International Journal of Biological Macromolecules 256 (2024) 128064
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules
journal homepage: www.elsevier.com/locate/ijbiomac
Microencapsulation of green coffee oil by complex coacervation of soy
protein isolate, sodium casinate and polysaccharides: Physicochemical
properties, structural characterisation, and oxidation stability
Jingyi Mu a, b, Rongsuo Hu a, c, 1, Yumei Tang a, c, 1, Wenjiang Dong a, c, d, *, Zhenzhen Zhang b, **
a
Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning, Hainan 571533, China
College of Food Science and Pharmacy, Xinjiang Agricultural University, Urumqi, Xinjiang 830052, China
Key Laboratory of Processing Suitability and Quality Control of the Special Tropical Crops of Hainan Province, Wanning, Hainan 571533, China
d
National Center of Important Tropical Crops Engineering and Technology Research, Wanning, Hainan 571533, China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Green coffee oil
Encapsulation
Soy protein isolate
Sodium carboxymethylcellulose
Oxidation kinetics analysis
This study developed a combination method between protein-polysaccharide complex coacervation and freezing
drying for the preparation of green coffee oil (GCO) encapsulated powders. Different combinations of soy protein
isolate, sodium caseinate, sodium carboxymethylcellulose, and sodium alginate were utilised as wall materials.
The occurrence of complexation between the biopolymers were compared to the final emulsion of the individual
protein and confirmed by fourier transform infrared spectrometry and X-ray diffraction. The mean diameter and
estimated PDI of GCO microcapsules were 72.57–295.00 μm and 1.47–2.02, respectively. Furthermore, the
encapsulation efficiency of GCO microcapsules was between 61.47 and 90.01 %. Finally, oxidation kinetics
models of GCO and its microcapsules demonstrated that the zero-order model of GCO microcapsules was found to
have a higher fit, which could better reflect the quality changes of GCO microcapsules during storage. Different
combinations of proteins and polysaccharides exhibited effective oxidative stability against single proteins
because of polysaccharide addition. This research revealed that soy protein isolate, sodium caseinate combined
with polysaccharides can be used as a promising microencapsulating agent for microencapsulation of GCO,
especially with sodium carboxymethylcellulose and sodium alginate, and provided useful information for the
potential use of GCO in the development of powder food.
1. Introduction
Lipids are located in the endosperm of coffee beans and are
composed mainly of triacylglycerols (75 %) and fatty acids (18 %), a
composition that is similar to that of edible vegetable oils [1]. The oil
extracted from green coffee beans is known as green coffee oil (GCO). It
is a natural source of valuable bioactive compounds, such as diterpene
esters, fatty acids, and unsaponifiable matter [2]. GCO has a potential to
create new goods, owing to its composition. GCO has antidiabetic,
antitumor, and antiangiogenic properties, and it inhibits the prolifera­
tion of cancer cells [3]. Despite its health benefits, GCO has a highly
unsaturated chemical structure, resulting in poor stability during food
processing and storage. This oil is highly sensitive to temperature, light,
and pH, which leads to decreased shelf life, as well as loss of sensory and
nutritional quality [2]. Therefore, protective stabilization technology is
urgently required to realize the feasibility of using GCO as a natural
functional oil in the food industry.
Microencapsulation is an extensive embedding technology typically
used for flavours, minerals, vitamins, oils, and bioactive substances in
the food industry. Some functional oils possess various unsaturated fatty
acids, such as fish oil, linseed oil, nut oil, and olive oil, which lead to
extremely poor oxidative stability. However, this can be mediated
through the application of microcapsule technology on these oils [4,5].
The use of microcapsule technology can substantially enhance their
oxidative stability and improve their characterisation. Additionally,
microcapsule technology can control the release rate of the core mate­
rial, cover its unpleasant smell, and reduce the toxic side effects.
Various methods, including emulsification, liposome formation, and
* Correspondence to: W. Dong, Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences, Wanning 571533, China.
** Corresponding author.
E-mail addresses: dongwenjiang.123@163.com, dongwenjiang.123@catas.cn (W. Dong), zhangzz2012@vip.sina.com (Z. Zhang).
1
This author contributed to the work equally and should be regarded as co-first author.
https://doi.org/10.1016/j.ijbiomac.2023.128064
Received 26 January 2023; Received in revised form 20 September 2023; Accepted 10 November 2023
Available online 14 November 2023
0141-8130/© 2023 Elsevier B.V. All rights reserved.
J. Mu et al.
International Journal of Biological Macromolecules 256 (2024) 128064
spray drying, have been used to prepare microcapsules. Researchers
have reported spray-dried emulsions using proteins as wall materials.
However, they found limited protection against biologically active
compounds. Proteins are very sensitive to high temperature conditions,
and flocculation increases at pH values near the isoelectric point. In
addition, the phospholipids used to make liposomes are susceptible to
acid hydrolysis and high shear forces, which may lead to rupture of
liposome membranes [6]. Owing to the high payload, the composite copreservation method of microencapsulation is more stable compared to
other techniques, such as emulsion formation, liposomes, and conven­
tional spray drying. Thus, complex co-preservation constitutes a unique
alternative to lipids due to high encapsulation efficiency and mild pro­
cessing conditions [6]. This associative phase separation phenomenon is
caused by changes in pH, temperature, ionic strength or solubility of the
dissolution medium [7].
Microencapsulation through complex coacervation has aroused
increasing attention in recent years owing to its practical applications in
the food, cosmetic and pharmaceutical industries [8]. Non-toxicity, low
cost and good encapsulating properties have made proteinpolysaccharide (Pr-Ps) complexes ideal wall materials for the encapsu­
lation of active ingredients using complex coacervation [7]. Several
polysaccharides, such as chitosan, gum arabic, sodium alginate (SA),
and sodium carboxymethylcellulose (CMC) and proteins such as whey
protein, gelatine, wheat protein, sodium caseinate (SC), and soy protein
isolate (SPI) are widely used in complex coacervation [4]. The combi­
nation of different wall materials might form a more effective and stable
carrier. Chen & Zhang [9] selected a combination of SPI and SA as wall
materials to prepare an oleogel with high oil-holding capacity and good
antibacterial properties. Wijaya et al. [10] observed physically stable
oleogels with high gel strength and high oil binding capacity using SC
and alginate (ALG) as structural framework. This combination played an
important role in thickening the interface. Thus, the combination of SA
with SPI and SC may have strong stability properties for effective pro­
tection and delivery of oils. Zhu et al. [11] revealed enhanced foaming
and foam stability of sodium caseinate (SC) with the addition of CMC.
Similarly, Wei et al. [12] found that CMC enhanced the stability of pea
protein dispersion at an acidified pH. Therefore, CMC was selected as
one of the model polysaccharides to combine with proteins and subse­
quently construct a stable delivery system.
There are a few studies on GCO microencapsulation. Albertina De
Oliveira et al. [13] used cashew gum and gelatine as complex co­
acervations to encapsulate GCO. They concluded that microparticles
containing 25 % GCO had an encapsulation rate of 85.57 %, which
resulted in a 6-fold lower GCO oxidation. Carvalho, Silva & Hubinger
[14] utilised electrostatic layer-by-layer deposition to prepare GCO
microcapsules by spray drying combined Hi-Cap100 and corn syrup/HiCap100. Subsequently, they prepared microparticles with the highest
oxidative stability. Furthermore, Da Silva Soares et al. [15] investigated
the effects of different combinations of modified starch or gum arabic
with maltodextrin on spray dried microencapsulated GCO. However,
spray drying has a high-temperature destructive effect on natural
functional oils compared to complex coacervation. Thus, its application
in oil delivery in the food industry is limited.
In this study, SPI and SC were combined with SA and CMC to form a
protective barrier, and a complex coacervation method was used to
prepare GCO microcapsules. (1) The physicochemical properties of GCO
microcapsules, such as encapsulation efficiency, particle size, bulk
density, and solubility, were evaluated. (2) The structural characteri­
sation of GCO microcapsules was analysed using scanning electron mi­
croscopy (SEM), confocal laser scanning microscopy (CLSM), Fourier
transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and
thermogravimetric analysis (TG). In addition, differential scanning
calorimetry (DSC) was used to evaluate the thermal properties of GCO
microcapsules. (3) The oxidative stability of the embedded GCO was
investigated through oxidation kinetics models and analysed to improve
the encapsulation effect of the microcapsules, which provided
conditions for the slow release of GCO during storage. This study could
provide a theoretical basis for the application of GCO microcapsules in
the food industry. Moreover, it provides a basis for improved techniques
in the preparation of foods enriched with protected bioactive
components.
2. Materials and methods
2.1. Chemicals and reagents
The GCO was provided by Jacques Yeti Aromatic Medicine Co., Ltd.
(Qingdao, China), the GCO was stored at − 80 ◦ C refrigerator and used
up within one month. Arabic gum, soybean protein isolate, Nile Red (for
fluorescence analysis, purity>95 %), and glutaraldehyde were pur­
chased from Aladdin (Shanghai Aladdin Biochemical Technology Co.,
Ltd., Shanghai, China). Whey protein isolate (American Hilmar WPI
9410, purity>89 %), sodium caseinate (purity>90 %), fluorescein iso­
thiocyanate (conjugated cascade, 90 % labelling rate), and rose-red B
isothiocyanate (conjugated cascade, 70 % labelling rate) were all pur­
chased from Shanghai Yuanye Biotechnology Co., LTD (Shanghai,
China). Milli-Q water was obtained from a Milli-Q Plus water system
(Millipore, Hetai, Shanghai, China). All other chemicals and reagents
were of analytical or chromatographic grade.
2.2. Preparation of protein and polysaccharide stock solutions
A protein stock solution (Pr, 1.0 %, w/v) was prepared by adding
protein (SPI and SC) powder into deionised water and agitating at 1200
rpm for 40 min at 25 ◦ C. Then, the pH of the protein solution was
adjusted to 7.0 using 0.01–1.0 M NaOH solution and stirred overnight to
promote dissolution. The polysaccharide stock solution (Ps, 1.0 %, w/v)
was prepared by dispersing polysaccharide (SA and CMC) powder into
deionised water, constantly stirring at 800 rpm for 24 h at 25 ◦ C, and
finally adjusting the pH to 7.0. These two solutions were centrifuged
(5000 xg, 10 min, 4 ◦ C) to remove insoluble particles and air bubbles.
They were subsequently stored at 4 ◦ C until use.
2.3. Determination of turbidity
In order to optimize the complex coacervation process, pH is regar­
ded as a crucial factor on account of its control on the degree of ioni­
zation of the functional groups [16]. In order to obtain the optimal pH
for the complex coacervation process between protein and poly­
saccharide, the turbidity were determined in the pH range of 2.0–7.0. PrPs mixtures were formulated at different protein-polysaccharide mixing
ratios (Table 1) at a total biopolymer concentration of 0.1 % (w/v). The
turbidity of these mixtures was measured as a function of pH (2.0–7.0)
using a microplate reader (Synergy H1, BioTek Instruments, Inc.
Winooski, VT, USA), and absorbance was recorded at 600 nm. The pH
value at which the highest light absorption occurred was regarded as the
optimum pH for complex coacervation.
Table 1
Formulations for determination of turbidity in composite wall materials (SPI/
CMC: soy protein isolate/sodium carboxymethylcellulose; SC/CMC: sodium
caseinate/sodium carboxymethylcellulose; SPI/SA: soy protein isolate/sodium
alginate; SC/SA: sodium caseinate/ sodium alginate).
Group
1
2
3
4
5
6
2
SPI/CMC
SC/CMC
SPI/SA
SC/SA
1:2
1:1
2:1
5:1
6:1
9:1
1:2
1:1
2:1
5:1
6:1
9:1
1:2
1:1
1.5:1
2:1
3.5:1
5:1
1:2
1:1
1.5:1
2:1
3.5:1
5:1
J. Mu et al.
International Journal of Biological Macromolecules 256 (2024) 128064
2.4. Preparation of Pr-Ps complex coacervate
2 min at 25 ◦ C. The mixture was filtered and washed 3 times using 20 mL
petroleum ether. After filtering and washing, the residue and filter paper
were dried at 60 ◦ C until they reached a constant weight. The surface oil
content was calculated as a weight percentage difference of the powder
pre- and post-extraction and washing. The total oil content (encapsu­
lated and surface oil) of microcapsules was determined according to the
method by Bakry, Huang, Zhai, & Huang [19] with minor modifications.
Encapsulation efficiency (EE) was calculated using Eq. (1):
The Pr-Ps complex coacervate was prepared according to Li, Wang &
Mei [17], with slight modification. The protein solution (1.0 %, w/v)
was added to the polysaccharide solution (1.0 %, w/v) at the optimum
mixing ratio (SPI/CMC of 5:1, SPI/SA of 3.5:1, SC/CMC of 5:1, SC/SA of
5:1). The mixed solution was transferred to a three-necked flask, incu­
bated at 50 ◦ C and placed in a thermostatically heated magnetic stirrer
(DF-101, Gongyi Yuhua Instrument Co., Henan, China). Then, an HCl
solution was used to slowly adjust the pH at a stirring rate of 400 r/min
for 30 min. To promote the formation of Pr-Ps complex coacervates, the
heating source was switched off. The mixture was naturally cooled to 25
± 1 ◦ C over 2 h. Next, it was cooled to 4 ◦ C in an ice water bath for 1 h.
The reaction system was then placed in an ice water bath for 30 min to
cool the solution to <15 ◦ C and end the complex coalescence reaction.
The pH of the system was adjusted to 6.0 using 1.0 % NaOH solution.
Then 5.0 mL glutaraldehyde (1.0 %) was added for 3 h curing. After
curing, a GCO microcapsule suspension was prepared, stratified, and
centrifuged, and the ratio of core to wall was 1:1. The supernatant was
separated, pre-chilled at − 80 ◦ C in an ultra-low temperature refrigerator
(RS552NRUAWW/SC, Suzhou Samsung Electronics Co., Zhejiang,
China) for 24 h and freeze-dried under vacuum (− 50 ◦ C, 20 Kbar for 48
h) (TFDX0.250, Shandong Yantai Zhongfu Cold Chain Equipment Co.,
Shandong, China). The resulting samples were placed in high-density
polyethylene bags and stored at 4 ◦ C for further use.
EE(%) =
PDI =
m0
Vb
(3)
ρT =
m0
Vt
(4)
2.6.4. Flowability and cohesiveness
The flowability and cohesiveness of the powder samples was
measured through Carr’s Index (CI) and the Hausner ratio (HR) [20] and
according to Eq. (5) and Eq. (6), respectively.
2.6. Physicochemical properties of GCO microcapsules
DI water (mL)
1:1
1:1
1:1
1:1
1:1
1:1
Add to
Add to
Add to
Add to
Add to
Add to
ρT
ρB
(5)
(6)
2.6.5. Solubility
The Solubility of the microparticles in water was determined ac­
cording to Francisco, et al. [21], with minor modifications. Samples (0.5
g) were placed in tubes with 50.0 mL deionised water and kept under
agitation in a shaker for 30 min. Next, this mixture was centrifuged at
3000 rpm for 10 min. An aliquot of the supernatant was transferred to
pre-weighed petri dishes and oven-dried at 105 ◦ C for 5 h. The solubility
(%) was calculated based on the weight difference.
Table 2
Formulations of green coffee oil microcapsules prepared through complex
coacervation (SPI-CGO: soy protein isolate-green coffee oil; SC-GCO: sodium
caseinate-green coffee oil; .SPICMC-GCO: soy protein isolate sodium
carboxymethylcellulose-green coffee oil; SCCMC-GCO: sodium caseinate sodium
carboxymethylcellulose-green coffee oil; SPISA-GCO: soy protein isolate sodium
alginate-green coffee oil; SCSA-GCO: sodium caseinate sodium alginate-green
coffee oil).
Wall/oil ratio
(ρT − ρB)
× 100
ρT
HR =
2.6.1. Determination of surface oil and encapsulation efficiency
The surface oil and encapsulation efficiency were determined as
previously described [18], with minor modifications. Surface oil content
was analysed using the gravimetric method. Petroleum ether (15.0 mL)
was added to 2.0 g powder in a test tube and processed in the vortex for
–
–
5:1
5:1
3.5:1
5:1
(2)
ρB =
CI =
Pr/Ps ratio
D[0, 9] − D[0, 1]
D[0, 5]
2.6.3. Bulk density and tapped density
The bulk density (ρB) and tapped density (ρT) were determined as
previously described [7], with minor modifications. Briefly, 1.0 g
microcapsule powder was weighed (m0) and gently loaded into a 10 mL
graduated glass cylinder. The volume (vb) of powder was recorded, and
the ρB was calculated according to Eq. (3). Then, the cylinder was tap­
ped on a flat surface until a constant volume (vt) was achieved. The ρT
was calculated according to Eq. (4):
As indicated in Table 2, the total biopolymer concentration of each
mixture was fixed at 1.0 % (w/v). According to the ratio of core material
(oil) and wall material (protein-polysaccharide mixture) is 1:1, a certain
amount of core material was slowly added to the prepared proteincoated solution., and the resultant mixtures were homogenised at
25 ◦ C (10,000 rpm, 5 min) using a digital display high-speed dispersion
homogeniser (FJ200-SH, Shanghai Specimen Model Factory, Shanghai,
China) to obtain a primary O/W emulsion. Then, the polysaccharide
coating materials were added and homogenised again (10,000 rpm, 5
min) to produce an oil-in-water emulsion. The pH of the emulsion was
adjusted to 3.0 (SPI/CMC of 5:1), 3.0 (SC/CMC of 5:1), 3.0 (SPI/SA of
2:1) and 2.5 (SC/SA of 5:1) using 0.1 M HCl under the stirring rate of
500 rpm at 45 ◦ C. After continuously stirring for 30 min, subsequent
preparation was conducted as described in Section 2.4.
Formulation
(1)
2.6.2. Analysis of particle size and polydispersity index (PDI)
The particle size distribution of microcapsules was measured
through laser diffraction using the Mastersizer 3000 laser particle size
analyser (Malvern Instruments, Malvern, UK). All samples were diluted
to a concentration of 1.0 % using deionised water to avoid multiscattering. Further, they were analysed at 25 ◦ C. The refractive indices
of the dispersant (deionised water) and the particle were 1.33 and 1.56,
respectively [18]. The PDI was calculated as shown in Eq. (2):
2.5. Production of GCO microcapsules through freeze-drying
SPI-GCO
SC-GCO
SPICMC-GCO
SCCMC-GCO
SPISA-GCO
SCSA-GCO
(Total oil/g⋅100 g − 1 − Surface oil/g⋅100 g − 1)
× 100
Total oil/g⋅100 g − 1
2.6.6. Moisture content
Moisture content analysis for GCO micropaticles was performed
through a moisture analyser that utilises infrared heating (model MB45,
OHAUS, Parsippany, New Jersey, USA). Each sample was determined in
triplicate, and the mean of three measurements was used for further
analysis.
100
100
100
100
100
100
3
J. Mu et al.
International Journal of Biological Macromolecules 256 (2024) 128064
2.7. FT-IR
screened (beam voltage acceleration = 10 kV).
The GCO, SPI, SC, CMC, SA, SPICMC, SCCMC, SPISA, SCSA, SPIGCO, SC-GCO, SPICMC-GCO, SCCMC-GCO, SPISA-GCO, and SCSAGCO powders were subjected to FT-IR analysis on a Nicolet 6700
Fourier transform infrared spectrometer (Thermo Fisher Corporation,
Waltham, Massachusetts, USA) according to Francisco et al. [21]. An
appropriate amount of KBr was separately mixed with dried microcap­
sules and ground to form disks at 10 tons pressure. The experimental
conditions were as follows: the range of the spectra was 400–4000 cm− 1
at a resolution of 4 cm− 1, the measurement temperature was 25 ◦ C, and
the total number of scans was 32.
2.11.3. CLSM
The encapsulation of oil droplets was observed as previously
described [17] using a confocal laser scanning microscope (Fluoview
FV10i, Olympus, Japan). The freshly prepared microcapsule suspensions
were stained using a blended fluorescent dye solution consisting of 1.0
mg/mL Nile red and 1.0 mg/mL fluorescein isothiocyanate (FITC).
Three microlitres of dyed microcapsule suspensions were then trans­
ferred onto a single concave glass slide and examined using a 63×
objective lens. The microscope was operated using different laser exci­
tation sources for Nile red (550 nm) and FITC (494 nm).
2.8. XRD
2.12. Oxidation kinetics models of GCO microcapsules
XRD patterns of wall materials and microcapsules were obtained
through the Ultima IV X-ray diffractometer (RIKEN Japan Ltd., Tsukuba,
Japan), where Cu and Kα radiation (40 kV and 40 mA) was used as an Xray source. For the measurements, samples were kept in glass sample
holders and subsequently scanned from 5o to 60o at a scan angular speed
of 2 θ/min at 5 o/min. X-ray diffraction patterns of samples were ana­
lysed using MDI Jade 6 software and calculated as relative crystallinity
(%) according to Eq. (7):
Zero-order and first-order kinetic models can describe the quality
changes of lipids in food during storage [23]. Therefore, the oxidative
stability of GCO microcapsules was studied when the oil had not visibly
leaked. Then, kinetic models were used to fit the oxidation stability of
GCO and GCO microcapsules numerically, as shown in Eq. (8) and Eq.
(9):
Relative crystallinity(%) =
Zero − order Kinect model equation C = kt + c0
Sum of total crystalline peak areas
× 100
Sum of total crystalline and amorphous peak areas
2.9. Thermogravimetric analysis (TGA)
First − order Kinect model equation lnC = lnc0 + kt
TGA was used as previously described [22] to determine the thermal
behaviour of microcapsule powders. This analysis was performed using
the TGA 4000 thermographic analyser (PerkinElmer, MA, USA). The
samples (5.0–10.0 mg) were sealed in a crucible and heated from 30 to
600 ◦ C at a heating rate of 20 ◦ C/min. Nitrogen gas was used as the
heating medium at a flow rate of 20 mL/min.
(8)
(7)
(9)
where c0 represents the initial peroxide value (POV, meq/kg), C repre­
sents the POV at t (meq/kg), k represents the reaction rate constant, and
t represents time (d).
2.13. Statistical analysis
All data were statistically analysed using variance and Duncan’s
multiple range tests. Additionally, triplicate analyses were carried out
on oil and microencapsule samples. Differences between means were
considered significant at 95 % (p < 0.05). Stat-Packets Statistical
Analysis software (SPSS Base 20.0, SPSS Inc., Chicago, Ill., USA) was
used for the analysis of variance.
2.10. DSC
The thermal behaviour of microcapsule powders was analysed using
a differential scanning calorimeter (DSC 25, TA Instruments, New Castle,
DE, USA) according to Da Silva Soares et al. [15] with slight modifica­
tion. The microcapsule powders (4.0 mg) were analysed over a tem­
perature range of 20 to 250 ◦ C at a rate of 20 ◦ C/min. An empty and
sealed crucible was used as a reference. The results were analysed using
DSC data analysis software (Mettler Toledo International, Zurich,
Switzerland).
3. Results and discussion
3.1. Effects of pH on turbidity during complex coacervation
Coacervates can be ascertained using turbidity during complex
coacervation [24]. The optimum conditions for the preparation of
SPICMC, SPISA, SCCMC, and SCSA were obtained through the turbidity
values of the four tested mixtures as a function of pH. When the ratio of
SPI to CMC was 5:1, the highest turbidity in this mixture was observed at
pH 3.0–3.5 (Fig. 1A). The increased turbidity was mainly a consequence
of the negatively charged CMC when the pH value was greater than its
isoelectric pint, which facilitated the complete coacervation between
SPI and CMC. The lower turbidity was observed in SPI-CMC mixtures at
1:2. The 1:1 and 2:1 ratio can be attributed to steric repulsion due to the
presence of excess CMC in the mixture. Similarly, higher ratios (6:1, 9:1,
w/w) for SPI to CMC also resulted in lower turbidity values. This result
can be attributed to the presence of and interaction among excess SPI
molecules. Based on these observations, the optimal ratio for SPI to CMC
was 5:1 at pH 3.0–3.5. To ensure the stability of GCO during
2.11. Microstructure observation of emulsion and microcapsules
2.11.1. Optical microscopy (OM)
The sample was placed at the centre of a clean glass slide and gently
covered with a coverslip. Then, an optical microscope (CX 21, Olympus,
Japan) equipped with a camera was applied to monitor the formation of
the microcapsules.
2.11.2. SEM
A scanning electron microscope (Phenom Prox, Founa Scientific In­
struments, Netherlands) was used to examine the microstructure of the
microcapsule powder. The microencapsulated powder was placed on a
double-adhesive tape-affixed specimen holder and vacuum coated with
a thin layer of gold. Finally, the sputtered coated specimens were
4
J. Mu et al.
International Journal of Biological Macromolecules 256 (2024) 128064
Fig. 1. Turbidity as a function of the pH of the system containing (A) SPI-CMC, (B) SPI-SA, (C) SC-CMC, and (D) SC-SA in different ratios and variations.
encapsulation and produce complex coacervates for further tests, the
optimal ratios for SPI to SA, SC to CMC, and SC to SA were 3.5:1, 5:1, and
5:1 at pH 3.0, 3.0, and 2.5, respectively (Fig. 1B-D).
the repulsive force in the system is strong, thus preventing the drastic
phase separation. Significant phase separation appeared at pH 3.5 and
3.0; precipitation occurred, and a clear solution was formed. It was
mainly a consequence of the negatively charged CMC when the pH value
was greater than its pl. This phase behaviour originated from the charge
neutralisation of SPI and CMC along with the pH corresponding to the
optimal electrical equivalence, which was consistent with the SCCMC
system at pH 3.0 and 3.5 [26].
3.2. Effects of pH on the phase behaviour of the oil dispersions
To better visualise the impact of pH on the appearance of GCO in two
different dispersions, a state diagram was constructed to represent the
phase behaviours of both SPI stabilised GCO emulsions and SPI-CMC
complex dispersions (Fig. 2A). The diagram was constructed as a func­
tion of pH (5.0–2.0) after maintaining the dispersion to reach equilib­
rium at 25 ◦ C for 24 h. One phase solution was observed at pH 5.0, 4.5,
and 2.0 away from the CMC of SPI. However, it transitioned to precip­
itation and a cloudy appearance as the pH decreased. Finally, it evolved
into condensed precipitates at pH 3.5–3.0 [25]. For SPI–CMC complex
dispersions, three distinguishable phases were presented in the state
diagram. Under weak acidic conditions (5.0, 4.5), one homogenous
phase was observed in the mixture, owing to repulsion from the same
charge of SPI and CMC (Fig. 2B). As the pH decreased to acidic condi­
tions (5.0–2.0), insoluble complexes (coacervates or precipitates) with
two distinct phase behaviours appeared. These complexes could account
for different structural properties. As the pH approached 4.0, co­
acervates formed precipitation and a cloudy appearance. This phase
behaviour originated from the attraction between the negative charge of
polyanionic CMC and the opposed patch on the surface of SPI. However,
3.3. Physicochemical properties of GCO microcapsules
3.3.1. Determination of EE
The EE and surface oil contents of the GCO microcapsules are listed
in Table 3. EE is a crucial quality parameter for the preparation of mi­
crocapsules, which determine the potential of wall material to encap­
sulate the core material in microcapsules. An effective packaging
method relies on achieving high retention of the core material inside the
wall material. Here, SPICMC-GCO, SPISA-GCO, SCCMC-GCO, and SCSAGCO treatments presented high EE with no significant difference (p <
0.05). Moreover, their EE was larger than that of the single-component
treatment group (SPI-GCO and SC-GCO, 61.47 % and 78.80 %, respec­
tively). The best treatments with relatively higher EE were SPISA-GCO
(89.12 %) and SCSA-GCO (90.01 %). Da Silva Soares et al. [15] used
sodium alginate and ovalbumin as wall material to microencapsulate
sacha inchi oil. Their obtained EEs were 86.89 % and 94.12 %,
5
J. Mu et al.
International Journal of Biological Macromolecules 256 (2024) 128064
Fig. 3. Particle size distribution of GCO microcapsules prepared with different
wall materials.
(SPISA-GCO) with increasing SA content. Furthermore, the D [4, 3] of
SPI treatment microcapsules was lower than that of SC treatment mi­
crocapsules (p < 0.05). Increased cross-linking between the free primary
amino groups in SC and anionic groups in GA is associated with more
uniform particle size distribution [28,29]. Therefore, we surmise that
particles composed of only SC exhibit large size and high PDI. In addi­
tion, GCO microcapsules are widely distributed in the range of
20.30–566.00 μm, the difference between D[4,3] and D[3,2] values is
great, the particle size of GCO microcapsules is non-uniform, and the
shape is irregular.
Fig. 2. (A) A phase diagram of SPI-CMC, SPI-SA, SC-CMC, and SC-SA complex
dispersions during acid titration. (B) The appearance of SPI-CMC = 5:1 complex
distribution as a function of pH (The appearance was observed after 24 h; (■)
represents turbid solution; ( ) represents precipitation and cloudy solution; ( )
represents precipitation and clear solution; SPI: soybean protein isolate; SC:
sodium caseinate; CMC: sodium carboxymethylcellulose; SA: sodium alginate).
respectively, similar to those found in this study. Lyu et al. [27] found
that encapsulation of a high oil concentration results in a low surface oil
concentration (11.99 %). This demonstrates that composite wall mate­
rials have a better GCO encapsulating effect than single components.
Here, SPISA and SCSA achieved the best GCO encapsulating effect.
Higher EE indicated a low GCO content on the surface of microcapsules,
which was not easily oxidised under the environment and guaranteed
the overall quality of microcapsule powder.
3.3.3. Bulk density, tapped density, flowability, and cohesiveness analysis
High bulk and tapped densities are desirable in transporting and
storing powder because its storage requires smaller containers. In
addition, high bulk density implies fewer powder cavities available for
air penetration, which helps in the prevention of lipid oxidation [30]. In
this study, the bulk densities of the powders were between 0.35 and
0.40 g/cm3, where formulation with SPISA had a significantly higher
value (0.40 g/cm3) (p < 0.05). In contrast, other formulations were
practically the same (Table 3). Bulk densities of microcapsules using SPI
and SC as wall material were similar to those of chia seed oil micro­
capsules produced by Bordón et al. [31]. Tapped densities of GCO mi­
crocapsules for all formulations ranged from 0.39 to 0.46 g/cm3. The
higher content of the solid could increase the bulk density of SPISAGCAO, and SPI combined with SA also contributed to the densest par­
ticles when compared to that of SPI-GCO and SC-GCO. CI and HR enable
the assessment of the flow behaviour of powder based on bulk and
tapped densities [32]. CI was utilised to evaluate flowability, whereas
3.3.2. Analysis of particle size and PDI
Particle size and PDI are crucial parameters for the characterisation
of microcapsule stability. As shown in Fig. 3, the particle size of mi­
crocapsules appeared to be a normal distribution. Uniform and
concentrated particle size distribution of microcapsules and a high span
value indicate a wide size distribution and high polydispersity. The
mean diameter and estimated PDI of GCO microcapsules were
72.57–295.00 μm and 1.47–2.02, respectively (Table 4). The particle
size of SPI-GCO microcapsules increased from 72.57 nm to 118.33 nm
Table 3
Mean values and standard deviations for the bulk densities, Carr’s index, Hausner ratio, solubility, moisture content, encapsulation efficiency and surface oil content of
GCO microcapsules prepared using different wall materials (SPI-CGO: soy protein isolate-green coffee oil; SC-GCO: sodium caseinate-green coffee oil; SPICMC-GCO:
soy protein isolate sodium carboxymethylcellulose-green coffee oil; SPISA-GCO: soy protein isolate sodium alginate-green coffee oil; SCCMC-GCO: sodium caseinate
sodium carboxymethylcellulose-green coffee oil; SCSA-GCO: sodium caseinate sodium alginate-green coffee oil).
Variables
SPI-GCO
SC-GCO
SPICMC-GCO
SPISA-GCO
SCCMC-GCO
SCSA-GCO
Surface oil (g/100 g)
EE (%)
Moisture content (%)
ρB (g/cm3)
ρT (g/cm3)
Carr’s index (%)
Hausner ratio
Solubility (%)
0.38 ± 0.08 a
61.47 ± 8.48c
4.20 ± 0.01a
0.35 ± 0.01b
0.46 ± 0.03ab
23.33 ± 3.75a
1.31 ± 0.07a
73.78 ± 0.99e
0.21 ± 0.04b
78.80 ± 4.00b
4.43 ± 0.02d
0.35 ± 0.03b
0.41 ± 0.03b
13.95 ± 2.56b
1.16 ± 0.03b
77.29 ± 0.99d
0.14 ± 0.06b
88.23 ± 0.48ab
4.94 ± 0.03b
0.34 ± 0.01b
0.39 ± 0.02b
13.65 ± 0.45b
1.16 ± 0.01b
81.40 ± 0.71c
0.16 ± 0.00b
89.12 ± 0.14a
5.01 ± 0.03a
0.40 ± 0.01a
0.48 ± 0.02a
18.02 ± 2.86ab
1.22 ± 0.44ab
83.77 ± 0.45ab
0.16 ± 0.02b
86.65 ± 1.53ab
4.96 ± 0.02ab
0.36 ± 0.02ab
0.41 ± 0.03b
14.33 ± 0.84b
1.17 ± 0.01b
85.27 ± 0.09a
11.99 ± 3.01b
90.01 ± 2.51a
4.63 ± 0.03c
0.37 ± 0.01ab
0.44 ± 0.02ab
17.03 ± 3.14ab
1.21 ± 0.05ab
82.13 ± 0.59bc
All values are mean ± standard deviation of three times. Means in the same column with different superscripts differ significantly: p < 0.05.
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International Journal of Biological Macromolecules 256 (2024) 128064
Table 4
Mean values and standard deviations for the particle size and PDI of GCO microcapsules prepared using different wall materials (SPI-CGO: soy protein isolate-green
coffee oil; SC-GCO: sodium caseinate-green coffee oil; SPICMC-GCO: soy protein isolate sodium carboxymethylcellulose-green coffee oil; SPISA-GCO: soy protein
isolate sodium alginate-green coffee oil; SCCMC-GCO: sodium caseinate sodium carboxymethylcellulose-green coffee oil; SCSA-GCO: sodium caseinate sodium
alginate-green coffee oil).
Formulation
D[4,3] (μm)
D[3,2] (μm)
PDI
SPI-GCO
SC-GCO
SPICMC-GCO
SPISA-GCO
SCCMC-GCO
SCSA-GCO
72.57 ± 13.43c
295.00 ± 33.47a
68.47 ± 3.20c
118.33 ± 8.58c
204.00 ± 27.76b
291.00 ± 20.99a
31.47 ± 4.22d
116.67 ± 4.50b
29.33 ± 0.71d
64.97 ± 1.02c
141.67 ± 31.12ab
168.67 ± 10.40a
1.82
1.67
2.07
1.77
1.47
1.95
± 0.13ab
± 0.06ab
± 0.34a
± 0.18ab
± 0.12b
± 0.04a
Dx (10) (μm)
Dx (50) (μm)
Dx (90) (μm)
20.30 ± 2.65c
56.83 ± 2.25b
21.00 ± 0.71c
40.33 ± 1.57b
85.57 ± 18.11a
104.37 ± 4.45a
51.13 ± 8.58c
284.33 ± 34.02a
47.83 ± 1.45c
88.03 ± 2.33c
184.33 ± 28.64b
248.00 ± 0.82a
119.03
539.00
115.03
196.00
354.00
566.00
±
±
±
±
±
±
25.18c
72.29a
19.37c
9.80c
37.21b
43.57a
All values are mean ± standard deviation of three times. Means in the same column with different superscripts differ significantly: p < 0.05.
HR was used to evaluate the cohesiveness of microcapsule powder.
Higher HR indicated that the powder was more cohesive and had a lower
flowability [32]. Here, CI means were in the range of 13.65–23.33 %.
SPICMC and SCCMC microcapsules had HR values of 1.16 and 1.17 and
CI values of 13.65 and 14.33, respectively. These results indicate that
the microcapsules had good flowability, according to the classification
scheme established by Dima et al. [33]. Thus, the flowability value
showed a significant increase as the CMC increased (p < 0.05), enabling
good fluidity in the microcapsules.
material are shown in Fig. 4A–F. GCO exhibited a strong vibrational
mode associated with C–H stretching at 2933 cm− 1, 2859 cm− 1, and
1747 cm− 1 (C–C stretching), and a strong fiat stretching at 3475 cm− 1
(O–H stretching). The fingerprint region below 1600 cm− 1 showed a
more intricate profile of identified absorption bands: 1461 cm− 1 (-CH2),
1241 cm− 1 (-CH3), 910 cm− 1 (-C-O-, ester), and 719 cm− 1 (fatty acids
with cis double bonds) [31]. The band at 1159 cm− 1 corresponded with
C-O-C stretching. This band was identified in bulk and micro­
encapsulated GCO but not in blank samples (Fig. 4). This characteristic
band was attenuated in the microcapsule, indicating that the oil was
located in the matrix [31]. Other bands identified in powders were
located at 3000–3500 cm− 1 (O–H stretching), 2933 cm− 1 (C–H
stretching), 1747 cm− 1 (ester, C–
–O stretching) and 1652–1681 cm− 1
–
(amide 1, C–O stretching). Furthermore, special features were observed
after coacervation, FT-IR can also be used to elucidate the interaction
between proteins and polysaccharides. Firstly, the bands identified at
1531 cm− 1 (amide II, N–H bending) and 1234 cm− 1 (amide III, C–N
stretching) in SPI, and at 1610 cm− 1 and 1608 cm− 1 (C–
–O stretching,
free carboxyl groups) in CMC and SA (Fig. 4A) were shifted towards a
higher wavenumber in the blank sample and microcapsules, which in­
dicates interactions between functional groups [39]. For the spectra of
the four agglutinates, such as SPICMC, SPISA, SCCMC, and SCSA
copolymer, the absorption band at 3291 cm− 1 and 3154 cm− 1 in SPI and
SC moved to 3552 cm− 1, 2559 cm− 1, 3556 cm− 1, and 3511 cm− 1,
respectively. It can be observed that a hydrogen bond was formed be­
tween O–H group in SPI and C–
–O group in CMC and SA. The peaks at
1660 cm− 1, 1531 cm− 1 and 1234 cm− 1 in amide 1, amide II and amide
III of SPI moved to 1677 cm− 1, 1540 cm− 1, and 1243 cm− 1 in SPICMC
copolymer. The results demonstrated that electrostatic interaction
existed between SPI and CMC. Similarly, the peaks at 1660 cm− 1, 1529
cm− 1, and 1232 cm− 1 corresponding to amide 1, amide II, and amide III
of SC shifted to 1677 cm− 1, 1538 cm− 1, and 1240 cm− 1 in SCCMC co­
polymers, which indicated that there was electrostatic interaction be­
tween SC and CMC. Secondly, all the spectra showed a broad band
around 3000–3600 cm− 1, demonstrating enhanced hydrogen bonding
compared with SPI and SC alone. This was also noted by Yang et al.
[28,29] on soy protein isolate-chitosan complex coacervation. The for­
mation of new bands was not observed for microcapsules, indicating
that no new chemical bonds between the core and wall materials were
formed, and mainly the predominance of physical interactions between
them, indicating that the complexation with GCO occurred on the sur­
face hydrophobic domain of the protein.
3.3.4. Solubility and moisture content
Solubility is an important instant property and a decisive factor for
determining the quality of microcapsules. Microcapsules may be sub­
jected to rehydration when used as a food ingredient. Therefore, poorly
soluble microcapsules may cause processing difficulties and economic
losses in the food industry [21]. High solubility leads to rapid adsorption
of the solvent, swelling, and rupture of the microparticles, thereby
favouring the release of compounds. The solubility of microcapsule was
found in the range of 73.78–85.27 % (Table 3). A maximum solubility of
85.27 % was observed for the microcapsule using SCCMC-GCO, which is
higher than that of SC alone (77.29 %). Similarly, lower solubility of SPIcontaining (73.78 %) microcapsules was observed when compared with
that of SPI-CMC/SA (81.40 %, 83.77 %). The higher solubility of CMC
and SA-containing microcapsules can be attributed to the high polarity
of the polysaccharide [34].
Appropriate stability of dried foods requires a moisture content of
3–10 g/100 g DW during storage [35]. Sugar has no affinity for the airwater interface. However, it can encourage protein-protein interactions,
enabling the microcapsule to maintain some moisture content. The
moisture content of GCO microcapsules ranged from 4.2 % to 5.0 %,
which meets the water requirement of microcapsules during storage,
because the water holding capacity of proteins was better than in an
amorphous state. However, the water content of SPI-GCO microcapsules
is lower than that of other GCO microcapsules. This may be related to
the high surface oil content of SPI-GCO microcapsules. Increased oil
droplets on the surface of the SPI-GCO microcapsules increase the hy­
drophobicity of the microcapsule surface and reduce the water vapor
permeability. As a result, more water is retained inside the microcap­
sules [36]. Lin et al. [37] demonstrated that moisture content in freezedried oil microcapsules is related to the presence of oil droplets. The oil
droplets act as a vapor transmission barrier to reduce water evaporation,
increase the hydrophobicity of the microcapsules, and limit the transfer
of water molecules [38].
3.4.2. XRD analysis of GCO microcapsules
XRD is a common method for evaluating the crystallinity or amor­
phous forms of powders. The presence of diffuse broad peaks in the XRD
spectra represents an amorphous structure, whereas crystalline mate­
rials exhibit a series of sharp peaks. In the SPI and SC spectra, two broad
and non-defined peaks appeared near angles 9◦ and 19◦ , revealed that
the encapsulation of GCO by SPICMC coacervate significantly affected
the semi-crystalline structure of blank capsule, and GCO microcapsule
has an amorphous structure with minimal crystallinity (Fig. 5A), the
3.4. Structural characterisation of GCO microcapsules
3.4.1. FT-IR analysis of GCO microcapsules
FT-IR is a powerful tool for the structural analysis of active molecules
and their polymers. This technique could show the molecular structure
and characteristic chemical bonds of each substance [19]. Here, FT-IR
was performed to prove whether GCO was effectively loaded into the
microcapsules. The FT-IR of microcapsules, wall materials, and core
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International Journal of Biological Macromolecules 256 (2024) 128064
Fig. 4. FT-IR spectra of GCO microcapsules prepared with different wall materials. (A: SPI-GCO; B: SC-GCO; C: SPICMC-GCO; D: SPISA-GCO; E: SCCMC-GCO; F:
SCSA-GCO).
XRD results also confirmed the findings of SEM. This was due to the
α-helixes in the polypeptide chain [35]. Similarly, the complex co­
acervates formed by SPICMC, SPISA, SCCMC, and SCSA had two broad
and non-defined peaks near angles 9◦ and 19◦ . Additionally, the peaks of
wall materials near 9◦ disappeared in GCO microcapsules. This may be
due to the intermolecular interaction with the matrix, which further
confirmed the formation of GCO-loaded microcapsules [40]. Six types of
microcapsules exhibited similar peak shapes, among which SPISA-GCO
exhibited sharper peaks, followed by SPICMC-GCO. The relative crys­
tallinity in the six GCO microcapsules ranged from 47.21 % to 62.19 %
(Fig. 5B). SPISA-GCO showed the highest relative crystallinity, followed
by the SPICMC-microencapsulated GCO. The increased sharpness of the
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International Journal of Biological Macromolecules 256 (2024) 128064
SPICMC, SCCMC, SPISA and SCSA at the same temperature. Here, the
chemical bonds broke and resulted in intermediates such as CO2 and
H2O. (4) At 460–600 ◦ C, the average weight loss was 11.21 %, primarily
because the internal GCO began to decompose. The temperature of the
maximum weight loss rate of GCO shifted from 424 ◦ C to 474 ◦ C as it was
encapsulated by the wall materials, which provided a protection layer.
These results indicate that the encapsulation process considerably
improved the thermal stability of GCO. This is mainly attributed to the
thermal resistance of the material used as the shell of the microcapsule,
resulting in slower heat transfer [43]. Furthermore, the mass loss of the
wall materials and microcapsules occurred at temperatures >220 ◦ C,
demonstrating the superior thermal stability of the products. The DTG
chart of all samples is shown in Fig. 6A, where the peak shape represents
the type of thermal event. GCO had endothermic peaks at 424.21 ◦ C,
whereas SPISA-GCO had endothermic peaks at 79.35, 303.13, 426.23,
and 474.47 ◦ C. This behaviour indicated that microencapsulation
influenced the displacement of endothermic peaks to a higher temper­
ature, which was related to the protective effect of wall material on
heating [42]. Other microcapsules exhibited similar shifts of the endo­
thermic peak. SCCMC-GCO, SPISA-GCO, and SCSA-GCO showed a
higher initial decomposition temperature than the other microcapsules,
indicating a better thermal stability.
3.4.4. DSC analysis of GCO microcapsules
Temperature, including Tg, considerably influenced the storage
stability of GCO microcapsules. Tg refers to the critical temperature
transitioning from a glass state to a highly elastic state [44]. When the
GCO microcapsules were in a glass state, the wall and core materials
were relatively stable, which is beneficial to storage of GCO microcap­
sules. When the storage temperature of the GCO microcapsules were
higher than Tg, the properties of the wall material and core material
changed. The glassy state quickly changed to a highly elastic state even
to a viscous flow state, which can result in an accelerated release rate of
the core material. In this study, DSC analysis demonstrated that the Tg of
microcapsules was 56.45–61.79 ◦ C (Fig. 6C). This temperature is higher
than room temperature; therefore, the GCO microcapsules were stable at
normal storage temperatures. The increased Tg of GCO microcapsules
may be attributed to the use of CMC and SA as wall materials, the higher
molecular weight of CMC and SA increased the Tg of GCO microcap­
sules. Furthermore, the range of degradation temperature (ΔTd),
degradation temperature peak (Td), and degradation enthalpy (ΔHd) of
the GCO and its microcapsules were calculated from the thermograms
(Table 5). The peak degradation temperature and enthalpy of SPI-GCO
(Td = 110.86 ◦ C, ΔHd = 123.17 J/g) were lower than those of the
SPICMC-GCO microcapsules (Td = 111.35 ◦ C, ΔHd = 138.06 J/g).
Similarly, the peak degradation temperature and enthalpy of SC-GCO
(Td = 107.18 ◦ C, ΔHd = 127.10 J/g) were lower than those of the
SCCMC-GCO microcapsules (Td = 111.77 ◦ C, ΔHd = 149.78 J/g). The
higher peak degradation temperature and the degradation enthalpy of
the SPICMC-GCO and SCCMC-GCO microcapsules indicated more
excellent thermal stability than those of the SPI-GCO and SC-GCO mi­
crocapsules. Both SPICMC-GCO and SCCMC-GCO microcapsules ach­
ieved better results than free biopolymers when compared with results
in the literature, where Td varied from 100 to 120 ◦ C [15]. This confirms
that the increase in thermal stability is due to the complexation of
polymers.
Fig. 5. XRD patterns of (A) wall materials and (B) GCO microcapsules.
diffraction peaks in the XRD pattern indicated an increase in the crys­
tallinity of samples. This further demonstrates a better stability in the
SPISA-GCO microcapsules. Amorphous materials are generally more
soluble and hygroscopic than crystalline materials, resulting in water
absorption during storage [41].
3.4.3. TGA of GCO microcapsules
TGA was used to assess the thermal stability of the treatments, which
reflected mass loss under continuous heating. The first derivative of the
TGA curve indicated the intensity of weight loss due to decomposition or
combustion at a certain temperature. This derivative thermogravimetric
(DTG) curve clearly reflected the initial reaction temperature, the tem­
perature of the maximum weight loss rate, and the final reaction tem­
perature (Fig. 6A–B). All GCO microcapsules had four weight loss stages,
(1) At temperatures ≤150 ◦ C, the weight loss was related to a loss of
moisture (2.64 %, 2.68 %, 2.68 %, 3.24 %, 2.18 %, and 2.80 %) by the
particles, with the low values indicating low moisture content. (2) At
temperatures >150 ◦ C, the weight loss was related to reactions of the
wall materials [42]. Two instances of mass loss, 9.89 % (96.31–86.42 %)
and 5.20 % (96.64–89.45 %), occurred at 200–250 ◦ C for SCCMC and
SCSA, respectively. (3) At 300–460 ◦ C, substantial peaks were observed
in the TGA curves. The weight loss of the samples was 58.10 % on
average, which was due to the combined decomposition of SPI, SC,
3.4.5. Emulsion and microstructure of GCO microcapsules
The morphology of emulsions (pH = 7.0) prepared with SPI, SC,
SPICMC, SPISA, SCCMC and SCSA stabilising solutions and imaged
through CLSM is shown in Fig. 7A–R. The protein was adsorbed on the
surface of the oil droplets shown in the dual-channel overlay images
(Fig. 7M-R), indicating that the emulsions were oil-in-water types [45].
Without polysaccharides, the SPI and SC solution resulted in spherical
oil droplets (red colour staining), whereas SPI-GCO was a bright green
layer thicker than SC-GCO. Therefore, there were more proteins
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International Journal of Biological Macromolecules 256 (2024) 128064
Fig. 6. Thermal curves were obtained through thermogravimetric analysis for (A) wall material and (B) GCO and its capsules. The square indicates an event
associated with GCO thermal degradation presence in capsules. The insert shows the highlight of the thermal curves from DTG samples. (C) DSC curves of GCO
microcapsules prepared with different wall materials.
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International Journal of Biological Macromolecules 256 (2024) 128064
around each droplet. These droplets were further surrounded by a
continuous phase rich in SPICMC and SCCMC (Fig. 7O-Q), potentially
indicating the beginning of the interaction between CMC and the pro­
tein. With SA, the protein and its interaction force were not strong, and
the green fluorescence was weak. This result indicates that the potential
binding ability of SA with SPI and SC was weak. CLSM was used to mark
the oil distribution of microcapsules, with green fluorescence repre­
senting the wall materials (Fig. 7A1-F1) and red fluorescence showing
the core materials (Fig. 7G1–L1). In this analysis, the particles had an
irregular structure. These results were in accordance with those
observed via SEM. The observed oil (in red) was uniformly embedded in
the protein matrix (in green), indicating that either SPI or SC success­
fully encapsulated the oil. This result further indicated that the soluble
small molecular fraction of SPI/SC and CMC/SA could encapsulate
microcapsule particles and work as wall materials. CLSM images proved
that GCO was successfully encapsulated with high efficiency (88 % ~ 69
%). Wang, Shi & Han [46] reported that the core-shell structure
benefited from an increased loading capacity and encapsulation effi­
ciency of microcapsules. Moreover, wall materials with homogeneous
thickness properties are beneficial to the improvement of microcapsule
mechanical strength and for enhancing their oxidative stability.
Table 5
Thermal properties of microcapsules containing GCO (SPI-CGO: soy protein
isolate-green coffee oil; SC-GCO: sodium caseinate-green coffee oil; SPICMCGCO: soy protein isolate sodium carboxymethylcellulose-green coffee oil;
SPISA-GCO: soy protein isolate sodium alginate-green coffee oil; SCCMC-GCO:
sodium caseinate sodium carboxymethylcellulose-green coffee oil; SCSA-GCO:
sodium caseinate sodium alginate-green coffee oil).
Formulation
ΔTd
ΔHd
Td
Tg
SPI-GCO
SC-GCO
SPICMC-GCO
SPISA-GCO
SCCMC-GCO
SCSA-GCO
56.45–145.77
58.82–148.67
61.39–157.80
61.65–160.09
60.77–140.27
61.79–152.69
123.17
127.10
138.06
148.48
149.78
149.98
110.86
107.18
111.35
109.88
111.77
108.21
56.45
58.82
61.39
61.65
60.77
61.79
ΔTd = Range of degradation temperature (◦ C).
ΔHd = Degradation enthalpy (J/g).
Td = Degradation temperature peak (◦ C).
Tg = Glass transition temperature (◦ C).
adsorbed on the surface of the oil droplets in SPI-GCO, which could
better stabilise the emulsion (Fig. 7M-N). SPICMC and SCCMC bind to
the oil-water interface, as evidenced by well-defined darker edges
Fig. 7. CLSM of (A-R) emulsion (pH = 7.0) and (A1-R1) GCO microcapsules prepared with different wall materials. (A-F show FITC-dyed microcapsules, G-L show
Nile red-dyed oil droplets, M-R show an overlay of G-L and A-F). (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
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International Journal of Biological Macromolecules 256 (2024) 128064
Fig. 8. Optical microscopy observation of GCO microcapsules prepared with different wall materials. (A: SPI-GCO × 100; B: SC-GCO × 100; C: SPICMC-GCO × 100;
D: SPISA-GCO × 100; E: SCCMC-GCO × 100; F: SCSA-GCO × 100; a: SPI-GCO-GCO × 400; b: SC-GCO-GCO × 400; c: SPICMC-GCO × 400; d: SPISA-GCO × 400; e:
SCCMC-GCO × 400; f: SCSA-GCO × 400).
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International Journal of Biological Macromolecules 256 (2024) 128064
Optical microscopy images of microcapsules are shown in Fig. 8; the
large red circles represent oil drops (Fig. 8A-F). When the emulsion was
acidified to pH 2.5–3.0, localised aggregates of large particles appeared,
and the oil inside was stained with Nile Red. The oil droplets were
successfully encapsulated, as demonstrated by the formation of a solid
bond of microcapsules due to the formation of complex coacervates
[47]. In addition, the type of wall material and its combination had a
considerable effect on the morphology. The microencapsulated particles
prepared with different wall materials were also characterised using
SEM (Fig. 9A-L). The GCO microcapsules had grainy-like structures of
different sizes without cracks. These were related to the interaction
between GCO and wall materials. In addition, SPICMC-GCO had smaller
pores than SPI at pH 3.0. This phenomenon was attributed to the higher
charge density of CMC, resulting in stronger electrostatic attraction with
SPI at the same pH, thus a more compact network structure. This result is
also applicable to SPI-GCO and SPISA-GCO. The microcapsule pores of
the latter are smaller, owing to the addition of SA at pH 2.5, which has a
good encapsulation efficiency for GCO. The surface of the microcapsule
remained intact with no apparent cracks or gaps, suggesting that in­
teractions between SPI and CMC, SPI, and SA, SPI, and CMC, as well as
SC and SA are sufficient to promote microcapsule formation [48]. These
results demonstrate that the complex coacervates of SPICMC, SPISA,
SCCMC, and SCSA can encapsulate microcapsule particles as wall
materials.
environmental conditions. The effect of different combinations of carrier
agents on the stability of GCO was evaluated with respect to storage time
(Fig. 10A). There was a slight increase of POV in GCO after 12 days,
whereas no significant difference was observed in GCO microcapsules.
The POV of GCO with SCSA or SPISA at 60 ◦ C was 38.17–41.67 meq
peroxide/kg oil after 30 days of storage. In contrast, GCO with SC or SPI
was 51.33–55.33 meq peroxide/kg oil. This revealed that SCSA-GCO
and SPISA-GCO had better oxidative stability compared to SC-GCO
and SPI-GCO. Furthermore, GCO with SCSA had higher encapsulation
efficiency, owing to the combined effect of small droplet size emulsions
and large particle size microcapsules that led to higher stability and
lower surface oil content. Larger particles covered the active oil drops
inside the medium effectively, providing maximum protection by
increasing the shell around the base material [49]. Higher surface oil
content can lead to the higher rate of oxidation [50]. Microcapsules from
SPI and SC had a greater amount of surface oil content when compared
to that of SPI and SC microcapsules, thus led to the higher rate of
oxidation. Hence, the possible reason behind an increase in peroxide
value for microcapsules prepared with single component may be due to
the higher surface oil content. As a result，the SA treatment resulted in
the lowest POV values compared to all other treatments. In particular,
SCSA-GCO and SPISA-GCO were promising formulates to inhibit
peroxide production. The changes in POV values were due to the
imbalance between production and degradation of hydroperoxides
during storage.
3.5. Lipid oxidation analysis of GCO microcapsules
3.5.2. Oxidation kinetics model of GCO microcapsules
Lipid oxidation kinetic models can reasonably predict the degree of
lipid oxidation deterioration [51]. A certain amount of oil leaked on the
surface of GCO microcapsules after storage at 60 ◦ C for 30 d. However,
no rancidity or peculiar smell appeared. Therefore, the study was carried
out on GCO and GCO microcapsules stored for 30 d. The POV oxidation
kinetic models in this period were constructed. The zero- and first-order
models were applied to fit the POV of GCO and its microcapsules. As
3.5.1. Changes of POV value in GCO microcapsules during accelerated
storage
The lipid oxidation of GCO microcapsules was analysed based on
POV. Over time, primary oxidation products, such as hydroperoxides
were formed. These compounds are reactive and responsible for unde­
sirable flavour in food products. Lipid oxidation is directly related to
microcapsule surface oil content, which is more susceptible to
Fig. 9. SEM of GCO microcapsules prepared with different wall materials. (A: SPI-GCO × 2300; B: SC-GCO × 2300; C: SPICMC-GCO × 2300; D: SPISA-GCO × 2300;
E: SCCMC-GCO × 2300; F: SCSA-GCO × 2300; G: SPI-GCO-GCO × 5600; H: SC-GCO-GCO × 5600; I: SPICMC-GCO × 5600; J: SPISA-GCO × 5600; K: SCCMC-GCO ×
5600; L: SCSA-GCO × 5600).
13
J. Mu et al.
International Journal of Biological Macromolecules 256 (2024) 128064
Table 6
Regression equations of POV for GCO and its microcapsules (GCO: green coffee
oil; SPI: soy protein isolate; SC: sodium caseinate; SPICMC-GCO: soy protein
isolate sodium carboxymethylcellulose-green coffee oil; SPISA-GCO: soy protein
isolate sodium alginate-green coffee oil; SCCMC-GCO: sodium caseinate sodium
carboxymethylcellulose-green coffee oil; SCSA-GCO: sodium caseinate sodium
alginate-green coffee oil).
Samples
GCO
SPI
SC
SPICMCGCO
SPISAGCO
SCCMCGCO
SCSA-GCO
Zero-order
First-order
Fitting equation
R2adj
Fitting equation
R2adj
C0 = 4.24405*t20.94048
C1 = 1.41171*t +
2.20833
C2 = 1.32044*t +
2.35119
C3 = 1.20536*t +
2.47024
C4 = 1.05456*t +
3.06548
C5 = 1.18452*t +
2.41667
C6 = 0.95437*t +
2.86905
0.80241
ln(C0) = 0.10082*t
+ 1.58385
ln(C1) = 0.06608*t
+ 1.85837
ln(C2) = 0.06489*t
+ 1.83222
ln(C3) = 0.06314*t
+ 1.79555
ln(C4) = 0.05862*t
+ 1.81490
ln(C5) = 0.06234*t
+ 1.79789
ln(C6) = 0.05853*t
+ 1.72311
0.99002
0.98904
0.99124
0.98744
0.98411
0.98662
0.98693
0.92899
0.93234
0.92933
0.93744
0.94173
0.93528
indicated in Table 6, the correlation coefficients of the first-order model
fitting equation of GCO were higher than those of the zero-order model
fitting equation. Moreover, the first-order model had a higher degree of
fit (Fig. 10B). The correlation coefficients of the zero-order model of
GCO microcapsules were all higher than those of the first-order model,
and the fitting degree of the zero-order model was higher. In addition,
the regression equations were linearly correlated and the correlation
degree was significant, indicating that the GCO microcapsules had better
oxidation stability (Fig. 10C). The zero-order reaction form a straight
line by plotting a quality factor (positive or negative) versus time, which
are independent of the reagent concentration, the rate of POV formation
was constant with the time and are independent of the concentration of
reacting oil. Table 6 showed the zero-order kinetic parameters of GCO
and microencapsulated GCO covered with SPI, SC and CMC, SA. A
higher rate constants of POV formation (K) indicated a higher oxygen
diffusion through the biopolymer matrix. The K of non-encapsulated
GCO (4.24) was significantly (p < 0.05) higher than that of micro­
encapsulated GCO with SPI\SA (1.41 and 1.32), and both of them were
higher than K of microencapsulated GCO covered with SPICMC, SPISA,
SCCMC, and SCSA mixtures (1.21, 1.05, 1.18, and 0.95, respectively),
these all reflected that GCO microcapsules achieved a greater protection
of GCO against oxidation. To verify the feasibility of the kinetic models,
a set of parallel test data with storage times of 6, 12, and 30 d was
randomly selected and compared with the predicted values obtained
from the optimised model. The relative error range values were subse­
quently calculated (Table 7). For SPI-GCO, the relative error between
the measured shelf life from the zero-order model and predicted shelf
life from the first-order model at 60 ◦ C was <14.14 %. These results
indicate that the established model can better reflect the quality changes
and predict the degree of lipid oxidation from GCO microcapsules dur­
ing storage.
4. Conclusion
This study described the use of four types of complex coacervates,
namely SPI-CMC, SPI-SA, SC-CMC, and SC-SA, for the microencapsula­
tion of GCO using complex coacervation. The GCO microcapsules were
produced under optimum conditions (pH = 3.0, SPI: CMC = 5:1, SPI: SA
= 3.5:1, SC: CMC = 5:1, SC: SA = 5:1) and presented favourable elec­
trostatic interactions. The microcapsules obtained with different mate­
rials presented high EE, indicating that adding polysaccharides could
improve the overall EE of microcapsules. FT-IR and XRD analysis
confirmed that the interaction between the wall materials and GCO, as
Fig. 10. Changes in POV values of GCO and its microcapsules during storage
(A), oxidation kinetic model of GCO and its microcapsules during storage, (B)
zero-order model and (C) first-order model.
14
J. Mu et al.
International Journal of Biological Macromolecules 256 (2024) 128064
Table 7
Validation of oxidation kinetics models of GCO microcapsules (GCO: green coffee oil; SPI: soy protein isolate; SC: sodium caseinate; SPICMC-GCO: soy protein isolate
sodium carboxymethylcellulose-green coffee oil; SPISA-GCO: soy protein isolate sodium alginate-green coffee oil; SCCMC-GCO: sodium caseinate sodium
carboxymethylcellulose-green coffee oil; SCSA-GCO: sodium caseinate sodium alginate-green coffee oil).
Samples
GCO
SPI
SC
SPICMC-GCO
SPISA-GCO
SCCMC-GCO
Optimal model
ln(C0) =0.10082*t
+ 1.58385
C1 = 1.41171*t +
2.20833
C2 = 1.32044*t +
2.35119
C3 = 1.20536*t +
2.47024
C4 = 1.05456*t +
3.06548
C5 = 1.18452*t +
2.41667
8.92
9.67
10.68
9.17
10.27
8.67
9.70
7.17
9.39
7.00
9.52
7.50
8.60
6.50
16.34
19.67
19.15
17.83
18.2
17.17
16.93
16.67
15.72
14.83
16.63
16.17
14.32
14.00
100.33
98.83
44.56
43.17
41.96
41.33
38.63
38.50
34.70
34.33
37.95
38.33
31.5
31.17
1.50–20.38
3.12–14.14
1.50–15.58
0.34–26.08
1.07–25.45
1.00–21.22
1.05–24.42
C6 = 0.95437*t + 2.86905
Storage for 6 d (predicted)
Storage for 6d
(experimental)
Storage for 12 d (predicted)
Storage for 12
d (experimental)
Storage for 30 d (predicted)
Storage for 30
d (experimental)
Range of 3 experiments of
relative error/%
well as the selected process and materials could successfully encapsulate
GCO, which ensures the protective effect of the oil. Furthermore, SEM
and OM analyses showed that five types of microcapsules had good
surface morphology. Additionally, CLSM results demonstrated that GCO
was encapsulated well into the microcapsules. TG and DSC analyses
indicated that GCO microcapsules had good thermal stability below
220 ◦ C, which could satisfy general food processing requirements (Tg of
microcapsules was 56.45–61.79 ◦ C). In addition, oxidative stability was
remarkably enhanced, and the applications of GCO were expanded by
complex coacervation. These results can provide an effective and safe
delivery system for encapsulating and stabilising GCO for application in
the food industry. However, further research is required to identify the
mechanism through which the protected oil would be released. Since the
wall material structure can overcome the serious impact of environ­
mental changes, it is necessary to study the efficiency and bioavailability
of bioactive compounds in green coffee oil, and further analyse the
bioavailability of micro-functional factors released by green coffee oil
microcapsules during oxidative storage. The wall material loaded with
bioactive substances can reduce its toxicity and off-target side effects, so
it can be further combined with cell models and animal models to
explore the anti-cancer cell proliferation ability of these composite wall
materials coated with green coffee oil, and further reveal the possible
signal pathways involved from the molecular level. The specific appli­
cation of green coffee oil microcapsules can be expanded in the food
field, such as the application of baked food, functional food, solid
beverage, and juice.
SCSA-GCO
Acknowledgment
We would like to thank the China Central Public-Interest Scientific
Institution (1630142022010), National Natural Science Foundation of
China (31872888), Key Research and Development Project of Yunnan
Province (202302AE090004), and the Chinese Academy of Tropical
Agricultural Sciences for Science and Technology Innovation Team of
National Tropical Agricultural Science Center (NO. CAT­
ASCXTD202304) for their financial support.
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CRediT authorship contribution statement
Jingyi Mu: Methodology, Validation, Writing – original draft.
Rongsuo Hu: Investigation, Supervision. Yumei Tang: Investigation,
Formal analysis, Supervision. Wenjiang Dong: Methodology, Software,
Formal analysis, Investigation, Writing – original draft, Writing – review
& editing, Visualization, Funding acquisition. Zhenzhen Zhang:
Methodology, Formal analysis, Supervision.
Declaration of competing interest
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as
potential conflicts of interest.
Data availability
The data that has been used is confidential.
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