Enzymatic synthesis of triacylglycerols of docosahexaenoic acid: Transesterification of its ethyl esters with glycerol.

The synthesis of docosahexaenoyl triacylglycerides at low temperature (e.g., 50°C) using biocatalysts of 6 commercial lipases adsorbed on hydrophobic supports was studied. In general, the triacylglyceride yields were very low with the exceptions of those produced with the enzymes from Candida antarctica fraction B, CALB (82%), and those produced with the enzyme from Pseudomonas fluorescens, PFL (57%). The reactions were performed under vacuum to remove the released ethanol. The yields varied widely when different derivatives of CALB were used, and they were higher when CALB adsorbed on hydrophobic supports was used (82%). One interesting by-product (18% of sn-2 monoacylglyceride of DHA) remained at the end of the synthetic process. CALB adsorbed on Sepabeads exhibited better activity and stability than did the commercial derivative Novozym 435. The best CALB biocatalyst preserved 90% of the activity after 30days under the reaction conditions.

Supercritical fluid extraction of oregano (Origanum vulgare) essentials oils: anti-inflammatory properties based on cytokine response on THP-1 macrophages.

Two fractions (S1 and S2) of an oregano (Origanum vulgare) extract obtained by supercritical fluid extraction have been used to test anti-inflammatory effects on activated human THP-1 cells. The main compounds present in the supercritical extract fractions of oregano were trans-sabinene hydrate, thymol and carvacrol. Fractions toxicity was assessed using the mitochondrial-respiration-dependent 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction method for several concentrations during 24 and 48 h of incubation. Concentrations higher than 30 microg/mL of both supercritical S1 and S2 oregano fractions caused a reduction in cell viability in a dose-dependent manner. Oxidized-LDLs (oxLDLs) activated THP-1 macrophages were used as cellular model of atherogenesis and the release/secretion of cytokines (TNT-alpha, IL-1beta, IL-6 and IL-10) and their respective mRNA expressions were quantified both in presence or absence of supercritical oregano extracts. The results showed a decrease in pro-inflammatory TNF-alpha, IL-1beta and IL-6 cytokines synthesis, as well as an increase in the production of anti-inflammatory cytokine IL-10. These results may suggest an anti-inflammatory effect of oregano extracts and their compounds in a cellular model of atherosclerosis.

Screening for bioactive compounds from algae.

In the present work, a comprehensive methodology to carry out the screening for novel natural functional compounds is presented. To do that, a new strategy has been developed including the use of unexplored natural sources (i.e., algae and microalgae) together with environmentally clean extraction techniques and advanced analytical tools. The developed procedure allows also estimating the functional activities of the different extracts obtained and even more important, to correlate these activities with their particular chemical composition. By applying this methodology it has been possible to carry out the screening for bioactive compounds in the algae Himanthalia elongata and the microalgae Synechocystis sp. Both algae produced active extracts in terms of both antioxidant and antimicrobial activity. The obtained pressurized liquid extracts were chemically characterized by GC-MS and HPLC-DAD. Different fatty acids and volatile compounds with antimicrobial activity were identified, such as phytol, fucosterol, neophytadiene or palmitic, palmitoleic and oleic acids. Based on the results obtained, ethanol was selected as the most appropriate solvent to extract this kind of compounds from the natural sources studied.

Subcritical water extraction and characterization of bioactive compounds from Haematococcus pluvialis microalga.

In this work, extraction and characterization of compounds with antioxidant and antimicrobial activity from Haematococcus pluvialis microalga in red phase have been carried out. To do this, subcritical water extraction (SWE) has been combined with analytical techniques such as HPLC-DAD, HPLC-QqQ-MS and GC-MS and in vitro assays (i.e., for antioxidant and antimicrobial activity). The effect of the extraction temperature (50, 100, 150 and 200 degrees C) and solvent polarity has been studied in terms of yield and activity of the extracts. Results demonstrate that the extraction temperature has a positive influence in the extraction yield and antioxidant activity. Thus, the extraction yield achieved with this process was higher than 30% of dry weight at 200 degrees C as extraction temperature. Moreover, the extract obtained at 200 degrees C presented the highest antioxidant activity by far, while temperature does not seem to significatively affect the antimicrobial activity. Chemical composition was determined by HPLC-DAD, HPLC-QqQ-MS and GC-MS. Short chain fatty acids turned out to be responsible of the antimicrobial activity, whereas the antioxidant activity was correlated to vitamin E (present exclusively in the 200 degrees C extract), together with simple phenols, caramelization products and possible Maillard reaction products obtained during the extraction at high temperatures.

Supercritical fluid fractionation of fatty acid ethyl esters from butteroil.

Countercurrent supercritical fractionation of the fatty acid ethyl esters from butteroil has been investigated. The main objective of the present study was to obtain extracts rich in short- and medium-chain fatty acid ethyl esters. To that end, transesterification of the original butteroil was used to transform the triacylglycerols into the corresponding fatty acid ethyl esters. Then, several supercritical fluid extractions were carried out at pressures ranging from 8.9 to 18.6 MPa and at 2 different temperatures (48 and 60 degrees C). The flow ratio of CO2 to butteroil was 15. Composition and yield of short- and medium-chain fatty acid ethyl esters was evaluated at different extraction conditions. Extracts containing approximately 70% short- and medium-chain fatty acid ethyl esters were obtained at 101 bar and 60 degrees C, and can be used as starting material for the production of highly valuable functional lipids.

Pressurized fluid extraction of bioactive compounds from Phormidium species.

In the search for new functional ingredients with potential use in the food industry, extracts of unknown species of microalgae, such as Phormidium species have been studied. Three solvents of different polarities (i.e., hexane, ethanol, and water) have been used to obtain pressurized liquid extracts with different compositions. Moreover, extractions were performed at four different extraction temperatures (50, 100, 150, and 200 degrees C) with 20 min as extraction time. Antioxidant activity of the extracts has been measured by the TEAC assay. In general, hexane and ethanol extracts showed a higher antioxidant capacity that was mainly attributed to carotenoid compounds, as the TEAC value trend seems to be similar to the carotenoid content of the extracts. On the other hand, the high antioxidant activity of the 200 degrees C water extracts is likely related to the presence of Maillard reaction compounds produced by thermal degradation of the sample. beta-Carotene, lutein, violaxanthin, and neoxanthin were identified in 150 degrees C ethanol extracts. Four different microbial species ( Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger) were used to screen the potential antimicrobial activity of the Phormidium sp. extracts. The most sensitive microorganism was the yeast, C. albicans, whereas the fungus, A. niger, was the most resistant. In general, no drastic differences were found for solvents and temperatures tested, showing a very diverse nature of the compounds responsible for the antimicrobial activity of these microalgae. In ethanol extracts, antimicrobial activity could be mainly attributed to the presence of terpenes (i.e., beta-ionone, neophytadiene) and fatty acids (i.e., palmitoleic and linoleic acids) in the samples. Toxicity studies carried out with the extracts evaluated in the present work showed a cellular toxicity lower than those of other cyanobacteria such as Spirulina plantensis.

Supercritical carbon dioxide extraction of compounds with antimicrobial activity from Origanum vulgare L.: determination of optimal extraction parameters.

Oregano leaves were extracted using a pilot-scale supercritical fluid extraction plant under a wide range of extraction conditions, with the goal of determining the extraction and fractionation conditions to obtain extracts with optimal antimicrobial activity. In this investigation, the essential oil-rich fractions were selectively precipitated in the second separator, and their chemical composition and antimicrobial activity were investigated. Gas chromatography-mass spectrometry analysis of the various fractions resulted in the identification of 27 compounds of the essential oil. The main components of these fractions were carvacrol, trans-sabinene hydrate, cis-piperitol, borneol, terpinen-4-ol, and linalool. Antimicrobial activity was investigated by the disk diffusion and broth dilution methods against six different microbial species, including two gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the supercritical fluid extraction fractions obtained showed antimicrobial activity against all of the microorganisms tested, although the most active fraction was the one obtained in experiment 5 (fraction was obtained with 7% ethanol at 150 bar and 40 degrees C). C. albicans was the most sensitive microorganism to the oregano extracts, whereas the least susceptible was A. niger. Carvacrol, sabinene hydrate, borneol, and linalool standards also showed antimicrobial activity against all of the microorganisms tested, with carvacrol being the most effective. Consequently, it was confirmed that essential oil from experiment 5, with the best antimicrobial activity, also presented the highest quantity of carvacrol.

Subcritical water extraction of nutraceuticals with antioxidant activity from oregano. Chemical and functional characterization.

In the present work, oregano leaves (Origanum vulgare L.) are explored as natural source of nutraceuticals with antioxidant activity. To do this, subcritical water extraction (SWE), a new environmentally friendly technique, is employed as extraction procedure and HPLC coupled to DAD is used for the chemical characterization of the extracts. Moreover, the radical scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) method and the determination of the total phenolic content (measured with the Folin test) are applied to evaluate the antioxidant activity of the extracts. The extraction of antioxidants from oregano leaves by SWE is studied considering different temperatures (25, 50, 100, 150 and 200 degrees C) to investigate the selectivity of the process. The highest antioxidant activity is observed for the extract obtained at the highest temperature, 200 degrees C (EC(50) equal to 10 microg/ml). Moreover, the extraction yield was also the highest (54% dry weight) at these extraction conditions. The total phenolic content showed no differences among the different extracts, concluding that the amount of phenolic compounds extracted was similar but the type and structure of the phenolics was different, providing in this way different antioxidant activity. Some compounds could be tentatively identified, proposing some probable chemical structures for some of them, such as flavanones, dihydroflavonols, favonols and flavones.

Chemical composition and antimicrobial activity of Rosmarinus officinalis L. essential oil obtained via supercritical fluid extraction.

The chemical composition and antimicrobial activity of essential oil-rich fractions obtained by supercritical CO2 extraction from Rosmarinus officinalis L. were investigated. Gas chromatography-mass spectroscopy analysis of these fractions resulted in the identification of 33 compounds of the essential oil. The main components of these fractions were alpha-pinene, 1,8-cineole, camphor, verbenone, and borneol, constituting ca. 80% of the total oil. The antimicrobial activity was investigated by the disc diffusion and broth dilution methods against six microbial species, including gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), a yeast (Candida albicans), and a fungus (Aspergillus niger). All of the essential oil-rich fractions obtained showed antimicrobial activity against all of the microorganisms tested, with inhibition zones and minimal bactericidal and fungicidal concentration values in the range of 17 to 33 mm and 2.25 to 0.25 mg/ml, respectively. The most active fraction was the one obtained in experiment 4 (4% ethanol as modifier; extraction pressure, 25 MPa; extraction temperature, 60 degrees C). S. aureus was found to be the most sensitive bacteria to the rosemary extracts, whereas the least susceptible was A. niger. alpha-Pinene, 1,8-cineole, camphor, verbenone, and borneol standards also showed antimicrobial activity against all the microorganisms tested, borneol being the most effective followed by camphor and verbenone. In that way, it was confirmed that essential oil from experiment 4, with the best antimicrobial activity, presented the highest quantity of camphor, borneol, and verbenone.

Truffle aroma characterization by headspace solid-phase microextraction.

In the present study, a headspace solid-phase microextraction (HS-SPME) combined to gas chromatography-mass spectrometry (GC-MS) has been used to fully characterize aroma of truffles of different species. A fiber of medium polarity (for flavors) was used to avoid discrimination towards very non-polar and polar volatile compounds. In a previous work, extraction conditions were optimized by means of an experimental design leading to the following conditions that were used in the present study: extraction temperature, 53 degrees C; extraction time, 13.6 min; and equilibrium time, 5 min. A comparison among different truffles species has been established in terms of qualitative and quantitative differences on volatile composition. By using the optimal extraction conditions and GC-MS it was possible to identify 89 compounds in two different truffle species such as Tuber aestivum and Tuber melanosporum. An attempt has been made in order to be able to determine the influence of different geographical origins on the aroma fraction of such fungi.

Isolation of antioxidant compounds from orange juice by using countercurrent supercritical fluid extraction (CC-SFE).

Antioxidants from orange juice are isolated by the use of countercurrent supercritical fluid extraction (CC-SFE) and characterized by reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) and diode-array detection (DAD). A pilot-scale SFE plant equipped with a packed column has been employed for countercurrent extraction and fractionation of raw orange juice with carbon dioxide. Several experiments have been performed in order to study the effect of the countercurrent conditions on the content of antioxidative compounds. In this study, the main variable that has been considered is the solvent-to-feed ratio (S/F) because it plays an essential role in the extraction efficiency. The values tested covered a wide range of sample and solvent (CO(2)) flow rates. In each experimental run, two different extracted fractions and the residual nonextracted juice were obtained and characterized. Different flavonoids have been identified in the fractions obtained after CC-SFE. The possibility of using this process for antioxidant compounds enrichment is discussed.

Countercurrent supercritical fluid extraction and fractionation of alcoholic beverages.

A procedure for the recovery of aromatic extracts from distilled alcoholic beverages by means of a countercurrent supercritical fluid extraction (CC-SFE) on a pilot plant scale is studied. The beverage is directly in contact with the carbon dioxide current in a packed column, and the extracts are recovered in two different fractionation cells, where the depressurization occurs. The proposed method allows the selective extraction of aromatic components of the brandy flavor, rendering a high-value concentrated extract and a colored residue without brandy aroma. The content in ethanol of the aromatic extract can be modified by tuning the extraction/fractionation conditions, rendering from 15 to 95% recovery. The effect of the main variables, including extraction pressure and quality of extracting CO(2), has been tested.

Combined use of supercritical fluid extraction, micellar electrokinetic chromatography, and reverse phase high performance liquid chromatography for the analysis of antioxidants from rosemary (Rosmarinus officinalis L.).

Antioxidants from rosemary were determined by the combined use of supercritical fluid extraction (SFE) prior to reverse-phase high-performance liquid chromatography (RP-HPLC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published HPLC procedure, both with diode array detection. The characterization of the different antioxidants was further done by HPLC-mass spectrometry. Advantages and drawbacks of HPLC and MEKC for analyzing the antioxidants found in the different extracts are discussed. From the results it is concluded that HPLC renders higher peak area and is better in its reproducibility than MEKC; both techniques provide similar analysis time reproducibility. The main advantage of MEKC is its much higher separation speed, which is demonstrated to be useful for the quick adjustment of SFE conditions, allowing rosemary fractions of higher antioxidative power to be obtained quickly. Moreover, the possibilities of this approach for following the degradation of antioxidants are discussed.

Tuning of mobile and stationary phase polarity for the separation of polar compounds by SFC.

The separation of polar compounds by supercritical-fluid chromatography (SFC) is reviewed. New developments in mobile and stationary phase tuning are reviewed for packed and packed capillary SFC. In terms of mobile phase polarity adjustment, new pure and multiple component fluids are presented. The approach of tuning the polarity of the stationary phase as a way of increasing the range of polar compounds analyzed by SFC using pure CO(2) is discussed using either silica-based or new materials as stationary phase. Chiral, liquid crystal and polymer-based stationary phases coated on particles are widely covered in this review as an interesting approach to separate polar compounds avoiding the major drawbacks associated to the use of modifiers in SFC.

Liquid chromatographic-mass spectrometric analysis of supercritical-fluid extracts of rosemary plants.

A two-step supercritical fluid extraction process of rosemary leaves, on a pilot plant scale, is proposed to divide the oleoresin into two fractions with different antioxidant activities and essential oil composition. Rosemary leaves were extracted by using different conditions of pressure and temperature as well as different conditions for fractionation of the extracts. Conditions can be tuned to selectively extract one antioxidant fraction with almost no residual aroma. In the present investigation, the antioxidant fraction was exhaustively studied in terms of antioxidant activity measurements as well as of chemical composition. An LC-MS method was adapted to perform the analysis and identification of the compounds responsible for the antioxidant activity of the extracts. Different extraction and fractionation conditions were studied in order to correlate the process conditions with the antioxidant activities obtained.