1 Josip Juraj Strossmayer University of Osijek, Faculty of Food Technology Osijek, Franje Kuhaca 20, HR-31000 Osijek, Croatia 2 MOL Hungarian Oil and Gas Company, H-2443 Szazhalombatta, P.O. Box 1, Hungary 3 Croatian Veterinary Institute Zagreb, Veterinary Department Vinkovci, Josipa Kozarca 24, HR-32100 Vinkovci, Croatia 4 Budapest University of Technology and Economics, Department of Chemical and Environmental Process Engineering,

We developed an efficient method for the lipase-catalyzed consecutive kinetic resolution of trans-1,2-cyclohexanediol in scCO2 with vinyl-acetate as acetyl donor catalyzed by a commercial immobilized Candida antarctica lipase B (CAL-B). The reaction was optimized in scCO2 in a batch reactor. The first acylation step is moderately enantioselective, preferring the formation of (1R,2R)-2-acetoxycyclohexane-1-ol, while the second acylation step is fully enantioselective. Michaelis–Menten type reaction constants and turnover number values were calculated. A combined extractor–enzymatic packed-bed reactor unit was designed with a residence time of a few seconds (the time requirement of a batch reaction was several hours). The reactions were performed at 10MPa and 45°C. The mean residence time in the enzymatic reactor was varied from 2 to 13s by changing the flow rate of the CO2. The implemented continuous reactor was optimized to achieve maximum productivity and enantiopure products. The optimum residence time in the reactor entirely confirmed the calculated operational parameters (calculated necessary residence time: 9.6s, measured mean residence time for full conversion: 9s). No loss of enzyme activity was observed after 28h at continuous operation.

Coronene was encapsulated in single-walled carbon nanotubes (SWNT) by vapor-phase filling at high (450 degrees C) and low (385 degrees C) temperature and by nanoextraction from supercritical carbon dioxide. The presence of coronene inside the tubes was demonstrated indirectly via the formation of double-walled nanotubes (DWNT). To this end several subsequent annealing steps were applied and monitored by Raman spectroscopy. Our results show that the encapsulation is successful with all three methods. However, high-temperature vapor filling produces adsorbed dicoronylene, the dimerized form of coronene, as a side reaction. In order to avoid dicoronylene contamination, we suggest to use low-temperature methods for the production of coronene-filled carbon nanotubes. Coronene (top) and dicoronylene (bottom) molecules.

The lipase-catalysed kinetic resolution of rac-4-phenyl-2-azetidinone was investigated in supercritical carbon dioxide (scCO2). Water (0.5mol equivalent) was used as nucleophilic donor. The effects of pressure and temperature were studied in a batch reactor (internal volume: 30mL). The optimum pressure and temperature of the β-lactam ring-opening reaction proved to be 14MPa and 70°C. Under optimum conditions, full conversion was achieved in 120h. The resulting (R)-β-phenylalanine (ee≥98%) and (S)-4-phenyl-2-azetidinone (ee≥99%) could be easily separated by scCO2 extraction of the (S)-β-lactam and subsequent washing of the enzyme with hot water to recover the amino acid.

Summary Our objectives were to establish a GC method capable of quantitative analysis of terpenoids without derivatisation and to examine the amount of β-sitosterol extracted from Morus alba L. leaf and stem bark by use of traditional organic solvent extraction and supercritical-fluid extraction (SFE). To measure β-sitosterol content without derivatization, GC-FID was used with 5-α-cholestan-3-one as internal standard. To identify terpenoid constituents, GC-MS was used; β-sitosterol, phytol, lanost-7-en-3-on, α-amyrin, β-amyrin, and lupeol were identified. We established that for Morus leaf the best SFE method for β-sitosterol was pilot scale SFE; the β-sitosterol content of this extract was higher than that of the hexane solvent extract. Among analytical SFE conditions, 200 bar for 90 min and 300 bar for 60 min resulted in extraction of the most β-sitosterol. For mulberry stem bark, solvent extraction with hexane and SFE at 400 bar and 40°C for 60 min proved the best methods.

Qualitative and quantitative analysis was performed on supercritical-fluid and conventional Soxhlet extracts of Betula pendula Roth., Alnus glutinosa (L.) Gaertn., and Platanus hybrida Brot. bark. The effect of the two extraction methods on extraction yields was compared. Lupeol and beta-sitosterol were identified in the bark extracts by TLC and by GC-MS. The main components were betulin and lupeol followed by beta-sitosterol; betulinic acid seemed to be a minor constituent. Betulin content was determined by RP-HPLC, with acetonitrile-water 80: 20 (v/v) as mobile phase. Comparison of the extraction methods showed that supercritical-fluid (scCO(2) + EtOH) and ethanolic Soxhlet extraction resulted in the highest extraction yields. Accumulation of betulin derivatives was higher in supercritical-fluid extracts (scCO(2) + EtOH) than in conventional Soxhlet extracts.

The effects of supercritical fluid extraction (SFE) conditions (pressure and temperature) on extraction yield and recoveries of biologically active components were studied using a 3(2) full factorial design. Pilot plant SFE experiments were performed in a 5 x 10(-3) m(3) Volume high-pressure vessel. The pressure and temperature were varied over the ranges of 100-450 bar and 40-60 degrees C, respectively. The yield and recoveries were compared to those obtained with n-hexane and ethanol (96%, v/v) extractions. The extract samples were analysed by TLC, TLC-densitometry, GC and HPLC methods.The obtained yields changed between 1.9 and 65.7 g kg(-1), according to the solvent power of the supercritical fluid. The recoveries of the different minor components were (g minor components kg(-1) dried raw material): 0.06-1.06 rotundifurane, 0.02-1.08 beta-sitosterol, 0.04-0.63 beta-amyrin, 0.87-2.71 casticin.By evaluation the designed experiments 450 bar and 45 degrees C were chosen as the best conditions within the ranges investigated. (C) 2008 Elsevier B.V. All rights reserved.

The effectiveness and selectivity of different extraction procedures (hydrodistillation, Soxhlet extraction with organic solvents and supercritical fluid extraction) was compared. The main volatile constituents (geraniol, nerol, geranial, neral, geranyl acetate and neryl acetate) of the traditional essential oil obtained by hydrodistillation and supercritical fluid extracts of Dracocephalum moldavica L. were analysed by the gas chromatographic–mass spectrometric method. The supercritical fluid extraction was carried out using fluid carbon dioxide. Extracts were collected by stepwise precipitation in two separators. Fractioned extraction was also performed by interrupting the extraction process at determined time intervals and collecting four succesive fractions from the second separator. The main volatile constituents of the essential oil were geraniol and geranyl acetate. The supercritical extract collected at the beginning of the extraction process was richer in geranyl acetate and neryl acetate and poorer in geraniol than the conventional essential oil. Copyright © 2006 John Wiley & Sons, Ltd.

In this work the supercritical carbon dioxide extraction of biologically active, high valued compounds such as carotenoids (lycopene, β-carotene), tocopherols, fatty acids and sitosterols from industrial tomato waste was investigated. The differences of two samples of different sources and the effect of air-dried and deep-frozen storages were also reported. The effects of extraction parameters (pressure and temperature) on the extraction yields and the compositions of the products were determined with statistical analysis. Supercritical fluid extraction was compared to conventional extraction processes of tomato pomace samples. The extracts were analysed by gas chromatography, by high-performance liquid chromatography and by TLC-densitometry. The extraction yields and the amounts of lycopene, tocopherols and sitosterols depend on the experimental conditions. The product obtained by supercritical CO2 extraction at 460bar and 80°C contained the highest concentration of carotenoids with 90.1% of lycopene, while products rich in tocopherols and phytosterol were obtained above 300bar and 40°C.

Pilot-scale supercritical fluid extraction of okra seeds was carried out, using carbon dioxide as solvent, at temperatures of 40, 50 and 60 degrees C and pressures of 150, 300 and 450 bar. Laboratory-scale Soxhlet extraction of the ground seeds was carried out with ethanol and n-hexane. The yields of supercritical fluid extraction and n-hexane Soxhlet extractions were similar. The ethanol Soxhlet extraction gave the highest yield, but the concentrations of beta-sitosterol and tocopherols in this extract were lower than in the supercritical fluid extraction product. The fatty acid profiles of the extracts were determined, and a high unsaturated/saturated ratio was observed. The fatty acid compositions were only slightly different for oils obtained by the different extraction methods. (c) 2005 Society of Chemical Industry.

Lipase-catalyzed acylation of 3-benzyloxypropane-1,2-diol with vinyl acetate as acyl donor using different lipases [porcine pancreas lipase (PPL), Lipase AK "Amano", Lipase PS "Amano", and crude enzymes from Trichoderma reesei RUT-C30, Thermoascus thermophilus (NRRL5208), Talaromyces emersonii (NRLL3221)] was studied in supercritical carbon dioxide (scCO(2)). In the reactions catalyzed by different lipases different amounts of monoacetate and diacetate products along with minor amounts of cyclic acetals forming from the diol and acetaldehyde were obtained. Application of Lipase AK led to the highest conversion (84.7%) and the highest enantiomeric excess values (ee(mononetates) = 38%, ee(diacetate) = 85%). Effect of water content of scCO(2) on the productivity and the enantiomer selectivity of the reactions with Lipase AK was also investigated. (c) 2005 Elsevier B.V. All rights reserved.

Antioxidant properties of marjoram (Origanum majorana L.) herb and extracts obtained with ethanol, n-hexane, and supercritical CO2 extraction are presented. Individual antioxidants, ursolic acid, carnosic acid, and carnosol, were quantified with high-performance liquid chromatography. The effects of different parameters (temperature and pressure) of high-pressure extraction on the yield of carnosol were studied. Furthermore, two marjoram herbs from Hungary and Egypt were compared measuring hydrogen-donating abilities with 1,1-diphenyl-2-picrylhydrazyl by spectrophotometric and the total scavenger capacities by chemiluminometric methods from the aqueous extracts of the herbs. The antioxidant activities of the solvent extracts were performed using the Rancimat method. The Egyptian herb and its extracts possessed better antioxidant activities than Hungarian ones. Applying supercritical CO2 extraction, the highest value of carnosol was obtained at 400 bar and 60 degreesC.

Volatile components of marjoram (Origanum majorana L.) essential oil obtained by hydrodistillation and extracts obtained by solvent extraction with ethyl alcohol and supercritical fluid extraction (SFE) were investigated. The compositions of volatile compounds in essential oil, ethanolic and SFE extracts were determined by GC and GC–MS. The antimicrobial properties of marjoram solvent extracts were investigated with microbiological tests against food borne fungi and bacteria strains. Extracts obtained by SFE at high pressure and temperature showed significantly stronger antimicrobial properties in comparison to the slight inhibitory effects of the ethanolic extract. The results support the notion that extracts obtained by SFE might have a role as flavourings and natural colourants as well as use as preservatives in food and cosmetic systems.

A novel, organic solvent free procedure was developed for separation of enantiomers of a racemate. Partial diastereomeric complex formation of trans-2-chloro-cyclohexan-1-ol and O,O′-dibenzoyl-(2R,3R)-tartaric acid monohydrate was performed in the melt of the alcohol. The highest resolution effiency was achieved, when the unreacted alcohol (R,R-enantiomer, e.e.=43.5%, yield=45.3%) was extracted with supercritical carbon dioxide at mild conditions (100 bar, 33 °C). Then in a second step the diastereomeric complex was in situ decomposed in the extractor (200 bar, 73 °C) and the S,S-enantiomer (e.e.=61.6%, yield=31.9%) was also extracted. These results are much better than those achieved by conventional methods. Decomposition temperature of the complex (73 °C) was much lower than that at atmospheric pressure (>100 °C), which indicates a great advantage of the supercritical conditions. The raffinate of the fractionated extraction was reused for new resolutions without further treatment. Effects of the amount of resolving agent and extraction pressure and temperature of the first extraction step were studied in details.

Cellulase enzyme production by Trichoderma reesei RUT C30 was examined on two different carbon sources, delignified pine pulp (Solka Floc, SF) and glucose. In order to determine the total amount of enzymes produced, besides measuring the extracellular cellulase and beta-glucosidase activities, intracellular enzyme activities were also determined. Cell disruption was performed using supercritical carbon dioxide.Cellulase activity could be detected only on SF, which is an inducer of cellulase production, whereas equal amounts of beta-glucosidase activity could be observed on both carbon sources. When examining the "total" (extra and intracellular) cellulase and beta-glucosidase activities achieved on SF carbon source, a ratio of 1:1.14 was found. This means that in the cell an optimal ratio of enzymes is produced but beta-glucosidase is partly cellbound. The results also emphasise the difference in the production of beta-glucosidase and the other components of the cellulase enzyme complex, that is, while the endo- and exoglucanases are inductive enzymes, beta-glucosidase enzyme is produced constitutively.

The aim of this work is to demonstrate our results on comparison of composition of essential oil fractions obtained by traditional steam distillation and supercritical fluid extraction. The plant materials for the various extraction methods were selected from the Lamiaceae, Apiaceae and Asteraceae families. For the supercritical fluid extraction (SFE) carbon dioxide was used as supercritical solvent. The extracts were collected by stage wise precipitation in two separators. The waxy product and extract rich in essential oil were collected in the 1st and in the 2nd separator respectively. The traditional water steam distillation (SD) was carried out in the special apparatus of the Hungarian Pharmacopoea (7th ed.). GC analysis was carried out on capillary silica fused columns coated with DB-1701 and the specific chiral columns coated with Rt-beta DEX m or Rt-beta DEX sm. Comparing the composition of steam distilled oils with that of volatile SFE fractions the following general characteristics were established. The SFE fractions were richer in monoterpene-esters and poorer in alcohols than the traditional essential oils (clary sage, lavander, moldavian dragonhead). Regarding the distribution of the monoterpene and sesquiterpene compounds, the SFE fractions contained sesquiterpenes in higher percentage than the distilled oils (Salvia fruticosa). Furthermore, the proportion of sesquiterpenes increased in SFE fractions collected successively with time (Salvia officinalis) similar to the ratio of oxygenated monoterpenes to monoterpene hydrocarbons (Rosmarinus officinalis). The phtalides of lovage (Satureja hortensis) did not show regular change during the supercritical extraction. In other cases it was verified that part of the mono- and sesquiterpenes were present originally in bound form (glycosides) in plants. Thus they appeared only in essential oil fractions after previous acidic treatment (Thymus, Origanum, Satureja species). During the super-critical extraction the azulenogene sesquiterpene lactones did not transform to azulenes (chamomile, yarrow), but SFE fractions of some Asteraceae plants contained sesquiterpene-gamma-lactones of unchanged structure.

For the production of thermostable endoglucanase from Clostridium thermocellulum the celC gene was cloned into Escherichia coli BL 21 (DE3) host. The recombinant E. coli was grown in shake flask cultures. The intracellular recombinant protein was extracted from the cells after applying supercritical CO2 cell disruption. The supercritical CO2 cell disintegration was optimized and then compared to the traditional ultrasonic cell disruption technique. With the supercritical cell disruption the cellulase recovery was approximately 17 % lower than that of obtained with sonication.

The very first application of supercritical fluid extraction (SFE) on enantioseparation of alcohols is discussed. Resolution of three chiral alcohols (trans-2-chloro-cyclohexanol, trans-2-bromo-cyclohexanol, and trans-2-iodo-cyclohexanol) were performed by partial complexation with (-)-O,O'-dibenzoyl-(2R,3R)-tartaric acid monohydrate (DBTA). DBTA formed diastereomeric complexes with all S,S-enantiomers stable enough to extract the unreacted alcohols with supercritical carbon dioxide. Resolution efficiency increased with the size of halogen substituents, and by the proper selection of molar ratio, pure (-)-R,R-trans-2-iodo-cyclohexanol (ee > 99%, yield: 39%) or (+)-S,S-trans-2-iodo-cyclohexanol (ee = 98%, yield: 8%) were prepared in one process step. Achieved resolution efficiency values were much higher in all resolution procedures than in any other known enantioseparation of these racemic compounds. The developed method offers an environmentally friendly, efficient alternative of currently applied resolution processes, also on a preparative scale.

Kinetic resolution of racemic 1-(benzofuran-2-yl)ethanols rac-1a–d was performed by lipase-catalyzed enantiomer selective acylation (E≫100) yielding (1R)-1-acetoxy-1-(benzofuran-2-yl)ethanes (R)-2a–d and (1S)-1-(benzofuran-2-yl)ethanols (S)-1a–d in highly enantiopure form. The degree of enantiomer selectivity for enzymatic alcoholysis/hydrolysis processes starting from racemic 1-acetoxy-1-(benzofuran-2-yl)ethane rac-2 was also tested under various conditions including supercritical CO2 medium. Racemization-free lipase-catalyzed ethanolysis of the (1R)-1-acetoxy-1-(benzofuran-2-yl)ethanes (R)-2a–d yielded almost quantitatively the enantiopure (1R)-1-(benzofuran-2-yl)ethanols (R)-1a–d.