Food Chemistry 148 (2014) 261267

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

Stevia rebaudiana Bertoni as a natural antioxidant/antimicrobial for high

pressure processed fruit extract: Processing parameter optimization
Francisco Jos Barba a, Mara Nieves Criado a, Clara Miracle Belda-Galbis a, Mara Jos Esteve b,
Dolores Rodrigo a,
a
Departamento de Conservacin y Calidad, Instituto de Agroqumica y Tecnologa de Alimentos (IATA-CSIC), Carrer del Catedrtic Agustn Escardino Benlloch 7, 46980 Paterna,
Valncia, Spain
b
Department of Nutrition and Food Chemistry, Universitat de Valncia, Avda. Vicent Andrs Estells, s/n 46100 Burjassot, Valncia, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Response surface methodology was used to evaluate the optimal high pressure processing treatment
Received 25 July 2013 (300500 MPa, 515 min) combined with Stevia rebaudiana (Stevia) addition (02.5% (w/v)) to guarantee
Received in revised form 7 October 2013 food safety while maintaining maximum retention of nutritional properties. A fruit extract matrix was
Accepted 9 October 2013
selected and Listeria monocytogenes inactivation was followed from the food safety point of view while
Available online 20 October 2013
polyphenoloxidase (PPO) and peroxidase (POD) activities, total phenolic content (TPC) and antioxidant
capacity (TEAC and ORAC) were studied from the food quality point of view. A combination of treatments
Keywords:
achieved higher levels of inactivation of L. monocytogenes and of the oxidative enzymes, succeeding in
Stevia rebaudiana Bertoni
High pressure processing
completely inactivating POD and also increasing the levels of TPC, TEAC and ORAC. A treatment of
Polyphenoloxidase 453 MPa for 5 min with a 2.5% (w/v) of Stevia succeeded in inactivating over 5 log cycles of L. monocyt-
Peroxidase ogenes and maximizing inactivation of PPO and POD, with the greatest retention of bioactive components.
Total antioxidant capacity 2013 Elsevier Ltd. All rights reserved.
Phenolic compounds
Listeria monocytogenes
Response surface methodology

1. Introduction combine this technology with the addition of preservatives of nat-

Increased consumer awareness of the relationship between diet
and health has encouraged the search for food preservation tech-
nologies that avoid the use of intense thermal treatments and
the use of chemical additives. They include high pressure process-
ing (HPP), which has achieved increasing interest over the past two
decades, given its potential to inactivate enzymes, such as poly-
phenoloxidase (PPO, EC 1.14.18.1) and peroxidase (POD, EC
1.11.1.7) (Terefe et al., 2013; Wang et al., 2012), as well as spoilage
and/or pathogenic microorganisms (Pina-Prez, Silva-Angulo,
Rodrigo, & Lpez, 2012; Saucedo-Reyes, Marco-Celdrn, Pina-
Prez, Rodrigo, & Martnez-Lpez, 2009; Sokoowska et al., 2013),
with a minimum impact on the nutritional and physicochemical
properties of foods (Barba, Esteve, & Frigola, 2010). However, this
technology on its own is less effective in terms of enzymatic and
microbial inactivation than conventional thermal treatments. A
possible alternative in order to complement its effectiveness is to
Corresponding author. Tel.: +34 963900022; fax: +34 963636301.
E-mail address: lolesra@iata.csic.es (D. Rodrigo).
ural origin (Snchez-Moreno, Plaza, de Ancos, & Cano, 2004).
Stevia rebaudiana (Stevia) Bertoni is a perennial shrub belonging
to the Asteraceae family that grows in tropical and subtropical
areas of South America, where its leaves have been used tradition-
ally as a natural sweetener for hundreds of years (Chaturvedula,
Upreti, & Prakash, 2011). Nowadays, the potential use and practical
implications of Stevia as a sweetener are shown in a number of pro-
cessed food, such as fruit drinks, biscuits and breads, as a sugar
substitute commercialized in countries such as France, Spain and
USA between others, because it contains steviol-glycosides, which
are low- or non-caloric ingredients, up to 100300 times sweeter
than sucrose (Lemus-Mondaca, Vega-Glvez, Zura-Bravo, & Ah-
Hen, 2012). In addition, dry Stevia leaves also contain minerals,
vitamins, phenolic compounds, avonoids and other antioxidant
compounds (Lemus-Mondaca et al., 2012; Tadhani, Patel, & Sub-
hash, 2007), with antimicrobial (Belda-Galbis et al., 2014; Tadhani
& Subhash, 2006) and antioxidant properties (Criado, Barba, Frgo-
la, & Rodrigo, in press; Muanda, Soulimani, Diop, & Dicko, 2011),
and with potential benecial effects on human health. Neverthe-
less, little data has been available about Stevia stability under dif-
ferent processing and storage conditions (Nehir El & Simsek, 2012).

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.10.048
262 F.J. Barba et al. / Food Chemistry 148 (2014) 261267
Taking into account the Stevia properties mentioned, its combi-
nation with HPP treatment could be a good strategy to improve the
microbial, nutritional and physicochemical quality of foods. In
this respect, Carbonell-Capella, Barba, Esteve, and Frgola (2013)
published a study carried out in fruit juice sweetened with Stevia,
focused on determining the optimal high pressureStevia concen-
tration combination in order to obtain the best retention of phys-
icochemical and nutritional quality in the beverage following
high pressure. However, microbial inactivation was not considered
in that study.
Response surface methodology (RSM) consists of a set of math-
ematical and statistical methods developed for modeling phenom-
ena and establishing combinations of a certain number of
experimental factors (variables) that will lead to optimum re-
sponses. With RSM, several variables are tested simultaneously
with a minimum number of trials, in accordance with special
experimental designs, which elucidates interactions between vari-
ables (Giovanni, 1983).
In the present study RSM was used as a tool to investigate the
combined effects of the various HPP processing parameters and
Stevia concentrations to obtain a fruit extract with the highest lev-
els of antioxidant compounds and the minimum oxidative reac-
tions, which in turn succeeded in reducing the microbial load of
the product by at least 5 log cycles.
2. Material and methods
2.1. Samples
2.1.1. Plant material
Oranges (Citrus sinensis, cultivar Salustiana), mango (Mangifera
indica) and papaya (Carica papaya) at commercial maturity were
purchased from a local supermarket and then stored at 4 C for
one day before the enzyme extraction procedure.
2.1.2. Enzyme crude extract preparation
POD and PPO were extracted using the method described by
Rodrigo, Rodrigo, Alvarruiz, and Frgola (1996) with some modi-
cations. Papaya, mango and orange fruits were washed, peeled
and cut into small pieces. A mixture of the fruit pulp (orange, man-
go and papaya in a proportion of 15:20:65, w/w/w, respectively)
was homogenized in a proportion of 1:1 (w/v) with 0.05 M sodium
phosphate buffer solution (Panreac Qumica, Barcelona, Spain) at
pH 7.0 in a blender for 5 min at 4 C. The buffer contained 1 M NaCl
(Scharlau, Barcelona, Spain) and 5% (w/v) polyvinylpolypyrrolidone
(SigmaAldrich Co. LLC, St. Louis, MO, USA). The homogenate was
ltered through a layer of cheesecloth and the residue was centri-
fuged at 20,199g for 30 min at 4 C with an Avanti J-25 centrifuge,
Beckman Instruments Inc. (Fullerton, California, USA.). The super-
natant constituted the enzyme extract.
2.1.3. Stevia infusion and sample preparation
Dried Stevia leaves were supplied by Anagalide S. A. (Spain) and
stored at room temperature. To prepare a stock solution of
8.33 0.01% (w/v), 100 mL of boiling mineral water was added to
the dried leaves (8.33 g) and the mixture was covered and allowed
to infuse for 30 min. The infusion was vacuum ltered using lter
paper (Whatman No. 1, Whatman International Ltd, UK) and the
ltrate obtained was stored in 2 mL vials at 40 C.
Different volumes of Stevia stock solution (3 and 6 mL) were
added to the crude enzyme extract (14 mL) to obtain nal Stevia
concentrations of 1.25% and 2.5% (w/v), respectively. The higher
Stevia concentration (2.5% (w/v)) was selected, taking into account
the sucrose concentration of commercial fruit-based beverages and
the sweetness equivalence of Stevia and sucrose (Savita et al.,
2004). A blank sample was formulated with 14 mL of crude extract
and 6 mL of water.
Total phenolic content (TPC), POD and PPO activities, antioxi-
dant capacity (TEAC and ORAC) and microbial inactivation of both
untreated (blank) and HPP-treated samples were evaluated. Anal-
yses were carried out immediately after the HPP treatments.
2.1.4. Microorganism
Listeria monocytogenes was the microorganism selected to as-
sess the antimicrobial effects of HPP and Stevia in view of the fact
that controlling its concentration and growth is an important chal-
lenge for food business operators because it is a ubiquitous, psy-
chrotrophic, facultative anaerobic foodborne pathogen that grows
in very hostile environments (Codex Alimentarius Commision.,
2002).
From a lyophilized pure culture provided by the Spanish Type
Culture Collection, a stock vial containing L. monocytogenes (CECT
4032) was generated following the method described by Sauce-
do-Reyes et al. (2009). The average cell concentration was ca.
7.6  109 cfu/mL. It was established by viable plate count using
Tryptic Soy Agar (TSA; Scharlau Chemie S. A., Spain) for the spread-
ing of samples.
2.2. Methods
2.2.1. HPP treatments
For each time and pressure condition, inoculated and un-inocu-
lated samples of enzyme extract, with and without Stevia, were
packed in polyethylene bags that were heat-sealed (MULTIVAC
Thermosealer, Switzerland) before being inserted in the pressure
vessel (High-Pressure Food Processor, EPSI NV, Belgium). HPP
treatments were performed in a pilot-scale unit (2.35 L vessel vol-
ume), with an operation pressure vessel of 600 MPa. The pressure
transmitting uid was a mixture of water and ethylene glycol
(70:30% (v/v)). The samples were pressurized at 300, 400 and
500 MPa, at ambient temperature (1822 C), for 5, 10 and
15 min. The pressure level, pressurization time and temperature
were controlled automatically. The compression rate was
300 MPa/min and the decompression time was less than 1 min.
The treatment time described in this study does not include
come-up and come-down times. All the treatments were applied
in triplicate.
After completing the treatment, the samples were removed
from the vessel and immediately transferred to an ice-water bath
and stored under refrigeration (3 1 C) until needed for analysis.
2.2.2. PPO and POD activities determination
PPO activity was measured by the method described by Giner,
Gimeno, Barbosa-Cnovas, and Martn (2001), with some modica-
tions. The method was based on measuring increase in absorbance
at 410 nm when 1950 lL of 0.05 M 1,2-dihydroxybenzene (pyro-
catechol; SigmaAldrich Co., LLC, USA) in phosphate buffer
(0.05 M, pH 7.00, with 1 M NaCl) as substrate reacted with
0.1 mL of enzyme extract in a plastic cuvette 1 cm long. The initial
colour of the sample was used as reference. The increase in absorp-
tion of the yellow product was recorded at 410 nm every 1 s for
2.5 min and was used for the quantication of enzyme activity.
Each PPO activity test was replicated three times at 25 C. One unit
of PPO activity was expressed as one absorbance increment (at
410 nm in the conditions in which the assay was carried out) per
min and mL of enzyme extract.
POD activity was measured spectrophotometrically. 200 lL of
extract was added to a tube with 3.8 mL of sodium phosphate buf-
fer (0.05 M, pH 7.0, with 1 M NaCl), agitated for 5 min and kept at
25 C for 7 min. The reaction began when 2.39 mL of this solution
was placed in a polystyrene 4.5 mL cuvette and mixed with
 F.J. Barba et al. / Food Chemistry 148 (2014) 261267 263

0.61 mL of a reaction mixture consisting of 2-methoxyphenol 2.2.4.2. Oxygen radical absorbance capacity (ORAC) assay. The ORAC
(guaiacol; SigmaAldrich Co., LLC, USA), H2O2 (SigmaAldrich Co., assay described by Ou, Hampsch-Woodill, and Prior (2001) was
LLC, USA) and bidistilled water. The cuvette concentration was used, with uorescein (FL) (SigmaAldrich, Germany) as the uo-
45 mM for H2O2 and 36 mM for guaiacol. The changes in absor- rescent probe. The automated ORAC assay was carried out on a
bance at 470 nm, using a Lan Optics Model PG1800 spectropho- Wallac 1420 VICTOR2 multilabel counter (Perkin-Elmer, USA) with
tometer (Labolan, Spain), were recorded every 1 s to 2.5 min. uorescence lters, for an excitation wavelength of 485 nm and an
Enzyme activity was calculated from the slope of the linear part emission wavelength of 535 nm. The measurements were made in
of the graph of absorbance versus time. Each POD activity test plates with 96 white at-bottom wells (Sero-Wel, Bibby Sterilin
was replicated three times at 25 C. One unit of POD activity was Ltd., UK). The reaction was performed at 37 C as the reaction
expressed as one absorbance increment (at 470 nm in the condi- was started by thermal decomposition of 2,20 -azobis(2-amidino-
tions in which the assay was carried out) per min and mL of en- propane) dihydrochloride (AAPH; SigmaAldrich Co., USA) in
zyme extract. 75 mM phosphate buffer (pH 7.0) because of the sensitivity of FL
In both cases, before and immediately after applying HPP, the to pH. The nal reaction tested and the concentrations of the differ-
enzymatic activities of the fruit extract (A) were measured and ent reagents were determined following Barba et al. (2013).
compared with the initial activities of untreated samples (A0).
The initial reaction rate was expressed as a percentage of relative
2.2.5. Microbial inactivation
activity (RA), calculated using the following equation (Eq. (1)):
The cellular density of treated samples, with and without Stevia,
% RA 100  A=A0 1 was determined, in terms of log10 (cfu/mL), before and after the
different HPP treatments, by viable plate count, using TSA (Schar-
where A and A0 are the current (after HPP) and the initial PPO and
lau Chemie S. A., Spain) for the sample spreading followed by an
POD activities in the untreated samples, respectively.
incubation period of 48 h at 37 C. The inactivation associated with
each of the treatments tested was established according to the dif-
2.2.3. Total phenolic content
ference existing between the counts obtained pre- and post-treat-
The amount of TPC was determined according to the method
ment. From each sample, two aliquots were diluted and spread
described by Georg, Brat, Alter, and Amiot (2005), with some
separately. The dilutions were done employing buffered peptone
modications. Briey, 10 mL of sample was homogenized with
water (Scharlau Chemie S. A, Spain). From each aliquot, two plates
50 mL of a mixture of acetone:water (70:30% (v/v)) for 30 min.
were spread. Thus, each count was obtained from four plates.
Mixture supernatants were then recovered by ltration (What-
man No. 2, Whatman International Ltd, UK) and constituted the
raw extracts (REs). REs (2 mL) were settled on an Oasis cartridge 2.3. Experimental design and statistical analysis
(Waters S. A. S., France). Interfering water-soluble components
(reducing sugars, ascorbic acid) were recovered with 2  2 mL of RSM was used to investigate the simultaneous effects of pres-
distilled water. The recovered volume of the washing extract sure, time and Stevia concentration on TPC, PPO and POD activities,
(WE) was carefully measured. microbiological inactivation and antioxidant capacity of the fruit
In order to eliminate vitamin C, heating was carried out on the extract. Face-centred central composite design was used with
washing extract (3 mL) for 2 h at 85 C and this led to the heated three levels (maximum, minimum and central) of each indepen-
washing extract (HWE). All extracts (RE, WE and HWE) were sub- dent variable, pressure (from 300 to 500 MPa), time (from 5 to
mitted to the FolinCiocalteu method, adapted and optimized (Bar- 15 min) and Stevia concentration (from 0 to 2.5% (w/v)), leading
ba, Esteve, Tedeschi, Brandolini, & Frgola, 2013). Gallic acid to 16 combinations (Table 1). Independent variable levels were se-
calibration standards with concentrations of 0, 100, 300, 500, 700 lected considering the sample and the operating condition of the
and 1000 ppm were prepared and 0.1 mL was transferred to boro- HPP equipment. The combinations included HPPStevia conditions
silicate tubes. 3 mL of sodium carbonate solution (2% (w/v)) with an intermediate level (central point) of the 3 variables repli-
(Scharlau Chemie S. A., Spain) and 100 lL of FolinCiocalteu re- cated 2 times, which was used to check the reproducibility and sta-
agent (1:1 (v/v)) were added to an aliquot of 100 lL from each gal- bility of the results. The experimental design was performed twice,
lic acid standard or sample tube. The mixture was vortexed and resulting in two blocks of experiments. Accordingly, samples were
allowed to stand at room temperature in the dark for 1 h. Absor-
bance was measured at 765 nm using a Perkin Elmer UV/Vis Lamb-
Table 1
da 2 spectrophotometer (Perkin-Elmer, Germany) and the results Experimental design matrix.
were expressed as mg of gallic acid equivalents (GAE)/L.
Run Pressure (MPa) Time (min) Stevia (% (w/v))
(X1) (X2) (X3)
2.2.4. Total antioxidant capacity
2.2.4.1. Trolox equivalent antioxidant capacity (TEAC) assay. For 2,20 - 1 500 15 0.00
2 400 15 1.25
azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay the 3 500 5 2.50
procedure followed the method of Re et al. (1999), based on the 4 500 15 2.50
capacity of a sample to inhibit the ABTS radical (ABTS+) (SigmaAl- 5 300 10 1.25
drich Co., USA) compared with a reference antioxidant standard 6 400 5 1.25
7a 400 10 1.25
(Trolox; SigmaAldrich Co., USA). The radical was generated using
8 400 10 0.00
440 lL of potassium persulfate (140 Mm) (SigmaAldrich Co., 9 300 5 2.50
USA). The solution was diluted with ethanol (Baker, The Nether- 10 400 10 2.50
lands) in order to obtain an absorbance of 0.70 0.02 units at 11 500 5 0.00
734 nm. Once the radical was formed, 2 mL of ABTS+ was mixed 12 300 5 0.00
13 300 15 0.00
with 100 lL of appropriately diluted sample and the absorbance 14 300 15 2.50
was measured at 734 nm for 20 min in a Perkin Elmer UV/Vis 15 500 10 1.25
Lambda 2 spectrophotometer (Perkin-Elmer, Germany) in accor- 16a 400 10 1.25
dance with Barba et al. (2013). The results, obtained from triplicate a
Central point.
analyses, were expressed as mM Trolox equivalents (TE).
264 F.J. Barba et al. / Food Chemistry 148 (2014) 261267

treated in duplicate and analysed in triplicate in all cases. Experi-
ments were randomized to minimize the systematic bias in the ob-
served responses due to extraneous factors and in order to increase
precision. The software generated regression coefcients for each
of the term combinations of the independent variables, and their
signicance was determined using the p-value generated by t-test.
The quadratic model for each response was as follows (Eq. (2)):
Y b0 b1 X 1 b2 X 2 b3 X 3 b11 X 21 b22 X 22 b33 X 23
b12 X 1 X 2 b13 X 1 X 3 b23 X 2 X 3
where Y represents the predicted response; Xi were the indepen-
dent variables; b0 was a constant that xed the response at the cen-
tral point of the experiment, and bii the regression coefcients for
the linear, quadratic and interaction effect terms.
The non-signicant terms (p > 0.05) were deleted from the sec-
ond-order polynomial model after an ANOVA test, and a new AN-
OVA was performed to obtain the coefcients of the nal
equation for better accuracy. For this purpose, the program always
conducts one search beginning at the centre of the experimental
region. Given that the starting point may affect whether a global
or local optimum is located, the optimization was also done using
additional searches starting at the best design point (design point
with highest predicted desirability), all design points, best vertex
(combination of low or high level of each factor with highest pre-
dicted desirability), or all vertices, in order to obtain the best result
amongst the set of searches that the program performs.
The experimental design and the data analysis were performed
using the software Statgraphics Centurion XV (Statpoint Technol-
ogies, Inc., USA).
3. Results and discussion
3.1. Effect of HPP and Stevia rebaudiana on PPO and POD activities,
TPC, total antioxidant capacity (ORAC and TEAC) and microbial counts
Effects of HPP treatments with and without Stevia on the PPO
and POD activities, TPC, total antioxidant capacity (TEAC and
ORAC) and on microbial inactivation in the fruit extract are shown
in Table 2.
With regard to oxidative enzymes, PPO and POD untreated ex-
tract activities in the absence of Stevia were 7.42 1.3 and
2.07 0.69 DAbsorbance/min mL of extract, respectively. These
values were in the range of others described previously in the lit-
erature, although the PPO and POD contents of fruits and vegeta-
bles might be affected by species, cultivar and maturity (Elez-
Martnez, Soliva-Fortuny, & Martn-Belloso, 2006; Gasull & Becerra,
2006).
Pressure inuenced residual PPO and POD activities. In the ab-
sence of Stevia the results showed that the residual PPO activity
in the fruit extract decreased to 33.8%, 16.4%, 12.3%, 3.1% and
2 2.6% after treatment at 300 MPa for 5 and 15 min, 400 MPa for
10 min and 500 MPa for 5 and 15 min, respectively. In the case of
POD the residual activity values were 27.5%, 22.3%, 20.0%, 16.1%
and 15.7%, at the same conditions, respectively, POD being more
resistant to HPP treatments than PPO. These differences were more
pronounced in the 500 MPa treatments, where the residual POD
activity in the fruit extract was 13% higher than residual PPO.
Several studies that have focused on the effect of HPP on PPO
and POD activities in various fruits and vegetables show that both
enzymes exhibit a wide range of pressure stability. In general
terms, the effectiveness of a treatment depends on the type of en-
zyme, pH, medium composition, temperature, time and pressure
level applied (Hendrickx, Ludikhuyze, Van den Broeck, & Weemaes,
1998). However, many studies show that PPO and POD present a
rather high pressure resistance. In relation to PPO from fruit
smoothies, HPP treatments of 450 MPa for 5 min and 600 MPa for
10 min resulted in 35% and 83% residual activity, respectively (Kee-
nan, Roessle, Gormley, Butler, & Brunton, 2012). With regard to
POD, for green peas, a treatment of 900 MPa for 10 min at room
temperature was needed to cause an 88% reduction of POD activity
(Quaglia, Gravina, Paperi, & Paoletti, 1996).
Due to the very high pressure level that is often necessary to
inactivate PPO and POD, several researches have proposed the
addition of natural enzyme inhibitors. The pressure inactivation
of PPO and POD is inuenced by the addition of salts, sugars or
other compounds, including antibrowning agents. Table 2 shows,
under the given experimental conditions, the capacity of Stevia to
inhibit both PPO and POD enzymes. To the best of our knowledge,
this is the rst time that the effect of Stevia on PPO and POD from
fruits has been reported. The results obtained shown that, in the
case of POD, Stevia enhanced the inactivation percentage achieved
by HPP The three-way ANOVA showed that pressure and Stevia
concentration had a signicant inuence on POD activity

Table 2
Effect of HPP and Stevia on enzyme activities (PPO and POD), total phenolic content, total antioxidant capacity (TEAC and ORAC) and the microbial load of the fruit extract.

Pressure Time Stevia PPO POD TPC TEAC ORAC log10 (Nf/N0)
MPa (min) % (w/v) % Residual activity % Residual activity (mg GAE/L) (mM TE) (mM TE) log10 (cfu/mL)
0 0 0 100 100 185.52 7.13 2.30 0.11 4.41 0.20 0
1.25 100 100 2261.13 41.74 16.62 0.21 16.91 0.83 0
2.5 100 100 4050.81 42.73 18.81 0.13 28.25 3.71 0
300 5 0 33.84 1.88 27.46 2.69 180.91 10.09 2.41 0.28 4.65 0.19 0.17 0.01
2.5 15.60 0.23 ND 4337.82 19.44 27.93 0.97 38.30 0.29 3.96 0.03
10 1.25 17.96 0.11 23.05 1.24 2300.70 22.90 14.90 1.84 21.50 0.30 -
15 0 16.37 0.23 22.29 0.04 177.17 11.62 2.16 0.46 4.75 0.24 5.72 0.02
2.5 16.54 0.69 ND 4777.89 56.94 29.63 3.18 40.90 0.52 >7+
400 5 1.25 15.51 0.11 20.55 1.3 2802.51 13.98 17.32 1.18 23.80 0.20 -
10 0 12.25 0.78 19.95 0.73 177.80 8.46 2.62 0.06 4.10 0.30 6.30 0.08
1.25 12.35 0.75a 18.52 0.27a 2847.11 146.08a 19.62 1.26a 23.65 0.35a -
2.5 11.90 0.13 ND 4151.48 25.50 30.07 0.8 39.30 0.40 >7+
15 1.25 12.07 0.36 17.50 0.01 2457.54 5.99 19.72 0.23 21.10 0.20 -
500 5 0 3.14 0.07 16.13 0.59 191.17 2.54 2.55 0.13 4.15 0.24 >7+
2.5 3.99 0.05 ND 4193.85 21.97 29.02 1.50 41.35 0.35 >7+
10 1.25 3.00 0.21 12.10 0.01 2357.64 18.41 19.38 0.12 22.30 0.20 -
15 0 2.56 0.21 15.66 0.71 188.18 8.12 2.61 0.28 3.60 0.14 >7+
2.5 1.87 0.25 ND 4142.25 52.88 31.88 1.45 40.70 0.35 >7+
a
Average of central point. PPO: Polyphenoloxidase. POD: Peroxidase. TPC: Total phenolic content. TEAC: Trolox equivalent antioxidant capacity. ORAC: Oxygen radical
antioxidant capacity. mg GAE/L: milligrams of gallic acid equivalents. mM TE: millimolar Trolox equivalents. Nf: Final cell concentration. N0: Initial cell concentration. -: Not
tested. +N0 = 7.84 0.17 log10 (cfu/mL). ND: not detected.
 F.J. Barba et al. / Food Chemistry 148 (2014) 261267
(p < 0.05), but in the case of PPO, the residual activity present in ex-
tracts supplemented with 2.5% (w/v) and subjected to 300 MPa for
5 min (the lowest treatment assayed) was signicantly less than
the activity present in extract without Stevia, subjected to the same
pressure and time combination (p < 0.05). Regardless of the HPP
treatment applied, complete inactivation of POD was achieved
when the Stevia concentration was 2.5% (w/v). In the case of PPO,
the maximum reduction achieved was 98.13% with the highest
treatment tested (500 MPa, 15 min, 2.5% (w/v) Stevia). In this case,
since the enzymeantibrowning agent interaction may alter the
pressure stability of the enzyme, the combination of chemical
and physical browning prevention treatments would enable the
use of less intense inactivation treatments. In view of the results
obtained, Stevia could act as an antibrowning agent, reducing oxi-
dation. Muanda et al. (2011) reported that the higher levels of anti-
oxidant activity in water extracts of Stevia were due to the
presence of phenolic and avonoid components. Bracht, lvarez,
and Bracht (1985) studied the effect of several natural products ex-
tracted from Stevia leaves on rat liver mitochondria. They also
found inhibition of oxidative phosphorylation including ATPase,
NADH-oxidase, succinate-oxidase, succinate dehydrogenase and
glutamate dehydrogenase activity, but to our best knowledge no
information is available about the effect of these natural com-
pounds on PPO and POD inactivation.
TPC is a parameter related with enzymatic oxidation. As previ-
ously reported, PPO and POD are the main enzymes involved in
oxidative reactions of phenolic compounds. Therefore, TPC was
determined in the untreated samples and immediately after apply-
ing HPP in order to determine its relationship with the enzyme
residual activities when Stevia was added at different concentra-
tions and HPP was applied. The concentration of TPC in unpro-
cessed samples without Stevia was 185.5 7.1 mg GAE/L. In the
presence of 1.25% and 2.5% (w/v) of Stevia it was 2261.1 41.7
and 4050.8 42.7 mg GAE/L, respectively. Thus the addition of Ste-
via increased TPC values signicantly (p < 0.05). Moreover, many
researchers have reported high levels of phenolic compounds in
Stevia water extracts. Muanda et al. (2011) obtained 20.85 mg
GAE/g (dry weight) and Kaushik, Narayanan, Vasudevan, Muthu-
kumaran, and Usha (2010) reported a polyphenol concentration
averaging 4.15% by weight of dried Stevia leaf. Although the oxida-
tion rate of polyphenols becomes greater as the pH of the solution
in which they are present increases, as a result of a greater propor-
tion of molecules in the form of phenolate (Singleton, 1987), our
results for TPC in fruit extract (neutral pH) are similar to those ob-
tained by Carbonell-Capella et al. (2013) in a fruit juice containing
mango, orange and papaya (acid pH).
In view of the results obtained (Table 2), only the Stevia concen-
tration had a signicant inuence (p < 0.05) on the TPC of treated
samples. These results are in close agreement with those published
by Barba et al. (2010) who demonstrated that phenolic compounds
were not signicantly affected by pressure and time when HPP was
applied to vegetable beverages. In addition, phenols appeared to be
relatively resistant to HPP, no signicant decrease was found for
any of the pressure/time combination applied. They even increased
after HPP (26% increase after HPP at 400 MPa for 10 min with 1.25%
(w/v) of Stevia). This increase in TPC may be due to improved
extractability of antioxidant compounds as a result of the breaking
down of the cell wall following HPP. In any case, these results are
in good agreement with those found in carrot and spinach (Jung,
Lee, Kim, & Ahn, 2013).
This relative resistance to HPP and even increase in TPC coin-
cides with a decrease in enzyme activities, since PPO and POD
are involved in the degradation of antioxidant compounds, includ-
ing phenolic compounds. Accordingly, in the present research, total
antioxidant capacity of the samples measured with the TEAC and
ORAC methods was also determined before and immediately after
265
applying HPP. Values for the TEAC assay in the untreated samples
with 0%, 1.25% and 2.5% (w/v) were 2.3 0.1, 16.6 0.2, and
18.8 0.1 mM TE, respectively. As can be observed, the Stevia con-
centration had a signicant inuence on the antioxidant capacity
values (p < 0.05). Values for the ORAC assay in the untreated sam-
ples were 4.4 0.2 mM TE, 16.9 0.8 and 28.2 3.7 mM TE when
the Stevia concentration was 0%, 1.25% and 2.5% (w/v). As for
HPP treated samples, only Stevia concentration had a signicant
inuence (p < 0.05) on antioxidant capacity measured with the
ORAC method. As can be seen in Table 2, ORAC values were signif-
icantly higher (p < 0.05) in the various samples in comparison with
the TEAC assay. This can be attributed to the formation and/or
extraction of antioxidant compounds such as polyphenols. Some
previous published studies have demonstrated that the ORAC
method has greater specicity and is capable of responding to a
greater number of antioxidant compounds than the TEAC assay
in fruit complex mixtures (Barba et al., 2013; Zulueta, Esteve, & Fri-
gola, 2009). In the present research, a stronger signicant correla-
tion (p < 0.05) was found between ORAC and TPC (R = 0.986) than
between TEAC and TPC (R = 0.903).
Finally, in food technology, quality and stability are bound up
with safety. Any preservation strategy, even if it involves the joint
application of different methodologies, must guarantee that the
food concerned does not jeopardize consumer health, at least dur-
ing the shelf-life time. Hence, it is important to assess the potential
of the preservation methods considered to inactivate food
pathogens.
In view of the results obtained (Table 2), it can be concluded
that HPP is an appropriate tool to reduce the concentration of L.
monocytogenes in the matrix developed. For a determined pressure,
the microbial inactivation was higher when treatment time was in-
creased, independently of the Stevia concentration used in the
sample formulation (Table 2). In addition, pressures higher than
300 MPa or times higher than 5 min were always able to inactivate
at least 5 log cycles, the standard proposed for any processing
strategy intended to guarantee the safety of fruit juices and similar
products (CFR, 2012), L. monocytogenes being the pertinent patho-
gen. Moreover, for any pressuretime combination, in the presence
of Stevia the number of inactivated log cycles was always greater
than those achieved in its absence (Table 2). For instance, the addi-
tion of 2.5% Stevia increased the inactivation reached after
300 MPa, 5 min, by almost 4 log cycles. This is due to the antimi-
crobial character of Stevia, which has also been reported by other
authors (Tadhani & Subhash, 2006).
3.2. Processing parameter optimization based on their effect on the
safety and quality of the formulated matrix
Generally, the objective of a preservation treatment is to stabi-
lize a product, guaranteeing its microbial safety while producing a
minimum loss of functional and nutritive components. For this rea-
son, the best processing conditions to treat a fruit puree extract
when HPP is combined with the addition of a natural ingredient
with antimicrobial and antioxidant properties were studied by
RSM.
Eqs. (3)(7) show the response function for each of the factors
studied (PPO, POD, TPC, TEAC and ORAC), while the dependence
of the factors on the independent variables (pressure (P), time (t),
Stevia concentration (S)) is shown in Fig. 1.
PPO 46:257  0:086  P corrected R2 0:72 3
PPO 40:842  0:051  P  2:144  S  5:264  S2 corrected R2 0:96 4
TPC 314:389 1655:040  S corrected R2 0:97 5
266 F.J. Barba et al. / Food Chemistry 148 (2014) 261267

intensity and increases the nutritional value (TPC and antioxidant

(a) (b)
capacity) of the product while at the same time avoiding the addi-

POD (% Residual activity)

PPO (% Residual activity)

20 24
tion of sugar to the food formulation.
16 20
16
12
12 4. Conclusions
8
8
4 The results suggested that the combination of HPP with Stevia
4
can be a useful tool in order to obtain safe and shelf-stable fruit-
0 0
300 500 5 15 0.0 2.5
based beverages with enhanced antioxidant and nutritional prop-
300 500 5 15 0.0 2.5
Pressure time Stevia Pressure time Stevia erties. Specically, a treatment of 453 MPa applied for 5 min com-
bined with 2.5% (w/v) Stevia inactivates at least 5 log cycles L.
(c) (d) monocytogenes and maximizes TPC content and antioxidant activ-
(X 1000,0)
5 40 ity while minimizing PPO and POD activities. This combination
leads to an effective enzyme activity decrease impossible to obtain
TPC (mg GAE/L)

TEAC (mM TE)

4 30
just by applying the same high pressure and time conditions.
3
20
HPP is an attractive food preservation technology that clearly
2 offers opportunities for horticultural and food processing indus-
1
10 tries to provide new functional foods of proven physical and nutri-
tional quality, thus increasing product added value, especially if it
0 0
is combined with the addition of Stevia, a natural ingredient that
300 500 5 15 0.0 2.5 300 500 5 15 0.0 2.5
Pressure time Stevia Pressure time Stevia
acts as a low-calorie sweetener and as an antioxidant and antimi-
crobial at the same time.
(e)
50
Acknowledgements
ORAC (mM TE)

40

30 The authors thank the Ministry of Economy and Competitive-

20
10
0 and JAE-Predoc grant, respectively. Francisco Jos Barba received
300 500 5 15 0.0 2.5
Pressure time Stevia
Fig. 1. Effect of Stevia concentration (% (w/v)), and HPP treatment conditions
(pressure (MPa) and time (min)) on enzyme activities (PPO and POD), total phenolic
content (TPC) and antioxidant capacity (TEAC and ORAC) of the samples analysed.
TEAC 2:470 14:636  S  1:497  S2
ORAC 4:433 14:344  S
In view of results obtained, factor time has no signicant effect
on the parameters studied showing that Stevia concentration and
pressure applied had strongest inuence on them. A multilinear
analysis of response surface design using the desirability approach
was used to optimize HPP conditions (pressure and time) and the
Stevia percentage used in the formulation of the samples to maxi-
mize phenolic compounds and antioxidant capacity, minimize PPO
and POD activity and inactivate 5 log cycles of L. monocytogenes.
The desirability function is an approach for solving the problem
of optimizing several responses and it is applied when various re-
sponses have to be considered at the same time. It is rst con-
structed for each individual response, and then it is possible to
obtain the overall desirability. The results obtained showed that
a Stevia concentration of 2.5% (w/v) and 453 MPa applied for
5 min were the conditions that optimized treatment with an over-
all desirability of 0.929. Moreover, according to the results ob-
tained, these conditions ensure the inactivation of 5 log cycles of
L. monocytogenes (Table 2). The response values predicted under
these conditions by the multiple response optimization were
7.3% residual activity for PPO and not-detected for POD, 4452 mg
GAE/L for TPC, 29.7 mM TE for TEAC and 40.3 mM TE for ORAC.
Therefore, the addition of Stevia rebaudiana to the fruit enzyme
extract, considered as a model, allows a decrease in HPP treatment
ness for its support through project number AGL2010-22206-
C02-01-ALI. Mara Nieves Criado and Clara Miracle Belda-Galbis
are especially grateful to the CSIC for providing their JAE-Tec grant
an employment contract from this project to carry out the study.
Finally, the authors also thank the company Anagalide, S. A. (Spain)
for providing them with dried Stevia leaves.
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