Enzymatic interesterification of structured lipids containing conjugated linoleic acid with palm stearin for possible margarine production

Structured lipids containing conjugated linoleic acid as a functional ingredient were blended with palm stearin in the ratios of 30 : 70, 40 : 60, 50 : 50, 60 : 40 and 70 : 30 (wt/wt). The blends were subjected to enzymatic interesterification by Candida antarctica lipase. After interesterification of the blends, changes in the physical properties of the products, including lower melting points and solid fat contents along with different melting behaviors, were evidenced. Analysis of triacylglycerols (TAG) of the interesterified blends showed a decrease in the concentration of high‐melting TAG. X‐ray diffraction analysis revealed, that all the reacted blends were predominantly in the β' crystal form. The mixture could be used for the formulation of margarines or other, similar products.     

Physical properties of lipase‐catalyzed interesterification of palm stearin with canola oil blends

Interesterified blends of hard palm stearin (IV of 11) and canola oil (hPS/CO) in ratios of 20 : 80, 30 : 70, 40 : 60, 50 : 50, 60 : 40 and 70 : 30 were prepared using immobilized Thermomyces lanuginosus lipase (Lipozyme TL IM). Comparison of physical properties was carried out between non‐interesterified and enzymatically interesterified products by monitoring their slip melting point (SMP), solid fat content (SFC), melting thermogram and polymorphism behavior. The Lipozyme TL IM‐catalyzed interesterification significantly modified the physical properties of the hPS:CO blends. The results showed that all the interesterified blends had lower SMP and SFC than their unreacted blends. The SMP result showed that the interesterified blends of hPS/CO 40 : 60, 50 : 50 and 60 : 40 could be useful for stick margarine and shortening applications, respectively. From the SFC analysis, the interesterified blends of hPS/CO 40 : 60 have SFC curves similar to vanaspati. The interesterified blends of hPS/CO 50 : 50 and 60 : 40 have SFC curves similar to margarines, puff pastry margarine and shortening. Interesterification had replaced the higher‐ and lower‐melting triacylglycerols by the middle‐melting triacylglycerols, yielding mixtures of lower SMP and SFC, compared to the original palm stearin. X‐ray diffraction analysis indicated the appearance of β' crystals in all the interesterified hPS/CO blends from predominantly β‐type oils.     

Conjugated linoleic acid production from linoleic acid by lactic acid bacteria

After screening 14 genera of lactic acid bacteria, Lactobacillus plantarum AKU 1009a was selected as a potential strain for CLA production from linoleic acid. Washed cells of L. plantarum with high levels of CLA production were obtained by cultivation in a nutrient medium with 0.06% (wt/vol) linoleic acid (cis-9,cis-12-octadecadienoic acid). Under the optimal reaction conditions with the free form of linoleic acid as the substrate, washed cells of L. plantarum produced 40 mg CLA/mL reaction mixture (33% molar yield) from 12% (wt/vol) linoleic acid in 108 h. The resulting CLA was a mixture of two CLA isomers, cis-9,trans-11 (or trans-9,cis-11)-octadecadienoic acid (CLA1, 38% of total CLA) and trans-9,trans-11-octadecadienoic acid (CLA2, 62% of total CLA), and accounted for 50% of the total FA obtained. A higher yield (80% molar yield to linoleic acid) was attained with 2.6% (wt/vol) linoleic acid as the substrate in 96 h, resulting in CLA production of 20 mg/mL reaction mixture [consisting of CLA1 (2%) and CLA2 (98%)] and accounting for 80% of total FA obtained. Most of the CLA produced was associated with the cells (ca. 380 mg CLA/g dry cells), mainly as FFA.     

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.     

Conjugated linoleic acid‐producing enzymes: A bioinformatics study

Linoleic acid isomerases (LAI) are enzymes responsible for conversion of linoleic acid (LA) to conjugated linoleic acids (CLA) in different isomeric forms. CLAs are well known for numerous beneficial effects as functional foods. Despite identification of several LAI producing‐bacteria and release of their LAI nucleotide sequences to Bio‐Banks, no related bioinformatics study on these important enzymes is addressed yet. To gain insights into the structural/functional and phylogentic relations of LAIs as well as recombinant production of the desired enzyme, we employed several bioinformatical tools to analyze their primary structure and physicochemical properties. The results indicated that LAIs produced in different bacterial strains might be divided in two distinct families (Propionibacterium acnes and myosin cross‐reactive antigen (MCRA)‐like LAIs) with specific isomerase activities. In another part of the study, physicochemical properties, solubility upon over expression in E. coli, disulfide bond formation, and potential glycosylation sites in LAI sequences were predicted using bioinformatics tools and the most appropriate strategy for recombinant production of each LAI was determined. Our predicted data may be useful for further experimental studies toward production of the desired LAI.     

Safety of conjugated linoleic acid (CLA) in overweight or obese human volunteers

The main objective of the study was to investigate the safety of conjugated linoleic acid (CLA) in healthy volunteers. The effect of CLA on body composition was also investigated. The trial design was a randomized, double‐blind placebo controlled study including 60 overweight or obese volunteers (body mass index (BMI) 27.5—39.0 kg/m²). The subjects were divided into two groups receiving 3.4 g CLA or placebo (4.5 g olive oil) daily for 12 weeks. The safety was evaluated by analysis of blood parameters and by clinical examinations at baseline and week 12. Vital signs and adverse events were registered at baseline, week 6, and week 12. Bio Impedance Assessment was applied for body composition measurements. 55 subjects completed the study. Adverse events occurred in 10% of the subjects. No difference in adverse events or other safety parameters was found between the treatment groups. Small changes in the laboratory safety data were not regarded as clinically significant. Moreover, no clinically significant changes in vital signs were observed in any of the groups. In the CLA group, mean weight was reduced by 1.1 kg (paired t‐test p = 0.005), while mean BMI was reduced by 0.4 kg/m²(p = 0.007). However, the overall treatment effect of CLA on body weight and BMI was not significant. There were no differences found between the groups with regard to efficacy parameters. The results indicate that CLA in the given dose is a safe substance in healthy populations with regard to the safety parameters investigated.     

Kinetics of lipase‐catalysed interesterification of triolein and caprylic acid to produce structured lipids

The influence of the molar ratio caprylic acid/triolein, enzyme concentration and water content on the kinetics of the interesterification reaction of triolein (TO) and caprylic acid (CA) were studied. The enzyme used was the 1,3‐specific Rhizomucor miehei lipase. Data modelling was based on a simple scheme in which the acid was only incorporated in positions 1 and 3 of the glyceride backbone. In addition, it was assumed that positions 1 and 3 of the triglycerides were equivalent and that the events at position 1 did not depend on the nature of the fatty acid in position 3 and vice versa. Monoglycerides and diglycerides were not detected during the experiments. This was attributed to the low water content of the immobilised enzyme particles. The value of the equilibrium constant, K, for the exchange of caprylic and oleic acids was 2.7, which indicated that the incorporation of caprylic acid into triglycerides was favoured compared with the incorporation of oleic acid. Simple first order kinetics could describe the interesterification reaction. Using this model and the calculated equilibrium constant, the apparent kinetic constants were calculated. The model fitted all the experimental data except for the CA/TO molar ratios larger than 6. Moreover, the interesterification reaction rate had a maximum value at CA/TO molar ratios of 4–6 mol mol^?1. Copyright © 2003 Society of Chemical Industry     

Trans‐free fats through interesterification of canola oil/palm olein or fully hydrogenated soybean oil blends

Binary blends of canola oil (CO) and palm olein (POo) or fully hydrogenated soybean oil (FHSBO) were interesterified using commercial lipase, Lypozyme TL IM, or sodium methoxide. Free fatty acids (FFA) and soap content increased and peroxide value (PV) decreased after enzymatic or chemical interesterification. No difference was observed between the PV of enzymatically and chemically interesterified blends. Enzymatically interesterified fats contained higher FFA and lower soap content than chemically prepared fats. Slip melting point (SMP) and solid‐fat content (SFC) of CO and POo blends increased, whereas those of CO and FHSBO blends decreased after chemical or enzymatic interesterification. Enzymatically interesterified CO and POo blends had lower SMP and SFC (at some temperatures) than chemically interesterified blends. The status was reverse when comparing chemically and enzymatically interesterified CO and FHSBO blends. The induction period for oxidation at 120°C of blends decreased after interesterification. However, chemically interesterified blends were more oxidatively stable than enzymatically interesterified blends. Interesterified blends of CO and POo or FHSBO displayed characteristics suited to application as trans‐free soft tub, stick, roll‐in and baker's margarine, cake shortening and vanaspati fat.     

The Production of an Experimental Table Margarine Enriched with Conjugated Linoleic Acid (CLA): Physical Properties

Even though conjugated linoleic acid (CLA) is known to have some beneficial effects on the human body, its consumption has decreased over the past 20 years due to the replacement of animal fats by vegetable oils. In this study, using the structured lipid (SL) containing CLA, an experimental table margarine enriched with CLA was produced and stored for 3 months at two temperatures prior to performing the relevant analyses. The GC results showed that the margarine fat had 10.6% CLA. The solid fat content was the highest in week 0 in all samples, which then decreased during storage but the hardness increased. An increment in dropping point was also observed in the samples. In week 0, all the samples had the β′ crystal as the predominant crystal form but a crystal transformation from β′ to β was observed during storage.     

Conjugated linoleic acid and oxidative stress

At the present time, conjugated linoleic acid (CLA) is the subject of a growing number of studies since it has been demonstrated to possess anticarcinogenic and antiatherogenic activities in experimental animal models and to increase in some pathological states in humans. In both situations, CLA has been claimed to be involved in oxidative stress, as an antioxidant in the first case and as a primary product of a free-radical attack on polyunsaturated fatty acids (PUFA) in the other. The controversial results are due mostly to a lack of a suitable methodology because the presence of conjugated dienes (CD) in lipid moiety has been taken for years as evidence of lipid peroxidation due to the occurrence of this structure in fatty acid hydroperoxides. We have recently developed a new methodology that consists of the extraction of fatty acids, including CD fatty acid hydroperoxides, by mild saponification and their separation and identification by high-performance liquid chromatography with diode array detector. Fatty acid analyses of liver homogenate, oxidized in vitro either with Fe-ADP or t-butyl hydroperoxide (t-ButylHP), of lamb and rats fed CLA at levels known to prevent carcinogenesis, showed that CLA and its metabolites steadily decreased during oxidative stress and that they are more prone to oxidation than their corresponding methylene-interrupted fatty acids. No significant antioxidant effect of CLA was detected in any model tested. However, CD fatty acid hydroperoxides increased in the t-ButylHP model but not in the Fe-ADP model, owing probably to the degradation of CD fatty acid hydroperoxides induced by this oxidative agent. In conclusion, CLA and its metabolites seem to behave, under oxidative stress, as regular PUFA. Thus, it is highly unlikely that the peculiar effects of CLA are directly related to interference in lipoperoxidative processes.     

Impact of Conjugated Linoleic Acid (CLA) on Skeletal Muscle Metabolism

Conjugated linoleic acid (CLA) has garnered special attention as a food bioactive compound that prevents and attenuates obesity. Although most studies on the effects of CLA on obesity have focused on the reduction of body fat, a number of studies have demonstrated that CLA also increases lean body mass and enhances physical performances. It has been suggested that these effects may be due in part to physiological changes in the skeletal muscle, such as changes in the muscle fiber type transformation, alteration of the intracellular signaling pathways in muscle metabolism, or energy metabolism. However, the mode of action for CLA in muscle metabolism is not completely understood. The purpose of this review is to summarize the current knowledge of the effects of CLA on skeletal muscle metabolism. Given that CLA not only reduces body fat, but also improves lean mass, there is great potential for the use of CLA to improve muscle metabolism, which would have a significant health impact.     

Optimization of Lipase-Catalyzed Fractionation of Two Conjugated Linoleic Acid (CLA) Isomers

The separation of two isomers of conjugated linoleic acid is highly significant since each exhibits different biochemical properties. The aim of this study was to investigate and optimize several factors affecting the esterification of l-menthol with the c9,t11-CLA isomer in an organic solvent-free system using lipase from Candida rugosa (Lipase AY-30). D-optimal design with 5 factors and 3 levels were employed to evaluate the effects of synthesis parameters; reaction time (8–24 h), temperature (30–50 °C), enzyme content (2–20 U/ml), substrate molar ratio of conjugated linoleic acid oil to l-menthol (2:1–1:2) and pH (6–8) on esterification of c9,t11-CLA with l-menthol. Based on the analysis of the residual amount of c9,t11-CLA in the free fatty acid fraction after just one-step esterification, the optimum synthesis conditions were as follows: reaction time 23.12 h, temperature 32.65 °C, enzyme amount 135.40 U, molar ratio of CLA oil to l-menthol at 1:1.7 and pH at 7.7; the lowest purity of c9,t11-CLA in free fatty acid fraction based on the total content of c9,t11 and t10,c12-CLA isomers was 8.6 %.     

Biodiesel production from mixtures of canola oil and used cooking oil

Used cooking oil (UCO) was mixed with canola oil at various ratios in order to make use of used cooking oil for production of biodiesel and also lower the cost of biodiesel production. Methyl and ethyl esters were prepared by means of KOH-catalyzed transesterification from the mixtures of both the oils. Water content, acid value and viscosity of most esters met ASTM standard except for ethyl esters prepared from used cooking oil. Canola oil content of at least 60% in the used cooking oil/canola oil feedstock is required in order to produce ethyl ester satisfying ASTM specifications. Although ethanolysis was proved to be more challenging, ethyl esters showed reduced crystallization temperature (−45.0 to −54.4 °C) as compared to methyl esters (−35.3 to −43.0 °C). A somewhat better low-temperature property of ester was observed at higher used cooking oil to canola oil ratio in spite of similar fatty acid compositions of both oils.     

Chemical and enzymatic interesterification of a beef tallow and rapeseed oil equal‐weight blend

A mixture of beef tallow and rapeseed oil (1:1, wt/wt) was interesterified using sodium methoxide or immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (Novozym 435) as catalysts. Chemical interesterifications were carried out at 60 and 90 °C for 0.5 and 1.5 h using 0.4, 0.6 and 1.0 wt‐% CH₃ONa. Enzymatic interesterifications were carried out at 60 °C for 8 h with Lipozyme IM or at 80 °C for 4 h with Novozym 435. The biocatalyst doses were kept constant (8 wt‐%), but the water content was varied from 2 to 10 wt‐%. The starting mixture and the interesterified products were separated by column chromatography into a pure triacylglycerol fraction and a nontriacylglycerol fraction, which contained free fatty acids, mono‐, and diacylglycerols. It was found that the concentration of free fatty acids and partial acylglycerols increased after interesterification. The slip melting points and solid fat contents of the triacylglycerol fractions isolated from interesterified fats were lower compared with the nonesterified blends. The sn‐2 and sn‐1,3 distribution of fatty acids in the TAG fractions before and after interesterification were determined. These distributions were random after chemical interesterification and near random when Novozym 435 was used. When Lipozyme IM was used, the fatty acid composition at the sn‐2 position remained practically unchanged, compared with the starting blend. The interesterified fats and isolated triacylglycerols had reduced oxidative stabilities, as assessed by Rancimat induction times. Addition of 0.02% BHA and BHT to the interesterified fats improved their stabilities.     

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.