Indian J Dairy Sci 71(3): 246-251
RESEARCH ARTICLE
Solvent fractionation technique paired with apparent solidification time (AST)
test as a method to detect palm olein and sheep body fat in ghee (clarified milk
fat)
Kamal Gandhi, Anil Kumar and Darshan Lal
Received: 18 December 2017 /Accepted: 25 May 2018
Abstract: Apparent solidification time (AST) test (min:sec) is
defined as the time taken by the melted fat to get apparently
solidified at a given temperature. It has been used to detect
adulteration of palm olein and sheep body fat when added singly
and in combination with ghee. The AST test was applied to
samples containing combination of adulterants before and after
solvent fractionation. The mixture of palm olein and sheep body
fat was detectable at 9+21 % level. After fractionation, the lower
level of 6+14 % was also found to be detectable. Thus, the solvent
fractionation technique when coupled with AST enhances the
sensitivity of AST Test and could be applied as a valuable
parameter for checking adulteration of palm olein and sheep body
fat in cow and buffalo ghee.
Keywords: Adulteration, Apparent solidification time, Ghee
(clarified milk fat), Palm olein, Sheep body fat
Introduction
Ghee (the clarified milk fat) is the most widely used milk product
in the Indian subcontinent and is considered as the supreme
cooking and frying medium (Gandhi et al. 2013). Ghee is the second
largest consumed dairy product after liquid milk because of its
Kamal Gandhi ()
Dairy Chemistry Division, ICAR-National Dairy Research Institute,
Karnal-132 001, Haryana
E-mail: kamalgandhindri@gmail.com
Tel:-0184-2259148; Fax:-0184- 0184-2250042; Mob. No.:
09729134444
Anil Kumar
Centre for Rural Development & Technology, Indian Institute of
Technology, Delhi, India
Darshan Lal
Dairy Chemistry Division, NDRI, Karnal
246
numerous benefits and the fact that it is an integral part of Indian
diet. India produces 900,000 tonnes of marketed ghee, valued at
Rs 85,000 million (Hazra, 2014). Ghee is an expensive product,
costing three times as much as edible vegetable oils (Gosewade
et al. 2017). Expensive oils and fats such as ghee are often
subjected to adulteration with cheaper fats in order to have an
economic advantage (Kumar et al. 2013a; Kumar et al. 2013b).
The adulteration of milk fat can start at the stage of milk itself.
Once milk fat has been isolated in the form of ghee, a variety of
materials including vegetable oils, animal body fats, mineral oils,
tailored fats, hydrogenated fats etc. are employed for its
adulteration. Various vegetable oils and hydrogenated fats are in
common use. Animal body fats available from the slaughterhouse,
being hard and firm can’t be detected visually in ghee even when
present in considerable proportion. By blending animal body fat
and vegetable oils, the composition can exactly be made to that
of ghee (Rangappa and Achaya, 1974). Animal body fats addition
in holy religious foods not only devoid people from their money
and health, but also affects their religious sentiments. Prevailing
state has damaged the image of dairy industry not only in India
but abroad also. Seriousness of the problem can be judged from
numerous cases of adulteration appearing in the newspaper.
Furthermore, the variable composition of milk fat as affected by
the season, species and the diet given to the animal further
aggravates the problem of detection of adulterants in it (Kumar
et al. 2002; Kumar et al. 2009; Kumar et al. 2010).
Detecting adulteration in milk fat has presumed a very critical
dimension in the prevailing preview especially in the regime of
worldwide competition, where the quality of milk and milk
products is not an alternative, but a requirement. Nevertheless,
establishing the wholesomeness of milk fat is a very intricate
phenomenon (Kumar et al. 2002; Boghra et al. 2004; Lal et al.
2005).
Methods currently employed for the detection of adulteration of
foreign fats in milk fat are to some extent able to help when
adulterant fats like body fats or vegetable oils are added
individually. However, when a mixture of body fats and vegetable
oils is added to milk fat, the complication arises because these
adulterants which differ in their chemical composition and physical
characteristics counteract the effect of each other on the changes
Indian J Dairy Sci 71(3): 246-251
brought about by them in the physicochemical properties of milk
fat. Thus, partitioning the pure and adulterated milk fat into a
solid and liquid fraction on the basis of crystallization, which
enriches the solid fraction with the body fat and the liquid fraction
with vegetable oil followed by the analysis of these fractions for
various parameters can be a novel approach for establishing the
purity of milk fat. Different types of fractionation processes have
been developed, which include melt, solvent, and detergent
fractionation; supercritical fluid extraction and short path
distillation. Solvent fractionation besides lowering the viscosity
of the liquid makes heat transfer easier, nucleation and growth
faster, ensures very low levels of entrained oil and thus is more
advantageous over dry fractionation technique (Timms, 2005).
Ghee because of the variable concentration of triglycerides in it,
melts over a wide range of temperature. It liquefies above 40oC
and becomes completely solidified below –40oC (Fox, 2016). Milk
fat is partially solidified at ambient temperatures. Earlier tests
based on partial solidification behaviour of milk fat such as
opacity test (Panda and Bindal, 1998) expressed as the time taken
by a melted fat sample to acquire certain opaqueness has been
employed as one of the criteria for detecting adulteration in ghee
with animal body fats. Although a simple test for field
applications, it requires the use of a spectrophotometer or
colorimeter. Moreover, opacity test suffers from another
drawback that it fails to detect the body fats in ghee in the
presence of vegetable oils. Therefore, in order to overcome such
limitations, in the present investigation it was proposed to study
the apparent solidification behaviour of the control as well as
adulterated and fractionated samples in terms of the time taken
by the melted fat to get apparently solidified at a given temperature,
referred to as apparent solidification time (AST), developed by
(Kumar et al. 2009, 2010).
Being cheaper, both sheep body fat and palm olein are being
added as adulterants in milk fat (Gandhi et al. 2014; Gandhi et al.
2015). Fractionation techniques have been integrated with other
parameters including iodine value (Gandhi et al. 2015), Reichert-
Meissl value (Gandhi et al. 2014), apparent solidification time
(Kumar et al. 2010), fatty acid profile using gas chromatography
(Kumar et al. 2015), Butyro-refractometer reading (Kumar et al.
2017; Gandhi et al. 2017 ) etc. in the past for the authentication of
milk fat. Therefore, keeping the above points in mind, the present
study was aimed to detect sheep body fat and palm olein in milk
fat using apparent solidification test coupled with solvent
fractionation technique.
Materials and Methods
Preparation of samples
Cow and buffalo ghee samples were prepared from their respective
pooled milk separately by creamery butter method (De, 2010).
Refined palm olein used for the study was of Ginni brand
manufactured and packed by Bunge India Pvt. Ltd., Mumbai.
247
The oil was procured locally and kept under refrigeration
condition (4-7°C) until its further use as an adulterant. For the
preparation of sheep body fat, the adipose tissues were procured
from the local slaughterhouse (Bakra Market, Karnal). The adipose
tissues after collection were washed thoroughly with running
tap water. After draining out the residual water, the adipose
tissues were heated at 140°C to 150°C on the direct flame in a
stainless steel vessel till a transparent liquefied animal body fat
was obtained. The liquid fat thus obtained was filtered through
6-8 fold muslin cloth followed by vacuum filtration using Whatman
No. 4 filter paper, filled in polyethylene bottle, cooled to room
temperature, capped and kept in a refrigerator (5-100C) for its
further use as the adulterant fat (Gandhi et al. 2014; Gandhi et al.
2015).
Preparation of adulterated ghee samples
For the preparation of adulterated ghee samples, pure ghee, palm
olein, and sheep body fat were heated and kept at 65-70°C for 10
min prior to 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, comprising
a total adulteration of 10 (3+7), 20 (6+14) & 30 (9+21) % levels,
respectively, thereby keeping a ratio of unsaturated to saturated
fatty acid in adulterated ghee samples as 30 to 70, as is normally
found in pure ghee. After the inclusion of adulterant oils and fats
to ghee, the samples were completely mixed (Gandhi et al. 2014;
Gandhi et al. 2015).
Selection 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 650C 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 500C for 5 min
(Upadhyay et al. 2014). For the selection of temperature-time
combination of the first solid fraction, the fractionation was carried
out at temperatures of 18, 15, 12 0C in a refrigerated water bath,
noting time (time 1) 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 4 0C, noting the

time (time 2) 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 (Fig. 1). From these
trials, a temperature-time combination of 150C/15 min was selected
for obtaining the first solid fraction, whereas a temperature-time
combination of 40C/3 hr was selected for obtaining the last liquid
fraction (Gandhi et al. 2014; Gandhi et al. 2015). Since the AST
test could not be applied to the last fraction, it was not of any use
in the present study.
Apparent Solidification Time (AST) test
The time taken by the melted fat samples to apparently get
solidiûed at a particular temperature is referred to as apparent
solidification time (AST) test. In this test, 3 g of the completely
melted fat samples were placed in test tubes (10 × 1.0 cm ID) and
maintained at 60°C for a period of 5 min. The test tubes were then
kept in a refrigerated water bath maintained at 18 ± 0.20C. The
test tubes were observed constantly until the apparent
solidiûcation of fat samples took place, which was conûrmed by
non-movement of fat samples on tilting the test tube. At this
stage, when the fat sample was apparently solidiûed, the time
taken for the same was recorded as AST using a stopwatch
(Kumar et al. 2010).
Statistical Analysis
Final results are displayed as means ± standard error from six
samples gathered bi-monthly over the period of 1 year. One way
ANOVA (P < 0.05) of the data obtained by the AST test to establish
the least significant difference with an appropriate posthoc test
(Fisher’s least significant difference test) between control and
adulterated samples was also carried out using SAS software
version 5.1.
Results and Discussion
AST values of pure ghee samples of both cow and buffalo show
a particular range. Any deviation from these values would give
an indication of adulteration of milk fat (Kumar et al. 2009). The
AST test was applied in the present study and the results
obtained from the AST values (min:sec) for pure ghee (cow &
Table 1 AST values (min: sec) of pure cow and buffalo ghee samples and adulterants fat/oil in different months of the year
Type of sample AST value (min:sec)
January March May July
Cow ghee 2:45 3:17 2:49 3:12
Buffalo ghee 2:36 2:50 2:45 2:43
Palm olein ND ND ND ND
Sheep body fat 0:33 0:36 0:43 0:48
ND: Not determined
*Data represent mean±SE of six determinations.
Values bearing different superscripts within the column differ significantly.
248
Indian J Dairy Sci 71(3): 246-251
buffalo), sheep body fat and palm olein samples collected
throughout the year after every two months of interval along
with their average values are given in Table 1. The AST value
(min: sec) for pure cow ghee ranged from 2:45 to 3:17 with an
average of 3:01, whereas that for pure buffalo ghee ranged from
2:29 to 2:50 with an average of 2:40 (Tables 1 and 2). When the
values of pure cow and buffalo ghee were combined together,
the AST values (min: sec) ranged from 2:29 to 3:17 with an average
of 2:50 (Table 2). The AST value (min: sec) for sheep body fat
ranged from 0:33 to 0:48 with an average of 0:39, while palm olein
did not solidify at analysing temperature (18oC) of AST test. This
indicated that of the two pure milk fats, the AST values were
observed to be higher for cow ghee. Sheep body fat showed
very low AST values which are much below the values of pure
cow and buffalo ghee samples, whereas palm olein did not show
any AST value as it did not solidify. Kumar et al. 2010 also reported
that body fats (goat and sheep body fat) showed lowest AST
values as they got completely solidified in the least time at all the
temperatures employed, whereas vegetable oils took highest
(infinite) time.
The results showed that the AST value (Table 1) for pure cow
ghee was lowest (2:45) in January and highest (3:17) in March.
For pure buffalo ghee, the lowest AST value (2:29) was found to
be in November, but highest (2:50) was observed to be in March
as noticed for pure cow ghee. The similar trend of variation
between AST values of cow and buffalo ghee was also observed
by earlier workers (Kumar et al. 2009; Kumar et al. 2010). AST
values of cow and buffalo ghee differed statistically (P<0.05)
from each other, as well as with the sheep body fat.
AST value of cow as well as buffalo ghee when adulterated with
palm olein individually at 5, 10 and 15 % levels were found to
increase and this increase was dependent upon the amount of
adulterant added to the pure cow and buffalo ghee. AST values
of cow and buffalo ghee added with palm olein and sheep body
fat, respectively at 15 % level differed significantly (P<0.05) from
that of pure cow and buffalo ghee. Sheep body fat adulterated to
both cow and buffalo ghee at the same levels (5, 10 and 15 %)
decreased the AST value. It was also observed that addition of
palm olein and sheep body fat in their mixture when added at 3+7
September November Average±SE*
3:08 2:59 3:01±0:05a
2:38 2:29 2:40±0:03b
ND ND -
0:39 0:35 0:39±0:02c
Indian J Dairy Sci 71(3): 246-251

(10), 6+14 (20) and 9+21(30) % levels in both types of ghee caused level of mixture adulterant added, greater was the effect on the
a decrease in the AST value. Addition at 9+21(30) level of mixture AST value of ghee samples.
of adulterants caused a significant difference (p<0.05) in the AST
values from that of the pure cow and buffalo ghee. Higher the

Table 2 AST values (min: sec) of cow and buffalo pure ghee and ghee adulterated with individual adulterants and mixture thereof.

Adulterant AST value (min:sec)

Level (%) Cow ghee Buffalo ghee Pooled(Cow+Buffalo) ghee
Range* Average±SE Range* Average±SE Range** Average±SE
Control 0 2:45 - 3:17 3:01±0:07a,x,l 2:29 - 2:50 2:40±0:02a,x,k 2:29 - 3:17
2:50±0:03a,x,l
PO 5 3:07- 3:35 3:22±0:04b 2:44 - 2:54 2:48±0:01a 2:44 - 3:35 3:05±0:02b
10 3:08 - 3:45 3:31±0:05bc 2:55 - 3:38 3:07±0:11b 2:55 - 3:45 3:19±0:04c
15 3:15 - 3:52 3:38±0:05c 3:01 - 3:35 3:15±0:08b 3:01 - 3:52 3:26±0:04c
SBF 5 2:33 - 3:25 2:56±0:18x 2:17 - 2:49 2:31±0:07x 2:17 - 3:25
2:43±0:06xy
10 2:25 - 3:23 2:47±0:20xy 2:08 - 2:29 2:20±0:03y 2:08 - 3:23 2:33±0:03y
15 2:01 - 2:56 2:26±0:20y 2:05- 2:14 2:09±0:01y 2:05 - 2:56 2:18±0:0z
PO+SBF 3+7(10) 2:47 - 3:35 3:05±0:14l 2:19 - 2:37 2:28±0:02l 2:19 - 3:35
2:46±0:03lm
6+14(20) 2:04 - 3:23 2:49±0:32lm 2:15 - 2:24 2:18±0:01m 2:15 - 3:23 2:34±0:05m
9+21(30) 2:03 - 2:45 2:27±0:10m 2:02 - 2:09 2:05±0:01n 2:02 - 2:45 2:16±0:03n

PO: Palm olein SBF: Sheep body fat

*Data represent mean ±SE of six determinations in alternate months over the period of 1 year.
Different superscripts (alphabetic) represent the significant difference within the column (for different levels of a similar type of
adulterant) from the control ghee samples at P < 0.05

Table 3 AST values (min: sec) of first solid (S15) fractions of pure ghee samples and ghee sample added with a combination
of adulterants.

Type of Type of Level of AST value

ghee adulterant fat/oil adulteration (%) (min: sec)
Range Average±SE
S15 S15
Cow Ghee Control 0 1:50 to 2:15 2:00±0:03a
PO+ SBF 3+7(10) 1:42 to 2:05 1:53±0:03ab
PO+ SBF 6+14(20) 1:28 to 1:52 1:44±0:03b
PO+ SBF 9+21(30) 1:11 to 1:39 1:24±0:04c
Buffalo Ghee Control 0 1:37 to 1:47 1:40±0:01k
PO+ SBF 3+7(10) 1:24 to 1:39 1:28±0:02l
PO+ SBF 6+14(20) 1:11 to 1:18 1:14±0:01m
PO+ SBF 9+21(30) 1:03 to 1:15 1:07±0:01n

Pooled Control 0 1:37 to 2:15 1:50±0:01w

(cow+buffalo)Ghee PO+ SBF 3+7(10) 1:24 to 2:05 1:41±0:03x
PO+ SBF 6+14(20) 1:11 to 1:52 1:29±0:02y
PO+ SBF 9+21(30) 1:03 to 1:39 1:15±0:02z
PO-Palm olein SBF- Sheep body fat
Data represent mean ±SE of six determinations in alternate month over the period of 1 year. Different superscripts (alphabetic)
represent the significant difference within the column (for different levels of combination of adulterant) from the control ghee
samples at P < 0.05.

249

Figure. 1 Fractionation approach: Optimising time-temp combination
On comparison with the overall range (2:29 to 3:17) for AST value
(min:sec) of both cow and buffalo pure ghee samples, the results
revealed that the adulteration of cow ghee with palm olein
individually could easily be detected at all the levels, while in
case of buffalo ghee, it could not be detected at any of the levels
studied. Failure of detection of adulteration of buffalo ghee with
palm olein as compared to cow ghee adulterated with it could be
ascribed to the wide variation of AST values for pure ghee
observed in this study for the two species including the seasonal
effects. On the other hand, sheep body fat added individually in
cow ghee samples could only be detected at 15% level, whereas
in the case of buffalo ghee, it could easily be detected at all the
levels except at 5% level (Table 2).
Further, it was observed from the results of the average AST
values (Table 2) of the ghee samples added with mixture of
adulterants at 3+7(10), 6+14(20) and 9+21(30) % levels that palm
olein along with sheep body fat could only be detected at the
level of 9+21(30) % in case of cow ghee samples. On the other
hand, in the case of buffalo ghee samples, all the levels studied
were easily detectable as the addition of even lower level of
3+7(10) caused a significant difference (p< 0.05) in the AST values
when compared with the control sample.
Pooling of the data of pure and adulterated cow and buffalo ghee
samples revealed that the addition of palm olein was detectable
at the levels of 10 and 15%, but not at 5% level. On the other
hand, sheep body fat added individually could not be detected
at 5 and 10 % levels, while higher level (15 %) was found to be
detectable. Likewise, a mixture of palm olein and sheep body fat
was not detectable at all the levels except at 9+21 % level.
As discussed earlier the fractionation of pure ghee samples and
the samples adulterated with a mixture of adulterants was carried
250
Indian J Dairy Sci 71(3): 246-251
out at 15oC for the enrichment of sheep body fat in first solid
fraction (S15) which was subsequently analyzed for AST value
and the results obtained are shown in Table 3.
The AST value ranged from 1:50 to 2:15 with an average of 2:00
for the first solid (S15) fraction of pure cow ghee, whereas the
similar values for the S15 fraction of pure buffalo ghee ranged
from 1:37 to 1:47 with an average of 1:40. It was observed from
the results that the AST values of the first solid (S15) fractions of
both cow and buffalo pure ghee were found to be lower than that
of the corresponding (unfractionated) pure cow and buffalo
ghee samples from which the fractions were obtained. Further, it
was also observed that the AST value of the first solid (S15)
fractions of ghee samples of both cow and buffalo adulterated
with mixture of palm olein and sheep body fat in at 3+7(10),
6+14(20) and 9+21(30) % levels decreased with the level of
adulteration which was probably because of enrichment of sheep
body fat as it possessed very low AST value as compared to
pure ghee samples. Addition at a lower level of 3+7(10) of palm
olein and sheep body fat caused a significant difference (p<0.05)
in the AST values of the buffalo ghee but not of cow ghee
indicating higher unsaturation in the cow ghee.
The low AST values for the S15 fraction of pure ghee samples as
compared to AST values of unfractionated pure ghee may be
possibly due to the reason that the fractionation process leads
to separation of triglycerides containing more of saturated fatty
acids and moving into first solid (S15) fraction on the basis of
their melting points.
Considering the overall range (1:37 to 2:15) of the AST value of
first solid (S15) fraction of both pure cow and buffalo ghee samples
as the basis and then comparing it with the average AST values
for the respective first solid (S15) fraction of cow and buffalo
Indian J Dairy Sci 71(3): 246-251
ghee samples added with mixture of palm olein and sheep body
fat at 3+7(10), 6+14(20)and 9+21(30) % levels of adulterations, it
was noted from the cow ghee samples that the adulteration of
palm olein and sheep body fat could not be detected at the level
of 3+7 (10) % and 6+14 (20) % levels, while 9+21 (30) % level
could easily be detected. On the other side, in the case of buffalo
ghee samples, all levels of the mixture of adulterants studied
could easily be detected.
On pooling of the data obtained on first solid (S15) fraction of
pure and adulterated cow and buffalo ghee samples and using
the overall range (1:37 to 2:15) of AST value was used as the
basis for comparison of AST value of first solid (S15) fractions of
adulterated ghee samples, it was observed that the lower level of
adulteration (3+7 % level) could not be detected, while the higher
levels of adulteration (6+14 and 9+21 % ) could easily be detected.
When the results obtained on average values of the AST value
of fractionated ghee samples were compared with those of
unfractionated ghee samples (Table 3) applied on pooled data of
cow and buffalo ghee samples, it was found that the fractionation
technique has offered an advantage in lowering the detection
limit using AST value because that level (6+14 %) of adulteration
with adulterants (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 first solid (S15) fractions.
The result corroborates the findings of the earlier workers (Kumar
et al. 2009) who also reported an increase in the sensitivity of the
AST test on fractionation.
Conclusions
From the above study, it may be concluded that apparent
solidification time determination is a simple technique and can
considerably help to detect the adulteration in ghee. The AST
test could be applied as a valuable parameter for checking
adulteration in milk fat, in particular for the detection of palm
olein addition in case of cow ghee and sheep body fat addition in
case of buffalo ghee. Palm olein was detectable at the levels of 10
and 15%, however, not at 5 % level in pooled samples. However,
sheep body fat added individually could not be detected at 5 and
10 % levels, while higher level (15 %) was found to be detectable.
Palm olein along with sheep body fat studied at all the levels
could not be detected in the case of cow ghee samples except at
9+21(30) % level. On the other hand, in the case of buffalo ghee
samples, all the levels considered were easily detectable.
Fractionation technique has provided advantage of increasing
the level of sensitivity of AST test by cutting down the detection
limit using AST value because that level (6+14) of adulteration
with adulterants (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 solid (S15) fraction obtained
at 150C.
251
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