Process Biochemistry 36 (2001) 933– 939

www.elsevier.com/locate/procbio

Cocoa butter equivalent through enzymic interesterification of

palm oil midfraction
Daniel Undurraga a, Andrés Markovits a,*, Sonia Erazo b
a
Escuela de Ingenierı́a Bioquı́mica, Uni6ersidad Católica de Valparaı́so, A6. Brasil 2147, Valparaı́so, Chile
b
Instituto de Quı́mica, Uni6ersidad Católica de Valparaı́so, A6. Brasil, Valparaı́so, Chile

Received 6 July 2000; received in revised form 31 August 2000; accepted 29 October 2000

Abstract

The production of a cocoa butter equivalent (CBE) through enzymic interesterification of palm oil midfraction (POMF) with
stearic acid in a solvent free system using Novo lipase Lipozyme™ as a catalyst was analyzed. A two level factorial design was
used to study the effect of the initial ratio of stearic acid– POMF, initial humidity of the enzyme preparation and the
enzyme–substrate ratio on the yield, mass productivity and specific productivity. Studies were carried out both in batch and in
a continuous packed bed reactor. The highest specific productivity obtained in shake flask was 0.0393 g/Batch Interesterification
Unit (BIU) h at a stearic acid–POMF ratio of 1.6 and enzyme– substrate ratio of 23 BIU/g. In the continuous packed bed reactor
the highest mass productivity observed was 1.54 g/g·h, using an enzymic load of 73 BIU/g. Unreacted fatty acids were separated
from the intereresterified products by short path distillation at 0.2 mbar and 140°C obtaining a product practically without free
fatty acids. Thermograms of the products obtained by scanning differential calorimetry were similar to cocoa butter (CB), but
exhibited several distinct peaks, due presumably to the presence of diglycerides and trisaturated triglycerides. © 2001 Elsevier
Science Ltd. All rights reserved.

Keywords: Interesterification; Lipases; Cocoa butter equivalent; Palm oil midfraction; Solvent free system; Diferential scanning calorimetry

1. Introduction composition, but its triglycerides derive from a source

The modification of fats and oils through interester-
ification reactions catalyzed by lipases has been studied
since the 1980’s. This is a method with significant
technical advantages over the traditional methods of
chemical interesterification. Among the advantages are
lower energy consumption, absence of isomerization by
products and better control of products.
The transformation of low-cost fats and oil triglyce-
rides through enzymic interesterification for the elabo-
ration of cocoa butter (CB) substitutes or equivalents
has been previously reported [1 – 5]. A substitute is a
type of fat, which does not have a similar chemical
composition, but presents a fusion profile similar to
CB. On the other hand, a cocoa butter equivalent
(CBE) is a type of fat that has a very similar chemical
* Corresponding author.
other than cocoa beans [6].
CBE contains approximately 40% 1-palmito, 2-olein,
3-sterin glycerol (POS), 27% of 1,3 distearin-
monooleate glycerol (SOS) and 21% of 1,3 dipalmitin-
2-monooleato glycerol (POP) and minor amounts of
other triglycerides. A suitable raw material for the
production of CBE in terms of cost, availability and
composition is palm oil midfraction (POMF), which is
obtained by double fractionation of palm oil. POMF
contains approximately 73% POP, 13% POS, 2% SOS
and 12% of other triglycerides.
The purpose of interesterification is to incorporate
selectively stearic residues into POMF triglycerides, in
positions 1 and 3 until a composition resembling CB
composition is obtained. The stearate donor could be
either free stearic acid or an ester of stearic acid,
however, for practical applications stearic acid is pre-
ferred due to its availability and low cost.
For that purpose, the enzyme used should be 1,3

0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 0 0 ) 0 0 2 6 0 - 0
934 D. Undurraga et al. / Process Biochemistry 36 (2001) 933–939
regiospecific, exchanging triglyceride residues only at
positions sn-1 and sn-3, and preferably immobilized
because in this form it is easily separated from the
products and is also more thermostable than the free
enzyme.
The interesterification reaction can be carried out in
a system containing triglycerides and stearic acid
only (‘solvent free system’) or the mixture could be
diluted with some suitable non-polar solvent. There are
distinct advantages and disadvantages in using
either system, but in food application processes solvent
free systems are clearly preferable. For technological
purposes, it is important to determine the parameters
that affect the productivity of a CBE and find
the optimal operation strategy that maximize produc-
tivity.
In this research, the production of a CBE through
enzymic interesterification of POMF with Lipozime™
in a solvent-free system using stearic acid as stearate
donor was studied. Two factorial experimental designs
were used to evaluate the effect of different parameters
on productivity.
The variables studied were the stearic acid– POMF
ratio, which affects the equilibrium and, therefore, the
time at which a composition similar to that of CB is
obtained; the enzyme– substrate ratio which directly
affects the rate of reaction, and the humidity or water
activity of the enzymic preparation, which have to be
over a minimum value to activate the enzyme but not in
such excess as to promote the hydrolysis of triglycerides
[7].
2. Materials and methods
2.1. Materials
The Novo-Nordisk commercial preparation of lipase
(triacyl glycerol hydrolase EC 3.1.1.3) called
Lipozyme™ (76.9 Batch Interesterification Unit (BIU)/
g) which presents 1, 3 regiospecificity, is immobilized on
a macroporous anionic exchange resin with a particle
diameter of 0.2–0.6 mm. One BIU is defined as micro-
moles of palmitic acid incorporated into triolein per
minute [8].
Refined, bleached and deodorized POMF was kindly
donated by The Palm Oil Research Institute of
Malaysia (PORIM). Stearic acid used had a purity over
99% (Riedel De Haen). Triolein (OOO) and trilinolein
(LiLiLi) were obtained from Sigma Chemical Co. (St.
Louis, MO). Other triglycerides such as POP, POO,
SOS, SOO, PLiP, PLiLi, SLiS and SLiLi were pro-
duced from the above standards by enzymic interester-
ification with stearic or palmitic acid. Acetone and
acetonitrile (Baker) were of high-performance liquid
chromatography (HPLC) grade.
2.2. Analysis of glycerides
The composition of diglycerides and triglycerides
were determined by HPLC in a HPLC Shimadzu deliv-
ery system (Shimadzu Scientific Instrument, Columbia,
MD) provided with refraction index detector. A CLC-
ODS C-18 4.6 mm ID and 15 cm long Shimpack
column (Shimadzu) was used along with a mixture of
3.5/l (v/v) acetone–acetonitrile as the mobile phase.
The samples were diluted in acetone up to a concentra-
tion of 2 g/l approximately. The injection volume was
of 20 ml. Detector and column were maintained at
35°C. The chromatograms were processed in a Shi-
madzu CR4A integrator.
2.3. Interesterification in batch reactor
A 23 factorial design (experimental design I) was used
to investigate the effect on productivity of the stearic
acid–POMF ratio (RS 1.2 and 1.4), the enzyme–sub-
strate ratio (RE 7.7 and 15.4 BIU/g) and the initial
humidity of the enzyme preparation (H 4.5 and 5.9%
d.w. basis).
Each experience consisted of a batch interesterifica-
tion reaction under conditions imposed by the experi-
mental design. Reactions were carried out in 50 ml
Erlenmeyer flasks with about 6 g of substrate each and
incubated with Lipozyme™ at 65°C in a rotary Shaker.
The initial humidity was fixed as follows, samples of
Lipozyme™ were dried in vacuum at 40°C overnight
and then placed into a constant temperature, constant
relative humidity closed chamber containing a satu-
rated solution of an inorganic salt, and maintained in
the chamber until reaching constant weight.
This usually occurred in about 12 h. Using different
salts the initial equilibrium humidity of the biocatalyst
could be fixed at the values of 4.5 and 5.9% d.w. basis.
Samples of about 10 mg were withdrawn at timed
intervals until equilibrium conditions were obtained.
Samples were diluted in acetone and analyzed by
HPLC. Time of reaction, yield and productivity were
recorded for each sample.
Time of reaction (tr) is defined as the time required
for the incorporation of stearate into POMF triglyce-
rides at CB level, quantified by a stearate index (SI) as
defined by Bloomer [1].
2[SOS] + [POS]
SI = (1)
2([POP] + [POS] +[SOS])
where [triglyceride] is the triglyceride weight fraction. SI
for CB and POMF were 0.52 and 0.1, respectively.
The yield (R or mass of triglycerides produced per
unit mass of initial substrate), mass productivity (PM or
mass of product/(time·initial mass of substrate)) and
specific productivity (PE or mass of product/(time
BIU)) were calculated as follows:
 D. Undurraga et al. / Process Biochemistry 36 (2001) 933–939
R
PM = (2)
(1+ RS)tr
R 1.4 was fed from the bottom using a peristaltic pump.
PE = (3)
Re(1+ RS)tr
2.4. Interesterification in a continuous reactor
In order to appreciate the effect of higher RE values
on productivity than those attainable in batch reactors,
a continuous interesterification system using a column
reactor packed with Lipozyme™ and an inert packing
(0.4 mm glass balls) was employed. The reactor con-
sisted of a 2.5-cm diameter and 25-cm long glass
Fig. 1. Time course of the interesterification in experiment 7 of
experimental design I, showing changes in triglyceride composition.
, POS;
, POP; , SOS.
Fig. 2. Time course of the interesterification in experiment 7 of
experimental design I, showing its degree of stearate incorporation
into triglycerides.
935
column. The packing was loaded up to 15 cm height
approximately. The substrate at a constant RS value of
All the experimental set up including the reactor, feed
and product lines and pump head were enclosed in an
insulating box whose interior was controlled at 70°C by
means of an air heating system. The feed rate was set to
obtain a product with a SI value of 0.529 0.01.
2.5. Separation of the product
The interesterified product was distilled in a short
path distillation unit KDL 4 (UIC GmbH) at P=0.2
mbar and T= 140°C. At these conditions, free fatty
acids were totally eliminated, but not diglycerides pro-
duced in the reaction.
2.6. Functional analysis
The distilled product was subjected to scanning dif-
ferential calorimetry analysis using a DSC-7 Perkin–
Elmer calorimeter. The samples were tempered by
heating them up to 80°C, fast cooling to 0°C and
keeping for 7 days at 25°C. For the analysis, around 15
mg samples were taken and heated at the rate of
5°C/min from 5 to 50°C.
3. Results and discussion
3.1. Interesterification in batch reactor
The course of the interesterification reaction in expe-
rience 7 (RS 1.2; RE 15.5 y H 5.9) is plotted in terms of
the composition of triglycerides in Fig. 1 and in terms
of level of incorporation of the stearic acid, in Fig. 2. In
these figures, it is observed that the desired composition
(with a SI value of 0.52) is achieved at 1.2 h approxi-
mately and equilibrium is reached at a SI value of 0.56.
Time of reaction, yield and productivity results were
adjusted to a linear model with interactions. Model
coefficients had a 95% level of confidence. The correla-
tions obtained were the following:
tr = 3.174−0.115 · RS − 1.906 · RE − 0.4035 · H
+ 0.2665 · RE · H (4)
R=0.9318− 0.0077 · RE − 0.006 · H (5)
PM = 0.2029+ 0.121 · RE + 0.0235 · H (6)
PE = 0.0157+ 0.0052 · RE + 0.0018 · H (7)
Symbols in italics stand for the coded variables (−1,
0, + 1).
Batch productivities slightly increased with H as a
result of the combined effect of increasing both transes-
terification and tryglyceride hydrolysis rates, water be-
936 D. Undurraga et al. / Process Biochemistry 36 (2001) 933–939
Fig. 3. Time course of the interesterification in experiment 4 of
experimental design II, showing changes in triglyceride composition.
, POS;
, POP; , SOS. 1.6 and RE 23.2) expressed as the variation in the
Fig. 4. Time course of the interesterification in experiment 4 of
experimental design II, showing its degree of stearate incorporation
into triglycerides.
ing a reactant of this later reaction (it is assumed that,
hydrolysis is an intermediate step of the whole inter-
esterification reaction [9]).
For the purpose of producing high quality CBE,
hydrolysis reactions pose another difficulty, the possible
production of diglycerides, which have a strong effect
in the fusion profile of the product. Fig. 7 is the
chromatogram of an interesterified product after short
path distillation. Several smaller distinct peaks corre-
sponding to compounds of lower retention time than
triglycerides can be observed. These compounds, which
make up approximately 9% of the mixture, are believed
to correspond to diglycerides. The presence of these
compounds has a pronounced negative effect on the
fusion characteristics of the product.
In order to asses the effect of higher RS y RE values
a 22 design (experimental design II; RS 1.2 and 1.6; RE
15.4 and 23.2) was carried out. Given the slight influ-
ence of initial humidity and to preclude possible hy-
drolytic effects the value of the initial humidity was not
varied but was fixed at the value 5.9% d.w. basis as
explained above. Results were as follows.
tr = 0.6176− 0.2258 · RE − 0.1763 · RS
+ 0.0922 · RE · RS (8)
R=0.8864− 0.0192 · RS (9)
PM = 0.6591+ 0.1972 · RE + 0.0807 · RS (10)
PE = 0.0336+ 0.0037 · RE + 0.0044 · RS (11)
The time course of the reaction of experiment 4 (RS
percentage of triglycerides is shown in Fig. 3, and
expressed as the variation of the level of incorporation
of stearic (SI) in Fig. 4. In both figures, it can be seen
that the times of reaction are shorter than those of first
design. Furthermore, a higher SI equilibrium value was
observed due to the higher RS values used in the second
design.
PM increases with higher values of RE as expected
from theory. The PE also increased with increasing
values of RE, although the effect was less pronounced
than in the first design. Theoretically there is no rela-
tion between these variables, and the variations ob-
served might be due to the decrease of humidity, either
by evaporation or desorption into the oil phase. In the
batch system humidity was not controlled and dehydra-
tion should be expected to increase with time. Therefore
the reaction rate should be higher at higher values of
RE, which corresponded to shorter reaction times, when
the humidity was still higher.
The effect of RS on PM is rather complex. An in-
crease of RS should cause a decrease in PM according to
Eq. (1). Nevertheless, RS also affects — inversely —
the time of reaction, therefore, the overall effect is
diminished. This would explain the low value of the
coefficient of RS in Eq. (10).
RE is the single factor that most affects productivity.
However, in batch reactors it is not practical to increase
its value over 25 BIU/g of substrate (32% in weight of
Lipozyme™) due to operational problems (poor mix-
ing, sampling difficulties etc.), therefore, in order to
investigate the effect of this variable at higher values a
continuous system was utilized.
Results of batch interesterification experiments car-
ried out by Blumer et al. in a model system consisting
of triolein and palmitic acid using Lipozyme™ were
similar to those of this work [10].
 D. Undurraga et al. / Process Biochemistry 36 (2001) 933–939 937

In summary the best results in batch systems were RE = 23.3 (BIU/g), a ratio between substrates, RE =1.6
obtained using the highest enzyme– substrate ratio, and an initial humidity of H=5.9% (d.w. basis). Under
these conditions values of PM, PE and R were 0.913
(g/h·g), 0.0393 [g/(BIU·h)] and 86.7%, respectively.
Table 1
Enzyme–substrate ratio (RE), residence time (~), yield (R), specific
productivity (PE) and mass productivity (PM) in continuous packed 3.2. Interesterification in continuous reactor
bed reactor
In order to prevent or to minimize dehydration of the
RE (UI/g) ~ (min) R PE (g/BIU·h) PM (g/g·h) biocatalyst, whose initial value was set at 5.9% as
73.13 14.0 0.86 0.0210 1.536
described above, the substrate fed to the reactor was
52.6 20.3 0.872 0.0204 1.074 saturated with water. In this manner, it was possible to
48.16 24.1 0.866 0.0187 0.901 maintain constant SI during at least six residence times.
Results obtained in the continuous system are shown in
Table 1. Extrapolating the results of the batch system
and based on previous experiences with the continuous
system it was possible to determine a suitable feed rate
(and residence time) to produce a SI value of 0.52 at the
reactor outlet. An increase in RE caused a decrease in
residence time (~), an increase in productivity but prac-
tically no effect on yield (Table 1).
A packed bed continuous reactor in a plug flow
regime behaves as a perfectly mixed batch reactor.
What occurs in a continuous reactor along its length is
equivalent to what occurs in a batch reactor along time.
Therefore, it should be possible to make a plot showing
the effect of RE on productivities along the whole range
(15.4− 73.1 BIU/g) as shown in Figs. 5 and 6. In these
figures, it can be observed that the behaviour at high
RE in the continuous reactor is not the extension of
what occurs at lower RE in the batch reactors, as might
have been expected.
These behaviours can be in part explained assuming
Fig. 5. Changes in mass productivity in the whole range of BIU/sub- that only a fraction of the enzymic activity loaded into
strate ratio studied.
, Batch reactor; , continuous packed bed the reactor is expressed, and in part, by considering
reactor.
that in the continuous reactor the loss of water was less
(better control) than in the batch system, thus reducing
or eliminating the dependence between PE and RE
found in the latter.
Non-expressed enzymic activity can be attributed to
external diffusion restrictions since the enzyme is ab-
sorbed on to the particle surface and not inside the
micropores [11].
It can be seen that the yield was nearly constant at
different RE values, which would indicate that humidity
was similar in all experiments in the continuous system,
because otherwise yield would have fluctuated widely
(humidity strongly affects yield).
The best results, as far as production was concerned,
were obtained with the highest enzyme–substrate ratio
RE = 73.1, RS = 1.4 and ~= 14 min, obtaining a mass
productivity of 1.54 [g/(g·h)] and a specific productivity
of 0.02 [g/BIU·h] with a yield of 86%. These results can
be compared with those obtained by Forsell et al. [7]
Fig. 6. Changes in specific productivity in the whole range of BIU/ who interesterified rape seed oil with lauric acid in a
substrate ratio studied.
, Batch reactor; , continuous packed bed solvent-free system using a packed bed continuous reac-
reactor. tor with Lipozyme™. The ratio between lauric
938 D. Undurraga et al. / Process Biochemistry 36 (2001) 933–939

Table 2
Triglyceride composition POMF, CB and CBE produced by inter-
esterification

Sample POP POS SOS Other TGs DGs

POMF 74.3 14.3 2.0 9.4 –

CB 23.4 42.8 27.5 3.6 2.7
CBE 23.4 38.5 20.2 8.2 9.7

3.3. Scanning differential calorimetry

As mentioned earlier, the chromatogram of the inter-

esterified product after distillation at vacuum to remove
impurities, still presented compounds other than
triglycerides, which are believed to correspond to
diglycerides. These latter strongly affect the fusion
characteristics of the products as can be observed in the
thermograms (Figs. 7–10). Table 2 shows the composi-

Fig. 7. Chromatogram of interesterified and distilled product.

Fig. 9. Thermogram of CB.

Fig. 8. Thermogram of POMF.

acid:rape seed oil acid used was 1:3. With a residence

time of 30 min, a conversion of 60% of the equilibrium
value was obtained, (continuous interesterification in
this work was carried out at 85% conversion) and mass
productivity and yield were of 1.34 [g/(g·h)] and 89%,
respectively. Svanholm [12] using soybean oil instead of
rape seed oil and a ratio between acid and oil of 2:5
obtained a conversion of 65% of the equilibrium value
and a specific productivity 1.8 [g/(g·h)]. These and the
present results indicate that conversion by an interester-
ification reaction with Lipozyme™ are similar irrespec-
tive of the substrates used. The yield of the reaction is
high (80–90%) and mass productivity is less than 2
[g/(g·h)]. Fig. 10. Thermogram of interesterified and distilled product.
 D. Undurraga et al. / Process Biochemistry 36 (2001) 933–939
Table 3
Scanning differential calorimetry parameters of POMF, CB and CBE produced by interesterification
Sample Onset temperature (°C)
POMF 21.47
CB 24.08
CBE (first peak) 16.24
Second peak 20.94
Third peak 25.07
tion of POMF, a sample of pure CB, and a sample of
CBE of this research, whose chromatogram is shown in
Fig. 7.
From the thermograms three characteristics for each
sample can be obtained: the onset temperature (TO),
peak temperature (TP) and the area of the curve under
each peak. These values for POMF, CB and a sample
of CBE of this study are shown in Table 3. The
thermogram of CB exhibts a narrow range of fusion
between 24 and 35°C with a steep descending slope.
The area under this peak is 88.7 J/g and represents the
heat of fusion of CB. In addition two smaller fusion
peaks are noted between 5 and 15°C and 15 and 23°C,
which are likely due to the crystal structures I and II of
CB. The thermogram of CBE resembles that of CB, but
a closer scrutiny indicates several important differences,
which could impair seriously its utilization in choco-
lates. As can be observed, CBE shows a ‘shoulder’
between 35 and 39°C in the descending portion of its
largest peak, due probably to trisaturated triglicerides
(PPS, PPP, PSS and others), which may have formed
during the interesterification reaction. These triglice-
rides have larger fusion temperature than the disatu-
rated triglicerides. In addition, it exhibits a fairly large
peak between 5 and 15°C, probably due to triglycerides
in the crystalline from I, and two smaller peaks between
15 and 25°C, which could be attributed to the presence
of diglycerides. It have been noted that the presence of
diglycerides in fats affects their fusion behaviour
[10,13].
4. Conclusions
POMF appears to be a suitable raw material for .
interesterification with stearic acid using Lipozyme™,
leading to products whose composition matches closely
that of CB. Nevertheless the fusion profile of the prod-
ucts of the reaction does not warrant their use as a
good quality CBE for the reasons discussed above and
939
Peak temperature (°C) Area (J/g)
29.76 107.86
31.04 86.48
18.67 2.47
22.33 1.01
29.73 56.84
there is little to be done to improve in this aspect.
Further purification of the reaction products to obtain
a high quality CBE, although technically feasible, could
render the overall process uneconomical.
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