Fatty Acid Ethyl Esters from Refined Ostrich Oil

LUCIA PINTILIE, ILEANA CATALINA PARASCHIV, IRINA LUPESCU, CRISTINA HLEVCA, GEORGETA RADULESCU,
RAMONA DANIELA PAVALOIU
National Institute for Chemical-Pharmaceutical Research and Development, 112 Vitan Av., 74373, Bucharest, Romania

The fatty acid ethyl esters were prepared by well-known chemical transesterification of fatty acid triglycerides
of refined ostrich oil with ethanol in alkaline catalysis, acid catalysis or two-step acid-base catalyzed
transesterification without prior cleavage of the free fatty acids. Determination of fatty acids in samples of
refined ostrich oil and of fatty acid ethyl esters (FAEE) was performed by gas chromatography.
Keywords: ostrich oil, FAEE, transesterification, fatty acid, fatty acid triglycerides

Fatty acid ethyl esters are used along with a proper diet of the free acids from the refined ostrich oil and two-step
to reduce blood triglyceride levels. They can help prevent transesterification in alkaline catalysis takes place. This
medical problems caused by clogged blood vessels, such process can be used for oils having a free acids value
as heart attacks and strokes[1]. Fatty acid ethyl esters are greater than 1%. [7-9].Purification of fatty acid esters
used as markers for monitoring alcohol abuse, providing a obtained from vegetables oils or animal fats is achieved
practical approach for evaluating a patient[2]. Huang by chromatographic methods, distillation method, low
Chifu[3] claims a new discovery on the antimicrobial temperature cr ystallization, enzymatic methods,
activity of the ethyl esters of fatty acids omega 3, omega 6 supercritical fluid extraction, urea complexation [10].
and omega 9 and the methyl esters of fatty acids omega This paper presents our results related to trans-
3, omega 6 and omega 9 against oral bacteria, discovery esterification of fatty acid triglycerides of refined ostrich
can be applied in the control and prevention of oral disease. oil with ethanol, in two step acid-base catalyzed
In general, the preparation of methyl or ethyl esters of fatty transesterification, in alkaline catalysis and acid catalysis,
acids is made by well-known transesterification of without prior cleavage of the free fatty acids.
triglycerides with alcohol, base catalyst [4, 5], the acid
catalyst [4-6] or two-step acid-base catalyzed trans- Experimental part
esterification (sulfuric acid-sodium hydroxide[7], sulfuric Refined ostrich oil
acid, sodium alkoxide [8, 9] without prior cleavage at free Ostrich oil was refined in our laboratories, with the
fatty acids, according to the reaction(scheme 1): following characteristics: density- 0.8760 - 0.9280 g
cm -3,(40 0C), free fatty acids: 0.14-2 mg KOH 1 g-1 oil,
peroxid value, 1-2.85 mE kg-1, moisture content 0.02-0.1%,
iodine value 62-72 g I 100g -1oil, saponification value 185-
205 mg KOH 1 g-1 oil.
Fatty acids contained in the refined ostrich oil are:
- saturated fatty acids (FS): lauric acid C12:0, myristic
acid C14:0, palmitic acid C16:0, stearic acid C18:0,
Scheme 1. Transesterification of triglycerides arachidic acid C20:0;
- monounsaturated fatty acids (MUFA): palmitoleic acid
Transesterification in alkaline catalysis is carried out in C16:1, oleic acid C18:1(n-9) (omega 9 fatty acid),
the presence of alkaline catalysts (sodium hydroxide, eicosenoic acid C20:1(n-9) (omega 9 fatty acid), erucic
potassium hydroxide, sodium methoxide) (catalyst aid C22:1 (n-9)(omega 9 fatty acid);
concentration of 0.5-5% by weight of the raw materials) in - polyunsaturated fatty acids (PUFA) : Acid alpha-
ethanolic solution at normal pressure and at the boiling linolenic acid (ALA) C18: 3(n-3) (omega 3 fatty acid),
point of ethanol. The reaction speeds higher than acid linoleic acid (LA) C18:2 (n-6)(omega 6 fatty acid), gamma-
catalysis. This method has the disadvantage that it requires linolenic acid (GLA) C18: 3 (n-6) (omega 6 fatty acid),
raw material to be anhydrous (water content below 0.8% arachidonic acid (AA) C20:4 (n-6) (omega 9 fatty acid).
by weight) and have a free acid content, expressed as Determination of fatty acids in samples of refined
acid number preferably less than 0.1 %. Transesterification ostrich oil and of fatty acid methyl esters (FAME) is
in acid catalysis is carried out in the presence of acid performed by gas chromatography, in accordance with EN
catalyst (catalyst concentration of 0.5-5% by weight of the ISO 15304, using a capillary column with a stationary phase
raw material) (sulfuric acid, hydrochloric acid, para- with high polarity, HP88-(88% cyanopropyl) ar yl
toluenesulfonic acid). This method allows processing polysiloxane. Gas chromatography is performed with a gas
animal fats with high free acid and water. It works in chromatograph-689N Agilent detector equipped with FID
ethanolic solution, at normal pressure, at the boiling point (flame ionization) and autosampler 7683B.
of ethanol. The reaction time is between 2-3 Progress of the transesterification was followed by TLG
hours.According to the literature, two step acid-base plates on Sigma-Aldrich, eluted with the solvent system : n
catalyzed transesterification is carried out as follows: the hexane:ethyl acetate:acetic acid : 90:10:1(v:v:v). The spots
first step, pre-treatment with acid, when the esterification were visualised by exposing the plates to iodine vapour.

* email: lucia.pintilie@gmail.com

REV. CHIM. (Bucharest) ♦ 65 ♦ No. 11 ♦ 2014 http://www.revistadechimie.ro 1275

 Two-step acid-base catalyzed transesterification: at 65-70oC. Next, the reaction mixture is cooled to 60oC,
sulfuric acid-sodium hydroxide (FAEE 1) ethanol solution of sodium ethoxide (16.75 g of sodium
Over a solution of ethanol abs.(470 mL) and refined dissolved in 343 mL ethyl alcohol abs.) was added and the
ostrich oil (1675 g, 1896 mL) heated to 65-70oC was added mixture was heated, under stirring at 65-70oC for 3 h. The
sulfuric acid (conc. 16.75 g, 9.1 mL). The reaction mixture end of transesterification was revealed by thin layer
was heated under vigorous stirring for 3 h at 65-70oC . Next, chromatography.The mixture was cooled to 60oC and
the reaction mixture is cooled to 60oC. Then, ethanol washed with 2000 mL water to remove the trace amounts
solution of sodium hydroxide (16.75 g of sodium hydroxide of catalyst. Shake it about 30 min. at the same temperature
dissolved in 343 mL ethyl alcohol abs.) was added and the and then let it separate. The top layer was dried over
mixture was heated, under stirring at 65-70oC for 3 h. The sodium sulfate anh., then filtered. 1420 g of oil containing
end of transesterification was revealed by thin layer ethyl esters of fatty acids were obtained. After
chromatography. The mixture was cooled to 60oC and crystallization from acetone at low temperatures, filtration
washed with 2000 mL water to remove the trace amounts and concentration there were obtained 1340 g oil
of catalyst. Shake it about 30 min. at the same temperature containing fatty acid ethyl esters.
and then let it separate. The top layer was dried over Alkaline- catalyzed transesterification: sodium
sodium sulfate anh., then filtered. 1497 g of oil containing hydroxide (FAEE 5)
ethyl esters of fatty acids were obtained. After Over a solution of ethanol abs.(470 mL) and refined
crystallization from acetone at low temperatures, filtration ostrich oil (1675 g, 1896 mL) heated to 65-70oC was added
and concentration there were obtained 1373 g oil ethanol solution of sodium hydroxide (16.75 g of sodium
containing fatty acid ethyl esters. hydroxide dissolved in 343 mL ethyl alcohol abs.) and then
Two-step acid-base catalyzed transesterification: the mixture was heated, under stirring, at 65-70oC for 3 h .
sulfuric acid- sodium ethoxide (FAEE 2) End of transesterification was revealed by thin layer
Over a solution of ethanol abs.( 470 mL) and refined chromatography. The mixture was cooled to 60oC and
ostrich oil (1675 g, 1896 mL) heated to 65-70oC was added washed with 2000 mL water to remove the trace amounts
sulfuric acid (conc. 16.75 g, 9.1 mL ). The reaction mixture of catalyst. Shake it about 30 min. at the same temperature
was heated under vigorous stirring for 3 hours at 65-70oC. and then let it separate.The top layer was dried over sodium
Next, the reaction mixture is cooled to 60oC. Then, ethanol sulfate anh., then filtered. 1350 g of oil containing ethyl
solution of sodium ethoxide ( 16.75 g of sodium dissolved esters of fatty acids were obtained. After crystallization
in 343 mL ethyl alcohol abs.) was added and the mixture from acetone at low temperatures, filtration and
was heated, under stirring at 65-70oC for 3 h. End of concentration there were obtained 1172 g oil containing
transesterification was revealed by thin layer chromato- fatty acid ethyl esters.
graphy. The mixture was cooled to 60oC and washed with Alkaline- catalyzed transesterification: sodium ethoxide
2000 mL water to remove the trace amounts of catalyst. (FAEE 6)
Shake it about 30 min. at the same temperature and then Over a solution of ethanol abs.(470 mL) and refined
let it separate. The top layer was dried over sodium sulfate ostrich oil (1675 g,1896 mL) heated to 65-70oC was added
anh., then filtered.1325 g of oil containing ethyl esters of ethanol solution of sodium ethoxide (16.75 g of sodium
fatty acids were obtained. After crystallization from dissolved in 343 mL ethyl alcohol abs.) and then the
acetone at low temperatures, filtration and concentration mixture was heated, under stirring at 65-70oC for 3 h. End
there were obtained 1256 g oil containing fatty acid ethyl of transesterification was revealed by thin layer
esters. chromatography. The mixture was cooled to 60oC and
Two-step acid-base catalyzed transesterification:para- washed with 2000 mL water to remove the trace amounts
toluenesulfonic acid monohydrat-sodium hydroxide of catalyst. Shake it about 30 min. at the same temperature
(FAEE 3) and then let it separate. The top layer was dried over
Over a solution of ethanol abs.(470 mL) and refined sodium sulfate anh., then filtered. 1450 g of oil containing
ostrich oil (1675 g, 1896 mL) heated to 65-70oC was added ethyl esters of fatty acids were obtained. After
para-toluenesulfonic acid monohydrat (16.75 g). The crystallization from acetone at low temperatures, filtration
reaction mixture was heated under vigorous stirring for 3 h and concentration there were obtained 1256 g oil
at 65-70oC. Next, the reaction mixture is cooled to 60oC. containing fatty acid ethyl esters.
Then, ethanol solution of sodium hydroxide (16.75 g of Acid-catalyzed transesterification:sulfuric acid (FAEE 7)
sodium hydroxide dissolved in 343 mL ethyl alcohol abs.) Over a solution of ethanol abs.(81.3 mL) and refined
was added and the mixture was heated, under stirring at ostrich oil (167.5 g, 189.6 mL) heated to 65-70 oC was
65-70oC for 3 h. End of transesterification was revealed by added sulfuric acid (conc. 8.4 g, 4.6 mL). The reaction
thin layer chromatography. The mixture was cooled to 60oC mixture was heated under vigorous stirring for 15 h at 65-
and washed with 2000 mL water to remove the trace 70oC. The mixture was cooled to 60oC and washed with
amounts of catalyst. Shake it for about 30 min. at the same 200 mL water to remove the trace amounts of catalyst.
temperature and then let it separate. The top layer was Shake it about 30 min. at the same temperature and then
dried over sodium sulfate anh., then filtered. 1600 g of oil let it separate. The top layer was dried over sodium sulfate
containing ethyl esters of fatty acids were obtained. After anh., then filtered. 117 g of oil containing a mixture of ethyl
crystallization from acetone at low temperatures, filtration esters of fatty acids and triglycerides were obtained.
and concentration there were obtained 1507 g of oil Acid-catalyzed transesterification:para-toluenesulfonic
containing fatty acid ethyl ester acid monohydrat(FAEE 8)
Two-step acid-base catalyzed transesterification: para- Over a solution of ethanol abs.(81.3 mL) and refined
toluenesulfonic acid monohydrat-sodium ethoxide ostrich oil(167.5 g, 189.6 mL) heated to 65-70oC was added
(FAEE 4) para-toluenesulfonic acid monohydrat (8.4 g).The reaction
Over a solution of ethanol abs.(470 mL) and refined mixture was heated under vigorous stirring for 15 h at 65-
ostrich oil (1675 g, 1896 mL) heated to 65-70oC it was 70oC. The mixture was cooled to 60oC and washed with
added para-toluenesulfonic acid monohydrat(16.75 g). The 200 mL water to remove the trace amounts of catalyst.
reaction mixture was heated under vigorous stirring for 3 h Shake it about 30 min. at the same temperature and then
let it separate. The top layer was dried over sodium sulfate
1276 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 65♦ No. 11 ♦ 2014
 Table 1
CONDITION FOR FATTY ACID METHYL
ESTERS PREPARATION FROM REFINED
OSTRICH OIL

anh., then filtered. 110 g of oil containing containing a h approximately 20 % of refined oil was transformed into
mixture of ethyl esters of fatty acids and triglycerides were fatty acid methyl esters. It was observed, also, the
obtained. disappearance of the corresponding spots of the free fatty
acids, mono and diglycerides. (Rf = 0.299; 0.260; 0.182).
Results and disscusions After acid transesterification step, the mixture was cooled
Refined ostrich oil was obtained in our laboratories, by to 60oC.
purification of crude oil through neutralization, washing, 2)base catalyzed transesterification
drying and fading. The crude oil was obtained by wet a) over the mixture obtained from the first step, heated
melting of fat from the back and belly obtained from Suraki to 60oC, was added sodium hydroxide (1% of the oil)
farmed ostriches.(Rasuceni, Giurgiu district) ethanolic solution, and was heated under stirring at the
Characteristics of the refined ostrich oil are: semisolid same temperature for 1.5-3 h.
mass, unctuous (20oC), pale yellow color, odorless, relative b) over the mixture obtained from the first step, heated
density: 0.8760 to 0.9280 (40 oC), free fatty acids: 0.14-2 to 60 oC, was added sodium ethoxide (1% of the oil)
mg KOH 1 g-1 oil, peroxide value,1-2.85 mE kg-1, moisture ethanolic solution, and was heated under stirring at the
content 0.02-0.1%, iodine value 62-72 g I 100 g -1 oil, same temperature for 1-2 h.
saponification value 185-205 mg KOH 1 g-1 oil. End of transesterification reaction was determinated by
A number of experimental studies were done: two step thin layer chromatography.(RfFAEE = 0.663).
acid-base catalysed transesterification of refined ostrich Conditions for preparation and the yield of fatty acid
oil under optimum conditions of ethanol to oil ratio 3:7 (v/ ethyl esters are presented in table 1.
v), temperature 65 and 70oC for acid and base trans- A number of experimental studies were done: base
esterification respectively, and catalyst concentration of 1 catalyzed transesterification of refined ostrich oil under
% (w/w) for acid (sulfuric acid or para-toluenesulfonic acid) optimum conditions of ethanol to oil ratio 3:7 (v/v),
and 1 % (w/w) for base (sodium hydroxide or sodium temperature 700C and catalyst concentration of 1 % (w/
ethoxide). This method allows processing refined ostrich w) for base (sodium hydroxide or sodium ethoxide). The
oil with high free acid and water. It was observed that the conditions for preparation and yield of fatty acid ethyl esters
presence of free fatty acids in refined oil, has not led to the are presented in table 1. This method has the disadvantage
formation of soap; that it requires raw material to be anhydrous (water content
Transesterification proceeds in two steps: below 0.8% by weight) and have a free acid content,
1) acid pretreatment step expressed as acid number preferably less than 0.1 %. It
The mixture of ethyl alcohol and refined ostrich oil in the was observed that, the presence of free fatty acids in
proportion of 3:7 (v/v) was introduced into the reaction refined oil, led to the formation of soap. From these reason
vessel and heated to 65-70oC with stirring, then added the the yield of FAEE (%) based on the starting oil is less lower
catalyst: than two step acid-base catalyzed transesterification.
a) a mixture of conc. sulfuric acid (1 % w/w) ;the mixture A number of experimental studies were done: acid
was heated for 3 or 4 h at the 65-70oC; catalyzed transesterification is carried out in the presence
b) para-toluenesulfonic acid (1 % w/w); the mixture of acid catalyst (catalyst concentration of 5% by weight of
was heated for 3 or 4 h at 65-70oC the raw material) (sulfuric acid, para-toluenesulfonic acid).
The evolution of reaction was monitored by thin layer Conditions for preparation and the yield of fatty acid ethyl
chromatography. (Rf oil = 0.519, Rf FAEE = 0.663). After 3 or 4 esters are presented in table 1.This method allows
REV. CHIM. (Bucharest) ♦ 65 ♦ No. 11 ♦ 2014 http://www.revistadechimie.ro 1277
 Table 2
COMPOSITION OF OSTRICH OILS AND OF
FAEEs (%)

Fig. 1. GC analysis of the product of

transesterification of refined ostrich oil in
two step acid-base catalyzed
transesterification ( sulfuric acid-sodium
hydroxide) FAEE 1

Fig.2. GC analysis of the product of

transesterification of refined ostrich oil in two
step acid-base catalyzed transesterification
(sulfuric acid- sodium ethoxide) FAEE 2

1278 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 65♦ No. 11 ♦ 2014

 Fig. 3. GC analysis of the product of
transesterification of refined ostrich oil in two
step acid-base catalyzed transesterification (para-
toluenesulfonic acid monohydrat -sodium
hydroxide) FAEE 3

Fig. 4. GC analysis of the product of

transesterification of refined ostrich oil in two
step acid-base catalyzed transesterification (para-
toluenesulfonic acid monohydrat- sodium
methoxide) FAEE 4

Fig. 5. GC analysis of the product of

transesterification of refined ostrich oil in
base catalyzed transesterification (sodium
hydroxide) FAEE 5

Fig. 6. GC analysis of the product of

transesterification of refined ostrich oil in
base catalyzed transesterification (sodium
ethoxide) FAEE 6

processing animal fats with high free acid and water. It Progress of the transesterification was followed by TLG
works in ethanolic solution, at normal pressure, at the 70oC. plates on Sigma-Aldrich, eluted with the solvent system : n
The reaction time is greater than 15 h. After 15 h the hexane:ethyl acetate :acetic acid : 90:10:1(v:v:v). The
transesterification is incomplete. The ethanolysis of the spots were visualised by exposing the plates to iodine
refined ostrich oil in acid catalysis (sulfuric acid or para- vapour.
toluenesulfonic acid) highlights the characteristic spot of The characteristics of the product “fatty acid ethyl
the starting material.(Rf oil = 0.519, Rf FAEE = 0.663) esters” are: oily liquid, colourless to pale yellow, odor-
REV. CHIM. (Bucharest) ♦ 65 ♦ No. 11 ♦ 2014 http://www.revistadechimie.ro 1279
characteristic, relative density: 0.8400 to 0.8931 g cm-3, The second stage of the transesterification was carried
refraction index: 1.4041 to 1.4951 out in the same reaction vessel, by adding the basic catalyst
Determination of fatty acids in samples of refined ostrich to the mixture resulting from the first stage of the
oil and of fatty acid methyl esters (FAEE) is performed by transesterification.
gas chromatography, in accordance with EN ISO 15304, This method allows processing refined ostrich oil with
using a capillary column with a stationary phase with high high free acid and water. It was observed that the presence
polarity, HP88-88% cyanopropyl) aryl polysiloxane. Gas of free fatty acids in refined oil, has not led to the formation
chromatography is performed with a gas chromatograph- of soap.
689N Agilent detector equipped with FID (flame ionization)
and autosampler 7683B. (table 2) (fig.1- 6) Acknowledgements This work had financial assistance from POS
CCE 2.1.1. CTR No 149/2010 “ Valorization of ostrich fat in highly
Conclusions valuable pharmaceutical, nutritional and cosmetics products”
Studies regarding the conversion of waste (fat ostrich)
in high added value product were carried out in order to References
obtain fatty acid ethyl esters. The Conversion of the crude 1. KOSHI R.R., Drug Forecast, 33, 2008, p. 271
product in fatty acid ethyl esters was performed by applying 2. LAPOSATA M., Clinical Chemistry, 43:8(B), 1997, p. 1527
a chemical transesterification. Fatty acid ethyl esters were 3. CHIFU H., ; WO 2011/056327
obtained by the well-known transesterification of fatty acid 4. GHITA D. A., CIOBANU M., SERBAN S., CODESCU M. C., ION O., Bv.
triglycerides of refined ostrich oil (obtained in our RO 109328,1994.
laboratories, by purification of crude oil through 5. UTAI K, ARAN H-K., PAVINEE C., PAIRAT S., Food Chemistry, 87, 415
neutralization, washing, drying and fading) with ethanol in (2004).
alkaline catalysis, acid catalysis, or two-step acid-base 6. ORCHIDEA R., Y-H JU, SHAIK R. V., ISMOJOWATI T. AND A.S. MUSFIL,
catalyzed transesterification without prior cleavage of RevistaVirtualpro Biodiesel,65, 2007, www.revista virtualpro.com
the free fatty acids. 7.SIDDARTH J.,SHARMA M.P., SHALINI R., Fuel Processing
Best conditions were obtained in the case of the two Technology,92, 2011, p. 32
step acid-base catalyzed transesterification, when yields 8 BRYAN R. M., STEVEN F. V.,Biomass and bioenergy, 34, 2010, p. 550
were obtained between 75-90%. 9 BRYAN R. M., STEVEN F. V.;Biomass and bioenergy, 37, 2012, p. 31
Transesterification was carried out in two steps in acid- 10. KAPOOR, R. AND PATIL, U.K.; International Food Research Journal,
base catalysis, without isolating the product obtained in 18, 2011, p. 493
the first step-acid pretreatment of the refined ostrich oil.
Manuscript received: 12.12.2013

1280 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 65♦ No. 11 ♦ 2014