Removal and Degradation of Phorbol Esters during Pre-treatment and Transesterification of <Emphasis Type="Italic">Jatropha curcas</Emphasis> Oil

Phorbol esters present in Jatropha curcas oil are toxic when consumed and are co-carcinogens. These could be a potential constraint in the widespread acceptance of Jatropha oil as a source of biodiesel. Phorbol esters were quantified in the fractions obtained at different stages of oil pre-treatment and biodiesel production. During degumming some phorbol esters were removed in the acid gums and wash water. This implies that the use of these acid gums in animal feed is not possible and care should be taken when disposing the wash water into the environment. Silica treatment did not decrease the phorbol esters, while stripping/deodorization at 260 °C at 3 mbar pressure with 1% steam injection completely degraded phorbol esters. Phorbol esters were not detected in stripped oil, fatty acid distillate, transesterified oil (biodiesel) and glycerine. The presence of possibly toxic phorbol ester degradation products in these fractions could not be ruled out.     

Quantification of Phorbol Esters in Jatropha curcas by HPLC‐UV and HPLC‐ToF‐MS with Standard Addition Method

Established analytic methods for the quantification of phorbol esters (PE), which are some toxic components in Jatropha curcas L., include HPLC with UV‐detection with the commercially available phorbol myristate acetate (PMA) as internal standard or HPLC coupled with MS detection with an external calibration, mostly also with PMA. The differences in the fatty acid side chains and connection to the base structure of PMA compared to PE leads to different UV absorption and MS ionization effects and cause problems for exact quantitative measurements. In this paper, a method is presented which combines both detection types and shows differences between both results. For this purpose, an extraction routine is performed on a PE‐containing seed oil to get a PE standard in high purity, which was used for a standard addition method on two real J. curcas oil samples, derived from Ghana and Mexico. Furthermore, a detection window of ±10 ppm for the high accurate ToF‐MS detection is set to eliminate isobaric interferences from co‐eluting material. Method evaluation of inter‐ and intra‐day variance as well as the recovery rate are performed and determined. With this method a limit of detection of 62 ng mL^?1 (UV) and 11 ng mL^?1 (MS) can be achieved. Practical Applications: Due to the good biological and technical properties of Jatropha curcas L., its seed oil seems perfect for the application as biodiesel feedstock. The toxicity on the other hand could cause problems when converting side products from the oil production to products of higher value. With the here described method an accurate and precise analysis procedure for the quantification of the toxic compounds namely, phorbol esters, could be applied for toxicity studies or routine checks in industry which is converting plant material from J. curcas, so that no toxic material is used for example as animal feed. In this paper, an exact and robust analysis method is described for the quantification of phorbol esters (PE) in Jatropha curcas L. seed oil. This method procedure includes the extraction of PE in methanol, chromatographic separation on a reverse phase C18 HPLC column and the quantification by standard addition method. For the standard addition method a highly pure PE standard is used, which is extracted and purified by semi preparative HPLC right before the measurements. The used detector for identification and quantification is UV set at 280 nm and ESI‐ToF‐MS with a ±10 ppm mass difference of the deprotonated and formate adduct pseudo molecular ion of PE.

     

Fatty acids of Thespesia populnea: Mass spectrometry of picolinyl esters of cyclopropene fatty acids

Thespesia populnea belongs to the plant family of Malvaceae which contain cyclopropane and cyclopropene fatty acids. However, previous literature reports vary regarding the content of these compounds in Thespesia populnea seed oil. In this work, the content of malvalic acid (8,9‐methylene‐9‐heptadecenoic acid) in the fatty acid profile of Thespesia populnea seed oil was approximately 7% by GC. Two cyclopropane fatty acids were identified, including dihydrosterculic acid. The methyl and picolinyl esters of Thespesia populnea seed oil were also prepared. The mass spectrum of picolinyl malvalate was more closely investigated, especially an ion at m/e 279, which does not fit the typical series of ions observed in picolinyl esters. It is shown that this ion is caused by cleavage at the picolinyl moiety and contains the fatty acid chain without the picolinyl moiety. This type of cleavage has previously not been observed prominently in picolinyl esters and may therefore be diagnostic for picolinyl esters of cyclopropene fatty acids. The NMR spectra of Thespesia populnea methyl esters are also discussed. Practical applications: The work reports the fatty acid composition of Thespesia seed oil whose derivatives have not yet been extensively utilized for industrial purposes, for example, biodiesel. Knowing this composition is essential for understanding potential uses and, for example, in case of biodiesel the fuel properties. Besides this issue, some data (mass spectrometry and NMR) crucial for obtaining the composition information are analyzed in detail. The biodiesel properties of methyl esters of Thespesia populnea will be reported separately.     

Methyl Esters (Biodiesel) from and Fatty Acid Profile of Gliricidia sepium Seed Oil

Increasing the supply of biodiesel by defining and developing additional feedstocks is important to overcome the still limited amounts available of this alternative fuel. In this connection, the methyl esters of the seed oil of Gliricidia sepium were synthesized and the significant fuel‐related properties were determined. The fatty acid profile was also determined with saturated fatty acids comprising slightly more than 35 %, 16.5 % palmitic, 14.5 % stearic, as well as lesser amounts of even longer‐chain fatty acids. Linoleic acid is the most prominent acid at about 49 %. Corresponding to the high content of saturated fatty acid methyl esters, cold flow is the most problematic property as shown by a high cloud point of slightly >20 °C. Otherwise, the properties of G. sepium methyl esters are acceptable for biodiesel use when comparing them to specifications in biodiesel standards but the problematic cold flow properties would need to be observed. The ¹H‐ and ¹³C‐NMR spectra of G. sepium methyl esters are reported.     

Use of the Product from Low Pressure Extraction (Crambe Seed Oil and Methyl Acetate) for Synthesis of Methyl Esters and Triacetin Under Supercritical Conditions

Methyl esters (ME) and triacetin production from the supercritical interesterification of the product from low pressure extraction (crambe seed oil and methyl acetate) are evaluated. Reactions are conducted at 300–375 °C for different residence times, at 20 MPa, and under these conditions the thermal stability of triacetin is evaluated. The effect of the free fatty acid (FFA) concentration (in oil) is determined. An increase in temperature favors the formation of ME and triacetin at shorter reaction times. At 375 °C, after 15 min a drop in the ME yield is observed and triacetin is not detected. A reduction in the triacetin concentration (reaching ≈99%) is observed at 375 °C. High FFA concentration (in oil) initially provided higher product generation, however, after 15 min no influence is observed. The highest ME yield (≈60%) is obtained at 300 °C, along with 1.22 wt% triacetin and ≈5.0 wt% unreacted compounds. Practical Applications: This paper reports new experimental data on an integrated process for the production of methyl esters from low pressure extraction (crambe seeds and methyl acetate) and supercritical reaction of the extraction mixture. The technique used allows the removal of a high quantity of oil from good quality crambe seeds. The viability of applying the integrated process to obtain oils with a high content of free fatty acids is verified, promoting the obtainment of relatively simple methyl esters. The procedure does not require oil purification and solvent recovery prior to the reaction.     

Reactive extraction and in situ esterification of Jatropha curcas L. seeds for the production of biodiesel

Jatropha curcas L. has recently been hailed as the promising feedstock for biodiesel production as it does not compete with food sources. Conventional production of biodiesel from J. curcas L. seeds involve two main processing steps; extraction of oil and subsequent esterification/transesterification to fatty acid methyl esters (FAME). In this study, the feasibility of in situ extraction, esterification and transesterification of J. curcas L. seeds to biodiesel was investigated. It was found that the size of the seed and reaction period effect the yield of FAME and amount of oil extracted significantly. Using seed with size less than 0.355 mm and n-hexane as co-solvent with the following reaction conditions; reaction temperature of 60 °C, reaction period of 24 h, methanol to seed ratio of 7.5 ml/g and 15 wt% of H₂SO₄, the oil extraction efficiency and FAME yield can reached 91.2% and 99.8%, respectively. This single step of reactive extraction process therefore can be a potential route for biodiesel production that reduces processing steps and cost.     

Investigating Some Physicochemical Properties and Fatty Acid Composition of Native Black Mulberry (<Emphasis Type="Italic">Morus nigra</Emphasis> L.) Seed Oil

The physicochemical properties of seed and seed oil obtained from the native black mulberry (Morus nigra L.) were investigated in 2008 and 2009. The results showed that the seed consisted of 27.5–33% crude oil, 20.2–22.5% crude protein, 3.5–6% ash, 42.4–46.6% carbohydrate and 112.2–152.0 mg total phenolics/100 g. Twenty different fatty acids were determined, with the percentages varying from 0.02% myristic acid (C14:0) to 78.7% linoleic acid (C18:2). According to the GC analysis of fatty acid methyl esters, linoleic acid (C18:2), followed by palmitic acid (C16:0), oleic acid (C18:1) and stearic acid (C18:0) were the major fatty acids, which together comprised approximately 97% of the total identified fatty acids. High C18:2 content (average 73.7%) proved that the black mulberry seed oil is a good source of the essential fatty acid, linoleic acid. Linolenic acid (C18:3) was also found in a relatively lower amount (0.3–0.5%). The α-tocopherol content was found to be between 0.17 and 0.20 mg in 100 g seed oil. The main sterols in the mulberry seed oil were β-sitosterol, Δ5-avenasterol, Δ5, 23-stigmastadienol, clerosterol, sitosterol and Δ5, 24-stigmastadienol. The present study stated that the native black mulberry seed oil can be used as a nutritional dietary substance and has great usage potential.     

Potential of <Emphasis Type="Italic">Jatropha curcas</Emphasis> as a Biofuel,Animal Feed and Health Products

Jatropha curcas is a multipurpose plant with numerous attributes. It can potentially become one of the world’s key energy crops. Its seed weighs 0.53–0.86 g and the seed kernel contains 22–27% protein and 57–63% lipid indicating good nutritional value. The seeds can produce crude vegetable oil that can be transformed into high quality biodiesel. Several methods for oil extraction have been developed. In all processes, about 75% of the weight of the seed remains as a press cake containing mainly carbohydrates, protein and residual oil and is a potential source of livestock feed. The highly toxic nature of whole as well as dehulled seed meal due to the presence of high levels of shells, toxic phorbol esters and other antinutrients prevents its use in animal diet. The genetic variation among accessions from different regions of the world and rich diversity among Mexican genotypes in terms of phorbol ester content and distinct molecular profiles indicates the potential for improvement of germplasm of Jatropha through breeding programs. The extracts of Jatropha display potent cytotoxic, antitumor, anti-inflammatory and antimicrobial activities. The possibilities on the exploitation potential of this plant through various applications have been explored.     

Fatty Acids of <Emphasis Type="Italic">Chthonomonas calidirosea</Emphasis>, of a Novel Class Chthonomonadetes from a Recently Described Phylum Armatimonadetes

A Gram-negative, aerobic, pink-pigmented, rod-shaped bacterium Chthonomonas calidirosea (strain T49^T) with an optimal temperature for growth of 68 °C, isolated from soil samples from Hell’s Gate in the Tikitere geothermal system (New Zealand), was the first cultivated bacterium of the novel phylum Armatimonadetes (formerly candidate division OP10). The lipid composition of C. calidirosea presents a number of unusual features both in the fatty acids and polar lipids. This contribution reports on the fatty acid profile of C. calidirosea. Transmethylation of bacterial biomass yielded fatty acid methyl esters and hydrocarbons, including squalene, partially hydrogenated squalenes, and diploptene. The only type of unsaturation found in C. calidirosea fatty acids was cis-Δ5, as revealed by GCMS of dimethyl disulfide (DMDS) adducts, and the lack of trans-unsaturation absorbance at 960–980 cm⁻¹ in the IR spectrum of fatty acids methyl esters. An unidentified component X with ECL 16.86 (BP1) and ECL 17.27 (BP20) was also observed, with molecular ion at m/z 282 (“17:1”). X did not form DMDS adducts, nor was affected by mild hydrogenation conditions, indicating the likely presence of a ring rather than unsaturation. The presence of a cyclopropane ring with cis-stereochemistry was confirmed by the ¹H-NMR spectrum. Hydrogenation of X in acetic acid resulted in formation of straight chain 17:0, 5-methyl- and 6-methyl-16:0 fatty acid methyl esters, thus confirming the structure of a novel 5,6-methylene hexadecanoic acid. The major fatty acids of a solid media-grown C. calidirosea were as follows (in weight % of total fatty acids): 16:0 (25.8), i17:0 (19.3), ai17:0 (13.5), 16:1∆5 (8.8), i17:1∆5 (6.8), 5,6-methylene 16:0 (5.2), i16:0 (4.4), 18:0 (3.6), 18:1∆5 (3.2).     

Preparative Separation of <Emphasis Type="Italic">cis</Emphasis>- and <Emphasis Type="Italic">trans</Emphasis>-Isomers of Unsaturated Fatty Acid Methyl Esters Contained in Edible Oils by Reversed-Phase High-Performance Liquid Chromatography

In order to measure exactly the trans-fatty acids content in food materials, a preparative group separation of cis- and trans-isomers of unsaturated fatty acid methyl esters (FAMEs) was achieved by an isocratic reversed-phase HPLC (RP-HPLC) method. The trans-isomers of 16:1, 18:1, 18:2, 18:3, 20:1 and 22:1 FAMEs were readily separated from the corresponding cis-isomers by a COSMOSIL Cholester C18 column (4.6 mm I.D. × 250 mm, Nacalai Tesque) or a TSKgel ODS-100Z column (4.6 mm I.D. × 250 mm, TOSOH), using acetonitrile as the mobile phase. This method was applied for determining the trans-18:1 fatty acid content in partially hydrogenated rapeseed oil. The methyl esters of cis- and trans-18:1 isomers of the oil were collected as two separate fractions by the developed RP-HPLC method. Each fraction was analyzed by gas chromatography (GC) for both qualitative and quantitative information on its positional isomers. By a combination of RP-HPLC and GC methods, a nearly complete separation of cis- and trans-18:1 positional isomers was achieved and the trans-18:1 fatty acid content was able to be evaluated more precisely than is possible by the direct GC method. The reproducibility of cis- and trans-18:1 isomers fractionated by the RP-HPLC method was better than 98%. These results suggested that the preparative RP-HPLC method developed in this study could be a powerful tool for trans-fatty acid analysis in edible oils and food products as an alternative to silver-ion chromatography.     

Analysis of hydroxylated fatty acids from plant oils

A novel process has been described recently for the preparation of hydroxylated fatty acids (HOFA) and HOFA methyl esters from plant oils. HOFA methyl esters prepared from conventional and alternative plant oils were characterized by various chromatographic methods (thin-layer chromatography, high-performance liquid chromatography, and gas chromatography) and gas chromatography-mass spectrometry as well as¹H and¹³C nuclear magnetic resonance spectroscopy. HOFA methyl esters obtained fromEuphorbia lathyris seed oil, low-erucic acid rapeseed oil, and sunflower oil contain as major constituents methylthreo-9,10-dihydroxy octadecanoate (derived from oleic acid) and methyl dihydroxy tetrahydrofuran octadecanoates, e.g., methyl 9,12-dihydroxy-10,13-epoxy octadecanoates and methyl 10,13-dihydroxy-9,12-epoxy octadecanoates (derived from linoleic acid). Other constituents detected in the products include methyl esters of saturated fatty acids (not epoxidized/derivatized) and traces of methyl esters of epoxy fatty acids (not hydrolyzed). The products that contain high levels of monomeric HOFA may find wide application in a variety of technical products.     

Chemical Composition and Characteristics of <Emphasis Type="Italic">Commiphora wightii</Emphasis> (Arnott) Bhandari Seed Oil

The seeds of Commiphora wightii (Arnott) Bhandari contain 9.8 ± 0.7% oil. The fatty acid composition and chemical properties of the extracted oil were determined. Gas liquid chromatography of the methyl esters of the fatty acids shows the presence of 46.62% saturated fatty acids and 51.40% unsaturated fatty acids. The fatty acid composition is as follows: capric acid 3.50%, myristic acid 14.51%, palmitic acid 6.68%, stearic acid 4.70%, arachidic acid 3.18%, behenic acid 14.05%, myristoleic acid 1.34%, palmitoleic acid 12.07%, oleic acid 14.15%, eicosenoic acid 0.11%, linoleic acid 22.34% and alpha linoleic acid 1.37%.     

Analyses of lipids and oxidation products by partition chromatography: Hydroxy fatty acids and esters

A liquid-partition chromatographic procedure was used to separate hydroxy fatty acids, their methyl esters, and reduced fatty ester hydroperoxides. Mixtures of methyl stearate, mono- and dihydroxystearate, and mixtures of the corresponding free fatty acids were easily separated. Chromatographic determinations for ricinoleate in castor oils compared favorably with the chemical and infrared analyses. The chromatographic procedure was used to separate hydroxy fatty acids inDimorphotheca andStrophanthus seed oils. The methyl ester of dimorphecolic acid, the principal hydroxy fatty ester ofDimorphotheca oil, behaved like reduced methyl linoleate hydroperoxide and showed a polarity intermediate between methyl 12-hydroxystearate and methyl 9,10-dihydroxystearate. The 9-hydroxy-12-octadecenoic ester ofStrophanthus oil had a larger retention volume than methyl ous hydroxy fatty esters isolated chromatographically. The diene content of the reduced hydroperoxides agrees well with values reported in the literature (1,5,16). The diene content of the chromatographed methyl dimorphecolate is higher than reported by Smithet al. (20) for their preparations but agrees well with the value reported by Chipault and Hawkins (6) for puretrans-trans conjugated methyl linoleate. The extinction coefficient of methyl 12-hydroxystearate at 2.8 μ is higher than that reported for ricinoleate and the absorption band is much sharper. Because of these two conditions no association of the hydroxyl groups is indicated. These results also confirm the purity of the hydroxy fatty esters obtained by LPC. This method has been a valuable adjunct to the study of various oxygen-containing fatty acid and esters and was used to characterize the hydroxy esters obtained from the hydrogenation of methyl linolenate hydroperoxides (9). This work offers a basis for the development of analytical methods to determine the hydroxy and other polar acid content of fatty glycerides and their derivatives.     

Quality of Biodiesel Prepared from Phorbol Ester Extracted <Emphasis Type="Italic">Jatropha curcas</Emphasis> Oil

Jatropha curcas seeds are rich in oil (28–32%), which can be converted to high quality biodiesel. The oil is non-edible due to the presence of toxic compounds, namely, phorbol esters (PEs). PEs have a number of agricultural/medicinal/pharmaceutical applications and hence their recovery generates a value added co-product towards the biodiesel production chain. This study aims to assess the effects of PE extraction on quality of both the residual oil and the biodiesel production from it. Two Approaches (1, use of an Ultra-turrax; and 2, use of a magnetic stirrer) were used with an effective treatment time of 2 and 5 min, resulting in 80 and 78% extraction of PEs, respectively. The phosphorus content was reduced by 70.2 and 75.8%, free fatty acids by 55.3 and 55.6%, and the fatty acid composition did not change in the residual oils. The peroxide value increased from 2.69 (untreated oil) to 3.01 and 3.49 mequiv O₂/kg in the residual oils following Approach 1 and Approach 2, respectively. The biodiesel prepared from both residual oils met European (EN 14214:2008) and American biodiesel standard (ASTM D6751-09) specifications. Oxidative stability indices for both the biodiesels were well within the permitted limit. It is concluded that PEs could be isolated in active forms for various applications by either of the two methods with a high yield and the residual oil can be processed to produce high quality biodiesel.     

Fatty acids,fatty alcohols,wax esters,and methyl esters fromCrambe abyssinica andLunaria annua seed oils

Crambe abyssinica andLunaria annua, members of the Cruciferae family, have seed oil glycerides containing ca. 55–65% of C₂₂ and C₂₄ unsaturated fatty acids. Fatty acids were prepared by saponification; fatty alcohols, by sodium reduction of glycerides; liquid wax esters, byp-toluenesulfonic acid-catalyzed reaction of fatty acids with fatty alcohols; and methyl esters, by reaction of fatty acids with diazomethane. Solid hydrogenated glyceride oils and wax esters were compared with several commercial waxes. Chemical and physical constants were determined for the seed oils and their derivatives. Position of unsaturation in theCrambe fatty acids was determined by gas chromatographic analysis of the permanganate-periodate degradation products. The major dicarboxylic acid was brassylic (C₁₃), proving the docosenoic acid to be erucic. Presented in part at the AOCS meeting in New Orleans, La., 1962. A laboratory of the No. Utiliz. Res. & Dev. Div., ARS, U.S.D.A.     

Toxic Compound,Anti-Nutritional Factors and Functional Properties of Protein Isolated from Detoxified Jatropha curcas Seed Cake

Jatropha curcas is a multipurpose tree, which has potential as an alternative source for biodiesel. All of its parts can also be used for human food, animal feed, fertilizer, fuel and traditional medicine. J. curcas seed cake is a low-value by-product obtained from biodiesel production. The seed cake, however, has a high amount of protein, with the presence of a main toxic compound: phorbol esters as well as anti-nutritional factors: trypsin inhibitors, phytic acid, lectin and saponin. The objective of this work was to detoxify J. curcas seed cake and study the toxin, anti-nutritional factors and also functional properties of the protein isolated from the detoxified seed cake. The yield of protein isolate was approximately 70.9%. The protein isolate was obtained without a detectable level of phorbol esters. The solubility of the protein isolate was maximal at pH 12.0 and minimal at pH 4.0. The water and oil binding capacities of the protein isolate were 1.76 g water/g protein and 1.07 mL oil/g protein, respectively. The foam capacity and stability, including emulsion activity and stability of protein isolate, had higher values in a range of basic pHs, while foam and emulsion stabilities decreased with increasing time. The results suggest that the detoxified J. curcas seed cake has potential to be exploited as a novel source of functional protein for food applications.     

The seed oil ofBernardia pulchella (Euphorbiaceae) —A rich source of vernolic acid

The fatty acids from the seed oil ofBernardia pulchella (Euphorbiaceae) have been analyzed by gas chromatography (GC) and GC-mass spectrometry (MS) analysis of their methyl esters. Vernolic acid is the main compound (91%), along with other usual fatty acids. In addition to the quantitation by GC analysis,¹H-nuclear magnetic resonance (NMR) signals from the seed oil have been used to estimate the total epoxy fatty acid content. The structure of vernolic acid has been proven by spectroscopic methods (infrared,¹H, and¹³C-NMR) and by GC-MS analysis of the corresponding silylated hydroxy-methoxy derivative. The 4,4-dimethyloxazoline derivatives of the fatty acid mixture have also been examined by GC-MS, and it was shown that this derivazation reaction is not suitable for the structure analysis of vernolic acid.     

Curupira tefeensis II: Occurrence of Acetylenic Fatty Acids

Four acetylenic fatty acids [11-octadecen-9-ynoic acid (E) (ximenynic acid), 11-octadecen-9-ynoic acid (Z), 9,11-octadecadiynoic acid and 13-octadecen-9,11-diynoic acid (E) (exocarpic acid)] were found in the seed oil of Curupira tefeensis (Olacaceae) as minor compounds. The mass spectra of the methyl esters (EI and CI mode), picolinyl esters and 4,4-dimethyloxazoline derivatives of these acids are discussed in detail. Furthermore the NMR data of 9,11-octadecadiynoic acid and 13-octadecen-9,11-diynoic acid are presented.     

GC-MS characterization (Chemical lonization and electron impact modes) of the methyl esters and oxazoline derivatives of cyclopropenoid fatty acids 1

The CI-(CH₄) mass spectra of the methyl esters and the EI mass spectra of the oxazoline derivatives of three cyclopropenoid fatty acids (malvalic, sterculic and α- hydroxy-sterculic acid) from the seed oil ofPachira aquatica were found to be useful for structure elucidation of such compounds. Furthermore, some hitherto unknown minor fatty acids were identified and the nuclear magnetic resonance data of the oil and α-hydroxysterculic acid methyl ester are presented.     

Curupira tefeensis (Olacaceae) — A Rich Source of Very Long Chain Fatty Acids

The fatty acid composition of the seed oil of Curupira tefeensis was analysed by capillary GC of their methyl esters. The gaschromatographic assignments were ensured by corresponding mass spectra. The oil is composed to more than 62% of very long chain fatty acids (>C18). Erucic acid is found to be the main component (35%). The position of the double bonds of the monounsaturated fatty acids (MUFAME) was verified after derivatization with dimethyl disulfide and subsequent GC/MS analysis. All identified MUFAME belong to the (n-9-)type. The UV-spectroscopical data show that approx. 1.2% conjugated acetylenic fatty acids occur in the oil. Furthermore IR- and NMR-spectroscopical investigations and the basic analyses of the seed were carried out.