Conjugated linoleic acid (CLA) production and lipase-catalyzed interesterification of purified CLA with canola oil
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400 Eur. J. Lipid Sci. Technol. 2008, 110, 400–404
Research Paper
Conjugated linoleic acid (CLA) production and lipase-
catalyzed interesterification of purified CLA with canola oil
Sayyed Amir Hossein Goli, Mahdi Kadivar, Javad Keramat, Mohammad Fazilati
Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan,
Iran
In this study, two important isomers of CLA, i.e. c9,t11 and t10,c12, were produced up to ca. 73% of total
fatty acids, employing alkali isomerization of safflower oil, followed by purification with only one-step urea
crystallization to 85.6%, while the recovery of the purification process was 35%. Interesterification (acid-
olysis) of purified CLA with canola oil was then conducted by Thermomyces lanuginosus lipase. The CLA
content incorporated into the triacylglycerols (TG) was 26.6 mol-% after 48 h of reaction time. Physical
and chemical properties of the TG were then changed according to the degree of substitution of oleic acid
in canola oil with CLA.
Keywords: Canola oil / Conjugated linoleic acid / Enzymatic interesterification / Production and purification
Received: November 6, 2007; accepted: January 3, 2008
DOI 10.1002/ejlt.200700267
1 Introduction Although CLA have several beneficial effects, the con-
sumption of CLA has decreased due to replacement of milk
Conjugated linoleic acids (CLA) are a group of positional and and animal fats by vegetable oils. Enzyme-catalyzed acidolysis
geometrical isomers of linoleic acid (LA) with a conjugated is an approach to increase the CLA content in structured
double bond system. The major natural sources of CLA are lipids (SL). Several researches of enzymatic interesterification
fat tissues of ruminants (meat and dairy products). The cis9,- of CLA with fats and oils were reported; Garcia et al. [16, 17]
trans11 (c9,t11) isomer is the most abundant natural isomer prepared SL from butter and fish oils with CLA by enzymatic
(about 75–90% of total CLA) which is also called rumenic acidolysis. Ortega et al. [18], using a lipase, incorporated CLA
acid [1]. Studies (in vivo and in vitro) have revealed biological in fully hydrogenated soybean oil; Lee et al. [19, 20] reported
activities of CLA including antioxidative, anticarcinogenic, the interesterification of CLA with soybean, sunflower and
antiatherosclerotic, antidiabetogenic and antiobesity proper- safflower oils. The altered composition of triacylglycerols in
ties, along with immune-enhancing effects [2–5]. Different SL (incorporation of CLA) provides different changes in
methods, such as dehydration of ricinoleic acid [6], photo- physical and chemical characteristics of SL compared to the
production of CLA [7], alkaline isomerization of LA or LA- initial lipid, which possibly improve the functional properties
rich oils [8–11], are used to synthesize CLA. Alkaline isom- of the oil.
erization of LA is usually used for commercial production of The objective of this study was to produce high-purity
CLA containing two isomers, c9,t11 (43–45%) and t10,c12 CLA from safflower oil and the incorporation of this func-
(43–45%), accompanied by small amounts of other CLA iso- tional ingredient into canola oil to prepare CLA-rich tria-
mers [5]; however, since the biological activity of the product cylglycerols (TG) by enzymatic interesterification and to
is due to the presence of both isomers, a purification step compare the TG with the starting lipid with respect to physi-
would be necessary. Urea-inclusion crystallization has been cal and chemical properties.
generally employed to concentrate useful polyunsaturated
fatty acids (PUFA) as well as CLA in edible oils [12–15].
2 Materials and methods
Correspondence: Mahdi Kadivar, Department of Food Science and
Technology, College of Agriculture, Isfahan University of Technology, Isfa-
han, 84156 83111, Iran. Safflower seed was prepared by Oilseed Research & Devel-
E-mail: mak120@mail.usask.ca opment Company (Tehran, Iran). CLA (mixture of c9,t11
Fax: 198-31-13912254 and t10,c12 isomers) and other fatty acid standards were
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.comEur. J. Lipid Sci. Technol. 2008, 110, 400–404 Enzymatic interesterification of CLA with canola oil 401
obtained from Sigma-Aldrich. Chemicals and solvents were with membrane filters (0.45 mm) in mother liquor; the mother
all of analytical grade and purchased from Merck. Immobi- liquor was transferred to a separatory funnel and acidified to
lized lipase from Thermomyces lanuginosus was a gift provided pH 3 with HCl (2 N). The CLA were extracted with hexane
by Novozymes (Tehran, Iran). Refined, bleached and deo- (three times) and the hexane layer was washed with deionized
dorized canola oil was obtained from Naz Vegetable Oils Co. water (twice). The washed hexane was dried over anhydrous
(Isfahan, Iran). sodium sulfate and removed by rotary vacuum evaporator; the
CLA content was determined by GC and the yield of CLA
2.1 Extraction of safflower oil purification was then calculated.
The oil was extracted using hexane as solvent. Hexane 2.5 CLA content in crude and purified mixtures
(1500 mL) was added to 500 g milled seeds and extraction
was performed for 24 h at room temperature; this operation To determine the CLA content, methylation was done as
was done twice to complete oil extraction from the seeds. The described by Boylston and Beitz [22] with slight modification.
solvent was then removed by rotary vacuum evaporator and Of borontrifluoride (BF3, 14% in methanol), 7 mL was added
the safflower oil was stored in dark container in a refrigerator to 300 mg CLA mixture and the mixture was methylated for
for the subsequent steps. 30 min at room temperature; 25 mL saturated NaCl solution
was added and the mixture was vortexed; then, 6 mL hexane
2.2 Fatty acid composition of safflower oil (to extract the methylated fatty acids) was added. The hexane
layer was dried with anhydrous sodium sulfate and quantified
To determine the fatty acid profile of safflower oil, the oil was by GC. Heptadecanoic acid (17:0) was used as an internal
methylated according to AOAC method [21]. Methylated standard to determine the amount of CLA.
samples (1 mL) were injected into a gas chromatograph
(CP9002; Chrompac) equipped with a flame ionization 2.6 Enzymatic interesterification
detector (FID) and the fatty acid methyl esters were separated
using FFAP-CB fused-silica WCOT (25 m60.32 mm Into a 50-mL Erlenmeyer flask with a screw cap, 10 g of a
60.3 mm) and helium gas as a carrier with an inlet pressure of substrate consisting of 40 : 60 wt/wt of purified CLA mixture
75 kPa. The temperature program was as follows: increasing to canola oil (molar ratio of CLA isomers to canola oil was
from 40 to 100oC at a rate of 10 7C/min and holding for equal to 0.57) were weighed. The reaction was started by
0.2 min, then increasing to 240 7C at 25 7C/min and holding adding 700 mg of the lipase (7% by total weight of the sub-
for 30 min at 240 7C. The temperatures of the injector and strate). The flask containing the reaction mixture was flushed
detector were 230 and 250 7C, respectively. with nitrogen, stoppered and incubated for 48 h in an orbital
shaker at 200 rpm and 55 7C. This reaction was also per-
2.3 CLA production formed in large amounts (100 g purified CLA mixture to
150 g canola oil) to provide sufficient TG for the following
CLA were produced as described by Kim et al. [9]. Briefly, experiments.
400 g safflower oil (75% LA) was added to 100 g sodium hy-
droxide (NaOH) dissolved in 320 g propylene glycol (180 7C) 2.6.1 Analyses
in an oil bath. The CLA isomers were formed when the mix-
ture was left to cool to 80 7C for 2 h; phosphoric acid (2 N) Samples (50 mL) were withdrawn at intervals (every 12 h).
was then added to adjust the mixture pH to 2.5–3.0, and CLA Methylation of the fatty acids was carried out according to
were recovered by extraction with hexane in a separatory Ortega et al. [18]. Total fatty acids (free 1 esterified) were
funnel. The hexane layer was washed twice with methanol/ determined by methylating the mixture with methanolic HCl
water (30%) solution and then evaporated by rotary vacuum (0.2 M), whereas methylation of the esterified fatty acids was
evaporator to obtain the CLA. The CLA content was deter- conducted by methanolic NaOH (0.1 M). The percentage of
mined by gas chromatography (GC). esterified CLA was determined by injection of methylated
samples (1 mL) into a gas chromatograph, using heptadeca-
2.4 CLA purification noic acid (17:0) as internal standard.
The CLA were purified according to Yang and Liu [11] with 2.7 Deacidification of TG by alkaline extraction
slight modification. Crude CLA mixture was added gradually
to urea dissolved in warmed methanol (70 7C) in a proportion Deacidification of the TG by alkaline extraction was carried
of 1 : 2 : 6 (oil/urea/methanol); then, the solution was allowed out according to Lee et al. [19]. After the incubation time,
to cool to room temperature for 4 h. The well-defined needles hexane was added and the reaction mixture was filtered
of the urea complexes were put into a refrigerator overnight at immediately through a 0.45-mm filter to remove the enzyme,
5 7C. The purified CLA were recovered by vacuum filtration and the hexane was removed by rotary vacuum evaporator.
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com402 S. A. H. Goli et al. Eur. J. Lipid Sci. Technol. 2008, 110, 400–404
The reaction mixture was mixed with 0.5 N KOH solution Table 1. Fatty acid compositions of safflower oil, crude CLA and
(20% ethanol, 20 mL) and hexane (40 mL) in a separatory purified CLA.
funnel with a stopcock. The upper phase (hexane layer) was
collected in a round-bottom flask and 3–4 drops of phe- Fatty acids Safflower Crude Purified
[%] oil CLA CLA
nolphthalein solution were added, and the titration with 0.5 N
KOH (20% ethanol) was conducted. Then, saturated NaCl Palmitic acid 5.5 4.5 0.1
(10 mL) solution was vigorously mixed in and the hexane Stearic acid 1.6 1.2 0
phase was isolated and passed through an anhydrous sodium Oleic acid 12.1 12.0 2.4
sulfate column. The hexane was evaporated by rotary vacuum Linoleic acid 75.5 3.5 7.3
evaporator and the TG were obtained. Linolenic acid 0.6 0 0
Cis9,trans11 – 35.6 37.0
2.8 Physical and chemical properties Trans10,cis12 – 37.2 48.5
Yield [%]§ – 80 35
Iodine value (IV), saponification value (SV) and % free fatty
§
acids (FFA) of the TG and canola oil were determined The yield of oil mixture, based on the previous step.
according to AOCS methods Cd 1-25, Cd 3-25 and Cd 3d-
63, respectively. The oxidative stability index (OSI) was purification step, about 100 g purified CLA mixture was
determined according to AOCS method Cd 12b-92 using a obtained from 320 g crude CLA, indicating a yield of 35%
743 Rancimat (Metrohm). The refractive index was reported (Table 1).
using a refractometer according to AOCS method Cc 7-25 Concentrates of PUFA are generally prepared by urea
[23]. The color was measured using a Texflash instrument inclusion or low-temperature fractional crystallization tech-
(Datacolor, International), and Hunter L ( 6 lightness/dark- niques. The urea fractionation of the fatty acids is mainly based
ness), a ( 6 redness/greenness), b ( 6 yellowness/blueness) on the degree of unsaturation, and there is an inverse relation
values were determined. between unsaturation and the formation of urea crystals [14].
All experiments were carried out in triplicate and each By adding a large amount of fatty acids to urea-saturated
parameter was reported as mean 6 standard deviation (SD). methanol at one time, a large amount of urea crystals without
fatty acids would be formed. Yang and Liu [11] suggested that,
2.9 Statistical analysis considering the yield and cost, the ratio of oil sample/urea/
methanol should be 1 : 2 : 5.5. By increasing the amount of
Statistical analysis was performed by Statistical Analysis Sys- urea, however, more desired fatty acids will be lost, whereas in
tem (SAS) software; calculated mean values were compared the presence of more methanol, more fatty acids will be retained
using the Least Significant Difference (LSD) test [24]. in the mother liquor; therefore, a higher proportion of metha-
nol volume was used. In the purification step, the ratio of crude
CLA/urea/methanol was 1 : 2 : 6, and under this condition, the
3 Results and discussion amount of total CLA increased from 73 to 85.5%. It is note-
worthy that, in the purification process, the c9,t11 isomer was
3.1 CLA production and purification enriched only to 3.9%, whereas this value for t10,c12 isomer
was 30.3%. As expected, after this step, saturated fatty acids
The fatty acid profile of the safflower oil showed that the oil along with oleic acid were eliminated from the purified CLA
sample can be categorized as high-LA seed oil that is free of and, conversely, the unsaturated fatty acid concentration (LA
any trace of CLA in its composition (Table 1). Moreover, it and CLA) increased (Table 1).
contains very low amounts of linolenic acid and provides a
good oxidative stability. Kim et al. [9] and Roche-Uribe and
Hernandez [25] used alkali isomerization of safflower oil as a 3.2 TG production
promising method to produce CLA. In this study, CLA
(considering only two major isomers) were produced by alkali In this study, a solvent-free method of interesterification was
isomerization up to ca. 73% of total fatty acids from safflower selected because, when an organic solvent is used, the product
oil, in which the c9,t11 and t10,c12 isomer contents were 35.6 must be bleached and deodorized and the hydrolysis reaction
and 37.2%, respectively. The yield of crude CLA was 80%, is increased [16]. Deacidification with alkaline extraction is a
meaning that from 400 g safflower oil, 320 g crude CLA was very effective approach to reduce FFA [26]. After the deaci-
obtained, in which about 73% of fatty acids were the CLA dification step, the yield of the TG was 40% based on total
isomers (c9,t11 and t10,c12 isomers), indicating a conversion substrate weight; this value might be due to much more soap
rate of 96.5% for LA. As expected, CLA isomers were pro- formed during deacidification of the TG, which tended to
duced mostly from LA isomerization, and the amounts of form a stable emulsion that was not easily separated into hex-
other fatty acids were not changed significantly; later, in the ane and aqueous phases, resulting in more wasted TG.
© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.comEur. J. Lipid Sci. Technol. 2008, 110, 400–404 Enzymatic interesterification of CLA with canola oil 403
Table 2. Physical and chemical characteristics of TG-canola
compared to canola oil.
Properties Canola oil SL-canola
a
Iodine value 106.6 6 0.2 115.0 6 0.2b
Saponification value 195.2 6 2.3a 177.7 6 4.7b
% FFA (based on oleic acid) 0.06a 1.3b
OSI [h] (110 7C) 8a 1b
Color
L (lightness/darkness) 63.7 6 0.002a 65.3 6 0.002a
a (redness/greenness) 0.4 6 0.02a 1.6 6 0.01b
b (yellowness/blueness) 15.5 6 0.003a 23.5 6 0.001b
Refractive index (25 7C) 1.472a 1.473a
Figure 1. Changes in fatty acid composition (mol-%) during acid-
olysis. (r) Palmitic acid, (u) stearic acid, (m) oleic acid, (n) LA, a, b
Values within each row showing a significant difference (p ,0.05).
(d) total CLA.
The oxidative stability index decreased in TG compared to
canola oil; this reduction was due to the removal or oxidation
of tocopherols, which naturally exist as antioxidants in vege-
table oils [19]. Moreover, replacement of oleic acid by CLA
makes the TG more susceptible; therefore, additional anti-
oxidants should be added into the TG to prevent oxidation.
With regard to the physical properties, the higher refrac-
tive index of the TG revealed a higher unsaturation degree of
the fatty acids compared to this value for canola oil. There was
a significant difference between the Hunter a and b values of
canola oil and TG. The TG had higher L, b and a values than
canola oil, indicating that the TG were lighter and more yel-
lowish than canola oil. One reason of the increment to yel-
Figure 2. Contents of CLA isomers esterified during a 48-h reac- lowness in TG may be the oxidation of tocopherols and their
tion. (m) c9,t11, (n) t10,c12. conversion to other compounds that can cause a more yel-
lowish color in TG-canola [19].
As expected, the fatty acid composition of canola oil was
changed after lipase-catalyzed acidolysis with a CLA mixture.
In TG, the major fatty acids in canola oil, namely oleic Acknowledgments
(56 mol-%), linoleic (26 mol-%) and linolenic (9 mol-%)
acids, decreased to about 43, 20 and 3.5 mol-%, respectively, We appreciate Isfahan University of Technology (IUT) for project
whereas the CLA content reached 26.6 mol-% in TG-canola. funding. We also thank Mr. B. Bahrami and Mr. H. Khoshoei for
As anticipated, since the content of the t10,c12 isomer was their technical assistance. The authors wish to thank Mr. H. Ali-
higher in the purified CLA mixture, a higher amount of this zadeh (Novozyme, Tehran, Iran) for kindly providing lipase for the
isomer was incorporated into TG (16.3 mol-%) compared to project.
the c9,t11 isomer (10.3 mol-%) at the end of the esterification
(Figs. 1, 2).
Table 2 presents the physical and chemical characteristics Conflict of interest statement
of TG-canola compared to canola oil. The iodine value,
defined as the number of grams of iodine absorbed by 100 g The authors have declared no conflict of interest.
of sample, was higher whereas the saponification value,
defined as the number of milligrams of potassium hydroxide
needed to saponify 1 g of sample, was lower (177.7) in TG References
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