Strategies for the enzymatic enrichment of PUFA from fish oil

PUFA from oil extracted from Nile perch viscera were enriched by selective enzymatic esterification of the free fatty acids (FFA) or by hydrolysis of ethyl esters of the fatty acids from the oil (FA‐EE). Quantitative analysis was performed using RP‐HPLC coupled to an evaporative light scattering detector (RP‐HPLC‐ELSD). The lipase from Thermomyces lanuginosus discriminated against docosahexaenoic acid (DHA) most, resulting in the highest DHA/DHA‐EE enrichment while lipase from Pseudomonas cepacia discriminated against eicosapentaenoic acid (EPA) most, resulting in the highest EPA/EPA‐EE enrichment. The lipases discriminated between DHA and EPA with a higher selectivity when present as ethyl esters (EE) than when in FFA form. Thus when DHA/EPA were enriched to the same level during esterification and hydrolysis reactions, the DHA‐EE/EPA‐EE recoveries were higher than those of DHA/EPA‐FFA. In reactions catalysed by lipase from T. lanuginosus, at 26 mol% DHA/DHA‐EE, DHA recovery was 76% while that of DHA‐EE was 84%. In reactions catalysed by lipase from P. cepacia, at 11 mol% EPA/EPA‐EE, EPA recovery was 79% while that of EPA‐EE was 92%. Both esterification of FFA and hydrolysis of FA‐EE were more effective for enriching PUFA compared to hydrolysis of the natural oil and are thus attractive process alternatives for the production of products highly enriched in DHA and/or EPA. When there is only one fatty acid residue in each substrate molecule, the full fatty acid selectivity of the lipase can be expressed, which is not the case with triglycerides as substrates.     

Concentration of Docosahexaenoic Acid (DHA) by Selective Alcoholysis Catalyzed by Lipases

The aim of this work was to obtain acylglycerols from tuna oil (23 % weight DHA) rich in docosahexaenoic acid (DHA) by selective ethanolysis, catalyzed by lipases. First, seven immobilized lipases were tested and the best DHA concentration and recovery in the acylglycerol fraction were attained with Lipozyme TL IM^® from Thermomyces lanuginosus, Lipozyme RM IM from Rhizomucor miehei, and lipase from Thermomyces lanuginosus immobilized on Immobead 150. As it is the cheapest, Lipozyme TL IM^® was selected to optimize the reaction conditions. The influence of temperature, reaction time, and ethanol/oil and lipase/oil ratios were studied. Under the optimized conditions (35 °C, ethanol/oil molar ratio 2.3, lipase/oil ratio 5 % weight and 48 h) and for a 56 % conversion, acylglycerols were obtained with a 45 % DHA concentration and 90 % recovery. In these optimized conditions the reaction was scaled up to 766 g of tuna oil and carried out in a batch stirred tank reactor, with the lipase contained in a cartridge filter attached to the stirring rod. The results were similar to those obtained on the smaller scale. The DHA-enriched acylglycerols were separated from the ethyl esters by evaporation of the latter in a short-path vacuum distiller, where the influence of distillation temperature was studied. At 170 °C DHA-rich acylglycerols (44 % DHA) were recovered in the residue with 94.5 % purity and 72 % recovery.     

Fractionation of urea-pretreated squid visceral oil ethyl esters

Ethyl esters of squid (Illex argentinus) visceral oil contained 11.8% eicosapentaenoic acid (EPA) and 14.9% docosahexaenoic acid (DHA). The esters were treated with urea to increase the contents of EPA and DHA. The non-urea complexing ethyl esters of squid visceral oil contained 28.2% EPA and 35.6% DHA. This mixture was fractionated by molecular distillation to further increase the EPA or DHA content. The fraction collected in the 110°C distillate had an EPA content of 39.0% with 0.26 g/100 g of cholesterol, while the 130°C distillate contained 65.6% DHA and 0.42 g/100 g of cholesterol. Ethyl esters prepared from visceral oil of squid Ommastrephes bartrami had 4.5% EPA and 12.7% DHA. After urea pretreatment, the EPA and DHA contents were raised to 10.1 and 30.0%, respectively. When this mixture was further fractionated by molecular distillation, 16.9% EPA with 0.35 g/100 g cholesterol was found in the 110°C distillate and 52.6% DHA with 0.70 g/100 g cholesterol was found in the 130°C distillate. Cholesterol in the squid visceral oil ethyl esters was concentrated in the final residue of molecular distillation when the polyunsaturated ethyl esters were enriched by the urea complexation method prior to molecular distillation. For example, the cholesterol content in the ethyl esters from O. bartrami squid visceral oil was 2.28 g/100 g originally. It was enriched to 64.15 g/100 g in the final residue from the molecular distillation.     

Lipase‐catalyzed alcoholysis of vegetable oils

Fatty acid alkyl esters were produced from various vegetable oils by transesterification with different alcohols using immobilized lipases. Using n‐hexane as organic solvent, all immobilized lipases tested were found to be active during methanolysis. Highest conversion (97%) was observed with Thermomyces lanuginosa lipase after 24 h. In contrast, this lipase was almost inactive in a solvent‐free reaction medium using methanol or 2‐propanol as alcohol substrates. This could be overcome by a three‐step addition of methanol, which works efficiently for a range of vegetable oils (e.g. cottonseed, peanut, sunflower, palm olein, coconut and palm kernel) using immobilized lipases from Pseudomonas fluorescens (AK lipase) and Rhizomucor miehei (RM lipase). Repeated batch reactions showed that Rhizomucor miehei lipase was very stable over 120 h. AK and RM lipases also showed acceptable conversion levels for cottonseed oil with ethanol, 1‐propanol, 1‐butanol and isobutanol (50‐65% conversion after 24 h) in solvent‐free conditions. Methyl and isopropyl fatty acid esters obtained by enzymatic alcoholysis of natural vegetable oils can find application in biodiesel fuels and cosmetics industry, respectively.     

Lipase selectivity toward fatty acids commonly found in fish oil

The main objective of this study was to compare the fatty acid selectivity of numerous commercially available lipases toward the most ubiquitous fatty acids present in fish oils in form of their corresponding ethyl esters. Special interest was taken in their ability to separate the n‐3 long‐chain polyunsaturated fatty acids (PUFA), mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), from the more saturated fatty acids as well as exploiting the putative discrimination between these highly valuable n‐3 PUFA. Hydrolysis of sardine oil ethyl esters in a Tris buffer solution by 12 microbial lipases is described. The results reveal that all of the lipases strongly discriminate against the n‐3 PUFA and prefer the more saturated fatty acids as substrates. Most of the lipases discriminate between EPA and DHA in favor of EPA, however, 2 bacterial lipases from Pseudomonas were observed to prefer DHA to EPA. Digestive lipolytic enzymes isolated from salmon and rainbow trout intestines displayed reversed fatty acid selectivity when their fish oil triacylglycerol hydrolysis was studied. Thus, the n‐3 PUFA including EPA and DHA were observed to be hydrolyzed at a considerably higher rate than the more saturated fatty acids.     

Separation of eicosapentaenoic acid and docosahexaenoic acid in fish oil by kinetic resolution using lipase

The objective of this study was to investigate the use of lipases as catalysts for separating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil by kinetic resolution. Transesterification of various fish oil triglycerides with a stoichiometric amount of ethanol by immobilized Rhizomucor miehei lipase under anhydrous solvent-free conditions resulted in a good separation. When free fatty acids from the various fish oils were directly esterified with ethanol under similar conditions, greatly improved results were obtained. By this modification, complications related to regioselectivity of the lipase and nonhomogeneous distribution of EPA and DHA into the various positions of the triglycerides were avoided. As an example, when tuna oil comprising 6% EPA and 23% DHA was transesterified with ethanol, 65% conversion into ethyl esters was obtained after 24 h. The residual glyceride mixture contained 49% DHA and 6% EPA (8:1), with 90% DHA recovery into the glyceride mixture and 60% EPA recovery into the ethyl ester product. When the corresponding tuna oil free fatty acids were directly esterified with ethanol, 68% conversion was obtained after only 8h. The residual free fatty acids comprised 74% DHA and only 3% EPA (25:1). The recovery of both DHA into the residual free fatty acid fraction and EPA into the ethyl ester product remained very high, 83 and 87%, respectively.     

Selective Ethanolysis of Fish Oil Catalyzed by Immobilized Lipases

Selective ethanolysis of fish oil was catalyzed by immobilized lipases and their derivatives in organic media. Lipases from Candida antarctica B (CALB), Thermomyces lanuginosa (TLL) and Rhizomucor miehei (RML) were studied. The three lipases were immobilized by anion exchange and hydrophobic adsorption. The discrimination between the ethyl ester of eicosapentaenoic acid (EE-EPA) and the ethyl ester of docosahexaenoic acid (EE-DHA) depends on the lipase, the immobilization support, the physico-chemical modifications of the immobilized lipase derivatives and on the solvents used. TLL and RML were much more selective than CALB. EE-EPA is released 20-fold faster than EE-DHA when ethanolysis was catalyzed, in cyclohexane, by TLL hydrophobically adsorbed on Sepabeads C18. The selectivity and stability of the different derivatives in these polar organic solvents were further improved after physico-chemical modification. The best results for activity-selectivity-stability were obtained in cyclohexane for TLL adsorbed on Sepabeads C18 and further modified via solid-phase physical modification with a polyethylenimine polymer. In this case, the initial selectivity was higher than 20, and a 80 % of EPA was released as ethyl ester after 3 h at 25 °C. At this conversion, mixtures of ethyl esters highly enriched in the ethyl ester of EPA with less than 5 % of the EE-DHA were obtained. TLL derivatives remained fully active after incubation for 24 h in anhydrous solvents.     

The preparation of concentrates of eicosapentaenoic acid and docosahexaenoic acid by lipase-catalyzed transesterification of fish oil with ethanol

The objective of this study was to investigate the use of lipases as catalysts for producing concentrates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil as an alternative to conventional chemical procedures. Transesterification of fish oil with ethanol was conducted under anhydrous solvent-free conditions with a stoichiometric amount of ethanol. Among the 17 lipases tested, the results showed that Pseudomonas lipases had the highest activity toward the saturated and monounsaturated fatty acids in the fish oil, much lower activity toward EPA and DHA and, at the same time, good tolerance toward the anhydrous alcoholic conditions. With 10 wt% of lipase, based on weight of the fish oil triacylglycerol substrate (15% EPA and 9% DHA initial content), a 50% conversion into ethyl esters was obtained in 24 h at 20°C, in which time the bulk of the saturated and monounsaturated fatty acids reacted, leaving the long-chain n-3 polyunsaturated fatty acids unreacted in the residual mixture as mono-, di-, and triacylglycerols. This mixture comprised approximately 50% EPA+DHA. Total recovery of DHA and EPA was high, over 80% for DHA and more than 90% for EPA. The observed fatty acid selectivity, favoring DHA as a substrate, was most unusual because most lipases favor EPA.     

Integrated Green and Enzymatic Process to Produce Omega-3 Acylglycerols from Echium plantagineum Using Immobilized Lipases

This work presents an original approach to develop an integrated process to improve the nutritional characteristics of natural oils, starting with the extraction from raw material by ecofriendly methods, followed by the production of novel acylglycerols using immobilized lipases. Specifically, 2-monoacylglycerols (2-MAG) enriched in omega-3 stearidonic acid (SDA) were synthesized by selective ethanolysis of extracted Echium plantagineum oil using the lipase from Thermomyces lanuginosus (TLL). Different reaction conditions were investigated to minimize acyl migration and to ensure the purity of final products. The biocatalyst produced in our laboratory by the immobilization of TLL on Sepabeads C-18 reached the maximum theoretical amount of 2-MAG (33%) in only 2 hour at mild reaction conditions (25 °C), achieving a product enriched in omega-3 SDA (up to 25%). Moreover, the produced biocatalyst exhibited higher stability than commercial lipases. The average activity after five cycles was 71%, allowing several reutilization cycles and developing a feasible enzymatic process. Finally, 2-MAG were used as starting material to synthesize structured triacyclglycerols (STAG) through transesterification with caprylic acid ethyl esters using the lipase from Rhizomucor miehei (RML) in solvent-free systems. The use of molecular sieves in combination with RML immobilized on Sepabeads C-18 was demonstrated to be an extraordinarily fast strategy to produce pure STAG (100% in 1 hour), four times higher than the activity showed by commercial biocatalyst. Thus, the enzymatic processes developed in this study open a range of possibilities to synthesize omega-3 acylglycerols with improved characteristics, proving the usefulness of immobilized lipases to produce novel lipids.     

Fractionation of squid visceral oil ethyl esters by short-path distillation

Squid visceral oil contains high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Its ethyl esters were fractionated by short-path distillation in this study. The elimination temperatures of squid visceral oil ethyl esters (SVOEE) ranged from 50 to 140°C, increasing with the carbon number of ethyl esters. The elimination temperature of cholesterol was higher than those of SVOEE. The SVOEE of Illex argentinus (SVOEE-A) was more advantageous as the raw material (feed) than that of Ommastrephes bartrami (SVOEE-B) for the isolation of EPA and DHA, because SVOEE-A contained less 20∶1 and 22∶1. When SVOEE-A originally containing 9.0% EPA, 14.7% DHA, and 1,121 mg/100 g of cholesterol was distilled from 50 to 150°C with 20°C interval, the 130°C distillate could give 15.5% EPA and 34.7% DHA with 99 mg/100 g of cholesterol, and the yield was 21.8%. The 150°C distillate could give 43.1% DHA with 496 mg/100 g of cholesterol. Furthermore, the distillates collected from 110 to 150°C contained 24.4 to 50.2% of EPA plus DHA, and their total yield was 58.3%. The final residue after 150°C distillation contained 77% of the total cholesterol in the initial SVOEE-A, and the yield was 6.0%.     

Applications of immobilized <Emphasis Type="Italic">Thermomyces lanuginosa</Emphasis> lipase in interesterification

The aim of this work was to investigate the catalytic functions of a new immobilized Thermomyces lanuginosa lipase in interesterification and to optimize the conditions of interesterification for the production of human milk fat substitutes (HMFS) containing n−3 PUFA by response surface methodology (RSM). Thermomyces lanuginosa lipase had an activity similar to that of immobilized Rhizomucor miehei lipase (Lipozyme RM IM) in the glycerolysis of sunflower oil, but the former had higher activity at a low reaction temperature (5°C). Thermomyces lanuginosa lipase was found to have much lower catalytic activity than Lipozyme RM IM in the acidolysis of sunflower oil with caprylic acid. However, the activity of T. lanuginosa lipase was only slightly lower than that of Lipozyme RM IM in the ester-ester exchange between tripalmitin (PPP) and the ethyl esters of EPA and DHA (EE). For this reason, the new immobilized T. lanuginosa lipase was used to produce HMFS from PPP by interesterification with EE. The optimization of major parameters was conducted with the assistante molar ratio of 5 (EE/PPP), a lipase load of 20 wt% (on substrates), and a reaction time of 20 h, with acyl incorporation up to 42%. The model generated significantly represented real relationships between the response (incorporation) and reaction parameters.     

Synergism of microwave irradiation and enzyme catalysis in synthesis of isoniazid

Isoniazid is a useful antitubercular drug widely employed in combination therapy with rifampicin. The synthesis of isoniazid from ethyl isonicotinate and hydrazine hydrate was studied in non‐aqueous media via lipase‐catalyzed hydrazinolysis under both conventional heating and microwave irradiation by using different supported lipases. Among three different commercial lipases used, namely Novozym 435 (Candida antarctica lipase), Lipozyme RM IM (Rhizomucor miehei lipase) and Lipozyme TL IM (Thermomyces lanuginosus lipase), Novozym 435 was found to be the most effective, with conversion of 54% for equimolar concentrations at 50 °C in 4 h. The rate of reaction as well as final conversion increased synergistically under microwave irradiation in comparison with conventional heating, which showed 36.4% conversion, even after 24 h, for the control experiment. Effects of various process parameters such as speed of agitation, catalyst loading, substrate concentration, product concentration and temperature were studied. A kinetic model is also described. Copyright © 2007 Society of Chemical Industry     

Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid

Because of the complexity of marine lipids, polyunsaturated fatty acid (PUFA) derivatives in highly purified form are not easily prepared by any single fractionation technique. The products are usually prepared as the ethyl esters by esterification of the body oil of fat fish species and subsequent physicochemical purification processes, including short-path distillation, urea fractionation, and preparative chromatography. Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils. At room temperature in the presence of Pseudomonas sp. lipase and a stoichiometric amount of ethanol without any solvent, efficient transesterification of fish oil was obtained. At 52% conversion, a concentrate of 46% eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) was obtained in excellent recovery as a mixture of mono-, di-, and triacylglycerols. The latter can be easily separated from the saturated and monounsaturated ethyl esters and converted into ethyl esters either by conventional chemical means or enzymatically by immobilized Candida antarctica lipase. Urea-fractionation of such an intermediary product can give an EPA+DHA content of approximately 85%.     

Synthesis of Fatty Acid Ethyl Ester from Acid Oil in a Continuous Reactor via an Enzymatic Transesterification

Synthesis of a fatty acid ethyl ester via the lipase‐catalyzed transesterification of acid oil and ethanol was investigated in a continuous reactor. Lipozyme TL IM was employed as the immobilized lipase. This immobilized lipase derived from Thermomyces lanuginosus was purchased from Novozymes (Seoul, Korea). The acid oil was prepared by the acidification of soapstock formed as a by‐product during the refining of rice bran oil. The parameters investigated were water content, temperature, and molar ratio of substrates. The relative activity of Lipozyme TL IM was assessed during the repeated use of the immobilized lipase. The water content of the substrate had a considerable effect on the yield and the optimum water content was 4 %. The optimum temperature and molar ratio of acid oil to ethanol were 20 °C and 1:4, respectively. The maximum yield of approximately 92 % was achieved under the optimum conditions. The corresponding compositions were 92 % fatty acid ethyl esters, 3 % fatty acids, and 5 % acylglycerols. When glycerol formed during the reaction was removed by intermittent washing with ethanol, the relative activity of lipase was maintained over 82 % for a total usage of 27 cycles. For a mean residence time of 4 h, the half‐life times of Lipozyme TL IM on the control (unwashed) and treatment (washed) were 39 and 45 cycles, respectively.     

Enzymatic and chemical synthesis of phosphatidylcholine regioisomers containing eicosapentaenoic acid or docosahexaenoic acid

Regioselective incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into phosphatidylcholine (PC) was carried out using enzymatic and chemical synthesis. Incorporation at the sn‐1 position was successfully achieved by lipase‐catalysed esterification of 2‐palmitoyl‐lysophosphatidylcholine (LPC), although in most cases, the enzymes incorporated EPA and DHA at lower rates than other fatty acids. For the incorporation of DHA, Candida antarctica lipase B was the only useful enzyme, while incorporation of EPA was efficiently carried out using either this enzyme or Rhizopus arrhizus lipase. The highest yields in the lipase‐catalysed reactions were obtained at the lowest water activity (close to 0). However, by carrying out the reactions at a higher water activity of 0.22, more EPA and DHA were incorporated. Esterification of 2‐palmitoyl‐LPC with pure EPA at this water activity converted 66 mol‐% of LPC to PC using Rhizopus arrhizus lipase as catalyst. When the fatty acid was DHA and the catalyst Candida antarctica lipase B, 45 mol‐% of PC was obtained. For incorporation of EPA and DHA at the sn‐2 position, phospholipase A₂ was used, but the reaction was very slow. Chemical coupling of 1‐palmitoyl‐LPC and EPA or DHA was more efficient, resulting in complete conversion of LPC.     

Esterification of glycerol with conjugated linoleic acid and long-chain fatty acids from fish oil

Free fatty acids from fish oil were prepared by saponification of menhaden oil. The resulting mixture of fatty acids contained ca. 15% eicosapentaenoic acid (EPA) and 10% docosahexaenoic acid (DHA), together with other saturated and monounsaturated fatty acids. Four commercial lipases (PS from Pseudomonas cepacia, G from Penicillium camemberti, L2 from Candida antarctica fraction B, and L9 from Mucor miehei) were tested for their ability to catalyze the esterification of glycerol with a mixture of free fatty acids derived from saponified menhaden oil, to which 20% (w/w) conjugated linoleic acid had been added. The mixtures were incubated at 40°C for 48h. The ultimate extent of the esterification reaction (60%) was similar for three of the four lipases studied. Lipase PS produced triacylglycerols at the fastest rate. Lipase G differed from the other three lipases in terms of effecting a much slower reaction rate. In addition, the rate of incorporation of omega-3 fatty acids when mediated by lipase G was slower than the rates of incorporation of other fatty acids present in the reaction mixture. With respect to fatty acid specificities, lipases PS and L9 showed appreciable discrimination against esterification of EPA and DHA, respectively, while lipase L2 exhibited similar activity for all fatty acids present in the reaction mixture. The positional distribution of the various fatty acids between the sn-1,3 and sn-2 positions on the glycerol backbone was also determined.     

Two-step enzymatic synthesis of docosahexaenoic acid-rich symmetrically structured triacylglycerols via 2-monoacylglycerols

Symmetrically structured triacylglycerols (TG) rich in docosahexaenoic acid (DHA) with caprylic acid (CA) at the outer positions were synthesized enzymatically form bonito oil in a two-step process: (i) ethanolysis of bonito oil TG to 2-monoacylglycerols (2-MG) and fatty acid ethyl esters, and (ii) reesterification of 2-MG with ethyl caprylate. Ethanolysis catalyzed by immobilized Candida antarctica lipase (Novozym 435) yielded 92.5% 2-MG with 43.5% DHA content in 2 h. The 2-MG formed were reesterified with ethyl caprylate by immobilized Rhizomucor miehei lipase (Lipozyme IM) to give structured TG with 44.9% DHA content [based on fatty acid composition with caprylic acid (CA) excluded] in 1 h. The final structured lipids comprised 85.3% TG with two CA residues and one original fatty acid residue, 13% TG with one CA residue and two original fatty acid residues, and 1.7% tricaprylolglycerol (weight percent). The amount of TG with two CA residues and one C₂₂ residue (22∶6=DHA, 22∶5, and 22∶4) was 51 wt%. The 1,3-dicapryloyl-2-docosahexaenoylglycerol to 1,2(2,3)-dicapryloyl-3 (1)-docosahexaenoylglycerol ratio (based on high-performance liquid chromatography peak area percentages) was greater than 50∶1. The recovery of TG as structured lipids after silica gel column purification was approximately 71%. Ethyl esters and 2-MG formed at 2 h of ethanolysis could be used to determine the positional distribution of fatty acids in the intial TG owing to the high 1,3-regiospecificity of Novozym 435 and the reduced acyl migration in the system.     

Conjugated linoleic acid (CLA) production and lipase‐catalyzed interesterification of purified CLA with canola oil

In this study, two important isomers of CLA, i.e. c9,t11 and t10,c12, were produced up to ca. 73% of total fatty acids, employing alkali isomerization of safflower oil, followed by purification with only one‐step urea crystallization to 85.6%, while the recovery of the purification process was 35%. Interesterification (acidolysis) of purified CLA with canola oil was then conducted by Thermomyces lanuginosus lipase. The CLA content incorporated into the triacylglycerols (TG) was 26.6 mol‐% after 48 h of reaction time. Physical and chemical properties of the TG were then changed according to the degree of substitution of oleic acid in canola oil with CLA.     

Evaluation of ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of triacylglycerols containing highly unsaturated fatty acids

The suitability of a recently proposed method based on ethanolysis with immobilized Candida antarctica lipase for regiospecific analysis of oils containing long-chain PUFA such as [PA and DHA has been evaluated using selected marine oils and regio-isomerically enriched synthetic TAG substrates. 1,3-Regios-electivity of the lipase was enhanced when the ethanolysis was conducted in a high excess of ethanol, typically 10–50 times by weight of the oil. This enabled the reaction to be conducted on a milligram scale. However, irrespective of the ethanol-to-oil ratio, C. antarctica lipase released FA from TAG at different rates depending on the degree of unsaturation and/or chain length of the FA. Differences in lipolysis rates were particularly significant for EPA and DHA, with EPA released faster than DHA. Although DHA can be measured with reasonable accuracy by ethanolysis with C. antarctica, the method requires further optimization before it can be adopted for reliable regiospecific analyses that are as accurate as those obtainable by ¹³C NMR analysis for all major FA occurring in oils rich in long-chain PUFA.     

Concentration of docosahexaenoic acid in glyceride by hydrolysis of fish oil withcandida cylindracea lipase

In an attempt to concentrate the content of DHA (docosahexaenoic acid) in a glyceride mixture containing triglyceride, diglyceride and monoglyceride, fish oil was hydrolyzed with six kinds of microbial lipase. After the hydrolysis, free fatty acid was removed and fatty acid components of the glyceride mixtures were analyzed. When the hydrolysis withCandida cylindracea lipase was 70% complete, the DHA content in the glyceride mixture was three times more than that in the original fish oil. The EPA (eicosapentaenoic acid) content became almost 70% of the original fish oil. Hydrolysis with other lipases did not result in an increase in the DHA content in the glyceride mixtures. Hydrolysis of DHA-rich tuna oil (DHA content is about 25%) withCandida cylindracea lipase resulted in 53% DHA in the glyceride mixture. The EPA content, however, remained close to that of the original tuna oil. In this report, the acyl chain specificity of lipases is evaluated in terms of hydrolysis resistant value (HRV). HRV is the ratio between the DHA contents in the glyceride mixture of hydrolyzed oil and original oil. HRV clearly indicates differences in hydrolysis between DHA and other fatty acids (e.g., saturated and monoenoic acids).