Production of triglycerides enriched in long-chain n-3 polyunsaturated fatty acids from fish oil

Processes that combine enzymic and physical techniques have been studied for concentrating and separating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil.Candida rugosa lipase was used in hydrolysis reactions to concentrate these acids in the glyceride fraction. By controlling the degree of hydrolysis, two products have been obtained, one enriched in total n-3(∼50%), the other enriched in DHA and depleted in EPA (DHA∼40%, EPA∼7%). The glyceride fraction from these reactions was recovered by evaporation and converted back to triglycerides by partial enzymic hydrolysis, followed by enzymic esterification. Both reactions were carried out withRhizomucor miehei lipase. DHA-depleted free fatty acids from aC. rugosa hydrolysis were fractionated to increase the EPA level (∼30%) and re-esterified to triglycerides by reaction with glycerol andR. miehei.     

Selective hydrolysis of polyunsaturated fatty acid-containing oil withGeotrichum candidum lipase

Polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can be concentrated in glycerides by hydrolyzing tuna oil withGeotrichum candidum lipase, the main components in the resulting oil being triglycerides. The reaction mechanism of this selective hydrolysis was investigated. Although the lipase acted well on the esters of oleic, linoleic, and α-linolenic acids, it did not affect the esters of γ-linolenic acid, arachidonic acid, EPA, and DHA as much. The action of PUFA-glycerides was mono-> di- > triglycerides. Furthermore, the condensation of PUFA-partial glycerides and PUFA occurred even in the presence of a large amount of water, and the partial glycerides converted to the triglycerides by transacylation. These results suggested that the PUFA-rich triglycerides were accumulated in the glyceride fraction by the following mechanism: The PUFA-partial glycerides generated by the hydrolysis were converted to PUFA-triglycerides by condensation and transacylation reactions. As the PUFA-triglycerides formed were the poor substrates of lipase, they were accumulated in the reaction mixture.     

Enzymatic enrichment of C20 cis-5 polyunsaturated fatty acids fromBiota orientalis seed oil

Enrichment ofcis-5 polyunsaturated fatty acids [20:3(5c,11c,14c), 4.3% and 20:4(5c,11c,14c,17c), 11.3%] fromBiota orientalis seed oil was carried out by lipase-catalyzed selective esterification and hydrolysis reactions. Lipases fromRhizomucor miehei (Lipozyme),Candida cylindracea and porcine pancreas were used. Lipozyme-catalyzed esterification ofBiota fatty acids withn-butanol inn-hexane allowed 20:3 and 20:4 (as fatty acids) to be enriched to a maximum level of 52.9%, and in the presence ofC. cylindracea lipase 61.5% enrichment was achieved. Esterification with pancreatic lipase was poor with low levels of enrichment of 20:3 and 20:4 (22%). A multigram scale esterification of the free fatty acids fromBiota seed oil by repeated treatment of the isolated fatty acid fraction withn-butanol inn-hexane in the presence ofC. cylindracea lipase furnished an enrichment yield of 72.5% of a mixture of 20:3 and 20:4 fatty acids. Urea fractionation of the free fatty acids ofBiota oil gave an initial enriched fraction of 20:3 (9.5%) and 20:4 (25.2%) which, upon treatment withC. cylindracea lipase inn-butanol andn-hexane, gave an enriched fraction of 85.3% of 20:3 and 20:4 fatty acids. Partial hydrolysis of the triglycerides ofBiota oil byC. cylindracea lipase in potassium phosphate buffer at 25°C resulted in a 2.8-fold enrichment ofcis-5 polyunsaturated fatty acids (40.8% of 20:3 and 20:4) as contained in the unhydrolyzed acylglycerol fractions.     

Lipase-assisted concentration of n-3 polyunsaturated fatty acids in acylglycerols from marine oils

Preparation of n-3 polyunsaturated fatty acid (PUFA) concentrates from seal blubber oil (SBO) and menhaden oil (MHO) in the form of acylglycerols was carried out by hydrolysis with a number of commercial microbial lipases. The lipases tested were Aspergillus niger, Candida cylindracea (CC), Chromobacterium viscosum, Geotrichum candidum, Mucor miehei, Pseudomonas sp., Rhizopus oryzae, and Rhizopus niveus. After lipase-assisted hydrolysis of oils, free fatty acids were removed, and fatty acid composition of the mixture containing mono-, di-, and triacylglycerols was determined. All lipases were effective in increasing the n-3 PUFA content of the remaining acylglycerols of both SBO and MHO. The highest concentration of n-3 PUFA was provided by CC lipase; 43.5% in SBO [9.75% eicosapentaenoic acid (EPA), 8.61% docosapentaenoic acid (DPA), and 24.0% docosahexaenoic acid (DHA)] and 44.1% in MHO (18.5% EPA, 3.62% DPA, and 17.3% DHA) after 40 h of hydrolysis. Thus, CC lipase appears to be most suitable for preparation of n-3 PUFA in the acylglycerol form from marine oils.     

The production of n‐3 long‐chain polyunsaturated fatty acids in transgenic plants

Long‐chain polyunsaturated fatty acids (LC‐PUFA) now have a proven role in human health and nutrition, including the n‐3 forms normally found in fish oils. Unfortunately, global fish stocks are now more than ever subject to over‐fishing and environmental pollution, indicating the need for an alternative source of fish oils. Recent efforts have focussed on the production of LC‐PUFA in transgenic plants to provide a sustainable and clean source of fish oils. The current progress in this area is considered, as well as the bottlenecks that remain to be overcome.     

Production of n-3 polyunsaturated fatty acid-enriched fish oil by lipase-catalyzed acidolysis without solvent

Fish oil rich in n-3 polyunsaturated fatty acid (n-3 PUFA) was prepared by nonsolvent enzymic acidolysis. n-3 PUFA-enriched fish oil contained 25% eicosapentaenoic acid (EPA) and 40% docosahexaenoic acid (DHA). In acidolysis of cod liver oil, EPA content of the original fish oil was reduced at 5 h, but DHA content of the fish oil increased. It was assumed that EPA in the fish oil was replaced by DHA to reach a new chemical equilibrium. Two-stage acidolysis, which was carried out under CO₂ replacement early (about 3 h) and also in vacuum at 5–24 h, was effective for reduction in the content of diacylglycerol, which was formed by reverse reaction, hydrolysis. This method has industrial significance because PUFA-enriched triacylglycerol is easily separated from the reaction mixture by molecular distillation. Bioreactors for fats and their derivatives, Part XIV.     

Synthesis of polyunsaturated fatty acid-enriched triglycerides by lipase-catalyzed esterification

This paper reports on the synthesis of triglycerides by enzymatic esterification of polyunsaturated fatty acids (PUFA) with glycerol. A PUFA concentrate obtained from cod liver oil was used to optimize the reaction to favor triglyceride synthesis with lipases. The type and amount of lipase and organic solvent, glycerol content, temperature, water content, and amount and time of addition of molecular sieves were studied. The optimal reaction mixture and conditions were: 9 mL hexane, 60°C, 0.5% (vol/vol) water, 1 g molecular sieves added after 24 h of reaction, glycerol/fatty acid molar ratio 1:3 and 100 mg of Novozym 435 (Novo Nordisk A/S) lipase. Under these conditions, an enriched triglyceride yiedl of 84.7% containing 27.4% eicosapentaenoic acid and 45.1% docosahexaenoic acid was obtained from a cod liver oil PUFA concentrate.     

Production of odd chain polyunsaturated fatty acids byMortierella fungi

A soil isolate,Mortierella alpina 1S-4, was found to show high production of odd chain polyunsaturated fatty acids (PUFAs) among various arachidonic acid-producingMortierella strains tested. The fungus mainly accumulated 5,8,11,M-cis-nonadecatetraenoic acid. With 5%n-hepta-decane and 1% yeast extract as growth substrates, the amount of C_19:4:4 acid accumulated reached 44.4 mg/g dry mycelia (0.68 mg/mL of culture broth). This value accounted for 11.2% of the total fatty acids in the extracted lipids from mycelia, and odd chain fatty acids comprised over 95% of the total mycelial fatty acids. The addition of sesamin, a specific inhibitor of A5 desaturation, caused an increase in C_19:3 acid and an accompanying decrease in C_19:4 acid. On the other hand, species ofMortierella that could not produce C-20 PUFAs accumulated C-17 acids, but no C-19 PUFAs, when grown with fatty substrates with an odd chain skeleton. The odd chain PUFAs were distributed in both neutral and polar lipids. The biosynthetic route to C_19:4 acid was presumed to mimic the n-6 route to arachidonic acid as follows: C_17:0 → C_17:1→ C_17:2→ C_17:3 → C_19:3 → C_19:4 acids. On leave from Suntory Ltd.     

Valorization of mullet roe by-products for the production of polyunsaturated fatty acids rich oils

This work examines the potential valorization of mullet roe by-products for the production of mullet roe oil using mild processes. Three different extraction methods with potential of scale-up for the food industry, namely pressure (PE), supercritical fluid extraction (SFE), and solvent extraction (SE) are examined. Mild temperature conditions to prevent oil oxidation and (wherever applicable) food-grade solvents are used. The oil yield, the composition of oils in fatty acids by GC-FID, the level of oil oxidation (peroxide value (PV), p-anisidine value (AV), K₂₃₂ K₂₆₈, TOTOX)) and the antioxidant activity (DPPH, ABTS) are determined. SE provided the highest oil recovery, followed by SFE and PE (68%, 28% and 10% respectively). The extracted oils had a high concentration of EPA and DHA and a total of 20.7%–24.3% of identified PUFAs among the fatty acids. Oxidation was the lowest in the SFE extracted oil followed by PE, PV was <2.5 meq_O2, AV≤10 and TOTOX <15 in all examined oil samples. Further research is needed to optimize processing conditions for the increase in oil recovery.     

Lipase-catalyzed esterification of glycerol and n-3 polyunsaturated fatty acid concentrate in organic solvent

Enzymatic synthesis of glycerides from glycerol and n-3 polyunsaturated fatty acid in organic solvent was studied. Optimal conditions for glyceride synthesis by lipases were established. Of the commercially available lipases that were investigated, lipase PS-30 fromPseudomonas sp. and lipase IM-60 fromMucor miehei resulted in the highest extent of esterification. Isooctane and hexane were particularly useful organic solvents in glyceride synthesis. The water content in the reaction mixture was of primary importance. For lipase PS-30 and lipase IM-60, optimal water contents were 5 and 1%, respectively. Lipases PS-30 and IM-60 manifested contrasting positional specificities in glyceride synthesis. Glycerides containing predominantly eicosapentaenoic acid and docosahexaenoic acid can be easily synthesized.     

Thermal degradation of long‐chain polyunsaturated fatty acids during deodorization of fish oil

Long‐chain polyunsaturated fatty acids (LC‐PUFA) of the n‐3 series, particularly eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid, have specific activities especially in the functionality of the central nervous system. Due to the occurrence of numerous methylene‐interrupted ethylenic double bonds, these fatty acids are very sensitive to air (oxygen) and temperature. Non‐volatile degradation products, which include polymers, cyclic fatty acid monomers (CFAM) and geometrical isomers of EPA and DHA, were evaluated in fish oil samples obtained by deodorization under vacuum of semi‐refined fish oil at 180, 220 and 250 °C. Polymers are the major degradation products generated at high deodorization temperatures, with 19.5% oligomers being formed in oil deodorized at 250 °C. A significant amount of CFAM was produced during deodorization at temperatures above or equal to 220 °C. In fact, 23.9 and 66.3 mg/g of C20 and C22 CFAM were found in samples deodorized at 220 and 250 °C, respectively. Only minor changes were observed in the EPA and DHA trans isomer content and composition after deodorization at 180 °C. At this temperature, the formation of polar compounds and CFAM was also low. However, the oil deodorized at 220 and 250 °C contained 4.2% and 7.6% geometrical isomers, respectively. Even after a deodorization at 250 °C, the majority of geometrical isomers were mono‐ and di‐trans. These results indicate that deodorization of fish oils should be conducted at a maximal temperature of 180 °C. This temperature seems to be lower than the activation energy required for polymerization (intra and inter) and geometrical isomerization.     

Enzymic enhancement of n−3 fatty acid content in fish oils

Commercially available fish oils with n−3 fatty acid contents ranging from 29 to 34% were converted enzymically, with Amano P lipase, to mixtures of glycerides with n−3 fatty acid contents ofca. 50%, in weight recovery yields of 23–50%, depending upon extraction procedures. Glyceride mixtures with n−3 fatty acid contents above 70% were obtained in yields of 14–21%. The processes are based on the relative stability of the ester linkages that involve n−3-fatty acyl groups and the regioselectivity of the enzyme toward acyl groups at the 1,3-positions of glycerol. This paper was presented at the 82nd AOCS Annual Meeting, May 12–15, 1991.     

Oil content and fatty acid composition of commercially important Turkish fish species

The oil content and fatty acid composition of commercially important Turkish fish species (anchovy,Engraulis encrasicholus; freshwater rainbow trout,Salmo gairdneri; and cultured salmon,S. salar) were determined. Palmitic (16∶0), palmitoleic (16∶1), oleic (18∶1), and docosahexaenoic (22∶6) acids were the most abundant fatty acids in all species. Eicosapentaenoic acid (20∶5) was twice as high in the anchovy oil as in the rainbow trout and salmon oils. Significant quantities of linoleic acid (18∶2) and docosahexaenoic acids (22∶6) were found in both rainbow trout and salmon samples. The individual fatty acid data obtained from rainbow trout and salmon were similar to each other. All three fish species contain high levels of n-3 polyunsaturated fatty acids and would be suitable for inclusion in the formulation of low-fat highly unsaturated diets.     

Essential Polyunsaturated Fatty Acids in Blood from Patients with and without Catheter-Proven Coronary Artery Disease

Coronary artery disease (CAD) is the leading cause of death worldwide. Statins reduce morbidity and mortality of CAD. Intake of n-3 polyunsaturated fatty acid (n-3 PUFAs), particularly eicosapentaenoic acid (EPA), is associated with reduced morbidity and mortality in patients with CAD. Previous data indicate that a higher conversion of precursor fatty acids (FAs) to arachidonic acid (AA) is associated with increased CAD prevalence. Our study explored the FA composition in blood to assess n-3 PUFA levels from patients with and without CAD. We analyzed blood samples from 273 patients undergoing cardiac catheterization. Patients were stratified according to clinically relevant CAD (n = 192) and those without (n = 81). FA analysis in full blood was performed by gas chromatography. Indicating increased formation of AA from precursors, the ratio of dihomo-gamma-linolenic acid (DGLA) to AA, the delta-5 desaturase index (D5D index) was higher in CAD patients. CAD patients had significantly lower levels of omega-6 polyunsaturated FAs (n-6 PUFA) and n-3 PUFA, particularly EPA, in the blood. Thus, our study supports a role of increased EPA levels for cardioprotection.     

Efficacy of Polyunsaturated Fatty Acids (PUFAs) on Impulsive Behaviours and Aggressiveness in Psychiatric Disorders

It is the focus of increasing interest to investigate the effects of long-chain n-3 and long-chain n-6 polyunsaturated fatty acids (LC n-3 PUFAs; LC n-6 PUFAs) on psychiatric symptoms in a transdiagnostic perspective. There is some evidence that low levels of LC n-3 PUFAs and a higher ratio of LC n-6 to LC n-3 PUFAs in plasma and blood cells are associated with aggressive and impulsive behaviours. Therefore, implementation of LC n-3 PUFAs may produce positive effects on hostility, aggression, and impulsivity in both psychiatric and non-psychiatric samples across different stages of life. A possible mechanism of action of LC n-3 PUFAs in conditions characterized by a high level of impulsivity and aggression is due to the effect of these compounds on the serotonin system and membrane stability. Studies that evaluated the effects of LC n-3 PUFAs on impulsivity and aggressiveness indicated that addition of rather low doses of these agents to antipsychotic treatment might reduce agitation and violent behaviours in psychosis, attention deficit hyperactivity disorder, personality disorders, and impulsive control and conduct disorders. The present review is aimed at examining and discussing available data from recent trials on this topic.     

Preparation of phospholipids highly enriched with n-3 polyunsaturated fatty acids by lipase

The immobilized 1,3-regiospecific Rhizomucor miehei lipase (Lipozyme™) was employed to catalyze the transesterification reaction (acidolysis) of 1,2-diacyl-sn-glycero-3-phosphatidylcholine with n-3 polyunsaturated fatty acids under nonaqueous solvent-free conditions. With a concentrate of 55% eicosapentaenoic acid (EPA) and 30% docosahexaenoic acid (DHA) and pure phosphatidylcholine from egg yolk, phospholipids of 32% EPA and 16% DHA content were obtained, presumably as a mixture of phosphatidylcholine and lysophosphatidylcholine. ³¹P nuclear magnetic resonance (NMR) analysis turned out to be a valuable technique to study the details of the reactions involved. It revealed that when 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine was transesterified with 98% pure EPA, a substantial amount of hydrolysis side reaction took place (39%), leading to a product mixture of 39% phosphatidylcholine, 44% lysophosphatidylcholine, and 17% sn-glycerol-3-phosphatidylcholine. The lysophosphatidylcholine constituent comprised 70% EPA, whereas the phosphatidylcholine component contained 58% EPA. The ³¹P NMR technique provided valid information about the mechanism of the reaction. It became evident that a high dosage of lipase containing 5% water afforded optimal conditions for the optimal extent of EPA incorporation into the phospholipids, under which the extent of hydrolysis side reaction remained relatively high.     

Lipase-catalyzed incorporation of n−3 polyunsaturated fatty acids into vegetable oils

The ability of immobilized lipases IM60 fromMucor miehei and SP435 fromCandida antarctica to modify the fatty acid composition of selected vegetable oils by incorporation of n−3 polyunsaturated fatty acids into the vegetable oils was studied. The transesterification was carried out in organic solvent with free acid and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as acyl donors. With free EPA as acyl donor, IM60 gave higher incorporation of EPA than SP435. However, when ethyl esters of EPA and DHA were the acyl donors, SP435 gave higher incorporation of EPA and DHA than IM60. When IM60 and free acid were used, the addition of 5 μL water increased EPA incorporation into soybean oil by 4.9%. With ethyl ester of EPA as acyl donor, addition of 2 μL water increased EPA incorporation by 3.9%. For SP435, addition of water up to 2μL resulted in increased EPA incorporation, but the incorporation declined when the added water exceeded this amount. The addition of water increased the EPA incorporation into Trisun 90 after 24 h reaction but not the reaction rate at early stages of the reaction.     

Denicking of fatty acid pitch

Unsaturated fatty acids are usually hydrogenated with a nickel catalyst. As a side reaction, nickel dissolves in the fatty acids and forms nickel soaps. On refining the crude fatty acid feedstock by distillation, these nickel soaps are enriched in the bottom fraction (pitch) to a concentration range of 2,500–5,500 ppm. With a heat value comparable to heavy fuel oil, the pitch has been used as fuel. However, due to new environmental restrictions, the nickel concentration must be considerably reduced to limit gas emission of nickel. In this study, solvent extraction was investigated as a separation method for denickling of this valuable oleochemical by-product. At a temperature of 80°C, the nickel content in the pitch was reduced from 2,900 ppm to 4 ppm in three extraction steps by means of 1 wt% citric acid in water. Based on experimental results, a preliminary process flowsheet is presented consisting of extraction, stripping, acid washing and electrolysis. An economic analysis, performed on the extraction process and a process based on direct combustion followed by electrostatic precipitation revealed that direct combustion is economically more advantageous. However, the compatibility of the extraction alternative can be considerably improved by process optimization. The type and concentration of the stripping and acid washing agents, phase ratios, temperature, ionic strength and the equilibrium pH of the aqueous and acid phases are important process parameters that should be considered in the optimization process.     

Increasing n-3 polyunsaturated fatty acid content of fish oil by temperature control of lipase-catalyzed acidolysis

An attempt was made to further increase the content of n-3 polyunsaturated fatty acid (n-3 PUFA) of fish oil by lipase-catalyzed acidolysis (reaction between fish oil and n-3 PUFA-enriched free fatty acid) without solvent. A bioreactor system was constructed composed of a water-jacketed packed-bed column and a substrate reservoir with a circulation pipeline between the packed-bed column and the reservoir. By keeping the temperature of the reservoir at −10°C (for the first 20 h), followed by −20°C (for the subsequent 40 h) during the batch acidolysis, crystals of free fatty acid appeared, which were removed intermittently by a cotton plug packed in the tip of the outlet pipe in the reservoir. The n-3 PUFA content of the triacylglycerol fraction increased a further 10% by the reduced temperature of the reservoir. Bioreactors for Enzymatic Reaction of Fats and Fatty Acid derivatives, Part XV.