Process for production of arachidonic acid concentrate by a strain of mortierella alpina

Various Mortierella alpina fungi were screened for their capacities to produce arachidonic acid. A strain of M. alpina was found to show the highest productivity. Arachidonic acid content of biomass and overall yield per litre of culture was highest in soya flour supplemented medium which produced dispersed mycelium. When the glucose concentration in the medium was varied from 30 to 100 g/L, biomass, lipid, arachidonic acid content of biomass and arachidonic acid yield increased with increasing glucose concentration. Several natural oils, when added to the growth medium, stimulated arachidonic acid production. After fermentation in a 20-L fermenter under optimal culture conditions, the arachidonic acid yield was 5.3 g/L, representing 34.2% w/w of total fatty acids and 13.7% w/w of biomass. An extract containing 72.5% w/w arachidonic acid was prepared from the recovered mycelium.     

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.     

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.     

Effect of mustard meal on the production of arachidonic acid by Mortierella elongata SC-208

A strain of Mortierella elongata SC-208 that was isolated from soil of a mustardseed extraction plant can produce arachidonic acid in significant amounts, and it was grown in three different media, one of which contained 0.5% deoiled mustard meal. The arachidonic acid content in the lipid part of dry mycelium (23.2 g/L) was as high as 33% w/w from the medium that contained mustard meal, and the overall yield of arachidonic acid was 0.49 g/L. The arachidonic acid contents in the phospholipid fraction and the triglyceride fraction were 39.5 and 30.2%, respectively.     

Production of dihomo-γ-linolenic acid byMortierella alpina 1S-4

The mycelial dihomo-γ-linolenic acid content of an arachidonic acid-producing fungus,Mortierella alpina 1S-4, was found to increase, with an accompanying marked decrease in its arachidonic acid content, on cultivation with sesame oil. The resultant mycelia were found to be a rich source of dihomo-γ-linolenic acid. This unique phenomenon was suggested to be due to specific repression of the conversion of dihomo-γ-linolenic acid to arachidonic acid by the oil. After fractionation of the oil with acetone into oil and non-oil fractions, it was found that the effective factor(s) was present in the non-oil fraction. In a study on optimization of the culture conditions for the production of dihomo-γ-linolenic acid byM. alpina 1S-4, a medium containing glucose, yeast extract and the non-oil fraction was found to be suitable for the production. Under the optimal conditions in a 50-1 fermentor, the fungus produced 107 mg of dihomo-γ-linolenic acid/g dry mycelia (2.17 g/l of culture broth). This value accounted for 23.1% of the total fatty acids in the lipids extracted from the mycelia. The mycelia were also rich in arachidonic acid (53.5 mg/g dry mycelia, 11.2%). Other major fatty acids in the lipids were palmitic acid (24.1%), stearic acid (7.0), oleic acid (20.1), linoleic acid (6.6) and γ-linolenic acid (4.1). On leave from Suntory Ltd.     

Near-Infrared Spectroscopy and Partial Least-Squares Regression for Determination of Arachidonic Acid in Powdered Oil

Near-infrared (NIR) spectroscopy was evaluated as a rapid method of predicting arachidonic acid content in powdered oil without the need for oil extraction. NIR spectra of powdered oil samples were obtained with an NIR spectrometer and correlated with arachidonic acid content determined by a modification of the AOCS Method. Partial Least-Squares regression was applied to calculate models for the prediction of arachidonic acid. The model developed with the raw spectra had the best performance in cross-validation (n = 72) and validation (n = 21) with a correlation coefficient of 0.965, and the root mean square error of cross-validation and prediction were both 0.50. The results show that NIR, a well-established and widely applied technique, can be applied to determine the arachidonic acid content in powdered oil.     

Lack of effects oftrans fatty acids on eicosanoid biosynthesis with adequate intakes of linoleic acid

The minimum requirement of linoleic acid to prevent effects of dietary C18trans fatty acids on eicosanoid biosynthesis in rats was assessed. In a first experiment, six groups of animals were fed diets with a high content oftrans fatty acids [20% of energy (en%)], and increasing amounts of linoleic acid (0.4 to 7.1 en%). In a second experiment, four groups of rats were fed diets designed to comparetrans fatty acids with saturated andcis-monounsaturated fatty acids of the same chain length at the 2 en% linoleic acid level. After 9–14 weeks the biosynthesis of prostacyclin by pieces of aorta and the biosynthesis of hydroxy-heptadecatrienoic acid and 12-hydroxy-eicosatetraenoic acid by platelets were measured. The fatty acid compositions of aorta phospholipid and platelet lipid were also determined. Both the prostacyclin-production by aorta pieces and the production of hydroxy-heptadecatrienoic acid and 12-hydroxy-eicosatetraenoic acid by platelets appeared to be a linear function of the arachidonic acid level in aorta phospholipid and platelet lipid, irrespective of thetrans fatty acid content in the diet. This indicates thattrans fatty acids do not directly influence enzymes involved in eicosanoid biosynthesis. In a direct comparison withcis-monounsaturated or saturated fatty acids with 2 en% linoleic acid in the diet, only a moderate reduction in arachidonic acid level in aorta phospholipids in the group fedtrans fatty acids was observed. The geometry of the double bond did not influence the arachidonic acid level in platelet lipid, although the diet rich in saturated fatty acids increased arachidonic acid levels significantly compared with all other diets. Neither prostacyclin-production nor hydroxy-heptadecatrienoic acid or 12-hydroxy-eicosatetraenoic acid-production were significantly affected bytrans fatty acids when 2 en% linoleic acid was present in the diet. Our study indicates that in rats 2 en% linoleic acid is sufficient to prevent effects of dietarytrans fatty acids on eicosanoid synthesis.     

Linoleic acid requirement of rats fedtrans fatty acids

The amount of linoleic acid required to prevent undesirable effects of C18trans fatty acids was investigated. In a first experiment, six groups of rats were fed diets with a high content oftrans fatty acids (20% of energy [en%]), and increasing amounts of linoleic acid (0.4 to 7.1 en%). In a second experiment, four groups of rats were fed diets designed to comparetrans fatty acids with saturated andcis-monounsaturated fatty acids of the same chain length at the 2 en% linoleic acid level. After 9–14 weeks, the oxygen uptake, lipid composition and ATP synthesis of heart and liver mitochondria were determined. The phospholipid composition of the mitochondria did not change, but the fatty acid compositions of the two main mitochondrial phospholipids were influenced by the dietary fats.Trans fatty acids were incorporated in all phospholipids investigated. The linoleic acid level in the phospholipids, irrespective of the dietary content of linoleic acid, increased on incorporation oftrans fatty acids. The arachidonic acid level had decreased in most phospholipids in animals fed diets containing 2 en% linoleic acid. At higher linoleic acid intakes, the effect oftrans fatty acids on the phospholipid arachidonic acid level diminished. However, in heart mitochondrial phosphatidylethanolamine,trans fatty acids significantly increased the arachidonic acid level. Despite these changes in composition, neither the amount of dietary linoleic acid nor the addition oftrans fatty acids influenced the mitochondrial function. For rats, a level of 2 en% of linoleic acid is sufficient to prevent undesirable effects of high amounts of dietary C18trans fatty acids on the mitochondrial function.     

γ-Linolenic acid-rich triacylglycerols derived from borage oil via lipase-catalyzed reactions

γ-Linolenic acid (GLA), a precursor of arachidonic acid, possesses physiological functions of modulating immune and inflammatory response. Highly purified GLA is desired both as a medicine and as an ingredient of cosmetics. In this work, urea fractionation and lipase-catalyzed reactions were employed for the enrichment of GLA in borage oil. GLA content in free fatty acids from saponified borage oil can be increased from 23.6 to 94% by the method of urea fractionation. Partial hydrolysis of borage oil catalyzed by immobilized Candida rugosa lipase raises GLA content in the unhydrolyzed acylglycerols from 23.6 to 52.1%. The IM-60 catalyzed acidolysis reaction between the GLA-rich free fatty acid and the unhydrolyzed acylglycerols increases the GLA content in the acylglycerols from 52.1 to 75%. The acylglycerols in the reaction product contains ca. 90% triacylglycerol. The effects of temperature, water content, substrate weight ratio, and organic solvents on the GLA content in the acylglycerols were examined.     

Influence of partially hydrogenated vegetable and marine oils on lipid metabolism in rat liver and heart

Partially hydrogenated marine oils containing 18∶1-, 20∶1- and 22∶1-isomers and partially hydrogenated peanut oil containing 18∶1-isomers were fed as 24–28 wt % of the diet with or without supplement of linoleic acid. Reference groups were fed peanut, soybean, or rapeseed oils with low or high erucic acid content. Dietary monoene isomers reduced the conversion of linoleic acid into arachidonic acid and the deposition of the latter in liver and heart phosphatidylcholine. This effect was more pronounced for the partially hydrogenated marine oils than for the partially hydrogenated peanut oil. The content oftrans fatty acids in liver phospholipids was similar in groups fed partially hydrogenated fats. The distribution of various phospholipids in heart and liver was unaffected by the dietary fat. The decrease in deposition of arachidonic acid in rats fed partially hydrogenated marine oils was shown in vitro to be a consequence of lower Δ6-desaturase activity rather than an increase in the peroxisomal β-oxidation of arachidonic acid. The lower amounts of arachidonic acid deposited may be a result of competition in the Δ6-desaturation not only from the C22-and C20-monoenoic fatty acids originally present in the partially hydrogenated marine oil, but also from C18- and C16-monoenes produced by peroxisomal β-oxidation of the long-chain fatty acids. Part of this work was presented at the ISF-AOCS Congress, New York City, 1980.     

In Vitro hydrolysis of fungal oils: Distribution of arachidonic acid-containing triacylglycerol molecular species

Four commercially prepared arachidonic acidrich oils from the fungus Mortierella alpina were analyzed by high-performance liquid chromatography and gas chromatography. The levels of arachidonic acid and the distribution of triacylglycerol (TG) molecular species varied significantly among these oils. The major arachidonate-containing TG species were AAA, LAA, DAA, OAA, PAA, SAA, OLA, PGA, PLA, POA, and SOA where the abbreviations A, D, G, L, O, P, and S represent arachidonic (20:4n-6), dihomo-γ-linolenic (20:3n-6), γ-linolenic (18:3n-6), linoleic (18:2n-6), oleic (18:1n-9), palmitic (16:0), and stearic (18:0) acids, respectively. In vitro incubation of the TG fractions, purified from these oils with porcine pancreatic lipase for 5 min, yields a mixture of intermediate products, such as 1,2- and 2,3-diacylglycerols (1,2- and 2,3-DG), 2-monoacylglycerol (2-MG) and free fatty acids (FFA), as well as residual TG. The degrees of hydrolysis varied significantly among the four oil preparations, ranging from 35 to 57%. The levels of arachidonic acid in the residual TG and 1,2(2,3)-DG were significantly higher than those in the original TG, whereas those in the FFA fraction were significantly lower than those in 1,2(2,3)-DG and 2-MG. Results from this study suggest that the bioavailability of arachidonic acid differs among fungal oils prepared by different suppliers. These differences could be attributed to the arachidonic acid content of the oil as well as to the association of arachidonic acid with other fatty acids in the same TG molecule.     

Profiling of arachidonic acid metabolites in rabbit platelets by radio gas chromatography

A method for profiling arachidonic acid metabolites by radio gas chromatography (GC) is described. The incubation mixture of rabbit platelets with [¹⁴C]arachidonic acid was purified on a Sep-Pak C₁₈ cartridge and derivatized with diazomethane,O-methylhydroxylamine and dimethylisopropylsilylimidazole. The recovery of total¹⁴C-radioactivity was 93.1±7.2%. Loss of radioactivity during derivatization was negligible. Baseline separations for [¹⁴C]arachidonic acid and its metabolites were obtained in a single run within 45 min by GC using a synchronized accumulating radioisotope detector (GC/SARD). The recovery of radioactivity from the GC column was virtually 100%. The chemical structures of the metabolites were confirmed by GC/mass spectrometry; peaks of arachidonic acid metabolites were assigned by comparison of the methylene unit values with those of radioactive peaks in GC/SARD analyses. The intra-assay coefficients of variation in GC/SARD analyses were less than 10%. The method was used to map the profile of arachidonic acid metabolites formed by rabbit platelets in the presence of indomethacin, baicalein or glutathione.     

Eicosapentaenoic acid inhibits tube formation of vascular endothelial cellsin vitro

We previously have described a quantitative angiogenesisin vitro model, in which endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures. Using this model, the effect of eicosapentaenoic acid (20∶5n−3) on tube formation was investigated. When the endothelial cells isolated from bovine carotid artery were treated for 2 days with 5 μg/mL of arachidonic acid (20∶4n−6), eicosapentaenoic acid or docosahexaenoic acid (22∶6n−3), these polyunsaturated fatty acids were extensively incorporated into cellular phospholipids. The content of arachidonic, eicosapentaenoic and docosahexaenoic acid increased from 9.58% to 23.29%, from 0.98% to 11.76% and from 6.88% to 18.40%, respectively. When the eicosapentaenoic acid-treated cells were cultured between collagen gels, the tube-forming ability of the cells was markedly inhibited. The inhibition was dose-dependent between 1.0 and 5.0 μg/mL of eicosapentaenoic acid. At 5.0 μg/mL of eicosapentaenoic acid the inhibition reached 76%. By contrast, arachidonic acid increased tube formation, and docosahexaenoic acid had no effect. To elucidate the mechanism of eicosapentaenoic acid induced inhibition ofin vitro tube formation, we examined the effect of the acid on the proliferation of endothelial cells. Eicosapentaenoic acid at any dose (<5.0 μg/mL) had no effect on the proliferation of endothelial cells cultured on plastic plates without collagen gel. However, when the cells were cultured between collagen gels, eicosapentaenoic acid inhibited cell growth in a dose-dependent manner with maximum inhibition being observed at 2.5 μg/mL. These data suggest that eicosapentaenoic acid suppresses tube formation of endothelial cells, at least in part,via its inhibitory effect on cellular proliferation. Thus eicosapentaenoic acid may act as an endogenous inhibitor of angiogenesis under various pathological conditions, including tumor growth and chronic inflammation.     

An efficient method for the purification of arachidonic acid from fungal single-cell oil (ARASCO)

PUFA, such as arachidonic acid (AA), have several pharmaceutical applications. An efficient method was developed to obtain high-purity arachidonic acid (AA) from ARASCO, a single-cell oil from Martek (Columbia, MD). The method comprises three steps. In the first step, AA was enriched from saponified ARASCO oil by low-temperature solvent crystallization using a polar, aprotic solvent, which gave a FA fraction containing 75.7% AA with 97.3% yield. The second step involved enriching AA content via lipase-catalyzed selective esterification of FA with lauryl alcohol. When a mixture of 1 g FA/lauryl alcohol (2∶1 mol/mol), 50 mg Candida rugosa lipase, and 0.33 g water was incubated at 50°C for 24 h with stirring at 400 rpm, the AA content in the unesterified FA fraction was as much as 89.3%, with ca. 90% yield. Finally, a solvent extraction procedure, in which acetonitrile was the extracting solvent, was used to enrich AA from FA fraction dissolved in n-hexane. The best results were obtained when 2 g FA was dissolved in 80 mL hexane and extracted twice, each time with 20 mL acetonitrile at −20°C, by allowing 2 h storage. This step gave a FA fraction containing 95.3% AA with 81.2% yield. By using this three-step process the AA content in the saponified single-cell oil (ARASCO) was increased from 38.8 to 95.3% with a total yield of ca. 71%.     

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%).     

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.     

Effect of antioxidant‐enriched foods on plasma: Phospholipid molecular species composition

The dietary supplementation with antioxidant‐enriched foods was evaluated for the phospholipid (PL) class and molecular species composition of human plasma. Twenty healthy subjects were supplied with α‐tocopherol (13.7 mg/day) and coenzyme Q₁₀ (19.4 mg/day) by enriched milk, a dessert, fruit juice and yogurt for 21 days. Phosphatidylcholine (85.2%) and sphingomyelin (10.9%) were the main PL in all samples. Differences among the contents of PL classes were not found. However, principal component analysis showed differences in the PL molecular species 2 h after the mid‐morning snack. An increase of phosphatidylinositol (PI) containing stearic/arachidonic (on average from 42.5 to 47.0%), stearic/docosahexaenoic (3.2 vs. 4.9%), oleic/arachidonic and palmitic/docosapentaenoic acid (2.4 vs. 3.7%) was observed. The decreasing species of PI were palmitic/linoleic (5.7 vs. 4.3%), palmitic/oleic (8.1 vs. 6.9%) and stearic/linoleic acid (17.4 vs. 13.8%) after the mid‐morning snack. Phosphatidylethanolamine (PE) species showed an opposite trend with respect to PI: A decrease was registered for stearic/arachidonic (17.0 vs. 15.8%), stearic/docosahexaenoic (7.2 vs. 4.9%), oleic/arachidonic and palmitic/docosapentaenoic acid (5.8 vs. 4.8%); an increase was observed for the PE species containing oleic/linoleic (5.5 vs. 7.5% on average after the mid‐morning snack), stearic/linoleic (19.7 vs. 23.4%) and stearic/oleic acid (11.4 vs. 13.9%).     

Enrichment of arachidonic acid: Selective hydrolysis of a single-cell oil fromMortierella withCandida cylindracea lipase

Three lipases, isolated previously in our laboratory, and a known lipase fromCandida cylindracea were screened for the enrichment of arachidonic acid (AA). The enzyme fromC. cylindracea was the most effective for the production of oil with high concentration of AA. When a single-cell oil fromMortierella alpina, containing 25% AA, was hydrolyzed with this lipase for 16 h at 35°C, the resulting glycerides contained 50% AA at 52% hydrolysis. After this, no further hydrolysis occurred, even with additional lipase. However, when the glycerides were extracted from the hydrolyzate and were hydrolyzed again with new lipase, the resulting oil contained 60% AA, with a recovery of 75% of its initial AA content. Triglycerides were the main components of the resulting oil. The release of each fatty acid from the oil depended on the hydrolysis rate of its ester. The fatty acid, whose ester is the poorest substrate for the enzyme, is concentrated in the glycerides.     

Arachidonic and eicosapentaenoic acids in developing gametophores and sporophytes of the moss,Mnium cuspidatum

The fatty acid composition of different parts of the moss,Mnium cuspidatum, which contains up to 35% arachidonic acid in its lipids, was studied through the annual cycle and especially during the period of rapid development of the reproductive parts. The content of 20∶4ω6 was highest in summer and lowest in winter; but for 20∶5ω3, the reverse was found. Levels of the acids, 20∶5ω3, 18∶3ω3 and 16∶3ω3 showed parallel fluctuations through the seasons of the year, and functionally they may substitute for each other. In contrast, 20∶5ω6 is at a high level when 18∶2ω6 is low. The latter acid accumulates in storage or dormant tissue and may be a reserve to form arachidonic acid for specific requirements in cell membranes when rapid growth resumes.     

Mechanism of decreased arachidonic acid in the renal cortex of rats with diabetes mellitus

The purpose of this study was to investigate the roles of decreased synthesis and increased consumption in the depression of arachidonic acid levels in renal cortex and glomeruli of rats with streptozotocin-induced diabetes mellitus. In diabetic rats, arachidonic acid was depressed 33.2% in renal cortex, 47.4% in liver and 66.1% in heart compared to values of control rats. Δ6 Desaturase activity was depressed in renal cortex, liver and heart of diabetic rats to 53.3, 55.5 and 63.7%, respectively, of control values. Δ5 Desaturase activity was also depressed 43.7, 55.5 and 47.6% in renal cortex, liver and heart of diabetic rats, respectively. In other rats the activities of five enzymes involved in the synthesis and esterification of arachidonic acid were measured in renal cortex and in isolated glomeruli. Both tissues from diabetic rats showed depressed activities of Δ5 and Δ6 desaturases, increased activities of long-chain acyl-CoA synthetase and 1-acyl-sn-glycero-3-phosphocholine acyltransferase and no change in the activity of elongase as compared to those in control tissues. Malondialdehyde, an end product of lipid peroxidation, was lower in the renal cortex of diabetic rats than in control rats, whereas β-oxidation of linoleic acid and arachidonic acid were similar in diabetic and in control rats. Basal and stimulated prostaglandin E² synthesis were significantly higher in isolated glomeruli from diabetic rats compared to those in control rats. In isolated tubules, prostaglandin E₂ synthesis was similarly low in both groups. From these data we conclude that the reduced level of arachidonic acid esterified in lipids of the kidney cortex is caused principally by depressed synthesis of arachidonic acid secondary to decreased activity of Δ5 and Δ6 desaturases. Increased consumption of arachidonic acid to support prostaglandin synthesis may have contributed to the depression of arachidonic acid in glomeruli but not in tubules.