Food Chemistry 230 (2017) 257–264

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

Effect of temperature and relative humidity on stability following

simulated gastro-intestinal digestion of microcapsules of Bordo grape
skin phenolic extract produced with different carrier agents
Luiza Siede Kuck, Júlia Lerina Wesolowski, Caciano Pelayo Zapata Noreña ⇑
Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, CEP 91501-970 Porto Alegre, RS, Brazil

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

Article history: The stability of microparticles of Bordo grape skin aqueous extract, produced by spray-drying and freeze-
Received 4 October 2016 drying using polydextrose (5%) and partially hydrolyzed guar gum (5%), was evaluated under accelerated
Received in revised form 16 February 2017 conditions (75 and 90% relative humidity, at 35, 45, and 55 °C for 35 days) and simulated gastrointestinal
Accepted 8 March 2017
digestion. The temperature had a significant effect on the reduction of phenolics content, with retentions
Available online 9 March 2017
varying from 82.5 to 93.5%. The retention of total monomer anthocyanins were in the range of 3.9–42.3%.
The antioxidant activity had a final retention of 38.5–59.5%. In the simulated gastrointestinal digestion, a
Keywords:
maximum release was observed for the phenolic compounds in the intestinal phase (90.6% for the spray-
Spray-drying
Freeze-drying
dried powder and 94.9% for the freeze-dried powder), as well as the antioxidant activity (69.4% for the
Anthocyanins spray-dried powder and 67.8% for the freeze-dried powder). However, a reduction of monomeric antho-
Antioxidant activity cyanins was observed in the intestinal phase.
Simulated gastrointestinal digestion Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction Although various types of encapsulating agents can be used in

Bordo grape is one of the varieties of Vitis labrusca L. most
important and most cultivated in Brazil and is characterized
mainly by its intense red-purple colour, a result of its high content
of polyphenols, especially anthocyanins (Lago-Vanzela, Da-Silva,
Gomes, García-Romero, & Hermosín-Gutiérrez, 2011). Phenolic
compounds have antioxidant activity, whose function is to neutral-
ize and prevent the formation of free radicals (Rice-Evans, Miller, &
Paganga, 1997), resulting in health benefits, such as cardioprotec-
tive capacity, anti-cancer, anti-diabetes, anti-microbial and anti-
inflammatory properties, besides acting against neurodegenerative
diseases, kidney disease and aging (Rodrigo, Miranda, & Vergara,
2011).
However, polyphenols are unstable in food matrices, under var-
ious conditions of processing and storage. Microencapsulation
aims to increase the shelf life of products, creating a barrier
between the encapsulated components and the environment
(Jiménez-Aguilar et al., 2011). There are several microencapsula-
tion techniques, including drying methods, such as spray-drying
and freeze-drying.
⇑ Corresponding author.
E-mail address: czapatan@ufrgs.br (C.P.Z. Noreña).
the microencapsulation processes, some characteristics should be
observed, including their ability to form films, biodegradability,
resistance to the gastrointestinal tract, viscosity, solids content,
hygroscopicity and cost (Silva, Stringheta, Teófilo, & Oliveira,
2013).
The partially hydrolyzed guar gum and polydextrose are highly
water soluble carbohydrates with low viscosity, prebiotic, being
potential encapsulating agents, little studied in the encapsulation
of food compounds.
Although the estimation of stability and shelf life is an impor-
tant aspect of using pigments as colouring agents and antioxidants
in food, products with long shelf life impair the experimental stud-
ies. Thus, accelerated tests can be applied, in which the product is
subjected to extreme conditions of storage, using one or more
acceleration factors (such as temperature and relative humidity)
at higher levels, so that the degradation rate is increased, resulting
in a shorter shelf life, thereby facilitating the stability experiments
(Richards, De Kock, & Buys, 2010).
It is also important to understand the role of the digestion pro-
cess, on the stability and bioavailability of these compounds, since
the polyphenols are ingested in the diet in the form of complex
dispersions immersed in a food matrix (Bermúdez-Soto, Tomás-
Barberán, & García-Conesa, 2007). Thus, in vitro tests can be
performed to verify the stability of a product under simulated
gastrointestinal conditions. The bioavailability of a compound is

http://dx.doi.org/10.1016/j.foodchem.2017.03.038
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
258 L.S. Kuck et al. / Food Chemistry 230 (2017) 257–264
related to its digestive stability, food matrix release, and efficiency
of its transepithelial passage (Tagliazucchi, Verzelloni, Bertolini, &
Conte, 2010).
The objective of this study was to evaluate the stability of
microparticles of Bordo grape skin aqueous extract obtained by
spray-drying and freeze-drying, under accelerated storage condi-
tions and simulated conditions of the gastrointestinal digestion,
using polydextrose and partially hydrolyzed guar gum as wall
materials.
2. Material and methods
2.1. General
The grapes were obtained directly from the producer in the city
of Cotiporã, Rio Grande do Sul, Brazil. The grape clusters were
selected, washed in water, packed in polyethylene bags and frozen
at
18 °C until use. The encapsulating agents were: polydextrose
(MasterSense Ing. Alim. Ltda., Brazil), partially hydrolyzed guar
gum (R & S Blumos, Com. Prod. Alimentícios Ltda., Brazil). The
ABTS (2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)),
Trolox (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic
acid), gallic acid, mucin, a-amylase, pepsin, pancreatin, lipase,
and bile salts were purchased from Sigma-Aldrich. All reagents
were analytical grade.
2.2. Preparation of microparticles
The grapes were thawed and blanched at 80 °C for 5 min and
cooled on ice/water bath for 3 min. After separating the seeds from
the pulp, the polyphenols were extracted from skins using water
acidified with citric acid (2%, w/v). Peels were mixed with a solvent
at a ratio of 1:3 (w/v), the mixture was ground in a blender and
kept in the dark for 20 h at room temperature. After this period,
the extract was filtered through Whatman filter paper No. 1 for
separating the solid residue. According to a previous study (Kuck
& Noreña, 2016), the dispersion was prepared using the extract
with the addition of 5% (w/v) of polydextrose and 5% (w/v) par-
tially hydrolyzed guar gum. Thereafter, dispersions were encapsu-
lated by spray-drying and freeze-drying.
The spray-drying was performed using a dual-fluid atomizer,
pneumatic type (Mini Spray Dryer LM MSDI 1.0 Labmaq, Brazil),
with a feed nozzle diameter of 1.0 mm. Feeding was carried out
using a peristaltic pump at a flow rate of 0.60 L h
1, drying air tem-
perature 140 °C, air pressure 3.5 kgf cm
2, and air flow rate
40.5 L h
1.
For freeze-drying, the dispersion was previously frozen in ultra-
freezer (Liotop UFR 30, Liobras, Brazil) at
68 °C for 24 h. Then, the
samples were placed in a freeze-dryer (Liotop L101, Liobras, Brazil)
and freeze-dried at
57 °C, at vacuum pressure less than
200 mmHg for 48 h. After freeze-drying, the samples were crushed
using a pestle and mortar.
2.3. Chemical determinations
The total phenolics were determined using the Folin-Ciocalteu
reagent (Singleton & Rossi, 1965). The readings were made in a
spectrophotometer (Genesys S10, Thermo Scientific) at 765 nm,
and the results were expressed as mg gallic acid equivalents
(GAE) per g sample on a dry basis.
The total monomeric anthocyanins (TMAC) were determined
according to the methodology described by Lee, Durst, and
Wrolstad (2005). The extract and the diluted powders were mixed
with buffer solutions pH 1.0 and 4.5, and the absorbance was mea-
sured in a spectrophotometer at 520 and 700 nm. The results were
expressed as mg malvidin-3,5-diglucoside per g sample on a dry
basis, using molar absorptivity 37,000 L cm
1mol
1, and molecular
weight 724.5 g mol
1.
The antioxidant activity was determined by ABTS assay accord-
ing to Re et al. (1999) with adaptations. For that, 1 ml ABTS stock
solution (7 mM) and 88 ll potassium persulfate (140 mM) were
mixed 16 h before analysis. ABTS solution was diluted in ethyl
alcohol and its absorbance was adjusted between 0.680 and
0.720. To determine the antioxidant activity, 30 ll of sample was
mixed with 3 ml of ABTS solution, and readings were performed
in a spectrophotometer at 734 nm after 6 min of reaction. The
results were expressed as lmol Trolox equivalents (TE) per g sam-
ple on dry basis.
All determinations were performed in triplicate.
2.4. Accelerated tests of storage stability
To evaluate the stability of the microparticles, 0.5 g of each
powder was placed into 2 ml cryogenic tubes covered with perfo-
rated Parafilm. The conditions used in the study were 75 and 90%
relative humidity at 35, 45, and 55 °C.
The relative humidity conditions were reproduced by
conditioning the samples in desiccators containing saturated
solutions of sodium chloride (75%) and barium chloride (90%).
The desiccators were placed in an incubator (411/FDP Ethik
Technology) at constant temperature (35, 45 and 55 °C) for
35 days. All experiments were performed in duplicate. The
analysis of total phenolics, total monomeric anthocyanins,
and antioxidant activity by ABTS assay were performed every
7 days.
2.4.1. Determination of kinetic parameters
To adjust the experimental data of the total monomeric antho-
cyanins during storage, the first order kinetic model was used, as
shown in the Eq. (1):
C ¼ C0 expð
ktÞ ð1Þ
where: C is the residual anthocyanins content at time t, C0 the initial
anthocyanins content, k is the rate constant (days
1) and t is time
(days).
The decimal reduction time (D) and half-lives (t1/2), indicating
the time required for 90 and 50% degradation, respectively, were
calculated according to the Eqs. (2) and (3):
lnð10Þ
D¼ ð2Þ
k
lnð2Þ
t1=2 ¼ ð3Þ
k
From the k values, the Arrhenius equation (4) was used to
describe the temperature dependence of the rate constant and to
estimate the activation energy (Ea)
 
Ea
k ¼ Aexp
ð4Þ
RT
where: A is the Arrhenius constant (days
1), Ea is the activation
energy (kJ mol
1), R is the universal gas constant (8.314 kJ kmol
1
K
1) and T is temperature (K).
The activation energy was calculated using the Eq. (5):
Ea
lnðKÞ ¼ lnðAÞ
ð5Þ
RT
The thermal resistance coefficient (z), which is the temperature
change required to change the D value by one logarithmic cycle,
 L.S. Kuck et al. / Food Chemistry 230 (2017) 257–264
was obtained by plotting the D values on a logarithmic scale versus
the corresponding temperature.
The Q10 temperature coefficient is a measure of the rate of
change of a chemical reaction as a consequence of increasing
the temperature by 10 °C, and was calculated according to the
Eq. (6):
 
kTþ10
Q 10 ¼ ð6Þ
kT
2.4.2. Determination of thermodynamic parameters
The values of the degradation constants and activation energy
were used to determined the thermodynamic parameters: Gibbs
free energy (ΔG–), enthalpy (ΔH–) and entropy (ΔS–), according
to the Eqs. (7–9):
 
k  hp
DG– ¼ R  T  ln
KB  T
DH – ¼ E a
R  T
 – 
DH
DG –
D S– ¼
T
where: KB is the Boltzmann constant (1.3806  1023 J K
1), and hp is
the Planck’s constant (6.6262  10
34 J s).
2.5. Simulation of gastrointestinal digestion
The simulation of gastrointestinal digestion (mouth, stomach
and small intestine) of the microparticles was carried out accord-
ing to Flores, Singh, Kerr, Pegg, and Kong (2014), with some
adjustments. The digestive juices (saliva, gastric, duodenal and
bile) were prepared on the day of the experiment. For the simu-
lated GI conditions, 3.5 g powder were placed in Erlenmeyer
flasks of 125 ml and incubated in shaking bath (Dubnoff
CT-232) at 37 °C at 60 rpm. To simulate digestion in the mouth,
6 ml of artificial saliva solution was added, and the mixture was
stirred for 5 min. Then, 12 ml of gastric juice was added and
the samples remained stirring for 2 h. Finally, 12 ml of duodenal
juice and 6 ml of bile juice were added to the Erlenmeyer, which
was kept under stirring for 2 h. An aliquot of 1.5 ml was removed
every hour, for 4 h. The samples were centrifuged at 6238g for
5 min at 20 °C and frozen-stored (
18 °C) until analysis. The
experiment was performed in duplicate. The contents of
phenolics and total monomeric anthocyanins and antioxidant
activity by ABTS were determined.
The percentage of release of total phenolics or total monomeric
anthocyanins and the antioxidant activity of the microparticles
during digestion were calculated as follows:
mg TPC or TMAC during digestion  100%
Releaseð%Þ ¼
mg TPC or TMAC of the microparticles
lmol TE during digestion  100%
Antioxidant activityð%Þ ¼
lmol TE of the microparticles
2.6. Statistical analysis
The results were submitted to ANOVA, Tukey’s multiple
comparison tests, and Pearson correlation, using the SAS
9.3 program. Estimation of the kinetic parameters was done
259
by non-linear regression analysis using Sigma Plot 8.0
software.
3. Results and discussion
3.1. Accelerated tests of storage stability
3.1.1. Effect of storage conditions on the content of total phenolics of
the microparticles
The total phenolics content decreased significantly during the
first 7 days of storage for all conditions studied (Fig. 1). The
freeze-drying and spray-drying methods can lead to the formation
of microspheres, which are particles formed by a continuous phase
of one or more polymers containing dispersed active compounds
(Mathiowitz, Kreitz, & Brannon-Peppas, 1999). In addition, film-
forming involves the quick formation of a thin and dense matrix
ð7Þ around the atomized droplets during spray drying, while moisture
is being removed at high rates, forming a protective barrier against
oxygen diffusion and maintaining the core temperature below
ð8Þ 100 °C in spite of the high temperatures employed during spray
drying (Anandharamakrishnan & Ishwarya, 2015). Polyphenols
might form hydrogen bonds with hydroxyl groups from polydex-
ð9Þ trose and guar gum partially hydrolyzed, resulting in a strong asso-
ciation between them. However, in this type of microparticle, some
of the compounds may be exposed to the environment, which
might have caused this largest drop of phenolic compounds in
the first 7 days.
After seven days, the phenolics content had stabilized, with no
significant differences over time (p > 0.05), except for the spray-
dried powder stored at 55 °C, which exhibited an increase in phe-
nolics after 21 days (Table S1, Supplementary material). These
increases at elevated temperatures have been observed in other
studies (Flores, Singh, & Kong, 2014), and are due to the ability of
these compounds to react with each other and hydrolyze to smal-
ler molecules (Sun-Waterhouse & Waterhouse, 2015), or polymer-
ize to form larger molecules, forming phenolic compounds of
higher or lower antioxidant activity.
When assessing the effect of temperature, relative humidity,
time and their interactions, a significant effect was observed
only for temperature (p < 0.05). It is known that phenolic
compounds are unstable at high temperatures, exhibiting signif-
icant decomposition, giving rise to degradation products which
may, in turn, be decomposed (Hamama & Nawar, 1991). The
reduction of the phenolics content during storage can be due
to oxidation (enzymatic or non-enzymatic), hydrolysis, and
polymerization with other molecules (Cao, Bi, Huang, Wu, &
Liao, 2012; Chang, Zuo, Chow, & Ho, 2006). Several factors can
influence such changes, including the chemical structures of
the phenolic compounds, the matrix in which they are dispersed,
physicochemical characteristics of the powders, such as surface
area, and processing and storage conditions (Mrkìc, Cocci, Dalla
Rosa, & Sacchetti, 2006; Sun-Waterhouse & Waterhouse, 2015).
ð10Þ In this study, different relative humidities and temperatures
were assessed.
The retention of phenolics ranged from 82.5 to 93.5%, and the
freeze-dried powders presented significantly higher retention
(p < 0.05) than the spray-dried powders stored at 35 °C, regardless
ð11Þ of the relative humidity. No significant differences were observed
between the other temperatures, while humidity had no effect
on the retention of phenolics in the microparticles. Significantly
lower retention (p < 0.05) was observed for the storage at 45 °C
when compared to 35 °C, with no significant difference (p > 0.05)
between the powders stored at 35 and 55 °C, which can be due
to the increased content of total phenolics at 55 °C, due to hydrol-
ysis and polymerization reactions at higher temperatures.
260 L.S. Kuck et al. / Food Chemistry 230 (2017) 257–264

Fig. 1. Effect of temperature (35, 45, and 55 °C) and relative humidity (75 and 90%) on total phenolics (mg gallic acid per g sample on dry basis) of the Bordo grape skin
phenolic extract microparticles produced with 5% polydextrose and 5% partially hydrolyzed guar gum by spray-drying (A) and freeze-drying (B).

Fig. 2. Effect of temperature (35, 45, and 55 °C) and relative humidity (75 and 90%) on total monomeric anthocyanins (mg malvidin-3,5-diglucoside) of the Bordo grape skin
phenolic extract microparticles produced with 5% polydextrose and 5% partially hydrolyzed guar gum by spray-drying (A) and freeze-drying (B).

3.1.2. Effect of storage conditions on the degradation of total
monomeric anthocyanins of the microparticles
Fig. 2 shows the temperature, relative humidity, and storage
time affected the reduction of the total monomeric anthocyanins.
ANOVA also indicated that the main effects of these factors were
important, as well as the effect of interactions between the tem-
perature and time (p < 0.05).
The retentions of the total monomeric anthocyanins (Table S2,
Supplementary material) were significantly lower (p < 0.05) with
increasing temperature. Zhang et al. (2012) have reported that
the degradation of anthocyanins is mainly due to the cleavage of
covalent bonds and oxidation reactions with increasing tempera-
ture, forming colourless compounds. In addition to degradation,
anthocyanins can undergo polymerization during prolonged stor-
age. The reactions responsible for browning and polymerization
of pigments may be enzymatic, through the activity of endogenous
enzymes not totally inactivated, and non-enzymatic reactions,
such as the condensation between anthocyanins and other pheno-
lic compounds and Maillard reaction (Wrolstad, Skrede, Lea, &
Enersen, 1990). The reducing sugars and proteins in the extract
can lead to the Maillard reaction, which typically occurs during
processing at high temperatures or during prolonged storage, lead-
ing to the condensation of anthocyanins with hydroxymethylfur-
fural, forming brown-coloured compounds (Tonon, Brabet, &
Hubinger, 2010). The method for analysis of monomeric antho-
cyanins by differential pH in this study determined the monomeric
anthocyanins rather than the polymeric anthocyanins. Thus, all
reduction of anthocyanins content does not necessarily mean
degradation, since part of the molecules may have undergone poly-
merization (Lee et al., 2005).
Regarding the relative humidity, the reduction of monomeric
anthocyanins was higher at 90% RH. Significantly lower retention
(p < 0.05) was observed for the microparticles stored at 35 °C and
90% relative humidity (34.9%), when compared to 75% relative
humidity (42.3%). Tonon et al. (2010) also observed a greater
reduction in the anthocyanins content of açai powder with
increased relative humidity, due to the higher molecular mobility
in foods, which facilitates the degradation reactions. The drying
method influenced the retention of monomeric anthocyanins only
at 35 °C and 75% relative humidity, with better retention for the
freeze-dried powder when compared to the spray-dried powder
(p < 0.05). Souza et al. (2014) studied the encapsulated Bordo grape
residue extract using maltodextrin as wall material, stored at 25 °C
and 32.8% relative humidity, and found losses of 19–30% of the
total anthocyanins within 120 days. However, it is worth noting
that the temperatures and relative humidities used in this study
were higher than those used by Souza et al. (2014).
When the results of anthocyanins were assessed within the
kinetic stability models, a first-order kinetic behaviour was
observed. This type of behaviour is characteristic of anthocyanins,
found in several studies of kinetic degradation of these compounds
(Cao et al., 2012; Flores et al., 2014; Souza et al., 2014). The results
of regression analysis showed that data were well adjusted to the
kinetic and thermodynamic models, once the coefficients of
 L.S. Kuck et al. / Food Chemistry 230 (2017) 257–264
determination were > 0.97 for all conditions. The kinetic and
thermodynamic parameters are shown in Table S2.
The rate constant (k) is a parameter which allows a predic-
tion of the thermal degradation of anthocyanins, and the smaller
the k value the greater the stability. The k values increased with
increased temperature and relative humidity. According to
Al-Zubeidy and Khalil (2007), increased degradation rate is
mainly due to the increase in the number of collisions between
molecules. The D values ranged from 25.7 to 95.5 days, and t1/2
values from 7.7 to 28.8 days and both decreased with increasing
temperature and relative humidity. The drying method had little
effect on D and t1/2 values, which were very close in all storage
conditions. Souza et al. (2014) found t1/2 from 233.5 to
461.2 days for the Bordo grape bagasse extract encapsulated
with maltodextrin (10–30%) stored at 25 °C and relative
humidity of 38.2% for 120 days.
The thermal resistance coefficient (z) values of the spray-dried
powders were 43.9 °C, and higher than the freeze-dried powders,
which showed z values of approximately 38 °C, indicating that
the spray-dried powder is more stable to temperature changes
than the freeze-dried powder.
Regarding the activation energy (Ea), the freeze-dried powders
had higher values (approximately 50 kJ mol
1) than the spray-
dried powders (approximately 44 kJ mol
1). According to
Heldman (2011), the activation energy constants of degradation
of anthocyanins vary from 35 to 125 kJ mol
1. In the reactions with
higher activation energies, the substrates tend to be degraded
more rapidly with little temperature changes at higher tempera-
tures. When comparing the activation energy of the powders, the
freeze-dried powder can be considered less stable than the
spray-dried when stored at higher temperatures. The Q10 values,
which indicate how much faster a reaction occurs when the
temperature is raised by 10 °C, were slightly superior for the
freeze-dried powders. The Q10 value for the degradation of natural
pigments is often near 2. Low Q10 values suggest the importance of
the molecular associations that can reduce the degradation
rate of anthocyanins, which can be confirmed by the determina-
tion of activation energy and other thermodynamic functions
(Al-Zubaidy & Khalil, 2007).
Although the z and Ea values indicate that the freeze-dried
powder was less stable to temperature changes, D and t1/2 values
were very similar between the two powders when higher temper-
atures were used, indicating little difference in stability in the tem-
peratures of this study. This result is confirmed by the final
retention, as mentioned above, which was significantly higher
(p < 0.05) for the freeze-dried powder at 35 °C, and did not differ
significantly between the powders in the other two temperatures
studied.
The microparticles produced by freeze-drying and spray-drying
had similar values of Gibbs free energy in all storage conditions,
ranging from 84.4 to 87.5 kJ mol
1. Similar Gibbs energy values
indicate that the monomeric anthocyanins degradation rate is
influenced by similar factors in the different treatments (Al-
Zubaidy & Khalil, 2007). The positive DG– values indicate that
the degradation of the monomeric anthocyanins occurred by a
non-spontaneous reaction (Mercali, Gurak, Schmitz, & Marczak,
2015). The Gibbs free energy is related to the balance and spon-
taneity of the process and reveals the increase in the total energy
of the system (Georgieva, Zvezdova, & Vlaev, 2012; Mercali et al.,
2015). The DG– values increased with increase in temperature
and anthocyanins degradation. Other authors have also observed
similar Gibbs free energy behaviour of anthocyanins degradation,
however with higher values. Martynenko and Chen (2016) found
ΔG– values from 102.17 to 106.0 kJ mol
1 when the anthocyanin
degradation was evaluated in dynamical hydrothermal treatment
(70–105 °C) of blueberries. Mercali et al. (2015) found DG– values
261
from 102.5 to 104.2 kJ mol
1 in anthocyanin degradation during
heating of jabuticaba juice from 70 to 90 °C.
The ΔH– values varied from 41.3 to 48.1 kJ mol
1, and the
microparticles produced by freeze-drying had higher values than
spray-drying in all storage conditions. Higher ΔH– values indicate
less stability of bioactive compounds to higher temperatures
(Karaaslan et al., 2014). Positive enthalpy values indicate that the
degradation of anthocyanins is an endothermic reaction (Al-
Zubaidy & Khalil, 2007). Other authors have observed positive
ΔH– values in anthocyanins degradation reactions (Martynenko &
Chen, 2016; Mercali et al., 2015).
The ΔS– values of freeze-dried microparticles varied from

112.1 to
113.6 J mol
1 K
1, while a range of
131.0 to

132.9 J mol
1 K
1 was observed for the spray-dried microparti-
cles. Negative ΔS– values indicate an increase in order, ie, the
material has undergone a physical or chemical rearrangement of
the initial structure, getting close to its thermodynamic equilib-
rium (Al-Zubaidy & Khalil, 2007; Georgieva et al., 2012). Both Ea
as ΔH– and ΔS– values were higher for the freeze-dried micropar-
ticles, which primarily means that the reaction rate constant of the
anthocyanins degradation in the freeze-dried powder are more
sensitive to temperature than of the spray-dried powder
(Karaaslan et al., 2014).
3.1.3. Effect of storage conditions on the antioxidant activity of the
microparticles
As shown in Fig. 3, there was a great reduction of the antioxi-
dant activity in the first 7 days of storage, followed by stabilization
or slow decrease, similar to that observed for total phenolics. When
assessing the effect of the factors, temperature, relative humidity,
time and their interactions, only temperature and time were signif-
icant (p < 0.05) by ANOVA.
The retention of the antioxidant activity ranged from 38.5 to
59.5% (Table S3, Supplementary material) and significantly
decreased (p < 0.05) with increasing temperature. The relative
humidity affected only the freeze-dried powder stored at 45 °C,
once significantly reduced retention was observed at 90% RH when
compared to 75% relative RH. Regarding the drying method, there
was no effect on the retention of the antioxidant activity of the
powders, except for those stored at 35 °C and 90% relative humid-
ity, with significantly higher retention (p < 0.05) for the spray-
dried powder when compared to the freeze-dried powder.
The reduction of the antioxidant activity during storage is asso-
ciated with a reduction in phenolics and monomeric anthocyanins
levels. The Pearson correlation analysis indicated a strong correla-
tion between antioxidant activity and total phenolics (0.89) and
total monomeric anthocyanins (0.92). Despite the great reduction
of monomeric anthocyanins, the reduction of the antioxidant activ-
ity was not of the same order of magnitude, as well as phenolic
compounds, which have not suffered major reductions. During
prolonged heating or storage, some phenolics degradation, includ-
ing anthocyanins, was observed, due to hydrolysis or polymeriza-
tion of molecules, forming new compounds which can
compensate completely or partially the loss of the antioxidant
activity, mainly due to the lower content of monomeric antho-
cyanins (Cao et al., 2012; Chang et al., 2006; Flores et al., 2014).
According to Tsai and Huang (2004), the formation of polymeric
anthocyanins in experiments using hibiscus did not significantly
affect the antioxidant activity measured by the radical ABTS.
Sadilova, Carle, and Stintzing (2007) analyzed the degradation of
the purified anthocyanin fraction of elderberry, black carrot, and
strawberry after heating, and observed higher retention of the
antioxidant activity when compared to the anthocyanins. The
authors suggested that this is due to the formation of degradation
products, which may vary and have different antioxidant activities
in different food matrices. However, the authors also emphasized
262 L.S. Kuck et al. / Food Chemistry 230 (2017) 257–264
Fig. 3. Effect of temperature (35, 45, and 55 °C) and relative humidity (75 and 90%) on the antioxidant activity (ABTS , lmol TE/g sample on dry basis) of the Bordo grape skin
phenolic extract microparticles produced with 5% polydextrose and 5% partially hydrolyzed guar gum by spray-drying (A) and freeze-drying (B).
that the degradation products do not fully compensate for the
degradation of anthocyanins, whereas there was a significant
reduction of antioxidant activity after the end of heating.
3.2. Simulation of gastrointestinal digestion
The effect of in vitro digestion on the release of phenolics from
the microparticles was divided into two phases. The first phase
corresponds to the upper gastrointestinal tract or gastric phase
(pH 1–3), which involves the steps of mouth and stomach in the
first and second hour of the experiment, while the second phase
involves the lower gastrointestinal tract or intestinal phase (pH
around neutrality), which covers the small intestine and corre-
sponds to the third and fourth hour of the experiment (Flores
et al., 2014). In relation to the phenolics bioavailability, it is known
that the aglycone can be absorbed from the small intestine, how-
ever, most of the polyphenols present in food are in the form of
esters, glycosides, or polymers, which should first be hydrolyzed
by intestinal enzymes or by colonic microflora, and then absorbed
in the intestine (Manach, Scalbert, Morand, Rémésy, & Jiménez,
2004). Little alterations were observed for the total phenolics, total
monomeric anthocyanins, and the antioxidant activity between
the two drying methods (freeze-drying and spray-drying), with a
more significant change in relation to digestion period (Fig. 4).
No significant difference (p > 0.05) was observed between the
spray-dried and freeze-dried microparticles in the release of phe-
nolics, monomeric anthocyanins, and antioxidant activity in a sin-
gle digestion time.
A great release of total phenolics was observed during the gas-
tric phase (first and second time) when a maximal release was
observed (approximately 80%), with a significant increase
(p < 0.05) during the intestinal phase. A release of 90.6% total phe-
nolics was observed for the spray-dried microparticles, while the
freeze-dried microparticles exhibited 94.9% release. Similar results
were found in other studies evaluating the stability of polyphenols
against simulated gastrointestinal digestion (Bouayed, Hoffmann,
& Bohn, 2011; Saikia, Mahnot, & Mahanta, 2015), and may be
due to the wall material used in the encapsulation (Saikia et al.,
2015), particularly the chain size and solubility in the pH of the
environment. Polydextrose and partially hydrolyzed guar gum
are highly water-soluble polymers, but have very low digestibility
in the small intestine due to their resistance to hydrolysis by the
enzymes present in the body, reaching almost intact to the colon
where they are partially or completely fermented by the micro-
biota (Cho & Samuel, 2009). Thus, it is believed that the release
of the phenolic compounds is due to the solubilization of the

microparticles since no hydrolysis of the polymer was observed
in the phase studied.
Polyphenols are sensitive to pH near neutrality in the small
intestine, and part of them can be transformed into other com-
pounds with different structural forms and chemical properties
during digestion (Bermúdez-Soto, Tomás-Barberán, & García-
Conesa, 2007). The hydrolysis and polymerization of some pheno-
lic compounds to form others can be due to the increase of the phe-
nolics content in the third and fourth hour of the experiment.
Anthocyanin degradation products are some of the compounds
that can contribute to this increase in the intestinal stage
(McDougall, Dobson, Smith, Blake, & Stewart, 2005).
Unlike total phenolics, anthocyanins showed a significant
reduction (p < 0.05) in the intestinal phase (third and fourth time).
Anthocyanins are highly sensitive to neutral or alkaline pH, which
has been observed by several authors who evaluated the stability
of anthocyanins during the digestive process (Bouayed et al.,
2011; McDougall et al., 2005; Pérez-Vicente, Gil-Izquierdo, &
García-Viguera, 2002). It is known that four molecular species of
anthocyanins exist in equilibrium (flavylium cation and three sec-
ondary structures: quinoidal base, carbinol pseudo base, and chal-
cone pseudo base), and one of the reasons for the low recovery of
anthocyanins may be the transformation of flavylium cation
(coloured) to pseudo base chalcone (colourless) in intestinal pH,
with subsequent cleavage of the ring and formation of ionized
chalcones (McDougall et al., 2005; Pérez-Vicente et al., 2002).
The reasons for the reduction in total monomeric anthocyanins
during the intestinal phase is unknown. One possibility is that
the high pH in this step leads to the formation of non-coloured
anthocyanins that are not quantified by spectrophotometric
method, and may subsequently be degraded, forming other com-
pounds, such as phenolic compounds with lower molecular size
(Pérez-Vicente et al., 2002).
The retention of antioxidant activity showed a similar beha-
viour to total phenolics, with a significant increase (p < 0.05) in
the intestinal phase (third and fourth time), with 69.4% and
67.8% retention for the spray-dried and freeze-dried microparti-
cles, respectively. The antioxidant activity of the phenolic com-
pounds is related to its pH and chemical structure in the
digestion step (Kamiloglu, Pasli, Ozcelik, Camp, & Capanoglu,
2015). According to Tagliazucchi, Verzelloni, Bertolini, and Conte
(2010), the antioxidant activity of phenolic compounds is pH-
dependent, and can have a higher protective effect against oxida-
tive stress in intestinal cells rather than in stomach cells, once
these compounds have greater sequestration capacity in the pH
conditions in the intestine. This increased antioxidant activity in
the intestinal phase was probably due to deprotonation of the
 L.S. Kuck et al. / Food Chemistry 230 (2017) 257–264
Fig. 4. Release of total phenolics (A), total monomeric anthocyanins (B), and antioxidant activity (C) during the simulated gastrointestinal digestion. The first and second
hours corresponded to the gastric phase (mouth and stomach), while the third and fourth hours corresponded to the intestinal phase (small intestine).
hydroxyl groups present in the aromatic ring in the transition from
acidic to alkaline medium (Bouayed et al., 2011).
Despite the great reduction of total monomeric anthocyanins in
the intestinal phase, the retention of antioxidant activity was close
to 70%. This behaviour was similar to that observed in the evalua-
tion of storage stability of the microparticles, probably due to the
formation of new hydrolysis compounds and polymerization of
the novel compounds.
4. Conclusions
The evaluation of the stability of the microparticles by acceler-
ated stability tests showed a significantly lower total phenolics
content during the first seven days of storage, while after 35 days
the retentions varied from 82.5 to 93.5%, with a significant effect
of temperature. The simulated gastrointestinal digestion showed
that most of the phenolic compounds were released in the gastric
phase. With respect to the total monomeric anthocyanins, the sta-
bility test indicated that temperature, relative humidity, and time
affected the anthocyanins contents, with retentions ranging from
3.9 to 42.3% after 35 days, and the losses with time exhibited
first-order kinetics. Simulated digestion tests indicated lower
monomeric anthocyanins levels in the intestinal phase due to alka-
line pH. Similar behaviour was observed for the antioxidant activ-
ity when compared to the total phenolics, however, the final
retention ranged from 38.5 to 59.5%. In the gastrointestinal simu-
lation, the retention of antioxidant activity was near 70% in the
intestinal phase. Despite the reduction of bioactive compounds,
better retentions of the antioxidant activity were observed, thus
further studies to assess the stability of these products in food
matrices are required.
Acknowledgment
The authors thank FAPERGS, CAPES, and CNPq – Brazil for
financial support and the R & S Blumos by donation of partially
hydrolyzed guar gum.
263
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.
03.038.
References
Al-Zubaidy, M. M. I., & Khalil, R. A. (2007). Kinetic and prediction studies of ascorbic
acid degradation in normal and concentrate local lemon juice during storage.
Food Chemistry, 101, 254–259.
Anandharamakrishnan, C., & Ishwarya, S. P. (2015). Spray drying techniques for food
ingredient encapsulation. New Jersey: Wiley-Blackwell (Chapters 3, 4, 7).
Bermúdez-Soto, M.-J., Tomás-Barberán, F.-A., & García-Conesa, M.-T. (2007).
Stability of polyphenol in chokeberry (Aronia melanocarpa) subjected to
in vitro gastric and pancreatic digestion. Food Chemistry, 102, 865–874.
Bouayed, J., Hoffmann, L., & Bohn, T. (2011). Total phenolics, flavonoids,
anthocyanins and antioxidant activity following simulated gastro-intestinal
digestion and dialysis of apple varieties: Bioaccessibility and potential uptake.
Food Chemistry, 128, 14–21.
Cao, X., Bi, X., Huang, W., Wu, J., & Liao, X. (2012). Changes of quality of high
hydrostatic pressure processed cloudy and clear strawberry juices during
storage. Innovative Food Science and Emerging Technologies, 16, 181–190.
Chang, Q., Zuo, Z., Chow, M. S. S., & Ho, W. K. K. (2006). Effect of storage temperature
on phenolics stability in hawthorn (Crataegus pinnatifida var. major) fruits and a
hawthorn drink. Food Chemistry, 98, 426–430.
Cho, S. S., & Samuel, P. (Eds.). (2009). Fiber ingredients: Food applications and health
benefits. Boca Raton: CRC Press (Chapters 6, 9).
Flores, F. P., Singh, R. K., Kerr, W. L., Pegg, R. B., & Kong, F. (2014). Total phenolics
content and antioxidant capacities of microencapsulated blueberry
anthocyanins during in vitro digestion. Food Chemistry, 153, 272–278.
Flores, F. P., Singh, R. K., & Kong, F. (2014). Physical and storage properties of spray-
dried blueberry pomace extract with whey protein isolate as wall material.
Journal of Food Engineering, 137, 1–6.
Georgieva, V., Zvezdova, D., & Vlaev, L. (2012). Non-isothermal kinetics of thermal
degradation of chitosan. Chemistry Central Journal, 6, 81–91.
Hamama, A. A., & Nawar, W. W. (1991). Thermal decomposition of some
phenolic antioxidants. Journal of Agricultural and Food Chemistry, 39,
1063–1069.
Heldman, D. R. (2011). Food preservation process design. San Diego: Academic Press
(Chapter 2, 4).
Jiménez-Aguilar, D. M., Ortega-Regules, A. E., Lozada-Ramírez, J. D., Pérez-Pérez, M.
C. I., Vernon-Carter, E. J., & Welti-Chanes, J. (2011). Color and chemical stability
of spray-dried blueberry extract using mesquite gum as wall material. Journal of
Food Composition, 24, 889–894.
264 L.S. Kuck et al. / Food Chemistry 230 (2017) 257–264
Kamiloglu, S., Pasli, A. A., Ozcelik, B., Camp, J. V., & Capanoglu, E. (2015). Influence of
different processing and storage conditions on in vitro bioaccessibility of
polyphenols in black carrots jams and marmalades. Food Chemistry, 186, 74–82.
Karaaslan, M., Yilmaz, F. M., Cesur, O., Vardin, H., Ikinci, A., & Dalgiç, A. C. (2014).
Drying kinetics and thermal degradation of phenolic compounds and
anthocyanins in pomegranate arils dried under vacuum conditions.
International Journal of Food Science & Technology, 49, 595–605.
Kuck, L. S., & Noreña, C. P. Z. (2016). Microencapsulation of grape (Vitis labrusca var.
Bordo) skin phenolic extract using gum Arabic, polydextrose, and partially
hydrolyzed guar gum as encapsulating agents. Food Chemistry, 194, 569–576.
Lago-Vanzela, E. S., Da-Silva, R., Gomes, E., García-Romero, E., & Hermosín-
Gutiérrez, I. (2011). Phenolic composition of the edible parts (flesh and skin)
of Bordô grape (Vitis labrusca) using HPLC-DAD-ESI-MS/MS. Journal of
Agricultural and Food Chemistry, 59, 13136–13146.
Lee, J., Durst, R. W., & Wrolstad, R. E. (2005). Determination of total monomeric
anthocyanin pigment content of fruit juices, beverages, natural colorants, and
wines by the pH differential method: collaborative study. Journal of AOAC
International, 88(5), 1269–1278.
Manach, C., Scalbert, A., Morand, C., Rémésy, C., & Jiménez, L. (2004). Polyphenols:
food sources and bioavailability. The American Journal of Clinical Nutrition, 79,
727–747.
Martynenko, A., & Chen, Y. (2016). Degradation kinetics of total anthocyanins and
formation of polymeric color in blueberry hydrothermodynamic (HTD)
processing. Journal of Food Engineering, 171, 44–51.
Mathiowitz, E., Kreitz, M. R., & Brannon-Peppas, L. (1999). Microencapsulation. In E.
Mathiowitz (Ed.), Encyclopedia of controlled drug delivery (pp. 493–553). New
York: John Wiley and Sons.
McDougall, G. J., Dobson, P., Smith, P., Blake, A., & Stewart, D. (2005). Assessing
potential bioavailability of raspberry anthocyanins using an in vitro digestion
system. Journal of Agricultural and Food Chemistry, 53, 5896–5904.
Mercali, G. D., Gurak, P. D., Schmitz, F., & Marczak, L. D. F. (2015). Evaluation of non-
thermal effects of electricity on anthocyanin degradation ohmic heating of
jaboticaba (Myrciaria cauliflora) juice. Food Chemistry, 171, 200–205.
Mrkìc, V., Cocci, E., Dalla Rosa, M., & Sacchetti, G. (2006). Effect of drying conditions
on bioactive compounds and antioxidant activity of broccoli (Brassica oleracea
L.). Journal of the Science of Food and Agriculture, 86, 1559–1566.
Pérez-Vicente, A., Gil-Izquierdo, A., & García-Viguera, C. (2002). In vitro
gastrointestinal digestion study of pomegranate juice phenolic compounds,
anthocyanins, and vitamin C. Journal of Agricultural and Food Chemistry, 50,
2308–2312.
Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Catherine, R. E. (1999).
Antioxidant activity applying an improved ABTS radical cation decolorization
assay. Free Radical Biology & Medicine, 26(9/10), 1231–1237.
Rice-Evans, C. A., Miller, N. J., & Paganga, G. (1997). Antioxidant properties of
phenolic compounds. Trends in Plant Science, 2(4), 152–159.
Richards, M., De Kock, H. L., & Buys, E. M. (2010). Multivariate accelerated shelf-life
test of low fat UHT milk. International Dairy Journal, 36, 38–45.
Rodrigo, R., Miranda, A., & Vergara, L. (2011). Modulation of endogenous antioxidant
system by wine polyphenols in human disease. Clinica Chimica Acta, 412,
410–424.
Sadilova, E., Carle, R., & Stintzing, F. C. (2007). Thermal degradation of anthocyanins
and its impact on color and in vitro antioxidant capacity. Molecular Nutrition &
Food Research, 51, 1461–1471.
Saikia, S., Mahnot, N. K., & Mahanta, C. L. (2015). Optimisation of phenolic extraction
from Averrhoa carambola pomace by response surface methodology and
its microencapsulation by spray and freeze-drying. Food Chemistry, 171,
144–152.
Silva, P. I., Stringheta, P. C., Teófilo, R. F., & Oliveira, I. R. N. (2013). Parameter
optimization for spray-drying microencapsulation of jaboticaba (Myrciaria
jaboticaba) peel extracts using simultaneous analysis of responses. Journal of
Food Engineering, 117, 538–544.
Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of total phenolics with
phosphomolybdic–phosphotungstic acid reagents. American Journal of Enology
and Viticulture, 16, 144–158.
Souza, V. B., Fujita, A., Thomazini, M., Silva, E. R., Lucon, J. F., Jr, Genovese, M. I., &
Favaro-Trindade, C. S. (2014). Functional properties and stability of spray-dried
pigments from Bordo grape (Vitis labrusca) winemaking pomace. Food
Chemistry, 164, 380–386.
Sun-Waterhouse, D., & Waterhouse, G. I. N. (2015). Spray-drying of green or gold
kiwifruit juice-milk mixtures; novel formulations and processes to retain
natural fruit colour and antioxidants. Food Bioprocess Technology, 8, 191–207.
Tagliazucchi, D., Verzelloni, E., Bertolini, D., & Conte, A. (2010). In vitro bio-
accessibility and antioxidant activity of grape polyphenols. Food Chemistry, 120,
599–606.
Tonon, R. V., Brabet, C., & Hubinger, M. D. (2010). Anthocyanin stability and
antioxidant activity of spray-dried açai (Euterpe oleracea Mart.) juice produced
with different carrier agents. Food Research International, 43, 907–914.
Tsai, P.-J., & Huang, H.-P. (2004). Effect of polymerization on the antioxidant
capacity of anthocyanins in Roselle. Food Research International, 37,
313–318.
Wrolstad, R. E., Skrede, G., Lea, P., & Enersen, G. (1990). Influence of sugar on
anthocyanin pigment stability in frozen strawberries. Journal of Food Science, 55
(4), 1064–1072.
Zhang, L., Zhou, J., Liu, H., Khan, M. A., Huang, K., & Gu, Z. (2012). Compositions of
anthocyanins in blackberry juice and their thermal degradation in relation to
antioxidant activity. European Food Research Technology, 253, 637–645.