Eicosapentaenoic acid (EPA) Production byMortierella alpina ATCC 32222

Mortierella alpina ATCC 32222 grew well at 11 degrees C, as well as at 25 degrees C in a liquid medium containing glucose or linseed oil and yeast extract. High Eicosapentaenoic acid (EPA) yield was obtained at 11 degrees C. M. alpina cells did not produce EPA at 25 degrees C in the absence of linseed oil, whereas at 11 degrees C, EPA accumulation was noted in the absence of linseed oil. When grown at 11 degrees C for 10 d in a medium containing 2% linseed oil as carbon source, the mycelium yielded 435 mg/L EPA (20 mg EPA/g dry mycelia) with 5.1% in lipid fraction. By gradually increasing the concentration of linseed oil to 4%, yield of biomass and EPA were increased to 43 g/L and 596 mg/L, respectively.     

Pythium irregulare Fermentation to Produce Arachidonic Acid (ARA) and Eicosapentaenoic Acid (EPA) Using Soybean Processing Co-products as Substrates

Arachidonic acid (ARA) and eicosapentaenoic acid (EPA) were produced by Pythium irregulare fungus using soybean cotyledon fiber and soy skim, two co-products from soybean aqueous processing, as substrates in different fermentation systems. Parameters such as moisture content, substrate glucose addition, incubation time, and vegetable oil supplementation were found to be important in solid-state fermentation (SSF) of soybean fiber, which is to be used as animal feed with enriched long-chain polyunsaturated fatty acids (PUFA). Soybean fiber with 8 % (dwb) glucose supplementation for a 7-day SSF produced 1.3 mg of ARA and 1.6 mg of EPA in 1 g of dried substrate. When soy skim was used as substrate for submerged fermentation, total ARA yield of 125.7 mg/L and EPA yield of 92.4 mg/L were achieved with the supplementation of 7 % (w/v) soybean oil. This study demonstrates that the values of soybean fiber and soy skim co-products could be enhanced through the long-chain PUFA production by fermentation.     

Biosynthesis of Unsaturated Fatty Acids via Mortierella Isabellina Cultivated in a Medium Containing Butanol

IntroductionThe biosynthesis of unsaturated fatty acidshasattracted more attention in recent years. Forexample,linoleic and linolenic acids are importantmaterials for pharmaceutical and food industries.Besides,they can be used to synthesize paint,printing ink and surfactant,etc. A number ofmicroorganisms have been studied as potentialcommercial sources to produce unsaturated fattyacids[1] .Agricultural and industrial products,by-products,etc. have been employed in thecultivation of those orga…     

Simultaneous quantification of total eicosapentaenoic acid,docosahexaenoic acid and arachidonic acid in plasma by high‐performance liquid chromatography–tandem mass spectrometry

A method for the simultaneous quantification of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) in human plasma by HPLC–tandem mass spectrometry (HPLC‐MS/MS) was developed and validated. Free and esterified forms of fatty acids were hydrolysed from plasma samples in the presence of an internal standard and subjected to liquid–liquid extraction. The chromatographic run time was 3.5 min per sample. The assay was linear from 0.5 to 300 mg/L (r² > 0.997, n = 18). Based on matrix addition, accuracy deviation was <15%, except for AA at 10 mg/L (30–90%), whereas precision was <8% for all fatty acids studied. The method was applied to the measurement of these omega‐3 fatty acids in a fish oil supplement study with healthy volunteers. Healthy males (n = 4) were administered a supplement containing 465 mg EPA and 375 mg DHA per capsule (Omacor®). A dose of two capsules was given daily over a 4 week period. Pre‐treatment concentrations varied between subjects for EPA (17–68 mg/L), DHA (36–63 mg/L) and AA (121–248 mg/L). During the dosing period EPA increased 460–480% from the baseline concentration, while DHA increased 150–160%. The EPA–AA ratio increased from 0.07–0.56 to 0.3–3.1 after 4 weeks of dosing. In conclusion, the method described could be suitable for monitoring EPA, DHA and AA in clinical studies that may aid in achieving optimal concentrations of these fatty acids in patients who could be at risk of sudden cardiac death. Copyright © 2010 John Wiley & Sons, Ltd.     

Aalysis of the fatty acid composition of the lipid classes in human blood serum under normal diet and when supplemented with fish oil

Total lipids have been extracted from human serum with chloroform–methanol 2:1 (v/v) and separated into individual classes by TLC. After transesterification the fatty acid methyl esters were analyzed by capillary gas chromatography on an FFAP column. The quantitation of ω-3 fatty acids has been performed using internal and external standards. Internal lipid standards for each lipid class were carried throughout the entire analytical procedure. Under normal diet eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are incorporated into the lipid classes to different extents: cholesterol esters; EPA, 6.5 ± 1.9 γ/ml serum; DHA, 4.3 ± 1.9 μg/ml: phospholipids; EPA, 5.9 ± 2.7 μg/ml; DHA, 31.8 ± 8.1 μg/ml. Fish oil supplementation leads to a 4 to 6-fold rise in EPA and to an approximately 2-fold rise in DHA.     

Isolation of 6,9,12,15-Hexadecatetraenoic Fatty Acid (16:4n-1) Methyl Ester from Transesterified Fish Oil by HSCCC

Fish oil is considered a healthy food due to the presence of large amounts of polyunsaturated fatty acids (PUFAs), especially in the form of n-3 fatty acids 5,8,11,14,17-eicosapentaenoic acid (20:5n-3; EPA) and 4,7,10,13,16,19-docosahexaenoic acid (22:6n-3; DHA). However, fish oil is known to contain many other PUFAs, some of which are uncommon and whose bioactivity is scarcely investigated. In this study, we isolated the rare PUFA 6,9,12,15-hexadecatetraenoic fatty acid (16:4n-1) which bears a double bond on the terminal carbon from fish oil in form of its methyl ester. We used high-speed counter-current chromatography (HSCCC) for the fractionation of 500 mg-portions of fatty acid methyl esters prepared from a fish oil capsule and investigated the fractions by GC/MS. Twenty-eight 13-mL fractions were collected and fatty acid methyl esters were detected in fractions 11–23. The elution was carried out in normal phase mode, providing the long-chained saturated and monoenoic fatty acids first. More than 100 fatty acids ranging from 10:0 to 26:0 could be identified in the HSCCC fractions, and most of them were polyunsaturated. The reproducibility of the HSCCC method was shown by repeated injection of the fish oil and the fractions containing 6,9,12,15-hexadecatetraenoic fatty acid (16:4n-1). The late eluting 16:4n-1 methyl ester was isolated in pure form and its structure was verified.

     

Control Mechanism for cis Double‐Bond Formation by Polyunsaturated Fatty‐Acid Synthases

Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) are essential fatty acids for humans. Some microorganisms biosynthesize these PUFAs through PUFA synthases composed of four subunits with multiple catalytic domains. These PUFA synthases each create a specific PUFA without undesirable byproducts, even though the multiple catalytic domains in each large subunit are very similar. However, the detailed biosynthetic pathways and mechanisms for controlling final‐product profiles are still obscure. In this study, the FabA‐type dehydratase domain (DH_FabA) in the C‐subunit and the polyketide synthase‐type dehydratase domain (DH_PKS) in the B‐subunit of ARA synthase were revealed to be essential for ARA biosynthesis by in vivo gene exchange assays. Furthermore, in vitro analysis with truncated recombinant enzymes and C₄‐ to C₈‐acyl ACP substrates showed that ARA and EPA synthases utilized two types of DH domains, DH_PKS and DH_FabA, depending on the carbon‐chain length, to introduce either saturation or cis double bonds to growing acyl chains.     

Preparation of Triacylglycerols Rich in Omega-3 Fatty Acids from Sardine Oil Using a Rhizomucor miehei Lipase: Focus in the EPA/DHA Ratio

The increasing evidence on the differential biochemical effects of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) raises the need of n-3 highly unsaturated fatty acid concentrates with different amounts of these fatty acids. In the present work, physicochemical and enzymatic techniques were combined to obtain acylglycerols, mainly triacylglycerols (TAG), rich in n-3 fatty acids. Sardine oil was obtained by washing sardine (Sardina pilchardus) mince with a NaHCO₃ solution, hydrolyzed in a KOH–ethanol solution, and concentrated with urea. The esterification reaction was performed in the stoichiometric proportion of substrates for re-esterification to TAG, with 10 % level of Rhizomucor miehei lipase based on the weight of substrates, without any solvent, during 48 h. This procedure led to approximately 88 % of acylglycerols, where more than 66 % were TAG and the concentration of n-3 fatty acids was higher than 60 %, the EPA and DHA ratio (EPA/DHA) was 4:1. The content of DHA in the unesterifed fraction (free fatty acids) increased from 20 to 54 %, while the EPA level in the same fraction decreased from 33 to 12.5 % (EPA/DHA ratio ≈1:4). Computational methods (density functional theory calculations) have been carried out at the B3LYP/6-31G(d,p) level to explain some of the experimental results.     

Production of Thermostable Lipase by Thermomyces lanuginosus on Solid-State Fermentation: Selective Hydrolysis of Sardine Oil

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.     

Analysis of polyunsaturated fatty acids in blood serum after fish oil administration.

Free and total fatty acids in the blood serum of patients with hyperlipoproteinemia have been analysed as their methyl esters by capillary gas chromatography using an FFAP column. In one-step reactions the free fatty acids in serum react with methanol-acetyl chloride (50:1, v/v) at 25 degrees C, the total fatty acids (free plus esterified) are transesterified with methanol-toluene-acetyl chloride (8:2:1, v/v) at 100 degrees C. The quantification of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is based on an internal standard (13,16,19-docosatrienoic acid) and on calibration standards. Under normal diet the concentrations of EPA and DHA are as follows (mean +/- S.D., n = 27): free EPA, 0.2 +/- 0.1 mg/dl; free DHA, 0.6 +/- 0.2 mg/dl; total EPA, 3.6 +/- 2.1 mg/dl; total DHA 11.4 +/- 3.1 mg/dl. Under a fish oil intake of 9 g per day, free and total EPA concentrations rise by ca. five- to six-fold, and free and total DHA concentrations by ca. two-fold.     

Optimization of Fatty Acid Determination in Selected Fish and Microalgal Oils

The use of ten fatty acid methyl ester reference standards coupled with a detailed quantification method was shown to significantly optimize the fatty acid determination of selected fish and microalgal oils when compared to methods that use only one reference standard (C19:0 or C23:0) as a relative response factor. When using the mixture of ten reference standards after transesterifying oils with NaOH/BF₃, determination of total fatty acids, eicosapentaenoic acid and docosahexaenoic acid improved by an average of 7.3, 11.5 and 8.4%, respectively. Furthermore, improvements of 13.9, 18.9 and 6.8% of total fatty acids, EPA and DHA, respectively, were obtained when using the mixture of reference standards for fatty acid determination after directly extracting and transesterifying oil contained in microalgal cells with a mixture of methanol, HCl and chloroform. Fatty acid methyl ester standards dissolved in isooctane showed <5% variability throughout 130 days of stability testing when stored at ?20 °C. The optimized method can be used for improving the quantification of fatty acids in both oils (fish and microalgal oils) and dry microalgal cells.     

Use of Isopropanol as a Modifier in a Hexane-Acetonitrile Based Mobile Phase for the Silver Ion HPLC Separation of Positional Isomers of Triacylglycerols Containing Long Chain Polyunsaturated Fatty Acids

A high resolution approach to silver ion HPLC was studied for the separation of positional isomers of triacylglycerols (TAGs) containing long chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and docosapentaenoic acid (DPA) in enzymatically synthesized structured TAGs. Isopropanol was used as a novel modifier in a hexane-acetonitrile based mobile phase for silver ion HPLC. Peak identification was based on HPLC-mass spectroscopy and selectivities of lipases. Positional isomers of TAGs containing one molecule of EPA, DHA, or DPA with saturated fatty acids (FAs) such as caprylic acid and palmitic acid were separated within 13 min using a gradient of hexane-isopropanol-acetonitrile as mobile phase. TAGs containing two or more EPA, DHA, or DPA were also separated from each other within 25 min, but their positional isomers were unresolved. The retention characteristics of the TAG were found to be related to the number of carbon atoms in the FAs present in addition to the number of double bonds and their isomeric configuration. One isomer with an unsaturated FA in the sn-2 position eluted faster than the other with the unsaturated FA in the sn-1 or 3 position. Species with longer chain FAs attached to TAGs with the same degree of unsaturation eluted faster than those that have shorter chain FAs. For example, docosapentaenoylhexadecanoyloctanoin (DPA/C16/C8) was eluted faster than dioctanoyldocosapentaenoin (DPA/C8/C8).     

Clean synthesis of biodiesel over solid acid catalysts of sulfonated mesopolymers

FDU-15-SO₃H, a solid acid material prepared from the sulfonation of FDU-15 mesoporous polymer, has been demonstrated to serve as an efficient catalyst in the esterification of palmitic acid with methanol as well as in the transesterification of fatty acid-edible oil mixture. FDU-15-SO₃H achieved an acid conversion of 99.0% when the esterification was carried out at 343 K with a methanol/palmitic acid molar ratio of 6:1 and 5 wt% catalyst loading. It was capable of giving 99.0% yield of fatty acid methyl esters (FAME) when the transesterification of soybean oil was performed at 413 K and the methanol/oil weight ratio of 1:1. FDU-15-SO₃H was further applied to the transesterification/esterification of the oil mixtures with a varying ratio of soybean oil to palmitic acid, which simulated the feedstock with a high content of free fatty acids. The yield of FAME reached 95% for the oil mixtures containing 30 wt% palmitic acid. This indicated the sulfonated mesopolymer was a potential catalyst for clean synthesis of fuel alternative of biodiesel from the waste oil without further purification.     

Quantification of eicosapentaenoic and docosahexaenoic acid geometrical isomers formed during fish oil deodorization by gas-liquid chromatography

Long-chain polyunsaturated fatty acids (LC-PUFAs) of the n-3 series and especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) have important biological properties. The main dietary sources of LC-PUFAs are fish and fish oil. Geometrical isomerization is one of the main reactions happening during the thermal treatment of polyunsaturated fatty acids. Refined fish oils are used to supplement food products in LC-PUFAs and the quality of these nutritional ingredients have to be controlled. In the present study, a suitable method for the quantification of EPA and DHA geometrical isomers in fish oils by gas-liquid chromatography (GC) is presented. A highly polar capillary column (CP-Sil 88, 100 m) operating under optimal conditions was used. Method selectivity was studied by GC-mass spectrometry. The performance characteristics of the quantification method were studied using samples of fish oil deodorized at 220 degrees C for 3 h. The linearity of the method was assessed by analyzing composite samples obtained by mixing fish oil deodorized at 220 degrees C with semi-refined fish oil (control). Precision was evaluated by analyzing the same samples in triplicate. Results showed that the validated method is suitable to quantify low amounts of geometrical (trans) isomers of EPA and DHA in refined fish oils. The limits of quantification of the EPA and DHA geometrical isomers are 0.16 and 0.56 g/100 g of fish oil, for EPA and DHA, respectively. Commercially available LC-PUFA oil samples were evaluated by using the validated method. The results show that the oils analyzed contain low amounts (<1% of total fatty acids) of geometrical isomers of EPA and DHA.     

Effects of Dietary α-Linolenic Acid Treatment and the Efficiency of Its Conversion to Eicosapentaenoic and Docosahexaenoic Acids in Obesity and Related Diseases

The essential fatty acid alpha-linolenic acid (ALA) is present in high amounts in oils such as flaxseed, soy, hemp, rapeseed, chia, and perilla, while stearidonic acid is abundant in echium oil. ALA is metabolized to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by desaturases and elongases in humans. The conversion of ALA to EPA and DHA is limited, and these long-chain n−3 polyunsaturated fatty acids (PUFAs) are mainly provided from dietary sources (fish and seafood). This review provides an overview of studies that explored the effects of dietary supplementation with ALA in obesity and related diseases. The obesity-associated changes of desaturase and elongase activities are summarized, as they could influence the metabolic conversion of ALA. Generally, supplementation with ALA or ALA-rich oils leads to an increase in EPA levels and has no effect on DHA or omega-3 index. According to the literature data, stearidonic acid could enhance conversion of ALA to long-chain n−3 PUFA in obesity. Recent studies confirm that EPA and DHA intake should be considered as a primary dietary treatment strategy for improving the omega-3 index in obesity and related diseases.     

深黄被孢霉生物转化十六醇合成油脂的研究

不饱和脂肪酸有重要的生理机能 ,寻找富含不饱和脂肪酸的新油源 ,以弥补动植物油源的不足已成为当前的研究热点 .高碳醇转化为高碳酸已有报道[1],但将其转化为不饱和脂肪酸的研究还少见报道 .利用产脂微生物合成不饱和脂肪酸是开发新油源的良好途径[2 ,3].本文用深黄被孢霉生物转化十六醇合成油脂 ,并分析了油脂中脂肪酸的组成 ,对转化条件也进行了初步研究 .1　实验部分将深黄被孢霉 (Ｍｏｒｔｉｅｒｅｌｌａｉｓａｂｅｌｌｉｎａ)于 2 8℃在种子培养液中活化 2 4ｈ ,然后接种于十六醇转化培养基上 ,于 2 8℃摇瓶发酵 96ｈ(摇瓶转速 1 50ｒ…     

Lipids Containing Polyunsaturated Fatty Acids Synthesized by Zygomycetes Grown on Glycerol

Several strains of Zygomycetes cultivated on glycerol produced mycelia rich in lipids containing higher amounts of neutral lipids (NL) than glycolipids plus sphingolipids and phospholipids (P), while biosynthesis of P in Mortierella ramanniana, Mucor sp., and Cunninghamella echinulata occurred though NL accumulation process was in progress. Polyunsaturated fatty acids (PUFA) concentration gradually decreased in all lipid fractions of M. ramanniana during growth. In contrast, in C. echinulata concentration of both linoleic and γ-linolenic acids increased with time, especially in P. Taking for granted that the main function of PUFA is associated to their participation in mycelial membranes, we could suppose that biosynthesis of these fatty acids is associated to mycelial growth. However, this is accurate only for some Zygomycetes, e.g., M. ramanniana. On the contrary, PUFA biosynthesis in C. echinulata persists after growth cessation, suggesting that in this species biosynthetic ability is not a strictly growth-associated process. Phosphatidyl-inositol and phosphatidyl-choline were the major P classes in C. echinulata and M. ramanniana, respectively. In M. ramanniana, a decrease of PUFA concentration was noticed even when mycelia were incubated in low temperature (conditions that normally favor PUFA biosynthesis), indicating that PUFA biosynthesis in this fungus is associated to primary metabolism.     

An Analysis of Oxidative Changes and the Fatty Acid Profile in Stored Poultry Sausages with Liquid and Microencapsulated Fish Oil Additives

This study deals with the fatty acid profile and oxidative changes (TBARS) in vacuum-packed (VP) or modified-atmosphere-packed (MAP) finely-comminuted poultry sausages with liquid fish oil and microencapsulated fish oil (MC) additives. An analysis of omega-3 fatty acids (EPA and DHA) showed that their content in the samples with the fish oil additive decreased from the initial value of 0.22 g∙100 g⁻¹ of the product to 0.18 g∙100 g⁻¹ (MAP) and 0.17 g∙100 g⁻¹ (VP), respectively. After in vitro digestion, the total EPA and DHA content in the sample with microencapsulated oil amounted to 0.17 g∙100 g⁻¹ of the product. The TBARS values showed the VP samples with both forms of the fish oil additive had the lowest values on the first day of storage. Storage of the samples for 21 days caused a slight increase in the degree of lipid oxidation. The research indicated that the forms of the oil additive did not have a negative influence on the sensory features or the physicochemical properties of the sausages. The EPA and DHA levels in samples with liquid fish oil and those with oil microcapsules were sufficient for the sausage producer to declare high content of these fatty acids in accordance with the current EC regulation.     

Harnessing Indigenous Plant Seed Oil for the Production of Bio-fuel by an Oleaginous Fungus,Cunninghamella blakesleeana- JSK2, Isolated from Tropical Soil

Cunninghamella blakesleeana- JSK2, a gamma-linolenic acid (GLA) producing tropical fungal isolate, was utilized as a tool to evaluate the influence of various plant seed oils on biomass, oleagenicity and bio-fuel production. The fungus accumulated 26 % total lipid of their dry biomass (2 g/l) and 13 % of GLA in its total fatty acid. Among the various plant seed oils tested as carbon sources for biotransformation studies, watermelon oil had an effect on biomass and total lipid increasing up to 9.24 g/l and 34 % respectively. Sunflower, pumpkin, and onion oil increased GLA content between 15–18 %. Interestingly, an indigenous biodiesel commodity, Pongamia pinnata oil showed tremendous effect on fatty acid profile in C. blakesleeana- JSK2, when used as a sole source of carbon. There was complete inhibition of GLA from 13 to 0 % and increase in oleic acid content, one of the key components of biodiesel to 70 % (from 20 % in control). Our results suggest the potential application of indigenous plant seed oils, particularly P. pinnata oil, for the production of economically valuable bio-fuel in oleaginous fungi in general, and C. blakesleeana- JSK2, in particular.