Production Of Gamma-linolenic Acid Research Articles

Unveiling the optimal path: High-pressure homogenization and D-phase emulsification methods for borage oil nanoemulsion design

Borage oil contains high levels of essential omega-6 fatty acids, which are important for skin structure and function. For this reason, this oil can be used for the treatment of cutaneous diseases including atopic dermatitis. This disease is associated with an abnormality in essential fatty acids (EFAs) metabolism, particularly in the production of gamma-linolenic acid, leading to impaired incorporation of EFAs into the phospholipid membrane. EFAs bioavailability can be enhanced by using a borage oil nano-based drug delivery system. In this study a borage oil nanoemulsion was prepared using high-pressure homogenization (HPH) and D-phase emulsification (DPE), respectively, high and low energy methods. A design space approach was performed to optimize the formulations. The optimized HPH nanoemulsion (HPH-NE) presented a particle size and zeta potential (ZP) of 425.3 ± 5.6 nm, and − 19.4 ± 3.7 mV, respectively. For the DPE nanoemulsion (DPE-NE) the particle size was 334.4 ± 5.2 nm, and the zeta potential was − 34.6 ± 1.0 mV. Furthermore, DPE-NE presented higher toxicity compared to HPH-NE, possibly related to the presence of the polyol. These nanoemulsions may improve the borage oil bioavailability for oral administration. Thus, they can be used as an adjuvant therapy in the treatment of atopic dermatitis.

Gamma-Linolenic and Pinolenic Acids Exert Anti-Inflammatory Effects in Cultured Human Endothelial Cells Through Their Elongation Products.

Omega-3 fatty acids (FAs) from oily fish reduce cardiovascular disease. This may be partly due to modulation of endothelial cell (EC) inflammation. Fish stocks are declining and there is a need for sustainable alternative FAs. Gamma-linolenic acid (GLA) and pinolenic acid (PLA) are plant-derived FAs, which can fulfil this role. EA.hy926 cells are exposed GLA and PLA prior to stimulation with tumor necrosis factor (TNF)-α. GLA and PLA are incorporated into ECs, resulting in increases in long-chain derivatives produced by elongase 5, dihomo-gamma-linolenic acid (DGLA), and eicosatrienoic acid (ETA). Both GLA and PLA (50µm) decrease production of soluble intercellular adhesion molecule-1 (sICAM-1), monocyte chemoattractant protein 1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES). However, decreases in these mediators are not seen after pre-treatment with GLA or PLA in elongase 5 silenced EA.hy926 cells. DGLA and ETA (10µm) decrease EC production of sICAM-1, MCP-1, RANTES, and IL-6. All FAs reduce adhesion of THP-1 monocytes to EA.hy926 cells. Both PLA (50µm) and ETA (10µm) decrease NFκBp65 phosphorylation. These effects suggest potential for GLA, PLA and their long-chain derivatives, DGLA and ETA, as sustainable anti-inflammatory alternatives to fish-derived FAs.

Functional characterization and substrate specificity analysis of Δ6-desaturase from marine microalga Isochrysis sp.

To express a Δ6-desaturase gene and produce gamma-linolenic acid (GLA) and stearidonic acid (SDA) in prokaryotic expression system (Escherichia coli), and analyze its substrate specificity in the omega-3 fatty acid biosynthetic pathway. Full-length ORF (1448bp) of Δ6Des-Iso was isolated from Isochrysis sp. and characterized using multiple sequence alignment, phylogenetic analysis, transmembrane domain, and protein tertiary structure. Δ6Des-Iso is a front-end desaturase consisting of three conserved histidine domains and a cytochrome b5 domain. Δ6Des-Iso was cloned and expressed in E. coli with the production of GLA and SDA. Recombinant E. coli utilized 27 and 8% of exogenously supplied alpha-linolenic acid (ALA) and linoleic acid (LA) to produce 6.3% of SDA and 2.3% of GLA, respectively, suggesting that isolated Δ6Des-Iso is specific to the omega-3 pathway. For the first time production of GLA and SDA in a prokaryotic system was achieved.

Seed oil quality of GA3 induced flowering evening primrose (Oenothera biennis L.).

Gamma-linoleic acid in the seed oil of evening primrose makes it nutritionally and pharmaceutically valuable. If evening primrose wants to be cultivated as an annual plant, sowing time is important. By the late sowing and depends to time most plants do not produce flower stem and stay in rosette stage until next spring. To solve this problem, the present study was performed. Seedling were transplanted to 4 kg plastic pots containing a mixture of leaf compost: local soil: perlite (1:2:1 ratio) and placed in outdoor conditions. Non-vernalized plants were then selected and treated with different temperature regimes (1, 2 and 3 weeks in 4-6oC) and gibberellic acid (GA3) application in different concentrations of 0, 500, 1000, 2000 ppm, separately. The results showed that although some physiological parameters were affected by low temperature, no flower stem was produced. In contrast, plants that were treated with GA3 produced flower stem. Time of flowering and the number of flowers were significantly affected by the concentrations of applied GA3. Although yield components of GA3 induced plants were higher than that of normal growth plant, the seed yield of them was low. Surprisingly, the gamma linolenic acid (GLA) percentage of seed oil of plants treated with 2000 ppm GA3 was significantly higher than that of non- treated plants. Finally, it can be concluded that, although the application of GA3 guarantees delayed sowing evening primrose flowering and better GLA production, but low seed yield is a subject that cannot be easily ignored in production.

Using LC-MS Based Methods for Testing the Digestibility of a Nonpurified Transgenic Membrane Protein in Simulated Gastric Fluid.

The digestibility of a nonpurified transgenic membrane protein was determined in pepsin, as part of the food safety evaluation of its resistance to digestion and allergenic potential. Delta-6-desaturase from Saprolegnia diclina, a transmembrane protein expressed in safflower for the production of gamma linolenic acid in the seed, could not be obtained in a pure, native form as normally required for this assay. As a novel approach, the endoplasmic reticulum isolated from immature seeds was digested in simulated gastric fluid (SGF) and the degradation of delta-6-desaturase was selectively followed by SDS-PAGE and targeted LC-MS/MS quantification using stable isotope-labeled peptides as internal standards. The digestion of delta-6-desaturase by SGF was shown to be both rapid and complete. Less than 10% of the initial amount of D6D remained intact after 30 s, and no fragments large enough (>3 kDa) to elicit a type I allergenic response remained after 60 min.

Optimization of Physiological Growth Conditions for Maximal Gamma‐linolenic Acid Production by Cunninghamella blakesleeana‐JSK2

AbstractEffects of various cultural conditions on biomass, lipid and Gamma‐linolenic acid (GLA) production were investigated in the oleaginous fungus Cunninghamella blakesleeana‐JSK2 isolated from soil. The GLA production was influenced by various factors such as growth condition (static and shaken), incubation time, pH, temperature, carbon and nitrogen sources. The results indicated that optimum GLA production (21 %) was obtained when the fungus was grown under shaken condition at 120 rpm for 6 days with optimum pH and temperature of 6 and 28 °C, respectively. Glucose and potassium nitrate enhanced the GLA production. Urea and sucrose were poor substances for biomass, lipid and GLA production.

Optimization of aeration and agitation rate for lipid and gamma linolenic acid production by Cunninghamella bainieri 2A1 in submerged fermentation using response surface methodology.

The locally isolated filamentous fungus Cunninghamella bainieri 2A1 was cultivated in a 5 L bioreactor to produce lipid and gamma-linolenic acid (GLA). The optimization was carried out using response surface methodology based on a central composite design. A statistical model, second-order polynomial model, was adjusted to the experimental data to evaluate the effect of key operating variables, including aeration rate and agitation speed on lipid production. Process analysis showed that linear and quadratic effect of agitation intensity significantly influenced lipid production process (P < 0.01). The quadratic model also indicated that the interaction between aeration rate and agitation speed had a highly significant effect on lipid production (P < 0.01). Experimental results showed that a lipid content of 38.71% was produced in optimum conditions using an airflow rate and agitation speed of 0.32 vvm and 599 rpm, respectively. Similar results revealed that 0.058 (g/g) gamma-linolenic acid was produced in optimum conditions where 1.0 vvm aeration rate and 441.45 rpm agitation rate were used. The regression model confirmed that aeration and agitation were of prime importance for optimum production of lipid in the bioreactor.

Biosynthesis of gamma‐linolenic acid and beta‐carotene by Zygomycetes fungi

Due to increasing demand for natural sources of both polyunsaturated fatty acids (PUFAs) and beta-carotene, 28 Zygomycetes fungal soil isolates were screened for their potential to synthesize these biologically active compounds. Although all fungi produced C18 PUFAs, only nine strains also formed beta-carotene. Although Actinomucor elegans CCF 3218 was the best producer of gamma-linolenic acid (GLA) (251 mg/L), Umbelopsis isabellina CCF 2412 was found to be the most valuable fungus because of the dual production of GLA (217 mg/L) and beta-carotene (40.7 mg/L). The calculated ratio of formed PUFAs provided new insight into activities of individual fatty acid desaturases involved in biosynthetic pathways for various types of PUFAs. The maximal activity of delta-9 desaturase was accompanied by high accumulation of storage lipids in fungal cells. On the other hand, maximal activity of delta-15 desaturase was found in strains synthesizing low amounts of oleic acid due to diminished delta-9 desaturase. Activities of delta-6 desaturase showed competition for fatty acids engaged in n3, n6, and n9 biosynthetic pathways. Such knowledge about fatty acid desaturase activities provides new challenges for the regulation of biotechnological production of PUFAs by Zygomycetes fungi.

Aeration effect on Spirulina platensis growth and γ-linolenic acid production

The influence of aeration on algal growth and gamma-linolenic acid (GLA) production in a bubble column photobioreactor was investigated. Studies were performed in a 20-L reactor at different aeration rates (0.2– 2.5 vvm). Static, continuous, and periodic operation of air resulted in 41.9%, 88.4%, and 108% air saturation of dissolved oxygen, for which the corresponding values of GLA were 2.3, 6.5, and 7.5 mg·g-1 dry cell weight, respectively. An increase in the aeration rate from 0.2 to 2.5 vvm enhanced both the specific growth rate and GLA content under periodic sparging in the bicarbonate medium. With a 6-fold increase in the aeration rate, the GLA content of the alga increased by 69.64% (5.6–9.5 mg· g-1 dry cell weight). In addition, the total fatty acid (TFA) content in dry biomass increased from 2.22% to 4.41%, whereas the algae maintained a constant GLA to TFA ratio within the aeration rate tested. The dependence of GLA production on the aeration rate was explained by interrelating the GLA production rate with the specific growth rate using the Luedeking and Piret mixed growth model.

Enhancing the production of gamma-linolenic acid in Hansenula polymorpha by fed-batch fermentation using response surface methodology

AbstractGamma-linolenic acid (GLA, C18:3Δ6,9,12) is an n-6 polyunsaturated fatty acid (PUFA) that has been used for the alleviation and treatment of a number of symptoms and diseases. Increasing GLA demand has led to a search for alternative producers and potential strategies for GLA production. Based on the successful performance of Hansenula polymorpha, a methylotrophic yeast, as a “cell factory” for the production of valuable bioproducts, a bioprocess development approach was implemented for GLA production in the recombinant yeast carrying the mutated Δ6-desaturase gene of Mucor rouxii. Using a substrate-feeding strategy under glycerol-limited conditions, the physical-chemical variables during the fed-batch fermentation of the recombinant H. polymorpha were optimised for GLA production through response surface methodology using a Box-Behnken design. The medium composition, including yeast extract and trace elements, and dissolved oxygen tension (DOT) were targeted. We found that DOT was the most effective variable for enhancing GLA yield. These results also suggest that the optimum conditions for GLA production are 28 % saturation of DOT, 1 g L−1 of yeast extract and 3.6 mL L−1 of the Pichia trace metals 1 (PTM1).

Fermentation Process Development of Recombinant Hansenula polymorpha for Gamma-Linolenic Acid Production

Development in the strain and the fermentation process of Hansenula polymorpha was implemented for the production of gamma-linolenic acid (GLA, C18:3 delta 6,9,12), which is an n-6 polyunsaturated fatty acid (PUFA) and has been reported to possess a number of health benefits. The mutated delta 6-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without utilization of methanol a high cell-density culture of the yeast recombinant carrying the delta 6-desaturase gene was achieved by fed-batch fermentation using glycerol-limited conditions. The delta 6-desaturated products, octadecadienoic acid (C18:2 delta6,9), GLA and stearidonic acid (C18:4 delta6,9,12,15), accumulated at high levels under the derepression condition. The GLA production was also optimized by adjusting specific growth rates. The results show that the specific growth rate affected both lipid content and fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates studied, the highest GLA concentration, which was at of 697 mg/l, was obtained in the culture with the specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor delta 6-desaturase gene was similar to that of blackcurrant oil with both containing similar proportions of n-3 and n-6 essential fatty acids.

Studies on Mucor racemosus Fermentation to Manufacture Gamma-linolenic Acid Functional Food Douchi

This study aims to maximize the yield of gamma-linolenic acid (GLA) by a filamentous fungus, Mucor racemosus, using fermentation Douchi. Optimized fermentation conditions includes inoculum size and temperature. GLA content in Douchi by M. racemosus greatly varied in terms of fermentation temperature, from zero to 3.38% in total fatty acids, which means apart from fermentation temperature some other factors must affect GLA content in Douchi. The optimal amount of inoculums was 5.30 × 107 spores/10 g cooked soybean. The optimal temperature for fermentation was about 26°C. Under the optimal fermentation conditions, the GLA production of M. racemosus could be enhanced by optimizing the Douchi fermentation condition. Douchi, which contains GLA, may be directly absorbed through diet. Thus, Douchi may be a good kind of healthy food for people.

Medium Optimization of Gamma Linolenic Acid Production in Mucor rouxii CFR -G15 using RSM

Omega-6 polyunsaturated fatty acids has both pharmaceutical and neutraceutical importance. Central composite rotatable design (CCRD), along with response surface methodology (RSM), was employed to optimize the medium components for maximum production of gamma linolenic acid (GLA) from Mucor rouxii CFR-G15. Nutritional parameter such as carbon (glucose) and nitrogen (yeast extract and ammonium nitrate) sources were focused in this study to enhance the GLA production. The optimal conditions for maximizing GLA production (18.55%) were at 65 g glucose per liter; 3.5 g yeast extract per liter, and 0.5 g ammonium nitrate per liter. Thus, by using CCRD with RSM, it is quite possible to determine the accurate values of the fermentation parameters where maximum production of GLA is achieved.

Application of Response Surface Method for Studying the Role of Dissolved Oxygen and Agitation Speed on Gamma-Linolenic Acid Production

To study the effect of agitation speed (rpm) and dissolved oxygen concentration (DO) on the production of gamma linolenic acid by Mucor sp. RRL001, a central composite design experiment was performed in a 5-L stirred tank bioreactor. The design consisted of a total of 10 runs consisting of runs at five levels for each factor and was divided in two blocks. The ANOVA analysis and Pareto chart of effects suggested agitation speed (p = 0.0142) linear effect and DO concentration (p = 0.0342) quadratic effects were significant factors with significant contribution to the response. The validation run based on the optimum production zone in response surface plot resulted in the maximum 350.3 mg l(-1) GLA yield as compared with model predicted value of 340.7 mg l(-1). The study suggests that agitation rate is having more pronounced effect on GLA yield than dissolved oxygen concentration by ensuring enhanced mass transfer and by preventing wall growth at elevated agitation speed. Also, it shows that higher GLA yields can be obtained in a simple medium at moderate oxygen saturation and that the Mucor sp. RRL001 is resistant to high agitation linked shear stress and suitable for GLA production at higher scale.

Effects of Metal Ion Concentrations on Lipid and Gamma Linolenic Acid Production by Cunninghamella sp. 2A1

The effects of different concentration of MgSO4.7H2O, FeCl3.6H2O, ZnSO4.7H2O, CuSO4.5H2O and MnSO4.5H2O on lipid and GLA production by Cunninghamella sp. 2A1 were studied in submerged culture fermentation. Cultivation was performed in 250 ml conical flask (100ml working volume), at 30°C and 250 rpm agitation for 120h. Mg2+, Fe2+ and Zn2+ have significant effect on lipid accumulation as inclusion of each ions resulted in a 64, 43 and 33% increase in lipid content compared to cultures which was cultivated with the omission of each ions, respectively. All three metal ions also affected GLA production by Cunninghamella sp. 2A1 with Zn2+ having the most profound effect with an increase of up to 74% of GLA yield (g GLA/g lipidless biomass) achieved. As omission of any single metal ions did not affect biomass concentration achieved, we concluded that metal ions components in production media may have affected lipid and GLA production by affecting pathways involved in the biosynthesis of lipid and GLA. This study reports the critical concentration of each metal ion for optimal lipid and GLA production by Cunninghamella sp. 2A1.

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

To facilitate a cost-effective preparation of spore inoculum with good capacity for gamma-linolenic acid (GLA) production from Mucor rouxii. Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi-synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30.7 g kg(-1) initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore-forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. The simple, low cost method of inoculum preparation can be applied for large-scale production of GLA-rich oils, which reduce a time constraint and variation in fatty acid composition.

Truncation Mutants Highlight a Critical Role for the N- and C-termini of the Spirulina Δ6 Desaturase in Determining Regioselectivity

The results of our previous study on heterologous expression in Escherichia coli of the gene desD, which encodes Spirulina Delta(6) desaturase, showed that co-expression with an immediate electron donor-either cytochrome b ( 5 ) or ferredoxin-was required for the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6) desaturase. Since a system for stable transformation of Spirulina is not available, studies concerning Spirulina-enzyme characterization have been carried out in heterologous hosts. In this present study, the focus is on the role of the enzyme's N- and C-termini, which are possibly located in the cytoplasmic phase. Truncated enzymes were expressed in E. coli by employing the pTrcHisA expression system. The truncation of the N- and C-terminus by 10 (N10 and C10) and 30 (N30 and C30) amino acids, respectively, altered the enzyme's regioselective mode from one that measures from a preexisting double bond to that measuring from the methyl end of the substrate.

Effect of two intermediate electron donors, NADPH and FADH2, on Spirulina Δ6-desaturase co-expressed with two different immediate electron donors, cytochrome b 5 and ferredoxin, in Escherichia coli

When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.

Revealing the complementation of ferredoxin by cytochrome b 5 in the Spirulina-∆6-desaturation reaction by N-terminal fusion and co-expression of the fungal-cytochrome b 5 domain and Spirulina-∆6-acyl-lipid desaturase

Spirulina-acyl-lipid desaturases are integral membrane proteins found in thylakoid and plasma membranes. These enzymes catalyze the fatty acid desaturation process of Spirulina to yield gamma-linolenic acid (GLA) as the final desaturation product. It has been reported that the cyanobacterial desaturases use ferredoxin as an electron donor, whereas the acyl-lipid desaturase in plant cytoplasm and the acyl-CoA desaturase of animals and fungi use cytochrome b (5). The low level of ferredoxin present in Escherichia coli cells leads to an inability to synthesize GLA when the cells are transformed with the Spirulina-(6) desaturase, desD, and grown in the presence of the reaction substrate, linoleic acid. In this study, Spirulina-(6) desaturase, encoded by the desD gene, was N-terminally fused and co-expressed with the cytochrome b (5) domain from Mucor rouxii. The product, GLA, made heterologously in E. coli and Saccharomyces cerevisiae, was then detected and analyzed. The results revealed the production of GLA by Spirulina-(6) desaturase fused or co-expressed with cytochrome b (5) in E. coli cells, in which GLA production by this gene cannot occur in the absence of cytochrome b (5). Moreover, the GLA production ability in the E. coli host cells was lost after the single substitution mutation was introduced to H52 in the HPGG motif of the cytochrome b (5) domain. These results revealed the complementation of the ferredoxin requirement by the fusion or co-expression of the fungal-cytochrome b (5) domain in the desaturation process of Spirulina-(6) desaturase. Furthermore, the free form of cytochrome b (5) domain can also enhance GLA production by the Spirulina-desD gene in yeast cells.

CHARACTERISATION AND EVALUATION OF SPECIES OF THE BORAGINACEAE FAMILY AS SOURCE OF GAMMA-LINOLENIC ACID FOR MEDITERRANEAN CONDITIONS

Trabajo presentado en XXVI International Horticultural Congress: The Future for Medicinal and Aromatic Plants, celebrado en Toronto (Canada) el 03 de enero de 2004