Improved method for the synthesis of trans-feruloyl-beta-sitostanol

Phytosterols and phytostanols are known to lower low-density lipoprotein-cholesterol (LDL-C) levels in humans by up to 15%, and at least two products, Benecol and Take Control, are now on the market as naturally derived fatty acid esters of phytostanols (stanol esters) and phytosterols (sterol esters), respectively. A synthetic process was developed to synthesize gram quantities of trans-feruloyl-beta-sitostanol from ferulic acid and beta-sitostanol, with high purity and yields of approximately 60%. The process involves (a) condensation of trans-4-O-acetylferulic acid with the appropriate phytostanol or phytostanol mixture in the presence of N,N-dicyclohexylcarbodiimide and 4-(dimethylamino)pyridine, (b) separation of the trans-4-O-acetylferuloyl products by preparative liquid chromatography, (c) selective deacetylation of the feruloyl acetate, and (d) chromatographic purification of the feruloylated phytostanols. The process was successfully applied to synthesize stanol trans-feruloyl esters from "Vegetable Stanols", a mixture of approximately 70:30 beta-sitostanol and beta-campestanol, in comparable purity and yield.     

Phase transitions, solubility, and crystallization kinetics of phytosterols and phytosterol-oil blends

The thermal properties, solubility characteristics, and crystallization kinetics of four commercial phytosterol preparations (soy and wood sterols and stanols) and their blends with corn oil were examined. Differential scanning calorimetry (DSC) revealed narrow melting peaks between 138 and 145 degrees C for all phytosterol samples, reversible on rescan. Broader and less symmetrical melting transitions at lower temperatures with increasing oil content were observed for two samples of phytosterol-oil admixtures. The estimated, from the solubility law, deltaH values (34.7 and 70.7 mJ/mg for wood sterols and stanols, respectively), were similar to the DSC experimental data. Fatty acid esters of soy stanols differing in the chain length of the acyl groups (C2-C12) exhibited suppression of the melting point and increase of the fusion enthalpy with increasing chain length of the acyl group; the propionate ester exhibited the highest melting point (Tm: 151 degrees C) among all stanol-fatty acid esters. Solubility of phytosterols in corn oil was low (2-3% w/w at 25 degrees C) and increased slightly with a temperature rise. Plant sterols appeared more soluble than stanols with higher critical concentrations at saturation. The induction time for recrystallization of sterol-oil liquid blends, as determined by spectrophotometry, depended on the supersaturation ratio. The calculated interfacial free energies between crystalline sediments and oil were smaller for sterol samples (3.80 and 3.85 mJ/m2) than stanol mixtures (5.95 and 6.07 mJ/m2), in accord with the higher solubility of the sterol crystals in corn oil. The XRD patterns and light microscopy revealed some differences in the characteristics among the native and recrystallized in oil phytosterol preparations.     

Simultaneous analysis of free phytosterols/phytostanols and intact phytosteryl/phytostanyl fatty acid and phenolic acid esters in cereals

An approach based on solid-phase extraction for the effective separation of free phytosterols/phytostanols and phytosteryl/phytostanyl fatty acid and phenolic acid esters from cereal lipids was developed. The ester conjugates were analyzed in their intact form by means of capillary gas chromatography. Besides free sterols and stanols, up to 33 different fatty acid and phenolic acid esters were identified in four different cereal grains via gas chromatography-mass spectrometry. The majority (52-57%) of the sterols and stanols were present as fatty acid esters. The highest levels of all three sterol and stanol classes based on dry matter of ground kernels were determined in corn, whereas the oil extract of rye was 1.7 and 1.6 times richer in fatty acid esters and free sterols/stanols than the corn oil. The results showed that there are considerable differences in the sterols/stanols and their ester profiles and contents obtained from corn compared to rye, wheat, and spelt. The proposed method is useful for the quantification of a wide range of free phytosterols/phytostanols and intact phytosteryl/phytostanyl esters to characterize different types of grain.     

Plant Sterols in Cereals and Cereal Products

The total plant sterol contents (free sterols and covalently bound structures) of the main cereals cultivated in Finland were determined. Furthermore, sterol contents were determined for different flour and bran fractions in the milling process of wheat and rye, as well as plant sterol contents in various milling and retail bakery products. The sample preparation procedure included acid and alkaline hydrolysis to liberate sterols from their glycosides and esters, respectively. Free sterols were extracted and, after recovery using solid‐phase extraction, derivatized to trimethylsilyl ethers for gas chromatography (GC) analysis. We used GC with a mass spectrometer (MS) for identification. When two cultivars of rye, wheat, barley, and oats grown in the same year were compared, the highest plant sterol content was observed in rye (mean content 95.5 mg/100 g, wb), whereas the total sterol contents (mg/100 g, wb) of wheat, barley, and oats were 69.0, 76.1, and 44.7, respectively. In addition, the 10 rye cultivars and breeding lines compared had total sterol contents of 70.7–85.6 mg/100 g. In the milling process of rye and wheat, the plant sterols fractionated according to the ash content of the corresponding milling product. In all cereal grain and milling product samples, sitosterol was the main sterol. The level of stanols differed in the different milling process samples; it was lower in the most refined rye and wheat flours (≈15%) than in the bran fractions (≈30% in the bran with 4% ash content). Rye bread with whole meal rye flour as the main or only ingredient was a good source of sterols. Sterol content was higher than that of wheat bread, whereas plant sterol content of other bakery products was affected by the type and amount of fat used in baking.     

Characterization of triterpene alcohol and sterol ferulates in rice bran using LC-MS/MS

Ferulic acid esters of triterpene alcohols and sterols in rice bran oil have been extensively studied and reported to possess important pharmacological actions. Inconsistent results on the numbers and structures of ferulates have been reported, primarily because of the analytical procedures employed. Conventional methods for analysis of phytosterol content in oil are carried out by characterization of trimethylsilylated derivatives (TMS) using GC-EI-MS after saponification of oils or individual compound isolated from oils. This study developed an LC-MS/MS method for the direct analysis of triterpene alcohol and sterol esters in rice bran oil. In addition to verifying the results of previous research, nine new relatively polar triterpene alcohol and sterol esters were characterized by their retention behaviors in LC and ESI-MS data from both negative- and positive-ion mode. This is the first evidence for the presence of hydroxylated ferulate esters and caffeate esters as part of gamma-oryzanol in rice bran. The method enables rapid and direct on-line characterization of triterpene alcohol and sterol esters in oils. LC-MS/MS equipped with reverse-phase LC and ESI-MS should be well-suited for identification and quantification of the polar metabolites of phytosterols in biological fluids after consumption of rice bran oil or other oils.     

Relationship Between Fecal Indicators in Sediment and Recreational Waters in a Danish Estuary

The European Union has introduced a new bathing water directive where future classification of recreational waters will be based on the microbial parameters Escherichia coli, and intestinal enterococci. Introduction of enterococci as a new quality parameter may pose a challenge in some areas because relatively less is known about these organisms compared to E. coli. In the present study, the relative abundance of intestinal enterococci, E. coli, and ten fecal sterol and stanol biomarkers were investigated in water and sediment at two estuarine beach sites affected by fecal pollution. In the bathing water, enterococci were relatively more abundant at low E. coli concentrations. In the sediment, enterococci were generally more abundant than E. coli with surface concentrations between 1.0?×?10² and 4.5?×?10³ CFU cm^?3. Enterococci populations were relatively similar in water and sediment, and were phenotypically different from that of nearby pollution sources. The putative human specific genetic marker esp in Enterococcus faecium was not detected in water or sediment samples despite occasional inputs of human waste from storm water overflows. Sterol and stanol profiles suggested a direct link between water and sediment pollution profiles on days with wind conditions that facilitated resuspension. Sediment resuspension may occur at wind speeds exceeding 6–8 m s^?1, and could contribute significantly to enterococci concentrations in the overlying water. The study emphasized that recontamination of the water column due to wind induced resuspension should be considered when evaluating indicator levels and microbial hazards in estuarine recreational waters.     

GC-based analysis of plant stanyl fatty acid esters in enriched foods

Approaches for the capillary gas chromatographic (GC) based analysis of intact plant stanyl esters in enriched foods were developed. Reference compounds were synthesized by enzyme-catalyzed transesterifications. Their identities were confirmed by means of mass spectrometry. Using a medium polar trifluoropropylmethyl polysiloxane stationary phase, long-chain plant stanyl esters could be separated according to their stanol moieties and their fatty acid chains. Thermal degradation during GC analysis was compensated by determining response factors; calibrations were performed for ten individual plant stanyl esters. For the analysis of low-fat products (skimmed milk drinking yogurts), the GC separation was combined with a "fast extraction" under acidic conditions. For fat-based foods (margarines), online coupled LC-GC offered an elegant and efficient way to avoid time-consuming sample preparation steps. The robust and rapid methods allow conclusions on both, the stanol profiles and the fatty acid moieties, and thus provide a basis for the authentication of this type of functional food ingredients.     

Simultaneous determination and differentiation of glycidyl esters and 3-monochloropropane-1,2-diol (MCPD) esters in different foodstuffs by GC-MS

The aim of this study was the development of a method for the simultaneous determination and differentiation of fatty acid esters of 3-monochloropropane-1,2-diol (3-MCPD esters) and glycidol (glycidyl esters) in different foodstuffs. The esters were isolated from fat-rich food samples using a single extraction step and separated from interfering substances. For differentiation of 3-MCPD esters and glycidyl esters the glycidol moiety was converted into 3-methoxypropane-1,2-diol (3-MPD) by acidic alcoholysis. Subsequent determination was achieved by isotope dilution GC-MS after transesterification using an isotope-labeled 3-MCPD ester as internal standard. During optimization of the procedure, critical parameters affecting simultaneous determination and differentiation of these analytes were investigated. Rapid ester cleavage and derivatization at ambient temperature proved to be essential for the simultaneous determination of these analytes. The method was validated for various fat-rich foodstuffs such as bakery products, sweets, gravy, and soup powders as well as edible fats and oils. LODs of 8 and 15 μg/kg (fat-rich foodstuffs) as well as 50 and 65 μg/kg (edible oils and fats) were obtained for 3-MCPD esters and glycidyl esters, respectively. Recoveries for 3-MCPD esters and glycidyl esters ranged within 98 ± 4 and 88 ± 2% in all tested foodstuffs (0.05-2.5 mg/kg) and within 99 ± 16 and 93 ± 13% for edible oils and fats (0.15-3 mg/kg) over a wide concentration range. These results proved an accurate and differentiated determination of 3-MCPD esters and glycidyl esters with successful application to the fast screening of samples, avoiding tedious and laborious sample preparation.     

Gas chromatography/electron-capture negative ion mass spectrometry for the quantitative determination of 2- and 3-hydroxy fatty acids in bovine milk fat

2- and 3-hydroxy fatty acids (2- and 3-OH-FAs) are bioactive substances reported in sphingolipids and bacteria. Little is known of their occurrence in food. For this reason, a method suitable for the determination of OH-FAs at trace levels in bovine milk fat was developed. OH-FAs (and conventional fatty acids in samples) were converted into methyl esters and the hydroxyl group was derivatized with pentafluorobenzoyl (PFBO) chloride to give PFBO- O-FA methyl esters. These derivatives with strong electron affinity were determined by gas chromatography interfaced to mass spectrometry using electron-capture negative ion in the selected ion monitoring mode (GC/ECNI-MS-SIM). This method proved to be highly sensitive and selective for PFBO-O-FA methyl esters. For the analysis of samples, two internal standards were used. For this purpose, 9,10-dideutero-2-OH-18:0 methyl ester (ISTD-1) from 2-OH-18:1(9 c) methyl ester as well as the ethyl ester of 3-PFBO-O-12:0 (ISTD-2) was synthesized. ISTD-1 served as a recovery standard whereas ISTD-2 was used for GC/MS measurements. The whole-sample cleanup consisted of accelerated solvent extraction of dry bovine milk, addition of ISTD 1, saponification, conversion of fatty acids into methyl esters by use of boron trifluoride, separation of the methyl esters of OH-FAs from nonsubstituted FAs on activated silica, conversion of OH-FAs methyl esters into PFBO-O-FA methyl esters, addition of ISTD-2, and measurement by GC/ECNI-MS-SIM. By this method, ten OH-FAs were quantified in bovine milk fat with high precision in the range from 0.02 +/- 0.00 to 4.49 +/- 0.29 mg/100 g of milk fat.     

Mass spectrometric identification and gas-liquid chromatographic determination of 2-chloroethyl esters of fatty acids in spices and foods.

The 2-chloroethyl esters of 5 fatty acids have been identified in spice and food samples by gas-liquid chromatography-mass spectrometry (GLC/MS). Twenty-four spice samples were analyzed for the 2-chloroethyl esters of fatty acids by AOAC official multiple residues pesticide procedure using GLC with microcoulometric detection. The esters of capric, lauric, myristic, palmitic, and linoleic acids have been identified at levels up to 1400 ppm. 2-Chloroethyl linoleate was the most abundant ester in all samples. Several foods analyzed by the same procedures showed levels of 2-chloroethyl linoleate as high as 35 ppm. Recoveries from fortified samples ranged from 84 to 98% for the various esters. A method using an acid-catalyzed esterification reaction was developed to rapidly determine the fatty acid content of these spices. GLC analysis with microcoulometric detection was used. Recoveries from fortified samples ranged from 92 to 110%. After 2 spice samples found to be free of 2-chloroethyl esters were fumigated with ethylene oxide, the level of 2-chloroethyl linoleate reached 77 ppm. All levels of 2-chloroethyl esters were confirmed by GLC/MS.     

基于高光谱与协同克里金的土壤耕作层含水率反演

为研究土壤耕作层(0~40 cm)含水率的空间分布情况,利用EO-1的Hyperion传感器高光谱数据,对研究区域(106°20′~109°19′E,40°19′~41°18′N)的表层(0~10 cm)含水率进行定量反演,并利用表层含水率反演结果作为协同克里金插值的协变量,同时利用103个采样点实测的耕作层含水率作为主变量,进行协同克里金插值。结果表明:通过特征指数法提取水分反演的敏感波段集中在1 295~2 224 nm波长区间,特征指数法模型校正的相关系数r0.5但模型验证的精度较低(r0.2);通过偏最小二乘法建模,模型校正的r0.8,模型验证的r0.5,效果较好;运用协同克里金插值时,将反演的表层含水率作为协变量,可以弥补主变量耕作层含水率样本点少,变异函数欠稳定的缺点,同时,所提取理论模型的块金值(C0)与基台值(C0+C)的比值均25%,随机因素比例小,模型稳健。此外,协同克里金插值方法与利用表层与耕作层含水率线性拟合进行预测相比,能够有效提高预测精度,决定系数r2和Nash效率系数(nash-sutcliffe modelling efficiency,NSE)分别提高72.6%和89.9%,因此,将高光谱反演与协同克里金方法相结合,可以综合两者优势,节约采样成本,提高预测效率。     

Solid Phase Extraction Method for Selective Determination of Phthalate Esters in the Aquatic Environment

The use of solid phase extraction and capillary GLC provides thebasis for the selective determination of phthalate ester plasticizers in rivers and marine water samples. Of the severalsolvent ratios (methanol in dichloromethane) used for selective elution of phthalate esters from the C18 solid phase glass catridge, the 50/50 ratio, CH₃OH in CH₂Cl₂ (v/v)gave the best result. The method was tested on river and marinewater samples that receive effluent from industries that use phthalate esters. The rivers and marine water samples were grossly polluted as several phthalate esters, for example, dimethyl (DMP), diethyl (DEP), dibutyl (DBP) and diethylhexyl (DEHP)were present at 0.03–2306±9.4 μg L^-1. A study on uncontaminated water was done to establish background levels.     

The effect of calcium nutrition on the sterol content of tobacco roots and their susceptibility to Phytophthora parasitica var. nicotianae

The influence of nutrient solutions containing low (25 mg/1) and high (250 mg/1) calcium (Ca) concentrations on the total lipid, free sterol, sterol glycoside and steryl ester content and on the subsequent susceptibility of three flue‐cured tobacco cultivars to black shank, incited by Phytophthora parasitica var. nicotianae was investigated. The total lipid, total sterol and free sterol content differed appreciably among root systems of the three cultivars grown at high Ca. Roots of plants grown at low Ca contained relatively higher proportions of steryl glycosides and lower proportions of free sterols than roots of plants grown at high Ca, although the absolute sterol concentrations also differed significantly among the roots of cultivars grown at low Ca. Black shank disease development was significantly lower in roots of susceptible plants grown at low Ca than in roots of the same cultivar grown at high Ca. The innate susceptibility of the cultivars were not related to the lipid or sterol content. The reduced disease development of plants grown at low Ca appeared to be associated with changes in membrane permeability, which may be correlated with changes in the various sterol fractions.     

Quantitative ester analysis in cachaca and distilled spirits by gas chromatography-mass spectrometry (GC-MS)

An analytical procedure for the separation and quantification of ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl lactate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, isoamyl octanoate, and ethyl laurate in cachaca, rum, and whisky by direct injection gas chromatography-mass spectrometry was developed. The analytical method is simple, selective, and appropriated for the determination of esters in distilled spirits. The limit of detection ranged from 29 (ethyl hexanoate) to 530 (ethyl acetate) microg L(-1), whereas the standard deviation for repeatability was between 0.774% (ethyl hexanoate) and 5.05% (isoamyl octanoate). Relative standard deviation values for accuracy vary from 90.3 to 98.5% for ethyl butyrate and ethyl acetate, respectively. Ethyl acetate was shown to be the major ester in cachaca (median content of 22.6 mg 100 mL(-1) anhydrous alcohol), followed by ethyl lactate (median content of 8.32 mg 100 mL(-1) anhydrous alcohol). Cachaca produced in copper and hybrid alembic present a higher content of ethyl acetate and ethyl lactate than those produced in a stainless-steel column, whereas cachaca produced by distillation in a stainless-steel column present a higher content of ethyl octanoate, ethyl decanoate, and ethyl laurate. As expected, ethyl acetate is the major ester in whiskey and rum, followed by ethyl lactate for samples of rum. Nevertheless, whiskey samples exhibit ethyl lactate at contents lower or at the same order of magnitude of the fatty esters.     

Extreme variability of steroid profiles in cow feces and pig slurries at the regional scale: implications for the use of steroids to specify fecal pollution sources in waters

Thirty-five samples of cow feces (cowpat and cow manure) and pig slurries subjected to different treatment processes and different storage times before land spreading were extracted and analyzed by gas chromatography-mass spectrometry to determine their fecal stanol profiles. The fresh pig slurry data presented here increase considerably the classical range of values obtained for steroid ratios, resulting in an overlap with the range for cow feces. These results lead to the inability to distinguish species source of feces on the basis of steroid ratios alone. The cause of these differences is not known, although it appears likely to be related to differences in the metabolism of animals in relation to their age and/or variations in diet, rather than to secondary mechanisms of steroid degradation during storage or/and treatment of the feces. Nevertheless, the specificity of steroids to serve as a tool to differentiate cow feces from pig slurries is restored by considering the fecal stanol profile, notably, the six most diagnostic stanol compounds, which are 5β-cholestan-3β-ol (coprostanol), 5β-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3β-ol (campestanol), 24-ethyl-5α-cholestan-3β-ol (sitostanol), 24-ethyl-5β-cholestan-3β-ol (24-ethylcoprostanol), and 24-ethyl-5β-cholestan-3α-ol (24-ethylepicoprostanol). In this study, chemometric analysis of the fingerprint of these six stanols using principal components analysis (PCA) distinguished pig slurries from cow feces. The application of PCA to the stanol profiles, as developed in this study, could be a promising tool for identifying the animal source in fecal contamination of waters.     

Chemical composition of abaca (Musa textilis) leaf fibers used for manufacturing of high quality paper pulps

The chemical composition of leaf fibers of abaca (Musa textilis), which are commonly used for high-quality paper pulp production, was thoroughly studied. The results revealed that the lignin content was 13.2% of the total fiber. The analysis of abaca fibers by pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS) released predominantly compounds arising from lignin and p-hydroxycinnamic acids, with high amounts of 4-vinylphenol. The latter compound was demonstrated to arise from p-coumaric acid by pyrolysis of abaca fibers in the presence of tetramethylammonium hydroxide, which released high amounts of p-coumaric acid (as the methyl derivative). Products from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) propanoid units, with a predominance of the latter (H:G:S molar ratio of 1.5:1:4.9), were also released after Py-GC/MS of abaca fibers. Sinapyl and coniferyl acetates, which are thought to be lignin monomer precursors, were also found in abaca. The extractives content of the abaca fiber (0.4%) was low, and the most predominant compounds were free sterols (24% of total extract) and fatty acids (24% of total extract). Additionally, significant amounts of steroid ketones (10%), triglycerides (6%), omega-hydroxyfatty acids (6%), monoglycerides (4%), fatty alcohols (4%), and a series of p-hydroxycinnamyl (p-coumaric and ferulic acids) esterified with long chain alcohols and omega-hydroxyfatty acids were also found, together with minor amounts of steroid hydrocarbons, diglycerides, alpha-hydroxyfatty acids, sterol esters, and sterol glycosides.     

Phytosterols in sea buckthorn (Hippopha? rhamnoides L.) berries: identification and effects of different origins and harvesting times.

Sterols in seeds, pulp/peel fractions, and whole berries of sea buckthorn (Hippopha? rhamnoides L.) samples belonging to two major subspecies (sinensis and rhamnoides) from Finland and China were analyzed as TMS derivatives by gas chromatography-mass spectrometry after saponification of the oils. The total sterol contents in the seeds, the fresh pulp/peel, and the whole berries were 1200-1800, 240-400, and 340-520 mg/kg, respectively. The corresponding values in the extracted oils were 12-23, 10-29, and 13-33 g/kg. Sitosterol constituted 57-76 and 61-83%, respectively, of the seed and pulp/peel sterols. The sterol content and composition showed little variation between subspecies and collection sites. Different harvesting dates showed significant effects on the levels of some sterols both in the seeds and in the pulp/peel. The sterol profiles obtained are useful for characterizing sea buckthorn and detecting adulterations of the valuable oils. The information provided by the present investigation is also important for further chemical investigation of sea buckthorn sterols and industrial utilization of the berries as a raw material of functional foods.     

可见-近红外光谱联合随机蛙跳算法检测生物柴油含水量

生物柴油是一种优质清洁柴油,可从各种生物质中提炼,其特有的优势受到越来越广泛的关注。该文应用可见-近红外光谱技术原理对生物柴油的含水率进行了检测。配置含水率分别为0、2.5%、5.0%、7.5%和10.0%的试验样品并获取可见-近红外光谱,进行主成分分析,观察不同含水率生物柴油的聚类性,并采用Random Frog算法进行特征波段的提取,最后采用随机蛙跳算法(Random Frog)挑选出的特征波段作为偏最小二乘回归(partial least squares regression,PLSR)和最小二乘支持向量机(least squares-support vector machine,LS-SVM)模型的输入量,建立生物柴油含水量的预测模型。结果发现:采用Random Frog提取出的8条特征波段(563、560、642、565、562、493、559和779 nm)所建立非线性模型LS-SVM所得到的结果较好,其中Random Frog-LS-SVM的结果中R均大于0.95,校正集均方根误差RMSEC=0.722,预测集均方根误差RMSEP=0.520。结果表明采用Random Frog-LS-SVM模型可以准确的预测生物柴油的含水量,为实际应用提供参考。     

Relation of acidity and sensory quality with sterol content of olive oil from stored fruit

Composition of the sterol fraction, fatty acid, acidity, and the sensorial evaluation of virgin olive oils were studied in two eastern Spanish varieties grown and processed under the same conditions. Fruits were stored at 5 degrees C and ambient temperature for different times. During fruit storage, there was no significant variation (P = 0.05) in fatty acid composition. However, the sterol composition of the oil varied markedly (in particular, there was an increase in stigmasterol), acidity increased, and there was a very significant decrease in sensorial quality. The stigmasterol content presented a high correlation with the acidity and sensory evaluation (P < 10(-)(6)). The total sterol content increased gradually with olive storage time. Oils with stigmasterol greater than campesterol are graded to a low level (lampant). It is of interest that sensorial quality is revealed by stigmasterol content, a fact unknown until now.     

Changes in key odorants of raw coffee beans during storage under defined conditions

During storage of raw coffee beans (green coffee) atypical odors may develop, which are suggested to influence the aroma of particularly the coffee beverage. To gain insight into the aroma compounds responsible for such odor changes, a comparative aroma extract dilution analysis was applied on unstored, raw Arabica coffee beans from Colombia (water content=11.75%) and on the same beans with a water content of 13.5%, which were stored for 9 months at 40 degrees C. In combination with the flavor dilution (FD) factors, the results of the identification experiments showed strong increases in (E)-beta-damascenone (cooked apple-like), 2-methoxy-4-vinylphenol (clove-like), and methyl 2-methyl- and methyl 3-methylbutanoate (fruity), whereas others, such as the earthy smelling 3-isopropyl-2-methoxypyrazine as well as 2-phenylethanol and 3-methoxyphenol, remained unchanged during storage. In addition, the previously unknown coffee odorant 2-methoxy-5-vinylphenol (intense smoky odor) increased significantly during storage. Quantitative measurements performed on raw coffee samples stored at various temperatures, water contents, and oxygen availabilities indicated that the significant increase of, in particular, the methyl esters of 2- and 3-methylbutanoic acid were responsible for the pronounced and fruity odor quality perceived in the stored green coffee, whereas the higher concentrations of 2-methoxy-4-vinylphenol and 2-methoxy-5-vinylphenol led to the more pronounced smoky, clove-like odor quality. On the basis of the results obtained, in particular the reduction of the water content in combination with lower temperatures can be suggested to avoid aroma changes in raw coffee beans caused by storage.