EXPERIMENT 9: DETERMINATION OF ACID VALUE OF FATS

2011-35493, 2011-85007
Biochemistry 34.1, HEJ, Sir Avvin Pelovello
I. Abstract

Lipids are biologically produced molecules that are relatively insoluble in water and soluble in polar and non-polar
organic solvents. Fatty acids are amphipathic, containing both polar and non-polar groups. Oils are long straight-chain
carboxylic acids composed of saturated and unsaturated fatty acids. In the experiment, the acid value of used
vegetable, lauric, and coconut oil were determined. The obtained average acid values (mg KOH/g) for the four oil
samples are 1.19, 2.18, 0.87, and 1.86. Based on the data, the rancidification of these used oils are apparent due
to the higher level of acid value as compared to the acceptable one at 0.6 mg KOH/g. This showed the occurrence of
rancidification, or the production of foul odor and taste in fats. There are three pathways for rancidity; oxidation of
peroxides (oxidative rancidity); hydrolysis of unsaturated fatty acids and production of free fatty acids (hydrolytic
rancidity); and microorganism activity (microbial rancidity).

II. Keywords: lipids, fatty acids, acid value of fats, oxidative rancidity, hydrolytic rancidity
III. Introduction IV. Experimental

Lipids are compounds that are defined based
on solubility. They have low solubility in water and high
solubility in nonpolar solvents. The nonpolar nature of
lipid is due to the large portion of hydrocarbons in the
molecule. Lipids have diverse biological functions,
including being highly concentrated energy sources,
membrane components (structure), and molecular
signals (Moore & Langley, 2008).
Biological lipids are either hydrophobic,
containing only nonpolar groups, or amphipathic,
possessing both polar and nonpolar groups (Garrett &
Grisham, 2010). Fatty acids are amphipathic lipids
because they have polar carboxylic acid group and a
long nonpolar hydrocarbon tail. They normally have
even number of carbon atoms. Presence of carbon-
carbon double bonds in a fatty acid chain makes it
unsaturated. While fatty acid with only single bonds is
saturated. Unsaturated fatty acids are usually in cis
configuration rather than trans (Campbell & Farrell,
2015).
Derivatives of fatty acids include fats and oils
which serve as stored forms of energy in living
organisms (Nelson & Cox, 2013). Fats and oils are
triglycerides or triacylglycerols. They are lipids that are
formed through ester linkages between three hydroxyl
groups (of glycerol) and fatty acids. The apparent
distinction between fat and oil is that the former is a
solid at room temperature making it saturated while
the latter is liquid at room temperature making it
unsaturated (Moore & Langley, 2008). Fatty acid
composition in fats and oils has been explored due to
its association with health related risks (Garrett &
Grisham, 2010).
The objective of the experiment is to
determine the acid value of commercially available
oils.
Primarily, 200 mL of fat solvent was prepared
by mixing 100 mL of absolute ethanol and 100 mL of
diethyl ether in a beaker. Covering the solution is
required due to its volatile trait. Then, 1.0 mL of fat
sample or used cooking oil was dissolved in 10 mL of
fat solvent. Next, 2 drops of phenolphthalein indicator
was added and it was mixed thoroughly. It was titrated
against 0.01 M KOH until a pale pink end point is
achieved. Four fat samples were analyzed and for
each sample, three trials were executed. The volumes
of the titrant consumed were noted. Finally, the acid
value of the fat for each trial was calculated using the
equation below.
()
=
()
V. Results and Discussion
Oils are naturally occurring esters long
straight-chain carboxylic acids. They belong to the
saponifiable group of lipids. Lipids are biologically
produced. They are relatively insoluble in water and
soluble in polar and non-polar organic solvents.
Triacylglycerol molecules mostly constitute edible oils.
Both saturated fatty acids, such as oleic acid and
linoleic acid, and unsaturated fatty acids, such as
palmitic acid and stearic acid, form these oils. A single
fatty acid that could be esterified up to three times into
glycerol backbone, or even different ones, lead to the
formation of these oils. (Atinafu & Bedemo, 2011).
Acid value (AV) is the amount of potassium
hydroxide in milligrams required to neutralize the free
acids presence in a gram of sample. It is generally a
measure of the free fatty acids in oil. Fatty acids are
normally found in the triglyceride form, but hydrolysis
of these fatty acids frees them and accounts to the the
decreased quality of oil. Acceptable levels for all oil finally with the presence and activity of
samples should be below 0.6 mg KOH/g (Atinafu & microorganisms (Luck & Lipinski, 2002).
Bedemo). Oxidative rancidity is due to the cleaving of
From the experiment, the following values unsaturated fatty acid double bonds that releases
were obtained from the titration of the used vegetable volatile aldehydes and ketones. This type of rancidity
oil with potassium hydroxide: commonly occurs with unsaturated fatty acids, both
mono- and poly-, as the lipids undergo oxidation
Table 1. Titration of used vegetable oil with KOH. reaction (Thomas, 2005).
density volumeKOH acid value Hydrolytic rancidity is due to the hydrolysis of
oil sample
(g/mL) (mL) (mg KOH/g) triglycerides, releasing free fatty acids and lower the
2.0 1.23 quality of the fat. This type of rancidity has catalytic
1 2.1 1.29 requirements to develop with the presence of water.
0.910 Short-chain fatty acids are commonly affected by this
(vegetable) 1.7 1.05
Ave: 1.19 type of rancidification (Freeman, 2000).
Microbial rancidity is due to the presence of
3.6 2.21
microorganisms such as bacteria and molds.
2 3.5 2.16
0.910 Enzymatic activity aid these microorganisms in
(vegetable) 3.5 2.16
metabolizing lipids and fats, producing the foul odor
Ave: 2.18
and taste (Allen & Hamilton, 1994).
2.0 1.11
Other values that are tested for the quality of
3 1.4 0.78
1.007 fats. One example is the peroxide value. This is a test
(lauric) 1.3 0.72
for the propensity of the fat to undergo oxidative
Ave: 0.87 rancidification. The measure of peroxides and
3.1 1.89 hydroperoxides formed in the initial stages of lipid
4
0.920 3.0 1.83 oxidation are determined through titration with iodide
(coconut)
Ave: 1.86 ion. High values of peroxide are indicative of a rancid
fat, but moderate and lower values may not be
The acid values were obtained by using the conclusive and correlated with rancidity (Grossi, et. al,
formula in the lab manual. Since the sample is oil, the 2015).
density was used to convert it to grams of fat. Another value is the anisidine value.
Hydroperoxides produce volatile aldehydes such as
hexanal and leaves the non-volatile part to the
() glyceride molecule. High values of anisidine is also an
=
() indication of the fat undergoing oxidation (FoodLab,
2012).
0.0020 0.01 56105.6
= The aforementioned values are also tested

0.910 1.00 with their respective tests, such as the peroxide value

= . / test for peroxides and p-anisidine value test for
anisidine value.
The results above show the amount of free
fatty acids present in the oils based on the titration VI. Conclusion and Recommendation
with potassium hydroxide. The acid values of these
oils are above the acceptable levels for consumption. Acid value refers to the amount of potassium
Hydrolysis of the fatty acids present in these used oils hydroxide in milligrams that is required to neutralize a
predominate in the sample, giving a higher amount of gram of fat sample.
free fatty acids and lower oil quality or rancidity. Fats undergo rancidification or production of
Rancidification is a general term for the foul odor and taste. There are three types: oxidative
process that causes a substance to become rancid or rancidity, hydrolytic rancidity, and microbial rancidity.
have unpleasant odor or taste. This is due to three Oxidative rancidity is due to the fats undergoing
pathways. One is through degradation by oxygen oxidation reaction and consequent elevation of
through a free radical process, another is through the peroxide values in the sample. Hydrolytic rancidity is
hydrolysis of triglycerides into free fatty acids, and due to the cleavage of double bonds in unsaturated
fatty acids, producing volatile aldehydes and ketones,
as well as free fatty acids. Microbial rancidity is due to Nelson, D., Cox, M. (2012). Leningher principles of
the presence of microorganisms and enzymatic biochemistry (6th edition). USA: W.H. Freeman
activity that break down fat. This results to a lower and Company.
quality of fats. The acceptable level for consumption
is at 0.6 mg KOH/g of acid value. Thomas, A. (2005). Ullmanns encyclopedia of
Results showed that the oil samples used in industrial chemistry, fats and fatty oils. Wiley-
the experiment have a higher acid value to that of the VCH, Weinheim.
acceptable level. This is due to the type of oil used. All
oil samples were used in the past, proving the lower
quality of these fats. Rancidification also occurs over
time, and repeated use of the fats can easily promote
processes for either of the three types of rancidity to
occur.
It is recommended to use fresh sample of oil
to test for the acid values for consumption. This will
ensure the quality of the commercially available edible
oils. Other values to test the quality of fats may also
be tested, such as peroxide value, anisidine value,
TBA value, and other important quality check systems.

VII. References

Allen, J.C., Hamilton, R.J. (1994). Rancidity in foods.

Springer-Verlag, GmbH.

Campbell, M., Farrel, S. (2013). Biochemistry (8th

edition). USA: Cengage Learning.

Freeman, I. P. (2000). Ullmanns encyclopedia of

industrial chemistry, margarines and
shortenings.

p-Anisidine value: (2012). Analysis system for fats and

oils. FoodLab Quality Control Systems for
Food and Beverage. Retrieved (May 19,
2016): http://www.cdrfoodlab.com/food-
analysis/anisidine-value-fats.html

Garret, R., and Grisham, C., (2010). Biochemistry (5th

ed.)

Grossi, M., Di Lecce, G., Arru, M., Gallina, Toschi, T.,

Rocco, B. (2015). Journal of food
engineering, an opto-electronic system for
in-situ determination of peroxide value and
total phenol content in olive oil.

Luck, E., Lipinski, G-W., (2002). Ullmanns

encyclopedia of industrial chemistry, foods,
food additives. Wiley-VCH, Weinheim.