Conjugated Linoleic Acid Methyl Ester Research Articles

Efficacy of methyl ester of conjugated linoleic acid (t10,c12 isomer) for sows and cows for reproduction.

A mixture of methylated conjugated linoleic acid (CLA) isomers (t10,c12 and c9,t11) in equal proportions is the subject of this assessment. The active substance is considered to be CLA (t10,c12) methyl ester (ME). The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) of EFSA previously issued an opinion on the safety and efficacy of the product, in which it could not conclude on the efficacy of this additive for sows for reproduction and for cows for reproduction. The European Commission asked EFSA to deliver an opinion on the efficacy of this additive for sows and cows for reproduction, based on additional data submitted by the applicant. The FEEDAP Panel has performed the assessment of the new data following an approach in line with the principles laid down in Regulation (EC) No 429/2008 and the relevant guidance documents. In relation to the data on efficacy in sows for reproduction, owing to methodological shortcomings of the study submitted, including the duration of the study and the limited biological relevance of the effect observed, the FEEDAP Panel cannot conclude on the efficacy of CLA (t10,c12)‐ME for sows for reproduction. The data related to dairy cows indicate that dietary CLA (t10,c12)‐ME supplementation in the late dry period and/or lactation period showed an increase of the probability of pregnancy and a reduction of time to conception in the same reproductive cycle. However, considering that the minimum duration of efficacy studies for reproductive parameters is of at least two reproductive cycles, the FEEDAP Panel is not in a position to conclude on the efficacy of the additive for cows for reproduction.

Response Surface Optimization of a Solvent Free Production of <i>cis</i>-9, <i>trans</i>-11 and <i>trans</i>-10, <i>cis</i>-12 Isomers of Conjugated Linoleic Acid Using High Linoleic Sunflower Oil

The most dominant and beneficial conjugated linoleic acids isomers (CLAs) with miscellaneous biological tasks are 9c11t-C18:2 and 10t12c-C18:2. The problem with most of the commercial CLA produced by non-optimized conventional approaches is the heterogeneity of their isomers and undesirable toxic by-products. In this study, optimization of the isomerization of the fatty acid methyl esters of the high linoleic sunflower oil was investigated through response surface methodology (RSM). The reaction temperature, the concentration of PEG400 and NaOCH3 had positive influence on the total conjugated linoleic acid methyl esters (CLAME) production as response. However, the effect of the polyethylene glycol 400 (PEG400) concentration was more significant on response than those of other factors (p i.e., based on the mass percentage of total fatty acid methyl esters mixture) was at 5% w/w NaOCH3, 1.06% w/w PEG400 and temperature of 140°C.

Rhodium‐catalysed cyclisation by addition of unsaturated 1,4‐dicarbonyl compounds to oleic acid methyl ester

The Rh‐catalysis allows the one‐step synthesis of Diels–Alder‐like cycloadducts from mono‐unsaturated fatty acid and 1,4‐dicarbonyl components. In this article we report on the addition of maleic acid‐, fumaric acid‐ and acetylene dicarboxylic acid dimethyl ester to oleic acid methyl ester. In previous studies, Rh(OAc)2 showed the highest selectivity of the investigated catalysts towards the 3,6‐disubstituted‐1,2,3,6‐tetrahydro‐phthalic acid derivatives (THPA) for the addition of maleic anhydride to oleic acid. Thus, it was also used in this study. The relative configurations of the final products were identified by comparison with the Diels–Alder products, resulting from conjugated linoleic acid methylester (CLAMe) and the mentioned 1,4‐dicarbonyl compounds.Practical applications: The Rh‐catalysis is an appropriate method to synthesise Diels–Alder‐like cycloadducts from mono‐unsaturated fatty acids derivatives and 1,4‐dicarbonyl compounds in a one‐step process. The resulting products are considered to be used as detergents, corrosion inhibitors, plasticiser or new monomeric building blocks, e.g. for polycondensation processes. The obtained polyesters can be used for several applications, e.g. for coatings or adhesives, and lead to new products with promising properties.Diels–Alder‐like cycloadducts can be obtained in a one‐pot reaction from mono‐unsaturated fatty acids and 1,4‐dicarbonyl compounds using Rh‐catalysts. In contrast to common Diels–Alder reactions, a conjugated diene is not mandatory as educt, but will be generated in situ from the mono‐unsaturated fatty acid compound. The configurations of the resulting cyclocadducts are determined by NMR‐measurements.

Optical fibre-based methodology for screening the effect of probiotic bacteria on conjugated linoleic acid (CLA) in curdled milk

A methodology based on optical fibre (OF) detection was developed for screening the potential of CLA production by Lactobacillus casei-01, Lactobacillus acidophilus La-5 and Bifidobacterium lactis B94 in probiotic curdled milk. The OF based methodology was validated by comparison with an analytical method based on gas chromatography–mass spectrometry (GC–MS) and it showed comparable linearity (between 5 and 130μg), accuracy and detection limits, which ranged from 1.92 to 2.56μg for CLA methyl ester and oleic acid methyl ester, respectively. Furthermore, the proposed OF based methodology was an advantageous analytical methodology for screening CLA production in probiotic curled milk, due to its compact design and effective cost of analysis.The application of the OF analytical method allowed to detect CLA in the probiotic curdled milk; the production of CLA by probiotic enzymes reached values of 297±19μgg−1 by 60days of ripening unfolding this potential functional food as very interesting from a technological point of view.

Effect of calcium salts of a mixture of conjugated linoleic acids containingtrans-10,cis-12 in the diet on milk fat synthesis in goats

Dietary supplements of conjugated linoleic acid (CLA) containing trans-10, cis-12 CLA decrease milk fat secretion in the lactating cow and sheep, but their effects on mammary lipogenesis in the goat are less well defined. Eight lactating goats were used in two 4 x 4 Latin-square experiments with 14 d experimental periods to examine the effects of calcium salts of CLA methyl esters (CaCLA) containing trans-10, cis-12 on milk fat synthesis. Experimental treatments consisted of incremental inclusion of 0, 30, 60 or 90 g of CaCLA/d (corresponding to 7.47, 14.9 and 22.4 g/d of trans-10, cis-12 CLA) offered during the first 10 d of each experimental period that replaced maize grain in concentrates (Experiment 1) or calcium salts of palm oil fatty acids (Experiment 2). Relative to the control, inclusion of 30, 60 or 90 g CaCLA/d in the diet reduced milk fat yield by 19.8, 27.9 and 32.3 % and 17.5, 39.0 and 49.3 % in Experiments 1 and 2, respectively. Decreases in milk fat were due to reductions in the secretion of fatty acids synthesised de novo rather than the uptake of fatty acids from the peripheral circulation. Indirect comparisons with the studies in the lactating cow indicated a lower efficacy of CaCLA supplements on mammary lipogenesis in the goat. In conclusion, CaCLA in the diet inhibits milk fat synthesis in the goat, responses that are dependent on the supply of dietary fatty acids, with evidence that the caprine is less sensitive to the anti-lipogenic effects of trans-10, cis-12 CLA compared with the bovine or ovine.

NMR properties of conjugated linoleic acid (CLA) methyl ester hydroperoxides

NMR data on lipid hydroperoxides is scarce. In this study, hydroperoxides were produced from methyl 9- cis,11- trans-octadecadienoate and from methyl 10- trans,12- cis-octadecadienoate by autoxidation in the presence of 20% of α-tocopherol. Ten different hydroperoxides were isolated from the autoxidation mixtures of the two conjugated linoleic acid (CLA) methyl esters by SPE and HPLC. The assignment of the 1H and 13C NMR spectra of these hydroperoxides was accomplished by 2D NMR experiments and by spectral simulations. Substitution of a hydroperoxyl group at the allylic position in CLA methyl esters induced a 53.93 ppm downfield shift on the hydroperoxyl-bearing carbon resonance. The effects on the olefinic α, β, γ, and δ carbon resonances were −3.45, +4.96, −1.22, and +4.42 ppm, respectively. Furthermore, the solvent effects of deuterochloroform, deuteroacetone, and deuterobenzene on the 13C resonances of the hydroperoxides suggest that deuterochloroform is the appropriate solvent for 13C NMR studies on mixtures of lipid hydroperoxides.

Production of trans-10, cis-12 Conjugated Linoleic Acid in Rice

Conjugated linoleic acid (CLA) has anti-carcinogenic and anti-atherosclerosis activity, and modulatory effects on the immune system and lipid metabolism. To produce a transgenic rice plant that can accumulate CLA, a linoleate isomerase gene that can convert linoleic acid to trans-10, cis-12 CLA was introduced and expressed under the control of seed-specific promoters from the oleosin and globulin genes. The fatty acid composition of the transgenic rice grain was analyzed by gas chromatography. Although there was no clear difference in the fatty acid composition between seeds from transformed versus untransformed plants, a peak of trans-10, cis-12 CLA methyl ester, which was not present in seeds from untransformed plants, was found in transformed plants. The trans-10, cis-12 CLA comprised an average of 1.3% (w/w) of the total fatty acids in seeds carrying the oleosin promoter in comparison to 0.01% (w/w) in seeds carrying the globulin promoter. In addition, approximately 70 and 28% of the total amount of the CLA isomer were present in the triacylglycerol and free fatty acid fractions, respectively. These results demonstrate the ability to produce fatty acid components of vegetable oils with novel physiological activities in crops.

π-Complex formation of conjugated linoleic acid with iron

Conjugated linoleic acid (CLA) is known to have several beneficial biological effects in animal models, including anticarcinogenic and antiatherosclerotic effects, antiobesity, and antioxidant activity. However, reports of its antioxidant activities have been inconsistent. In this study, we investigated the possible occurrence of π-bonding between CLA and iron. CLA methyl ester was reacted with triiron dodecacarbonyl and confirmed to form π-complexes with iron tricarbonyl. This study may suggest the possible involvement of CLA in oxidation by way of interacting with iron.

Fatty acid composition and productive traits of broiler fed diets containing conjugated linoleic acid

An experiment was carried out to evaluate the transfer of dietary CLA to broiler chicken tissues (breast, drumstick meat, skin, and abdominal fat) and its effect on productive traits and on carcass yields of birds. Cobb 500 females (n=360), divided into three groups, received from 22 d to slaughtering age (47 d) a grower diet supplemented with 2% conjugated linoleic acid (CLA) source containing 60% CLA methyl esters (CLA2) or 4% CLA source (CLA4). The control group had no supplementation. The addition of CLA source to chicken diet decreased the content of monounsaturated fatty acid (MUFA) (oleic and palmitoleic acids) in breast and drumstick meat. The deposition of CLA in muscles significantly increased as the dietary CLA increased, whereas only little amounts of CLA were detected in the control group. Arachidonic acid (ARA) content was significantly depressed and linearly related to the addition of CLA to the chicken diet. Other non-CLA polyunsaturated fatty acids (PUFA) were little affected by the dietary CLA supplementation. Saturated fatty acids (myristic and stearic acids) significantly increased about 30% in abdominal fat pad of both treated groups enhancing the firmness of abdominal fat. Productive performances-as well as carcass yields-were similar across dietary treatment of birds.

Preparation of a t,t conjugated linoleic acid methylester (CLA-Me) isomers mixture from synthetic CLA by methylation with BF3/methanol.

Mixtures of t,t conjugated linoleic acid methylester (t,t CLA-Me) isomers were prepared from synthetic CLA, consisting of 47.8% t10,c12 CLA; 45.5% c9,t11 CLA; 2.0% t,t CLA; and 4.7% others, by methylation with BF(3)/methanol (designated TT-TC/CT) in conjunction with purification at -68 degrees C for 24 h. The amount or composition of the TT-TC/CT was greatly affected by the concentration of BF(3) in methanol and the duration of methylation. The methylation of 50 mg of synthetic CLA for 30 min with 1 mL of 7.0% BF(3)/methanol produced a TT-TC/CT (21.54 mg) with the composition of 1.3% t12,t14; 5.9% t11,t13; 42.7% t10,t12; 44.0% t9,t11; 5.0% t8,t10; and 1.1% t 7,t9 CLA, whereas the methylation for 60 min with 14.0% BF(3)/methanol produced a TT-TC/CT (28.62 mg) with the composition of t,t CLA isomers different from that of TT-TC/CT by methylation for 30 min with 7.0% BF(3)/methanol. A large quantity of TT-TC/CT (14.15 g) with the composition similar to that of TT-TC/CT prepared from 50 mg of synthetic CLA was also prepared from 25 g of synthetic CLA. The purity of TT-TC/CT samples was greater than 98%. These results suggest that TT-TC/CT with a purity greater than 98% was easily prepared from synthetic CLA by BF(3)-catalyzed methylation, and the amount and composition of t,t CLA isomers of TT-TC/CT samples could be controlled by methylation conditions.

Screening of Conjugated Linoleic Acid Producing Lactic Acid Bacteria from Fecal Samples of Healthy Babies

This study was carried out to obtain conjugated linoleic acid (CLA) producing lactic acid bacteria for further study on the enzymes related to the production of CLA which has gained considerable attention and on the development as a probiotic culture. Total 34 lactic acid bacteria were isolated from 19 feces samples of healthy babies. CLA forming ability was measured spectrophotometrically by the modification of linoleate 12-cis, 11-trans-isomerase activity measuring method, and CLA of the cultures were extracted, methylated, and examined by HPLC analysis. CLA methyl ester of only one culture showing the highest value of CLA forming ability could be detected by HPLC analysis. The culture was found to be Gram positive, rods and catalase negative. It grows at 45°C but not at 15°C, and was identified to be Lactobacillus fermentum on the basis of the biochemical characteristics and the utilization of substrates. These results provide an efficient experimental method to screen CLA producing lactic acid bacteria. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 7 : 1031-1035)

Hydroperoxide formation during autoxidation of conjugated linoleic acid methyl ester

The aim of this study was to investigate whether hydroperoxides are formed in the autoxidation of conjugated linoleic acid (CLA) methyl ester both in the presence and absence of α-tocopherol. The existence of hydroperoxide protons was confirmed by D2O exchange and by chemoselective reduction of the hydroperoxide groups into hydroxyl groups using NaBH4. These experiments were followed by nuclear magnetic resonance (NMR) spectroscopy. The 13C and 1HNMR spectra of a mixture of 9-hydroper-oxy-10-trans,12-cis-octadecadienoic acid methyl ester (9-OOH) and 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid methyl ester (13-OOH), which are formed during the autoxidation of methyl linoleate, were studied in detail to allow the comparison between the two linoleate hydroperoxides and the CLA methyl ester hydroperoxides. The 13CNMR spectra of samples enriched with one of the two linoleate hydroperoxide isomers were assigned using 2D NMR techniques, namely Correlated Spectroscopy (COSY), gradient Heteronuclear Multiple Bond Correlation (gHMBC), and gradient Heteronuclear Single Quantum Correlation (gHSQC). The 13C and 1H NMR experiments performed in this study show that hydroperoxides are formed during the autoxidation of CLA methyl ester both in the presence and absence of α-tocopherol and that the major isomers of CLA methyl ester hydroperoxides have a conjugated monohydroperoxydiene structure similar to that in linoleate hydroperoxides.

Lipase-catalyzed fractionation of conjugated linoleic acid isomers.

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9, trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.

Preparation, separation, and confirmation of the eight geometrical cis/trans conjugated linoleic acid isomers 8,10- through 11,13-18:2.

Conjugated linoleic acid (CLA) mixtures were isomerized with p-toluenesulfinic acid or I2 catalyst. The resultant mixtures of the eight cis/trans geometric isomers of 8,10-, 9,11-, 10,12-, and 11,13-octadecadienoic (18:2) acid methyl esters were separated by silver ion-high-performance liquid chromatography (Ag+-HPLC) and gas chromatography (GC). Ag+-HPLC allowed the separation of all positional CLA isomers and geometric cis/trans CLA isomers except 10,12-18:2. However, one of the 8,10 isomers (8cis, 10trans-18:2) coeluted with the 9trans,11cis-18:2 isomer. There were differences in the elution order of the pairs of geometric CLA isomers resolved by Ag+-HPLC. For the 8,10 and 9,11 CLA isomers, cis,trans eluted before trans,cis, whereas the opposite elution pattern was observed for the 11,13-18:2 geometric isomers (trans,cis before cis,trans). All eight cis/trans CLA isomers were separated by GC on long polar capillary columns only when their relative concentrations were about equal. Large differences in the relative concentration of the CLA isomers found in natural products obscured the resolution and identification of a number of minor CLA isomers. In such cases, GC-mass spectrometry of the dimethyloxazoline derivatives was used to identify and confirm coeluting CLA isomers. For the same positional isomer, the cis,trans consistently eluted before the trans,cis CLA isomers by GC. High resolution mass spectrometry (MS) selected ion recording (SIR) of the molecular ions of the 18:1, 18:2, and 18:3 fatty acid methyl esters served as an independent and highly sensitive method to confirm CLA methyl ester peak assignments in GC chromatograms obtained from food samples by flame-ionization detection. The high-resolution MS data were used to correct for the nonselectivity of the flame-ionization detector.

Autoxidation of the furan fatty acid ester, methyl 9,12‐epoxyoctadeca‐9,11‐dienoate

AbstractThe objective of this study was to identify autoxidation products of methyl 9,12‐epoxyoctadeca‐9,11‐dienoate (F9,12). Previous work has shown that F9,12 is a product both of autoxidation and singlet oxygen oxidation of the methyl ester derivative of conjugated linoleic acid (CLA). F9,12, 95% pure, was synthesized from methyl ricinoleate. The synthetic F9,12 was heated at 50°C in sealed tubes containing air. Each tube contained 6 mg F9,12 and 1 mg methyl stearate as an internal standard. Samples were taken at 4.5, 7, 23, 46.5, 69.5, and 93 h. The oxidized F9,12 was dissolved in isooctane and analyzed by gas chromatography (GC), GC‐direct deposition‐Fourier transform infrared spectroscopy, and GC‐electron ionization mass spectrometry. CLA methyl ester was oxidized in a similar manner. Under these conditions, the half‐lives of CLA and F9,12 were 40 and 35 h, respectively. Oxidation products of F9,12 that were identified included: 5‐hexyl‐2‐furaldehyde (I), methyl 8‐oxooctanoate (II), methyl 13‐oxo‐9,12‐epoxytrideca‐9,11‐dienoate (III), methyl 8‐oxo‐9,12‐epoxy‐9,11‐octadecadienoate (IV), and methyl 13‐oxo‐9,12‐epoxy‐9,11‐octadecadienoate (V).