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Designing multiple bioactives loaded emulsions

Cite this: DOI: 10.1039/d0fo00021c
for the formulations for diets of elderly
Milda Keršienė, Ina Jasutienė, Viktorija Eisinaitė, Petras Rimantas Venskutonis
and Daiva Leskauskaitė *

Received 3rd January 2020,
Accepted 15th February 2020
DOI: 10.1039/d0fo00021c
rsc.li/food-function
In this study, a stable double emulsion loaded with essential bioactives for the elderly was prepared using
a two-step mechanical emulsification process. Vitamins B6, B12, and C and anthocyanin-rich black cho-
keberry pomace extract were added in the inner water phase and vitamins A and D3 were added in the oil
phase of the double emulsion. The loaded emulsion showed excellent creaming stability (<96%), even dis-
tribution of droplet size, and high viscosity during 30 days of storage. The fair encapsulation efficiency
(75.00–99.37%) and the encapsulation stability (74.00–95.98%) of the bioactives during storage for
30 days indicate the successful protection of vitamins and anthocyanin-rich black chokeberry pomace
extract from environmental factors. For all vitamins, controlled release during digestion of double emul-
sion was observed. At the end of the duodenal phase, approximately 100% of the vitamins were released
from the loaded emulsions. However, the release kinetics of vitamins under gastric conditions differed:
71.75% of vitamin C, 37.5% of vitamin B9, 20% of vitamin B12, 70% of vitamin D3, and 50% of vitamin A
were released at the end of the gastric stage of digestion. 26.38% of the black chokeberry pomace extract
was released in the gastric fluids and 46.95% of the extract was found in the soluble part of the digesta at
the end of the duodenal phase. The study demonstrates that a multiple bioactives-loaded double emul-
sion can be successfully used for formulations for elderly people’s diets to deliver priority bioactives.

Introduction The fortification of food with vitamins and phenolic antiox-

Elderly consumers require a more nutrient-dense diet in order
to meet their nutritional needs because aging considerably
decreases energy demands. Several options for elderly diets
have been suggested, such as nutrient-dense foods, fortified
foods, and dietary supplements.1 During the fortification of
food, nutrients or non-nutrient bioactive compounds are
added to the products.2 Fortified food can become a successful
tool for managing the nutrient intake of the elderly.
The EURRECA Network of Excellence identified 10 priority
micronutrients for the elderly: vitamin D, folate, vitamin B12,
vitamin C, iron, calcium, zinc, selenium, iodine, and copper.3
The latest discoveries regarding the positive effects of dietary
polyphenols on age-related neurodegenerative diseases,4,5 also
show the importance of these natural molecules in the
elderly’s diets. Many berries and even by-products of their pro-
cessing are noteworthy sources of vitamins and polyphenolic
antioxidants. The biorefining processes of berry pomace have
been successfully employed for obtaining valuable products.6,7
Department of Food Science and Technology, Kaunas University of technology,
Radvilėnų pl. 19, 50254 Kaunas, Lithuania. E-mail: daiva.leskauskaite@ktu.lt;
Tel: +370 67523721
idants has several limitations because these bioactive com-
pounds may degrade and/or undergo undesirable changes
during processing and storage due to their high sensitivity to
heat, oxygen, pH, and other environmental conditions.
Another challenge when adding these bioactive compounds
into foods is related to their uniform distribution throughout
the product, because the amounts of added compounds are
usually very small. Currently, encapsulation is recognised as
the best strategy for incorporating bioactive compounds into
fortified food products.8 Water in oil in water (W1/O/W2)
double emulsion (DE) is one of the most attractive systems for
the encapsulation of various bioactive compounds. The advan-
tage of this system comes from its unique morphology, as it is
a general multifunctional carrier able to encapsulate different
hydrophilic and lipophilic molecules in the same particle.9
The high viscosity of the system is another advantage of the
DE. Following National Dysphagia Diet classification of dys-
phagia fluids DE correspond to the III level (honey), therefore
they can be successfully used in foods for elderly people with
dysphagia, a disorder of the swallowing mechanism.10,11
In recent years, double emulsions have been widely studied
as carriers for bioactive compounds. Gharehbeglou et al.12
encapsulated oleuropein within double nano-emulsions stabil-
ized by pectin-whey protein concentrate complexes. Double

This journal is © The Royal Society of Chemistry 2020 Food Funct.


Paper
emulsions with resveratrol ethanol/water solution entrapped in
the inner water phase were characterised by high encapsula-
tion efficiency, good stability, and appropriate rheological
behaviour.13 Additionally, Aditya et al.14 developed a W1/O/W2
emulsion for simultaneously encapsulating hydrophilic cate-
chin and hydrophobic curcumin. Double emulsions have also
been reported to be good matrixes for the encapsulation of
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vitamin C15 and vitamin B12.16 In our previous research,
stable food-grade double emulsions with encapsulated beet-
root juice were prepared, with high encapsulation efficiency
(>98%) of the colourant betalain using milk proteins and poly-
glycerol polyricinoleate.17
When looking for the suitable encapsulation vehicles, the
most important parameter of the system is the protection of
the encapsulated bioactive compound until it reaches the tar-
geted physiological sites. To our knowledge, there are few
studies reporting double emulsions as providing effective pro-
tection for vitamins under gastrointestinal conditions. The
encapsulation of vitamin B12 in double emulsions exhibited
greater than 96% efficiency and prevented vitamin losses
during in vitro gastric digestion.18 In another study, Andrade
et al.19 incorporated hydrophobic vitamin D3 and hydrophilic
vitamin B12 molecules into DEs and demonstrated that the
physical properties of the internal phase of the DEs influenced
lipid digestion and the kinetics of vitamins transfer during
in vitro digestion.
Furthermore, in vitro digestion studies of DEs uploaded
with polyphenol-rich extracts suggest that the release of encap-
sulated compounds depends on the composition of the DE,
the method of double emulsion preparation and the nature of
the encapsulated compounds. Huang and Zhou20 studied
anthocyanin-rich Des and found that the double emulsion
could control the release of anthocyanin-rich black rice extract
in the stomach with the total release of anthocyanin within
the first 20 min of intestinal digestion. The double emulsion
with entrapped beet extract reported by Kaimainen et al.21
showed a gradual release of bioactive compounds during
gastric digestion.
The aim of this study was to formulate a stable bioactives-
loaded double emulsion for the fortification of the elderly’s
food. Essential bioactives for the elderly were chosen for
encapsulation in different phases of the double emulsion. The
internal water phase was loaded by hydrophilic vitamin B6,
vitamin B12, vitamin C, and anthocyanin-rich black choke-
berry pomace extract (BCPE). The oil phase was loaded by lipo-
philic vitamin A and vitamin D3. Milk proteins and polygly-
cerol polyricinoleate were used as hydrophilic and lipophilic
emulsifiers, respectively. The overall stability of the micronutri-
ents-loaded emulsion was characterised during storage for 30
days. The kinetics of the release of bioactive molecules during
the in vitro digestion of the double emulsion were also exam-
ined. Simulated gastro-intestinal digestion is widely employed
in many fields of food and nutritional sciences, as conducting
human trials are often costly, resource intensive, and ethically
disputable.22 In vitro alternatives are successfully used for
building new hypotheses before conducting in vivo studies.
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Therefore, in this study, the kinetics of proteolysis was ana-
lysed during the in vitro gastric and intestinal digestion of the
protein formulations. Characterisation of bioactives release
from loaded double emulsions during in vitro digestion might
be important when designing food products for older consu-
mers. To our knowledge, this is the first attempt to formulate
a stable multiple bioactives-loaded double emulsion to simul-
taneously deliver priority bioactives for the elderly.
Materials and methods
Chemicals and materials
Milk protein concentrate (My Protein, Northwick, UK) contain-
ing 80.0% protein, 1.5% fat, and 5.0% carbohydrates was used
as a water-soluble emulsifier. Polyglycerol polyricinoleate
(PGPR) was used as a lipophilic emulsifier. Refined deodorized
rapeseed oil ‘Tyras’ (Obelių Aliejus, Lithuania) was used for
the oil phase. Sodium chloride (>99.7%) was purchased from
Eurochemicals (Vilnius, Lithuania).
Fresh black chokeberry pomace, after pressing, was freeze-
dried, ground, and defatted by supercritical carbon dioxide in
a pilot-scale system (Applied Separation, Allentown, USA).
Black chokeberry pomace extract (BCPE) was recovered from
the residue by extracting with ethanol (EtOH) pressurized to
10.3 MPa in an Accelerated Solvent Extractor ASE 350 (Dionex,
Sunnyvale, CA, USA). For this purpose, the residue was mixed
with diatomaceous earth in 10 mL extraction cells, which were
fixed with cellulose filters at the top and at the bottom. EtOH
was evaporated in a rotary evaporator and the obtained extracts
were stored at −20 °C. PLE-EtOH parameters were selected
based on previous studies.23,24
Vitamin A palmitate 1 MIU g−1 oily liquid, vitamin D3 1
MIU g−1 oily liquid, folic acid powder of 99.9% purity, and
vitamin B12 0.1% powder were obtained from DSM Nutritional
Products Ltd (Basel, Switzerland). Vitamin C powder of 99.9%
purity was obtained from the Myprotein Co. (The Hut Group,
Cheshire, UK). HPLC-grade solvents were used for analysis.
Acetonitrile (ACN) and trifluoroacetic acid (TFA) were obtained
from Sigma-Aldrich (St Louis, USA) and n-hexane from Avantor
Performance Materials (Gliwice, Poland).
Preparation of DE
Two types of double emulsions were prepared. Empty DE was
made without BCPE extract and vitamins. Loaded DE was
made with BCPE and water-soluble vitamins in the inner water
phase and fat-soluble vitamins in the oil phase. For that
purpose, two types of inner water phases (W1) were produced:
0.5 wt% of sodium chloride was dissolved in distilled water
(for empty DE); 0.5 wt% of sodium chloride, 35 g per 100 g of
BCPE, 27 mg per 100 g folic acid, 16 650 mg per 100 g vitamin
B12, and 2670 mg per 100 g vitamin C were dissolved in dis-
tilled water (for loaded DE). For the oil phase (O), 3 wt% of
PGPR was dispersed in the rapeseed oil (empty DE).
Additionally, fat-soluble vitamins (7 mg per 100 g of vitamin D
and 12 mg per 100 g of vitamin A) added during the oil phase
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for the loaded DE preparation. In the oil phase, the emulsion ðCin  Cf day x Þ
ES ¼ 100% ð2Þ
was heated and mixed at 50 °C for 30 minutes, then cooled to Cin
room temperature. For the outer water phase (W2), 14 wt% of
where Cin is the amount of particular compound, initially
milk protein solution (completely dissolved) was used.
added to the emulsion, Cf day 0 is the amount of particular
For the primary emulsion preparation, 20 wt% of the
compound, found in supernatant after preparation, Cf day x
inner water phase was mixed with 80 wt% of the oil phase and
is the amount of particular compound, found in supernatant
homogenized with Ultra-Turrax (IKA® T-18 basic, Staufen,
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after storage at 4 °C in the in the fridge for 14 and 30 days.

Germany) for 8 min at 15 000 rpm. Later, primary (W/O) emul-
The amount of oil soluble vitamins was measured after
sion (30 wt%) was dropped in the outer water phase (70 wt%)
full enzymatic hydrolysis of the DE. For that purpose, 5 g of
and homogenized at a lower speed of 11 000 rpm for 5 min.
the DE was mixed with 5 mL 0.004% pepsin solution in
water, 200 µL 6 M HCl, and 10 mL of water and kept at 37 °C
for 2 h in a water bath. pH was adjusted to 2.7. Then, 15 mL
Characterisation of DE of lipase (0.82 g in 30 mL) and pancreatin solution (0.3 g in
Creaming stability. DE were poured into glass tubes (25 ml) 30 mL) was added, pH was corrected to 6.6 with 800 µL 2 N
and stored for 30 days at a temperature of 4 °C. Phase separ- NaOH, and samples have been kept at 37 °C for 3 h in the
ation was monitored during all periods of storage and cream- water bath.
ing stability was expressed as the ratio of the separated phase EE and ES for oil-soluble vitamins were calculated using the
volume to the initial volume of the DE sample. following equations:
Droplet size and distribution. The average oil droplet size
Cf day 0
and distribution were measured by static light scattering in a EE ¼ 100% ð3Þ
Cin
Malvern Mastersizer 2000 (Malvern Instruments, UK) with the
following parameters: 1.45 – refractive index of droplets; 1.33 – Cf day x
ES ¼ 100% ð4Þ
refractive index of water; 0.01 – absorption. The results were Cin
expressed as the average of the volume-weighted mean globule
where Cin is the amount of particular compound, initially
size (d[4,3]). All measurements were replicated at least three
added to the emulsion, Cf day 0 is the amount of particular e
times.
compound recovered from the oil phase after preparation, Cf
Rheological characteristics. Rheological characteristics were
day x is the amount of particular compound recovered from the
measured at 25 °C with a Physica MCR 502 rheometer (Anton
oil phase after storage at 4 °C in the in the fridge for 14 and 30
Paar, Messtechnik, Stuttgart, Germany) equipped with a CF41
days.
Cryo-Compact Circulator (Julabo, Ziedbach, Germany). The
Determination of anthocyanins by UPLC-MS. Quantitative
plate-plate system (20 mm diameter and 1 mm gap) was used.
analyses of anthocyanins were carried out on a Waters
The shear rate was increased from 0.1 to 500 s−1. Data were
AQCUITY ultra-performance liquid chromatography system
analyzed using the Herschel–Bulkley model in order to obtain
equipped with a Waters TQ-S triple quadrupole mass detector
the viscosity factor (K), yielding stress (τ0), and flow behaviour
(Waters Corp., Milford, MA, USA). The equipment consisted of
index (n). All measurements were replicated at least three
a quaternary solvent manager, sample manager, and column
times.
heater, interfaced with a mass spectrometer equipped with an
Encapsulation efficiency and stability. The encapsulation
ESI source operating in the positive mode. Instrument control
efficiency (EE) was calculated as the ratio of particular com-
and data processing were performed using MassLynx™ soft-
pound (Cy-3-galactoside or water soluble vitamins) amount
ware. Separations were performed on an Acquity BEH, C18
entrapped in the inner water phase (W1) and the amount of
column (2.1 × 100 mm, particle size 1.7 μm) (Waters Corp.,
particular compound initially added to the emulsion.21,25
Ireland) and the autosampler and analytical column oven were
Encapsulation stability (ES) was calculated as the ratio of par-
maintained at 10 °C and 40 °C, respectively. The mobile phase
ticular compound amount which remained entrapped in the
involved A – 1% formic acid in ultra-pure water – and B – 100%
inner water phase (W1) during storage to its amount initially
acetonitrile. The gradient was held at 3% B, raised to 25% B
added into the emulsion. For the extraction of incorporated
over 7 minutes, and then increased to 100% B for 8 minutes
compounds, 9 g of the emulsion was weighted into a centrifu-
and returned to initial conditions over the next 3 minutes. The
gal tube together with 36 g of NaCl solution (5.84 g L−1) and
column was equilibrated for 3 minutes before the next ana-
the mixture was centrifuged at 3000 rpm for 30 min using the
lysis. The flow rate was 0.4 mL min−1 and the injection volume
Sorvall Legend XFR (Thermo Scientific, Vanta, Finland) centri-
was 2 μL. Mass spectrometric data of the eluted compounds
fuge. The lower aqueous phase was collected and treated with
from the column were acquired in a single run. Nitrogen was
Carrez clarification reagents, first filtered through a cotton
used as both drying and nebulizing gas and the flow con-
filter, then through a 0.45 µm pore size syringe filter. EE and
ditions were 1000 L h−1 and 150 L h−1 for desolvation and at
ES were calculated using the following equations:
the cone, respectively. The desolvation temperature was set at
ðCin  Cf day 0 Þ 500 °C. Capillary and cone voltages were 1.5 kV and 20 eV,
EE ¼ 100% ð1Þ
Cin respectively. Quantification was performed using the external

This journal is © The Royal Society of Chemistry 2020 Food Funct.


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standard (cyanidin-3-galactoside). A standard stock solution
was prepared in methanol and subsequently diluted to
working concentrations.
Determination of water soluble vitamins B6, B12 and C.
A Shimadzu Prominence series (Shimadzu Corp., Kyoto
Japan) HPLC system with an Atlantis dC 18 5 μm
4.6 × 150 mm column (Waters, Ireland) was used for the sep-
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aration and quantification of vitamins. The mobile phase
involved A – 0.1% TFA in H2O and B – 0.1% TFA in ACN.
The time program was B 0% → 3% (0.0–5.0 min) → 15%
(6.0 min) → 20% (10.0 min) → 100% (12.0 min) → 100%
(25.0 min). The flow rate was 1.4 mL min−1, the column
temperature was 30 °C, and the injection volume was 20 μL.
Vitamin C was recorded at 210 nm and vitamins B9 and B12
at 280 nm using an SPD-M20A diode array detector
(Shimadzu Corp., Kyoto Japan). Quantification of vitamin
content was done using the calibration curves of standard
solutions.
Determination of fat soluble vitamins A and D3. After enzy-
matic hydrolysis, the samples were clarified with Carrez
reagents and filtered through a cotton filter. Then, 20 mL of
the sample solution was poured into the Chromabond XTR
(70 mL 14.5 g) column (Macherey-Nagel, GmbH & Co.,
Düren, Germany) and allowed to absorb for 15 min. No
washing was performed. The vitamins were eluted using
100 mL of n-hexane containing 5 mg BHT (2,6-di-tert-butyl-p-
cresol). The eluates were evaporated to dryness using a
vacuum evaporator (IKA RV 10 Rotary Evaporator, Germany).
Before HPLC analysis, the dry residue was dissolved in 1 mL
of the mobile phase ACN : H2O (100 : 5 v/v) and filtered
through a 0.45 µm pore size syringe filter. A Shimadzu
Prominence series (Shimadzu Corp., Kyoto Japan) HPLC
system with a Nucleodur C18 Isis 5 μm 2.0 × 125 mm
column (Macherey-Nagel, GmbH & Co. Germany) was used
for the separation and quantification of fat-soluble vitamins.
This involved mobile phase ACN : H2O (100 : 5 v/v), a flow rate
of 0.2 mL min−1, a column temperature of 25 °C, and an
injection volume of 20 μL. Vitamin D was recorded at
275 nm and vitamin A at 325 nm using an SPD-20A UV/VIS
detector (Shimadzu Corp., Kyoto Japan). The calibration
curves of retinyl palmitate (Sigma Aldrich, Switzerland) and
cholecalciferol (Sigma Aldrich, Poland) were used to quantify
the vitamins.
Characterisation of DE degradation and bioactives release
during digestion
In vitro digestion model. The simulated gastrointestinal
digestion model operated under adult conditions.26 Digestion
juices were prepared as previously described.22 5.0 ± 0.05 g of
the DE was weighted into the beaker with 2.0 ± 0.05 g of glass
beads (3 mm diameter) to improve mixing. Simulated saliva
fluids (SSF) without amylase were added to a final volume of
10 mL of the sample to initiate the oral step, which lasted
2 min. A temperature-controlled water bath with continuous
shaking at 100 rpm (GFL 1092, Thermolab®, Germany) was
used.
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Then, the gastric step was initiated by adding simulated
gastric fluids (SGF) containing pepsin (2000 U mL−1 of
digest) and the final digest volume was adjusted up to 20 mL.
After 2 h of incubation, the final intestinal step was initiated
by adding simulated intestinal fluids (SIF), supplemented
with pancreatin (100 U mL−1 of digest), lipase (2000 U ml−1
of digest), and bile salts (10 mM of digest). The final
volume of the sample was 40 mL. The target gastric pH
was between 2 and 3 and the duodenal pH was between 6.5
and 7.
To obtain a digestion profile, seven beakers were used and
their contents were analysed at different times of digestion.
The digestion was stopped after 5, 60, and 120 min during the
gastric stage (G5, G60, and G120) and 125, 180, 210, and
240 min during the intestinal stage (D5, D60, D90, and D120).
Gastric samples were neutralized to a pH of 7.0 ± 0.1 and the
digestion process of intestinal samples was stopped by cooling
the sample to 0–4 °C in ice water. After the reaction was
stopped, samples were centrifuged at 4000 rpm at +4 °C (MPW
Med. Instruments MPW-260RH) and filtered. The soluble frac-
tion of the digest was collected, frozen, and stored at −18 °C
until analyses.
Degradation of DE during in vitro digestion was character-
ised by morphology, particle size, degree of proteolysis and
degree of lipolysis. The amount of anthocyanins, vitamins A,
D, B12, B9 and C was determined in the digesta obtained after
each step of digestion. Digestion procedure was performed
twice.
Microscopy. Polarized light microscopy was used to
examine the morphology of the DE during digestion. Drops
of digesta obtained at different times of digestion were
placed on a microscope slide and gently covered with a cover-
slip. The microstructure of the DE was observed using an
Axio Scope.A1 (Carl Zeiss Microscopy GmbH, Germany)
microscope with an AxioCam MRc camera (Carl Zeiss
Microscopy GmbH, Germany). The images were recorded at a
magnification of ×100.
Proteolysis degree. The N-terminal amines were determined
as described by Larsen et al.27 In brief, digesta obtained at
different times of digestion were mixed 1 : 1 with 24% TCA
and kept on ice for at least 30 min, followed by centrifugation
at 14 000g at 4 °C for 20 min. The obtained supernatants were
used for the fluorescamine analysis of primary amines.28 For
that, the sample (30 µL) with TCA was mixed with 900 µL of
0.1 M sodium tetraborate buffer ( pH 8.0), followed by the
addition of 300 µL of 0.2 mg mL−1 fluorescamine in dry
acetone. Fluorescence was measured on a FLUOstar Omega
microplate reader (BMG LABTECH, Germany) using exci-
tation and emission wavelengths of 390 and 480 nm, respec-
tively. Quantification was achieved by calculating leucine
equivalents using an external leucine standard curve. The
degree of proteolysis was calculated using the following
equation:
h
DHprotein ¼ 100% ð5Þ
htot
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where h is the amount of N-terminal amines at each time
point of in vitro digestion; htot is the total amount of
N-terminal amines, determined after full hydrolysis with HCL
Lipolysis degree. A pH-stat automatic titration unit
(Metrohm, USA Inc.) was used to monitor the pH and maintain
it at pH 7.0 by titrating appropriate concentrations of NaOH
solution to neutralize any free fatty acids (FFA) formed. The
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Results and discussion
Characteristics of BCPE
After the evaporation of ethanol, the BCPE was a resinous
dark-coloured substance. It contained 82.7 ± 0.1 mg g−1
of polyphenolics, which were measured using Folin–
Ciocalteu’s reagent and expressed in gallic acid equivalents:

volume of NaOH (0.25 M NaOH) added to the samples was
recorded and used to calculate the concentration of free fatty
acids generated by lipolysis. The lipolysis degree was expressed
as the percentage of free fatty acids released. This was calculated
from the number of moles of NaOH required to neutralize the
FFA divided by the number of moles of FFA that could be pro-
duced from the triacylglycerols if they were all digested (assum-
ing 2 FFA produced per 1 triacylglycerol molecule):29
316.6 ± 7.0 mg g−1 procyanidins, determined spectrophoto-
metrically; 43.41 ± 1.38 mg g−1 cyanidin-3-O-galactoside;
3.53 ± 0.08 mg g−1 cyanidin-3-O-arabinoside; 3.42 ± 0.01 mg g−1
cyanidin-3-O-glycoside; and 1.92 ± 0.01 mg g−1 cyanidin-3-O-
xyloside quantified by UPLC. The DPPH radical and ABTS cation
radical scavenging capacity of BCPE were 198 ± 0.01 and
249 ± 1.4 mg Trolox equivalents per g, respectively. Consequently,
the BCPE may be considered rich in bioactive antioxidants.

V ðNaOHÞ  NðNaOHÞ  MðlipidÞ

DHlipid ¼ 100
αðFFAÞ  mðlipidÞ  2
where V(NaOH) is the volume (mL) of NaOH consumed during
the titration; N(NaOH) is the NaOH normality (eq per L);
M(lipid) is the molecular weight (g mol−1) of the triglycerides
in the oil (728 g mol−1 in this study); α(FFA) is the mean
degree of dissociation of FFA carboxylic groups (α = 0.54);
m(lipid) is the oil mass (g).
Statistical analysis
The results were expressed as calculated means and standard
deviations. Statistical analysis was carried out using ANOVA at
p < 0.05.
ð6Þ Encapsulation stability and physicochemical changes of DE
during storage
The structure of the DE is very complex and easily destroyed
during the manufacturing and storage processes. It is clear
that the successful encapsulation of bioactive compounds is
possible only in a stable DE. In this study, the stability of the
DE was evaluated by creaming stability, droplet size, and the
rheological characteristics of the DE during storage for 30
days. Data are presented in Table 1. It is obvious that the
creaming stabilities of the empty and loaded DEs were very
high. For both emulsions, the first signs of instability were
observed 30 days after their preparation, although high cream-
ing stability values were recorded. For the loaded emulsion,

Table 1 Physical properties of DE during storage

Storage time, days

Sample 1 3 7 14 30

Creaming stability, %
Empty DE 100 ± 0.00aB 100 ± 0.00aB 100 ± 0.00aB 100 ± 0.00aB 99.2 ± 0.03bA
Loaded DE 100 ± 0.00aB 100 ± 0.00aB 100 ± 0.00aB 100 ± 0.00aB 96.0 ± 0.05aA
κ, Pa s n

Empty DE 3.04 ± 0.21aD 2.21 ± 0.15aC 1.83 ± 0.48aC 1.17 ± 0.00aB 0.45 ± 0.01aA
Loaded DE 12.70 ± 0.55bB 13.92 ± 0.14bB 16.21 ± 0.74bC 10.53 ± 0.73bA 10.12 ± 1.07bA
n
Empty DE 0.58 ± 0.09aA 0.61 ± 0.01bA 0.59 ± 0.01bA 0.62 ± 0.07bA 0.76 ± 0.01bB
Loaded DE 0.50 ± 0.05aBC 0.53 ± 0.03aC 0.47 ± 0.01aBC 0.48 ± 0.00aBC 0.41 ± 0.02aA
τ0, Pa
Empty DE 4.25 ± 0.12aE 1.54 ± 0.18aD 0.86 ± 0.06aC 0.33 ± 0.04aB 0.01 ± 0.00aA
Loaded DE 52.09 ± 2.11bD 40.95 ± 3.56bC 21.45 ± 2.54bB 8.41 ± 0.32bA 6.06 ± 0.0 bA
R2
Empty DE 0.993 0.999 0.999 0.999 0.998
Loaded DE 0.939 0.958 0.988 0.999 0.981
Apparent viscosity at shear rate 50 s−1, Pa s
Empty DE 0.629 ± 0.013aD 0.585 ± 0.002aC 0.527 ± 0.009aD 0.333 ± 0.001aB 0.120 ± 0.002aA
Loaded DE 3.350 ± 0.156bE 2.875 ± 0.064bD 2.655 ± 0.035bC 1.615 ± 0.106bB 1.160 ± 0.000bA
Droplet size (d43), µm
Empty DE 61.66 ± 0.93bAB 65.80 ± 2.31bB 62.10 ± 1.94bAB 64.11 ± 2.16bAB —
Loaded DE 37.65 ± 0.77aC 31.02 ± 1.03aA 31.34 ± 1.03aA 32.53 ± 0.59aB —
Span
Empty DE 2.588 2.575 2.909 3.827 —
Loaded DE 4.650 4.046 3.674 4.565 —

Values are reported as means ± standard deviation; lower case letters indicate significant (p < 0.05) differences in characteristics between empty
and loaded double emulsions and upper case letters indicate significant (p < 0.05) differences in double emulsion characteristics during storage.

This journal is © The Royal Society of Chemistry 2020 Food Funct.


Paper
this was 96.0% and for the empty DE, it was 99.2%.
Immediately after the preparation of the DEs, the volume-
weighted mean diameter (d43) of the empty DE was
61.66 ± 0.93 µm. Upon loading bioactive compounds into the
W1 and O phases of the DE, the size of the droplets reduced
and became 37.65 ± 0.77 µm. Other researchers have recorded
the reduction of droplet size in the DEs after encapsulation
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when the DEs were loaded with simultaneously hydrophilic
catechin and hydrophobic curcumin.15 This phenomenon was
explained by the surface activity of these polyphenols.
According to Akhtar et al.30 flavonoids can decrease the
droplet size in DEs due to their impact on the decrease of
interfacial tension at the oil–water interface. Zembyla et al.31
reported complex formation at the interface between polyphe-
nol particles adsorbing from the oil side and whey proteins co-
adsorbing from the aqueous side of the interface, which
strengthens the mechanical properties of the adsorbed film.
Such an interpretation may also be appropriate for our DEs
loaded with anthocyanin-rich BCPE extract. A slight increase
in droplet size for the empty DE and a slight decrease for the
loaded DE were recorded during storage. Striking differences
between the viscosities of the DEs could explain such
behaviour.
The Herschel–Bulkley equation was adjusted to the rheolo-
gical behaviour of the studied DEs because this model fitted
well to the shear stress and shear rate data (R2 ≥ 0.98). The
rheological parameters presented in Table 1 reveal that all DEs
typically yield pseudoplastic fluids with τ0 > 1 and n < 1. The
highest viscosity factor and the highest yielding stress were cal-
culated for the loaded DE immediately after preparation. The
empty DE showed the smallest K and τ0 values and the largest
n values, indicating lower pseudoplasticity. Such differences
could be due to the presence of considerable amounts of
pectin in BCPE loaded in the inner water phase of the emul-
sion. However, during storage, the viscosity factor and the
yield stress of both emulsions significantly ( p > 0.05)
decreased. The simultaneous transportation of dissolved mole-
cules and water from W1 to W2 through the oil layer32 can
explain such behavior of DE. The changes in droplet size of
DEs during storage can also be considered an indicator of
such a phenomenon. However, even when the emulsions’ visc-
osities decreased during storage, no water phase release or oil
phase creaming occurred.
Encapsulation efficiency of bioactive compounds during
storage of DE
Obtaining the therapeutic benefits of the DE loaded with bio-
active compounds requires the high entrapment of these com-
pounds. In literature, terms such as “yield”, “encapsulation
efficiency”, “incorporation efficiency”, and “loading efficiency”
have often been used interchangeably for emulsion-based
encapsulation systems.33 In this study, the initial encapsula-
tion efficiency (EE) and encapsulation stability (ES) of freshly
prepared DE after 14 and 30 days of storage at 4 °C were used
as an indices of the stability and efficiency of DEs as an encap-
sulation system for bioactive compounds.
Food Funct.
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Food & Function
Table 2 Encapsulation efficiency (EE) and encapsulation stability (ES) of
loaded DE
Encapsulation stability, %
Encapsulation After 14 days After 30 days
Item efficiency, % of storage of storage
BCPE (in terms of 96.56 ± 0.04a 96.22 ± 0.14b 95.98 ± 0.41b
cy-3-galactoside)
Vitamin C 76.63 ± 0.79a 75.63 ± 0.83ab 74.00 ± 0.81b
Vitamin B9 75.00 ± 0.88a 50.00 ± 0.85b 45.00 ± 0.87c
Vitamin B12 84.00 ± 2.12a 83.60 ± 1.98a 78.00 ± 2.08b
Vitamin A 99.37 ± 5.93a 93.75 ± 6.19a 90.00 ± 7.32a
Vitamin D3 98.52 ± 10.43a 69.57 ± 12.30b 78.26 ± 11.90b
Values are mean ± SD of three independent samples, lower case letters
indicate significant (p < 0.05) differences in double emulsion
characteristics during storage.
For BCPE, high EE value (96.56%) was recorded in freshly
prepared DE. The ES for the study period was also high and
ranged from 96.22% after 14 days of storage of the DE to
95.98% after 30 days of storage (Table 2). Similarly, Velderrain-
Rodríguez et al.34 found high EE of phenolic-rich extract from
mango peel in the DE made with PGPR and Tween 20
or lecithin as surfactants; this was 98.65 ± 1.14% and
96.11 ± 1.37% for freshly prepared DE with Tween 20 and
lecithin, respectively, but the DE with Tween 80 showed better
ES during storage. There are also studies affirming the ability
of DEs with macromolecular emulsifiers to retain the encapsu-
lated polyphenols during the emulsification process and
storage. Bamba et al.35 reported DEs stabilized by whey pro-
teins as a promising system for coencapsulating total pheno-
lics and anthocyanins from blueberry pomace with EE above
80%. Estévez et al.36 encapsulated polyphenols from grape
seed extract in DEs stabilised by electrostatic complexes made
of sodium caseinate and different polysaccharides and
reported that electrostatic complexes were efficient stabilizers
of DEs entrapping procyanidins. Pasrija et al.37 reported the
EE of green tea polyphenols at 60–76% and 40–70% depending
on encapsulation methods (freeze drying and spray drying).
Despite the wide range of reported EE of polyphenol-rich
extracts in DE, scientists suggest that the physical character-
istics of the DE seem to be crucial for the ability of the DE to
retain phenolics, anthocyanins, procyanidins, and other com-
pounds from the extracts.
In our study, the EE values of water-soluble vitamins in DE
were lower than those of BCPE. Although the initial EE values
for the water-soluble vitamins were quite similar, between 76
and 84%, trends of ES versus time differed. For vitamin C,
there were no significant changes in ES throughout the storage
period. A slight decrease in ES was recorded for vitamin B12
after 30 days of DE storage. The concentration of encapsulated
vitamin B9 showed a significant reduction after 14 days of DE
storage, from 75 to 50%, and a moderate reduction after 30
days of storage to 45%.
We couldn’t find much published data about the EE of
water-soluble vitamins in DEs. The successful encapsulation of
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Food & Function
folic acid in spray-dried DE was reported by Assadpour et al.38
DE formulations in this study contained an internal nano/
micro-emulsion composed of a water-in-oil (W/O) system with
folic acid present in the water phase, re-emulsified within an
aqueous phase of pectin-whey protein complexes. The average
EE of folic acid was approximately 88.3% and depended on
whey proteins and dispersed phase content. Kheynoor et al.39
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observed changes in the entrapment efficiency of vitamin C as
a function of time in the DE made using PGPR, Tween 80, and
natural sweetener as emulsifiers. They showed that the EE of
vitamin C decreased from 86.52 to 70.02% after 30 days, which
is very close to the results of our study.
Monodisperse food-grade DE containing vitamin B12 in the
inner water phase was prepared with high encapsulation
efficiencies (90–94%) using sodium carboxymethylcellulose
and Tween 20 as stabilizers in a two-step process involving
mechanical agitation and membrane emulsification.16 Similar
emulsification efficiency was reported by Fechner et al.,40 who
encapsulated vitamin B12 in DE stabilized by PGPR and
sodium casein-dextran conjugates. Such a variety of data on
the ability of DEs to retain water-soluble vitamins suggests that
encapsulation efficiency depends on the method of DE prepa-
ration and the nature of the emulsifiers and stabilizers used in
the composition of the DE.
In our study, the incorporation of vitamins A and D3 in the
oil phase of the DEs gave them significant protection during
storage. The EE of vitamin D3 in the freshly prepared DE
showed the highest value, 98.52%, even though the ES of
vitamin D in the DE rapidly decayed during storage at 4 °C.
When bioactive compounds are added into the emulsions,
their release can be affected by their interaction with the inter-
face. Andrade and Corredig41 showed that DE containing
PGPR and β-lactoglobulin or sodium caseinate at interfaces
resulted in better stability over the storage period when 0.05%
(w/w) of vitamin D3 or phytosterol were added in the oil phase.
The authors suggested that phytosterols and vitamin D3 may
interact with the emulsifiers at the interface, affecting the
physico-chemical properties of the DE and possibly the release
of vitamin D3. In our study, there is also the possibility of
interaction between the PGPR present in the O/W1 interface
and vitamin D3 in the O phase, which can enhance the entrap-
ment efficiency of the emulsion.
The encapsulation efficiency of vitamin A was also high
(99.37%) and only a slight decrease in ES was recorded during
all periods of storage. At previous attempt at the microencap-
sulation of vitamin A into emulsion systems showed the
importance of the oil used in preparing the emulsion in order
to have oxidative stable core material.42 The chemical stability
of encapsulated vitamin A was successfully improved by the
presence of vitamin E and other antioxidants.43 In our study,
the high stability of vitamin A could have been driven by the
presence of polyphenolic antioxidants from BCPE, as well as
vitamin C in the inner water phase of the DEs. In our previous
study, we proved the ability of BCPE to inhibit lipid oxidation
during storage when extract is added in the inner water phase
of the DE. We presume that this behaviour of BCPE explains
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the lack of changes in the encapsulation stability of vitamin A
after 30 days of storage.
Degradation of DE during in vitro digestion
First, the physical degradation of the empty and loaded DEs
was recorded by measuring the particle size distribution and
observing the microstructure at different stages of digestion
(Fig. 1). The empty and loaded DEs showed the same pattern
of particle size distribution in the gastrointestinal environ-
ment. As already mentioned in the first part of this study, a
monomodal distribution of particle size was observed for both
fresh and undigested emulsions, with slightly smaller particles
for the loaded DE. When DEs were incubated with gastric
fluids for 120 min (G120), the monomodal distribution of par-
ticle size reverted to a binomial distribution for both DEs; still,
the values of average particle size (d43 and d32) were very
similar to those of the DEs before digestion. No significant
differences were recorded between the average size of oil dro-
plets (d43) in the empty and loaded DEs. For the empty DE,
this was 54.86 ± 19.74 µm and for the loaded DE, this was
51.28 ± 8.63 µm. Changes in the particle size distribution of
the DEs were noticed at the very beginning of the duodenal
phase of digestion (D5). The addition of duodenal fluids con-
taining surface active substances, such as lipase, phospholi-
pids, and bile salts, induced a wider distribution of droplet
sizes. In both DEs, a great increase in d43 was registered at the
beginning of the duodenal stage, which seemed to further
increase in the duodenal environment. At the end of the duo-
denal phase, both DEs showed a significant population of
larger droplets, with an average size (d43) of 146.77 ± 51.51 µm
for the empty DE and 154.86 ± 19.74 µm for the loaded DE. We
presume that these results indicate the aggregation of oil dro-
plets during the duodenal stage of digestion.
Our results on the aggregation of droplets as the DEs pass
through the simulated duodenal stage were somewhat similar
to those reported by other authors.16,44 There are studies on
O/W emulsions showing an increase in droplet size in gastroin-
testinal conditions due to the aggregation of oil droplets.45,46
Moreover, there are data that the extent of oil droplets aggrega-
tion is higher in O/W emulsions with milk proteins as an
emulsifier than those made with lecithin.47
Light microscopy was used to observe changes in the physi-
cal appearance of the loaded DE during digestion. The results
are presented in Fig. 1C. The appearance of the DE before
digestion (0) confirmed the morphology of the DE with the
internal water phase surrounded by oil droplets that were dis-
persed in the outer water phase. The DE droplets were still
observed at the end of the gastric stage of digestion (G120).
Single oil droplets with bigger inner water droplets could be
seen up to the end of the duodenal phase of digestion (D120).
It is possible that the coalescence of the inner water phase
could influence the release of bioactive compounds present in
this phase.
A timed study of protein and lipid hydrolysis during in vitro
digestion was conducted to understand the effect of the degra-
dation process of DEs on the release of hydrophilic and hydro-
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Fig. 1 Particle size distribution of empty DE (A) and loaded DE (B) and microstructure of loaded DE (C) as function of time during gastric (G) and
intestinal (D) in vitro digestion: 0 – before digestion; G120 – gastric phase at 120 min of digestion, D5 – duodenal phase at 5 min and D120 – duo-
denal phase at 120 min of digestion.

phobic bioactive compounds from the different phases of the
DEs. The results are presented in Table 3.
Again, as in the study of changes in the physical properties
of the DEs during digestion, no significant differences
between the proteolysis degree of empty and loaded DE were
recorded. The result of the protein hydrolysis in the DEs
showed that the degradation of proteins took place immedi-
ately after adding gastric fluids into the DEs (G5) and gradually
increased during incubation under gastric conditions for
120 min. At the end of the gastric stage (G120), the values of
proteolysis degree for the empty and loaded DEs were
21.14 ± 1.47% and 19.72 ± 1.01%, respectively. At the very
beginning of the second step of protein hydrolysis with duo-
denal fluids (D5), protein degradation increased drastically, up
to 52.21 ± 1.40% for the empty DE and 49.99 ± 1.75% for the
loaded DE. Further incubation of the DEs with duodenal fluids
had no effect on the proteolysis degree. These findings are in
agreement with the results about changes in particle size in
DEs under gastrointestinal conditions. The significant increase
in particle size was observed at the beginning of the duodenal
digestion stage and was due to the extended degradation of pro-
teins on the interfacial layer O/W2 at this point of digestion.
The extent of lipid digestion was also examined by measur-
ing the lipolysis degree during the duodenal stage of the diges-

Food Funct. This journal is © The Royal Society of Chemistry 2020


Food & Function
Table 3 Protein and lipid hydrolysis as function of time during gastric
(G) and intestinal (D) in vitro digestion
Proteolysis degree (%) Lipolysis degree (%)
Stage of
digestion Empty DE Loaded DE Empty DE Loaded DE
G5 10.61 ± 1.39a 10.99 ± 0.74a — —
— —
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G60 12.80 ± 1.89a 15.16 ± 1.85a
G120 21.14 ± 1.47a 19.72 ± 1.01a — —
D5 52.21 ± 1.40b 49.99 ± 1.75b 9.0 ± 3.25a 7.2 ± 2.25a
D60 — — 15.0 ± 4.86a 12.8 ± 2.98a
D90 — — 46.0 ± 5.48a 41.2 ± 4.86a
D120 52.21 ± 1.68b 49.68 ± 1.45b 79.0 ± 6.54a 65.0 ± 5.24b
Values are reported as means ± standard deviation; lower case letters
indicate significant (p < 0.05) differences in characteristics between
empty and loaded double emulsions.
tion of DEs (Table 3). Lipid hydrolysis profiles of the empty
and loaded DEs followed a very similar model during the first
60 min of the duodenal stage. No significant differences
between the lipolysis degrees of the emulsions were observed.
The continued presence of emulsions under intestinal con-
ditions indicated significant ( p < 0.05) differences in lipid
digestion. After 90 min of the duodenal stage, the lipolysis
degree of the empty DE was 46%, while for the loaded DE the
value of hydrolysis was lower at 41.2%. This trend continued
until the end of the duodenal stage, suggesting slower lipolysis
of oil in the loaded DE. To explain such results, we should con-
sider the differences in viscosity of the empty and loaded DEs,
as presented in Table 1. There are some reports that the diges-
tion and the final release of the bioactive compounds depend
on the viscosity of the aqueous phase of emulsions.48–50 We
presume that in the more viscous DE, the access of lipase to
the surface of oil droplets could be suppressed, thereby
slowing down the rate of lipid digestion.
The release kinetics of bioactive compounds during digestion
of DE
Finally, the release kinetics of bioactive compounds loaded to
the different phases of the DEs during digestion were
measured. These data are presented in Fig. 2 as the ratio of
individual bioactive compound content released into the
soluble phase of the digesta to the content of the compound
in the DEs.
For all vitamins, the controlled release from the DEs during
digestion was observed. At the end of the duodenal phase,
approximately 100% of vitamins were released from the loaded
DE. However, the release kinetics of different vitamins under
gastrointestinal conditions differed. For vitamin C, the release
was very fast under gastric conditions; at the end of the gastric
stage, 71.75% of added vitamin C was released. After adding
the duodenal fluids, the amount of released vitamin C was
around 98%. For vitamins B9 and B12, the observed release at
the end of the gastric stage was considerably lower: 37.5% and
20%, respectively. Then, at the beginning of the duodenal
stage, 75–80% of these vitamins were released and their
release increased as the digestion progressed. For vitamins
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Fig. 2 Release of vitamins and anthocyanins from DE as function of
time during gastric (G) and intestinal (D) in vitro digestion: G120 –
gastric phase at 120 min of digestion, D5 – duodenal phase at 5 min and
D120 – duodenal phase at 120 min of digestion. Values are reported as
means ± standard deviation; lower case letters indicate significant ( p <
0.05) differences between different stages of in vitro digestion.
loaded in the oil phase of the DEs, about 70% of vitamin D3
and about 50% of vitamin A were released within 120 min of
gastric digestion. When duodenal fluids were added, the
release of vitamins D3 and A further increased and was com-
plete at the end of the duodenal stage of digestion. The
relationship between the structural changes of the DEs and
the vitamins’ release can be seen during the simulated diges-
tion process. Our results show that the degradation of the DE
structure was limited during the gastric stage and became
more intense after the addition of duodenal fluids, increasing
as the duodenal stage progressed. That could explain why the
DEs demonstrated a poor release of vitamins B9 and B12 and
a moderate release of vitamins D3 and A during the gastric
stage of digestion. Most of the release of these vitamins was
observed during the first 5 min of the duodenal stage, with
complete release at the end of digestion.
Recent studies on nano-encapsulated folic acid within DEs
showed the lowest release rate in acid conditions and the
highest release in alkaline conditions.38 This finding accords
with our results regarding the slower release of vitamin B9
under gastric conditions. There are few studies on the release
behaviour of vitamin B12 from DEs under gastrointestinal con-
ditions and those that exist have conflicting results. Giroux
et al.18 reported that less than 4.4% of vitamin B12 released
after 120 min of gastric digestion of a DE containing PGPR
and sodium caseinate as emulsifiers. Andrade et al.19 incorpor-
ated hydrophobic vitamin D3 and hydrophilic vitamin B12 in a
DE prepared with PGPR and sodium caseinate. They showed
that vitamin D3 had no influence on the control release
capacity of the system in relation to vitamin B12, which was
mostly released under gastric conditions. However, a signifi-
cantly lower release of vitamin B12 at the end of the gastric
stage was recorded in the samples with a gelled inner water
phase. Our findings also suggest that the physical properties
Food Funct.

Paper
of the inner water phase are of great importance for the
release of hydrophobic compounds during digestion. In our
study, the viscosity of the loaded DE was significantly higher
in comparison with the empty DE (Table 1) due to the action
of BCPE added in the inner phase of the DE. However, our sug-
gestion regarding the more controlled release of vitamins B9
and B12 from the viscous inner phase of the DEs under gastric
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conditions was not true for the release of vitamin C.
The release of bioactive compounds present in BCPE was
defined as the rate of cy-3-galactoside content released in the
soluble phase of digesta during in vitro digestion to the total
content of cy-3-galactoside based on the complete destruction
of the structure of the DEs. Cy-3-galactoside was outlined as
the predominant anthocyanin in BCPE. The controlled release
of cy-3-galactoside during the gastric stage was determined.
After 120 min of incubation with gastric fluids, the percentage
of released cy-3-galactoside from the DEs was 26.38%.
Immediately after the addition of duodenal fluids, the release
of cy-3-galactoside from the inner water phase of the DEs
increased up to 46.95%. The further incubation of the DEs
with duodenal fluids assisted the additional release of cy-3-
galactoside in the soluble part of the digesta. However, the
complete release of cy-3-galactoside from the DEs was not
achieved after 120 min of incubation with duodenal fluids.
The degradation of anthocyanins is affected by environ-
mental conditions. Under gastric conditions (low pH), antho-
cyanins prevail in the stable form of flavylium cation, while
under duodenal conditions (higher pH), the anthocyanins
degrade due to conversion into quinonoidal, hemiketal, and
chalcone forms (Clifford, 2000).51 According to Pérez-Vicente
et al.,52 the concentration of total anthocyanins remains con-
stant throughout gastric digestion and then declines during
intestinal juice treatment due to degradation.
An in vitro digestion study was carried out to evaluate the
anthocyanin release characteristics of a DE made with PGPR
and gum arabic as emulsifiers.20 The researchers reported a
very limited release of anthocyanins after the incubation of the
DE with gastric fluids and a total release within the first
20 min of DE incubation with duodenal fluids. These results
differ from those of our studies, where milk proteins were
used in the preparation of the DE for the encapsulation of
anthocyanins. There were attempts to compare the release of
phenolics from gum arabic and milk protein microcapsules.53
Gum arabic microcapsules had a higher release rate of pheno-
lics during in vitro digestion compared with whey protein
microcapsules. Proteins, peptides, and amino acids have been
reported to interact with anthocyanins.54,55 Several reports also
suggest the delay of anthocyanins degradation in duodenal
fluids due to their interactions with proteins and
polyphenols.56,57 The results obtained in these studies demon-
strate that the presence of the proteins in the encapsulation
system of an anthocyanin successfully supports sustained-
release issues.
Our results on protein hydrolysis kinetics during the diges-
tion of DEs suggest that milk proteins are digested at different
rates under gastrointestinal conditions. The results on
Food Funct.
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Food & Function
changes in particle size and lipid hydrolysis indicate the degra-
dation of the DE structure during digestion. Thus, some of the
anthocyanins that were released from the DEs under duodenal
conditions formed complexes with proteins and the products
of their hydrolysis. Some anthocyanins, after their release from
the DEs, may undergo degradation. Our results show that
60.5% of the total loaded anthocyanins in the DE were
released in the duodenal fluids after 120 min of digestion.
This means that the amount of released anthocyanins
exceeded the amount of anthocyanins degraded under duo-
denal conditions and complexed with proteins.
These results suggest that the double emulsion developed
in this study can restrict the release of vitamin B9, vitamin
B12, and anthocyanins during the gastric stage of digestion
and provide the total release of vitamins and moderate release
of anthocyanins during the duodenal stage of digestion.
Consequently, further research should be carried out to formu-
late a double emulsion that can limit the release of vitamins
C, D, and A under gastric conditions to ensure better avail-
ability of these bioactive compounds.
Conclusions
Six essential bioactives for the elderly were encapsulated into
different phases of a double emulsion. Vitamins B6, B12, and C
and anthocyanin-rich black chokeberry pomace extract were
added in the inner water phase, while vitamins A and D3 were
added in the oil phase. The characterisation of creaming stabi-
lity, droplet size, and the viscosity of the double emulsion, as
well as the encapsulation efficiency and encapsulation stability
of the bioactives, indicate good stability and successful protec-
tion of the bioactive compounds from environmental factors.
Furthermore, the in vitro digestion of the loaded emulsion
showed limited structural degradation during the gastric stage
and more intense degradation after the addition of duodenal
fluids. That explains the restricted release of bioactives into the
gastric fluids and the nearly complete release at the end of the
duodenal phase. The release kinetics of black chokeberry
pomace extract were distinguished from those of the other
bioactives. Only 46.95% of added extract was found in the
digesta at the end of the duodenal phase. Such characteristics
of double emulsions loaded by multiple essential bioactives for
the elderly make them suitable for food applications as contain-
ers and preservatives of bioactives. They can be used for the
simultaneous delivery of priority micronutrients for the elderly.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This research was funded by the European Regional
Development Fund according to the supported activity
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Food & Function
‘Research Projects Implemented by World-Class Researcher
Groups’ under Measure No. 01.2.2-LMT-K-718.
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