Tocochromanol content and composition in Jatropha curcas seeds

Physic nut (Jatropha curcas L.) is a promising seed oil source for biodiesel production. Natural antioxidants play a major role in maintaining oxidative stability of oils and they also have important food and industrial applications. Among them, tocochromanols are the most abundant in seeds. The objective of this research was to evaluate the variation for tocochromanol content and profile in a germplasm collection of 52 accessions of J. curcas. Seeds collected in two different periods, August and November of 2009, were analysed for tocochromanol content. Additionally, the dynamics of tocochromanol accumulation in developing seeds was studied. Total seed tocochromanol content averaged 307.2 mg kg⁻¹ in August and 303.7 mg kg⁻¹ in November, whereas total oil tocochromanol content averaged 507.4 mg kg⁻¹ in August and 500.8 mg kg⁻¹ in November. The tocochromanol fraction was made up of 15.4% gamma-tocopherol, 83.8% gamma-tocotrienol, and 0.8% delta-tocotrienol in August and 18.0% gamma-tocopherol, 80.4% gamma-tocotrienol, and 1.6% delta-tocotrienol in November. Genotype × environment effects were identified for tocochromanol content but not for the proportion of major tocochromanol homologues, which showed a high positive correlation between both environments. Developing seeds contained primarily alpha-tocopherol and gamma-tocopherol at early stages of development, with gamma-tocotrienol and delta-tocotrienol being practically undetectable. Gamma-tocotrienol content remained practically undetectable till 66 DAP and then increased pronouncedly to final levels of 177.1 mg kg⁻¹ (74.8% of the total tocochromanol content). The powerful antioxidant and health-promoting properties of gamma-tocotrienol encourages further studies on selection for the tocopherol/tocotrienol ratio in Jatropha and on the potential of tocochromanols as high added-value products derived from Jatropha seed oil production.     

In vitro regeneration from petiole explants of non-toxic Jatropha curcas

Jatropha curcas, a multipurpose shrub has acquired significant economic potential as biodiesel plant. The seeds or pressed cake is toxic due to the presence of toxic substances and is not useful as food/fodder despite having the best protein composition. A simple, efficient, and reproducible method for plant regeneration through direct organogenesis from petiole explants of non-toxic J. curcas was developed using Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (57.61%), and number of shoot buds (4.98) per explant were obtained when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 μM TDZ. The Induced shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM 6-benzyl aminopurine (BA), and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation and subsequent elongation was achieved on MS medium supplemented with 2.25 μM BA and 8.5 μM IAA. The elongated shoots could be rooted on half-strength MS medium with 15 μM IBA, 11.4 μM IAA and 5.5 μM NAA with more than 90% survival rate.     

Value added waste of Jatropha curcas residue: Optimization of protease production in solid state fermentation by Taguchi DOE methodology

The oil extraction of Jatropha curcas created the large amount of the by-product from its seeds. An application of solid-state fermentation (SSF) was considered to be of value to these raw materials. This study investigated the potential of a utilization of deoiled J. curcas seed cake as substrate for protease productions by Aspergillus oryzae. While various parameters for SSF was conventionally individually optimized, five parameters were simultaneously examined based on Taguchi method. The effect of three different levels of five factors, including moisture content of substrate, inoculums size, incubation temperature, type of porous substrate and incubation time were examined. The optimum conditions for the protease production by A. oryzae obtained from this experiment were 45% moisture content of substrate, 10% inoculums size, 30 °C incubation temperature, deoiled J. curcas seed cake mixed with cassava bagasse ratio 4:1 as porous substrate at 84 h of incubation time. By adjusting the conditions to these optimum levels, the protease production increased up to 4.6 times as many as the protease yield from the non-optimizing experiment. The use of statistical approach, Taguchi method, provided a satisfactory outcome in defining the optimum conditions for protease production by A. oryzae. Further, the utilization of deoiled J. curcas seed cake as substrate for SSF was proven as the suitable practice for this agricultural waste, in order to develop for an industrial use.     

Methyl ester of Sal oil (Shorea robusta) as a substitute to diesel fuel—A study on its preparation, performance and emissions in direct injection diesel engine

Many research reported vegetable oil as a potential substitute for diesel engines with its ester form known as biodiesel. The biodiesel can be prepared by different process using vegetable oil and alcohol. The common process used for biodiesel preparation is known as transesterification. This paper presents the transesterification of Sal oil (Shorea robusta) into Sal oil methyl ester (SOME) and its performance in direct injection diesel engine. Several process parameters such as catalyst quantity, molar ratio of alcohol, reaction temperature and reaction time were studied and the optimized process conditions are amount of catalyst (NaOH) - 0.25 wt%, alcohol (methanol) - 150% excess, reaction temperature - 65 °C and reaction time - 1.5 h. The studies with SOME as fuel in the direct injection diesel engine shows that the exhaust emissions such as CO, HC and NO_x are reduced by 25%, 45% and 12%, respectively compared to diesel without significant difference in thermal efficiency. Based on this study it is concluded that the SOME can be used as fuel without any modifications in the engine and hence this biodiesel can be a potential substitute to standard diesel fuel.     

Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes

An efficient and reproducible protocol was established for genetic transformation in Jatropha curcas through microprojectile bombardment. Decotyledonated embryos from mature seeds were pre-cultured for 5 days and elongated embryonic axis was subjected to bombardment for the optimization of physical parameters. The frequency of transient gus expression and survival of putative transformants were taken into consideration for the assessment of physical parameters. Statistical analysis reveal that microcarrier size, helium pressure and target distance had significant influence on transformation efficiency. Among different variables evaluated, microcarrier size 1 μm, He pressure 1100 and 1350 psi with a target distance of 9 and 12 cm respectively were found optimum by co-relating microcarrier size, helium pressure and target distance on the frequency of gus expression and survival of putative transformants. Selection of putative transformants was done with increasing concentrations (5-7 mg L⁻¹) of hygromycin. The integration of desired gene into Jatropha genome was confirmed with PCR amplification of 0.96 and 1.28 kb bands of hptII and gus gene respectively from the T₀ transgenics and Southern blot analysis using PCR amplified DIG labeled hptII gene as a probe. A successful attempt of genetic transformation was made with optimized conditions using particle gene gun and establishing a stable transformation in J. curcas with 44.7% transformation efficiency. The procedure described will be very useful for the introgression of desired genes into J. curcas and the molecular analysis of gene function.     

Predicting Jatropha curcas seed-oil content, oil composition and protein content using near-infrared spectroscopy—A quick and non-destructive method

The potential of near-infrared reflectance spectroscopy (NIRS) to estimate the oil content, fatty acid composition, and protein content of Jatropha curcas seeds was studied. Seventy-four intact kernels from various sources were scanned by NIRS. All samples were analyzed for oil content (hexane extractions), fatty acid composition (gas chromatography), and protein content (Kjeldahl). Calibration equations were developed for oil content, individual fatty acids (oleic C18:1, linoleic C18:2, stearic C18:0 and palmitic C16:0), and protein content. The performance of the calibration equations was evaluated through external and cross-validation. The results showed that NIRS was a reliable, accurate and nondestructive technique to estimate oil and protein contents, as well as oleic and linoleic fatty acid concentrations in J. curcas kernels; NIRS provides a rapid, simple, and cost-effective alternative method for screening intact J. curcas kernels.     

Regeneration in Jatropha curcas: Factors affecting the efficiency of in vitro regeneration

Factors influencing in vitro regeneration through direct shoot bud induction from hypocotyl explants of Jatropha curcas were studied in the present investigation. Regeneration in J. curcas was found to be genotype dependent and out of four toxic and one non-toxic genotype studied, non-toxic was least responsive. The best results irrespective of genotype were obtained on the medium containing 0.5 mg L⁻¹ TDZ (Thidiazuron) and in vitro hypocotyl explants were observed to have higher regeneration efficiency as compared to ex vitro explant in both toxic and non-toxic genotypes. Adventitious shoot buds could be induced from the distal end of explants in all the genotypes. The number of shoot buds formed and not the number of explants responding to TDZ treatment were significantly affected by the position of the explant on the seedling axis. Explants from younger seedlings (≤15 days) were still juvenile and formed callus easily, whereas the regeneration response declined with increase in age of seedlings after 30 days. Transient reduction of Ca²⁺ concentrations to 0.22 g L⁻¹ in the germination medium increased the number of responding explants.Induced shoot buds, upon transfer to MS medium containing 2 mg L⁻¹ Kn (Kinetin) and 1 mg L⁻¹ BAP (6-benzylamino purine) elongated. These elongated shoots were further proliferated on MS medium supplemented with 1.5 mg L⁻¹ IAA (indole-3-acetic acid) and 0.5 mg L⁻¹ BAP and 3.01-3.91 cm elongation was achieved after 6 weeks. No genotype specific variance in shoot elongation was observed among the toxic genotypes except the CSMCRI-JC2, which showed reduced response. And for proliferation among the toxic genotypes, CSMCRI-JC4 showed highest number of shoots formed. Among the rest, no significant differences were observed. The elongated shoot could be rooted by pulse treatment on half-strength MS medium supplemented with 2% sucrose, 3 mg L⁻¹ IBA (indole-3-butyric acid), 1 mg L⁻¹ IAA, 1 mg L⁻¹ NAA (α-naphthalene acetic acid) and subsequent transfer on 0.25 mg L⁻¹ activated charcoal medium. The rooted plants could be established in soil with more than 90% success. No significant differences were observed in rooting of shoots in the different toxic genotypes. However, rooting response was reduced in non-toxic genotype as compared to toxic genotypes.     

Jatropha curcas seed cake as substrate for production of xylanase and cellulase by Aspergillus niger FGSCA733 in solid-state fermentation

Jatropha curcas seed-cake was evaluated for use as a solid state fermentation substrate for production of cellulolytic and xylanolytic enzymes by Aspergillus niger. Supplementation of the seedcake with 10% thatch grass (Hyperrhaenia sp.) resulted in a fivefold increase in xylanase production. Ammonium chloride supplementation increased production of xylanase by 13%. Under the same conditions, cellulase production was not influenced by supplementation with grass or the nitrogen sources used. Maximum xylanase was produced at 25 °C whilst cellulase was maximally produced at 40 °C. Highest xylanase activity was obtained when the cultures had an initial pH of 3 whereas cellulase was maximally produced at an initial pH of 5. Under optimised conditions, 6087 U and 3974 U of xylanase and cellulase respectively were obtained per gram of substrate. Zymograms of crude enzyme extracts showed six active bands ranging from 20 kDa to 43 kDa for cellulase and a 31 kDa active band for xylanase.     

Preparation of biodiesel from Idesia polycarpa var. vestita fruit oil

The feasibility of producing biodiesel from Idesia polycarpa var. vestita fruit oil was studied. A methyl ester biodiesel was prepared from refined I. polycarpa fruit oil using methanol and potassium hydroxide (KOH) in an alkali-catalyzed transesterification process. The experimental variables investigated in this study were catalyst concentration (0.5–2.0 wt.% of oil), methanol/oil molar ratio (4.5:1 to 6.5:1), temperature (20–60 °C) and reaction time (20–60 min). A maximum yield of over 99% of methyl esters in I. polycarpa fruit oil biodiesel was achieved using a 6:1 molar ratio of methanol to oil, 1.0% KOH (% oil) and reaction time for 40 min at 30 °C. The properties of I. polycarpa fruit oil methyl esters produced under optimum conditions were also analyzed for specifications for biodiesel as fuel in diesel engines according to China Biofuel Systems Standards. The fuel properties of the I. polycarpa fruit oil biodiesel obtained are similar to the No. 0 light diesel fuel and most of the parameters comply with the limits established by specifications for biodiesel.     

Conversion of starch from dry common beans (Phaseolus vulgaris L.) to ethanol

Dry common beans (Phaseolus vulgaris L.) were evaluated for potential conversion of starch to ethanol. Eight varieties of beans with average starch content of 46% (db) were assayed in a laboratory-scaled process based upon the commercial corn dry grind fermentation process. Ethanol yield was 0.43-0.51 g ethanol/g glucose (0.19-0.23 g ethanol/g beans). The average ethanol yield for the eight bean types was 92% of maximum theoretical yield, demonstrating that starch from beans could be efficiently converted to ethanol. Ethanol concentration obtained from 20% (w/w) solids loading was 3.5-4.4% (w/v). The residual fermentation solids contained, on a dry basis, 37.1-43.6% crude protein, 10.8-15.1% acid detergent fiber and 19.1-31.3% neutral detergent fiber.     

Assessment of genetic stability in micropropagules of Jatropha curcas genotypes by RAPD and AFLP analysis

Jatropha curcas (Euphorbiaceae), a drought resistant non edible oil yielding plant, has acquired significant importance as an alternative renewable energy source. Low and inconsistent yields found in field plantations prompted for identification of high yielding clones and their large scale multiplication by vegetative propagation to obtain true to type plants. In the current investigation plantlets of J. curcas generated by axillary bud proliferation (micropropagation) using nodal segments obtained from selected high yielding genotypes were assessed for their genetic stability using Randomly Amplified Polymorphic DNA (RAPD) and Amplified Fragment Length Polymorphism (AFLP) analyses. For RAPD analysis, 21 out of 52 arbitrary decamer primers screened gave clear reproducible bands. In the micropropagated plantlets obtained from the 2nd sub-culture, 4 out of a total of 177 bands scored were polymorphic, but in the 8th and 16th sub-cultures (culture cycle) no polymorphisms were detected. AFLP analysis revealed 0.63%, 0% and 0% polymorphism in the 2nd, 8th and 16th generations, respectively. When different genotypes, viz. IC 56557 16, IC 56557 34 and IC 56557 13, were assessed by AFLP, 0%, 0.31% and 0.47% polymorphisms were found, respectively, indicating a difference in genetic stability among the different genotypes. To the best of our knowledge this is the first report on assessment of genetic stability of micropropagated plantlets in J. curcas and suggests that axillary shoot proliferation can safely be used as an efficient micropropagation method for mass propagation of J. curcas.     

Variation in oil content and fatty acid profile of Calophyllum inophyllum L. with fruit maturity and its implications on resultant biodiesel quality

Variation in oil content and fatty acid profile (FAP) of Calophyllum inophyllum; a potential biodiesel feedstock species were studied at different maturity stages and biodiesel quality parameters were estimated based on the FAP. A steady increase in oil content was observed with maturity. Variation in palmitic (16:0) and linoleic (18:2) acids followed exactly opposite trends where palmitic acid content has decreased and linoleic acid content has increased 77 days after anthesis. Oleic acid (18:1) content has shown a steady increase. Stearic acid (18:0) content remained steady up to 68 days after anthesis and then felt slightly in 77 days after anthesis. Linoleic and eicosanoic acids were found to exist in low concentrations demonstrated very little compositional variation with fruit maturity. Estimated biodiesel parameters of all maturity stages were found to comply with industrial standards. Even though 48 days after anthesis had the most ideal FAP for biodiesel production, ∼77 days after anthesis is preferred point to harvest due to higher oil content.     

Optimizing mechanical oil extraction of Jatropha curcas L. seeds with respect to press capacity, oil recovery and energy efficiency

The objective of this study was to optimize the mechanical oil extraction of Jatropha curcas seeds by increasing the efficiency of oil recovery and decreasing oil residues in press cake. The experiments were carried out with mechanical screw press type - Komet D85-1G. Four setups were created by parameter combination of two different screws (16 and 21.5 mm choke ring size), with two different press cylinders (1 and 1.5 mm bore size), three different nozzles (8, 10 and 12 mm nozzle diameter) and three rotational speeds (low, medium and high). Oil recovery reduced when rotational speed increases for all setups; highest oil was 89.4% (m/m). The oil recovery was increasing when energy input increased and decreasing when seed material throughput increased. The relations between energy input and seed material throughput followed a strict pattern, which correlated with oil recovery. This correlation can be used for determining the optimal operation parameters.     

Microscopy techniques and image analysis for evaluation of some chemical and physical properties and morphological features for seeds of the castor oil plant (Ricinus communis)

Ricinus communis seed is a source of protein and oil with a high potential to use as animal's feedstock and biodiesel production. However, the oil yield and the extraction efficiency depend on the process conditions applied, as well as on the physical, chemical and structural properties of the seed, which have not been fully investigated. Hence, the objective of this study was to evaluate some chemical and physical properties of R. communis seed as well as to describe and quantify the macro and microstructure of this raw material by microscopy techniques and image analysis. Chemical analysis confirmed the seeds’ high contents of protein (28.48 ± 0.25%) and fat (51 ± 0.31%). On the other hand, the values of geometric mean diameter (8.95 ± 0.05 mm), bulk density (538 ± 11 kg/m³), and true density (1458 ± 27 kg/m³), among others, were higher than the ones reported about similar oils seeds. Microstructural studies showed that the endosperm cells presented an ovoid shape, as obtained from the aspect ratio results (AR = 1.28 ± 0.17), and a cell density of 570 ± 10 cell/mm², resulting in a porous structure, while the embryo cells had a cell density of 4903 ± 2 cell/mm², and an AR of 2.41 ± 0.48, related to a more compact structure (rectangular form) in this part of the seed. Regarding to lipids bodies (lb), they were only visible in the endosperm cells, showing a circular shape (AR = 1.16 ± 0.1), and a mean cell density of 9.57 ± 2.40 lipid bodies/μm², associated to protein as observed by the mineral presence (K, P, Mg and S) as determined by the energy dispersive X-ray analysis. Microscopy techniques and images analysis were efficient tools for the characterization of macro and microstructure of seeds and the data obtained integrate numerical information that could be useful for thermal and mechanical processing of R. communis seed, as well as for the design process equipment.     

Enzymatic epoxidation of Sapindus mukorossi seed oil by perstearic acid optimized using response surface methodology

As a novel renewable resource, Sapindus mukorossi seed oil (SMSO) with an iodine value of 84.86 g/100 g, and containing 51.0 ± 0.9% oleic acid (18:1), 6.6 ± 0.6% linoleic acid (18:2), 1.1 ± 0.3% linolenic acid (18:3), and 23.1 ± 0.9% eicosanoic acid (20:1), was epoxidized using hydrogen peroxide as oxygen donor and stearic acid as active oxygen carrier in the presence of immobilized Candida antarctica lipase B. The effect of the amount of stearic acid on the enzymatic epoxidation was investigated. Response surface methodology (RSM) was used to study and optimize the effects of variables (reaction temperature, enzyme load, mole ratio of H₂O₂/CC-bonds, and reaction time) on the epoxy oxygen group content (EOC) of epoxidized SMSO. Results showed that stearic acid as active oxygen carrier could enhance the enzymatic epoxidation of SMSO. The variables of reaction temperature and enzyme load were the most significant in the process. A two second-order model was satisfactorily fitted the data (R² = 0.9723) with non-significant lack of fit. The optimum EOC of epoxidized SMSO was 4.6 ± 0.3% under the conditions of 50.0 °C, 7.0 h, 2.00% (relative to the weight of SMSO) enzyme load, and 4:1 mole ratio of H₂O₂/CC-bonds.     

Rice bran, a potential source of biodiesel production in Indonesia

Biodiesel is a biodegradable, renewable, non-toxic and environmentally friendly alternative fuel. The cost of raw materials comprises 60-88% of the production cost in commercial biodiesel (fatty acid methyl esters, FAMEs) production. Therefore, the use of low-cost raw material as a substrate and an in situ process for biodiesel production are being preferred. In this case, rice bran, which contains 13.5% oil, was an interesting substrate. In situ esterification of high-acidity rice bran with methanol and sulfuric acid catalyst was investigated. The individual and interaction effects of methanol to rice bran ratio, sulfuric acid catalyst concentration and reaction time on purity and recovery of biodiesel were discussed. Our results suggest that under the following operation conditions: methanol to rice bran ratio of 5 mL/g, sulfuric acid concentration in methanol of 1.5 vol.%, and reaction time of 60 min, an in situ esterification operated on rice bran could yield FAMEs with a high purity and recovery. By applying an in situ esterification with n-hexane/water extractions, Indonesia will be succesfull in obtaining biodiesel from rice bran up to 96,000 ton per year.     

Preparation of shikonin by hydrolyzing ester derivatives using basic anion ion exchange resin as solid catalyst

An efficient catalytic hydrolysis approach was developed to prepare shikonin from its ester derivatives with anion exchange resins as solid catalyst. The performance of seven anion exchange resins including D290, D296, D261, D280, 201×7, D301-G and D301-R has been evaluated. The research results indicate that D290 resin is the most appropriate for preparation of shikonin. The reaction conditions such as reaction temperature, reaction time, catalyst amount and catalyst reuse have been studied. The maximum yield of shikonin reached 93.74 ± 2.99% under optimal conditions of hydrolysis temperature 20 °C, hydrolysis and desorption time both 30 min and resin amount 10% (w/v). HPLC chromatograms of samples, obtained after the new catalytic process, demonstrated that conversion of shikonin ester derivatives into shikonin performed well.     

The effects of onion and salt treatments on essential oil content and composition of Rosa damascena Mill

Due to the low oil content of Rosa damascena Mill., on one hand, and the wide spectrum of its application, on the other hand, it is desirable to find ways to increase the oil yield and also its quality. Therefore, in the present study the effects of chopped onion (1.0, 1.5, 2.0 and 4.0 g per 220 g of rose flower) and sodium chloride (11.0, 15.5, 22.0 and 30.0 g per 220 g of rose flower) which were added to rose flowers before distillation, were investigated on the quality and quantity of the oil. The quantity was measured as the ratio of oil to rose flower (w/w, %) and the quality was evaluated using the oil composition especially the content of monoterpene alcohols and hydrocarbons. Compared with corresponding control sample, treating with 1.5 g of chopped onion increased the oil content by 41% but it resulted in a lower quality; whereas in the presence of 1.0 g of onion, an increase of 30% and a decrease of 18% were observed in the percentage of monoterpene alcohols and hydrocarbons, respectively without any considerable change in the oil content. However, the effect of sodium chloride was somehow different from that of onion; such that in the optimal amount (22.0 g), the salt had no considerable effect on the quality but an increase of 21% in the oil yield.     

Avenanthramide content and related enzyme activities in oats as affected by steeping and germination

Samples from three different oat (Avena sativa L.) cultivars, Vista, Gem and Dane were steeped and germinated in a pilot plant malting system. The content of avenanthramides, antioxidant compounds unique to oats, and some unknown compounds as well as the activities of the avenanthramide-synthesising enzyme hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) and the avenanthramide-oxidizing enzyme phenoloxidase (PO) were measured. An increase in avenanthramide content of germinated seeds, as compared to raw grains, was observed for Dane (125%, p < 0.001) and for Vista (29%, p = 0.007). The HHT activity increased 62% (p = 0.014) in Dane, whereas no change was detected in Vista and Gem. The PO activity decreased slightly during the germination process for Gem (p < 0.001) and Vista (p = 0.005). Many of the unknown compounds increased significantly during germination in all three cultivars, and one of them was identified to be the avenanthramide N-(4′-hydroxy-3′-methoxy-(E)-cinnamoyl)-5-hydroxy-4-methoxyanthranilic acid. This study indicates that a highly controlled steeping and germination process can be a valuable method to increase the content of endogenous avenanthramides in oats.     

Changes in phytochemical, antimicrobial and free radical scavenging activities of the Peruvian pepper tree (Schinus molle L.) as influenced by fruit maturation

The effect of stage of maturity on total lipids, fatty acids, yields and essential oil composition and their antimicrobial and free radical scavenging activities of the Schinus molle fruits was investigated. The content of total lipids varied from 2.87 to 5.35% (w/w, dw) and were rich in unsaturated fatty acids particularly linoleic acid. As maturation progress, the essential oil yield dropped from 5.18% to 1.15%. Monoterpene hydrocarbons with α-phellandrene (35.15-40.38%), limonene + β-phellandrene (21.47-36.62%), β-myrcene (7.61-24.96%) and α-pinene (1.92-2.58%) were found to be the main components. At the same time, the essential oils were evaluated for their antimicrobial and free radical scavenging activities. They were found to be active against Bacillus subtilis, Bacillus cereus, Salmonella typhymurium and Escherichia coli but they do not inhibit the growth of Candida albicans. Conversely, they showed very weak activity against the DPPH radical. In both assay, the oil derived from the intermediate stage was reported as more efficient.