Effect of ionic liquids,organic solvents and supercritical CO2 pretreatment on the conformation and catalytic properties of Candida rugosa lipase

The objective of this work is to assess the structure and activity of Candida rugosa lipase (CRL) pretreated with seventeen ionic liquids (ILs), five organic solvents and super-critical carbon dioxide (SC-CO₂). The results revealed that anion selection of ILs showed generally much greater effects on CRL esterification activity than cation choice, and CRL pretreated by ILs with strong water miscible properties showed very low esterification activity. The highest CRL activity treated with ILs [Hmim][PF6] was obtained with the value of 45078.0 U/g-protein. Furthermore, the CRL activities pretreated with five conventional organic solvents were also examined and the values increased with the log P decrease of organic solvents when log P was lower than 2.0. Finally, the CRL activities were respectively 1.2- and 1.3-fold higher over the untreated ones after pretreatment with sub- and super-critical CO₂ under the pressures of 6 MPa and 15 MPa at 40 °C for 20 min. Further analyses via FT-IR demonstrated that the high activity of CRL pretreated with ILs, organic solvents and SC-CO₂ was probably caused by the changes of CRL secondary structure. In conclusion, the results in this work will be helpful for us to choose the suitable reaction medium in CRL biocatalysis and biotransformation reactions.     

Pilot-scale supercritical carbon dioxide extractions for the recovery of triacylglycerols from microalgae: a practical tool for algal biofuels research

We describe the preliminary extractions from a pilot-scale supercritical carbon dioxide (SC-CO₂) extractor for the isolation of algal lipids suitable for small-scale conversion to liquid hydrocarbon fuels. Flowable oils were recovered from SC-CO₂ extractions of lyophilized Nannochloropsis granulata. The extracted oils were determined to be composed primarily of triacylglycerols (TAG) by liquid chromatography–mass spectrometry analysis. Gravimetric lipid yield was increased significantly from 15.56 to 28.45 mg g^?1 ash-free dry weight (AFDW) with an increase in temperature from 50°C to 70°C, at 35 MPa over 270 min. Varying pressure had no significant effect on lipid yield. Liquid chromatography–mass spectrometry analysis of the SC-SO₂ extracts indicated that the TAG profile remained constant regardless of extraction pressure, and analysis of fatty acid methyl esters (FAME) revealed a uniform profile throughout all extraction conditions. Our optimized gravimetric lipid yields from N. granulata (28.45 mg g^?1 AFDW) were approximately half of the yields obtained by Soxhlet extraction with hexane (57.53 mg g^?1 AFDW); however, the FAME yields were similar regardless of extraction technique (18.23 mg FAME g^?1 and 17.35 mg FAME g^?1 AFDW from SC-CO₂ extraction and hexane extraction, respectively). Further extractions with Botryococcus braunii indicated that fatty acid extraction by SC-CO₂ was as efficient as hexane extraction. These results highlight the suitability of SC-CO₂ for large-scale oil extraction of microalgae for biofuel or biojet analyses due to its selectivity for TAG extraction.     

Optimization of supercritical carbon dioxide extraction of silkworm pupal oil applying the response surface methodology

Supercritical carbon dioxide extraction (SC-CO₂) of oil from desilked silkworm pupae was performed. Response surface methodology (RSM) was applied to optimize the parameters of SC-CO₂ extraction. The effects of independent variables, including pressure, temperature, CO₂ flow rate, and extraction time, on the yield of oil were investigated. The statistical analysis showed that the pressure, extraction time, and the quadratics of pressure, extraction time, and CO₂ flow rate, as well as the interactions between pressure and temperature, and temperature and flow rate, showed significant effects on oil yield. The optimal extraction condition for oil yield within the experimental range of the variables researched was at 324.5 bar, 39.6 °C, 131.2 min, and 19.3 L/h. At this condition, the yield of oil was predicted to be 29.73%. The obtained silkworm pupal oil contained more than 68% total unsaturated fatty acids, and alpha-linolenic acid (ALA) accounted for 27.99% in the total oil.     

Development of a protocol for supercritical carbon dioxide extraction of ubiquinone-10 from dried biomass of <Emphasis Type="Italic">Pseudomonas diminuta</Emphasis>

Ubiquinone (Coenzyme Q10, CoQ10), a yellow-to-orange-colored lipophilic substance having nutraceutical value, was extracted from dried biomass of Pseudomonas diminuta using supercritical carbon dioxide (SC-CO₂). The effect of different operational parameters (temperature, pressure, and extraction time) and addition of co-solvent on SC-CO₂ extraction of CoQ10 was studied in detail. The solubility parameter of CoQ10, CO₂, and CO₂ with ethanol and methanol as co-solvents was calculated and validated with experimental results. Theoretically, ethanol and methanol had significant effect as co-solvent, and the difference between the two was only marginal. A maximum recovery of 22.33% was obtained using pure SC-CO₂ at 40 °C, 150 bar, and run time of 60 min. Ethanol as co-solvent at 3 mL/g of dried biomass at 40 °C and 150 bar increased the recovery from 22.33 to 68.57%. Further optimization of the extraction conditions by Box–Behnken design effectively increased the recovery to 96.2%. The optimized conditions were a temperature of 38 °C, pressure of 215 bar, and run time of 58 min.     

Pretreatment of sugarcane bagasse using supercritical carbon dioxide combined with ultrasound to improve the enzymatic hydrolysis

This work evaluates the pretreatment of sugarcane bagasse combining supercritical carbon dioxide (SC-CO₂) and ultrasound to enhance the enzymatic hydrolysis of pretreated bagasse. In a first step the influence of process variables on the SC-CO₂ pretreatment to enhance the enzymatic hydrolysis was evaluated by mean of a Plackett–Burmann design. Then, the sequential treatment combining ultrasound + SC-CO₂ was evaluated. Results show that treatment using SC-CO₂ increased the amount of fermentable sugar obtained of about 280% compared with the non-treated bagasse, leading to a hydrolysis efficiency (based on the amount of cellulose) as high as 74.2%. Combining ultrasound + SC-CO₂ treatment increased about 16% the amount of fermentable sugar obtained by enzymatic hydrolysis in comparison with the treatment using only ultrasound. From the results presented in this work it can be concluded that the combined ultrasound + SC-CO₂ treatment is an efficient and promising alternative to carry out the pretreatment of lignocellulosic feedstock at relatively low temperatures without the use of hazardous solvents.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

Extractability of polyphenols of sunflower seed in various solvents

The extractability of chlorogenic acid from defatted sunflower seed flour in water and salt solutions at different pH values and also in aqueous organic solvents was determined. It increased with increase in pH and at pH 8 in water nearly 70% chlorogenic acid was removed in a single extraction, while NaCl did not increase the extraction, and, MgCl₂ and CaCl₂ increased it, especially at higher concentrations. Methanol, ethanol, isopropanol and acetone, at 20% concentration in water, caused the maximum extraction of polyphenol. These organic solvents without added water were poor solvents for the extraction of polyphenol from the flour.     

Identification of anti-lung cancer extract from Chlorella vulgaris C-C by antioxidant property using supercritical carbon dioxide extraction

In this study, supercritical carbon dioxide extraction (SC-CO₂) technology was developed to extract the active components (such as antioxidants) from a novel microalga, Chlorella vulgaris C-C, due to its superior advantages over conventional solvent or ultrasonic extraction methods. Using SC-CO₂ and ultrasonic extractions, the polyphenol contents of C. vulgaris C-C were 13.40 and 0.46 (mg_{gallic acid}/g lyophilized extract), respectively. The flavonoid content obtained from SC-CO₂ (3.18 mg_quercetin/g lyophilized extract) was also significantly higher than from ultrasonic extraction (0.86 mg_quercetin/g lyophilized extract). Investigation of C. vulgaris C-C extract from SC-CO₂ indicates strong antioxidant activities in radical scavenging, ferric reducing power and metal chelating abilities. In cell proliferation assay, the extract of C. vulgaris C-C inhibits human lung cancer H1299, A549, and H1437 cells in a dose-dependent manner. In addition, the treatment with extracts of C. vulgaris C-C effectively reduced the migration of tumor cells, suggesting the potential of using the C. vulgaris C-C extract to inhibit lung cancer metastasis.     

Durum wheat by-products as natural sources of valuable nutrients

This review reports the use of wheat milling by-products for the extraction of high quality oil and vitamin E including our results on the exploitation of durum wheat bran as a valuable source of important healthful compounds. Wheat oil can be used as an ingredient in food, pharmaceutical or cosmetic preparations because it contains important bioactive compounds such as vitamin E, carotenoids and unsaturated fatty acids. Different methods are used for oil recovery from plant materials, such as solvent extraction, mechanical pressing or the eco-friendly supercritical carbon dioxide (SC-CO₂) extraction technology. By using SC-CO₂, we obtained an oil from durum wheat (Triticum durum Desf.) bran and optimized the extraction conditions to increase oil and vitamin E yields. Wheat bran, which is composed of pericarp, aleurone layer and germ, is discarded during the early stages of durum wheat milling processes to obtain a final product (semolina) that is stable over time. Maximum oil and vitamin E yields were obtained when a durum wheat bran matrix with particle size of ~30 mesh and a moisture content of 2.6 % was used. The optimal conditions for oil extraction were: 300–350 bar, 60–70 °C, and 4 l min^?1 gaseous CO₂ flow rate for 1 h. The chemical composition (vitamin E forms, carotenoids, quinones, lipids and fatty acids) of the SC-CO₂ extracted oil was analyzed and compared to that of the oil extracted by Soxhlet using hexane as solvent. The findings here reported highlight the importance of durum wheat bran as a rich source of valuable natural nutrients.     

Omega-3 fatty acids concentrate production by enzyme-catalyzed ethanolysis of supercritical CO<Subscript>2</Subscript> extracted oyster oil

The separation of oil by a suitable technique from the Pacific oyster muscle is important for the utilization of the oil as a ω-3 polyunsaturated fatty acids (ω-3 PUFAs) source and production of bio-functional peptides/ oligosaccharides from oil-free residue. This study was conducted to prepare ω-3 PUFAs concentrate from supercritical carbon dioxide (SC-CO₂) extracted Pacific oyster oil by enzyme-catalyzed ethanolysis reactions. SC-CO₂ extractions were done at different temperatures and pressures to optimize suitable extraction conditions and extracted oils were compared with Soxhlet (n-hexane) extracted oil to evaluate the yield and quality. Oil extracted by SC-CO₂ at optimized conditions was used for ethanolysis reaction catalyzed by immobilized sn-1,3 specific lipases, namely Novozymes-435, Lipozyme TLIM, and Lipozyme RMIM to produce 2-monoacylglycerols (2-MAG) rich in ω-3 PUFAs. The optimum temperature and pressure for SC-CO₂ extractions of oyster oil was 50°C and 30 MPa. In this condition, the yield of oil was 5.96% and the acid, peroxide, free fatty acid, and p-anisidine values were 4.49 mg KOH/g, 4.72 meq/kg, 3.42%, and 10.03, respectively. The ω-3 PUFAs content significantly increased in 2-MAG obtained from Novozymes 435, Lipozyme TLIM, and Lipozyme RMIM to 43.03 ± 0.36, 45.95 ± 0.29, and 40.50 ± 0.77%, respectively (p < 0.05). A thin layer chromatography (TLC) analysis confirmed the production and separation of 2-MAG in the ethanolysis process. The ratio of total ω-3 to ω-6 fatty acids was almost twice in 2-MAG of SC-CO₂ extracted oyster oil. SC-CO₂ extracted Pacific oyster oil can be used for sn-1,3 specific lipases catalyzed ethanolysis to produce ω-3 PUFAs rich in 2-MAG.     

Inactivation of Alternaria brassicicola spores by supercritical carbon dioxide with ethanol entrainer

Supercritical carbon dioxide (SC-CO₂) was used to inactivate fungal spores of Alternaria brassicicola. The inactivation conditions were optimized using response surface methodology (RSM). When the SC-CO₂-entrainer (ethanol) system was applied to fungal spores, the treatment time required for the complete inactivation of fungal spores was substantially reduced.     

Sterols of the lichen Pseudevernia furfuracea

A mixture of C₂₇, C₂₈ and C₂₉ sterols was isolated from the lichen Pseudevernia furfuracea and characterized by means of GLC and MS. Mono-, di- and tri-unsaturated sterols were identified as well as a small amount of fully saturated sterols (stanols). Only a part of the total sterols present in the lichen tissue was easily extractable with organic solvents. Another portion was only obtained after saponification of the lichen residue remaining after extraction with organic solvents. The composition of these two fractions difrered considerably, the former contained predominantly 5a,8a-epidioxy-5a-ergosta-6,22-dien-3β-ol (ergosterol peroxide) and 24-ethylcholesta-5,22-dien-3β-ol while in the latter 24-ethylcholesta-5,22-dien- 3β-ol and C₂₈ triene sterols were the main components.     

Dextrin and salt as impurities of histological dyes

Summary The dextrin and salt contents of 18 common histological dyes (usually obtained from more than one supplier) were estimated by solvent extraction of dye from the commercial sample with organic solvents such as ethanol, iso-propanol and methanol. The procedure is also suitable for preparing large quantities of dextrin and salt-free dyes. The dye contents of the commercial dye samples varied widely:many commercial dyes were pure but nearly half the samples contained less than 75% dye. The solvent insoluble residues from 10 commercial pyronin and rhodamine dyes were studied in more detail. Tests for dextrin and for NH ₄ ⁺ , Cl^– and SO ₄ ^2– ions were applied and the dextrin:salt ratio determined by combustion. Only 3 of the residues contained appreciable amounts of salt. Certain samples contained organic materials other than dextrin.     

Application of Supercritical Fluid Extraction in Biotechnology

ABSTRACT

In the present paper recent investigations on the applications of supercritical fluid extraction (SCE) from post fermentation biomass or in situ extraction of inhibitory fermentation products as a promising method for increasing the yield of extraction have been reviewed. Although supercritical CO₂ (SC-CO₂) is unfriendly, or even toxic, for some living cells and precludes direct fermentation in dense CO₂, it does not rule out other useful applications for in situ extraction of inhibitory fermentation products and fractional extraction of biomass constituents. This technique is a highly desirable method for fractional extraction of biomass constituents, and intracellular metabolites due to the potential of system modification by physical parameters and addition of co-solvents to selectively extract compounds of different polarity, volatility and hydrophilicity without any contamination.     

Deactivation of isoamylase and β-amylase in the agitated reactor under supercritical carbon dioxide

The current research examines the impact of agitation on deactivation of isoamylase and β-amylase under supercritical carbon dioxide (SC-CO₂). Our experimental results showed that the activity of either enzyme decreased with increasing pressure or speed of agitation. The degree of enzymatic deactivation caused by pressure became more prominent in the presence of agitation, suggesting that the agitation plays an important role in enzymatic deactivation in SC-CO₂ environment. Moreover, the enzymatic deactivation behavior associated with agitation and pressure was further quantitatively analyzed using a proposed inactivation kinetic model. Our analysis indicated that isoamylase and β-amylase exhibited significantly different relationships between the inverse of percentage residual activity and the product of number of revolution per time and time elapsed under pressurized carbon dioxide. We believe that the outcome from this work may provide a better understanding of the effects of agitation and pressure in enzyme deactivation behavior under SC-CO₂.     

Supercritical carbon dioxide pretreatment of corn stover and switchgrass for lignocellulosic ethanol production

Supercritical CO₂ (SC-CO₂), a green solvent suitable for a mobile lignocellulosic biomass processor, was used to pretreat corn stover and switchgrass at various temperatures and pressures. The CO₂ pressure was released as quickly as possible by opening a quick release valve during the pretreatment. The biomass was hydrolyzed after pretreatment using cellulase combined with β-glucosidase. The hydrolysate was analyzed for the amount of glucose released. Glucose yields from corn stover samples pretreated with SC-CO₂ were higher than the untreated sample’s 12% glucose yield (12 g/100 g dry biomass) and the highest glucose yield of 30% was achieved with SC-CO₂ pretreatment at 3500 psi and 150 °C for 60 min. The pretreatment method showed very limited improvement (14% vs. 12%) in glucose yield for switchgrass. X-ray diffraction results indicated no change in crystallinity of the SC-CO₂ treated corn stover when compared to the untreated, while SEM images showed an increase in surface area.     

Application of supercritical CO2 for extraction of polyisoprenoid alcohols and their esters from plant tissues

In this study, a method of supercritical fluid extraction (SFE) with carbon dioxide of polyisoprenoids from plant photosynthetic tissues is described. SFE was an effective extraction method for short- and medium-chain compounds with even higher yield than that observed for the “classical extraction” method with organic solvents. Moreover, SFE-derived extracts contained lower amounts of impurities (e.g., chlorophylls) than those obtained by extraction of the same tissue with organic solvents. Elevated temperature and extended extraction time of SFE resulted in a higher rate of extraction of long-chain polyisoprenoids. Ethanol cofeeding did not increase the extraction efficiency of polyisoprenoids; instead, it increased the content of impurities in the lipid extract. Optimization of SFE time and temperature gives the opportunity of prefractionation of complex polyisoprenoid mixtures accumulated in plant tissues. Extracts obtained with application of SFE are very stable and free from organic solvents and can further be used directly in experimental diet supplementation or as starting material for preparation of semisynthetic polyisoprenoid derivatives, e.g., polyisoprenoid phosphates.     

Solvent effect on the synergic extraction of thulium(III) with acetylacetone and 1,10-phenanthroline

The synergic extraction equilibrium of Tm(III) with acetylacetone (Hacac) and 1,10-phenanthroline (phen) in various organic solvents has been studied. The adduct formation constants, β_s, for Tm(acac)₃^? phen, were determined in heptane, cyclohexane, carbon tetrachloride, benzene and chloroform. The solvent effect on β_s is explained in connection with the activity coefficients of the neutral ligand, the chelate, and the adduct in the organic solvent. The activity coefficients can be calculated from the corresponding solubility parameters on the basis of the regular solution theory, and the solubility parameters of the solutes were estimated from their two-phase partition coefficients. It is demonstrated that β_s in different organic solvents except those having a specific interaction with the solute, such as chloroform, can be calculated by the present approach.     

Starch hydrolysis using enzyme in supercritical carbon dioxide

Summary The enzymatic hydrolysis reaction with supercritical carbon dioxide(SC-CO₂) as a reaction medium to make glucose from starch was investigated. The reaction rate was enhanced at higher temperature and pressure, especially near the critical point of the CO₂. The -amylase and glucoamylase were found to be active in a SC-CO₂.     

Accumulation of Xanthine Derivatives in the Culture Broths of Bacteria Incubated with Decoyinine

The present report describes the successful isolation procedure and some physico-chemical properties of an unknown blue-violet fluorescent substance existing in spinach leaves. Through the isolation steps of water extraction of leaves (200 kg), active carbon adsorption, extraction by organic solvents, and column chromatographies with various adsorbents and ion exchange resins, a purified compound was obtained in colorless planeary crystal of long parallelogram (40 mg; mp. 195°C; C₁₃H₁₇O₉N₁). The UV-, IR- and NMR-spectra, the results of organic tests, and behaviors on chromatographies and electrophoreses are presented.