Direct methylation procedure for converting fatty amides to fatty acid methyl esters in feed and digesta samples

Two direct methylation procedures often used for the analysis of total fatty acids in biological samples were evaluated for their application to samples containing fatty amides. Methylation of 5 mg of oleamide (cis-9-octadecenamide) in a one-step (methanolic HCl for 2 h at 70 degrees C) or a two-step (sodium methoxide for 10 min at 50 degrees C followed by methanolic HCl for 10 min at 80 degrees C) procedure gave 59 and 16% conversions of oleamide to oleic acid, respectively. Oleic acid recovery from oleamide was increased to 100% when the incubation in methanolic HCl was lengthened to 16 h and increased to 103% when the incubation in methoxide was modified to 24 h at 100 degrees C. However, conversion of oleamide to oleic acid in an animal feed sample was incomplete for the modified (24 h) two-step procedure but complete for the modified (16 h) one-step procedure. Unsaturated fatty amides in feed and digesta samples can be converted to fatty acid methyl esters by incubation in methanolic HCl if the time of exposure to the acid catalyst is extended from 2 to 16 h.     

Contribution of primary organic matter to the fatty acid pool in agricultural soils

Fatty acids as major compounds of soil lipids may affect many soil properties, but the input and turnover rates in soil are largely unknown. The objective of this study was to identify and quantify fatty acids in soils as a result of input from primary sources such as plant residues, farmyard manure and soil organisms, and to evaluate the corresponding turnover- and stabilization processes. The concentrations of n-C_10:0 to n-C_34:0 fatty acids were determined in the Ap horizon of a Phaeozem with long-term cropping of rye and maize and the treatments ‘Unfertilized’ (‘U’) and fertilized with ‘Farmyard manure’ (‘FYM’). The most important primary sources of fatty acids such as rye and maize stubble and roots, soil micro- and mesofauna, and the applied FYM were also investigated. The quantification of fatty acids by gas chromatography/mass spectrometry (GC/MS) showed that long-term FYM application led to larger concentrations of n-alkyl fatty acids in the plots grown with rye (‘U’: 48.1 μg g⁻¹, ‘FYM’: 57.7 μg g⁻¹, **P≤0.01, n=3) and maize (‘U’: 17.0 μg g⁻¹, ‘FYM’: 23.4 μg g⁻¹, ***P≤0.001, n=3). The observed bimodal fatty acid distribution in soils from n-C_10:0 to n-C_21:0 and from n-C_21:0 to n-C_34:0 with a predominance at n-C_16:0 and at n-C_28:0 was apparently due to input from crop residues, soil organisms and FYM. The short-chain lengths may have originated from the investigated primary sources. The major contributors to the long-chain lengths, with a maximum at n-C_28:0, were rye stubble and FYM. A change in mono-culture from rye to maize, 38 years prior to sampling, led to a decrease in fatty acid concentrations by factors of about 2.8 (‘U’) and 2.5 (‘FYM’). Therefore, rye-derived fatty acids and soil tillage had a larger impact on fatty acid pools than the input of primary organic matter. The changes in fatty acid distributions and pools under the consideration of the quantified input of primary organic matter led to the conclusion that the short-chained fatty acids were more rapidly decomposed than the long-chains.     

Fractionation of humic acid by organic solvents

TJURIN (1) had proposed the use of organic solvent to fractionate humus. In his method, he extracted soils with benzene-alcohol (1 : 1) mixture in the SOXHLET's apparatus. The extract was dried at 80°C and weighed.     

Succession of microbiota estimated by phospholipid fatty acid analysis and changes in organic constituents during the composting process of rice straw

The succession of responsible microbiota during the composting process of rice straw (RS) was studied for 145 d in relation to the changes in the organic constituents of RS. During the composting process, the C / N ratio of RS decreased from 56 to 22. On a C basis, the relative contents of lipid, water-soluble organic matter (WSOM), hemicellulose, cellulose, and lignin fractions in RS changed from 5.6, 8.9, 32.9, 17.9, and 34.0%, to 7.3, 5.8, 30.7, 3.8, and 51.1%, respectively, indicating that the cellulose fraction was mainly decomposed in the composting process. Biomass C accounted for 18.3% (on day 75 when the total amount of phospholipid fatty acids (PLFAs) reached a peak) and 11.5% (at the end of composting) of the total C of RS under the composting process. As for PLFAs, the biomarkers of fungi and Gram-negative bacteria predominated in the RS material used. At the thermophilic stage (the first 2 weeks), biomarkers of Gram-positive bacteria and actinomycetes predominated. After the thermophilic stage, biomarkers of other Gram-positive bacteria became dominant. Finally, at the curing stage, the proportion of the biomarkers of Gram-negative bacteria and eukaryotes increased, indicating the co-contribution of Gram-positive and Gram-negative bacteria and fungi in the decomposition process at this stage. The trans / cis ratio of 16 : 1ω7 PLFA of RS under the composting process ranged from 0.18 to 0.30, indicating that the composting process of RS prepared a significantly lower environmental stress (p < 0.01) compared to the decomposition of RS in a submerged paddy soil.     

Authentication of organic feed by near-infrared spectroscopy combined with chemometrics: a feasibility study

Organic products tend to retail at a higher price than their conventional counterparts, which makes them susceptible to fraud. In this study we evaluate the application of near-infrared spectroscopy (NIRS) as a rapid, cost-effective method to verify the organic identity of feed for laying hens. For this purpose a total of 36 organic and 60 conventional feed samples from The Netherlands were measured by NIRS. A binary classification model (organic vs conventional feed) was developed using partial least squares discriminant analysis. Models were developed using five different data preprocessing techniques, which were externally validated by a stratified random resampling strategy using 1000 realizations. Spectral regions related to the protein and fat content were among the most important ones for the classification model. The models based on data preprocessed using direct orthogonal signal correction (DOSC), standard normal variate (SNV), and first and second derivatives provided the most successful results in terms of median sensitivity (0.91 in external validation) and median specificity (1.00 for external validation of SNV models and 0.94 for DOSC and first and second derivative models). A previously developed model, which was based on fatty acid fingerprinting of the same set of feed samples, provided a higher sensitivity (1.00). This shows that the NIRS-based approach provides a rapid and low-cost screening tool, whereas the fatty acid fingerprinting model can be used for further confirmation of the organic identity of feed samples for laying hens. These methods provide additional assurance to the administrative controls currently conducted in the organic feed sector.     

Rapid determination of free fatty acids in poultry feed lipid extracts by SB-ATR FTIR spectroscopy

A simple, rapid, and reproducible method has been developed for the quantitative determination of free fatty acid (FFA) content in lipids extracted from poultry feeds by Fourier transform infrared (FTIR) spectroscopy with the use of a single-bounce attenuated total reflectance (SB-ATR) accessory. An FTIR calibration curve was prepared by gravimetrically adding oleic acid (15-37%) to pure refined, bleached, and deodorized (RBD) canola oil and measuring the area of the COOH absorption band at 1710 cm-1. The oil from each of 12 poultry feed formulations was extracted using conventional Soxhlet extraction, and after evaporation of the solvent, the FFA content was determined by the conventional AOCS titrimetric procedure and by the SB-ATR/FTIR method. The SB-ATR/FTIR FFA predictions were related to those determined by the AOCS titrimetric method by linear regression, producing an R value of 0.999 and a SD of +/-0.28% FFA. Time-course spectra collected as lipids extracted into hexane indicated that a 15 min extraction was adequate to obtain a representative sample for FFA determination, with further extraction resulting in little, if any, change in the proportion of FFA in the lipid extract. Only a small volume of the hexane extract ( approximately 20 mL) yielded sufficient material for the SB-ATR/FTIR analysis. Thus, by shortening the extraction time and taking a small sample so as to reduce solvent removal time, the SB-ATR/FTIR procedure provides a very simple and rapid means of determining the FFA content of poultry feed lipids.     

基于近红外分析技术检测大豆脂肪酸含量的研究

为探索近红外光谱技术在大豆脂肪酸测试中的应用,寻找一种快速的检测方法。以黑龙江省各地的25份大豆品种为材料,采用Perten8620型近红外光谱仪对搜集到的样品进行扫描并得到光谱数据,采用多元线性回归(MLR)和偏最小二乘法(PLS)对试验数据进行了多元统计分析。结果表明：在1700～2300 nm范围内检测大豆脂肪酸含量是可靠的,并且PLS模型的性能优于MLR模型。该文还对近红外仪中的滤光片组合作了初步探讨,表明不同的滤光片组合对测量精度有一定影响。     

乳酸菌发酵使木薯淀粉残渣饲料化研究

以处理木薯淀粉加工残渣,减轻环境压力为目的,对木薯渣接种乳酸菌复合系SFC-2,并添加不同量的蔗糖进行发酵,通过测定其发酵过程中的生化变化研究了其饲料化效果.结果显示:在发酵开始时接种处理的乳酸菌数为5×108,cfu/g,而对照处理没有检测到:发酵结束后,接种的3个糖水平处理的外观发酵品质比未接种处理好,但糖浓度低时丁酸积累较多,乳酸积累少,接种并添加4%蔗糖的处理乳酸积累量最高,发酵物中乳酸积累达18.7 g/kg, 接种并添加8%蔗糖的处理丁酸积累最少,仅为0.141 g/kg.相同蔗糖浓度下,接种处理的丁酸积累量均低于对照,不接种对照处理中均检测到了腐败菌分解蛋白质产生的甲胺,而接种的则没有.木薯渣材料和发酵物中的氰化物含量均远低于国家安全标准.采用乳酸菌发酵法使木薯渣饲料化是一个可行的途径.     

Determination of organic acid anions in precipitation by ion chromatography exclusion

A method for the determination of organic acid anions in precipitation samples is described. The technique of ion chromatography exclusion (ICE) is used to identify and quantify the more commonly found anions of the acids citric, formic and acetic in central Illinois rainwater. By using a two column system consisting of a separator and suppressor, method detection limits (MDL) of 0.03 mg L^?1 for each of the three anions are obtained. The total analysis time for quantitation is 24 min. Samples collected from early spring rains contained primarily formate and acetate ions with levels of concentration ranging from 0.03 to 0.44 mg L^?1. The organic acids formic and acetic contributed approximately 8% to the total acidity in the precipitation samples analyzed. Precision averaged 5% as the relative standard deviation (RSD) and analyte spikes of natural precipitation samples revealed a mean recovery of greater than 99%. A preservation technique using chloroform (CHCl₃) addition is discussed with results indicating its necessity in maintaining original organic anion concentrations.     

Changes in the fatty acid profile of the subcutaneous fat of swine throughout fattening as affected by dietary conjugated linoleic acid and monounsaturated fatty acids

The fatty acid profile of the subcutaneous fat of pigs and its evolution throughout fattening as affected by dietary conjugated linoleic acid (CLA), monounsaturated fatty acids (MUFA), and their interaction (CLAxMUFA) were studied. Three levels (0, 1, and 2%) of an enriched CLA oil (28% cis-9, trans-11 and 28% trans-10, cis-12 CLA) were combined with two levels of MUFA (low, 19% average; and high, 39% average) for pig feeding (288 gilts). Subcutaneous shot-biopsies were taken from 48 animals at the beginning of the trial (S1, 70 kg), 14 days later (S2, 80 kg), and at slaughter (S3, 107 kg). Inclusion of CLA in the diet caused an increase during fattening in cis-9, trans-11 CLA, trans-10, cis-12 CLA, and saturated fatty acids (SFA) contents of pig backfat and a decrease in MUFA and polyunsaturated fatty acids (PUFA). MUFA supplementation also led to a MUFA enrichment of backfat. The interaction CLAxMUFA affected the SFA content. The rates of accumulation of CLA isomers, SFA, and MUFA throughout the trial did not follow a linear behavior, such rates being higher from S1 to S2 than from S2 to S3. These rates were also influenced by dietary CLA and MUFA levels. The increase in the ratio of saturated to unsaturated fatty acids of backfat caused by dietary CLA might be balanced by supplementation of pig diets with MUFA.     

Temperature and substrate controls on microbial phospholipid fatty acid composition during incubation of grassland soils contrasting in organic matter quality

Soil incubations are often used to investigate soil organic matter (SOM) decomposition and its response to increased temperature, but changes in the activity and community composition of the decomposers have rarely been included. As part of an integrated investigation into the responses of SOM components in laboratory incubations at elevated temperatures, fungal and bacterial phospholipid fatty acids (PLFAs) were measured in two grassland soils contrasting in SOM quality (i.e. SOM composition), and changes in the microbial biomass and community composition were monitored. Whilst easily-degradable SOM and necromass released from soil preparation may have fuelled microbial activity at the start of the incubation, the overall activity and biomass of soil microorganisms were relatively constant during the subsequent one-year soil incubation, as indicated by the abundance of soil PLFAs, microbial respiration rate (r), and metabolic quotient (qCO₂). PLFAs relating to fungi and Gram-negative bacteria declined relative to Gram-positive bacteria in soils incubated at higher temperatures, presumably due to their vulnerability to disturbance and substrate constraints induced by faster exhaustion of available nutrient sources at higher temperatures. A linear correlation was found between incubation temperatures and the microbial stress ratios of cyclopropane PLFA-to-monoenoic precursor (cy17:0/16:1ω7c and cy19:0/18:1ω7c) and monoenoic-to-saturated PLFAs (mono/sat), as a combined effect of temperature and temperature-induced substrate constraints. The microbial PLFA decay patterns and ratios suggest that SOM quality intimately controls microbial responses to global warming.     

Lipase-catalyzed synthesis of fatty acid diethanolamides.

Diethanolamides are nonionic emulsifiers widely used in industries such as cosmetics and as corrosion inhibitors. Candida antarctica lipase (Novozym 435) was used to catalyze the amidation of various fatty acids with diethanolamine. Contents of fatty acids, metal ions, and water affected the yields of diethanolamides. Hexanoic acid was the best substrate among all acyl donors. Yields of hexanoyl diethanolamide (HADEA), lauroyl diethanolamide (LADEA), and oleoyl diethanolamide (OADEA), obtained after 24 h of lipase-catalyzed reaction at 50 degrees C and 250 rpm with 90 mM fatty acid and 360 mM diethanolamine in acetonitrile, were 76.5, 49.5, and 12.1%, respectively. Addition of 1 mM metal salts increased the yields of HADEA and LADEA. Kinetic analysis showed that the yields of HADEA and LADEA in lipase-catalyzed reactions were largely associated with the rate of the forward reaction constant k(1). Anhydrous enzyme was found to be the best for the amidation reaction. Study on the enzyme operational stability showed that C. antarctica lipase retained 95 and 85% of the initial activity for the syntheses of HADEA and LADEA, respectively (even after repeated use for 10 days). The reaction runs smoothly without the use of hazardous reactants, and the developed method is useful for the industrial application.     

分提橄榄油中主要脂肪酸成分傅里叶变换红外光谱法测量（英）

为了测量从橄榄油中分提的高、低熔点油脂的脂肪酸成分,在4 000～600 cm-1 的范围测量了31个含有不同脂肪酸成分植物油的傅里叶变换红外光谱,用于建立偏最小二乘（PLS）回归分析校正模型。在油脂的傅里叶变换红外光谱变量和脂肪酸组成变量之间建立了交叉验证的PLS校正模型。为了校正油酸和亚油酸含量,在4?000～600 cm-1 的频率范围,经平滑,二阶导数,规范化处理的红外光谱获得了最好的交叉验证校正模型和最佳的预测结果。PLS校正模型预测结果表明,与高熔点橄榄油（油酸,72.29%,亚油酸,9.98%）相比,低熔点橄榄油含有较高的油酸含量和亚油酸含量（油酸,77.46%,亚油酸,12.51%）,预测的结果与气相色谱测量的结果有很好的一致性。建立的PLS校正模型预测橄榄油的不饱和脂肪酸含量具有较好的相关性。该研究为分提油脂质量的判别评价提供了便捷的方法。     

Synthesis of fatty acid esters by recombinant Staphylococcus epidermidis lipases in aqueous environment

Various flavor esters were obtained by using recombinant lipases from Staphylococcus epidermidis as a catalyst in an aqueous environment. These esters were enzymatically synthesized to overcome the problems associated with chemical processes. This study showed that the S. epidermidis lipases could catalyze ester synthesis from decyl alcohol and fatty acids of different chain length. The wild-type and mutant lipases (M419A and V649I) could efficiently catalyze the synthesis of decyl alcohol esters of unsaturated fatty acids. In contrast, the yield of decyl laurate was better by wild-type and mutant enzyme V6491, but mutant enzyme M419A only favored the synthesis of decyl myristate. The esterification of oleic acid and various carbon-chain-length alcohols from ethanol to hexadecanol increased up to decanol by wild-type and M419A mutant enzymes and reached an optimum for dodecanol by V6491 mutant enzyme. The enzyme is potentially useful in food industries such as dairy product flavoring.     

高温分解与乳酸菌分步发酵提高秸秆饲料消化率及适口性

以提高秸秆饲料的消化率和适口性为目的,该研究1)以干玉米秸秆为材料,接种木质纤维素分解菌复合系WDC2,进行高温分解发酵;2)以乳酸菌复合系SFC-2为接种物,进行乳酸菌液体发酵;3)将乳酸菌发酵液以质量体积比1∶1比例均匀喷洒到发酵秸秆中,制备发酵秸秆饲料。该研究从营养学和分子生态学角度探讨了高温分解与乳酸菌分步发酵提高秸秆饲料消化率和及适口性的可行性。结果表明,经高温分解发酵,秸秆中微生物多样性丰富,不含致病菌。以南阳黄牛为研究对象,秸秆的干物质、中性洗涤纤维和酸性洗涤纤维体外消化率分别提高了13.94%、22.56%和21.12%,采食量提高21.71%;部分分解的秸秆经乳酸菌发酵液吸附后,粗蛋白增加36.17%。秸秆的高温分解发酵与乳酸菌的乳酸发酵相结合的饲料制作工艺,即提高了秸秆的消化率和营养价值,也改善了秸秆的适口性。     

Selectivity of Candida antarctica B lipase toward fatty acid and (Iso)propanol substrates in esterification reactions in organic media

Fatty acid (FA) selectivity of immobilized Candida antarctica B lipase was assessed as influenced by various cosubstrate systems for ester synthesis. Reaction mixtures contained a homologous series of even-chain n-acyl donor (C(4)(-)(16)) substrates (FA or their methyl esters, FAME) and a single alcohol cosubstrate (propanol, 2-propanol, or their acetate derivatives) in hexane. Multiple FA optima were often observed, with preferences for C(6) (or C(4)) followed by C(14) and sometimes C(10). The degree of selectivity among acyl donors was modest (up to 1.28-2.60, based on ratios of selectivity constants) and was dependent on the choice of cosubstrate system. Acyl group selectivity ranged up to 1.31-1.36 for [FA + alcohol], 1. 48-2.60 for [FAME + alcohol], 1.30-1.72 for [FA + alcohol acetate], and 1.28-1.88 [FAME + alcohol acetate] reaction systems. General shifts in selectivity were observed between short-chain (C(4)(-)(8)) and long-chain (C(10)(-)(16)) FA as groups with propanol cosubstrate, whereas shifts in reaction selectivity were observed toward specific FA(s) for 2-propanol cosubstrate. Selectivity among a series of alcohol cosubstrates ranged up to 13-fold in esterification reactions with C(6) FA.     

Amino acid composition of soil organic matter

This study investigated the amino acid composition of soil organic matter extracted from ten surface soils in addition to surface soils from two long-term cropping systems [continuous corn (CCCC), corn-soybean-corn-soybean (CSCS), and corn-oats-meadow-meadow (COMM)] at two sites in Iowa: the Clarion-Webster Research Center (CWRC) and the Galva-Primghar Research Center (GPRC). Results showed that, with the exception of asparagine pluse aspartic acid and glutamine plus glutamic acid, the other 13 amino acids studied, expressed as perecentages of total amino acids extracted, were generally very uniform among the soils. The total amino acids extracted from the ten soils were significantly correlated with organic carbon (C) ( and clay content (, but not with total nitrogen (N), pH, or sand content. Expressed as percentages or organic C and N in soils, the amounts extracted ranged from 10.9% to 32.4% and from 12.0% to 27.4%, respectively. The amino acid N identified, expressed as percentages of organic N extracted, ranged from 32% to 50% and the C/N ratios of the extracted organic matter ranged from 10.1 to 14.9. The type of rotation did not significantly affect the total amino acid content of the soils from the same N treatment, but it did affect the total amino acid content of soils from the control plots. The total amino acids measured under the different crop rotations at the CWRC site were in the order: COMM>CCCC>CSCS. The order for the GPRC site was: CSCS>COMM>CCCC. The amino acid N identified, expressed as percentages of organic N extracted from soils at the CWRC site, ranged from 33.1% to 50% and for the GPRC site ranged from 26.5% to 51.4%. The C/N ratios of the organic matter extracted ranged from 10.4 to 14.1 and from 6.5 to 14.3 for the soils from CWRC and GPRC sites, respectively. Received: 26 May 1997     

Sorption of dissolved organic nitrogen by acid subsoil horizons and individual mineral phases

Dissolved organic matter is important in translocation and export of nutrients from forest ecosystems. Its mobility in soil is restricted by sorption to mineral surfaces which depends on its chemical properties. Carboxyl and hydroxyl groups form strong bondings to mineral surfaces, whereas the role of N‐containing functional groups in the sorption process is less well understood. We examined in laboratory experiments the binding of dissolved organic matter from the forest floor to amorphous Al(OH)₃, goethite, kaolinite, and illite and to subsoils in order to compare the sorption and desorption of dissolved organic C with that of dissolved organic N. The mineral samples were equilibrated with acidic solutions of organic matter at pH 4. In the equilibrium solutions organic C and N and their contribution to two operationally defined fractions, namely the so‐called hydrophilic and hydrophobic fractions, were determined. We measured neutral and acidic amino sugars to discover the nature of the binding of organic N. Within the hydrophilic and hydrophobic fractions, the sorption and desorption of organic C and N did not differ, indicating that there was no preferential binding of N‐containing compounds. The hydrophilic fraction contained more N and sorbed less than the hydrophobic fraction, and so the overall retention of organic N by the mineral phases and subsoils was smaller than that of organic C. Among the amino sugar compounds, muramic acid was preferentially removed from the solution, whereas the neutral amino sugars were sorbed similar to organic C. The results suggest that the sorption of N‐containing compounds is favoured by acidic groups and not by amino groups.     

Hydrocarbon and fatty acid composition of cheese as affected by the pasture vegetation type

The determination of the geographical origin of dairy products is an ongoing issue. In this paper the effects of botanical diversity of two pastures on the hydrocarbon and fatty acid composition of cheese fat were studied, over 2 years of experimentation. Two areas in the Italian southwestern Alpine region, dominated by Trifolium alpinum (T) and Festuca nigrescens (F) vegetation, respectively, were chosen, and milk obtained from cows grazing on these pastures was used to produce a semihard traditional cheese. Cheese samples showed a significantly different composition of most linear hydrocarbons, odd-chain (C15, C17, and C17:1) and unsaturated (trans-11,cis-15-C18:2, C18:3, C20:4n-6, C20:4n-3, and 20:5n-3) fatty acids, according to pasture type. The ratio between C(29) and C(27) linear hydrocarbons, unlike the absolute content of the single molecules, showed a good discriminating ability between the two pastures and was little affected by the natural variability due to the climatic and environmental factors.     

Triacylglycerol and fatty acid compositions of French virgin olive oils. Characterization by chemometrics

There is no data concerning the fatty acid and triacylglycerol composition of French virgin olive oil. Thus, these compositions were determined using 564 samples coming from four olive harvests (1998-1999 to 2000-2001). Among these 564 samples, 372 came from the four main French cultivars (Aglandau, Cailletier, Picholine, and Salonenque) and from both of the oldest French protected designations of origin: "Nyons" (cv. Tanche) and "Vallée des Baux". The fatty acid compositions took the different isomeric monounsaturated fatty acids (C16:1 and C18:1) into account. The eicosenoic acid is gondoic acid (20:1n-9) and was determined by dimethyl disulfide adduct using GC/MS. The use of propionitrile as a mobile phase for the HPLC analysis of the triacylglycerols led to better resolutions between triacylglycerols than those resolutions obtained with the mix of solvents recommended by the normalized method (acetone/acetonitrile). Of the samples, 88 had a 9-heptadecenoic acid level (17:1n-8) higher than 0.3% and 33 had a linolenic acid level higher than 0.9%, which are maximal values accepted by the International Olive Oil Council and the European Union. A linear discriminant analysis was carried out on 372 samples with the SAS system and particularly with STEPISC and CANDISC procedures. Variables (n = 37) representing the different fatty acids, the sum of saturated, monounsaturated, and polyunsaturated fatty acids, squalene, and triacylglycerols were used, thus allowing us to classify samples into six groups defined with 100% of well classified samples. These results constitute an original data bank that can be used to identify the origin of virgin olive oils.