International Journal of Biological Macromolecules 140 (2019) 682–696

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Antioxidant and antibacterial activities of omega-3 rich oils/curcumin

nanoemulsions loaded in chitosan and alginate-based microbeads
Ayat F. Hashim a, Said F. Hamed a, Hoda A. Abdel Hamid b, Kamel A. Abd-Elsalam c, Iwona Golonka d,
Witold Musiał d, Ibrahim M. El-Sherbiny e,⁎
a
Fats and Oils Department, National Research Centre, Egypt
b
Chemistry Department, Faculty of Science, Ain Shams University, Egypt
c
Agricultural Research Center – Plant Pathology Research Institute, 9 Gamaa St., 12619 Giza, Egypt
d
Department of Physical Chemistry, Wroclaw Medical University, Poland
e
Center of Materials Science (CMS), Zewail City of Science and Technology, Egypt

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

Article history: Omega-3 fatty acids can be considered as potential alternative therapeutic agents because of their antimicrobial
Received 31 May 2019 and antioxidant properties. Two investigated omega-3 rich oils (flaxseed or fish) have been nanoemulsified with
Received in revised form 12 July 2019 and without the natural antioxidant (curcumin, Cur) followed by their incorporation into crosslinked polymeric
Accepted 9 August 2019
microbeads. The microbeads were developed from chitosan (CS), alginate (AL) and their combination (CSAL). Re-
Available online 09 August 2019
sults indicated that the mean droplet diameter of the plain and Cur-loaded nanoemulsions ranged from 62.3 to
Keywords:
111.29 nm. The microbeads produced from AL, CS and their combination without Cur had predominantly shriv-
Omega-3 rich oils eled surfaces compared to Cur-loaded ones. Addition of Cur was found to enhance oxidative stability, encapsula-
Nanoemulsions tion efficiency, loading capacity, and antioxidant activity of the formulated microbeads. Plain fish oil revealed
Microbeads more antibacterial activity than plain flaxseed oil. Fish oil nanoemulsion-in-AL microbeads had more antibacte-
Oxidative stability rial activity than nanoemulsions of flaxseed oil-in-AL, fish oil-in-CS and the combined (CSAL) microbeads. How-
Antioxidant activity ever, flaxseed oil nanoemulsion-in-CS microbeads showed higher antibacterial activity than nanoemulsions of
Antibacterial activity fish oil-in-CS, flaxseed oil-in-AL and the combined microbeads. The obtained results suggested the suitability
of the formulated nanoemulsions-loaded microbeads to be used in food and pharmaceuticals products.
© 2019 Elsevier B.V. All rights reserved.

1. Introduction
Omega-3 fatty acids are vital not only for normal growth and matu-
rity but also for their positive impacts on brain, heart, eyes, skin, joints as
well as mood and behavior [1,2]. Besides, omega-3 fatty acids are in-
volved in the prevention of coronary artery diseases, hypertension, dia-
betes, arthritis, autoimmune disorders and cancer [3]. They possess
numerous biological activities, such as anti-inflammatory [4], antioxi-
dant [5], antimicrobial [6], and neuroprotective [7] activities. However,
due to their unsaturated nature, omega-3 fatty acids are chemically un-
stable and susceptible to oxidation, resulting in the production of free
radicals and unpleasant tastes and off-flavors, which affect negatively
on the shelf-life, sensory properties as well as on the general acceptabil-
ity of the food products [8].
Adding antioxidant is one of the most effective ways to protect oil
systems from oxidation [9]. However, the concern related to possible
adverse effects of synthetic antioxidants is increasing. Hence, there is a
growing demand for using antioxidants of natural origin for better
⁎ Corresponding author.
E-mail address: ielsherbiny@zewailcity.edu.eg (I.M. El-Sherbiny).
health [10]. Cur, a polyphenolic compound, is widely used due to its
pharmacological effects, such as being antioxidant, anti-inflammatory,
anticancer and antivirus [11–13]. It was also demonstrated that Cur
has antioxidant properties in edible oils. Besides, polyphenolics have
been used as antioxidants to increase the shelf-life of microencapsu-
lated flaxseed oil [14]. Cur can also be applied in both corn oil and oil
in water (O/W) emulsion systems to inhibit lipid oxidation [15].
Microencapsulation technology has been also used as an applicable
method to develop and improve the biological and functional character-
istics of the oils [16]. Microencapsulation can also partially prevent the
omega-3 fatty acids oxidation and prolongs their shelf-life as well as
displaying a practical solution for their stabilization and enhanced deliv-
ery in food products [17]. In this context, microencapsulation is a pro-
cess in which tiny particles of the active and/or sensitive component,
such as pure fatty acids, known as the core, are packaged within an en-
capsulating matrix [18]. Some of the commonly used coating materials
for the microencapsulation of omega-3 fatty acids are proteins, lipids,
cellulose and polysaccharides. The application of polysaccharides-
based edible coatings, such as chitosan (CS) and alginate (AL), and
enriched with antimicrobials or antioxidant has been proved to form
rigid and stable gels [19].

https://doi.org/10.1016/j.ijbiomac.2019.08.085
0141-8130/© 2019 Elsevier B.V. All rights reserved.
 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696
Nowadays, AL is widely used in food, textile and pharmaceutical in-
dustry. It is generally applied as the thickening and gelling agents in the
presence of divalent cations, such as Ca+2 and Zn+2, and also as stabiliz-
ing, suspending and emulsifying agents [20]. However, the leakage of
the core materials due to the loose AL-Ca network limits its wide use
in such applications [21]. This can be improved via further coating of
the AL-Ca beads with a layer of polycations, such as CS. CS has a unique
property called the permeation enhancing effect, which makes it a good
carrier system. It increases permeation by opening epithelial tight junc-
tions, which is important for the movement of hydrophilic compounds
[22]. AL and CS are nontoxic, biodegradable and mucoadhesive food
grade materials [23]. CS being cationic and AL being anionic allow
them to form a good polyelectrolyte complex (PEC)-based hydrogel
[24]. It is also worth to mention that the hydrogel obtained via polyelec-
trolyte crosslinking of AL and CS was used to provide materials useful
for various medical and pharmaceutical applications, where the ob-
tained systems demonstrated enhanced stability compared to those ob-
tained from a single polymer [25].
Essential fatty acids can be encapsulated using different methods,
such as spray-drying [26], simple and complex coacervation [27], emul-
sion extrusion [28] and supercritical fluid precipitation [29] resulting in
their successful incorporation in product formulations. It was
established a technique to encapsulate tuna oil in CS through using ul-
trasonic atomization and freeze drying [30]. In another study, the stabil-
ity of flaxseed oil was improved during storage through its
encapsulation within nanoemulsion-loaded AL microgels [31].
Calcium-AL beads coated with CS have been used to encapsulate shark
liver oil and control its leakage from capsules [32]. CS/AL complex was
used as the encapsulating agent for peppermint oil [33]. The polymeric
complex showed good antibacterial activity against both gram positive
and gram negative bacterial strains. In another research study, Whey
protein and gum Arabic have been used to encapsulate fish oil using a
combination of complex coacervation and thermal crosslinking pro-
cesses [34].
Conjugated linoleic acid (CLA) was encapsulated using polymeric
matrices as wall materials, and also the microparticles was studied oxi-
dative ability by some methods, including determination of the perox-
ide value [35]. In a reported study, it was confirmed that the main
factor associated with the oxidation of oil encapsulated by spray drying
is the increased availability of oxygen during the formation of oil emul-
sion with the carrier. This is followed by using high temperature in the
drying chamber (about 200 °C) upon evaporating the carrier solvent
from the ready emulsion [36]. Besides, it was reported that encapsula-
tion of fish oil along with ginger essential oil could improve the oxida-
tive stability of fish oil encapsulates [37]. It was observed the
protection of eicosapentaenoic acid (EPA) and docosahexaenoic acid
(DHA) by encapsulation within CS using ultrasonic atomizer [30].
CS-AL systems have been widely studied for nanoparticle formation
for drug delivery, but only a few studies have examined encapsulation
of oily compounds into these systems. CS-AL nanocapsules containing
turmeric oil was carried out by emulsification of turmeric oil in aqueous
sodium alginate solution and gelification with calcium chloride and CS,
followed by solvent removal [38]. CS-AL nanocapsules were synthesized
using an ionic gelation method and the size of the particles was below
300 nm with good stability [22].
In order to get the maximum health benefit and to minimize omega-
3 rich oils oxidation; two main strategies have been followed in the cur-
rent study: (1) addition of antioxidant (Cur) to fish and flaxseed oils,
and (2) encapsulating these oils into microbeads. Therefore, the main
objective of this research work was to formulate omega-3 rich
nanoemulsions loaded in microbeads to: (a) study the potential effect
of wall materials (CS, AL and their combination) as food ingredients
on omega-3 rich oils microbeads properties, (b) evaluate the oxidative
stability and encapsulation efficiency of these microbeads in the pres-
ence and absence of Cur, and (c) investigate the antibacterial efficiency
of the prepared microbeads.
683
2. Materials and methods
2.1. Raw materials
Flaxseed oil was obtained as a crude oil from the oil extraction unit
(National Research Centre, Egypt). Fish oil was purchased from Holland
& Barrett (Amsterdam, Netherland). Polyoxyethylene glycol sorbiton
monooleate (Tween-80) was obtained from Biotech Co. (Canada). Po-
tassium iodide, sodium hydroxide and ethanol were supplied by El-
Nasr Company for Pharmaceutical Chemicals (Egypt). Sodium thiosul-
fate (Na2S2O3) was provided by Sisco Research Laboratories (India).
CS was purchased from Acros (Morris Plains, NJ, USA) with a degree of
deacetylation and average MW of 85% and 100.000 Da, respectively. So-
dium alginate (AL) from brown algae (91%) was received from
Lobachemia (India). Cur was purchased from Bio-Basic (Canada). 2,2-
diphenyl-1-picrylhydrazyl (DPPH, 90%) was purchased from Sigma
(St. Louis, MO, USA). Acetic acid was supplied by BDH Chemicals (En-
gland). Calcium chloride dihydrate (99.99%) was obtained from SDFCL
(India). Dimethyl sulphoxide (DMSO) was received from Research Lab
(India). All other solvents and reagents were purchased from various
suppliers and used without further purification.
2.2. Determination of the physico-chemical characteristics of omega-3 rich
oils (flaxseed or fish)
Physico-chemical characteristics of the oils (refractive index, perox-
ide, acid, iodine, saponification value and fatty acid composition) were
determined according to the American oil Chemists Society (AOCS) [39].
2.3. Preparation of nanoemulsion-in-microbeads and Cur-loaded
nanoemulsion-in-microbeads
In the current study, the two investigated omega-3 rich oils (flax-
seed and fish) have been nanoemulsified with and without Cur followed
by their incorporation into crosslinked polymeric microbeads. The poly-
meric microbeads were developed from CS, AL or their combination
(CSAL) using NaOH or CaCl2 aqueous solutions as the crosslinking
agents (Scheme 1).
2.3.1. Preparation of nanoemulsion and Cur-loaded nanoemulsion
The emulsification process was performed in two stages of homoge-
nization. In the first stage, omega-3 rich oils (flaxseed or fish) was used
as the dispersed phase (4(% with 96 ml distilled water (DW) containing
2% Tween 80 as the continuous phase. The three components were ho-
mogenized with the aid of a high speed homogenizer (X520, CAT
Ingenieurburo M. Zipperer, GmbH, Germany) for 3 min at 16.000 rpm
to form a coarse emulsion. Then, in the second stage, the coarse emul-
sion with and without Cur (Cur, 5 mg in 10 ml absolute ethanol) was
ultrasonicated using a bench scale sonicator (Sonics & Materials Inc.
53 Church Hill Rd. Newtown, CT USA) at 60% of full power amplitude
(60 W) for 10 min and using a probe with a diameter of 22.5 mm.
2.3.2. Incorporation of the nanoemulsion and Cur-loaded nanoemulsion
into polymeric microbeads
The developed oil (flaxseed or fish) nanoemulsion with and without
Cur was incorporated into crosslinked polymeric microbeads. The poly-
meric microbeads were developed from CS, AL or their combination
through coacervation method.
(a) For CS microbeads, 3 g of CS was dissolved in 50 ml of acetic acid
(1% v/v) and filtered through Whatman paper no. 1. Then, both CS solu-
tion and the previously prepared oil (flaxseed or fish) nanoemulsion
were mixed and stirred together. The mixture was then taken into sy-
ringe and added dropwise to 2% NaOH solution to form microbeads.
(b) For AL microbeads, 3 g of AL was mixed properly with 50 ml distilled
water with continuous stirring. After attaining the complete dissolution,
AL solution was mixed with stirring with the pre-prepared oil (flaxseed
684 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696
Scheme 1. An illustration for incorporation of the developed omega-3 rich oils (flaxseed or fish) nanoemulsions and their Cur-loaded forms into hydrophilic polymeric microbeads based
on CS, AL or their combination (CSAL) using NaOH or CaCl2 aqueous solutions as the crosslinking agents.
or fish) nanoemulsion. Then, the mixture was added dropwise to 3%
aqueous CaCl2 solution with a gentle agitation at room temperature to
form the microbeads. (c) For (CSAL) combination microbeads, 30 ml
aqueous AL solution (8% w/v) was mixed with a pre-determined vol-
ume of the oil (flaxseed or fish) nanoemulsion with constant stirring.
Then, 20 ml of CS solution (2.5% w/v) dissolved in 1% (v/v) acetic acid
was added slowly to the prepared mixture. The resulting mixture was
then added dropwise to 3% CaCl2 aqueous solution with gentle agitation
followed by allowing the formed microbeads to stand in the solution for
about 10 min before their collection via filtration.
All the three types of formed microbeads (CS, AL or CSAL) were
washed twice with DW (30 ml) and air dried in un-covered Petri dishes.
2.4. Encapsulation efficiency and encapsulation load
Encapsulation efficiency (EE) and encapsulation load (EL) were de-
termined according to the method mentioned by [40] with some mod-
ification. Fifty milliliters of n-hexane were added to 3 g of microbeads in
100 ml beaker followed by shaking for 2 min at room temperature for
the extraction of surface oil. The mixture was filtered through a
Whatman filter paper no. 1, and the microbeads collected on the filter
were rinsed three times with 20 ml of hexane. Then, the solvent was
evaporated at reduced pressure until constant weight. The non-
encapsulated oil (surface oil) was determined by mass difference be-
tween the initial clean flask and that containing the extracted oil residue
[41]. Total oil was assumed to be equal to the initial oil, since prelimi-
nary tests revealed that all the initial oil was retained, which was ex-
pected, since both flaxseed and fish oils are non-volatile. EE and EL
were calculated from Eqs. (1) and (2), respectively.
EE% ¼ ðTO–SO=TOÞ  100
EL% ¼ ðTO–SO=TW Þ  100
where TO is the total oil content, SO is the surface oil content, and TW is
the total weight of microbeads.
2.5. Oxidative stability
The prepared microbeads were examined for their oxidative stability
by two different ways; first through measuring the antioxidant activity
(using the DPPH assay) and second by following up the primary oxida-
tion products (measured as peroxide value, PV).
2.5.1. Antioxidant activity assessment
The antioxidant activity assay using the stable 2,2`-diphenyl-1-
picrylhydrazyl free radical (DPPH•) was carried out according to Kim
et al. [42]. Briefly, 2 g of dried milled microbeads were stirred in
100 ml ethanol for 1 h, and then filtered using Whatman no. 1 paper.
The residue was re-extracted with another 100 ml ethanol for 15 min.
Then, 5 μl of the filtrate was added to 1 ml pure methanol and 2 ml of
freshly prepared 0.13 mM DPPH• solution in ethanol. The sample solu-
tions were vigorously shaken on a vortex for 30 s and then immediately
placed in a UV/VIS spectro-photometer (T80 UV/VIS Spectrometer, PG
Instruments Ltd., UK). The absorbance was measured at 516 nm after
exactly 30 min against pure ethanol. Blank was made as above by re-
placing the test sample with 5 μl ethanol. The percentage radical scav-
enging activity (RSA %) was calculated from the following equation:
RSA% ¼ ðAbB −AbS =AbB Þ  100
where, AbB and AbS are the absorbance values of the blank and sample,
respectively. The measurements were carried out in duplicates.
2.5.2. Peroxide value
The prepared microbeads were stored for a period of 30 days at 4 °C.
Extraction of flaxseed or fish oil was performed according to a previ-
ously reported method [43] with some amendments. Briefly, 4 g of the
investigated microbeads were weighted and placed into a 100 ml bea-
ker, suspended in 50 ml of n-hexane, and shaken until complete disso-
lution over 1 h. The mixture was filtered and the residue was re-
ð1Þ extracted with another 50 ml hexane for 15 min, and the solvent was
evaporated using rotary extraction system under reduced pressure at
50 °C. The oxidation stability of extracted oil was evaluated by measur-
ð2Þ
ing the peroxide value, which is an indication on occurrence or not of
any oxidation processes during storage [44]. The peroxide value was
followed up at 10 days intervals using iodometric titration method ac-
cording to the official methods of analysis.
2.6. Thermo gravimetric analysis
Thermal characteristics of the prepared microbeads was evaluated at
the Faculty of Pharmacy, Wroclaw medical university, Poland by
 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696
thermogravimetric analysis (TGA) in a thermo-balance TG 209 F1
Libra® at a heating rate of 10 °C/min, with sample weights of 7 to
10 mg, under nitrogen flow (20 ml/min). The samples were investigated
in the temperature range from 25 °C to 1000 °C.
2.7. Assessment of antibacterial activity
The gram-negative bacteria (Escherichia coli, Salmonella typhmerium,
Yersinia enterocolitica, and Pseudomonas aeruginosa), and gram-positive
bacteria (Staphylococcus aureus, Bacillus cereus, and Listeria
monocytogenes) were obtained from dairy microbiological Lab (National
Research Centre, Egypt). The bacterial strains were grown on nutrient
agar (Lab-Lemco powder 1.0 g/l, yeast extract 2.0 g/l, peptone 5.0 g/l, so-
dium chloride 5.0 g/l and agar 15.0 g/l at pH of 7.4 ± 02 at 25 °C). The
well diffusion method was used to determine the antibacterial activity
of the tested microbeads [45]. Test samples with two concentrations
(90 and 180 mg/ml) were dispersed in DMSO. Then, 50 μl of the dis-
persed microbeads were pipetted into created wells to reveal the most
sensitive microorganisms against investigated microbeads. DMSO was
used as a negative control. The plates were incubated at 37 °C for 24 h
after that the inhibition zones development were examined. A caliper
was used to measure the inhibition zones. Three replicates were carried
out at least for each assessment. The inoculum's concentration was 6.5
× 105 CFU and the reaction of the microorganisms with antibacterial
samples was determined by the size of the inhibitory zone.
2.8. Physico-chemical characterization of nanoemulsions and Cur-loaded
nanoemulsions-in-microbeads
2.8.1. Determination of fatty acids composition
A Hewlett Packard, HP 6890 gas chromatograph equipped with a
flame ionization detector (FID) was used. A capillary column of 30.0 m
× 530 μm, 1.0 μm thickness, and polyethylene glycol phase INNO Wax
was used. Carrier gas (N2) with a flow rate of 15 ml/min was used
with average velocity 89 cm/s (8.2 psi). Injector temperature was ad-
justed at 280 °C, detector temperature at 280 °C and the column tem-
perature was maintained at 240 °C. Programmed temperature starting
from 100 °C to reach a maximum of 240 °C was used for eluting the
fatty acid methyl esters. The identification of the peaks was done as
compared with chromatograms of standard fatty acids methyl esters
(Sigma, USA).
2.8.2. Particle size and zeta potential measurements
The hydrodynamic size and zeta potential of the prepared oil (flax-
seed or fish) nanoemulsions and Cur-loaded nanoemulsions were ob-
tained using dynamic light scattering (DLS) instrument (PSS, Santa
Barbara, CA, USA) using the 632 nm line of a HeNe laser as the incident
light with an angel 90° and Zeta potential with external angel 18.9°
(Central laboratory, National Research Center). Zeta potential provides
information about the potential difference between the dispersion me-
dium and the stationary layer of the fluid attached to the dispersed par-
ticle, which helps to expect the stability of the nanoemulsion. Samples
were prepared by 0.2 g/5 ml millipore water and sonication for 5 min.
2.8.3. FTIR spectroscopy
FTIR spectra of the formed microbeads were acquired on Thermo
Scientific Nicolet iS50 (Faculty of Pharmacy, Wroclaw medical univer-
sity, Poland) with a number of scans of 65 and 16 cm−1 resolution
within the range of 4000–400 cm−1.
2.8.4. X-ray diffraction (XRD)
XRD of the prepared microbeads was recorded using Panalytical–
Empyrean, Netherlands equipped with CuKα Radiation (λ = 1.5406
Å). The measurements were performed at a scanning rate of 0.1° in
the 2θ range from 5° to 70° and step time of 1 s.
685
2.8.5. Morphological studies
The obtained microbeads were placed on a microscope slide covered
with a glass cover slip. Then, microscopy images of the samples were
obtained using an optical microscope (Olympus BX50, Tokyo, Japan)
with 20 × objective lens.
A stereo microscope (Motic SMZ-171 Series) was also used to image
and particularly determine the mean diameters of the prepared
microbeads. The mean diameter for a certain number (6–10) of
microbeads of each formulation was determined using the following
equation:
Mean diameter ¼ Sum of the diameters of microbeads=n
where, n is the total number of measured microbeads (6–10).
High resolution transmission electron microscope (HR-TEM, Tecnai
G20, FEI, Netherlands) was also used for the purpose of imaging, crystal
structure revelation and elemental analysis (qualitative and semi-
quantitative analysis). Two different modes of imaging were used; the
bright field at electron accelerating voltage 200 kV using lanthanum
hexaboride (LaB6) electron source gun and the diffraction pattern imag-
ing. Eagle CCD camera with (4 k*4 k) image resolution was used to ob-
tain and collect transmitted electron images. Besides, the surface
morphology and shape of the optimized microbeads were investigated
by scanning electron microscopy (SEM) (quanta fei 250 republic
Czech). In addition, atomic force microscopy was also used to investi-
gate the surface morphology of the developed microbeads. AFM
Nanoman microscope was also used with a tapping mode injection
and with leverage of a resonant frequency of 330 kHz. The images
were scanned at a speed of 1 line/s. During the measurements, the am-
plitude was suppressed by 30%.
2.9. Statistical analysis
All the experimental data were subjected to one way variance anal-
ysis (ANOVA) using Statistica 6.0 software. Mean values were compared
using Duncan's multiple range test (DMRT) and judged at P ≤ 0.05 level.
3. Results and discussion
Omega-3 fatty acids are associated with beneficial health outcomes
including reduced cardiovascular disease [1–3] and improvement of
brain development [4]. Microencapsulation can be considered as a key
technique to package sensitive ingredients, such as omega-3 rich oils
in the form of microbeads within different wall materials and enhanced
delivery in food and pharmaceuticals products [17]. CS, AL and their
combinations have been used in the present study as microencapsulat-
ing wall materials due to their desirable biocompatibility, non-toxicity,
low cost and their abundance in nature. The developed microbeads
were investigated for their stability, encapsulation efficiency as well as
their antioxidant and antibacterial activities.
3.1. Physico-chemical properties of investigated omega-3 rich oils (flaxseed
or fish)
The physical and chemical attributes of oils can directly affect their
shelf-life and functionality. Parameters like the acid value (AV), perox-
ide value, oxidation stability and the color are basic criteria which are
used to evaluate the quality characteristics of the oils [46]. The investi-
gated flaxseed oil showed a yellow-brown color with a pleasant odor,
while the fish oil was typically a shade of yellow with fishy smell. As
shown in Table 1, the refractive indexes indices of flaxseed and fish
oils were measured at 25 ± 1 °C to be 1.47 and 1.45, respectively.
Also, the acid value was determined to be 1.62 and 0.12 mg KOH/g oil,
for flaxseed and fish oils, respectively. AV measures the amount of free
fatty acids present and increments in AV reveal hydrolysis of the triglyc-
erides of the oil due to chemical or enzymatic deteriorations. The low AV
686 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696

Table 1
Physico-chemical properties of the investigated omega-3 rich oils (flaxseed or fish). 100 Flaxseed nanoemulsion
Parameters Values mean Flaxseed Cur nanoemulsion
Fish Cur nanoemulsion
Flaxseed oil Fish oil
80 Fish nanoemulsion
Refractive index at 25 ± 1 °C 1.47 1.45
Peroxide value (meq. O2/kg oil) 1.46 3.86
Acid value 1.62 0.12
60
Iodine value (Hanus) 167.5 195.176
Saponification value 203.5 159.03
Fatty acids Content (%)
Palmitic acid (C16:0) 5.5197 2.31 40
Plamitoleic acid (C16:1) – 9.22
Stearic acid (C18:0) 4.37 –
Oleic acid (C18:1) 12.68 9.75
20
Linoleic acid (C18:2) 14.74 20.35
Linolenic acid (C18:3) 62.69 –
EPA – 27.44
DHA – 18.166 0
Others – 12.764 0 50 100 150 200 250 300 350 400

Size

indicated the refreshness of the two investigated oils. PV of flaxseed and
fish oils before microencapsulation was found to be 1.46 and 3.86 meq
O2/kg oil, respectively (Table 1). According to the Codex Alimentarius
Standard (1999) for edible fats and oils, AV and PV of our investigated
oils reflect their suitability to be used for edible or pharmaceutical use
[47].
The saponification value of flaxseed and fish oil was found to be
203.5 and 159.03 mg KOH/g oil, respectively. Besides, the iodine value
of flaxseed and fish oil was found to be 167.5 and 195.17 g I2/100 g
oil, respectively. The high iodine value reflected a high level of unsatu-
rated fatty acids which are very important from a nutritional point of
view. In our case, the unsaturated fatty acids include the presence of
high concentration of linoleic and linolenic acid, EPA and DHA with all
settled health importance [1–7]. As shown in Table 1, the main fatty
acids in flaxseed oil were oleic, linoleic and linolenic acid with 12.68%,
14.74% and 62.69%, respectively. Regarding fish oil, the main omega-3
fatty acids were EPA and DHA with amounts of 27.44% and 18.16%,
respectively.
3.2. Size and stability of plain and Cur-loaded nanoemulsions
Plain and Cur-loaded nanoemulsions were prepared using
ultrasonication. Both particle size and stability were investigated using
a DLS technique. The mean diameter of the plain and Cur-loaded
nanoemulsions ranged from 62.3 to 111.29 nm (Table 2). As apparent
from the Fig. 1, both plain and Cur-loaded fish oil nanoemulsions have
almost attained same size (62.3–64.1 nm). However, in the case of flax-
seed oil, the Cur-loaded nanoemulsion demonstrated a larger size
(111.29 nm) than that of its plain nanoemulsion (71.2 nm). The fre-
quent size measurements also demonstrated that both plain and Cur-
loaded nanoemulsions have survived the stability tests.
In colloidal systems the zeta potential is the electric potential differ-
ence across the ionic layer around a charged colloid ion. It is worth to
mention that zeta potential of the microbeads is one of the essential
characteristics of a dispersed system in terms of dispersion stability. Re-
sults indicated that zeta potential of plain and Cur-loaded
Table 2
Average zeta potential, polydispersity index and mean diameter of plain and Cur-loaded
nanoemulsions.
Sample Avg. zeta Polydispersity
potential index
Flaxseed nanoemulsion −20.92 0.228
Flaxseed Cur −20.73 0.181
nanoemulsion
Fish nanoemulsion −20.57 0.199
Fish Cur nanoemulsion −14.31 0.181
Fig. 1. Particle size distribution of plain and Cur-loaded nanoemulsions.
nanoemulsions were found to be between −20.92 and −14.31 mV
confirming the stability of the developed nanoemulsions. Such amount
of zeta potential of the produced nanoemulsions may resist coagulation
or flocculation. DLS results showed also that the polydispersity indices
of plain and Cur-loaded nanoemulsions were in the range from 0.181
to 0.023 which reveal an acceptable uniformity in particles distribution
[48].
3.3. FTIR analysis
FTIR analysis was performed to investigate the possibility of complex
formation and interaction of major functional groups involved in the
oils encapsulation. As apparent from the Fig. 2, FTIR spectrum of pure
CS shows a broad band at 3362 cm−1 that corresponds to the amine
and hydroxyl groups. The characteristic absorption peaks appeared
around 2877 cm−1 was caused by -OH stretching, where the absorption
band of the carbonyl (C=O) stretching of the secondary amide (amide I
band) was noted at 1647 cm−1, and the peak appeared at 1596 cm−1
could be attributed to the N\\H bending vibration. The peaks at 1424
and 1373 cm−1 belong to the N\\H stretching of the amide and the
ether bonds, respectively. The peaks observed at 1061 and 1022 cm−1
were related to the secondary hydroxyl group (characteristic peak of
-CH-OH in cyclic alcohols, C\\O stretching), and the primary hydroxyl
group (characteristic peak of -CH2-OH in primary alcohols, C\\O
stretch), respectively. FTIR spectrum of sodium alginate (AL) showed a
stretching frequency for carboxylic acid groups at 2927 cm−1. The
peaks observed at 3349 cm−1, 2922 cm−1, 1609 cm−1, 1080 cm−1
and 1022 cm−1 were assigned to alcoholic OH stretching, OH stretching
for carboxylic groups, carboxylate salt asymmetric stretching, and C\\O
stretching of ether, respectively. The band noted around 1022 cm−1 (C-
O-C stretching) may be attributed to the AL saccharide structure. In ad-
dition, the bands appeared at 1609 and 1405 cm−1 are assigned to
asymmetric and symmetric stretching of the AL carboxylate salt groups.
The FTIR spectrum of Cur showed a characteristic stretching band of
O\\H at 3509 cm−1. The peak at 2922 cm−1 represents the C\\H
stretching while the peak at 1615 cm−1 was assigned to C_C symmet-
ric aromatic ring stretching. The peak at 1500 cm−1 represents C_O,
Mean while enol C\\O peak was obtained at 1271 cm−1 and the benzoate
diameter trans-C-H vibration was noted at 965 cm−1. The main vibrational
71.2 bands of fish oil were noted at 2935 cm−1 and 2852 cm−1 correspond-
111.29 ing to asymmetric and symmetric stretching (CH2) for total
polysaturated fatty acids (PUFAs), respectively. Also a stretching ester
62.3 carbonyl (–C=O) was observed at 1730 cm−1 and several bands at
64.1
1366 cm−1, 920 cm−1 and 716 cm−1 were also observed. The
 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696 687

Fig. 2. FTIR spectra of CS, AL, Cur, flaxseed oil, fish oil as well as the developed oil-encapsulated plain and Cur-loaded CS, AL and CSAL microbeads.

fingerprint region between 1462 and 704 cm−1 of fish oil spectrum was
also identified. The different functional groups present in the flaxseed
oil were confirmed by their various peaks. For instance, the peaks of
CH2 group were observed at 2858 and 2922 cm−1 while the peak at
3011 cm−1 was due to the presence of =C–H group, where the C_O
and C_C stretching were noted at 1736 cm−1 and 1654 cm−1,
respectively.
FTIR spectra were analyzed to investigate the potential interactions
in the microbeads. For instance, both PUFAs and ester carbonyl peaks
of omega-3 rich oils (flaxseed or fish) were detected in CS and AL
microbeads spectra at 2922 cm−1 and 1749 cm−1. These results con-
firmed the presence of oils in the developed microbeads. The FTIR spec-
tra also confirm the absence of any strong chemical interaction between
encapsulated oils and wall polymeric materials, which demonstrates
that the oils are physically entrapped in the polymeric microcapsules.
This conclusion is in agreement with the literature [49].
The FTIR spectra of CSAL microbeads (Fig. 2e–f) clearly revealed that
the amino groups of CS and the carboxyl groups of AL are interacting to-
gether to form a strong polyelectrolyte complex (PEC) [24]. The peak
appeared at 3349 cm−1 indicates the presence of hydroxyl and amino
groups from AL and CS and becomes broad upon complexation of the
two polymers [50]. The peak at 1609 cm−1 of CSAL microbeads indicates
the association of the carboxylate groups of AL with CS, and the peak
noted at 1086 cm−1 is attributed to the amino groups of CS. It was
also evident that the major peaks present in the spectra of CS/AL are
prominent in the combined microbeads. This indicates the strong inter-
action between CS and AL in oil-loaded microbeads. Besides, the new
band appeared around 1240 cm−1 confirms the occurrence of the
688 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696

electrostatic interaction between the protonated amino groups of CS

and the dissociated carboxylate groups of AL.

3.4. XRD

Fig. 3 shows the XRD patterns of CS, AL as well as all the developed
oil-loaded and Cur/oil-loaded CS, AL and CSAL microbeads. As apparent
from Fig. 3(a), CS, a polysaccharide, is partially crystalline due to its rel-
ative regular chain and it demonstrated two reflections appeared at 2θ
= 10.87° and 20.03°. From the Fig. 3, the XRD patterns of oil-loaded
microbeads revealed that the microbeads with different oils were very
similar. This suggests that the XRD results were less influenced by the
type and composition of the oil. In the case of CS microbeads Fig. 3(a),
the peak at 20° became broader upon oil encapsulation, which indicated
that the crystallization extent of CS was reduced during microbeads for-
mation. AL (Fig. 3b) exhibited two typical crystalline diffraction peaks at
13.46° and 21.25°. These two crystalline diffraction peaks were shifted
to 8.27° and 19.34° in the XRD pattern of AL microbeads (Fig. 3b).
From Fig. 3, it can be depicted that either CS and/or AL microbeads,
sharp new peaks were noted at around 2θ = 32° and 44°, which may
be attributed to the presence of oil.
It was observed from Fig. 3 that the addition of Cur hadn't changed
the XRD diffraction pattern of CS, AL or their combination. This confirms
the encapsulation of Cur in the crosslinked network of AL and CS. It was
showed that the crystalline peaks of Cur were not noted in Cur-loaded
AL aldehyde–gelatin nanogels, and the pattern was very similar to that
of Cur-free nanogel. This suggested that the addition of Cur had not
change the nature of plain nanogel [51]. This reported observation is
in agreement with what has been noted in the present study.

3.5. Morphological studies

The size and morphology of plain oil nanoemulsions and Cur-loaded

oil nanoemulsion were investigated with the aid of TEM microcopy as
shown in Fig. 4. Both nanoemulsions (plain and Cur-loaded) have
attained spherical shapes of their droplets with average size of
12–36 nm.
The morphology of the developed microbeads was also observed by
optical microscopy as in Fig. 5. The shape of the microbeads was found
to be spherical with shiny appearance due to their oily surface. Some
of the microbeads demonstrated formation of tails which may be attrib-
uted to the forces that the droplets are exposed to at the oil–water inter-
face [52]. The mean diameter of the developed microbeads of different
formulations was determined under a stereo microscope, and the re-
sults are shown in Table 3. As apparent from Table 3, no significant dif-
ference was found between the mean diameters of all the developed
omeg-3 rich oils-loaded microbeads. All the microbeads containing
nanoemulsion (fish or flaxseed) as a core material have depicted a
spherical shape. As can be noted from the table, all plain
nanoemulsions-loaded microbeads have demonstrated off-white
color, however, addition of Cur to AL showed a yellowish color of the
resulting microbeads. In the case of CS microbeads incorporating Cur-
loaded nanoemulsions led to a reddish color due to the acidic medium
Fig. 3. X-ray diffraction analysis of CS, AL, as well as the developed oil-encapsulated plain
of CS solution during the preparation. and Cur-loaded CS, AL and CSAL microbeads.
Fig. 5 demonstrates the optical, stereo and SEM micrographs of the
developed plain and Cur-loaded microbeads showing their microstruc-
ture. From the SEM micrographs, it can be noted that all the formed have a crystal form with different sizes (Fig. 5b,d,f). This is in agreement
microbeads have spherical morphology. Also, the microbeads based on with the results obtained by Kolanowski et al. [54] who observed spher-
AL, CS and their combination had predominantly shriveled surfaces ical structure of the microencapsulates with different sizes obtained via
(Fig. 5a,c,e). According to Re´ et al. [53], such imperfections can be re- spray drying of fish oil. In another study, it was reported that different
sulted due to the droplet collapse during the initial stages of drying, degrees of formation of surface indentations for fish oil-containing mi-
when there is a slow process of film formation. On the other hand, the crocapsules produced from protein and dextrin wall materials [55].
Cur-loaded microbeads had a smooth surface, which indicates better AFM was used to obtain information about the topographical fea-
surface properties. Such smooth surface can be a result of faster drying, tures of the developed microbeads, such as morphology and roughness.
which also reveals the better encapsulation efficiency and the lower Fig. 6 depicts the AFM micrographs of the developed fish oil-CSAL
lipid oxidation observed in these microbeads. Cur-loaded microbeads microbeads and the corresponding Cur-loaded fish oil-CSAL
 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696 689

(a) (b)

(c) (d)

Fig. 4. TEM micrographs of (a) flaxseed nanoemulsion, (b) fish nanoemulsion, (c) Cur-loaded flaxseed nanoemulsion, and (d) Cur-loaded fish nanoemulsion.

Fig. 5. Optical, stereo and SEM micrographs of the developed plain and Cur-loaded microbeads showing their microstructure; (a) fish oil-CS microbeads (b) Cur-loaded fish oil-CS
microbeads, (c) fish oil-AL microbeads, (d) Cur-loaded fish oil-AL microbeads, (e) fish oil-CSAL microbeads, and (f) Cur-loaded fish oil-CSAL microbeads.
690 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696

Table 3
Mean diameters of the developed microbeads as determined with the aid of stereo microscope (n = 6).

microbeads. The fish oil-CSAL microbeads have sharp, irregular and
spiky projections on the surface, indicating a coarse or rough texture.
In the case of the Cur-loaded fish oil-CSAL microbeads, they demon-
strated smooth-edged projections and grooves (Fig. 6b). The AFM im-
ages also indicated that the Cur was successfully deposited on the
microbeads during the development process.
3.6. Encapsulation efficiency and encapsulation load
Encapsulation efficiency (EE) and encapsulation load (EL) were cal-
culated from the surface oil determined for each type of the developed
microbeads. As apparent from Fig. 7, EE varied from 83 to 99% and
was considerably influenced by the type of wall material. The combined
CSAL microbeads showed the highest EE and EL. This may be attributed
to the strong ionotropic gelation resulting through ionic interactions be-
tween the positively charged amino groups of chitosan and the carbox-
ylic residues of the alginate results in the formation of a membrane
surrounding the surface of the AL-Ca microbeads, and consequently re-
ducing their porosity. This is in agreement with the results published by
[38,56]. It has been also reported by Shu & Zhu [57] who demonstrated
that more CS coated on the surface of AL capsules reduced their porosity.
Moreover, it was stated that a successful microencapsulation system is

Fig. 6. AFM micrographs of (a) fish oil-CSAL microbeads and (b) Cur-loaded fish oil-CSAL microbeads.
 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696 691

Fig. 7. The encapsulation efficiency (EE) and the encapsulation loading (EL) of the developed microbeads with different wall materials. Vertical bars represent standard deviation from the
mean values.

evaluated on the basis of its EE values along with the storage stability of
its omega-3 oil microcapsules [17].
As apparent from Fig. 7, AL microbeads displayed the lowest EE and
EL which may be due to high-porosity and loose network. Besides, there
is non-significant difference (P ≤ 0.05) found in EE and EL between flax-
seed and fish oils encapsulated with different wall materials. It was also
noted that addition of Cur increased both EE and EL of AL and CS-based
microbeads.
3.7. Oxidative stability
3.7.1. Antioxidant activity
Fig. 8 shows the absorbance decay of DPPH solution after reaction
with the oils microbeads extracts and the calculated radical scavenging
percentage over time. As shown in Fig. 8 there are variable absorbency
responses with each extract. Fig. 8(a, b) clarified that fish Al and fish
Al Cur had the greatest decrease in absorbance among all fish samples
(hence, highest (RSA %) over time. However, fish CS and fish CS Cur
had the highest absorbencies and smallest RSA %. After 15,000 s there
was a scavenging activity of about 70% for either AL or fish AL Cur ex-
tracts. Regarding fish oil samples the decreasing order of RSA % was
fish-AL-Cur N fish-AL N fish-CSAL-Cur N fish-CSAL N fish-CS-Cur ≥ fish-
CS. This decreasing order proved greater antioxidant properties of sam-
ples containing curcumin compared to those without curcumin. It was
reported that Cur had strong inhibitory effect against fish oil oxidation
[58].
The DPPH absorbance decay and the RSA % of flaxseed microbeads
extracts are presented in Fig. 8(c, d). After 15,000 s, RSA % there was a
scavenging activity of about 20% for either flax Al and flax Al Cur
microbeads extracts. The decreasing order of RSA % was flaxseed AL-
Cur N flaxseed-AL N flaxseed-CS-Cur ≥ flaxseed-CSAL-Cur N flaxseed-CS
N flaxseed-CSAL. Samples containing CS or CS-Cur are shown to have
the lowest antioxidant properties; this may be due to the formation of
a polyphenol-CS complex that retards the release of Cur effectively.

Fig. 8. Antioxidant activity of (a,b) for fish microbeads and (c,d) for flaxseed microbeads.
692 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696
Pablo and coworkers [59] agreed with our results and attributed the
lower polyphenols release to the stronger interactions in the
polyphenols-CS complex of the coated beads. The pronounced higher
RSA % of fish oil microbeads than that of flaxseed oil may be attributed
to the already added amount of α-tocopherol (2.3 mg/1000 g oil) in
fish oil capsules by the manufacturer (see Section 2.1).
3.7.2. Peroxide value
The oxidative stability of the omega-3 rich oils (flaxseed and fish)
entrapped in the microbeads was evaluated through following up the
formation of primary oxidation products (peroxide value) during stor-
age at 4 °C for 30 days. The peroxide value increased as lipid oxidation
proceeded and significantly (P b 0.05) rose in all microbeads with in-
creasing storage time. PV variations in the microbeads of different wall
materials are shown in Fig. 9. PV of flaxseed and fish oils before micro-
encapsulation was found to be 1.46 and 3.86 meq O2/kg oil, respectively.
On the other hand, the PV of microencapsulated oils immediately after
drying (Day 0) ranged from 1.51 to 2.65 meq O2/kg oil and 3.87 to
4.25 meq O2/kg oil for flaxseed and fish oil, respectively. This increase
in PV value for omega-3 rich oils may be first as a result of the somewhat
high temperature that the oils exposed to during ultrasonication and
second to the contact with oxygen during the drying process at room
temperature.
In agreement with our results Heinzelmann and Franke [60] stated
that a consequence of the homogenization process is that the tempera-
ture of emulsion increases which may lead to an increased oxidation of
polyunsaturated fatty acids (PUFA). Also, it was revealed that contact
between fish oil and oxygen during the emulsion production process
may lead to acceleration of oxidation changes in emulsion before the
drying process. They added that it is impossible to prevent this contact
completely [61].
The AL-based microbeads showed the least oxidative stability
highest peroxide concentration 18.13 and 16.90 meq.O2/kg oil for flax-
seed and fish oils, respectively after 30 days. As presented in Fig. 9 oxi-
dative stability was strongly influenced by the wall material
combination. It was noted that CSAL microbeads gave the highest pro-
tection against oil oxidation in comparison with the CS and AL
microbeads. This may be due to the polyelectrolyte complexation
20
18
16
14
12
0 Day
10
10 Day
20 Day
30 Day 8
0
Flaxseed CS Flaxseed CS Flaxseed AL Flaxseed AL
Cur Cur
Fig. 9. Oxidative stability of the oils entrapped in microbeads as evaluated by peroxide value (PV) method. Vertical bars represent the stander deviation of the mean values.
between cationic CS and anionic AL. It was also found that crosslinking
of CS and AL in hydrogel enhanced the stability compared to those ob-
tained with a single polymer [25].
Current results are in a good agreement with encapsulation effi-
ciency, where individual CS and AL microbeads showed lower encapsu-
lation efficiency than their combination. The lower encapsulation
efficiency suggests a higher amount of oil present on the microbeads
surface. This non-encapsulated oils, when in contact with oxygen, are
much more susceptible to lipid oxidation than the encapsulated oils. Be-
sides, it was observed that addition of Cur to omega-3 rich oils (flaxseed
or fish) has improved the oxidative stability of oils. Huang et al. [58] in-
dicated in their study that Cur has a strong inhibitory effect against fish
oil oxidation and under experimental condition of 4 °C, Cur depicted
similar antioxidant activity to α-tocopherol, and they was concluded
that Cur has antioxidant properties in edible oil. Antioxidants have
been used to increase the shelf-life of microencapsulated linseed oil
[14]. Cur can also be applied in both corn oil and oil in water (O/W)
emulsion systems to inhibit lipid oxidation [15]. Application of antioxi-
dants is one of the technically simplest ways of reducing fat oxidation
[9]. In another study, it was demonstrated that the oxidation stability
of the flaxseed oil droplets could be improved by adding caseinate mol-
ecules as a natural antioxidant [31].
3.8. Thermogravimetric analysis
The thermogravimetric analysis of the CS, AL, Cur and the prepared
microbeads in a nitrogen atmosphere from ambient temperature to
1000 °C are shown in Table 4. As apparent from the Fig. 10, two peaks
were recorded in the thermogram of pure CS. The first event occurred
at around 43 °C to 154 °C associated to water evaporation with 6.16%
of weight loss. Prolonging evaporation above 100 °C can be due to the
inner hydrogen bonding in CS chains. The main CS thermal degradation
was observed at around 254 °C to 438 °C with 62.33% weight loss. AL ex-
hibited two-step decomposition, the salt decomposed by dehydration at
25–199.21 °C with 10.39% weight loss, followed by decomposition at
199.21–591.34 °C with 47.82% weight loss to form Na2CO3 and the
final step involved Na2CO3 residue at 600–750 °C. The TGA thermogram
Flaxseed CSAL Flaxseed CSAL Fish CS Fish CS Cur Fish AL Fish AL Cur Fish CSAL Fish CSAL Cur
Cur
 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696 693

Table 4
Thermal decomposition data of the prepared microbeads under N2 atmosphere.

Microbeads Flax With Cur Microbeads Fish With Cur

Weight loss (%) Temp. °C Weight loss (%) Temp. °C Weight loss (%) Temp. °C Weight loss (%) Temp °C

CS 8.06 199.508 8.26 199.586 CS 1.28 84.826 9.20 154.222

39.14 384.508 36.48 379.586 1.09 104.826 47.81 394.222
22.34 589.508 20.57 594.586 52.57 384.826 8.50 539.222
8.33 894.508 5.24 784.586 11.37 624.826 1.60 624.222
4.71 989.508 12.39 989.586 21.18 989.826 11.95 994.222
17.43 999.4 17.06 999.4 12.51 999.4 20.83 999.4
AL 9.08 179.49 7.24 169.513 AL 39.32 279.572 40.35 284.605
17.56 314.49 8.43 244.513 13.87 344.572 10.74 339.605
14.14 374.49 11.50 319.513 11.64 419.572 14.17 424.605
14.99 429.49 12.40 374.513 7.85 584.572 7.36 604.605
20.39 534.49 15.35 429.513 4.57 989.572 4.71 974.605
3.47 804.49 19.80 594.513 22.73 999.4 22.65 999.4
1.83 939.49 2.35 744.513
1.06 969.49 1.94 989.513
17.45 999.49 20.99 999.4
CSAL 15.89 244.561 7.30 184.533 CSAL 5.45 159.84 38.75 274.605
11.63 319.561 8.65 244.533 25.97 299.84 12.19 334.605
12.62 374.561 11.36 319.533 18.48 364.82 12.23 424.605
15.06 429.561 10.48 369.533 19.36 534.79 7.54 604.605
18.62 584.561 16.91 429.533 8.91 974.78 4.12 989.605
2.46 794.561 18.74 589.533 21.65 999.4 25.15 999.4
2.19 989.561 4.05 989.533
21.52 999.4 22.51 999.4

of Cur showed that the main decomposition starts at around 266 °C and
continued up to 440 °C.
In the case of flaxseed oil, TGA thermograms of CS or AL and their
combination microbeads showed mainly three major weight losses.
The first one was noted before 200 °C with weight loss of
8.09–15.89%, which is due to the elimination of water molecules in
microbeads. Prolonging water evaporation above 100 °C was related
to formation of network which led to preventing of water molecules
movements. The second peak appeared around 200–390 °C with weight
loss 12.62–39.14% due to the dehydration of saccharide rings of

Fig. 10. TGA curves of (a) flaxseed CS/Cur, fish CS/Cur, (b) flaxseed AL/Cur, fish AL/Cur, (c) flaxseed CSAL/Cur and fish CSAL/Cur.
694 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696

polymers and the oxidation of unsaturated fatty acids [62]. The third
one was noted at around 390–590 °C with weight loss 14.99–22.34%,
and it is attributed to depolymerisation and main processes of decom-
position (combustion) which gives rise to a complete volatilization
[63]. Maximum weight loss occurred at the temperature range of
217–600 °C. Similar results were observed for fish oil microbeads. The
increased thermal stability indicates the formation of stronger and
harder CSAL microbeads networks as a result of crosslinking. Smitha
et al. [64], while studying AL-CS complex as interchange membranes,
found similar results with an endothermic peak around 200 °C,
confirming the formation of amide linkages in an ionic complex struc-
ture (-COO– + NH+ 3 ) with concluding that the incorporation of CS into
the AL–Ca2+ matrix does not affect considerably the overall thermal
stability.
Upon comparing the TGA curves of AL, CS and their combined
microbeads after incorporation of Cur addition, some differences were
noted. For instance, the TGA results suggest that the Cur addition im-
proved the overall thermal stability of microbeads, which is in agree-
ment with literature. For instance, Rezaei and Nasirpourb [65]
depicted that the thermal stability of almond gum/polyvinyl alcohol
nanofibers was slightly increased after incorporation of Cur and Cur-
β-cyclodextrin complex into it. Besides, Parize et al. [66] reported a
high degradation temperature of 321 °C for Cur as a result of its interac-
tions with the used polymer (CS) that contribute to the thermal
stability.
Comparing fish and flaxseed oils showed that the fish oil-AL
microbeads confer more antibacterial activity than flaxseed oil-AL
microbeads against gram positive and gram negative bacteria. Flaxseed
oil-CS microbeads, however, have showed more antibacterial activity
than fish oil-CS microbeads. In the combined microbeads, both fish-
CSAL and flaxseed-CSAL had a little difference in the inhibition zone
for gram negative bacteria, but in the case of gram positive bacteria,
the fish oil-CSAL microbeads revealed a better activity than the flaxseed
oil-CSAL.
Generally, incorporation of Cur into fish oil-CS, fish oil-AL and their
combination microbeads has improved the overall activity against
gram negative bacteria but reduced the activity towards gram positive
strains. On the other hand, addition of Cur in the case of flaxseed oil-
based microbeads led to a reduction in the activity against both gram
negative and gram positive bacteria. The most sensitive bacteria in
gram negative strains are Y. enterocolitica while in gram positive Listeria
monocytogenes is the most sensitive one. The present study clearly indi-
cated that the prepared microbeads have excellent antibacterial activity
against gram negative and gram positive bacteria. These properties of
the microbeads have potential uses to improve the shelf-life of food
products. The study also revealed that integration of these FAs into
food preservatives may suppress bacterial growth, and making them
potential agents for improving food safety [67].
4. Conclusion

Oil (fish and flaxseed) nanoemulsions with and without Cur-loaded

3.9. Antibacterial activity
Antibacterial activities of omega-3 rich oils (flaxseed and fish), Cur,
CS, AL, and their combination were assessed through an agar well diffu-
sion method. The zone of inhibition values of the samples against the
growth of selected bacteria are given in Table 5. The potential of samples
was evaluated against gram-negative bacteria (Escherichia coli, salmo-
nella typhmerium, Yersinia enterocolitica, and Pseudomonas aeruginosa),
and gram-positive bacteria (Staphylococcus aureus, Bacillus cereus, and
Listeria monocytogenes). It was revealed that fish oil has more antibacte-
rial activity than flaxseed oil. Fish oil-AL microbeads, and Cur-loaded
fish oil-AL microbeads showed the best performance against gram pos-
itive and gram negative bacteria. Fish oil-AL microbeads had more anti-
bacterial activity than that based on CS and CSAL combination.
However, flaxseed oil CS-microbeads depicted higher antibacterial ac-
tivity than AL-based microbeads and that based on CSAL combination.
microbeads were prepared and evaluated. DLS data of the plain and Cur-
loaded nanoemulsions revealed that they had small mean droplet diam-
eters (ranged from 62.3 to 111.29 nm), monodal particle size distribu-
tions (from 0.181 to 0.023) and zeta potentials ranged between
−20.92 and −14.31 mV. This DLS data favors stability of the developed
nanoemulsions. Characterization of the developed microbeads using
FTIR and XRD confirmed the presence of loaded oils and Cur within
the microbeads. Also, FTIR and XRD demonstrated that both Cur and
loaded oils were just physically entrapped in the polymeric microbeads
and there was no chemical interaction. The morphology of the plain and
Cur-loaded oil nanoemulsions as determined by TEM demonstrated
spherical shapes of the droplets. This spherical morphology was also
confirmed by SEM, optical microscopy and stereo-microscopy. Also,
the microbeads based on AL, CS and their combination had predomi-
nantly shriveled surfaces. The topographical features taken by AFM
showed that fish oil-CSAL microbeads have sharp, irregular and spiky

Table 5
Antibacterial activities (diameters of inhibition zones) of crude oil, CS, AL, Cur and the prepared microbeads.

Treatments Inhibition zone (mm)

Y. enterocolitica S. typhimurium P. aeruginosa E. coli L. monocytogenes S. aureus B. cereus

90 180 90 180 90 180 90 180 90 180 90 180 90 180

Fish oil 7.3 gh 13.7 ef 12.3 de 13.0 cd 7.2 ij 11.8 f 0.00 i 14.2 cd 0.00 e 12.3 fg 0.00 e 8.5 e 8.2 gh 9.8 ef
Flaxseed oil 6.2 h 11.8 fg 7.2 f 8.2 f 10.5 fgh 12.3 f 5.7 h 6.5 f 9.5 d 11.5 g 0.00 e 0.00 g 9.2 fgh 10.7 de
CS 6.3 h 11.3 g 7.8 f 8.8 ef 0.00 k 5.8 h 9.3 d 12.0 de 0.00 e 0.00 h 0.00 e 0.00 g 7.0 hi 11.3 de
AL 9.7 ef 13.7 ef 10.7 e 14.3 bcd 5.8 j 9.2 g 7.7 efg 13.3 cd 8.5 d 12.8 ef 0.00 e 9.0 e 12.3 cde 14.2 bc
Cur 11.7 cd 12.8 efg 13.5 bcd 15.2 b 12.5 de 16.2 c 13.5 c 13.7 cd 12.5 c 13.8 de 14.0 b 15.3 bc 11.2 def 13.5 c
Fish CS 12.2 cd 13.3 ef 8.5 f 13.0 cd 12.7 de 15.0 d 0.00 i 13.8 cd 16.5 a 17.3 c 0.00 e 0.00 g 13.2 abcd 14.7 abc
Fish Cur CS 11.8 cd 18.5 cd 13.0 cd 14.7 bc 10.5 fgh 12.2 f 0.00 i 10.5 e 0.00 e 0.00 h 0.00 e 0.00 g 0.00 j 7.3 g
Fish AL 15.5 a 20.7 b 14.5 abc 15.2 b 15.5 bc 16.3 c 15.2 b 16.8 ab 16.5 a 21.0 b 14.7 ab 17.7 a 14.7 ab 16.2 a
Fish Cur AL 14.8 ab 19.8 bc 15.7 a 16.0 ab 18.7 a 20.0 a 17.7 a 18.7 a 14.8 b 18.0 c 15.2 a 15.8 b 15.0 a 15.8 a
Fish CS AL 12.3 cd 13.0 efg 12.8 cd 15.7 ab 12.0 ef 16.8 c 7.5 fg 14.2 cd 0.00 e 18.3 c 10.3 c 14.3 bc 9.8 fg 11.5 d
Fish Cur CS AL 15.7 a 18.2 cd 15.5 ab 15.8 ab 8.8 hi 19.7 a 9.2 de 12.2 de 8.7 d 12.5 fg 0.00 e 0.00 g 5.7 i 10.8 de
Flaxseed CS 0.00 i 23.0 a 0.00 g 10.2 e 11.0 efg 18.5 b 5.5 h 10.7 e 16.5 a 22.7 a 0.00 e 6.5 f 10.2 efg 15.7 ab
Flaxseed Cur CS 12.5 cd 13.3 ef 7.7 f 12.7 d 9.3 gh 13.8 e 9.8 d 13.5 cd 9.5 d 14.3 d 0.00 e 0.00 g 12.5 bcd 13.8 c
Flaxseed Al 12.8 cd 17.8 d 13.8 abcd 14.5 bc 15.7 b 16.3 c 9.0 def 18.0 a 13.0 c 17.5 c 7.3 d 10.7 d 14.2 abc 15.0 abc
Flaxseed Cur AL 11.2 de 12.8 efg 13.0 cd 15.5 b 13.8 cd 14.3 de 6.7 gh 15.2 bc 9.0 d 14.2 d 11.2 c 14.2 c 14.0 abc 14.7 abc
Flaxseed CSAL 13.2 bc 13.8 e 0.00 g 17.3 a 11.0 ef 17.0 c 7.5 fg 12.2 de 13.0 c 13.0 ef 0.00 e 0.00 g 8.2 gh 8.7 fg
Flaxseed Cur CS AL 8.8 fg 12.7 efg 13.8 abcd 15.3 b 12.5 de 14.7 de 0.00 i 10.8 e 0.00 e 12.2 fg 0.00 e 0.00 g 8.2 gh 10.8 de
 A.F. Hashim et al. / International Journal of Biological Macromolecules 140 (2019) 682–696
projections on the surface, indicating a coarse or rough texture.
Whereas, the corresponding Cur-loaded fish oil-CSAL microbeads dem-
onstrated smooth-edged projections and grooves. It was also noted that
addition of Cur improved both encapsulation efficiency (EE) and encap-
sulation loading (EL) of the core oils. The antioxidant activity (measured
by DPPH assay) and the overall thermal stability of microbeads (mea-
sured by TGA) were generally enhanced more in Cur-loaded
microbeads. The antibacterial activity of the investigated microbeads
showed that incorporation of Cur into fish oil-CS, fish oil-AL and their
combination microbeads has improved the overall activity against
gram negative bacteria but reduced the activity towards gram positive
strains. On the other hand, addition of Cur in the case of flaxseed oil-
based microbeads led to a reduction in the activity against both gram
negative and gram positive bacteria. So, we can finally conclude that ul-
trasound emulsification and electrostatic crosslinking between CS and
AL is a suitable formulation procedure for the encapsulation of omega-
3 rich oils.
Acknowledgment
This research was financed and supported by National Research Cen-
tre (NRC), Egypt. We also, gratefully thank our Fats and Oils Depart-
ment' colleagues as well as the Academy of Scientific Research and
Technology (ASRT) (Egypt) and faculty of pharmacy, Wroclaw Univer-
sity (Poland) for providing insight and expertise that greatly assisted
this research. Authors would like to thank Dr. I. Khalil for his help with
drawing the graphical abstract.
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