Protection of mono and polyunsaturated fatty acids from

grapeseed oil by spray drying using green biopolymers as wall

material

Diego Mauricio Sánchez-Osorno1,2*, Angie Vanesa Caicedo Paz1, María Camila López-
Jaramillo2, Aída Luz Villa3, Julián Paul Martínez-Galán1*
1
Grupo de Investigación Alimentación y Nutrición Humana-GIANH, Escuela de Nutrición y Dietética,
Universidad de Antioquia, Cl. 67, No 53-108, Medellín
2
Grupo de Investigación e Innovación Ambiental. GIIAM, Institución Universitaria Pascual Bravo, Cl. 73, No
73a-226, Medellín 050034, Colombia
3
Grupo Catálisis Ambiental, Universidad de Antioquia, Cl. 67, No 53-108, Medellín

*Correspondence: [email protected]; [email protected]

ABSTRACT
Grapeseed oil that is obtained from agro-industrial waste, has a high value for various industries,
including food, pharmacology and cosmetics. This oil has shown health benefits such as
cardioprotective, anticancer, antimicrobial, and anti-inflammatory properties. The health benefits
have been mainly associated with the monounsaturated (MUFA) and polyunsaturated (PUFA) fatty
acids. In this sense, it has been observed that grapeseed oil can be easily modified by environmental
factors such as oxygen, high temperatures and light, showing the instability and easy degradation of
grapeseed oil. The degradation of these oils is expressed directly in changes in flavor and nutritional
value. The objective of this study was to encapsulate grapeseed oil using a spray drying technique
to conserve its lipidic profile. In addition, green biopolymers, as wall materials of fatty acids, such
as microcrystalline cellulose and whey protein are used in the encapsulation process. Powder
recovery of the grapeseed oil microcapsules ranged from 65-70%. The encapsulation efficiency of
the microcapsules varied between 80-85%. The FTIR analysis showed chemical interactions that
demonstrate chemisorption between the grapeseed oil and the encapsulating material, while the
SEM micrographs show a correct encapsulation in a spherical shape. Gas chromatography showed
that the lipid profile of grapeseed oil is preserved thanks to microencapsulation. Release tests
showed 80% desorption within the first three hours at pH 5.8. Overall, whey protein and
microcrystalline cellulose could be used as a wall material to protect grapeseed oil to potential
application in controlled delivery of fatty acids microcapsules.
Keywords: fatty acids, spray drying, grapeseed oil, encapsulation, microcrystalline cellulose, whey
protein isolated

1. INTRODUCTION
Nowadays, there is a growing trend towards the use of healthy foods to preserve or improve health
or to prevent various types of diseases. This trend of consumers has been reflected in the food
industry that seeks to respond to these market needs, thus offering functional and nutraceutical
foods. One of the most notorious changes in healthy eating has been the substitution of fats of
animal origin for vegetable oils since the latter have healthier characteristics. In this sense, grape
seed oil exhibits a high concentration of polyphenols, which are antioxidant substances that function
as preservatives in food or as protective agents at the cellular level against oxidative damage from
free radicals (Böger, Georgetti, & Kurozawa, 2018).
Grapeseed oil has high content of monounsaturated fatty acids (MUFA), and polyunsaturated fatty
acids (PUFA) (Tangolar, Özoǧul, Tangolar, & Torun, 2009). These fatty acids have been associated
with health benefits such as antimicrobial, cardiac protector, anti-inflammatory, and anticancer
(Kapoor, Kapoor, Gautam, Singh, & Bhardwaj, 2021). It is possible to improve the nutritional value
of some food products by incorporating grape seed oil in various matrices, thus taking advantage of
the beneficial effects it has on health. However, some industrial processes such as the use of high
temperatures, other compounds present in food, light and oxygen, could generate a degradation or
modification of grape oil since it is chemically unstable and susceptible to oxidative degradation
giving as unwanted flavors, colors or aromas result in addition to the loss of its nutritional value
(Zhong et al., 2019).
Microencapsulation is a technology that seeks to protect bioactive compounds. In the case of lipids,
it can delay oxidation, reduce volatility and improve the stability of oils and aromas.
Microencapsulation consists of coating a particle that is in a solid, liquid or gaseous state, said
coating consists of a thin layer that isolates the particle from external factors and keeps it
completely contained within the wall of the capsule. The microencapsulation principle protects a
bioactive compound from external factors that can generate degradation, creating a physical barrier
between the active compounds and the outside (Mishra, 2015). Although there are many methods
that allow the encapsulation of bioactive compounds, spray drying is the most widely used, mainly
in industries such as food, pharmaceuticals, and cosmetics because it is an inexpensive, flexible and
easily scalable process. This technique allows a liquid mixture, in which the bioactive compound
and the encapsulating agent are found, to become fine droplets that will be dehydrated with the help
of a stream of hot air to finally obtain microparticles in the form of powders. By having the
bioactive compounds in powder form and protected, it is possible to improve storage, transport and
their dosage in food formulations (Mohammed, Tan, Manap, Muhialdin, & Hussin, 2020).
There is a wide variety of encapsulating agents, however, there is a growing inclination for
environmentally friendly encapsulating agents during their extraction, production and final disposal
process. In this sense, obtaining encapsulating agents from agro-industrial waste becomes a great
alternative to contribute to the environment. Whey protein concentrate is an encapsulating material
extracted from dairy industry waste with good emulsifying properties, high solubility and low
viscosity. This material has a wide range of solubility from pH 2 to pH 9. However, the pH of the
solution has a tendency to affect the nature and distribution of the net charge of proteins. Normally,
proteins are more soluble at low pH (acids) or high pH (alkalines), due to the excess of charges of
the same sign, which produces repulsion between the molecules, thus contributing to a high
solubility (Gao et al., 2019). Due to its wide range of solubility, whey protein can be considered an
immediate release encapsulating material. As for cellulose, it is extracted from agro-industrial waste
such as wood, banana rachis, oil palm rachis, among others (Osorno, D. M. S., & Castro, 2018).
Therefore, recent investigations have focused on the development of cellulose micro-particles,
known as microcrystalline cellulose (MCC) with prominent characteristics, making it useful for a
wide range of materials and products (Guyton A. C., 2011; Kumar, De La Luz Reus-Medina, &
Yang, 2002).
MCC is a partially depolymerized pristine cellulose derivative conventionally prepared through acid
hydrolysis approach to eliminate the disordered regions, whereas, the ordered domains that present
a higher resistance to the hydrolysis process remain intact (Holilah et al., 2021). The obtained MCC
shows short particles and exhibits high crystallinity index, improved density, large surface area, and
increased thermal stability (Holilah et al., 2021; O’Connor & Schwartz, 1993). Besides that, the
presence of several accessible and active hydroxyl side chemical groups on the surface allowed
MCC to be functionalized through multiple energetic chemical groups such as nitrate esters,
nitramines, tetrazoles and azides to design innovative high-value biopolymers for advanced
energetic materials (Peng, Zhang, & Zhang, 2019) with the ability to interact with other
encapsulating agents thanks to its availability of OH groups and its hydrophilicity.
To preserve the lipid profile of grapeseed oil over time, this work aimed to use green biopolymers
to protect the monounsaturated and polyunsaturated fatty acids present in grapeseed oil and thus
preserve its nutritional characteristics. For this purpose, a whey protein (WPI) to microcrystalline
cellulose weight ratio of 3:1 was evaluated in order to protect the grapeseed oil using spray drying
as the selected encapsulation method.
2. Materials and methods

2.1. Materials
In brief, 280 g of cotton linter sheet and HCl solution (1 N) a 5L flask was used (60 min room
temperature) and then it was brought to the boil for another 1.5–2 h under constant stirring. The
mixture was cooled and filtered. The white residue obtained was washed and then air-dried (yield
>85%). A mixture of an aqueous solution of sodium hydroxide and dry powder was prepared with
constant application of stirring. The previous mixture was left to stand between 4 and 12 hours at
room temperature. Ethanol was added until a white precipitate was obtained, which normally occurs
when a concentration between 50 and 60% of ethanol is reached. The solid obtained was air dried
and passed through a US#20 screen. The sieved material was dried to less than 6% moisture using
temperatures between 45–50 °C (yield > 90%) (Kumar et al., 2002).
Whey protein isolate (WPI) with 80% protein content on dry basis was supplied by Bell Chem
International S.A.S. (Colombia). Grapeseed oil was supplied by Distriol (Brasil). Absolute ethanol
of analytical grade was from Merck (USA). Deionized water (Milli-Q) was used for all preparation.
2.2. Preparation of emulsions
WPI and MCC were dissolved in water under stirring by using an Ultra-Turrax T-25 homogenizer
(IKA model Bioblock Scientific, Medellín, Colombia) for 5 min at room temperature and then
grape seed oil was added. The emulsion was prepared by adding the lipid component to the aqueous
phase using an Ultra-Turrax operated at 25000 rpm for 10 min, then passed to spray drying. The
encapsulating agent vs grapeseed oil weight ratio was 3: 1.
2.3. Spray-drying conditions
Immediately after preparation of the feed solution, encapsulation was carried out using a Mini
Spray Dryer (Buchi B-290), nozzle diameter of 0.5 mm; drying chamber: diameter of 35 cm and
height 90 cm. Parameters: pump (10%); aspirator (100%); flow rate (600 L/h); inlet temperature
(180 °C); and outlet temperature (100 °C).
2.4. Solubility
The solubility of spray-dried microparticles was measured according to Jadafi (2019) with some
modifications. The standard technique for dairy powders was used, initially preparing a 5% (w/w)
solution in distilled water. To obtain a good dispersion, stirring was used at 750 rpm for 30 min at
20 °C. An aliquot of 40 mL was taken and centrifuged for 10 min at 1000 rpm by using an CD-
0412-50 centrifuge. 5 g of the supernatant were taken, dried at 105 ± 5 °C for 24 h, then transferred
to a desiccator and reweighed. The amount of soluble material in the supernatant was calculated and
expressed as solubility percentage (Mahdi Jafari, Masoudi, & Bahrami, 2019).
2.5. Porosity
Powder porosity was measured according to Jadafi 2019 in which, to determine the bulk density, 2
g of microcapsules were taken and placed in a cylinder (10 mL). The cylinder was tapped several
times on a rubber surface. The apparent density was determined with the mass-occupied volume
relationship. Then, the true density was calculated by filling approximately 1 g of spray-dried whey
powders in a burette containing toluene (Mahdi Jafari et al., 2019). Then, rise in toluene level (mL)
was measured and true density was calculated according to equation 1:

weight of powder sample (g)

True density= (Equation 1)
rise∈toluene volume (mL)

Finally, the porosity of the powder samples was calculated using the relationship between the bulk
and the true density of the powders as shown in the equation 2 (Bhusari, Muzaffar, & Kumar,
2014):

( bulk density )
Porosity=(1− ) (Equation 2)
( true density )
2.6. Surface free oil content and microencapsulation efficiency
The amount of unencapsulated oil (i.e. free oil or surface oil) presents at the surface of the powders
was measured using a method described by Tan et al. (2005) with some modifications. For this
purpose, 2 g of microparticles were taken and mixed with 15 mL of hexane, placed in a glass vial
with a lid and stirred at room temperature for 2 min using a vortex to extract the free oil. Followed
by a decantation and filtration process. Subsequently, the washed microparticles were rinsed with
hexane 3 times using 20 mL hexane. The residual solvent was removed by drying at 60°C until
brought to a constant temperature. The weight difference in the powder before and after hexane
extraction was used to calculate the free oil content as a percentage. Microencapsulation efficiency
(EE) was calculated using the equation 3.

Microencapsulation efficiency =
[ total oil ]
( total oil−surface oil )
×100 (Equation 3)

2.7. Fourier transform infrared spectroscopy (FTIR)

FTIR measurements were performed in triplicate using an Alpha Platinum FTIR-ATR spectrometer
(range: 4000-600 cm−1; number of scans: 144; zero filling factor: 4; resolution: 4 cm −1; mode:
absorbance). The powder was settled on the optic window with a diamond crystal.
2.8. Microcapsule characterization
The morphology of the spray-dried microcapsules was observed with a scanning electron
microscope (SEM, JSM-590LV, JEOL) operating at 15 kV. The microcapsules were coated with
gold and taken under the microscope. Additionally, the internal morphology of the microparticles
was observed by initially freezing the microparticles with nitrogen and then fracturing them. The
interior morphology of the wet status microcapsules was also observed using a transmission
electron microscope (TEM, JEM 1011 JEOL) at an accelerating voltage of 120 kV. The
microparticle samples were diluted in hexane (1:100 vol/vol) and the suspension was kept in an
ultrasonic bath (Unique, model USC – 1800, Colombia) for 30 min. Subsequently, the suspension
was deposited on a copper grid coated with carbon film. The images were observed at a
magnification of 120 k (Leung et al., 2005).
2.9. Particle size distribution
SEM micrographs (1000x magnification) were analyzed using ImageJ software. For this analysis, at
least 140 particles were taken to measure their diameter. From these measurements, a global
average was performed and the size distribution curves were constructed.
2.10. Gas chromatography
Analysis of the composition of grapeseed oil, before and after encapsulation, was performed by
means of gas chromatography-mass spectrometry (GC-MS: Varian CP 3800 and CP-Sil 8 CB Low
Bleed/MS column (30 m × 0,25 mm × 0,5 µm)). Equipment conditions: injector 250°C; helium 1.5
ml/min; oven from 50°C to 240°C at 3°C/min. The composition of the oil was determined by mass
spectrometry (ion trap, temperature at 220°C; temperature other than 80°C, transfer line
temperature at 240°C).
Gas chromatography (GC) system was used to perform the analysis of FAME in hexane (Agilent
Technologies 6890, Hewlett-Packard, Avondale, PA) equipped with a split/splitless injector. The
flow rate 128 mL/min; temperature 150°C (5 min) to 220°C (30 min), rate of 4°C/min. Temperature
of injection 250°C.
2.10.1. Fatty acid profile of grape seed oil
The FAMEs were analyzed by gas chromatography according to the American Oil Chemists`
Society Method as reported for composition oil described in the section 2.2.1.(AOAC, 2005). The
analysis of fatty acid profile was carried out by gas chromatography (GC). FFA methyl esters were
analyzed on an Agilent 6890N gas chromatograph with flame ionization detector (FID), TR-CN100
capillary column, 60 m x 250 µm x 0. 20 µm ID, split/split less injector with a Split ratio 100:1,
injection volume 1.0 uL, injector temperature 260°C, oven temperature program, start 90°C (7 min)
to 240°C (15 min) at a rate of 5°C/min, detector temperature 300°C. He carrier gas at a flow rate of
1.1 mL/min. Standard (37-component FAME Mix, Supelco) was used for fatty acid identification.
The results are presented as relative amount of each fatty acid (% of FA/100 g of sample). The
concentrations of total SFA, MFA and PFA were calculated as the sum of the corresponding
families. The percentage of total fat was determined as the sum of SFA, MFA and PFA.
2.10.2. Fatty acid profile of microencapsulated grape seed oil
The microcapsules of grape seed oil were taken as a solid matrix, therefore, prior to the
determination of the fatty acid profile, acid hydrolysis on the oil microcapsules was carried out
using a SOXCAP 2047. Then the fat extraction was performed using the SOXTEC 2050 in
accordance to (FOSS, 2017), after extraction, hexane was added with a pasteur pipette and the
contents were transferred to an erlenmeyer where the methylation process was continued. This
procedure was repeated three times. The methylation process was followed as described above.
2.11. Controlled Release
To determine the release profile of the microcapsules, the dynamic dialysis method was used. In
which, grapeseed oil capsules (3 mg mL−1) were scattered in PBS (phosphate buffer solution) 100
mM at pH 1 (stomach) and pH 5.8 (intestine). This experiment was performed using dialysis bags,
37 °C, incubation in absolute ethanol (30 mL) and stirred at 100 rpm. 1 mL aliquots were
withdrawn at various time intervals and the same volume was replaced with absolute ethanol. The
cumulative release rate was obtained by the ratio of cumulative release amount to the amount of
grapeseed oil encapsulated.
2.12. Statistical analysis
The data collected is reported as the average of tests performed in triplicate. ANOVA was used for
the analysis of results (SPSS program version 10.0 for Windows). Duncan's multiple range test was
used to test for differences between means.
3. RESULTS AND DISCUSSION
3.1. Solubility
The solubility of produced microcapsules containing grapeseed oil was 65 % ± 0.26. This solubility
value could be due to the inlet temperature used in the spray dryer in which inlet values of 180°C -
190°C decreased the solubility of the whey protein. The low solubility can be related to the
formation of a surface layer over the powder particles and thereby reducing the diffusion rate of
water molecules through the surface of particles (Mahdi Jafari et al., 2019). Additionally, the low
solubility of microcrystalline cellulose in wall material reduces the solubility of microcapsules due
to the cellulose-protein and protein-protein interactions increase because the electrostatic forces are
lowest and less water interact with the protein molecules (Pelegrine & Gasparetto, 2005).
3.2. Porosity
Porosity is an important factor in determining the functionalities of an agglomerates. In this sense,
reconstitution of powders can be determined taking into account their porosity. Generally, dried
powders with a lower porosity are desirable in order to increase their storage stability (O’Connor &
Schwartz, 1993). The porosity of powder sample was 0.45% ± 0.28. The high porosity value could
be due to formation of more vacuoles inside powder particles at higher temperatures which results
in a lower bulk density and then, a higher porosity (Saurí et al., 2014). An increase in porosity
related to the addition of WPI was observed during the spray-drying process. This increase in
porosity may be related to the increase in particle size which increases with increasing WPI.
3.3. Surface free oil content and microencapsulation efficiency (EE)
The visible ultraviolet spectroscopy technique was used to determine the % encapsulation
efficiency. The concentrations of the grapeseed oil were determined using the standard graph (y =
0.0339x + 0.0332, R2 = 0.9921). EE% was found in 86.9% of the grape seed oil microencapsulated.
Samples were analysed in triplicates. This suggests that WPI and MCC are very well suited for
encapsulation of hydrophobic compounds.
Fourier transform infrared spectroscopy (FT-IR)
FT-IR analysis was applied to assess the properties of microcapsules of grapeseed oil. The spectra
of the grapeseed oil, WPI, MCC, and the microcapsules are presented in Fig 1.

Figure 1 FTIT spectra

MCC spectrum shown the representative peaks in which the region from 3400 to 3500 cm _1 and
absorption at 2900 cm-1 is due to stretching of –OH groups and CH 2 groups respectively (Jonoobi et
al., 2011). The absorption band at 1163cm-1 and the peak at 896 cm -1 correspond to C–O–C
stretching and associated to C–H rock vibration of cellulose (anomeric vibration, specific for β-
glucosides) observed in MCC (Alemdar & Sain, 2008). Additional bands are shown in table 1.

Whey protein spectrum revealed fats and oils regions in the region between 3000 and 2800 cm−1
which corresponds to the C-H stretching vibrations of carbonyl groups of triglycerides (Upadhyay,
Jaiswal, & Jha, 2018). Other peaks related with fatty acids are located at 2924 and 2854
cm−1(Botelho, Reis, Oliveira, & Sena, 2015). The main vibrational bands analyzed in the region
between 1700 and 1200 cm−1 were fatty acids, polysaccharides and proteins. Between 1700 and
1500 cm−1 it is possible to find the Amide I (ν C=O, ν CeN) in 1640 cm−1 and Amide II in 1550
cm−1 (δ NeH, ν CeN), related to peptide bonds (CO-NH) (Andrade et al., 2018). The region
dominated by polysaccharide peaks is located at 1200-900 cm −1 (El Darra et al., 2017). Another
important band are shown in table 1.

In the microencapsulated grapeseed oil deconvolution was used to find the main bands of the
different encapsulant agent and the oil as well as to find the formation of new peaks. In the grape
seed oil spectrum, it is showed the adsorption bands for common triglyceride and fatty acid, the
main peaks are showed in table 1.

Main bands corresponding to MCC, WPI and grapeseed oil was observed in the microcapsule
spectra and no new peaks were found. In the microcapsule spectrum two regions received special
attention that are related to fatty acids specially for linoleic and oleic fatty acids. The first lipid
region located between 3050-2800 cm-1 and the second lipid region located between 1800 and 1350
cm-1. In these regions, it is possible to find the bands mainly associated with lipids. The band at
3006 cm-1 results from the C-H stretching vibration of HC=CH groups which could be a useful
indicator of the different degrees of unsaturation in acyl chains of phospholipids (Dogan, Siyakus,
& Severcan, 2007). The absorption band of carbonyl stretches of carboxyl at 1747 cm -1 exhibited
the presence of lipids (Dokken & Davis, 2007), assigned to C=O stretching vibration of ester groups
in triacylglycerols (Szalontai, Kóta, Nonaka, & Murata, 2003).

These results are in agreement with other studies which reported the IR regions of edible oil for
unsaturated fatty acid featured around 2900 cm -1, as well as oleic acid and linoleic acid profiles
(Sherazi, Mahesar, Bhanger, Van De Voort, & Sedman, 2007).

Tabla 1 FTIR funcional groups

Micro Cristalin Celullose - MCC

Setting Waveleght (cm-1) Vibration of functional groups

1 3400 to 3500 Stretching of –OH

6 2900 Groups and CH2
11 1645 Absorption of water
13 1425 Intermolecular hydrogen of aromatic ring group
17 1163 C–O–C Stretching
22 896 C–H Rock vibration
2 3009 C-H stretching vibration of the cis-double bond (=CH)
4 2954 Asymmetric stretching vibration of methyl (-CH3) group
Methylene (-CH2) band vibration (symmetric and asymmetric
5 2924 and 2852
stretching)
9 1743 Carbonyl (C=O) functional group
Grape Seed Oil

10 1654 Cis C=C

12 1465 Bending vibration of CH3 and CH2 aliphatic groups
14 1417 Rocking vibration of CH bond of cis-disubstituted alkenes
15 1377 Symmetric bending vibration of CH3
16 1228 and 1155 Vibration of stretching mode from C-O group in esters
18 1111 vibration of fatty acids (-CH bending and –CH deformation)

Methylene (-CH2) rocking vibration (overlapping), vibration of cis-

23 721
disubstituted olefins

Between 3000 and

3 C-H stretching vibrations of carbonyl groups of triglycerides
2800
5 2924 stretch vibrations of -C-H(CH2) group of fatty acids
Whey Protein

7 2854 stretch vibrations of -C-H(CH3) group of fatty acids

8 1750 C=O stretch of ester or carboxylic acid group
19 1080 OH stretch of carbohydrates
20 1073 ν OH
21 1030 ν C-O of alcohols)

3.4. Morphological analysis

Figure 2 shows the different micrographs (SEM) of the microcapsules. All the samples observed
show a superficial shrinkage of the particles, similar to the illustrations described by other authors
(Yang & Shan, 2021). It has been reported that the use of optimal encapsulation conditions
produces particles with smooth surfaces. However, when changing the encapsulating materials, it is
necessary to modify the encapsulation conditions. The cellulose fiber used in encapsulation
processes produce rough surfaces (Meneguin et al., 2020) which would explain the roughness
present in the obtained particles.
Additionally, we believe that there is an incidence of temperature on surface roughness and particle
shrinkage as reported by Yang 2021, where three states associated with temperature are identified.
At the beginning of the drying process, in the water drop formed, there is a greater evaporation of
water from the drop compared to the wall material. At this point, it can be determined that the
pressure inside the drop increases, which generates an expansion in volume. In the next stage, the
drying and subsequent hardening of the wall material occurs, producing a cavity inside the particle.
In this way, we have a particle with low permeability but subjected to high temperatures, thus
allowing the internal pressure of the particle to be maintained. Finally, in the last stage of drying,
the temperature decreases causing a decrease in the internal pressure of the particle causing the
surface of the particle to contract and the morphology observed in the microparticles of this
research is obtained (Yang & Shan, 2021).
The microcapsules do not show cracks or fractures which is important for the protection and
retention of the encapsulated oil. However, when evaluating the internal morphology (Fig. 2D), a
correct wall formation is observed. Additionally, in the hollow part there is evidence of droplets
embedded in the matrix, this being typical of the spray-drying process (Ramos, Silveira Júnior, &
Prata, 2021). The wall thickness obtained is consistent at the drying temperatures used, exhibiting a
good thickness in the wall material (Ramos et al., 2021).

Figure 2 A) X 1000; B) X 2000; C) X 6000; D) Microcapsules Internal morphology

Particle size distribution of microcapsules is shown in Figure 3. A heterogeneous distribution can be
observed with a peak that represents a predominant size, but only indicated the presence of
micrometric particles and exhibited monomodal behaviour.

Figure 3 Microcapsule’s particle size distribution

The understanding of the internal structure of the microparticle as well as the dispersion of the
particles within the matrix of the wall material was analyzed by transmission electron microscopy
(TEM). Qualitative analysis of the microparticles was performed based on TEM micrographs
(Figure 4). The distribution of grape seed oil within the coating material, green polymers (WPI and
MCC), can be seen in figure 4. As it can be seen, the grapeseed oil (light gray color) was
concentrated in the centre of the particle, being surrounded by the wall material (dark gray color)
(Fernandes et al., 2021). These results are according with SEM results where the formed wall has a
good formation.

Figure 4 Microcapsules TEM micrograph

 3.5. Gas chromatography
Fatty acid profiles (Figure 5) of the grape seeds oil could be observed in Table 2. The results fall
within similar ranges to the literature reports (Beveridge, Girard, Kopp, & Drover, 2005). The fatty
acid compositions of the non-encapsulated and encapsulated oil were 16.5% and 21.6% saturated
fatty acids (SFAs); 25.67% and 25.47% monounsaturated fatty acids (MUFAs); 57.81% and
52.89% polyunsaturated fatty acids (PUFAs).

Figure 5 Fatty acid gas chromatographic profiles; left: grapeseed oil; right: grapeseed oil microcapsules

For both, non-encapsulated and encapsulated grape seed oil, among the observed SFAs, the
predominant fatty acid was palmitic acid (C16:0) followed by stearic acid. Low concentrations of
other SFAs were found as follow: C14:0, myristic acid; C17:0, margaric acid; C20:0, arachidic
acid; and C22:0, behenic acid.
Oleic acid (C18:1) was the primary MUFA with 25.37% and 25.08% for non-encapsulated and
encapsulated grape seed oil respectively. When performing the PUFA analysis, it was found that
linoleic acid (C18:2) was the main fatty acid, accounting for 51.64% and 47.30% of total PUFAs
for non-encapsulated and encapsulated grape seed oil respectively. These results are in agreement
with previous study on the fatty acid compositions of other grape seeds (Crews et al., 2006).
MUFAs and PUFAs fatty acids are the predominant fatty acids observed in the fatty acid profile of
grape seeds (Table 2). However, the minimum value of the PUFA/SFA ratio recommended is 0.45
(HMSO, 1994), which is lower than those found in this study. The highest PUFA/SFA ratio was
obtained from grape seed oil non encapsulated (3.50), whereas the lowest values were found for
grape seed oil encapsulated (2.44). MUFAs have been paid attention during past decades due to the
beneficial effects on cardiovascular heart disease (Kalogeropoulos, Andrikopoulos, & Hassapidou,
2004). Low-density lipoproteins cause the accumulation of plaque in the arteries or arteriosclerosis,
this problem can be reduced with a diet rich in MUFA. Since grapeseed oil is high in MUFAs, it
can be made a part of your daily diet.
Table 2 Fatty acid profiles of grape seed oil encapsulated and non-encapsulated

Nº Peak Fatty acid Grapeseed oil (%) Microcapsules-Grapeseed oil (%)

2 C6:0 0.10 0.00
 3 C8:0 0.10 0.08
4 C10:0 0.12 1.11
6 C12:0 0.11 0.30
8 C14:0 0.08 1.04
12 C16:0 11.11 13.41
14 C17:0 0.09 0.14
16 C18:0 3.77 4.58
22 C20:0 0.37 0.37
28 C22:0 0.46 0.51
35 C24:0 0.19 0.09
TOTAL SATURATED FAT 16.509 21.628
13 C16:1 0.09 0.19
18 C18:1n9c 25.37 25.08
24 C20:1n9 0.43 0.21
TOTAL MONOINSATURATED FAT 25.678 25.473
20 C18:2n6c 51.64 47.30
23 27C18:3n3 6.17 5.60
TOTAL POLYINSATURATED FAT 57.813 52.899
TOTAL FAT 100 100

3.6. Controlled release

In order to improve uniformity in the release of the microencapsulated grapeseed oil, and to reduce
particle aggregation, the buffer used was adjusted to 24% ethanol. Figure 6 indicates the release of
both the grapeseed oil-loaded microcapsules at pH 1 and 5.8. When comparing the release obtained
at both pHs, a better release is observed at pH 5.8. It was observed that during the first three hours
and at a pH of 5.8, the microparticles of WPI and MCC released 80% of the encapsulated grapeseed
oil. At pH 1, the release was around 20% for grapeseed oil. This might be attributed to the
interaction among cellulose fibres which tend to bond with each other at acidic pH. The previous
results suggest that the encapsulation system (WPI and MCC) is sensitive to pH, which has
applications in pharmacology and functional foods. In this case, the grapeseed oil released from the
microcapsules (WPI-MCC) can be released in greater quantity in the small intestine or in the blood
system (at pH 5.8-7.4) compared to the grapeseed oil that can be released in the stomach (at pH 1-
2). It is conceivable that much of the grapeseed oil will be absorbed into the blood system more
efficiently than when the oil is not encapsulated in the microparticles.
This result is in coherence with FTIR results where we found physic interactions among wall
materials and weak interactions with grapeseed oil. Additionally, the little release at pH 1 is
consistent with the cellulose reaction at low pH where cellulose tends to bind. While the cellulose,
at high pH, tends to open the net.
 Figure 6 Microcapsule´s-controlled release at pH 1 (left); pH 5.8 (right)

4. CONCLUSION
Matrices made by using whey protein and microcrystalline cellulose offer important possibilities for
microencapsulation of grapeseed oil with higher encapsulation efficiency of 85%. The
microcapsules produced by green polymers (WP:MCC of 3:1) have an optimum
microencapsulation process. FTIR microcapsules analysis shown that no new peaks were formed
which is evidence of physic sorption and weak interactions among the employed encapsulant agents
and among grapeseed oil. Morphological analysis shown a good thickness of the wall of the
microcapsules confirming a correct encapsulation of grapeseed oil.
These microcapsules can be used to controlled release system based on pH. At low pHs release is
retarded while at high pHs speed up the release. In this sense, these microcapsules can be used to
release bioactive compounds in the small intestine where pH is above 5.8 and food remains there
for around 3 hours.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or
not-for-profit sectors.
CRediT authorship contribution statement

Diego Mauricio Sánchez-Osorno: Investigation, Formal analysis, Writing - original draft,

Writing - review & editing. Angie Vanesa Caicedo Paz: Investigation - review & editing,
Supervision. María Camila López-Jaramillo: Conceptualization, review & editing. Aída Luz
Villa: Conceptualization, Writing - review & editing . Julián Paul Martínez-Galán:
Conceptualization, Writing - review & editing, Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
Acknowledgment
We would like to acknowledge DYFOMECO research group, Universidad de Antioquia for their
help in providing MCC. We would like to acknowledge Minciencias-Colombia announcement 848
de 2019

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