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Food Chemistry 127 (2011) 447454

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Inuence of ultra-high pressure homogenisation on antioxidant capacity,


polyphenol and vitamin content of clear apple juice
ngela Surez-Jacobo a, Corinna E. Rfer b, Ramn Gervilla a, Buenaventura Guamis a,
Artur X. Roig-Sagus a, Jordi Saldo a,
a
Centre Especial de Recerca Planta de Tecnologia dels Aliments, Departament de Cincia Animal i dels Aliments, XaRTA, TECNIO, MALTA-Consolider, Facultat de Veterinria, edici V,
Universitat Autnoma de Barcelona, Bellaterra, 08193 Barcelona, Spain
b
Department of Safety and Quality of Fruit and Vegetables, Federal Research Centre for Nutrition and Food, Max Rubner-Institute, Haid-und-Neu-Strasse 9, D-76131
Karlsruhe, Germany

a r t i c l e

i n f o

Article history:
Received 15 June 2010
Received in revised form 15 October 2010
Accepted 30 December 2010
Available online 9 January 2011
Keywords:
Apple juice
Ultra-high pressure homogenisation
Antioxidant
Polyphenols

a b s t r a c t
Ultra-high pressure homogenisation (UHPH) is a recently developed technology and is still under study to
evaluate its effect on different aspects of its application to food products. The aim of this research work
was to evaluate the effect of UHPH treatments on quality characteristics of apple juice such as antioxidant
capacity, polyphenol composition, vitamin C and provitamin A contents, in comparison with raw (R) and
pasteurised (PA) apple juice. Several UHPH treatments that include combinations of pressure (100, 200
and 300 MPa) and inlet temperatures (4 and 20 C) were assayed. Apple juice was pasteurised at 90 C
for 4 min. Antioxidant capacity was analysed using the oxygen radical antioxidant capacity (ORAC),
2,2-diphenyl-1-picrylhydrazyl (DPPH), trolox equivalent antioxidant capacity (TEAC), ferric reducing
antioxidant power (FRAP) assay while total phenolic content was determined by the FolinCiocalteau
assay. According to the FRAP and DPPH assays, UHPH processing did not change apple juice antioxidant
capacity. However, signicant differences were detected between samples analysed by TEAC and ORAC
assays. In spite of these differences, high correlation values were found between the four antioxidant
capacity assays, and also with total polyphenol content. The analysis and quantication of individual phenols by HPLC/DAD analytical technique reects that UHPH-treatment prevented degradation of these
compounds. Vitamin C concentrations did not change in UHPH treated samples, retaining the same value
as in raw juice. However, signicant losses were observed for provitamin A content, but lower than in PA
samples.
UHPH-treatments at 300 MPa can be an alternative to thermal treatment in order to preserve apple
juice quality.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
The growing demand for high quality and minimally treated
foods has generated an impulse in the development of several
non-thermal processing methods such as: ionising radiation, pulsed
light, pulsed electric elds, supercritical gases pasteurisation, UV
radiations and high pressure processing (HPP); and all of these have
been developed as feasible substitutes for thermal processing (Ortega-Rivas, Zrate-Rodrguez, & Barbosa-Cnovas, 1998).
Food producers are increasingly interested in developing new
products by application of new technologies that offer an increase
or preserve certain health protecting compounds. Thermal pasteurisation, commonly applied to liquids food products, has presented some drawbacks such as undesirable biochemical and
Corresponding author. Tel.: +34 935814731; fax: +34 935812006.
E-mail address: jordi.saldo@uab.cat (J. Saldo).
0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.12.152

nutritional changes in processed products (colour changes, avour


and aroma decreases, and vitamin losses) which affect the quality
of the nal product and make it unattractive to the consumer (Choi
& Nielsen, 2005).
UHPH is a new non-thermal processing technique, which is
based on the same principle as conventional homogenisation used
in the dairy industry (Hayes & Kelly, 2003; Thiebaud, Dumay, Picart, Guiraud, & Cheftel, 2003) but it works at signicantly higher
pressures (>200 MPa). Above this pressure, the elimination of
spoilage and pathogenic microorganisms is carried out effectively
and without affecting the nutritional value of the food products,
making this system a real substitute for the traditional thermal
processing. At the moment, the food industry has been searching
new food technologies for developing a continuous process. In this
sense the application of pressure by means of a continuous dynamic technology such as UHPH, could be an effective alternative
to treatment for liquid and pumpable foods.

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. Surez-Jacobo et al. / Food Chemistry 127 (2011) 447454

The effects of UHPH on some spoilage and pathogenic microorganisms have been studied in foods and model systems (Briez,
Roig-Sagus, Hernndez, Lpez, & Guamis, 2007; Campos & Cristianini, 2007; Cruz et al., 2007; Surez-Jacobo, Gervilla, Guamis, RoigSagus, & Saldo, 2009; Vachon, Kheadr, Giasson, Paquin, & Fliss,
2002). The reduction or destruction of microorganisms by UHPH
is probably due to several physical phenomena such as pressure
drop, cavitation, shear stress, turbulence and collision that could
increase the permeability or rupture of the cell membrane causing
cell death (Hayes & Kelly, 2003; Middelberg, 1995; Popper & Knorr,
1990). Besides its ability to lowering the initial microbial load,
UHPH minimises heat stress during the treatment while reducing
the adverse effects of heat on food properties or constituents. In
addition, UHPH is expected to modify protein and polysaccharide
properties, produce ner emulsions, inhibit microorganisms and
enzymes, and could have the potential to produce value added or
novel food products (Hayes, Fox, & Kelly, 2005; Paquin, 1999).
On the other hand, clear apple juice is one of the most popular
study topics due to its high demand in Spain. Its typical amber-like
colour is commercially desirable for the Spanish consumers. Additionally, there is much scientic literature dealing with the benecial effects that the consumption of apple juice offers to human
health (Gliszczynska-Swiglo & Tyrakowska, 2003; Tsao, Yang, Xie,
Sockovie, & Khanizadeh, 2005). Increasing attention is being paid
to the role of bioactive compounds in apple juice as well as in other
juices, because they possess a possible health protecting capacity.
The majority of the antioxidant capacity of a fruit may be attributed to compounds such as vitamin C, vitamin E, b-carotene, and
polyphenolic compounds, e.g. avanols, avonols, and anthocyanins (Kaur & Kapoor, 2001).
Different studies have been conducted in order to evaluate the
antioxidant capacity in fruit and vegetable extracts. In these investigations several assays have been applied, including FRAP, DPPH,
TRPA, ORAC, LDL oxidation and so on. However different trends between the assays have been found (Nilsson et al., 2005; Oszmianski
& Wojdylo, 2007; Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, & Hawkins Byrne, 2006). Hence it is pertinent to use several assays instead of a single one to evaluate and compare the
antioxidant activity in fruits and vegetable extracts.
Due to the lack of data in the eld of the effects of UHPH technology on these potential properties, the aim of this research work
was to evaluate the effect of UHPH treatments (100, 200 and
300 MPa at two inlet temperatures 4 and 20 C) on the antioxidant
capacity, polyphenol and vitamin content of clear apple juice in
comparison with raw and pasteurised juice.
2. Material and methodos
2.1. Apple juice supply and production
Fresh apple juice was supplied from Cal Valls (Vilanova de Bellpuig, Spain). Apples (cv. Golden Delicious) were crushed to obtain
apple juice. Pectolytic enzymes were added to increase yield production and maintained for 1.5 h at 4 C. Afterwards, apple juice
was passed through a lter press to obtain a clear apple juice
and it was maintained at 4 C until treatments (Fig. 1). The juice
was UHPH processed at the CERPTA Pilot Plant (Universitat Autnoma de Barcelona, Bellaterra, Spain).
2.2. Chemical reagents
2,20 -azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), potassium persulphate (K2S2O8), 2,2-diphenyl1-picrylhydrazyl (DPPH), Folin Ciocalteau reagent and b-carotene
were purchased from Sigma Aldrich (St. Louis, USA).

Trolox [()-6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid], polyphenolic standards (chlorogenic acid, phloretin


and quercetin, gallic acid monohydrate), metaphosphoric acid,
DTT (DL-dithiothreitol), ascorbic acid, a,a0 -azodiisobutyramidine
dihydrochloride (ABAP), 2,4,6-tri-2-pyridyl-1,3,5-triazine (TPTZ),
uorescein sodium salt and sulfuric acid (HPLC grade) were purchased from Fluka Chemie (Buchs, Switzerland).
Acetone, formic acid, acetonitrile, hexane and dichloromethane,
methanol and tert-c-butyl-methylether (TBME) were purchased
from Merck (Darmstadt, Germany).
2.3. UHPH treatment and pasteurisation of apple juice
Apple juice samples were processed using an ultra-high pressure homogenizer (Model/DRG No. FPG 11300:400 Hygienic
Homogenizer, Stansted Fluid Power Ltd., Harlow, UK) at a ow rate
of 100 L h1. This equipment consisted of a high-pressure ceramic
valve able to withstand 400 MPa. The high-pressure system consisted of two intensiers, which were driven by a hydraulic pump.
To minimise temperature retention after treatment, one spiral type
heat-exchanger (Garva, Barcelona, Spain) located behind the
homogenisation valve was used. Moreover, the inlet temperature
(Ti), the temperature just before the high-pressure valve (T1), the
temperature right after the high-pressure valve (T2) and the outlet
temperature (T0) were monitored during the experiment.
Apple juice was then UHPH-treated at 100, 200 and 300 MPa
(single-stage) with inlet temperatures of 4 and 20 C at each level
of pressure. Apple juice samples were collected in sterile containers and stored at 4 C until analysis. PA apple juice was obtained
using a tubular heat exchanger at 90 C for 4 min, and was bottled
without refrigeration (it was supplied by the juice processing company, Cal Valls). Samples were stored at 4 C until analysis. The full
experiment was conducted independently three times.
2.4. Physical and physicochemical assays
Raw and treated juices were equilibrated at 20 C to measure
pH and titrable acidity (TA), simultaneously. TA was carried out
using an automatic titrator (model Titrando 842, Metrohm, Herisau, Switzerland). Results were expressed as grams of malic acid
per litre of juice (g MA L1) (International Federation of Fruit Juice
Producers., 1996). Total soluble solids (TSS) were measured by
using a Spectronic Instruments refractometer (Rochester, NY,
USA) at 20 C, results are reported as Brix. Titrable acidity, total
soluble solids, total and reducing sugar, dry matter and density
were also measured according to IFFJP methods (International Federation of Fruit Juice Producers, 1996). Viscosity was measured by
using a Haake RheoStress1 rheometer (Thermo Fisher Scientic,
Inc., Karlsruhe, Germany), with concentric cylinders geometry
(Z34). Flow curves were recorded at 4 C at a shear rate ranging
from 0 to 120 s1 to calculate Newtonian sample viscosity, expressed as Pa s.
2.5. Antioxidant capacity measurements
2.5.1. Antioxidant capacity determined by stable radical method
(DPPH)
Radical scavenging activity of apple juice against stable DPPH
was determined according to the method proposed by Bondet,
Brand-Williams, and Berset (1997), where the stable free radical
DPPH is reduced to the corresponding hydrazine by reaction with
hydrogen donors. Extract (50 ll) was mixed with 1.95 mL of a
methanolic solution of DPPH. The samples were kept in the dark
for 1 h at room temperature before measurement of the decrease
in the absorption at 515 nm. Determinations were performed using
a Biochrom LibraS21 spectrophotometer (Biochrom Ltd. Cam-

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Apples
Golden delicious var.
Sanitizing
Crushing
Juice Extraction

Enzymatic
treatment
1.5 h 4 C

Apple pomace

Cloudy apple juice

Decantation
1.5 h 4 C
Filtration

UHPH treatment
Inlet Temperature: 4 and 20 C
Pressure:
100 MPa, 200 MPa, 300 MPa

Raw juice
(R)

Thermal treatment
90 C/ 4 min
Hot filling

Storage at 4 C
Fig. 1. Apple juice production ow chart.

bridge, England) with eight cuvettes. Trolox solutions within the


range of 1001000 lM were used for calibration. A new trolox calibration curve was made for each assay. Samples were diluted in
methanol to t into the concentration range of trolox calibration
curve and the results were expressed as trolox equivalents (TE)
where one TE equals the net protection produced by 1 lM Trolox.
2.5.2. Antioxidant capacity determined by trolox equivalent
antioxidant capacity (TEAC)
The TEAC assay was performed as described by Stracke, Rfer,
Weibel, Bub, and Watzl (2009). Samples were diluted in PBS to
t into the concentration range of trolox calibration curve and
the results were expressed as trolox equivalents (TE).
2.5.3. Antioxidant capacity determined by ferric reducing antioxidant
power (FRAP)
Again, the FRAP assays was performed as described by Stracke,
Rfer, Weibel et al. (2009). Samples were diluted in double distiled
water to t into the concentration range of trolox calibration curve
and the results were expressed as TE.
2.5.4. Antioxidant capacity determined by oxygen radical antioxidant
capacity (ORAC)
As stated above the ORAC assay was performed as described by
Stracke, Rfer, Weibel et al. (2009) by using a multimode microplate reader Mithras LB 940 (Berthold technologies GmbH & Co.,
Germany). Quantication of raw data was analysed by using
MIKROWIN 2000 software v. 4.41 (Mikrotek Laborsysteme GmbH,
Germany). The area under the uorescence decay curves (AUC)
was calculated by using the integral of kinetic measurement function called KITG(MEA) in this software. The net AUC corresponding
to a sample was calculated by subtracting the AUC of the blank
(BK). ORAC values for the apple samples were expressed as TE.

2.6. Total phenolic compounds (FolinCiocalteau assay)


The phenolic compounds were determined by using FolinCiocalteau reagent (Singleton, Orthofer, & Lamuela-Raventos, 1999).
Apple juice samples were ltered through a 0.45 lm M.E cellulose
membrane (Teknokroma, Barcelona, Spain). One ml of the ltered
solution was mixed with 5 mL of 0.2 N Folin Ciocalteau reagent.
Four ml of a saturated sodium carbonate solution (7.5%) were
added to the mixture and then shaken. The absorbance of the solution at 765 nm was measured after 2 h with a double beam spectrophotometer CECIL 9000 Series (CECIL instruments Ltd.,
Cambridge, England). Quantication was based on the standard
curve ranged from 10100 mg L1 of gallic acid prepared daily. Results were expressed as mg of gallic acid equivalent (GAE) per litre
of juice (mg GAE L1).

2.7. Identication and quantication of polyphenolic compounds


HPLC analysis of polyphenols was done as described by Stracke,
Rfer, Weibel et al. (2009). After centrifugation the apple juice
samples were directly subjected to HPLC/DAD analysis. For identication of the polyphenols without reference standards a HPLC/
MS/MS analysis was performed with a HP 1200 series HPLC (Agilent Technologies, Waldbronn, Germany) equipped with an autoinjector, quaternary HPLC pump, column heater, and UV detector
using the same separation conditions used for HPLC/DAD. The
HPLC system was directly coupled to a hybrid triple-quadrupole/
linear ion trap mass spectrometer (QTrap 3200; Applied Biosystems, Darmstadt, Germany) equipped with a TurboIonSpray
source. Analytes were detected in the negative ion mode at a
vaporiser temperature of 700 C and ion spray voltage of 4.5 kV.
Spectral data were recorded with N2 (CAD = 4) as collision gas
and a declustering potential of 30 V. Data acquisition was per-

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Table 1
Characterization of apple juice: raw (R), pasteurised (PA) and UHPH treated at 4 and 20 C inlet temperatures.
Parameters

pH
TSSA (Brix)
TAA (g MA L1)
Reducing sugars
(g Glucose100 mL1)
Total sugars (g Glucose
100 mL1)
Viscosity (mPa s)

Treatments
R

100 MPa at
4 C

100 MPa at
20 C

200 MPa at
4 C

200 MPa at
20 C

300 MPa at
4 C

300 MPa at
20 C

PA

3.89 0.02a
13.67 0.23a
2.88 0.08a
9.26 1.05a

3.87 0.02a
13.67 0.23a
3.04 0.03a
9.20 0.64a

3.87 0.01a
13.70 0.17a
3.03 0.02a
9.51 0.25a

3.87 0.01a
13.47 0.31a
3.00 0.03a
9.22 0.47a

3.88 0.02a
13.51 0.19a
3.01 0.01a
9.34 0.47a

3.92 0.02a
13.40 0.40a
2.88 0.01a
9.13 0.79a

3.92 0.02a
13.47 0.31a
2.90 0.02a
9.06 0.46a

3.85 0.04a
13.22 0.50a
3.02 0.03a
8.26 1.82a

11.92 1.37a

12.01 0.53a

12.51 0.18a

11.78 0.57a

11.99 0.05a

12.15 1.25a

12.00 0.31a

11.34 0.12a

3.68 0.10a

3.77 0.02abc

3.77 0.02ba

3.74 0.04a

3.81 0.08ba

3.85 0.10cb

3.87 0.08c

3.75 0.06ba

Values are means standard deviations (SD) of triplicate analysis from 3 different productions. Values in the same row with different superscripts differ signicant (P < 0.05).
A
TSS, total soluble solids; TA, titrable acidity.

formed in the EMS mode. Spectra were scanned in the mass range
of m/z 100800. Data collection and handling was done with Analyst 1.5. For the LC the same conditions as described were used.
Quantication was performed according to an external standard
method using the available reference standards. For polyphenol
compounds where no standards were attainable, the quantication
was based on a representative standard of the same polyphenol
class. The concentrations of coumaroyl and caffeoyl derivatives
were calculated using chlorogenic acid, quercetin glycosides using
quercetin and phloretin glycosides using phloretin.
2.8. Vitamins
2.8.1. Vitamin C
Ascorbic acid and total vitamin C (ascorbic plus dehydroascorbic acid) were determined by HPLC according to SnchezMoreno et al. (2003). A total of 50 mL of apple juice were
homogenised with 40 mL of extraction solution (3% metaphosphoric acid plus 8% acetic acid). The resulting mixture was centrifuged, ltered and adjusted up to 100 mL. Samples were
ltered through a 0.45 lm membrane lter, and 20 lL of each
extract were analysed. Results were expressed as mg of ascorbic
acid per 100 mL.
Ten mL of the resulting mixture were taken to react with 2 mL
of a solution 20 mg mL1 DL-dithiothreitol (reducing reagent) for
2 h at room temperature in the dark. Samples were ltered through
a 0.45 lm membrane lter, and 20 lL of each extract was analysed.
Results were expressed as mg of total vitamin C per 100 mL.
A Dionex HPLC pump, a thermostatted column compartment
and a UVVis detector operated at a wavelength of 245 nm were
used for analysis. Separation of ascorbic acid was performed on a
reversed phase C18 Hypersil ODS (5 lm) column (250  4.6 i.d.
mm) (Teknokroma). The solvent system used was a 0.01% solution
of H2SO4. The ow rate was set to 1.0 mL min1. Calibration curves
were made with a minimum of 7 concentrations. Chromatographic
data were collected and recorded by Chromeleon software (Dionex
Corporation, Sunnyvale, USA).
2.8.2. b-Carotene
b-Carotene extraction and analysis was conducted according to
Stracke, Rfer, Bub et al. (2009). Apple juice (5 ml) was used. The
carotenoid extract was reconstituted with 150 lL of acetone/dicloromethane (90:10 v/v containing 0.1% BHT). Quantication was
achieved using b-carotene as external standard. Calibration curves
were made with a minimum of 5 concentration levels at 450 nm.
The nal results were expressed as retinol equivalents (RE) by
using the following formula:

REunit 6lg of b-Carotene

2.9. Statistical analyses


Statistical analyses were performed using the ANOVA procedure of SAS Institute (2004) and P < 0.05 was used to determine
statistical signicance. Tukey test was performed for data comparison. All results were obtained from triplicate analysis of three
independent experiments (n = 9). Tests for association between
the concentration of a compound (or the total polyphenolic content) and the antioxidant activity measured by the different assays
were run using standard Pearson correlation.
3. Results
Temperature data, monitored during the UHPH processing was
reported in a previous research work (Surez-Jacobo, Gervilla, Guamis, Roig-Sagus, & Saldo, 2010).
TSS, TA, pH, total and reducing sugar contents of raw, UHPHtreated and PA apple juice are listed in Table 1. For all these parameters there were no differences due to treatment. TSS amounted to
13.5 0.4 Brix, TA equalled 2.97 0.03 g MA L1 and pH was
3.88 0.02, showing the low pH characteristics of apple juice.
Reducing and total sugar were 9.1 0.8 and 12.0 0.6 g glucose100 mL1 of juice.
Viscosity changed only slightly due to treatment (Table 1). Maximum viscosity was observed for 300 MPa at 20 C treated juice
(3.87 0.08 MPa s) and minimum for raw (R) apple juice
(3.68 0.10 MPa s). In general, the UHPH treatments did not affect
the juice acidity and overall composition, which has the advantage
of preserving the characteristics of apple juice.
3.1. Effect of UHPH on antioxidant capacity and polyphenol content of
apple juice
The antioxidant capacity of apple juice treated by UHPH was
measured using different antioxidant assays (DPPH, TEAC, FRAP
and ORAC). The antioxidant capacity of apple juices is mainly
attributed to polyphenolic compounds and to a lower extent to
ascorbic acid content. Thus, total polyphenolic content was evaluated by using the FolinCiocalteau method. The results are presented in Fig. 2.
No signicant differences were found between R and UHPH
samples analysed by DPPH and FRAP methods, however signicant
differences were detected in comparison with PA juice samples.
The DPPH and FRAP values obtained ranged from 334 22 to
769 38 lM TE and 421 22 and 902 32 lM TE in UHPH-treated
and PA juices, respectively. In the TEAC assays the lowest values
were found for UHPH treatments performed at 20 C. ORAC test
showed the lowest values for R and 100 MPa treated samples. Of

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. Surez-Jacobo et al. / Food Chemistry 127 (2011) 447454


Antioxidan Capacity (ORAC)
Antioxidant Capacity (TEAC)
Antioxidant Capacity (FRAP)
Antioxidant Capacity (DPPH)
Total phenolics (Folin-Ciocalteau)

2000

Antioxidant Capacity ( M TE)

bc

ab

60

abc

55

abc

Total phenolics (mg GAE L-1)

2500

1500

50
**

1000

500

ab

ab

45
***

PA

30
0

20
0

Pa
/2

Pa
/2

C
0
M
Pa
/2

30
0

Pa
/4

Pa
/4
M
20
0

10
0

C
Pa
/4
M
10
0

40

Fig. 2. Antioxidant Capacity [lM Trolox equivalents (TE)] and total phenolic contents [mg of gallic acid equivalents (GAE) L1] of apple juices raw (R), pasteurised (PA) and
treated by ultra high pressure homogenisation (UHPH) at two different inlet temperatures (4 and 20 C). Treatments with same letter index for ORAC and TEAC series did not
differ signicantly (P < 0.05) () indicate values with signicant differences (P < 0.05) between samples in total phenols assay. () Indicate values with signicant differences
between samples in FRAP assay. () Indicate values with signicant differences between samples in DPPH assay.

the several treatments examined in this study, the antioxidant


capacity of PA samples was signicantly (519%) higher than the
rest of treatments by all antioxidant tests assayed.
As shown in Fig. 2, total polyphenolic concentrations were statistically signicant not affected by the UHPH-treatment. This content ranged from 49.3 0.9 to 52.4 0.8 mg GAE L1. In the apple
juices, several polyphenols such as hydroxycinnamic acids, dihydrochalcones and avonols were identied. The polyphenol proles
obtained are shown in Table 2. The highest concentrations were
found for the hydroxycinnamic acids with 64.9%, (4-caffeinoylquinic acid, 3-p-coumaroylquinic acid, chlorogenic acid, 4-p-coumaroylquinic acid, 5-p-coumaroylquinic acid and caffeic acid
derivatives), followed by 21.6% avonols (quercetin 3-galactoside,

quercetin 3-glucoside, quercetin 3-xyloside, quercetin 3-arabinoside and quercetin 3-rhamnoside) and 13.6% dihydrochalcones
(phloretin 20 -xyloglucoside and phloridzin). Neither catechin nor
epicatechin were detected. Chlorogenic acid was the dominant
constituent of hydroxycinnamic acids (26.5% of total amount).
For this compound it was observed statistically signicant differences between [R < 100 MPa (Ti = 4 C)  100 MPa (Ti = 20 C) 
200 MPa (Ti = 4 C) < 200 MPa (Ti = 20 C)  300 MPa (Ti = 4 C) <
300 MPa (Ti = 20 C)  PA]. This means that for chlorogenic acid
similar concentrations were found for UHPH and PA juices which
were, however, statistically signicant different from R juices. Signicant differences were also observed for 4-caffeinoylquinic acid
[R  100 MPa (Ti = 20 C)  200 MPa (Ti = 4 C)  300 MPa (Ti =

Table 2
Polyphenol content (mg L1) of apple juices: raw (R), pasteurised (PA) and UHPH treated at 4 and 20 C inlet temperatures.
Treatments
Polyphenol content
(mg L1)

100 MPa at
4 C

100 MPa at
20 C

200 MPa at
4 C

200 MPa at
20 C

300 MPa at
4 C

300 MPa at
20 C

PA

Hydroxycinnamic derivatives
4-Caffeinoylquinic acid
3-p-Coumaroylquinic acid
Chlorogenic acid
4-p-Coumaroylquinic acid
5-p-Coumaroylquinic acid

1.42 0.22a
0.92 0.24a
2.75 0.48a
1.22 0.18a
0.57 0.06a

1.53 0.10ab
0.90 0.16a
3.26 0.16ab
0.88 0.32a
0.67 0.23a

1.47 0.13a
1.17 0.32a
3.29 0.13ab
0.93 0.22a
0.53 0.18a

1.53 0.07a
1.03 0.14a
3.40 0.28ab
0.87 0.29a
0.59 0.24a

1.58 0.12ab
1.34 0.26a
3.58 0.18bc
0.98 0.30a
0.55 0.20a

1.50 0.09a
1.21 0.25a
3.60 0.54bc
0.90 0.30a
0.55 0.26a

1.62 0.08ab
1.24 0.26a
4.09 0.05c
1.02 0.18a
0.61 0.31a

1.81 0.08b
0.87 0.18a
4.17 0.06c
1.01 0.05a
0.92 0.24a

Caffeic acid derivatives

0.62 0.07a

0.88 0.28a

0.92 0.19a

0.87 0.26a

0.98 0.25a

1.02 0.34a

0.99 0.16a

0.73 0.09a

Dihydrochalcones
Phloretin-xyloglucoside
Phloridzin

0.92 0.13ab
0.72 0.17a

0.95 0.01ab
0.74 0.05a

1.04 0.15b
0.88 0.20a

0.99 0.04ab
0.82 0.22a

1.01 0.08ab
0.88 0.20a

0.90 0.08ab
0.92 0.19a

0.96 0.06ab
0.82 0.06a

0.83 0.06a
1.01 0.05a

0.37 0.03a
0.28 0.08a
0.17 0.02a
0.20 0.02abc
1.75 0.13a
12.58 0.20ab

0.41 0.05a
0.26 0.03a
0.17 0.03a
0.18 0.04ab
1.72 0.08a
12.96 0.77ab

0.38 0.08a
0.29 0.09a
0.19 0.05a
0.20 0.09abc
1.72 0.13a
12.89 0.92ab

0.41 0.06a
0.26 0.05a
0.18 0.04a
0.19 0.05ab
1.70 0.10a
13.63 0.84ab

0.42 0.05a
0.22 0.09a
0.21 0.03a
0.29 0.04c
1.74 0.20a
13.51 1.46ab

0.49 0.17a
0.21 0.15a
0.18 0.09a
0.28 0.02bc
1.80 0.09a
14.30 0.18b

Flavonols
Quercetin-3-galactoside
Quercetin-3-glucoside
Quercetin-3-xyloside
Quercetin-3-arabinoside
Quercetin-3-rhamnoside
Total

0.24 0.03a
0.34 0.10a
0.18 0.03a
0.16 0.03a
1.64 0.18a
11.69 1.43a

1.01 1.09a
0.32 0.06a
0.20 0.04a
0.28 0.02bc
1.67 0.14a
14.50 1.33b

Values are means (SD) of triplicate analysis from 3 different productions (n = 9). Values in the same row with different superscripts differ signicantly (P < 0.05).

452

. Surez-Jacobo et al. / Food Chemistry 127 (2011) 447454

4 C) 6 100 MPa
(Ti = 4 C)  200 MPa
(Ti = 20 C)  300 MPa
(Ti = 20 C) < PA] and quercetin-3-arabinoside [R < 100 MPa
(Ti = 20 C)  100 MPa (Ti = 4 C)  200 MPa (Ti = 4 C)  200 MPa
(Ti = 20 C) < 300 MPa (Ti = 4 C) 6 300 MPa (Ti = 20 C) < PA]. However, for the other polyphenols no signicant changes could be
found (Table 2).
The total polyphenol content obtained ranged from
11.7 1.4 mg L1 in R to 14.5 1.3 mg L1 in PA apple juice. Significant differences were found as follow: [R 6 100  200  300 MPa
(Ti = 4 C)  100  200 MPa (Ti = 20 C) < 300 MPa (Ti = 20 C) 
PA] (Table 2).
3.2. Effect of UHPH on vitamins of apple juice
Although ascorbic acid was found in low levels in all samples,
its content did not change as a result of different pressure intensities applied by UHPH and/or by thermal treatment (Table 3). The
average content of ascorbic acid of the samples analysed was
0.46 0.01 mg L1. Dehydroascorbic acid is a compound that can
be converted into ascorbic acid and, therefore, is considered to exhibit vitamin C activity although it lacks its antioxidant capacity.
No signicance differences were found in total vitamin C content
of R and UHPH-treated samples, but their content was nearly 8times higher than in PA samples (Table 3).
b-Carotene concentrations were also evaluated. The results obtained are expressed as RE. Signicant differences between different homogenisation pressure intensities and inlet temperature
applied were observed. R apple juice had the highest b-carotene
content (11.38 0.59 lg L1). For the UHPH-treatments the following values were observed: for 100200 MPa at 4 C (Ti)
9.4 0.9 lg L1; for 300 MPa at 4 C and 100200300 MPa at
20 C 7.6 0.5 lg L1. PA samples had the lowest concentrations
of only 6.8 0.4 lg L1 (Table 3). The losses observed based on
the R juice were in the following order: 18% for the 100200 MPa
at 4 C (Ti) treatments 33% for the 300 MPa at 4 C and 100200
300 MPa at 20 C treatments, and 40% for PA treated juice.
3.3. Relationship among compounds and the antioxidant activities
measured
To better understand the effect of UHPH technology on the different compounds analysed, linear correlation coefcients were

Table 3
Ascorbic acid, total Vitamin C, b-carotene and retinol equivalent of apple juices: R, PA
and UHPH treated at 4 and 20 C inlet temperatures.
Treatment

Ascorbic acid
(mg L1)

Total vit C l
(mg L1)

b-Carotene
(lg L1)

Retinol
equivalent
(lg L1)

R
100 MPa
at 4 C
100 MPa
at
20 C
200 MPa
at 4 C
200 MPa
at
20 C
300 MPa
at 4 C
300 MPa
at
20 C
PA

0.44 0.05a
0.45 0.05a

13.54 1.76b
12.16 0.66b

11.38 0.59d
9.71 1.00cd

1.90 0.10d
1.62 0.18cd

0.46 0.05a

13.12 1.92b

7.75 1.00ab

1.29 0.17ab

0.46 0.07a

12.34 1.40b

9.11 0.81bc

1.52 0.14bc

0.45 0.04a

12.94 1.76b

7.48 0.09ab

1.25 0.02ab

0.46 0.05a

12.58 1.51b

7.46 0.85ab

1.24 0.14ab

0.48 0.05a

12.98 1.76b

7.67 0.35ab

1.28 0.06ab

0.46 0.07a

1.51 0.28a

6.82 0.43a

1.14 0.07a

Values are means (SD) of triplicate analysis from 3 different productions (n = 9).
Values in the same column with different superscripts differ signicantly (P < 0.05).

calculated to explore the relationships of these compounds with


the antioxidant capacity after treatments. Data of the FRAP assay
were statistically signicant positively correlated with total polyphenolic content, the 4-caffeinoylquinic acid, chlorogenic acid,
and the total polyphenol concentration (84%, 95%, 77%, 74%,
respectively) (P < 0.01) as well as inversely correlated with total
vitamin C content (96%, P < 0.05). Results of the ORAC assay were
positively correlated with the chlorogenic acid (81%, P < 0.05) and
polyphenol content (86%, P < 0.01) and inversely correlated with
the b-carotene content (0.63%, P < 0.05). On the other hand, TEAC
and DPPH assays were inversely correlated with the vitamin C content (90% and 91% respectively) (P < 0.05).
A strong correlation among FRAP and ORAC, TEAC and DPPH assays (74%, 83%, and 90%, respectively) (P < 0.05) was observed.
ORAC assay also shows correlation between TEAC and DPPH (69%
and 77%). Positive correlation between TEAC and DPPH was also
found (78%, P < 0.01).

4. Discussion
The present study was made to reduce the lack of existing data
regarding the inuence of UHPH treatment on the quality properties of apple juice after treatment and to identify the optimal treatment to avoid the quality deterioration during processing.
The main juice components, together with pH, did not change
due to treatment, being no difference among UHPH-treated samples, neither with R or PA samples. These results are in agreement
with Butz, Fernndez-Garca, and Tauscher (2002) who evaluated
the inuence of HPP treatment over nutritional quality of orange,
apple, carrot and tomato juices and did not nd remarkable
changes after treatments. Although changes in viscosity are far below sensory detection level, a slight increase can be attributed to
UHPH treatment.
To fully elucidate a complete prole of antioxidant capacity of
UHPH-treated apple juice, different antioxidant capacity assays
were used. DPPH, TEAC and FRAP assays, were recommended as
easy, accurate and rapid methods for measuring antioxidant capacity in fruit and vegetable juices. On the other hand, ORAC assay is a
widely used procedure for measuring the antioxidant capacity of
biological samples, it is sensitive, precise and robust within accepted criteria (Ou, Hampsch-Woodill, & Prior, 2001), and was thus
included in the characterisation of the effects of UHPH-treatment
on apple juice.
Antioxidant capacity values were highest in the ORAC assay
(ORAC > TEAC > FRAP > DPPH). However, same tendency in the results was observed for the FRAP and DPPH test. In contrast, significant differences were observed by the TEAC and ORAC assay
(Fig. 2). R and low intensity UHPH treatments showed lower ORAC
& TEAC values than P and high intensity UHPH. This difference
among assays can be attributed to the fact that food antioxidant
capacity measurement depends on several factors, including the
nature of the technology, which free radical generator oxidants is
used, and the complexity of the molecular structure evaluated
(Frankel & Meyer, 2000; Rice-Evans, Miller, & Paganga, 1996).
DPPH, TEAC and FRAP methods are based on the same reaction
principle: single electron transfer. These assays measure the specic oxidant-reducing power not necessarily equivalent to the
antioxidant capacity. Nevertheless, DPPH method has been applied
to measure the antioxidant capacity in fruit and vegetables
although it seems less sensitive than the other methods for hydrophilic antioxidants (Gil, Tomas-Barberan, Hess-Pierce, Holcroft, &
Kader, 2000). The advantage of ORAC assay is that it is the only
one that combines the total inhibition time and the percentage
of the free radical damage by the antioxidant into a single value,
ensuring that all the antioxidants have reacted with the radical

. Surez-Jacobo et al. / Food Chemistry 127 (2011) 447454

generated. The inhibition is reected in the protection against the


uorescence change in ORAC assay (Fig. 3). However, this method
can not be considered a total antioxidant activity assay since the
assay is performed in aqueous solution, and thus primarily measures hydrophilic antioxidant activity against peroxyl radical (Ou
et al., 2001). Despite the different results of the antioxidant capacities results, the correlation analysis showed high correlations between the four assays. It seems that the synergy effects or
interactions among antioxidants may affect the results and give
rise to different activities in the methods evaluated.
The total phenolic content of the juices was not affected by the
different UHPH treatments, in comparison with the sum of the
individual polyphenols (13.3 1.3 mg L1) quantied by HPLC/
DAD, these spectrophotometric results revealed a 3.7-fold overestimation. Similar overestimation was also observed by Schilling
et al. (2008). The responsible compounds for the increased measurements of polyphenolic compounds are not known, but it is
probable that the results do not correspond to polyphenols at all.
It is possible that unidentied nonphenolic compounds present
in juice are capable of react with the FolinCiocalteau reagent (Folin Ciocalteau assay, like DPPH, TEAC and FRAP assays, are based on
electron transfer reactions). These compounds could be attributed
to carbohydrate breakdown products, enediols or reductones,
Maillard reaction products (Van Buren, De Vos, & Pilnik, 1976).
Treatment of pulp with pectolytic enzymes was effective in
increasing the amount of this unknown material in the juice
(Van Buren et al., 1976).
The low polyphenolic content in R and low-pressure UHPH
samples can be attributed to the enzymatic oxidation, producing
not measurable high molecular weight compounds. UHPH at high
pressure can inactivate polyphenol oxidase in the same way as
can be done by thermal treatments (Saldo, Surez-Jacobo, Gervilla,
Guamis, & Roig-Sagus, 2009; Schilling et al., 2008).
Chlorogenic acid was the major polyphenol found in apple juice
followed by 4-caffeinoylquinic acid, and the avonol, quercetin-3rhamnoside. In fact, chlorogenic and 4-caffeinoylquinic acid were
highly correlated with FRAP and ORAC assays (95 and 75%,
P < 0.01 and P < 0.05). Quercetin-3-arabinoside was subjected to
correlation analysis due to the signicant differences observed,
however, these differences could be neglected because their values
are low compared with the rest of compounds. The total polyphenol content was lower than reported in other studies, probably due
to the juice extraction method and processing. Pectolytic enzymes
were used to obtain a higher juice yield before pressing by degrading pectins in the cell walls (Fig. 1). Before addition of pectolytic
enzymes, the pulp must be aerated to allow the oxidation of phenols (reducing their content), because these compounds may inhi-

100 MPA

200 MPA

300 MPA

Relative Fluorescense intensity

bit pectolytic enzymes. van der Sluis, Dekker, Skrede, and Jongen
(2002) found that conventional processing of apple juices resulted
in great avonoid losses. Van Buren et al. (1976) found that chlorogenic acid concentrations in juices were lower when pulps or
juices had been allowed to undergo enzymatic oxidation (PPO
activity), being less than the observed for other avonoids. In this
sense, juice-processing conditions affect the polyphenol content.
In addition, the polyphenol content is determined by the variety, ripeness grade, climate and cultural conditions of apples used
to make the juice (Stracke, Rfer, Weibel et al., 2009; Tsao, Yang,
Young, & Zhu, 2003; van der Sluis, Dekker, de Jager, & Jongen,
2001).
UHPH has no effect on the ascorbic and dehydroascorbic acid
functionality, keeping the same activity as in R. However, the pasteurisation caused a signicant decrease in the total vitamin C content derived by the intensity and time of the thermal treatment
applied. The strongest thermal effect on vitamin C, observed in
the PA samples, reduced its content to around 88% of original value
(Table 3). In fact, vitamin C content is inversely correlated (97%,
P < 0.01) with the hydroxymethylfurfural concentration (a good index for thermal treatment) (Saldo et al., 2009).
Ascorbic acid content has an inuence on the antioxidant capacity of fruit juices; however, the dehydroascorbic acid does not and,
therefore, does not contribute to the total antioxidant capacity
(less than 1%) (Gardner, White, McPhail, & Duthie, 2000). This
means that ascorbic acid in apple juice just represents a minimal
fraction of the total antioxidant capacity. Vitamin C was correlated
inversely with FRAP, TEAC and DPPH antioxidant capacity assays.
For b-carotene signicant differences between UHPH treatments in function of Ti were observed. However, these differences
were subtle compared to thermal treatment. The losses could be
due to the fact that carotenoids are sensitive to light, acids and,
thermal treatment that promotes oxidation and structural changes
such a cistrans isomerization. According to Rodriguez-Amaya
(2003) oxidation depends on availability of oxygen, water activity,
presence of antioxidants, exposure to light, presence of metals, enzymes and severity of treatments (destruction of the cellular structure that protects the carotenoids, increase of surface area,
duration and temperature of heat treatment). The effect of UHPH
on carotenoids has not been intensely studied. However, it is supposed that oxidation reactions could have occurred during processing as was observed by Pereda, Ferragut, Quevedo, Guamis, and
Trujillo (2007) when lipid oxidation increased as a consequence
of UHPH treatment. The reactions occurring during carotenoid oxidation are not as well understood as lipid oxidation but it could initially involve epoxidation and the formation of apocarotenoids as a
result of the attack of terminal double bond, yielding low mass
compounds similar to those obtained in fatty acid oxidation (Rodriguez-Amaya, 2003).

BK

1.00E+04

5. Conclusion

5.00E+03

0.00E+00

453

50
Kinetic cycles (time)

100

Fig. 3. ORAC decay curve of uorescence of UHPH-treated samples. 100, 200 and
300 MPa at 20 C inlet temperature in comparison with a blank (BK). Values are
means of 3 replicates.

In the present study, raw and pasteurised apple juices were


compared to UHPH-treated to evaluate the impact of this technology on their quality characteristics. Several assays were performed,
including analysis of the antioxidant capacity, the total and individual polyphenol content, as well as the vitamin C and b-carotene
concentrations. UHPH at 300 MPa both inlet temperatures were
the best conditions to preserve the antioxidant capacity and polyphenolic compounds in apple juice. On the contrary, at these conditions the b-carotene content is reduced due to oxidation during
the processing, more studies must be carried on in order to understand the b-carotene degradation. UHPH treatment does not disturb the original ascorbic acid and dehydroascorbic acid content
in apple juice. Hence, UHPH is a quite suitable technology to pre-

454

. Surez-Jacobo et al. / Food Chemistry 127 (2011) 447454

serve bioactive nutrients in fruit juices without additional thermal


damage.
Acknowledgements
We thank to Joan Miquel Quevedo for his valuable technical
support in the CERPTA pilot plant and Serap Demirel is appreciated
for her valuable technical assistance in the carotenoid analysis.
The authors acknowledge the Ministerio de Educacin y Ciencia
(AGL2006-09607/ALI) for the nancial support given to this investigation. The author Surez-Jacobo gratefully acknowledges the
nancial support for her doctoral studies from the CONACyT (Mexico) Fellowship program.
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