The incorporation of n-3 polyunsaturated fatty acids into acylglycerols of borage oil via lipase-catalyzed reactions

The purpose of this work was to add n-3 polyunsaturated fatty acids (PUFA) into the acylglycerols of borage oil. The acidolysis reaction between borage oil and n-3 PUFA was carried out with lipase (Lipozyme IM-60) in organic solvent. The effects of temperature, solvent, and water content on the reaction product were investigated. For the acidolysis reaction between acylglycerols (product of the selective hydrolysis of borage oil, catalyzed by immobilized Candida rugosa lipase) and n-3 PUFA, the total content of n-3 and n-6 PUFA in acylglycerols was 72.8% after a reaction time of 18 h. The contents of γ-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid were 26.5, 19.8, and 18.1%, respectively. By properly controlling the reaction time, acylglycerols with ca. 70–72% PUFA and a ratio of n-3 PUFA to n-6 PUFA from 0–1.09 can be obtained.     

The ratio of n-6 to n-3 polyunsaturated fatty acids in the rat diet alters serum lipid levels and lymphocyte functions

Previous studies have reported that feeding rats diets rich in fish oils, which contain high proportions of the n-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic and docosahexaenoic acids, results in lowering of blood lipid levels and suppression of lymphocyte functions testedex vivo andin vivo. The effects of other n-3 PUFA, such as α-linolenic acid, which is found in high proportions in linseed oil, are not as well documented. Therefore, in the present study, weanling male rats were fed for six weeks on one of five high-fat (20% by weight) diets made by mixing together sunflower and linseed oils; the resulting blends had n-6/n-3 PUFA ratios of 112.5:1 (pure sunflower oil), 14.8:1, 6.5:1, 0.8:1, and 0.33:1 (pure linseed oil); the levels of all other components in the diet were identical. The final body weight and total dissectable fat were lowest in rats fed the pure linseed oil diet. Serum cholesterol, triacylglycerol and nonesterified fatty acid concentrations decreased as the n-6/n-3 PUFA ratio of the diet decreased. The fatty acid composition of the serum and of spleen lymphocytes was influenced by the diet fed-there was a progressive decrease in the proportions of linoleic and arachidonic acids and a progressive increase in the proportion of α-linolenic acid as the n-6/n-3 PUFA ratio of the diet decreased. Eicosapentaenoic and docosahexaenoic acids were detected in the serum but not in spleen lymphocytes. Inclusion of α-linolenic acid in the diet resulted in significant suppression of spleen lymphocyte proliferation in response to the T-cell mitogen concanavalin A and in spleen lymphocyte natural killer cell activity, both measuredex vivo. The localized graft vs. host response, a measure of cellmediated immunityin vivo, progressively decreased as the n-6/n-3 PUFA ratio of the diet decreased. Thus, this study shows that dietary α-linolenic acid results in lowered blood lipid levels and suppressed lymphocyte functionsex vivo andin vivo. With respect to these effects, α-linolenic acid is as potent as dietary fish oil.     

High-purity γ-linolenic acid from borage oil fatty acids

High-purity γ-linolenic acid (GLA) was obtained by employing a modified low-temperature solvent crystallization process, followed by a lipase-catalyzed esterification, to borage oil fatty acid. By applying a two-stage solvent crystallization process to the borage oil fatty acid, GLA content was increased from 23.4 to 92.1% with a yield of 89.3%. After the esterification of GLA-rich fatty acid with butanol catalyzed by Lipozyme IM-60, GLA content in the fatty acid was further raised from 92.1 to 99.1%. The overall yield of the combined process was 72.8%. The effects of operation parameters on the Lipozyme IM-60 catalyzed esterification between fatty acid and alcohol were systematically investigated.     

Urea-based fractionation of seed oil samples containing fatty acids and acylglycerols of polyunsaturated and hydroxy fatty acids

The selectivity and efficiency of urea complex (UC) formation-based fractionation of free fatty acids (FFA) were examined. A rapid, simple, and inexpensive procedure recently developed for urea fractionation was applied to lipid mixtures containing various polyunsaturated and hydroxy FFA species. Urea treatment proved useful for isolating polyunsaturated FFA (PUFA) from FFA derived from fish, borage, and linseed oils by removal of saturated and monounsaturated FFA, but was not effective for isolating hydroxy FFA from the FFA derived from castor, Lesquerella, and Dimorphotheca oils. In situations where FFA within the crystalline or UC phase were rich in PUFA, the urea/FFA mole ratio of the UC was relatively higher, with lower recovery of FFA in this phase. The distribution of urea between the crystalline phase and the solvent was not significantly affected by the FFA composition of feed nor the overall ratio of FFA to urea. It was strongly dependent on the overall mass fraction of solvent. Phospholipids and mono-, di-, and triacylglycerols were poor templates for UC formation relative to FFA. Their inclusion in acylglycerol mixtures containing FFA reduced UC formation.     

Cyclic fatty acid yields from linseed oil

Increased yields of saturated cyclic fatty acids which are fluid at −50C have been obtained from linseed oil. Depending on reaction conditions, yields varied from 20–42 g of cyclic acids per 100 g of linseed oil. Solvent ratios of 6, 3, and 1.5∶1; catalyst concentrations of 10, 30, 60, and 100%; and reaction temps of 225, 275, 295, and 325C were evaluated. Ethylene glycol and diethylene glycol were compared as reaction solvents. In general, high solvent ratios favored high cyclic acid yields at the lower reaction temperature, but as the temperature increased the effect of solvent ratio decreased. Increasing the percentage excess of sodium hydroxide increased the cyclic acid yield. Diethylene glycol gave higher yields than ethylene glycol at comparable conditions. Presented at the AOCS meeting in Chicago, Ill., October, 1961. A laboratory of the No. Utiliz. Res. & Dev. Div., ARS, U.S.D.A.     

γ-Linolenic acid concentrates from borage and evening primrose oil fatty acidsvia lipase-catalyzed esterification

γ-Linolenic acid (GLA, all-cis 6,9,12-octadecatrienoic acid) has been enriched from fatty acids of borage (Borago officinalis L.) seed oil to 93% from the initial concentration of 20% by lipase-catalyzed selective esterification of the fatty acids withn-butanol in the presence ofn-hexane as solvent. The immobilized fungal lipase preparation, Lipozyme, used as biocatalyst, preferentially esterified palmitic, stearic, oleic and linoleic acids and discriminated against GLA, which was thus concentrated in the unesterified fatty acids fraction. In the absence of hexane, concentrate containing about 70% GLA was obtained. When the reaction conditions, optimized for borage oil fatty acids, were applied to fatty acids of evening primrose (Oenothera biennis L.) oil, concentrates containing 75% GLA were obtained. From both oils, GLA concentrates were prepared efficiently in short reaction times (1–3 h) at 30–60°C. The process can be applied for the production of GLA concentrates for dietetic purposes.     

Enrichment of γ-linolenic acid from borage oil via lipase-catalyzed reactions

Three lipase-catalyzed reactions were utilized to enrich γ-linolenic acid in borage oil: (i) selective hydrolysis in isooctane by Candida rugosa lipase immobilized on microporous polypropylene, (ii) selective esterification of free fatty acid from saponified borage oil and n-butanol by Lipozyme IM-20, and (iii) acidolysis of the products of the previous two reactions, that is, unhydrolyzed acylglycerols and unesterified free fatty acid. In the selective hydrolysis, γ-linolenic acid content could be raised from 23.6 mol% in borage oil to 51.7% in the unhydrolyzed acylglycerols. On the other hand, γ-linolenic acid content in free fatty acid could be increased to 87% after selective esterification. Products with 65% γ-linolenic acid in their acylglycerols were obtained by means of the acidolysis reaction.     

Enzymatic modification of evening primrose oil: Incorporation of n−3 polyunsaturated fatty acids

Immobilized lipase SP435 fromCandida antaractica was used as a biocatalyst for the modification of the fatty acid composition of evening primrose oil by incorporating n−3 polyunsaturated fatty acid (PUFA) and eicosapentaenoic acid (EPA). Transesterification (ester-ester interchange) was conducted in organic solvent or without solvent, with EPA ethyl ester (EEPA) as the acyl donor. Products were analyzed by gas-liquid chromatography (GLC). After 24-h incubation in hexane, the fatty acid composition of evening primrose oil was markedly changed to contain up to 43% EPA. The amount of 18:2n−6 PUFA was reduced by 32%, and the saturated fatty acid content was also reduced. The effects of incubation time, molar ratio, enzyme load, and reaction medium on mol% EPA incorporation were also studied. Generally, as the incubation time (up to 24 h), molar ratio, and enzyme load increased, EPA incorporation also increased. Evening primrose oil, containing EPA and γ-linolenic acid (18:3n−6) in the same glycerol backbone, was successfully produced and may be more beneficial for certain applications than unmodified oil.     

Essential fatty acids alter the activity of manganese-superoxide dismutase in rat heart

The effects of oil-derived dietary essential fatty acids on the activities of mitchondrial Mn-SOD (manganese-superoxide dismutase) and cytosolic cupric zinc-superoxide dismutase (Cu/Zn-SOD) were investigated in rat heart. A control group of rats was fed a stock diet for 29 d, and a second group was fed on a fat-free diet. Three other groups were fed fat-free diets that were supplemented with (i) borage oil, which is rich in linoleic (18∶2n−6) and γ-linolenic (18∶3n−6) acids, (ii) fungal oil, which is rich in γ-linolenic, but low in linoleic acid, or (iii) evening primrose oil, which is rich in linoleic acid and low in γ-linolenic acid. An increase in the percentage composition of arachidonic acid (20∶4n−6) in both the choline and ethanolamine phospholipids, together with a decrease in linoleic acid in ethanolamine phospholipids, were found in heart membranes after feeding the rats with diets containing borage oil or fungal oil as compared to those fed the stock diet. The respective activities of Mn-SOD in rats fed the borage or fungal oil diets were also significantly higher than in rats fed the stock diet alone. No change in cytosolic Cn/Zn-SOD activity was observed. Dietary supply of linoleic acid-rich evening primrose oil resulted in an increased proportion of choline phospholipid linoleic acid without any changes in arachidonic acid content or in the activity of Mn-SOD. By contrast, a reduction in the activity of Mn-SOD was detected in rats fed a fat-free diet. These results show that the activity of heart mitochondrial Mn-SOD is influenced by dietary essential fatty acids, whereas the activity of cytosolic Cu/Zn-SOD remained unaffected.     

Effects of Rice Bran Oil Enriched with n-3 PUFA on Liver and Serum Lipids in Rats

Lipase-catalyzed interesterification was used to prepare different structured lipids (SL) from rice bran oil (RBO) by replacing some of the fatty acids with α-linolenic acid (ALA) from linseed oil (LSO) and n-3 long chain polyunsaturated fatty acids (PUFA) from cod liver oil (CLO). In one SL, the ALA content was 20% whereas in another the long chain n-3 PUFA content was 10%. Most of the n-3 PUFA were incorporated into the sn-1 and sn-3 positions of triacylglycerol. The influence of SL with RBO rich in ALA and EPA + DHA was studied on various lipid parameters in experimental animals. Rats fed RBO showed a decrease in total serum cholesterol by 10% when compared to groundnut oil (GNO). Similarly structured lipids with CLO and LSO significantly decreased total serum cholesterol by 19 and 22% respectively compared to rice bran oil. The serum TAGs level of rats fed SLs and blended oils were also significantly decreased by 14 and 17% respectively compared to RBO. Feeding of an n-3 PUFA rich diet resulted in the accumulation of long chain n-3 PUFA in various tissues and a reduction in the long chain n-6 PUFA. These studies indicate that the incorporation of ALA and EPA + DHA into RBO can offer health benefits.     

Dietary saturated fat level alters the competition between α-linolenic and linoleic acid

Male weanling rats were fed semi-synthetic diets high in saturated fat (beef tallow) vs high in linoleic acid (safflower oil) with or without high levels of α-linolenic acid (linseed oil) for a period of 28 days. The effect of feeding these diets on cholesterol content and fatty acid composition of serum and liver lipids was examined. Feeding linseed oil with beef tallow or safflower oil had no significant effect on serum levels of cholesterol. Serum cholesterol concentration was higher in animals fed the safflower oil diet than in animals fed the beef tallow diet without linseed oil. Feeding linseed oil lowered the cholesterol content in liver tissue for all dietary treatments tested. Consumption of linseed oil reduced the arachidonic acid content with concomitant increase in linoleic acid in serum and liver lipid fractions only when fed in combination with beef tallow, but not when fed with safflower oil. Similarly, ω3 fatty acids (18∶3ω3, 20∶5ω3, 22∶5ω3, 22∶6ω3) replaced ω6 fatty acids (20∶4ω6, 22∶4ω6) in serum and liver lipid fractions to a greater extent when linseed oil was fed with beef tallow than with safflower oil. The results suggest that the dietary ratio of linoleic acid to saturated fatty acids or of 18∶3ω3 to 18∶2ω6 may be important to determine the cholesterol and arachidonic acid lowering effect of dietary α-linolenic acid.     

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

The γ-linolenic acid (Z,Z,Z-6,9,12-octadecatrienoic acid, GLA) present in borage oil free fatty acids was concentrated in esterification reactions that were catalyzed by several preparations of the acyl-specific lipase ofGeotrichum candidum. In this manner, a 95% recovery of the GLA originally present in borage oil (25% GLA) was obtained as a highly enriched fatty acid fraction with a GLA content of >70%. Other fatty acids concentrated in this fraction were the monounsaturated fatty acids with chainlengths of C-20 and longer that were present in the oil. An immobilized preparation ofG. candidum on silica gel also was used for the enrichment of GLA in borage oil. In this instance, a 75% recovery of GLA was obtained, and the supported lipase was reusable (three cycles) with minimal loss in activity. Presented in part at the 84th Annual Meeting of the American Oil Chemists’ Society, Anaheim, California, May 1993.     

Enzymatic fractionation of fatty acids: Enrichment of γ-linolenic acid and docosahexaenoic acid by selective esterification catalyzed by lipases

Immobilized lipase preparations from seedlings of rape (Brassica napus L.) andMucor miehei (lipozyme) used as biocatalysts in esterification and hydrolysis reactions discriminate strongly against γ-linolenic and docosahexaenoic acids/acyl moieties. Utilizing this property, γ-linolenic acid contained in fatty acids of evening primrose oil has been enriched seven to nine-fold, from 9.5 to almost 85% by selective esterification of the other fatty acids with butanol. Similarly, docosahexaenoic acid of cod liver oil has been enriched four to five-fold, from 9.4 to 45% by selective esterification of fatty acids (other than docosahexaenoic acid) with butanol. As long as the reaction is stopped before reaching equilibrium, very little of either γ-linolenic acid or docosahexaenoic acid are converted to butyl esters, which results in high yields of these acids in the unesterified fatty acid fraction.     

Purification of Stearidonic Acid from Modified Soybean Oil by Argentation Silica Gel Column Chromatography

The objective of this study was to purify stearidonic acid (SDA, 18:4 ω-3) from modified soybean oil containing a mixture of over 20 fatty acids (23% SDA). Interest in obtaining purified fractions of SDA arises from reported health benefits associated with polyunsaturated fatty acids (PUFA), such as cardiovascular disease prevention. In addition, SDA may also provide improved stability characteristics since its unsaturation index is less than longer PUFA, such as eicosapentaenoic acid (EPA, 20:5 ω-3). First, a chemical ethanolysis of modified soybean oil was performed to transform the triacylglycerols into fatty acid ethyl esters (FAEE). Then, the FAEE were fractionated and SDA-EE was purified by argentation silica gel (10% AgNO₃) open column chromatography, which allows selectivity based on degree of unsaturation. Different FAEE sample loads and mobile phases were explored until the best purification of SDA-EE was achieved. The solvents used were hexane and hexane:acetone mixtures (99 and 95%). Under the optimal conditions, a fraction with high SDA-EE purity (96%) was obtained with 77% yield. Besides, it was possible to obtain another fraction enriched in α-linolenic acid-EE (37% purity and 68% yield) and γ-linolenic acid-EE (22% purity and 61% yield). A scaled-up process resulted in 840 mg of final product composed of 97% SDA-EE with 71% yield.     

Wiederveresterung von Konzentraten mehrfach ungesättigter Fettsäuren

Reesterification of Polyunsaturated Fatty Acid Concentrates Fatty acids of the n-6 series such as γ-linolenic acid (C18:3) and of the n-3 series such as α-linolenic (C18:3), stearidonic (C18:4), eicosapentaenoic (C20:5) and docosahexaenoic acid (C22:6) are of great nutritional interest. These acids can be obtained by urea fractionation in concentrated form from natural sources like blackcurrant seed oil, borage oil, evening primrose oil, linseed oil and fish oil, respectively. Certain dietary applications require a reesterification of these fatty acid concentrates with glycerol to triglycerides. Industrial scale methods of reesterification could not be applied as such for the present polyunsaturated fatty acid systems. Therefore reaction conditions had to be adapted to these highly sensitive substances. A one step reesterification method, using ZnCl₂ as catalyst, was optimized for three different fatty acid concentrates composed of the above mentioned acids. Under the given reaction conditions, it could be observed that α-linolenic acid is much more sensitive to polymerization than γ-linolenic acid. Different purification methods of the crude triglycerides have been evaluated, obtaining best results by liquid chromatography methods, in particular with respect to decoloration of the final products.     

Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

Microbial conversion of an oil containing α-linolenic acid to an oil containing eicosapentaenoic acid

Mycelia of arachidonic acid-producing fungi belonging to the genusMortierella were found to convert an oil containing α-linolenic acid to an oil containing 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). This conversion was observed when they were grown in a medium containing the oil, glucose and yeast extract at 28 C. On the screening of various oils, linseed oil, in which α-linolenic acid amounts to about 60% of the total fatty acids, was found to be the most suitable for EPA production. Under the optimal culture conditions, a selected strain,Mortierella alpina 20-17, converted 5.1% of the α-linolenic acid in the added oil into EPA, the EPA production reaching 1.35 g/l of culture broth (41.5 mg/g dry mycelia). This value corresponded to 7.1% (by weight) of the total fatty acids in the extracted lipids. The lipid was also found to be rich in arachidonic acid (12.3%). Other major fatty acids in the lipid were palmitic acid (4.4%), stearic acid (3.2%), oleic acid (13.5%), linoleic acid (13.7%), α-linolenic acid (38.5%) and γ-linolenic acid (0.9%).     

The isolation of omega-3 polyunsaturated fatty acids and methyl esters of fish oils by silver resin chromatography

Multigram quantities of the highly unsaturated ω3 component from samples of fish oil fatty acids and esters were isolated by silver resin chromatography. An XN1010 resin column saturated with silver ions was utilized. Polyunsaturated fatty acid (PUFA) esters from fish oil concentrate (FOC) were fractionated based on the number of double bonds by using solvent programming (acetonitrile in methanol). Larger samples (4–9 g) of FOC acids and esters and menhaden acids and esters were enriched in ω3 polyunsaturates to 82–99% (95–99% total PUFA) by use of a large 100% silver resin column and isocratic elution with 30, 35 or 45% acetonitrile in acetone.     

Differential effects of dietary linoleic and α-linolenic acid on lipid metabolism in rat tissues

Comparative effects of feeding dietary linoleic (safflower oil) and α-linolenic (linseed oil) acids on the cholesterol content and fatty acid composition of plasma, liver, heart and epididymal fat pads of rats were examined. Animals fed hydrogenated beef tallow were used as isocaloric controls. Plasma cholesterol concentration was lower and the cholesterol level in liver increased in animals fed the safflower oil diet. Feeding the linseed oil diet was more effective in lowering plasma cholesterol content and did not result in cholesterol accumulation in the liver. The cholesterol concentration in heart and the epididymal fat pad was not affected by the type of dietary fatty acid fed. Arachidonic acid content of plasma lipids was significantly elevated in animals fed the safflower oil diet and remained unchanged by feeding the linseed oil diet, when compared with the isocaloric control animals fed hydrogenated beef tallow. Arachidonic acid content of liver and heart lipids was lower in animals fed diets containing safflower oil or linseed oil. Replacement of 50% of the safflower oil in the diet with linseed oil increased α-linolenic, docosapentaenoic and docosahexaenoic acids in plasma, liver, heart and epididymal fat pad lipids. These results suggest that dietary 18∶2ω6 shifts cholesterol from plasma to liver pools followed by redistribution of 20∶4ω6 from tissue to plasma pools. This redistribution pattern was not apparent when 18∶3ω3 was included in the diet.     

Enrichment of eicosapentaenoic acid and docosahexaenoic acid in saponified menhaden oil

Eicosapentaenoic acid (EPA, 20∶5n−3) and docosahexaenoic acid (DHA, 22∶6n−3) in free fatty acids (FFA) derived from saponified menhaden oil were concentrated by the solubility differences of FFA-salts in organic solvent. FFA-salts were formed by adding NaOH to a solution containing FFA. A Buchner funnel was used to separate solid phases from liquids containing FFA-salts. FFA that are rich in EPA and DHA can be recovered from the liquid phase by the addition of 12 N HCl. The effects of reaction time, the amount of NaOH, and solvent used on the concentration of EPA and DHA were systematically investigated. With a total volume of 112 mL, made up of 1.85% 15 N NaOH, 88.1% acetone, and 10.0% FFA, a reaction temperature of 30°C, and a reaction time of 1 h, the resulting liquid phase contained 65.4 wt% EPA and DHA, with a corresponding yield of 41.5%. By replacing the acetone with a mixture of 45% acetone and 55% acetonitrile and then storing the liquid phase at −70°C overnight, the content and yield of EPA and DHA in the final liquid phase were 61.4 wt% and 66.2%, respectively.