Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification

Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield. To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but not with DHA. These results suggest that lauryl DHA was generated only by esterification.     

Purification of γ-linolenic acid from borage oil by a two-step enzymatic method

γ-Linolenic acid (GLA) was purified from borage oil by a two-step enzymatic method. The first step involved hydrolysis of borage oil (GLA content, 22.2 wt%) with lipase, Pseudomonas sp. enzyme (LIPOSAM). A mixture of 3 g borage oil, 2 g water, and 5000 units (U) LIPOSAM was incubated at 35°C with stirring at 500 rpm. The reaction was 91.5% complete after 24 h. The resulting free fatty acids (FFA) were extracted from the reaction mixture with n-hexane (GLA content, 22.5 wt%; recovery of GLA, 92.7%). The second step involved selective esterification of borage-FFA with lauryl alcohol by using Rhizopus delemar lipase. A mixture containing 4 g borage-FFA/lauryl alcohol (1:2, mol/mol), 1 g water, and 1000 U lipase was incubated at 30°C for 20 h with stirring at 500 rpm. Under these conditions, 74.4% of borage-FFA was esterified, and the GLA content in the FFA fraction was enriched from 22.5 to 70.2 wt% with a recovery of 75.1% of the initial content. To further elevate the GLA content, unesterified fatty acids were extracted, and esterified again in the same manner. By this repeated esterification, GLA was purified to 93.7 wt% with a recovery of 67.5% of its initial content.     

Ultrasonic extraction of ferulic acid from Ligusticum chuanxiong

The extraction of ferulic acid, a pharmacologically active ingredient from the root of Ligusticum chuanxiong, with ultrasonic extraction was investigated. Percolation and supercritical fluid extraction (SFE) were employed to make comparisons with ultrasonic extraction. Three variables, which included the concentration of solvent, the ratio of solvent volume/sample (mL/g), and extraction time, were found to have a great influence on ultrasonic extraction. The optimum extraction was with pure ethanol with a solvent volume/sample ratio 8:1 (mL/g) and extraction time of 30 min. Under the optimum extraction conditions, the extraction yield could reach 8.8% which was higher than that using SFE with ethanol as co-solvent and a content of ferulic acid of 0.7%; both the yield and the content were higher than those obtained by percolation. Ultrasonic extraction significantly shortened the time required for the extraction process. Overall, ultrasonic extraction was shown to be highly efficient in the extraction of ferulic acid from Ligusticum chuanxiong.     

Lipase-catalyzed esterification of lactic acid with straight-chain alcohols

Enzymatic synthesis of esters of lactic acid and straight-chain alcohols with different chain lengths (C₆–C₁₈) were investigated in batch reactions with hexadecanol (C₁₆) as the model alcohol. Cyclohexane was the best solvent for higher ester yields, and the best biocatalyst was the immobilized Candida antarctica lipase B (Novozym 435) as well as the textile-immobilized Candida sp. lipase. A method was established to obtain ester yields in the range of 71 to 82% for the different alcohols, and the most favorable conditions for the esterification reaction using Novozym 435 were an equimolar ratio of lactic acid to alcohol, each at a concentration of 120 mM each; a 50°C reaction temperature; 190 rpm shaking speed; and the addition of 100 mg molecular sieves (4 Å) for drying. The ester yield increased with increasing lipase load, and a yield of 79.2% could be obtained after 24 h of reaction at 20 wt% of Novozym 435. The immobilized Candida sp. lipase prepared in the laboratory also could be used to produce esters of lactic acid and straight-chain alcohols, but it had a much lower activity than Novozym 435 with a temperature optimum of 40°C.     

Biosynthetic pathway of diepoxy bicyclic FA from linoleic acid by Clavibacter sp. ALA2

The biosynthetic pathway of two bicyclic FA, 12∶17, 13∶17-diepoxy-9(Z)-octadecenoic acid (DEOA) and 7-hydroxy-12∶17, 13∶17-diepoxy-9(Z)-octadecenoic acid (hDEOA), by Clavibacter sp. ALA2 was investigated. When cultivated with linoleic acid as a substrate, the strain produced 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA), DEOA, and hDEOA as well as other FA. To clarify the synthetic route to these bicyclic FA, the strain was cultivated with purified THOA as a starting substrate. THOA was consumed almost completely by the strain with sequential generation of DEOA and hDEOA. Moreover, the strain produced hDEOA when cultivated with purified DEOA. Therefore, it was confirmed that THOA was a precursor of these bicyclic FA and that hDEOA was generated from DEOA. Based on our previously reported result that linoleic acid is first converted to 12,13-dihydroxy-9(Z)-octadecenoic acid (DHOA) and the present results, the overall biosynthetic pathway for the diepoxy bicyclic FA from linoleic acid was postulated as: linoleic acid→DHOA→THOA→DEOA→hDEOA.     

Enzymatic synthesis of trierucin from high-erucic acid rapeseed oil

The lipase fromCandida rugosa has been shown to discriminate against erucic acid. Advantage of this property has been taken to produce trierucin from high-erucic acid rapeseed (HEAR) oil. A method has been developed for extracting erucic acid from the oil as dierucin and subsequently enzymatically converting it to trierucin. Unrefined HEAR oil was hydrolyzed with lipase fromC. rugosa to produce a mixture of free fatty acids and dierucin. Precipitation and filtration from cold ethanol gave 73% pure dierucin, free of fatty acids. This dierucin was treated in two ways to produce trierucin. First, in the presence of an immobilized lipase and a known amount of water, some trierucin is produced by interesterification. Second, a more efficient route to trierucin utilizedRhizopus arrhizus lipase to completely hydrolyze dierucin to erucic acid, which was then combined with an appropriate amount of dierucin in the presence of an immobilized lipase to produce trierucin in a quantitative yield. Partly presented at the AOCS Annual Meeting held in Toronto, May 10–14, 1992.     

Microwave-assisted extraction of chlorogenic acid from flower buds of Lonicera japonica Thunb.

An efficient microwave-assisted extraction (MAE) technique has been developed to recover chlorogenic acid from flower buds of Lonicera japonica Thunb. The yield of chlorogenic acid rapidly reached 6.14% within 5 min under the optimal MAE conditions, i.e. 50% ethanol as extraction solvent, 1:10 (w/v) of the solid/liquid ratio and 60 °C of extraction temperature. The MAE showed obvious advantages in terms of short duration and high efficiency to recover chlorogenic acid from raw plant materials in comparison with conventional heat-reflux extraction. The mechanism of the enhanced extraction by microwave assistance was discussed by observing cell destruction of plant material after MAE treatment by scanning electron microscopy. The results showed that the plant materials were significantly destroyed due to the cell rupture after MAE treatment.     

Fatty Acid Desaturation and Elongation Reactions of <Emphasis Type="Italic">Trichoderma</Emphasis> sp. 1-OH-2-3

The fatty acid desaturation and elongation reactions catalyzed by Trichoderma sp. 1-OH-2-3 were investigated. This strain converted palmitic acid (16:0) mainly to stearic acid (18:0), and further to oleic acid (c9-18:1), linoleic acid (c9,c12-18:2), and α-linolenic acid (c9,c12,c15-18:3) through elongation, and Δ9, Δ12, and Δ15 desaturation reactions, respectively. Palmitoleic acid (c9-16:1) and cis-9,cis-12-hexadecadienoic acid were also produced from 16:0 by the strain. This strain converted n-tridecanoic acid (13:0) to cis-9-heptadecenoic acid and further to cis-9,cis-12-heptadecadienoic acid through elongation, and Δ9 and Δ12 desaturation reactions, respectively. trans-Vaccenic acid (t11-18:1) and trans-12-octadecenoic acid (t12-18:1) were desaturated by the strain through Δ9 desaturation. The products derived from t11-18:1 were identified as the conjugated linoleic acids (CLAs) of cis-9,trans-11-octadecadienoic acid and trans-9,trans-11-octadecadienoic acid. The product derived from t12-18:1 was identified as cis-9,trans-12-octadecadienoic acid. cis-6,cis-9-Octadecadienoic acid was desaturated to cis-6,cis-9,cis-12-octadecatrienoic acid by this strain through Δ12 desaturation. The broad substrate specificity of the elongation, and Δ9 and Δ12 desaturation reactions of the strain is useful for fatty acid biotransformation.     

Biosynthesis of tetrahydrofuranyl fatty acids from linoleic acid by <Emphasis Type="Italic">clavibacter</Emphasis> sp. ALA2

Clavibacter sp. ALA2 converts linoleic acid into many novel oxygenated products including hydroxy FA and tetrahydrofuranyl unsaturated FA (THFA). One of them was tentatively identified by GC-MS as 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA) (Hou, C.T., H.W. Gardner, and W. Brown, J Am. Oil Chem. Soc. 78∶1167–1169, 2001). We have separated and purified 12,13,16-THOA from its isomer, 12,13,17-THOA, by silica gel column chromatography and by preparative TLC. Its structure was then confirmed by proton and ¹³C NMR analyses. Purified 12,13,16-THOA was used as a substrate to study the biosynthesis of THFA. Within 24 h of incubation, cells of strain ALA2 converted 12,13,16-THOA to both 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid (12-hydroxy-THFA) and 7,12-dihydroxy-13,16-epoxy-9(Z)-octadecenoic acid (7,12-dihydroxy-THFA). The relative abundance of 7,12-dihydroxy-THFA increased with incubation time, whereas that of 12,13,16-THOA and of 12-hydroxy-THFA decreased. Therefore, the biosynthetic pathway of THFA from linoleic acid by strain ALA2 is as follows: linoleic acid→12,13-dihydroxy-9(Z)-octadecenoic acid→12,13,16-THOA→12-hydroxy-THEA→7,12-dihydroxy-THFA.     

Lipase PS-catalyzed transesterification of citronellyl butyrate and geranyl caproate: Effect of reaction parameters

Pseudomonas sp. lipase PS was immobilized by adsorption and tested for its ability to catalyze the synthesis of citronellyl butyrate and geranyl caproate by transesterification in n-hexane. The reaction parameters investigated were: enzyme load, effect of substrate concentration, added water, temperature, time course, organic solvent, pH memory, and enzyme reuse. Yields as high as 96 and 99% were obtained for citronellyl butyrate and geranyl caproate, respectively, with 300 units (approx. 15% w/w of reactants) of lipase PS. Increasing amounts of terpene alcohol inhibited lipase activity, while excess acyl donor (triacylglycerol) concentration enhanced ester production. Optimal yields were obtained at temperatures from 30–50°C after 24-h incubation time. Yields of 90 and 99% were obtained for citronellyl and geranyl esters, respectively, with 2% added water. Solvents with log P values ≥ 2.5 showed the highest conversion yields. pH 7 and 6–8 seemed to be ideal for citronellyl butyrate and geraniol caproate, respectively. The lipase remained active after reusing 12 times.     

Solvent extraction of rare earths from chloride medium with mixtures of 1-phenyl-3-methyl-4-benzoyl-pyrazalone-5 and sec-octylphenoxyacetic acid

In the present study, the extraction of rare earths with mixtures of 1-phenyl-3-methyl-4-benzoyl-pyrazalone-5 (HPMBP, HA) and sec-octylphenoxyacetic acid (CA12, H₂B₂) in benzene has been studied from chloride medium. The synergistic enhancement coefficient decreases with increasing atomic numbers of rare earths. The synergistic extraction of lanthanum and neodymium has been studied with the methods of slope analysis and constant mole. The extracted complexes are determined as LaH₂ClA₂B₂ and NdH₃ClA₃B₂, respectively. The equilibrium constants, formation constants, and the thermodynamic functions are calculated. Furthermore, the different extraction effects on rare earths have been employed to discuss the possibilities for the separation of rare earths.     

Oil content and fatty acid composition of seed oil from guayule plants with different chromosome numbers

Guayule, a perennial desert plant, is being developed for domestic production of natural rubber, a strategic commodity for which the United States presently depends totally on foreign sources. At present, rubber alone is not sufficient to make guayule a commercial crop, and additional revenues are being sought from by-products. Because guayule flowers profusely during several years of growth before it is harvested for rubber, seed may also contribute to the economics of guayule production. Seed from 120 plants, including 20 genotypes with 36, 37, 54 and 72 chromosomes, were analyzed for oil content and fatty acid composition. Oil content ranged from 17.1 to 30.5%. On average, seed from diploid and aneuploid plants (with 36 and 37 chromosomes) contained 40.4% more oil than the seed from polyploid plants. The oil consisted of four fatty acids—palmitic (8.7–11.5%), stearic (3.7–6.2%), oleic (6.5–13.9%) and linoleic (69.1–80.2%)—at all ploidy levels. Guayule seed oil was similar to the seed oil from high-linoleic safflower varieties. The use of genetic variation to increase seed yield and seed oil will depend on the absence of negative correlation between oil and rubber production.     

Lipase-catalyzed synthesis of lesquerolic acid wax and diol esters and their properties

Lesquerolic acid was and α,Ω-diol esters were synthesized via immobilizedRhizomucor miehei lipase-(Lipozyme) catalyzed esterification of lesquerolic acid and alcoholysis of lesquerella oil. For each wax ester synthesis, when alcohol substrate was present at a slight (ca. 20%) stoichiometric excess and water content was kept low, over 94% of the hydroxy acyl groups were esterified. The extent of reaction and the ratio of monoester to diester produced for α,Ω-diol reactions was controlled by the solubility of diol in the medium. This latter quantity increased as alcoholysis proceeded due to the formation of partial glycerides and monoesters, which increased the polarity of the medium. Alcoholysis reactions were significantly slower when the medium diol content was above saturation. As the diol chainlength increased, diol solubilization decreased, the ratio of monoester to diester decreased, and the extent of hydrolysis increased. Alcoholysis reactions involving either fatty alcohols or diols suffered from acyl migration, which lowered the purity of lesquerolic acid esters. Several lesquerolic acid esters, synthesized on a preparative scale and purified via column chromatography, were evaluated for their properties: density, viscosity, and melting point. Potential applications for lesquerolic acid esters are discussed.     

Enzymatic fractionation of fatty acids: Enrichment of γ-linolenic acid and docosahexaenoic acid by selective esterification catalyzed by lipases

Immobilized lipase preparations from seedlings of rape (Brassica napus L.) andMucor miehei (lipozyme) used as biocatalysts in esterification and hydrolysis reactions discriminate strongly against γ-linolenic and docosahexaenoic acids/acyl moieties. Utilizing this property, γ-linolenic acid contained in fatty acids of evening primrose oil has been enriched seven to nine-fold, from 9.5 to almost 85% by selective esterification of the other fatty acids with butanol. Similarly, docosahexaenoic acid of cod liver oil has been enriched four to five-fold, from 9.4 to 45% by selective esterification of fatty acids (other than docosahexaenoic acid) with butanol. As long as the reaction is stopped before reaching equilibrium, very little of either γ-linolenic acid or docosahexaenoic acid are converted to butyl esters, which results in high yields of these acids in the unesterified fatty acid fraction.     

Fatty acid composition, extraction, fractionation, and stabilization of bullfrog (Rana catesbeiana) oil

The oil extracted from the fat-storage organ (fat body) of the bullfrog (Rana catesbeiana) was characterized for its fatty acid composition. The main fatty acids were palmitic (18.1%), stearic (4.1%), myristic (2.7%), oleic (31.7%), and linoleic (12.9%) acids. Long-chain polyunsaturated fatty acids were also present in significant amounts, i.e., eicosapentaenoic (1.5%) and docosahexaenoic (4.7%), and were probably derived from the fish meal content of the diet. A partially fractionated oil was extracted from the homogenized and frozen fat body with an oleic acid content of 43.2%. The natural alkaloid boldine, added at 0.5 mg/g oil level, improved the oxidative stability by a factor ranging from 1.7 to 2.4, as assessed by the Oil Stability Index method between 90 and 110°C. The stabilization effect of boldine was higher than that of naringenin, morin, and quercitin and for the synthetic antioxidant butylated hydroxytoluene at the same concentration level.     

Identification and Synthesis of Volicitin and Related Components from Beet Armyworm Oral Secretions

Oral secretion of beet armyworm caterpillars (BAW), when applied to damaged tissues of corn seedlings, induces the seedlings to emit volatile compounds that attract the natural enemies of the caterpillars. The key elicitor present in BAW oral secretions is N-[17-hydroxylinolenoyl]-L-glutamine (volicitin). Analysis of the oral secretion showed that it also contained N-[17-hydroxyolinoleoyl]-L-glutamine, free 17-hydroxylinolenic, and 17-hydroxylinoleic acid, the glutamine conjugates of linolenic and linoleic acid as well as free linolenic and linoleic acid. Here we present the identification and synthesis of the hydroxy acids and of glutamine conjugates.     

Supercritical CO2 extraction of glycosides from Stevia rebaudiana leaves: Identification and optimization

The aim of this work was to optimize the glycoside composition of Stevia rebaudiana leaves using supercritical fluid extraction (SFE). A Box-Behnken statistical design was used to evaluate the effect of various values of pressure (150–350 bar), temperature (40–80 °C) and concentration of ethanol-water mixture (70:30) as co-solvent (0–20%) by CO₂ flow rate of 15 g min⁻¹ for 60 min. The most effective variables were co-solvent concentration (P < 0.005) and temperature (P ≤ 0.005). Evaluative criteria for both dependent variables (stevioside and rebaudioside A yields) in the model was assigned maximum. Optimum extraction conditions were elicited as 211 bar, 80 °C and 17.4% which yielded 36.66 mg/g stevioside and 17.79 mg/g rebaudioside A. Total glycosides composition were close to those obtained using conventional water extraction (64.49 mg/g) and a little higher than ethanol extraction (48.60 mg/g) demonstrating challenges for industrial scale application of SFE.     

Studies of the fatty acid specificity of the lipase fromRhizomucor miehei toward 20:1n-9, 20:5n-3, 22:1n-9 and 22:6n-3

The fatty acid specificity of the lipase fromRhizomucor miehei toward 20:1n-9, 20:5n-3, 22:1n-9 and 22:6n-3 has been determined by comparing the alcoholysis (byn-propanol) of various mixtures of C₂₀ and C₂₂ fatty acids (FFA) or the corresponding ethyl esters (FAEE) inn-heptane. For all the fatty acids examined, the degree of conversion was much higher when using FFA rather than FAEE. When comparing the experiments with either single FAEE or FAEE mixtures, it was found for all four fatty acids that the degree of conversion depended on whether the FAEE was alone or together with other fatty acids in the reaction mixture. The lipase showed a strong specificity toward 20:1n-9, whereas the polyunsaturated fatty acids were much poorer substrates, especially 22:6n-3. The degrees of conversion for the two n-3 fatty acids show a clear preference for 20:5n-3 over 22:6n-3, not only when present alone but also in the different mixtures examined. The results obtained in the present experiments therefore suggest that when using the lipase fromR. miehei for enrichment of fish oils with n-3 fatty acids, it should not only be possible to diminish the content of 20:1 and 22:1 present in the outer positions in the triacylglycerols, but also to incorporate relatively more 20:5n-3 than 22:6n-3 into the triglycerides.