ABSTRACT: Conjugated linoleic acid l-menthyl ester was hydrolyzed in water by the lipase from Candida rugosa with the addition of an organic solvent. The degree of hydrolysis (yield) greatly improved when a tertiary alcohol, such as t-butyl alcohol, was added. However, the addition of a less polar solvent, such as hexane, decreased the degree of hydrolysis, and some water-miscible solvents, such as acetone, caused inactivation of the lipase. With the addition of t-butyl alcohol, the reaction mixture formed a one- or two-phase system, and the mixing ratio of substrates and t-butyl alcohol determined the number of phases. Although the degree of hydrolysis at 10 d was higher in the one-phase system, the initial reaction rate was generally lower. Meanwhile, the reaction was much faster in the two-phase system while maintaining a moderate degree of hydrolysis.
SpringerPlus. 12/2012; 1(1):67.
ABSTRACT: We isolated two new microorganisms capable of converting vegetable oil to several rare unsaturated fatty acids and rare unsaturated
fatty alcohols from a soil sample. The strains were identified as belonging to the same genus and species, Aeromonas hydrophila. The rare unsaturated fatty acids and rare unsaturated fatty alcohols were accumulated as a wax ester form by the strains.
Compared to other strains, the A. hydrophila isolates effectively decreased fatty acid chain lengths and converted rapeseed oil, which is rich in 9-C18:1 fatty acid,
into rare fatty acids, such as 7-C16:1 fatty acid and 5-C14:1 fatty acid. Furthermore, the A. hydrophila isolates converted the resulting fatty acids to rare unsaturated fatty alcohols, such as 7-C16:1 fatty alcohol and 5-C14:1
fatty alcohol. The isolates also converted safflower oil, which is rich in 9,12-C18:2 fatty acid, to 7,10-C16:2 fatty acid,
5,8-C14:2 fatty acid, 9,12-C18:2 fatty alcohol, 7,10-C16:2 fatty alcohol, and 5,8-C14:2 fatty alcohol. 7,10,13-C16:3 fatty
acid, 9,12,15-C18:3 fatty alcohol, and 7,10,13-C16:3 fatty alcohol were also converted from linseed oil, which is rich in
9,12,15-C18:3 fatty acid, by the A. hydrophila isolates. These fatty acids and fatty alcohols are rarely found in natural oils. Since decreasing fatty acid carbon chain
lengths from the carboxyl end and reducing unsaturated fatty acids to unsaturated fatty alcohols are both difficult reactions
to accomplish by chemical means, we suggest that these A. hydrophila isolates may facilitate introduction of new bioprocess for producing rare unsaturated fatty acids and rare unsaturated fatty
alcohols, especially fatty alcohols harboring more than two double bonds.
Journal of Oil & Fat Industries 04/2012; 86(12):1189-1197. · 1.77 Impact Factor
ABSTRACT: Purification of arachidonic acid (AA) from Mortierella alpina single-cell oil was attempted. The process comprised three steps: (i) preparation of FFA by nonselective hydrolysis of the
oil with Alcaligenes sp. lipase; (ii) elimination of long-chain saturated FA from the resulting FFA by urea adduct fractionation; and (iii) enrichment
of AA through lipase-catalyzed selective esterification with lauryl alcohol (LauOH). In the third step, screening of industrially
available lipases indicated that Burkholderia cepacia lipase (Lipase-PS, Amano Enzyme Inc., Aichi, Japan) acted on AA more weakly than on other FA and was the most effective for
enrichment of AA in the FFA fraction. When the FFA obtained by urea adduct fractionation were esterified with 2 molar equivalents
of LauOH at 30°C for 16 h in a mixture with 20% water and 20 units (U)/g-mixture of Lipase-PS, the esterification reached
39% and the content of AA in the FFA fraction was raised from 61 to 86 wt%. To further increase the content of AA, unesterified
FFA were allowed to react again under the same conditions as those in the first selective esterification except for the use
of 50 U/g Lipase-PS. A series of procedures raised the content of AA to 97 wt% with a 49% recovery based on the initial content
in the single-cell oil. These results indicated that the three-step process for selective esterification with Lipase-PS was
effective for purifying AA from the single-cell oil.
Journal of Oil & Fat Industries 04/2012; 82(11):833-837. · 1.77 Impact Factor
ABSTRACT: Acid oil is a by-product in the neutralization step of vegetable oil refining and is an alternative source of biodiesel fuel.
A model substrate of acid oil, which is composed of TAG and FFA, was used in experiments on the conversion to FAME by immobilized
Candida antarctica lipase. FFA in the mixture of TAG/FFA were efficiently esterified with methanol (MeOH), but the water generated by the esterification
significantly inhibited methanolysis of TAG. We thus attempted to convert a mixture of TAG/FFA to FAME by a two-step process
comprising methyl esterification of FFA and methanolysis of TAG by immobilized C. antarctica lipase. The first reaction was conducted at 30°C in a mixture of TAG/FFA (1∶1, wt/wt) and 10 wt% MeOH using 0.5 wt% immobilized
lipase, resulting in efficient esterification of FFA. The reaction mixture after 24 h was composed of 49.1 wt% TAG, 1.3 wt%
FFA, 49.1 wt% FAME, and negligible amounts of DAG and MAG (<0.5 wt%). The reaction mixture was then dehydrated and used as
a substrate for the second reaction, which was conducted at 30°C in a solution of the dehydrated mixture and 5.5 wt% MeOH
using 6 wt% immobilized lipase. The activity of the lipase increased gradually when the reaction was repeated by transferring
the enzyme to a fresh substrate mixture. The activity reached a maximum after 6 cycles, and the content of FAME achieved was
>98.5 wt% after a 24-h reaction. The immobilized lipase was very stable in the first-and second-step reactions and could be
used for >100 d without significant loss of activity.
Journal of Oil & Fat Industries 04/2012; 82(11):825-831. · 1.77 Impact Factor
ABSTRACT: An industrially available preparation of astaxanthin (Ax) from Haematococcus pluvialis contained 41.6 wt% acylglycerols and 24.9 wt% FFA in addition to 14.6 wt% Ax, which was a mixture of free and FA ester forms
(free Ax/Ax monoesters/Ax diesters=4.9∶80.3∶14.8, by mol). Enrichment of Ax by a two-step process was attempted. The first
step was hydrolysis of acylglycerols with Candida rugosa lipase: A mixture of 1.0 kg H. pluvialis cell extracts, 1.0 L water, and 50 U/g-reaction mixture of the lipase was agitated at 30°C for 42 h. The degree of hydrolysis
of acylglycerols reached 94.4%, but Ax esters were not hydrolyzed. Removal of FFA from the resulting oil layer by molecular
distillation enriched the content of Ax esters to 40.8 wt5 (named Ax40). The second step was enzymatic conversion of Ax esters
to free Ax, which successfully proceeded in the presence of ethanol (EtOH). When a mixture of 50.0 g Ax40, 8.2 g EtOH (5 molar
equiv. against FA), 58.2 mL water, and 1500 U/g-mixture of Pseudomonas aeruginosa lipase was stirred at 30°C for 68 h, the free Ax content increased to 89.3 mol%. Free Ax was efficiently recovered by precipitation
with n-hexane. The purity of Ax was thereby raised to 70.2 wt% with a 63.9% overall recovery of the initial content in the cell
Journal of Oil & Fat Industries 04/2012; 80(10):975-981. · 1.77 Impact Factor
ABSTRACT: A commercial product of CLA contains almost equal amounts of cis-9,trans-11 (c9,t11)-CLA and trans-10,cis-12 (t10,c12)-CLA. We attempted to enrich the two isomers by a two-step selective esterification using Candida rugosa lipase that acted on c9,t11-CLA more strongly than on t10,c12-CLA. An FFA mixture containing CLA isomers was esterified with an equimolar amount of lauryl alcohol in a mixture of 20%
water and the lipase. When the esterification of total FA reached 50%, two isomers were fractionated in a good yield: t10,c12-CLA was enriched in FFA, and c9,t11-CLA was recovered in lauryl esters. The FFA were esterified again to enrich t10,c12-CLA. At 27.3% esterification of total FA, the t10,c12-CLA content in FFA increased to 64.8 wt% with 89.3% recovery: The ratio of the content of t10,c12-CLA to that of two isomers was 95.9%. Lauryl esters obtained by the single esterification were employed for enrichment
of c9,t11-CLA. After the esters were hydrolyzed, the resulting FFA were esterified again with lauryl alcohol. At 62.0% esterification
of total FA, the c9,t11-CLA content in lauryl esters increased to 73.3 wt% with 79.4% recovery: The ratio of the content of c9,t11-CLA to that of two isomers was 95.6%. In a 600-g-scale purification, molecular distillation was effective in separating
the reaction mixture into lauryl alcohol, FFA, and lauryl ester fractions.
Journal of Oil & Fat Industries 04/2012; 79(3):303-308. · 1.77 Impact Factor
ABSTRACT: We attempted to synthesize high-purity structured triacylglycerols (TAG) with caprylic acid (CA) at the 1,3-positions and
a polyunsaturated fatty acid (PUFA) at the 2-position by a two-step enzymatic method. The first step was synthesis of TAG
of PUFA (TriP), and the second step was acidolysis of TriP with CA. Candida antarctica lipase was effective for the first reaction. When a reaction medium of PUFA/glycerol (3∶1, mol/mol) and 5% immobilized Candida lipase was mixed for 24 h at 40°C and 15 mm Hg, syntheses of TAG of γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic
acids reached 89, 89, 88, and 83%, respectively. In these reactions, the lipase could be used for at least 10 cycles without
significant loss of activity. In the second step, the resulting trieicosapentaenoin was acidolyzed at 30°C for 48h with 15
mol parts CA using 7% of immobilized Rhizopus delemar lipase. The CA content in the acylglycerol fraction reached 40 mol%. To increase the content further, the acylglycerols were
extracted from the reaction mixture with n-hexane and were allowed to react again with CA under conditions similar to those of the first acidolysis. After three successive
acidolysis reactions, the CA content reached 66 mol%. The content of dicapryloyl-eicosapentaenoyl-glycerol reached 86 wt%
of acylglycerols, and the ratio of 1,3-dicapryloyl-2-eicosapentaenoyl-glycerol to 1(3),2-dicapryloyl-3(1)-eicosapentaenoyl-glycerol
was 98∶2 (w/w). In this reaction, the lipase could be used for at least 20 cycles without significant loss of activity. Repeated
acidolysis of the other TriP with CA under similar conditions synthesized 1,3-dicapryloyl-2-γ-linolenoyl-glycerol, 1,3-dicapryloyl-2-arachidonoyl-glycerol,
and 1,3-dicapryloyl-2-docosahexaenoyl-glycerol in yields of 58, 87, and 19 wt%, respectively.
Journal of Oil & Fat Industries 04/2012; 78(6):611-616. · 1.77 Impact Factor
ABSTRACT: Human milk fat contains 20–25% palmitic acid, and about 70% of the fatty acid is esterified to the 2-position of triglycerides.
It was also reported that arachidonic acid (AA) accelerated the growth of preterm infants. Thus, we attempted the synthesis
of 1,3-arachidonoyl-2-palmitoyl-glycerol by acidolysis of tripalmitin with AA using 1,3-specific Rhizopus delemar lipase. When a mixture of 10 g tripalmitin/AA (1∶5, w/w) and 0.7 g immobilized Rhizopus lipase was incubated at 40°C for 24 h with stirring, the AA content in glycerides reached 59 mol%. The immobilized lipase
could be used five times without a decrease in the extent of acidolysis. Glycerides were extracted from the reaction mixture
with n-hexane, and regiospecific analysis was performed. As a result, the AA contents at the 1,3- and 2-positions were 56.9 and
3.2 mol%, respectively. It was therefore confirmed that the fatty acids at the 1,3-positions of triglyceride were exchanged
for AA. High-performance liquid chromatography showed that the contents of triarachidonin, 1,3-arachidonoyl-2-palmitoyl-glycerol,
and 1(3)-arachidonoyl-2,3(1)-palmitoyl-glycerol were 7.3, 75.9, and 12.4 wt%, respectively.
Journal of Oil & Fat Industries 04/2012; 77(1):89-93. · 1.77 Impact Factor
ABSTRACT: A newly developed 1,3-positionally specific thermostable lipase from Fusarium heterosporum (named R275A lipase) was immobilized on Dowex WBA for the production of structured lipid by acidolysis of tripalmitin (PPP)
with oleic acid (OA). The immobilized catalyst was fully activated by pretreatment at 50°C in a PPP/OA mixture containing
2% water. The pretreatment caused concomitant hydrolysis, but the hydrolysis was repressed using a substrate without water
in the subsequent reactions. The optimal reaction conditions were determined as follows: A mixture of PPP/OA (1∶2, w/w) and
8% immobilized lipase catalyst was incubated at 50°C for 24 h with shaking at 130 oscillations/min. The acidolysis reached
50% under these conditions, and the contents of triolein, 1,3-dioleoyl-2-palmitoyl-glycerol, 1(3),2-dioleoyl-3(1)-palmitoyl-glycerol,
1(3),2-palmitoyl-3(1)-oleoyl-glycerol, 1,3-dipalmitoyl-2-oleoyl-glycerol, and PPP in the reaction mixture were 8, 36, 4, 28,
1, and 6 mol%, respectively. The stabilities of immobilized R275A lipase catalyst and two immobilized catalysts containing
Rhizopus delemar or Rhizomucor miehei lipases were compared under the conditions mentioned above, with the catalysts being transferred to fresh substrate every
24 h. The half-life of the R275A lipase catalyst was 370 d, which was significantly longer than those of Rhizopus and Rhizomucor lipase catalysts.
Journal of Oil & Fat Industries 04/2012; 78(2):167-172. · 1.77 Impact Factor
ABSTRACT: Docosahexaenoic acid (DHA)-rich oil has been industrially produced by selective hydrolysis of tuna oil with a lipase that acts weakly on DHA. The free fatty acids (FFAs) generated in this process as by-products contain a high DHA concentration (46wt%) but are treated as industrial waste. This study attempted to reuse these by-product FFAs using a one-pot process, and succeeded in producing triacylglycerols (TAGs) through the esterification of the by-product FFAs with glycerol using immobilized Rhizomucor miehei lipase. Regiospecific analysis of the resulting TAGs showed that the content of DHA at the sn-1(3) position (51.7mol%) was higher than the content of DHA at the sn-2 position (17.3mol%). The DHA distribution in TAGs synthesized in this study was similar to the DHA distribution in TAGs from seal oil.
New Biotechnology 01/2011; 28(1):7-13. · 2.76 Impact Factor
ABSTRACT: Commercially available conjugated linoleic acid (CLA) is a mixture of two main isomers. Fractionation of the two isomers was performed by a lipase-catalyzed esterification of CLA with L-menthol. In this study, a GC analytical method was developed to simultaneously determine the degree of esterification and fatty acid (FA) compositions of CLA in the free fatty acid (FFA) and ester forms without separation of the FFA and the ester. The methylation of the oil phase of the reaction mixture was performed using trimethylsilyldiazomethane in a mixture of toluene/methanol. Only FFA was quantitatively methylated, whereas the other compounds were little changed. A GC analysis using a polar column was performed to simultaneously determine the degree of esterification and the fatty acid compositions.
Journal of oleo science 01/2011; 60(8):445-8. · 1.42 Impact Factor
ABSTRACT: Candida antarctica lipase has generally been known to be nonregiospecific. This report, however, showed that its regiospecificity was linearly correlated to the index of polarity of the reaction mixture, which was calculated based on the dielectric constant of the components. Thus, it was strongly indicated that the regiospecificity depended on the polarity of the environment; the higher the polarity, the stricter the regiospecificity. The highest 1(3)-regiospecificity was obtained in the transesterification of oil with ethanol among other alcohols investigated. Although methanol, which is more polar than ethanol, was expected to give highest regiospecificity, it was fatal to the lipase. The transesterification of oil with ethanol (1:10, wt/wt) at 30 degrees C for three hours efficiently accumulated 2-monoacylglycerols without significant fatty acid (FA) specificity. Thus, it was successfully applied to regiospecifically analyze FA composition of triacylglycerols containing saturated and unsaturated FAs of C4-C24.
New Biotechnology 06/2009; 26(1-2):23-8. · 2.76 Impact Factor
ABSTRACT: The effect of linoleic acid-menthyl ester (LAME) on lipid metabolism were assessed in HepG2 cells. It is well known that high level of apolipoprotein (apo) B100 in the serum is risk for atherosclerosis. Although linoleic acid (LA) treatment and LA plus L-mentol treatment increased apo B100 secretion, LAME treatment significantly decreased apo B100 secretion in HepG2 cells compared with control medium. The hypolipidemic effect of LAME was attributable to the suppression of triglyceride synthesis in HepG2 cells. It is also known that the risk of coronary heart disease is negatively related to the concentration of serum apo A-1. In the present study, LAME treatment increased apo A-1 secretion as compared with LA treatment in HepG2 cells. These results suggest that mentyl-esterification of fatty acids may be beneficial in anti-atherogenic dietary therapy.
Journal of oleo science 02/2009; 58(4):171-5. · 1.42 Impact Factor
02/2008: pages 59 - 82; , ISBN: 9780470385869
ABSTRACT: Acid oil, a by-product of vegetable oil refining, was enzymatically converted to fatty acid methyl esters (FAME). Acid oil
contained free fatty acids (FFA), acylglycerols, and lipophilic compounds. First, acylglycerols (11wt%) were hydrolyzed at
30°C by 20units Candida rugosa lipase/g-mixture with 40wt% water. The resulting oil layer containing 92wt% FFA was used for the next reaction, methyl
esterification of FFA to FAME by immobilized Candida antarctica lipase. A mixture of 66wt% oil layer and 34wt% methanol (5mol for FFA) were shaken at 30°C with 1.0wt% lipase. The degree
of esterification reached 96% after 24h. The resulting reaction mixture was then dehydrated and subjected to the second esterification
that was conducted with 2.2wt% methanol (5mol for residual FFA) and 1.0wt% immobilized lipase. The degree of esterification
of residual FFA reached 44%. The degree increased successfully to 72% (total degree of esterification 99%) by conducting the
reaction in the presence of 10wt% glycerol, because water in the oil layer was attracted to the glycerol layer. Over 98%
of total esterification was maintained, even though the first and the second esterification reactions were repeated every
24h for 40days. The enzymatic process comprising hydrolysis and methyl esterification produced an oil containing 91wt%
FAME, 1wt% FFA, 1wt% acylglycerols, and 7wt% lipophilic compounds.
Journal of Oil & Fat Industries 10/2007; 84(11):1015-1021. · 1.77 Impact Factor
Annals of the New York Academy of Sciences 12/2006; 613(1):362 - 365. · 3.15 Impact Factor
ABSTRACT: Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.
Bioscience Biotechnology and Biochemistry 03/2006; 70(2):355-62. · 1.28 Impact Factor
ABSTRACT: Commercially available preparations of CLA are composed of almost equal amounts of 9-cis,11-trans (9c,11t)-CLA and 10-trans,12-cis (10t,12c)-CLA. Each isomer was fractionated and enriched, for availability as a food supplement, by a process comprising selective
esterification with l-menthol by Candida rugosa lipase, distillation, and n-hexane extraction. The first selective esterification of CLA isomers was conducted with an equimolar amount of l-menthol of 30°C. The oil phase of the reaction mixture was fractionated into an l-menthyl ester fraction (9c,11t-CLA rich) and an FFA fraction (10t,12c-CLA rich) by distillation. The FFA fraction was esterified again with an equimolar amount of l-menthol to enrich 10t,12c-CLA. The 10t,12c-CLA preparation was obtained as the resulting FFA fraction by distillation. 10t,12c-CLA was enriched to 91% with 40% recovery. To enrich 9c,11t-CLA, the l-menthyl ester fraction in the first esterification was chemically hydrolyzed, and the resulting FFA were esterified again
with an equimolar amount of l-menthol. The 9c, 11t-CLA preparation was obtained by chemical hydrolysis of the resulting l-methyl ester fraction, followed by n-hexane extraction. 9c,11t-CLA was enriched to 94% with 42% recovery. This effective process for purification of CLA isomers using l-methol is applicable to the production of food supplements.
Journal of Oil & Fat Industries 01/2006; 83(2):93-99. · 1.77 Impact Factor
ABSTRACT: Production of MAG with CLA using Penicillium camembertii mono- and diacylglycerol lipase (referred to as lipase) was attempted for the purpose of expanding the application of CLA.
The commercial product of CLA (referred to as FFA-CLA) is a FFA mixture containing almost equal amounts of 9cis,11trans (9c,11t)-CLA and 10t,12c-CLA. Esterification of FFA-CLA with glycerol without dehydration achieved 84% esterification but produced almost equal amounts
of MAG and DAG. Esterification with dehydration not only achieved a high degree of esterification but also suppressed the
formation of DAG. When a mixture of FFA-CLA/glycerol (1∶2, mol/mol), 1% water, and 200 units/g-mixture of P. camembertii lipase was agitated at 30°C for 72 h with dehydration at 5 mm Hg, the degree of esterification reached 95% and the contents
of MAG and DAG were 90 and 6 wt%, respectively. This reaction system may be applied to the industrial production of MAG with
Journal of Oil & Fat Industries 01/2005; 82(9):619-623. · 1.77 Impact Factor
ABSTRACT: TAG (MLM) with medium-chain FA (MCFA) at the 1,3-positions and long-chain FA (LCFA) at the 2-position, and TAG (LMM) with
LCFA at the 1(3)-position and MCFA at 2,3(1)-positions are a pair of TAG regioisomers. Large-scale preparation of the two
TAG regioisomers was attempted. A commercially available FFA mixture (FFA-CLA) containing 9-cis, 11-trans (9c, 11t)- and 10t,12c-CLA was selected as LCFA, and caprylic acid (C8FA) was selected as MCFA. The MLM isomer was synthesized by acidolysis of acyglycerols (AG) containing two CLA isomers with
C8FA: A mixture of AG-CLA/C8 FA (1∶10, mol/mol) and 4 wt% immobilized Rhizomucor miehei lipase was agitated at 30°C for 72 h. The ratio of MLM to total AG was 51.1 wt%. Meanwhile, LMM isomer was synthesized by
acidolysis of tricaprylin with FFA-CLA: A mixture of tricaprylin/FFA-CLA (1∶2, mol/mol) and 4 wt% immobilized R. miehei lipase was agitated at 30°C for 24 h. The ratio of LMM to total AG was 51.8 wt%. MLM and LMM were purified from 1,968 and
813 g reaction mixtures by stepwise short-path distillation, respectively. Consequently, MLM was purified to 92.3% with 49.1%
recovery, and LMM was purified to 93.2% with 52.3% recovery. Regiospecific analyses of MLM and LMM indicated that the 2-positions
of MLM and LMM were 95.1 mol% LCFA and 98.3 mol% C8 FA, respectively. The results showed that a process comprising lipase reaction and short-path distillation is effective for
large-scale preparation of high-purity regiospecific TAG isomers.
Journal of Oil & Fat Industries 10/2004; 81(11):1013-1020. · 1.77 Impact Factor