Journal of Food Composition and Analysis 96 (2021) 103703

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Journal of Food Composition and Analysis

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Original Research Article

Fortification of wheat flour and oil with vitamins B12 and D3: Effect of
processing and storage
Seema R. Bajaj , Rekha S. Singhal *
Food Engineering and Technology Department, Institute of Chemical Technology, Nathalal Parekh Marg, Matunga (E), Mumbai, 400 019, India

A R T I C L E I N F O A B S T R A C T

Keywords: Fortification of commodity foods has been accepted as a successful strategy to overcome micronutrient de­
Enrichment ficiencies. This work evaluates the stability of vitamin B12 and vitamin D3 during different processing techniques
Cyanocobalamin and/or storage. Kinetic behavior of vitamins B12 and D3 degradation in fortified whole wheat flour (FWWF)
Cholecalciferol
during storage at varying temperature and relative humidity (RH) was evaluated. An increase in temperature
Whole wheat flour
accelerated the degradation of both vitamins in FWWF during storage. However, RH affected the degradation of
Rice bran oil
Frying and baking vitamin D3 but not that of vitamin B12. Products prepared from FWWF showed highest retention of vitamin B12 in
Storage and stability chapatti (91 %) and vitamin D3 in cake (84 %), respectively. Retention of vitamins B12 and D3 was lowest (14 %
Degradation kinetics and 10 % respectively) during frying. Further stability of vitamin D3 was evaluated during multiple frying cycles
Chemical compounds studied in this article: of vitamin D3-fortified rice bran oil (FRBO) in batter based and dough based products. Absorption of vitamin D3
Cyanocobalamin (PubChem CID: 5311498 in both the products reduced significantly and progressively after the first frying cycle. Dough based products
Cholecalciferol (PubChem CID: 5280795) showed better absorption of vitamin D3 from FRBO during frying. This work will help to establish suitable
fortification limits in the wheat flour and edible oil considering processing and storage losses.

1. Introduction
Vitamin D is a fat soluble vitamin which acts both as nutrient and
hormone. It captured attention of the world after its reported pandemic
deficiency (Alkhatatbeh et al., 2017). The requirement of vitamin D3 or
cholecalciferol is met either through exposure of skin to UVB rays
and/or from foods of animal origin such as fish, egg, and liver. Vitamin
B12 is important for neurocognitive development in the body and sour­
ces of vitamin B12 are also limited to foods of animal origin (Green et al.,
2017). Further, economic viability and religious beliefs are responsible
for lower consumption of food products of animal origin. This makes
vegans and vegetarians vulnerable to the deficiency of vitamin D as well
as vitamin B12 as has been evident from lower levels of serum vitamin D
(Sharma et al., 2017; Crowe et al., 2011) and vitamin B12 (Yajnik et al.,
2006; Davey et al., 2003) in this population segment than meat and fish
eaters. The recommended dietary allowance (RDA) of vitamin B12 and
vitamin D for Indian healthy adult is 1 μg/ day and 400 IU (10 μg)/ day,
respectively (Nutrient requirements and dietary allowances for Indians,
2011).
A serum level of 25-hydroxyvitamin D concentration below 50 nmol/
L is indicative of deficiency of vitamin D (Sowah et al., 2017). Deficiency
of vitamin D can cause rickets in children, and osteomalacia and oste­
oporosis in adults due to weaker bones and muscle. It is also associated
with diabetes, cardiovascular diseases, infectious diseases and cancer
(Alkhatatbeh et al., 2017). Saraf et al. (2016) reported on the deficiency
of vitamin D in pregnant women and infants to be widespread across
many parts of the world. Vitamin D deficiency is reported to be an
epidemic in western part of India wherein 75 % of the study population
showed lower vitamin D levels (Akhtar, 2016). Other relevant studies
showed prevalence of vitamin D deficiency to the extent of 56 % among
the elderly in Hyderabad (Suryanarayana et al., 2018), and 40 % in
mixed population of Punjab (28 % males and 53 % females) (Bachhel
et al., 2015). A recently conducted national nutrition survey in India has
shown the prevalence of vitamin D deficiency among preschool school
children of 1–4 years (14 %), children of age 5–9 years (18 %), and
adolescent aged 10–19 years (24 %) (Comprehensive nutrition national
survey 2016-2018: National report, 2019).
Pernicious anemia, gastric disease or surgery, intestinal disease,
pancreatic disease, malnutrition, vegetarian and vegan diet, and chronic
alcoholism can cause deficiency of vitamin B12. Vitamin B12 deficiency is
associated with haematological disorders, Alzheimer’s disease, depres­
sion, and neural tube defect (Green et al., 2017). Deficiency of vitamin

* Corresponding author.
E-mail address: rs.singhal@ictmumbai.edu.in (R.S. Singhal).

https://doi.org/10.1016/j.jfca.2020.103703
Received 3 February 2020; Received in revised form 13 September 2020; Accepted 27 October 2020
Available online 4 November 2020
0889-1575/© 2020 Elsevier Inc. All rights reserved.
S.R. Bajaj and R.S. Singhal
B12 is marked by serum B12 levels < 149 pml/ L (Hannibal et al., 2016).
Green et al. (2017) reviewed many studies and found that 2.5–26% of
the general population is affected by sub-clinical deficiency of vitamin
B12 all over the world. Margalit et al. (2018) reported a higher preva­
lence of vitamin B12 deficiency in men (25.5 %) than in women (18.9 %).
A study conducted in south India revealed 35 % prevalence of vitamin
B12 deficiency with vegetarians (54 %) at a greater risk than population
consuming mixed diet (31 %) (Sivaprasad et al., 2016). The overall
prevalence of vitamin B12 deficiency is 47 % in north India (Singla et al.,
2019) and 53.4 % in rural women in Haryana (Das et al., 2019). A
recently conducted national nutritional survey has shown pre-school
children (14 %) aged 1–4 years, school-age children (17 %) aged 5–9
years, and adolescents (31 %) aged 10–19 years to show deficiency of
vitamin B12 (Comprehensive nutrition national survey 2016-2018: Na­
tional report, 2019).
Fortification of commodity foods is a precautionary public health
initiative to protect vulnerable populations from deficiency. Its success
is visible in countries like India, Switzerland, USA, and Canada where
fortification of commodity food products like milk, margarine, flour, and
oil is reported to improve the levels of serum B12 and D3 and to reduce
prevalence of deficiency in many studies (Sirohi et al., 2018).
Fortification of wheat flour with different micronutrients is reported
to be an effective approach due to its wide consumption across the world
(Allen et al., 2010; Garrod et al., 2019). Fortification of wheat flour with
both vitamin D3 and vitamin B12 could help to reduce the prevalence of
deficiency of these vitamins. Wheat flour is normally transported and
stored under uncontrolled conditions wherein it is exposed to extreme
environmental conditions. The stability and the rate of degradation of
micronutrients are also influenced by packaging material and storage
conditions viz. temperature and humidity (Hemery et al., 2019) as well
as the subsequent processing (Jakobsen and Knuthsen, 2014).
In situ produced cyanocobalamin has been reported to more stable
than hydroxycobalamin during bread making with 23 % and 44 %
degradation, respectively (Edelmann et al., 2016). Another report has
suggested in situ fortification of flour to show vitamin B12 content in the
order of bran > whole wheat flour > wheat flour (33, 87, and 155 ng/g
respectively) (Xie et al., 2018). A better retention of vitamin D3 than that
of D2 in fortified flat bread has been shown with both being stable until
60 min of fermentation but degrading rapidly during baking (Tabibian
et al., 2017). The effect of food matrix is evident from the fact that the
retention of vitamin D3 in fortified wheat bread is better than fortified
rye bread (Jakobsen and Knuthsen, 2014).
There is a wide variation in the climatic conditions of India. Western
regions of India have a dry and hot weather during which summer
temperature reaches above 45 ◦ C and humidity reduces to less than 30
%. Coastal regions of India have hot and humid weather in summer and
very high humidity (>75 %) during rainy season. Degradation of vita­
mins during storage has been shown to follow first order kinetics from
which the rate of degradation or stability can be determined during
storage (Bajaj and Singhal, 2020). It is essential to know rate of degra­
dation of added micronutrients during storage of wheat flour for a
successful fortification program.
Fried foods are universally popular due to their appealing color,
attractive flavor and crispy texture. Effect of frying, baking, and
steaming on the retention of vitamins A, D3, and E in different fish va­
rieties has shown lower retention during frying in comparison with
steaming and baking with vitamin D3 showing higher degradation than
vitamins A and E (Szlinder-Richert, and Malesa-Ciećwierz, 2018). In
another study, cooking and frying in vegetable oil has shown 17 % and
31 % degradation of fortified vitamin D3 (Saghafi et al., 2018). Similarly,
cocoyam tubers are documented to show better retention of vitamins A,
D, E, and K during drying than that during frying (Omotosho, 2015).
Fortification of edible oils with vitamin D3 efficiently combats the
prevalence of vitamin D deficiency (Yang et al., 2013). It is cost effective
and can be adopted by small, medium, and large scale oil manufacturers
(Walters et al., 2019). Voluntary fortification of oil with vitamin D has
2
Journal of Food Composition and Analysis 96 (2021) 103703
been introduced in 2002 and limits of fortification have been doubled in
2010 due to its effectiveness in controlling deficiency (Boucher, 2018).
Holland et al. (1991) studied fried food in vitamin E fortified oil and
found vitamin E content to be significantly high in fried foods fried
therein. Hence, oil can be a good medium for fortification with fat sol­
uble vitamins. However, oil is used repeatedly during frying both in the
industry as well as domestic use. Since repeated frying of oil leads to
oxidation and polymerization, the fat soluble vitamins present therein
are also unstable (Hrncirik, 2010). This necessitates the determination
of vitamin D3 in the fried food as well as frying oil over multiple frying
cycles.
In India, fortification of oils with vitamin D is permitted up to 4.5 IU/
g (Food safety standard regulations for fortification of food, 2018),
which could provide 25–30 % of the daily requirement at a cost of
around 0.08 – 0.15 Rs (5–10 $)/ kg of oil (0.1 – 0.2 % increase in the
retail cost) (Chaudhry, 2018). Vitamin B12 fortification in wheat flour is
permitted up to 10 μg/ kg flour (Food safety standard regulations for
fortification of food, 2018), which could increase the cost marginally by
0.20− 0.25 Rs (14–18 $)/kg (Sirohi et al., 2018). This will increase retail
cost of wheat flour by 0.05 – 0.06 %. Hence food fortification is a sus­
tainable, effective, viable and inexpensive method to combat wide­
spread vitamin B12 and D3 deficiencies. However, the efficacy of these
fortifications is influenced by the stability of these vitamins during
processing and storage.
To the best of our knowledge, there are no reports on storage stability
of vitamin B12 and D3 in double fortified whole wheat flour and their
comparative retention in various food products after processing. The
present work aimed at assessing the stability of fortified vitamin B12 and
vitamin D3 in widely consumed staples such as whole wheat flour as a
function of storage temperature and humidity, and in products viz.
bread, cake, cookies, chapatti, and poori subjected to a wide range of food
processing conditions. It also investigates the stability of fortified
vitamin D3 in rice bran oil during multiple frying cycles of traditional
Indian dough based (poori) and batter based (pakora) fried products.
2. Materials and methods
2.1. Materials
Rice bran oil, whole wheat flour, chickpea flour, powdered sugar,
and fat were purchased from local market of Matunga, Mumbai, India.
Food grade vitamin B12 (cyanocobalamin) was a gift sample from Paras
Organics Pvt. Ltd., Navi Mumbai, India. Vitamin D3 (cholecalciferol)
was obtained as a gift sample from Fermenta Biotech Ltd., Mumbai,
India. TLC Silica gel 60 F254 Aluminum sheet plate 20 × 20 cm2 and
syringe filters (0.22 μm) were purchased from local suppliers of Merck
Millipore, Mumbai, India. Potassium hydroxide pellets, magnesium
chloride, sodium nitrite, potassium nitrite, 2-propanol, formic acid, so­
dium bi-carbonate, and ammonium hydroxide were obtained from S. D.
Fine Chemicals Pvt. Ltd., Mumbai, India. HPLC grade methanol and
acetonitrile were procured from Merck, Mumbai, India.
The standard vitamin B12 and vitamin D3 were stored in aluminum
coated air sealed pouch and this pouch was kept in self sealable poly­
ethylene pouch to avoid any moisture absorption. The pouches were
stored at 4 ◦ C in dark.
2.2. Methods
2.2.1. Fortification of whole wheat flour with vitamins B12 and D3 and its
storage stability
Whole wheat flour was fortified with vitamin B12 (150 μg/g flour)
and vitamin D3 (150 μg/g flour) and mixed uniformly in a Hobart N50 5-
quart mixer (Mumbai, India) using a flat blade for 15 min. This dual
fortified flour (500 g) was stored in air tight plastic containers of di­
mensions 10*10*12 cm length*width*height to replicate the household
storage conditions of flour, and stored under different combinations of
S.R. Bajaj and R.S. Singhal
temperature and relative humidity (RH) to mimic the effect of various
climatic conditions. The relative humidity conditions were kept constant
by using saturated saline solutions of magnesium chloride (RH 33 %),
sodium nitrite (RH 63 %) and potassium nitrite (RH 93 %) in desiccators.
Samples were kept in their respective desiccators and subjected to
storage in dark under temperature controlled cabinets to maintain
temperature. The combinations of temperature and relative humidity
used were: 25 ◦ C/33 % RH, 25 ◦ C/63 % RH, 25 ◦ C/93 % RH, 45 ◦ C/33 %
RH, 45 ◦ C/63 % RH, and 45 ◦ C/93 % RH. Analysis was carried out for
120 days (0, 15, 30, 45, 60, and 120 days) as average shelf life of
packaged wheat flour available in the Indian market is four months.
After withdrawing the sample for analysis, the containers were packed
and stored again under similar conditions until next analysis.
The higher concentrations of vitamin B12 and vitamin D3 were added
to the flour for analytical quantification purpose considering losses
during processing and storage. All the samples were analyzed on dry
weight basis.
2.2.2. Analysis of vitamin B12
Vitamin B12 was extracted from the whole wheat flour by mixing 10 g
of flour with 20 mL of methanol:deionised water (1:1) in an amber
colored conical flask with stopper on. The flask was kept on a rotary
shaker at room temperature (30 ± 1 ◦ C) for 30 min followed by soni­
cation for 15 min in a bath sonicator. This mixture was centrifuged at
5000 g for 15 min at 25 ◦ C. The supernatant was filtered through
Whatman® filter paper 1. This extract was stored in amber colored glass
bottle at 4 ◦ C prior to analysis. Extract was analyzed for concentration of
vitamin B12 using high pressure thin layer chromatography (HPTLC)
(CAMAG Linomat 5 autosampler, CAMAG TLC scanner 3, and winCATS
1.2.2 software, Switzerland) by a previously developed and validated
method (Bajaj and Singhal, 2019). The extraction recovery was deter­
mined by adding known quantity of vitamin B12 (100 μg/ g of flour) into
the flour and it was analyzed for concentration of vitamin B12 therein.
The loss occurring during analysis was calculated to determine extrac­
tion recovery. The extraction recovery (77 %) was always considered
during determination of vitamin B12. The results obtained for method
validation are given in the Table 1.
2.2.3. Analysis of vitamin D3
Vitamin D3 was extracted from whole wheat flour (10 g) by alcoholic
saponification using 5 mL potassium hydroxide (30 %) and 15 mL
ethanol on a water bath at 70 ◦ C for 30 min with intermittent shaking.
Chloroform (5 mL) was added to this saponified mixture and centrifuged
at 1500 g for 5 min at 20 ◦ C. The chloroform phase at the bottom was
collected in amber colored glass bottle. The extract was dried using ni­
trogen gas flushing to evaporate chloroform and the extract was
reconstituted in methanol. This sample was stored at 4 ◦ C prior to
analysis. Vitamin D3 in oil samples was analyzed similarly by saponi­
fying 0.25 g oil and 1 mL of 30 % potassium hydroxide:ethanol (1:3) and
extraction with 1 mL chloroform.
Vitamin D3 was analyzed using reverse phase high pressure liquid
chromatography (HPLC) (Dionex, Ultimate 3000 LC system, Thermo
Fisher Scientific) using the method of Kazmi et al. (2007) with slight
Table 1
Linearity range, correlation coefficient, regression equation, limit of detection,
and limit of quantification for vitamin B12 and vitamin D3.
Parameters Vitamin B12 Vitamin D3
Method HPTLC HPLC
Linearity range 0− 700 ng /spot 0–100 μg/injection
Correlation coefficient (R2) 0.98 0.99
Regression equation y = 8911x + 1004 y = 2.394x
Limit of detection 0.085 μg 6.820 μg
Limit of quantification 0.284 μg 22.750 μg
The results are average of n = 3.
3
Journal of Food Composition and Analysis 96 (2021) 103703
modifications. The sample (20 μL) was injected on C18 column (Waters
Spherisorb®5 μm, 4.6*250 mm, Ireland) using methanol:acetonitrile
(1:1) as the mobile phase at a flow rate of 1 mL/min for 12 min at 25 ◦ C
and using diode-array detector at 265 nm. External standard was used
for validation. Freshly prepared standard stock was used as external
standard during analysis and analysis was carried out in the dark to
avoid any error. The extraction recovery (97 %) was always considered
during determination. A standard graph prepared and method was
validated for limit of detection and limit of quantification (Table 1).
2.2.4. Kinetic calculations
A general reaction rate expression for degradation kinetics was
expressed earlier (Bajaj and Singhal, 2020):
− d[C]/dt = k[C]m (1)
C: Quantitative value of the product of degradation under
consideration
k: Reaction rate constant
m: Order of the reaction
Integration of Eq. (1) for first order equation can be written as:
lnCt /C0 = − kt (2)
C0: Concentration of vitamin B12 or D3 at time zero
Ct: Concentration of vitamin B12 or D3 at time ‘t’
t: Storage time in days
The time required for degradation to half of its original concentration
is called half-life. Half-life was calculated using the Eq. 3, given below:
t12 = 0.693/k (3)
/
2.2.5. Preparation and analysis of different products from fortified whole
wheat flour
Universally popular products such as cake, bread, and cookies as well
as Indian traditional products such as chapatti (Indian unleavened baked
flat bread) and poori (Indian unleavened fried flat bread) were prepared
with double fortified whole wheat flour and evaluated for retention of
vitamin B12 and vitamin D3.
2.2.5.1. Preparation of chapattis. Whole wheat flour and water (1:0.75)
were mixed in a Hobart mixer to prepare dough of desired consistency.
The dough balls (30 g) were prepared and rolled on a wooden flat
platform with rolling pin to a thickness of 2 mm and a diameter of 15 cm.
The chapattis so rolled were baked on a preheated flat metal pan at
200− 220 ◦ C on both sides followed by flame puffing. The total baking
time was 80–90 s.
2.2.5.2. Preparation of pooris. Whole wheat flour and water (1:0.7)
were mixed to prepare dough in a Hobart mixer and dough balls (20 g)
were rolled on a wooden flat platform to a thickness of 3 mm and
diameter of 7 cm. Oil was taken in a deep pan and heated to 170− 180
◦
C. Pooris were fried in this heated oil on both sides and removed
immediately after puffing and change of color to light brown. The total
frying time was around 20− 30 s.
2.2.5.3. Preparation of cakes. Egg was excluded from cake recipe to rule
out the effect of the indigenously present vitamins D3 and B12. The
preparation of eggless cake requires the use of additives which can alter
the pH of cake batter and the cake baked therefrom. Our earlier work
showed pH to show a significant correlation with degradation of vitamin
B12 (Bajaj and Singhal, 2020). Hence three different cake formulations
were developed on the basis of an earlier report (Godefroidt et al., 2019)
to determine interference, if any, of the ingredients on the stability of
vitamin B12 and vitamin D3 during the cake preparation. Model cake
(control) was prepared without any additives (fortified whole wheat
flour 100 g, sugar 50 g, fat 50 g, lecithin 0.5 g, and water 25 mL). Two
formulations were used to prepare sample cakes as reported by
S.R. Bajaj and R.S. Singhal
Godefroidt et al. (2019). Formulation 1 [flour (100 g), sugar (50 g), fat
(50 g), sodium bi-carbonate (2 g), tartaric acid (0.5 g), lecithin (0.5 g),
and water (25 mL)] and Formulation 2 [flour (100 g), sugar (50 g), fat
(50 g), sodium bi-carbonate (2 g), lecithin (0.5 g), and water (25 mL)]
were used to prepared cake batter. Fat, lecithin, and sugar were whipped
together followed by addition of water and mixing at medium speed in
Hobart mixer for 5 min to get a homogenous mix. Flour and remaining
dry ingredients were incorporated in this mixture to prepare the cake
batter of desired consistency. Mixing was carried out in single direction
to avoid rupture of air bubbles. Batter pH was determined on digital pH
meter. Batter was poured into a mould and cake was baked in a pre­
heated oven at 175− 180 ◦ C for 25 min. The pH of the baked cake was
determined on digital pH meter by mixing 10 g cake in 30 mL deionized
water.
2.2.5.4. Preparation of breads. The bread was prepared by straight
dough method (Shah et al., 2018). Yeast (2 g), sugar (12 g), and common
salt (1.5 g) were dissolved in cold water (70 mL) while the emulsifier,
diacetyl tartaric acid ester of monoglyceride or DATEM (0.4 g) was
added in fat (3 g). These ingredients were mixed with 100 g flour in a
Hobart mixer. The dough was kneaded repeatedly on slab to achieve
better puffing of dough and fine texture of bread. Proofing was carried
out in two stages (2 h and 1.5 h) at ambient temperature followed by
baking in a preheated oven at 200− 205 ◦ C for 20 min.
2.2.5.5. Preparation of cookies. The cookies were prepared with (sam­
ple) and without (model) sodium bi-carbonate and baking soda to
evaluate the effect of pH on stability of vitamin B12 and D3 during cookie
baking. An earlier published recipe was used to prepare the cookies
(Singh et al., 2008). The dry ingredients (fortified flour 100 g, common
salt 1 g, baking powder 0.5 g, and sodium bi carbonate 1 g) were mixed
in Hobart mixer for 2 min and kept aside. Fat (40 g) and sugar (30 g)
were creamed in a Hobart mixer and water (20 mL) was added to it with
constant mixing for 5 min to obtain a homogenous mixture. Dry in­
gredients were then added to prepare dough. The dough was sheeted
using a rolling pin to the thickness of 4 mm and cookies were cut with a
cookie die of 50 mm diameter. The cookies were transferred to a pre­
viously greased tray and baked at 200− 205 ◦ C for 12 min in a preheated
oven. Cookie dough and product were analyzed for pH following method
explained in section 2.2.5.3.
All the products were prepared in triplicates, cooled on metal rack,
and stored immediately at 4 ◦ C in aluminum zip lock pouches till further
analysis. The products were ground in a lab scale mixer grinder and
analyzed for retention of vitamin B12 and vitamin D3 using methods
explained in sections 2.2.2 and 2.2.3, respectively, as above.
Moisture content of samples was determined by hot air oven method
at 105 ◦ C. The moisture content on dry weight basis was calculated using
formula given in the Eq. 4:
Md = (Mw × 100)/
(100 − Mw )
Where, Md is moisture content on dry weight basis and Mw is moisture
content on wet weight basis.
True retention of vitamin after processing was calculated using for­
mula given in the Eq. 5 below:
(μg ofvitamin in product on dry weight basis × 100)
True retention (%) =
(μg of vitamin added in raw material on dry weight basis − extraction loss in μg)
Journal of Food Composition and Analysis 96 (2021) 103703
The poori fried oil was analyzed for concentration of vitamin D3 and
vitamin B12 by using method discussed in the section 2.2.3 and 2.2.2
respectively to evaluate any transfer of vitamin D3 and vitamin B12 from
dough to the oil during frying.
2.2.6. Fortification and analysis of rice bran oil
Vitamin D3 (250 μg/g of oil) was added to rice bran oil and kept at
ambient temperature for 12 h to ensure complete dissolution of vitamin
D3 in oil. Vitamin D3 content was analyzed using method outlined in
section 2.2.3. This fortified oil was used further for frying.
2.2.7. Preparation and analysis of dough and batter based fried products
and fried oils
Dough based product poori was prepared as per the method
explained earlier in section 2.2.5.2. Batter based product pakora (Indian
fried snack) was prepared from chickpea flour batter. Chickpea flour
(100 g), salt (2 g), red chilli powder (1 g), and water (70 mL) were mixed
to prepare batter. Fortified rice bran oil was heated to 170− 180 ◦ C and
batter was dropped into oil using table spoon in the form of small balls of
1.5–2 cm diameter. It was fried on slow flame until golden brown color
and then removed immediately. Frying of pakora required 60− 70 s.
Oil was used repeatedly for five consecutive frying cycles. After every
frying cycle, oil was allowed to cool down to room temperature and oil
sample was collected for analysis. Further the oil was heated for next
frying cycle. Samples of poori and pakora from each frying cycle were
cooled and kept separately in an aluminum foil zip lock bag at 4 ◦ C till
analysis.
Poori and pakora samples were ground in a lab scale mixer grinder.
Oil content of fried products was determined using pet ether as a solvent
using Soxhlet apparatus (Socs Plus SCS 6, Pelican Equipments, India) for
2 h. The oil content was calculated gravimetrically. Vitamin D3 from all
the product and oil samples was extracted and analyzed using methods
explained in section 2.2.3 as above. Retention of vitamin D3 was
calculated as per gram oil to compare the results.
2.2.8. Statistical analysis
IBM® SPSS® Statistics Version 20.0.0 was used for statistical anal­
ysis. A one-way ANOVA at p < 0.05 was used followed by LSD and
Duncan’s new multiple range tests. Each experiment was done in trip­
licate and average values were taken for the calculations. Kinetic data
were analyzed by regression analysis using MS Excel.
3. Results and discussion
3.1. Degradation of vitamin B12 in fortified whole wheat flour during
storage
(4) The temperature and relative humidity during food storage are well
known to influence the shelf life and retention of nutrients. Fig. 1(a) and
(b) represent the degradation kinetics of vitamin B12 during storage at
25 ◦ C and 45 ◦ C, respectively, at relative humidities of 33 %, 63 %, and
93 %. An increase in storage temperature accelerated the degradation of
vitamin B12 at all the three relative humidities evaluated in this work.
(5)
4
S.R. Bajaj and R.S. Singhal Journal of Food Composition and Analysis 96 (2021) 103703

Fig. 1. Degradation of vitamin B12 in fortified whole wheat flour during storage Fig. 2. Degradation of vitamin D3 in fortified whole wheat flour during storage
at different percent relative humidity (RH) at (a) 25 ◦ C, and (b) 45 ◦ C (n=3, SD is at different percent relative humidity (RH) at (a) 25 ◦ C, and (b) 45 ◦ C (n=3, SD is
indicated using error bars). indicated using error bars).

However relative humidity showed a significant effect on degradation of
vitamin B12 only at 45 ◦ C [Fig. 1(b)] with highest loss of 51.4 % being
recorded at 45 ◦ C/93 % RH as against 15 % at 25 ◦ C/93 % RH, both after
120 days of storage.
Hemery et al. (2019) also reported a positive correlation between
degradation of vitamin B12 and temperature. However, a negative cor­
relation between relative humidity and degradation of vitamin B12was
recorded in his work wherein 23 % loss of vitamin B12 at 40 ◦ C/85 % RH
and 63 % loss of vitamin B12at 40 ◦ C/65 % RH were reported after
6-month-storage. The findings in the present work are not in the com­
plete agreement with this earlier report. The reason could be the use of
different packaging materials used in the two studies, and also the fact
that the earlier study did not include data at very low relative humidity
(33 % RH). While air tight plastic containers were used in the present
work, Hemery et al. (2019) used paper bags for their study.
The half-life (t1/2) for loss of vitamin B12 was similar at 25 ◦ C at all
the three relative humidities under study (Table 2). The lowest half-life
of vitamin B12 during storage in fortified flour was recorded as 139 days
at 45 ◦ C/63 % RH and 45 ◦ C/93 % RH which suggest that wheat flour
can be effectively fortified with vitamin B12.
Table 2
Rate constant (k) (day− 1) and half life (t1/2) (days) of degradation of vitamin B12
and vitamin D3 in fortified whole wheat flour during storage under different
conditions.
Vitamin B12 Vitamin D3
3.2. Degradation of vitamin D3 in fortified whole wheat flour during
storage
Both temperature and relative humidity positively affected the
degradation of vitamin D3 during storage of fortified whole wheat flour
[Fig. 2 (a and b)]. The presence of double bonds in the structure of
vitamin D3 makes it vulnerable to oxidation. A study conducted on
storage stability of crystalline vitamin D3 at 25 and 40 ◦ C and 45 % RH
and 85 % RH reported it to remain stable at 25 ◦ C/45 % RH but degrade
at 25 ◦ C/85 % RH, 40 ◦ C/45 % RH, and 40 ◦ C/85 % RH. This was
attributed to the oxidation reaction in the side chain of vitamin D3
molecule (Grady and Thakker, 1980). Our findings are in accordance
with this earlier report. Lower storage temperature and relative hu­
midity indicated lower rate of degradation [Fig. 2(a)] and higher
half-life (Table 2). The highest degradation rate was observed at 45
◦
C/93 % RH [Fig. 2(b)] with lowest half-life of 63 days (Table 2).
The stability of vitamin D3 was lower than vitamin B12 during storage
in fortified whole wheat flour which indicated higher susceptibility of
vitamin D3 to degrade at high temperature and humidity in comparison
to vitamin B12. The degradation of vitamin D3 after 120 days storage at
25 ◦ C was 42 %, 47 %, and 50 % at 33 %, 63 %, and 93 % RH,
respectively, whereas it was 65 %, 71 %, and 77 % at 45 ◦ C, and 33 %, 63
%, and 93 % RH, respectively. To the best of our knowledge, no such
work has been reported till date on dual fortification of wheat flour with
vitamins B12 and D3.

Storage k (day− 1) at t1/2 (days) k (day− 1) at t1/2 (days) 3.3. Retention of vitamin B12 and vitamin D3 in processed products from
condition (% at at
fortified whole wheat flour
RH)
25 ◦ C 45 ◦ C 25 45 25 ◦ C 45 ◦ C 25 45
◦
C ◦
C ◦
C ◦
C Degradation of naturally occurring vitamins B12 and vitamin D3
33 0.001 0.004 693 173 0.004 0.008 173 87 during different processing techniques has been earlier shown to be
63 0.001 0.005 693 139 0.005 0.009 139 77 product and process specific in foods of animal origin (Jakobsen and
93 0.001 0.005 693 139 0.006 0.011 116 63
Knuthsen, 2014; Nishioka et al., 2011). However, degradation of these
The results are average of n = 3. micronutrients in many fortified products is yet to be explored. Whole

5
S.R. Bajaj and R.S. Singhal Journal of Food Composition and Analysis 96 (2021) 103703

wheat flour is a widely consumed commodity food in the form of various
products prepared through commonly used processing techniques such
as baking and frying. The effect of baking on the retention of vitamins D3
and B12 from fortified wheat flour in products such as cakes, bread,
cookies and chapattis, and frying as in the case of pooris is presented in
Table 3. The stability of vitamin B12 (Garrod et al., 2019) and D3
(Jakobsen and Knuthsen, 2014) in bread and cake has been evaluated
earlier, but there are no scientific reports on the retention of vitamin B12
and vitamin D3 in cookies, chapattis, and pooris prepared from fortified
whole wheat flour to the best of our knowledge.
Vitamin B12 is reported to be stable during cooking (Allen et al.,
2010) and does survive baking too (Garrod et al., 2019). Among all the
products, retention of vitamin B12 was found to be the maximum at 90.6
% in chapattis (Table 3). This could be due to very low baking time of
80− 90 s and also immediate cooling due to its flat surface and low width
as compared to that of other products. The retention of vitamin B12 in
wheat bread was found to be 79.7 % which agreed closely with retention
of 77 % as reported by Winkels et al. (2008) (Table 3). Tabibian et al.
(2017) reported the degradation rate of fortified vitamin to increase
with an increase in processing temperature. Cakes, breads, and cookies
were exposed to high temperature for longer time which did result in
lower retention of vitamin B12 in these products as compared to
chapattis.
Leavening agents are used alone or in combination with an acid in
eggless bakery products to provide aeration to the product (Godefroidt
et al., 2019). Addition of these compounds changes the pH of the cake
batter and cookie dough. Strongly acidic and alkaline conditions are
reported to degrade vitamin B12 rapidly (Bajaj and Singhal, 2020).
Hence the effect of chemical ingredients used in cake and cookies on the
stability of fortified vitamins was evaluated. This was the basis of using
formulation 1 (sodium bicarbonate + tartaric acid) and formulation 2
(sodium bicarbonate) for preparation of cakes. On similar lines, cookies
were prepared with and without sodium bicarbonate. Model sample for
both the product was prepared without any addition of any chemical
agents. The changes in the pH of the batter/dough and product were not
found to be significantly different among the formulations (Table S2
from supplementary file). The differences observed in the retention of
vitamin B12 between the control and the formulated cakes and cookies
were not very large (Table 3).
The lowest retention of vitamin B12 was recorded in pooris prepared
by frying. During frying, water escapes as steam and is replaced by the
frying oil which is at very high temperature. This explains the reasons
for poor retention of vitamin B12 in pooris. The wide difference between
confidence intervals and actual value (Table 3) for retention of vitamin
B12 during frying is result of the fluctuations in processing parameters
during frying which can generate instability in the results. The fried oil
was also evaluated for concentration of vitamin B12 to confirm the
possibility of transfer of vitamin B12 from dough to frying oil during
frying, but no detectable amount of vitamin B12 was seen in the oil.
Frying has been reported to degrade vitamin C in potato at higher rate
than baking and boiling (Ruiz-Roso, 1998).
Vitamin D3 undergoes isomerization and/or oxidation during heat­
ing which results in its degradation (Mahmoodani et al., 2017). Among
the various products made from fortified wheat flour, the retention of
vitamin D3 was the maximum (> 80 %) in cake followed by cookies,
bread, chapatti, and poori (Table 3). An earlier report showed 64 %
retention of vitamin D3 in the cakes prepared with fortified margarine
(Jakobsen and Knuthsen, 2014). The retention of vitamin D3 in cakes
was higher in the present work in comparison to earlier published work.
There is difference in the temperature and time of baking in present
work (175− 180 ◦ C for 25 min) in comparison with the temperature and
time (175 ◦ C for 60 min) used by Jakobsen and Knuthsen (2014) which
resulted in better retention of vitamin D3 in the present work. Cake and
cookies have high fat content which may have provided a good medium
for dissolution of vitamin D3 and hence protected it from degradation
during baking. No significant effect of cake and cookie formulation was
seen on the retention of vitamin D3 in control products and that prepared
with additives (Table 3). Besides, vitamin D3 is known to be stable under
alkaline conditions (Kendrick, 2007).
The retention of vitamin D3 was lower in bread and chapattis both of
which were prepared from fortified whole wheat flour. One of the rea­
sons for this observation could be the higher temperatures during
baking, exposure of dough/product to oxygen causing oxidative degra­
dation, and low fat content in these products. Preparation of bread is a
longer process and dough is exposed to oxygen in the atmosphere during
different processing steps viz. kneading and proofing. Chapattis has wide
surface area hence exposure of the product to the air is more. Lower fat
content may have caused higher oxidation of vitamin D. Jakobsen and
Knuthsen (2014) showed lower retention of vitamin D3 in rye bread (69
%) than in wheat flour bread (85 %) which was attributed to the acidic
isomerization of vitamin D to isotachysterol. The degradation of vitamin
D3 was very rapid in pooris which can be due to the migration of vitamin
D3 during frying. Retention of vitamin D3 in pooris showed wide dif­
ference between actual value and confidence interval (Table 3), similar
to that of retention of vitamin B12, which indicated that the variation in
the processing parameters caused instability in the retention during
frying. It is hypothesized that vitamin D3 may have migrated from dough
to oil during frying due to its lipophilic nature. This possibility was
confirmed by analyzing fried oil for vitamin D3 content. Absorption of
vitamin D3 in fried oil from dough was recorded 10 ± 2.11 percent of
that present in the dough before frying. Hence products fortified with fat
soluble vitamins are not suitable for processing operations like frying.
However, encapsulated vitamin D is used for fortification of food in the
food industry. Encapsulation increases stability of vitamins (Wilson and
Shah, 2007) and use of encapsulated vitamin D may give better retention
during processing. It is necessary to collect data on stability of

Table 3
Percent retention of vitamin B12 and vitamin D3 in products prepared from fortified whole wheat flour.
Confidence interval for B12 Confidence interval for D3
Percent retention of (95%) Percent retention of (95%)
Time and temperature of
Product vitamin B vitamin D
cooking Lower Upper Lower Upper
12 3
bound bound bound bound

Pooris 170− 180 ◦

C, 20− 30 s 13.9 ± 0.7 h − 78.3 − 7.9 9.5 ± 1.0 e − 77.0 − 17.3
Chapattis 200− 220 ◦
C, 80− 90 s 90.6 ± 1.0a 9.4 78.3 28.5 ± 1.0d − 58.0 20.6
Cake (control)* 175− 180 ◦
C, 25 min 35.8 ± 0.9c − 56.4 23.5 84.2 ± 0.6a − 2.3 76.3
Cake sample 1** 175− 180 ◦
C, 25 min 33.7 ± 1.4d − 58.5 21.4 84.9 ± 0.6a − 0.9 77.0
Cake sample 2*** 175− 180 ◦
C, 25 min 32.1 ± 0.7e − 60.2 19.7 83.5 ± 1.4a − 3.0 75.6
Bread 200− 205 ◦
C, 20 min 79.7 ± 0.3b − 12.6 67.3 40.2 ± 0.8c − 46.4 32.3
Cookie (control)* 200− 205 ◦
C, 12 min 24.5 ± 0.4f − 67.7 12.2 65.3 ± 1.8b − 21.2 57.5
Cookie 200− 205 ◦
C, 12 min 23.4 ± 1.4fg − 68.8 11.1 64.2 ± 1.8 b − 22.4 56.3
sample****

Values with different letters are significantly different at p<0.05. Values are average of n=3 ±SD. s: second (*Prepared without any additives, ** prepared with sodium
bicarbonate, tartaric acid, and lecithin; *** prepared with sodium bicarbonate and lecithin; **** prepared with sodium bicarbonate and baking soda).

6
S.R. Bajaj and R.S. Singhal
micronutrients in a wide range of fortified products and its successful
implementation.
3.4. Degradation of vitamin D3 during multiple frying cycles
The global market size of edible oil is more than 165 million tons of
which rice bran oil has a share of 1.2 million tons of market size. India is
the largest producer of rice bran oil in the world. Rice bran oil is popular
edible oil due to its good oxidative stability and the presence of health
promoting compounds such as tocopherols, tocotrienols, phytosterols,
and γ-oryzonal (Maszewska et al., 2018). It also showed good solubility
of vitamin D3 over other oils (data not shown). Hence rice bran oil was
selected for this study. Frying oils are used for multiple frying cycles,
both in the households and industrial sectors. Hence this study was
designed to study the effect of multiple frying cycles using the
commonly used dough based food matrix (poori) and a batter based food
matrix (pakora). Fig. 3(a) represents concentration of vitamin D3 in
control (non-fried) oil and fried oil during the course of five consecutive
frying cycles in both the products. A rapid reduction of vitamin D3 in the
frying oil immediately after the first cycle of frying for both food
matrices was seen. Frying leads to many chemical reactions in the oil
such as oxidation and polymerization which are known to degrade fat
soluble vitamins in oil (Hrncirik, 2010). Migration of moisture and other
constituents from the food to the oil also causes degradation of vitamins
in oil during frying (Hrncirik, 2010). The degree of unsaturation of the
oil, type of food material, and heating the oil at high temperatures in the
presence of air are some of the major contributing factors to the progress
of degradative reactions in the oil during frying which degrade the
Fig. 3. Effect of frying cycles (FC) on the concentration of (a) vitamin D3 (μg/ g
of oil) in the frying oil, (b) vitamin D3 (μg/g of oil) in the oil absorbed in the
fried product, and (c) vitamin D3 (mg/100 g of product) in the fried product
(pooris and pakoras) (n=3, SD is indicated using error bars).
7
Journal of Food Composition and Analysis 96 (2021) 103703
fortified vitamins in oil (Holownia et al., 2001).
Vitamin D3 was extracted by saponification from product and
analyzed. The results obtained were total vitamin D3 content of the
product. The moisture content and oil absorption for both the dough
based and batter based products were different. Hence efforts were made
to study the concentration of vitamin D3 in the oil absorbed by the fried
products during each frying cycle and the results so obtained were
represented as vitamin D3 /g oil [Fig. 3(b)]. From the consumer
perspective, the concentration of vitamin D3 was expressed per unit
weight of the product (food + oil) [Fig. 3(c)]. Moisture content of pooris
ranged between 31–39 % and for pakoras it was 50–54 % on dry weight
basis (Table S3 in the supplementary file). Oil absorption rate increased
consecutively during each frying cycle for both the products (Fig. 4). The
increase in the absorption of oil in the course of multiple frying cycles is
a function of increased viscosity, polymerization, and oxidation of oil
during frying (Debnath et al., 2012). Absorption of oil was recorded
higher in pooris than in the pakoras. Both pooris and pakoras showed
highest absorption of vitamin D3 during first frying cycle [Fig. 3(b)]
beyond which the concentration of vitamin D3 decreased steadily up to
fifth frying cycle. This was evident both from the concentration of
vitamin D3 in the oil absorbed by the products after each frying cycle
[Fig. 3(b)] as well as in the product itself [Fig. 3(c)]. This indicates that
the effect of fortification of edible oils with vitamin D3 is substantially
and consecutively reduced during successive frying cycles. Holland et al.
(1991) reported on migration of vitamin E from the frying oil to the
product during deep fat frying in an effort to increase the content of
vitamin E of the fried product. Considering fortification limits for
vitamin D3 (10 μg/100 g oil), 100 g fried pooris and pakoras each can
supply approximately 10 % and 3% of the RDA, respectively, but only
after the first frying cycle.
Vitamin D3 absorption was lower in pakoras than in pooris which may
be because of difference in the moisture content of food matrix, frying
time, and cooling time of the products. Pooris required less time for
frying (20− 30 s) and cooling than pakoras (60− 70 s) due to difference in
the geometry of the products. This reduced heat exposure of the pooris
and retained higher concentration of vitamin D3 than the pakoras. These
results indicate the efficiency of frying oil to transfer vitamin D3 into the
fried product. Fried foods are vastly popular, and hence fortification of
oil can be nutritionally advantageous.
Fortification of commodity food products will be effective only if
fortified nutrient survives processing. Whole wheat flour and rice bran
oil are commonly used in preparation of many food products all over the
world. These products undergo many operations during processing
wherein the processing parameters and the conditions of subsequent
storage influence the content of the vitamin fortificants. This work
documents the processing and/or storage losses of fortified vitamin B12
and vitamin D3 to establish suitable fortification limits of commonly
consumed raw materials such as whole wheat flour and edible oil. Pooris
Fig. 4. Concentration of oil in fried products during multiple frying cycles (FC)
(n=3, SD is indicated using error bars).
S.R. Bajaj and R.S. Singhal
and pakoras showed 40 % and 13 % absorption of vitamin D3 during first
frying cycle, respectively. Overages of approximately 250 IU vitamin D/
100 g oil can meet 100 % RDA through pooris. Chapatti is a staple food in
many parts of India. Vitamin B12 degradation is negligible in chapattis
and hence the current guidelines of 10 μg of vitamin B12/kg of wheat
flour are suitable for fortification. However, vitamin D3 fortification of
wheat flour will need approximately overages of 300 IU/kg flour to meet
RDA.
4. Conclusion
The present study highlighted that an increase in the storage tem­
perature increased the rate of degradation of vitamin B12 and vitamin D3
in fortified whole wheat flour during storage. Relative humidity posi­
tively affected the degradation of vitamin D3 in whole wheat flour
during storage. Retention of vitamin B12 and vitamin D3 varied in
different food products prepared from fortified whole wheat flour with
maximum retention of vitamin B12 in chapatti (91 %) and vitamin D3 (84
%) in cake. Fortified micronutrients degraded rapidly during frying and
showed lowest retention of both the vitamins in pooris. Fortification of
edible oil demonstrated significant migration in both the dough based
and batter based product only after the first frying cycle with better
migration in dough based product than the batter based one. Pooris
absorbed 40 % of the fortified vitamin D3 from oil during the first frying
cycle. Successive frying cycles decreased the migration substantially and
consecutively during subsequent frying cycles.
CRediT authorship contribution statement
Seema R. Bajaj: Methodology, Data curation, Writing - original
draft, Software, Validation, Visualization, Investigation. Rekha S. Sin­
ghal: Conceptualization, Supervision, Writing - review & editing.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
The authors are grateful to the University Grants Commission,
Government of India, for providing financial assistance during the
course of this investigation.
Fellowship number: F.25-1/2014-15(BSR)/No.F.5-62/2007(BSR)
dated on 16th Feb 2015
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jfca.2020.103703.
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