Omega-3 rich oils from microalgae: A chitosan mediated in situ transesterification method.

Microalgae are a precious source of polyunsaturated fatty acids (PUFA), however extraction is difficult due to the peculiar microalgae cell structure. Here we describe a new method based on the application of chitosan nanoaggregates as CO2 responsive emulsifier, used to promote the swelling of algae cell wall and the formation of a large oil - ethanol interphase area during the ethanolysis. Tests were carried out with Pseudokirchneriella subcapitata and Nannochloropsis sp. at different biomass/ethanol/chitosan ratios. CO2 was added to trigger demulsification to promote an easy recovery of the lipid fraction. The highest yields in PUFA were obtained with Nannochloropsis sp. (207.9 mg/g of oil) using 0.4% wt of chitosan and 1:10 biomass:ethanol ratio; 43.6 mg/g of linolenic acid were obtained from Pseudokirchneriella subcapitata. Overall, because the method employs ethanol, a generally recognized as safe (GRAS) solvent, and food grade additives, it is suited for the preparation of PUFA supplements.

Improvement of biodiesel production from palm oil by co-immobilization of Thermomyces lanuginosa lipase and Candida antarctica lipase B: Optimization using response surface methodology.

Candida antarctica lipase B (CALB) and Thermomyces lanuginose lipase (TLL) were co-immobilized on epoxy functionalized silica gel via an isocyanide-based multicomponent reaction. The immobilization process was carried out in water (pH 7) at 25 °C, rapidly (3 h) resulting in high immobilization yields (100%) with a loading of 10 mg enzyme/g support. The immobilized preparations were used to produce biodiesel by transesterification of palm oil. In an optimization study, response surface methodology (RSM) and central composite rotatable design (CCRD) methods were used to study the effect of five independent factors including temperature, methanol to oil ratio, t-butanol concentration and CALB:TLL ratio on the yield of biodiesel production. The optimum combinations for the reaction were CALB:TLL ratio (2.1:1), t-butanol (45 wt%), temperature (47 °C), methanol: oil ratio (2.3). This resulted in a FAME yield of 94%, very close to the predicted value of 98%.

Optimization, characterization and rheological behavior study of pectin extracted from chayote (Sechium edule) using ultrasound assisted method.

Pectin is one of high-value functional food ingredients. Better knowledge of properties of chayote pectin would help to promote the application of the chayote as a pectin source. In this study, ultrasound-assisted extraction (UAE) was used to extract chayote pectin. The extraction parameters were optimized by a Box-Behnken response surface design. The highest yield (6.19%) was obtained at a liquid/solid ratio of 50 mL/g, ultrasonic temperature of 70 °C and ultrasonic time of 40 min as optimized extraction conditions. The chemical properties, spectral information and rheological properties of pectin extracted with UAE under the optimum conditions (PEUO) were measured and the results indicated that the PEUO exhibited a low degree of esterification, high molar mass (2.47 × 106 g/mol) and suitable foaming capacity. The PEUO featured a stronger antioxidant capacity compared to commercially apple pectin and the PEUO solutions (1%, 2%, 3% and 5%) showed a non-Newtonian behavior. Hence, PEUO may provide beneficial characteristics to find use in food industry.

High-methylated pectin from walnut processing wastes as a potential resource: Ultrasound assisted extraction and physicochemical, structural and functional analysis.

The ultrasound-assisted extraction of pectin from walnut processing waste was optimized by Box-Behnken design. The highest extraction yield (12.78 ± 0.83%) was obtained at ultrasound power of 200 W, sonication time of 10 min, pH of 1.5 and LS ratio of 15 v/w. The resulting pectin in these conditions was rich in galacturonic acid (69.44%) and was high in degree of esterification (59.21%) which was confirmed by NMR and FTIR spectra. In addition, the molecular weight distribution analysis showed that the obtained pectin had a heterogeneous natural with low molecular weight (6.30-158.48 kDa). The XRD spectrum of the walnut pectin showed an amorphous structure with few crystalline portions. Furthermore, walnut green husk pectin had good emulsifying properties, water and oil holding capacities, and radical-scavenging activity. Given that the obtained result, the extraction of pectin from this by-product in addition to being able to reduce environmental problems, it can also provide financial benefits for the walnut production sector.

Disrupting cholesterol esterification by bitter melon suppresses triple-negative breast cancer cell growth.

Triple negative breast cancer (TNBC) is aggressive with a worse prognosis. We have recently shown that bitter melon extract (BME) treatment was more effective in inhibition of TNBC tumor growth in mouse models as compared to ER positive breast tumor growth. Aberrant dysregulation of lipid metabolism is associated with breast cancer progression, however, anti-cancer mechanism of BME linking lipid metabolism in breast cancer growth remains unexplored. Here, we observed that accumulation of esterified cholesterol was reduced in BME treated TNBC cell lines as compared to control cells. We next evaluated expression levels of acyl-CoA: cholesterol acyltransferase 1 (ACAT-1) in TNBC cells treated with BME. Our results demonstrated that BME treatment inhibited ACAT-1 expression in TNBC cells. Subsequently, we found that sterol regulatory element-binding proteins-1 and -2, and FASN was significantly reduced in BME treated TNBC cell lines. Low-density lipoprotein receptor was also downregulated in BME treated TNBC cells as compared to control cells. We further demonstrated that BME feeding reduced tumor growth in TNBC mammospheres implanted into NSG mice, and inhibits ACAT-1 expression. To our knowledge, this is the first report demonstrating BME suppresses TNBC cell growth through ACAT-1 inhibition, and have potential for additional therapeutic regimen against human breast cancer.

Cell wall pectin methyl-esterification and organic acids of root tips involve in aluminum tolerance in Camellia sinensis.

Tea plant (Camellia sinensis (O.) Kuntze) can survive from high levels of aluminum (Al) in strongly acidic soils. However, the mechanism driving its tolerance to Al, the predominant factor limiting plant growth in acid condition, is still not fully understood. Here, two-year-old rooted cuttings of C. sinensis cultivar 'Longjingchangye' were used for Al resistance experiments. We found that the tea plants grew better in the presence of 0.4 mM Al than those grew under lower concentration of Al treatments (0 and 0.1 mM) as well as higher levels treatment (2 and 4 mM), confirming that appropriate Al increased tea plant growth. Hematoxylin staining assay showed that the apical region was the main accumulator in tea plant root. Subsequently, immunolocalization of pectins in the root tip cell wall showed a rise in low-methyl-ester pectin levels and a reduction of high-methyl-ester pectin content with the increasing Al concentration of treatments. Furthermore, we observed the increased expressions of C. sinensis pectin methylesterase (CsPME) genes along with the increasing de-esterified pectin levels during response to Al treatments. Additionally, the levels of organic acids increased steadily after treatment with 0.1, 0.4 or 2 mM Al, while they dropped after treatment with 4 mM Al. The organic acids secretion from root followed a similar trend. Similarly, a gradual increase in malate dehydrogenase (MDH), citrate synthase (CS) and glycolate oxidase (GO) enzyme activities and relevant metabolic genes expression were detected after the treatment of 0.1, 0.4 or 2 mM Al, while a sharp decrease was resulted from treatment with 4 mM Al. These results confirm that both pectin methylesterases and organic acids contribute to Al tolerance in C. sinensis.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina / Toxicidade do óleo essencial de cravo e seu éster acetato de eugenila contra Artemia salina

Abstract The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of “green chemistry”. Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL–1) and ester (LC50= 0.1178 µg.mL–1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Resumo A produção de compostos via esterificação enzimática possui grande interesse científico e tecnológico devido às inúmeras inconveniências relacionadas com a catálise ácida, principalmente por estes sitemas não se adequarem ao atual termo “tecnologias limpas”. Além disso, produtos naturais como o óleo de cravo, apresentam compostos com excelentes potenciais biológicos. Compostos bioativos são quase sempre tóxicos em altas doses. A avaliação da letalidade em um organismo animal menos complexo pode ser usada para um monitoramento simples e rápido, servindo também para a identificação de compostos com potencial atividade inseticida contra larvas de insetos vetores de doenças. Neste sentido, foi determinada a toxicidade frente a Artemia salina do óleo essencial de cravo e do seu derivado acetato de eugenila obtido por esterificação enzimática com lipase Novozym 435. A conversão da reação de síntese de acetato de eugenila foi de 95,6%. Os resultados referentes à avaliação da toxicidade frente ao microcrustáceo Artemia salina demonstraram que tanto o óleo (LC50= 0,5993 µg.mL–1) quanto o éster (LC50= 0,1178 µg.mL–1) apresentam elevado potencial toxicológico, sendo que o éster apresenta aproximadamente 5 vezes mais toxicidade em relação ao óleo. Estes resultados demonstram o potencial emprego do óleo de cravo e de acetato de eugenila em formulações de inseticidas.

Toxicity of clove essential oil and its ester eugenyl acetate against Artemia salina.

The production of compounds via enzymatic esterification has great scientific and technological interest due to the several inconveniences related to acid catalysis, mainly by these systems do not fit to the concept of "green chemistry". Besides, natural products as clove oil present compounds with excellent biological potential. Bioactives compounds are often toxic at high doses. The evaluation of lethality in a less complex animal organism can be used to a monitoring simple and rapid, helping the identification of compounds with potential insecticide activity against larvae of insect vector of diseases. In this sense, the toxicity against Artemia salina of clove essential oil and its derivative eugenyl acetate obtained by enzymatic esterification using Novozym 435 as biocatalyst was evaluated. The conversion of eugenyl acetate synthesis was 95.6%. The results about the evaluation of toxicity against the microcrustacean Artemia salina demonstrated that both oil (LC50= 0.5993 µg.mL-1) and ester (LC50= 0.1178 µg.mL-1) presented high toxic potential, being the eugenyl acetate almost 5 times more toxic than clove essential oil. The results reported here shows the potential of employing clove oil and eugenyl acetate in insecticide formulations.

Nanobioconjugates of Candida antarctica lipase B and single-walled carbon nanotubes in biodiesel production.

Carboxylated single-walled carbon nanotubes (SWCNTCOOH) were used as support for covalent immobilization of Candida antarctica lipase B (CaL-B) using linkers with different lengths. The obtained nanostructured biocatalysts with low diffusional limitation were tested in batch mode in the ethanolysis of the sunflower oil. SWCNTCOOH-CaL-B proved to be a highly efficient and stable biocatalyst in acetonitrile (83.4% conversion after 4h at 35°C, retaining >90% of original activity after 10 cycles).

PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination.

Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48-/- pollen grains. In contrast, the PME activity was lower in pme48-/-, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48-/- with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca(2+) necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

Boosting accumulation of neutral lipids in Rhodosporidium kratochvilovae HIMPA1 grown on hemp (Cannabis sativa Linn) seed aqueous extract as feedstock for biodiesel production.

Hemp seeds aqueous extract (HSAE) was used as cheap renewable feedstocks to grow novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 isolated from Himalayan permafrost soil. The yeast showed boosted triglyceride (TAG) accumulation in the lipid droplets (LDs) which were transesterified to biodiesel. The sonicated HSAE prepared lacked toxic inhibitors and showed enhanced total lipid content and lipid yield 55.56%, 8.39±0.57g/l in comparison to 41.92%, 6.2±0.8g/l from industrially used glucose synthetic medium, respectively. Supersized LDs (5.95±1.02µm) accumulated maximum TAG in sonicated HSAE grown cells were visualized by fluorescent BODIPY (505/515nm) stain. GC-MS analysis revealed unique longer carbon chain FAME profile containing Arachidic acid (C20:0) 5%, Behenic acid (C22:0) 9.7%, Heptacosanoic acid (C27:0) 14.98%, for the first time in this yeast when grown on industrially competent sonicated HSAE, showing more similarity to algal oils.

A more robust model of the biodiesel reaction, allowing identification of process conditions for significantly enhanced rate and water tolerance.

A more robust kinetic model of base-catalysed transesterification than the conventional reaction scheme has been developed. All the relevant reactions in the base-catalysed transesterification of rapeseed oil (RSO) to fatty acid methyl ester (FAME) were investigated experimentally, and validated numerically in a model implemented using MATLAB. It was found that including the saponification of RSO and FAME side reactions and hydroxide-methoxide equilibrium data explained various effects that are not captured by simpler conventional models. Both the experiment and modelling showed that the "biodiesel reaction" can reach the desired level of conversion (>95%) in less than 2min. Given the right set of conditions, the transesterification can reach over 95% conversion, before the saponification losses become significant. This means that the reaction must be performed in a reactor exhibiting good mixing and good control of residence time, and the reaction mixture must be quenched rapidly as it leaves the reactor.

Biodiesel production from transesterification of palm oil with methanol over CaO supported on bimodal meso-macroporous silica catalyst.

Calcium oxide-loaded porous materials have shown promise as catalysts in transesterification. However, the slow diffusion of bulky triglycerides through the pores limited the activity of calcium oxide (CaO). In this work, bimodal meso-macroporous silica was used as a support to enhance the accessibility of the CaO dispersed inside the pores. Unimodal porous silica having the identical mesopore diameter was employed for the purpose of comparison. Effects of CaO content and catalyst pellet size on the yield of fatty acid methyl esters (FAME) were investigated. The basic strength was found to increase with increasing the CaO content. The CaO-loaded bimodal porous silica catalyst with the pellet size of 325µm achieved a high %FAME of 94.15 in the first cycle, and retained an excellent %FAME of 88.87 after five consecutive cycles.

A green recyclable SO(3)H-carbon catalyst derived from glycerol for the production of biodiesel from FFA-containing karanja (Pongamia glabra) oil in a single step.

Simultaneous esterification and transesterification method is employed for the preparation of biodiesel from 7.5% free fatty acid (FFA) containing karanja (Pongamia glabra) oil using water resistant and reusable carbon-based solid acid catalyst derived from glycerol in a single step. The optimum reaction parameters for obtaining biodiesel in >99% yield by simultaneous esterification and transesterification are: methanol (1:45 mole ratio of oil), catalyst 20wt.% of oil, temperature 160°C and reaction time of 4h. After the reaction, the catalyst was easily recovered by filtration and reused for five times with out any deactivation under optimized conditions. This single-step process could be a potential route for biodiesel production from high FFA containing oils by simplifying the procedure and reducing costs and effluent generation.

Fomiroid A, a novel compound from the mushroom Fomitopsis nigra, inhibits NPC1L1-mediated cholesterol uptake via a mode of action distinct from that of ezetimibe.

Hypercholesterolemia is one of the key risk factors for coronary heart disease, a major cause of death in developed countries. Suppression of NPC1L1-mediated dietary and biliary cholesterol absorption is predicted to be one of the most effective ways to reduce the risk of hypercholesterolemia. In a screen for natural products that inhibit ezetimibe glucuronide binding to NPC1L1, we found a novel compound, fomiroid A, in extracts of the mushroom Fomitopsis nigra. Fomiroid A is a lanosterone derivative with molecular formula C30H48O3. Fomiroid A inhibited ezetimibe glucuronide binding to NPC1L1, and dose-dependently prevented NPC1L1-mediated cholesterol uptake and formation of esterified cholesterol in NPC1L1-expressing Caco2 cells. Fomiroid A exhibited a pharmacological chaperone activity that corrected trafficking defects of the L1072T/L1168I mutant of NPC1L1. Because ezetimibe does not have such an activity, the binding site and mode of action of fomiroid A are likely to be distinct from those of ezetimibe.

Effect of metal ions on the hydrolytic and transesterification activities of Candida rugosa lipase.

In order to study the effect of metal ions on lipase activity, hydrolytic and transesterification activities of Candida rugosa lipase were investigated in presence of alkali (Na⁺ and K⁺), alkaline earth (Ca⁺² and Ba⁺²) and transition (Cr⁺³, Fe⁺³, Co⁺², Cu⁺² and Ni⁺²) metal ions. Maximum enhancement in hydrolytic activity of lipase was observed by Ca⁺², and in transesterification activity by Cr⁺³ and Co⁺². The kinetics of the lipase catalyzed transesterification (methanolysis and ethanolysis) reactions were also studied, and the activation energies of methanolysis and ethanolysis were reduced from 10.16 and 10.24 kcal mol⁻¹, respectively, to 5.41 and 7.55 kcal mol⁻¹, respectively, when reactions were performed in presence of Co⁺². Thus, in lipase catalyzed transesterification Cr⁺³ or Co⁺² could be added to the assay in order to produce the biodiesel in relatively shorter reaction duration.

Biodiesel synthesis by TiO2-ZnO mixed oxide nanocatalyst catalyzed palm oil transesterification process.

Biodiesel is a promising alternating environmentally benign fuel to mineral diesel. For the development of easier transesterification process, stable and active heterogeneous mixed metal oxide of TiO2-ZnO and ZnO nanocatalysts were synthesized and exploited for the palm oil transesterification process. The synthesized catalysts were characterized by XRD, FT-IR, and FE-SEM studies for their structural and morphological characteristics. It was found that TiO2-ZnO nanocatalyst exhibits good catalytic activity and the catalytic performance was greatly depends on (i) catalyst concentration (ii) methanol to oil molar ratio (iii) reaction temperature and (iv) reaction time. A highest 98% of conversion was obtained at the optimum reaction parameters with 200 mg of catalyst loading and the biodiesel was analyzed by TLC and (1)H NMR techniques. The TiO2-ZnO nanocatalyst shows good catalytic performance over the ZnO catalyst, which could be a potential candidate for the large-scale biodiesel production from palm oil at the reduced temperature and time.

"One-step production of biodiesel from Jatropha oil with high-acid value in ionic liquids" [Bioresour. Technol. 102 (11) (2011)].

Catalytic conversion of un-pretreated Jatropha oil with high-acid value (13.8 mg KOH/g) to biodiesel was studied in ionic liquids (ILs) with metal chlorides. Several commercial ILs were used to catalyze the esterification of oleic acid. It was found that 1-butyl-3-methylimidazolium tosylate {[BMIm][TS]} had high catalytic activity with 93% esterification rate for oleic acid at 140 °C but only 63.7% Jatropha biodiesel yield at 200 °C. When ZnCl2 was added to [BMIm][TS], a maximum Jatropha biodiesel yield of 92.5% was achieved at 180 °C. Addition of metal ions supplied Lewis acidic sites in ILs promoted both esterification and transestrification reactions. It was also found that the transition metal ions performed higher catalytic activity in transestrification than the ions of Group A. Mixture of [BMIm][TS] and ZnCl2 was easily separated from products for reuse to avoid producing pollutants.

Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation.

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.

Transesterification of rapeseed oil for biodiesel production in trickle-bed reactors packed with heterogeneous Ca/Al composite oxide-based alkaline catalyst.

A conventional trickle bed reactor and its modified type both packed with Ca/Al composite oxide-based alkaline catalysts were studied for biodiesel production by transesterification of rapeseed oil and methanol. The effects of the methanol usage and oil flow rate on the FAME yield were investigated under the normal pressure and methanol boiling state. The oil flow rate had a significant effect on the FAME yield for the both reactors. The modified trickle bed reactor kept over 94.5% FAME yield under 0.6 mL/min oil flow rate and 91 mL catalyst bed volume, showing a much higher conversion and operational stability than the conventional type. With the modified trickle bed reactor, both transesterification and methanol separation could be performed simultaneously, and glycerin and methyl esters were separated additionally by gravity separation.