The most unique property of CRL is that TAG is the dominate component in the glyceride fraction, which consists of
monoacylglycerol (MAG), diacylglycerol (DAG), and TAG, despite
over 50% hydrolysis of the oil [ 1]. Generally, DAG and MAG are
accumulated in the reaction mixture when TAG hydrolysis is
catalyzed by lipase.
Hydrolysis is not the only reaction that occurs during CRL
catalyzation. Esterification and interesterification also occur,
and result in di-PUFA TAG and/or tri-PUFA TAG (Fig. 2, page 62).
The rate at which PUFA-containing glycerides are produced by
hydrolysis depends on the number of PUFA: mono-PUFA TAG
> di-PUFA TAG > tri-PUFA TAG. Hydrolysis kinetics of non-PUFA
glycerides is faster than that of PUFA-containing glycerides.
Therefore, DHA-rich and TAG-rich oil is obtained.
lOWER tEMpERAtURE, lOWER
sAtURAtED FAtty AcID cONtENt
Generally, the optimal temperature of a lipase-catalyzed reaction is approximately 40°C. CRL shows a unique property when
the reaction temperature drops. Hydrolysis catalyzed by CRL at
around 20°C results in lower contents of saturated fatty acids
(SFA) in the glyceride fraction compared with those at 40°C,
even though there is no difference in the rate of hydrolysis and
DHA content in the glyceride fraction [ 2]. In addition, TAG content in the glyceride fraction is slightly higher at 20°C than that
at 40°C. For example, when the hydrolysis of tuna oil that contains 23.6% DHA and 17.4% palmitic acid (PAL) was catalyzed by
CRL at 20°C or 40°C, DHA was concentrated to more than 47%
at either temperature, but the content of PAL was 5.9% and
10.2%, at 20°C and 40°C, respectively (Fig. 3, page 63).
Although SFAs play an important role as a source of calories, the modern SFA-rich diet often results in excessive calorie intake. The co-ingested SFA is preferably as low as possible
when omega- 3 PUFAs are ingested. This technique is suitable
for manufacturing health foods or supplements comprising
Animal oils, including fish oils, generally contain various
amounts of cholesterols (Chol). There are two forms of Chol:
free and esterified. The esterified form, which binds with fatty
acid, is more difficult to remove from oil components than the
free form. It is important to reduce the amount of free form
before conducting the CRL catalyzed reaction, since the esterified form is generated during the reaction.
Distillation is an efficient way to reduce the amount of free-form Chol. Due to a molecular weight (MW) difference between
free-form Chol (MW: 386.7) and TAG (MW: approximately 900),
short path distillation can be used to separate Chol from the oil
component. This method results in a relative decrease in Chol
content compared with the content that is obtained without
distillation pretreatment. For example, when tuna oil that contains 0.34% Chol was catalyzed by CRL, the Chol content was
0.48% after refining (Fig. 4, page 63). On the other hand, when
distillation was carried out before the CLR-catalyzed reaction,
the Chol content decreased to 0.05%—and the amount of
Chol remained lower level after CRL catalyzed reaction and
refining [ 3].
NOt EAsy tO EstERIFy glycEROl/DHA
Using CRL to synthesize glycerides enzymatically from glycerol
and DHA is not efficient. When TAG is hydrolyzed by CRL, DHA is
enriched due to the weakness of acting on DHA. Incorporating
DHA into the glycerol moiety by CRL-catalyzed esterification is
difficult due to the weak action on DHA.
Although other lipases from Penicillium, Pseudomonas or
Rhizomucor have been proposed for synthesizing glycerides
from glycerol and DHA [ 4], they don’t have enough specificity
toward DHA to incorporate it into the glycerol. For these organisms to synthesize DHA-enriched TAG, the DHA must be purified
before incorporating it into the glycerol moiety.
Hirouchi Yuji and Doisaki Nobushige are researchers at
Nippon Suisan Kaisha, Ltd. They work on processing fish oils,
microbial oils or functional lipids, especially concentrating
PUFAs and refining oils. Hirouchi’s email address is
[ 1] Shimada, Y., K. Maruyama, M. Nakamura, S. Nakayama,
A. Sugihara, and Y. Tominaga, J. Am. Oil Chem. Soc. 72:
[ 2] Ikemoto, H., N. Doisaki, and Y. Umehara, US 20130123525
[ 3] Doisaki, N., H. Ikemoto, K. Hata, S. Tokiwa, and K.
Matsushima, US 20140066644 A1 (2014).
[ 4] Li, Z. Y. and O.P. Ward, J. Am. Oil Chem. Soc. 70: 745–749