Indian J. Dairy Sci.
68(4), 2015
RESEARCH ARTICLE
Iodine value integrated with solvent fractionation technique
as a tool for detecting palm olein and sheep body fat
adulteration in ghee (clarified milk fat)
Kamal Gandhi, Anil Kumar and Darshan Lal
Received : 28 December 2014 / Accepted : 07 June 2015
Abstract Iodine value for pure cow ghee ranged from
35.53 to 41.24 with an average of 38.69, whereas that for pure
buffalo ghee ranged from 30.14 to 36.48 with an average of
34.10. The iodine value for pure cow ghee was lowest (35.53)
in January and highest (41.24) in July. For pure buffalo ghee
also, lowest iodine value (30.14) was observed to be in
January and highest (36.48) was noticed to be in July, as
observed for pure cow ghee. Using iodine value as parameter,
palm olein and sheep body fat added individually could not
be detected at any of the levels studied in pooled samples
of ghee. Mixture of palm olein and sheep body fat was
detectable only at 9+21(30) percent level. After fractionation,
lower level of 6+14(20) percent was also detectable.
Keywords : Ghee, iodine value, adulteration, fractionation,
palm olein, sheep body fat
Introduction
Ghee production accounts for the largest segment of milk
consumption and utilization pattern in India. Ghee plays an
important role in the economics, nutrition, flavor and physico-
chemical properties of milk and milk products. Ghee, being a
costly constituent, is highly prone to adulteration with
cheaper fats such as vegetable oils, animal body fats,
hydrogenated fats, interesterified fats and inedible mineral
oils, etc., especially during lean season. In recent years, the
problem of adulteration has assumed a very serious dimension.
Kamal Gandhi (
), Anil Kumar and Darshan Lal Cow and buffalo ghee samples were prepared from their
Division of Dairy Chemistry, National Dairy Research Institute,
Karnal (Haryana), India.
Kamal Gandhi
GCMMF Ltd. Anand
Email:kamalgandhi4444@gmail.com Mob. No. 07351444292
347
Humanity has gone to the back seat and even the holy foods
are not spared. Such a situation has tarnished the image of
the dairy industry, not only in India, but abroad also. The
problem of adulteration is further complicated by the fact that
the composition of milk fat varies with the species, season
and the diet given to the animals. In the past, several
techniques have been developed to detect milk fat adulteration,
yet it is realized that they have their own limitations in
detecting the type and level of all types of adulterants
particularly in view of multiple component adulteration
malpractices. Partitioning the pure and adulterated milk fat in
to solid and liquid fractions on the basis of crystallization,
which enriches the solid fraction with the body fat and that
of liquid fraction with vegetable oils, followed by the analysis
of these fractions for iodine value, can be a new approach for
unlocking the problem of detecting adulterants in milk fat.
Solvent fractionation has advantages over dry fractionation in
that besides lowering viscosity of the liquid, it makes heat
transfer easier, nucleation and growth faster, and there are
very low levels of entrained oil. Sheep body fat and palm
olein both being cheaper are suspected to be used as
adulterants in milk fat (Gandhi, 2014). Therefore, keeping the
above points in mind, the present study was aimed to detect
sheep body fat and palm olein in milk fat using iodine value
integrated with solvent fractionation technique.
Material and Methods
Preparation of samples
respective pooled milks separately by creamery butter method
(De, 2010). The palm olein was procured locally and kept
under refrigeration condition (4-7°C) till its further use as
adulterant. For the preparation of sheep body fat, the adipose
tissues were procured from the local slaughter house (Bakra
Market ,Karnal) and were heated at 140°C to 150°C on direct
flame in a stainless steel vessel till a transparent liquefied
animal body fat was obtained. The liquid fat thus obtained
Indian J. Dairy Sci. 68(4), 2015
was filtered, cooled to room temperature, capped and kept in
a refrigerator (5-10°C) for its further use as the adulterant fat.
Pure ghee (cow & buffalo), sheep body fat and palm olein
samples were collected throughout the year after every two
months of interval.
Preparation of Adulterated Ghee Samples
For the preparation of adulterated ghee samples, pure ghee,
palm olein and sheep body fat were heated and maintained
at 65-70°C for 10 min before mixing. The adulterant fats/oils
were added to ghee individually as well as in their
combinations. At individual levels, these adulterants were
separately added to ghee at 5, 10 and 15% levels. In case of
their combined mixtures, palm olein (@ 3, 6 & 9%) and sheep
body fat (@ 7, 14 & 21%) were added in ghee, representing
a total adulteration of 10 (3+7), 20 (6+14) & 30 (9+21) %
levels, respectively, thereby maintaining a ratio of unsaturated
to saturated fatty acid in adulterated ghee samples as 30 to
70, as is generally found in pure ghee. After the addition of
adulterant oils and fats to ghee, the samples were thoroughly
mixed.
Optimisation of suitable temperature-time combination for
solvent fractionation
Preliminary trials were conducted with pure ghee samples
(cow and buffalo separately) and adulterant oil and fat (palm
olein and sheep body fat) with a purpose of obtaining two
temperatures in a successive manner so as to get two solids
and one liquid fraction so that the first solid fraction is sheep
body fat enriched while the last liquid fraction is palm olein
enriched. For this, 30 g samples (pure ghee and adulterants)
were melted and equilibrated at 65°C for 5 min in 100 ml
graduated glass tubes. These were then dissolved in 60 ml
acetone previously warmed in a water bath followed by
equilibrating the mixture at 50°C for 5min. For the selection of
temperature-time combination of the first solid fraction, the
fractionation was carried out at temperatures of 18, 15, 12 °C
in a refrigerated water-bath, noting time at each temperature
till maximum amount of sheep body fat solidifies while the
pure cow and pure buffalo ghee either do not solidify or
solidify to the least extent. After the desired temperature-time
combination was selected for obtaining the first solid fraction,
temperature-time for the successive fractionation step was
standardized so as to get the last liquid fraction enriched
with the palm olein. For this purpose, preliminary trials were
conducted at temperatures of 8, 6 and 40C, noting time at
each temperature till all the palm olein and minimum of low
melting triglycerides fraction of pure cow ghee and pure
buffalo ghee appear in the last liquid fraction. From these
trials, temperature-time combination of 15° C/15 min was
selected for obtaining the first solid fraction, whereas a
temperature-time combination of 4°C/ 3 hr was selected for
348
obtaining the subsequent solid and liquid fractions.
Iodine Value
Iodine Value of the samples was determined by standard
method (BIS, 1981).
Statistical Analysis
Results are presented as means ± standard error from six
replicates of each experiment. P-value < 0.05 was used to
denote significant differences among mean values determined
by analysis of variance (ANOVA) with a subsequent least
significant difference (LSD) test was applied to test for any
significant differences (P<0.05) in the mean values as described
by Snedecor and Cochran (1994).
Results and Discussion
The results on iodine values for pure ghee (cow & buffalo),
sheep body fat and palm olein samples collected throughout
the year after every two months of interval are depicted in
Figure 1, while their average values along with standard error
are depicted in Figure 2.
It can be seen from the Table 1 that the iodine value for pure
cow ghee ranged from 35.53 to 41.24 with an average of 38.69,
whereas that for pure buffalo ghee ranged from 30.14 to 36.48
with an average of 34.10. When the values of pure cow and
buffalo ghee were pooled together, the iodine values ranged
from 30.14 to 41.24 with an average of 36.39 (Table 1). The
iodine value for palm olein ranged from 59.37 to 68.21 with an
average of 62.86, while sheep body fat it ranged from 47.27
to 51.08 with an average of 48.91. (Figure 1 and Figure 2).
From the results, it can be seen that the iodine value for pure
cow ghee was lowest (35.53) in January and highest (41.24)
in July. For pure buffalo ghee also, the lowest iodine value
(30.14) was observed to be in January and highest (36.48) was
noticed to be in July, as observed for pure cow ghee (Figure
1). The similar seasonal variation in iodine value was also
observed by earlier workers (Lal 2005, Kumar 2013) for both
cow and buffalo milk fats, but they have reported different
minimum and maximum values for different months of a year.
Analysis of variance of data for iodine value revealed that
pure ghee (cow and buffalo) and adulterant oil and fat (palm
olein and sheep body fat) differed significantly (P<0.05) from
each other (Figure 2). Comparison among all the pure samples
of ghee, and adulterant fat and oil revealed that pure cow and
pure buffalo ghee, palm olein and sheep body fat differed
significantly from each other (P<0.05). Adulterants (palm olein
and sheep body fat) showed the higher iodine values as
compared to pure ghee samples (Figure 2). Of the two pure

Figure 1 : Iodine values of pure ghee samples and
adulterants fat and oil in different months of the year
milk fats, the average iodine value was observed to be higher
for cow ghee. The similar trend of variation between iodine
values of cow and buffalo ghee as noticed in the present
study was also observed by earlier workers (Bector and
Narayanan 1974, Lal and Naryanan 1984, Kumar 2002, Lal
2005). However, Singhal (1980) observed an opposite trend
and reported higher iodine values for buffalo ghee than cow
ghee. Palm olein showed very high iodine value, almost 1.7
times to that of pure cow and buffalo ghee samples.
Samples represented with different letters are significantly
different (p < 0.05) from each other.
Similarly, sheep body fat also showed higher iodine value,
about 1.30 times to that of pure cow and buffalo ghee samples.
The iodine values were found to increase in cow as well as
buffalo ghee samples with the addition of palm olein and
sheep body fat individually at 5, 10 and 15 percent levels and
in their combinations at 3+7(10), 6+14(20) and 9+21(30) percent
levels and this increase was dependent upon the amount of
adulterants added to the pure cow and buffalo ghee. Higher
the level of adulterant added, greater was the increase in the
iodine value of ghee samples. It was also observed that
addition of palm olein caused more increase in the iodine
value of ghee as compared to that caused by addition of
sheep body fat.
Considering the overall range (30.14 to 41.24) for iodine value
of both cow and buffalo pure ghee samples, the results
obtained on the basis of comparison of average values showed
that the adulteration of cow ghee with palm olein individually
could only be detected at 15% level, while in buffalo ghee it
remained undetectable at all the levels studied. Whereas, sheep
body fat added individually in cow and buffalo ghee samples
could not be detected at all the levels studied (Table 1).
Further, when the results of the average values (Table 1) of
the ghee samples added with mixture of adulterants at 3+7
(10), 6+14 (20) and 9+21 (30) were compared, it was noticed
349
Indian J. Dairy Sci. 68(4), 2015
Figure 2 : Average Iodine values of pure ghee
samples and adulterant fat and oil
from the results that palm olein in the presence of sheep
body at the lower level i.e. 3+7 (10) percent remained
undetectable, while higher levels i.e. 6+14 (20) and 9+21 (30)
percent could be detected easily in cow ghee, while in
buffalo ghee it remained undetectable at all the levels studied.
Pooling of the data of pure and adulterated cow and buffalo
ghee samples revealed that both palm olein and sheep body
fat added individually could not be detected at any of the
levels studied. Mixture of palm olein and sheep body fat was
detectable only at 9+21 (30) percent level.
As described earlier, the pure ghee samples and the samples
adulterated with mixture of adulterants were subjected to
fractionation at 15°C and 4°C for enrichment of sheep body
fat in first solid (S15) fraction and palm olein in last liquid (L4)
fraction. The first and last fractions were also analyzed for
iodine value so as to know whether fractionation increases
the sensitivity of detection of adulteration. The results
obtained on iodine value of first solid (S15) and last liquid
(L4) fractions along with their respective pooled values are
shown in Table 2.
The iodine value ranged from 34.11 to 40.90 with an average
of 37.64 for the first solid (S15) fraction of pure cow ghee,
whereas the similar values for S15 fraction of pure buffalo
ghee ranged from 30.04 to 35.12 with an average of 33.28. The
iodine value of the last liquid (L4) fraction of pure cow ghee
ranged from 34.74 to 41.85 with an average of 38.82, while that
of L4 of pure buffalo ghee ranged from 31.87 to 36.24 with an
average of 34.76 (Table 2). It was observed from the results
that the iodine value of the first solid (S15) fractions of both
cow and buffalo pure ghee were lower and that of last liquid
(L4) fractions were found to be higher than the corresponding
(unfractionated) pure cow and buffalo ghee samples from
which the fractions were obtained. Last liquid (L4) fractions
obtained from pure cow and buffalo ghee samples and samples
added with combinations of adulterants showed the higher
Indian J. Dairy Sci. 68(4), 2015

iodine value as compared to corresponding first solid (S15)
fractions. Further, it was also observed that the iodine value
of the first solid (S15) and last liquid (L4) fractions of ghee
samples adulterated with mixture of palm olein and sheep
body fat at 3+7(10), 6+14(20) and 9+21(30) percent levels
increased with the level of adulteration, as both the adulterants
had higher iodine value compared to pure ghee.
Considering the overall range of the iodine value for first
solid (S15) fractions (30.04 to 40.90) of both pure cow and
buffalo ghee samples together as the basis and then
comparing it with the average iodine values for the first solid
(S15) fraction of cow ghee and buffalo ghee samples added
with mixture of palm olein and sheep body fat at 3+7(10),
6+14(20) and 9+21(30) percent levels, it was observed that the
lower level of adulteration done at 3+7(10) percent levels
could not be detected both in case of cow and buffalo ghee,
6+14(20) level was detectable in cow ghee but not in buffalo

Table 1 Iodine values of cow and buffalo pure ghee and ghee adulterated with individual adulterants and mixture thereof.
Adulterant Level (%) Iodine value
Cow ghee Buffalo ghee Pooled
(Cow+Buffalo) ghee
Range* Average±SE Range* Average±SE Range** Average±SE
Control 0 35.53-41.24 38.69±0.87 30.14-36.48 34.10±0.93 30.14-41.24 36.39±0.84
PO 5 36.77-42.59 39.90±0.89 31.69-38.07 35.54±0.93 31.69-42.59 37.72±0.86
10 38.01-43.94 41.11±0.91 33.24-39.65 36.98±0.93 33.24-43.94 39.04±0.88
15 39.24-45.29 42.31±0.93 34.79-41.24 38.42±0.94 39.24-45.29 40.36±0.90
SBF 5 36.16-41.87 39.32±0.87 31.00-37.21 34.85±0.91 31.00-41.87 37.08±0.84
10 36.80-42.51 39.96±0.87 31.85-37.94 35.59±0.89 31.85-42.51 37.77±0.83
15 36.96-42.67 40.12±0.87 32.71-38.67 36.32±0.87 32.71-42.67 38.22±0.82
PO+SBF 3+7 37.12-42.74 40.13±0.86 31.80-37.94 35.51±0.90 31.80-42.74 37.82±0.83
6+14 38.70-44.24 41.57±0.85 34.40-40.43 37.90±0.88 34.40-44.24 39.73±0.83
9+21 40.29-45.73 43.01±0.85 36.52-42.40 39.80±0.86 36.52-45.73 41.40±0.82
PO-Palm oleinSBF-sheep body fat *Data represent mean±SE of six determinations
**Data represent mean±SE of twelve determinations.

Table 2 Iodine values of first solid (S15) and last liquid (L4) fractions of pure ghee samples and ghee
sample added with combination of adulterants
Type of Type of Level of Iodine value
ghee adulterant fat/oil adulteration (%) Range Averages'
S15 L4 S15 L4
Cow Ghee Control 0 34.11-40.90 34.74-41.85 37.64±1.11 38.82±1.16
PO+ SBF 3+7 36.35-42.43 38.34-45.15 39.58±0.98 42.63±1.06
PO+ SBF 6+14 38.86-48.93 42.93-52.85 43.47±1.57 47.47±1.44
PO+ SBF 9+21 40.13-49.82 43.78-54.73 46.37±1.53 50.99±2.01

Buffalo Ghee Control 0 30.04-35.12 31.87-36.24 33.28±0.80 34.76±0.71

PO+ SBF 3+7 32.43-41.47 36.12-45.16 36.68±1.36 40.66±1.38
PO+ SBF 6+14 37.84-44.28 39.98-48.23 40.49±1.10 44.32±1.27
PO+ SBF 9+21 38.08-48.12 42.85-52.46 43.11±1.51 47.74±1.52
Pooled
(cow+buffalo)Ghee Control 0 30.04-40.90 31.87-41.85 35.46±0.95 36.79±0.93
PO+ SBF 3+7 32.43-42.43 36.12-45.15 38.13±1.12 41.64±1.15
PO+ SBF 6+14 37.84-48.93 39.98-52.85 42.10±0.94 45.90±1.06
PO+ SBF 9+21 38.08-49.82 42.85-54.73 44.62±1.42 49.36±1.58
PO-Palm oleinSBF-sheep body fat S15-First solid fraction L4-Last liquid fraction

350

ghee and higher level at 9+21(30) percent could easily be
detected in both types of ghee.
On the other hand, keeping in view of the overall range of
the iodine value for last liquid (L4) fractions (31.87 to 41.85)
of both pure cow and buffalo ghee samples together as the
basis and then comparing it with the average iodine values
for the last liquid (L4) fraction of cow ghee and buffalo ghee
samples added with mixture of palm olein and sheep body fat
at 3+7(10), 6+14(20) and 9+21(30) percent levels, the results
revealed that all the levels (3+7(10), 6+14(20) and 9+21(30)
percent studied) could easily be detected in cow and buffalo
ghee, except at 3+7(10) level in buffalo ghee. This indicated
that using the iodine value of last liquid (L4) fractions, all the
levels of adulteration studied for the mixture of palm olein and
sheep body fat could easily be detected in cow ghee.
On pooling of the data obtained on first solid (S15) fraction
and last liquid (L4) of pure as well as adulterated cow and
buffalo ghee samples and using the overall range of iodine
value as 30.04 to 40.90 for S15 factions and 31.87 to 41.85
for last liquid (L4) fractions of both pure cow and buffalo
ghee as the basis for comparison of iodine value of first solid
(S15) fractions and last liquid fraction (L4) of cow and buffalo
adulterated ghee samples, it was observed that the lower level
of adulteration done at 3+7(10) percent of palm olein and sheep
body fat could not be detected, while higher levels of 6+14(20)
and 9+21(30) percent the same could easily be detected.
On comparing the results obtained on the iodine value of
fractionated ghee samples with these of unfractionated ghee
samples, based on the pooled data of cow and buffalo ghee
together, it may be inferred that fractionation technique has
offered advantage in lowering the detection limit using iodine
value because even that level of adulteration (6+14 percent of
palm olein and sheep body fat totaling to 20 percent) which
could not be detected in case of unfractionated ghee samples,
was found to be detectable on the basis of both first (S15)
and last liquid (L4) fractions. This is because of the reason
that the fractionation process helped in partitioning of
triglycerides containing more of saturated fatty acids in to
first solid (S15) fraction and triglycerides containing more of
unsaturated fatty acids in to last liquid (L4) fraction on the
basis of their melting points.
Conclusions
It may be concluded from the above part of the study that
351
Indian J. Dairy Sci. 68(4), 2015
iodine value is not a very useful parameter for checking
adulteration in milk fat for the detection of palm olein as
against other vegetable oils reported by other workers. Palm
olein and sheep body fat added individually could not be
detected even at the level of 15 percent levels in pooled ghee
samples. Mixture of palm olein and sheep body fat was
detectable only at 9+21 (30) percent levels. Fractionation
technique has offered advantage of increasing the sensitivity
of iodine value by lowering the detection limit because even
that level (6 and 14 percent, respectively of palm olein and
sheep body fat) which could not be detected in case of
unfractionated ghee samples, was found to be detectable on
the basis of iodine value of both first solid and last liquid
(L4) fractions.
Acknowledgements
The first author acknowledges the Institute fellowship from
ICAR and infrastructural facilities received from National Dairy
Research Institute, Karnal, Haryana (India) during the course
of research.
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