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Received: 12 February 2020    Revised: 2 November 2020    Accepted: 28 December 2020

DOI: 10.1111/jfpp.15231

ORIGINAL ARTICLE

Inclusion of vitamin D3 (free or liposome) into white chocolate

and an investigation of its stability during storage

Zohreh Didar

Department of Food Science and

Technology, Neyshabur Branch, Islamic Azad
University, Neyshabur, Iran
Correspondence
Didar Zohreh, Assistant Professor of Food
Science, Department of Food Science and
Technology, Neyshabur Branch, Islamic Azad
University, Neyshabur, Iran.
Abstract
This study is focused on the addition of vitamin D3 in two forms (free or liposome) to
white chocolate. Film dispersion-homogenization was chosen to prepare liposomes
containing vitamin D3 in order to be added to white chocolate (3 µg/10 g). Quality
assessment of liposome was performed. In vitro release of vitamin D3 from liposome
structure showed that vitamin D release in SGF and SIF were 7 ± 0.5% and 72.1 ± 1%,
respectively. The results showed that there was better retention of vitamin D3 in
fortified samples with liposomes than free vitamin D3 (p < .05). Organoleptic charac-
terization was performed by 10 panelists and hedonic test scale where the panelists
scored from 1 to 7 for different attributes. Accordingly, the addition of vitamin D3
(free or liposome) had no remarkable impact on sensory parameters of the resulted
white chocolate.
Fortified white chocolate with liposome had satisfactory stability and sensory acceptance.

Practical applications
Preparing liposome containing vitamin D3 through film dispersion-homogenization
method provides liposome with suitable particle distribution, PDI, and zeta potential.
The inclusion of vitamin D into white chocolate in liposome generates fine stability
of vitamin D during storage. As well as being a suitable protection against gastric
media, the white chocolate fortified with free vitamin D3 is good release into intesti-
nal simulated condition. Furthermore, fortified white chocolate has suitable sensory
acceptance. Adding vitamin D3 into white chocolate leads to a functional chocolate
with suitable organoleptic characteristics.

1 |  I NTRO D U C TI O N
Vitamin D plays important roles in bone homeostasis and health
(Chansathirapanich et al., 2016). To keep serum 25(OH)D at the de-
sirable level, 15 μg/day of vitamin D is recommended for the age
of <70 and 20 μg/day of vitamin D is enough at the age of 71 (Institute
of Medicine, 2012). Hypovitaminosis D is a worldwide issue (Mithal
et al., 2009). Vatandost et al. (2018) reported the overall prevalence
of vitamin D deficiency in Iran was equal to 0.56. Subgroup anal-
ysis revealed that 0.64 of women and 0.44 of men were suffering
from vitamin D deficiency (Vatandost et al., 2018). Chailurkit et al.
(2011) reported that vitamin D deficiency is common in Thailand
(64.6% of people in the capital city, Bangkok) (Chailurkit et al., 2011).
Receiving vitamin D either from food or supplements could be one
of the efficient ways to improve vitamin D status. Several studies
focused on fortification of foodstuffs such as the addition of vita-
min D into frying oil (corn, sunflower, and canola oils) (Saghafi et al.,
2018), ice cream (Chansathirapanich et al., 2016), cheese (Tippetts
et al., 2011), Cheddar cheese-like matrix, yogurt, and ice cream
(Kazmi et al., 2007), and milk (Omidvar et al., 2012).
Several challenges still face the fortification of foods with
vitamin D including high hydrophobicity, which prevents direct
dispersion of the vitamin in food products particularly foods con-
taining some moisture content. In addition, chemical degradation

J Food Process Preserv. 2021;45:e15231. wileyonlinelibrary.com/journal/jfpp |

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https://doi.org/10.1111/jfpp.15231
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of vitamin D, variable oral bioavailability, susceptibility to light,
oxygen, high temperature that cause vitamin isomerization and
degradation into its inactive forms are other challenges which
interfere with the fortification of foods with vitamin D (Glowka
et al., 2019). An effective approach for improving the bioavailabil-
ity, distribution, solubility, and protection against oxidation, UV
light and processing conditions is the encapsulation of vitamin D
(Öztürk, 2017). One of the most suitable encapsulation forms for
vitamin D is liposome, which is thanks to its flexibility in composi-
tion and size as well as its promising high biocompatibility with an-
imal tissue, it mimics with the natural plasma membrane (Maurya
et al., 2020).
Chocolate is a popular product among people. It has three main
classes including dark, milk, and white (Afaokwa, 2010). White
chocolate formulation includes sugar, milk solids, and cocoa but-
ter (Lončarević et al., 2018). Due to the absence of cocoa in white
chocolate formulation, this product has low functionality than other
chocolate types.
Several carriers were investigated for the inclusion of vitamin
D. Maurya and Aggarwal (2019) studied four lipid carrier formula-
tions for the encapsulation of vitamin D3 and reported acceptable
stability of lipid nanocarrier in various conditions (temperature and
humidity, refrigerated condition, and NaCl concentration) as well
as desirable sensory acceptability in Lassi’ (A milk-based bever-
age) fortified with nanocarrier of vitamin D3 (Maurya & Aggarwal,
2019). Golfomitsou et al. (2018) investigated edible oil in water
(o/w) nanoemulsions as carriers of vitamin D (D3: cholecalciferol)
for the fortification of milk. Accordingly, fortified milk was sta-
ble to gravitational separation for at least 10 days (Golfomitsou
et al., 2018). Mohammadi et al. (2014) reported satisfactory phys-
ical properties of liposomes of vitamin D as well as good stability
of vitamin D in liposome structure during storage over a span of
30 days at 4°C (Mohammadi et al., 2014).
This study is focused on the addition of vitamin D3 in two forms
(free or liposome) to white chocolate and an investigation on the re-
tention of vitamin D3 during storage and simulated gastric and intes-
tinal condition.
2 |  M ATE R I A L A N D M E TH O DS
2.1 | Materials
The main formulation for white chocolate manufacturing was as fol-
lows: sugar (497.6 g/kg), cocoa butter (Shirin asal Co, Iran) (348.4 g/
kg), whole milk powder (Pegah Co, Iran) (149.3  g/kg), sunflower
lecithin (4.5 g/kg), vanilla powder (0.2 g/kg), which are purchased
from a confectionary shop. This formulation was considered as the
control sample (Lončarević et al., 2018). Adding vitamin D3 (free or
liposome), adjusted to the final amount of vitamin D3 in white choco-
late formulation, was equal to 3 µg/per serving (10 g). All chemicals
used in the present study were purchased from Merck Company
(Germany).
2.2 | Vitamin liposomes preparation
Film dispersion-homogenization method was applied for liposome
preparation. Briefly, 30 mg of phospholipids, 10 mg of cholesterol,
and 10 mg of vitamin D3 was dissolved in 5 ml of ethanol which is
lipid phase. Thereafter, the lipid solution was dried by rotary evapo-
ration at 35°C to form a film. Then, 200 ml of distilled water was
used to hydrate lipid, also liposomes were condensed to 100 ml.
Finally, liposomes were sonicated (5 min) in an ice-bath and were
homogenized in triplicate (high-pressure homogenization, IKA,
Germany) (Bi et al., 2019). A specific volume of the newly prepared
liposomal dispersion was used for analysis (particle size, zeta poten-
tial, and loading efficiency) and the rest was freeze-dried (Operon,
Korea) and stored at –20°C.
2.3 | Characterization of liposome
2.3.1 | Particle size and zeta potential determination
Particle size analysis was carried out applying Particle Size Analyzer
(Cordouan model Vasco 3, France). Span index was calculated as an in-
dicator of the uniformity of the liposomes using the following equation:
Dv90 − Dv10
Span index =
Dv50
where Dv10, Dv50, and Dv90 are the equivalents to volume di-
ameters at 10%, 50%, and 90% cumulative volumes, respectively
(Mehanna et al., 2014). The zeta potential of liposome were mea-
sured using a Zetasizer (CAD, Zeta Compact Model, France), as de-
scribed by Golfomitsou et al. (2018).
2.3.2 | Assessment loading efficiency of vitamin D3
containing liposomes
To determine the percentage of vitamin D3 loading into liposome struc-
ture, centrifugation method was followed (Fathi & Varshosaz, 2013;
Maurya & Aggarwal, 2019). One mL of liposome was transferred into
a centrifugal concentrator tube and centrifuged (10,000 rpm, 10 min,
25°C). The amount of total and free vitamin D in the filtrate phase was
measured using HPLC (KNAUER, SMART, Germany). The loading ef-
ficiency was determined by the following formula (Xiang et al., 2008).
Loading efficiency = (quantity of vitamin D in liposomes∕
quantity of vitamin D used in the formulation) × 100.
2.3.3 | Fourier-transform infrared spectroscopy
(FTIR)
Fourier-transform infrared spectroscopy was applied to interpret
the structure of the components and revealed the functional groups
present in materials.
ZOHREH
The Fourier-transform infrared spectroscopy (FTIR) spec-
tra analysis was carried out for cholesterol, lecithin, vitamin D3 ,
and liposome. This analysis was conducted by a Perkin-Elmer
FTIR spectrophotometer (model Spectroma2) in the range of
4,000–500 cm-1.
2.3.4 | Sample preparation
To manufacture white chocolate, the melted fat components (in-
cluding 20% of the total cocoa butter) and the dry powders (sugar,
whole milk powder) were mixed until homogeneous mixture was
formed, meanwhile heating was done during mixing so as the
mixture reach the temperature of 40°C. Following this step, the
chocolate mass was first prerefined on a pilot-scale of 3-roll re-
finer (Lehmann, Aalen, Germany), and then was mixed again and
warmed to 50°C. Dry conching was the next stage of white choc-
olate making which was about 45 min and the remaining cocoa
butter (80% of the total), lecithin was added. The total conching
time was 360  min at 60°C. After conching, vitamin D3 (free or
liposome) (3 µg/10 g) was added to chocolate mass at 32°C–33°C.
Then, the mass was mixed for about 5  min. Afterward, a three-
step tempering process (33°C–35°C, 24°C–25°C, and 25°C–26°C)
was done. Subsequently, the molding and vibration process were
carried out at 27°C–30°C. After 20  min of cooling at 5°C, the
process was finished. The samples were stored at temperatures
of 13°C–15°C at a cold and dark condition prior to the analysis
(Toker et al., 2018).
In food fortification by nanoemulsions, the total quantity of an
additive depends on the fraction of the additive in the dispersed
phase, the fraction of the phase in the delivery system, the amount
of the delivery system added to the food material and also the serv-
ing size of the product (Walker et al., 2015). Considering this, adding
vitamin D3 was adjusted as the final chocolate vitamin D3 was equal
to 3 µg/10 g.
2.3.5 | Extraction and HPLC analysis of Vit D
Vit D content determination was performed according to the
method outlined by Kazmi et al. (2007). The first step was the sa-
ponification which was carried out by mixing 1 g of the diluted
sample (1:3) with 0.5 ml of 60% KOH. The total quantity of vita-
min D3 was determined by high-performance liquid chromatogra-
phy (HPLC) using HPLC equipment (KNAUER, SMART, Germany)
equipped with an auto injector, C18 column (VDSTHER) (250
× 4.6 nm). The mobile phase was methanol: acetonitrile: water
(45:45:10). The volume of the injected sample was 50 μl and the
flow rate was 1.0 ml/min. The elution of vitamin D3 was detected
at 254 and 228 nm on an ultraviolet detector. The specific concen-
tration of Vit D reference standard was utilized for Vit D identifi-
cation and qualification in the samples.
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2.3.6 | Vitamin D3 retention
In order to evaluate the stability of vitamin D3 during storage time,
the fortified samples were kept at ambient temperature in normal
circumstances of chocolate storage in packed forms and in specific
intervals of time (30, 60, 90, and 120 days), then, the chocolate sam-
ples were analyzed for quantification of vitamin D3 into samples
which was performed by high-performance liquid chromatography
as described above. The results reported as µg/10 g vitamin D3 in
chocolate samples.
2.3.7 | In vitro evaluation profile and kinetic of
vitamin D3 release from liposome structure
The release of Vit D from liposome was studied using a dialysis bag
immersed into simulated gastric media (pH 1.2) and simulated intes-
tinal media (pH 7.4), prepared as a method outlined by Maurya and
Aggarwal (2019). A 2 ml of Vit D-liposome was poured into a dialysis
bag and was placed into 98 ml of rotating simulated gastric media for
2 hr. Then, the same dialysis bag was placed for the following 7 hr
into 98 ml of rotating simulated intestinal media. The concentration
of Vit D released in both simulated media was quantified at regular
interval (0, 1, 2, 3, 4, 5, 6, 7, 8, and 9 hr) using high-performance liq-
uid chromatograph (HPLC) as described above.
To interpret the release pattern of vitamin D3 from liposome,
four mathematical models (zero degree, first degree, Higuchi, and
Korsmeyer–peppas models) were used and, having compared RMSE
and R 2, the best fitted model was chosen.
Zero-order model is described in the following equation:
Ct = C0 + K0 t.
Ct is the amount of compound released at time t; and C0 is the
initial concentration of compound at time t = 0; K0 is the zero-order
rate constant.
First-order model, was described using the following equation:
log C = log C0 − K1 t ∕ 2.303.
K1 is the first-order rate equation expressed in time-1 or per
hour; C0 is the initial concentration of the compound; C is the per-
centage of compound remaining at time t.
Higuchi model is in the basis of the release from an insoluble ma-
trix in basis on diffusion
Q = KH × t1 ∕ 2 where, KH is the Higuchi dissolution constant.
And Korsmeyer–peppas model basis is the material release
through simple diffusion and transition II.
( )
Log Mt ∕M∞ = log Kkp + n log t
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Mt is the quantity of compound released in time t, M∞ is the quan-
tity of compound released after time ∞, n is the diffusional exponent
or compound release exponent, Kkp is the Korsmeyer release rate
constant (Gouda et al., 2017).
2.3.8 | Organoleptic characteristics
Organoleptic parameters included (appearance: blooming, color,
surface brightness; texture: firmness; taste and aroma: aftertaste,
sweetness, acidity, bitter, metallic taste, and overall acceptance) of
the white chocolate samples were evaluated by 10 panelists. Prior to
organoleptic evaluation, panelists were educated to describe their
sensory experiences using the following definitions (appearance:
blooming, color, surface brightness; texture: firmness; taste and
aroma: aftertaste, sweetness, acidity, bitter, metallic taste, and over-
all acceptance). The trained panelists evaluated three different sam-
ples of chocolate simultaneously (control, free vitamin D included,
liposome vitamin D included in chocolates). The chocolate samples
were served in a random order in opaque plastic cups which were
identified with a different three-digit code. About 10 g of chocolate
samples was served to the panelists, who cleansed their palates with
water and crackers between assessments. Responses were recorded
using a hedonic scale where the panelists scored from 1 to 7 for dif-
ferent attributes (Toker et al., 2018).
2.4 | Statistical analysis
All the data were shown as the mean of three times with standard
deviations. An analysis of variance (ANOVA) was performed apply-
ing the SPSS software version 16.0 (SPSS Inc., Chicago, IL, USA).
Duncan's multiple range tests were used to determine the significant
differences at 95% confidence (p < .05).
F I G U R E 1   Histogram of size dispersion of liposome by intensity (left), number (right)
TA B L E 1   Particle size distribution of liposome
Dv10 Dv50 Dv90
213.85 ± 9.2 354.91 ± 11.2 676.26 ± 22.5
3 | R E S U LT S A N D D I S CU S S I O N S
3.1 | Particle size distribution of liposome
Having prepared the liposome, its size distribution was analyzed
using a particle size analyzer (Cordouan model Vasco 3, France) and
the results were shown as DV10, DV50, and DV90 (Figure 1 and
Table 1). Dv10 value, interpreted as 10% of the total particles, was
smaller than the value. Interpretation was used to describe dv50 and
dv90. The span index is an indicator for the uniformity of the lipo-
somal preparation, the smaller the span index, the narrower the size
distribution. The values of DV10, DV50, and DV90 for the prepared
liposomes were 213.85 ± 9.2, 354.91 ± 11.2, 676.26 ± 22.5, respec-
tively, and the span index was 1.3 ± 0.001. Polydispersity index (PDI)
in the present study was 0.265 ± 0.1 Other researchers reported
various PDI for liposomes of vitamin D. Dalmoro et  al.  (2019) re-
ported the PDI of D3-loaded nanoliposomes equal to 0.40 ± 0.07.
There are many parameters which impact the particle sizes of li-
posomes. Preparation method is one of the remarkable factors af-
fecting the particle size of liposomes. Polydispersity index (PDI)
measured in this study was 0.265 ± 0.1. PDI is related to the homo-
geneity and stability of an emulsion. The lower quantity of the PDI
indicated better stability of an emulsion (Li & Chiang, 2012). The low
quantity of the PDI in the present study revealed suitable emulsion
formation for liposome preparation.
3.2 | Zeta potential of liposome
Zeta potential is an important factor in the stability of nanoemul-
sions which is influenced by physic-chemical properties of nanopar-
ticles. Observations exhibited that zeta potential and mean mobility
of liposome were −27.62 mV and −2.19 µm s–1 V–1 cm–1, respectively.
Mohammadi et al. (2014) applied thin-film hydration-sonication
Span index Z average Polydispersity index
1.3 ± 0.001 306.24 ± 6.8 0.265 ± 0.1
ZOHREH
method to prepare liposomes and reported that the zeta poten-
tial of the prepared liposomes varied between −29 and −42.9 mv.
According to this report, the zeta potential is suitable for liposomes
by about −25 mv (Mohammadi et al., 2014).
Figure 2 depicted the motilities distribution of liposome. Higher
quantity of zeta potential is because of the more repulsive force be-
tween drops and lower tendency to adhere each other that resulted
in more stability of emulsion (Mao et al., 2009).
3.3 | Evaluation of vitamin D loading
The results showed that the loading efficiency of vitamin D3 into
liposomes was equal to 68.28 ± 1.2%. Figure 3 depicted vitamin
F I G U R E 2   Mobility's distribution of liposome
F I G U R E 3   HPLC chromatogram of vitamin D3 in liposome
F I G U R E 4   The FTIR spectrum of
vitamin D3 (A), lecithin (B), cholesterol (C)
and liposome (D)
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D3 measured in liposome. Bi et al. (2019) reported that the per-
centage of encapsulation efficiency of vitamin D in liposomes was
62.2 ± 0.9% (Bi et al., 2019).
3.4 | FTIR
FTIR spectra of the cholesterol, lecithin, vitamin D3, and liposome
are shown in Figure 4. The peaks at 3,350 cm–1 and 3,450 cm–1 is
due to the presence of free and bonded hydroxyl (OH) and may be
as a result of the formation of weak hydrogen bonds (Mukherjee
et al., 2008). Cholesterol and lecithin had bands in the range of
3,500 cm–1 and 3,200 cm–1 and the bands were very similar to each
other. Vitamin D3 showed peak at 3,442 cm–1 (3,200–3,550 cm–1)
which could be attributed to the O–H bands. The peak observed at
2,914 (2,850–2,960 cm–1) was attributed to C–H bands and the peak
at 1,235 cm–1 (1,110–1,250 cm–1) was ascribed to the presence of
C–C bands (Thoke Sagar et al., 2013).
3.5 | Release profile and kinetic from liposome in
gastric simulated media (SGF) and intestinal simulated
media (SIF)
The rate of vitamin D3 release from liposome at a simulated condi-
tion of gastric media and intestinal media are shown in Table 2.
In vitro release study, it was shown that 7 ± 0.5% of vitamin D3
was released from liposomes in gastric simulated condition (120 min)
and 72.1 ± 1% in simulated intestinal condition (next 7 hr) (Table 2).
In general, it is assumed that components are released by sev-
eral processes such as (i) diffusion through the particle matrix, (ii)
degradation, and (iii) diffusion through micro channels that are
formed by erosion (Lamprecht et al., 2002). In the present study,
reasonable release of vitamin D in simulated intestinal condition
was observed (72.1 ± 1%) which was higher than similar reports.
Kiani et al. (2016) reported 9.6% of vitamin D3 release occurred
after 2 hr in simulated gastric fluid at 37°C, and over the next 7 hr
in the intestine media, approximately 16.2% of the vitamin D3 was
released from the nanocapsules. Hence, this low release of the vi-
tamin D3 was attributed to the rigid external shell of nanocarriers
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TA B L E 2   % Release of vitamin D3 from liposome structure in TA B L E 3   Kinetic parameters of vitamin D3 release in SGF and
stimulated gastric media (2 hr) and stimulated intestinal media (next SIF
7 hr)
Model
Time (min) % Release Medium parameters

0 0 Zero-order model SGF k 0.082

k
30 4.2 ± 1.1 RMSE 2.2
60 5.3 ± 1.2j R2 0.93
i
90 6.1 ± 1.5 SIF k 0.0031
120 7 ± 0.5h RMSE 2.4
180 14 ± 2g R2 0.89
f
240 22 ± 2.1 First-order model SGF k 0.088
e
300 31 ± 2.3 RMSE 2.2
360 45 ± 3.1d R2 0.92
c
420 52 ± 2.2 SIF k 0.004
480 66 ± 2.1b RMSE 2.1
540 72.1 ± 1a R2 0.89

Note: Different superscript letters show the significant differences Higuchi model SGF k 1.52
between the samples (p < .05). RMSE 1.1
R2 0.96
SIF k 0.91
which were resistant to digestion and acted as a barrier for the
RMSE 1.5
diffusion of vitamins during vitro release experiments (Kiani et al., 2
R 0.94
2016). In the present study, the higher release at intestinal simu-
Korsmeyer–peppas SGF k 0.0857
lated condition might be due to the lipophilic nature of wall ma-
model n 0.197
terials of liposomes (lecithin and cholesterol) which is digested by
enzymes in intestinal media. RMSE 0.12
2
The assessment of vitamin-release kinetic patterns and vi- R 0.97
tamin-release data were analyzed using zero order, first order, SIF k 0.0870
Higuchi, and Korsmeyer–peppas kinetic models. The results are n 0.191
showed in Table 3. The data showed that the most suitable model RMSE 0.14
for interpreting the release kinetic of vitamin D3 from liposome is R 2
0.98
Korsmeyer–peppas model with the highest R 2 (0.97 for SGF and
0.98 for SIF) and the lowest RMSE (0.12 for SGF and 0.14 for SIF)
(Table 3). Korsmeyer–peppas kinetics involves combining the dif- fortified with liposomes (Table 4). The reduction of fortified vita-
fusion and erosion mechanism for bioactive components release min D content (in free form) has been reported by some researches.
(Rudra et al., 2010). Chansathirapanich et al. (2016) reported that vitamin D3 content
in fortified ice cream had reduced during storage time (28 days)
(Chansathirapanich et al., 2016). The stability of liposome form
3.6 | Vitamin D3 retention of vitamin D was also pointed out in some reports. Mohammadi
et al. (2014) showed good stability of vitamin D in liposomes espe-
Vitamin D3 stability and the retention in final product during stor- cially in dark conditions and at low temperatures (more than 95%
age time (0–120 days) at normal circumstances (25°C in packed retention). The high stability of vitamin D in liposomes was attrib-
form) were evaluated and the results are shown in Table 4. The uted to the presence of cholesterol in liposome structure which
exact amount of vitamin D3 addition to white chocolate formula- reduces the permeability of liposome membrane; therefore, en-
tion was 3 µg/10 g and the measured quantity of vitamin D3 after trapped materials cannot leak out easily (Mohammadi et al., 2014).
preparation was 2.98 ± 0.01 µg/10 g which demonstrated no sig- Cholecalciferol (vitamin D) is a fat-soluble vitamin that is susceptible
nificant difference between the sample prepared immediately and to light, heat, and air. In this study, the addition of vitamin into white
the samples stored for 30, 60, and 90 and 120 days of vitamin D chocolate formulation was carried out at mild temperatures (after
in liposome form (p < .05) (Table 4). However, the vitamin D3 con- couching and temperature about 25°C–35°C). The storage condi-
tent of chocolate samples lessened after 120 days of storage time in tion was the normal condition of chocolate storage including ambi-
the samples fortified with free vitamin D3 compared to the sample ent temperature (25°C), away from light and air (packed samples).
ZOHREH |
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TA B L E 4   Vitamin D3 content in chocolate samples

Storage time Vitamin D3 Storage time Vitamin D3

Chocolate sample (days) (µg/10 g) Chocolate sample (days) (µg/10 g)

0 2.98 ± 0.01a 0 2.98 ± 0.01a
Chocolate samples fortified with 30 2.98 ± 0.01a Chocolate samples fortified 30 2.97 ± 0.01a
vitamin D3 liposome 60 2.98 ± 0.01 a with free vitamin D3 60 2.97 ± 0.01a
90 2.97 ± 0.01a 90 2.96 ± 0.01a
120 2.96 ± 0.01a 120 2.94 ± 0.01ab

Note: Means and standard deviation (SD) for triplicate vitamin D3 content over the study period for each chocolate sample. Different superscript
letters show the significant differences between the samples (p < .05).

TA B L E 5   Organoleptic properties of white chocolate

White chocolate enriched White chocolate enriched

Sensory properties Control with free vitamin D with vitamin D liposome

Appearance Blooming 1 ± 0.01a 1 ± 0.01a 1 ± 0.01a

a a
Surface brightness 6.65 ± 0.2 6.65 ± 0.2 6.65 ± 0.2a
Color 6.71 ± 0.04a 6.71 ± 0.04a 6.71 ± 0.04a
a a
Texture Firmness 6.76 ± 0.01 6.75 ± 0.01 6.75 ± 0.01a
Taste and aroma Last taste in mouth 4.22 ± 0.03a 4.22 ± 0.02a 4.22 ± 0.02a
a a
Sweetness 6.85 ± 0.05 6.85 ± 0.03 6.86 ± 0.05a
Acidity 1 ± 0.00a 1 ± 0.00a 1 ± 0.00a
a a
Bitterness 1.11 ± 0.02 1.11 ± 0.03 1.11 ± 0.02a
Metallic taste 1.1 ± 0.01a 1.1 ± 0.01a 1.1 ± 0.01a
a a
Overall Acceptability 6.82 ± 0.15 6.82 ± 0.13 6.82 ± 0.11a

Note: Different superscript lowercase letters show the significant differences between the samples (p < .05).

Therefore, the reduction in vitamin D3 after preparation and during 4 | CO N C LU S I O N S

storage time was negligible (Table 4).
3.7 | Sensory evaluation
The addition of vitamin D3 either in free form or liposome had no
remarkable impact on sensory parameters of the resulted white
chocolate (Table 5). Sensory parameters of chocolate included ap-
pearance: blooming, color, surface brightness; texture: firmness; taste
and aroma: aftertaste, sweetness, acidity, bitterness, metallic taste,
and overall acceptance. There was no significant difference between
the two forms of vitamin D3 added to white chocolate formulation
(Table 5).
According to some reports, adding vitamin D3 to some food
products had adverse impact on sensory characteristics. Yeh et al.
(2017) reported panelists and consumers detected flavor differ-
ences between skim milks fortified with water-dispersible vitamin
A or vitamin A and D (Yeh et al., 2017). But Maurya and Aggarwal
(2019) reported that there was no significant difference in sensory
properties of the control and vitamin D fortified in Lassi (A milk-
based beverage) (Maurya & Aggarwal, 2019).
Preparing functional white chocolate by adding (3 µg/10 g) vitamin
D (free or liposome form) was the focus of this study. Film disper-
sion-homogenization method was chosen to prepare liposomes
containing vitamin D3. Measurement particle size distribution,
zeta potential, loading efficiency, and FTIR were adopted to as-
sess the quality of the resulted liposomes. The results showed that
Polydispersity index was equal to 0.265  ± 0.1 and Z average was
306.24  ± 6.8. Zeta potential and mean mobility of liposome were
−27.62 mV and −2.19 µm s–1 V–1 cm–1, respectively. According to the
results, the selected method was suitable for liposome prepara-
tion. Furthermore, FTIR analysis affirmed incorporation of vitamin
D3 into liposome structure. Also, loading efficiency was equal to
68.28  ± 1.2%. In vitro release of vitamin D3 from liposome struc-
ture, it was shown that the magnitude of vitamin D release in gastric
and intestinal simulated condition were 7 ± 0.5% and 72.1 ± 1%, re-
spectively, and the release kinetic follows Korsmeyer–peppas model
(both in SGF and SIF). Vitamin D3 retention during storage time (0,
30, 60, 90, and 120 days) was determined and the results imply that
there was better retention of vitamin D3 in fortified samples with
liposomes than free vitamin D3 (p < .05).
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8 of 9       ZOHREH
C O N FL I C T O F I N T E R E S T
The authors have declared no conflicts of interest for this article.
DATA AVA I L A B I L I T Y S TAT E M E N T
The author confirm that data supporting the findings of this study
are available within the article.
ORCID
Zohreh Didar  https://orcid.org/0000-0001-6268-6376
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