Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.     

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.     

Interesterification kinetics of triglycerides and fatty acids with modified lipase inn-hexane

The kinetics of lipase-catalyzed interesterification of triglycerides and fatty acids in organic media was studied. First, the lipase Saiken 100,Rhizopus japonicus, was modified by surfactant to form an enzyme precipitate in aqueous solution, which was well dispersed in organic solvents. This modified lipase catalyzed the interesterification of tripalmitin and stearic acid. The enzyme has 1,3-positional specificity and does not distinguish between stearic and palmitic acids. The kinetic model developed to describe the interesterification reaction system is based on mass balance of two consecutive second-order reversible reactions. The reaction rate constant, k, was determined by solving the differential rate equations of the reaction system and by expressing the value of k as a function of concentrations of the substrates with time. The model gave satisfactory results. The best value of the specific reaction rate constant k* that fits all experimental data was 1.2 · 10⁻⁵ [L²/(mmol · mg biocatalyst · h)] under the reaction conditions in this study.     

Esterification of polyunsaturated fatty acids by various forms of immobilized lipase from Rhizomucor miehei

Ethyl esterification specificity of a lipase from Rhizomucor miehei for polyunsaturated fatty acids (PUFA) was compared at 1 and 100 mM to study molecular recognition of PUFA. The chemical shift of methylene adjacent to carboxyl groups in the nuclear magnetic resonance spectrum of docosahexaenoic acid (DHA) in ethanol moved to a lower magnetic field as the concentration of DHA increased, suggesting that the degree of dissociation of DHA decreased. Specificity constants or apparent second-order rate constants (V _max/K _m or catalytic power) for 1 mM esterification by immobilized lipases were higher than the native lipase. Immobilized hydrophobic carrier of low mass transfer resistance for the esterification substrate may improve maximal velocity and affinity for the substrate. Higher specificity constants for 1 mM substrates were observed using immobilized lipases fixed on an anion exchange resin with glutaraldehyde and on a cation exchange carrier with carbodiimide. Activity yields measured with 1 mM PUFA substrate were high. For the substrates at a concentration of 100 mM, higher specific constants with these bifunctional reagents were not observed but higher activity yields were found.     

Synthesis of triglycerides from fish oil fatty acids

The generally accepted methods for the synthesis of triglycerides are unsatisfactory when they are applied to highly unsaturated systems such as those present in fish oil. The methods either fail to g ive sufficiently high yields, or they are prohibitive in cost when applied to large scale production. Of the numerous reactions studied, the most feasible was the zinc-catalyzed esterification of fish oil fatty acids with glycerol. Thinlayer chromatography (TLC) showed that this reaction gave yields of 75–78% triglycerides. Gas-liquid chromatography (GLC) demonstrated the composition of the triglycerides was essentially the same as that of the original fatty acids.     

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.     

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.     

Resistance of certain long-chain polyunsaturated fatty acids of marine oils to pancreatic lipase hydrolysis

When whale oil triglycerides were subjected to pancreatic lipase hydrolysis, eicosapentaenoic and docosahexaenoic acids were found mainly in the di- and triglyceride products, suggesting that they are in the 1,3-positions but resistant to the action of the lipase. Their presence in the 1,3-positions was confirmed. Their resistance to pancreatic lipase hydrolysis was demonstrated by analysis of the products of the enzyme action on: (a) a concentrate of highly unsaturated whale oil triglycerides; (b) the latter after randomization; and (c) synthetic 1,2-di-octadecenoyl-3-eicosapentaenoyl glycerol. Docosapentaenoic acid was also shown to be present in the 1,3-position of whale oil triglycerides but was not lipase resistant. It is postulated that the presence of a double bond near the carboxyl group exercises an inhibitory effect, or that the location of the double bonds in the resistant acids places their terminal methyl groups close to the carboxyl, producing a steric hindrance effect.     

The metabolic fate of fatty acids derived from dietary triglycerides

The metabolic fates of dietary tricaprylin, trimyristin, tripalmitin, triolein, and trilinolein at the 15% level were followed with tracer doses of the corresponding C¹⁴-labeled acids. Distribution of the label in respiratory C¹⁴O₂ and in fatty acids of adipose tissue and liver lipids as well as the fatty acid composition of these unfractionated tissue lipids led to the following conclusions: Tissue fatty acid compositional homeostasis is limited mainly by the degrees to which dietary fatty acids can be converted to endogenous fatty acids. Other factors, such as their effects on lipogenesis and the relative degrees to which they are catabolized and stored, also play roles.     

Positional specificity of gastric hydrolysis of long-chain n−3 polyunsaturated fatty acids of seal milk triglycerides

Long-chain n−3 polyunsaturated fatty acids (n−3 PUFA) of marine oils are important dietary components for both infants and adults, and are incorporated into milks following maternal dietary intake. However, little is known about the hydrolysis of these PUFA from milk triglycerides (TG) by lipases in suckling young. Seals, like humans, possess gastric lipase; however, the milk lipids of seals and sea lions are almost devoid of the readily hydrolyzable medium-chain fatty acids, and are characterized by a large percentage (10–30%) of n−3 PUFA. Gastric hydrolysis of milk lipids was studiedin vivo in suckling pups of three species (the California sea lion, the harp seal and the hooded seal) in order to elucidate the actions and specificity of gastric lipases on milk TG in relation to fatty acid composition and TG structure. Regardless of milk fat content (31–61% fat) or extent of gastric hydrolysis (10–56%), the same fatty acids were preferentially released in all three species, as determined by their relative enrichment in the free fatty acid (FFA) fraction. In addition to 16∶1 and 18∶0, these were the PUFA of 18 carbons and longer, except for 22∶6n−3. Levels of 20∶5n−3 were most notably enriched in FFA, at up to five times that found in the TG. Although 22∶6n−3 was apparently also released from the TG (reduced in the diglyceride), it was also notably reduced in FFA. Positional analysis of milk TG based on the products of Grignard hydrolysis revealed that these PUFA, including 22∶6n−3, were preferentially esterified at the α-position of the TG, and that the fatty acids not released during gastric hydrolysis were located at thesn-2 position. The extreme reduction of 22∶6n−3 and enrichment of 20∶5n−3 in FFA is discussed. Results from this study are consistent with reports that gastric lipase acts stereo-specifically to release fatty acids at the α-positions (sn−3,sn−1). We conclude that the n−3 PUFA in milk are efficiently hydrolyzed by gastric lipase and that this has important implications for digestion of milks enriched in PUFA by neonates in general. Based on a paper presented at the Symposium on Milk Lipids held at the AOCS Annual Meeting, Baltimore, MD, April 1990; part of this work is from the doctoral dissertation by S.J.I., University of Maryland, 1988.     

Surface tension of fatty acids and triglycerides

Surface tension as a function of temperature was measured for four fatty acids (lauric, myristic, palmitic, and oleic) and two triglycerides (tricaprylin and tripalmitin). These surface tension measurements were performed using a K12 vers. 3.1 (Krüss GmbH) tensiometer at temperatures from 20 (or the melting point of each substance) to 90°C. The constants for a van der Waals-type correlation as well as for a linear equation are presented. Both equations are quite accurate, presenting mean deviations not exceeding 0.570%. Such correlation constants are valuable in the design or evaluation of processing equipment, especially that involving gas-liquid contact such as distillation and stripping columns, deodorizers, reactors, and equipment for physical refining.     

Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids

Lipase hydrolysis was evaluated as a means of selectively enriching long-chain ω3 fatty acids in fish oil. Several lipases were screened for their ability to enrich total ω-3 acids or selectively enrich either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA). The effect of enzyme concentration, degree of hydrolysis, and fatty acid composition of the feed oil was studied. Because the materials that were enriched in long-chain ω3 acids were either partial glycerides or free fatty acids, enzymatic reesterification of these materials to triglycerides by lipase catalysis was also investigated. Hydrolysis of fish oil by eitherCandida rugosa orGeotrichum candidum lipases resulted in an increase in the content of total ω3 acids from about 30% in the feed oil to 45% in the partial glycerides. The lipase fromC. rugosa was effective in selectively enriching either DHA or EPA, resulting in a change of either the DHA/EPA ratio or the EPA/DHA ratio from approximately 1:1 to 5:1. Nonselective reesterification of free fatty acids or partial glycerides that contained ω3 fatty acids could be achieved at high efficiency (approximately 95% triglycerides in the product) by using immobilizedRhizomucor miehei lipase with continuous removal of water.     

Industrial chemical uses of polyunsaturated fatty acids

Production of vegetable, animal and marine oils containing more than about 40% unsaturated fatty acids totaled 15,000 million pounds in 1968, almost on the scale of petrochemical production. The greater share (64%) of this nonfossil oil production was directed toward food uses, the remainder toward industrial and animal feed uses. The variety of chemical reactions carried out on these unsaturated fatty acid products include hydrogenation, interesterification, dimerization, sulfation, formation of nitrogen compounds, epoxidation, alkaline cleavage and oxidative ozonolysis. Some of these reactions have been developed at Utilization Research and Development Divisions of the Agricultural Research Service, U.S. Department of Agriculture. Research is continuing in developing new reactions for potential industrial application. An example is reductive ozonolysis of unsaturated fatty esters to produce monofunctional aldehydes and bifunctional aldehyde esters. Presented at the ISF-AOCS World Congress, Chicago, September 1970. No. Market. Nutr. Res. Div., ARS, USDA.     

The production of fish oils enriched in polyunsaturated fatty acid-containing triglycerides

A simple concentration technique was developed and used for the production of fish oils highly enriched with respect to the polyunsaturated triglycerides. The method involves the rapid solidification of fish oil droplets in liquid nitrogen followed by extraction with acetone at −60°C. The combined percentage ofcis-5,8,11,14,17-eicosapentaenoic acid (20:5) andcis-4,7,10,13,16,19-docosahexaenoic acid (22:6) after enrichment of a crude South African Anchovy (Engraulis capensis) oil was 57.4. A maximum percentage of 66.0 was attained for n-3 fatty acids after enrichment of a crude Chilean fish oil. A maximum yield of 26.0% was achieved for a crude sardine (Sardina pilchardus) oil. Triglycerides containing only saturated fatty acids or a combination of saturated and monoenoic acids were totally removed by the process, as assessed by silver-ion high-performance liquid chromatography of the triglyceride oils. This process permits the production of significant quantities of highly unsaturated triglycerides, which may be used in physiological and oxidative studies or for structural analysis of these triglycerides, many of which are present at extremely low concentrations in the original oils.     

Polymorphism of mixed triglycerides containing odd fatty acids

Paris of C _n C_(n+1)C _n (i.e., C₁₆C₁₇C₁₆ and C₁₈C₁₉C₁₈) and C _n C_(n-1)C _n triglycerides (C ₁₆ C ₁₅ C ₁₆ and C ₁₈ C ₁₇ C ₁₈ ) triglycerides,n even, were prepared to compare with C _n C_(n+2)C _n and C _n C_(n-2)C _n glycerides, respectively, in polymorphic behavior. It was found that C _n C_(n+1)C _n compounds were β′ tending and C _n C_(n-1)C _n compounds β tending in line with their all-even counterparts. The glyceride C₁₁C₁₃C₁₁ is β′ stable, like C₁₀C₁₂C₁₀ and C₁₆C₁₈C₁₆, and seems closely related to them in physical behavior and diffraction characteristics.     

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.     

Comparison of radiolytic compounds from saturated and unsaturated triglycerides and fatty acids

Formation of certain radiolysis products from palmitic acid, oleic acid, tripalmitin and triolein has provided a means for comparing the radiolytic effects in saturated and unsaturated triglycerides and fatty acids. These substances were chosen to represent the major constituents of fat found in beef. Fractionation and concentration of radiolytic compounds from the irradiated samples was accomplished by the means of size exclusion chromatography. Quantitative and qualitative analyses were performed using a combined GC/MS computer system. In addition to the primary radiolytic compounds, recombination products of relatively high molecular weight and various propanediol diesters from the corresponding glyceryl moities were identified. Quantitative analyses indicated a greater yield of various radiolytic compounds from free fatty acids than from the corresponding triglycerides. Similarly, radiolytic compounds were produced in greater quantities from the saturated fats than the unsaturated fats. Most of the radiolytic compounds identified in this study have not been previously reported.     

The biosynthesis of polyunsaturated fatty acids in plants

This paper is a review of some of the work being done at the author's laboratory. The phospholipids and glycolipids of the alga,Chlorella vulgaris, have been implicated in fatty acid transformations such as chain elongation and desaturation. Labeling studies with [¹⁴C] acetate have shown that newly synthesized galactosyl glycerides have mainly saturated fatty acids. Subsequent to de novo synthesis, a series of alterations of fatty acid structure takes place within the same glycolipid molecules. The specific incorporation of [¹⁴C] oleic acid intoChlorella phosphatidyl choline provides a convenient model system for studying the lipid dependent desaturation of oleic to linoleic acid. The inhibitor of fatty acid desaturation, sterculic acid, only inhibits the conversion of oleate into linoleate if added before the precursor fatty acid has been incorporated into a complex lipid. Studies with isomeric monoenoic fatty acids have suggested that there are two enzymes which catalyze the formation of linoleic from oleic acid. One measures the position of the second double bond from the carboxyl group, the other, from the methyl end of the chain. The latter enzyme probably requires the complex lipid substrate.     

Effect of polyunsaturated fatty acids in obese mice

Genetically obese (ob/ob) mice display a variety of metabolic differences from lean litter mates. In the obese state, fatty acid desaturation-elongation in brown adipose tissue mitochondria is apparently altered, resulting in differences in membrane fatty acid composition. This change in membrane lipid environment appears to influence GDP binding and there-fore the activity of the proton conductance pathway associated with regulation of energy expenditure in these animals. In liver, binding of insulin to the nuclear membrane is increased by feeding a high polyunsaturated/saturated (P/S) diet fat. Consumption of a high P/S diet decreased mRNA levels for fatty acid synthase, acetyl-CoA carboxylase, malic enzyme, and pyruvate kinase in obese and lean animals. Expression of mRNA for these lipogenic enzymes was higher in obese animals and suggests that obese mice may be resistant to polyunsaturated fatty acid feedback control of gene expression.     

Concentration and stabilization of n-3 polyunsaturated fatty acids from sardine oil

A simple and relatively inexpensive procedure to obtain 90% eicosapentaenoic acid + docosahexaenoic acid concentrates from sardine oil involved a two-step winterization of the oil (10 and 4°C), followed by saponification and selective precipitation of saturated and less unsaturated free fatty acids by an ethanolic solution of urea. Antioxidant effects of butylated hydroxytoluene, dl-α-tocopherol, and two natural antioxidants, quercetin and boldine, added to the concentrate (0.5% wt/vol), were compared in the Rancimat at 60°C. dl-α-Tocopherol was unable to inhibit concentrate oxidation. Butylated hydroxyanisole and butylated hydroxytoluene had induction periods of 1.7–1.8 h compared to the control (1.0 h). A mixture of quercetin + boldine (2:1 w/w) significantly increased the induction period to 4.5 h.