Purification of conjugated linoleic acid isomers through a process including lipase-catalyzed selective esterification.

Article abstract of DOI:10.1271/bbb.67.1429

A mixture of conjugated linoleic acids (CLAs) was prepared by alkali conjugation of high purity linoleic acid. The preparation contained 45.1 wt% cis-9, trans-11 (c9,t11)-CLA, 46.8 wt% trans-10, cis-12 (t10,c12)-CLA, and 5.3 wt% other CLAs. A process comprising Candida rugosa lipase-catalyzed selective esterification with lauryl alcohol, molecular distillation, and urea adduct fractionation under strict conditions in ethanol was very effective for purification of c9,t11- and t10,c12-CLAs. In particular, the urea adduct fractionation efficiently eliminated CLAs except c9,t11- and t10,c12-isomers. Purification of c9,t11- and t10,c12-CLAs from 1.0 kg of the CLA mixture increased the c9,t11-CLA purity to 93.1% with 34% recovery of the initial content, and increased the t10,c12-CLA purity to 95.3% with 31% recovery.

Full text of DOI:10.1271/bbb.67.1429

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Purification of Conjugated Linoleic Acid Isomers
through a Process Including Lipase-catalyzed
Selective Esterification
Toshihiro NAGAOa, Yoshie YAMAUCHI-SATOb, Akio SUGIHARAc, Toshio IWATAb, Koji
NAGAOd, Teruyoshi YANAGITAd, Shuji ADACHIe & Yuji SHIMADAa
a Osaka Municipal Technical Research Institute1-6-50 Morinomiya, Joto-ku, Osaka
536-8553, Japan
b Rinoru Oil Mills Co., Ltd.2-13-12 Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan
c Faculty of Engineering, Tokushima Bunri University1314-1 Sido, Sanuki, Kagawa
769-2101, Japan
d Department of Applied Biological Sciences, Saga University1 Honjo-machi, Saga
840-8502, Japan
e Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto
UniversitySakyo-ku, Kyoto 606-8502, Japan
Published online: 22 May 2014.
To cite this article: Toshihiro NAGAO, Yoshie YAMAUCHI-SATO, Akio SUGIHARA, Toshio IWATA, Koji NAGAO, Teruyoshi
YANAGITA, Shuji ADACHI & Yuji SHIMADA (2003) Purification of Conjugated Linoleic Acid Isomers through a Process
Including Lipase-catalyzed Selective Esterification, Bioscience, Biotechnology, and Biochemistry, 67:6, 1429-1433, DOI:
10.1271/bbb.67.1429
To link to this article: http://dx.doi.org/10.1271/bbb.67.1429
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Biosci. Biotechnol. Biochem., 67 (6), 1429–1433, 2003
Note
Puriˆcation of Conjugated Linoleic Acid Isomers through a Process Including
Lipase-catalyzed Selective Esteriˆcation
4
Toshihiro NAGAO,1 Yoshie YAMAUCHI-SATO,2 Akio SUGIHARA,3 Toshio IWATA,2 Koji NAGAO
,
†
1,
Teruyoshi YANAGITA,4 Shuji ADACHI,5 and Yuji SHIMADA
1Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan
2Rinoru Oil Mills Co., Ltd., 2-13-12 Nihonbashi, Chuo-ku, Tokyo 103-0027, Japan
3Faculty of Engineering, Tokushima Bunri University, 1314-1 Sido, Sanuki, Kagawa 769-2101, Japan
4Department of Applied Biological Sciences, Saga University, 1 Honjo-machi, Saga 840-8502, Japan
5Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
Received December 16, 2002; Accepted February 24, 2003
expenditure.11–14) These studies called a great deal of
attention to the fractionation of CLA isomers.
Lipases from Geotrichum candidum and Candida
A mixture of conjugated linoleic acids (CLAs) was
prepared by alkali conjugation of high purity linoleic
acid. The preparation contained 45.1 wt cis-9, trans
11 ( 9, 11)-CLA, 46.8 wt trans-10, cis-12 ( 10, 12)-
CLA, and 5.3 wt other CLAs. A process comprising
%
-
c
t
%
t
c
rugosa were recently reported to recognize
CLA more readily than 10, 12-CLA, and an
enzymatic method with these lipases was eŠective for
the separation of 9, 11- and 10,
12-CLAs.15,16)
In particular, we successfully puriˆed 9, 11- and
10, 12-CLAs to 95 based on the total content of
c9,t11-
%
t
c
Candida rugosa lipase-catalyzed selective esteriˆcation
with lauryl alcohol, molecular distillation, and urea
adduct fractionation under strict conditions in ethanol
c
t
t
c
c
t
was very eŠective for puriˆcation of
10, 12-CLAs. In particular, the urea adduct fractiona-
tion e‹ciently eliminated CLAs except 9, 11- and
10, 12-isomers. Puriˆcation of 9, 11- and 10, 12-
CLAs from 1.0 kg of the CLA mixture increased the
9, 11-CLA purity to 93.1 with 34 recovery of the
initial content, and increased the 10, 12-CLA purity to
95.3 with 31 recovery.
c9,t11- and
t
c
z
t
c
the two isomers by a process composed of C. rugosa
lipase-catalyzed selective esteriˆcation with lauryl
alcohol (LauOH), molecular distillation, and urea
adduct fractionation.16) However, the two puriˆed
c
t
t
c
c
t
t
c
c
t
%
%
c
preparations, especially
t
10,
c
12-CLA preparation,
9, 11-
and t10,c
12-isomers.16) In addition, the two puriˆed
t
contained nearly 10 of CLAs other than
c
t
z
%
%
preparations contained oleic acid. These con-
taminants are undesirable when studying phys-
icochemical and physiological properties of the two
CLAs. We thus attempted to purify the two CLAs
without contamination by other isomers. This paper
reports that high purity of the two CLAs can be
obtained by improving our previous process as fol-
lows: a CLA mixture prepared by alkali conjugation
of highly puriˆed linoleic acid is used as a starting
material, and urea adduct fractionation is done
under strict conditions with ethanol.
Key words: conjugated linoleic acid; Candida rugosa
lipase; esteriˆcation; urea adduct fractio-
nation; molecular distillation
Conjugated linoleic acid (CLA) is a group of C18
fatty acids (FAs) containing a pair of conjugated
double bonds in either the cis or trans conˆguration.
A commercialized product contains almost equal
amounts of cis-9, trans-11 (
c9,t11)-CLA and trans-
10, cis-12 ( 10, 12)-CLA. The mixture of CLA
t
c
isomers has various physiological activities, such as
reduction of the incidence of cancer,1–3) decrease in
body fat content,4–6) beneˆcial eŠects on atheroscle-
rosis,7,8) and improvement of immune function.9) It
We previously fractionated
CLAs from a commercially available CLA mixture.
Because the product contained 17.0 wt oleic acid
that disturbed an e‹cient puriˆcation of the two
isomers, the purities of 9, 11- and 10, 12-CLA
increased to only 78.8 and 83.5
, respectively.16)
To eliminate this drawback, a mixture of CLA
c9,t11- and t10,c12-
z
was recently reported that
c
9,
t
11-CLA had antican-
c
t
t
c
cer activity,10) and that
t
10,
c
12-CLA participated in
z
z
the decrease of body fat and the increase of energy
†
To whom correspondence should be addressed. Tel:
＋
81-6-6963-8073; Fax:
81-6-6963-8079; E-mail: shimaday omtri.city.osaka.jp
＋ ＠
Abbreviations: CLA, conjugated lioleic acid;
c
,
cis
;
t
,
trans; LauOH, lauryl alcohol; FA, fatty acid; FFA, free fatty acid; FALE, fatty
acid lauryl ester; TAG, triacylglycerol

1430
T. NAGAO et al.
isomers was prepared by alkali conjugation of high
purity linoleic acid (purity, 97.1 ; Tokyo Kasei
FALEs. The
late 1-2 increased to 78.1 wt
content in FALEs of residue 1-2 reached 85.1 wt
Distillate 1-2 and residue 1-2 were used for further
t
10,
c
12-CLA content in FFAs of distil-
z
z
, and the 9, 11-CLA
c t
Kogyo Co., Ltd., Tokyo). A mixture of 1.8 kg of
propylene glycol and 0.6 kg of KOH was heated to
z
.
170
acid, and the mixture was agitated at 170
with nitrogen gas blown through it. After the mixture
was cooled to 90 C, its pH was adjusted at 1 with
9
C. To the mixture was added 1.2 kg of linoleic
puriˆcation of
tively.
Puriˆcation of
t
10,
c
12- and
c9,t11-CLAs, respec-
9
C for 1 h
t
10,
c
12-CLA was done according
º
9
to Scheme 1. A mixture of 510 g of distillate 1-2,
235 g of LauOH (equimolar amount to FFA-CLA),
concentrated HCl and 1700 ml of water was added.
Free fatty acids (FFAs) were extracted three times
186 ml of water, and 30 U g-reaction mixture of C.
W
with 1200 ml of
n
-hexane, and the organic solvent
rugosa lipase was incubated at 30
liter reactor with agitating at 270 rpm (esteriˆcation
degree, 20.1 ). The oil layer (708 g) was ˆrst put
9C for 16 h in a 2-
was then removed with an evaporator (yield,
1.15 kg). The CLA mixture (referred to as FFA-
z
CLA) contained 45.1 wt
10, 12-CLA, and 5.3 wt
C. rugosa lipase was purchased from Meito
z
z
c
9,
t
11-CLA, 46.8 wt
z
through molecular distillation to recover LauOH-
free FFAs. Because LauOH could not completely be
removed by one-time distillation, it was removed by a
two-step distillation. The ˆrst distillation was done at
t
c
other CLA isomers.
Sangyo Co., Aichi. One unit (U) of the lipase activity
was deˆned as the amount of enzyme that liberated
120
9
C and 0.2 mm Hg, and 204 g of distillate 2-1
1
m
mole FA per minute in hydrolysis of olive oil.17)
(LauOH content, 98.5 wt
z
) and 498 g of residue 2-1
Olive oil, LauOH, and urea were obtained from
Wako Pure Chemical Industry Co., Osaka. The
other reagents were of analytical grade.
(LauOH content, 2.4 wt
tion of residue 2-1 at 135
almost all LauOH in the distillate 2-2 (25 g; 44.0 wt
LauOH, 56.0 wt FFAs). Separation of residue 2-2
z
) were obtained. Distilla-
9
C and 0.2 mm Hg removed
z
FFA was measured by titration with 0.1 to 1.0 N
KOH, and the degree of esteriˆcation was calculated
from the amount of FA consumed during a reaction.
FA composition was analyzed as described previous-
z
into FFAs and FALEs was attempted but the fractio-
nation was not complete. Final distillation of residue
2-2 (467 g) was therefore done at 190
Hg to obtain FFA-rich fraction: distillate 2-3 (310 g)
consisted of 0.3 wt LauOH, 91.6 wt FFAs, and
8.1 wt FALEs, and residue 2-3 (152 g) was com-
posed of 12.6 wt FFAs and 87.4 wt FALEs. The
FFA composition in distillate 2-3 was 0.6 wt oleic
acid, 3.4 wt 9, 11-CLA, 86.3 wt 10, 12-CLA,
CLAs except 9, 11- and 10, 12-
9
C and 0.2 mm
ly.16) In brief, FFAs were methylated in 3 ml of 5
z
HCl methanol by heating at 70
9
C for 5 min, and FA
lauryl esters (FALEs) were converted to FA methyl
esters in 3 ml of methanol containing 1 Na-methy-
late by heating at 70 C for 15 min. The FA methyl
z
z
W
z
z
z
z
9
z
esters were analyzed with a Hewlett-Packard 5890
gas chromatograph (Avondale, PA, USA) connected
z
z
c
t
z
t
c
and 9.7 wt
c
t
t
c
×
to a DB-23 capillary column (0.25 mm 30 m; J & W
isomers.
Scientiˆc, Folsom, CA, USA). The contents of
LauOH, FFA, and FALE were measured by gas
chromatography with a DB-5 capillary column
The ˆnal puriˆcation step of
removal of FALEs and CLAs except
10,
t
10,
c
12-CLA is
9, 11- and
c
t
t
c
12-isomers. We previously did urea adduct frac-
(0.25 mm
×
10 m; J & W Scientiˆc).16)
tionation in methanol. The fractionation completely
removed FALEs, but not impurities of CLA. Hence,
urea adduct fractionation of distillate 2-3 under the
strict conditions was preliminarily attempted. A
mixture of diŠerent amounts of distillate 2-3, 10 g of
urea, 50 ml of ethanol, and 4 ml of water was stirred
A puriˆcation process of
c
9,t11- and t10,c12-
CLAs is shown in Scheme 1. Selective esteriˆcation
for fractionation of the two isomers was done under
the conditions that were found to be optimal in our
previous paper.16) A mixture of 1000 g of FFA-CLA,
664 g of LauOH (equimolar amount to FFA-CLA),
in a water bath set at 65
solved completely. The mixture was cooled gradually
to 5 C over ca. 10 h, and the resulting precipitates
9C until all substances dis-
416 ml of water, and 20 U g-reaction mixture of C.
W
rugosa lipase was agitated at 30
9
C and 270 rpm for
9
16 h in a 2-liter reactor (MDS-U; Marubishi Bioen-
gineering Co., Ltd., Tokyo) (esteriˆcation degree,
were removed by ˆltration. To the ˆltrate was added
50 ml of 0.1 N HCl, and FFAs were extracted twice
with 50 ml of n-hexane. After removal of n-hexane
47.1
z). The oil layer (1587 g) was put on a molecular
distillation apparatus (Wipren Type 2-03; Sinko
Pantec Co., Ltd., Hyogo). The ˆrst distillation at
with an evaporator, the composition in the extracts
was analyzed (Table 1). FALEs were completely
removed in the precipitates under all conditions.
When 5 g of distillate 2-3 was fractionated with 10 g
120
297 g of distillate 1-1 containing 98.7 wt
and 1276 g of residue 1-1. The residue 1-1 was then
distilled at 190 C and 0.2 mm Hg: Distillate 1-2
(516 g) was composed of 9.9 wt LauOH, 84.3 wt
FFAs, and 5.8 wt FALEs, and residue 1-2 (751 g)
was composed of 10.0 wt FFAs and 90.0 wt
9
C and 0.2 mm Hg separated the oil layer into
z
LauOH
of urea, CLAs other than
c9,t11- and t10,c12-
9
isomers were most e‹ciently removed. Meanwhile,
the recovery of FFAs were decreased with smaller
amounts of distillate 2-3. From these results, the
z
z
z
z
z
ratio of distillate 2-3 to urea was ˆxed at 1:2, wt wt.
W

Puriˆcation of Conjugated Linoleic Acid Isomers
1431
Scheme 1. Puriˆcation Process of cis-9, trans-11 (c9,t11)-Conjugated Linoleic Acid (CLA) and trans-10, cis-12 (t10,c12)-CLA.
A mixture of CLA isomers (FFA-CLA) prepared by alkali conjugation of high purity linoleic acid was used as a starting material.
FFA, free fatty acid; FALE, fatty acid lauryl ester; TAG, triacylglycerol.
Based on the preliminary experiment, urea adduct
fractionation of distillate 2-3 was done as follows. A
mixture of 275 g of distillate 2-3, 550 g of urea,
2750 ml of ethanol, and 220 ml of water was heated
content of CLAs other than
isomers from 9.7 to 1.3 wt
c9,t11- and t10,c12-
z
, and the purity of
t
10,c12-CLA reached 95.3z (Table 2). The content
of
and
10,
t
10,
c
12-CLA based on the total content of
c9,t11-
at 65
9
C to completely resolve all components. The
t
10, 12-isomers was 96.9 Recovery of
c
z.
9
mixture was gradually cooled to 5 C over ca. 10 h.
t
c
12-CLA by a series of puriˆcation procedures
After removal of the resulting precipitate, 3 liter of
0.1 N HCl was added to the ˆltrate. Non-adducted
would become 31z of the initial content, if a part of
distillate 2-3 used for the preliminary urea adduct
fractionation was also used for this large-scale puriˆ-
cation.
FFAs were extracted three times with 200 ml of
n-
hexane and the solvent was then removed by evapo-
ration (yield, 137 g). This fractionation decreased the
Another isomer, c9,t11-CLA, enriched in FALEs

1432
T. NAGAO et al.
of residue 1-2 (FALEs content, 90.0 wt
z
) was next
residue 3-2 (366 g) consisted of 4.6 wt
95.4 wt FALEs. To purify FALEs, 300 ml of 0.5 N
KOH (20 ethanol solution) was added to residue
3-2, and FALEs were then extracted three times with
400 ml of -hexane. -Hexane was removed with an
z FFAs and
puriˆed according to Scheme 1. Enzymatic hydroly-
sis of FALE was attempted, but commercially availa-
ble lipases tested did not recognize FALE as a sub-
strate. The residue 1-2 was therefore hydrolyzed with
NaOH in the presence of ethanol: A mixture of 150 g
of residue 1-2, 600 ml of ethanol, 60 ml of water, and
z
z
n
n
evaporator, and 334 g of extracts were obtained.
The extracts (FALEs) were hydrolyzed with NaOH
in the presence of ethanol as described in the previous
section, and the hydrolysis products (LauOH and
9
45 g of NaOH was heated at 60 C for 30 min with
stirring and blowing of nitrogen gas. After the reac-
tion, 600 ml of water was added to the mixture, and
its pH was adjusted to 2 with concentrated HCl.
FFAs and LauOH were extracted three times with
FFAs) were recovered by n-hexane extraction under
acidic conditions. To the mixture (329 g) was added
55 g of saŒower oil (Rinoru Oil Mills Co., Ltd.,
200 ml of
n
-hexane, and the solvent was removed by
Tokyo), and distillation was done at 120
0.2 mm Hg: Distillate 4-1 (128 g) contained 97.7 wt
LauOH, and residue 4-1 (253 g) was composed of
1.4 wt LauOH, 76.2 wt FFAs, 0.6 wt FALEs,
and 21.8 wt triacylglycerols (TAGs). Distillation
for removal of LauOH was then done at 135 C and
0.2 mm Hg: Distillate 4-2 (7.9 g) consisted of
37.5 wt LauOH and 62.5 wt FFAs, and the
residue 4-2 (242 g) was composed of 0.2 wt
LauOH, 76.7 wt FFAs, 0.6 wt FALEs, and
22.5 wt TAGs. FFAs were ˆnally puriˆed by distil-
lation of residue 4-2 at 200 C and 0.2 mm Hg: Distil-
late 4-3 (166 g) consisted of 0.3 wt LauOH,
99.4 wt FFAs, and 0.3 wt FALEs, and residue
4-3 (74 g) was composed of 25.7 wt FFAs, 1.4 wt
9
C and
evaporation. This procedure was done ˆve times in
total, and 750 g of residue 1-2 was converted to a
mixture of FFAs and LauOH (yield, 714 g). To the
mixture was added 46 g of LauOH to set the ratio of
z
z
z
z
z
FFAs LauOH at 1:1, mol mol. The mixture of FFA
9
W
W
LauOH (760 g) was incubated at 30
9
C for 16 h with
W
10 U g-reaction mixture of C. rugosa lipase in the
z
z
W
presence of 20
The oil layer recovered (717 g) was distilled at 120
and 0.2 mm Hg: Distillate 3-1 (110 g) contained
97.3 wt LauOH, and residue 3-1 consisted of
4.0 wt LauOH, 33.7 wt FFAs, and 62.3 wt
FALEs. To further remove LauOH and FFAs,
residue 3-1 was distilled at 190 C and 0.2 mm Hg:
The distillate 3-2 (231 g) was composed of 10.4 wt
LauOH, 79.7 wt FFAs, and 9.9 wt FALEs, and
z
water (esteriˆcation degree, 53.0
z
9C
).
z
z
z
z
z
z
9
z
z
z
z
z
9
z
z
z
FALEs, and 72.9 wt
puriˆed fraction of 9,
processes, the content of
total content of 9, 11- and
, and purity of 9,
(Table 2). Recovery of
of the initial content.
A previous puriˆcation process could not eliminate
z
TAGs. Distillate 4-3 is the
z
z
c
t11-CLA. Through a series of
c
9,
t
t
11-CLA based on the
10, 12-isomers reached
11-CLA increased to
9, 11-CLA by this
c
t
c
Table 1. Free Fatty Acid (FFA) Composition after Urea Adduct
Fractionation
96.4
93.1
z
z
c
t
c
t
a
Fatty acid composition (wt
CLA
z)
puriˆcation was 34
z
Amount of
distillate 2-3b
(g)
Recovery
of FFA
(
z)
oleic acid and CLAs except
c9,t11- and t10,c12-
18:1
c
9,
t
11
t
10,c
12 Othersc
isomers.16) The use of FFA-CLA originating the high
purity of linoleic acid and the adoption of urea ad-
duct fractionation under strict conditions with
ethanol were found to be eŠective for a preparation
4
5
6
8
10
ndd
nd
nd
nd
0.4
2.9
3.0
3.3
3.6
3.6
95.6
95.9
94.7
93.2
91.7
1.5
1.1
2.0
3.2
4.4
45.0
54.6
67.3
77.5
84.2
of high purity of c9,t11- and t10,c12-CLAs. We are
convinced that the puriˆed CLA isomers are useful
for studies of their physicochemical and physiologi-
cal properties.
a
CLA, conjugated linoleic acid;
c
,
cis
;
t
,
trans
oleic acid (18:1), 3.4 wt
12-CLA, and 9.7 wt other CLAs.
11-CLA, 10, 12-CLA, and unidentiˆed CLA.
0.1 wt ).
.
b
FFA composition in distillate 2-3: 0.6 wt
9, 11-CLA, 86.3 wt 10,
Total content of 9,
z
z
c
t
z
c
t
c
z
c
c
c
c
d
Not detected (
º
z
Table 2. Composition in Puriˆed Preparations of
t
10,
c
12- and
CLA
c9,t11-CLAs
FFA (wt
z
)
LauOH
(wt
FALE
(wtz)
Preparation
FFA-CLAa
z)
18:1
18:2
c
9,
t
11
t
10,
c
12
c
9,
c
11
c
10,
c
12
Others
—
0.4
0.3
2.0
nd
1.5
0.8
nd
0.9
45.1
3.0
93.1
46.8
95.3
3.5
1.3
nd
nd
1.3
0.6
nd
2.7
0.7
0.4
—
nd
0.3
t
10,
9,
c
12-CLA
c
t
11-CLA
a
Starting material that was prepared by alkali conjugation of 97.1
z
linoleic acid in the presence of propylene glycol.
LauOH, lauryl alcohol; FALE, fatty acid lauryl ester; 18:2, linoleic acid. See Table 1 for other abbreviations.

Puriˆcation of Conjugated Linoleic Acid Isomers
1433
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Inhibition of benzo( )pyrene-induced mouse fore-
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