Production of eicosapentaenoic acid bySaprolegnia sp. 28YTF-1

Saprolegnia sp. 28YTF-1, isolated from a freshwater sample, is a potent producer of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). The fungus used various kinds of carbon sources, such as starch, dextrin, sucrose, glucose, and olive oil for growth, and olive oil was the best carbon source for EPA production. The EPA content reached 17 mg/g dry mycelium (0.25 mg/L) when the fungus was grown in a medium that contained 2.5% olive oil and 0.5% yeast extract, at pH 6.0 and 28°C for 6 d with shaking. Accompanying production of arachidonic acid (AA; 3.2 mg/g dry mycelia, EPA/AA = 5.1) and other ω6 polyunsaturated fatty acids was low. Both EPA content and EPA/AA ratio increased in parallel by lowering growth temperature. Triglyceride was the major mycelial lipid (ca. 84%), but EPA comprised only 2.2% of the total fatty acids of this lipid. About 40% of the EPA produced was found in polar lipids, such as phosphatidylethanolamine (EPA content, 28.2%), phosphatidylcholine (13.6%), and phosphatidylserine (21.2%).     

The production potential of eicosapentaenoic and arachidonic acids by the red algaPorphyridium cruentum

The red microalgaPorphyridium cruentum is a new source for eicosapentanoic acid (EPA) and arachidonic acid (AA) fatty acids of potential pharmaceutical value. The conditions leading to a high content of either fatty acid were investigated. The highest EPA content was obtained under conditions resulting in high growth rate (2.4% of ash free dry weight in Strain 1380-1d). High AA content was obtained under slow growth conditions and was maximal in th stationary phase or under nitrogen starvation (2.9%). Strain 1380-la had the highest content (1.9%) of arachidonic acid under exponential growth conditions. By imposing nitrogen starvation, it was possible to obtain a lipid mixture which may be separated into AA and EPA rich fractions.     

Preparation of eicosapentaenoic acid (EPA) concentrate fromPorphyridium cruentum

The polyunsaturated fatty acid eicosapentaenoic acid (EPA) has attracted increased attention due to its pharmaceutical properties. The main source is marine fish oil which contains approximately 15% EPA. However, pharmaceutical applications of EPA will probably require higher concentrations, perhaps as high as 90%. The red microalgaPorphyridium cruentum is a potential source, because its EPA content approaches 44.1% of the total fatty acids. Three methods were attempted for EPA concentration and arachidonic acid (AA) removal from the oil of this alga. Separation of the glycolipids, formation of a urea inclusion complex and reverse phase chromatography on C-18 Sep-Pak filters resulted in an EPA concentrate of 97% purity. Similar methods resulted in an AA concentrate of 80% purity.     

Production of eicosapentaenoic acid by a bacterium isolated from mackerel intestines

Optimization of culture conditions for the growth rate, 5,8,11,14,17-cis-Eicosapentaenoic acid (EPA) content and EPA productivity of a bacterium isolated from Pacific mackerel intestines was investigated by use of a culture medium containing 1.00 wt% peptone and 0.50 wt% yeast extract in an artificial sea water (ASW). Cultivation temperature affected the growth rate and cellular EPA content of the bacterium. The cellular EPA content at 8°C was as great as 16.8 mg/g of dry cells, which was more than two times greater than that at 25°C (7.3 mg/g of dry cells), although the growth rate showed a maximum at 25°C. Both the yield of bacterial cells and the cellular EPA content at 25°C reached maximum values when the pH of the culture medium was nearly 7.0 and when the concentration of ASW was 100% (v/v). Under optimum culture conditions [25°C pH 7.0 and 100% (v/v) ASW], the amount of EPA accumulated in the cellular lipids reached 45.6 mg/L of culture broth after 8 hr.     

Growth and eicosapentaenoic acid production byPhaeodactylum tricornutum in batch and continuous culture systems

Maximum specific growth rate (μ_max) ofPhaeodactylum tricornutum increased with increasing culture reactor surface-to-volume ratio. Values for μ_max of 0.647, 0.377 and 0.339 day⁻¹ were observed for the 75-mL tube, 5.6-L tank and the 16-L tank, respectively. Higher biomass was achieved in the 75-mL batch culture tube under continuous light as compared with light cycle conditions. Palmitic acid, palmitoleic acid and eicosapentaenoic acid (EPA) accounted for over 60% of total fatty acids in the batch tube culture, with EPA content increasing to a maximum after three days. In chemostat cultures, run at dilution rates of 0.15 day⁻¹ (0.45 of μ_max) and 0.3 day⁻¹ (0.9 of μ_max), cell concentration reached a steady state of 2.18 and 0.7 g/L, respectively, while contents of EPA per liter of culture at steady state were 100.9 and 82.5 mg/L, respectively. At both dilution rates, EPA content of total fatty acids was the same (35.0–35.2%). At a dilution rate of 0.3 day⁻¹, the continuous culture system manifested productivities of 0.51 g/L/d and 25.1 mg/L/d for biomass and EPA, respectively.     

Effects of agingmortierella mycelium on production of arachidonic and eicosapentaenoic acids

Arachidonic acid and eicosapentaenoic acid (EPA) are important intermediates of eicosanoid metabolism and are presently the subject of extensive nutritional and medical research. The effects of mycelial aging on production of these fatty acids were investigated as part of a research program directed toward examining the feasibility of economically producing these products by fungal fermentation. Arachidonic acid content ofM. alpina ATCC 32222 increased from 4.1–8.3% to 13–16% during aging while lipid content of mycelium increased from 14–18% to 33–45%. Maximum lipid content produced in biomass during storage declined as harvesting time was increased from 3 to 6 days while maximum arachidonic acid content in lipid increased. Maximum lipid and arachidonic acid was produced during aging at pH 8, whereas arachidonic acid content of lipids was highest in mycelium aged at pH 6. EPA content ofM. elongata NRRL 5513 biomass increased during aging, reaching a maximum after 22–28 days. When the pH of the culture prior to harvesting was adjusted in the range of pH 4–9, pH values for development of maximum EPA in biomass and in lipids during storage were found to be 6 and 7, respectively. Temperature of aging had little effect on arachidonic acid or EPA content.     

Validation of a method for gas chromatographic analysis of eicosapentaenoic acid and docosahexaenoic acid as active ingredients in medicinal products

A gas chromatographic method for the determination of all-cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and all-cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) as active ingredients in medicinal products was developed and validated. In accordance with the rules governing medicinal products in the European Community, the method establishes relations between label claims of active ingredients and known reference standards. A routine for examining instrument status is proposed. The relative standard deviation was 1% (n=26) for determination of the empirical response factors of EPA ethyl ester and DHA ethyl ester relative to the internal standard, C23:0 methyl ester. This experiment included two columns and EPA and DHA standards from two different suppliers and was carried out over a five-month period. Repeatability (n=6) for low and medium concentrates of glycerides and high concentrates of ethyl esters, expressed as coefficient of variation, was 4, 0.7 and 0.7%, respectively. Accuracy (n=6) determined as percent recovery was better than 98% for all sample types. Analytical results from a twelve-month stability study of the high concentrate are shown. Presented at the AOCS Annual Meeting in Chicago, IL, May 1991.     

Production of an eicosapentaenoic acid-containing oil by a Δ12 desaturase-defective mutant ofMortierella alpina 1S-4

A Δ12 desaturase-defective mutant of an arachidonic acid (AA)-producing fungus,Mortierella alpina 1S-4, converted α-linolenic acid (18:3ω3) to 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid (EPA). On submerged cultivation at 20°C for 10 d in a 5-L fermentor containing medium comprising 1% glucose, 1% yeast extract and 3% (vol/vol) linseed oil, EPA production amounted toca. 1 g/L culture broth (64 mg/g dry mycelium), which accounted forca. 20% of the total mycelial fatty acids. AA content was 26 mg/g dry mycelium (0.4 g/L), accounting for 7.8% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (4.5%), oleic acid (20.4%), linoleic acid (10.0%), 18:3ω3 (20.3%) and lignoceric acid (4.3%). Most of the EPA produced (ca. 90 mol%) was in triglyceride form.     

Lipase-catalyzed modification of borage oil: Incorporation of capric and eicosapentaenoic acids to form structured lipids

Two immobilized lipases, nonspecific SP435 from Candida antarctica and sn-1,3 specific IM60 from Rhizomucor miehei, were used as biocatalysts for the restructuring of borage oil (Borago officinalis L.) to incorporate capric acid (10:0, medium-chain fatty acid) and eicosapentaenoic acid (20:5n-3) with the free fatty acids as acyl donors. Transesterification (acidolysis) reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The fatty acid profiles of the modified borage oil were different from that of unmodified borage oil. Higher incorporation of 20:5n-3 (10.2%) and 10:0 (26.3%) was obtained with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase. However, SP435 lipase was able to incorporate both 10:0 and 20:5n-3 fatty acids at the sn-2 position, but the IM60 lipase did not. Solvents with log P values between 3.5 and 4.5 supported the acidolysis reaction better than those with log P values between −0.33 and 3.0.     

Production of arachidonic acid by filamentous fungus, Mortierella alliacea strain YN-15

A filamentous fungus producing significant levels of arachidonic acid (AA, C20∶4n−6) was isolated from a freshwater pond sample and assigned to the species Mortierella alliacea. This strain, YN-15, accumulated AA mainly in the form of triglyceride in its mycelia. An optimized culture in 25 L of medium containing 12% glucose and 3% yeast extract yielded 46.1 g/L dry cell weight, 19.5 g/L total fatty acid, and 7.1 g/L AA by 7-d cultivation in a 50-L jar fermenter. Assimilation of soluble starch by YN-15 was notably enhanced by the addition of oleic acid, soybean oil, ammonium sulfate, or potassium phosphate to a starch-based medium. Using starch as a main carbon source in the pre-pilot scale cultivation improved the production of AA by up to 5.0 g/L. Mortierella alliacea strain YN-15 is therefore a promising fungal isolate for industrial production of AA and other polyunsaturated fatty acids.     

Lipase-assisted acidolysis of high-laurate canola oil with eicosapentaenoic acid

The production of structured lipids via acidolysis of high-laurate canola oil (Laurical 15) with EPA in hexane was carried out using lipase from Pseudomonas sp. The optimal reaction conditions used 4% lipase, at a mole ratio of oil to EPA of 1∶3 at 45°C over 36 h. The positional distribution of FA on the glycerol backbone of unmodified oil indicated that lauric acid was mainly located at the sn-1,3 positions. Stereospecific analysis of the oil modified with EPA showed that lauric acid remained mostly esterified to the sn-1,3 positions of the TAG molecules and that EPA was also primarily in the sn-1,3 positions of the TAG molecules. Thus, the resultant structured lipids may have optimal value for use in applications where quick energy release and EPA supplementation are required.     

Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid

Because of the complexity of marine lipids, polyunsaturated fatty acid (PUFA) derivatives in highly purified form are not easily prepared by any single fractionation technique. The products are usually prepared as the ethyl esters by esterification of the body oil of fat fish species and subsequent physicochemical purification processes, including short-path distillation, urea fractionation, and preparative chromatography. Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils. At room temperature in the presence of Pseudomonas sp. lipase and a stoichiometric amount of ethanol without any solvent, efficient transesterification of fish oil was obtained. At 52% conversion, a concentrate of 46% eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) was obtained in excellent recovery as a mixture of mono-, di-, and triacylglycerols. The latter can be easily separated from the saturated and monounsaturated ethyl esters and converted into ethyl esters either by conventional chemical means or enzymatically by immobilized Candida antarctica lipase. Urea-fractionation of such an intermediary product can give an EPA+DHA content of approximately 85%.     

Incorporation of eicosapentaenoic and docosahexaenoic acids into groundnut oil by lipase-catalyzed ester interchange

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were incorporated into groundnut oil by interesterification with a 1,3-specific lipase fromMucor miehei. The resultant EPA and DHA concentrations of the groundnut oil were 9.5 and 8.0%, respectively.     

Concentration of eicosapentaenoic acid by selective esterification using lipases

The aim of this work was to increase the content of EPA in FFA extracts from a commercial oil (43.1% EPA) and from Phaeodactylum tricornutum oil, a single-cell oil, by selective enzymatic esterification. Initially, the FFA extract was esterified with lauryl alcohol using nine lipases. All the lipases concentrated EPA in the unesterified FFA fraction. The criterion used to choose the best lipase was maximization of the dimensionless effectiveness factor (F_AE). This factor grouped the concentration factor (ratio between the EPA concentrations in the FFA fractions before and after esterification) with EPA recovery in the final FFA fraction. Experiments were carried out to correlate F_AE and the degree of esterification (ED, percentage of initial FA converted to lauryl esters). Lipase AK from Pseudomonas fluorescens was the most effective for concentrating EPA. Studies, of the optimal temperature, substrate molar ratio, solvent/substrate ratio, and treatment intensity (product of the lipase mass and the reaction time) were also carried out using the lipase. The maximum F_AE was obtained when the ED was 60%: EPA concentration was 72%, and recovery was 73%. Finally, this lipase was used to concentrate EPA from a FFA extract from P. tricornutum (23% EPA). The content of EPA in the unesterified FFA fraction increased to 71% at 78% ED (recovery of EPA, 75.5%). Comparison of the results of obtained with the two FFA extracts seemed to indicate that the selectivity of Lipase AK for EPA depended on the content of EPA, with higher contents of EPA in the initial FFA mixture reducing the selectivity for EPA.     

Recovery of eicosapentaenoic acid from fungal mycelia by solvent extraction

Utilization of lipids containing eicosapentaenoic acid (EPA) produced by microorganisms requires processes for their efficient recovery from microbial cells. Recovery of EPA from mycelia of the fungusPythium irregulare by solvent extraction with hexane-isopropanol (HIP) in a pilot-plant colloid mill was investigated. Extraction efficiencies of 96% for lipid and EPA were achieved with a 3∶2 (vol/vol) HIP mixture by milling wet, filtered mycelia for 5 min at a solvent/dry solids ratio of 100 L/kg. The process yielded a crude extract that contained up to 96% lipid and an EPA content as high as 24% (with no selectivity for EPA).     

富里酸诱导对三角褐指藻EPA合成积累的影响

探究不同富里酸质量浓度对三角褐指藻二十碳五烯酸(EPA)合成积累的影响。结果表明，在质量浓度20 mg/L富里酸诱导下，三角褐指藻EPA含量和产量均达到最大值，为19.81 g/100 g和738.91 mg/L，较无富里酸诱导EPA含量和产量分别提高1.12和1.50倍。外源富里酸作用提高了三角褐指藻胞内过氧化物酶(POD)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)的酶活，使色素(叶绿素、类胡萝卜素)和总多酚抗氧化组分比例增加，活性氧(ROS)和丙二醛(MDA)水平降低，整体抗氧化能力获得显著提升，有效阻抑EPA的氧化分解；富里酸作用同时还上调了EPA合成通路去饱和酶(Δ⁵-FADS、Δ⁶-FADS、Δ¹²-FADS、Δ¹⁵-FADS、Δ¹⁷-FADS)和延长酶(Δ⁶-ELOVL)等系列关键酶，使其上调表达量较无富里酸作用分别增加0.33、0.94、0.33、0.58、1.15和0.70倍，为EPA高效合成积累提供前驱物和能量。     

富里酸诱导对三角褐指藻EPA合成积累的影响

探究不同富里酸作用浓度对三角褐指藻二十碳五烯酸（EPA）合成积累的影响。结果表明,在20 mg/L富里酸诱导下,三角褐指藻EPA含量和产量均达到最大值,为19.81 g/100 g和738.91 mg/L,较无富里酸诱导EPA含量和产量分别提高2.01和2.54倍。外源富里酸作用提高了三角褐指藻胞内过氧化物酶（POD）、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）酶活,使得色素和多酚抗氧化组分比例增加,活性氧（ROS）和丙二醛（MDA）水平降低,整体抗氧化能力获得显著提升,有效阻抑EPA的氧化分解;富里酸作用同时还上调了EPA合成通路脱饱和酶（Δ5-FADS、Δ6-FADS、Δ12-FADS、Δ15-FADS、Δ17-FADS）和延长酶（Δ6-ELOVL）等系列关键酶,使其上调表达量较无富里酸作用分别增加1.27、1.81、1.24、1.45、1.43和1.46倍,为EPA高效合成积累提供前驱物和能量。富里酸作用效果协同强化了三角褐指藻EPA合成积累效率。     

Autoxidation of synthetic isomers of triacylglycerol containing eicosapentaenoic acid

Several triacylglycerols (TAG) that contained eicosapentaenoic acid (EPA) were chemically synthesized and stored at 25°C to assess the influence of TAG structure on oxidative stability and formation of oxidation products. Oxidative stability was evaluated by oxygen consumption during storage of the TAG. Autoxidation products of TAG were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Results showed that a 2:1 (mole/mole) mixture of trieicosapentaenoylglycerol (EEE) and tripalmitoylglycerol (PPP) was most susceptible to autoxidation. The oxidative stability of TAG that contained EPA and palmitic acid was negatively correlated with the moles of EPA in a single TAG molecule. When TAG with one EPA and two other fatty acids were oxidized, chainlength of constituent fatty acids hardly affected the oxidative stability of EPA-containing TAG molecules, except for stearic acid. HPLC and LC-MS analyses showed that monohydroperoxides were major oxidation products regardless of type of TAG. Bis- and tris-hydroperoxides were formed during autoxidation of EEE and dieicos-apentaenoylpalmitoylglycerol. Monohydroperoxy epidioxides were found in all autoxidized TAG. These observations suggested that TAG structure affected the oxidation of TAG with highly unsaturated fatty acids.     

Oxidation of synthetic triacylglycerols containing eicosapentaenoic and docosahexaenoic acids: Effect of oxidation system and triacylglycerol structure

Thirteen synthetic triacylglycerols (TAG) containing eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) were oxidized in the presence of 2,2′-azobis(2,4-dimethyl-valeronitrile) (AMVN) and 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) as aqueous and nonaqueous radical initiators to investigate the influence of TAG structure and oxidation system on the oxidative stability of TAG that contain highly unsaturated fatty acids (HUFA). A 2:1 (mol/mol) mixture of trieicosapentaenoylglycerol and tripalmitoylglycerol was most susceptible to the AMVN-initiated oxidation among three types of TAG that contained EPA and palmitic acid (2:1, mol/mol). Compared with 1,2 (or 2,3)-dieicosapentaenoyl-3(or 1)-palmitoylglycerol (EEP) and 1,3-dieicosapentaenoyl-2-palmitoylglycerol (EPE), the oxidative rate of EEP was somewhat higher. A similar result was obtained for DHA-containing TAG. The oxidative rate of TAG that contained EPA and palmitic acid (1:2, mol/mol) showed a positive correlation with the amount of EPA in a single TAG molecule. Moreover, in the nonaqueous system, the oxidative rate of EPA-containing TAG was affected by unsaturation and carbon chainlength of constituent fatty acids. In the AAPH-initiated oxidation in the aqueous system, the oxidative rate of TAG with EPA and palmitic acid was higher with the increased quantity of EPA in a single TAG molecule. Also, constituent fatty acids modified the oxidative rate of EPA-containing TAG in an aqueous system. The glycerol position of EPA and DHA also affected the oxidative rate of the TAG. EPA and DHA located at the 1,2 (or 2,3)-position of glycerol were more oxidizable than those at the 1,3-position during AAPH-initiated oxidation. Thus, 1,2(or2,3)-dipalmitoyl-3(or 1)-eicosapentaenoylglycerol was oxidized faster than 1,3-dipalmitoyl-2-eicosapentaenoylglycerol. These observations suggest that the oxidative stability of TAG that contain HUFA could be modulated by the oxidation system and TAG structure.     

Production of high yields of docosahexaenoic acid by Schizochytrium sp. strain SR21

The culture conditions for high-yield production of docosahexaenoic acid (DHA) by Schizochytrium sp. strain SR21 were investigated in a fermenter. With increasing carbon (glucose) and nitrogen (corn steep liquor and ammonium sulfate) sources (up to 12% glucose) in the medium, DHA productivity increased without a decrease in growth rate, i.e., 2.0, 2.7, and 3.3 g DHA/L/d with 6, 10, and 12% glucose, respectively. Eventually, 48.1 g dry cells/L and 13.3 g DHA/L were produced in 4 d with 12% glucose. DHA productivity was decreased with 15% glucose, i.e., 3.1 g/L/d. With 12% glucose, the lipid content was 77.5% of dry cells, and DHA content was 35.6% of total fatty acids. The lipid was composed of about 95% neutral lipid and 5% polar lipid. In polar lipids, the contents of phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylinositol were 74, 11, and 5%, respectively. The PC profile was simple, 70% of PC molecules were 1-palmitoyl-2-DHA-PC and 1.2-di-DHA-PC. These results indicate that Schizochytrium sp. strain 21 is an excellent source for microbial DHA production, including not only the acid form of DHA but also 2-DHA-PC.