Polyunsaturated fatty acids production with a solid-state column reactor

To investigate the potential production of polyunsaturated fatty acids (PUFAs), a solid-state column reactor of rice bran with Mortierella alpina was used. The optimal conditions for PUFAs production were rice bran supplementation with 3.75% (ww(-1)) nitrogen source at initial moisture content 57%, initial pH 6-7, aeration, and incubation at 20 degrees C for 5 days and then at 12 degrees C for 7 days. Each gram of substrate carbon yielded 127 mg of total PUFAs, 12 mg of eicosapentaenoic acid (EPA), 6 mg of arachidonic acid (AA), 5mg of alpha-linolenic acid (ALA), and 117 mg of linoleic acid (LA) after 12 days incubation. Aeration enhanced the productions of AA, EPA, and total PUFAs. Supplementation of the nitrogen source on the fourth day and then a shift to lower temperature on the fifth day increased EPA production.     

Production of Dihomo-gamma-Linolenic Acid by a Delta5-Desaturase-Defective Mutant of Mortierella alpina 1S-4

A mutant, which has low Delta5-desaturase activity, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was shown to be a novel potent producer of dihomo-gamma-linolenic acid (DHGA). On submerged culture under optimal conditions for 6 days at 28 degrees C in a 10-liter fermentor, the mutant produced 3.2 g of DHGA per liter of culture broth (123 mg/g of dry mycelia), which accounted for 23.4% of the total mycelial fatty acids. Mycelial arachidonic acid amounted to only 19 mg/g of dry mycelia (0.5 g/liter of culture broth), which accounted for 3.7% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (11.0%), stearic acid (12.8%), oleic acid (22.7%), linoleic acid (8.9%), gamma-linolenic acid (6.5%), and lignoceric acid (7.8%). More than 97 mol% of the DHGA produced was found in the triglyceride fraction irrespective of the growth temperature employed (12 to 28 degrees C).     

Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Pythium ultimum

Several strains of species of the fungal genus Pythium, and of Phytophthora cinnamomi, were screened for content of the polyunsaturated fatty acids (PUFAs) arachidonic acid (AA) and eicosapentaenoic acid (EPA). The aim of the investigation was to establish alternative sources of these PUFAs, which are of importance in human nutrition. As a relatively prolific producer of EPA and AA, P. ultimum strain #144 was selected for a study of conditions that enhance their production over baseline levels that are present in the fungus when cultured for 6 d at 25 degrees C with rotary shaking (120 r.p.m.) in Vogel's medium containing sucrose as the carbon substrate. The levels of AA and EPA under these conditions were 133 +/- 27 and 138 +/- 25 mg l-1 (n = 5), respectively. Maximal production of these fatty acids was accomplished by the following sequence of steps. (1) Incubate the cultures for 6 d after inoculation under the conditions described above. Then (2) add glucose to the cultures (2%, w/v, final concentration) and incubate for a further 6 d at 13 degrees C. Under these conditions, the AA content of the mycelium was 205% higher than baseline levels and the EPA content was 198% higher. (3) Allow the cultures to remain stationary for 10 d which increases the AA content to 253% above baseline levels and the EPA content by 236%. Using such a procedure, 322 mg AA l-1 and 383 mg EPA 1-1 were produced.     

Polyunsaturated fatty acids production by solid-state fermentation on polyurethane foam by Mortierella alpina

Polyunsaturated fatty acids (PUFAs) are essential in healthy diets and their production is extremely important. Natural sources of PUFAs includes animal and aquatic products such as marine fish oil, however there are several limitations such as the decrease of fish stocks throughout the world. Thus, microbial oils are a preferable source of PUFAs. Herein, it was studied the production of PUFAs by Mortierella alpina under solid-state fermentation (SSF) using polyurethane foam as inert substrate and synthetic medium or lignocellulosic hydrolysate as source of C, N, and other nutrients. Several parameters of fermentation conditions were evaluated as carbon source, inductors addition, ratio C/N and temperature. The highest amount of total PUFAs per mass of solid (535.41 ± 24.12 mg/g), linoleic acid (129.66 ± 5.84 mg/g), and α-linoleic acid (401.93 ± 18.10 mg/g) were produced when the culture medium contained 20 g/L glucose, 10% (w/v) linseed oil, the C/N ratio was adjusted to 25 and the incubation temperature was 25°C for 3 days decreasing to 16°C on the remaining 4 days of fermentation. In addition, a hemicellulosic hydrolysate can be used as low-cost substrate to produce PUFAs, although the production was lower than the achieved with synthetic medium. SSF showed an interesting technology for microbial PUFAs production.     

Physiological studies of eicosapentaenoic acid production in the marine microalga Glossomastix chrysoplasta

We describe the characterization of the microalga Glossomastix chrysoplasta, an eicosapentaenoic acid (EPA) producer in the Pinguiophyceae class, Chromophyte division. Growth conditions were selected to optimize algal growth and EPA production. EPA represented up to 30% of the fatty acid content of Glossomastix chrysoplasta, at levels of 22 mg EPA per gram dry weight. Up to 72% of the EPA was produced as glycolipids, components of structural lipids. The optimal growth conditions in continuous culture were found to be greater than 500 micromol photons/m(2) . s light intensity, 0.33/day dilution rate, pH 7.20-7.45, and a temperature of 18-20 degrees C. Macronutrient studies indicated the limiting nutrient to be bicarbonate or dissolved carbon dioxide, and consequently decreasing pH increased EPA production.     

A Novel Delta5-Desaturase-Defective Mutant of Mortierella alpina 1S-4 and Its Dihomo-gamma-Linolenic Acid Productivity

A novel Delta5-desaturase-defective mutant was derived from an arachidonic acid-producing fungus, Mortierella alpina 1S-4, after treating the parental spores with N-methyl-N'-nitro-N-nitrosoguanidine. The mutant produced only a trace (about 1%) amount of arachidonic acid, and the ratio of dihomo-gamma-linolenic acid (DGLA) to total fatty acids in each lipid class was markedly high, accounting for as much as 60% in phosphatidylcholine. Under submerged batch culture conditions, the mutant produced 2.4 g of DGLA per liter (43.3% of total fatty acids) when grown at 28 degrees C for 7 days in a 5-liter jar fermentor. The other major (more that 1%) fatty acids were palmitic acid (21.2%), stearic acid (9.6%), oleic acid (14.3%), linoleic acid (4.4%), and gamma-linolenic acid (5.8%). About 80 mol% of the DGLA produced was found in triacylglycerol.     

Production and recovery of polyunsaturated fatty acids-added lipids from fermented canola

Canola flake was investigated as a potential substrate for fungal conversion to produce polyunsaturated fatty acids (PUFAs)-added oil in a 7l fermenter. The results showed that yields of total oil were reduced 9-22% compared to initial oil in the canola flake, but as high as 445mg/l arachidonic acid (ARA, C20:4n6) and 67mg/l eicosapentaenoic acid (EPA, C20:5n3) were produced. The percentages of ARA and EPA of total fatty acids in this fermented oil were 15.5% and 2.3%, respectively. Supercritical CO(2) extraction was then investigated for the lipid recovery from fermented canola flake, and extraction kinetics were modeled. The feasibility was demonstrated for production of PUFAs in a laboratory-scale fermentor using canola flake as a single nutrient, and for lipid extraction using supercritical CO(2).     

Association of calmodulin inhibition, erythrocyte membrane stabilization and pharmacological effects of drugs

Isolated liver cells from rats fed a diet deficient in essential fatty acids were used to study the oxidation, esterification and, especially, the desaturation and chain elongation of [1-14C]linoleic acid. 14C-labelled arachidonic acid (20:4) and smaller amounts of eicosatrienoic acid (20:3) were recovered mainly in the phospholipids, while gamma-linolenic acid (18:3) was found in both the phospholipids and the triacylglycerol fraction. Lactate strongly increased the formation of arachidonic acid, which was found mainly in the phosphatidylcholine and the phosphatidylinositol fractions. Lactate reduced the amounts of gamma-linolenic acid. Glucagon and (+)-decanoylcarnitine reduced the formation of arachidonic acid, and (+)-decanoylcarnitine increased the incorporation of gamma-linolenic acid especially, in the triacylglycerol fraction. Increasing concentrations of the [1-14C]linoleic acid substrate increased the formation of arachidonic acid and of the other chain-elongated or desaturated fatty acids. Lactate also stimulated the formation of arachidonic acid in liver cells from animals fed adequate amounts of essential fatty acids. It is suggested that dietary and hormonal factors which can change the intracellular levels of malonyl-CoA may influence both the ratio of arachidonic acid/gamma-linolenic acid formed and the total amounts of desaturated and chain-elongated fatty acids formed from linoleic acid.     

Arachidonic acid production by the red alga Porphyridium cruentum

The single-celled alga, Porphyridium cruentum, was assessed by means of chromatographic separation and mass spectral analysis of its fatty acids to be a potentially competetive source of arachidonic (5,8,11,14-eicosatetraenoic) acid. Models for both cell growth and production of the prostaglandin precursor at various temperatures and light intensities are presented. Increasing the light intensity within the range 1700-8000 lux increases the cell growth rate without affecting the arachidonic acid yield per cell; increasing the cultivation temperature from 18 degrees C to ca. 32 degrees C lowers the yield of arachidonic acid per cell but increases the rate of its production per unit volume and time. The increase of the weight ratio of arachidonic:palmitic acids at low temperatures is interpreted as a means of controlling the microviscosities of cellular membranes. In addition, the arachidonic acid content of cells decreases with the culture's age, despite increases in unit cell dry weight. The maximum rate of 0.46 mg arachidonic acid L(-1) h(-1) was calculated by means of the model to occur at ca. 32 degrees C and 8000 lux in liquid cultures of 12 x 10(9) cells/L. Estimates of the cost of producing arachidonic acid by means of this alga range from $0.15/g to $1.00/g of arachidonic acid. Cells grown at 18 degrees C in the presence of 0.3% linoleic acid swelled and produced gorlic (13-cyclopent-2-enyltridec-6-enoic) acid and another compound not normally observed. An estimated threefold increase of arachidonic acid content also occurred, but no significant lipogenesis was induced at 23 degrees C in the presence of 1% kerosene or 0.3% palmitic, stearic, oleic, or linoleic acids.     

Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media

Mortierella isabellina cultivated in nitrogen-limited media presented remarkable cell growth (up to 35.9 g/l) and high glucose uptake even with high initial sugar concentrations (e.g. 100 g/l) in media. After nitrogen depletion, significant fat quantities were accumulated inside the fungal mycelia (50-55%, wt/wt oil in dry biomass), resulting in a notable single cell oil production of 18.1 g/l of culture medium. Total dry biomass and lipid yields presented greatly increased values (0.34 and 0.17 g respectively per gram of glucose consumed). The microbial lipid produced contained gamma-linolenic acid (GLA) at a concentration of 3.5+/-1.0%, wt/wt, which corresponded to 16-19 mg GLA per gram of dry microbial mass and a maximum concentration of 0.801 g GLA per liter of culture medium.     

Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation

Effects of medium components and culture conditions on biomass and lipid production of Trichosporon fermentans were studied. The optimal nitrogen source, carbon source and C/N molar ratio were peptone, glucose and 163, respectively. The favorable initial pH of the medium and temperature were 6.5 and 25 degrees C. Under the optimized conditions, a biomass of 28.1 g/l and a lipid content of 62.4% could be achieved after culture for 7 days, which were much higher than the original values (19.4 g/l and 50.8%) and the results reported by other groups. T. fermentans could grow well in pretreated waste molasses and a lipid yield of 12.8 g/l could be achieved with waste molasses of 15% total sugar concentration (w/v) at pH 6.0, representing the best result with oleaginous microorganisms on agro-industrial residues. Addition of various sugars to the pretreated molasses could efficiently enhance the accumulation of lipid and the lipid content reached as high as above 50%. Similar to vegetable oils, the lipid mainly contains palmitic acid, stearic acid, oleic acid and linoleic acid and the unsaturated fatty acids amount to about 64% of the total fatty acids. The microbial oil with an acid value of 5.6 mg KOH/g was transesterified to biodiesel by base catalysis after removal of free fatty acids and a high methyl ester yield of 92% was obtained.     

Fatty acid and carotenoid production by Sporobolomyces ruberrimus when using technical glycerol and ammonium sulfate

The production of carotenoids, lipid content, and fatty acid composition were all studied in a strain of Sporobolomyces ruberrimus when using different concentrations of technical glycerol as the carbon source and ammonium sulfate as the nitrogen source. The total lipids represented an average of 13% of the dry weight, and the maximum lipids were obtained when using 65.5 g/l technical glycerol (133.63 mg/ g). The optimal conditions for fatty acid production were at 27 degrees C using 20 g of ammonium sulfate and a pH range from 6 to 7, which produced a fatty acid yield of 32.5+/-1 mg/g, including 1.27+/- 0.15 mg of linolenic acid (LNA), 7.50+/-0.45 mg of linoleic acid (LLA), 5.50+/-0.35 mg of palmitic acid (PA), 0.60+/-0.03 mg of palmitoleic acid (PAL), 1.28+/-0.11 mg of stearic acid (SA), 9.09+/-0.22 mg of oleic acid, 2.50+/-0.10 mg of erucic acid (EA), and 4.25+/-0.20 mg of lignoceric acid (LCA), where the palmitic, oleic, and linoleic acids combined formed about 37% of the total fatty acids. The concentration of total carotenoids was 2.80 mg/g when using 20 g of ammonium sulfate, and consisted of torularhodin (2.70 mg/g) and beta-carotene (0.10 mg/ g), at 23 degrees C and pH 6. However, the highest amount with the maximum specific growth rate was obtained (micromax=0.096 h(-1)) with an ammonium sulfate concentration of 30 g/l.     

Effects of polyunsaturated fatty acids and gonadotropin on prostaglandin series E production in a primary testis cell culture system for the European sea bass

Prostaglandin series E (PGE) production by mixed populations of isolated sea bass Dicentrarchus labrax testicular cells was measured after culture at 20° C in L-15 for up to 24 h in the absence or presence of arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and/or gonadotropin (hCG). AA stimulated a significant dose- and time-dependent increase in PGE production. EPA stimulated PGE production only during the first 6 h although levels were 26·4% those induced with AA; DHA reduced PGE production. Although hCG alone had no effect, it enhanced AA-induced PGE formation and suppressed EPA-induced PGE production. These results suggest that essential PUFAs modulate significantly testicular PGE production in vitro and therefore may have important effects on steroidogenesis and spermiation in the male European sea bass.     

Characterization of an enriched lipoxygenase extract from Aspergillus niger in terms of specificity and nature of flavor precursors production

Aspergillus niger was grown for 6 days, and the harvested biomass was homogenized; the resultant supernatant, considered as the crude enzymatic extract, was enriched by ammonium sulfate precipitation. The extract was assayed for its lipoxygenase (LOX) activity using a wide range of polyunsaturated fatty acids (PUFAs), including linoleic, linolenic and arachidonic acids, as substrates. Two pH maxima were determined at 5.0, 10.5. The K_m and V_max values indicated that the microbial LOX displayed preferential substrate specificity towards linolenic acid at low pH. The microbial LOX demonstrated preferential substrate specificity towards free fatty acids over the acyl esters of linoleic acid. It was shown that the LOX activity of A. niger produced all monohydroperoxy regioisomers of the PUFAs, and there was a predominance of conjugated diene hydroperoxides. Significant production of the unconjugated 10-hydroperoxides of both linoleic and linolenic acids was obtained by the LOX activity. The amounts of 10-hydroperoxides ranged from 15 to 21% of total produced isomers, for linolenic and linoleic acids, respectively. The greatest proportion of the 10-regioisomer was attributed to the maximum activity at pH 5.0. Four major hydroperoxy-eicosatetraenoic acid (HPETE) regioisomers were isolated from the bioconversion of arachidonic acid, including the 8-, 9-, 12- and 15-HPETE, which accounted for approximately 97% of total isomers.     

Production of Dihomo-γ-Linolenic Acid by a Δ5-Desaturase-Defective Mutant of Mortierella alpina 1S-4

A mutant, which has low Δ5-desaturase activity, of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, was shown to be a novel potent producer of dihomo-γ-linolenic acid (DHGA). On submerged culture under optimal conditions for 6 days at 28°C in a 10-liter fermentor, the mutant produced 3.2 g of DHGA per liter of culture broth (123 mg/g of dry mycelia), which accounted for 23.4% of the total mycelial fatty acids. Mycelial arachidonic acid amounted to only 19 mg/g of dry mycelia (0.5 g/liter of culture broth), which accounted for 3.7% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (11.0%), stearic acid (12.8%), oleic acid (22.7%), linoleic acid (8.9%), γ-linolenic acid (6.5%), and lignoceric acid (7.8%). More than 97 mol% of the DHGA produced was found in the triglyceride fraction irrespective of the growth temperature employed (12 to 28°C).     

Anti-HCV activities of selective polyunsaturated fatty acids

HCV infection can lead to chronic infectious hepatitis disease with serious sequelae. Interferon-alpha, or its PEGylated form, plus ribavirin is the only treatment option to combat HCV. Alternative and more effective therapy is needed due to the severe side effects and unsatisfactory curing rate of the current therapy. In this study, we found that several polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) are able to exert anti-HCV activities using an HCV subgenomic RNA replicon system. The EC(50) (50% effective concentration to inhibit HCV replication) of AA was 4microM that falls in the range of physiologically relevant concentration. At 100microM, alpha-linolenic acid, gamma-linolenic, and linoleic acid only reduced HCV RNA levels slightly and saturated fatty acids including oleic acid, myristic acid, palmitic acid, and steric acid had no inhibitory activities toward HCV replication. When AA was combined with IFN-alpha, strong synergistic anti-HCV effect was observed as revealed by an isobologram analysis. It will be important to determine whether PUFAs can provide synergistic antiviral effects when given as food supplements during IFN-based anti-HCV therapy. Further elucidation of the exact anti-HCV mechanism caused by AA, DHA, and EPA may lead to the development of agents with potent activity against HCV or related viruses.     

Enzymes for transgenic biosynthesis of long-chain polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) are important for the normal development and function of all organisms, and are essential in maintaining human health. Impaired PUFA metabolism is thought to be associated with pathogenesis of many chronic diseases. Dietary supplementation of PUFAs, such as gamma-linolenic acid, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid, which bypass the defective or dysfunctional steps of the biosynthetic pathway has been found to significantly alleviate the symptoms of the disease. These findings have drawn a great deal of interest from general public and food manufacturers. As the demand of these beneficial PUFAs has drastically increased in recent years, there are also increasing efforts in finding the alternate sources of PUFAs that are more economical and sustainable. One option is to modify the oil-seed crops to produce PUFAs through genetic engineering technique. This review examines the isolation, identification and expression of genes encoding the enzymes required for the biosynthesis of the above mentioned PUFAs in plants.     

Rhodomonas salina (Cryptophyta) pastes as feed for Brachionus plicatilis (Rotifera)

Rotifers are an important live feed for first feeding larvae of many fish species. The use of concentrated algae cells in the mass culture of the rotifer Brachionus plicatilis (Brachionidae) has opened new horizons for research on this organism. Pastes of Rhodomonas salina (Pyrenomonadaceae) obtained either by centrifugation or flocculation with chitosan were preserved, with or without vitamin C, at -20 degrees C for four weeks and were evaluated biochemically (proteins, lipids, pigments and fatty acids contents) and subsequently, were used to feed the rotifer Brachionus plicatilis at a ratio of 25 mg/L/day. Four different microalgae pastes were prepared: (1) centrifuged and preserved with vitamin C (CV), (2) centrifuged and preserved without vitamin C (C), (3) flocculated and with vitamin C (FV) and (4) flocculated without vitamin C (F). All treatments showed similar contents of proteins and total lipids with respect to control culture (a fresh culture of R. salina), with mean values of 40.0 +/- 2.32% and 12.0 +/- 1.45%, respectively. The pheophytin a/chlorophyll a ratio, a general indicator of the chemical status of microalgal concentrates, was similar (0.09-0.11) between centrifuged pastes and control culture, but was found to be higher in flocculated pastes (1.28-1.48). The fatty acid profile varied with respect to the control culture, mainly in the proportion of the essential polyunsaturated fatty acids (PUFAs): eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Total PUFAs, EPA and DHA contents were statistically similar between centrifuged pastes and control culture (PUFAs: 47%, EPA: 4% and DHA: 4.7%), whereas values obtained for flocculated pastes were significantly lower. The rotifers grew equally well when fed with centrifuged pastes or control culture (maximum density: 320 rotifers/mL; instantaneous growth rate: 0.23 rotifers/day, fecundity: 1.49 eggs/female and productivity: 43 x 10(3) rotifers/L/day. No significant effect of vitamin C was found when used as a paste preservative. We concluded that centrifugation is an effective harvesting method, and that freezing to -20 degrees C for four weeks (no vitamin added), may help maintain the nutritional quality of R. salina paste, similar to fresh microalgae and can be offered to Brachionus plicatilis.     

gamma-Linolenic acid production by solid-state fermentation of Mucorales strains on cereals

Oleaginous fungi of the genus Mucorales were screened for gamma-linolenic acid (GLA) production on solid substrates containing moistened cereals. Cunninghamella elegans CCF 1318 produced the highest yields of GLA when cultivated on barley. Substrate moisture and cultivation temperature proved critical for effective GLA production. Vegetable oil supplied to the cultures improved GLA production. Rotating bottles and plastic bags were used as cultivation vessels to reproduce the conditions found in rotating drums and tray bioreactors, respectively. After 11 days of cultivation at 21 degrees C, C. elegans produced 14.2 mg of GLA per gram of dry substrate, composed of a mixture of barley, spent malt grains (SMG) and peanut oil. GLA represented 15.6% of the total fatty acids in the lipid extract.     

Cytotoxicity of combined essential fatty acids on a human prostate cancer cell line

In this study the effect of single and concomitantly added n-6 or n-3 polyunsaturated fatty acids (PUFAs) was investigated on human prostate cells. Data obtained from the single fatty acids (FAs) experiments showed that except for oleic acid (OA), arachidonic (AA) and linoleic acid (LA), which had very little (less than 10% cells dead) effect on the cells, an increase in dead cells was observed at physiological concentrations of, eicosapentaenoic acid (EPA), gamma-linolenic acid (GLA) and alpha-linolenic acid (ALA). However, this was not the case when combining these acids at physiological concentrations. A slight increase in cell death was only obtained with three combinations of ALA, namely with AA, OA, or GLA. Other combinations with ALA, such as with LA or EPA, had respectively no effect on cell number or increased the cell number by causing less cells to die. Other PUFAs combinations tested, did not show the three groups mentioned with ALA, but only the last two types, namely, no effect, or a decrease in the amount of cell death. The latter might mean that the FA combination had stimulated the cells, since a decrease in the amount of dead cells was observed. Therefore, it is concluded that the characteristics of combined FAs may differ from single FAs, which may explain some controversies in the literature and in response to treatments.