Article

Effects of high hydrostatic pressure on the overall

quality of Pêra-Rio orange juice during shelf life

Paz Spira1, Antonio Bisconsin-Junior1 , Amauri Rosenthal2 and

Magali Monteiro1

Abstract
The effect of high hydrostatic pressure on antioxidant activity, total phenolic compounds, physicochemical
characteristics, color, pectin methylesterase activity, and microbiological count were evaluated during the
shelf life of Pêra-Rio orange juice. Pressurized (520 MPa, 60  C, for 360 s), non-processed and pasteurized
(95  C/30 s) orange juice were compared at zero time of storage. Pressurized and pasteurized juices were
studied during a refrigerated 90-day shelf life. Pressurization did not cause expressive change in physico-
chemical characteristics of Pêra-Rio orange juice along shelf life, but significantly reduced pectin methyles-
terase residual activity to 13% and microbiological counts below detection levels up to 68 days of storage,
with small counts (30.0  10 CFU/mL mesophilic aerobic bacteria and 20.7  10 CFU/mL yeast and mold) at
90 days, capable of ensuring the juice’s stability along shelf life. Lightness (L*) and b* values were signifi-
cantly reduced by high hydrostatic pressure during shelf life, while a* values were significantly higher.
Ascorbic acid decreased around 80% during shelf life. Antioxidant activity remained stable after processing
and during storage.

Keywords
High hydrostatic pressure, orange juice, shelf life, antioxidant activity, physicochemical characteristics,
microbiological count
Date received: 8 June 2017; accepted: 20 February 2018

Comércio Exterior (SECEX), 2017; United States

INTRODUCTION
Department of Agriculture (USDA), 2017). The
Brazil is the largest producer and exporter of orange Brazilian citriculture numbers are expressive—out of
juice worldwide, withholding 61.3% of the global pro- five glasses of orange juice consumed worldwide,
duction in the 2015/2016 harvest. Of the nearly 300 three come from the Brazilian production
million orange boxes, weighing 40.8 kg each, produced (CITRUSBR, 2017).
in the 2015/2016 harvest, 15% were consumed in In recent years, a new trend has been observed regard-
natura and 85% were destined to the industry. Brazil ing orange juice consumption of NFC rather than
produced 531 mil ton of frozen concentrated orange FCOJ. This could be explained by the fact that the
juice (FCOJ) and 240 mil ton of not from concentrate NFC juice is exposed a shorter time to high temperatures
(NFC) FCOJ equivalent orange juice in the 2015/2016 when compared to FCOJ, resulting in less drastic flavor
harvest. The country exports 98% of the orange juice it and aroma modifications (Janzantti et al., 2011).
produces, mostly to European countries (70%), the
United States (25%), and Japan (3%) Instituto de 1
Department of Food and Nutrition, School of Pharmaceutical
Economia Agrı́cola (IEA), 2017; Secretaria de Science, São Paulo State University – UNESP, Araraquara, Brazil
2
Embrapa Food Technology, Av. das Américas, 29501, Rio de
Food Science and Technology International 0(0) 1–12
Janeiro, RJ, Brazil
! The Author(s) 2018 Reprints and permissions: Corresponding author:
sagepub.co.uk/journalsPermissions.nav Magali Monteiro, Department of Food and Nutrition, Rodovia
DOI: 10.1177/1082013218768997 Araraquara-Jau, km 01, Campus, 14800-903 Araraquara-SP,
journals.sagepub.com/home/fst Brazil.
Email: [email protected]
Food Science and Technology International 0(0)

Thermal treatment is still the most used processing five levels and 17 experiments was used (Rodrigues and
treatment for orange juice due being able to destroy Iemma, 2009). The increase in pressure (100 to 600
microorganisms and inactivate enzymes. However, the MPa), temperature (30 to 60  C), and time (30 to
intense time/temperature conditions may reduce natural 360 s) promoted the reduction on the PME residual
and characteristic flavor and ascorbic acid (AA), affect- activity from CCD (15.4% to 107.9%) and aerobic
ing the juice’s overall quality (Janzantti et al., 2011). microorganism count of orange juice. The optimal con-
The fruit juice industry has been looking into innova- dition (520 MPa, 60  C, for 360 s) was defined based on
tive technologies, which use minimal heat treatment and its capacity to reduce PME residual activity below 20%
are able to produce juice with more natural-like charac- and microbial count lower than 2 log CFU/mL.
teristics, in order to preserve flavor and nutritional However, these were not necessarily the same condi-
aspects, complying with the consumption trend that tions for maintaining optimal vitamin C levels and bio-
searches for healthy and flavorful products (Deliza active compounds activity (Bisconsin-Junior et al.,
et al., 2005). High hydrostatic pressure (HHP) reduces 2015). A different study (Torres et al., 2015), which
orange juice contaminating microflora and the enzyme evaluated the effects of pressure, temperature, and
pectin methylesterase’s (PME) activity (Nienaber and time on orange juice with combinations ranging from
Shellhammer, 2001) by using pressure instead of high 250 to 500 MPa, 25 to 65  C and 1 to 30 min, found that
temperature, therefore preserving its sensory and nutri- a 6.5 log cycle reduction was obtained at 400 MPa for
tional aspects. Several PME isoenzymes are associated 3 min at 25  C. However, in order to inactivate at least
with cloud loss, attributed its endogenous activity, 90% of PME activity the ideal conditions were 500
which demethoxylates soluble pectins causing calcium MPa, at 50  C for 15 min, corroborating with idea
pectates precipitation and clarification of the juice that enzyme inactivation requires more severe condi-
(Versteeg et al., 1980). Cloud stability is an important tions, as shown by the optimization study.
quality aspect of orange juice, since it positively affects The aim of this study was to evaluate the effect of
turbidity, flavor, and color characteristics of the juice. HHP treatment on the quality aspects of the Pêra-Rio
A low PME residual activity, however, could still pre- orange juice during shelf life, thus offering knowledge-
serve cloud stability during the shelf life of the juice able insight to the citrus industry, enabling it to be
(Bisconsin-Junior et al., 2014). produced and commercialized both domestically and
In addition, color, soluble solids, pH, carotenoid abroad.
content, and bioactive compounds of orange juice are
not considerably affected by HHP (Baxter et al., 2005; MATERIALS AND METHODS
Bull et al., 2004; Timmermans et al., 2011; Vervoort
Chemicals and materials
et al., 2011).
Although HHP is a well researched technology, to Citrus pectin, ABTS diammonium salt, 2,4,6-tris(2-pyr-
the best of our knowledge, the shelf life study of the idyl)-s-triazine, 2,2-diphenyl-1-picrylhydrazyl, 6-
Pêra-Rio orange juice has not yet been developed. This hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
technology is still not being used by the Brazilian juice (Trolox), and gallic acid were obtained from Sigma-
industry, which presents itself as a whole range of Aldrich (St Louis, MO); AA and glucose purchased
opportunities. Bull et al. (2004) reported that Valencia from Merck (Darmstadt, Alemanha); Folin-Ciocalteu
orange juice treated at 600 MPa, 20  C for 60 s did not reagent from Imbralab (Ribeirão Preto, SP, Brazil);
affect AA significantly, however a different study found potassium persulfate from Fluka (Steinheim,
11% reduction in AA of the juice treated at 100 MPa, Germany); methanol from JT Baker (Philipsburg, PA);
60  C during 300 s (Sánchez-Moreno et al., 2005). sodium carbonate, sodium hydroxyl, potassium sodium
Sánchez-Moreno et al. (2005) reported that after 400 tartrate tetrahydrate, cupric sulfate pentahydrate, and
MPa, 40  C for 60 s, orange juice showed no significant potassium phosphate dibasic from Labsynth (Diadema,
reduction on vitamin C, but presented higher extract- SP, Brazil); oxalic acid and 2,6-dichloroindophenol
ability of carotenoids (54%) and flavanones (34%), and sodium salt hydrate from Vetec (Rio de Janeiro, RJ,
no significant difference on antioxidant activity. Brazil); PetrifilmTM used for counting mesophilic aerobic
The HHP conditions applied in this work were bacteria, thermotolerant and total coliforms, mold and
chosen through the results of an optimization study yeast was obtained from 3MTM (St. Paul, MN).
(Bisconsin-Junior et al., 2014), which applied the
response surface methodology to evaluate the effect of
Orange juice
pressure, temperature and time on PME activity and
total counts of aerobic microorganisms, yeasts, and Oranges from the Pêra-Rio variety were provided by a
molds. A central composite design (CCD) of three inde- citrus industry from Araraquara, SP, Brazil. The fruit
pendent variables (pressure, time and temperature) with were cultivated in the region of Bauru, SP, Brazil

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 Spira et al.

(22 25’ 59’’ S; 49 10’ 31’’ W), during the 2012/2013
Pectin methylesterase
harvest. The oranges were processed at JBT FoodTech
Citrus System in Araraquara, SP. Before the extraction, The PME activity was evaluated according to
the fruit and the equipment were sanitized using water Hagerman and Austin (1986), as reported by
aspersion, and later submerged in a sodium hypochlor- Bisconsin-Junior et al. (2014). Orange juice and NaCl
ite solution (100 mg of chlorine/L) for 10 min. The (8.8%, w/v) were homogenized (4.5:15, w/v) and cen-
extraction itself was performed in the JBT 391B extrac- trifuged (18,000 g/20 min at 4  C). The supernatant was
tor, using ideal configuration for obtaining NFC (not collected and used as the enzymatic extract. The sub-
from concentrate) juice and premium juice extractor strate was composed of citrus pectin solution (0.5%,
settings with a UFC-35 finisher (0.25 mm sieve). w/v), bromothymol blue (0.01%, w/v) in potassium
phosphate buffer (0.003 M) and distilled water. The
substrate and enzymatic extract were adjusted to pH
HHP treatment
7.5. Substrate was added to the enzymatic extract
For the HHP treatment, the orange juice (100 mL) was (20 mL) and the pectin hydrolysis reaction was moni-
packaged in heat sealed, polyethylene (PE) bags tored by the absorbance decrease at 620 nm using a
(Selovac 200B II, Selovac, São Paulo, Brazil). The spectrophotometer (Evolution 220, Thermo Scientific,
juice was pressurized, according to the experimental USA), and water as blank. PME activity analyses were
design, in a Stansted Food Lab 9000 (Stansted Fluid performed in triplicate, at 25  C and results expressed
Power, Stansted, UK). The treatment conditions were as PME residual activity (%).
previously defined in an optimization study (Bisconsin-
Junior et al., 2014). The chosen pressurization condi-
Microbiological analysis
tions were 520 MPa, 60  C, for 360 s, which are capable
of producing orange juice with residual enzyme PME Orange juice (10 mL) was added to 90 mL sterilized buf-
activity lower than 20% and aerobic microorganism fered peptone water (BPW). After homogenization, ali-
count lower than 2 log CFU/mL. The HHP equipment quots were serially diluted in BPW and 1 mL of each
has a 500 mL pressure vessel with a maximum nominal dilution was inoculated onto PetrifilmTM 3MTM plates
operation pressure of 900 MPa and a temperature range for thermotolerant and total coliforms, mesophilic aer-
from 20  C to 90  C. The temperature in the vessel is obic bacteria, yeast, and mold counts. The thermoto-
controlled by liquid circulation in the outer layer con- lerant and total coliforms, and aerobic microorganisms
nected to a heating–cooling system. The pressure trans- count were performed after incubation at 35  1  C for
mitting fluid used was 70% (v/v) ethanol. The 48  3 h and the yeast and mold count after incubation
compression rate was 3.5 MPa/s and the decompression at 25  1  C for 120  6 h. The minimum level of detec-
time was less than 10 s. During the treatment, the tem- tion was 10 CFU/mL (AOAC, 2011). The analyses were
perature and pressure inside the pressure vessel were performed in triplicate.
monitored (Bisconsin-Junior, et al., 2015).
Color measurement
Pasteurization of the orange juice
Konica Minolta CM 600D (Konica Minolta Sensing,
The orange juice was pasteurized using the Armfield Osaka, Japan) spectrophotometer was used (Meléndez-
FTD25D SSHE (Armfield, UK) tubular heat exchan- Martı́nez et al., 2005) to measure color (D65 light
ger at 95  C for 30 s (Braddock, 1999), and later cooled source, 10 observation angle, 8 mm opening). The
to 20  C. Afterwards, the juice was packaged aseptically orange juice was placed in a quartz cell of 10 mm
in PE flasks (500 mL). optic path length (50  38  10 mm). L* (lightness/
darkness), a* (redness/greenness), and b* (yellowness/
blueness) parameters were evaluated. Additionally,
Physicochemical analysis
chroma (color saturation), Hue angle, and total color
The physicochemical parameters of the non-processed, difference were calculated. The analyses were per-
pasteurized, and pressurized orange juice were evalu- formed at 25  C, in quintuplicate.
ated. Soluble solids (method no 932.12), total titratable
acidity (method no 942.15), pH (method no 945.27),
Assessment of bioactive compounds
total and reducing sugars (method no 925.36) were ana-
and antioxidant capacity
lyzed according to Association of Official Analytical
Chemists (AOAC, 2011), and ratio was calculated (sol- Ascorbic acid. Ascorbic acid analysis was based on the
uble solids/total titratable acidity). All analyses were reduction of 2,6-dichloroindophenol (method no
performed in triplicate. 967.21) (AOAC, 2011). The analyses were performed

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in triplicate and the results were expressed as mg of results were expressed as mmol Trolox/100 mL of
AA/100 mL of orange juice. orange juice.

Extraction of bioactive compounds. The extraction Shelf life. The pressurized and pasteurized orange
was based on the procedure described by Asami et al. juices were stored under the same refrigerated condi-
(2003). Orange juice (5.00 mL) and a methanol:water tions at zero (after juice characterization), 23, 45, 68
solution (10 mL, 80:20, v/v) were vortexed (1 min), soni- and 90 days, which corresponded to 0, 25, 50, 75 and
cated (15 min), and centrifuged (10,000 g/20 min at 100 of the shelf life of NFC (ASTM, 1993). The physi-
20  C). The supernatant was collected and the residue cochemical parameters, extraction and quantification
was submitted to the extraction procedure once again of bioactive compounds, and antioxidant activity were
under the same conditions. Then supernatants were evaluated during shelf life. The non-processed juice was
joined and submitted to total phenolic compounds also analyzed at zero time (juice characterization).
(TPC) and antioxidant activity analyses.
Statistical analysis
Total phenolic compounds. The TPC were determined
as described by Asami et al. (2003) and Singleton et al. Results were expressed as mean  standard deviation of
(1999). Orange juice extract (0.4 mL) was added to the three replicate analyses and submitted to ANOVA and
Folin-Ciocalteu reagent (0.12 mL). After 6 min, a Tukey (zero time) or Student’s t test (shelf life), both at
sodium carbonate solution (4 mL, 70 g/L) was added p  0.05, using the SigmaStat 4.0 software (Systat
and the volume fixed to 10 mL with water. The absorb- Software Inc., San Jose, CA). Linear regression ana-
ance was measured at 730 nm using a spectrophotom- lyses and correlation were performed for shelf life
eter (Evolution 220, Thermo Scientific, EUA) and results using the Origin 8 software (Origin Lab,
compared to a gallic acid calibration curve, with con- Northampton, MA).
centrations ranging from 72 to 200 mg/L. The analyses
were performed in triplicate and results expressed as mg RESULTS AND DISCUSSION
of gallic acid equivalent/100 mL of orange juice.
PME activity
A correction factor was calculated to discount AA,
since it responds to the Folin-Ciocalteu reaction. The pressurization conditions were chosen based on its
Ascorbic acid standard solutions were prepared in con- capacity to reduce PME activity below 20%. The initial
centrations that corresponded to the same range count for PME activity of the non-processed orange
found in orange juice, and submitted to Folin- juice was considered 100% of residual activity and
Ciocalteu reaction. The results, expressed as mL of under these conditions HHP successfully decreased
gallic acid equivalent/100 mL juice provided a correc- PME residual activity to 13%, nearing the residual
tion factor of 0.389, which was deducted from the pre- activity foreseen (15%) in a previous optimization
viously obtained TPC values. study (Bisconsin-Junior et al., 2014). The pasteuriza-
tion was able to reduce residual PME activity to 4%
Antioxidant activity. The antioxidant activity was eval- (Table 1).
uated with ABTSþ, as reported by Rufino et al. (2010). Figure 1 shows linear regression of each parameter
ABTS (5.0 mL, 7 mmol/L) was added to potassium per- for HHP and pasteurized orange juice and confidence
sulfate (88 mL, 140 mmol/L) to form the ABTS radical interval, in order to illustrate trends regarding PME
solution. The solution was stored protected from light residual activity and color parameters during shelf
for 16 h to ensure the complete formation of a stable life. The heat treatment was more effective at reducing
radical. The ABTS radical solution was diluted with PME residual activity than HHP, however both juices
ethanol up to an absorbance of 0.70  0.05 at 753 nm. remained stable under 20% of residual PME activity
Three solutions of orange juice extract:ethanol were during shelf life. The enzyme’s activity is responsible
prepared (1:3; 2:5 and 1:2 v/v). A 30 mL aliquot of for a great part in orange juice’s quality loss, causing
each orange juice extract:ethanol solutions from non- reduction in viscosity and cloud as well as separation of
processed, pasteurized and pressurized juice was mixed phases. Orange juice pressurized at 800 MPa at 25  C
with 3 mL of the ABTS radical solution. Absorbance for 60 s resulted in 4% residual PME activity, showing
was measured at 753 nm after 6 min of reaction in a stability for over 90 days at 4  C and 37  C storage
spectrophotometer (Evolution 220, Thermo Scientific, (Nienaber and Shellhammer, 2001). Orange juice pro-
USA). Calibration curves built with Trolox ethanolic cessed at 700 MPa for 60 s resulted in 18% PME resi-
solutions (100–1600 mmol/L) were used. All the antioxi- dual activity, also stable for over 50 days at 4  C
dant activity analyses were performed in triplicate and (Goodner et al., 1998).

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 Spira et al.

Table 1. Residual PME activity (%), microbiological count and color parameters of pressurized (HHP), pas-
teurized and non-processed orange juice at zero time of storage

Parameter Non-processed HHP Pasteurized

Residual PME activity (%) 100.00  6.85 13.19  2.15 4.24  0.29
Mesophilic aerobic (CFU/mL) 1.03  102  2.1  10 ND ND
Yeast and mold (CFU/mL) 3.6  10  1.5  10 ND ND
Total coliform (CFU/mL) <10 ND ND
Thermotolerant coliform (CFU/mL) ND ND ND
L* 41.38c  0.16 42.47b  0.27 45.68a  0.07
a* 1.78a  0.03 1.32b  0.11 1.21b  0.05
b* 16.13c  0.24 18.35b  0.42 22.21a  0.12
Chroma 16.22c  0.23 18.40b  0.41 22.24a  0.12
Hue angle 83.70c  0.17 85.87b  0.43 86.87a  0.13
Total color difference 0.00c  0.00 2.52b  0.77 7.52a  0.13
pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
formation  units; ND: not  detected; ffiChroma ¼ a2 þ b2 ; Hue angle ¼ arctangent (b*/a*); total color
CFU: colonyqffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
0 2 0 2  0 2
difference ¼ L  L  0 þða  a  0Þ þ b  b  0 .
Values are mean  standard deviation (n ¼ 3). Different letter in each row indicate significant difference in Tukey test
(p  0.05).

the non-processed juice. The same was observed for

Microbiological quality of orange juice
b* values, meaning an increase in yellow color. HHP
Total coliform count was below detection levels for all and pasteurized juices showed a slight increase in a*
the juices at characterization and along shelf life values and a slight decrease in b* values during shelf
(AOAC, 2011). The mesophilic aerobic count in the life, being significantly different (p  0.05) over time
non-processed juice was 10.2  102 CFU/mL, and (Table 1, Figure 1). Confidence interval could not be
yeast and mold 3.67  102 CFU/mL. After treatment, showed for a* parameter because of visual overlapping
both pressurized and pasteurized juice effectively data. No changes in L* values for Navel oranges, pres-
reduced microbiological count below detection levels. surized at 400 MPa, 40  C for 60 s were found (Sánchez-
At each period of time during shelf life the pasteur- Moreno et al., 2005). An increase in yellow color (b*)
ized and pressurized juice were plated and microbio- after juice pasteurization of Navel oranges was
logical count was performed. Counts were below observed (Cortés et al., 2008). The same trend regard-
detection levels up to 68 days of storage for both ing color change during shelf life (slight L* value
juices. At 90 days of storage (estimated shelf life), pas- decrease, a* value increase and b* value decrease)
teurized juice had mesophilic aerobic bacteria growth of (Figure 1) was also reported by Timmermans et al.
12.3  10 CFU/mL and 2.3  10 CFU/mL for yeast and (2011) using Valencia, Pêra and Baladi oranges
mold, while pressurized juice had 30.0  10 CFU/mL during 68 days of storage at 5  C. Pomegranate juice
and 20.7  10 CFU/mL of mesophilic aerobic bacteria treated at 450 MPa for 90, 150 s and 550 MPa showed
and yeast and mold growth, respectively, but there was an increase in a* values (Varela-Santos et al., 2012),
no visual indication of microbiological growth in the similar to our results.
juices. At 99 days (110% of shelf life) both HHP and Chroma and Hue angle were higher in HHP and
pasteurized orange juices showed visible microbiological pasteurized juices (p  0.05), remaining mostly stable
growth, indicating that they had lost their quality and during shelf life, and presenting a slight decrease over
that the estimated shelf life was of 90 days. time for both juices (Table 1, Figure 1). Total color
difference indicates the magnitude of the non-processed
juice’s color difference in relation to the pressurized and
Color
pasteurized juice. Both HHP and pasteurized juices
Both HHP and pasteurized orange juices exhibited a exhibited total color difference value higher than 2,
slightly lower lightness (L*) compared to the non- which indicates that it is possible to visually notice
processed juice (p  0.05), and values remained stable the color difference among the juices, as reported by
during shelf life, with only a slight reduction at 90 days Francis and Clydesdale (1975). Similar results were
for the HHP juice. The a* values were significantly obtained by Cortés et al. (2008), which observed an
higher for HHP and pasteurized juice compared to increase in chroma after pasteurization of orange

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(a)
30

PME residual activity (%)

20

10

0
HHP
P
Confidence interval of 95%

0 20 40 60 80 100
Time (days)

(b) 50 (c) 5

4
48
3
46
2

44 1

0
L*

a*

42
–1
40
–2

38 –3
HHP
Pasteurized –4 HHP
36 Confidence interval of 95% P
–5
0 20 40 60 80 100 0 20 40 60 80 100
Time (days) Time (days)

(d) 30 (e) 30

25 25

20 20
Chroma
b*

15 15

10 10
HHP HHP
P P
Confidence interval of 95% Confidence interval of 95%
5 5
0 20 40 60 80 100 0 20 40 60 80 100
Time (days) Time (days)

Figure 1. Residual PME activity (%), L*, a*, b* and Chroma parameters for pressurized (HHP) and pasteurized (P)
orange juice during shelf life.

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 Spira et al.

juice and high intensity pulsed electric field. An increase stability in all parameters, as well as no significant dif-
in Hue angle was also observed after pasteurization of ferences between the juices (p > 0.05), indicating that
orange juice (Lee and Coates, 2003). Total color differ- both HHP and pasteurization did not impact the juice
ence was significantly higher in treated pomegranate shelf life. Orange juice submitted to HHP and thermal
juice sample at 350, 450 and 550 MPa compared to a treatment from Valencia and Navel oranges, stored at 4
control untreated sample, and a decrease was observed and 10  C, showed no significant difference in physico-
during storage (Varela-Santos et al., 2012). chemical parameters between both juices and untreated
Additionally, a study conducted with pumpkin puree juice, as well as no changes during storage (p > 0.05)
treated with HHP found that color was better preserved (Bull et al., 2004). Similarly, orange juice pasteurized
(total color difference lower than 2) when using lower at 92  C for 30 s showed no significant changes during
pressure levels (400 MPa rather than 600 MPa) and 32 weeks of storage for pH and total titratable acidity
treatment time (200 s rather than 600 s) (González- (Wibowo et al., 2015).
Cebrino et al., 2015).
Bioactive compounds and antioxidant activity
Physicochemical analysis
Ascorbic acid. The non-processed orange juice
The physicochemical characteristics for the non-pro- showed higher AA levels (p  0.05) compared to
cessed, pressurized and pasteurized orange juice at the pressurized and pasteurized juice, as expected
zero time are in Table 2. Soluble solids were similar (Table 2). The pasteurized juice presented higher AA
among the non-processed, pressurized and pasteurized than the pressurized juice (p  0.05). Plaza et al. (2006)
orange juice (p  0.05). The non-processed juice exhib- reported that HHP reduced AA levels of Valencia
ited higher total titratable acidity (p  0.05) and no dif- oranges in 5% and Sánchez-Moreno et al. (2005)
ference was found between the pasteurized and reported AA reduction of 8% in Navel oranges sub-
pressurized juices (p > 0.05). Both pressurized and pas- mitted to 400 MPa, at 40  C for 60 s. The higher tem-
teurized juices (p > 0.05) showed higher ratio compared perature and time conditions applied in this work (520
to the non-processed juice (p  0.05). There was no sig- MPa, 60  C, 360 s) resulted in a higher reduction of
nificant difference in pH for all juices. Reducing and AA levels (16%). Similarly, pasteurization reduced
total sugar exhibited similar values, with significant dif- AA levels in 13%, lower than a 17% decrease as
ferences for pressurized juice (p  0.05) (Table 2). reported by Elez-Martı́nez et al. (2006), and higher
As reported by Bull et al. (2004) and Sánchez- than the reduction of 8% reported by Sánchez-
Moreno et al. (2005), pasteurization also led to an Moreno et al. (2005) after pasteurizing orange juice
increase in soluble solids in Navel and Valencia at 90  C for 60 s.
oranges, similar to our results. A reduction in total It should be noticed that our results for AA levels for
titratable acidity was also observed after pasteurization non-processed, pressurized and pasteurized orange
using Navel oranges (Sánchez-Moreno et al., 2005). juice were in the same range of Brazilian (Stella et al.,
Figure 2 shows the shelf life results for both pasteur- 2011) and Spanish (Meléndez-Martı́nez et al., 2007)
ized and pressurized orange juice which exhibited orange juice.

Table 2. Physicochemical characteristics, total phenolic content and antioxidant activity of pressurized (HHP), pasteur-
ized and non-processed orange juice at zero time of storage

Parameter Non-processed HHP Pasteurized

Soluble solids ( Brix) 9.5b  0.1 9.5b  0.0 9.7a  0.1

Total titratable acidity (g citric acid/100 mL) 0.9a  0.0 0.6b  0.0 0.6b  0.0
Ratio 10.9b  0.1 16.0a  0.1 16.0a  0.1
pH 4.06a  0.01 4.07a  0.04 4.05a  0.04
Reducing sugar (g glucose/100 mL) 3.7a  0.0 3.5b  0.0 3.7a  0.0
Total sugar (g glucose/100 mL) 7.1a  0.0 6.6b  0.0 7.1a  0.1
Ascorbic acid (mg/100 mL) 38.3a  0.3 32.2c  0.2 33.3b  0.3
Total phenolic compounds 52.85a  2.8 52.76a  3.9 52.62a  2.2
(mg gallic acid equivalent/100 mL)
ABTS (mmol Trolox/100 mL) 302.3a  2.0 299.1a  2.4 294.7a  8.3
Values are mean  standard deviation (n ¼ 3) and same letter in each row did not indicate significant difference in Tukey test (p  0.05).

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(a) 11.0 (b) 7

Reducing sugar (g glucose/100ml)

10.5
5
Soluble solids (°Brix)

10.0 4

3
9.5

2
9.0
HHP 1 HHP
P P
Confidence interval of 95% Confidence interval of 95%
8.5 0
0 20 40 60 80 100 0 20 40 60 80 100
Time (days) Time (days)

(c) 1.2 (d) 12

Total titratable acidity (g citric acid/100ml)

1.0 Total sugar (g glucose/100ml) 10

0.8 8

0.6 6

0.4 4

0.2 2 HHP
HHP
P P
Confidence interval of 95% Confidence interval of 95%
0.0 0
0 20 40 60 80 100 0 20 40 60 80 100
Time (days) Time (days)

(e) 6

5
pH

HHP
P
Confidence interval of 95%
2
0 20 40 60 80 100
Time (days)

Figure 2. Physicochemical characteristics for pressurized (HHP) and pasteurized (P) orange juice during shelf life.

8
 Spira et al.

Figure 3 shows AA levels during storage. Both pres- holding times, was effective in maintaining total anti-
surized and pasteurized juice exhibit the same strong oxidant activity levels in pumpkin puree.
reduction over time (p  0.05), reducing around ABTS shows a slight increase during shelf life and
70–80% of AA levels in 90 days. Ascorbic acid degrad- overall antioxidant activity for both juices maintained
ation is due to many factors, including type of process- high values along shelf life (Figure 3). It has been
ing, storage conditions, packaging, oxygen, and light reported that HHP can either increase or maintain
(Shaw and Moshonas, 1991; Teixeira and Monteiro,
2006). The results obtained in our study indicate that
a very strong decrease in AA levels occurred during
shelf life. Similar results were obtained using Valencia (a)
50
and Navel oranges, which presented a reduction in AA
during a three-month storage at 4  C (Bull et al., 2004). 40

Ascorbic acid (mg/100ml)

Nienaber and Shellhammer (2001) attributed AA level
reduction to its oxidation over time. 30

Total phenolic compounds. The results for TPC are in 20

Table 2. Non-processed, pasteurized and pressurized

10
orange juice showed no significant difference at zero
time, indicating that TPC is resistant to both HHP 0
and thermal treatment. Some studies suggest that HHP
P
TPC may even increase with processing, when antioxi-
0 20 40 60 80 100
dants can be more extracted (Chen et al., 2015; Varela- Time (days)
Santos et al, 2012).
Both pressurized and pasteurized orange juice exhib- (b)
80
ited the same behavior during shelf life regarding TPC,
(mg gallic acid equivalent/100ml)

with a slight decrease over time, and no significant dif- 70

Total phenolic compounds

ference between them (p > 0.05). Comparative literature

on the effects of HHP on TPC for orange juice is scarce; 60

however, a few studies have showed this influence on

50
different fruit juices. A study using papaya beverage
treated with HHP and pasteurization, stored at 4  C 40
showed similar results, with comparable small decrease
rate over time, although HHP retained TPC better than 30 HHP
P
pasteurization (Chen et al., 2015). Similarly, a study Confidence interval of 95%
with mulberry juice, compared thermal treatment 20
0 20 40 60 80 100
(85  C, 15 min) with HHP (500 MPa, 10 min), and Time (days)
found that HHP was able to retain more TPC (Wang
(c) 700
et al., 2016). Varela-Santos et al. (2012) found that
pomegranate juice processed with HHP showed slight 600
decrease during storage of 35 days at 4  C.
ABTS (mmol trolox/100ml)

500

Antioxidant activity. Results of antioxidant activity are 400

given in Table 2. Antioxidant activity of pressurized,
non-processed and pasteurized orange juice using 300

ABTS radical assay ranged from 294.7 to 302.3 mmol 200

Trolox/100 mL, with no significant difference between
pressurized and pasteurized orange juice (p > 0.05). The 100 HHP
P
antioxidant activity levels obtained with ABTS were in Confidence interval of 95%
0
the range of those reported by Stella et al. (2011) for 0 20 40 60 80 100
Brazilian orange juice. Other studies showed that anti- Time (days)
oxidant activity was not significantly affected after
HHP at 440 MPa, 40  C for 60 s and pasteurization Figure 3. Ascorbic acid (mg/100 mL), total phenolic com-
(Gil-Izquierdo et al., 2002; Sánchez-Moreno et al., pounds (mg gallic acid equivalent/100 mL), and antioxidant
2005). Gonzáles-Cebrino et al. (2015) were able to activity (mmol Trolox/100 mL) in pressurized (HHP) and
show that HHP, while using different pressures and pasteurized (P) orange juice during shelf life.

9
Food Science and Technology International 0(0)

antioxidant levels, depending of pressure and time con- ORCID iDs

ditions (Casquete et al., 2015). Antonio Bisconsin-Junior http://orcid.org/0000-0001-
9474-1691
Antioxidant activity, TPC, and AA Magali Monteiro http://orcid.org/0000-0002-5436-2598
correlation. Correlation among antioxidant activity,
TPC, and AA was determined for HHP and pasteur-
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