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A study was conducted to investigate the short-term safety of dietary conjugated linoleic acid (CLA) in overweight Japanese male volunteers. The study design was a randomized, double-blind placebo-controlled trial including 60 healthy overweight volunteers (body mass index (BMI), 25 approximately 35 kg/m(2)). The subjects were randomly assigned to three groups: 5.4 g CLA-triacylglycerol (3.4 g as CLA), 10.8 g CLA-triacylglycerol (6.8 g as CLA) and placebo (10.8 g safflower oil) daily for 12 weeks. The CLA-triaclyglycerol contained 9c,11t- and 10t,12c-isomers at an equal proportion. The safety was evaluated by analyses of blood parameters and by clinical examinations at the baseline, and at 6 and 12 weeks, including vital signs and adverse effects. All subjects completed the study. The occurrence of adverse events tended to be higher in the CLA groups than in the placebo group, but all of the adverse events were mild to moderate, within normal ranges, and temporary. Serum aspartate aminotransferase (AST) activity did not differ significantly between the groups at 12 weeks, but in the high CLA group it was slightly increased from the baseline. Serum alanine aminotransferase (ALT) activity was higher in the high CLA group than in the placebo group after 12 weeks and was higher than at the baseline in both CLA groups. However, statistical analysis of the population of apparently healthy volunteers who had normal blood parameters at the baseline revealed that AST and ALT levels did not differ significantly among the 3 groups at 12 weeks. Moreover, no clinically significant changes in vital signs were observed in any of the groups. These results indicate that CLA at a dose of 3.4 g/day is a safe dietary level in healthy Japanese populations in terms of the parameters examined.

Commercially available preparations of CLA are composed of almost equal amounts of 9-cis,11-trans (9c,11t)-CLA and 10-trans,12-cis (10t,12c)-CLA. Each isomer was fractionated and enriched, for availability as a food supplement, by a process comprising selective esterification with l-menthol by Candida rugosa lipase, distillation, and n-hexane extraction. The first selective esterification of CLA isomers was conducted with an equimolar amount of l-menthol of 30°C. The oil phase of the reaction mixture was fractionated into an l-menthyl ester fraction (9c,11t-CLA rich) and an FFA fraction (10t,12c-CLA rich) by distillation. The FFA fraction was esterified again with an equimolar amount of l-menthol to enrich 10t,12c-CLA. The 10t,12c-CLA preparation was obtained as the resulting FFA fraction by distillation. 10t,12c-CLA was enriched to 91% with 40% recovery. To enrich 9c,11t-CLA, the l-menthyl ester fraction in the first esterification was chemically hydrolyzed, and the resulting FFA were esterified again with an equimolar amount of l-menthol. The 9c, 11t-CLA preparation was obtained by chemical hydrolysis of the resulting l-methyl ester fraction, followed by n-hexane extraction. 9c,11t-CLA was enriched to 94% with 42% recovery. This effective process for purification of CLA isomers using l-methol is applicable to the production of food supplements.

We have developed an efficient esterification for the synthesis of triacylglycerol (TAG) containing conjugated linoleic acids (CLA) using a blend of two powdered lipases. Two pairs of blended lipases promoted the esterification. Rhizomucor miehei lipase, plus Alcaligenes sp. lipase and Penicillium cammembertii MAG and DAG lipase plus Alcaligenes sp. lipase were used. At the optmal ratio of two lipases, the content of TAG containing CLA (TAG-CLA) in all glycerols reached 82–83% after 47 h using 1 wt% of lipases. With R. miehei lipase plus Alcaligenes sp. lipase, the reaction time to obtain ca. 60% of TAG-CLA was one-third of that needed with R. miehei lipase alone. The optimal ratio of two lipases differed between these two pairs. The optimal ratio was 70–80 wt% of R. miehei lipase in the last stage of the reaction, whereas it was over a wide range of 10–90 wt% for P. camembertii lipase. In the blend of R. miehei lipase plus Alcaligenes sp. lipase, activity remained very high after 10 cycles of esterification (every 47 h) and could be used in the industrial production of TAG-CLA.

Production of MAG with CLA using Penicillium camembertii mono- and diacylglycerol lipase (referred to as lipase) was attempted for the purpose of expanding the application of CLA. The commercial product of CLA (referred to as FFA-CLA) is a FFA mixture containing almost equal amounts of 9cis,11trans (9c,11t)-CLA and 10t,12c-CLA. Esterification of FFA-CLA with glycerol without dehydration achieved 84% esterification but produced almost equal amounts of MAG and DAG. Esterification with dehydration not only achieved a high degree of esterification but also suppressed the formation of DAG. When a mixture of FFA-CLA/glycerol (1∶2, mol/mol), 1% water, and 200 units/g-mixture of P. camembertii lipase was agitated at 30°C for 72 h with dehydration at 5 mm Hg, the degree of esterification reached 95% and the contents of MAG and DAG were 90 and 6 wt%, respectively. This reaction system may be applied to the industrial production of MAG with unstable CLA.

Polyglycerol fatty acid monoesters are used widely as food emulsifiers, and conjugated linoleic acid (CLA) has various physiological activities. Hence, production of polyglycerol CLA monoester was attempted through a lipase-catalyzed reaction. A commercially available free fatty acid mixture (referred to as FFA-CLA) containing 9cis,11trans (9c,11t)-CLA and 10t,12c-CLA was used as a source of CLA, and its esterification with diglycerol was studied. Screening of several lipases showed that Penicillium camembertii lipase was suitable for production of diglycerol fatty acid monoesters (DGFAMEs). When a mixture of FFA-CLA/diglycerol (1:2, mol/mol), 100 U/g-mixture of the lipase, and 2% water was agitated at 30 °C for 24 h with dehydration at 5 mm Hg using a vacuum pump, the degree of esterification reached 93%, and the ratio of DGFAMEs/diglycerol fatty acid diesters was 1.9:1 (wt/wt). The synthesized DGFAMEs were purified to 97% in a 69% yield by short-path distillation. This process comprising lipase-catalyzed esterification and short-path distillation may be effective for industrial production of an emulsifier for use in nutraceutical applications.

Conjugated linoleic acid (CLA) has various physiological activities, and a commercial product is a mixture of free fatty acids (named FFA-CLA) which contains almost equal amounts of 9-cis, 11-trans (9c,11t) and 10t,12c isomers. We attempted to efficiently produce monoacylglycerol (MAG) of CLA by lipase-catalyzed esterification. Study on effect of reaction temperature clarified that synthesis of diacylglycerols (DAGs) from MAGs was repressed at low temperature. When FFA-CLA was esterified at 5 °C with 5 molar equivalents of glycerol using 200 U/g mixture of Penicillium camembertii lipase in the presence of 2% water, the degree of esterification reached 89.6% after 45 h and the contents of MAGs and DAGs were 87.0 and 4.5 wt.%, respectively. Triacylglycerols were not synthesized in this Penicillium lipase-catalyzed esterification. After the esterification was conducted for 20 h (the degree of esterification, 80.8%), dehydration was started by evaporation at 5 mmHg using a vacuum pump. The degree of esterification increased concomitantly with dehydration and reached 94.5% after 16 h (36 h in total). The contents of MAGs (main components, 1(3)-isomers) and DAGs were 92.7 and 2.9 wt.%, respectively. Fatty acid compositions in MAGs synthesized with and without dehydration were the same as that in FFA-CLA. These results showed that the esterification system with dehydration is effective for producing MAGs in a high yield.

We attempted to produce MAG of CLA through lipase-catalyzed esterification of a FFA mixture containing CLA (referred to as FFA-CLA) with glycerol. Screening of lipases showed that MAG-CLA was produced efficiently at 5°C with Penicillium camembertii, Rhizopus oryzae, and Candida rugosa lipases. Among them, C. rugosa lipase was selected because the lipase is widely used as a catalyst for oils and fats processing. The reaction was conducted with agitation of a 300-g mixture of FFA-CLA/glycerol (1∶5, mol/mol), a 200-U/g mixture of C. rugosa lipase, and 2% water. When the reaction was conducted at 30°C, the esterification scarcely proceeded, owing to inhibition of the reaction by glycerol. But the reaction at 5°C eliminated the inhibition and produced MAG efficiently: The degree of esterification reached 93.8% after 58 h, and MAG content in the reaction mixture was 88.4 wt%. To reduce the reaction time, the reactor was connected with a vacuum pump after 24 h, and the reaction was continued with dehydration at 5 mm Hg. The degree of esterification reached 94.7% after 24 h of dehydration (48 h in total), and MAG content increased to 93.0 wt%. Candida rugosa lipase acted a little more strongly on cis-9, trans-11 CLA than on trans-10,cis-12 CLA, but the contents of the two isomers in MAG obtained from a 48-h reaction were the same as the contents in FFA-CLA.

The autoxidation processes of the cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) isomers of CLA were separately observed at ca. 0% RH and different temperatures. The t10,c12 CLA oxidized faster than the c9,t11 isomer at all tested temperatures. The first half of the oxidation process of t10,c12 CLA obeyed an autocatalytic-type rate expression, but the latter half followed first-order kinetics. On the other hand, the entire oxidation process of c9,t11 CLA could be expressed by the autocatalytic-type rate expression. The apparent activation energies and frequency factors for the autoxidation of the isomers were estimated from the rate constants obtained at various temperatures based on the Arrhenius equation. The apparent activation energies for the CLA isomers were greater than those for the nonconjugated n−6 and n−3 PUFA or their esters. However, the enthalpyentropy compensation held during the autoxidation of both the CLA and PUFA. This suggested that the autoxidation mechanisms for the CLA and PUFA were essentially the same.

It was attempted to prepare cis-9, trans-11 conjugated linoleic acid (c9,t11-CLA) and t10,c12-CLA concentrates that can be used as foods. A free fatty acid mixture (FFA-CLA) containing almost equal amounts of c9,t11- and t10,c12-CLAs was esterified with glycerol using immobilized Rhizomucor miehei lipase, and the resulting acylglycerols (Gly-CLA) were purified by molecular distillation. Contents of c9,t11- and t10,c12-CLAs in Gly-CLA were the same as those in FFA-CLA: c9,t11-CLA, 33.7 wt%; t10,c12-CLA, 34.5 wt%. Gly-CLA was first hydrolyzed with an equal weight of water and 1.0 U/g-mixture of Candida rugosa lipase, and c9,t11-CLA-rich FFAs were prepared by molecular distillation: purity of c9,t11-CLA based on the total content of c9,t11- and t10,c12-CLAs, 72.9%. Meanwhile, purity of t10,c12-CLA in acylglycerols was 65.0%. To further increase the purity, the acylglycerols were hydrolyzed again with 15 U/g-mixture of C. rugosa lipase, resulting in enrichment of t10,c12-CLA in acylglycerols (purity of t10,c12-CLA, 80.4%). Non-selective hydrolysis of t10,c12-CLA-rich acylglycerols with 200 U/g-mixture of C. rugosa lipase produced t10,c12-CLA-rich FFAs (purity of t10,c12-CLA, 81.5%). In addition, c9,t11-CLA-rich FFAs were successfully esterified with glycerol using immobilized R. miehei lipase, and c9,t11-CLA-rich acylglycerols can be synthesized (purity of c9,t11-CLA, 73.0%). The process was composed of reactions with C. rugosa and R. miehei lipases, which can be used for production of foods, and molecular distillation. Hence, the c9,t11- and t10,c12-CLA concentrates can be used as foods.

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.

A commercial product of CLA contains almost equal amounts of cis-9,trans-11 (c9,t11)-CLA and trans-10,cis-12 (t10,c12)-CLA. We attempted to enrich the two isomers by a two-step selective esterification using Candida rugosa lipase that acted on c9,t11-CLA more strongly than on t10,c12-CLA. An FFA mixture containing CLA isomers was esterified with an equimolar amount of lauryl alcohol in a mixture of 20% water and the lipase. When the esterification of total FA reached 50%, two isomers were fractionated in a good yield: t10,c12-CLA was enriched in FFA, and c9,t11-CLA was recovered in lauryl esters. The FFA were esterified again to enrich t10,c12-CLA. At 27.3% esterification of total FA, the t10,c12-CLA content in FFA increased to 64.8 wt% with 89.3% recovery: The ratio of the content of t10,c12-CLA to that of two isomers was 95.9%. Lauryl esters obtained by the single esterification were employed for enrichment of c9,t11-CLA. After the esters were hydrolyzed, the resulting FFA were esterified again with lauryl alcohol. At 62.0% esterification of total FA, the c9,t11-CLA content in lauryl esters increased to 73.3 wt% with 79.4% recovery: The ratio of the content of c9,t11-CLA to that of two isomers was 95.6%. In a 600-g-scale purification, molecular distillation was effective in separating the reaction mixture into lauryl alcohol, FFA, and lauryl ester fractions.

Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.

We have recently shown that alpha-eleostearic acid (alpha-ESA), a conjugated linolenic acid, has a stronger antitumor effect than conjugated linoleic acid (CLA), both in vitro and in vivo. In this study, the oxidative stability of alpha-ESA was examined compared with linoleic acid (LA), alpha-linolenic acid (LnA), and CLA. Thin layers of the FA (LA, 9Z,11 E-CLA, 10E,12Z-CLA, LnA, and alpha-ESA) were auto-oxidized at 37 degrees C, and the FA remaining, the absorbed oxygen volume, the lipid hydroperoxide content, and the TBARS content were determined. The oxidation rate of alpha-ESA was faster than that of the unconjugated FA and CLA (9Z, 11 E-CLA and 10E, 12Z-CLA). However, the lipid hydroperoxide and TBARS contents following alpha-ESA oxidation were low, suggesting production of only small amounts of rapid-reacting secondary oxidation products. Furthermore, the oxidative stability of conjugated FA (CLA and CLnA) in which the carboxylic acid group was esterified with triacylglycerol was greater than that of the FFA. Addition of an antioxidant (alpha-tocopherol) also increased the stability of the conjugated FA to a level similar to that of the unconjugated FA.

We evaluated the effect of cis-9, trans-11 (9c,11t) and trans-10, cis-12 (10t,12c) conjugated linoleic acid (CLA) on the immune system in C57BL/6J mice. Mice were fed experimental diets containing 0% CLA (controls), 1% 9c,11t-CLA, 1% 10t,12c-CLA or a 1:1 mixture (0.5% + 0.5%) of these two CLA isomers for 3 wk. Relative spleen weights of all CLA fed mice were greater than the controls. Spleen lymphocytes isolated from the mice fed 10t,12c-CLA produced more immunoglobulin (Ig)A and IgM but not IgG when stimulated with concanavalin A (ConA) compared with controls. IgA production from unstimulated spleen lymphocytes was greater in the 10t, 12c-CLA group than in controls. Conversely, 9c,11t-CLA did not affect the production of any of the Ig subclasses. Lymphocytes isolated from 9c,11t-CLA fed mice produced more tumor necrosis factor-alpha than the control group. The proportion of B cells in the spleen lymphocyte population was significantly lower in the 9c,11t-CLA group, and higher in the 10t,12c-CLA group than in the controls. Compared with the control group, the percentage of CD4(+) T cells was lower in the 10t,12c-CLA group, and the percentage of CD8(+) T cells was higher in the 9c,11t-CLA group. Furthermore, the percentage of CD8(+) T cells was higher in the 1:1 mixture group than in controls. The CD4(+)/CD8(+) ratio was lower in the 1:1 mixture group than in controls. These results suggest that 9c,11t and 10t,12c-CLA can stimulate different immunological effects and that the simultaneous intake of the two isomers can change the T cell population.