Kinetics of photoirradiation-induced synthesis of soy oil-conjugated linoleic acid isomers

Photoirradiation of soy oil with UV/visible light has been shown to produce significant amounts of trans,trans conjugated linoleic acid (CLA) isomers through conversion of various synthesized intermediate cis,trans isomers. The objective of this study was to determine the kinetics of CLA isomers synthesis to better understand the production of various isomers. Soy oil was irradiated with UV/visible light for 144 h in the presence of an iodine catalyst and CLA isomers analyzed by gas chromatography (GC). Arrhenius plots were developed for the conversion of soy oil linoleic acid (A) to form cis-, trans/trans-, cis-CLA (B), conversion of cis-, trans/trans-, cis-CLA to form trans,trans-CLA (C) with respect to B, and formation of trans,trans-CLA isomers with respect to C. The kinetics of consumption of linoleic acid (LA) to form cis-, trans/trans-, cis-CLA was found to be of second-order with a rate constant of 9.01 x 10-7 L/mol s. The rate of formation of cis-, trans/trans-, cis-CLA isomers depends on the rate of formation from LA and its rate of consumption to form trans,trans-CLA isomers. The conversion of cis-, trans/trans-, cis-CLA isomers to trans,trans-CLA isomers was found to be of first-order with a rate constant of 2.75 x 10-6 s-1. However, the formation of thermodynamically stable trans,trans-CLA isomers (C) with respect to C was found to be a zero-order reaction with a rate constant of 10.66 x 10-7 mol/L s. The consumption of LA was found to be the rate-determining step in the CLA isomers formation reaction mechanism. The findings provide a better understanding of the mechanism of CLA isomers synthesis by photoirradiation and the factors controlling the ratio of various isomers.     

Photocatalytic production and processing of conjugated linoleic acid-rich soy oil

Daily intake of conjugated linoleic acid (CLA), an anticarcinogenic, antiatherosclerotic, antimutagenic agent, and antioxidant, from dairy and meat products is substantially less than estimated required values. The objective of this study was to obtain CLA-rich soybean oil by a customized photochemical reaction system with an iodine catalyst and evaluate the effect of processing on iodine and iodo compounds after adsorption. After 144 h of irradiation, a total CLA yield of 24% (w/w) total oil was obtained with 0.15% (w/w) iodine. Trans,trans isomers (17.5%) formed the majority of the total yield and are also associated with health benefits. The isomers cis-9,trans-11 and trans-10,cis-12 CLA, associated with maximum health benefits, formed approximately 3.5% of the total oil. This amount is quite significant considering that total CLA obtained from dairy sources is only 0.6%. ATR-FTIR, 1H NMR, and GC-MS analyses indicated the absence of peroxide and aldehyde protons, providing evidence that secondary lipid oxidation products were not formed during the photochemical reaction. Adsorption processing vastly reduced the iodine and iodocompounds without CLA loss. Photocatalysis significantly increased the levels of CLA in soybean oil.     

Determination of the conjugated linoleic acids in cow's milk fat by Fourier transform Raman spectroscopy

The collective term "conjugated linoleic acid" or "CLA" generally refers to a mixture of conjugated positional and geometric isomers of linoleic (cis-9,cis-12-octodecadienoic) acid. In nature, these isomers are mainly formed in the rumen by biohydrogenation of polyunsaturated fatty acids. This study concerns a first trial of CLA determination in cow's milk fat by Raman spectroscopy. The spectra of pure cis-9-oleic, cis-9,cis-12-linoleic, cis-9,trans-11-linoleic, and trans-10,cis-12-linoleic acids have been examined in comparison with the spectra of selected milk-fat samples containing between 0 and 3% of CLA. The trial of CLA determination by Raman spectroscopy on cow milk fat has reached its objective with the two following results. First, the examination of the Raman spectra allows to identify three specific Raman signals of the chemical bonds associated to the cis,trans conjugated C=C in the rumenic and trans-10,cis-12-octodecadienoic acids at 1652, 1438, and 3006 cm(-1). Second, the calibration of Raman spectrometer for the CLA determination has indicated that these three specific signals suit very well for the accurate and reliable measurement of CLA concentration in milk fat. To our knowledge, the present study is the first successful attempt to determine the CLA content of milk fat by a spectrophotometric method.     

Effect of antioxidants on soy oil conjugated linoleic acid production and its oxidative stability

Conjugated linoleic acid (CLA)-rich soy oil can be produced by photoisomerization of soy oil linoleic acid to produce a soy oil with up to 20% CLA. Recent studies indicate that mixed soy tocopherols added to refined bleached deodorized (RBD) oil produced significant increase in soy CLA yield during soy oil linoleic acid photoisomerization. However, the effect of common synthetic free radical scavenging antioxidants and specific tocopherols on CLA yield and its oxidative stability is not known. Therefore, this investigation evaluated the effects of various antioxidant systems on soy oil CLA yield and oxidative stability. Soy oil with added antioxidants consisting of combinations of mixed tocopherols (MT), ascorbyl palmitate (AP), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) was photoisomerized to produce CLA- rich soy oil. The CLA content was determined by GC-FID analysis and oxidative stability by peroxide value (PV). The soy oil in the presence of TBHQ, MT alone and MT with 500 ppm of AP produced significantly greater CLA yields and improved oxidative stability compared to a control without added antioxidants (p < 0.05). However, added mixed tocopherols produced the greatest CLA yield and also reduced PV relative to the control. Tocopherols in the form of α-, γ- and δ-tocopherols were then each examined as to their relative effect on CLA yields and PV. The largest increase in CLA yield was obtained with 1800 ppm of γ-tocopherols with reduced PV. Mixed tocopherols, TBHQ and γ-tocopherols can be used to increase CLA yield and reduce PV of soy oil during linoleic acid photoisomerization.     

Differential incorporation of conjugated linoleic acid isomers into egg yolk lipids

The incorporation pattern of conjugated linoleic acids (CLA) isomers into the egg yolk of hens in relation to that in the diet was studied. Silver-ion high-performance liquid chromatography (Ag-HPLC) was used to separate individual CLA isomers. It was found that the isomeric distribution pattern in the egg yolk lipids was different from that in the dietary fat. Total cis/trans isomers accounted for 81.2% of total CLA incorporated into the egg yolk, which was in contrast to the value of 92.0% of total CLA in the diet. Total cis/cis isomers accounted for 3.8% total CLA in the diet but they were 6.6% of the total CLA in the egg yolk lipids. In contrast, total trans/trans isomers were 12.2% of the total CLA isomers in the egg yolk lipids, whereas they were only 4.2% of total CLA in the diet. The results showed that total trans/trans-CLA was preferentially incorporated into the egg yolk, whereas the incorporation of total cis/trans-CLA isomers was partially discriminated. Within each group, the incorporation of individual isomers into the egg yolk lipids was also selective. cis-9,trans-11/trans-9,cis-11 and cis-10,trans-12/trans-10,cis-12 were the two major isomers in the diet. Ag-HPLC analysis showed that the former was preferentially transferred into the egg yolk compared with the latter. It was observed that supplementation of CLA in the diet of laying hens decreased the concentration of oleic acid (18:1n-9), arachidonic acid (20:4n-6), and docosahexaenoic acid (22:6n-3) but increased that of linolenic acid (18:3n-3), stearic acid (18:0), and palmitic acid (16:0) in the egg yolk, suggesting that CLA may inhibit Delta6 and Delta9 desaturases.     

Effect of the degree of processing on soy oil conjugated linoleic acid yields

Photoirradiation processing can be used to rapidly synthesize conjugated linoleic acid (CLA) in large quantities in soy oil. The objective of this study was to evaluate the effect of the level of refining of soy oil on CLA yields and oxidative properties after photoirradiation. Crude, alkali-refined, alkali-refined bleached, and alkali-refined bleached and deodorized (RBD) soy oils were photoirradiated in a pilot-plant processing system for 12 h with 0.35% iodine catalyst at 47 degrees C. RBD soy oil gave the highest total CLA yield of 16.3% of total oil with 4.3% cis, trans- and trans, cis-CLA isomers. Oxidative stability as measured by weight gain during incubation at 64 degrees C showed that iodine decreased the induction time of soy oil samples by 2-4 days. Photoirradiation processing further decreased the induction time by 2 days as a result of loss of total tocopherols. Iodine addition increased the titratable acidity in all the samples of soy oil. However, the level of refining affects this increase of titratable acidity, and RBD soy oil was found to be the most stable. The study indicates that RBD soy oil was the most suitable candidate for photoirradiation processing. Thus, soy oil should be alkali-refined, bleached, deodorized, and then photoprocessed followed by a secondary adsorption step to remove the iodine catalyst to obtain a RBD CLA-rich soy oil.     

Determining milk isolated and conjugated trans-unsaturated fatty acids using fourier transform Raman spectroscopy

The feasibility of Raman spectroscopy in combination with partial least-squares (PLS) regression for the determination of individual or grouped trans-monounsaturated fatty acids (trans-MUFA) and conjugated linoleic acids (CLA) in milk fat is demonstrated using spectra obtained at two temperature conditions: room temperature and after freezing at -80 °C. The PLS results displayed capability for direct semiroutine quantification of several individual CLA (cis-9,trans-11 and trans-10,cis-12 C18:2) and trans-MUFA (trans-4-15 C18:1) in minor concentrations (below 1.0 g/100 g of milk fat). Calibration models were based on reference data cross-correlation or determined by specific scattering signals in the Raman spectra. Distinct bands for trans-MUFA (1674 cm(-1)) and CLA (1653 cm(-1)) from the trans isolated and cis,trans conjugated C ═ C bonds were identified, as well as original evidence for the temperature effect (new bands, peak shifts, and higher intensities) on the Raman spectra of fatty acid methyl ester and triacylglyceride standards, are supplied.     

Conjugated linoleic acid in processed cheeses during the manufacturing stages

Conjugated linoleic acid (CLA) is a naturally occurring micronutrient in milk fat and dairy products consisting of a group of geometric and positional isomers. The purpose of this study was to assess the level and type of CLA isomers found in two commercial processed cheeses (portions and slices) as well as to monitor their evolution during the different manufacturing stages. Total CLA concentrations ranged from 7.5 to 7.9 mg/g of fat, and rumenic acid (cis-9,trans-11 C18:2), the isomer responsible for the biological functions, represented >80% of total CLA. trans-11,cis-13 and trans-11,trans-13 were, with approximately 4% each, the second main CLA isomers. trans-trans isomers accounted for <10% of total CLA. The processing parameters used in this research had negligible effects on the CLA content of processed cheese and did not modify the isomer profile in these dairy products, thereby confirming the stability of rumenic acid during manufacturing.     

Changes in the fatty acid profile of the subcutaneous fat of swine throughout fattening as affected by dietary conjugated linoleic acid and monounsaturated fatty acids

The fatty acid profile of the subcutaneous fat of pigs and its evolution throughout fattening as affected by dietary conjugated linoleic acid (CLA), monounsaturated fatty acids (MUFA), and their interaction (CLAxMUFA) were studied. Three levels (0, 1, and 2%) of an enriched CLA oil (28% cis-9, trans-11 and 28% trans-10, cis-12 CLA) were combined with two levels of MUFA (low, 19% average; and high, 39% average) for pig feeding (288 gilts). Subcutaneous shot-biopsies were taken from 48 animals at the beginning of the trial (S1, 70 kg), 14 days later (S2, 80 kg), and at slaughter (S3, 107 kg). Inclusion of CLA in the diet caused an increase during fattening in cis-9, trans-11 CLA, trans-10, cis-12 CLA, and saturated fatty acids (SFA) contents of pig backfat and a decrease in MUFA and polyunsaturated fatty acids (PUFA). MUFA supplementation also led to a MUFA enrichment of backfat. The interaction CLAxMUFA affected the SFA content. The rates of accumulation of CLA isomers, SFA, and MUFA throughout the trial did not follow a linear behavior, such rates being higher from S1 to S2 than from S2 to S3. These rates were also influenced by dietary CLA and MUFA levels. The increase in the ratio of saturated to unsaturated fatty acids of backfat caused by dietary CLA might be balanced by supplementation of pig diets with MUFA.     

Influence of feeding soybean oil on conjugated linoleic acid content in beef

Forty-eight steers were used to study the influence of feeding soybean oil (SO) on the conjugated linoleic acid (CLA) content of beef. Steers were fed either a control diet containing 954 g/kg of dry matter (DM) corn-based concentrate (CTL) or a control diet supplemented with SO at 20 (SO2) or 40 g/kg (SO4) of diet DM for 105 days. Adipose tissue samples were collected from the M. longissimus dorsi (LD) and from the M. semitendinosus (ST) on days 0 and 63 of the experiment. Adipose and muscle tissue samples were collected from the LD and ST immediately after slaughter. Feeding 40 g/kg of DM as SO increased the proportions of trans-C(18:1) in beef lipid as compared to CTL and SO2 treatments. The C(18:2) cis-9, trans-11 isomer of CLA as a proportion of total fat was not different in adipose and muscle across treatments. Supplementing SO increased C(18:2) trans-10, cis-12 CLA in adipose tissue of the LD. Supplementing high-grain finishing diets with SO is not an effective strategy to enhance the C(18:2) cis-9, trans-11 isomer of CLA in beef.     

Positional distribution of conjugated linoleic acid in triacylglycerol of Saccharomyces cerevisiae

Saccharomyces cerevisiae was cultivated in the presence of cis-9,trans-11 or trans-10,cis-12 isomers of free conjugated linoleic acid (CLA), and the effects of the isomers on the regioisomerisms of triacylglycerol (TAG) of the yeast were elucidated. Both isomers constituted about 34% of all fatty acids and increased drastically the number of different TAG species. Nearly all of the species contained CLA in at least one sn-position. In the most abundant species analyzed (20% of total species), the cis-9,trans-11 isomer appeared in combination with monounsaturated fatty acids (C16:1, C:18:1) whereas trans-10,cis-12 isomer was most frequently present with a medium chain fatty acid (C10:0 or C12:0) in the sn-2 position and C16:0 in one of the end positions (14% of total species). With either isomer, the amount of TAG species in which CLA encompassed all sn-positions was ca. 4%. Thus, S. cerevisiae can be used to produce edible single cell oil characterized by very heterogeneous distribution of CLA among the different TAG species.     

Antioxidant evaluation and oxidative stability of structured lipids from extravirgin olive oil and conjugated linoleic acid

Structured lipid (SL) was synthesized from extravirgin olive oil (EVOO) and conjugated linoleic acid (CLA) via a lipase-catalyzed reaction. CLA provides a variety of health benefits, but it is not consumed in free fatty acid form. The synthesized SL olive oil contained 42.5 mol % CLA isomers, and the major isomers were cis-9,trans-11-CLA (16.9 mol %) and trans-10,cis-12-CLA (24.2 mol %). The antioxidant activity determined by the radical scavenging capacity with the 2,2-diphenyl-1-picrylhydrazyl radical was lower in SL olive oil than in EVOO. The oxidative stability was also lower in SL olive oil since it had a higher peroxide value, rho-anisidine value, and 2-thiobarbituric acid reactive substances values during 20 days of storage at 60 degrees C. This observation could be due to the reduction in the natural phenolic compounds (97%) and tocopherols (56%), and the incorporated CLA with two conjugated double bonds in the SL olive oil. The oxidative stability of SL olive oil was increased by added rosemary extracts at concentrations of 100, 200, and 300 ppm. The present study suggests that the SL olive oil may be a suitable way to incorporate or deliver CLA into human diets. However, the addition of a proper antioxidant would be required for improving its oxidative stability.     

Comparison of methylation procedures for conjugated linoleic acid and artifact formation by commercial (trimethylsilyl) diazomethane

Four different methods for methylating conjugated linoleic acid (CLA) were compared. The HCl/MeOH and BF(3)/MeOH methods were tested under different time and temperature combinations. Increasing temperature and/or incubation time for either method decreased the cis-9,trans-11 and trans-10,cis-12 isomers, but trans-9,trans-11/trans-10,trans-12 isomers and artifacts (allylic methoxide) were increased. In addition, the triacylglyceride form of CLA was tested using the above methods and NaOMe at various temperatures for 20 min. The NaOMe did not generate methoxy artifacts. However, there were impurities in GC after methylation with NaOMe as well as with BF(3)/MeOH. The (trimethylsilyl)diazomethane method, which is a mild and easy alternative, was tested. Free forms of fatty acids were easily, but not completely, methylated by this method. Also, the method generated artifacts (trimethylsilyl CLA esters) and impurities (trimethylsilyl) that would interfere with short-chain fatty-acid analysis by GC.     

Effect of diet on the deposition of n-3 fatty acids, conjugated linoleic and C18:1trans fatty acid isomers in muscle lipids of German Holstein bulls

This study examined the effects of feeding diets rich in either n-3 or n-6 polyunsaturated fatty acids (PUFA) on the fatty acid composition of longissimus muscle in beef bulls. Thirty-three German Holstein bulls were randomly allocated to either an indoor concentrate system or periods of pasture feeding (160 days) followed by a finishing period on a concentrate containing linseed to enhance the contents of n-3 PUFA and conjugated linoleic acids (CLA) in beef muscle. The relative proportion and concentration (mg/100 g fresh muscle) of n-3 fatty acids in the phospholipid and triglyceride fractions were significantly increased (p < or = 0.05) in muscle lipids of pasture-fed bulls. The pasture feeding affected the distribution of individual CLA isomers in the muscle lipids. The proportion of the most prominent isomer, CLA cis-9,trans-11, was decreased from 73.5 to 65.0% of total CLA in bulls fed on concentrate as compared to pasture. The second most abundant CLA isomers were CLA trans-7,cis-9 and CLA trans-11,cis-13 in bulls fed on concentrate and pasture, respectively. Diet had no effect on the concentration of C18:1 trans-11. In contrast, the concentration of the C18:1 trans-13/14, trans-15, and trans-16 isomers in the muscle lipids was up to two times higher in pasture-fed as compared to concentrate-fed bulls. Pasture feeding enhanced the concentration of n-3 fatty acids, but the diet had no effect on the concentration of CLA cis-9,trans-11.     

Effect of diet and dietary fatty acids on the transformation and incorporation of C18 fatty acids in double-muscled Belgian Blue young bulls

Three groups of double-muscled Belgian Blue young bulls were fed during different stages of production diets differing in the proportions of linolenic and linoleic acid by including linseed in the concentrate or giving grass silage as main linolenic acid suppliers. Samples of rumen and abomasal contents and of the longissimus thoracis, subcutaneous fat, and liver were taken to analyze the fatty acid pattern with emphasis on the individual trans (t) C18:1 fatty acids and cis-9,trans-11 conjugated linoleic acid (c9t11CLA). Trans C18:1 isomers represented up to 20 g/100 g of total fatty acids in rumen and abomasal contents, whereas the accumulation of c9t11CLA was limited. Total trans C18:1 content in subcutaneous fat and intramuscular fat of the longissimus thoracis comprised 8.4 and 5.2 g/100 g of total fatty acids, respectively, with t11C18:1 being the most abundant one. Compared to rumen contents, subcutaneous and intramuscular fat were enriched in c9t11CLA and contained fewer tC18:1 isomers, resulting in a higher c9t11CLA/t11C18:1 ratio (0.04, 0.22, and 0.22, respectively). This result suggests that the endogenous synthesis of c9t11CLA in adipose tissue by the Delta(9)-desaturase was more important than its ruminal production.     

Formation and evolution of monoepoxy fatty acids in thermoxidized olive and sunflower oils and quantitation in used frying oils from restaurants and fried-food outlets

The formation and evolution of monoepoxy fatty acids, arising from oleic and linoleic acids, were investigated in olive oil and conventional sunflower oil, representatives of monounsaturated and polyunsaturated oils, respectively, during thermoxidation at 180 degrees C for 5, 10, and 15 h. Six monoepoxy fatty acids, cis-9,10- and trans-9,10-epoxystearate, arising from oleic acid, and cis-9,10-, trans-9,10-, cis-12,13-, and trans-12,13-epoxyoleate, arising from linoleic acid, were analyzed by gas chromatography after oil derivatization to fatty acid methyl esters. Considerable amounts, ranging from 4.29 to 14.24 mg/g of oil in olive oil and from 5.10 to 9.44 mg/g of oil in sunflower oil, were found after the heating periods assayed. Results showed that the monoepoxides quantitated constituted a major group among the oxidized fatty acid monomers formed at high temperature. For similar levels of degradation, higher contents of the monoepoxides were found in olive oil than in sunflower oil. Ten used frying oils from restaurants and fried-food outlets in Spain were analyzed to determine the contents of the monoepoxides in real frying oil samples. Levels ranged from 3.37 to 14.42 mg/g of oil. Results show that, for similar degradation levels, the monoepoxides were more abundant in the monounsaturated oils than in the polyunsaturated oils.     

trans-10,cis-12-conjugated linoleic acid isomer exhibits stronger oxyradical scavenging capacity than cis-9,trans-11-conjugated linoleic acid isomer

Although studies have been performed to test whether conjugated linoleic acid (CLA) acts as an antioxidant, the results were not conclusive. In addition, the CLA widely used in previous research contains 43% cis-9,trans-11 isomer, 45% trans-10,cis-12 isomer, and 10 other minor isomers. The objective of this study was to investigate the antioxidant activity of cis-9,trans-11- and trans-10, cis-12-CLA isomers using high-purity CLA isomers (>98%) by total oxyradical scavenging capacity assay (Winston, G. W.; Regoli, F.; Dugas, Jr., A. J.; Fong, J. H.; Blanchard, K. A. Free Radical Biol. Med. 1998, 24, 480-493). At all concentrations (2-200 microM), t10, c12-CLA performed as an antioxidant with a 15-min lag phase, which was more effective than c9,t11-CLA and alpha-tocopherol at lower concentrations (2 and 20 microM). On the other hand, c9,t11-CLA possessed weak antioxidant activity at 2 and 20 microM, whereas at 200 microM it acted as a strong pro-oxidant, which suggests that discrepancies of the results of the previous studies on the antioxidant properties of CLA may be due to the balance of the antioxidant properties of t10,c12-CLA and the pro-oxidant properties of c9,t11-CLA in different oxidation conditions.     

Enrichment of conjugated linoleic acid in oats (Avena sativa L.) by microbial isomerization

A method for microbial isomerization of oat linoleic acid to conjugated linoleic acid (CLA) was developed. The method includes hydrolysis of oat lipids in aqueous flour slurries by the endogenous oat lipase. Then, the flour slurry containing free linoleic acid is utilized as a substrate for the isomerization reaction carried out by resting cells of Propionibacterium freudenreichii ssp. shermanii. The isomerization reaction progressed most effectively when, after the lipid hydrolysis period, the pH of the slightly acidic oat slurry was elevated to 8.0-8.5 and maintained at this range. With slurries containing 5% (w/v) oat flour, the amounts of CLA formed per dry matter were up to 10.1 mg/g corresponding to 102 mg/g lipids or 0.44 mg/mL slurry. Increments in the flour content up to 15% increased the volumetric production of CLA to 0.85 mg/mL. The proportion of the cis-9,trans-11 isomer was 80% of the total CLA formed. CLA could be concentrated into the solid material of the oat slurry by acidification.     

Conjugated linoleic acid regulates phosphorylation of PPARγ by modulation of ERK 1/2 and p38 signaling in human macrophages/fatty acid-laden macrophages

Stimulation of macrophages by a variety fatty acids causes activation of MAP kinases (MAPKs). The consequences arising from down-regulation of MAPKs may be a limitation in the activity of PPARγ, which is modulated by a modification catalyzed by these kinases. Phosphorylation of MAP kinases-ERK1/2 and p38 as well as PPARγ was determined by real-time polymerase chain reaction and Western blotting in human macrophages cultured with conjugated linoleic acids (CLAs). We demonstrated that CLA isomers alter MAP kinase phosphorylation and PPARγ activation. Phosphorylation of ERK1/2 was diminished in cells cultivated with cis-9,trans-11 CLA, whereas phosphorylation of p38 was reduced by trans-10,cis-12 CLA. PPARγ was phosphorylated mainly by ERK1/2, and consequently, PPARγ phosphorylation was suppressed mainly by cis-9,trans-11 isomer. In human adipocytes, cis-9,trans-11 C 18:2 raised the activation of PPAR and several of its downstream target genes. We suggest that a similar process may also occur in human macrophages.