Dairy cows as a class of ruminants have evolved an extremely efficient
digestive system, principally for utilizing poor quality forages found
in natural diet. Although moderate levels of performance can be achieved
from diets containing only forage, high producing dairy cows require additional
energy and protein from supplementary sources. This is particularly true
during early lactation, where appetite is usually reduced to such an extent
that the cow cannot eat enough to satisfy nutrient requirements for high
Traditional sources of supplementing energy, high in starch can be rapidly
fermented in the rumen leading to lowered pH (Jouany, 2006) which is detrimental
to forage digesting bacteria. Fats can be used to formulate diets with
very high-energy concentration, as they have the advantage of creating
space within the diet, due to high energy content. In order to increase
the energy density of concentrates and meet the fat requirement of crossbred
and high producing animals, a non-conventional, cost effective and non-toxic
source of fat is essential.
During refining of edible oils, free fatty acids are removed by treating
them with sodium hydroxide and sulphuric acid. The free fatty acids thus
removed by centrifugation along with variable amount of triglycerides
and other minor fat-soluble constituents are termed acid oils. These are
available at a cheaper price. These acid oils posses the potential to
be used as a lipid source in ruminant rations. Though acid oils from various
sources are available sunflower acid oil was chosen for this study as
it is abundantly available in the study area (India).
However, feeding of free or unprotected fats beyond 3-4% level leads
to reduction in microbial activity resulting in depression of fibre digestion
in ruminants (Alexander et al., 2002). Hence, protection of fats
is an alternative way of feeding fat to these animals. Therefore, techniques
that would allow the use of fat in the form that is not harmful to microbes
or retard digestion (Ramana Reddy et al., 2003) is gaining much
While calcium salts was preferred to protect rapeseed oil (Kowalski,
1997), canolamide was found to be suitable for canola oil (Loor et
al., 2002), Sutton et al. (1983) concluded protein encapsulated
formaldehyde treatment was suitable to linseed oil and coconut oil. Keeping
this concept in view, a study was conducted to chemically characterize
the sunflower acid oil as well as to identify the suitable method of protection
of acid oil against rumen degradation.
MATERIALS AND METHODS
Procurement of Samples
Sunflower acid oil samples were collected in airtight container containing
0.05% of butylated hydroxy anisole, from six different batches of processing
from an oil mill and stored in cool and dark place.
Chemical Characterization of Sunflower Acid Oil
The samples were analyzed for their chemical composition viz., moisture,
crude protein and total ash as per the method of AOAC (1995). The remaining
part (i.e.,) 100- (Moisture+crude protein+total ash) was assumed as ether
extract. The samples were also analyzed for their chemical characteristics
viz., acid value, saponification number, unsaponifiable matters, peroxide
value and free fatty acids content was carried out by adopting the method
of AOAC (1995).
Preparation of Protected Forms of Sunflower Acid Oil
The sunflower acid oil was subjected to three methods of protection
viz., Aldehyde treated protein encapsulated form was prepared by the method
described by Sutton et al. (1983), calcium soaps was prepared by
precipitation method as described by Alexander et al. (2002) and
fatty acyl amides of sunflower acid oil were synthesized by mixing 100
g of sunflower acid oil and 100 g of ethanolamine as described by Loor
etal. (2002). The final amide products were examined in thin
layer chromatography as per Bilyk et al. (1991) to confirm the
absence of triglycerides and free fatty acids, which indicated that the
conversion of sunflower acid oil fatty acids to amide formation was complete.
Evaluation of Protection Against Rumen Degradation of Sunflower Acid
Oil by Thin Layer Chromatography
The three protected forms of sunflower acid oil (100 mg) and un protected
sunflower acid oil (100 mg) was evaluated by incubating (39°C) in
rumen fluid (10 mL) for 24 h and evaluating the extent of rumen lipolysis
(24 h) by measuring the free fatty acids production using thin layer chromatography
as per the procedure described by Gulati etal.(1997).
Un incubated samples (0 h) served as control to compare the extent of
rumen lipolysis by 24 h of incubation.
At the end of 24 h 0.5 mL of 5 N HCl and 4 mL of distilled water were
added to both unincubated and incubated tubes, shaken vigorously and allowed
to stand for 2-4 h until the two phases were clearly distinguished. The
lower organic phase was filtered to remove suspended solids. The filtrate
was evaporated to dryness by keeping it in the oven at 60°C for 24
h. After drying, 2 mL of chloroform/methanol (2:1 v/v) was added. Accurately
20 μL of the mixture was spotted on silica gel F254 plate
(aluminum sheet thickness 0.2 mmx10x20 cm) and placed in the solvent system
made up of petroleum ether: diethyl ether: acetic acid (84/15/1 v/v/v).
The separated lipids were visualized by keeping it in an iodine chamber.
The relative intensity and sizes of the free fatty acids spots in both
the incubated and unincubated reaction mixtures were estimated semi-qualitatively
by following a hundred point scale score for measuring the intensity of
the free fatty acids spots and measuring the surface area of the spot
to determine the size of the free fatty acid zone. While one hundred points
were assigned to the free fatty acids spot of the unincubated zero and
unprotected fat, its relative points were assigned to other spots depending
upon their intensity measured visually. The surface areas of the free
fatty acids spots were calculated by formula:
Surface area = 3.14 x a x b
Duplicate measurements for score and surface areas were carried out by two
persons for six runs. The data obtained in different parameters were subjected
to Complete Randomized Design (CRD) of statistical analysis as per the procedure
of Snedecor and Cochran (1994).
Sunflower acid oil contained low level of moisture, crude protein and
total ash. Sunflower acid oil has moderate level of acid value and free
fatty acids necessitating use of anti-oxidant. A high saponification number
of sunflower acid oil indicates its potential for converting into soaps.
The peroxide value and unsaponifiable matters were at moderate level in
sunflower acid oil (Table 1).
Protected forms of fat prepared from sunflower acid oil were evaluated
by thin layer chromatography to find out the efficiency of protected fat.
The relative intensity and size of free fatty acid zone present on the
chromatographic field was used as the criteria of evaluation, as intensity
and size of free fatty acid zone are inversely related to level of protection.
The lower intensity and smaller size of the free fatty acid zone were
considered to be better level of protection (Gulati et al., 1997).
Within the treatments of unprotected, aldehyde treated, calcium soaps
and amide, the score for intensity as well as surface area at 24 h incubated
samples were consistently higher than their respective 0 h samples (Table 2).
||Average chemical composition* of sunflower acid oil (% FMB) and
important quality parameters (Mean±SE)
|*: Mean of six observations, FMB: Fresh Matter Basis
||Score for intensity and relative surface area of free fatty acids
zone (Mean±SE*) measured from thin layer chromatography plate
for various forms of protected and unprotected sunflower acid oil
at 0 or 24 h of incubation
|*: Mean of six observations, Mean with different superscripts
within a row differ significantly (p<0.01)
Across the treatments, the score for the intensity of free fatty acid
of calcium soaps at 0 h was the best and significantly (p<0.01) different
from rest. Similarly among 24 h of incubation, the intensity of free fatty
acid zone of calcium soaps was the significantly (p<0.01) lower than
the rest treatments. Fatty acyl amides of sunflower acid oil showed next
significantly (p<0.01) better performance at 0 as well as at 24 h of
incubation. Unprotected sunflower acid oil had the lowest values (p<0.01).
Similar to the intensity of zone, measurement of surface area of the
free fatty acid zones reveals that calcium soaps at 0 h of incubation
as well as at 24 h of incubation had significantly (p<0.01) lowest
surface area than rest treatments (Table 2).
The results of this study are comparable to the reported values for constituents
in sunflower acid oil (Alexander et al., 2002). The moisture level
below 2.5% is desirable as higher moisture content permits the formation
of rust, which will accelerate autocatalytic (non enzymatic) oxidative
rancidity. Moisture in the presence of high levels of free fatty acids
and high temperature will also promote autocatalytic hydrolysis of glycerides
(Gunstone et al., 1994). Thus, with low level of moisture (0.74%),
sunflower acid oil do not pose threat for oxidative rancidity.
The insoluble impurities like hair, fleshing grease, phospholipids, trace
metals and protein as contaminants or adulterants may be associated with
many blend compounds. Insoluble impurities cause not only the fall in
the energy value of oil/fat, but also leads to blockage in feed mill equipments
like fat spray nozzles (Howard, 1984). The negligible quantity of crude
protein and total ash in the sunflower acid oil indicate that the study
material is unadulterated and free from contaminants.
The observations of the present investigation were comparable to the
reported acid value, Saponification number (Alexander et al., 2002).
The higher observed saponification number might be due to the presence
of higher proportions of short chain fatty acids in acid oils which might
be the consequence of degradation of polyunsaturated fatty acids during
the refining process of crude oil by heating acid oxidation (Wiseman et
al., 1992). The mean peroxide value of 8.74 was within the reported
range of 1.61 to 10.11 (meq oxygen kg-1) for sunflower acid
oil (Vila and Esteve-Garcia, 1996b). Peroxide value is the measure of
peroxides contained in the oil. The degree of oxidation resulting from
processing has often been assessed in terms of peroxide value. Vila and
Esteve-Garcia (1996b) have reported that inclusion of sunflower acid oil
having peroxide values up to 10.11 meq.oxygen kg-1 did not
cause any deleterious effect when added in the poultry ration at 10% level.
The sunflower acid oil values obtained in the current study were below
this level and hence, could be considered to be safe. More over the sunflower
acid oil is mixed with antioxidants upon arrival to the laboratory to
prevent further oxidation.
Unsaponifiable matters refer to the material, which is soluble in petroleum
ether but does not react with sodium or potassium hydroxide to form soaps.
This includes wide variety of compounds such as sterols, pigments, fat-soluble
vitamins, fatty alcohols, fatty-fatty esters (condensation products),
waxes, mineral oils, pesticides etc. Unsaponifiable matters contribute
very little to the energy value of feed fat. The unsaponifiable matters
estimated in the current study were similar to the value reported by Wiseman
et al. (1992).
The mean Free Fatty Acid (FFA as oleic acid) level of sunflower acid
oil recorded in the present study were lower than the value (58.22%) reported
by Alexander et al. (2002) and (57.885%) Vila and Esteve-Garcia,
(1996a). However, lower values (38.80%) were reported by Wiseman et
al. (1992). The variation in FFA content might be due to the difference
in the method of processing and refining of crude oils. Free fatty acids
refer to the fatty acids not esterified to glycerol. In whole fats, presence
of high levels of free fatty acids may be an indication of improper storage
and/or handling of fat. Hydrolysis may occur as either enzymatic lipolysis
during storage or prior to rendering or as autocatalysing hydrolysis.
The latter is often associated with oxidative rancidity. Hence, addition
of anti-oxidants is recommended to all feed fats to prevent rancidity
particularly in the presence of high levels of free fatty acids.
Consistently higher score recorded for 24 h incubated samples than their
respective 0 h samples might be due to the rumen metabolism and relative
dissociation of samples in the rumen fluid.
The data suggest that the calcium soaps offer best protection, as even
after 24 h of incubation, the samples had the most desirable faint intensity
and lower surface area of free fatty acids zone, which indicate that the
calcium soaps undergo lowest metabolism in the rumen than its counterparts.
Based on the intensity and surface area of the free fatty acids zone
on the chromatographic field, calcium soaps of sunflower acid oil was
selected as the potential protected fat to be used as concentrated energy
source in the rations of dairy cows.
The authors gratefully acknowledge the Tamil Nadu Veterinary and Animal
Sciences University authorities for the facilities rendered in conducting
this study as a part of M.V.Sc. dissertation.