[Show abstract][Hide abstract] ABSTRACT: The sterilization of microfluidic chips is a vital step of the fabrication process prior to the customer use in biomedical applications. The aim of this study was to analyze the influence of different sterilization techniques and to compare the characteristics of the material before and after sterilization of polymethylmethacrylate (PMMA) microchips. For this, supercritical carbon dioxide (SC-CO2) along with standard sterilization methods such as ultraviolet (UV), heat (autoclaving), ethylene oxide (EtO) and hydrogen peroxide (H2O2) were applied. The treated microchips were analyzed by Scanning Electron Microscopy, Differential Scanning Calorimetry, Fourier Transform Infrared Spectroscopy and Laser Scanning Microscopy in order to ascertain any changes in the chemical structure and surface morphology. The optimum sterilization parameters for SC-CO2 were elicited as 120 bar, 40°C and 60 min which provided complete sterility and did not alter the main properties of the polymer along with EtO and H2O2 sterilizations unlike heat and UV treatments. However, surface roughness and microchannel profiles were negatively affected. Although complete sterility was achieved, each protocol has its own strengths and weaknesses.
Full-text · Article · Jan 2016 · Journal of Supercritical Fluids The
[Show abstract][Hide abstract] ABSTRACT: In this paper, the extraction conditions yielding the highest steviol glycosides from the leaves of Stevia rebaudiana were determined using microwave (MAE) and ultrasonically assisted extraction (UAE) and biological activities of the extracts along with analyses of the residue from the extraction process were investigated. Under optimum conditions, 21.21mg glycosides/g dried leaf in MAE and 14.90mg glycosides/g dried leaf in UAE were quantified by HPLC analyses. After extraction, total chlorophyll, carotenoid contents and total dietary fibers were quantified as 15.14mg/100g, 2.93mg/100g and 6.5% in the raffinate phase. The total phenols were determined as 80.13 and 86.47mg gallic acid/g extract, whereas the total flavonoids were 111.16 and 126.70mg quercetin/g extract and DPPH radical scavenging activities were 91.39 and 92.40%, respectively. The extracts exhibited no cytotoxicity against healthy cell line and macrophage RAW 264.7 cells. The IC50 values were 68μg/ml and >100μg/ml for MAE and UAE. Overall, obtained results suggest that stevia extracts and its residue can be utilized holistically on industrial scale.
No preview · Article · Dec 2015 · Industrial Crops and Products
[Show abstract][Hide abstract] ABSTRACT: Blueberries are best known for their high content of flavonoids and other phenolic compounds which provide health benefits. The objective of this study was to investigate the effect of microwave, pressurized solvent, and supercritical carbon dioxide (SC-CO2) extraction techniques on the anthocyanin content of the water extracts obtained from dry whole blueberries. Effect of the blueberry extracts on selected cancer cell lines was also investigated. Neat and ethanol entrained SC-CO2 extracts did not contain detectable amount of anthocyanin. Microwave power did not have a significant effect on anthocyanin recovery. The blueberry juice (BJ) prepared by homogenization of fresh berries followed by microfiltration had the highest anthocyanin content among the extracts examined in this study. However, the blueberry water extracts prepared using other extraction techniques had a higher total phenolic content than the BJ. The effect of the blueberry extracts on the cancer cell lines was dose-dependent and varied with the cell type and the composition of the extract. BJ had a similar IC50 to the commercial cancer treatment drug doxorubicin for the cancer cell line MCF7. Further research is needed to elucidate the mechanism underlying the effect of blueberry extract composition on the viability of different cancer lines.
No preview · Article · Nov 2015 · European Food Research and Technology
[Show abstract][Hide abstract] ABSTRACT: Enzyme activity enhancement represents a great opportunity for biotechnological production, while recovery of most enzymes from media, possible loss of catalytic activity in reactions, and denaturation occur. In this study, the activity and stability enhancement, kinetic parameters, and thermal inactivation for cellulose hydrolysis of supercritical carbon dioxide (SC-CO2)-treated and untreated cellulase from Trichoderma longibrachiatum as a model enzyme are presented, and the activity enhancement capability of SC-CO2 for cellulase after consecutive enzymatic reactions is discussed. The pH and temperature stability were pH 5 and 50 °C for the enzymatic reaction, whereas the kinetic parameter values, V
max and K
m, were calculated using the Michaelis–Menten model. The optimal operational parameters were determined to be 54 °C, 180 bar, and 10 g/min CO2 flow rate for 120 min, yielding 48.3 % higher activity (9.27 μmol/ml/min) than for untreated enzyme. In addition, SC-CO2-treated cellulase with the highest activity was immobilized on NaY zeolite, and consecutive reactions were carried out. The presented results suggest that enzymes as catalysts in biochemical applications can be improved by using supercritical fluids as potential media.
[Show abstract][Hide abstract] ABSTRACT: Biomicrofluidics is an emerging field at the cross roads of microfluidics and life sciences which requires intensive research efforts in terms of introducing appropriate designs, production techniques, and analysis. The ultimate goal is to deliver innovative and cost-effective microfluidic devices to biotech, biomedical, and pharmaceutical industries. Therefore, creating an in-depth understanding of the transport phenomena of cells and biomolecules becomes vital and concurrently poses significant challenges. The present article outlines the recent advancements in diffusion phenomena of cells and biomolecules by highlighting transport principles from an engineering perspective, cell responses in microfluidic devices with emphases on diffusion- and flow-based microfluidic gradient platforms, macroscopic and microscopic approaches for investigating the diffusion phenomena of biomolecules, microfluidic platforms for the delivery of these molecules, as well as the state of the art in biological applications of mammalian cell responses and diffusion of biomolecules.
No preview · Article · Sep 2015 · Biomicrofluidics
[Show abstract][Hide abstract] ABSTRACT: Leather is a porous material composed of a three-dimensional weave of collagen fiber bundles. The high pressure tanning (HPT) of sheepskins with valonea tannin in supercritical CO2 was approached to investigate the diffusion process through the skin matrix. Uptake of vegetable tanning agent (VTA) was analyzed at 100 bar and 32 °C with varying tanning times (2–8 h). Shrinkage temperature (Ts) as thermal stability of the tanned collagen and filling coefficient (%) of pressurized vegetable tanning (PVT) were also analyzed. The best results were obtained at 8 h treatment yielding 83.77% of VTA uptake and a filling coefficient of 54.97%. PVT experiments showed a satisfactory conversion of the skins to leather in terms of the thermal stability. Scanning electron microscopy (SEM) of the tanned skins showed that high pressure did not alter the fiber structure and morphology negatively. The proposed technique has high potential to be deployed to industrial scale.
Full-text · Article · Jul 2015 · Journal of Supercritical Fluids The
[Show abstract][Hide abstract] ABSTRACT: In this study, the effect of binary and ternary systems, solvent/feed (S/F) ratio (20–100 w/w), time (2–3 h) and pressure (10–200 bar) were tested with subcritical and supercritical CO2 to produce valonea tannin from acorn cups. The best yield of phenolic contents (PCs) of the valonea tannin (34.73 ± 0.02 wt%) was obtained by H2O/CO2 binary system under the conditions of S/F = 100 (w/w) at 100 bar for 3 h with supercritical CO2. Phase equilibria of H2O/CH3OH/CO2 ternary system with different solvent fractions at different temperatures and pressures were also imaged in the high pressure view cell, where liquid–liquid–vapor phase behaviors were observed for all ratios of H2O and CH3OH from 60 to 100 °C and 60 to 250 bar. High tannin content isolated in a short time will decrease the waste load and provide great advantage in terms of reducing the cost of the tannin used in leather industry.
Full-text · Article · May 2015 · Separation and Purification Technology
[Show abstract][Hide abstract] ABSTRACT: Olive pomace is a by-product of olive oil production and an important biomass for the Mediterranean countries. The aim of this study was to optimize the oil extraction from olive pomace by supercritical CO2 and utilize the remaining biomass by hydrolysis. A Box–Behnken statistical design was used to evaluate the effect of temperature (30–80 °C), pressure (120–300 bar) and CO2 flow rate (10–20 g/min) for 60 min. The most effective variable was pressure (p < 0.005) and optimum extraction conditions were elicited as 68 °C, 280 bar and 20 g/min flow rate yielding 4.8% of oil. Additionally, the effect of particle size was investigated on extraction efficiency. Indeed, the oil yield was increased to 13.8% by decreasing the particle size from 2000 to 850–500 μm. For utilization of remaining biomass, both supercritical CO2 (SC-CO2) and hexane de-oiled pomaces were subjected to acid and enzymatic hydrolyses. Hydrolyses with 2% of sulfuric acid for 90 min yielded 12.30 and 12.65 g/l reducing sugar from SC-CO2 and hexane treated biomasses, whereas the highest reducing sugar concentrations achieved with enzymatic hydrolysis with a substrate amount of 6 g, 20–27% Cellic CTec2/Novozyme 188 for 24 h were 12.72 g/l and 10.13 g/l, respectively. A holistic engineering approach is proposed where supercritical CO2 can be used as a main process to extract remaining oil in olive pomace and as a pretreatment loosen the structure in order to obtain liquor which can be converted to a biofuel.
No preview · Article · Feb 2015 · Journal of Supercritical Fluids The
[Show abstract][Hide abstract] ABSTRACT: Enzymatic reactions with supercritical carbon dioxide (SC-CO2) have received increased attention during the last decade. The objective of this study was to alter the activity and stability of Bacillus sp. protease under SC-CO2 conditions. The activity and stability of protease were evaluated according to the effects of operational variables, temperature (28-80 degrees C), pressure (60-300 bar), CO2 flow (2-10 g/min) and process duration (60-180 min). Best conditions were identified as 300 bar, 54 degrees C, 6 g/min CO2 flow and 120 min of process time efficient 54.4% (417.50 mu mol/ml/min) as against to the untreated enzyme. Activity enhancement was observed with the raise of pressure while low flow rates decreased the activity by 5.1% with a flow rate of 2 g/min under supercritical conditions. Potential mechanisms for pressure stimulated activation and stabilization were investigated by NMR, SEM, FTIR, SOS-PAGE and XPS analyses. As a result, applications of SC-CO2 medium for enzymatic processes are expected to become sustainable and important with economical synthetic and environmentally friendly protocols.
No preview · Article · Nov 2014 · Biochemical Engineering Journal
[Show abstract][Hide abstract] ABSTRACT: The objectives of this work were to obtain steviol glycosides of S. rebaudiana leaves, possessing natural and noncaloric sweetener properties, using subcritical water extraction; to assess optimum extraction conditions; to determine biological activities of Stevia extracts and to characterize the raffinate phase. A Box-“Bhenken” statistical design was used to evaluate the effects of various values of temperature (100-150 °C), time (30-60 min) and flow rate (2-6 ml/min) at a pressure of 230 bar applying a solid/liquid ratio of 1:10 (m:v). The most effective parameter was temperature (p < 0.005). Optimum extraction conditions were elicited as 125 °C, 45 min, 4 ml/min flow rate which yielded 38.67 mg/g stevioside and 35.68 mg/g rebaudioside A. The total phenolic, flavonoid contents and DPPH free radical scavenging activity were found as 48.63 mg gallic acid/g extract, 29.81 mg quercetin/g extract and 92.50%, respectively. After extraction, total chlorophyll, carotenoid contents and dietary fibers were quantified as 31.91 mg/100 g, 5.71 mg/100 g and 4.98% in the raffinate phase. Hence, both extract and raffinate phases of S. rebaudiana leaves can be utilized as sources of natural sweeteners, fibers and coloring agents in the industry.
No preview · Article · Nov 2014 · Journal of Supercritical Fluids The
[Show abstract][Hide abstract] ABSTRACT: Mediterranean countries contribute highly on world peach production and tonnes of waste leaves are released due to pruning. The aim of this study was to investigate the utilization possibilities of the leaves by supercritical fluid extraction. A statistical design was used to evaluate the effect of temperature (40-80 degrees C), pressure (150-300 bar) and concentration of ethanol as co-solvent (6-20%) at a flow rate of 15 g/min and for a duration of 60 min. The most effective variables were found as pressure and co-solvent ratio (p < 0.005). Optimum extraction conditions were elicited as 60 degrees C, 150 bar and 6% co-solvent yielding a total phenol content of 79.92 mg GAE/g extract, EC50 value of 232.20 mu g/ml and a radical scavenging activity of 53.25% which was higher than the value obtained by conventional solvent extraction method (32.23%). Consequently, Prunus persica L. leaves were found as a potential phenolic source for industrial applications.
No preview · Article · Aug 2014 · Journal of Supercritical Fluids The
[Show abstract][Hide abstract] ABSTRACT: The miniaturization of continuous processes has been of interest in the academia and industry which is reflected by the increase in scientific publications and patent disclosures in the last decade. The aim of this study was to evaluate the patenting trends regarding enzyme related microfluidic applications in order to observe the progress of science and technology. The mapped patents have been classified as "immobilization method", "biomolecule screening systems", "integrated process development" and "microreactor design". Half of the patent disclosures were filed by academia, whereas the other half was from industrial research which complies with the shift in microfluidics from academic and industrial research to commercial applications. Immobilization procedures carried out at room temperatures such as formulation of silica matrices using sol-gel technique, incorporation of novel hybrid materials, the integration of supercritical fluids and microfluidics, employing ionic liquids as wall-less microreactors, designing low cost, high performance microfluidic devices were the highlights which can pose challenges in various life science applications. The increasing trend is expected to continue and the presented state-of-the-art in enzyme related microfluidic applications have the potential to enhance industry's capabilities for designing innovative systems which would demonstrate significant economic, societal and environmental benefits.
No preview · Article · Jun 2014 · Biochemical Engineering Journal
[Show abstract][Hide abstract] ABSTRACT: Echinacea species is provided as dietary supplements for various infectious and immune related disorders and has a potential role in cancer prevention. The aim of this study was to optimize the extraction of total flavonoids using different extraction methods and investigate the cytotoxic effects on various cancer cell lines (CaCo-2, MCF-7, A549, U87MG, and HeLa) and VERO (African green monkey) as a non-cancerous cell line. Box-Behnken statistical design was used to evaluate the effect of pressure (100-200 bar), temperature (40-80 degrees C) and ethanol as co-solvent (6-20 wt%) at a flow rate of 15 g/min for 60 min in supercritical CO2 extraction and the effect of temperature (60-100 degrees C), time (5-15 min) and power (300-900W) in microwave-assisted extraction. Optimum extraction conditions were elicited as 300 bar, 80 degrees C and 13% co-solvent yielding 0.472 mg rutin equivalent total flavonoids/g extract in SC-CO2 extraction, whereas 60 degrees C, 10 min and 300W yielded the highest (0.202 mg rutin equivalent) total flavonoids in microwave-assisted extraction. Additional trials with subcritical water (0.022 mg/g) and Soxhlet extraction with methanol (0.238 mg/g) yielded lower flavonoid contents. The exposures upto 50 mu g/ml of extracts revealed no significant inhibition on the proliferation of both tested cancer cells and healthy VERO cells.
No preview · Article · May 2014 · Journal of Supercritical Fluids The
[Show abstract][Hide abstract] ABSTRACT: The aim of this work was to optimize the extraction parameters of tannins from the acorns and the tanning properties. Experiments were planned to evaluate the effect of solvent/feed ratio (20–100 ml/g), time (2–10 h) and solvent mixture ratio (methanol–water) (0–100%) on extraction yields, tannin contents and total tannin amounts. Valonea tannin obtained under optimum conditions was applied to leather tanning process where filling coefficient (%) and shrinkage temperature (Ts) of the leathers were examined. Optimum extraction parameters were elicited as 100 ml/g for solvent/feed ratio, 6 h of process duration and solvent mixture ratio of 62% methanol–38% water. The filling coefficient of the leathers tanned with extracted valonea (57.81%) and the shrinkage temperature (75.5 °C) was superior to the commercial valonea (52.83%, 73 °C) implying the significant impact of process optimization. The tanning properties of the extracted valonea were satisfactory for the vegetable tanning in leather industry.
No preview · Article · Feb 2014 · Industrial Crops and Products
[Show abstract][Hide abstract] ABSTRACT: An efficient on-chip enzyme immobilized monolith microreactor for glucose 6-phosphate dehydrogenase and its extension to cellulase catalyzed reactions is reported. The monoliths were fabricated using the sol-gel technique, where two different silica precursors were investigated, tetraethoxysilane (TEOS) and ethylene glycol modified silane (EGMS) by determining the activity and aging of the enzymes in the gels. Subsequently catalytic reactions were conducted in continuous flow microreactors and the performance of the system was evaluated by applying various flow rates (5, 10, 15, 20 mu l/min). The addition of polyethylene oxide (PEO) reduced shrinkage of the gels during a period of 4 days after gel preparation suggesting a strengthened gel skeleton leading to a reduced channeling within the porous structure. In the microfluidic systems, the highest conversion rates achieved were 20.0% and 28.8% at a flow rate of 5 mu l/min with TEOS-PEO and EGMS-PEO gels. Continuous reaction exhibited better yield than batch-wise operation using same volume/enzyme ratios which can be attributed to favorable enzyme substrate affinities. However, increase in the flow rate resulted in reduced conversion rates due to lower residence times. Consequently, the applied technique not only provided shorter preparation times but also sustained the stability of immobilized enzymes.
No preview · Article · Dec 2013 · The Chemical Engineering Journal
[Show abstract][Hide abstract] ABSTRACT: Posidonia oceanica residues were extracted with supercritical CO2 in order to isolate phenolic compounds. The process was optimized by developing a mathematical model based on mass transfer mechanism consisting of adsorption of supercritical fluid on the solid particles, desorption of solute and convective transfer of solute phase along the column. Henry relation between solute concentrations on the surface of the solid (Cs) and in the solid (q) was approximated in order to describe the adsorption/desorption equilibrium. The model parameters such as solid-liquid film mass transfer coefficient (kf), molecular diffusivity coefficient (DAB) and axial dispersion (Dax) were estimated using empirical methods. The linear driving force model was applied to improve the yield of total phenolic acid recovery. The optimum parameters were elicited as 25 MPa, 323.15 K and a co-solvent mass ratio of 20% yielding 34.97 μg per gram of dry feed and the model satisfactorily described the extraction yield which can be used for scale-up purposes.
No preview · Article · Oct 2013 · Journal of Supercritical Fluids The