Chemoenzymatic conversion of linoleic acid into conjugated linoleic acid

Article abstract of DOI:10.1021/jf903521b

An efficient chemoenzymatic method for preparing conjugated linoleic acid (CLA) using free linoleic acid (LA) as a substrate is described. In the first step, LA was transformed into 10-hydroxy-cis-12octadecenoic acid (HA) by the whole cells of Lactobacillus plantarum after 48 h of incubation. The preincubation of whole cells with 0.03% LA resulted in a better yield of HA (480 mg/g) compared to cells grown without LA. In a second fast microwave step, HA was converted to cis-9, trans-11-octadecadienoic acid in the presence of iodine as a catalyst over a silica gel surface. The advantage of this method in preparing cis-9,.trans-11 CLA is simple via the whole cell bioconversion of LA into HA via L plantarum followed by the fast microwave-assisted synthesis of cis-9, trans-11 CLA in higher yields.

Full text of DOI:10.1021/jf903521b

1646 J. Agric. Food Chem. 2010, 58, 1646–1652
DOI:10.1021/jf903521b
Chemoenzymatic Conversion of Linoleic Acid into
Conjugated Linoleic Acid
,†
*
†,‡
A
YHAN S. DEMIR
AND
FARAH N. TALPUR
†Department of Chemistry, Middle East Technical University, 06531Ankara, Turkey and
‡National Center of Excellence in Analytical Chemistry, University of Sindh, 76080 Jamshoro, Pakistan
An efficient chemoenzymatic method for preparing conjugated linoleic acid (CLA) using free linoleic acid
(LA) as a substrate is described. In the first step, LA was transformed into 10-hydroxy-cis-12-
octadecenoic acid (HA) by the whole cells of Lactobacillus plantarum after 48 h of incubation. The
preincubation of whole cells with 0.03% LA resulted in a better yield of HA (480 mg/g) compared to cells
grown without LA. In a second fast microwave step, HA was converted to cis-9,trans-11-octadecadi-
enoic acid in the presence of iodine as a catalyst over a silica gel surface. The advantage of this method
in preparing cis-9,trans-11 CLA is simple via the whole cell bioconversion of LA into HA via L. plantarum
followed by the fast microwave-assisted synthesis of cis-9,trans-11 CLA in higher yields.
KEYWORDS: Chemoenzymatic; CLA; Lactobacillus plantarum; microwave irradiation; LA
INTRODUCTION
isomerization, or genetic engineering/bioengineering (13). Tradi-
tional organic synthesis is highly capital-intensive and results in
an isomeric mixture of CLA. However, low yields, extensive
purification steps, and the inseparability of isomers all limit the
commercial use of most chemical methods (14). Therefore,
biotechnology offers new alternatives to traditional lipid manu-
facturing methods. A rumen bacterium, Butyrivibrio fibrisolven,
was the first identified for converting LA to cis-9,trans-11 CLA,
due to the presence of linoleic acid isomerase activity (15). Since
then, there have been numerous reports that a large number of
benign bacteria, which are naturally present in the environment
and intestinal tracts of ruminants, can convert LA to
CLA (16-18). Further analyses with Lactobacillus species
washed cells as model strains have revealed the involvement of
a hydroxy fatty acid, 10-hydroxy-12-octadecanoic acid, as an
intermediate for CLA production (19). However, the production
of CLA was greatly influenced by aerobic conditions. Because of
the growth inhibitory action of LA at low concentrations, the
yields of CLA remain poor, as LA is required for any feasible
production process. In addition, some of the CLA-producing
bacteria transform the wanted isomers further to secondary
compounds (20, 21). Therefore, the chemoenzymatic conversion
that combines the flexibility of chemical synthesis and the high
selectivity of enzymatic conversions is a powerful approach to
obtain individually pure isomers (22).
Conjugated linoleic acid (CLA) is a collective term that refers
to a mixture of the positional and geometric isomers of linoleic
acid (LA) with conjugated double bonds at carbons 10 and 12 or 9
and 11, in all of the possible cis and trans combinations. It was
first isolated and identified by Pariza and co-workers from a
fraction of grilled beef that possessed antimutagenic activity (1).
Since then, interest has increased markedly because of its dis-
covered physiological activities, such as the reduction of cancer
incidence (2), a decrease in body fat content (3), profitable
effects on atherosclerosis (4), and strengthening of the immune
system (5).
Recently, it was discovered that each isomer has different
bioactivities: cis-9,trans-11 CLA shows anticancer activity (6);
trans-10,cis-12 CLA shows activities that decrease body fat
content (6, 7), increase energy expenditure (8), and suppress the
development of hypertension (9). The cis-9,trans-11 isomer is
predominantly found in foods sourced from ruminants. The
formation of this conjugated polyunsaturated system was cata-
lyzed by ruminal bacteria (10). Gaullier et al. (11) assessed the
effects of the supplementation of 3.4 g/day of CLA (1:1 ratio
9- and 10-CLA in triglyceride form) in healthy, overweight adults
and revealed a decrease in body weight and body fat mass along
with increased circulating lipoprotein, thrombocytes, and aspar-
tate aminotransferase. Human intake of CLA from natural food
sources, such as dairy (0.55% total fat) and beef (0.60% total fat),
is approximately 10% of the suggested recommended value (12).
To achieve the estimated optimum dietary CLA levels of
approximately 3-4 g/day, it would be necessary to increase
dietary animal fat, which would increase saturated fat
intake. Therefore, efforts have been made to produce CLA
through organic synthesis, microbial fermentation, enzymatic
Herein we describe a chemoenzymatic method for preparing
cis-9,trans-11 CLA by using a two-step approach. In the first step,
linoleic acid is converted into 10-hydroxy-12-octadecenoic acid
by the whole cell reaction of Lactobacillus plantarum bacteria
followed by the fast microwave production of cis-9,trans-11 CLA
via the dehydration of hydroxyoctadecenoic acid in the presence
of iodine as a catalyst. Microwave techniques offer a reduction in
the amount of solvents and hazardous substances required, along
with a more efficient use of energy with minimum time and better
yields in synthesis (23).
*Author to whom correspondence should be addressed [telephone
(90) 312-2103242; fax (90) 312-2101280; e-mail asdemir@metu.edu.tr].
pubs.acs.org/JAFC
Published on Web 01/08/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 3, 2010 1647
Figure 1. (A) Time course for the conversion of linoleic acid into hydroxyoctadecenoic acid by the whole cells of L. plantarum under optimized conditions. (B)
Effect of preincubation time and concentration on hydroxyoctadecenoic acid conversion. The incubations were performed in triplicate, and the values are
shown in the figure as an average.
Figure 2. 1H NMR spectrum of 10- hydroxy-cis-12-octadecenoic acid (HA) produced from LA by the whole cell incubation of L. plantarum.
MATERIALS AND METHODS
cells were grown twice under aerobic conditions at 30 °C in MRS broth, as
described previously (24).
Chemicals. LA (cis-9,cis-12-octadecadienoic acid) and CLA mixture
of cis-9trans-11 and trans-10,cis-12 CLA was purchased from Sigma-
Aldrich (St. Louis, MO). The other chemicals and reagents (analytical
grade, J. T. Baker, Deventer, The Netherlands) that were used in the
present study are commercially available.
Whole Cell Reaction Conditions. A pure active culture was prepared
by picking a single colony from the MRS agar plate and growing it in
100 mL of MRS broth for 24 h, supplemented with 0.03% LA. After
24 h of preincubation, a 250 μgmL-1 LA complex with BSA (0.5 mg of BSA/
mg of LA) was added to the MRS culture. The whole cell reaction was
further continued for 48 h at 30 °C with gentle shaking (120 strokes/min)
under aerobic conditions. All of the incubations were performed in triplicate.
Culture of Cells. L. plantarum isolated from tulum cheese, as reported
earlier (19), was obtained from Kafka’s University in Kars, Turkey. The

1648 J. Agric. Food Chem., Vol. 58, No. 3, 2010
Demir and Talpur
Lipid Extraction and Isolation of the Reaction Products. Lipids
were extracted from the reaction mixture with chloroform/methanol
(1:2, v/v) according to the procedure of Bligh and Dyer (25). The reaction
product isolation was carried out by column chromatography on silica gel
60 (40-63 μm) with petroleum ether/diethyl ether/acetic acid (70:30:1) as
eluting solvents. TLC was conducted on aluminum sheets that were
precoated with silica gel 60F 254, in which any possible spots were
visualized by phosphomolybdic acid (Sigma).
1H NMR and 1H-1H COSY Analysis. All of the NMR experiments
were performed on a Bruker DPX 400 NMR instrument. The chemical
shifts δ are reported in parts per million relative to CDCl3 (1H, δ 7.27),
(
13C, δ 77.0), and CCl4 (13C, δ 96.4) as internal standard.
LC-MS Analysis of Fatty Acid Methyl Esters (FAME). The
FAME derivative of free CLA was prepared as reported earlier (26),
followed by analysis with Theromo Finnigan LC-MS equipped with a
Surveyor MSQ Mass detector (San Jose, CA). The mass analysis was
performed in atmospheric pressure chemical ionization (APCI) mode with
a probe temperature of 450 °C, corona current of 5 μA, and cone voltage of
30 V, and the sheath gas was high-purity nitrogen operated at 55 psi. The
mass spectrometer was set to scan positive ions over a range of m/z
140-350 at 200 spans. Two Chromsphere Lipid 5 columns (250 ꢀ
4.6 mm.) connected in series were used for the separation. The mobile
phase was hexane and isopropanol (90:10) at a flow rate of 0.7 mL/min.
The photodiode array detector was set at a primary wavelength of 233 nm
to detect conjugated dienes.
Microwave Reaction Conditions. One millimole of isolated and
purified 10-hydroxy-cis-12-octadecenoic acid was placed in a vial contain-
ing 200 mg of silica and iodine crystals (1-5%). Then, the vial was heated
for 60-120 min at 30-50 °C and 800 W by using a Milestone-Start
microwave apparatus. Subsequently, the sample was dissolved in 10 mL of
chloroform, vortexed for 1 min, and filtered. For the removal of iodine, the
sample was shaken for 10 s with 5 mL of 0.01 N Na2S2O3, in which this step
was repeated until a transparent solution was obtained. The solvent was
removed under vacuum, and prepared TLC with petroleum ether/diethyl
ether/acetic acid (70:30:1) as the eluent system yielded the product.
RESULTS AND DISCUSSION
Transformation of LA by the Whole Cells of L. plantarum. The
whole cells of L. plantarum cultivated in an MRS medium
containing LA was investigated for the production of 10-hydro-
xy-cis-12-octadecenoic acid (HA). This was conducted because
whole-cell conversion has advantages, such as greater resistance
to environmental perturbations and a lower effective enzyme
cost, all while eliminating the enzyme purification and extraction
steps (27). The inhibitory effect of LA on bacterial growth has
been reported by many authors (20, 28) who have demonstrated
that there are different tolerances according to strain. In the
present paper, the LA tolerance was evaluated by the addition
of increasing LA concentrations (0, 50, 100 150, 200, 250, and
300 μg mL-1) in MRS agar plates where the bacteria were spread.
Although previous studies (28) have reported bacterial growth
inhibition by using a lower fatty acid level (25 μg mL-1), in the
present study L. plantarum was able to grow in the presence of
higher LA concentrations (250 μg mL-1). This fact is supported
by previous explanations (29) wherein L. plantarum strains were
able to adaptto different environmentsand could grow to high cell
Figure 3. 1H-1H COSY spectrum of 10- hydroxy-cis-12-octadecenoic
acid (HA) produced from LA by the whole cell incubation of L. plantarum.
Table 1. Effect of the Amount of Iodine and Reaction Conditions on the
Conversion of 10-Hydroxy-cis-12-octadecenoic Acid into CLA
entry
solventa
I2 (%) time (min) temperatureb (°C) conversionc (%)
1
2
CH3COOH
1
5
60
60
30
30
-
43
3
4
CH3CN
CH3Cl
1
5
60
60
50
50
-
38
5
6
7
1
5
5
60
60
50
50
50
-
25
37
120
densities. However, a higher concentration of LA (300 μg mL-1
)
inhibited the growth of the cells and decreased HA production.
To determine the optimal incubation time for HA conversion,
HA formation was monitored from 0 to 56 h of incubation at an
interval of 8 h in MRS broth supplemented with 250 μg mL-1. As
can be seen in Figure 1A, the cells that incubated for 48 h formed
8
9
neat
1
5
60
60
50
50
-
40
a One milliliter of each solvent was used. b MW temperature monitored and
controlled with Terminal 320 from MLS-Milestone. c Conversion determined from the
1H NMR spectra.
Scheme 1

Article
J. Agric. Food Chem., Vol. 58, No. 3, 2010 1649
Figure 4. (A) Mass spectrum of cis-9,trans-11 CLA produced via 10-hydroxy-cis-12-octadecenoic acid (HA) by microwave irradiation under optimized conditions.
(B) HPLC-diode array detection of cis-9,trans-11 CLA produced under microwave irradiation. For MS and HPLC conditions, see Materials and Methods.
more HA (480 mg/g of LA) than the cells of other incubation
times, followed by an insignificant decrease in 56 h. Recently, Yu
et al. (28) have also described an LA conversion into 10-hydroxy-
12-octadecenoic acid by using the whole cells of Stenophomonas
nitireducens in a bioreactor.
The higher productivity for HA conversion has already been
reported (17) for the washed cells of L. acidophilus when pre-
incubated with LA (0.05 wt %/vol) versus those cells that are
obtained by cultivation without LA. With such findings in mind,
the effect of the preincubation time on the production of HA was
investigated in the present study by varying the incubation time
from 8 to 32 h and the LA concentration from 0.005 to 0.05% at
30 °C. As shown in Figure 1B, the highest conversion was
obtained after 24 h of incubation in broth supplemented with
0.03% LA. It was found that the addition of a greater amount of
LA exerted an inhibitory effect on HA production, as lessHA was
produced in the presence of 0.05% LA than 0.03% LA, which is
in agreement with the studies of the antimicrobial effect of free
LA on propionic acid bacteria as reported by Kishino et al. (17)
and Jiang et al. (29).
1
Identification of HA. 1H NMR and H-1H COSY analyses
were carried out to identify the positions and geometric config-
urations of double bonds in HA. The 1H NMR spectrrum of HA
is shown in Figure 2. The signal at 3.5 ppm suggested the existence

1650 J. Agric. Food Chem., Vol. 58, No. 3, 2010
Demir and Talpur
Figure 5. 1H NMR spectrum and structure of cis-9,trans-11-octadecadienoic acid (CLA) produced from 10-hydroxy-cis-12-octadecenoic acid (HA) by
microwave irradiation under optimized conditions.
of a hydroxyl group, and the signals H-12 (5.4 ppm, m, 1H) and
H-13 (5.5 ppm, m, 1H) were identified as the protons on the
double bond. The 1H-1H COSY spectrum showed that there is
one methylene group between the double bond and the carbon
bonding to the hydroxyl group (Figure 3). Therefore, a double
bond was thought to be located at the Δ12-position. The coupling
constants between H-12 and H-13 were calculated as 10.90 Hz, as
described for AB spin proton coupling (30), inturnindicating that
the double bonds are in the cis configuration. From these results,
HA was identified as 10- hydroxy-cis-12-octadecenoic acid.
Conversion of 10-Hydroxy-cis-12-octadecenoic Acid into CLA.
The transformation of LA to CLA is not the one-step isomeriza-
tion of a nonconjugated diene to a conjugated diene. The
transformation rather involves the production of hydroxy fatty
acids, that is, 10-hydroxy-trans-12-octadecaenoic acid and
10-hydroxy-cis-12-octadecaenoic acid (17). Such a conversion
of CLA was reported within the past decade by using several
bacteria, including Bifibobacteria, Lactobacillus, and Propionio-
bacterium (31). However, the potential factors being conducted
for the production of CLA highlighted two critical variables to be
tested. First, it was considered that the growth stage might be a
determinant factor. A second potential factor affecting CLA
production was considered to be oxygen availability, in which
these factors markedly affect CLA production (32). Therefore,
the present work aimed to use a simple, clean, and convenient
microwave procedure for CLA production. In the current study,
10-hydroxy-cis-12-octadecenoic acid was treated in catalytic
quantities of iodine under microwave irradiation (MW) on silica
gel. The MW irradiation in 60 min results in the production of
CLA in 43% yield. First, we investigated the effect of the reaction
conditions (Table 1) on the transformation of 10-hydroxy-cis-12-
octadecenoic acid that was induced by various amounts of iodine.
We found that 1% of iodine was insufficient to induce dehydra-
tion (entries 1, 3, 5, and 8), whereas the solvent importantly
influenced the elimination process when 5% of iodine was used.
After 60 min in CH3CN or CH3COOH, maximum conversion
occurred (entries 2 and 4). Transformation in CHCl3 was ob-
served within 120 min, whereas a mild temperature of 30 °C was
found to be sufficient for maximum conversion with CH3COOH.
Finally, we performed the reaction under solvent-free condi-
tions, and in a typical experiment (Scheme 1), 10-hydroxy-cis-12-
octadecenoic acid (1 mmol) and various amounts of iodine (1 and
5%) were left for different times at 50 °C. In the presence of 1%
iodine, no transformation took place after 60 min, whereas a
maximum yield of CLA (40 mg/g) was achieved when 5% iodine
was used (entry 9). Moreover, no CLA product formation was
observed when the samples were conventionally heated in the
presence of iodine (1 and 5%) in CH3CN, CH3COOH, and
CHCl3 for 60 min. The acceleration of reactions by microwave
exposure results from material-wave interactions leading to
thermal effects and specific (nonpurely thermal) effects. Clearly,
the combination of these two contributions could be responsible

Article
J. Agric. Food Chem., Vol. 58, No. 3, 2010 1651
Figure 6. 1H-1H COSY spectrum of cis-9,trans-11-octadecadienoic acid (CLA) produced from 10-hydroxy-cis-12-octadecenoic acid (HA) by microwave
irradiation under optimized conditions.
for the observed effects. It has been shown that higher boiling
points could be observed when polar solvents are submitted to
microwave irradiation conditions when compared with conven-
tional heating, in which this effect is called “superheating”. In the
case of solid-state reactions in MW irradiation, atomic level
excitation has been reported, which results in the generation of
surface defects in solid particles. The generation of these surface
defects enables increased ion motion and thereby helps in the easy
excitation of these ions to higher energy levels (33).
The present MW method is faster compared to the earlier
reported methods involving microorganisms, photoirradiation,
or chemical isomerization techniques for CLA produc-
tion (13, 15, 28). As is evident from the literature, the use of
microwaves offers improvements, such as a reduced time of
reaction, a cleaner product due to fewer side reactions, and the
use of minimal quantities of solvent. Thus, microwave-
assisted synthesis is more economical and environmentally
friendly (34).
Recently, Staver et al. (35) reported iodine as an efficient
catalysis for the transformation of alcohols into corresponding
alkenes by undergoing a dehydration reaction under solvent-free
conditions. Furthermore, iodine has long been recognized
as one of the extremely effective catalysts for the cis-trans
isomerization of olefins. It has been suggested that the isomeriza-
tion process itself involves rotation around the σ-bond, after the
reaction of I• with the π-bond. According to this mechanism, the
rate-determining step is either the formation of the carbon radical
or the rotation around the σ-carbon-carbon bond (36).
Identification of CLA. The mass spectra of the isolated methyl
ester of CLA lead to the formation of highly abundant [M þ H]þ
ions (m/z 295, 100%) followed by the main fragment of [M - 32]þ
m/z 263 due to methanol loss (Figure 4A). The gap of 18 mass
units between the ions from m/z 263 to 245 shows the loss of a
water molecule. After the removal of the water molecules, a series
for methylene-interrupted dienes was detected (m/z 243, 229,
215, 201, 183, and 165). The LC diode array chromatogram
(Figure 4B) equipped with two Chromsphere Lipid 5 columns in
series showed a single peak at a wavelength of 233 nm, which is
1
specific for diene determination. Moreover, H NMR analysis
was carried out to identify the geometric configurations of CLA
(Figure 5). The deduced structure was further confirmed by
1H-1H COSY analysis (Figure 6). The signals for H-9 (5.20
ppm), H-12 (5.57 ppm), H-10 (5.86 ppm), and H- 11 (6.20 ppm)
suggested the existence of double bonds. Other signals were
identified as shown in Figure 4. NMR δH (CDCl3) 6.20 ppm
(1H, dd, J = 15.03, 10.97 Hz, dCH;CHd), 5.86 (1H, t,
J = 10.90 Hz, dCH;CHd), 5.57 (1H, dt, J = 14.79, 7.29,
-CHdCH-), 5.20 (1H, dt, J = 10.87, 6.98 z, -CHdCH-), 2.30
(2H, t, J = 7.50 Hz, -COCH3), 2.13 (2H, dt, J = 7.71, 6.89 Hz,
-CH2;CHd), 2.08 (2H, dt, J = 7.71, 6.73 Hz, dCH;CH2-),
1.62 (2H, m, -CH2CH2CH2-), 1.39 (4H, m, -CH2CH2CH2-),
1.30 (12H, m, -CH2CH2CH2-), 0.90 (3H, t, J = 6.99 Hz, -CH3).
These 1H spin couplings are in agreement with the literature (37).
The spin-spin coupling constant between H-9 and H-10 was
10.9 Hz, which suggests that the double bond between H-9 and H-
10 is in the cis configuration. The coupling constant between H-11
and H-12 was 15.00 Hz, which indicates the trans configuration.
These results show that CLA is a cis/trans-conjugated octadeca-
dienoic acid. The IR analysis of CLA also showed characteristic
peaks at 990 and 945 cm-1, which suggested that CLA is a cis/
trans isomer and, from these results, CLA was identified as cis-9,
trans-11-octadecadienoic acid.

1652 J. Agric. Food Chem., Vol. 58, No. 3, 2010
Demir and Talpur
In conclusion, a highly selective, fast, economical, and envir-
onmentally friendly chemoenzymatic method for the preparation
of cis-9,trans-11 CLA is described via the simple whole cell
incubation of L. plantarum of LA into 10-hydroxy-cis-12-octa-
decenoic followed by the microwave production of cis-9,trans-11
CLA in higher yields.
(17) Kishino, S.; Ogawa, J.; Omura, Y.; Matsumura, K.; Shimizu, S.
Conjugated linoleic acid production from linoleic acid by lactic acid
bacteria. J. Am. Oil Chem. Soc. 2002, 79, 159–163.
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Eisenmenger, M. Biocatalysis of linoleic acid to conjugated linoleic
acid. Lipids 2006, 41, 771–776.
(19) Ogawa, J.; Matsumura, K.; Kishino, S.; Omura, Y.; Shimizu, S.
Conjugated linoleic acid (CLA) accumulation via 10-hydroxy-12-octa-
decaenoic acid during microaerobic transformation of linoleic acid by
Lactobacillus acidophilus. Appl. Environ. Microbiol. 2001, 67, 1246–1252.
(20) Kim, Y. J.; Liu, R. H.; Bond, D. R.; Russell, J. B. Effect of linoleic acid
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ACKNOWLEDGMENT
€ €
€
We are grateful to Dr. Abamuslum Guven from Kafka’s
University in Kars, Turkey, for a generous donation of the
Lactobacillus plantarum species.
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Received for review October 8, 2009. Revised manuscript received
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