Enhancement of arachidonic acid production by Mortierella alpina 1S-4

The effect of mineral addition on arachidonic acid (AA) production by Mortierella alpina 1S-4 was evaluated. At first, the addition of minerals such as sodium, potassium, calcium, and magnesium was examined in flask cultures, and then the addition of phosphorus with the optimal amounts of the minerals was investigated in a 10-L jar-fermenter. As a result, 1.5% soy flour medium with the addition of 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O, and 0.05% MgCl₂·6H₂O was found to enhance the AA yield 1.7-fold over that without mineral addition. When 1% yeast extract with the above mineral mixture was used, the AA yield was enhanced 1.35-fold over that without minerals. We also verified that an increase in the polar lipid content occurred in the case of only KH₂PO₄ addition, and that the above-mentioned increase in the AA yield was due to the minerals themselves, not a pH buffer effect.     

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.     

Effects of mineral addition on the growth morphology of and arachidonic acid production by Mortierella alpina 1S-4

The culture conditions for arachidonic acid (AA) production by Mortierella alpina 1S-4 were investigated by means of a morphological study with the aims of obtaining a high AA yield and scale-up. In a 50-L jar fermentor study, a medium containing 3.1% soy flour and 1.8% glucose with 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% CaCl₂·2H₂O and 0.05% MgCl₂·6H₂O was found to be optimum. The AA yield reached 9.8 g/L/7 d, and the major morphology was small pellets (1–2 mm). However, in the case of the only addition of KH₂PO₄, the major morphology was filaments. The apparent viscosity increased to 2240 cp, thereby requiring a high agitation speed to maintain adequate oxygen tension, which caused mycelial damage due to shear stress and therefore a decrease in the AA yield. When a medium with Na₂SO₄, CaCl₂, and MgCl₂ was used, the major morphology was large pellets (2–3 mm), and mass transfer limitation through the pellet wall caused a decrease in the AA yield. Based on these results, a scale-up study was carried out under the optimal conditions described above. An AA yield of 10.9 g/L/8 d was obtained in a 10-kL industrial fermentor, and the major morphology was small pellets.     

Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate

A fungal isolate Wuji-H4 with a dense-lobe rosette growth pattern on malt extract agar was identified as Mortierella alpina Peyronel. It was capable of producing 504 mg/L of arachidonic acid (AA) in the screening medium. Its AA content accounted for 42.4% of the total fatty acids. The AA yield was raised to 1,817 mg/L by a step-by-step approach, which uncovered that the preferred carbon source, nitrogen source, and temperature for fungal growth and lipid production were soluble starch, urea, and 24°C, respectively. Productivity was further optimized by exploiting the interactions between the constituents of the medium by the response surface method. A partial factorial design, followed by steepest ascent analysis, was carried out to locate the general vicinity of the optimal level of each nutrient. The response surface of AA production in this optimal region was then approximated with a full quadratic equation obtained from a three-factor/five-level central composite rotatable design. Maximum AA yield was predicted to occur in a medium that contained 99.7 g/L of soluble starch, 12.6 g/L of yeast extract, and 3.0 g/L of KH₂PO₄. Upon verification, the average experimental yield of AA (3,885 mg/L) was not significantly different from the predicted AA yield (3,940 mg/L), indicating that the response surface method had succeeded in exploiting the AA production potential of this new fungal isolate.     

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

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

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Enzymatic enrichment of arachidonic acid from Mortierella single-cell oil

An attempt was made to enrich arachidonic acid (AA) from Mortierella single-cell oil, which had an AA content of 25%. The first step involved the hydrolysis of the oil with Pseudomonas sp. lipase. A mixture of 2.5 g oil, 2.5 g water, and 4000 units (U) Pseudomonas lipase was incubated at 40°C for 40 h with stirring at 500 rpm. The hydrolysis was 90% complete after 40 h, and the resulting free fatty acids (FFA) were extracted with n-hexane (AA content, 25%; recovery of AA, 91%). The second step involved the selective esterification of the fatty acids with lauryl alcohol and Candida rugosa lipase. A mixture of 3.5 g fatty acids/lauryl alcohol (1:1, mol/mol), 1.5 g water, and 1000 U Candida lipase was incubated at 30°C for 16 h with stirring at 500 rpm. Under these conditions, 55% of the fatty acids were esterified, and the AA content in the FFA fraction was raised to 51% with a 92% yield. The long-chain saturated fatty acids in the FFA fraction were eliminated as urea adducts. This procedure raised the AA content to 63%. To further elevate the AA content, the fatty acids were esterified again in the same manner with Candida lipase. The repeated esterification raised the AA content to 75% with a recovery of 71% of its initial content.     

Production of 8,11,14,17-cis-eicosatetraenoic acid by Δ5 desaturase-defective mutants of an arachidonic acid-producing fungus, Mortierella alpina

Δ5 Desaturase-defective mutants of an arachidonic acid-producing fungus, Mortierella alpina 1S-4, accumulate large amounts of 8,11,14,17-cis-eicosatetraenoic acid (20:4ω3) when grown with linseed oil. One of the mutants, the S14 strain, produced 1.65 mg of 20:4ω3 per mL of culture medium (corresponding to 66.0 mg/g dry mycelia and 11.6% of total cellular fatty acids) when grown in a medium containing 1% glucose, 1% yeast extract, and 4% linseed oil methyl ester at 28°C for 2 d, and then at 16°C for 7 d. In a bench-scale fermentation in a 5-L jar fermenter, 20:4ω3 production reached 1.60 g/L of culture medium on the eighth day (corresponding to 77.3 mg/g dry mycelia and 26.0% of total cellular fatty acids). The cellular lipids of the S14 strain comprised 75.8% triacylglycerol (TG), 6.7% diacylglycerol, and 13.3% phospholipids (PL). The percentage of 20:4ω3 was higher in PL than in TG, and highest in phosphatidylcholine (32.6%).     

Metabolic flux analysis on arachidonic acid fermentation

The analysis of flux distributions in metabolic networks has become an important approach for understanding the fermentation characteristics of the process. A model of metabolic flux analysis of arachidonic acid (AA) synthesis in Mortierella alpina ME-1 was established and carbon flux distributions were estimated in different fermentation phases with different concentrations of N-source. During the exponential, decelerating and stationary phase, carbon fluxes to AA were 3.28%, 8.80% and 6.97%, respectively, with sufficient N-source broth based on the flux of glucose uptake, and those were increased to 3.95%, 19.21% and 39.29%, respectively, by regulating the shifts of carbon fluxes via fermentation with limited N-source broth and adding 0.05% NaNO₃ at 96 h. Eventually AA yield was increased from 1.3 to 3.5 g·L⁻¹. These results suggest a way to improve AA fermentation, that is, fermentation with limited N-source broth and adding low concentration N-source during the stationary phase. __________ Translated from Journal of Chemical Engineering of Chinese Universities, 2007, 21(2): 316–321 [译自: 高校化学工程学报]     

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.     

An efficient method for the enrichment of the arachidonic acid methyl ester from Mortierella alpina-derived crude oils

An improved method was developed for enriching arachidonic acid (AA) methyl ester from microbial oil by two-step low-temperature wet fractionation. The effects of solvent, operating temperature, and solvent-to-fatty acid methyl esters (FAMEs) ratio on the enrichment of AA were investigated. The best results were achieved when n-hexane was used as solvent. With operating temperatures in the range ?30 °C to ?80 °C and a FAMEs-to-solvent ratio of 1:5 (v/v), the proportion of AA methyl ester isolated could be increased to 83.76 ± 2.78% with a yield of 52.89%. The total recovery of AA methyl ester would be further increased to 90.84% by recrystallization of the solid phases. The 20C, 22C saturated fatty acids were enriched by n-hexane or petroleum ether at ?30 °C, with concentrations increased 7.5-fold or 7.2-fold compared with their original levels, respectively. In addition, a method that combined alkali and acid catalysis of the transmethylation was the most conducive to the preparation of polyunsaturated fatty acid methyl esters.     

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.     

Antioxidative effect of the main sinapic acid derivatives from rapeseed and mustard oil by‐products

Amongst oilseeds, rapeseed and mustard are rich sources of phenolic compounds, which also prominent in the by‐products of their respective oil processing or in commercial rapeseed and mustard press cakes. These cakes are rich sources of sinapic acid derivatives, which could be extracted as free sinapic acid or sinapine, the choline ester of sinapic acid. Sinapic acid is a widely investigated antioxidative compound. However, the main compound in the press cakes is present as sinapine. Investigations on the free‐radical‐scavenging activity of sinapic acid and sinapine indicate that sinapine had a significant but lower activity as compared to sinapic acid. Apart from this, sinapic acid, sinapine and different tocopherols were compared as antioxidants for inhibition of the formation of lipid oxidation products in purified rapeseed oils. The oxidation at 40 °C was monitored by the formation of hydroperoxides and propanal. The experiments indicate that in contrast to tocopherol mixtures addition of sinapic acid causes increasing inhibition of hydroperoxide formation when enhancing the concentration from 50 to 500 μmol/kg oil. Sinapine was not able to inhibit the formation of hydroperoxides, compared to sinapic acid. This indicates that sinapic acid‐rich extracts, as compared to sinapine‐rich fractions, could better inhibit the lipid oxidation in bulk lipid systems.     

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.     

Determination of the fatty acid composition of the oil in intact-seed mustard by near-infrared reflectance spectroscopy

Near-infrared reflectance spectroscopy (NIRS) was used to estimate the fatty acid composition of the oil in intact-seed samples of Ethiopian mustard (Brassica carinata Braun) within a mutation breeding program that produced seeds with variable fatty acid compositions. Five populations, from 1992 to 1996 crops, were included in this study; and NIRS calibration equations for major fatty acids (palmitic, stearic, oleic, linoleic, linolenic, eicosenoic, and erucic) were developed within each single population. Furthermore, global calibration equations, including samples from the five populations, were developed. After external validation, the NIRS technique permitted us to obtain a reliable and accurate nondestructive estimation of the fatty acid composition of the oil, especially for the major acids—oleic, linoleic, linolenic, and erucic. For these, the r ² in external validation was higher than 0.95 by using both single-and multipopulation equations, and higher than 0.85 for the remaining fatty acids. Moreover, the multipopulation equations provided an accurate estimation of samples from a population not represented in the calibration data set, with values of coefficient of determination in validation (r ²) from 0.80 (palmitic and eicosenoic acids) to 0.97 (erucic acid). The ability of NIRS to discriminate among different fatty acid profiles was mainly due to changes within six spectral regions, 1140–1240, 1350–1400, 1650–1800, 1880–1920, 2140–2200, and 2240–2380 nm, all of them associated with fatty acid absorbers. Thus, NIRS can be used to estimate the fatty acid composition of Ethiopian mustard seeds with a high degree of accuracy, provided that calibration equations be developed from calibration sets that include large variability for the fatty acid composition of the oil.     

Effect of rosehip extraction process on oil and defatted meal physicochemical properties

The physicochemical properties of oil from Rosa affinis rubiginosa seeds were analyzed after extraction by (i) organic solvent, (ii) cold pressing, and (iii) cold pressing assisted by enzymatic pretreatment using a mixture of the Novo-Nordisk A/S products Cellubrix (cellulase and hemicellulase activities) and Olivex (pectinase, cellulase, and hemicellulase activities). There were no significant differences in oil quality parameters, such as iodine value, refractive index, saponification value, unsaponifiable matter, and FA profile, when applying any of the three extraction processes. Although significant variations were observed in FFA content (acid value) and PV of the oil obtained by both of the cold-pressing oil extraction processes, these results were lower than the maximum value established from the Codex Alimentarius Commission. All-trans-retinoic acid content improved by 700% in rosehip oil obtained through cold pressing, with and without enzymatic pretreatment, in comparison with organic solvent extraction. This result is quite important for cosmetic oil because all-trans-retinoic acid is the main bioactive component responsible for the regenerative properties of this oil.     

Conjugated linoleic acid production from linoleic acid by lactic acid bacteria

After screening 14 genera of lactic acid bacteria, Lactobacillus plantarum AKU 1009a was selected as a potential strain for CLA production from linoleic acid. Washed cells of L. plantarum with high levels of CLA production were obtained by cultivation in a nutrient medium with 0.06% (wt/vol) linoleic acid (cis-9,cis-12-octadecadienoic acid). Under the optimal reaction conditions with the free form of linoleic acid as the substrate, washed cells of L. plantarum produced 40 mg CLA/mL reaction mixture (33% molar yield) from 12% (wt/vol) linoleic acid in 108 h. The resulting CLA was a mixture of two CLA isomers, cis-9,trans-11 (or trans-9,cis-11)-octadecadienoic acid (CLA1, 38% of total CLA) and trans-9,trans-11-octadecadienoic acid (CLA2, 62% of total CLA), and accounted for 50% of the total FA obtained. A higher yield (80% molar yield to linoleic acid) was attained with 2.6% (wt/vol) linoleic acid as the substrate in 96 h, resulting in CLA production of 20 mg/mL reaction mixture [consisting of CLA1 (2%) and CLA2 (98%)] and accounting for 80% of total FA obtained. Most of the CLA produced was associated with the cells (ca. 380 mg CLA/g dry cells), mainly as FFA.     

Electrophoretic and functional properties of mustard seed meals and protein concentrates

Defatted meals and protein concentrates from five varieties of mustard seeds (four Brassica spp. and one Sinapis alba) were analyzed for polypeptide composition and functional properties. Nonreducing gel electrophoresis showed that Brassica seeds lacked the 135- and 50-kDa polypeptides that were present in the seeds of the S. alba variety. On the other hand, the 29-kDa polypeptide found in the Brassica seeds was absent from the seed of the S. alba variety. Under reducing conditions, the 135 kDa was not detected in the S. alba variety and the intensity of the 50-kDa polypeptide was severely reduced; in contrast, the intensity of the 29-kDa polypeptide in the Brassica seeds was not affected. Meals from yellow seeds had significantly higher (P≤0.05) protein contents than meals from the brown seeds. The emulsifying activity indexes (EAI) of meals and protein concentrates from the Brassica seeds were significantly higher (P≤0.05) than those obtained for similar products from S. alba sees. It was concluded that the disulfide-bonded 50- and 135-kDa polypeptides may have contributed to increased rigidity of S. alba meal proteins, which resulted in poor EAI when compared to the Brassica meals, which do not contain these polypeptides.     

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%).