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Article history:
Received 14 May 2013
Received in revised form
30 September 2013
Accepted 23 November 2013
Stability of vitamin D2 in milk was determined in vitamin D2 fortied milk. Reverse phase high pressure
liquid chromatography was used to determine the vitamin D2 loss during processing, packaging and
under light. The percentage losses during pasteurization, boiling and sterilization were demonstrated to
be statistically insignicant. Milk was stored for seven days in both glass and plastic bottles under
refrigerated temperature, non signicant loss of vitamin D2 was observed, whereas, when stored in
polyethylene pouches signicant loss was observed as vitamin D2 decreased from 596.66 to 548.04 IU.
This clearly indicated that vitamin D2 was sorbed up by polyethylene material during storage resulting in
its loss. Milk samples were stored for 32 h under three different light intensities (14,852,970 and
4455 lux). Non signicant loss of vitamin D2 was observed in glass packaging, whereas signicant loss
was observed in polyethylene pouches. In milk fortied with both calcium and vitamin D2, non significant effect of calcium was observed on the loss of vitamin D2.
2013 Elsevier Ltd. All rights reserved.
Keywords:
Vitamin D2
Pasteurization
Sterilization
Light
Packaging
1. Introduction
Vitamins are vital food ingredients for maintaining good health
in humans; lack of a sufcient amount of any of them can cause
serious diseases (Riaz, Asif, & Ali, 2009). The human diet does not
always contain the amount of vitamins needed for normal development and maintenance of body functions (Gomez & Jose, 2006).
For this reason, several food products are fortied with vitamins,
mainly milk and milk products.
Vitamin D is a fat-soluble vitamin recognized for its importance
in skeletal health (Ceglia, 2009). Vitamin D is present in animal
foods as cholecalciferol (vitamin D3). Ergocalciferol (vitamin D2)
has plant origin, where it is converted from the provitamin,
ergosterol. The two vitamin D forms differ by the side chain to the
sterol skeleton and in the hydroxylated products (Mawer et al.,
1998).
Numerous studies have shown that the vitamin D status is far
from optimal in many countries all over the world. The main reason
for this is lack of sufcient solar Ultra Violet Blue (295e315 nm). If
ones exposure to sunlight is limited, vitamin D deciency may
develop, and the need for supplementation, either as an oral
422
Cow and buffalo milk were collected from Cattle yard, National
Dairy Research Institute (Karnal, India). Encapsulated vitamin D2
(100,000 IU/g) was obtained from DSM Nutritional Products
(Singapore) and calcium phosphate was obtained from Himedia
(Himedia, Mumbai, India). All solvents and reagents employed in
this study were HPLC-grade. Water, hexane and pyrogallol were
purchased from Rankem (New Delhi, India). Acetonitrile, methanol
and chloroform were procured from Sigma Aldrich (St. Louis, MO,
USA). Potassium hydroxide, Ammonium hydroxide and HCl (AR
grade) were procured from Fisher Scientic (Mumbai, India). Ethyl
alcohol (99.9%) was procured from Jiangsu Huaxi International
(China).
2.8. Recovery
% Recovery of vitamin D2 using above developed method from
vitamin D2 fortied milk samples (600 IU/L) was estimated. The %
recovery of vitamin D2 from fortied milk samples was calculated
against the standardized chromatographic conditions. % Recovery
was calculated as
% recovery
X
100
Y
where,
X vitamin D2 recovered after analysis
Y vitamin D2 added to milk
423
424
Table 1
Effect of heat treatment on vitamin D2 retention in milk.
Vitamin D2 content in milk (IU/L)
Samples
CTRL
CTRL VD600
CTRL VD600 CaP600
Raw milk
Pasteurized milk
Boiled milk
Sterilized milk
ND
594.28 2.22aA
593.13 2.79aA
ND
591.30 3.40aA
592.04 4.23aA
ND
589.97 2.76aA
588.43 2.51aA
ND
590.01 1.61aA
588.49 2.04aA
Table 2
Effect of storage in glass bottles at 4e7 C on vitamin D2 retention in milk.
Storage time/
SampleY
(CTRL + VD600)
(CTRL + VD600 + CaP600)
3rd day
aA
599.80 3.23
599.72 3.84aA
5th day
aA
598.40 2.50
598.19 3.31aA
7th day
aA
596.82 2.22
596.89 2.72aA
594.94 1.61aA
595.15 1.46aA
Table 3
Effect of storage in plastic bottles at 4e7 C on vitamin D2 retention in milk.
Storage time/
SampleY
(CTRL + VD600)
(CTRL + VD600+CaP600)
3rd day
5th day
7th day
597.60 3.47aA
597.66 3.73aA
597.36 2.72aA
597.29 3.48aA
593.82 4.14aA
593.46 3.81aA
592.98 3.77aA
592.63 3.09aA
425
Table 4
Effect of storage in polyethylene pouches at 4e7 C on vitamin D2 retention in milk.
Vitamin D2 content in milk (IU/L)
Storage time/
SampleY
0 day
CTRL + VD600
CTRL + VD600 + CaP600
3rd day
aA
5th day
bA
566.10 11.90
568.84 13.43bA
596.66 3.16
595.78 4.02aA
7th day
bcA
548.04 6.25cA
547.82 5.66cA
554.12 8.40
554.79 9.17bcA
Table 5
Effect of light on vitamin D2 retention in milk packed in glass bottles.
Light intensity
1485 lux
2970 lux
4455 lux
CTRL + VD600
CTRL + VD600 + CaP600
CTRL + VD600
CTRL + VD600 + CaP600
CTRL + VD600
CTRL + VD600 + CaP600
0h
597.00
597.05
597.42
597.17
598.36
597.37
2h
4.49aA
4.15aA
4.72aA
3.67aA
3.19aA
4.54aA
596.22
596.22
596.73
597.51
597.06
596.51
4h
4.26aA
4.26aA
4.29aA
3.49aA
2.79aA
4.45aA
595.42
596.33
596.01
597.13
596.62
595.83
8h
3.58aA
3.27aA
4.35aA
3.17aA
2.98aA
4.23aA
595.39
597.59
595.77
596.83
595.36
594.21
16 h
3.72aA
1.61aA
4.40aA
3.78aA
2.60aA
4.03aA
594.97
594.35
594.61
595.47
594.56
594.83
2.90aA
2.32aA
3.93aA
3.80aA
2.93aA
4.12aA
32 h
594.76
594.62
594.21
594.30
594.97
594.16
3.04aA
2.66aA
4.18aA
3.05aA
2.97aA
4.66aA
Table 6
Effect of light on vitamin D2 retention in milk packed in polyethylene pouches.
Light intensity
1485 lux
2970 lux
4455 lux
CTRL + VD600
CTRL + VD600 +CaP600
CTRL + VD600
CTRL + VD600 +CaP600
CTRL + VD600
CTRL + VD600 +CaP600
0h
594.12
594.06
596.10
596.22
595.44
597.18
2h
1.44aA
1.44aA
1.56aA
1.80aA
3.36aA
3.00aA
584.04
584.22
579.9
579.66
579.06
578.58
4h
9.00aA
8.64aA
7.44aA
7.56aA
7.68aA
8.16aA
567.42
567.72
564.42
564.36
563.94
563.82
8h
6.24bA
5.64bA
6.96bA
7.32bA
4.56bA
4.56bA
547.32
547.38
544.62
544.20
545.22
544.38
15.48cA
15.60cA
13.44cA
13.32cA
15.12cA
15.24cA
16 h
538.38
538.62
537.72
536.34
529.26
528.72
7.68cA
8.28cA
6.84cA
6.84cA
8.88cA
8.64cA
32 h
532.38
532.38
529.74
529.56
528.30
528.54
6.84cA
6.84cA
4.8cA
5.04cA
3.36cA
3.12cA
426
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