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International Journal of Food Science and Technology 2022, 57, 6531–6539 6531

Original article
Development and quality evaluation of multivitamin fortified
low-fat biscuits

Monalisha Pattnaik* & Hari Niwas Mishra

Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur West Bengal, 721302, India
(Received 21 May 2022; Accepted in revised form 21 July 2022)

Summary Multivitamin encapsulated nanoparticles that were produced by using a lipid-based emulsion template
were incorporated to fortify biscuits to obtain a low-fat nutrient-enriched food product. The present study
involves the development of biscuits containing vitamins (A, D, B9 and B12) both in free (C1) and encap-
sulated form (C2). The quality attributes of the developed biscuits were further evaluated by determining
their change in functional groups, textural properties, rheological characteristics and physical morphology.
The storage modulus (G’) was higher than loss modulus (G"), suggesting a viscous nature of the dough
(C1 and C2); meanwhile, C2 had a harder and a crispier texture than C1 (hardness: 30.06 N > 15.84 N;
fracturability: 15.84 > 26.18 N). FTIR spectra of C2 showed higher retention of nutritional attributes in
baking conditions as compared to C1. SEM images demonstrate that C2 has a regular/spherical-shaped
morphology (size: 8.3 mm) with minimal fissures sustaining the baking conditions than C1 (size: 6.7 mm).
The cumulative release (%) for all the vitamins of biscuits containing encapsulated nanoparticles (C2) was
relatively low (i.e. ca. 1.1–2.6 times), indicating a good bioavailability and thermal stability of the fortified
food product.
Keywords Encapsulation, fortification, low-fat biscuits, multivitamin, nanoparticles.

in any food product, it has to be adequately stable to

Introduction
About >80% of the total Indian population suffers
from micronutrient deficiencies predominantly vitamin
A, B12, folate and calcium (Ritchie et al., 2018). Cal-
cium deficiency is usually linked to vitamin D defi-
ciency possibly due to dietary patterns or lack of
sufficient sunlight exposure. This results in poor bone
health, rickets in children and osteomalacia in adults
(Sizar et al., 2021). Additionally, anaemia in pregnant
women and nonpregnant women of reproductive age
is quite prevalent (Ministry of Health and Family Wel-
fare (MoHFW), Government of India, UNICEF and
Population Council, 2019). These deficiencies cause
several complications including mental, physical
defects and even mortality. One of the prime reasons
for the existing deficiencies is the uptake of improper
diets along with poor absorption of the vitamins in the
body (Das et al., 2013). To address this, food fortifica-
tion is performed at several processing stages that
involve addition or enrichment of two or more vita-
mins into the food product. Edible oils are commonly
utilised as fortification vehicles. To prevent the degra-
dation of vitamins during fortification of multivitamins
*Correspondent: E-mail: monalisha.pattnaik21@iitkgp.ac.in
sustain severe processing conditions without compro-
mising the sensorial attributes (Das et al., 2013).
Microcapsules have been highly recommended as a
promising nutrient delivery system for food industries
because of their exclusive benefits such as active ingre-
dient protection, controlled release, thermostability,
masking of odour, colour and taste (Yang et al., 2020;
Pattnaik et al., 2021). Encapsulation through a lipid-
based emulsion template is a promising technique that
allows enclosing the vitamins in a layer of polymers,
protecting them from adverse environmental condi-
tions (Pattnaik & Mishra, 2021a, 2021b). Lipid-based
nanoparticles (liposomes) are self-assembled phospho-
lipids that offer unique properties of enclosing both
water and fat-soluble components in their membrane
and core, respectively, thereby improving bioavailabil-
ity (Sercombe et al., 2015).
Biscuits/cookies are liked and consumed by the popu-
lation of all ages. They are produced by mixing starch
and/or protein together through a kneading process.
They have an extended shelf-life that favours large-scale
production and wider distribution (Rebellato et al.,
2015). The gaining awareness amongst the masses
towards a healthy lifestyle has urged the consumption
of more low-fat biscuits. Several studies on the

doi:10.1111/ijfs.15992
Ó 2022 Institute of Food, Science and Technology (IFSTTF).

6532 Fortified Multi-Vitamin Low-Fat Biscuits M. Pattnaik and H. N. Mishra
fortification of flour have been reported in the literature
(Rebellato et al., 2015; Bajaj & Singhal, 2021). Fortifica-
tion of biscuits by encapsulating vitamins has proved to
be advantageous in entrapping thermolabile compounds
in its compartments with less degradation and better
stability in extreme processing conditions such as high
temperature (baking) and different pH treatments
(Canizales et al., 2018). Also, it helps in enhancing the
functionality of food (Tripathy et al., 2021). In this
study, the dough is fortified by encapsulated multivita-
min nanoparticles. Later the effect of baking conditions
on the dough and biscuit characteristics have been stud-
ied. The vitamins are quite sensitive to different pH con-
ditions result in poor absorption in the gastrointestinal
tract. The feasibility of utilising the developed biscuits
for enhancing the absorption of vitamins was deter-
mined through a simulated in vitro model by investigat-
ing the vitamins’ release profile.
Materials and methods
Materials
Refined rice bran oil, flaxseed oil and peanut oil were
procured from AAK KAMANI PVT limited, Mumbai
Maharashtra. For phospholipid (PL), soy lecithin was
purchased from the local market. Synthetic vitamins,
that is, folic acid (B9), vitamin A (retinyl acetate) vita-
min D3 and vitamin B12 were purchased from Sisco
Research Laboratories (SRL) Pvt. Ltd, India. All the
chemicals were purchased of analytical grade and stan-
dards from Sigma-Aldrich, India.
Preparation of encapsulated multivitamin nanoparticles
Encapsulated fat and water-soluble vitamins (A, D, B9
and B12) were prepared in a two-stage emulsion pro-
cess in the ratio (20:80 – step1: step2). The oil blend
concentration was kept constant at 30% (v/v). The oil
blend consisted of refined edible oils of rice bran oil,
flaxseed oil, peanut oil as mentioned in Pattnaik and
Mishra (2021a, 2021b) (fatty acid composition: total
saturated fatty acid (TSFA): 17.87; monounsaturated
fatty acid (MUFA) as oleic acid (%): 42.19; polyunsat-
urated fatty acids (PUFA) as linolenic acid (%): 10.4;
as linoleic acid (%): 29.51). The concentration of vita-
mins used in this study as per 25% RDA (Food Safety
and Standards (Fortification of Foods) Regulations,
Government of India, 2018; ICMR-National Institute
of Nutrition, 2020): vitamin A = 9.9 μg g−1 oil; vita-
min D = 0.16 μg g−1 oil; vitamin B12 = 0.625 μg; vita-
min B9 = 55 μg.
Step1: Preparation of single emulsion (W1/O)
Emulsion1 was prepared by mixing soy lecithin with
vegetable oil blend at a ratio of 1 (w/v): 3 (v/v).
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Firstly, lecithin was added to the lipid and the mixture
was kept in the water bath shaker (make: REICO) for
a time duration of 10 min at 60 °C temperature. After
complete solubilisation of lecithin in the lipid, hydra-
tion (water as 1/5th of the lecithin-lipid solution) was
done dropwise for the next 5 min in the water bath
shaker (100 rpm) without any temperature control.
The water-soluble vitamins (i.e. vit B9, B12) were mixed
into the water during the hydration step, while fat-
soluble vitamins (i.e. vit A, D) were added to the lipid
during the first 10 min duration.
Step 2: Preparation of double emulsion (W1/O/W2)
The prepared single emulsion (W/O) was added to the
polymer solution containing trehalose and milk protein
isolate (MPI) at fixed total solids of 45%, where the
ratio between milk protein isolate and trehalose was
1.7: 1 (w/v) to form a double emulsion (W1/O/W2).
The mixture was ultrasonicated (Model: Uwave1000;
Make: Sineo Microwave Chemistry Technology
(China) Co., Ltd, probe diameter: 13 mm; power:
150 W/50 g solution; frequency: 27 kHz) for 5 min.
The encapsulated particles in the emulsion (E2) had a
particle size, PDI and zeta potential of 195
1.01 nm,
0.336
0.01 and − 28
0.47 mV, respectively. The
double emulsion was then prefrozen at −20 °C for
24 h and it was freeze-dried (Model: LYODEL-0555;
Make: DELVAC Pumps Pvt. Ltd.) for 10 h at
−40 °C. The freeze-dried particles were stored in an
airtight pouch under refrigerated conditions until anal-
ysis.
Preparation of multivitamin enriched low-fat biscuits
The formulation for low-fat biscuits was based on the
method described by Manley (2001) with some modifi-
cations. The ingredients used for low-fat biscuits were
as follows (on 100 g flour basis): 100 g wheat flour,
20 g sugar, 15 g fat/oil, 0.5 g salt, 1 g baking powder
and ca. 30 g water. For fortified biscuits (C2), fat/oil
was replaced with 15% encapsulated multivitamin
nanoparticles (containing vitamins ca. 15–30% Recom-
mended dietary allowance (RDA). The control biscuits
(C1) were prepared by externally adding water and
fat-soluble vitamins to the oil and water during the
mixing step, respectively. All the dry ingredients were
mixed and fat and sugar were added and further
blended/kneaded in presence of water until the forma-
tion of dough. The process consisted of dough mixing,
dough kneading, dough relaxation, dough sheeting,
cutting and baking. The dough was kept covered for
30 min at 25 °C. The dough was cut with rings of
4 cm diameter and baked in a baking oven (Make:
S.D. Instruments & Equipments, Kolkata, India) at
160 °C for 15 min. The baked biscuits were cooled
and stored in plastic pouches for further analysis.
Ó 2022 Institute of Food, Science and Technology (IFSTTF).

Quality parameters of the developed biscuits
Texture analysis
The hardness, fracturability, springiness, gumminess
and chewiness were measured by CT3 texture analyser
Ametex Brookfield (USA). The hardness of a sample
is indicative of its freshness, while the crispiness
parameter characterises its inner crumb structure and
baking characteristics. Biscuits were located beneath
the probe and clamped to the analyser bed using low-
pressure clips. The probe then penetrated the sample
to the target distance (3 mm) and the texture profile of
characteristics was recorded through the interface
package. The conditions were kept as: test speed
0.5 mm s−1, pretest speed 1 mm s−1, return speed
0.5 mm s−1, trigger load 2–3.2 N, probe 2 mm stain-
less steel cylinder probe and fixture TA-BT-KI. All the
measurements were replicated six times and the mean
values are reported.
FTIR spectroscopy
FTIR spectrometer (Spectrum-100; Perkin Elmer, Wal-
tham, MA, USA) was used to identify the functional
groups on the biscuit samples containing both encap-
sulated vitamins and free vitamins.
Scanning electron microscopy
A field emission scanning electron microscope
(FESEM) (JSM-7610F; JEOL, Tokyo, Japan) was
used to study the morphology of the biscuit samples at
magnifications of 1.5 K X. All the samples were
spread on carbon stubs and gold-coated in an auto-
finer for 2 min for high conductivity.
Rheology
The dough’s rheological characteristics were deter-
mined by using Anton Paar modular compact rheome-
ter MCR 52 (Graz, Austria). The plate-plate geometry
(diameter: 25 mm; gap: 1 mm) was selected to assess
the stress and temperature sweep tests, as mentioned
in Tang & Liu (2017) with some alteration. For stress
sweep, the linear viscoelasticity region of a strain of
0.1 to 200% was determined at 1 Hz frequency. There-
after, a frequency sweep test was performed to esti-
mate storage modulus (G´) and loss modulus (G") at
the obtained strain value (%) in the range of 0.1 to 50
rad s−1. Similarly, the temperature sweep was per-
formed at strain 0.1 % and frequency 1 Hz to record
the storage modulus (G´) and loss modulus (G") as a
function of time and temperature heated from 30 to
100 °C at 2 °C min−1.
In vitro sequential digestion
The biscuits (C1 contained free vitamins, and C2 con-
tained encapsulated vitamins) were added to simulated
gastric (SGF) and intestinal (SIF) conditions by
Ó 2022 Institute of Food, Science and Technology (IFSTTF).
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Fortified Multi-Vitamin Low-Fat Biscuits M. Pattnaik and H. N. Mishra 6533
following the method reported in Liu et al. (2020) with
slight modifications. SGF is comprised of sodium chlo-
ride (2 g L−1) and pepsin (3.2 g L−1). The pH value was
adjusted to 1.2 using hydrochloric acid and sodium
hydroxide. SIF consisted of pancreatic (4.8 mg mL−1),
bile extract (5 mg mL−1), calcium chloride 750 mM and
sodium chloride (7.5 M). The simulated fluid was
adjusted to pH 7 by using sodium hydroxide and
hydrochloric acid. The biscuit samples were mixed SGF
in the ratio of 1:4 w/v (biscuit samples: SGF) and sub-
jected to a water bath shaker at 37 °C, @ 100 rpm for
2 h. After SGF digestion, the gastric chyme was col-
lected and mixed with SIF in the ratio of 1:1 v/v and
shaken for another 2 h. The sampling was done at
proper intervals and placed in 30-mL tubes.
For extraction of vitamins, the method reported by
Kucukkolbasi et al. (2013) was followed with some
modifications. Briefly, 1 mL from the withdrawn sam-
ples was added to 0.01% TFA and methanol (1:1 v/v).
The biscuits (C1 and C2) were ground before quantify-
ing the degraded vitamin concentration. All vitamins
from the ground samples (ca. 1.5 g) were then
extracted with 0.01% TFA and methanol (1:1 v/v).
Subsequently, the mixture (M1) was sonicated for
15 min (Make: Hielscher, Germany; conditions: 100%
amplitude, frequency 30 Hz, tip diameter 0.3 mm) in
an ice bath. Then, chloroform (ratio 1:4 v/v, chloro-
form: M1) was added to the resultant solution and
vortexed for another 5 min. Finally, the solution was
centrifuged at 3500 rpm for 30 min. The supernatant
was collected and heated for 5 min at 60 °C to evapo-
rate the chloroform from the solution. The quantifica-
tion of vitamins was done by HPLC and the
cumulative release rate was calculated by eqn 1.
released vitamin
Cumulative release rate ð%Þ ¼  100
total vitamin
(1)
Statistical analysis
All the experiments were conducted in triplicate. The
data were analysed by analysis of variance (ANOVA),
while a post hoc test of the experimental results was per-
formed by Duncan’s multiple range test on SPSS 19.0
statistical software (SPSS Inc., Chicago, IL, USA). The
graphs were plotted with the help of Origin 2018. And
P values <0.05 were considered statistically significant.
Results and discussion
Effect of fat type on the rheology of the dough
The storage modulus (G’) is a measure of the solid or
elastic character of the dough, whereas the loss
International Journal of Food Science and Technology 2022

6534 Fortified Multi-Vitamin Low-Fat Biscuits M. Pattnaik and H. N. Mishra
modulus (G") denotes the liquid or viscous character.
The rheological characteristics of the dough made
from both liquid oil (with free vitamins) and encapsu-
lated lipid particles (with vitamins) are summarised in
Fig. 1(i) a, b. From Fig. 1i(a), it is evident that G’ val-
ues are higher than G" until the cross-over point indi-
cating a visco-elastic behaviour. The cross-over point
was observed at 7 and 13 rad s−1 for C1 and C2,
respectively. The viscous component became more
prominent than the elastic component when angular
frequency was higher than cross-over frequency. In
case of C1, a huge difference between G’ and G" was
noticed than C2, suggesting a more viscous nature.
This might be due to the release of liquid oil from the
gluten networks during or after the kneading process
of the dough making, which was absent in C2 because
of the presence of encapsulated particles that shielded
the core material.
The temperature sweep of the dough is shown in
Fig. 1(ii) a,b with different biscuit types (C1- with liq-
uid oil; C2- encapsulated lipid particles). The
Figure 1 (i) Frequency sweep of the dough, (a) C1-free vitamin (b) C2-encapsulated nanoparticles. (ii) Temperature sweep of the fortified bis-
cuits, (a) C1-free vitamins (b) C2-encapsulated nanoparticles.
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rheological properties can be described in three distinc-
tive stages. The first stage was observed from 30 to
65 °C for C1 and 30–70 °C for C2 where G’ and G"
slightly decreased, which might be due to the hydroly-
sis of starch. The second stage begins after the first,
that is, 65 and 70 °C for C1 and C2, respectively. At
this stage, the gelatinisation of starch occurs, where
the amylose chains mix with the aqueous phase,
increasing the dough elasticity and viscosity (Tang &
Liu (2017). This fact is supported by the sharp
increase in G’ and G" (@ 90.33 for C1 and @ 70 for
C2). After the gelatinisation is over, the third stage
occurs, in which G’ and G" gradually decrease due to
starch degradation. An interesting trend was perceived
on adding encapsulated lipid particles in C2, and the
transition temperature was delayed in comparison to
C1 containing liquid oil (70 vs. 65 °C). Analogous rhe-
ological reports were demonstrated by Tang & Liu
(2017) in their study. It has been reported that dough
made from semi-solid/solid fat provided better struc-
tures than liquid oil. As the crystals in semi-solid/solid
Ó 2022 Institute of Food, Science and Technology (IFSTTF).

fat separate from liquid oil during dough mixing, it
envelopes the protein-membrane allowing more crys-
tals to attach to air bubbles. During baking, these
crystals melt and the protein membrane incorporates
into the surface of the bubbles as they expand, dictat-
ing better structure and resistance (Jacob & Leelavathi,
2007). Mamat & Hill (2014) delineated similar effects
of fat type and baking conditions during the produc-
tion of biscuits/cookies.
Effect of fat type on the textural properties of biscuits
In texture profile analysis (TPA), about 50%–80% of
the food sample is compressed between the test probe to
mimic the human-biting perception in the first and sec-
ond cycle. The TPA results of the biscuits (C1 and C2)
are shown in Table 1 where hardness, fracturability,
springiness, gumminess and chewiness values are given.
From Table 1, it is evident that C2 has higher hardness
than C1, which is due to the presence of encapsulated
multivitamin nanoparticles. The dough prepared with
high solid contents (C2) is stiffer as compared to the
dough made from liquid oil (C1) because liquid oil
tends to disperse on mixing throughout the dough sys-
tem disrupting the gluten networks, thereby producing
a softer dough (Mamat & Hill, 2014). The fracturability
value indicates that C2 has a crispier texture than C1
(26.18 N > 15.84 N). Springiness values are similar for
both the biscuits (C1 and C2), which describes the
recovery behaviour between the two cycles. As the bis-
cuits produced from encapsulated particles had a crispy
texture, the energy required to disintegrate them until
they can be swallowed was less than biscuits produced
from free liquid oil. The results are in agreement with
the gumminess and chewiness values (gumminess: 4.55
vs. 1.77 N; chewiness: 1.3 vs. 0.41 for C1 and C2,
respectively). A similar increasing trend in the textural
properties on raising the solid contents in fat or decreas-
ing the fat content was reported by Mamat & Hill
(2014) and Sudha et al. (2007). Mamat & Hill (2014)
stated that the dough made of palm mid-fraction
(PMF) needed more strength as it was harder to com-
press. Meanwhile, biscuit dough made from palm olein
was softer and required minimum force to compress it
to the desired extent.
Table 1 Textural properties of biscuits made from liquid oil and encapsulated oil
Time (min) Hardness (N) Fracturability (N)
C1
15 15.84
0.53c 15.84
0.51d
C2
15 30.06
0.41a 26.18
0.45b
Expressed as mean
standard deviation; different letter lower case in the column indicates significant difference (P < 0.05).
Ó 2022 Institute of Food, Science and Technology (IFSTTF).
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Fortified Multi-Vitamin Low-Fat Biscuits M. Pattnaik and H. N. Mishra 6535
Effect of baking conditions on functional groups and
morphology of biscuits
FTIR spectroscopy provides an idea about the molec-
ular structures of the samples that can be assessed
from the absorption bands. FTIR spectra of the bis-
cuits (C1 and C2) are shown in Fig. 2(i). The triglyc-
erides are the primary component present in the fat/oil
that predominates over the spectral region 500–
1500 cm−1. The major application of FTIR was to
determine the characteristics of the biscuits retained
after the baking. The spectral peaks obtained at a dif-
ferent wavelength for both the biscuits (C1 and C2)
are nearly close to each other, indicating that the oil
did not change much even after the encapsulation and
baking process. Proteins have a characteristic absorp-
tion band between 1650 and 1654, 1531 and 1539, and
1238 and 1464 cm−1 for amide I, amide II and amide
III, respectively (Qu et al., 2015; Srivastava & Mishra,
2021). In this study, the peak at 1379–1361 cm−1
denoted –CN bending vibrations (amide III) in the
peptide bonds. Similarly, 1630–1680 cm−1 peak region
belonged to amide groups (Matheus et al., 2006). The
characteristic band mentioned in both the biscuits was
found to be present at same locations, indicating that
the composition of biscuits at the molecular structure
level is quite appropriate. In other words, it was
hypothesised that the high temperature of baking
might have resulted in a change in molecular structure
of the biscuits made from encapsulated particles. The
broadening of the peaks indicates some interaction
occurring in the biscuit (mainly hydrogen bond inter-
action) or the compositional heterogeneity of the bis-
cuit (C2). Peaks between wave number 3500 and 3200
indicate stretching of O-H bonds, whereas between
3200 and 2800 show the stretching vibrations of C-H
bonds. The wave number 2925 cm−1 corresponds to
asymmetric stretching of the methylene (–CH2) group
and at 2854 cm−1 reflects symmetric stretches of the
methylene group (Sim & Ting, 2012). It is more promi-
nent in C1 due to the presence of free oil in the bis-
cuits. However, most of the peaks are obtained in the
fingerprint region of the IR spectra (1500–2000 cm−1),
which denotes the stretching and rocking of C=C
bonds. However, the spectra below 1000 cm−1
Springiness (mm) Gumminess (N) Chewiness (mJ)
0.23
0.02g 4.55
0.1m 1.04
0.01a
0.21
0.02k 1.77
0.08n 0.41
0.02c
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6536 Fortified Multi-Vitamin Low-Fat Biscuits M. Pattnaik and H. N. Mishra

C1
(i) (a)

2927
C2

1746.33
2855

1164
1097
1458
1379
3432

2363

718
853
2050
Absorbance

3414

991
1083
2927

1163
1663

1468
1361

920

572
708
760
852
2050
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber (cm-1)

B9
2919

(ii) B12
2861

1746

VD
VA
1155
1456
3006

1379

725
1691
Absorbance

1604
1486

1341
1411
3410
3544

3314

1691
1691

835
916

763
979
3433

2941

1671
1575
2136

1474 1499
2931
2855

2357

977

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm -1)

(b)

Figure 2 (a) FTIR spectra of (i) fortified

biscuits (ii) vitamins. *C1 – biscuit with free
vitamins; C2 – biscuits with encapsulated
vitamin nanoparticles; B9 – vitamin B9; B12
– vitamin B12; VD – vitamin D3; VA –
vitamin A. (b) Scanning electron microscopy
(SEM) images of fortified biscuits (i) C1;
(ii) C2. *C1 – biscuit with liquid oil and free
vitamins; C2 – biscuits with encapsulated oil
and vitamin.

belonged to the strong bonding regions and bending in C2 (Fig. 2b(ii)). The increase in particle size in the
of heavier atoms like aromatic rings, C-Cl, etc., that case of C2 might also be due to the different composi-
were mostly found in the vitamins (see Fig. 2(ii)). The tional formulation of the encapsulated particles along
FTIR spectra findings of C2 and vitamins validate with oil that has attributed to the possible agglomera-
higher retention and thermal stability of nutritional tion (although the biscuit preparation method was
attributes as compared to C1. The akin findings were similar for both). The images also reveal that the
reported by Srivastava & Mishra (2021) where the encapsulated particles contributed to a more
cookies produced from microencapsulated oil blend regular/spherical-shaped morphology with minimal fis-
not only survived the baking heat but also their chemi- sures sustaining the baking conditions than C1. This
cal composition remained unaltered during baking. suggests that the phenomenon of encapsulation has
The morphology of the biscuits produced from liq- provided a protective effect to the oil from releasing
uid oil (C1) and encapsulated particles (C2) are shown onto the surfaces (Pattnaik & Mishra, 2020). Con-
in Fig. 2b. The topography demonstrates the particle versely, C1 had an irregular structure with a sticky
size varied from 6.7 mm to 8.3 mm as the liquid fat/ nature due to the free oil breaking out from the gluten
oil (Fig. 2b(i)) was replaced by encapsulated particles network on the surface. A similar trend was observed

International Journal of Food Science and Technology 2022 Ó 2022 Institute of Food, Science and Technology (IFSTTF).

by Srivastava & Mishra (2021) during the development
of cookies from microencapsulated oil powder.
Release of vitamins in simulated gastrointestinal
conditions
The stability of vitamins in simulated gastro-intestinal
conditions is shown in Fig. 3. The first 120 min corre-
sponds to the simulated gastric conditions (SGF) with
pH 1.2, where the biscuits with free vitamins (C1)
showed a cumulative release of 1.2–2.6 times more
than the biscuits containing encapsulated vitamins
(C2). The time scale from 120 min to 270 min corre-
sponds to simulated intestinal conditions (SIF), where
the digested chyme from the SGF is further digested.
A similar trend of sustained release was observed in
SIF conditions where the release from C1 biscuits was
1.1–1.7 times higher than from C2 biscuits. Amongst
all the vitamins, free vitamin B12 and vitamin A were
very liable to the simulated conditions as there were
100% and 93% releases, respectively, towards the end
of the experimentation. Meanwhile, the encapsulation
of multivitamins in a lipid-emulsion-based template
provided effective protection against pH conditions
that aided controlled release, which is essential for
increasing the bioavailability and stability. Bajaj
et al. (2021) reported a slow in vitro release of vitamin
B12 and D3 after co-encapsulating in gum acacia, Hi-
Cap® 100 and maltodextrin than control samples. Sim-
ilarly, Ramalho et al. (2021) demonstrated the excel-
lent protection of poly(lactic-co-glycolic acid) (PLGA)
in enhancing the bioavailability of vitamin B9 and B12
than free vitamins. Supporting literature was eluci-
dated by Zhang et al. (2022) where the emulsion
Figure 3 Cumulative release (%) of vitamins during sequential
in vitro digestion. *C1 – biscuit with free vitamins; C2 – biscuits with
encapsulated vitamin nanoparticles; B9 – vitamin B9; B12 – vitamin
B12; VD – vitamin D3; VA – vitamin A.
Ó 2022 Institute of Food, Science and Technology (IFSTTF).
13652621, 2022, 10, Downloaded from https://ifst.onlinelibrary.wiley.com/doi/10.1111/ijfs.15992 by Agricultural University of Faisalabad, Wiley Online Library on [30/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Fortified Multi-Vitamin Low-Fat Biscuits M. Pattnaik and H. N. Mishra 6537
encapsulating vitamin A in alginate and gelatine gels
provided sufficient stability in the gastric fluid followed
by its release in the intestinal juices (cumulative
release = 75%).
Conclusion
In the present study, vitamin (A, D, B9 and B12)
nanoparticles were synthesised by encapsulating them
in a lipid-based emulsion template. Lipid-based emul-
sion nanoparticles were further dried into powder
form, which was then used for the fortification of bis-
cuits. The biscuits were produced from both free vita-
mins, C1 (as control), and encapsulated nanoparticles,
C2, to investigate the efficacy of the nanoparticles.
The similar FTIR spectral peaks of C2 with vitamins
suggested better nutrient retention. The sticky mor-
phology of the C1 biscuits was indicative of the
leaked oil/lipid from the fissures during baking. Over-
all, the C2 biscuits provided a harder and crispier tex-
ture along with a protective coating to the vitamins.
The in vitro release studies validate the combined
findings of the biscuit produced from encapsulated
nanoparticles (C2) evincing the benefits of encapsula-
tion over free vitamins in sustaining the high baking
temperature. Moreover, the established properties of
fortified biscuits (from encapsulated nanoparticles)
imply its novel application to be used in bakery
industries without compromising the nutritional and
sensorial attributes.
Funding
Thankful to the Indian Institute of Technology
Kharagpur, Ministry of Human Resource Develop-
ment (MHRD for providing Fellowship to the first
author. Authors also acknowledge the Scheme for Pro-
motion of Academic and Research Collaboration, an
MHRD (The Ministry of Human Resource Develop-
ment) initiative (https://sparc.iitkgp.ac.in/) for the
award of a project (No.: SPARC/2018-2019/P369/SL;
Project Code: P369; Proposal-Id: 369) entitled Lipo-
somes for control release of health-promoting factors
such as multivitamins (vit D, A, B9 and B12), omega 3
fatty acids and bioactives (bacosides).
Conflicts of interest
We declare no financial or other conflicts of interest in
our work. This work is original research and the data
presented in the manuscript have not been published
elsewhere.
Ethical approval
Ethics approval was not required for this research.
International Journal of Food Science and Technology 2022

6538 Fortified Multi-Vitamin Low-Fat Biscuits M. Pattnaik and H. N. Mishra
Author contributions
Monalisha Pattnaik: Conceptualization (equal); data
curation (lead); formal analysis (lead); investigation
(lead); methodology (lead); validation (lead); visualiza-
tion (lead); writing – original draft (lead). Hari Niwas
Mishra: Conceptualization (equal); resources (lead);
supervision (lead); writing – review and editing (sup-
porting).
Peer review
The peer review history for this article is available at
https://publons.com/publon/10.1111/ijfs.15992.
Data availability statement
All data generated or analyzed during this study are
included in this published article.
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