Journal of Oleo Science

Copyright ©2009 by Japan Oil Chemists’ Society

J. Oleo Sci. 58, (11) 549-555 (2009)

High Oleic Enhancement of Palm Olein via

Enzymatic Interesterification
Siew Wai Lin＊ and Saw Mei Huey
Analytical and Quality Development Unit, Product Development and Advisory Services Division, Malaysian Palm Oil Board (6, Persiaran
Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, MALAYSIA)

Abstract: Acidolysis to incorporate oleic acid into refined, bleached and deodorized (RBD) palm olein (IV
56) using various lipases (enzymes) as catalysts to increase the oleic content of the oil was investigated.
Immobilised lipases (lipase PLG, Lipozyme TL IM, Lipozyme RM IM and Novozym 435) and non-
immobilised lipase (lipase PL) were used in this study to compare the effectiveness of the selected lipases in
catalyzing the reaction to produce a high oleic oil. The results showed that the TAG of OLO/OOL content
was increased at least 4 fold and OOO content was increased at least 3 fold when a 5% enzyme load was
used. Lipase PL showed the greatest increase in tri-unsaturated triacylglycerols (TAGs) content. A pilot
scale experiment conducted using TL IM enzyme, followed by recovery of the oil and fractionation allows
the production of oils with varying oleic contents. A high oleic content of 56% was achievable.

Key words: high oleic oil, acidolysis, lipases, RBD palm olein, triacylglycerol composition

1 INTRODUCTION
Oleic acid (also called cis–9–octadecenoic acid) is a
mono–unsaturated fatty acid found in animal and vegetable
oils. It occurs naturally in greater quantities than any
other fatty acid. It is an omega–nine fatty acid, and consid-
ered one of the healthier fatty acids in the diet. High oleic
(HO) oils such as HO sunflower oil fetch higher premium
over the normal oil.
Numerous studies have been carried out to investigate
the benefits of HO oils, particularly against coronary heart
disease. High concentrations of oleic acid can lower blood
levels of cholesterol, and raise levels of high–density
lipoproteins (HDLs) while lowering low–density lipopro-
teins (LDLs), known as the “bad” cholesterol1,2). It has been
reported that oleic acid protects against LDL oxidation,
which has been suggested to play an important role in
atherogenosis 3–5) . Therefore, high oleic oils have a real
opportunity to substitute existing oils of higher saturated
fatty acids, while providing certain functionality and nutri-
tive values.
Oleic acid can be incorporated into oils to increase the
oleic content of the mother oil to offer desirable properties
related to both health benefits and stability characteristics.
Several researchers have also studied the incorporation of
oleic acid into oils especially butterfat6–8), tristearin9,10), soy-
bean oil 11) and tallow12) to produce more value–added lipids.
Palm oil and palm olein have moderate contents of oleic
acid. Increasing the oleic contents by selective breeding
and /or genetic engineering takes many years of research
and is nowhere near commercial ventures as yet. A much
faster route will be via enzymatic acidolysis, which forms
the research shown in this paper.
Lipases are enzymes that hydrolyze ester bonds of long
chain fatty acids and alcohols and also catalyze esterifica-
tion and transesterification (acidolysis, alcoholysis and
interesterification). They are powerful tools for syntheses
of structured lipids (SLs) which are triacylglycerols (TAGs)
modified to change the fatty acid (FA) composition and/or
the positional distribution in the glycerol backbone.
Although chemical interesterification catalyzed by metal
alkoxides is simple and inexpensive, it is not capable of
modifying specific positions due to the random nature of
the reactions. The 2–position of the TAGs in palm oil is
preferentially occupied by an unsaturated fatty acid, main-
ly oleic acid. Therefore, 1.3–specific enzymes are more
favourable for positionally specific modification of palm oil
products, retaining the oleic acid at the 2–position of the
TAGs. The advantages of enzymatic reactions are selectiv-
ity, mild reaction conditions, little or no unwanted side
reactions or by–product isomerisation, ease of product

＊
Correspondonce to: Siew Wai Lin, Analytical and Quality Development Unit, Product Development and Advisory Services Division,
Malaysian Palm Oil Board, 6, Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, MALAYSIA
E-mail: Siew@mpob.gov.my
Accepted July 21, 2009 (received for review April 28, 2009)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/

549
 W.L. Siew and M.H. Saw
recovery, easy control over the process and less waste dis-
posal13–15).
Most of the work on acidolysis of oils have been on other
oils. Very little work has been on palm oil products, in par-
ticular with respect to using oleic acid. In this work, sever-
al enzymes were tried, with the final selection of one of the
enzymes for a pilot scale study.
2 MATERIALS AND METHODS
2.1 Materials
The substrates used were refined, bleached and deodor-
ized palm olein (iodine value 56) and oleic acid (88% purity).
Refined, bleached and deodorized palm olein (IV 56) and
standard refined, bleached and deodorized palm oil were
purchased from Sime Darby Plantations Sdn Bhd.,
Malaysia. Oleic acid was purchased from Merck Sdn. Bhd.,
Malaysia. The catalysts used were lipases such as lipase
PL, lipase PLG, Lipozyme TL IM, Lipozyme RM IM and
Novozym 435. Lipase PL (Alcaligenes sp.) and lipase PLG
(Alcaligenes sp. immobilized on granulated diatomaceous
earth) were a generous gift from Meito Sangyo Co., Ltd.,
Japan. Lipozyme TL IM (Thermomyces lanuginosus lipase
immobilized on a granulated silica), Lipozyme RM IM (Rhi-
zomucor miehei lipase immobilized on an anion–exchange
resin) and Novozym 435 (Candida antartica fraction B
lipase immobilized on a macroporous resin) were a present
from Novozymes A/S, Bagsvaerd, Denmark. The reagents
used for high performance liquid chromatography (HPLC)
analysis were acetone (99.8% purity, Merck) and acetoni-
trile (99.8% purity, Scharlau). All solvents were of liquid
chromatography grade and were purchased from local sup-
pliers.
2.2 Methods
2.2.1 Reaction protocol
Acidolysis of refined, bleached and deodorized palm olein
(IV 56) was carried out to incorporate oleic acid into the
glycerol backbone, catalysed by the chosen lipases. Reac-
tion was monitored by reduction in POP content of the oil
(Fig. 1).
The optimum temperature for enzymes may vary, how-
ever, in order to avoid crystallisation or solidification of the
oils, a higher temperature of 60℃ was selected. Palm oil
products have higher melting points, and during the reac-
tion, higher melting TAGs may be formed, resulting in
some solidification if temperature of reaction is too low.
The enzymes used in this study were Meito PLG (immobi-
lized), Meito PL (non–immobilized) and Lipozyme TL IM
(immobilized). The amount of substrate, palm olein:oleic
acid used was 3:1 mol/mol. This ratio was selected based
on earlier trials indicating reasonable amounts of oleic acid
could be incorporated, and also based on cost effectiveness.
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J. Oleo Sci. 58, (11) 549-555 (2009)
Fig. 1 Time Course Profile of Reaction at 60˚C Based on
POP Content (%).
A dosage of 5% was chosen for comparison, being the com-
mon dosage used for immobilised type enzymes. For the
non–immobilised enzymes, this dosage may be considered
too high. However, for the purpose of a quick comparison,
the dosage of enzymes is kept at 5%. When the need to
study or effectively select the most appropriate enzyme for
commercial application, further consideration should be
taken into account the most appropriate dosage or process.
The resultant fatty acids remaining in the oil had to be
removed by short path distillations. These experiments
were carried out using the rotary evaporator for 14 hours
for time course profile. No vacuum was applied. Samples
were collected every two h. by removing a small aliquot
and filtering over cotton wool. The oil is retained for HPLC
analysis of the TAGs. TAG data obtained from 8 h reaction
were compared and shown in Figs. 2a–d.
The pilot scale experiment (1.8 kg) was carried out at
60℃ using TL IM enzyme in a 5kg reactor fitted with a
stirrer. Reaction was carried out for 8 h. The enzyme load
was 5% w/w. After reaction, the oil was filtered through a
vacuum filter flask. Free fatty acids (FFA) were removed by
short path distillations using a VTA 70 from VTA GmbH
Germany. The major part of the equipment was construct-
ed from glass. The heating of the evaporator was provided
by a jacket circulated with thermal oil from an oil bath.
The main components are short path evaporator integral
condenser, cooling trap for filling of liquid nitrogen and the
vacuum system which is included oil diffusion pump and
rotary vane pump. The evaporator surface is 0.04 m2, with
vacuum achieved up to 1 x 10–3 mbar. 1.5 kg of oil was pre-
heated to 80℃ and taken into the distillation column by a
pump. The residue (Oil) and distillate (FFA) were collected
in round bottomed flasks at temperature of 160℃.
2.2.2 Fractionation
Fractionation was carried out on 100g sample, using a
small container, fitted with a stirrer. The container was
placed in a waterbath, where the temperature control is as
High Oleic Enhancement of Palm Olein via Enzymatic Interesterification

Figs. 2a-d Tri-unsaturated, Mono-saturated, Mono-unsaturated

and Tri-saturated TAGs Content in RBD Palm Olein
(IV 56) at 60℃.

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J. Oleo Sci. 58, (11) 549-555 (2009)
 W.L. Siew and M.H. Saw

follows: After fractionation, the oil is filtered through a fil- 3 RESULTS AND DISCUSSION
tered paper, under vacuum. Filtration is carried out under 3.1 Laboratory trials
cool conditions to avoid remelting of crystals. The incorporation of oleic acid in an oil depends on the
type of enzyme being used, the ratio of oil to oleic acid as
Fractionation at 8℃ well as optimum conditions. In this study, oil to oleic acid
Step Temperature, ℃ Duration, min ratio of 3:1 was chosen, as being more cost effective, taking
1 65 30 into account cost of oleic acid. A higher oleic acid ratio
2 35 30 would no doubt provide more oleic acid into the triacyl-
3 20 40 glycerols, however, the mixture of fatty acids that need to
4 12 40 be removed would be higher. Thus, after some considera-
5 8 140 tion, it was estimated that the 3:1 would provide sufficient
oleic for the reaction.
Fractionation at 10℃ The TAG profiles of the acidolysed samples were com-
Step Temperature, ℃ Duration, min pared. From the HPLC analysis, TAGs were grouped into
1 65 30 tri–unsaturated, tri–saturated, mono–unsaturated and
2 35 30 mono–saturated TAGs. Graphs of the tri–unsaturated,
3 25 30 tri–saturated, mono–unsaturated and mono–saturated
4 15 40 TAGs before and after reactions with different types of
5 10 140 enzymes were plotted.
The reaction was monitored by evaluating the reduction
Fractionation at 15℃ in POP triacylglycerol, the main triacylglycerol in the oil.
Step Temperature, ℃ Duration, min This method was used, as other TAGs with oleic contents
1 65 30 may not be reflective of the overall incorporation of the
2 35 30 oleic acid. Although the best way is to monitor the oleic
3 25 40 content of the oil directly after reaction, it can only be car-
4 18 40 ried out after removal of the fatty acids present in the oil.
5 15 140 Thus, a more direct method was utilised by checking the
POP content by HPLC. In actual fact all the TAGs were
2.2.3 HPLC analyses monitored, but for the purpose of this paper, the POP
The TAG composition of samples were determined by reduction is illustrated.
reversed–phase HPLC from Gilson, France using refractive Figure 1 shows the reduction of the main triacylglycerol
index detector from Waters 2410, USA and two commer- component (POP) over 14 h. The results indicate that after
cially packed LiChrospher100 RP–18 column encapped (25 8–10 h. equilibrium is reached with the 5% dosage at 60℃
cm each) with 5 mm particle size from Merck Sdn. Bhd., for PL, PLG and TL IM, but not for RM IM and N435. Table
Malaysia. Oil samples of about 0.040 g were weighed and 1 shows that except for RM IM and N435, most other
diluted in acetone for injection into the HPLC column. The enzymes attained equilibrium with almost similar oleic
TAG composition were eluted with a mobile phase of ace- contents at 60˚C. PL can achieve a higher oleic content
tone:acetonitrile (7:3) at a flow rate of 1 mL/min. The under similar conditions, indicating the higher efficiency
method of analysis was modified from AOCS Official or activity of the enzyme. Both RM IM and N435 require
Method Ce 5c–93 (AOCS Official Method 1995). The TAG high reaction temperature, thus oleic content tends to be
peaks were identified according to the reference standards lower. Also, if PL were to be used, the equilibrium time is
and comparison with information on palm oil available in shorter with the 5% dosage. It is possible to use a lower
the literature16). Quantification was based on area %. In dosage, and should be considered in further studies. When
the analysis, the TAG of POP referred to mixtures of POP using TL IM, the temperature within 50–70℃ gave almost
and PPO, and do not differentiate between the isomers. similar results.
Palm olein IV 56 consists mainly of disaturated (POP and
PPO) and monosaturated (POO and OPO) TAGs, which are
28.9% and 23.7% respectively. There is at least 4 fold
increase in oleic–linoleic–oleic or oleic–oleic–linoleic TAGs
contents (OLO) and 3 fold increase in triolein (OOO) con-
tent. Lipozyme® TL IM gives a comparable yield of 12.6%
of triolein and can work as well as lipase PLG.
Figures 2a –d show the different classes of TAG content
in the refined, bleached and deodorized palm olein (IV 56)

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 High Oleic Enhancement of Palm Olein via Enzymatic Interesterification

Table 1 Properties of Sample after Reaction with Different Enzymes at Different

Temperature.
Sample SMP ( ˚C) IV C18:1 (%)
Palm Olein (IV56)
25.5 56.2 41.9
(Before purification)
50˚C PL 33.1 67.0 53.6
PLG 33.2 64.5 51.2
TLIM 29.5 65.5 52.2
RMIM 25.0 63.3 49.9
N435 25.3 63.0 49.0
60˚C PL 34.3 66.0 53.3
PLG 29.9 64.7 51.7
TLIM 32.5 64.9 50.6
RMIM 24.7 64.6 50.7
N435 25.9 64.3 50.9
70˚C PL 34.5 66.0 52.9
PLG 33.4 64.8 50.5
TLIM 32.6 65.1 51.5
RMIM 25.9 64.8 51.1
N435 27.0 65.4 51.6

after incorporating oleic acid. Lipase PL showed the high-
est increase in tri–unsaturated TAGs. The activity of lipase
PL is higher as it is a non–immobilized enzyme when com-
pared to the immobilized enzymes. Although by using
Lipase PL will generate the highest yield in tri–unsaturat-
ed TAGs, but tri–saturated TAGs content is also the high-
est among the 3 types of enzymes used. Therefore, at room
temperature, the esterified oil by lipase PL will tend to
crystallize faster than the other two samples, resulting the
oil becoming half solidified in room temperature. Lipase
PLG and Lipozyme TL IM showed comparable results in
tri–unsaturated TAGs. However, Lipozyme RM IM and
Novozym 435 showed a much lower tri–unsaturated TAGs
content.
Mono–saturated TAGs are of POO, and PLO, with SOO
and PLL being minor components. The highest POO tends
to come from N435 and RM IM, while for the mono–unsat-
urated TAGs, the lowest POP comes from PL and TL IM.
In terms of saturated TAGs being formed, PL, PLG and TL
IM provide the highest contents, resulting from the break-
down in POP, where the palmitic acid is re–esterified with
the small amount of OPO normally present. As the cheap-
est enzyme for use is TL IM, further studies on pilot scale
were carried out with this enzyme. As it is in the immo-
bilised form, it is easier to carry out the reaction.
3.2 Pilot scale experiments
A pilot scale experiment was carried out using TL IM
enzyme, followed by short path distillations to remove the
free fatty acids. The final oil was analysed, and further
fractionated at different temperatures of 8, 10 and 15 ˚C.
The oils obtained illustrate compositions of high oleic palm
oil products which are achievable with enzymatic process-
ing and fractionation. Table 2 shows the fatty acid compo-
sitions of the original olein, and the final products
obtained. The iodine values have been modified from IV 56
to 67.3, and with further fractionation, improved to 72.0 to
73.6. This amount to about 33% increase in the oleic con-
tent from 42.1% to 55.8%.
4 CONCLUSIONS
Lipase–catalyzed acidolysis (interesterification) reac-
tions can be used to incorporate oleic acid into palm olein
to increase its oleic content. From the study, the OLO con-
tent was increased at least 4 fold and the triolein (OOO)
content was increased at least 3 fold. An increment of 33%
oleic acid was achievable, resulting in high oleic content of
55–56% oleic in palm oil products. Currently, the available
palm olein which has the highest oleic content is one with
iodine value about 67, having oleic of only 49%. Further
improvements could be obtained if fractionation is opti-
mised to a lower temperature. Also, there is further need
to optimise reaction conditions for any one of the enzymes,
if to be utilised in commercial operation, as the paper only

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J. Oleo Sci. 58, (11) 549-555 (2009)

Table 2 Fatty Acid Composition of Palm Olein IV 56, Enzymatically Modified Palm Olein and New Fractions at 8, 10 and 15˚C.

W.L. Siew and M.H. Saw

Sample Yield
C12:0 C14:0 C16:0 C18:0 C20:0 SFA C16:1 C18:1 MUFA C18:2 C18:3 PUFA IV
Olein IV 56 – 0.2 1.1 40.7 4.0 0.4 46.4 0.2 41.9 42.1 11.2 0.3 11.5 56.2
Modified olein – 0.6 0.9 31.6 3.5 0.3 36.9 0.2 50.0 50.2 12.8 0.2 13.0 65.6
Olein 8 70.5 0.6 0.8 25.1 2.7 0.3 29.5 0.2 55.8 56.0 14.5 0.2 14.7 73.6
Olein 10 72.5 0.6 0.8 25.7 2.7 0.3 30.1 0.2 55.3 55.5 14.3 0.2 14.5 72.9
Olein 15 73.8 0.6 0.8 26.2 2.8 0.3 30.7 0.2 54.9 55.1 14.0 0.2 14.2 72.0
 High Oleic Enhancement of Palm Olein via Enzymatic Interesterification
serve to provide a general estimate of the possible proper-
ties of a high oleic oil from palm products through enzy-
matic process.
ACKNOWLEDGEMENTS
The authors thank the Director–General of MPOB for
permission to publish this work; Director of Product Devel-
opment and Advisory Services Division for their support
and other assistant research officers of the Analytical and
Quality Development Unit for their technical assistance.
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