Comparison of the Chemical Characteristics of Two Butter Oils with
Different Fatty Acid Composition
Rahman Ullah1, *Muhammad Nadeem1, M. W. Azeem2 and Shakeel Ahmad1
1
Department of Dairy Technology, University of veterinary and Animal Sciences, Lahore
2
United Industries Ltd. Faisalabad
*Corresponding Author: muhammad.nadeem@uvas.edu.pk
Abstract: This study investigated the chemical characteristics of butter oil either prepared by the
modification of milk fat by feeding 2% calcium salts of fatty acids or dry fractionation of cream.
Milk fat from the feeding trial and the low melting fraction of cream extracted at 10oC was
turned into butter oil, compared with standard butter oil and designated as BOFT and BOFF and
SBO. Both the modification strategies induced major changes in the fatty acid composition of the
milk fat, feeding calcium salts of fatty acids decreased the short-chain fatty acids whereas they
increased in the olein based butter oil (P<0.05). Feed and fractionation induced oleic acid and
linoleic acid in BOFT and BOFF were 28.56%, 31.83% and 2.89%, 3.24%, respectively. The
cholesterol content of SBO, BOFT and BOFF were 285, 278 and 346 mg/100 gram. Iodine value
of BOFT, BOFF and SBO was 42.78, 45.73 and 34.96. Peroxide value, anisidine value and
conjugated dienes of three moths stored BOFT and BOFF were higher than the control (P<0.05)
with no difference in free fatty acids, color, smell taste and overall acceptability score. Peroxide
value, anisidine value and conjugated dienes were non-significant up to 1-month of storage
period. The findings suggest that butter oil modified fatty acid composition can be prepared from
the milk fat with modified fatty acid composition with increased health benefit, acceptable
sensory attributes with moderate storage stability.
Keywords: Calcium salts of fatty acids, unsaturated fatty acids, oxidative stability
1
INTRODUCTION
In the subcontinent butter oil is regarded as superior fat over vegetable oils and fats for
cooking, frying, baking and coating of chapatti (unleavened bread). Due to the higher
atherogenic index of milk fat, increased mortalities with cardiovascular diseases, awareness
of nutrition related health disparities, people have started to avoid milk and milk products
(Hansel et al., 2007). Dairy products are criticized on account of higher content of dietary
cholesterol (Williams, 2000). Heart Associations around the globe direct to limit the intake of
saturated fatty acids and cholesterol (British Heart Foundation, 2005; European Heart
Network, 2005). Regular consumption of milk and dairy products has been associated with
cardiovascular disease (Honda et al., 2007). The high incidences of mortalities from
cardiovascular disease has led to the necessity of studying milk and milk products, as a fact
they contain a bigger concentration of C12:0, C14:0 and C16:0 and lower concentration of
unsaturated fatty acids as compared to other edible fats (Rudel and Morris, 1973). These fatty
acids are atherogenic, and increase the risk of cardiovascular disease by increasing plasma
cholesterol and low density lipoproteins (Lokuruka, 2007). For the reduction of saturated
fatty acids and cholesterol from milk and its products, large number of strategies has been
developed; manipulation of bovine feeding regime can have a great impact on the extent of
saturated fatty acids and bioactive compounds (Abu Ghazaleh and Holmes, 2009). Feeding
rumen protected fatty acids and dry fractionation of milk fat considerably increased
unsaturated fatty acids in milk and milk fat (Fahey et al., 2002; Nadeem et al., 2013a). Dry
fractionation of milk fat is superior to solvent fractionation due to the higher cost of solvents
and health hazards associated with them (Reddy, 2010). To our knowledge, chemical
characteristics of butter oils prepared from milk fat whose fatty acid composition has been
2
modified by feeding calcium salts of fatty acids and fractionation has not been studied in
detail previously and the comparison of their chemical characteristics of the same has not
been performed so far. This objective of this investigation was to compare the butter oils with
changed fatty acids with standard butter oil on the basis of certain chemical and sensory
characteristics.
MATERIALS AND METHODS
Butter Oil from Milk of Cows Fed on Calcium Salts of Fatty Acids. Fatty acid composition
of milk fat was modified by feeding calcium salts of fatty acids at the rate of 2% to twelve
randomly stratified cows in two groups. The feed comprised 55% forages and 45% concentrate
(Iso-caloric and iso-nitroenous) with free excess to water, equal number of installers, same barn
conditions, the experiment was conducted for sixty days, detail of feed is mentioned in Table-1.
Butter Oil by Dry Fractionation of Milk Fat. Cream with 40% fat content was purchased from
Haleeb Foods Ltd. Kasur, heated to 63oC for 1-hr, cooled down to 10oC in 2-hrs, held at this
temperature for 3-hrs to promote the crystallization, filtered over Buckner filtration assembly
connected with a vacuum pump (Buchi). The crystallized cream was filtered at 600-mm Hg
Pressure, stearin remained over the filter paper, while the low melting fraction was collected
from the Buckner flask, repeated five times to minimize the errors, pooled and converted into
butter and finally into butter oil (Reddy, 2010).
Experimental Plan. Two types of butter oils; butter oil manufactured from milk fat obtained
from the milk of cows that were fed on 300-grams calcium salts of fatty acids (BOFT) and the
butter oil from the low melting fraction of unmodified milk fat fractionated at 10oC (BOFF) were
compared with each other for chemical characteristics.
3
Analysis. Fatty acid composition of butter oils were determined by transformation into fatty acid
methyl esters. The fatty acid methyl esters were prepared by sodium methoxide
transesterification technique in iso-octane, the supernatant FAME layer was injected at a 1µL
concentration into the GC fitted with flame ionization detector (Perkin Elmer Instrument, Auto
system XL) using SP-2380 fused silica capillary column (30m x 0.25mm Supelco Bellefonte,
PA) using nitrogen (1.5 mL/minute) as a carrier gas (Qian, 2003). Fatty acids were identified and
quantified by using FAME Mix GLC-30, Supelco). Iodine value, peroxide anisidine values, free
fatty acids, moisture, unsaponifiable matter, and slip melting point was determined by the
standard methods (AOCS, 1995) Color was checked in 5 inch quartz cell in Lovibond
Tintometer (Tinto Meter Corporation Salisbury, England). Cholesterol determination was
performed as per method of (Rudel and Morris, 1973). Schaal oven test was performed by
keeping 10±0.1 gram samples in triplicate beakers at 63±2 0C in an oven for five days; peroxide
value was measured as indication of oxidative stability after five days. Oxidation products in the
form of conjugated dienes were measured by dissolving one gram sample of butter oil in
isooctane and absorbance was measured at 232 nm in the ultra violet region on
spectrophotometer (Schimadzu, Japan) as per methods of (IUPAC, 1987). Induction period was
measured by using Metrohm Rancimat (679) 2.5±0.1 gram samples were directly weighed into
the reaction vessels, oxidized at 120 oC under steady stream of oxygen by following the protocol
as given in instruction manual of Metrohm Corporation Limited, Switzerland (Metrohm, 1993).
Sensory evaluation was performed by a panel of 10-trained judges on a 9-point scale in the
sensory evaluation booths (Larmond, 1987). All determinations were carried out in triplicate and
data was expressed as Mean ± SD. The data was analyzed by using completely randomized
design and significant difference (P<0.05) among the treatments was calculated by using LSD
test (Steel et al., 1997).
RESULTS AND DISCUSSION
Chemical Characteristics of Butter Oils with Modified Fatty Acid Profile. The results of
chemical characteristics of butter oils with modified fatty acid composition are given in Table-2.
Modification of fatty acid composition through feeding and fractionation did not have any effect
on free fatty acids, color and unsaponifiable matter of the butter oils (P>0.05). Major changes
4
were observed around iodine value and melting point in both types of butter oils. Iodine value of
BOFT and BOFF increased whereas melting point decreased as a function of fatty acid
modification (P<0.05). The increase in iodine value or decrease in melting point was in the order
of BOFF > BOFT > SBO. The increase in iodine value and decline in melting point of the butter
oils was due to the enhancement of unsaturated fatty acids. Iodine value and melting point are
correlated with each other, fats having higher iodine value show lower melting point (Fereidoon,
2005). Enhancement of unsaturated fatty acids in butter fat through feeding rumen protected fatty
acids increased iodine value and decreased solid fat index of the modified version of the milk fat
(Gonzalez et al., 2003). The increase in unsaturated fatty acids of milk fat through manipulation
in the ration of cows has also been reported by (Mallia et al., 2008). Feeding unsaturated fatty
acids in protected form did not have any influence on the concentration of cholesterol in milk
(P>0.05). Our findings were also corroborated with previously reported results. (Brzoska and
Sala, 2001) also reported a non-significant effect of feeding regimes on the cholesterol content of
milk. (Nadeem et al., 2013a) did not find any variation in the cholesterol of the milk when
Sahiwal cows were fed on calcium salts of soybean oil fatty acids. Cholesterol content of the low
melting fraction of milk fat was considerably higher than the control. The rise in cholesterol
content of the olein fraction could be connected to the migration of cholesterol to olein fraction,
this could also happened due to the affiliation of cholesterol with low melting triglycerides of the
milk fat. The migration of the cholesterol from milk fat to the low melting fractions has been
observed by Nadeem et al. (2013b).
Fatty Acid Composition of Butter Oils with Modified Fatty Acid Profile. The results
regarding fatty acid composition of butter oils prepared from milk of cows fed on calcium salts
of fatty acids and fractionated cream are presented in Table-3. Feeding and fractionation had a
5
major effect on the fatty acid composition of butter oils (P<0.05) with some difference in the
pattern of re-adjustment of the fatty acids in the modified version of the fats. The major
difference was observed around the short chain fatty acids, feeding increased the extent of short
chain fatty acids while fractionation showed opposite trend of their enhancement. However, both
the techniques showed common trends of the declining of medium chain fatty acids and
enhancement of unsaturated fatty acids. The increase in unsaturated fatty acids of the butter oils
were in the order of BOFF > BOFT > SBO. The drop in atherogenic fatty acids in the reformed
fats is beneficial from health point of view as they have been implicated in the uplifting of
harmful LDL cholesterol. The relatively higher concentration of unsaturated fatty acids in the
fractionation process over the feeding could be attributed to the lower fractionation temperature
employed in this investigation. Fractionation temperature had a great effect on the fatty acid
composition of milk fat fractions. Fatty acid composition of the low and high melting fractions of
milk fat collected at different temperatures was different (Reddy, 2010). The fatty acid
composition of milk fat fractions was considerably different from the parent milk fat (vanAken et
al., 1999; Chen et al., 2004; Nadeem et al., 2013b).
Storage Stability of Butter Oils at Ambient Temperature. Table-4 describes the results of
storage stability of butter oils with altered fatty acid composition. Fatty acid modification did not
have any effect on the generation of free fatty acids in the fats during storage period of 60-days.
The rise in the extent of free fatty acids in the control and modified butter oils was in a nonlinear fashion without any specificity to a treatment. Free fatty acids content of the butter oils
increased with progression of the storage period. The intensification of free fatty acids during the
storage period could be correlated to the presence of moisture, metal contamination and presence
of lipases etc. (McSweeney and Fox, 2003). Free fatty acids of fats and oils usually increase as
6
the storage period progresses (Erickson, 1999). Extent of free fatty acids is connected with shelf
life of fats and oils, higher concentrations commonly confer objectionable odors to the stuffs.
Peroxide value of the treatments and control increased slowly and steadily in a classical
manner during 60-days of storage. The increase in peroxide value of the stored butter oils and
control was dependent upon the concentration of unsaturated fatty acids, therefore, BOFF
generated the higher extent of the primary oxidation products, the order of rise of peroxide
value during the storage period was in the order of BOFF > BOFT > SBO. The extent of
unsaturated fatty acids were strongly correlated with peroxide value, Fig. 3 (R2=0.9998). The
connection between the type and extent of unsaturation has been reported in the literature
(Anwar et al., 2007; Gulla and Waghray, 2011). Determination of peroxide value provides
magnitude of the amount of primary oxidation products generated as a result of free radical
mechanism (McGinely, 1991). The peroxide value of butter oil increased during ambient
storage of 90-days (Nadeem et al., 2013c).
Anisidine value of the three months stored butter oil prepared from fractionated milk fat
was greater than the butter oil produced from milk of cows fed on calcium salts of fatty acids
and standard butter oil. Anisidine value increased steadily with the progression of the storage
period, the oxidation rate of oleic acid and linoleic acid is 10 and 25 times higher than the
oxidation rate of stearic acid (Baer et al., 2001). This could be the justification of the shoot
up of anisidine value of BOFF over the other samples. The concentration of oleic and linoleic
acid was relatively higher in BOFF than BOFT and control. Anisidine value is a measure of
the intensity of photochemical reaction between aldehydes and anisidine (Anwar et al.,
2010). Measurement of anisidine value gives magnitude of the extent of the secondary
oxidation products generated during the oxidative breakdown of fats and oils (Pritchard,
7
1991). The peroxide and anisidine values are generally linked with keeping quality, fats and
oils showing higher magnitudes will have poor storage stability. Milk fat with modified fatty
acid composition showed poor oxidative stability as compared to un-modified milk fat
(Gonzalez et al., 2003). Anisidine value of canola oil increased during storage (Chatha
2011).
The oxidation products went on increasing throughout the storage period of 60-days.
BOFF possessed the higher concentration of unsaturated fatty acids over BOFT and SBO, the
extent of oxidation products as a function of the breakdown of unsaturated fatty was in the
order of BOFF > BOFT > SBO. It is evident from the results of Table-4 that the storage
period up to 30-days was non-significant for free fatty acids, peroxide value, anisidine value
and conjugated dienes. To determine the oxidative stability of canola oil, (Chatha 2011)
included conjugated dienes an important parameter.
Oxidative and Thermal Stability in Accelerated Autoxidation. The results of induction period
are given in Figure-1, the induction period of BOFF, BOFT and SBO was different from each
other (P<0.05) and found in the order of SBO > BOFT > BOFF. The variation in the induction
periods was due to the difference in the fatty acid composition of two butter oils and the control,
the higher the extent of unsaturated fatty acids, lower was the induction period. We compared the
oxidation rate of stearic acid, oleic acid and linoleic acid, the oxidation rates were 1: 10: 25,
respectively. The higher proportion of linoleic acid in BOFF could also be reason for lower
induction period of BOFF and BOFT over the control. Measurement of induction period
quantifies the oxidative and thermal stability of fats and oils in the accelerated oxidation
conditions (Anwar et al., 2003). The higher induction periods are usually associated with better
and prolonged keeping quality and vice versa. The induction period of vegetable oils without any
8
additives was dependent on the fatty acid composition, stuffs having higher degree of
unsaturation revealed lower induction periods (Anwar et al., 2007). The peroxide value of butter
oils with different fatty acid composition and control in the accelerated oxidation chamber (63oC
for 5-days) was in the order of BOFF > BOFT > SBO. The significant difference could be
attributed to the variation in the fatty acid composition, fats having higher content of unsaturated
fatty acids suffered from serious oxidation. (Nadeem et al., 2013b) found a strong correlation
between the extent of unsaturation and oxidizabilty of olein fraction of milk fat. Measurement of
peroxide value in the accelerated oxidation chamber provides useful information regarding the
capacity of a fat towards the autoxidation (Mahuya et al., 2008).
Sensory Characteristics. The results of sensory characteristics of butter oils with different fatty
acid composition are given in Table-5. Modification of the fatty acid composition by
manipulating the ration of cows and fractionation of cream did not have any effect on color,
smell, taste and overall acceptability of butter oils. The non-variation in the sensory
characteristics of butter oils could be attributed to; in BOFF the concentration of short chain fatty
acids was higher than the native milk fat (P<0.05) though the content of short chain fatty acids in
BOFT was less than the parent milk fat, yet the decreased limits were not detected by the judges.
The second reason for non-significant difference could be the non-variation in the peroxide value
of experimental and control butter oils. (Nadeem et al., 2013b) recorded a non-significant
difference in the sensory attributes of fresh olein based ice creams.
Conclusion. Fatty acid composition of butter oil prepared from milk of cows fed on calcium
salts of fatty acids, olein based butter oil and standard butter oil was different from each other
(P<0.05). Peroxide and anisidine values of both the butter oils were not different from the control
9
up to 1-month of storage period. The overall acceptability score of the fresh experimental butter
oils were similar to the standard butter oil.
Acknowledgements
The authors are highly obliged to Sheikh Pervez Ahmed Anwel, General Manager
Works, United Industries Limited Kashmir Road Nishatabad, Faisalabad, for his great help in the
smooth running and completion of this experiment.
10
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