Journal of the American Oil Chemists' Society (v.75, #11)
Microbial synthesis of chiral intermediates for β-3-receptor agonists
by Ramesh N. Patel; Amit Banerjee; Linda Chu; David Brozozowski; Venkata Nanduri; Laszlo J. Szarka (pp. 1473-1482).
Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of β-3-receptor agonists. These include: (i) the microbial reduction of 4-benzyloxy-3-methanesulfonylamino-2′-bromoacetophenone 1 to the corresponding (R)-alcohol 2 by Spingomonas paucimobilis SC 16113. In the biotransformation process, a reaction yield of >85% and an optical purity of 99.5% were obtained for the desired (R)-alcohol 2; (ii) the enzymatic resolution of racemic α-methyl phenylalanine amide, 3, and α-methyl-4-hydroxy-phenylalanine amide, 5, by amidase from Mycobacterium neoaurum ATCC 25795 to prepare the corresponding (S)-amino acids 4 and 6. Reaction yields of 49.9 and 49 M% (theoretical maximum yield 50 M%) and optical purities of 99 and 94% were obtained for the desired (S)-amino acids 4 and 6, respectively; (iii) the asymmetric hydrolysis of methyl-(4-methoxyphenyl)-propanedioic acid, ethyl diester, 7, to the corresponding (S)-monoester 8 by pig liver esterase. A reaction yield of 96 M% and an optical purity of 96% were obtained for (S)-monoester 8 when reactions were carried out in a biphasic system containing 10% ethanol at 10°C.
Keywords: Asymmetric hydrolysis; biocatalysis; chiral intermediates; pig liver esterase; β-3-receptor agonist; resolution using amidase; stereoselective reduction
Production of polyhydroxy fatty acids from linoleic acid by Clavibacter sp. ALA2
by Ching T. Hou; Harold Gardner; Wanda Brown (pp. 1483-1487).
Hydroxy fatty acids are important industrial materials. We isolated a microbial culture, Clavibacter sp. ALA2, which converts linoleic acid to many polyhydroxy fatty acids. Structures of the products were determined as: 12,13,17-trihydroxy-9(Z)-octadecenoic (THOA, main product), 12-[5-ethyl-2-tetrahydrofuranyl]-7,12-dihydroxy-9(Z)-dodecenoic (ETDDA), and 12-[5-ethyl-2-tetrahydrofuranyl]-12-hydroxy-9(Z)-dodecenoic (ETHDA) acid. The yield of THOA was 25% and the relative amount of the products were THOA/ETDDA/ETHDA =9:1.3:1. The structures of the hydroxy unsaturated fatty acids resemble those of plant self-defense substances.
Keywords: Biocatalysis; biotransformation; enzymes; hydroxy unsaturated fatty acids; microbes
Application of potato lipid acyl hydrolase for the synthesis of monoacylglycerols
by Alasdair R. Macrae; Jean E. Visicchio; Alexandra Lanot (pp. 1489-1494).
Protein extracts from potato tubers contain a lipid acyl hydrolase (LAH) with an unusual selectivity. The component responsible for the enzyme activity is a group of closely related glycoproteins, known as patatin. Potato LAH catalyzes the rapid hydrolysis of monoacylglycerols (MAG), but in contrast expresses only low activity with di- and triacylglycerols. The selectivity of the LAH can be exploited for the synthesis of MAG from fatty acids and glycerol in microaqueous reaction systems. Oleic, linoleic, linolenic, capric, lauric, and myristic acids can be used as reactants, and in each case the reaction products contain >95 mol% MAG and <5 mol% diacylglycerol. By removing water from the reaction mixture by distillation under vacuum, excellent conversions of fatty acids into MAG are possible. Low conversions are observed with palmitic and stearic acids, because of the necessity to use a high temperature (70°C) to maintain a liquid reaction mixture. Potato LAH is rapidly inactivated at 70°C in the microaqueous reaction systems. Potato LAH also catalyzes the selective synthesis of monoesters from oleic acid and simple diols. With oleic acid and diglycerol, monoesters are again the main reaction products, but 17 mol% diesters are also formed.
Keywords: Diglycerol; diol; fatty acid; glycerol; lipase; lipid acyl hydrolase; monoacylglycerol; patatin; potato
Production of 8,11,14,17-cis-eicosatetraenoic acid (20:4ω-3) by a Δ5 and Δ12 desaturase-defective mutant of an arachidonic acid-producing fungus Mortierella alpina 1S-4
by Hiroshi Kawashima; Eiji Sakuradani; Nozomu Kamada; Kengo Akimoto; Kyoko Konishi; Jun Ogawa; Sakayu Shimizu (pp. 1495-1500).
A Δ5 and Δ12 desaturase-defective mutant of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, produced 8,11,14,17-cis-eicosatetraenoic acid (20:4ω3) intracellularly when grown with linseed oil. Dihomo-γ-linolenic acid was the only C20 polyunsaturated fatty acid (4.9 wt% of total mycelial fatty acids) other than 20:4ω3. AA and 5,8,11,14,17-cis-eicosapentaenoic acid were not detected. The mycelial lipids consisted of 82.2% (by mol) triacylglycerol (TG), 7.1% diacylglycerol, 8.9% phospholipids (PL), and 1.9% free fatty acids. The percentage of 20:4ω3 was higher in PL (30.1%) than in TG (11.6%), and highest in phosphatidylcholine (38.9%). Under the optimal conditions with a 5-L jar fermenter, 20:4ω3 production amounted to 97.4 mg/g dry mycelia with a mycelial yield of 23 g/L on the twelfth day (corresponding to 2.24 g/L medium and 37.1% of total mycelial fatty acids).
Keywords: Δ5 Desaturase; Δ12 desaturase; eicosatetraenoic acid (20:4ω3); Mortierella alpina ; mutant
Enhancement of arachidonic acid production by Mortierella alpina 1S-4
by Kenichi Higashiyama; Toshiaki Yaguchi; Kengo Akimoto; Shigeaki Fujikawa; Sakayu Shimizu (pp. 1501-1505).
The effect of mineral addition on arachidonic acid (AA) production by Mortierella alpina 1S-4 was evaluated. At first, the addition of minerals such as sodium, potassium, calcium, and magnesium was examined in flask cultures, and then the addition of phosphorus with the optimal amounts of the minerals was investigated in a 10-L jar-fermenter. As a result, 1.5% soy flour medium with the addition of 0.3% KH2PO4, 0.1% Na2SO4, 0.05% CaCl2·2H2O, and 0.05% MgCl2·6H2O was found to enhance the AA yield 1.7-fold over that without mineral addition. When 1% yeast extract with the above mineral mixture was used, the AA yield was enhanced 1.35-fold over that without minerals. We also verified that an increase in the polar lipid content occurred in the case of only KH2PO4 addition, and that the above-mentioned increase in the AA yield was due to the minerals themselves, not a pH buffer effect.
Keywords: Arachidonic acid; arachidonic acid yield; lipid composition; mineral addition; morphology; Mortierella alpina
Lipase-catalyzed synthesis of kojic acid esters in organic solvents
by Kuan-Ju Liu; Jei-Fu Shaw (pp. 1507-1511).
Kojic acid is an inhibitor of bacteria, viruses, and fungi. It is used for inhibiting the browning effect of tyrosinase in the food and cosmetic industries. To improve its lipophilic properties, Pseudomonas cepacia lipase and Penicillium camembertii lipase were used for catalyzing the esterification of kojic acid to synthesize kojic acid monolaurate and kojic acid monooleate. These products showed a 69.5% inhibitory effect on tyrosinase in hydrophobic organic solvent. The yields of kojic acid esters were affected by enzymes, substrates, organic solvent, and temperature. Lauric and oleic acids were the best substrates for esterification among various fatty acids tested. CaCl2 and MnCl2 stimulate Pseudomonas cepacia lipasecatalyzed esterification by 7.0%. On the contrary, MgCl2, SrCl2, and ZnCl2 inhibited the reaction. The best pH of buffer for lipase pretreatment was pH 6.0. Pseudomonas and Penicillium lipases can be reused for the synthesis of kojic acid esters. After reaction at 40°C for 10 d, the Penicillium and Pseudomonas lipases still retained 57.0% and 92.0% of their initial activities, respectively.
Keywords: Esterification; kojic acid; kojic acid ester; lipase; organic solvent
Lipase-catalyzed synthesis of chiral triglycerides
by Ian C. Chandler; Paul T. Quinlan; Gerald P. McNeill (pp. 1513-1518).
Under certain reaction conditions, the acidolysis of tripalmitin with oleic acid using immobilized lipase from Rhizomucor miehei resulted in a higher level of monosubstituted oleoyldipalmitoyl (OPP) triglycerides than had been predicted according to kinetic modeling. The reaction products were subjected to chiral analysis by high-performance liquid chromatography (HPLC), which indicated that the enzyme was more active at the sn-1 position of the triglyceride than at the sn-3 position, resulting in synthesis of the chiral triglyceride 1-oleoyl-2,3-dipalmitoyl-sn-glycerol. A kinetic model was developed and was correlated with the HPLC method to provide a simple means to predict the stereoselectivity of lipase-catalyzed reactions. By using the model, the stereoselectivity of immobilized Rhizomucor miehei lipase was found to depend strongly on the initial water activity (a w) of the reaction mixture, with greater selectivity occurring at lower a w. The sn-1 selectivity was essentially maintained using various solvents, or without solvent, when a w was kept constantly low. Variation in the fatty acid composition of the triglyceride indicated that shorter-chain fatty acids result in greater stereoselectivity, while variation of the chainlength of the free fatty acid indicated an enhancement by the longest chainlength. The stereoselectivity of this lipase was confirmed using a new 13C nuclear magnetic resonance method. By using immobilized R. miehei lipase at low a w approximately 80% of the chiral triglyceride found in the reaction mixture was the sn-1 enantiomer, at high reaction conversion.
Keywords: Acidolysis; chain length; chiral; free fatty acid; lipase; solvent type; source organism; stereoselectivity; triglyceride; water activity
Evaluation of immobilized modified lipase: Aqueous preparation and reaction studies in n-hexane
by Kenneth D. Green; Mitsutoshi Nakajima (pp. 1519-1526).
Lipase Saiken 100 (Rhizopus japonicus) and its immobilized form displayed very poor activity (hydrolysis and interesterification) in microaqueous n-hexane solutions. Enzyme modification by the addition of stearic acid or sorbitan monostearate significantly improved activity. A ceramic carrier (SM-10) was used to immobilize modified lipase Saiken (stearic acid, sorbitan monostearate, and lecithin) and was found to further enhance hydrolysis and interesterification rates in n-hexane. In addition, the biocatalysts were re-used for four consecutive batch reactions with no significant shortfall in activity. Reaction rates were also greatly affected by the total reaction water content. Careful control of the biocatalyst water content prior to use and additional reaction water were required to optimize activity and minimize hydrolytic diglyceride byproducts. Hydrolysis and interesterification reaction rates were favored with immobilized biocatalyst water contents of 6.25 and 0.43 wt% with additional reaction water contents of 600 and 20 mg/L, respectively.
Keywords: Fats and oils; n-hexane; hydrolysis; immobilized lipases; interesterification; modification; stearic acid
Optimization of the reaction conditions in the lipase-catalyzed synthesis of structured triglycerides
by U. Schmid; U. T. Bornscheuer; M. M. Soumanou; G. P. McNeill; R. D. Schmid (pp. 1527-1531).
Structured triglycerides of the ABA-type, containing one type of fatty acid (A) in the sn-1 and sn-3 positions and a second type of fatty acid (B) in the sn-2 position of the glycerol, were synthesized using lipases. The highest yields and purities were achieved in a two-step process, where a triglyceride of the B-type was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn-1,3-regiospecific lipases yielding the corresponding 2-monoglyceride (2-MG). Using this strategy, e.g., 2-monopalmitin (2-MP) was obtained in up to 88% yield at >95% purity by crystallization. Esterification of 2-MP with oleic acid resulted in the formation of 1,3-oleyl-2-palmitoyl-glycerol in up to 72% yield containing 94% palmitic acid in the sn-2 position. The best lipases were from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus. Water activity, solvent, and carrier for lipase immobilization strongly influenced the yield and purity of the products in both steps. Furthermore, 2-MG from fish oil were produced by alcoholysis in up to 84% yield at >95% purity.
Keywords: Alcoholysis; betapol; DHA; EPA; fish oil; immobilization; lipase; 2-monopalmitin; structured triglyceride; water activity
Solvent-free enzymatic synthesis of structured lipids from peanut oil and caprylic acid in a stirred tank batch reactor
by Ki-Teak Lee; Casimir C. Akoh (pp. 1533-1537).
Structured lipids were synthesized by transesterification of peanut oil and caprylic acid in a stirred-batch reactor. Different substrate molar ratios (1:1 to 1:4, peanut oil/caprylic acid) were used. The reaction was performed for 72 h at 50°C catalyzed by IM60 lipase from Rhizomucor miehei (10 g, 2% w/w substrate) in the absence of organic solvent. The highest incorporation of caprylic acid was obtained with a 1:2 molar ratio (peanut oil/caprylic acid) after 72 h reaction. With a 1:2 molar ratio, the incorporation increased by 28% from 1:1. On the other hand, a 1:4 molar ratio gave the lowest incorporation during the reaction. The effect of different mixing speeds (200, 640, or 750 rpm) on reaction was studied with a 1:2 substrate molar ratio for 24 h. A high incorporation of caprylic acid (14.3 mol%) was obtained at 640 rpm, while 200 rpm gave the lowest incorporation (2.2 mol%), suggesting that good mixing is essential in a stirred-batch reactor. After 24 h of reaction at different rpm, IM60 lipase was recovered, washed with hexane, and reacted with substrates to study its stability after reaction at different mixing speeds. The results showed that caprylic acid incorporation was similar (24.9, 24.3, 24.2 mol%) at 200, 640, and 750 rpm, respectively. When 20 g of IM60 lipase (4% w/w substrate) instead of 10 g was used in a 1:1 substrate molar ratio reaction, the incorporation of caprylic acid increased by 26% after 72 h. To study enzyme reuse, 10 g of IM60 lipase was used in a 1:1 substrate molar ratio for 24 h at 640 rpm. The incorporation of caprylic acid gradually decreased with increased number of reuses. During five times of reuse, 15, 13.9, 9.6, 6.7, and 9.7 mol% of caprylic acid were incorporated into peanut oil, respectively.
Keywords: Caprylic acid; peanut oil; solvent-free; stirred-batch reactor; structured lipids; transesterification
Large-scale purification of γ-linolenic acid by selective esterification using Rhizopus delemar lipase
by Yuji Shimada; Norihito Sakai; Akio Sugihara; Hiroyuki Fujita; Yo Honda; Yoshio Tominaga (pp. 1539-1544).
γ-Linolenic acid (GLA) is a physiologically valuable fatty acid, and is desired as a medicine, but a useful method available for industrial purification has not been established. Thus, large-scale purification was attempted by a combination of enzymatic reactions and distillation. An oil containing 45% GLA (GLA45 oil) produced by selective hydrolysis of borage oil was used as a starting material. GLA45 oil was hydrolyzed at 35°C in a mixture containing 33% water and 250 U/g-reaction mixture of Pseudomonas sp. lipase; 91.5% hydrolysis was attained after 24 h. Film distillation of the dehydrated reaction mixture separated free fatty acids (FFA; acid value 199) with a recovery of 94.5%. The FFA were selectively esterified at 30°C for 16 h with two molar equivalents of lauryl alcohol and 50 U/g of Rhizopus delemar lipase in a mixture containing 20% water. The esterification extent was 52%, and the GLA content in the FFA fraction was raised to 89.5%. FFA and lauryl esters were not separated by film distillation, but the FFA-rich fraction contaminated with 18% lauryl esters was recovered by simple distillation. To further increase the GLA content, the FFA-rich fraction was selectively esterified again under similar conditions. As a result, the GLA content in the FFA fraction was raised to 97.3% at 15.2% esterification. After simple distillation of the reaction mixture, lauryl esters contaminating the FFA-rich fraction were completely eliminated by urea adduct fractionation. When 10 kg of GLA45 oil was used as a starting material, 2.07 kg of FFA with 98.6% GLA was obtained with a recovery of 49.4% of the initial content.
Keywords: Borage oil; distillation; hydrolysis; γ-linolenic acid; Pseudomonas sp. lipase; purification; Rhizopus delemar lipase; selective esterification; urea adduct
Synthesis of polyfunctional glycerol esters: Lipase-Catalyzed esterification of glycerol with diesters
by P. Villeneuve; T. A. Foglia; T. J. Mangos; A. Nuñez (pp. 1545-1549).
The enzymatic esterification of glycerol with dicarboxylic acids or esters was studied to produce mono- and/or diesterified glycerol adducts. Such materials are useful synthons in the synthesis of biodegradable polymers and surfactants. In this work two strategies were studied for preparing these prepolymeric glycerol derivatives: the lipase-catalyzed esterification of free glycerol with diacids or esters and the reaction of supported or protected glycerol with diesters. For example, reaction of isopropylidene glycerol with dimethyl sebacate gave a >95% yield of isopropylidene glycerol-monomethyl sebacate ester. Reaction of glycerol supported on silica with dimethyl adipate gave a 40% yield of glycerol-monomethyl adipate ester. Best yields of glycerol-mono- and diesters (70% and 10%, respectively) were obtained by direct esterification of free glycerol with a diester in a solvent-free system containing small amounts of water (<4%).
Keywords: Dicarboxylic acids; dimethyl esters; glycerolysis; partial glycerides; polyesters; prepolymers
Separation of eicosapentaenoic acid and docosahexaenoic acid in fish oil by kinetic resolution using lipase
by Gudmundur G. Haraldsson; Björn Kristinsson (pp. 1551-1556).
The objective of this study was to investigate the use of lipases as catalysts for separating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil by kinetic resolution. Transesterification of various fish oil triglycerides with a stoichiometric amount of ethanol by immobilized Rhizomucor miehei lipase under anhydrous solvent-free conditions resulted in a good separation. When free fatty acids from the various fish oils were directly esterified with ethanol under similar conditions, greatly improved results were obtained. By this modification, complications related to regioselectivity of the lipase and nonhomogeneous distribution of EPA and DHA into the various positions of the triglycerides were avoided. As an example, when tuna oil comprising 6% EPA and 23% DHA was transesterified with ethanol, 65% conversion into ethyl esters was obtained after 24 h. The residual glyceride mixture contained 49% DHA and 6% EPA (8:1), with 90% DHA recovery into the glyceride mixture and 60% EPA recovery into the ethyl ester product. When the corresponding tuna oil free fatty acids were directly esterified with ethanol, 68% conversion was obtained after only 8h. The residual free fatty acids comprised 74% DHA and only 3% EPA (25:1). The recovery of both DHA into the residual free fatty acid fraction and EPA into the ethyl ester product remained very high, 83 and 87%, respectively.
Keywords: DHA; EPA; esterification; ethanolysis; fish oil; kinetic resolution; lipase; Lipozyme IM; n-3 fatty acids; Rhizomucor miehei lipase
Transesterification of trimethylolpropane and rapeseed oil methyl ester to environmentally acceptable lubricants
by Esa Uosukainen; Yu-Yen Linko; Merja Lämsä; Tommi Tervakangas; Pekka Linko (pp. 1557-1563).
Biodegradable trimethylolpropane [2-ethyl-2-(hydroxymethyl)-1,3-propanediol] esters of rapeseed oil fatty acids were synthesized by transesterification with rapeseed oil methyl ester both by enzymatic and chemical means, both in bench and pilot scales. Nearly complete conversions were obtained with both techniques. A reduced pressure of about 2 to 5 kPa, to remove the methanol formed during transesterification, was critical for a high product yield. The quantity of added water was also critical in the biocatalysis. Candida rugosa lipase was used as biocatalyst and an alkaline catalyst in chemical transesterifications. In biocatalysis the maximum total conversion to trimethylolpropane esters of up to 98% was obtained at 42°C, 5.3 kPa, and 15% added water. The maximum conversion of about 70% to the tri-ester was obtained at the slightly higher temperature of 47°C. The reaction time was longer in the biocatalysis, but considerably higher temperatures were required in chemical synthesis. In the chemical synthesis tri-ester yields increased when the temperature was first held at 85 to 110°C for 2.5 h and subsequently increased to up to 120°C for 8 h. The trimethylolpropane esters obtained were tested as biodegradable hydraulic fluids and compared to commercially available hydraulic oils. The hydraulic fluids based on trimethylolpropane esters of rapeseed oil had good cold stability, friction and wear characteristics, and resistance against oxidation at elevated temperatures.
Keywords: Biocatalysis; 2-ethyl-2-(hydroxymethyl)-1,3-propanediol; hydraulic oil; lipase; lubricant; rapeseed oil; trimethylolpropane; transesterification
Purification of ethyl docosahexaenoate by selective alcoholysis of fatty acid ethyl esters with immobilized Rhizomucor miehei lipase
by Yuji Shimada; Kazuaki Maruyama; Akio Sugihara; Takashi Baba; Sadao Komemushi; Shigeru Moriyama; Yoshio Tominaga (pp. 1565-1571).
Ethyl docosahexaenoate (E-DHA) is efficiently enriched by the selective alcoholysis of ethyl esters originating from tuna oil with lauryl alcohol using immobilized lipase. Alcoholysis of ethyl esters by immobilized Rhizopus delemar lipase raised the E-DHA content in the unreacted ethyl ester fraction from 23 to 49 mol% in 90% yield. However, the content of ethyl eicosapentaenoate (E-EPA) was higher than the initial content. Hence we attempted to screen for a suitable lipase to decrease the E-EPA content, and chose Rhizomucor miehei lipase. Several factors affecting the alcoholysis of ethyl esters were investigated, and the reaction conditions were determined. When alcoholysis was performed at 30°C with shaking in a mixture containing ethyl esters/lauryl alcohol (1:3, mol/mol) and 4 wt% of the immobilized R. miehei lipase, the E-DHA content in the ethyl ester fraction was increased and the E-EPA content was decreased. By alcoholyzing ethyl esters in which the E-DHA content was 45 mol% (E-tuna-45) for 26 h, the E-DHA content was increased to 74 mol% in 71% yield and the E-EPA content was decreased from 12 to 6.2 mol%. To investigate the stability of the immobilized lipase, batch reactions were carried out continually by replacing the reaction mixture with fresh E-tuna-45/lauryl alcohol (1:3, mol/mol) every 24 h. The decrease in the alcoholysis extent was only 17% even after 100 cycles of reaction. It was found that increasing the proportion of lauryl alcohol increased the conversion of E-EPA to lauryl-EPA. When an ethyl ester mixture in which the E-DHA content was 60 mol% (E-tuna-60) was alcoholyzed for 24 h with 7 molar equivalents of lauryl alcohol, the E-DHA content was raised to 93 mol% with 74% yield and the E-EPA content was reduced from 8.6 to 2.9 mol%.
Keywords: Ethyl docosahexaenoate; immobilized enzyme; lipase; Rhizomucor miehei ; selective alcoholysis; tuna oil
Production of specific-structured lipids by enzymatic interesterification in a pilot continuous enzyme bed reactor
by X. Xu; S. Balchen; C. E. Høy; J. Adler-Nissen (pp. 1573-1579).
Production of specific-structured lipids (interesterified lipids with a specific structure) by enzymatic interesterification was carried out in a continuous enzyme bed pilot scale reactor. Commercial immobilized lipase (Lipozyme IM) was used and investigations of acyl migration, pressure drop, water dependence, production efficiency, and other basic features of the process were performed. The extent of acyl migration (defined as a side reaction) occurring in the present enzyme bed reactor was compared to that in a pilot batch reactor. The continuous enzyme bed reactor was better than the batch reactor in minimizing acyl migration. Generally the former produced about one-fourth the acyl migration produced by the latter at a similar extent of incorporation. Pressure drop and production efficiency were evaluated in order to obtain a suitable yield in one reaction step. High incorporation was favored by high substrate ratios between acyl donors and oils, requiring long reaction times on the enzyme bed. Under these conditions, the pressure drop of the reactor was modeled statistically and theoretically. Residence time, water content, and effects of mass transfers were also investigated. Incorporation of medium-chain fatty acids increased with increased residence time. Approximately 40% of lipase activity was lost after a 4-wk run. External mass transfer was not a major problem in the linear flow range, but internal mass transfer did impose some transfer limitations.
Keywords: Acyl migration; enzyme bed reactor; incorporation; lipase-catalyzed interesterification; Lipozyme IM; pressure drop; Rhizomucor miehei ; specific-structured lipids
Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction
by Yuji Shimada; Nobuhiro Fukushima; Hiroyuki Fujita; Yo Honda; Akio Sugihara; Yoshio Tominaga (pp. 1581-1586).
A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.
Keywords: Borage oil; Candida rugosa lipase; γ-linolenic acid; selective hydrolysis
Trans fatty acids in Canadian margarines: Recent trends
by W. M. N. Ratnayake; G. Pelletier; R. Hollywood; S. Bacler; D. Leyte (pp. 1587-1594).
The fatty acid composition and the trans fatty acid content of the top-selling 109 Canadian margarines were determined by a combined capillary gas-liquid chromatography/infrared spectroscopy method. The 109 brands accounted for 68% of the margarine brands sold in Canada and represented 74% of the market share. The mean level of total trans content in tub margarines (n=79) was 18.8% (g/100 g fatty acids) and ranged from 0.9 to 46.4%. The most frequent occurrence of trans in tub margarines was in the 15–20% range; 48 of the 79 tub brands were in this range but seven brands contained more than 40% trans. The trans content of hard margarines (n=30) ranged from 16.3 to 43.7% and the mean value was 34.3%. In 20 of the 109 brands, the levels of trans,trans isomers of linoleic acid exceeded the maximum level of 1% recommended for Canadian margarines. The levels of cis,trans/trans,cis isomers of linoleic acid were also high; 78 brands contained more than 1% and in 16 brands, the levels were in the 6–7% range. Linoleic acid content in the 109 brands ranged from 1.0 to 45.2% and averaged 18.3%. In 33 samples, linoleic acid was below the level of 5% recommended by an ad hoc committee of Health Canada. Moreover, in these, the total trans content exceeded 30%, and trans polyunsaturated fatty acid level was greater than 5%. There were eight margarines prepared from nonhydrogenated fat and their total trans content was below 2.5%. From the trans content and market share of each of the margarine brands, the average intake of trans fatty acids from margarine was estimated as 0.96 g/person/d. The intake of trans fatty acids in Canada from various sources was previously estimated by us as 8.4 g/person/d. Thus it is suggested that only 11% of the dietary trans fatty acids are supplied by margarines and the majority of trans fatty acids in the Canadian diet is derived from hidden fats in fast foods and bakery products.
Keywords: Fatty acids; gas chromatography; hydrogenated vegetable oils; cis-trans isomers; linoleic acid; α-linolenic acid; margarine
Antioxidant and radical-scavenging activities of buckwheat seed components
by R. Przybylski; Y. C. Lee; N. A. M. Eskin (pp. 1595-1601).
The search for endogenous components in food ingredients exhibiting antioxidant activity has been intensified in order to eliminate synthetic antioxidants. Tocopherols are widely used as natural antioxidants, although their protective ability is not always sufficient. Buckwheat seed components were evaluated for antioxidant and free radical-scavenging activities using solvents of different polarities to isolate components from hulls and groats. Components extracted from buckwheat hulls were pro-oxidant in canola oil. Antioxidant activity of extracts from buckwheat groats increased when more polar solvents were used for extraction. The highest activity was observed for the methanolic extract. Radical-scavenging activity of buckwheat extracts was analyzed with DPPH (2,2-diphenyl-2-picryl-hydrazyl). This activity increased when the more polar solvents were used for extraction, with the highest activity observed for the methanolic extract. It was also observed that the radical scavenging effectiveness of extracts was concentration dependent. Analysis revealed the presence of tocopherols in the hexane extract, while methanolic extracts were rich in phenolic acids and flavonoids.
Keywords: Antioxidants; buckwheat; DPPH; phenolics; radicals; scavengers; solvents; stability; tocopherols
Effects of storage conditions on lipid oxidation in infant formulas based on several protein sources
by A. J. Angulo; J. M. Romera; M. Ramírez; A. Gil (pp. 1603-1607).
The time course of several lipid oxidation parameters in infant formulas was the main focus of this study. Infant formulas based on different protein sources (milk protein, isolated soy protein, and hydrolyzed milk protein) were stored at different temperatures (32 and 55°C) in sealed containers in air for 1 yr. Levels of thiobarbituric acid-reactive substances and 7-ketocholesterol, and losses of essential fatty acids and tocopherols were measured to monitor lipid oxidation. Tocopherol losses and 7-ketocholesterol concentrations were better lipid oxidation parameters than the others. Their concentration and losses depended on type of infant formula, storage temperature, and time.
Keywords: Antioxidants; cholesterol oxidation; infant formulas; lipid oxidation
Relation of fat bloom in chocolate to polymorphic transition of cocoa butter
by J. Bricknell; R. W. Hartel (pp. 1609-1615).
A special chocolate with spray-dried sugar (50:50 w/w sucrose/20 Dextrose Equivalent corn syrup solids) was made to study the polymorphic changes in cocoa butter crystals using X-ray diffraction. Anhydrous milk fat (AMF) and high-, middle-, and low-melting milk fat fractions were used to replace 2% (w/w) of cocoa butter. Chocolates were tempered, and the consistency of temper among chocolate samples was verified by a temper meter. Chocolates were cycled between 19 and 29°C at 6-h intervals to induce fat bloom. The special chocolates were analyzed by X-ray spectroscopy and colormeter.X-ray analysis on the special chocolates showed polymorphic transition from the βV to the βVI form of cocoa butter. After a lag phase, the percentage of the βVI form rapidly increased. However, the sample made with the high-melting milk fat fraction transformed slowly to βVI. Visual bloom appeared rapidly on the special chocolates made with AMF, middle- and low-melting fractions, whereas visual bloom was very slow to appear on the special chocolates made with high-melting milk fat fraction and on the cocoa butter control. The commercial chocolate responded consistently; the control bloomed rapidly, the AMF exhibited some bloom resistance, and the high-melting fraction inhibited bloom. Despite the βV to βVI transition, the control chocolates with amorphous sugar did not bloom. Since the only difference in the chocolates was sugar microstructure, differences in bloom formation were caused by the microstructure, not the polymorphic transition.
Keywords: Bloom; chocolate; milk fat; milk fat fractions; polymorphic transition
Thermal analysis of isothermal crystallization kinetics in blends of cocoa butter with milk fat or milk fat fractions
by Serpil Metin; Richard W. Hartel (pp. 1617-1624).
The kinetics of isothermal crystallization of binary mixtures of cocoa butter with milk fat and milk fat fractions were evaluated by applying the Avrami equation. Application of the Avrami equation to isothermal crystallization of the fats and the binary fat blends revealed different nucleation and growth mechanisms for the fats, based on the Avrami exponent. The suggested mechanism for cocoa butter crystallization was heterogeneous nucleation and spherulitic growth from sporadic nuclei. For milk fat, the mechanism was instantaneous heterogeneous nucleation followed by spherulitic growth. For milk fat fractions, the mechanism was high nucleation rate at the beginning of crystallization, which decreased with time, and plate-like growth. Addition of milk fat fractions did not cause a significant change in the suggested nucleation and growth mechanism of cocoa butter.
Keywords: Avrami equation; cocoa butter; crystallization kinetics; DSC; fats; fractionation; isothermal crystallization; milk fat; milk fat fractions
Physico-chemical characteristics of palm-based oil blends for the production of reduced fat spreads
by Habi Mat Dian Noor Lida; A. Rahim Md Ali (pp. 1625-1631).
The effect of blending and interesterification on the physicochemical characteristics of fat blends containing palm oil products was studied. The characteristics of the palm-based blends were tailored to resemble oil blends extracted from commercial reduced fat spreads (RFS). The commercial products were found to contain up to 20.4% trans fatty acids, whereas the palm-based blends were free of trans fatty acids. Slip melting point of the blends varied from 26.0–32.0°C for tub, and 30.0–33.0°C for block RFS. Solid fat content at 5 and 10°C (refrigeration temperature), respectively, varied from 10.9–19.7% and 8.5–17.6% for tub, and 28.2–38.6% and 20.8–33.5% for block RFS. Melting enthalpy of the tub RFS varied from 35.0–54.3 J/g and that of block RFS varied from 58.0–75.4 J/g. To produce block RFS, 65% palm oil (PO) and 18% palm kernel olein (PKOo) could be added in a ternary blend with sunflower oil (SFO), but only 47% PO and 10% PKOo are suggested for tub RFS. Higher proportion of PO, i.e., 72% for block RFS and 65% for tub RFS, could be used after the ternary blend was interesterified. Although a ternary blend of palm olein (POo)/SFO/PKOo was not suitable for RFS formulation, after interesterification as much as 90% POo and 26% PKOo could be used in the block RFS formulation. For tub RFS a maximum of 30% POo was found suitable.
Keywords: Chemical interesterification; differential scanning calorimetry; palm kernel olein; palm oil; palm olein IV60; reduced fat spreads; ternary blends
The influence of chemical interesterification on the physicochemical properties of complex fat systems. 3. Rheology and fractality of the crystal network
by Alejandro G. Marangoni; Dérick Rousseau (pp. 1633-1636).
Chemically interesterified and noninteresterified lard-canola oil (LCO) and palm oil-soybean oil blends ranging from 100% hardstock to 50%:50% hardstock/vegetable oil (w/w) were evaluated for hardness index (HI) using cone penetrometry and viscoelastic properties, such as shear storage modulus G′, using controlled-stress rheometry. The HI and G′ of palm oil decreased upon addition of soybean oil, and chemical interesterification did not affect the HI or G′ of palm oil or palm oil-soybean oil blends. The HI and G′ of lard decreased upon addition of canola oil, while chemical interesterification led to increases in both the HI and G′ of lard and LCO blends. All these effects were nonsolid fat content-related, since solid fat content did not change substantially upon chemical interesterification. The microstructure of the fat crystal network in lard and palm oil was quantified rheologically using fractal analysis. Chemical interesterification did not affect the fractal dimension of the fat crystal networks in palm oil or lard (2.82 and 2.88, respectively). The rheological properties of the macroscopic systems were not affected by the spatial distribution of mass within their fat crystal networks. Moreover, our results suggest that the increases in G′ observed in lard upon chemical interesterification are potentially due to changes in the properties of the particles which make up the network (crystal habit).
Keywords: Chemical interesterification; cone penetrometry; fractal; lard; palm oil; viscoelasticity
The seed fatty acid composition and the distribution of Δ5-olefinic acids in the triacylglycerols of some taxaceae (Taxus and Torreya)
by Robert L. Wolff; Frédérique Pédrono; Anne M. Marpeau; William W. Christie; Frank D. Gunstone (pp. 1637-1641).
The fatty acid compositions of the seeds from three Taxus (yew) species and one Torreya species belonging to the Taxaceae family [Taxus cuspidata (Japanese yew), T. chinensis (Chinese yew), T. baccata (English yew), and Torreya grandis (Chinese nutmeg yew)] have been established. These compositions were compared with those previously published for T. canadensis (Canadian yew) and Torreya nucifera. In Taxus species, as well as in Torreya species, Δ5-olefinic acids are present in the seed lipids from all species analyzed. In Taxus, 5,9-18:2 (taxoleic) acid is the prominent Δ5-olefinic acid. It represents between 9.5 and 16.2% of total fatty acids. Other Δ5-olefinic acids that occur in low amounts are 5,9,12-18:3 (<3.5%), 5,11-20:2 (<0.3%), 5,11,14-20:3 (<2.2%), and 5,11,14,17-20:4 (<0.3%) acids. In Torreya species, the major Δ5-olefinic acid is 5,11,14-20:3 (sciadonic) acid (between 6.7 and 11.2%). In contrast to Taxus species, the 5,9-18:2 and 5,9,12-18:3 acids are scarce in Torreya species: less than 0.1%. Also, the 9,12,15-18:3 acid content is significantly lower in Torreya than in Taxus. The prominence of taxoleic acid among Δ5-olefinic acids in the seed lipids is a unique characteristic of the genus Taxus that isolates it from all other Coniferophytes analyzed so far. However, this feature is not shared by other Taxaceae species, such as Torreya, and with regard to their seed fatty acid compositions, the family Taxaceae appears particularly heterogeneous. Our observations favor the hypothesis that in Gymnosperm seeds, there might exist two Δ5-desaturases, one specific for unsaturated acids with a Δ9-ethylenic bond (active in Taxus but not in Torreya), and the other specific for unsaturated acids with a Δ11-ethylenic bond (active in Torreya but not in Taxus). Our data also highlight the importance of the elongase(s) in the metabolism of fatty acids in Gymnosperm seeds. 14-Methylhexadecanoic acid, a habitual component of Pinaceae and Ginkgo biloba seed lipids, could not be detected in the Taxaceae studied here. 13C nuclear magnetic resonance spectroscopy of the oils from both genera has confirmed that Δ5-olefinic acids are apparently excluded from the internal position of triacylglycerols, which is a characteristic common to all Gymnosperm species analyzed so far, and consequently of great antiquity in their life history.
Keywords: Fatty acid composition; Δ5-olefinic acids; sciadonic acid; seed lipids; taxoleic acid; Taxaceae; taxonomy; Taxus ; Torrreya
Hydroxytyrosol and tyrosol as the main compounds found in the phenolic fraction of steam-exploded olive stones
by J. Fernández-Bolaños; B. Felizón; M. Brenes; R. Guillén; A. Heredia (pp. 1643-1649).
The lignocellulosic by-products, whole stones, and seed husks obtained from processing pitted table olives and oil olives were pretreated under various conditions of steam explosion, with and without previous acid impregnation. The various water-soluble noncarbohydrate compounds generated during steam explosion, such as sugar degradation compounds (furfural and hydroxymethylfurfural), lignin degradation compounds (vanillic acid, syringic acid, vanillin, and syringaldehyde) and the simple phenolic compounds characteristic of olive fruit (tyrosol and hydroxytyrosol), were identified. The amount of hydroxytyrosol solubilized was higher than that of the other compounds, and increased with increasing steaming temperature and time. This suggests its presence as a structural component of the olive stone.
Keywords: Hydroxytyrosol; lignin; lignocellulosic by-products; olive seed husks; phenolic compounds; steam explosion; tyrosol; whole olive stones
Identification of trans-3,5-dimethoxystilbene in commercial soy protein isolates
by W. L. Boatright; J. St. Pyrek; J. Song; A. D. Crum (pp. 1651-1655).
A previously unidentified component of the lipid extracts from commercial soy protein isolates (SPI) was analyzed by gas chromatography-mass spectrometry (GC-MS), high resolution mass spectrometry (HRMS), ultraviolet-visible spectroscopy, and GC-Fourier transform infrared spectrometry (FTIR). All these data, together with mass spectra of derivatives obtained by hydrogenation, indicated the structure of an unsymmetrical dimethoxystilbene. Subsequently, standard trans-3,5-dimethoxystilbene, synthesized according to established procedures, was found to have identical retention times and spectra by GC-MS and GC-FTIR with the compound isolated from commercial SPI. Laboratory SPI prepared from Probst, Stressland, and Burlison variety soybeans contained no detectable amounts of either trans-3,5-dimethoxystilbene or dehydroabietinal.
Keywords: Dehydroabietinal; trans-3,5-dimethoxystilbene; Fourier transform infrared spectrometry; mass spectrometry; nuclear magnetic resonance; protein; soy protein
Lipids of Haliphthoros philippinensis: An oomycetous marine microbe
by H. Kim; S. R. Gandhi; R. A. Moreau; J. D. Weete (pp. 1657-1665).
Lipids of the marine oomycetous microbe Haliphthoros philippinensis were characterized by chromatographic and spectroscopic techniques. Total lipid content of this organism was relatively low and not very responsive to manipulation of the culture conditions. Neutral lipid comprised 21% of the total lipid and the polar lipids were mainly phosphatidylcholine (44%), phosphatidylethanolamine (15%), and a ceramide-phosphorylethanolamine (19%). Palmitic (16:0) was the primary saturated fatty acid at 25% of the total fatty acids, and arachidonic acid (20:4n-6, ARA) and eicosapentaenoic acid (20:5n-3, EPA) were the major unsaturated fatty acids at 19 and 21%, respectively. Fucosterol was the principal sterol at 59% of the total sterols. The effects of several cultivation variables on growth and EPA production by this species were investigated. Among those tested, glucose and sodium glutamate were the most efficient carbon and nitrogen sources for growth, respectively. When the mycelium was cultivated for 6 d to produce biomass under optimal growth conditions, and then transferred to low temperature for an additional 13 d without glucose, the EPA content reached 31% of the total fatty acids and the yield was 203 mg/L. When the same experiment was performed with glucose supplementation during the low-temperature phase, EPA composed 27% of total fatty acids and yield reached 316 mg/L, or a 285% increase over that from mycelium cultured for 6 d at 24°C, and 56% over that cultured at 16°C for 13 d. ARA production did not respond accordingly.
Keywords: Arachidonic acid; eicosapentaenoic acid; fatty acids; fucosterol; Haliphthoros philippinensis ; lipid; Oomycete; phospholipid; sphingolipid; sterols
Similarities in the lipid class profiles of oils from Atlantic and Pacific dogfish livers
by S-J. Kang; M. C. A. Timmins; R. G. Ackman (pp. 1667-1672).
Liver oils from Atlantic and Pacific dogfish (Squalus acanthias) have been compared for lipid classes, fatty acids of the total oil and of important lipid classes, and details of the alkyl chains in the 1-O-alkyl-2,3-diacylglycerol ethers (DAGE). In general there were few striking differences, confirming the view of biologists that these sharks are one species. The Pacific dogfish liver oil had a higher content (41.2%) of DAGE than the oil from Atlantic dogfish (18.2%). Both oils had all common and expected fatty acids in the proportions usual for marine oils, but they differed in the eicosenoic chains of the glycerol ethers (GE). The Pacific oil was unusual in having low but similar proportions of two alkyl chain isomers, 20:1n-11 and 20:1n-9. The Atlantic oil was very high in the 20:1n-11 isomer, which is usually lower than 20:1n-9 in the fatty acids of most regional marine oils. Unexpectedly, the DAGE of both oils had further unusual 20:1 isomer proportion in the GE chain, with 20:1n-7 >20:1n-9. Minor oddities in the fatty acids may reflect different basic food sources.
Keywords: 1-O-Alkyl-2,3-diacylglycerol ethers; dogfish liver oil; fatty acids; glycerol ethers; shark liver oil
Studies on lipid and fatty acid compositions of puffer livers from Indian coastal waters with seasonal variation
by Alok K. Hazra; Somiranjan Ghosh; Shivaji Banerjee; Biswapati Mukherjee (pp. 1673-1678).
The puffer fishes Chelonodon patoca, Sphaeroides oblongus, Lagocephalus lunaris, and L. inermis of Indian coastal waters are wasted in huge quantity. The livers of these fishes were investigated for their lipid contents and fatty acid compositions in different seasons. It was found that monsoon season is the suitable time to obtain the maximal lipids (40.1–48.8%) from their livers, an amount similar to cod liver lipid content (39.5–55.0%). The fatty acids were mostly saturates and monoenes (60–70%). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) concentrations (7–12%) were high during monsoon season. Neutral lipids were the predominant lipid class (>80%) and comprised triglycerides (277–674 mg/g) and cholesterol (0.6–3.1 mg/g). Quality indices of puffer liver lipids, e.g., specific gravity, refractive index, acid value, iodine value, saponification value and unsaponifiable matter, were evaluated. Puffer liver lipids were quantitatively and qualitatively comparable to other commercially important marine fish oils. The overall study suggests the possibility of future commercial utilization of liver lipids from puffer, an unconventional, cheap, and easily available source.
Keywords: DHA; DPA; EPA; fatty acids; liver lipids; puffer fish; quality index; seasonal variation
5α,8α-Epidioxycholest-6-en-3-β-ol from three cone snails of the Indian ocean
by M. Aknin; I. Viracaoundin; R. Faure; E. M. Gaydou (pp. 1679-1681).
Three cone snail species (Conus ebraeus, C. leopardus, C. tessulatus, family Conidae) have been investigated for their sterolic fraction. Besides cholesterol, 5α,8α-epidioxycholest-6-en-3-β-ol has been isolated from each species and the structure determined using two-dimensional nuclear magnetic resonance. The occurrence of 5α,8α-epidioxycholest-6-en-3-β-ol in high amounts in the crude extract (0.5%), constitutes an interesting source of this compound.
Keywords: Cholesterol; cones; Conus ebraeus ; Conus leopardus ; Conus tessulatus ; 5α,8α-epidioxycholest-6-en-3-β-ol; sterols
Physical refining of rice bran oil in relation to degumming and dewaxing
by B. K. De; D. K. Bhattacharyya (pp. 1683-1686).
Physical refining of rice bran oil (RBO) with acidity between 4.0 and 12.4% has been investigated in relation to degumming and dewaxing pretretments. It appears that physical refining after combined low-temperature (10°C) degumming-dewaxing produces good-quality RBO with respect to color, free fatty acid, oryzanol, and tocopherol content.
Keywords: Degumming; dewaxing; physical refining; rice bran oil
Differential extraction of eleostearic acid-rich lipid-protein complexes in tung seeds
by John M. Dyer; Fuqiang Tang; Dorselyn C. Chapital; Alan R. Lax; Hurley S. Shepherd; Ding S. Shih; Armand B. Pepperman (pp. 1687-1690).
Lipid-protein complexes were identified in the 104,000×g supernatant fraction of developing tung seeds. Incubation of this fraction with linoleoyl-CoA promoted an increase of chloroform-extractable lipids in a time-dependent manner. High-performance liquid chromatography analysis indicated that the extracted lipids were similar to mature tung oil triglycerides. Differential extraction using chloroform or chloroform/methanol indicated that linoleoyl-CoA promoted extraction of pre-existing lipids rather than de novo synthesis. An increase in extractable lipids was also observed after incubation with proteinase K. Isolation of lipid-protein complexes by sucrose density centrifugation and analysis of proteins by gel electrophoresis revealed several proteins specifically associated with this lipid fraction.
Keywords: Aleurites fordii ; differential extraction; eleostearic acid; linoleoyl-CoA; lipid-protein complexes; triglycerides; tung
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