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.     

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.     

Degumming and bleaching ofLesquerella fendleri seed oil

A lesquerella species (Lesquerella fendleri) being investigated as a domestic source of seed oil containing hydroxy fatty acids shows good agronomic properties and is being tested in semi-commercial production.Lesquerella fendleri seeds contain 25% oil, of which 55% is lesquerolic acid (14-hydroxy-cis-11-eicosenoic). Oils produced in pilot-plant quantities by screw press, prepress-solvent extraction and extrusion-solvent extraction processes have been refined in the laboratory by filtering, degumming and bleaching. Two American Oil Chemists’ Society (AOCS) standard bleaching earths and two commercial earths were compared for effectiveness in bleaching these dark, yellow-red, crude lesquerella oils. Free fatty acids (1.3%), iodine value (111), peroxide value (<4 meq/kg), unsaponifiables (1.7%) and hydroxyl value (100) were not significantly affected by degumming and bleaching, but phosphorus levels of 8–85 ppm in the crude oils were reduced to 0.5–1.1 ppm in the degummed and bleached oils. Crude oils had Gardner colors of 14, which were reduced to Gardner 9–11 in the degummed and bleached oil, depending on bleach type and quantity used. AOCS colors in the range of 21–25R 68–71Y were obtained. By including charcoal in the bleaching step, a considerably lighter oil could be obtained (Gardner 7).     

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.     

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.     

Hemp seed oil: Minor components and oil quality

Owing to its high content of omega‐6/omega‐3 fatty acids and bioactive minor components with antioxidant activities, hemp seed oil is now recognized for its health benefits by a large number of consumers. This paper primarily discusses the profile of minor components in hemp seed oil and their beneficial and adverse effects on oil quality. While tocopherols, polyphenols and phytosterols prevent oxidative deterioration of hemp seed oil, the high amount of chlorophyll can be detrimental to oil quality.     

Pilot plant extraction of oil fromVernonia galamensis seed

Vernonia galamensis seed containing 40–42% oil and 30–34% epoxy acid, (cis-12,13-epoxy-cis-9-octadecenoic) was processed to oil and meal. Seed conditioning, pressing and solvent extraction research were conducted in pilot facilities at the French Oil Mill Machinery Co. (Piqua, OH). The robust lipase system was successfully inactivated by treating 200 lb. batches ofV. galamensis seed in a cooker/conditioner at 195–200°F and >10% moisture. Conditioned seed was mechanically pressed and the press discharge cone setting was varied during operation from 1/32″ to 3/32″ to demonstrate the feasibility of both full pressing and prepressing. Prepressing successfully reduced oil level in the press cake to ca. 20%. Press cake was extracted with hexane in a 1.5-ft³ batch-type, four-stage percolation unit with a 6″ square extraction cross section. Solvent extraction reduced oil level in the defatted meal to 1–2%. The defatted meal was desolventized and toasted. Excessive foaming of the vernonia oil extract made complete solvent stripping in the oil stripping unit difficult.     

Epoxidation ofLesquerella andLimnanthes (Meadowfoam) oils

Lesquerella gordonii (Gray) Wats andLimnanthes alba Benth. (Meadowfoam) are species being studied as new and alternative crops. Triglyceride oil from lesquerella contains 55–60% of the uncommon 14-hydroxy-cis-11-eicosenoic acid. Meadowfoam oil has 95% uncommon acids, includingca. 60%cis-5-eicosenoic acid. Both oils are predominantly unsaturated (3% saturated acids), and have similar iodine values (90–91), from which oxirane values of 5.7% are possible for the fully epoxidized oils. Each oil was epoxidized withm-chloro-peroxybenzoic acid, and oxirane values were 5.0% (lesquerella) and 5.2% (meadowfoam). The epoxy acid composition of each product was examined by gas chromatography of the methyl esters, which showed that epoxidizedL. gordonii oil contained 55% 11,12-epoxy-14-hydroxyeicosanoic acid, and epoxidized meadowfoam oil contained 63% 5,6-epoxyeicosanoic acid, as expected for normal complete epoxidation. Mass spectrometry of trimethylsilyloxy derivatives of polyols, prepared from the epoxidized esters, confirmed the identity of the epoxidation products and the straightforward nature of the epoxidation process. Synthesis and characterization of these interesting epoxy oils and derivatives are discussed.     

Fatty acids and triacylglycerols of cherry seed oil

Cherry seed oil, from the Rosaceae family, prunoid subfamily, is characterized by the existence of about 10% α-eleostearic acid. The structure of the acid was proven by H and¹³C nuclear magnetic resonance. The triacylglycerols of this oil were identified and quantitated by highperformance liquid chromatography by means of several types of detectors. α-Eleostearic acid was not found in the seeds of previously studied prunoids (almond, peach, apricot and plum). The main fatty acids found in the seeds of cherry and other prunoids were linoleic (L), oleic (O) and palmitic acids, and the major triacylglycerols were LLO, LOO and OOO. These chemical data support the botanical relationship within the prunoid subfamily and show the proximity of cherry to the Chrysobalanaceae family.     

Estimating soybean seed protein and oil concentration before harvest

Temperature and precipitation during the growing season have been shown to influence the protein and oil composition of the soybean [Glycine max L. Merr.] seed. A method based on these parameters was developed to estimate protein and oil concentrations of the seed before harvest. This method was developed with protein and oil data and temperature and precipitation data from the Uniform Soybean Tests, Southern Region, for the years 1975 to 1983. Classification and regression “tree-based” analyses were used to determine the month and numeric value (“splitting point”) of the environmental variable that correctly classified the variation from median protein and oil composition for the 126 location-years. Temperature in September was most influential in determining the splitting point for three of the four variables. Oil concentrations from the location-years were separated into low vs. high median-based boundary categories most readily by the September sum of minimum temperatures. Total protein and oil concentrations from the location-years were classified best by September growing degree days. Protein-to-oil ratios were best separated by the September mean minimum temperature. The August mean maximum temperature best separated protein concentration. These data demonstrate that temperature during specific months of the crop year were useful in estimating the final concentration of protein and oil in the seed and could be used by seed processors to estimate seed composition before harvest.     

Fatty acid variation in seed oil amongOcimum species

Content, fatty acid composition, and glyceride profile of oil from seeds of seven basil (Ocimum sp.) chemotypes were determined. The species studied includedO. basilicum, O. canum, O. gratissimum, andO. sanctum. The oil content ranged from 18 to 26%, with triglycerides comprising between 94 and 98% of extracted neutral lipids. The major acylated fatty acids were linolenic (43.8–64.8%), linoleic (17.8–31.3%), oleic (8.5–13.3%), and palmitic acid (6.1–11.0%). Linolenic acid was similar among the fourO. basilicum chemotypes (57–62%), highest inO. canum (65%), and lowest inO. sanctum (44%). Basil seed oil appears suitable as an edible oil or can be used for industrial purposes, and could be processed in the same way as linseed oil. Preliminary calculations estimate that a hectare of basil could produce from 300 to 400 kg of seed oil.     

Minor components responsible for flavor reversion of soybean oil

Edible refined, bleached and deodorized (RBD) soybean oil was fractionated by silicic acid column chromatography to identify minor components responsible for flavor reversion. Minor components from oil eluted with diethyl ether/n-hexane (1:1) were compared with those from corn and canola oils. All vegetable oils contain free fatty acids, diglycerides and sterols as major ingredients in this fraction. However, unusual triglycerides consisting of 10-oxo-8-octadecenoic acid and 10-and 9-hydroxy octadecanoic acids were detected in RBD and crude soybean oils.     

Nutritional and toxicological evaluation of rubber seed oil

Rubber (Hevea brasiliensis) seed oil (RSO) is available in India (Ca. 4500 tons per year) and is used mainly as a drying oil. The oil does not contain any unusual fatty acids, and it is a rich source of essential fatty acids C_18∶2 and C_18∶3 that make up 52% of its total fatty acid composition. Acute toxic potential in rats and the systemic effects and nutritional quality were assessed in a 13 week feeding study in weanling albino rats using a diet containing RSO or groundnut oil (GNO) (as the control) at a 10% level as the sole source of dietary fat. RSO did not manifest any acute toxic potential. Food consumption, growth rate and feed efficiency ratio of rats fed RSO were similar to those fed GNO. The digestibility of this oil was found to be 97%, as compared to 94% for GNO. There were no macroscopic or microscopic lesions in any of the organs which could be ascribed to the RSO incroporation in the diet. Thus the current data show that RSO could be used for edible purposes. However, it will be necessary to process the oil to achieve deodorization and to remove free fatty acids to make it organoleptically acceptable.     

Investigation of the technological properties ofNigella sativa (black cumin) seed oil

Three commercially cultivatedNigella sativa seed varieties of Turkish origin were analyzed, and the characteristics and constituents of the seed oils were reported. Presence of lipase enzyme in seed results in enzymatic hydrolysis at ordinary temperature; the free acid content of oil may increase up to 40% or higher. Black cumin seed oil might serve as a source of semi-drying oil and fatty acids of technical grade, and the removal of free fatty acids from oil and the recovery of fatty acids were investigated.     

The triglyceride composition,structure, and presence of estolides in the oils ofLesquerella and related species

Members of the genusLesquerella, native to North America, have oils containing large amounts of hydroxy fatty acids and are under investigation as potential new crops. The triglyceride structure of oils from twenty-fiveLesquerella species in the seed collection at our research center has been examined after being hydrolysis-catalyzed by reverse micellar-encapsulated lipase and alcoholysis-catalyzed by immobilized lipase. These reactions, when coupled with supercritical-fluid chromatographic analysis, provide a powerful, labor-saving method for oil triglyceride analysis. A comprehensive analysis of overall fatty acid composition of these oils has been conducted as well.Lesquerella oils (along with oils from two other Brassicaceae:Physaria floribunda andHeliophilia amplexicaulis) have been grouped into five categories: densipolic acid-rich (Class I); auricolic acid-rich (Class II); lesquerolic acid-rich (Class III); an oil containing a mixture of hydroxy acids (Class IV); and lesquerolic and erucic acid-rich (Class V). The majority of Class I and II triglycerides contain one or two monoestolides at the 1- and 3-glycerol positions and a C₁₈ polyunsaturated acyl group at the 2-position. Most Class III and IV oil triglycerides contain one or two hydroxy acids at the 1- and 3-positions and C₁₈ unsaturated acid at the 2-position. A few of the Class III oils have trace amounts of estolides. The Class V oil triglycerides are mostly pentaacyl triglycerides and contain monestolide and small amounts of diestolide. Our triglyceride structure assignments were supported by¹H nuclear magnetic resonance data and mass balances.     

Extraction and characterization ofDimorphotheca pluvialis seed oil

Dimorphotheca pluvialis is increasingly recognized as an interesting industrial new oilseed crop because it contains up to 60% of the unusual fatty acid dimorphecolic acid (9-hydroxy,10t,12t-18∶2) (DA) for which new applications are being developed. In this paper, the yield, composition and quality are evaluated for dimorphotheca oils (DMO) which were recovered by pressing, conventional solvent extraction and supercritical carbon dioxide extraction (SCE). Mechanical pressing of the seeds required high temperatures and resulted in an oil recovery of only 40%, whereas the extraction protocols yielded more than 95%. Oil recovery by pressing of winged seed was even more difficult than that of unwinged seeds; hence, solvent extraction of winged seeds was preferred. The dark-colored DMO, recovered by expelling or by extraction with organic solvents, needed further refining to remove pigments and gums, whereas the light yellow-colored SCE DMO did not require further refining. SCE oil had a low phospholipid content (11 mg P/kg). Pressed oil (95 mg P/kg) and hexaneor pentane-extracted DMO (200 mgP/kg) had much higher phospholipid contents. Peroxide andp-anisidine values were low for freshly recovered oils, but increased after storage, especially in the SCE oil, due to the low concentration of natural antioxidants in SCE DMO, such as tocopherols. The DA content of the oils recovered by the various techniques showed only minor differences, except that supercritical carbon dioxide had slightly decreased solubilizing power for tri- and di-dimorphecolin as compared to hexane and pentane.     

Phenolic compounds and some quality parameters of pumpkin seed oil

Pumpkin seed oil has become a recognized source of phenolic compounds. The main aim of this paper was to evaluate the concentration of phenolic compounds and their extraction from pumpkin seed oil. The total phenolics content (TPC) measured in the pumpkin seed oil samples ranged from 24.71 to 50.93 mg GAE/kg of oil. The individual phenolics were tyrosol, vanillic acid, vanillin, luteolin and sinapic acid. Hexane and acetone were the best solvents for the washing step, and methanol for the elution of the phenolics in the solid‐phase extraction (diol‐SPE), whereas bleaching caused a significant increase in the TPC obtained (24.5–30.7%). Additionally, some other oil characteristics were evaluated. The mean oxidative stability of the oils (OSI) was around 4 h, with 5.43 h for the most stable oil. The maximum antioxidant capacity measured by the reduction of the DPPH radical was 62%, which was comparable to 0.16 mM Trolox equivalent. The color of the oil was expressed by L*a*b* coefficients and its hue and saturation. Whereas all samples had similar lightness, their rates of green, red, yellow and blue color were different. Moreover, TPC correlated negatively with lightness, b* and saturation (–0.49, –0.48, and –0.43), and positively with a* and hue (0.58 and 0.52).     

A method to estimate soybean seed protein and oil concentration before harvest

Temperature and precipitation variables during linear seed fill are known to be environmental determinants of protein and oil composition of the soybean [Glycine max (L.) Merr.] seed. However, the contribution of other precipitation and temperature events during the growing season and a method that would determine the precipitation and temperature variables most related to protein and oil concentration values of the seed has not been fully explored. The former was evaluated by comparing monthly temperature and precipitation variables of the growing seasons to protein and oil data for the years 1959 to 1996 from three locations listed in the Uniform Soybean Tests, Northern Region. The data set comprised locations from Maturity Groups II and III and consisted of 186 location-years. Classification and regression “tree-based” analysis were conducted to determine the month, environmental variable, and “splitting” points that correctly classified most of the 186 location-years for below-vs.-above-median protein or oil composition. The protein concentrations from the location-years were separated into these two median-boundary categories most readily by temperature variables from the months of April and August. The oil concentrations from the location-years were classified best by August and September temperature variables and precipitation in May and September. The sum of protein and oil concentrations from the location-years were best separated by August and July temperature variables and precipitation in May and July. The protein-to-oil ratios from the location-years were best separated by September precipitation and July and June temperature variables. These data demonstrate that tree-based models can use monthly temperature and precipitation variables during linear seed fill and other specific months of the crop year and relate them to the final protein and oil concentration in the seed. These results could be used by the processing industry to estimate seed composition before harvest.     

Characteristics and some potential applications of date palm (Phoenix dactylifera L.) seeds and seed oil

The seeds of four date palm (Phoenix dactylifera L.) cultivars, Dekel Noor, Zahidi, Medjool and Halawy, grown in the Arava Valley of southern Israel were analyzed for their inorganic and some organic constituents. The following average values were obtained for the four cultivars on a dry-weight basis: protein 5.60%, oil 8.15%, fiber 16.13%, and ash 1.13%. Analysis of the mineral elements in the ash gave the following average values: Ca, 1.55%; Na, 0.97%; Mg, 8.07%; K, 27.60%; Fe, 0.73%; Cu, 0.13% and Mn, 0.08%. The oil exhibited the following characteristics (average for the four cultivars): acid value ∼1.04, iodine value 49.5, saponification value 221.0, and unsaponifiable matter 0.8%. Gas-liquid chromatography revealed that the major unsaturated fatty acid was oleic acid (42.3%), while the main saturated fatty acid was lauric (21.8%). Myristic, palmitic and linoleic acids were also found, average values being 10.9, 9.6 and 13.7%, respectively. Potential uses for date seed oil are considered for cosmetic, pharmaceutical and related specialty products, and to a lesser degree for food products.     

Cultural practices for lesquerella production

WhileLesquerella fendleri has good potential of becoming a domestic, water-efficient source of hydroxy fatty acids in the United States, little research has been done outside of Arizona to determine its environmental response and cultural requirements. This study examined the effects of planting date, irrigation rate and fertilizer on growth and yield in Oregon, and it also evaluated tolerance to existing herbicides as a first step in developing cost-effective weed control programs. An October planting failed, while a March planting generally out-yielded a May planting. Seed yield was maximized at irrigation plus precipitation in the range of 959 to 1111 mm. Fertilizer response was indefinite, although response to phosphorus sometimes occurred. Herbicides with excellent potential include trifluralin and benefin pre-plant incorporated, and fluazifop post-emergent. Potential herbicides also include pendimethalin preemergent, and oxyfluorfen, dicamba and clopyralid postemergent. In addition to further studies on herbicide, irrigation, fertilizer and planting date effects, studies to optimize other cultural practices (e.g., row width, plant density, seed source, varietal development) that increase oil yield and quality while reducing input cost are required if lesquerella production is to occur in Oregon.