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Microalgae are primary producers of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids in marine environment and so they are considered promising alternative sources of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs). Nannochloropsis gaditana is an oleaginous cultivated microalga with an important content of triglycerides and polar lipids which include interesting EPA amounts. In this work, new methods using pressurized liquids for simultaneous extraction and fractionation of neutral and polar lipids from wet microalgal biomass are proposed. Pressurized liquid extraction (PLE) using different solvents, combined or not with pre-treatment of the alga with enzymes, allows one to enrich and fractionate neutral and polar lipids such as triacylglycerols, diacylglycerols, monoacylglycerols, free fatty acids and glycolipids. The use of enzymatic pre-treatment did not show a significant increase in the lipid yield, though a different fractionation of the microalgal lipids was achieved. PLE with a single solvent (hexane at 120 °C) rendered a lipid yield of 17.6%, slightly higher than the traditional Folch method (16.9%), but with a less-toxic solvent and faster extraction technique. In addition, PLE with hexane attained a high triacylglycerols fraction (17.2% ± 3.0). The optimal four-step sequential method developed using pressurized liquids reached a good fractionation characterized by a high content of glycolipids (29.2% ± 2.0) and monoacylglycerols (17.3% ± 1.9), with a lipid recovery of 14.0% ± 1.3 for the original biomass. The analysis by GC/MS of fractions obtained with each method and solvent combination, showed a different composition in fatty acids of fractionated neutral and polar lipid species, especially in saturated and omega-3 fatty acids. Therefore, the proposed fast PLE methods allow the simultaneous extraction and fractionation of the neutral and polar lipids of Nannochloropsis gaditana and produce enriched EPA fractions (up to 53%).

To develop greener extraction alternatives for microalgae biomass, ultrasound assisted extraction (UAE) and pressurized liquid extraction (PLE) with different biobased solvents were investigated, demonstrating that both techniques are useful alternatives for algal lipid extraction. Specifically, Nannochloropsis gaditana lipids were extracted by UAE and PLE at different temperatures and extraction times with sustainable solvents like 2-Methyltetrahydrofuran (2-MeTHF) and its mixtures with ethanol and other alcohols. The best oil yields for both PLE and UAE of N. gaditana were achieved with the mixture of 2-MeTHF:ethanol (1:3), reaching yields of up to 16.3%, for UAE at 50 °C and up to 46.1% for PLE at 120 °C. Lipid composition of the extracts was analyzed by HPLC-ELSD and by GC-MS to determine lipid species and fatty acid profile, respectively. Different fractionation of lipid species was achieved with PLE and solvent mixtures of different polarity. Thus, for the extraction of glycolipids, ethanolic extracts contained higher amounts of glycolipids and EPA, probably due to the higher polarity of the solvent. The optimized method was applied to microalgae Isochrysis galbana and Tetraselmis chuii showing the potential of mixtures of biobased solvents like 2-methyl-THF and ethanol in different proportions to efficiently extract and fractionate lipids from microalgal biomass.

In this work, microencapsulation of omega-3 lipids from sustainable sources, including oilseeds such as Salvia hispanica L., Echium plantagineum L. and Camelina sativa L., microalgal lipids (from Nannochloropsis gaditana) and enzymatically produced fatty acid ethyl esters (FAEEs), was successfully done by spray drying and their outcomes studied. Previous to microencapsulation process, lipid extracts from new omega-3 sources (chia, camelina and echium oilseeds and wet microalgal biomass of Nannochloropsis) were produced using Pressurized Liquid Extraction. The best microencapsulation efficiency was achieved for chia FAEEs (76.9%), while the lowest was obtained for camelina oil (58.8%) and Nannochloropsis gaditana lipids (57.0%) microcapsules, which also showed low emulsion stability. Analysis by GC-MS showed that microencapsulation process did not negatively affect omega-3 fatty acid profile of microencapsulated extracts. The percentage of omega-3 in each used lipid was similar before and after spray drying (no significant differences at 5% level). Under the optimal conditions, produced microcapsules ranged from 1.5 to 30 μm and showed spherical shape and smooth surface without cracks. Thus, the proposed method provides an original approach to produce omega-3 microencapsulated lipid extracts from different nature, with high microencapsulation efficiency and payload, for potential applications in the development of foods enriched in omega-3 fatty acids, Authors thank the Spanish Ministry of Education, Culture and Sport for the pre-doctoral contract (FPU 2013–01796) granted to Natalia Castejón. The authors gratefully acknowledge the financial support from Comunidad de Madrid provided through project ALGATEC-CM (P2018/BAA-4532), co-financed by the European Social Fund. Authors thank Camelina Company (Spain) for kindly provide camelina seeds, Technology Crops Int. (UK) for providing echium seeds and Novozymes (Denmark) for donating commercial lipases. The assistance of Javier Agundez (ICP–CSIC, Spain) for getting SEM images is also acknowledged. Authors also thank AlgaEnergy S.A. (Spain) and Dr. Federico Witt for kindly provide Nannochloropsis gaditana biomass

The importance of squalene in both the cosmetic and pharmaceutical industries motivated the development of new technologies to purify squalene from different sources, one of such technologies being the supercritical fluid extraction using carbon dioxide (CO2). Knowledge of phase equilibria behavior is essential for the design of these processes. In this work, vapor−liquid equilibria of the binary system squalene + CO2 at high pressures (10 to 35) MPa and at T = (313, 323, and 333) K were measured using a variable-volume view cell. Experimental compositions of both the liquid and vapor phases were compared with previous data available in the literature. Additionally, the results obtained in this work were employed to evaluate the capability of a group contribution based equation of state in the phase equilibria modeling of the squalene + CO2 mixture.

BACKGROUND: Enzymatic esterification of phytosterols with fatty acids from butterfat in equimolecular conditions to produce phytosteryl esters was performed in solvent-free medium. Commercial and immobilized Candida rugosa lipases were used as biocatalysts for the reaction. RESULTS: By this methodology, under simple and mild reaction conditions (without solvents, 50 °C and short reaction times), 94% and 99% (w/w) of phystosteroyl esters were obtained in 48 h and 9 h with the commercial and the immobilized lipase, respectively. The effects of temperature, fatty acid specificity, enzyme amount and residual activity of each lipase were also evaluated. CONCLUSIONS: The phytosteryl esters from butterfat produced in this study are expected to have lower melting point, improved oil and fat solubility and bioavailability compared to that of their corresponding free phytosterols. Copyright © 2008 Society of Chemical Industry

Enzymatic esterification and supercritical fluid extraction was used to produce phytosterol esters from soybean oil deodorizer distillates. The raw material was first subjected to a two-step enzymatic reaction; the product obtained mainly comprised fatty acid ethyl esters, tocopherols and phytosterol esters, together with minor amounts of squalene, free fatty acids, free sterols and triacylglycerols. The phytosterol esters were then purified from this mixture using supercritical carbon dioxide. Experimental extractions were carried out in an isothermal countercurrent column (without reflux), with pressures ranging from 200 to 280 bar, temperatures of 45-55 °C and solvent-to-feed ratios from 15 to 35 kg/kg. Using these extraction conditions, the fatty acid esters were completely extracted and, thus, the fractionation of tocopherols and phytosterol esters was studied. At 250 bar, 55 °C and a solvent-to-feed ratio of 35, the phytosterol esters were concentrated in the raffinate up to 82.4 wt-% with satisfactory yield (72%).

Supercritical carbon dioxide extraction to separate phytosterol esters from fatty acid esters and tocopherols was simulated and optimized using the group contribution equation of state. Experimental extraction data at 328 K, pressures ranging from 200 to 280 bar and solvent-to-feed ratio around 25, was employed to verify the performance of the thermodynamic model. The raw material is the product obtained after a two-step enzymatic reaction carried out on soybean oil deodorizer distillates, and contains mainly fatty-acid ethyl esters, tocopherols and phytosterol esters. The extraction process was simulated using model substances to represent the complex multicomponent feed material. Nonlinear programming techniques were applied to find out optimal process conditions for a steady-state countercurrent process with partial reflux of the extract. The process optimization procedure predicts that a product with 94.2 wt % of phytosterol ester purity and 80% yield could be achieved. © 2009 American Institute of Chemical Engineers AIChE J, 2009

We conducted a near quantitative esterification of phytosterols from soybean oil deodorizer distillate with conjugated linoleic acid. We used a 1:1 molar ratio of sterols to conjugated linoleic acid. For that matter, stepwise addition of sterols was investigated. Total sterols were divided into several portions and added sequentially to the reaction mixture. Using this methodology, purities of up to 80% steryl esters were obtained that consumed more than 90% of the total conjugated linoleic acid. In addition, the effects of temperature, amount, and stability of lipase were also evaluated.

This work explores the capabilities of UNIFAC-based models to predict the solubility of different solid solutes in subcritical water as a function of temperature. The original UNIFAC, its modified (Dortmund) version and the A-UNIFAC model, which explicitly includes association effects between groups, are applied to calculate the solubility of solid compounds in subcritical water in the temperature range 298–500 K. The comparison between the three models is carried out using polycyclic aromatic hydrocarbons (PAHs) as test substances and for which reliable physical properties and experimental solubility data are available in the literature. The results obtained indicate that modified UNIFAC (Dortmund) provides the best representation of the aromatic compounds’ solubility as a function of temperature. In addition, the application of the A-UNIFAC model confirms the hypothesis that a decrease in the level of association between the subcritical water molecules (in accordance with the dielectric constant decrease with temperature), greatly improves the solubility of hydrophobic organic compounds.

In the present work, an HPLC method is proposed to simultaneously detect and quantify water- and fat-soluble vitamins, phenolic compounds, carotenoids and chlorophylls in a single run, by using an ultradeactivated C18 column and gradient separation using trifluoroacetic acid, water and methanol. It is shown that the HPLC method provides baseline separation of all these compounds with good resolution values in 40 min. Moreover, other figures of merit of the method show a good linear response and low detection limits for all the compounds considered in the present study. Furthermore, the usefulness of this method is demonstrated via its successful application to the analysis of different beverages from different natural origin (orange, strawberry, apple, peach pineapple, plum and blackcurrant juices, soybean milk, beers) without the need of any previous sample preparation. A good correlation is also found by comparing the total phenol content (measured by Folin-Ciocalteu method) with the sum of total phenolic compounds obtained using the proposed HPLC method. By using statistical tools, the main compounds associated with antioxidant activity of the extracts (measured by 1,1-diphenyl-2-picrylhydrazyl radical scavenging) were assessed.

Ethanolysis of a raffinate product-obtained after distillation of olive oil-catalyzed by three commercial lipases (from Candida antarctica, Rhizomucor miehei and Thermomyces lanuginosa) was studied. Uni-response model derived from a generalized Michaelis–Menten mechanism was utilized to describe the rates of formation of ethyl esters from the fatty acids present in the precursor oil. The rate constants for the ethanolysis of triacylglycerols and steryl esters under the reaction conditions were compared. Moreover, inactivation of three lipases was evaluated. Because the half-life of the enzyme is comparable to or even shorter than the half-life of the reaction, the intrinsic reaction rate and enzyme deactivation must both be considered in modeling the kinetics.

A new, specially designed column has been developed for fractionation of supercritical fluid extract of rosemary by using a preparative supercritical fluid chromatography system (Prep-SFC). The column evaluated in this work was prepared using a new packing method consisting of a combination of slurry and supercritical CO2 with commercial silica particles coated with a stationary phase commonly used in gas chromatography, such as SE-54 (5% phenyl-, 95% methylsilicone). The new packing procedure provided columns with reasonable efficiencies, with high stability and useful at high-pressure range. A 25 cm x 10 mm i.d. column packed with silica particles coated with 3% of SE-54 was prepared, and its separation power was tested for isolating fractions with high antioxidant and/or antimicrobial activity from a supercritical rosemary extract. The SFC conditions were selected based on a previous work done with a commercial LC-Diol packed column (130 bar, 80 degrees C), and different percentages of modifier in the mobile phase were tested (5 and 10%). Two cyclones were employed to collect the fractions which were then characterized by HPLC-diode array detection (DAD), GC, and in vitro antioxidant and antimicrobial assays. The use of coated packed columns allowed the fractionation of a complex mixture of rosemary supercritical extract with a minimum amount of modifier in the mobile phase (5% ethanol). At the optimum conditions it was possible to obtain two very active fractions in terms of antioxidant and antimicrobial activity, with no residual rosemary aroma and with improved activities compared to the original supercritical extract.

The purification of squalene from residues of the olive oil deodorization process using countercurrent supercritical carbon dioxide extraction was studied. The raw material employed is a by-product obtained after distillation and ethylation of olive oil deodorizer distillates. This by-product contains mainly squalene and fatty acid esters. The Group Contribution Equation of State was employed to simulate the separation process and to design the experimental extractions, which were carried out in an isothermal countercurrent column, without reflux, at 343 K, pressures ranging from 150 to 230 bar and a solvent-to-feed ratio around 13. Satisfactory agreement was found between experimental and calculated yields and phase compositions, obtaining a raffinate product with a squalene concentration up to 90 wt.%. Finally, the thermodynamic model was employed to find out optimal process conditions to enhance squalene recovery, including partial reflux of the extract product and recirculation of the supercritical solvent in a continuous countercurrent extraction column.

The possibility of using oregano leaves as a new source of natural antioxidants has been investigated. In the present study, oregano leaves were extracted using a pilot-scale SFE plant under a wide range of extracting conditions (at different extraction pressures and temperatures and considering the addition of ethanol as modifier) with the objective of knowing not only the best extraction conditions but also the variables that control the process in terms of extraction and fractionation of products of high antioxidant activity. The different fractions obtained at each experimental condition tested were characterized in terms of chemical composition by using liquid chromatography–diode array detection (LC–DAD) and liquid chromatography–mass spectrometry (LC–MS). Nine compounds have been tentatively identified as belonging to a specific family of compounds (flavone, flavanone and flavonol); their aglycon structures have been proposed using UV spectra profile while a tentative pattern of substitution, based on their mass spectra and molecular ion (MH+) major fragments, has been also given. Antioxidant activity was measured using two different in vitro assays (1,1-diphenyl-2-picrylhydrazyl (DPPH) antiradical test and bleaching β-carotene method).

A supercritical fluid extract of rosemary has been fractionated under supercritical conditions by using a preparative-SFC system. In this work, the optimum conditions have been evaluated to achieve a selective isolation of the compounds responsible for both, antioxidant and antimicrobial activities. A 25 cm × 10 mm i.d. LC-Diol packed column (dp = 5 μm) has been used and the separation took place at 80 °C of column temperature, 130 bar of pressure, and 10% of ethanol as modifier of the mobile phase (CO 2 ). Two cyclones were employed to collect the fractions which were subsequently characterized by HPLC-DAD, GC, and in vitro antioxidant and antimicrobial assays. By a careful selection of the separation conditions it is possible to obtain two different fractions, one enriched with antioxidant and antimicrobial compounds (with an improvement of about 20% and 40% of antioxidant and antimicrobial activity, respectively, compared to the original extract) collected in cyclone 2 and with no residual rosemary aroma and another one containing the essential oil.

Supercritical CO2 extracts of the marine diatom Chaetoceros muelleri (gracilis) have been investigated for their potential use as food preservatives, namely, as antimicrobials. A screening of different pressures and temperatures for supercritical CO2 extraction was assayed in order to determine the main factors controlling the yield and antimicrobial activity of the extracts. Since the potential antimicrobial activity of these CO2 extracts is mainly induced by the lipidic fraction, HPLC with evaporative light scattering detection (HPLC-ELSD) and GC with flame ionization detection (GC-FID) were used to identify lipid families and fatty acids, respectively. Antimicrobial activity of the extracts was measured against Staphyloccocus aureus, Escherichia coli and Candida albicans. Possible correlations between antimicrobial activity of extracts and their chemical composition were investigated, concluding that the total triglycerides and the DPA content seem to be the main parameters controlling the antimicrobial activity of the studied extracts.

Specially designed chromatographic columns were evaluated for antioxidant compounds separation by supercritical fluid chromatography (SFC) with pure CO2. Columns studied involved the use of commercial octadecylsilica particles (ODS) and silica particles coated with a stationary phase commonly used in gas chromatography, such as CW20M (polyethylene glycol) of high polarity. In order to select the appropriate chromatographic conditions to elute antioxidant compounds of phenolic structure, carnosic acid was selected as a model molecule and a theoretical study of the solubility of carnosic acid was performed. Carnosic acid was eluted using CO2 as mobile phase (without modifiers) with a pressure programming up to 37 MPa (and at different temperatures) but only when coated particles were used. No elution was possible when ODS particles were tested, probably due to the strong interactions of the acid group of the molecule with the active sites remaining in the column. A separation of a complex mixture of a rosemary antioxidant extract (obtained by supercritical fluid extraction) using the columns designed in the present work is shown.

An HPLC method with evaporative light scattering detection (ELSD) for the simultaneous analysis of various lipid classes, particularly alkoxyglycerols and acylglycerols with very similar structure and polarity, has been developed. These lipid classes are frequently found in numerous fats and oils such as shark liver oils and can serve as substrates for lipase-catalyzed reactions. This method utilizes a silica column and a gradient elution of isooctane, methyl tert-butyl ether and 2-propanol in different proportions. Separation between squalene, sterol esters, and fatty acid ethyl esters has been achieved in a time of analysis slightly higher than 8 min. In addition, a good resolution between 1,3-diacylglycerols and free sterols was also attained in the same run, with a broad range of concentrations. Excellent precision regarding the retention times was obtained. The limit of detection for the different lipid classes studied was below 1 microg. Intra-day and inter-day variation of retention times and areas was also evaluated. The relative standard deviation of intra-day variation for retention times and areas never exceeded of 0.1 and 10, respectively. The HPLC-ELSD method was also optimized to separate and quantify the hydrolysis products of alkoxyglycerols and acylglycerols (mono-esterified and non-esterified alkoxyglycerols and mono-esterified and di-esterified acylglycerols) at the same time, rendering a useful method for the study of lipase-catalyzed reactions and a wide variety of fats and oils. The present methodology not only separates 18 different lipid classes with a good reproducibility, but it is also able to estimate the relative proportion in which they are found in a broad range of concentrations.

Different supercritical fluid extraction conditions were tested on rosemary leaves using a pilot-plant-scale extractor. Each of them provided two separated fractions which were characterized chemically by liquid chromatography–diode-array detection–mass spectrometry using electrospray in the positive mode. Twelve compounds were identified, including phenolic diterpenes and flavonoids. Owing to the lack of available standards, only carnosic acid could be quantified. The antioxidant activity of the extracts was determined by the 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) test and the β-carotene bleaching test was used to study the differences in behavior of antioxidants in an emulsified medium for some of the extracts obtained. The results of the correlation studies to examine the relationship between the antioxidant activity (EC50), obtained by using the DPPH test, and the concentration of all the compounds detected in the extracts showed that carnosic acid was the more correlated compound, with a coefficient of correlation of −0.87. Using forward stepwise multiple linear regression, carnosic acid, methyl carnosate and carnosol were the compounds selected to predict the mentioned activity, with a value of 0.95 for the coefficient of determination.

In this work, different extracts from the microalga Spirulina platensis are obtained using pressurized liquid extraction (PLE) and four different solvents (hexane, light petroleum, ethanol and water). Different extraction temperatures (115 and 170 degrees C) were tested using extraction times ranging from 9 to 15 min. The antioxidant activity of the different extracts is determined by means of an in vitro assay using a free radical method. Moreover, a new and fast method is developed using micellar electrokinetic chromatography with diode array detection (MEKC-DAD) to provide a preliminary analysis on the composition of the extracts. This combined application (i.e., in vitro assays plus MEKC-DAD) allowed the fast characterization of the extracts based on their antioxidant activity and the UV-vis spectra of the different compounds found in the extracts. To our knowledge, this work shows for the first time the great possibilities of the combined use of PLE-in vitro assay-MEKC-DAD to investigate natural sources of antioxidants.

This article describes a method to determine standard pesticides in a model solution based on very large volume injection in capillary gas chromatography using a temperature-programmable injector (PTV). The influences of sample volume, speed of sample introduction, flow rate during sampling, and both initial and final PTV temperatures on the performance of the procedure are considered. Experimental values for sample volume and introduction speed were selected to make it possible subsequently to perform an eventual liquid chromatatographic preseparation for on-line GC analysis. The technique was applied to the analysis of 10 pesticides and allowed the direct introduction into the PTV of sample volumes as high as 10 mL. Relative standard deviation values for the absolute peak areas obtained from the direct introduction of standard mixtures containing the pesticides at 1 ppm and 10 ppb were in general 15%., Financial support by CICYT, Projects AMB95-0138-C02-02 and ALI95-0052, is gratefully acknowledged. FJS thanks the Ministerio de Educacion y Ciencia for his grant

6 páginas, 4 figuras, 2 tablas., Lipase-catalyzed ethanolysis of two short-chain triradylglycerols, namely tributyrin and 2,3-dibutyroil-1-O-alkylglycerols, have been studied. Much faster rate of reaction for the ethanolysis of tributyrin than that of 2,3-dibutyroil-1-O-alkylglycerols was attained. A kinetic model for the rate of release of ethyl butyrate and for the inactivation of the lipase has been also studied. The parameter corresponding to the release of ethyl butyrate was one order of magnitude higher for ethanolysis of tributyrin than the corresponding of 2,3-dibutyroil-1-O-alkylglycerols. On the contrary, the stability of Novozym 435 during ethanolysis of 2,3-dibutyroil-1-O-alkylglycerols was higher than the corresponding of tributyrin. At the reaction conditions under study, both ethanolysis reactions take place with high selectivity and yield monoesterified alkylglycerols and sn-2 monobutyrin as the main acylglycerols in the reaction mixtures., This work was supported by the projects AGL2006-02031/ALI and AGL2008-05655 by Ministerio de Ciencia (Spain) and also by Comunidad Autonoma de Madrid (ALIBIRD, project number S-505/AGR-0153) and Consolider-Ingenio FUN-C-FOOD (CSD2007- 00063). A predoctoral fellowship for Luis Vázquez, provided by Comunidad Autonoma de Madrid, is also acknowledged.

The supercritical removal of ethanol from alcoholic beverages (brandy, wine, and cider) was studied using the GC-EoS model to represent the phase equilibria behavior of the CO2 + beverage mixture. Each alcoholic drink was represented as the ethanol + water mixture with the corresponding ethanol concentration (35 wt% for brandy, 9-12 wt% for different wines and 6 wt% for cider). The thermodynamic modeling was based on an accurate representation of the CO2 + ethanol and CO2 + water binary mixtures, and the CO2 + ethanol + water ternary mixture. The GC-EoS model was employed to simulate the countercurrent supercritical CO2 dealcoholization of the referred beverages; the results obtained compared good with experimental data from the literature. Thus, the model was used to estimate process conditions to achieve an ethanol content reduction from ca. 10 wt% to values lower than 1 wt%. The model results were tested by carrying out several extraction assays using wine, in a 3 m height packed column at 308 K, pressures in the range of 9-18 MPa and solvent to wine ratio between 9 and 30 kg/kg. © 2010 Elsevier B.V. All rights reserved.

An antioxidant-enriched extract (RE) was obtained from rosemary (Rosmarinus officinalis) by supercritical fluid extraction to be used as an ingredient to design functional foods. The optimized mixture (42 mg RE g(-1) sunflower oil) was submitted to in vitro digestion and absorption tests (using Caco2 cells) to investigate the effect of these processes on its DPPH scavenging activity and also whether its major abietanes (tricyclic diterpenes) might be bioaccessible and bioavailable. Results indicated that supplementation of the rosemary extract with sunflower oil and lecithin (37 mg g(-1)) enhanced abietanes micellation (almost 2-fold). In vitro digestion of the mixture including RE, sunflower oil, and lecithin reduced 50% the bioaccesibility in terms of antioxidant activity. Bioavailability was 31%. It was evidenced that this activity was not due to the original levels of carnosol, carnosic acid, and methyl carnosate (which only 47% remained after digestion) but due to their derivatives and digestion products.

The objective of this research was to evaluate the antimicrobial activity of carbon dioxide extracts of the unicellular biflagellated green alga Dunaliella salina against Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger. The effects of different extraction pressures ranging from 185 to 442 bar and extraction temperatures ranging from 9.8 to 45.2 degrees C on the extracts' composition and consequently on their antimicrobial activities were investigated. The extracts were analyzed by gas chromatography-mass spectrometry in order to identify the compounds responsible for the antimicrobial activity detected. Fourteen different volatile compounds and several fatty acids were identified. The highest antimicrobial activity was obtained using 314 bar and 9.8 degrees C. Under these conditions, the presence of an indolic derivative that had never been reported in D. salina was detected in the extract, together with polyunsaturated fatty acids and compounds related to carotene metabolism, such as beta-ionone and neophytadiene, with known antimicrobial activity.

Three different parameters (temperature, solvent, and extraction time) were studied regarding to pressure liquid extraction (PLE) of antioxidant and antimicrobial compounds from Spirulina platensis. Two different antioxidant methods, β-carotene bleaching method and DPPH• (2,2-Diphenyl-1-picrylhydrazyl hydrate) free radical scavenging assay, were used to determine the optimal PLE conditions for antioxidants extraction. The selected conditions were as follows: extraction temperature equal to 115 ◦C, extraction time equal to 15 min and ethanol as extracting solvent. The main antioxidant compounds found in this extract were identified as zeaxanthin, a myxoxanthophyll-like compound and very polar phenolic compounds. Moreover, antimicrobial activity of different PLE fractions was tested against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 11775, Candida albicans ATCC 60193, and Aspergillus niger ATCC 16404. Data obtained showed the hexane and petroleum ether extracts were slightly more active than ethanolic extracts. As for water extracts, none of them were active against the microorganisms tested. Data indicated that both 115 and 170 ◦C were the best extraction temperatures conditions in order to optimize the extraction of antimicrobial compounds, whereas 9 min was the optimal extraction time. Besides, C. albicans was the most sensitive microorganism to all Spirulina PLE extracts., MH thanks Ministerio de Ciencia y Tecnolog´ıa for a FPI grant. Authors thank Ministerio de Ciencia y Tecnolog´ıa (project AGL2002-04621-C02-02) for financial support.

Spirulina platensis microalga has been extracted on a pilot scale plant using supercritical fluid extraction (SFE) under various extraction conditions. The extraction yield and the antioxidant activity of the extracts were evaluated in order to select those extracts with both the highest antioxidant capacity and a good extraction yield. These extracts were characterized using LC coupled to diode array detection (DAD) and LC coupled to mass spectrometry (MS) with two different interfaces, atmospheric pressure chemical ionization (APCI) and electrospray (ESI) which allowed us to perform tandem MS by using an ion trap analyzer. The best extraction conditions were as follows: CO2 with 10% of modifier (ethanol) as extraction solvent, 55 degrees C (extraction temperature) and 220 bar (extraction pressure). Fractionation was achieved by cascade depressurization providing two extracts with different activity and chemical composition. Several compounds have been identified in the extracts, corresponding to different carotenoids previously identified in Spirulina platensis microalga along with chlorophyll a and some degradation products. Also, the structure of some phenolic compounds could be tentatively identified. The antioxidant activity of the extracts could be attributed to some of the above mentioned compounds.

An experimental design has been used to optimize the extraction of antioxidants from the microalga Spirulina platensis using accelerated solvent extraction (ASE) with four different solvents (hexane, petroleum ether, ethanol and water). The optimization of the main variables involved in the ASE process (extraction temperature and time) has been done by means of a full factorial (three levels) design using, as responses, the extraction yield and the antioxidant activity of the extracts (determined as EC50, i.e., efficient concentration, using an in vitro assay based on a free radical method). The parameters of the model, for each response variable, were estimated by multiple linear regression (MLR). The statistical analysis of the results provided mathematical models that allowed prediction of the behaviour of the different responses selected, as a function of the main variables involved in the process. It was observed that the optimum conditions that maximize yield and minimize EC50 depend on the polarity of the solvent used to perform the extractions. Extraction temperature had an enormous influence in both responses while the effect of extraction time was almost negligible. Ethanol was finally selected as the extracting solvent for its GRAS (Generally Recognized as Safe) status and because it provides higher yields with medium antioxidant activities. The results presented in this work show the possibility of using a fast and easy process to recover natural antioxidants from natural sources such as microalgae., M.H. thanks MCYT for a FPI grant. Authors thank MCYT (Project AGL2002-04621-C02-02) for financial support.

Subcritical water extraction at several temperatures ranging from 25 to 200 °C has been studied to selectively extract antioxidant compounds from rosemary leaves. An exhaustive characterization of the fractions obtained using subcritical water at different temperatures has been carried out by LC-MS, and the antioxidant activities of the extracts have been measured by a free radical method (DPPH). Results indicate high selectivity of the subcritical water toward the most active compounds of rosemary such as carnosol, rosmanol, carnosic acid, methyl carnosate, and some flavonoids such as cirsimaritin and genkwanin. The antioxidant activity of the fractions obtained by extraction at different water temperatures was very high, with values around 11.3 μg/mL, comparable to those achieved by SFE of rosemary leaves. A study of the effect of the temperature on the extraction efficiency of the most typical rosemary antioxidant compounds has been performed. Keywords: Antioxidant activity; antioxidant compounds; rose...

An on-line reversed-phase LC-GC method involving the use of a Programmed Temperature Vaporizer (PTV) as interface is used for the rapid analysis of sterols. The experimental procedure requires only the use of commercial HPLC and GC equipments and a Rheodyne valve. The LC preseparation can be performed satisfactorily even with aqueous eluents containing up to 25% water. Factorial experimental design is applied to investigate the sensitivity achievable in the analysis with some variables (adsorbent material used in the PTV, particle size and plug length) being considered at two different levels. As a result, the sensitivty can be increased by a factor as large as 35 merely by performing the analysis under the experimental conditions giving the highest response. The use of the proposed configuration allows the transfer of up to 1050 μL from LC into the GC system, 1400 μL/min being the speed of sample introduction. The proposed procedure was succesfully applied to the analysis of real samples.

In this work some of the newest trends in food processing are reviewed. This revision intends to provide an updated overview (including works published until February 2001) on the newest food processes, including food manufacturing, preservation, and control. Modern processes for food and food ingredients manufacturing based on membrane technology, super-critical fluid technology, and some applications of biotechnology are presented, mainly applied to obtain functional foods, "all-natural" enriched foods, probiotics and prebiotics. Also included is a critical assessment concerning non-thermal preservation techniques used for food preservation, such as high hydrostatic pressure, pulsed electric fields, ultrasound, pulsed light, hurdle systems, etc. Finally, a group of new analytical techniques (i.e., molecular techniques such as Polymerase Chain Reaction (PCR), food image analysis, and biosensors) and their use for food and process control is reviewed.