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Article history:
Received 16 November 2011
Received in revised form 1 March 2012
Accepted 10 March 2012
Available online 30 March 2012
Keywords:
Chilli
Carotenoids
Non-aqueous extract
Antioxidant activity
Microencapsulates
a b s t r a c t
Antimicrobial, antioxidant, and pro-vitamin properties have been attributed to Capsicum based on the
carotenoid and polyphenolic compound content. The aim of this study was to obtain and characterize
non-aqueous extracts of Capsicum annuum L. (chilli) using three different oils (corn, sunower, and safower) as well as their microencapsulates. Corn extract showed better antioxidant activity and total
carotenoid content (as b-carotene) than sunower and safower extracts. The extracts were encapsulated
by spray-drying with a biopolymers:extract-solution ratio of 4:1 (w/w). The biopolymers were gum
Arabic and maltodextrin. In addition, the encapsulation efciency, mean particle size, morphology, water
activity, moisture content, and stability were evaluated for the microcapsules. The retention of antioxidant activity after encapsulation varied from 76% to 80% of its activity in the oily extract, whereas the
preservation of carotenoids in microcapsules was between 84% and 86%.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Capsicum annuum L. (chilli) is widely used for culinary and
industrial purposes due to its characteristic avour and colour
(Topuz et al., 2009). Antimicrobial, antioxidant/free-radical, and
pro-vitamin properties have been attributed to C. annuum, given
its content of polyphenolic compounds and carotenoids
(Acero-Ortega et al., 2005; Hornero-Mendez et al., 2000; Topuz
and Ozdemir, 2007). Chillies are good sources of carotenoids;
approximately 25 different carotenoids have been identied in
C. annuum L. grossum Sendt, including b-carotene, a-carotene,
b-cryptoxanthin, zeaxanthin, luteine, capsanthin, capsorubin,
and cryptocapsin, which are the most abundant components
(Collera-Ziga et al., 2005). Provitamin A, carotenoids (bcarotene, a-carotene, and b-cryptoxanthin), and xanthophylls
may be important for the prevention of age-related macular degeneration and cataracts (Matsufuji et al., 1998; Seddon et al., 1994).
The antioxidant activity of the carotenoid pigments is due to the
following characteristics of their structure: (a) the polyene carbon
chain, which permits the incorporation of free radicals by addition
mechanisms, which slows propagation, and (b) functional groups
on the b-rings, such as keto groups as well as the esteried form,
Corresponding author. Tel.: +52 5557296000x62464.
E-mail address: liliana.alamilla@gmail.com (L. Alamilla-Beltrn).
0260-8774/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2012.03.032
30
extraction media may be a novel option for non-aqueous extraction processes that have potential advantages in health, safety,
and ecologically-related issues. To date, the use of edible oils as
non-polar dissolvents for the extraction of carotenoids from Capsicum has not been explored. Once carotenoids are extracted, isomerization (Xianquan et al., 2005) and oxidative reactions may occur
as a consequence of the exposure of carotenoids to ambient temperature and oxygen as well as the presence of unsaturated double
bonds in the molecular structure of carotenoids, which causes molecule fractioning and the production of apocarotenoids (Maoka
et al., 2001) or epoxides. For this reason, the extracts must be
carefully handled. However, one possible approach for minimizing
or inhibiting deteriorative reactions of carotenoids in extracts of
Capsicum is by protecting the extracts in microcapsules.
Microencapsulation through spray-drying is an effective technique that allows for the protection of food ingredients, such as
carotenoids (Rocha et al., 2012) and polyphenols (Fang and Bhandari, 2010), from chemical deterioration and environmental factors
by converting liquids into functional powders that can be incorporated into various formulations. This protection was performed
using wall materials, such as sugars, gums, proteins, natural and
modied polysaccharides, lipids, and synthetic polymers (Gharsallaoui et al., 2007; Fang and Bhandari, 2010; Rocha et al., 2012).
It has been reported that both protein and lipid fractions that are
part of the molecular structure of gum Arabic provide emulsifying
properties, lm formation, low viscosity and high water solubility
(Yadav et al., 2007). Similarly, it has been postulated that more
hydrophobic polypeptide chains adsorbed at the surface of the oil
droplets with hydrophilic carbohydrate blocks attached to the
chain protruding out into solution can provide a stronger steric
barrier that prevents droplet aggregation and coalescence (Islam
et al., 1997). Maltodextrin exhibits various favourable characteristics, including high hydrosolubility, low emulsifying capacity, low
retention of volatiles, and high protection against oxidation (Buffo
and Reineccius, 2000). During microencapsulation by spray-drying,
dry particles are obtained in the form of powders or agglomerates
that can enhance the shelf-life of food products (Soottitantawat
et al., 2005). Based on these studies, the objective of this work
was to explore the extraction of carotenoids from chilli peppers
by using non-aqueous media, such as edible oils, and the preservation of the extracted compounds in the form of microcapsules,
which could be used as an ingredient in food processing.
2. Materials and methods
2.1. Materials
The C. annuum L. grossum Sendt (chilli) used for this work as
well as the corn (Maceite), sunower (Patrona) and safower
(Oleico) oils were purchased from a local market in Mexico City,
Mexico. The wall materials used for microencapsulation were
gum Arabic, food grade E 414 (Distribuidora Qumica LEFE S.A de
C.V., Mexico City, Mexico) and maltodextrin 20 DE (CPI Ingredientes S.A de C.V, State of Mexico, Mexico). Potassium persulfate
(K2S2O8) and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid
(ABTS) radical were purchased from Sigma Chemical Company,
St. Louis, MO, USA.
2.2. Methods
2.2.1. Extraction of carotenoids from C. annuum L. grossum Sendt
(chilli)
Dried chilli peppers were selected and manually cleaned to
eliminate foreign matter. The stems were also removed prior to
dry grinding. The resulting powder was sieved using a 25 mesh
Ast0 Astf
h
i
%Inhibition
Adt0 Adtf
Ast0
Adt 0
where Ast0 and Astf are the absorbance of sample at initial and nal
times; Adt0 and Adtf are the absorbance of dissolvent at initial and
nal times.
A standard curve was constructed using different concentrations of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Sigma Chemical Company, St. Louis, MO, USA). The
results were presented as lmol Trolox/mL. The antioxidant activity
determined for pure oils were subtracted from the antioxidant
activity of the extracts.
2.2.3. The Folin test
The Folin test for NAEC and NAEC microcapsules was determined
using the FolinCiocalteau reagent (Sun et al., 2007). The reagent
was diluted 10 times with deionized water. Each NAEC was diluted
at a ratio of 1:5 with ethanol, and a 0.1 mL aliquot was mixed with
0.75 mL of the diluted FolinCiocalteau reagent. After the reaction
solution had been incubated at room temperature (RT) for 5 min,
0.75 mL (60 g/L) sodium bicarbonate solution was added and thoroughly mixed. The mixture was incubated at RT for 90 min and then
ltered by using a 0.45 lm syringe lter (Corning, Germany). The
absorbance of the solution was then determined at 750 nm. Gallic
acid was used as standard reference and the results were expressed
as gallic acid equivalents (lg) per mL of extract.
2.2.4. Extraction of carotenoids with fatty acids
Carotenoid extracts of chilli powder were obtained through
homogenization with stearic and oleic acids at 70 C for 5 min to
evaluate carotenoid afnity with saturated or unsaturated fatty
acids. This temperature of extraction was selected due to the fact
that stearic acid is in a solid state below 70 C.
2.2.5. Preparation of oil-in-water (o/w) emulsions and the spraydrying process
Gum Arabic (GA; 10 g) and maltodextrin (MD; 10 g) were
dissolved in 100 mL of distilled water using a mixer (Stir-Pak
31
Eo
AT AS
AT
x100
where AT and AS are the total content of NAEC and the content of
extractable portion of encapsulated NAEC, respectively.
2.2.11. Encapsulation efciency (EE)
The EE was calculated as the ratio between the initial content of
total carotenoid present in the capsules and the total carotenoid
content in the extract used to produce them. The EE was analysed
based on b-carotene as a target chemical compound that has been
reported as the major component in chilli (Collera-Ziga et al.,
2005). The total carotenoid content in extracts and microcapsules
(as b-carotene content) was determined using the procedure described in Section 2.2.7.
2.2.12. Determination of the mean particle size of microcapsules
A particle size analyzer (Mastersizer 2000, Malvern Instruments
Ltd., Malvern, Worcestershire, UK) was used to determine the
Sauter mean diameter (d3,2) of the particles (Jafari et al., 2007).
2.2.13. Stability tests for microencapsulates
The stability of microencapsulated NAEC was determined by
measuring the antioxidant activity (Section 2.2.6) and water activity (Section 2.2.8) of the microcapsules after 60 d of storage at
20 5 C.
2.2.14. Statistical analysis
A one-way analysis of variance (ANOVA) with 2a = 0.05 signicance level was applied to all results. The Tukeys test was used to
compare differences between mean values of individual samples
(Minitab 15.0 software, Minitab Inc., USA).
32
mL. These results suggested that saturated fatty acids had a better
afnity for carotenoids and exhibited enhanced stability compared
to extracts obtained by using unsaturated fatty acids, despite both
types having hydrocarbon chains of 18 carbon atoms. Carotenoids
consist of a skeleton of 40 unsaturated carbon atoms, which provides
hydrophobic specicity, and differences in the antioxidant activity of
extracts may be due to a higher chemical afnity of carotenoids for
saturated fatty acids than for unsaturated fatty acids in the oils. This
may be due to differences in the molecular conformation of
saturated and unsaturated fatty acids, which have one or more rigid
torsions in their hydrocarbon chains due to the inability to present
bond rotation. In the case of saturated fatty acids, the extended form
(linear) conguration prevails because this structure has the lowest
energy (Lehninger et al., 1995; Kaneko et al., 1998).
Hyeon and Young (2008) evaluated the antioxidant activity of
seed and pericarp of red pepper. Extraction was performed with
70% ethanol for 3 h, and antioxidant activity was measured using
various chemical assays, including the ABTS+ method. The seed
showed a radical scavenging activity of 29% at 1000 lg/mL extract,
while pericarp had 51% activity. In our study, the NAEC of corn,
sunower, and safower oils showed 90%, 75%, and 60% radical
scavenging by ABTS+, respectively. The values observed by Hyeon
and Young (2008) were lower than those obtained with NAEC,
which may be due to the different mechanisms of action that phenolic compounds, avonoids, and carotenoids have in the presence
of the ABTS radical. The NAEC were obtained at moderate temperature and extraction time (60 C and 5 min), which further reduced
the possibility of compound degradation and represents a technological advantage for food-related applications, since volatile solvents were not used for the extraction.
Table 1 shows the results of the Folin test and antioxidant activity, which demonstrated that the highest temperature used for
extraction (80 C) and longer time to homogenization (10 min)
caused a negative effect on the compounds detected. The highest
antioxidant activity was observed in extracts made with corn oil.
Therefore, the compounds detected by the Folin reagent play an
important role in the antioxidant activity of non-aqueous extracts
of chilli.
3.2. Antioxidant activity, carotenoid content, and Folin test of
microencapsulated NAEC
All microencapsulated NAEC collected from the drier retained
7680% of the antioxidant activity of the respective extracts (Table
Table 1
Conditions of extraction, antioxidant activity, and Folin test of NAEC.
Oils used for extraction
Temperature (C)
Time (min)
Antioxidant Activity of
NAEC (lmol Trolox/mL)
Corn
60
60
70
70
80
80
5
10
5
10
5
10
78.5 0.7a
83.1 2.0a
68.2 1.6b
55.9 0.5c
61.3 2.3d
52.2 2.4e
57.3 0.7a
63.5 0.6b
40.6 0.4c
55.8 1.4a
45.7 0.4d
47.4 1.8d
Sunower
60
60
70
70
80
80
5
10
5
10
5
10
67.3 1.1a
60.9 0.4b
43.7 2.5c
46.3 1.8c
51.4 2.0d
38.4 0.2e
50.3 0.4a
57.9 0.8b
36.4 1.4c
40.2 1.9c
35.6 0.3c
26.6 0.5d
Safower
60
60
70
70
80
80
5
10
5
10
5
10
51.7 0.1a
36.9 0.9b
47.1 1.0c
35.4 0.4b
48.3 0.3c
42.1 0.6d
32.3 0.1a
27.9 0.4b
30.8 0.7a
37.2 0.3c
32.8 0.2a
18.3 0.4d
Statistical analysis was performed by the type of oil used, and different letters in the same column indicate signicant difference at p 6 0.05.
33
Corn
Sunower
Safower
72.0 0.5a
159.1 1.8c
231.1 2.3e
68.4 0.6b
153.1 0.4c
221.4 1.1f
64.4 1.0b
144.3 0.5d
208.6 6.0f
Statistical analysis was performed by the type of oil used for extraction, and different letters indicate signicant difference at p 6 0.05.
a
Isochromatic families are CR: capsanthin and capsorubin; CY: zeaxanthin, bcryptoxanthin, and b-carotene.
Table 3
Characteristics of microencapsulated NAEC obtained from three different extracts.
Corn
Sunower
Safower
80.5 3.1a
60.3 0.5c
139.9 0.04e
200.3 0.6g
4.0 0.05j
0.15 0.01l
90.7 0.5m
86.6 1.4n
11.8 1.5o
80.0 3.1a
58.8 0.6c
126.9 1.1f
185.8 0.5h
5.0 0.1k
0.18 0.01l
90.4 0.6m
83.9 1.5n
11.1 1.4o
76.5 3.1b
53.3 0.2d
122.4 0.9f
175.8 0.7i
3.9 0.03j
0.19 0.01l
91.6 0.6m
84.2 1.0n
12.7 0.5o
Statistical analysis was performed by the type of oil used for extraction, and different letters indicate a signicant difference at p 6 0.05.
a
Values based on the antioxidant activity with respect to the non-aqueous extracts.
b
Isochromatic families are CR: capsanthin and capsorubin; CY: zeaxanthin, b-cryptoxanthin, and b-carotene expressed as lg/mL of extract.
34
Fig. 1. Scanning electron microscopy (SEM) micrographs of the microcapsules obtained from extracts using (a) corn oil, (b) sunower oil, and (c) safower oil.
The viscosity of the rened oils, which constitute the oil phase of
emulsions, was found to be 51.44 (25 C), 48.98 (25 C), and 52
(30 C) cP for corn, sunower, and safower oils, respectively
(Abromovic and Klofutar, 1998; Khtoon and Krishna, 1998; Rogers
et al., 2011).
Fig. 2. Distribution of particle size for the microcapsules obtained from extracts
using corn oil, sunower oil, and safower oil.
35
evaluated at time zero. The yellow and red fractions as well as total
carotenoid content are shown in Table 4. Total carotenoid content
in the microencapsulated NAEC decreased by 22.1%, 22.7%, and
27.6% for NAEC prepared with corn, sunower, and safower oils,
respectively, during the storage period. The carotenoids from
microcapsules of NAEC prepared with corn oil had the highest antioxidant activity, which may be due to different fatty acid composition of the oils. Corn oil has a higher proportion (12.2%) of
saturated fatty acids compared to sunower (10%) and safower
(7.42%) oils, according to Mexican Standard References (NMX-F030-SCFI-2005; NMX-F-265-SCFI-2005; NMX-161-SCFI-2007),
which makes it less susceptible to degradation by oxidation. The
oxidation of fatty acids is a chain reaction due to various factors,
such as temperature, light, and the presence of metals. Once this
reaction is initiated in the fatty acids, it continues in the carotenoids (co-oxidation) (Takahashi et al., 2003). Saturated fatty acids
are more stable than polyunsaturated and monounsaturated fatty
acids, suggesting that carotenoids are oxidated more rapidly by
polyunsaturated oils than by monounsaturated oils, and much less
rapidly by saturated fatty acids. Therefore, the only way by which
carotenoids can undergo greater oxidation in oil containing more
saturated fatty acids is if the saturated oils were oxidated before
contacting the carotenoids (Omara-Alwala et al., 1985; Bezbradica
et al., 2005; Sachindra and Mahendrakar, 2005). Changes in antioxidant activity and total carotenoid content in the stored microcapsules could also be related to degradation reactions of lipids and
carotenoids by complex mechanisms of co-oxidation (Takahashi
et al., 2003) or to the presence of MD (D-glucose) and GA (that
has a protein portion), which may trigger the Maillard reaction that
usually occurs during food processing and storage (Pitalua et al.,
2010).
Corn oil has good oxidative stability, which is partially attributed to the nonrandom distribution of fatty acids on triglycerides.
It has been determined that 98% of the fatty acids esteried in the
sn-2 position of corn oil triglycerides are unsaturated, leaving the
outer sn-1 and sn-3 positions with possibilities of binding saturated fatty acids and the unsaturated acids that may still be present
in the oil. Therefore, since the outer positions of the triglycerides
are more reactive, the polyunsaturated fatty acids in the sn-2 position present a certain degree of protection from oxidation (OBrien,
2004).
The extractable fraction of the oil phase, which has greater
exposure to oxygen, was more susceptible to the oxidation processes than the encapsulated fraction (Shimada et al., 1991;
Velasco et al., 2000) which is less exposed to oxygen, in the NAEC
microcapsules prepared with sunower and safower oils. The
opposite was observed for microcapsules prepared with corn oil,
even though a similar percentage of non-encapsulated oil was
present for the three types of NAEC microcapsules. This observation could be due to the above mentioned lower proportion of
unsaturated fatty acids present in corn oil compared with sunower and safower oils.
The loss of antioxidant activity and total carotenoid content of
the microencapsulates may be directly related to the amount of
extractable or non-encapsulated oil containing carotenoids in the
microcapsules, which is exposed to oxygen and causes oxidation
of carotenoids. In addition, the presence of open pores in the wall
of microcapsules allows accessibility of oxygen to NAEC structures.
Mrquez-Ruiz et al. (2003) reported that the extractable oil fraction in matrices containing an oily phase is affected by numerous
variables, but in some cases, the extractable oil was more prone
to oxidation than the non-extractable fraction.
The delivery and controlled release of carotenoids from microcapsules are decisive in understanding the effects of the active
agent for a particular application and impact their availability.
The release of bioactives from capsules is affected by the chemical
36
Table 4
Characteristics of NAEC microcapsules after storage for 60 d at 20 5 C.
Corn
a
Sunower
a
72.0 0.8
59.2 0.1c
96.8 1.2d
156.0 3.9g
0.17 0.01l
3.06 0.3m
Safower
a
68.1 2.2
54.3 0.03c
89.3 0.5e
143.6 0.1h
61.3 3.4b
51.3 0.04c
75.9 0.5f
127.2 1.0i
0.18 0.01l
5.50 0.2n
0.15 0.0l
5.02 0.07n
Statistical analysis was performed by the type of oil used for extraction, and different letters indicate a signicant difference at p 6 0.05.
a
Values based on the antioxidant activity with respect to the non-aqueous
extracts.
b
Isochromatic families are CR: capsanthin and capsorubin; CY: zeaxanthin,
b-cryptoxanthin, and b-carotene expressed as lg/mL of extract.
37