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La presente invención se refiere a un nuevo procedimiento de obtención de aceites o grasas con alto contenido antioxidante, debido a la acción de compuestos de naturaleza fenólica y en concreto debido al hidroxitirosol y esteres grasos de hidroxitirosilo.

[EN] The invention relates to a novel method for producing oils and fats with a high anti-oxidant content, as a result of the action of compounds of a phenolic nature, and more concretely, as a result of hydroxytyrosol and fatty esters of hydroxytyrosyl., [ES] La presente invención se refiere a un nuevo procedimiento de obtención de aceites o grasas con alto contenido antioxidante, debido a la acción de compuestos de naturaleza fenólica y en concreto debido al hidroxitirosol y esteres grasos de hidroxitirosilo.

Several works have measured free radical scavenging capacity of nut oils, since they may become a significant source of dietary fat. However, they have not considered kinetic parameters, what was the first aim of this work. Also, it was studied the possible relation between values of free radical scavenging capacity DPPH{radical dot} and oxidative stability (Rancimat method) in different nut (hazelnut, peanut, pistachio, walnut and almond) oils. The ranking of antioxidant capacity of nut oils, by both assays, was: pistachio > hazelnut > walnut > almond > peanut. A significant correlation was found between DPPH and Rancimat methods assays. Tocopherols appear to be the responsible compounds of this antioxidant capacity being neglictible the contribution of polyphenols. An interference effect of phospholipids, present in methanolic fraction of nut oils, was observed in the determination of polyphenols in nut oils by Folin and ortho-diphenols assays. © 2008 Elsevier Ltd. All rights reserved.

The bitterness of the main compounds identified in the phenolic extract of virgin olive (Olea europaea L.) oils has been sensory-tested. The aldehydic form of oleuropein aglycone (AOA) was responsible for this attribute. Correlations between the sensory bitterness and concentrations of secoiridoid derivatives, analyzed separately or in different combinations, were obtained for olive oils from different olive varieties. The best correlation obtained corresponds to AOA content (r=0.96; P=1.83×10−17) in the concentration range of 0.03 to 0.5 mmol/kg. AOA concentrations ≥0.5 mmol/kg produce sensory saturation of this attribute. The correlation with AOA concentration was better than that with the absorbance of the phenolic extract at 225 nm. Therefore, the equation obtained allows the evaluation of the bitterness in virgin olive oils by HPLC analysis of the phenolic extract using detection at 280 nm.

[EN]The invention relates to a method of preparing a wide range of hydroxytyrosol esters. According to the invention, the compounds are synthesised by reacting synthetic hydroxytyrosol or natural hydroxytyrosol compounds, oleuropein or oleuropein aglycones (which are found in olive oil, vegetable water, olive marc or olive leaves) with an acylating agent which is a compound containing at least one acylic group R, wherein R is H, an alkyl radical with between 1 and 31 linear, branched or cyclic carbon atoms, an alkenyl radical with up to 31 linear, branched or cyclic carbon atoms or an aryl group. The invention also relates to the esters prepared with the inventive method, which can be used as an additive in food and cosmetic products as well as in pharmaceutical preparations. [ES]El objeto de la presente invención es un procedimiento para la preparación de una amplia gama de ésteres de hidroxitirosol. La síntesis de los compuestos se lleva a cabo por reacción de hidroxitirosol sintético o de los compuestos naturales hidroxitirosol, oleuropeína o aglucones de la oleuropeína (que se encuentran en el aceite de oliva, alpechín, orujo de oliva u hojas de olivo) con un agente acilante que es un compuesto que contiene al menos un grupo acílico de resto R, donde R es H, un radical alquilo de entre 1 y 31 átomos de carbono, sea lineal, ramificado o cíclico, un radical alquenilo de hasta 31 átomos de carbono también lineal, ramificado o cíclico ó un grupo arilo. Los ésteres preparados mediante el procedimiento son también objeto de la invención y pueden utilizarse como aditivo en productos alimenticios y cosméticos, así como en preparaciones farmaceúticas.

A procedure for the simultaneous determination of long-chain aliphatic aldehydes, and aliphatic and triterpenic waxes in virgin olive oils is described. A fraction containing these compounds was isolated from the oil using solid-phase extraction on silica-gel cartridges. The fraction was analyzed by capillary GC on 35%-dimethyl–65%-diphenylpolysiloxane phase using on-column injection. In extra virgin olive oils, the long-chain aliphatic aldehydes with even carbon atom numbers from C22 to C30 were identified by comparison of retention times and mass spectra with those of synthesized standards. The concentration of total aldehydes ranged from 20.2 to 108.0 mg/kg—n-hexacosanal being the most abundant aldehyde. The determination of aliphatic waxes was achieved with similar or better precision than that of the EU official methods.

The analysis of triacylglycerols has great importance as quality control and origin determination aid in olive oils analytical methodologies. New improvements in the analysis of triacylglycerols by high-performance liquid chromatography in olive oils are developed in order to increase the separation between LLL/OLLn and OLL/OOLn critical pairs used for detection of seed oils in olive oils. Elution with acetone/acetonitrile (55:45) using gradient temperature and isothermal elution with propionitrile were investigated, in comparison with the European Union official method. The best resolution was achieved using propionitrile at 20ºC. Although the HPLC profile was similar using propionitrile and acetone/acetonitrile, differences in minor triacylglycerols contributing to each HPLC peak were encountered. The precision of the method was good., El análisis de triacilgliceroles tiene gran importancia como herramienta en el control de calidad y en la determinación del origen de los aceites de oliva. Nuevas mejoras en el análisis de triacilgliceroles en aceite de oliva mediante cromatografía líquida de alta eficacia se han desarrollado para mejorar la separación entre las parejas críticas LLL/OLLn y OLL/OOLn usadas en la detección de aceites de semilla en aceite de oliva. Eluciones con acetona/acetonitrilo (55:45) usando gradiente de temperatura y elución con propionitrilo en condiciones isotérmicas han sido investigadas en comparación con el método oficial de la Unión Europea. La mejor resolución se consiguió usando propionitrilo como eluyente a 20ºC. Aunque el perfil cromatográfico obtenido usando propionitrilo fue similar al obtenido usando acetona/acetonitrilo, se encontraron diferencias en los triacilgliceroles minoritarios que contribuyen a cada pico cromatográfico. La precisión del método fue buena.

A simple and precise analytical method was developed for the simultaneous determination of squalene and methyl, ethyl, propyl, and butyl esters of fatty acids present in olive and olive pomace oils. A fraction containing squalene and fatty acid alkyl esters was isolated from the oil by solid phase extraction on silica gel cartridges and quantitatively analyzed by gas chromatography. A modification of the procedure allowed the isolation of squalene and esters separately. Repeatability and recovery of the method were good. The method was applied to extra and lampant virgin olive oil categories and also to oils obtained from olive pomace by second centrifugation and solvent extraction. Extra virgin olive oils contained low amounts of fatty acid methyl and ethyl esters, while oils obtained from altered olive or olive pomace showed high concentrations of fatty acid alkyl esters, mainly ethyl esters. Correlation between oil acidity and ethyl esters concentration was poor.

Olives (Olea europaea) of the Manzanilla and Verdial varieties, harvested at the green mature stage of ripening, were heated at 30, 40, 45, and 50 °C during 24 h and at 40 °C during 24, 48, and 72 h, respectively. Just after treatments, oils were physically extracted from the olives. Olive heating promotes a reduction of oil bitterness in direct relationship to the time and temperature used. Fruit heating at ≤40 °C during 24 h did not produce significant changes of acidity, UV absorption, peroxide index, panel test score, or oxidative stability of the obtained oils. Both longer treatments at 40 °C and heating at >40 °C yielded oils with less oxidative stability. Oils obtained from olives heated at ≥40 °C showed higher concentrations of chlorophylls and carotenes. For each olive variety, a good correlation between oil bitterness and content of hydroxytyrosol secoiridoid derivatives was found.

A simple analytical method for the quantitative determination of phenols, flavones, and lignans in virgin olive oils was developed. The polar fraction was isolated from small amounts of oil sample (2.5 g) by solid-phase extraction (SPE) using diol-phase cartridges, and the extract was analyzed by reversed-phase HPLC coupled with diode array UV detection. Chromatographic separation of pinoresinol, cinnamic acid, and 1-acetoxypinoresinol was achieved. Repeatability (RSD < 6.5%), recovery (> 90%), and response factors for each identified component were determined. SPE on amino-phase cartridges was used for isolating acidic phenols and as an aid for phenol identification. For the first time, 2-(4-hydroxyphenyl)ethyl acetate was detected in olive oils. The aldehydic structure of the ligstroside aglycon was confirmed by NMR spectroscopy. The colorimetric determination of total o-diphenolic compounds by reaction with molybdate was consistent with their HPLC determination. Differences between results obtained by liquid−liquid extraction and SPE were not statistically significant.

The evolution of 1,3- and 1,2-isomers of diacylglycerols (DGs) in olive oils obtained from healthy olives and the influence of the olive quality was studied. Healthy olive fruits yielded oils containing almost exclusively 1,2-isomers whereas altered olives produced oils with significant amounts of 1,3-isomers. Virgin olive oils obtained from various olive cultivars and stored at different temperatures showed triacylglycerol hydrolysis and diacylglycerol isomerization depending on the acidity and temperature. The results indicated that the relationship between acidity and total diacylglycerol content has scarce utility for detecting mild refined oil in virgin olive oil. On the other hand, the 1,3-/1,2-DG isomers ratio is useful for assessing the genuineness of virgin olive oils with low acidities during the early stages of storage.

Hydroxytyrosol (2-(3,4-dihydroxyphenyl)ethanol), a phenolic compound present in extravirgin olive oil, has been reported to contribute to the prevention of cardiovascular disease. The present study describes an accurate and reproducible reversed-phase HPLC method to measure hydroxytyrosol in plasma. This compound was extracted from acidified plasma by solid-phase extraction using an Oasis HLB copolymer. The plasma sample was rinsed with water and methanol in water (5:95; v/v). Hydroxytyrosol was eluted with methanol, which was subsequently evaporated under a nitrogen stream. Analysis by HPLC with diode array-UV detection was carried out using a C18 column and a gradient elution with acidified water and methanol/acetonitrile (50:50; v/v). The method was validated by the analyses of plasma samples spiked with pure hydroxytyrosol, obtaining a linear correlation (0.9986) and precision with a coefficient of variation ranging from 0.79 to 6.66%. The recovery was 100%, and the limit of detection was 37 ng/mL. The oral administration of hydroxytyrosol to rats and its subsequent detection in plasma showed that the method is suitable for pharmacokinetic studies.

[EN] The official regulations for the control of the olive and olive pomace oils of the European Union (EU) and lnternational Olive Oil Council (IOOC) include the determination of fatty acids in order to be appjied to several puriíy criteria. The determination of fatty acids require the preparation of the fatíy acid methyl esters (FAME) for the subsequent analysis by gas chromatography with good precision and reproducibility. Among the methods used in the laboratories of both the industries and the official institutions looking after the olive oil control, the ones selected were: 1) cold methylation with methanolic potash and 2) hot methylation with sodium methylate followed by acidification with sulphuric acid in methanol and heating. A statistical assessment of the precision characteristics were performed on the determination of fatty acids using both methods by a collaborative trial following the directions included in the AOAC regulation (AOAC 1995). In oils with low acidities, the results obtained for both methylation methods were equivalent. However, the olivepomace oil sample (acidity 15.5%) showed significative differences between the fatty acid compositions obtained using both methylation methods. Finally, the methylation with the acidic+basic method did not yield an increase of the frans-isomers of the fatty acids., [ES] Los métodos oficiales para el control del aceite de oliva y de orujo de oliva de la Unión Europea (UE) y del Comité Oleícola Internacional (COI) incluyen la determinación de ácidos grasos en la aplicación de varios criterios de pureza. La determinación de ácidos grasos requiere la preparación de los ésteres metílicos de los ácidos grasos (FAME) y su posterior análisis mediante cromatografía de gases con una buena repetibilidad y reproducibilidad. Entre los muchos métodos usados por los laboratorios de la industria y de los organismos oficiales de control, se seleccionaron los siguientes: 1) metilación en frío con potasa metanólica y 2) metilación en caliente con metilato sódico seguido de acidificación con ácido sulfúrico en metano1 y calentamiento. Se realizó una evaluación estadística de la precisión de la composición de ácidos grasos obtenidos usando ambos métodos de metilación, mediante un estudio colaborativo siguiendo las indicaciones recogidas por la AOAC (AOAC 1995). En aceites con baja acidez, los resultados obtenidos usando ambos métodos de metilación fueron equivalentes. Sin embargo, en la muestra de aceite de orujo crudo de oliva (acidez 15.5%) se apreciaron diferencias significativas en la composición de ácidos grasos obtenidos usando ambos métodos. Finalmente, el uso del método de metilación en caliente no dio lugar a un aumento de la concentración de isómeros trans.

[EN] The fraudulent addition of hazelnut oil to olive oil can be only detected in high proportions (20-25%) using the A-stigmastenol and the difference between triacylglycerols of equivalent carbon number 42, determined experimentally by HPLC and calculated theoretically from the fatty acid composition (AECN42). A new method lies on a sequential comparison of the values of several algorithms with a database built with data obtained from genuine virgin olive oils. The algorithms are: LLLexp en función de %L; (LLL/OLLn)exp - (LLL/OLLn)teor, vs AECN44 and (ECN441LLL), vs %L; being: LLLnexpO LLnexpa nd ECN44ew the percentage of triacylglycerols determined by HPLC, LLLth, OLLnthWr and EcN44th,, the percentage of those calculated theoretically from the fatty acid wmposition; AECN44 the difference between the experimental and theoretica~v alue of ECN44; and finally %L the percentage of linoleic acid. The database has been built considering the values obtained from olive oils of diierent fatty acid composition and from admixtures between them. The method allows the detection of low percentages of hazelnut oil in olive oii ( 5%)., [ES] La adición fraudulenta de aceite de avellana en aceite de oliva puede ser detectada sólo en altas proporciones (20–25 %), usando el D7-estigmastenol y la diferencia entre los triglicéridos con número de carbono equivalente igual a 42, determinados experimentalmente por HPLC y teóricamente a partir de la composición de ácidos grasos (DECN42). Se propone un nuevo método que consiste en la comparación de los valores de varios algoritmos con una base de datos de valores experimentales obtenidos de aceites de oliva virgen genuinos. Estos algoritmos son: LLLexp en función de %L; (LLL/OLLn)exp - (LLL/OLLn)teor en función de DECN44 y (ECN44/LLL)exp en función de %L; siendo LLLexp, OLLnexp, y ECN44exp los porcentajes de los triglicéridos obtenidos por HPLC; LLLteor , OLLnteor, y ECN44teor los porcentajes de éstos calculados teóricamente a partir de la composición de ácidos grasos; DECN44 la diferencia entre el valor experimental y teórico del ECN44; y finalmente %L el porcentaje de ácido linoleico. La base de datos se ha recopilado considerando los valores obtenidos de aceites de oliva de diferentes composiciones de ácidos grasos y mezclas entre ellos. El método permite la detección de pequeños porcentajes (5 %) de aceites de avellana en aceites de oliva.

The free diacylglycerols (DAGs) in adipose tissue are involved in the metabolism of stored lipids and hence are related to the supply of fatty acids for other tissues. This paper describes a simple, fast, and reproducible method for the identification and quantification of different molecular species of DAGs in human adipose tissue. The method comprised solid-phase extraction on a diol-bonded phase column combined with capillary GC analysis of silylated DAG derivatives on a polar phase (65% phenylmethylsilicone). Separation of the DAGs was achieved based on chain length, isomeric structure (1,2- and 1,3-DAGs), and degree of unsaturation. The main DAGs were 1,2-OO, 1,2-OP, 1,2-LO and 1,2-LP. The composition was corroborated by analysis of the component fatty acids of the DAGs, 18:1(n-9), 16:0, and 18:2(n-6) being the three major fatty acids obtained.

[ES] Se presenta un método para la determinación conjunta de esteróles y dialcoholes triterpénicos (eritrodiol y uvaol) en aceites de oliva que consta de las etapas siguientes: obtención del insaponificable, fraccionamiento mediante cromatografía líquida de alta eficacia (CLAE) en régimen isocrático con detector de índice de refracción, preparación de derivados sililados y análisis por cromatografía de gases en columna capilar. Se comparan cuatro procedimientos que utilizan el análisis por cromatografía de gases de las fracciones de esteróles y de esteróles + dialcoholes obtenidas tanto por CLAE como por CCF. Se lleva a cabo un análisis colaborativo para la determinación de esteróles comparando los métodos CLAE-esteroles+dialcoholes y CCFesteroles (método oficial). Los resultados mostraron que por el primer procedimiento la determinación de A7-esteroles se realiza con mayor repetibilidad y reproducibilidad, aunque los valores resultantes son más altos. No se encontraron diferencias en los parámetros de precisión ni en los valores de los restantes esteróles. Las determinaciones de eritrodiol+uvaol por los métodos CLAE-esteroles+dialcoholes y CCF-esteroles +dialcoholes (método oficial) resultaron con similar precisión., [EN] A method is developed for the analysis of sterols together with triterpenic alcohols (erythrodiol and uvaol) in vegetable oils. The method involves obtention of the unsaponifiable matter, isocratic HPLC separation with refractive index detector, preparation of silylderivatives, and analysis by capillary GC. Four procedures involving GC analysis of sterol and sterol+dialcohol fractions obtained by HPLC and CCF separations are compared. A collaborative trial comparing HPLC-sterols+dialcohols and CCF-sterols (official) methods is carried out. For A7-sterols, higher values and better repeatability and reproducibility were observed in the first method, but no differences for the remainder sterols were found. The erythrodiol+uvaol determinations by HPLC-sterols+dialcohols and CCF-sterols+dialcohols (official) methods resulted in similar precision.

[ES] En este trabajo se ha estudiado la viabilidad de la segunda centrifugación utilizada como sistema para mejorar el agotamiento de los orujos producidos por el nuevo sistema de elaboración de aceite de oliva virgen por centrifugación en dos fases así como su influencia en las principales características analíticas de los productos y subproductos que intervienen en el proceso. Para ello se han analizado las muestras obtenidas en los ensayos comparativos de primera y segunda centrifugación en fresco y de primera, segunda y tercera centrifugación de masas de aceitunas centrifugadas y almacenadas. Se ha realizado también un ensayo de decoloración de los aceites obtenidos, con el fin de ver cómo afectan estos procesos a los distintos sistemas de refinación (física y química). Los resultados obtenidos ponen de manifiesto, fundamentalmente, la necesidad de realizar la segunda centrifugación en fresco y a moderadas temperaturas para obtener aceites con parámetros de calidad y pureza dentro de los límites establecidos en la reglamentación para aceites de oliva virgen., [EN] In this work, the feasibility of a second centrifugation to improve the extraction of the olive paste, in the new two phases system for the extraction of virgin olive oil, has been studied. The study of its influence on the characteristics of the products and byproducts obtained has been carried out. The samples of comparative assays between the first and second centrifugation of recently obtained olive paste and between the first, with second and third centrifugation of stored olive paste has been analyzed. Therefore, a decoloration assay has been developed to study its influence on the different refination processes (physical and chemical procedures). Results obtained evidence the importance of achieving the second centrifugation with the recently obtained olive paste and under moderate temperatures. In these conditions, it is possible to obtain oils with parameters of quality and purity into the legal limits.

[ES] Algunos de los métodos analíticos oficiales de la Unión Europea y del Consejo Oleícola Internacional para determinar características de los aceites de oliva no presentan datos sobre su precisión. Con la participación de 22 laboratorios españoles del gobierno y de la industria, se ha llevado a cabo un estudio colaborativo de los métodos analíticos siguientes: absorción de luz UV, a 232 nm (K232), composición de ácidos grasos, trilinoleína, y triglicéridos con número equivalente de carbonos 42 (NEC42). Los datos de repetibilidad y reproducibilidad se han obtenido mediante estudios estadísticos de los resultados según la norma ISO 5275/1986 (E). También se ha estudiado el efecto de la purificación de los aceites en columna de silica gel sobre los valores de K232 y triglicéridos. Finalmente se han comparado las composiciones de ácidos grasos obtenidas utilizando los métodos de reacción con potasa alcohólica en frío y de reacción en caliente con metilato sódico., [EN] Some analytical methods for the determination of olive oil characteristics included in the European Union and International Olive Oil Council regulations lack precision data. To overcome such inconvenient a collaborative trial was set up including 22 laboratories from governmental institutions and industries. UV absorption at 232 nm, fatty acid composition, trilinolein and triglycerides with 42 equivalent carbon number were the tried methods. Repeatability and reproducibility data were obtained using the statistical method included in the International Standard ISO 5275/1986 (E).The effect of oil purification through a silica gel column on K232 and triglycerides was also studied. Finally for fatty acid determination, methylations by cold methanolic potash and by heating with methylated sodium were compared.

[ES] Se presentan los resultados de un estudio colaborativo realizado con la participación de laboratorios de la Administración Pública y de industrias del Sector Oleícola con el objeto de determinar la precisión de los métodos analíticos que se utilizan para la cuantificación de ciertos componentes de los aceites de oliva. Esteroles totales, composición esterólica, isómeros trans de ácidos grasos, ceras y estigmastadienos han sido determinados en muestras de aceites de oliva virgen, de oliva y de orujo de oliva, mediante la aplicación de los métodos analíticos indicados en el reglamento CEE 2568/91 de la Unión Europea y posteriores modificaciones, así como en el Documento TC20/11 del Consejo Oleícola Internacional (1993). Se ha efectuado un análisis estadístico de los resultados de acuerdo con la norma ISO 5725-1986 (E)., [EN] The results of a collaborative study with the participation of Governmental and Olive Oil Industry laboratories is presented. This study was carried out to determine the precision of the analytical methods used for quantifying certain components of olive oils. Samples of virgin olive oil, olive oil and residue olive oil were analysed for total sterols, sterolic composition, trans isomers from fatty acids, waxes and stigmastadienes, using the analytical methods included in the Commission Regulation EEC 2568/91 of the European Union and modifications therein, and in Document TC20/11 of the International Olive Oil Council (1993). The results have been statistically procesed according to the standard ISO 5725-1986 (E).

[ES] Entre los métodos analíticos aceptados oficialmente por la Unión Europea para su aplicación a los aceites de oliva y los aceites de orujo de oliva, las determinaciones de esteroles, trilinoleína, isómeros trans de los ácidos grasos y ceras han mostrado algunas dificultades en su aplicación. El estudio de dichos problemas ha permitido establecer diversas modificaciones y recomendaciones que facilitan la práctica de los métodos y mejoran la fiabilidad de los resultados., [EN] Among the analytical methods officially adopted by the European Union to be applied to olive oils and olive pomace oils, some troubles have been found in the application of the procedures for the determination of sterols, trilinolein, trans isomers of fatty acids and waxes. The study of such difficulties suggests several modifications and recommendations facilitating the performance of methods and improving the reliability of results.

The lipid composition of rat caecal mucosa, including the fatty acid composition of phospholipids and triacylglycerols, has been examined by capillary gas chromatography. Thirty-seven peaks were resolved, ranging in chain length from 12 to 24 carbon atoms. Preliminary identification of fatty acids by comparison with authentic standards was confirmed by gas chromatography—mass spectrometry using electron-impact ionization. The neutral and polar components were examined. Fatty acid methyl esters were quantified in absolute amounts with respect to the percentage of total phospholipid and triacylglycerols. The results show significantly higher levels of 16:0, 18:0, 18:1(n − 9), 18:1(n − 7), 18:2(n − 6) and 20:4(n − 6) in phospholipids, and higher levels of 16:0, 18:1(n − 9) and 18:2(n − 6) in triacylglycerols. On the other hand, analysis of caecal triacylglycerols revealed sn-glycerol-palmitate-oleate-palmitate, sn-glycerol-palmitate-linoleate-palmitate and sn-glycerol-palmitate-linoleate-oleate as major components.

Pyrolysis-gas chromatography-mass spectrometry studies of fulvic and humic acids from different soils: Calcic Rhodoxeralf, Typic Xerochrept, Typic Chromoxeret, Andic Hapludult, Humic Haplorthod, Typic Haplorthod and a Canadian Orthic Humo-Ferric Podzol were made. Samples were pyrolyzed at 700ªC for 10 sec, and low boiling point compounds were separated in a Chromosorb 102 column. Peaks were identifies with a mass spectrometer coupled to the gas chromatograph. Major components from soil humic acids were related to proteins, namely methane, ethene, ethane, propene, propane, butene, acetonitrile, benzene, pyridine, pyrrol, toluene, and phenol, and to polysaccharides, such as acetaldehyde, acetone, furan, methylfuran, 2-butanone, and furfural. Soil fulvic acids gave pyrograms with major peaks of methane, ethene, acetaldehyde, acetone, furan, acetic acid, 2-butanone, benzene, toluene, and furfural, which are characteristic for soil polysaccharides. Bromomethane, an unusual compound, was found in the humic fractions from podzolic soils.