GLA Content Research Articles

Large‐scale purification of γ‐linolenic acid by selective esterification using Rhizopus delemar lipase

Abstractγ‐Linolenic acid (GLA) is a physiologically valuable fatty acid, and is desired as a medicine, but a useful method available for industrial purification has not been established. Thus, large‐scale purification was attempted by a combination of enzymatic reactions and distillation. An oil containing 45% GLA (GLA45 oil) produced by selective hydrolysis of borage oil was used as a starting material. GLA45 oil was hydrolyzed at 35°C in a mixture containing 33% water and 250 U/g‐reaction mixture of Pseudomonas sp. lipase; 91.5% hydrolysis was attained after 24 h. Film distillation of the dehydrated reaction mixture separated free fatty acids (FFA; acid value 199) with a recovery of 94.5%. The FFA were selectively esterified at 30°C for 16 h with two molar equivalents of lauryl alcohol and 50 U/g of Rhizopus delemar lipase in a mixture containing 20% water. The esterification extent was 52%, and the GLA content in the FFA fraction was raised to 89.5%. FFA and lauryl esters were not separated by film distillation, but the FFA‐rich fraction contaminated with 18% lauryl esters was recovered by simple distillation. To further increase the GLA content, the FFA‐rich fraction was selectively esterified again under similar conditions. As a result, the GLA content in the FFA fraction was raised to 97.3% at 15.2% esterification. After simple distillation of the reaction mixture, lauryl esters contaminating the FFA‐rich fraction were completely eliminated by urea adduct fractionation. When 10 kg of GLA45 oil was used as a starting material, 2.07 kg of FFA with 98.6% GLA was obtained with a recovery of 49.4% of the initial content.

γ‐Linolenic acid in zygomycetous fungi: Syzygites megalocarpus

AbstractThe fatty acids of over 150 species and isolates of zygomycetous fungi were analyzed, and it was found that γ‐linolenic acid (GLA) composed 35 to 62% of the total fatty acids in several species, i.e., Circenella simplex, Mucor indicus, Syzygites megalocarpus (ATCC 18025), and Zygorhynchus moellierie A (UAMH 1556). Further study of S. megalocarpus showed that the total lipid content of the mycelium could be increased from 9.8% of the dry biomass to 20 to 25% when grown in a medium with a high carbon/nitrogen ratio. Under these conditions, the GLA content of the triacyglycerols increased during culture development, even during the stationary phase, but remained relatively constant in the phospholipid fraction. Nonsaponifiable lipid represented 4% of the total lipid, and the major sterol among 14 others detected was ergosterol at 52% of the total. Phospholipids composed 7% of the total lipid with phosphatidylethanol‐amine and phosphatidylcholine representing 53 and 39% of the total, respectively.

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

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

Inoculum effects on the production of γ-linolenic acid by the shake culture of Cunninghamella echinulata CCRC 31840

The effects of using the fragmented mycelia as inoculum on the growth of and lipid and γ-linolenic acid (GLA) production by the sporulation-deficient fungal strain Cunninghamella echinulata CCRC 31840 were examined in shake cultures. In a 5-day culture sown with 1.6% inoculum, the biomass increased 51.5%. Its lipid content increased 69.0% when the floc massiveness of the inoculum was increased from 27,000 to 81,000 colony forming units per milliliter (CFU ml −1). This in turn resulted in a synergistic increase of GLA yield from 358 to 860 mg l −1 whereas the GLA content of the lipid was virtually unaffected (11.2–13.4%). The maximum GLA yield of 1,349 mg l −1 was obtained in a 5-day culture when the production medium was pitched at the 4% level with an inoculum preparation in which 1.0 g of mycelia have been fragmented to a floc massiveness of 83,000 CFU ml −1 by vortexing for 4 min.

Lipid and γ-linolenic acid production by Mucor inaquisporus

The production of lipid and γ-linolenic acid (GLA) was studied in Mucor inaquisporus. In batch culture on ethanol, rapid lipid accumulation occurred during logarithmic growth phase of the culture and a direct proportional relationship was demonstrated between specific growth rate of the culture and GLA content of the lipid. The highest value of GLA in the lipid (36% w/w) was reached at the highest growth rate in batch culture. It has been shown that M. inaquisporus is suitable for GLA production in continuous culture. GLA content in biomass of continuous culture (2·7% w/w) was comparable to the highest GLA amount in biomass achieved in batch culture.

Enhancement of eicosapentaenoic acid (EPA) and γ‐linolenic acid (GLA) production by manipulating algal density of outdoor cultures of Monodus subterraneus (Eustigmatophyta) and Spirulina platensis (Cyanobacteria)

The effect of algal density on cell growth and composition with special reference to the production of eicosapentaenoic acid (EPA) and γ‐linolenic acid (GLA) was investigated in semi-continuous outdoor cultures of Monodus subterraneus and Spirulina platensis. In both species, an exponential decrease in specific growth rate and a positive skewed pattern in biomass productivity were associated with an increase in density. In M. subterraneus, the highest EPA cell content (3·8% of dry weight) occurred at the optimal density of c. 4gl−1 i.e. the density that yields the highest output rate of biomass per culture volume or area. In S. platensis, high density was associated with a decrease in total fatty acid content, but the relative abundance of GLA increased. GLA content was therefore fairly stable at c. 1% of dry weight throughout the range of algal density tested. Fatty acid desaturation seemed to be associated with an increase in culture density of S. platensis, resulting in an increase in the proportion of the fatty acids 16:1 and GLA and a decrease in 16:0 and 18:2. Using a flat plate reactor with a narrow light‐path and intensive stirring which facilitated high cell concentration, we obtained maximal EPA productivity of 58·9mg1−1 day−1 in M. subterraneus and maximal GLA productivity of 26·4 mg1−1 day−1 in S. platensis. The former is the highest EPA production rate reported to date for any microalga, and the latter is the first report on GLA productivity by microalgae. The data presented herein suggest that maintaining very high, yet optimal culture densities (i.e. 4−10gl−1) in enclosed reactors represents an efficient operating mode for enhancing productivity of desired polyunsaturated fatty acids.

Application of Maxblend Fermentor ® for microbial processes

Application of the Maxblend ® impeller (which was originally developed for mixing high viscous liquids during chemical reactions) to a bioreactor (Maxblend Fermentor ® [MBF]) was investigated. In comparison with turbine impeller fermentor (TBN), the MBF showed higher mixing capacity for high viscous liquids and consequently, the mixing time (θ m) was less than 50% of that observed with the TBN. Furthermore, by combining the Maxblend ® impeller with a spiral sparger, the k L a was much higher than that of the TBN with a ring sparger. During the fermentative production of hyaluronic acid (a biopolymer) in TBN, there was a nonhomogeneous pH distribution inside the fermentor when the viscosity of the broth was high. Consequently, the pH in some parts of the fermentor was lower than the optimum pH for hyaluronic acid production. On the other hand, when MBF with spiral sparger was used, the pH distribution inside the fermentor was homogeneous throughout the cultivation, resulting to about 20% increase in the hyaluronic acid productivity compared with a TBN. In the case of γ-linolenic acid (GLA) production by Mortierella ramanniana, although the power consumption for liquid agitation was lower in the MBF equipped with a spiral sparger than in the TBN, small hard pellets were formed in former and this resulted in a higher GLA content of the lipid. Furthermore, in contrast with the pellets of various sizes formed in the TBN, the sizes of the pellets formed in the MBF were uniform. This was because the shear stress distribution was more uniform inside the MBF than in the TBN under the same agitation conditions.

Effectiveness of natural oils as sources of γ-linolenic acid to correct peripheral nerve conduction velocity abnormalities in diabetic rats: modulation by thromboxane A 2 inhibition

Reduced nerve conduction velocity (NCV) in experimental diabetes can be prevented by evening primrose oil (EP), which is rich in γ-linolenic acid (GLA). This study examined the efficacy of natural GLA sources, blackcurrant (BC), borage (BO) and fungal (FU) oils, compared with EP, in correcting motor and sensory NCV deficits in streptozotocin-diabetic rats, and any potential contribution of thromboxane (TX) A 2 synthesis using the TX antagonist, ZD1542, alone and jointly with GLA-rich oils. Sciatic motor NCV, 20% reduced by 8 weeks of diabetes, was partially (16%) corrected by 2 weeks ZD1542 treatment. 1% BC, BO, FU and EP dietary supplementation caused 11%, 32%, 41% and 53% NCV ameliorations, respectively. A 2% EP diet, more closely matching the GLA intake from the other oils, caused 67% correction. Joint oil/ZD1542 treatment produced further motor NCV improvements for BC and, particularly, BO. A 13% sensory saphenous NCV deficit in diabetic rats was ameliorated by 31%, 24%, 49%, 81%, 70% and 94% for ZD1542, BC, BO, FU, EP and 2% EP, respectively. Joint ZD1542-oil treatment further improved NCV, particularly for BO. Therefore, efficacy against experimental diabetic neuropathy is not predictable from the GLA content of natural oils, EP consistently outperforming BC, BO and FU. Increased TXA 2 with diabetes made a minor contribution to NCV deficits, but blockade improved the response to BO.

Production of γ-Linolenic Acid by the Fungus Cunninghamella echinulata CCRC 31840

The effect of various carbon and nitrogen sources and carbon/nitrogen (C :N) ratios on the γ-linolenic acid (GLA) production by Cunninghamella echinulata CCRC 31840 was examined in shake cultures. On the same weight base, soluble starch was the most preferred carbon source for the growth of this fungal strain and also yielded the largest amount of GLA per unit volume. In the lactose medium, growth of C. echinulata CCRC 31840 was very poor but the most unsaturated lipid was produced. Ammonium nitrate and urea were the better nitrogen sources for growth than potassium nitrate, ammonium chloride, and ammonium sulfate. C. echinulata CCRC 31840 grown on KNO 3 acquired the richest lipid content (35.4%) ; however, the content of GLA in the lipid (7.9%) was much lower than that grown on urea (25.7%). An initial C :N ratio of approximately 33-48.5 favored lipid accumulation in biomass without affecting the GLA content of the lipid. A maximum GLA yield of 964 mg/L was obtained after 5 days of incubation at 20 °C in a medium containing 10% soluble starch, 0.5% yeast extract, 0.11% NH 4 NO 3 , 0.1% KH 2 PO 4 , and 0.025% MgSO 4 .7H 2 O.

γ-linolenic acid production by Cunninghamella japonica in solid state fermentation

The production of lipid and γ-linolenic acid (GLA) by Cunninghamella japonica was studied in solid state fermentations (SSF) on cereal substrates. A number of cereals were used as sources of nutrients for fungal SSF and the substrate which favoured the highest accumulation of lipid and GLA in fermented substrate was determined. The treatment of cereals, addition of nutrients to the grain and thickness of the solid substrate influenced fungal growth, lipid and GLA production. Rice and millet gave the highest yields of GLA in the biomass (7·03–7·87%, w/w). The GLA content in the lipid obtained was 20% of the total fatty acids.

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ‐linolenic acid extracted from fungi

AbstractThe effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF‐1384) containing γ‐linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two‐step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol‐extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n‐butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two‐step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n‐butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.

Optimization and scale-up of γ-linolenic acid production by Mortierella ramanniana MM 15-1, a high γ-linolenic acid producing mutant

Optimum conditions for cultivation of Mortierella ramanniana mutant MM 15-1 were investigated with an aim of producing a lipid which contains high γ-linolenic acid (GLA). The optimum initial glucose concentration and pH control level were 300 g/ l and 4.0, respectively. Although both pellet and filamentous forms of this mutant were observed during the cultivation, the pellets accumulated lipid with higher GLA content than the filamentous forms. The effects of culture conditions on pellet formation were therefore investigated in a 30- l jar fermentor. The results showed that with an inoculum spore concentration of 5.0 × 10 3/ml and an agitation speed of 800 rpm, 0.15–0.5 mm pellets were formed and consequently, the GLA content of the lipid increased to a very high value of 18.3%. Furthermore, when this process was scaled up to a 10-kl fermentor, using the impeller tip velocity as the scale-up parameter, both the pellet formation and the lipid production as well as the GLA content of the lipid were consistent with those obtained in the 30- l jar fermentor.

γ-Linolenic acid production by a low temperature-resistant mutant of Mortierella ramanniana

As a result of breeding experiments using low temperature growth as an index, Mortierella ramanniana mutant MM 15-1, which produces lipids with a high γ-linolenic acid (GLA) content, was obtained. The results of cultivation experiments in Erlenmeyer flasks showed that although the total lipid produced by the MM15-1 mutant was about 14% lower than that of the parent strain ( M. ramanniana IFO 8187), the GLA content of the lipid was 16.5%, which represents about a 2-fold increase over that of the parent strain. In particular, there was a significant increase in the GLA proportion of the phospholipid fraction in the MM15-1 mutant. Furthermore, the results of the lipid composition analysis after phospholipid fractionation by TLC showed that there was a significant increase in the GLA proportions in the phosphatidylethanolamine (PE) and phosphatidylinositol (PI) of the MM 15-1 mutant. When the MM 15-1 mutant was cultivated in a 600- l fermentor, it produced lipid with 18.3% GLA. This was a 2-fold higher than the value obtained with the parent strain under the same cultivation conditions.

Antithrombotic activity of a symmetrical triglyceride with eicosapentaenoic acid and γ-linolenic acid in guinea pig mesenteric microvasculature

The antithrombotic effect of a synthetic symmetrical triglyceride having eicosapentaenoic acid (EPA) at positions 1 and 3, and γ-linolenic acid (GLA) at position 2 was investigated. Administration of the triglyceride significantly increased thrombus formation time and thrombotic occlusion time induced by light irradiation and a fluorescent dye in guinea pigs after 14 days administration compared to that of soybean oil. The antithrombotic effect of the triglyceride was similar to that of EPA ethyl ester. Administration of the triglyceride increased GLA, dihomo-γ-linolenic acid (DGLA) and EPA contents in plasma and the liver, and the ratio of DGLA to arachidonic acid. These results might be responsible for this antithrombotic effect.

Fatty acid selectivity of lipases: γ‐linolenic acid from borage oil

AbstractThe γ‐linolenic acid (Z,Z,Z‐6,9,12‐octadecatrienoic acid, GLA) present in borage oil free fatty acids was concentrated in esterification reactions that were catalyzed by several preparations of the acyl‐specific lipase ofGeotrichum candidum. In this manner, a 95% recovery of the GLA originally present in borage oil (25% GLA) was obtained as a highly enriched fatty acid fraction with a GLA content of >70%. Other fatty acids concentrated in this fraction were the monounsaturated fatty acids with chainlengths of C‐20 and longer that were present in the oil. An immobilized preparation ofG. candidum on silica gel also was used for the enrichment of GLA in borage oil. In this instance, a 75% recovery of GLA was obtained, and the supported lipase was reusable (three cycles) with minimal loss in activity.

Optimization of γ-linolenic acid (GLA) production inSpirulina platensis

The cyanobacteriumSpirulina platensis is one of the most promising sources of the polyunsaturated fatty acid γ-linolenic acid (GLA). The GLA content ofSpirulina can be enhanced by cultivation under light-dark cycles in the laboratory or outdoors. Thus, in strain BP, the GLA content increased from 1.2 to 1.6% when cultivated under light-dark cycles. Moreover, in the derived mutant Z19, the GLA content reached 2.4% when cultivated outdoors. To the best of our knowledge, this is the highest GLA content ever reported for any alga.

Enrichment of γ‐linolenic acid from evening primrose oil and borage oilvia lipase‐catalyzed hydrolysis

AbstractLipase‐catalyzed selective partial hydrolysis of evening primrose (Oenothera biennis L.) seed oil and borage (Borago officinalis L.) seed oil led to an increase in the level of γ‐linolenic acid (GLA; 18∶3n−6) in the unhydrolyzed acylglycerols. Thus, in evening primrose oil, the GLA level could be raised from 9.4% in the starting material to 46.5% in the unhydrolyzed acylglycerols by means of a lipase fromCandida cylindracea. Selective hydrolysis of borage oil with Pancreatin led to an increase in the GLA content from 20.4% in the oil to 33.5% in the unhydrolyzed acylglycerols. Partial hydrolysis of borage oil with lipase fromC. cylindracea raised the GLA content of the acylglycerols to 47.8%.

Effects of menatetrenone on prednisolone-induced bone loss in rats

This study was carried out to evaluate the effect of menatetrenone, a vitamin K 2 with 4 isoprene units, on prednisolone-induced bone loss. Three experiments were performed in rats which received menatetrenone as a dietary supplement. In experiment 1, a soluble form of prednisolone, dissolved in drinking water, was administered to rats at 7 mg/kg/day for 9 weeks. The length, dry weight, and bone density of femurs and tibiae, as well as urinary γ-carboxyglutamic acid (Gla) content, were significantly lower in the prednisolone-control group than in the intact group. Menatetrenone (17 mg/kg/ day) significantly inhibited the decrease in these bone parameters, especially in tibiae, and completely inhibited the decrease in urinary Gla content. In experiments 2 and 3, prednisolone (10 mg/kg), dissolved in cottonseed oil, was given to rats intramuscularly three times a week for 4 and 10 weeks, respectively. In experiment 2, bone length, bone strength and calcium content in the femur were reduced by 4-week prednisolone treatment. These reductions were significantly improved by menatetrenone (21 mg/kg/day). In experiment 3, 10-week prednisolone treatment reduced bone length and the calcium and hydroxyproline content of the femur. Menatetrenone (0.4, 10, and 50 mg/kg/day) significantly inhibited the reduction of calcium content in the femur. These results suggest that menatetrenone may inhibit the bone loss induced by corticosteroid treatment.

Production and partial purification of γ-linolenic acid and some pigments fromSpirulina platensis

The polyunsaturated fatty acid γ-linolenic acid (GLA, 18:3ω6) is of potential pharmaceutical value. The cyanobacteriumSpirulina platensis could become an excellent source for this fatty acid, provided that GLA content could be increased and a GLA concentrate could be obtained at a low cost. Increasing the cell concentration inSpirulina platensis enhanced the fatty acid content and thus the GLA content. This effect was used to further enhance the GLA content of GLA-overproducing strains. Separation of the galactolipids and their purification via urea complexes formation, resulted in a GLA concentrate of over 90% purity.

Alterations of Gammacarboxyglutamic Acid in Formic Acid and Guanidine Extracts in Phosphate-Deficient Rat Bone

We developed rickets and osteomalacia in rats by means of a low phosphorus, normal calcium and normal vitamin D diet, causing severely inhibited mineralization. The concentrations of gammacarboxyglutamic acid (Gla) in 10% formic acid and 5M guanidine extracts were studied in normal and phosphate-deficient rat bone. Although the Gla concentration in the formic acid extract was constant for both groups, it decreased in the guanidine extract of the phosphate-deficient group. The Gla content of the guanidine extract reflected a lower concentration of Gla-containing proteins, one of which, matrix Gla protein (MGP), acts as a mineral deposition inhibitor. Thus, the production of MGP decreased in the impaired mineralization of bone. The quantification of Gla in formic acid and guanidine extract is useful in studying the Gla-containing proteins, osteocalcin and MGP.