LWT - Food Science and Technology 87 (2018) 47e53

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Effect of microwaves and ultrasound on bioactive compounds and

microbiological quality of blackberry juice
Brenda Pe
rez-Grijalva a, Mo
nica Herrera-Sotero b, Rosalva Mora-Escobedo a,
Julio C. Zebadúa-García a, Eryck Silva-Herna
ndez b, Rosa Oliart-Ros c, Claudia Pe

rez-Cruz d,

n-Gero
nimo b, *
Rosa Guzma
a
Escuela Nacional de Ciencias Biolo
gicas, Instituto Polit

pez Mateos”, Campus Zacatenco, Ciudad de M

ecnico Nacional, Unidad Profesional “Adolfo Lo exico,
Mexico
b
sicas, Universidad Veracruzana, Xalapa, Veracruz, Mexico
Instituto de Ciencias Ba
c
Unidad de Investigacio
n y Desarrollo de Alimentos, Instituto Tecnolo
gico de Veracruz, Veracruz, Veracruz, M

exico
d
Departamento de Farmacología, CINVESTAV-IPN, Campus Zacatenco, Ciudad de M
exico, Mexico

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

Article history: Blackberry mash added with citric acid (0e500 mg/kg) was treated with microwaves (0e60 s), and the
Received 24 March 2017 resulting juice was evaluated to determine the effects on bioactive compounds and antioxidant activity
Received in revised form using surface response methodology. This methodology allowed the selection of the parameters for the
20 July 2017
microwave treatment and concentrations of citric acid that yielded the highest content of total poly-
Accepted 19 August 2017
Available online 23 August 2017
phenols, monomeric anthocyanins and antioxidant activity of the juice. Mash samples were sonicated
(40% amplitude, 20 KHz, 10 min) and stored at 5
C þ 1
C for a month. Then the contents of total
polyphenols and monomeric anthocyanins, as well as microbiological quality were analyzed. Analysis of
Keywords:
Blackberry juice
polyphenols was carried out by UPLC-DAD in the raw juice and the sample selected by surface response
Microwaves methodology and treated with ultrasound. The highest levels of polyphenols and monomeric anthocy-
Ultrasound anins in juice were obtained at a time of 60 s of microwave processing and a concentration of 500 mg/kg
Polyphenols citric acid in the blackberry mash. Phenolic acids, flavonols and cyanidin were detected in both samples.
Microbiological quality During storage, juice processed with ultrasound retained more than 90% of monomeric anthocyanins and
showed a significant microbial load reduction, agreeing with the quality parameters established by the
Mexican standards. This study highlights the potential application of microwaves and ultrasound to
increase the functional value of blackberry juice.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Nowadays consumers have a particular interest in beverages
that provide bioactive compounds with healthful effects, such as
anthocyanins, which are polyphenolic compounds with anti-
inflammatory, anti-carcinogenic, and antioxidant properties (Dai
& Mumper, 2010; Zafra-Stone et al., 2007). Berries, used in the
production of beverages, are a good source of these compounds.
However, it has been reported that industrial processing decreases
the content of anthocyanins in the final product (Hager, Howard, &
* Corresponding author. Instituto de Ciencias Ba
sicas, Universidad Veracruzana,


Av. Dr. Luis Castelazo Ayala S/N, Col. Industrial Animas, 91190, Xalapa, Veracruz,
Me
xico.
E-mail address: roguzman@uv.mx (R. Guzma
n-Gero
nimo).
Prior, 2008; Howard, Prior, Liyanage, & Lay, 2012). Since anthocy-
anins have so many beneficial effects on human health, it is relevant
to explore the application of alternative technologies in juices
prepared from anthocyanin-rich fruits e like blackberry e to in-
crease their content of natural antioxidants and functional value.
An important step in the industrial preparation of fruit juices is
blanching. Previous reports suggest that microwave blanching may
be used to obtain fruit juice with a high quantity of polyphenolic
compounds (Gerard & Roberts, 2004), since microwaves produce a
quick and uniform heating, minimizing changes in flavonoids such
as anthocyanins (De Ancos, Cano, Hernandez, & Monreal, 1999).
On the other hand, it is well known that the traditional process
for juice production induces changes in anthocyanins and increases
polymerized compounds in berry juices (Brownmiller, Howard, &
Prior, 2008; Li, Walker, & Faubion, 2011). It has been reported

http://dx.doi.org/10.1016/j.lwt.2017.08.059
0023-6438/© 2017 Elsevier Ltd. All rights reserved.
48 B. P
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that low pH values and temperature decrease anthocyanin poly-
merization reactions during storage. Acidic conditions keep an-
thocyanins in the form of red flavylium cation, which plays an
important role in the antioxidant capacity and bioavailability of
anthocyanins (Lapidot, Harel, Akiri, Granit, & Kanner, 1999;
Pacheco-Palencia, Mertens-Talcott, & Talcott, 2010; Van Acker
et al., 1996). In addition, organic acids such as citric acid favor the
presence of monomeric anthocyanins and decrease polymerization
(Li et al., 2011; Nicoue
, Savard, & Belkacemi, 2007).
Another important step involved in the food processing is
pasteurization, a thermal treatment widely used in juice industry to
obtain a safe product from the microbiological point of view.
However, anthocyanins and many bioactive compounds are
degraded by traditional pasteurization processes. Several authors
report that ultrasound is an alternative technology to traditional
thermal treatments in the reduction of microbial load (Aadil, Zeng,
Han, & Sun, 2013) and to minimize changes in bioactive compounds
such as anthocyanins (Tiwari, O'Donnell, Patras, Brunton, & Cullen,
2009). Give the above, a combination of microwaves and ultra-
sound represents a promising new technology to improve the
extraction of polyphenols during the blanching of fruit mash with
microwaves, while ultrasound processing could be applied to the
resulting juice for microbial inactivation, as well as to maintain the
functional value over the shelf life of the product. Therefore, the
aim of the present study was to evaluate the combined effect of
microwaves and ultrasound to enhance the functional value of
blackberry juice. The first part of this research was to evaluate the
combined effect of microwave processing and the addition of citric
acid of blackberry mash on the content of polyphenols, anthocya-
nins, color density, percent polymeric color and antioxidant ca-
pacity of blackberry juice, using an experimental design based on
surface response methodology. The second part focused in studying
the effect of ultrasound on the chemical composition and micro-
biological quality of blackberry juice during storage in refrigeration,
as compared to the initial conditions analyzed before microwave
blanching and citric acid addition.
2. Materials and methods
2.1. Material
Blackberries (Rubus americanus (Pers.) Britton) at ripe stage
were obtained from a local market in Xalapa, State of Veracruz,
Mexico. Frozen fruits were placed in plastic trays and stored
at 20
C.
2.2. Microwaves processing
In the present study, microwaves were used to blanch black-
berry mash. Blackberries were thawed at 20
C and crushed using a
mortar. The resulting blackberry mash (50 g) was placed on petri
dishes (10 cm diameter x 1 cm height) and heated in a microwave
oven (Panasonic, 2450 MHz, 453 W). Microwave energy (E) was
calculated for each sample according to the Buffler method (Buffler,
1993, pp. 157e159) using equation E ¼ Wt/m, where W is micro-
wave oven power; t is time of microwave exposure, and
m ¼ quantity of sample (Ortiz, Dorantes, Galindez, Guzma
n, 2003).
Immediately after the thermal treatment, the surface temperature
of each sample was recorded with a Rayinger ST non-contact
thermometer. After heating, the samples were cooled in ice water
until the temperature dropped to 20
C ± 1
C. The juice was ob-
tained by centrifugation.
2.3. Response surface methodology
Response surface methodology was applied to establish the ef-
fect of microwave time (s) and citric acid (mg/kg) on the poly-
phenols content (gallic acid equivalent, mg/L), monomeric
anthocyanins (cyanidin-3-glucoside equivalent, mg/L), color den-
sity, percent polymeric color and antioxidant capacity by DPPH and
FRAP assay (mM trolox equivalent) of blackberry mash. The levels
of independent variables microwave (0e60 s) and citric acid
(0e500 mg/kg) were selected according to preliminary experi-
ments. A central composite design of 13 runs with five replicate
runs at center point was performed. The statistical data analysis
and response surface plots were carried out using a Design Expert
statistical package 10.0. Experimental values were fitted to a
second-order polynomial model and regression coefficients were
obtained.
2.4. Ultrasound processing
The blackberry juice sample with the highest content of total
polyphenols and anthocyanins, as showed by the surface response
analysis, was selected for the ultrasound treatment. 80 mL of this
juice were processed in an ultrasonic homogenizer at frequency of
20 KHz and power of 750 W (Cole-Palmer Instrumental Company,
VCX-750, USA) with a 13 mm diameter probe, operating at ampli-
tude of 32 mm with 5 s on and 5 off pulse during a time of 10 min.
The probe was submerged 25 mm deep into the juice. The juice was
placed into a water bath at 20
C to control temperature. Then, the
juice was stored at 5
C ± 1
C during 30 days in an amber glass
container. The ultrasound conditions for the present study were
selected according to previous reports dealing with the application
of ultrasound in berry juices (Tiwari, O’Donnell, et al., 2009; Tiwari,
Cullen, & O'Donnell, 2009).
2.5. Total polyphenols
Total polyphenols content was estimated by the Folin-Cicalteu
assay as described by Singleton and Rossi (1965). The juice was
mixed with 1N Folin-Ciocalteau reagent and then saturated sodium
carbonate solution and water were added to the mixture. After
60 min, the absorbance was measured at 760 nm. A calibration
curve was performed with gallic acid. The concentration of total
phenolics was expressed as mg of gallic acid equivalent per liter of
juice (mg GAE/L). All samples were prepared by triplicate.
2.6. Monomeric anthocyanins, color density and percent polymeric
color analysis
The content of monomeric anthocyanins was evaluated using
the pH differential method (Giusti & Wrolstad, 2001). The absor-
bance was measured in a lambda 35 UV-VIS spectrophotometer
(Perkin Elmer, Inc. Shelton, CT, USA) at 515 and 700 nm. Juice
samples were dissolved with 0.025 M potassium chloride buffer, pH
1.0 and 0.4 M sodium acetate buffer, pH 4.5. Then the anthocyanins
content was expressed as mg of cyanidin-3-glucoside equivalent
per liter of juice (mg C3GE/L), using the extinction coefficient of
26,900 L x cm1 x mol1, and a molecular weight of 449.2 g/mol.
For the percent polymeric color and color density analysis, 0.2 mL of
0.90 M potassium metabisulfite was added to 2.8 mL of diluted
sample, and 0.2 mL of distilled water was added to 2.8 mL diluted
sample (control). Samples were recorded at 420 nm, 515 nm, and
700 nm.
 B. P
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Color density was calculated as follows:
Color density ¼ [(A420nm e A700nm) þ (A515nm e A700nm)]  DF(dilutionfactor)
Percent polymeric color was calculated using the following
equations:
Polymeric color ¼ [(A420 nm e A700 nm) þ (A515nm e A700nm)]  DF(2)
Percent polymeric color ¼ (polymeric color/color density)  100(3)
2.7. Antioxidant capacity
2.7.1. DPPH assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) method was used to
determine antioxidant activity (Brand-Williams, Cuvelier, & Berset,
1995). The DPPH reagent was prepared by placing 3.9 mg DPPH into
a beaker and taking the volume up to 100 mL with 80% methanol;
0.1 mL of juice was added with 2.9 mL of this DPPH solution and the
absorbance was recorded at 571 nm after 30 min. Quantification
was performed using a trolox standard curve (0.1e0.8 mM). Results
were expressed as mM trolox equivalent (TE).
2.7.2. FRAP assay
Analysis by Ferric Reducing Antioxidant Power (FRAP) was
carried out following the method proposed by Benzie & Strain
(1996). A juice sample (0.1 mL) was mixed with 3 mL of FRAP re-
agent. Then, the mix was incubated for 4 min at room temperature,
and the absorbance at 593 nm was recorded. Results were
expressed in mM TE, using a standard curve (0.1e0.8 mM) (Benzie
& Strain, 1996; Giusti & Wrolstad, 2001).
2.8. Polyphenols extraction and UPLC-DAD analysis
Several authors were taken as reference (Cuevas Rodríguez
et al., 2010; Fan-Chiang & Wrolstad, 2005; Zou et al., 2012). Poly-
phenols extraction was performed from 1 g of lyophilized juice. The
samples were mixed with methanol/water/acetone and then kept
at 4
C for 2 h. Later, they were centrifuged at 12,000 rpm for 15 min
to 4
C. The supernatant was concentrated by rotary evaporation at
40
C to remove methanol. Then, anthocyanins were hydrolyzed
with 2N HCl (1:2, v:v) during 45 min at 100
C (Giusti, Rodriguez-
Saona, Griffin, & Wrolstad, 1999). The concentrate was partitioned
with ethyl acetate (1:1, v/v). This extract was analyzed by UPLC-
DAD. Analysis was performed in the 1100 UPLC (Agilent technolo-
gies Inc., Santa Clara, CA.) chromatograph coupled to diode array
detector and a reversed-phase column (ACQUITY UPLC BEH C-18,
1.7 mm, 2.1 mm  50 mm). The mobile phase was composed of
eluent A (2% acetic acid in water, v/v) and eluent B (water:-
acetonitrile:acetic acid 68:30:2, v:v:v) using a flow rate of 0.2 mL/
min, and injection volume of 10 mL. The gradient program was done
according to Fang, Li, Pan, & Huang, 2007. Anthocyanins were
determinate at 510 nm, while phenolic compounds at 280, 325 and
360 nm. The analysis was done by triplicate.
2.9. Microbiological analysis
Mold and yeast analysis were carried out by the plate count
49
method, as established by the Official Mexican Standards (NOM-
(1)
111-SSA1-1994) for food products destined for human consump-
tion. This method is based on the inoculation of a known quantity
of sample into a specific selective medium (Potato dextrose agar),
acidified at pH 3.5 and incubated at a temperature of 25 ± 1
C and
examined for growth after 2e7 days. This results in the growth of
colonies that are specific for the microorganism in question. The
colonies were counted by multiplying the dilution factor, to obtain
the total plate count (CFU/mL).
The analysis for aerobic mesophiles was carried out according to
the method described in the Mexican Standards (NOM-092-SSA1-
1994). The analysis for aerobic mesophiles was carried out ac-
cording to the method described in the Mexican Standards (NOM-
092-SSA1-1994). Several dilutions were prepared and stored in
petri dishes with triptone-yeast extract agar (standard count agar).
Then the samples were incubated at 35 ± 1
C for 48 h. Count of
colonies was done by multiplying the dilution factor to obtain total
plate count.
2.10. Statistical analysis
Response surface data obtained were analyzed using stepwise
multiple regression procedures with the help of the software
Design Expert 7.0. Correlation analyses of the data were performed
using XLSTAT 7.0.
3. Results and discussion
3.1. Total polyphenols, monomeric anthocyanins, percent polymeric
color and color density
The effect of microwave time and citric acid content on
dependent variables is shown in Table 1. The content of total
polyphenols and monomeric anthocyanins for untreated black-
berry juice were of 1610 mg GAE/L and 290 mg C3GE/L, respectively.
Table 2 summarizes the results of each dependent variable with
their determination coefficients (R2 > 0.80). The statistical analysis
indicates that a linear model (p < 0.001) fitted well the total
polyphenols data. The linear model (p < 0.001) was also adequate
for monomeric anthocyanins, percent polymeric color and color
density. Total polyphenols, monomeric anthocyanin and percent
polymeric color in blackberry juice showed a positive effect
(p < 0.05) for the linear terms heating time (A) and citric acid (B);
whereas color density was significantly affected by the linear term
heating time.
Juice samples obtained from the microwaved blackberry mash
showed higher levels of total polyphenols as compared to the
control sample (without treatment) (Fig. 1a). Our results agree with
previous research by other authors, who reported that microwave
blanching had a positive effect on the content of polyphenols when
apple mash was microwaved prior to juice extraction (Gerard &
Roberts, 2004).
In addition, in the present study a significant increase in
monomeric anthocyanins was observed in the juice when mashed
blackberries were heated with microwaves for 60 s (Fig. 1b). Pre-
vious studies reported that microwaves permitted an efficient and
rapid extraction of anthocyanins from red raspberry (Sun, Liao,
50 B. P
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Table 1
Experimental design in term of experimental variables (independent variables) and the observed response values (dependent variables).

No. Independent variables Dependent variables

Microwave treatments Citric acid content Total polyphenols Monomeric Percent polymeric color Color density DPPH FRAP
mg/kg mg GAE/L Anthocyanin C3GE mg/L mM TE mM TE

1 0 0 1610 290 7.36 5.08 11.2 16.7

2 60 0 2702 707 3.33 7.34 9.00 23.8
3 0 500 2000 418 6.34 5.72 10.6 18.6
4 60 500 2837 833 3.05 7.96 13.0 25.6
5 0 250 1862 476 5.6 6.24 10.4 18.7
6 72 250 2683 692 3.26 7.4 12.5 23.7
7 30 0 1970 495 5.61 6.56 9.20 17.7
8 30 603 2187 595 3.97 6.89 10.2 21.6
9 30 250 2165 538 4.4 6.59 9.30 20.3
10 30 250 2164 557 4.55 6.57 9.43 20.4
11 30 250 2162 550 4.49 6.67 9.59 20.3
12 30 250 2183 563 4.22 6.61 9.36 20.3
13 30 250 2159 534 4.59 6.54 9.55 20.2

Table 2
Regression coefficients of the response surface models for polyphenols, monomeric anthocyanins, percent polymeric color, color density, antioxidant activity by DPPH and
FRAP.

Factora Total Monomeric anthocyanin Percent polymeric Color density DPPH FRAP
Polyphenols C3GE mg/L color mM TE mM TE
mg GAE/L

Intercept 1716 385 6.45 5.64 11.9 16.8

Linear
A-Heating time 12.8** 4.7** 0.04** 0.02** 0.09 0.08**
B-Citric acid 0.41* 0.19* 1.80* 8.63 3.81** 4.62**
Quadratic
A2 e e e e 1.22 e
B2 e e e e 3.39 e
Interactions
AB e e e e 1.50 e
R2 0.88 0.82 0.83 0.80 0.91 0.86
F value 37.2 23.2 24.3 20.3 13.6 30.1
Prob > F 0.00 0.00 0.00 0.00 0.00 0.00

*Significant at < 0.05.**Significant at < 0.001.

a
Experimental values.

Wang, Hu, & Cheng, 2007). It seems that microwaves cause
expansion with subsequent rupture of the cell walls leading to a
better extraction of antioxidant compounds.
Also, a positive effect on the content of monomeric anthocya-
nins in blackberry juice was observed at higher concentrations of
citric acid (Fig. 1b). In the same way, it has been reported that the
addition of this organic acid increased the retention of a higher
level of anthocyanins in blue corn cookies (Li et al., 2011). This may
be due to a decrease in the degradation of anthocyanins caused by
citric acid (Dao, Takeoka, Edwards, & Berrios, 1998).
On the other hand, previous reports indicate that anthocyanin
concentration has a positive effect in color stability. In this research,
when blackberry mash was treated at longer heating times it
showed the maximum values of color density (data not shown). In
the same way, De Ancos et al. (1999) reported an enhancement of
color density in strawberry puree processed with microwaves.
Regarding percent polymeric color, it decreased at longer
heating times and lower citric acid values (Fig. 1c). Similar results
were found by Price and Wrolstad (1995), who showed that mi-
crowaves blanching of Royal Okanogan Huckleberry fruit resulted
in a significant decrease of the polymeric color of the juice. In
addition, studies performed in red raspberry extracts show that
microwaves do not affect the anthocyanin profile and no degra-
dation of these compounds was observed (Sun, Liao, Wang, Hu, &
Cheng, 2007). Losses of monomeric anthocyanins and a raise in
polymeric anthocyanins have been reported for blackberry
products, juices in particular. Some authors report that the con-
ventional thermal treatment of blackberry juice produces a
decrease of these compounds (Hager et al., 2008). Previous studies
suggest that polymeric anthocyanins are less bioavailable due to its
high molecular weight. Hence the importance of minimize the
formation of these compounds in blackberry juice.
Therefore, a daily glass of blackberry juice (250 mL) obtained by
microwaving the mash for 60 s and adding 500 mg kg1 of citric
acid provides 709 mg and 208 mg of total polyphenols and
monomeric anthocyanins, respectively. If we consider the total
polyphenols and anthocyanins recommended intake to be 1 g/day
and 2.5 mg/kg/day (Clifford, 2000; Scalbert & Williamson, 2000),
respectively, one serving (250 mL) of the experimental blackberry
juice accounts for 70.9% and 118% of the daily polyphenols and
anthocyanins recommendation. In contrast, a daily glass of control
juice provides with only 25.7% and 39.8% of the total recommended
amounts of polyphenols and anthocyanins, respectively.
3.2. Antioxidant capacity by DPPH and FRAP assay
Several methods have been developed to evaluate the antioxi-
dant capacity in vitro, such as the free radical scavenging capacity
(DPPH) and the ferric reducing antioxidant power (FRAP). Both
antioxidant assays are fast, inexpensive, simple and reproducible.
Antioxidant capacity evaluated by DPPH and FRAP assay of fresh
blackberry juice was 11.2 and 16.7 mM TE, respectively. The
 B. P
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Fig. 1. Response surfaces of total polyphenols, monomeric anthocyanins, percent polymeric color and antioxidant activity by FRAP assay of blackberry juice processed with mi-
crowaves. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

statistical analysis of the experimental design shows that the 1998).

antioxidant activity of the blackberry juice evaluated by DPPH assay
was significantly influenced (p < 0.05) by the citric acid concen-
tration (linear and quadratic term) as well as the interaction term
AB (Table 2). The antioxidant activity of blackberry juice, as deter-
mined by the FRAP assay, showed a positive effect by the linear
term of heating time and citric acid; in other words, the FRAP values
increased with longer microwave treatment times and citric acid
concentrations (Fig. 1d). The enhancement of antioxidant activity
could be attributed to a better extraction of antioxidant compounds
such as monomeric anthocyanins in microwaved blackberry mash.
Other authors have also reported an increase of antioxidant ca-
pacity in berry products after processing with alternative technol-
ogies, which could be due to a better extractability of polyphenolic
compounds (Patras, Brunton, Da Pieve, & Butler, 2009). It is well
known that very high rates of microwaves lead to rapid energy
transfers and high rates of heating. Therefore, compounds present
in foods such as anthocyanins that have additional beneficial effects
on human health can be better preserved.
On the other hand, a negative correlation was found between
percent polymeric color and color density. Other authors have also
reported an inverse correlation between these parameters
(Cevallos-Casals & Cisneros-Zevallos, 2004; Tsai, Huang, & Huang,
2004). In the same way, the total polyphenols and monomeric
anthocyanins showed a positive correlation with antioxidant ca-
pacity, as evaluated by the FRAP assay. Previous studies reported
that antioxidant activity of blackberry is highly correlated with
anthocyanin content (Elisia, Hu, Popovich, & Kitts, 2007).
3.4. Identification of polyphenos in juice
Polyphenols in blackberry extract were identified by UPLC-DAD.
The identification was carried out by comparison of retention time
(Table 4) with the standards. In both juices caffeic, ferullic, gallic
and p-cumaric acids were identified, as well as flavonols like
quercetin and catechin, and anthocyanidins like cyanidin and

Table 3
3.3. Correlation of total polyphenols, monomeric anthocyanins,
Phenolic compounds identified by UPLC-DAD in unprocessed blackberry juice
color density, percent polymeric color and antioxidant capacity (control juice) and processed with microwaves and ultrasound (MUJ).

Retention time lmax Compound Juice control MUJ

It has been reported that the conventional processing of fruit
min nm
juice leads to losses of anthocyanin content and color. Results in
3.14 280 Catequin þ þ
Table 3 show that total polyphenols and monomeric anthocyanins
8.07 325 Pherulic acid þ þ
are positively correlated to color density, with coefficients of 0.94 0.07 280 Gallic acid þ þ
and 0.99, respectively. This agrees with Rommel, Wrolstad, and 4.13 325 Caffeic acid þ þ
Heatherbell (1992), and suggests a positive contribution of antho- 9.13 510 Cyanidin þ þ
cyanins in the color of the product. The increase in stability can be 9.79 510 Pelargonidin þ þ
12.3 360 Quercetin þ þ
attributed to self-association (Dao, Takeoka, Edwards, & Berrios,
52 B. P
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Table 4
Pearson correlations between total polyphenols, monomeric anthocyanins, percent polymeric color, color density and antioxidant activity of blackberry juice obtained prior to
the microwave treatment and addition of citric acid to the mash.

Total polyphenols Monomeric anthocyanins Percent polymeric color Color density DPPH FRAP

Total polyphenols 1
Monomeric anthocyanins 0.962 1
Percent polymeric color 0.983 0.981 1
Color density 0.948 0.993 0.964 1
DPPH 0.973 0.967 0.994 0.946 1
FRAP 0.989 0.958 0.989 0.932 0.987 1

pelargonidin. This pattern was consistent with previous reports on
blackberry (Mertz, Cheynier, Ga
nata, & Brat, 2007; Cuevas-
Rodriguez et al., 2010). In addition, cyanidin-3-glucoside exhibi-
ted the highest antioxidant activity among the 14 anthocyanins of
blackberry tested by Elisia et al. (2007). It is important to say that
cyanidin-3-glucoside has showed several biological properties such
as hypoglycemic effect in diabetic mice and anti-obesity effect in an
animal model of metabolic syndrome (Sun et al., 2012; Tsuda,
2008).
3.5. Juice stability
Regarding stability of blackberry juice, currently our research
group is carrying out studies on microbiological safety and physi-
cochemical quality after the application of microwaves and ultra-
sound. These studies suggest that with the application of these
technologies it is possible to obtain a stable product with a good
physicochemical stability that is shelf-stable for a month. At the
end of storage, retention of total polyphenols and monomeric an-
thocyanins in the blackberry juice was 87 y 90%, respectively
(Table 5). Similar data were found by Tiwari, Cullen, et al. (2009). In
contrast, previous studies reported that the conventional treatment
of blackberry fruit resulted in loss of antioxidant activity in clarified
non-pasteurized blackberry juice (Hager et al., 2008).
On the other hand, ultrasound treatment caused a significant
reduction of molds, yeasts and aerobic mesophiles in blackberry
juice (pH ¼ 3.1) as compared to the control (Table 6). At day 7, the
control sample had an uncountable content of mesophiles and
molds, making it unsuitable for human consumption; instead, the
experimental juice showed a microbial count acceptable for con-
sumption. These results suggest that ultrasound power can be used
for inactivation of microorganisms in blackberry juice. It could be
attributed to the mechanical or/and physical effects of ultrasound
in microorganisms, since the propagation of power ultrasound in a
liquid induces bubble cavitation due to pressure changes (Tiwari,
O’Donnell, et al., 2009), these micro-bubbles collapse violently
and shock waves radiated by collapsing bubbles could be strong
enough to shear and break cell walls and membrane structures
(Earnshaw, Appleyard, & Hurst, 1995). Also, electric discharges and
production of free radicals and electronically excited species asso-
ciated with the collapse of cavities could also cause chemical and
biological effects. Previous reports suggested that ultrasound can
be used as an alternative technology for pasteurization of fruit
juices (Valero et al., 2007; Yuan, Hu, Yue, Chen, & Lo, 2009; Tiwari,
Cullen, et al., 2009).
4. Conclusion
Microwave blanching may be used in blackberry mash prior to
juice extraction in order to increase the content of antioxidant
compounds, such as polyphenols, in the final product. Ultrasound is
a technology with potential to reduce the microbial load in black-
berry juice and to preserve bioactive compounds during refriger-
ated storage. In summary, microwaves in combination with
ultrasound technologies present an alternative in the processing of
fruit juices with high content of antioxidant compounds and good
microbial quality, as demanded by consumers and industry.

Table 5 Acknowledgements
Total polyphenols and monomeric anthocyanins retention percentage during stor-
age of the blackberry juice processed with microwaves and ultrasound (MUJ).
rez-Grijalva is grateful to the graduate program in
Brenda Pe

n (%)
Retentio Days Food Science from ENCB-IPN and CONACyT by the scholarship
0 7 15 22 30 granted. This project was supported by PROMEP (103-5/12/464)
and CONACyT, Me
xico (SEP- CONACyT 2014-242860).
Total polyphenols 100 97 93 90 87
Monomeric anthocyanins 100 99 98 97 90
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