Carbohydrate Polymers 127 (2015) 252–263

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Saffron and beetroot extracts encapsulated in maltodextrin,

gum Arabic, modified starch and chitosan: Incorporation
in a chewing gum system
Charikleia Chranioti, Aspasia Nikoloudaki, Constantina Tzia ∗
Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Str.,
Polytechniou, Zografou, 15780 Athens, Greece

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

Article history: Maltodextrin (MD-21DE), gum Arabic (GA), gum Arabic–modified starch (GA–MS), modified
Received 8 December 2014 starch–chitosan (MS–CH) and modified starch–maltodextrin–chitosan (MS–MD–CH) were used as agents
Received in revised form 12 March 2015 for beetroot and saffron coloring-extracts microencapsulation by freeze drying. The produced powders
Accepted 13 March 2015
were evaluated in terms of coloring strength (E) during storage at 40 ◦ C for 10 weeks and a first-order
Available online 30 March 2015
kinetic was applied. Color parameters (L* , a* , b* , C* and E* ) and water sorption behavior was also stud-
ied. Moreover, incorporation of the powders in a chewing gum model system was conducted. The type of
Keywords:
encapsulating agent significantly (P < 0.05) affected the studied parameters with the order of protection
Saffron extract
Beetroot extract
in both extracts being as follows: MD > GA > GA–MS > MS–CH > MS–MD–CH. The water sorption study
Encapsulation revealed that MD and GA kept their structural integrity up to water activities of 0.66 and 0.82, respec-
Freeze drying tively. The chewing gum samples produced with coloring extracts encapsulated in GA–MS showed the
Color stability greatest a* (for beetroot) and b* (for saffron) values indicating a better protection.
Chewing gum © 2015 Elsevier Ltd. All rights reserved.

1. Introduction
Color is one of the most important characteristics of foods, being
considered as a quality indicator that determines their acceptance
(Azeredo, 2009). Although chemical food additives such as artificial
colorants have been widely applied for coloring purposes of food
products, their use is still a controversial issue in the food indus-
try due to their toxicological potential on human health (Mizutani,
2009; Reyes, Valim, & Vercesi, 1996). The consumption of artificial
colorants has been associated with the development of allergies
in children (Inomata, Osuna, Fujita, Ogawa, & Ikezawa, 2006) and
the increased risk of cancer (Sasaki et al., 2002). Therefore, legisla-
tive actions and consumer concerns have resulted in an increased
interest toward replacement of chemical colorants with natural
pigments, which are considered not only to be harmless but also
to possess functional properties that can exert beneficial effect to
human health (Moreira et al., 2012; Rutkowska & Stolyhwo, 2009).
Beetroot and saffron are basic natural sources of pigments
which are typically used as colorants in quite a wide range of food
products. Beetroot pigments consist of two major water soluble
∗ Corresponding author. Tel.: +0030 2107723165; fax: +0030 2107723163.
E-mail address: tzia@chemeng.ntua.gr (C. Tzia).
fractions, betacyanins that confer the red-violet color and betax-
hantins, a yellow–orange colorant also present in beetroot in lesser
proportion than betacyanins (Pitalua, Jimenez, Vernon-Carter, &
Beristain, 2010). They are typically used at levels of 4–25 mg/kg
in a wide range of dairy and confectionery products as well as in
meat substitutes. However, their use in foods is limited due to their
poor stability when exposed to heat and light (Serris & Biliaderis,
2001).
On the other hand, saffron pigments include crocins, a group
of water soluble carotenoids, which are glycosyl esters of 8,8-
diapocarotene-8,8-dioic acid (or crocetin) with biologically activity
on human health (Tarantilis, Tsoupras, & Polissiou, 1995). Saffron
pigments are typically used at levels of 1–260 ppm in a wide range
of culinary, bakery and confectionery preparations as well as in
alcoholic and non alcoholic beverages. However, as being highly
unsaturated components, they are prone to oxidation and isom-
erization reactions that lead to losses of coloring strength and
nutritive value (Tsimidou & Biliaderis, 1997). Therefore, a protec-
tive encapsulation technique is needed for the feasible usage of the
afore-mentioned natural pigments in food products.
Among the common encapsulation techniques used are spray
(Akhavan, Jafari, Ghorbani, & Assadpoor, 2014) and freeze–drying
of the oil-in-water prepared emulsion containing the encapsulat-
ing agent and the pigment-core (Lim, Tan, Bakar, & Ng, 2011).

http://dx.doi.org/10.1016/j.carbpol.2015.03.049
0144-8617/© 2015 Elsevier Ltd. All rights reserved.
 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263
Microencapsulation by freeze drying leads to products with excel-
lent sensory characteristics and preserved biofunctionality since
changes associated with high temperatures are minimized, due
to the low temperatures involved in the process (Minemoto,
Adachi, & Matsuno, 2001). Moreover, the selection of the encap-
sulation agent is very crucial for an efficient process and it
depends on the final application of the encapsulated pigment. In
our previous works, various edible polymers have been exam-
ined (plain and in mixtures) in fennel oleoresin (Chranioti & Tzia,
2013, 2014a) and saffron volatile flavor substances (Chranioti,
Papoutsakis, Nikoloudaki, & Tzia, 2012) encapsulation. Among
the studied encapsulating agents, plain gum Arabic (GA), binary
blends of gum Arabic–modified starch (GA–MS) and chitosan-
modified starch (MS–CH) as well as a ternary one of modified
starch–maltodextrin–chitosan (MS–MD–CH) were selected as they
displayed relatively high encapsulation efficiencies while plain
maltodextrin (MD) was chosen since it is colorless and provides
good protection against oxidation (Chranioti & Tzia, 2014b).
Although there have been some studies on encapsulation of
beetroot (Pitalua et al., 2010) and saffron (Cormier, Dufresne, &
Dorion, 1995; Dufresne et al., 1999; Khazaei, Jafari, Ghorbani, &
Hemmati Kakhki, 2014) extracts, the use of the afore-mentioned
mixtures of agents has not been investigated.
Therefore, the aim of this study was to produce natural colorants
encapsulated in different mixtures of agents with the use of freeze
drying technique and then: (i) to examine the color stability of the
obtained encapsulated colorants, (ii) to study their sorption behav-
ior at various relative humidity environments and (iii) to evaluate
their incorporation in a chewing gum model system.
2. Materials and methods
2.1. Materials
Dried stigmas of saffron were provided directly from the ‘Coop-
erative of saffron, Krokos Kozanis’ while the beetroot vegetable
was purchased from a local market. Modified Starch (MS, Clear-
gum CO-01, an octenyl-succinylated starch), maltodextrin DE-21
(MD, from Waxy Maize) and gum Arabic (GA) were obtained from
Chemicotechnica S.A. Chitosan (CH) high molecular weight (viscos-
ity of 800,000 cps, food-grade, water soluble, odorless and tasteless
powder as stated by the manufacturer) was obtained from Sigma-
Aldrich Chemical Co. Sorbitol and mannitol were purchased from
Cargill whereas gum base and lecithin were kindly donated from
Kraft Foods and Biotrek—Greece, respectively.
2.2. Preparation of aqueous saffron and beetroot extracts
Saffron (1 g) was extracted with distilled water (50 mL) under
continuous shaking in an ultrasound water bath (Elma, S 30H, Elma-
sonic) at T = 25 ◦ C for 60 min and at fixed-frequency of 30 kHz, while
beetroots were washed, peeled and extracted with water in a com-
mercial juice extractor. Both saffron and beetroot aqueous extracts
were filtered and kept in the dark at −30 ◦ C until used.
2.3. Microencapsulation of saffron and beetroot extracts by
freeze–drying
Plain GA and MD, binary (1:1) blends of GA–MS and MS–CH plus
a ternary (1:1:1) one of MS–MD–CH were selected as encapsulating
agents. An aliquot of 15 g of GA, MD and MS agent was dispersed
individually in distilled water to a final volume of 100 mL, while
a 2% chitosan solution (CH) in 1% glacial acetic acid was prepared.
A fixed weight ratio (w/w) of 0.33 (extract:agent) was tested (Ahn
et al., 2008; Hogan, McNamee, O’Riordan, & O’Sullivan, 2001). The
extracts were dissolved into the agents under stirring for 15 min
253
at 600 rpm in a rotor–stator. The resulting solutions were frozen
overnight at −30 ◦ C (Shimada, Roos, & Karel, 1991) and lyophilized
in a freeze dryer (Christ Alpha 1–4 LD Plus) at P = 0.017 mbar and
T = −57 ◦ C for 48 h. The freeze-dried encapsulated extracts were
converted into powder with help of a pestle and mortar.
2.4. Kinetic studies of beetroot and saffron pigment degradation
Freeze-dried beetroot and saffron powders were stored in
brown bottles with a screw cap and kept at 40 ◦ C with 20% RH
(relatively humidity) as directly measured with a hygrometer for a
period of 10 weeks in order to determine the effect of storage tem-
perature on the retention of betacyanins and carotenoids levels,
respectively. The degradation of natural pigments was expressed as
coloring strength (E) and followed by periodic absorbance measure-
ments of the reconstituted powder (0.2 g) in aqueous solution with
distilled water (10 mL, stirring for 10 min and immediate measure-
ment of the absorbance at certain wavelength). The absorbance was
measured with a spectrophotometer (DMS 80, Varian Techtron PTY,
LTD, Belrose, Australia) at max = 537 nm, the maximum absorp-
tion wavelength of betacyanin (Pasch & von Elbe, 1975) and
max = 440 nm, the maximum absorption wavelength of crocin (ISO,
1993). The coloring strength, (E), was calculated as follows (Alonso,
Varon, Gomez, Navarro, & Salinas, 1990):
AV
1%
Emax = (1)
pdC
where A is the absorbance at the max , V is the quantity of solvent
added (mL), p is the weight of the sample (g), d is the pathlength of
the cell (cm) and C is a constant, the value of which is 100 cm2 /g.
Measurements were carried out in triplicate and reaction rate
constants (k) and half-life periods (t1/2 ) were determined by apply-
ing a first-order reaction model to the data (Tsimidou & Tsatsaroni,
1993). Powder samples were withdrawn every 2 weeks for a period
of 10 weeks for betacyanins and carotenoids degradation kinetic
analysis.
2.5. Color change during storage
L∗ (lightness), a∗ (redness to green), and b∗ (yellow to blue)
color parameters were measured immediately after freeze–drying
(zero time) and after 12 weeks of storage at 40 ◦ C and the mean
of three replicates was reported. Color measurements were per-
formed directly on the encapsulated powdered products using a
colorimeter (Minolta CR 200, Tokyo, Japan). The parameters of
chroma (C* ) and total color differences (E* ) were also calculated
as follows:
 
2 1/2
C ∗ = (a∗ )2 + (b∗ ) (2)
 2  2  2 1/2
E ∗ = L0∗ − L∗ + a∗0 − a∗ + b∗0 − b∗ (3)
2.6. Water sorption studies
Approximately 0.4 g of freeze dried microencapsulated saffron
and beetroot samples was placed in small glass desiccators (10 cm
diameter) which contained different saturated salt solutions at
ambient temperature (25 ◦ C). The prepared saturated solutions of
LiCl, MgCl2 , K2 CO3 , Mg(NO3 )2 ·6H2 O, KI and KCl provided water
activity (aw ) levels of 0.11, 0.31, 0.42, 0.51, 0.66 and 0.82, respec-
tively (Labuza, 1984). The samples were weighed until equilibrium
was attained for a period of 15–20 days. The initial moisture con-
tent of the samples was measured on a dry weight basis by drying
the sample in a vacuum oven at 100 ◦ C until constant weight
was obtained (AOAC, 2000). The water activity (aw ) was mea-
sured using Aqualab 3TE water activity meter (Decagon Devices
254 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263
Inc. Pullman, WA, USA) according to manufacturer instructions.
The sorption isotherms which indicate the variation of water activ-
ity and moisture content were plotted and fitted to well-known
sorption isotherm models such as Brunauer–Emmett–Teller (BET)
and Guggenheim Anderson–de Boer (GAB) (Van den Berg & Bruin,
1981). The BET model is described by the following equation:
aw 1 aw (k − 1)
= +
(1 − aw ) m mm k mm k
where m is the equilibrium moisture content (g H2 O/g dry weight),
k is a constant and mm is the BET monolayer value. These two
constants can be calculated from the linear regression of the exper-
imental data.
The GAB isotherm model is described by the following equation:
m CKaw
= (5)
mm (1 − Kaw ) (1 − Kaw + CKaw )
where m is the equilibrium moisture content (g H2 O/g dry weight),
C and K are constants and mm is the GAB monolayer value. This
model can also be presented as a second-order polynomial equation
for data-fitting purposes, according to the following equation:
aw
= ˛(aw )2 + ˇaw + 
m
The model that was considered most suitable was based on the
high coefficient of determination (R2 ) and the low mean relative
percentage deviation modulus (E), defined as follows:
100  mi − mpi
N reaction kinetics for beetroot and saffron’s coloring strength degra-
E=
N mi
i=1
where mi is the experimental value, mpi is the predicted value and
N is the population of experimental data. It is generally assumed
that a good fit is obtained when E < 5%.
2.7. Scanning electron microscopy
In order to study the morphological properties of the freeze-
dried encapsulated extracts, a scanning electron microscope
(Quanta 200, FEI with LFD Detector) was used. Particles were loaded
onto a specimen stub, coated with gold with a sputter coater. Exam-
inations were made at 25–30 kV with ×500 magnification.
2.8. Food application—Incorporation as natural colorants in a
chewing gum model system
2.8.1. Preparation of chewing gum model
The chewing gum ingredient formulation consisted of gum
base (29.48 g/100 g), sorbitol powder (37.24 g/100 g), glycerin
(7.06 g/100 g), saturated sorbitol solution (25.82 g/100 g) and
lecithin (0.40 g/100 g). For chewing gum preparation the gum base
was initially melted (raised to 98–104 ◦ C). The extracts (pure or
encapsulated) were diluted in the lecithin solution and then added
to the molten gum base under mixing and after 2 min the mixture
was allowed to cool. During cooling, the saturated sorbitol solu-
tion was added (under mixing for 2 min) followed by addition of
about 50% of the sorbitol powder and further mixing for 2 min. At
approximately 75 ◦ C the remaining sorbitol powder was added and
mixed for 2 min. Finally, the glycerin was added and further mixed
for 1 min (Potineni & Peterson, 2008). Twelve different formula-
tions of chewing gum were designed based on two types of extracts
(beetroot and saffron) either in pure or encapsulated form.
2.8.2. Color stability test of chewing gum samples
The chewing gum model samples were stored at 25 and
40 ◦ C and during storage the changes in color parameters were
monitored. The color measurements were performed in tripli-
cate directly on the chewing gum samples using a colorimeter
as described in section 2.5. The L* parameter was taken into
account for samples with both samples, while a* and b* param-
eters were taken into account for beetroot and saffron samples,
respectively.
(4)
2.9. Statistical analysis
Statistical analysis of measurements results was carried out
with the Statistica software (Statsoft 224 Inc., Tulsa, USA). Analy-
sis of variance was performed by ANOVA procedure and significant
differences (P < 0.05) between the means were determined by Dun-
can’s multiple range test. Moreover, multivariate analysis was
conducted to correlate the studied parameters by means of prin-
cipal component analysis (PCA) and multiple linear correlation
(Pearson’s correlation).
3. Results and discussion
3.1. Storage stability evaluation
(6)
The stability of the freeze dried microencapsulated beetroot
and saffron extracts was evaluated in terms of coloring strength
(E) during 10 weeks storage at 40 ◦ C. A linear relationship was
observed from the plots of the ln(E) vs time, implying first-order
dation (Fig. 1). The rate constant (k) and half-life period (t1/2 ) of
the microencapsulated powders were determined and are pre-
sented in Table 1. During 10 weeks of storage at 40 ◦ C, MS–CH
and MS–MD–CH samples showed higher reaction rate constant
and, consequently, shorter half-life in both extracts (23.61, 26.52
and 30.77, 29.01 weeks for beetroot and saffron, respectively). The
half-life was found not to be significantly affected by the type of
extract used, presenting mean values of 40.43 and 40.30 weeks in
the case of beetroot and saffron, respectively. Overall, MD exhibited
the greatest protection against stability accompanied also by high
half-life period (t1/2 ) with values of 53.03 and 60.03, for beetroot
and saffron, respectively.
Previous research have shown that the degradation of natu-
ral colorants such as betalains follows first order kinetics during
the storage (Saenz, Tapia, Chavez, & Robert, 2009; Serris &
Biliaderis, 2001). Similar first-order kinetic responses have also
been reported for saffron pigment oxidation under various water
activity and temperature conditions (Alonso et al., 1990; Selim,
Tsimidou, & Biliaderis, 2000; Tsimidou & Biliaderis, 1997; Tsimidou
& Tsatsaroni, 1993).
The coloring strength stability was strongly influenced
(P < 0.05) by the encapsulating agent combination. In particu-
lar, the protection order proved as follows: MD > GA > GA–MS >
MS–CH > MS–MD–CH, for both extracts. Similar observations for
the protective effect of MD and GA on natural pigments against heat
during storage have been reported by other researchers (Nayak &
Rastogi, 2010). In particular, Cai and Corke (2000) observed that
the use of MD (20 or 25DE) provided better storage stability for
Amaranthus betacyanin during storage at 25 ◦ C for 16 weeks. Saenz
et al. (2009), also, found that MD protected the betacyanin content
of cactus pear when stored at 60 ◦ C for 44 days. In another work,
Ferrari, Germer, Alvim, and Maurício de Aguirre (2013) found that
the use of MD or GA reinforced the stability of spray-dried antho-
cyanins stored at 25 and 35 ◦ C and relative humidity of 32.8% for a
period of 150 days. Recently, Khazaei et al. (2014) concluded that
encapsulation of saffron petal’s extract with freeze drying tech-
nique and MD and GA as wall materials can protect anthocyanins
content during storage at 35 ◦ C for a period of 10 weeks.
 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263 255

8,5
(a)

GA
8,0 MD
GA-MS
7,5
ln (E5371%)
MS-CH
MS-MD-CH
7,0

6,5

6,0
0 2 4 6 8 10 12
Storage time (wee ks)

8,5 (b)

GA
8,0
MD
7,5 GA-MS
ln (E4401%)

MS-CH
7,0 MS-MD-CH

6,5

6,0

5,5
0 2 4 6 8 10 12
Storage time (wee ks)

Fig. 1. First-order degradation plots of coloring strength (ln E vs time) for freeze dried microencapsulated beetroot (a) and saffron (b) extracts prepared with different agents
during storage at 40 ◦ C for 10 weeks.

The stabilizing effect of encapsulation on natural pigments
against heat during storage is well studied. In particular, Jafari,
He, and Bhandari (2007) reported that encapsulating agents can
act as physical obstacles that decrease the effects of oxygen,
light, heat and moisture on the encapsulated ingredients. Lim-
itation or absence of oxygen, which is the most deteriorative
agent, may as well decrease the negative effect of heat on natural
pigments (Jackman, Yada, Tung, & Speers, 1987). Moreover, con-
cerning moisture content, decrease in its value which occurs during
encapsulation (Jafari, Assadpoor, He, and Bhandari (2008)) is
another protective factor, that acts on reducing molecular mobility
and increasing the viscosity of the encapsulating agent in the glassy
state (Tonon, Brabet, & Hubinger, 2010).
3.2. Color stability of freeze dried microencapsulated beetroot
and saffron extracts
In order to investigate the effect of storage on the color of
the obtained encapsulated powders, color measurement was per-
formed. The color parameters (a* , b* , L* , C* and E* ) of the microen-
capsulated beetroot and saffron extracts immediately after produc-
tion and after 10 weeks storage at 40 ◦ C are presented in Table 2.

Table 1
Regression analysis of coloring strength in freeze dried microencapsulated beetroot and saffron extracts prepared with different agents during storage at 40 ◦ C for 10 weeks.

Sample Beetroot Saffron

−1
k (week ) t1/2 (weeks) R2
k (week−1 ) t1/2 (weeks) R2

GA 0.082 50.12 0.975 0.117 34.59 0.992

MD 0.079 53.03 0.983 0.069 60.03 0.879
GA–MS 0.091 44.66 0.977 0.086 47.12 0.958
MS–CH 0.163 23.61 0.876 0.114 30.77 0.987
MS–MD–CH 0.139 26.52 0.941 0.119 29.01 0.969

GA: gum Arabic; MD: maltodextrin; GA–MS: gum Arabic and modified starch; MS–CH: modified starch and chitosan; MS–MD–CH: modified starch, maltodextrin and chitosan,
k: rate constant and t1/2 : half-life period.
256 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263

Table 2
Color parameters (a* , b* , L* , C* and E* ) of freeze dried microencapsulated beetroot and saffron extracts prepared with different agents after production (0) and after storage
at 40 ◦ C for 10 weeks (t).

Sample L* a* b* C* E*

L0∗ Lt∗ a∗0 a∗t b∗0 b∗t C0∗ Ct∗

Beetroot
GA 64.64 ± 1.15a 54.80 ± 0.34a 35.27 ± 0.45b 25.22 ± 0.20e −2.91 ± 0.17b 14.35 ± 0.06a 35.39 ± 0.44b 29.02 ± 0.17a 22.28 ± 0.55b
MD 61.03 ± 4.61a 59.84 ± 1.01d 37.77 ± 1.12c 26.55 ± 0.53d −4.05 ± 0.10a 11.54 ± 0.41c 37.99 ± 1.11c 28.95 ± 0.65a 19.76 ± 1.24a
GA–MS 60.48 ± 3.56a 57.56 ± 0.39b 34.13 ± 1.86b 23.54 ± 0.41c −3.57 ± 0.37ab 14.26 ± 0.49a 34.32 ± 1.88b 27.52 ± 0.61b 21.23 ± 1.51ab
MS–CH 65.58 ± 3.94a 61.33 ± 0.24e 25.78 ± 1.54a 20.49 ± 0.37b 4.43 ± 1.00c 20.30 ± 0.15b 26.16 ± 1.68a 28.85 ± 0.37a 17.61 ± 0.24c
MS–MD–CH 64.46 ± 1.95a 58.83 ± 0.09c 27.92 ± 0.87a 17.43 ± 0.13a 3.90 ± 0.36c 20.45 ± 0.15b 28.19 ± 0.90a 26.87 ± 0.19b 20.46 ± 0.75ab

Saffron
GA 89.79 ± 0.67a 61.46 ± 0.12d −2.49 ± 0.08e 1.32 ± 0.03a 41.71 ± 0.40ab 31.58 ± 0.51a 41.78 ± 0.40ab 31.61 ± 0.51a 30.33 ± 0.89a
MD 89.09 ± 3.33a 59.88 ± 0.29c −6.60 ± 0.05b 2.47 ± 0.31b 44.71 ± 1.89b 31.85 ± 0.12a 45.19 ± 1.87b 31.95 ± 0.15a 33.18 ± 3.57ab
GA–MS 90.98 ± 0.48a 56.95 ± 0.80b −6.15 ± 0.13c 2.77 ± 0.40b 42.38 ± 0.15ab 30.82 ± 0.57d 42.82 ± 0.16ab 30.95 ± 0.60a 37.03 ± 0.53b
MS–CH 74.93 ± 3.09b 58.79 ± 0.16a −5.69 ± 0.13d −1.11 ± 0.07c 39.71 ± 3.63a 26.06 ± 0.03c 40.12 ± 3.58ac 14.44 ± 0.03b 21.73 ± 3.90c
MS–MD–CH 83.32 ± 0.55c 58.85 ± 0.41a −7.45 ± 0.05a 1.24 ± 0.07a 36.30 ± 0.74c 21.81 ± 0.20b 37.06 ± 0.73c 21.84 ± 0.20c 29.74 ± 0.41a

Mean of three replicates ± standard error. a,b,c,d Values with different letters in the same column differ significantly (P < 0.05). GA: gum Arabic; MD: maltodextrin; GA–MS:
gum Arabic and modified starch; MS–CH: modified starch and chitosan; MS–MD–CH: modified starch, maltodextrin and chitosan.

Based on the initial values of a* and b* parameters, in beetroot
case, MD, GA and GA–MS presented a purple shade of red color
in comparison with MS–CH and GA– MS–CH which showed a pink
one. The same trend was also observed for saffron samples, in which
MD, GA and GA–MS had an intense yellow color while MS–CH
and GA–MS–CH showed a pale shade of yellow. The initial vari-
ations among the samples were related to the different nature of
the agents used (Desobry, Netto, & Labuza, 1997).
In the case of beetroot, all powders had a high value of color
parameter a* which is attributed to their betacyanin content.
Jimenez-Aguilar et al. (2011) have similarly correlated a* value with
cyanin content in spray-dried blueberry microencapsulated prod-
ucts using mesquite gum. After 10 weeks of storage, the parameter
a* showed a decrease in all samples with MS–MD–CH presenting
the highest (P < 0.05) decrease of 37.52%. A slight reduction in light-
ness, expressed by L* parameter, was also observed in all beetroot
samples, but without being significantly affected by the type of the
agent used for encapsulation.
Concerning saffron samples, b* parameter, which characterizes
the extent of yellow color, best represented the observed color
changes, since this color was dominant. A decrease in its value with
storage time was recorded, with MS–CH and MS–MD–CH samples
suffering the greater reduction of 34.37 and 39.91%, respectively,
while MD showed the lowest reduction of 28.76%. A more pro-
found decrease in lightness (L* parameter) was observed compared
to beetroot samples, with reduction levels varying from 29.36
(MS–MD–CH) to 37.40 (GA–MS), respectively.
The chroma (C* ) and total color difference (E* ), which are
a combination of the chromatic coordinates, were also analyzed
(Table 2). These parameters are extensively used to evaluate the
color changes in foods during processing (Goncalves, Pinheiro,
Abreu, Branda, & Silva, 2007; Karabulut, Topcu, Duran, Turan, &
Bólent, 2007; Skrede et al., 1997). Parameter C* which indicates
the dullness/vividness of the product, ranging from 0 to 100, was
decreased with storage time in all samples, irrespectively of the
agent used. Based on the E* values, corresponding to the color
difference between initial and final time for a specific formulation,
the samples suffered significant changes during storage. High val-
ues of E* parameter means more color changes during treatment
or storage (Maskan, 2006).
Overall, both in beetroot and saffron samples, the type of the
agent significantly (P < 0.05) affected almost all the examined color
parameters with MD offering the best protection. The parameter a*
and b* best characterized the observed color changes in beetroot
and saffron samples, respectively. Similar observations have been
reported by other researchers as well. Desobry, Netto, and Labuza
(1999) and Desobry et al. (1997) who colorimetrically evaluated the
microencapsulation of carotenoids in maltodextrin systems also
found that L* (lightness) and a* (redness) were the most sensitive
parameters. Moreover, parameter a* was the most sensitive one
when studying the retention of betalains, encapsulated in gum Ara-
bic, during storage (Pitalua et al., 2010). In addition, Sutter, Buera,
and Elizalde (2007) and Spada, Norenab, Marczaka, and Tessaro
(2012) concluded that a* parameter best represented the color
changes in carotenoids encapsulated in a mannitol-phosphate and
starch matrix, respectively.
3.3. Water sorption isotherms of freeze dried microencapsulated
beetroot and saffron extracts
The water sorption isotherms describing the relationship
between water activity and equilibrium moisture content
(Shrestha, Howes, Adhikari, & Bhandari, 2007) are useful for food
products to predict their shelf life stability (Martinelli, Gabas, &
Telis-Romero, 2007).
The experimental moisture adsorption isotherms of the
microencapsulated saffron and beetroot extracts at room tempera-
ture (25 ◦ C) are given in Fig. 2. The sorption isotherms of all samples
were of sigmoidal shape. This form of isotherm is typical for most
food systems including encapsulated natural colorants (Desobry
et al., 1997; Nayak & Rastogi, 2010; Pavon-Garcia et al., 2011;
Rascón, Beristain, García, & Salgado, 2011; Serris & Biliaderis, 2001).
The model that best fit to the experimental data (water activ-
ity versus moisture content) for all samples was the GAB model,
considering both the lowest value of the average relative deviation
(E) and the highest coefficient of determination (R2 ).
Similar observations on the fitting of sorption models like BET
and GAB have also been reported on freeze-dried beetroot pig-
ments (Serris & Biliaderis, 2001) and spray-dried carotene pigments
(Wagner & Warthesen, 1995). Moreover, Nayak and Rastogi (2010)
and Pavon-Garcia et al. (2011) have obtained similar results dur-
ing their sorption studies on spray-dried anthocyanins and natural
colorants from Justicia spicigera plant, respectively.
The resulting parameters values of the BET and GAB model for
both saffron and beetroot microencapsulated extracts are given
in Table 3. The parameter of monolayer value (mm ) is of partic-
ular interest, as it indicates the amount of water that is strongly
adsorbed to specific sites and is considered as the optimum
value at which a food is more stable against microbial spoilage
(Labuza, 1984). In the present study, the estimated monolayer
value (mm ) fell within the range of 3.27–14.40 g H2 O/100 g dry
weight and 4.70–11.33 g H2 O/100 g dry weight for freeze dried
 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263 257

(a) GA
35
MD
30

g H2O / 100g dry weight

GA-MS
25 MS-CH

20 MS-MD-CH

15

10

0
0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90

water activity (a w)

(b) GA
35 MD
30 GA-MS
g H2O / 100g dry weight

MS-CH
25
MS-MD-CH
20

15

10

0
0,00 0,10 0,20 0,30 0,40 0,50 0,60 0,70 0,80 0,90

water activity (a w)

Fig. 2. Water isotherms for freeze dried microencapsulated beetroot (a) and saffron (b) extracts at various water activities.

Table 3
Water isotherm model (BET and GAB) parameters for freeze dried microencapsulated beetroot and saffron extracts prepared with different agents.

Sample BET model GAB model

2
mm C R E% mm K C R2 E%

Beetroot
GA 5.46 −7.04 0.884 14.97 10.74 0.78 19.83 0.926 4.82
MD 3.73 −7.44 0.845 16.55 7.34 0.78 14.45 0.933 4.36
GA–MS 3.27 −5.77 0.908 14.78 9.59 0.62 21.06 0.943 5.65
MS–CH 6.04 −17.08 0.905 8.95 14.40 0.38 18.95 0.953 6.46
MS–MD–CH 4.61 −6.03 0.848 18.28 10.12 0.73 19.21 0.951 4.02

Saffron
GA 5.43 −6.34 0.894 15.35 9.70 0.81 40.54 0.980 2.77
MD 4.69 −19.36 0.956 8.66 6.18 0.93 34.62 0.986 3.33
GA–MS 6.49 −19.25 0.930 9.84 9.58 0.80 32.37 0.940 6.57
MS–CH 5.71 −12.50 0.913 33.21 11.13 0.56 26.88 0.949 5.20
MS–MD–CH 5.24 −7.64 0.845 19.03 11.33 0.75 16.60 0.888 7.06

mm : Monolayer moisture content; C, K: model constants; aw : water activity; R2 : coefficient of determination; E: mean relative deviation modulus.

microencapsulated beetroot and saffron extracts, respectively,
depending on the equation used for estimation and on the type of
the encapsulating agent. Regarding the type of the encapsulating
agent and based on GAB model, MS–CH and MS–MD–CH presented
the highest mm values of 14.40 and 11.33 g H2 O/100 g dry weight,
for beetroot and saffron samples, respectively. Serris and Biliaderis
(2001) obtained lower values of mm (3.03–6.31 g H2 O/100 g dry
weight) for beetroot pigments encapsulated in maltodextrin and
pullulan by freeze drying. This could be attributed to the different
nature of the agents used that affects their adsorption behavior.
For biopolymers, the process not only involves adsorption but
also structural changes (crystalline or amorphous) due to swelling,
physical and/or chemical changes occurring during the drying pro-
cess (Pérez-Alonso, Beristain, Lobato-Calleros, Rodríguez-Huezo,
& Vernon-Carter, 2006). The parameter C of the GAB model
is related to the heat of adsorption of water on the powders.
258 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263

Fig. 3. Physical characteristics of freeze dried microencapsulated beetroot (a) and saffron (b) extracts prepared with different agents at various water activities during the
determination of the moisture adsorption isotherms.

Lewicki (1997) stated that C values should fall within the range of
5.67 ≤ C < ∞ for describing properly an isotherm mathematically. In
our case, the observed C values fell within the aforementioned limit.
Particularly, GA–MS followed by GA presented the highest C val-
ues of 21.06 and 19.83 for beetroot samples while GA showed
also the highest C value of 40.54 for saffron samples. The param-
eter K provides a measure of the interaction of the molecules in
the multilayers with the adsorbent, and tends to fall between the
energy value of the molecules in the monolayer and that of liquid
water. When K equal 1 the multilayers have the properties of liquid
water (Pérez-Alonso et al., 2006). The values of K for both beetroot
and saffron microencapsulated extracts (Table 3) fell within the
range of 0.24 < K ≤ 1, which according to Lewicki (1997) describe
properly an isotherm mathematically. Among the studied encap-
sulating agents, MS–CH presented the lowest K values of 0.38 and
0.56 for beetroot and saffron samples, respectively, while GA and
MD showed similar values for both extracts. Concerning the GAB
parameters (especially K), similar values have been reported by
other researchers as well (Pérez-Alonso et al., 2006; Rascón et al.,
2011).
The physical characteristics of the powders subjected to vari-
ous humidity levels demonstrated that there was apparent phase
change in the produced samples (Fig. 3). In particular, both GA and
MD microencapsulated beetroot and saffron extracts at aw = 0.82
were in a liquid form of high viscosity while MD presented a liquid
form also at aw = 0.66 (Fig. 3). Similar observations have also been
reported in other research works (Rascón et al., 2011; Righetto &
Netto, 2005), according to which carotenoids and juice from acerola
encapsulated in MD and/or GA at aw ≥ 0.74 became unable to keep
their structural integrity leading to collapse and liquefaction.
 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263 259

Fig. 4. Representative outer topography of MD (a), GA (b), GA–MS (c), MS–MD–CH (d) and MS–CH (e) freeze dried microencapsulated saffron extract (magnification ×500).

3.4. Microstructure of freeze dried microencapsulated saffron 3.5. Multivariate analysis

extracts
The outer topographies of freeze-dried encapsulated saffron
extracts were assessed by SEM (Fig. 4). SEM examination showed
an amorphous glass-like formation, which according to some
researchers is considered that protects the entrapped molecules
from exposure to heat and oxygen (Goubet, Le Quere, & Voiley,
1998; Kaushik & Roos, 2007; Porzio, 2004; Roos, 1995). Similar
images, characteristic of freeze-dried encapsulated in GA and MD
pigments are reported by other researchers as well (Khazaei et al.,
2014; Sousdaleff et al., 2013). The analysis, also, showed that MD
presented less particle differences in comparison to GA, in which
the differences in size of its particles may be attributed to its highest
bulk density and lowest special volume (Khazaei et al., 2014).
The results obtained based on the principal component analysis
(PCA) are presented in Fig. 5. In the case of beetroot samples, the two
principal components accounted for 93.45% of the variance in the
data (Fig. 5a). The first principal component (71.62% of the total data
inertia) was positive for Et , a∗t and t1/2 while the parameters b∗t and
k presented negative values. Consequently, this component (PC1)
was probably related to the degree of betalains degradation. Con-
cerning saffron samples, the two principal components accounted
for 86.06% of the variance in the data (Fig. 5b). The first principal
component (PC1) was positive for k while parameters Et , b∗t , Ct∗ and
a∗t , t1/2 and E* presented negative values. As in the case of bee-
root, it could be interpreted in saffron as the indicative axis of the
carotenoids degradation.
260 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263
Fig. 5. Principal component analysis of the coloring strength and color parameters
of freeze dried microencapsulated beetroot (a) and saffron (b) extracts prepared
with different agents.
According to the multiple linear correlation, in the case of beet-
root samples, the highest Pearson correlation coefficients were
observed between k and t1/2 (−0.99), Et and t1/2 (0.98) and between
Et and b∗t (−0.98). The coloring strength (Et ) showed the highest
correlation coefficient with a∗t (0.97) and a negative relationship
with k (−0.94). In contrast, it displayed low correlation coefficients
with the other color parameters; Et and Ct∗ (0.50), Et and E*
(0.52) and Et and Lt∗ (−0.49). The halt life half-life period (t1/2 )
showed a positive correlation with a∗t (0.91) and a negative one
with b∗t (−0.95). Among the color parameters the stronger correla-
tions were observed between Lt∗ and E* (−0.94) and between a∗t
and b∗t (−0.94). Concerning the saffron samples, the highest Pear-
son correlation coefficients were also observed between k and t1/2
(−0.98), b∗t and Ct∗ (0.99) and between a∗t and E* (−0.98). The col-
oring strength (Et ) showed the highest correlation coefficient with
b∗t (0.97) and Ct∗ (0.95), while displayed low correlation coefficients
with the other color parameters; Et and a∗t (0.67), Et and E* (0.63)
and Et and Lt∗ (−0.34).
In the current study, based on the multivariate analysis, colori-
metric analysis proved to be a useful tool for evaluating pigment
degradation from the natural colorants sources. Other studies
have also shown a strong relationship between the color param-
eters (such as a* ) and pigments content (carotenoid) (Bengtsson,
Namutebi, Alminger, & Svanberg, 2008; Ramakrishnan & Francis,
1973; Spada et al., 2012; Takahata, Noda, & Nagata, 1993).
3.6. Application in chewing gum model—Color stability test
The evolution of color parameters of the chewing gum samples
with freeze dried microencapsulated beetroot and saffron extracts
during storage at 25 and 40 ◦ C is presented in Figs. 6 and 7. The
color parameters of the prepared chewing gum samples with both
beetroot and saffron extracts depended significantly (P < 0.05) on
the form of the extract (pure or encapsulated), the type of agent
and the storage temperature.
Storage temperature affected significantly (P < 0.05) the L*
parameter; increase in storage temperature resulted in a decrease
in L* values in all samples (Figs. 6a and c and 7a and c). Concerning
a* color parameter (Fig. 6b and d), it decreased with storage time
and temperature, suffering greater decrease when samples were
stored at 40 ◦ C compared to 20 ◦ C. In particular, it was observed that
a* color parameter almost disappeared after two weeks of storage
at 40 ◦ C (0.41, 0.83, −0.37 and −0.45 for pure extract, GA, MS–CH
and MS–MD–CH, respectively). The chewing gum samples pre-
pared with GA–MS were found to offer the best protection against
color degradation at storage temperature or even at accelerated
temperature, followed by MD ones.
Concerning b* color parameter (Fig. 7b and d), it decreased with
time regardless of the storage temperature. A similar behavior
of GA–MS protection was observed in both saffron and beetroot
samples. In particular, the chewing gum samples with the encap-
sulated in GA–MS saffron extracts presented the greatest (P < 0.05)
b* values (average values of 36.20 and 38.76 at 25 and 40 ◦ C, respec-
tively). The high b* values observed in chewing gum samples with
incorporation of GA–MS saffron extract and stored at 40 ◦ C may be
attributed only to the inherent color of the GA matrix and chewing
gum base since the color of the extract was already diminished.
Overall, color parameters of the prepared chewing gum sam-
ples as well as their evolution during storage depended greatly on
the type of agent used and storage conditions (time and temper-
ature). Among the agents used, GA–MS provided the best color
stability to the examined extracts. The chewing gum samples
produced with coloring extracts encapsulated in GA–MS showed
the greatest a* (for beetroot) and b* (for saffron) values indicat-
ing a better protection. Similar observations have been reported
by other researchers as well. Marcolino, Zanin, Durrant, Benassi,
and Matioli (2011) also received positive results with regard to
color stability when incorporating curcumin, encapsulated in b-
cyclodextrin, into a yogurt food system. According to Gomes, Petito,
Costa, Falcão, and de Lima Araújo (2014) the red bell pepper extract
encapsulated in b-cyclodextrin could simulate the color of com-
mercial colorants in yogurt, providing high color stability compared
to the crude bell pepper extract. Moreover, pasta prepared with
potassium norbixinate and ice cream prepared with curcumin,
both colorants encapsulated in MD with freeze drying technique,
resulted in greater color stability compared with samples prepared
with non-encapsulated colorants (Sousdaleff et al., 2013). The cur-
rent incorporation study demonstrated the feasilbilty of production
 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263 261

(a) (b)
85 35
Pure extract Pure extract
80 30
MD MD
75 GA 25 GA
L * parameter

α * parameter
70 GA-MS 20 GA-MS
MS-CH 15 MS-CH
65
MS-MD-CH MS-MD-CH
60 10

55 5
0
50
0 5 10 15
0 5 10 15 -5
Storage time (days) Storage time (days)

(c) (d)
35
85 Pure extract
Pure extract 30
80 MD
MD 25
75 GA
GA
L * parameter

α * parameter
20 GA-MS
70 GA-MS
MS-CH 15 MS-CH
65
MS-MD-CH 10 MS-MD-CH
60
5
55
0
50
0 5 10 15
0 5 10 15 -5
Storage time (days) Storage time (days)

Fig. 6. Changes of L∗ (Lightness), and a∗ (redness) color parameters of chewing gum samples prepeared with freeze dried microencapsulated beetroot extracts during storage
at 25 and 40 ◦ C. (a) L∗ parameter of beetroot samples stored at 25 ◦ C, (b) a∗ parameter of beetroot samples stored at 25 ◦ C, (c) L∗ parameter of beetroot samples stored at 40 ◦ C
and (d) a∗ parameter of beetroot samples stored at 40 ◦ C.

90 (a) 45 (b)
Pure extract Pure extract
88
40
MD MD
86
GA 35 GA
L * parameter

b * parameter

84
GA-MS GA-MS
82 30
MS-CH MS-CH
80
MS-MD-CH 25 MS-MD-CH
78
20
76
74 15
0 5 10 15 0 5 10 15
Storage time (days) Storage time (days)

90 (c) 45 (d)
Pure extract Pure extract
85 40
MD MD
80 GA GA
35
b * parameter
L * parameter

75 GA-MS GA-MS
30
70 MS-CH MS-CH
MS-MD-CH 25 MS-MD-CH
65

60 20

55 15
0 5 10 15 0 5 10 15
Storage time (days) Storage time (days)

Fig. 7. Changes of L∗ (Lightness) and b∗ (yellowness) color parameters of chewing gum samples prepeared with freeze dried microencapsulated saffron extracts during
storage at 25 and 40 ◦ C. (a) L∗ parameter of saffron samples stored at 25 ◦ C, (b) b∗ parameter of saffron samples stored at 25 ◦ C, (c) L∗ parameter of saffron samples stored at
40 ◦ C and (d) b∗ parameter of saffron samples stored at 40 ◦ C.
262 C. Chranioti et al. / Carbohydrate Polymers 127 (2015) 252–263
of freeze dried microencapsulated beetroot and saffron extracts
that can attain their coloring strength and thus could be used as
food-grade colorants. However, the incorporation procedure needs
to be evaluated for other kinds of foods (such as those of high
acidity), so as to verify the extension of their use.
4. Conclusion
MD along with GA proved to be effective agents for beetroot
and saffron coloring extracts microencapsulation by freeze drying.
MD presented the greatest protection against heat during storage
accompanied also by high half-life period (t1/2 ) with average val-
ues of 53.03 and 60.03 weeks for beetroot and saffron, respectively.
The water sorption study showed that MD and GA retained their
structural integrity up to water activities of 0.66 and 0.82, respec-
tively. The incorporation study demonstrated higher stability for
food model of low moisture such as chewing gum prepared with
extracts encapsulated in GA–MS. To conclude, microencapsulation
by freeze drying could be suggested not only as a suitable method
for color stabilizing of beetroot and saffron extracts, but also as a
feasible mean of production of food-grade colorants.
Acknowledgment
Charikleia Chranioti is grateful to the State Scholarships Foun-
dation of Greece (IKY) for financial support.
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.carbpol.
2015.03.049.
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