1
Production of Monoacylglycerols (MAG) via
Lipase Catalyzed Esterification of Glycerol with
Fatty Acids of Skipjack Tuna
Norhafizah Sakrani, Nur Adillah Mohd Anuar, Syahirah Onn,
Faculty of Chemical Engineering, Universiti Teknologi Mara
Abstract Monoacylglycerols (MAG) rich in
polyunsaturated fatty acids (PUFA) composing of
eicosapentaenoic acid (EPA) and docosahexaenoic (DHA) were
successfully produced under the process of glycerolysis of
skipjack tuna oil with lipase; Lip.nr-68 from Geobacillus
thermodenitrificans immobilized on Accurel EP-100 (IM-AK).
The reaction factors that influenced MAG synthesis were the
glycerol to fatty acid mole ratio, amount of skipjack tuna oil,
amount of enzyme, and temperature. Tert-Butyl methyl ether
(MTBE) was selected as the most suitable organic solvent for
this reaction. The optimum conditions for MAG synthesis were
determined to be 3.0:1.0 of the mole ratio of glycerol to
skipjack tuna oil, 10 %w/v of skipjack tuna oil in MTBE, 30
wt% of IM-AK based on tuna oil, and water added 4 wt% in
glycerol. Under 45C temperature, the content of MAG was
found to be 24.6% with 56.0 wt% PUFA (EPA and DHA) after
undergoing for 24h of reaction. In this process, the IM-AK
stability was also studied. After incubating in MTBE at 45 C
for 24h, the hydrolytic activity of the enzyme was 88% and
80% of the initial activity. The Vmax and Km values of the
lipase-catalyzed glycerolysis of skipjack tuna oil in MTBE for
IM-AK were determined to be 2.71 and 19.47 mM/min.
Keywordsmonoacylglycerol (MAG), esterification, fish oil,
lipase, skipjack tuna
I. INTRODUCTION
Fish oil can be obtained from the tissues of oily fish that
contains omega-3 fatty acids eicosapentaenoic acid (EPA) and
docosahexaenoic (DHA) that is believed to have ability on
reducing inflammation in the body and have other health benefits
like treating hypertriglyceridemia. The benefits and effects of this
oil are still under research studies. Skipjack tuna or also known as
Katsuwonus pelamis has been chosen to produce
monoacylglycerols (MAG) as it is currently one of the major
sources of polyunsaturated fatty acids (PUFA). The fish oil from
Skipjack tuna contains approximately 5.7% EPA and 18.8% DHA
which are distributed in mixed triacylglycerols (TAG).
Monoacylgycerols are a class of glycerides which composed
of a molecule of glycerol bonded to a fatty acid via esterification.
MAG are non-ionic emulsifiers that is stable and has conditioning
properties that makes it popular to be use in bakery products, dairy
products and also confectionary. Additionally, MAG also is used in
pharmaceutical industry act as binders in tablets and as emollients
for transdermal, slow-release drugs. The goods that MAG brings
and their derivatives as surface active additives in a wide range of
foods, significant attention has recently been given to improve the
synthesis of MAG.
Production of MAG can be synthesized at high temperature
using metallic kind of catalysts. Commercial MAG is widely
manufactured by glycerolysis of fats and oils and usually is
accelerated by the use of inorganic alkaline catalysis, such as
NaOH or Ca(OH)2 at 220-260oC. Unfortunately, unwanted side
products might occur in random manner that require extensive
purification of the products. Higher temperature is not suitable to
be applied to the chemical reaction as MAG containing protein that
can be denatured easily. There are studies turned out revealing
synthesis of MAG enriched in PUFA such as alcoholysis of TAG
with ethanol using 1,3-regiospecific lipase or glycerolysis of fish
oil using an immobilized lipase.
Under decision of using lipase; Lip.nr-68 esterification of
glycerol Skipjack tuna oil as substrate, a list of solvents were first
evaluated for the synthesis of MAG. All parameters needed were
first studied to ensure high yield of MAG is obtained as well with
the quantity of PUFA inside it.
II. METHODOLOGY
A. Materials Preparation
Crude skipjack tuna oil with 4.7% water content, Lipase (Lip.nr-
68) with water content of 0.04%, Microporous polypropylene
powder, phosphate buffer, Tert-Butyl methyl ether, Acetone,
Hexane, Chloroform, Methanol, Silica gel, Distilled water,
Anhydrous sodium sulfate, Accurel EP-100 (particle size <
400m), and all other chemicals and solvents used were of reagent
grade or analytical grade.
B. Methods
Wash the Skipjack tuna fish thoroughly to remove all the
unwanted substances and the crude oil was obtained by a
conventional pressing method. The refined oil was achieved
through degumming, neutralization, bleaching, and deodorizing.
Lipase (Lip.nr-68) was purified by Geobacillus
thermodenitrificans with water content of 0.04%. Hydrolytic
activity of the immobilized lipase was determined by the modifies
cupric acetate method of Lee and Rhee (1993). One unit of
hydrolytic activity was defined as the amount of enzyme which
liberates 1 micro mole equivalent of palmitic acid from palm olein
in 1 min at 30OC.
Esterification was conducted in a 50 mL vessel of 0.1M
phosphate buffer (pH 7) containing approximately 50 U/mL Lipase
AK stirring at 500 rpm using a magnetic stirrer for 10 minutes.
Then, 50 mL of 0.1 mol/L phosphate buffer (pH 7) was added and
the suspension was filtered through a Buchner funnel by vacuum.
The immobilized enzyme (IM-AK) was washed with 50 mL of
buffer to remove the soluble enzyme.
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The mixture of 5 g skipjack tuna oil with required amounts of

glycerol and amount of tert-butanol were incubated in a capped 20
mL flask at the designed conditions under 400 rpm shaker. The
reaction mixture was composed of fatty acid, glycerol and lipases
solution. It continues by withdrawing 0.20 mL of reaction mixture
and the lipase was removed by filtration then the solvent was
removed by vacuum. All samples were stored at -20oC before
analysis

The product collected after reaction under optimal conditions

is subjected to solvent removal under vacuum. The product in this
stage is named as crude MAG. The crude MAG in 0.1 g was
dissolves in different solvents or mixtures with 30 mL. Acetone
and hexane were commonly used with the ratio of 1:1. The fraction
was conducted under different temperatures. Based on melting
points of different MAG fractions, the following three temperatures
were selected for evaluation including 10, 4, and 0oC. The samples
afterwards were centrifuged at the same temperature for 30 minutes
under 10,000 rpm. The supernatant was removed and the solids Fig 1: Time course of glycerolysis by IM-AK in MTBE with the reaction
were washed several times with the same solvent left cooled to the mixture contained 2 ml of 10% w/v tuna oil and 1.78 g of glycerol with 4
same temperature. The liquid parts were collected together and the wt%. The amount of IM-AK used was 0.6 g with 0.46 U/mg. the reaction
was carried out at 300 rpm and 45C for 24 h
solvents were removed by a vacuum evaporator for both solids and
liquids. The two fractions were then weighted and used for further Table 1: Fatty acid compositions of species of tuna oil and reaction product
analysis. after TLC separation

The MAG selectivity was expressed as the percentage of

MAGs to total acylglycerols (MAGs + DAGs + TAGs) in the
reaction mixture. It was analyzed with a thin layer chromatography
with flame ionization detector for the content of TAG, 1,2(2,3)-
DAG, 1,3-DAG, MAG and free fatty acids (FFA). The samples
diluted in chloroform and methanol (2:1) was spotted onto the
chromarod and developed for 35 minutes in a mixture of benzene,
chloroform and acetic acid with ratio of 50:20:0.7 as developing
solvent. After developing and drying, the rods were subjected to
scanning with FID. The experiment was performed in triplicate.

The fatty acid compositions of acylglycerol species were

determined by converting into fatty acid methyl esters (FAME)
followed by GC analysis. After evaporating excessive solvent of
the sample, the mixture was applied to normal TLC-plate with
silica gel and developed in benzene, chloroform and acetic acid
(50:20:0.7). After drying, the MAG band was scraped off and
methylated with 0.5% NaOH in methanol (1000 L) for 1 min. The
n-hexane layer was washed with 200 L distilled water and dried
over anhydrous sodium sulfate. Analysis was carried out with a gas
chromatograph. The carrier gas used was helium set at a flow rate
of 0.5 mL/min (15 psi) and operated in a spit ratio of 50:1.
Response factors were determined using a standard mixture of
FAME. Duplicate analysis was carried out for all the analysis and
the average was used in the paper.
The SPSS program analysis was used for data analysis.
Analysis of variance and t-test were used to evaluate the
significance and difference of data. Values are expressed as
means standard deviations.
III. RESULTS AND DISCUSSION
A. MAG Production under Optimal Condition
The optimal conditions for MAG production were decided as
tuna oil concentration of 10 %w/v in MTBE, the mole ratio of
glycerol to tuna oil about 3:1, water content in glycerol with 4 wt%
and using IM-AK 30 wt% of tuna oil. The temperature was
controlled at 45C. The reaction time course is given in Figure 1.
The yield of MAG was 24.5 wt% and PUFA (EPA and DHA)
content was 56.0 wt% after 24h incubation. The reaction products
were separated by TLC. The fatty acid profiles of each band
(glyceride species) were determined by GC table 1. As shown,
monodocosahexylglycerol and monooleylglycerol was the
predominant MAG in the products.
* Major fatty acids identified
** Fatty acid compositions of tuna oil at the sn-2 position before
glycerolysis
IV. CONCLUSION
Glycerolysis of Skipjack tuna oil was investigated to produce
MAG enrich with PUFA by introducing lipase; Lip.nr-68 from
Geobacillus thermodenitrificans immobilized on Accurel EP-100
(IM-AK). It turned out that this species of tuna oil is suitable in
producing high yield MAG with high content of PUFA especially
EPA and DHA. Thus, it can be proceed to next research as the
health benefits of MAG and DAG is really have a great impact.
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