Recovery of hydroxy fatty acids from lesquerella oil with lipases

Two hydroxy acids, lesquerolic (53 wt%) and auricolic (4%), are present at significant quantities inLesquerella fendleri seed oil. Results reported here indicate the selective release of hydroxy fatty acids during hydrolysis of this oil catalyzed byRhizopus arrhizus lipase. For example, hydroxy acids composed 85–90 wt% of the free fatty acids released during lipolysis, as compared to 54% present overall in the oil. In addition, over 80% of the lesquerolic acid is released from the triglycerides. The reason for this lipase’s success was determined to be its 1,3-positional specificity. The vast majority of lesquerella oil’s hydroxy acids is at the 1- and 3-positions of its triglycerides, as confirmed by the compositional analysis of partial glycerides formed during lipolysis.     

Preparative chromatographic isolation of hydroxy acids fromLesquerella fendleri andL. gordonii seed oils

To conduct product development research onLesquerella seed oils, we explored methods to obtain >100 g quantities of lesquerolic (14-hydroxy-cis-11-eicosenoic) acid. Preliminary experiments with open-column silica gel chromatography showed thatL. fendleri oil could be separated into 3 triglyceride (TG) fractions. The first (10%) contained nonhydroxy 16-(13%) and 18-carbon acids (65% 18∶1,2,3). The second fraction (15%) contained monolesquerolins (39% lesquerolic acid). The major TG fraction (73%) was mainly dilesquerolins (66% lesquerolic acid) showing that a hydroxy acid-enriched TG oil was obtainable by this procedure. Silica gel chromatography easily separatedL. fendleri fatty acid methyl esters (FAME) into a hydroxy-free ester fraction (40–44%) consisting largely of 18∶1 (39%), 18∶2 (19%) and 18∶3 (31%), and a hydroxy ester fraction (56–60%) that was largely methyl lesquerolate (94%) with small amounts of auricolate (5%) (14-hydroxy-cis-11,cis-17-eicosadienoate) and traces of 18-carbon hydroxy esters. This process for isolating the hydroxy FAME ofLesquerella oil was scaled up 15-to 100-fold with a preparative high performance liquid chromatograph. Thirty-gram samples ofL. gordonii FAME were dissolved in eluting solvent, pumped onto the high performance liquid chromatography (HPLC) silica column and eluted with 97∶3 hexane/ethyl acetate. In an 8-hr period, up to 200 g of methyl lesquerolate could be obtained with a purity >98%, the only contaminants being methyl auricolate and methyl ricinoleate. Presented at the AOCS meeting in Phoenix, AZ, May 1988. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Medium-chain fatty acid-rich glycerides by chemical and lipase-catalyzed polyester-monoester interchange reaction

Medium-chain triglycerides (MCT) that contain caprylic acid (C_8:0) and capric acid (C_10:0) have immense medicinal and nutritional importance. Coconut oil can be used as a starting raw material for the production of MCT. The process, based on the interchange reaction between triglycerides and methyl esters of medium-chain fatty acids by chemical catalyst (sodium methoxide) or lipase (Mucor miehei) catalyst, appears to be technically feasible. Coconut oils with 25–28.3% (w/w) and 22.1–25% (w/w) medium-chain fatty acids have been obtained by chemical and lipase-catalyzed interchange reactions. Coconut olein has also been modified with C_8:0 and C_10:0 fatty acids, individually as well as with their mixtures, by chemical and lipase-catalyzed interchange reactions. Coconut olein is a better raw material than coconut oil for production of mediumchain fatty acid-rich triglyceride products by both chemical and lipase-catalyzed processes.     

Acidolysis of several vegetable oils by mycelium-bound lipase of Aspergillus flavus link

The ability of mycelium-bound lipase of a locally isolated Aspergillus flavus to modify the triglyceride structure of vegetables oils was studied. The catalysis involved the acidolysis of vegetable oils, such as palm olein, coconut oil, cotton-seed oil, rapeseed oil, corn oil and soybean oil, with selected fatty acids (FA). The reactions were followed against time, and the percentages of FA incorporated were determined by gas chromatography. Percentage of FA incorporated after 20-h reaction was in the range of 13 to 18%. Reaction between cottonseed oil with lauric acid gave the highest percentage of incorporation (18%), followed by soybean oil with lauric acid (16%) and coconut oil with oleic acid (16%). The results indicated that the hydrolytic affinity of A. flavus lipase demonstrates an acyl group specificity toward short-chain FA (C₈–C₁₀). Changes in triglyceride profiles of each oil were also monitored by reverse-phase high-pressure liquid chromatography. In all products, there were increases in the concentrations of several existing triglycerides and formation of new triglycerides. The melting points of all acidolyzed vegetable oils were determined by differential scanning calorimetry, and significant changes in melting profiles were noted.     

Selectivity of lipases: isolation of fatty acids from castor,coriander, and meadowfoam oils

The lipase‐catalyzed hydrolysis of castor, coriander, and meadowfoam oils was studied in a two‐phase water/oil system. The lipases from Candida rugosa and Pseudomonas cepacia released all fatty acids from the triglycerides randomly, with the exception of castor oil. In the latter case, the P. cepacia lipase discriminated against ricinoleic acid. The lipase from Geotrichum candidum discriminated against unsaturated acids having the double bond located at the Δ‐6 (petroselinic acid in coriander oil) and Δ‐5 (meadowfoam oil) position or with a hydroxy substituent (ricinoleic acid). The expression of the selectivities of the G. candidum lipase was most pronounced in lipase‐catalyzed esterification reactions, which was exploited as part of a two‐step process to prepare highly concentrated fractions of the acids. In the first step the oils were hydrolyzed to their respective free fatty acids, in the second step a selective lipase was used to catalyze esterification of the acids with 1‐butanol. This resulted in an enrichment of the targeted acids to approximately 95—98% in the unesterified acid fractions compared to the 70—90% content in the starting acid fractions.     

Chemical survey and erucic acid content of commercial varieties of nasturtium,Tropaeolum majus L.

Nasturtium (Tropaeolum majus L.) oil contains the highest levels of erucic acid of known seed oils (75–80%). A significant portion of the acid is attached to the 2-position of the glycerol, and trierucin is a major component (ca. 50%) of the oil. Seeds from eleven varieties of commercially available garden nasturtium (T. majus) were screened for oil content, erucic acid levels and fatty acid distribution. Oil contents ranged fromca. 6 to 11%, and erucic acid levels in the oils ranged from 62 to 80%. One sample ofT. speciosum was also analyzed, and contained 28% oil, fatty acids from C₁₆ to C₂₈ and triglycerides up to C₇₂.     

Search for new industrial oils. VI. Seed oils of the genusLesquerella

Fatty acid composition of seed oil from 14 species of the genusLesquerella has been determined by gas-liquid chromatography. All but two species contain hydroxyeicosenoie acid in amounts ranging from 45 to 74%. The remaining two species contain about 50% C₁₈ hydroxy acids, but none of the C₂₀ hydroxy acid. Presented at the spring meeting of the American Oil Chemists' Society, St. Louis, Missouri, May 1–3, 1961. A laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture.     

Vegetable oil raw materials

Vegetable oils that are important to the chemical industry include both edible and industrial oils, which contribute 24% and 13.5%, respectively, compared to 55% for tallow, to the preparation of surfactants, coatings, plasticizers, and other products based on fats and oils. Not only the oils themselves but also the fatty acids recovered from soapstock represent a several billion pound resource. Coconut oil is imported to the extent of 700-1,000 million pounds per year. Its uses are divided about equally between edible and industrial applications. Safflower oil has a relatively small production, but 15–25% of the oil goes into industrial products. Soybean oil, the major edible oil of the world, is produced in the United States at the rate of 11,000 million pounds per year with more than 500 million pounds going into industrial uses, representing 5% of the total production. Castor oil is imported to the extent of about 100 million pounds per year. Linseed oil production has declined drastically over the last 25 years but still amounts to about 100 million pounds per year. Oiticica and tung oils are imported in lesser amounts than castor and linseed oils. New crops that have industrial potential, as well as the traditional vegetable oil crops, include seed oils from crambe,Limnanthes, Lesquerella, Dimorphotheca, Vernonia, andCuphea plants. Crambe oil contains up to 65% erucic acid. Oil fromLimnanthes contains more than 95% of fatty acids above C₁₈.Lesquerella oil contains hydroxy unsaturated acids resembling ricinoleic acid from castor oil.Dimorphotheca oil contains a conjugated dienol system.Vernonia oils contain as much as 80% epoxy acids. TheCuphea oils contain a number of short chain fatty acids. Of these, crambe,Limnanthes, andVernonia are probably the most developed agronomically. Competition between vegetable oils and petrochemicals for the traditional fats and oil markets has been marked over the past 25 years, but prices for petrochemicals have accelerated at a greater rate than those for vegetable oils; and, it is now appropriate to reexamine the old as well as the new markets for fatty acids.     

1,3-specific lipolysis ofLesquerella fendleri oil by immobilized and reverse-micellar encapsulated enzymes

Three types of reaction systems, all batch-mode, were employed for production of hydroxy (lesquerolic and auricolic) fatty acidsvia 1,3-specific lipolysis ofLesquerella fendleri oil: “Free”Rhizopus arrhizus or immobilizedRhizomucor miehei lipase (Lipozyme?) in reverse micelles (System 1), Lipozyme suspended in lesquerella oil/isooctane mixture (System 2) and a suspension of water and freeR. miehei lipase in lesquerella oil/isooctane (System 3). The objective was to find the system that best maximized yield (i.e., percent hydrolysis), the proportion of hydroxy acids among the free acids liberated (hydroxy acid “purity”), and recovery/reuse of lipase activity, and that could be easily adapted into a large-scale process. System 1 provided the largest percent hydrolysis (55%) and hydroxy acid purity (85%), but of the three systems would be the most difficult to scale up. Thus, System 1 would be the most desirable reaction system only when small batch sizes are to be processed. System 3 yielded 47.2% hydrolysis, but the hydroxy acid purity was at most 73%, making it the least desirable of the three systems to employ. System 2 yielded moderate extents of hydrolyses (30–40%) and large hydroxy acid purity initially (80–83%), but the purity decreased slightly in the latter stages of the reaction due to acyl migration. System 2 was the system most easily adaptable to a large-scale process, making it the method of choice. For System 2 reactions, only when the medium was initially saturated with water and water consumed by the reaction was continuously replaced could 30–40% hydrolysis be achieved. External mass transfer limitations for Lipozyme-catalyzed reactions were not present when the solution’s water content was not above saturation, and its kinematic viscosity, controlled by the temperature and the proportion of isooctane, was below 41 centistokes.     

Hydroxy acids and estolide triglycerides ofHeliophila amplexicaulis L.f. Seed oil

Thirty percent of the fatty acids fromHeliophila amplexicaulis seed oil are hydroxy acids, primarily lesquerolic acid (14-hydroxy-cis-11-eicosenoic acid) with a trace of a new fatty acid, 16-hydroxy-cis-13-docosenoic acid. The hydroxy acids in the oil are found exclusively in the 1 and/or 3 positions of the triglycerides and are completely acylated with C₂₀ or C₂₂ saturated or monoenoic acids. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

A detailed triglyceride analysis ofLesquerella fendleri oil: Column chromatographic fractionation followed by supercritical fluid chromatography

The triglyceride structure of oil fromLesquerella fendleri, a potential new U.S. crop, rich in C₂₀ hydroxy fatty acids, was examined by silica gel column chromatographic fractionation followed by supercritical fluid chromatography. The analysis confirmed previous findings derived by our research group, but provided further detail. The analysis demonstrated the presence of trihydroxy triglyceride, which contained all of the oil’s C₁₈ hydroxy acyl groups (present at less than 0.5% in the oil). Lipolysis indicated that these groups were located solely at the 2-position. In addition, a strong correlation was detected between the presence of α-linolenic (18:3^9,12,15) and auricolic (20:2^11,17 OH¹⁴) acids in triglycerides.     

High-performance liquid chromatography and spectroscopic studies on fish oil oxidation products extracted from frozen atlantic mackerel

The formation of stable hydroxy derivatives from hydroperoxides produced during the oxidation of linoleic acid methyl ester and fish oil were studied by reverse-phase high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The oxidation products identified were mixtures of four isomeric hydroxy derivatives: 13-hydroxy-9-cis,11-trans-octadecadienoic, 13-hydroxy-9-trans,11-trans-octadecadienoic, 9-hydroxy-10-trans,12-cis-octadecadienoic, and 9-hydroxy-10-trans,12-trans-octadecadienoic acids. The presence of hydroxy compounds was confirmed by ¹³C NMR, which gave rise to a hydroxy carbon peak at 87 ppm, and by GC-MS, which showed three peaks corresponding to isomeric mixtures of trimethylsilyl ethers of the oxidized linoleic acid methyl ester. The mass spectra scans of the three peaks showed that they represent isomers of molecular weight 382 and are consistent with the molecular formula C₂₂H₄₂O₃Si. In oil extracted from stored frozen mackerel, 13-hydroxy-9-cis,11-trans-octadecadienoic acid was more prominent compared to the model lipid systems. HPLC offered a sensitive means of detection of hydroxy compounds produced both in the initiation and latter stages of oxidation. The effect of antioxidants added to the fish mince prior to storage can also be monitored by HPLC. Thus, the monitoring of lipid oxidation hydroxy derivatives by HPLC is of practical value in the efficient processing and quality control of fish, fish oils, and other fatty foodstuffs in order to enhance the acceptability, nutritional, and safety aspects.     

Gas-liquid chromatography of triglycerides from erucic acid oils and fish oils

By critically selecting optimum operating conditions, quantitative gas-liquid chromatography of triglycerides has been extended to molecules containing substantial amounts of C₂₀, C₂₂, and C₂₄ fatty acids. The triglycerides of four erucic acid oils (water cress, rapessed, nasturtium, andLunaria annua) and two fully hydrogenated fish oils (menhaden and tuna) have been quantitatively analyzed by this technique. The average fatty acid chain length calculated from the triglyceride composition of each oil agreed closely with that determined by GLC of its respective methyl esters. Several conclusions about the triglyceride composition of the fats analyzed are discussed. Winner, AOCS Bond Award. Presented at the AOCS Meeting in Cincinnati, October 1965.     

Triacylglycerol composition of oil from <Emphasis Type="Italic">Pistacia atlantica</Emphasis> fruit growing in Algeria

The distribution of FA between the sn-2 and sn-1,3 positions of TAG from Pistacia atlantica fruit oil of Algeria has been determined. Unsaturated FA showed a preference for the internal position. Linoleic and oleic acids occurred predominantly in the sn-2 position with lesser amounts evenly distributed between the sn-1 and sn-3 positions, as generally found in vegetable oils. The oil was found to contain TAG that were trisaturated (0.93%), disaturated (15.06%), monosaturated (44.64%), and triunsaturated (38.10%). The distribution of the TAG calculated using the lipase hydrolysis technique is slightly different from that determined with HPLC. This is particularly true for trioleoyl and trilinoleoylglycerols. In contrast, the agreement between theory and experiment is good for TAG containing two palmitoyl and one oleoyl, one oleoyl and two linoleoyl, and one palmitoyl and two oleoyl chains.     

Glyceride structure ofCardamine impatiens L. Seed oil

A group of unusual triglycerides, in which one of the acyl groups is a vicinal dihydroxy acid with one of the hydroxyl groups acetylated, has been isolated fromCardamine impatiens L. (Cruciferae) seed oil. Hydrolysis of these triglycerides with castor bean lipase facilitated isolation and identification of a mixture of C₁₈, C₂₀, C₂₂, and C₂₄ hydroxy acetoxy fatty acids. Pancreatic lipase hydrolysis data revealed that these monoacetylated dihydroxy acid residues are esterified exclusively with one of the α-positions of the glycerol moiety. The remaining acyl groups are comprised of ordinary C₁₈ unsaturated acids (which occupy 98% of the β-position), palmitic acid, and C₂₀, C₂₂, and C₂₄ monoenoic fatty acids. No. Utiliz. Res. Dev. Div., ARS, USDA.     

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.     

The structure of the glycerides of ergot oils

The oils from sclerotia or from suitable mycelial cultures ofClaviceps purpurea (ergot) contain up to 44% of ricinoleic acid but no free hydroxyl groups. This is due to the presence of, besides normal triglycerides, tetra-acid, penta-acid and hexa-acid triglycerides. These contain respectively one, two and three ricinoleic acids esterified to glycerol, these in turn being acylated at their hydroxy groups with normal long-chain fatty acids. By suitable complementary use of TLC, GLC and lipase hydrolysis techniques, the proportions, compositions and structures of these novel triglyceride classes were determined. Four types of positional specificities in fatty acid combinations could be shown by our procedures. These are discussed and, on the basis of our results, some tentative proposals as to possible biosynthetic mechanisms are advanced. Dedicated to the late Prof. T. P. Hilditch, and presented to the Symposium on Analysis of Natural Fat Triglycerides, AOCS Meeting, Houston, April 1965.     

Utilization of acid oils in making valuable fatty products by microbial lipase technology

Microbial lipase-catalyzed hydrolysis, esterification, and alcoholysis reactions were carried out on acid oils of commerce such as coconut, soybean, mustard, sunflower, and rice bran for the purpose of making fatty acids and various monohydric alcohol esters of fatty acids of the acid oils. Neutral glycerides of the acid oils were hydrolyzed byCanadida cylindracea lipase almost completely within 48 h. Acid oils were converted into fatty acid esters of short- and long-chain alcohols like C₄, C₈, C₁₀, C₁₂, C₁₆, and C₁₈ in high yields by simultaneous esterification and alcoholysis reactions withMucor miehei lipase as catalyst. Acid oils of commerce can be utilized as raw materials in making fatty acids and fatty acid esters using lipase-catalyzed methodologies.     

Enrichment of lesquerolic and auricolic acids from hydrolyzed Lesquerella fendleri and L. gordonii oil by crystallization

Lesquerolic and auricolic acids were obtained from hydrolyzed lesquerella oil by a low-temperature crystallization procedure. The lesquerolic and auricolic fatty acid fraction was enriched from 55–59% to 85–99% with high yields (94%). Washing the free fatty acids with pH 6.0 buffer provided reproducible crystallizations of those hydroxy fatty acids. In contrast, when hydrolyzed oil from Lesquerella fendleri was not buffer-washed, there was, in most cases, no separation of hydroxy fatty acids by crystallization. This crystallization procedure is suitable for a large-scale separation process of the hydroxy fatty acids from nonhydroxy fatty acids obtained from hydrolyzed lesquerella oil.     

Production of a very low saturate oil based on the specificity of Geotrichum candidum lipase

Lipase B (GCB) produced by the fungus Geotrichum candidum CMICC 335426 is known for its high specificity towards cis-Δ9 unsaturated fatty acids. The wild-type lipase (not genetically modified) as well as the lipase obtained by heterologous expression of the corresponding gene in Pichia pastoris (genetically modified) were studied in a process aiming to produce an oil containing very little saturated fatty acids (SAFA). The approach described in this paper is based on the selective hydrolysis of sunflower oil (12% SAFA) using the G. candidum type B (GCB) lipases. Depending on the lipase input, up to 60% w/w degree of hydrolysis was obtained within 6–8 h. Because of the high specificity of the GCB lipases (specificity factor ∼30), the level of unsaturates in the free fatty acid fraction was >99% w/w. In contrast with literature data, no loss of specificity was observed, even at the highest degree of hydrolysis obtained. Though both GCB lipases are stable at 30°C, the rate of hydrolysis decreased considerably during the process. Product inhibition as well as time-dependent deactivation (half-life ≈2 h) were shown to be involved. After separation of the oil phase, the unsaturated free fatty acids were recovered from the mixture by evaporation and reconverted to triglycerides by enzymatic esterification with glycerol. Because the GCB lipases have a very low efficiency for esterification, this reaction was carried out with immobilized Rhizomucor miehei lipase. Under continuous removal of the water generated during the process, >95% triglycerides were obtained in less than 24 h. Standard deodorization resulted in an odorless, colorless, and tasteless oil with less than 1% SAFA.