This study investigated the thermal stability of vitamin D fortified groundnut oil. Vitamin D extraction and quantification methods were optimized. The oil was tested for vitamin D concentration after heating to temperatures from room temperature to 140°C for an hour. Ascorbyl palmitate was found to be a more effective antioxidant than vitamin E for maintaining vitamin D levels, showing a two-fold increase in vitamin D concentration compared to vitamin E. Further studies could explore other antioxidants and oils for vitamin D fortification.

1. thermal stability study of vitamin d fortified oil
Darshana Nadar
Msc FST-1-15018
Supervisor : Dr Azza Silotry Naik
1

2. Graphical Abstract
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3. 3

4. Significance of fortification
Fortification in India
Need for Vitamin D fortification
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5. 8 out of 10
INDIANS SUFFER
VITAMIN D
DEFICIENCY
Vitamin D Status in India and
around the world.
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6.  Vitamin D Chemistry
 Vitamin D Sources : Plant and animal sources
 Role of Vitamin D in the body: calcium absorption, fight against
infection, Depression, cancer
Ref: Novak & Potts, 1997)
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7. Serum 25 [OH]D Level Interpretation
< 20ng/mL Vitamin D deficiency
21-29 ng/mL Vitamin D insufficiency
>30 ng/mL Vitamin D Sufficiency
>150 ng/mL Vitamin D toxicity
Vitamin D transformation in body
Levels of 25[OH]D in the body
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8. RDA
 ICMR-2010, 400 I.U/day i.e 10µg ( Not specific age group)
 U.S.D.A – 600 I.U/day i.e 15µg ( 1-70 years, both male and female)
 1 I.U-0.025 µg
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9. Product Brand Name Concentration of vitamin D
100g
Bread spread
or butter
Amul lite 5µg
Margarine Delicious table
margarine
10-15µg/ qt
Milk Amul slim & trim 0.5µg
Sunflower oil Sundrop 15 µg
Refined
sunflower oil
Sunrich 5µg
Refined
sunflower oil
Sweekar 5µg
Products fortified with Vitamin D in Indian
Market
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10. Fortification strategy.
 A person should consume 70 gram/day oil and 15µg/day
So,0.214 µg/ml of vitamin D
 Products available in market provide 5µg/100 grams oil
So, 0.05µg/mL of vitamin D
 FSSAI – 0.112 µg/ml of Vitamin D
 The concentration of vitamin D fortified Oil in this study is 50 µg/mL
which exceeds the RDA values
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11. Oil Fortification cost
 Mainly Fortification Cost in premix
 10-12 Paise per kg of oil
 Testing and Monitoring cost
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12. Objective of the study
 Maximize the recovery of vitamin D using HPLC
 Study thermo oxidative stability of vitamin D Fortified oil
 Study thermo oxidative stability of vitamin D Fortified oil
in presence of antioxidants
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13. 13

14. MATERIALS
 Cholecalciferol or vitamin D3 (FBL,Thane,India)
 Antioxidants: Tocopherol ( Godrej, Mumbai), Ascorbyl Palmitate(
Kohinoor, Khopoli,India)
 Solvents : Acetonitrile, Methanol, Ethanol, water
( Merck, New jersey, USA)
 Groundnut oil ( Brand Name: Svitty, kamani oils, Khopoli, India)
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15. HPLC
 C -18 guard column (4.6 mm × 150 i.d., particle size 5 μm)
 UV- detector- 265nm
 Injection Volume 100 to 200 μL ( Actual Volume 20 μL)
 Mobile Phase Acetonitrile: Methanol (8:2)
 Stationary Phase: silica
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16. Statistical Analysis
 Empower pro ( Waters, Bangalore, India)
 Graph Pad ( La jolla, U.S.A)
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17. Method
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18. 18

19. y = 57815x - 161785
R² = 0.9821
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
0 10 20 30 40 50 60
AUC
Concentration of Vitamin D3( µg/ml)
Standard graph (using AOAC Method 2002.05) 10-50µg/ml)
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20. Solvent Optimization
Ethanol showed higher percent of recovery than Methanol. Hence
Ethanol was the solvent of choice for the study
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21. Volume optimization
Volume of ethanol added to
extract D3
% recovery
2.5 40.472
Re-extract
Total = 2.5 + Re extract
27.41
67.883
5 67.52
10 80
10 ml Volume of ethanol showed highest percent recovery of
Vitamin D3.
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22. Thermal Stability Study
0
5
10
15
20
25
30
35
40
45
GO-34 GO-50 GO-80 GO-110 GO-140 GO-170
ConcentrationofvitaminD3(µg/ml)
Temperature ºC
* *
*
P- 0.0001
P- 0.0115 P-0.0004
At 110ºC groundnut oil showed drastic decrease in vitamin D3 concentration
At 140ºC there was an increase in vitamin D3 concentration in oil this might be due to thermal isomerization
of vitamin D3 to pre-vitamin D3 (Mahmoodani, Perera, Fedrizzi, Abernethy, & Chen, 2017).
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23. Hypothesis:
1. If Pre Vitamin D and Vitamin D have same retention time
2. If Pre Vitamin D and Vitamin D do not have same retention time.
At 110º C, the vitamin D content is low, it might be because vitamin D is
present in its pre-form and cannot be detected as they have different
retention time. At 140ºC, the pre-form is getting converted to vitamin D which
is detected and therefore there is an increase in vitamin D content at 140 ºC
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24. Thermal Stability study using Vitamin E
antioxidant
The reason behind not much increase in the concentration is antioxidant vitamin E is
completely evaporated at a high temperature on groundnut oil (Huang et al., 1995).
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
110-D
110-D&E
Concentration of vitamin D3 (µg/ml)
Sample
*
P-0.0035
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25. Thermal Stability study using Ascorbyl
palmitate antioxidant
0 0.5 1 1.5 2 2.5 3
110-D
110-D-AP
Concentration of vitamin D3 (µg/µL)
Samples
Ascorbyl palmitate 0.02% w/v was incorporated in 50µg/ml of
vitamin D fortified oil. 3 fold increase in the concentration of vitamin
D3 when compared to control samples
*
P-0.0001
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26. Correlation of Vitamin D & Vitamin D + E and Vitamin D +
AP fortified oil
0 0.5 1 1.5 2 2.5 3
CONTROL
VITAMIN E
ASCORBYL PALMITATE
Concentration of Vitamin D3 (µg/ml)
Samples
*
P-0.001
Ascorbyl palmitate showed 2 fold more antioxidant ability than Vitamin E in
groundnut oil
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28.  Extraction method was optimized
 Thermal stability status of groundnut oil fortified with vitamin D
was studied ranging from room temperature to near frying
temperature for an hour
 Vitamin D fortified oil along with two antioxidants was tested
 In Groundnut oil Ascorbyl palmitate showed better antioxidant
capacity than vitamin E
 Ascorbyl Palmitate showed two fold increase in Vitamin D3
Concentration than Vitamin E i.e AP 2.59 and Vitamin E
1.49µg/ml
 Vitamin D3 fortified oil was formulated
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30.  Synergistic effect under study
 Confirmation of hypothesis by LC-MS
 Antioxidant capacity can be studied in other temperatures
 Other Natural antioxidants can be incorporated to study its capacity in
Vitamin D fortified oil
 Different edible oils like sunflower oil, sesame oil, rape seed oil etc
can be formulated with vitamin D3 and studied.
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