Food Bioprocess Technol (2014) 7:2159–2177
DOI 10.1007/s11947-013-1163-z
ORIGINAL PAPER
Canola Oil Encapsulated by Alginate and Its Combinations
with Starches of Low and High Amylose Content:
Effect of Quercetin on Oil Stability
Dongxiao Sun-Waterhouse & Wei Wang &
Geoffrey I. N. Waterhouse
Received: 20 May 2013 / Accepted: 15 July 2013 / Published online: 30 July 2013
# Springer Science+Business Media New York 2013
Abstract Co-extrusion encapsulation in combination with
antioxidant (quercetin, vitamin E or BHT) addition was
employed to reduce deterioration of unsaturated canola oil.
Alginate only (A), alginate–Novation 2300 starch with 1.4 %
amylose (A-N), and alginate–Gelose 80 starch with 89.8 %
amylose (A-G) were used as encapsulants, and storage trials of
encapsulated oil beads were conducted at 20 °C and 38 °C.
Microscopy examination confirmed that the freshly prepared
1 %A, A-N, and A-G beads generally had spherical shape,
with the 1 %A beads possessing smoother surfaces. The
average diameter and wall thickness of the freshly prepared
beads decreased in the order of A-G (380 μm) > 1 %A
(348 μm)>A-N (310 μm) beads, and 1 %A (70 μm)>A-N
(60 μm)>A-G (50 μm) beads, respectively. After the treat-
ment at pH 3 for 2 h, these three types of beads remained intact
but shrank significantly with the A-G beads exhibiting the
largest contraction. From the bead storage trials, primary oil
oxidation dominated over secondary oil oxidation and oil
hydrolytic rancidity. Quercetin was more powerful in sup-
pressing primary oil oxidation than vitamin E and comparable
to BHT. Overall, the three types of shell formulations were
suitable for protecting unsaturated oil, with the A-N and A-G
shells forming the best and worst barriers, respectively. The
1 %A shell could be an alternative to A-N for a prolonged
storage at 38 °C. Fourier-transform infrared spectra of the
beads were consistent with the bead formulations, indicating
roughly similar chemical composition before and after stor-
ages despite some detected spectroscopic differences. High-
performance liquid chromatography analyses showed the
D. Sun-Waterhouse (*) : W. Wang : G. I. N. Waterhouse
School of Chemical Sciences, The University of Auckland,
Private Bag 92019, Auckland, New Zealand
e-mail: dx.sun-waterhouse@auckland.ac.nz
D. Sun-Waterhouse
e-mail: dxsun72@hotmail.com
differences in the degradation pathways of added quercetin
and the changes of the alginate or alginate-starch shell during
storage. Results suggest that core oil stability depends on a
complex interplay between shell components, added antioxi-
dants, and storage conditions. Encapsulated oil beads provide a
convenient vehicle for delivering health-promoting unsaturated
oil, antioxidants, and starch fiber polysaccharides to consumers
either as a supplement or food ingredient. Alginate-starch shells
can be formed to offer different protective barriers with differ-
ent physical characteristics and nutritional value.
Keywords Alginate . Amylose content . Canola oil .
Co-extrusion encapsulation . Quercetin . Starch
Introduction
The positive roles of plant-based bioactives such as phenolics,
dietary fiber (DF), and unsaturated fatty acids (FAs) to human
health are widely recognized (Ratnayake et al. 2000; Kumar
and Goyal 2008; Warren et al. 2009; van Kempen et al. 2010).
Food products that carry broad spectrum of bioactives and do
not possess undesirable taste or odor are highly demanded by
consumers (Verbeke 2010). Microencapsulation technologies
can deliver bioactive rich products to satisfy this demand and
offer consumption options, either in supplement form for
direct intake or as an ingredient for food applications (Fang
and Bhandaria 2010; Shinde et al. 2013).
Co-extrusion is a microencapsulation process in which a
continuous coating (termed “shell”) is created as a physical
barrier to protect a liquid dispersion (termed “core”) from detri-
mental environments (Sun-Waterhouse et al. 2011a, 2012b).
Selecting materials that can form a robust shell with controlled
release properties is essential to maximize the delivery of target
bioactives to consumers (Sanguansri and Augustin 2006).
Polysaccharides possess moderately low oxygen permeability
2160
thus are often used as encapsulants (Whistler and BeMiller
1997; Rayment et al. 2009). Alginate is a linear 1,4-linked
copolymer of β-D-mannuronic acid and α-L-guluronic acid
(Draget et al. 2006) and has pH-sensitive properties with a
low degree of shrinkage distortion (Hoad et al. 2004; Rayment
et al. 2009; Georg Jensen et al. 2012). The pKa for mannuronic
acid and guluronic acid is 3.38 and 3.65, respectively (Draget
et al. 2006). Alginate gels are formed via hydrogen bonds when
pH is <pKa of uronide residues and in the presence of divalent
cations, e.g., Ca2+ (Rayment et al. 2009; Sun-Waterhouse et al.
2011a, 2012b).
Edible coatings attract interest today as efficient and safe
techniques for controlling the deterioration and extending the
shelf-life of food products. Mixed gels are often used to create
shell walls or coatings, in order to achieve controlled release
properties for core bioactives, increase encapsulation efficien-
cy, and extend product shelf-life (Sun-Waterhouse et al.
2012b; Poverenov et al. 2013). Using DF polymers such as
starch as encapsulants will impart corresponding DF function-
ality which increases the nutritional value of the microbead
products (van Kempen et al. 2010; Chung et al. 2011). The
combined use of alginate and starches for shell wall enables
controllable modification of the characteristics of encapsulat-
ed beads (Roy et al. 2009; Onyido et al. 2012). Starch is the
major source of glucose and caloric energy to human, and its
nutritional value is greatly influenced by its composition,
physical structure, and processing methods (Regmi et al.
2011). Resistant starch positively contributes to colonic and
bowel health in the way similar to DFs (Granfeldt et al. 1993;
van Kempen et al. 2010; Regmi et al. 2011). Starches have
been used as a binder in foods and pharmaceuticals, and they
form gels via hydrogen bonds (Fazilah et al. 2011). Starch
contains linear amylose and branched amylopectin in which
glucose building blocks are linked primarily by α-(1→4)
bonds and secondarily by α-(1→6) bonds. The ratio of
amylose and amylopectin plays an important role in starch
digestion, because of their different structural characteristics
(e.g., amylose molecules are unbranched) and consequently
different response to pancreatic amylase. High-amylose
starch generally has weaker digestibility than low-amylose
starch (Bello-Pérez et al. 2004; Chung et al. 2011; Regmi
et al. 2011). Amylose as a diluent and inhibitor can nega-
tively affect the swelling of starch and preserve its granule
integrity (Noranizan et al. 2010; Chung et al. 2011). The
formation of complexes between amylose molecules and
surface compounds like fatty acids is possible, and sub-
stances such as phenolics may have an effect on starch
digestion (Bello-Pérez et al. 2004). Alginate–starch com-
posite shell materials are expected to be highly beneficial
for oil encapsulation, though limited work has been report-
ed to date in the area of co-extruded unsaturated plant oil
microbeads.
Food Bioprocess Technol (2014) 7:2159–2177
Unsaturated plant oils are an important component of a
healthy human diet but have poor stability and are highly
susceptible to rancidity. Fortifying these oils with antioxidants
is an established practice to improve oil stability. Synthetic
antioxidants such as butylated hydroxytoluene (BHT) were
once popular but are no longer recommended due to their
increased health risks (Kahl and Kappus 1993). Naturally
occurring antioxidants such as vitamin E and phenolics are
now in favor. Phenolics have demonstrated various health
benefits and can suppress lipid oxidation via donating hydro-
gen atoms to lipid peroxyl radicals which interferes with the
initiation or propagation of primary oxidation (Ahn et al.
2008; Warren et al. 2009).
The main aim of this study was to establish the best
combination of shell materials and added antioxidants for
preserving unsaturated oil quality during storage and to estab-
lish the viability of encapsulated beads for delivering bioac-
tives such as unsaturated oil, fiber polysaccharides, and anti-
oxidants to consumers. The co-extrusion encapsulation of
canola oil containing added antioxidants (quercetin, vitamin
E, and BHT) is systematically examined using 1 % alginate
only (1 %A), alginate–Novation 2300 starch (very-low-amy-
lose starch) (A-N), or alginate–Gelose 80 starch (very-high-
amylose starch) (A-G) as encapsulants. Canola oil was select-
ed as a model unsaturated plant oil, as it is widely used for
cooking and possesses health benefits that are associated with
its high unsaturated FA contents, i.e., 62.4 % monounsaturated
FAs, 31.3 % polyunsaturated FAs, and 6.3 % saturated FAs
(Ackman 1990). Emphasis is placed on the physicochemical
characteristics of the encapsulated oil beads, and the storage
stability of the encapsulated canola oil at 20 °C or 38 °C.
Fourier-transform infrared (FT-IR) spectroscopy and high-
performance liquid chromatography (HPLC) are used to ex-
amine the potential compositional changes in the oil beads
during storage trials.
Materials and Methods
Chemicals and Materials
Canola oil (Simply, Malaysia) was purchased from a local
market (Countdown, Auckland, New Zealand) and stored in
the dark at 4 °C in a fridge (SRS 535NW, Samsung, Korea) until
use. Sodium alginate was purchased from Danisco, Auckland,
New Zealand. Novation 2300 starch and Gelose 80 starch were
obtained from National Starch Food Innovation. Quercetin,
rutin, catechin, gallic acid, BHT, α-tocopherol (vitamin E),
Folin-Ciocalteu’s phenol reagent (2 N), calcium chloride,
active carbon, p-anisidine, tridecanoic acid, sodium azide,
sodium acetate (anhydrous), sodium chloride, calcium chloride
dihydrate, magnesium chloride hexahydrate, and manganese
Food Bioprocess Technol (2014) 7:2159–2177
chloride tetrahydrate were purchased from Sigma-Aldrich Inc.,
St Louis, MO, USA. HPLC-grade acetonitrile was purchased
from J.T. Baker, Phillipsburg, NJ, USA. Glacial acetic acid,
absolute ethanol, chloroform, concentrated HCl (36 %), acetoni-
trile, dimethyl sulfoxide, n-hexane (95 %), isooctane, methanol,
potassium iodide, potassium dichromate, potassium hydrogen
phthalate, sodium hydroxide, sodium carbonate, sodium
thiosulphate, and sulfuric acid were from Ajax Finechem,
Auckland, New Zealand. PEG 400 (carbowax™) was purchased
from AMCD Australia Ltd, Mulgrave, Victoria, Australia.
Ethanol (96 %v/v, food grade) was purchased from Anchor
Ethanol Ltd, New Zealand. Ammonium chloride was
purchased from May & Baker, Dagenham, England. Iodine
solution was purchased from ACROS ORGANICS, New
Jersey, USA. Soluble starch, sodium sulphite, and potassium
chloride were purchased from AnalaR, BDH laboratory
chemicals, Poole, England. Cyclohexane (analytical grade) was
purchased from Biolab (Aust) Ltd, Scoresby, Australia.
Megazyme amylose/amylopectin assay kit (K-AMYL 07/11)
containing lectin concanavalin A (ConA), amyloglucosidase plus
fungal α-amylase, glucose reagent buffer, glucose oxidase, and
peroxidase (GOPOD), D-glucose standard solution, and starch
reference sample were purchased from Megazyme Int, Ireland.
Phenolphthalein indicator was purchased from Scharlau
Chemie, Barcelona, Spain.
Analysis of the Amylose Content of Starch
The amylose content of Novation 2300 and Gelose 80 starches
was measured following the instructions contained in the
Megazyme amylose/amylopectin assay kit (K-AMYL
07/11, Megazyme Int). Prior to analysis, pre-treatment
using 96 % (v/v) ethanol was performed to remove lipids
in the starch samples (to avoid interference that causes
underestimation of the amylose content). The amylose con-
tent (wt%) of starch was estimated as the ratio of absor-
bance (Abs) of the lectin concanavalin A (ConA) superna-
tant to total starch, as described by the following equation:
ðConA SupernatantÞ
Amylose wt% ¼ Abs510 Abs510ðTotal StarchÞ  6:15
9:2  100 , where
Abs510 is the absorbance at 510 nm and 6.15 and 9.2 are dilution
factors for the ConA and total starch solutions preparation.
Novation 2300 starch and Gelose 80 starch contain either an
extremely low and high amylose content, respectively, thus
some modifications were required to the standard assay proce-
dure in order to achieve a detectable and accurate UV absor-
bance at 510 nm. For the Novation 2300 starch, the sample
weight was increased to 30 mg (1.5 times that of the initial
20 mg required in the standard procedure), and the dilution
degree by ConA solvent after the ethanol pre-treatment step
was reduced (e.g., the final volume after dilution was changed
from 25 to 5 mL). All the other steps and the initial amounts of
other reagents were kept the same. The amylose content in
2161
Novation 2300 starch was calculated using this equation:
ðConA SupernatantÞ
Amylose wt% ¼ Abs510
Abs510ðTotal StarchÞ  6:15 1
9:2  51:5  100 . For
Gelose 80 starch, half of the initial amount of starch that
was required by the standard procedure (e.g., 10 mg in-
stead of 20 mg) was used for the amylose content analysis
without changing the remaining steps and the initial
amounts of other reagents. The equation used for calculat-
ing the amylose content in Gelose 80 starch was as follows:
ðConA SupernatantÞ
Amylose wt% ¼ Abs510
Abs510ðTotal StarchÞ  6:15
9:2  2  100 .
Preparation of Encapsulated Canola Oil Beads
Preparation of Encapsulant Formulations
The alginate solutions were prepared 1 day before use.
Sodium alginate solution (1 wt%, called “1 %A”) was
prepared by dissolving the required amount of Na-alginate
in pre-boiled distilled water (85 °C, 50 % final volume),
mixed at 5000 rpm for 1 min using a Silverson L5T high
shear mixer (Silverson Machines Ltd., Chesham, Bucks,
UK). The 50 % volume of distilled water (room temperature,
RT) was added and the resulting solution subjected to further
mixing with the Silverson high shear mixer (5,000 rpm for
1 min) and then stored overnight in the fridge (4 °C). A 0.67 %
sodium alginate solution was prepared in the same manner.
Novation 2300 starch or Gelose 80 starch solution (1 wt%)
was prepared by slowly adding the required amount of each
starch to the 50 % of total required volume distilled water
(80 °C) with gentle stirring (speed at 3) using a magnetic
stirring hotplate. Once the starch had dispersed, the remaining
50 % volume of distilled water (room temperature) was added
and the resulting solution mixed for a further 1 min. These
starch solutions were prepared freshly each day 2 h prior to
use to allow good hydration.
The alginate–Novation 2300 starch (called “A-N”) and
alginate–Gelose 80 starch (called “A-G”) solutions were pre-
pared by mixing carefully the 1 wt% alginate solution with the
1 wt% starch solution at a volume ratio of 2:1 and gentle
stirring with a magnetic stirrer (speed at 3 for 2 min) to obtain
homogenous shell solutions.
Preparation of Canola Oil Fortified with Quercetin, Vitamin
E, or BHT
Quercetin dihydrate (200 ppm) was first mixed with PEG 400
(2 wt%, final concentration) in a beaker, to which canola oil was
slowly added. The beaker containing canola oil and quercetin
was then placed in an ice bath and the mixture homogenized
using the Silverson high shear mixer (2,000 rpm for 1 min,
followed by 5,000 rpm for 2 min). Fortification of BHT or
vitamin E into canola oil was conducted in the same way but in
the absence of PEG.
2162
Encapsulation of Canola Oil Using Co-extrusion Technology
The canola oil with or without a fortifying antioxidant was
encapsulated using an Inotech Encapsulator (Inotech
Encapsulation AG, Dottikon, Switzerland), following the
procedures of Sun-Waterhouse et al. (2011a) with some
modifications. The core fluid (unfortified or fortified oil)
in a glass syringe and the shell fluid (i.e., 1 %A, A-N or A-G
solution) in a plastic syringe were simultaneously pumped
into a nozzle (200 μm) and sprayed out under a vibration
frequency about 1,750 Hz and a voltage about 1.5 kV,
forming regular-sized microdroplets that were dispersed
into a 3 wt% CaCl2 solution (200 mL) to form beads.
After hardening in the CaCl2 solution, the beads were
collected on a 50-μm nylon mesh, washed with water at
30 °C, and then transferred to 50 mL falcon tubes (wrapped
with aluminum foil) and then freeze-dried using a freeze
dryer (Labconco, Total Lab Systems Ltd, New Zealand).
After preliminary encapsulation trials, the optimal condi-
tions, i.e., core and shell feed flow rate of 30 and 200 mL/h,
respectively, and hardening at 4 °C for 2 h, were selected.
The unencapsulated oil (control) was prepared by placing
the same batch of canola oil in the same type of glass
syringe (as the core fluid) subjected to the same encapsula-
tion process in the absence of a shell fluid in the plastic
syringe and then harvested in the same manner.
Storage Trials
Unencapsulated canola oil and the freeze-dried encapsulated
canola oil beads were stored in sealed glass scintillation vials
(20 mL) with the same headspace ratio. These vials were
flushed with N2, tightly capped, wrapped with aluminum foil,
and stored (away from light) for 30 or 60 days in a cool room
at room temperature (20±2 °C) or at 38±1 °C (in an incubator
oven, MIR 162, Sanyo Electric Co., Ltd, Osaka, Japan). Two
subsamples from each vial were withdrawn on Day 0, 30, or
60, with a portion being used for analyses, and the remaining
beads in the vials being flushed with N2 and kept in a freezer
at −20 °C.
Quercetin-Fortified Oil-Encapsulated Beads Subjected
to Acidified Water at pH 3
Milli-Q water was acidified by adding a 0.5 % HCl solution
(prepared from concentrated HCl) to until pH 3. The pH was
monitored using a pH meter (Schott Instrument pH Meter Lab
850, Xylem Inc, Germany). Fresh quercetin-containing beads
(initially suspended in the CaCl2 solution) were transferred
onto a 50-μm nylon mesh and partially dried by absorbing
excess water with a paper towel held underneath the nylon
mesh. The partially dried beads (300 mg, accurately weighed)
Food Bioprocess Technol (2014) 7:2159–2177
were immediately placed into the acidified water (5 mL) at
pH 3 for 2 h with gentle and regular shaking (which simulated
the acidic food matrix and human stomach). After this pH
treatment, the wet beads were collected and subjected to
optical microscopy examinations and the residual aqueous
solution subjected to total phenolic content analysis by the
Folin-Ciocalteu assay (to examine the potentially released
quercetin and/or its derived products from the oil beads during
the acidic treatment).
Water Activity of the Freeze-Dried Beads
The water activity (aw) of the freeze-dried quercetin-fortified
canola oil beads was measured using a dew point
water activity meter (AquaLab 4TE, Decagon Devices Inc.,
Pullman, WA, USA) at 25 °C. Duplicate of each type of bead
were measured in triplicate.
Microscopic Examination of Encapsulated Oil Beads
Optical microscopy examination on the appearance, size,
and shell wall thickness of the encapsulated beads were
conducted using a Nikon Eclipse E600 microscope
(Nikon Corporation, Chiyoda-ku, Tokyo, Japan) equipped
with a Nikon Coolpix 995 3.34 mega pixel camera (×10,
Nikon corporation, Chiyoda-ku, Tokyo, Japan). A small
portion of wet beads were transferred onto a glass slide
using a plastic dropper. One drop of 3 % CaCl2 solution
was added to the beads before they were covered with a
cover slide for microscopy examination.
Environmental scanning electron microscopy (ESEM) was
used to examine the partially dried encapsulated beads. A
small amount of wet encapsulated beads suspended in the
3 % CaCl2 solution were collected, placed onto a 50 μm nylon
mesh, and then partially dried through absorbing excess water
with a paper towel placed beneath the nylon mesh. The
partially dried beads were directly examined by an environ-
mental scanning electron microscope (FEI-Quanta-200, FEI,
Hillsboro, OR, USA). The images were taken at an electron
gun accelerating voltage of 10 kV, at a sample temperature of
2 °C, and a relative humidity of 95 %.
Fourier-Transform Infrared (FT-IR) Spectroscopy
FT-IR spectra of the ingredients used to prepare the oil beads
(canola oil, quercetin dihydrate, sodium alginate, Novation
2300 starch, Gelose 80 starch, and water), as well as derived
freeze-dried beads, were obtained using a Perkin Elmer®
Spectrum 100 FT-IR spectrometer (equipped with a universal
ATR sampling attachment). The FT-IR spectra within the
frequency regions of 4,000–650 cm−1 were collected at a
resolution of 4 cm−1 with 16 accumulated scans under dim
Food Bioprocess Technol (2014) 7:2159–2177
light at room temperature. Prior to analysis, spectra were
normalized using SigmaPlot (version 11.0).
Oil Stability Evaluation
Extraction of Canola Oil from the Encapsulated Beads
and Extraction of Phenolics from the Obtained Oil
The extraction of canola oil from the encapsulated beads
followed the method of Sun-Waterhouse et al. (2012b) with
some modifications. Freeze-dried beads (2 g) were firstly
ground in a mortar with pestle and mixed with 20 mL of KCl
solution (0.88 %w/v). After mixing using a MT19 vortex
mixer (Chiltern International, Slough, UK, operating at
Speed 4), chloroform (20 mL) and methanol (20 mL) were
added. The resultant mixture was homogenized using a homog-
enizer (12,000 rpm for 2 min; CH 6005 model, Luzern,
Switzerland). The chloroform layer was collected and trans-
ferred into a glass vial wrapped with aluminum foil. The
extraction step was repeated. The resultant chloroform layers
were collected, combined, evaporated using a Labconco
RapidVap® Concentrator (40 min at 40 °C and10 kPa; Model
79100–01, Labconco Corp., Kansas City, Missouri, USA) un-
der N2, dried in the Ultra-Low Cold Trap Centrivap®
Centrifugal Concentrator (Model 78100–01, Labconco Corp.,
Kansas City, MO) at 40 °C for 4 h under vacuum, and stored in
a freezer (−80 °C) until analysis.
The phenolics in the extracted oils were isolated using the
method of Sun-Waterhouse et al. (2011a) with some modifi-
cations. An aliquot of extracted oil (0.5 g) was dissolved in n-
hexane (1.25 mL), before the 80 %v/v aqueous methanol
(0.5 mL) was added. The resultant mixture was vigorously
mixed for 2 min using the MT19 vortex mixer (Speed 4) and
centrifuged at 3,000 rpm for 3 min (Centrifuge 5702,
Eppendorf, Hamburg, Germany), and then the methanolic
phase was collected using a glass pipette and transferred to a
vial wrapped with aluminum foil. This methanolic extraction
was repeated three times. The combined methanolic extracts
were dried in the Ultra-Low Cold Trap Centrivap®
Concentrator at 40 °C for 4 h under vacuum and stored in a
freezer at −80 °C until analysis.
Peroxide Value (PV) Determination
The PVs of the oils were determined (AOCS 1998a) following
the procedures of Sun-Waterhouse et al. (2012b) and expressed
as peroxide milliequivalent (meq) per kilogram of oil.
p–Anisidine Value (p-AV) Determination
The p-AVs of the oils were determined (AOCS 1998b),
following the procedures of Sun-Waterhouse et al. (2012b). A
spectrophotometer (SpectraMax Plus 384, MDS Analytical
2163
Technologies, Hawthorn, Australia) was used to measure the
absorbance at 350 nm.
Totox Value Calculation
Totox values were calculated from the PVs and p-AVs of the
oil samples using the equation of Totox value=2PV+p-AV
(O’Connor et al. 2007).
Free Fatty Acid Determination
FFAs were determined using the direct titration method of
AOCS (2000) and following the procedures of Sun-
Waterhouse et al. (2012b). FFA content was expressed as
“grams FFA as oleic acid per 100 g oil.”
Iodine Value (IV) Determination
IV is based on the reactions between iodine and fatty acids. IV
was determined using the AOCS Official Method (1997) and
expressed as grams I2/100 g oil.
Analysis of Total Extractable Phenolic Content
The total extractable phenolic content (TEPC) was determined
by the Folin-Ciocalteu assay (Singleton et al. 1997) and
expressed as “milligrams gallic acid equivalents (GAE) per
kilogram oil.”
High-Performance Liquid Chromatography (HPLC) Profiling
of the Phenolics in Oil Beads
An analytical HPLC (HP Agilent 1100 series, Agilent
Technologies, USA) equipped with a G1313A autosampler,
a G1322A degasser, a G1311A Quaternary pump, a G1316A
column compartment, and a Phenomenex Luna C18 column
(4.6×250 nm, 5 μm particle size) was used to analyze the
phenolics including quercetin in the encapsulated canola oil
beads at 28 °C (Phani et al. 2010). The mobile phases consist
of (A) 1 % acetic acid (45.0 %), (B) acetonitrile (15.0 %), and
(C) methanol (40.0 %), with the flow rate of 1.0 mL/min. The
injection volume was 20 μL, and total run time was 30 min.
The wavelength detection range was 200 to 540 nm.
The phenolic extracts (∼1 g) obtained in section on
“Extraction of Canola Oil from the Encapsulated Beads and
Extraction of Phenolics from the Obtained Oil” were
reconstituted in 80 %v/v aqueous methanol (0.5 mL),
vortexed, and centrifuged (3,000 rpm for 3 min). The
resultant supernatant was filtered through a 0.45 μm filter
(Nylon Membranes; Supelco, Bellefonte, PA, USA) and trans-
ferred to brown vials for HPLC analysis. The undissolved
quercetin was recovered from the residues obtained by
2164
centrifugation and filtration. Individual phenolics were iden-
tified based on their retention time and absorbance maximum
(λmax) by comparison with those of authentic standards. The
concentrations of non-flavonoid phenolics present at 280 nm,
and flavonoids present at 370 nm were estimated using a catechin
or quercetin external standard, respectively. Quercetin was
quantified at 370 nm using rutin hydrate (25 ppm) as the internal
standard. The concentration of quercetin was calculated
using this equation: Concentrationquercetin ¼ Peak Areaquercetin 
Concentrationrutin Þ=Peak Areaquercetin.
Statistical Analysis
All data are expressed as mean±standard deviation of three
replicates. Analyses were carried out using MINITAB 15
(Minitab Inc., Pennsylvania, USA) statistical software with
one-way ANOVA followed by Tukey’s multiple comparison
test at p<0.05.
Results and Discussion
Characteristics of the Encapsulated Canola Oil Beads Before
and After the Treatment at pH 3
Chemical analyses on the two starch ingredients revealed that
the Novation 2300 starch had 1.4 % amylose whilst the
Gelose 80 starch had 89.8 % amylose. During the preparation
of the 1 % starch solutions, the Novation 2300 starch solution
exhibited a very different pasting pattern and had a much
higher viscosity, compared with the Gelose 80 starch solution.
Encapsulated oil beads prepared using1 %A, A-N, or A-G
showed minimal breakage before and after freeze-drying
(2±0.5 % and 4±0.5 %, respectively).
Microscopy examinations (Fig. 1) confirmed the integrity of
the co-extruded canola oil beads, with no cracks or damage
evident. ESEM images (Fig. 1a, b, and c, left hand column)
revealed that the alginate alone beads (1 %A) were rounder
with smoother surfaces compared with the alginate–starch
beads. Optical microscopy examination confirmed that the
freshly prepared 1 %A beads (Fig. 1a, middle column) had
near-perfect spherical shape, whilst the freshly prepared A-N
(Fig. 1b, middle column) and A-G (Fig. 1c, middle column)
beads were more ovoid in shape. The average diameter of beads
decreased in the order of A-G (380 μm)>1 %A (348 μm)>A-
N (310 μm). The three types of bead all had perfectly spherical
cores. The average wall thickness decreased in the order of
1 %A (70 μm)>A-N (60 μm)>A-G (50 μm). The alginate–
starch beads contained a number of small individual “spherical
features” in the shell wall, with the A-G beads having a higher
density of the spherical features. These spherical features in the
shell wall are likely to be oil droplets or possibly air pockets.
After the treatment in the acidified water at pH 3 for 2 h, all the
Food Bioprocess Technol (2014) 7:2159–2177
three types of beads remained intact but shrank significantly,
i.e., bead size reduction increased in the order of 1 %A beads
(decreased by 13 %)<A-N beads (decreased by 21 %)<A-G
beads (decreased by 29 %) (Fig. 1a, b, and c, right hand
column). As negligible breakage of beads occurred during the
pH 3 treatment, it is concluded that the three types of encapsu-
lated beads prepared in this study were chemically and me-
chanically robust.
To evaluate the viability of the co-extrusion encapsulation
method for bead preparation, the total phenolics (including the
fortified quercetin and its degradation products) released from
the beads into the pH 3 solutions were analyzed by the Folin-
Ciocalteu assay (Table 1). In the absence of added quercetin,
the total phenolic content of acidified water after immersing
the 1 %A, A-N, and A-G beads was ∼0.179±0.003 μg GAE/
mg bead, which can be attributed to reducing substances
including sugars, vitamins, and nitrogen compounds (i.e.,
non-phenolic compounds) in the beads which reacted with
the Folin-Ciocalteu reagent (Everette et al. 2010). After
subtracting this background reading, only marginal phenolic
contents were detected for the quercetin-containing oil beads
encapsulated by 1 %A, A-N, and A-G shells (Table 1). This
indicates that negligible amounts of quercetin and/or its de-
rived phenolic products were leached out of the beads during
the pH 3 treatment, which is also consistent with the lack of
bead rupture seen in Fig. 1 after the pH 3 treatment. The dense
alginate or alginate–starch networks prevented diffusion of
quercetin and its derived phenolics through the shell and
hence prevented phenolic leaching.
The release characteristics of the encapsulated beads strong-
ly depended on the shell wall formulation. Alginate is sensitive
to pH change, thus rendering it suitable for creating intestinal
delivery systems (Whistler and BeMiller 1997). Solution pH is
expected to have a significant effect on the alginate shell, due to
the ionization of its carboxylate groups (Whistler and BeMiller
1997; Rayment et al. 2009). At pH 3, alginate beads tend to
shrink due to strong hydrogen bonding interactions or
intermolecular lactonization, converting to alginic acid, and
forming a viscous gel which retards the loss of encapsulated
phenolics (Rayment et al. 2009; Li et al. 2011).
The extracted oil content of the freeze-dried quercetin-
fortified oil beads was the lowest for the A-N beads (55.2 g/
100 g beads) and similar for the 1 %A beads (78.3 g/100 g
beads) and A-G beads (72.0 g/100 g beads) (Table 1).
Differences in the oil content of the three types of beads can
be attributed to the different shell wall thicknesses. The water
activities of the three types of freeze-dried beads were similar
and <0.5, which was consistent with their free-flowing behav-
ior and indicating a low risk of microbial spoilage and good
shelf life (Sun-Waterhouse et al. 2012a). The water activity of
the A-N beads (0.399) was slightly higher than the 1 %A
beads (0.352) and A-G beads (0.368). This partially explains
the low extracted oil content of the A-N beads.
Food Bioprocess Technol (2014) 7:2159–2177 2165

A, A+O+Q

Before pH treatment After pH 3 for 2 h

B, A+N+O+Q Before pH treatment After pH 3 for 2 h

C, A+G+O+Q Before pH treatment After pH 3 for 2 h

Fig. 1 ESEM micrographs (magnification ×400; left, before pH treat- encapsulated by a 1 % alginate (A+O+Q), b alginate–Novation 2300
ment), and optical microscopy images (magnification ×10; middle, starch (A+N+O+Q), and c alginate–Gelose 80 starch (A+G+O+Q)
before pH treatment; right, after pH 3 for 2 h) of fresh (wet) oil beads

Mixed biopolymer gels are expected to behave differently
to gels of a single polymer (Piculell et al. 1995). The three
types of beads produced in this study exhibited some differ-
ences in their surface characteristics (e.g., smoothness and
surface area), bead size, and textural properties. Low-
amylose starch may interfere with the Ca2+-alginate bonding
in the shell wall and reduce electrostatic repulsion between
alginate chains, thus explaining the smaller size of A-N beads
compared with the alginate alone and A-G beads (Xu et al.
2005). Surface characteristics are important parameters for
gauging the protective properties of shell walls and the stabil-
ity of core substances of encapsulated beads (Stojanovic et al.
2012). A regular smooth surface, free of defects, uniform bead
size distribution, and shell thickness generally offer better
protection against detrimental agents or reduced loss of core
substances (Rajam et al. 2012). The hardness of beads is likely
to influence their stability during storage and food processing
(Nualkaekul et al. 2012). The addition of starch to alginate is
expected to increase the density, elasticity, and hardness of the
bead shell, as the starch matrix can acquire higher mechanical
2166
Table 1 Total phenolic content in the aqueous solutions used for the
pH 3 treatment, the extracted oil from Day 0 beads, and water activity of
freeze-dried beads
Bead Total phenolic content Extracted oil Water activity
of the beads after the from Day 0 of freeze-dried
treatment at pH 3 for beads (g/100 beads (aw)
2 h (GAE/mg beads) dried bead)
A+O+Q 0.213±0.001a 78.3±0.2a 0.352±0.001b
A+N+O+Q 0.195±0.001b 55.2±0.4c 0.399±0.001a
A+G+O+Q 0.182±0.001c 72.0±0.1b 0.368±0.001ab
Data expressed as mean±standard deviation. Different lowercase letters
(within the same column) indicate statistically significant differences at
p<0.05
GAE gallic acid equivalents, A alginate (1 %), O canola oil, Q
quercetin, N Novation 2300 starch, G Gelose 80 starch
strength in the presence of alginate gel (Onyido et al. 2012).
Such effects depend on the type of starch and the amylose
content (Li et al. 2012). Moreover, a more viscous starch
would slow diffusion of Ca2+ required for the formation of
the “egg-box” structures during the co-extrusion encapsula-
tion process, thus in turn influencing the alginate cross-linking
(Maiti et al. 2012). In general, amylose contributes to gel
strength whereas amylopectin increases gel viscosity.
Amylose molecules can easily form gels due to their linear
structure which favors hydrogen bonding, whereas amylopec-
tin molecules are branched resulting in a lower level of hy-
drogen bonding and weaker gel strengths (Jane et al. 1999;
Sandhu and Singh 2007; Li and Yeh 2001). Noranizan et al.
(2010) reported a linear relationship between starch gel
strength and the ratio of amylose/amylopectin and that gels
are stronger at low ratios of amylose to amylopectin. The
linear nature of amylose allows the chains to pack closely
and a higher amylose-containing shell matrix is likely to be
more robust resulting in a slower release of core substances
(Wing et al. 1988). Thus, different amylose contents in
Novation 2300 and Gelose 80 starches are expected to strong-
ly influence the properties of the alginate networks containing
these starches. High-amylose starch like Gelose 80 starch is
expected to show less swelling and shear thinning, and smaller
gel strength than low-amylose Novation 2300 starch (Chung
et al. 2011). In summary, large difference in amylose content
between the two starches is expected to cause considerable
differences in packing density, pore structure, and mechanical
properties of the alginate–starch bead shells, which ultimately
determines the bead characteristics and the rate of core oil or
core antioxidant release.
Stability of Core Oil and Total Extracted Phenolic Content
(TEPC) in Beads After Storage
The PV, p-AV, Totox, FFA, and TEPC values of unencap-
sulated and encapsulated canola oil samples after 0, 30, or
Food Bioprocess Technol (2014) 7:2159–2177
60 days storages at room temperature and 38 °C are summarized
in Tables 2 and 3, respectively. The PVs of each oil increased
with storage time, and the increase was greater at 38 °C. Under
the same storage conditions, the PV increased with storage was
smaller when quercetin or BHT was added to the oil, compared
with vitamin E. The quercetin-containing encapsulated oil had a
lower PV than the control oil (unencapsulated oil). At RT
(Table 2), the PV decreased in the order 1 %A∼A-G>A-N after
30 days, and A-G>1 %A>A-N after 60 days. At 38 °C
(Table 3), the PV decreased in the order 1 %A>A-G>A-N
after 30 days, and A-G>A-N and 1 %A after 60 days. Thus, the
shell formulation and storage temperature and time affected the
PV readings. A-N appeared to be the best encapsulant for the 60-
day storage at 38 °C.
The p-AVs of the control and encapsulated oils generally
increased after the storages at either RT or 38 °C. The ability
to suppress the secondary oil oxidation was lower for vitamin E
compared with quercetin or BHT. For the quercetin-containing
oil beads, the p-AV at RT was slightly lower for 1 %A than
for A-N or A-G after 30 days but similar after 60 days. At
38 °C, the p-AV of A-N was marginally higher than 1 %A or
A-G after 30 days. After 60 days, the p-AV decreased in the
order of A-N>A-G>1 %A. Thus, storage conditions, shell
formulations, and types of added antioxidants all affected the
extent of secondary oxidation. The 1 %A shell demonstrated a
better performance than the two alginate–starch shells against
oil secondary oxidation at both RT and 38 °C. Totox values
provide an estimation of an oil’s overall oxidation status. The
current Totox value calculations revealed that primary oil oxi-
dation was dominant over secondary oil oxidation for the
encapsulated oils. All the Totox values increased when the
storage conditions became harsher (i.e., longer storage time or
higher storage temperature or both). After 60 days at 38 °C, the
use of co-extrusion encapsulation in the absence or presence of
quercetin kept the Totox value <30 (the legal acceptable limit,
O’Connor et al. 2007), except for the 1 %A bead without added
quercetin which had a Totox value of 30.9). A-N was the best
shell material for protecting against oil oxidation. The 1 %A
could be a viable alternative for 60 days storage at 38 °C. A-G
appeared to be the worst shell material.
The FFA of canola oil was low on Day 0 (<0.08 %) and then
increased with storages at RT and 38 °C. The FFA increase
reflects moisture capture and permeability of the different shell
formulations. The three types of antioxidants were comparable
for suppressing hydrolytic rancidity, with BHT generally being
the worst. In the presence of added quercetin, similar FFA levels
were detected for beads prepared using the three types of
encapsulants. The 1 %A beads had slightly higher FFAs than
the alginate–starch beads under the applied storage conditions.
Clearly, some interplay exists between shell formulation and
storage conditions on FFAs, which is associated with the
moisture-capturing properties of the bead shells (Hung and
Slinger 1981; Sun-Waterhouse et al. 2011a, b).
Food Bioprocess Technol (2014) 7:2159–2177 2167

Table 2 Peroxide value, p-anisidine value, free fatty acid content, iodine value, and total extracted phenolic content of control and encapsulated oils
after storages at room temperature

Oil sample Storage (day) PV (meq/kg oil) p-AV Totox FFA (g/100 g oil) IV (g I2/100 g oil) TEPC (mg GAE/kg oil)

Control 0 4.99±0.14C,a 1.57±0.02C,c 11.6±0.20C,a 0.08±0.00C,a 185±0.51A,ab 21.2±0.13C,e

30 9.66±0.32B,a 1.87±0.01B,bc 21.2±0.45B,a 0.22±0.00B,a 180±0.50B,c 23.0±0.82B,cd
60 13.4±0.21A,b 2.66±0.05A,a 29.4±0.30A,b 0.39±0.01A,a 176±0.41C,ab 26.9±0.35A,de
A+O 0 4.79±0.13C,a 1.33±0.07C,de 10.9±0.19C,b 0.08±0.00C,a 177±0.50A,b 25.2±0.00A,de
30 7.91±0.12B,bc 1.98±0.06B,ab 17.8±0.17B,b 0.19±0.00B,ab 176±0.35A,d 26.2±0.62A,c
60 12.8±0.00A,bc 2.23±0.07A,b 27.8±0.07A,bc 0.41±0.01A,a 172±0.30B,b 22.5±0.49B,e
A+O+Q 0 4.89±0.00C,a 1.17±0.06C,de 12.0±0.06C,a 0.08±0.00C,a 172±0.32A,bc 48.7±0.12A,b
30 7.03±0.29B,cd 1.83±0.05B,bc 15.9±0.41B,c 0.17±0.01B,b 171±0.33A,e 32.3±0.27C,b
60 9.01±0.21A,fg 2.20±0.08A,d 20.2±0.30A,cd 0.20±0.00A,c 159±0.48B,c 37.8±0.39B,c
A+O+VE 0 4.79±0.13C,a 1.50±0.01C,cd 11.1±0.19C,ab 0.08±0.00C,a 187±0.00A,a 28.2±0.23B,d
30 7.93±0.54B,bc 2.07±0.15B,ab 17.9±0.76B,b 0.15±0.00B,bc 162±0.35B,f 26.0±0.20C,c
60 12.2±0.33A,c 2.48±0.07A,ab 26.9±0.47A,c 0.20±0.02A,c 125±0.67C,ef 43.2±1.37A,b
A+O+BHT 0 4.90±0.28C,a 1.45±0.06C,cd 11.3±0.40C,ab 0.08±0.00C,a 130±0.00A,d 32.3±0.57B,cd
30 7.61±0.35B,c 1.66±0.06B,c 16.9±0.39B,bc 0.17±0.00B,b 127±0.00B,h 26.8±0.76C,c
60 11.2±0.26A,d 1.85±0.12A,c 24.3±0.37A,cd 0.29±0.01A,bc 121±0.89C,ef 35.1±1.79A,cd
A+N+O 0 4.89±0.14C,a 1.39±0.01C,d 11.2±0.20C,ab 0.08±0.00C,a 158±0.45B,c 26.4±0.76A,de
30 8.85±0.50B,b 1.89±0.00B,b 19.6±0.71B,ab 0.15±0.00B,bc 196±0.00A,a 17.5±1.26C,e
60 11.0±0.53A,d 2.43±0.07A,ab 24.4±0.75A,cd 0.33±0.02A,b 150±0.22C,cd 21.9±0.55B,e
A+N+O+Q 0 4.89±0.14C,a 2.03±0.05B,a 11.8±0.20C,a 0.08±0.00C,a 185±0.45B,ab 38.6±0.62A,c
30 6.36±0.59B,de 2.24±0.14A,a 15.0±0.83B,c 0.14±0.00B,bc 189±0.45A,b 30.9±0.09C,bc
60 8.47±0.00A,g 2.16±0.11A,bc 19.1±0.11A,d 0.19±0.01A,c 182±0.49C,a 32.3±0.25B,d
A+N+O+VE 0 4.89±0.00C,a 1.32±0.00C,d 11.1±0.00C,ab 0.08±0.00C,a 185±0.45A,ab 24.5±0.41B,de
30 8.08±0.60B,b 1.91±0.09B,b 18.1±0.85B,b 0.17±0.00B, b 176±0.45B,d 24.8±0.09B,cd
60 11.5±0.55A,d 2.68±0.13A,a 25.7±0.78A,c 0.20±0.02A,c 116±0.22C,f 59.6±0.08A,a
A+N+O+BHT 0 4.89±0.00C,a 1.70±0.05B,bc 11.1±0.05C,ab 0.08±0.00C,a 170±0.45A,bc 30.2±0.00B,cd
30 6.62±0.00B,d 1.82±0.02A,bc 15.1±0.02B,c 0.15±0.00B,bc 145±0.70B,g 21.6±0.61C,cd
60 9.25±0.66A,f 1.84±0.02A,c 20.3±0.93A,d 0.19±0.01A,c 134±0.37C,e 33.2±0.00A,cd
A+G+O 0 4.79±0.13C,a 1.82±0.03C,b 11.4±0.19C,ab 0.08±0.00C,a 176±0.00B,bc 26.3±0.76A,de
30 9.83±0.00B,a 1.90±0.01B,b 21.6±0.01B,a 0.19±0.00B,ab 196±0.75A,a 20.8±0.76C,d
60 13.3±0.82A,b 2.29±0.04A,b 28.9±1.16A,b 0.28±0.01A,bc 164±0.89C,bc 23.7±0.69B,de
A+G+O+Q 0 4.89±0.00C,a 1.81±0.03B,b 11.6±0.03C,a 0.08±0.00C,a 188±0.45A,a 52.7±0.07A,a
30 6.97±0.62B,cd 2.02±0.02A,ab 16.0±0.88B,c 0.15±0.02B,bc 170±0.67B,e 50.1±0.36B,a
60 9.67±0.78A,f 2.12±0.09A,bc 21.5±1.10A,cd 0.22±0.02A,c 143±0.60C,d 22.6±0.08C,e
A+G+O+VE 0 4.89±0.00C,a 1.08±0.03C,e 10.9±0.03C,b 0.08±0.00C,a 171±0.89B,bc 28.2±0.97B,d
30 7.76±0.34B,c 1.98±0.02B,ab 17.5±0.48B,b 0.13±0.00B,c 173±0.67B,de 26.3±0.90B,c
60 14.3±0.68A,a 2.71±0.05A,a 31.3±0.76A,a 0.23±0.02A,bc 125±0.89A,ef 38.3±0.11A,c
A+G+O+BHT 0 4.89±0.14C,a 1.28±0.08C,de 11.1±0.20C,ab 0.08±0.00B,a 120±0.52B,d 30.6±0.53B,cd
30 6.01±0.53B,e 1.58±0.02B,c 13.6±0.75B,d 0.21±0.01A,a 143±0.45A,g 20.8±0.78C,d
60 10.6±0.84A,e 2.09±0.01A,bc 23.3±1.19A,cd 0.23±0.02A,bc 117±0.45C,f 33.9±0.75A,cd

A–C values of the same analysis for the same oil sample but different storage periods (Days 0, 30, and 60) with different letters are significantly different
(P<0.05). a–h values of the same analysis and on the same storage day but for different oil samples with different letters are significantly different
(P<0.05).
PV peroxide value, p-AV p-anisidine value, FFA free fatty acid, IV iodine value, TEPC total extracted phenolic content, GAE gallic acid equivalents,
A alginate (1 %), O canola oil, Q quercetin, VE vitamin E, N Novation 2300 starch, G Gelose 80 starch

Iodine value (IV) estimates the degree of oil unsaturation and generally decreases with storage time before reaching a plateau
potential oil stability (Toscano et al. 2012). The unsaturation of (Toscano et al. 2012). In this study, the IV values of the control
oil decreases due to oxidation (Sun-Waterhouse et al. 2011b). IV and encapsulated oils decreased after storage at RT or 38 °C. The
2168 Food Bioprocess Technol (2014) 7:2159–2177

Table 3 Peroxide value (PV), p-anisidine value (p-AV), free fatty acid (FFA) content, iodine value (IV), and total extracted phenolic content
(TEPC) of control and encapsulated oils after storages at 38 °C

Oil sample Storage (day) PV (meq/kg oil) p-AV Totox FFA (g/100 g oil) IV (g I2/100 g oil) TEPC (mg GAE/kg oil)

Control 0 4.99±0.14C,a 1.57±0.02C,c 11.6±0.20C,ab 0.08±0.00C,a 185±0.69A,ab 21.2±0.12B,f

30 11.3±0.23B,a 2.10±0.03B,d 24.8±0.33B,a 0.28±0.01A,b 170±0.50B,ab 19.6±0.17C,h
60 16.5±0.10A,a 3.91±0.07A,bc 36.9±0.14A,a 0.22±0.01B,bc 151±0.12C,b 26.1±0.28A,g
A+O 0 4.79±0.13C,a 1.33±0.07C,de 10.9±0.19C,b 0.08±0.00B,c 177±0.15A,c 25.2±0.00B,d
30 8.06±0.17B,d 2.78±0.07B,bc 18.9±0.24B,bc 0.33±0.00A,a 175±0.15B,a 24.1±0.31C,g
60 13.8±0.30A,c 3.31±0.03A,d 30.9±0.42A,b 0.29±0.01B,a 142±0.18C,cd 28.3±0.04A,fg
A+O+Q 0 4.89±0.00C,a 1.17±0.06C,ef 12.0±0.06C,a 0.08±0.00B,a 172±0.33A,d 48.7±0.08A,b
30 8.66±0.25B,c 2.38±0.02B,cd 19.7±0.35B,b 0.24±0.01A,c 151±0.22B,bc 38.5±0.34B,e
60 10.1±0.15A,f 2.98±0.02A,e 23.2±0.21A,d 0.25±0.01A,b 126±0.16C,d 32.6±0.18C,f
A+O+VE 0 4.79±0.13C,a 1.50±0.01C,cd 12.1±0.19C,a 0.08±0.00C,a 182±0.00A,b 26.6±0.23C,de
30 9.62±0.63B,b 2.46±0.01B,c 21.7±0.89B,ab 0.19±0.00B, d 126±0.67B,e 50.0±0.39B,c
60 13.7±0.87A,c 3.70±0.01A,cd 31.1±1.23A,b 0.23±0.02A,bc 118±0.67C,de 74.9±1.39A,c
A+O+BHT 0 4.90±0.27C,a 1.45±0.06C,cd 11.3±0.38C,b 0.08±0.00C,a 130±0.00A,g 30.2±0.57C,d
30 8.24±0.59B,cd 2.22±0.09B,cd 18.7±0.83B,bc 0.22±0.01B,c 118±0.67B,ef 49.7±0.08B,cd
60 12.6±0.35A,c 3.42±0.00A,cd 28.6±0.50A,bc 0.31±0.01A,a 113±0.60C,e 73.1±0.36A,c
A+N+O 0 4.99±0.14C,a 1.39±0.01C,d 11.2±0.20C,b 0.08±0.00C,a 160±0.45C,f 26.4±0.76B,de
30 7.50±0.51B,de 3.60±0.09B,a 18.6±0.72B,bc 0.33±0.00A,a 170±0.45A,ab 32.2±1.10A,f
60 11.2±0.86A,e 4.86±0.12A,a 27.3±1.22A,c 0.27±0.01B,b 165±0.00B,a 23.6±0.50C,h
A+N+O+Q 0 4.99±0.14C,a 2.03±0.05C,a 11.8±0.20C,ab 0.08±0.00C,a 168±0.35A,e 38.6±0.62C,c
30 7.05±0.89B,e 2.84±0.01B,b 16.9±1.26B,c 0.22±0.01A,c 145±0.35B,c 71.7±0.63A,b
60 10.7±0.50A,ef 4.18±0.17A,b 25.6±0.71A,cd 0.19±0.01B,c 119±0.69C,de 60.2±0.69B,d
A+N+O+VE 0 4.89±0.00C,a 1.32±0.00C,de 11.1±0.00C,b 0.08±0.00B,a 168±0.45A,e 24.4±0.41C,e
30 9.07±0.68B,bc 2.88±0.02B,b 21.0±0.96B,ab 0.24±0.02A, c 123±0.60B,ef 48.3±0.38B,cd
60 15.3±0.76A, b 4.61±0.18A,a 35.2±1.07A,a 0.25±0.01A,b 120±0.59B,c 54.3±0.48A,de
A+N+O+BHT 0 4.89±0.00C,a 1.70±0.05C,bc 11.5±0.05C,b 0.08±0.00B,a 143±0.45A,g 30.1±0.00C,d
30 7.65±0.00B,de 2.09±0.01B,d 17.4±0.01B,d 0.24±0.02A,c 110±0.13B,f 43.5±0.17B,d
60 12.9±0.16A,cd 3.80±0.04A,c 29.6±0.23A,b 0.26±0.02A,b 98.1±0.57C,f 52.1±0.09A,e
A+G+O 0 4.79±0.13C,a 1.82±0.03C,b 11.4±0.19C,ab 0.08±0.00C,a 178±0.00A,c 26.3±0.76A,de
30 8.07±0.00B,d 2.85±0.10B,b 19.0±0.10B,bc 0.31±0.01A,ab 154±0.00B,b 24.7±0.68B,g
60 12.2±1.10A,d 4.70±0.00A,a 29.1±1.56A,b 0.26±0.03B,b 125±0.57C,d 26.8±0.39A,g
A+G+O+Q 0 4.89±0.00C,a 1.81±0.03C,b 11.6±0.03C,ab 0.08±0.00B,a 187±0.25A,a 52.7±0.07C,a
30 7.90±0.43B,d 2.33±0.11B,cd 18.1±0.61B,c 0.23±0.01A,c 140±0.28A,cd 77.3±0.63A,a
60 11.8±0.67A,de 3.82±0.05A,c 27.4±0.75A,c 0.22±0.02A,bc 120±0.48C,de 70.5±0.18B,cd
A+G+O+VE 0 4.89±0.01C,a 1.08±0.03C,f 10.9±0.03C,b 0.08±0.00B,a 170±0.60A,de 29.1±0.97B,d
30 9.18±0.36B,b 2.43±0.05B,c 20.8±0.51B,b 0.20±0.03A,cd 148±0.22B,bc 31.2±0.38B,f
60 16.1±0.17A,a 4.25±0.15A,ab 36.5±0.28A,a 0.22±0.01A,bc 125±0.68C,d 81.1±2.05A,b
A+G+O+BHT 0 4.99±0.14C,a 1.28±0.08C,e 11.1±0.20C,b 0.08±0.00C,a 130±0.32B,h 31.5±0.33C,cd
30 8.38±0.63B,cd 2.29±0.01B,cd 19.1±0.89B,b 0.22±0.00B,c 138±0.35B,d 34.2±0.13B,ef
60 13.0±0.48A,cd 3.60±0.09A,cd 29.6±0.68A,b 0.26±0.01A,b 99.5±0.33C,ef 86.3±0.48A,a

A–C values of the same analysis for the same oil sample but different storage periods (Days 0, 30, and 60) with different letters are significantly different
(P<0.05). a–h values of the same analysis and on the same storage day but for different oil samples with different letters are significantly different
(P<0.05).
PV peroxide value, p-AV p-anisidine value, FFA free fatty acid, IV iodine value, TEPC total extracted phenolic content, GAE gallic acid equivalents,
A alginate (1 %), O canola oil, Q quercetin, VE vitamin E, N Novation 2300 starch, G Gelose 80 starch

canola oil used in this study had an initial IV value of 185 g I2/ for the BHT-containing oil encapsulated with A-N and A-G, 98.1
100 g oil) on Day 0. After 60 days at 38 °C, the IV of encapsu- and 99.5 g I2/100 g oil, respectively). In terms of preserving the
lated oil decreased considerably (with the lowest values observed unsaturation of encapsulated oils, BHT was the least effective,
Food Bioprocess Technol (2014) 7:2159–2177
with quercetin exhibiting better or comparable protection to
vitamin E. In the presence of quercetin, the IVs of the A-G beads
were the lowest under the storage conditions of this study. The
A-N beads had slightly higher IVs than the 1 %A beads at RT,
but the trend reversed at 38 °C. Some of the encapsulated canola
oils exhibited a relatively high IV on Day 30 which might result
from the presence of interfering substances to the IV assay, such
as newly generated double bond-containing intermediates (e.g.,
aldehydes and ketones) formed during oil oxidation.
The TEPC values for the three types of beads behaved
differently during storages. The presence of an added antioxi-
dant led to a higher TEPC reading. In the presence of quercetin
and after 60 days storage, the TEPC values of the A-G beads
(70.5 mg GAE per kg oil) and A-N beads (60.2 mg GAE per kg
oil) were considerably higher than that of the 1 %A beads
(32.6 mg GAE per kg oil) at 38 °C. Following storage at RT,
TEPC values decreased in the order 1 %A (37.8 mg GAE per kg
oil)>A-N (32.3 mg GAE per kg oil)>A-G (22.6 mg GAE per
kg oil). The relationship between storage temperature and shell
formulation is strong. A higher storage temperature (38 °C) did
not necessarily lead to a lower TEPC value for the same type of
encapsulated oil. Thus, it is likely that several action processes
affected the detected TEPC value rather than a single mecha-
nism involving consuming antioxidant to suppress oil deterio-
ration. The detected TEPC values result from the self-
degradation of antioxidants under the applied storage conditions,
the consumption of antioxidants for preserving oil, and also the
extractability of antioxidant and derived antioxidant products
from the canola oils encapsulated by different shell matrices.
Lipid rancidity involves complex mechanisms, and oil sta-
bility depends on chemical and physical factors such as light,
temperature, pH, transition metal ion concentration, FA com-
position, and availability of oxygen (Frankel et al. 2002).
Quercetin is potent antioxidant (Nakamura et al. 2000). Both
BHT and tocopherols are lipophilic (fat-soluble) organic com-
pounds and contain phenolic components. These three antiox-
idants impacted differently on the PV, p-AV, FFA, and IV
results possibly due to their different structure-function
properties. With storage (especially at 38 °C), a greater amount
of antioxidants were consumed to protect oil against deteriora-
tion. Concomitant with the decrease in these antioxidants was
the likely simultaneous generation of active phenolic interme-
diates (thereby some TEPC readings were relatively high at
38 °C). In summary, primary oxidation dominated over sec-
ondary oxidation and hydrolytic rancidity for the encapsulated
oils. Overall, the A-N formulation formed the best shell for
preventing oil deterioration.
HPLC Analysis of Phenolics in the Encapsulated
Quercetin-Containing Canola Oil Beads
HPLC analyses revealed different decomposition pathways
for quercetin that resulted from the interplay between shell
2169
formulation and storage conditions (Table 4 and Fig. 2). This
was evidenced by the difference in the quantity of quercetin,
the rate of quercetin loss, and the presence of flavonoid and
non-flavonoid phenolics that were derived from quercetin.
The absorption spectra of flavonoids generally consist of
two distinctive bands with the maxima at 280 and 370 nm,
while flavan-3-ol and benzoic acid derivatives only have a
maximum at 280 nm (Sun-Waterhouse et al. 2011a, b;
Zvezdanović et al. 2012). Quercetin can be quantified at
370 nm wavelength using rutin as an internal standard, which
had retention times of ∼7.1–7.4 and 3.4–3.6 min, respectively.
On Day 0, quercetin was detected in all the three types of
encapsulated oil (3.12–3.38 μg/g dried bead), with the 1 %A
beads having a marginally lower amount of quercetin
(Fig. 2a). A flavonoid at retention time 4.1 min (Flavonoid
IV) was also found in all these three encapsulated beads. The
quantity of this flavonoid was inversely proportional with the
amount of quercetin, suggesting flavonoid IV might derive
from quercetin during the encapsulation process.
After 30 days at RT no quercetin was detected, but two
flavonoids with retention time 3.7 min (flavonoid III) and
4.1 min (flavonoid IV) were found in all the three types of
encapsulated oil. The amount of flavonoid III was slightly
lower in the 1 %A beads (0.71 μg/g dried bead) than in the
two alginate–starch beads (0.90 μg/g dried bead). After
30 days at 38 °C, no flavonoids including quercetin were
detected in all the three types of encapsulated oil, although a
non-flavonoid phenolic compound (retention time 3.2 min)
was found in the 280 nm chromatograms whose concentration
followed the order of A-N>1 %A>A-G beads. This trend
agreed with the TEPC findings in the earlier oil chemical
analyses. Two non-phenolic compounds were observed in
the 280 nm chromatograms at retention times of 4.7 and
10.2 min. The appearance of non-phenolic products in the
HPLC profiles of the encapsulated beads was possibly asso-
ciated with the instability of the shell under harsh storage
conditions.
After 60 days at RT, no quercetin was detected, but flavo-
noid II (retention time 3.3 min) and a non-flavonoid phenolic
compound at 3.2 min were found in all the three types of
encapsulated oil. The amount of the non-flavonoid phenolic
compound at 3.2 min decreased in the order of A-N>A-
G>1 %A beads. A non-phenolic compound at 10.2 min was
found under these storage conditions. After 60 days at 38 °C,
no quercetin was detected; instead, flavonoid IV (at 4.1 min)
and a flavonoid at 3.1–3.2 min (flavonoid I) were found in all
the three types of encapsulated oil (Fig. 2b). A number of non-
phenolic compounds in the 280 nm chromatograms were
found dependent on the shell formulation—4, 10, and 11 were
for the 1 %A, A-N, and A-G beads, respectively. The algi-
nate–starch shell seemed to have a lot more non-phenolic
compounds than the alginate alone shell, with the A-G beads
having an extra compound at 14.8 min. This suggests that the
2170 Food Bioprocess Technol (2014) 7:2159–2177

Table 4 Amount of identified compounds in encapsulated quercetin-containing oil beads

Storage Bead Amount of each identified compound (μg/g dried bead)

Day 0 A+O+Q 370 nm, 7.1–7.4 min quercetin (3.12 μg/g), 4.0–4.1 min flavonoid IV (0.76 μg/g), a shoulder peak at
3.3 min (flavonoid II)
280 nm, no phenolics detected
A+N+O+Q 370 nm, 7.1–7.4 min, quercetin (3.38 μg/g), 4.0–4.1 min Flavonoid IV (0.72 μg/g), a shoulder peak at
3.3 min (flavonoid II)
280 nm, no phenolics detected
A+G+O+Q 370 nm, 7.1–7.4 min, quercetin (3.32 μg/g), 4.0–4.1 min flavonoid IV (0.73 μg/g), a shoulder peak at
3.3 min (flavonoid II)
280 nm, no phenolics detected.
Day 30, RT A+O+Q 370 nm, no quercetin detected, 3.7 min flavonoid III (0.71 μg/g), 4.1 min flavonoid IV (0.29 μg/g)
280 nm, no phenolics detected
A+N+O+Q 370 nm, no quercetin detected, 3.7 min flavonoid III (0.90 μg/g), 4.1 min flavonoid IV (0.21 μg/g)
280 nm, no phenolics detected
A+G+O+Q 370 nm, no quercetin detected, 3.7 min flavonoid III (0.90 μg/g), 4.1 min flavonoid IV (0.19 μg/g)
280 nm, no phenolics detected
Day 30, 38 °C A+O+Q 370 nm, no quercetin and other flavonoid detected
280 nm, 3.2 min a phenolic compound (3.34 μg/g), with unknown non-phenolic compounds at
4.7 and 10.2 min
A+N+O+Q 370 nm, no quercetin and other flavonoid detected
280 nm, 3.2 min a phenolic compound (4.55 μg/g), with unknown non-phenolic compounds at
4.7 and 10.2 min
A+G+O+Q 370 nm, no quercetin and other flavonoid detected
280 nm, 3.2 min a phenolic compound (3.00 μg/g), with unknown non-phenolic compounds at
4.7 and 10.2 min
Day 60, RT A+O+Q 370 nm, no quercetin detected but a shoulder peak at 3.3 min (flavonoid II, smaller than Day 0)
280 nm, 3.2 min a phenolic compound (1.66 μg/g). A non-phenolic compound at 10.2 min
(smaller than Day 30, 38 °C)
A+N+O+Q 370 nm, no quercetin detected but a shoulder peak at 3.3 min (flavonoid II, smaller than Day 0)
280 nm, 3.2 min a phenolic compound (2.54 μg/g). A non-phenolic compound at 10.2 min
(smaller than Day 30, 38 °C)
A+G+O+Q 370 nm, no quercetin detected but a shoulder peak at 3.3 min (flavonoid II, smaller than Day 0)
280 nm, 3.2 min a phenolic compound (1.89 μg/g). A non-phenolic compound at 10.2 min
(smaller than Day 30, 38 °C)
Day 60, 38 °C A+O+Q 370 nm, no quercetin detected, 4.0–4.1 min flavonoid IV (0.39 μg/g), with a small peak at
3.1–3.2 min flavonoid I
280 nm, no phenolics detected, with unknown non-phenolic compounds at 4.2, 4.6–4.7, 8.4,
and 10.2 min
A+N+O+Q 370 nm, no quercetin detected, 4.0–4.1 min flavonoid IV (0.43 μg/g), with a small peak at
3.1–3.2 min flavonoid I
280 nm, no phenolics detected, with unknown non-phenolic compounds at 4.2, 4.6–4.7, 5.5, 5.8, 6.4,
6.8, 8.4, 10.2, 12.5, and 13.9 min
A+G+O+Q 370 nm, no quercetin detected, 4.0–4.1 min flavonoid IV (0.44 μg/g), with a small peak at 3.1–3.2
min flavonoid I
280 nm, no phenolics detected, with unknown non-phenolic compounds at 4.2, 4.6–4.7, 5.5, 5.8, 6.4,
6.8, 8.4, 10.2, 12.5, 13.9, and 14.8 min

compounds at retention times 5.5, 5.8, 6.4, 6.8, 12.5, 13.9, and
14.8 min might originate from the starch ingredient.
The chemical structure of quercetin ultimately determines
its stability and interactions with canola oil and polysaccharide
polymers as well as its susceptibility to oxidation/degradation
formulations (Rice-Evans et al. 1996; Lin et al. 2007; Sun-
Waterhouse et al. 2011a, 2012b). Quercetin has the hydroxyl
group at C-3 on the C-ring, adjacent to the 2,3-double bond
and the 4-carbonyl, which is easily oxidized due to electron
donation by the ketone structure at C-4 (Rice-Evans et al.
Food Bioprocess Technol (2014) 7:2159–2177 2171

1996; Sun-Waterhouse et al. 2011a). Possible phenolic prod- and 2,5,7,3',4'-pentahydroxy-3,4-flavandione (Buchner et al.
ucts resulting from quercetin decomposition included non- 2006; Zenkevich et al. 2007). Quercetin degradation in the
flavonoid benzoic acids such as protocatechuic acid and other presence of oxygen can be substantially reduced if quercetin is
phenolics such as 1,3,5-trihydroxybenzene (phloroglucinol) bound to an encapsulant polymer. This may explain why the

a
A+O+Q, Day 0, 280 nm

A+O+Q, Day 0, 370 nm

A+N+O+Q, Day 0, 280 nm

A+N+O+Q, Day 0, 370 nm

3.320

4.211

A+G+O+Q, Day 0, 280 nm

A+G+O+Q, Day 0, 370 nm

3.306

4.219

Fig. 2 High-performance liquid chromatograms (at 280 and 370 nm) for 1 % alginate (A+O+Q), alginate–Novation 2300 starch (A+N+O+Q), and
alginate–Gelose 80 starch (A+G+O+Q) encapsulated beads on a Day 0 and b Day 60 at 38 °C
2172 Food Bioprocess Technol (2014) 7:2159–2177

b
A+O+Q, Day 60, 38 C, 280 nm

A+O+Q, Day 60, 38 C, 370 nm

A+N+O+Q, Day 60, 38 C, 280 nm

6.431

A+N+O+Q, Day 60, 38 C, 370 nm

A+G+O+Q, Day 60, 38 C, 280 nm

6.878

A+G+O+Q, Day 60, 38 C, 370 nm

Fig. 2 (continued)

fresh encapsulated beads here still contained reasonable quer- (i.e., at 38 °C), quercetin was lost. The differences in bead
cetin contents. During oil encapsulation, storages and heating characteristics including size, shape, and surface attributes and
Food Bioprocess Technol (2014) 7:2159–2177 2173

a b

Gelose 80 starch A+O+Q day 60 38ºC

Absorbance (arbitrary units)

Absorbance (arbitrary units)

Novation 2300 starch
A+O+Q day 60 RT

Sodium alginate

A+O+Q day 30 38ºC

Water

Quercetin dihydrate A+O+Q day 30 RT

A+O+Q day 0
Canola oil

4000 3500 3000 2500 2000 1500 1000 4000 3500 3000 2500 2000 1500 1000

Wavenumber (cm-1 ) Wavenumber (cm-1)

c d

A+N+O+Q day 60 38ºC A+G+O+Q day 60 38ºC

Absorbance (arbitrary units)
Absorbance (arbitrary units)

A+N+O+Q day 60 RT
A+G+O+Q day 60 RT

A+N+O+Q day 30 38ºC

A+G+O+Q day 30 38ºC

A+N+O+Q day 30 RT

A+G+O+Q day 30 RT

A+N+O+Q day 0

A+G+O+Q day 0

4000 3500 3000 2500 2000 1500 1000 4000 3500 3000 2500 2000 1500 1000

Wavenumber (cm )
-1
Wavenumber (cm -1 )

Fig. 3 Normalised FT-IR absorbance spectra for a ingredients, and (A+N+O+Q), and d alginate–Gelose 80 starch (A+G+O+Q). The
freeze-dried encapsulated quercetin-canola oil beads before and after spectra have been offset vertically for clarity
storages: b 1 % alginate (A+O+Q), c alginate–Novation 2300 starch
2174
hence their different degrees of susceptibility to oxidation, all
influenced the HPLC results.
FT-IR Analysis of Encapsulated Canola Oil Beads
FT-IR was used to characterize the ingredients used in this
study, i.e., alginate, canola oil, quercetin, water, Novation
2300 starch, and Gelose 80 starch (Fig. 3a) and examine any
potential composition changes in the three types of freeze-
dried quercetin-containing oil beads (Fig. 3b, c, and d).
Freeze-dried beads were used for FT-IR analyses to reduce
spectral interference from water.
FT-IR spectra for all ingredients (Fig. 3a) were consistent with
the literature spectra of canola oil, quercetin, and carbohydrates
(including alginate and starch) (Mathlouthi and Koenig 1986;
Vlachos et al. 2006; Capron et al. 2007; Sun-Waterhouse et al.
2012a; Che Man and Rohman 2012; Krishnakumar et al. 2012).
Canola oil has several characteristic bands (Che Man and
Rohman 2012; Vlachos et al. 2006): 3,009 cm−1 (C-H stretching
vibration of the cis-double bond =CH), 2,925 and 2,854 cm−1
(symmetric and asymmetric stretching vibration of the aliphatic
CH2 group), 1,746 cm−1 (ester carbonyl group of triglycerides),
1,465 cm−1 (bending vibrations of CH2 and CH3 aliphatic
groups), 1,377 cm−1 (bending vibrations of CH2 groups), 1,238
and 1,163 cm−1 (stretching vibration of the C–O ester groups),
and 723 cm−1 (overlap of the CH2 rocking vibration and out-of-
plane vibration of cis-disubstituted olefins). Alginate has bands
at 1,200–800 cm−1 and 1,160–1,130 cm−1 due to (C–OH) side
groups and (C–O–C) glycosidic bond stretching vibrations. The
1,616 and 1,415 cm−1 bands are assigned to the asymmetric –
COO- and symmetric –COO- stretching modes of the carboxyl-
ate groups in alginate (Caykara et al. 2005). The characteristic
absorption for α- and β-linkages at 834 and 898 cm−1,
respectively, of carbohydrates could not be unambiguously
identified in this study (Mathlouthi and Koenig 1986; Caykara
et al. 2005). Quercetin absorbs strongly in the 1,700–700 cm−1
region (Sun-Waterhouse et al. 2012a; Krishnakumar et al. 2012;
Lu et al. 2011)—1,618 cm−1 (ring C–C stretch of phenyl ring),
1,339 cm−1 (in-plane C–O stretching vibration combined with
the ring stretch of phenyl ring), 1,664 cm−1 (vibrations of C=O
group), 3,400 cm−1 (O–H stretching), 1,450 cm−1 (C=O
stretching), 1,262–1,168 cm−1 (C–O–C antisymmetrical
stretching), and 1,130–1,014 cm−1 (C–O–C symmetrical
stretching), which is associated with the dihydrate form of the
quercetin ingredient. A flat and broad band at 3,100–3,600 cm−1
was observed in the quercetin spectrum of this study. Water has
an intense band in this region at 1,635 cm−1 due to H–O–H
bending modes.
FT-IR spectra (Fig. 3b, c, and d) of the quercetin-containing
1 %A, A-N, and A-G beads were dominated by signals from
canola oil with the co-occurrence of some signals from algi-
nate, water, and starch. The spectra of the three types of freeze-
dried oil beads were consistent with their composition where
Food Bioprocess Technol (2014) 7:2159–2177
canola oil is the main component on the mass basis. The
absence of major changes in the FTIR spectra of encapsulated
oil beads during storages at RT or 38 °C indicates the good
chemical and thermal stability of beads. Unambiguous assign-
ment of FT-IR absorbance signals due to quercetin in the
spectra of the 1 %A, A-N, and A-G beads was not possible,
because of the low concentration of quercetin in the freeze-
dried beads. However, reaction between quercetin or its deg-
radation products and the encapsulating matrix over storages
may also contribute to the non-observation of FT-IR signals
for quercetin (Sun-Waterhouse et al. 2012a). For all the three
types of oil beads, the decreased oil signals were generally
accompanied by the increased water signals. The band at
3,009 cm−1 that is associated with the degree of unsaturation
and assigned to the C–H stretching vibration of cis-double
bond =CH of canola oil (Madankar et al. 2013) was still
intense for the 1 %A beads but almost disappeared for the
two alginate–starch beads after 60 days at 38 °C. The absorp-
tion FT-IR signals caused by starch addition was weakly
detected (Fig. 2c and d), e.g., the 998–1,000 cm−1 absorption
signals that are the major absorption band of Novation 2300 or
Gelose 80 starch and associated closely with the hydrogen
bonding of the hydroxyl group at C6 and the water content of
a starch-containing system (Capron et al. 2007).
Polysaccharide polymer gels are generally a cross-linked
hydrophilic network matrix with a porous structure, formed
via chemical interactions (e.g., covalent bonds) or physical
interactions (e.g., non-covalent hydrogen bonding, hydropho-
bic, and ionic interactions) (Whistler and BeMiller 1997). For
the alginate-alone gels, hydrogen bonding or electrostatic
interactions are the only significant interactions for stabilising
the polymer network (Andresen and Smidsmd 1977), thereby
a monotonous decrease of the elastic modulus with increasing
temperature. For the alginate–starch beads, hydrophobic in-
teractions also occur and are likely strengthened when the
temperature increases (Oakenfull and Fenwick 1977).
Blending two polysaccharides modifies the rheological prop-
erties of mixed gel (Choi and Yoo 2008). In this study, the
presence of starch in alginate network could weaken the
intermolecular forces between the alginate and water (Tan
et al. 2009; Roy et al. 2009). Starch might enhance the tensile
strength but decrease the solubility of alginate–starch shell
(Fazilah et al. 2011; Maiti et al. 2012). Amylose is the key
component involved in water absorption, swelling, and gela-
tion of starch in food processing. The crystalline structure of
the starch low in amylose (e.g., waxy starches) is easily
disrupted (Jane et al. 1999), thus exhibiting lower syneresis
than normal or high-amylose starches (Singh et al. 2007; Kaur
et al. 2008). Starch rich in amylose has a lower degree of
gelatinisation, because of its high crystallinity (Juarez-Garcia
et al. 2006; Chung et al. 2011). Thus, the different amylose
contents in Novation 2300 and Gelose 80 starches account for
the different behaviors observed for the A-N and A-G beads.
Food Bioprocess Technol (2014) 7:2159–2177
It is worth noting that starch digestibility should be consid-
ered when a starch is selected as an encapsulant component,
especially in the case that consumers have high risk of diabetes
and obesity diseases. High-amylose starch generally has lower
digestibility leading to lower glucose concentrations in blood
and insulin responses, compared with low-amylose starch (Li
et al. 2012; Regmi et al. 2011). In this study, the Totox values
of the A-N and A-G oil beads, in the absence and presence of
quercetin, were all <30. The A-N was the best shell to suppress
oil deterioration, but the A-G shell is possibly a more suitable
shell for the encapsulated oil bead products that are designed
for regular consumption by the diabetes or obesity patients.
Conclusions
Co-extrusion encapsulation in combination with antioxidant
addition can effectively suppress the deterioration of unsatu-
rated canola oil during storage at 20 °C or 38 °C. Alginate or
alginate–starch mixtures can be used to successfully encapsu-
late canola oil, producing spherical or ovoid shaped beads of
good chemical stability. For encapsulated oil beads stored at
20 °C or 38 °C, primary oxidation dominated over the sec-
ondary oxidation and hydrolytic rancidity. Quercetin was a
more effective antioxidant than vitamin E but comparable to
BHT in slowing oil rancidity. For oils containing quercetin,
the A-N shell appeared to be the best shell in preventing oil
deterioration, while the 1 %A and A-G were still acceptable
shell formulations (their encapsulated oil had a Totox value
<30). FT-IR studies further confirmed the good chemical and
thermal stability of these three types of encapsulated beads.
After the acidic treatment at pH 3 for 2 h, these three types
of beads remained intact but shrank significantly, i.e., the
reduction of bead size in the increasing order of 1 %A
(∼45 μm)<A-N (∼60 μm)<A-G (∼96 μm) beads. HPLC
profiling detailed the different changes of added quercetin
after encapsulation process and over the four different stor-
ages, including the retention and loss of quercetin, the co-
occurrence of quercetin-derived products (flavonoids versus
non-flavonoid phenolics). In the 280-nm chromatograms,
there existed different non-phenolic compounds as a result
of the interplay between storage conditions and shell formu-
lation, which might originate from alginate alone or alginate–
starch shell. Concept product “quercetin-containing unsaturat-
ed oil beads encapsulated with alginate–starch” can simulta-
neously deliver health-promoting unsaturated oil, quercetin
antioxidant, and starch fiber polysaccharides in a stable and
convenient format for consumers. Oil beads encapsulated by
A-G may be suitable for consumers who have diabetes or
obesity risks.
Acknowledgments We acknowledge research funding from The Uni-
versity of Auckland and Plant and Food Research.
2175
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