Previous reports showed that vitamin E in palm oil consists of various isomers of tocopherols and tocotrienols [alpha-tocopherol (alpha-T), alpha-tocotrienol, gamma-tocopherol, gamma-tocotrienol, and delta-tocotrienol), and this is normally analyzed using silica column HPLC with fluorescence detection. In this study, an HPLC-fluorescence method using a C30 silica stationary phase was developed to separate and analyze the vitamin E isomers present in palm oil. In addition, an alpha-tocomonoenol (alpha-T1) isomer was quantified and characterized by MS and NMR. (alpha-T1 constitutes about 3-4% (40+/-5 ppm) of vitamin E in crude palm oil (CPO) and is found in the phytonutrient concentrate (350+/-10 ppm) from palm oil, whereas its concentration in palm fiber oil (PFO) is about 11% (430+/-6 ppm). The relative content of each individual vitamin E isomer before and after interesterification/transesterification of CPO to CPO methyl esters, followed by vacuum distillation of CPO methyl esters to yield the residue, remained the same except for alpha-T and gamma-T3. Whereas alpha-T constitutes about 36% of the total vitamin E in CPO, it is present at a level of 10% in the phytonutrient concentrate. On the other hand, the composition of gamma-T3 increases from 31% in CPO to 60% in the phytonutrient concentrate. Vitamin is present at 1160+/-43 ppm, and its concentrations in PFO and the phytonutrient concentrate are 4,040+/-41 and 13,780+/-65 ppm, respectively. The separation and quantification of alpha-T1 in palm oil will lead to more in-depth knowledge of the occurrence of vitamin E in palm oil.
In this study, a novel continuous reactor has been developed to produce high quality methyl esters (biodiesel) from palm oil. A microporous TiO2/Al2O3 membrane was packed with potassium hydroxide catalyst supported on palm shell activated carbon. The central composite design (CCD) of response surface methodology (RSM) was employed to investigate the effects of reaction temperature, catalyst amount and cross flow circulation velocity on the production of biodiesel in the packed bed membrane reactor. The highest conversion of palm oil to biodiesel in the reactor was obtained at 70 °C employing 157.04 g catalyst per unit volume of the reactor and 0.21 cm/s cross flow circulation velocity. The physical and chemical properties of the produced biodiesel were determined and compared with the standard specifications. High quality palm oil biodiesel was produced by combination of heterogeneous alkali transesterification and separation processes in the packed bed membrane reactor.
The objective of this research is to investigate the potential of transesterification of crude palm oil (CPO) to biodiesel at 30 degrees C. The mass transfer limitations problem crucial at 30 degrees C due to the viscosity of CPO has been addressed. The process parameters that are closely related to mass transfer effects like enzyme loading, agitation speed and reaction time were optimized. An optimum methanol to oil substrate molar ratio at 6.5:1 was observed and maintained throughout the experiments. The optimum operating condition for the transesterification process was found at 6.67 wt% of enzyme loading and at 150 rpm of agitation speed. The corresponding initial reaction and FAME yield obtained at 6 h were 89.29% FAME yield/hr and 85.01%, respectively. The 85% FAME yield obtained at 30 degrees C operation of CPO transesterification shows that the process is potentially feasible for the biodiesel synthesis.
Current study presents RSM based optimized production of biodiesel from palm oil using chemical and enzymatic transesterification. The emission behavior of biodiesel and its blends, namely, POB-5, POB-20, POB-40, POB-50, POB-80, and POB-100 was examined using diesel engine (equipped with tube well). Optimized palm oil fatty acid methyl esters (POFAMEs) yields were depicted to be 47.6 ± 1.5, 92.7 ± 2.5, and 95.4 ± 2.0% for chemical transesterification catalyzed by NaOH, KOH, and NaOCH3, respectively, whereas for enzymatic transesterification reactions catalyzed by NOVOZYME-435 and A. n. lipase optimized biodiesel yields were 94.2 ± 3.1 and 62.8 ± 2.4%, respectively. Distinct decrease in particulate matter (PM) and carbon monoxide (CO) levels was experienced in exhaust emissions from engine operating on biodiesel blends POB-5, POB-20, POB-40, POB-50, POB-80, and POB-100 comparative to conventional petroleum diesel. Percentage change in CO and PM emissions for different biodiesel blends ranged from -2.1 to -68.7% and -6.2 to -58.4%, respectively, relative to conventional diesel, whereas an irregular trend was observed for NOx emissions. Only POB-5 and POB-20 showed notable reductions, whereas all other blends (POB-40 to POB-100) showed slight increase in NOx emission levels from 2.6 to 5.5% comparative to petroleum diesel.
Methyl esters from vegetable oils have attracted a great deal of interest as substitute for petrodiesel to reduce dependence on imported petroleum and provide an alternate and sustainable source for fuel with more benign environmental properties. In the present study biodiesel was prepared from sunflower seed oil by transesterification by alkali-catalyzed methanolysis. The fuel properties of sunflower oil biodiesel were determined and discussed in the light of ASTM D6751 standards for biodiesel. The sunflower oil biodiesel was chemically characterized with analytical techniques like FT-IR, and NMR ((1)H and (13)C). The chemical composition of sunflower oil biodiesel was determined by GC-MS. Various fatty acid methyl esters (FAMEs) were identified by retention time data and verified by mass fragmentation patterns. The percentage conversion of triglycerides to the corresponding methyl esters determined by (1)H NMR was 87.33% which was quite in good agreement with the practically observed yield of 85.1%.
The aim of this work was to study the potential of biofuel and biomass processing industry side-products as acid catalyst. The synthesis of carbon cryogel from lignin-furfural mixture, prepared via sol-gel polycondensation at 90°C for 0.5h, has been investigated for biodiesel production. The effect of lignin to furfural (L/F) ratios, lignin to water (L/W) ratios and acid concentration on carbon cryogel synthesis was studied. The carbon cryogels were characterized and tested for oleic acid conversion. The thermally stable amorphous spherical carbon cryogel has a large total surface area with high acidity. Experimental results revealed the optimum FAME yield and oleic acid conversion of 91.3wt.% and 98.1wt.%, respectively were attained at 65°C for 5h with 5wt.% catalyst loading and 20:1 methanol to oleic acid molar ratio. Therefore, carbon cryogel is highly potential for heterogeneous esterification of free fatty acid to biodiesel.
In view of several disadvantages as well as adverse effects associated with the use of chemical processes for producing esters, alternative techniques such as the utilization of enzymes on multi-walled carbon nanotubes (MWCNTs), have been suggested. In this study, the oxidative MWCNTs prepared using a mixture of HNO3 and H2SO4 (1:3 v/v) were used as a supportive material for the immobilization of Candida rugosa lipase (CRL) through physical adsorption process. The resulting CRL-MWCNTs biocatalysts were utilized for synthesizing geranyl propionate, an important ester for flavoring agent as well as in fragrances. Enzymatic esterification of geraniol with propionic acid was carried out using heptane as a solvent and the efficiency of CRL-MWCNTs as a biocatalyst was compared with the free CRL, considering the incubation time, temperature, molar ratio of acid:alcohol, presence of desiccant as well as its reusability. It was found that the CRL-MWCNTs resulted in a 2-fold improvement in the percentage of conversion of geranyl propionate when compared with the free CRL, demonstrating the highest yield of geranyl propionate at 6h at 55°C, molar ratio acid: alcohol of 1:5 and with the presence of 1.0g desiccant. It was evident that the CRL-MWCNTs biocatalyst could be reused for up to 6 times before a 50% reduction in catalytic efficiency was observed. Hence, it appears that the facile physical adsorption of CRL onto F-MWCNTs has improved the activity and stability of CRL as well as served as an alternative method for the synthesis of geranyl propionate.
The use of pseudo-infinite methanol in increasing the rate of esterification and transesterification reactions was studied using oil palm trunk (OPT) and sugarcane bagasse (SCB) derived solid acid catalysts. The catalysts were prepared by incomplete carbonisation at 400°C for 8h, followed by sulfonation at 150°C for 15h and characterised using TGA/DTA, XRD, FT-IR, SEM-EDS, EA and titrimetric determinations of acid sites. Under optimal reaction conditions, the process demonstrated rapid esterification of palmitic acid, with FAME yields of 93% and 94% in 45min for OPT and SCB catalysts, respectively. With the process, moisture levels up to 16.7% accelerated the conversion of low FFA oils by sulfonated carbon catalysts, through moisture-induced violent bumping. Moisture assisted transesterification of palm olein containing 1.78% FFA and 8.33% added water gave FAME yield of 90% in 10h, which was two folds over neat oil.
Lipase-catalyzed production of triethanolamine-based esterquat by esterification of oleic acid (OA) with triethanolamine (TEA) in n-hexane was performed in 2 L stirred-tank reactor. A set of experiments was designed by central composite design to process modeling and statistically evaluate the findings. Five independent process variables, including enzyme amount, reaction time, reaction temperature, substrates molar ratio of OA to TEA, and agitation speed, were studied under the given conditions designed by Design Expert software. Experimental data were examined for normality test before data processing stage and skewness and kurtosis indices were determined. The mathematical model developed was found to be adequate and statistically accurate to predict the optimum conversion of product. Response surface methodology with central composite design gave the best performance in this study, and the methodology as a whole has been proven to be adequate for the design and optimization of the enzymatic process.
The wet biomass microalgae of Nannochloropsis sp. was converted to biodiesel using direct transesterification (DT) by microwave technique and ionic liquid (IL) as the green solvent. Three different ionic liquids; 1-butyl-3-metyhlimidazolium chloride ([BMIM][Cl], 1-ethyl-3-methylimmidazolium methyl sulphate [EMIM][MeSO4] and 1-butyl-3-methylimidazolium trifluoromethane sulfonate [BMIM][CF3SO3]) and organic solvents (hexane and methanol) were used as co-solvents under microwave irradiation and their performances in terms of percentage disruption, cell walls ruptured and biodiesel yields were compared at different reaction times (5, 10 and 15 min). [EMIM][MeSO4] showed highest percentage cell disruption (99.73%) and biodiesel yield (36.79% per dried biomass) after 15 min of simultaneous reaction. The results demonstrated that simultaneous extraction-transesterification using ILs and microwave irradiation is a potential alternative method for biodiesel production.
Pretreatment of the high free fatty acid rubber seed oil (RSO) via esterification reaction has been investigated by using a pilot scale hydrodynamic cavitation (HC) reactor. Four newly designed orifice plate geometries are studied. Cavities are induced by assisted double diaphragm pump in the range of 1-3.5 bar inlet pressure. An optimised plate with 21 holes of 1mm diameter and inlet pressure of 3 bar resulted in RSO acid value reduction from 72.36 to 2.64 mg KOH/g within 30 min of reaction time. Reaction parameters have been optimised by using response surface methodology and found as methanol to oil ratio of 6:1, catalyst concentration of 8 wt%, reaction time of 30 min and reaction temperature of 55°C. The reaction time and esterified efficiency of HC was three fold shorter and four fold higher than mechanical stirring. This makes the HC process more environmental friendly.
Palm oil is one of the most important edible oils in the world. Its composition (rich in palmitate and oleate) make it suitable for general food uses but its utility could be increased if its fatty acid quality could be varied. In this study, we have modified a palm olein fraction by transesterification with the n-3 polyunsaturated fatty acids, alpha-linolenate or eicosapentaenoic acid (EPA). Evaluation of the potential nutritional efficacy of the oils was made using chondrocyte culture systems which can be used to mimic many of the degenerative and inflammatory pathways involved in arthritis. On stimulation of such cultures with interleukin-1alpha, they showed increased expression of cyclooxygenase-2, the inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-1beta and the proteinase ADAMTS-4. This increased expression was not affected by challenge of the cultures with palm olein alone but showed concentration-dependent reduction by the modified oil in a manner similar to EPA. These results show clearly that it is possible to modify palm oil conveniently to produce a nutraceutical with effective anti-inflammatory properties.
Rosmarinic acid (RA) is a highly valued natural phenolic compound that is very commonly found in plants of the families Lamiaceae and Boraginaceae, including Coleus blumei, Heliotropium foertherianum, Rosmarinus officinalis, Perilla frutescens, and Salvia officinalis. RA is also found in other members of higher plant families and in some fern and horned liverwort species. The biosynthesis of RA is catalyzed by the enzymes phenylalanine ammonia lyase and cytochrome P450-dependent hydroxylase using the amino acids tyrosine and phenylalanine. Chemically, RA can be produced via methods involving the esterification of 3,4-dihydroxyphenyllactic acid and caffeic acid. Some of the derivatives of RA include melitric acid, salvianolic acid, lithospermic acid, and yunnaneic acid. In plants, RA is known to have growth-promoting and defensive roles. Studies have elucidated the varied pharmacological potential of RA and its derived molecules, including anticancer, antiangiogenic, anti-inflammatory, antioxidant, and antimicrobial activities. The demand for RA is therefore, very high in the pharmaceutical industry, but this demand cannot be met by plants alone because RA content in plant organs is very low. Further, many plants that synthesize RA are under threat and near extinction owing to biodiversity loss caused by unscientific harvesting, over-collection, environmental changes, and other inherent features. Moreover, the chemical synthesis of RA is complicated and expensive. Alternative approaches using biotechnological methodologies could overcome these problems. This review provides the state of the art information on the chemistry, sources, and biosynthetic pathways of RA, as well as its anticancer properties against different cancer types. Biotechnological methods are also discussed for producing RA using plant cell, tissue, and organ cultures and hairy-root cultures using flasks and bioreactors. The recent developments and applications of the functional genomics approach and heterologous production of RA in microbes are also highlighted. This chapter will be of benefit to readers aiming to design studies on RA and its applicability as an anticancer agent.
Conjugated linoleic acid (CLA)-rich triacylglycerols (TAG) have received significant attention owing to their health promoting properties. In this study, CLA-rich TAG were successfully synthesized by an immobilized mutant lipase (MAS1-H108A)-catalyzed esterification of CLA-rich fatty acids and glycerol under vacuum. MAS1-H108A was first immobilized onto ECR1030 resin. Results showed that the lipase/support ratio of 41 mg/g was suitable for the immobilization and the thermostability of immobilized MAS1-H108A was greatly enhanced. Subsequently, the immobilized MAS1-H108A was employed for the synthesis of CLA-rich TAG and 95.21% TAG with 69.19% CLA was obtained under the optimized conditions. The TAG content (95.21%) obtained by immobilized MAS1-H108A is the reported highest value thus far, which was significantly higher than that (9.26%) obtained by Novozym 435 under the same conditions. Although the TAG content comparable to the results obtained in this study could also be obtained by Novozym 435, the used enzyme amount is approximately 5-fold of the immobilized MAS1-H108A. Additionally, the immobilized MAS1-H108A exhibited excellent recyclability during esterification retaining 95.11% of its initial activity after 10 batches. Overall, such immobilized mutant lipase with superior esterification activity and recyclability has the potential to be used in oils and fats industry.
An approach was made to enhance the halophilic lipase secretion by a newly isolated moderate halophilic Marinobacter litoralis SW-45, through the statistical optimization of Plackett-Burman (PB) experimental design and the Face Centered Central Composite Design (FCCCD). Initially, PB statistical design was used to screen the medium components and process parameters, while the One-factor-at-a-time technique was availed to find the optimum level of significant parameters. It was found that MgSO4 · 7H2 O, NaCl, agitation speed, FeSO4 · 7H2 O, yeast extract and KCl positively influence the halophilic lipase production, whereas temperature, carbon source (maltose), inducer (olive oil), inoculum size, and casein-peptone had a negative effect on enzyme production. The optimum level of halophilic lipase production was obtained at 3.0 g L-1 maltose, 1% (v/v) olive oil, 30 °C growth temperature and 4% inoculum volume (v/v). Further optimization by FCCCD was revealed 1.7 folds improvement in the halophilic lipase production from 0.603 U ml-1 to 1.0307 U ml-1 . Functional and biochemical characterizations displayed that the lipase was significantly active and stable in the pH ranges of 7.0-9.5, temperature (30-50 °C), and NaCl concentration (0-21%). The lipase was maximally active at pH 8.0, 12% (w/v) NaCl, and 50 °C temperature. Besides, M. litoralis SW-45 lipase was found to possess the promising industrial potential to be utilized as a biocatalyst for the esterification.
Tyrosinase inhibitors have become increasingly important targets for hyperpigmentation disease treatment. Kojic monooleate (KMO), synthesized from the esterification of kojic acid and oleic acid, has shown a better depigmenting effect than kojic acid. In this study, the process parameters include the speed of high shear, the time of high shear and the speed of the stirrer in the production of nanoemulsion containing KMO was optimized using Response Surface Methodology (RSM), as well as evaluated in terms of its physicochemical properties, safety and efficacy. The optimized condition for the formulation of KMO nanoemulsion was 8.04 min (time of high shear), 4905.42 rpm (speed of high shear), and 271.77 rpm (speed of stirrer), which resulted in a droplet size of 103.97 nm. An analysis of variance (ANOVA) showed that the fitness of the quadratic polynomial fit the experimental data with large F-values (148.79) and small p-values (p < 0.0001) and an insignificant lack of fit. The optimized nanoemulsion containing KMO with a pH value of 5.75, showed a high conductivity value (3.98 mS/cm), which indicated that the nanoemulsion containing KMO was identified as an oil-in-water type of nanoemulsion. The nanoemulsion remains stable (no phase separation) under a centrifugation test and displays accelerated stability during storage at 4, 25 and 45 °C over 90 days. The cytotoxicity assay showed that the optimized nanoemulsion was less toxic, with a 50% inhibition of cell viability (IC50) > 500 μg/mL, and that it can inhibit 67.12% of tyrosinase activity. This study reveals that KMO is a promising candidate for the development of a safe cosmetic agent to prevent hyperpigmentation.
Recent studies on biocatalysis in water-organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a(w)) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.
Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme's enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50°C, after 12-16h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.
Fatty acid methyl esters (FAME) were produced from palm oil using eggshell modified with magnesium and potassium nitrates to form a composite, low-cost heterogeneous catalyst for transesterification. The catalyst, prepared by the combination of impregnation/co-precipitation was calcined at 830 °C for 4 h. Transesterification was conducted at a constant temperature of 65 °C in a batch reactor. Design of experiment (DOE) was used to optimize the reaction parameters, and the conditions that gave highest yield of FAME (85.8%) was 5.35 wt.% catalyst loading at 4.5 h with 16:1 methanol/oil molar ratio. The results revealed that eggshell, a solid waste, can be utilized as low-cost catalyst after modification with magnesium and potassium nitrates for biodiesel production.
The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.