Production of Conjugated Linoleic Acid by Microwave-Assisted and Ultrasound-Assisted Alkali Isomerization: Effects of Microwave Power and Ultrasound Amplitude

Conjugated linoleic acid (CLA) is commercially produced by alkali isomerization of linoleic acid (LNA). However, this method constitutes a relatively high content of undesirable CLA isomers. In present study, microwave-assisted and ultrasound-assisted alkali isomerization techniques were applied for production of CLA as an alternative to traditional alkali isomerization. This study was aimed to evaluate the isomerization degree of LNA, by using various process conditions such as microwave power, ultrasound amplitude, and their reaction times. The best conditions for LNA isomerization were a microwave power of 700 W and a reaction time of 6 h for microwave-assisted alkali isomerization and an ultrasound amplitude of 100% and a reaction time of 6 h for ultrasound-assisted alkali isomerization. Under determined conditions, microwave-assisted alkali isomerization (97.21%) resulted in a higher isomerization degree compared to ultrasound-assisted alkali isomerization (76.98%) while the content of undesirable CLA isomers in ultrasound-assisted alkali isomerization (0.62%) was lower than that of microwave-assisted alkali isomerization (1.87%). This study showed that application of the both techniques resulted in equal amounts of desirable CLA isomers. The content of desirable CLA isomers was 47.09% cis-9, trans-11 and 48.25% trans-10, cis-12 for microwave-assisted alkali isomerization and 36.34% cis-9, trans-11 and 40.02% trans-10, cis-12 for ultrasound-assisted alkali isomerization.     

碱法异构化亚油酸甲酯制备共轭亚油酸

该文探索在乙二醇溶剂中以氢氧化钠碱法异构化亚油酸甲酯制备共轭亚油酸的方法,适宜的异构化条件为m(氢氧化钠)∶m(亚油酸甲酯)∶m(乙二醇)=1∶5∶8.4,170℃反应4 h,亚油酸甲酯转化率和共轭亚油酸产率分别为92.4%和89.3%。该法比目前常用的亚油酸原料法的优越之处体现在:以氢氧化钠取代常用碱氢氧化钾,降低成本;以亚油酸甲酯取代亚油酸为原料,避免了原料本身大量消耗碱;反应初期避免原料大量皂化,减小了体系的传质阻力,因而减少了溶剂乙二醇消耗量。     

Rapid synthesis of conjugated linoleic acid from fruit processing residues seed oil by alkali-dimethyl sulfoxide isomerization

Rapid synthesis of conjugated linoleic acid (CLA) by the alkali-dimethyl sulfoxide (DMSO) isomerization method is presented in this study. Seeds from fruit processing residues in Thailand were screened as an alternative potential source of linoleic acid (LA)-rich oil. Passion fruit with 72.10% LA and 26.18% oil in the seeds was selected as starting oil. The Alkali-DMSO isomerization was carried out by mixing DMSO with alkali hydroxides and polyhydric alcohols. Under a mass ratio of 1:0.5:1:1 oil/NaOH/propylene glycol/DMSO, at 180°C, production of the major isomers of CLA (c9,t11, and t10,c12) was complete within 3 min, resulting in a 20-fold increase in productivity compared to the conventional method. The reaction time decreased because of an increment in the basic strength of the alkali-DMSO catalyst. CLA from alkali-DMSO isomerization and commercial supplements showed a similar isomer distribution and yielded similar CC50 values when evaluated against the Vero cell line. This method might be an alternative in industrial production as it is fast and simply applied to the conventional system. In addition, seed oils from fruit processing residues can be used as an alternative to traditional LA-rich oils.     

Direct Isomerization of Linoleic Acid to Conjugated Linoleic Acids (CLA) using Gold Catalysts

Supported gold catalysts, e.g., Au on Al₂O₃, Fe₂O₃, CeO₂, MnO₂, TiO₂, ZrO₂, activated carbon, titanium silicalite TS‐1, were prepared and used for the isomerization of linoleic acid (cis‐9,cis‐12‐octadecadienoic acid) to conjugated linoleic acids (CLA) in the presence of hydrogen at 165 °C in a batch reactor. The best results were obtained using a catalyst with 2 wt % Au on TS‐1, which exhibits a high selectivity (78 %) towards CLA. The two biologically active target CLA isomers, i.e., cis‐9,trans‐11‐CLA and trans‐10,cis‐12‐CLA, were the main products. During the isomerization of linoleic acid to CLA, consecutive reactions also took place. These were the hydrogenation of linoleic acid and CLA to monounsaturated octadecenoic acids and the further hydrogenation of monounsaturated acids to stearic acid. Thus, gold catalysts are capable of isomerizing linoleic acid to CLA and hydrogenating their double bonds to an extent that depends on the Au catalyst used.     

Enzymatic Synthesis of Diacylglycerols Enriched with Conjugated Linoleic Acid by a Novel Lipase from <Emphasis Type="Italic">Malassezia globosa</Emphasis>

Diacylglycerols (DAG) of conjugated linoleic acid (CLA) were prepared by esterification of glycerol with fatty acids enriched with CLA (FFA–CLA, >95%) in the presence of a novel lipase from Malassezia globosa (SMG1). Lipase SMG1 is strictly specific to mono- and diacylglycerols but not triacylglycerols, which is similar to the properties of lipase from Penicillium camembertii (lipase G 50), but lipase SMG1 showed preference on the production of DAG with the reaction proceeding. Low temperature was beneficial for the conversion of FFA–CLA into acylglycerols, the degree of esterification reached 93.0% when the temperature was 5 °C. The maximum DAG content (53.4%) was achieved at 25 °C. The rate of DAG synthesis increased as the enzyme loading increased. However, at lipase amounts above 240 U/g mixtures, no significant increases in DAG concentration were observed. The molar ratio of FFA–CLA to glycerol and initial water content were optimized to be 1:3 (mol/mol) and 3%. Lipase SMG1 showed no regioselectivity because the contents of 1,3-DAG and 1,2-DAG were 43.1% and 21.2% based on total content of acylglycerols. By calculating the ratio of 9c, 11t-CLA to 10t, 12c-CLA, it was indicated that lipase SMG1 showed a little preference to 10t, 12c-CLA at the sn-1(3) position of monoacylglycerols (MAG), while no selectivity for 9c, 11t-CLA at the sn-2 position of DAG was obviously found.     

Body Compositional Changes and Growth Alteration in Chicks from Hens Fed Conjugated Linoleic Acid

Effects of feeding conjugated linoleic acid (CLA) to hens on progeny chick development and composition at hatch (NHC) and three weeks of age (TWC) were assessed. CLA (0 or 0.5%, composed of mixed isomers of cis-9,trans-11 or trans-10,cis-12-CLA) was fed to hens with either safflower (SO) or olive oil (OO) (3 or 3.5%) to assure successful hatch for 2 weeks prior to collection for incubation. Maternal CLA feeding had no effect on hatchability, but improved egg fertility (p < 0.05). Maternal feeding of CLA with SO increased 21 day-old progeny growth, while CLA with OO decreased growth (oil*CLA, p < 0.05). In 25 day-old chicks (TWC), but not NHC, maternal CLA decreased the proportion of total body water (p < 0.05) and increased body ash (p < 0.05). While monounsaturated fatty acids were decreased and saturated fatty acids increased in eggs and NHC from hens fed CLA, no differences in fatty acid composition were observed in chicks at 25 days of age from hens fed CLA. Maternal CLA feeding resulted in the presence of c9,t11 and t10,c12-CLA in NHC, but only c9,t11 in the TWC. In conclusion, hens fed CLA led to improved fertility and altered body composition at 3 weeks of age.     

红花油制取共轭亚油酸的研究

以新疆克拉玛依厂的红花油为原料,以丙三醇为溶剂,在KOH的碱性催化作用下,将红花油先转化成亚油酸钾;再利用异构化反应制得浅黄色的共轭亚油酸产品。对产品进行了薄层层析法和紫外分光光度法定性分析,并定量分析了红花油异构化反应的程度。本研究对红花油的深加工将起到很好的推动作用。     

Conjugated linoleic acid (CLA) production and lipase‐catalyzed interesterification of purified CLA with canola oil

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.     

Two-Step Production of Oil Enriched in Conjugated Linoleic Acids and Diacylglycerol

A two-step process was used to produce diacylglycerol-enriched structured lipid that contained mainly c9,t11 and t10,c12 isomers of conjugated linoleic acids (CLA). First, a structured triacylglycerol (TAG) was synthesized by lipase-catalyzed acidolysis of corn oil with CLA. This structured triacylglycerol contained 30.4 mol% CLA with 45.5% of the CLA mostly located at sn-1,3 positions of the glycerol backbone. Then, lipase-catalyzed glycerolysis was conducted between structured triacylglycerol and glycerol to produce diacylglycerol-enriched structured lipid. The final product contained 6.8% monoacylglycerol, 31.5% diacylglycerol and 61.1% TAG after 48 h reaction. The selected chemical (fatty acid composition, the content of mono-, di-, and triacylglycerol in the reaction product) and physical properties (melting profile) were determined by hihg-performance liquid chromatography (HPLC), gas chromatography (GC), and differential scanning calorimetry (DSC).     

Selective Enrichment of Conjugated Linoleic Acid Isomers in Their Mixtures Using Combined Chemical and Enzymatic Methods

The aim of this study was to selectively enrich t10,c12-conjugated linoleic acid (t10,c12-CLA) and c9,t11-CLA in commercial CLA mixtures using a combination of urea crystallization and lipase-catalyzed esterification. The objective of the urea fractionation is to remove saturated and monounsaturated fatty acids (FA) from the CLA mixtures. CLA-enriched free FA (FFA) mixtures containing 53.8 wt% t10,c12-CLA and 39.1 wt% c9,t11-CLA were produced from the CLA mixtures containing ~34 wt% each of the two CLA isomers by a urea crystallization using methanol and the urea-to-FA weight ratio of 2.5:1. The CLA-enriched FFA mixtures were partially esterified with dodecan-1-ol in a recirculating packed-bed reactor using an immobilized lipase from Candida rugosa to further enrich the t10,c12-CLA and c9,t11-CLA in an FFA fraction and an FA dodecyl ester fraction, respectively, under the optimal conditions, i.e., temperature, 20 °C; FA-to-dodecan-1-ol molar ratio, 1:1; water content, 2 wt% of total substrates; residence time, 5 min; and reaction time, 24 h (for t10,c12-CLA enrichment) and 12 h (for c9,t11-CLA enrichment). After the reaction, an FFA fraction with 72.6 wt% t10,c12-CLA was obtained. Another FFA fraction with 62.0 wt% c9,t11-CLA was recovered after the saponification of the FA dodecyl ester fraction. The yields of t10,c12-CLA and c9,t11-CLA in the FFA fractions were 43.6 and 21.5 wt%, respectively, based on their initial weights in the CLA mixtures.     

Preparation of conjugated linoleic acid from safflower oil

Synthetically prepared mixtures of conjugated linoleic acid (CLA) are widely used in animal and cell culture studies to investigate the potential effects of the Δ9c, 11t-18:2 isomer found in food products from ruminant animals. Alkali isomerization of linoleic acid is a common method used in the synthesis of a mixture of CLA isomers containing predominantly the Δ9c, 11t-18:2 and Δ10t, 12c-18:2 isomers. Some biological activity might also be mediated by the Δ10t, 12c-18:2 isomer. Currently few published methodologies exist describing procedures for the enrichment of these two isomers. A method is described herein to take advantage of an inexpensive oil, safflower oil, for use in synthesis of CLA and a procedure to enrich the Δ10t, 12c-18:2 isomer.     

Rumenic Acid Significantly Reduces Plasma Levels of LDL and Small Dense LDL Cholesterol in Hamsters Fed a Cholesterol- and Lipid-Enriched Semi-Purified Diet

Conjugated linoleic acids (CLAs) consist of a series of positional and geometrical isomers of linoleic acid. CLA have been reported to beneficially affect cardiovascular risk factors in animal models. In order to assess the role of individual CLA isomers on lipoprotein cholesterol concentration, 30 hamsters were fed for 12 weeks an hyperlipidic diet containing pure cis-9,trans-11 CLA (c9,t11) or pure trans-10, cis-12 CLA (t10,c12) isomers given alone or as a mixture. Plasma total cholesterol, LDL and HDL cholesterol concentrations were significantly lower in the c9,t11 CLA isomer fed hamsters relative to the Control group, with the most substantially effect on LDL cholesterol (−56%; P < 0.05). Plasma triacylglycerol concentrations did not differ significantly regarding those two groups. Plasma cholesterol parameters showed a tendency to decrease in the t10,c12 CLA isomer and CLA mixture fed hamsters compared with the Control group, but differences were not significant. For the first time, the atherogenic fraction of small dense LDL was investigated. Plasma small dense LDL cholesterol concentration was lower in the c9,t11 CLA relative to Control, while the t10,c12 and CLA mixture groups showed only a non significant tendency to decrease. Taken together, these data indicate that feeding rumenic acid (c9,t11 CLA) may beneficially affect lipoprotein profile in hamster fed a cholesterol- and lipid-enriched semi-purified diet, when t10,c12 CLA isomer or CLA mixture would be less active.     

Dynamic Features of the Rumen Metabolism of Linoleic Acid,Linolenic Acid and Linseed Oil Measured in Vitro

The lipid quality of ruminant products is largely determined by the extent of rumen microbial biohydrogenation (BH) of polyunsaturated fatty acids (FAs) and the substances formed thereby. In vitro batch incubations with mixed rumen bacteria were tracked over 24 h to characterize the profiles and kinetics of the BH products from three lipid sources: pure linoleic acid (c9,c12–18:2), pure linolenic acid (c9,c12,c15–18:3) and linseed oil (mainly c9,c12,c15–18:3 in triacylglycerols). After 24 h of incubation biohydrogenation was more complete for c9,c12–18:2, which gave mainly stearic acid (18:0), than for c9,c12,c15–18:3, which yielded mainly trans-18:1 FAs. This suggests inhibition of the final BH step (18:1 to 18:0). Incubations of c9,c12–18:2 resulted in high levels of carbon 10- and 12-desaturated 18:1, t10,c12- and c9,t11-CLAs. Incubations of c9,c12,c15–18:3 resulted in high levels of t11–18:1, carbon 13- and 15-desaturated 18:1 as well as t11,c15–18:2 and 11,13-CLAs. A comparative study of linolenic acid and linseed oil kinetics revealed that the BH process was not significantly slowed by the esterification of polyunsaturated FAs, but may have been limited by the isomerization step in which the cis12 double bond goes to the trans11 position. The disappearance rates of c9,c12–18:2 and c9,c12,c15–18:3 ranged from 23.6 to 44.6%/h. The wide variety of BH intermediates found here underlines the large number of possible BH pathways. These data help provide a basis for dynamic approaches to BH processes.     

Effect of <Emphasis Type="Italic">trans</Emphasis>8, <Emphasis Type="Italic">cis</Emphasis>10+<Emphasis Type="Italic">cis</Emphasis>9, <Emphasis Type="Italic">trans</Emphasis>11 Conjugated Linoleic Acid Mixture on Lipid Metabolism in 3T3-L1 Cells

Evidence suggests that minor isomers of conjugated linoleic acid (CLA), such as trans8, cis10 CLA, can elicit unique biological effects of their own. In order to determine the effect of a mixture of t8, c10+c9, t11 CLA isomers on selected aspects of lipid metabolism, 3T3-L1 preadipocytes were differentiated for 8 days in the presence of 100 μM linoleic acid (LA); t8, c10+c9, t11 CLA; t10, c12+c9, t11 CLA or purified c9, t11 CLA. Whereas supplementation with c9, t11 and t10, c12+c9, t11 CLA resulted in cellular triglyceride (TG) concentrations of 3.4 ± 0.26 and 1.3 ± 0.11 μg TG/μg protein, respectively (P < 0.05), TG accumulation following treatment with CLA mixture t8, c10+c9, t11 was significantly intermediate (2.5 ± 0.22 μg TG/μg protein, P < 0.05) between the two other CLA treatments. However, these effects were not attributable to an alteration of the Δ⁹ desaturation index. Adiponectin content of adipocytes treated with t8, c10+c9, t11 mixture was similar to the individual isomer c9, t11 CLA, and both the t8, c10+c9, t11 and c9, t11 CLA groups were greater (P < 0.05) than in the t10, c12+c9, t11 CLA group. Overall, these results suggest that t8, c10+c9, t11 CLA mixture affects TG accumulation in 3T3-L1 cells differently from the c9, t11 and t10, c12 isomers. Furthermore, the reductions in TG accumulation occur without adversely affecting the adiponectin content of these cells.     

固体超强酸SO_4~(2-)/TiO_2催化合成亚油酸乙酯

用两相滴定沉淀法制备了SO2-4/TiO2固体超强酸催化剂,得到了适合亚油酸酯化的催化剂制备工艺条件:硫酸浸渍浓度0 75mol/L,浸渍时间4h,焙烧温度450℃,焙烧时间4h。首次将该催化剂用于亚油酸的酯化反应催化合成亚油酸乙酯,考察了物料比、反应时间、催化剂用量对亚油酸与乙醇酯化反应的影响规律,最佳反应条件为:n(无水乙醇)/n(亚油酸)=4,w(催化剂)=3%(相对于亚油酸),反应时间8h,亚油酸转化率可达93%。     

A novel method for solvent fractionation of two CLA isomers

Conjugated linoleic acid (CLA) is commercially available as a mixture consisting of almost equal amounts of the cis-9,trans-11-CLA (c9, t11) and trans-10, cis-12-CLA (t10, c12) isomers. Separation of the two isomers is highly significant since each exhibits different biochemical properties. Highly efficient separation could be accomplished by crystallization in acetone (solvent) of the two CLA isomers (solutes) in the presence of medium-chain fatty-acid (MCFA) additives. The relative concentration ratios of the two CLA isomers in the solvent-crystallized materials varied depending on which MCFA were added. Addition of lauric and decanoic acids resulted in the crystals predominantly containing t10,c12, whereas octanoic acid yielded those predominantly containing c9,t11. We have confirmed that onetime solvent crystallization using decanoic acid and octanoic acid additives increased the t10,c12 and c9,t11 concentrations, and that repeated solvent crystallization resulted in the ratio of c9,t11 to t10,c12 of at least 4∶96 or 98∶2.     

Fatty acid profiles in tissues of mice fed conjugated linoleic acid

The incorporation of vaccenic acid (VA, 0.5 and 1.2%), conjugated linoleic acid (CLA, mixture of primarily c9,t11‐ and t10,c12‐CLA, 1.2%), linoleic acid (LA, 1.2%) and oleic acid (OA, 1.2%) into different tissues of mice was examined. The effects on the fatty acid composition of triacylglycerols (TAG) and phospholipids (PL) in kidney, spleen, liver and adipose tissue were investigated. VA and CLA (c9,t11‐ and t10,c12‐CLA) were primarily found in TAG, especially in kidney and adipose tissue, respectively. Conversion of VA to c9,t11‐CLA was indicated by our results, as both fatty acids were incorporated into all the analyzed tissues when a diet containing VA but not c9,t11‐CLA was fed. Most of the observed effects on the fatty acid profiles were seen in the CLA group, whereas only minor effects were observed in the VA groups compared with the OA group. Thus, CLA increased n‐3 polyunsaturated fatty acids (PUFA) in PL from kidney and spleen and lowered the ratio of n‐6/n‐3 PUFA in these tissues. Furthermore, CLA increased C₂₂ PUFA in the PL fraction of kidney, spleen and liver, but reduced the level of arachidonic acid in PL of liver and spleen and lowered the Δ⁹‐desaturation indexes in all analyzed tissue TAG.     

Incorporation of conjugated linoleic acid and vaccenic acid into lipids from rat tissues and plasma

The objective of this study was to determine the incorporation of conjugated linoleic acid (CLA) into triacylglycerols (TAG) and phospholipids (PL) of tissues and plasma, and to interpret the role of dietary‐derived vaccenic acid (VA) in increasing the tissue content of CLA (c9,t11) and the influence on the fatty acid profile. We fed five groups of rats semi‐purified diets with varying levels of CLA and VA: control butter with low CLA (c9,t11) and VA; control butter added 5% CLA (c9,t11); control butter added 5% Tonalin [equal amount of CLA (c9,t11) and CLA (t10,c12)]; control butter added 5% VA; butter with high CLA (c9,t11) and VA (H‐CLA), for 3 weeks. The highest incorporation of CLA (c9,t11) was found in adipose tissue, and the lowest was observed in liver. Low intake of CLA (c9,t11) combined with high intake of VA resulted in a higher incorporation of CLA (c9,t11) in tissues due to the conversion of VA to CLA (c9,t11), compared to feeding CLA (c9,t11) without VA. However, in enterocytes, the proportion of CLA (c9,t11) was low after feeding VA, indicating no or only a minor conversion of VA to CLA (c9,t11) in the intestine. The incorporation of CLA (t10,c12) into TAG from plasma and tissues was generally much lower than that of the CLA (c9,t11) isomer, except in the enterocyte TAG, which had similar proportions of the two isomers.     

A recommended esterification method for gas chromatographic measurement of conjugated linoleic acid

The effect of the methylation method on isomerization of conjugated linoleic acid (CLA) in gas chromatographic analysis was studied. Among methylation methods examined, the magnitude of isomerization of CLA was greatest with BF₃ catalyst, followed by HCI and H₂SO₄ catalyst. Short-time methylation did not extensively change the CLA composition in all methods, and c,t and t,c isomers were essentially maintained, while the appearance of t,t isomers and unknown peaks was practically restricted. After 120 min of methylation, there was essentially no conversion in the H₂SO₄ method, in contrast to a marked change in the BF₃ method. The antioxidants butylated hydroxytoluene, ascorbic acid, β-carotene, and α-tocopherol did not suppress conversion, while dimethylsulfoxide (DMSO) and dimethylformamide (DMF) attenuated the changes in CLA composition. Suppression was more effective in the H₂SO₄ method than in the BF₃ method. Thus, methylation with H₂SO₄ in the presence of a proper amount of DMSO or DMF is recommended for esterification of CLA.     

天然植物油碱性异构为共轭亚油酸的制备条件优化

采用KOH为催化剂,丙二醇为溶剂,天然植物油为原料,碱性异构法转化为共轭亚油酸。根据二次正交组合实验设计,研究了反应温度、反应时间、天然植物油用量等诸因素的一次、二次及其交互作用对共轭亚油酸选择性的影响,得到相应数学模型和适宜的制备条件:当m(丙二醇)∶m(KOH)=3∶1,搅拌速度500r min时,氮气保护,最适反应温度173 5℃,最佳反应时间2 85h,最优配方m(天然植物油) m(KOH)=1 55,此时共轭亚油酸的选择性≥93 6%。