Research Paper Conjugated linoleic acid (CLA) production and lipasecatalyzed interesterification of purified CLA with canola oil

Transcription

1 400 Eur. J. Lipid Sci. Technol. 2008, 110, Research Paper Conjugated linoleic acid (CLA) production and lipasecatalyzed 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 (acidolysis) 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 /ejlt Introduction Conjugated linoleic acids (CLA) are a group of positional and geometrical isomers of linoleic acid (LA) with a conjugated double bond system. The major natural sources of CLA are fat tissues of ruminants (meat and dairy products). The cis9,- trans11 (c9,t11) isomer is the most abundant natural isomer (about 75 90% of total CLA) which is also called rumenic acid [1]. Studies (in vivo and in vitro) have revealed biological activities of CLA including antioxidative, anticarcinogenic, antiatherosclerotic, antidiabetogenic and antiobesity properties, along with immune-enhancing effects [2 5]. Different methods, such as dehydration of ricinoleic acid [6], photoproduction of CLA [7], alkaline isomerization of LA or LArich oils [8 11], are used to synthesize CLA. Alkaline isomerization of LA is usually used for commercial production of CLA containing two isomers, c9,t11 (43 45%) and t10,c12 (43 45%), accompanied by small amounts of other CLA isomers [5]; however, since the biological activity of the product is due to the presence of both isomers, a purification step would be necessary. Urea-inclusion crystallization has been generally employed to concentrate useful polyunsaturated fatty acids (PUFA) as well as CLA in edible oils [12 15]. Correspondence: Mahdi Kadivar, Department of Food Science and Technology, College of Agriculture, Isfahan University of Technology, Isfahan, , Iran. mak120@mail.usask.ca Fax: Although CLA have several beneficial effects, the consumption of CLA has decreased due to replacement of milk and animal fats by vegetable oils. Enzyme-catalyzed acidolysis is an approach to increase the CLA content in structured lipids (SL). Several researches of enzymatic interesterification of CLA with fats and oils were reported; Garcia et al. [16, 17] prepared SL from butter and fish oils with CLA by enzymatic acidolysis. Ortega et al. [18], using a lipase, incorporated CLA in fully hydrogenated soybean oil; Lee et al. [19, 20] reported the interesterification of CLA with soybean, sunflower and safflower oils. The altered composition of triacylglycerols in SL (incorporation of CLA) provides different changes in physical and chemical characteristics of SL compared to the initial lipid, which possibly improve the functional properties of the oil. The objective of this study was to produce high-purity CLA from safflower oil and the incorporation of this functional ingredient into canola oil to prepare CLA-rich triacylglycerols (TG) by enzymatic interesterification and to compare the TG with the starting lipid with respect to physical and chemical properties. 2 Materials and methods Safflower seed was prepared by Oilseed Research & Development Company (Tehran, Iran). CLA (mixture of c9,t11 and t10,c12 isomers) and other fatty acid standards were

2 Eur. J. Lipid Sci. Technol. 2008, 110, Enzymatic interesterification of CLA with canola oil 401 obtained from Sigma-Aldrich. Chemicals and solvents were all of analytical grade and purchased from Merck. Immobilized lipase from Thermomyces lanuginosus was a gift provided by Novozymes (Tehran, Iran). Refined, bleached and deodorized canola oil was obtained from Naz Vegetable Oils Co. (Isfahan, Iran). 2.1 Extraction of safflower oil The oil was extracted using hexane as solvent. Hexane (1500 ml) was added to 500 g milled seeds and extraction was performed for 24 h at room temperature; this operation was done twice to complete oil extraction from the seeds. The solvent was then removed by rotary vacuum evaporator and the safflower oil was stored in dark container in a refrigerator for the subsequent steps. 2.2 Fatty acid composition of safflower oil To determine the fatty acid profile of safflower oil, the oil was methylated according to AOAC method [21]. Methylated samples (1 ml) were injected into a gas chromatograph (CP9002; Chrompac) equipped with a flame ionization detector (FID) and the fatty acid methyl esters were separated using FFAP-CB fused-silica WCOT (25 m60.32 mm 60.3 mm) and helium gas as a carrier with an inlet pressure of 75 kpa. The temperature program was as follows: increasing from 40 to 100 o C at a rate of 10 7C/min and holding for 0.2 min, then increasing to 240 7C at 25 7C/min and holding for 30 min at 240 7C. The temperatures of the injector and detector were 230 and 250 7C, respectively. 2.3 CLA production CLA were produced as described by Kim et al. [9]. Briefly, 400 g safflower oil (75% LA) was added to 100 g sodium hydroxide (NaOH) dissolved in 320 g propylene glycol (180 7C) in an oil bath. The CLA isomers were formed when the mixture was left to cool to 80 7C for 2 h; phosphoric acid (2 N) was then added to adjust the mixture ph to , and CLA were recovered by extraction with hexane in a separatory funnel. The hexane layer was washed twice with methanol/ water (30%) solution and then evaporated by rotary vacuum evaporator to obtain the CLA. The CLA content was determined by gas chromatography (GC). 2.4 CLA purification The CLA were purified according to Yang and Liu [11] with slight modification. Crude CLA mixture was added gradually to urea dissolved in warmed methanol (70 7C) in a proportion of 1 : 2 : 6 (oil/urea/methanol); then, the solution was allowed to cool to room temperature for 4 h. The well-defined needles of the urea complexes were put into a refrigerator overnight at 5 7C. The purified CLA were recovered by vacuum filtration with membrane filters (0.45 mm) in mother liquor; the mother liquor was transferred to a separatory funnel and acidified to ph 3 with HCl (2 N). The CLA were extracted with hexane (three times) and the hexane layer was washed with deionized water (twice). The washed hexane was dried over anhydrous sodium sulfate and removed by rotary vacuum evaporator; the CLA content was determined by GC and the yield of CLA purification was then calculated. 2.5 CLA content in crude and purified mixtures To determine the CLA content, methylation was done as described by Boylston and Beitz [22] with slight modification. Of borontrifluoride (BF 3, 14% in methanol), 7 ml was added to 300 mg CLA mixture and the mixture was methylated for 30 min at room temperature; 25 ml saturated NaCl solution was added and the mixture was vortexed; then, 6 ml hexane (to extract the methylated fatty acids) was added. The hexane layer was dried with anhydrous sodium sulfate and quantified by GC. Heptadecanoic acid (17:0) was used as an internal standard to determine the amount of CLA. 2.6 Enzymatic interesterification Into a 50-mL Erlenmeyer flask with a screw cap, 10 g of a substrate consisting of 40 : 60 wt/wt of purified CLA mixture to canola oil (molar ratio of CLA isomers to canola oil was equal to 0.57) were weighed. The reaction was started by adding 700 mg of the lipase (7% by total weight of the substrate). The flask containing the reaction mixture was flushed with nitrogen, stoppered and incubated for 48 h in an orbital shaker at 200 rpm and 55 7C. This reaction was also performed in large amounts (100 g purified CLA mixture to 150 g canola oil) to provide sufficient TG for the following experiments Analyses Samples (50 ml) were withdrawn at intervals (every 12 h). Methylation of the fatty acids was carried out according to Ortega et al. [18]. Total fatty acids (free 1 esterified) were determined by methylating the mixture with methanolic HCl (0.2 M), whereas methylation of the esterified fatty acids was conducted by methanolic NaOH (0.1 M). The percentage of esterified CLA was determined by injection of methylated samples (1 ml) into a gas chromatograph, using heptadecanoic acid (17:0) as internal standard. 2.7 Deacidification of TG by alkaline extraction Deacidification of the TG by alkaline extraction was carried out according to Lee et al. [19]. After the incubation time, hexane was added and the reaction mixture was filtered immediately through a 0.45-mm filter to remove the enzyme, and the hexane was removed by rotary vacuum evaporator.

3 402 S. A. H. Goli et al. Eur. J. Lipid Sci. Technol. 2008, 110, The reaction mixture was mixed with 0.5 N KOH solution (20% ethanol, 20 ml) and hexane (40 ml) in a separatory funnel with a stopcock. The upper phase (hexane layer) was collected in a round-bottom flask and 3 4 drops of phenolphthalein solution were added, and the titration with 0.5 N KOH (20% ethanol) was conducted. Then, saturated NaCl (10 ml) solution was vigorously mixed in and the hexane phase was isolated and passed through an anhydrous sodium sulfate column. The hexane was evaporated by rotary vacuum evaporator and the TG were obtained. 2.8 Physical and chemical properties Iodine value (IV), saponification value (SV) and % free fatty acids (FFA) of the TG and canola oil were determined according to AOCS methods Cd 1-25, Cd 3-25 and Cd 3d- 63, respectively. The oxidative stability index (OSI) was determined according to AOCS method Cd 12b-92 using a 743 Rancimat (Metrohm). The refractive index was reported using a refractometer according to AOCS method Cc 7-25 [23]. The color was measured using a Texflash instrument (Datacolor, International), and Hunter L ( 6 lightness/darkness), a ( 6 redness/greenness), b ( 6 yellowness/blueness) values were determined. All experiments were carried out in triplicate and each parameter was reported as mean 6 standard deviation (SD). 2.9 Statistical analysis Statistical analysis was performed by Statistical Analysis System (SAS) software; calculated mean values were compared using the Least Significant Difference (LSD) test [24]. 3 Results and discussion 3.1 CLA production and purification The fatty acid profile of the safflower oil showed that the oil sample can be categorized as high-la seed oil that is free of any trace of CLA in its composition (Table 1). Moreover, it 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 promising method to produce CLA. In this study, CLA (considering only two major isomers) were produced by alkali isomerization up to ca. 73% of total fatty acids from safflower oil, in which the c9,t11 and t10,c12 isomer contents were 35.6 and 37.2%, respectively. The yield of crude CLA was 80%, meaning that from 400 g safflower oil, 320 g crude CLA was obtained, in which about 73% of fatty acids were the CLA isomers (c9,t11 and t10,c12 isomers), indicating a conversion rate of 96.5% for LA. As expected, CLA isomers were produced mostly from LA isomerization, and the amounts of other fatty acids were not changed significantly; later, in the Table 1. Fatty acid compositions of safflower oil, crude CLA and purified CLA. Fatty acids [%] Safflower oil Crude CLA Purified CLA Palmitic acid Stearic acid Oleic acid Linoleic acid Linolenic acid Cis9,trans Trans10,cis Yield [%] The yield of oil mixture, based on the previous step. purification step, about 100 g purified CLA mixture was obtained from 320 g crude CLA, indicating a yield of 35% (Table 1). Concentrates of PUFA are generally prepared by urea inclusion or low-temperature fractional crystallization techniques. The urea fractionation of the fatty acids is mainly based on the degree of unsaturation, and there is an inverse relation between unsaturation and the formation of urea crystals [14]. By adding a large amount of fatty acids to urea-saturated methanol at one time, a large amount of urea crystals without fatty acids would be formed. Yang and Liu [11] suggested that, considering the yield and cost, the ratio of oil sample/urea/ methanol should be 1 : 2 : 5.5. By increasing the amount of urea, however, more desired fatty acids will be lost, whereas in the presence of more methanol, more fatty acids will be retained in the mother liquor; therefore, a higher proportion of methanol volume was used. In the purification step, the ratio of crude CLA/urea/methanol was 1 : 2 : 6, and under this condition, the amount of total CLA increased from 73 to 85.5%. It is noteworthy that, in the purification process, the c9,t11 isomer was enriched only to 3.9%, whereas this value for t10,c12 isomer was 30.3%. As expected, after this step, saturated fatty acids along with oleic acid were eliminated from the purified CLA and, conversely, the unsaturated fatty acid concentration (LA and CLA) increased (Table 1). 3.2 TG production In this study, a solvent-free method of interesterification was selected because, when an organic solvent is used, the product must be bleached and deodorized and the hydrolysis reaction is increased [16]. Deacidification with alkaline extraction is a very effective approach to reduce FFA [26]. After the deacidification step, the yield of the TG was 40% based on total substrate weight; this value might be due to much more soap formed during deacidification of the TG, which tended to form a stable emulsion that was not easily separated into hexane and aqueous phases, resulting in more wasted TG.

4 Eur. J. Lipid Sci. Technol. 2008, 110, 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 Figure 1. Changes in fatty acid composition (mol-%) during acidolysis. (r) Palmitic acid, (u) stearic acid, (m) oleic acid, (n) LA, (d) total CLA. Figure 2. Contents of CLA isomers esterified during a 48-h reaction. (m) c9,t11, (n) t10,c12. 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 (56 mol-%), linoleic (26 mol-%) and linolenic (9 mol-%) acids, decreased to about 43, 20 and 3.5 mol-%, respectively, whereas the CLA content reached 26.6 mol-% in TG-canola. As anticipated, since the content of the t10,c12 isomer was higher in the purified CLA mixture, a higher amount of this isomer was incorporated into TG (16.3 mol-%) compared to the c9,t11 isomer (10.3 mol-%) at the end of the esterification (Figs. 1, 2). Table 2 presents the physical and chemical characteristics of TG-canola compared to canola oil. The iodine value, defined as the number of grams of iodine absorbed by 100 g 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 compared to canola oil. These changes were expected due to the replacement of oleic acid by CLA. Due to excess amounts of fatty acids used as substrate, TG-canola should show a high FFA value. The FFA percentage of the TG indicated that the alkaline deacidification procedure successfully removed most of the FFA, which were unreacted CLA or fatty acids hydrolyzed from the canola oil. Iodine value a b Saponification value a b % FFA (based on oleic acid) 0.06 a 1.3 b OSI [h] (110 7C) 8 a 1 b Color L (lightness/darkness) a a a (redness/greenness) a b b (yellowness/blueness) a b Refractive index (25 7C) a a a, b Values within each row showing a significant difference (p,0.05). 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 vegetable oils [19]. Moreover, replacement of oleic acid by CLA makes the TG more susceptible; therefore, additional antioxidants should be added into the TG to prevent oxidation. With regard to the physical properties, the higher refractive 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 yellowish than canola oil. One reason of the increment to yellowness in TG may be the oxidation of tocopherols and their conversion to other compounds that can cause a more yellowish color in TG-canola [19]. Acknowledgments We appreciate Isfahan University of Technology (IUT) for project funding. We also thank Mr. B. Bahrami and Mr. H. Khoshoei for their technical assistance. The authors wish to thank Mr. H. Alizadeh (Novozyme, Tehran, Iran) for kindly providing lipase for the project. Conflict of interest statement The authors have declared no conflict of interest. References [1] S. Gnadig, Y. Xue, O. Berdeaux, J. M. Cheardigni, J. L. Sebedio: Conjugated linoleic acid (CLA) as a functional ingredient. In: Functional Dairy Products. Eds. T. M. Saadholm, M. Saarela, CRC Press, Cambrige (UK) 2003, pp [2] N. L. Flintoff-Dye, S. T. Omaye: Antioxidant effects of conjugated linoleic acid isomers human low-density lipoproteins. Nutr Res. 2005, 25, 1 12.

5 404 S. A. H. Goli et al. Eur. J. Lipid Sci. Technol. 2008, 110, [3] G. S. Kelly: Conjugated linoleic acid: A review. Alter Med Rev. 2001, 6, [4] K. W. J. Wahle, S. D. Heys, D. Rotondo: Conjugated linoleic acids: Are they beneficial or detrimental to health? Prog Lipid Res. 2004, 43, [5] Y. W. Wang, P. J. H. Jones: Conjugated linoleic acid and obesity control: Efficacy and mechanisms. Int J Obesity. 2004, 28, [6] L. Yang, Y. Huang, H. Q. Wang, Z. Y. Chen: Production of conjugated linoleic acids through KOH-catalyzed dehydration of ricinoleic acid. Chem Phys Lipids. 2002, 119, [7] R. R. Gangidi, A. Proctor: Photochemical production of conjugated linoleic acid from soybean oil. Lipids. 2004, 39, [8] O. Berdeaux, L. Voinot, E. Angioni, P. Juaneda, J. L. Sebedio: A simple method of preparation of methyl trans-10,cis-12 and cis-9,trans-11 octadienoates from methyl linoeate. J Am Oil Chem Soc. 1998, 75, [9] Y. J. Kim, K. W. Lee, H. Kim, H. J. Lee: The production of high-purity conjugated linoleic acid (CLA) using two-step urea-inclusion crystallization and hydrophilic arginine-cla complex. J Food Sci. 2003, 68, [10] D. W. L. Ma, A. A. Wierzbicki, C. J. Field, M. T. Clandinin: Preparation of conjugated linoleic acid from safflower oil. J Am Oil Chem Soc. 1999, 76, [11] T. S. Yang, T. T. Liu: Optimization of production of conjugated linoleic acid from soybean oil. J Agric Food Chem. 2004, 52, [12] D. G. Hayes: Effect of temperature programming on the performance of urea inclusion compound-based free fatty acid fractionation. J Am Oil Chem Soc. 2006, 83, [13] Y. J. Kim, R. H. Liu: Selective increase in conjugated linoleic acid in milk fat by crystallization. J Food Sci. 1999, 64, [14] A. R. Medina, A. G. Gimenez, F. G. Camacho, J. A. Sanchez Perez, E. M. Grima, A. C. Gomez: Concentration and purification of stearidonic, eicosapentaenoic, and docosahexaenoic acids from cod liver oil and the marine microalga Isochrysis galbana. J Am Oil Chem Soc. 1995, 72, [15] U. N. Wanasundara, F. Shahidi: Concentration of omega 3- polyunsaturated fatty acids of seal blubber oil by urea complexation: Optimization of reaction conditions. Food Chem. 1999, 65, [16] H. S. Garcia, J. M. Storkson, M. W. Pariza, C. G. Hill: Enrichment of butteroil with conjugated linoleic acid via enzymatic interesterification (acidolysis) reactions. Biotechnol Lett. 1998, 20, [17] H. S. Garcia, J. A. Arcos, D. J. Ward, C. G. Hill: Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil. Biotechnol Bioeng. 2000, 70, [18] J. Ortega, A. Lopez-Hernandez, H. S. Garcia, C. G. Hill: Lipase-mediated acidolysis of fully hydrogenated soybean oil with conjugated linoleic acid. J Food Sci. 2004, 69, 1 6. [19] J. H. Lee, M. R. Kim, H. R. Kim, I. H. Kim, K. T. Lee: Characterization of lipase-catalyzed structured lipids from selected vegetable oils with conjugated linoleic acid: Their oxidative stability with rosemary extracts. J Food Sci. 2003, 68, [20] J. H. Lee, J. A. Shin, J. H. Lee, K. T. Lee: Production of lipasecatalyzed structured lipids from safflower oil with conjugated linoleic acid and oxidation studies with rosemary extracts. Food Res Int. 2004, 37, [21] AOAC: Official Methods of Analysis of AOAC International.17 th Edn. AOAC, Gaithersburg, MD (USA) [22] T. D. Boylston, D. C. Beitz: Conjugated linoleic acid and fatty acid composition of yogurt produced from milk of cows fed soy oil and conjugated linoleic acid. J Food Sci. 2002, 67, [23] AOCS: Official Methods and Recommended Practices of the AOCS. 5 th Edn. AOCS Press, Champaign, IL (USA) [24] SAS Institute: SAS/STAT User s Guide. Statistical Analysis Systems Institute, Inc., Cary, NC (USA) [25] A. Roche-Uribe, E. Hernandez: Solvent-free enzymatic synthesis of structured lipids containing CLA from coconut oil and tricaprylin. J Am Oil Chem Soc. 2004, 81, [26] K. L. Lee, C. C. Akoh: Characterization of enzymatically synthesized structured lipids containing eicosapentaenoic, docosahexanoic, and caprylic acids. J Am Oil Chem Soc. 1998, 75,