24c 11.79±0.02d 11.36±0.07c 7.54±0.06b 5.50±0.20a
indicate significant differences 56 11.12±0.07d 10.97±0.07c 11.86±0.07f 11.58±0.07e 7.12±0.07b 4.42±0.07a
(p<0.05)

lar pulses of 4 µs at 200 Hz and a maximum treatment
temperature of 50 ◦ C. Most differences between HIPEF
and heat treatments can be explained through the temper-
atures reached throughout processing. Ascorbic acid is a
well known sensitive bioactive compound and therefore
both treatment and storage temperatures can greatly affect
the rates of its degradation.
The effects of HIPEF processing and thermal processing
on the concentration of vitamin C during storage at 4 and
22 ◦ C are illustrated in Fig. 5. The highest vitamin C reten-
tion throughout storage was achieved in HIPEF-treated OJ.
During 56 days storage, vitamin C content of processed
and unprocessed OJ decreased exponentially. Vitamin C
content of the juice processed with HIPEF and stored at
4 ◦ C was 49.2% at 56 day, but it felt below the RDI in
only 14 days (44.5%) when stored at 22 ◦ C. For thermally-
processed juice retention was 42.8% and 44.7% at 14 day
and 7 day storage at 4 and 22 ◦ C, respectively. Results of
Sánchez-Moreno et al. [22], Yeom et al. [23] and Min et al.
[24] are in accordance with ours, reporting higher levels of
vitamin C retention in orange juice processed with HIPEF
compared to thermally-processed juice. In addition, high
storage temperature showed to be detrimental on vitamin
C retention, according to other published studies [23, 40,
41].
Degradation of vitamin C could be responsible for the
greater changes in color found in heat-pasteurized juice
stored at 22 ◦ C. Ascorbic acid acts as an oxygen scav-
enger for the removal of molecular oxygen in enzymatic
reactions, but at high temperatures it may react forming car-
328
Fig. 5 Effects of HIPEF treatment and heat pasteurization (TT) on
the vitamin C retention of orange juice throughout storage at 4 and
22 ◦ C
bonylic compounds that are responsible for non-enzymatic
browning [42]. In this regard, Manso et al. [43] reported
that addition of supplementary L-ascorbic acid in order to
minimize its loss in OJ can be counterproductive so that
color changes are promoted, especially at high processing
and storage temperatures.
Antioxidant capacity
The antioxidant capacity of OJ is related to the amount and
composition of bioactive compounds such as vitamin C,
carotenoids or flavanones [22, 44]. The effects of process-
ing and storage conditions on the DPPH radical scavenging
capacity (RSC) are shown in Fig. 6. Inhibition of DPPH· at
0 day storage for untreated, traditionally heat-pasteurized
and HIPEF-treated OJ was 41.4%, 30.6% and 35.2%, re-
spectively. These values are in accordance with the initial
loss in vitamin C attributed to each treatment (Fig. 5). There
is substantial evidence for the antioxidant activity of vita-
min C, which acts as a scavenging agent of reactive oxygen
and nitrogen species before they participate in oxidative
Fig. 6 Effects of HIPEF treatment and heat pasteurization (TT) on
the DPPH· radical scavenging capacity of orange juice throughout
storage at 4 and 22 ◦ C
reactions [45]. In a recent work, no significant differences
were found between untreated and HIPEF-treated DPPH·
radical scavenging capacity [22]. In our study, though, no
statistically significant (p<0.05) differences could be ob-
served between the estimated means for DPPH· inhibition
of untreated and HIPEF-processed OJ during storage.
The antioxidant capacity of OJ slightly depleted with
time irrespective of the storage temperature. However, the
magnitude of the changes in the free-radical scavenging
capacity could not be associated with the variation ob-
served in vitamin C contents during storage, suggesting
that other antioxidant compounds were more stable than
ascorbic acid to processing. Changes in the composition of
bioactive compounds could be a possible explanation for
these observations but little research has been developed
in this field. Sánchez-Moreno et al. [22] speculated about
an unexpected release of carotenoids and flavanones from
vegetable cells suspended in the cloud of OJ due to HIPEF
processing but no differences between untreated and treated
samples were detected. No information is available about
the changes in the composition of these compounds during
storage of OJ processed with novel processing technologies
in comparison to traditional processing.
Conclusions
HIPEF processing (35 kV/cm for 1,000 µs with 4-µs bipo-
lar pulses at 200 Hz) can produce stable OJ with a shelf-life
comparable to that achieved with thermal processing (90 ◦ C
for 1 min) but retained higher nutritional value during stor-
age. Treating OJ with HIPEF was not as effective as heat
pasteurization in terms of microbiological stability at the
process parameters applied. However, HIPEF processing
ensured safety of OJ during at least 56 days in combina-
tion with refrigerated storage (4 ◦ C) and also produced a
juice rich in vitamin C (more than 100% of the recom-
mended daily intake in a 250 ml serving size) during this
same period. HIPEF-treated OJ also retained 2.7 and 2.5
times more vitamin C than heat-treated and untreated OJ
throughout 56 days storage at 4 ◦ C respectively. Vitamin C
retention in OJ processed with HIPEF was a 10% higher
than that achieved in a juice processed with traditional pas-
teurization. Hence the better color preservation achieved
due to the reduction of both anaerobic (heat-induced) and
aerobic (oxygen-mediated) browning processes. Despite
vitamin C degradation, antioxidant capacity was kept al-
most stable throughout storage, evidencing that HIPEF pro-
cessing, might have a beneficial effect on the amounts of
other bioactive compounds in OJ. Furthermore, the HIPEF
treatment was more effective than thermal processing in
inactivating OJ peroxidase and achieved slightly lower but
irreversible inactivation of PME.
More information is needed to cast light on the effect
of different HIPEF treatments on the shelf life of fruit
juices. In addition, further research dealing with the effects
of HIPEF treatments on enzymes should be addressed to
reach higher levels of inactivation and to make feasible a

scale-up of HIPEF technology for the juice processing in-
dustry. In depth research should be carried out to investigate
new prospects for the use of HIPEF processing in order to
preserve the nutritional value of fresh products.
Acknowledgements This work was supported by the Departament
d’Universitats, Recerca i Societat de la Informació of the General-
itat de Catalunya (Spain) and the Consejerı́a de Educación of the
Comunidad Autónoma de Madrid (Spain) through the project CAM
07G/0040/2000. P. Elez-Martı́nez thanks the Universitat de Lleida
(Spain) for the pre-doctoral grant.
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