LWT - Food Science and Technology 56 (2014) 421e426

Contents lists available at ScienceDirect

LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Vitamin D2 stability in milk during processing, packaging and storage

Ravinder Kaushik 1, Bhawana Sachdeva 2, Sumit Arora*
Dairy Chemistry Division, National Dairy Research Institute, Karnal 132001, Haryana, India

a r t i c l e i n f o

a b s t r a c t

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

* Corresponding author. Tel.: 91 0184 2259156 (O), 91 9896054444 (M);

fax: 91 0184 2250042.
E-mail addresses: ravinder_foodtech2007@rediffmail.com (R. Kaushik),
bhaona.31s@gmail.com (B. Sachdeva), sumitak123@gmail.com (S. Arora).
1
Tel.: 91 9416962729 (M).
2
Tel.: 91 8930691698 (M).
0023-6438/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.lwt.2013.11.029

supplement or through innovations in food fortication is

compelling. In sunnier countries this is due to clothing habits and
avoidance of sunshine. Low serum vitamin D concentrations indicating deciency have been found in all age groups and in North
America and in different parts of Europe (Ovesen, Brot, & Jakobsen,
2003). There are limited dietary sources of vitamin D, including cod
liver oil, fatty sh such as salmon, as well as small amounts found in
egg yolks (Byrdwell et al., 2011). Vitamin D contents of whole milk
provide only 1% of the daily value of vitamin D (Holden, 2009).
Vitamins are commonly used to supplement milk products,
either to restore production losses, or to further enhance their
availability to potentially at-risk groups (WHO/FAO, 2006). Micronutrients share a general liability when exposed to thermal stress
(Riaz et al., 2009). Such potential losses during production are to be
distinguished from those further accruing under post-production
storage conditions. Overall nutrient losses are therefore generally
compensated by the addition of an overage during manufacture,
designed to be vitamin and product specic (Mays, 1982).
Tanner et al. (1988) reported signicant discrepancies between
analyzed values of vitamin D and fortication in uid milk samples,
thus this matter merits attention. Such inconsistencies between
claimed and measured values in vitamin D content have been
associated with chemical breakdown and/or processing effects
(Renken & Warthesen, 1993). Vitamin D stability studies are limited
and several contradictory results have been reported. Degradation
of vitamins depends on specic conditions during the culinary
process e.g., temperature, oxygen, light, moisture, pH and during
heat treatments (Leskova et al., 2006). Kreutler (1980) reported that

422

R. Kaushik et al. / LWT - Food Science and Technology 56 (2014) 421e426

vitamin D3 was remarkably stable to light, heat and oxidation.

Kutsky (1981) reported that vitamin D3 was unstable to oxidation
and light, but stable to acid and alkali. Pike and Brown (1984) reported that it was unstable to irradiation and acid while remaining
stable to oxidation and alkali. Cremin and Power (1985) reported
that vitamin D3 was unstable to oxidation, light and acid. Vitamin D
was not stable to heat, light and oxidation must be taken into account (Blanco, Fernandez, & Gutierrez, 2000). Vitamin D was stable
to oxidation, acid and unstable to heat, moisture and trace minerals.
It was destroyed by over treatment by UV light (Charlton & Ewing,
2007).
It is quite clear that individual studies have provided quite
contradictory data and it appears that its stability is strongly
dependent on the food in which it is present. Upreti, Mistry, and
Warthesen (2002), Wagner, Sidhom, Whiting, Rousseau, and
Veith (2008), and Ganesan, Brothersen, and McMohan (2011)
examined fortication of pasteurized processed cheese with
vitamin D, and reported no loss of vitamin D3 over 9 months of
storage. Vitamin D3 appears to be stable in cheese during both
short-term (Banville, Vuillemard, & Lacroix, 2000) and long-term
storage (Kazmi, Veith, & Rousseau, 2007; Wagner et al., 2008).
Riaz et al. (2009) reported that vitamin D3 was stable during
extrusion, pelleting and is stable to oxidation, light and heat. Processing and cooking conditions cause variable losses of vitamins.
Losses vary widely according to cooking method and type of food.
The ability of a vitamin to retain its activity during storage and
under chemical and physical stress refers to its stability.
Vitamin D may have toxic effects above a certain threshold
concentration (Upreti et al., 2002). It was therefore, important to
determine the stability of vitamin D during processing, packaging
and subsequent storage. This will help in delivering the desired
amount of vitamin D2 in fortied milk, which will be benecial to
both the consumers and milk industry.

actinic glass vessels. Vitamin D2 was added to milk at the level of

600 IU/L based on its recommended daily allowance (ICMR, 2009).
The vitamin D2 solution was rst mixed in an aliquot of toned milk
and then mixed to the bulk milk. The contents were then agitated
for complete mixing. Vitamin D2 and calcium (600 ppm calcium)
fortied milk was also prepared to determine any effect of calcium
on vitamin D2 recovery.
2.4. Processing treatments
Milk samples were pasteurized at 63  C for 30 min, boiled and
sterilized at 121  C for 15 min at 15 psi pressure in air tight glass
bottles. The samples were immediately cooled to 4  C. After 2 h of
storage at 4  C, milk samples were analyzed for vitamin D2 content
and loss of vitamin D2 was calculated.
2.5. Storage stability of vitamin D2
The loss (%) of vitamin D2 in fortied toned milk during storage
was determined. The milk samples were fortied, pasteurized and
packed in glass and plastic bottles and polyethylene pouches and
stored at 4  C for seven days. Vitamin D2 content was analyzed at
0 day and after 3rd day, 5th day and 7th day of storage.

2.6. Effect of light on vitamin D2

The effect of light on loss (%) of vitamin D2 in fortied milk was
determined. The milk samples were fortied, pasteurized and
packed in glass bottles and polyethylene pouches and stored at
refrigeration temperature (4  C) under three light intensities viz.
1485, 2970 and 4455 lux. The loss of vitamin D2 under light was
estimated after 2, 4, 8, 16 and 32 h exposure of light.

2. Materials and methods

2.1. Materials and reagents

2.7. Analysis of vitamin D2

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).

Vitamin D2 was analyzed using the method described by Kazmi

et al. (2007). Vitamin D2 in fortied milk was isolated by alkaline
saponication followed by liquideliquid extraction with n-hexane.
The vitamins extracted into hexane were concentrated by evaporation under nitrogen. The extract was further puried using silica
cartridge and injecting it in the C-18 column to determine vitamin
D2 by RP-HPLC with UV detection (lmax 254 nm). Vitamin D2 content was determined using the calibration curve drawn using ve
concentrations 1, 2, 3, 4 and 5 ng/100 mL. Calibration curves were
generated by plotting the peak area of standard vitamin D2 versus
the theoretical concentration.

2.2. Preparation of toned milk

Cow milk and buffalo milk were mixed in 1:1 ratio and cream
was removed for the preparation of toned milk. The fat and solid
non fat (SNF) were set to 3.0% and 8.5%, respectively by adding
cream/skim milk using Pearson square method.
2.3. Fortication of milk with vitamin D2
A solution of 10,000 IU/mL solution was prepared by dissolving
1 g of microencapsulated vitamin D2 in 10 mL of water. The vitamin
D2 solution was sonicated for 5.0 min in an ultrasonicator (SN-1,
Toshcon Industries, Haridwar, India) for uniform mixing. The
vitamin D2 solution was stored below 20  C in dark or in low

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

R. Kaushik et al. / LWT - Food Science and Technology 56 (2014) 421e426

2.9. Statistical analysis

Means (n 3), standard error mean (SEM), linear regression
analysis and 95% condence intervals were calculated using
Microsoft Excel 2007 (Microsoft Corp., Redmond, WA). Data was
subjected to a single way analysis of variance (ANOVA).

423

vitamin D concentration in both fortied whole and skim milk.

Vitamin D in milk was unaffected by pasteurization, boiling or
sterilization (Hartman & Dryden, 1974). Liu (2003) showed that
vitamin D was stable in skim milk at high temperature low time
pasteurization (72  C for 15 s) and ultra high temperature processing. Wagner et al. (2008) found no changes in vitamin D content after heating the cheese at 100 and 232  C.

3. Results and discussion

Toned milk was fortied with vitamin D2 singly and in combination with calcium (calcium phosphate) and subjected to different
heat treatments viz. pasteurization (63  C/30 min), boiling and
sterilization (121  C/15 min/15 psi); light exposure treatment
(1485, 2970 and 4455 lux/0, 2, 4, 8, 16 and 32 h) and storage under
refrigerated conditions (4e7  C) and at 37  C in glass bottles, plastic
bottles and polyethylene pouches. Effect of these processing conditions on vitamin D2 retention in fortied milk was then evaluated
and vitamin D2 chromatogram is presented in Fig. 1.
3.1. Effect of heat treatments on vitamin D2 retention in milk
The retention of vitamin D2 at different heat treatments was
estimated. Three heat treatments which are generally used for milk
processing were selected to evaluate retention of vitamin D2 in milk
were
1) Pasteurization (63  C/30 min)
2) Boiling
3) Sterilization (121  C/15 min at 15 psi)
It is evident from Table 1 that vitamin D2 content of raw (control) and pasteurized milk was negligible as no peak of vitamin D2
was observed during analysis. Vitamin D2 content of raw, pasteurized, boiled and sterilized fortied milk samples did not signicantly differ (P > 0.05) from each other, suggesting that there was
negligible losses of vitamin D2 of these heat treatments upon milk.
In fortied milk non signicant difference (P > 0.05) could be
observed between vitamin D2 and vitamin D2 calcium fortied
samples on heating, with 0.95 and 1.15% loss of vitamin D2 during
pasteurization, loss of 1.45% and 1.32% on boiling and losses of 1.67%
and 1.92% on sterilization of the fortied milk suggested that
vitamin D2 fortication along with calcium did not have any signicant effect on vitamin D2 loss in fortied milk occurring during
heat processing.
Similar results were observed by Wagner et al. (2008), who
reported that heating of milk at 72  C for 16 s did not affect the

3.2. Retention of added vitamin D2 in fortied milk during storage

3.2.1. Effect of storage in glass bottles on retention of vitamin D2 in
milk
The retention of vitamin D2 in both vitamin D2 and vitamin
D2 calcium fortied toned milk, packed in glass bottles and stored
under refrigerated conditions (4e7  C) was determined after 3rd,
5th and 7th day of pasteurization.
As shown in Table 2, vitamin D2 content at zero day was 599.80
and 599.72 IU, respectively for vitamin D2 and vitamin D2 calcium
fortied milk. After seven days storage the vitamin D2 (content)
was 594.94 and 595.15 IU for vitamin D2 and vitamin D2 calcium
fortied milk, respectively. No signicant difference was observed
in vitamin D2 content between 0, 3rd, 5th and after 7th day of
storage. It is evident from Table 2 that vitamin D2 was not affected
during storage in both vitamin D2 and vitamin D2 calcium fortied milk samples.
Losses of 0.033, 0.27, 0.53 and 0.84% of vitamin D2 were
observed in 0, 3rd, 5th and 7th day of storage of fortication in
vitamin D2 fortied milk and 0.047, 0.30, 0.52 and 0.81% for 0, 3rd,
5th and 7th day in calcium vitamin D2 fortied milk. The
observed decrease in vitamin D2 content after seven days storage
was not signicant compared to vitamin D2 at day zero.

3.2.2. Effect of storage in plastic bottle on retention of vitamin D2 in

milk
The fortied milk was pasteurized and packed in plastic bottles
and then stored under refrigerated conditions (4e7  C) for seven
days. Stability of vitamin D2 was determined after 3rd, 5th and 7th
day of storage to determine the effect of both storage and the
packaging material. Vitamin D2 content at zero day was 599.60 and
599.66 IU, respectively for vitamin D2 and vitamin D2 calcium
fortied milk (Table 3). After seven days storage the vitamin D2
(content) was 592.98 and 592.63 IU for vitamin D2 and vitamin
D2 calcium fortied milk, respectively. No signicant difference
(P > 0.05) was observed in vitamin D2 content between 0, 3rd, 5th
and after 7th day of storage.

Fig. 1. HPLC chromatogram of vitamin D2.

424

R. Kaushik et al. / LWT - Food Science and Technology 56 (2014) 421e426

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

Data are presented as means  SEM (n 3). ND not detected.

a,b
Means within rows with different lowercase superscript are signicantly different (P < 0.05) from each other.
A,B
Means within columns with different uppercase superscript are signicantly different (P < 0.05) from each other.

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)

Vitamin D2 content in milk (IU/L)

0 day

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

Data are presented as means  SEM (n 3).

aeb
Means within rows with different lowercase superscript are signicantly different (P < 0.05) from each other.
AeB
Means within columns with different uppercase superscript are signicantly different (P < 0.05) from each other.

Losses of 0.40, 0.44, 1.03 and 1.17% of vitamin D2 were observed

for 0, 3rd, 5th and 7th day of fortication in vitamin D2 fortied milk
and 0.39, 0.45, 1.09 and 1.23% for 0, 3rd, 5th and 7th day in calcium
and vitamin D2 fortied milk. The % loss of vitamin D2 increased with
storage up to seven days. However, loss of vitamin D2 after 3rd, 5th
and 7th day of pasteurization did not differ signicantly (P > 0.05)
from % loss of vitamin D2 observed on zero day.
3.2.3. Effect of storage in polyethylene pouches on retention of
vitamin D2 in milk
The fortied milk was pasteurized and packed in polyethylene
pouches and then stored under refrigerated conditions (4e7  C) for
seven days. Stability of vitamin D2 was determined after 3rd, 5th
and 7th day of storage to determine the effect of both storage and
the packaging material.
It is evident from Table 4 that vitamin D2 was signicantly
affected (P < 0.05) during storage in both the vitamin D2 and vitamin
D2 calcium fortied milk samples. Vitamin D2 content at zero day
was 599.66 and 599.78 IU of vitamin D2 and vitamin D2 calcium
fortied milk samples, respectively. After 3rd day, vitamin D2 content signicantly reduced in both vitamin D2 and vitamin
D2 calcium fortied milk samples. On further storage, i.e. after
seven day storage, there was signicant reduction (P < 0.05) in the
vitamin D2 content as compared to its content on zero and 3rd day.
Reduction of 0.56, 5.65, 7.65 and 8.66% of vitamin D2 were
observed on 0, 3rd, 5th and 7th day, respectively in vitamin D2 fortied milk and 0.70, 5.19, 7.54 and 8.70% for 0, 3rd, 5th and 7th day,
respectively in vitamin D2 calcium fortied milk. The % loss of
vitamin D2 after 3rd, 5th and 7th day of pasteurization was signicantly different (P < 0.05) from % loss of vitamin D2 observed on zero
day. Brown and Wnuk (1988) reported absorption and/or loss of

essential oils, vitamins and critical avoring compounds of fruit juices

packed in polyethylene. Citrus peel oil readily diffuses through the
containers inner polyethylene coating and absorbed by polyethylene.
Stability of vitamin D in dairy and dairy products has been
reviewed by several workers. 100 Percent retention of vitamin D in
skim milk powder was reported by Bauernfeind and Allen (1963)
during storage for one year at 25, 37 and 45  C. Their data indicated, correlation between moisture pickup and vitamin loss during long periods of storage under eld conditions and emphasized
the importance of adequate packaging of the enriched product
under stress storage conditions. Banville et al. (2000) reported that
vitamin D was stable in cheese for 3e5 months of ripening, but
decreased thereafter. Holick, Shao, Liu, and Chen (1992) evaluated
the stability of vitamin D in milk during storage (at 4  C) for seven
days and found no signicant decrease in vitamin D content after
seven day storage. Upreti et al. (2002) reported no detectable
deterioration of vitamin D in fortied cheese during 9 months at
room and refrigerated temperatures. Liu (2003) studied the effect
of storage of vitamin D fortied milk in glass and plastic bottles and
reported higher values of vitamin D in milk packed in glass bottles
when compared to plastic bottles. Kazmi et al. (2007) reported that
vitamin D was stable in cheese, yogurt and ice cream and did not
degrade as a result of processing or during the expected shelf life of
these products.
3.3. Effect of light on stability of added vitamin D2 in glass bottles
for different time duration
The effect of light on retention of vitamin D2 fortied milk
packed in glass bottles under refrigerated conditions (4e7  C) using
three different intensities viz. 1485, 2970 and 4455 lux was

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)

Vitamin D2 content in milk (IU/L)

0 day

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

Data are presented as means  SEM (n 3).

aeb
Means within rows with different lowercase superscript are signicantly different (P < 0.05) from each other.
AeB
Means within columns with different uppercase superscript are signicantly different (P < 0.05) from each other.

R. Kaushik et al. / LWT - Food Science and Technology 56 (2014) 421e426

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

Data are presented as means  SEM (n 3).

aeb
Means within rows with different lowercase superscript are signicantly different (P < 0.05) from each other.
AeB
Means within columns with different uppercase superscript are signicantly different (P < 0.05) from each other.

Table 5
Effect of light on vitamin D2 retention in milk packed in glass bottles.
Light intensity

Vitamin D2 content in milk (IU/L)

Duration of exposure/
SampleY

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

Data are presented as means  SEM (n 3).

aeb
Means within rows with different lowercase superscript are signicantly different (P < 0.05) from each other.
AeB
Means within columns with different uppercase superscript are signicantly different (P < 0.05) from each other.

determined and the results are presented in Table 5. Statistically

similar vitamin D2 content was observed in fortied milk samples
after 0, 2, 4, 8, 16 and 32 h storage under light intensity of 1485,
2970 and 4455 lux. For all three light intensities and at different
time intervals of light exposure, there was non signicant difference between vitamin D2 content of vitamin D2 fortied milk and
milk fortied with both calcium vitamin D2, stored in glass
bottles.
3.4. Effect of light on stability of added vitamin D in polyethylene
pouches for different time durations
Effect of light on retention on vitamin D2 fortied milk stored in
polyethylene pouches under refrigerated conditions (4e7  C) using
three different intensities viz. 1485, 2970 and 4455 lux and the
results are given in Table 6. There was signicant (P > 0.05) difference in vitamin D2 content in all milk samples after 4 h at three
light intensities. For all three light intensities and at different time
intervals of light exposure, there was non signicant difference
(P > 0.05) between vitamin D2 content of vitamin D2 fortied milk
and milk fortied with both calcium vitamin D2, stored in polyethylene pouches. The % loss of vitamin D2 after 4 h light exposure

under different light intensities had signicant difference (P < 0.05)

from % loss of zero hour vitamin D2 content.
Disagreement with respect to vitamin D stability has also been
reported. Cremin and Power (1985) and Kutsky (1981) reported
that vitamin D was unstable to light, whereas Kreutler (1980) reported that vitamin D3 was remarkably stable to light, heat and
oxidation. Renken and Warthesen (1993) reported a slight loss of
vitamin D in fortied milk upon exposure to light. As described
earlier under Section 3.3, the effect of light on stability of vitamin D
in fortied milk stored in glass bottle, no signicant decrease in
vitamin D2 content was observed, however, signicant decrease
was observed in vitamin D2 content of vitamin fortied milk stored
in polyethylene pouches. Decrease in vitamin D2 was not due to
exposure to light but was due to the packaging material (polyethylene) used. The microstructure of polymers is porous and hydrophobic in nature which attracts not only the fat but also the fatsoluble components.
In the present study, the difference in vitamin D content of
fortied milk, stored in polyethylene pouches and glass bottles
could be ascribed to the sorption of vitamin D by polyethylene.
Paredes (1996), also indicated that the maximum sorption of
vitamin A (another fat-soluble vitamin) was 63% by low density

Table 6
Effect of light on vitamin D2 retention in milk packed in polyethylene pouches.
Light intensity

Vitamin D2 content in milk (IU/L)

Duration of exposure/
SampleY

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

Data are presented as means  SEM (n 3).

aeb
Means within rows with different lowercase superscript are signicantly different (P < 0.05) from each other.
AeB
Means within columns with different uppercase superscript are signicantly different (P < 0.05) from each other.








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

R. Kaushik et al. / LWT - Food Science and Technology 56 (2014) 421e426

polyethylene bottle and upto 55% for by high density polyethylene

(HDPE) plastic after one hour of contact with fortied milk.
4. Conclusion
From above results, it can be concluded that vitamin D2 is stable
in milk during heat treatments (pasteurization, boiling and sterilization). Vitamin D2 was stable during storage at refrigerated
temperature (4e7  C) in glass and plastic bottles, whereas in
polyethylene pouches the loss was signicantly higher. Vitamin D2
was stable upon exposure to three light intensities (1485, 2970 and
4555 lux) when stored in glass bottles, whereas in polyethylene
pouches there was signicant reduced in vitamin D2 content.
Acknowledgments
This study is part of the DBT-project nancially supported by the
Department of Biotechnology (Ministry of Science and Technology,
New Delhi, India).
References
Banville, C., Vuillemard, J. C., & Lacroix, C. (2000). Comparison of different methods
for fortifying cheddar cheese with vitamin D. International Dairy Journal, 10,
375e382.
Bauernfeind, J. C., & Allen, L. E. (1963). Vitamin A and D enrichment of nonfat dry
milk. Journal of Dairy Science, 46, 245e254.
Blanco, D., Fernandez, M. P., & Gutierrez, M. D. (2000). Simultaneous determination
of fat soluble vitamins and provitamins in dairy products by liquid chromatography with a narrowbore column. Analyst, 125(3), 427e431.
Brown, M. T, & Wnuk, A. J. (1998). Barrier laminates for the retention of essential oils
vitamins and avors in citrus beverages and a method of making said laminate and
leak-tight containers therefrom. US Patent (19), no. 4,753,832.
Byrdwell, W. C., Exler, J., Gebhardt, S. E., Harnly, J. M., Holden, J. M., Horst, R. L., et al.
(2011). Liquid chromatography with ultraviolet and dual parallel mass spectrometric detection for analysis of vitamin D in retail fortied orange juice.
Journal of Food Composition and Analysis, 24, 299e306.
Ceglia, L. (2009). Vitamin D and its role in skeletal muscle. Current Opinion in Clinical
Nutrition and Metabolic Care, 12, 628e633, 9685.
Charlton, S. J., & Ewing, W. N. (2007). The vitamin directory. England: Context
Products Ltd.
Cremin, F. M., & Power, P. (1985). Vitamins in bovine and human milks. In P. F. Fox
(Ed.), Developments in dairy chemistryd3 (p. 346). Essex, England: Elsevier
Applied Science Publishers.
Ganesan, B., Brothersen, C., & McMohan, D. J. (2011). Fortication of cheddar cheese
with vitamin D does not alter cheese avour perception. Journal of Dairy Science, 94, 3708e3714.

Gomez, M., & Jose. (2006). The role of insulin-like growth factor I components in the
regulation of vitamin D. Current Pharmaceutical Biotechnology, 7(2), 125e132.
Hartman, A. M., & Dryden, L. P. (1974). Vitamins in milk and milk products. In
B. H. Webb, A. H. Johnson, & J. A. Alford (Eds.), Fundamentals of dairy chemistry
(2nd ed.). New York: AVI/Van Nostrand Reinhold.
Holden, J. (2009). USDA national nutrient database. http://www.nal.usda.gov/fnic/
foodcomp/search/ Accessed 16.05.11.
Holick, M. F., Shao, Q., Liu, W. W., & Chen, T. C. (1992). The vitamin D content of
fortied milk and infant formula. New England Journal of Medicine, 326(18),
1178e1181.
ICMR. (2009). Nutrient requirements and recommended dietary allowances for Indians. A report of Expert Group of the Indian Council of Medical Research.
Kazmi, S. A., Vieth, R., & Rousseau, D. (2007). Vitamin D3 fortication and quantication in processed dairy products. International Dairy Journal, 17, 753e759.
Kreutler, P. A. (1980). Nutrition in perspective. Englewood Cliffs, NJ, USA: PrenticeHall.
Kutsky, R. J. (1981). Vitamin D. In Handbook of vitamins, minerals and hormones (pp.
191e198). New York, NY: Van Nostrand Reinhold Co.
Leskova, E., Kubikova, J., Kovacikova, E., Kosicka, M., Porubska, J., & Kosicka, M.
(2006). Vitamin losses: retention during heat treatment and continual changes
expressed by mathematical models. Journal of Food Composition and Analysis, 19,
252e276.
Liu, Y. (2003). Beta lactoglobulin complexed vitamin A and vitamin D in skim milk:
Shelf life and bioavailability (Ph.D. thesis). Raleigh, N.C.: North Carolina State
University.
Mawer, E. B., Jones, G., Davies, M., Still, P. E., Byford, V., Schroeder, N. J., et al. (1998).
Unique 24-hydroxylated metabolites represent a signicant pathway of metabolism of vitamin D2 in humans: 24-hydroxyvitamin D2 and 1, 24dihydroxyvitamin D2 detectable in human serum. Journal of Clinical Endocrinology & Metabolism, 83(6), 2156e2166.
Mays, D. L. (1982). Nutrition overages and methodology trends. Journal of the Association of Ofcial Analytical Chemists, 65(6), 1495e1499.
Ovesen, L., Brot, C., & Jakobsen, J. (2003). Food contents and biological activity of 25hydroxyvitamin D: a vitamin D metabolite to be reckoned with? Annals of
Nutritional Metabolism, 47, 107e113.
Paredes, E. M. (1996). The effect of polymeric packaging materials upto vitamin A and
D losses from uid non-fat milk (M.Sc. thesis). North Carolina State University.
Pike, R. L., & Brown, M. L. (1984). Nutrition, an integrated approach. New York, NY,
USA: Wiley.
Renken, S. A., & Warthesen, J. J. (1993). Vitamin D stability in milk. Journal of Food
Science, 58, 552e556.
Riaz, M. N., Asif, M., & Ali, R. (2009). Stability of vitamins during extrusion. Critical
Reviews in Food Science and Nutrition, 49, 361e368.
Tanner, J. T., Smith, J., Debaugh, P., Angyal, G., Villalobus, M., Bueno, M. P., et al.
(1988). Survey of vitamin content of fortied milk. Journal of the Association of
Ofcial Analytical Chemists, 71(3), 607e610.
Upreti, P., Mistry, V. V., & Warthesen, J. J. (2002). Estimation and fortication of
vitamin D3 in pasteurized process cheese. Journal of Dairy Science, 85, 3173e
3181.
Wagner, D., Sidhom, G., Whiting, S. J., Rousseau, D., & Veith, R. (2008). The
bioavailability of vitamin D from fortied cheeses and supplements is equivalent in adults. Journal of Nutrition, 138(7), 1365e1371.
WHO/FAO. (2006). Guidelines on food fortication with micronutrients. World Health
Organization and Food and Agriculture Organization of the United Nations.